Development of dual casein kinase 1δ/1ε (CK1δ/ε) inhibitors for treatment of breast cancer

Article abstract of DOI:10.1016/j.bmc.2017.12.020

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers.

Full text of DOI:10.1016/j.bmc.2017.12.020

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of dual casein kinase 1d/1e (CK1d/e) inhibitors
for treatment of breast cancer
Andrii Monastyrskyi a, Napon Nilchan a, Victor Quereda b, Yoshihiko Noguchi a, Claudia Ruiz b,
Wayne Grant b, Michael Cameron b, Derek Duckett b, William Roush a,
⇑
a Department of Chemistry, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
b Department of Molecular Medicine, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Casein kinase 1d/
e
have been identified as promising therapeutic target for oncology application, includ-
Received 20 October 2017
Revised 9 December 2017
Accepted 14 December 2017
Available online xxxx
ing breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of
purine scaffold inhibitors that led to identification of two new CK1d/ inhibitors 17 and 28 displaying low
nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast can-
cer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of
principle animal xenograft studies against human cancers.
e
Keywords:
Casein kinase 1 delta and epsilon
Kinase
Ó 2017 Elsevier Ltd. All rights reserved.
Inhibitor
Structure-activity relationship
Breast cancer
1. Introduction
and the activation of Wnt transcription targets.13 At the same time,
forced expression of kinase defective CK1d mutants blocks SV40-
The casein kinase 1 (CK1) family consists of six monomeric ser-
ine/threonine-specific protein kinases ( , d, 1, 2 and
3).1 All
six human CK1 isoforms are highly homologous in their active site,
with the delta (CK1d) and epsilon (CK1 ) isoforms sharing 98%
driven cellular transformation in vitro and mammary carcinogene-
a
e,
c
c
c
sis in vivo.14
The Wnt/b-catenin pathway has known roles in breast cancer,
where: (i) Wnt signaling can contribute to triple negative breast
cancer; (ii) nuclear b-catenin connotes metastasis and poor out-
come; and (iii) b-catenin contributes to mutant ErbB2-driven
breast cancer.15 Importantly, gain-of-function (e.g., in b-catenin)
e
sequence identity in their protein kinase domains. Elsewhere in
the enzyme, such as in the C-terminal, and non-catalytic domains,
significant variances exist between isoforms.2 CK1 kinases play
crucial roles in regulating a variety of cellular growth and survival
processes including circadian rhythm,3 membrane trafficking,4
DNA damage repair,5 cytoskeleton maintenance,6 and notably
and loss-of-function (e.g., in CK1a APC and AXIN) mutations preva-
lent in other cancers are not found in breast cancer. Recent discov-
eries from our laboratories establish casein kinase 1 delta (CK1d) as
an essential regulator of b-catenin activity that is overexpressed
and amplified in human breast cancer.16
Wnt signaling.7 A recent study showed that CK1d/CK1
e might be
involved in the etiology of addictive behavior and that their inhibi-
tion prevents relapse-like alcohol drinking.8 Importantly, abnormal
regulation of these two CK1 family members is implicated in
CK1d and CK1
e are eminently tractable for small molecule tar-
geted drug discovery.17 Among the small molecules reported to
human cancers and several known CK1d and/or CK1
e
substrates
inhibit CK1d/CK1e
are CKI-7,18 (R)-CR8 and (R)-DRF053,19
control tumor cell growth, apoptosis, metabolism and differentia-
tion.9 For instance, both isoforms are overexpressed in pancreatic
ductal adenocarcinoma,10 ovarian cancer11 and chronic lympho-
cytic leukemia.12 Interestingly, expression of constitutively active,
IC261,20 D4476,21 LH846,22 benzimidazole 5,23 PF480056724 and
PF670462.3a,24 Unfortunately, most of these compounds are not
specific inhibitors of CK1d/CK1e and in some cases their
pharmacological effects are now known to be (or are suspected
myristoylated CK1e in mammary epithelial cells is sufficient to
to be) due to non-selective target engagement.20b,25 At the same
drive cell transformation in vitro via stabilization of b-catenin
time, the contribution of CK1d and CK1
not fully understood and the non-selective nature of previously
reported CK1d/ inhibitors has impeded pharmacological
validation of these kinases as anti-cancer targets.3a,20b
e to human cancer is still
e
⇑
Corresponding author.
E-mail address: roush@scripps.edu (W. Roush).
https://doi.org/10.1016/j.bmc.2017.12.020
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

2
A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Nonetheless, available data clearly shows that targeted inhibition
CK1d/e was also encountered by Shokat and colleagues when
of CK1d/CK1
e
is a viable, highly attractive anticancer strategy yet
developing analog-sensitive kinase technology.27
to be fully developed.26
Herein we report structure-activity and structure-property rela-
tionship (SAR and SPR) studies that led to identification of dual
selective CK1d/e inhibitors (including SR-3029) with physicochem-
ical properties and in vitro and in vivo pharmacokinetic parame-
ters suitable for use in murine xenograft studies against breast
cancer.26
In research targeting the development of novel therapeutics for
treatment of metastatic and resistant forms of cancer, the Roush
laboratory at Scripps Florida identified highly potent and selective
purine-derived dual inhibitors of CK1d and CK1
the NIH’s MLPCN program (Fig. 1).25 These agents induce prolifer-
ative arrest and rapid apoptosis of CK1d/CK1 -expressing human
e under the aegis of
e
luminal B, HER2+ and triple negative cancer cells ex vivo and SR-
3029 induces tumor regression in orthotopic xenograft models
in vivo.26 Specifically, genetic knockdown or pharmacological inhi-
bition of CK1d using SR-3029 blocks b-catenin nuclear localization
and TCF transcriptional activity, induces rapid apoptosis and pro-
vokes tumor regression in patient derived orthotopic models of tri-
ple negative breast cancer.25,26 Mechanistic studies are consistent
with the premise that CK1d controls breast cancer tumorigenesis
via its effect on b-catenin, which plays known roles as an effector
of aberrant Wnt signaling in human breast cancer.26
Noteworthy, profiling of 442 kinases with SR-3029 (and three
close analogs) confirmed their high selectivity vs. the plethora of
kinases whose activity is impaired by the purportedly specific Pfi-
zer CK1d inhibitor (PF670462).25 The four off-target kinases that
are inhibited by SR-3029 largely have no known function. High
selectivity of N9-arylsubstituted purine scaffold could be explained
2. Results and discussion
2.1. Synthetic chemistry
The general procedure for the synthesis of purine-based CK1d/
e
inhibitors is based on a previously reported sequence25,28 which
we improved by incorporating a more efficient method for aryla-
tion of the N9 nitrogen of dichloropurine (Scheme 1). Thus, N9 aryl
purine intermediates (permutations of structure 6) were generated
by copper (I) mediated coupling of commercially available
dichloropurine 4 with a variety of symmetrical or unsymmetrical
diaryliodonium salts 5.29 This reaction provides 6 in 65%–85% yield
– a substantial improvement relative to the previously employed
Chan-Lan coupling (yield 0–30%).25 The diaryliodonium salts were
accessed via one-step procedures from aryl iodides 1 and arenes 2
or boronic acids 3.30 Subsequently, substituted benzimidazoles 9
and various amines were introduced at C6 and C2 of 6, respec-
by the unique structural features of the CK1d/
ically, relatively small size of the gatekeeper residue Met82 in case
of both CK1d/ creates large hydrophobic pocket which is occupied
e active site. Specif-
tively. This procedure provided the targeted CK1d/e inhibitors with
e
excellent regioselectivity and good yields (70–90%).25 The substi-
tuted benzimidazoles (9) were accessed as previously described
from o-dianilines, 7.25,31
by N9 aryl residue of the inhibitors. This structural feature of
SR-653234
SR-3029
2.2. Structure-activity relationship (SAR) studies
F
We synthesized analogs in an iterative fashion using the chem-
istry outlined in Scheme 1, refining our compound design on the
basis of biochemical and biological data obtained from prior
rounds of tested inhibitors. Compounds with low nanomolar bio-
chemical affinity (IC50 < 100 nM, 50 mM ATP concentration) were
assessed in cell proliferation assays vs. MDA-MB-231 breast cancer
cell lines using 3-day MTT and longer term clonogenicity assays
(see Section 4 for details).25
F
HN
N
N
HN
N
N
NH
NH
N
N
N
N
N
N
N
N
O
O
Our SAR optimization efforts commenced with the synthesis of
agents with various N9 alkyl and aryl substitution (R1) (Table 1).
Alkyl substitution of the purine N9 position (11, 12) resulted in
F
S
Fig. 1. Structures of HTS hit SR-653234 and optimized probe SR-3029.
the complete loss of CK1d/e activity. Consequently, subsequent
Scheme 1. General strategy for the synthesis of N9-arylsubstituted CK1d/e inhibitors.
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Table 1
SAR data for CK1d/e inhibitors with alkyl and aryl substituents at N9 (R1).
№
R1
CK1d
CK1
e
MDA-MB-231 EC50 (nM)b
IC50 (nM)a
IC50 (nM)a
c
11
12
–Me
–Et
>10,000
4010
44
>10,000
6560
260
–
c
–
SR-3029 (13)
26
c
14
15
16
535
4520
48
395
990
80
–
c
–
101
68
17
53
145
18
19
20
10
16
38
c
525
110
250
135
–
6280
9790
21
230
175
a
Biochemical assay.
Cellular proliferation assay.
Not determined.
b
c
SAR efforts focused on aryl R1 substituents. Since we looked at only
one example of a N9-difluorophenyl substitution in our original
publication,25 other mono- and disubstituted fluorophenyl analogs
were explored. Notably, all the analogs with a N9 4-fluorophenyl
substitution resulted in an approximately 10-fold loss of activity
against CK1d compared to SR-3029 (14, 15, 19, Table 1). Other
difluorophenyl analogs 2,3-difluorophenyl 16, 3,5-difluorophenyl
17 and 2,5-difluorophenyl 18 showed similar activity to SR-3029
with 18 having a slightly better biochemical profile.
Introduction of other substituents at the ortho position of the
N9 aromatic moiety (R1), such as in inhibitors 20 and 21, resulted
in a modest reduction of biochemical activity (2-4-fold) but sub-
stantial loss of cell-based activity (EC50 = 6–10 lM, Table 1).
While performing the initial rounds of SAR optimization, we
noticed a moderate correlation between the calculated logD
(pH = 7.4) values of tested analogs and their antiproliferative
properties (Fig. 2, library of 224 inhibitors from Bibian et al.25
and related patent32). In particular, the majority of poorly active
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

4
A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
18
16
14
12
10
8
throughout this compound set. Compounds 36, 37 and 38, which
have an unsubstituted benzimidazole ring, showed decreased
CK1d/ inhibition (IC50 vs CK1d = 824–1566 nM) compared to other
analogs in this set (Table 3). Substitution of the benzimidazole with
either 5-chloro (39–41) or 5-methoxy (45–47) groups led to a
active EC50 < 750 nM
e
2–3-fold increase in CK1d/
nM) relative to the unsubstituted benzimidazole, derivatives 36–
38. Further improvement of CK1d/ inhibitor activity was achieved
e inhibition (IC50 vs CK1d = 336–624
e
through the incorporation of two fluorine atoms into the 5 and 6
positions of the benzimidazole ring (48, 49). Finally, the most
active compounds (IC50 vs CK1d < 100 nM) in this series were
obtained through the introduction of a 5,6-dichloro substitution
pattern (42–44) into the benzimidazole unit. These three analogs
were additionally tested in a cell based assay vs. MDA-MB-231
breast cancer cell lines, where they showed excellent antiprolifer-
ative properties (EC50 < 6 nM). Interestingly, compound 42 is
almost 100-fold more potent in the cell-based assay than in
in vitro tests against purified CK1d. This observation suggests that
42 may possibly be engaging additional kinases that contribute to
cell-based activity of this compound. This assumption, however,
remains to be validated experimentally.
6
4
2
0
1
2
3
4
5
LogD
70
inactive EC50 > 750
Lastly, inclusion of morpholine or piperazine units at C2 of the
60
50
40
30
20
10
0
purine scaffold generally conferred greater CK1d/e inhibition activ-
ity than compounds with N-methylpiperazine units at this
position.
In addition to SAR studies performed on the benzimidazole ring
itself, we also examined the possibility of using benzimidazole iso-
steres or replacing the benzimidazoles with different heterocycles
(Table 4). The benzimidazole unit (R5) was replaced with struc-
turally similar benzoxazole (50) and benzothiazole (51) units.
Despite evincing excellent biochemical activity (51, IC50 vs
CK1d = 9 nM), these two compounds were inactive in the cell based
assay (EC50 > 10 lM). Other inhibitors with benzimidazole replace-
ments, such as phenyl-substituted imidazole (52), oxazole (53) and
thiazole (54), were synthesized and tested in biological assays. Of
-2
0
2
4
6
these three compounds, 52 showed good activity against CK1d/
e
LogD
(IC50 vs CK1d = 214 nM). Unfortunately 52 displayed markedly
diminished potency in cell-based assays against MDA-MB-231
cancer cells (EC50 = 1400 nM). Replacing the benzimidazole
with 1-methyl-4-(pyridin-3-yl)piperazine (55) or 1-methyl-4-
phenylpiperazine (56) led to improvement in biochemical activity
Fig. 2. Correlation between calculated logD (pH 7.4) and antiproliferative proper-
ties (cell based assay, EC50, nM) of CK1d/ inhibitors available library (total 224
inhibitors including compounds from Bibian et al.25 and related patent31). A:
probability of logD distribution for active compounds (EC50 < 750 nM, n = 52). B:
probability of logD distribution for inactive compounds (EC50 > 750 nM, n = 172).
e
(IC50 of 56 vs CK1d/e = 50 nM).
Sadly, these compounds also displayed a significant disparity
between biochemical and cell-based potency (EC50 of 56 vs.
MDA-MB-231 = 7160 nM). Replacement of the benzimidazole
analogs (EC50 > 750 nM) were more lipophilic (logD > 4) than those
agents with significant activity. The logD distribution of com-
pounds with EC50 < 750 nM and compounds with EC50 > 750 nM
also suggests that partition coefficients peak around 2.5 for active
and 3.8 for inactive analogs (Fig. 2A, B).
unit with an imidazole decreased CK1d/
significantly (57). Finally, eschewing the benzimidazole in favor
of 2-pyridyl (58), 3-pyridyl (59), 4-methyl-2-pyridyl (60),
3-methyl-2-pyridyl (61) or 2-flurophenyl (62) groups resulted in
a series of highly potent CK1d/ inhibitors, but which were inactive
against MDA-MB-231 breast cancer cell lines (EC50 > 10 M).
e inhibition activity
a
In an effort to lower the lipophilicity of the CK1d/e inhibitors,
we synthesized and tested a series of N9 heteroaryl-substituted
analogs (Table 2). Although N9 thiophene substitution resulted in
inhibitors with low nanomolar values in both biochemical and cell
based assays (22 and 23), an unsubstituted thiophene is generally
considered to be a metabolic liability.33 To avoid this potential
problem, we selected alternate groups that could be used at the
purine N9 position (R1) to lower lipophilicity while, hopefully,
e
l
We tested a subset of eleven compounds for their ability to per-
meate cell membranes (PAMPA assay, see Section 4 for details).
The majority of the analogs tested possessed low permeability of
Papp ꢀ 2.5ꢁ10ꢂ6 cm/s (Table 4), suggesting that this property could
be a significant contributor to the disconnect between biochemical
and cell-based activity for this analogs series.
maintaining CK1d/e inhibitory activity. Replacement of the thio-
phene ring by a variety of pyridyl groups retained the logD values
We completed this series of SAR studies of the purine core by
varying the substitution at the C2 position (Table 5). Analogs 63
and 64 retained good potency but lost antiproliferative activity.
Next, the morpholine unit of SR-3029 was exchanged for a
thiomorpholine (65) and its oxidized variant (66), which led to a
2–3-fold decrease in CK1d/e biochemical and cell based activities.
We also introduced a number of different piperidines at the R4
position to obtain compounds 67, 68, 73, 75 and 76. Each of these
agents displayed moderate biochemical activity against CK1d/e.
in the targeted range (ꢀ3.5), however all these compounds were
less potent against CK1d/e (IC50 > 143) compared to either 23 or
SR-3029.
Contemporaneously, we sought to vary the substitution on the
benzimidazole unit (R5). The data presented in Table 3 are for one
series in which the purine N9 substituent was held constant as m-
pyridyl. Additionally three different amines (N-methylpiperazine,
morpholine and piperazine; Table 3) were utilized at purine C2
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 2
SAR and clogD data for CK1d/e inhibitors with heteroaryl substituents at N9 (R1).
№
R1
R6
CK1d
CK1
e
MDA-MB-231
EC50 (nM)b
LogDc
IC50 (nM)a
IC50 (nM)a
22
N-Me
47
125
75
2.6
23
24
25
26
27
28
29
30
31
32
O
54
105
1145
500
860
1905
315
385
660
245
860
43
3.0
1.8
2.8
2.4
3.3
3.0
3.3
3.0
3.4
2.7
d
N-Me
O
700
335
260
440
175
260
295
145
1120
–
d
–
d
N-Me
O
–
d
–
N-Me
O
28
d
–
d
N-Me
O
–
8
d
N-Me
–
d
33
O
>9900
1195
–
3.1
d
34
35
N-Me
295
385
245
–
3.2
3.5
d
O
1195
–
a
b
c
Biochemical assay.
Cellular proliferation assay.
Calculated with Pipeline Pilot workflow application (Accelrys) at pH 7.4.
Not determined.
d
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

6
A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 3
SAR data for CK1d/e inhibitors with a substituted benzimidazole unit (R5).
№
R5
R6
CK1d
CK1
e
MDA-MB-231
EC50 (nM)b
IC50 (nM)a
IC50 (nM)a
c
36
N-Me
1565
1155
825
2955
–
c
37
38
39
O
2095
1840
630
–
c
NH
N-Me
–
c
335
–
c
40
41
42
43
44
O
520
430
100
50
715
915
320
105
255
–
c
NH
N-Me
O
–
<1
4
NH
80
6
c
45
46
47
48
N-Me
O
610
520
625
200
335
975
1765
660
–
c
–
c
NH
–
N-Me
1040
60
49
O
175
340
a
Biochemical assay.
Cellular proliferation assay.
Not determined.
b
c
The best compound in this series was 68, which evinced an IC50 of
69 nM against CK1d and 133 nM in the MDA-MB-321 cellular
assay. Extending the morpholine unit by two or three carbons from
the purine core resulted in compounds 70 and 71, both of which
were are approximately 20-fold less active than SR-3029. Finally,
tetrahydropyran derivative 69 and butyl analog 72 did not show
any improvement in activity (69, IC50 vs CK1d = 321 nM; 72, IC50
vs CK1d = 1031 nM).
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Table 4
SAR and apparent cell permeability data for CK1d/e inhibitors with bioisosters or heterocyclic replacements of the benzimidazole unit (R5).
№
R5
CK1d
CK1
e
MDA-MB-231
EC50 (nM)b
PAMPAc
IC50 (nM)a
IC50 (nM)a
Pappꢁ10ꢂ6
13
44
260
26
0.4
50
51
52
42
9
150
115
280
>10,000
>10,000
1400
<0.1
<0.1
<0.1
215
d
53
54
>10,000
>10,000
>10,000
>10,000
–
<0.1
<0.1
d
–
55
56
57
245
50
380
54
1850
0.3
7.1
0.1
7160
180
205
>10,000
d
58
59
60
61
85
98
80
37
–
>10,000
>10,000
6460
6.8
d
d
–
–
d
d
–
–
d
d
–
>10,000
–
d
62
89
–
>10,000
<0.1
a
b
c
Biochemical assay.
Cellular proliferation assay.
Permeability, Parallel Artificial Membrane Permeability Assay (PAMPA), see Section 4 for details.
Not determined.
d
2.3. Physicochemical and ADME properties
ranitidine as controls and analyzed by HPLC-MS/MS. A large set
of the most promising CK1d/ inhibitors was additionally tested
e
In addition to testing inhibitory activity against CK1d/
antiproliferative activity against MDA-MB-231 breast cancer cell
lines, standard physicochemical properties including logP, logD7.4
and TPSA (total polar surface area) were assessed for all newly syn-
thesized analogs. Compounds were prioritized on the basis of both
their biological data and physicochemical properties, and high pri-
ority agents were assessed for in vitro drug metabolism and phar-
macokinetics (DMPK, including aqueous solubility, microsomal
stability and CYP450 inhibition; Table 6, PAMPA data for selected
compounds are in Table 4).
e
and
for hepatic microsomal stability (human, mouse and rat) and cyto-
chrome P450 inhibition as described previously.25 Briefly, the com-
pounds were incubated together with hepatic microsomes and
NADPH was used to initiate enzymatic oxidation. Acetonitrile
was then added to quench the reaction at different time points
and processed samples were analyzed using LC-MS/MS. Cyto-
chrome P450 inhibition (CYP1A2, CYP2C9, CYP2D6, and CYP3A4)
was evaluated in human liver microsomes using four selective
,
marker substrates in the presence or absence of 10 lM test com-
pound and reported as percentage inhibition (see Section 4 for
Kinetic aqueous solubility in PBS buffer at pH 7.4 was deter-
mined using an HPLC-based protocol and reported as the average
of two measurements (see Section 4 for details). Apparent cell per-
meability was determined using the standard parallel artificial
membrane permeability assay (PAMPA)34 using propanolol and
details).
The calculated distribution coefficients (logD7.4) was in the
acceptable range (1 < log D < 4) for the majority of new analogs,
with the exception of 18, 51 and 62 (Table 6). This represents an
improvement over the initial set of CK1d/
e
inhibitors,25 as the
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

8
A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 5
SAR data for CK1d/e inhibitors with other amine substituents at purine position C2 (R4).
№
R4
CK1d
CK1
e
MDA-MB-231
EC50 (nM)b
IC50 (nM)a
IC50 (nM)a
13
44
260
26
63
64
55
76
170
205
580
185
65
66
185
175
310
370
130
145
67
230
205
190
68
69
70
69
50
130
320
830
375
1230
1290
c
–
c
71
915
1310
–
c
72
73
1030
1180
2010
1200
–
625
74
75
76
160
150
620
305
265
655
175
390
195
a
Biochemical assay.
Cellular proliferation assay.
Not determined.
b
c
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
Table 6
ADME properties of selected CK1d/e inhibitors.
№
clogD
Solubility
Microsome stability, min
(H/M/R)c
CYP% inhibitiond
1A2
pH 7.4a
(mM)b
2C9
2D6
3A4
13
12
16
17
18
22
23
25
28
29
31
40
42
43
44
49
50
51
58
59
61
62
Sunitinib
3.6
2.7
3.7
3.7
4.4
2.6
3.0
2.8
3.0
3.3
3.4
3.1
2.6
3.9
2.3
2.9
3.5
4.4
3.1
3.4
3.4
4.3
0.1
–
18/5/17
16/3/5
17/5/10
26/5/27
12/3/6
13/2/4
15/2/6
25/5/9
36/4/11
20/5/9
32/12/9
9/3/10
5/1/4
12
40
23
ꢂ48
ꢂ41
46
91
96
62
49
89
42
73
41
84
95
21
53
31
96
44
91
ꢂ3
14
47
ꢂ2
8
37
76
72
38
64
34
62
86
44
74
18
86
60
88
–
85
ꢂ38
ꢂ12
ꢂ17
74
e
0.4
0.1
0.3
e
–
47
53
67
47
50
10
79
67
80
e
–
0.1
28
ꢂ1
0.4
0.1
61
ꢂ9
97
30
e
–
1.6
e
–
55/16/26
19/5/18
22/7/9
16/3/–e
30/9/–e
90
e
e
e
e
2.8
1.1
0.1
0.1
–
–
–
91
59
66
68
74
37
85
92
61
52
31
74
35
67
62
31
42
1
18
ꢂ12
38
e
e
–
–
–
e
e
–
e
–
6/2/–e
ꢂ31
e
–
15/6/–e
46/13/30
ꢂ51
a
b
c
Calculated with Pipeline Pilot workflow application (Accelrys) at pH 7.4.
Solubility in pH 7.4 phosphate buffered saline.
Microsome stability using human, mouse and rat liver microsomes, with sunitinib as the reference, half life in 1 mg/ml hepatic microsomes.
Cytochrome P450 inhibition assay.
Not determined.
d
e
average logD value of our compound collection was reduced by
approximately 1 order of magnitude. Despite improvements in
lipophilicity, the majority of the compounds that were advanced
to in vitro DMPK assessment possessed poor aqueous solubility
While analog 28 is the most soluble compound in this series (28
M, selected for PO administration), both compounds 17 and 28
possess acceptable stability in human, mouse and rat liver micro-
somes and a favorable CYP inhibition profile (Table 6) when com-
pared to SR-3029 (13).
l
(less than 0.5 lM). Fortunately, during our iterative analog synthe-
sis efforts, we observed that aqueous solubility could be improved
by appending a piperazine unit to the scaffold in lieu of a morpho-
line (i.e., 28, Table 6). Importantly, agent potency was not affected
greatly by this substitution (though morpholine analogs are
slightly more potent than their N-methylpiperazine counterparts).
Hepatic microsomal stability was considered to be an important
factor when selecting candidates for in vivo testing. We sought
compounds with half-lives resembling the FDA-approved small
molecule tyrosine kinase inhibitor sunitinib, which was used as
positive control (t1/2 = 46, 13 and 30 min in human, mouse and
rat microsomes, respectively). Among the compounds advanced
to metabolic stability studies, 43, 28, 31 and 51 emerged as the
most stable candidates (Table 6). Agent stability was marginally
reduced or stayed comparable relative to reference compound
SR-3029 for the thiophene (22, 23), benzylpyridine (61) and 2,3-
difluorophenyl analogs 16 and 18.
2.4. Molecular modelling
Docking studies were performed using the co-crystal structure
of CK1d and PF670462 (PDB ID: 3UYT)36 to predict the binding
modes of designed and synthesized inhibitors. The original co-
crystal structure was refined using the Protein Preparation
Wizard37 implemented in the Maestro 11.1 (Schrödinger Release
2017-2) interface, and invalid atom types were corrected using this
same wizard. A receptor grid was generated from the refined struc-
ture using default values. The docked models for PF670462 were in
good agreement with the reported crystal structures coordinates
(see Supporting information for details). Designed inhibitors were
docked into the grid using Glide 7.438 in standard precision (SP)
mode, without any constraints. The proposed binding pose of 17
within the CK1d active site is shown in Fig. 3A,B.
Finally, CYP inhibition for a set of the most active compounds was
assessed. It is well-known that 3-substituted pyridine-containing
compounds can act as CYP3A4 inhibitors.35 Indeed, N9 m-pyri-
dine-containing analogs 25, 40, 43 and 49 were the most potent
inhibitors of all four cytochrome P450s tested (1A2, 2C9, 2D6, and
3A4, Table 6). However, it has also been reported that CYP3A4 inhi-
bition can be decreased through the introduction of ortho- halogen
or alkyl substitution into the pyridine.35 On this basis, we synthe-
sized a series of 2-fluoro (30, 31), 2-chloro (32, 33) and 2-methyl
(34, 35) substituted pyridines, which displayed significantly
improved CYP inhibition profile (31, <30% inhibition against all four
As revealed by docking studies, the key contacts between 17
and the binding pocket of CK1d are two hydrogen bonding interac-
tions between the purine scaffold and Leu85 in the hinge region
(Fig. 3B). The benzimidazole moiety projects into the solvent-
exposed area and also makes additional contacts with the same
residue Leu85, (Fig. 3A and B), while the N9 m-fluorophenyl ring
occupies a relatively large hydrophobic pocket created by the gate-
keeper residue Met82. An image of 28 docked in CK1d active site is
presented in Fig. 3C. The binding mode of this agent closely mirrors
the pose adopted by 17 (Fig. 3B, C). Specifically, 28 is positioned in
a manner analogous to 17 so as to maintain two key interactions
with the hinge region (Leu85) of CK1d. Additionally, as predicted
by Glide, the N9 para-pyridyl ring of 28 projects into the kinase
hydrophobic pocket and picks up additional stabilizing interac-
tions with Tyr56 (Fig. 3C). Despite this prediction, we observed a
tested CYP substrates at 10
tions led to reduced CK1d/
l
M, Table 6). However, these modifica-
e
potency in some cases (32, 33, 35).
Based on the combination of biological activity and ADME prop-
erties, we selected 17 and 28 to advance into in vivo PK testing.
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

10
A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
C
A
B
MET-82
MET-82
TYR-56
LEU-85
LEU-85
Fig. 3. A, docking pose of 17 (green) in the CK1d active site (surface representation); B, docking pose 17 (green) in the CK1d active site (mesh representation). C, docking pose
of 28 (grey) in the active site of CK1d. Hydrogen bond interactions are shown in red.
4-fold decrease in CK1d inhibition activity for 28 (Table 2). We
hypothesize that reduced inhibitory activity compared to 17 could
be the result of an undesirable repulsive interaction between gate-
keeper Met80 and the pyridyl unit of 28.
coverX scanEDGE Kinase Assay Panel). Analog 17 was exception-
ally selective under the assay conditions, with only CK1d being
inhibited >90% of the 97 kinases tested (Fig. 4, i.e., 5.2% of active
kinase remaining at 10 mM). The only other off-target kinase,
FLT3 (17% of active kinase remaining at 10 mM), was previously
shown by us not to be responsible for the potent antiprolifera-
2.5. Kinase selectivity
tive effects demonstrated by the purine-based CK1d/
e
inhibitors.25 The 95 remaining kinases in this assay showed
greater than 35% control activity at 10 mM (see Supporting infor-
mation for details).
In order to address the question of selectivity, kinase binding
was performed using compound 17 (at 10 mM concentration)
against a panel of 97 targets distributed across the kinome (Dis-
Fig. 4. Dendrogram presentation of results of DiscoverRX scanEDGE kinase binding selectivity analysis of compound 17. Data are presented for all kinases that have <35%
control activity at 10 mM (% control is the percentage of kinase remaining bound to the bead-bound active-site ligand in the presence of the inhibitor), represented as red
circles on kinome tree and >35% control activity 10 mM, represented as green circles.
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
2.6. In vivo pharmacokinetics
The pharmacokinetic properties of 17 and 28 were assessed in
male C57Bl6 mice following IP administration at 20 mg/kg (17)
and IV and PO administration at 1 mg/kg and 10 mg/kg, respec-
tively (28, Table 7, route of administration was selected based on
solubility data, Table 6). Compound 17 showed good exposure after
IP administration (Fig. 5). The plasma concentration was main-
tained above 1 mM for more than 3 h and above 100 nM for about
7 h (EC50 MDA-MB-231 = 68 nM), albeit a short half-life (Fig. 5,
Table 7). At the same time, 28 exhibited a modest in vivo half-life
(1.3 h) and a high volume of distribution (9.1 L/kg) following intra-
venous administration. The apparent oral bioavailability of 28 after
PO dosing was approximately 16% (Table 7), marginally better than
SR-3029 (13), despite improvement in aqueous solubility.
3. Conclusions
We have developed a series of potent and selective purine-
based CK1d/e inhibitors with excellent antiproliferative activities.
Fig. 5. Plasma concentration versus time profile for 17 (points are the average of 3
mice) following IP administration at dose of 20 mg/kg using 10/10/80 DMSO/Tween
80/water.
A set of the most active compounds was also subjected to extensive
physicochemical testing for solubility, permeability, and microso-
mal stability in an effort to predict their in vivo profile. These
efforts led to the identification of 17 and 28 that has physical,
in vitro and in vivo PK properties suitable for use in xenograph
studies of human cancer. Such studies in mice are planned and will
be reported in due course.
The structures of all compounds were verified via 1H NMR, 13C
NMR, 19F NMR and HPLC/HRMS. The purity of isolated products
was determined using an LC-MS instrument (Agilent 1260 Infinity
series LC with 500 Ion Trap MS) equipped with KinetexÒ
5 lm EVO
C18 100 Å LC Column 100 ꢃ 4.6 mm (Phenomenex) column. Elu-
tion was performed using the following conditions: 2% (v/v) ace-
tonitrile (+0.1% FA) in 98% (v/v) H2O (+0.1% FA), ramped to 98%
acetonitrile over 8 min, and holding at 98% acetonitrile for 1 min
with a flow rate of 1.75 mL/min; UV absorption was detected from
200 to 950 nm using a diode array detector. The purity of each
compound was ꢄ95% based on this analysis.
4. Experimental
4.1. Material and methods
All reagents were purchased from commercial suppliers and
were used without further purification. Dichloromethane, diethyl
ether, N,N-dimethylformamide and tetrahydrofuran were dried
by being passed through a column of desiccant (activated A-1 alu-
mina). Triethylamine and diisopropyl amine was purified by distil-
lation from calcium hydride. Reactions were either monitored by
thin layer chromatography or analytical LC-MS. Thin layer chro-
matography was performed on Kieselgel 60 F254 glass plates
pre-coated with a 0.25 mm thickness of silica gel. TLC plates were
visualized with UV light and/or by staining with ninhydrin solu-
tion. Normal phase column chromatography was performed on a
Biotage Isolera automated flash system. Compounds were loaded
NMR spectra were recorded at ambient temperature on a 400 or
700 MHz Bruker NMR spectrometer in DMSO d6. All 1H NMR data
are reported in parts per million (ppm) downfield of TMS and were
measured relative to the signals for dimethyl sulfoxide (2.50 ppm).
All 13C NMR spectra are reported in ppm relative to the signals for
dimethyl sulfoxide (39.5 ppm) with 1H decoupled observation. 19F
NMR experiments were performed with 1H decoupling. Data for 1H
NMR are reported as follows: chemical shift (d, ppm), multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), inte-
gration, and coupling constant (Hz), whereas 13C NMR analyses
were obtained at 101 or 176 MHz and reported in terms of chem-
ical shift. NMR data was analyzed and processed by using MestRe-
Nova software. High-resolution mass spectra were recorded on a
spectrometer (ESI-TOF) at the University of Illinois Urbana-Cham-
paign Mass Spectrometry Laboratory.
onto pre-filled cartridges filled with KP-Sil 50 lm irregular silica.
For microwave reactions, a Biotage Initiator Microwave system
was used. Some of the final products were isolated by reverse-
phase HPLC using Shimadzu Prep LC system with photodiode array
detector, Waters SunFire C18 OBD Prep Column, 100 Å, 10 mm, 30
mm ꢃ 250 mm. Compounds were eluted using a gradient elution
of 90/10 to 0/100 A/B over 10 min at a flow rate of 50.0 mL/min,
where solvent A was water (+0.1% TFA) and solvent B was acetoni-
trile/methanol (1:1).
The synthesis and analysis of 2,6-dichloro-9-aryl-9H-purines 6
and benzimidazoles
9
were previously described.25,28,29
Diaryliodonium salts 5 were prepared according to the reported
procedures.30 Compounds 11–76 were newly synthesized and
described in the Supporting Information.
Table 7
Mouse PK data for selected CK1d/e inhibitors 28 (PO dosing), 17 (IP dosing) and SR-3029 (13) (PO dosing).
№
Routea
Dose
Cmax
Cl
AUC
t1/2 (h)
% F
(mg/kg)
(mM)
(ml/min/kg)
(mMꢁh)
13
28
17
PO
PO
IP
3
10
20
1.1
0.7
7.1
49
2.4
0.32
15.6
0.9
1.3
1.0
13
16
–
c
–
43b
a
b
c
10/10/80 DMSO/Tween 80/water.
Assumes 100% absorption.
Not determined.
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

12
A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.2. General procedure for preparation of compounds 11–76
5 mL Biotage microwave vial was charged with 2,6-
4.5. Methods for in vitro and in vivo PK studies
A
4.5.1. Solubility
dichloropurine-9-(aryl/alkyl)-purine (1 eq, 0.35 mmol), 6, 2-(ami-
nomethyl)-benzimidazole, 9, (or the benzylamine corresponding
to R5CH2NH2 in Scheme 1) (1.1 eq 0.39 mmol), freshly distilled
N,N-diisopropylethylamine (5 eq, 1.77 mmol, 0.31 mL), and iso-
propanol (1.4 mL. 0.25 M). The vial was sealed with a microwave
cap, and then the reaction mixture was heated to 90 °C for 30
min in microwave reactor. The completion of the reaction was
confirmed by LC-MS and resulting product 10 was collected by
filtration. In cases where the product was soluble in isopropanol,
the reaction mixture was concentrated and used in the next step
without further purification. A large excess (>30 eq) of amine
corresponding to R4 (Scheme 1, N-methyl piperazine, morpho-
line, etc.) was then added to the vial containing the concentrated
crude mixture or collected solid. The vial was resealed and the
reaction was heated to 130 °C for 30 min in the microwave unit
(monitored by LC-MS). The cooled reaction mixture was concen-
trated on a rotary evaporator, then the crude product was puri-
fied by flash chromatography using 10 g Biotage column
(gradient, 0%–10% MeOH in DCM over 25 column volumes). Col-
lected fractions were washed with solution of saturated NH4Cl to
remove remaining amine (monitored by TLC using ninhydrin
stain). In some cases (as specified), reverse-phase HPLC was used
after the normal phase column chromatography to obtain pro-
duct with purity of > 95%.
Compounds from 10 mM DMSO stock solutions were
introduced to pre-warmed pH 7.4 PBS in a 96-well plate. The final
DMSO concentration was 1% and the plate was maintained at 37 °C
for 24 h on an orbital shaker. The samples were centrifuged
through a Millipore Multiscreen Solvinter 0.45 mm low binding
PTFE hydrophilic filter plate and analyzed by HPLC with peak area
compared to standards of known concentration.
4.5.2. Permeability
An assessment of permeability was done using a commercial
PAMPA (Parallel Artificial Membrane Permeability Assay) kit from
BD Biosciences (Cat# 353015).34 Compound (5mM) was added to
300 ml PBS in the bottom donor plate and 200 ml of blank PBS
was added to the top receiver plate. The plates were incubated in
an orbital shaker temperature at 37 °C for 5 h, aliquots were taken
from the donor and receiver plates and the concentration of drug
was determined. Compound permeability was calculated using
the equation
h
i
CAðtÞ
ln 1 ꢂ
Ceq
ꢀ
ꢀ
ꢁ
ꢁ
Papp ¼ ꢂ
1
1
A
A ꢅ
þ V ꢅ t
VD
where Papp is expressed in units of cm/s, CA(t) is drug concentration
in the acceptor at time t, VD is donor well volume, VA is acceptor
well volume, A is the area of the filter (0.3 cm2), t is time in seconds.
4.3. Biochemical assays
4.5.3. Hepatic microsomal stability
Microsome stability was evaluated by incubating 1 mM com-
pound with 1 mg/ml hepatic microsomes (human, rat, or mouse)
in 100 mM PBS, pH 7.4. The reactions were held at 37 °C with con-
tinuous shaking. The reaction was initiated by adding NADPH,
1 mM final concentration. The final incubation volume was
300 ml and 40 ml aliquots were removed at 0, 5, 10, 20, 40, and
60 min. The removed aliquot was added to 160 ml acetonitrile to
stop the reaction and precipitate the protein. NADPH dependence
of the reaction was evaluated in parallel incubations without
NADPH. At the end of the assay, the samples are centrifuged
through a 0.45 mm filter plate (Millipore Solventer low binding
hydrophilic plates, cat# MSRLN0450) and analyzed by LC-MS/MS.
The data was log transformed and results are reported as half-life.
CK1d inhibitor IC50 values were measured by using a time-
resolved fluorescence resonance energy transfer (TR-FRET) assay.
Briefly, final assay concentrations for CK1d (Signal Chem), Ulight
peptide substrate (ULight-Topo-IIa(Thr1342) peptide, Perkin
Elmer) and ATP were 2 nM, 200 nM and 50
The reaction was performed at room temperature in a 10
l
M respectively.
l final
l
volume (384-well low volume plate, Greiner) containing: 50 mM
HEPES, pH 7.5, 5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 1
mM dl-dithiothreitol, 0.01% Triton X-100 and 5% DMSO (Sigma-
Aldrich). After 10 min, the reaction was terminated by addition
of 10
ll of 4 nM Eu-anti-p-Topo-IIa (Cat:TRF-0218, PerkinElmer)
in Lance Detection Buffer (Cat: CR97-100, PerkinElmer). The flu-
orescent signal was detected using an EnVision plate reader (Per-
kinElmer). Ten point dose–response curves with 3–10-fold
4.5.4. P450 inhibition
dilutions starting from 10 lM for each compound was generated
Cytochrome P450 inhibition was evaluated in human liver
microsomes using four selective marker substrates (CYP1A2, phe-
naceten demethylation to acetaminophen; CYP2C9, tolbutamide
hydroxylation to hydroxytolbutamide; CYP2D6, bufuralol hydrox-
ylation to 40-Hydroxybufuralol; and CYP3A4, midazolam hydroxy-
lation to 10-hydroxymidazolam) in the presence or absence of
10 mM test compound. The reaction is initiated by the addition of
1 mM NADPH and stopped after ten min by the addition of 2-times
volume of acetonitrile containing dextrorphan as an internal stan-
dard. The concentration of each marker substrate is approximately
its Km.39 Furafylline, sulfaphenazole, quinidine, and ketoconazole
were included to each run to validate that the assay could identify
selective inhibitors of each isoform.
in duplicate and data fit to a four parameter logistic (GraphPad
Prism 5).
4.4. Cell culture and proliferation assays of CK1d/e inhibitors
Human MDA-MB-231 breast cancer cells were cultured in Dul-
becco’s Modified Eagle Medium (DMEM, LifeTechnologies) supple-
mented with 10% fetal bovine serum and 1% penicillin/
streptomycin at 37 °C, 5% CO2. To evaluate the anti-proliferative
activity of newly synthesized CK1d/e inhibitors against MDA-MB-
231 breast cancer cells, cells were plated into a 384-well plate.
10 point dose-response curves with 3–10-fold dilutions starting
from 10
l
M for each compound was generated in duplicate includ-
4.5.5. Pharmacokinetics
ing DMSO control. Cell proliferation was measured 72 h after
compound treatment using CellTiter-Glo (Promega) according to
the manufacturer’s instructions. EC50 values were determined by
nonlinear regression and a four-parameter algorithm (GraphPad
Prism 5).
All procedures described are covered under existing protocols
and have been approved by the Scripps Florida IACUC to be con-
ducted in the Scripps vivarium, which is fully AAALAC accredited.
Pharmacokinetics were determined in n = 3 male C57Bl/6 mice.
Compounds were dosed as indicated in the text via intravenous
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020

A. Monastyrskyi et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13
5. Hoekstra MF, Liskay RM, Ou AC, et al. Science. 1991;253:1031.
6. Li G, Yin H, Kuret J. J Biol Chem. 2004;279:15938.
7. (a) Price MA. Genes Dev. 2006;20:399;
(b) Peters JM, McKay RM, McKay JP, et al. Nature. 1999;401:345.
8. Perreau-Lenz S, Vengeliene V, Noori HR, et al. Neuropsychopharmacology.
2012;37:2121.
9. Knippschild U, Wolff S, Giamas G, et al. Onkologie. 2005;28:508.
10. Brockschmidt C, Hirner H, Huber N, et al. Gut. 2008;57:799.
11. Kaucka M, Plevova K, Pavlova S, et al. Cancer Res. 2013;73:1491.
12. Rodriguez N, Yang J, Hasselblatt K, et al. EMBO Mol Med. 2012;4:952.
13. Kim SY, Dunn IF, Firestein R, et al. PLoS ONE. 2010;5:e8979.
14. Hirner H, Gunes C, Bischof J, et al. PLoS ONE. 2012;7:e29709.
15. (a) Dey N, Barwick BG, Moreno CS, et al. BMC Cancer. 2013;13:537;
injection via tail vein or oral gavage. 25 ll of blood was collected
via a small nick in the tail using heparin coated hematocrit capil-
lary tubes which were sealed with wax and kept on ice until
plasma was generated by centrifugation using a refrigerated cen-
trifuge equipped with a hematocrit rotor. Dose levels are provided
in the text. Time points for determination of pharmacokinetic
parameters were 5 m, 15 m, 30 m, 1 h, 2 h, 4 h, 6 h, and 8 h. Plasma
concentrations were determined via LC-MS/MS using a nine point
standard curve between 0.4 and 2000 ng/ml prepared in mouse
plasma. Pharmacokinetic analysis was done with WinNonLin,
Pharsight Inc. using a noncompartimental model.
(b) Khramtsov AI, Khramtsova GF, Tretiakova M, et al. Am
2010;176:2911;
J Pathol.
(c) Khalil S, Tan GA, Giri DD, et al. PLoS ONE. 2012;7:e33421;
4.6. LogD calculations
(d) Schade B, Lesurf R, Sanguin-Gendreau V, et al. Cancer Res. 2013;73:4474.
16. Rosenberg LH, Lafitte M, Quereda V, et al. Sci Transl Med. 2015;7:a202.
17. Knippschild U, Krüger M, Richter J, et al. Front Oncol. 2014;4.
18. Chijiwa T, Hagiwara M, Hidaka H. J Biol Chem. 1989;264:4924.
19. (a) Bettayeb K, Oumata N, Echalier A, et al. Oncogene. 2008;27:5797;
(b) Oumata N, Bettayeb K, Ferandin Y, et al. J Med Chem. 2008;51:5229.
20. (a) Behrend L, Milne DM, Stoter M, et al. Oncogene. 2000;19:5303;
(b) Cheong JK, Nguyen TH, Wang H, et al. Oncogene. 2011;30:2558.
21. (a) Rena G, Bain J, Elliott M, et al. EMBO Rep. 2004;5:60;
cLogD values were calculated with Pipeline Pilot workflow
application (Accelrys) at pH 7.4 as previously described.40
Acknowledgments
(b) MacLaine NJ, Oster B, Bundgaard B, et al. J Biol Chem. 2008;283:28563.
22. Lee JW, Hirota T, Peters EC, et al. Angew Chem Int Ed Engl. 2011;50:10608.
23. Bischof J, Leban J, Zaja M, et al. Amino Acids. 2012;43:1577.
24. Walton KM, Fisher K, Rubitski D, et al. J Pharmacol Exp Ther. 2009;330:430.
25. Bibian M, Rahaim RJ, Choi JY, et al. Bioorg Med Chem Lett. 2013;23:4374.
26. Rosenberg LH, Lafitte M, Quereda V, et al. Sci Transl Med. 2015;7.
27. Zhang C, Lopez MS, Dar AC, et al. ACS Chem Biol. 2013;8:1931.
28. Ding S, Gray NS, Ding Q, et al. Tetrahedron Lett. 2001;42:8751.
29. Niu HY, Xia C, Qu GR, et al. Org Biomol Chem. 2011;9:5039.
We thank Benjamin Huffman and Alexander Burns for contribu-
tions to the synthesis of several CK1d/e inhibitors in this work. We
also thank the Scripps Florida NMR facility and Xiangming Kong for
assistance. This work was supported in part by NIH NCI grant
R01CA175094 (W.R.R., and D.R.D.) and NIH NCI NRSA postdoctoral
fellowship F32CA200105 (A.M.).
30. (a) Bielawski M, Aili D, Olofsson B. J Org Chem. 2008;73:4602;
(b) Bielawski M, Malmgren J, Pardo LM, et al. ChemistryOpen. 2014;3:19.
31. Casagrande M, Barteselli A, Basilico N, et al. Bioorg Med Chem. 2012;20:5965.
32. Roush WR, Rahaim R, Bibian M. Wee1 degradation inhibitors. Google Pat. 2016.
33. Gramec D, Masic LP, Dolenc MS. Chem Res Toxicol. 2014;27:1344.
34. Chen X, Murawski A, Patel K, et al. Pharm Res. 2008;25:1511.
35. Zlokarnik G, Grootenhuis PDJ, Watson JB. Drug Discov Today. 2005;10:1443.
36. Long AM, Zhao H, Huang X. J Med Chem. 2012;55:10307.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.12.020.
References
37. Sastry GM, Adzhigirey M, Day T, et al. J Comput Aided Mol Des. 2013;27:221.
38. (a) Friesner RA, Banks JL, Murphy RB, et al. J Med Chem. 2004;47:1739;
(b) Friesner RA, Murphy RB, Repasky MP, et al. J Med Chem. 2006;49:6177;
(c) Halgren TA, Murphy RB, Friesner RA, et al. J Med Chem. 2004;47:1750.
39. Testino Jr SA, Patonay G. J Pharm Biomed Anal. 2003;30:1459.
40. Csizmadia F, Tsantili-Kakoulidou A, Panderi I, et al. J Pharm Sci. 1997;86:865.
1. Cheong JK, Virshup DM. Int J Biochem Cell Biol. 2011;43:465.
2. Fish KJ, Cegielska A, Getman ME, et al. J Biol Chem. 1995;270:14875.
3. (a) Badura L, Swanson T, Adamowicz W, et al.
2007;322:730;
J Pharmacol Exp Ther.
(b) Gallego M, Virshup DM. Nat Rev Mol Cell Biol. 2007;8:139.
4. Pooler AM, Usardi A, Evans CJ, et al. Neurobiol Aging. 2012;33:e27.
Please cite this article in press as: Monastyrskyi A., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.12.020