Discovery of potent and bioavailable GSK-3β inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.038

Here we report on the discovery of a series of maleimides which have high potency and good selectivity for GSK-3β. The incorporation of polar groups afforded compounds with good bioavailability. The most potent compound 34 has an IC₅₀ of 0.6 nM for GSK-3β, over 100-fold selectivity against a panel of other kinases, and shows efficacy in rat osteoporosis models. The X-ray structure of GSK-3β protein with 34 bound revealed the binding mode of the template and provided insights for future optimization opportunities.

Full text of DOI:10.1016/j.bmcl.2010.01.038

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1693–1696
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and bioavailable GSK-3b inhibitors
a
a
a
b
b
c
Leyi Gong a, , Don Hirschfeld , Yun-Chou Tan , J. Heather Hogg , Gary Peltz , Zafrira Avnur , Pete Dunten
*
a Department of Medicinal Chemistry, Roche Palo Alto, Palo Alto, CA 94304, USA
b Center for Genetics and Genomics Roche Palo Alto, Palo Alto, CA 94304, USA
c Lead Discovery, Roche Palo Alto, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we report on the discovery of a series of maleimides which have high potency and good selectivity
for GSK-3b. The incorporation of polar groups afforded compounds with good bioavailability. The most
potent compound 34 has an IC50 of 0.6 nM for GSK-3b, over 100-fold selectivity against a panel of other
kinases, and shows efficacy in rat osteoporosis models. The X-ray structure of GSK-3b protein with 34
bound revealed the binding mode of the template and provided insights for future optimization
opportunities.
Received 31 October 2009
Revised 7 January 2010
Accepted 11 January 2010
Available online 25 January 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
GSK-3b inhibitors
Glycogen synthase kinase inhibitors
Osteoporosis
Kinase
In the US nearly 1 million fractures occur annually in people
over the age of 65, mostly due to osteoporosis. The need to develop
novel drugs that stimulate bone formation and thereby increase
bone mass is an important research area for therapeutic interven-
tion in the treatment of osteoporosis. Combined genetic and geno-
mic studies revealed that a Wnt interacting protein (Frzb1) was a
key element influencing peak bone density in humans as well as
in mice.1 Furthermore, glycogen synthase kinase-3b (GSK-3b), a
serine/threonine protein kinase, has been identified as a regulatory
enzyme in this pathway. Reported here is our effort to use small
molecules to define the role of GSK-3b inhibition in the Wnt path-
way and the resulting effect on bone density in a murine model of
osteoporosis.
The investigation was focused on the exploration of the SAR
around the maleimide template with the aim of improving kinase
selectivity, particularly against PKC, and improving in vivo
bioavailability.
Compounds 8–26 were synthesized following the general route
depicted in Scheme 1.
Acylation of N-methylindole 4 with oxalyl chloride was carried
out in diethyl ether to give compound 5, which was treated with
O
Cl
NO2
N
N
N
O
N
O
Cl
N
N
Human GSK-3 has two isozymes (a and b) sharing 95% homol-
N
Cl
N
O
N
N
ogy around the catalytic domain. GSK-3 is a ubiquitous kinase that
participates in a multitude of cellular processes, such as cell mem-
brane-to-nucleus signaling, gene transcription, translation, cyto-
skeletal structuring and cell cycle progression and survival.2,3
Therefore, there are potentially multiple therapeutic benefits by
inhibiting GSK-3.
A number of chemical classes of GSK-3 inhibitors with varying
potencies have been reported in the literature (Fig. 1).4–8
Our discovery effort to identify a potent class of GSK-3b inhib-
itors with oral bioavailability and in vivo efficacy was initiated
from the directed screening of an in-house PKC inhibitor library
containing bis-indolyl maleimides. Representative examples are
shown in Figure 2.
N
N
N
R
Paullone
CT-98024
SB-415286
IC50 = 100 nM
IC50= 0.56 nM
IC50 = 77 nM
N
O
O
O
N
O
R2
R3
N
N
N
N
N
O
R1
N
NMe2
Lilly-GSK-3
J&J GSK-3
IC50 = 7 nM
IC50 = 1-5 nM
* Corresponding author. Tel.: +1 650 354 7187; fax: +1 650 354 2442.
E-mail address: leyi.gong@roche.com (L. Gong).
Figure 1. Different chemical classes of GSK-3 inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.038

1694
L. Gong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1693–1696
N
N
Cl
N
N
O
O
O
O
O
O
N
O
O
N
O
F
F
O
NO2
O
(i)
+
NO2
O
O
N
N
N
N
N
N
N
36
N
N
3
O
O
N
1
2
S
O
O
F
F
PKC: 22 nM
GSK: 20 nM
PKC: 1050 nM
GSK: 10 nM
PKC: 6000 nM
GSK: 90 nM
(ii)
(iii)
N
O
N
N
N
O
Figure 2. Examples of the screen hits from PKC inhibitor library.
37
O
38
N
O
Cl
O
F
(iv)
O
O
(ii)
(i)
N
O
+
Y
O
N
N
X
N
O
X
4
5
6
34
O
O
O
N
O
O
Scheme 3. Reaction conditions and yields: (i) Scheme 1 (ii–iii); (ii) TiCl3 in acetone,
rt, 18 h, 83%; (iii) 2,2-dimethyldioxolane-4-carboxaldehyde,9 Na(OAc)BH3, CH2Cl2,
rt, 18 h, 25%; (iv) pTsOH (cat.), MeOH, 50 °C, 18 h, 66%.
(iii)
X
X
N
Y
N
Y
7
8-26
Table 1
Enzymatic activity of monoindolyl maleimidesa
Scheme 1. Reaction conditions and yields: (i) (COCl)2 in Et2O at 0 °C; (ii) Et3N in
CH2Cl2 at 0 °C, 30%; (iii) NH3 (aq) in DMF, 80–90%.
N
O
O
phenyl acetic acid 6 and triethylamine in CH2Cl2 at 0 °C to afford
the corresponding furan-2,5-dione analogs 7. A solution of 7 in
DMF and aqueous ammonia was heated to 140 °C for 5 h to afford
80–90% of the maleimide analogs 8–26.
Scheme 2 described the synthesis of compounds 30–32 from a
commercially available chiral precursor.
Compounds 33–35 were prepared via the route shown in
Scheme 3, starting from 3-nitrophenylacetic acid. The reduction
of the nitro group in 36, followed by reductive amination and
deprotection, afforded compound 34.
The maleimide series identified from the screening exhibited
dual inhibition against both PKC and GSK-3b. A close examination
of the initial SAR of compounds 1–3 (Fig. 2) reveals two elements
differentiate PKC from GSK. First, PKC potency requires two-indolyl
rings attached to the maleimide whereas GSK potency could be
maintained with one indolyl ring and one monocyclic ring such
as phenyl. Second, PKC inhibition is favored by the presence of a
basic nitrogen moiety whereas it is not essential for GSK activity.
Based on these observations, the strategy we chose was to first
confirm our hypothesis that GSK selectivity may be obtained by
removing the second indole group, and then to optimize a mono-
indolyl maleimide for potency (Table 1).
Ar
N
Compound
Ar
GSK3b/PKCa
IC50, nMa
8
9
2-Cl-Ph
3-NH2-Ph
3.6/760
1.8/1900
2.4/5500
6.6/6600
22/380
22/8900
42/>30,000
730/NA
10
11
12
13
14
15
2,4-Di-OMe-Ph
3,4-Di-OMe-Ph
2,5-Di-OMe-Ph
2-Thiophene
3-Pyridyl
4-Ph-Ph
Compounds were characterized by mass spectral, 1H NMR, elemental analysis
and mp. IC50 values are an average of multiple determinations (n P 2). Assay
conditions are described in Ref. 10; N.A. = no data obtained.
a
Table 2
Enzyme activity of phenyl analogs with linkers
H
N
O
O
In general, a number of substitution patterns can be tolerated at
the phenyl ring as exemplified in compounds 8–11 where 2-, 3-,
R
N
a
O
Compound
R
GSK3b IC50 (nM)
O
O
(iii)
O
O
O
O
(i)-(ii)
16
17
18
19
20
–SPh
–NHPh
–OPh
–N(BOC)Ph
–N-morpholine
2000
350
2800
3100
8200
O
O
+
TsO
O
O
O
27
28
N
N
O
O
O
Compounds were characterized by mass spectral, 1H NMR, elemental analysis
and mp. IC50 values are an average of multiple determinations (n P 2). Assay
conditions are described in Ref. 10; N.A. = no data obtained.
a
(iv)
O
O
O
N
N
O
O
29
30
3,4-, and 2,4- substitutions are all potent for GSK-3b but 2,5-substi-
tution (12) resulted in a less active compound. Thiophene 13 and
pyridine 14 are tolerated by GSK-3b and have very good selectivity
Scheme 2. Reaction conditions and yields: (i) NMP, K2CO3, 96 °C, 18 h, 68%; (ii)
LiOH–H2O (4 equiv) in MeOH/H2O (10:1), >99%; (iii) Scheme 1 (i–iii); (iv) pTsOH
(cat.), MeOH, 63%.

L. Gong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1693–1696
1695
Table 5
Solubility and Rat oral PK
Table 3
Enzyme activity of substituted indolyl analogs
Compound
Solubility
g/ml)a
Dose
(mpk)
Cmax
g/ml)
AUC
(lg h/ml)
(
l
(
l
O
21
32
34
35
<0.5
2.8
104
42
10b
10
10
25
BQLc
0.40
0.11
0.11
BQLc
1.06
0.35
0.10
N
O
X
O
N
a
b
c
Aqueous solubility: pH 6.6.
The route of administration was IP in mice.
BQL: Below Quantification Limit of 4 ng/ml.
a
Compound
X
GSK3b/PKCa IC50 (nM)
21
22
23
24
25
26
H
2-Me
6-F
5-Cl
6-Me
4-OMe
90/6000
220/NA
31/4200
6.4/1400
35/2900
1300/NA
a
Compounds were characterized by mass spectral, 1H NMR, elemental analysis
and mp. IC50 values are an average of multiple determinations (n P 2). Assay
conditions are described in Ref. 10; N.A. = no data obtained.
against PKC whereas biphenyl 15 has reduced activity. Compounds
16–19 demonstrate that placing a linker between the maleimide
and phenyl rings results in less active inhibitors (Table 2). The N-
linked morpholine analog 20 is also weak.
Our focus then shifted to substitution of the indole ring. Substi-
tution at position 5 or 6 enhanced the potency with the effect being
more pronounced at position 5 whereas the 2-Me and 4-OMe ana-
logs were less active (Table 3).
Having attained the desired potency and selectivity, the next
challenge was to achieve good bioavailability. In the course of eval-
uating this series, compound 21 was found to be extremely insol-
uble in addition to being extensively metabolized. We attributed
both problems to the highly lipophilic nature of the series and
we envisaged that reduction of the lipophilicity by incorporating
a polar functionality would improve both the solubility and the
metabolic stability. Compounds 30–35 were synthesized based
on this rationale.
Figure 3. X-ray of 34 bound to GSK-3b.
Incorporation of a number of polar or ionizable groups at the 3-
position of the phenyl ring afforded compounds that maintained
GSK-3b potency and PKC selectivity (Table 4). More gratifying
was that solubility and metabolic stability were improved as com-
pared to compound 21 (Table 5).
Shown in Figures 3 and 4 is the X-ray structure of GSK-3b with
compound 34 occupying the ATP binding pocket.11 The structure
revealed several key interactions by the compound with GSK-3b.
Table 4
Enzyme activity of substituted phenyl analogs with polar groups
H
N
O
O
O
diol =
X
O
Figure 4. Surface representation of 34 bound to GSK-3b.
R
N
With regard to polar interactions, the maleimide NH acts as a H-
bond donor interacting with Asp133 carbonyl group and one of
the carbonyl groups from the maleimide acts as a H-bond acceptor
to interact with Val135 NH. Together, they form the hinge interac-
tion as seen in most kinase inhibitors. The other carbonyl group
from the maleimide interacts with Asp200 through water. The side
chain shows additional polar interactions: the NH with Asn186 via
water and the terminal OH with Thr138 via water. As shown in Fig-
ure 4, the indole and phenyl rings stack on top of each other and
occupy the hydrophobic part of ATP pocket. In addition, the phenyl
a
Compound
X
R
GSK3b/PKCa IC50 (nM)
30
31
32
33
34
35
H
H
F
H
F
O-diol (R)
O-diol (S)
O-diol (R/S)
NH(CH2)3OH
NH-diol (R/S)
NH-diol (R/S)
3.2/1000
8.2/1400
1.8/2200
1/740
0.6/400
0.4/260
Cl
Compounds were characterized by mass spectral, 1H NMR, elemental analysis
and mp. IC50 values are an average of multiple determinations (n P 2). Assay
a
conditions are described in Ref. 10; N.A. = no data obtained.

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L. Gong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1693–1696
Table 6
Kinase selectivity profile of compound 34
a
a
Kinase
Kd
(lM)
Ratio over GSK3b
Kinase
Kd
(l
M)
Ratio over GSK3b
GSK3b
0.000053
0.0015
0.010
0.012
0.016
0.032
0.041
0.069
0.080
0.087
0.11
1
28
180
230
300
CDK9
ERK5
TTK
CDK5
PRKR
AURKC
PLK1
AURKA
ERK3
AURKB
PLK3
CDK11
ERK1
ERK2
ERK4
CDK8
0.41
5.6
6.7
6.8
14
23
23
40
40
40
40
40
40
40
40
40
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
GSK3
CLK2
a
PCTK1
CLK1
ERK8
600
770
CDK7
PCTK2
CDK3
DYRK1B
CLK4
PCTK3
CDK2
STK16
CLK3
1300
1500
1600
>2000
>2000
>2000
>2000
>2000
>2000
0.11
0.15
0.23
0.31
PLK4
0.32
a
Binding constant (Kd) Determination: Quantitative affinity measurement (11-point curve) using Ambit’s competition binding assay.12
References and notes
1. Chan, A.; van Bezooijen, R. L.; Lowil, C. W. G. M. Curr. Opin. Invest. Drugs 2007, 8,
293.
2. Wauwe, J. V.; Haefner, B. Drug News Perspect. 2003, 16, 557.
3. Kulkarni, N. H.; Wei, T.; Kumar, A.; Dow, E. R.; Stewart, T. R.; Shou, J.; N’cho, M.;
Sterchi, D. L.; Gitter, B. D.; Higgs, R. E.; Halladay, D. L.; Engler, T. A.; Martin, T. J.;
Bryant, H. U.; Ma, Y. L.; Onyia, J. E. J. Cell. Biochem. 2007, 102, 1504.
4. Bain, J.; McLauchlan, H.; Elliott, M.; Cohen, P. Biochem. J. 2003, 371, 199.
5. Nuss, J.M.; Harrison, S.D.; Ring, D.B.; Boyce, R.S.; Pfister, K.B.; Goff, D.; Johnson,
K.; Subramanian, S.; Wagman, A.S.; Zhou, X.A.; Ramurthy, S.; Renhowe, P.A.;
Seely, L. WO9965897, 1999.
6. Nuss, J. M.; Harrison, S. D.; Ring, D. B.; Boyce, R. S.; Pfister, K. B. WO0220495,
2002.
7. Engler, T. A.; Henry, J. R.; Malhotra, S.; Cunningham, B., et al J. Med. Chem. 2004,
47, 3934.
8. Zhang, H.; Bonaga, L. V. R.; Ye, H.; Derian, C. K.; Damiano, B. P.; Maryanoff, B. E.
Bioorg. Med. Chem. Lett. 2007, 17, 2863.
9. Kumont, V. R. Helv. Chim. Acta 1983, 66, 814.
Figure 5. Compound 34’s anabolic effect in rat (OVX) model.13
10. The GSK-3b inhibition assay was described in Gong, L.; Peltz, G. Grupe, A. U.S.
Patent 6,479,490, 2002. In detail, it was performed in 50
well polypropylene plate, each reaction containing 20 mM magnesium
chloride, 40 M ATP, 2 mM DTT, 88.5 M biotinylated and phosphorylated
CREB-peptide substrate (biotin-KRREILSRRPS(PO4)YR-OH, and
33P] ATP
(1 Ci). 15 l of the test compounds in DMSO (various concentrations) were
added and the reaction mixture was incubated at 30 °C for 1 h. The reaction
was stopped by transferring 25 of the reaction mixture to
phosphocellulose plate containing 130 l of 1.85% phosphoric acid. The free
ll reactions in a 96
ring from the inhibitor also has an edge/face interaction with
Phe67.
Compound 34 was screened against 317 kinases at Ambit Bio-
sciences. In this panel, compound 34 was found to inhibit only
l
l
[c-
l
l
l
l
l
a
36 kinases with >90% inhibition at 10 lM. The Kds of these 36 ki-
radionucleotides in the membrane were washed off under vacuum with 1.85%
phosphoric acid (five times). After the last wash, the plate was transferred to an
adaptor plate and 50 ll of scintillation cocktail (Microscint-20, Packard, cat.
#20-133) were added to each well and the amount of radioactivity was
counted in a top counter.
nases were then determined and the results are shown in Table
6. Overall, compound 34 shows good selectivity over other kinases.
Combined with its reasonable oral exposure and desired po-
tency against GSK-3b, 34 was chosen for in vivo evaluation in
our rat osteoporosis model. It was administrated at 30 mpk daily
for 3 weeks to ovariectomized (Ovx) rats to assess its ability to in-
crease the bone mineral density (BMD).13 Compound 34 showed a
pronounced anabolic effect at multiple locations (Fig. 5) and the re-
sults are comparable to the effect of known anabolic hormone, bo-
vine PTH.
11. Crystallographic data for the structure in this paper has been deposited with
the RCB Protein Data Bank with PDB ID: 1r0e.
12. Karaman, M. W.; Herrgard, S.; Zarrinkar, P. P., et al Nat. Biotechnol. 2008, 26,
127.
13. OVX model was described in Day-Lollini, P. A.; Gong, L. US20030176484, 2003.
In detail, three month old rats were ovariectomized (OVX) and administered
either bovine parathyroid hormone (AminoAcids1-34) (bPTH) or compound 34
once
a day by mouth starting at least 4 weeks post-ovariectomy and
continuing until final sacrifice after 3 weeks of daily treatment. Control
groups, both sham (rats that were not ovariectomized) and OVX, received
vehicle only. Bovine parathyroid hormone, bPTH, was tested at 40 mg/kg
(optimal dose) was an internal positive control for anabolic activity. The bone
mineral density of the right femur was determined using the High Resolution
Software on a QDR-4500W Bone Densitometer™ (Hologic, Waltham, Mass.).
The animals were scanned by placing them on a plexiglass block in a supine
position such that the right leg was perpendicular to the main body and the
tibia was perpendicular to the femur. The increase in the bone density was
expressed as % change at the end of 3 week experiment.
Starting from a focused screen of a PKC inhibitor library, we
have discovered a novel series of monoindolyl maleimides by dial-
ing out PKC activity and improving GSK-3b activity. The chemistry
effort has resulted in compound 34, an orally active, potent and
selective GSK-3b inhibitor. In vivo studies with 34 have demon-
strated that GSK-3b plays a role in influencing bone density in a
murine model of osteoporosis.