Discovery of novel potent and highly selective glycogen synthase kinase-3β (GSK3β) inhibitors for Alzheimers Disease: Design, synthesis, and characterization of pyrazines

Article abstract of DOI:10.1021/jm201724m

Glycogen synthase kinase-3β, also called tau phosphorylating kinase, is a proline-directed serine/threonine kinase which was originally identified due to its role in glycogen metabolism. Active forms of GSK3β localize to pretangle pathology including dystrophic neuritis and neurofibrillary tangles in Alzheimers disease (AD) brain. By using a high throughput screening (HTS) approach to search for new chemical series and cocrystallization of key analogues to guide the optimization and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and bloo-brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy for Alzheimers disease.

Full text of DOI:10.1021/jm201724m

Article
pubs.acs.org/jmc
Discovery of Novel Potent and Highly Selective Glycogen
Synthase Kinase-3β (GSK3β) Inhibitors for Alzheimer’s Disease:
Design, Synthesis, and Characterization of Pyrazines
Stefan Berg,*,† Margareta Bergh,† Sven Hellberg,† Katharina Hogdin,† Yvonne Lo-Alfredsson,†
̈
Peter Soderman,† Stefan von Berg,† Tatjana Weigelt,† Mats Ormo,‡ Yafeng Xue,‡ Julie Tucker,§
̈
̈
Jan Neelissen,∥ Eva Jerning,⊥ Yvonne Nilsson,# and Ratan Bhat▽
†Department of Medicinal Chemistry, AstraZeneca R&D, Innovative Medicines CNS & Pain Sodertalje, SE-151 85 Sodertalje,
̈
̈
̈
̈
Sweden
‡Discovery Sciences, AstraZeneca R&D Molndal, SE-43183 Molndal, Sweden
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§Discovery Sciences, AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.
∥Department of DMPK, ⊥Department of Neuroscience, AstraZeneca R&D, Innovative Medicines CNS & Pain Sodertalje, SE-151 85
̈
̈
Sodertalje, Sweden
̈
̈
#Department of Translational Sciences, AstraZeneca R&D Sodertalje, SE-151 85 Sodertalje, Sweden
▽Strategy, AstraZeneca R&D, Innovative Medicines CNS & Pain Sodertalje, SE-151 85 Sodertalje, Sweden
̈
̈
̈
̈
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̈
̈
S
* Supporting Information
ABSTRACT: Glycogen synthase kinase-3β, also called tau phos-
phorylating kinase, is a proline-directed serine/threonine kinase
which was originally identified due to its role in glycogen meta-
bolism. Active forms of GSK3β localize to pretangle pathology
including dystrophic neuritis and neurofibrillary tangles in
Alzheimer’s disease (AD) brain. By using a high throughput
screening (HTS) approach to search for new chemical series
and cocrystallization of key analogues to guide the optimiza-
tion and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and
blood−brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy
for Alzheimer’s disease.
Increased levels of total GSK3β have not been consistently
observed in AD brain, however, active forms of GSK3β localize
to pretangle pathology3 including dystrophic neuritis and neuro-
fibrillary tangles (NFT) in AD brain. Neurons undergoing
granulovascular degeneration4 also contain active GSK3β. A
spatial and temporal pattern of increased GSK3β expression
coinciding with the progression of NFT and neurodegeneration
has also been observed. Taken together, these studies suggest
that there is an increase in the active form of GSK3β in
degenerating neurons and pretangles. In preclinical studies,
abnormal increased activity of GSK3β has been shown to be
associated with a multitude of adverse events linked to micro-
tubule dysfunction, neuritic dystrophy and cytoskeletal damage,
cognitive deficits, and amyloid production.5 Overexpression of
GSK3β in the brain of conditional transgenic mice resulted in
tau hyperphosphorylation and somatodendritic localization
of tau.6 Given the significant role of GSK3β in a variety of
INTRODUCTION
■
Alzheimer’s disease1 is a chronic neurodegenerative disorder
affecting more than 25 million people worldwide. The disease is
characterized by progressive cognitive decline such as loss of
memory and orientation capability. Two major pathological
hallmarks are observed in the AD brain. These are extracellular
amyloid deposits,1 consisting primarily of β-amyloid which is
produced by proteolytic cleavage of the amyloid precursor
protein, and intracellular neurofibrillary tangles,2 primarily
composed of hyperphosphorylated tau protein.
Glycogen synthase kinase-3β, also called tau phosphorylating
kinase, is a proline-directed serine/threonine kinase which was
originally identified due to its role in glycogen metabolism.
GSK3β is abundant in the brain where it is localized primarily
in neurons. GSK3β phosphorylates tau, a microtubule associ-
ated protein, at sites which are abnormally hyperphosphory-
lated in AD. This leads to detachment of tau from the micro-
tubules. The detached tau protein accumulates and aggregates
in the somatosensory compartment of neurons, leading to the
formation of paired helical filaments and subsequently neuro-
fibrillary tangles and neuronal death.
Special Issue: Alzheimer's Disease
Received: December 21, 2011
Published: April 10, 2012
© 2012 American Chemical Society
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effects linked to mechanism in AD and other central nervous
system (CNS) disorders, GSK3β inhibition using small-
molecule inhibitors for treating particular disease states will
be worth testing in the clinic.
Many research groups7 have reported diverse GSK3β
inhibitors over the last 10 years, but few have been potent
and selective. Paullones were an early compound class where
compound 1 (Figure 1, alsterpaullone8,9) was shown to act as
Figure 2. Compound 5.
To investigate the structure−activity relationships and
understand the selectivity vs CDK2 (a kinase with close
homology within the ATP binding pocket) and other kinases,
we synthesized a number of pyrazine analogues.
The synthesis of the kinase inhibitors is outlined in Schemes
1−7. The preparation of sulphonamides 8−28 is outlined in
Scheme 1. Commercially available bromophenyl sulfonyl
chlorides were treated with the appropriate amine to give the
bromophenyl sulfonamides 6a,b,f−i,k−p. The synthesis of
bromides 6c−e is outlined in Scheme 2. Compounds 8−17
were prepared in a one-pot reaction from the corresponding
bromides 6a−j via the in situ formed boronic acids and Suzuki
reaction with the 6-bromopyrazines (33, 34a−b, and 35a−b,
Scheme 3). The boronic acids 7a−g were isolated with an
acidic workup and subjected to Suzuki reactions to give
compounds 18−28. Data for compounds 8−28 are presented
in Tables 1−3.
Figure 1. Reported GSK3 inhibitors.
Bromides 6c−e were synthesized as described in Scheme 2.
The precursor anilines 30a−c were prepared by treating
the appropriate sulfonyl halide with 1-methylpiperazine. The
sulfonyl halides were either commercially available (29a)
or synthesized from either the sulfonic acid (29b) or the
aniline (29c). The sulfonic acid 29b was treated with thionyl
chloride, and the aniline 29c was subjected to direct sul-
fonylation using chlorosulfonic acid. The anilines 30a−c
were converted to the bromides 6c−e via a Sandmeyer
reaction by treating the formed diazonium salt with HBr and
CuBr.
The 6-bromo-pyrazines (33, 34a−b, and 35a−b) required
for the synthesis of compounds 8−28 were prepared from
methyl 3-amino-6-bromo-pyrazine-2-carboxylate13 or the com-
mercially available methyl 2-amino-5-bromobenzoate or methyl
2-amino-5-bromo-pyridine-3 - carboxylate and the correspond-
ing aniline (32a−c) as outlined in Scheme 3. Anilines (32a−c)
were prepared by reductive amination of Boc-protected 3-
aminopyridine-4-carbaldehyde with pyrrolidine or N,N-dime-
thylammonium hydrochloride using NaBH(OAc)3 (31a−b),
followed by deprotection of the Boc-group using trifluoroacetic
acid (TFA) in dichloromethane.
Analogues 37−38 were prepared in a one-pot reaction
(Scheme 4) first treating bromides 36a−b with n-BuLi and
B(OiPr)3 to form the corresponding boronic acid followed by a
Suzuki reaction with 3-amino-6-bromo-N-pyridin-3-ylpyrazine-
2-carboxamide (33). The intermediate bromides (36a−b) were
prepared by heating 4-bromobenzoic acid in refluxing thionyl
chloride to give 4-bromobenzoyl chloride followed by treat-
ment with the corresponding amine in DCM at 0 °C. Data for
compounds 37−38 are presented in Table 1.
Compound 41 was prepared (Scheme 5) by diazotation of
compound 18 followed by hydrolysis to give the pyrazinone 39.
This compound was treated with phosphoryl chloride to give
the chloride 40, which was reduced to 41 by heating with
Pd(PPh3)4 and NaCO2H in DMF at 100 °C. Data for com-
pounds 41 are presented in Table 2.
an adenosine triphosphate (ATP) competitive GSK3β
inhibitor, however, compound 1 was also found to inhibit
cyclin dependent kinase 2 (CDK2), a kinase with close
homology within the ATP binding pocket. Another early
cluster of compounds are the maleimide analogues where
compound 2 (SB-21676310) from SmithKline Beecham repre-
sents one of the first potent GSK3β inhibitors reported,
inhibiting GSK3β at an IC50 of 9 nM. We reported in 2003
the crystal structure of the highly selective ATP competi-
tive GSK3β inhibitor 3 (AR-A01441811) with an IC50 of
104 nM.
More recently, Takeda Pharmaceuticals12 has shown efficacy
in a transgenic mouse model with the GSK3β inhibitor 4 (6-
methyl-N-[3-[[3-(1-methylethoxy)propyl]carbamoyl]-1H-pyra-
zol-4-yl]pyridine-3-carboxamide), suggesting that a GSK3β
inhibitor can prevent tau pathology in the brain.
In this report, we describe the discovery, characterization,
and structure−activity relationship of a novel series of pyrazine
analogues as potent GSK3β inhibitors. We have used
cocrystallization of key pyrazine analogues to guide the
synthesis toward highly selective inhibitors which may serve
as a new treatment strategy for Alzheimer’s disease.
CHEMISTRY
■
By using a high throughput screening approach to search for
new chemical series for the GSK3β therapeutic target, we found
several chemically diverse series showing moderate to high
potency (nM−μM), These series were also found active after
retesting in the primary assay (GSK3β enzyme).
One of the series, the pyrazines, initially showed good
biochemical and physicochemical properties, and this initial
lead was used for further optimization. One of the early actives
from the high throughput screening campaign was compound 5
(Figure 2) in the pyrazine series, a GSK3β inhibitor which
showed acceptable potency (41 nM) when the HTS solution
was retested.
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a
Scheme 1
a
Reagents: (a) NHRR′, tetrahydrofuran (THF), dioxane, or dichloromethane (DCM); (b) n-BuLi, B(OiPr)3, THF, −78 °C; (c) (1) n-BuLi,
B(OiPr)3, THF, −78 °C, (2) 33, Pd(dppf)Cl2, Na2CO3 (aq); (d) 33, 34a−b, or 35a−b, Pd(dppf)Cl2, Na2CO3 (aq), toluene/EtOH, 1,2-
b
c
dimethoxymethane (DME), or THF. Position of R3 o-,m-, p- relative to bromine/pyrazine. For structures, see Tables 1−3.
a
Scheme 2
a
Reagents: (a) (1) 1-methylpiperazine, THF, rt to 60 °C, (2) HCl, 110 °C; (b) (1) SOCl2, (2) 1-methylpiperazine THF/DCM; (c) (1) HSO3Cl,
0−80 °C, (2) 1-methylpiperazine, THF; (d) NaNO2, HBr/H2O, and CuBr.
Compounds 44−46 were prepared (Scheme 6) by amidation
of intermediate 43 with 2-methoxyaniline, 4-methoxyaniline, or
1,2,4-thiadiazol-5-amine in the presence of HOBt, TBTU, and
DIPEA in DMF or using a mixture of DIPEA, HOBt, and
bromo-tris-pyrrolidinophosphoniumhexafluorophosphate (Py-
BroP) in DCM. Intermediate 43 was synthesized by a Suzuki
reaction of 4-[(4-methylpiperazin-1-yl)sulfonyl]phenylboronic
acid (7a) and methyl 3-amino-6-bromo-2-pyrazinecarboxylate
to give the methyl ester intermediate 42 that was subsequently
hydrolyzed with LiOH in a mixture of THF and H2O at 50 °C.
Data for compounds 44−46 are presented in Table 3.
Compounds 49−51 were synthesized by a Suzuki coupling
of 4-[(4-methylpiperazin-1-yl)sulfonyl]phenylboronic acid (7a)
with bromides 48a−c as outlined in Scheme 7. The inter-
mediate bromides 48a−c were prepared by amidation of 3-amino-
6-bromo-pyrazine-2-carboxylic acid (47) with 3-methoxyaniline,
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a
Scheme 3
a
Reagents: (a) pyrrolidine and HOAc or dimethylammonium hydrochloride, NaBH(OAc)3, DCM or 1,2-dichloroethane; (b) TFA, DCM;
(c) 3-aminopyridine, methyl 3-amino-6-bromo-2-pyrazinecarboxylate, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 70 °C; (d) 3-amino-6-bromopyrazine-
2-carboxylic acid or methyl 2-amino-5-bromo-pyridine-3-carboxylate, 1-hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N′,
N′-tetramethyluronium tetrafluoroborate (TBTU), diisopropylethylamine (DIPEA), CH3CN; (e) Et3Al, methyl-2-amino-5-bromobenzoate,
3-aminopyridine, DCM reflux; (f) N,N′-diisopropylcarbodiimide (DIPC), HOBt, N-methylmorpholine-N-oxide (NMO), N,N′-dimethylformamide
(DMF).
a
Scheme 4
a
Reagents: (a) (1) SOCl2, reflux, (2) 1-methylpiperazine or N1,N1,N2-trimethylethane-1,2-diamine, DCM, 0 °C; (b) (1) n-BuLi, B(OiPr)3, THF,
b
−78 °C, (2) 33, Pd(dppf)Cl2, Na2CO3 (aq), THF, 70 °C. For structures see Table 1.
2-methoxyethylamine, or 3-methoxypropylamine using either a
mixture of Et3N, HOBt and TBTU in a mixture of DMF and
CH3CN or HOBt, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDCI) in CH3CN. Data for compounds 49−
51 are presented in Table 3.
form hydrogen bonds to the backbone NH of Val135 and the
backbone carbonyl of Asp133 in the ATP site.
Furthermore, the carbonyl in the amide bond of compound
18 likely forms an internal hydrogen bond with the aniline
group on the pyrazine ring, thus directing the pyridine ring
toward the conserved Lys85 at the inner part of the ATP
pocket. This would thus facilitate the formation of a hydrogen
bond between the pyridine nitrogen and the terminal amino
group of Lys85.
The 1-methylpiperazine sulfonamide is directed out toward
the solvent (Figure 3B) ,adopting an upwards directed
conformation. In this conformation, the sulfonamide function-
ality directly faces the guanidine group of Arg141. The sulfon-
amide group occupies a subpocket that is formed by the salt
bridge between Glu137 and Arg141. The phenyl ring linking
the sulfonamide to the pyrazinyl core is slightly twisted relative
to the pyrazine ring (∼20−30° anticlockwise).
RESULTS AND DISCUSSION
■
To investigate the chemical space defining GSK3β potency and
kinase selectivity, three piperazinyl sulfonamide analogues 8, 9,
and 18 were designed and synthesized (Table 1). Potency for
the para-substituted sulfonamide analogue (18) increased 8-
fold (Ki 4.9 nM) compared to compound 5, whereas potency
for the meta (8) and ortho (9) isomers decreased to 79 and
300 nM, respectively. Many known GSK3β-inhibitors were also
potent inhibitors of CDK2, a proline directed serine/threonine
kinase showing high sequence similarity14 to GSK3β in the
ATP binding site, hence selectivity versus CDK2 was routinely
measured for all synthesized analogues in our GSK3β program.
The three piperazine sulfonamides showed >100-fold selectivity
versus CDK2. The solubility for 18 was found to be acceptable
(35 μM) in the solubility assay likely due to protonation of
the basic pyrazine nitrogen. We measured the permeability in
Caco2 cells and obtained a Papp for 18 of 14 × 10−6 cm/s,
indicating a putative good blood−brain barrier (BBB) pene-
trance and bioavailability for this compound.
Combining information from the potency data and the
crystal structure, we chose to fix the position of the large
solubilizing substituent, R3, in the para position of the phenyl
ring.
Exchanging the 1-methylpiperazine for a piperidine (20) or a
morpholine (22) increased the potency for GSK3β, resulting in
a Ki of 0.4 and 0.67 nM, respectively. This increase in potency
might be due to the removal of a potential repulsive interaction
between the positively charged side chain of Arg141 and the
basic pyrazine functionality in compound 18. This also resulted
in a selectivity increase vs CDK2 of up to 550-fold for
compound 20. When the ionizable nitrogen in the 6-membered
aliphatic ring was replaced with a carbon atom (compound 20),
the solubility dropped below 1 μM. This was also the case for
High resolution X-ray crystal structures of our lead pyrazine
analogues bound to the GSK3β protein were generated. The
structure confirmed the binding mode of the analogues in the
ATP pocket of GSK3β. The X-ray crystal structure of com-
pound 18 bound to GSK3β (Figure 3A) indicated that a
pyrazine nitrogen and one of the adjacent anilino hydrogens
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Table 1. GSK3β Inhibition, CDK2 Selectivity, Solubility, and Caco2 Permeability Data for Pyrazines with Modifications in the
a
Part of the Molecule Pointing out Towards the Solvent
a
Positions other than para substitution of R3 are indicated explicitly in the table by ortho or meta. * indicates point of attachment. Values are means
of at least two experiments performed in duplicates. In Caco2 assay, n = 1. nd denotes no data.
the 5-membered pyrrolidine analogue 19, for which the poten-
cy toward GSK3β and the selectivity against CDK2 were
acceptable.
most likely due to a reduction in planarity while permeability
likely increased due to an increase in lipophilicity.
To gain a deeper understanding of the selectivity against
CDK2, X-ray crystal structures for compound 23 bound to
GSK3β and CDK2 were generated. See Figure 4A,B.
Comparison of the sequences and structures of the ATP site
in CDK2 and GSK3β (Figure 5) reveals a difference in amino
acid sequence and protein structure at the phenyl subsite,
especially in the areas around Pro136/Glu137/Thr138 in
GSK3β (His84/Gln85/Asp86 in CDK2) and Ile62 in GSK3β
(Ile10 in CDK2).
Introduction of a small R1 substituent ortho to the pyrazine
ring resulted in an increase in GSK3β potency compared to
18. Compounds 10, 11, and 12 gave a 4−10-fold increase in
GSK3β potency and a further increase in selectivity against
CDK2 up to 3700-fold. The dimethyl analogue 13 showed the
highest selectivity against CDK2 (>20000-fold) achieved within
the pyrazine series. Solubility for this compound increased,
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a
Scheme 5
a
Reagents: (a) NaNO2, H3PO2, H2O, 50 °C; (b) POCl3, 140 °C; (c) Pd(PPh3)4, NaCO2H, DMF, 100 °C.
Table 2. GSK3β Inhibition, CDK2 Selectivity, Solubility, and Caco2 Permeability Data for Sulfonamide Pyrazines with Core
a
Modifications
compd
R8
X
Y
GSK3β Ki (nM)
CDK2 Ki (nM)
selectivity vs CDK2 (fold)
solubility (μM)
permeability Caco2 (10−6 cm/s)
27
28
41
NH2
NH2
H
C
N
N
C
C
N
120
64
52000
4900
433
77
50
4
nd
nd
25
370
>30000
>81
119
a
Values are means of at least two experiments performed in duplicates. In Caco2 assay, n = 1. nd Denotes no data.
a
Scheme 6
a
Reagents: (a) methyl 3-amino-6-bromo-2-pyrazinecarboxylate, Pd(dppf)Cl2, Na2CO3 (aq) or K4PO3 (aq), THF or dimethyl sulfoxide (DMSO)/
H2O or DME/H2O, 50−160 °C; (b) LiOH, THF/H2O, 50 °C; (c) 2-methoxyaniline or 4-methoxyaniline, HOBt, TBTU, DIPEA, DMF; (d) 1,2,4-
b
thiadiazol-5-amine, DIPEA, HOBt, PyBroP, DCM. For structures see Table 3.
The presence of Pro136 in GSK3β leads to a difference in
main chain conformations, making this area of the pocket in
GSK3β significantly larger (in the vertical direction) than in
CDK2 (areas indicated by yellow dashed circles in Figure 5).
We observed from the X-ray structures that the phenyl ring of
compound 23 is in a nonplanar arrangement with respect to the
pyrazine ring (dihedral angle of +21 in GSK3β and −13 in
CDK2). Thus, we concluded that the selectivity for GSK3β
may be a result of the GSK3β ATP site being able to leverage a
nonplanar binding mode and a concomitant increase in three-
dimensional bulk.
On the bases of the X-ray crystal structures, we
hypothesized that compounds with R1 substituents further
accentuate the nonplanarity, leading to the observed increase
in selectivity vs CDK2 (compounds 11−13 in Table 1).
Compounds with a substituent in the R2 position (14 R2 = F
and 15 R2 CH3) resulted in potencies and selectivities
comparable to compound 18.
In an attempt to reduce the size of our analogues, the
primary sulfonamide 21 was synthesized. This resulted both in
a drop in potency (12 nM) and selectivity (8-fold). In addition,
the permeability was reduced to below detection level, most
likely due to the introduction of two additional hydrogen bond
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Table 3. Inhibition, Selectivity, Solubility, and Permeability Data for Sulfonamide Pyrazines with Modifications in the Part of
a
the Molecule Pointing Towards the Conserved Salt Bridge
a
* indicates point of attachment. Values are means standard error of the mean (SEM) of at least two experiments performed in duplicates. In
Caco2 assay, n = 1. nd denotes no data.
a
Scheme 7
a
Reagents: (a) 3-methoxyaniline, Et3N, HOBt, TBTU, DMF/CH3CN; (b) 2-methoxyethylamine or HOBt, EDCI, CH3CN; (c) 3-
methoxypropylamine, HOBt, TBTU, DIPEA, DMF; (d) [4-[(4-methyl-1-piperazinyl)sulfonyl]phenyl]boronic acid (7a), Pd(dppf)Cl2, Na2CO3,
b
THF/H2O, MW 70 °C. For structures see Table 3.
donors. Introduction of an amino containing carbon chain on
the sulfonamide (23) substantially increased solubility
(>2500 μM). By introducing a second substituent on the
sulfonamide (ethyl, 24), the permeability was increased while
retaining potency and solubility. Compound 16, which lacks the
solubilizing amino functionality, gave a lower solubility. The
corresponding amide analogues showed comparable potency
and selectivity profiles to the sulfonamides. Compounds 37 and
38 showed a potency of 3.1 and 16 nM and a 600- and 250-fold
selectivity vs CDK2, respectively. Solubility was substantially
higher for the amides compared to the sulfonamides, however,
the permeability decreased for both compounds. Acceptable poten-
cy, selectivity, solubility, and permeability were also achieved with
the aminoalkyl ether 17 (Ki 18 nM).
nitrogen and the NH of the amide functionality. An alternative
explanation could be that the replacement of nitrogen with
carbon may have an electronic effect with an indirect impact on
the hydrogen bonding strength to the hinge region. Removal of
the second nitrogen resulted in the phenyl analogue 27 with an
Ki of 120 nM, a 24-fold decrease compared to compound 18.
When the amino group on the pyrazine was removed, com-
pound 41 R8 = H, the potency for GSK3β was reduced dra-
matically (Ki 370 nM). The solubility for the three compounds
varied between 4 and 119 μM.
The modifications in compound 27 and 41 reduced the
number of hydrogen bond interactions available to the back-
bone of the hinge region of GSK3β, and possibly also the hydro-
gen bonding strength, which explains the dramatic drop in
potency for the two compounds.
The chemical scope for the core pyrazine was also inves-
tigated. Compared to compound 18, the corresponding pyri-
dine analogue 28 showed a 12-fold drop in potency (Table 2).
This might be due to a conformational effect caused by loss of
the partial internal hydrogen bond between the pyrazine
Highly potent inhibitors were also found through the syn-
thesis of the 4-substituted benzyl aminopyridines 25 (Ki 0.22 nM)
and 26 (Ki 0.48), which show moderate to good selectivity vs
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Figure 5. X-ray crystal structure of compound 23 in the GSK3β ATP
site (blue surface) aligned with CDK2 ATP site (red surface).
with the polar pyrrolidine ring extending toward and partially
filling the ribose binding site, resulting in a 22-fold increase
in potency compared to compound 18. This site is occupied
by a water molecule in the crystal structure of compound 18.
The presence of the pyrrolidine ring has also resulted in
a slight upward movement of the phenyl ring and thus the
attached sulfonamide and methylpiperazine moieties (∼1 Å
at the sulfur) and an increase in the twist between the
pyrazine and the phenyl rings to about 40° (vs about 20°
for compound 18), resulting in a 580-fold CDK2 selectivity
(Figure 6B). This change led to better van der Waals
interaction to the side chain of Ile62 (part of the Gly-rich
loop). Furthermore, the “space-filling” effect of the
pyrrolidine ring in the ribose binding area (e.g., the van
der Waals interaction to the glycine-rich loop) may also
contribute to the increase in binding affinity compared
to compound 18. Potency was thus improved by interacting
with the ribose site. The alignment of compound 18 and
25 in the ATP site of GSK3β (Figure 6B) shows that,
although the pyridine nitrogen is in the same position
in both compounds, the solubilizing piperazine ring is
shifted slightly up and out toward the solvent area for
compound 25.
Figure 3. X-ray crystal structure of compound 18 in the GSK3β ATP
site. (A) Top view; (B) side view. Resolution 2.6 Å. Figures were
prepared using VIDA 4.0.3 from OpenEye Scientific Software, 9
Bisbee Court, Suite D, Santa Fe, NM 87508.
CDK2 (Table 3). Although permeability was relatively low, the
solubility was acceptable for 25.
Figure 6A shows the X-ray crystal structure of the
benzylpyrrolidine 25 in the ATP site of GSK3β. The overall
binding mode is the same as that observed for compound 18,
To investigate whether the 3-pyridine ring could be
replaced with a substituted phenyl ring, the ortho (44),
meta (49), and para (45) methoxy isomers were synthesized.
Figure 4. (A) X-ray crystal structure of compound 23 in the GSK3β ATP site, resolution 2.5 Å. (B) X-ray crystal structure of compound 23 in the
CDK2 ATP site, resolution 1.63 Å.
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Figure 6. (A) X-ray crystal structure of compound 25 in the GSK3β
ATP site. Resolution 2.6 Å. (B) Alignment of compound 18 (pink)
and 25 (blue) in the ATP site.
Figure 7. (A) X-ray crystal structure of compound 51 bound in the
GSK3β ATP site. Resolution 2.8 Å. (B) Superimposition of compound
18 (pink), 25 (blue), and 51 (green).
The ortho analogue, where the methoxy group likely extends
toward the ribose binding site, showed the highest potency
(12 nM) of the three analogues with 160-fold selectivity
against CDK2, however, both the permeability and solubility
were low. The meta and para analogues showed a 16- and
140-fold drop in potency compared to 18. Thus the
conclusion was that neither the meta nor the para methoxy
could reach Lys85 and form a productive hydrogen bond.
The 5-membered heteroaromatic thiadiazole analogue 46
showed a high potency of 0.99 nM, 44-fold selectivity against
CDK2, and good permeability. Finally, the corresponding
methoxy substituted aliphatic amides were made. Potency
and selectivity is observed to increase with the methoxy-
carbon chain length. The 2-methoxyethyl analogue 50 is
substantially less potent (90 nM) than the 3-methoxypropyl
analogue 51 (22 nM).
Initial docking studies suggested that the alkoxy mod-
ification could replace the pyridine of compound 18, with the
alkoxy function extending sufficiently to be within hydro-
gen bond distance of Lys85. The observed potency for the
alkoxy analogues appeared to support this docking hypoth-
esis. However, subsequent X-ray crystal structures showed
a different binding mode, leading us to reject our initial
hypothesis. The methoxypropyl substituent (51) instead
extends toward the ribose binding area of the GSK3β ATP
pocket (Figure 7A). The folded-back conformation of
the methoxypropyl group resembles the pyrrolidine group
in compound 25 (Figure 7B), i.e., “space-filling” the ribose
pocket and also stabilizing the twist between the pyrazine and
the phenyl rings of the ligand (similar twisting of ∼40° is
observed for both compound 25 and 51). This interaction
apparently compensates, at least partly, for loss of the
hydrogen bond to Lys85 via the 3-pyridine in compound 18
and 25.
Inhibition of Tau Phosphorylation. To evaluate whether
analogues from the pyrazine series inhibit GSK3β-mediated tau
phosphorylation in cells, 3T3 fibroblasts were engineered to stably
express four-repeat tau protein. Transfected cells were treated with
vehicle (0.1% Me2SO) or with increasing concentrations (100 nM
to 50 μM) of compounds 11, 12, 14, 16, 18, 19, 22, 23, and 25
and harvested 4 h after treatment. The effect on tau phos-
phorylation in the presence or absence of serum was determined
by Western blotting. Detection was carried out using a phos-
phospecific antibody, which detects an epitope on tau (Ser(P)-
396), a site which is specifically phosphorylated by GSK3β in cells.
Total levels of tau in the samples were determined by stripping the
blot and reprobing with a phosphorylation independent antibody
to total tau protein (Tau5). The bands were quantified by
densitometric analysis. The levels of Ser(P)-396 were not affected
by changes in serum. Tested compounds inhibit tau phosphor-
ylation in the transfected cells in a dose-dependent fashion,
exhibiting IC50s ranging from 5 to 87 nM, as shown in Table 4.
Pan-Kinase Selectivity. Compound 18 was evaluated for
pan-kinase selectivity (10 μM) at the University of Dundee.
The compound shows a good overall selectivity versus 26
kinases (Figure 8). The CDK2 Ki was determined in-house and
was shown to be 510 nM, corresponding to a 110 fold
selectivity versus CDK2
Blood−Brain Barrier Permeability. To investigate
whether our compounds were suitable for in vivo testing of
GSK3β inhibition in the brain, compound 18 and its analogues
14, 16, and 22 were tested for blood−brain barrier permeability
in a bovine endothelial cell assay.15 All four compounds showed
a good permeability in this assay, indicating that a consider-
able total brain exposure can be expected when dosed in vivo
(Table 5).
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a
Table 4. Inhibition of Tau Phosphorylation in 3T3 Cells
Table 5. Blood−Brain Barrier (BBB) Permeability Data in
a
Bovine Endothelial Cells
a
* indicates point of attachment. n = 1.
CONCLUSIONS
■
Inhibition of tau kinases may have therapeutic effects on the
progression of chronic degenerative diseases such as Alzheimer's
and other tauopathies. In this report, we have described a new
structural class of GSK3β inhibitors. The pyrazine structural class
has generated several highly potent and selective inhibitors
showing good solubility and permeability in both the Caco2 and
BBB assays and thus predicting good bioavailability and brain
penetrance. Several pyrazine analogues are suitable for testing of
inhibition of tau phosphorylation in the brain, and the inhibitors
have potential as novel therapeutic agents for the treatment of
neurodegenerative diseases.
a
* indicates point of attachment. Values are means
SEM of
at least two experiments performed in duplicates. nd denotes no
data.
EXPERIMENTAL PROCEDURES
■
Synthesis details for key compounds. All solvents used were com-
mercially available and were used without further purification. Starting
materials used were either available from commercial sources or
prepared according to literature procedures.
1
The H and 13C nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker 400 at 400 and 100 MHz, or on a Varian Gemini
300 spectrometer operating at 300 and 75 MHz or on a Bruker Avance
III 500 spectrometer operating at 500 and 126 MHz, respectively.
Spectra were recorded on a NMR spectrometer fitted with a probe of
suitable configuration. Spectra were recorded at ambient temperature
unless otherwise stated. Chemical shifts are given in ppm down- and
upfield from trimethylsilane (TMS) (0.00 ppm). The following
reference signals were used: TMS δ 0.00, or the residual solvent signal
of DMSO-d6 δ 2.50 (1H), δ 39.51 (13C), CD3OD δ 3.30 (1H), δ 49.00
(13C), CDCl3 δ 7.26 (1H), δ 77.16 (13C), CD3CN δ 1.94 (1H), δ
118.26 (13C), D2O δ 4.79 (1H), or TFA δ 11.50 (1H), δ 164.2 (13C),
(unless otherwise indicated). Resonance multiplicities are denoted s, d,
t, q, m, br, and app for singlet, doublet, triplet, quartet, multiplet, broad,
and apparent, respectively.
The analytical purity and the mass spectra (MS) for the test
compounds were recorded utilizing thermospray (TS) (Finnigan MAT
SSQ 7000, buffer: 50 nM NH4OAc in CH3CN:H2O; 3:7), electron
impact (EI) (Finnigan MAT SSQ 710) or electro spray (ES) (LC-MS;
LC: Waters 2790, column XTerra MS C8 2.5 μm 2.1 mm × 30 mm,
buffer gradient H2O + 0.1%TFA:CH3CN + 0.04%TFA; MS:
micromass ZMD) ionization techniques.
Figure 8. Kinase activity at 10 μM. Effect of compound 18 on the
activities of 26 protein kinases in vitro. Protein kinases were assayed
in the presence of 10 μM compound 18 or vehicle (DMSO). The
enzymatic activity was measured in the presence of 0.1 mM ATP.
Kinase activities are given as the mean of triplicate determinations.
AMPK, AMP-activated protein kinase; Chk, checkpoint kinase;
CKII, casein kinase-2; JNK, c-Jun N-terminal kinase; Lck,
lymphocyte c-Src kinase; MAPK, mitogen-activated protein kinase;
MAPKAPK-2, mitogen activated protein kinase-activated protein
kinase-2; MEK1, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase-1; MSK1, mitogen- and stress-
activated protein kinase-1; p70 S6K, p70 ribosomal protein S6
kinase; PDK1, 3-phosphoinositide-dependent protein kinase-1;
PhosK, phosphorylase kinase; PKA, protein kinase A; PKBa, protein
kinase B; PKCa, protein kinase C; PRAK, p38-regulated/activated
kinase; ROCKII, Rho-dependent protein kinase II; SAPK2a, stress-
activated protein kinase-2a; SAPK2b, stress-activated protein kinase-
2b; SAPK3, stress-activated protein kinase-3; SAPK4, stress-
activated protein kinase-4; SGK, serum- and glucocorticoid-induced
kinase; CSK, carboxyl-terminal Src kinase; DYRK1A, dual-specificity
tyrosine phosphorylation-regulated kinase; CK1, casein kinase-2;
RSK1, ribosomal S6 kinase-1.
Preparative high-performance liquid chromatography (HPLC) was
run on a Waters autopurifier HPLC with a diode array detector.
Column: Xterra MS C8, 19 mm × 300 mm, 10 μm. Narrow gradients
with MeCN/water were used at a flow rate of 20 mL/min.
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High resolution mass spectra (HRMS) were recorded on a
Micromass Q-Tof (Waters MS Technologies, Manchester, UK) equip-
ped with a LockSpray source and connected to an Acquity ultra
performance liquid chromatography (UPLC) system with a photo-
diode array detectors (PDA) detector (Waters Corp., Milford, USA).
All analyses were acquired using positive mode electrospray ionization
(ESI+) in full scan, and leucine enkephalin (Sigma) was used as
the lock mass (m/z 556.2771) at a concentration of 0.9 pmol/μL and
a flow rate of 150 μL/min with a 1:10 split, ion source:waste, or a
flow rate of 10 μL/min. Cone Voltage was set to 54 to achieve
approximately 200 counts for leucine enkephalin. Nitrogen was used as
the nebulizing and desolvation gas, with the source operated at 120 °C
and the desolvation gas at 300 °C. Capillary voltage was 3000 V, cone
voltage 30 V, collision energy 5 V. Argon was used as the collision gas.
Chromatographic separation was achieved with either a 2.3 min
gradient from 5 to 95% acetonitrile (11 mM formic acid, 1 mM NH4+
formate) over an ACQUITY UPLC BEH C18 1.7 μm, 2.1 mm ×
50 mm column (Waters) maintained at 50 °C and run at a flow rate of
0.85 mL/min with a 1:5 split, ion source:waste, or a 2 min linear
gradient from 100% A (A: 10 mM HCOOH) to 100% B (B: MeCN)
at a flow rate of 0.4 mL/min with a 1:4 split prior to the ion source.
Analytes were diluted in MeOH or DMSO to a suitable concentration
for the LC-MS analysis, generally 10 μM, and 0.1 μL were injected.
Accurate masses of all analytes were obtained from the protonated
molecule [M + H]+ and were within 5 ppm mass error (difference
between observed protonated mass and calculated protonated mass).
Microwave heating was performed in a Creator or Smith Syn-
thesizer single-mode microwave cavity producing continuous irradi-
ation at 2450 MHz at the indicated temperature in the recommended
microwave tubes.
gradient of ethyl acetate/heptane, (1:100), to ethyl acetate/methanol
(10:1). Hydrochloric acid in diethyl ether (1 M, 5 mL) was added to a
solution of the base in a mixture of dichloromethane and methanol (3:1,
3 mL). The formed precipitate was collected by filtration, washed with
diethyl ether, and dried in vacuo to give 90 mg (48% yield) of the title
compound. 1H NMR (400 MHz, D2O) δ 9.40 (m, 1H), 8.59 (m, 1H),
8.55 (m, 1H), 8.36 (s, 1H), 8.03 (ddd, J = 9, 6, 1 Hz, 1H), 7.78 (s, 1H),
7.50 (s, 1H), 3.90 (d, J = 12 Hz, 2H), 3.60 (d, J = 11 Hz, 2H), 3.18 (m,
4H), 2.93 (s, 3H), 2.52 (s, 3H), 2.38 (s, 3H).
3-Amino-6-[4-[[(2-methoxyethyl)amino]sulfonyl]phenyl]-N-
3-pyridinyl-2-pyrazinecarboxamide (16). Compound 16 was
prepared in 33% yield from 4-bromo-N-(2-methoxyethyl)-
benzenesulfonamide17 (6i) using a similar procedure to that of 13.
Purification was by column chromatography using dichloromethane/
methanol (98:2), followed by a second column chromatography using
1
n-heptane/ethyl acetate (1:1) followed by ethyl acetate (100%). H
NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.03 (s, 1H), 8.99 (d,
1H), 8.42−8.50 (m, 2H), 8.38 (dd, 1H), 8.18−8.26 (m, 1H), 7.88 (d,
2H), 7.75−7.86 (m, 2H), 7.45 (dd, 1H), 3.18 (s, 3H), 2.94 (q, 2H),
(2H under the water peak).
3-Amino-6-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}-N-
pyridin-3-ylpyrazine-2-carboxamide Hydrochloride (18). A
suspension of 4-[(4-methylpiperazin-1-yl)sulfonyl]phenylboronic
acid18 (0.199 g, 0.70 mmol), 33 (0.200 g, 0.68 mmol), and
Pd(dppf)Cl2 × CH2Cl2 (0.029 g, 0.036 mmol) in a mixture of
toluene (10 mL), ethanol (2 mL), and Na2CO3 (2 M, 1 mL) was
heated at 80 °C overnight. Silica was added, and the solvent was
evaporated. The product was purified by column chromatography
using dichlorometane/methanol (93:7) to give 0.244 g (79% yield) of
1
the base as a pale-yellow solid; mp 220−229 °C (decomp). H NMR
(400 MHz, DMSO-d6) δ 9.85 (br s, 1H), 8.88 (br s, 1H), 8.75 (s, 1H),
8.45 (d, J = 4 Hz, 1H), 8.30 (m, 1H), 8.07 (d, J = 8 Hz, 2H), 7.88 (d,
J = 8 Hz, 2H), 7.37 (dd, J = 8, 5 Hz, 1H), 3.37 (m, 4H), 2.92 (m, 4H),
2.56 (m, 3H). Hydrochloric acid in diethyl ether (1 M, 0.81 mL) was
added to a solution of 3-amino-6-{4-[(4-methylpiperazin-1-yl)-
sulfonyl]phenyl}-N-pyridin-3-ylpyrazine-2-carboxamide (0.096 g,
0.21 mmol) in a mixture of dichloromethane and methanol
(0.95:0.05, 8 mL). The yellow precipitate was removed by filtration,
washed with diethyl ether, and dried in vacuo to give the title
compound as a yellow solid; mp 217−223 °C (decomp).
Thin layer chromatography (TLC) was performed on Merck TLC-
plates (silica gel 60 F254) and UV visualized the spots. Column chro-
matography was performed using Merck silica gel 60 (0.040−0.063 mm)
unless otherwise specified.
The purity of the test compounds was greater than 95% unless
specified otherwise.
1-[(4-Bromo-2,5-dimethylphenyl)sulfonyl]-4-methylpipera-
zine16 (6f). 2-Bromo-1,4-dimethylbenzene, (2.5 g, 13.5 mmol) was
cooled to 0 °C, and chlorosulfonic acid (2.2 mL, 33 mmol) was added
slowly. The reaction mixture was stirred at 0 °C for 15 min at room
temperature for 10 min and subsequently heated at 80 °C for 3 h. The
reaction mixture was cooled to room temperature and slowly added to an
ice/water mixture. The formed precipitate was dissolved in a mixture of
dichloromethane and tetrahydrofuran (10:1) and washed with saturated
sodium bicarbonate (aq). The organic phase was dried over sodium sulfate
and filtered, and the solvent was removed in vacuo to give 1.3 g of the
crude sulfonyl chloride that was dissolved in tetrahydrofuran (20 mL) and
cooled to 0 °C. 1-Methylpiperazine (2.5 mL, 22.6 mmol) was added, and
stirring was continued for 30 min at room temperature. Saturated sodium
hydrogencarbonate (aq 20 mL) was added, and the mixture was extracted
with dichloromethane. The combined organic phases were dried over
sodium sulfate and filtered, and the solvent was removed in vacuo. The
resulting residue was purified by column chromatography using a gradient
of methanol in ethyl acetate (0−50%) as the eluent to give 1.50 g (32%
yield) of the title compound. 1H NMR (400 MHz, DMSO-d6) δ 7.71 (m,
2H), 3.02 (m, 4H), 2.48 (s, 3H), 2.37 (s, 3H), 2.32 (m, 4H), 2.14 (s, 3H).
3-Amino-6-{2,5-dimethyl-4-[(4-methylpiperazin-1-yl)-
sulfonyl]phenyl}-N-pyridin-3-ylpyrazine-2-carboxamide Hy-
drochloride (13). Triisopropylborate (0.40 mL, 1.73 mmol) was
added to a solution of 1-[(4-bromo-2,5-dimethylphenyl)sulfonyl]-4-
methylpiperazine16 (6f) (0.200 g, 0.58 mmol) in anhydrous
tetrahydrofuran (10 mL) at −78 °C under an atmosphere of nitrogen
followed by dropwise addition of n-butyllithium (1.2 mL, 1.80 mmol,
1.5 M) over 30 min. The resulting mixture was stirred at −78 °C for
1 h, hydrochloric acid (1 M, 1.8 mL, 1.8 mmol) was added, and the
reaction mixture was allowed to warm to room temperature. Sodium
carbonate (0.382 g, 3.6 mmol) was added, followed by addition of 33
(0.40 g, 0.14 mmol) and Pd(dppf)Cl2 (40 mg, 0.033 mmol). The resulting
mixture was heated at 70 °C overnight. Silica was added, the solvent was
evaporated, and the residue was purified by chromatography using a
3-Amino-6-[4-(morpholin-4-ylsulfonyl)phenyl]-N-(pyridin-3-
yl)pyrazine-2-carboxamide (22). Compound 22 was prepared in
18% yield from 4-(morpholin-4-ylsulfonyl)phenylboronic acid18 (7e)
using a similar procedure to that of 18. Purification was by column
chromatography using dichloromethane/methanol (100:1). HRMS:
measured m/z [M + H]+ = 441.1342 (theoretical, 441.1345).
3-Amino-6-[4-({[2-(dimethylamino)ethyl]amino}sulfonyl)-
phenyl]-N-pyridin-3-ylpyrazine-2-carboxamide hydrochloride
(23). Compound 23 was prepared in 52% yield from (4-{[2-
(dimethylamino)ethyl]sulfamoyl}phenyl)boronic acid18 (7f) using a
similar procedure to that of 18. 1H NMR (400 MHz, D2O) δ 8.90 (s,
1H), 8.25 (m, 2H), 8.08 (s, 1H), 7.66 (m, 1H), 7.56 (d, 2H), 7.31 (d,
2H), 3.07 (m, 8H), 1.11 (t, 6H).
3-Amino-6-{4-[(4-methylpiperazin-1-yl)sulfonyl]phenyl}-N-
[4-(pyrrolidin-1-ylmethyl)pyridin-3-yl]pyrazine-2-carboxamide
Hydrochloride (25). Compound 25 was prepared in 23% yield from
4-[(4-methylpiperazin-1-yl)sulfonyl]phenylboronic acid18 and 34a using a
similar procedure to that of 18. Purification was by column chromato-
graphy using a gradient of ethyl acetate to ethyl acetate/methanol (1:1).
1H NMR (400 MHz, D2O) δ 9.01 (s, 1H), 8.79 (d, J = 6 Hz, 1H), 8.75
(m, 1H), 8.15 (m, 3H), 7.83 (d, J = 9 Hz, 2H), 4.68 (s, 2H), 3.90 (d, J =
14 Hz, 2H), 3.56 (d, J = 12 Hz, 2H), 3.39 (m, 4H), 3.20 (t, J = 12 Hz,
2H), 2.85 (s, 3H), 2.79 (m, 2H), 2.03 (m, 4H).
tert-Butyl 4-(Pyrrolidin-1-ylmethyl)pyridin-3-ylcarbamate16
(31a). tert-Butyl 4-formylpyridin-3-ylcarbamate19 (1.03 g, 4.64 mmol)
was dissolved in 1,2-dichloroethane (20 mL) under an atmo-
sphere of nitrogen. Pyrrolidine (0.41 mL, 4.9 mmol) and acetic acid
(0.27 mL, 4.72 mmol) were added, and the reaction mixture was
stirred for 1 h. Sodium triacetoxyborohydride (1.27 g, 6 mmol) was
added, and stirring was continued for 10 h. Sodium hydroxide (1 M,
5 mL, 5 mmol) was added, and the phases were separated. The aqueous
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phase was extracted with dichloromethane, the combined organic phases
were dried over sodium sulfate, and the solvent was evaporated. The
product was purified by column chromatography using a gradient of
methanol in dichloromethane (100:2−100:10) to give 900 mg (70%
yield) of the title compound as an oil. 1H NMR (400 MHz, CDCl3) δ
9.83 (s, 1H), 9.21 (s, 1H), 8.16 (d, J = 5 Hz, 1H), 6.96 (d, J = 5 Hz,
1H), 3.66 (s, 2H), 2.49 (m, 4H), 1.81 (m, 4H), 1.52 (s, 9 H).
3-Amino-6-bromo-N-(3-methoxypropyl)pyrazine-2-carbox-
amide (48c). 3-Methoxy-propylamine (2.1 mL, 20.5 mmol) was
added to a mixture of 2-amino-5-bromonicotinic acid (3.0 g, 10.0 mmol),
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate (4.42 g,13.8 mmol), 1-hydroxybenzotriazole hydrate (2.54 g,
16.6 mmol), and N-ethyl-N,N-diisopropylamine (4.8 mL, 27.6 mmol)
in anhydrous N,N-dimethylformamide (25 mL). The reaction
mixture was stirred overnight. The solvent was evaporated, and the
product was purified by column chromatography using dichloro-
methane/methanol (95:5) as the eluent to give a 3.52 g (42%
4-(Pyrrolidin-1-ylmethyl)pyridin-3-amine16 (32a). Trifluoro-
acetic acid (3 mL, 39 mmol) was added to a stirred solution of 31a
(1.0 g, 3.6 mmol) in dichloromethane (20 mL), and the resulting
mixture was stirred for 30 min. The solvent was removed in vacuo.
Ethyl acetate (5 mL) was added and removed in vacuo, and this
procedure was repeated three times. The residue was dissolved in
methanol (50 mL), and DOWEX-OH was added until the methanolic
solution was basic. Filtration and removal of the solvent in vacuo gave
1
yield) of the title compound. H NMR (400 MHz, DMSO-d6) δ
8.64 (m, 1H), 8.33 (s, 1H), 7.70 (br s, 2H), 3.37 (t, J = 6 Hz, 2H),
3.31 (t, J = 7 Hz, 2H), 3.24 (s, 3H), 1.75 (quin, J = 7 Hz, 2H).
3-Amino-N-(3-methoxypropyl)-6-{4-[(4-methylpiperazin-1-
yl)sulfonyl]phenyl}pyrazine-2-carboxamide Hydrochloride
(51). Compound 51 was prepared as a yellow solid in 19% yield
from 1-[(4-bromophenyl)sulfonyl]-4-methylpiperazine21 and 48c
using a similar procedure to that of 13. Purification was by column
chromatography using a gradient of dichloromethane/methanol/
triethyl amine (9:1:0.1) in methanol (0−100%). 1H NMR (400
MHz, DMSO-d6) δ 11.02 (br s, 1H), 8.97 (s, 2H), 8.46 (d, J = 9 Hz,
2H), 7.82 (d, J = 9 Hz, 2H), 3.82 (d, J = 13 Hz, 2H), 3.41 (m, 6H),
3.27 (s, 3H), 3.15 (m, 2H), 2.72 (m, 5H), 1.81 (m, 2H).
1
0.573 mg (90% yield) of the title compound. H NMR (400 MHz,
CD3OD) δ 7.92 (s, 1H), 7.75 (d, J = 5 Hz, 1H), 7.05 (d, J = 5 Hz,
1H), 3.61 (s, 2H), 2.49 (m, 4H), 1.79 (m, 4H).
3-Amino-6-bromo-N-pyridin-3-ylpyrazine-2-carboxamide16
(33). Methyl 3-amino-6-bromo-2-pyrazinecarboxylate20 (1.0 g,
4.3 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (645 μL, 4.3 mmol)
were added at 70 °C to 3-aminopyridine (10 g, 106 mmol), and the
resulting mixture was stirred for 4 h. The reaction mixture was diluted
with water (75 mL) and extracted with dichloromethane. The combined
organic phases were washed with aqueous saturated ammonium chloride
and dried over magnesium sulfate, and the solvent was evaporated in
vacuo. The product was purified by column chromatography using
dichloromehane/ethanol (9:1) to give 750 mg (59% yield) of the title
compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.50 (br s,
1H), 8.82 (d, J = 3 Hz, 1H), 8.43 (dd, J = 5 and 1 Hz, 1H), 8.31 (s, 1H),
8.23 (ddd, J = 8, 3, 2 Hz, 1H), 7.34 (dd, J = 8, 5 Hz, 1H).
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis details for nonkey compounds, GSK3β and CDK2 X-
ray data collection and refinement statistics, protein expression
and purification of GSK3β and CDK2 used for crystallization,
crystallization of GSK3β and CDK2, data collection and
structure determination. This material is available free of charge
via the Internet at http://pubs.acs.org.
3-Amino-6-bromo-N-[4-(pyrrolidin-1-ylmethyl)pyridin-3-yl]-
pyrazine-2-carboxamide.16 (34a). 3-Amino-6-bromopyrazine-2-
carboxylic acid20 (148 mg, 0.68 mmol), 32a (100 mg, 0.56 mmol),
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate
(288 mg, 0.89 mmol), 1-hydroxybenzotriazole hydrate (118 mg,
0.87 mmol), and N,N-diisopropylethylamine (0.2 mL, 1.15 mmol)
were suspended in anhydrous acetonitrile (8 mL) and stirred under an
atmosphere of nitrogen at room temperature for 12 h. The solvent
was removed in vacuo, and the residue was partitioned between
dichloromethane and saturated aqueous sodium bicarbonate. The
organic phase was dried over sodium sulfate, and the solvent was
evaporated in vacuo. The product was purified by column chro-
matography using a gradient of ethyl acetate in n-heptane (1:1−4:1) to
give 210 mg (98% yield) of the title compound as a light-brown solid.
1H NMR (400 MHz, DMSO-d6) δ 11.97 (s, 1H), 9.41 (s, 1H), 8.46 (s,
Accession Codes
PDB ID GSK3β: compound 18, 4acd; compound 23, 4acc;
compound 25, 4acg; compound 51, 4ach. PDB ID CDK2:
compound 23, 4acm.
AUTHOR INFORMATION
Corresponding Author
*Phone: +46855326000. E-mail: Stefan.berg@astrazeneca.com.
■
Notes
The authors declare no competing financial interest.
1H), 8.30 (d, J = 5 Hz, 1H), 7.84 (br s, 2H), 7.34 (d, J = 5 Hz, 1H),
3.77 (s, 2H), 2.57 (m, 4H), 1.84 (m, 4H).
ACKNOWLEDGMENTS
We thank all colleagues in the Department of DMPK AstraZeneca
■
1-(4-Bromobenzoyl)-4-methylpiperazine16 (36a). 4-Bromo-
benzoic acid (3.0 g, 14.9 mmol) was dissolved in refluxing thionyl
chloride (35 mL), and the solution was heated at reflux for 1 h and
then cooled to room temperature. The solvent was evaporated,
coevaporated with toluene (3 × 40 mL), and the resulting solid
dried in vacuo. The solid was dissolved in dichloromethane (18
mL), cooled in an ice-bath, and 1-methylpiperazine (1.5 mL, 13.6
mmol) added dropwise to give a solid. Dichloromethane and
aqueous potassium carbonate were added, and the aqueous phase
was extracted with dichloromethane. The combined organic phases
were dried over Na2SO4 and filtered, and the solvent was
Sodertalje who helped to generate the in vivo permeability data
̈
̈
presented, Fanny Bjarnemark and Ylva Roddis, Department of
Medicinal Chemistry, AstraZeneca Sodertalje, for generation of
̈
̈
HRMS data and Susanne Olofsson and Alexandra Bernlind,
Department of Medicinal Chemistry, AstraZeneca Sodertalje, for
valuable NMR support, I. Janssen, C. Dartsch, and R. Svensson,
AstraZeneca Biotech Laboratories, for production of GSK3β
protein, Tomas Lundqvist, Department of Structure & Biophysics,
̈
̈
AstraZeneca, Molndal, for his contribution in data collection and
̈
1
initial structure determination of GSK3β complexes, Judith
Stanway for expression of CDK2 in baculovirus infected insect
cells, Malcolm Anderson for purification of CDK2 protein for
crystallization, Claire Brassington for CDK2 crystallization and
soaking, and Andrew Pannifer, Helen McMiken, and the staff at the
ESRF for assistance with CDK2 X-ray diffraction data collection.
evaporated to give 3.86 g (91% yield) of the title compound. H
NMR (300 MHz, DMSO-d6) δ 7.64 (d, J = 8 Hz, 2H), 7.34 (d, J =
8 Hz, 2H), 3.59 (m, 4H), 2.34 (m, 4H), 2.21 (s, 3H).
3-Amino-6-{4-[(4-methylpiperazin-1-yl)carbonyl]phenyl}-N-
pyridin-3-ylpyrazine-2-carboxamide Hydrochloride (37). Com-
pound 37 was prepared in 26% yield from 36a using a similar
procedure to that of 13. 1H NMR (300 MHz, DMSO-d6,) δ 11.41 (br
s, 1H), 11.11 (s, 1H), 9.40 (s, 1H), 9.06 (s, 1H), 8.90 (d, J = 9 Hz,
1H), 8.69 (d, J = 5 Hz, 1H), 8.38 (d, J = 8 Hz, 2H), 8.06 (dd, J =
9, 6 Hz, 1H), 7.59 (d, J = 8 Hz, 2H), 3.39 (m, 4H), 3.13 (m, 2H), 2.77
(s, 3H), 2.50 (m, 2H).
ABBREVIATIONS USED
Aβ, β-amyloid; AD, Alzheimer’s disease; ATP, adenosine triphos-
phate; BBB, blood−brain barrier; n-BuLi, normal-butyl lithium;
■
9118
dx.doi.org/10.1021/jm201724m | J. Med. Chem. 2012, 55, 9107−9119

Journal of Medicinal Chemistry
Article
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CDK2, cyclin dependent kinase 2; CNS, central nervous system;
DIPEA, N,N-diisopropylethylamine; DBU, 1,8-diazabicyclo[5.4.0]-
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LC, liquid chromatography; MS, mass spectra; MOPS, morpho-
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MW, microwave; PDA, photodiode array detectors; PHFs, paired
helical filaments; PyBroP, bromo-tris-pyrrolidino phosphoniumhex-
afluorophosphate; SEM, standard error of the mean; TBTU,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMS,
trimethylsilane; TS, thermospray; UPLC, ultra performance liquid
chromatography
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