Lead optimization of novel p53-MDM2 interaction inhibitors possessing dihydroimidazothiazole scaffold

Article abstract of DOI:10.1016/j.bmcl.2012.11.091

With the aim of discovering potent inhibitors of the p53-MDM2 interaction and thus obtaining a potent anticancer drug, we have pursued synthesis and optimization of dihydroimidazothiazole derivatives, which have been discovered via scaffold hopping by mimicing the mode of interaction between MDM2 and Nutlins. Upon the discovery we encountered a problem involving the chemical instability of the scaffold, that is, susceptibility to oxidation which led to imidazothiazole. In order to solve this problem and to obtain further potent compounds, we executed medicinal research and thus furnished the optimal compounds by incorporating the methyl group onto the C-6 position to avoid the oxidation, and by modifying the C-2 moiety of the additional proline motif, which furnished high potency. The incorporation of the pyrrolidine moiety at the C-2 position raised another hydrophobic interaction site with MDM2 protein, which was generated by the induced-fitting observed by co-crystal structure analysis. These optimal molecules showed significant improvement in potency when compared with the early lead (+)-1 or Nutlin-3a.

Full text of DOI:10.1016/j.bmcl.2012.11.091

Bioorganic & Medicinal Chemistry Letters 23 (2013) 728–732
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead optimization of novel p53-MDM2 interaction inhibitors possessing
dihydroimidazothiazole scaffold
Masaki Miyazaki a, , Hiroyuki Naito a, Yuuichi Sugimoto a, Haruko Kawato a, Tooru Okayama a,
⇑
Hironari Shimizu 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, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of discovering potent inhibitors of the p53-MDM2 interaction and thus obtaining a potent
anticancer drug, we have pursued synthesis and optimization of dihydroimidazothiazole derivatives,
which have been discovered via scaffold hopping by mimicing the mode of interaction between
MDM2 and Nutlins. Upon the discovery we encountered a problem involving the chemical instability
of the scaffold, that is, susceptibility to oxidation which led to imidazothiazole. In order to solve this
problem and to obtain further potent compounds, we executed medicinal research and thus furnished
the optimal compounds by incorporating the methyl group onto the C-6 position to avoid the oxidation,
and by modifying the C-2 moiety of the additional proline motif, which furnished high potency. The
incorporation of the pyrrolidine moiety at the C-2 position raised another hydrophobic interaction site
with MDM2 protein, which was generated by the induced-fitting observed by co-crystal structure anal-
ysis. These optimal molecules showed significant improvement in potency when compared with the early
lead (+)-1 or Nutlin-3a.
Received 28 October 2012
Revised 14 November 2012
Accepted 21 November 2012
Available online 1 December 2012
Keywords:
MDM2
p53
Dihydroimidazothiazole
Protein–protein interaction inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Human murine double minute 2 (MDM2) is a negative regulator
of the p53 tumor suppressor protein, and is overexpressed in
cancer cells which has wild-type p53 genes.1 MDM2 binds to the
N-terminal transcriptional activation domain of p53, and promotes
export of p53 from nucleus to cytoplasm, thereby promoting its
proteosomal degradation via ubiquitination through E3 ligase
activity.2–4 There have been many reports regarding the relevance
between MDM2 inhibition and growth inhibition of cancer cells.
About 50% of human cancers have wild-type p53. Especially, it is
noteworthy that the rate of wild-type genes is more than 90% in
leukemia and sarcoma cell lines.5,6 Thus, a p53-MDM2 inhibitor
should serve as novel molecular target drug by the new mecha-
nism. In fact, various small molecule inhibitors, which mimic the
three hydrophobic residues of p53, that is, side chains of Phe19,
Trp23, and Leu26,7–9 have been reported.10–13 The p53-MDM2
binding inhibitory activity (IC50) of these compounds in the reports
p53-MDM2 binding inhibitory activity.14 Compound (rac)-1 plac-
ing cis-bischlorophenyl moieties at the C-5 and C-6 position of
dihydroimidazothiazole displayed potent activity (IC50 = 0.26 lM)
(Fig. 1). The enantiomers were separated by using chiral HPLC,
and we confirmed that one of them ((+)-1) possessed high potency.
However, upon further exploratory research, it became clear that
the dihydroimidazothiazole ring was susceptible to oxidation,
and easily provided imidazothiazole, for instance at the final ami-
dation step, as shown in Figure 2.15 Thus, we pursued the further
optimization of our lead (rac)-1 to furnish more potent and stable
compounds. We report herein the synthesis, biological evaluation
and SARs by the further optimization of dihydroimidazothiazole
derivatives.
Since i-propyl group was optimal for the C-3 substituent as we
reported in a previous paper, we continued to utilize the substitu-
ent for the position. With regard to the C-2 position, various sub-
stituents with both acyclic amide and cyclic one, encompassing
4- to 6-membered rings, were designed and synthesized.
In order to prevent the oxidation of the imidazoline moiety to
imidazole, we modified the scaffold not to eliminate both protons
at the C-5 and C-6 positions. Thus, we envisioned to incorporate
the methyl group to the C-6 position not affecting the interaction
with MDM2. Although an asymmetric carbon was generated, this
trial worked well, resulting in a stable compound without spoiling
the affinity (Fig. 3), as we envisaged.
are roughly 0.01–1
lM on a cell free assay, among which potent
inhibitor reported first was Nutlin-3a whose IC50 was 0.09
lM.
We reported recently that novel dihydroimidazothiazole deriv-
atives, which were discovered by analyzing research in the mode of
interaction between MDM2 and the Nutlins, showed a potent
⇑
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.2012.11.091

M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 728–732
729
O
O
Cl
Cl
Cl
O
NH
N
O
N
N
N
N
S
NH
cis
O
N
O
Cl
(+)-1
(potent enantiomer)
(rac)-1
Nutlin-3a
IC50 (µM):
0.26
0.16
0.09a
Figure 1. Structure and IC50s of our early lead and Nutlin-3a.
aLit. data (see Ref. 10).
Oxidized to imidazothiazole ring.
O
O
Cl
Cl
Cl
condensation
with amine
3
N
2
O
OH
N
N
5
(rac)-1
S
N H
S 1
cis
+
Cl
N
N
6
(~13%)
(50%)
(rac)-2
3
(inactive by-product)
Figure 2. Instability of the scaffold; oxidation occurred in the condensation step.
((+)-4) was obtained as a co-crystal with
anol, which was then collected by filtration. Then, the crystal was
basified to give the free diamine. On the other hand, the filtrate
L
-(+)-tartaric acid in eth-
Phe19 pocket
Trp23 pocket
Leu26 pocket
O
Cl
Cl
O
N
was basified and then recrystallized with
D-(À)-tartaric acid, gave
N
N
*
S
NH
cis
the salt of (À)-diamine ((À)-4) -tartrate in the same manner as
D
*
for (+)-4. The salt was again basified to provide (À)-diamine
((À)-4). The optical purities of those enantiomers were analyzed
using chiral HPLC (CHIRALPAKÒ AS-H, eluant:hexane-IPA),18 and
turned out to be >99% ee. The absolute conformation was deter-
mined by co-crystal structure analysis of the final product 14b
with MDM2 protein. All diamines were led to the final products
using the same manner as used for compound (rac)-1 reported
previously.14 For example, as shown in Scheme 2, diamines 4 were
Incorporation of the methyl moiety
to the opposite side of affinity pockets.
Figure 3. Designs to prevent oxidation referred from predicted affinity model.
reacted with CS2, followed by thiazole formation with a-chloro-b-
The vicinal diamine intermediate (rac)-4 needed for the forma-
tion of 6-methyldihydroimidazothiazole was synthesized by utiliz-
ing the method of Pansare and others.16 Optical resolution by
diastereomeric salt formation using chiral tartaric acids (Scheme
1)17 was performed for cis-racemate diamine ((rac)-4); (+)-diamine
ketoester, providing 6-methyl-dihydroimidazothiazole derivatives
(6a–c). Although two kinds of cyclized compounds (6-methyl or
5-methyl) might have been generated, only one isomer was ob-
tained which turned out to provide strong potency (compound
8b). The selectivity was attained presumably by the steric
Cl
OH
O
(b)
(c)
NH
2
HO
OH
O
Cl
Cl
Cl
Cl
NH
OH
2
OH
O
NH
NH
2
(a)
Cl
2
HO
cis
D
-(-)-tartrate)
(b)
OH
crude mixture
+
NH
NH
2
2
O
OH
(
L
-(+)-tartrate)
(rac)-4
Cl
Cl
Cl
(b)
NH
NH
2
2
NH
NH
2
2
Cl
(+)-4
(-)-4
Scheme 1. Optical resolution of (rac)-4 using
aReagents and conditions: (a)
L
-(+)- or
D
-(À)-tartaric acid.a
L
-(+)-tartaric acid, EtOH, reflux; (b) NaOHaq; (c)
D-(À)-tartaric acid, EtOH, reflux.

730
M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 728–732
O
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
N
O
OH
NH2
NH2
N
N
N
N
(a)
(b)
(c)
(d)
S
S
cis
cis
S
cis
cis
See Table 1.
N
H
b
(rac)-4
(+)-4
7a-c
5a-c
6a-c
(-)-4
(e) from 7b
O
N
Cl
Cl
N
N
S
O
O R2
18b R2: -t-Bu
19b R2: -H
(g)
(f)
See Table 2.
Scheme 2. Synthesis of C-6 substituted dihydroimidazothiazole derivatives from the cis-diamines 4.a
aReagents and conditions: (a) CS2, EtOH, reflux; (b) ethyl 2-chloro-4-methyl-3-oxopentanoate, EtOH, reflux; (c) NaOHaq, EtOH 60 °C; (d) HNR2, EDC/HCl, HOBt, Et3N or DIPEA,
DMF; (e) t-Butyl-L-prolinate hydrochloride, EDC/HCl, HOBt, DIPEA, DMF; (f) TFA, CHCl3; (g) HNR2, EDC/HCl, HOBt, Et3N or DIPEA, DMF or CH2Cl2.
bThe compounds notated with [a] are the racemate derived from (rac)-4, and the compounds notated with [b] and [c] are the chiral isomer derived from (+)-4 and (À)-4.
Table 1
Inhibition of the p53-MDM2 binding interaction (1)
hindrance of the methyl group. Absolute structure was later deter-
mined by the same method, that is, co-crystallization with MDM2
protein, as described for diamine (+)-4. Hydrolysis of the ester gave
the corresponding carboxylic acids (7a–c), then following EDC
mediated amidation with the appropriate amines, furnished the
amide derivatives (8a–17b). (2S)-N,N-dimethylazetidine-2-carbox-
amide, the starting material of the azetidine moiety of 17b, was
synthesized from chiral nitrile derivative via hydrolysis.19,20
Compounds 20b–25b were also designed because the proline
variant 14b showed a very strong activity. To investigate SAR of
proline derivatives, amide moieties having branched or ring types
referred to our previous research were introduced at the terminal
position.
The result of SAR for the C-2 position (amide site) is displayed in
Table 1. IC50s were measured by p53-MDM2 plate binding assay21
or HTRF-based assay.22 In the beginning, we investigated the effect
of the C-6 methyl group for the activity with the variant having 2-
oxopiperazine at the C-2 position. Compound 8a showed a potency
O
Cl
1
R
N
N
S
cis
Cl
b
Compda
R1
Derived diamine
IC50
(lM)
O
8a
8b
8c
*
(rac)-4
(+)-4
(-)-4
0.41
0.092
1.5
N
NH
*
*
NH2
9a
(rac)-4
(rac)-4
0.78
1.1
N
H
10a
11a
11b
(rac)-4
(+)-4
0.042
0.026
*
*
N
N
12a
13a
(rac)-4
(rac)-4
0.20
almost equivalent to (rac)-1 (IC50 = 0.41 lM). Each of the enantio-
mers (8b and 8c) synthesized from chiral diamines ((+)-4 and
O
*
N
N
0.94
H
(À)-4) were then evaluated. Compound 8b derived from diamine
(+)-4 possessed potent activity (IC50 = 0.092 lM), in contrast, the
14a
14b
(rac)-4
(+)-4
0.059
0.0092
*
N
IC50 of the other isomer 8c was found to be 1/16 times weaker
O
(IC50 = 1.5 M). Since the final products 8a–c were obtained in high
l
N
yield without forming imidazothiazole derivatives23 at the last
amidation step, it was proved that introducing the methyl group
to the C-6 position was effective for the avoidance of ring-oxida-
tion. Moreover, since enantiomer 8b showed a high potency rather
than the dimethyl variant (+)-1, the design is more favorable for
p53-MDM2 inhibition.
*
N
O
15a
16a
(rac)-4
0.74
0.17
N
*
O
N
In comparison of the types of amides, compound 11a having a
N,N-dimethylcarbamoyl group, showed the highest potency, thus
the dialkyl amides were more advantageous in respect to the activ-
ity than the other class of substituents. Efficacy of compound 11b,
chiral variant synthesized from diamine (+)-4, furthermore im-
(rac)-4
(+)-4
N
*
N
0.14c
0.09d
O
17b
N
proved (IC50 = 0.026 lM).
Whilst pyrrolidine derivatives (12a and 13a) remained moder-
Nutlin-3a
ate activity, compound 14a possessing N,N-dimethylcarbamoyl-
a
Compounds notated with [a] are the racemate/diastereomer, and the com-
L
-proline moiety, provided
Moreover, chiral variant (14b)23 showed significant improvement
(IC50 = 0.0092 M), and this was the most potent molecule.
Compound 15a, placed with -proline moiety gave less (IC50
a high potency (IC50 = 0.059 lM).
pounds notated with [b] or [c] are the chiral isomer.
b
p53-MDM2 plate binding assay.
HTRF assay.
Lit. data (see Ref. 10).
c
l
d
D
=

M. Miyazaki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 728–732
731
Table 2
0.74
(16a and 17b) having a N,N-dimethylcarbamoyl substituent dis-
played modest activity at around 0.2 M.
lM). Other compounds possessing 4- or 6-membered rings
Inhibition of the p53-MDM2 binding interaction (2)
l
O
Cl
Co-crystal structure analysis of dihydroimidazothiazoles and
MDM2 was performed. The structure of the complex of compound
14b/MDM2 is displayed in Figure 4. This crystallographic analysis
revealed that two cis-bischlorophenyl groups and i-propyl moiety
had the same roles for three hydrophobic residues (Trp23, Leu26
and Phe19) of p53, as we expected. Furthermore, introducing the
5-membered substituent at the C-2 position, it was observed that
the pyrrolidine ring provided a new 4th affinity site, which was
generated by the induced fitting with MDM2. It is considered that
this induced fitting makes the molecule 14b about 10 times more
potent than Nutlin-3a for the cell-free activity. The stacking model
of co-crystal structure of 14b and Nutlin-2, in which MDM2 loop is
distorted by the induced-fitting with the proline ring, can explain
that the three-dimensional structure is actually changed.
N
N
N
S
O
R3
Cl
Compd
R3
IC50
(l
M)a
Compd
R3
IC50 (l
M)a
O
O
O
*
*
N
N
N
N
N
20b
0.046
0.026
23b
0.079
0.023
NH
*
*
21b
22b
24b
25b
O
N
N
*
*
N
NH
0.066
0.037
a
Through the X-ray structure analysis, we found out that dihy-
droimidazothiazole derivatives efficiently bound to the surface of
MDM2 only by hydrophobic interaction. Although the amide part
of the proline moiety seemed to direct to outside of MDM2 protein,
p53-MDM2 plate binding assay.
branched type (20b), but also 6-membered substituents like
morpholine (21b) or N-methylpiperazine (24b) possessed a high
potency (IC50s = 0.023–0.026 lM). This data suggests that struc-
compound 15a,
tency than that of
D
-proline variant, showed 1/12 times weaker po-
-proline derivative 14a. This SAR provided us
L
tural capability at the C-2 carboxamide position is extensive, thus
it would be possible for the improvement of physicochemical prop-
erties, that is solubility, hydrophobicity, and metabolic stability, in
keeping with the potent p53-MDM2 inhibitory activity. These
parameters should be very important to achieve antitumor efficacy
in vivo. Further investigation and rational drug design are now on
going, containing and utilizing the information of the co-crystal
analysis.
another opportunity and motivation for derivatizing the part to
improve the efficacy or physicochemical property. The induced-fit-
ting could also explain the reason why neither 6- nor 4-membered
rings showed high activity; both of them would not form the fitting
efficiently like 5-membered rings. The result of derivatizing the
amide part of the proline moiety is shown in Table 2. Not only
In conclusion, we executed lead optimization of our early lead,
dihydroimidazothiazole derivatives, for p53-MDM2 inhibitory
activity. The prevention of the oxidation of the scaffold, where
we could prepare optically active diamines efficiently, was accom-
plished by placing the methyl group at the C-6 position, without
compromising potency. As a result of co-crystal structure analysis,
a novel hydrophobic pocket was identified via induced-fitting of
the pyrrolidine ring attached to the C-2 position of the scaffold.
Compound 14b showed 10 times more potent than Nutlin-3a,
which can be explained by the increased affinity to MDM2. From
these results, it was confirmed that our dihydroimidazothiazole
derivatives possessed superior deposition as p53-MDM2 binding
inhibitors. Further optimization is ongoing to improve the efficacy
and physicochemical properties, and the results will be reported in
due course.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.091.
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:
IPA = 80:20 (v/v), Flow rate: 1.0 ml/min, rt; (+)-4: 7.47 min., [a] +69° (c = 1.0,
D
MeOH, 22 °C); (À)-4: 6.66 min, [
a
]
À60° (c = 1.0, MeOH, 22 °C).
D
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21. Details of p53-MDM2 plate binding assay; The compounds in 3-fold dilutions
were incubated with human 20 ng/ml His-p53 immobilized on a Nickel-coated
plate. 200 ng/ml Human GST-tagged MDM2, ATP, FLAG-tagged ubiquitin, E1,
and E2 (UbcH5c) were added to the plate. After 60 min, the reaction was
quenched by washout reaction mixture, and then HRP-labeled anti-FLAG
antibody and AP-labeled anti-GST antibody were added. Ubiquitination and
binding between p53 and MDM2 were detected by chemiluminescence.
22. Details of HTRF-based assay: The compounds in 3-fold dilutions were incubated
with 2.5 nM GST-tagged human MDM2 (25-108, L33E) and 2.5 nM His-tagged
p53 (1–132) with detection by time-resolved fluorescence spectroscopy.
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