Structure-activity relationship study of 4-substituted piperidines at Leu26 moiety of novel p53-hDM2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.03.078

Led by the structural information of the screening hit with mDM2 protein, a structure modification of Leu26 moiety of the novel p53-hDM2 inhibitors was conducted. A structure-activity relationship study of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles.

Full text of DOI:10.1016/j.bmcl.2016.03.078

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2735–2738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship study of 4-substituted piperidines
at Leu26 moiety of novel p53–hDM2 inhibitors
a
a
a
a
b
Yuan Tian a, , Yao Ma , Craig R. Gibeau , Brian R. Lahue , Gerald W. Shipps Jr. , Corey Strickland ,
⇑
Stéphane L. Bogen b
a Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
b Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Led by the structural information of the screening hit with mDM2 protein, a structure modification of
Leu26 moiety of the novel p53–hDM2 inhibitors was conducted. A structure–activity relationship study
of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 23 January 2016
Accepted 22 March 2016
Available online 23 March 2016
Keywords:
p53
hDM2
SAR
The p53 tumor suppressor protein plays a critical role in the
DNA damage response1,2 and is defective in more than 50% of
human tumors.3,4 Murine mDM2 (or hDM2, the human isoform)
is the main regulator of p53 stability and subjects it to degrada-
tion.5–10 There is a need for effective inhibitors of the hDM2 protein
in order to treat or prevent cancer, other disease states associated
with cell proliferation, diseases associated with hDM2, or diseases
caused by inadequate p53 activity.11,12 In the past few years, small
molecule inhibitors of the hDM2–p53 protein–protein interaction
appear to offer an attractive strategy for cancer therapy.13–16 The
crystal structure of mDM2 bound to p53 revealed that mDM2
has a deep hydrophobic cleft on which the p53 peptide binds as
The structure of compound 1 in complex with mDM2 protein
was solved using X-ray diffraction data (Fig. 1). On the basis of
the crystal structure, that the piperidine serves as the central core
to hold three subunits in different directions: 4-CF3-nicotinamide
group occupies Phe19 pocket, 4-CF3-phenoxy group inserts deep
into Trp23 pocket and 4-aryl piperizine binds with Leu26 pocket.
The conformation of the central piperidine core and the stereo-
chemistry of C3 position are critical to maintain the potency.
Trp23 and Phe19 binding pockets are sensitive to ligand modifica-
tion and 4-CF3-phenoxy and 4-CF3-nicotinamide were found as
optimal groups for these two binding pockets respectively.
an amphipathic
mentarity between the mDM2 cleft and the hydrophobic face of
the p53 helix. In particular, three residues of p53 peptide:
a
helix.17 The interface relies on the steric comple-
F3C
a
F3C
Phe19, Trp23, and Leu26 insert deep into the mDM2 cleft. Thus,
inhibition of p53–mDM2 interaction could be achieved by intro-
ducing a small molecule inhibitor to mimetic p53 peptide, which
has the appropriate three dimensional structure to impact these
hydrophobic binding pockets of mDM2 target protein. Our
research group recently reported the discovery of 3,3-disubstituted
piperidine 1 with hDM2 binding activity.18 To capitalize on this
novel chemical structures of p53–hDM2 inhibitors, a series of lead
optimization efforts were directed to improve upon the potency
and drug properties.19,20 Herein, we focus on the structure
modification on Leu26 binding pockets in our lead series.
3
O
N
N
N
N
1
2
O
N
O
FP IC50 = 2.3 µM
TdF Kd = 0.5 µM
Cell viab. IC50 > 18µM
From the X-ray structure and preliminary SAR study, it was
envisioned that two areas could be explored to improve binding
affinity for this new chemotype. The first strategy relied on
restricting the conformation of the central core;21 the second was
to optimize binding to the Leu26 pocket by targeting interactions
⇑
Corresponding author. Tel.: +1 617 992 2368.
E-mail address: yuan.tian2@merck.com (Y. Tian).
http://dx.doi.org/10.1016/j.bmcl.2016.03.078
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

2736
Y. Tian et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2735–2738
the sterically hindered ester with NaSEt gave acid 8. The final tar-
get compounds were synthesized through the amide coupling
reaction of acid 8 with the corresponding piperidine building
blocks.
Initially, analogs 10, 11 and 12 with phenyl or substituted phe-
nyl groups as Ar group were tested in FP assay and the biological
data (IC50) are summarized in Table 1. From the direct comparison
of 10a–c and 11a–c, the sulfur and oxygen linker were superior to
methylene linker. In terms of substitutions on the phenyl ring,
there were no significant electronic effects when methyl-,
chloro- and cyano-groups were applied. However, in general, the
meta-substitutions show better potencies than ortho- and
para-substitutions. For example, compound 12b was 10 times
more potent than its regioisomers 12a and 11a, and meta-cyano
analog 14b was one of the most potent compounds in this series.
Since the oxygen linker had comparable biological activity and
could potentially be more metabolically stable than the sulfur
Table 1
Figure 1. Co-crystal structure of compound 1 with mDM2.
Enzymatic potency of the analogs with substituted phenyl group
Compd #
Piperidine
FP IC50 Compd #
(nM)
Piperidine
FP IC50
(nM)
with His96 and Tyr100 residues of mDM2 protein. 4-Pyridyl piper-
izine group of compound 1 showed potential for stacking with
p–p
His96 as well as edge-to-face interaction with Tyr100. Thus, our
group decided to modify the piperazine linker of compound 1 to
probe its effect on such interactions. A comprehensive SAR study
of 4-substituted piperidine analogs on Leu26 moiety is described
herein.
The syntheses of the target compounds are shown in Scheme 1.
Starting from the commercial material 3-piperidone methyl
carbamate (2), the enantiomerically pure 2-R–3-S-isomer 3 was
synthesized and resolved in 4 steps.21 Catalytic hydrogenation
and deprotection of the methyl carbamate with iodotrimethyl-
silane yielded compound 5. Subsequently, amide coupling with
4-trifluoromethyl-nicotinic acid (6) followed by demethylation of
O
O
Me
10a
2414 12b
1981 13a
5831 13b
668
N
H
N
H
S
O
Cl
10b
10c
2764
2095
N
H
N
H
H C
2
O
Cl
Cl
O
2
O
CO2Me
F3C
a
O
CO2Me
N
CF3
N
H
N
H
(S)
4 steps
(R)
Me
Me
Me
N
N
CO2Me
CO2Me
CO2Me
4
3
O
O
11a
11b
11c
12a
8766 13c
9359 14a
25,801 14b
6599 14c
4112
8714
399
F3C
O
CO2Me
N
H
N
H
F3C
b
O
CO2Me
c
N
O
CO2H
CF3
N
CF3
O
H
S
5
N
CN
N
7
6
N
H
N
H
Ar
X
O
CN
CN
H C
2
N
F3C
O
CO2H
N
F3C
O
O
N
H
N
H
e
X
d
O
N
O
Ar
CF3
CF3
O
O
N
H
N
N
8
9
Me
1919
Scheme 1. Reagents and conditions: (a) H2, Pd/C, MeOH, 23 °C, 93%; (b) TMSI, DCM,
23 °C, 55%; (c) HATU, acid 6, DIEA, DMF, 23 °C, 82%; (d) NaSEt, DMF, 80 °C, 75%;
(e) HATU, DIEA, DMF, 23 °C, 60–85%.
N
H
N
H

Y. Tian et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2735–2738
2737
linker, further investigation was directed towards the oxygen lin-
ker. Subsequently, the SAR studies were focused on the modifica-
tion of the aromatic ring occupying the Leu26 pocket. A variety
of six-membered-ring aromatics were investigated and the biolog-
ical data are summarized in Table 2. When phenyl ring was
replaced with 2-pyridyl, the potency was dramatically improved
from 2.4 lM of compound 10a to 0.15 lM of compound 15a. On
the other hand, 3 or 4-pyridyl analog 15b or 15c were less potent
than their regioisomer 15a. When the second nitrogen was
introduced on the pyridyl ring, the enzymatic potency dropped
(compounds 16–19).
X-ray structure of compound 15a reveals that 4-substituted
piperidine occupies the Leu26 pocket (Fig. 2). The pyridyl ring
has strong
p–p stacking with His96 and edge to face interaction
with Tyr100 of mDM2 protein. X-ray structure also showed that
there additional space in Leu26 pocket could accommodate small
substitution on pyridyl ring, especially on 6-position. Thus, an
extensive SAR study was conducted (Table 3).
Initially, a set of 6-substituted pyridyl analogs 20a–f were
tested and the cyano functionality showed the most promising
result. Moving cyano substitution around the pyridyl ring indi-
cated that 4-CN analog 20h was most potent among four of its
regioisomers 20f–i. On the contrary, for H-bond donating polar
substituents, the SAR differed. Comparing four regioisomers of
aminomethyl analogs 20j–m, which were the reduced products
from the corresponding cyano compounds, the 6-position was
most active. This SAR was consistent with the corresponding
primary amides 20n–q, culminating with 6-carboxamide 20n
(111 nM) and its equipotent monomethyl analog 20r (114 nM).
Not surprisedly, at the 6-position, the less polar carboxylic ester
(20s) was less potent than the amide group due to the lack of
H-bond donor. Among the set of substitutions of pyridyl ring,
6-carboxylic acid was found most effective with compound 20t
being the most potent (Table 3, FP IC50 = 42 nM).
Figure 2. X-ray co-crystal structure of compound 15a with mDM2 protein.
Table 3
Enzymatic potency of the analogs of 20a–t
R
N
O
N
F3C
O
O
N
N
O
CF3
Compound 20t was tested in cell proliferation (cell viability)
assays using multiple cell lines and was found to have selective
inhibition of cell growth in cancer cells expressing wild-type p53
15a
(R = H)
Compd #
R
FP IC50 (nM) Compd #
R
FP IC50 (nM)
20a
20b
20c
20d
20e
20f
20g
20h
20i
6-Me
6-OMe
6-CF3
6-Cl
5027
1146
2039
2359
1863
686
492
293
2399
20k
20l
5-CH2NH2
4-CH2NH2
3-CH2NH2
6-CONH2
5-CONH2
4-CONH2
3-CONH2
6-CONHMe 114
6-CO2Me
6-CO2H
801
Table 2
4026
7744
111
144
8721
4276
Enzymatic potency of the analogs with different aromatic group
20m
20n
20o
20p
20q
20r
20s
20t
Compd # Piperidine
FP IC50 (nM) Compd # Piperidine
FP IC50 (nM)
6-Br
6-CN
5-CN
4-CN
3-CN
N
O
O
N
392
42
10a
2414
148
267
224
16
17
18
19
334
20j
6-CH2NH2 173
N
H
N
H
N
N
(osteosarcoma SJSA-1 cell line IC50 = 4.65
HCT-116 IC50 = 8.65 M), but not cancer cells expressing mutant
p53 (SKUT-1 uterine cancer) and null p53-null cells (HCT-116)
with IC50 > 100 M. More importantly, compound 20t was also
evaluated for inhibition of a panel of CYP’s (3A4, 2D6, 2C9;
IC50s > 50 M) and found to be devoided of CYP liabilities,
lM, colorectal cell line
O
N
l
O
15a
15b
15c
1065
l
N
H
N
H
l
N
overcoming a major limitation in our earlier research on this lead
N
N
series.21,22
O
O
N
N
In conclusion, structure-based design was applied on the SAR
development of Leu26 moiety of the lead series of p53–hDM2 inhi-
bitors. Focusing on the ligand–target interaction in Leu26 pocket,
4-substituted piperidines were extensively investigated including
different linkages between the piperidine and aromatic rings,
numerous heterocyclic alternatives, and a variety of the substitu-
tion patterns on the aromatic ring. The potency was optimized
from sub-micro molar to double digital nano molar in the
enzymatic assay. In addition, compound 20t showed micro molar
cellular potency in a few p53-related cell lines and offered
376
N
H
N
H
N
O
O
525
N
H
N
H

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Y. Tian et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2735–2738
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