Substituted piperidines as HDM2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.01.026

Novel small molecule HDM2 inhibitor, substituted piperidine, was identified. Initial SAR study indicated potential for several position optimizations. Additional potency enhancement was achieved by introducing a sidechain off the aromatic ring. DMPK study

Full text of DOI:10.1016/j.bmcl.2014.01.026

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1026–1030
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted piperidines as HDM2 inhibitors
⇑
Yao Ma , Brian R. Lahue, Gerald W. Shipps Jr., Jeseca Brookes, Yaolin Wang
Discovery and Preclinical Sciences, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel small molecule HDM2 inhibitor, substituted piperidine, was identified. Initial SAR study indicated
potential for several position optimizations. Additional potency enhancement was achieved by introduc-
ing a sidechain off the aromatic ring. DMPK study of one of the active compounds has shown a moderate
oral PK and reasonable bioavailability.
Received 4 November 2013
Revised 8 January 2014
Accepted 9 January 2014
Available online 17 January 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
HDM2
MDM2
p53
Substituted piperidine
Small molecule inhibitor
Cancer
Oncology
The tumor suppressor protein p53 plays a central role in main-
taining the integrity of the genome in a cell. It regulates the expres-
sion of a diverse array of genes responsible for DNA repair, cell
cycle and growth arrest, and apoptosis.1–3 As with murine
MDM2, the human homologue HDM2 acts to down-regulate p53
activity in an autoregulatory manner in which the cellular level
of each protein is controlled by the other.4–6 Attenuating this neg-
ative feedback loop could have a critical effect on cell homeostasis.
It was found that in many types of human tumors, HDM2 was
overexpressed and p53 function was decreased.7,8 To restore the
function of wt-p53 in tumor cells by inhibition of HDM2 should
result in decreased proliferation and apoptosis thus inhibiting
tumor growth, which offers great therapeutic potential for the
treatment of a variety of cancers.9,10
The co-crystal structure of the HDM2 N-terminal domain bound
to a p53-derived peptide revealed a major pocket of interaction
driven by three nearby amino acid residues on one face of a helix,
corresponding to p53 residues Phe19, Leu26, and Trp23.11 It was
anticipated that a small molecule could mimic this interaction
and antagonize the HDM2 protein, freeing p53 to perform its
downstream oncogenic functions. Despite the large surface area
of protein–protein interaction (PPI) interfaces and relative com-
plexity, several potent low molecular weight p53/HDM2 inhibitors
have been discovered to date and were recently reviewed.12,13
Herein, we report a series of novel HDM2 small molecule antago-
nists that selectively inhibit proliferation of several wild-type
p53 human cancer cells. SAR trends identified within this chemo-
type for substituents correlating to the three aforementioned resi-
dues on p53 guided the optimization of this series.
From our in-house high-throughput screening (HTS) platform,
several hits (1–3) were identified which inhibit the p53/HDM2
protein–protein interaction with IC50 values in the low single digit
micromolar range (Fig. 1). These geminally disubstituted piperi-
dines (4) were shown to bind to HDM2’s p53 binding pocket based
on a fluorescence polarization (FP) peptide displacement assay.14
Synthesis of this class of compounds was straightforward
(Scheme 1) and has been detailed in previously published patent
applications.15,16 Key intermediates 6 were readily prepared in
racemic form through the Bargellini reaction of ketone 5 with
the corresponding phenol in the presence of chloroform under ba-
sic conditions.15–17 Extensive optimization of the reaction condi-
tions identified the order of addition of the reagents as a key
factor in producing intermediates 6 in the best yields. Depending
on the stability of the phenol to the basic reaction conditions, add-
ing it after all the other reagents could considerably improve the
yield of desired products 6. Intermediates 6 were subsequently
subjected to amidation, deprotection, and acylation to produce
fully substituted final compounds 4.
Compound 1, chosen as the starting point for optimization
based on the frequency with which its substituents appeared in
hits within the initial HTS results, was resynthesized from the
corresponding common intermediate carboxylic acid 6. Palla-
dium-catalyzed hydrogenolysis of 6 in the presence of di-tertbutyl
dicarbonate and Hunig’s base gave 7 as the diisopropylethylamine
salt, which was used directly in an amidation with 1-(2-pyridin-2-
⇑
Corresponding author.
E-mail address: yaoma1@yahoo.com (Y. Ma).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.026

Y. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1026–1030
1027
O
O
O
O
R2'
R2
O
O
O
O
O
O
CF3
CH3
N
N
N
N
N
CF3
HO
CF3
O
CF3
N
N
d, e
a, b, c
N
O
N
O
N
O
N
N
O
CF3
CF3
CF3
CF3
N
N
N
N
FP IC50: 2.3 µM
FP IC50: 1.5 µM
2
1
6a
10
11
O
O
O
R2'
N
N
O
CF3
N
N
O
CF3
O
HN
O
CF3
N
O
R1
X
X
R2
N
O
N
O
R
(X = OH, NH2)
N
O
(X = O, NH)
CF3
Cl
CF3
N
R3
N
N
FP IC50: 1.4 µM
3
4
11'
12
0
Scheme 3. R2/R2 (amine and sidechain) optimization. Reagents and conditions: (a)
(Z)-tert-butyl N,N0-diisopropylcarbamimidate; (b) H2, Pd/C; (c) acid, EDC, HOBt; (d)
4 N HCl in 1,4-dioxane; (e) PS-CDI; HOBt, amine.
Figure 1. Substituted piperidines as novel HDM2 inhibitors.
R2'
O
O
O
HOOC
O Ar
N
Ar
R2
O
O
a
N
O
N
O
CF
3
N
N
O
CF
3
N
N
N
N
N
HCl
R3
Ph
8
Ph
N
NH
3
5
6
4
R
13
14
O
S
O
Scheme 1. Synthesis of substituted piperidine 4. Reagents and conditions: (a)
phenol (ArOH), CHCl3, NaOH, 0–40 °C.
Alkyl
R
R3:
R
Scheme 4. R3 optimization.
yl)piperazine to give 8 (Scheme 2). Further elaboration by acidic
BOC deprotection and subsequent amide formation with 4-(trifluo-
romethyl)nicotinic acid led to final compounds 9.
For ease of handling and purification as well as to enable rapid
0
optimization at R2/R2 (see 4), intermediate acid 6a was esterified18
which, after hydrogenolysis of the benzyl group and amide forma-
tion with 4-(trifluoromethyl)nicotinic acid, gave intermediate 10
(Scheme 3). Ester hydrolysis of 10 with HCl produced the corre-
Table 1
R1 SAR: In vitro activity (refer to general structures 9)
Compound
R1
Cl
IC50
21
(lM)
sponding acid which underwent EDC-promoted amidation with
0
various amines to generate diamides 11. Further R2/R2 optimiza-
9a
tion was carried out through extension of a sidechain off the ortho
position of the piperazine-linked phenyl ring (110), most effectively
by O-alkylation or N-acylation leading to final compounds 12.
Similarly, common intermediate 13, used for R3 optimization,
was synthesized by BOC de-protection of the corresponding piper-
azine amide 8 (Scheme 4). The resulting free amine enabled the
broad profiling of R3 substituents and the generation of SAR
Cl
9b
5.2
9c
9d
9e
Cl
F
6.7
6
Br
1.9
O
F
F
N
O
N
N
O
Ar
O
HO
O
Ar
O
9f
2.9
1.4
41
N
Br
a
b
N
O
6
N
O
9g
9h
Cl
S
7
8
Ar
O
9i
9j
48
33
N
N
O Ar
N
CH3
O
CF
3
c, d
N
O
CF
CH3
CF3
2
1
1.5
2.3
9
1
3
N
Scheme 2. R1 (aryl) optimization. Reagents and conditions: (a) 5% Pd/C, H2, BOC2O,
iPr2NEt (b) Carbonyldiimidazole, polymer-supported (PS-CDI), HOBt, 1-(pyridin-2-
yl)piperazine, THF (c) 4 N HCl in 1,4-dioxane (d) PS-CDI, HOBt, 4-(trifluoro-
methyl)nicotinic acid, DMF.
(a) All compounds were tested as HCl salts; (b) Fluorescent polarization was
measured by reading the plate using the Analyst AD (Molecular Device). IC50 was
determined as described in Zhang et al.14

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Y. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1026–1030
Table 3
through subsequent N-functionalization to give final compounds
14.
R2 sidechain SAR: In vitro activity (refer to general structure 12)a
As depicted in the above syntheses, our initial optimization plan
centered around various substituents at three motifs attached to
intermediate 4 (R1, R2, R3). R1 variation was derived from original
phenols and, although several steps were involved after incorpo-
rating R1, the very mild reaction conditions employed allowed for
profiling various phenolic substituents (Table 1).
Compound
Side chain
IC50 (lM)
O
11m
12a
12b
0.56
0.97
0.59
OH
O
Overall, substitution of the R1 aryl ring was better-tolerated in
the meta- and para-positions compared to the corresponding
ortho-substituted analogs as exemplified by chlorinated com-
pounds 9a–9c. Replacement of the para-chloro substituent with
p-fluoro (9d) or p-bromo (9e), respectively, demonstrated that
while decreased electron density of the aryl ring was tolerated, a
larger group may be preferred in the para-position. This trend
was confirmed with disubstituted analogs 9f and 9g, in which
the addition of a meta-fluoro substituent was tolerated (compare
9e–9f) or modestly improved (compare 9c–9g) IC50 values. How-
ever, several analogs disubstituted in the form of heterobicyclic
compounds like 9h exhibited substantial losses in enzymatic activ-
ity. The para-substituent was further examined in the context of
O
12c
12d
12e
12f
0.33
0.38
0.37
0.54
O
NH
O
NH
O
NH
O
O
N
NH
12g
12h
0.67
0.59
O
NH
O
Table 2
R2 SAR: In vitro activity of various amides (refer to general structures 11)a
N
Compound
R2R20 N
IC50
2.3
2.8
18
(lM)
NH
O
12i
12j
1.08
0.63
N
N
N
N
1
N
N
NH
O
11a
11b
N
N
N
N
O
N
O
N
NH
O
N
N
11c
N
N
35
O
NH
N
12k
0.27
HN
11d
11e
11f
4.2
36
12
O
N
N
a
Compounds were tested under similar conditions as described in Table 1.
H3C
N
N
N
N
N
size and polarity–an exercise which revealed the preference for rel-
atively small, hydrophobic groups like methyl (2) and trifluoro-
methyl (1) over larger, non-polar phenyl (9i) or small, polar
methoxy (9j) moieties. Based on its overall profile and likelihood
to withstand metabolic degradation, compound 1 was selected
for further optimization.
11g
11h
11i
39
1.4
3.7
N
N
By fixing compound 10s substituents at both R1 and R3 (refer to
general structure 4) as p-trifluoromethylphenol and 4-trifluorom-
N
11j
1.4
1.5
ethylnicotinamide, respectively (Fig. 1), a thorough investigation
0
of the R2/R2 amine substituent was undertaken. While the pyridine
Cl
nitrogen of 1 did not appear to be critical as illustrated by equipo-
tent phenyl analog 11a, the addition of a second ring-nitrogen (11b
and 11c) led to significant losses in activity (Table 2). Saturation of
the terminal ring was tolerated (11d) however, acyl (11e), small al-
kyl (11f), and one-methylene homologated (11g) variants were sig-
nificantly less active. Modifications to the linking piperazine ring
were well-tolerated as partially unsaturated 11h, ring-contracted
pyrrolidine 11i, and annulated tetrahydroisoquinoline 11j all
maintained the activity of 1. The complete replacement of the
piperazine ring by one or two methylenes (11k and 11l, respec-
11k
HN
11l
3.5
HN
N
N
11m
0.56
O
N
N
11n
11o
>50
23
tively) led to similarly active compounds. This variety of active
0
O
O
substituents at the R2/R2 position offered considerable opportuni-
ties for later DMPK optimization. From an enzymatic activity
standpoint, it was not until terminal ring-substitution was ex-
plored that a jump in activity was unveiled. 2-Methoxy analog
N
N
a
Compounds were tested under similar conditions as described in Table 1.

Y. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1026–1030
1029
Table 4
Table 5
R3 SAR: In vitro activity of (refer to general structure 14)a
DMPK of the 12c active enantiomer
Compound
R1
IC50
2.3
(
lM)
Route Dose
(mpk)
AUC 24 h
M h)
Cmax
M)
Tmax
(h)
Bioavailability
(%)
(l
(
l
O
O
F
F
F
Full Mouse PK
F
F
1
IV
PO
20
20
3.4
1.1
N
0.7
1
32
F
F
CYP Inhibition
14a
14b
6.5
14
Co-incubation (IC50
,
lM)
Pre-incubation (IC50, lM)
3A4
4.9
2D6
>30
2C9
>30
3A4
0.3
2D6
>30
2C9
>30
F
F
containing additional heteroatoms (12c). Such modifications via a
tethered sidechain to pick up additional interactions would be con-
sistent with those of other published HDM2/P53 inhibitors.19
When the phenolic oxygen atom was replaced with an anilino-
nitrogen, amide-based sidechains of analogous lengths were also
tolerated (compare 12b with 12d and 12c with 12f). Basic
amine-containing sidechains of similar length were well-tolerated
(12g–12i) as were select heterocycles like isoxazole 12j and hydan-
toin 12j. This general tolerability for added polarity within this
sidechain offered an opportunity to improve the overall physical
properties of the compounds without sacrificing enzymatic
activity.
14c
14d
6.5
6.9
14e
5.1
O
14f
3.0
22
Structural modifications around the R3 amide in 1 were under-
taken to generate SAR trends in an effort to further improve activ-
ity (Table 4). Removal of the apparently important R3 pyridine
nitrogen resulted in a 3-fold loss in activity (compare 1 with
14a). Subsequent replacement of the amide carbonyl with a meth-
ylene to give 14b led to a significant loss in enzymatic activity,
indicating the possible importance of non-basic nitrogen in the
piperadine core of this series. However, further profiling of basic
amine-linked substituents identified smaller aliphatic groups like
14c–14e could retain the in vitro activity of benzoic acid derivative
14a, an indication that perhaps the amide carbonyl served more to
orient the ortho trifluoromethyl moiety than to moderate the basi-
city of the piperidine nitrogen atom. Attempts to combine the
amide linkage of 1 and 14a with the simple aliphatic sidechains
of 14c–14e revealed that a substituent as simple as a tert-butyl
group could maintain the enzymatic activity of 14a when posi-
tioned the appropriate distance from the carbonyl (14f) but was
much less active when just one carbon too short (14g). Amides hin-
dered at the alpha carbon to the carbonyl regained their ability to
inhibit the HDM2/p53 protein–protein interaction when coupled
with an aromatic substituent, as in 14h. Our efforts to replace
the amide functionality with similarly substituted sulfonamides
proved largely unfruitful (14i–14l, compare 14a with 14j). Only
moderate activity was observed with smaller aliphatic sulfon-
amide (14m).
O
O
14g
14h
1.9
Cl
O
S
O
14i
14j
14k
26
41
31
O
S
S
O
F
F
F
O
O
Cl
O
S
O
14l
50
Cl
O
S
14m
6.9
O
Chiral HPLC separation of racemate 12c cleanly afforded two
enantiomers, the active isomer of which displayed an IC50 of
160 nM. DMPK analysis of this compound indicated a moderate
oral exposure in mice with a reasonable bioavailability (32%, Ta-
ble 5). While this compound did not inhibit cytochrome P450
2D6 and 2C9 isoforms, it exhibited both direct and mechanism-
based inhibition on isoform 3A4. Further optimization of this series
is ongoing and will be reported on in the future.
a
Compounds were tested under similar conditions as described in Table 1.
11m represented a 5-fold improvement over pyridyl progenitor 1
and clearly defined an SAR path to follow since the analogous 3-
methoxy (11n) and 4-methoxy (11o) compounds were less active
in this assay.
With the preference for a heteroatom-linked sidechain in the
Overall a new chemical series, substituted piperidines, was
identified as inhibitors of the HDM2/p53 protein–protein interac-
tion. Initial optimization efforts have generated useful SAR trends
at several binding positions. Further improvements to this scaffold
should furnish a potent HDM2 inhibitor with an improved overall
DMPK profile.
0
ortho position on the R2/R2 phenyl ring determined (see 11m),
the size and polarity of this substituent were profiled in an effort
to further improve biochemical activity (Table 3). While shortening
of the sidechain to the phenol 12a led to a twofold loss in activity,
longer substituents were tolerated (see 12b), especially those

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Y. Ma et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1026–1030
11. Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.;
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