Discovery of highly potent human deoxyuridine triphosphatase inhibitors based on the conformation restriction strategy

Article abstract of DOI:10.1021/jm300416h

Human deoxyuridine triphosphatase (dUTPase) inhibition is a promising approach to enhance the efficacy of thymidylate synthase (TS) inhibitor based chemotherapy. In this study, we describe the discovery of a novel class of human dUTPase inhibitors based on the conformation restriction strategy. On the basis of the X-ray cocrystal structure of dUTPase and its inhibitor compound 7, we designed and synthesized two conformation restricted analogues, i.e., compounds 8 and 9. These compounds exhibited increased in vitro potency compared with the parent compound 7. Further structure-activity relationship (SAR) studies identified a compound 43 with the highest in vitro potency (IC₅₀ = 39 nM, EC₅₀ = 66 nM). Furthermore, compound 43 had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1 breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage.

Full text of DOI:10.1021/jm300416h

Article
pubs.acs.org/jmc
Discovery of Highly Potent Human Deoxyuridine Triphosphatase
Inhibitors Based on the Conformation Restriction Strategy
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: Human deoxyuridine triphosphatase (dUT-
Pase) inhibition is a promising approach to enhance the
efficacy of thymidylate synthase (TS) inhibitor based chemo-
therapy. In this study, we describe the discovery of a novel
class of human dUTPase inhibitors based on the conformation
restriction strategy. On the basis of the X-ray cocrystal
structure of dUTPase and its inhibitor compound 7, we
designed and synthesized two conformation restricted
analogues, i.e., compounds 8 and 9. These compounds
exhibited increased in vitro potency compared with the parent compound 7. Further structure−activity relationship (SAR)
studies identified a compound 43 with the highest in vitro potency (IC50 = 39 nM, EC50 = 66 nM). Furthermore, compound 43
had a favorable oral PK profile and exhibited potent antitumor activity in combination with 5-fluorouracil (5-FU) in the MX-1
breast cancer xenograft model. These results suggested that a dUTPase inhibitor may have potential for clinical usage.
Since dUTPase can also hydrolyze FdUTP,18 increased
dUTPase activity induces resistance to 5-FU and its analogues.
INTRODUCTION
Thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-
■
Genetically induced expression of dUTPase confers resistance
FU), its analogues, and folate analogues are widely used in
clinical chemotherapy.1−4 However, acquired and intrinsic
to 5-fluoro-2′-deoxyuridine (FdUrd) in human tumor
cells,19−21 and small interfering RNA (siRNA) silencing of
resistance still remains a limitation to the clinical use of TS
inhibitors.5 Therefore, novel therapeutic strategies to improve
dUTPase in SW620 and MCF-7 cells significantly enhances the
growth inhibition activity of FdUrd by perturbing (F)dUTP/
the efficacy of TS inhibitor based chemotherapy are urgently
required.
TS inhibitors lead to rapid depletion of the cellular 2′-
deoxyuridine 5′-triphosphate (dTTP) pool, inducing thymine-
less death. In addition, TS inhibition results in the
accumulation of 2′-deoxyuridine 5′-monophosphate (dUMP),
dTTP levels.12 On the other hand, a high level of dUTPase
expression is associated with resistance to not only 5-FU and its
analogues but folate analogues.22
Several clinical studies show that nuclear dUTPase levels
correlate with poor survival outcomes in colorectal cancer23 and
hepatocelluar carcinoma (HCC).24 These findings demonstrate
which may subsequently lead to increased levels of 2′-
deoxyuridine 5′-triphosphate (dUTP).6,7 dUTP can be
that human dUTPase inhibitors may be useful as clinical
medicines that enhance anticancer activity of TS inhibitors.
Thus, efforts to obtain efficient dUTPase inhibitors have
misincorporated into DNA in place of dTTP during replication
and repair by DNA polymerase.8 High cellular dUTP/dTTP
resulted in the development of several compounds (Figure
ratios induce futile cycles of misincorporation, which eventually
1).25−34 These inhibitors had low Ki value against Escherichia
lead to DNA strand breaks and cell death. This is one of the key
antitumor mechanisms of TS inhibitors.9−13
coli or Plasmodium faliciparum dUTPase and were effectively
used for solving the X-ray structure of the enzyme. However,
these inhibitors appear to be ineffective for chemotherapeutic
usage owing to high polarity or insufficient human dUTPase
dUTPase catalyzes the hydrolysis of dUTP to dUMP and has
two functions in nucleotide metabolism: it decreases the
number of intracellular dUTP pools to prevent misincorpora-
tion of uracil instead of thymine into DNA, and it supplies the
inhibition activity.
substrate dUMP for TS, which is responsible for an important
de novo nucleotide metabolism pathway in DNA syn-
Received: March 25, 2012
Published: May 18, 2012
thesis.14−17
© 2012 American Chemical Society
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Figure 1. Structure of previously reported human dUTPase inhibitors.
a
Scheme 1. Synthesis of Compound 8
a
Reagents and conditions: (a) PhSO2Cl, Et3N, CH2Cl2, room temp, 2 h; (b) (1) NBS, AIBN, CCl4, reflux, 0.5 h, (2) (TMS)2uracil, I2, DCE, reflux, 3
h.
We previously reported the development of highly potent
dUTPase inhibitors.35−37 Among them, compound 6 not only
significantly enhances the growth inhibition activity of FdUrd
against HeLa S3 cells in vitro but also shows robust antitumor
CHEMISTRY
■
The target compounds in this study were synthesized as
depicted in Schemes 1−6. Compound 8 was synthesized by the
method shown in Scheme 1. Briefly, the commercially available
amine 10 was treated with benzenesulfonyl chloride to give
compound 11. After treatment of compound 11 with N-
bromosuccinimide (NBS) in the presence of 2,2′-azobisisobu-
tyronitrile (AIBN), the obtained benzyl bromide was coupled
with (TMS)2uracil to give 8.
The synthesis of compound 9 is shown in Scheme 2. Moffat
oxidation of commercially available compound 12 and
subsequent Horner−Emmons olefination afforded compound
13. After reduction of the olefin moiety of compound 13 using
palladium on carbon under hydrogen atmosphere, treatment of
activity in combination with 5-FU in vivo.
In this paper, we have described a new class of dUTPase
inhibitors. We designed and synthesized conformation
restricted dUTPase inhibitors based on the reported X-ray
cocrystal structures (PDB code 3ARN36) and evaluated the
inhibition activity against human dUTPase and in vivo
antitumor activity in combination with 5-FU.
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a
Scheme 2. Synthesis of Compound 9
a
Reagents and conditions: (a) (1) TFA, pyridine, EDC, DMSO, toluene, room temp, 30 min, (2) diethyl phosphonoacetate, NaH, THF, 75 °C, 1 h;
(b) H2, Pd/C, AcOEt, room temp, 4 h; (c) 2.0 M LiBH4 in THF, THF, room temp, 16 h; (d) ethyl (triphenylphosphoranylidene)acetate, toluene,
reflux, 18 h; (e) 1.0 M DIBAL-H in THF, THF, −78 °C, 2 h ; (f) (1) 4 N HCl/dioxane, room temp, 50 min, (2) PhSO2Cl, Et3N, MgO, THF, H2O,
room temp, 2 h; (g) (1) CBr4, PPh3, THF, room temp,1 h, (2) (TMS)2uracil, I2, DCE, reflux, 3 h.
a
Scheme 3. Synthesis of Compound 20 and Its Derivatives 23−25
a
Reagents and conditions: (a) 3-MeOPhSO2CI, Et3N, CH2Cl2, room temp, 2 h; (b) (1) NBS, AIBN, CCl4, reflux, 0.5 h, (2) (TMS)2uracil, I2, DCE,
reflux, 3 h; (c) 3-BzOPhSO2Cl, Et3N, CH2Cl2, room temp, 2 h; (d) (1) 40% MeNH2 in MeOH, room temp, 20 min, (2) RX, K2CO3, DMF, 90 °C,
16 h.
the resulting compound 14 with lithium borohydride (LiBH4)
afforded compound 15. The Wittig reaction of compound 15
with ethyl (triphenylphosphoranylidene)acetate, followed by
reduction of the ester group of the resulting compound 16,
afforded alcohol 17. Removal of the Boc group from compound
17 and treatment of the resulting amine with benzenesulfonyl
chloride afforded compound 18. After bromination of
compound 18 with carbon tetrabromide and triphenylphos-
phine, coupling of the resulting brominated compound with
(TMS)2uracil gave compound 9.
Compound 20 and its derivatives 23−25 were synthesized in
two or three steps, as shown in Scheme 3. These compounds
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a
Scheme 4. Synthesis of Compound 27 and Its Derivatives 30−32
a
Reagents and conditions: (a) (1) 4 N HCl/dioxane, room temp, 50 min, (2) 3-MeOPhSO2Cl, Et3N, MgO, THF, H2O, room temp, 2 h; (b) (1)
CBr4, PPh3, THF, room temp, 1 h, (2) (TMS)2uracil, I2, DCE, reflux, 3 h; (c) (1) 4 N HCI/dioxane, room temp, 50 min, (2) 3-BzOPhSO2Cl, Et3N,
MgO, THF, H2O, room temp, 2 h; (d) (1) 40% MeNH2 in MeOH, room temp, 20 min, (2) RX, K2CO3, DMF, 90 °C, 16 h.
a
Scheme 5. Synthesis of Compounds 38−40
a
Reagents and conditions: (a) 4-(bromomethyl)benzenesulfonyl chloride, Et3N, CH2Cl2, 0 °C, 1 h; (b) (TMS)2uracil, I2, DCE, reflux, 5 h.
were synthesized in a manner similar to that shown in Scheme
1.
the production of compound 43. Compounds 44 and 45 were
synthesized in a similar manner.
Compound 27 and its derivatives 30−32 were synthesized
from intermediate 17, as shown in Scheme 4. These
compounds were synthesized in a manner similar to that
shown in Scheme 2.
The synthesis of compounds 38−40 is shown in Scheme 5.
Procedures for the synthesis of chiral amines 36b−c are
detailed in the Supporting Information. Briefly, chiral amine
36a was treated with 4-(bromomethyl)benzenesulfonyl chlor-
ide, and the obtained benzyl bromide was coupling with
(TMS)2uracil to give compounds 38−40. Compounds 39 and
40 were synthesized in a similar manner.
In Scheme 6, synthesis of compounds 43−45 is detailed.
Procedures for the synthesis of intermediates 41a−c are
detailed in the Supporting Information. The Mitunobu reaction
of 41a with N-3-benzoyluracil provided compound 42a.
Deprotection of the benzoyl and MOM group of 42a led to
RESULTS AND DISCUSSION
■
Design of the Conformation Restricted Analogues of
Compound 7. The conformational restriction of a flexible
ligand has often been a promising strategy to increase the
potency for a given target protein in drug development because
the entropic loss associated with the ligand adopting a preferred
binding conformation can be minimized.38−41 We previously
reported the X-ray cocrystal structure of human dUTPase with
compound 7 (PDB code 3ARN36) that has potent inhibition
activity against human dUTPase (IC50 = 3.9 μM) (Figure 2).
From this X-ray cocrystal structure, we confirmed that
compound 7 adopts a folded conformation and is stacked
between the uracil ring and its terminal phenyl ring. The central
flexible linker moiety, i.e., -O-CH2-CH2-CH2-C(Me)2-, aided
formation of the bent structure. We previously noted that the
terminal phenyl ring of 7 occupies the same region as the
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a
Scheme 6. Synthesis of Compounds 43−45
a
Reagents and conditions:(a) N-3-benzoyluracil, PPh3, DEAD, THF, room temp, 2 h; (b) (1) 40% MeNH2 in MeOH, room temp, 1 h, (2) 4 N
HCI/dioxane, dioxane, room temp, 4 h.
Figure 3. Design of the conformation restricted analogues as human
dUTPase inhibitors.
Figure 2. X-ray cocrystal structure of compound 7 (cyan) with human
dUTPase (PDB code 3ARN36).
Phe158 residue of the enzyme.35 Mol and co-workers reported
that capping each of the enzyme’s active sites by the flexible C-
terminal tail is critical for human dUTPase activity.42 On the
basis of this structural information, we proposed that this
flexible linker moiety can be conformationally restricted into
the bioactive conformation by replacing it with a p-phenylene
ring or a trans-alkenylene group, in which the positions of the
uracil and the terminal phenyl ring should be similar to those in
compound 7. On the basis of this hypothesis, we designed
conformation restricted analogues 8 and 9 that have a rigid p-
phenylene ring or a trans-alkenylene group (Figure 3).
We performed a molecular modeling study of conformation
restricted analogues 8 and 9 based on the crystallographic pose
of compound 7 (Figure 4A and Figure 4B). In modeling
analysis, the aromatic stacking interaction between the uracil
moiety and the terminal phenyl rings was conserved (Figure
4C).
FdUrd against Hela S3 cells compared with the parent
compound 7 as expected (Table 1).
Further Improvement of Compounds 8 and 9. Next, to
identify further potent inhibitors, we investigated the
substitution effect at the terminal phenyl ring of compounds
8 and 9. In our previous work describing the structure−activity
relationship (SAR) of compound 7, we discovered that
introduction of a hydrophobic alkoxy group at the meta-
position of the terminal phenyl ring of 7 is highly conducive to
human dUTPase inhibitory activity36 (approximately 2 orders).
Accordingly, using the same strategy, we designed and
synthesized compounds 20, 23−25, 27, and 30−32 having
an alkoxy group at the meta-position of the terminal phenyl
ring.
Introduction of bulky lipophilic groups such as cyclo-
propylmethoxy, cyclopentyloxy, and 2,2-difluoroethoxy group
into the meta-position of the terminal phenyl ring dramatically
increased both enzyme inhibition activity and enhanced the
growth inhibition activity of FdUrd against Hela S3 cells as
expected (Table 2). It is suggested that the meta-subsutituent
on their terminal phenyl ring contributes important hydro-
phobic interaction with the enzyme.
We assayed the enzyme inhibition activity of compounds 8
and 9 and their in vitro growth inhibition in combination with
FdUrd against Hela S3 cells. Conformation restricted analogues
8 and 9 demonstrated an almost 2-fold increment in enzyme
inhibition activity and enhanced growth inhibition activity of
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Figure 4. (A) Proposed model for compound 8 (green) binding to the active site pocket. (B) Proposed model for compound 9 (orange) binding to
the active site pocket. (C) Overlay of compounds 7 (cyan), 8, and 9 complexed to human dUTPase.
respectively. Unexpectedly, compounds 38−40 were approx-
imately 4-fold to 13-fold less active than compounds 23−25
(Table 3). The terminal phenyl ring of the reverse sulfonamide
compounds 38−40, which is essential for efficient dUTPase
inhibition activity, may not have been located at a suitable
position in the active site of the enzyme because of the rigidity
of compounds. On the other hand, compounds 43−45 retained
enzyme inhibition activity compared with parent compounds
30−32 (Table 4). These compounds had favorable phys-
icochemical properties (both compounds were solidified).
Among them, compound 43 has the greatest in vitro potency
(IC50 = 39 nM, EC50 = 66 nM). On the other hand, compound
43 alone had little effect on cell growth (IC50 = 38.2 μM).
Therefore, we selected compound 43 for further study.
In Vivo Pharmacokinetics (PK) Profile of Compound
43. Compound 43 was evaluated for preliminary pharmacoki-
netics profile in mice. These results are summarized in Table 5.
Compound 43 was well absorbed into mice plasma after oral
administration. These PK data showed that compound 43 has a
desirable profile for in vivo study.
Table 1. Bilological Activity of Compounds 7, 8, and 9
ac
,
bc
,
compd
IC50 (μM)
EC50 (μM)
7
8
9
3.90 0.63
1.80 0.20
1.60 0.05
5.10 0.31
2.60 0.07
2.60 0.36
a
Concentration of each compound required to inhibit 50% of the
b
amount of [5-3H]dUMP produced by human dUTPase. Concentra-
tion of each compound that reduces the T/C (%) of FdUrd (1 μM)
c
against HeLa S3 to half in 24 h. IC50 and EC50 data represent the
average of three independent assays, and errors are given as standard
deviations.
Although these compounds have favorable biological activity,
none of these could be solidified, making them difficult for
further development. As described in our previous paper, the
conversion of the linker part of compound 33 to an alternative
linker resolved this physicochemical issue without disrupting
enzyme inhibition activity. Furthermore, introduction of an
(R)-monomethyl group at the benzyl position of compound 34
significantly contributed to its increased enzyme inhibition
activity (Figure 5). This result suggested that the substituent of
the (R)-tail is more likely to maintain close contact with the
enzyme.
Antitumor Activity of Compound 43 against the MX-1
Breast Cancer Xenograft Model in Mice. We next
evaluated the antitumor activity of compound 43 in
combination with 5-FU in vivo. Compound 43 was
administered orally with a continuous infusion of 5-FU into
the MX-1 xenograft model in mice. Compound 43 dramatically
increased the efficacy of 5-FU (inhibition rate IR = 68%)
On the basis of the SAR in our previous study, we designed
and synthesized compounds 38−40 and 43−45 with (R)-
methylated benzyl groups, as shown in Schemes 5 and 6,
Table 2. Bilological Activity for Meta-Substituted Analogues of Compounds 8 and 9
ac
,
bc
,
compd
R1
R2
IC50 (μM)
EC50 (μM)
20
23
24
25
27
30
31
32
CH3
1.27 0.07
3.13 0.44
0.14 0.02
0.12 0.005
0.10 0.03
0.45 0.04
0.071 0.005
0.20 0.005
0.049 0.002
cyclopropylmethyl
cyclopentyl
0.073 0.012
0.031 0.002
0.073 0.009
0.34 0.02
2,2-difluoroethyl
CH3
cyclopropylmethyl
cyclopentyl
0.028 0.004
0.033 0.003
0.026 0.001
2,2-difluoroethyl
a
b
Concentration of each compound required to inhibit 50% of the amount of [5-3H]dUMP produced by human dUTPase. Concentration of each
c
compound that reduces the T/C (%) of FdUrd (1 μM) against HeLa S3 to half in 24 h. IC50 and EC50 data represent the average of three
independent assays, and errors are given as standard deviations.
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Figure 5. (A) Replacement of the linker moiety conferred favorable physicochemical properties in our chemical series. (B) (R)-Monomethylated
compound showed increased inhibition enzyme activity.
Table 3. Biological Data of Compounds 38−40
Table 5. Pharmacokinetic Profile of 43 after Oral
Administration (po)
a
compd
AUC0−t (μM·h)
Cmax (μM)
T1/2 (h)
Tmax (h)
43
19.5
19.5
0.89
0.5
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.
ac
,
bc
,
compd
R
IC50 (μM)
EC50 (μM)
38
39
40
cyclopropylmethyl
cyclopentyl
0.82 0.02
0.43 0.06
0.41 0.05
0.93 0.09
0.61 0.02
0.61 0.03
and 0.5% HPMC) treated group (Table 6, Figure 6A, Figure
6B). On the other hand, compound 43 exhibited no antitumor
activity when administered alone (Table 6). These results
indicate that this conformation restricted dUTPase inhibitor
may provide high efficacy for treating cancer patients when
used in combination with TS inhibitor.
2,2-difluoroethyl
a
Concentration of each compound required to inhibit 50% of the
b
amount of [5-3H]dUMP produced by human dUTPase. Concentra-
tion of each compound that reduces the T/C (%) of FdUrd (1 μM)
against HeLa S3 to half in 24 h. IC50 and EC50 data represent the
c
average of three independent assays, and errors are given as standard
deviations.
CONCLUSION
■
We have described the discovery of conformation restricted
human dUTPase inhibitors. On the basis of the X-ray cocrystal
structure of lead compound 7 and human dUTPase, we
designed and synthesized compounds 8 and 9 having rigid
linkers such as a p-phenylene ring or a trans-alkenylene group
instead of the flexible linker. Modeling study of these
compounds suggested that the terminal phenyl ring can be
located at the same region as that of compound 7. In biological
assay, these compounds show more potent activity than the
parent compound 7, as expected. Further elaboration of the
SAR around compounds 8 and 9 gave a highly potent human
dUTPase inhibitor 43. Compound 43 is one of the most potent
human dUTPase inhibitors known so far, and it shows excellent
antitumor activity in combination with 5-FU in the MX-1
xenograft model. Development of this inhibitor may validate
dUTPase inhibitors as a potential new regiment in the
combination chemotherapy with TS inhibitors for treating
human cancer.
Table 4. Biological Data of Componds 43−45
ac
,
bc
,
compd
R
IC50 (μM)
EC50 (μM)
43
44
45
cyclopropylmethyl
cyclopentyl
0.039 0.003
0.041 0.003
0.082 0.007
0.066 0.002
0.15 0.01
2,2-difluoroethyl
0.096 0.009
a
Concentration of each compound required to inhibit 50% of the
b
amount of [5-3H]dUMP produced by human dUTPase. Concentra-
tion of each compound that reduces the T/C (%) of FdUrd (1 μM)
c
against HeLa S3 to half in 24 h. IC50 and EC50 data represent the
average of three independent assays, and errors are given as standard
deviations.
EXPERIMENTAL SECTION
Chemistry. All commercially available reagents and solvents were
used without further purification unless otherwise specified. All
■
without causing significant weight loss or toxicity compared
with the vehicle (2.5% DMA, 2.5% Tween 80, 10% Cremophor,
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Table 6. Antitumor Activity of Compound 43 in Combination with 5-FU in the MX-1 Xenograft Model
a
b
c
drug
dose (mg kg−1 day−1
)
treatment
TV, mean SD (mm3)
RTV, mean SD
IR (%)
control
5-FU
2048 693.7
1645 322.6
2819 776.3
746 301.1
11.4 3.74
9.13 1.35
14.7 2.54
15
CI
20
−29
68
43
300
po
5-FU/43
15/300
CI/po
3.66 1.30**,##
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). ∗∗: p < 0.01, Dunnett's test compared with the control group. ##: p < 0.01, Student's t-test compared with the 5-FU group. Inhibition rate
(IR) of tumor growth on day 15 on the basis of RTV was calculated according to the following formula: IR (%) = [1 − (mean RTV of the treated
group)/(mean RTV of the control group)] × 100.
N-(2-p-Tolylpropan-2-yl)benzenesulfonamide (11). To a
stirred solution of 10 (280 mg, 1.88 mmol) in CH2Cl2 (3.5 mL)
were added Et3N (250 μL, 1.79 mmol) and benzenesulfonyl chloride
(205 μL, 1.60 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, filtered, 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 (121 mg, 0.42 mmol, 22%) as a colorless oil.
1H NMR (270 MHz, CDCl3) δ 1.62 (6H, s), 2.34 (3H, s), 7.03−7.19
(8H, m), 7.31−7.34 (1H, m).
N-(2-(4-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-
phenyl)propan-2-yl)benzenesulfonamide (8). A mixture of 11
(121 mg, 0.42 mmol), N-bromosuccinimide (78.0 mg, 0.44 mmol),
and AIBN (2.5 mg, 0.015 mmol) in CCl4 (3.0 mL) was heated to
reflux at 90 °C for 0.5 h. The mixture was cooled to room temperature,
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 to afford a crude brominated
product.
To a suspension of 2,4-bis(trimethylsilyloxy)pyrimidine (150 mg,
0.58 mmol) in 1,2-dichloroethane (2.0 mL) was added a solution of
the crude brominated product in 1,2-dichloroethane (1.0 mL) at room
temperature. The resulting mixture was heated to reflux at 95 °C for 3
h. After cooling to room temperature, the mixture was poured into
Figure 6. (A, top) Antitumor activity of 43 in combination with 5-FU
in the MX-1 xenograft model. Relative tumor volume (RTV) is
expressed as the mean SD of at least three independent experiments.
(B, bottom) Body weight change (%) is expressed as the mean SD.
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 (39.1 mg, 0.098 mmol, 23% from 11) as a colorless gum.
1H NMR (270 MHz, CDCl3) δ 1.60 (6H, s), 4.84 (2H, s), 5.53 (1H,
brs), 5.72 (1H, d, J = 7.9 Hz), 7.09−7.19 (3H, m), 7.27−7.38 (4H, m),
7.42−7.52 (1H, m), 7.65−7.69 (2H, m), 8.45 (1H, brs). 13C NMR
(100 MHz, DMSO-d6) δ 29.7, 49.8, 57.2, 101.3, 125.8, 126.1, 126.8,
128.5, 131.4, 134.8, 143.2, 145.3, 145.7, 151.0, 163.7. Anal. Calcd for
C20H21N3O4S: C, 60.13; H, 5.3; N, 10.52. Found: C, 60.00; H, 5.35;
N, 10.33.
(E)-Ethyl 4-(tert-Butoxycarbonylamino)-4-methylpent-2-
enoate (13). To a mixture of tert-butyl 1-hydroxy-2-methylpropan-
2-ylcarbamate 12 (38.5 g, 203 mmol), trifluoroacetic acid (11.3 mL,
152 mmol), pyridine (24.6 mL, 305 mm) in DMSO (160 mL) and
toluene (160 mL) was added EDC (116.8 g, 609 mmol) at room
temperature. The resulting mixture was stirred at this temperature for
30 min and then poured into H2O. The aqueous layer was extracted
with EtOAc two times. The combined organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated under reduced
pressure to give a crude aldehyde.
To a stirred suspension of NaH (55% in oil, 10.7 g, 245 mmol) in
THF (150 mL) was slowly added ethyl diethylphosphonoacetate (49.6
mL, 248 mmol) at 0 °C, and the resulting mixture was stirred at room
temperature for 1 h. Then a solution of the crude aldehyde in THF
(100 mL) was added to the mixture, and the whole was stirred at 75
°C for 1 h. The reaction mixture was quenched by the addition of
saturated aqueous NH4Cl, and the aqueous layer was extracted with
EtOAc two times. The combined organic layer was washed with brine,
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 are given in parts per million
(ppm, δ) with tetramethylsilane as the internal standard, and coupling
constants (J) are 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 were recorded either on a
JEOL JMS-700 (FAB) or a on Waters micromass Q-Tof-2 (TOF)
instrument. 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% to 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 using a Shim-pack XR-ODS column (3.0 mm × 50
mm, 2.2 μm) with an 8 min linear gradient from 10% to 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 is denoted as tR.
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Journal of Medicinal Chemistry
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dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel,
eluting with hexane/EtOAc = 7/3 to afford the title compound (47.4
g, mmol, 91%, from 12) as a colorless oil. 1H NMR (270 MHz,
CDCl3) δ 1.21−1.29 (3H, m), 1.41 (6H, s), 1.43 (9H, s), 4.11−4.23
(2H, m), 4.68 (1H, brs), 5.84 (1H, d, J = 15.8 Hz), 7.00 (1H, d, J =
15.8 Hz).
Ethyl 4-(tert-Butoxycarbonylamino)-4-methylpentanoate
(14). A mixture of 13 (47.4 g, 184 mmol) and 10% palladium on
activated carbon (3.0 g) in EtOAc (200 mL) was stirred under a
hydrogen atmosphere (balloon) at room temperature for 4 h. The
reaction mixture was filtered with a pad of Celite, and the pad was
washed with EtOAc. The filtrate was concentrated under reduced
pressure to give the title compound (40.0 g, 154 mmol, 84%) as a
colorless oil. 1H NMR (270 MHz, CDCl3) δ 1.24 (3H, t, J = 7.1 Hz),
1.26 (6H, s), 1.46 (9H, s), 1.96−2.05 (2H, m), 2.28−2.34 (2H, m),
4.12 (2H, q, J = 7.1 Hz), 4.43 (1H, brs). HRMS (FAB) calcd for
C13H26NO4 [M + H]+ 260.1862, found 260.1857.
residue was partitioned between EtOAc and H2O. The 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 hexane/EtOAc = 1/1 to
afford the title compound (128 mg, 0.57 mmol, 23% from 17) as a
1
colorless oil. H NMR (270 MHz, CDCl3) δ 1.20 (6H, s), 1.59−1.64
(2H, m), 2.01−2.17 (2H, m), 4.05−4.09 (2H, m), 4.52 (1H, brs),
5.61−5.64 (2H, m), 7.50−7.55 (3H, m), 7.86−7.90 (2H, m). HRMS
(FAB) calcd for C14H20NO3S [M − H]− 282.1164, found 282.1192.
(E)-N-(7-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-meth-
ylhept-5-en-2-yl)benzenesulfonamide (9). To a solution of 18
(160 mg, 0.56 mmol) in THF (2.5 mL) were added PPh3 (223 mg,
0.85 mmol) and CBr4 (280 mg, 0.84 mmol) at room temperature, and
the resulting mixture was stirred for 1 h. The mixture was concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel, eluting with hexane/EtOAc = 3/1) to
give a crude brominated compound.
To a suspension of 2,4-bis(trimethylsilyloxy)pyrimidine (79.0 mg,
0.31 mmol) in 1,2-dichloroethane (1.0 mL) was added a solution of
the crude brominated compound in 1,2-dichloroethane (1.0 mL) and
iodine (catalyst) at room temperature. The resulting mixture was
heated to reflux at 95 °C for 3 h. After cooling to room temperature,
the mixture was poured into H2O/saturated aqueous Na2S2O3 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 (32.0 mg, 0.085 mmol, 15% from 18) as a white foam. 1H
NMR (270 MHz, CDCl3) δ 1.17 (6H,s), 1.61−1.66 (2H, m), 2.06−
2.17 (2H, m), 4.27 (2H, d, J = 6.2 Hz), 4.52 (1H, brs), 5.41−5.51 (1H,
m), 5.64−5.71 (1H, m), 5.71 (1H, dd, J = 8.1, 2.7 Hz), 7.16 (1H, d, J =
8.1 Hz), 7.46−7.55 (3H, m), 7.86−7.91 (2H, m), 8.33 (1H, brs). 13C
NMR (100 MHz, CDCl3) δ 26.8, 27.7, 41.6, 49.6, 56.9, 102.3, 123.3,
126.9, 128.9, 132.2, 136.1, 143.4, 143.9, 150.8, 163.8. Anal. Calcd for
C18H23N3O4S·0.3H2O: C, 56.47; H, 6.21; N, 10.98. Found: C, 56.76;
H, 6.08; N, 11.04.
tert-Butyl 5-Hydroxy-2,2-dimethylpyrrolidine-1-carboxylate
(15). To a solution of 14 (17.4 g, 67.0 mmol) in THF (200 mL) was
added LiBH4 (2.0 M, 55.4 mL, 111 mmol) at room temperature. The
reaction mixture was stirred at this temperature for 16 h. Then to the
mixture was added water dropwise at the same temperature. The
aqueous layer was 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 hexaneEtOAc = 3/7
to afford the title compound (5.90 g, 27.4 mmol, 41%) as a white solid.
1H NMR (270 MHz, DMSO-d6) δ 1.40 (6H, s), 1.47 (9H, s), 1.51−
1.59 (2H, m), 1.96−2.05 (2H, m), 5.17−5.21 (1H, m). HRMS (FAB)
calcd for C11H20NO3 [M − H]− 214.1443, found 214.1440.
(E)-Ethyl 6-(tert-Butoxycarbonylamino)-6-methylhept-2-
enoate (16). To a solution of 15 (940 mg, 4.37 mmol) in toluene
(20 mL) was added ethyl (triphenylphosphoranylidene)acetate (1.74
g, 5.0 mmol), and the resultant was heated to reflux at 110 °C for 18 h.
The mixture was concentrated under reduced pressure and the residue
was purified by column chromatography on silica gel, eluting with
hexane/EtOAc = 8/1 to afford the title compound (543 mg, 1.90
3-Methoxy-N-(2-p-tolylpropan-2-yl)benzenesulfonamide
(19). To a stirred solution of 10 (350 mg, 2.35 mmol) in CH2Cl2 (3.5
mL) were added Et3N (401 μL, 2.88 mmol) and 3-methoxybenze-
nesulfonyl chloride (398 μL, 2.53 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, filtered, 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 (179 mg, 0.56 mmol, 24%) as a white
solid. 1H NMR (270 MHz, CDCl3) δ 1.64 (6H, s), 2.28 (3H, s), 3.76
(3H, s), 4.85 (1H, brs), 6.95−7.00 (3H, m), 7.04−7.06 (1H, m),
7.14−7.17 (2H, m), 7.26−7.29 (2H, m). HRMS (FAB) calcd for
C17H20NO3S [M − H]− 318.1164, found 318.1153.
1
mmol, 43%) as a colorless oil. H NMR (270 MHz, CDCl3) δ 1.25−
1.31 (9H, m), 1.43 (9H, s), 1.78−1.85 (2H, m), 2.14−2.20 (2H, m),
3.38 (1H, brs), 4.17 (2H, q, J = 7.3 Hz), 5.79−5.86 (1H, m), 6.93−
7.02 (1H, m). HRMS (FAB) calcd for C15H28NO4 [M + H]+
286.2018, found 286.1998.
(E)-tert-Butyl 7-Hydroxy-2-methylhept-5-en-2-ylcarbamate
(17). To a stirred solution of 16 (530 mg, 1.86 mmol) in THF (10
mL) was slowly added DIBAL-H in THF (1.0 M, 9.30 mL, 9.30
mmol) at −78 °C. The reaction mixture was stirred at −78 °C for 2 h.
Brine and saturated aqueous Rochelle’s salt were added to the mixture
at −78 °C, and the resultant was stirred at room temperature for 16 h.
The aqueous layer was 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 hexane/
EtOAc = 3/1 to afford the title compound (452 mg, 1.86 mmol,
quantitative) as a colorless oil. 1H NMR (270 MHz, DMSO-d6) δ 1.13
(6H, s), 1.36 (9H, s), 1.58−1.62 (2H, m), 1.87−1.93 (2H, m), 3.84−
3.89 (2H, m), 4.53 (1H, t, J = 5.4 Hz), 5.41−5.59 (2H, m), 6.35 (1H,
brs). HRMS (FAB) calcd for C13H26NO3 [M + H]+ 244.1913, found
244.1933.
( E ) - N - ( 7 - H y d r o x y - 2 - m e t h y l h e p t - 5 - e n - 2 - y l ) -
benzenesulfonamide (18). A solution of 17 (486 mg, 2.00 mmol)
in 4 N HCl/dioxane (5.0 mL) was stirred at room temperature for 50
min. The mixture was concentrated under reduced pressure, and the
residue was coevaporated with toluene four times and dissolved in
THF (3.2 mL) and H2O (800 μL). To this mixture were added Et3N
(585 μL, 4.20 mmol), MgO (400 mg, 9.93 mmol), and
benzenesulfonyl chloride (293 μL, 2.30 mmol) at room temperature,
and the resultant was stirred at the same temperature for 2 h. The
mixture was filtered, and the filter cake was washed with EtOAc and
H2O. The filtrate was concentrated under reduced pressure, and the
N-(2-(4-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-
phenyl)propan-2-yl)-3-methoxybenzenesulfonamide (20). 20
was prepared from 19 (174 mg, 0.54 mmol) as described for the
1
preparation of 8, colorless gum (21 mg, 0.053 mmol, 9%). H NMR
(270 MHz, CDCl3) δ 1.63 (6H, s), 3.77 (3H, s), 4.84 (2H, s), 5.24
(1H, brs), 5.72 (1H, d, J = 7.8 Hz), 6.94−7.34 (9H, m), 8.82 (1H,
brs). 13C NMR (100 MHz, CD3OD) δ 30.5, 51.7, 56.0, 58.6, 102.6,
112.6, 119.1, 120.0, 127.5, 128.4, 130.7, 135.8, 145.3, 146.3, 147.1,
152.8, 160.8, 166.7. HRMS (TOF) calcd for C21H24N3O5S [M + H]+
430.1437, found 430.1443. HPLC purity: 96.8%, tR = 4.82 min
(method B).
3-(N-(2-p-Tolylpropan-2-yl)sulfamoyl)phenyl Benzoate (21).
To a stirred solution of 10 (1.49 g, 9.98 mmol) in CH2Cl2 (25 mL)
were added Et3N (1.46 mL, 10.5 mmol) and 3-(chlorosulfonyl)phenyl
benzoate (2.08 g, 7.01 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, filtered, and concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel, eluting with hexane/EtOAc = 4/1 to
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afford the title compound (2.07 g, 5.05 mmol, 51%) as a colorless
gum. 1H NMR (270 MHz, CDCl3) δ 1.67 (6H, s), 2.26 (3H, s), 4.88
(1H, brs), 6.98−7.01 (2H, m), 7.17−7.20 (2H, m), 7.33−7.39 (2H,
m), 7.48−7.57 (4H, m), 7.64−7.71 (1H, m), 8.18−8.22 (2H, m).
HRMS (FAB) calcd for C23H22NO4S [M − H]− 408.1270, found
408.1282.
Hz), 6.41 (1H, tt, J = 54.2, 3.5 Hz), 7.02−7.17 (5H, m), 7.24−7.32
(3H, m), 7.67 (1H, d, J = 7.8 Hz), 8.06 (1H, brs), 11.32 (1H, brs). 13C
NMR (CDCl3) δ 29.8, 50.7, 58.3, 67.3 (t, J = 24.1 Hz), 102.6, 112.4,
113.3 (t, J = 242.3 Hz), 118.9, 120.2, 126.4, 127.4, 130.0, 133.9, 143.9,
144.1, 145.1, 151.0, 157.5, 163.8. HRMS (TOF) calcd for
C22H24F2N3O5S [M + H]+ 480.1405, found 480.1411. HPLC purity:
96.8%, tR = 7.65 min (method A).
3-(Cyclopropylmethoxy)-N-(2-p-tolylpropan-2-yl)-
benzenesulfonamide (22a). A solution of 21 (287 mg, 0.70 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 and was then
dissolved in DMF (4.0 mL). To the mixture were added K2CO3 (193
mg, 1.40 mmol) and (bromomethyl)cyclopropane (74.7 μL, 0.77
mmol) at room temperature. The resulting mixture was stirred at 90
°C for 16 h and then poured into H2O. The aqueous layer was
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 = 4/1 to afford the title
(E)-N-(7-Hydroxy-2-methylhept-5-en-2-yl)-3-methoxybenze-
nesulfonamide (26). 26 was prepared from 17 (475 mg, 1.95 mmol)
as described for the preparation of 18, colorless oil (95.0 mg, 0.66
mmol, 34%). 1H NMR (270 MHz, CDCl3) δ 1.19 (6H, s), 1.54−1.63
(2H, m), 2.00−2.09 (2H, m), 2.18 (3H, s), 4.49 (1H, brs), 5.52−5.57
(2H, m), 7.46−7.60 (4H, m), 7.84−7.93 (2H, m). HRMS (FAB) calcd
for C15H22NO4S [M − H]− 312.1270, found 312.1279.
(E)-N-(7-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-meth-
ylhept-5-en-2-yl)-3-methoxybenzenesulfonamide (27). 27 was
prepared from 26 (205 mg, 0.65 mmol) as described for the
1
preparation of 9, white foam (35.0 mg, 0.086 mmol, 13%). H NMR
(270 MHz, CDCl3) δ 1.19 (6H,s), 1.61−1.66 (2H, m), 2.06−2.18
(2H, m), 3.85 (3H, s), 4.27 (2H, d, J = 6.1 Hz), 4.54 (1H, brs), 5.41−
5.50 (1H, m), 5.64−5.72 (2H, m), 7.07 (1H, dd, J = 8.3, 2.7 Hz), 7.15
(1H, d, J = 8.1 Hz), 7.36−7.47 (3H, m), 8.30 (1H, brs). 13C NMR
(100 MHz, CDCl3) δ 26.7, 27.7, 41.6, 49.5, 55.6, 56.8, 102.3, 111.7,
118.3, 118.9, 123.3, 130.0, 136.1, 143.8, 144.6, 150.8, 159.7, 163.8.
HRMS (TOF) calcd for C19H26N3O5S [M + H]+ 408.1593, found
408.1598. HPLC purity: 96.4%, tR = 5.08 min (method B).
1
compound (224 mg, 0.62 mmol, 89% from 21) as a white solid. H
NMR (270 MHz, CDCl3) δ 0.32−0.38 (2H, m), 0.62−0.70 (2H, m),
1.20−1.29 (1H, m), 1.63 (6H, s), 2.28 (3H, s), 3.74 (2H, d, J = 7.0
Hz), 4.84 (1H, brs), 6.93−7.04 (4H, m), 7.11−7.14 (2H, m), 7.25−
7.30 (2H, m). HRMS (FAB) calcd for C20H24NO3S [M − H]−
358.1477, found 358.1465.
3 - ( C y c l o p e n t y l o x y ) -N - ( 2 - p - t o l y l p r o p a n - 2 - y l ) -
benzenesulfonamide (22b). 22b was prepared from 21 (573 mg,
1.40 mmol) as described for the preparation of 22a, white solid (519
(E)-3-(N-(7-Hydroxy-2-methylhept-5-en-2-yl)sulfamoyl)-
phenyl Benzoate (28). 28 was prepared from 17 (450 mg, 1.85
mmol) as described for the preparation of 18, light brown gum (320
1
mg, 1.39 mmol, 99%). H NMR (270 MHz, CDCl3) δ 1.63 (6H, s),
1
mg, 0.79 mmol, 43%). H NMR (270 MHz, CDCl3) δ 1.22 (6H, s),
1.64−1.69 (2H, m), 1.72−1.90 (6H, m), 2.28 (3H, s), 4.67−4.83 (1H,
m), 4.80 (1H, brs), 6.93−7.00 (3H, m), 7.11−7.26 (5H, m). HRMS
(FAB) calcd for C21H26NO3S [M − H]− 372.1633, found 372.1659.
3-(2,2-Difluoroethoxy)-N-(2-p-tolylpropan-2-yl)-
benzenesulfonamide (22c). 22c was prepared from 21 (573 mg,
1.40 mmol) as described for the preparation of 22a, white solid (510
1.58−1.64 (2H, m), 2.04−2.09 (2H, m), 4.03−4.05 (2H, m), 5.07
(1H, brs), 5.57−5.62 (2H, m), 7.39−7.43 (1H, m), 7.50−7.70 (4H,
m), 7.78−7.82 (2H, m), 8.17−8.21 (2H, m). HRMS (FAB) calcd for
C21H24NO5S [M − H]− 402.1375, found 402.1406.
(E)-3-(Cyclopropylmethoxy)-N-(7-hydroxy-2-methylhept-5-
en-2-yl)benzenesulfonamide (29a). 29a was prepared from 28
(310 mg, 0.77 mmol) as described for the preparation of 22a, colorless
oil (248 mg, 0.70 mmol, 91%). 1H NMR (270 MHz, CDCl3) δ 0.33−
0.39 (2H, m), 0.62−0.70 (2H, m), 1.20 (6H, s), 1.22−1.30 (1H, m),
1.59−1.64 (2H, m), 2.01−2.10 (2H, m), 3.84 (2H, d, J = 7.0 Hz), 4.07
(2H, d, J = 4.1 Hz), 4.42 (1H, brs), 5.60−5.64 (2H, m), 7.05−7.09
(1H, m), 7.35−7.47 (3H, m). HRMS (FAB) calcd for C18H26NO4S
[M − H]− 352.1583, found 352.1587.
1
mg, 1.38 mmol, 99%). H NMR (270 MHz, CDCl3) δ 1.65 (6H, s),
2.27 (3H, s), 4.08 (2H, td, J = 13.2, 4.3 Hz), 4.90 (1H, brs), 6.07 (1H,
tt, J = 55.1, 4.1 Hz), 6.93−7.04 (4H, m), 7.11−7.14 (2H, m), 7.27−
7.36 (2H, m). HRMS (FAB) calcd for C18H20F2NO3S [M − H]−
368.1132, found 368.1161.
3-(Cyclopropylmethoxy)-N-(2-(4-((2,4-dioxo-3,4-dihydropyr-
i m i d i n - 1 ( 2 H ) - y l ) m e t h y l ) p h e n y l ) p r o p a n - 2 - y l ) -
benzenesulfonamide (23). 23 was prepared from 22a (230 mg,
0.64 mmol) as described for the preparation of 8, colorless gum (113
(E)-3-(Cyclopentyloxy)-N-(7-hydroxy-2-methylhept-5-en-2-
yl)benzenesulfonamide (29b). 29b was prepared from 28 (600 mg,
1.49 mmol) as described for the preparation of 22a, yellow oil (292
1
mg, 0.24 mmol, 38%). H NMR (270 MHz, DMSO-d6) δ 0.30−0.36
(2H, m), 0.54−0.61 (2H, m), 1.19−1.24 (1H, m), 1.45 (6H, s), 3.79
(2H, d, J = 6.9 Hz), 4.76 (2H, s), 5.60 (1H, d, J = 7.8 Hz), 6.97−7.06
(5H, m), 7.20−7.26 (3H, m), 7.69 (1H, d, J = 7.8 Hz), 8.02 (1H, brs),
11.32 (1H, brs). 13C NMR (100 MHz, DMSO-d6) δ 3.1, 10.0, 29.7,
49.7, 57.2, 72.4, 101.2, 111.6, 118.1, 125.8, 126.8, 129.7, 134.7, 144.4,
145.4, 145.6, 151.0, 158.3, 163.7. Anal. Calcd for
C24H27N3O5S·0.3H2O: C, 60.69; H, 5.86; N, 8.85. Found: C, 60.68;
H, 5.71; N, 8.96.
1
mg, 0.79 mmol, 53%). H NMR (400 MHz, CDCl3) δ 1.20 (6H, s),
1.59−1.64 (2H, m), 1.75−2.00 (8H, m), 2.05−2.10 (2H, m), 4.05−
4.09 (2H, m), 4.19 (1H, brs), 4.40−4.44 (1H, m), 5.61−5.64 (2H, m),
7.00−7.04 (1H, m), 7.35−7.47 (3H, m). HRMS (FAB) calcd for
C19H28NO4S [M − H]− 366.1739, found 366.1762.
(E)-3-(2,2-Difluoroethoxy)-N-(7-hydroxy-2-methylhept-5-en-
2-yl)benzenesulfonamide (29c). 29c was prepared from 28 (600
mg, 1.49 mmol) as described for the preparation of 22a, yellow oil
(426 mg, 1.17 mmol, 79%). 1H NMR (400 MHz, CDCl3) δ 1.20 (6H,
s), 1.59−1.64 (2H, m), 2.01−2.17 (2H, m), 4.05−4.09 (2H, m), 4.23
(2H, td, J = 12.9, 4.2 Hz), 4.49 (1H, brs), 5.61−5.64 (2H, m), 6.11
(1H, tt, J = 55.1, 3.9 Hz), 7.08−7.11 (1H, m), 7.37−7.45 (3H, m).
HRMS (FAB) calcd for C16H22F2NO4S [M − H]− 362.1238, found
362.1269.
3-(Cyclopentyloxy)-N-(2-(4-((2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl)methyl)phenyl)propan-2-yl)benzenesulfonamide
(24). 24 was prepared from 22b (247 mg, 0.66 mmol) as described for
the preparation of 8, pale yellow foam (22.5 mg, 0.047 mmol, 7%). 1H
NMR (270 MHz, DMSO-d6) δ 1.45 (6H, s), 1.58−1.70 (6H, m),
1.89−1.95 (2H, m), 4.72−4.76 (1H, m), 4.76 (2H, s), 5.60 (1H, d, J =
7.9 Hz), 6.94−7.09 (5H, m), 7.18−7.27 (3H, m), 7.69 (1H, d, J = 7.9
Hz), 8.03 (1H, brs), 11.3 (1H, brs). 13C NMR (100 MHz, DMSO-d6)
δ 23.6, 29.7, 32.2, 49.8, 57.3, 79.2, 101.3, 112.6, 118.0, 119.0, 125.8,
126.8, 129.7, 134.8, 144.4, 145.4, 145.6, 151.0, 157.4, 163.7. Anal.
Calcd for C25H29N3O5S: C, 62.09; H, 6.04; N, 8.69. Found: C, 61.87;
H, 6.04; N, 8.65.
(E)-3-(Cyclopropylmethoxy)-N-(7-(2,4-dioxo-3,4-dihydropyr-
imidin-1(2H)-yl)-2-methylhept-5-en-2-yl)benzenesulfonamide
(30). 30 was prepared from 29a (248 mg, 0.70 mmol) as described for
1
the preparation of 9, colorless gum (108 mg, 0.24 mmol, 34%). H
NMR (270 MHz, DMSO-d6) δ 0.30−0.34 (2H, m), 0.53−0.56 (2H,
m), 1.01 (6H, s), 1.15−1.24 (1H, m), 1.39−1.45 (2H, m), 1.91−1.95
(2H, m), 3.85 (2H, d, J = 6.9 Hz), 4.16 (2H, d, J = 5.1 Hz), 5.30−5.58
(2H, m), 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), 11.30 (1H, brs). 13C NMR (100
MHz, CDCl3) δ 3.2, 10.0, 26.7, 27.6, 41.6, 49.5, 56.8, 73.1, 102.2,
112.2, 118.8, 119.0, 123.3, 129.9, 136.1, 143.8, 144.5, 150.8, 159.1,
3-(2,2-Difluoroethoxy)-N-(2-(4-((2,4-dioxo-3,4-dihydropyri-
m i d i n - 1 ( 2 H ) - y l ) m e t h y l ) p h e n y l ) p r o p a n - 2 - y l ) -
benzenesulfonamide (25). 25 was prepared from 22c (240 mg,
0.65 mmol) as described for the preparation of 8, white foam (32.7
1
mg, 0.068 mmol, 10%). H NMR (270 MHz, DMSO-d6) δ 1.47 (6H,
s), 4.35 (2H, td, J = 14.7, 3.3 Hz), 4.77 (2H, s), 5.61 (1H, d, J = 7.8
5492
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163.9. HRMS (TOF) calcd for C22H30N3O5S [M + H]+ 448.1906,
found 448.1909. HPLC purity: 95.1%, tR = 6.05 min (method B).
(E)-3-(Cyclopentyloxy)-N-(7-(2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl)-2-methylhept-5-en-2-yl)benzenesulfonamide
(31). 31 was prepared from 29b (125 mg, 0.34 mmol) as described for
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 (107 mg, 0.24 mmol, 68%) as a colorless
1
gum. H NMR (400 MHz, CDCl3) δ 0.29−0.35 (2H, m), 0.60−0.67
1
the preparation of 9, colorless gum (24.5 mg, 0.053 mmol, 16%). H
(2H, m), 1.17−1.28 (1H, m), 1.42 (3H, d, J = 7.1 Hz), 3.69 (2H, dd, J
= 7.1, 2.4 Hz), 4.46 (1H, quin, J = 7.1 Hz), 4.91 (2H, s), 5.16 (1H,
brs), 5.76 (1H, d, J = 7.9, 2.0 Hz), 6.59−6.68 (3H, m), 7.04 (1H, t, J =
8.2 Hz), 7.14 (1H, d, J = 7.9 Hz), 7.23−7.28 (2H, m), 7.69 (2H, d, J =
8.2 Hz), 8.95 (1H, brs). 13C NMR (100 MHz, DMSO-d6) δ 3.1, 10.2,
23.5, 49.9, 53.0, 71.8, 101.5, 112.2, 112.7, 118.1, 126.7, 127.7, 129.0,
140.8, 144.7, 145.4, 145.6, 151.0, 158.4, 163.7. Anal. Calcd for
C23H25N3O5S·0.3H2O: C, 59.93; H, 5.60; N, 9.12. Found: C, 60.05; H,
5.28; N, 9.01. [α]25 +41.4 (c 0.20, MeOH).
NMR (400 MHz, CDCl3) δ 1.19 (6H, s), 1.58−1.70 (4H, m), 1.74−
1.88 (4H, m), 1.89−1.97 (2H, m), 2.07−2.15 (2H, m), 4.27 (2H, d, J
= 6.3 Hz), 4.74 (1H, brs), 4.77−4.82 (1H, m), 5.41−5.51 (1H, m),
5.64−5.70 (1H, m), 5.72 (1H, d, J = 7.7 Hz), 7.02 (1H, dd, J = 8.1, 1.5
Hz), 7.16 (1H, d, J = 7.7 Hz), 7.33−7.45 (3H, m), 8.69 (1H, brs). 13C
NMR (100 MHz, CDCl3) δ 24.0, 26.8, 27.7, 32.7, 41.6, 49.6, 56.8,
79.9, 102.3, 113.3, 118.5, 119.9, 123.3, 130.0, 136.2, 143.7, 144.3,
150.6, 158.3, 163.5. Anal. Calcd for C23H31N3O5S·0.9H2O: C, 57.82;
H, 6.92; N, 8.79. Found: C, 57.46; H, 6.53; N, 8.42.
(R)-N-(1-(3-(CycDlopentyloxy)phenyl)ethyl)-4-((2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)methyl)benzenesulfonamide (39).
39 was prepared from 37b (191 mg, 0.44 mmol) as described for
(E)-3-(2,2-Difluoroethoxy)-N-(7-(2,4-dioxo-3,4-dihydropyri-
midin-1(2H)-yl)-2-methylhept-5-en-2-yl)benzenesulfonamide
(32). 32 was prepared from 29c (110 mg, 0.30 mmol) as described for
1
the preparation of 38, colorless gum (50 mg, 0.11 mmol, 25%). H
1
the preparation of 9, colorless gum (23.0 mg, 0.050 mmol, 17%). H
NMR (270 MHz, DMSO-d6) δ 1.18 (3H, d, J = 7.0 Hz), 1.54−1.64
(6H, m), 1.83−2.10 (2H, m), 3.25−3.43 (1H, m), 4.26−4.29 (1H, m),
4.62−4.70 (1H, m), 4.87 (2H, s), 5.62 (1H, d, J = 8.2 Hz), 6.56−6.53
(2H, m), 6.70 (1H, s), 6.97 (1H, t, J = 7.8 Hz), 7.29−7.33 (2H, m),
7.58−7.62 (2H, m), 8.16 (1H, d, J = 8.2 Hz), 8.30 (1H, brs). 13C
NMR (100 MHz, DMSO-d6) δ 23.5, 23.6, 32.2, 49.7, 53.0, 78.4, 101.5,
113.2, 113.5, 117.9, 126.7, 127.6, 129.0, 140.8, 144.6, 145.4, 145.6,
151.0, 157.4, 163.7. Anal. Calcd for C24H27N3O5S·0.5H2O: C, 60.23;
NMR (270 MHz, CDCl3) δ 1.19 (6H, s), 1.58−1.70 (2H, m), 2.06−
2.17 (2H, m), 4.23−4.28 (4H, m), 4.63 (1H, brs), 5.41−5.51 (1H, m),
5.64−5.70 (2H, m), 6.11 (1H, tt, J = 54.8, 3.8 Hz), 7.09−7.17 (2H,
m), 7.40−7.55 (3H, m), 8.40 (1H, brs). 13C NMR (100 MHz, CDCl3)
δ 26.8, 27.7, 41.5, 49.7, 57.0, 67.4 (t, J = 24.0 Hz), 102.3, 112.6, 113.3
(t, J = 240.7 Hz), 118.9, 120.2, 123.4, 130.3, 135.9, 143.8, 144.9, 150.7,
157.8, 163.6. Anal. Calcd for C20H25F2N3O5S·H2O: C, 50.52; H, 5.72;
N, 8.84. Found: C, 50.74; H, 5.40; N, 8.60.
H, 5.90; N, 8.78. Found: C, 60.32; H, 5.65; N, 8.49. [α]25 +33.4 (c
D
(R)-4-(Bromomethyl)-N-(1-(3-(cyclopropylmethoxy)phenyl)-
ethyl)benzenesulfonamide (37a). To a stirred solution of (R)-1-
(3-(cyclopropylmethoxy)phenyl)ethanamine hydrochloride 36a (363
mg, 1.60 mmol) in CH2Cl2 (3.0 mL) were added Et3N (670 μL, 4.81
mmol) and 4-(bromomethyl)benzenesulfonyl chloride (450 mg, 1.67
mmol) at 0 °C, and the resultant was stirred at the same temperature
for 1 h. The mixture was poured into H2O and extracted with EtOAc.
The organic layer was washed with 1 N HCl, brine, dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel, eluting with hexane/
EtOAc = 3/1 to afford the title compound (188 mg, 0.44 mmol, 28%)
as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 0.31−0.34 (2H, m),
0.63−0.65 (2H, m), 1.19−1.28 (1H, m), 1.43 (3H, d, J = 6.8 Hz),
3.66−3.70 (2H, m), 4.48 (1H, quin, J = 7.8 Hz), 4.57 (2H, s), 4.69
(1H, brs), 6.60−6.64 (2H, m), 6.68−6.71 (1H, m), 7.08 (1H, td, J =
7.6, 1.2 Hz), 7.37−7.40 (2H, m), 7.68−7.71 (2H, m). HRMS (FAB)
calcd for C19H21BrNO3S [M − H]− 422.0426, found 422.0414.
(R)-4-(Bromomethyl)-N-(1-(3-(cyclopentyloxy)phenyl)ethyl)-
benzenesulfonamide (37b). 37b was prepared from 36b (387 mg,
1.60 mmol) as described for the preparation of 37a, white solid (419
mg, 0.96 mmol, 60%). 1H NMR (400 MHz, CDCl3) δ 1.44 (3H, t, J =
6.6 Hz), 1.58−1.64 (2H, m), 1.72−1.89 (6H, m), 4.43 (2H, s), 4.46
(1H, quin, J = 6.8 Hz), 4.63 (1H, brs), 4.69 (1H, d, J = 7.1 Hz), 6.57−
6.61 (2H, m), 6.66−6.70 (1H, m), 7.05 (1H, t, J = 8.3 Hz), 7.38 (2H,
dd, J = 8.3, 2.6 Hz), 7.66−7.70 (2H, m). HRMS (FAB) calcd for
C20H23BrNO3S [M − H]− 436.0582, found 436.0585.
0.45, MeOH).
(R)-N-(1-(3-(2,2-Difluoroethoxy)phenyl)ethyl)-4-((2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)methyl)benzenesulfonamide
(40). 40 was prepared from 37c (158 mg, 0.36 mmol) as described for
1
the preparation of 38, colorless gum (84.6 mg, 0.18 mmol, 50%). H
NMR (400 MHz, CDCl3) δ 1.44 (3H, d, J = 6.8 Hz), 4,00−4.10 (2H,
m), 4.48−4.54 (1H, m), 4.87 (1H, d, J = 6.8 Hz), 4.91(2H, s), 5.76
(1H, dd, J = 8.1, 1.7 Hz), 6.05 (1H, tt, J = 55.4, 4.1 Hz), 6.57 (1H, s),
6.69 (1H, dd, J = 8.3, 2.7 Hz), 6.73−6.77 (1H, m), 7.09−7.15 (2H,
m), 7.24−7.28 (1H, m), 7.67 (3H, d, J = 8.3 Hz), 8.34 (1H, brs). 13C
NMR (100 MHz, DMSO-d6) δ 23.6, 49.9, 52.8, 66.2 (t, J = 24.0 Hz),
101.6, 112.4, 112.7, 114.1 (t, J = 242 Hz), 119.2, 126.7, 127.6, 129.2,
140.6, 140.9, 145.0, 145.6, 151.0, 157.3, 163.7. Anal. Calcd for
C21H21F2N3O5S: C, 53.16; H, 4.67; N, 8.86. Found: C, 53.34; H, 4.32;
N, 8.77. [α]25 +38.8 (c 0.50, MeOH).
D
(R,E)-5-(3-Benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
N - ( 1 - ( 3 - ( c y c l o p r o p y l m e t h o x y ) p h e n y l ) e t h y l ) - N -
(methoxymethyl)pent-3-ene-1-sulfonamide (42a). To a solution
of 41a (3.60 g, 9.39 mmol) in THF (60 mL) were added N-3-
benzoyluracil (3.10 g, 14.3 mmol) and PPh3 (3.91 g, 14.9 mmol) at
room temperature. To the mixture was slowly added a solution of
DEAD in toluene (2.2 M, 6.41 mL, 14.1 mmol) in THF (2.0 mL), and
the whole mixture was stirred at room temperature for 2 h. The
mixture was concentrated under reduced pressure, and the concentrate
was purified by column chromatography on silica gel, eluting with
hexane/EtOAc = 1/1 to afford the title compound (4.92 g, 8.46 mmol,
(R)-4-(Bromomethyl)-N-(1-(3-(2,2-difluoroethoxy)phenyl)-
ethyl)benzenesulfonamide (37c). 37c was prepared from 36c (380
mg, 1.60 mmol) as described for the preparation of 37a, colorless oil
(337 mg, 0.78 mmol, 49%). 1H NMR (400 MHz, CDCl3) δ 1.43 (3H,
d, J = 6.9 Hz), 3.97−4.14 (2H, m), 4.44−4.52 (1H, m), 4.56 (2H, s),
4.87 (1H, d, J = 6.9 Hz), 6.04 (1H, tt, J = 57.0, 4.0 Hz), 6.59−6.61
(1H, m), 6.69−6.77 (2H, m), 7.13 (1H, t, J = 7.9 Hz), 7.36−7.40 (2H,
m), 7.64−7.69 (2H, m). HRMS (FAB) calcd for C17H17BrF2NO3S [M
− H]− 432.0081, found 432.0077.
1
90%) as a colorless gum. H NMR (270 MHz, CDCl3) δ 0.33−0.37
(2H, m), 0.63−0.67 (2H, m), 1.22−1.32 (1H, m), 1.66 (3H, d, J = 7.3
Hz), 2.58−2.66 (2H, m), 3.04−3.12 (2H, m), 3.24 (3H, s), 3.80 (2H,
d, J = 7.0 Hz), 4.28−4.34 (3H, m), 4.68 (1H, d, J = 10.5 Hz), 5.09−
5.13 (1H, m), 5.53−5.69 (1H, m), 5.76−5.83 (2H, m), 6.82−6.88
(1H, m), 6.95−6.99 (2H, m), 7.22−7.64 (5H, m), 7.92−7.96 (2H, m).
HRMS (FAB) calcd for C30H35N3NaO7S [M + Na]+ 604.2093, found
604.2127.
(R,E)-5-(3-Benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
N-(1-(3-(cyclopentyloxy)phenyl)ethyl)-N-(methoxymethyl)-
pent-3-ene-1-sulfonamide (42b). 42b was prepared from 41b (306
mg, 0.77 mmol) as described for the preparation of 42a, colorless gum
(420 mg, 0.71 mmol, 92%). 1H NMR (270 MHz, CDCl3) δ 1.53−1.68
(5H, m), 1.75−1.94 (6H, m), 2.59−2.67 (2H, m), 3.03−3.15 (2H, m),
3.24 (3H, s), 4.32−4.37 (3H, m), 4.68 (1H, d, J = 10.6 Hz), 4.74−4.78
(1H, m), 5.10 (1H, q, J = 7.3 Hz), 5.56−5.67 (1H, m), 5.77−5.81
(2H, m), 6.78−6.82 (1H, m), 6.93−6.95 (2H, m), 7.21−7.26 (1H, m),
(R)-N-(1-(3-(Cyclopropylmethoxy)phenyl)ethyl)-4-((2,4-
d i o x o - 3 , 4 - d i h y d r o p y r i m i d i n - 1 ( 2 H ) - y l ) m e t h y l ) -
benzenesulfonamide (38). To a suspension of 2,4-bis-
(trimethylsilyloxy)pyrimidine (130 mg, 0.51 mmol) in 1,2-dichloro-
ethane (2.5 mL) was added a solution of 37a (143 mg, 0.34 mmol) in
1,2-dichloroethane (1.0 mL) and iodine (catalyst) at room temper-
ature. The resulting mixture was heated to reflux at 95 °C for 5 h. After
cooling to room temperature, the mixture was poured into H2O/
saturated aqueous Na2S2O3 and extracted with EtOAc two times. The
5493
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Article
7.31 (1H, d, J = 8.1 Hz), 7.43−7.56 (2H, m), 7.62−7.70 (1H, m),
7.92−7.96 (2H, m). HRMS (FAB) calcd for C31H37N3NaO7S [M +
Na]+ 618.2250, found 618.2248.
(Montreal, Canada). Ligand structures were built using MOE builder
tool, part of the MOE suite, and were subjected to MMFF94x energy
minimization until rmsd gradient was <0.05 kcal mol−1 Å−1.
Hydrogen atoms were added to the X-ray cocrystal structure of
dUTPase and compound 7 (PDB code 3ARN35), and the energy of
the structure was minimized keeping fixed the atoms of the mainframe.
The models 8 and 9 were constructed on the basis of crystal structure
conformation of compound 7 followed by energy minimization. A part
of the flexible linker (-O-CH2-CH2-CH2-) of compound 7 has enough
planarity to perform the modeling (dihedral angle is 35°). Molecular
graphics were prepared using the PyMOL Molecular Graphics System,
(R,E)-5-(3-Benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
N-(1-(3-(2,2-difluoroethoxy)phenyl)ethyl)-N-(methoxymethyl)-
pent-3-ene-1-sulfonamide (42c). 42c was prepared from 41c (173
mg, 0.44 mmol) as described for the preparation of 42a, colorless gum
(211 mg, 0.36 mmol, 81%). 1H NMR (270 MHz, CDCl3) δ 1.67 (3H,
d, J = 7.3 Hz), 2.60−2.68 (2H, m), 3.03−3.12 (2H, m), 3.25 (3H, s),
4.19 (2H, td, J = 13.2, 4.3 Hz), 4.28−4.36 (3H, m), 4.69 (1H, d, J =
10.6 Hz), 5.09−5.14 (1H, m), 5.57−5.67 (1H, m), 5.77−5.90 (2H,
m), 6.09 (1H, tt, J = 55.1, 4.1 Hz), 6.85 (1H, dd, J = 8.6, 3.0 Hz), 7.01
(1H, s), 7.07 (1H, d, J = 7.8 Hz), 7.25−7.29 (2H, m), 7.43−7.56 (2H,
m), 7.62−7.70 (1H, m), 7.92−7.96 (2H, m). HRMS (FAB) calcd for
C28H31F2N3NaO7S [M + Na]+ 614.1748, found 614.1741.
version 1.2r3pre (Schrodinger, LLC).
̈
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 gel-filtered 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).
(R,E)-N-(1-(3-(Cyclopropylmethoxy)phenyl)ethyl)-5-(2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)pent-3-ene-1-sulfona-
mide (43). A solution of 42a (4.90 g, 8.42 mmol) in 40% MeNH2 in
MeOH (100 mL) was stirred at room temperature for 1 h. The
mixture was concentrated under reduced pressure, and the residue was
coevaporated with toluene two times. This residue was dissolved in 4
N HCl/dioxane (30.0 mL). The resulting mixture was stirred at room
temperature for 4 h and then poured into saturated aqueous NaHCO3.
The aqueous layer was extracted with CHCl3/MeOH (10/1) three
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 CHCl3/EtOAc = 3/1 to afford the title compound (2.73 g, 6.30
mmol, 75% from 42a) as a colorless gum. 1H NMR (400 MHz,
CDCl3) δ 0.33−0.37 (2H, m), 0.62−0.68 (2H, m), 1.22−1.31 (1H,
m), 1.53 (3H, d, J = 7.0 Hz), 2.36−2.47 (2H, m), 2.66−2.73 (1H, m),
2.79−2.86 (1H, m), 3.80 (2H, d, J = 6.8 Hz), 4.20−4.29 (2H, m), 4.59
(1H, quin, J = 7.0 Hz), 4.66 (1H, brs), 5.42−5.49 (1H, m), 5.54−5.61
(1H, m), 5.71 (1H, dd, J = 7.8, 2.0 Hz), 6.80−6.83 (1H, m), 6.86−
6.92 (2H, m), 7.12 (1H, d, J = 7.8 Hz), 7.24−7.29 (1H, m), 8.25 (1H,
brs). 13C NMR (100 MHz, CDCl3) δ 3.2, 10.2, 24.0, 26.5, 49.3, 52.5,
53.7, 72.8, 102.4, 112.8, 113.6, 118.4, 125.6, 129.9, 132.1, 143.8, 144.3,
150.7, 159.4, 163.7. Anal. Calcd for C21H27N3O5S: C, 58.18; H, 6.28;
dUTPase Inhibition Assay. In vitro dUTPase inhibition assays
were conducted by measuring the production of [5-3H]dUMP from
[5-3H]dUTP. Breifly, 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 15 000
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.
N, 9.69. Found: C, 57.89; H, 6.34; N, 9.53. [α]25 +17.4 (c 0.42,
D
MeOH).
(R,E)-N-(1-(3-(Cyclopentyloxy)phenyl)ethyl)-5-(2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)pent-3-ene-1-sulfonamide (44).
44 was prepared from 42b (420 mg, 0.71 mmol) as described for the
preparation of 43, colorless gum (89.0 mg, 0.20 mmol, 28%). 1H NMR
(270 MHz, CDCl3) δ 1.53 (3H, d, J = 6.8 Hz), 1.61−1.71 (2H, m),
1.77−1.93 (6H, m), 2.33−2.48 (2H, m), 2.69−2.85 (2H, m), 4.22−
4.25 (2H, m), 4.51−4.58 (2H, m), 4.75−4.79 (1H, m), 5.47−5.68
(2H, m), 5.70 (1H, dd, J = 7.8, 2.2 Hz), 6.74−6.87 (3H, m), 7.12 (1H,
d, J = 8.0 Hz), 7.21−7.26 (1H, m), 8.28 (1H, brs). 13C NMR (100
MHz, DMSO-d6) δ 23.5, 24.1, 26.0, 32.2, 48.1, 51.2, 52.8, 78.4, 101.1,
113.3, 113.8, 118.0, 125.9, 129.4, 130.5, 145.0, 145.6, 150.7, 157.7,
163.7. Anal. Calcd for C22H29N3O5S·0.5H2O: C, 57.88; H, 6.62; N,
9.20. Found: C, 57.76; H, 6.33; N, 9.14. [α]25D +17.9 (c 0.48, MeOH).
(R,E)-N-(1-(3-(2,2-Difluoroethoxy)phenyl)ethyl)-5-(2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)pent-3-ene-1-sulfonamide (45).
45 was prepared from 42c (210 mg, 0.35 mmol) as described for the
preparation of 43, colorless gum (70.0 mg, 0.16 mmol, 46%). 1H NMR
(270 MHz, CDCl3) δ 1.54 (3H, d, J = 6.9 Hz), 2.36−2.48 (2H, m),
2.68−2.87 (2H, m), 4.14−4.25 (4H, m), 4.61 (1H, quin, J = 6.9 Hz),
4.94 (1H, brs), 5.42−5.60 (2H, m), 5.72 (1H, d, J = 6.5 Hz), 6.10 (1H,
tt, J = 55.1 Hz, 4.1 Hz), 6.81−7.06 (3H, m), 7.13 (1H, d, J = 7.9 Hz),
7.26−7.31 (1H, m), 8.64 (1H, brs). 13C NMR (100 MHz, CDCl3) δ
24.0, 26.5, 49.4, 52.5, 53.5, 67.2 (t, J = 24.1 Hz), 102.4, 112.8, 113.5,
113.6 (t, J = 244.0 Hz), 119.8, 125.6, 130.2, 131.8, 144.0, 144.8, 150.8,
158.1, 163.9. HRMS (TOF) calcd for C19H24F2N3O5S [M + H]+
444.1405, found 444.1412. HPLC purity: 96.2%, tR = 4.90 min
inhibition rate (%) = 1 − [(amount of [5‐3H]dUMP in the
presence of test solution (dpm))
/(amount of [5‐3H
]dUMP as control (dpm))] × 100
IC50 (μM), the concentration of inhibitor yielding 50% inhibition rate,
was obtained from the concentration−inhibition rate curve.
In Vitro Cell Inhibition Assays. HeLa S3 (human cervix
adenocarcinoma) cells were cultured in RPMI-1640 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 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 (%),
which is the ratio of OD540 with drug treatment to OD540 without
drug: T/C (%) = [(OD540 of treated well)/(OD540 of nontreated
well)] × 100. The IC50 (μM) for the cytotoxicity of the test compound
is the concentration yielding 50% T/C, which was calculated from
concentration−T/C (%) curve.
(method B). [α]25 +16.4 (c 0.22, MeOH).
D
Molecular Modeling Studies. All molecular modeling was
performed using the Molecular Operating Environment (MOE,
version 2010.10), developed by Chemical Computing Group, Inc.
In Vitro Cell Inhibition Assays in Combination with FdUrd.
HeLa S3 cells were seeded in 96-well plates (1500 cells/0.18 mL) and
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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 was calculated from the
concentration− T/C (%) curve as a concentration of each compound
that reduces the T/C (%) of FdUrd (1 μM) against HeLa S3 cells to
half in 24 h.
Pharmacokinetic Studies. Experiments were conducted with 6-
week-old to 9-week-old male Balb/c-A mice. Compound 43 was
administered to mice orally at a dose of 50 mg/kg in a solution
containing 2.5% DMA/2.5% Tween 80 and 10% Cremophor EL. The
concentration of 43 in the plasma was determined by ultraperformance
liquid chromatography (UPLC).
Evaluation of Antitumor Efficacy of Compound 43. Five-
week-old Balb/cA JcL-nu mice were obtained from Clea Japan, Inc.
(Tokyo, Japan). MX-1 human breast carcinoma (Japanese Foundation
for Cancer Research) was maintained by subcutaneous (sc) trans-
plantation in mice. Briefly, tumors were excised and fragments
(approximately 2 mm in diameter) were implanted sc using a trocar.
After implantation, the animals were divided into four groups and
treated either with vehicle (2.5% DMA, 2.5% Tween 80, 10%
Cremophor, and 0.5% HPMC), 5-FU (15 mg kg−1 day−1) by
continuous infusion using osmotic pump for 14 days, and compound
43 (300 mg kg−1 mg−1) by po for 14 days or with a combination of 5-
FU (15 mg kg−1 day−1) and compound 43 (300 mg kg−1 mg−1).
Tumor size and body weight were measured twice weekly. Tumor
volume (TV) was estimated with the formula TV (mm3) = length
(mm) × width (mm) × width (mm) × 0.5. 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 day 0). Body weight change (%) on day 15 was calculated
according to the following formula: body weight change (%) = [(body
weight on day 15) − (body weight on day 0)]/(body weight on day 0)
× 100.
FU, 5-fluorouracil; FdUrd, 5-fluoro-2′-deoxyuridine; FdUTP, 5-
fluoro-2′-deoxyuridine 5′-triphosphate; HCC, hepatocelluar
carcinoma; SAR, structure−activity relationship
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and characterization data for compounds
36b−c and 41a−c. This material is available free of charge via
the Internet at http://pubs.acs.org.
Accession Codes
The PDB code of compound 7 with human dUTPase is
3ARN.36
AUTHOR INFORMATION
Corresponding Author
*Phone: 81-29-865-4527. Fax: 81-29-865-2170. E-mail: m-
fukuoka@taiho.co.jp.
■
Notes
The authors declare no competing financial interest.
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
staff 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.
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ABBREVIATIONS USED
■
dUTPase, deoxyuridine triphosphatase; dUTP, 2′-deoxyuridine
5′-triphosphate; dTTP, thymidine 5′-triphosphate; dUMP, 2′-
deoxyuridine 5′-monophosphate; TS, thymidylate synthase; 5-
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