(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine: A wingless β-catenin agonist that increases bone formation rate

Article abstract of DOI:10.1021/jm9014197

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt β-catenin cellular messaging system. Hit-tolead in

Full text of DOI:10.1021/jm9014197

6962 J. Med. Chem. 2009, 52, 6962–6965
DOI: 10.1021/jm9014197
(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-
4-yl)methanamine: A Wingless β-Catenin
Agonist That Increases Bone Formation Rate
Jeffrey C. Pelletier,*,† Joseph T. Lundquist IV,†
Adam M. Gilbert, Nipa Alon, Frederick J. Bex,‡
Bheem M. Bhat,‡ Mattew G. Bursavich,
Valerie E. Coleburn,‡ Luciana A. Felix,† Daniel M. Green,†
Paula Green,‡ Diane B. Hauze,† Yogendra P. Kharode,‡
Ho-Sun Lam,‡ Susan R. Lockhead,§ Ronald L. Magolda,†
Jeanne J. Matteo,‡ John F. Mehlmann,† Colleen Milligan,‡
Richard J. Murrills,‡ Jennifer Pirrello,‡ Sally Selim,‡
Michael C. Sharp,‡ Ray J. Unwalla,† Matthew D. Vera,†
Jay E. Wrobel,† Paul Yaworsky,‡ and Peter V. N. Bodine‡
Figure 1. Structures, Dkk-1 mediated TCF-luciferase activity, and
GSK-3β inhibition of 1 and 2.
†Chemical Sciences, ‡Tissue Repair and §Drug Safety & Metabolism,
Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426,
and Chemical Sciences, Wyeth Research, 401 N. Middletown Road,
Pearl River, New York 10965
Received September 23, 2009
Abstract: A high-throughput screening campaign to discover small
molecule leads for the treatment of bone disorders concluded with
the discovery of a compound with a 2-aminopyrimidine template
that targeted the Wnt β-catenin cellular messaging system. Hit-to-
lead in vitro optimization for target activity and molecular proper-
ties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-
yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5
has excellent pharmacokinetic properties and showed a dose de-
pendent increase in the trabecular bone formation rate in ovariec-
tomized rats following oral administration.
Figure 2. Docked structure of 1 in the active site of GSK-3β Yellow
dotted lines indicate H-bond interactions with residues in the hinge
region (see refs 9 and 10 for docking/structure details).
Hence, clinically relevant data indicate the important role of
Dkk-1 in attenuating the Wnt β-catenin anabolic process and
suggest that pharmacologic inhibition of Dkk-1 may lead to
bones of greater strength for the treatment of osteoporosis and
other diseases of low bone mass or quality.4
Toidentifysmall molecules thatinhibit the action of Dkk-1,
a high-throughput assay was conducted with the corporate
collection using an osteosarcoma cell line transfected with
Wnt-3a and Dkk-1. In addition, a TCF-luciferase response
element and a renilla standard were transfected for light
readouts of active compounds and control readout, respec-
tively.5 The 2-aminopyrimidine phenylsulfonamide 1 was
identified as a weak agonist in this assay (EC50 = 7.0 μM).
An N-methyl analogue, 2, also potentiated the TCF-luciferase
response in this assay at roughly the same potency (Figure 1).
Resynthesized compounds 1 and 2 showed similar TCF-
luciferase activity in HTS and benchtop formats.
The 2-aminopyrimidine motif is common to numerous
kinase inhibitors.6 Inhibition of the glycogen synthase ki-
nase-3β (GSK-3β), an enzyme involved in β-catenin phos-
phorylation, would provide a false positive signal in the TCF-
luciferase assay.7 Therefore, compound evaluation in a GSK-
3β enzyme assay was studied side by side with target evalua-
tion.8 Moderately potent enzyme inhibition was achieved with
1 (IC50 = 0.20 μM). To understand the key ligand features
that are responsible for this activity, 1 was docked9 in the
active site of previously disclosed X-ray structure of GSK-
3β.10 As shown in Figure 2, the docked structure shows that
ligand recognition is achieved by critical hydrogen bond
interactions within the hinge region. The model also suggested
The canonical Wnta β-catenin cellular messaging system
controls several physiological events including bone home-
ostasis.1 Bone forming osteoblasts express the proteins LRP5
and Fzd on the surface membrane. These behave as corecep-
tors for the soluble peptide agonist Wnt-3a. Once stimulated
with Wnt-3a, internal concentrations of free β-catenin rise and
enter the nucleus and recruit T-cell factor (TCF). Transcrip-
tional events follow and result in the production of additional
anabolic gene products. An additional soluble extracellular
protein, Dkk-1, antagonizes this process by simultaneously
binding to the cell surface receptors Kr2 and LRP5, effectively
inhibiting Wnt-3a binding to LRP5. In addition, the Kr2/
LRP5/Dkk-1 complex undergoes endocytosis to remove
LRP5 from the cell membrane, thereby nullifying its function.2
Reports have described a familial gain-of-function mutation
on the LRP5 receptor that results in bones of normal size and
shape but with significantly increased density.3 The mutation
occurs at a single position on the LRP5 receptor, G171V, and
results in a protein with substantially less affinity for Dkk-1
while affinity for Wnt-3a remains essentially unchanged.
*To whom correspondence should be addressed. Phone: (484) 865-
2912. Fax: (484) 865-9399. E-mail: Pelletj@wyeth.com.
a Abbreviations: Wnt, wingless; LRP5, low-density lipoprotein re-
ceptor-related protein 5; Fzd, frizzled; TCF, T-cell factor; Dkk-1,
Dickkopf-1; Kr2, Kremen-2; GSK-3β, glycogen synthase kinase 3β;
OVX, ovariectomized rat; BFR, bone formation rate; qd, once per day;
hPTH, human parathyroid hormone; KO, knockout mouse; wt, wild
type mouse.
r
pubs.acs.org/jmc
Published on Web 10/26/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 6963
Scheme 1. Preparation of Compoundsa
Table 1. TCF-Luciferase, GSK-3β, and Solubility Data for Com-
pounds 1-6
a Conditions: (a) 2-methylthiophene, n-BuLi, then DDQ (80%);
(b) sulfanilamide or N0-methylsulfanilimide, toluenesulfonic acid, 1,4-
dioxane, 95 ꢀC (80-88%); (c) DMF-DMA, 85 ꢀC (100%); (d) urea,
NaOEt, EtOH, ref (54%); (e) POCl3, ref (84%); (f) RR0NH, DMSO,
60 ꢀC (69-70%).
that alkylation of the 2-amino group to remove the H-bond
donor would diminish enzyme activity. This was the case with
2 (see Figure 1). These experiments suggest that 2 potentiates
the TCF response through Dkk-1 inhibition and not through
a mechanism related to kinase inhibition. Hence, a structure-
activity relationship (SAR) study centered on 2 was estab-
lished.
Compounds were synthesized according to the routes shown
in Scheme 1. 4-(Thiophenyl)pyrimidines 1 and 2 were prepared
by the method of Bursavich.11 4-(Naphthyl)pyrimidines 3-6
were obtained from the corresponding 2-naphthylmethyl ke-
tone which was reacted with dimethylformamide dimethylace-
tal to provide the dimethylvinylogous amide.12 Reaction of this
product with urea under basic conditions gave the pyrimidinone
sodium salt which was converted to the 2-chloropyrimidine
with phosphorus oxychloride. Final products were obtained
when the chloride was displaced with secondary amines.
Attempts to improve primary assay potency and reduce
potential metabolic activity led to replacement of the methyl-
thiophene in 1 with a 2-naphthyl function (e.g., 3). This
structural change also led to enhanced GSK-3β inhibiton
and substantially reduced solubility. It was assumed that
replacement of the sulfanilamide functionality with a fully
reduced cyclic structure appended with distal H-bond donors
would retain TCF-luciferase activity, reduce GSK-3β activity
via full alkylation of the exocyclic nitrogen, improve solubility
by interfering with the π-stacking characteristics of the phenyl
group, and maintain the cLogP between 3 and 4. The 4-amino-
piperidine group satisfied these requirements and, in fact, led
to an active molecule, 4, with high solubility and low GSK-3β
activity. Additional standard SAR studies showed the methy-
lamine 5 to have the most potent activity in the primary assay,
low kinase inhibition potential, and high solubility (Table 1).
In vitro pharmaceutical properties13 of 5 indicated it would
be an excellent candidate for in vivo pharmacokinetic evalua-
tion in ovariectomized (OVX) rats (PAMPA membrane
a Efficacy for all six compounds ranged from 2.0 to 6.0 relative signal
increase compared to DMSO alone. b Values represent the average of at
least two experiments done in triplicate. c SD valuess are within (25% of
the EC50/IC50.
d See ref 8 for assay details. e See ref 13 for assay details.
permeability, Pe = 5.9 ꢀ 10-6 cm/s; rat liver microsome
stability, t1/2 > 30 min at 1.0 μM). Hence, 5 was administered
to OVX rats (single dose iv and po). Pharmacokinetic para-
meters and plasma drug concentration-time curves are
shown in Table 2 and Figure 3, respectively.
The PK profile indicated that 5 was a candidate for in vivo
pharmacological evaluation with once a day dosing. Hence,
OVX rats14 were treated orally with 5 (po, vehicle = 0.5%
methylcellulose/2% Tween-80, qd, 28 days) at four doses.
Trabecular bone formation rate (BFR) in the tibia was
established in all dose groups at the end of the in-life portion
of the study. A clear dose response and activity as low as
0.3 mg/kg/day were observed (Figure 4).
To confirm activity via the Wnt pathway, the calvariae of
wild type (wt) and Dkk-1 knockout (KO) mice were treated
with 5 once a day for 7 days (DMSO solution, sc injection).15
The KO animals were not expected to respond because of the
inherent inabilityto inhibit a missing target protein, while wild

6964 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Pelletier et al.
Table 2. Single Dose Pharmacokinetic Parameters for Compound 5 in
OVX Rats
type animals with fully expressed Dkk-1 were expected to
show a pharmacological response. Calvariae from wt mice
treated with 5 showed statistically increased BFR, while
similarly treated KO animals were no different from control
(Figure 5). This indicates that 5 is acting via the Wnt β-catenin
pathway and most likely through inhibition of Dkk-1.
In conclusion, 2-aminopyrimidines that potentiate the Wnt
β-catenin cellular signaling pathway were discovered via high-
throughput screening. Lead optimization led to a compound
with enhanced in vitro activity. Compound 5 was shown to
possess excellent pharmaceutical and PK properties, and the
compound was able to enhance the bone formation rate in
OVX rats following oral adminstration.
compound 5
parameter
vehicle
dose (mg/kg)
po admin
iv admin
2% MC/0.5% T80a
10
DMSO
2.0
8.2
plasma t1/2 (h)
Tmax (h)
Cmax (ng/mL)
5.6
4.7
277
3990
78
AUC0-¥ (ng h/mL)
1029
3
F (%)
Clp ((mL/min)/kg)
Vss (L/kg)
32
22
a MC = methylcellulose. T80 = Tween-80.
Acknowledgment. We thank Tarek Mansour and John
Ellingboe for support of this program. We thank the Depart-
ment of Chemical Technologies for compound characteriza-
tion and pharmaceutical profiling.
Supporting Information Available: Experimental details and
characterization data for all compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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