C-Glucosides with heteroaryl thiophene as novel sodium-dependent glucose cotransporter 2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.05.048

Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted derivatives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Full text of DOI:10.1016/j.bmc.2013.05.048

Bioorganic & Medicinal Chemistry 21 (2013) 5561–5572
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C-Glucosides with heteroaryl thiophene as novel sodium-dependent
glucose cotransporter 2 inhibitors
Yuichi Koga a, Shigeki Sakamaki a, Mitsuya Hongu a, Eiji Kawanishi a, Toshiaki Sakamoto a,
Yasuo Yamamoto a, Hirotaka Kimata b, Keiko Nakayama b, Chiaki Kuriyama b, Yasuaki Matsushita b,
Kiichiro Ueta b, Minoru Tsuda-Tsukimoto c, Sumihiro Nomura a,
⇑
a Medicinal Chemistry Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
b Pharmacology Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
c DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been
developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring
in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory
activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted deriva-
tives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associ-
ated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects
in high-fat diet fed KK (HF-KK) mice.
Received 17 April 2013
Revised 23 May 2013
Accepted 24 May 2013
Available online 4 June 2013
Keywords:
Type 2 diabetes
SGLT2 inhibitor
Thiophene
Ó 2013 Elsevier Ltd. All rights reserved.
Glucoside
Canagliflozin
1. Introduction
distributed in the intestine, heart, and trachea, while SGLT2 is ex-
pressed solely in the kidney.10 By enhancing glucose excretion into
The achievement and maintenance of near-normal glycemia re-
duces the risk of diabetes complications.1,2 Despite lifestyle and
pharmacological interventions, glucose levels increase over time
in type 2 diabetes, probably as a consequence of declining pancre-
atic b-cell function.3 The progressive nature of type 2 diabetes
makes it difficult to maintain good glycemic control with several
glucose-lowering agents,4 and as a result, it requires the gradual
dose escalation, the use of combination therapies or insulin.5
Therefore, there is a need for agents with a newer and complemen-
tary mechanism of action which can be used throughout the life of
urine, SGLT2 inhibitors should lead to a significant loss of calories.
Their potential advantage would be reducing blood glucose and
leading to weight loss; thus, they could be used at any stage of type
2 diabetes.11 In fact, several specific and potent SGLT2 inhibitors
have been developed.12–15
Canagliflozin (1), one of potent SGLT2 inhibitors, is being stud-
ied in clinical trials, and a more thorough understanding of its
derivatives is required. To explore the structure–activity relation-
ship (SAR) of the aryl substituent at thiophene ring on 1, we devel-
oped synthetic strategies for C-glucosides with heteroaryl
thiophene 2 and evaluated these compounds on SGLT2 activity
and urinary glucose excretion (UGE). Herein, we report the synthe-
sis and the biological result of 2.
patients with type
2 diabetes. Inhibitors of sodium-glucose
cotransporters (SGLTs) is an attractive approach to such need, as
their action of reducing blood glucose is independent from insu-
lin.6 The kidney contributes to glucose homeostasis by reabsorbing
approximately 180 g of glucose from the glomerular filtrate each
day, and SGLT2 expressed in the early convoluted segment (S1)
of the proximal tubule mediates 80–90% of renal glucose reabsorp-
tion.7–9 The remaining 10–20% of renal glucose reabsorption occurs
through SGLT1, which is expressed in the more distal, straight sec-
tion of the proximal tubule (S3).9 Besides the kidney, SGLT1 is
2. Chemistry
We initially planned to synthesize C-glucosides 2 bearing a het-
eroaromatic ring using a previously reported strategy,12,13 and
compound 2a was prepared as shown in Scheme 1. 2-(3-Pyridyl)
thiophene (3) was treated with n-butyllithium, followed by
addition of 5-bromo-2-methylbenzaldehyde to give diarylcarbinol
4. One of the reducing agents of diarylcarbinols is a combination of
sodium tetrahydroborate and trifluoroacetic acid along with
⇑
Corresponding author. Tel.: +81 48 433 2511; fax: +81 48 433 2611.
E-mail address: nomura.sumihiro@mw.mt-pharma.co.jp (S. Nomura).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.048

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Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
Me OH
Me
Br
N
N
N
S
S
a
b
S
Br
3
4
5
Me
OH
Me
N
N
S
S
c, d, e
f
MeO
O
OH
OH
OH
O
HO
HO
OH
OH
6
2a
Scheme 1. Synthesis of 2a. Reagents and conditions: (a) n-BuLi, THF, À78 °C, then 5-bromo-2-methyl-benzaldehyde, À78 °C, 67%; (b) NaBH(OAc)3, TFA, 0 °C to rt, quant.; (c)
n-BuLi, THF–toluene, À78 °C; (d) 2,3,4,6-tetrakis-O-trimethylsilyl- -gluconolactone, À78 °C; (e) MeSO3H, MeOH, À78 °C to rt, 2.3%; (f) Et3SiH, BF3ÁEt2O, CHCl3, 0 °C, 30%.
D
R1
R1
R1
S
S
X
S
R2
R2
a
b
OAc
OAc
OAc
O
OH
OH
O
O
AcO
AcO
HO
OAc
OAc
OAc
OH
7 (R1=Me,X=Cl)
8 (R1=Me,X=Br)
9 (R1=Cl, X=Br)
10b-d, 10f, 10g
2b-d, 2f, 2g
Scheme 2. Syntheses of 2b–2d, 2f, 2g. Reagents and conditions: (a) 7, Pd2(dba)3, P(tert-Bu)3ÁHBF4, CsF, 5-tri(n-butyl)stannylpyrimidine, dioxane, 100 °C, 41% (for 10b) or 8,
PdCl2(PPh3)2, CuI, 2-tri(n-butyl)stannylpyrimidine, NMP, 100 °C, 66% (for 10c) or 8, Pd(PPh3)4, CuI, 5-tri(n-butyl)stannylthiazole, dioxane, reflux, 23% (for 10d) or 9, Pd(PPh3)4,
CsF, pyridine-3-boronic acid or 6-fluoropyridine-3-boronic acid pinacol ester, DME, reflux, 53–81% (for 10f, 10g); (b) NaOMe, MeOH–THF, rt, 55–100%.
hydrogen gas evolution.16 To avoid gas evolution, sodium triacet-
oxyborohydride was used instead of sodium tetrahydroborate.
With this combination, compound 4 was rapidly reduced to yield
aglycon 5. Lithium halogen exchange of 5 with n-butyllithium fol-
in a manner similar to 2e. The chlorothiophene derivative 7 was
obtained by protection of the hydroxyl groups with acetic anhy-
dride. The bromothiophene derivative 8 was synthesized by
hydrogenolysis of 7 with palladium on carbon and triethylamine
in methanol–tetrahydrofuran, followed by bromination with
bromine.
The synthetic route to 9 is outlined in Scheme 4. While the tert-
butyl ester group of 16 did not tolerate conditions of lithium halo-
gen exchange with n-butyllithium or tert-butyllithium, we found
that aryllithium was generated from 16 successfully using 2,4,6-
trimethylphenyllihium (mesityllithium), which has a less nucleo-
philic character.17 In addition, Barbier-type conditions had a good
result. In particular, a mixture of 16 and 2,3,4,6-tetrakis-O-trimeth-
lowed by addition of 2,3,4,6-tetrakis-O-trimethylsilyl-D-glucono-
lactone12 generated an anomeric mixture of lactols, which were
converted in situ to the desilylated methyl ether 6 by treatment
with methanesulfonic acid in methanol. Unfortunately, the lithia-
tion of 3-pyridylthiophene derivative 5 was problematic and this
reaction resulted in a low yield (2.3%). Finally, the C-glucoside
derivative 2a was obtained by stereoselective reduction of 6 using
a combination of triethylsilane and boron trifluoride etherate. The
stereochemistry of 2a was determined as the b-configuration
based on the coupling constant between anomeric C–H and adja-
cent C–H (J = 9.5 Hz) in the 1H NMR spectrum.
ylsilyl-D-gluconolactone was added dropwise to mesityllithium in
tetrahydrofuran, followed by addition of methanesulfonic acid in
methanol to generate desilylated methyl ether 17 as an amorphous
powder (89.9% pure by HPLC). Acetylation of 17 with acetic anhy-
dride and recrystallization from toluene–n-hexane gave 18 with a
purity of 98.7% by HPLC. The tert-butyl ester 18 was converted into
carboxylic acid using formic acid, and treated with oxalyl chloride,
then 2-bromothiophene and aluminum trichloride to afford 19. Fi-
nally, intermediate 9 was obtained by reduction of the carbonyl
group of 19 to the benzyl alcohol using sodium tetrahydroborate,
followed by simultaneous reduction of the resultant hydroxyl
group and the anomeric methoxy group with triethylsilane and
boron trifluoride etherate in acetonitrile–water.
To avoid the problematic lithium halogen exchange reaction,
we designed new approaches to compounds 2 using palladium cat-
alyzed coupling reactions of C-glucosides having halogenothioph-
ene. The coupling reactions of halogenothiophene derivatives 7, 8
or 9 with the heteroaryl stannanes, boronic acid, or boronate ester
proceeded smoothly to give O-acetyl protected C-glucosides 10 in
moderate to good yields as shown in Scheme 2. C-glycosides 2b–
2d, 2f, and 2g were obtained by methanolysis of the corresponding
10b–10d, 10f, and 10g.
The synthetic route to intermediates 7 and 8 is outlined in
Scheme 3. Benzoic acid 11 was treated with oxalyl chloride, fol-
lowed by addition of 2-chlorothiophene and aluminum trichloride
to give ketone 12. Reduction of 12 with triethylsilane and boron
trifluoride etherate in acetonitrile–chloroform afforded thiophene
aglycon 13. The aglycon 13 was converted into b-C-glucoside 14
C-Glucoside 2e was synthesized according to the method de-
scribed previously by our group using palladium-catalyzed cou-
pling of aglycon 24 with glucal boronate 2518 as shown in
Scheme 5. The coupling of 2-bromothiophene (20) with pyrazole

Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
5563
Me
O
Me
O
Me
S
S
a, b
c
OH
Cl
Cl
Br
Br
S
Br
S
11
12
13
Me
OH
Me
Cl
Cl
h
d, e, f, g
OH
OH
OAc
O
O
HO
AcO
OAc
OAc
14
7
Me
Me
S
S
Br
i
j
OAc
OAc
OAc
OAc
O
O
AcO
AcO
OAc
OAc
15
8
Scheme 3. Syntheses of intermediates 7, 8. Reagents and conditions: (a) (COCl)2, DMF, CHCl3, rt; (b) AlCl3, 2-chlorothiophene, CH2Cl2, 0 °C to rt, 81%; (c) Et3SiH, BF3ÁEt2O,
CH3CN–CHCl3, 0 °C to rt, 100%; (d) n-BuLi, THF–toluene, À78 °C; (e) 2,3,4,6-tetrakis-O-trimethylsilyl- -gluconolactone, À78 °C; (f) MeSO3H, MeOH, À78 °C to rt; (g) Et3SiH,
BF3ÁEt2O, CHCl3, 0 °C, 57%; (h) Ac2O, pyridine, 4-dimethylaminopyridine, CHCl3, rt, 75%; (i) H2, Pd-C, Et3N, MeOH–THF, rt, 72%; (j) Br2, pyridine, CHCl3, 0 °C to rt, 93%.
D
Cl
Cl
CO2tBu
CO2tBu
Cl
Br
CO2tBu
c
a, b
MeO
O
MeO
O
OH
OH
OAc
OAc
HO
AcO
16
OH
OAc
17
18
Cl
O
Cl
S
S
Br
Br
d, e, f
g, h
MeO
O
OAc
OAc
OAc
OAc
O
AcO
AcO
OAc
OAc
19
9
Scheme 4. Synthesis of intermediate 9. Reagents and conditions: (a) 2,3,4,6-tetrakis-O-trimethylsilyl-
À78 °C to rt, 87%; (c) Ac2O, iPr2NEt, 4-dimethylaminopyridine, toluene, 0 °C to rt; 73%; (d) HCO2H, 50 °C; (e) (COCl)2, DMF, CH2Cl2, rt; (f) AlCl3, 2-bromothiophene, CH2Cl2,
À78 °C to 0 °C, 79%; (g) NaBH4, EtOH–THF, 0 °C; (h) Et3SiH, BF3ÁEt2O, CH3CN–H2O, 0 °C to rt, 66%;
D
-gluconolactone, mesityllithium, THF, À78 °C; (b) MeSO3H, MeOH,
gave 2-(pyrazol-1-yl)thiophene (21).19 Lithiation of 21 with
3. Results and discussion
n-butyllithium, followed by addition of 5-bromo-2-chlorobenzal-
dehyde, generated an inseparable mixture of 22 and 23. Under
reaction conditions of triethylsilane and boron trifluoride etherate,
only 22 was reduced to afford a mixture of 23 and 24, which was
easily separated by column chromatography to yield desired 24.
Aglycon 24 was coupled with glucal-boronate 25 in the presence
of catalytic dichlorobis(triphenylphosphine)palladium, followed
by the stereoselective hydroboration, oxidation using hydrogen
peroxide in alkaline condition, and the deprotection of O-silyl
groups with tetra-n-butylammonium fluoride to afford C-glucoside
2e.
We evaluated the effects of the heteroaryl thiophene deriva-
tives 2 (Fig. 1) on human SGLT2 (hSGLT2) activity and on UGE in
male Sprague–Dawley (SD) rats per 200 g of body weight over
24 h. The SARs of the representative compounds are shown in Ta-
ble 1. 3-Pyridyl (2a) or 2-pyrimidyl (2c) derivative showed higher
hSGLT2 inhibitory activities than 5-pyrimidyl derivative 2b. 5-
Membered heteroaryl derivatives (2d and 2e) also retained good
hSGLT2 inhibitory activities. For R1 substituents, chlorine atom
(2f) possessed better in vitro potency than methyl group (2a).
Next, we were interested in the C-glucosides with fluorinated

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Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
S
S
a
b
Br
N
N
20
21
Cl
OH
Cl
OH
Cl
Br
S
S
N
c
S
N
N
N
N
N
Br
Br
22
23
24
Cl
N
S
N
O
O
B
d, e, f, g
OH
OH
O
O
HO
OTIPS
O
O
OH
Si
t-Bu
t-Bu
2e
25
Scheme 5. Synthesis of 2e. Reagents and conditions: (a) pyrazole, NaH, CuO, pyridine, reflux, 67%; (b) n-BuLi, THF, À78 °C, then 5-bromo-2-chlorobenzaldehyde, À78 °C; (c)
Et3SiH, BF3ÁEt2O, CH2Cl2, 0 °C to rt, 29% from 21; (d) PdCl2(PPh3)2, 25, DME–Na2CO3 aq, reflux; (e) BH3ÁTHF, THF, 0 °C; (f) H2O2 aq–NaOH aq, 0 °C to rt; (g) n-Bu4NF, THF, 60 °C,
39%.
R1
Table 1
SAR of 2a–2g
Me
OH
S
S
F
Het
R1
S
R2
OH
OH
OH
OH
O
O
HO
HO
OH
OH
O
OH
HO
1
2
OH
Het: Heteroaromatic ring
Figure 1. Structures of C-glucosides with thiophene ring.
Compd
R1
R2
hSGLT2 IC50 (nM)
1.0
UGEa (mg/day)
2023
2a
Me
N
N
heteroaryl thiophene, considering the structural similarity with 1.
6-Fluoro-3-pyridyl derivative 2g possessed comparable hSGLT2
inhibitory activity to 1 and showed the highest UGE effect among
2a–2g.
N
2b
2c
2d
Me
Me
Me
7.3
1.7
2.4
N.D.b
2220
N.D.b
N
N
We next determined the selectivity of 2g for glucose transport-
ers. hSGLT1 or hSGLT2 were stably expressed in Chinese hamster
ovary-K (CHOK) cells and we investigated the inhibitory effects
N
N
of 2g on the uptake of [14C]
a-methyl-D-glucopyranoside (AMG)
in these cell lines.20 The IC50 values were 750 nM for hSGLT1 and
2.0 nM for hSGLT2, respectively. We next evaluated the inhibitory
effect of 2g on facilitated glucose transporter 1 (GLUT1) in L6 myo-
S
2e
2f
Cl
Cl
Cl
1.5
0.8
2.0
1940
2140
2666
N
blast cells. Compound 2g inhibited [3H]-2-deoxy-
D
-glucose (2-DG)
uptake mediated by GLUT1 transporters21 by only 19% at 10
l
M.
N
N
Taken together, these results identified compound 2g as a potent
and selective inhibitor of hSGLT2.
F
2g
Oral administration (30 mg/kg) of 2g to male SD rats induced
UGE over 24 h by 2666 mg/200 g body weight. Following intrave-
nous and oral doses of 3 and 10 mg/kg, respectively, to male SD
rats, AUC0–inf,po, t1/2,po, and oral bioavailability were determined
to be 10391 ng h/mL, 3.53 h, and 27.4%, respectively (Table 2).
These results suggest that the exposure to 2g is sufficient to inhibit
SGLT2 activity, and inhibition of SGLT2 in renal tubules after oral
administration of 2g is likely to continuously suppress glucose
reabsorption. Since most of the filtered glucose is reabsorbed by
SGLT2 in the renal tubules, this novel compound 2g would be use-
ful as an anti-diabetic agent.
F
1
Me
2.2
3696
a
Each compound was orally administered at a dose of 30 mg/kg to male Spra-
gue–Dawley (SD) rats. Urinary glucose excretion (UGE) data over 24 h were nor-
malized per 200 g body weight.
b
N.D.: not determined.
Single oral administration of 2g at 3 mg/kg remarkably reduced
blood glucose levels without influencing food intake in hyperglyce-
mic high-fat diet fed KK (HF-KK) mice (Fig. 2). There was a 43%

Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
5565
Table 2
pyridylthiophene derivative 2g (TA-3404) has potential for the
treatment of type 2 diabetes.
Pharmacokinetic (PK) parameters of 2g in male Sprague–Dawley (SD) rats following
intravenous and oral administrations
Compd
2g
2g
5. Experimental
5.1. Chemistry
Dose (mg/kg)
Route
Cmax (ng/mL)
tmax
3
iv
10
po
964
3.0
All reactions were carried out under inert gas or with a CaCl2
drying tube, and reaction mixtures were stirred magnetically. All
reagents and solvents were purchased from commercial suppliers
and used without further purification unless otherwise noted.
Reaction products were monitored by TLC using 0.25 mm E. Merck
silica gel plates (60 F254) and were visualized using UV light or 5%
phosphomolybdic acid in 95% ethanol. Melting points were mea-
sured by a BÜCHI model B-545 instrument and were uncorrected.
NMR spectra were collected on JEOL JNM-ECX400P and Varian
UNITY INOVA500 spectrometers. Chemical shifts are reported in
parts per million (ppm) downfield from internal reference tetra-
methylsilane standard; coupling constants (J value) are given in
hertz (Hz). MS (APCI) spectra were obtained on Finnigan MAT
SSQ7000C or ThermoQuest LCQ Advantage, eluting with 10 mM
AcONH4/MeOH. MS (GC) spectra were measured on a Shimadzu
GCMS-QP2010. Analytical HPLC spectra were reported using Agi-
lent 1100 with a UV detector measuring absorbance at 210 nm.
Elemental analyses were conducted by Medicinal Chemistry Re-
search Laboratories, Mitsubishi Tanabe Pharma.
AUC0-inf (ng h/mL)
t1/2 (h)
CLtot (mL/h/kg)
Vdss (mL/kg)
F (%)
11371
3.22
264
10391
3.53
1000
27.4
reduction in blood glucose level versus vehicle at 6 h. Therefore, 2g
(TA-3404) would control hyperglycemia in the therapy of type 2
diabetes.
4. Conclusion
We have demonstrated the synthesis and biological evaluation
of C-glucosides with heteroaryl thiophene. Among the tested com-
pounds, 2g showed a highly potent and selective inhibition for
hSGLT2 with favorable pharmacokinetic profiles and remarkably
increased UGE. In addition, oral administration of 2g induced
anti-hyperglycemic effects in HF-KK mice. The 6-fluoro-3-
Figure 2. Effects of single oral dosing of 2 g on blood glucose levels and food intake in high-fat diet fed KK (HF-KK). Data are expressed as the mean SEM (n = 6): ⁄⁄P <0.01,
⁄⁄⁄P <0.001 versus vehicle.

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Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
5.1.1. (5-Bromo-2-methylphenyl)(5-pyridin-3-yl-thiophen-2-yl)
methanol (4)
1H), 6.84 (d, J = 3.4 Hz, 1H), 7.15 (d, J = 7.9 Hz, 1H), 7.33 (m, 1H),
7.39 (dd, J = 7.7, 4.8 Hz, 1H), 7.42–7.46 (m, 2H), 7.93 (m, 1H),
8.44 (dd, J = 4.7, 1.1 Hz, 1H), 8.79 (d, J = 1.8 Hz, 1H). Mass (APCI)
m/z: 458 (M+H).
To
a stirred solution of 3-pyridylthiophene (3) (1.22 g,
7.54 mmol) in tetrahydrofuran (30 mL) at À78 °C under argon
atmosphere was added dropwise n-butyllithium (2.6 N n-hexane
solution, 2.91 mL, 7.54 mmol). After being stirred at 0 °C for
10 min, the mixture was cooled again at À78 °C, and to the mixture
was added dropwise a solution of 5-bromo-2-methyl-benzalde-
hyde (1.50 g, 7.54 mmol) in tetrahydrofuran (5 mL). The resultant
mixture was stirred at the same temperature for 1 h. The reaction
mixture was quenched with saturated aqueous ammonium chlo-
ride solution, and extracted with ethyl acetate. The organic layer
was washed with brine, dried over sodium sulfate prior to filtra-
tion, and the solvent was evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
5.1.4. 1-(b-D-Glucopyranosyl)- 4-methyl-3-[5-(3-pyridyl)-2-
thienylmethyl]benzene (2a)
To a stirred solution of 6 (46 mg, 0.10 mmol) in chloroform
(5 mL) was added triethylsilane (0.06 mL, 0.40 mmol) followed
by boron trifluoride diethyletherate (0.05 mL, 0.40 mmol) at
À78 °C. The mixture was allowed to warm to 0 °C, and stirred for
3 h. The reaction mixture was quenched with saturated aqueous
sodium hydrogen carbonate solution, and extracted with ethyl ace-
tate–tetrahydrofuran. The organic layer was washed with brine,
dried over magnesium sulfate prior to filtration, and the solvent
was evaporated under reduced pressure. The residue was purified
by silica gel column chromatography (5–10% methanol in chloro-
form) followed by trituration with diethyl ether to give tilted com-
pound 2a (13 mg, 30%) as a pale brown powder. 1H NMR (DMSO-
d6) d: 2.27 (s, 3H), 3.13–3.40 (m, 4H), 3.44 (m, 1H), 3.69 (m, 1H),
3.97 (d, J = 9.5 Hz, 1H), 4.14 (d, J = 15.9 Hz, 1H), 4.18 (d,
J = 15.9 Hz, 1H), 4.41 (t, J = 5.7 Hz, 1H), 4.72 (d, J = 5.6 Hz, 1H),
4.90 (d, J = 4.8 Hz, 2H), 6.86 (d, J = 3.4 Hz, 1H), 7.10–7.17 (m, 2H),
7.23 (s, 1H), 7.39 (dd, J = 7.6, 4.7 Hz, 1H), 7.45 (d, J = 3.5 Hz, 1H),
7.94 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 3.5 Hz, 1H), 8.80 (d, J = 1.8 Hz,
1H). Mass (APCI) m/z: 428 (M+H). HPLC: 97.2% (tR = 6.8 min,
L-column ODS 4.6 Â 150 mm, CH3CN/20 mM phosphate buffer
(pH 6.5) (30/70), 1 mL/min of flow rate). Anal. Calcd for
(33% ethyl acetate in n-hexane) to give titled compound
4
(1.81 g, 67%) as a yellowish brown caramel. 1H NMR (CDCl3) d:
2.24 (s, 3H), 2.96 (br s, 1H), 6.15 (br s, 1H), 6.82 (dd, J = 3.7,
0.7 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 3.7 Hz, 1H), 7.24
(m, 1H), 7.28 (dd, J = 8.1, 2.2 Hz, 1H), 7.80 (m, 1H), 7.82 (d,
J = 2.2 Hz, 1H), 8.44 (dd, J = 4.9, 1.6 Hz, 1H), 8.68 (dd, J = 2.4,
0.7 Hz, 1H). Mass (APCI) m/z: 360/362 (M+H).
5.1.2. 3-[5-(5-Bromo-2-methylbenzyl)thiophen-2-yl]pyridine
(5)
To a stirred solution of 4 (1.62 g, 4.49 mmol) in trifluoroacetic
acid (20 mL) at 0 °C under argon atmosphere was added portion-
wise sodium triacetoxyborohydride (4.76 g, 22.5 mmol). After
being stirred at the same temperature for 5 min, the resultant mix-
ture was allowed to warm to room temperature and stirred for
90 min. The reaction mixture was quenched with 10% aqueous so-
dium hydroxide at 0 °C, extracted with diethyl ether. The organic
layer was washed with brine, dried over magnesium sulfate prior
to filtration, and the solvent was evaporated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy (20% ethyl acetate in n-hexane) to give titled compound 5
(1.63 g, quant.) as a colorless solid. 1H NMR (DMSO-d6) d: 2.28 (s,
3H), 4.11 (s, 2H), 6.73 (dt, J = 3.5, 1.1 Hz, 1H), 7.06 (d, J = 8.1 Hz,
1H), 7.18 (d, J = 3.7 Hz, 1H), 7.24–7.36 (m, 3H), 7.78 (m, 1H), 8.47
(dd, J = 4.8, 1.5 Hz, 1H), 8.80 (d, J = 1.7 Hz, 1H). Mass (APCI) m/z:
344/346 (M+H).
C
23H25NO5SÁ0.2AcOEtÁ0.5H2O: C, 62.88; H, 5.86; N, 2.93; S, 7.06.
Found: C, 62.94; H, 6.15; N, 3.08; S, 7.00.
5.1.5. 4-Methyl-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-3-
[5-(5-pyrimidyl)-2-thienylmethyl]benzene (10b)
To a mixture of 7 (100 mg, 0.18 mmol) and 5-(tri-n-butylstan-
nyl)pyrimidine (100 mg, 0.27 mmol) in 1,4-dioxane (5 mL) were
added
tris(dibenzylideneacetone)dipalladium(0)
(8.3 mg,
0.009 mmol), tri-tert-butylphosphine tetrafluoroborate (10.5 mg,
0.036 mmol) and cesium fluoride (69 mg, 0.45 mmol), then the
mixture was stirred at 100 °C for 3 h under argon atmosphere.
After being cooled to room temperature, the insoluble was filtered
off, and the filtrate was extracted with ethyl acetate, washed with
brine, and dried over sodium sulfate prior to filtration. The solvent
was evaporated under reduced pressure, and the residue was puri-
fied by silica gel column chromatography (25–40% ethyl acetate in
n-hexane) to give titled compound 10b (266 mg, 41%) as colorless
crystals. 1H NMR (DMSO-d6) d: 1.72 (s, 3H), 1.93 (s, 3H), 2.00 (s,
3H), 2.02 (s, 3H), 2.26 (s, 3H), 4.05–4.16 (m, 3H), 4.19 (s, 2H),
4.64 (d, J = 9.6 Hz, 1H), 4.99 (t, J = 9.5 Hz, 1H), 5.06 (t, J = 9.6 Hz,
1H), 5.35 (t, J = 9.5 Hz, 1H), 6.89 (d, J = 3.5 Hz, 1H), 7.17 (s, 2H),
7.20 (s, 1H), 7.60 (d, J = 3.5 Hz, 1H), 9.01 (s, 2H), 9.05 (s, 1H). Mass
(APCI) m/z: 597 (M+H).
5.1.3. 1-(1-Methoxyglucopyranosyl)-4-methyl-3-[5-(3-pyridyl)-
2-thienylmethyl] benzene (6)
To a stirred solution of 5 (1.78 g, 5.17 mmol) in tetrahydrofuran
(8 mL) and toluene (16 mL) at À78 °C under argon atmosphere was
added dropwise n-butyllithium (2.6 N n-hexane solution, 1.96 mL,
5.07 mmol), and the mixture was stirred at the same temperature
for 30 min. To the mixture was added a solution of 2,3,4,6-tetrakis-
O-trimethylsilyl-D-gluconolactone (2.19 g, 4.70 mmol) in toluene
(8 mL) dropwise at À78 °C, and the mixture was stirred at the same
temperature for 3 h. Subsequently, to the mixture was added a
solution of methanesulfonic acid (0.91 mL, 14.1 mmol) in metha-
nol (20 mL), and the resulting mixture was allowed to warm to
room temperature and stirred overnight. Under ice-cooling, to
the mixture were added saturated aqueous sodium hydrogen car-
bonate solution and water, and the mixture was extracted with
ethyl acetate. The organic layer was washed with brine, dried over
magnesium sulfate prior to filtration, and the solvent was evapo-
rated under reduced pressure. The residue was purified by silica
gel column chromatography (5–10% methanol in chloroform) to
give titled compound 6 (49 mg, 2.3%) as a pale brown foam. 1H
NMR (DMSO-d6) d: 2.27 (s, 3H), 2.92 (m, 1H), 2.96 (s, 3H), 3.22
(m, 1H), 3.38 (m, 1H), 3.50–3.64 (m, 2H), 3.74 (m, 1H), 4.13 (d,
J = 15.7 Hz, 1H), 4.21 (d, J = 16.1 Hz, 1H), 4.49 (t, J = 5.9 Hz, 1H),
4.64 (d, J = 7.2 Hz, 1H), 4.67 (d, J = 5.1 Hz, 1H), 4.92 (d, J = 5.5 Hz,
5.1.6. 4-Methyl-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-3-
[5-(2-pyrimidyl)-2-thienylmethyl]benzene (10c)
To a mixture of 8 (1.20 g, 2.01 mmol) and 2-(tri-n-butylstan-
nyl)pyrimidine (1.11 g, 3.01 mmol) in N-methyl-2-pyrrolidone
(24 mL) were added dichlorobis(triphenylphosphine)palladium
(141 mg, 0.20 mmol) and copper(I) iodide (38 mg, 0.20 mmol),
then the mixture was stirred at 100 °C for 3.5 h under argon atmo-
sphere. After being cooled to room temperature, to the mixture
were added ethyl acetate and water, and the insoluble was filtered
off. The organic layer was separated, washed with brine, and dried
over sodium sulfate prior to filtration. The solvent was evaporated
under reduced pressure, and the residue was purified by silica gel
column chromatography (33–50% ethyl acetate in n-hexane) to
give titled compound 10c (787 mg, 66%) as a colorless amorphous

Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
5567
powder. 1H NMR (DMSO-d6) d: 1.74 (s, 3H), 1.92 (s, 3H), 2.00 (s,
3H), 2.01 (s, 3H), 2.26 (s, 3H), 4.04–4.15 (m, 3H), 4.17 (d,
J = 16.2 Hz, 1H), 4.20 (d, J = 16.2 Hz, 1H), 4.65 (d, J = 10.0 Hz, 1H),
4.98 (t, J = 9.5 Hz, 1H), 5.06 (t, J = 9.6 Hz, 1H), 5.35 (t, J = 9.5 Hz,
1H), 6.86 (d, J = 3.5 Hz, 1H), 7.17 (s, 1H), 7.18 (s, 2H), 7.29 (t,
J = 5.0 Hz, 1H), 7.76 (d, J = 3.5 Hz, 1H), 8.73 (d, J = 5.0 Hz, 2H). Mass
(APCI) m/z: 597 (M+H).
sulfate prior to filtration, and the solvent was evaporated under re-
duced pressure. The residue was dissolved in ethyl acetate
(1500 mL), and the mixture was treated with activated carbon
and NH–silica gel (450 mL). The insoluble was filtered off and the
filtrate was evaporated under reduced pressure. To the residual so-
lid was added ethyl alcohol (1000 mL), and the mixture was re-
fluxed for 30 min and then stirred at room temperature
overnight. The pale yellow crystals were collected by filtration.
To the crystals was added methanol (1000 mL), and the mixture
was refluxed for 1 h and then stirred at room temperature over-
night. The colorless crystals were collected by filtration, washed
with methanol and dried under reduced pressure to give 10g
(35.8 g, 81%). Mp 161–162 °C. 1H NMR (DMSO-d6) d: 1.75 (s, 3H),
1.93 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H), 4.07–4.15 (m, 3H), 4.26 (d,
J = 15.8 Hz, 1H), 4.30 (d, J = 15.8 Hz, 1H), 4.72 (d, J = 9.8 Hz, 1H),
4.97 (t, J = 9.7 Hz, 1H), 5.08 (t, J = 9.6 Hz, 1H), 5.36 (t, J = 9.5 Hz,
1H), 6.92 (d, J = 3.5 Hz, 1H), 6.99 (dd, J = 8.0, 2.3 Hz, 1H), 7.33 (dd,
J = 8.3, 2.0 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H),
7.69 (d, J = 3.7 Hz, 1H), 7.77 (dd, J = 7.5, 2.3 Hz, 1H), 7.97 (q,
J = 8.2 Hz, 1H). Mass (APCI) m/z: 634/636 (M+H). HPLC: 99.2%
(tR = 12.5 min, L-column ODS 4.6 Â 150 mm, CH3CN/20 mM phos-
phate buffer (pH 6.5) (60/40), 1 mL/min of flow rate). Anal. Calcd
for C30H29ClFNO9S: C, 56.83; H, 4.61; Cl, 5.59; F, 3.00; N, 2.21; S,
5.06. Found: C, 56.80; H, 4.47; Cl, 5.60; F, 2.91; N, 2.29; S, 4.93.
5.1.7. 4-Methyl-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-3-
[5-(5-thiazolyl)-2-thienylmethyl]benzene (10d)
To a mixture of 8 (500 mg, 0.84 mmol) and 5-(tri-n-butylstan-
nyl)thiazole (626 mg, 1.67 mmol) in 1,4-dioxane (5 mL) were
added
tetrakis(triphenylphosphine)palladium
(193 mg,
0.17 mmol) and copper(I) iodide (48 mg, 0.26 mmol), then the
mixture was stirred at refluxed temperature for 2 h under argon
atmosphere. After being cooled to room temperature, to the mix-
ture was added aqueous potassium fluoride solution, and the mix-
ture was stirred at the same temperature for 30 min, and diluted
with ethyl acetate. The insoluble was filtered off, and the organic
layer was separated, washed with brine, and dried over sodium
sulfate prior to filtration. The solvent was evaporated under re-
duced pressure, and the residue was purified by silica gel column
chromatography (33–50% ethyl acetate in n-hexane) to give titled
compound 10d (117 mg, 23%) as a colorless amorphous powder.
1H NMR (DMSO-d6) d: 1.72 (s, 3H), 1.92 (s, 3H), 2.00 (s, 3H), 2.01
(s, 3H), 2.25 (s, 3H), 4.04–4.13 (m, 3H), 4.14 (s, 2H), 4.65 (d,
J = 9.8 Hz, 1H), 4.98 (t, J = 9.8 Hz, 1H), 5.06 (t, J = 9.6 Hz, 1H), 5.35
(t, J = 9.5 Hz, 1H), 6.79 (d, J = 3.4 Hz, 1H), 7.17 (s, 3H), 7.22 (d,
J = 3.5 Hz, 1H), 8.00 (s, 1H), 8.99 (s, 1H). Mass (APCI) m/z: 602
(M+H).
5.1.10. 1-(b-D-Glucopyranosyl)-4-methyl-3-[5-(5-pyrimidyl)-2-
thienylmethyl]benzene (2b)
To a solution of compound 10b (260 mg, 0.44 mmol) in metha-
nol (5 mL) and tetrahydrofuran (2 mL) was added sodium methox-
ide (28% methanol solution, 6 drops), and the mixture was stirred
at room temperature for 1 h. The solvent was evaporated under re-
duced pressure, and the residue was purified by silica gel column
chromatography (0–10% methanol in chloroform) to give titled
compound 2b (160 mg, 86%) as a colorless powder. 1H NMR
(DMSO-d6) d: 2.27 (s, 3H), 3.12–3.30 (m, 4H), 3.44 (dt, J = 11.8,
6.2 Hz, 1H), 3.70 (ddd, J = 11.8, 5.7, 2.1 Hz, 1H), 3.97 (d, J = 9.3 Hz,
1H), 4.17 (d, J = 15.9 Hz, 1H), 4.20 (d, J = 15.9 Hz, 1H), 4.42 (t,
J = 5.7 Hz, 1H), 4.73 (d, J = 5.7 Hz, 1H), 4.91 (d, J = 5.1 Hz, 1H),
4.92 (d, J = 4.6 Hz, 1H), 6.93 (d, J = 3.6 Hz, 1H), 7.13 (d, J = 7.7 Hz,
1H), 7.16 (dd, J = 7.7, 1.5 Hz, 1H), 7.24 (s, 1H), 7.58 (d, J = 3.6 Hz,
1H), 9.02 (s, 2H), 9.05 (s, 1H). Mass (APCI) m/z: 429 (M+H). HPLC:
98.6% (tR = 3.4 min, Sumipax ODS F210SLP 4.6 Â 50 mm, 0.05% TFA
in CH3CN/H2O (25/75), 1 mL/min of flow rate). Anal. Calcd for
5.1.8. 4-Chloro-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-3-
[5-(3-pyridyl)-2-thienylmethyl]benzene (10f)
To a mixture of 9 (300 mg, 0.49 mmol) and 3-pyridylboronic
acid (179 mg, 1.46 mmol) in 1,2-dimethoxyethane were added tet-
rakis(triphenylphosphine)palladium (58 mg, 0.049 mmol) and ce-
sium fluoride (443 mg, 2.91 mmol), then the mixture was stirred
at refluxed temperature under nitrogen atmosphere overnight.
After being cooled to room temperature, to the mixture were
added ethyl acetate and saturated aqueous sodium hydrogen car-
bonate solution. The organic layer was separated, washed with
brine, dried over sodium sulfate prior to filtration. The solvent
was evaporated under reduced pressure, and the residue was puri-
fied by silica gel column chromatography (50–80% ethyl acetate in
n-hexane), followed by trituration with methanol to give titled
C
22H24N2O5SÁ1.0H2O: C, 59.18; H, 5.87; N, 6.27; S, 7.18. Found: C,
59.31; H, 5.59; N, 6.19; S, 7.20.
compound 10f (157 mg, 53%) as
a
colorless solid. 1H NMR
(DMSO-d6) d: 1.72 (s, 3H), 1.92 (s, 3H), 1.99 (s, 3H), 2.01 (s, 3H),
4.05–4.17 (m, 3H), 4.29 (s, 2H), 4.71 (d, J = 9.8 Hz, 1H), 4.97 (t,
J = 9.8 Hz, 1H), 5.08 (t, J = 9.3 Hz, 1H), 5.36 (t, J = 9.3 Hz, 1H), 6.90
(d, J = 3.6 Hz, 1H), 7.31 (dd, J = 8.2, 2.1 Hz, 1H), 7.39 (d, J = 2.1 Hz,
1H), 7.40 (dd, J = 7.7, 4.6 Hz, 1H), 7.473 (d, J = 3.6 Hz, 1H), 7.474
(d, J = 8.2 Hz, 1H), 7.94 (dd, J = 7.7, 4.6 Hz, 1H), 8.46 (dd, J = 4.6,
1.5 Hz, 1H), 8.81 (d, J = 2.6 Hz, 1H). Mass (APCI) m/z: 616/618
(M+H).
5.1.11. 1-(b-D-Glucopyranosyl)-4-methyl-3-[5-(2-pyrimidyl)-2-
thienylmethyl]benzene (2c)
Compound 2c (313 mg, 55%) was prepared according to the
method described for the synthesis of 2b using 10c (787 mg,
1.32 mmol). 1H NMR (DMSO-d6) d: 2.26 (s, 3H), 3.13–3.29 (m,
4H), 3.41–3.47 (dt, J = 11.7, 5.8 Hz, 1H), 3.69 (dd, J = 11.6, 5.3 Hz,
1H), 3.97 (d, J = 9.5 Hz, 1H), 4.15 (d, J = 16.1 Hz, 1H), 4.19 (d,
J = 16.1 Hz, 1H), 4.43 (t, J = 5.9 Hz, 1H), 4.73 (d, J = 5.6 Hz, 1H),
4.92 (d, J = 4.7 Hz, 2H), 6.89 (d, J = 3.7 Hz, 1H), 7.13 (d, J = 7.9 Hz,
1H), 7.15 (dd, J = 7.9, 1.3 Hz, 1H), 7.24 (s, 1H), 7.29 (t, J = 4.8 Hz,
1H), 7.76 (d, J = 3.7 Hz, 1H), 8.73 (d, J = 4.8 Hz, 2H). Mass (APCI)
m/z: 429 (M+H). HPLC: 99.6% (tR = 5.4 min, L-column ODS
4.6 Â 150 mm, CH3CN/20 mM phosphate buffer (pH 6.5) (30/70),
1 mL/min of flow rate). Anal. Calcd for C22H24N2O5S: C, 61.67; H,
5.65; N, 6.54; S, 7.48. Found: C, 61.56; H, 5.44; N, 6.50; S, 7.36.
5.1.9. 4-Chloro-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)- 3-
(5-(6-fluoro-3-pyridyl)-2-thienylmetyl)benzene (10g)
A suspension of 9 (43.0 g, 69.6 mmol), 2-(2-(6-fluoro)pyridine)-
4,4,5,5-tetramethyl-1,3-dioxaborolane (23.3 g, 104 mmol), cesium
fluoride (63.4 g, 418 mmol) and tetrakis(triphenylphosphine)palla-
dium(0) (8.04 g, 6.96 mmol) in 1,2-dimethoxyethane (1300 mL)
was vigorously stirred at reflux for 3 h under argon atmosphere.
The reaction mixture was poured into saturated aqueous sodium
hydrogen carbonate solution (1200 mL), and the mixture was ex-
tracted with ethyl acetate (1000 mL) twice. The combined organic
layers were washed with brine (600 mL), dried over magnesium
5.1.12. 1-(b-D-Glucopyranosyl)-4-methyl-3-[5-(5-thiazolyl)-2-
thienylmethyl]benzene (2d)
Compound 2d (84 mg, quantitative) was prepared according to
the method described for the synthesis of 2b using 10d (115 mg,

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Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
0.19 mmol). 1H NMR (DMSO-d6) d: 2.25 (s, 3H), 3.12–3.29 (m, 4H),
3.44 (dt, J = 11.6, 5.9 Hz, 1H), 3.67–3.72 (dd, J = 11.6, 6.6 Hz, 1H),
3.96 (d, J = 9.5 Hz, 1H), 4.12 (d, J = 16.1 Hz, 1H), 4.15 (d,
J = 16.1 Hz, 1H), 4.43 (t, J = 5.8 Hz, 1H), 4.73 (d, J = 5.8 Hz, 1H),
4.92 (d, J = 4.8 Hz, 2H), 6.82 (d, J = 3.5 Hz, 1H), 7.13 (d, J = 7.7 Hz,
1H), 7.15 (dd, J = 7.9, 1.1 Hz, 1H), 7.20 (d, J = 3.5 Hz, 1H), 7.22 (s,
1H), 8.01 (s, 1H), 8.99 (s, 1H). Mass (APCI) m/z: 434 (M+H). HPLC:
90.7% (tR = 1.8 min, Sumipax ODS D-210SLP 4.6 Â 50 mm, 0.05%
TFA in CH3CN/H2O (35/65), 1 mL/min of flow rate). Anal. Calcd
for C21H23NO5S2Á0.12CHCl3Á0.8H2O: C, 54.87; H, 5.39; Cl, 2.76; N,
3.03; S, 13.87. Found: C, 54.90; H, 5.13; Cl, 2.82; N, 2.96; S, 13.31.
in vacuo, dissolved in dichloromethane (650 mL), and to the mix-
ture was added 2-chlorothiophene (21.8 mL, 236 mmol). To the
mixture was added portion-wise aluminum chloride (26.6 g,
200 mmol) at 0 °C. The resultant mixture was allowed to warm to
room temperature, and stirred at the same temperature overnight.
The reaction mixture was poured into ice–water, and extracted with
chloroform. The organic layer was separated, washed with satu-
rated aqueous sodium hydrogen carbonate solution, and brine,
and dried over sodium sulfate prior to filtration. The solvent was
evaporated under reduced pressure, and the residue was dissolved
in methanol on heating, and treated with activated carbon. The
insoluble was filtered off, and the filtrate was evaporated under
reduced pressure. The residue was recrystallized from methanol
to give titled compound 12 (52 g, 81%) as pale yellow powder. 1H
NMR (DMSO-d6) d: 2.22 (s, 3H), 7.30 (d, J = 4.2 Hz, 1H), 7.33–7.36
(m, 2H), 7.66–7.68 (m, 2H). Mass (APCI) m/z: 314/316/318 (M+H).
5.1.13. 4-Chloro-1-(b-D-glucopyranosyl)-3-[5-(3-pyridyl)-2-
thienylmethyl]benzene (2f)
Compound 2f (45 mg, 86%) was prepared according to the
method described for the synthesis of 2b using 10f (72 mg,
0.12 mmol). 1H NMR (DMSO-d6) d: 3.07–3.30 (m, 4H), 3.45 (dt,
J = 11.8, 6.2 Hz, 1H), 3.70 (ddd, J = 11.8, 5.7, 1.5 Hz, 1H), 4.03 (d,
J = 9.3 Hz, 1H), 4.26 (d, J = 15.9 Hz, 1H), 4.30 (d, J = 15.9 Hz, 1H),
4.44 (t, J = 6.2 Hz, 1H), 4.85 (d, J = 6.2 Hz, 1H), 4.95 (t, J = 5.1 Hz,
2H), 6.93 (d, J = 3.6 Hz, 1H), 7.28 (dd, J = 8.2, 2.1 Hz, 1H), 7.40
(ddd, J = 8.2, 4.6, 1.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.45 (s, 1H),
7.46 (d, J = 3.6 Hz, 1H), 7.95 (ddd, J = 7.7, 2.6, 1.5 Hz, 1H), 8.46
(dd, J = 5.1, 1.5 Hz, 1H), 8.81 (d, J = 1.5 Hz, 1H). Mass (APCI) m/z:
448/450 (M+H). HPLC: 98.2% (tR = 3.7 min, Sumipax ODS D-
210SLP 4.6 Â 50 mm, 0.05% TFA in CH3CN/H2O (20/80), 1 mL/min
of flow rate). Anal. Calcd for C22H22ClNO5SÁ0.5H2O: C, 57.83; H,
5.07; Cl, 7.76; N, 3.07; S, 7.02. Found: C, 57.63; H, 4.81; Cl, 7.88;
N, 3.08; S, 7.02.
5.1.16. 2-(5-Bromo-2-methylbenzyl)-5-chlorothiophene (13)
To a solution of 12 (47.3 g, 150 mmol) in chloroform (470 mL)
and acetonitrile (470 mL) was added triethylsilane (52.3 g,
450 mmol) and then dropwise boron trifluoride diethyletherate
(63.9 g, 450 mmol) at 0 °C. The resultant mixture was stirred at the
same temperature for 1 h, allowed to warm to room temperature
and stirred overnight. The reaction mixture was basified with satu-
rated aqueous sodium hydrogen carbonate solution at 0 °C, and ex-
tracted with chloroform. The organic layer was washed with brine,
and dried over sodium sulfate prior to filtration. The solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (n-hexane) to give tilted com-
pound 13 (45 g, quantitative) as a pale yellow oil. 1H NMR (DMSO-
d6) d: 2.21 (s, 3H), 4.09 (s, 2H), 6.72 (br d, J = 3.6 Hz, 1H), 6.95 (d,
J = 3.6 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.35 (dd, J = 8.2, 2.6 Hz, 1H),
7.39 (d, J = 2.6 Hz, 1H). Mass (GC) m/z: 300/302/304 (M+). HPLC:
98.0% (tR = 4.2 min, Sumipax ODS D-210SLP 4.6 Â 50 mm, 0.05%
TFA in CH3CN/H2O (75/25), 1 mL/min of flow rate). Anal. Calcd for
5.1.14. 4-Chloro-3-(5-(6-fluoro-3-pyridyl)-2-thienylmethyl) -1-
(b-D
-glucopyranosyl)benzene (2g)
Compound 10g (35.5 g, 56.0 mmol) was dissolved in methanol
(350 mL)—tetrahydrofuran (700 mL). To the mixture was added so-
dium methoxide (28% methanol solution, 0.11 mL, 0.56 mmol), and
the mixture was stirred at room temperature for 17 h under argon
atmosphere. To the mixture was added silica gel (260 mL), and the
solvent was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (0–10% methanol in
chloroform) to give a colorless solid. The solid was dissolved in tet-
rahydrofuran (1000 mL), and the mixture was treated with activated
carbon. The insoluble was filtered off and the filtrate was evaporated
under reduced pressure. The residue was recrystallized from ethyl
acetate (1000 mL) to give titled compound 2g (23.3 g, 89%) as color-
less crystals. 1H NMR (DMSO-d6) d: 3.12 (td, J = 9.03, 5.08 Hz, 1H),
3.18 (dd, J = 8.91, 5.22 Hz, 1H), 3.22–3.27 (m, 2H), 3.45 (ddd,
J = 11.8, 6.10, 5.98 Hz, 1H), 3.70 (ddd, J = 12.0, 5.38, 1.44 Hz, 1H),
4.03 (d, J = 9.47 Hz, 1H), 4.24 (d, J = 15.5 Hz, 1H), 4.29 (d,
J = 15.5 Hz, 1H), 4.45 (t, J = 5.86 Hz, 1H), 4.85 (d, J = 5.94 Hz, 1H),
4.95 (d, J = 4.81 Hz, 1H), 4.96 (d, J = 4.49 Hz, 1H), 6.94 (d,
J = 3.69 Hz, 1H), 6.99 (dd, J = 8.10, 2.17 Hz, 1H), 7.29 (dd, J = 8.26,
2.01 Hz, 1H), 7.42 (d, J = 8.34 Hz, 1H), 7.45 (d, J = 1.77 Hz, 1H), 7.68
(d, J = 3.69 Hz, 1H), 7.76 (dd, J = 7.70, 2.41 Hz, 1H), 7.97 (q,
J = 8.18 Hz, 1H). Mass (APCI) m/z: 483/485 (M + NH4). HPLC: 99.9%
(tR = 10.7 min, L-column ODS 4.6 Â 150 mm, CH3CN/20 mM phos-
phate buffer (pH 6.5) (35/65), 1 mL/min of flow rate). Anal. Calcd
for C22H21ClFNO5S: C, 56.71; H, 4.54; Cl, 7.61; F, 4.08; N, 3.01; S,
6.88. Found: C, 56.60; H, 4.49; Cl, 7.34; F, 4.09; N, 2.97; S, 6.56.
C12H10BrClS: C, 47.78; H, 3.34; Br, 26.49; Cl, 11.75; S, 10.63. Found:
C, 47.64; H, 3.34; Br, 25.76; Cl, 11.62; S, 10.52.
5.1.17. 3-[5-Chloro-2-thienylmethyl]-1-(1-b-D-glucopyranosyl)-
4-methylbenzene (14)
To a stirred solution of 13 (15.4 g, 51.1 mmol) in tetrahydrofuran
(150 mL) and toluene (300 mL) at À78 °C under argon atmosphere
was added n-butyllithium (1.6 N n-hexane solution, 32.3 mL,
51.1 mmol) dropwise over a period of 5 min, and the mixture was
stirred at the same temperature for 20 min. To the mixture was
added a solution of 2,3,4,6-tetrakis-O-trimethylsilyl-D-gluconolac-
tone (23.8 g, 51.1 mmol) in toluene (150 mL) dropwise over a period
of 20 min at À78 °C, and the mixture was stirred at the same temper-
ature for 1 h. Subsequently, to the mixture was added a solution of
methanesulfonic acid (9.94 mL, 153 mmol) in methanol (300 mL),
and the resulting mixture was allowed to warm to room tempera-
ture and stirred overnight. Under ice-cooling, to the mixture were
added saturated aqueous sodium hydrogen carbonate solution
(200 mL) and water (150 mL), and the mixture was extracted with
ethyl acetate (600 mL) twice. The combined organic layers were
washed with brine, dried over sodium sulfate prior to filtration,
and the solvent was evaporated under reduced pressure. The residue
was dissolved in dichloromethane (200 mL), and to the mixture was
added triethylsilane (24.4 mL, 153 mmol) and then dropwise boron
trifluoride diethyletherate (19.4 mL g, 153 mmol) at À78 °C under
argon atmosphere. The resultant mixture was allowed to warm to
0 °C and stirred for 2 h. The reaction mixture was basified with sat-
urated aqueous sodium hydrogen carbonate solution (250 mL) at
0 °C, and extracted with dichloromethane (150 mL). The organic
layer was washed with brine (150 mL), and dried over sodium sul-
fate prior to filtration. The solvent was evaporated under reduced
5.1.15. (5-Bromo-2-methylphenyl)-(5-chlorothiophen-2-
yl)methanone (12)
To a stirred suspension of 5-bromo-2-methylbenzoic acid (11)
(39 g, 181 mmol) in chloroform (390 mL) was added oxalyl chloride
(20.7 mL, 218 mmol) dropwise at room temperature, followed by
N,N-dimethylformamide (1 drop). The resultant mixture was stirred
at the same temperature overnight. The mixture was concentrated

Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
5569
pressure, and the residue was purified by silica gel column chroma-
tography (0–10% methanol in chloroform) to give tilted compound
14 (11.2 g, 57%) as a colorless amorphous powder. 1H NMR
(DMSO-d6) d: 2.22 (s, 3H), 3.11–3.28 (m, 3H), 3.40–3.47 (m, 1H),
3.66–3.72 (m, 1H), 3.95 (d, J = 9.5 Hz, 1H), 4.04 (d, J = 15.9 Hz, 1H),
4.09 (d, J = 15.9 Hz, 1H), 4.42 (t, J = 5.8 Hz, 1H), 4.70 (d, J = 5.8 Hz,
1H), 4.91 (d, J = 4.7 Hz, 1H), 4.91 (d, J = 5.1 Hz, 1H), 6.66 (d,
J = 3.7 Hz, 1H), 6.91 (d, J = 3.7 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 7.14
(dd, J = 7.7, 1.5 Hz, 1H), 7.18 (br s, 1H). Mass (APCI) m/z: 402/404
(M+NH4).
sodium pyrosulfate solution (90 mL) and saturated aqueous
sodium hydrogen carbonate solution (90 mL), extracted with
chloroform (100 mL) twice. The combined organic layers were
washed with brine, and dried over magnesium sulfate prior to fil-
tration. The solvent was evaporated under reduced pressure, and
the residue was purified by silica gel column chromatography
(10–40% ethyl acetate in n-hexane) followed by recrystallization
from methanol to give titled compound 8 (8.63 g, 93%) as a color-
less solid. Mp 155–157 °C. 1H NMR (DMSO-d6) d: 1.72 (s, 3H), 1.93
(s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 2.22 (s, 3H), 4.08 (s, 2H), 4.09
(m, 3H), 4.63 (d, J = 9.8 Hz, 1H), 4.97 (t, J = 9.6 Hz, 1H), 5.05
(t, J = 9.6 Hz, 1H), 5.25 (t, J = 9.5 Hz, 1H), 6.62 (d, J = 3.7 Hz, 1H),
7.03 (d, J = 3.7 Hz, 1H), 7.12 (s, 1H), 7.16 (s, 2H). Mass (APCI) m/z:
614/616 (M+NH4). HPLC 98.0% (tR = 14.4 min, L-column ODS
4.6 Â 150 mm, CH3CN/20 mM phosphate buffer (pH 6.5) (60/40),
1 mL/min of flow rate). Anal. Calcd for C26H29BrO9S: C, 52.27; H,
4.89; Br, 13.37; S, 5.37. Found: C, 52.29; H, 4.69; Br, 13.14; S, 5.35.
5.1.18. 3-[5-Chloro-2-thienylmethyl]-4-methyl-1-(2,3,4,6-tetra-
O-acetyl-b-D-glucopyranosyl)benzene (7)
To a stirred solution of 14 (11 g, 28.6 mmol), acetic anhydride
(27 mL, 286 mmol), and pyridine (23.1 mL, 286 mmol) in chloro-
form (110 mL) was added 4-(dimethylamino)pyridine (349 mg,
2.86 mmol) at room temperature. The resultant mixture was stir-
red at the same temperature for 1 h. The solvent was evaporated
under reduced pressure, and the residue was poured into water
(330 mL). The mixture was extracted with ethyl acetate (220 mL)
twice. The combined organic layers were washed with 10% copper
sulfate solution (280 mL) twice, and brine (280 mL), and dried over
magnesium sulfate prior to filtration. The solvent was concen-
trated in vacuo, and the residue was recrystallized from ethanol
(150 mL) to give titled compound 7 (11.9 g, 75%) as a colorless so-
lid. Mp 157–158 °C. 1H NMR (DMSO-d6) d: 1.93 (s, 3H), 2.00 (s, 3H),
2.02 (s, 3H), 2.22 (s, 3H), 4.06 (m, 4H), 4.12 (m, 1H), 4.63 (d,
J = 9.8 Hz, 1H), 4.97 (t, J = 9.6 Hz, 1H), 5.05 (t, J = 9.6 Hz, 1H), 5.35
(t, J = 9.5 Hz, 1H), 6.64 (d, J = 3.9 Hz, 1H), 6.92 (d, J = 3.7 Hz, 1H),
7.12 (s, 1H), 7.16 (s, 2H). Mass (APCI) m/z: 570/572 (M + NH4).
HPLC: 97.8% (tR = 9.2 min, L-column ODS 4.6 Â 150 mm, CH3CN/
20 mM phosphate buffer (pH 6.5) (65/35), 1 mL/min of flow rate).
Anal. Calcd for C26H29ClO9S: C, 56.47; H, 5.29; Cl, 6.41; S, 5.80.
Found: C, 56.43; H, 5.24; Cl, 6.32; S, 5.65.
5.1.21. tert-Butyl 2-chloro-5-(1-methoxy-D-
glucopyranosyl)benzoate (17)
Mesityl bromide (64.43 g, 323.62 mmol) was dissolved in tetra-
hydrofuran (1100 mL), and the mixture was cooled in a dry ice–
acetone bath under argon atmosphere. To the mixture was added
dropwise n-butyllithium (2.71 N n-hexane solution, 119.4 mL,
323.62 mmol) over a period of 15 min below À64 °C (internal tem-
perature), and the resultant suspended mixture was stirred at the
same temperature for 1 h. Then, a solution of tert-butyl 5-bromo-
2-chlorobenzoate 16 (67.4 g, 231.16 mmol) and 2,3,4,6-tetrakis-
O-trimethylsilyl-D-gluconolactone (110 g, 235.78 mmol) in tetra-
hydrofuran (540 mL) was added dropwise to the reaction mixture
over a period of 1 h below À63 °C (internal temperature), and the
orange mixture was further stirred at the same temperature for
30 min. A solution of methanesulfonic acid (66 mL, 1017.10 mmol)
in methanol (660 mL) was added dropwise to the reaction mixture
over a period of 5 min, and the mixture was stirred at room tem-
perature for 40 h. Under ice-water cooling, to the mixture was
added saturated aqueous sodium hydrogen carbonate solution
(1500 mL). The organic layer was concentrated to about half vol-
ume under reduced pressure, and the residue was extracted with
ethyl acetate (1200 mL, 800 mL and 500 mL) three times. The com-
bined organic layers were washed with brine (1000 mL), dried over
magnesium sulfate, and the solvent was evaporated under reduced
pressure. The resultant residue was dissolved in toluene (250 mL),
and the mixture was added dropwise to n-hexane (3000 mL) under
vigorous stirring. After being stirred for 30 min, the precipitate was
collected by filtration, washed with n-hexane and soon dried under
reduced pressure at room temperature to give 17 (81.59 g, 87%) as
a colorless powder. 1H NMR (DMSO-d6) d: 1.56 (s, 9H), 2.87 (dd,
J = 9.0, 7.4 Hz, 1H), 2.93 (s, 3H), 3.22 (m, 1H), 3.38 (m, 1H), 3.53
(m, 1H), 3.59 (m, 1H), 3.75 (m, 1H), 4.61 (t, J = 5.9 Hz, 1H), 4.79
(d, J = 5.1 Hz, 1H), 4.90 (d, J = 7.2 Hz, 1H), 5.00 (d, J = 5.0 Hz, 1H),
7.51 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 8.4, 2.0 Hz, 1H), 7.76 (d,
J = 1.9 Hz, 1H). Mass (APCI) m/z: 422/424 (M + NH4). HPLC 89.9%
(tR = 9.7 min, L-column ODS 4.6 Â 150 mm, CH3CN/20 mM phos-
phate buffer (pH 6.5) (30/70), 1 mL/min of flow rate). Anal. Calcd
for C18H25ClO8: C, 53.40; H, 6.22; Cl, 8.76. Found: C, 52.69; H,
6.12; Cl, 8.05.
5.1.19. 4-Methyl-1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-
3-[2-thienylmethyl]benzene (15)
A vigorous stirred suspension of 7 (11.9 g, 21.5 mmol), 10% pal-
ladium on carbon (3.5 g), and triethylamine (24 mL, 172 mmol) in
tetrahydrofuran (120 mL) and methanol (360 mL) was hydroge-
nated at room temperature for 18 h. The insoluble was removed
by filtration, washed with tetrahydrofuran (500 mL). The filtrate
and the washing were combined, and the solvent was evaporated
under reduced pressure. The residue was dissolved in chloroform
(150 mL), washed with 5% aqueous citric acid solution (100 mL),
saturated aqueous sodium hydrogen carbonate solution (100 mL),
and water (100 mL), and dried over sodium sulfate prior to filtra-
tion. The solvent was concentrated under reduced pressure, and
the residue was recrystallized from methanol to give titled com-
pound 15 (8.03 g, 72%) as a colorless solid. Mp 124 °C. 1H NMR
(DMSO-d6) d: 1.71 (s, 3H), 1.92 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H),
2.22 (s, 3H), 4.04–4.09 (m, 2H), 4.10–4.16 (m, 3H), 4.62 (d,
J = 9.8 Hz, 1H), 4.96 (t, J = 9.7 Hz, 1H), 5.05 (t, J = 9.6 Hz, 1H), 5.35
(t, J = 9.5 Hz, 1H), 6.75 (dd, J = 3.4, 1.0 Hz, 1H), 6.93 (dd, J = 5.1,
3.4 Hz, 1H), 7.12 (br s, 1H), 7.15 (s, 2H), 7.32 (dd, J = 5.1, 1.1 Hz,
1H). Mass (APCI) m/z: 536 (M+NH4). HPLC 98.0% (tR = 8.5 min, L-
column ODS 4.6 Â 150 mm, CH3CN/20 mM phosphate buffer (pH
6.5) (60/40), 1 mL/min of flow rate). Anal. Calcd for C26H30O9S: C,
60.22; H, 5.75; S, 6.18. Found: C, 60.17; H, 5.75; S, 6.15.
5.1.22. tert-Butyl 2-chloro-5-(1-methoxy-2,3,4,6-tetra-O-acetyl-
D-glucopyranosyl)benzoate (18)
5.1.20. 3-[5-Bromo-2-thienylmethyl]-4-methyl-1-(2,3,4,6-tetra-
To a mixture of 17 (81.3 g, 200.82 mmol) in toluene (1300 mL)
O-acetyl-b-
To a stirred 0 °C solution of 15 (8.03 g, 15.5 mmol) in chloro-
form (70 mL) was added solution of bromine (385 mg,
2.41 mmol) in chloroform (7 mL) dropwise over a period of
10 min. The mixture was poured into a mixture of 10% aqueous
D
-glucopyranosyl)benzene (8)
were added successively acetic anhydride (133 mL, 1405 mmol),
N,N-diisopropylethylamine (245 mL, 1405 mmol) and 4-(dimethyl-
amino)pyridine (4.89 g, 40 mmol) at 0 °C. The resultant mixture
was stirred at room temperature for 1 h. The mixture was poured
into water (1300 mL) and extracted with ethyl acetate (500 mL)
a

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Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
twice. The combined organic layers were successively washed with
20% aqueous phosphoric acid solution (700 mL), water (300 mL)
and saturated aqueous sodium hydrogen carbonate solution
(600 mL), dried over magnesium sulfate and treated with activated
carbon. The insoluble was filtered off and the filtrate was evapo-
rated under reduced pressure. The residue was dissolved in toluene
(150 mL), and to the mixture were added successively n-hexane
(600 mL) and seed crystals under stirring. After being stirred over-
night, the crystals were collected by filtration, washed with tolu-
ene–n-hexane (40–160 mL) and dried under reduced pressure to
give titled compound 18 (83.75 g, 73%) as colorless crystals. Mp
116–118 °C. 1H NMR (DMSO-d6) d: 1.56 (s, 9H), 1.91 (s, 3H), 1.92
(s, 3H), 2.03 (s, 3H), 2.06 (s, 3H), 3.07 (s, 3H), 4.05 (m, 1H), 4.22–
4.34 (m, 2H), 4.84 (d, J = 10.1 Hz, 1H), 5.20 (t, J = 9.8 Hz, 1H), 5.44
(t, J = 9.8 Hz, 1H), 7.52 (dd, J = 8.5, 2.1 Hz, 1H), 7.60 (d, J = 8.5 Hz,
1H), 7.65 (d, J = 2.1 Hz, 1H). Mass (APCI) m/z: 590/592 (M+NH4).
HPLC 98.7% (tR = 10.0 min, L-column ODS 4.6 Â 150 mm, CH3CN/
20 mM phosphate buffer (pH 6.5) (60/40), 1 mL/min of flow rate).
Anal. Calcd for C26H33ClO12: C, 54.50; H, 5.81; Cl, 6.19. Found: C,
54.23; H, 5.79; Cl, 6.08.
mixture was added sodium tetrahydroborate (6.47 g, 171 mmol).
The mixture was stirred at the same temperature for 2 h. Further-
more, to the mixture was added sodium tetrahydroborate (1.3 g,
34.2 mmol), and the resultant mixture was stirred at the same
temperature for 30 min. The mixture was poured into hydrochloric
acid (0.5 N, 900 mL), diluted with water (1000 mL) and extracted
with ethyl acetate (1500 mL, 500 mL) twice. The combined organic
layers were washed with brine and saturated aqueous sodium
hydrogen carbonate solution, and dried over magnesium sulfate
prior to filtration. The solvent was evaporated under reduced pres-
sure to give yellow foam. The resultant foam was dissolved in ace-
tonitrile (1000 mL) and water (4.1 mL, 228 mmol), and the mixture
was cooled in an ice-water bath under argon atmosphere. To the
mixture were added triethylsilane (109 mL, 684 mmol) and then
dropwise boron trifluoride diethyletherate (58 mL, 456 mmol) over
a period of 10 min. After being stirred at the same temperature for
30 min, the mixture was allowed to warm to room temperature
and stirred overnight. The resultant mixture was poured into satu-
rated aqueous sodium hydrogen carbonate solution (1400 mL)–ice,
and extracted with ethyl acetate (1500 mL, 500 mL) twice. The
combined organic layers were washed with brine (1000 mL), dried
over magnesium sulfate and treated with activated carbon and sil-
ica gel (300 mL). The insoluble was filtered off and the filtrate was
evaporated under reduced pressure. The residue was recrystallized
from methanol (400 mL) to give titled compound 9 (46.53 g, 66%)
as colorless crystals. Mp 142–143 °C. 1H NMR (DMSO-d6) d: 1.73
(s, 3H), 1.93 (s, 3H), 2.01 (s, 3H), 2.02 (s, 3H), 4.10 (m, 3H), 4.20
(s, 2H), 4.70 (d, J = 9.8 Hz, 1H), 4.95 (t, J = 9.6 Hz, 1H), 5.08 (t,
J = 9.6 Hz, 1H), 5.36 (t, J = 9.6 Hz, 1H), 6.69 (d, J = 3.7 Hz, 1H), 7.04
(d, J = 3.7 Hz, 1H), 7.31 (dd, J = 8.3, 1.9 Hz, 1H), 7.34 (d, J = 1.4 Hz,
1H), 7.46 (d, J = 8.2 Hz, 1H). Mass (APCI) m/z: 634/636 (M+NH4).
HPLC: 95.1% (tR = 10.9 min, L-column ODS 4.6 Â 150 mm, CH3CN/
20 mM phosphate buffer (pH 6.5) (65/35), 1 mL/min of flow rate).
Anal. Calcd for C25H26BrClO9S: C, 48.60; H, 4.24; Br, 12.93; Cl,
5.74; S, 5.19. Found: C, 48.87; H, 4.15; Br, 12.80; Cl, 5.75; S, 5.11.
5.1.23. 2-Chloro-5-(1-methoxy-2,3,4,6-tetra-O-acetyl-D-
glucopyranosyl)phenyl 5-bromo-2-thienyl ketone (19)
A solution of 18 (83.4 g, 145.5 mmol) in formic acid (300 mL)
was stirred at 50 °C for 30 min. The solvent was evaporated and re-
moved by azeotropic distillation with toluene under reduced pres-
sure to give 2-chloro-5-(1-methoxy-2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl)benzoic acid (ESI-Mass m/z 515/517 (MÀH)) as a
colorless foam. The benzoic acid was dissolved in dichloromethane
(600 mL), and to the mixture were added oxalyl chloride (16.5 mL,
189.15 mmol) and N,N-dimethylformamide (5 drops). The mixture
was stirred at room temperature for 2 h. The solvent was evapo-
rated and removed by azeotropic distillation with toluene under
reduced pressure to give 2-chloro-5-(1-methoxy-2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl)benzoyl chloride as a pale yellow syrup.
This benzoyl chloride and 2-bromothiophene (30.84 g,
189.15 mmol) were dissolved in dichloromethane (700 mL), and
the mixture was cooled in a dry ice-acetone bath, and to the mix-
ture was added aluminum chloride (97 g, 727.5 mmol). After being
stirred for 10 min, the resultant mixture was allowed to warm
gradually to ice-water temperature and stirred at same tempera-
ture for 1 h. The reaction mixture was poured into ice-water
(1500 mL), and extracted with chloroform (800 mL). The organic
layer was washed with brine and saturated aqueous sodium
hydrogen carbonate solution, and dried over magnesium sulfate
prior to filtration. The solvent was evaporated under reduced pres-
sure, and the residue was dissolved in chloroform (300 mL)–etha-
nol (400 mL) and the mixture was treated with activated carbon.
The insoluble was filtered off and the filtrate was evaporated under
reduced pressure. The residue was recrystallized from ethanol
(400 mL) to give titled compound 19 (75.7 g, 79%) as pale yellow
crystals. Mp 162–163 °C. 1H NMR (DMSO-d6) d: 1.87 (s, 3H), 1.92
(s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 3.13 (s, 3H), 4.04 (m, 1H), 4.20–
4.30 (m, 2H), 4.92 (d, J = 10.1 Hz, 1H), 5.21 (t, J = 9.8 Hz, 1H), 5.44
(t, J = 9.8 Hz, 1H), 7.16 (d, J = 4.0 Hz, 1H), 7.46 (d, J = 4.1 Hz, 1H),
7.53–7.60 (m, 2H), 7.67 (d, J = 8.9 Hz, 1H). Mass (APCI) m/z: 680/
682 (M + NH4). HPLC: 87.2% (tR = 11.6 min, L-column ODS
4.6 Â 150 mm, CH3CN/20 mM phosphate buffer (pH 6.5) (60/40),
1 mL/min of flow rate). Anal. Calcd for C26H26BrClO11S: C, 47.18;
H, 3.96; Br, 12.07; Cl, 5.36; S, 5.39. Found: C, 47.75; H, 3.93; Br,
11.31; Cl, 5.39; S, 4.53.
5.1.25. 1-Thiophen-2-yl-1H-pyrazole (21)
Sodium hydride (60% in mineral oil, 4.0 g, 100 mmol) was
washed with n-hexane three times, and treated with pyridine
(20 mL). To the mixture was added a solution of pyrazole (6.81 g,
100 mmol) in pyridine (20 mL) at 0 °C under argon atmosphere.
When the evolution of hydrogen gas was ceased, 2-bromothio-
phene 20 (16.3 g, 100 mmol) and copper oxide (500 mg,
6.29 mmol) were added to the mixture, and the resultant mixture
was stirred at refluxed temperature for 17 h. After being cooled to
room temperature, the reaction mixture was diluted with diethyl
ether (200 mL), and filtered through a Celite pad. The filtrate was
washed with water (100 mL) and 10% copper sulfate solution
(150 mL) twice. The organic layer was dried over magnesium sul-
fate prior to filtration, and the solvent was evaporated under re-
duced pressure. The residue was purified by silica gel column
chromatography (17% ethyl acetate in n-hexane) to give titled
compound 21 (10.0 g, 67%) as a pale yellow oil. 1H NMR (CDCl3)
d: 6.43 (dd, J = 2.4, 1.8 Hz, 1H), 6.93–7.05 (m, 3H), 7.67 (br d, 1H),
7.80 (dd, J = 2.4, 0.6 Hz, 1H). Mass (APCI) m/z: 151 (M+H).
5.1.26. 1-[5-(5-Bromo-2-chlorobenzyl)thiophen-2-yl]-1H-
pyrazole (24)
To a stirred solution of 21 (3.0 g, 20 mmol) in tetrahydrofuran
(200 mL) was added a solution of n-butyllithium (1.58 N n-hexane
solution, 12.6 mL, 19.9 mmol) at À78 °C dropwise over a period of
10 min under argon atmosphere, and the mixture was stirred at
the same temperature for 1 h. To the mixture was added dropwise
a solution of 5-bromo-2-chlorobenzaldehyde (4.37 g, 19.9 mmol)
in tetrahydrofuran (15 mL). The resultant mixture was stirred at
the same temperature for 30 min. The reaction mixture was
5.1.24. 1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-4-chloro-
3-(5-bromo-2-thienylmethyl)benzene (9)
A solution of 19 (75.5 g, 114 mmol) in ethanol (755 mL) and tet-
rahydrofuran (450 mL) was cooled in an ice–water bath, and to the

Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
5571
quenched with saturated aqueous ammonium chloride solution
(300 mL), and extracted with ethyl acetate (200 mL) twice. The or-
ganic layer was washed with brine (100 mL), dried over sodium
sulfate prior to filtration, and the solvent was evaporated under re-
duced pressure. The residue was purified by silica gel column chro-
matography (10–40% ethyl acetate in n-hexane) to give an
inseparable 3:2 mixture of 22 and 23 (5.55 g) as a pale yellow oil
which was taken onto the next step. The obtained pale yellow oil
(5.04 g, 19.6 mmol) was treated with dichloromethane (100 mL),
and cooled to 0 °C. To the mixture was added triethylsilane
(6.50 mL, 40.8 mmol), followed by boron trifluoride diethylether-
ate dropwise over a period of 5 min. The resultant mixture was al-
lowed to warm to room temperature, and stirred for 3 h, and
quenched with saturated aqueous sodium hydrogen carbonate
solution at 0 °C. The mixture was extracted with dichloromethane
(80 mL) twice, and the combined organic layers were washed with
water (50 mL), dried over sodium sulfate prior to filtration. The sol-
vent was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (0–40% ethyl acetate
in n-hexane) to give titled compound 24 (1.85 g, 29% in 2 steps) as
a colorless solid. 1H NMR (CDCl3) d: 4.21 (s, 2H), 6.50 (t, J = 2.3 Hz,
1H), 6.81 (d, J = 3.9 Hz, 1H), 7.12, (d, J = 3.9 Hz, 1H), 7.44 (d,
J = 8.5 Hz, 1H), 7.51 (dd, J = 8.5, 2.4 Hz, 1H), 7.64 (d, J = 2.3 Hz,
1H), 7.69 (d, J = 2.3 Hz, 1H), 8.33 (d, J = 2.4 Hz, 1H). Mass (APCI)
m/z: 353/355/357 (M+H). HPLC: 99.8% (tR = 3.2 min, Sumipax
ODS D-210SLP 4.6 Â 50 mm, 0.05% TFA in CH3CN/H2O (70/30),
1 mL/min of flow rate). Anal. Calcd for C14H10BrClN2S: C, 47.55;
H, 2.85; Br, 22.59; Cl, 10.02; N, 7.92; S, 9.07. Found: C, 47.39; H,
2.72; Br, 22.01; Cl, 9.97; N, 7.84; S, 8.90.
reduced pressure, and the residue was purified by silica gel column
chromatography (0–10% methanol in chloroform) followed by
recrystallization from ethyl acetate–diethylether to give titled
compound 2e (383 mg, 39% in 3 steps) as colorless crystals. 1H
NMR (DMSO-d6) d: 3.07–3.19 (m, 2H), 3.21–3.28 (m, 2H), 3.45
(m, 1H), 3.69 (m, 1H), 4.02 (d, J = 9.5 Hz, 1H), 4.20 (m, 2H), 4.45
(t, J = 5.9 Hz, 1H), 4.85 (d, J = 5.8 Hz, 1H), 4.95–4.96 (m, 2H), 6.49
(dd, J = 2.3, 1.9 Hz, 1H), 6.76 (d, J = 3.7 Hz, 1H), 7.10 (d, J = 3.7 Hz,
1H), 7.28 (dd, J = 8.2, 2.4 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.44 (d,
J = 1.9 Hz, 1H), 7.64 (d, J = 2.3 Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H). Mass
(APCI) m/z: 437/439 (M+H). HPLC 99.8% (tR = 10.2 min, L-column
ODS 4.6 Â 150 mm, CH3CN/20 mM phosphate buffer (pH 6.5) (30/
70), 1 mL/min of flow rate). Anal. Calcd for C20H21N2O5ClS: C,
54.98; H, 4.84; Cl, 8.11; N, 6.41; S, 7.34. Found: C, 54.90; H, 4.63;
Cl, 8.19; N, 6.40; S, 7.42.
5.2. Pharmacology
5.2.1. Sodium-dependent glucose uptake in CHO cells
expressing hSGLT1 and hSGLT2
Parental CHOK cells expressing hSGLT1 and hSGLT2 were used
in these experiments. For the uptake assay, cells were seeded into
24-well plates, and were post-confluent on the day of assay.
Cells were rinsed one time with 400 lL Assay Buffer (137 mM
NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM HEPES, 20 mM
Tris Base, pH 7.4), and were pre-incubated with the solutions of
compounds (250
was initiated by addition of 50
(16.7 Ci; final concentration, 0.3 mM for CHOK-hSGLT1 and
l
L) for 10 min at 37 °C. The transport reaction
lL
AMG/14C-AMG solution
l
0.5 mM for CHOK-hSGLT2, respectively) and incubated for
120 min at 37 °C. After the incubation, the AMG uptake was halted
by aspiration of the incubation mixture followed by immediate
washing three times with PBS. The cells were solubilized in 0.3 N
5.1.27. 4-Chloro-1-(b-D-glucopyranosyl)-3-(5-(2-pyrazol-1-yl)-2-
thienylmethyl)benzene (2e)
To a mixture of 24 (800 mg, 2.40 mmol) and boronic acid ester
25 (2.65 g, 3.60 mmol) in 1,2-dimethoxyethane (24 mL) were
NaOH of 300 lL and the radioactivity associated with the cells
added
dichlorobis(triphenylphosphine)palladium
(84 mg,
was monitored by a liquid scintillation counter (Quantasmart™
(Packard, Boston, MA, USA)). Inhibitory concentration of 50%
(IC50) was calculated by nonlinear least squares analysis using a
four-parameter logistic model (Prism version 4; GraphPad Soft-
ware, San Diego, CA, USA).
0.12 mmol) and 2 M aqueous sodium carbonate solution (6 mL),
then the mixture was stirred at refluxed temperature for 1.5 h un-
der argon atmosphere. The mixture was cooled to room tempera-
ture and extracted with ethyl acetate, washed with water, brine,
and dried over sodium sulfate prior to filtration. The solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (0–7% ethyl acetate in n-hex-
ane), and the obtained material was further purified by NH–silica
gel column chromatography (0–5% ethyl acetate in n-hexane) to
give a colorless viscous oil (1.25 g). The obtained oil was treated
with tetrahydrofuran (20 mL), and to the mixture was added bor-
ane–tetrahydrofuran complex (1 M solution in tetrahydrofuran,
4.5 mL) dropwise at 0 °C. After being stirred at 0 °C overnight, to
the mixture were added 30% aqueous hydrogen peroxide solution
(6 mL) and 3 N aqueous sodium hydroxide solution (6 mL), and
the resultant mixture was allowed to warm to room temperature,
and stirred for 2 h, and poured into saturated aqueous ammonium
chloride solution. The mixture was extracted with ethyl acetate,
and the organic layer was washed with 10% aqueous sodium thio-
sulfate solution, brine, and dried over sodium sulfate prior to filtra-
tion. The solvent was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography (0–5%
ethyl acetate in n-hexane) to give colorless amorphous powder
(788 mg). The obtained powder was treated with tetrahydrofuran
(16 mL), and to the mixture was added tetra-n-butylammonium
fluoride (1 M solution in tetrahydrofuran, 5.35 mL) dropwise under
argon atmosphere, and the mixture was stirred at 60 °C for 2 h.
After being cooled to room temperature, the mixture was poured
into 1% hydrochloric acid solution, and extracted with ethyl ace-
tate. The organic layer was washed with brine, and dried over so-
dium sulfate prior to filtration. The solvent was evaporated under
5.2.2. 2-DG uptake in L6 myoblast cells
The rat skeletal muscle cell line, L6 (JCRB9081), was obtained
from Health Science Research Resources Bank (HSRRB, Osaka, Ja-
pan). L6 myoblast cells were maintained in Dulbecco’s modified
Eagle’s medium containing 5.6 mM glucose supplemented with
10% FBS. Cells were seeded in 24-well plates at a density of
3.0 Â 105 cells/well and cultured for 24 h in an atmosphere of 5%
CO2 at 37 °C before the experiment. Prior to the transport experi-
ment, cells were rinsed twice with KRPH buffer (pH 7.4, 150 mM
NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.25 mM CaCl2, 10 mM HEPES,
2.9 mM Na2HPO4), and were pre-incubated with compounds
(250
was initiated by addition of 50
nal concentration, 750 M) and incubated for 15 min at room tem-
l
l) for 5 min at room temperature. The transport reaction
l
l [3H]-2-DG solution (0.625
l
Ci; fi-
l
perature. After the incubation, the 2-DG uptake was halted by
aspiration of the incubation mixture. Cells were immediately
washed three times with ice-cold PBS and were solubilized in
300 lL of 0.3 N NaOH. The radioactivity associated with the cells
was determined by a liquid scintillation counter (Quantasmart™
(Packard, Boston, MA, USA)).
5.2.3. UGE Study
Male SD rats aged 4–5 weeks were obtained from Japan SLC
(Shizuoka, Japan) and were used for experiments at 6 weeks of
age after acclimation period. The animals were divided into exper-
imental groups matched for body weight (n = 3). The compounds

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Y. Koga et al. / Bioorg. Med. Chem. 21 (2013) 5561–5572
were prepared in vehicles as suspension or solution. UGE studies
were performed after two-day acclimation period in metabolic
cages. The compounds or vehicle were orally administered at a
dose of 30 mg/kg in 0.2% CMC/0.2% Tween 80. Urine samples were
collected for 24 h using metabolic cages to measure urinary glu-
cose excretion. Urine glucose contents were determined by an
enzymatic assay kit (UGLU-L, Serotec, Hokkaido, Japan). All ani-
mals were allowed free access to a standard pellet diet (CRF1; Or-
iental Yeast Co., Ltd, Tokyo, Japan) and tap water.
measurement ANOVA followed by Student’s t-test (EXSAS, Arm
Systex Co. Ltd). Probabilities less than 5% (P <0.05) were considered
to be statistically significant.
References and notes
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5.2.4. Single oral dosing study
Male KK/Ta Jcl mice aged 9 weeks were obtained from CLEA Ja-
pan Inc. (Tokyo, Japan) and kept on a standard diet (CRF-1; 5.7%
(w/w) fat, 3.59 kcal/g, Oriental Yeast Co., Ltd, Tokyo, Japan), 20-
week-old mice were fed with a high-fat diet (60 kcal %, Research
Diets, Inc., New Brunswick, NJ) for 4 weeks. The experiment was
carried out at the age of 24 weeks. Male C57BL/6N mice aged
11 weeks were obtained from Charles River Laboratories Japan,
INC. (Yokohama, Japan) and were also used in this study. The ani-
mals were divided into experimental groups matched for body
weight and blood glucose levels, which were measured in the fed
state on the day of the experiment.
The compounds (3 mg/kg) or vehicle (0.2% CMC/0.2% Tween 80)
were orally administered at a volume of 10 mL/kg. The blood sam-
ples were collected from the tail vein before and at 1, 2, 4, 6 and
24 h after the administration.
The blood glucose level was determined using commercially
available kits based on the glucose oxidase method (Glucose CII-
Test Wako; Wako Pure Chemical Industries, Osaka, Japan). Data
are expressed as means SEM. Area under the curve for blood glu-
cose levels (AUC0–24 hBG) was calculated by the trapezoidal
rule. Differences between groups were analyzed by repeated
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