(1 S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio- d -glucitol (TS-071) is a potent, selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor for type 2 diabetes treatment

J. Med. Chem. 2010, 53, 3247–3261 3247

DOI: 10.1021/jm901893x

(1S)-1,5-Anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol (TS-071) is a

Potent, Selective Sodium-Dependent Glucose Cotransporter 2 (SGLT2) Inhibitor for Type 2 Diabetes

Treatment

Hiroyuki Kakinuma,*^,†Takahiro Oi,^†Yuko Hashimoto-Tsuchiya,^†Masayuki Arai,^‡Yasunori Kawakita,^‡Yoshiki Fukasawa,^‡

Izumi Iida,^‡Naoko Hagima,^‡Hiroyuki Takeuchi,^‡Yukihiro Chino,^‡Jun Asami,^§Lisa Okumura-Kitajima,^§Fusayo Io,^§

Daisuke Yamamoto,^§Noriyuki Miyata,^§Teisuke Takahashi,^§Saeko Uchida,^§and Koji Yamamoto^§

^†Medicinal Chemistry Laboratories, ^‡Drug Safety and Pharmacokinetics Laboratories, and ^§Molecular Function and Pharmacology Laboratories,

Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan

Received December 22, 2009

Derivatives of a novel scaffold, C-phenyl 1-thio-D-glucitol, were prepared and evaluated for sodium-

dependent glucose cotransporter (SGLT) 2 and SGLT1 inhibition activities. Optimization of substi-

tuents on the aromatic rings afforded five compounds with potent and selective SGLT2 inhibition

activities. The compounds were evaluated for in vitro human metabolic stability, human serum protein

binding (SPB), and Caco-2 permeability. Of them, (1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-

4-methylphenyl]-1-thio-^D-glucitol (3p) exhibited potent SGLT2 inhibition activity (IC₅₀=2.26 nM),

with 1650-fold selectivity over SGLT1. Compound 3p showed good metabolic stability toward cryo-

preserved human hepatic clearance, lower SPB, and moderate Caco-2 permeability. Since 3p should

have acceptable human pharmacokinetics (PK) properties, it could be a clinical candidate for treating

type 2 diabetes. We observed that compound 3p exhibits a blood glucose lowering effect, excellent

urinary glucose excretion properties, and promising PK profiles in animals. Phase II clinical trials of 3p

(TS-071) are currently ongoing.

Introduction

Sodium-dependent glucose transporters (SGLTs) present

on the chronic membrane of the intestine and kidney play an

important role in the absorption and reabsorption of glucose.¹³

Approximately 180 g of plasma glucose per day is filtered in

the glomerulus in the kidney, 99% of which is reabsorbed in

the proximal tubule.^14,15Sodium-dependent glucose trans-

porter 2 (SGLT2) is mainly expressed in epithelial cells of the

early S1 segment of the renal cortical tubule and is likely the

major transporter for this reabsorption process.^16,17

Phlorizin (1), a natural nonselective SGLT inhibitor, was

used in proof of concept experiments in several diabetic

animal models (Figure 1). It was shown to advance glucose

excretion and lower fasting and postprandial blood glucose

levels without hypoglycemic side effects.^18,19Tanabe Seiyaku

was the first to conduct a clinical trial of T-1095, which is a

prodrug of an active phlorizin analogue.^20-23This finding led

the company to develop aryl and heteroaryl O-glucosides for

treatment of type 2 diabetes and obesity in the early 2000s.^24-27

It was, however, recognized that in vivo instability of O-gluco-

sides against glucosidases resulted in need of high dosage.

Subsequently, Bristol-Myers Squibb²⁸and Kotobuki²⁹dis-

closed C-aryl glucosides in 2001, which appear to have potent

inhibition and good stability in vivo. Consequently, a number

of companies have pursued C-aryl glucosides.³⁰At present,

dapagliflozin (2) (Bristol-Myers Squibb),^31,32ASP1941

(Kotobuki/Astellas),³³and canagliflozin (Johnson & Johnson/

Mitsubishi Tanabe)³⁴with C-aryl glucosides are undergoing

phase III clinical trials. At least three compounds including BI

10773 (Boehringer Ingelheim),³⁵CSG452 (Chugai/Roche),³⁶

The number of patients diagnosed with type 2, non-insulin-

dependent, diabetes mellitus (NIDDM^a) is increasing.^1,2The

International Diabetes Federation estimates that more than 250

million people worldwide had diabetes in 2007. This total is

expected to rise to 380 million by 2025.³NIDDM can cause

diabetic complications including blindness (retinopathy), renal

failure (nephropathy), the need for lower limb amputation

(neuropathy), and increased risk of a cardiovascular event.⁴

The treatment of NIDDM can prevent or delay the risk of

complications, thus decreasing overall medical costs and helping

maintain quality of life. Good control of blood glucose can be

achieved by a controlled diet and physical exercise. Diabetes can

also be managed by orally active drugs including sulfonylurea,⁵

biganide,⁶R-glucosidase inhibitors,⁷thiazolidinediones,^8,9and

DPP4 inhibitors.^10-12Since combination therapy is usually the

most effective approach for controlling hyperglycemia, there is

need for novel antidiabetic agents with unique modes of action to

complement existing therapies and prevent potential side effects.

*Corresponding author. Hiroyuki Kakinuma Ph.D., Medicinal

Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd. 1-403 Yoshi-

no-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan. Phone: þ81-

48-669-3108. Fax: þ81-48-652-7254. E-mail: hiroyuki.kakinuma@po.

rd.taisho.co.jp.

^aAbbreviations: SGLT2, sodium-dependent glucose cotransporter 2;

NIDDM, noninsulin dependent diabetes mellitus; PPTS, pyridinium

p-toluenesulfonate; STZ, streptozotocin; SAR, structure-activity rela-

tionships; PK, pharmacokinetics; AUC, area under the curve; LLOQ,

lower limit of quantification.

r

2010 American Chemical Society

Published on Web 03/19/2010

pubs.acs.org/jmc

3248 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

Scheme 1

Figure 1

and LX4211 (Lexicon)^37,38are currently in phase II clinical

trials.

We have also given considerable attention to 5-thioglucose-

derived SGLT2 inhibitors, which have a sulfur atom in place

of the oxygen atom in the glucose ring. This glucose analogue

has provided important information regarding the metabolic

stability of several 5-thio-O-glycosides. For example, 5⁰-thio-

N-acetyllactosamine is 200 times more resistant to digestion

by β-galactosidase,³⁹and methyl R-5⁰-thiomaltoside is not

hydrolyzed at all by glucoamylase from Rhizopus niveus.⁴⁰

Thus, we had initially used O-aryl 5-thio-β-glucoside 4 as a

SGLT inhibitor and demonstrated that it increased urinary

glucose excretion and lowered blood glucose in Zucker fatty

rats.^41,42However, more metabolically robust and effective

compounds were required, in order to achieve lower dose

in vivo. Thus, we developed a novel synthetic strategy for

preparing C-phenyl 1-thio-^D-glucitol derivatives. These com-

pounds produced a blood glucose lowering effect and had

excellent pharmacokinetics properties. Here, we focused on

the synthesis of C-phenyl 1-thio-^D-glucitol 3a-3s and the

evaluation and selection of the clinical candidate, 3p, as a

selective SGLT2 inhibitor.

(a) MeNHNH₂, AcOH, DMF (70%); (b) 3,4-DHP, TsOH H₂O,

3

CHCl₃; (c) NaOMe, MeOH; (d) BnBr, NaH, DMF (96%, 3 steps); (e)

PPTS, EtOH (100%); (f) DMSO, Ac₂O (82%).

Scheme 2

Chemistry

ꢀ

Bozo et al. reported that condensation of 2,3,4,6-tetra-O-

acetyl 5-thio-^D-glucopyranosyl bromide with 4-cyanoben-

zenethiol in the presence of K₂CO₃gives a mixture of R and

β-S-5-thio-^D-glucopyranoside of 4-cyanothiophenol (68%),

together with a small amount of C-phenyl R- and β-5-thio-^D-

glucopyranoside derivatives.⁴³This strategy for the prepara-

tion of C-phenyl 1-thio-^D-glucitol was not suitable for our

purposes, due to its lack of general versatility. Our prepara-

tion of C-aryl β-glucoside was inspired by the report that

addition of an aryl organolithium reagent to a sugar lactone

givesa mixture oflactols, which is reduced selectively toafford

C-aryl β-glucoside.^31,44It was expected that this approach

would be applicable to the preparation of C-phenyl 1-thio-^D-

glucitol; therefore, thiolactone 8 was believed to be an im-

portant intermediate. The first synthesis of tetra-O-benzyl

derivative 7 and thiolactone 8 was reported by Yuasa.⁴⁵

Compound 7 was prepared by acid hydrolysis of methyl

tetra-O-benzyl-5-thio-^D-glucoside in moderate yield (50%).

We therefore prepared 8 in accordance with Scheme 1, due to

the availability of 7. 5-Thio-^D-glucose penta-O-acetate 5b can

be prepared in 8 steps from commercially available ^D-glucur-

ono-3,6-lactone (5a).^41,46Anomeric acetyl group of 5b was

selectively removed with hydrazine acetate⁴⁷or a combination

of methylhydrazine and acetic acid as described previously.⁴⁸

(f) n-BuLi, THF, -78 °C, then 10; (g) Et₃SiH, BF₃OEt₂, CH₃CN/

CHCl₃.

The resulting anomeric hydroxyl group of 5c was protected

with 3,4-dihydro-2H-pyranyl ether in the presence of p-tolue-

nesulfonic acid monohydrate. Then, the acetyl group was

ꢀ

removed by Zemplen deacetylation, and the resulting hydro-

xyl groups were protected by a benzyl group using benzyl-

bromide with sodium hydride to generate 6. The tetra-

hydropyranyl group was removed by treating 6 with PPTS,

and finally DMSO oxidation of 7 provided 8 in 79% total

yield.

The aglycon portions 12a-12d and 12g-12i were synthe-

sized as shown in Scheme 2. After lithium halogen exchange

of 9, benzaldehyde derivatives 10a-10d and 10g-10i⁴⁹were

added to give 11a-11d and 11g-11i in 58-90% yield.

Reduction of alcohol 11 using triethylsilane and boron

trifluoride diethyletherate gave 12a-12d and 12g-12i in

43-99% yield.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3249

Scheme 4

Scheme 3

(i) (COCl)₂, DMF (cat), CHCl₃; (j) cumene, AlCl₃, CHCl₃; (k)

Et₃SiH, BF₃OEt₂, CH₃CN/CHCl₃; (l) Br₂, AcOH.

Scheme 5

(m) MeI, K₂CO₃, n-Bu₄NI, DMF; (h) 4-ethylbenzoylchroride, AlCl₃,

CHCl₃; (k) Et₃SiH, BF₃OEt₂, CH₃CN/CHCl₃.

C-Phenyl 1-thio-^D-glucitols 3a-3s were prepared as out-

lined in Scheme 6. Compounds 20a-20s were obtained by

adding thiolactone 8 to Grignard reagents prepared from

compounds 12a-12s and magnesium powder. Each product

was found to be a single isomer, but the anomeric configration

could not be determined. The hydroxyl group of 20a-20s was

reduced β-stereoselectively to afford compounds 21a-21s. In

this reaction, the R-isomer was formed as a byproduct in a

yield of several percent, but could be decreased using the

following conditions. First, boron trifluoride diethyletherate

was selected as the Lewis acid. Second, the most favor-

able solvent was chosen, and found to be acetonitrile or a

mixture of acetonitrile and chloroform. For example, 21p was

obtained with a ratio of 4/96 of R/β in 77% yield. Finally, the

benzyl ether of compound 21 was removed by catalytic

hydrogenation with palladium hydroxide under a hydrogen

atmosphere to afford 3a-3e, 3g-3i, and 3p-3r. Alterna-

tively, compounds 3f, 3j-3o, and 3s were obtained by removal

of the benzyl group using Lewis acid conditions to prevent

reduction of the chloride.

(h) Br₂, Fe (cat), CHCl₃; (i) (COCl)₂, DMF (cat), CHCl₃; (j) AlCl₃,

CHCl₃; (k) Et₃SiH, BF₃OEt₂, CH₃CN/CHCl₃for 12e-12p; NaBH₄,

AlCl₃, THF for 12q.

The aglycon portions 12e, 12f³¹and 12j-12q were synthe-

sized as shown in Scheme 3. Commercially available 4-meth-

oxy-2-methylbenzoic acid 13, brominated using bromine in

the presence of a catalytic amount of Fe, generated a 1:1 mix-

ture of 3- and 5-bromo derivatives. The desired isomer 14 was

isolated after two recrystallizations from MeOH. Friedel-

Crafts reaction of a corresponding substituted benzene (Ph-

R³) was carried out using the acid chlorides prepared from 14

and commercially available 15 and 16 with oxalyl chloride to

afford 17e, 17f, and 17j-17q, respectively (43-99%). Reduc-

tion of 17e, 17f, and 17j-17p using triethylsilane and boron

trifluoride diethyletherate gave aglycons 12e, 12f, and

12j-12p (47-96%). Compound 17q, however, was resistant

to the silane reduction conditions, and so was reduced by a

combination of sodium borohydride and aluminum chloride

to provide 12q in good yield (90%).

Results and Discussion

Compound 12r was prepared as shown in Scheme 4.

Friedel-Crafts reaction of cumene using the acid chloride

prepared from 13 afforded 17r. Compound 17r was re-

duced under silane reducing conditions, and the obtained

diphenylmethane derivative was brominated to give 12r.

Compound 12s was synthesized as shown in Scheme 5.

After methylation of commercially available 18 with iodo-

methane under basic conditions, Friedel-Crafts reaction of

19 was performed using 4-ethylbenzoyl chloride to afford 17s.

Subsequent reduction of 17s using the same conditions de-

scribed above gave the aglycon portion 12s.

To assess the in vitro potency of SGLT2 inhibition and its

selectivity for SGLT1, the inhibitory activities of compounds

on SGLTs were determined by a cell-based assay in which

inhibition of the rate of glucose uptake was monitored with

CHO-K1 cells expressing human SGLT1 and SGLT2.

We were interested in using thioglucose instead of glucose

because the ring sulfur atom has little charge, whereas the ring

oxygen atom possesses a high negative-charge density.⁵⁰If the

binding site interacts hydrophobically with the upper face of

the sugar ring, the thioglucose might be a better ligand than its

ring oxygen analogue. We selected 3-(4-ethoxybenzyl)phenyl

3250 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

Scheme 6

(p) Mg, THF; (q) Et₃SiH, BF₃OEt₂, CH₃CN, or CH₃CN/CHCl₃; (r) Pd(OH)₂/H₂(for 3a-3e, 3g-3i, 3p-3r) or AlCl₃in anisole (for 3f, 3j-3n) or

CF₃CO₂H, Me₂S, m-cresol, 1,2-ethanedithiol, TfOH (for 3o, 3s).

Table 1. hSGLT1 and hSGLT2 Inhibitory Activity^a

SGLT2 (nM)

mean (95% CI)

SGLT1 (nM)

mean (95% CI)

cmpds

R¹

R²

R³

T1/T2 selectivity

1

27.8 (21.8-35.3)

73.6 (51.4-105)

283 (268-298)

246 (162-374)

8.8

355

3a

3b

3c

3d

3e

3f

H

OEt

OMe

Me

26100 (20300-33700)

14600 (11500-18500)

565 (510-627)

H

OH

OMe

F

51.6

42.2

847

H

13.4 (11.3-15.8)

9.40 (5.87-15.0)

2.29 (1.76-2.99)

1.77 (0.95-3.30)

17.4 (15.9-19.0)

37.9 (26.4-54.4)

10.8 (6.84-17.1)

1.68 (1.08-2.60)

1.37 (0.97-1.95)

1.78 (0.88-3.63)

4.01 (1.75-9.17)

18.8 (11.0-32.1)

1.16 (0.73-1.85)

2.26 (1.48-3.43)

1.71 (1.19-2.46)

2.68 (2.15-3.34)

1.51 (0.75-3.04)

H

7960 (7180-8820)

671 (230-1960)

H

Me

Cl

293

684

H

1210 (798-1840)

4040 (1200-13600)

100000 (66500-151000)

4270 (1560-11600)

260 (72.5-931)

3g

3h

3i

OH

OMe

H

232

H

2640

395

OMe

Cl

3j

155

153

3k

3l

H

209 (80.2-545)

602 (473-767)

H

Cl

Et

iPr

338

3m

3n

3o

3p

3q

3r

3s

H

8160 (4860-13700)

35600 (31900-39800)

391 (239-641)

3990 (2690-5920)

2830 (1540-5200)

17300 (14100-21100)

3340 (2710-4110)

2040

1890

337

H

Cl

tBu

SMe

OEt

Et

H

OMe

Me

Cl

1770

1650

6400

2210

iPr

Et

^aIC₅₀values for SGLT1 and 2 activities represent the mean and the 95% confidence intervals (CI) of at least three determinations.

derivative3aasa simplelead compoundbecause ofits aglycon

structure. According to previous reports,^31,42the positions

of substituents on aromatic rings should be important. Com-

pound 3a showed moderate inhibition activity (73.6 nM for

SGLT2) and was 355 times more selective for SGLT2 than for

SGLT1 (Table 1). First, we investigated substitution effects

onthe centralbenzene moiety. The introduction of a methoxy,

fluoro, methyl, or chloro group in R²(3c-3f) increased

SGLT2 inhibition potency. Compound 3e with a methyl

group and 3f with a chloro group showed strong inhibition

activity toward SGLT2 at 2.29 and 1.77 nM, respectively.

However, the introduction of a hydroxyl group in R²(3b)

decreased potency (283 nM), suggesting that the hydrogen

donating property of a hydroxyl group in R²may be undesir-

able. The substitution of R¹with a hydroxyl (3g) or methoxy

(3h and 3i) group resulted in 2-4 times greater potency toward

SGLT2 compared to 3a. Surprisingly, 3h had decreased

SGLT1 inhibition activity compared with 3a and resulted in

a 2640-fold greater selectivity for SGLT2 than for SGLT1.

However, the selectivity for SGLT2 (395-fold) of the di-

methoxy analogue (3i) was same as that of 3a.

We next studied the structure-activity relationships (SAR)

of para-position derivatives of the distal benzene moiety while

retaining the chloro group in the central benzene ring. From

comparison of compounds 3f and 3j-3o in Table 1, except for

3n with a tertiary butyl group, compounds with a methyl,

ethyl, isopropyl, methoxy, ethoxy, or methylthio substituent

strongly inhibited SGLT2 activity and showed IC₅₀values of

less than 5 nM. In this series, compound 3m(with anisopropyl

group) and 3n (with a tertiary butyl group) showed high

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3251

Table 2. In Vitro Human Metabolic Stability of Verapamil and 3m,

3p-3s

(% residual)

cryopreserved

hepatocytes^b

cmpds

microsomes^a

verapamil

NT^c

96

3.9 ( 0.3

22.4 ( 3.5

69.0 ( 7.0

4.7 ( 1.3

6.8 ( 0.9

3.7 ( 1.3

3m

3p

3q

3r

96

90

91

89

3s

^aTest compounds (5 μmol/L) were incubated with human liver

microsomes (1 mg/mL protein) in the presence of an NADPH-generat-

ing system for 15 min at 37 °C. ^bTest compounds (5 μmol/L) were

incubated with human cryopreserved hepatocytes (1.0 ꢀ 10⁶cells/mL)

for 240 min at 37 °C. Each value represents the mean ( SD of triplicate

samples. ^cNT, not tested.

Figure 2. Suppression of glucose AUC_{0-8 h}in STZ-induced dia-

betic rats versus vehicle control, following an oral dose of 0.3 or

3.0 mg/kg of 3m, 3p, 3s (n = 6) and 3q, 3r (n = 5). Data are expressed

as means ( SE.

Table 3. Serum Protein Binding of 3m, 3p-3s in Human

serum protein

binding (%)^a

selectivity for SGLT2 over SGLT1 (2040- and 1890-fold,

respectively).

cmpds

3m

3p

3q

3r

99.7 ( 0.0

96.2 ( 0.2

98.3 ( 0.1

99.1 ( 0.1

99.4 ( 0.0

Finally, we combined the substituents in R¹, R², and R³and

optimized SGLT2 inhibition potency and selectivity for SGLT2.

We selected the methoxy group for R¹because of its expected

high SGLT2 selectivity. A methyl and a chloro group were

chosen for R², and an ethyl, an ethoxy, and an isopropyl group

for R³were selected due to their expected potency and selectivity

for SGLT2 inhibition activity, based on the above results.

Compounds 3p-3s strongly inhibited SGLT2 activity, with

IC₅₀values of less than 3 nM, showing that 3p-3s are 10 times

more potent than phlorizin (1). On the other hand, their SGLT1

inhibitory activities were lower (IC₅₀values were greater than

2.8 μM), and their selectivity for SGLT2 was 1650- to 6400-fold,

which is the same as or greater than the selectivity of dapagli-

flozin (2, 1200-fold)³¹and phlorizin (1, 8.8-fold).

3s

^aSerum protein binding was determined by equilibrium dialysis at

1 μg/mL compounds. Each value represents the mean ( SD of triplicate

samples.

which has a Cl_humanof 11.8 ( 5.0 mL/min/kg and a F_humanof

20 ( 12%.⁵¹Further, we compared 3a with 4 (R^A=H, R^B=

4-OEt) which had the same aglycon of 3a. The stability of 3a

was greater than that of 4 against cryopreserved hepatocytes

(data not shown). Although interesting information was

known that O-5-thioglucoside linkage would be resistant to

bacterial glucosidases,^39,40O-aryl 5-thio-β-glucoside 4 may be

less stable in human tissues. In contrast, 22.4 ( 3.5% of 3m

and 69.0 ( 7.0% of 3p remained after 240 min incubation with

human cryopreserved hepatocytes. These results indicate that

the clearance of 3m and 3p may be slower than that of vera-

pamil in humans. Serum protein binding (SPB) also impacts

both PK and exposure to the therapeutic target. Human SPB

of 3p was 96.2%, indicating that the free fraction of 3p was

3.8%, which is 13 times higher than that of 3m (free fraction of

3m was 0.3%) (Table 3). It was expected that compound 3p

would be readily absorbed via the intestine, based on Caco-2

assay results (14.4 ( 2.2 cm/s ꢀ 10⁶). This result was compar-

able with that of a positive control, propranolol (9.1 ( 0.5 cm/

s ꢀ 10⁶).⁵²These results suggested that 3p would have good

absorption and distribution profiles and would be stable in

human tissues. We therefore selected and administered 3p

orally and intravenously to rats and dogs at1 mg/kg (Table 4).

The bioavailability (F=35.3%) and total clearance (36.3 (

2.67 mL/min/kg) of 3p in rats were moderate. On the other

hand, C_maxwas 914 ( 73.4 ng/mL at 0.67 h in dogs, with

excellent bioavailability (92.7%) and a slow clearance rate of

3.18 ( 0.260 mL/min/kg. The volume of distribution at steady

state (Vdss) was 0.8 ( 0.06 L/kg, similar to total body water in

dog (ca. 0.6 L/kg), indicating modest extravascular distribu-

tion. Compared to the disclosed PK parameters of dapagli-

flozin (2) in dogs,⁵³the bioavailability (F), Vdss, and T_max

were similar to those of 3p. The half-life (T_1/2) of 3p, however,

was shorter than that of 2. This may be attributed to the

considerably slower clearance rateof2 (1.5 ( 0.2 mL/min/kg).

The potency of 3q (1.71 nM for SGLT2, 2830 nM for

SGLT1) was the same as that of 3s (1.51 nM for SGLT2, 3340

nM for SGLT1). Comparable potencies were also observed

for 3e and 3f, indicating that having a methyl or chloro group

in R²produces equivalent effects. This result indicates that

a steric, rather than electronic, effect in R²may influence

SGLT2 inhibition activity. The selectivity of 3m and 3r for

SGLT2 was 4.6- and 3.9-fold greater than that of 3l and 3q,

respectively. From these results, it appears that a methoxy

group in R¹and a bulky isopropyl group in R³contribute to a

greater selectivity for SGLT2, and that a methyl and a chloro

group in R²increase potency toward SGLT2.

We selected 3m and 3p-3s, which had strong SGLT2 inhi-

bition potency (IC₅₀less than 5nM) and high SGLT2 selec-

tivity (morethan 1500-fold). These compounds lowered blood

glucose in streptozotocin (STZ)-induced diabetic rats when

orally administered at 0.3 or 3 mg/kg (Figure 2). Since these

compounds showed promise for the effective treatment of

type 2 diabetes, they were evaluated further.

Our intention was to identify compounds having favorable

pharmacokinetics (PK) profiles in humans. Metabolic stabil-

ity affects PKand plays a major roleindrug clearance, with an

increase in metabolic stability leading to a decrease in clear-

ance. We therefore studied the stabilities of 3m and 3p-3s

using human liver microsomes and hepatocytes. Table 2

shows that all five compounds were resistant to human micro-

somal degradation. However, 3q-3s were largely decom-

posed after 240 min incubation with human cryopreserved

hepatocytes, comparable to that of a control drug, verapamil,

3252 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

Table 4. Pharmacokinetic Parameters of 3p after Oral and Intravenous

Administration to Rats and Dogs

the substituents on the aromatic rings generated potent

SGLT2 selective inhibitors (3m, 3p-3s) which lowered blood

glucose in STZ-induced diabetic rats. In order to estimate the

pharmacological effects in humans, we conducted further

in vitro evaluations and found that compound 3p was 1650-

fold more selective for SGLT2 with an IC₅₀of 2.26 nM than

for SGLT1 and the highest metabolic stability toward

degradation by human cryopreserved hepatocytes. Com-

pound 3p also had the lowest human serum protein binding

of the five compounds tested. Further, the Caco-2 perme-

ability of 3p was comparable with that of propranolol. For

these reasons, it was anticipated that compound 3p would

have good PK profiles in humans. Compound 3p ((1S)-1,5-

anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-

1-thio-^D-glucitol; TS-071) was therefore chosen as a clinical

candidate. In order to confirm the pharmacology of 3p, PK

profiles and urinary glucose excretion effects were evaluated

in animals. Compound 3p showed excellent urinary glucose

excretion and PK profiles in dogs. Provided that compound

3p has desirable PK profiles in humans, 3p will be useful for

treating type 2 diabetes by means of SGLT2 inhibition. These

results demonstrate the utility of thioglucose as a SGLT2

inhibitor, and indicate that thioglucose works as a glucose

analogue. Compound 3p is currently undergoing phase II

clinical trials.

cmps (species)

3p (rats)^a

3p (dogs)^a

2 (dogs)^b

dose (mg/kg)

1

6.6

C

_max(ng/mL)

_max(h)

35.7 ( 17.0

0.50 ( 0.00

2.93 ( 2.00

35.3

914 ( 73.4

0.67 ( 0.29

4.07 ( 0.25

92.7

10 700 ( 1600

0.6 ( 0.4

7.4 ( 1.2

83 ( 2

0.80 ( 0.1

1.5 ( 0.2

T

T_1/2(h)

F (%)

Vd_ss(L/kg)

Cl (mL/min/kg)

2.63 ( 0.57

36.3 ( 2.67

0.80 ( 0.06

3.19 ( 0.26

^aEach data represents mean ( SD of three animals. ^bPK parameters

of dapagliflozin (2) were quoted from the ref 53.

Table 5. Distribution of 3p to the Kidney in Rats

concentration (ng/mL or g)^a

time (h)

plasma

kidney

kidney/plasma ratio

1

23.1 ( 3.2

13.9 ( 3.9

NC^b

562 ( 199

488 ( 123

NC

23.9 ( 5.1

35.3 ( 0.8

NC

4

24

^aConcentrations of 3p in plasma and kidney in rats were measured at

1, 4, and 24 h after oral administration at 1 mg/kg. Each value represents

mean ( SD of three animals. ^bNC, (not calculated) means limit of

detection.

Table 6. Enhancement of Urinary Glucose Excretion after Oral Glu-

cose Loading in Zucker Fatty Rats and Beagle Dogs

urinary glucose output (mg/24 h)

Experimental Section

rats^a

dogs^b

Chemistry. Melting points were determined on a Yanaco MP-

500D melting point apparatus and are uncorrected. All reac-

tions were monitored using TLC plates Silicagel gel 60F₂₅₄

(Merck). 200, 300, and 600 MHz NMR spectra were obtained

using a Varian Gemini 200, Varian Unity Inova 300, or JEOL

JNM-ECA600, respectively. Chemical shifts are reported in

parts per million relative to tetramethylsilane (TMS) used as

an internal standard. Electron impact (EI) mass spectra were

taken on a Perkin-Elmer Sciex API-300 mass spectrometer.

Electrospray ionization (ESI) mass spectra were taken on a

Micromass Platform LC mass spectrometer. Elemental analyses

were performed on a Perkin-Elmer 2400 elemental analyzers.

Silica gel column chromatography was performed on Silica gel

60 (Kanto Chemical) or NH-silica gel chromatorex 1020 (Fuji

Silicia), using the solvent systems (volume ratios) indicated

below. Purity of the test compounds was determined to be

>95% by HPLC (shiseido capcell pak UG120 4.6 ꢀ 150 mm,

acetonitrile/water=4/6, 1 mL/min of flow rate at 40 °C, and

detection at 220 nm) or elemental analyses.

3p (1 mg/kg)

vehicle control

normal control

179.4 ( 15.27

6.07 ( 3.58

0.98 ( 0.08

20129 ( 1870

;

7.64 ( 1.95

^aCompound 3p (1 mg/kg) was administered orally to Zucker fatty

rats (n = 8, mean body weight, approximately 380 g) 0.5 h prior to 2 g/kg

glucose loading. Urine samples were collected for 24 h using metabolic

cages. ^bCompound 3p (1 mg/kg) was administered orally to beagle dogs

(n = 10, body weight, 8.1-9.6 kg) 1 h prior to 2 g/kg glucose loading.

Urine samples were collected for 24 h using metabolic cages. Each value

represents mean ( SD.

Furthermore, kidney/plasma ratio was 24-35-fold higher,

indicating that appreciable amounts of 3p are distributed

to the target organ (kidney) (Table 5). In contrast, 3p was

excreted within 24 h in rats and was not detected in nontarget

organs such as liver, heart, and brain 24 h after administra-

tion at 3 mg/kg, p.o. Most metabolites of [¹⁴C] 3p in rats were

excreted to bile. (data not shown) Compound 3p shouldbewell-

distributed to kidney and be eliminated primarily by meta-

bolism in liver.

In Zucker fatty rats after oral glucose loading, a single oral

dose of 3p (1 mg/kg) increased urinary glucose excretion to 30

times that of the vehicle control and 180 times that of normal

control Zucker lean rats over 24 h. Experiments in dogs

showed over 2600-fold enhancement of the urinary glucose

level after 1 mg/kg oral administration of 3p (Table 6). This

may be attributed to higher bioavailability of 3p in dogs

(Table 4).

General Methods for Removal of the Benzyl Group (Method A).

(1S)-1,5-Anhydro-1-[3-(4-ethoxybenzyl)phenyl]-1-thio-^D-glu-

citol (3a). A mixture of (1S)-1,5-anhydro-2,3,4,6-tetra-O-ben-

zyl-1-[3-(4-ethoxybenzyl)phenyl]-1-thio-^D-glucitol (21a) (550

mg, 0.732 mmol), 20% palladium hydroxide on carbon of

Degussa type (550 mg) in 15 mL of ethyl acetate and 15 mL of

ethanol was stirred under a hydrogen atmosphere at room

temperature for 48 h. The insoluble components were filtered

off using a Celite pad and the filtrate was concentrated. The

residue was purified by silica gel column chromatography

(CHCl₃/MeOH=10/1) to give 0.5 hydrate of 3a as a colorless

powder (60 mg, 21%). mp 110.0-113.0 °C. ¹H NMR (300

MHz, MeOH-d₄) δ 1.35 (t, J=7.0 Hz, 3 H), 2.92-3.03 (m,

1 H), 3.19-3.28 (m, 1 H), 3.59 (dd, J= 9.1, 10.2 Hz, 1 H),

3.69-3.78 (m, 3 H), 3.88 (s, 2 H), 3.90-4.04 (m, 3 H), 6.80 (d,

J=8.7 Hz, 2 H), 7.04-7.11 (m, 3 H), 7.14-7.25 (m, 3 H). MS

(ESI) m/z 413 (MþNa), 389 (M-H). Anal. Calcd for

Conclusion

We described the synthesis of C-phenyl 1-thio-^D-glucitols

3a-3s and revealed the SAR for the substitution of various

groups on the aromatic rings. A methoxy group adjacent to

the sugar moiety was important for expression of SGLT2

selectivity, whereas a chloro or methy group para to the sugar

moiety strongly increased SGLT2 potency. Optimization of

(C₂₁H₂₆O₅S 0.5H₂O) C, 63.14; H, 6.81. Found C, 63.38; H,

3

6.73.

(1S)-1,5-Anhydro-1-[3-(4-ethoxybenzyl)-4-hydroxyphenyl]-1-

thio-^D-glucitol (3b). Compound 3b (0.117 g, 66%) was prepared

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3253

as a colorless powder from 21b (0.566 g, 0.660 mmol) according

to the method described for the synthesis of 3a. (Method A) mp

95.0-100.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 1.35 (t, J=6.9

Hz, 3 H), 2.89-2.98 (m, 1 H), 3.16-3.24 (m, 1 H), 3.56 (dd, J=

9.0, 10.2 Hz, 1 H), 3.60-3.76 (m, 3 H), 3.83 (s, 2 H), 3.88-3.95

(m, 1 H), 3.98 (q, J=6.9 Hz, 2 H), 6.69-6.80 (m, 3 H), 6.96-7.04

(m, 2 H), 7.06-7.15 (m, 2 H). MS (ESI) m/z 424 (MþNa), 405

Anal. Calcd for (C₂₁H₂₆O₆S 1.2H₂O) C, 58.92; H, 6.69. Found

3

C, 58.87; H, 6.40.

(1S)-1,5-Anhydro-1-[5-(4-ethoxybenzyl)-2-methoxyphenyl]-1-

thio-^D-glucitol (3h). Compound 3h (0.037 g, 7.2%) was prepared

as a colorless powder from 21h (0.87 g, 1.11 mmol) according to

the method described for the synthesis of 3a. (Method A) mp

130.0-130.5 °C. ¹H NMR (600 MHz, MeOH-d₄) δ 1.33 (t,

J=7.1 Hz, 3 H), 2.90-2.98 (m, 1 H), 3.22 (t, J=8.9 Hz, 1 H),

3.51-3.61 (m, 1 H), 3.65-3.74 (dd, J = 6.4, 11.5 Hz, 1 H),

3.76-3.84 (m, 6 H), 3.91 (dd, J=3.7, 11.5 Hz, 1 H), 3.96 (q,

J=7.1 Hz, 2 H), 4.31 (brs, 1 H), 6.77 (d, J=8.7 Hz, 2 H), 6.85 (d,

J=8.7 Hz, 1 H), 7.00 (dd, J=2.3, 8.7, Hz, 1 H), 7.04 (d, J=8.7

Hz, 2 H), 7.16 (brs, 1 H). MS (ESI) m/z 443 (MþNa). Anal.

(M-H). Anal. Calcd for (C₂₁H₂₆O₆S 1.1H₂O) C, 59.17; H,

3

6.67. Found C, 59.14; H, 6.32.

(1S)-1,5-Anhydro-1-[3-(4-ethoxybenzyl)-4-methoxyphenyl]-1-

thio-^D-glucitol (3c). Compound 3c (0.385 g, 66%) was prepared

as a colorless powder from 21c (0.880 g, 1.13 mmol) according to

the method described for the synthesis of 3a. (Method A) mp

89.0-95.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 1.35 (t, J=7.0

Hz, 3 H), 2.91-3.01 (m, 1 H), 3.18-3.25 (m, 1 H), 3.57 (dd,

J=9.0, 10.3 Hz, 1 H), 3.68-3.76 (m, 3 H), 3.79 (s, 3 H), 3.84 (s, 2

H), 3.89-4.02 (m, 3 H), 6.76 (d, J=8.7 Hz, 2 H), 6.88 (d, J=8.7

Hz, 1 H), 7.03-7.11 (m, 3 H), 7.17 (dd, J=2.3, 8.6 Hz, 1 H). MS

(ESI) m/z 443 (MþNa), 419 (M-H). Anal. Calcd for

Calcd for (C₂₂H₂₈O₆S H₂O) C, 60.25; H, 6.90. Found C, 60.00;

3

H, 6.76.

(1S)-1,5-Anhydro-1-[2,4-dimethoxy-5-(4-ethoxybenzyl)phenyl]-

1-thio-^D-glucitol (3i). Compound 3i (0.156 g, 66%) was prepared

as a colorless powder from 21i (0.428 g, 0.527 mmol) according

to the method described for the synthesis of 3a. (Method A) mp

158.0-160.0 °C. ¹H NMR (600 MHz, MeOH-d₄) δ 1.34 (t,

J=7.0 Hz, 3 H), 2.88-2.99 (m, 1 H), 3.22 (t, J=8.8 Hz, 1 H),

3.51-3.59 (m, 1 H), 3.66-3.79 (m, 4H), 3.81 (s, 3 H), 3.84 (s,

3 H), 3.88-4.01 (m, 3 H), 4.21-4.32 (m, 1 H), 6.57 (s, 1 H), 6.75

(d, J=8.7 Hz, 2 H), 7.03 (s, 1 H), 7.04 (d, J=8.7 Hz, 2 H). MS

(ESI) m/z 468 (MþNH₄), 449 (M-H). Anal. Calcd for (C₂₃H₃₀-

(C₂₂H₂₈O₆S H₂O) C, 60.25; H, 6.90. Found C, 60.49; H, 6.75.

3

(1S)-1,5-Anhydro-1-[3-(4-ethoxybenzyl)-4-fluorophenyl]-1-

thio-^D-glucitol (3d). Compound 3d (0.305 g, 75%) was prepared

as a colorless powder from 21d (0.767 g, 0.997 mmol) according

to the method described for the synthesis of 3a. (Method A) mp

60.0-65.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 1.35 (t, J=7.0

Hz, 3 H), 2.91-3.03 (m, 1 H), 3.19-3.25 (m, 1 H), 3.58 (dd, J=

9.0, 10.3 Hz, 1 H), 3.68-3.79 (m, 3 H), 3.86-4.04 (m, 5 H),

6.77-6.82 (m, 2 H), 6.95-7.04 (m, 1 H), 7.07-7.12 (m, 2 H),

7.15-7.24 (m, 2 H). MS (ESI) m/z 431 (MþNa). Anal. Calcd for

O₇S 0.5H₂O) C, 60.11; H, 6.80. Found C, 60.22; H, 6.61.

3

(1S)-1,5-Anhydro-1-[4-chloro-3-(4-methoxybenzyl)phenyl]-1-

thio-^D-glucitol (3j). Compound 3j (0.100 g, 19%) was prepared as

a colorless powder from 21j (1.01 g, 1.31 mmol) according to the

method described for the synthesis of 3f. (Method B) mp

71.0-74.0 °C. ¹H NMR (600 MHz, MeOH-d₄) δ 2.96-3.01

(m, 1 H), 3.21-3.25 (m, 1 H), 3.57 (dd, J=8.9, 10.3 Hz, 1 H),

3.66-3.74 (m, 3 H), 3.75 (s, 3 H), 3.93 (dd, J=3.7, 11.5 Hz, 1 H),

3.98-4.05 (m, 2 H), 6.82 (d, J=8.7 Hz, 2 H), 7.09 (d, J=8.7 Hz, 2

H), 7.18-7.22 (m, 2 H), 7.32 (d, J=8.3 Hz, 1 H). MS (ESI) m/z

428 (MþNH₄), 430 (Mþ2þNH₄). Anal. Calcd for (C₂₀H₂₃O₄-

ClS) C, 58.46; H, 5.46. Found C, 58.36; H, 5.55.

(C₂₁H₂₅FO₅S 0.5H₂O) C, 60.41; H, 6.28. Found C, 60.22; H,

3

6.09.

(1S)-1,5-Anhydro-1-[3-(4-ethoxybenzyl)-4-methylphenyl]-1-

thio-^D-glucitol (3e). Compound 3e (0.181 g, 33%) was prepared

as a colorless powder from 21e (1.04 g, 1.36 mmol) according to

the method described for the synthesis of 3a. (Method A) mp

105.0-107.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 1.35 (t, J=

6.9 Hz, 3 H), 2.17 (s, 3 H), 2.93-3.03 (m, 1 H), 3.19-3.28 (m,

1 H), 3.59 (dd, J=9.0, 10.2 Hz, 1 H), 3.68-3.79 (m, 3 H), 3.89 (s,

2 H), 3.93 (dd, J=3.7, 7.9 Hz, 1 H), 3.97 (q, J=6.9 Hz, 2 H),

6.74-6.82 (m, 2 H), 6.96-7.04 (m, 2 H), 7.05-7.15 (m, 3 H). MS

(ESI) m/z 422 (MþNH₄), 403 (M-H). Anal. Calcd for (C₂₂H₂₈-

(1S)-1,5-Anhydro-1-[4-chloro-3-(4-methylbenzyl)phenyl]-1-

thio-D-glucitol (3k). Compound 3k (0.183 g, 23%) was prepared

as a colorless powder from 21k (1.54 g, 2.04 mmol) according to

the method described for the synthesis of 3f. (Method B) mp

151.0-153.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 2.28 (s, 3 H),

2.94-3.02 (m, 1 H), 3.18-3.26 (m, 1 H), 3.57 (dd, J=8.9, 10.2

Hz, 1 H), 3.65-3.77 (m, 3 H), 3.93 (dd, J=3.7, 11.4 Hz, 1 H),

4.02 (s, 2 H), 7.02-7.10 (m, 4 H), 7.16-7.24 (m, 2 H), 7.29-7.35

(m, 1 H). MS (ESI) m/z 412 (MþNa), 414 (Mþ2þNa). Anal.

O₅S 0.4H₂O) C, 64.18; H, 7.05. Found C, 64.13; H, 6.94.

3

General Methods for Removal of the Benzyl Group (Method B).

(1S)-1,5-Anhydro-1-[4-chloro-3-(4-ethoxybenzyl)phenyl]-1-thio-

D-glucitol (3f). To a solution of 21f (1.58 g, 2.01 mmol) in anisole

(15 mL) at 4 °C was added AlCl₃(2.68 g, 21.1 mmol). The

reaction mixture was allowed to warm to room temperature,

stirred for 0.5 h, then poured into ice. The resulting mixture was

extracted with ethyl acetate twice, and the combined organic

layers were washed with 1 M hydrochloric acid, saturated

aqueous NaHCO₃, and brine, and then dried over MgSO₄and

concentrated. The residue was purified by silica gel column

chromatography (CHCl₃/MeOH=10/1) to give 3f (0.16 g, 20%)

as a colorless powder. mp 79.0-83.0 °C. ¹H NMR (300 MHz,

MeOH-d₄) δ 1.36 (t, J=7.0 Hz, 3 H), 2.94-3.03 (m, 1 H), 3.22

(t, J=8.2 Hz, 1 H), 3.57 (dd, J=9.0, 10.3 Hz, 1 H), 3.65-3.78

(m, 3 H), 3.89-4.05 (m, 5 H), 6.80 (d, J=8.7 Hz, 2 H), 7.08 (d,

J=8.7 Hz, 2 H), 7.16-7.23 (m, 2 H), 7.32 (d, J=8.3 Hz, 1 H).

MS (ESI) m/z 447 (MþNa), 449 (Mþ2þNa). Anal. Calcd

Calcd for (C₂₀H₂₃ClO₄S 0.5H₂O) C, 59.74; H, 5.97. Found C,

3

59.66; H, 5.90.

(1S)-1,5-Anhydro-1-[4-chloro-3-(4-ethylbenzyl)phenyl]-1-thio-

D-glucitol (3l). Compound 3l (0.299 g, 26%) was prepared as a

colorless powder from 21l (2.13 g, 2.77 mmol) according to the

method described for the synthesis of 3f. (Method B) mp

142.0-144.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 1.20 (t, J=

7.6 Hz, 3 H), 2.59 (q, J =7.6 Hz, 2 H), 2.94-3.03 (m, 1 H),

3.18-3.27 (m, 1 H), 3.57 (dd, J=9.1, 10.3 Hz, 1 H), 3.66-3.78

(m, 3 H), 3.93 (dd, J=3.6, 11.5 Hz, 1 H), 4.03 (s, 2 H), 7.06-7.11

(m, 4 H), 7.17-7.25 (m, 2 H), 7.33 (d, J=8.1 Hz, 1 H). MS (ESI)

m/z 431 (MþNa), 433 (Mþ2þNa). Anal. Calcd for (C₂₁H₂₅-

for (C₂₁H₂₅ClO₅S 0.7H₂O) C, 57.65; H, 6.08. Found C, 57.56;

ClO₄S 0.4H₂O) C, 60.61; H, 6.25. Found C, 60.55; H, 6.13.

3

H, 6.00.

(1S)-1,5-Anhydro-1-[4-chloro-3-[4-(propan-2-yl)benzyl]phenyl]-

1-thio-^D-glucitol (3m). Compound 3m (0.280 g, 26%) was pre-

pared as a colorless powder from 21m (2.01 g, 2.57 mmol)

according to the method described for the synthesis of 3f.

(1S)-1,5-Anhydro-1-[5-(4-ethoxybenzyl)-2-hydroxyphenyl]-1-

thio-^D-glucitol (3g). Compound 3g (0.142 g, 37%) was prepared

as a colorless powder from 21g (0.810 g, 0.945 mmol) according

to the method described for the synthesis of 3a. (Method A) mp

145.0-150.0 °C. ¹H NMR (600 MHz, MeOH-d₄) δ 1.37 (t,

J=7.0 Hz, 3 H), 2.94-3.05 (m, 1 H), 3.22-3.29 (m, 1 H), 3.58-

3.62 (m, 1 H), 3.69-3.88 (m, 4 H), 3.90-4.04 (m, 3 H), 4.33 (d,

J=10.6 Hz, 1 H), 6.71 (d, J=8.2 Hz, 1 H), 6.76-6.90 (m, 3 H),

7.03-7.15 (m, 3 H). MS (ESI) m/z 429 (MþNa), 405 (M-H).

1

(Method B) mp 72.0-83.0 °C. H NMR (300 MHz, MeOH-

d₄) δ 1.21 (d, J=6.8 Hz, 3 H), 2.76-2.92 (m, 1 H), 2.94-3.03 (m,

1 H), 3.19-3.27 (m, 1 H), 3.58 (dd, J = 9.2, 10.1 Hz, 1 H),

3.66-3.79 (m, 3 H), 3.94 (dd, J=3.6, 11.4 Hz, 1 H), 4.03 (s, 2 H),

7.06-7.15 (m, 4 H), 7.17-7.26 (m, 2 H), 7.30-7.35 (m, 1 H). MS

(ESI) m/z 445 (MþNa), 447 (Mþ2þNa), 421 (M-H). Anal.

3254 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

Calcd for (C₂₂H₂₇ClO₄S 0.5H₂O) C, 61.17; H, 6.53. Found C,

3

(1S)-1,5-Anhydro-1-[4-chloro-5-(4-ethylbenzyl)-2-methoxy-

phenyl]-1-thio-^D-glucitol (3s). Compound 3s (0.35 g, 24%) was

prepared as a colorless powder from 21s (2.65 g, 3.31 mmol)

according to the method described for the synthesis of 3o.

(Method C) ¹H NMR (600 MHz, MeOH-d₄) δ 1.19 (t, J=7.6

Hz, 3 H), 2.59 (q, J=7.6 Hz, 2 H), 2.93-3.00 (m, 1 H), 3.23 (t,

J=8.9 Hz, 1 H), 3.56 (dd, J=8.9, 10.3 Hz, 1 H), 3.67-3.87 (m,

5 H), 3.90-4.02 (m, 3 H), 4.22-4.37 (m, 1 H), 6.99 (s, 1 H),

7.03-7.11 (m, 4 H), 7.23 (s, 1 H). MS (ESI) m/z 461 (MþNa).

61.06; H, 6.44.

(1S)-1,5-Anhydro-1-[3-(4-tert-butylbenzyl)-4-chlorophenyl]-1-

thio-^D-glucitol (3n). Compound 3n (0.101 g, 18%) was prepared

as a colorless powder from 21n (1.04 g, 1.30 mmol) according to

the method described for the synthesis of 3f. (Method B) mp

94.0-100.0 °C. ¹H NMR (300 MHz, MeOH-d₄) δ 1.29 (s, 9 H),

2.90-3.05 (m, 1 H), 3.23 (t, J=8.7 Hz, 1 H), 3.58 (dd, J=8.7,

10.1 Hz, 1 H), 3.64-3.80 (m, 3 H), 3.94 (dd, J=3.5, 11.4 Hz,

1 H), 4.04 (s, 2 H), 7.10 (d, J=8.2 Hz, 2 H), 7.16-7.37 (m, 5 H).

MS (ESI) m/z 454 (MþNH₄), 456 (Mþ2þNH₄). Anal. Calcd for

(C₂₃H₂₉ClO₄S) C, 63.22; H, 6.69. Found C, 62.83; H, 6.64.

General Methods for Removal of the Benzyl Group (Method C).

(1S)-1,5-Anhydro-1-[4-chloro-3-[4-(methylsulfanyl)benzyl]phenyl]-

1-thio-^D-glucitol (3o). To a stirred mixture of 21o (3.00 g,

3.81 mmol) in trifluoroacetic acid (1.5 mL), dimethylsulfide

(9.0 mL), m-cresol (2.4 mL), and 1,2-ethanedithiol (0.6 mL)

was added trifluoromethane sulfonic acid (3.0 mL) at -15 °C.

After stirring for 20 min, the mixture was poured into a mixture

of ice and saturated aqueous NaHCO₃. The resulting mixture

was extracted with ethyl acetate, and the organic layer was

washed with saturated aqueous NaHCO₃twice, brine, and then

dried over MgSO₄and concentrated. The residue was purified

by silica gel column chromatography (CHCl₃/MeOH = from

100/1 to 10/1) to give a residue, which was recrystallized from

MeOH/water=2/1 to afford 3o (0.865 g, 53%) as a colorless

powder. ¹H NMR (600 MHz, MeOH-d₄) δ 2.43 (s, 3 H),

2.95-3.03 (m, 1 H), 3.23 (t, J=8.7 Hz, 1 H), 3.58 (dd, J=8.9,

10.3 Hz, 1 H), 3.68-3.77 (m, 3 H), 3.93 (dd, J=3.2, 11.5 Hz,

1 H), 4.00-4.09 (m, 2 H), 7.09-7.13 (m, 2 H), 7.15-7.19 (m, 2

H), 7.21 (dd, J=2.3, 8.3 Hz, 1 H), 7.23 (d, J=2.3 Hz, 1 H), 7.33

(d, J = 8.3 Hz, 1 H). MS (ESI) m/z 449 (MþNa), 451

(Mþ2þNa), 425 (M-H) 427 (Mþ2-H). Anal. Calcd for

Anal. Calcd for (C₂₂H₂₇ClO₅S H₂O) C, 56.70; H, 6.50. Found

3

C, 56.40; H, 6.45.

2,3,4,6-Tetra-O-acetyl-5-thio-^D-glucopyranose (5c). To a solu-

tion of 1,2,3,4,6-penta-O-acetyl-5-thio-^D-glucopyranose (5b)^41,46

(42.0 g, 103 mmol) in DMF (300 mL) was added a mixture of

methylhydrazine (5.76 g, 125 mmol), AcOH (7.50 g, 125 mmol),

and DMF (125 mL), and the resulting mixture was stirred at

room temperature for 2 h, followed by adding an additional

mixture of 0.967 g methylhydrazine, 1.26 g AcOH, and 21 mL

DMF and stirring for additional 1 h. The reaction mixture was

diluted with 400 mL of ethyl acetate and poured into a mixture of

brine (1.0 L) and ethyl acetate (1.0 L), followed by separation.

The organic layer was washed with 0.5 M hydrochroric acid (400

mL), brine (400 mL), then dried over MgSO₄and concentrated.

The residue was purified by silica gel column chromatography

(hexane/ethyl acetate = 65/35) to give 26.5 g of 5c for R/β

1

anomeric mixture (91.4/8.6) as a colorless crystal. H NMR of

R anomer (300 MHz, CDCl₃) δ 2.02, 2.04, 2.08, 2.08 (each s, each

3H), 3.70 (ddd, J=3.3, 5.0, 8.3 Hz, 1 H), 4.08 (dd, J=3.3, 12.0 Hz,

1 H), 4.38(dd, J=5.0, 12.0 Hz, 1 H), 5.15-5.19 (m, 2 H), 5.31(dd,

J=9.6, 10.9 Hz, 1 H), 5.55 (t, J=9.6 Hz, 1 H).

2,3,4,6-Tetra-O-benzyl-5-thio-^D-glucono-1,5-lactone (8). To a

stirred solution of 2,3,4,6-tetra-O-acetyl-5-thio-^D-glucopyra-

nose (5c) (2.0 g, 5.49 mmol) in 40 mL of CHCl₃were added

3,4-dihydro-2H-pyran (1.5 mL, 16.5 mmol) and p-toluenesulfo-

nic acid monohydrate (104 mg, 0.549 mmol) at room tempera-

ture, then the mixture was stirred at room temperature for 1 h.

The reaction mixture was poured into saturated aqueous NaH-

CO₃, and the layers were separated. The aqueous layer was

extracted with CHCl₃. The combined organic layers were

washed with brine, dried over MgSO₄, and concentrated. The

residue was purified by silica gel column chromatography

(hexane/ethyl acetate = 1/1) to give 2.56 g of tetrahydro-2H-

pyran-2-yl 2,3,4,6-tetra-O-acetyl-5-thio-^D-glucopyranose as a

pale yellow amorphous, which was taken up into 40 mL of

anhydrous methanol and treated with a 25 wt % solution of

sodium methoxide in methanol (0.11 mL, 0.55 mmol). The

resultant mixture was stirred at room temperature for 3 h. After

neutralization by the addition of dry ice, the mixture was

concentrated. The residue was taken up into DMF (20 mL)

and added to a suspension of sodium hydride (1.3 g, 32.9 mmol;

60% oil) in DMF (4 mL) dropwise at 0 °C. After stirring at room

temperature for 20 min, the mixture was cooled to 4 °C and

treated with benzyl bromide (5.6 g, 32.9 mmol). The resulting

mixture was stirred at room temperature for 12 h and quenched

by the addition of methanol (5 mL). After an additional 30 min

stirring at room temperature, the reaction mixture was poured

into ice water and extracted with ethyl acetate. The organic layer

was washed with brine, dried over MgSO₄, and concentrated.

The residue was purified by silica gel column chromatography

(hexane/ethyl acetate=6/1) to give compound 6 (3.36 g, 96% for

three steps). A mixture of 6 (3.30 g, 5.15 mmol) and pyridinium

p-toluenesulfonate (518 mg, 2.06 mmol) in ethanol (58 mL) was

stirred at 80 °C for 2 h. The resulting mixture was cooled to room

temperature and concentrated. The residue was dissolved in

ethyl acetate and washed with saturated aqueous NaHCO₃and

brine, dried over MgSO₄, and concentrated. The residue was

purified by silica gel column chromatography (hexane/ethyl

acetate=3/1) to give 2,3,4,6-tetra-O-benzyl-5-thio-^D-glucopyr-

anose (7) (2.89 g, quant) as a colorless crystal. ¹³C NMR (125

MHz, CDCl₃) δ 41.3, 67.8, 71.6, 73.0, 73.2, 75.6, 76.2, 81.9, 82.9,

(C₂₀H₂₃ClO₄S₂0.5H₂O) C, 55.09; H, 5.56. Found C, 54.90;

3

H, 5.55.

(1S)-1,5-Anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methyl-

phenyl]-1-thio-^D-glucitol (3p). Compound 3p (0.281 g, 81%) was

prepared as a colorless powder from 21p (0.630 g, 0.792 mmol)

according to the method described for the synthesis of 3a.

(Method A) mp 155.0-157.0 °C. ¹H NMR (600 MHz, MeOH-

d₄) δ 1.35 (t, J=6.9 Hz, 3 H), 2.17 (s, 3 H), 2.92-3.01 (m, 1 H),

3.24 (t, J=8.7 Hz, 1 H), 3.54-3.60 (m, 1 H), 3.72 (dd, J=6.4, 11.5,

Hz, 1 H), 3.81 (s, 3 H), 3.83 (s, 2 H), 3.94 (dd, J=3.7, 11.5 Hz,

1 H), 3.97 (q, J=6.9 Hz, 2 H), 4.33 (brs, 1 H), 6.77 (d, J=8.3 Hz,

2 H), 6.76 (s, 1 H), 6.99 (d, J=8.3 Hz, 2 H), 7.10 (s, 1 H). MS (ESI)

m/z 452 (MþNH₄). Anal. Calcd for (C₂₃H₃₀O₆S 0.5H₂O) C,

3

62.28; H, 7.06. Found C, 62.39; H, 7.10.

(1S)-1,5-Anhydro-1-[5-(4-ethylbenzyl)-2-methoxy-4-methyl-

phenyl]-1-thio-^D-glucitol (3q). Compound 3q (0.61 g, 68%) was

prepared as a colorless powder from 21q (1.68 g, 2.16 mmol)

according to the method described for the synthesis of 3a.

1

(Method A) H NMR (300 MHz, MeOH-d₄) δ 1.19 (t, J=7.6

Hz, 3 H), 2.17 (s, 3 H), 2.58 (q, J=7.6 Hz, 2 H), 2.91-3.02 (m, 1

H), 3.19-3.28 (m, 1 H), 3.58 (dd, J=9.2, 10.3 Hz, 1 H), 3.67-3.89

(m, 7 H), 3.94 (dd, J=3.7, 11.5 Hz, 1 H), 4.25-4.39 (m, 1 H), 6.76

(s, 1 H), 6.96-7.09 (m, 4 H), 7.12 (s, 1 H). MS (ESI) m/z

441(MþNa), 417 (M-H). Anal. Calcd for (C₂₃H₃₀O₅S 0.3H₂O)

3

C, 65.16; H, 7.27. Found C, 65.18; H, 7.32.

(1S)-1,5-Anhydro-1-[2-methoxy-4-methyl-5-[4-(propan-2-yl)-

benzyl]phenyl]-1-thio-^D-glucitol (3r). Compound 3r (0.31 g,

83%) was prepared as a colorless powder from 17r (0.69 g,

0.87 mmol) according to the method described for the synthesis

1

of 3a. (Method A) H NMR (600 MHz, MeOH-d₄) δ 1.21 (d,

J=6.9 Hz, 6 H), 2.17 (s, 3 H), 2.80-2.86 (m, 1 H), 2.94-2.99 (m,

1 H), 3.25 (m, 1 H), 3.58 (dd, J=9.2, 10.1 Hz, 1 H), 3.72 (dd,

J=6.6, 11.5 Hz, 1 H), 3.77-3.91 (m, 6 H), 3.94 (dd, J=3.7, 11.5

Hz, 1 H), 4.28-4.38 (m, 1H), 6.76 (s, 1 H), 7.00 (d, J=8.3 Hz, 2

H,) 7.08 (d, J=8.3 Hz, 2 H), 7.13 (s, 1 H). MS (ESI) m/z 455

(MþNa), 431 (M-H). Anal. Calcd for (C₂₄H₃₂O₅S 0.6H₂O) C,

3

65.01; H, 7.55. Found C, 65.07; H, 7.49.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3255

84.4, 127.5, 127.7, 127.8, 127.9, 128.0, 128.3, 128.4, 128.5, 137.8,

138.3, 138.8.

mol) and 10g (7.0 g, 0.0244 mol) as a colorless oil. ¹H NMR (300

MHz, CDCl₃) δ 1.40 (t, J=7.0 Hz, 3 H), 3.82 (s, 2 H), 4.00 (q,

J=7.0 Hz, 2 H), 5.12 (s, 2 H), 6.78-6.87 (m, 3 H), 6.98-7.10 (m,

3 H), 7.27-7.50 (m, 6 H).

A mixture of 7 (2.82 g, 5.07 mmol) and acetic anhydride

(39 mL) in DMSO (47 mL) was stirred at room temperature for

12 h. The reaction mixture was poured into ice water and

extracted with ethyl acetate. The organic layer was washed with

water, saturated aqueous NaHCO₃, and brine, then dried over

MgSO₄and concentrated. The residue was purified by silica gel

column chromatography (hexane/ethyl acetate=6/1) to give 8

2-Bromo-4-(4-ethoxybenzyl)-1-methoxybenzene (12h). Com-

pound 12h (12.2 g, 82%, 2 steps) was prepared according to

the method described for the synthesis of 12a using 9 (8.81 g,

0.0488 mol) and 10h (10.0 g, 0.0465 mol) as a colorless powder.

¹H NMR (300 MHz, CDCl₃) δ 1.40 (t, J=7.0 Hz, 3 H), 3.83 (s, 2

H), 3.86 (s, 3 H), 4.01 (q, J=7.0 Hz, 2 H), 6.78-6.85 (m, 3 H),

7.03-7.10 (m, 3 H), 7.35 (d, J=2.2 Hz, 1 H). MS (EI) m/z 320

(M^þ), 322 (Mþ2).

1

(2.3 g, 82%) as a colorless oil. H NMR (200 MHz, CDCl₃) δ

3.70 (d, J=4.8 Hz, 2 H), 3.86-4.02 (m, 2 H), 4.09-4.22 (m, 2 H),

4.40-4.68 (m, 7 H), 4.83 (d, J=11.4 Hz, 1 H), 7.12-7.41 (m, 20 H).

General Methods for Synthesis of Aglycon 12a-12s. 1-Bromo-

3-(4-ethoxybenzyl)benzene (12a). To a stirred solution of 4-bromo-

phenetole (9) (2.87 g, 0.0143 mol) in THF (30 mL) at -78 °C was

added dropwise a 2.6 M hexane solution of n-BuLi (5.8 mL, 0.0150

mol). After stirring for 30 min, a THF (15 mL) solution of

3-bromobenzaldehyde (10a) (2.65 g, 0.0143 mol) was added drop-

wise at -78 °C. After stirring for 15 min, the reaction mixture

was allowed to warm to room temperature, and then, saturated

aqueous NH₄Cl was added. The resulting mixture was extracted

with ethyl acetate twice, and the combined organic layers were

washed with brine, dried over MgSO₄, and concentrated. The

residue was purified by silica gel column chromatography (hexane/

ethyl acetate=from 7/1 to 5/1) to give 3.94 g of a colorless oil. This

was taken up into 11 mL of CHCl₃and 11 mL of CH₃CN. The

mixture was cooled to -60 °C and Et₃SiH (4.09 mL, 0.0256 mol)

1-Bromo-2,4-dimethoxy-5-(4-ethoxybenzyl)benzene (12i). Com-

pound 12i (1.80 g, 51%, 2 steps) was prepared according to the

method described for the synthesis of 12a using 9 (2.34 g, 0.0117

mol) and 10i (3.11 g, 0.0127 mol) as a colorless powder. ¹H

NMR (200 MHz, CDCl₃) δ 1.39 (t, J=7.0 Hz, 3 H), 3.80 (s, 2 H),

3.82 (s, 3 H), 3.88 (s, 3 H), 4.00 (q, J=7.0 Hz, 2 H), 6.47 (s, 1 H),

6.75-6.85 (m, 2 H), 7.02-7.12 (m, 2 H), 7.17 (s, 1 H). MS (EI)

m/z 350 (M^þ), 352 (Mþ2).

4-Bromo-1-chloro-2-(4-methoxybenzyl)benzene (12j). Com-

pound 12j (3.88 g, 74%) was prepared according to the method

described for the synthesis of 12p using 5-bromo-2-chloroben-

zoic acid (16) (4.00 g, 0.0170 mol) and anisole (1.88 mL, 0.0173

mol) as a colorless gum. ¹H NMR (300 MHz, CDCl₃) δ 3.80 (s, 3

H), 3.99 (s, 2 H), 6.82-6.89 (m, 2 H), 7.06-7.13 (m, 2 H), 7.19-

7.30 (m, 3 H).

4-Bromo-1-chloro-2-(4-methylbenzyl)benzene (12k). Compound

12k (2.86 g, 57%) was prepared according to the method

described for the synthesis of 12p using 16 (5.00 g, 0.0212 mol)

and toluene (2.48 mL, 0.0234 mol) as a colorless oil. ¹H NMR

(200 MHz, CDCl₃) δ 2.33 (s, 3 H), 4.02 (s, 2 H), 7.03-7.16 (m,

4 H), 7.18-7.32 (m, 3 H). MS (EI) m/z 294 (M^þ), 296 (Mþ2).

4-Bromo-1-chloro-2-(4-ethylbenzyl)benzene (12l). Compound

12l (2.98 g, 47%) was prepared according to the method

described for the synthesis of 12p using 16 (5.00 g, 0.0212 mol)

and then BF₃Et₂O (1.47 mL, 0.0116 mol) were added sequen-

3

tially. After stirring for 1 h, the reaction mixture was allowed to

warm to room temperature, and then, saturated aqueous Na₂CO₃

was added. The resulting mixture was extracted with CHCl₃, and

the organic layer was washed with brine, dried over MgSO₄, and

concentrated. The residue was purified by silica gel column

chromatography (hexane/ethyl acetate=10/1) to give 12a (2.84

g, 68%, 2 steps) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ

1.40 (t, J=7.0 Hz, 3 H), 3.88 (s, 2 H), 4.01 (q, J=7.0 Hz, 2 H), 6.83

(d, J=8.9 Hz, 2 H), 7.07 (d, J=8.9 Hz, 2 H), 7.09-7.18 (m, 2 H),

7.29-7.34 (m, 2 H).

1

and ethylbenzene (2.86 mL, 0.0234 mol) as a colorless oil. H

NMR (200 MHz, CDCl₃) δ 1.23 (t, J=7.7 Hz, 3 H), 2.63 (q,

J=7.7 Hz, 2 H), 4.02 (s, 2 H), 7.04-7.18 (m, 4 H), 7.18-7.32 (m,

3 H). MS (EI) m/z 308 (M^þ), 310 (Mþ2).

1-(Benzyloxy)-4-bromo-2-(4-ethoxybenzyl)benzene (12b).

Compound 12b (5.88 g, 61%, 2 steps) was prepared according

to the method described for the synthesis of 12a using 9 (5.12 g,

4-Bromo-1-chloro-2-[4-(propan-2-yl)benzyl]benzene (12m).

Compound 12m (3.28 g, 48%) was prepared according to the

method described for the synthesis of 12p using 16 (5.00 g,

0.0212 mol) and cumene (3.24 mL, 0.0234 mmol). ¹H NMR (200

MHz, CDCl₃) δ 1.24 (d, J=7.0 Hz, 6 H), 2.80-2.97 (m, 1 H),

4.02 (s, 2 H), 7.06-7.32 (m, 7 H).

1

0.0255 mol) and 10b (10.0 g, 0.0345 mol) as a colorless oil. H

NMR (200 MHz, CDCl₃) δ 1.40 (t, J=6.8 Hz, 3 H), 3.90 (s, 2 H),

4.01 (q, J = 6.8 Hz, 2 H), 5.03 (s, 2 H), 6.72-6.85 (m, 3 H),

7.02-7.13 (m, 2 H), 7.15-7.43 (m, 7 H).

4-Bromo-2-(4-ethoxybenzyl)-1-methoxybenzene (12c). Com-

pound 12c (3.12 g, 41%, 2 steps) was prepared according to

the method described for the synthesis of 12a using 9 (4.99 g,

4-Bromo-2-(4-tert-butylbenzyl)-1-chlorobenzene (12n). Com-

pound 12n (4.44 g, 62%) was prepared according to the method

described for the synthesis of 12p using 16 (5.00 g, 0.0212 mol)

1

0.0248 mol) and 10c (5.08 g, 0.0236 mol) as a colorless oil. H

1

and t-butylbenzene (2.90 g, 0.0216 mol) as a colorless oil. H

NMR (200 MHz, CDCl₃) δ 1.31 (s, 9 H), 4.03 (s, 2 H), 7.11 (d,

J=7.9 Hz, 2 H), 7.22-7.37 (m, 5 H).

NMR (300 MHz, CDCl₃) δ 1.40 (t, J=7.0 Hz, 3 H), 3.79 (s, 3 H),

3.85 (s, 2 H), 4.01 (q, J=7.0 Hz, 2 H), 6.72 (d, J=8.6 Hz, 1 H),

6.81 (d, J=8.7 Hz, 2 H), 7.09 (d, J=8.7 Hz, 1 H), 7.13 (d, J=2.5

Hz, 1 H), 7.27 (dd, J=2.5, 8.6 Hz, 1 H).

4-Bromo-1-chloro-2-[4-(methylsulfanyl)benzyl]benzene (12o).

Compound 12o (2.83 g, 41%) was prepared according to the

method described for the synthesis of 12p using 16 (5.00 g,

4-Bromo-2-(4-ethoxybenzyl)-1-fluorobenzene (12d). Compound

12d (2.46 g, 33%, 2 steps) was prepared according to the method

described for the synthesis of 12a using 9 (10.4 g, 0.0517 mol)

and 10d (10.0 g, 0.0493 mol) as a colorless amorphous substance.

¹H NMR (200 MHz, CDCl₃) δ 1.40 (t, J=7.0 Hz, 3 H), 3.88 (s,

2 H), 4.01 (q, J=7.0 Hz, 2 H), 6.79-6.96 (m, 3 H), 7.05-7.16 (m,

2 H), 7.19-7.32 (m, 2 H). MS (EI) m/z 309 (M^þ), 311 (Mþ2).

4-Bromo-2-(4-ethoxybenzyl)-1-methylbenzene (12e). Com-

pound 12e (2.34 g, 96%) was prepared according to the method

described for the synthesis of 12p using 15 (4.29 g, 0.0199 mol)

and phenetole (2.52 g, 0.0209 mol) as a colorless oil. ¹H NMR

(300 MHz, CDCl₃) δ 1.40 (t, J=7.0 Hz, 3 H), 2.18 (s, 3 H), 3.86

(s, 2 H), 4.00 (q, J=7.0 Hz, 2 H), 6.76-6.87 (m, 2 H), 6.94-7.07

(m, 3 H), 7.17-7.30 (m, 2 H). MS (EI) m/z 304 (M^þ), 306 (Mþ2).

1-(Benzyloxy)-2-bromo-4-(4-ethoxybenzyl)benzene (12g). Com-

pound 12g (8.80 g, 86%, 2 steps) was prepared according to the

method described for the synthesis of 12a using 9 (7.30 g, 0.0366

1

0.0212 mol) and thioanisole (2.60 mL, 0.0220 mol). H NMR

(300 MHz, CDCl₃) δ 2.47 (s, 3 H), 4.01 (s, 2 H), 7.06-7.14 (m,

2 H), 7.17-7.32 (m, 5 H).

1-Bromo-5-(4-ethoxybenzyl)-2-methoxy-4-methylbenzene (12p).

To a stirred suspension of 5-bromo-4-methoxy-2-methylbenzoic

acid (14) (4.93 g, 0.0201 mol) in CHCl₃(15 mL) was added oxalyl

chloride (1.79 mL, 0.0209 mol) and DMF (0.02 mL). The

resulting mixture was stirred for 2 h. An additional amount of

oxalyl chloride (0.5 mL, 5.83 mmol) was added, and the mixture

was stirred for 15 h. The mixture was concentrated, and the

residual colorless solid was dissolved in CHCl₃(30 mL). To this

solution were added phenetole (2.50 g, 0.0205 mol) and then

AlCl₃(2.71 g, 0.0203 mol) portionwise so that the temperature

did not exceed -4 °C. After being stirred at 5 °C for 3 h, the

mixture was poured into ice water and extracted with CHCl₃

3256 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

three times. The combined organic layers were washed with 1 M

hydrochloric acid, water, and brine, then dried over MgSO₄and

concentrated. The residual solid was crystallized from a 4:1

mixture of hexane and ethyl acetate to give 5.75 g (82%) of

(5-bromo-4-methoxy-2-methylphenyl)(4-ethoxyphenyl)methan-

one (17p) as a colorless crystal. ¹H NMR (200 MHz, CDCl₃) δ

1.45 (t, J=7.0 Hz, 3 H), 2.33 (s, 3 H), 3.95 (s, 3 H), 4.11 (q, J=7.0

Hz, 2 H), 6.79 (s, 1 H), 6.87-6.97 (m, 2 H), 7.51 (s, 1 H),

7.69-7.80 (m, 2 H).

mol) and Fe (0.20 g, 3.61 mmol) in CHCl₃(10 mL) was added

bromine (3.87 mL, 0.076 mol) dropwise at 5 °C, and the resulting

mixture was allowed to warm to room temperature and stirred

overnight. The mixture was diluted with CHCl₃(600 mL) and

washed with 200 mL of 10% sodium hydrogen sulfate twice,

then with brine, dried over MgSO₄, and concentrated to give a

pale yellow solid which was recrystallized from methanol twice

to give 14 (4.96 g, 34%). ¹H NMR (300 MHz, DMSO-d₆) δ 2.53

(s, 3 H), 3.90 (s, 3 H), 7.05 (s, 1 H), 7.99 (s, 1 H), 12.82 (brs, 1 H).

MS (ESI) m/z 243, 245 (M-H).

To a stirred solution of 17p (5.75 g, 0.0165 mol) in 30 mL of

CHCl₃and 30 mL of CH₃CN at 4 °C were added Et₃SiH

1-Bromo-4-chloro-2-methoxybenzene (19). To a suspension

of 2-bromo-5-chlorophenol (18) (34.3 g, 0.165 mol), K₂CO₃

(22.8 g, 0.165 mol), and n-Bu₄NI (0.60 g, 1.65 mmol) in DMF

(95 mL) was added iodomethane (12.9 mL, 0.206 mol). After

stirring the mixture for 3 h, ice water was added, and the

resulting mixture was extracted with ethyl acetate twice. The

combined organic layers were washed with brine, dried over

MgSO₄, and concentrated. The residue was purified by silica gel

column chromatography (hexane/ethyl acetate=95/5) to give 19

(33.3 g, 91%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ

3.89 (s, 3H), 6.71-6.94 (m, 2H), 7.45 (d, J=8.4 Hz, 1H).

General Methods for Coupling Reaction with Grignard

Reagents. 2,3,4,6-Tetra-O-benzyl-1-C-[3-(4-ethoxybenzyl)phenyl]-

5-thio-^D-glucopyranose (20a). A stirred suspension of magnesium

(137 mg, 5.63 mmol) and 1-bromo-3-(4-ethoxybenzyl)benzene

(12a) (1.38 g, 4.73 mmol) in THF (5 mL) was treated with

1,2-dibromoethane (0.05 mL) and heated to reflux for 2 h. After

cooling to room temperature, the mixture was stirred for 4 h. To

the resulting Grignard reagent at 0 °C was added a solution of 8

(1.25 g, 2.25 mmol) in 5.0 mL of THF. The reaction mixture was

stirred at room temperature for 1 h and poured into saturated

aqueous NH₄Cl. The resulting mixture was extracted with ethyl

acetate twice, and the combined organic layers were washed with

brine, dried over MgSO₄, and concentrated. The residue was

purified by silica gel column chromatography (hexane/ethyl acetate

=4/1) to give 20a (0.580 g, 34%) as a colorless oil. ¹H NMR (300

MHz, CDCl₃) δ 1.38 (t, J=7.0 Hz, 3 H), 3.04 (s, 1 H), 3.48-3.57

(m, 1 H), 3.64 (dd, J=2.6, 9.8 Hz, 1 H), 3.74 (d, J=10.1 Hz, 1 H),

3.88-4.16 (m, 8 H), 4.41 (d, J=10.1 Hz, 1 H), 4.52 (s, 2 H), 4.65 (d,

J = 10.7 Hz, 1 H), 4.79-4.96 (m, 3 H), 6.67-6.79 (m, 4 H),

7.00-7.35 (m, 22 H), 7.47-7.55 (m, 2 H). MS (ESI) m/z 789

(MþNH₄).

(6.57 mL, 0.0411 mol), then BF₃Et₂O (3.14 mL, 0.0248 mol),

3

and the resulting mixture was warmed to room temperature and

stirred for 12 h. The mixture was poured into saturated aqueous

NaHCO₃and extracted with CHCl₃twice. The combined

organic layers were washed with brine, dried over MgSO₄, and

concentrated. The residue was purified by silica gel column

chromatography (hexane/ethyl acetate=15/1) to give a colorless

oily compound 12p (5.50 g, 99%). ¹H NMR (300 MHz, CDCl₃)

δ 1.40 (t, J=7.0 Hz, 3 H), 2.19 (s, 3 H), 3.82 (s, 2 H), 3.87 (s, 3 H),

4.00 (q, J=7.0 Hz, 2 H), 6.71 (s, 1 H), 6.77-6.83 (m, 2 H), 6.95-

7.04 (m, 2 H), 7.24 (s, 1 H). MS (EI) m/z 335 (M^þ), 337 (Mþ2).

1-Bromo-5-(4-ethylbenzyl)-2-methoxy-4-methylbenzene (12q).

Compound 17q (90.43 g, 0.27 mol), which was prepared accord-

ing to the method described for the synthesis of 17p using 14 and

ethylbenzene, was dissolved in THF (450 mL) and cooled to

4 °C. To the solution was added NaBH₄(32.41 g, 0.82 mol), then

AlCl₃(36.17 g, 0.271 mmol) was added portionwise under a

nitrogen atmosphere. The reaction mixture was stirred for 3 h at

50 °C, cooled to 4 °C, then poured into ice water cautiously to

avoid a rise in temperature. The resulting mixture was extracted

with ethyl acetate. The organic layer was washed with 1 M

hydrochloric acid, water, saturated aqueous NaHCO₃, and

finally brine, and then dried over MgSO₄and concentrated.

The residue was purified by silica gel column chromatography

(hexane/ethyl acetate=1/1) to give 12q (77.0 g, 90%). ¹H NMR

(300 MHz, CDCl₃) δ 1.22 (t, J=7.6 Hz, 3 H), 2.20 (s, 3 H), 2.61

(q, J=7.6 Hz, 2 H), 3.85 (s, 2 H), 3.87 (s, 3 H), 6.71 (s, 1 H),

6.97-7.14 (m, 4 H), 7.27 (s, 1 H). MS (EI) 318 (M^þ).

1-Bromo-2-methoxy-4-methyl-5-[4-(propan-2-yl)benzyl]benzene

(12r). Compound 17r (4.53 g, 56%) was prepared according to

the method described for the synthesis of 17p using 4-methoxy-

2-methylbenzoyl chloride prepared from 13 (5.00 g, 30.08 mmol)

and cumene (4.60 mL, 33.08 mmol). Compound 17r (4.53 g, 16.9

mmol) was reduced with Et₃SiH (8.10 mL, 0.0506 mol) and

2,3,4,6-Tetra-O-benzyl-1-C-[4-(benzyloxy)-3-(4-ethoxybenzyl)-

phenyl]-5-thio-^D-glucopyranose (20b). Compound 20b (1.00 g,

42%) was prepared according to the method described for the

synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12b (2.15 g, 5.41

BF₃Et₂O (3.14 mL, 0.0338 mol) according to the reduction

3

1

method described for the synthesis of 12p to give 1-(4-iso-

propylbenzyl)-4-methoxy-2-methylbenzene (3.79 g) as a pale

yellow oil. To an AcOH (30 mL) solution of 1-(4-isopropyl-

benzyl)-4-methoxy-2-methylbenzene (3.79 g, 14.89 mmol) at

4 °C was added bromine (0.76 mL, 14.89 mmol). The mixture

was stirred at 100 °C for 2 h and allowed to cool to room

temperature, then was diluted with ethyl acetate. The resulting

solution was washed with saturated aqueous NaHCO₃, brine,

and then dried over MgSO₄and concentrated. The residue was

purified by silica gel column chromatography (hexane/ethyl

mmol) as a yellow oil. H NMR (300 MHz, CDCl₃) δ 1.38 (t,

J=7.0 Hz, 3 H), 2.97 (s, 1 H), 3.46-3.55 (m, 1 H), 3.63 (dd,

J=2.7, 9.6 Hz, 1 H), 3.80 (d, J=10.3 Hz, 1 H), 3.86-4.11 (m, 8

H), 4.42 (d, J=10.3 Hz, 1 H), 4.52 (s, 2 H), 4.64 (d, J=10.4 Hz,

1 H), 4.79-4.95 (m, 3 H), 5.07 (s, 2 H), 6.68-6.77 (m, 4 H), 6.90

(d, J=9.0 Hz, 1 H), 7.02-7.35 (m, 25 H), 7.42-7.48 (m, 2 H).

MS (ESI) m/z 895 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[3-(4-ethoxybenzyl)-4-methoxy-

phenyl]-5-thio-^D-glucopyranose (20c). Compound 20c (1.59 g,

74%) was prepared according to the method described for the

synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12c (1.73 g, 5.40

mmol) as a colorless amorphous. ¹H NMR (600 MHz, CDCl₃)

δ 1.37 (t, J=6.9 Hz, 3 H), 2.97 (s, 1 H), 3.50 (s, 1 H), 3.63 (dd,

J=2.8, 10.1 Hz, 1 H), 3.78-3.88 (m, 5 H), 3.89-3.98 (m, 5 H),

4.01-4.10 (m, 2 H), 4.41 (d, J=10.1 Hz, 1 H), 4.52 (s, 2 H), 4.64

(d, J=10.6 Hz, 1 H), 4.80-4.94 (m, 3 H), 6.71-6.76 (m, 4 H),

6.84 (d, J=8.7 Hz, 1 H), 7.03-7.20 (m, 7 H), 7.23-7.34 (m, 13

H), 7.41 (d, J=2.3 Hz, 1 H), 7.45-7.49 (m, 1 H). MS (ESI) m/z

819 (MþNa).

1

acetate = 15/1) to give 12r (4.21 g, 80%) as a yellow oil. H

NMR (300 MHz, CDCl₃) δ 1.23 (d, J=7.0 Hz, 6 H), 2.21 (s, 3

H), 2.81-2.92 (m, 1 H), 3.85 (brs, 2 H), 3.87 (s, 3 H), 6.71 (s, 1 H),

6.98-7.06 (m, 2 H), 7.10-7.16 (m, 2 H), 7.28 (s, 1 H). MS (EI)

322 (M^þ), 334 (Mþ2).

1-Bromo-4-chloro-5-(4-ethylbenzyl)-2-methoxybenzene (12s).

Compound 12s (4.46 g, 27%) was prepared according to the

method described for the synthesis of 12p using 4-ethylbenzoic

acid (6.78 g, 45.1 mmol) and 19 (10.0 g, 45.1 mmol). ¹H NMR

(300 MHz, CDCl₃) δ 1.22 (t, J=7.6 Hz, 3 H), 2.62 (q, J=7.6 Hz,

2 H), 3.87 (s, 3 H), 3.97 (s, 2 H), 6.91 (s, 1 H), 7.04-7.18 (m, 4 H),

7.32 (s, 1 H). MS (EI) 338 (M^þ), 340 (Mþ2), 342 (Mþ4).

5-Bromo-4-methoxy-2-methylbenzoic acid (14). To a stirred

suspension of 4-methoxy-2-methylbenzoic acid (13) (10 g, 0.060

2,3,4,6-Tetra-O-benzyl-1-C-[3-(4-ethoxybenzyl)-4-fluorophenyl]-

5-thio-^D-glucopyranose (20d). Compound 20d (1.83 g, 86%) was

prepared according to the method described for the synthesis of

20a using 8 (1.50 g, 2.70 mmol) and 12d (1.67 g, 5.41 mmol) as a

1

colorless amorphous substance. H NMR (300 MHz, CDCl₃)

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3257

δ 1.37 (t, J=7.0 Hz, 3 H), 3.00 (s, 1 H), 3.46-3.53 (m, 1 H), 3.62

(dd, J=2.8, 9.8 Hz, 1 H), 3.79 (d, J=10.3 Hz, 1 H), 3.83-4.09

(m, 8 H), 4.45 (d, J=10.0 Hz, 1 H), 4.51 (s, 2 H), 4.64 (d, J=10.7

Hz, 1 H), 4.79-4.94 (m, 3 H), 6.68-6.78 (m, 4 H), 6.97-7.20 (m,

8 H), 7.25-7.34 (m, 13 H), 7.43-7.50 (m, 2 H). MS (ESI) m/z

807 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-3-(4-ethylbenzyl)phenyl]-

5-thio-^D-glucopyranose (20l). Compound 20l (3.03 g, 80%) was

prepared according to the method described for the synthesis of

20a using 8 (2.67 g, 4.81 mmol) and 12l (2.98 g, 9.62 mmol) as

a colorless amorphous. ¹H NMR (300 MHz, CDCl₃) δ 1.19 (t,

J=7.7 Hz, 3 H), 2.58 (q, J=7.7 Hz, 2 H), 3.00 (s, 1 H), 3.45-3.53

(m, 1 H), 3.62 (dd, J=2.9, 9.9 Hz, 1 H), 3.79-4.17 (m, 7 H),

4.43-4.53 (m, 3 H), 4.64 (d, J=10.6 Hz, 1 H), 4.78-4.95 (m,

3 H), 6.68-6.77 (m, 2 H), 7.01-7.54 (m, 25 H). MS (ESI) m/z

807 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[3-(4-ethoxybenzyl)-4-methyl-

phenyl]-5-thio-^D-glucopyranose (20e). Compound 20e (1.53 g,

50%) was prepared according to the method described for the

synthesis of 20a using 8 (2.20 g, 3.93 mmol) and 12e (1.20 g, 3.93

mmol) as a colorless amorphous. ¹H NMR (300 MHz, CDCl₃) δ

1.37 (t, J=7.0 Hz, 3 H), 2.23 (s, 3 H), 3.02 (s, 1 H), 3.48-3.57 (m,

1 H), 3.64 (dd, J=2.7, 9.9 Hz, 1 H), 3.81-4.17 (m, 9 H), 4.44 (d,

J=10.1 Hz, 1 H), 4.52 (s, 2 H), 4.62-4.72 (m, 1 H), 4.78-4.96

(m, 3 H), 6.66-6.82 (m, 4 H), 6.95 (d, J=8.7 Hz, 2 H), 7.08-7.38

(m, 19 H), 7.41-7.47 (m, 2 H). MS (ESI) m/z 798 (MþNH₄), 779

(M-H).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-3-[4-(propan-2-yl)benzyl]-

phenyl]-5-thio-^D-glucopyranose (20m). Compound 20m (2.65 g,

65%) was prepared according to the method described for the

synthesis of 20a using 8 (2.80 g, 5.07 mmol) and 12m (3.28 g,

10.13 mmol) as a yellow powder. ¹H NMR (300 MHz, CDCl₃)

δ 1.19 (d, J=6.8 Hz, 6 H), 2.77-2.89 (m, 1 H), 3.01 (s, 1 H),

3.45-3.54 (m, 1 H), 3.62 (dd, J=2.7, 9.9 Hz, 1 H), 3.79-4.20 (m,

7 H), 4.46 (d, J=10.3 Hz, 1 H), 4.51 (s, 2 H), 4.64 (d, J=10.7 Hz,

1 H), 4.79-4.95 (m, 3 H), 6.69-6.76 (m, 2 H), 7.05-7.21 (m,

8 H), 7.24-7.37 (m, 15 H), 7.39-7.46 (m, 1 H), 7.52 (d, J=2.3

Hz, 1 H). MS (ESI) m/z 821 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-3-(4-ethoxybenzyl)-

phenyl]-5-thio-^D-glucopyranose (20f). Compound 20f (3.80 g,

89%) was prepared according to the method described for the

synthesis of 20a using 8 (3.00 g, 5.40 mmol) and 12f (3.52 g, 10.8

mmol) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.38 (t,

J=7.0 Hz, 3 H), 3.01 (s, 1 H), 3.45-3.54 (m, 1 H), 3.62 (dd, J=

2.8, 10.0 Hz, 1 H), 3.77-4.13 (m, 9 H), 4.46 (d, J=10.3 Hz, 1 H),

4.51 (s, 2 H), 4.64 (d, J=10.7 Hz, 1 H), 4.78-4.94 (m, 3 H),

6.68-6.79 (m, 4 H), 7.00-7.52 (m, 23 H). MS (ESI) m/z 823

(MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[3-(4-tert-butylbenzyl)-4-chloro-

phenyl]-5-thio-^D-glucopyranose (20n). Compound 20n (1.73 g,

79%) was prepared according to the method described for the

synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12n (1.82 g, 5.40

mmol) as a colorless gum. ¹H NMR (300 MHz, CDCl₃) δ 1.26

(s, 9 H), 3.01 (s, 1 H), 3.45-3.55 (m, 1 H), 3.62 (dd, J=2.7, 9.7

Hz, 1 H), 3.80-4.18 (m, 7 H), 4.42-4.54 (m, 3 H), 4.64 (d, J=

10.7 Hz, 1 H), 4.78-4.96 (m, 3 H), 6.68-6.76 (m, 2 H), 7.04-

7.38 (m, 23 H), 7.40-7.56 (m, 2 H). MS (ESI) m/z 835 (MþNa),

811 (M-H).

2,3,4,6-Tetra-O-benzyl-1-C-[2-(benzyloxy)-5-(4-ethoxybenzyl)-

phenyl]-5-thio-^D-glucopyranose (20g). Compound 20g (1.48 g,

59%) was prepared according to the method described for the

synthesis of 20a using 8 (1.60 g, 2.88 mmol) and 12g (2.29 g, 5.76

mmol) as a pale yellow powder. ¹H NMR (300 MHz, CDCl₃) δ

1.38 (t, J=6.9 Hz, 3 H), 3.52-3.70 (m, 2 H), 3.79-4.08 (m, 9 H),

4.51 (s, 2 H), 4.59-4.73 (m, 3 H), 4.80-4.93 (m, 3 H), 5.09 (s,

2 H), 6.64-7.40 (m, 32 H). MS (ESI) m/z 895 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[5-(4-ethoxybenzyl)-2-methoxy-

phenyl]-5-thio-^D-glucopyranose (20h). Compound 20h (1.71 g,

79%) was prepared according to the method described for the

synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12h (1.74 g, 5.41

mmol) as a pale yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 1.37

(t, J=7.1 Hz, 3 H), 3.54-3.70 (m, 2 H), 3.78-4.09 (m, 12 H),

4.51 (s, 2 H), 4.58-4.72 (m, 2 H), 4.81-4.98 (m, 3 H), 6.67-6.90

(m, 5 H), 6.95-7.35 (m, 22 H). MS (ESI) m/z 819 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[5-(4-ethoxybenzyl)-2,4-dimethoxy-

phenyl]-5-thio-^D-glucopyranose (20i). Compound 20i (1.76 g,

79%) was prepared according to the method described for the

synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12i (1.80 g, 5.12

mmol) as a colorless amorphous. ¹H NMR (300 MHz, CDCl₃) δ

1.36 (t, J=7.0 Hz, 3 H), 3.50-3.70 (m, 2 H), 3.74-4.11 (m, 15

H), 4.51 (s, 2 H), 4.57-4.68 (m, 2 H), 4.77-4.97 (m, 3 H), 6.44 (s,

1 H), 6.64-6.80 (m, 4 H), 6.98-7.36 (m, 21 H). MS (ESI) m/z

849 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-3-[4-(methylsulfanyl)-

benzyl]phenyl-5-thio-^D-glucopyranose (20o). Compound 20o

(2.00 g, 92%) was prepared according to the method described

for the synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12o (1.77

1

g, 5.40 mmol) as a colorless amorphous. H NMR (300 MHz,

CDCl₃) δ 2.42 (s, 3 H), 3.01 (s, 1 H), 3.45-3.55 (m, 1 H), 3.62

(dd, J=2.7, 9.9 Hz, 1 H), 3.79-4.14 (m, 7 H), 4.43-4.55 (m,

3 H), 4.64 (d, J=10.7 Hz, 1 H), 4.77-4.96 (m, 3 H), 6.69-6.76

(m, 2 H), 7.01-7.38 (m, 23 H), 7.40-7.53 (m, 2 H). MS (ESI) m/z

825 (MþNa), 801 (M-H).

2,3,4,6-Tetra-O-benzyl-1-C-[5-(4-ethoxybenzyl)-2-methoxy-

4-methylphenyl]-5-thio-^D-glucopyranose (20p). Compound 20p

(2.20 g, 75%) was prepared according to the method described

for the synthesis of 20a using 8 (2.00 g, 3.61 mmol) and 12p (2.71

g, 8.08 mmol) as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 1.37

(t, J=6.9 Hz, 3 H), 2.21 (s, 3 H), 3.51-4.20 (m, 14 H), 4.51 (s,

2 H), 4.60-4.75 (m, 2 H), 4.78-4.99 (m, 3 H), 6.59-7.43 (m, 26

H). MS (ESI) m/z 812 (MþNH₄).

2,3,4,6-Tetra-O-benzyl-1-C-[5-(4-ethylbenzyl)-2-methoxy-4-

methylphenyl]-5-thio-^D-glucopyranose (20q). Compound 20q

(2.25 g, 48%) was prepared according to the method described

for the synthesis of 20a using 8 (3.25 g, 5.87 mmol) and 12q (3.75

g, 11.74 mmol) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ

1.17 (t, J=7.7 Hz, 3 H), 2.22 (s, 3 H), 2.49-2.62 (m, 2 H), 3.53-

4.16 (m, 12 H), 4.51 (s, 2 H), 4.58-4.74 (m, 2 H), 4.80-4.98 (m,

3 H), 6.71-6.86 (m, 2 H), 6.89-7.03 (m, 3 H), 7.06-7.41 (m, 21

H). MS (ESI) m/z 817 (MþNa), 793 (M-H).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-3-(4-methoxybenzyl)-

phenyl]-5-thio-^D-glucopyranose (20j). Compound 20j (1.93 g,

91%) was prepared according to the method described for the

synthesis of 20a using 8 (1.50 g, 2.70 mmol) and 12j (1.68 g, 5.40

mmol) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 3.00 (s,

1 H), 3.45-3.54 (m, 1 H), 3.62 (dd, J=2.9, 10.0 Hz, 1 H), 3.74 (s,

3 H), 3.80-4.12 (m, 7 H), 4.42-4.54 (m, 3 H), 4.64 (d, J=10.9

Hz, 1 H), 4.77-4.96 (m, 3 H), 6.68-6.80 (m, 4 H), 7.03-7.37 (m,

21 H), 7.39-7.51 (m, 2 H). MS (ESI) m/z 809 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-3-(4-methylbenzyl)-

phenyl]-5-thio-^D-glucopyranose (20k). Compound 20k (2.50 g,

67%) was prepared according to the method described for the

synthesis of 20a using 8 (2.68 g, 4.84 mmol) and 12k (2.86 g, 9.68

mmol) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 2.28 (s,

3 H), 3.00 (s, 1 H), 3.45-3.54 (m, 1 H), 3.62 (dd, J=2.6, 9.9 Hz,

1 H), 3.79-4.15 (m, 7 H), 4.43-4.53 (m, 3 H), 4.64 (d, J=10.7

Hz, 1 H), 4.76-4.96 (m, 3 H), 6.69-6.77 (m, 2 H), 6.98-7.54 (m,

25 H). MS (ESI) m/z 793 (MþNa).

2,3,4,6-Tetra-O-benzyl-1-C-[2-methoxy-4-methyl-5-[4-(propan-

2-yl)benzyl]phenyl]-5-thio-^D-glucopyranose (20r). Compound

20r (1.14 g, 23%) was prepared according to the method des-

cribed for the synthesis of 20a using 8 (3.30 g, 5.94 mmol) and

1

12r (4.00 g, 12.0 mmol) as a yellow amorphous substance. H

NMR (300 MHz, CDCl₃) δ 1.18 (d, J=6.7 Hz, 6 H), 2.23 (s,

3 H), 2.76-2.88 (m, 1 H), 3.52-4.16 (m, 12 H), 4.51 (s, 2 H),

4.57-4.69 (m, 2 H), 4.81-4.96 (m, 3 H), 6.71-6.84 (m, 2 H),

6.90-7.38 (m, 24 H). MS (ESI) m/z 831 (MþNa), 807 (M-H).

2,3,4,6-Tetra-O-benzyl-1-C-[4-chloro-5-(4-ethylbenzyl)-2-

methoxyphenyl]-5-thio-^D-glucopyranose (20s). Compound 20s

(3.12 g, 74%) was prepared according to the method described

3258 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

for the synthesis of 20a using 8 (2.86 g, 5.15 mmol) and 12s

(3.50 g, 10.30 mmol) as a pale yellow crystal. H NMR (300

MHz, CDCl₃) δ 1.18 (t, J=7.5 Hz, 3 H), 2.57 (q, J=7.5 Hz, 2 H),

3.51-3.71 (m, 2 H), 3.83 (s, 3 H), 3.88-4.09 (m, 6 H), 4.50 (s,

2 H), 4.58-4.70 (m, 2 H), 4.79-4.96 (m, 4 H), 6.75 (d, J=7.0 Hz,

2 H), 6.85-7.39 (m, 24 H).

General Methods for Reduction of Anomeric Hydroxyl

Group. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-(4-ethoxy-

benzyl)phenyl]-1-thio-^D-glucitol (21a). To an CH₃CN (6 mL)

solution of 2,3,4,6-tetra-O-benzyl-1-C-[3-(4-ethoxybenzyl)phenyl]-

5-thio-^D-glucopyranose (20a) (570 mg, 0.743 mmol) was added

acetate. ¹H NMR (300 MHz, CDCl₃) δ 1.38 (t, J=6.9 Hz, 3 H),

3.00-3.15 (m, 1 H), 3.50 (t, J=8.9 Hz, 1 H), 3.70 (dd, J=3.0,

9.9 Hz, 1 H), 3.75-3.80 (m, 2 H), 3.82-3.99 (m, 6 H), 4.06 (d,

J=15.6 Hz, 1 H) 4.47-4.53 (m, 3 H), 4.59 (d, J=10.6 Hz, 1 H),

4.82-4.88 (m, 2 H), 4.89 (d, J=10.6 Hz, 1 H), 6.70 (d, J=6.9 Hz,

2 H), 6.74 (d, J=8.7 Hz, 2 H), 7.03 (d, J=8.7 Hz, 2 H), 7.09-7.37

(m, 21 H). MS (ESI) m/z 807 (MþNa).

1

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[2-(benzyloxy)-

5-(4-ethoxybenzyl)phenyl]-1-thio-^D-glucitol (21g). Compound

21g (0.210 g, 99%) was prepared from 20g (0.216 g, 0.247 mmol)

according to the method described for the synthesis of 21a. ¹H

NMR (300 MHz, CDCl₃) δ 1.36 (t, J=6.9 Hz, 3 H), 2.97-3.17

(m, 1 H), 3.47-3.62 (m, 1 H), 3.62-3.75 (m, 1 H), 3.76-4.03

(m, 8 H), 4.40-4.67 (m, 5 H), 4.82-5.12 (m, 5 H), 6.62-7.42 (m,

32 H). MS (ESI) m/z 879 (MþNa).

Et₃SiH (0.71 mL, 4.46 mmol) and then BF₃Et₂O (0.38 mL, 2.97

3

mmol) at -15 °C, and the mixture was stirred for 1 h. The reac-

tion was quenched by addition of saturated aqueous NaHCO₃

and extracted with ethyl acetate twice. The combined organic

layers were washed with brine, dried with MgSO₄, and concen-

trated. The residue was purified by silica gel column chroma-

tography (hexane/ethyl acetate=9/1) to give 21a (330 mg, 59%).

¹H NMR (600 MHz, CDCl₃) δ 1.38 (t, J=7.0 Hz, 3 H), 3.04 (s,

1 H), 3.48-3.57 (m, 1 H), 3.64 (dd, J=2.6, 9.8 Hz, 1 H), 3.74 (d,

J=10.1 Hz, 1 H), 3.88-4.16 (m, 8 H), 4.41 (d, J=10.1 Hz, 1 H),

4.52 (s, 2 H), 4.65 (d, J=10.7 Hz, 1 H), 4.79-4.96 (m, 3 H),

6.67-6.79 (m, 4 H), 7.00-7.35 (m, 22 H), 7.47-7.55 (m, 2 H).

MS (ESI) m/z 773 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[5-(4-ethoxybenzyl)-

2-methoxyphenyl]-1-thio-^D-glucitol (21h). Compound 21h (0.87

g) was prepared as a pale yellow oil from 20h (1.71 g, 2.15 mmol)

according to the method described for the synthesis of 21a. ¹H

NMR (600 MHz, CDCl₃) δ 1.37 (t, J=7.1 Hz, 3 H), 3.07-3.15

(m, 1 H), 3.54 (t, J = 9.2 Hz, 1 H), 3.62-3.99 (m, 11 H),

4.47-4.62 (m, 6 H), 4.84-4.93 (m, 3 H), 6.61-7.41 (m, 27 H).

MS (ESI) m/z 803 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[5-(4-ethoxybenzyl)-

2,4-dimethoxyphenyl]-1-thio-^D-glucitol (21i). Compound 21i

(0.358 g, 30%) was prepared as a yellow oil from 20i (1.22 g,

1.48 mmol) according to the method described for the synthesis

of 20a. ¹H NMR (300 MHz, CDCl₃) δ 1.35 (t, J=6.9 Hz, 3 H),

3.06-3.15 (m, 1 H), 3.52 (t, J=8.9 Hz, 1 H), 3.64-3.98 (m, 14

H), 4.45-4.62 (m, 4 H), 4.71 (s, 2 H), 4.84-4.93 (m, 3 H), 6.45 (s,

1 H), 6.63-6.72 (m, 4 H), 6.99-7.38 (m, 21 H). MS (ESI) m/z

828 (MþNH₄).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-(benzyloxy)-

3-(4-ethoxybenzyl)phenyl]-1-thio-^D-glucitol (21b). Compound

21b (566 mg, 58%) was prepared as a colorless powder from

20b (1.00 g, 1.15 mmol) according to the method described for

the synthesis of 21a following recrystallization from hexane/

ethyl acetate. ¹H NMR (300 MHz, CDCl₃) δ 1.38 (t, J=6.9 Hz,

3 H), 3.02-3.14 (m, 1 H), 3.51 (t, J=8.7 Hz, 1 H), 3.66-4.05 (m,

10 H), 4.47 (d, J=10.3 Hz, 1 H), 4.52 (s, 2 H), 4.59 (d, J=10.4 Hz,

1 H), 4.83-4.94 (m, 3 H), 5.06 (s, 2 H), 6.64-6.74 (m, 4 H),

6.84-6.91 (m, 1 H), 6.96-7.38 (m, 27 H). MS (ESI) m/z 874

(MþNH₄).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-3-(4-

methoxybenzyl)phenyl]-1-thio-^D-glucitol (21j). Compound 21j

(1.01 g, 55%) was prepared as a colorless powder from 20j

(1.87 g, 2.37 mmol) according to the method described for the

synthesis of 21a following recrystallization from ethyl acetate.

¹H NMR (300 MHz, CDCl₃) δ 3.01-3.16 (m, 1 H), 3.50 (t,

J=8.9 Hz, 1 H), 3.66-3.72 (m, 1 H), 3.76 (s, 3 H), 3.76-3.99 (m,

6 H), 4.02-4.14 (m, 1 H), 4.46-4.53 (m, 3 H), 4.59 (d, J=10.7

Hz, 1 H), 4.82-4.95 (m, 3 H), 6.63-6.82 (m, 4 H), 7.01-7.36 (m,

23 H). MS (ESI) m/z 788 (MþNH₄).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-(4-ethoxybenzyl)-

4-methoxyphenyl]-1-thio-^D-glucitol (21c). Compound 21c (930

mg, 60%) was prepared as a colorless amorphous from 20c (1.58

g, 1.98 mmol) according to the method described for the synthe-

1

sis of 21a. H NMR (600 MHz, CDCl₃) δ 1.37 (t, J=6.9 Hz,

3 H), 3.05-3.11 (m, 1 H), 3.50 (t, J=8.9 Hz, 1 H), 3.70 (dd,

J=2.8, 9.6 Hz, 1 H), 3.76-3.84 (m, 6 H), 3.84-3.92 (m, 3 H),

3.92-3.99 (m, 3 H), 4.45 (d, J=10.1 Hz, 1 H), 4.52 (s, 2 H), 4.59

(d, J=10.6 Hz, 1 H), 4.85 (s, 2 H), 4.89 (d, J=11.0 Hz, 1 H),

6.67-6.75 (m, 4 H), 6.83 (d, J=8.3 Hz, 1 H), 7.02-7.18 (m, 8 H),

7.22-7.35 (m, 14 H). MS (ESI) m/z 803 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-3-(4-

methylbenzyl)phenyl]-1-thio-^D-glucitol (21k). Compound 21k

(1.54 g, 63%) was prepared as a colorless powder from 20k

(2.50 g, 3.24 mmol) according to the method described for the

synthesis of 21a following recrystallization from hexane/ethyl

acetate. ¹H NMR (300 MHz, CDCl₃) δ 2.30 (s, 3 H), 3.04-3.13

(m, 1 H), 3.46-3.54 (m, 1 H), 3.66-4.13 (m, 8 H), 4.47-4.53 (m,

3 H), 4.59 (d, J=11.0 Hz, 1 H), 4.84-4.92 (m, 3 H), 6.67-6.72

(m, 2 H), 7.02 (s, 4 H), 7.08-7.35 (m, 21 H). MS (ESI) m/z 772

(MþNH₄), 774 (Mþ2þNH₄).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-(4-ethoxybenzyl)-

4-fluorophenyl]-1-thio-^D-glucitol (21d). Compound 21d (767 mg,

49%) was prepared as a colorless powder from 20d (1.60 g, 2.04

mmol) according to the method described for the synthesis of

1

21a following recrystallization from hexane/ethyl acetate. H

NMR (300 MHz, CDCl₃) δ 1.38 (t, J=7.0 Hz, 3 H), 3.03-3.13

(m, 1 H), 3.46-3.54 (m, 1 H), 3.66-4.00 (m, 10 H), 4.45-4.53

(m, 3 H), 4.59 (d, J = 10.7 Hz, 1 H), 4.84-4.93 (m, 3 H),

6.65-6.77 (m, 4 H), 6.96-7.34 (m, 23 H). MS (ESI) m/z 791

(MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-3-(4-

ethylbenzyl)phenyl]-1-thio-^D-glucitol (21l). Compound 21l (2.13

g, 72%) was prepared as a pale yellow powder from 20l (3.03 g,

3.86 mmol) according to the method described for the synthesis

of 21a following recrystallization from hexane/ethyl acetate. ¹H

NMR (300 MHz, CDCl₃) δ 1.19 (t, J=7.8 Hz, 3 H), 2.58 (q,

J=7.8 Hz, 2 H), 3.04-3.13 (m, 1 H), 3.50 (t, J=8.7 Hz, 1 H),

3.66-4.14 (m, 8 H), 4.46-4.53 (m, 3 H), 4.59 (d, J=10.7 Hz,

1 H), 4.84-4.92 (m, 3 H), 6.66-6.72 (m, 2 H), 7.00-7.36 (m, 25

H). MS (ESI) m/z 791 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-(4-ethoxybenzyl)-

4-methylphenyl]-1-thio-^D-glucitol (21e). Compound 21e (1.04 g,

69%) was prepared as a colorless powder from 20e (1.53 g, 1.96

mmol) according to the method described for the synthesis of

21a. ¹H NMR (300 MHz, CDCl₃) δ 1.38 (t, J=7.0 Hz, 3 H), 2.22

(s, 3 H), 3.06-3.14 (m, 1 H), 3.52 (t, J=8.9 Hz, 1 H), 3.68-4.00

(m, 10 H), 4.46-4.54 (m, 3 H), 4.60 (d, J=10.7 Hz, 1 H), 4.84-

4.93 (m, 3 H), 6.67-6.76 (m, 4 H), 6.92-6.98 (m, 2 H), 7.08-

7.35 (m, 21 H). MS (ESI) m/z 782 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-3-[4-(propan-

2-yl)benzyl]phenyl]-1-thio-^D-glucitol (21m). Compound 21m (2.01 g,

77%) was prepared as a colorless powder from 20m (2.65 g, 3.31

mmol) according to the method described for the synthesis of 21a

following recrystallization from hexane/ethyl acetate. ¹H NMR (300

MHz, CDCl₃) δ 1.20 (d, J= 7.0 Hz, 6 H), 2.77-2.89 (m, 1 H),

3.05-3.13 (m, 1 H), 3.51 (t, J=8.9 Hz, 1 H), 3.66-4.15 (m, 8 H),

4.46-4.54 (m, 3 H), 4.59 (d, J=10.7 Hz, 1 H), 4.83-4.92 (m, 3 H),

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-3-(4-

ethoxybenzyl)phenyl]-1-thio-^D-glucitol (21f). Compound 21f

(2.62 g, 71%) was prepared as a colorless powder from 20f

(3.79 g, 4.73 mmol) according to the method described for the

synthesis of 21a following recrystallization from hexane/ethyl

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3259

6.65-6.72 (m, 2 H), 7.04-7.37 (m, 25 H). MS (ESI) m/z 805

(MþNa).

and 75 μL of uptake buffer containing test compounds (methyl-

R-^D-glucopyranoside containing [¹⁴C] methyl-R-D-glucopyra-

noside (0.1 mM for SGLT1 inhibition, 1 mM for SGLT2 inhi-

bition), 140 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂,

10 mM HEPES/5 mM Tris, pH 7.4) were added, and the uptake

reaction was performed at 37 °C for 30 min (SGLT1) or for 1 h

(SGLT2). After the reaction, cells were washed with 200 μL of

washing buffer (10 mM methyl-R-^D-glucopyranoside, 140 mM

choline chloride, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM

HEPES/5 mM Tris, pH 7.4) twice and dissolved in 75 μL of

0.25 M NaOH. Opti phase Super Mix (Perkin-Elmer Corpora-

tion) was added and mixed well with the sample, then radio-

activity was measured with a Micro Beta Trilux (Perkin-Elmer

Corporation). Uptake buffer containing no test compound

was used as a control. Another uptake buffer containing

choline chloride in place of NaCl was also prepared for back-

ground.

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-(4-tert-butyl-

benzyl)-4-chlorophenyl]-1-thio-^D-glucitol (21n). Compound 21n

(1.05 g, 64%) was prepared as a colorless powder from 20n (1.68

g, 2.07 mmol) according to the method described for the synthe-

sis of 21a following recrystallization from hexane/ethyl acetate.

¹H NMR (300 MHz, CDCl₃) δ 1.26 (s, 9 H), 3.05-3.20 (m, 1 H),

3.51 (t, J=8.9 Hz, 1 H), 3.63-3.93 (m, 6 H), 3.93-4.03 (m, 1 H),

4.06-4.17 (m, 1 H), 4.45-4.54 (m, 3 H), 4.59 (d, J=10.6 Hz,

1 H), 4.84-4.93 (m, 3 H), 6.69 (dd, J = 1.5, 8.0 Hz, 2 H),

7.04-7.38 (m, 25 H). MS (ESI) m/z 819 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-3-[4-(methyl-

sulfanyl)benzyl]phenyl]-1-thio-^D-glucitol (21o). Compound 21o

(1.20 g, 62%) was prepared as a colorless powder from 20o

(1.98 g, 2.46 mmol) according to the method described for the

synthesis of 21a following recrystallization from hexane/ethyl

acetate. ¹H NMR (600 MHz, CDCl₃) δ 2.42 (s, 3 H), 3.05-3.12

(m, 1 H), 3.51 (t, J=8.9 Hz, 1 H), 3.70 (dd, J=3.0, 9.9 Hz, 1 H),

3.74-3.82 (m, 2 H), 3.82-3.92 (m, 3 H), 3.93-4.01 (m, 1 H),

4.07 (d, J=15.1 Hz, 1 H), 4.48-4.54 (m, 3 H), 4.59 (d, J=10.6

Hz, 1 H), 4.82-4.87 (m, 2 H), 4.89 (d, J=10.6 Hz, 1 H), 6.70 (d,

J = 7.3 Hz, 2 H), 7.00-7.38 (m, 25 H). MS (ESI) m/z 804

(MþNH₄).

In order to determine IC₅₀values, each test compound was

tested at six suitable concentrations. The concentration at which

glucose uptake was inhibited by 50% (IC₅₀value) compared to

glucose uptake in the control (100%) was calculated.

Hypoglycemic Effect in STZ-induced Diabetic Rats. Male

Sprague-Dawley rats (7-weeks old, Charles River, Japan) were

intravenously injected with STZ (50 mg/kg, Sigma) to induce

hyperglycemia. One week later, a group of 5 or 6 nonfasted STZ

rats were given an oral dose (0.3 or 3 mg/kg) of one of test

compounds 3m and 3p-3s. Blood was collected from the orbital

venous sinus under ether anesthesia at serial time points, and

plasma glucose levels were measured using a Glucose C2 Test

Wako (Wako pure Chemical Industries, Japan). Male SD rats

(8 weeks old) not treated with STZ were used as the normal

control.

Urinary Glucose Excretion in Zucker Fatty Rats. After fasting

for 17 h, Zucker fatty rats (10 weeks old, mean body weight,

approximately 380 g, Charles River Japan) were given a single

oral dose (1 mg/kg) of 3p 30 min prior to a 2 g/kg glucose load.

Urine was collected for 24 h using metabolic cages, and glucose

content was measured using Glucose C2 Test Wako. Male ZF-

lean rats (10 weeks old) were used as the normal control.

Urinary Glucose Excretion in Beagle Dogs. After fasting for

21 h, beagle dogs (7 to 12 months old, body weight, 8.1-9.6 kg,

Charles River, Japan) were given a single oral dose (1 mg/kg) of

3p 1 h prior to a 2 g/kg glucose load. Urine was collected for 24 h

using metabolic cages, and glucose content was measured using

Glucose C2 Test Wako.

Metabolic Stability. Human Liver Microsomal Stability. Test

compounds 3m and 3p-3s (5 μM) were incubated with 1.0 mg

protein/mL of human liver microsomes (Xenotech LLC, USA)

in 0.255 M phosphate buffer containing 0.5% (w/v) KCl (pH

7.4) and NADPH-generating system (2.4 mM MgCl₂, 1.5 mM

glucose-6-phosphate, 0.18 U/mL glucose-6-phosphate dehydro-

genase, 0.16 mM NADP^þ) at 37 °C for 15 min. Reactions were

quenched by the addition of DMSO, and supernatants were

analyzed by LC/MS/MS.

Human Cryopreserved Hepatocyte Stability. Test compounds

3m and 3p-3s (5 μM) were incubated with 1 ꢀ 10⁶human cryo-

preserved hepatocytes (In Vitro Technologies, USA) in pre-

pared Hank’s Balanced Salt Solution (KAC, Japan) at 37 °C for

240 min. Reactions were quenched by the addition of DMSO,

and supernatants were analyzed by LC/MS/MS.

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[5-(4-ethoxybenzyl)-

2-methoxy-4-methylphenyl]-1-thio-^D-glucitol (21p). Compound

21p (0.640 g, 77%) was prepared as a colorless syrup from 20p

(0.840 g, 1.0 mmol) according to the method described for the

synthesis of 21a. ¹H NMR (600 MHz, CDCl₃) δ 1.35 (t, J=6.9

Hz, 3 H), 2.21 (s, 3 H), 3.02-3.21 (m, 1 H), 3.55 (t, J=9.4 Hz,

1 H), 3.69-3.72 (m, 1 H), 3.74-3.97 (m, 10 H), 4.00-4.05 (m,

1 H), 4.45-4.56 (m, 3 H), 4.60 (d, J=10.6 Hz, 2 H), 4.86 (s, 2 H),

4.90 (d, J=10.6 Hz, 1 H), 6.58-6.76 (m, 4 H), 6.90 (d, J=7.3 Hz,

1 H), 7.09-7.19 (m, 5 H), 7.23-7.35 (m, 16 H). MS (ESI) m/z

812 (MþNH₄).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[5-(4-ethylbenzyl)-

2-methoxy-4-methylphenyl]-1-thio-^D-glucitol (21q). Compound

21q (1.68 g, 76%) was prepared as a colorless oil from 20q

(2.25 g, 2.83 mmol) according to the method described for the

synthesis of 21a. ¹H NMR (300 MHz, CDCl₃) δ 1.15 (t, J=7.5

Hz, 3 H), 2.22 (s, 3 H), 2.54 (q, J=7.5 Hz, 2 H), 3.06-3.16 (m,

1 H), 3.49-4.07 (m, 11 H), 4.45-4.65 (m, 5 H), 4.84-4.94 (m,

3 H), 6.69-6.76 (m, 3 H), 6.89-6.99 (m, 4 H), 7.07-7.19 (m,

5 H), 7.22-7.35 (m, 14 H). MS (ESI) m/z 801 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[2-methoxy-4-

methyl-5-[4-(propane-2-yl)benzyl)phenyl]-1-thio-^D-glucitol (21r).

Compound 21r (0.69 g, 62%) was prepared as a colorless amor-

phous from 20r (1.14 g, 1.41 mmol) according to the method

described for the synthesis of 21a. ¹H NMR (300 MHz, CDCl₃)

δ 1.16 (d, J=6.8 Hz, 3 H), 2.23 (s, 3 H), 2.70-2.86 (m, 1 H),

3.03-3.20 (m, 1 H), 3.55 (t, J=8.9 Hz, 1 H), 3.64-4.08 (m, 10

H), 4.43-4.66 (m, 5 H), 4.80-4.95 (m, 3 H), 6.67-6.78 (m, 3 H),

6.89-7.00 (m, 4 H), 7.05-7.19 (m, 5 H), 7.21-7.37 (m, 14 H).

MS (ESI) m/z 815 (MþNa).

(1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-chloro-5-(4-ethyl-

benzyl)-2-methoxyphenyl]-1-thio-^D-glucitol (21s). Compound

21s (2.65 g, 87%) was prepared as a pale yellow crystal from

20s (3.12 g, 3.83 mmol) according to the method described for

1

the synthesis of 21a. H NMR (300 MHz, CDCl₃) δ 1.14 (t,

J=7.8 Hz, 3 H), 2.54 (q, J=7.8 Hz, 2 H), 3.06-3.16 (m, 1 H),

3.53 (t, J=8.8 Hz, 1 H), 3.64-4.13 (m, 10 H), 4.46-4.65 (m,

5 H), 4.83-4.95 (m, 3 H), 6.64-6.72 (m, 2 H), 6.87-7.35 (m, 24

H). MS (ESI) m/z 821 (MþNa).

Serum Protein Binding. Human blood was obtained from

healthy male and female volunteers by venous puncture with-

out anticoagulants, as approved by the institutional ethics

committee. The serum was obtained by centrifugation. Com-

pounds 3m and 3p-3s (1 μg/mL) were added to serum and

dialyzed against 0.05 M sodium phosphate buffer (pH 7.4)

containing 0.07 M sodium chloride. Dialysis was performed at

37 °C for 6 h using a 96-well equilibrium dialysis equipment. The

concentrations of the test compounds in serum and buffer were

analyzed by LC/MS/MS.

Glucose Uptake Inhibition Assay. Chinese hamster ovary - K1

cells stably expressing human SGLT2 (NM_003041) and human

SGLT1 (NM_000343) were used for the sodium dependent

glucose transport inhibition test. Cells were incubated in

20 μL of pretreatment buffer solution (140 mM choline chloride,

2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES/5 mM

Tris, pH 7.4) for 20 min. The pretreatment buffer was removed

3260 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Kakinuma et al.

Caco-2 Monolayer Permeability. Caco-2 cells were obtained

from the American Type Culture Collection (Rockville, MD).

Cells were grown in Dulbecco’s modified Eagle medium

(DMEM) supplemented with 10% fetal bovine serum, 1%

L-glutamine, 1% nonessential amino acids, and a 1% antibio-

tic-antimycotic mixture (10 000 U/mL penicillin G, 10 000 μg/

mL streptomycin sulfate, and 25 μg/mL amphotericin B in

0.85% saline) at 37 °C in culture flasks in a humidified

5% CO₂atmosphere. For cell permeability assessment, cells

(passage number 50 to 60) were seeded on 24-well cell culture

inserts (Transwell, 0.3 μm pores, 6.5 mm id) and cultured for

21 days. The integrity of the cell monolayer was evaluated by

measuring transepithelial electric resistance (TEER) and ¹⁴C-

mannitol permeability before the transport experiment. Hank’s

balanced salt solution (HBSS) was used in all experiments after

adjusting the pH to 6.5 for the apical side and the pH 7.4 for the

basolateral side. Compound 3p or propranolol was dissolved in

HBSS (pH 6.5) at a final concentration of 10 μM. After 20 min

incubation of both sides of the monolayer with drug-free HBSS,

3p or propranolol contained HBSS (pH 6.5) was added to the

apical side of the monolayers. Samples (50 μL) were taken from

the basolateral side at 30, 60, 90, and 120 min after dose admini-

stration. The volume of the basolateral side was maintained by

adding HBSS (pH 7.4). All experiments were performed in

triplicate at 37 °C. Concentrations of compound 3p and pro-

pranolol were determined by LC-MS/MS.

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The apparent permeability coefficients (P_app) for each well

were calculated using the following equation:

P_appðcm=sÞ ¼ ðΔcVR=ΔtAÞco

Where ΔcVR/Δt is the cumulative amount transported as a

function of time (seconds), VR is the volume of the receiver

chamber (mL), A is the surface area of the cell monolayer (cm²),

and co is the concentration of the compound at t=0.

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H.; Hickey, G. J.; Kowalchick, J. E.; Leiting, B.; Lyons, K.;

Marsilio, F.; McCann, M. E.; Patel, R. A.; Petrov, A.; Scapin,

G.; Patel, S. B.; Roy, R. S.; Wu, J. K.; Wyvratt, M. J.; Zhang, B. B.;

Zhu, L.; Thornberry, N. A.; Weber, A. E. (2R)-4-Oxo-4-[3-(tri-

fluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-R]pyrazin-7(8H)-yl]-

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Pharmacokinetics. Plasma Concentrations. After administra-

tion of compound 3p, 0.3 (rats) or 2 (dogs) mL blood samples

were taken from a vein and drawn into a tube, and the plasma

was separated by centrifugation. To a 50 μL aliquot of plasma

sample, 25 μL of I.S. methanol solution (100 ng/mL) and 500 μL

of 10 mM ammonium acetate solution were added, and the

samples were vortexed. The samples were loaded into SPE

cartridges (OASIS HLB, Waters USA), washed, then eluted

with acetic acid/methanol (0.1/100). The eluates were evapo-

rated to dryness, reconstituted with 100 μL of CH₃CN/10 mM

ammonium acetate (20/80), and a 10 μL aliquot of the sample

was analyzed by LC/ESI-MS/MS. The lower limit of quantifi-

cation (LLOQ) of plasma was 0.3 ng/mL.

Kidney Concentrations. After oral administration of com-

pound 3p, rats were decapitated following blood sampling at

1, 4, and 24 h post dose. The kidneys were immediately excised,

weighed, and 4-fold saline was added, followed by homogeniza-

tion. To a 50 μL aliquot of plasma or kidney homogenate

sample, 200 μL of I.S. CH₃CN/MeOH (9/1) solution (250 ng/

mL) was added, and the sample was vortexed. A 10 μL aliquot of

the supernatant was analyzed by LC-MS/MS system. LLOQs of

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Acknowledgment. We thank Dr. Yasuhiro Nakai, Dr.

Nagaaki Sato and Dr. Tomomi Ota for assistance in writing

this paper and for useful discussions. We also thank

Ms. Hisayo Sekine and Ms. Noriko Takahashi for providing

elementalanalysesandHPLCanalysesofthe testcompounds.

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Article Doi

(1 S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio- d -glucitol (TS-071) is a potent, selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor for type 2 diabetes treatment

DOI: 10.1021/jm901893x

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