Pivotal role of an aliphatic side chain in the development of an HDM2 inhibitor

Article abstract of DOI:10.1021/ml500019s

Introduction of an aliphatic side chain to a key position of a novel piperidine-based HDM2 inhibitor scaffold resulted in significant potency gains, enabling further series progression.

Full text of DOI:10.1021/ml500019s

Letter
pubs.acs.org/acsmedchemlett
Pivotal Role of an Aliphatic Side Chain in the Development of an
HDM2 Inhibitor
Yao Ma,*,† Brian R. Lahue,† Craig R. Gibeau,† Gerald W. Shipps, Jr.,† Stephane L. Bogen,‡ Yaolin Wang,‡
́
Zhuyan Guo,‡ and Timothy J. Guzi†
†Discovery and Preclinical Sciences, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United
States
‡Discovery and Preclinical Sciences, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United
States
S
* Supporting Information
ABSTRACT: Introduction of an aliphatic side chain to a key
position of a novel piperidine-based HDM2 inhibitor scaffold
resulted in significant potency gains, enabling further series
progression.
KEYWORDS: HDM2, MDM2, p53, substituted piperidine, protein−protein interaction, small molecule inhibitor
he tumor suppressor protein p53 plays a central role in
molecular weight inhibitors have also been described.16,17
Recently, we have reported the discovery of a novel series of
substituted piperidines as small molecule antagonists of HDM2,
which selectively inhibit proliferation of wild-type p53 cancer
cell lines (Figure 1).18,19 It has historically been challenging to
overcome the large surface contacts of protein−protein
interactions with a small molecule inhibitor. Herein we report
a breakthrough made in our efforts to further optimize this
substituted piperidine series through chemical modifications at
the piperidine 2-position.
Tmaintaining the integrity of the genome in a cell. It
Chemistry. The synthesis of this series of 2-substituted
piperidine-based p53-HDM2 inhibitors was carried out in a
fashion analogous to that described previously for 2-
unsubstituted compounds 1 and 2 (Figure 1).18,19 Beginning
with N-benzyl-3-piperidone 3, which was readily converted to
the corresponding methyl carbamate 4 in a single step under
conditions described by Brubaker,20 the resulting ketone was
subjected to an enamine alkylation with allyl bromide to
provide 5 (Scheme 1). The requisite quaternary center was
then introduced through a Bargellini reaction18,21,22 with 4-
trifluoromethylphenol to give racemic carboxylic acid 6, which
was converted to corresponding methyl ester for ease in
handling. Chiral HPLC conditions were developed to resolve 7
to a single enantiomer (refer to Supporting Information for
details). The carbamate protecting group on 7 was removed
through the action of trimethylsilyliodide, and the resulting free
Figure 1. Novel piperidine-based HDM2 inhibitors.
regulates the expression of a diverse array of genes responsible
for DNA repair, cell cycle and growth arrest, and apoptosis.1−5
The oncogene HDM2 is a ubiquitin protein ligase that
negatively regulates tumor suppressor p53 and lessens its
transcriptional activity as well as promoting p53 protein
degradation.6−9 Antagonists of the tumor suppressor p53-
HDM2 interaction represent an attractive and novel mecha-
nism in oncological therapeutics. Disrupting this HDM2-p53
protein−protein interaction with a small molecule is hypothe-
sized to increase intracellular levels of p53 leading to decreased
proliferation and increased apoptosis of tumor cells, which
offers great therapeutic potential for the treatment of
cancers.10−13 Crystallographic studies of MDM2- (the murine
homologue of human HDM2) and p53-derived peptides bound
together have revealed a deep hydrophobic binding pocket
involving residues Phe19, Trp23, and Leu26 of p53.14 Since the
discovery of Roche’s Nutlin imidazoline series, which
antagonizes this HDM2−p53 interaction,15 several potent low
Received: January 16, 2014
Accepted: February 24, 2014
Published: February 24, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthesis of the 2-Substituted Piperidine Core
and Key Analogues
Table 1. Biochemical and Cellular Activities of Compounds
9−14
a
a
a
Reagents and conditions: (a) methyl chloroformate, Et3N, CHCl3 0
°C to r.t., 57%; (b) 1. pyrrolidine, benzene reflux; 2. allyl bromide,
MeCN, 70 °C, 67%; (c) 4-trifluoromethylphenol, NaOH, CHCl3, 0−
40 °C; (d) TMS-CHN2, MeCN, MeOH, 0 °C to r.t., ∼40% overall
yield from 5; (e) chiral HPLC (see Supporting Information for the
condition), 27%; (f) iodotrimethylsilane, CH2Cl2, r.t., >95%; (g) 4-
trifluoronicotinic acid, HATU, Et2NiPr, CH2Cl2, rt to 45 °C, >85%;
(h) KOH, EtOH, H2O, 65 °C, 88%; (i) HATU, 1-[2-(2-methoxy−
ethoxy)-phenyl]-piperazine, Et2NiPr, CH2Cl2, r.t. to 40 °C, 53%.
a
Scheme 2. Modification of the Olefin
a
All compounds were tested as HCl salts. Fluorescent polarization was
measured by reading the plate using the Analyst AD (Molecular
Device). IC50 was determined as described in Zhang et al.23
The allyl substituent in the 2-position of the piperidine ring
in compound 9 served as a synthetic handle for further
elaboration at this position (Scheme 2). Reduction of the olefin
in 9 by hydrogenation led to compound 10. Polarity was
introduced at this position in the form of two hydroxyl groups
through dihydroxylation of the double bond in 9 to give diols
11, which in turn were converted to alcohol 12 through in situ
sodium periodate-promoted cleavage followed by reduction of
the resulting aldehyde.
Results and Discussion. While it was anticipated that
improvements to the in vitro potency and concomitant cell-
based activity of C2-unsubstituted compounds such as 1318
could be made through modifications of the largely hydro-
phobic binding interactions with the HDM2 protein,
a
Reagents and conditions: (a) H2, Pd/C, EtOAc, 87%; (b) OsO4,
NMO, THF, H2O, r.t., ∼18% for combined two isomers; (c) 1. OsO4,
NaIO4, 2,6-lutidine, dioxane, H2O, r.t.; 2. NaBH4, MeOH, r.t., 25%.
amine was subjected to standard amidation conditions with 4-
trifluoromethylnicotinic acid to give intermediate 8a. Ester
hydrolysis of 8a provided carboxylic acid 8b, which underwent
amidation with the corresponding piperazine building block to
give final compound 9, the absolute stereochemistry of which
was confirmed by analysis of its X-ray cocrystal structure with
HDM2.
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ACS Medicinal Chemistry Letters
Letter
Table 2. Cytochrome P450 Inhibition of Select HDM2
Inhibitors
cytochrome P450 inhibition
coincubation (IC50, μM) preincubation (IC50, μM)
compound
3A4
2D6
2C9
3A4
2D6
2C9
13
9
4.9
>30
>30
>30
>30
>30
>30
>30
>30
0.3
>30
>30
>30
>30
>30
>30
>30
>30
2.1
<0.3
7.5
11a
12
>30
13.3
0.9
kcal/mol) was equivalent to that of the ring-flipped
conformation, within the resolution (5 kcal/mol) of the Sybyl
software used to perform the calculations (Figure 2). However,
the binding conformation of the 2-allyl analogue 9 was
calculated to be more than 10 kcal/mol more favorable than
the nonbinding conformer (17.6 vs 27.7 kcal/mol), likely due
to the unfavorable steric interactions of the two amides with the
adjacent axial allyl substituent in the latter. As a result, by
introducing the side chain at the piperidine-2 position,
exchange between ring conformations has been significantly
slowed down with the preferred binding conformation existing
predominantly in the absence of HDM2. Locking the
orientation of the substituents on the piperidine core in this
way improved the binding affinity of 2-substituted compounds
such as 9 considerably.
As potent HDM2 inhibitors, this series of compounds
exhibited a ∼3× improvement in the cell Titer-Glo assay with
SJSA-1 osteosarcoma cells compared to well-studied Nutlin-3a
(Figure 3).15,16 As with Nutlin-3a, these compounds were only
active against wild-type (wt)-p53 cell lines like SJSA-1 with no
activity observed in the mutant-p53 cell lines, such as the
endometrial cancer cell line SKUT-1, indicating on-target
mechanism-based antiproliferative activity. Thus, a ∼30×
toxicity window was achieved between the wt-p53 and
mutant-p53 cancer cell lines.
Figure 2. Impact of side chain substitution on the piperidine ring
conformations.
optimization of the overall DMPK profile was expected to
require the introduction of polar substituents to the inhibitor in
such a way as to not interfere with binding to HDM2. The
addition of an allyl group as a starting point for synthetic
diversification as described in the previous paragraph was
targeted to do just this; however, introduction of this simple
aliphatic side chain itself provided an unexpected yet significant
impact on binding efficiency. Compared to unsubstituted
analogue 13, the addition of allyl (9) and n-propyl (10) groups
at C2 of the piperidine core provided a 4- and 7-fold
improvement, respectively, in the biochemical fluorescence
polarization (FP) peptide displacement assay23 (Table 1). As a
result of this intrinsic potency gain, activity of these compounds
in the cellular viability assay with osteosarcoma SJSA-1 cells
translated to similar levels of improvement with both 9 and 10
displaying IC50 values around 1 μM. As anticipated, the
incorporation of more polar functional groups such as diols
11a/b and the primary alcohol 12 did not negatively impact the
binding affinity with biochemical IC50 values from 16 to 24 nM.
In the absence of binding activities with the protein, the
piperidine core as a six-membered cyclic ring should have
several conformations coexisting in solution; however, only one
of them was found to fit into HDM2’s p53-binding pocket. The
energy required for the 2-unsubstituted piperidine 13 to adopt
the chair conformation necessary for binding to HDM2 (17.5
As anticipated, DMPK analysis of compound 9 indicated a
similar cytochrome P450 (CYP) profile to that of the 2-
unsubstituted compound 13 as both lipophilic molecules did
not inhibit CYP 2D6 and 2C9 isoforms but exhibited both
direct (coincubation IC50 = 2.1−4.9 μM) and metabolism/
mechanism-based CYP 3A4 inhibition (coincubation IC50/
preincubation IC50 ≈ 7−16-fold) (Table 2). The introduction
Figure 3. Novel HDM2 inhibitors selectively inhibit wt-p53 cancer cell lines.
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ACS Medicinal Chemistry Letters
Letter
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ASSOCIATED CONTENT
* Supporting Information
■
(19) Ma, Y.; Lahue, B.; Shipps, G.; Wang, Y.; Bogen, S.; Voss, M.;
Nair, L.; Tian, Y.; Doll, R.; Guo, Z.; Strickland, C.; Zhang, R.; McCoy,
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piperidines that increase p53 activity and the uses thereof. U.S. Pat.
Appl. Publ. US2008004287, 2008.
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Experimental procedures and LC−MS characterization data for
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(Y.M.) E-mail: yaoma1@yahoo.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Authors would like to acknowledge Diane Rindgen, Hui Wan,
Anthony Soares, and Shiying Chen for processing and
interpreting CYP data.
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