Structure-guided development of deoxycytidine kinase inhibitors with nanomolar affinity and improved metabolic stability

Article abstract of DOI:10.1021/jm501124j

Recently, we have shown that small molecule dCK inhibitors in combination with pharmacological perturbations of de novo dNTP biosynthetic pathways could eliminate acute lymphoblastic leukemia cells in animal models. However, our previous lead compound had a short half-life in vivo. Therefore, we set out to develop dCK inhibitors with favorable pharmacokinetic properties. We delineated the sites of the inhibitor for modification, guided by crystal structures of dCK in complex with the lead compound and with derivatives. Crystal structure of the complex between dCK and the racemic mixture of our new lead compound indicated that the R-isomer is responsible for kinase inhibition. This was corroborated by kinetic analysis of the purified enantiomers, which showed that the R-isomer has >60-fold higher affinity than the S-isomer for dCK. This new lead compound has significantly improved metabolic stability, making it a prime candidate for dCK-inhibitor based therapies against hematological malignancies and, potentially, other cancers.

Full text of DOI:10.1021/jm501124j

Article
pubs.acs.org/jmc
Structure-Guided Development of Deoxycytidine Kinase Inhibitors
with Nanomolar Affinity and Improved Metabolic Stability
Julian Nomme,†,∞,# Zheng Li,§,∥,∞ Raymond M. Gipson,§,∥,∞ Jue Wang,§,∥,∞ Amanda L. Armijo,§,∥
Thuc Le,§,∥ Soumya Poddar,§,∥ Tony Smith,§,∥ Bernard D. Santarsiero,‡ Hien-Anh Nguyen,†
Johannes Czernin,§,∥ Anastassia N. Alexandrova,⊥ Michael E. Jung,⊥ Caius G. Radu,*,§,∥
and Arnon Lavie*,†
‡
†Department of Biochemistry and Molecular Genetics, and Center for Pharmaceutical Biotechnology, University of Illinois at
Chicago, Chicago, Illinois 60607, United States
∥
⊥
§Department of Molecular and Medical Pharmacology, Ahmanson Translational Imaging Division, and Department of Chemistry
and Biochemistry, University of California Los Angeles, Los Angeles, California 90095, United States
S
* Supporting Information
ABSTRACT: Recently, we have shown that small molecule
dCK inhibitors in combination with pharmacological pertur-
bations of de novo dNTP biosynthetic pathways could
eliminate acute lymphoblastic leukemia cells in animal models.
However, our previous lead compound had a short half-life in
vivo. Therefore, we set out to develop dCK inhibitors with
favorable pharmacokinetic properties. We delineated the sites
of the inhibitor for modification, guided by crystal structures of dCK in complex with the lead compound and with derivatives.
Crystal structure of the complex between dCK and the racemic mixture of our new lead compound indicated that the R-isomer is
responsible for kinase inhibition. This was corroborated by kinetic analysis of the purified enantiomers, which showed that the R-
isomer has >60-fold higher affinity than the S-isomer for dCK. This new lead compound has significantly improved metabolic
stability, making it a prime candidate for dCK-inhibitor based therapies against hematological malignancies and, potentially, other
cancers.
and 2 can be divided into four distinct structural parts (Figure
1A). Part A is the pyrimidine ring, which is connected by a linker
(part B) to a 5-subsituted-thiazole ring (part C), which in turn is
connected to a phenyl ring (part D). Conceptually, each of these
parts can be modified to attain desired “druglike” properties. In
previous work, we focused on the thiazole portion of the
inhibitor. The crystal structure of dCK with one of the early
compounds suggested that the ring 5-position could accom-
modate hydrophobic substituents, which led to the discovery that
a propyl group at the 5-position is strongly favored over a methyl
group.8,9
INTRODUCTION
■
Deoxycytidine kinase (dCK) is a deoxyribonucleoside kinase
capable of phosphorylating deoxycytidine, deoxyadenosine, and
deoxyguanosine to their monophosphate forms using either ATP
or UTP as phosphoryl donors.1 Phosphorylation by dCK is
responsible for converting salvaged deoxycytidine into deoxy-
cytidine monophosphate (dCMP), a precursor for both dCTP
and dTTP pools. Apart from the physiological role of generating
dNTPs, dCK plays a crucial role in activating multiple nucleoside
analog prodrugs that are widely used in anticancer and antiviral
therapy.2 Recently, we3,4 and others5 identified a requirement for
dCK in hematopoiesis in lymphoid and erythroid progenitors.
The kinase has also been implicated in regulating the G2/M
transition in response to DNA damage in cancer cells.6 More
recently, we have shown that partial inhibition of dCK activity,
combined with perturbations of nucleotide de novo synthesis
pathways, was synthetically lethal to acute lymphoblastic
leukemia cells but not to normal hematopoietic cells.7 These
aspects of dCK’s biology, and its potential role as a new
therapeutic target in cancer, prompted us to develop small
molecule inhibitors of its enzymatic activity.
To guide and rationalize the medicinal chemistry efforts in
other parts of the molecule, we solved the crystal structures of
human dCK with several of the inhibitors we developed. The
crystal structures illuminate the relationship between the enzyme
structure, the small molecule structure, and its inhibition
potency. In the first part of this manuscript we report the in
app
vitro binding affinities (IC50 and Kiapp), cellular IC50 values,
and crystal structures of dCK in complex with compounds that
differ in the pyrimidine and phenyl rings. Unfortunately, despite
nanomolar affinity for dCK, when tested in a liver microsomal
assay, these compounds exhibited low metabolic stability (data
In earlier publications8,9 we reported the discovery of hit
compounds from a high throughput screen and subsequent
optimization of the molecules to lead compounds 1 and 2
(numbered 36 and 37, respectively, in ref 8). Lead compounds 1
Received: July 23, 2014
© XXXX American Chemical Society
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Figure 1. dCK inhibitors lead compounds. (A) Schematic representa-
tion of lead compounds 1 and 2. These compounds are composed of
four parts. Part A indicates the pyrimidine ring, and part B is the linker
connecting to a 5-substituted-thiazole ring (part C), which is followed
by a phenyl ring (part D). Compounds 1 and 2 differ at the substituent
present at the phenyl meta position (Rm). (B) In vitro (IC50app and Kiapp
and cell (IC50) properties for 1 and 2.
)
not shown). This shortcoming was recapitulated by pharmaco-
kinetic studies in mice.8,7
To identify inhibitors with improved in vivo properties, we set
out to explore additional chemical modifications, specifically,
those that maintain the low nanomolar binding affinity of the
lead compounds. In the second part of the manuscript, we report
novel chiral derivatives of our inhibitors. Crystal structures of
these chiral compounds bound to dCK played a key role in
elucidating the chirality of the active form of the inhibitor. By
combining organic chemistry intuition with detailed structural
information on the target−inhibitor complex, we have identified
a lead compound that retains the nanomolar affinity for dCK but
has gained significant in vivo metabolic stability. This compound
could play a vital role in any therapeutic strategy based on
induction of DNA replication stress overload by perturbing a
cancer cell’s dNTP pools.
Figure 2. Modifications to the pyrimidine ring. (A) Schematic
representation of compound 3 that has a single exocyclic amino group
and of compound 4 that has a ring nitrogen atom between the two
exocyclic amino groups. (B) In vitro (IC50app and Kiapp) and cell (IC50)
properties for 3 and 4. (C) Overlay of the dCK−4 (orange, PDB code
4Q18) and dCK-1 (green, PDB code 4L5B) structures with a focus on
the pyrimidine ring. Note the ∼0.4 Å shifted position of 4 relative to 1
that is due to the presence of a water molecule (orange sphere). Binding
of this water molecule is made possible by the ring N atom in compound
4.
RESULTS AND DISCUSSION
■
The Inhibitor’s Pyrimidine Ring Appears To Be Already
Optimized for the Interaction with dCK. The pyrimidine
ring (part A of the molecules, Figure 1A) was predicted to be the
part of the molecule most difficult to improve. This is because, as
observed in the crystal structures of dCK in complex with lead
compounds 1 and 2 (PDB codes 4L5B and 4KCG, respectively),
the inhibitor’s pyrimidine ring binds to dCK at a position nearly
identical to that adopted by the pyrimidine ring of the
physiological substrate dC, making several hydrogen bonds,
hydrophobic, and π−π stacking interactions (Supporting
Information Figure S1). This binding mode suggested an already
quite optimized enzyme−pyrimidine ring interaction. For
compounds 1 and 2, both pyrimidine ring exocyclic amino
groups formed hydrogen-bonding interactions with side chains
of Glu53, Gln97, and Asp133. Hence, not surprisingly,
simultaneous removal of both amino groups resulted in complete
loss of dCK inhibition.8 In contrast, removal of a single amino
group to generate compound 3 (Figure 2A), which is identical to
1 except for having a single exocyclic amino group in the
pyrimidine ring (Figure 1A), resulted in similarly tight binding
affinity as measured for 2 (Figures 1B and 2B). To explain how
the affinity of 3 for dCK is maintained with only a single exocyclic
amino group, we sought the crystal structure of the complex, but
unfortunately, we were unable to obtain diffraction quality
crystals. We speculate that the sole exocyclic amino group
present in compound 3 is oriented in the dCK active site such
that it maintains its interaction with Asp133, since only in that
orientation can the neighboring pyrimidine ring N atom
maintain its interaction with the side chain of Gln97 (Supporting
Information Figure S1). The conclusion here is that the
interaction with Glu53 made by an exocyclic amino group,
when present, provides only moderate additional binding energy.
While a single exocyclic pyrimidine ring amino group is sufficient
for a tight interaction with dCK, in our CEM cell-based assay
compound 3 exhibited a much-increased IC50 value (21.8 nM,
Figure 2B) relative to compound 2 (4.9 nM, Figure 1B). This
B
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result showcases the importance of evaluating the interaction
between an inhibitor and its target in using both an enzymatic in
vitro assay and a cell-based assay. Because of the reduced
inhibition of dCK activity of 3 in the cell-based assay, all future
compounds contained the two exocyclic amino groups.
Next, we assessed the importance of the position of the
pyrimidine ring N atoms by synthesizing compound 4 (Figure
2A). This compound was measured to bind with ∼50-fold higher
IC50app relative to the very similar lead compound 1 (Figure 1A),
which only differs in the position of one pyrimidine ring nitrogen
atom. We solved the 2.0 Å resolution crystal structure of the
dCK−4 complex to understand how this subtle change so
drastically impacted the interaction with the enzyme (see Table 1
for the data collection and refinement statistics).
All of the examined compounds bind to the open state of the
enzyme, which is also the catalytically incompetent state (for a
discussion about the open and closed states of dCK, see refs 10
and 11). Inhibitors bind within a deep cavity, with the pyrimidine
ring of the inhibitors positioned deepest and occupying the same
position occupied by the pyrimidine ring of the nucleoside
substrate.8,9 While preventing the binding of the nucleoside
substrate, our inhibitors do not interfere with binding of
nucleotide to the phosphoryl donor-binding site. In fact, all
crystal structures of dCK in complex with inhibitors also
contained UDP at the donor site.
app
Despite significantly different IC50 values between com-
pound 1 (14.5 nM) and compound 4 (754 nM), the pyrimidine
ring of these related molecules interacts with the enzyme via very
similar hydrophobic and polar interactions. The latter include
Glu53, Gln97, and Asp133. However, the entire molecule 4 is
displaced about 0.4 Å away from the floor of the binding cavity
relative to compound 1. (Figure 2C and Supporting Information
Figure S2). The crystal structure suggests that the factor
responsible for this shift is the recruitment of a water molecule
(orange sphere, Figure 2C) by the pyrimidine ring N present in
compound 4. In contrast, for compound 1 the CH group in this
position eliminates the potential for a hydrogen bond. This water
molecule is also held in place through interactions with Arg104
and Asp133. Hence, despite formation of this additional water-
mediated interaction with the enzyme, the displacement away
from the enzyme caused by allowing the water molecule to bind
at that position ultimately reduces the binding affinity of 4.
On the basis of these results, we decided to maintain the
original structure of the pyrimidine ring and to focus on the other
parts of the molecule as potential modification sites. We next
examined the effect of various substituents at different phenyl
group positions (part D of the molecule, Figure 1A).
Figure 3. Modifications to the phenyl ring meta position. (A) Schematic
representation of compounds 5 and 6 that differ by the nature of the
meta position substituent. (B) In vitro (IC50app and Kiapp) and cell (IC50)
properties for 5 and 6. (C) Overlay of the dCK−5 (magenta, PDB code
4Q19) and dCK−6 (pale green, PDB code 4Q1A) structures with a
focus on the phenyl ring meta position. The tighter binding of 6 relative
to 5 can be rationalized by the interaction of the longer meta substituent
(position highlighted with a gray background) with S144/S146 of dCK.
resolution, respectively (Table 1). The structure of dCK in
complex with compound 5 reveals that the hydroxyl group at the
phenyl group meta position does not make any inhibitor−
enzyme interactions. In contrast, the structure of dCK in
complex with compound 6 shows that the hydroxyethoxy at this
position is able to interact with the side chains of Ser144 and
Ser146 (Figure 3C and Supporting Information Figure S3). We
attribute this added interaction to the superior binding of
compound 6 versus compound 5.
Longer Alkyl Chains with Polar Groups at the Phenyl
Meta Position Increase Binding Affinity. Previously, we
reported that a compound with no phenyl ring substituents, but
otherwise identical to compound 1, showed very modest potency
in our CEM cell based assay (IC50 = 37 nM 8). Adding a hydroxyl
group at the meta position decreased the IC50 in that assay by
about half (compound 5, previously compound 31,3 Figure 3).
The effect of adding the longer hydroxyethoxy group at that
position (compound 6, previously compound 323) was more
impressive, yielding an IC50 of ∼1 nM (Figure 3). We are aware
that primary hydroxyls as in 6 are prone to oxidation or
glucuronidation,12 but these studies do inform us as to the
importance of the type of substituent at the phenyl meta position.
To understand the difference in affinities to dCK between
compounds 5 and 6, we determined the structures of dCK in
complex with these molecules, solved at 2.09 and 1.9 Å
In terms of the importance of substituents at the phenyl meta
position, it is clear that having none or a short one such as a
hydroxyl (compound 5) diminishes the interaction with dCK.
On the other hand, the binding affinity measured by both the in
vitro kinetic assay and by the cell-based CEM assay of larger
substituents (as present in compounds 1, 2, and 6) are
comparable. Previous crystal structures of dCK in complex
with compound 1 (PDB code 4L5B) and 2 (PDB code 4KCG)
also show an interaction between the substituent at the phenyl
meta position and the enzyme, this time to Ser144. Additional
side chains such as 2-fluoroethoxy poly(ethylene glycol) (n = 2)
(PEG)2 (S16, S17, S19), 2-hydroxyethyl (PEG)2 (S11), 2-
methoxyethyl (PEG)2 (S20, S22, S23, S25−S29), and 2-(4,6-
diaminopyrimidine-2-thio)ethyl (PEG)2 (S10) substituents
were well tolerated at the meta position (data not shown and
Supporting Information Table S1).
D
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Figure 4. Modifications to the phenyl ring para position. (A) Schematic representation of compounds 7 and 8 that differ by the nature of the para
position substituent. (B) In vitro (IC50app and Kiapp) and cell (IC50) properties for 7 and 8. (C) Overlay of the dCK−7 (teal, PDB code 4Q1B) and dCK−
8 (beige, PDB code 4Q1C) structures with a focus on the phenyl ring para position. The inhibitors bind very similarly; the meta position substituents
make a direct interaction with the enzyme, but the para substituent does not. The very similar IC50app and Kiapp values of 7 and 8 are explained by the lack
of direct interactions to the enzyme via the para position. In contrast, the presence of a para position substituent lowers the cell-based determined IC50
value.
a
Table 2. Human Microsomal Intrinsic Clearance Assay
a
compd
verapamil
NADPH-dependent CLint (μL min−1 mg−1
)
NADPH-dependent T1/2 (min)
comment
201
0.0
11.5
>240
4.1
high clearance control
low clearance control
warfarin
1
561
870
142
419
254
22.7
2
2.7
15a (Murphy et al.)
9(R/S)
16.3
5.5
10(R/S)
9.1
12R
102
a
Test concentration of compounds was 1 μM.
We conclude that the precise nature of the substituent at the
phenyl ring’s para position such as 2-fluoroethoxy (S4, S14,
S18), fluoro (S5, S6), methoxymethyl terminated (PEG)2 (S21,
S24), and N-substituted methanesulfonamide (S29, S30) were
relatively well tolerated (data not shown and Supporting
Information Table S1). Groups attached to the thiazole like 4-
pyridinyl (S7), meta monosubstituted phenyl (S17), and 3,5-
disubstituted phenyl ring (S31) substituents were also tolerated
(data not shown and Supporting Information Table S1).
Therefore, while not directly important for the binding affinity,
having even a small substituent at the phenyl group para position
improves the relevant cell-based measurements. As a result, most
subsequent compounds contained the methoxy group at that
position.
The Nature of the Thiazole Ring Substituent Dictates
Metabolic Stability. In previous work we demonstrated that
the nature of the substituent at the thiazole ring 5-position (part
C of the molecule, Figure 1A) plays a crucial role in binding
affinity.9 In short, we compared having no substituent at that
position to having a methyl, ethyl, or propyl. We found that
propyl dramatically improved the binding affinity, and as a result,
compounds with a propyl at the 5-position became our lead
compounds (i.e., compounds 1 and 2, Figure 1). Interestingly,
compounds with a small/no substituent at the thiazole 5-position
were observed to bind two inhibitor molecules per dCK active
site, to binding sites that we refer to as position 1 and position 2.
In contrast, the tighter binding propyl-containing molecules were
phenyl meta position is not critical as long as it contains a polar
group that can extend to the proximity of Ser144/Ser146.
The Substituent at the Phenyl Group Para Position
Plays a Minor Role in Binding. To determine the importance
of substituent at the phenyl group para position, we prepared
compound 7 (previously compound 283), which only differs
from compound 2 by lacking a para position substituent (Figure
4A). The in vitro measured binding affinity values (IC50app; Kiapp
)
of compound 7 are nearly identical to that of 2 (Figure 4B),
indicating that substituents at the para position are not required
for tight binding. This is explained by the crystal structures of
dCK in complex with compounds 7 and 8 (previously compound
303), which show a nearly identical binding mode, very similar to
that observed for compound 2 (Figure 4C and Supporting
Information Figure S4). The crystal structures also reveal that no
significant inhibitor−enzyme interactions occur via the para
substituent, if present. This conclusion is supported by the
properties of compound 8, which in contrast to the methoxy
group in compounds 1 and 2 has the longer hydroxyethoxy group
but similar binding affinity. Hence, the in vitro binding affinities
are largely unchanged between having no substituent at the
phenyl group para position, having a methoxy, or the longer
hydroxyethoxy. However, we did notice a ∼10-fold difference
between compounds 7 and 8 in the CEM cell-based assay, with
compound 7 being less potent. Furthermore, substituents at the
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a
Scheme 1. Synthesis Route for Methyl Linker Compound 11(R/S)
a
Reagents and conditions: (a) (NH4)2S (20% in H2O), pyridine, Et3N, 60 °C, 85%; (b) 4-bromopentane-2,3-dione, EtOH, reflux, 95%; (c) 13-
chloro-2,5,8,11-tetraoxatridecane, Cs2CO3, DMF, 50 °C, 89%; (d) DIBAL-H, DCM, −78 °C, 92%; (e) SOCl2, DCM, 0 °C to rt; (f) 4,6-diamino-2-
mercaptopyrimidine, K2CO3, DMF, 75 °C, 65% in last two steps.
observed to bind with a single inhibitor molecule, at position 1,
per dCK active site.9 This revealed that binding of two molecules
is not required for high affinity. In our previous report, we
analyzed the implication of single versus double binding of
inhibitor molecules to dCK and concluded that inhibition of
dCK is primarily caused by the binding of the inhibitor at
position 1, whereas the molecule bound at position 2 does not
appreciably enhance the inhibition.
However, when tested for metabolic stability, we discovered
that the propyl-group-containing compounds 1 and 2 are less
stable relative to those having the shorter methyl group, e.g.,
compound 15a as reported by Murphy et al. (Table 2). We also
explored the activity of cyclopropyl and phenyl groups at the
thiazolyl 5-position (Supporting Information Table S1 and data
not shown). The cyclopropyl analog (S27) had a good IC50
value, but it failed in the PET L-FAC assay.8 The phenyl analog
(S28) demonstrated poor affinity. Hence we were forced to
revert to the methylthiazole ring substituent despite a weaker
interaction with dCK. To compensate for the loss of affinity
provided by the thiazole propyl group, we searched for a
compensating modification that would restore the in vitro
binding affinity while maintaining acceptable metabolic stability.
For that purpose, we decided to explore modifications on the
linker moiety (part B of the compounds, Figure 1A).
Chemistry of Racemic Linker Modified Compounds.
The −SCH2− group acts to link the pyrimidine and thiazole
rings of our compounds. We tested a variety of alternatives to this
linker, such as its deuterated analog (−SCD2−), for the purpose
of a kinetic isotope study. We reasoned that if the linker was
implicated in hydrolytic metabolism, then, because of the kinetic
isotope effect, a deuterated (−SCD2−) analog would show an
improvement in metabolic stability. The deuterium analogs (S1,
S8, S9, S13) had affinity similar to their isotopologues, as
expected (Supporting Information Table S1 and data not
shown). However, the deuterated compounds failed to show
an improvement in the PET L-FAC liver assay, indicating that a
hydrolytic mechanism is probably not involved in the
metabolism of the −SCH2− linker. We also tested the
replacement of the sulfur atom of the −SCH2− group with a
methylene group (−CH2CH2−). Replacing the sulfur atom of
the linker with a carbon atom resulted in a considerable decrease
in dCK affinity and metabolic stability (Supporting Information
Table S1 and data not shown). We next tested a linker in which
the methylene was substituted to contain a methyl group
(−SCH(CH3)−). These racemic methyl-linker compounds
showed very promising biological results and increased
metabolic stability (see Supporting Information Schemes 1 and
2 for the synthesis of compounds 9 and 10). Therefore, we
carefully examined the synthetic route in an attempt to reduce
the synthetic steps and improve the total yield. We succeeded in
developing a six-step synthetic route toward 11 in an overall yield
of 43% (Scheme 1). Commercially available 3-hydroxy-4-
methoxybenzonitrile A was subjected to an aqueous ammonium
sulfide solution under basic conditions to provide thioamide B.
Cyclization to form the thiazole core of C was achieved via
condensation of thioamide B with 4-bromopentane-2,3-dione13
in refluxing ethanol. Introduction of a PEG chain into the phenyl
ring of compound D with 13-chloro-2,5,8,11-tetraoxatridecane14
under basic conditions was achieved in 89% yield. Reduction of
the resulting ketone-containing compound with diisobutylalu-
minum hydride (DIBAL-H) afforded racemic secondary alcohol
E in high yield. Alcohol E was converted to the respective
chloride F with thionyl chloride. The acyl chloride was reacted in
crude form with 4,6-diamino-2-mercaptopyrimidine to generate
product 11R/S.
The Chirality of the Linker Methyl Group Is a Critically
Important Determinant of Binding Affinity. The −S−
CH(CH3)− linker was introduced to a compound that contained
the propyl group at the thiazole ring 5-position (compound 9)
and to a compound that, instead of the propyl group, contained a
methyl (compound 10) (Figure 5A). As mentioned above, the
rationale for compound 10 was the predicted improvement in
F
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Figure 5. Modifications to the linker. (A) Schematic representation of compounds 9 and 10. Both compounds were synthesized as the racemic mixture
(R/S); the addition of a methyl group (arrow) to the methylene linker group makes these compounds chiral. Whereas 9 has a propyl group at the
thiazole ring 5-position (Rt), 10 has a methyl group. (B) In vitro (IC50app and Kiapp) and cell (IC50) properties for 9 and 10. (C) The propyl group at the
thiazole ring makes 9 bind as a single molecule to binding site position 1 of dCK (see text for details). Notably, despite forming the enzyme−inhibitor
with racemic 9, in the crystal structure we observe only the R-isomer (compound 9 in yellow, PDB code 4Q1D, Fo − Fc omit map in blue contoured at
2σ). A theoretical model of the S-isomer (gray) demonstrates that only the R-isomer fits the electron density. (D) The methyl group at the thiazole ring
permits two molecules of 10 to bind to dCK: one to position 1 and one to position 2. In position 1 we observe only the R-isomer (10R-P1, cyan, PDB
code 4Q1E; Fo − Fc omit map contoured at 2σ in green). A theoretical model of the S-isomer at position 1 (gray) clearly demonstrates that only the R-
isomer fits the electron density (red arrow). (E) In position 2 we observe only the S-isomer (10S−-P2, plum, PDB code 4Q1E; Fo − Fc omit map
contoured at 1.5σ in green). A theoretical model of the R-isomer at position 2 (gray) clearly demonstrates that only the S-isomer fits the electron density
(red arrow).
metabolic stability. Interestingly, whereas compounds with a
propylthiazole ring previously showed tighter binding to dCK
compared to the analogous methylthiazole compounds, we now
measured better binding with the methyl-containing compound
10 to the propyl-containing compound 9 (Figure 5B). Hence,
the proximity of the thiazole-ring substituent (propyl or methyl)
to the methyl-linker substituent resulted in the larger propyl
group being not as accommodating in the dCK active site.
Despite the improved in vitro binding parameters for 10 over 9,
the cell-based assay yielded similar IC50 values, yet consistent
with 10 being superior (Figure 5B).
crystal structure of dCK in complex with compounds 9 and 10
(solved at 2.0 and1.85 Å resolution, respectively, Table 1). As
expected, compound 9 binds as a single molecule to dCK,
specifically at position 1, because of the presence of the propyl
group in the thiazole ring. Interestingly, despite the fact that a
racemic mixture of 9 was used to form the complex to dCK, the
crystal structure provides unambiguous evidence for the R-
isomer binding at position 1 (Figure 5C and Supporting
Information Figure S5). Likewise, inspection of the structure
of the complex between racemic 10 and dCK shows that the R-
isomer occupies the most relevant position 1 binding site (Figure
5D and Supporting Information Figure S5). Since compound 10
contains the methyl substituent in the thiazole ring, which allows
for a molecule to also occupy position 2, we observe compound
10 at that position as well. However, whereas it is the R-isomer of
Both compounds 9 and 10 were prepared as racemic mixtures;
the introduced linker-methyl group makes that position a new
chiral center (arrow, Figure 5A). To elucidate which of the two
enantiomers is the active dCK inhibitor, we determined the
G
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Figure 6. The R-isomer is the relevant isomer regarding dCK inhibition. (A) Schematic representation of compounds 11S, 11R, and 12R (R or S
designate the chirality of the linker methylene carbon; arrows point at the added methyl group). (B) In vitro (IC50app and Kiapp) and cell (IC50) properties
for 11S, 11R, and 12R. The R-isomer of both 11 and 12 is responsible for the observed inhibition of the enzyme. (C) dCK was crystallized in the
presence of enantiomerically pure 12R, and the enzyme−inhibitor complex structure was solved (PDB code 4Q1F). Fo − Fc omit map (1.6σ) for the
position 1 binding site clearly shows the presence of 12R (brown). Despite the thiazole methyl group in 12R (which is compatible with molecules also
binding to position 2), we do not observe a second 12R molecule at position 2. This is consistent with the results with compound 10 (Figure 5) that
showed that only the S-isomer binds to positon 2.
10 that binds to position 1, it is the S-isomer that binds to
position 2 (Figure 5E and Supporting Information Figure S5).
We previously concluded that position 1 is the critical binding
site for this family of inhibitors. This would suggest that the
measured in vitro inhibition values of racemic 10 are reflecting
the preferential binding of the R-isomer. To test this, we
synthesized compound 11, which is a slight modification of 10
(the nature of the phenyl group substituents) but notably had the
racemic mixture separated to yield the pure isomers 11R and 11S
(Figure 6A). We determined the in vitro binding affinities of the
enantiomerically pure compounds and observed that 11S has
∼400-fold weaker binding affinity relative to 11R (Figure 6B).
This result provides clear evidence that the R-form is responsible
for the tight interaction with dCK. This result also validates our
structure-based interpretation that position 1 is the one most
relevant inhibitor binding site for dCK inhibition and that
position 2 is occupied because of the high concentration of the
inhibitor used in the crystallization setups.
Enantioselective Synthesis of Chiral Molecules. Having
discovered that the R-isomers of compounds 9, 10, and 11 are
responsible for the dCK inhibition, we set out to develop an
asymmetric synthesis (Scheme 2). The chiral synthesis
developed by our group for compound 12R, which is a close
analog of 10, features a chiral Corey−Bakshi−Shibata (CBS)
reaction15 of ketone D. Chiral alcohol E was synthesized
according to this method with an enantiomeric excess of 96%, as
determined via chiral HPLC. Employing mesic or tosic
anhydride to give the sulfonates under different basic conditions
such as Et3N, pyridine, or DMAP resulted in elimination to the
alkene, presumably due to the stability of the secondary benzylic-
like carbocation. The use of trifluoroacetic anhydride (TFAA) at
0 °C converted alcohol E into the corresponding trifluoroacetate
(TFA) F without a significant decrease in the % ee of the ester.
Finally, compound F was reacted with 4,6-diamino-2-mercapto-
pyrimidine to generate 12R in 61% yield over two steps with an
enantiomeric excess of 40%. Presumably, a portion of the
reaction occurs via a direct SN2 pathway, while another part
occurs via an SN1 pathway, and thereby racemized material was
obtained. Chiral resolution via recrystallization generated 12R
with an enantiomeric excess of over 90%. Likewise, (S)-(−)-2-
methyl-CBS-oxazaborolidine was used in the CBS reduction to
synthesize 12S.
Characterization of Enantiomerically Pure 12R. Com-
pound 12R (Figure 6A) was measured to have very similar in
vitro binding affinities to 11R (Figure 6B). Significantly, just as
the affinity of 11S was much reduced relative to 11R, the affinity
to dCK of 12S was much reduced relative to 12R. This reiterated
the preference of dCK for compounds that contain the R-isomer
of the linker.
We solved the dCK−12R complex crystal structure. We
expected 12R to bind only at position 1 based on the previous
structure with compound 10 (observing 10R bound at position
1) and the kinetic results using enantiomerically pure 11S, 11R,
12S, and 12R (observing higher affinities for the R-isomers) and
since the crystals were formed with the enantiomerically pure
12R. Additionally, lacking the S-isomer, we expected a vacant
position 2 binding site. Indeed, the crystal structure of the dCK−
12R complex revealed a single inhibitor molecule at position 1
(Figure 6C). This result suggests that the R-isomer has very low
affinity to the binding site at position 2. Notably, while the
interaction between the R-isomer and dCK is limited to the
position 1 binding site, this does not diminish the binding affinity
for the enzyme.
Determinant of Chiral Selectivity. What could be behind
the dramatic selectivity of the dCK position 1 binding site for the
R-isomers of the inhibitors? Likewise, what prevents the R-
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a
Scheme 2. Asymmetric Synthesis Route of 12R
a
Reagents and conditions: (a) (NH4)2S (20% in H2O), pyridine, Et3N, 60 °C, 85%; (b) 4-bromopentane-2,3-dione, EtOH, reflux, 96%; (c) N-(2-
bromoethyl)methanesulfonamide, Cs2CO3, DMF, 50 °C, 82%; (d) (R)-(+)-2-methyl-CBS-oxazaborolidine, BH3−THF complex, THF, −78 °C,
77%, (96% ee); (e) TFAA, DCM, 0 °C, (f) 4,6-diamino-2-mercaptopyrimidine, DMF, 80 °C, 61% in last two steps.
isomer from binding at position 2, while this binding site is
compatible with the binding of the S-isomer? The simple
explanation would involve steric considerations relating the
inhibitor and enzyme, where the chiral methyl group of the linker
clashes with enzyme residues in the case of one isomer but not
the other. However, inspection of the crystal structures solved
with compounds 10(R/S) and 12R does not support this
interpretation; we could model the S-isomer bound to position 1
(Figure 5D) and the R-isomer bound at position 2 (Figure 5E)
with no apparent clashes.
Comparison of the binding mode between 10R and 10S
reveals that the relative orientation of the pyrimidine ring to the
thiazolephenyl part is strikingly different between the R and S
isomers (Figure 7A and Figure 7B). That is, by a change of the
angles of the linker that connects the pyrimidine ring to the
thiazole ring, each isomer has adjusted its conformation to best fit
its binding site (i.e., induced fit). This demonstrates that the
enzyme dictates the relative orientations between the pyrimidine
ring, linker, and the thiazolephenyl rings. It also shows that the
relative orientation between thiazole and phenyl rings (being
coplanar) is largely unchanged, not surprising because of the
resonance between the rings.
between the chiral methyl of the linker and the thiazole ring
methyl group for 10R in position 1 is 4.2 Å (Figure 7C), for the
modeled 10S bound to position 1, that distance would be an
unfavorable 2.5 Å (Figure 7D). Likewise, whereas the observed
distance between the chiral methyl and the thiazole methyl for
10S in position 2 is 4.4 Å (Figure 7E), for the modeled R-isomer
adopting the same conformation as 10S, that distance would be
an unfavorable 2.6 Å (Figure 7F). Hence, the strict chiral
selection to either position 1 or position 2 is due to the enzyme
dictating a particular inhibitor orientation that is vastly different
between the binding sites. In the case of position 1, that
orientation is not compatible with the S-isomer, and for position
2, that orientation is not compatible with the R-isomer.
Using computer simulations, we obtained a qualitative
estimate of the conformational penalty incurred by 10R and
10S upon binding with the protein. The conformational penalty
is the energy difference between the preferred solution-phase
geometry of a substrate and the geometry that it assumes upon
binding: ΔE = Esolution − Ebound. Each enantiomer was docked
with the solvated protein at position 1 and allowed to equilibrate
(see details in Experimental Section and Supporting Information
Figure S6). The equilibrated, docked inhibitor structures were
removed from the protein, and their energies were assessed with
the semiempirical PDDG/PM3 method.16−21 Unbound struc-
tures of 10R and 10S were optimized in implicit solvent to
To further probe the observed chiral selectivity, we
constructed a theoretical model of 10S binding at position 1
with the same orientation as 10R. Whereas the observed distance
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Figure 7. Chiral selectivity is due to conformational selection by the enzyme’s binding site. (A) Observed orientation of 10R (cyan) at position 1 (10R-
P1, PDB code 4Q1E) and 10S (plum) at position 2 (10S-P2) upon dCK binding. (B) 10S overlaid on 10R based on the thiazole ring. Note the different
relative orientations of the thiazole and pyrimidine rings between 10R and 10S. (C) The conformation of 10R (10R-P1) is dictated by the position 1
binding site. In this conformation the distance between the chiral linker methyl group and the thiazole ring methyl group is 4.2 Å. (D) The theoretical
model of 10S binding with the same conformation as 10R in position 1 (10S-P1) shows that the homologous distance is reduced to 2.5 Å. (E) The
conformation of 10S (10S-P2) is dictated by the position 2 binding site. In this conformation the distance between the chiral linker methyl group and the
thiazole ring methyl group is 4.4 Å. (F) The theoretical model of 10R binding with the same conformation as 10S in position 2 (10R-P2) shows that the
homologous distance is reduced to 2.6 Å. (G) For 10R-P1, the observed torsion angle between the thiazole ring and the linker is −59°. Scanning possible
torsion angles shows that this value represents a low energy conformation of 10R. (H) For 10S-P1, the observed torsion angle is 189°. This value
corresponds to a high-energy conformation. (I) For 10S-P2, the observed torsion angle is −326°. Scanning possible torsion angles shows that this value
is at a low energy conformation of 10S. (J) For 10R-P2, the observed torsion angle is 147°. This value corresponds to a high-energy conformation.
determine their low-energy solution-phase conformations. As
with the bound structures, energies of the unbound structures
were assessed with PDDG/PM3. The resulting energies were
used to obtain qualitative conformational penalties for each
enantiomer. The conformational penalty for 10S was almost
twice the conformational penalty for 10R (45 kcal/mol larger
penalty for 10S), further demonstrating that 10R needs to
undergo a much less unfavorable structural rearrangement in
order to bind with the protein at position 1.
Another way of considering this issue is to examine the energy
of the inhibitor as a function of rotation around the bond that
connects the thiazole ring to the chiral linker atom (bond marked
with ∗ in Figure 7C−F). For 10R bound to dCK at position 1, the
observed dihedral angle that specifies this rotation is −59° and
fits a low energy conformation (Figure 7G). In contrast, the
modeled S-isomer at this binding site would have a torsion angle
of 189°, which is clearly a high-energy conformation (Figure
7H). The same pattern is observed for position 2, with the S-
isomer binding to dCK with a torsion angle of −326°, which is a
J
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low energy conformation, while the modeled R-isomer at that
position is a high-energy conformation (Figure 7I and Figure 7J).
Hence, the chiral selectivity does not come directly from the
enzyme sterically favoring one isomer over the other. Rather, the
enzyme dictates a particular conformation, and the selectivity
comes from one isomer being able to adopt that particular
conformation, whereas the energy penalty for the other isomer
precludes its binding.
In addition to explaining the chiral selectivity for the
compounds discussed here, this understanding can be used for
the design of chiral molecules that bind to either binding site.
Specifically, the prediction would be that replacing the thiazole
methyl group with a hydrogen atom would eliminate any steric
clash to the chiral methyl group, and hence either isomer could
bind to either inhibitor binding site.
Improved Metabolic Stability of 12R. We first determined
the metabolic stability of 12R in a standard microsomal liver
clearance assay. The NADPH-dependent T1/2 of 12R was ∼37-
fold longer than that of our previous lead compound 2 (Table 2).
We then tested compound 12 in mice, using our previously
described positron emission tomography (PET) assay.8 Whereas
our earlier lead compound 2 retained only ∼25% inhibition of
dCK activity 4 h after dosing by intraperitoneal injection,3
compound 12 (given as the racemic mixture) exhibited >50%
inhibition of dCK activity at this time point (Figure 8A).
Furthermore, 8 h after treatment with compound 12, dCK
inhibition was still above 30%. We then determined the
pharmacokinetic properties of compound 12 to compare with
our previous lead compounds 1 and 2.7,8 As shown in Figure 8B,
the pharmacokinetic properties of compound 12 were
significantly improved relative to the previously published values
for compounds 1 and 2.7,8 Collectively, these findings
demonstrate that introduction of the chiral linker plus
replacement of the thiazole ring propyl substituent by a methyl
group yields a dCK inhibitor with improved metabolic stability.
CONCLUSION
■
Structural and inhibition studies of the compounds discussed
here, performed using both the purified recombinant enzyme
and a cell-based assay, revealed and rationalized the essential
determinants for binding to dCK and also guided the type and
placement of substituents. This informed the development of the
initial leads, compounds 1 and 2. These compounds contain a
propyl group at the 5-position of the thiazole ring, since, as
shown earlier, the propyl substituent provides improved affinity
for dCK compared to compounds with a methyl group at that
position. Unfortunately, this affinity-strengthening propyl group
compromised the metabolic stability relative to compounds
containing a methyl group at that position. This forced us to
revert to the weaker-binding, but more metabolically stable,
scaffold of a methyl group at the thiazole ring. With the goal of
improving metabolic stability, we tested a chiral methylene
methyl sulfur linker between the thiazole and pyrimidine
moieties. This linker was found to confer two positive effects:
(1) in terms of affinity for dCK, the modified linker compensated
for the lack of the thiazole propyl group, and (2) the compounds
exhibited improved metabolic stability. The interaction of dCK
with compounds containing this linker is specific to the R-isomer.
This was proven by the dCK-inhibitor crystal structure and by
comparing the binding affinities of the R versus S enantiomers.
The new lead compound 12R is a promising dCK inhibitor,
which by perturbing the dNTP pools and inducing DNA
replication stress overload could be used in combination with
other drugs to specifically trigger synthetic lethality in cancer
cells.
EXPERIMENTAL SECTION
■
Materials. General laboratory reagents were purchased from Fisher
(Pittsburgh, PA, USA) and Sigma-Aldrich (St. Louis, MO, USA).
Nucleotides were obtained from Sigma. All inhibitors were synthesized
at UCLA. Chiral Technologies Inc. (800 North Five Points Road, West
Chester, PA 19380, USA) performed the separation of R and S
enantiomers.
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 chromatography was performed using
E. Merck silica gel 60 (230−400 mesh) with compressed air. 1H and 13
C
NMR spectra were recorded on a ARX500 (500 MHz), Avance 500
(500 MHz), or Avance 300 (300 MHz) spectrometers. Chemical shifts
are reported in parts per million (ppm, δ) using the residual solvent peak
as the reference. 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
Figure 8. In vivo evaluation of compound 12. (A) Quantification of PET
probe, 18F-L-FAC, uptake in the liver of C57Bl/6 female mice treated
with compounds 12 (25 mg/kg) via intraperitoneal injection. Dose
formulation: 50% PEG/Tris, pH 7.4. Data are mean values SEM for at
least n = 5 mice/time point. (B) Plasma pharmacokinetic profile of
compound 12. C57Bl/6 female mice were dosed via intraperitoneal
injection with 50 mg/kg compound 12 formulated in 50% PEG/Tris,
pH 7.4. Data are mean values SEM for n = 4 mice/time point.
K
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infused using direct loop injection from a Waters Acquity UPLC into the
multimode 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 mm × 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 for each described compound.
Percent enantiomeric excess (% ee) values were determined via chiral
HPLC with a CHIRALPAK IA-3/IA polysaccharide-based immobilized
type column (3 μm, 4.6 mm × 150 mm) with inline Knauer UV (310
nm) detector. Mobile phase: A, 0.1% TFA in hexanes; B, 0.1% TFA in
propanol. Eluent gradient: 50% phase A and 50% phase B. Chromato-
grams were collected by a GinaStar (Raytest USA, Inc.; Wilmington,
NC, USA) analog to digital converter and GinaStar software (Raytest
USA, Inc.).
Scheme 1. 3-Ethoxy-4-hydroxybenzothioamide (B). To a
mixture of 3-ethoxy-4-hydroxybenzonitrile A (2.50 g, 15.3 mmol) in
pyridine (35 mL) and triethylamine (2.5 mL) was added ammonium
sulfide solution (20 wt % in H2O, 15.65 mL, 46.0 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 Na2SO4, concentrated in vacuo, and
purified by flash column chromatography over silica gel (3:1 ethyl
acetate/hexanes) to yield B (2.56 g, 13.0 mmol, 85%) as a yellow solid.
1H NMR (300 MHz, CDCl3) δ 7.68 (d, J = 2.1 Hz, 1H), 7.48 (br s, 1H),
anhydrous Na2SO4, and concentrated in vacuo to give the desired
alcohol E (978 mg, 2.3 mmol, 92%) as a pale yellow solid. 1H NMR (500
MHz, CDCl3) δ 7.44 (d, J = 2.0 Hz, 1H), 7.33 (dd, J = 8.5, 2.0 Hz, 1H),
6.89 (d, J = 8.5 Hz, 1H), 4.91 (q, J = 6.5 Hz, 1H), 4.22−4.17 (m, 2H),
4.13 (q, J = 7.0 Hz, 2H), 3.91−3.86 (m, 2H), 3.76−3.72 (m, 2H), 3.69−
3.61 (m, 4H), 3.55−3.51 (m, 2H), 3.35 (s, 3H), 2.37 (s, 3H), 1.52 (d, J =
6.0 Hz, 3H), 1.44 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
164.3, 155.1, 150.0, 149.0, 127.2, 125.8, 119.3, 113.8, 111.0, 71.8, 70.8,
70.6, 70.4, 69.5, 68.7, 64.6, 64.4, 58.9, 24.0, 14.7, 10.7.
4-(1-Chloroethyl)-2-(3-ethoxy-4-(2-(2-(2-methoxyethoxy)-
ethoxy)ethoxy)phenyl)-5-methylthiazole (F). To a stirred solution
of alcohol E (425 mg, 1.0 mmol) in CH2Cl2 (8 mL) was added thionyl
chloride (0.78 mL, 10.0 mmol) slowly at 0 °C. The mixture was allowed
to warm to 23 °C and stirred for 1 h. After concentration in vacuo to
remove residual solvent, the resulting crude residue was used directly for
next step without any further purification because of the instability of
chloride F.
2-((1-(2-(3-Ethoxy-4-(2-(2-(2-methoxyethoxy)ethoxy)-
ethoxy)phenyl)-5-methylthiazol-4-yl)ethyl)thio)pyrimidine-
4,6-diamine (( )-9). A mixture of crude chloride F from the previous
step, 4,6-diamino-2-mercaptopyrimidine (625 mg, 4.0 mmol), and
K2CO3 (552 mg, 4.0 mmol) in DMF (7 mL) was stirred at 70 °C for 1 h.
The solution was cooled, concentrated in vacuo, and purified by flash
column chromatography over silica gel (25:1 dichloromethane/
methanol) to give the desired product ( )-9 (357 mg, 0.65 mmol,
65% in two steps) as a white solid. 1H NMR (500 MHz, CDCl3) δ 7.49
(d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.5, 2.0 Hz, 1H), 6.90 (d, J = 8.5 Hz,
1H), 5.24 (s, 1H), 5.02 (q, J = 7.0 Hz, 1H), 4.58 (s, 4H), 4.22−4.18 (m,
2H), 4.15 (q, J = 7.0 Hz, 2H), 3.91−3.87 (m, 2H), 3.78−3.75 (m, 2H),
3.69−3.63 (m, 4H), 3.56−3.53 (m, 2H), 3.37 (s, 3H), 2.50 (s, 3H), 1.81
(d, J = 7.0 Hz, 3H), 1.46 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 170.7, 163.8, 163.2 (2), 153.3, 149.9, 149.1, 127.9, 126.8,
119.4, 114.0, 111.3, 80.6, 71.9, 70.9, 70.7, 70.6, 69.7, 68.9, 64.7, 59.1,
37.7, 22.0, 14.8, 11.6; HRMS-ESI (m/z) [M + H]+ calcd for
C25H35N5O5S2H, 550.2158; found 550.2169.
Scheme 2. 3-Hydroxy-4-methoxybenzothioamide (B). To a
mixture of 3-hydroxy-4-methoxybenzonitrile A (3.00 g, 20.11 mmol) in
pyridine (30 mL) and triethylamine (3 mL) was added ammonium
sulfide solution (20 wt % in H2O, 20.7 mL, 60.3 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 Na2SO4, concentrated in vacuo, and
purified by flash column chromatography over silica gel (3:1 ethyl
acetate/hexanes) to yield B (3.13 g, 17.1 mmol, 85%) as a yellow solid.
1H NMR (500 MHz, acetone-d6) δ 8.77 (br s, 1H), 8.65 (br s, 1H), 7.85
7.28 (dd, J = 8.5, 2.1 Hz, 1H), 7.11 (br s, 1H), 6.89 (d, J = 8.5 Hz, 1H),
6.03 (s, 1H), 4.21 (q, J = 6.9 Hz, 2H), 1.47 (t, J = 6.9 Hz, 3H); 13C NMR
(125 MHz, acetone-d6) δ 200.5, 150.3, 145.8, 131.0, 121.0, 114.0, 112.6,
64.3, 14.1.
1-(2-(3-Ethoxy-4-hydroxyphenyl)-5-methylthiazol-4-yl)-
ethan-1-one (C). A mixture of thioamide B (1.50 g, 7.6 mmol) and 4-
bromopentane-2,3-dione (2.04 g, 11.4 mmol) in ethanol (40 mL) was
stirred under refluxing conditions for 4 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 C
(2.00 g, 7.2 mmol, 95%) as a white solid. 1H NMR (300 MHz, CDCl3) δ
7.47 (d, J = 1.8 Hz, 1H), 7.35 (dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 8.1
Hz, 1H), 5.93 (s, 1H), 4.23 (q, J = 7.2 Hz, 2H), 2.77 (s, 3H), 2.71 (s,
3H), 1.50 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 196.0,
162.8, 148.9, 148.0, 146.3, 142.9, 125.9, 120.5, 114.8, 109.4, 64.9, 29.5,
14.9, 13.6.
1-(2-(3-Ethoxy-4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-
phenyl)-5-methylthiazol-4-yl)ethan-1-one (D). To a solution of
thiazole intermediate C (1.66 g, 6.0 mmol) in DMF (35 mL) were added
Cs2CO3 (3.13 g, 9.6 mmol) and 13-chloro-2,5,8,11-tetraoxatridecane
(2.19 g, 12.0 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 Na2SO4, and
concentrated in vacuo, and the crude residue was purified by flash
column chromatography over silica gel (1:1 ethyl acetate/hexanes) to
(s, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.56 (dd, J = 8.5, 2.3 Hz, 1H), 6.94 (d, J
= 8.5 Hz, 1H), 3.88 (s, 3H); 13C NMR (125 MHz, acetone-d6) δ 200.7,
150.5, 145.7, 132.4, 119.5, 114.8, 110.2, 55.5.
1-(2-(3-Hydroxy-4-methoxyphenyl)-5-methylthiazol-4-yl)-
ethan-1-one (C). A mixture of thioamide B (2.75 g, 15.0 mmol) and 4-
bromopentane-2,3-dione (4.03 g, 22.5 mmol) in ethanol (70 mL) was
stirred under refluxing conditions for 4 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 C
(3.79 g, 14.4 mmol, 96%) as a white solid. 1H NMR (500 MHz, DMSO-
d6) δ 9.53 (br s, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.26 (dd, J = 8.5, 2.0 Hz,
1H), 6.98 (d, J = 8.5 Hz, 1H), 3.80 (s, 3H), 2.66 (s, 3H), 2.57 (s, 3H);
13C NMR (125 MHz, DMSO-d6) δ 195.2, 162.5, 150.1, 148.5, 147.1,
1
yield desired ketone D (2.26 g, 5.3 mmol, 89%) as a white solid. H
NMR (500 MHz, CDCl3) δ 7.48 (d, J = 2.0 Hz, 1H), 7.38 (dd, J = 8.5,
2.0 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 4.24−4.20 (m, 2H), 4.17 (q, J = 7.0
Hz, 2H), 3.93−3.89 (m, 2H), 3.79−3.75 (m, 2H), 3.70−3.63 (m, 4H),
3.57−3.53 (m, 2H), 3.37 (s, 3H), 2.77 (s, 3H), 2.71 (s, 3H), 1.47 (t, J =
7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 196.0, 162.5, 150.8, 149.4,
149.0, 143.1, 126.9, 119.8, 114.0, 111.4, 72.1, 71.1, 70.8, 70.7, 69.7, 69.0,
64.9, 59.2, 29.5, 15.0, 13.6.
142.7, 125.6, 118.2, 112.9, 112.5, 55.9, 29.4, 13.2.
N-(2-(5-(4-Acetyl-5-methylthiazol-2-yl)-2-methoxyphenoxy)-
ethyl)methanesulfonamide (D). To a solution of thiazole inter-
mediate C (1.58 g, 6.0 mmol) in DMF (35 mL) were added Cs2CO3
(3.13 g, 9.6 mmol) and N-(2-bromoethyl)methanesulfonamide (2.18 g,
10.8 mmol). The mixture was stirred for 72 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 Na2SO4, and
concentrated in vacuo, and the crude residue was purified by flash
1-(2-(3-Ethoxy-4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-
phenyl)-5-methylthiazol-4-yl)ethan-1-ol (E). To a stirred solution
of ketone D (1.06 g, 2.5 mmol) in CH2Cl2 (35 mL) cooled to −78 °C
was added slowly diisobutylaluminum hydride (1.0 M in THF, 10 mmol,
10 mL). The mixture 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
L
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column chromatography over silica gel (3:2 ethyl acetate/hexanes) to
yield desired ketone D (1.89 g, 4.9 mmol, 82%) as a white solid. H
Escherichia coli BL21 C41(DE3) cells using a pET-14b vector; the cells
were grown in 2xYT medium and induced with 0.1 mM IPTG for 4 h at
310 K. The cells were harvested, and the pellet was lysed by sonication.
The lysate was cleared by centrifugation at 30 000 rev/min for 1 h at 277
K, and the supernatant was loaded onto a 5 mL HisTrap nickel-affinity
column (GE Healthcare). The column was washed with 300 mL of a
buffer composed of 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 30 mM
imidazole. The bound protein was eluted with the same buffer but
containing 250 mM imidazole and was further purified by gel filtration
using an S-200 column in a buffer consisting of 25 mM HEPES, pH 7.5,
200 mM sodium citrate, 2 mM EDTA, 3 mM DTT. The protein
fractions were pooled, concentrated, aliquoted, flash-frozen in liquid
nitrogen, and stored at 193 K until use.
Kinetic Assay. The phosphorylation activity of dCK was determined
using a spectroscopic NADH-dependent enzyme-coupled assay.2,23 All
measurements were taken in triplicate at 310 K in a buffer consisting of
100 mM Tris, pH 7.5, 200 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.8
mM phosphoenolpyruvate, 0.4 mM NADH with 50 nM dCK, and 1 mM
ATP. IC50app and Kiapp were determined as described by us,9 and all data
were fitted using the KaleidaGraph software.
1
NMR (500 MHz, CDCl3) δ 8.00 (s, 1H), 7.51 (d, J = 2.0 Hz, 1H), 7.46
(dd, J = 8.5, 2.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 4.25−4.20 (m, 2H),
3.90 (s, 3H), 3.60−3.55 (m, 2H), 3.03 (s, 3H), 2.76 (s, 3H), 2.70 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 195.8, 162.5, 151.5, 148.9, 147.8,
143.1, 126.4, 121.1, 112.4, 111.7, 69.1, 55.9, 42.7, 40.6, 29.4, 13.4.
(S)-N-(2-(5-(4-(1-Hydroxyethyl)-5-methylthiazol-2-yl)-2-
methoxyphenoxy)ethyl)methanesulfonamide (E). To a stirred
solution of (R)-(+)-2-methyl-CBS-oxazaborolidine (6.7 mL of a 1.0 M
solution in toluene, 6.7 mmol) in THF (26 mL) at −78 °C under Ar was
added borane−tetrahydrofuran complex (4.4 mL of a 1.0 M solution in
THF, 4.4 mmol) followed by a solution of D (284 mg, 0.74 mmol) in
THF (14 mL). After addition of the D solution with syringe pump for 6
h, the reaction mixture was stirred for another 20 min at −78 °C. H2O
(10 mL) and MeOH (5 mL) were added, and the mixture was allowed to
warm to room temperature. 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 Na2SO4, and concentrated in vacuo, and the crude
residue was purified by flash column chromatography twice over silica
gel with 3:2 ethyl acetate/hexanes and 40:1 dichloromethane/methanol
as washing system separately to yield alcohol E (221 mg 0.57 mmol,
77%, 96% ee) as a white solid. 1H NMR (500 MHz, acetone-d6) δ 7.57
(d, J = 2.0 Hz, 1H), 7.46 (dd, J = 8.5, 2.0 Hz, 1H), 7.05 (d, J = 8.5 Hz,
1H), 6.26 (br s, 1H), 5.02−4.95 (m, 1H), 4.21 (t, J = 5.5 Hz, 2H), 3.88
(s, 3H), 3.57 (dt, J = 5.5, 5.5 Hz, 2H), 3.04 (s, 3H), 2.48 (s, 3H), 1.50 (d,
J = 6.0 Hz, 3H); 13C NMR (125 MHz, acetone-d6) δ 162.9, 156.1, 151.3,
148.4, 127.1, 126.8, 119.7, 112.1, 111.4, 68.6, 64.1, 55.3, 42.6, 39.6, 23.0,
10.0.
(S)-1-(2-(4-Methoxy-3-(2-(methylsulfonamido)ethoxy)-
phenyl)-5-methylthiazol-4-yl)ethyl 2,2,2-Trifluoroacetate (F).
To a stirred solution of alcohol E (221 mg, 0.57 mmol) in CH2Cl2
(13 mL) was added trifluoroacetic anhydride (0.66 mL, 2.9 mmol)
slowly at 0 °C. After being stirred at 0 °C for 30 min, the mixture was
allowed to warm to 23 °C and stirred for another 30 min. After
concentration in vacuo to remove residual solvent, the resulting crude
residue was used directly for next step without any further purification
because of the instability of the desired trifluoroacetate F.
(R)-N-(2-(5-(4-(1-((4,6-Diaminopyrimidin-2-yl)thio)ethyl)-5-
methylthiazol-2-yl)-2-methoxyphenoxy)ethyl)methane-
sulfonamide (10R) and (S)-N-(2-(5-(4-(1-((4,6-Diaminopyrimi-
din-2-yl)thio)ethyl)-5-methylthiazol-2-yl)-2-methoxyphenoxy)-
ethyl)methanesulfonamide (12S). A mixture of crude chloride F
from the previous step and 4,6-diamino-2-mercaptopyrimidine (112 mg,
0.86 mmol) in DMF (5 mL) was stirred at 80 °C for 1 h. The solution
was cooled, concentrated in vacuo, and purified by flash column
chromatography over silica gel (25:1 dichloromethane/methanol) to
give the couple of enantiomers 12R and 12S (178 mg, 0.35 mmol, 40%
ee of 12R, 61% total yield in two steps) as a white solid. Recrystallization
of the enantiomers with MeOH/acetone solvent system gave the 12R
with >93% ee. 1H NMR (500 MHz, acetone-d6) δ 7.55 (d, J = 2.0 Hz,
1H), 7.48 (dd, J = 8.5, 2.0 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H), 6.26 (br s,
1H), 5.60−5.55 (m, 4H), 5.37 (s, 1H), 5.30 (q, J = 7.0 Hz, 1H), 4.23 (t, J
= 5.5 Hz, 2 H), 3.89 (s, 3H), 3.58 (dt, J = 5.5, 5.5 Hz, 2H), 3.05 (s, 3H),
2.52 (s, 3H), 1.74 (d, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6)
δ 168.0, 163.5 (2), 162.9, 153.6, 150.6, 147.8, 126.6, 126.2, 119.5, 112.3,
110.4, 79.0, 67.9, 55.7, 41.9, 36.1, 30.7, 22.2, 11.2; HRMS-ESI (m/z) [M
+ H]+ calcd for C20H26N6O4S3H, 511.1256; found 511.1259; 12R
[α]19D +340.0 (c 0.12, acetone) (93% ee).
IC50 Determinations. These were performed in CCRF-CEM acute
lymphoblastic leukemia cells as previously described.8,9
PET Studies. PET studies to determine % inhibition of dCK activity
in vivo were performed as previously described.8,9
Human Microsomal stability Assays. These assays were
performed by Cyprotex (Watertown, MA) according to standard
operating protocols.
Plasma Pharmacokinetics of Compounds 10 and 12 in Mice.
These measurements were performed as previously described.8,9 Briefly,
C57Bl/6 female mice were treated with the dCK inhibitors via
intraperitoneal injection. The drugs were administered in 50%
polyethylene glycol (PEG 400)/50 mM Tris-HCl, pH 7.5. Five minutes
after drug injection, whole blood (∼75 μL) was obtained at various time
points from the retro-orbital sinus using hematocrit capillary tubes.
Samples were centrifuged at 20 000g for 5 min, and the supernatant (5
μL) was transferred into a clean tube. Calibration standards were
prepared by spiking various amounts of 11 and 12 in 5 μL of supernatant
from the plasma of untreated mice to obtain final concentrations
between 0.001 to 100 pmol/μL. Samples and the calibration standards
were mixed with 500 μL ice-cold acetonitrile/water (50/50, v/v)
containing an internal standard (1). All of the samples were evaporated
to dryness in a vacuum centrifuge. The residue was reconstituted in 100
μL of acetonitrile/water (50/50, v/v). Samples (5 μL) were injected
onto a reverse phase column (Agilent ZORBAX rapid resolution high
definition Eclipse Plus C18, 2.1 mm × 50 mm, 1.8 μm) equilibrated in
water acetonitrile/formic acid, 95/5/0.1, and eluted (200 μL/min) with
an increasing concentration of solvent B (acetonitrile/formic acid 100/
0.1, v/v: min/% acetonitrile; 0/5, 2/5, 8/80, 9/80, 10/5, 12/5). The
effluent from the column was directed to an electrospray ion source
(Agilent Jet Stream) connected to a triple quadrupole mass
spectrometer (Agilent 6460 QQQ) operating in the positive ion
MRM mode. The ion transitions for 1, 11, and 12 are 476.2−334.5,
550.2−408.2, and 511.1−369.1 respectively. The peak areas for 11 and
12 were normalized to the peak area of the internal standard, and the
plasma concentrations were computed using the standard curves
generated by calibration standards spiked in plasma from untreated
mice. Approximated values of the area under the curve (AUC), half-life
(T1/2), maximum concentration in the plasma (Cmax), and time to reach
the maximum concentration (Tmax) were calculated using Boomer/
Multi-Forte PK functions from Microsoft Excel.24,25
Protein Expression and Purification. Protein expression and
purification were performed exactly as described by us.9 In brief, we used
the S74E-C4S-dCK variant, which is the human dCK protein where four
solvent-exposed cysteines are mutated into serines (C4S). We showed
that the C4S mutant generates better quality crystals without altering the
three-dimensional conformation of the enzyme or its enzymatic
activity.22 Additionally, the enzyme contained the mutation of Ser74
to glutamic acid (S74E); this mutation serves to mimic the
phosphorylated state of this residue. When we refer to dCK in this
report, we mean the C4S-S74E-dCK variant. dCK was expressed in
Crystallization, X-ray Data Collection, and Refinement.
Crystals of human dCK in complex with inhibitors and UDP were
grown at 285 K using the hanging-drop vapor-diffusion method. All
dCK-inhibitor complexes were prepared as follows: 1 μL of dCK protein
at 10−17 mg/mL in complex with a 2.5-fold molar excess of inhibitor,
and 2 mM UDP and 5 mM MgCl2 were mixed with 1 μL of reservoir
buffer solution. The reservoir solution consisted of 0.9−1.5 M trisodium
citrate dehydrate and 25 mM HEPES, pH 7.5. Prior to data collection,
crystals were soaked in mineral oil for cryoprotection. Diffraction data
for dCK in complex with compounds 4−8 were collected on the Life
M
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Journal of Medicinal Chemistry
Article
Sciences Collaborative Access Team (LS-CAT) beamline 21-ID-G.
Data for all other complexes (compounds 9−12) were collected using
the in-house X-ray source (Rigaku RU-200 rotating anode) with a R-
AXIS IV++ image plate detector. Data were processed and scaled with
XDS and XSCALE.26 Structures were determined by molecular
replacement with MOLREP27 using the dCK structure (PDB entry
4JLN 9) as a search model. Refinement was conducted using
REFMAC,28 and model building was conducted using Coot.29 All
inhibitor coordinates and library descriptions were generated using the
PRODRG server.30 All data sets were perfectly twinned, and iterative
refinements were carried out using REFMAC with the Twin option
active. Data collection and refinement statistics are listed in Table 1.
Structural figures were prepared using the PyMOL Molecular Graphics
in this study. This intellectual property has been patented by the
University of California and licensed to Sofie Biosciences, a
company that both C.G.R. and the University of California own
equity in. In addition, C.G.R., M.E.J., A.L.A, and A.L. are co-
inventors of the dCK inhibitors used in this study. This
intellectual property has been patented by the University of
California and optioned to Triangle Therapeutics Inc., a
company that C.G.R. and M.E.J. own equity in. This material
is based upon work supported by the National Science
Foundation under Equipment Grant CHE-1048804 (A.L.),
Developmental Project Award from the In Vivo Cellular and
Molecular Imaging Center National Cancer Institute P50
CA86306 Award (C.G.R), National Cancer Institute Grant
5U54 CA119347 (C.G.R.), and National Institutes of Health
Grant R01 EB013685 (A.L.).
System (version 1.6.0, Schrodinger).
̈
Modeling. The S-isomer in position 1 and the R-isomer in position 2
were generated by flipping the chirality of the linker carbon using
Maestro, version 9.1, Schrodinger, LLC, 2010. This program was also
̈
used to generate the torsion scans around the bond connecting the chiral
linker carbon and the thiazole ring (torsion angle defined by CAC−
CBC−CBB−NAO).
ACKNOWLEDGMENTS
■
We thank Dr. Nagichettiar Satyamurthy for his expert chemistry
advice. We thank Larry Pang and Liu Wei for their assistance with
PET/CT imaging studies, Dr. Sam Sadeghi and the Ahmanson
Translational Imaging Division Cyclotron crew for producing
18F-L-FAC, and Dr. Jennifer M. Murphy for her assistance in
synthesizing compounds 3 and 4. We also thank Carolann
Gemski for help in editing the manuscript. This work was
supported by National Institutes of Health [Grant R01
EB013685 to A.L.] and In Vivo Cellular and Molecular Imaging
Centers Award National Institutes of Health [Grant P50
CA86306 to C.G.R.] and the U.S. National Cancer Institute
[Grant 5U54 CA119347 to C.G.R.]. Use of the Advanced
Photon Source, an Office of Science User Facility operated for
the U.S. Department of Energy (DOE) Office of Science by
Argonne National Laboratory, was supported by the U.S. DOE
under Contract DE-AC02-06CH11357. Use of the LS-CAT
Sector 21 was supported by the Michigan Economic Develop-
ment Corporation and the Michigan Technology Tri-Corridor
(Grant 085P1000817).
Equilibration simulations were performed using the MCPRO 2.0
software package31 with the OPLS-AA17 force field. The protein was
solvated in a 30 Å cap of TIP4P water molecules.16 The protein
backbone and all bond lengths within the protein were held fixed. Angles
and torsions within 11 Å of the center of the bound molecule were
allowed to vary. All degrees of freedom of the bound molecule were
sampled. Equilibration began with 5 × 106 configurations of solvent-
only moves, followed by 10 × 106 configurations in which the protein
and bound molecule were sampled, with additional solvent sampling at
every tenth configuration. Equilibrations were performed using
Metropolis Monte Carlo in the NPT ensemble at 1 atm and 25 °C.
For the unbound structures, optimizations were performed using OPLS-
AA. Implicit solvent was simulated with the generalized Born/surface
area (GB/SA) method.19,21 Energies were assessed using the PDDG/
PM3 method32 in the BOSS software package.31
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic schemes for compounds 9 and 10; general binding
information for 1 to dCK; omit maps for compounds 4−10; in
vitro biological data in CEM cells for compounds S1−S31;
spectroscopic data for compounds 3, 4, 9, 10, 11R, 11S, and 12S.
This material is available free of charge via the Internet at http://
pubs.acs.org.
ABBREVIATIONS USED
■
dCK, deoxycytidine kinase; dNTP, deoxyribonucleotide triphos-
phate; dC, deoxycytidine; dA, deoxyadenosine; dG, deoxygua-
nosine; ATP, adenosine triphosphate; UTP, uridine triphos-
phate; PK, pharmacokinetic; PET, positron emission tomog-
raphy; 18F-FDG, 2-18F-fluoro-2-deoxy-D-glucose; 18F-L-FAC,
18F-L-1-(2′-deoxy-2′-fluoroarabinofuranosyl) cytosine; PD,
pharmacodynamics; PEG, polyethylene glycol; MPEG, methoxy
polyethylene glycol; DIBAL-H, diisobutylaluminum hydride;
Rochelle’s salt, sodium potassium tartrate; CBS, Corey−Bakshi−
Shibata; TFA, trifluoroacetate; DMAP, 4-(N,N-dimethylamino)-
pyridine
Accession Codes
PDB codes for complexes 4−10 and 12R are the following: 4Q18
(4), 4Q19 (5), 4Q1A (6), 4Q1B (7), 4Q1C (8), 4Q1D (9),
4Q1E (10), and 4Q1F (12R).
AUTHOR INFORMATION
Corresponding Authors
■
*C.G.R.: e-mail, CRadu@mednet.ucla.edu; phone, 310-825-
1205.
*A.L.: e-mail, lavie@uic.edu; phone, 312-355-5029.
Present Address
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#J.N.: Institut des Technologies Avancee
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