Hit to lead studies on (hetero)arylpyrimidines-Agonists of the canonical Wnt-β-catenin cellular messaging system

Article abstract of DOI:10.1016/j.bmcl.2009.10.093

A series of (hetero)arylpyrimidines agonists of the Wnt-β-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3β inhibition indicating that the Wnt-β-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated β-catenin formation in bone.

Full text of DOI:10.1016/j.bmcl.2009.10.093

Bioorganic & Medicinal Chemistry Letters 20 (2010) 366–370
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hit to lead studies on (hetero)arylpyrimidines—Agonists of the canonical
Wnt-b-catenin cellular messaging system
a
a
c
c
Adam M. Gilbert a, , Matthew G. Bursavich , Nippa Alon , Bheem M. Bhat , Frederick J. Bex ,
Michael Cain d, Valerie Coleburn c, Virginia Gironda c, Paula Green c, Diane B. Hauze b,
Yogendra Kharode c, Girija Krishnamurthy f, Matthew Kirisits a, Ho-Sun Lam c, Yao-Bin Liu c,
Sabrina Lombardi a, Jeanne Matteo c, Richard Murrills c, John A. Robinson c, Sally Selim c,
Michael Sharp c, Raymond Unwalla b, Usha Varadarajan e, Weiguang Zhao c, Paul J. Yaworsky d
*
a Chemical Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, United States
b Chemical Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, United States
c Tissue Repair, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, United States
d Tissue Repair, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, United States
e Biological Technologies, Wyeth Research, 87 Cambridge Park Dr Cambridge, MA 02140, United States
f Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of (hetero)arylpyrimidines agonists of the Wnt-b-catenin cellular messaging system have been
prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-
1 and tk-Renilla. Selected compounds show minimal GSK-3b inhibition indicating that the Wnt-b-catenin
agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show
in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphor-
ylated b-catenin formation in bone.
Received 17 September 2009
Revised 20 October 2009
Accepted 21 October 2009
Available online 25 October 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Wnt-b-catenin agonist
(Hetero)arylpyrimidines
Osteoporosis
Calvaria
Activation of the Wnt-b-catenin signaling pathway has been
shown to play important roles in development, tissue regeneration,
stem cell control, tumor progression and metastases.1,2 This path-
way has also been implicated in the regulation of bone homeosta-
sis. The Wnt co-receptor LRP5 is central to this function where
both gain- and loss-of-function mutations have been described in
humans resulting in high bone mass or an osteoporosis pseudogli-
oma syndrome, respectively.3–5 Murine models of both genetic
conditions have been successfully generated.6,7 Pharmacologic
inhibition of either Dickkopf-1 (Dkk-1) or Sclerostin, two LRP5-
binding negative regulators of Wnt-b-catenin signaling, results in
increased bone mineral density in rodents.8,9 These data provide
additional evidence that agonists of Wnt-b-catenin activity could
yield an osteogenic agent.
Disheveled which in turn inactivates a protein complex comprising
Axin, Adenomatous Polyposis Coli, and Glycogen Synthase Kinase-
3b (GSK-3b). The resulting repression of GSK-3b activity leads to an
accumulation of unphosphorylated b-catenin enabling it to trans-
locate to the nucleus and form a transcriptional co-activator
complex with T-cell factor/lymphoid enhancer-binding factor
(TCF/LEF). Extracellularly, this mechanism can be antagonized by
several secreted molecules including Dkk-1, Sclerostin and the
Secreted Frizzled Related Proteins.
Both pharmacologic and genetic data support that the Wnt-b-
catenin pathway is a key mediator of the normal adaptive response
to mechanical loading in bone.11–13 As a result, agents capable of
mimicking the beneficial effects of Wnt-b-catenin activation on
the skeleton would represent a novel approach for the treatment
of osteoporosis and other bone disorders. In this Letter, we report
on the hit to lead studies on a class of (hetero)arylpyrimidines:
agonists of the Wnt-b-catenin pathway and their use as potential
anabolic agents to increase bone mass starting from 1, a (het-
ero)arylaminopyrimdine found from high-throughput screening.
Compounds 4 are generally prepared according to Scheme 1.14
Thus 2-chloropyrimidine 2 when reacted with aryl/heteroaryl
Signaling by the Wnt-b-catenin pathway has been extensively
studied in many systems.1,2,10 Briefly, secreted Wnt ligands bind
to cells via Frizzled receptors and the LRP5 or LRP6 co-receptors.
This ligand-receptor interaction activates the cytoplasmic protein
* Corresponding author.
E-mail address: gilbera@wyeth.com (A.M. Gilbert).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.093

A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 366–370
367
Table 1
H
N
Cl
N
Cl
N
N
Ar2
Wnt-b-catenin activities of compounds 1, 5–9
a
b
n
R
H
N
N
N
N
N
R1
Ar1
Ar1
N
2
3
4
Scheme 1. Reagents and conditions: (a) (1) Ar1Li, Et2O, À78 to 0 °C; (2) DDQ, THF,
23 °C; (b) H2N(CHR)nAr2, NMP, 90 °C or H2N(CHR)nAr2, NaH, DMSO, 80 °C.
N
1, 5-9
b
lithiums in Et2O produces the corresponding 4-aryl/heteroaryl
compounds.15,16 Target compounds 4 are prepared by reacting 3
with amines in hot NMP or by first deprotonating the amine with
NaH in DMSO and then heating with 3 in hot DMSO.
Compd
R1
TCFa
EC50
(20
l
M)
(lM)
1
5
6
7
8
9
N-(3-(1H-Imidazol-1-yl)propane)
N-(2-(Pyridin-4-yl)ethane)
N-(2-(Pyridin-3-yl)ethane)
N-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)propyl)
N-(2-(1H-Indol-3-yl)ethane)
N-(S)-3-(1H-Indol-3-yl)-2-propan-1-ol amine
2.9
4.3
2.8
2.1
7.5
2.2
4.2
6.8
4.6
29.6
2.7
N
H
N
N
N
FTCc
N
a
U2OS: Wnt-3a/Dkk-1/TCF-Luci fold induction at 20 lM. Values are the average
of 2 or 3 runs. Error 20%.
b
U2OS: Wnt-3a/Dkk-1/TCF-Luci EC50. Values are the average of 2 or 3 runs. Error
20%.
FTC: EC50 determination failed to converge.
N
c
1
Wnt-3a/Dkk-1/TCF-Luci EC 50: 4.2 µM (2.9 FI @ 20 µM)
GSK-3β IC50: 51 µM
produces a less potent compound (7) while the ethylindole deriva-
tive (8) gives a 2.5-fold more efficacious analog of 1 (8: TCF
(20 lM): 7.5; EC50: 2.7 lM). The more complex 9 containing a prop-
anolindole substituent possesses similar efficacy to 1 but the EC50
fails to converge.
Table 2 outlines the effects of varying the 4-pyrimidine position
on compounds similar to 1. Changing R2 from a pyridine-4-yl to a
pyridine-3-yl results in a slight increase in efficacy (10: TCF
Compounds were assayed using U2OS cells transfected with
Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla as a signal normalizer
compared to control U2OS cells transfected with Wnt-3a, TCF-
Luciferase and tk-Renilla.17 The signal from the TCF-luciferase is re-
duced in the presence of the inhibitor Dkk-1. Compounds that
modulate Dkk-1 activity or act downstream of the Wnt-3a/Dkk-1
complex (i.e., GSK-3b inhibition) cause an increase in the luciferase
signal. For all compounds in this manuscript, EC50s were deter-
(20
compound (11: EC50: 46.3
indole) shows M-potency with large fold-induction (13: TCF
(20 M): 10.0; EC50: 4.1 M).
l
M): 4.7) while a 3-NO2-phenyl group gives a much less potent
l
M). Compound 13 where R1 = 3-(1H-
mined as well as the fold-induction response at 20 lM compared
to the control U2OS cells. A [32P]-GSK-3b was used to assay com-
pounds for Wnt/b-catenin activity downstream (of, from) Wnt-
3a/Dkk-1.
l
l
l
Of the 4-(1H-imidazole) indole compounds 15–17, the 2-
(benzo[b]thiophene) analog 16 possesses the best combination of
potency and efficacy (EC50: 3.3 lM, TCF (20 lM): 3.7). Several
phenethyl analogs were also prepared, but they showed poor
Wnt-b-catenin agonism.
To follow-up compound 16 which shows lM potency and mod-
erate efficacy in the Wnt-3a/Dkk-1/TCF-Luci assay, 2-(naphthyl)-2-
Compound 1 shows a robust TCF-Luciferase response at 20
l
M as
well as a lM-EC50 (1: TCF (20 lM): 2.9; EC50: 4.2 lM) (Table 1). Since
arylamino-pyrimidines are well-known kinase inhibitors,18 1 was
assayed for GSK-3b inhibition but little kinase inhibition was seen
(IC50: 51 l
M). Modulation of the R1 substituent to ethylpyridines
(5 and 6) produces compounds of comparable efficacy/potency to
1. The replacement of R1 with a propyl(dimethylpyrazole) group
(benzo[b]thiophene)-derivatives were prepared according to the
Table 2
Wnt-b-catenin activities of compounds 1, 10–11
H
N
N
R1
N
R2
1, 10-17
R1
R2
TCFa (20
l
M)
EC50 (lM)
b
Compd
1
10
11
12
13
14
15
16
17
CH2-1H-Imidazole
CH2-1H-Imidazole
CH2-1H-Imidazole
4-Pyridine
4-(Pyridin-4-yl)
4-(Pyridin-3-yl)
4-(3-Nitrophenyl)
4-(Pyridin-3-yl)
4-(Pyridin-3-yl)
4-(pyridin-3-yl)
4-(Pyridin-3-yl)
2-(Benzo[b]thiophene)
2-(Naphthyl)
2.9
4.7
3.6
3.4
10.0
5.1
3.1
3.7
1.1
4.2
8.8
46.3
20.2
4.1
12.2
28.8
3.3
3-(1H-Indole)
3-(2-Methyl-1H-indol-5-ol)
4-(1H-Imidazole)
4-(1H-Imidazole)
4-(1H-Imidazole)
12.1
a
U2OS: Wnt-3a/Dkk-1/TCF-Luci fold induction at 20
U2OS: Wnt-3a/Dkk-1/TCF-Luci EC50. Values are the average of 2 or 3 runs. Error 20%.
l
M. Values are the average of 2 or 3 runs. Error 20%.
b

368
A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 366–370
with NaH followed by the addition of an alkylating agent to pro-
duce 30.
O-Substituted derivatives in Table 3 show moderate Wnt-3a/
Dkk-1/TCF-Luci efficacy at 20 M, but most of the compounds
were not as potent as and efficacious as 16. This was true for alkyl,
aryl and alkynyl derivatives 25–29. The exception was hydroxyl
analog 25. Analogs of 16 where the imidazole nitrogen is alkylated
show similar to greater efficacy compared to the O-substituted
analogs as a number of substituents are tolerated (31, 32: benzyl,
33: propynyl, 34: acetamide, 35, 36: 3-propanitrile), but compara-
tively weaker potency to 25 (see Table 4).
b
H2N
HO
a
H2N
NH
· 2HCl
NH
N
N
O
MeO
O
l
18
19
H
N
H
N
c
d
Trit
Trit
Trit
Trit
N
N
N
N
21
MeO
O
HO
20
H
N
Example 25 represents a compound with good properties of a
chemical lead based on 1. It has robust Wnt-3a/Dkk-1/TCF-Luci
efficacy @ 20 lM (FI 4.9), good potency (EC50: 6.8 lM) and an
acceptable ligand efficiency. The calculated physical properties
are in line for a molecule with good drug-like properties. Good
aqueous solubility, moderate PAMPA permeability and good rat li-
ver microsome stability is also seen.
e
H2N
f
Trit
Trit
N
NH
N
N
R1O
R1O
22
23
H
N
N
NH
H
N
N
N
N
R1O
R2
NH
N
N
24
HO
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 60 °C; (b) Trit–Cl, TEA, MeCN,
23 °C; (c) LiAlH4. THF 0 to 23 °C; (d) NaH, R1X, DMF, 50 °C; (e) HCl, THF, reflux; (f)
Scheme 1, step b.
25
Wnt-3a/Dkk-1/TCF-Luci fold induction at 20 µM: 4.9
chemistry outlined in Schemes 2 and 3. Histidine 18 may be con-
verted to methyl ester 19 using standard conditions. After being
bis(tritylated) to produce 20, reduction with LiAlH4 produces the
primary alcohol 21. Alkylation with various alkyl and aryl bro-
mides produces 22. Deprotection with HCl yields the derivatized
histidines 23 which were incorporated into products 4 using the
procedure of Scheme 1, Step b. Analogs where the imidazole nitro-
gen is substituted are prepared by reacting compounds like 25
Wnt-3a/Dkk-1/TCF-Luci EC50: 6.8 µM
LE: 0.27
LELP: 8.9
MW: 345.40
clogP: 2.4
TPSA: 87
Aqueous Solubility: 65 µg/mL
PAMPA: 0.13 x 10 -6 cm/s
RLM t1/2 ≥ 80 min
H
N
H
N
In order to establish the osteogenic activity of this series, 1 was
evaluated in vivo via local subcutaneous injection over the right side
of the calvaria of wild type C57BL/6 mice.11 As a positive control, an
inhibitor of GSK-3b (GSKi), 3-(3-chloro-4-hydroxyphenylamino)-4-
(2-nitrophenyl)-1H-pyrole-2,5-dione),19 was administered at 1 mg/
kg/day as previously described.11 Vehicle alone was used as the neg-
ative control. Using standard dynamic histomorphometry measure-
ments, the mineral apposition rate of new bone formation was
N
N
a
R1
N
NH
N
N
N
N
HO
HO
R2
R2
25
30
Scheme 3. Reagents and conditions: (a) NaH, R1X, THF/DMSO, 23 °C.
Table 3
Wnt-b-catenin activities of compounds 25–29
Table 4
Wnt-b-catenin activities of compounds 31–38
H
N
N
H
N
N
NH
N
N
R1
N
R1O
N
N
R2
HO
R2
25-29
31-36
Compd R1
R2
TCFa
EC50
M)
b
Compd R1
R2
3,5-Difluorobenzyl 2-(Benzo[b]thiophene) 1.7
3,5-Difluorobenzyl 2-(Naphthyl) 2.0
2-(Benzo[b]thiophene) 3.0
2-(Naphthyl) 3.9
2-(Benzo[b]thiophene) 4.1
2-(Naphthyl) 5.5
TCFa (20
l
M) EC50 (lM)
b
(20
l
M)
(
l
31
32
33
34
35
36
16.0
FTC
14.6
FTC
FTC
FTC
25
26
27
28
H
Ethyl
2-(Naphthyl)
2-(Naphthyl)
2-(Naphthyl)
2-(Naphthyl)
4.9
1.9
2.2
1.6
6.8
14.4
20.8
10.9
Prop-2-ynyl
2-Acetamide
3-Propanitrile
3-Propanitrile
Methylenecyclohexyl
(2-Fluoro-3-
(trifluoromethyl)benzyl
Prop-2-ynyl
29
2-(Benzo[b]thiophene) 3.5
FTC
a
a
Values are the average of 2 or 3 runs. Error 20%.
FTC: EC50 determination failed to converge.
Values are the average of 2 or 3 runs. Error 20%.
FTC: EC50 failed to converge.
b
b

A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 366–370
369
calculated exactly as previously described.11 All doses of 1 showed a
significant increase in anabolic bone activity over vehicle with levels
equivalent to that attained by the GSKi positive control in all but the
lowest tested dose of 1 (Fig. 1A). Mechanistically, this anabolic bone
activity could be ascribed to activated Wnt-b-catenin signaling as
visualizedby theimmunohistochemicaldetectionof non-phosphor-
ylated b-catenin in the osteoblastic cells lining the periosteal surface
of the calvaria (Fig. 1B–E). Furthermore, using the same in vivo mod-
el, 25 was also shown to be an anabolic bone agent (Fig. 1F). Together
these data support that 1 and 25 are both Wnt-b-catenin pathway
agonists and efficacious osteogenic agents in vivo.
In conclusion, we have disclosed a series of (hetero)arylpyrimi-
dines that act as agonists of the Wnt-b-catenin pathway. While we
currently don’t know their exact mechanism of action, these com-
pounds don’t function via inhibition of GSK-3b. Moreover, exam-
ples of this equity show osteogenic in vivo activity in mouse
calvaria as well as immunohistochemical data indicating that
Wnt-b-catenin activity is activated in bone.
*
*
3.0
2.5
2.0
A
*
1.5
1.0
0.5
Veh
GSKi
0.03
0.1
Compound 1 (mg/kg/day)
Acknowledgments
We thank Dr. John Ellingboe for support of this work and the
Chemical Sciences Compound Properties group for providing phys-
icochemical profiling data on all analogs disclosed in this work.
Periosteal
Surface
B
D
C
E
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Compound 25 (mg/kg/day)
Figure 1. Compounds 1 and 25 are osteogenic in vivo. (A) Quantitative dynamic
histomorphometry was performed on murine calvaria following 7 days of
treatment. Mineral apposition rates were calculated and are presented as an index
1
of anabolic, osteoblast activity. Veh, vehicle; GSKi, Glycogen synthase kinase-3b
*
inhibitor;19 p <0.01 versus vehicle. (B–D) Immunohistochemical detection of non-
phosphorylated b-catenin is enhanced in compound-treated calvaria suggesting
that Wnt-b-catenin signaling is activated. Positive cells are identified by the pink
staining as indicated by the arrowheads. Vehicle (B), GSKi (C), 0.3 mg/kg/day 1 (D),
1.0 mg/kg/day 1 (E). (F) Compound 25 is osteogenic in the same assay showing
significantly elevated mineral apposition rates versus vehicle.
14. All newly prepared compounds were characterized by reversed phases-HPLC/
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1H NMR.
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