Design, synthesis, and evaluation of DNA topoisomerase II-targeted nucleosides

Article abstract of DOI:10.1016/j.bmc.2017.06.001

We developed novel nucleoside-based topoisomerase II selective inhibitors and showed that small structural units, such as catechols, are essential for DNA topoisomerase II inhibitory activity. Moreover, nucleoside analogues containing TBS and 1,3-dithian

Full text of DOI:10.1016/j.bmc.2017.06.001

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of DNA topoisomerase II-targeted
nucleosides
Hironobu Matsumoto b, Mitsuaki Yamashita a, Teruyuki Tahara b, Shinya Hayakawa a, Shun-ichi Wada c,
Kiyoshi Tomioka b, Akira Iida a,
⇑
a School of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan
b Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
c Osaka University of Pharmaceutical Science, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We developed novel nucleoside-based topoisomerase II selective inhibitors and showed that small struc-
tural units, such as catechols, are essential for DNA topoisomerase II inhibitory activity. Moreover, nucle-
oside analogues containing TBS and 1,3-dithian moieties had potent and selective DNA topoisomerase II
inhibitory activities. In further experiments, compound 25b having a beta configuration of the thymine
moiety showed relatively strong growth inhibitory activity against cancer cell lines, and was more potent
against all cancer cell lines than compound 26b, which carries a thymine moiety in the alpha
configuration.
Received 8 March 2017
Revised 30 May 2017
Accepted 1 June 2017
Available online xxxx
Keywords:
DNA topoisomerase
Nucleoside
Ó 2017 Published by Elsevier Ltd.
Antiproliferative activity
Cancer
1. Introduction
II (topo II).5–8 However, little is still known about how 1 and 2
interact with either tubulin or topo II, although extensive investi-
DNA topoisomerases are essential enzymes that mediate the
topological adjustments required for DNA replication, transcrip-
tion, recombination, and repair, and chromatin assembly.1,2 There-
fore, targeted inhibitors of these enzymes are of considerable
interest as potential novel antitumor agents. Although large num-
bers of natural and/or synthetic topoisomerase inhibitors have
been discovered,3 no reported antitumor nucleosides target DNA
topoisomerases. In our ongoing investigations of biologically active
compounds, we designed and synthesized novel topoisomerase II-
targeted nucleosides that are essentially hybrids of podophyllo-
toxin analogues and nucleosides, and we report our initial assess-
ments of efficacy and specificity. To our knowledge, this class of
DNA topoisomerase II inhibitors with nucleoside motifs is entirely
novel.
gations of structure–activity relationships of 1 strongly indicate
structural preferences for topo II inhibitors9,10 over antimicro-
tubule agents. Specifically, (1) a b-configuration at C4, (2) a bulky
substitution at C4, (3) a free hydroxy group at C40, (4) a 2
a,3b-con-
figuration at C2 and C3 on the D ring, (5) free rotation of the E-ring
are likely determinants of enzyme preferences. Among these struc-
tural characteristics, the 40-hydroxy group has been considered
critical for topo II inhibition.11–14 However, previous synthetic
studies on 4b-arylamino derivatives of 1 suggest that even the
40-hydroxy group may tolerate structural modification.15 Accord-
ingly, Miyahara et al. showed that a nitrosourea derivative of
30,40-didemethoxy-30,40-dioxo-4-deoxypodophyllotoxin with
a
pendant E-ring that was oxidized to an ortho-quinone was a more
potent inhibitor of topo II than compound 2.16 Similar observations
were reported by Gantchev et al.,17 and in other studies, compound
2 was oxidized to the ortho-quinone via the O-demethylation pro-
duct (catechol) by enzymatic transformations in cells.18,19 In these
studies, following synthesis of ortho-quinone by electrolysis of 2 in
the absence of oxygen, improved topo II inhibitory activity was
demonstrated. These studies indicate that the pendant E-ring pre-
fers the ortho-quinone structure to the 40-hydroxy form for topo II
inhibition. Previously we reported topo II inhibitory activity of
structurally simpler deoxypodophyllotoxin aza-analogues that lack
4-substituents (Fig. 2).20 Among these, the ortho-quinone
Podophyllotoxin (1)4 is a natural lignan from the Podophyllum
plant and its clinically used semisynthetic derivatives etoposide
(2) and teniposide (3) are well known potent anticancer com-
pounds (Fig. 1). Despite structural similarities, the modes of action
of these compounds differ widely. Specifically, whereas compound
1 inhibits microtubule assembly by interacting with tubulin, com-
pounds 2 and 3 inhibit the catalytic activity of DNA topoisomerase
⇑
Corresponding author.
E-mail address: iida@nara.kindai.ac.jp (A. Iida).
http://dx.doi.org/10.1016/j.bmc.2017.06.001
0968-0896/Ó 2017 Published by Elsevier Ltd.
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
2. Results and discussion
H
O
R
O
The present retrosynthesis route is shown in Scheme 1. In this
study, TBS-protected butenolide 7, which is an equivalent of
O
O
H
OH
HO
H
H
H
OH
4
deoxyribose, was derived from
D-mannitol as a chiral building
O
O
O
O
3
O
O
block. We previously demonstrated examples of natural product
synthesis using this chiral building block,21–23 and stereoselective
introduction of the dithian 6 at the 30 position followed by acetyla-
tion and decoration of the nucleobase at the 10 position yielded
compound 4.
2
H
H
H
O
O
MeO
OMe
MeO
OMe
4'
The dithiane compound 9 was synthesized in single step from
commercially available 3,4-dialkoxy benzaldehyde, and subse-
quent stereoselective Michael reaction with butenolide 7 gave
the only erythro adducts 10, which was then desulfurized under
radical conditions to give the lactone form 11. Subsequently,
DIBAL-H reduction of the lactone followed by acetylation with
acetic anhydride and pyridine afforded the acetate 13. Finally,
introduction of the thymine moiety to 13 using the Silyl-Hilbert-
Johnson reaction24 afforded epimers 14 and 15 (Scheme 2). Details
of isolation protocols for the alpha forms 15a and 15b and the beta
forms 14a and 14b are described in the experimental section. In
addition, stereochemical characteristics of these compounds were
determined in comparisons of NMR spectra (See Supplementary
data).
OMe
OH
1 podophyllotoxin
2 etoposide: R = Me
3
teniposide: R =
S
Fig. 1. Podophyllotoxin, etoposide, and teniposide.
R (E ring) =
In further synthetic steps, debenzylation of 14a was performed
by catalytic hydrogenation over 10% Pd–C to afford 16a quantita-
tively, and subsequent treatment of 16a with TBAF afforded desily-
lated 17a. Alternatively, oxidation of 16a with manganese dioxide
(IV) gave ortho-quinone 18a (Scheme 3). Compounds 14b and 15b
were converted to 19b and 20b, respectively (Scheme 4).
The conversion of supercoiled plasmid DNA to relaxed DNA by
human Topo II was examined in the presence of 17a and 19b
(Fig. 3). In determinations of topoisomerase II enzyme inhibition
activity of the catechol 17a, no inhibitory activity was observed
MeO
OMe MeO
OMe
H
OMe
OH
H
N
4
O
A
B
C
D O
O
O
H
R
MeO
OH
MeO
O
OH
O
at 100
l
M, whereas amounts of supercoiled plasmid DNA were
M (IC50 of 17a, 176 M).
dose–dependently increased from 150
l
l
To our knowledge, this is the first example of a nucleoside ana-
Fig. 2. Deoxypodophyllotoxin aza-analogue.
logue that inhibits topoisomerase II, albeit with moderate efficacy.
In contrast, 19b showed no inhibitory activity even at 200 lM,
indicating that incorporation of the catechol structure improves
inhibitory activity.
derivative showed potent topo II inhibition despite the absence of
the 4-substitution, and differed from compounds 1, 2, and 3. In
contrast, topo II inhibitory activity was completely abolished in
the 40-hydroxy aza-derivative, regardless of the presence of the
40-hydroxy group in the E-ring, indicating that the ortho-benzo-
quinone unit is the smallest promising structural determinant of
topo II inhibition. Given this structural preference in the pendant
E-ring, we designed a new type of topo II inhibitor by incorporating
an ortho-quinone structure. In addition, substrates of DNA topoiso-
merase is DNA, which is composed of nucleosides. Therefore, we
hypothesized that nucleosides containing structural unit for the
topo II inhibition such as catechol or ortho-quinone could be
promising topo II inhibitors.
Contrasting inhibitory activities of 17a and 19b against DNA
topoisomerase II closely matched the data from previous experi-
ments with deoxypodophyllotoxin aza-analogues. Furthermore,
flavonoids containing the catechol moiety and catechins contain-
ing the trihydroxyphenyl moiety showed DNA topoisomerase II
inhibitory activity.25,26 These observations suggest that small
structural units, such as catechol structures, are essential for
DNA topoisomerase II inhibitory activity.
In assessments of DNA topoisomerase I inhibitory activity of the
synthetic nucleoside 17a, all supercoiled plasmid DNA super
helixes were converted to relaxed DNA even at 200 lM. These
Scheme 1. Retrosynthesis of nucleoside analogues (4).
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 2. Synthesis of nucleoside analogues.
Scheme 3.
observations indicate that 17a is a selective inhibitor of DNA topoi-
somerase II that does not interfere with DNA topoisomerase I
(Fig. 4).
In further experiments, inhibition patterns of the synthesized
nucleoside 17a were inferred from comparisons with the topo II
poison etoposide, which is known to induce DNA strand breaks.27
In these experiments, linear DNA was detected following
incubation of 10 units of enzyme with 200-
l
M etoposide, whereas
no linear DNA was observed in the presence of the same concentra-
tion of 17a. Therefore, 17a was considered a catalytic inhibitor that
does not cleave DNA strands (Fig. 5).
DNA topoisomerase II inhibitory activities of the dimethoxy
analogues 10b and 11b were more potent than that of 17a
(Fig. 6). Hence, DNA topoisomerase II inhibitory activity reflects
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 4.
Fig. 3. Inhibitory effects of nucleoside analogues on DNA topoisomerase II. Lane a: supercoiled DNA alone; Lane b: control (no drug, 1 unit of Topo II); Lanes c, d, and e: 200,
175, and 150 M of 17a, respectively; Lane f: supercoiled DNA alone; Lane g: b: control (no drug, 1 unit of Topo II); Lane h: 200 M of 19a. SC: supercoiled DNA. RLX: relaxed
l
l
DNA topoisomers. NC: nicked circle DNA.
catechol structures and other structural factors, warranting further
studies of structure–activity relationships. Accordingly, com-
pounds 21b–27b were synthesized according to the methods used
for the synthesis of 17a with some modifications (Scheme 5).
DNA topoisomerase II inhibitory activities of the present syn-
thesized nucleoside analogues are shown in Table 1. Among these,
lactone derivatives 10b, 11b, 14b, 15b, 16a, 18a, 25b, 26b, and 27b
contained TBS and/or the 1,3-dithian moiety, and showed higher
activities than the other compounds. However, DNA topoisomerase
II inhibitory activities of the lactone derivatives 19b, 20b, and 22b,
which contained neither TBS nor 1,3-dithian moieties, were
>200 lM. Moreover, compound 25b has a beta configuration of
the thymine moiety and showed higher inhibitory activity than
26b, which carries a thymine moiety in the alpha configuration.
Based on these results, we speculate that the higher inhibitory
effects by introduction of nucleobases may come from increasing
affinity for the enzyme.
In further experiments, we used the COMPARE analysis to con-
firm the anticancer effect of 25b and 26b and estimate correlation
coefficient between the fingerprints of the test compounds and
those of the various reference compounds. It is well established
in a panel of 39 human cancer cell lines (JFCR39) that a pair of com-
pounds potentially share the same mode of action when they have
Fig. 4. Inhibitory effects of compounds 17a and 25b on DNA topoisomerase I. Lane
a: supercoiled DNA alone; Lane b: control (no drug, 6 unit of Topo I); Lanes c and f:
200 lM of 25b and 17a. respectively; Lane d and e are data of compounds not
concerning about this report. SC: supercoiled DNA. RLX: relaxed DNA topoisomers.
NC: nicked circle DNA.
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Fig. 5. Effect of compound 17a on Topo II-mediated DNA cleavage. Lane a: DNA alone; Lane b: control (no drug, 6 unit of Topo II); Lanes c: 200
l
M of etoposide and 10 unit of
Topo II; Lanes d: 200
lM of etoposide and 5 unit of Topo II; Lanes e: 200 lM of 17a and 10 unit of Topo II; Lanes f: 200 lM of 17a and 5 unit of Topo II. SC: supercoiled DNA.
PRLX: partially relaxed DNA topoisomers. RLX: relaxed DNA topoisomers. NC: nicked circle DNA. LIN: linear DNA.
Fig. 6. Inhibitory effects of synthetic intermediates on DNA topoisomerase II. Lane a: supercoiled DNA alone; Lane b: control (no drug, 1 unit of Topo II); Lanes c, d, e and f:
175, 150, 125 and 100
lM of 11b, respectively; Lane g, h, i and j: 70, 50, and 25
l
M of 10b. SC: supercoiled DNA. RLX: relaxed DNA topoisomers. NC: nicked circle DNA.
fingerprints, the growth inhibitory profiles across JFCR39, closely
resembling each other. The COMPARE analysis is carried out by cal-
culation of the Pearson correlation coefficient (r value) between
the fingerprints of compounds X and Y.28–30 Although compounds
18a showed best DNA topoisomerase II inhibitory activities in
Table 1, antiproliferative activities were not evaluated due to its
instability. Fig. 7 shows 50% growth inhibitory concentrations
(GI50) relative to the control. In these investigations, compound
25b showed relatively strong growth inhibitory activity against a
larger number of cancer cell lines, and was more potent against
all cancer cell lines than compound 26b. Moreover, these results
were consistent with respective DNA topoisomerase II inhibitory
activities of these compounds, and 25b was selective of DNA topoi-
somerase II, with no effects on DNA topoisomerase I (Fig. 4). The
COMPARE analysis revealed that the fingerprint of compound
25b and 26b correlated with that of ellipticine, which is known
as DNA Topoisomerase II inhibitor and DNA-Intercalation Drugs
nucleic acid breakage and subsequent cell death.31 The detailed
mechanisms of nucleoside analogues containing TBS and the 1,3-
dithian moiety is still unclear at this stage. Further studies are nec-
essary to elucidate their effects against TopoII inhibition and
growth inhibitory cancer cell lines.
3. Conclusion
In conclusion, we developed a novel series of nucleoside-based
selective topoisomerase II inhibitors. Important structural units
included the orthoquinone structure and catechol structures, and
these were specific for DNA topoisomerase II inhibitory activity.
Finally, nucleoside analogues containing TBS and the 1,3-dithian
moiety were potent DNA topoisomerase II inhibitors. These data
warrant further studies of structure–activity relationships of other
topoisomerase II inhibitors with nucleoside structures.
(25b: r = 0.492, 26b: r = 0.611). Generally, an
r value of
0.5 < r < 0.75 between two agents suggests they might have a sim-
ilar mechanism of action. These data suggest that at least com-
pound 26b may have a similar mode of action to ellipticine. It is
reported that ellipticine acts by stimulating topoisomerase II-
mediated DNA breakage. It is likely that formation of a ternary
complex between topoisomerase II, DNA, and drug is critical for
4. Experimental section
4.1. General
1H and 13C NMR spectra were taken in CDCl3. Chemical shift val-
ues are expressed in ppm relative to internal tetramethylsilane.
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 5.
Table 1
wave-numbers of maximum absorption peaks of IR spectroscopy
are presented in cmꢀ1. MS (EI) is presented in m/z. Extracts were
washed with brine and then dried over sodium sulfate. Silica gel
column chromatography was used for purification. Purified human
IC50 values of synthesized compounds against recombinant Topo II.a
Compound
IC50
56
(l
M)
Compound
IC50 (lM)
Etoposideb
10b
11b
14b
15b
19b
20b
21b
22b
25b
26b
27b
>200d
NIc
placenta DNA topoisomerases I (2 U/mL) and II
a (2 U/mL), kineto-
55.7 5.5
137 4.0
99.1 8.3
89.2 2.0
35.2 9.9
2.43 0.9
>200e
NIc
plast DNA, etoposide and 40-(9-acridinylamino)-methanesulfonm-
anisidide (m-AMSA) were purchased from TopoGen, Inc. (Colum-
bus, Ohio). Supercoiled pUC19 and pBR322 plasmid DNA and Hin-
dIII restriction endonuclease were obtained from Toyobo (Osaka).
8.1 3.2
18.5 1.6
64.5 10.3
16a
18a
a
IC50 values were determined by interpolation from plots of enzyme activity vs.
inhibitor concentrations. IC50 values are presented as the means SD of at least
three independent experiments.
4.2. DNA topoisomerase II mediated DNA relaxation
b
Positive control.
No inhibition at 200 mM.
23.7% 10.6% inhibition at 200 mM.
19.8% 5.7% inhibition at 200 mM.
DNA topoisomerase II activity was measured by assessing relax-
ation of supercoiled pBR 322 plasmid DNA. Reaction mixture con-
tained 50 mM Tris-HCl (pH 8), 120 mM KCl, 10 mM MgCl2, 0.5 mM
ATP, 0.5 mM dithiothreitol, pBR 322 plasmid DNA (198 ng), the
indicated drug concentrations (less than 10% DMSO) and 1 U of
DNA topoisomerase II in a final volume of 20 mL. Reaction mixtures
were incubated for 30 min at 37 °C and stopped by addition of 5 mL
of a mixture of 5% sarkosyl, 0.0025% bromophenol blue and 25%
c
d
e
Coupling constants J values are presented in Hz. Abbreviations are
as follows: s, singlet; d, doublet; t, triplet; m, multiplet. IR spec-
troscopy of oil sample was measured as neat liquid film. The
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Fig. 7. Growth inhibitory profiles of 25b and 26b across JFCR39 cell lines. Cell growth inhibition was assessed by measuring changes in total cellular protein levels following
treatment for 48 h with given test compounds using the sulforhodamine B colorimetric assay. Fingerprints were produced by processing 50% growth inhibition (GI50) values
using a computer. The x-axis represents the logarithm of GI50 values for the 39 cell lines. CNS, central nervous system.
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H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
glycerol. Reaction products were electrophoresed on a 1% agarose
gel in TAE (Tris-acetate-EDTA) running buffer at 1.5 v/cm for
3.5 h. Gels were stained with ethidium bromide (0.5 mg/mL) for
60 min, and destained in water for 30 min. For the visualizing
and quantitative analyses of DNA topoisomerase II activity, the gels
were directly scanned with fluoroimage analyzer FLA-2000 (Fuji
Photo Film Co., Ltd.) and the area representing supercoiled DNA
was calculated. Concentrations for 50% inhibition (IC50) were deter-
mined by interpolation from plots of DNA topoisomerase II activity
versus inhibitor concentration. The IC50 values are means from at
least three independent experiments.
4.6. Synthesis
4.6.1. 2-(3,4-Bis(benzyloxy)phenyl)-1,3-dithiane (9a)
To a stirred solution of 8a (12.0 g, 37.7 mmol) and SnCl2 (7.15 g,
1.0 eq) in THF (150 mL) was added 1,3-propanedithiol (4.5 mL,
1.2 eq). The reaction mixture was refluxed for 1.5 h and the solvent
was removed in reduced pressure. The residue was taken up in
benzene (300 mL ꢁ 2) and the organic layer was washed with
10% NaOH (300 mL ꢁ 2) and brine (300 mL), dried over Na2SO4,
reduced to dryness to afford 16.9 g of crude colorless powder.
The mixture was recrystallized from hexane-benzene (95:5) to
afford a colorless needle (9a) (13.9 g, 90%): 1H NMR (CDCl3): 1.91
(dddd, 1H, J = 3.1, 6.1, 12.5, 15.6 Hz), 2.13–2.18 (m, 1H), 2.87–
2.91 (m, 2H), 3.01–3.07 (m, 2H), 5.09 (s, 1H), 5.14 (s, 2H), 5.15 (s,
2H), 6.88 (Ar, 1H), 6.99 (Ar, 1H), 7.12 (Ar, 1H), 7.28–7.47 (Ar,
10H). 13C NMR (CDCl3): 25.0, 32.1, 51.0, 71.0, 114.4, 114.8, 120.8,
127.2, 127.5, 127.8, 128.5, 132.3, 137.1, 137.2, 149.1. IR (KBr):
1508, 1015, 737 cmꢀ1. MS (EI) m/z: 408 [M+]. Anal. Calcd for
C24H24O2S2: C, 70.55; H, 5.92. Found: C, 70.29; H, 5.80.
4.3. DNA topoisomerase II mediated DNA cleavage
Reactions (20 mL) containing 30 mM Tris-HCl (pH 7.6), 60 mM
NaCl, 8 mM MgCl2, 3 mM ATP, 15 mM mercaptoethanol, 2 mL of a
drug solution (10% DMSO), 6 U of DNA topoisomerase II and
200 ng of pBR322 plasmid DNA were incubated at 37 °C for
30 min. Samples were terminated by the addition of 2 mL of a solu-
tion containing 5% sodium dodecyl sulfate (SDS) and 2.5 mg/mL of
proteinase K and incubated at 37 °C for 60 min. The samples were
electrophoresed through a 1% agarose gel in TBE (Tris-borate-
EDTA) running buffer containing 0.1% SDS. Both agarose gel and
running buffer contained 0.5 mg/mL of ethidium bromide.
4.6.2. (4S,5S)-4-(2-(3,4-Bis(benzyloxy)phenyl)-1,3-dithian-2-yl)-5-
(((tert-butyldimethylsilyl)oxy)methyl)dihydrofuran-2(3H)-one (10a)
To a solution of 9a (6.00 g, 14.7 mmol) in THF (100 mL) at
ꢀ78 °C under Ar atmosphere was added n-BuLi (10.0 mL,
16.2 mmol, 1.60 M in hexane) during 10 min. The resulting mix-
ture was stirred for 40 min at same temperature and 6 h at –
30 °C to complete deprotonation (from a yellow solution to a green
solution). To the solution was added a solution of 721 (4.02 g,
17.6 mmol) in THF (10 mL) at ꢀ78 °C over 10 min. The resulting
mixture was stirred at ꢀ30 °C for 12 h and quenched by adding
sat. NH4Cl, followed by extraction with EtOAc (300 mL ꢁ 2). The
combined organic layer was washed with brine (200 mL), dried
over Na2SO4, and concentrated in vacuo. The residue was purified
4.4. DNA topoisomerase I mediated DNA relaxation
Reactions were carried out in the same manner as described for
the Topo II relaxation assay except that reaction mixtures con-
tained 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, 150 mM NaCl, 0.1%
bovine serum albumin (BSA), 0.1 mM spermidine, 5% glycerol
and pUC19 plasmid DNA (200 ng).
by silica gel chromatography (200 g) hexane-EtOAc (5:1) as an elu-
ent to give a yellow foam (10a) (7.93 g, 91%): [
23
a
]
+13.2 (c 5.0,
D
EtOH). 1H NMR (CDCl3): ꢀ0.01 (s, 3H), ꢀ0.00 (s, 3H), 0.84 (s, 9H),
1.75–1.88 (m, 2H), 2.42–2.55 (m, 5H), 3.12 (dd, 1H, J = 1.9,
11.3 Hz), 3.63 (dd, 1H, J = 2.2, 11.3 Hz), 4.58–4.59 (m, 1H), 5.19
(s, 2H), 5.21 (s, 2H), 6.93 (Ar, 1H), 7.28–7.51 (m, 12H). 13C NMR
(CDCl3): ꢀ5.75, ꢀ5.61, 18.1, 24.5, 25.7, 26.8, 31.5, 48.6, 62.0, 64.8,
71.0, 80.3, 114.4, 116.7, 122.6, 127.3, 127.4, 127.9, 128.6, 131.9,
4.5. Determination of cell growth inhibition profiles
This experiment was carried out at the Cancer Chemotherapy
Center, Japanese Foundation for Cancer Research. The screening
panel consisted of the following 39 human cancer cell lines
(JFCR39): breast cancer HBC-4, BSY-1, HBC-5, MCF-7, and
MDAMB-231; brain cancer U251, SF-268, SF-295, SF-539, SNB-75,
and SNB-78; colon cancer HCC2998, KM-12, HT-29, HCT-15, and
HCT-116; lung cancer NCI-H23, NCI-H226, NCI-H522, NCI-H460,
A549, DMS273, and DMS114; melanoma LOX-IMVI; ovarian cancer
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3; renal can-
cer RXF-631L and ACHN; stomach cancer St-4, MKN1, MKN7,
MKN28, MKN45, and MKN74; and prostate cancer DU-145 and
PC-3. Inhibition of cell growth was assessed by measuring changes
in total cellular protein levels following 48 h treatment with a
given test compound, using the sulforhodamine B colorimetric
assay. The molar concentration of a test compound required for
50% growth inhibition (GI50) of cells was calculated as reported
previously. A detailed method is described elsewhere.28–30 COM-
PARE analysis was performed by calculating the Pearson correla-
tion coefficient (r) between the GI50 mean graphs of compounds
X and Y using the following formula: r = ((xi ꢀ xm) (yi ꢀ ym))/((xi -
ꢀ xm)2 (yi ꢀ ym)2)1/2, where xi and yi are Log GI50 of the two com-
pounds, respectively, for each cell line, and xm and ym are the
mean values of xi and yi, respectively (n = 39). The Pearson correla-
tion coefficients were used to determine the degree of similarity.
The larger the coefficient is, the higher the similarity between X
and Y is.32
137.0, 148.2, 148.4, 176.2. IR (KBr): 1778, 1504, 1258, 1130 cmꢀ1
.
MS (EI): m/z 636 [M+]. Anal. Calcd for C35H44O5S2Si: C, 66.00; H,
6.96. Found: C, 65.85; H, 6.91.
4.6.3. (4S,5S)-4-(3,4-Bis(benzyloxy)benzyl)-5-(((tert-
butyldimethylsilyl)oxy)methyl)dihydrofuran-2(3H)-one (11a)
To a solution of 10a (4.90 g, 7.69 mmol) and n-Bu3SnH (8.3 mL,
30.8 mmol) in benzene (15 mL) was added 2,20-azobisisobutyloni-
trile (AIBN, 50 mg). The reaction mixture was wormed to 80 °C and
stirred for 1.5 h. The reaction mixture was evaporated to dryness
under reduced pressure to afford 13.9 g of crude oil. The residue
was purified by silica gel chromatography (150 g) with hexane-
EtOAc (80/20) as an eluent to give a colorless oil (11a) (3.55 g,
24
87%): [a]
+15.9 (c 1.53, EtOH). 1H NMR (CDCl3): ꢀ0.05 (s, 6H),
D
0.88 (s, 9H), 2.16–2.20 (m, 1H), 2.62–2.68 (m, 4H), 3.46 (dd, 1H,
J = 3.1, 11.3 Hz), 3.73 (dd, 1H, J = 3.1, 11.3 Hz), 4.16–4.17 (m, 1H),
5.15 (s, 2H), 5.16 (s, 2H), 6.67 (Ar, 1H), 6.71 (Ar, 1H), 6.88 (Ar,
1H), 7.30–7.46 (m, 10H). 13C NMR (CDCl3): ꢀ5.68, ꢀ5.61, 18.1,
25.7, 34.8, 38.0, 39.3, 64.1, 71.3, 84.2, 115.3, 116.1, 121.8, 127.3,
127.3, 127.8, 127.9, 128.5, 128.5, 131.6, 137.1, 137.3, 147.9,
148.8, 176.7. IR (KBr): 1778, 1512, 1258, 837 cmꢀ1. MS (EI) m/z:
532 [M+]. Anal. Calcd for C32H40O5Si: C, 72.14; H, 7.57. Found: C,
71.86; H, 7.38.
Please cite this article in press as: Matsumoto H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.001

H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
4.6.4. 1-((2R,4S,5S)-4-(3,4-Bis(benzyloxy)benzyl)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (14a)
4.6.6. 1-((2R,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(3,4-
dihydroxybenzyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4
(1H,3H)-dione (16a)
DIBAL (5.63 mL, 8.45 mmol, 1.5 mol/L in toluene) was added
to a solution of 11a (3.00 g, 5.63 mmol) in THF (10 mL) at
ꢀ78 °C under Ar atmosphere. The resulting mixture was stirred
for 1.5 h at the same temperature. The reaction mixture was
quenched by the addition of H2O and then 5% HCl was added,
followed by extraction with EtOAc (200 mL ꢁ 2). The organic
layer was washed with brine (200 mL), dried over Na2SO4, and
concentrated in vacuo to give a colorless solid (12a) (3.68 g).
This product was used for next reaction without further purifica-
tion because of its instability.
Acetic anhydride (15 mL) and DMAP (10 mg) was added to a
solution of 12a in pyridine (20 mL) at 0 °C to room temperature.
The resulting mixture was stirred for 21 h at the same tempera-
ture. The reaction mixture was cooled in an ice-water bath,
quenched by the addition of H2O (200 mL), extracted with EtOAc
(200 mL ꢁ 2). The organic layer was washed with 5% HCl
(150 mL ꢁ 3), NaHCO3 (150 mL ꢁ 2), and brine (150 mL), dried
over Na2SO4, and concentrated in vacuo to a colorless oil (13a). This
product was used for next reaction without further purification
because of its instability.
A solution of thymine (1.42 g, 2.0 eq) and N,O-Bis(trimethylsi-
lyl)acetamide (8.4 mL, 6.0 eq) in 1,2-dichloroethane (15 mL) was
refluxed for 1 h and cooled to room temperature. To the mixture
was added a solution of acetate (13a) (5.63 mmol) in 1,2-dichlor-
oethane (10 mL), followed by the addition of TMSOTf (1.5 mL,
1.3 eq). After 3 h, the reaction was quenched by adding sat.
NaHCO3 and stirred for 10 min. The mixture was filtered, and fil-
trate was extracted with CHCl3 (200 mL ꢁ 2). The organic layer
was washed with brine (200 mL), dried over Na2SO4 and concen-
trated to give 3.90 g of a mixture. The residue was purified by
silica gel chromatography (200 g) with hexane-Et2O (15:85) as
To a solution of 14a (180 mg, 0.280 mmol) and AcOH (0.3 mL) in
EtOH (0.4 mL) was hydrogenated at atmospheric pressure over 10%
palladium on carbon (150 mg) for 24 h. The catalyst was removed
by filtration, and the solution was evaporated. The residue was
purified by silica gel chromatography (hexane-Et2O : 30/70) to
afford 16a (107 mg, 95%): 1H NMR (CDCl3): 0.10 (s, 3H), 0.11 (s,
3H), 0.93 (s, 9H), 1.90 (s, 3H), 2.00–2.03 (m, 1H), 2.13–2.19 (m,
1H), 2.36–2.46 (m, 2H), 2.70 (dd, 2H, J = 4.3 Hz, 12.9 Hz), 3.71
(dd, 1H, J = 2.8 Hz, 11.6 Hz), 3.79–3.81 (m, 1H), 3.98 (dd, 1H,
J = 2.2 Hz, 11.6 Hz), 5.97 (dd, 1H, J = 4.0 Hz, 6.4 Hz), 6.51 (Ar, 1H),
6.71 (Ar, 1H), 6.79 (Ar, 1H), 7.65 (s, 1H), 9.70 (br s, 1H). 13C NMR
(CDCl3): ꢀ5.40, ꢀ5.33, 12.5, 18.5, 25.9, 37.4, 38.8, 39.1, 63.1, 85.5,
86.2, 110.3, 115.6, 116.2, 120.7, 131.5, 136.3, 142.8, 144.1, 150.8,
164.7. IR (KBr): 3270, 1690 cmꢀ1. MS (FAB) m/z: 463 [M+H+]. HRMS
(FAB) Calcd. for C23H35O6N2Si, 463.2264; Found 463.2272.
4.6.7. 1-((2R,4S,5S)-4-(3,4-Dihydroxybenzyl)-5-(hydroxymethyl)
tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (17a)
To a solution of 16a (129 mg, 0.206 mmol) in THF (0.5 mL) was
added TBAFꢂ3H2O (196 mg, 3.0 eq). The reaction mixture was stir-
red for 3 h at room temperature. The reaction mixture was
quenched by the addition of H2O (10 mL). The solvent was
removed in reduced pressure, and extracted with EtOAc
(10 mL ꢁ 2). The combined organic layer was washed with brine
(15 mL), dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel chromatography (6 g) with EtOAc as an
eluent to give a colorless oil (17a) (57.4 mg, 80%): 1H NMR (CD3-
OD): 1.86 (s, 3H), 2.06 (ddd, 1H, J = 3.1, 6.9, 13.5 Hz), 2.17 (ddd,
1H, J = 6.9, 13.5 Hz), 2.48–2.56 (m, 2H), 2.66 (dd, 1H, J = 9.8,
17.0 Hz), 3.55 (dd, 1H, J = 3.4, 12.2 Hz), 3.77–3.80 (m, 2H), 6.03
(dd, 1H, J = 3.1, 6.9 Hz), 6.52 (dd, 1H, J = 1.9, 8.0 Hz), 6.64 (d, 1H,
J = 1.9 Hz,), 6.68 (d, 1H, J = 8.0 Hz). 13C NMR (CD3OD): 12.5, 38.5,
40.0, 62.4, 86.3, 87.8, 110.7, 116.4, 116.9, 121.2, 132.4, 138.4,
144.8, 146.3, 152.3, 166.6. IR (KBr): 3220.9, 1685.7 cmꢀ1. MS
(FAB) m/z: 549 [M+H+]. HRMS (FAB) Calcd. for C17H20N2O6,
348.1321; Found: 349.1410.
an eluent to give a colorless solid (14a) (1.45 g, 40%, from
25
11a): [a]
+15.9 (c 1.53, EtOH). 1H NMR (CDCl3): 0.09 (s, 6H),
D
0.93 (s, 9H), 1.92 (s, 3H), 1.98–2.00 (m, 1H), 2.10–2.14 (m,
1H), 2.46–2.52 (m, 2H), 2.66–2.71 (s, 3H), 3.55 (dd, 1H, J = 2.8,
11.6 Hz), 3.72–3.73 (m, 1H), 3.90 (dd, 1H, J = 2.2, 11.6 Hz), 5.14
(s, 2H), 5.15 (s, 2H), 6.09 (dd, 1H, J = 4.3, 7.0 Hz), 6.66 (Ar, 1H),
6.71 (Ar, 1H), 6.87 (Ar, 1H), 7.28–7.46 (Ar, 10H), 7.55 (d, 1H,
J = 1.2 Hz), 9.25 (br s, 1H). 13C NMR (CDCl3): ꢀ5.42, ꢀ5.41,
12.6, 18.4, 25.9, 37.8, 38.9, 62.9, 71.2, 71.3, 84.6, 85.5, 110.2,
115.2, 115.9, 121.6, 127.3, 127.8, 127.8, 128.5, 132.4, 135.7,
4.6.8. 1-((2R,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-((3,4-
dioxocyclohexa-1,5-dien-1-yl)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (18a)
To a mixture of 16a (20.0 mg, 0.0432 mmol), MnO2 (3.76 mg,
0.216 mmol), MS 4A (21.6 mg, powder) in CH2Cl2 (1 mL) was
refluxed for 3 h. After cooling, the reaction mixture was filtered
through a pad of CelliteÒ 535, and concentrated under reduced
pressure to give yellow oil. The oil was purified by silica gel chro-
matography using hexane-Et2O (1:1) as the eluent to afford 18a
(80 mg, 87%) as a brown solid: UV–Vis (MeOH) kmax 236 (3462),
254 (3278), 282 (2854), 392 (286). IR (KBr): 1771, 1690, 1273,
137.1, 137.3, 147.7, 148.7, 150.4, 164.1. IR (KBr): 1686 cmꢀ1
.
MS (FAB) m/z: 642 [M+]. Anal. Calcd for C37H46N2O6Si: C,
69.13; H, 7.21; N, 4.36. Found C, 69.00; H, 7.29; N, 4.20.
4.6.5. 1-((2S,4S,5S)-4-(3,4-Bis(benzyloxy)benzyl)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (15a)
1196 cmꢀ1. MS (FAB) m/z: 461 [M+H+]. HRMS (FAB) Calcd. for C23
33N2O6Si, 461.2108; Found: 461.2115.
-
25
A colorless solid (15a) (543 mg, 15%, from 11a). [
a]
+12.0 (c
H
D
9.10, EtOH). 1H NMR (CDCl3): 0.04 (s, 3H), 0.06 (s, 3H), 0.90 (s,
9H), 1.51–1.57 (m, 1H), 1.92 (s, 3H), 2.34–2.39 (m, 1H), 2.47–
2.49 (m, 1H), 2.51–2.55 (m, 1H), 2.73 (dd, 1H, J = 5.8, 13.4 Hz),
3.50 (dd, 1H, J = 4.0, 11.3 Hz), 3.64 (dd, 1H, J = 3.7, 11.3 Hz),
3.92–3.95 (m, 1H), 5.11 (s, 2H), 5.14 (s, 2H), 5.97 (dd, 1H,
J = 5.8, 7.4 Hz), 6.63 (Ar, 1H), 6.69 (Ar, 1H), 6.85 (Ar, 1H), 7.14
(s, 1H), 7.23–7.34 (Ar, 6H), 7.40–7.43 (Ar, 4H), 10.08 (br s, 1H).
13C NMR (CDCl3): ꢀ5.66, ꢀ5.56, 12.4, 18.0, 25.7, 37.7, 38.7,
41.3, 64.0, 71.0, 71.2, 85.0, 85.8, 110.5, 115.1, 127.1, 127.1,
127.6, 127.6, 128.3, 132.5, 135.0, 137.1, 137.2, 147.6, 148.6,
150.4, 164.3. IR (KBr): 3175, 1686 cmꢀ1. MS (FAB) m/z: 642
[M+]. HRMS (FAB) Calcd. for C37H46N2O6Si, 642.3125; Found:
642.3134.
4.6.9. 2-(3,4-Dimethoxyphenyl)-1,3-dithiane (9b)33
To a stirred solution of 3,4-dimethoxybenzaldehyde (15.0 g,
90.3 mmol) and SnCl2 (17.1 g, 90.3 mmol) in THF (100 mL) was
added 1,3-propanedithiol (10.8 mL, 108.3 mmol). The reaction
mixture was refluxed for 1.5 h and the solvent was removed in
reduced pressure. The residue was taken up in Et2O (500 mL ꢁ 2)
and the organic layer was washed with 10% NaOH (500 mL ꢁ 2)
and brine (200 mL), dried over Na2SO4, reduced to dryness to afford
25 g of crude colorless powder. The mixture was recrystallized
from hexane-benzene (9:1) to afford a colorless needle (9b)
(22.4 g, 97%): mp 90–91 °C. 1H NMR (CDCl3): 1.91 (dddd, 1H,
J = 3.1, 6.1, 12.5, 15.6 Hz), 2.13–2.18 (m, 1H), 2.87–2.91 (m, 2H),
Please cite this article in press as: Matsumoto H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.001

10
H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3.01–3.07 (m, 2H), 3.87 (s, 3H), 3.90 (s, 3H), 5.09 (s, 1H), 5.14 (s,
2H), 5.15 (s, 2H), 6.88 (Ar, 1H), 6.99 (Ar, 1H), 7.12 (Ar, 1H), 7.28–
7.47 (Ar, 10H). 13C NMR (CDCl3): 25.0, 32.1, 51.0, 71.0, 114.4,
114.8, 120.8, 127.2, 127.5, 127.8, 128.5, 132.3, 137.1, 137.2,
149.1. IR (KBr): 1508, 1015, 737 cmꢀ1. MS (EI) m/z: 408 [M+]. Anal.
Calcd for C12H10O2S2: C, 56.22; H, 6.29. Found: C, 56.16 H, 6.32.
ture. The reaction mixture was cooled in an ice-water bath,
quenched by the addition of H2O (200 mL), extracted with EtOAc
(500 mL ꢁ 2). The combined organic layer was washed with 5%
HCl (100 mL ꢁ 3), NaHCO3 (100 mL ꢁ 3), and brine (100 mL), dried
over Na2SO4, and concentrated in vacuo to a pale yellow oil (13b).
This product was used for next reaction without further purifica-
tion because of its instability.
4.6.10. (4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(2-(3,4-
dimethoxyphenyl)-1,3-dithian-2-yl)dihydrofuran-2(3H)-one (10b)
To a solution of 9b (2.00 g, 7.8 mmol) in THF (16 mL) at ꢀ78 °C
under Ar atmosphere was added n-BuLi (5.8 mL, 8.5 mmol, 1.5 M in
hexane) during 5 min. The resulting mixture was stirred for 10 min
at same temperature (from a yellow solution to a slightly yellow
suspension) and then for 80 min at ꢀ20 °C to complete deprotona-
tion. To the suspension was added a solution of 7 (2.14 g,
9.4 mmol) in THF (4 mL) at ꢀ78 °C. The resulting mixture was stir-
red at ꢀ78 °C for 50 min and at ꢀ30 °C for 20 min and quenched by
adding sat. aq. NH4Cl, followed by extraction with Et2O
(20 mL ꢁ 2). The combined organic layer was washed with brine
(20 mL), dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel chromatography (330 g) with benzene-
Et2O (97.5/2.5 to 90/10) as an eluent to give a colorless crystal
(10b) (3.02 g, 80%): mp 153–154 °C (decomp.). 1H NMR (CDCl3):
ꢀ0.01 (6H, d, J = 2.75 Hz), 0.84 (6H, s), 1.83–1.98 (2H, m), 2.56
(1H, dd, J = 10.0, 18.3 Hz), 2.66–2.74 (4H, m), 2.89 (1H, d,
J = 4.30 Hz), 2.93–2.96 (1H, m), 3.24 (1H, d, J = 11.3 Hz), 3.73 (1H,
dd, J = 2.2, 11.3 Hz), 3.89 (s, 3H), 3.91 (s, 3 h), 4.76 (1H, d,
J = 3.1 Hz) 6.88 (1H, d, J = 8.6 Hz), 7.51 (2H, ddd, J = 2.2, 2.45,
11.2 Hz). 13C NMR (CDCl3): ꢀ5.68, ꢀ5.53, 18.2, 24.6, 25.8, 27.0,
27.1, 31.6, 48.7, 55.9, 56.1, 62.2, 65.0, 80.4, 110.9, 112.1, 122.2,
131.5, 148.4, 149.2, 176.1. IR (KBr): 1774 cmꢀ1. MS (EI) m/z: 487
[M+]. Anal. Calcd for C23H36O5S5Si: C, 56.99; H, 7.49. Found: C,
56.74; H, 7.26.
A solution of thymine (3.31 g, 2.0 eq) and N,O-Bis(trimethylsi-
lyl)acetamide (19.4 mL, 6.0 eq) in 1,2-dichloroethane (50 mL) was
refluxed for 1 h and cooled to room temperature. To the mixture
was added a solution of acetate (13b) (13.1 mmol) in 1,2-dichlor-
oethane (5.0 mL), followed by the addition of TMSOTf (3.4 mL,
1.3 eq). After 3 h, the reaction was quenched by adding sat.
NaHCO3 and stirred for 10 min. The organic layer was dried over
Na2SO4 and concentrated to give 6.15 g of a mixture. The residue
was purified by silica gel chromatography (200 g) with hexanes-
Et2O (1:9) as an eluent to give a pale yellow foam (3.65 g,
14b:15b = 1:1.2, 57% from 11b). This mixture was inseparable in
this step, so 19b and 20b were isolated after removal of TBS group.
Reintroduction of TBS group gave pure 14b and 15b, respectively.
See below.
4.6.13. 1-((2R,4S,5S)-4-(3,4-Dimethoxybenzyl)-5-(hydroxymethyl)
tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (19b)
To a solution of mixture of 14b and 15b (497 mg, 1.01 mmol,
14b:15b = 1:1.2,) in THF (10 mL) was added TBAFꢂ3H2O (959 mg,
3.0 eq). The reaction mixture was stirred for 3 h at room tempera-
ture. The reaction mixture was quenched by the addition of H2O
(20 mL). The solvent was removed in reduced pressure, and
extracted with EtOAc (100 mL ꢁ 2). The combined organic layer
was washed with brine (100 mL), dried over Na2SO4, and concen-
trated in vacuo to yield 615 mg of a colorless oil. The residue was
purified by silica gel chromatography (30 g) with EtOAc as an elu-
ent to give a colorless oil (19b) (156 mg, 41%): 1H NMR (acetone-
d6): 1.79–1.97 (m, 1H), 1.84 (s, 3H), 2.39 (ddd, 1H, J = 6.70, 11.3,
13.1 Hz), 2.56–2.68 (m, 2H), 2.84 (dd, 1H, J = 5.5, 12.9 Hz), 3.46
(dd, 1H, J = 4.85, 11.9 Hz), 3.63 (dd, 1H, J = 0, 11.9 Hz), 3.75
(s,3H), 3.77 (s, 3H), 3.91 (br s, 1H), 4.12–4.15 (m, 1H), 6.05 (dd,
1H, J = 6.70, 7.95 Hz), 6.73 (Ar, 1H), 6.83 (Ar, 1H), 6.84 (Ar, 1H),
7.51 (s, 1H), 10.13 (br s, 1H). 13C NMR (acetone-d6): 12.7, 38.4,
39.5, 42.3, 56.3, 63.9, 86.5, 86.9, 111.0, 113.1, 113.9, 121.8, 133.8,
137.0, 149.2, 150.6, 151.7, 164.9. IR (KBr): 3418, 3182, 3044,
4.6.11. (4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(3,4-
dimethoxybenzyl)dihydrofuran-2(3H)-one (11b)
To a solution of 10b (15 g, 30.9 mmol) and n-Bu3SnH (23.9 mL,
124 mmol) in benzene (30 mL) was added 2,20-azobisisobutyloni-
trile (AIBN, 100 mg). The reaction mixture was wormed to 80 °C
and stirred for 1.5 h. The reaction mixture was evaporated to dry-
ness under reduced pressure to afford 37 g of crude oil. The residue
was purified by silica gel chromatography (2 kg) with hexane-
EtOAc (80/20) as an eluent to give a colorless oil (11b) (9.02 g,
80%): 1H NMR (CDCl3): ꢀ0.22 (s, 6H), 0.85 (s, 9H), 2.24 (dd, 1H,
J = 13.1 Hz), 2.68–2.73 (m, 4H), 3.55 (dd, 1H, J = 2.7, 11.3 Hz),
3.76 (dd, 1H, J = 3.01, 11.3 Hz), 3.85 (s, 6H), 4.24 (m, 1H), 6.65 (s,
1H), 6.68 (dd, 1H, J = 8.1 Hz), 6.79(d, 1H, J = 8.1 Hz). 13C NMR
(CDCl3): ꢀ5.76, ꢀ5.68, 18.0, 25.6, 34.8, 38.0, 39.4, 55.7, 64.1, 84.2,
111.3, 111.8, 120.6, 130.8, 147.7, 148.9, 176.5. IR (KBr):
1774 cmꢀ1. MS (EI) m/z: 380 [M+]. HRMS (EI) Calcd. for C20H32O5Si,
380.2019; Found 380.2032.
.
1693 cmꢀ1 MS (EI) m/z: 376 (M+). HRMS (EI) Calcd. for
C19H24N2O6, 376.1634; Found 376.1628.
4.6.14. 1-((2S,4S,5S)-4-(3,4-Dimethoxybenzyl)-5-(hydroxymethyl)
tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (20b)
A colorless oil (20b) (220 mg, 58%). 1H NMR (CD3OD): 1.86 (s,
3H), 2.04–2.09 (m, 1H), 2.17–2.23 (m, 1H), 2.55–2.63 (m, 2 H),
2.76 (dd, 1 H, J = 5.2 Hz, 12.5 Hz), 3.55 (dd, 1 H, J = 3.4 Hz,
12.5 Hz), 3.78–3.82 (m, 2 H), 3.79 (s, 3 H), 3.82 (s, 3 H), 6.04 (dd,
1 H, J = 3.4 Hz, 7.1 Hz), 6.76 (Ar, 1 H), 6.85 (Ar, 2 H), 7.96 (s, 1H).
13C NMR (CD3OD): 12.4, 38.7, 40.01, 40.02, 56.5, 56.6, 62.4, 86.4,
87.8, 110.8, 113.3, 114.0, 122.3, 134.0, 138.5, 149.2, 150.6, 152.4,
166.6. IR (KBr): 3418, 3179, 3059, 1693 cmꢀ1. MS (EI) m/z: 376
[M+]. HRMS (EI) Calcd. for C19H24N2O6, 376.1634; Found 376.1638.
4.6.12. (4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(3,4-
dimethoxybenzyl)tetrahydrofuran-2-yl acetate (14b)
DIBAL (13.6 mL, 19.7 mmol, 1.5 mol/L in toluene) was added to
a solution of 11b (5.0 g, 13.1 mmol) in THF (30 mL) at ꢀ78 °C under
Ar atmosphere. The resulting mixture was stirred for 1.5 h at the
same temperature. The reaction mixture was quenched by the
addition of H2O and then 5% HCl (36 mL) was added, followed by
extraction with EtOAc (500 mL ꢁ 2). The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo to give a
colorless oily 12b (5.76 g). This product was used for next reaction
without further purification because of its instability.
4.6.15. 1-((2R,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(3,4-
dimethoxybenzyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4
(1H,3H)-dione (14b)
To a stirred solution of 19b (76.0 mg, 0.202 mmol) and imida-
zole (19.2 mg, 0.283 mmol) in CH2Cl2 (1 mL) was added TBSCl
(36.5 mg, 0.242 mmol). The reaction mixture was stirred at 0 °C
for 6 h and H2O was added. The reaction mixture was extracted
with CHCl3 (50 mL ꢁ 2) and washed with brine (50 mL). The com-
bined organic extracts were evaporated and dried with Na2SO4;
Acetic anhydride (50 mL) and DMAP (10 mg) were added to a
solution of 12b in pyridine (50 mL) at 0 °C to room temperature.
The resulting mixture was stirred for 19 h at the same tempera-
Please cite this article in press as: Matsumoto H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.001

H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
yield of a colorless oil. The residue was purified by silica gel col-
with hexane-EtOAc (10/90) as an eluent to give a white foam (22b)
(48.7 mg, 70%): 1H NMR (CDCl3): 2.29 (dd, 1H, J = 6.4, 17.1 Hz), 2.49
(br s, 1H), 2.65–2.78 (m, 4H), 3.45 (dd, 1H, J = 12.4 Hz), 3.74 (dd,
1H, J = 12.4 Hz), 3.83 (s, 6H), 4.25 (m, 1H), 6.34 (H, 1H), 6.67 (H,
1H), 6.77 (H, 1H). 13C NMR (CDCl3): 35.0, 37.6, 38.9, 55.8, 55.9,
63.1, 85.2, 111.4, 111.8, 120.7, 130.5, 147.9, 149.1, 176.6. IR
(KBr): 3394.5, 1770.5, 1515.9 cmꢀ1. MS (EI) m/z: 266 [M+]. HRMS
(EI) Calcd. for C14H18O5, 266.1154; Found 266.1150.
umn chromatography (2 g) with hexane-EtOAc (90/10) as an elu-
25
ent to give a white foam (14b) (86.0 mg, 87%): [
a]
+10.4 (c
D
1.05, CHCl3). 1H NMR (CDCl3): 0.02 (s, 3H), 0.04 (s, 3H), 0.88 (s,
9H), 1.66 (m, 1H), 1.92 (s, 3h), 2.52–2.60 (m, 3H), 2.78 (dd, 1H,
J = 4.0, 11.9 Hz), 3.52–3.65 (m, 1H), 3.66–3.68 (m, 1H), 3.83 (s,
3H), 3.84 (s, 3H), 3.98–4.01 (m, 1H), 5.98 (dd, 1H, J = 5.8, 7.7 Hz),
6.65 (Ar, 1H), 6.76 (Ar, 1H), 7.19 (s, 1H), 9.61 (br, 1H). 13C NMR
(CDCl3): ꢀ5.60, ꢀ5.49, 12.5, 18.4, 25.9, 38.1, 39.2, 41.3, 55.7, 55.8,
63.1, 84.7, 85.6, 110.2, 111.4, 111.7, 120.5, 131.8, 135.0, 147.6,
149.0, 150.4, 164.2. IR (KBr): 3175, 3047, 1693 cmꢀ1. MS (FAB)
m/z: 513 [M+Na+], 491 [M+H+]. HRMS (FAB) Calcd. for C25H38N2-
NaO6Si, 513.2397; Found 513.2407.
4.6.19. 1-((2R,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(2-
(3,4-dimethoxyphenyl)-1,3-dithian-2-yl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (25b)
DIBAL (1.0 mL, 1.55 mmol, 1.5 mol/L in toluene) was added to a
solution of 10b (500 mg, 1.03 mmol) in THF (9.0 mL) at ꢀ78 °C
under Ar atmosphere. The resulting mixture was stirred for 0.5 h
at the same temperature. The reaction mixture was warmed to
ꢀ50 °C for 1 h and quenched by the addition of H2O (10 mL) and
then 1 mol/L HCl (20 mL) was added, followed by extraction with
Et2O (20 mL ꢁ 2). The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo to give a colorless oily
23b (548 mg). This product was used for next reaction without fur-
ther purification because of its instability.
Acetic anhydride (2.0 mL) was added to a solution of 23b
(548 mg, 1.13 mmol) in pyridine (2.0 mL) at 0 °C to room temper-
ature. The resulting mixture was stirred for 18 h at the same tem-
perature. The reaction mixture was cooled in an ice-water bath,
quenched by the addition of H2O (20 mL), extracted with EtOAc
(20 mL ꢁ 2). The combined organic layer was washed with 5%
HCl (20 mL ꢁ 3), NaHCO3 (20 mL), and brine (20 mL), dried over
Na2SO4, and concentrated in vacuo to yield 612 mg of a slightly yel-
low oil (24b). This product was used for next reaction without fur-
ther purification because of its instability.
To a stirred solution of 24b (1.00 g, 1.70 mmol) and N,O-Bis
(trimethylsilyl)acetamide (2.49 mL, 10.2 mmol) in 1,2-Dichlor-
oethane (18 mL) was refluxed for 1.5 h. The reaction mixture was
cooled at room temperature, and added to trimethylsilyl trifluo-
romethanesulfonate (2.26 mmol, 1.3 eq.). The mixture was
refluxed for 17 h. Sat. aq. NH4Cl were added to the reaction mixture
successively, and the solution was added saturated NaHCO3, stirred
for 10 min. The resulting solution was taken organic layer was fil-
tered, and a precipitate was removed, and concentrated in vacuo.
The residue was purified by silica gel chromatography (117 g) with
hexanes-EtOAc (hexane 100 to 40/60) as eluent to give the color-
less oil (87.5 mg, 25b:26b = 1:1.2, 50%, 3 steps). The reaction mix-
ture was separated to 25b (beta form, 48 mg) and 26b (alfa form,
40 mg) by using chiral stationary phase (CHIRALFLASH IA, DAICEL,
Japan) with hexane-2-propanol (hexane/2-propanol = 100 to
50/50) as eluent. The stereochemistry of those compounds were
determined by comparison of the NOESY spectrum (See Supple-
mentary data).
4.6.16. 1-((2S,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(3,4-
dimethoxybenzyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4
(1H,3H)-dione (15b)
To a stirred solution of 20b (100 mg, 0.266 mmol) and imida-
zole (25.3 mg, 0.372 mmol) in CH2Cl2 (1 mL) was added TBSCl
(48.0 mg, 0.319 mmol). The reaction mixture was stirred at 0 °C
for 6 h and H2O was added. The reaction mixture was extracted
with CHCl3 (50 mL ꢁ 2) and washed with brine (50 mL). The com-
bined organic extracts were evaporated and dried with Na2SO4;
yield of a colorless oil. The residue was purified by silica gel col-
umn chromatography (2 g) with hexane-Et2O (3/7) as an eluent
to give a white foam (15b) (100 mg, 77%): [a]
25+9.12 (c 2.35,
D
CHCl3). 1H NMR (CDCl3): 0.09 (s, 6H), 0.91 (s, 6H), 1.89 (s, 1H),
2.06 (ddd, 1H, J = 4.6, 7.7, 13.1 Hz), 2.19 (ddd, 1H, J = 6.7, 7.5,
14.1 Hz), 2.52–2.58 (m, 2H), 2.70–2.74 (m, 1H), 3.61 (dd, 1H,
J = 2.8, 11.6 Hz), 3.61 (dd, 1H, J = 2.2, 11.6 Hz), 6.11 (dd, 1H,
J = 4.6, 6.7 Hz), 6.65 (dd, 1H, 6.68 (dd, 1H, J = 1.8, 7.9 Hz), 7.56 (s,
1H), 9.26 (br, 1H). 13C NMR (CDCl3): ꢀ5.46, ꢀ5.43, 12.5, 18.4,
25.9, 38.1, 39.0, 39.2, 55.7, 55.8, 63.1, 85.6,, 110.2, 111.4, 111.8,
120.5, 131.8, 135.6, 147.6, 149.0, 150.4, 164.2. IR (KBr): 3175,
3047, 1693 cmꢀ1. MS (FAB) m/z: 513 [M+Na+], 491 [M+H+]. HRMS
(FAB) Calcd. for C25H38N2NaO6Si, 513.2397; Found 513.2382.
4.6.17. (4S,5S)-4-(2-(3,4-Dimethoxyphenyl)-1,3-dithian-2-yl)-5-
(hydroxymethyl)dihydrofuran-2(3H)-one (21b)
To a solution of 10b (220 mg, 0.454 mmol) in THF (0.5 mL) was
added TBAFꢂ3H2O (429 mg, 3.0 eq). The reaction mixture was stir-
red for 3 h and the H2O was added. The solvent was removed in
reduced pressure. The residue was taken up in EtOAc (20 mL ꢁ 2)
and the organic layer was washed with brine (20 mL), dried over
Na2SO4, reduced to dryness to afford 263.5 mg of crude yellow
foam. The residue was purified by silica gel column chromatogra-
phy (7.5 g) with hexane-EtOAc (10/90) as an eluent to give a white
foam (21b) (159 mg, 95%): 1H NMR (CDCl3): 1.80 (br s, 1H), 1.87–
1.91 (m, 1H), 1.96–1.99 (m, 1H), 2.59–2.76 (m, 5H), 2.95 (m, 1H),
3.03 (dd, 1H, J = 5.4, 18.3 Hz), 3.19 (dd, 1H, J = 10.7 Hz), 3.66 (dd,
1H, J = 10.7 Hz), 3.90 (s, 3H), 3.91 (s, 3H), 4.79 (m, 1H), 6.89 (Ar,
1H), 7.50 (Ar, 1H), 7.52 (Ar, 1H). 13C NMR (CDCl3): 24.6, 27.0,
27.1, 31.5, 48.4, 55.9, 56.1, 61.9, 64.3, 80.8, 111.0, 112.1, 122.1,
131.4, 148.5, 149.3, 176.1. IR (KBr): 3491, 1771 cm -1. MS (EI) m/
z: 370 [M+]. HRMS (EI) Calcd. for C17H22O5S2, 370.0909; Found
370.0917.
4.6.20. 1-((2R,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(2-
(3,4-dimethoxyphenyl)-1,3-dithian-2-yl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (25b)
[a]
25 +7.77 (c 3.52, CHCl3). 1H NMR (CDCl3): 0.48 (s, 3H), 0.48 (s,
D
3H), 0.88 (s, 9H), 1.76–1.79 (m, 1H), 1.86 (s, 3H), 1.86–1.92 (m, 2H),
2.62–2.72 (m, 4H), 2.77–2.86 (m, 1H), 2.87–2.88 (m, 1H), 3.25 (d,
1H, J = 11.0 Hz), 3.79 (d, 1H, J = 11.0 Hz), 3.89 (s, 3H), 3.89 (s, 3H),
4.39 (m, 1H), 5.91 (t, 1H, J = 7.3, 7.3 Hz), 6.88 (Ar, 1H), 7.48 (s,
1H), 7.52–7.54 (Ar, 2H). 13C NMR (CDCl3): ꢀ5.40, ꢀ5.30, 12.4,
18.3, 24.7, 25.9, 27.2, 27.3, 35.4, 51.5, 55.9, 55.9, 62.2, 65.1, 80.8,
85.4, 110.4, 110.9, 112.2, 122.3, 132.0, 135.5, 148.3, 149.1, 150.1,
163.8. IR (KBr): 3175, 3047, 1693 cmꢀ1. MS (FAB) m/z: 617 [M
+Na+], 595 [M+H+]. HRMS (FAB) Calcd. for C28H42N2NaO6S2Si,
617.2151; Found 617.2141.
4.6.18. (4S,5S)-4-(3,4-Dimethoxybenzyl)-5-(hydroxymethyl)
dihydrofuran-2(3H)-one (22b)
To a solution of 11b (100 mg, 0.263 mmol) in THF (0.5 mL) was
added TBAFꢂ3H2O (249 mg, 3.0 eq). The reaction mixture was stir-
red for 3 h and the H2O was added. The solvent was removed in
reduced pressure. The residue was taken up in EtOAc (20 mL ꢁ 2)
and the organic layer was washed with brine (20 mL), dried over
Na2SO4, reduced to dryness to afford 89.0 mg of crude yellow foam.
The residue was purified by silica gel column chromatography (7 g)
Please cite this article in press as: Matsumoto H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.001

12
H. Matsumoto et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.6.21. 1-((2S,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(2-
(3,4-dimethoxyphenyl)-1,3-dithian-2-yl)tetrahydrofuran-2-yl)-5-
Aid for Scientific Research on Innovative Areas, Scientific Support
Programs for Cancer Research, from The Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan.
methylpyrimidine-2,4(1H,3H)-dione (26b)
25
[
a]
ꢀ23.1 (c 2.64, CHCl3). 1H NMR (CDCl3): 0.00 (s, 3H), 0.01 (s,
D
3H), 0.86 (s, 9H), 1.89 (s, 3H), 1.82–1.92 (m, 2H), 2.15–2.22 (m, 1H),
2.31–2.37 (m, 1H), 2.63–2.77 (m, 4H), 2.93–2.98 (m, 1H), 3.59 (d,
1H, J = 10.4 Hz), 3.87 (s, 3H), 3.88 (d, 1H, J = 10.4 Hz), 3.89 (s, 3H),
4.52 (m, 1H), 6.16 (dd, 1H, J = 6.4 Hz, 8.0 Hz), 6.86 (Ar, 1H), 7.11
(s, 1H), 7.49–7.51 (Ar, 2H). 13C NMR (CDCl3): ꢀ5.61, ꢀ5.43, 12.6,
18.2, 24.8, 25.8, 27.3, 27.4, 34.5, 51.2, 55.8, 55.9, 62.3, 66.3, 80.1,
85.0, 110.6, 110.8, 112.1, 122.1, 132.2, 135.3, 148.3, 149.2, 150.1,
163.8. IR (KBr): 3175, 3047, 1693 cmꢀ1. MS (FAB) m/z: 617 [M
+Na+], 595 [M+H+]. HRMS (FAB) Calcd. for C28H42N2NaO6S2Si,
617.2151; Found 617.2152.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.001.
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Acknowledgments
In vitro antiproliferative activities of compounds 25b and 26b
against human cancer cell lines were examined by the Screening
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Please cite this article in press as: Matsumoto H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.001