1,2,3-Triazole-containing uracil derivatives with excellent pharmacokinetics as a novel class of potent human deoxyuridine triphosphatase inhibitors

Article abstract of DOI:10.1021/jm3004174

Deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as a 5-fluorouracil-based combination chemotherapy. We describe the design and synthesis of a novel class of human dUTPase inhibitors, 1,2,3-triazole-containing u

Full text of DOI:10.1021/jm3004174

Article
pubs.acs.org/jmc
1,2,3-Triazole-Containing Uracil Derivatives with Excellent
Pharmacokinetics as a Novel Class of Potent Human Deoxyuridine
Triphosphatase Inhibitors
Hitoshi Miyakoshi,†,§ Seiji Miyahara,†,‡ Tatsushi Yokogawa,† Kanji Endoh,† Toshiharu Muto,†
Wakako Yano,† Takeshi Wakasa,† Hiroyuki Ueno,† Khoon Tee Chong,† Junko Taguchi,†
Makoto Nomura,† Yayoi Takao,† Akio Fujioka,† Akihiro Hashimoto,† Kenjirou Itou,† Keisuke Yamamura,†
Satoshi Shuto,‡ Hideko Nagasawa,§ and Masayoshi Fukuoka*,†
†Drug Discovery 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: Deoxyuridine triphosphatase (dUTPase) has
emerged as a potential target for drug development as a 5-
fluorouracil-based combination chemotherapy. We describe
the design and synthesis of a novel class of human dUTPase
inhibitors, 1,2,3-triazole-containing uracil derivatives. Com-
pound 45a, which possesses 1,5-disubstituted 1,2,3-triazole
moiety that mimics the amide bond of tert-amide-containing
inhibitor 6b locked in a cis conformation showed potent
inhibitory activity, and its structure−activity relationship
studies led us to the discovery of highly potent inhibitors
48c and 50c (IC50 = ∼0.029 μM). These derivatives
dramatically enhanced the growth inhibition activity of 5-
fluoro-2′-deoxyuridine against HeLa S3 cells in vitro (EC50 = ∼0.05 μM). In addition, compound 50c exhibited a markedly
improved pharmacokinetic profile as a result of the introduction of a benzylic hydroxy group and significantly enhanced the
antitumor activity of 5-fluorouracil against human breast cancer MX-1 xenograft model in mice. These data indicate that 50c is a
promising candidate for combination cancer chemotherapies with TS inhibitors.
can mediate resistance to 5-FU by preventing the misincorpora-
tion of dUTP and FdUTP into DNA in cancer cells.6−8
Therefore, we have been developing dUTPase inhibitors to
enhance the antitumor effect of 5-FU based chemotherapy
because dUTPase has been considered to be an attractive and
potential target in a new strategy for 5-fluorouracil-based
combination chemotherapy.
Although there have been extensive efforts made to develop
dUTPase inhibitors (Figure 1),9−18 to the best of our knowledge,
none of them has been evaluated in clinical trials. We have
already reported the development of amide-type human
dUTPase inhibitors, 5−7, which have potent inhibitory activity
and drug-like properties compared to known compounds 1−
4.19−21 Among these, compound 5 has shown the most potent
inhibitory activity (IC50 = 0.021 μM) and the greatest ability to
enhance antitumor activity of 5-FU in vivo.20 During the
development of the dUTPase inhibitors, our initial screening led
to the identification of the simple amide 6b as a hit compound
INTRODUCTION
■
Deoxyuridine triphosphatase (dUTPase) specifically recognizes
deoxyuridine triphosphate (dUTP) among the natural nucleo-
side triphosphates and hydrolyzes it to deoxyuridine mono-
phosphate (dUMP) and pyrophosphate.1 This enzyme is
thought to be responsible for two biological roles, (1) decreasing
the intracellular dUTP pools to prevent misincorporation of
uracil instead of thymine into DNA and (2) supplying dUMP as a
substrate for thymidylate synthase (TS) for conversion to
thymidine monophosphate (dTMP), an essential precursor for
the de novo pathway of DNA synthesis.2
TS inhibitors decrease the intracellular thymidine triphos-
phate (dTTP) pools, resulting in thymineless death concurrently
with increasing the dUMP pools, which results in expansion of
intracellular dUTP pools. When 5-fluorouracil (5-FU) or its
derivatives are used as a TS inhibitor, intracellular 5-fluoro-2′-
deoxyuridine triphosphate (FdUTP) pool is also increased. The
expansion of dUTP and FdUTP pools causes uracil and 5-FU
misincorporation into DNA, which is one of the key mechanisms
of the cytotoxity induced by TS inhibitors.3−5 Accordingly, the
expression of dUTPase which decomposes dUTP and FdUTP
Received: March 25, 2012
© XXXX American Chemical Society
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Figure 1. Structural formulas of dUTPase inhibitors.
Figure 2. Triazole-replacement strategy for compound 6b and molecular modification for the SAR study.
with a favorable level of lipophilicity and molecular weight (MW,
391; clogP, 2.17). Subsequent structure−activity relationship
(SAR) studies of amide-containing uracil derivatives revealed
that the tert-amide compound 6b had almost 12-fold more
potent inhibitory activity compared to the sec-amide compound
6a.19 We predicted from a conformational analysis by NMR that
the cis conformational preference of the tert-amide moiety of 6b
may contribute to its greater potency.19 On the basis of this
hypothesis, we designed 1,2,3-triazole derivatives by replacing
the amide moiety of 6b with a conformationally restricted
surrogate of the cis amide to further improve the inhibitory
potency. These derivatives could be readily synthesized from
alkyne intermediates and azide intermediates (Figure 2). From
the SAR studies focused on 1,2,3-triazole-containing uracil
derivatives, we have identified highly potent inhibitors of human
dUTPase with IC50 values in the 10−8 M range. We also report
that compound 50c has a desirable pharmacokinetic profile in
mice and clearly enhances the antitumor activity of a TS inhibitor
in vivo.
evaluation as human dUTPase inhibitors, and their ability to
enhance the growth inhibition activity of a TS inhibitor against
cancer cells in vitro and in vivo.
CHEMISTRY
■
Alkyne intermediates 9a−b and 11 were synthesized as shown in
Scheme 1. Compounds 9a−b were prepared by the Mitsunobu
reaction of commercially available 4-pentyn-1-ol 8b or 4-pentyn-
1-ol 8c and 3-N-benzoyluracil,22 followed by the removal of the
benzoyl group with methylamine. Compound 11 was synthe-
sized in two steps. Commercially available 3-butyn-1-ol 8a was
treated with chloromethylmethylether to provide 10. The
methoxymethyl (MOM) group of 10 was activated with BCl3
and was then treated with (TMS)2Ura and a catalytic amount of
iodine to give 11.23
The synthesis of azide intermediates 14a−d is shown in
Scheme 2. The phenolic group of 12a−b was alkylated with
(bromomethyl)cyclopropane to provide 13c−d. Commercially
available 13a−b and intermediates 13c−d were treated with
MsCl and then with NaN3 to give 14a−d.
In this paper, we report the details of the design and synthesis
of novel 1,2,3-triazole-containing uracil compounds, their
B
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Azide intermediates 22a−d were synthesized as shown in
Scheme 3. Commercially available 15 was converted to ethyl
ester 16. The phenolic group of 16 was then benzylated, and the
ethyl ester was hydrolyzed to give 17. Carboxylic acid 17 was
activated by SOCl2, and reaction of the acyl chloride with (R) or
(S)-4-benzyl-2-oxazolidine activated by n-BuLi provided 18a−b.
Evans asymmetric alkylation was performed on 18a−b to afford
chiral derivatives 19a−d, respectively.24 Removal of their benzyl
groups generated the unmasked phenol groups which were then
alkylated to provide 20a−f. Reduction of 20a−f with LiBH4
produced 21a−f. The specific rotation of 21f was almost identical
Scheme 1. General Synthetic Method for Preparation of
Alkyne Intermediates 9a−b and 11
a
with reference data [21f, [α]25 = −16.1 (c 1.09, CHCl3);
D
reference data,25 [α]25D = −16.6 (c 1.09, CHCl3)]. Compounds
21a−d were then converted to the corresponding azides 22a−d.
Synthesis of azide intermediates 34a−i is shown in Scheme 4.
Commercially available 23 was treated with chloromethylme-
thylether to give 24. After the hydrolysis of 24 to give 25, a
condensation reaction with MeNHOMe·HCl afforded the
Weinreb amide 26. Intermediate 26 was converted to the
propiophenone derivative 27 by a Grignard reaction, and a
subsequent Wittig reaction with MePPh3Br provided 28.
Compound 28 was converted to chiral derivatives 29a−b by
asymmetric dihydroxylation using AD-mixα or AD-mixβ. The
primary hydroxyl group of 29a−b was then acylated by the
appropriate acyl chloride, and subsequent removal of the MOM
protecting group gave crystalline compounds 31a−c. The optical
purities of 31a and 31b were improved to 96% ee by repeated
crystallizations (three times). A single crystal X-ray structure of
31c showed that this analogue possesses the desired S
configuration (Figure 3). The phenolic group of 31a−b was
then alkylated via a Mitsunobu reaction or by treatment with
a
Reagents and conditions: (a) N-3-Bz-uracil, PPh3, DIAD, THF, room
temp, 2 h then MeNH2, MeOH, room temp, 1 h; (b) MOMCl, DIEA,
CH2Cl2, room temp, 5 h; (c) BCl3, CH2Cl2, room temp, 1.5 h then
(TMS)2Ura, I2, DCE, 90 °C, 2 h.
Scheme 2. General Synthetic Method for Preparation of Azide
Intermediates 14a−d
a
a
Reagents and conditions: (a) (bromomethyl)cyclopropane, K2CO3,
DMF, 90 °C, 5 h; (b) MsCl, TEA, CH2Cl2, room temp, 30 min then
NaN3, DMF, 50 °C, 12 h.
a
Scheme 3. General Synthetic Method for Preparation of Azide Intermediates 22a−d
a
Reagents and conditions: (a) H2SO4, EtOH, reflux, 4 h; (b) BnBr, K2CO3, DMF, 90 °C, 5 h then aq NaOH, EtOH, 60 °C, 4 h; (c) SOCl2, DMF,
50 °C, 2 h then (R)- or (S)-4-benzyl-2-oxazolidine, n-BuLi, THF, 0 °C, 2 h; (d) EtI or MeI, NaHMDS, THF, −78 to −20 °C, 4−6 h; (e) Pd(OH)2/
C, H2, MeOH, room temp, 1 h then (bromomethyl)cyclopropane or MeI, K2CO3, DMF, 70−90 °C, 2 h; (f) LiBH4, THF, 0 °C, 6 h; (g) MsCl, TEA,
CH2Cl2, room temp, 30 min then NaN3, DMF, 60 °C, 12 h.
C
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a
Scheme 4. General Synthetic Method for Preparation of Azide Intermediates 34a−i
a
Reagents and conditions: (a) MOMCl, DIEA, CH2Cl2, room temp, 5 h; (b) aq NaOH, EtOH, 60 °C, 4 h; (c) MeNHOMe·HCl, EDC·HCl, HOBt,
TEA, DMF, room temp, 6 h; (d) EtMgBr, THF, room temp, 3 h; (e) MePPh3Br, NaHMDS, THF, room temp, 12 h; (f) AD-mixα or AD-mixβ, t-
BuOH, H2O, 0 °C, 3 h; (g) BzCl or 4-Br-BzCl, TEA, CH2Cl2, room temp, 2 h; (h) aq AcOH, TFA, 50 °C, 3 h; (i) alkylbromide, K2CO3, DMF, 90
°C, 2−5 h or alcohol, PPh3, DIAD, THF, room temp, 3 h; (j) aq NaOH, MeOH, 50 °C, 2 h; (k) MsCl, TEA, CH2Cl2, room temp, 30 min then
NaN3, DMF, 90 °C, 12 h.
Figure 3. ORTEP view of single-crystal X-ray structure of 31c.
alkyl halide to give the corresponding derivatives 32a−i. The
benzoyl group of 32a−i was removed by alkaline hydrolysis to
give 33a−i, and these compounds were subsequently converted
to the corresponding azides 34a−i.
Azide intermediates 43 and 44 were synthesized as shown in
Scheme 5. Commercially available 35 was treated with sulfuric
acid in EtOH to give the ethyl ester, and this was followed by
alkylation to give 36. After the hydrolysis of the ethyl ester of 36,
a condensation reaction of the resulting acid with MeNHO-
Me·HCl afforded the Weinreb amide 38. Compound 38 was
converted to the propiophenone derivative 39 via a Grignard
reaction, and 39 was then subjected to a Wittig reaction to
provide 40. Compound 40 was converted to chiral derivatives
41a−b by asymmetric dihydroxylation using AD-mixα or AD-
D
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a
Scheme 5. General Synthetic Method for Preparation of Azide Intermediates 43, 44
a
Reagents and conditions: (a) H2SO4, EtOH, reflux, 4 h then (bromomethyl)cyclopropane, K2CO3, DMF, 90 °C, 5 h; (b) aq NaOH, EtOH, 60 °C,
4 h; (c) MeNHOMe·HCl, EDC·HCl, HOBt, TEA, DMF, room temp, 6 h; (d) EtMgBr, THF, room temp, 3 h; (e) MePPh3Br, NaHMDS, THF,
room temp, 12 h; (f) AD-mixα or AD-mixβ, t -BuOH, H2O, 0 °C, 3 h; (g) 2-Cl-4-NO2-BzCl, TEA, CH2Cl2, room temp, 3 h; (h) aq NaOH, MeOH,
50 °C, 2 h then MsCl, TEA, CH2Cl2, room temp, 30 min then NaN3, DMF, 90 °C, 12 h; (i) MsCl, TEA, CH2Cl2, room temp, 30 min then NaN3,
DMF, 90 °C, 12 h.
mixβ, respectively. Intermediate 41a was treated with 2-chloro-4-
nitro-benzoylchloride to afford crystalline 42, whose optical
purity was improved to >99% ee by repeated crystallizations
(three times). The benzoyl group of 42 was then removed by
hydrolysis, and this was followed by sequential reactions with
MsCl and NaN3 to give azide 43. Compound 41b was directly
converted to 44 without acylation and crystallization.
The 1,2,3-triazole derivatives were prepared by a ruthenium-
catalyzed cycloaddition reaction26 of alkyne intermediates (9a−
b, 11) with azide intermediates (14a−e, 22a−d, 34a−i, 43, 44)
as shown in Scheme 6.
dUTPase based on the cocrystal structure of compound 7 and
human dUTPase (PDB code 3ARA). (see Figure S1 in the
Supporting Information). The modeling study showed that
uracil and one of the terminal phenyl rings of 6b were attributed
to its potent and specific interaction with dUTPase. Accordingly,
to improve the inhibitory potency, we designed a new scaffold
with 1,5-disubstituted 1,2,3-triazoles as a viable amide surrogate
for a locked cis conformation (Figure 2).27,28 To carry out SAR
studies focusing on the 1,2,3-triazole-containing uracil structure,
we synthesized various uracil derivatives. We then examined in
detail the influence of linker length and various N-alkyl moieties
on the inhibitory activity.
Replacement of a tert-Amide Bond of 6b with a 1,2,3-
Triazole Ring. First, we synthesized a 1,2,3-triazole derivative
45a that corresponds to the tert-amide containing uracil
derivative 6b as a conformationally cis-locked compound (Figure
2). We also prepared analogues of 45a to examine the effect of
linker length on inhibitory potency (Table 1).
Scheme 6. General Synthetic Method for Preparation of
Triazole Derivatives 45a−b (see Table 1), 46a−b (see Table
1), 47a−g (see Table 2), 48a−d (see Table 3), 49a−h (see
a
Table 5) and 50a−d (see Table 6)
The human dUTPase inhibitory potency of the triazole-
containing uracil derivative 45a (IC50 = 1.3 μM) was comparable
Table 1. Human dUTPase Inhibitory Activity of Triazole-
Containing Uracil Derivatives 45a−b, 46a−b, and tert-Amide
Containing Uracil Compound 6b
a
Reagents and conditions: (a) azide, Cp*RuCl(COD), dioxane, 80
°C, 3 h. R7: various alkyl groups. X: CH2 or O, m = 1 or 2
RESULTS AND DISCUSSION
■
Molecular Design and Synthesis of dUTPase Inhibitors
Bearing a 1,2,3-Triazole Ring. The SAR studies of amide-
containing uracil compounds revealed that tert-amide compound
6b has almost 12-fold more improvement in the human
dUTPase inhibitory activity over sec-amide compound 6a. The
1H NMR spectrum and NOE data of 6b revealed that 6b exists as
a mixture of two conformational isomers at 25 °C (cis:trans =
2:3).19 These results suggest that the cis conformation of 6b
should make an important contribution to its potency. To gain
further evidence, we performed a docking study with human
dUTPase and 6b (cis isomer), and this suggested that the cis
conformation of 6b successfully interacts with the catalytic site of
a
IC50 (μM)
6b
1.3 0.068
1.3 0.052
0.74 0.011
>10
45a
45b
46a
46b
1.6 0.019
a
Enzyme inhibition assays are tested at 10 μM or below. IC50 values
are shown as the mean SE (n = 3)
E
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to that of the tert-amide containing uracil compound 6b (IC50 =
1.3 μM). Compound 45b with a tetramethylene linker also
showed potent inhibitory activity (IC50 = 0.74 μM). These data
clearly showed that the inhibitory potency of 6b is retained even
if its tert-amide bond is replaced by the “cis-locked” 1,2,3-triazole
ring and that both trimethylene and tetramethylene linkers are
also applicable to triazole compounds. On the other hand, 46a−
b, having a 1-N-diphenylmethyl triazole moiety, had somewhat
weaker inhibition potency compared to 45a−b (IC50 = 1.6−10
μM).
0.21 μM) showed almost 6-fold greater potency than the initial
compound 45a.
The Substituent Effect of Lower Alkyl Groups at the
Benzylic Position of 47g on Inhibitory Potency. To
improve the potency of 47g, we investigated the effects of alkyl
substitution at the benzylic position on inhibitory potency
(Table 3). On the basis of the comparison of inhibitory potency
Table 3. Human dUTPase Inhibitory Activity of Triazole-
Containing Uracil Derivatives 48a−d and 47g
Structural Simplification of the 2,2-Diphenylethyl
Moiety of 45a−b and Substituent Effects of the Terminal
Phenyl Ring. 1,2,3-Triazole-containing compounds such as
45a−b showed potent enzyme inhibitory activity as expected.
However, these compounds are not suitable as lead compounds
owing to the following unfavorable properties: (1) relatively high
molecular weight and (2) synthetic difficulties in various
derivatization reactions due to the diphenyl ethyl moiety.
Therefore, on the basis of the chemical structures of 45a−b
(Table 1), we next performed SAR study on the derivatives with a
monophenylethyl moiety in order to solve these issues (Table 2).
a
R9
IC50 (μM)
47g
48a
48b
48c
48d
0.21 0.0026
0.058 0.0019
0.87 0.015
Me (R)
Me (S)
Et (R)
Et (S)
0.029 0.00047
0.72 0.022
Table 2. Human dUTPase Inhibitory Activity of Triazole-
Containing Uracil Derivatives 47a−g and 45a−b
a
IC50 values are shown as the mean SE (n = 3)
of 45b (IC50 = 0.74 μM) to 47b (IC50 = 3.5 μM), we introduced
some lower alkyl groups at the benzylic position of 47g for
further improvement in the inhibitory potency.
We synthesized compounds 48a−d, by introduction of a
methyl or ethyl group stereoselectively at the benzylic position of
47g, and evaluated their potencies. From this evaluation, 48a and
48c having the R configuration were identified as highly potent
inhibitors of human dUTPase (48a, IC50 = 0.057 μM; 48c, IC50 =
0.029 μM). In contrast, 48b and 48d having the S configuration
clearly exhibited weaker potency compared to the R isomers
(48b, IC50 = 0.87 μM; 48d, IC50 = 0.72 μM, eudismic ratio = 15−
25).
A Solution to the Metabolic Instability of 48c and the
Substituent Effects at the m- Position of the Terminal
Phenyl Ring on Human dUTPase Inhibitory Activity. We
next performed a pharmacokinetics study of the highly potent
analogue 48c to estimate its feasibility for in vivo evaluation via
oral administration. From this study, we determined that 48c was
metabolically unstable in mice although its absorption was
acceptable (Figure 4).
a
n
R8
IC50 (μM)
45a
45b
47a
47b
47c
47d
47e
47f
1.3 0.052
0.74 0.011
>10
1
2
2
2
2
2
2
H
H
3.5 0.023
5.7 0.030
4.1 0.065
>10
2-MeO
3-MeO
4-MeO
2-(cyclopropyl)CH2O
3-(cyclopropyl)CH2O
0.39 0.0044
0.21 0.0026
47g
a
Enzyme inhibition assays are tested at 10 μM or below. IC50 values
are shown as the mean SE (n = 3)
The inhibitory potency of 47a without one of the terminal
phenyl group of 45a is clearly diminished (IC50 > 10 μM).
However, in the tetramethylene-linked series, the degree of
reduced inhibitory potency of 47b (almost 5-fold) was
significantly smaller than that of 47a. These data suggested
that 47b having the tetramethyene linker between the uracil ring
and the triazole ring is a promising lead compound. Accordingly,
we next synthesized compounds 47c−e, which possess a
methoxy group at various positions on the terminal phenyl
ring of 47b. The 2-methoxy derivative 47c (IC50 = 5.7 μM) and
3-methoxy derivative 47d (IC50 = 4.1 μM) retained moderate
potency comparable to 47b. On the other hand, the introduction
of the methoxy group at the p-position of the terminal phenyl
ring of 47b diminished its inhibitory potency. We further
designed and synthesized 47f−g bearing a larger alkyloxy group
such as a (cyclopropyl)methoxy group at the o- or m- position of
its terminal phenyl ring. The resulting compounds both showed
significantly potent inhibitory activity. In particular, 47g (IC50 =
Although the metabolites of 48c were not identified, we
presumed that the instability of 48c was associated with
metabolism at its phenylethyl moiety. We expected that
stereoselective introduction of a hydroxyl group into the benzylic
position of 48c should improve its metabolic stability in mice by
preventing metabolism at the phenylethyl moiety. On the basis of
this consideration, we synthesized 49a and performed its
pharmacokinetics study in mice. This study revealed that the
metabolic stability of 49a in mice was dramatically improved and
the AUC value of 49a was almost 15-fold higher than that of 48c
(Figure 5, Table 4). Fortunately, 49a also retained its potent
human dUTPase inhibitory activity (IC50 = 0.15 μM, Table 5)
and its desirable lipophilicity (clogP 2.18).
We next designed and synthesized 49b−h in order to optimize
the enzyme inhibitory potency of the 1,2,3-triazole containing
uracil derivatives and evaluated (Table 5). The results of this
study showed that 49b (IC50 = 0.21 μM), having a cyclo-
pentyloxy group, and 49h (IC50 = 0.15 μM), having a
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Table 5. Human dUTPase Inhibitory Activity of Triazole-
Containing Uracil Derivatives 49a−h
Figure 4. Pharmacokinetic profile of 48c after oral administration dose
at 50 mg/kg in Balb/cA mice (n = 2, ♂). Compound 48c was
administered as a solution (2.5% DMA, 2.5% Tween 80, 10%
Cremophor EL). The pharmacokinetic parameters of 48c are shown
in Table 4. M-1, M-2, M-3, and M-4 represent the metabolites of 48c.
The HPLC analysis chart at 0.5 h after the oral administration is also
shown in Figure S2 in the Supporting Information.
a
Enzyme inhibition assays are tested at 1.0 μM or below. IC50 values
are shown as the mean SE (n = 3).
Table 6. Human dUTPase Inhibition Activity of Triazole-
Containing Uracil Derivatives 50a−d and 49a
Figure 5. Pharmacokinetic profile of 49a after oral administration dose
at 50 mg/kg in Balb/cA mice (n = 2, ♂). Compound 49a was
administered as a solution (2.5% DMA, 2.5% Tween 80, 10%
Cremophor EL). Pharmacokinetics parameters of 49a are shown in
Table 4. M-1, M-2, and M-3 represent the metabolites of 49a. The
HPLC analysis chart at 0.5 h after oral administration is also shown in
Figure S2 in the Supporting Information.
a
Table 4. Pharmacokinetics Parameters of 48c and 49a after
R or S
X
Y
IC50 (μM)a
Oral Administration Dose at 50 mg/kg in Balb/cA mice (n =
a
49a
50a
50b
50c
50d
S
R
S
S
R
CH2
CH2
O
H
H
H
F
0.15 0.0030
>1.0
2, ♂)
AUC0−t (μM·h)
T1/2 (h)
Tmax (h)
Cmax (μM)
0.17 0.0074
0.067 0.0017
0.35 0.0049
CH2
CH2
48c
49a
4.56
0.5−1
1−2
0.5
0.5
8.69
F
67.37
62.06
a
a
Enzyme inhibition assays are tested at 1.0 μM or below. IC50 values
Each compound was administered as a solution (2.5% DMA, 2.5%
are shown as the mean SE (n = 3).
Tween 80, 10% Cremophor EL).
(cyclobutyl)methoxy group, retained the inhibitory activity.
However, none of these compounds had more potent inhibitory
activity than 49a.
group of 49a was replaced by an oxygen atom, showed potent
inhibitory activity (IC50 = 0.17 μM) comparable to 49a. We next
examined compound 50c having a fluoro group at the p-position
of the terminal phenyl ring because the introduction of a fluoro
group at the same position of sulfonamide-containing uracil
compound 5 significantly increased its potency compared to the
compound without the fluoro group in our previous report.20 As
we expected, compound 50c showed highly potent inhibitory
activity (IC50 = 0.067 μM, Table 6). The potency of R isomer 50d
(distomer) was weaker than that of S isomer 50c (eutomer)
(eudismic ratio = 5.2).
Optimization of Compound 49a. To develop compounds
with potent inhibitory activity and a desirable pharmacokinetic
profile, we performed a SAR study of the derivatives of 49a. Thus,
we examined the influence of the linker between the uracil ring
and the triazole ring, and the stereochemistry of benzylic position
of 49a (Table 6). Compound 50a (distomer), which is an
enantiomer of 49a (eutomer), clearly exhibited weaker enzyme
inhibitory potency (IC50 > 1.0 μM) as seen in the difference of
potency of 48c and 48d. Compound 50b, in which a methylene
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Although the novel dUTPase inhibitors reported in this paper
are not triphosphate mimics of a natural substrate dUTP, these
compounds exhibited potent human dUTPase inhibitory
activity. In particular, 48a, 48c, and 50c exhibited high potency
with IC50 values in the 10−8 M range. Such low IC50 value
suggests that the active site of the enzyme can recognize these
compounds better than the natural substrate dUTP (the
concentration of a substrate dUTP in the enzyme inhibition
assays is 0.1 μM). Therefore, elucidation of the enzyme
inhibition mode by these compounds is crucial to discover
further potent and clinical applicable inhibitors of human
dUTPase. We previously described the cocrystal X-ray structure
of pyrrolidylsulfonamide-containing uracil derivative 7 with
human dUTPase.19 This X-ray structure showed that the uracil
ring of 7 occupies the uracil recognition region and that one of
the terminal phenyl rings of 7 occupies a hydrophobic region
which is formed by Val65, Ala90, Ala98, and Val112 residues of
the active site and makes a stacking interaction with its own uracil
ring (Figure S1 in the Supporting Information). From these
results, the uracil rings and the terminal phenyl rings of these
triazole-containing uracil derivatives were proposed to make
similar interactions with human dUTPase. We further proposed
that the high potency of eutomers (48a, 48c, and 50c) can be
attributed to the efficient insertion of their phenyl ring into the
hydrophobic region of the enzyme and that the improvement in
potency observed by the introduction of a (cyclopropyl)methoxy
group into the terminal phenyl ring can be attributed to an
enhancement of hydrophobic interactions between the inhibitor
and human dUTPase.
Human dUTPase Inhibitors Enhance the Growth
Inhibition Activity of FdUrd in Vitro. We performed in
vitro growth inhibition experiments with certain of our novel
dUTPase inhibitors. These studies were done on HeLa S3 cells in
combination with 5-fluoro-2′-deoxyuridine (FdUrd) to evaluate
the chemotherapeutic enhancing effects of the dUTPase
inhibitors. We performed cell proliferation assay by treatment
with FdUrd at the concentration of 1 μM combined with various
concentrations of the dUTPase inhibitors. T/C values (%) were
obtained as the ratio of cell density determined by the crystal
violet assay with drug treatment to without drug treatment for 24
h. To show the enhancement effect of the dUTPase inhibitor for
growth inhibition activity of FdUrd, the EC50 (μM) value was
calculated from concentration−T/C (%) curve as the concen-
tration reducing the T/C (%) value by treatment with only
FdUrd (T/C 70−80%) by half.
(Figure 6, Table 8). Pharmacokinetics and enzyme inhibitory
activity data (Table 6) showed that 50c had a desirable profile for
Figure 6. The pharmacokinetic profile of 50c after oral administration
dose at 50 mg/kg in Balb/cA mice (n = 2, ♂). Compound 50c was
administered as a solution (2.5% DMA, 2.5% Tween 80, 10%
Cremophor EL). The pharmacokinetic parameters of 50c are shown
in Table 8. M-1, M-2, and M-3 represents the metabolites of 50c.
an in vivo study of mice and that the pharmacokinetic profile of
50c (AUC: 57.3 μM·h) in mice was superior to that of 5 (AUC:
24.9 μM·h)20 reported previously.
We next evaluated the enhancing effect of 50c for antitumor
activity of 5-FU against in vivo breast cancer xenograft composed
of MX-1 cells in vivo in mice. The details of the protocol for in
vivo evaluation in mice are described in the Experimental
Section. Compound 50c alone exhibited no antitumor activity in
vivo, which was consistent with feeble in vitro cytotoxicity of 50c
alone (Table 7). On the other hand, compound 50c significantly
enhanced the antitumor activity of 5-FU without significant body
weight loss (Figure 7, Table 9).
Table 7. Enhancing Effect of dUTPase Inhibitors for Growth
Inhibition Activity of FdUrd against HeLa S3 Cells in Vitro
cytotoxicity EC50
EC50 (μM) With 1 μM
dUTPase IC50
a
b
c
(μM)
FdUrd
(μM)
45a
47g
48c
50c
>100
1.1 0.022
0.26 0.0046
0.05 0.00049
0.07 0.0013
1.3 0.052
0.21 0.0026
0.029 0.00047
84 2.1
22 0.13
41 0.25
0.067 0.0017
a
b
Cytotoxity of dUTPase inhibitors against HeLa S3 cells (72 h). EC50
We selected four representative compounds (45a, 47g, 48c,
50c) from each group of derivatives reported in this paper for
evaluation of enhancement activity. These compounds exhibited
feeble growth inhibition activity against HeLa S3 cell by
themselves (EC50 = 23−100 μM). However, these compounds
dramatically enhanced the growth inhibition activity of FdUrd (1
μM) against HeLa S3 cells (EC50 = 0.05−1.1 μM). Moreover, the
degree of the enhancement correlated well with the potency of
dUTPase inhibition. On the basis of these data, the ability of
these compounds to enhance the growth inhibition mediated by
FdUrd clearly can be ascribed to their ability to inhibit dUTPase
in HeLa S3 cells.
values show the concentration at which the dUTPase inhibitor reduces
the T/C (%) value of FdUrd (1 μM, 70−80%) against HeLa S3 cells
c
(24 h) by half. EC50 values are shown as the mean SE (n = 3). IC50
values show dUTPase inhibitory activity as the mean SE (n = 3).
Table 8. Pharmacokinetics Parameters of 50c after Oral
Administration Dose at 50 mg/kg in Balb/cA mice (n = 2, ♂)
a
AUC0−t (μM·h)
T1/2 (h)
1−2
Tmax (h)
Cmax (μM)
50c
57.28
1
49.80
a
Compound 50c was administered as a solution (2.5% DMA, 2.5%
Tween 80, 10% Cremophor EL).
Human dUTPase Inhibitor 50c Enhances the Anti-
tumor Effect of 5-FU in Vivo. We performed a pharmacoki-
netics study on the highly potent dUTPase inhibitor 50c to
estimate the feasibility for an in vivo study of mice performed via
oral administration. As expected, 50c was well absorbed and
metabolically stable in mice, comparable to the properties of 49a
CONCLUSIONS
A 1,2,3-triazole-containing uracil compound 45a was designed as
an analogue of tert-amide-containing 6b wherein the 1,2,3-
■
H
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Journal of Medicinal Chemistry
Article
against the MX-1 xenograft model in mice. These data indicate
that our novel dUTPase inhibitors should open the way to
unprecedented beneficial chemotherapeutic strategies for cancer
patients when used in combination with TS inhibitors.
EXPERIMENTAL SECTION
■
All commercially available starting materials and solvents were reagent
grade. Silica gel column chromatography was performed on Merck silica
gel 60 (230−400 mesh). 1H NMR spectra were measured at 270 MHz
on a JEOL JNM-EX270 or 400 MHz on a JEOL JNM-LA400. 13C NMR
spectra were measured at 100 MHz on a JEOL JNM-LA400. Chemical
shifts were recorded in parts per million (ppm, δ) and were reported
relative to the solvent peak or internal tetramethylsilane peak. High-
resolution mass spectra (HRMS) were measured with a JEOL JMS-700
(FAB) or Waters micromass Q-Tof-2 (TOF). All NMR spectrum data
and MS spectrum data of intermediates and tested compounds were
presented in the Supporting Information. Chemical purities of the tested
compounds were determined by combustion analysis or HPLC analysis
and confirmed ≥95% purity. Combustion analyses (C, H, N) were
performed on a Thermo Electron Corp. Flash EA 1112 series, and the
values were within 0.4% of the theoretical values and were presented in
the Supporting Information. Chemical purities of some tested
compounds were determined by HPLC analysis with a SHIMADZU
Prominence HPLC system, and the values are presented in the
Supporting Information. Optical rotations were measured by a
HORIBA SEPA-200 polarimeter and are presented in the Supporting
Information. The clogP values were calculated using the ACD/LogP
algorithm (ACD/Laboratories release 10.00, version 10.01).
Figure 7. (A) Efficacy of 50c for enhancing the antitumor activity of 5-
FU against breast cancer xenograft MX-1 in mice. Relative tumor
volume (RTV) is reported as mean SD of at least three independent
experiments. (B) Body weight change (%) is expressed as mean SD.
dUTPase Inhibition Assay. In vitro dUTPase inhibition assays
were conducted by measuring the production of [5-3H]dUMP from
[5-3H]dUTP according to the reported method.19 Briefly, a total of 0.2
mL solution, containing 0.02 mL of 1 μM dUTP (including 588 Bq/mL
[5-3H]dUTP), 0.05 mL of a 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, followed by centrifugation at 15000 rpm for 2
min. An aliquot (150 μL) of the supernatant thus obtained by
centrifugation was analyzed using an Atlantis dC18 column (manufac-
tured by Waters Corp., 4.6 mm × 250 mm) and high-performance liquid
chromatography (manufactured by Shimadzu Corp., Prominence). The
inhibitory rate of the compound was calculated from the formula shown
below. IC50 (μM), which the concentration of the inhibitor yielding 50%
inhibition rate, was obtained from the concentration−inhibitory rate
curve.
triazole moiety mimics the amide bond of 6b locked in a cis
conformation. Compound 45a possessed potent human
dUTPase inhibitory activity, comparable to 6b. We focused on
47b, which is the monophenyl analogue of 45a, as a lead
compound because of its lower molecular weight and ease of the
molecular modification. The SAR studies of the lead compound
47b led to the discovery of 48a, 48c, and 50c, which have high
potency (IC50 = 0.029−0.067 μM). The affinity of these
compounds for dUTPase was proposed to be superior to that
of the natural substrate dUTP because their IC50 values were
lower than the concentration of a substrate dUTP in the enzyme
inhibition assays. These novel triazole-containing uracil deriva-
tives markedly enhanced the growth inhibition activity of FdUrd
against HeLa S3 cells in vitro with IC50 values in the 10−7−10−8
M range. To the best of our knowledge, a pharmacokinetics study
of 50c in mice revealed that the AUC value of 50c is the highest
among the previously reported dUTPase inhibitors. It is
noteworthy that 50c, which exhibited high human dUTPase
inhibitory potency and a desirable pharmacokinetic profile in
mice, significantly enhanced the antitumor activity of 5-FU
Table 9. in Vivo Enhancing Efficacy of 50c for Antitumor Activity of 5-FU against Breast Cancer Xenograft MX-1 in Mice
a
b
c
dose (mg/kg/day)
treatment
TV (mm3, mean SD)
RTV (mean SD)
IR (%)
control
5-FU
2047.62 693.70
1644.48 322.60
2474.95 534.68
300.68 103.20
11.41 3.74
9.13 1.35
14.06 3.35
15
ci
20.0
50c
300
po
−23.2
84.9
d
5-FU/50c
15/300
ci/po
1.72 0.55
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 based on the RTV was calculated according to the following formula: IR (%) = [1 − (mean
d
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.
I
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Journal of Medicinal Chemistry
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3
plantation in mice. For the experiment, tumors were excised and
fragments (∼2 mm in diameter) were implanted subcutaneously by
trocar with fragment of human tumor. After implantation, the animals
were divided into four groups and treated with either vehicle (2.5%
DMA, 2.5% Tween 80, 10% cremophor EL, and 0.5% HPMC), 5-FU
(15 mg/kg/day) by continuous infusion using osmotic pump for 14
days, compound 50c (300 mg/kg/day, once daily) by po for 14 days, or
a combination of 5-FU (15 mg/kg/day) and compound 50c (300 mg/
kg/day, once daily) for 14 days. Tumor size and body weight were
measured twice a week. Tumor volume (TV) were 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). The inhibition rate of tumor growth
on day 15 on the basis of RTV was calculated according to the following
formula:
⎣
⎡
inhibitory rate (%) = 1 − amount of [5‐ H]dUMP in presence
(
of test solution (dpm)
)
amount of [5‐3H]
(
⎦
⎤
dUMP as control (dpm) × 100
)
Evaluation of in Vitro Cytotoxicity. HeLa S3 cells (human ovarian
carcinoma) were grown in the RPMI1640 medium supplemented with
10% fetal bovine serum (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. After 24 h, various concentration of
test compounds were added to the corresponding plates at the volume of
20 μL per well, the plates were incubated for 72 h, and then cell
proliferation was determined by the crystal violet assay. Optical density
at 540 nm (OD540) was measured by a plate reader. Then, we calculated
the T/C (%) value which is the ratio of the OD540 with drug treatment to
without drug [T/C (%) = (OD540 of treated well/OD540 of nontreated
well) × 100]. The EC50 (μM) for the cytotoxicity of the test compound
was the concentration yielding 50% T/C value, which was calculated
from the concentration−T/C (%) curve.
⎛
⎞
⎟
⎠
mean RTV of the treated group
mean RTV of the control group
inhibition rate (%) = ⎜1 −
⎝
× 100
Evaluation of the Enhancement of the Growth Inhibition
Activity of FdUrd in Vitro. 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, five different concentrations
of test compounds and FdUrd (1 μM) were added to the corresponding
plates at the volume of 20 μL per well. After 24 h incubation, thymidine
(30 μM) was added to the plates at the volume of 10 μL per well. After
further 48 h incubation, the cell density was measured and the T/C (%)
values were obtained as mentioned above. The EC50 for the
enhancement of the growth inhibition activity of FdUrd was calculated
from the concentration−T/C (%) curve as the concentration at which
the test compound reduced the T/C (%) value for the treatment of 1 μM
FdUrd by half.
Ethics Statement. All animal experiments were performed with the
approval of the Animal Ethics Committees of Animal Care and Use.
Evaluation of Pharmacokinetics Profile. Balb/cA mice were also
used to study the pharmacokinetic properties of the inhibitors. Male
Balb/cA mice were supplied by CLEA Japan, Inc. (Tokyo, Japan). Blood
samples were collected at five time points (30 min, 1 h, 2 h, 4 h, 6 h) after
a single oral dose of 50 mg/kg inhibitor in a solution (2.5%DMA, 2.5%
Tween 80, 10% Cremophor EL). The quantitation of inhibitor plasma
levels was analyzed by reverse-phase ultraperformance liquid
chromatography (0.35 mL/min 10 mM ammonium acetate/acetonitrile
gradient, 10 cm, 2.1 mm i.d. Acquity BEH Shield RP18 column, UV
detection 268 or 270 nm). Ultraperformance liquid chromatography
condition was shown in below.
The body weight change was calculated according to the following
formula:
body weight change (%)
[(body weight) − (body weight on day 0)]
=
× 100
(body weight on day 0)
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details and analytical data for intermediates and
test compounds, details of the binding model of 6b with human
dUTPase, and HPLC charts of the pharmacokinetics studies
described in the article. This material is available free of charge via
the Internet at http://pubs.acs.org.
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
■
System: Waters ultraperformance liquid chromatography
Column: ACQUITY BEH Shield RP18 2.1 mm × 100 mm
Column temperature: 40 °C
Mobile phase: A = 10 mM ammonium acetate, B = acetonitrile
Flow rate: 0.35 mL/min
We thank Dr. K. L. Kirk (NIDDK, NIH), Dr. Tadafumi Terada
(Taiho Pharmaceutical Co. Ltd.), and Dr. Kazuharu Noguchi
(Taiho Pharmaceutical Co. Ltd.) for very helpful suggestions and
Satoko Tanaka, Emi Inaba, and the staff of the Drug Discovery
Research Center of Taiho Pharmaceutical Co. Ltd. for their
technical assistance.
Detection: UV 268 or 270 nm
Gradient condition:
min A (%) B (%)
ABBREVIATIONS USED
■
0.00 95
0.40 95
6.00 20
6.50 20
6.51 95
9.00 95
5
5
80
80
5
dUTPase, deoxyuridine triphosphatase; TS, thymidylate syn-
thase; 5-FU, 5-fluorouracil; dUTP, deoxyuridine triphosphate;
dUMP, deoxyuridine monophosphate; dTMP, thymidine mono-
phosphate; dTTP, thymidine triphosphate; FdUTP, 5-fluoro-2′-
deoxyuridine triphosphate; (TMS)2Ura, bistrimethylsilyluracil;
DIAD, diisopropyl azodicarboxylate; DIEA, N-ethyldiisopropyl-
amine; NaHMDS, sodium bis(trimethylsilyl)amide; EDC·HCl,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlor-
ide; HOBt, 1-hydroxybenzotriazole; TEA, triethylamine;
Cp*RuCl(COD), chloro(pentamethylcyclopentadienyl)-
(cyclooctadiene)ruthenium(II); FdUrd, 5-fluoro-2′-deoxyuri-
dine; SE, standard error; SD, standard deviation; TV, tumor
5
The pharmacokinetic parameters were derived from the mean plasma
concentrations at each time point using the WinNonlin computer
program (Pharsight).
Evaluation of Antitumor Efficacy of Compound 50c. Balb/cA
JcL-nu mice (five weeks of ages) were obtained from Clea Japan, Inc.
(Tokyo, Japan). MX-1 human breast carcinoma (Japanese Foundation
For Cancer Research) was maintained by subcutaneous (sc) trans-
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dx.doi.org/10.1021/jm3004174 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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volume; RTV, relative tumor volume; IR, inhibition rate; FBS,
fetal bovine serum; HPMC, hydroxypropyl methylcellulose
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