Synthesis and discovery of N -carbonylpyrrolidine- or N -sulfonylpyrrolidine-containing uracil derivatives as potent human deoxyuridine triphosphatase inhibitors

Article abstract of DOI:10.1021/jm201627n

Recently, deoxyuridine triphosphatase (dUTPase) has emerged as a potential target for drug development as part of a new strategy of 5-fluorouracil-based combination chemotherapy. We have initiated a program to develop potent drug-like dUTPase inhibitors based on structure-activity relationship (SAR) studies of uracil derivatives. N-Carbonylpyrrolidine- and N-sulfonylpyrrolidine- containing uracils were found to be promising scaffolds that led us to human dUTPase inhibitors (12k) having excellent potencies (IC₅₀ = 0.15 μM). The X-ray structure of a complex of 16a and human dUTPase revealed a unique binding mode wherein its uracil ring and phenyl ring occupy a uracil recognition region and a hydrophobic region, respectively, and are stacked on each other. Compounds 12a and 16a markedly enhanced the growth inhibition activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells in vitro (EC₅₀ = 0.27-0.30 μM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemotherapeutic strategies when used in combination with TS inhibitors.

Full text of DOI:10.1021/jm201627n

Article
pubs.acs.org/jmc
Synthesis and Discovery of N-Carbonylpyrrolidine- or
N-Sulfonylpyrrolidine-Containing Uracil Derivatives as Potent
Human Deoxyuridine Triphosphatase Inhibitors
Hitoshi Miyakoshi,†,§ Seiji Miyahara,†,‡ Tatsushi Yokogawa,† Khoon Tee Chong,† Junko Taguchi,†
Kanji Endoh,† Wakako Yano,† Takeshi Wakasa,† Hiroyuki Ueno,† Yayoi Takao,† Makoto Nomura,†
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: Recently, deoxyuridine triphosphatase (dUT-
Pase) has emerged as a potential target for drug development
as part of a new strategy of 5-fluorouracil-based combination
chemotherapy. We have initiated a program to develop potent
drug-like dUTPase inhibitors based on structure−activity
relationship (SAR) studies of uracil derivatives. N-Carbonyl-
pyrrolidine- and N-sulfonylpyrrolidine-containing uracils were
found to be promising scaffolds that led us to human dUTPase
inhibitors (12k) having excellent potencies (IC50 = 0.15 μM).
The X-ray structure of a complex of 16a and human dUTPase
revealed a unique binding mode wherein its uracil ring and
phenyl ring occupy a uracil recognition region and a hydro-
phobic region, respectively, and are stacked on each other.
Compounds 12a and 16a markedly enhanced the growth inhibition activity of 5-fluoro-2′-deoxyuridine against HeLa S3 cells in
vitro (EC50 = 0.27−0.30 μM), suggesting that our novel dUTPase inhibitors could contribute to the development of chemo-
therapeutic strategies when used in combination with TS inhibitors.
dUMP and pyrophosphate.14−16 This enzyme is thought to be
INTRODUCTION
■
Thymidylate synthase (TS) inhibitors,1−3 including 5-fluoroura-
responsible for two biological roles: (1) decreasing the
intracellular dUTP pools to prevent the uracil misincorporation
cil (5-FU) and its derivatives, are widely used in the treatment
instead of thymine into DNA and (2) supplying dUMP as a
of a range of cancers. However, the clinical antitumor effect of
substrate for TS to be converted to thymidine monophosphate
TS inhibitors is often restricted by the occurrence of intrinsic or
(dTMP), an essential precursor for the de novo pathway of
acquired drug resistance. Some reports have shown that the
DNA synthesis.17 Therefore, dUTPase plays an important role
effect of TS inhibitors can be influenced by a number of fac-
tors.4−7 For example, Ladner et al. found that deoxyuridine
in maintenance of the nucleotide homeostasis by strict regula-
tion of the cellular dUTP/dTTP ratio during DNA replication
and repair. In addition, it has been reported that dUTPase is
also able to hydrolyze 5-fluoro-2′-deoxyuridine triphosphate
(FdUTP).18 Accordingly, dUTPase expression can mediate
resistance to 5-FU by preventing the misincorporation of
FdUTP and dUTP into DNA in cancer cells (Figure 1).19−21
Indeed, siRNA-mediated suppression of human dUTPase signi-
ficantly enhanced cell growth inhibition effect of 5-fluoro-2′-
deoxyuridine (FdUrd) against colon and breast cancer cell
lines.22 It also has been reported that low intratumoral levels of
nuclear dUTPase expression in clinical specimens are associated
triphosphatase (dUTPase, EC 3.6.1.23) could be an important
determinant for the antitumor effect of TS inhibitors.8
TS inhibitors decrease the intracellular dTTP (thymidine
triphosphate) pools (thymineless death) concurrently with in-
creasing the deoxyuridine monophosphate (dUMP) pools,
which results in expansion of intracellular deoxyuridine
triphosphate (dUTP) pools. dUTP is one of the noncanonical
nucleoside triphosphates (NTPs) which cause DNA damage by
being incorporated into DNA.9 Because DNA polymerase
cannot recognize the difference between dTTP and dUTP,10
the expansion of dUTP pools causes uracil misincorporation
into DNA, which is one of the key mechanisms of the cyto-
toxity induced by TS inhibitors.11−13 dUTPase specifically
recognizes dUTP among natural NTPs and hydrolyzes it to
Received: December 1, 2011
Published: March 9, 2012
© 2012 American Chemical Society
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Figure 1. Proposed mechanism of the enhancing effect of dUTPase inhibitors for the antitumor effect of TS inhibitors by inducing severe DNA
damage.
Figure 2. Structural formula of dUTPase inhibitors.
with better response to 5-FU-based chemotherapy, longer
time to progression, and greater survival in colorectal
cancer.8 Furthermore, Takatori et al. have reported that nuclear
dUTPase activation significantly correlates with a poor prognosis
in hepatocellular carcinoma patients,23 and Kawahara et al. also
have reported that the expression of dUTPase may predict the
metastatic potential of colorectal cancer.24 Consequently,
dUTPase has emerged as a potential target for chemotherapeutic
drug development.
Although several previous efforts have been made to develop
dUTPase inhibitors (Figure 2),25−34 to the best of our
knowledge, none of them is evaluated in clinical trials. In our
own research, we focused on the specificity of the uracil ring for
dUTPase and prepared a library of uracil derivatives to be
screened as potential inhibitors. We then focused on com-
pounds obtained from the first screening to develop potent
drug-like dUTPase inhibitors. Such dUTPase inhibitors would
provide a new strategy for combination chemotherapy with TS
inhibitors. In this paper, we report the design and synthesis of
novel uracil compounds, their evaluation as human dUTPase
inhibitors, and their abilities to enhance the growth inhibition
activity of FdUrd against cancer cells. We also discuss the
mechanism of inhibition of these new inhibitors based on X-ray
analysis of the crystal structure of a complex of the dUTPase
inhibitor and human dUTPase.
CHEMISTRY
■
Pyrrolidine derivatives 8a−j were synthesized according to the
method of Li et al.35 In brief, chiral N-carboethoxy-proline
methyl esters 6a and 6b were treated with the appropriate
Grignard reagents to provide 7a−j. These were then converted
to the corresponding amines 8a−j by removal of the
ethoxycarbonyl group (Scheme 1).
Amide-containing uracil derivatives 11a−w and N-carbon-
ylpyrrolidine-containing uracil derivatives 12a−l were prepared
by a condensation reaction between a known carboxylic acid
936 or 1036 and the appropriate amine (8a−f, or a known amine)
(Scheme 2).
The synthesis of N-sulfonylpyrrolidine-containing uracil
derivatives 16a−f is shown in Scheme 3. Pyrrolidine derivatives
8g−l were treated with 3-chloropropanesulfonyl chloride to pro-
vide 13a−f, followed by reaction with AcONa to give 14a−f.
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Scheme 1. General Synthetic Method for Preparation of
Pyrrolidine Derivatives 8a−j
RESULTS AND DISCUSSION
■
a
Molecular Design of dUTPase Inhibitors. First, we
addressed the strict substrate specificity of dUTPase14 for uracil
by preparing our own library of uracil derivatives for screening
as dUTPase inhibitors. In this library, we identified a hit
compound 5 (IC50 = 97 μM) which had moderate but superior
or comparable inhibitory activity to those of the known
dUTPase inhibitors such as 1b and 3 (Table 1). This finding
enforced us to perform further structure−activity relationship
(SAR) studies focused on amide or sulfonamide-containing
uracil structures in which the linker and amine moiety of hit
compound 5 were modified for structural optimization to
improve inhibitory potency (Figure 3).
Structure−Activity Relationship Study of Amide-
Containing Uracil Derivatives. Human dUTPase inhibitory
activities of amide-containing uracil derivatives are shown in
Table 1. Among the derivatives having an ethylene linker (n = 1),
11i (IC50 = 7.3 μM) showed an almost 2-fold greater potency as
a dUTPase inhibitor compared to the substrate dUTP-mimic 1b.
Among the derivatives having a trimethylene linker (n = 2), 11q
(IC50 =16 μM) and 11r (IC50 = 2.5 μM) having the N-2,2-
diphenylethyl moiety possessed moderate potencies. In partic-
ular, 11s (IC50 = 1.3 μM) and 11w (IC50 = 1.1 μM), bearing
both a diphenylmethyl and a tert-amide group, have relatively
high and comparable potency that is not influenced by the pres-
ence or absence of the tert-alcohol group. These derivatives also
have a favorable level of lipophilicity (clogP: 1.83−2.17).
SAR study of these amide-containing uracil derivatives
showed clearly that the tert-amide compound 11s had almost
a 12-fold more potent inhibitory activity compared to the sec-
a
Reagents and conditions: (a) R1 MgBr, THF, 0 °C, 3 h, 60−81%; (b)
KOH, MeOH, reflux, 12 h, 85−97%.
Scheme 2. General Synthetic Method for the Preparation of
Derivatives 5 and 11a−w (See Table 1) and 12a−l (See
Table 2)
a
a
Reagents and conditions: (a) amine, EDC·HCl, HOBt, DMF, room
1
temp, 3 h, 14%−quant. R2: various amino groups. R3: H or OH.
amide compound 11q. Interestingly, the H NMR spectrum
and NOE data of 11s in DMSO-d6 revealed that 11s existed as
mixture of two conformational isomers at 25 °C (cis:trans = 2:3)
(Figure 4, see also Figure S1 in the Supporting Information).
Although it is not apparent, the existence of the cis-isomer of
tert-amide 11s may make some contribution to binding to the
enzyme. We may suppose that the extra hydrophobic interaction
of the tert-amide methyl group could be the key to its higher
activity.
Scheme 3. General Synthetic Method for the Preparation of
Derivatives 16a−f (See Table 3)
a
Structure−Activity Relationship Study of N-Carbon-
ylpyrrolidine-Containing Uracil Derivatives. Next, we
envisaged that reducing the entropy loss associated with
binding of the bulky alkyl amino chain present in 11s could
increase binding to the enzyme and translate into increased
inhibitory activity. On the basis of this hypothesis, we designed
and synthesized N-carbonylpyrrolidine-containing uracil deriv-
atives that have enhanced rigidity of the side chain relative to
11s because of the reduced number of rotatable bonds.
We were pleased that our prediction proved to be correct.
In fact, certain of the N-carbonylpyrrolidine derivatives had
remarkably increased inhibitory activity. This structural adjust-
ment also resulted in a more favorable level of lipophilicity
(Table 2). A comparison of the potency of 12a (S configuration)
to that of 12b (R configuration) revealed the influence of the
stereochemistry at the C-2 position of the pyrrolidine ring. Thus,
the S enantiomer, 12a, was a highly potent inhibitor (IC50 = 0.29
μM). By comparison, the potency of 12b (R configuration, IC50 =
10 μM, eudismic ratio = 34) was significantly weaker than that
of 12a. On the basis of this result, various derivatives having the
S configuration were synthesized for evaluation. Compound 12k,
wherein the hydroxyl group is absent, also had higher potency
than 12a. This result clearly suggests that the terminal hydroxyl
a
Reagents and conditions: (a) Cl(CH2)3SO2Cl, TEA, CH2Cl2, room
temp, 2 h, 73%−quant; (b) AcONa, NaI, DMF, 90 °C, 12 h, 63−70%;
(c) MeNH2, MeOH, room temp, 1 h, 92−99%; (d) N-3-Bz-uracil,
PPh3, DIAD, THF, room temp, 2 h then MeNH2, MeOH, room temp,
1 h, 55−73%.
After removal of the acetyl group, alcohol compounds 15a−f
were coupled with 3-N-benzoyluracil37 by a Mitsunobu reaction.
Removal of the benzoyl group with methylamine provided 16a−f.
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Table 1. Human dUTPase Inhibitory Activity of Amide-
Containing Uracil Derivatives 5, 11a−w, and Reference
Compounds 1−4
Figure 3. Structural formula of hit compound 5 (X = CO) and
molecular modification for the SAR study.
than an ethylene linker (n = 1) because the potency of 12l
bearing an ethylene linker was much less than that of 12a.
We next evaluated the effect of aromatic substituents R2 on
potency by comparing the activities of compounds 12c−j to
12a. Although the inhibitory activities of the analogues with
electron-withdrawing groups such as F and Cl on the phenyl
ring were comparable to that of 12a, the parent compound 12a
remained the best activity, while introduction of a electron-
donating methoxy group into any position of the terminal
phenyl ring obviously lowered inhibitory activity (12g−i). The
thiophenyl isosteric isomer 12j was as potent as the parent 12a.
Structure−Activity Relationship Study of N-Sulfonyl-
pyrrolidine-Containing Uracil Derivatives. We also de-
signed and synthesized several N-sulfonylpyrrolidine-containing
uracil derivatives to examine the effects of structural changes in
the sulfonamide series (Table 3). It is interesting that, in a series
of sulfonamide derivatives, the R enantiomer (16a) was a
significantly more potent inhibitor of dUTPase (IC50 = 0.32 μM)
than the corresponding S enantiomer (16b; IC50 = 9.4 μM,
eudismic ratio = 29), in contrast to the N-carbonylpyrrolidine-
containing uracil series. Introduction of a fluorine atom into the
m- or p-position of the terminal phenyl ring of 16a did not
diminish the highly potent inhibitory activity (16c−d, IC50 =0.28−
0.31 μM). However, introduction of a methoxy group into the o- or
m-position clearly reduced potency, similar to the results observed
with the N-carbonylpyrrolidine derivatives.
The strategies describe above have allowed us to develop
highly potent dUTPase inhibitors N-carbonylpyrrolidine-
containing uracil derivatives such as 12a (IC50 = 0.29 μM)
and N-sulfonylpyrrolidine-containing derivatives such as 16a
(IC50 = 0.32 μM). In particular, the most potent compound,
12k (IC50 = 0.15 μM), was a remarkably more potent inhibitor
of dUTPase (ca. 100−2000-fold) than previously described
dUTPase inhibitors such as compounds 1−3. These novel
dUTPase inhibitors also have drug-like properties that meet the
standards of Lipinsky’s rule-of-five (for example: 12a clogP,
1.82; molecular weight, 433.5; H-bond donors, 2; H-bond
acceptors, 7; 16a clogP, 1.92; molecular weight, 469.6; H-bond
donors, 2; H-bond acceptors, 8).38 Among previously described
dUTPase inhibitors, compound 4 was reported to be the most
potent.30 However, 4 does not satisfy Lipinsky’s rule-of-five
(molecular weight, 587.54 >500; H-bond acceptors, 16 >10).
Human dUTPase Inhibitors Enhances Growth Inhi-
bition 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 com-
bination with 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 inhibitor.
T/C (%) value was obtained as the ratio of cell density determined
a
b
See Experimental Section. Except compounds 1a−b, 2−3, and 5,
enzyme inhibition assay are tested at 30 μM or below. IC50 values are
shown as the mean SE (n = 3). Reference data 1a,25 1b,26 2,27 3,29
c
and 4.30 N.T. = not tested
group of N-carbonylpyrrolidine-containing uracil derivatives does
not contribute to their potency as dUTPase inhibitors. These
studies also revealed that the trimethylene linker (n = 2) between
the uracil ring and carbonyl moiety of the amide group is better
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Figure 4. Conformational preference of amide compounds 11q (trans) and 11s (trans and cis).
Table 2. Human dUTPase Inhibitory Activity of
N-Carbonylpyrrolidine-Containing Uracil Derivatives
12a−l and 11s
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,
EC50 (μM) value was calculated from concentration-T/C (%)
curve as the concentration reducing by half the T/C (%) value
by treatment with only FdUrd (T/C 70−80%).
We chose four representative compounds (11r, 11s, 12a, and
16a) from each derivative reported in this paper (Table 4).
Table 4. The Enhancing Effect of dUTPase Inhibitors for
Growth Inhibition Activity of FdUrd against HeLa S3 Cells
in Vitro
a
b
n
S or R
R2
R3
clogP
IC50 (μM)
11s
12a
12b
12c
12d
12e
12f
12g
12h
12i
2.17
1.82
1.82
1.92
1.92
3.01
3.01
1.65
1.65
1.65
1.17
2.81
1.60
1.3 0.068
2
2
2
2
2
2
2
2
2
2
2
1
S
R
S
S
S
S
S
S
S
S
S
S
Ph
Ph
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
0.29 0.015
10 0.30
cytotoxicity EC50
EC50 (μM) with 1 μM
dUTPase IC50
a
b
c
(μM)
FdUrd
(μM)
3-F-Ph
0.35 0.017
0.60 0.014
0.58 0.027
11r
11s
12a
16a
>100
>100
>100
>100
3.0 0.10
2.5 0.071
1.3 0.068
0.29 0.015
4-F-Ph
1.2 0.037
3-Cl-Ph
4-Cl-Ph
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
3-thienyl
Ph
0.27 0.0041
0.30 0.0050
c
>1.0
0.32 0.018
c
>1.0
a
b
Cytotoxity of dUTPase inhibitors against HeLa S3 cells (72 h). EC50
c
>1.0
value shows the concentration at which the dUTPase inhibitor reduces
c
>1.0
by half the T/C (%) value of FdUrd (1 μM, 70−80%) against HeLa S3
c
12j
0.23 0.0033
0.15 0.0094
cells (24 h). EC50 values are shown as the mean SE (n = 3). IC50
12k
12l
value shows dUTPase inhibitory activity as the mean SE (n = 3).
c
Ph
OH
>1.0
a
b
None of the compounds inhibited growth of HeLa S3 cells by
themselves at the concentration of 100 μM for 72 h. However,
low concentration of each of these compounds dramatically
enhanced the FdUrd (1 μM) mediated inhibition of growth of
HeLa S3 (EC50 = 0.27−3.0 μM). Moreover, the degree of the
enhancement correlated well with the potency of dUTPase
inhibition. On the basis of these results, 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.
See Experimental Section. IC50 values are shown as the mean SE
c
(n = 3). Except 12b, enzyme inhibition assay are tested at 1.0 μM or below.
Table 3. Human dUTPase Inhibitory Activity of
N-Sulfonylpyrrolidine-Containing Uracil Derivatives
16a−f and 11s
X-Ray Structure of a Complex of 16a and dUTPase.
Our novel dUTPase inhibitors that possess a uracil ring and
either a chiral diphenylmethyl pyrrolidylamide or sulfonamide
moiety are quite different in structure from the natural sub-
strate dUTP. Nevertheless, they are highly potent inhibitors of
dUTPase, suggesting that the active site of the enzyme can
recognize these structures as readily as dUTP (the concen-
tration of a substrate dUTP in the enzyme inhibition assays is
0.1 μM). Therefore, elucidation of the binding mode of these
derivatives to the active site of dUTPase by using X-ray diffrac-
tion data should facilitate development of additional potent
inhibitors for clinical use. Accordingly, high-resolution data
(1.7 Å) was obtained from a cocrystal of human dUTPase with
the highly potent N-sulfonylpyrrolidine containing inhibitor
16a. This allowed the visualization of the electron density map
a
b
S or R
R2
clogP
IC50 (μM)
11s
16a
16b
16c
16d
16e
16f
2.17
1.92
1.92
2.02
2.02
1.75
1.75
1.3 0.068
R
S
Ph
Ph
0.32 0.018
9.4 0.085
R
R
R
R
3-F-Ph
0.28 0.0026
0.31 0.0071
4-F-Ph
c
2-MeO-Ph
>1.0
c
3-MeO-Ph
>1.0
a
b
See Experimental Section. IC50 values are shown as the mean SE
(n = 3). Except 16b, enzyme inhibition assay are tested at 1.0 μM or
c
below.
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of the enzyme−inhibitor complex. A summary of statistics for
data collection and refinement are shown in Table S1 in the
Supporting Information.
Figure 5A shows the principal polar interactions between
human dUTPase and 16a in the cocrystal structure. The
Figure 6. Superimposition of 16a (blue stick) on the catalytic site of
X-ray structure of dUTPase (light-pink ribon) with α,β-imino-dUTP
1b (red stick) (PDB code: 2HQU).39 The green residue is Phe158 of a
C-terminal tail. Ordering of the C-terminal tails upon the active sites is
essential for initiation of dUTPase catalysis.14,39
structure, become ordered upon ligand binding to each of
the active sites between two subunits. The ordering of the
C-terminal tails is essential for the trimeric dUTPase enzyme to
exhibit enzymatic function.14,39 In detail, the phenyl ring of
Phe158 in this flexible tail is located in a hydrophobic region
formed by Val65, Ala90, Ala98, and Val112 and is stacked with
the uracil ring of a substrate. This stacking interaction is essen-
tial for dUTPase catalysis. Details of Figures 5A and 6 suggest
that one of the terminal phenyl rings of 16a occupies the
hydrophobic region instead of the phenyl ring of Phe158. It
follows that the potency of 16a as a dUTPase inhibitor may be
attributed to this stable interaction between its phenyl ring and
the hydrophobic region of the catalytic pocket, an interaction
that inhibits the ordering the C-terminal tails required for en-
zyme activation.
The Effects of Stereochemistry at C-2 Position of
Pyrrolidine Ring of N-Sulfonylpyrrolidine-Containing Ura-
cil Derivatives on Human dUTPase Inhibition Activity. SAR
studies for N-carbonylpyrrolidine-containing uracil derivatives
and N-sulfonylpyrrolidine-containing uracil derivatives suggest
that the stereochemistry at C-2 position of their pyrrolidine
rings markedly influences their potencies as inhibitors of
dUTPase. Initially, we tried to evaluate the effect of streo-
chemistry on the potency of enzyme inhibition activity by
docking studies with dUTPase-enantiomeric sulfonamide
inhibitors 16a (R) and 16b (S) based on the X-ray cocrystal
structure of human dUTPase and 16a. However, the order of
the derived factors of contact surface areas of 16a and 16b with
human dUTPase (16a, 565.5 Å2; 16b, 590.9 Å2) proved to be
opposite from what would be expected from their observed
relative potencies (16a, 0.32 μM; 16b, 9.4 μM). (see Figure S2
in the Supporting Information). We next considered the posi-
tion of pyrrolidine ring of 16a in the active site of dUTPase.
Tyr105 is a critical residue that discriminates between the 2′-
deoxyribose moiety of a substrate dUTP from the ribose moiety
of UTP. The rigidity of the Tyr105 position is one of the
pivotal factors that contribute to the narrow substrate specificity
of dUTPase.14 This rigidity is sustained by hydrogen bonding
between the phenol group of Tyr105 and carbonyl group of
Asn108. However, this hydrogen bond faces the solvent region
Figure 5. Binding of 16a (blue stick) in the catalytic site of human
dUTPase. (A) Polar interactions. Distances [Å] are indicated. Waters
are shown as small spheres. Red stick depicted α,β-imino dUTP 1b in
the human dUTPase:α,β-imino dUTP 1b structure (PDB code:
2HQU)39 superimposed on the 16a:human dUTPase. (B) Compar-
ison of 16a (blue stick) with α,β-imino dUTP 1b (red stick).
structure of α,β-imino dUTP 1b in human dUTPase:α,β-imino
dUTP 1b complex (PDB code: 2HQU)39 is also superimposed
on the cocrystal structure of human dUTPase and 16a. It is
interesting to note that this superimposition reveals very little
overlap between 1b and 16a. Only the uracil rings overlap
closely, occupying the uracil recognition region of the enzyme
(Figure 5B). These data also reveal that the sulfonamide and
diphenylmethanol moieties of 16a do not interact with the
amino acid residues that recognize the sugar and triphosphate
moieties of 1b. Furthermore, one of the terminal phenyl rings
of 16a is located in a hydrophobic region, formed by Val65,
Ala90, Ala98, and Val112 residues of the enzyme, and is stacked
with its own uracil ring in the active site of the enzyme. Figure
5B clearly shows that the orientation of 16a bound to the
enzyme is different from that of the substrate dUTP mimic 1b.
Superimposition of 16a in the cocrystal structure of human
dUTPase and 16a on the X-ray structure of α,β-imino-dUTP
1b in the active site of dUTPase is shown in Figure 6. It is
known that the flexible C-terminal tails of the third subunit of
trimeric dUTPase, which are disordered in the uncomplexed
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and is exposed to surrounding water. Fernandez et al. proposed
that the hydrophobic groups of ligands would exclude sur-
rounding water from the peripheral region of the hydrogen
bond, which plays a pivotal role for the catalytic functions by a
structural maintenance (wrapping) effect to stabilize a ligand−
enzyme complex.40−42 The X-ray structure of 16a suggests
that its pyrrolidine ring is proximal to Tyr105 and stabilizes a
ligand−enzyme complex by removing water molecules
(wrapping) from the peripheral region of Tyr105−Asn108
pair (Figure 7). In contrast, the binding model of 16b indicates
(0.1 μM) in the enzyme inhibition assays. It is particularly
worth noting that 12a and 16a markedly enhanced the growth
inhibition activity of FdUrd against HeLa S3 cells at low
concentrations, having EC50 values in the 10−7 M range. We
also achieved crystallization of dUTPase with 16a and obtained
an X-ray analysis of the complex. The X-ray structure revealed
that the high potency of 16a can be attributed to two factors:
(1) it is bound to the substrate recognition site and (2) its
phenyl group occupies the hydrophobic pocket (formed by
Val65, Ala90, Ala98, and Val112) and forms a stacking interac-
tion with its own uracil. Finally, these results suggest that our
novel dUTPase inhibitors should open the way to new bene-
ficial chemotherapeutic strategies for cancer patients when used
in combination with TS inhibitor.
EXPERIMENTAL SECTION
■
General Methods and Materials. 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 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 JEOL JMS-700 (FAB) or
Waters micromass Q-Tof-2 (TOF). Chemical purities of 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 values were within 0.4% of the theoretical values. Chemical
purities of tested compounds 11i and 11r were determined by HPLC
analysis with SHIMADZU Prominence HPLC system, and the values
are presented in the Supporting Information. Optical rotations were
measured by HORIBA SEPA-200 polarimeter. clogP values shown in
Tables 1−3 were calculated ACD/LogP algorithm (ACD/Laboratories
Release 10.00, version 10.01).
General Procedure for the Synthesis of 7a−7j (Method A).
Exemplified for (S)-Ethyl 2-(Bis(3-fluorophenyl)(hydroxy)-
methyl)pyrrolidine-1-carboxylate (7a). A THF solution of 3-
fluorophenylmagnesium bromide (1.0 M, 20 mL, 20 mmol) was added
dropwise over 5 min to a solution of N-carboethoxy-L-proline methyl
ester 6a (1.0 g, 5.0 mmol) in THF (30 mL) at 0 °C. After being stirred
at the same temperature for 3 h, the reaction was quenched by
addition of saturated aq NH4Cl (10 mL). The separated organic phase
was washed with brine (10 mL), dried over Na2SO4, and concentrated
to dryness in vacuo. The residue was purified by a silica gel column
chromatography (25−33% AcOEt in hexane) to give 7a (1.41 g, 78%,
a colorless solid). 1H NMR (CDCl3) δ 0.89−0.99 (1H, m), 1.24 (3H,
t, J = 7.0 Hz), 1.50−1.63 (1H, m), 1.86−1.97 (1H, m), 2.02−2.16
(1H, m), 2.96−3.06 (1H, m), 3.41−3.51 (1H, m), 4.12−4.15 (2H, m),
4.83 (1H, dd, J = 4.3, 8.9 Hz), 6.94−7.09 (2H, m), 7.12−7.33 (6H, m).
FAB-HRMS m/z [M+Na]+: calcd for C20H21F2NO3Na, 384.1387;
found, 384.1385
Figure 7. X-ray structure of 16a (blue stick and sphere) in the catalytic
site of dUTPase. The pyrrolidine group of 16a likely excludes
surrounding water from the backbone hydrogen bond (Tyr105−
Asn108).
that its pyrrolidine ring is not found in the neighborhood of the
Tyr105−Asn108 pair. Therefore, 16b cannot induce a wrapping
effect of the Tyr105−Asn108 pair (see Figure S2 in the
Supporting Information). These considerations suggest that the
greater potency of 16a compared to 16b can be attributed to
stabilization of the 16a−enzyme complex by dehydration of the
peripheral region of Tyr105−Asn108 pair that is covered by
the pyrrolidine ring of 16a in the complex. In the case of
N-carbonylpyrrolidine derivatives 12a and 12b, because we could
not solve their X-ray crystal structures with human dUTPase
and amide structure is quite different from sulfonamide, further
studies are necessary to rationalize the effects of the stereo-
chemistry on their inhibitory activity.
CONCLUSIONS
■
Using SAR studies of the linker and amine moieties of lead
compound 5, identified from preliminary screening of our own
library of uracil derivatives, we identified a tert-amide-
containing uracil derivative 11s that is a potent inhibitor of
dUTPase (IC50 = 1.3 μM). Further structural optimizations led
to the development of the N-carbonylpyrrolidine-containing
uracil derivatives 12a (IC50 = 0.29 μM), 12k (IC50 = 0.15 μM),
General Procedure for the Synthesis of 8a−8j (Method B).
Exemplified for (S)-Bis(3-fluorophenyl)(pyrrolidin-2-yl)-
methanol (8a). Potassium hydroxide (2.19 g, 39 mmol) was added
to a solution of 7a (1.41 g, 3.9 mmol) in MeOH (10 mL). The mixture
was heated at reflux for 12 h. The mixture was evaporated to give a
residue, which was dissolved in water (10 mL) and then extracted with
CHCl3 (20 mL). The organic phase was washed with water (10 mL)
and brine (10 mL), dried over K2CO3, and concentrated in vacuo. The
residue was coevaporated with toluene (10 mL × 3) to give 8a (1.07 g,
95% as a colorless gum). 1H NMR (DMSO-d6) δ 1.36−1.45 (2H, m),
1.51−1.63 (2H, m), 2.40 (1H, brs), 2.75−2.88 (2H, m), 4.26 (1H, t,
J = 7.6 Hz), 5.35 (1H, brs), 6.94−7.02 (2H, m), 7.25−7.45 (6H, m).
FAB-HRMS m/z [M + H]+: calcd for C17H18F2NO, 290.1356; found,
290.1380
and N-sulfonylpyrrolidine-containing derivative 16a (IC50
=
0.32 μM), which have excellent potencies and good drug-like
properties as human dUTPase inhibitors. Their inhibitory
activities were markedly more potent (>1000-fold) than those
of the known dUTPase inhibitors 2 and 3 which possess drug-
like properties. In addition, the degree of recognition of 12k by
dUTPase was estimated to be almost comparable to that of the
natural substrate dUTP because the IC50 values were of the
same order of magnitude as that of the concentration of dUTP
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General Procedure for the Synthesis of 5 and 11a−11w
(Method C). Exemplified for N-tert-Butyl-4-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)butanamide (5). A mixture of 4-(2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)butanoic acid 1036 (135 mg,
0.68 mmol), tert-butylamine (143 μL, 1.36 mmol), EDC·HCl (196
mg, 1.02 mmol), and HOBt (110 mg, 0.82 mmol) in DMF (4.0 mL)
was stirred at room temperature for 3 h. The reaction mixture was
evaporated, and the residue was purified by a silica gel column
chromatography (1−3% MeOH in CHCl3) to give 5 (34 mg, 20% as
temperature for 1 h. The reaction mixture was evaporated, and the
residue was purified by a silica gel column chromatography (50−75%
AcOEt in hexane) to give 15e (473 mg, 95% as a colorless solid).
1H NMR (CDCl3) δ 1.34−1.44 (1H, m), 1.62−1.76 (1H, m), 1.83−1.99
(3H, m), 2.12−2.26 (1H, m), 2.57 (1H, quint, J = 7.3 Hz), 2.69 (1H,
quint, J = 7.3 Hz), 2.98 (1H, dt, J = 7.6, 10.8 Hz), 3.30 (1H, s), 3.61−
3.74 (3H, m), 5.27 (1H, dd, J = 3.8, 8.64 Hz), 7.22−7.36 (6H, m),
7.42−7.46 (2H, m), 7.52−7.56 (2H, m). FAB-HRMS m/z [M − H]−:
calcd for C20H24NO4S, 374.1426; found, 374.1428
1
a colorless solid). H NMR (DMSO-d6) δ 1.21 (9H, s), 1.69−1.80
General Procedure for the Synthesis of 16a−f (Method H).
Exemplified for (R)-1-(3-(2-(Hydroxydiphenylmethyl)-
pyrrolidin-1-ylsulfonyl)propyl)pyrimidine-2,4(1H,3H)-dione
(16a). A suspension of 15e (406 mg, 1.08 mmol), triphenylphosphine
(567 mg, 2.16 mmol), and N-3-benzoyluracil (233 mg, 1.08 mmol)
was added a toluene solution of diisopropyl azodicarboxylate (1.9 M,
1.14 mL, 2.16 mmol) at room temperature, the mixture was stirred at
room temperature for 2 h. The reaction mixture was evaporated, and
the residue was purified by a silica gel column chromatography (1−2%
MeOH in CHCl3) to give (R)-3-benzoyl-1-(3-(2-(hydroxydiphenyl-
methyl)pyrrolidin-1-ylsulfonyl)propyl)pyrimidine-2,4(1H,3H)-dione
as a mixture with triphenylphosphine oxide. The mixture was dissolved
in a MeOH solution of methylamine (9.8 M, 4 mL) and stirred at
room temperature for 1 h. The reaction mixture was evaporated, and
the residue was purified by a silica gel column chromatography (2−4%
MeOH in CHCl3) to give 16a (355 mg, 70% as a colorless foam).
1H NMR (DMSO-d6) δ 1.62−1.67 (3H, m), 1.81−1.84 (2H, m),
1.98−2.12 (2H, m), 2.35−2.40 (1H, m), 3.22−3.24 (1H, m), 3.40−
3.64 (3H, m), 5.22 (1H, d, J = 6.2 Hz), 5.57 (1H, d, J = 7.6 Hz), 5.65
(1H, brs), 7.05−7.53 (11H, m), 11.3 (1H, brs). 13C NMR (CDCl3) δ
22.99, 26.00, 29.11, 47.25, 49.50, 50.64, 67.17, 81.17, 102.51, 126.68,
126.99, 127.35, 127.37, 128.17, 128.20, 144.11, 144.60, 145.76, 150.80,
163.37. Anal. Calcd for C24H27N3O5S·0.3H2O: C, 60.69; H, 5.86; N, 8.85.
(2H, m), 2.01 (2H, t, J = 7.3 Hz), 3.63 (2H, t, J = 7.0 Hz), 5.52
(1H, dd, J = 2.4, 7.8 Hz), 7.39 (1H, brs), 7.57 (1H, d, J = 7.8 Hz),
11.18 (1H, brs). 13C NMR (CDCl3) δ 24.81, 28.67, 33.22, 47.70, 51.18,
102.27, 144.82, 151.44, 164.05, 170.92; Anal. Calcd for C12H19N3O3: C,
56.90; H, 7.56; N, 16.59. Found: C, 56.74; H, 7.67; N, 16.41
General Procedure for the Synthesis of 12a−l (Method D).
Exemplified for (S)-1-(4-(2-(Hydroxydiphenylmethyl)-
pyrrolidin-1-yl)-4-oxobutyl)pyrimidine-2,4(1H,3H)-dione (12a).
A mixture of 10 (40 mg, 0.22 mmol), (S)-(−)-α,α-diphenyl-2-
pyrrolidinemethanol (56 mg, 0.22 mmol), EDC·HCl (57 mg, 0.3 mmol),
and HOBt (35 mg, 0.26 mmol) in DMF (2.0 mL) was stirred at room
temperature for 3 h. The reaction mixture was evaporated, and the residue
was purified by a silica gel column chromatography (1−3% MeOH in
CHCl3) to give 12a (62 mg, 65% as a colorless foam). 1H NMR (CDCl3)
δ 1.00−1.09 (1H, m), 1.54−1.65 (1H, m), 1.90−2.09 (4H, m), 2.19−2.40
(2H, m), 3.01−3.11 (1H, m), 3.35−3.44 (1H, m), 3.50−3.60 (1H, m),
3.67−3.77 (1H, m), 5.13 (1H, dd, J = 5.7, 8.4 Hz), 5.69 (1H, dd, J = 1.9,
7.8 Hz), 6.70 (1H, s), 7.14 (1H, d, J = 7.8 Hz), 7.21−7.42 (10H, m), 8.68
(1H, brs). 13C NMR (CDCl3) δ 23.27, 23.87, 29.80, 31.02, 47.72, 48.75,
67.39, 81.57, 102.13, 127.34, 127.40, 127.57, 127.87, 127.98, 143.52,
145.02, 146.06, 150.83, 163.54, 173.77. Anal. Calcd for C25H27N3O4·H2O:
C, 66.50; H, 6.47; N, 9.31. Found: C, 66.78; H, 6.11; N, 9.23; [α]25
=
D
Found: C, 60.40; H, 5.66; N, 8.71; [α]25 = −13.14 (c 0.38, CHCl3)
−102.58 (c 1.08, CHCl3)
D
General Procedure for the Synthesis of 13a−f (Method E).
Exemplified for (R)-(1-(3-Chloropropylsulfonyl)pyrrolidin-2-yl)-
diphenylmethanol (13e). To a solution of (S)-(−)-α,α-diphenyl-2-
pyrrolidinemethanol 8k (500 mg, 2.0 mmol) and triethylamine (554 μL)
in CH2Cl2 (10 mL), was added 3-chloropropanesulfonyl chloride
(287 μL, 2.4 mmol) at 0 °C, and the mixture was stirred at room
temperature for 2 h. Saturated aq NaHCO3 (5 mL) was added and
partitioned. The organic layer was washed with water (5 mL) and
brine (5 mL), dried over Na2SO4, and filtered. The filtrate was evapo-
rated to give a residue, which was purified by a silica gel column
chromatography (10−20% AcOEt in hexane) to give 13e (800 mg,
dUTPase Inhibition Assay. In vitro dUTPase inhibition assays
were conducted by measuring the production of [5-3H]dUMP from
[5-3H]dUTP. Briefly, 0.2 mL in total of a 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 cen-
trifugation at 15000 rpm for 2 min. An aliquot (150 μL) of the super-
natant thus obtained by centrifugation was analyzed using an Atlantis
dC18 column (manufactured by Waters Corp., 4.6 mm × 250 mm)
and a high-performance liquid chromatograph (manufactured by Shimadzu
Corp., Prominence). The inhibitory rate of the compound was calculated
from the formula shown below. IC50 (μM), the concentration of inhibitor
yielding 50% inhibition rate, was obtained from concentration−inhibitory
rate curve.
1
quant as a colorless solid). H NMR (CDCl3) δ 1.39−1.44 (1H, m),
1.66−1.77 (1H, m), 1.88−2.00 (1H, m), 2.04−2.24 (3H, m), 2.55−
2.70 (2H, m), 2.94−3.03 (1H, m), 3.01 (1H, s), 3.47−3.56 (2H, m),
3.66−3.75 (1H, m), 5.28 (1H, dd, J = 4.1, 8.9 Hz), 7.23−7.36 (6H, m),
7.41−7.46 (2H, m), 7.50−7.54 (2H, m). FAB-HRMS m/z [M − H]−:
calcd for C20H23ClNO3S, 392.1087; found, 392.1067
General Procedure for the Synthesis of 14a−f (Method F).
Exemplified for (R)-3-(2-(Hydroxydiphenylmethyl)pyrrolidin-1-
ylsulfonyl)propyl Acetate (14e). A mixture of 13e (800 mg,
2.0 mmol), sodium acetate (492 mg, 6.0 mmol), and sodium iodide
(300 mg, 2.0 mmol) in DMF (8 mL) was stirred at 90 °C for 12 h.
The mixture was cooled to room temperature and diluted with AcOEt
(10 mL) and toluene (10 mL) and then was washed with water
(20 mL × 2) and brine (20 mL). The organic layer was dried over
Na2SO4 and filtered. The filtrate was evaporated to give a residue,
which was purified by a silica gel column chromatography (20−40%
AcOEt in hexane) to give 14e (584 mg, 70% as a colorless solid).
1H NMR (CDCl3) δ 1.41−1.48 (1H, m), 1.67−1.77 (1H, m), 1.90−1.97
(3H, m), 2.06 (3H, s), 2.16−2.23 (1H, m), 2.37−2.48 (1H, m), 2.54−
2.62 (1H, m), 2.94−3.03 (1H, m), 2.99 (1H, s), 3.68−3.76 (1H, m), 4.00
(2H, t, J = 6.2 Hz), 5.31 (1H, dd, J = 3.5, 8.4 Hz), 7.22−7.35 (6H, m),
7.44 (2H, d, J = 7.6 Hz), 7.53 (2H, d, J = 8.1 Hz). FAB-HRMS
m/z [M − H]−: calcd for C22H26NO5S, 416.1532; found, 416.1560
General Procedure for the Synthesis of 15a−f (Method G).
Exemplified for (R)-3-(2-(Hydroxydiphenylmethyl)pyrrolidin-1-
ylsulfonyl)propan-1-ol (15e). A mixture of 14e (584 mg, 1.4 mmol)
in MeOH solution of methylamine (9.8 M, 5 mL) was stirred at room
3
Inhibitory rate(%) = [1 − (amount of[5‐ H]dUMP
in presence of test solution(dpm))
3
/(amount of[5‐ H]dUMP as control(dpm))] × 100
Evaluation of in Vitro Cytotoxicity. HeLa S3 cells (human
ovarian carcinoma) was grown in 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 a volume
of 20 μL per well, and 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 plate reader. Then, we
calculated T/C (%) value, which was the ratio of OD540 with drug
treatment to without drug [T/C (%) = (OD540 of treated well/OD540
of nontreated well) × 100]. The EC50 (μM) for the cytotoxicity of the
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test compound was the concentration yielding 50% T/C value, which
was calculated from concentration-T/C (%) curve.
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Evaluation of the Enhancement of 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
concentration of test compounds and FdUrd (1 μM) were added to
the corresponding plates at a volume of 20 μL per well. After 24 h
incubation, thymidine (30 μM) was added to the plates at a volume of
10 μL per well. After further 48 h incubation, cell density was mea-
sured and T/C (%) values ware obtained as mentioned above. The
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treatment of 1 μM FdUrd.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and analytical data for intermediates and
test compounds except for the compounds described in the
Experimental Section and details of 1H NMR spectrum of 11s,
X-ray crystallography diffraction data and refinement statistics,
and docking study of 16b in the catalytic site of human
dUTPase described in the manuscript. This material is available
free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID code of 16a with human dUTPase: 3ARA.
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. 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. We also thank the staff of
Pharmaceutical Industry Beamline BL32B2 at Spring 8 for help
with the X-ray structure data collection.
ABBREVIATIONS USED
■
dUTPase, deoxyuridine triphosphatase; TS, thymidylate
synthase; 5-FU, 5-fluorouracil; NTPs, nucleoside triphosphates;
siRNA, small interfering RNA; Bn, benzyl; THF, tetrahydrofu-
ran; EDC·HCl, N-(3-dimethylaminopropyl)-N′-ethylcarbodi-
imide hydrochloride; HOBt, 1-hydroxybenzotriazole; DMF,
N,N-dimethylformamide; TEA, triethylamine; Bz, benzoyl;
DIAD, diisopropyl azodicarboxylate; SAR, structure−activity
relationship; NOE, nuclear Overhauser effect; clogP, calculated
logP
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