Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists with improved cellular activity

Article abstract of DOI:10.1016/j.bmcl.2006.04.009

The disruption of the p53-Hdm2 protein-protein interaction induces cell growth arrest and apoptosis. We have identified the 1,4-benzodiazepine-2,5-dione scaffold as a suitable template for inhibiting this interaction by binding to the Hdm2 protein. Several compounds have been made with improved potency, solubility, and cell-based activities.

Full text of DOI:10.1016/j.bmcl.2006.04.009

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3463–3468
Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists
with improved cellular activity
Kristi Leonard,* Juan Jose Marugan, Pierre Raboisson, Raul Calvo, Joan M. Gushue,
Holly K. Koblish, Jennifer Lattanze, Shuyuan Zhao, Maxwell D. Cummings,
Mark R. Player, Anna C. Maroney and Tianbao Lu*
Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, PA 19341, USA
Received 14 February 2006; revised 31 March 2006; accepted 3 April 2006
Available online 2 May 2006
Abstract—The disruption of the p53-Hdm2 protein–protein interaction induces cell growth arrest and apoptosis. We have identified
the 1,4-benzodiazepine-2,5-dione scaffold as a suitable template for inhibiting this interaction by binding to the Hdm2 protein.
Several compounds have been made with improved potency, solubility, and cell-based activities.
Ó 2006 Elsevier Ltd. All rights reserved.
p53 is a multifunctional protein that regulates cell prolif-
eration by induction of cell growth arrest or apoptosis in
response to DNA damage and/or stress stimuli.1 Muta-
tions in the p53 gene occur in about 50% of all human
tumors,2 and these mutations facilitate carcinogenesis.
However, there are an estimated 50% of human tumors
that have wild-type p53, although its function appears to
be inactivated. The human double minute 2 protein
(Hdm2) inhibits the transcriptional activity of p53 by
binding to its transactivation domain and producing
an autoregulatory feedback loop.3 Blocking this pro-
tein–protein interaction raises the levels of p53 and
brings the cell to growth arrest and apoptosis. The dis-
ruption of the binding between these two proteins is
therefore an attractive therapeutic target for the treat-
ment of wild-type p53 tumors.4
extra donor hydrogen bond interaction between the
amino group and the carbonyl of Val93 of Hdm2, as
shown in Figure 2. However, 2 was found to be less
potent than 1 on the p53 inducible gene 3 (PIG-3)
production cell-based assay, which is typically used as
a downstream protein marker to monitor levels of
p53.8 We hypothesize that this reduction of cell activity
of 2 versus 1 is a consequence of the zwitterionic charac-
ter of 2, which limits the cross-membrane permeability
and adversely modulates the availability of the com-
pound to its intended target Hdm2. To obtain com-
pounds that combine optimized potency, solubility,
and cell-permeability characteristics, we synthesized
novel 1,4-benzodiazepine-2,5-diones bearing (i) different
solubilizing groups in position 1 and (ii) functionalized
benzyl groups in position 4.
Although the strategy of inhibiting the p53-Hdm2 inter-
action has been demonstrated as feasible by a number of
groups,5 only a few have disclosed small molecules that
specifically block this pathway.6 We have recently
reported the 1,4-benzodiazepine-2,5-dione 1 (Fig. 1) as
a potent inhibitor of the p53-Hdm2 interaction.7 We
now report the improved potency observed from the
introduction of an ortho amino functionality to the ben-
zylic ring 2. We believe this increased potency (2 vs 1
FP-IC50 = 0.25 and 0.85 lM, respectively) is due to an
The synthesis of the target diazepines is reported in
Schemes 1–7. The synthesis of the benzodiazepine scaffold
could be achieved by the Ugi reaction, as shown in
Scheme 4, or starting from isatoic anhydride (Scheme
7). The synthesis of various benzylamines necessary for
the Ugi reaction is reported in Schemes 1–3. Accordingly,
benzaldehydes 3a–d were reduced to the corresponding
alcohols 4a–d using either sodium borohydride for those
compounds with a methylene group in the benzylic posi-
tion or methyl lithium for compounds with a methyl
group in the benzylicposition. Alcohols 4a–d were reacted
with phthalimide under Mitsunobu conditions to gener-
ate intermediates 5a–d, which were deprotected with
hydrazine to yield amines 6a–d.
Keywords: Hdm2; p53; Benzodiazepinedione.
*
Corresponding authors. Tel.: +1 610 458 8959; e-mail addresses:
kleonar1@prdus.jnj.com; tlu3@prdus.jnj.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.009

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K. Leonard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3463–3468
Cl
Compds
R
FP
IC50 μM
PIG-3 production @
R
O
1.1 μM 3.3 μM 10 μM 30 μM
I
N
Cl
1
2
H
0.85
0.25
60
79.7
208.7
484.3
N
O
NH2
44
77
O
OH
Figure 1. IC50 and JAR cell normalized PIG-3 production (ELISA, ng/mg) of our previously published benzodiazepinediones.
OH
OH
Gln72
O
ii
i
I
O
O
O
Cl
Cl
NO2
13
14
O
N
N
O
NH2
iii
N
H
O
NH2
O
O
Val93
Leu54
Cl
Cl
NO2
NO2
Cl
Cl
15
6f
Figure 2. Schematic for the binding of the benzodiazepinedione 2 to
Hdm2.
Scheme 3. Reagents and conditions: (i) concd H2SO4, KNO3, 0 °C to
rt, 16 h; (ii) DPPA, THF, t-BuOH, reflux, 3 h; (iii) TFA, CH2Cl2, rt,
1 h.
R1 R2
R1
O
R1 OH
R2
O
i or ii
N
H
iii
Cl
O
Cl
Cl
C-
N+
O
O
3a-d
4a-d
5a-d
I
H
OH
6a-f, i, ii
R1
R1 R2
+
+
NH
Cl
iv
NH2
NO2 Me
F
O
O
a
c
Cl
H
b
R2
16
17
18
6a-d
Me
d
Scheme 1. Reagents and conditions: (i) NaBH4, THF, rt, 1 h; (ii)
MeLi, THF, À78 °C to rt, 0.5 h; (iii) phthalimide, Ph3P, DIAD, THF,
rt, 3 h; (iv) H2NNH2, THF, 80°C, 1 h.
O
O
R1
R1
I
I
N
N
iii
Cl
Cl
or iv, v
O
N
N
O
O
R2
O
O
H
F
F
H
i,ii
N
iii, iv
N
H
19
20
F
Cl
Cl
O
Cl
F
F
F
9
8
O
7
R1
I
vi
N
NO2
NO2
O
Cl
vi
v
F
F
N
NH2
N
H
R2
O
F
Cl
Cl
21-32
(for R1 and R2, see table 1, 2 and 3)
F
F
10
6e
Scheme 2. Reagents and conditions: (i) MeLi, THF, À78 °C to rt, 1 h;
(ii) phthalimide, Ph3P, DIAD, THF, À78 °C to rt, 0.5 h; (iii) H2NNH2,
THF, 80 °C, 1 h; (iv) pyridine, trifluoroacetic anhydride, 0 °C to rt,
16 h; (v) concd H2SO4, KNO3, 0 °C to rt, 16 h; (vi) LiOH, THF–H2O,
rt, 3 days.
Scheme 4. Reagents and conditions: (i) MeOH, rt, 2 days; (ii) AcCl, rt
to 60 °C, 1 h; (iii) chromatographic separation of diastereoisomers, Br-
R2, Bu4NI, K2CO3, THF, 80 °C, 2 h; (iv) 1-bromo-4-chlorobutane,
K2CO3, THF, 80 °C, 2 h; (v) 1-methylpiperazine, K2CO3, THF, 80 °C,
2 h; (vi) NH4Cl, Zn0, AcOH, 80 °C, 3 h.

K. Leonard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3463–3468
3465
O
O
O
i
ii
H
i, ii
OH
OH
iii, iv
OH
H
Cl
Cl
N
Cl
Cl
N
Cl
Cl
Cl
N
Cl
Cl
OH
33
34
39
40
41
OH
v
iv
Cl
iii
OH
NH2
Cl
NH2
Cl
O
Cl
O
Cl
N
N
35
36
42
43
Scheme 5. Reagents and conditions: (i) borane-methyl sulfide
complex, THF, 0 °C to reflux, 16 h; (ii) DDQ, CH2Cl2, rt, 4 h; (iii)
3-bromopropene, CsCO3, THF, reflux, 3 h; (iv) NaBH4, THF, rt, 2 h;
(v) SOCl2, CH2Cl2, rt, 1 h.
Scheme 6. Reagents and conditions: (i) n-BuLi, diisopropylamine,
DMF, THF, À78 °C to rt, 1 h; (ii) NaBH4, THF, rt, 1 h; (iii) NH3,
Cu0, EtOH, 120 °C, 6 h; (iv) HCl (gas), SOCl2, CH2Cl2, rt, 1 h.
was deprotected with lithium hydroxide to give the
target benzylamine 6e.
The synthesis of the amine 6e is reported in Scheme 2.
Aldehyde 7 was reacted with methyl lithium followed
by Mitsunobu reaction with phthalimide to give
intermediate 8, which was successively deprotected with
hydrazine, followed by reprotection with trifluoroacetic
anhydride to give amide 9. Subsequent nitration of 9
with potassium nitrate in concentrated sulfuric acid
provided the corresponding nitro derivative 10, which
The synthesis of cyclopropylamine 6f is depicted in
Scheme 3. The commercially available acid 13 was
nitrated with concentrated nitric acid to give 14,
followed by a Curtius rearrangement to produce
carbamate 15, which was deprotected in the presence
of TFA to give amine 6f.
Cl
O
O
O
I
O
I
I
O
i
ii
OH
NH2
N
H
N
H
O
O
NH2
44
45
46
O
O
I
NH
I
NH
O
iii
iv
Cl
Cl
N
N
H
O
47
48
O
v
O
vi
Cl
Cl
Cl
N
vii
NH2
O
OH
O
O
O
I
I
I
N
N
N
Cl
Cl
Cl
N
N
N
O
O
O
O
O
O
O
O
O
49
50
51
Scheme 7. Reagents and conditions: (i) triphosgene, DIEA, THF-CH2Cl2, rt, 16 h; (ii) amino-(4-chlorophenyl)acetic acid methyl ester, NEt3, DMF,
100 °C, 10 h; (iii) t-BuOK, THF, rt, 1 h; (iv) 1-bromo-2-(2-methoxyethoxy)ethane, K2CO3, THF, 80 °C, 2 h; (v) 43, CsCO3, THF, 80 °C, 3 h; (vi) 36,
CsCO3, THF, 80 °C; (vii) Cl2PdP(Ph3)2, NaBH4, THF, rt, 1 h.

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K. Leonard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3463–3468
The benzylamines 6a–f were engaged in a four-com-
ponent Ugi reaction with 4-chlorobenzaldehyde 17,
Boc-protected anthranilic acid 16, and 1-isocyanocy-
clohexene 18 (reaction inducer), which after treat-
ment with acetyl chloride and in situ cyclization
provided the target diazepinediones 19 as diastereo-
meric mixtures. Chromatographic separation of dia-
stereomers and subsequent alkylation with the
corresponding solubilizing groups afforded nitroderiv-
atives 20, which were reduced with zinc, to produce
target compounds 21–32, as racemic mixtures
(Scheme 4).
In Scheme 6, 2,5-dichloropyridine 39 was converted to
the corresponding aldehyde 40 in the presence of LDA
and DMF. Reduction of the aldehyde with sodium
borohydride gave alcohol 41, which was treated with
ammonia to selectively displace the chloride in position
2 leading to intermediate 42. Transformation of the ben-
zylic alcohol to the chloride 43 was achieved by treat-
ment with thionyl chloride.
Alkylation of diazepine 48 with the two chloro deriva-
tives 43 and 36 afforded 4-substituted derivatives 49
and 50, respectively (Scheme 7). Subsequent unmasking
of the phenol was achieved by treatment of 50 with sodi-
um borohydride and dichloro bis (triphenylphos-
phine)palladium (II) to give phenol 51.
Iodoisatoic anhydride 45 (Scheme 7) was used as
starting material in an alternative route to the
synthesis
of
substituted
benziodiazepinediones.
Accordingly, 45 was reacted with (amino)(4-chloro
phenyl)acetic acid methyl ester to provide the
3-substituted benzodiazepine core 47 followed by suc-
cessive substitution at the N4-position and then at
the N1-position (Scheme 7). Schemes 5 and 6 show
the synthesis of the benzylic chloride intermediates.
In Scheme 5, commercially available 4-chloro salicylic
acid 33 was reduced to the corresponding alcohol
with borane-methyl sulfide complex and then reoxidi-
zed to the aldehyde 34 using DDQ. Protection of
the phenol with allyl bromide and reduction back
to the alcohol yielded 35. Finally, conversion of the
alcohol to the chloride using thionyl chloride pro-
duced 36.
Tables 1–3 show the IC50 values obtained in an FP as-
say8 and JAR cell normalized PIG-3 production.9 The
introduction of a methyl group on the benzylic position
of 52 to give 2 resulted in a 3-fold improvement in FP-
IC50 value (Table 1). Although this is a significant in-
crease in potency, there is no difference between the cel-
lular PIG-3 production of these two compounds at 1.1
or 3.3 lM. We hypothesized that this is due to the zwit-
terionic character of these compounds, which limits
their cell permeability. Accordingly, the replacement of
the valeric acid side chain of 2 with a non-charged solu-
bilizing group, such as the methoxyethoxyethyl side
chain of 21, led to a decrease in potency in the FP assay,
but showed better results in the PIG-3 production exper-
Table 1. FP-IC50 and JAR cell normalized PIG-3 production (ELISA, ng/mg) of analogues bearing ethylene glycol in position 1 as the solubilizing group
R1
O
N
I
Cl
N
R2
O
Compound
R2
R1
FP-IC50
(lM)
PIG-3 (lM) at
1.1
47
3.3
10
Cl
O
O
52
2
0.81
74
77
196
OH
OH
NH2
NH2
NH2
OH
O
Cl
Cl
Cl
Cl
Cl
0.25
0.70
2.44
44
O
O
O
O
21
49
50
51
78
218
74
375
99
O
62.3
O
O
O
>12.5
N
11.6
NH2

K. Leonard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3463–3468
3467
Table 2. FP-IC50 and JAR cell normalized PIG-3 production (ELISA, ng/mg) of analogues bearing a morpholino side chain in position 1 as a
solubilizing group
R1
O
N
I
Cl
N
O
R2
Compound
R2
R1
FP-IC50 (lM)
PIG-3 (lM) at
1.1
44
3.3
10
Cl
O
2
22
23
27
28
26
24
25
0.25
0.79
3.6
2.6
2.7
8.6
25
77
234
184
110
54
NH2
NH2
NH2
OH
Cl
Cl
O
79
55
37
34
395
375
313
111
N
O
N
Cl
F
O
N
N
N
N
N
NH2
Cl
HO
Cl
O
O
O
O
NH2
F
Cl
Cl
NH2
19
iment, indicating that this compound is able to penetrate
the cell, unlike 2 and 52. The replacement of the ortho-
anilino substituent of 21, a hydrogen bond donor, with
the hydroxy group of 49 caused no significant loss of
activity (assuming 3-fold loss of activity due to the lack
of a benzylic methyl group). A significant decrease in
activity is observed, however, with the protected
hydroxyl group of 50, as well as more soluble 2-ami-
no-5-chloro-pyridine 51.
group is replaced by a methyl group (25), or a fluoro
group (26), a loss of activity is also observed. Similarly,
introduction of an electron-withdrawing fluoro substi-
tuent para to the amino group (27) resulted in a loss
of more than 3-fold potency. Similar loss of potency
was observed when the functional group in the para
position of the amine is an electron-donating hydroxyl,
as in 28.10
Table 3 shows compounds with solubilizing chains
bearing N-methylpiperazine or dimethylamino groups.
Compounds with morpholino side chains as a solubiliz-
ing group are shown in Table 2. The introduction of
the morpholino group attached to the template with
a two-carbon (22) or three-carbon (23) linker yielded
an increase in PIG-3 production. Further modifications
to the benzylic moiety of these morpholine derivatives
were attempted to improve cell potency. Replacing
the methyl group on the benzylic position with a cyclo-
propyl and moving the amino hydrogen bond donor to
the meta position, as in 24 result in a significant loss of
activity. When the ortho-amine hydrogen bond donor
Although, introduction of
a
butyl and propyl
dimethylamines 29 and 31, and the butylpiperazine 32
led to compounds with reduced cell activity, 4-methyl-
piperazinopropyl derivative 30 was found to be equipo-
tent to the methoxyethoxyethyl 21 and morpholinoethyl
22 derivatives.
In summary, we have reported novel 1,4-benzodiaze-
pine-2,5-diones with potent disrupting capacity of the
binding between p53 and Hdm2. The SAR analysis

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K. Leonard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3463–3468
Table 3. FP-IC50 and JAR cell normalized PIG-3 production (ELISA, ng/mg) of analogues bearing N-methylpiperazine or dimethylamine functional
groups in position 1 as solubilizing groups
R1
O
N
I
Cl
N
R2
O
Compound
R2
R1
FP-IC50 (lM)
PIG-3 (lM) at
1.1
47
3.3
10
Cl
O
N
52
29
31
30
32
0.81
2.83
12.2
5.41
5.66
74
79
196
OH
NH2
NH2
NH2
NH2
NH2
Cl
Cl
Cl
Cl
32
36
57
43
À136
À92
385
32
N
N
68
N
198
125
N
N
Lukacs, C.; Klein, C.; Fotouhi, N. A.; Liu, E. A. Science
2004, 303, 844.
of the benzylic position shows that the amino group
in the ortho position can be replaced by a hydroxyl
group, but not by methyl or fluoro, validating our
hypothesis that hydrogen bond donor groups in this
position can establish an extra interaction with the
carbonyl of Val93 of Hdm2. Moreover, the replace-
ment of the valeric acid solubilizing group of 2 with
methoxyethoxyethyl (21), morpholinoethyl (22), and
4-methylpiperazinopropyl (30) resulted in the discov-
ery of new lead compounds with improved cell-based
activity as measured by the level of PIG-3
production.
7. (a) Grasberger, B. L.; Lu, T.; Schubert, C.; Parks, D. J.;
Carver, T. E.; Koblish, H. K.; Cummings, M. D.; LaFrance,
L. V.; Milkiewicz, K. L.; Calvo, R. R.; Maguire, D.;
Lattanze, J.; Franks, C. F.; Zhao, S.; Ramachandren, K.;
Bylebyl, G. R.; Zhang, M.; Manthey, C. L.; Petrella, E. C.;
Pantoliano, M. W.; Deckman, I. C.; Spurlino, J. C.;
Maroney, A. C.; Tomczuk, B. E.; Molloy, C. J.; Bone, R.
F. J. Med. Chem. 2005, 48, 909; (b) Parks, D. J.; LaFrance,
L. V.; Calvo, R. R.; Milkiewicz, K. L.; Gupta, V.; Lattanze,
J.; Ramachandren, K.; Carver, T. E.; Petrella, E. C.;
Cummings, M. D.; Maguire, D.; Grasberger, B. L.; Lu, T.
Bioorg. Med. Chem. Lett. 2005, 15, 765; (c) Raboisson, P.;
Marugan, J. J.; Schubert, C.; Koblish, H. K.; Lu, T.; Zhao,
S.; Player, M. R.; Maroney, A. C.; Reed, R. L.; Huebert, N.
D.; Lattanze, J.; Parks, D. J.; Cummings, M. D. Bioorg.
Med. Chem. Lett. 2005, 15, 1857.
References and notes
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8. The evaluation of the inhibitory activity of our com-
pounds is based on the displacement of a peptide
substrate attached in the N-terminus with fluorescein
(FP assay).
3. (a) Picksley, S. M.; Lane, D. P. Bioessays 1993, 15, 689; (b)
Piette, J.; Nell, H.; Marechal, V. Oncogene 1995, 15, 1001.
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9. In order to confirm that these compounds were stimulat-
ing p53 activity, we measured the induction of PIG-3 (p53
inducible gene 3) Polyak, K.; Xia, Y.; Zweier, J. L.;
Kinzler, K. W.; Vogelstein, B. Nature 1997, 389, 300, JAR
choriocarcinoma cells, which overexpress both Hdm2 and
p53, were treated with various antagonists and cellular
PIG-3 protein was measured by ELISA (Exalpha, Water-
town, MA). Results greater than 100 ng PIG-3 normalized
to total protein were considered positive for specific
activation of p53.
5. (a) Bottger, A.; Bottger, V.; Sparks, A.; Liu, W. L.;
Howard, S. F.; Lane, D. P. Curr. Biol. 1997, 7, 860; (b)
Blaydes, J. P.; Gire, V.; Rowson, J. M.; Wynford-Thomas,
D. Oncogene 1997, 14, 1859; (c) Wasylyk, C.; Salvi, R.;
Argentini, M.; Dureuil, C.; Delumeau, I.; Abecassis, J.;
Debussche, L.; Wasylyk, B. Oncogene 1999, 18, 1921.
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M.; Yin, D.; Liu, Z. Cancer Lett. 2002, 183, 69;
(b) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.;
Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.;
10. Compound 28 was isolated as a byproduct of the
reduction of the nitro functional group in the synthesis
of compound 27.