Discovery of a novel class of potent human deoxyuridine triphosphatase inhibitors remarkably enhancing the antitumor activity of thymidylate synthase inhibitors

Article abstract of DOI:10.1021/jm201628y

Inhibition of human deoxyuridine triphosphatase (dUTPase) has been identified as a promising approach to enhance the efficacy of 5-fluorouracil (5-FU)-based chemotherapy. This study describes the development of a novel class of dUTPase inhibitors based on the structure-activity relationship (SAR) studies of uracil derivatives. Starting from the weak inhibitor 7 (IC ₅₀ = 100 μM), we developed compound 26, which is the most potent human dUTPase inhibitor (IC₅₀ = 0.021 μM) reported to date. Not only does compound 26 significantly enhance the growth inhibition activity of 5-fluoro-2′-deoxyuridine (FdUrd) against HeLa S3 cells in vitro (EC ₅₀ = 0.075 μM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound 26 is a promising candidate for clinical development.

Full text of DOI:10.1021/jm201628y

Article
pubs.acs.org/jmc
Discovery of a Novel Class of Potent Human Deoxyuridine
Triphosphatase Inhibitors Remarkably Enhancing the Antitumor
Activity of Thymidylate Synthase Inhibitors
Seiji Miyahara,†,‡ Hitoshi Miyakoshi,†,§ Tatsushi Yokogawa,† Khoon Tee Chong,† Junko Taguchi,†
Toshiharu Muto,† Kanji Endoh,† Wakako Yano,† Takeshi Wakasa,† Hiroyuki Ueno,† Yayoi Takao,†
Akio, Fujioka,† Akihiro Hashimoto,† Kenjirou Itou,† Keisuke Yamamura,† Makoto Nomura,†
Hideko Nagasawa,§ Satoshi Shuto,‡ and Masayoshi Fukuoka*,†
†Tsukuba Research Center, Taiho Pharmaceutical Co. Ltd., Okubo 3, Tsukuba, Ibaraki 300-2611, Japan
‡Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
§Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
S
* Supporting Information
ABSTRACT: Inhibition of human deoxyuridine triphospha-
tase (dUTPase) has been identified as a promising approach to
enhance the efficacy of 5-fluorouracil (5-FU)-based chemo-
therapy. This study describes the development of a novel class
of dUTPase inhibitors based on the structure−activity
relationship (SAR) studies of uracil derivatives. Starting from
the weak inhibitor 7 (IC50 = 100 μM), we developed
compound 26, which is the most potent human dUTPase
inhibitor (IC50 = 0.021 μM) reported to date. Not only does
compound 26 significantly enhance the growth inhibition activity of 5-fluoro-2′-deoxyuridine (FdUrd) against HeLa S3 cells in
vitro (EC50 = 0.075 μM) but also shows robust antitumor activity against MX-1 breast cancer xenograft model in mice when
administered orally with a continuous infusion of 5-FU. This is the first in vivo evidence that human dUTPase inhibitors enhance
the antitumor activity of TS inhibitors. On the basis of these findings, it was concluded that compound 26 is a promising
candidate for clinical development.
it hydrolyzes dUTP to dUMP and inorganic pyrophos-
phate.14−16 This enzyme serves two functions in nucleotide
metabolism: first, it decreases the amount of intracellular dUTP
pools to prevent the misincorporation of uracil instead of
thymine into DNA, and second, it supplies the substrate dUMP
for TS, which is responsible for an important de novo
nucleotide metabolism pathway in DNA synthesis.17 In
addition, it has been reported that dUTPase can hydrolyze
FdUTP.18Accordingly, increased dUTPase activity induces
resistance to TS inhibitors. Basic research on the relationship
between the sensitivity of TS inhibitors and expression of
dUTPase has been reported previously. Elevation of dUTPase
activity correlates with resistance to FdUrd in colorectal and
breast cancer cell lines,19−21 and small interfering RNA-
mediated suppression of human dUTPase enhances the growth
inhibition activity of FdUrd against SW620 and MCF-7 cells by
expansion of the dUTP pool in vitro.12
INTRODUCTION
■
Thymidylate synthase (TS) inhibitors including 5-fluorouracil
(5-FU) and its derivatives are widely used in clinical cancer
chemotherapy.1−4 Although TS inhibitors are significantly
beneficial, a large percentage of tumors exhibit intrinsic or
acquired drug resistance.5 Therefore, novel therapeutic targets
and strategies are urgently required to overcome the frequent
occurrence of drug resistance.
TS inhibitors lead to rapid depletion of the cellular 2′-
deoxyuridine 5′-triphosphate (dTTP) pool. Depletion of dTTP
pool induces thymineless death. In addition, TS inhibition
results in the accumulation of 2′-deoxyuridine 5′-mono-
phosphate (dUMP), which may subsequently lead to increased
levels of 2′-deoxyuridine 5′-triphosphate (dUTP).6,7 Both
dUTP and the 5-FU metabolite 5-fluoro-2′-deoxyuridine 5′-
triphosphate (FdUTP) can be misincorpotated into DNA in
place of dTTP during replication and repair by DNA
polymerase.8 High cellular (F)dUTP/dTTP ratios induce futile
cycles of misincorporation; these cycles eventually lead to DNA
strand breaks and cell death. This is one of the key antitumor
mechanisms of TS inhibitors.9−13
On the basis of the analysis of human tissue specimens,
Ladner and co-workers reported that nuclear dUTPase
overexpression may correlate with resistance to TS inhibitor-
dUTPase is a preventive DNA repair enzyme with exquisite
specificity for dUTP among canonical nucleoside triphosphates;
Received: December 1, 2011
Published: February 16, 2012
© 2012 American Chemical Society
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based cancer chemotherapy and poor prognosis in colorectal
cancer.22 Furthermore, Takatori and co-workers have reported
that nuclear dUTPase activation significantly correlates with
poor survival outcomes in patients with hepatocellular
carcinoma (HCC).23 These findings demonstrate that human
dUTPase inhibitors could be clinically useful agents that
enhance the antitumor activity of TS inhibitors.
In this report, we describe the discovery of a novel class of
dUTPase inhibitors with increased inhibition activity (IC50
values in the 10−8 M range). Furthermore, we found that
compound 26 is the first human dUTPase inhibitor that
remarkably enhances the antitumor activity of a TS inhibitor in
vivo. Our present study provides compelling evidence that the
small molecule dUTPase inhibitor may provide a promising
novel cancer therapeutic strategy.
Thus, efforts to obtain efficient dUTPase inhibitors have
resulted in the development of several compounds (Figure 1).
RESULT AND DISCUSSION
■
Identification of an Initial Lead Compound 9. Human
dUTPase has an exquisite substrate specificity only for dUTP
among the canonical nucleoside triphosphates. Thus, human
trimeric dUTPase specifically recognizes a uracil ring in the
active site pockets located at the three subunit interfaces of the
dUTPase trimer.35,36 On the basis of this fact, we prepared our
own N-1 substituted uracil derivatives library and examined the
enzyme inhibition activity of constituent compounds to identify
novel scaffolds (hit compounds) that could be used for
designing potent human dUTPase inhibitors. Throughout this
study, we found several classes of weak dUTPase inhibitors.
Among these compounds, we focused our attention on 1-((2-
(tert-butyldimethylsilyloxy)ethoxy)methyl)pyrimidine-2,4-
(1H,3H)-dione 7 (IC50 = 100 μM) as a prototype compound
that could be used to develop efficient inhibitors because of its
low molecular weight and amenability to derivatization.
Substructure searches of compound 7 led to the
identification of compound 8 and 9 that have still low
molecular weight and enable further structural modification
(Figure 2). In enzyme inhibition assays, these compounds
Figure 1. Chemical structures of BM-dUTP 1,24 dUPNPP 2,25 5′-O-
trityl-2′-deoxyuridine 3,29 acyclouridine derivative 4,31 oxime-linkage
compound 5,33 and our previous inhibitor 6.34.
For example, nonhydrolyzable isosteres 1 and 2 of the substrate
dUTP have been synthesized in which the oxygen atom
between the α- and β-phosphates of the triphosphate moiety of
dUTP is replaced by a methylene or an imino group.24,25 These
compounds had a low Ki value against Escherichia coli dUTPase
and were effectively used in kinetic studies26,27 and for solving
the X-ray crystal structures of the enzyme from various
species.28 However, these nonhydrolyzable inhibitors appear to
be ineffective for chemotherapeutic uses owing to high polarity
and consequent poor cell membrane permeability. In addition,
enzyme inhibitors without a phosphate or its isostere such as 5′-
O-substituted deoxyuridine derivatives 329,30 and acyclouridine
derivatives 431,32 have been reported. Although these
compounds have inhibition activity against the dUTPase of
Plasmodium faliciparum, their inhibition activity against human
dUTPase is weak. Stivers and co-workers have synthesized
oxime-linkage compounds such as 533 that only moderately
inhibited human dUTPase.
Figure 2. Structures of compound 7, 8, and 9.
exhibited increased dUTPase inhibition activity (IC50 = 3.1, 3.9
μM) compared with those of the hit compound 7 and the
previously reported inhibitors 2 and 4 (Table 1).
To investigate whether compounds 8 and 9 enhance the cell
growth inhibition activity of FdUrd, we performed a growth
inhibition assay in combination with FdUrd in HeLa S3 cells.
The EC50 value represents the concentration of each
compound that reduces the T/C (%) value of FdUrd (1 μM)
against HeLa S3 cells to half in 24 h. Compound 8 did not
enhance the growth inhibition activity of FdUrd in vitro. The
acidity of compound 8 may have led to the lack of cell
membrane permeability. On the other hand, compound 9
clearly enhanced the growth inhibition activity of FdUrd (1
μM) in a concentration dependent manner (EC50 = 5.1 μM) in
vitro (Figure 3). Treatment with 9 alone had little effect on cell
In our previous paper, we reported the discovery of N-1
substituted uracil derivatives, such as compound 6, as dUTPase
inhibitor with a unique binding mode and potent enzyme
inhibition activity (IC50 values in the 10−7 M range in vitro).34
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the amide NH group of Gly110. The sulfonamide NH group of
compound 9 interacts with the hydroxyl group of Tyr105 via
two water molecules. In Figure 4B, compound 9 was
superimposed on the X-ray cocrystal structure of dUPNPP 2
with human dUTPase. Compound 9 adopts a folded
conformation in the active site of the enzyme that is similar
to 6, as reported in our previous paper.34 The uracil ring of
compound 9 is accommodated in the conserved uracil binding
site, and its terminal phenyl ring is located in the relatively
hydrophobic region (formed by Val65, Ala90, Ala98, and
Val112) of the enzyme.
The C-terminal residues of each subunit of dUTPase, which
are invariably disordered in the uncomplexed structure (open
form), become ordered upon dUTP binding to each of the
active sites (closed form) (Figure 5).35,36 It may be crucial for
the enzymatic activity of human dUTPase that the flexible C-
terminal tail locates to each ligand binding active site, where the
uracil ring of the substrate and the phenyl group of Phe158 in
the enzyme are stacked. The terminal phenyl ring of compound
6 prevented the alignment of the flexible phenyl group of
Phe158 to the active site without any interaction between the
inhibitor and the triphosphate-binding pocket.34 X-ray cocrystal
structures show that compound 9 binds in a similar manner to
compound 6, with the terminal phenyl ring occupying a region
similar to that of Phe158. Therefore, the C-terminal tail that
includes Phe158 cannot be detected in the active site.
Table 1. Enzyme Inhibition Activities of Human dUTPase
Inhibitors
a
compd
IC50 (μM)
2
4
7
8
9
15.2 1.4
>100
100
3.1 0.30
3.9 0.63
a
Concentration of each compound required to inhibit 50% of the
amount of [5-3H]dUMP produced by human dUTPase. IC50 data
represent the average of three independent assays unless otherwise
stated. Errors are given as standard deviations.
Figure 3. Enhancing effect of compound 9 for growth inhibition
activity of FdUrd (1 μM) against HeLa S3 cells.
Improvement of the Initial Lead Compound 9. Part 1:
Substitution at the Phenyl Ring. We next investigated the
substituent effects at the terminal phenyl ring of compound 9.
A series of analogues 9 (10−16) were synthesized starting from
commercially available compound 17 (Scheme 1). Reduction of
nitro and ester groups of compound 17 with lithium aluminum
hydride (LiAlH4) followed by treatment with N-carbobenzox-
yoxysuccinimide afforded 18, which was further treated with
chloromethyl methyl ether (MOMCl) to afford 19. After
activation of the methoxymethyl (MOM) group of 19 by boron
trichloride (BCl3), treatment with 2,4-bis(trimethylsilyloxy)-
pyrimidine {(TMS)2uracil} afforded 20. Removal of the
carbobenzyloxy group and subsequent sulfonylation afforded
9−15. Compound 16 was synthesized from intermediate 19 by
removal of the carbobenzyloxy group, sulfonylation of the
primary amine, benzoyl group deprotection by methylamine,
growth (T/C 96% at 30 μM). These results suggest that
compound 9 enhanced the growth inhibition activity of FdUrd
by inhibiting human dUTPase in HeLa S3 cells.
To elucidate the binding mode of compound 9 to human
dUTPase, an X-ray crystal structure of the trimeric human
dUTPase complexed with compound 9 was solved at 1.8 Å
resolution. This clearly demonstrated the defined binding mode
in which compound 9 is accommodated in the active site
located at the three subunit interfaces between each subunit of
the dUTPase trimer. Some interactions of compound 9 with
the enzyme in the active site were observed as shown in Figure
4A. Uracil O4 form two hydrogen bonds with the amide NH
group of Val 112 and the carbonyl group of Gly97 via a water
molecule, and it forms another hydrogen bond with the amide
NH group of Gly99. Uracil N-3 interacts with the carbonyl
group of Gly110, and uracil O2 forms a hydrogen bond with
Figure 4. (A) X-ray cocrystal structure of compound 9 (green) with human dUTPase (PDB code: 3ARN). Water molecules are shown as small
spheres. Compound 9 forms key hydrogen bond contacts (yellow dotted lines) with the enzyme. (B) Overlay of compound 9 and X-ray cocrystal
structure of dUPNPP 2 (magenta) with human dUTPase (PDB code: 2HQU). Phe158 is highlighted in orange.
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Figure 5. A proposed dUTPase inhibition mechanism by compound 9.
a
Scheme 1. Synthesis of Compound 9−16
a
Reagents and conditions: (a) (1) 2.4 M LiAlH4, THF, 45 °C, 16 h, (2) N-carbobenzoxyoxysuccinimide, CH2Cl2, 4 h; (b) MOMCl, N,N-
diisopropylehtylamine, CH2Cl2, room temp, 2 h; (c) (1) 1.0 M BCl3 in CH2Cl2, CH2Cl2, room temp, 2 h, (2) (TMS)2uracil, I2, 1,2-dichloroethane,
reflux, 1.5 h; (d) H2, 10%Pd/C, MeOH, room temp, 4 h; (e) RSO2Cl, Et3N, CH2Cl2, room temp, 16 h; (f) (1) H2, 10%Pd/C, MeOH, room temp,
14 h, (2) 3-BzOPhSO2Cl, Et3N, CH2Cl2, room, temp, 2 h; (g) (1) 40% MeNH2 in MeOH, room temp, 20 min, (2) (bromomethyl)cyclopropane,
K2CO3, DMF, 90 °C, 16 h; (h) (1) 1.0 M BCl3 in CH2Cl2, CH2Cl2, room temp, 1.5 h, (2) (TMS)2uracil, I2, 1,2-dichloroethane, reflux, 3 h.
alkylation of the resulting phenol moiety, and coupling with
(TMS)2uracil after activation of the MOM group with BCl3.
It was found that introduction of a substituent (chloro or
methoxy group) at the m-position of the terminal phenyl ring
of 9 retained or enhanced its enzyme inhibition activity
compared with the unsubstituted compound 9 (Table 2).
Among them, compound 11 with its m-methoxy group showed
more potent inhibition (IC50 = 1.2 μM) than 9. All compounds
enhanced the growth inhibition activity of FdUrd (1 μM) in
HeLa S3 cells (Table 2). The EC50 value of these compounds
significantly correlated with their enzyme inhibition activity,
indicating that the degree of reduction in the cellular dUTPase
level could be responsible for the enhancement of the growth
inhibition of FdUrd.
Compound 16, containing a cyclopropylmethoxy group at
the m-position, exhibited an approximately 110-fold increase in
potency (IC50 = 0.035 μM) compared with the lead compound
9. Introduction of a cyclopropylmethoxy group at the m-
position of the terminal phenyl ring may enhance the
hydrophobic interaction between the compound and enzyme.
Improvement of the Lead Compound 9 Part 2:
Exploration of Another Linker Moiety. Although com-
pound 16 has favorable biological activity, it could not be
solidified, making it unsuitable as a drug candidate. In addition,
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Table 2. Substitution Effects at the Terminal Phenyl Ring of Compound 9 on Human dUTPase Inhibition Activity
a
b
compd
R
IC50 (μM)
EC50 (μM)
9
H
3.9 0.63
19.5 2.8
1.2 0.08
18.2 1.4
9.4 0.34
4.5 0.29
5.7 0.32
0.035 0.002
5.1 0.32
14.1 0.64
1.1 0.07
14.9 1.7
7.93 0.29
3.96 0.11
4.97 0.35
0.18 0.01
10
11
12
13
14
15
16
o-CH3O, p-Me
m-CH3O
p-CH3O
o-Cl
m-Cl
p-Cl
m-cyclopropylmethoxy
a
b
Concentration of each compound required to inhibit 50% of the amount of [5-3H]dUMP produced by human dUTPase. Concentration of each
compound that reduces the T/C (%) value of FdUrd (1 μM) against HeLa S3 to half in 24 h. In all cases, IC50 and EC50 data represent the average of
three independent assays, and errors are given as standard deviations.
Figure 6. Compound 24 showed good physicochemical property in our chemical series.
Table 3. Biological Data of Compounds 24, 25R/S, 25R, 25S, and 26
a
b
compd
R1
R2
stereo
X
IC50 (μM)
EC50 (μM)
24
Me
Me
Me
H
Me
H
H
H
H
H
F
0.34 0.03
0.062 0.001
0.040 0.007
2.5 0.34
0.45 0.06
0.14 0.01
25R/S
25R
25S
26
rac
R
H
0.066 0.005
4.15 0.57
Me
S
Me
H
R
0.021 0.003
0.075 0.002
a
b
Concentration of each compound required to inhibit 50% of the amount of [5-3H]dUMP produced by human dUTPase. Concentration of each
compound that reduces the T/C (%) value of FdUrd (1 μM) against HeLa S3 to half in 24 h. In all cases, IC50 and EC50 data represent the average of
three independent assays, and errors are given as standard deviations.
a
Scheme 2. Synthesis of Compounds 24, 25R/S, 25R, 25S, and 26
a
Reagents and conditions: (a) (1) 1.0 M BCl3 in CH2Cl2, CH2Cl2, room temp, 1.5 h, (2) (TMS)2uracil, I2, 1,2-dichloroethane, reflux, 3 h.
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analogues of compound 9 such as compounds 10−15 had
similar unfavorable physicochemical properties. Thus, we
designed additional novel compounds with an alternative linker
to improve physicochemical properties and found that
compound 24 was easily solidified (Figure 6). Hence, although
compound 24 had slightly less enzyme inhibition activity (IC50
= 0.34 μM) than compound 16, we selected 24 as an alternative
lead compound.
Enhancing Activity of Compound 26 for Antitumor
Effect of 5-FU against the MX-1 Breast Cancer Xenograft
Model in Mice. Compound 26 (300 mg/kg/day) was
administered orally in combination with continuous infusion
of 5-FU (15 mg/kg/day) against the MX-1 xenograft model in
mice. Compound 26 showed significant potency (IR = 83%) in
enhancing the antitumor effect of 5-FU (Table 5, Figure 7A).
To investigate the substituent effect at the p-position of the
terminal phenyl ring and the dimethyl group at the benzyl
position of compound 24 on the enzyme inhibition activity, we
synthesized monomethyl compounds 25R/S, 25R, 25S, and 26
(Table 3). Procedures for the synthesis of compounds 27, 28,
29R, 29S, and 29R/S are detailed in the Supporting
Information. These compounds were activated with BCl3 and
then reacted with (TMS)2uracil to give target analogues 24,
25R/S, 25R, 25S, and 26 (Scheme 2).
Racemic monomethyl compound 25R/S showed markedly
increased inhibition activity against human dUTPase (IC50
=
0.062 μM). Therefore, we further synthesized the S- and R-
enantiomers at the benzyl position of compound 25R/S to
identify the eutomer. R-Enantiomer 25R was revealed to be the
eutomer (IC50 = 0.040 μM), and the eudismic ratio was
relatively large. This large eudismic ratio suggested that the
(R)-asymmetric center may be essential for the bioactive form,
and the terminal phenyl moiety of the inhibitors plays an
important role in the pharmacophore.
We further found that compound 26, which has an
introduced fluorine moiety at the p-position of the terminal
phenyl group of compound 25R, also exhibit highly potent
enzyme inhibition activity (IC50 = 0.021 μM) and highly potent
enhancement of the growth inhibition activity of FdUrd in vitro
(EC50 = 0.075 μM). Compound 26 alone had little effect on
cell growth (IC50 = 28.3 μM).
In Vivo Pharmacokinetics (PK) Profile of Compound
26. We selected compound 26 because of its in vitro potency
and evaluated preliminary pharmacokinetics (PK) profile in
mice (Table 4). Compound 26 was well absorbed after oral
Figure 7. (A) Antitumor activity of 26 in combination with 5-FU in
the MX-1 xenograft model. Relative tumor volume (RTV) is expressed
as mean SD of at least three independent experiments. (B) Body
weight change (%) is expressed as mean SD.
Although 5-FU treatment at 15 mg/kg/day had little antitumor
effect, combination with 26 completely prevented the tumor
growth without significant weight loss, as compared with the
vehicle-treated group (Figure 7B). Compound 26 alone
exhibited no antitumor activity in vivo (Table 5). This is the
first in vivo evidence of a human dUTPase inhibitor enhancing
the antitumor activity of a TS inhibitor.
Table 4. Pharmacokinetic Profile of 26 after Oral
a
Administration (po)
AUC0−t (μM·h)
Cmax (μM)
T1/2 (h)
Tmax (h)
CONCLUSION
■
26
24.9
15.1
1.4
0.5
We have described the discovery of a novel class of human
dUTPase inhibitors. Starting from low affinity ligand 7, we
developed compound 9 whose terminal phenyl ring is located
in a position similar to that of Phe158 in the active form and
which mimics the stacked mode of the uracil ring of the
substrate dUTP with that of Phe158. Further SAR studies of
compound 9 led to compound 26 that has the greatest in vitro
a
Balb/cA male mice (n = 2) was dosed at 50 mg/kg. The po
formulation contained 2.5% DMA, 2.5% Tween 80, and 10%
Cremophor EL.
administration in mice plasma. These PK data showed that
compound 26 has a favorable profile for in vivo study.
Table 5. Antitumor Activity of Compound 26 in Combination with 5-FU in the MX-1 Xenograft Model
a
b
c
drug
dose (mg/kg/day)
treatment
TV (mm3, mean SD)
RTV (mean SD)
IR (%)
control
5-FU
2048 693.7
1645 322.6
2698 764.6
299.7 204.3
11.4 3.74
9.13 1.35
14.9 3.88
15
300
CI
20
26
po
−30.9
83.0
d,e
5-FU/26
15/300
CI/po
1.94 1.01**,##
a
b
Tumor volume (TV) on day 15 was calculated according to the following formula: TV (mm3) = (width)2 × (length)/2. Relative tumor volume
(RTV) on day 15 was calculated as the ratio of TV on day 15 to that on day 0 according to the following formula: RTV = (TV on day 15)/(TV on
c
day 0). Inhibition rate (IR) of tumor growth on day 15 on the basis of RTV was calculated according to the following formula: IR (%) = [1 −
d
e
(mean RTV of the treated group)/(mean RTV of the control group)] × 100. **p < 0.01 Dunnet test as compared with the control group. **p <
0.01 Student’s t test as compared with the 5-FU group.
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(2H, m), 3.35 (3H, s), 3.51 (2H, t, J = 6.2 Hz), 4.61 (2H, s), 4.71 (1H,
brs), 5.05 (2H, s), 7.30−7.36 (5H, m). HRMS (FAB) calcd for
C16H24NO4 [M − H]− 294.1705, found 294.1732.
potency (IC50 = 0.021 μM, EC50 = 0.075 μM) reported to date.
Furthermore, compound 26 displayed drastic tumor growth
inhibition when combined with 5-FU against the MX-1
xenograft model in mice. On the basis of these findings, we
expect that human dUTPase inhibitors will ultimately lead to
the establishment of a landmark cancer therapeutic strategy,
providing hope for cancer patients.
Benzyl 5-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
methoxy)-2-methylpentan-2-ylcarbamate (20). To a stirred
solution of 19 (369 mg, 1.25 mmol) in CH2Cl2 (1.0 mL) was
gradually added BCl3 in CH2Cl2 (1.0 M, 330 μL, 0.33 mmol) at 0 °C,
and the resulting mixture was stirred at room temperature for 2 h. The
reaction mixture was concentrated under reduced pressure, and the
residue was then dissolved in 1,2-dichloroethane (10 mL). To the
mixture was added 2,4-bis(trimethylsilyloxy)pyrimidine (256 mg, 1.00
mmol) and iodine (10.0 mg, 0.039 mmol) at room temperature, and
the mixture was heated to reflux at 93 °C for 1.5 h. The reaction
mixture was cooled to room temperature, satd aq Na2S2O3 solution
(10 mL) was then added, and the resulting mixture was extracted with
ethyl acetate (30 mL) two times. The combined organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel eluted with (CHCl3/MeOH = 19/1)
followed by C18 reverse-phase column chromatography eluted with
(MeOH/H2O = 1/1) to obtain the title compound (206 mg, 0.55
EXPERIMENTAL SECTION
■
Chemistry. All commercially available reagents and solvents were
used without further purification unless otherwise specified. All
reactions were performed under an inert nitrogen atmosphere unless
otherwise specified. 1H NMR spectra were recorded on a JEOL JNM-
EX-270 (270 MHz) or JEOL JNM-LA-400 (400 MHz) spectrometer,
and 13C NMR spectra were recorded on a JEOL JNM-LA-400 (100
MHz) spectrometer. Chemical shifts were given in parts per million
(ppm, δ) with tetramethylsilane as the internal standard, and coupling
constant (J value) is given in hertz (Hz). Splitting patterns and
apparent multiplicities are designated as s, singlet; d, doublet; dd,
double doublet; t, triplet; dt, doublet triplet; q, quartet; quin, quintet;
m, multiplet; brs, broad singlet. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 F254 precoated plates (Merck).
Column chromatography was performed on Merck silica gel 60 (230−
400 mesh). Optical rotation was determined using Horiba SEPA-200
polarimeter. High-resolution mass spectra (HRMS) was recorded
either on JEOL JMS-700 (FAB) or on Waters micromass Q-Tof-2
(TOF). The purity of all final compounds was determined by
combustion analysis or high pressure liquid chromatography (HPLC);
purity of at least 95% was found. Elemental analyses were performed
using a Thermo Electron Corporation Flash EA 1112 series. Analytical
HPLC was performed on a Shimazu Prominence system using L-
column 2 ODS column (4.6 mm × 150 mm, 3 μm) with an 8 min
linear gradient from 10−80% acetonitrile/10 mM phosphate buffer
(pH 6.5) and a flow rate of 1.3 mL/min with UV detection at 220 nm
(method A) and a Shim-pack XR-ODS column (3.0 mm × 50 mm, 2.2
μm) with an 8 min linear gradient of 10−80% acetonitrile/10 mM
phosphate buffer (pH 6.5) and a flow rate of 0.8 mL/min with UV
detection at 220 nm (method B). The retention time of compound
peak in HPLC was denoted tR.
1
mmol, 44%) as a colorless gum. H NMR (270 MHz, CDCl3) δ 1.28
(6H, s), 1.52−1.61 (2H, m), 1.63−1.74 (2H, m), 3.52 (2H, t, J = 5.9
Hz), 4.75 (1H, brs), 5.04 (2H, s), 5.10 (2H, s), 5.75 (1H, d, J = 8.1
Hz), 7.26−7.36 (6H, m). HRMS (FAB) calcd for C19H26N3O5 [M +
H]+ 376.1872, found 376.1881.
1-((4-Amino-4-methylpentyloxy)methyl)pyrimidine-2,4-
(1H,3H)-dione (21). A mixture of 20 (120 mg, 0.32 mmol) and 10%
palladium on activated carbon (30.0 mg) in MeOH (4.0 mL) was
stirred at room temperature for 4 h under a hydrogen atmosphere.
The precipitate was removed by filtration through a pad of Celite and
washed with MeOH. The combined filtrate was concentrated under
reduced pressure to afford crude amine (77.5 mg, quant.). The crude
1
amine 21 was used for the next step without further purification. H
NMR (270 MHz, DMSO-d6) δ 0.97 (6H, s), 1.20−1.27 (2H, m),
1.47−1.53 (2H, m), 3.16−3.46 (2H, m), 3.95 (2H, brs), 5.05 (2H, s),
5.59 (1H, dt, J = 8.0, 1.8 Hz), 7.67 (1H, dt, J = 8.0, 1.8 Hz). HRMS
(FAB) calcd for C11H20N3O3 [M + H]+ 242.1505, found 242.1542.
N-(5-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-2-
methylpentan-2-yl)benzenesulfonamide (9). To a stirred sol-
ution of 21 (45.0 mg, 0.19 mmol) in CH2Cl2 (1.5 mL) was added
Et3N (34 μL, 0.24 mmol) and benzenesulfonyl chloride (28 μL, 0.22
mmol) at 0 °C, and the resulting mixture was stirred at room
temperature for 16 h. The mixture was poured into H2O and extracted
with EtOAc two times. The combined organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by column chromatography on
silica gel eluting with (EtOAc) to afford the title compound (40.1 mg,
0.11 mmol, 56%) as a colorless gum. 1H NMR (270 MHz, DMSO-d6)
δ 1.01 (6H, s), 1.34−1.46 (4H, m), 3.26−3.32 (2H, m), 5.00 (2H, s),
5.60 (1H, d, J = 7.8 Hz), 7.41 (1H, brs), 7.49−7.54 (3H, m), 7.64 (1H,
d, J = 7.8 Hz), 7.78−7.81 (2H, m), 11.30 (1H, brs). 13C NMR (100
MHz, CDCl3) δ 23.8, 27.5, 39.2, 56.6, 69.5, 76.6, 103.1, 126.5, 128.8,
132.1, 143.4, 143.5, 151.2, 163.8. Anal. Calcd for C17H23N3O5S: C,
53.53; H, 6.08; N, 11.02. Found: C, 53.94; H, 6.27; N, 11.07.
Benzyl 5-Hydroxy-2-methylpentan-2-ylcarbamate (18). To a
stirred solution of LiAlH4 in THF (2.4 M, 71 mL, 170 mmol) was
slowly added a solution of methyl 4-methyl-4-nitropentanoate 17
(10.0 g, 57.1 mmol) over 30 min at room temperature, and the
mixture was stirred at 45 °C for 16 h. The reaction mixture was cooled
to 0 °C, and H2O was then added dropwise. The resultant precipitate
was removed by filtration and washed with MeOH (100 mL) and
THF (900 mL). The combined filtrate was concentrated under
reduced pressure. The residue was dissolved in CH2Cl2 (100 mL). To
the mixture was added N-carbobenzoxyoxysuccinimide (18.0 g, 72.7
mmol), and the resulting mixture was stirred at room temperature for
4 h. The reaction mixture was concentrated under reduced pressure,
and the residue was then purified by column chromatography on silica
gel eluting with (hexane/EtOAc = 1/1) to afford the title compound
1
(14.0 g, 55.7 mmol, 98%) as a colorless oil. H NMR (270 MHz,
CDCl3) δ 1.11 (6H, s), 1.50−1.61 (4H, m), 3.57−3.65 (2H, m), 4.71
(1H, brs), 5.21 (2H, s), 7.33−7.39 (5H, m). HRMS (FAB) calcd for
C14H22NO3 [M + H]+ 252.1600, found 252.1593.
N-(5-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-2-
methylpentan-2-yl)-2-methoxy-4-methylbenzenesulfonamide
(10). 10 was prepared from 21 (45.1 mg, 0.19 mmol) as described for
1
the preparation of 9; white foam (22.1 mg, 0.052 mmol, 27%). H
Benzyl 5-(Methoxymethoxy)-2-methylpentan-2-ylcarba-
mate (19). To a stirred solution of 18 (14.0 g, 55.7 mmol) in
CH2Cl2 (100 mL) was added N,N-diisopropylethylamine (20 mL, 115
mmol) and MOMCl (4.7 mL, 61.9 mmol) at room temperature, and
the resulting mixture was stirred at room temperature for 2 h. The
mixture was poured into satd aq NH4Cl and extracted with CHCl3 two
times. The combined organic layer was washed with satd aq NH4Cl
and brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel eluting with (hexane/EtOAc = 7/3) to afford the
NMR (400 MHz, CD3OD) δ 1.08 (6H, s), 1.43−1.49 (2H, m), 1.52−
1.56 (2H, m), 2.41 (3H, s), 3.37−3.40 (2H, m), 3.93 (3H, s), 5.10
(2H, s), 5.69 (1H, d, J = 7.8 Hz), 6.84−6.87 (1H, m), 6.97−6.99 (1H,
m), 7.60 (1H, d, J = 8.0 Hz), 7.66 (1H, d, J = 7.8 Hz). 13C NMR (100
MHz, CDCl3) δ 21.9, 23.9, 27.6, 39.1, 56.1, 56.4, 69.7, 76.7, 103.1,
112.8, 121.5, 128.5, 129.1, 143.2, 145.1, 150.8, 155.8, 163.0. Anal.
Calcd for C19H27N3O6S·0.3H2O: C, 52.96; H, 6.46; N,9.75. Found: C,
52.99; H, 6.31; N, 9.47.
N-(5-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-2-
methylpentan-2-yl)-3-methoxybenzenesulfonamide (11). 11
was prepared from 21 (45.0 mg, 0.19 mmol) as described for the
1
title compound (8.80 g, 29.8 mmol, 54%) as a colorless oil. H NMR
(270 MHz, CDCl3) δ 1.31 (6H, s), 1.57−1.62 (2H, m), 1.69−1.73
2976
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Article
1
preparation of 9; white foam (30.0 mg, 0.073 mmol, 38%). H NMR
(400 MHz, CD3OD) δ 1.12 (6H, s), 1.46−1.54 (4H, m), 3.37−3.41
(2H, m), 3.84 (3H, s), 5.10 (2H, s), 5.70 (1H, d, J = 7.9 Hz), 7.09−
7.14 (1H, m), 7.39−7.43 (3H, m), 7.60 (1H, d, J = 7.9 Hz). 13C NMR
(100 MHz, CDCl3) δ 23.9, 27.6, 39.3, 55.6, 56.8, 69.6, 76.7, 103.2,
111.8, 118.3, 119.1, 130.0, 143.2, 144.6, 150.9, 159.7, 163.1; Anal.
Calcd for C18H25N3O6S: C, 52.54; H, 6.12; N, 10.21. Found: C, 52.35;
H, 6.30; N, 9.98.
8.13−8.17 (2H, m). HRMS (FAB) calcd for C21H26NO6S [M − H]−
420.1481, found 420.1511.
3-(Cyclopropylmethoxy)-N-(5-(methoxymethoxy)-2-methyl-
pentan-2-yl)benzenesulfonamide (23). A solution of 22 (630 mg,
1.49 mmol) in 40% MeNH2 in MeOH (4.0 mL) was stirred at room
temperature for 20 min. The mixture was concentrated under reduced
pressure, and the residue was coevaporated with toluene two times.
This residue was dissolved in DMF (4.0 mL). To the mixture was
added K2CO3 (373 mg, 2.70 mmol) and (bromomethyl)cyclopropane
(156 μL, 1.61 mmol) at room temperature. The resulting mixture was
stirred at 90 °C for 16 h. After cooling to room temperature, the
mixture was poured into H2O, extracted with EtOAc two times. The
combined organic layer was washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel eluting with (hexane/EtOAc =
4/1) to afford the title compound (405 mg, 1.09 mmol, 73%) as a
N-(5-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-2-
methylpentan-2-yl)-4-methoxybenzenesulfonamide (12). 12
was prepared from 21 (30.1 mg, 0.12 mmol) as described for the
1
preparation of 9; white foam (35.2 mg, 0.086 mmol, 72%). H NMR
(270 MHz, CD3OD) δ 1.08 (6H, s), 1.46−1.56 (4H, m), 3.40−3.44
(2H, m), 3.86 (3H, s), 5.10 (2H, s), 5.70 (1H, d, J = 7.9 Hz), 7.03
(1H, dd, J = 6.8, 2.0 Hz), 7.60 (2H, d, J = 7.9 Hz), 7.78 (2H, dd, J =
6.8, 2.0 Hz). 13C NMR (100 MHz, CDCl3) δ 23.9, 27.7, 39.3, 55.6,
56.5, 69.6, 76.7, 103.2, 114.1, 129.0, 135.2, 143.3, 151.0, 162.5, 163.3.
Anal. Calcd for C18H25N3O6S·0.5H2O: C, 51.42; H, 6.23; N, 9.99.
Found: C, 51.74; H, 6.01; N, 9.80.
1
colorless oil. H NMR (270 MHz, DMSO-d6) δ 0.30−0.36 (2H, m),
0.53−0.60 (2H, m), 1.04 (6H, s), 1.18−1.24 (1H, m), 1.40−1.48 (4H,
m), 3.20 (3H, s), 3.26−3.34 (2H, m), 3.85 (2H, d, J = 6.8 Hz), 4.48
(2H, s), 7.11−7.14 (1H, m), 7.31−7.47 (4H, m). HRMS (FAB) calcd
for C18H28NO5S [M − H]− 370.1688, found 370.1716.
2-Chloro-N-(5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
methoxy)-2-methylpentan-2-yl)benzenesulfonamide (13). 13
was prepared from 21 (45.3 mg, 0.19 mmol) as described for the
3-(Cyclopropylmethoxy)-N-(5-((2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)methoxy)-2-methylpentan-2-yl)-
benzenesulfonamide (16). To a stirred solution of 23 (120 mg,
0.32 mmol) in CH2Cl2 (500 μL) was added BCl3 in CH2Cl2 (1.0 M,
120 μL, 0.12 mmol) at 0 °C, and the resulting mixture was stirred at
room temperature for 1.5 h. The mixture was concentrated under
reduced pressure, and the residue was dissolved in 1,2-dichloroethane
(2.0 mL). The mixture was then added to a mixture of 2,4-
bis(trimethylsilyloxy)pyrimidine (124 mg, 0.48 mmol) and iodine (3.0
mg, 0.012 mmol) in 1,2-dichloroethane (1.0 mL) at room temper-
ature. The resultant mixture was heated to reflux at 95 °C for 3 h. After
cooling to room temperature, the mixture was poured into H2O and
extracted with CHCl3 two times. The combined organic layer was
washed with satd aq Na2S2O3, brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel eluting with (EtOAc) to afford
1
preparation of 9; white foam (24.3 mg, 0.058 mmol, 31%). H NMR
(270 MHz, CD3OD) δ 1.11 (6H, s), 1.50−1.59 (4H, m), 3.40−3.43
(2H, m), 5.10 (2H, s), 5.70 (1H, d, J = 7.8 Hz), 7.44−7.47 (1H, m),
7.50−7.62 (3H, m), 8.04−8.07 (1H, m). 13C NMR (100 MHz,
CDCl3) δ 23.9, 27.5, 39.2, 57.0, 69.6, 76.7, 103.2, 127.3, 130.4, 131.2,
131.4, 133.2, 140.7, 143.2, 150.8, 163.0. Anal. Calcd for
C17H22ClN3O5S·H2O: C, 47.06; H, 5.58; N, 9.68. Found: C, 47.36;
H, 5.22; N, 9.49.
3-Chloro-N-(5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
methoxy)-2-methylpentan-2-yl)benzenesulfonamide (14). 14
was prepared from 21 (30.3 mg, 0.13 mmol) as described for the
1
preparation of 9; white foam (26.8 mg, 0.064 mmol, 49%). H NMR
(270 MHz, CD3OD) δ 1.13 (6H, s), 1.52−1.55 (4H, m), 3.41−3.46
(2H, m), 5.11 (2H, s), 5.70 (1H, d, J = 7.9 Hz), 7.53−7.63 (3H, m),
7.82−7.86 (2H, m). 13C NMR (100 MHz, CDCl3) δ 23.9, 27.7, 39.3,
57.0, 69.5, 76.7, 103.3, 125.0, 127.0, 130.3, 132.3, 135.0, 143.3, 145.3,
151.1, 163.5. HRMS (TOF) calcd for C17H23ClN3O5S [M + H]+
416.1047, found 416.1048. HPLC purity: method B = 99.3%, tR = 5.28
min.
1
the title compound (109 mg, 0.24 mmol, 75%) as a white foam. H
NMR (270 MHz, DMSO-d6) δ 0.30−0.34 (2H, m), 0.53−0.58 (2H,
m), 1.01 (6H, s), 1.15−1.24 (1H, m), 1.36−1.48 (4H, m), 3.32−3.39
(2H, m), 3.85 (2H, d, J = 6.9 Hz), 5.00 (2H, s), 5.60 (1H, d, J = 7.8
Hz), 7.09−7.13 (1H, m), 7.30−7.45 (4H, m), 7.64 (1H, d, J = 7.8 Hz),
11.3 (1H, brs). 13C NMR (100 MHz, CDCl3) δ 3.2, 10.0, 23.9, 27.5,
39.3, 56.6, 69.5, 73.1, 103.1, 112.3, 118.8, 119.0, 129.9, 143,4, 144.6,
151.2, 159.1, 163.7. Anal. Calcd for C21H29N3O6S·H2O: C, 53.72; H,
6.65; N, 8.95. Found: C, 54.09; H, 6.33; N, 8.96.
N-(2-(3-(Cyclopropylmethoxy)phenyl)propan-2-yl)-3-((2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)propane-1-sul-
fonamide (24). 24 was prepared from 27 (6.72 g, 18.1 mmol) as
described for the preparation of 16; white solid (3.38 g, 7.49 mmol,
41%). 1H NMR (270 MHz, CDCl3) δ 0.30−0.35 (2H, m), 0.62−0.73
(2H, m), 1.22−1.31 (1H, m), 1.74 (6H, s), 1.96−2.08 (2H, m), 2.81
(2H, t, J = 7.0 Hz), 3.55−3.62 (2H, m), 3.81 (2H, d, J = 7.6 Hz), 4.63
(1H, brs), 5.10 (2H, s), 5.76 (1H, dd, J = 7.8, 1.1 Hz), 6.76−6.88 (1H,
m), 7.05−7.10 (2H, m), 7.24−7.30 (2H, m), 8.35 (1H, brs). 13C NMR
(100 MHz, DMSO-d6) δ 3.1, 10.1, 23.9, 29.8, 51.3, 57.2, 66.4, 71.9,
76.2, 101.6, 112.0, 112.5, 117.6, 128.9, 144.8, 148.6, 151.0, 158.3,
163.6. Anal. Calcd for C21H29N3O6S: C, 55.86; H, 6.47; N, 9.31.
Found: C, 55.55; H, 6.50; N, 9.30.
N-(1-(3-(Cyclopropylmethoxy)phenyl)ethyl)-3-((2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)methoxy)propane-1-sulfona-
mide (25R/S). 25R/S was prepared from 29R/S (548 mg, 1.53
mmol) as described for the preparation of 16; colorless gum (248 mg,
0.57 mmol, 37%). 1H NMR (270 MHz, CDCl3) δ 0.31−0.38 (2H, m),
0.59−0.69 (2H, m), 1.20−1.38 (1H, m), 1.52 (3H, d, J = 6.8 Hz),
1.80−1.98 (2H, m), 2.51−2.88 (2H, m), 3.49−3.53 (2H, m), 3.88
(2H, d, J = 7.0 Hz), 4.58 (1H, quin, J = 6.8 Hz), 4.84 (1H, d, J = 6.8
Hz), 5.06 (2H, s), 5.76 (1H, dd, J = 8.1, 2.2 Hz), 6.8−6.91 (3H, m),
7.21−7.29 (2H, m), 8.69 (1H, brs). 13C NMR (100 MHz, DMSO-d6)
δ 3.0, 10.1, 23.6, 24.1, 49.1, 52.6, 66.3, 71.9, 76.1, 101.7, 112.3, 113.0,
118.1, 129.4, 144.7, 145.7, 151.0, 158.7, 163.6. Anal. Calcd for
4-Chloro-N-(5-((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
methoxy)-2-methylpentan-2-yl)benzenesulfonamide (15). 15
was prepared from 21 (45.3 mg, 0.19 mmol) as described for the
1
preparation of 9; white foam (34.1 mg, 0.082 mmol, 43%). H NMR
(270 MHz, CD3OD) δ 1.12 (6H, s), 1.45−1.54 (4H, m), 3.42−3.46
(2H, m), 5.11 (2H, s), 5.69 (1H, d, J = 7.8 Hz), 7.52−7.55 (2H, m),
7.60 (1H, d, J = 7.8 Hz), 7.82−7.85 (2H, m). 13C NMR (100 MHz,
CDCl3) δ 23.9, 27.7, 39.4, 56.9, 69.5, 76.7, 103.3, 128.4, 129.2, 138.6,
142.0, 143.4, 150.9, 163.1. Anal. Calcd for C17H22ClN3O5S·0.5H2O: C,
48.05; H, 5.46; N, 9.89. Found: C, 47.75; H, 5.17; N, 9.69.
3-(N-(5-(Methoxymethoxy)-2-methylpentan-2-yl)sulfamoyl)-
phenyl benzoate (22). A mixture of 19 (586 mg, 2.00 mmol) and
10% palladium on activated carbon (330 mg) in MeOH (7.0 mL) was
stirred at room temperature for 14 h under a hydrogen atmosphere.
The precipitate was removed by filtration through a pad of Celite and
washed with MeOH. The combined filtrate was concentrated under
reduced pressure, and the residue was coevaporated with THF two
times.
To a solution of the above residue in CH2Cl2 (4.0 mL) was added
Et3N (362 μL, 2.60 mmol) and 3-(chlorosulfonyl)phenyl benzoate
(682 mg, 2.30 mmol) at 0 °C, and the resulting mixture was stirred at
room temperature for 2 h. The mixture was poured into H2O and
extracted with EtOAc two times. The combined organic layer was
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel eluting with (hexane/EtOAc = 3/1) to afford the
title compound (630 mg, 1.49 mmol, 75% from 19) as a colorless oil.
1H NMR (270 MHz, DMSO-d6) δ 1.07 (6H, s), 1.45−1.52 (4H, m),
3.20 (3H, s), 3.32−3.40 (2H, m), 4.48 (2H, s), 7.52−7.79 (8H, m),
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Article
C20H27N3O6S: C, 54.90; H, 6.22; N, 9.60. Found: C, 55.28; H, 6.25;
N, 9.41.
detector. The data, extending to 1.8 Å (3ARN), were integrated and
scaled using the CrystalClear software package (Rigaku Corporation).
Structure Determination and Refinement. The structure of
dUTPase complexed with compound 9 was solved by molecular
replacement using the program AMoRe37 from the CCP4 suite.38 The
search model was based on human dUTPase structure (PDB ID:
1Q5U). The structure of dUTPase complexed to compound 9 was
refined using REFMAC5.39 Manual rebuilding of the models and
electron density map interpretation were carried out using Turbo-
Frodo.40 The final model has R values of Rcryst = 17.2%, Rfree = 19.7%.
A summary of statistics from the data collection and refinement is
given in Table 6.
(R)-N-(1-(3-(Cyclopropylmethoxy)phenyl)ethyl)-3-((2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)propane-1-sul-
fonamide (25R). 25R was prepared from 29R (219 mg, 0.61 mmol)
as described for the preparation of 16; colorless gum (106 mg, 0.24
1
mmol, 39%). H NMR (270 MHz, CDCl3) δ 0.31−0.38 (2H, m),
0.59−0.67 (2H, m), 1.19−1.30 (1H, m), 1.52 (3H, d, J = 6.8 Hz),
1.78−2.00 (2H, m), 2.51−2.89 (2H, m), 3.44−3.59 (2H, m), 3.88
(2H, d, J = 7.0 Hz), 4.56 (1H, quin, J = 6.8 Hz), 5.06 (2H, s), 5.17
(1H, d, J = 6.8 Hz), 5.77 (1H, d, J = 7.8 Hz), 6.79−6.92 (3H, m),
7.20−7.28 (2H, m), 9.12 (1H, brs). 13C NMR (100 MHz, CDCl3) δ
3.2, 10.2, 10.7, 23.7, 30.7, 50.2, 59.6, 66.9, 72.8, 76.3, 103.1, 113.1,
113.7, 118.9, 129.8, 143.2, 151.1, 159.3, 163.7. Anal. Calcd for
C20H27N3O6S: C, 54.90; H, 6.22; N, 9.60. Found: C, 54.71; H, 6.46;
Table 6. X-Ray Crystallography Data and Refinement
Statistics
N, 9.57. [α]25 + 20.7 (c 1.00, MeOH).
D
(S)-N-(1-(3-(Cyclopropylmethoxy)phenyl)ethyl)-3-((2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)propane-1-sul-
fonamide (25S). 25S was prepared from 29S (530 mg, 1.48 mmol)
as described for the preparation of 16; colorless gum (291 mg, 0.67
dUTPase with compd 9 (PDB ID: 3ARN)
Crystal Data
P212121 cell dimensions (Å)
a = 82.40, b = 83.15,
c = 89.59
1
mmol, 45%). H NMR (400 MHz, DMSO-d6) δ 0.25−0.29 (2H, m),
0.50−0.55 (2H, m), 1.14−1.20 (1H, m), 1.32 (3H, d, J = 7.5 Hz),
1.66−1.75 (2H, m), 2.49−2.56 (1H, m), 2.73−2.80 (1H, m), 3.26−
3.39 (2H, m), 3.75 (2H, d, J = 7.1 Hz), 4.33 (1H, quin, J = 7.5 Hz),
4.95 (2H, s), 5.57 (1H, d, J = 8.0 Hz), 6.74 (1H, dd, J = 8.3, 2.4 Hz),
6.85 (1H, d, J = 7.6 Hz), 6.90 (1H, s), 7.16 (1H, t, J = 8.1 Hz), 7.58
(1H, d, J = 8.0 Hz), 7.69 (1H, d, J = 8.5 Hz), 11.3 (1H, brs). 13C NMR
(100 MHz, DMSO-d6) δ 3.1, 10.2, 23.7, 24.1, 49.2, 52.7, 66.3, 71.9,
76.1, 101.7, 112.3, 113.0, 118.2, 129.4, 144.8, 145.8, 151.1, 158.7,
163.7. Anal. Calcd for C20H27N3O6S: C, 54.90; H, 6.22; N, 9.60.
resolution range (Å)
60.94−1.80 (1.86−1.80)
400353
no. observed reflections
no. unique reflections
completeness of data (%)
57479
99.6 (99.1)
0.040 (0.119)
29.3 (11.8)
a
Rsym
I/σI
Refinement Statistics
resolution range (Å)
58.53−1.80 (1.85−1.80)
17.2 (25.70)
51657 (3946)
19.8 (31.60)
2911 (187)
0.032
b
Found: C, 54.73; H, 6.18; N, 9.61. [α]25 −22.4 (c 1.10, MeOH).
́
Rcryst (no o cutoff) (%)
no. reflections in work set
D
(R)-N-(1-(3-(Cyclopropylmethoxy)-4-fluorophenyl)ethyl)-3-
((2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)propane-
1-sulfonamide (26). 26 was prepared from 28 (145 mg, 0.39 mmol)
as described for the preparation of 16; colorless gum (54.9 mg, 0.12
c
́
Rfree (no o cutoff) (%)
no. reflections in test set
d
rmsd bonds (Å)
1
mmol, 31%). H NMR (270 MHz, CDCl3) δ 0.31−0.38 (2H, m),
d
rmsd angles (deg)
2.443
0.59−0.69 (2H, m), 1.20−1.38 (1H, m), 1.52 (3H, d, J = 6.8 Hz),
1.80−1.98 (2H, m), 2.51−2.88 (2H, m), 3.53 (2H, t, J = 5.9 Hz), 3.88
(2H, d, J = 7.0 Hz), 4.57 (1H, quin, J = 6.8 Hz), 4.72 (1H, brs), 5.06
(2H, s), 5.77 (1H, dd, J = 8.1 Hz, 1.6 Hz), 6.85−7.11 (3H, m), 7.24−
7.27 (1H, m), 8.53 (1H, brs). 13C NMR (100 Mz, CDCl3) δ 3.3, 10.2,
23.7, 24.0, 50.3, 53.2, 66.9, 74.4, 76.5, 103.2, 113.7 (d, J = 6.6 Hz),
116.3 (dd, J = 19.0, 5.0 Hz), 118.7 (t, J = 4.1 Hz), 139.1, 143.3 (d, J =
11.6 Hz), 147.1 (d, J = 10.8 Hz), 151.1, 152.1 (d, J = 246.5 Hz), 163.6.
Anal. Calcd for C20H26FN3O6S: C, 52.74; H, 5.75; N, 9.23. Found: C,
no. nonhydrogen atoms
3427
no. protein/inhibiter/water/Mg atoms
average B-factor (Å2) for all atoms
2922/78/421/6
17.21
average B-factor (Å2) for
16.26/14.65/24.24/25.67
protein/inhibitor/water/Mg
a
Rsym = ∑∑j |Ij − ⟨I⟩|/∑⟨I⟩, where Ij is the recorded intensity of the
jth reflection and ⟨I⟩ is the average intensity over multiple recordings.
b
Rcryst = ∑h||Fo(h)| − |Fc(h)||/∑h|Fo(h)|, where Fo(h) and Fc(h) are
52.56; H, 5.48; N, 9.23. [α]25 + 13.9 (c 1.00, CHCl3).
c
D
observed and calculated structure factors. Rfree values are calculated
Cloning, Expression and Purification of Recombinant
Human dUTPase. The cDNA of human dUTPase was subcloned
into the expression vector pET19b. The construct was then
transformed into E. coli BL21(DE3) cells (Novagen) in Luria broth
at 37 °C. Protein expression was induced with 0.01 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) at an optical density of 0.6 at 595 nm.
The cell pellet was resuspended in ice-cold lysis buffer containing 50
mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM dithiothreitol
(DTT). After sonication, the disrupted debris was removed by
centrifugation. The supernatant was applied to Ni-NTA affinity gels,
and the 6 × His-Tag was removed by digestion with enterokinase in 50
mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM DTT for 12 h. The
protein solutions used for crystallization were gelfiltrated in a buffer
containing 50 mM Tris-HCl pH 7.5, 50 mM NaCl, and 1 mM DTT
on a preparative grade Superdex 75 column (GE Healthcare Life
Sciences).
Crystallization and Data Collection. The protein solution
prepared for cocrystallization contained 20 mg/mL dUTPase, 0.1
mM compound 9, and 10 mM MgCl2. Crystallization experiments
were carried out by the hanging-drop vapor diffusion method. Crystals
were obtained at 25 °C from the reservoir solution consisting of 15%−
20% PEG 4000, 50 mM Tris-HCl pH 7.5, 15% (v/v) glycerol, and 50
mM MgCl2. X-ray data were collected from a cryo-cooled (100 K)
crystal at Pharmaceutical Industry Beamline (BL32B2) (Harima,
Japan) using a RAXIS V (Rigaku Corporation) imaging plate (IP) area
for a randomly selected 5% of the data which were omitted the
refinement. Root mean square deviation from ideal/target geo-
metries.
d
dUTPase Inhibition Assay. In vitro dUTPase inhibition assays
were conducted by measuring the production of [5-3H]dUMP from
[5-3H]dUTP. Briefly, 0.2 mL of a solution containing 0.02 mL of 1 μM
dUTP (including 588 Bq/mL [5-3H]dUTP), 0.05 mL of 0.2 M Tris
buffer solution (pH 7.4), 0.05 mL of 16 mM magnesium chloride, 0.02
mL of 20 mM 2-mercaptoethanol, 0.02 mL of a 1% aqueous solution
of fetal bovine serum-derived albumin, 0.02 mL of varying
concentrations of test compound solutions or pure water as a control,
and 0.02 mL of a solution of human dUTPase was reacted at 37 °C for
15 min. After the reaction, the solution was immediately heated at 100
°C for 1 min to terminate the reaction and then centrifuged at 15000
rpm for 2 min. An aliquot (150 μL) of the supernatant thus obtained
by centrifugation was analyzed using an Atlantis C18 column
(manufactured by Waters Corp., 4.6 mm × 250 mm) and a high-
performance liquid chromatograph (manufactured by Shimadzu Corp.,
Prominence). The inhibition rate of the compound was determined
according to the formula shown below. IC50 (μM), the concentration
2978
dx.doi.org/10.1021/jm201628y | J. Med. Chem. 2012, 55, 2970−2980

Journal of Medicinal Chemistry
Article
of inhibitor yielding 50% inhibition rate, was obtained from
concentration−inhibition rate curve.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 81-29-865-4527. Fax: 81-29-865-2170. E-mail: m-
fukuoka@taiho.co.jp.
3
Inhibitory rate(%) = 1 − [(amount of[5‐ H]
dUMP in presence of test solution(dpm))
Notes
3
/(amount of[5‐ H]dUMP as control(dpm))] × 100
The authors declare no competing financial interest.
In Vitro Cell Inhibition Assays. HeLa S3 (human cervix
adenocarcinoma) cells were cultured in RPMI1640 supplemented
with 10% FBS. Exponentially growing cells were seeded in 96-well
plates (1500 cells/0.18 mL) and incubated at 37 °C in a humidified
5% CO2 atmosphere for 24 h. Vehicle-control (DMSO) and test
compounds (1−100 μM) were added to the plates at a volume of 20
μL per well, and the plates were incubated for 72 h. Cell proliferation
was determined by the Crystal Violet assay. Optical density at 540 nm
(OD540) was measured by plate reader. Then, we calculated T/C (%)
value, which was the ratio of OD540 with drug treatment to without
drug {T/C (%) = (OD540 of treated well/OD540 of nontreated well) ×
100}. The IC50 (μM) for the cytotoxicity of the test compound was
the concentration yielding 50% T/C value, which was calculated from
concentration−T/C (%) curve.
In Vitro Cell Inhibition Assays in Combination with FdUrd.
HeLa S3 cells were seeded in 96-well plates (1500 cells/0.18 mL) and
incubated at 37 °C in a humidified 5% CO2 atmosphere as described
above. After 24 h, vehicle-control (DMSO) and test compounds (1−
100 μM) in combination with FdUrd (1 μM) were added to the plates
at a volume of 20 μL per well and incubated for 24 h. Then, thymidine
(30 μM) was added to the plates at a volume of 10 μL per well and
incubated for 48 h. Cell proliferation was determined by the Crystal
Violet assay as mentioned above. The EC50 value was calculated from
the concentration−T/C (%) curve as a concentration of each
compound that reduces the T/C (%) value of FdUrd (1 μM) against
HeLa S3 cells to half in 24 h.
ACKNOWLEDGMENTS
■
We thank Dr. Tadafumi Terada and Dr. Kazuharu Noguchi
(Taiho Pharmaceutical Co. Ltd.) for valuable comments and
Satoko Tanaka for technical assistance. We are grateful to the
staffs of The Pharmaceutical Industry Beamline (BL32B2) for
collecting the X-ray crystallography diffraction data and the staff
of Tsukuba Research Center of Taiho Pharmaceutical Co., Ltd.
ABBREVIATIONS USED
■
dUTPase, deoxyuridine triphosphatase; dUTP, 2′-deoxyuridine
5′-triphosphate; dTTP, thymidine 5′-triphosphate; dUMP, 2′-
deoxyuridine 5′-monophosphate; TS, thymidylate synthase; 5-
FU, 5-fluorouracil; FdUrd, 5-fluoro-2′-deoxyuridine; FdUTP, 5-
fluoro-2′-deoxyuridine 5′-triphosphate; SAR, structure−activity
relationship; MOM, methoxymethyl
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and characterization data for compounds
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Accession Codes
PDB code of compound 9 with human dUTPase: 3ARN.
2979
dx.doi.org/10.1021/jm201628y | J. Med. Chem. 2012, 55, 2970−2980

Journal of Medicinal Chemistry
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