5,6,7,8-Tetrahydropyrido[4,3-d]pyrimidines as novel class of potent and highly selective CaMKII inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.005

A novel series of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines containing substituted phenyl sulfonamide are synthesized and evaluated for their inhibitory activity against CaMKII. Substituents on the phenyl group had significant impact on CaMKII inhibition, in particular, the inhibitory activity of 8p was 25-fold higher than that of KN-93, a known CaMKII inhibitor. Michaelis-Menten analysis of a representative compound suggested that the synthesized pyrimidines are calmodulin non-competitive inhibitors. Finally, 8p exhibited more than 100-fold higher selectivity for CaMKII over five types of off-target kinases.

Full text of DOI:10.1016/j.bmcl.2010.09.005

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6696–6698
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5,6,7,8-Tetrahydropyrido[4,3-d]pyrimidines as novel class of potent
and highly selective CaMKII inhibitors
⇑
Shigehiro Asano , Masafumi Komiya, Nobuyuki Koike, Erina Koga, Shogo Nakatani, Yoshiaki Isobe
Research Division, Dainippon Sumitomo Pharma Co., Ltd, 3-1-98 Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2010
Revised 31 August 2010
Accepted 1 September 2010
Available online 9 September 2010
A novel series of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines containing substituted phenyl sulfonamide
are synthesized and evaluated for their inhibitory activity against CaMKII. Substituents on the phenyl
group had significant impact on CaMKII inhibition, in particular, the inhibitory activity of 8p was 25-fold
higher than that of KN-93, a known CaMKII inhibitor. Michaelis–Menten analysis of a representative
compound suggested that the synthesized pyrimidines are calmodulin non-competitive inhibitors.
Finally, 8p exhibited more than 100-fold higher selectivity for CaMKII over five types of off-target kinases.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Calmodulin-dependent protein kinase II
Inhibitors
5,6,7,8-Tetrahydropyrido[4,3-d]pyrimidines
Calcium (Ca2+) signaling is known as a critical component of bio-
logical pathways. Ca2+ is also an important second messenger for
multiple intracellular processes, including apoptosis, ion channel
and cell cycle regulation, and cellular response to oxidative stress.1
Ca2+/calmodulin-dependent protein kinases (CaMKs), which are
ubiquitous serine/threonine protein kinases that are activated by
Ca2+-bound calmodulin and classified into three subtypes (I, II, and
IV), modulate many cellular functions in response to intracellular
Ca2+ levels.2 CaMKII, a member of CaMKs family, assembles into a
O
N
O
HO
N
S
OMe
Me
Cl
1 (KN-93)
O
S
O
HN
Cl
N
complex of dodecamers with four isoforms (a, b, c, and d), and each
N
N
H
subunit is comprised of three main parts; catalytic, regulatory, and
association domains.3 Upon binding Ca2+/calmodulin in the pres-
ence of ATP/Mg2+, CaMKII undergoes a rapid autophosphorylation
on Thr286/Thr287 located within the autoinhibitory domain. The acti-
vated CaMKII maintains considerable enzyme activity even without
Ca2+/calmodulin.4 This autophosphorylation has been reported to
cause a dramatic increase in the affinity of the enzyme for Ca2+/cal-
modulin.5 CaMKII is well established for its effects on modulating
synapticplasticityand processes like learningand memory.6 In addi-
tion, CaMKII plays a role in osteoclast differentiation and bone
resorption,7 and active CaMKII is known to enhance proliferation
and cytotoxic activity of T cells.8
2 (HTS hit compound)
Figure 1. Structures of 1, a representative CaMKII inhibitor, and 2, a hit compound
identified by HTS.
(Fig. 1).11 The IC50 of 2 toward CaMKII was evaluated as 11
lM,
approximately sevenfold weaker than that of 1. Based on compound
2 we conducted a structure–activity relationship (SAR) study, and
herein report the design, synthesis, and biological activity of a novel
series of pyrimidyl sulfonamides.
The general synthesis of the pyrimidines 6–9 are shown in
Scheme 1. Compounds 6–8 were prepared by simple replacement
reaction of the dichloride 3 with appropriate nucleophiles. The first
replacement reaction with the corresponding benzenesulfona-
mides (5) proceeded completely at position 4 of the pyrimidine
ring. Compound 3 is commercially available or can be prepared
easily according to a synthetic methods in the literature.12 Cata-
lytic reduction of the benzyl (Bn) group in compound 8, followed
by reductive amination using formaldehyde and NaBH(OAc)3
afforded the N-methylated compound 9.
As calmodulin-competitive inhibitor 1 (KN-93, Fig. 1)9 and auto-
camtide-2-related inhibitory peptide (AIP)10 have been reported as
well-known CaMKII inhibitors, we considered CaMKII to be a good
target for the development of anti-inflammatory agents. Recently,
we launched a HTS campaign to find novel structural CaMKII
inhibitors, and compound 2 was identified as a hit compound
⇑
Corresponding author. Tel.: +81 6 6466 5185.
E-mail address: shigehiro-asano@ds-pharma.co.jp (S. Asano).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.005

S. Asano et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6696–6698
6697
O
O
N
O
O
N
S
R2
N
S
R2
N
Cl
R1
X
R1
X
X
Y
a
b or c
N
N
Cl
Y
N
Cl
Y
N
Z
Bn
3
4
6
8
9
X=Y=H
X, Y=
X, Y=
N
d
7 X, Y=
Me
N
Scheme 1. Synthesis of pyrimidines. Reagents and conditions: (a) R2NHSO2R1 (5), K2CO3, NMP, 100 °C; (b) PhCH2CH2NH2, K2CO3, NMP, 100 °C; (c) PhCH2CH2OH, NaH, THF, rt;
(d) (1) 10%—Pd/C, H2 (0.45 MPa), CF3CO2H, MeOH, rt; (2) HCHO, NaBH(OAc)3, dichloroethane, MeOH, rt.
Table 2
First, we investigated the effects of various pyrimidine moieties
Effects of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines on CaMKII
on CaMKII inhibition. The inhibitory activity of compounds 2, 6a,b,
7, 8a,b, and 9 for CaMKII are shown in Table 1. The results of mono-
cyclic pyrimidines (2 and 6a,b) suggested that the phenyl group
shown as R1 into the sulfonamide was important for CaMKII inhi-
bition. We then focused our efforts on conversion of the mono-cyc-
lic pyrimidine to a fused-pyrimidine. The tetrahydroquinazoline
compound 7 abolished CaMKII inhibition compared to the HTS
hit compound 2. Therefore, we introduced a basic amine into posi-
tion 6 on the tetrahydroquinazoline ring based on the tertiary ami-
no moiety in 1. Among the tetrahydropyrido[3,4-d]pyrimidine
compounds, 8a,b exhibited inhibitory activity with single micro
molar IC50, while compound 9 showed no activity. These findings
suggest that the benzyl group at the nitrogen atom of the piperi-
dine moiety could be important in filling the hydrophobic region
of the enzyme. As compound 8b exhibited potent activity threefold
that of 8a, we conducted a detailed SAR study on the phenyl group
substituents (R1).
Next, we turned our attention to the effects of different substit-
uents on both the phenyl moiety (R1) and the nitrogen atom (R2).
The results for compounds 8a–q are summarized in Table 2.
Although the methyl compound 8c had an activity comparable to
that of the hydrogen compound 8a, the propyl compound 8d
showed a complete loss of activity. These results indicate that ste-
ric hindrance produced a significant impact on CaMKII inhibition.
In order to optimize the substituent on the phenyl moiety (R1),
we chose the chlorine atom as electron-withdrawing group and
the methyl group as electron-donating one. The order of potency
was meta (8f) > ortho (8e) > para (8b) in chlorine atom substituted
compounds, whereas ortho (8l) was better than meta (8m) or para
(8n) in methyl substitution. As the suitable position was different
with the chlorine atom and the methyl group, we combined these
O O
S
R2
N
R1
N
N
N
Z
Compd
R1
R2
Z
CaMKII
IC50a l
M
8a
8c
8d
8e
8f
8b
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
1
Ph
Ph
Ph
H
Me
CH2CH2CH3
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
NH
NH
NH
NH
NH
NH
9.7
9.3
>30
0.92
0.42
3.2
1.0
0.20
3.3
0.16
1.8
0.53
2.4
2.7
3.2
0.063
0.76
1.6b
11
2-Cl–C6H4
3-Cl–C6H4
4-Cl–C6H4
2,3-Cl2–C6H4
3,4-Cl2–C6H4
1-Naphtyl
2-Naphtyl
2-Naphtyl
2-Me–C6H4
3-Me–C6H4
4-Me–C6H4
4-OMe–C6H4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3-Cl–2-Me–C6H4
3-Cl–4-Me–C6H4
2
a
Compound inhibitory activity for CaMKII enzyme.
In-house data.
b
findings and made bis-substituted compounds. As expected, com-
pound 8p,13 considered as the best bis-substituted compound,
showed potent activity much higher than that of single substituted
compounds, including 8f and 8l. Compound 8q, with a methyl
group not in the best position, gave over 12-fold weaker activity
than 8p. Unfortunately, compound 8o, which had 4-methoxyben-
zensulfonamide as a common partial structure with 1, exhibited
Table 1
Effects of pyrimidines on CaMKII
O O
moderate inhibitory activity (IC50 = 3.2 lM).
S
HN R1
In the case of di-chlorophenyl compounds, meta- and para-sub-
stitution (8g) was better than ortho- and meta-substitution (8h).
However, the tendency was different from that of 8p or 8q, sug-
gesting that sterically small substituents might be better to place
at the ortho position. As representative of the di-cyclic group, we
prepared the naphthyl compounds 8i and 8j. The 2-naphtyl com-
pound 8j was more effective than the 1-naphtyl one 8i (8j,
X
N
Y
N
N
H
Compd
R1
X, Y
CaMKII
IC50 (lM)
a
1
2
1.6b
11
>60
27
>60
9.7
3.2
>10
4-Cl–C6H4
Me
Bn
4-Cl–C6H4
Ph
4-Cl–C6H4
Ph
–H, –H
–H, –H
–H, –H
–CH2CH2CH2CH2–
–CH2N(Bn)CH2CH2–
–CH2N(Bn)CH2CH2–
–CH2N(Me)CH2CH2–
IC50 = 0.16 lM). These findings were similar to those obtained with
6a
6b
7
8a
8b
9
di-chlorophenyl compounds. Unfortunately, using oxygen as the
linker atom (Z), gave decreased activity (8j vs 8k). Among the com-
pounds prepared, 8p showed the most potent inhibitory activity
for CaMKII with an IC50 value of 0.063 lM, that is, 170-fold
improvement from compound 2.
a
We next investigated CaMKII inhibition pattern of fused-pyrim-
idines. Kinetics analysis of CaMKII inhibition by compound 8j using
Compounds inhibitory activity for CaMKII enzyme.
In-house data.
b

6698
S. Asano et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6696–6698
Table 3
Kinase selectivity
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
Compd
CaMKII
Kinase selectivity
IC50 M)
a
IC50
(l
M)
(l
CaMKIV
MLCK
p38a
Akt1
PKC
2
8j
8p
11
0.16
0.063
Ntb
>60
>60
51
0.90
36
Ntb
11
11
Ntb
8.5
30
Ntb
22
21
a
Compounds inhibitory activity for CaMKII enzyme.
Nt: not tested.
b
Supplementary data
0
0
100
200
300
400
500
600
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.005.
[CaM] (nM)
Figure 2. Michaelis–Menten plot of CaMKII inhibition by various concentrations
(0 (d), 62.5 (s), 125 (.), 250 (5) M) of compound 8j. The inhibition kinetics
l
References and notes
analysis of CaMKII by compound 8j were performed SigmaPlot Enzyme Kinetic
Module. We calculated the goodness of fit to the enzyme inhibition model of
competitive, non-competitive, and un-competitive following equations V = Vmax/
{1 + (Km/S)(1 + I/Ki)}, V = Vmax/(1 + Km/S)(1 + I/Ki), and V = Vmax/(1 + I/Ki + Km/S),
respectively (Vmax: the maximum velocity. Km: Michaelis constant for the varied
substrate. S: concentration of the varied substrate. I: concentration of the test
substance. Ki: inhibition constant.). The coefficient of determination of non-
competitive was best and Ki value of 8j was calculated with 930 nM.
1. (a) Bootman, M. D.; Collins, T. J.; Peppiatt, C. M.; Prothero, L. S.; MacKenzie, L.;
De Smet, P.; Travers, M.; Tovey, S. C.; Seo, J. T.; Berridge, M. J.; Ciccolini, F.; Lipp,
P. Semin. Cell Dev. Biol. 2001, 12, 3; (b) Howe, C. J.; Lahair, M. M.; Mccubrey, J. A.;
Franklin, R. A. J. Biol. Chem. 2004, 279, 44573.
2. (a) Hook, S. S. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 471; (b) Braun, A. P.;
Schulman, H. Annu. Rev. Physiol. 1995, 57, 417.
3. (a) Hanson, P. I.; Schulman, H. Annu. Rev. Biochem. 1992, 61, 559; (b) Colbran, R.
J.; Soderling, T. R. Curr. Top. Cell. Regul. 1990, 31, 181; (c) Gaertner, T. R.;
Kolodziej, S. J.; Wang, D.; Kobayashi, R.; Koomen, J. M.; Stoops, J. K.; Waxham,
M. N. J. Biol. Chem. 2004, 279, 12484.
4. (a) Schworer, C. M.; Colbran, R. J.; Keefer, J. R.; Soderling, T. R. J. Biol. Chem.
1988, 263, 13486; (b) Miller, S. G.; Patton, B. L.; Kennedy, M. B. Neuron 1988, 1,
593; (c) Thiel, G.; Czernik, A. J.; Gorelick, F.; Nairn, A. C.; Greengard, P. Proc. Natl.
Acad. Sci. 1988, 85, 6337; (d) Ikeda, A.; Okuno, S.; Fujisawa, H. J. Biol. Chem.
1991, 266, 11582.
5. Meyer, T.; Hanson, P. I.; Stryer, L.; Schulman, H. Science 1992, 256, 1199.
6. Wayman, G. A.; Lee, Y. S.; Tokumitsu, H.; Silva, A.; Soderling, T. R. Neuron 2008,
59, 914.
7. Ang, E. S.; Zhang, P.; Steer, J. H.; Tan, J. W.; Yip, K.; Zheng, M. H.; Joyce, D. A.; Xu,
J. J. Cell. Physiol. 2007, 212, 787.
8. Lin, M. Y.; Zal, T.; Ch’en, I. L.; Gascoigne, N. R. J.; Hedrick, S. M. J. Immunol. 2005,
174, 5583.
9. Sumi, M.; Kiuchi, K.; Ishikawa, T.; Ishii, A.; Hagiwara, M.; Nagatsu, T.; Hidaka, H.
Biochem. Biophys. Res. Commun. 1991, 181, 968.
Michaelis–Menten plot indicated that 8j was a typical non-com-
petitive inhibitor (Fig. 2) with Ki value calculated using analysis
software as 926 nM.14 This finding indicates that our compounds
can be considered as calmodulin non-competitive inhibitors. Inter-
estingly, compounds 1 and 8j displayed different inhibition pat-
terns in spite of their structural similarities (aryl-sulfonamide
moiety). We speculate that the aminopyrimidine moiety in com-
pound 8j plays an important role in the inhibition pattern of CaM-
KII enzyme.
Finally, we examined the selectivity profile of 2, 8j, and 8p
against five types of kinase. The results are shown in Table 3. The
selectivity of the 2-naphtyl compound 8j against myosin light
chain kinase (MLCK), which belongs to the CaM kinase family,
was not good enough. Fortunately, compound 8p, the most potent
compound, showed significantly high selectivity against five off-
10. Ishida, A.; Kameshita, I.; Okuno, S.; Kitani, T.; Fujisawa, H. Biochem. Biophys. Res.
Commun. 1995, 212, 806.
11. A detailed description of the assay is provided in the Supplementary data.
12. (a) Sekiya, T.; Hiranuma, H.; Kanayama, T.; Hata, S. Eur. J. Med. Chem. 1980, 15,
317; (b) Aratake, S.; Ueno, K.; Miki, I.; Iwase, M.; Muramatsu, K.; Yano, H. J.P.
Patent 162,673, 2005.
target kinases, CaMKIV, MLCK, p38a, Akt1, and PKC.
13. Compound 8p: mp 208–210 °C (dec); 1H NMR (DMSO-d6, 400 MHz) d 2.45 (3H,
s), 2.47–2.53 (2H, m), 2.64 (2H, m), 2.69 (2H, br t, J = 7.0 Hz), 3.10 (2H, m), 3.17
(2H, br s), 3.67 (2H, br s), 6.62 (1H, br s), 7.02 (1H, t, J = 7.6 Hz), 7.17 (2H, m),
7.21 (1H, m), 7.30 (2H, m), 7.24–7.37 (5H, m), 7.44 (1H, dd, J = 7.6, 1.0 Hz), 7.89
(1H, dd, J = 7.6, 1.0 Hz), 11.25 (1H, br s); 13C NMR (DMSO-d6, 100 MHz) d 16.8,
26.2, 34.2, 41.3, 47.8, 49.1, 61.0, 104.7, 125.8, 126.7, 127.1, 127.9, 128.0, 128.3,
130.6, 132.8, 134.3, 138.0, 138.8, 145.6, 151.1, 162.1; IR (ATR) 3307, 1664,
1626 cmꢀ1. Anal. Calcd for C29H30ClN5O2S: C, 63.55; H, 5.52; N, 12.78; S, 5.85;
Cl, 6.47. Found: C, 63.20; H, 5.42; N, 12.55; S, 5.68; Cl, 6.29.
14. SigmaPlot for Windows Version 10.0.
In summary, we herein disclose a novel class of pyrimidine-
based CaMKII inhibitors. Key SAR as well as the synthetic methods
for these inhibitors are described. In particular, we found that com-
pound 8p inhibits CaMKII with an IC50 value of 0.063
substantially superior to that of the known CaMKII inhibitor 1
(IC50 = 1.0 M, Table 1). We therefore expect 8p to be a novel lead
lM, which is
l
compound in the development of treatments for inflammatory dis-
eases, including rheumatoid arthritis.