Core modification of substituted piperidines as Novel inhibitors of HDM2-p53 protein-protein interaction

Article abstract of DOI:10.1016/j.bmcl.2014.02.055

The discovery of 3,3-disubstituted piperidine 1 as novel p53-HDM2 inhibitors prompted us to implement subsequent SAR follow up directed towards piperidine core modifications. Conformational restrictions and further functionalization of the piperidine core were investigated as a strategy to gain additional interactions with HDM2. Substitutions at positions 4, 5 and 6 of the piperidine ring were explored. Although some substitutions were tolerated, no significant improvement in potency was observed compared to 1. Incorporation of an allyl side chain at position 2 provided a drastic improvement in binding potency.

Full text of DOI:10.1016/j.bmcl.2014.02.055

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1983–1986
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Core modification of substituted piperidines as Novel inhibitors
of HDM2–p53 protein–protein interaction
b
b
a
b
a
Weidong Pan a, , Brian R. Lahue , Yao Ma , Latha G. Nair , Gerald W. Shipps Jr. , Yaolin Wang ,
⇑
Ronald Doll a, Stéphane L. Bogen a,
⇑
a Merck Research Laboratories, Early Development and Discovery Sciences, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
b 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:
The discovery of 3,3-disubstituted piperidine 1 as novel p53–HDM2 inhibitors prompted us to implement
subsequent SAR follow up directed towards piperidine core modifications. Conformational restrictions
and further functionalization of the piperidine core were investigated as a strategy to gain additional
interactions with HDM2. Substitutions at positions 4, 5 and 6 of the piperidine ring were explored.
Although some substitutions were tolerated, no significant improvement in potency was observed com-
pared to 1. Incorporation of an allyl side chain at position 2 provided a drastic improvement in binding
potency.
Received 23 January 2014
Revised 18 February 2014
Accepted 20 February 2014
Available online 28 February 2014
Keywords:
HDM2
p53
Ó 2014 Elsevier Ltd. All rights reserved.
Protein–protein interaction
Cancer
Cancer is a disease of uncontrolled cell growth of various tissues
and organs in the body that is a leading cause of death. The Inter-
national Agency for Research on Cancer recently announced that
an estimated 14.1 million new cancer cases and 8.2 million can-
cer-related deaths occurred worldwide in 2012. According to the
American Cancer Society, about 1.6 million new cancer cases are
expected to be diagnosed in 2013, with an estimated half million
deaths in the US alone. Even with the use of conventional chemo-
therapy and newly approved targeted therapies, cancer still repre-
sents an unmet medical need.1
The tumor suppressor p53 plays many critical roles in surveying
and responding to various stress and damage signals. It regulates the
cell growth, migration, apoptosis, angiogenesis, metabolism, devel-
opment and stromal matrix cellular environment.2 The loss of p53
function predisposes cells to a cancerous state.3 HDM2 is a major
negative regulator of p53 activity by repressing p53 transcriptional
activity through its binding to p53 and targeting p53 degradation in
the proteosome through its ubiquitin E3 ligase activity.
anti-proliferative and proapoptotic functions. In more than 50% of
human cancers, p53 is inactivated due to overexpression of HDM2
protein,5 making the development of small molecule inhibitors of
HDM2–p53 a very attractive cancer therapy.6 Thus, in 2004, scien-
tists at Roche reported the discovery of a series of 4,5-dihydroim-
idazolines with Nutlin-3a as the first small molecule interfering
with protein–protein interaction of HDM2–p53.7 Since then, others
have started reporting the discovery of additional potent small
molecule inhibitors.8 Similarly, our group identified and recently
reported the discovery and optimization of gem-disubstituted
piperidines 1a and 1b (Fig. 1) as small molecule inhibitors of the
HDM2–p53 protein–protein interaction.9
R
O
N
N
The possibility of targeting the p53 binding pocket of HDM2
with a small molecule antagonist was raised in 1997 when the
crystal structure of a p53 peptide binding to HDM2 revealed three
surface pockets that bind p53 residues Phe19, Trp23 and Leu26.4
An inhibitor capable of blocking this HDM2–p53 interaction would
free p53 from negative regulation by HDM2, thereby promoting its
O
O
F3C
2
4
5
N
N
O
6
CF3
1a R= Me
1b R= -(CH2)2OMe
⇑
Corresponding authors. Tel.: +1 908 740 4580.
E-mail address: Stephane.bogen@merck.com (S.L. Bogen).
Figure 1. gem-Disubstituted piperidines.
http://dx.doi.org/10.1016/j.bmcl.2014.02.055
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1984
W. Pan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1983–1986
5a R5b
Satisfyingly, leads 1a and 1b displayed similar in vitro perme-
5a R5b
R
R
ability as Nutlin 3a as well as some evidence of oral absorption
in rodents that warranted further investigation.10 During the
course of our SAR investigation, the team envisioned the potential
of gaining additional binding affinity by functionalizing the piper-
idine core. To that end, tolerability for substitutions on the 4, 5, and
6 positions were probed with both small and large groups. As de-
scribed earlier,9a the team capitalized on the Bargellini reaction11
of protected 3-piperidinone with 4-(trifluoromethyl)phenol to effi-
ciently assemble the gem-disubstituted piperidine core of inhibitor
1. To exploit the same chemistry, ring expansion methodology of
prolinols to 3-hydroxypiperidines reported by Cossy12 was identi-
fied as a very attractive way to prepare the substituted piperidi-
nones required for our SAR exploration.
a
b
O
N
O
OH
4a-c
N
N
Bn
O
O
3a R5a=R5b=Me
2
3b R5a=H, R5b=Me
3c R5a=Me, R5b=H
OH
Bn
O
R5a
R5b
h
R5a
R5b
e, f, g
c, d
N
N
6a-c
Boc
5a-c
O
O
O
O
OH
Boc
i
F3C
Probing the 5 position of piperidine with compounds 9, 10 and
11 required preparation of 5-substituted-3-hydroxy-N-benzylpy-
peridines 5a–c described in Scheme 1.
N
N
R5a
R5b
N
7a-c
O
O
The poor selectivity and diastereoselectivity observed during
the alkylation of pyroglutamic acid derivative 2 with MeI was ben-
eficial to provide in one step multiple key intermediates. Precur-
sors 3a–c were easily separated by column chromatography and
individually subjected to reductive conditions using LAH. Treat-
ment of 3-substituted prolinols 4a–c with trifluoroacetic anhy-
dride in THF followed by addition of triethylamine and sodium
hydroxide provided 5-substituted-3-hydroxy-N-benzylpyperidines
5a–c.11 Depending on the steric hindrance of starting prolinols,
reaction time of up to 4 days was necessary to get complete con-
sumption of starting material and formation of piperidines 5a–c
in quantitative yield. Extensive optimization of the Barguellini
reaction conditions favored the use of N-Boc protected piperi-
done.9c Thus, 3-hydroxy-N-benzylpiperidines 5a–c were subjected
to Palladium-catalyzed hydrogenolysis in the presence of di-ter-
tbutyl dicarbonate and subsequently oxidatized with Dess–Martin
periodinane13 to provide the key piperidones 6a–c in good yield.
Bargellini reaction of 6a–c with the 4-(trifluoromethyl) phenol
afforded in one step the 2,2-disubstituted carboxylic acids 7b,c as
F3C
8a-c
O
R5a
R5b
N
Boc
O
N
j, k
N
9 R5a=Me, R5b=H
O
O
10 R5a=H, R5b=Me
11 R5a=R5b=Me
F3C
R5a
R5b
N
N
O
CF3
Scheme 1. Synthesis of 5-substituted piperidine inhibitors. Reagents and condi-
tions: (a) LiHMDS, THF, MeI, À78 °C, 58%; (b) LAH, THF, reflux, 100%; (c) TFAA, Et3N,
À78 to 60 °C (up to 4 days); (d) NaOH (2.5 M), 100%; (e) H2, Pd(OH)2/C; (f) THF,
NaOH, (Boc)2O; (g) Dess–Martin, 55–70%; (h) 4-CF3-phenol, CHCl3, 0–40 °C,
40–72%; (i) HATU, DIPEA, 1-(2-(2-methoxyethoxy)phenyl)piperazine hydrochlo-
ride, 41–60%; (j) 4.0 N HCl/dioxane, 100%; and (k) 4-CF3-nicotinic acid/HATU,
45–50%.
Table 1
FP and antiproliferative assay results for compounds 1a, 1b, 9–11, 15–17 and 24a
R7
O
N
F3C
N
O
O
R4
R5a
N
N
O
R5b
R6
CF3
Compds
R4
R5a
R5b
R6
R7
FP IC50
(
lM)
SJSA-1 IC50 (lM)
1a
1b
9
10
11
15
16
17
24
H
H
H
H
H
H
4F-Ph
H
H
H
H
Me
H
Me
H
H
H
H
H
H
H
Me
Me
Ph
H
H
H
H
H
H
H
H
H
Ph
Me
0.6
0.3
4.0
2.7
1.0
3.0
3.9
>10
5.6
5.5
4.7
—
—
—
—
—
—
—
–(CH2)2–OMe
–(CH2)2–OMe
–(CH2)2–OMe
–(CH2)2–OMe
Me
–(CH2)2–OMe
–(CH2)2–OMe
Me
Cyclopropyl
a
Fluorescence polarization (FP) peptide displacement assay value were determined as described in Zhang et al.15 For comparison Nutlin-3a fluorescence polarization (FP)
peptide displacement IC50 assay value obtained in house was 0.07 M. In our antiproliferative assay, Nutlin-3a had an IC50 value of 1.9 M using osteosarcoma SJSA-1 cells.
l
l

W. Pan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1983–1986
1985
a mixture of diastereomers and 7a as a racemic mixture. Interme-
diates 7a–c were directly reacted with 1-(2-(2-methoxyeth-
oxy)phenyl)piperazine hydrochloride under standard peptide
coupling conditions to afford compounds 8a–c. Further elaboration
to 9, 10 and 11 was achieved via acidic BOC deprotection and sub-
sequent amide formation with 4-(trifluoromethyl)-nicotinic acid.
Reverse phase and chiral HPLC conditions were developed to iso-
late each of the diastereomers or enantiomers.
Targets 15–17 (Table 1) were prepared according the sequence
depicted in Scheme 2. Using phenylpyrrolidine acids 12a–c as
starting materials, esterification followed by reduction and protec-
tion group exchange gave prolinols 13a–c, which were successfully
engaged in ring expansion methodology described earlier to gener-
ate substituted-3-hydroxy-N-benzylpyperidines 14a–c. Finally, the
analogous sequence as the one described in Scheme 1 was fol-
lowed to prepare compounds 15–17.
cyclopropanation conditions provided 20m and 20M as a mixture
of diastereomers in a 1:11 ratio, respectively.15 The major isomer
20M was subjected to the protection group exchange and reduc-
tion sequence to generate precursor 21 in 70% overall yield. The
highly functionalized prolinol 21 was successfully engaged in ring
expansion methodology to afford 3-hydroxy-N-benzylpyperidines
22 in 79% isolated yield. Protection group swapping necessary for
the Barguellini reaction required a chemoselective hydrogenolysis
of the benzyl group of 22 and was achieved using 10% Pd/C in EtOH
with one equivalent of HCl (2 N, Et2O). After treatment with di-ter-
tbutyl dicarbonate, Boc-protected hydroxy-piperidine 23 was ob-
tained in 98% overall yield. Target 24 was prepared following a
sequence analogous to the one described in Scheme 1.
To assess the potency of these modified geminally disubstituted
piperidines, we evaluated their ability to bind to HDM2’s p53 bind-
ing pocket using a fluorescence polarization (FP) peptide displace-
ment assay.16
Fused bicyclic ring system 24 was synthesized according to
Scheme 3. Cyclopropanation precursor 19 was prepared from pyro-
glutamic derivative 18 via lithium triborohydride amide reduction
and trifluoroacetic anhydride-mediated elimination.14 Furukawa’s
Most of the substitutions studied at position 5 of the piperidine
were tolerated. The dimethyl substituted compound 11 (IC50
=
1.0 M, Table 1) was the most potent and had HDM2 binding po-
l
tency within 2–3 fold of compounds 1a,b. With the assumption
that position 4, 5 and 6 of piperidine were close to the protein sur-
face, the team anticipated that potency enhancements could be
further achieved through modification of the hydrophobic
interaction of the piperidine moiety with the HDM2 protein. Unfor-
tunately, efforts to functionalize position 5 with a large hydropho-
bic group was unfruitful. Although the phenyl group in compound
15 was still tolerated, the compound was less active compared to
1a and 11. Position 4 and 6 of the piperidine ring seemed less tol-
erant to modification as incorporation of an aromatic moiety in
compounds 16 and 17 resulted in a loss in potency compared to
15. Rigidification of the piperidine core was also investigated as a
strategy to gain additional interactions with HDM2 by locking
the conformation of the piperidine ring. Incorporation of a small,
fused cylopropyl ring was thought attractive. Chemistry challenges
allowed exploration of a fused ring at position 5 and 6 only.
Unfortunately, compound 24 showed only moderate activity with
R4
R5
R5
R6
R4
OH
a,b
c,d
e,f
R6
N
Bn
N
COOH
Boc
12a R6= Ph, R5=R4= H
12b R6= H, R5= Ph, R4= H
12c R6=R5= H, R4= 4F-Ph
13a R6= Ph, R5=R4= H
13b R6= H, R5= Ph, R4= H
13c R6=R5= H, R4= 4F-Ph
R4
OH
R5
15, 16, 17
N
R6
Bn
14a R6= Ph, R5=R4= H
14b R6= H, R5= Ph, R4= H
14c R6=R5= H, R4= 4F-Ph
Scheme 2. Reagents and conditions: (a) TMS–CH2N2, MeOH, Et2O (b) 4.0 N HCl/
dioxane, (c) BnBr, DCM, DIPEA (d) LAH, THF, reflux, 85–94% from (a) to (d); (e) TFAA,
Et3N, À78 to 60 °C; (f) NaOH (2.5 M); and (f) H2, Pd(OH)2/C.
an IC50 of 5.6 lM.
While we anticipated that potency enhancements could be
achieved through modification of the hydrophobic interaction of
the piperidine moiety with the HDM2 protein, our efforts to func-
tionalize positions 4, 5 and 6 of the piperidine core proved largely
unsuccessful. However, our team recently reported that the incor-
poration of an allyl substituent at position 2 of the piperidine pro-
vided compound 25 with four fold gain in potency.17 Similarly, in
antiproliferative assay, 25 displayed similar levels of improvement
in activity compared to early leads 1a and 1b with an IC50 of
a,b
c
CO2Et
O
CO2Et
CO2Et
+
N
N
N
Boc
Boc
Boc
18
20
m (minor)
19
OH
d,e,f
g,h
CO2Et
N
1.0
lM in osteosarcoma SJSA-1 cells.
N
N
Bn
Boc
Bn
OH
20
22
M (major)
21
O
O
N
O
OH
N
i,j
N
N
NBoc
O
O
O
O
F3C
F3C
23
N
N
O
N
N
O
CF3
CF3
24
25
Scheme 3. Synthesis of fused ring on 5 and 6 position of piperidine inhibitor.
Reagents and conditions: (a) LiBHEt3, PhMe, À40 °C; (b) DIPEA, DMAP, TFAA; (c)
CH2I2/Et2Zn; (d) 4.0 N HCl; (e) BnBr, DMF; (f) LAH, 70% from (d) to (f); (g) TFAA,
Et3N; (h) NaOH, 79% from (g) to (h); (i) H2, 10% Pd/C, 2 N HCl in ether, 98%; and (j)
(Boc)2O, NaOH, THF, 98%.
FP IC50 (uM)= 0.04 ; SJSA-1 IC50 (uM)= 1.0
Based on this new finding, the allyl moiety is envisioned to be used
as a handle to incorporate functional diversity to probe potential

1986
W. Pan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1983–1986
8. (a) Hu, C.-Q.; Hu, Y.-Z. Curr. Med. Chem. 2008, 15, 1720; (b) Patel, S.; Player, M.
R. Expert Opin. Invest. Drugs 2008, 17, 1865; (c) Zhao, Y.; Yu, S.; Sun, W.; Liu, L.;
Lu, J.; McEachern, D.; Shargary, S.; Bernard, D.; Li, X.; Zhao, T., et al J. Med. Chem.
2013, 56, 5553; (d) Gonzalez-Lopez de Turiso, F.; Sun, D.; Rew, Y.; Bartberger,
M. D.; Beck, H. P.; Canon, J.; Chen, A.; Chow, D.; Correll, T. L.; Huang, X., et al J.
Med. Chem. 2013, 56, 4053; (e) Zak, K.; Pecak, A.; Rys, B.; Wladyka, B.; Domling,
A.; Weber, L.; Holak, T. A.; Dubin, G. Expert Opin. Ther. Patents 2013, 23, 425; (f)
Vu, B.; Wovkulich, P.; Pizzolato, G.; Lovey, A.; Ding, Q.; Jiang, N.; Liu, J.; Zhao, C.;
Glenn, K.; Wen, Y.; Tovar, C.; Packman, K.; Vassilev, L.; Graves, B. ACS Med.
Chem. Lett. 2013, 4, 466; (g) Ding, Q.; Zhang, Z.; Liu, J.; Jiang, N.; Zhang, J.; Ross,
T. M.; Chu, X.; Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Filipovic, Z.;
Higgins, B.; Glenn, K.; Packman, K.; Vassilev, L.; Graves, B. J. Med. Chem. 2013,
56, 5979.
9. (a) Ma, Y.; Lahue, R. B.; Shipps, W. G., Jr.; Brookes, J.; Wang, Y. Bioorg. Med.
Chem. Lett. 2014, 24, 1026; (b) Wang, Y.; Zhang, R.; Ma, Y.; Lahue, B.; Shipps, G.
U.S. Pat. Appl. Publ. 2008, US2008004286.; (c) 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, M.; Pan, W.; Siegel, E.; Gibeau, C. U.S. Pat. Appl. Publ. 2008,
US2008004287.
for additional surface interactions and fine tune physiochemical
properties of the lead molecules.
In conclusion, systematic functionalization of position 4, 5 and 6
of the piperidine ring was explored. Although most of substitutions
studied were tolerated, incorporation of a gem-dimethyl group at
position 5 in compound 11 retained potency level comparable ver-
sus 1a,b. Recent reports from our group demonstrated that incor-
poration of an allyl side chain at position 2 provided a drastic
improvement in binding potency; it would suggest that positions
5 and 2 of our newly discovered HDM2–p53 inhibitors can be used
for additional SAR optimization. Further work directed towards the
incorporation of functional diversity will be reported shortly.
References and notes
10. Nutlin 3a: Caco2 permeability = 126 nM/s. Compound 1a: Caco2
1. American Cancer Society, Surveillance Research, 2013.
2. (a) Mandinova, A.; Lee, S. W. Sci. Transl. Med. 2011, 3, 64rv1; (b) Raycroft, L.;
Wu, H. Y.; Lozano, G. Science 1990, 249, 1049.
permeability = 158 nM/s, mouse PK (PO/IV, 20/20 mpk) AUC = 1.9
F = 19%. Compound 1b: Caco2 permeability = 203 nM/s, mouse PK (PO/IV, 20/
20 mpk) AUC = 1.1 M h; F = 32%.
lM h;
l
3. Hainaut, P.; Hollstein, M. Adv. Cancer Res. 2000, 77, 81.
11. Cvetovich, R. J.; Chung, J. Y. L.; Kress, M. H.; Amato, J. S.; Matty, L.; Weingarten,
M. D.; Tsay, F.-R.; Li, Z.; Zhou, G. J. Org. Chem. 2005, 70, 8560; Bargellini, G. Gazz.
Chim. Ital. 1906, 36, 329; Rychnovsky, S. D.; Beauchamp, T.; Vaidyanathan, R.;
Kwan, T. J. Org. Chem. 1998, 63, 6363.
12. Cossy, J.; Dumas, C.; Michel, P.; Pardo, D. G. Tetrahedron Lett. 1995, 36,
549.
4. (a) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.;
Pavletich, N. Science 1996, 274, 948; (b) Chen, J.; Marechal, V.; Levine, A. J. Mol.
Cell. Biol. 1993, 13, 4107; (c) Bottger, A.; Bottger, V.; Garcia-Echeverria, C.;
Chene, P.; Hochkeppel, H. K.; Sampson, W.; Ang, K.; Howard, S. F.; Picksley, S.
M.; Lane, D. P. J. Mol. Biol. 1997, 269, 744.
5. (a) Vousden, K. H.; Lane, D. P. Rev. Mol. Cell Biol. 2007, 8, 275; (b) Bond, G. L.; Hu,
W.; Levine, A. J. Curr. Cancer Drug Targets 2005, 5, 3; (c) Wade, M.; Wang, Y. V.;
Wahl, G. M. Trends Cell Biol. 2010, 20, 299.
6. (a) Poyurovsky, M. V.; Prives, C. Genes Dev. 2006, 20, 125; (b) Brown, C. J.; Lain,
S.; Verma, C. S.; Fersht, A. R.; Lane, D. P. Nat. Rev. Cancer 2009, 9, 862.
7. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovie, Z.; Kong, N.;
Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303, 844.
13. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
14. Yu, J.; Truc, V.; Riebel, P.; Hierl, E.; Mudryk, B. Tetrahedron Lett. 2005, 46, 4011.
15. Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron 1968, 24, 53.
16. Zhang, R.; Mayhood, T.; Lipari, P.; Wang, Y.; Durkin, J.; Syto, R.; Gesell, J.;
McNemar, C.; Windsor, W. Anal. Biochem. 2004, 331, 138.
17. Ma, Y.; Lahue, R. B.; Gibeau, R. C.; Shipps, W. G., Jr.; Bogen, S. L.; Guo, Z.; Guzy,
T.; Wang, Y. ACS Med. Chem. Lett. 2013. accepted for publication.