Discovery of potent and orally active p53-MDM2 inhibitors RO5353 and RO2468 for potential clinical development

Article abstract of DOI:10.1021/ml400359z

The development of small-molecule MDM2 inhibitors to restore dysfunctional p53 activities represents a novel approach for cancer treatment. In a previous communication, the efforts leading to the identification of a non-imidazoline MDM2 inhibitor, RG7388, was disclosed and revealed the desirable in vitro and in vivo pharmacological properties that this class of pyrrolidine-based inhibitors possesses. Given this richness and the critical need for a wide variety of chemical structures to ensure success in the clinic, research was expanded to evaluate additional derivatives. Here we report two new potent, selective, and orally active p53-MDM2 antagonists, RO5353 and RO2468, as follow-ups with promising potential for clinical development.

Full text of DOI:10.1021/ml400359z

Letter
pubs.acs.org/acsmedchemlett
Discovery of Potent and Orally Active p53-MDM2 Inhibitors RO5353
and RO2468 for Potential Clinical Development
Zhuming Zhang,*,†,⊥ Xin-Jie Chu,*,†,⊥ Jin-Jun Liu,† Qingjie Ding,† Jing Zhang,† David Bartkovitz,†
Nan Jiang,† Prabha Karnachi,† Sung-Sau So,† Christian Tovar,§ Zoran M. Filipovic,§ Brian Higgins,§
Kelli Glenn,∥ Kathryn Packman,§ Lyubomir Vassilev,§ and Bradford Graves‡
‡
§
∥
†Discovery Chemistry, Discovery Technologies, Discovery Oncology, and Non-Clinical Development, Roche Pharma Research,
Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, New Jersey 07110, United States
S
* Supporting Information
ABSTRACT: The development of small-molecule MDM2 inhib-
itors to restore dysfunctional p53 activities represents a novel
approach for cancer treatment. In a previous communication, the
efforts leading to the identification of a non-imidazoline MDM2
inhibitor, RG7388, was disclosed and revealed the desirable in vitro
and in vivo pharmacological properties that this class of pyrrolidine-
based inhibitors possesses. Given this richness and the critical need
for a wide variety of chemical structures to ensure success in the
clinic, research was expanded to evaluate additional derivatives.
Here we report two new potent, selective, and orally active p53-
MDM2 antagonists, RO5353 and RO2468, as follow-ups with
promising potential for clinical development.
KEYWORDS: MDM2, p53, wild-type, small molecule, apoptosis, cancer
umor suppressor p53 is a potent transcription factor that
Tis activated in response to cellular stress and regulates
downstream genes controlling cell cycle arrest and apopto-
sis.1−4 Dysfunction of the p53 pathway is the most frequent
alteration observed in human cancers.5 MDM2 is the primary
negative regulator of p53 through binding to its transactivation
domain and promoting proteosomal degradation.6−8 In tumor
cells with wild-type p53 (∼50%), reactivation of the p53
pathway by inhibition of MDM2 with small molecules has been
considered as potentially an attractive novel therapeutic
approach for cancer treatment.9,10 Currently, several small-
molecule MDM2 inhibitors including RG7112 and RG7388
(Figure 1) are undergoing clinical evaluations.11−14 To
maximize the chance of success in the clinic and derisk any
potential idiopathic toxicity associated with specific chemo-
types, continued research efforts are required to expand
chemodiversity and identify potent and selective MDM2
antagonists with desirable in vitro ADMET and in vivo
pharmacokinetic properties. Here we report the discovery of
RO5353 and RO2468, two new highly potent and selective
MDM2 inhibitors with potential for clinical development.
Our exploration initially led to the identification of a potent
and selective MDM2 inhibitor RO8994 (Figure 1), which was
found to be highly efficacious against established human tumor
xenografts in nude mouse models.15 Two key structural
elements of RG7388 were preserved in RO8994. First, it was
established that the stereochemical configuration of the
pyrrolidine core structure in which the two aryl rings (“A”
Figure 1. Chemical structures of RG7388, RO8994, and analogues I−
V. Binding modes for RG7388 and RO8994 are shown.
and “B”) adopt a “Trans” orientation was very important for
optimal binding to MDM2.14 The architecture of spiroindoli-
none-3,3′-pyrrolidine series (as exemplified by MI-219) was
first reported by Ding et al.16−18 Consistent with our findings,
this group recently published their latest findings in which the
original stereochemistry was found to be unstable and
Received: September 13, 2013
Accepted: December 29, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400359z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
converted over time to the more stable “Trans” configuration as
shown in RO8994.19 Second, a methoxy para-benzoic acid
moiety in RG7388 (“C” in Figure 1) was transferred to
RO8994, in which a slight modification was made by converting
the terminal carboxylic acid to a carboxamide moiety. In
contrast to aliphatic groups,19,20 the aromatic group (“C”)
proved to be critical for both RG7388 and RO8994 as it
stabilized the molecule metabolically, significantly improved
cellular potency/selectivity, PK profiles, and in vivo effi-
cacy.14,15
To quickly assess the effects of bioisosteric replacements in
scaffolds I−V (Figure 1), compounds 1−13 were prepared
initially as racemic mixtures of two enantiomers (Figure 2). All
On the basis of the crystal structures, the 3-chloro-2-
fluorophenyl group binds to the Leu26 pocket on MDM2,
while the neopentyl group occupies the Phe19 pocket (Figure
1).14,15 As expected from the binding models with the Michigan
oxindole compounds,16 the 6-chloroxindole moiety in RO8994
is buried into the deep, narrow Trp23 pocket, and its NH
moiety forms a hydrogen bond with a backbone carbonyl of
MDM2 for enhanced binding affinity.15 Since the interaction in
the Trp23 pocket appears to be the most critical, further
exploration of bioisosteric replacements on the phenyl moiety
of 6-chlorooxindole, while preserving other important architec-
tural features in RO8994 for optimal binding and pharmaco-
logical properties, was prioritized.21 Thus, we describe our work
in detail on selected analogues of 4-, 5-, and 7-azaoxindole (I−
III), 2-chloropyrrolo[2,3-d]pyrimidin-6-one (IV), and 2-
chlorothienyl[3,2-b]pyrrol-5-one (V) (Figure 1).
Recently a practical synthesis of RO8994 has been
established.22 Utilizing these new methods, azaoxindole, 2-
chloropyrrolo[2,3-d]pyrimidin-6-one, and 2-chlorothienyl [3,2-
b]pyrrol-5-one analogues in I−V were efficiently synthesized as
outlined in Scheme 1. The key step is the cycloaddition
reaction of VI with common intermediate VII catalyzed by
lithium hydroxide to form thermodynamically more stable
stereochemical isomer as shown in the structure VIII.15,22
Other regio- or stereochemical isomers of compound VIII were
also formed but could be readily separated by chromatography.
Scheme 1. Racemic Synthesis of Azaoxindole I−III, 2-
Chloropyrrolo[2,3-b]pyrimidin-6-one IV, and 2-
a
Chlorothienyl[3,2-b]pyrrol-5-one V Analogues
Figure 2. Chemical structures of analogues 1−19 in I−V.
analogues display potent biochemical affinity with IC50 values
<100 nM in the binding assay (Table 1). In cellular
antiproliferative MTT assays, their potencies against wild-type
p53 cell line SJSA vary over a wide range from IC50 = 22.7 to
0.009 μM, but all appear to be selective against a mutant p53
cell line, SW480. Lack of the chloro substituent at the R1
position (compounds 1, 2, and 5) clearly led to weaker binding
affinity in the binding assay and significant loss in cellular
potency against SJSA compared to corresponding compounds
with R1 = Cl (3, 4, and 6). The antiproliferative potencies
(Table 1) of compounds 6, 7, 10, and 11 against SJSA are
generally slightly weaker compared to analogues 3, 4, 8, and 9,
indicating the placement of heteroatom N at position 7 as
shown in scaffolds III and IV is not favorable. Nevertheless,
compounds 4, 12, and 13 as racemic mixtures were found to
a
Reagents and conditions: (a) LiOH, THF, 40 °C; (b) LiOH, THF,
H2O, rt; (c) CDI, NH4OH, THF, rt; (d) chiral SFC separation; P, Q,
and W as defined in Figure 1.
B
dx.doi.org/10.1021/ml400359z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 1. Activity Results for Analogues 1−13 and RO8994 in a Biochemical Binding Assay and Human Cancer Cell
a
Proliferation Assays
compd
1
2
3
4
5
6
7
8
9
10
25
11
45
12
33
13
40
RO8994
b
HTRF IC50 (nM)
52
140
2.11
13.4
47
17
52
18
11
46
52
5
c
SJSA IC50 (μM)
22.7
>30
0.043
>30
0.014
12.8
1.37
>30
0.045
15.4
0.067
5.26
0.061
>30
0.042
5.9
0.10
>30
0.09
10.7
0.009
0.011
7.87
0.013
5.19
d
SW480 IC50 (μM)
29.3
a
b
c
d
IC50 was determined by one experiment performed in duplicate. 0.2% BSA in the buffer. Wild-type p53 cancer cell line. Mutant p53 cancer cell
line.
display cellular potency and selectivity comparable to RO8994.
These compounds were separated into chiral enantiomers 14−
19 by chiral super fluid chromatography (SFC) (Figure 2).
The potent activity of 4, 12, and 13 was confirmed by
assessment of their enantiomers 14, 15, and 16 (Tables 2 and
Table 2. Affinity of Analogues 14−19 and RO8994 in a
a
Biochemical Binding Assay
compd
14 15 16
17
18
19
RO8994
5
HTRF IC50 (nM)
6
7
7
5764
57
177
a
IC50 was determined by one experiment performed in duplicate.
Figure 3. Crystal structure of 16 bound to MDM2. The 2-
chlorothienyl[3,2-b]pyrrol-5-one group is buried in the Trp23 pocket.
The 3-chloro-2-fluorophenyl group occupies the Leu26 pocket. This
structure is available at the PDB (code 4LWV).
Table 3. Antiproliferative Activity of Analogues 14-19 in
Human Cancer Cell lines
a
compd
14
15
16
18
19
RO8994
b
MTT IC50 (μM)
0.015
742
0.003
5300
0.007
1023
0.18
88
0.85
8
0.02
312
c
Table 4. Human Liver Microsomal Stability and Mean Single
Dose PK Parameters of Compounds 14, 15, and 16 and
RO8994 in C57 Male Mice by Oral and IV Dosing
selectivity
a
IC50 was determined by one experiment performed in duplicate.
b
Average IC50 of three wt-p53 cancer cell lines (SJSA, HCT116, and
c
RKO). Ratio of average IC50 of two mutant p53 cell lines (SW480
and MDA-MB-435) and average IC50 of three wild-type p53 cell lines
(same as above).
compd
14
15
16
RO8994
a
HLM_CL (mL/min/kg)
PO dose (mg/kg)
10.2
5
2.0
10
7.5
25
10
PO AUC/dose (μg·hr/mL/mg/kg)
PO Cmax (μg/mL)
IV dose (mg/kg)
4.2
2.1
2
0.08
0.3
1.5
1.3
2.5
9.9
3.0
92
3.7
5.8
0.64
5.8
7.1
92
3). In the binding assay, 14 (IC50 = 6 nM) is 960-fold more
potent than its enantiomer 17 (Table 2). In contrast, 15 (IC50
= 7 nM) and 16 (IC50 = 6 nM) are only 8- and 30-fold more
potent than their enantiomers 18 and 19. In antiproliferative
assays (Table 3), 14, 15, and 16 display high potency against a
panel of wild-type p53 cancer cell lines (SJSA, HCT116, and
RKO) and high selectivity relative to mutant cell lines (SW480
and MDA-MB-435). Compounds 18 and 19 displayed
moderate cellular activity with much less selectivity (Table
3). Absolute stereochemical configuration of the more active
enantiomers 15 and 16 were confirmed by a crystal structure of
16 bound to MDM2 (Figure 3).
2.5
CL (mL/min/kg)
t1/2 (h)
1.8
3.0
46
68.6
1.68
32
b
F (%)
a
For HLM_CL: low clearance < 6.5; 6.5 < medium clearance < 35;
b
high clearance > 35. Oral bioavailability.
Since compounds 14, 15, and 16 exhibit substantial cellular
antiproliferative potency/selectivity and compare favorably to
RO8994, these analogues were evaluated for their in vivo
pharmacokinectic characteristics. Compound 15 displays low
plasma exposure orally and a high clearance rate in mice. In
contrast, both compounds 14 and 16 demonstrate excellent PK
profiles (Table 4). Both 14 (RO2468) and 16 (RO5353)
achieve impressive in vivo efficacy against established human
SJSA1 osterosarcoma xenoftrafts in nude mice (Figures 4 and
5) at doses and exposures comparable to RO8994. Both 14 and
16 displayed tumor regression at 10 mg/kg. In a similar study,
RO8994 exhibited regression at 6.25 mg/kg.15 No significant
weight loss was observed, consistent with the nongenotoxic p53
activation mechanism.
Figure 4. Oral in vivo efficacy profile of 14 (RO2468) on nude mice
implanted with SJSA1 osteosarcoma. Tumor stasis was observed at 3
mg/kg QD and tumor regression was observed at 10 mg/kg QD.
C
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ACS Medicinal Chemistry Letters
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In summary, our investigation of bioisosteric replacements of
the 6-chlorooxindole group in RO8994 led to the identification
of two highly potent, selective, and orally active p53-MDM2
inhibitors, RO2468 or RO5353, with promising potential for
clinical development.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental section, including X-ray crystal data of
16. Detailed results in HTRF biochemical binding affinity,
MTT cellular antiproliferative assays, human liver microsomal
stability, and in vivo animal model. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(Z.Z.) Phone: (215) 628-7076. E-mail: zzzhang_3000@
yahoo.com.
■
*(X.-J.C.) E-mail: xinjiechu@gmail.com.
Author Contributions
⊥Z.Z. and X.-J.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
SFC purification by Theodore Lambros, detailed H and NOE
NMR analysis for determination of stereochemical config-
uration by Gino Sasso, and high-resolution mass by George
Perkins are greatly acknowledged.
■
1
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