Identification and Profiling of a Selective and Brain Penetrant Radioligand for in Vivo Target Occupancy Measurement of Casein Kinase 1 (CK1) Inhibitors

Article abstract of DOI:10.1021/acschemneuro.7b00155

To enable the clinical development of our CNS casein kinase 1 delta/epsilon (CK1δ/e) inhibitor project, we investigated the possibility of developing a CNS positron emission tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on the initial CK1δ/e inhibitor HTS hits with the goal of identifying a compound that would fulfill a set of recommended PET ligand criteria. We identified [³H]PF-5236216 (9) as a tool ligand that meets most of the key CNS PET attributes including high CNS MPO PET desirability score and kinase selectivity, CNS penetration, and low nonspecific binding. We further used [³H]-9 to determine the binding affinity for PF-670462, a literature CK1δ/e inhibitor tool compound. Lastly, [³H]-9 was used to measure in vivo target occupancy (TO) of PF-670462 in mouse and correlated TO with CK1δ/e in vivo pharmacology (circadian rhythm modulation).

Full text of DOI:10.1021/acschemneuro.7b00155

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Article
Identification and Profiling of a Selective and Brain Penetrant Radioligand for
In Vivo Target Occupancy Measurement of Casein Kinase 1 (CK1) Inhibitors
Travis T Wager, Paul Galatsis, Ramalakshmi Y. Chandrasekaran, Todd W Butler, Jianke Li, Lei
Zhang, Scot Mente, Chakrapani Subramanyam, Shenping Liu, Angela C Doran, Cheng Chang,
Katherine Fisher, Sarah Grimwood, Joseph R Hedde, Michael Marconi, and Klaas Schildknegt
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 13 Jun 2017
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Identification and Profiling of a Selective and Brain Penetrant Radioligand
for In Vivo Target Occupancy Measurement of Casein Kinase 1 (CK1)
Inhibitors
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Travis T. Wager*, Paul Galatsis, Ramalakshmi Y. Chandrasekaran, Todd W. Butler, Jianke Li,
Lei Zhang, Scot Mente, Chakrapani Subramanyam, Shenping Liu, Angela C. Doran, Cheng
Chang, Katherine Fisher, Sarah Grimwood, Joseph R. Hedde, Michael Marconi, Klaas
Schildknegt
Pfizer Worldwide Research and Development: Worldwide Medicinal Chemistry, 1 Portland,
Cambridge, MA 02139 and 558 Eastern Point Rd, Groton, CT 06340 (PG, LZ, RYC, TWB, JL,
SC, SM, SL, TTW); Pharmacokinetics, Dynamics, and Metabolism, 610 Main Street,
Cambridge, MA 02139 and 558 Eastern Point Rd, Groton, CT 06340 (ACD, CC); Internal
Medicine Research Unit, 1 Portland, Cambridge, MA 02139 (KF, SG, JRH, MM);
Radiochemistry, Pharmaceutical Sciences, 558 Eastern Point Rd, Groton, CT 06340 (KS)
*Tel: 857ꢀ225ꢀ2840. Fax 860ꢀ686ꢀ6052. Eꢀmail: travis.t.wager@pfizer.com.
KEYWORDS ꢀ Casein kinase 1, CK1, CK1δ/ε, delta, epsilon, kinase inhibitor, circadian rhythm,
radiotracer, target occupancy
a
Abbreviations: ADME, absorption, distribution, metabolism and excretion; AUC, area under the
curve; CK1, casein kinase 1; CK1i, Casein Kinase 1 Inhibitor; C , average drug concentration;
ave
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C , efficacious concentration; Cmax, maximum drug concentration; Cmpd, compound; CNS,
eff
central nervous system; circadian rhythm, CR; dark:dark, D:D; HLM, human liver microsomes;
Inh, inhibitor; MDR, multiꢀdrug resistance; MPO, multiꢀparameter optimization; Pꢀgp, Pꢀ
glycoprotein; SAR, structureꢀactivity relationship; wild type, WT.
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Abstract: To enable the clinical development of our CNS casein kinase 1 delta/epsilon
(CK1δ/ε) inhibitor project we investigated the possibility of developing a CNS positron emission
tomography (PET) radioligand. For this effort, we focused our design and synthesis efforts on
the initial CK1δ/ε inhibitor HTS hits with the goal of identifying a compound that would fulfill a
3
set of recommended PET ligand criteria. We identified [ H]PFꢀ5236216 (9) as a tool ligand that
meets most of the key CNS PET attributes including high CNS MPO PET desirability score and
3
kinase selectivity, CNS penetration and low nonꢀspecific binding. We further used [ H]ꢀ9 to
determine the binding affinity for PFꢀ670462, a literature CK1δ/ε inhibitor tool compound.
3
Lastly, [ H]ꢀ9 was used to measure in vivo target occupancy (TO) of PFꢀ670462 in mouse and
correlated TO with CK1δ/ε in vivo pharmacology (circadian rhythm modulation).
Introduction
A retrospective analysis of historical Pfizer Phase 2 programs led to the ‘three pillars of
1
survival’ model. In this model, the highest probability of achieving success in clinical studies
occurred when the preclinical to clinical translation of the three pillars was met: 1. exposure at
the target site of action, 2. binding to the pharmacological target, and 3. expression of
pharmacology. For CNS targets, PET ligands are the ‘gold standard’ for measuring central target
engagement (pillar 2) but the identification of suitable PET ligands has been challenging. The
identification of PET ligands, until very recently, was one of serendipity. Historically,
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compounds identified with the best potency towards the desired target were considered as the
most likely radiotracers, while little attention was paid to physicochemical properties, ADME
attributes and biological target density (Bmax). Recently, a rigorous analysis of known PET
tracers was undertaken to aid in the rational design and selection of radioligands for in vivo
target occupancy measurement. The data from this analysis informed the development of a
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prospective PET ligand design tool (PET CNS MPO). This set of CNS PET criteria enable a
focused compound design with properties best aligned to deliver PET ligands. These criteria
coupled with previous understanding around maximum concentration of target binding sites
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(
Bmax) and binding affinity (K ) requirement, formed the basis for our radioligand development
d
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strategy. Here, we set out to identify a CK1δ/ε selective [ H]ꢀradioligand to: a) provide key
brain Bmax and biodistribution information and inform the probability of the development of a
PET tool for this brain kinase target; b) allow for binding affinity and in vivo CK1δ/ε enzyme
target occupancy (TO) determination, which in turn could be used to establish a correlation
between target occupancy and in vivo circadian rhythm modulation.
Specifically, we set out to identify a radioligand tool compound that would satisfy the
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following criteria: 1) structurally amenable to [ H] and PET labeling ([ C] or [ F]); 2) physicalꢀ
chemical properties as defined by CNS PET MPO > 3/6; 3) potent affinity for CK1δ/ε and
ꢀ6
minimal offꢀtarget pharmacology; 4) alignment of ADME attributes (Papp AB > 5 x 10 cm/sec,
low transporter liability (Pgp and Bcrp efflux ratio < 2), and good brain availability (AUC
C /C ≥ 0.3); 5) moderate unbound drug fractions in brain and plasma (Fu,b > 0.05, Fu,p
b,u p,u
>
0.15). Herein, we report on the key compounds prepared, and the studies used to assess their
potential as CK1 target engagement ligands for in vivo target occupancy (TO) tracers and PET
tracers.
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Results and Discussion
We recently reported the discovery of casein kinase delta and epsilon (CK1δ/ε) inhibitor
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pyrazolylꢀpyridine chemotype 1 in a HTS screening campaign. Structurally, compound 1 was
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amenable for [ H] and PET radionuclide ([ C] and[ F]) incorporation, thus, represented a viable
starting point for target occupancy (TO) radioligand development. Compound 1 was a dual
inhibitor of CK1δ and CK1ε that had doubleꢀdigit nM enzyme affinity for CK1δ (27 nM) and
was roughly 5ꢀfold less potent at the structurally related isoform CK1ε (Table 1). Whole cell
potency was moderate at 126 nM for CK1δ and 1068 nM for CK1ε. Overall, compound 1 had
desirable physicalꢀchemical properties based on the CNS MPO desirability score (4.8/6) and thus
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had a high probability of having alignment of ADME attributes (Table 1).
Further, the
physicalꢀchemical properties reside in favorable range for PET consideration using the recently
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disclosed CNS PET MPO desirability tool. However, 1 lacked the desired offꢀtarget selectivity
profile as it showed potent activity at p38 which resulted in a less than 5ꢀfold selectivity ratio
(p38/CK1δ). File mining and medicinal chemistry efforts around 1 led to the discovery and
profiling of closeꢀin analogs 2ꢀ5 (Table 1). Substitution of the pyridine ring with a methyl group
at C2 or C3 (compounds 2 and 3) lead to a decrease in CK1δ/ε affinity. However, 2 had a
modest improvement in selectivity over p38 (9ꢀfold). This result is consistent with previous
studies that have shown that substitution at C2 of the pyridine with an amino group induces a
8
peptide flip in the hinge region of p38.
Improvement in both potency and selectivity was
achieved by incorporating a hydroxyl group on the C2 methyl substituent of 2 to yield 4. The
improvement in selectivity, may be a result of the primary hydroxyl of 4 interacting with the
4
carbonyl of Pro87 of CK1δ, which is not present in p38. Conversion of the primary alcohol of
4
to a methylated amine yielded compound 5. Compound 5 had similar CK1 inhibitory effects
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as 4 and had improved CNS PET MPO desirability score (5.6/6) and p38 selectivity (203 fold).
The favorable physicochemical properties improved CK1δ/ε potency and selectivity prompted
further profiling and optimization of this chemotype.
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Table 1. SAR for CK1 enzyme and whole cell and p38 enzyme
CNS MPO a
PET MPO)
hCK1δ
hCK1ε
50
hCK1δ
50
hCK1ε
50
p38
P38/
CK1δ
Cmpd
b
b
c
c
(
IC (nM)
IC (nM)
EC (nM)
EC (nM)
IC (nM)
50
50
4.8
(5.0)
27
(20ꢀ35)
126
(107ꢀ149)
143
(99ꢀ208)
1068
(708ꢀ1611)
127
(60ꢀ269)
1
2
3
4
5
9
4.6
37
(n=2)
164
(137ꢀ197)
424
(n=1)
340
(n=1)
ND
ND
(
4.9)
4.5
45
(21ꢀ91)
203
(140ꢀ294)
488
(n=1)
ND
ND
29
203
(5.0)
.3
5.0)
.8
(5.6)
5
11
(7ꢀ17)
68
(48ꢀ96)
135
(90ꢀ200)
853
(710ꢀ1025)
333
(n=1)
(
5
15
(11ꢀ20)
76
(61ꢀ95)
45
(28ꢀ71)
308
(218ꢀ434)
3040
(n=1)
5
a
Calculated CNS MPO and CNS PET MPO desirability scores were obtained using the published
2
, 6, 7
algorithm (maximum value of 6) .
Calculated physicochemical properties were obtained
using standard commercial packages: BioByte, version 4.3, for ClogP calculations,
b
ACD/Laboratories, version 12.1, for ClogD at pH 7.4, and pKa. Purified human CK1δ and
c
CK1ε in vitro enzymes assays. Human CK1δ and CK1ε mediated PER3 nuclear translocation
in cells was measured. Each experiment performed in triplicate, mean S.E.M. and data reported
as a mean inhibitory activity with (95% CI) from at least three separate experiments unless
otherwise noted.
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The next round of design focused on improving potency by rigidifying the molecule and
locking it into a bioactive conformation. Substitutions at both C2 and C3 of the pyridine were
tolerated and led to the hypothesis that further potency improvements could be achieved by
cyclizing the amino group of compound 5 onto the C3 position of the pyridine to yield aza
isoindoline 6 (Scheme 1).
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Scheme 1. Cyclic amine design strategy
Along the way to the aza isoindoline 6, key synthetic intermediates were also profiled in the
binding, selectivity and ADME assays. The Nꢀbenzylimide 7 was the first compound included in
this profiling and provided a clear indication that the pyridineꢀdione was not well tolerated
(Table 2). This result is not unexpected, in that, the additional carbonyl at the C2 position could
lead to an electronic clash with the hinge backbone carbonyl of Leu85. In sharp contrast to 7,
lactam intermediate 8 displayed good CK1δ inhibitory potency (IC = 8 nM) and selectivity vs.
5
0
p38 (70 fold). Improved CK1δ/ε potency were observed with the Nꢀmethylated lactam 9 (PFꢀ
236216). Compound 9 was the first compound that we identified that exhibited single digit nM
5
potency in the CK1δ enzyme assay (8 nM) and maintained good CK1δ whole cell potency (36
nM) in this series. Importantly, 9 achieved >100ꢀfold selectivity vs. p38 while maintaining an
excellent CNS MPO desirability score (6/6) and a PET MPO desirability score (5.5/6). The
final targeted compound 6 had a similar CNS PET MPO desirability score as 9. However, it was
less active in the CK1 enzyme assays and whole cell assays.
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Table 2. SAR for CK1 enzyme and whole cell and p38 enzyme
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hCK1δ
hCK1ε
IC50c
hCK1δ
EC50b
(nM)
hCK1ε
EC50c
p38
IC50
(nM)
CNS MPO a
PET MPO)
P38/
CK1δ
Cmpd
IC50b
(
(
nM)
(nM)
(nM)
5.7
43
232
(113ꢀ478)
369
(161ꢀ836)
1541
(n=2)
>1884
(nꢀ2)
6
7
8
>44
ND
(
(
(
5.5)
.2
2.9)
.8
(15ꢀ118)
677
(853ꢀ
1
4
>4000
>9000
>10000
ND
3
299)
5
10
(9ꢀ12)
56
(50ꢀ64)
107
(93ꢀ123)
630
(567ꢀ701)
701
(n=2)
70
5.9)
9
6.0
(5.5)
8
36
(31ꢀ43)
58
(49ꢀ68)
318
(282ꢀ351)
>
861
>114
(PFꢀ5236216)
(7ꢀ9)
a
Calculated CNS MPO and CNS PET MPO desirability scores were obtained using the published
2
, 6, 7
algorithm (maximum value of 6).
Calculated physicochemical properties were obtained
using standard commercial packages: BioByte, version 4.3, for ClogP calculations,
b
ACD/Laboratories, version 12.1, for ClogD at pH 7.4, and pKa. Purified human CK1δ and
c
CK1ε in vitro enzymes assays. Human CK1δ and CK1ε mediated PER3 nuclear translocation in
cells was measured. Each experiment performed in triplicate, mean S.E.M. and data reported as
a mean inhibitory activity with (95% CI) from at least three separate experiments unless
otherwise noted.
To gain a better understanding of the origin of the increased potency and subtype selectivity
of 9, we obtained an Xꢀray structure of 9 bound to CK1δ (Figure 1). The pyridine ring of 9
forms a single point Hꢀbond acceptor interaction (2.8 Å) with the backbone NH of Leu85 hinge
residue. The fluorophenyl group pokes into a hydrophobic pocket that forms adjacent to the
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gatekeeper (Met82) residue and also has a favorable stacking interaction between the pꢀfluoro
phenyl group and Met82. Presumably, these favorable interactions contributed to the overall
improvement in potency and kinome selectivity. The ‘free’ pyrazole nitrogen of 9 forms a waterꢀ
mediated Hꢀbond interaction with the conserved Lys38 residue.
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Figure 1. Xꢀray coꢀcrystal structure showing the binding of CK1δ/ε inhibitor 9 to CK1δ (PDB
code INSERT HERE).
From this radioligandꢀspecific SAR effort, 5 and 9 were identified as the best two
radioligand leads, and followꢀup work was warranted. Compound 5 and 9 were screened in a
100 kinase target promiscuity panel to understand selectivity beyond p38, (Figure 2), see
supporting information for more details (Table s1). Four targets were identified as offꢀtarget hits
for either 5 or 9 at 1 ꢀM, including p38. Followꢀup IC work confirmed ZC3, CK1α, p38 and
5
0
ZC1 as offꢀtarget kinases (Table 3). For both 5 and 9 CK1α was the most active offꢀtarget
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pharmacology observed with enzyme IC values of 1059 and 630 nM, respectively. Overall,
5
0
both compounds exhibited excellent selectivity (≥ 70 fold) in this 100 kinase promiscuity panel.
CK1δ
ZC3
CK1α
p38
ZC1
0% at 1 ꢀM
50%
100%
Cmpd
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9
Figure 2. A) Percent inhibition of a 100 kinase target promiscuity panel at 1 µM for compound
and 9. Heat map colors for this continuum: green, 0% inhibition; yellow, 50% inhibition; red,
5
100% inhibition.
a
Table 3. Offꢀtarget selectivity profiling IC format and selectivity
5
0
%Inh
(
Selectivity
1ꢀM) CK1δ/target
Cmpd Kinase
IC (nM)
50
5
5
5
5
CK1α
p38
1059
3040
34
21
21
ꢀ1
71
203
ZC1
ZC3
>10000
>10200
>667
>680
9
9
9
9
CK1α
p38
630
861
51
37
65
42
79
108
109
197
ZC1
ZC3
873
1573
a
IC determination for the four targets hit in the promiscuity panel with inhibition >30% for
5
0
compound 9, along with the matched set for compound 5.
Previously, we demonstrated that ADME attributes are a critical component for the selection
of a CNS PET tracer. The set of ADME attributes identified includes passive permeability,
transporter liability, fraction unbound drug in plasma and brain compartments and CNS
penetration. To further differentiate between 5 and 9 we profiled them in inꢀvitro and inꢀvivo
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5
5
5
5
5
6
ADME assays. Compound 5 had moderate human liver microsome clearance (HLM) (18
mL/min/mg) and high rat liver microsome clearance (RLM) (64.5 mL/min/mg); in contrast 9 had
low HLM and RLM clearance (Table 4). Both compounds had favorable high passive
0
1
2
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8
9
0
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3
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8
9
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7
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9
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8
9
0
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2
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5
6
7
8
9
0
ꢀ6
permeability (Papp AB > 10 x 10 cm/sec) values. Transporter liability was higher for 5 (Pgp
BA/AB = 2.2 and Bcrp BA/AB = 4.2) than 9 (Pgp and Bcrp BA/AB ≤ 2). The fraction of
unbound drug for both 5 and 9 was within an acceptable range (≥ 0.05) in plasma and brain
tissue. Lastly, both compounds had good brain penetration as assessed by AUC C /Cp,u in
b,u
mouse. One potential advantage of 9 was that it had significantly higher free brain concentration
(Cmax,b,u) at the fixed dose of 56 mg/kg, achieving a free brain concentration of 8685 nM.
Table 4. ADME property summary of for 5 and 9
c
Mouse
Fu,p
Rat
Fu,b
C
AUC0ꢀ∞ AUC0ꢀ∞
a
b
d
f
max,b,u
Cmpd
5
Papp
g
HLM
18
RLM
64.5
<39
Pꢀgp
2.2
Bcrp
b
C /Cp
C /C
b,u p,u
[nM]
17.9
30
4.2
0.39
0.38
0.09
0.24
1480
3.1
0.7
0.7
9
<9
1.2
1.9
8685
0.7
a
b
Human liver microsomal clearance (mL/min/mg). Rat liver microsomal clearance
c
ꢀ6
d
(
mL/min/mg). Passive permeability (Papp AB x 10 cm/sec) determined by RRCK. Borst
f
g
MDR1 Efflux Ratio (BA/AB). Mouse Bcrp Efflux Ratio (BA/AB). Mouse Cmax,b,u values are
reported as maximum free drug concentration in brain at a 56 mg/kg dose, s.c.
While both compounds showed favorable properties as radioligand leads, compound 9
was selected for additional evaluation based on slightly better in vitro casein kinase inhibitor
(CK1i) potency and higher Fu,b to minimize the risk of nonꢀspecific binding. As shown in
3
Scheme 2, 9 was synthesized using standard alkylation chemistry. Treatment of 8 with [ H]ꢀMeI
3
in the presence of Cs CO yielded [ H]ꢀ9 with high specific activity.
2
3
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Scheme 2. Synthesis of [ H]ꢀ9
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Pilot experiments were performed to determine the optimal experimental parameters for
performing the in vitro TO, including extract concentrations, relative binding to different brain
regions, time and temperature for incubation. Similar to immunolocalization data, CK1δ/ε is
expressed throughout the brain with the greatest concentration in the hypothalamus. Saturation
binding experiments in the hypothalamus were performed to assess crossꢀspecies Bmax and K as
d
shown in Figure 3. CK1δ/ε Bmax values were comparable between rodent species (rat 12.5 nM
and mouse 10.7 nM), but lower in the monkey (6.7 nM). Binding potential (K ) values were
d
double digit nM (10.2 nM) for rodent species and single digit nM (3.0 nM) in monkey. The
Bmax/K ratio was low (<10 fold) for all species with the highest ratio observed in the monkey
d
(2.2 fold). The CK1δ/ε Bmax (3ꢀ5 nM) is in a reasonable range for PET consideration; however,
additional potency improvement (i.e. >5 fold) would be necessary to show sufficient specific
3
binding in PET imaging. Continued profiling of the suitability of [ H]ꢀ9 for in vivo TO
measurement in rodents was pursued in order to develop a relationship between TO and
circadian rhythm activity.
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Bmax
fmol/mg)
Bmaxb
(nM)
Kd
(nM)
Cmpd
9
Species
Rat
a
a
Bmax/Kd
1.2
(
250.2
(236.4ꢀ264.9)
10.2
(8.6ꢀ11.7)
12.5
10.7
6.7
214.2
10.4
(8.2ꢀ12.6)
Mouse
Monkey
1.0
(
198.7ꢀ229.6)
135
(126.0ꢀ144.0)
3.0
(2.3ꢀ3.7)
2.2
a
3
Figure 3. Saturation [ H]ꢀ9 binding curves for rat, mouse, and monkey brain hypothalamus
tissue. Each experiment was performed in triplicate; mean S.E.M. Bmax and K values were
d
b
determined by GraphPad Prism analyses. Bmax (fmol/mg) values were converted to nM
9
concentrations assuming 50 mg protein/g wet tissue.
Previously, we reported the in vivo pharmacology of PFꢀ670462 in rodent circadian
10, 11
rhythm models.
PFꢀ670462 treatment showed a dose response lengthening of the period in a
phaseꢀspecific manner and extended the duration of PER2ꢀmediated transcriptional feedback.
3
Using [ H]ꢀ9 we investigated the brain exposure to TO relationship and associated target
3
engagement to the pharmacodynamic response (circadian rhythm phase delay). [ H]ꢀ9 showed a
robust specific binding window enabling inꢀvivo TO studies. A doseꢀresponsive increase in TO
was observed for the CK1 inhibitor PFꢀ670462 (Table 5). PFꢀ670462 achieved a 100% TO with
a dose of 100 mg/kg (s.c.) and 81% TO with 10 mg/kg (s.c.). The estimated free brain
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concentration required to achieve 50% TO of the enzyme was 62 nM and is in line with the
whole cell CK1δ inhibitory activity (WC EC = 58 nM). In general, ≥50% CK1δ/ε in vivo TO
5
0
resulted in a 2 hr CR phase delay (Figure 4A). Figure 4B is a representative example of the
pharmacodynamic response of PFꢀ670462 in mouse after administering 32 mg/kg (s.c., 3 days of
dosing). In vivo CR modulation demonstrated expression of CK1i pharmacology (Pillar 3).
Together these results enabled us to assess the relationship of target engagement of CK1
inhibitor PFꢀ670462 in brain with the associated pharmacokinetic and pharmacodynamic end
points.
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Table 5. CK1i brain receptor occupancy from brain binding techniques in mouse for PFꢀ670462
3
using [ H]ꢀ9
Rat
Cb,u
CK1δ/ε
IVTOa
Treatment
(mg/kg, sc)
Compound
(nM)
(%)
100
1022
210
78
100
81
10
3.2
31
b
EC50
62 ± 27 nM
a
Brain region evaluated: rat hypothalamus (mean ± SEM, n = 3–4). Rat in vivo TO analysis with
3
[ H]ꢀ9 and E fixed at 100%.
max
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Figure 4. A) Brain region evaluated: rat hypothalamus brain receptor occupancy curves from
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the in vivo TO protocol in rat. Rat IVTO analysis with [ H]ꢀ9 as tracer and Emax fixed at 100%.
The black triangle data points represent actual measured TO and brain PK from individual
animals. The solid line is the dataꢀfitted TO model. The blue circle represents exposure, Cave
(over 4hrs) and TO (67% IVTO) to yield a 4 hr phase shift in dark: dark (D:D) mouse in vivo
circadian rhythm model (32 mg/Kg s.c. after 3ꢀdays dosing of PFꢀ670462). B) Representative
scaled temperature actograms for PFꢀ670462 reveal significant period lengthening by PFꢀ670462
in WT animals after 3 d of dosing, previously reported. The phase shift depicted was ꢀ3.70 hours
following dosing for 3 days in DD.
Conclusions
As part of our clinical development strategy for the CK1i program, we investigated the
possibility of developing a CNS PET tracer. For this effort we leveraged the initial CK1i HTS
hits to fuel our design and synthesis plans with the goal of identifying a compound that would
fulfill a set of recommended PET ligand design criteria, Table 5. In the initial triage of potential
starting points, we focused on the Nꢀmethyl pyrazole chemotype in part due to ease of
3
11
incorporating a [ H] and [ C] labels. Initial SAR yielded amine 5 that had a double digit IC
50
for CK1δ and was over 100 fold selective for CK1 over other kinases. Additional SAR work
yielded a novel synthetic intermediate 9 that was single digit IC for CK1δ and greater than 70
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fold selective over related CK1α isoform. The physicochemical properties of 9 were in
favorable range for a PET ligand (CNS PET MPO > 3/6) with an overall CNS PET MPO
desirability score of 5.5/6. Consistent with high passive permeability and low transporter
10
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liabilities (Pgp and Bcrp), brain penetration was excellent (mouse AUC C /Cp,u = 0.7). Using
b,u
Fu,b as a surrogate for nonꢀspecific binding, 9 was predicted to have low nonꢀspecific binding and
3
3
was confirmed in vivo using [ H]ꢀ9. Using [ H]ꢀ9 saturation curves for rat, mouse, and monkey
for brain hypothalamus tissue, experimental Bmax, and K values were determined. In monkey,
d
the Bmax and K values were 6.7 nM and 3.0 nM, respectively and a B /K ratio of 2.2. While 9
d
max
d
does not meet the Bmax/K criteria for CNS PET ligand development, it proved to be instrumental
d
in generating a fundamental understanding of TO to free brain concentration for the CK1i tool
3
compound PFꢀ670462 (EC = 62 nM). Further, [ H]ꢀ9 was used to connect TO and the CR
50
1
behavioral output (Pillar 3). In an in vivo CR model a 32 mg/kg s.c. dose yielded a free brain
exposures sufficient to occupy 67% of the CK1 enzyme which resulted in a 3.7 hr phase shift.
Further work will be required to improve CK1 potency (∼5 fold) while retaining the desirable
PET attributes of 9 to identify a CNS PET ligand.
Table 5. Summary of CNS PET attributes for 9.
a
Property
Attribute
Target Value
>3
Value
Physicochemical
Properties
PET MPO
5.5
Synthetic
Enabled Label
Sites
3
11
[ H], [ C],
19
1 site
2 sites
[
F]
Papp
MDR ER
Bcrp ER
> 10
≤ 2.5
≤ 2.5
30
1.2
1.9
Brain
Permeability
C /C
b,u p,u
≥ 0.3
0.7
Fu,p
Fu,b
≥ 0.15
≥ 0.05
≥ 0.15 and ≥ 0.05
>30x
0.38
0.24
Yes
>70x
Nonꢀspecific
Binding
Fu,b and F
u,p
Pharmacology Selectivity
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Bmax/Kd
≥ 10x
2.2
a
Color code: red, fail to meet target value and green, meet target value.
Chemistry
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The preparation of compounds 6ꢀ9 is outlined in Scheme 3. Suzuki coupling of 3ꢀfluoroꢀ4ꢀ
4
iodopicolinonitrile with pinacol boronate 10 proceeded in modest yield to give the diarylated
pyrazole 11. Treatment of 11 with potassium cyanide afforded the dinitrile 12 via SNAr
displacement in quantitative yield. The nitriles were then hydrolyzed with 5N aqueous
potassium hydroxide at 100 °C. Upon cooling, the dipotassium salt of the diacid product 13
precipitated from solution allowing for isolation via filtration. Refluxing the diacid salt in a 1:1
mixture of acetic anhydride and acetic acid gave the phthalic anhydride 14 which was isolated
after concentration by trituration with diethyl ether. This material was used without further
purification due to its reactivity with water and instability on silica gel. Compounds 7, 15 and 16
were accessed by the addition of primary amines into the anhydride and then subsequent in situ
ring closure using acetic acid/acetic anhydride. Selective reduction of the less hindered carbonyl
with zinc dust in hot acetic acid yielded the lactams 8 and 9.
a
Scheme 3. Synthesis of 7, 8, and 9
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a
Reagents and Conditions: (a) 3ꢀfluoroꢀ4ꢀiodopicolinonitrile, 1.2 equiv of Cs CO , DMF,
2
3
Pd (dba) , 50 °C, 6 h (59%); (b) KCN, DMSO, 50 °C, 2 h (quant.); (c) 5N aq KOH, 100 °C, 24 h
2
3
(
88%); (d) Ac O, HOAc, THF, 110 °C, 3 h; (e) Ac O, HOAc, BnNH (7, 75%), or NH OH (15,
2 2 2 4
9
2%) or MeNH (16, 93%), 80ꢀ120 °C, 2 h; (f) 5 equiv of Zn dust, HOAc, 110 °C, 1.5ꢀ4.5 h, (8,
2
4
1%),(9, 53%)
Azaisoindoline 6 was prepared as outlined in Scheme 4. Protection of azaisoindoline 17 as the
sulfonamide 18 was achieved by treatment with methanesulfonyl chloride. Nꢀoxide 19 was
prepared via mCPBA oxidation. Chlorination with oxalyl chloride gave the desired regioisomer
20 in 44% yield with 14% of the corresponding 2ꢀchloro isomer which was readily separated by
chromatography. Suzuki coupling with pinacol boronate 10 afforded the protected azaisoindoline
template 21. Cleavage of the methylsulfonamide in a 1:1 mixture of refluxing 48% hydrobromic
acid and acetic acid provided the unprotected azaisoindoline 22. Subsequent reductive amination
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6
with aqueous formaldehyde and sodium triacetoxyborohydride cleanly yielded the Nꢀmethylated
product 6.
a
Scheme 4. Synthesis of 6
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
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0
O
Cl
N
O
O
a
b
c
O
N
S
O
NH
N S
O
N+
O-
N
S
N
N
17
18
19
20
N
N
N
N
N
N
d
e
f
O
O
S
N
NH
N
F
F
F
N
N
N
2
1
22
6
a
Reagents and Conditions: (a) methanesulfonyl chloride, DIPEA, methylene chloride, ꢀ10 °C to
rt, 18 h (quant.); (b) 2 equiv of mCPBA, methylene chloride, rt, 20 h (95%); (c) oxalyl chloride,
DMF, rt, 18 h (44%); (d) 1.2 equiv of 10, 3 equiv of LiOH, DMF, 4 mol% of Pd(dppf)Cl , 100
2
°
C, 2 h (59%); (e) 48% aq. HBr, HOAc, reflux, 18 h (63%); (f) 37% aq. formaldehyde,
methylene chloride, STAB, rt, 18 h (73%)
Experimental section
General Information. All solvents and reagents were obtained from commercial sources and
were used as received. All reactions were followed by TLC (TLC plates F254, Merck) or LCMS
(liquid chromatography−mass spectrometry) analysis. Varian 300, 400, or 500 MHz instruments
1
13
were used to obtain H and C NMR spectra in deuterated solvents. All proton shifts are
reported in δ units (ppm) and were measured relative to signals for chloroform (7.27 ppm),
1
3
methanol (3.31 ppm) or deuterium oxide (4.75 ppm). All C shifts are reported in δ units (ppm)
1
relative to the signals for chloroform (77.0 ppm) or methanol (49.2 ppm) with H decoupling. All
coupling constants (J values) are reported in hertz (Hz). GCMS were recorded on an HP 6890
GC system equipped with a 5973 mass selective detector (stationary phase, HPꢀ1, fused silica, 12
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m × 0.202 mm × 0.33 ꢁm; temperature limits, −60 to 325 °C; ramp rate = 30 °C/min; solvent
delay = 0.4 min). Analytical analyses by UPLC were performed on a Waters Acquity system
with PDA detection (UV 210 nm) at 45 °C and flow rate 0.5 mL/min, with a gradient of 95/5
buffer/acetonitrile (0−7.55 min), 10/90 buffer/acetonitrile (7.55−7.85 min), 95/5
buffer/acetonitrile (8.10−10.30 min) using the following columns and buffers: Waters BEH C8
column (2.1 × 100 mm, 1.7 um) with 50 mM sodium perchlorate/0.1% phosphoric acid or 10
mM ammonium bicarbonate as buffer; Waters BEH RP C18 column (2.1 × 100 mm, 1.7 um) or
Waters HSS T3 (2.1 × 100 mm, 1.8 um) column with 0.1% methanesulfonic acid buffer. Mass
spectra were recorded on a Micromass ADM atmospheric pressure chemical ionization
instrument (MS, APCI). Highꢀresolution mass spectra were obtained on an Agilent 6620 LCꢀMS
TOF equipped with a Zorbax Eclipse column (50 mm × 4.6 mm, 1.8 ꢁm XDBꢀC18) using 0.1%
formic acid aqueous as mobile phase A1 and acetonitrile containing 0.1% formic acid as mobile
phase B1. Column chromatography was carried out on silica gel 60 (32−60 mesh, 60 Å) or on
prepacked Biotage or ISCO columns. The purities of final compounds 6ꢀ9 as measured by UPLC
were found to be above 95%.
10
11
12
13
14
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19
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3ꢀFluoroꢀ4ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)picolinonitrile
(11).
3ꢀ(4ꢀ
4
Fluorophenyl)ꢀ1ꢀmethylꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole (10) (14.5
g, 48.0 mmol) and 3ꢀfluoroꢀ4ꢀiodopicolinonitrile (9.92 g, 40.0 mmol) were combined in 100 mL
DMF and treated with Cs CO (19.90 g, 61.2 mmol). The resulting suspension was sparged with
2
3
nitrogen for 20 min and treated with tris(dibenzylideneacetone)dipalladium(0) (1.51 g, 1.6
mmol) in a single portion. The nitrogen sparging was continued for an additional 20 min, and
stirring of the dark suspension was continued for 30 min at rt. The reaction mixture was warmed
to 50 ºC for 6 h and was allowed to cool to rt overnight. The thick slurry was added to 150 mL
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EtOAc, the suspension was diluted with 50 mL 50% saturated aqueous sodium chloride, treated
with decolorizing carbon, and stirred 1h at rt. The mixture was filtered through celite, the layers
were separated, and the organic layer was washed with 3 x 30 mL 50% saturated aqueous
0
1
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0
NaCl. The organic layer was dried over anh. MgSO and was concentrated in vacuo to give 18 g
4
of a pasty orange solid. The solid was dissolved in a minimum amount of methylene chloride,
loaded onto a 100 g SNAP cartridge, and the crude material was eluted over a 340 g SNAP
cartridge with a 5ꢀ80% EtOAc/heptane gradient over 4.8 L. The appropriate fractions were
combined and concentrated. During concentration, a white solid precipitated. This was
collected to give 6.93 g (59%) of the title compound product as a white solid. APCI MS m/z
1
2
97.0 (M+1). H NMR (CDCl , 400 MHz,): δ 8.28 (d, J = 5.1, 1H), 7.84 (d, J = 2.9, 1H), 7.40
3
(dd, J = 5.3, 2.2, 2H), 7.25 (dd, J = 5.8, 4.9, 1H), 7.09 (dd, J = 8.6, 8.6, 2H), 4.02 (s, 3H).
+
HRMS: calcd for C H F N 297.0946 (M + H) , found 297.0941.
1
6
10
2
4
4ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridineꢀ2,3ꢀdicarbonitrile
(12).
Fluorocyanopyridine 11 (10.00 g, 33.8 mmol) was dissolved in DMSO (100 mL) with gentle
heating. Potassium cyanide (2.35 g, 35.0 mmol) was added and the mixture was heated to 50 ºC
and stirred. After 2 h, the reaction was cooled in an ice bath and 0.1N aqueous NaOH (50 mL)
was added. The resulting slurry was stirred for 5 min, the solids were collected, rinsed with water
1
and airꢀdried to yield 10.34 g (quant) of the title compound as a light cream colored solid. H
NMR (CDCl , 400 MHz,): δ 8.56 (d, J = 5.2, 1H), 8.02 (s, 1H), 7.33 (dd, J = 8.9, 5.2, 2H), 7.24
3
(
d, J = 5.5, 1H), 7.06 (dd, J = 8.6, 8.6, 2H), 4.02 (s, 3H). HRMS: calcd for C H FN 304.0993
17 10 5
+
(M + H) , found 304.0991.
ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)pyridineꢀ2,3ꢀdicarboxylic
4
acid,
dipotassium salt (13). Pyridinedinitrile 12 (10.34 g, 34.1 mmol) and aqueous KOH (28.3 g, 504
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mmol, dissolved in 100 mL water) were heated at 100 ºC for 24 h. The mixture was cooled in ice
to give a thick white slurry. The material was warmed back to rt, filtered and air dried to yield
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1
2.47 g (88%) of the title compound as a cream colored solid. LCMS m/z 342.1 (M+1). H NMR
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(
D O, 400 MHz,): δ 8.15 (d, J = 5.1, 1H), 7.77 (s, 1H), 7.39 (dd, J = 9.0, 5.5, 2H), 7.08 (dd, J =
2
9.0, 9.0, 2H), 6.99 (d, J = 5.1, 1H), 3.92 (s, 3H).
ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)furo[3,4ꢀb]pyridineꢀ5,7ꢀdione (14).
4
Diacid 13 (5.00 g, 12.0 mmol) was slurried with acetic acid (35 mL) and acetic anhydride (12.5
mL) and heated to 110 ºC for 3h. The resulting homogeneous mixture was cooled and
concentrated, slurried with 50 mL diethyl ether and reꢀconcentrated to yield the title compound
as a sticky yellow solid. This material was used without purification in the next step. An
analytical sample was purified by chromatography (ISCO 12g, heptane/EtOAc 6/4 to 0/1 in 10
1
min) to give a white solid. GCMS m/z 323.0 (M). H NMR (CDCl , 400 MHz,): δ 8.78 (d, J =
3
5
.3, 1H), 8.30 (s, 1H), 7.41 (dd, J = 8.8, 5.3, 2H), 7.34 (d, J = 5.3, 1H), 7.11 (dd, J = 8.6, 8.6,
2
H), 4.08 (s, 3H).
6ꢀBenzylꢀ4ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ5Hꢀpyrrolo[3,4ꢀb]pyridineꢀ
5
,7(6H)ꢀdione (7). Phthalic anhydride 14 (472 mg, 1.46 mmol) and benzyl amine (0.167 mL,
1
.53 mmol) in acetic acid (4.7 mL) were heated at reflux for 18 h, cooled and then concentrated
to a brown solid. This material was stirred with diethyl ether (20 mL) for 10 min and then
filtered to yield 450 mg (75%) of title compound as a tan colored solid. LCMS m/z 413.5 (M+1).
1
H NMR (CDCl , 400 MHz,): δ: 8.57 (d, J = 5.2, 1H), 8.15 (s, 1H), 7.26ꢀ7.42 (m, 7H), 7.13 (d, J
3
13
=
5.3, 1H), 7.01 (dd, J = 8.6, 8.6, 2H), 4.87 (s, 2H), 4.00 (s, 3H). C NMR (CDCl , 100 MHz): δ
3
1
66.4, 165.6, 162.8 (d, J FꢀC = 248.1), 153.8, 152.8, 150.1, 139.9, 135.8, 134.4, 130.2 (d, J FꢀC
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.1), 128.6ꢀ128.5 (m, doublet and two singlets overlapping), 127.9, 127.8, 122.1, 115.7 (d, J FꢀC
+
=
21.4), 111.8, 41.7, 39.3. HRMS: calcd for C H FN O 413.1408 (M + H) , found 413.1408.
24 17
4
2
4ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ5Hꢀpyrrolo[3,4ꢀb]pyridineꢀ5,7(6H)ꢀdione
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(15). Phthalic anhydride 14 (190 mg, 0.588 mmol) and 28ꢀ30% aqueous ammonium hydroxide
(4 mL) were heated to a vigorous reflux for 30 min and then concentrated. The residue was
dissolved in 1:1 acetic acid/acetic anhydride (20 mL) and heated at 120 ºC for 2hr and
concentrated. The residue was dissolved in EtOAc and run through a short silica gel plug to
afford 176 mg (92%) of title compound as a white solid which was used without further
1
purification. LCMS m/z 323.1 (M+1). H NMR (MeOHꢀd , 400 MHz,): δ 8.62 (d, J = 5.3, 1H),
4
8.26 (s, 1H), 7.42 (dd, J = 9.0, 5.3, 2H), 7.27 (d, J = 5.3, 1H), 7.10 (dd, J = 9.0, 9.0, 2H), 4.02 (s,
3H).
4
ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ6ꢀmethylꢀ5Hꢀpyrrolo[3,4ꢀb]pyridineꢀ
,7(6H)ꢀdione (16). Phthalic anhydride 14 (275.3 g, 659.4 mmoles), acetic acid (2.06 L) and
5
acetic anhydride (138 mL, 1456 mmol) were heated at 110 ºC for 1 hr, cooled to 80 ºC and then
methylamine (2.0M/THF, 659 mL; 567.1 g) was added over 20 min, maintaining the temperature
at 80 ºC. A white smoke was observed during the addition. The reaction was heated to 100 ºC
overnight and concentrated to approximately 500 mL giving a clear yellow solution. Water (200
mL) was added over 10 min with stirring. Acetic acid (~ 100 mL) and an additional 200 mL of
water were added to aid stirring. The solids were removed by filtration using a Buchner funnel
with cloth and washed with 250 mL water. The material was air dried for 1h then dried in a
vacuum oven at 60 ºC overnight to yield 206.5 g (93.1%) of title compound as a light yellow
1
solid. LCMS m/z 337.1 (M+1). H NMR (CDCl , 400 MHz,): δ 8.61 (d, J = 5.5, 1H), 8.22 (s,
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H), 7.41 (dd, J = 9.0, 5.5, 2H), 7.1 (d, J = 5.3, 1H), 7.07 (dd, J = 8.8, 8.8, 2H), 4.05 (s, 3H),
+
.26 (s, 3H). HRMS: calcd for C H FN O 337.1095 (M + H) , found 337.1093.
18 14
4
2
ꢀ(3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ6,7ꢀdihydroꢀ5Hꢀpyrrolo[3,4ꢀb]pyridinꢀ5ꢀ
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11
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one (8). Phthalimide 15 (176 mg, 0.55 mmol) and zinc dust (178 mg, 2.73 mmol) in glacial
acetic acid (2.4 mL) were heated at 110 °C for 1.5h. After cooling, the mixture was filtered
through celite with HOAc rinse and concentrated to yield a sticky orange solid. Silica gel
chromatography using a 100% methylene chloride to 10% MeOH/ methylene chloride gradient
over 10 min gave the title compound (120 mg, 71%) as a white solid. LCMS m/z 309.1 (M+1).
1
H NMR (CDCl 400 MHz): δ 8.40 (d, J = 5.4, 1H), 8.21 (s, 1H), 7.37ꢀ7.49 (m, 2H), 6.95ꢀ7.03
3,
1
3
(m, 3H), 6.14 (br s, 1H), 4.74 (s, 2H), 3.98 (s, 3H). C NMR (MeOHꢀd , 100 MHz): δ 171.1,
4
1
3
3
4
67.1, 164.3 (d, JFꢀC = 246.5), 152.7, 151.0, 142.3, 136.4, 131.7 (d, JFꢀC = 8.8), 130.7 (d, JFꢀC
.8), 126.0, 123.7, 116.6 (d, JFꢀC = 22.0), 114.2, 47.7, 39.3. HRMS: calcd for C H FN O
2
=
17 13
4
+
09.1146 (M + H) , found 309.1144.
ꢀ[3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl]ꢀ6ꢀmethylꢀ6,7ꢀdihydroꢀ5Hꢀpyrrolo[3,4ꢀ
b]pyridinꢀ5ꢀone (9). To a flask equipped with overhead stirring was added 16 (202.0 g, 600.6
mmoles) and zinc dust (<10 micron) (357.0 g, 5.41 moles) followed by acetic acid (2.02 L). The
mixture was heated to 105 ºC and stirred for 4.5 hr, then cooled to rt. Celite was added to the
reaction mixture, and this was filtered to remove the zinc The filter pad was rinsed with EtOAc
and the filtrate was concentrated to ~ 300 mL, then 200 mL of water was added. The resulting
solid was collected, rinsed with water, and dried under vacuum overnight to give 103.2 g (53%)
1
of the title compound as a solid. LCMS m/z 323.2 (M+1). H NMR (CDCl , 400 MHz): δ 8.38
3
(d, J = 5.5, 1H), 8.28 (s, 1H), 7.41 (dd, J = 8.8, 5.5, 2H), 7.03 (dd, J = 8.6, 8.6, 2H), 6.96 (d, J =
1
3
5.2, 1H), 4.44 (s, 2H), 4.01 (s, 3H), 3.24 (s, 3H). C NMR (MeOHꢀd , 100 MHz): δ 165.2, 164.5
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(
d, JFꢀC = 247.4), 159.1, 152.1, 150.0, 142.2, 138.9, 131.9 (d, JFꢀC = 8.8), 129.5 (d, JFꢀC = 3.0),
1
3
6
27.0, 126.9, 116.9 (d, JFꢀC = 22.1), 112.4, 50.7, 39.5, 29.7. HRMS: calcd for C H FN O
18 15 4
+
23.0508 (M + H) , found 323.1298.
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ꢀ(Methylsulfonyl)ꢀ6,7ꢀdihydroꢀ5Hꢀpyrrolo[3,4ꢀb]pyridine (18). Methanesulfonyl chloride
(0.97 mL, 12.4 mmol) was added dropwise to a ꢀ10 ºC solution of diisopropylꢀethylamine ( 5.16
mL, 31.1 mmol) and 17 (2.0 g, 10.0 mmol) in methylene chloride (20 mL). The mixture was
stirred overnight at rt, diluted with methylene chloride (300 mL) and washed with saturated
aqueous sodium bicarbonate. The organic phase was separated, dried (MgSO ) and filtered
4
through a short silica gel pad with EtOAc rinse. The filtrate was concentrated to yield 2.15 g
(quantitative) of title compound as a solid which was used without further purification. LCMS
1
m/z 199.2 (M+1). H NMR (CDCl 400 MHz): δ 8.54 (d, J = 4.9, 1H), 7.61 (d, J = 7.8, 1H), 7.25
3,
(dd, J = 7.8, 5.1, 1H), 4.75 (AB quartet, J AB = 2.1, ꢂνAB = 7.8, 4H), 2.94 (s, 3H). HRMS: calcd
+
for C H N O S 199.0536 (M + H) , found 199.0530.
8
10
2
2
6
ꢀ(Methylsulfonyl)ꢀ6,7ꢀdihydroꢀ5Hꢀpyrrolo[3,4ꢀb]pyridine 1ꢀoxide (19). mꢀChloroperbenzoic
acid (70% purity, 2.94 g, 11.9 mmol) was added to a solution of 18 (2.15 g, 10.84 mmol) in
methylene chloride (43 mL). After 2h a gummy solid had formed and LCMS showed ~50 %
conversion. Chloroform (20 mL) was added to help solubility and an additional equivalent of mꢀ
chloroperbenzoic acid (2.8 g) was added and the stirring was continued for another 18h at rt to
drive the reaction to completion. The crude reaction mixture was loaded onto a silica gel plug,
flushed with EtOAc (500 mLꢀ discarded) and then eluted with 50% MeOH/EtOAc (2L) to give
the desired product. Concentration yielded the title compound (2.22 g, 95%) as a white solid.
1
LCMS m/z 215.0 (M+1). H NMR (CDCl 400 MHz): δ 8.16 (d, J = 6.5, 1H), 7.29 (dd, J = 7.5,
3
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.5, 1H), 7.18 (d, J = 7.8, 1H), 4.82ꢀ4.89 (m, 4H), 2.96 (s, 3H). HRMS: calcd for C H N O S
8
10
2
3
+
15.0485 (M + H) , found 215.0480.
ꢀChloroꢀ6ꢀ(methylsulfonyl)ꢀ6,7ꢀdihydroꢀ5Hꢀpyrrolo[3,4ꢀb]pyridine (20). Oxalyl chloride
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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(
0.415 mL, 4.67 mmol) was added dropwise to a 0 ºC suspension of 19 (500 mg, 2.33 mmol) in
DMF (40 mL). The resulting mixture was warmed to rt and stirred for 18 hr. Water was added
slowly to quench the excess oxalyl chloride and the mixture was extracted into methylene
chloride (250 mL). The extract was washed with brine, dried (MgSO ) and concentrated to yield
4
a brown gum which was then purified by silica gel chromatography using a heptanes (100%) to
5
0% EtOAc/heptanes gradient. Regioisomer 2ꢀchloroꢀ6ꢀ(methylsulfonyl)ꢀ6,7ꢀdihydroꢀ5Hꢀ
pyrrolo[3,4ꢀb]pyridine (76 mg, 14%) eluted from the column first followed by the desired title
1
compound (240 mg, 44%, white solid). H NMR (CDCl 400 MHz): δ 8.45 (d, J = 5.5, 1H), 7.26
3,
(d partially obscured by residual CHCl peak, 1H), 4.80 (br s, 4H), 2.97 (s, 3H). HRMS: calcd
3
+
for C H ClN O S 233.0146 (M + H) , found 233.0143.
8
9
2
2
4
ꢀ[3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl]ꢀ6ꢀ(methylsulfonyl)ꢀ6,7ꢀdihydroꢀ5Hꢀ
4
pyrrolo[3,4ꢀb]pyridine (21). 20 (100 mg, 0.43 mmol), pinacol borane 10, (156 mg, 0.516
mmol), LiOH (30.9 mg, 1.29 mmol) and dichloro 1,1’ꢀbis(diphenylphosphino)ferrocene
palladium (II) (35.1 mg, 0.043 mmol) were combined as solids and degassed by evacuating and
back filling with nitrogen gas (3 times). DMF (30 mL) was added and the mixture was heated for
2
hr at 100 ºC. Following cooling to rt, EtOAc was added and the mixture was filtered (celite)
with EtOAc rinse. The filtrate was washed with water and brine, dried (MgSO ) and
4
concentrated. Silica gel chromatography using a 40ꢀ80% EtOAc/heptanes gradient afforded 102
1
mg (64%) of title compound as a light brown solid. LCMS m/z 373.1 (M+1); H NMR (CDCl
3,
4
00 MHz): δ 8.39 (d, J = 5.5, 1H), 7.52 (s, 1H), 7.5 (dd, J = 8.6, 5.5, 2H), 6.99ꢀ7.03 (m, 3H),
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.68 (br s, 2H), 4.39 (br s, 2H), 3.99 (s, 3H), 3.16 (s, 3H). HRMS: calcd for C H FN O S
1
8
17
4
2
+
373.1129 (M + H) , found 373.1128.
4ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)isoindoline (22). A mixture of 21 (1.40g,
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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.76 mmol) in 48% aq. hydrobromic acid (15 mL) and glacial acetic acid (15 mL) was refluxed
for 18h, After cooling to rt, the mixture was diluted with water (10ml) and brought to pH ~12
with 1N aq. NaOH. The aqueous was extracted into 3:1 chloroform/isopropyl alcohol (4 x 50
ml), dried over anh. Na SO , filtered and concentrated. The crude product was purified by
2
4
chromatography (ISCO 220 g, 2M ammonia in methanol in methylene chloride from 0% to
0%). Pure product fractions were combined, concentrated and recrystallized from methylꢀtꢀ
1
butylether to yield 696 mg (62.9%) of title compound as a gray solid. LCMS m/z 295.4 (M+1);
1
H NMR (CDCl 400 MHz): δ 8.29 (d, J = 5.07, 1H), 7.44 (s, 1H), 7.31ꢀ7.39 (m, 2H), 6.93ꢀ7.04
3
,
(m, 2H), 6.90 (d, J = 5.07, 1H), 4.23 (s, 2H), 3.96 (s, 3H), 3.93 (s, 2H), 2.25 (s, 1H). HRMS:
+
calcd for C H FN 295.1354 (M + H) , found 295.1348.
1
7
15
4
4
ꢀ(3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀmethylꢀ1Hꢀpyrazolꢀ4ꢀyl)ꢀ2ꢀmethylisoindoline
(6).
Sodium
triacetoxyborohydride (639 mg, 3.02 mmol) was added to a solution of 22 (222 mg, 0.754 mmol)
and 37% aqueous formaldehyde solution (0.12 mL, 1.55 mmol) in methylene chloride (5 mL).
After stirring at rt for 20 min, the mixture was concentrated and the residue was partitioned
between EtOAc and sat NaHCO , the organics were washed with brine, dried (MgSO ) and
3
4
treated with decolorizing carbon, filtered (celite) and concentrated to yield 171 mg (73%) of a
1
light yellow glassy solid. H NMR (CDCl 400 MHz): δ 8.29 (d, J = 5.1, 1H), 7.45 (s, 1H), 7.40
3
,
(dd, J = 9.0, 5.5, 2H), 7.02 (dd, J = 9.0, 9.0, 2H), 6.91 (d, J = 5.1, 1H), 4.00 (s, 3H), 3.98 (s, 2H),
1
3
3.69 (s, 2H), 2.54 (s, 3H). C NMR (CDCl , 100 MHz): δ 162.55 (d, J = 248.0), 162.5,
3
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48.6, 148.0, 135.9, 132.2, 130.7, 129.5 (d, JF-C = 8.1), 129.0 (d, JF-C =2.9), 122.0, 116.2, 115.4
+
(d, JF-C = 21.3), 61.6, 59.1, 42.4, 39.1. HRMS: calcd for C H FN 309.1510 (M + H) , found
18 17
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[
H]ꢀ9 Preparation. A sealed ampule containing a solution of tritiated methyl iodide (500 mCi,
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0 Ci/mmol, 6.3 ꢀmol) in 0.5 mL of DMF was cooled in an iceꢀbath, opened and placed under
positive nitrogen pressure. A solution of 8 (5.3 mg, 17.2 ꢀmol, 2.7 eq) in 0.5 mL of DMF was
added followed by solid cesium carbonate (8 mg, 24.6 ꢀmol, 3.9 eq). The ice bath was removed
and the reaction mixture was stirred at room temperature for 4 hours. The reaction was
quenched by addition of 0.5 mL of water and 1 mL of brine, and the resulting solution was
extracted with 3 x 4 mL of EtOAc. The combined organic layers were concentrated in vacuo to
yield 35 mCi of nonꢀvolatile radioactivity that contained low radiochemical purity [3H]ꢀ9. 22
mCi of this crude product was purified by reverse phase HPLC according to the following
method: Column:Luna C18, 5 ꢀm, 2.0 x 50 mm, Column temperature: ambient, Mobile phase A:
0.1% formic acid in H2O, Mobile phase B: AcN, Injection volume, solvent: 20 ꢀL, can,
Gradient: 0 min 15% B, 1 min 15% B, 8 min 60% B, 8.1 min 15% B, Run time: 12 min,
Detection: 210 nm. Desired product fractions were combined and concentrated to yield 4.8 mCi
of >98% radiochemical purity [3H]ꢀ9. Product specific activity was determined to be 70.7
Ci/mmol by mass spectrometry.
CK1δ/ε Enzyme Cell Free (Biochemical Assay). The CK1δ and CK1ε kinase assays were
performed in a 20 µl final volume in buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl , 1
2
mM DTT, 100 µg/mL BSA, 1 mM TCEP, 1.25% Glycerol, 0.0125% P104, 2.5 mM KCl, 10 µM
ATP, and 42 µM peptide substrate PLSRTLpSVASLPGL (Flotow et al.,1990). The final
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enzyme concentrations were 2 nM for CK1δ and 2.5 nM for CK1ε. Assays were run in a panel
format in the presence of 1 µL of compounds or 4% DMSO. The reactions were incubated for
60 min at 25°C for CK1δ and 85 min at 25°C for CK1ε, followed by detection with use of 20 µL
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of KinaseꢀGlo Plus Assay reagent according to the manufacturer’s instructions. Luminescent
output was measured on the PerkinꢀElmer Envision plate reader.
CK1δ/ε Whole Cell – WCA (Translocation Assay). COSꢀ7 cells were maintained at 37°C in
5% CO in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine
2
serum. Logꢀphase cells were dislodged with 5ꢀminute treatment of TrypLE Express and viable
cell count was determined with Cedex cell counter. Cells were diluted in DMEM medium to a
density of 1.5E5 viable cells/mL in 2/3 of the final volume of final transfected cell mix. Cells
were cotransfected with two plasmid DNAs, mPER3ꢀGFP in pd2EGFPꢀN1 vector and human
CK1 in pcDNA4/hisA vector (for CK1ε at a ratio of 1:5 respectively for CK1δ at a ratio of 1:11
respectively), using Lipofectamine 2000 reagent according to the manufacturer's
recommendations. The transfection mix contained approximately 0.83 ug/mL of DNA and 6
uL/mL of Lipofectamine 2000, in a total of 1/3 of the final transfection volume in OptiꢀMEM
medium. After 20 min at room temperature, the cell mix was combined with the DNA
transfection mix, per manufacturer instructions. Transfected cell suspension (50 mL) was
dispensed per well by multidrop dispenser into Greiner 384ꢀwell Cellcoat (PDL) plates.
Compounds were diluted with OptiꢀMEM I to a 4X concentration before addition to plated cells.
Inhibitors of CK1δ/ε were tested at a 11 concentrations to evaluate their ability to inhibit
mPER3ꢀGFP translocation to the nucleus. After overnight exposure at 37°C in a CO incubator,
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cells were fixed by the addition of 12% paraformaldehyde in phosphateꢀbuffered saline (PBS)
with 20% sucrose to a final concentration of 4%, and then the cells were incubated for 30 min at
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room temperature. The fixative was removed, and cells were washed with PBS and then stained
with 0.4 µg/ml Hoechst dye in blocking buffer containing 4% goat serum and 0.1% TritonX for
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hour. In some experiments, GFP enhancing antibodies were used when needed for signal
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amplification. Cells were washed again with PBS and stored at 4°C in PBS or scanned
immediately with the Cellomics ArrayScan VTI.
Animals
All procedures involving animals were conducted with the approval of the Pfizer
Institutional Animal Care and Use Committee. Male C57BL/6J mice from Jackson Laboratories,
9ꢀ12 weeks of age, were housed on a 12 hour light/dark cycle and given food and water ad
libitum. Animals were allowed to acclimate for 5 days after arrival and were always used within
10 days of arrival.
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[
H]ꢀ9 Preparation. [ H]ꢀ9 was prepared by the Pfizer Radiochemical Synthesis Group in
Groton, CT. Studies were performed using lot 00703442ꢀ029ꢀrvc001, 70.7 Ci/mmol, 0.770
3
mCi/mL in ethanol. The [ H]ꢀ9 dosing solution was prepared by diluting the appropriate amount
of stock solution in 0.9% saline to achieve a 100 ꢀCi/mL solution.
Dosing. Mice received a 10 mL/kg subcutaneous (s.c.) injection of PFꢀ670462 at 30 minutes
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prior to euthanasia. They then received a 3 mL/kg intraorbital injection of [ H]ꢀ9 at 1 minute
prior to euthanasia. PFꢀ670462 (100 mg/kg, s.c.) was used to determine the level of nonꢀspecific
binding. Note that all doses were corrected for salt content of the drug.
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[
H]ꢀ9 Saturation Binding Procedure. Homogenized hypothalamus was added to assay buffer
(50 mM Tris, 2 mM MgCl2, pH 7.4, 20 mg/ml ) and incubated with twelve different
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concentrations of [ H]ꢀ9 at 4 °C for 30 min. The reaction was terminated by rapid filtration
through 0.5% PEI soaked GF/B filters using a 96ꢀwell Brandel harvester. The filters were
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