Development of new deoxycytidine kinase inhibitors and noninvasive in vivo evaluation using positron emission tomography

Article abstract of DOI:10.1021/jm400457y

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using ¹⁸F-L-1-(2′-deoxy-2′-FluoroArabinofuranosyl) Cytosine (¹⁸F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC₅₀ = ～1-12 nM) using the ¹⁸F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

Full text of DOI:10.1021/jm400457y

Article
pubs.acs.org/jmc
Development of New Deoxycytidine Kinase Inhibitors and
Noninvasive in Vivo Evaluation Using Positron Emission Tomography
Jennifer M. Murphy,‡,†,§, ○ Amanda L. Armijo,‡,†,§ Julian Nomme,‡,∥ Chi Hang Lee,†,§
Quentin A. Smith,⊥ Zheng Li,†,§ Dean O. Campbell,†,§,▲ Hsiang-I Liao,†,§ David A. Nathanson,†,§
Wayne R. Austin,†,§ Jason T. Lee,†,§,◆ Ryan Darvish,†,§ Liu Wei,†,§ Jue Wang,†,§ Ying Su,∥
Robert Damoiseaux,# Saman Sadeghi,†,§ Michael E. Phelps,† Harvey R. Herschman,†,△
Johannes Czernin,†,§ Anastassia N. Alexandrova,⊥ Michael E. Jung,⊥ Arnon Lavie,*,∥
and Caius G. Radu*,†,§
§
⊥
†Department of Molecular and Medical Pharmacology, Ahmanson Translational Imaging Division, Department of Chemistry and
Biochemistry, California NanoSystems Institute, △Department of Biological Chemistry, University of California, Los Angeles, 650
#
Charles E. Young Dr. S., Los Angeles, California 90095, United States
∥Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Avenue, M/C 669, Chicago,
Illinois 60607, United States
S
* Supporting Information
ABSTRACT: Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK)
in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication
stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and
survival stimulated our interest in developing small molecule dCK inhibitors. Following a high
throughput screen of a diverse chemical library, a structure−activity relationship study was carried
out. Positron Emission Tomography (PET) using 18F-L-1-(2′-deoxy-2′-FluoroArabinofuranosyl)
Cytosine (18F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based
on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent
compounds in cell culture (IC50 = ∼1−12 nM) using the 18F-L-FAC PET pharmacodynamic assay
identified compounds demonstrating superior in vivo efficacy.
decitabine, fludarabine, cytarabine, clofarabine); phosphoryla-
tion by dCK is critically required for the activation of these
prodrugs.8 Recently, dCK was implicated in the regulation of
the G2/M checkpoint in cancer cells in response to DNA
damage.9 The role of dCK in hematopoiesis and cancer has led
to our interest in developing a small molecule inhibitor of this
kinase. Such dCK inhibitors could represent new therapeutic
agents for malignancies and immune disorders. To our
knowledge, few dCK inhibitors have been reported,10−12 and
only one13 has been demonstrated to inhibit dCK activity in
vivo.
Positron emission tomography (PET) is a noninvasive in
vivo imaging technique widely used for diagnosing, staging,
restaging, and therapy monitoring of various diseases.14,15
While PET using the radiotracer 2-18F-fluoro-2-deoxy-D-glucose
(18F-FDG)16,17 has become an important diagnostic and
treatment monitoring tool in cancer,18−21 another emerging
application of PET concerns its use in drug discovery and
development. Thus, by facilitating faster and more effective
INTRODUCTION
■
Mammalian cells rely on two major pathways for the
production and maintenance of deoxyribonucleotide triphos-
phates (dNTPs) for DNA replication and repair: the de novo
pathway and the nucleoside salvage pathway.1 The de novo
pathway produces dNTPs from glucose and amino acids. The
nucleoside salvage pathway produces dNTPs from preformed
deoxyribonucleosides present in the extracellular environment.1
The first enzymatic step in the cytosolic deoxyribonucleoside
salvage pathway is catalyzed by deoxycytidine kinase (dCK)
and by thymidine kinase 1 (TK1).2 dCK catalyzes 5′-
phosphorylation of deoxycytidine (dC), deoxyguanosine
(dG), and deoxyadenosine (dA) to their monophosphate
forms, exhibiting the highest affinity for dC.3 The mono-
phosphate deoxyribonucleotides are subsequently phosphory-
lated to their corresponding di- and triphosphate forms by
other kinases.4,5 We have shown that dCK and TK1 play
important roles in hematopoiesis by regulating dNTP biosyn-
thesis in lymphoid and erythroid progenitors.6,7 In addition to
its physiological role in nucleotide metabolism, dCK
phosphorylates several clinically important antiviral and
anticancer nucleoside analog prodrugs (e.g., gemcitabine,
Received: March 28, 2013
© XXXX American Chemical Society
A
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Figure 1. Structures and IC50 values determined using the 3H-dC uptake assay in L1210 cells for the initial HTS hits (1 and 2) and for commercially
available compounds containing similar structural scaffolds (3−7).
a
Table 1. In Vitro Biological Data in L1210 and CEM Cells for Compounds 8−14
IC50 (μM) L1210 cells
IC50 (μM) CEM cells
compd
R1
R2
R3
R4
a Y = CH
b Y = N
a Y = CH
b Y = N
8
H
H
H
H
H
OCH2CH2F
OCH3
OCH3
H
H
H
H
H
H
0.808 ( 0.406)
0.538 ( 0.014)
0.513 ( 0.100)
2.381 ( 0.042)
0.330 ( 0.160)
1.445 ( 0.060)
5.469 ( 1.336)
1.612 ( 0.543)
0.528 ( 0.015)
1.226 ( 0.450)
3.201 ( 0.566)
0.603 ( 0.140)
2.649 ( 0.902)
0.421 ( 0.075)
0.230 ( 0.042)
0.251 ( 0.020)
1.960 ( 1.001)
0.197 ( 0.109)
1.041 ( 0.084)
2.367 ( 0.238)
0.534 ( 0.012)
0.506 ( 0.138)
0.512 ( 0.409)
1.922 ( 0.573)
0.297 ( 0.020)
0.849 ( 0.183)
9
OCH2CH2F
OCH2CH2F
OCH2CH2F
OCH2CH2CH2F
OCH2CH2F
H
10
11
12
13
14
OCH2CH3
CH3
OCH3
OCH3
H
H
b
b
OCH2CH2F
OCH3
ND
ND
a
Inhibitory activity measured by 3H-deoxycytidine (3H-dC) uptake in murine L1210 cells and in CCRF-CEM human cells. Values reported are the
b
mean SD of at least n = 2 independent experiments. ND = not determined (compound was not synthesized).
decision-making early in the drug discovery/development
process, PET could accelerate the advancement of promising
candidates and reduce failures.22−24 For instance, PET can be
used to demonstrate the need to modify lead candidates early
in the drug discovery process by enabling noninvasive
evaluations of drug pharmacodynamic (PD) and/or pharma-
cokinetic (PK) properties. In the specific context of our drug
discovery and development program centered on dCK, PET
could play a particularly important role given the availability of
validated PET biomarkers to assess dCK activity in vivo. These
B
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a
Table 2. In Vitro Biological Data in L1210 and CEM Cells for Compounds 15−18
IC50 (μM) L1210 cells
IC50 (μM) CEM cells
b Y = Et
compd R1
R2
R3
a Y = Me
b Y = Et
c Y = Pr
a Y = Me
c Y = Pr
15
16
17
18
H
F
OCH3
OCH2CH2F
0.035 ( 0.015)
0.595 ( 0.163)
0.395 ( 0.134)
0.255 ( 0.021)
0.030 ( 0.077)
0.620 ( 0.170)
0.265 ( 0.163)
0.510 ( 0.014)
0.003 ( 0.000)
0.385 ( 0.262)
0.170 ( 0.099)
0.175 ( 0.007)
0.018 ( 0.012)
0.150 ( 0.099)
0.230 ( 0.134)
0.092 ( 0.048)
0.007 ( 0.002)
0.162 ( 0.019)
0.083 ( 0.043)
0.011 ( 0.038)
0.003 ( 0.000)
0.173 ( 0.157)
0.037 ( 0.020)
0.031 ( 0.024)
H
F
F
H
H
H
H
F
a
Inhibitory activity measured by 3H-deoxycytidine (3H-dC) uptake in murine L1210 and in CCRF-CEM human cells. Values reported are the mean
SD of at least n = 2 independent experiments.
PET PD biomarkers of dCK activity include a series of 18F-
fluoro-arabinofuranosylcytosine analogs substrates of dCK
developed by our group25 which include 18F-1-(2′-deoxy-2′-
FluoroArabinofuranosyl) Cytosine (18F-FAC)26 and 18F-L-1-
(2′-deoxy-2′-FluoroArabinofuranosyl) Cytosine (18F-L-FAC).27
Herein we describe the development of potent dCK inhibitors
and demonstrate their in vivo efficacy using 18F-L-FAC PET as
a noninvasive and clinically applicable PD biomarker.
fluoroethoxy side-chain on the phenyl ring was considered for
eventual 18F-radiolabeling purposes. Substitutions around the
phenyl ring with respect to the position of the fluoroethoxy
side-chain were also examined. Moving the fluoroethoxy side-
chain from the para position in 8a to the meta position in 9a
increased the inhibitory activity approximately 2-fold. It was
also apparent that alkoxy substituents in the para position were
better than alkyl moieties, since compound 11a had
substantially lower activity than either the methoxy 9a or
ethoxy 10a analogs. Compound 12a, which contains a side-
chain that was extended by one carbon to give a fluoropropoxy
group at the meta position, gave slightly greater inhibitory
activity, albeit not a significant increase from compounds 9a
and 10a. Substitution at the ortho position of the phenyl ring,
e.g. in compounds 13a and 14a, resulted in substantially lower
dCK inhibitory activity, an approximate 10-fold decrease in
potency was observed for compound 14a when compared to
9a. A general synthetic scheme for compounds in Table 1 can
be found in the Supporting Information (Scheme S2).
While the presence of fluorine in the small molecule may
eventually enable the synthesis of an 18F-isotopolog of the dCK
inhibitor, fluorine introduction also affects nearly all the
physical and ADME (adsorption, distribution, metabolism,
and excretion) properties of a compound.29 The capacity of
fluorine to enhance metabolic stability has become increasingly
apparent in recent years.30 Thus, a series of compounds were
synthesized which contained fluorine attached directly on the
aromatic ring of the inhibitors rather than linked by an ethoxy
side-chain (compounds 16−18, Table 2). For each compound
in this series, a set of three derivatives (a−c) were synthesized;
in each case the group on the 5-position of the thiazole was
either a methyl, ethyl, or propyl substituent. For compounds
15a−c the fluoroethoxy side-chain was retained at the meta
position of the phenyl ring, as was a methoxy group at the para
position due to the favorable inhibitory results from the initial
SAR in Table 1.
RESULTS AND DISCUSSION
■
Identification of Lead Compound 15c. To identify new
small molecule inhibitors of dCK, we performed a high
throughput screen (HTS) of a set of selected chemical libraries
totaling ∼90,000 small molecules. We screened the library for
dCK inhibitory function using a Firefly luciferase-coupled assay
with recombinant human dCK enzyme.28 In this assay,
inhibition of dCK prevents ATP depletion by dCK, thus
resulting in higher luminescent signals in positive wells. The
screen yielded two hit compounds, 1 and 2, which were
validated to inhibit the uptake of tritiated deoxycytidine (3H-
dC) with micromolar potency in the L1210 murine leukemia
cell line (Figure 1).
Based on these results, five commercially available com-
pounds containing similar structural scaffolds were tested; their
IC50 values against L1210 cells were determined by measuring
inhibition of 3H-dC uptake (Figure 1). Strikingly, compounds 6
and 7 were inactive, suggesting that the bis-amino functionality
on the pyrimidine ring is crucial for dCK inhibition. Based on
these results, we initiated a structure−activity relationship
(SAR) study to develop a lead structure, which could be further
optimized to compounds with potent in vivo activity.
We initially studied two main structural classes of
compounds, pyrimidines and 1,3,5-triazines (Table 1). Two
cell lines were used to determine the IC50 values: the L1210
murine leukemia cells and the CCRF-CEM human acute T-
lymophoblastic leukemia cells. In nearly all cases, substitution
of the pyrimidine ring with the 1,3,5-triazine motif reduced
dCK inhibitory activity; in some instances an approximate 2-
fold reduction in potency was observed. Consequently, the
pyrimidine motif was utilized as the preferred scaffold to
advance. At this stage of the SAR, the presence of a
Increasing nonpolar functionality at the 5-position of the
thiazole resulted in increasing inhibitory activity (Table 2). The
IC50 values in CCRF-CEM cells illustrate the same trend in
potency as observed in L1210 cells with one exception; set 16
shows little difference between the methyl, ethyl, or propyl
C
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a
Scheme 1. Synthesis of Compounds 15a−c
a
aReagents and conditions: (a) 1-bromo-2-fluoroethane, Cs2CO3, DMF, 99%; (b) (NH4)2S (20% in H2O), pyridine, Et3N, quantitative; (c) ethyl 3-
bromo-2-oxobutanoate, EtOH; (d) ethyl 3-bromo-2-oxopentanoate, EtOH; (e) ethyl 3-bromo-2-oxohexanoate, EtOH; (f) DIBAL-H, CH2Cl2; (g)
1,1,1,3,3,3-hexabromoacetone, PPh3, CH3CN; (h) 4,6-diamino-2-mercaptopyrimidine, NaOH, EtOH.
Figure 2. Binding of 15a to human dCK. (A) Ribbon diagram of a dCK monomer (light blue) with the two observed molecules of 15a bound (blue
and cyan spheres, respectively) at the active site. The nucleotide UDP (red) was also present in the complex. (B) The interactions between 15a and
dCK. Polar interactions are indicated as broken black lines. The two phosphate groups of UDP (top right) demonstrate the relative orientation of
15a-I (blue) and 15a-II (cyan) to the nucleotide. (C) The methyl group of the 15a-I and 15a-II thiazole ring (red arrows) stack against each other,
occupying a hydrophobic pocket.
substituents. However, for all compounds tested against L1210
cells, the propyl substituent yielded better inhibitory activity
than the corresponding methyl derivatives. The best example in
L1210 cells was the 12-fold increase in activity when comparing
compound 15c to compound 15a. In addition, comparisons
between the propyl substituents against their respective methyl
derivatives in CCRF-CEM cells also showed an increasing
inhibitory trend in activity: 6-fold (compare 17c to 17a) or 3-
fold (compare 18c to 18a). The most drastic effect from
modifications at the 5-position of the thiazole ring was the
change exhibited from 9a in Table 1 to 15c in Table 2, where
the substitution of a hydrogen for a propyl moiety resulted in a
180-fold increase in potency in L1210 cells. In addition,
removal of the fluoroethoxy side-chain (e.g., compound series
16−18) resulted in a significant decrease in potency in both cell
lines. Compound 15c, the most potent compound in this series,
contains both the fluoroethoxy side-chain at the meta position
on the phenyl ring and also a propyl group at the 5-position of
the thiazole ring.
Synthesis. Compounds 15a−c were synthesized in six steps
(Scheme 1). The commercially available 3-hydroxy-4-methox-
ybenzonitrile 19 was functionalized via alkylation with 1-
bromo-2-fluoroethane in DMF with cesium carbonate as the
base to obtain the nitrile 20 in 99% yield. Subjection of 20 to an
aqueous ammonium sulfide solution under basic conditions
afforded the thioamide 21 in excellent yield.31 Cyclization to
D
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Figure 3. (A) The complete thermodynamic cycle relating the binding energies to the perturbation of molecule A into molecule B. ΔGprotein(A →
B) denotes the change in free energy upon perturbation of A into B in the solvated inhibitor−protein complex, while ΔGwater(A → B) denotes the
free energy change when the perturbation takes place in water alone. The difference in free energies of binding, ΔΔGbinding, is equal to the change
in free energy when molecule A binds with the protein [ΔGbinding(A)] subtracted from the change in free energy when molecule B binds
[ΔGbinding(B)]. Because the sum of all components in a complete thermodynamic cycle must equal zero, ΔΔGbinding is therefore also equivalent
to ΔGprotein(A → B) − ΔGwater(A → B). (B) Computational model of compound 15c (orange) in complex with dCK. Binding pocket residues
Glu 53, Gln 97, Arg 114, and Asp 133 are shown explicitly, while the remainder of the protein is illustrated as a ribbon structure. (C) Free energy
changes (kcal/mol) associated with the perturbation of the alkyl chain at the 5-position of the thiazole. ΔGprotein is the change in free energy for
the solvated inhibitor−protein complex. ΔGwater is the free energy change for the inhibitor in water alone. The change in free energy upon binding
is denoted as ΔΔGbinding.
form the thiazole core of 15a−c was achieved via condensation
of thioamide 21 with the respective ethyl 3-bromo-2-
oxoalkanoate32 in refluxing ethanol.33 Reduction of the
resulting compounds with diisobutylaluminum hydride afforded
the respective alcohols 23a−c in 88−99% yield. The alcohols
23a−c were converted to the respective bromides 24a−c under
mild conditions34 in 74−80% yield. Finally, nucleophilic
displacement of the bromide with 4,6-diamino-2-mercaptopyr-
imidine35 generated the desired products 15a−c in 71−87%
yield.
X-ray Crystal Structure of Compound 15a Bound to
Human dCK. X-ray crystallographic studies of compound 15a
were initiated to obtain information about its binding to dCK.
Detailed analysis of the dCK-inhibitor interactions for this
series of compounds was performed (Nomme, unpublished
results). In short, the crystal structure of the dCK:15a complex
was solved at 1.9 Å resolution (Figure 2). Human dCK, a dimer
of two identical subunits with a molecular weight of ∼30 kDa
per monomer, can bind either ATP or UTP as the phosphoryl
donor for catalysis; in addition, dCK can adopt an open or
closed conformation.36,37,3 In complex with 15a, the enzyme
adopts the open conformation. We observed two 15a
molecules in each protomer of the dimeric enzyme (shown
in blue (15a-I) and cyan (15a-II), Figure 2A). Note that
binding of 15a to dCK does not preclude nucleotide binding
(UDP is shown in red, Figure 2A). The parallel orientation
between 15a-I and 15a-II allows for optimal π−π stacking
interactions between the phenyl and thiazole rings of each
molecule.
moiety of 15a-I and residues E53, Q97, and D133 in the dCK
nucleoside binding site are illustrated in Figure 2B. Figure 2C
illustrates the hydrophobic pocket that exists, via V55, L82, and
F96, around the methyl group of compound 15a. This figure
demonstrates that the pocket will accept larger substituents,
explaining the increased trend in potency obtained for
compounds 15b and 15c.
Monte Carlo-Based Computational Modeling. A
Monte Carlo38 (MC)-based computational modeling approach
using the free energy perturbation (FEP) method39,40 was used
to further investigate the inhibitory effects of alkyl chain
lengthening at the 5-position of the thiazole. FEP allows
calculation of the difference in binding energy of two molecules.
The perturbation of molecule A into molecule B in a complex
with a protein [ΔGprotein(A → B)] and in solution alone
[ΔGwater(A → B)] is part of a complete thermodynamic cycle
(Figure 3A). Because the sum of all components in such a cycle
must equal zero, the binding energy difference may be
calculated as the difference in free energies:
ΔΔGbinding = ΔGbinding(B) − ΔGbinding(A)
= ΔGprotein(A → B) − ΔGwater(A → B)
Models of structures 15b and 15c (Figure 3B) each in a
monomeric complex with dCK and in solution alone were
equilibrated using MC. The equilibrated structure of 15c was
subsequently perturbed into the structure of 15b (“shrinking”
the propyl chain into an ethyl) and vice versa (“growing” the
ethyl chain into a propyl) using FEP. These calculations were
performed using the MCPRO 2.041 software package. The free
energy changes for these perturbations are illustrated in Figure
3C. Averaging the ΔΔGbinding obtained from the two
While two molecules of 15a bind in the active site, it appears
that 15a-I forms more key interactions and shorter hydrogen
bond distances than 15a-II (Figure 2B). The extensive
hydrogen-bond network that exists between the pyrimidine
E
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a
Table 3. In Vitro Biological Data in CEM Cells for Compounds 25−37
compd
R1
R2
R3
Y
IC50 (nM) CEM cells
25
26
27
28
29
30
31
32
33
34
35
36
37
H
H
F
H
F
OCH2CH2OH
OCH2CH2OH
OCH2CH2OH
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
2.45 ( 0.778)
1.07 ( 0.230)
2.83 ( 1.628)
11.58 ( 3.353)
8.01 ( 0.230)
2.59 ( 1.146)
H
H
H
H
F
OCH2CH2NHSO2CH3
OCH2CH2NHSO2CH3
OCH2CH2OH
H
H
H
H
H
H
H
H
OCH2CH2OH
OCH3
b
OH
18.62
OCH3
OCH2CH2CH2OH
OCH2CH2OH
1.55 ( 0.354)
1.15 ( 0.762)
2.90 ( 0.300)
2.85 ( 0.071)
1.44 ( 0.538)
4.89 ( 2.014)
OCH3
OCH3
OCH2CH2OH
OCH3
OCH2CH(CH3)OH
OCH2C(CH3)2OH
OCH2CH2NHSO2CH3
NH2
NH2
NH2
OCH3
OCH3
a
Inhibitory activity measured by 3H-deoxycytidine (3H-dC) uptake in CCRF-CEM human cells. Values reported are the mean SD of at least n = 2
b
independent experiments. Value reported for n = 1.
simulations indicates that the propyl chain of 15c confers a
1.210 kcal/mol more favorable free energy of binding in
comparison to the ethyl chain of 15b; this favorable effect is
due to desolvation. The change in free energy upon extension
of the alkyl chain is unfavorable both in the complex with the
protein and in water alone (positive ΔG for chain lengthening,
negative ΔG for chain shortening); however, the magnitude of
the unfavorable ΔG is larger in solvent. The fact that this
produces an overall favorable ΔΔG of binding suggests that the
propyl chain is better able to exclude water from the interior
cavity of the protein, allowing a greater association between the
protein and the inhibitor.
Lead Optimization and SAR. Based on the potency trend
in Table 2 and the existence of a hydrophobic pocket around
the 5-position of the thiazole ring of 15a, all further compounds
in the SAR were made with the propyl chain installed at that
position, to increase nonpolar interactions between the dCK
enzyme pocket and the inhibitors. The fluorine atom
terminating the ethoxy side-chain was substituted for a hydroxyl
or sulfonamide group, with the goal of improving the
molecule’s solubility properties as well as potential hydrogen
bonding interactions that might exist in the active site.
Moreover, since inhibitory activity in L1210 and CCRF-CEM
cells demonstrated the same trend in potency, the SAR for all
subsequently synthesized compounds were examined only in
CCRF-CEM cells. The results are summarized in Table 3.
Compounds 25−27 showed excellent (1−2 nM) potency
against CCRF-CEM cells (Table 3). Substitution of the end-
chain hydroxyl for a methyl sulfonamide resulted in a decrease
in inhibitory activity of about 3-fold (compare 27 to 29) or 5-
fold (compare 25 to 28). The initial SAR in Table 1 indicated
that the presence of an alkoxy substituent at the para position
led to increased inhibitory activity; therefore, the methoxy
group was reinstalled at the para position. As expected, removal
of the ethoxy side-chain (e.g., compound 31) resulted in a
substantially lower inhibitory activity, reinforcing the data
observed for compounds 16−18 (Table 2). The presence of
the methoxy moiety at the para position, in addition to the
hydroxylethoxy side chain at the meta position, generated
compound 33, which has an inhibitory potency of 1 nM. To
our surprise, removal of one of the amino groups from the
pyrimidine ring led to a mere 2.5-fold decrease in inhibitory
activity (compare 33 to 34). Initially, we observed that removal
of both amino groups from the pyrimidine ring resulted in
complete loss of inhibitory activity (compounds 6 and 7, Figure
1); however, the presence of one amino group can provide
suitable key hydrogen bonding interactions to inhibit the
enzyme. Compound 32, which contains a side-chain that has
been extended by one carbon to give a hydroxylpropoxy group,
was also synthesized. However, this modification resulted in
slightly decreased inhibitory activity in comparison with the
hydroxylethoxy group. While compound 33 was a potent
compound in cell culture, the presence of a primary hydroxyl
group in the molecule raised concerns of a metabolic liability as
a consequence of potential oxidation or glucuronidation.42
Thus, compounds 35−37 were synthesized to decrease the
possibility of metabolic degradation of 33. Eight of these
compounds in Table 3, whose IC50 values were lower than 15a
and whose structural properties suggested that they would have
the best in vivo efficacy, were selected for further investigation.
Steady-State Kinetic Analysis of Selected dCK
Inhibitors. In order to confirm that the cell-based values
truly reflect the potency of the compounds we also determined
the Kiapp values for select compounds using steady-state kinetic
F
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assays. The cell-based assays indicated that compound 15a was
6−12-fold (depending on the cell line used for the assay) less
potent than compound 15c (Table 2). Correspondingly, the
steady-state data showed a 6-fold higher Kiapp value for
compound 15a (Table 4). Likewise, the low nanomolar IC50
Table 4. Steady-State Kinetics of Selected dCK Inhibitors
compd
Kiapp (nM)
error (nM)
R2
a
15a
9.5
1.5
0.8
0.5
2.3
0.3
0.7
0.5
0.974
0.998
0.982
0.988
a
15c
36
37
a
Data from Nomme et al.
observed in CEM cells for compounds 36 and 37 (Table 3) was
recapitulated in the steady-state kinetics derived Kiapp values for
these compounds (Table 4). Hence, we conclude that our cell-
based assays are providing us with relatively accurate data as to
the strength of the interactions between the compounds and
dCK.
Evaluation of in Vivo Inhibition of dCK Activity via a
New PET PD Assay. The nucleoside analog PET probe 18F-L-
FAC is a high affinity substrate for dCK, which can be used to
noninvasively estimate dCK enzymatic activity in vivo.27
A
schematic depicting the mechanism by which 18F-L-FAC
accumulates in cells in a dCK-specific manner is shown in
Figure 4A. We reasoned that 18F-L-FAC PET could be used to
rapidly identify the most potent dCK inhibitors based on their
effectiveness at inhibiting the accumulation of the 18F-labeled
dCK substrate PET tracer in various tissues. For the in vivo
PET PD assay we selected dCK inhibitors that demonstrated
3
1−12 nM inhibitory activity in the cell culture H-dC uptake
assay (Table 3). Mice were treated with a single dose (50 mg/
kg) of a given dCK inhibitor administered by intraperitoneal
injection. Control mice received vehicle (40% Captisol in
water) injections. Four hours later, treated mice were injected
intravenously with 18F-L-FAC; one hour after probe injection,
mice were imaged by mPET/CT. The readout for the PET PD
assay was the reduction in the accumulation of 18F-L-FAC in
dCK-positive tissues in dCK inhibitor versus vehicle treated
mice. Previously, we showed that 18F-L-FAC accumulates in a
dCK-dependent manner into various tissues such as the
thymus, spleen, bone marrow, and liver.27 Accumulation in
the bladder is a result of nonenzymatic renal clearance of the
unmetabolized probe. Since the reproducibility in the dCK-
dependent tissue retention of 18F-L-FAC was most consistent
in the liver,27 we chose to quantify 18F-L-FAC liver retention in
order to compare the in vivo efficacy of the various dCK
inhibitors. Optimal conditions for the PET PD assay were
determined by performing a dose escalation and time course
study using compound 33 (Supporting Figure S4).
Figure 4. In vivo evaluation of dCK inhibitors via PET analysis. (A)
Schematic of the mechanism by which 18F-L-FAC accumulates in dCK
expressing cells. (B) Representative transverse images of 18F-L-FAC
PET/CT liver scans of C57Bl/6 mice treated with compounds 15a,
36, and 37. (C) Quantification of 18F-L-FAC uptake in the liver for a
sample of inhibitors with low nanomolar in vitro potency. Data are
mean values SEM for at least n = 3 mice/group. *, P < 0.03. (D)
Representative images and quantification of 18F-L-FAC PET/CT scans
of CCRF-CEM tumor bearing NSG mice that were treated with
vehicle or compound 36. Data are displayed as box and whisker plots
for at least n = 4 mice/group. *, P < 0.0012.
Results from the 18F-L-FAC mPET/CT scans are summar-
ized in Figure 4. Transverse PET images of the 18F-L-FAC liver
scans for mice treated with either vehicle or compounds 15a,
36, or 37 are shown in Figure 4B. Figure 4C illustrates the
uptake of 18F-L-FAC in the livers of mice treated dCK
inhibitors. The more efficacious compounds induced a greater
reduction in the 18F-L-FAC uptake relative to vehicle treatment,
as a result of their greater inhibition of dCK-mediated
phosphorylation of its 18F-labeled substrate. Note the
approximate 30% decrease in 18F-L-FAC signal compared to
vehicle control induced by compounds 28, 29, 36, and 37,
indicating their superior in vivo efficacy relative to the other
dCK inhibitor candidates. In addition, compounds 30 and 32
show about a 20% decrease in probe uptake. Compound 33,
one of the most potent dCK inhibitors in the cell culture assay
(Table 3), showed poor in vivo efficacy in the 18F-L-FAC liver
PET assay, presumably due to its poor PK properties. As
hypothesized, substitution of the hydroxyl group at the end of
the ethoxy chain (e.g., compound 33) for the metabolically
stable methylsulfonamide (compounds 28, 29, and 37) or
hindering the hydroxyl group (compound 36) proved advanta-
geous for in vivo efficacy. Compounds 36 and 37 have the
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lowest IC50 values among all the efficacious compounds and
were chosen for further study. Here we focus on compound 36,
while compound 37 will be described in a subsequent
publication (Nathanson, unpublished results).
X-ray Crystal Structure of Compound 36 Bound to
Human dCK. X-ray crystallographic studies of compound 36
were initiated to obtain information about its binding to dCK.
The crystal structure of the dCK:36 complex was solved at 1.94
Å resolution (Figure 6 and Table 5). Similar to our
observations for compound 15a (Figure 2), in the case of 36,
the enzyme also adopts the open conformation. We observed
one 36 molecule (green) in each protomer of the dimeric
enzyme (Figure 6A). This is in contrast to the observation of
two molecules bound per active site when the substituent at the
5-position is smaller than the propyl present in 36 (Figure 2,
determinants of single versus double molecule binding to the
dCK active site was analyzed by Nomme, unpublished results).
Note that binding of 36 to dCK does not preclude nucleotide
binding (UDP is shown in red, Figure 6A). The specific
dCK:36 interactions are shown in Figure 6B. These include an
extensive hydrogen-bond network between the pyrimidine
moiety of 36 and residues E53, Q97, and D133 in the dCK
nucleoside binding site, as well as several hydrophobic
interactions.
Next we determined the efficacy of compound 36 at
inhibiting dCK activity in tumor tissues in vivo. Mice bearing
CCRF-CEM tumor xenografts were treated with compound 36
four hours prior to injection of 18F-L-FAC (Figure 4D). One
hour after the 18F-L-FAC injection, mice were imaged by
mPET/CT. The retention of 18F-L-FAC in tumor xenografts
from mice treated with compound 36 was reduced by about
30% compared to the retention of 18F-L-FAC in tumors from
vehicle treated mice (Figure 4D). To complement the PET
assay, the pharmacokinetics of compound 36 was determined
using standard analytical techniques, and the approximated
values are reported in Figure 5.
CONCLUSION
■
The identification of potent small molecule human dCK
inhibitors that demonstrate in vivo target inhibition is reported.
Optimization of inhibitory activity was achieved by extending
an alkyl chain from the 5-position of the thiazole ring. In vivo
efficacy was improved by manipulation of the ethoxy side-chain
present at the meta position of the phenyl ring. The utility of
PET as a powerful tool for noninvasive measure of target
inhibition and, consequently, as a measure of lack of target
inhibition (most likely due to substrate metabolism in vivo) is
also presented. Although the major clinical applications of PET
are primarily for central nervous system (CNS) and oncology-
based diagnostics/therapeutics, PET is playing an increasingly
important role in drug development, given the capability of this
molecular imaging platform to address key challenges that
include evaluation of biodistribution, absorption, target affinity,
plasma binding, metabolism, and dosing.43 Here we used the
Figure 5. Pharmacokinetic profile of compound 36. C57Bl/6 female
mice were dosed with compound 36 via intraperitoneal injection. Dose
formulation: 10% DMSO and 40% Captisol in water. Data are mean
values SEM for n = 4 mice/time point.
Figure 6. Crystal structure of the dCK:36 complex. (A) Ribbon diagram of a dCK monomer (light blue) with the single observed molecule of 36
bound (green spheres) at the active site. The nucleotide UDP (red spheres) was also present in the complex. (B) Detail of the interactions between
36 and dCK. dCK residues involved in polar and hydrophobic interactions with 36 are colored in light blue and gray, respectively. Polar interactions
are indicated as broken black lines.
H
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an 18F radioisotope could generate a positron-emitting version
of the therapeutic candidate that can be detected and quantified
noninvasively throughout the body of living individuals by PET
imaging. This work will be the subject of a future
communication.
Table 5. Data Collection and Refinement Statistics
parameter
comments
36 + UDP
complex
PDB codes
4L5B
data collection statistics
X-ray source and detector
LS-CAT ID-G
MARCCD 300
0.97856
EXPERIMENTAL SECTION
■
High-Throughput Screen. Recombinant human dCK at a
concentration of 1 μM was incubated with 10 μM of drug, 10 μM
of dC, and 0.5 μM ATP with 50 mM Tris (pH 7.6), 5 mM MgCl2, and
2 mM DTT. The reaction was incubated at 37 °C for 4 h before
adding CellTiter-Glo (Promega): Briefly, 40 μL of dCK enzyme were
dispensed into 384 well plates (Greiner, Bahlingen, Germany) using a
multidrop 384 (Thermo, Turku, Finnland) at concentration of 12.5
μg/mL; compounds were added using a Beckman-Coulter Biomek FX
(Beckman Coulter, Brea, CA) equipped with a 500 nL custom pin tool
(V&P Scientific, San Diego, CA). Columns 1, 2, 23, and 24 received
only DMSO instead of any drugs. In addition, no dCK was added to
column 23 and 24 as these columns served as additional controls (see
below). After 30 min incubation at 37 °C, dC and ATP were added to
a final concentration of 10 μM and 0.5 μM, respectively, for columns
1−22 using the multidrop in a volume of 10 μL. For columns 23 and
24 the following controls were used: 10 μL of a 2.5 μM ATP solution
containing the following additional controls was added: for wells A-
D23 1 μM dCTP, for wells E-H23 10 μM dCTP, for wells I-L23 10
μM L-FAC, for wells F-P23 10 μM FAC, for wells A-D24 0.5 μM ATP
standard, for wells E-H24 0.1 μM ATP standard, for wells I-L24 1 μM
DCK only, and for wells F-P24 10 μM UTP was added, respectively.
These controls were included on each plate to exclude equipment
failure. This was followed by a 4 h incubation at 37 °C and addition of
25 μL of Cell titer glo reagent (Promega, Fitchburg, WI) by multidrop
followed by reading on an Acquest plate reader (Molecular Devices,
Sunnyvale, CA). The libraries used were custom sets of compounds
from the compound manufacturers Asinex (Winston-Salem, NC) and
Enamine (Monmouth Jct., NJ). These sets consisted of compounds
selected extensively for drug-likeness using the Lipinski rule of five,
rotatable bonds, and maximal diversity using custom clustering
algorithms.
Chemistry. General Procedures. Unless otherwise noted,
reactions were carried out in oven-dried glassware under an
atmosphere of nitrogen using commercially available anhydrous
solvents. Solvents used for extractions and chromatography were not
anhydrous. 4,6-Diamino-2-mercaptopyrimidine was obtained from
drying the hydrate over dynamic vacuum at 110 °C for 20 h. All other
reagents obtained from commercial suppliers were reagent grade and
used without further purification unless specified. Reactions and
chromatography fractions were analyzed by thin-layer chromatography
(TLC) using Merck precoated silica gel 60 F254 glass plates (250 μm).
Visualization was carried out with ultraviolet light, vanillin stain,
permanganate stain, or p-anisaldehyde stain. Flash column chromatog-
raphy was performed using E. Merck silica gel 60 (230−400 mesh)
with compressed air. 1H and 13C NMR spectra were recorded on
ARX500 (500 MHz) or Avance500 (500 MHz) spectrometers.
Chemical shifts are reported in parts per million (ppm, δ) using the
wavelength (Å)
temperature (K)
100
a
resolution (Å)
1.94 (1.94−2.05)
number of reflections
observed
206005
unique
40954
completeness (%)
Rsym (%)
99.2 (98.1)
4.5 (71.8)
17.05 (2.05)
P 41
average I/σ(I)
space group
unit cell (Å)
a = b
68.67
c
120.02
refinement statistics
refinement program
twinning fraction
Rcryst (%)
REFMAC5
0.5
18.8
Rfree (%)
23.3
resolution range (Å)
protein molecules per a.u.
number of atoms
protein (ProtA, ProtB)
inhibitor
30−1.94
2
1932, 1925
32 × 2
25 × 2
75
UDP
water
r.m.s. deviation from ideal
bond length (Å)
bond angles (deg)
average B-factors (Å2) /chain
protein (ProtA, ProtB)
inhibitor (ProtA, ProtB)
UDP (ProtA, ProtB)
waters
0.011
1.647
47.1, 47.3
45.1, 43.4
47.8, 44.7
42.9
Ramachandran plot (%)
most favored regions
additionally allowed regions
generously allowed regions
disallowed regions
89.1
10.5
0.5
0.0
a
Last shell in parentheses.
radiotracer 18F-L-FAC as a PET PD biomarker to compare the
in vivo efficacies of candidate dCK inhibitors, first identified
and characterized by potency in cell culture assays. Moreover,
we used PET to provide estimates of in vivo target inhibition in
CCRF-CEM xenograft mouse models by one of our most
promising compounds, 36. The ability of another promising
compound, 37, to elicit a significant pharmacological response
against CCRF-CEM tumors with minimal toxicity to normal
tissues was evaluated by our group and is described in a
separate publication. Further optimization offering improve-
ments to the PK and solubility properties of our best dCK
inhibitors will be addressed in subsequent studies. In addition,
the presence of fluorine on the aromatic ring of one of our most
promising dCK inhibitors, 29, makes it amenable to 18F
radiolabeling. Synthesizing a small molecule dCK inhibitor with
1
residual solvent peak as the reference. DMSO-d6 (δ 2.50 ppm for H;
δ 39.5 ppm for 13C) was used as the solvent and reference standards
unless otherwise noted. The coupling constants, J, are reported in
Hertz (Hz), and the resonance patterns are reported with notations as
the following: br (broad), s (singlet), d (doublet), t (triplet), q
(quartet), and m (multiplet). Electrospray mass spectrometry data
were collected with a Waters LCT Premier XE time-of-flight
instrument controlled by MassLynx 4.1 software. Samples were
dissolved in methanol and infused using direct loop injection from a
Waters Acquity UPLC into the Multi-Mode Ionization source. The
purity of all final compounds was determined to be >95%. Analytical
HPLC analysis was performed on a Knauer Smartline HPLC system
with a Phenomenex reverse-phase Luna column (5 μm, 4.6 × 250
mm) with inline Knauer UV (254 nm) detector. Mobile phase: A:
0.1% TFA in H2O, B: 0.1% TFA in MeCN. Eluent gradient is specified
I
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for each described compound in the Supporting Information. All
chromatograms were collected by a GinaStar (raytest USA, Inc.;
Wilmington, NC, USA) analog to digital converter and GinaStar
software (raytest USA, Inc.).
stirred for 1 h at 40 °C when, by TLC analysis, all starting material had
been consumed. The solvent was removed in vacuo, and the crude
residue was purified by flash column chromatography over silica gel
(10:3 hexanes:ethyl acetate) to give the desired bromide 24a (1.84 g,
1
General Procedure for the Synthesis of Compounds 15a−c.
3-(2-Fluoroethoxy)-4-methoxybenzonitrile (20). To a solution of
3-hydroxy-4-methoxybenzonitrile 19 (3.0 g, 20.1 mmol) in DMF (100
mL) was added Cs2CO3 (10.5 g, 32.2 mmol) and 1-bromo-2-
fluoroethane (5.1 g, 40.2 mmol). The mixture was stirred for 18 h at
50 °C. After concentration to remove residual solvent, the resulting
residue was washed with brine and extracted with ethyl acetate. The
organic layer was washed with water three times, dried over anhydrous
MgSO4 and concentrated in vacuo to yield crude 20 (3.91 g, 20.03
mmol, 99%) as a cream-colored solid. 1H NMR (500 MHz, CDCl3) δ:
7.28 (dd, J = 8.5, 2.0 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.90 (d, J = 8.5
Hz, 1H), 4.83−4.81 (m, 1H), 4.73−4.71 (m, 1H), 4.28−4.26 (m, 1H),
4.23−4.21 (m, 1H), 3.89 (s, 3H); 13C NMR (125 MHz, CDCl3) δ:
153.6, 148.1, 127.3, 119.1, 116.5, 111.9, 103.8, 82.3 (d, JCF = 170.5
Hz), 68.7 (d, JCF = 20.3 Hz), 56.1.
5.1 mmol, 80%) as a light brown solid. H NMR (500 MHz, CDCl3)
δ: 7.50 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.5, 2.0 Hz, 1H), 6.88 (d, J =
8.0 Hz, 1H), 4.81 (dt, J = 47.0, 4.0 Hz, 2H), 4.59 (s, 2H), 4.36 (dt, J =
27.5, 4.0 Hz, 2H), 3.90 (s, 3H), 2.46 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ: 164.1, 151.2, 148.1, 148.0, 131.7, 126.4, 120.4, 111.6, 111.5,
82.4 (d, JCF = 169.9 Hz), 68.4 (d, JCF = 20.5 Hz), 55.9, 25.8, 11.4.
2-(((2-(3-(2-Fluoroethoxy)-4-methoxyphenyl)-5-methylthia-
zol-4-yl)methyl)thio)pyrimidine-4,6-diamine (15a). 4,6-Diami-
no-2-mercaptopyrimidine (336 mg, 2.36 mmol) and NaOH (94 mg,
2.36 mmol) were stirred in ethanol (20 mL) for 10 min at 23 °C. To
the reaction mixture was added a solution of bromide 24a (710 mg,
1.97 mmol) in hot ethanol (16 mL), and the resulting mixture was
stirred for 3 h at 70 °C. The solution was cooled, concentrated in
vacuo, and purified by flash column chromatography over silica gel
(100:5 dichloromethane:methanol) to give the desired product 15a
(590 mg, 1.40 mmol, 71%) as a pale yellow solid. 1H NMR (500 MHz,
DMSO-d6) δ: 7.36 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.5
Hz, 1H), 6.09 (brs, 4H), 5.12 (s, 1H), 4.72 (dt, J = 48.0, 3.5 Hz, 2H),
4.32 (s, 2H), 4.25 (dt, J = 30.5, 3.5 Hz, 2H), 3.78 (s, 3H), 2.43 (s,
3H); 13C NMR (125 MHz, DMSO-d6) δ: 168.3, 163.9 (2), 163.3,
3-(2-Fluoroethoxy)-4-methoxybenzothioamide (21). To a
mixture of 20 (3.86 g, 19.8 mmol) in pyridine (41 mL) and
triethylamine (3 mL) was added ammonium sulfide solution (20% wt.
in H2O, 13.52 mL, 39.6 mmol). The mixture was stirred for 18 h at 60
°C. The reaction mixture was cooled and concentrated in vacuo to
remove residual solvent. The resulting residue was washed with brine
and extracted with ethyl acetate. The organic layer was dried over
anhydrous MgSO4 and concentrated in vacuo to yield 21 (4.5 g, 19.8
150.9, 149.5, 148.3, 129.1, 126.4, 119.9, 112.7, 110.5, 83.2 (d, JCF
=
165.9 Hz), 79.5, 68.5 (d, JCF = 18.7 Hz), 56.1, 27.9, 11.7; HRMS-ESI
(m/z) [M + H]+ calcd for C18H20FN5O2S2 H, 422.1121; found
422.1136.
2-(((5-Ethyl-2-(3-(2-fluoroethoxy)-4-methoxyphenyl)thiazol-
4-yl)methyl)thio)pyrimidine-4,6-diamine (15b). 1H NMR (500
MHz, DMSO-d6) δ: 7.37 (dd, J = 8.0, 2.0 Hz, 1H), 7.36 (s, 1H), 7.02
(d, J = 8.5 Hz, 1H), 6.13 (brs, 4H), 5.13 (s, 1H), 4.72 (dt, J = 47.5, 4.0
Hz, 2H), 4.34 (s, 1H), 4.25 (dt, J = 30.5, 4.0 Hz, 2H), 3.79 (s, 3H),
2.87 (q, J = 7.5 Hz, 2H), 1.17 (t, J = 7.5 Hz, 3H); 13C NMR (125
MHz, DMSO-d6) δ: 168.2, 163.8 (2), 163.5, 151.0, 148.4, 148.3, 136.9,
1
mmol, quantitative) as a yellow-orange solid. H NMR (500 MHz,
acetone-d6) δ: 8.81 (brs, 1H), 8.74 (brs, 1H), 7.73 (s, 1H), 7.72 (dd, J
= 8.5, 2.0 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 4.79 (dt, J = 48.0, 4.0 Hz,
2H), 4.32 (dt, J = 29.5, 4.0 Hz, 2H), 3.89 (s, 3H); 13C NMR (125
MHz, acetone-d6) δ: 200.4, 152.9, 147.2, 131.8, 121.5, 113.6, 110.8,
82.7 (d, JCF = 167.3 Hz), 68.5 (d, JCF = 19.6 Hz), 55.4.
Ethyl 2-(3-(2-Fluoroethoxy)-4-methoxyphenyl)-5-methyl-
thiazole-4-carboxylate (22a). A mixture of thioamide 21 (1.50 g,
6.5 mmol) and ethyl 3-bromo-2-oxobutanoate (2.72 g, 13.0 mmol) in
ethanol (32 mL) was stirred under refluxing conditions for 2.5 h. The
resulting mixture was cooled and concentrated in vacuo to remove
residual solvent. The crude residue was purified by flash column
chromatography over silica gel (10:3 hexanes:ethyl acetate) to yield
the desired thiazole intermediate 22a (1.45 g, 4.3 mmol, 65%) as a
126.5, 119.9, 112.7, 110.5, 83.3 (d, JCF = 165.9 Hz), 79.5, 68.5 (d, JCF
=
18.8 Hz), 56.1, 28.0, 19.9, 17.1; HRMS-ESI (m/z) [M + H]+ calcd for
C19H22FN5O2S2 H, 436.1277; found 436.1263.
2-(((2-(3-(2-Fluoroethoxy)-4-methoxyphenyl)-5-propylthia-
zol-4-yl)methyl)thio)pyrimidine-4,6-diamine (15c). 1H NMR
(500 MHz, acetone-d6) δ: 7.53 (d, J = 2.0 Hz, 1H), 7.46 (dd, J =
8.5, 2.0 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 5.63 (brs, 4H), 5.38 (s, 1H),
4.80 (dt, J = 48.0, 4.0 Hz, 2H), 4.45 (s, 2H), 4.34 (dt, J = 29.5, 4.0 Hz,
2H), 3.87 (s, 3H), 2.91 (t, J = 7.5 Hz, 1H), 1.66 (qt, J = 7.5, 7.5 Hz,
2H), 0.97 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, acetone-d6) δ:
169.2, 164.0 (2), 163.9, 163.6, 151.4, 149.0, 148.5, 134.6, 126.9, 119.8,
112.1, 111.1, 82.8 (d, JCF = 167.5 Hz), 79.5, 68.6 (d, JCF = 19.5 Hz),
55.3, 28.1, 25.2, 13.0; HRMS-ESI (m/z) [M + H]+ calcd for
C20H24FN5O2S2 H, 450.1434; found 450.1432.
1
light brown solid. H NMR (500 MHz, DMSO-d6) δ: 7.40 (dd, J =
8.5, 2.0 Hz, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H),
4.72 (dt, J = 48.0, 4.0 Hz, 2H), 4.31−4.22 (m, 2H), 4.28 (q, J = 7.0 Hz,
2H), 3.81 (s, 3H), 2.67 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6) δ: 162.9, 162.1, 151.4, 148.2, 143.9, 141.9,
125.5, 120.5, 112.6, 110.8, 83.1 (d, JCF = 165.9 Hz), 68.3 (d, JCF = 19.0
Hz), 60.8, 56.0, 14.5, 13.3.
(2-(3-(2-Fluoroethoxy)-4-methoxyphenyl)-5-methylthiazol-
4-yl)methanol (23a). To a stirred solution of intermediate 22a (860
mg, 2.5 mmol) in CH2Cl2 (30 mL) cooled to 0 °C was added slowly
diisobutylaluminum hydride (1.0 M in THF, 10 mmol, 10 mL). The
reaction was allowed to warm to 23 °C and stirred for 1 h. The
mixture was cooled to 0 °C and slowly quenched with a saturated
aqueous solution of Rochelle’s salt. The cloudy solution was stirred for
1 h at 23 °C until the solution became clear again. The resulting
solution was extracted with ethyl acetate, washed with brine, dried over
anhydrous magnesium sulfate, and concentrated in vacuo to give the
desired alcohol 23a (654 mg, 2.2 mmol, 88%) as a pale yellow solid.
1H NMR (500 MHz, DMSO-d6) δ: 7.39 (d, J = 2.0 Hz, 1H), 7.36 (dd,
1-(5-(4-(((4,6-Diaminopyrimidin-2-yl)thio)methyl)-5-pro-
pylthiazol-2-yl)-2-methoxyphenoxy)-2-methylpropan-2-ol
1
(36). H NMR (500 MHz, MeOD) δ: 7.51 (d, J = 2.0 Hz, 1H), 7.39
(dd, J = 8.5, 2.0 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 5.48 (s, 1H), 5.32
(s, 1H), 4.48 (s, 2H), 3.89 (s, 3H), 3.86 (s, 2H), 2.88 (t, J = 7.5 Hz,
2H), 1.67 (qt, J = 7.5, 7.5 Hz, 2H), 1.33 (s, 6H), 0.98 (t, J = 7.5 Hz,
3H); 13C NMR (125 MHz, MeOD) δ: 168.8, 165.2, 163.8 (2), 151.2,
148.9, 148.0, 135.4, 126.4, 119.7, 111.8, 110.7, 79.2, 77.0, 69.6, 55.2,
48.4, 27.9, 27.8, 25.0, 24.9, 12.6; HRMS-ESI (m/z) [M + H]+ calcd for
C22H29N5O3S2 H, 476.1790; found 476.1772.
N-(2-(5-(4-(((4,6-Diaminopyrimidin-2-yl)thio)methyl)-5-pro-
p y l t h i a z o l - 2 - y l ) - 2 - m e t h o x y p h e n o x y ) e t h y l ) -
methanesulfonamide (37). 1H NMR (500 MHz, DMSO-d6) δ:
7.41 (dd, J = 7.5, 2.0 Hz, 1H), 7.39 (s, 1H), 7.25 (t, J = 6.0 Hz, 1H),
7.05 (d, J = 8.5 Hz, 1H), 6.13 (brs, 4H), 5.15 (s, 1H), 4.39 (s, 2H),
4.07 (t, J = 5.5 Hz, 2H), 3.80 (s, 3H), 3.36 (dt, J = 5.5, 5.5 Hz, 2H),
3.15 (d, J = 5.5 Hz, 1H), 2.98 (s, 3H), 2.84 (t, J = 7.5 Hz, 2H), 1.58
(qt, J = 7.5, 7.5 Hz, 2H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (125
MHz, DMSO-d6) δ: 168.3, 163.9 (2), 163.7, 151.1,149.1, 148.3, 135.0,
126.5, 119.9, 112.7, 110.6, 79.5, 68.3, 60.2, 42.4, 31.2, 28.2, 28.0, 25.4,
J = 8.5, 2.0 Hz, 1H), 7.02 (d, J = 8.5 Hz, 1H), 5.04 (t, J = 5.5 Hz, 1H),
4.73 (dt, J = 48.0, 3.5 Hz, 2H), 4.46 (d, J = 5.5 Hz, 2H), 4.25 (dt, J =
30.0, 3.5 Hz, 2H), 3.79 (s, 3H), 2.41 (s, 3H); 13C NMR (125 MHz,
DMSO-d6) δ: 162.7, 153.2, 150.8, 148.2, 129.5, 126.5, 119.8, 112.5,
110.4, 83.1 (d, JCF = 165.9 Hz), 68.4 (d, JCF = 18.5 Hz), 57.3, 55.9,
11.2.
4-(Bromomethyl)-2-(3-(2-fluoroethoxy)-4-methoxyphenyl)-
5-methylthiazole (24a). To a solution of 23a (1.90 g, 6.4 mmol) in
acetonitrile (30 mL) was added PPh3 (2.5 g, 9.6 mmol) followed by
hexabromoacetone (1.70 g, 3.2 mmol) at 23 °C. The mixture was
J
dx.doi.org/10.1021/jm400457y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
13.9; HRMS-ESI (m/z) [M + H]+ calcd for C21H28N6O4S3 H,
525.1412; found 525.1404.
i.p.). Blood samples (approximately 70 μL) were collected through
retro-orbital bleeding into heparinized tubes at 5 min, 15 min, 30 min,
35 min, 40 min, 45 min, 1 h, 2 h, 4 h, and 6 h. The blood samples were
centrifuged at 20,000g for 5 min to isolate plasma. One mL of
acetonitrile was added to 30 μL of plasma. The supernatant was
transferred to new tubes and was evaporated using a SpeedVac.
Samples were then resuspended in 50 μL of neat DMSO, and
supernatant was transferred to LC/MS sample vials. Samples were
then run on an Agilent 6460 Triple Quad LC/MS.
Statistical Analyses. All statistics presented as means of biological
replicates with standard error of the mean ( SEM), standard deviation
( SD), or box plots with max and min whiskers. P-value significances
were calculated using one sample Student’s t test function in GraphPad
Prism 5 (GraphPad Software).
dCK Uptake Assay Performed in Cell Culture. The murine
leukemic line L1210 was a gift from Charles Dumontet (Universite
Claude Bernard Lyon I, Lyon, France). The human lymphoma line
CCRF-CEM was provided by Margaret Black (Washington State
University). All L1210 and CCRF-CEM cell lines were cultured in
RPMI medium 1640, supplemented with 5% FCS in a 5% CO2 37 °C
incubator. For the uptake assays cells were seeded at a density of
50,000 cells/well in Millipore MultiScreen GV 96 well plates. 0.25 μCi
of 3H-dC (Moravek Biochemicals) were added to the cells
simultaneously with concentrations of dCK inhibitor at a final volume
of 100 μL/well. After 1 h at 37 °C, cells were washed four times with
ice cold phosphate-buffered saline (PBS) using the Millipore Vacuum
Manifold. The amount of incorporated probe was measured by
scintillation counting with the PerkinElmer Microbeta.
Protein Expression and Purification. Details on C4S S74E dCK
variant expression and purification are detailed in Nomme et al.
Crystallization, X-ray Data Collection, and Refinement.
Crystallization, data collection, and structure determination of dCK
in complex with 15a and 36 were performed following the general
procedure as detailed in Nomme et al. Specifically for compound 36,
crystals of dCK in complex with UDP, MgCl2 and a 2.5-fold excess of
the 36 inhibitor were grown using the hanging drop vapor diffusion
method at 12 °C. The reservoir solution contained 0.9−1.5 M
trisodium citrate dehydrate and 25 mM HEPES (pH 7.5). Diffraction
data were collected at the Advanced Photon Source, Argonne National
Laboratory on Life Sciences-Collaborative Access Team (LS-CAT)
beamlines 21 ID-G.
́
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and spectroscopic data for compounds 8−
14, 16−18, and 25−36; synthetic schemes for 8a, 8b, and 37;
dose escalation/time course for PET assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
Figures 2 and 3: dCK + 15a + UDP Code: 4JLK; Figure 6:
dCK + 36 + UDP Code: 4L5B
AUTHOR INFORMATION
Corresponding Author
*Phone: 310-825-1205. E-mail: CRadu@mednet.ucla.edu
(C.G.R.). Phone: 312-355-5029. E-mail: Lavie@uic.edu (A.L.).
■
Kinetic Assay. Steady-state kinetic assay and data fitting were
performed as described in Nomme et al.
Computational Modeling. All simulations were performed using
the MCPRO 2.0 package.41 Initial coordinates were obtained from the
X-ray structure of dCK in complex with compound 15c. The protein
was solvated in a 30 Å water cap, represented by the TIP4P44 classical
water model. Solute atoms represented by the OPLS-AA force field45
were used. Equilibrations were performed using Metropolis Monte
Carlo (MC) in the NPT ensemble at 25 °C and 1 atm. The backbone
of the protein and all bond lengths within the protein were fixed;
angles and torsions within 11 Å from the center of the bound molecule
were sampled. All degrees of freedom of the inhibitor compound were
sampled during equilibration simulations. Equilibration consisted of 5
× 106 configurations of sampling in which only solvent moves were
allowed and of 10 × 106 subsequent configurations for the protein-
inhibitor complex and for the lone inhibitor in solution. The
equilibrated systems were then subject to free energy perturbation
(FEP)/MC simulations. These simulations consisted of 14 perturbing
steps of double-wide sampling. During FEP, the system underwent 5 ×
106 configurations of solvent equilibration, followed by 10 × 106
configurations of full equilibration, and 25 × 106 configurations of data
collection. All degrees of freedom of the inhibitor were sampled except
those bonds undergoing perturbation. The perturbed bond lengths
were systematically varied from the original to the final length.
In Vivo MicroPET/CT Imaging Studies. Animal studies were
approved by the UCLA Animal Research Committee and were carried
out according to the guidelines of the Department of Laboratory
Animal Medicine at UCLA. For the PET liver assay, C57BL/6 mice
were intraperitoneally (i.p.) injected with the indicated amounts of
dCK inhibitor (resuspended in 40% Captisol) 4 h prior to intravenous
injection of 70 μCi of 18F-L-FAC. For the tumor xenograft assay, NOD
scid IL-2 receptor gamma chain knockout (NSG) bearing subcuta-
neous CCRF-CEM tumor xenografts were injected with 50 mg/kg of
compound 36 or vehicle. Four hours post-treatment mice were
injected intravenously with 70 μCi of 18F-L-FAC. For all mPET/CT
studies, a 1 h interval was allowed between probe administration and
mPET/CT scanning (Inveon, Siemens Medical Solutions USA Inc.;
microCAT, Imtek Inc.). Static mPET images were acquired for 600 s.
Images were analyzed using OsiriX Imaging Software Version 3.8.
Pharmacokinetic Studies. C57Bl/6 female mice, 8 weeks of age,
were injected with a single dose of indicated compounds (50 mg/kg,
Present Addresses
○UCLA, Crump Institute for Molecular Imaging CNSI, 570
Westwood Plaza, Building 114, Box 951770, Mail Code:
177010, Los Angeles, CA 90095-1770.
▲Agensys, Inc. 1800 Stewart Ave, Santa Monica, CA, 90404.
◆Department of Radiology, Memorial Sloan-Kettering Cancer
Center, New York, NY 10021, USA.
Author Contributions
‡These authors contributed equally.
Notes
The authors declare the following competing financial
interest(s): C.G.R., J.C. and M.E.P. are co-founders of Sofie
Biosciences, a molecular diagnostic company. They hold equity
in Sofie Biosciences. The University of California also holds
equity in Sofie Biosciences. Intellectual property that C.G.R.
and J.C. invented and which was patented by the University of
California has been licensed to Sofie Biosciences. The
University of California has patented additional intellectual
property invented by C.G.R., J.C., H.L., D.N., J.M.M., A.L.A.,
M.E.J., and A.L.
ACKNOWLEDGMENTS
■
We are extremely grateful to Dr. Nagichettiar Satyamurthy for
his expert chemistry advice and support in designing the
synthetic schemes, without his help this project would not have
been possible. We thank Larry Pang and Michelle Tom for
their assistance with PET/CT imaging studies, the UCLA
Biomedical Cyclotron crew for producing 18F-L-FAC, and
Radha Bathala for her assistance in synthesizing compound
intermediates. We would also like to acknowledge Jong Lee at
the UCLA Molecular Screening Shared Resource for assistance
with the high-throughput screening. J.M.M. and D.N. thank the
Scholars in Oncologic Molecular Imaging program for
postdoctoral fellowships. This work was funded by In Vivo
K
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Cellular and Molecular Imaging Centers Developmental
Project Award National Institutes of Health P50 CA86306
(C. G. Radu, H. R. Herschman, and W. R. Austin) and the US
National Cancer Institute grant 5U54 CA119347 (C. G. Radu).
R.D. would like to acknowledge support for the Molecular
Screening Shared Resource from the National Cancer Center
Support grant P3016042-35 (J. Gasson).
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ABREVIATIONS USED
■
(13) Jessop, T. C.; Tarver, J. E.; Carlsen, M.; Xu, A.; Healy, J. P.;
Heim-Riether, A.; Fu, Q.; Taylor, J. A.; Augeri, D. J.; Shen, M.; Stouch,
T. R.; Swanson, R. V.; Tari, L. W.; Hunger, M.; Hoffman, I.; Keyes, P.
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Nouraldeen, A.; Wilson, A. G. E.; Foushee, A. M. D.; Jhaver, K.; Finch,
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Advances in Molecular Imaging and an Appraisal of PET/CT
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ADA, adenosine deaminase; dCK, deoxycytidine kinase; dNTP,
deoxyribonucleotide triphosphate; 18F-FAC, 18F-1-(2′-deoxy-
2′-FluoroArabinofuranosyl) Cytosine; TK1, thymidine kinase
1; HTS, high throughput screen; dC, deoxycytidine; dG,
deoxyguanosine; dA, deoxyadenosine; 18F-FDG, 2-18F-fluoro-2-
deoxy-D-glucose; 18F-L-FAC, 18F-L-1-(2′-deoxy-2′-FluoroArabi-
nofuranosyl) Cytosine; 3H-dC, 3H-deoxycytidine; ATP, ad-
enosine triphosphate; UTP, uridine triphosphate; UDP, uridine
diphosphate; E53, glutamic acid 53; D133, aspartic acid 133;
Q97, glutamine 97; V55, valine 55; L82, leucine 82; F96,
phenylalanine 96; mPET/CT, micro positron emission
tomography/computed tomography; PK, pharmacokinetic;
PD, pharmacodynamic; DMF, dimethylformamide; DIBAL-H,
diisobutylaluminum hydride; EtOH, ethanol; Rochelle’s Salt,
sodium potassium tartrate; DMSO, dimethylsulfoxide; PBS,
phosphate-buffered saline; FCS, fetal calf serum
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