Structure and property based design of pyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase with activity in vivo

Article abstract of DOI:10.1021/ml400197u

In this letter, we describe the design, synthesis, and structure-activity relationship of 5-anilinopyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase. Property-based optimization of early leads using the 7-oxetan-3-yl amino group led to a series of matched molecular pairs with lower lipophilicity, decreased affinity for human plasma proteins, and reduced binding to the hERG ion channel. Agents in this study were shown to modulate pAKT^S129, a direct substrate of CK2, in vitro and in vivo, and exhibited tumor growth inhibition when administered orally in a murine DLD-1 xenograft.

Full text of DOI:10.1021/ml400197u

Letter
pubs.acs.org/acsmedchemlett
Structure and Property Based Design of Pyrazolo[1,5-a]pyrimidine
Inhibitors of CK2 Kinase with Activity in Vivo
James E. Dowling,* Marat Alimzhanov, Larry Bao, Michael H. Block, Claudio Chuaqui, Emma L. Cooke,
Christopher R. Denz, Alex Hird, Shan Huang, Nicholas A. Larsen, Bo Peng, Timothy W. Pontz,
Caroline Rivard-Costa, Jamal Carlos Saeh, Kumar Thakur, Qing Ye, Tao Zhang, and Paul D. Lyne
AstraZeneca, Oncology Innovative Medicines Unit, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: In this letter, we describe the design, synthesis,
and structure−activity relationship of 5-anilinopyrazolo[1,5-
a]pyrimidine inhibitors of CK2 kinase. Property-based
optimization of early leads using the 7-oxetan-3-yl amino
group led to a series of matched molecular pairs with lower
lipophilicity, decreased affinity for human plasma proteins, and
reduced binding to the hERG ion channel. Agents in this study
were shown to modulate pAKTS129, a direct substrate of CK2,
in vitro and in vivo, and exhibited tumor growth inhibition
when administered orally in a murine DLD-1 xenograft.
KEYWORDS: CK2 kinase, pyrazolo[1,5-a]pyrimidine, matched molecular pair, oxetane
he serine/threonine protein kinase CK2, a tetrameric
complex containing two catalytic (α or α′) and two
cellular activity. In subsequent designs, we proposed to release
the constraint in 1b and introduce a new conformational
constraint, exemplified by indoline 2, that would enforce the
crystallographically observed cisoid conformation of the
acetamide in 1 (Figure 1). Compound 2 is a potent inhibitor
T
regulatory (β) subunits, controls cell growth, proliferation, and
evasion of apoptosis by phosphorylation of a range of substrates
in critical cellular signaling pathways including PI3K
(phosphatidylinositol 3-kinase)/AKT (protein kinase B),
NFκB (nuclear factor kappa-light-chain-enhancer of activated
B cells) and Wnt (wingless type MMTV integration site
family).1−3 Overexpression of the CK2α subunit correlates with
tumor aggressiveness and disease severity in certain cancers,
while compensatory increases in CK2α′ levels have been
observed in response to RNAi treatment in mice.4,5 Several
academic and industrial research groups have been actively
engaged in developing small molecule inhibitors of CK2 to
provide further pharmacological validation of the compelling in
vitro and in vivo data amassed to date.6 The recent discovery of
CX-4945, a selective, orally available inhibitor of CK2 by
researchers from Cylene, represents an important first step in
evaluating the clinical potential of this novel target in man.7 We
have recently described the design of a series of conformation-
ally constrained inhibitors of CK2 containing the pyrazolo[1,5-
a]pyrimidine nucleus.8 Members of this series of compounds
exhibited potent inhibition of the enzyme, possessed a high
degree of kinase selectivity, and depleted cellular levels of
pAKTS129, a direct substrate of CK2 believed to hyperactivate
the AKT pathway.9 Although our attempts to enhance both
cellular potency and physical properties in this series were
unsuccessful, these studies resulted in an understanding of the
structure−property relationships within the scaffold and
provided additional insights into ligand−receptor binding. In
particular, we found that N-methylation of the acetamide of 1a,
to give ring-constrained analogue 1b, preserved enzymatic and
Figure 1. Design of conformationally constrained acetylindoline 2 and
ortho-substituted anilines (3).
Received: May 23, 2013
Accepted: July 3, 2013
© XXXX American Chemical Society
A
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of CK2 in biochemical and cellular assays; however, it exhibits
low aqueous solubility at physiological pH (sol. = 4 μM) and
high intrinsic clearance when incubated with rat liver
microsomes (CLint = 120 μL/min/mg). We postulated that
either the N-acetyl group or the methylene groups within the
newly introduced five-membered ring represented potential
sites of metabolism and chose instead to focus on other
variations of the pharmacophore. The potency of 2 established
that the position ortho to the acetamide could be function-
alized, and we subsequently prepared a series of N-(3-
aminoaryl)acetamide analogues (3) to evaluate the scope of
substituents that might be accommodated on the aryl ring
(Figure 1). Herein, we describe our efforts to build on the
enzymatic potency and kinase selectivity profile of 1a, while
striving for an acceptable balance of physical properties and
ADME characteristics that would enable us to carry out
pharmacodynamic and disease model studies using an orally
administered agent.
C6 alkyl substituted derivatives, the methyl (7b) isopropyl (7c)
and cyclopropyl (7d) analogues exhibit comparable activity in a
mechanistic cellular assay that measures levels of the CK2
substrate pAKTS129 in an ELISA format. Introduction of a
nitrogen atom in the aniline ring, to afford pyridyl analogues 7g
and 7h, reduced cellular potency relative to 7b, with a negligible
effect on solubility. We subsequently evaluated the effect of
varying the substituent at the hinge-binding N7 position and
found that five-membered heterocyclic substituents (7k,l)
preserved enzymatic activity but, with the exception of N-
methyl imidazole (7l), adversely affected solubility. With the
enzymatic and mechanistic cellular activity of compounds in
this series routinely reading out at or below the lower limits of
detection in the respective assays, we began to guide our
structure−activity relationship (SAR) studies with the aid of
phenotypic assays using colorectal (HCT-116, DLD-1) and
other cultured cell lines.
An X-ray cocrystal structure determination of 7l with
recombinant human CK2 at 2.2 Å resolution revealed the
inhibitor to bind as expected with N2 of the pyrazolo[1,5-
a]pyrimidine core and the C7 NH interacting with the hinge
region of the ATP-binding pocket (Figure 2).12 As noted
previously, critical to the high binding affinity of this inhibitor
class is the engagement with a buried water molecule adjacent
to the gatekeeper residue (Phe113). The energetically disfavored
cisoid conformation adopted by the acetamide enables the
carbonyl group to coordinate with the water molecule and with
Lys68 and permits formation of a hydrogen bond between the
acetamide NH and Asp175. The sum of these interactions, in
addition to further coordination of the water molecule by the
cyano group (3.0 Å) appears sufficient to compensate for the
higher energy form of the acetamide. Deletion of the 3-cyano
group from 7b to give des-cyano analogue 8 (not shown)
results in a >100-fold reduction in enzymatic potency (CK2
IC50 = 0.27 μM) and loss of measurable cellular activity
(pAKTS129 IC50 > 3.3 μM). A similar potency decrease was
observed with the des-cyano analogue of indole 1a.8 In
addition, we believe the increased potency of the anilino-
compounds relative to 1a is due to steric reinforcement of the
cisoid amide geometry by the ortho-substituent. Preorganiza-
tion of the amide in a conformation required for recognition by
the kinase presumably decreases the entropy loss upon binding
of the ligand and results in higher affinity. This substitution may
be essential for increased cellular activity as well; the growth
inhibitory effects (HCT-116 GI50s) reported by Polaris for a
series of related analogues lacking substitution ortho to the
acetamide are 2−3 log units weaker than those in this study.13
The N-methyl group of the imidazole in 7l is positioned at
the interface with the solvent-accessible surface and suggests
that further substitution at this position could be undertaken.
Through the intermediacy of hydroxyethyl derivatives 9a and
9b, we prepared a series of amine-containing analogues
(Scheme 2). While our earlier attempts to modify indole 1a
by introducing hydrophilic groups on the C7 substituent
resulted in decreased cellular potency, we were initially
encouraged to find that N-substituted analogues of 7l possessed
high enzymatic and cellular activity (Table 2). In the pyrazole
series (10a and 11a-c), these modifications resulted in a
The synthesis of 2 and related analogues was carried out by
adapting the convergent approach described in our earlier work
(Scheme 1).8 Installation of functionality at C7 was
Scheme 1a
a
Reagents and conditins: (a) amine, EtOH, 25 °C; (b) Pd(OAc)2,
Xantphos, Cs2CO3, DMA (N,N-dimethyl acetamide)/H2O (4:1), 150
°C, μwave, 0.5 h; (c) Pd2(dba)3, Xantphos, Cs2CO3, DMA (N,N-
dimethyl acetamide)/H2O (4:1), 150 °C, μwave, 0.5 h; (d) Pd2(dba)3,
di-tert-butyl(2′,4′6′-triisopropylbiphenyl-2-yl)phosphine, Cs2CO3,
DMA/H2O (4:1), 140 °C, μwave, 0.5 h; (e) KF, NMP, 150 °C 5 h.
accomplished by displacement of the 7-chloro group in 5,7-
dichloro-3-cyano-pyrazolo[1,5-a]pyrimidine (4) to afford inter-
mediates of general structure 5. Substitution at C5 was
subsequently achieved by palladium catalyzed10 or KF-
promoted coupling of 5 with the requisite N-(3-aminoaryl)-
acetamide (6) to furnish the desired analogues (7a−l).
Introduction of N-(3-aminophenyl)acetamides bearing
chloro (7a) and small alkyl groups (7b−d) at the 6-position
increased the half-maximum inhibitory potency below the limit
of detection (IC50 < 3 nM) in our enzymatic assay, which
measures the inhibition of recombinant human full length
CK2α mediated phosphorylation of a synthetic peptide
substrate at Km ATP concentration (Table 1).11 Among the
modest five- to 10-fold decrease in cellular potency (pAKTS129
)
yet maintained activity in the phenotypic assay in the sub-100
nM range. Despite possessing high aqueous solubility and low
intrinsic clearance, the plasma concentrations of 11b−d
following a single oral dose in the rat were extremely low
B
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Table 1. CK2 Biochemical and Cellular Potency for Compounds 7a−l
microsomal
b
a
a
a
a
a
a
compd CK2α IC50 (nM) pAKTS129 IC50 (nM) HCT-116 GI50 (nM) sol. (μM) LogD Hu PPB (% free)
stability
hERG IC50 (μM)
7a
7b
7c
7d
7e
7f
<3
<3
<3
<3
<3
<3
<3
<3
<3
<3
<3
<3
ND
2
300
81
<1
8
3.1
2.4
3.0
3.1
2.3
2.8
2.1
2.1
2.9
3.0
2.1
0.8
3
22
15
56
39
36
19
50
<4
47
31
<4
20
>33
21
8
ND
3
67
<1
<1
5
4
>33
19
21
2
2
57
13
9.6
15
12
2.4
4.3
12
21
16
2
12
7
ND
>33
19
7g
7h
7i
42
6
ND
300
600
410
ND
110
18
7
33
8
<1
5
11
7j
16
7k
7l
3
1
ND
ND
10
52
a
b
Mean value of two experiments. Deviations were within < 25%. Intrinsic clearance (CLint) determined from rat liver microsome incubations
(μL/min/mg); ND = not determined.
Scheme 2a
a
Reagents and conditions: (a) 2-(4-amino-1H-pyrazol-1-yl)ethanol,
iPr2NEt, EtOH, 25 °C; (b) 2-(4-amino-1H-imidazol-1-yl)ethanol,
Et3N, EtOH, 50 °C; (c) NMP, 140 °C, 5 h; (d) 4-methylbenzene-1-
sulfonyl chloride, Et3N, CH2Cl2, 0 °C, 12 h; (e) amine, MeCN, 25 °C,
12 h.
Figure 2. Human CK2 kinase in complex with 7l (PDB code: 4GUB)
as determined at 2.2 Å resolution. The structure is oriented with the
N-terminal lobe toward the top left, the C-terminal lobe toward the
bottom right, and residues of the hinge region on the bottom left. The
electron density (2Fo−Fc), contoured at 1σ, is shown as a wire mesh.
Polar interactions ≤3.0 Å are indicated as dotted lines and bound
water molecules are shown as red spheres.14
Table 2. Enzymatic/Cellular Activity and ADME/Physical
Property Data for 10a,b and 11a−f
CK2α
IC50 (nM)
HCT-116
GI50 (μM)
a
a
a
a
b
compd
sol. (μM) LogD CLint
10a
10b
11a
11b
11c
11d
11e
11f
<3
<3
<3
<3
<3
<3
<3
<3
0.08
0.04
0.07
0.10
0.97
0.65
0.65
0.67
22
1.8
1.6
2.3
2.5
1.3
1.5
1.7
1.4
17
43
80
65
31
25
30
63
83
860
3
(Cmax < 0.1 μM). This may be rationalized in part by
considering the low measured permeability of 11b (Papp A−B =
0.2 × 10−6 cm/s) and high efflux (efflux ratio = 100) in MDR1
expressing LLCK cells. Similarly functionalized imidazoles (10b
and 11d-f) exhibited a more significant drop off in cellular
potency and were not profiled in vivo.
We next focused on a strategy for achieving further reduction
in the lipophilicity of 7b and related compounds that would not
rely on introduction of basic groups. In recent years, oxetanes
10
84
110
120
a
Mean value of at least two experiments. Deviations were within <
25%. Intrinsic clearance determined from rat liver microsome
b
incubations (μL/min/mg).
C
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Table 3. Enzymatic/Cellular Activity and ADME/Physical Property Data for 12a−e
a
a
a
a
a
a
b
compd
CK2α IC50 (nM)
pAKTS129 IC50 (nM)
HCT-116 GI50 (μM)
Sol. (μM)
LogD
Hu PPB (% free)
CLint
hERG IC50 (μM)
12a
12b
12c
12d
12e
<3
<3
<3
<3
<3
ND
28
21
6
0.97
23
13
22
11
8
2.2
1.6
1.5
2.2
2.1
12
28
26
20
21
<4
17
6
ND
>33
>33
>33
>33
0.28
0.61
0.059
0.074
<4
<4
3
a
b
Mean value of at least two experiments. Deviations were within < 25%. Intrinsic clearance determined from rat liver microsome incubations (μL/
min/mg); ND = not determined.
Figure 3. Relationship between lipophilicity (logD), human plasma protein binding (log Ki), and microsomal stability (CLint) and the N7 substituent
in a series of matched molecular pairs. Markers are colored by the C5 substituent and arranged vertically according to N7 group.
have emerged in the synthetic and medicinal chemistry
literature as a versatile functional group with the ability to
modulate basicity, confer metabolic stability, and to reduce
lipophilicity in drug-like molecules.15,16
A series of N7 3-oxetanyl analogues (12a−e) was prepared
to compare the effect of this substitution on potency, physical
properties, and ADME characteristics. Consistent with our
expectations, in a series of matched molecular pairs, this
modification lowered logD by ∼0.8 units, significantly
decreased the fraction of compound bound by human plasma
proteins, increased metabolic stability in rat liver microsome
and hepatocyte incubations, and reduced activity toward the
hERG ion channel (Table 3 and Figure 3). These compounds
are potent inhibitors of CK2 (IC50 < 3 nM) but are 10-fold less
potent in the mechanistic and phenotypic assays than their N7
cyclopropyl counterparts and, as a consequence, oxetane 12b
possesses comparable activity to the isostructural N7 cyclobutyl
derivative 7i (pAKT IC50 = 0.03 μM).
D
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However, when the N7 oxetane group is combined with an
aniline bearing a cyclopropyl group ortho to the acetamide, as
in 12d and 12e, cellular potency is improved, presumably due
to an overall increase in lipophilicity. The pharmacokinetic
profiles of 12d (F = 47%) and 7b (F = 25%) in the rat (10 mg/
kg, PO) are characterized by good bioavailability and low
clearance although the plasma half-life of 12d (t1/2 = 1.4 h) is
decreased relative to that of 7b (t1/2 = 2.6 h) and may be
attributed to increased in vivo clearance (CL = 23 mL/min/
kg). Dissapointingly, the favorable PK characteriristics of 12d
were not shared by other analogues in this series; oxetanes 12b
and 12e exhibit reduced oral bioavailability (F < 5%) and
plasma levels (Cmax < 1 μM). These results may be explained, at
least in part, by considering the aqueous solubility data for
12a−e, which show little, if any, enhancement relative to the
cyclopropyl matched pairs.
model studies. Treatment of murine DLD-1 AKT1 over-
expressing xenografts with a single dose of 7b (10 mg/kg, PO)
results in inhibition of pAKTS129 at the 3 and 8 h time points,
but the effect is not sustained at 24 h. Treatment with 7e (12.5
mg/kg, PO) also depletes pAKTS129 at the early time points
and appears to have a partial effect at 24 h as determined by
Western blot analysis (Figure 5). Dose/response plots
Kinase selectivity profiling of 7b against a panel of 402
kinases revealed a high degree of selectivity (Figure 4).17
Figure 5. (a) Western blot analysis of pAKTS129 levels in tumor cell
lysates following administration of 7b (10 mg/kg, PO) and 7e (12.5
mg/kg, PO) in a DLD-1 AKT1 overexpressing murine xenograft
model. (b) Dose/response plot illustrating pAKTS129 levels (red) in
tumor cell lysates, as determined by ELISA, versus plasma
concentration of 7b (blue).
illustrating pAKTS129 inhibition following treatment with 7b,
as measured by ELISA of tumor cell lysates, indicate residual
inhibition of pAKTS129 at later time points, when no detectable
drug remains in plasma. Compound 7b showed limited tumor
growth inhibition when tested using a once daily 10 mg/kg oral
dose (TGI = 28%) or a twice daily 5 mg/kg oral dose (TGI =
37%) for 14 days in disease model studies using a murine DLD-
1 xenograft.
In particular, we have demonstrated that 3-amino oxetane
derivatives exhibit reduced lipophilicity, decreased affinity for
plasma proteins, and reduced hERG ion channel binding.
Additional studies to determine the relationship between
exposure and pharmacodynamic response for these and related
agents in vivo will be described in subsequent communica-
tions.19
Figure 4. Kinome-wide selectivity profile of 7b. Red spheres highlight
kinases for which the residual activity remaining after treatment with
the inhibitor at a concentration of 0.1 μM/L was less than 45% of
DMSO control using a competition binding assay that measures the
ability of a compound to compete with an immobilized, active-site
directed ligand (KINOMEscan, Discoverx, Inc., San Diego, CA).18
Limited off-target activity (10 kinases with >40% inhibition
following treatment with 7b at a concentration of 0.1 μM/L)
was observed against members of the CMGC family, including
isoforms of the dual-specificity tyrosine-regulated kinases
(Dyrk) and CAMK kinases such as the death-associated
protein kinase (Dapk) and histone interacting protein kinase
(Hipk) families. Biochemical IC50 determinations revealed an
activity pattern similar to that of indole 1a from our earlier
work with 7b exhibiting submicromolar activity against Hipk2
(IC50 = 40 nM), Dyrk3 (IC50 = 70 nM), and PIM3 (IC50 = 150
nM) with weaker effects on GSK3β (IC50 = 2.6 μM) and
CDK2 (IC50 = 7.2 μM).
ASSOCIATED CONTENT
■
S
* Supporting Information
Representative experimental procedures for synthesis of
analogues, biochemical and cellular assays, and X-ray structure
determination. This material is available free of charge via the
Internet at http://pubs.acs.org.
On the basis of their physical properties, pharmacokinetic
profiles in rodents and dog, and potency against a range of
cancer cell lines, 7b and 7e were chosen as probes to assess the
potential of the series in pharmacodynamic (PD) and disease
E
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ACS Medicinal Chemistry Letters
Letter
(16) Burkhard, J. A.; Wuitschik, G.; Rogers-Evans, M.; Muller, K.;
̈
AUTHOR INFORMATION
Corresponding Author
*(J.E.D.) Tel: 781-839-3900. E-mail: james.dowling@
astrazeneca.com.
Notes
■
Carreira, E. M. Oxetanes as versatile elements in drug discovery and
synthesis. Angew. Chem., Int. Ed. 2010, 49 (48), 9052−9067.
(17) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;
Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.;
Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.;
Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.;
Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase
inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127−132.
(18) Image generated using TREEspot Software Tool and reprinted
with permission from KINOMEscan, a division of DiscoveRx
Corporation, © DISCOVERX CORPORATION 2010.
(19) Dowling, J. E.; Alimzhanov, M.; Bao, L.; Chuaqui, C.; Denz, C.;
Ferguson, A.; Graff, C.; Liu, Z.-Y.; Lyne, P.; Racicot, V.; Wu, A.; Wu,
J.; Ye, Q.; Cooke, E. L. Discovery and characterization of AZ968, a
potent and selective inhibitor of CK2 kinase with effects on AKT
signaling in vivo. Poster presented at the 103rd Annual Meeting of the
American Association for Cancer Research, March 31−April 4, 2012.
Chicago, IL; Abstract No. 3907.
The authors declare no competing financial interest.
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