Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01550

GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics.

Full text of DOI:10.1021/acs.jmedchem.5b01550

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Article
Discovery of Isonicotinamides as Highly Selective, Brain Penetrable
and Orally Active Glycogen Synthase Kinase-3 Inhibitors
Guanglin Luo, Ling Chen, Catherine R Burton, Hong Xiao, Prasanna Sivaprakasam, Carol M Krause,
Yang Cao, Nengyi Liu, Jonathan Lippy, Wendy J Clarke, Kimberly Snow, Joseph Raybon, Vinod Arora,
Matt Pokross, Kevin Kish, Hal A. Lewis, David R. Langley, John E. Macor, and Gene M. Dubowchik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01550 • Publication Date (Web): 11 Jan 2016
Downloaded from http://pubs.acs.org on January 12, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Journal of Medicinal Chemistry
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Discovery of Isonicotinamides as Highly Selective, Brain
Penetrable and Orally Active Glycogen Synthase Kinase-3
Inhibitors
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Guanglin Luo*, Ling Chen, Catherine R. Burton, Hong Xiao, Prasanna Sivaprakasam, Carol M. Krause,
Yang Cao, Nengyin Liu, Jonathan Lippy, Wendy J. Clarke, Kimberly Snow, Joseph Raybon, Matt
Pokross, Kevin Kish, Hal A. Lewis, David R. Langley, John E. Macor, and Gene M. Dubowchik
For Table of Contents Use Only
Table of Contents Graphic
=
18
H
(
R
N
)
O
α
IC50
:
nM
nM
nM
GSK-3 /
9.8/5.5
nM
β
GSK3
:
IC 320
pTau
=
50
N
29
R
F
(
)
H
α
:
IC50
GSK-3 /
5.2/1.7
nM
β
N
:
IC 180
pTau
=
50
R
33
Cl
(
)
HN
α
:
33
demonstrated excellent kinase
IC50
GSK-3 /
5.9/2.0
nM
β
Compound
selectivity
:
IC 200
the Ambit radial
above
pTau
50
graph
R
by
O
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Discovery of Isonicotinamides as Highly Selective,
Brain Penetrable and Orally Active Glycogen Synthase
Kinaseꢀ3 Inhibitors
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Guanglin Luo*, Ling Chen, Catherine R. Burton, Hong Xiao, Prasanna Sivaprakasam, Carol M.
Krause, Yang Cao, Nengyin Liu, Jonathan Lippy, Wendy J. Clarke, Kimberly Snow, Joseph Raybon,
Vinod Arora, Matt Pokross, Kevin Kish, Hal A. Lewis, David R. Langley, John E. Macor, and Gene M.
Dubowchik
BristolꢀMyers Squibb Research & Development, BristolꢀMyers Squibb Company, 5 Research Parkway,
Wallingford, CT, 06492.
Guanglin.Luo@bms.com
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
ABSTRACT: GSKꢀ3 is a serine/threonine kinase that has numerous substrates. Many of these proteins
are involved in the regulation of diverse cellular functions, including metabolism, differentiation,
proliferation, and apoptosis. Inhibition of GSKꢀ3 may be useful in treating a number of diseases
including Alzheimer’s disease (AD), type II diabetes, mood disorders, and some cancers, but the
approach poses significant challenges. Here we present a class of isonicotinamides that are potent,
highly kinaseꢀselective GSKꢀ3 inhibitors, members of which demonstrated oral activity in a triple
transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis
of these compounds bodes well for their further exploration as tool compounds and therapeutics.
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Journal of Medicinal Chemistry
KEYWORDS (Word Style “BG_Keywords”). If you are submitting your paper to a journal that requires
keywords, provide significant keywords to aid the reader in literature retrieval.
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BRIEFS (WORD Style “BH_Briefs”). If you are submitting your paper to a journal that requires a brief,
provide a oneꢀsentence synopsis for inclusion in the Table of Contents.
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INTRODUCTION
Alzheimer’s disease (AD) is the most common form of dementia and affects over 30 million people
worldwide. An estimated 5.2 million Americans have AD, and it is the fifth leading cause of death in
Americans age 65 or older.1 AD is histopathologically defined by neuronal loss, the presence of
intracellular neurofibrillary tangles (NFTs), the formation of extracellular senile plaques, and the
occurrence of cerebral amyloid angiopathy.2,3 There is currently no disease modifying treatment
available. While extensive preclinical and clinical efforts have focused on betaꢀamyloid (Aß) related
intervention, a related hypothesis is that hyperphosphorylation of tau protein leads to the destabilization
of microtubules, and then aggregate to form NFTs.
Glycogen synthase kinaseꢀ3 (GSKꢀ3) is a prolineꢀdirected serine/threonine kinase that is involved in
the regulation of various physiological functions such as metabolism, proliferation and apoptosis, by
carrying out the phosphorylation of multiple protein substrates.4 GSKꢀ3 exists in two isoforms, GSKꢀ3α
(51 kDa) and GSKꢀ3ß (47 kDa), that share 84% overall identity and 98% identity within their respective
catalytic domains.5 Both primary isoforms are ubiquitously expressed, with high levels observed in the
brain.6,7 In most brain areas, GSKꢀ3β is the predominant isoform.8 There is evidence to suggest that the
GSKꢀ3β isoform is the key kinase required for hyperphosphorylation of tau.9,10 However, some studies
suggest that GKSꢀ3α and GSKꢀ3β share very similar, if not entirely redundant functions in a number of
cellular processes.11 GSKꢀ3 is one of a small number of kinases that are constitutively active, and its
activity generally results in a reduction of substrate function. The substrates on which it functions are
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numerous, with at least 50 being well defined, but perhaps as many as 100 or more in total.12 Given its
central role in cellular differentiation and metabolism, acting on a large number of substrates, it’s not
surprising that GSKꢀ3 is highly regulated. This is enforced by multiple mechanisms including substrate
priming, activating phosphorylation, physical sequestration, and most prominently, inhibitory
phosphorylation. The activity of GSKꢀ3β is significantly reduced by phosphorylation at Serꢀ9 in the Nꢀ
terminal domain, most notably by protein kinase B (PKB or AKT).13 This inhibitory pathway has been
proposed to result in neuroprotection, neurogenesis and favorable outcomes following pharmacological
treatment in various mood disorders. Hyperphosphorylation of tau by GSKꢀ3 has been demonstrated
both in cell culture and in in vivo studies.14 Hyperphosphorylated tau disengages from microtubules
resulting in destabilization of these filaments with concomitant negative effects on intracellular
structures and transport mechanisms.15 In addition, the uncomplexed, hyperphosphorylated tau
assembles into paired helical filaments (PHFs) that aggregate to produce the stereotypic intracellular
NFTs associated with AD.16ꢀ18 Other potential pathological consequences of overꢀactivation of GSKꢀ3
include neuroinflammation and apoptosis.19,20 Furthermore, GSKꢀ3 has been demonstrated to be
involved in mechanisms underlying memory and learning, and dysregulation of GSKꢀ3 may explain
some of the early cognitive deficits observed in AD.21 These results and observations suggest that
inhibition of GSKꢀ3 activity may be useful in the treatment of both the neuropathologic and
symptomatic aspects of AD, as well as other neurodegenerative diseases. Recently, a nonꢀATPꢀ
competitive, irreversible GSKꢀ3 inhibitor, Tideglusib, was tested in Phase II studies for AD.22
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GSKꢀ3 inhibitors have also been studied as potential agents for the treatment of mood disorders23 and
metabolic diseases, such as Type II Diabetes24 and bone density improvement.25 Looming over these
attractive opportunities for therapeutic intervention has been the perceived risk of hyperplasia resulting
from interference in one of GSKꢀ3’s most prominent roles: phosphorylation of betaꢀcatenin, leading to
its eventual destruction in the proteasome.26 However over 50 years of clinical experience with lithium
for treating bipolar disorder, which is now believed to work through GSKꢀ3 inhibition, firmly
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Journal of Medicinal Chemistry
establishes that these patients do not have increased cancer risks. Interestingly, several studies have
shown an antiꢀcancer effect of GSKꢀ3 inhibition, potentiating the effect of standard chemotherapy
against a number of tumor types including pancreatic cancer and leukemias.27 Initial Phase I clinical
data has recently been reported for a combination of Pemetrexed with a GSKꢀ3 inhibitor, LY2090314.28
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Over the past two decades a number of highly diverse classes of GSKꢀ3 inhibitors have been
discovered.29,30 Representative GSKꢀ3 inhibitors are shown in Chart 1. SBꢀ216763 (1) was an early
maleimide derivative that showed reduction of tau hyperphosphorylation in rat brain.31 Another
maleimide derivative BIPꢀ135 (2) has recently shown activity in models of bipolar disorder and spinal
muscular atrophy.32 Compound 3 showed a good brain/plasma (b/p) ratio and reduced tau
hyperphosphorylation in vivo.33,34 Compound 4 was disclosed in 2009 as a potent and selective GSKꢀ3
inhibitor that inhibited tau phosphorylation in rat primary neuronal cells and mouse brain tissue.35
Compounds 5, 6, and 7 were recently disclosed to be brain penetrable but no efficacy data has so far
been reported.36ꢀ38 Unfortunately, most GSKꢀ3 inhibitors that are claimed to be selective have only been
tested against a limited set of kinases. We believe that when targeting such a critical regulatory enzyme
such as GSKꢀ3, especially for a nonꢀoncology indication, kinase selectivity may be of paramount
importance.
Chart 1. Selected GSKꢀ3 inhibitors
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In a recent publication we disclosed a series of GSKꢀ3 inhibitors, exemplified by compound 8 (Figure
1).39 Modeling and xꢀray structural studies showed that the left side aminopyridine bound to the hinge
region of GSKꢀ3 and the right side amide carbonyl made a hydrogen bond with Lysꢀ85 (K85). We were
interested in replacing the lactam with pyridine, in which the pyridyl nitrogen would act as the Hꢀbond
acceptor, and which might improve CNS penetration. To further simplify the synthesis and to give the
rigid tricyclic scaffold a bit more flexibility, we also replaced the pyrrole portion with an amide group
(Figure 1).
Figure 1. Design of the 3ꢀpyridinyl isonicotinamides.
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Journal of Medicinal Chemistry
RESULTS AND DISCUSSION
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The proposed isonicotinamides were synthesized by two general strategies (Scheme 1). In the initial
approach, commercial isonicotinic acid dichloride was reacted with various 3ꢀaminopyridines to
generate the 2ꢀchloroisonicotinamide intermediates, which were then subjected to Buchwald amidation
without purification,40 to afford the desired products. For broader structural diversity or larger scale
reactions, we developed a second, more convergent approach. Starting with 2ꢀamino isonicotinic acid or
its methyl ester, monoꢀacylation of the amino group was not feasible (Figure 1). Instead, double
acylation and the formation of the mixed anhydride always occurred. To solve this issue, methyl 2ꢀ
amino isonicotinate was treated with excess acyl chloride (2.1 equiv.) to afford the bisꢀacylated
intermediate 9. Subsequent treatment with 4 equivalents of aqueous LiOH achieved complete
conversion to the monoꢀacylated acid 10, which upon neutralization with 4 equiv. of aqueous HCl,
precipitated out as a white solid. The acid could directly react with many 3ꢀaminopyridines under
various amide formation conditions, or be converted to the acyl chloride to react with more electron
deficient 3ꢀaminopyridines.
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Scheme 1. Synthesis of 3ꢀPyridinyl Isonicotinamides
Reagents and Conditions: (a) CH2Cl2, i-Pr2NEt, rt, 18 h; (b) cyclopropanecarboxyamide, Pd(OAc)2, K2CO3, Xantphos, dioxane, 150
oC, 2 h; (c) cyclopropanecarbonyl chloride (2.1 equiv), CH2Cl2, i-Pr2NEt, 0 oC, 1 h (88%); (d) LiOH (4 equiv), THF-MeOH (10:1), rt,
18 h (79%); (e) 3-aminopyridine, HATU, DMF, rt (or 85 oC), i-Pr2NEt, 18 - 48 h; (f) oxalyl chloride (1.2 equiv), DMF (cat.), rt, 30
min, then 3-aminopyridine, Et3N, rt, 2 h
.
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Compounds were initially tested in GSKꢀ3ß/α competition assays (GSKꢀ3ß/α IC50) with a
fluorescently labeled small molecule at its Kd, and a cellular phosphorylated Tau (pTau) lowering assay
(pTau IC50).41 While a modest level of isoform selectivity was often seen with particular compounds in
the enzyme assay, none of the compounds described herein could be described as truly selective in this
respect. We also developed a similar assay in a vinblastineꢀresistant carcinoma (KBV) cell line that
over expresses human tau protein. Testing compounds with or without the Pꢀglycoprotein (Pgp)
inhibitor tarquidar allowed us to assess Pgpꢀrelated efflux. A resulting KBV ratio > 2 would suggest a
low chance of significant brain penetration.
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Data for an initial set of isonicotinamides is shown in Table 1. To our delight, the unsubstituted
pyridine 11 showed good enzymatic inhibition of GSKꢀ3. Various substitutions at the 4ꢀposition were
well tolerated and usually improved enzyme inhibition and cell potency.
Table 1. GSKꢀ3ß/α Affinity and pTau for compounds 11ꢀ18
GSK3ß a /α
IC50 (nM)
KBV
ratio
0.4
0.8
0.7
ꢀ
pTau
IC50(nM)b
1400
1600
10000
ꢀ
Compd.
R
11
12
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15
16
17
18
H
24(±3.0)/3.9
37(±9.4)/34
490(±100)/210
51(±18)/43
2ꢀF
2ꢀOH
2ꢀCN
4ꢀOMe
4ꢀCl
4ꢀCF3
4ꢀPh
3.4(±0.42)/4.5
11(±2.1)/4.5
19(±2.5)/6.3
9.8(±3.6)/5.5
240
1.4
1.2
1.3
1.3
260
960
320
aValues are means of at least three experiments but data for GSK3α was not repeated bValues are means of at least two experiments.
;
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The proposed binding model for this series of compounds was confirmed by solving the Xꢀray coꢀ
crystal structures of select inhibitors with GSKꢀ3β.42 Figure 2 shows the details of key interactions
observed with compound 15 binding in the active site, with respect to ADP.43 The 2ꢀaminopyridine
makes hydrogen bonds with the hinge V135 backbone amide and the 3ꢀaminopyridine sp2 nitrogen
forms a key hydrogen bond with the K85 amine. Comparison with bound ADP suggested the 4ꢀposition
of the 3ꢀaminopyridine as a suitable vector to target the sugar pocket to optimize the potency and PK
properties of this class of compounds. The bound structures also indicated that positions 4ꢀ and possibly
5ꢀ of the 3ꢀaminopyridine should be more accessible for substitution than the more sterically crowded 2ꢀ
and 6ꢀpositions. Two conserved structural water molecules observed in the back pocket were found to
effectively bridge compound 15 through the central carbonyl oxygen.
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Figure 2: Xꢀray coꢀcrystal structure of compound 15 in the kinase domain of GSKꢀ3β overlaid onto the
Xꢀray structure of ADP bound GSKꢀ3β complex (PDB ID 1J1C)43. The ligands are rendered as a stick
model in green carbons for ADP and magenta carbons for compound 15. Figure 2 was made using
pymol.44
The 4ꢀmethyl ether compound 15 (Table 1) significantly increased GSKꢀ3 inhibition and cellular
pTau lowering. We then further explored the SAR with regard to the alkoxy group. Various 4ꢀalkoxyꢀ3ꢀ
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pyridylamine intermediates were prepared as shown in Scheme 2. While direct substitution of the
chloride by primary alcohols or phenol was straightforward, secondary alcohols such as iꢀPrOH failed.
On the other hand, Mitsunobu coupling worked smoothly with 2ꢀpropanol followed by reduction of the
nitro group via hydrogenation to afford the intermediate for compound 20. The final compounds were
prepared following the general sequence described in Scheme 1.
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Scheme 2. Synthesis of 4ꢀalkoxyꢀpyridinꢀ3ꢀamines
Reagents and conditions: (a) alcohol/phenol, DMF, K2CO3, 65 oC, 18 h; (b) Pd/C/hydrogen (1 atm.), MeOH; (c) iꢀPrOH, Ph3P, DIAD, rt, 5 h.
As shown in Table 2, all ether derivatives demonstrated single digit nM binding affinity towards
GSKꢀ3. In particular, the neopentyl ether 23 showed subꢀnM inhibition and very potent cellular pTau
lowering (IC50 = 36 nM). Unfortunately, the two most potent compounds in the cell assay, 23 and 24,
had poor metabolic stability, particularly in rodent liver microsomes (Table 2). The hydroxyethyl ether
25 had good potency and metabolic stability, but a high KBV efflux ratio (3.8). Data for compounds 15,
19, 21, and 22 supported further profiling.
Table 2. Data for Compounds 15 and 19ꢀ25
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pTau
GSK3ß a /α
IC50 (nM)
KBV Met.Stab.
ratio
Compd
R
IC50
(M/R/H)c
1
(nM)b
2
3
4
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23
OMe
OCH2CF3
OiPr
3.4(±0.42)/4.5
2.5(±0.98)/2.2
1.9(±0.81)/1.4
4.0(±1.3)/2.7
4.2(±1.1)/1.1
1.0(±1.1)/0.74
240
260
ꢀ
1.4
1.4
1.5
1.6
2.1
1.8
59/82/92
57/90/100
0/23/65
OEt
170
200
36
13/65/89
15/69/97
0/1/29
OPh
OCH2tBu
OCH2ꢀ
Cyclopropyl
24
25
2.4(±0.86)/1.8
65
1.2
3.8
0/35/81
O(CH2)2OH 4.2(±0.55)/3.2
270
50/80/88
aValues are means of at least three experiments but data for GSK3α was not repeated
remaining after 10 min with 0.5 µM compounds incubated in liver microsomes.
;
bValues are means of at least two experiments. cValues are percentage
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Since we were targeting some level of chronic inhibition of GSKꢀ3, a master regulatory kinase, we
considered a high degree of selectivity over offꢀtarget kinases to be highly desirable. For broad kinase
screening, we utilized the Ambit panel of almost 400 kinases, where selected compounds were tested at
1 µM.45 As represented by the Ambit radial graphs shown in Figure 3,41 the thiophenolactam 8 had very
poor kinase selectivity, while the isonicotinamide methyl ether 15 was dramatically improved. These
results were in agreement with IC50 data from a smaller inꢀhouse panel of 30 kinases, which were
generally greater than the concentrations tested at 2 µM or 50 µM.46
8
15
Figure 3. Ambit Radial Graphs of compounds 839, and 15. This is a singleꢀpoint assay panel with a
control for each kinase. Each line represents remaining activity of a single kinase in the presence of 1
µM test compound. Starting from the center, the circles denote 83%, 33% and 0% of control binding
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remaining.45 The kinases are grouped into families differing by line color. As shown for 15, the two teal
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lines at nearly 0% represent GSKꢀ3α and β.
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8
9
We decided to explore SAR around the phenyl group of compound 18. These analogs were prepared
by Suzuki coupling with a common iodide intermediate 26 (Scheme 3), which was prepared from the
carboxylic acid 10.
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Scheme 3. Synthesis of 27 – 45
o
o
Reagents and conditions: (a) oxalyl chloride (1.2 equiv), DMF (cat.), 0 C, 2 h, then 4ꢀiodopyridinꢀ3ꢀamine, Et3N, 0 C ꢀ rt. (b) aryl boronic acid, Na2CO3
(2.0 M), Pd(PPh3)4, dioxane, 110 oC (microwave), 30 min.
As shown in Table 3, a single fluorine was wellꢀtolerated at the 4ꢀposition (29), but less so at the 2ꢀ
and 3ꢀpositions (27 and 28). Other 4ꢀsubstituted analogs (29 ꢀ 36) showed good enzyme and cell
potencies with the exception of the tꢀbutyl compound (36). Generally poor rodent metabolic stability
was seen for most examples, but several showed promising human stability. Nitrile 32 looked good
with respect to metabolic stability, however, as with other examples such as 25 in which polarity was
introduced in this region, it was a strong Pgp substrate. The 2ꢀCF3 and 2ꢀCl derivatives, as well as
various 2,4ꢀdiꢀsubstituted analogs (37 – 43), showed improved GSKꢀ3 activities but had very poor
metabolic stability. No advantage was seen with 2,5ꢀ or 2,3ꢀdiꢀfluoro substitution (44 and 45).
Table 3. Data for compounds 18 and 27ꢀ45
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Journal of Medicinal Chemistry
1
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8
pTau
GSK3ß a /α
IC50 (nM)
KBV Met.Stab.
Compd
R
IC50
ratio
(M/R/H)c
(nM)b
9
18
27
28
29
30
31
32
33
34
H
2ꢀF
9.8(±3.6)/5.5
13(±4.2)/3.3
16(±11)/ꢀ
320
290
480
180
77
1.3
2.0
1.5
1.3
1.8
1.3
4.0
1.2
1.1
50/88/93
33/85/86
40/76/75
61/85/72
14/64/51
36/74/44
69/90/88
64/89/76
39/75/0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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42
43
44
45
46
47
48
49
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51
52
53
54
55
56
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60
3ꢀF
4ꢀF
5.2(±2.2)/1.7
2.1(±0.95)/0.45
11(±3.5)/6.9
4.7(±1.3)/1.9
5.9(±2.7)/2.0
5.9(±2/3)/3.1
4ꢀOMe
4ꢀOCF3
4ꢀCN
4ꢀCl
270
96
200
180
4ꢀ
OCHF2
35
36
37
38
39
40
41
42
4ꢀMe
4ꢀtBu
4.7(±1.6)/1.1
19(±8.9)/19
120
800
28
0.83
1.3
3/33/25
0/15/1
2ꢀCF3
2ꢀCl
2.0(±0.69)/0.66
3.7(±1.1)/0.88
1.9(±1.2)/0.34
2.2(±1.2)/2.1
1.2(±1.1)/0.28
1.4
0/4/7
61
1.1
1/27/16
48/87/66
8/41/22
2/32/34
0/11/13
2,4ꢀF2
2,4ꢀCl2
2ꢀClꢀ4ꢀF
62
0.83
2.1
47
29
1.0
2ꢀCF3ꢀ4ꢀ 0.87(±1.2)/0.17
F
14
1.2
43
44
45
2ꢀFꢀ4ꢀCl
2,5ꢀF2
2.8(±0.91)/1.2
20(±5.4)/15
10(±4.8)/6.4
75
0.91
1.7
36/80/63
30/63/82
29/62/52
770
230
2,3ꢀF2
1.1
aValues are means of at least three experiments but data for GSK3α was not repeated
remaining after 10 min with 0.5 µM compounds incubated in liver microsomes.
;
bValues are means of at least two experiments. cValues are percentage
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Ambit screening with almost 400 kinases of the phenyl compounds, 18, 29, and 33, showed them to
have excellent kinase selectivity (Figure 4).47 These compounds showed appreciable inhibitory activity,
at least in these assays, only to the two GSKꢀ3 isoforms.47
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18
29
33
Figure 4. Ambit Radial Graphs for compounds 18, 29, and 33.
It appears that a combination of the central amide linker, which participates in waterꢀmediated
hydrogen bonding in the rear of the ATP pocket, and the strongly tilted 4ꢀphenyl group occupying the
large sugar pocket in GSKꢀ3β are primarily responsible for the high degree of kinase selectivity
observed. Notably, the back pocket region of kinases varies in size and shape partly due to the nonꢀ
conserved nature of the gateꢀkeeper residue. Also, the hydrophilic sugar pocket has been used to achieve
selectivity,48 as the residues that make up this pocket are also not highly conserved. A surface
representation of 18 bound in the GSKꢀ3β kinase domain (2.9Å) is shown in Figure 5. The modeled
waterꢀmediated hydrogen bond network proposed for this class of compounds is also shown in the inset.
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Journal of Medicinal Chemistry
Figure 5: Surface representation of compound 18 bound to the ATPꢀbinding site of GSKꢀ3β showing
the 4ꢀphenyl group of the 3ꢀaminopyridine occupying the sugar pocket. The two conserved structural
water molecules which were not detected in the Xꢀray structure of GSKꢀ3β:compound 18 complex most
likely due to the low resolution (3.0Å) were modeled , and are shown in the inset.
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Consistent with their favorable cellular KBV ratios, compounds 18, and 33, had very good Cacoꢀ2
permeabilities with no observed active efflux (Table 4). In addition, both compounds showed excellent
inherent bilayer permeability in the PAMPA assay.
Table 4. Permeability data for compounds 18 and 33.
Compound Cacoꢀ2 Pc AꢀB, Cacoꢀ2 Pc BꢀA, Efflux Ratio PAMPA Pc
PAMPA Pc
(pH 5.5),
nm/sec.
526
nm/sec.
nm/sec.
(pH 7.4),
nm/sec.
483
319
354
301
279
0.95
0.80
18
33
1260
904
We tested selected compounds in a tripleꢀtransgenic mouse AD model.49 These mice show elevated
pTau levels and develop mild functional deficits, plaques and tangles that are hallmarks of AD. It is
important to note that, when tested, these compounds showed very high clearance in mice (for
compound 29, Cl = 133 mL/min/kg, or > 100% of hepatic blood flow), primarily due to facile
hydrolysis of the pyridylꢀ2ꢀaminocarboxamide and pyridyl Nꢀoxidation of the 3ꢀaminopyridine. When
incubated in mouse serum at 37oC, 24% amide cleavage from compound 18 was observed over 3 h,
while only trace levels were detected in human serum. No degradants from cleavage of the core amide
were seen. Less rapid in vivo clearance was seen for compound 18 in rat (24 mL/min/kg) and dog (9.6
mL/min/kg). We believe that this rapid clearance in mice resulted in a high degree of variability seen in
plasma and brain exposures of compounds dosed PO in these studies50, and limits the extent of PK/PD
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interpretation possible in this species. In addition, compound 18 had IC50s all higher than 20 µM in the
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CYP panel and higher than 80 µM in the hERG flux assay.
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8
9
Compound 18 showed significant activity at 30 mg/kg po in two independent studies in this model
(results for one study are shown in Figure 6). The effect was comparable to the maximal pTau reduction
achievable with LiCl at its MTD (250 mg/kg) and persisted to 24 h (pTau levels shown are normalized
to total Tau). It’s important to note that the maximal pTau reduction achievable with GSKꢀ3 inhibition
in this model is 50%, presumably due to the action of other kinases. Only a trend toward activity was
seen at 3 and 10 mg/kg. Similar results were obtained with compound 29 (data not shown). In the same
model, the alkoxyphenyl compounds, 15, 19, 21, and 22, were inactive at 30 mg/kg p.o. Subsequent
studies showed 18 to have somewhat robust but variable brain exposures (440 ± 362 nM at 2 h) and a
b/p ratio of 0.2ꢀ0.3.50 The alkoxy analogs, on the other hand, demonstrated poor oral exposures and no
detectable brain levels (e.g., compound 21 had plasma exposure of 47 nM at 30 mg/kg, PO).
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In two separate timeꢀcourse/doesꢀresponse studies, compound 33 showed significant activity at 10
mg/kg and 30 mg/kg, PO, at various time points (Figure 7). At 1 and 3 mg/kg, significant activity was
also seen at 2 h. Brainꢀtoꢀplasma levels were higher in comparison with 18 (≥ 0.5), but interꢀanimal
variability was large, complicating interpretation.50
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Journal of Medicinal Chemistry
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Figure 6. Dose response (3, 10 mg/kg at 5 h) and time course (30 mg/kg) of 18 in the tripleꢀtransgenic
mouse AD model49. The blue bar represents vehicle only and the red bar shows maximum pTau
reduction by LiCl (250 mg/kg ip).
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Figure 7. Dose response of 33 at 1, 3, 10 and 30 mg/kg po and time course at both 10 and 30 mg/kg po
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2
3
in the mouse AD model.
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8
CONCLUSION
9
In summary, we have discovered a novel class of potent GSKꢀ3 inhibitors such as 18, 29 and 33 that
show remarkable kinase selectivity in broad panel screening. Despite high in vivo clearance, these
compounds showed good in vivo activity, lowering pTau levels in a triple transgenic AD mouse model.
With their very high level of kinase selectivity, radiolabeled versions of these molecules would be
useful tools in further studies. These and further SAR studies around this chemotype will be reported in
due course.
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EXPERIMENTAL PROCEDURES
General Chemistry Details. All commercially available reagents and solvents were used without
further purification unless otherwise stated. All reactions were carried out under an inert atmosphere of
dry nitrogen in oven or flameꢀdried glassware unless otherwise stated. Flash column chromatography
was performed using 40ꢀ60 ꢁm Silica Gel 60 (EMD Chemicals, Inc.) as the stationary phase, or preꢀ
packed columns from ISCO Inco., Biotage, or Thomson Instrument Co. 1H NMR spectra were recorded
on a Bruker 400 or 500 MHz machine with tetramethylsilane or residual protiated solvent used as a
reference. 13C NMR were recorded on a Bruker DRXꢀ500 instrument operating at 125 MHz with
12
residual C solvent used as a reference. Low resolution mass spectra were recorded using a Waters
Micromass ZQ with electrospray ionization. High resolution mass spectra were recorded using a Waters
Micromass LCT time of flight mass spectrometer with electrospray ionization. The purity of all
compounds was determined by either Liquid chromatography–mass spectrometry (LCMS) or analytical
HPLC and was confirmed to be greater than 95% purity.
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Journal of Medicinal Chemistry
2-Chloro-N-(pyridin-3-yl)isonicotinamide (11i). In a 10 mL roundꢀbottom flask was dissolved 2ꢀ
chloroisonicotinoyl chloride (227.3 mg, 1.291 mmol) in methylene chloride to give a colorless solution.
Pyridinꢀ3ꢀamine (134 mg, 1.42 mmol) was added, and a precipitate formed. Hunig's Base (0.248 mL,
1.421 mmol) was added and all solids dissolved. The mixture was stirred at rt for 18 h. The mixture was
purified by flash column chromatography using silica gel and eluting with a gradient up to 8%
methanol/methylene chloride affording the desired product (248.4 mg, 82%) as a white solid: MS (ESI)
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1
(m/z): 234 (M+H)+; H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 2.5 Hz, 1H), 8.62 (dd, J = 5.1, 0.6 Hz,
1H), 8.47 (dd, J = 4.8, 1.4 Hz, 1H), 8.30 (d, J = 7.5 Hz, 1H), 8.20 (s, 1H), 7.85 ꢀ 7.79 (m, 1H), 7.69 (dd,
J = 5.1, 1.5 Hz, 1H), 7.40 (dd, J = 8.3, 4.8 Hz, 1H).
1
2-Chloro-N-(4-methoxypyridin-3-yl)isonicotinamide (15i): MS (ESI) (m/z): 264 (M+H)+; H NMR
(400 MHz, CDCl3) δ 9.41 (s, 1H), 8.58 (s, 1H), 8.52 (d, J = 5.1 Hz, 1H), 8.29 (d, J = 5.6 Hz, 1H), 7.75
(s, 1H), 7.63 (dd, J = 5.1, 1.4 Hz, 1H), 6.86 (d, J = 5.6 Hz, 1H), 3.97 (s, 3H).
2-Chloro-N-(4-phenylpyridin-3-yl)isonicotinamide (18i): MS (ESI) (m/z): 310 (M+H)+.
2-Chloro-N-(4-(2,2,2-trifluoroethoxy)pyridin-3-yl)isonicotinamide (19i): MS (ESI) (m/z): 332
(M+H)+.
1
2-Chloro-N-(4-isopropoxypyridin-3-yl)isonicotinamide (20i): MS (ESI) (m/z): 292 (M+H)+; H NMR
(400 MHz, CDCl3) δ 9.33 (s, 1H), 8.64 (s, 1H), 8.47 (dd, J = 5.1, 0.5 Hz, 1H), 8.20 (d, J = 5.6 Hz, 1H),
7.70 (d, J = 0.7 Hz, 1H), 7.56 (dd, J = 5.1, 1.5 Hz, 1H), 6.80 (d, J = 5.7 Hz, 1H), 4.70 (hept, J = 6.1 Hz,
1H), 1.37 (d, J = 6.1 Hz, 6H).
1
2-Chloro-N-(4-ethoxypyridin-3-yl)isonicotinamide (21i): MS (ESI) (m/z): 278 (M+H)+; H NMR
(400MHz, CHLOROFORMꢀd) δ 9.51 (s, 1H), 8.61 ꢀ 8.55 (m, 1H), 8.41 (br. s., 1H), 8.32 (d, J=5.5 Hz,
1H), 7.80 ꢀ 7.75 (m, 1H), 7.62 (dd, J=5.0, 1.5 Hz, 1H), 6.86 (d, J=5.5 Hz, 1H), 4.25 (q, J=7.0 Hz, 2H),
1.53 (t, J=7.0 Hz, 3H).
2-Chloro-N-(4-(cyclopropylmethoxy)pyridin-3-yl)isonicotinamide (24i): MS (ESI) (m/z): 304
1
(M+H)+; H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H), 8.65 (s, 1H), 8.51 (dd, J = 5.1, 0.5 Hz, 1H), 8.24
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(d, J = 5.6 Hz, 1H), 7.74 (d, J = 0.7 Hz, 1H), 7.60 (dd, J = 5.1, 1.5 Hz, 1H), 6.80 (d, J = 5.6 Hz, 1H),
3.96 (d, J = 7.1 Hz, 2H), 1.38 ꢀ 1.23 (m, 1H), 0.72 ꢀ 0.63 (m, 2H), 0.40 ꢀ 0.33 (m, 2H).
2-Chloro-N-(4-(2-hydroxyethoxy)pyridin-3-yl)isonicotinamide (25i): MS (ESI) (m/z): 294 (M+H)+; 1H
NMR (400 MHz, MeOD) δ 9.06 (s, 1H), 8.57 (dd, J = 5.1, 0.6 Hz, 1H), 8.29 (d, J = 5.8 Hz, 1H), 7.95 (d,
J = 0.7 Hz, 1H), 7.84 (dd, J = 5.2, 1.5 Hz, 1H), 7.15 (d, J = 5.8 Hz, 1H), 4.30 ꢀ 4.24 (m, 2H), 4.01 ꢀ 3.94
(m, 2H).
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2-(Cyclopropanecarboxamido)-N-(pyridin-3-yl)isonicotinamide (11). Into a 5 mL microwave tube
under a nitrogen atmosphere was added 11i (50.5 mg, 0.216 mmol), cyclopropanecarboxamide (55.2
mg, 0.648 mmol), and potassium carbonate (44.8 mg, 0.324 mmol) in dioxane (2 mL) (degassed) to give
a colorless suspension. Pd(OAc)2 (7.28 mg, 0.032 mmol) and XANTPHOS (37.5 mg, 0.065 mmol) were
added. The vial was sealed and heated at 150°C for 2 h. Volatile components were removed in vacuo.
The residue was suspended in 3 mL dimethylformamide and filtered. The solution was purified by prepꢀ
1
HPLC to afford the desired product (5 mg, 8%): MS (ESI) (m/z): 283 (M+H)+; H NMR (500 MHz,
DMSO) δ 11.06 (s, 1H), 10.74 (s, 1H), 8.92 (d, J = 2.4 Hz, 1H), 8.55 (s, 1H), 8.53 (d, J = 5.0 Hz, 1H),
8.36 (dd, J = 4.7, 1.5 Hz, 1H), 8.19 (ddd, J = 8.3, 2.5, 1.5 Hz, 1H), 7.57 (dd, J = 5.1, 1.5 Hz, 1H), 7.43
(ddd, J = 8.3, 4.7, 0.6 Hz, 1H), 2.14 ꢀ 1.99 (m, 1H), 0.93 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide (15): MS (ESI) (m/z): 313
1
(M+H)+; H NMR (500 MHz, DMSO) δ 11.02 (s, 1H), 10.07 (s, 1H), 8.57 (s, 1H), 8.54 (s, 1H), 8.50 (d,
J = 5.0 Hz, 1H), 8.38 (d, J = 5.6 Hz, 1H), 7.57 (d, J = 4.5 Hz, 1H), 7.19 (d, J = 5.7 Hz, 1H), 3.90 (s, 3H),
2.05 (tt, J = 6.9, 5.5 Hz, 1H), 0.92 ꢀ 0.80 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-phenylpyridin-3-yl)isonicotinamide (18): HRMS (ESI) (m/z):
1
Calcd for C21H19O2N4: 359.1503 (M+H)+; found: 359.1492; H NMR (500 MHz, DMSO) δ 11.00 (s,
1H), 10.46 (s, 1H), 8.63 (s, 1H), 8.58 (d, J = 5.0 Hz, 1H), 8.46 (d, J = 4.5 Hz, 2H), 7.55 ꢀ 7.48 (m, 3H),
7.48 ꢀ 7.43 (m, 2H), 7.40 (tt, J = 6.2, 1.4 Hz, 2H), 2.04 (tt, J = 7.1, 5.3 Hz, 1H), 0.90 ꢀ 0.80 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(2,2,2-trifluoroethoxy)pyridin-3-yl)isonicotinamide (19): MS
1
(ESI) (m/z): 381 (M+H)+; H NMR (500 MHz, MeOD) δ 11.83 (s, 1H), 11.01 (s, 1H), 9.34 (s, 2H), 9.32
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Journal of Medicinal Chemistry
(d, J = 5.2 Hz, 1H), 9.25 (d, J = 5.7 Hz, 1H), 8.33 (d, J = 4.3 Hz, 1H), 8.13 (d, J = 5.7 Hz, 1H), 5.76 (q, J
= 8.7 Hz, 2H), 2.85 (dq, J = 6.6, 5.7 Hz, 1H), 1.69 ꢀ 1.64 (m, 4H).
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2-(Cyclopropanecarboxamido)-N-(4-isopropoxypyridin-3-yl)isonicotinamide (20): MS (ESI) (m/z):
341 (M+H)+; H NMR (500 MHz, DMSO) δ 11.03 (s, 1H), 9.86 (s, 1H), 8.60 (s, 1H), 8.55 (s, 1H), 8.51
7
8
1
9
(d, J = 5.1 Hz, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.54 (s, 1H), 7.18 (d, J = 5.8 Hz, 1H), 4.80 (dt, J = 12.1, 6.0
Hz, 1H), 2.10 ꢀ 2.01 (m, 1H), 1.31 (d, J = 6.0 Hz, 6H), 0.90 ꢀ 0.84 (m, 4H).
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2-(Cyclopropanecarboxamido)-N-(4-ethoxypyridin-3-yl)isonicotinamide (21): MS (ESI) (m/z): 327
(M+H)+; 1H NMR (400MHz, CHLOROFORMꢀd) δ 9.58 (s, 1H), 8.70 (s, 1H), 8.50 ꢀ 8.41 (m, 3H), 8.35
(d, J=5.5 Hz, 1H), 7.59 (dd, J=5.0, 1.5 Hz, 1H), 6.87 (d, J=5.5 Hz, 1H), 4.24 (q, J=7.0 Hz, 2H), 1.67 ꢀ
1.60 (m, 1H), 1.59 ꢀ 1.54 (m, 3H), 1.17 ꢀ 1.12 (m, 2H), 0.99 ꢀ 0.94 (m, 2H).
2-(Cyclopropanecarboxamido)-N-(4-(cyclopropylmethoxy)pyridin-3-yl)isonicotinamide (24): MS
1
(ESI) (m/z): 353 (M+H)+; H NMR (400 MHz, DMSO) δ 11.03 (s, 1H), 9.98 (s, 1H), 8.57 (s, 2H), 8.51
(d, J = 5.1 Hz, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.56 (d, J = 5.0 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H), 4.01 (d, J
= 6.8 Hz, 2H), 2.10 ꢀ 1.98 (m, 1H), 1.27 ꢀ 1.23 (m, 1H), 0.86 (d, J = 6.1 Hz, 4H), 0.59 ꢀ 0.51 (m, 2H),
0.42 ꢀ 0.32 (m, 2H).
2-(Cyclopropanecarboxamido)-N-(4-(2-hydroxyethoxy)pyridin-3-yl)isonicotinamide (25): MS (ESI)
1
(m/z): 343 (M+H)+; H NMR (500 MHz, DMSO) δ 11.04 (s, 1H), 8.75 (s, 1H), 8.54 (s, 1H), 8.51 (d, J =
5.0 Hz, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.55 (d, J = 4.4 Hz, 1H), 7.19 (d, J = 5.7 Hz, 1H), 4.16 (t, J = 4.8
Hz, 2H), 3.78 ꢀ 3.71 (m, 2H), 2.06 (s, 1H), 0.91 ꢀ 0.85 (m, 4H).
Methyl
2-(N-(cyclopropanecarbonyl)cyclopropanecarboxamido)isonicotinate
(9).
Cyclopropanecarbonyl chloride (3.79 mL, 41.4 mmol) was added to a solution of methyl 2ꢀ
aminoisonicotinamide (3.0 g, 19.72 mmol) and Hünig's base (7.23 mL, 41.4 mmol) in methylene
chloride (150 mL) at 0 °C. The reaction was stirred at 0 °C for 1 hour before quenching with water.
The mixture was diluted with methylene chloride and washed with NaHCO3 (sat.) and water. The
organic layer was separated, dried (Na2SO4), filtered and concentrated. Flash column chromatography,
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eluting with ethyl acetate in hexane (0 to 50%) afforded the desired product (4.99 g, 88%) as a white
1
2
3
solid: MS (ESI) (m/z): 311 (M+Na)+.
4
5
6
7
8
9
2-(Cyclopropanecarboxamido)isonicotinic acid (10). A mixture of LiOH (8.65 mL, 69.2 mmol, 8 M
in water) and 9 (4.99 g, 17.31 mmol) in tetrahydrofuran (20 mL) and methanol (2mL) was stirred at
room temperature for 18 h. The volatile components were removed in vacuo and the residue acidified by
adding 1N HCl. The solid was filtered and washed with water. The white solid was used without further
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purification after drying under high vacuum for several hours (2.81 g, 79%): H NMR (400MHz,
DMSOꢀd6) δ 11.02 (s, 1H), 8.56 (s, 1H), 8.48 (dd, J=5.3, 0.8 Hz, 1H), 7.51 (dd, J=5.0, 1.5 Hz, 1H),
2.06 ꢀ 1.98 (m, 1H), 0.88 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-phenoxypyridin-3-yl)isonicotinamide (22): In a 5 mL roundꢀ
bottomed flask was 10 (34 mg, 0.165 mmol) in CH2Cl2 (1 mL) to give a white suspension. DMF (2.55
µl, 0.033 mmol) (one drop) and oxalyl chloride (0.017 mL, 0.198 mmol) were added. The mixture was
stirred at rt for 30 min. 4ꢀPhenoxypyridinꢀ3ꢀamine (30.7 mg, 0.165 mmol) and Et3N (0.069 mL, 0.495
mmol) were added. The mixture was stirred at rt overnight. The reaction mixture was concentrated,
redissolved in 1.5 mL MeOH, and purified by prepꢀHPLC to afford the desired product (21.3 mg, 35%):
1
MS (ESI) (m/z): 375 (M+H)+; H NMR (500 MHz, DMSO) δ 11.02 (s, 1H), 10.41 (s, 1H), 8.71 (s, 1H),
8.55 (s, 1H), 8.52 ꢀ 8.43 (m, 1H), 8.34 (d, J = 5.6 Hz, 1H), 7.54 ꢀ 7.50 (m, 1H), 7.50 ꢀ 7.45 (m, 2H), 7.31
ꢀ 7.25 (m, 1H), 7.20 ꢀ 7.15 (m, 2H), 6.79 (d, J = 5.6 Hz, 1H), 2.09 ꢀ 1.99 (m, 1H), 0.88 ꢀ 0.79 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(trifluoromethyl)pyridin-3-yl)isonicotinamide (17): MS (ESI)
1
(m/z): 351 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 11.04 (s, 1H), 10.80 ꢀ 10.64 (s,br, 1H), 8.85 (s,
1H), 8.78 (d, J=4.9 Hz, 1H), 8.59 (s, 1H), 8.53 (d, J=5.2 Hz, 1H), 7.85 (d, J=4.9 Hz, 1H), 7.56 (dd,
J=5.0, 1.4 Hz, 1H), 2.12 ꢀ 2.01 (m, 1H), 0.92 ꢀ 0.82 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(neopentyloxy)pyridin-3-yl)isonicotinamide (23): In a 5 mL vial
was 4ꢀ(neopentyloxy)pyridinꢀ3ꢀamine (24 mg, 0.133 mmol) and 10 (27.5 mg, 0.133 mmol) in DMF (1
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Journal of Medicinal Chemistry
mL) to give a tan solution. HATU (101 mg, 0.266 mmol) and Hunig's Base (0.047 mL, 0.266 mmol)
were added, and the mixture was stirred at rt for 18 h. The reaction mixture was purified by prepꢀHPLC
to afford the desired product (32 mg, 65%): MS (ESI) (m/z): 369 (M+H)+; 1H NMR (400 MHz, DMSO)
δ 10.98 (s, 1H), 9.97 (s, 1H), 8.56 (s, 1H), 8.51 (dd, J = 5.1, 0.6 Hz, 1H), 8.49 (s, 1H), 8.36 (d, J = 5.6
Hz, 1H), 7.53 (dd, J = 5.1, 1.4 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H), 3.77 (s, 2H), 2.12 ꢀ 1.99 (m, 1H), 0.96
(s, 9H), 0.90 ꢀ 0.83 (m, 4H).
1
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2-(Cyclopropanecarboxamido)-N-(2-fluoropyridin-3-yl)isonicotinamide (12): MS (ESI) (m/z): 301
(M+H)+; 1H NMR (500 MHz, DMSO) δ 11.06 (s, 1H), 10.61 (s, 1H), 8.57 (d, J = 0.5 Hz, 1H), 8.53 (dd,
J = 5.1, 0.6 Hz, 1H), 8.20 (ddd, J = 9.6, 7.8, 1.7 Hz, 1H), 8.15 ꢀ 8.09 (m, 1H), 7.56 (dd, J = 5.1, 1.6 Hz,
1H), 7.48 ꢀ 7.40 (m, 1H), 2.11 ꢀ 2.00 (m, 1H), 0.92 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(2-hydroxypyridin-3-yl)isonicotinamide (13): MS (ESI) (m/z): 299
(M+H)+; 1H NMR (500 MHz, DMSO) δ 12.18 (s, 1H), 11.08 (s, 1H), 9.44 (s, 1H), 8.61 ꢀ 8.44 (m, 2H),
8.30 (dd, J = 7.3, 1.8 Hz, 1H), 7.51 (dd, J = 5.1, 1.6 Hz, 1H), 7.23 (dd, J = 6.6, 1.8 Hz, 1H), 6.42 ꢀ 6.22
(m, 1H), 2.05 (dq, J = 7.5, 5.0 Hz, 1H), 0.93 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(2-cyanopyridin-3-yl)isonicotinamide (14): MS (ESI) (m/z): 308
(M+H)+; 1H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 8.62 (s, 1H), 8.51 (d, J = 4.9 Hz, 2H), 8.21 (dd, J
= 8.4, 1.3 Hz, 1H), 7.73 (dd, J = 8.4, 4.6 Hz, 1H), 7.64 (dd, J = 5.1, 1.5 Hz, 1H), 2.09 ꢀ 1.99 (m, 1H),
0.91 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-iodopyridin-3-yl)isonicotinamide (26). In a 250 mL roundꢀ
bottomed flask was 10 (776.5 mg, 3.77 mmol) in CH2Cl2 (30 mL) to give a white suspension. After
cooling to 0 °C, DMF (0.029 mL, 0.377 mmol) and oxalyl chloride (0.363 mL, 4.14 mmol) were added.
The mixture was stirred at 0 °C for 2 h. LCMS showed the desired chloride (as methyl ester form). 4ꢀ
Iodopyridinꢀ3ꢀamine (829 mg, 3.77 mmol) was added at 0 °C, followed by Et3N (2.1 mL, 15.06 mmol).
The mixture became clear to a tan solution. It was concentrated to a tan oil. The residue was purified by
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FCC up to 10% MeOH/CH2Cl2 (contains 1% AcOH). The product peak was pooled and concentrated to
a tan solid (100%) which was used without further characterizations.
1
2
3
4
5
6
N-(4-chloropyridin-3-yl)-2-(cyclopropanecarboxamido)isonicotinamide (16): MS (ESI) (m/z): 317
(M+H)+; H NMR (400 MHz, CDCl3) δ 9.60 (s, 1H), 8.76 (s, 1H), 8.71 (d, J = 0.7 Hz, 1H), 8.49 (dd, J
7
8
1
9
= 5.1, 0.7 Hz, 1H), 8.42 (s, 1H), 8.38 (d, J = 5.3 Hz, 1H), 7.59 (dd, J = 5.1, 1.6 Hz, 1H), 7.42 (d, J = 5.2
Hz, 1H), 1.69 ꢀ 1.58 (m, 1H), 1.20 ꢀ 1.13 (m, 2H), 1.00 ꢀ 0.93 (m, 2H).
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2-(Cyclopropanecarboxamido)-N-(4-(2-fluorophenyl)pyridin-3-yl)isonicotinamide (27): In a 5 mL
microwave tube was 26 (36.6 mg, 0.090 mmol), (2ꢀfluorophenyl)boronic acid (20.07 mg, 0.143 mmol),
and Na2CO3 (0.134 mL, 0.269 mmol) in dioxane (0.8 mL) to give a tan suspension under nitrogen. 1,1'ꢀ
bis(diphenylphosphino)ferrocenepalladium(II) dichloride,toluene (3.69 mg, 4.48 µmol) was added
under nitrogen. The vial was sealed and heated at 140°C for 45 min. It was filtered, and purified by
1
prepꢀHPLC to afford the desired product (7.2 mg, 21%): H NMR (500 MHz, DMSO) δ 10.98 (s, 1H),
10.41 (s, 1H), 8.70 (s, 1H), 8.58 (d, J = 5.0 Hz, 1H), 8.43 (d, J = 5.2 Hz, 1H), 8.40 (s, 1H), 7.49 (d, J =
5.0 Hz, 1H), 7.47 ꢀ 7.38 (m, 2H), 7.35 ꢀ 7.22 (m, 3H), 2.03 (dq, J = 7.1, 5.4 Hz, 1H), 0.89 ꢀ 0.81 (m,
4H).
2-(Cyclopropanecarboxamido)-N-(4-(3-fluorophenyl)pyridin-3-yl)isonicotinamide (28): MS (ESI)
(m/z): 377 (M+H)+; 1H NMR (500 MHz, DMSO) δ 11.00 (s, 1H), 10.47 (s, 1H), 8.66 (s, 1H), 8.59 (d, J
= 5.0 Hz, 1H), 8.49 ꢀ 8.43 (m, 2H), 7.52 (d, J = 5.0 Hz, 1H), 7.51 ꢀ 7.44 (m, 1H), 7.40 (d, J = 4.9 Hz,
1H), 7.35 (d, J = 8.0 Hz, 2H), 7.27 ꢀ 7.21 (m, 1H), 2.08 ꢀ 1.99 (m, 1H), 0.89 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(4-fluorophenyl)pyridin-3-yl)isonicotinamide (29): HRMS (ESI)
1
(m/z): Calcd for C21H18O2N4F: 377.1408 (M+H)+; found: 377.1398; H NMR (500 MHz, DMSO) δ
11.00 (s, 1H), 10.42 (s, 1H), 8.65 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 8.50 ꢀ 8.41 (m, 2H), 7.56 (dd, J = 8.7,
5.5 Hz, 2H), 7.49 (d, J = 5.0 Hz, 1H), 7.40 (d, J = 4.7 Hz, 1H), 7.35 ꢀ 7.24 (m, 2H), 2.10 ꢀ 1.98 (m, 1H),
0.88 ꢀ 0.82 (m, 4H).
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2-(Cyclopropanecarboxamido)-N-(4-(4-methoxyphenyl)pyridin-3-yl)isonicotinamide (30): MS (ESI)
(m/z): 389 (M+H)+; 1H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 10.38 (s, 1H), 8.60 (s, 1H), 8.54 (d, J
= 5.0 Hz, 1H), 8.47 (d, J = 5.0 Hz, 2H), 7.47 (dd, J = 9.8, 6.9 Hz, 3H), 7.43 (d, J = 6.2 Hz, 1H), 7.05 ꢀ
7.01 (m, 2H), 3.79 (s, 3H), 2.10 ꢀ 1.96 (m, 1H), 0.91 ꢀ 0.79 (m, 4H).
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7
8
9
2-(Cyclopropanecarboxamido)-N-(4-(4-(trifluoromethoxy)phenyl)pyridin-3-yl)isonicotinamide (31):
MS (ESI) (m/z): 443 (M+H)+; 1H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 10.46 (s, 1H), 8.71 (s, 1H),
8.61 (d, J = 5.1 Hz, 1H), 8.46 (d, J = 5.2 Hz, 1H), 8.44 (s, 1H), 7.66 (s, 1H), 7.64 (s, 1H), 7.55 (d, J = 5.0
Hz, 1H), 7.48 (s, 1H), 7.46 (s, 1H), 7.38 (d, J = 5.0 Hz, 1H), 2.10 ꢀ 1.98 (m, 1H), 0.92 ꢀ 0.79 (m, 4H).
N-(4-(4-cyanophenyl)pyridin-3-yl)-2-(cyclopropanecarboxamido)isonicotinamide (32): MS (ESI)
(m/z): 384 (M+H)+; 1H NMR (500 MHz, DMSO) δ 11.01 (s, 1H), 10.50 (s, 1H), 8.72 (s, 1H), 8.61 (d, J
= 5.0 Hz, 1H), 8.46 (d, J = 5.1 Hz, 1H), 8.40 (s, 1H), 7.95 ꢀ 7.90 (m, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.53
(d, J = 5.1 Hz, 1H), 7.39 (d, J = 4.0 Hz, 1H), 2.09 ꢀ 1.99 (m, 1H), 0.86 (dtd, J = 8.0, 5.0, 3.1 Hz, 4H).
N-(4-(4-chlorophenyl)pyridin-3-yl)-2-(cyclopropanecarboxamido)isonicotinamide (33): HRMS (ESI)
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11
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(m/z): Calcd for C21H18O2N4Cl: 393.1113 (M+H)+; found: 393.1104; H NMR (500 MHz, DMSO) δ
11.01 (s, 1H), 10.44 (s, 1H), 8.67 (s, 1H), 8.58 (d, J = 5.0 Hz, 1H), 8.52 ꢀ 8.40 (m, 2H), 7.56 ꢀ 7.51 (m,
4H), 7.50 (d, J = 5.1 Hz, 1H), 7.41 (d, J = 4.0 Hz, 1H), 2.04 (dq, J = 7.5, 5.0 Hz, 1H), 0.92 ꢀ 0.80 (m,
4H).
2-(Cyclopropanecarboxamido)-N-(4-(4-(difluoromethoxy)phenyl)pyridin-3-yl)isonicotinamide (34):
1
MS (ESI) (m/z): 425 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 11.00 (s, 1H), 10.42 (s, 1H), 8.66 (s,
1H), 8.57 (d, J=4.9 Hz, 1H), 8.46 (d, J=5.2 Hz, 1H), 8.43 (s, 1H), 7.59 (d, J=8.5 Hz, 2H), 7.49 (d, J=4.9
Hz, 1H), 7.41 (d, J=5.2 Hz, 1H), 7.31 ꢀ 7.25 (m, 3H), 2.08 ꢀ 1.99 (m, 1H), 0.88 ꢀ 0.82 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(4-(methyl)phenyl)pyridin-3-yl)isonicotinamide (35): MS (ESI)
(m/z): 373 (M+H)+; 1H NMR (500 MHz, DMSO) δ 11.00 (s, 1H), 10.38 (s, 1H), 8.62 (s, 1H), 8.56 (d, J
= 5.0 Hz, 1H), 8.51 ꢀ 8.42 (m, 2H), 7.47 (d, J = 5.0 Hz, 1H), 7.42 (d, J = 7.8 Hz, 3H), 7.27 (d, J = 7.9
Hz, 2H), 2.32 (s, 3H), 2.10 ꢀ 1.99 (m, 1H), 0.90 ꢀ 0.80 (m, 4H).
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N-(4-(4-(tert-butyl)phenyl)pyridin-3-yl)-2-(cyclopropanecarboxamido)isonicotinamide (36): MS
1
2
3
4
1
(ESI) (m/z): 415 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 11.00 (s, 1H), 10.38 (s, 1H), 8.65 (s, 1H),
5
6
8.55 (d, J=5.2 Hz, 1H), 8.49 ꢀ 8.43 (m, 2H), 7.54 ꢀ 7.46 (m, 5H), 7.41 (d, J=4.6 Hz, 1H), 2.08 ꢀ 1.99 (m,
7
8
1H), 1.30 (s, 9H), 0.87 ꢀ 0.82 (m, 4H).
9
2-(Cyclopropanecarboxamido)-N-(4-(2-(trifluoromethyl)phenyl)pyridin-3-yl)isonicotinamide
(37):
10
11
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13
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23
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27
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MS (ESI) (m/z): 427 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 10.95 (s, 1H), 10.19 (s, 1H), 8.72 (s,
1H), 8.56 (d, J=4.9 Hz, 1H), 8.39 (d, J=5.2 Hz, 1H), 8.31 (s, 1H), 7.83 (d, J=7.6 Hz, 1H), 7.73 ꢀ 7.67
(m, 1H), 7.65 ꢀ 7.59 (m, 1H), 7.40 (d, J=7.6 Hz, 1H), 7.36 (d, J=5.2 Hz, 1H), 7.20 (dd, J=5.0, 1.4 Hz,
1H), 2.05 ꢀ 1.98 (m, 1H), 0.86 ꢀ 0.81 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(2-chlorophenyl)pyridin-3-yl)isonicotinamide (38): MS (ESI)
(m/z): 393 (M+H)+; 1H NMR (500MHz, DMSOꢀd6) δ 11.02 (s, 1H), 10.58 (s, 1H), 8.94 (s, 1H), 8.72 (d,
J=5.2 Hz, 1H), 8.44 (d, J=5.2 Hz, 1H), 8.37 (s, 1H), 7.71 (d, J=5.5 Hz, 1H), 7.60 ꢀ 7.56 (m, 1H), 7.48 ꢀ
7.40 (m, 3H), 7.30 (dd, J=5.2, 1.2 Hz, 1H), 2.03 (quin, J=6.2 Hz, 1H), 0.86 ꢀ 0.83 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(2,4-difluorophenyl)pyridin-3-yl)isonicotinamide (39): MS (ESI)
(m/z): 395 (M+H)+; 1H NMR (500MHz, DMSOꢀd6) δ 10.99 (s, 1H), 10.40 (br. s., 1H), 8.73 (s, 1H), 8.58
(d, J=4.9 Hz, 1H), 8.45 (d, J=4.9 Hz, 1H), 8.39 (s, 1H), 7.51 ꢀ 7.43 (m, 2H), 7.39 ꢀ 7.29 (m, 2H), 7.18
(td, J=8.5, 2.4 Hz, 1H), 2.09 ꢀ 2.00 (m, 1H), 0.89 ꢀ 0.80 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(2,4-dichlorophenyl)pyridin-3-yl)isonicotinamide (40): MS
1
(ESI) (m/z): 427 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 10.98 (s, 1H), 10.34 (br. s., 1H), 8.77 (s,
1H), 8.58 (d, J=5.2 Hz, 1H), 8.44 (d, J=4.9 Hz, 1H), 8.35 (s, 1H), 7.73 (d, J=1.8 Hz, 1H), 7.51 (dd,
J=8.2, 2.1 Hz, 1H), 7.43 (d, J=5.2 Hz, 1H), 7.39 (d, J=8.2 Hz, 1H), 7.31 (d, J=4.0 Hz, 1H), 2.06 ꢀ 1.99
(m, 1H), 0.89 ꢀ 0.82 (m, 4H).
N-(4-(2-chloro-4-fluorophenyl)pyridin-3-yl)-2-(cyclopropanecarboxamido)isonicotinamide (41): MS
1
(ESI) (m/z): 393 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 10.98 (s, 1H), 10.32 (br. s., 1H), 8.75 (s,
1H), 8.57 (d, J=4.9 Hz, 1H), 8.43 (d, J=5.2 Hz, 1H), 8.35 (s, 1H), 7.55 (dd, J=9.0, 2.6 Hz, 1H), 7.45 ꢀ
7.39 (m, 2H), 7.34 ꢀ 7.27 (m, 2H), 2.05 ꢀ 1.99 (m, 1H), 0.87 ꢀ 0.83 (m, 4H).
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Journal of Medicinal Chemistry
2-(Cyclopropanecarboxamido)-N-(4-(4-fluoro-2-(trifluoromethyl)phenyl)pyridin-3-yl)isonicotinamide
1
2
3
1
(42): MS (ESI) (m/z): 445 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 10.96 (s, 1H), 10.17 (br. s., 1H),
4
5
6
7
8
8.77 (s, 1H), 8.55 (d, J=5.2 Hz, 1H), 8.41 (d, J=4.9 Hz, 1H), 8.28 (s, 1H), 7.75 (dd, J=9.3, 2.6 Hz, 1H),
7.60 (td, J=8.5, 2.6 Hz, 1H), 7.46 (dd, J=8.7, 5.6 Hz, 1H), 7.36 (d, J=4.9 Hz, 1H), 7.23 (dd, J=5.2, 1.2
Hz, 1H), 2.06 ꢀ 1.96 (m, 1H), 0.87 ꢀ 0.81 (m, 4H).
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N-(4-(4-chloro-2-fluorophenyl)pyridin-3-yl)-2-(cyclopropanecarboxamido)isonicotinamide (43): MS
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(ESI) (m/z): 411 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 11.00 (s, 1H), 10.42 (br. s., 1H), 8.75 (s,
1H), 8.57 (d, J=4.9 Hz, 1H), 8.45 (d, J=5.2 Hz, 1H), 8.39 (s, 1H), 7.55 (dd, J=10.1, 2.1 Hz, 1H), 7.50 ꢀ
7.43 (m, 2H), 7.41 ꢀ 7.32 (m, 2H), 2.08 ꢀ 1.99 (m, 1H), 0.90 ꢀ 0.82 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(2,5-difluorophenyl)pyridin-3-yl)isonicotinamide (44): MS (ESI)
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(m/z): 395 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 10.99 (s, 1H), 10.46 (br. s., 1H), 8.72 (s, 1H),
8.60 (d, J=4.9 Hz, 1H), 8.45 (d, J=5.2 Hz, 1H), 8.40 (s, 1H), 7.51 (d, J=5.2 Hz, 1H), 7.40 ꢀ 7.25 (m,
4H), 2.07 ꢀ 1.98 (m, 1H), 0.87 ꢀ 0.82 (m, 4H).
2-(Cyclopropanecarboxamido)-N-(4-(2,3-difluorophenyl)pyridin-3-yl)isonicotinamide (45): MS (ESI)
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(m/z): 395 (M+H)+; H NMR (500MHz, DMSOꢀd6) δ 10.97 (s, 1H), 8.75 (s, 1H), 8.57 (d, J=4.9 Hz,
1H), 8.43 (d, J=5.2 Hz, 1H), 8.39 (s, 1H), 7.51 (d, J=4.9 Hz, 1H), 7.46 (q, J=8.5 Hz, 1H), 7.34 (d, J=4.6
Hz, 1H), 7.30 ꢀ 7.19 (m, 2H), 2.07 ꢀ 1.99 (m, 1H), 0.87 ꢀ 0.81 (m, 4H).
GSK3ß/3α Kinase Assay. Inhibitors were evaluated in a competitive binding assay for their ability to
bind to GSK3ꢀα and GSKꢀ3β. Test compound was incubated in assay buffer (20 mM Hepes, pH 7.4, 10
mM MgCl2, 0.015% Brijꢀ35, 4 mM DTT, 0.05 mg/mL BSA) in the presence of a 1nM GSTꢀGSK3a or
GSTꢀGSK3B, a fluorescently labeled small molecule at Kd (GSK3ꢀα = 15 nM , GSKꢀ3β = 6 nM) and
0.2 nM antiꢀGST labeled detection antibody. After a 1 hour incubation at room temperature,
homogenous timeꢀresolved florescence (HTRF) was monitored on a Envision plate reader (Ex 340 nm;
Em 520/615 nm). Inhibition data were calculated by comparison of the no enzyme control reactions for
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100% inhibition and vehicleꢀonly reactions for 0% inhibition. Dose response curves were generated to
determine the concentration required to inhibit 50% of the kinase activity (IC50).
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Tripple Transgenic Mouse Model.49 Mice were handled strictly according to BristolꢀMyers Squibb
(BMS) Animal Care and Use Committee guidelines. 3xTg mice were obtained from Dr. Frank LaFerla
at the University of California, Irvine. The mice were bred onto the C57BL/6 background by Dr. Mark
Mattson (National Institute on Aging) and breeders from those congenic mice were obtained by BMS,
where they were bred as homozygotes. Mice were housed with a 6 AMꢀ6 PM light/dark cycle and
allowed free access to food and water. Animals were used for study between the ages of five to seven
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months.
Test compounds were dosed at 30mg/kg as a nanosuspension in 2% HPCꢀ
SSL/0.15%SLS/97.85% water by oral gavage. LiCl was administered at 250 mg/kg by i.p. injection in
water. Animals were euthanized by guillotine five hours postꢀdose. Brains were dissected into
hemispheres, flash frozen in liquid nitrogen and stored at ꢀ80C until processing. Each hemisphere was
homogenized in TꢀPER tissue protein extraction buffer (Thermo Scientific, Rockford, IL) supplemented
with phosphatase inhibitor cocktail sets I and II (SigmaꢀAldrich, St. Louis, MO), complete protease
inhibitor EDTAꢀfree (Roche, Indianapolis, IN) 7uM pepstatin (SigmaꢀAldrich, St. Louis, MO) and
1mM PMSF (Sigma, St. Louis, MO) prakasamusing a PotterꢀElvehjem homogenizer. Homogenate was
centrifuged at 100,000 x g for 30 min at 4C. Cleared homogenate was frozen and stored at ꢀ80C.
Commercial total Tau human (KHB0041) and Tau[S396] human (KHB7031) ELISA kits (Invitrogen,
Grand Island, NY) were used as directed to determine total and S396 phosphorylated human Tau levels
in the cleared homogenates. Each sample was tested in triplicate and compound treated animal groups
were compared to vehicle treated animal groups with statistical analysis using student t test.
ACKNOWLEDGMENT We thank Spectrix chemists for all the final compound purification and
characterizations.
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Journal of Medicinal Chemistry
SUPPORTING INFORMATION AVAILABLE Synthesis and characterizations of the
aminopyridine intermediates from Scheme 2; Xꢀray crystal studies of 15 and 18 bound to GSK3β,
biological assays, and related Ambit graphs. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding author: Phone: 203ꢀ677ꢀ6640. Fax: 203ꢀ677ꢀ7702. Eꢀ
mail: guanglin.luo@bms.com.
ABBREVIATIONS USED
GSKꢀ3, glycogen synthase kinaseꢀ3; AD, Alzheimer’s disease; Aß, betaꢀamyloid; NFTs,
neurofibrillary tangles; PKB(AKT), protein kinase B; PHFs, paired helical filaments; b/p ratio,
brain/plasma ratio; pTau, phosphorylated Tau; Pgp, Pꢀglycoprotein; HPLC, high pressure liquid
chromatography; LCMS, liquid chromatography mass spectrometry; FCC, flash column
chromatography; NMR, muclear magnetic resonance; pTau, phosphorylated Tau; SAR, structureꢀ
activity relationship; po, taken orally; ip, intraperitoneal.
REFERENCES
(1) 2013 Alzheimer’s Disease Facts and Figures. Alzheimer’s & Dementia 2013, 9, 208ꢀ245.
(2) Hardy, J. Alzheimer's disease: the amyloid cascade hypothesis: an update and reappraisal. J.
Alzheimer’s Dis. 2006, 9, 151ꢀ153.
(3) Zaghi, J.; Goldenson, B.; Inayathullah, M.; Lossinsky, A. S.; Masoumi, A.; Avagyan, H.;
Mahanian, M.; Bernas, M.; Weinand, M.; Rosenthal, M. J.; EspinosaꢀJeffrey, A.; de Vellis, J.; Teplow,
D. B.; Fiala, M. Alzheimer disease macrophages shuttle amyloidꢀbeta from neurons to vessels,
contributing to amyloid angiopathy. Acta Neuropathol. 2009, 117, 111ꢀ124.
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