Discovery of novel dihydroimidazothiazole derivatives as p53-MDM2 protein-protein interaction inhibitors: Synthesis, biological evaluation and structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2012.08.086

Starting with Nutlins as an initial lead, we designed and generated bicyclic scaffolds aiming to place cis-bischlorophenyl moiety at the equivalent location where the hydrophobic interaction with MDM2 could be expected. As a result, we discovered novel MDM2 inhibitors possessing a dihydroimidazothiazole scaffold. Further exploration of the side chains on the dihydroimidazothiazole scaffold aided by molecular modeling resulted in compounds exhibiting almost comparable in vitro potency to Nutlin-3a.

Full text of DOI:10.1016/j.bmcl.2012.08.086

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6338–6342
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel dihydroimidazothiazole derivatives as p53–MDM2
protein–protein interaction inhibitors: Synthesis, biological evaluation
and structure–activity relationships
Masaki Miyazaki a, , Haruko Kawato a, Hiroyuki Naito a, Masahiro Ikeda a, Masaya Miyazaki b,
⇑
Mayumi Kitagawa b, Takahiko Seki b, Setsuko Fukutake b, Masashi Aonuma b, Tsunehiko Soga a
a Lead Discovery & Optimization Research Laboratories II, R&D Division, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Oncology Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting with Nutlins as an initial lead, we designed and generated bicyclic scaffolds aiming to place cis-
bischlorophenyl moiety at the equivalent location where the hydrophobic interaction with MDM2 could
be expected. As a result, we discovered novel MDM2 inhibitors possessing a dihydroimidazothiazole scaf-
fold. Further exploration of the side chains on the dihydroimidazothiazole scaffold aided by molecular
modeling resulted in compounds exhibiting almost comparable in vitro potency to Nutlin-3a.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 22 June 2012
Revised 10 August 2012
Accepted 22 August 2012
Available online 30 August 2012
Keywords:
MDM2
p53
Dihydroimidazothiazole
Protein–protein interaction inhibitor
The p53 tumor suppressor protein plays an important role in
the growth suppression and apoptosis to cancer cells.1 About 50%
of human cancers have wild-type p53,2 whose functions are regu-
lated by an overexpression or an amplification of human murine
double minute 2 (MDM2) gene.3,4 MDM2 combines with the N-ter-
minal transcriptional activation domain of p53, and promotes ex-
port of p53 from nucleus to cytoplasm, thereby promoting
proteasomal degradation of p53 via ubiquitination through its E3
ligase activity.5–7 Thus, activation of the p53 function by the inhi-
bition of the protein–protein interaction of p53–MDM2 is regarded
as an effective approach in cancer therapy. In fact, there have been
many reports regarding the relevance between MDM2 inhibition
and growth inhibition of cancer cells.8–10
In the last ten years, various small molecules (Fig. 1) which inhi-
bit p53–MDM2 interactions have been reported.11–16 The p53–
MDM2 binding inhibitory activity (IC50) of these compounds in
the reports are roughly 0.1–10
potent inhibitor reported first was Nutlin-3a (1) whose IC50 was
0.09 M. AM-8553 (3), which was just reported recently, had im-
lM on a cell free assay, among which
l
proved potency considerably by lead optimization researches from
their HTS hit compounds.17 We also focused our attention on these
compounds and investigated further in order to obtain a novel and
more potent molecule as a p53–MDM2 interaction inhibitor.
We report herein the synthesis of novel p53–MDM2 interaction
inhibitors having a dihydroimidazothiazole scaffold, together with
their p53–MDM2 binding inhibitory activities.
O
O
H
O
N
O
N
OH
OH
HO
Cl
Cl
NH
O
N
Cl
OH
NH
O
F
N
O
Cl
Cl
N
N
H
O
Cl
3 (AM-8553)
1 (Nutlin-3a)
2 (MI-219)
Figure 1. Previously reported small molecules as p53–MDM2 inhibitors.
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5740 3640.
E-mail address: miyazaki.masaki.p3@daiichisankyo.co.jp (M. Miyazaki).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.086

M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6338–6342
6339
cis-bischlorophenyl moiety
Trp23 pocket
1
R
Cl
Cl
Br
N
O
2
OH
N
3
N
2
R
5
6
N
S
cis
cis
O
N
N
O
Br
bicyclic scaffold
(dihydroimidazothiazole ring)
Phe19 pocket
Leu26 pocket
4 (Nutlin-2)
Figure 2. Designs of novel scaffold having a bicyclic skeleton.
O
O
Cl
Cl
Cl
Cl
Cl
Cl
R1
N
Cl
Cl
R1
N
H
N
(a)
(b)
O
(c)
OH
(d)
NH2
NH2
S
S
cis
cis
S
cis
cis
N
H
N
N
5
6
7a-g
8a-g
O
O
Cl
O
R1
O
R1
O
Cl
Cl
R1
(e)
(f)
O
O
O
N
N
N
O
O
O
O
S
NH
cis
b
10
11a-g
12a-g
9a-g
compda
R1
11a
b
methyl
ethyl
c
i-propyl
n-propyl
c-propyl
t-butyl
d
e
f
g
MeOCH2-
Scheme 1. Synthesis of C-3 substituted dihydroimidazothiazoles 9a–g. aReagents and Conditions: (a) CS2, EtOH, reflux, 70%; (b) Compound 12a–g, EtOH, reflux, 42–85%; (c)
NaOHaq., EtOH, reflux,; (d) 2-oxopiperazine, EDC/HCl, Et3N, CH2Cl2, 30–75% (2 steps); (e) (i) R1-COCl, Pyridine, CH2Cl2, (ii) EtOH, reflux; (f) SO2Cl2, CH2Cl2, 56–58% from 10.
b11e and 11g were just synthesized from 10. The others were utilized commercially available.
The interaction between p53 and MDM2 depends on van der
Waals’ forces, which is mainly accomplished by the intervention
of three hydrophobic residues of p53, that is side chains of
Phe19, Trp23, and Leu26.18–20 Therefore, proficient filling of the
hydrophobic pockets is very important to furnish potent inhibitors.
Specifically, the substituents should be carefully placed on a scaf-
fold so as to let them fit the pockets efficiently. Compounds dis-
played in Figure 1 are considered to apply the concept of the
drug design. The co-crystal structure of MDM2 and Nutlin-2 (4)
supports the analysis, in which two 4-bromophenyl groups and
ethoxyphenyl moiety on Nutlin-2 corresponded to the three
hydrophobic residues of p53.11
In the beginning of our research to obtain a novel scaffold for
p53–MDM2 interaction inhibitors, we analyzed the mode of inter-
action between MDM2 and bis-(4-chlorophenyl) groups of Nutlin-
3a (1). And we tried to place the cis-bischlorophenyl structures in
proper positions by synthesizing and evaluating various bicyclic
scaffolds having the moieties. We discovered that dihydroimidazo-
thiazole derivatives hold the inhibitory activity (IC50 <1
lead structure is exemplified in Figure 2.
lM). The
Then, our medicinal effort was moved to the optimization of the
substituent at the C-3 position. Syntheses of the C-3 variants are
depicted in Scheme 1. cis-1,2-Bis(4-chlorophenyl)ethane-1,2-dia-
mine (5) was synthesized in accordance with the literature
method.21 Cyclization with CS2,22 followed by thiazole formation
with
a
-chloro-b-ketoesters (12a–g),23 provided dihydroimidazo-
thiazole derivatives (7a–g). Compounds 11a–c and 11f are com-
mercially available, while 11e and 11g were prepared via
conventional methods using Meldrum’s acid.24 Hydrolysis then
gave the corresponding carboxylic acids (8a–g). EDC mediated
amidation with oxopiperazine25 furnished the amide derivatives
(9a–g) for screening.
As shown in Scheme 2, variants for the amide portion in place of
2-oxopiperazine were also synthesized (13–19).

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M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6338–6342
Table 1
O
O
Cl
Cl
Cl
Cl
Cell free p53–MDM2 inhibitory activity of analogues 9a–g
OH
R2
N
N
N
N
(a)
S
S
cis
cis
O
Cl
R1
O
N
N
N
S
NH
cis
8c
9c, 13-19
Cl
R2
compda
R1
IC50 (lM)
O
9a
9b
9c
9d
9e
9f
9g
Nutlin-3
Methyl
Ethyl
i-Propyl
n-Propyl
c-Propyl
t-Butyl
2.7
1.8
0.26
3.1
9.5
3.0
7.1
*
*
*
N
N
9c
15
16
O
NH
*
N
N
O
N
13
14
neutral
N
H
O
MeOCH2-
*
0.18b
N
N
a
O
All compounds were racemate.
In house data.
b
*
N
*
N
*
NH
17
18
19
N
OH
basic
N
(9e), methoxymethyl (9g) and pivaloyl (9f) groups also reduced
the activities (IC50s = 1.8–9.5 M) (Table 1).
N
l
Next, the result of the SAR for C-2 position (amide site), in
which C-3 substituent was fixed to the i-propyl group, is depicted
in Table 2. We introduced neutral or basic 6-membered rings into
C-2, referring to Nutlin’s side chain, for the purpose of evaluating
the efficiency of the newly generated scaffold. In this connection,
the optimization of the C-2 position will be reported elsewhere
in due course. As for neutral substituents, compound 15 having a
morpholino group showed potency almost equivalent to 9c
Scheme 2. Synthesis of C-2 substituted dihydroimidazothiazoles 13–19. aReagents
and Conditions: (a) amine, EDC/HCl, HOBt, Et3N, DMF or CH2Cl2, 30–64%.
We evaluated 9a–g to investigate the influence of C-3 moiety on
the activity, wherein the capacity of interacting pockets could be
assessed. IC50 values of these compounds were measured by an
ELISA in which GST-tagged MDM2 binds to p53 immobilized to
the surface of a 96-well plate. As a result, compound 9c bearing
(IC50 = 0.34
variants gave less potency at around 1
for N,N-dimethylcarbamoylpiperidine (14) diminished (IC50
4.4 M). Compounds 17 and 18, which have basic substituents,
were less active (IC50 = 1.8 and 1.2 M). On the other hand, com-
l
M). N-acetylpiperazine (13) or homopiperazine (16)
lM on IC50, whilst the one
i-propyl moiety only showed high potency (IC50 = 0.26
is almost comparable to the one of Nutlin-3 (racemate, IC50
0.18 M). Other compounds possessing smaller substituents than
lM), which
=
=
l
l
l
i-propyl groups such as methyl (9a) and ethyl (9b) showed weak
activity. In addition, substituents such as n-propyl (9d), c-propyl
pound 19 possessing 2,5-dimethylpiperazine, which is a rather
Table 2
Cell free p53–MDM2 inhibitory activity of analogues 13–19
O
Cl
2
R
N
N
S
cis
Cl
compda
R2
IC50
(l
M)
compda
R2
IC50
1.8
(lM)
O
*
*
N
N
N
9c
0.26
17
N
N
NH
OH
*
*
*
*
N
N
13
0.84
18
19
1.2
O
O
N
N
N
NH
14
15
16
4.4
0.14
*
*
N
N
0.34
1.1
O
O
Nutlin-2
0.14b
N
H
a
All compounds were racemate.
Lit. data (see Ref. 11).
b

M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6338–6342
6341
Table 3
two chlorophenyl groups and the i-propyl group of (+)-9c are fitted
with the three MDM2 pockets efficiently.
Chiral separation of compound 9c
O
With regard to the substituent at the C-2 position, it seems that
there is a slight difference between our lead and Nutlin’s. Com-
pound 9c with 2-oxopiperazine as Nutlin-3 kept potent activity,
whilst compound 17 having hydroxyethylpiperazine like Nutlin-2
(4) reduced the activity, which was inconsistent with Nutlin’s
SAR. For dihydroimidazothiazoles, incorporation of basic moiety
at the C-2 position resulted in reduction of activity exemplified
by compounds 17 and 18, while improvement of activity was ob-
served by introducing a weak basic moiety demonstrated by com-
pound 19. Thus, there seems to be more space and opportunity for
derivatization at the C-2 position, and for further improvement of
potency and physicochemical properties as well.
Cl
O
CHIRALCEL OD-H
Hexane-IPA
N
N
N
S
NH
(+)-9c
+ (-)-9c
cis
(enantiomers)
(+/-)-9c
(racemate)
Cl
compd
IC50 (lM)
(+/À)-9c
(+)-9c
0.26
0.16
(À)-9c
11.9
Nutlin-3a
Nutlin-3b
0.09a
13.6a
In conclusion, we discovered novel inhibitors of the p53–MDM2
interaction possessing a dihydroimidazothiazole scaffold. Espe-
cially 2-oxopiperazine (9c) and 2,5-dimethylpiperazine (19) vari-
ants possessed high activity, which was almost equivalent to
Nutlin’s. Moreover, since p53–MDM2 inhibitory activity was dras-
tically influenced by altering substituents at the C-2 or C-3 posi-
tions, further optimization aiming to discover more potent
inhibitors is considered feasible. Investigation and further optimi-
zation are now underway, and the results will be reported in due
course.
a
Lit. data (see Ref. 11).
weaker basic moiety, showed the most potent activity
(IC50 = 0.14 M).
l
These dihydroimidazothiazole derivatives upon which we are
reporting thus far were racemates, and each enantiomer could be
separated by using HPLC with a chiral column. With regard to Nut-
lin, each enantiomer was obtained by optical resolution of the
racemate, and only one of these showed potency.11 Thus we also
separated compound 9c [equal to (+/À)-9c] using chiral HPLC (CHI-
RALCELÒ OD-H, eluant: hexane-IPA), and acquired each enantio-
mer ((+)-9c and (À)-9c)26 (Table 3). Compound (+)-9c possessed
potent activity, in contrast, the IC50 of the other isomer (À)-9c
was found to be 1/85 times weaker. This result suggested the same
tendency as Nutlin’s SAR11; hence the mode of placing the two hal-
ophenyl groups with the dihydroimidazothiazole scaffold was con-
sidered to be almost equivalent to the Nutlin’s imidazole as
expected, although the absolute configurations of (+)-9c and (À)-
9c are yet to be determined. Co-crystal structure analysis of the
dihydroimidazothiazoles and MDM2 has not been conducted yet,
however, the mode of the interaction of our lead would be inter-
preted as follows: From our drug design which referred to the
structural configuration of Nutlins and the result of optical resolu-
tion, it is considered that Nutlin-3a and our active lead (+)-9c make
an interaction to MDM2 protein in the same manner. As shown in
Figure 3, (+)-9c illustrated with an estimated configuration, was
posed by superposition to the co-crystal structure of MDM2/Nut-
lin-2 (PDB code: 1RV1) using docking calculation. In this model,
Acknowledgment
The authors thank the SBDD group in our laboratory for per-
forming the modeling study.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.086.
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6342
M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6338–6342
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