Substituted 3-imidazo[1,2-a]pyridin-3-yl-4-(1,2,3,4-tetrahydro-[1,4] diazepino-[6,7,1-hi]indol-7-yl)pyrrole-2,5-diones as highly selective and potent inhibitors of glycogen synthase kinase-3

Article abstract of DOI:10.1021/jm049768a

Glycogen synthase kinase-3 (GSK3) is involved in signaling from the insulin receptor. Inhibitors of GSK3 are expected to effect lowering of plasma glucose similar to insulin, making GSK3 an attractive target for the treatment of type 2 diabetes. Herein we report the discovery of a series of potent and selective GSK3 inhibitors. Compounds 7-12 show oral activity in an in vivo model of type II diabetes, and 9 and 12 have desirable PK properties.

Full text of DOI:10.1021/jm049768a

3
934
J . Med. Chem. 2004, 47, 3934-3937
Su bstitu ted 3-Im id a zo[1,2-a ]p yr id in -3-yl-
-(1,2,3,4-tetr a h yd r o-[1,4]d ia zep in o-
6,7,1-h i]in d ol-7-yl)p yr r ole-2,5-d ion es a s
4
[
High ly Selective a n d P oten t In h ibitor s of
Glycogen Syn th a se Kin a se-3
Thomas A. Engler,* J ames R. Henry,*
Sushant Malhotra, Brian Cunningham,
Kelly Furness, J oseph Brozinick,
Timothy P. Burkholder, Michael P. Clay,
J oshua Clayton, Clive Diefenbacher, Eric Hawkins,
Philip W. Iversen, Yihong Li, Terry D. Lindstrom,
Angela L. Marquart, J ohnathan McLean,
David Mendel, Elizabeth Misener, Daniel Briere,
J ohn C. O’Toole, Warren J . Porter, Steven Queener,
J on K. Reel, Rebecca A. Owens, Richard A. Brier,
Thomas E. Eessalu, J ill R. Wagner,
F igu r e 1. Bis-aryl maleimide GSK3 inhibitors.
Robert M. Campbell, and Renee Vaughn
Recently, several groups have reported a variety of
bis-aryl maleimide (BAM) inhibitors of GSK3 (Figure
1). BAMs GF 109203x and Ro 31-8220 were shown to
inhibit GSK3 with IC50 values of 170-360 nM and 3-79
nM, respectively, depending on the source of GSK3.
SmithKline Beecham reported SB-216763 and SB-
Lilly Research Laboratories, A Division of
Eli Lilly & Company, Lilly Corporate Center,
Indianapolis, Indiana 46285
Received March 24, 2004
Abstr a ct: Glycogen synthase kinase-3 (GSK3) is involved in
signaling from the insulin receptor. Inhibitors of GSK3 are
expected to effect lowering of plasma glucose similar to insulin,
making GSK3 an attractive target for the treatment of type 2
diabetes. Herein we report the discovery of a series of potent
and selective GSK3 inhibitors. Compounds 7-12 show oral
activity in an in vivo model of type II diabetes, and 9 and 12
have desirable PK properties.
4
15286 to inhibit GSK3-R with an IC50 of 34 and 77 nM,
1
0
respectively, while J &J PRD reports a GSK3-â IC50
of 34 nM IC50 for their macrocyclic BAM.
1
1a
While the
above compounds display varying degrees of GSK3
inhibition, no pharmacokinetic or in vivo efficacy data
1
2
were presented. Herein we report the results of an
effort to develop a new class of orally available and
efficacious GSK3 inhibitors, the 3-imidazo[1,2-a]pyridin-
Type 2, or noninsulin-dependent diabetes mellitus
3
7
-yl-4-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-
-yl)pyrrole-2,5-diones (7-12).
(
NIDDM), afflicts 120 million people worldwide and is
1
estimated to rise to over 200 million by the year 2010.
NIDDM accounts for approximately 90% of all cases of
diabetes and is characterized by an inability of the body
to effectively respond to insulin secreted by the pan-
The synthesis of our inhibitors is outlined in Scheme
. The 1,7-azaannulated indole 3 can be prepared in a
1
four-step process starting with the appropriately sub-
1
3,18
stituted 7-formylindole
(1). Reductive amination
2
creas, or insulin resistance. GSK3 is a serine-threonine
with ethanolamine and Pd/C gives the desired amino-
alcohol which can be protected as the BOC derivative
kinase encoded by two independent genes, GSK3-R and
3
-
â. Purified GSK3-R and -â show similar biochemical
2
. Activation of the alcohol with mesyl chloride followed
and substrate properties and display high sequence
by base-induced cyclization gives the desired annulated
4
homology, 85% overall and 95% in the catalytic domain.
1
4
indole 3 in good yield. Treatment with oxalyl chloride
GSK3 was first identified over 20 years ago as one of
several kinases that phosphorylates glycogen synthase
GS), the enzyme that catalyzes the last step in glycogen
1
4
and methanol gives the required oxalate 4, which can
be coupled with the appropriate amide acetamide 5a 15
or 5b under basic conditions to give the protected BAM
(
synthesis. This phosphorylation step, in contrast to most
1
6
6
.
Removal of the BOC group allows for incorporation
5
signaling pathways, inhibits the action of GS. It was
of a variety of acyl groups to give 7-12.
later shown that signaling from the insulin receptor
inactivates GSK3 via AKT-catalyzed phosphorylation of
The 5-fluoro-7-formylindole (1, R2 ) F) required for
1
7
the synthesis of 12 can be prepared from 13 via a two-
step sequence utilizing the Bartoli indole synthesis18 as
shown in Scheme 2.
6
serine #9 on the amino terminus of GSK3. Thus,
inhibitors of GSK3 would be expected to have some of
the same effects as insulin, such as its ability to activate
glycogen synthase and stimulate the conversion of
glucose to glycogen, thereby lowering plasma glucose.
Abnormal regulation of GSK3 has also been demon-
strated in insulin resistant rodent and human muscle
The 6-methylimidazopyridine 5b required for the
preparation of compound 9 can be prepared in a two-
step process starting with commercial 2-amino-5-meth-
ylpyridine as outlined in Scheme 3.
Inhibition of GSK3-â by compounds 7-12 was deter-
mined in both biochemical and cellular assays. In the
biochemical assay, a standard filter binding protocol was
used measuring the ability of recombinant GSK3-â to
phosphorylate phospho-CREB (cAMP response element
7
tissue. Because of these facts, GSK3 has become an
8
attractive target for the potential treatment of NIDDM.
*
To whom correspondence should be addressed. TAE: Engler_
Thomas@lilly.com; J RH: J RHenry@lilly.com.
1
0.1021/jm049768a CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/23/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3935
Ta ble 1. Biochemical and Cellular Assay Dataa
Sch em e 1a
no.
GSK3-â IC50 (nM)
P-Tau IC50 (nM)
NT
staurosporine
56 ( 6.9
1.3 ( 0.5
2.0 ( 0.4b
5.2 ( 0.8
1.3 ( 0.1b
1.6 ( 0.3
1.1 ( 0.3
c
7
8
9
1
1
1
15
1.6
c
b
41 ( 26
0
1
2
2.6 ( 0.22
12 ( 2.6
0.7 ( 0.26
a
Data represent mean values of at least three independent
determinations ((SE) unless otherwise noted. b N ) 2. N ) 1.
c
Ta ble 2. Kinase Activities for Compounds 7-12 (IC50s in µM)
kinase
7
8
9
10
11
12
GSK-3â
Cdk4
0.0013 0.002 0.0052 0.0013 0.0016 0.0011
3.3
4.8
18
1.7
1.5
1.8
Cdk2
3.4
5.4
>20
0.37
>20
NTa
>20
9.5
>20
>20
NT
5.3
1.1
2.1
1.0
2.8
PKCâII
AKT1
PDK-1
KDR
0.80
>20
6.5
0.56
>20
4.3
>20
3.1
>20
>20
>20
>20
0.42
>20
6.2
>20
1.2
>20
>20
>20
>20
>20
>20
>20
2.0
>20
>20
13.4
>20
>20
>20
>20
>20
>20
2.5
>20
TGFâ-R2 >20
hPKAâ
CAMKII
MLK7
p38
>20
>20
>20
>20
>20
>20
a
>20
Reagents and conditions (yields for 12): (a) Ethanolamine,
AcOH, NaBH(OAc)3, 93%; (b) Boc2O, CH3CN, 0 °C, 86%; (c)
methanesulfonyl chloride, Et3N, CH2Cl2, 0 °C; (d) NaH, DMF,
a
Not tested.
0
-
5
°C, 76% (two steps); (e) oxalyl chloride, CH2Cl2, 0 °C, then
78 °C, NaOMe, 98%; (f) 2-imidazo[1,2-a]pyridin-3-yl-acetamide
a , KOt-Bu, THF or DMF; (g) HCl/dioxane, CH2Cl2, 66% (two
sensitivity of this assay,22 and so a more accurate
measure of a compound’s true potency can be found in
the cellular assay. In the P-Tau assay we see a larger
differentiation in activities ranging from 0.7 to 41 nM.
Compound 9, bearing a 6-methyl group on the imidazo-
pyridine, is the least potent inhibitor in both assays and
is 25-fold less active in the P-Tau assay than the parent
steps). (h) carbonyl chloride, MeOH, Et3N, rt, 60%.
Sch em e 2a
8
. The piperidine (8, 12) and morpholine (10) ureas were
found to be favored substituents on the aliphatic ring
nitrogen over other groups such as N,N-dimethylurea
(
11) and the isopropyl carbamate (7). Further, fluorine
a
Reagents and conditions: (a) n-BuOH, TsOH (cat.), toluene,
substitution on the 5-position of the indole in 12 resulted
in a modest activity enhancement over the parent
compound 8.
The group of inhibitors was then run through a
diverse kinase panel to examine their selectivity pro-
files. As can be seen in Table 2, all compounds are very
selective for GSK3. The most cross reactivity occurs with
the cyclin dependent kinases cdk2, cdk4, and also with
PKCâII, although selectivity ratios are still very good
reflux, 95%; (b) vinylmagnesium bromide, THF, -40 °C, then THF,
.5 N HCl, 55%.
0
Sch em e 3a
a
23
Reagents and conditions: (a) (E)-4,4-Dimethoxy-but-2-enoic
even for compounds with the highest cross reactivity.
acid ethyl ester, CH3CN, H2O, TsOH, reflux; (b) NH3, MeOH,
00 °C, 22% two steps.
For example, compound 12 shows an IC50 of 0.42 µM
against PKCâII, but this is still nearly 400-fold less
potent than its activity against GSK3-â. As a further
measure of selectivity, compound 10 was screened
against a second panel of 36 kinases at concentrations
of 0.1 and 0.5 µM, and the results are shown in Figure
2. No cross reactivity was seen at 0.1 µM, and only weak
activity (>60% of negative control) against PKCR and
CDK3 was seen at 0.5 uM. Given the potency of 10, this
represents at least a 500-fold selectivity window for
GSK3-â over this panel of kinases.
1
3
3
binding protein) at serine-129 with ATP[γ- P] in the
presence of test compound.1
ing inhibition of GSK3-R was also routinely run, but
-12 showed no significant ability to discriminate
9,20
A similar assay measur-
7
between the two isoforms of GSK3 (data not shown).
To the best of our knowledge, no isoform specific
inhibitors of GSK3 have been reported, probably due
to their high sequence homology (vida supra). A cellular
ELISA (P-Tau) assay measured a test compound’s
ability to block GSK3-â dependent phosphorylation of
serine-396 of the Tau protein in SY5Y cells.21
It is clear from the data in Table 1 that all compounds
are very potent inhibitors of GSK3-â in the biochemical
assay with IC50s ranging from 1 to 5 nM. In contrast,
the IC50 of the standard kinase inhibitor staurosporine
is 56 nM. Activities e1 nM are below the limits of
After determining their in vitro properties, the com-
pounds were subjected to in vivo testing using Zucker
1
2
diabetic fatty (ZDF) rats. The ZDF rat is an offshoot
of the Zucker fatty rat model, which displays the marked
skeletal muscle insulin resistance of the parent Zucker
strain, combined with induction of diabetes at approxi-
mately 8 weeks of age due to pancreatic â-cell failure.24

3
936 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Letters
F igu r e 2. Kinase specificity for compound 10. Single point determinations were run in duplicate. Kinase activity is given as
percent of negative control at 0.1 µM (blue bars) and 0.5 µM (red bars). Kinases are of human origin unless otherwise noted. (m)
)
mouse, (r) ) rat, (y) ) yeast.
Ta ble 3. ZDF Rat in Vivo Efficacy of 7-12
fore, a compound’s potency in the P-Tau cellular assay
was a reasonable predictor of its oral in vivo efficacy.
Compound 12 was selected for further study in an oral
glucose tolerance test. In this dose-response study, ZDF
rats were treated with 12 via oral gavage (dose range
%
glucose
% of insulin
(10 units) response
@ 10 mg/kg
reduction
@ 10 mg/kg
TMED
(mg/kg)
no.
7
8
9
1
1
1
58
69
37
78
53
61
105
124
75
119
104
124
4.27 ( 1.14
2.22 ( 0.45
NDa
0
.1-3 mg/kg, qd) with compound for 8 days. On the
eighth day, the rats received a 1.25 g/kg glucose chal-
lenge, and the animal’s blood glucose was measured over
a 2 h period. Compound 12 had a TMED of 0.49 ( 0.15
mg/kg in this study, proving to be a very potent orally
active agent.
0
1
2
1.44 ( 0.25
NDa
1.5 ( 0.3
a
Not determined. The study failed to yield a significant TMED
within the dose range tested (0.3-10 mg/kg).
Pharmacokinetic parameters of compounds 9 and 12
were determined in female rats dosed orally at 10 mg/
kg and intravenously at 2 mg/kg. As can be seen in
Table 4, compound 9 demonstrated oral bioavailability
of 45%, while compound 12 gave an oral bioavailability
of 23%. The acceptable pharmacokinetic parameters of
9 and 12 would suggest that the superiority of 12 in
the ZDF rat study is due primarily to differences in
GSK3 activity as reflected in the P-Tau cellular activity.
In conclusion, we have developed a series of novel,
highly potent GSK3 inhibitors. These compounds show
a high degree of selectivity against both serine/threonine
and tyrosine kinases and have been shown to be highly
efficacious oral agents for the reduction of blood glucose
in the ZDF rat model of NIDDM. Finally, compound 12
has desirable in vivo and pharmacokinetic properties.
Additional studies with this compound will be reported
in due course.
As an initial filter, rats were dosed via oral gavage with
1
0 mg/kg of the compound of interest and plasma
glucose levels were measured 4 h postdose. Percent
glucose reduction could then be determined by compari-
son to vehicle-treated controls. A positive control group
was dosed with 10 units of insulin as an internal
standard for percent glucose reduction. Due to vari-
ability in response rates, we found it valuable to
compare a compound’s glucose reduction as a percentage
of the positive control group insulin response. To select
the most potent and efficacious GSK3 inhibitors, 7 day
dose-response studies were conducted to determine
threshold minimal effective doses (TMEDs). We defined
TMED as the dose where a statistically significant
reduction of blood glucose was achieved relative to
untreated controls, with the further condition that the
glucose reduction be at least 50% of the insulin-treated
control group.
The results of the ZDF rat studies are given in Table
. All compounds performed well at the 10 mg/kg dose,
Ack n ow led gm en t. We thank Donald B. Bennett,
Timothy I. Meier, and J ames A. Cook for performing
3
with all but one lowering glucose as well as 10 units of
insulin. The TMED shows more separation between the
compounds and reveals one general trend. The least
potent compounds in the P-Tau cellular assay (7, 9, and
the K determinations, Kathleen M. Valasek for help
with the P-Tau ELISA assay validation, Shenaz Khan
for early biochemical assays, Gene Kogut for scaling up
some syntheses, Viswanath Devanarayan for statistical
analysis, and Hongqi Tian (Array Biopharma) for help
with the preparation of 4 (R2 ) F).
i
1
7
1) are also the least efficacious, with only carbamate
yielding a significant result. In contrast, the three
most potent compounds in the P-Tau assay (8, 10, and
2) gave the best results in the dose response study,
1
Su p p or t in g In for m a t ion Ava ila b le: Synthetic pro-
cedures and characterization for compounds 7-12. This mate-
2
5
showing TMEDs between 1.4 and 2.2 mg/kg. There-
Ta ble 4. Pharmacokinetic Parameters of Compounds 9 and 12
iv clearance
(mL/min/kg)
volume of
distribution (l)
oral AUC 0-24 h
(ng‚h/mL)
oral
bioavailability
no.
half-life (IV), h
half-life (oral), h
9
1
1
1.1
ND
2.8
17
12
1
1
4793
3550
45 ( 4
23 ( 4
2

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 3937
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i
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2
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(
J M049768A