Structure-based design and synthesis of novel macrocyclic pyrazolo[1,5-a] [1,3,5]triazine compounds as potent inhibitors of protein kinase CK2 and their anticancer activities

Article abstract of DOI:10.1016/j.bmcl.2007.11.074

A series of macrocyclic derivatives has been designed and synthesized based on the X-ray co-crystal structures of pyrazolo[1,5-a] [1,3,5]triazines with corn CK2 (cCK2) protein. Bioassays demonstrated that these macrocyclic pyrazolo[1,5-a] [1,3,5]triazine

Full text of DOI:10.1016/j.bmcl.2007.11.074

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 619–623
Structure-based design and synthesis of novel macrocyclic
pyrazolo[1,5-a] [1,3,5]triazine compounds as potent inhibitors
of protein kinase CK2 and their anticancer activities
Zhe Nie,a,* Carin Perretta,a Philip Erickson,a Stephen Margosiak,b Jia Lu,b April Averill,b
Robert Almassyc and Shaosong Chua,*
aDepartment of Medicinal Chemistry, Polaris Pharmaceuticals Inc., San Diego, CA 92121, USA
bDepartment of Protein Chemistry, Polaris Pharmaceuticals Inc., San Diego, CA 92121, USA
cDepartment of Structural Biology, Polaris Pharmaceuticals Inc., San Diego, CA 92121, USA
Received 19 October 2007; revised 18 November 2007; accepted 19 November 2007
Available online 28 November 2007
Abstract—A series of macrocyclic derivatives has been designed and synthesized based on the X-ray co-crystal structures of pyraz-
olo[1,5-a] [1,3,5]triazines with corn CK2 (cCK2) protein. Bioassays demonstrated that these macrocyclic pyrazolo[1,5-a] [1,3,5]tri-
azine compounds are potent CK2 inhibitors with Ki around 1.0 nM and strongly inhibit cancer cell growth with IC50 as low as
ꢀ100 nM.
Ó 2007 Elsevier Ltd. All rights reserved.
Protein kinase CK2 is a highly conserved and pleiotro-
pic serine/threonine kinase that plays key roles in cell
growth, proliferation, and survival. It is localized in
both the cytoplasm and the nucleus of normal cells,
but is particularly more abundant in the nucleus com-
partment of cancer cells.1 It is comprised of a heterotet-
rameric structure of a2b2, aa0b2 or a02b2 where the a or
a0 subunits are catalytic, while the b subunit provides
the anchor for assembly of the scaffold and interacts
with CK2’s various protein substrates. The a and a0 sub-
units are highly homologous for a large part of the ami-
no acid sequence, only differing significantly in the
extreme C terminus where homology is essentially
non-existent.2 CK2 has been observed to be frequently
up-regulated in a wide variety of human cancers exam-
ined.3–5 Targeted overexpression of CK2 catalytic sub-
units results in tumoriogenesis in animal and cellular
models, and CK2 has been demonstrated to increase a
cell’s oncogenic potential by sensitizing a cell to trans-
formation by other oncogenic proteins.6 Down-regula-
tion of CK2 activity has been demonstrated to
decrease cellular proliferation as well as to induce apop-
tosis in cancer cells.7 Various approaches have been em-
ployed to study the cancer related bio-functions of CK2,
such as, the use of small molecule inhibitors, dominant
negative overexpression of kinase inactive mutants,8
anti-sense CK2a oligodeoxynucleotide,7 as well as small
interfering RNA molecules (siRNA).9 Recent studies by
Ahmed and co-workers10 demonstrated that antisense
CK2a caused cancer cell death in vitro as well as in a
PC3 xenograft model due to induction of apoptosis.
Complete eradication of the tumor was observed with
higher concentrations of the antisense agents. The effect
on normal and non-cancer cells however was minimal
under the same conditions. Clearly, data presented by
these studies support the rationale of inhibiting CK2
as a potential approach for cancer therapy. Specific,
O
O
CN
HN
NH
N
N
N
N
N
N
HN
N
N
H
HN
N
N
H
10
1
CK2 Ki = 0.26 nM
IC50= 2.3 μM (HCT116)
IC50= 5.1 μM (PC3)
CK2 Ki = 24 nM
IC50= 0.29 μM (HCT116)
IC50= 0.88 μM (PC3)
Keywords: CK2; Kinase; Anticancer.
*
Corresponding authors. Tel.: +1 858 7313565 (Z.N.); e-mail
addresses: zhe.nie@takedasd.com; shaosong.chu@takedasd.com
Figure 1. Pyrazolo[1,5-a] [1,3,5]triazine and macrocyclic derivative.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.074

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Z. Nie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 619–623
potent, and cell-permeable inhibitors of CK2 have been
developed in the past,11 with Ki values in the submi-
cromolar range (the best being 20 nM).11b These com-
pounds have also shown a remarkable pro-apoptotic
effect by down-regulating CK2 activity when tested
against multiple cancer cell lines. Therefore, develop-
ment of cell-permeable CK2 inhibitors should lead to
potential anti-cancer drugs.
Recently, we described pyrazolo[1,5-a] [1,3,5]triazines 1
(Fig. 1) as potent inhibitors of protein kinase CK2
(Ki < 1 nM).12 However, these compounds exhibited
only relative weak cellular activity against prostate and
colon cancer cell lines with IC50 at low lM levels. The
nature of a near planar molecular structure for this class
of compounds as observed in the X-ray co-crystal struc-
ture (Fig. 2) presumably produces poor membrane per-
meability and thus poor cellular activity.
As we discussed in the preceding paper,12 the cis-acet-
amide group provided two strong hydrogen bond inter-
actions with protein residues Asp175 and Lys68, which
induced the C2-phenyl group to adopt an extended con-
Figure 2. cCK2a with pyrazolo[1,5-a] [1,3,5]triazine inhibitor binding
to the active site (PDB code: 2PVN).
CN
CN
CN
a,b
c,d
N
N
N
NC
N
NH2
N
N
SH
HO
H
3
2
CN
CN
f
g
e
N
N
N
N
N
N
S
Cl
N
N
S
HO
5
4
CN
CN
CN
NH2
i
h
N
N
N
N
N
N
N
N
N
N
H
HN
N
N
S
S
HN
HN
N
O
O
8
6
7
CO2Me
NH2
O
k,l
j
NH
N
N
N
N
N
N
N
H
HN
N
N
H
N
HN
9
10
Scheme 1. Reagents and conditions: (a) NaH, HCO2Et, Et2O, reflux; (b) NH2NH2ÆHCl, EtOH/H2O, reflux; (c) (CO2Et)NCS, EtOAc, reflux; (d)
NH4OH, EtOAc, rt; (e) BnBr, i-Pr2NEt, NMP; (f) N,N-dimethylaniline, POCl3, reflux; (g) cyclopropylamine, NMP, rt; (h) mCPBA, CH2Cl2; (i)
benzene-1,3-diamine, acetic acid, 70 °C; (j) (1) HCl (g), MeOH; (2) HCl, MeOH, reflux; (k) NaOH, MeOH, reflux; (l) HATU, i-Pr2NEt, NMP,
sonicate.

Z. Nie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 619–623
621
formation and bury into the hydrophobic back pocket.
It was observed that the methyl group of cis-acetamide
treated with HCl gas in methanol at room temperature
overnight. However, after brief reflux in methanol, the
methyl ester 9 was the only product isolated. The ester
was then saponified. The intra-molecular cyclization
was readily achieved by treatment with the coupling re-
˚
was only about 4 A away from the nitrogen atom of
C8-nitrile group. Therefore, an appropriately placed
spatial alkyl chain would provide a linkage between
C8 and the acetamide to form a macrocyclic system.
Table 1. Cell activity of macrocyclic pyrazolotriazines
By design, macrocyclic pyrazolotriazines could be bene-
ficial in several aspects. First, an alkyl chain would fit
into the deep hydrophobic cavity composed of hydro-
phobic residues, such as Val95, Phe113, Val53, and
Ile174, to provide maximal interactions between ligand
and protein. Second, the conformationally constrained
cis-acetamide could still interact with Asp175 and
Lys68 to form two strong hydrogen bonds for enhanced
potency as well as significantly increased metabolic sta-
bility of the acetamide, facilitating in vivo animal stud-
ies. Third and most importantly, an alkyl side chain in
the macrocyclic system would form a bulge out from
the molecule and change the planar characteristics of
non-cyclized pyrazolotriazines to improve their cell
membrane permeability.
O
NH
N
N
N
N
H
N
O
HN
R1
N
R2
14
Compound NR1R2
Ki (nM) HCT116
PC3
IC50 (lM) IC50
(lM)
In direct comparison, compound 10 was synthesized by
following the procedure described in Scheme 1. 1,5-Dic-
yanopentane was treated with NaH followed by ethyl-
formate to give an intermediate, 2-formyl-
heptanedinitrile, which was then cyclized with hydra-
zine, providing 4-substituted amino pyrazole 2. Using
a similar procedure as described earlier,12 pyrazolotri-
azine 3 was obtained by reacting 2 with thioisocyanate
in refluxing EtOAc, followed by cyclization in the pres-
ence of base. Benzylation and chlorination afforded
compounds 4 and 5. The C4-chloro group was then dis-
placed with cyclopropylamine to provide compound 6.
Sulfone derivative 7 was obtained by treatment of 6 with
2.5 equivalents of mCPBA, which was then substituted
with benzene-1,3-diamine in acetic acid to give 8. A mix-
ture of imine and ester was initially obtained when 8 was
14a
14b
1.3
1.4
0.25
0.19
0.5
N
N
N
N
0.23
N
14c
14d
14e
14f
5.2
3.6
7.4
4.0
0.24
0.33
0.27
0.13
0.38
0.60
0.33
0.21
N
H
N
OH
N
N
H
N
N
N
14g
14h
2.0
2.2
0.083
0.13
0.12
0.14
N
N
N
N
14i
14j
3.4
0.8
0.30
0.44
0.62
ND
N
N
N
NH
14k
14l
2.4
1.4
0.15
0.18
ND
ND
N
N
N
˚
Figure 3. cCK2a active site complex with 10, at 2.0 A resolution (PDB
code: 3BE9).
ND, not determined.

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Z. Nie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 619–623
CN
CO2Me
NH2
NH2
N
N
a
b
N
N
O
N
N
O
HN
N
O
N
H
N
H
HN
N
O
11
12
O
CO2H
NH2
NH
N
N
N
N
c
N
N
N
H
N
O
HN
N
HN
N
H
R1
N
R2
OH
O
14
13
Scheme 2. Reagents and conditions: (a) (1) HCl (g), MeOH; (2) HCl, MeOH, reflux; (b) NaOH, MeOH, reflux; (c) (1) HATU, i-Pr2NEt, NMP,
sonicate; (2) HNR1R2, HATU, NMP.
agent, HATU, in the presence of base in NMP to pro-
vide macrocyclic pyrazolotriazine 10.
one-pot two-coupling procedure, in which 1.5 equiva-
lents of coupling reagent HATU was added in a dilute
solution of 13 to form the macrocyclic system followed
by additional 1.0 equivalent of HATU and excess of cor-
responding amines to give desired compounds 14.
˚
The 2.0 A co-crystal structure of compound 10 in com-
plex with cCK2a revealed that the alkyl linker of the
macrocyclic derivative fits quite elegantly into the
hydrophobic cavity at the back of the deep pocket, mak-
ing all the hydrophobic interactions with protein as pre-
dicted by modeling. The amide group still made two
hydrogen bonds with the salt bridge of cCK2a
(Asp175 and Lys68) without interruption by the four
carbon chain linkage (Fig. 3).
Bioassays clearly demonstrated that replacing the C4-
cyclopropyl group with a substituted phenyl group re-
gained enzyme inhibitory potency. At the meantime,
the substituent groups of the meta-amide greatly in-
creased the aqueous solubility, which, in turn, greatly
enhanced cellular activity of these macrocyclic pyrazol-
otriazines against both human prostate (PC3) and colon
cancer (HCT116) cell lines (Table 1).
MTT cell based assay13 demonstrated that compound 10
was about 10-fold more potent than compound 1, even
though it lost ꢀ100-fold enzyme inhibitory potency
(Fig. 1). Clearly, the significantly improved cellular
activity of 10 is most likely the result of enhanced mem-
brane permeability imparted by the less planar, more
three-dimensional structure imparted by the macrocyclic
lactam ring system.
In summary, we have described the design and synthesis
of a novel class of macrocyclic pyrazolo[1,5-a] [1,3,5]tri-
azines as potent CK2 protein kinase inhibitors. These
compounds strongly inhibit cell growth in vitro in both
human prostate and colon cancer cell lines. Compounds
with potent CK2 and cancer cell inhibitory activity are
currently being evaluated in animal xenograft models
for their anti-tumor activities in vivo.
Encouraged by the improvement of cellular activity, a
series of macrocyclic pyrazolotriazine derivatives (Table
1) was designed and synthesized. Based on the available
co-crystal structures, meta-position of the aniline points
to the solvent exposed region. Therefore, incorporation
of meta-amide groups might enhance their enzyme
inhibitory potency and increase solubility.
References and notes
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Preparation of this series of compounds is illustrated in
Scheme 2. Compound 11 was obtained by following a
similar procedure as described in Scheme 1. The nitrile
group was then converted to the ester. Upon saponifica-
tion, the di-acid compound 13 was obtained. The
cyclization and amide formation were carried out in a

Z. Nie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 619–623
623
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