Design and synthesis of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.027

Cyclin-dependent kinase 5 (CDK5) is a serine-threonine protein kinase that plays a significant role in neuronal development. In association with p25, CDK5 abnormally phosphorylates a number of cellular targets involved in neurodegenerative disorders. Usin

Full text of DOI:10.1016/j.bmcl.2009.10.027

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6591–6594
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors
a
b
c
a
a
Matthew R. Kaller a, , Wenge Zhong , Charles Henley , Ella Magal , Thomas Nguyen , David Powers ,
*
Robert M. Rzasa a, Weiya Wang c, Xiaoling Xiong a, Mark H. Norman a
a Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
b Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
c Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclin-dependent kinase 5 (CDK5) is a serine-threonine protein kinase that plays a significant role in neu-
ronal development. In association with p25, CDK5 abnormally phosphorylates a number of cellular tar-
gets involved in neurodegenerative disorders. Using active site homology and previous structure–
activity relationships, a new series of potent CDK5 inhibitors was designed. This report describes the opti-
mization of 6-oxo-1,6-dihydropyridines as CDK5 inhibitors.
Received 22 July 2009
Revised 6 October 2009
Accepted 7 October 2009
Available online 13 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
Neurodegenerative disorders
CDK5 inhibitor
6-Oxo-1,6-dihydropyridines
A number of studies have demonstrated the essential role of
cyclin-dependent kinase 5 (CDK5) in the early development of
the central nervous system.1 CDK5 is ubiquitously expressed in
all tissues, but its highest expression and associated kinase activity
are observed in the CNS. Deregulation of CDK5 is implicated in sev-
eral neurodegenerative disorders.1,2 As a result, CDK5 inhibitors
have potential therapeutic use for diseases such as Alzheimer’s dis-
ease,3 amyotrophic lateral sclerosis,4 and ischemic stroke.5
Our initial effort began with a high throughput screening cam-
paignthatledtotheidentificationofaseriesof acyclicthiazole-ureas
as potent CDK2 and CDK5 inhibitors.6 After obtaining a co-crystal
structure of CDK2 and acyclic urea 1, we developed an active site
homology model of CDK5 to guide the design and synthesis of con-
formationally constrained inhibitors (Fig. 1). Recently we reported
the investigations of 3,4-dihydro-1H-quinazolin-2-ones7 (2, CDK5
IC50 = 79 nM) and quinilon-2H-ones8 (3, CDK5 IC50 = 54 nM) as
potent CDK5 inhibitors. To further explore this line of strategy, we
envisioned keeping the donor-acceptor binding portion of the mol-
ecule intact while reducing molecular weight. Additionally, we
sought to improve the solubility of the molecules. Towards this
end, we developed a series of potent inhibitors based on the 6-
oxo-1,6-dihydropyridine core (4, Fig. 1). This report will profile the
design, synthesis, and biological activity of 6-oxo-1,6-dihydropyri-
dines as CDK5 inhibitors.
Typically, 5 was heated with dimethylamino dimethylacetal
(DMF-DMA) to give substituted ethyl 2-dimethylaminomethyl-
ene-3-oxoalkonates 6 in good yields. Treatment of 6 with sodium
acetoacetamide in THF at 60 °C gave the desired 6-oxo-1,6-dihy-
dropyridine 7, also in good yield.9 Subsequent bromination and
thiazole formation with the appropriate thioamides in EtOH at
80 °C furnished the final products 4, 11–19.8 This synthetic scheme
proved to be highly flexible and tolerated a large variety of
substituents.
H
N
H
N
O
N
O
N
N
N
N
H
S
S
N
N
1
2
N
N
S
H
H
N
O
N
N
O
EtO
S
O
N
The general synthesis of 6-oxo-1,6-dihydropyridines analogues
began with the appropriately substituted ethyl acetoacetates 5.
4
3
N
N
* Corresponding author. Tel.: +1 805 447 8004; fax: +1 805 480 3016.
E-mail address: mkaller@amgen.com (M.R. Kaller).
Figure 1. CDK5 inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.027

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M. R. Kaller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6591–6594
O
O
H
N
N
O
R1
O
O
NH2
R1
OEt
R1
OEt
i
ii
EtO
O
O
O
O
5
6
7
H
N
R1
O
O
N
H
N
R1
O
O
EtO
EtO
O
iv
iii
S
R2
4, 11-19
Br
8
v
H
H
R1
O
N
O
N
R1
N
O
EtO
vi
EtO
S
S
O
N
21-25
20
R3
Cl
N
N
Scheme 1. Reagents and conditions: (i) (CH3)2NCH(OCH3)2, neat, 100 °C, 2–4 h; (ii) NaH, THF, 60 °C, 12 h; (iii) 5,5-dibromobarbituric acid (0.55 equiv), THF, reflux, 2–4 h; (iv)
thioisonicotinamide, EtOH, 80 °C, 12 h; (v) 2-chloropyridine-4-carbothioamide, EtOH, 80 °C, 12 h; (vi) RNH2, 120 °C or NaOMe in refluxing MeOH.
H
H
N
H
N
N
O
N
O
N
O
N
i
R1O
iii
X
O
S
S
S
O
O
O
26-32
14
9 X = OH
10 X = imidazole
ii
N
N
N
Scheme 2. Reagents and conditions: (i) KOH (s), EtOH/water, 120 °C, 10 min, microwave; (ii) CDI, DIPEA, CH2Cl2/DMF, 16 h; (iii) excess alcohol, 150 °C, neat, 10 min,
microwave.
Compounds with substituents on the pyridyl ring (21–25) were
made from the common intermediate 20 (Scheme 1). 2-Chloroiso-
nicotinamide was treated with Lawesson’s reagent to give 2-chlo-
ropyridine-4-carbothioamide11 which was used to prepare 20. The
chloropyridine 20 was then heated with the appropriate amines or
sodium methoxide to give the desired pyridyl amines 21–24 and
methoxy pyridine 25 in moderate yield.12 Compound 25 was pre-
pared by reacting 20 with excess sodium methoxide in refluxing
MeOH.
Outlined in Scheme 2 is the general synthesis of esters 26–32
from intermediate 14. Treatment of 14 with KOH (5 equiv) in aque-
ous EtOH for 10 min at 120 °C in the microwave synthesizer ef-
fected hydrolysis9 and yielded acid 9, which was converted to
the corresponding acyl imidazole 10 with carbonyl diimidazole
in the presence of N,N-diisopropylethylamine in good yield. The
acyl imidazole 10 was heated with excess commercially available
alcohols at 150 °C for 10 min in a microwave synthesizer to pro-
vide the corresponding esters 26–32.10
were determined from dose–response curves and are reported in
Tables 1–4 as the average of at least three replications. In addition
to CDK5, the compounds were screened in an HTRF human CDK2/
E2 assay that was run in the presence of 50 lM ATP and 0.5 lM
histone-H1 as the substrate. Most analogues displayed only mod-
est selectivity over CDK2, with the aryl sulfone series showing
the best selectivity (up to 70-fold).
Our SAR exploration began with the finding that compound 4
was a modest CDK5 inhibitor (IC50 = 131 nM). To follow up with
this encouraging result, we initially focused on expanding the
SAR of substituents at the 2 position of the 6-oxo-1,6-dihydropyr-
idine core (Table 1). It was quickly determined that a wide range of
alkyl substituents were tolerated. For example, an isopropyl group
or a cyclopropyl group at this position gave inhibitors 14 and 15
with comparable potency to 4. Interestingly, inhibitors 12 and 13
with an ethyl group and an n-propyl group, respectively, were
found to be more potent than 4, with the former showing >30-fold
improvement. The introduction of electron withdrawing groups or
sterically bulky groups at the position led to reduction in activity as
demonstrated by compounds 11 and 16. Selected compounds from
this subset of analogs were evaluated in rats for in vivo PK proper-
ties. In general, the compounds displayed moderate to high in vivo
The analogues prepared for this study were tested for their abil-
ity to inhibit purified human CDK5/p25 in a homogenous time-re-
solved fluorescence assay run in the presence of 25
lM ATP using
histone-H1 as the phosphorylation substrate.13 The IC50 values

M. R. Kaller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6591–6594
6593
Table 1
Table 3
SAR of the 2 position of substituted 6-oxo-1,6-dihydropyridines
SAR at the 3 position of the pyridine-4-yl moiety
H
R1
O
N
O
N
H
R1
O
N
O
N
EtO
EtO
S
S
R2
N
N
Compound
R1
R2
CDK5/p25 (IC50, nM)a
Compound
R1
CDK5/p25 (IC50, nM)a
4
20
21
22
23
24
25
CH3
CH3
CH3
CH3
CH3
i-C3H7
i-C3H7
H
Cl
NH2
NHCH2CH(Me)2
NH(CH2)2N(Et)2
NHCH3
131 66
160b
4
11
12
13
14
15
16
CH3
CF3
C2H5
n-C3H7
i-C3H7
c-C3H4
BnOCH2
131 66
11489 3429.9
4.1b
20b
14 10
139 37
28 14
672 37
55 14
157 131
198 55
779 234
OCH3
a
At least three independent experiments were performed for each compound to
a
At least three independent experiments were performed for each compound to
determine the IC50 values.
determine the IC50 values.
b
One experiment was performed to determine the IC50 value.
b
One experiment was performed to determine the IC50 value.
Table 4
SAR of different esters at the 3 position of 6-oxo-1,6-dihydropyridines
Table 2
H
SAR of pyridin-4-yl replaced 6-oxo-1,6-dihydropyridines
N
O
N
R1O
H
S
R1
O
N
O
N
O
EtO
S
N
R2
Compound
R1
CDK5/p25 (IC50, nM)a
Compound
R1
R2
CDK5/p25 (IC50, nM)a
131 66
14
26
C2H5
CH2CH2Ph
157 131
119b
4
CH3
N
27
28
112 20
N
O
O
O
O
647 410
N
S
S
S
17
18
19
CH3
3.3 1.1
1.4 0.2
5.3 3.7
29
73 30
N
O
O
n-C3H7
BnOCH2
30
31
36 16
53 34
N
N
a
At least three independent experiments were performed for each compound to
determine the IC50 values.
32
47
6
N
a
At least three independent experiments were performed for each compound to
clearance and poor to moderate brain exposure. Among these
inhibitors, a notable exception, 14 showed a measured total brain
concentration of 4640 ng/g one half hour after a 2 mg/kg IV dose in
rats, an 8–10-fold improvement in brain exposure over other mea-
sured compounds.
determine the IC50 values.
b
One experiment was performed to determine the IC50 value.
arylsulfones led to significant improvements in potency.8 This SAR
trend was also observed in the current series (Table 2). In general,
arylsulfones provided potent inhibitors in the low nanomolar
Previous studies in the quinilon-2H-one series of CDK5 inhibi-
tors have shown that the replacement of the 4-pyridyl group with

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M. R. Kaller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6591–6594
range. For example, phenylsulfone derived compounds 17 and 18
displayed IC50 values of 3.3 nM and 1.4 nM, respectively. It is worth
noting that the arylsulfone replacements greatly improved the po-
tency of molecules with less than ideal groups at the 2 position.
This was illustrated by compound 19 which displayed >150-fold
improvement in potency than the corresponding 4-pyridyl inhibi-
tor 16.
The arylsulfone derived inhibitors were very potent; however,
they gave lower brain exposure (17, 44 ng/g at 0.5 h, 2 mg/kg IV
dose in rats) in comparison to the 4-pyridyl substituted molecules.
This is not that surprising considering the higher polar surface area
for these compounds along with lower permeability (typically 2–
6 Â 10À6 cm/s) these compounds demonstrated in comparison to
the 4-pyridyl substituted molecules. Therefore, we focused our fur-
ther exploration on the pyridyl compounds. Table 3 shows selected
examples where the 2 position of the 4-pyridyl was substituted.
Initially, this position was explored to mitigate possible CYP 450
inhibition due to the 4-pyridine; however it was soon evident that
this additional substitution could also offer other benefits. The
chloro derivative 20 had comparable potency to parent molecule
4, while the aminopyridine compound 21 gave a sixfold improve-
ment in activity. Although inhibitor 23 showed comparable activ-
ity to 4, the other two alkylaminopyridine compounds 22 and 24
demonstrated a 10- and 5-fold increase in potency, respectively.
In contrast to the aminopyridines, alkoxypyridines, such as 25,
proved to be less potent.
Table 4 summarizes the effects of replacing the ethyl ester. It
should be noted here that these esters can be viewed as vinylogous
carbamates and therefore are more stable than typical esters. This
is consistent with the observation that the hydrolysis of these es-
ters required very forcing conditions during synthesis (KOH,
5 equiv, in aqueous EtOH for 10 min at 120 °C in the microwave
synthesizer). Based on this observation, we reasoned that it was
worth exploring a variety of ester analogs. We quickly found that
the hydrophobic alkyl ester analogs, exemplified by the phenethyl
ester 26, provided no improvement in potency in comparison to
the ethyl ester. Drawing analogy to the SAR in the acyclic thiazole
urea series for improving potency and solubility, we incorporated
alkylamino groups into the ester sidechain. We found that a large
number of substituted or ring constrained aminoethyl esters were
potent CDK5 inhibitors (27 and 29–31, Table 4). Interestingly,
substituted aminopropyl ester 32 was found to be a potent inhib-
itor, while its ring constrained variant 28 was 13-fold less potent.
Based on previous crystal structures of 1 and molecular model-
ing of 2 we propose that the 6-oxo-1,6-dihydropyridine series
binds in a similar U-shape configuration where in the 6-oxo-1,6-
dihydropyridine is involved in a donor-acceptor hydrogen bonding
network with Leu83 (Fig. 2). In this model, the thiazole-pyridine
extends down to make an interaction with the Lys33-Asp145 salt
bridge of the ATP binding site. In addition, Lys89 also makes a
hydrogen bond with the carbonyl of the ester. These binding inter-
actions reconcile well with the observed SAR in this series.
In summary, guided by an active site homology model and SAR
from our early studies in the 3,4-dihydro-1H-quinazolin-2-one and
quinilon-2H-ones series,7,8 we were able to develop a new series of
6-oxo-1,6-dihydropyridines as potent CDK5 inhibitors. We ex-
plored the 2 position of the 6-oxo-1,6-dihydropyridine core and
determined that small alkyl substituents were optimal. Further-
more, we found that the 4-pyridyl group on the thiazole ring was
a good compromise for maximizing potency and maintaining ade-
Figure 2. A proposed binding mode for 6-oxo-1,6-dihydropyridine (27)–CDK2
complex. Nitrogen atoms are shown in blue, oxygen atoms are shown in red, sulfur
atoms are shown in yellow, and carbon atoms of active site residues are shown in
gray. Carbon atoms of compounds 27 are shown in green.
quate brain exposure. The initial ethyl ester could be replaced with
esters that also provided modestly improved potency and im-
proved solubility.
Acknowledgments
We are grateful to Vellarkad Viswanadhan and Kristin Andrews
for their modeling support. In addition, the authors would like to
thank Jiandong Zhang and Timothy Osslund, of the molecular
structure group, for their support.
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