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pubs.acs.org/jmcPublished on Web 08/06/2010r 2010 American Chemical Society

J. Med. Chem. 2010, 53, 6355–6360 6355

DOI: 10.1021/jm100332n

Discovery of Canagliflozin, a Novel C-Glucoside with Thiophene Ring, as Sodium-Dependent

Glucose Cotransporter 2 Inhibitor for the Treatment of Type 2 Diabetes Mellitus 1

Sumihiro Nomura,* ,† Shigeki Sakamaki,

† Mitsuya Hongu,

† Eiji Kawanishi,

† Yuichi Koga,

† Toshiaki Sakamoto,

†

Yasuo Yamamoto, † Kiichiro Ueta,

‡ Hirotaka Kimata,

‡ Keiko Nakayama,

‡ and Minoru Tsuda-Tsukimoto

§

† Medicinal Chemistry Research Laboratories,

‡ Pharmacology Research Laboratories, and

§ DMPK Research Laboratories,

Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama, Japan

Received March 11, 2010

We discovered that C-glucosides 4 bearing a heteroaromatic ring formed metabolically more stable inhibitors for sodium-dependent glucose cotransporter 2 (SGLT2) than the O-glucoside, 2 (T-1095). A novel thiophene derivative 4b-3 (canagliflozin) was a highly potent and selective SGLT2 inhibitor and showed pronounced anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Introduction

The incidence of type 2 diabetes mellitus (T2DM a ) is

markedly increasing in Westernized societies and some devel- oping countries.

2-4 However, at present, no single agent is

capable of achieving acceptable, long-lasting blood glucose control in the majority of patients.

5 Accordingly, there is a

strong incentive to develop novel drugs with improved effi- cacy and safety. Plasma glucose is filtered in the glomerulus and then

reabsorbed in the proximal tubules in the kidney. Renal glucose reabsorption is mediated predominantly by SGLT2 and to a lesser extent by SGLT1.

6-8 In the normoglycemic

state, all filtered glucose is transported from the tubular lumen to the blood. However, under hyperglycemic conditions, the reabsorption process is saturated and urinary glucose excre- tion (UGE) increases linearly.

9 An SGLT2 inhibitor, 2 (T-

1095, Figure 1), enhanced UGE and consequently lowered blood glucose levels in diabetic animal models independent of insulin action.

10,11 SGLT1 is distributed in the intestine, heart

and trachea besides the kidney, while SGLT2 is located solely in the kidney.

12 Therefore, selective SGLT2 inhibitors would

be desirable for anti-diabetic agents. Given that SGLT2 inhi- bition lowers plasma glucose levels in an insulin-independent fashion, SGLT2 inhibitors are not predicted to be associated with hypoglycemia, which is a major concern for the current therapieswithinsulinandsulfonylureas.Inaddition, increases in urinary caloric loss due to UGE predict that SGLT2 inhibitors will not be associated with weight gain that is often seen with other classes of currently approved antihyperglyce- mic agents. As a consequence of these anticipated properties, identification of novel SGLT2 inhibitors became a goal for medicinal chemistry.

13-16

Orally active 2 is an ester prodrug of active metabolite 1 (T- 1095A), which enhances the resistance against hydrolysis by β-glucosidase in the intestine.10 Nevertheless, the O-glucoside part of 2 is at least hydrolyzed to its aglycon in vivo (data not shown). The C-glucoside 3 was disclosed as SGLT2 inhibitor by Bristol-Myers Squibb Co.

17,18 Accordingly, to explore

novel C-glucosides metabolically more stable than O-gluco- sides, we evaluated a series of C-glucosides 4 bearing a heteroaromatic ring.

Chemistry

We describe herein the syntheses of C-glucosides 4a-4e bearing a heteroaromatic ring. The synthetic route is outlined in Scheme 1. We took advantage of the synthetic strategy outlined by Deshpande et al.

19-21 Aglycons 5a-5d were dis-

solved in tetrahydrofuran and toluene, and treated with n-butyllithium at -78 �C to generate aryllithium, followed by addition of 2,3,4,6-tetra-O-trimethylsilyl-β-D-gluconolactone.22

Aglycon 5e underwent deprotonation with 1 equiv of n-butyl- lithium at the benzylic position rather than lithium-bromine exchange. Additional amounts of n-butyllithium did not induce lithiation at all. The aryllithium of 5e was generated by tert-butyllithiumasthe secondequivalent.The resultinganome- ric mixture of lactolswas immediately convertedintodesilylated methyl ethers 6a-6e by addition of methanesulfonic acid in methanol.Finally,C-glucosidederivatives4a-4ewereobtained by stereoselective reduction of 6a-6e using a combination of triethylsilane and boron trifluoride etherate in methylene chloride.

23,24 The stereochemistry of 4a-4e was determined as

β-configuration by the coupling constant between anomeric C-H and adjacent C-H (J ≈ 9.5 Hz) in the 1H NMR spectrum. Synthetic routes to aglycons 5a-5e are shown in Scheme 2.

Aglycon 5a-1 having furan was prepared by a coupling reaction of bromophenyllithium and furaldehyde in diethyl ether followed by reduction of hydroxyl group with iodotri- methylsilane, in situ generated by chlorotrimethylsilane and sodiumiodideinacetonitrile.

25 Thiopheneaglycons5b-1,2and3

were synthesized by Friedel-Crafts acylation of corresponding

*To whom correspondence should be addresses. Phone: þ81-48-433-2503. Fax: þ81-48-433-8150. E-mail: [email protected].

a Abbreviations: SGLT, sodium-dependent glucose cotransporter;

HF-KK, high-fat diet fed KK; T2DM, type 2 diabetes mellitus; UGE, urinary glucose excretion; SD, Sprague-Dawley; SAR, structure- activity relationship; GLUT, facilitated glucose transporter; AMG, R-methyl-D-glucopyranoside; CHOK, Chinese hamster ovary-K; 2-DG, 2-deoxy-D-glucose.

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6356 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 Nomura et al.

benzoyl chloride and thiophene in methylene chloride, followed by reduction of ketone with triethylsilane and boron tri- fluoride etherate in methylene chloride. Bromine-substituted phenylpyrazole

26 was lithiated with n-butyllithium and reacted

with bromobenzaldehyde in diethyl ether, and the resulting

alcohol was reduced using sodium triacetoxyborohydride in trifluoroacetic acid

27 to give pyrazole derivative 5c-1. Pyridinyl

aglycon5d-1wassynthesizedbyacouplingreactionofWeinreb amide and pyridyllithium

28 in diethyl ether, followed by a

cross-coupling of triethylaluminum with bromopyridine in

Figure 1. Structures of some O- and C-glucosides.

Scheme 1. Syntheses of 4a-4ea

a Reagents and conditions: (a) n-BuLi, THF-toluene, -78 �C; (b) n-BuLi, then tert-BuLi, THF-toluene, -78 �C; (c) 2,3,4,6-tetra-O-trimethylsilyl-

β-D-gluconolactone, toluene, -78 �C; (d) MeSO3H, MeOH; (e) Et3SiH, BF3 3 OEt2, CH2Cl2, -78 to 0 �C.

Scheme 2. Syntheses of Aglycons 5a-5ea

a Reagents and conditions: (a) n-BuLi, Et2O, -78 �C, then 5-ethyl-2-furaldehyde, -78 �C; (b) TMSCl, NaI, CH3CN, 0 �C; (c) AlCl3, CH2Cl2, 0 �C;

(d) Et3SiH, BF3 3 OEt2, CH2Cl2, 0 �C; (e) n-BuLi, Et2O, -78 �C, then 5-bromo-2-chlorobenzaldehyde, -78 �C; (f ) NaBH(OAc)3, TFA, 0 �C; (g) n-BuLi, Et2O, -78 �C, then 5-bromo-2-chloro-N-methoxy-N-methylbenzamide, -78 �C; (h) Et3Al, Pd(PPh3)4, CeCl3, THF, 30 �C; (i) NH2NH2 3 H2O, KOH, ethylene glycol, 190 �C; ( j) 2-amino-40-fluoroacetophenone hydrochloride, EDC, HOBt, Et3N, CH2Cl2; (k) Lawesson’s reagent, 1,4-dioxane, 100 �C.

Article Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6357

tetrahydrofuran and reduction of the carbonyl group with hydrazine hydrate and potassium hydroxide in ethylene glycol.

29 To provide thiazole derivative 5e-1, ketoamide was

cyclized using Lawesson’s reagent in 1,4-dioxane.

Results and Discussion

Effects of furan, thiophene, pyrazole, pyridine and thiazole derivatives 4 (Figure 1) were evaluated on human SGLT2 (hSGLT2) activity and on urinary glucose excretion (UGE) in male Sprague-Dawley (SD) rats per 200 g of body weight over 24 h. The structure-activity relationship (SAR) of the representative compounds are shown in Table 1. Ethylthio- phene derivative 4b-1 possessed good hSGLT2 inhibitory po- tency compared with the corresponding furan 4a-1 or pyridine 4d-1 derivative. Also, phenylthiophene derivative 4b-3 showed higher hSGLT2 inhibitory activity and UGE effect than the corresponding pyrazole 4c-1 or thiazole 4e-1 derivative. There- fore, we selected the novel thiophene derivatives for further

optimization. For R 1 substituents, halogeno (chloro of 4b-2) or

lower alkyl (methyl of 4b-3) group provided better in vitro potency than proton (4b-1). Substituted aryl (4-fluorophenyl of 4b-3) group was preferred to alkyl (ethyl of 4b-1) or halogeno (chloroof4b-2) groupasR

2 substituentsforinvivopotencyand

chemical stability (data not shown). From these thiophene deri- vatives,compound4b-3(canagliflozin,TA-7284/JNJ-28431754) was selected as a clinical candidate. Table 2 displays effects of 1, 2, and 4b-3 on hSGLT1,

hSGLT2, facilitated glucose transporter 1 (GLUT1) activity and UGE in rats. The inhibitory effects of 1 and 4b-3 on the uptake of [

14 C]R-methyl-D-glucopyranoside (AMG) were in-

vestigated in Chinese hamster ovary-K (CHOK) cells stably expressing either hSGLT1 or hSGLT2. IC50 values of 1 and 4b- 3 were 240 and 910 nM for hSGLT1, and 5.2 and 2.2 nM for hSGLT2, respectively. Since GLUT1 mediates the uptake of glucose in almost all tissues, selectivity of versus GLUT1 was determined using L6 myoblast cells. Neither compound inhi- bited the incorporation of [

3 H]2-deoxy-D-glucose (2-DG)

mediated by GLUT1 predominantly expressed in L6 myo- blast cells.

30 Namely, compound 4b-3 was identified as a

potent and selective inhibitor of hSGLT2. Oral administration at 30 mg/kg of 2 and 4b-3 to male SD

rats induced glucose excretion over 24 h by 422 and 3,696 mg, respectively, per 200 g body weight. Pharmacokinetic studies revealed a much higher exposure of 4b-3 following oral admin- istration (Table 3). Following intravenous and oral doses of 3 and 10 mg/kg, respectively, to male SD rats, AUC0-inf,po, t1/2,po, and oral bioavailability were determined to be 35,980 ng 3 h/mL, 5.2 h, and 85%, respectively. Thus, inhibition of SGLT2 in renal tubules after oral dosing of 4b-3 is likely to continuously suppress reabsorption of glucose. The extensive UGE would reflect excellent pharmacokinetic properties of 4b-3 in vivo as well as high potency of SGLT2 inhibition. Since most of the filtered glucose is reabsorbed by SGLT2 in the renal tubules, the novel compound would be useful for an anti-diabetic agent. Single oral administration of 4b-3 at 3 mg/kg remarkably

reduced blood glucose levels without influencing food intake in hyperglycemic high-fat diet fed KK (HF-KK) mice (Figure 2A). There was a 48% reduction in blood glucose level versus vehicle at 6 h. In contrast, compound 4b-3 only slightly affected blood glucose levels in normoglycemic mice (Figure 2B). Therefore, this compound would control hyper- glycemia in the therapy of T2DM with low risk of hypogly- cemia. In brief, compound 4b-3 is a highly potent and selective

inhibitor for hSGLT2 with favorable pharmacokinetic profiles, and remarkably increases urinary glucose excretion. In addi- tion, oral administration of 4b-3 induced anti-hyperglycemic

Table 1. SAR of the Representative C-Glucosides with Heteroaromatic Ring

a

a Each compound was orally administered at a dose of 30 mg/kg

to male Sprague-Dawley (SD) rats. Urinary glucose excretion (UGE) data over 24 h were normalized per 200 g body weight.

b N.D.: not

determined.

Table 2. hSGLT1, hSGLT2, Facilitated Glucose Transporter 1 (GLUT1) Inhibitory Activity, and Rat Urinary Glucose Excretion (UGE) Data for 1, 2, and 4b-3

IC50 (nM)

compd hSGLT1 hSGLT2 GLUT1 UGE a (mg/day)

1 240 5.2 >10000

2 b

422

4b-3 910 2.2 >10000 3696 a Compound 2 or 4b-3 was orally administered at a dose of 30 mg/kg

to male SD rats. UGE data over 24 h were normalized per 200 g body weight.

b An ester prodrug 2 is rapidly converted to active metabolite 1 in

vivo, thus in vitro hSGLT inhibitory activities of 2 are not shown.

Table 3. Pharmacokinetic (PK) Parameters of 1 and 4b-3 in Male Sprague-Dawley (SD) Rats Following Intravenous and Oral Admin- istrations

compd 1 2 a

4b-3 4b-3

dose (mg/kg) 1 10 3 10

route iv po iv po

Cmax (ng/mL) 80 2513

tmax (h) 1.3 5.0

AUC0-inf (ng 3 h/mL) 153 304 12703 35980 t1/2 (h) 1.3 2.2 5.0 5.2

CLtot (mL/h/kg) 7506 236

Vdss (mL/kg) 11390 1357

F (%) 20 85 a Because a prodrug 2 is very rapidly converted to 1 in vivo, PK

parameters of 1 are shown.

6358 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 Nomura et al.

effects in HF-KK mice. Currently, the thiophene derivative 4b-3 (canagliflozin) is being developed for the treatment of type 2 diabetes mellitus.

Experimental Section

All reactions were carried out under inert gas or with CaCl2 tube, and reaction mixtures werestirred magnetically. Allreagents

and solvents were purchased from commercial suppliers and used without further purification unless otherwise noted. Reaction products were monitored by TLC using 0.25 mm E. Merck silica gel plates (60 F254) and were visualized using UV light or 5% phosphomolybdic acid in 95% EtOH. NMR spectra were col- lected on JEOL JNM-ECX400P and Varian UNITY INOVA500 spectrometers. Chemical shiftsaregiven inparts per million (ppm) downfield from internal reference tetramethylsilane standard; coupling constants (J value) are given in hertz (Hz). Elemental analyses were conducted by Medicinal Chemistry Research Laboratories, Mitsubishi Tanabe Pharma. Melting points were measured by a B€uchi model B-545 instrument and were uncor- rected. Infrared spectra were measured on Perkin-Elmer PARA- GON1000. APCI-MS spectra were obtained on Finnigan MAT SSQ7000CorThermoQuestLCQAdvantage,eluting with10mM AcONH4/MeOH. GC-MS spectra were measured on Shimadzu GCMS-QP2010. Analytical HPLC spectra were reported using Agilent 1100 with a UV detector measuring absorbance at 210 nm. All compounds were found to be >95% pure by HPLC analysis unless otherwise noted.

1-(β-D-Glucopyranosyl)-4-methyl-3-(5-(4-fluorophenyl)-2-thienyl- methyl)benzene (4b-3). 5-Bromo-2-methylbenzoic acid

17,18 (44.43 g,

206.6 mmol) was suspended in dichloromethane (600 mL), and to the mixture were added oxalyl chloride (20.0 mL, 229 mmol) and N, N-dimethylformamide (0.74 mL, 9.55 mmol). The mixture was stirred at room temperature for 6 h (clear solution). The solvent was evaporated under reduced pressure to give 5-bromo-2-methyl- benzoyl chloride as an oil. This compound and 2-(4-fluoro- phenyl)thiophene

31 (36.83 g, 206.6 mmol) were dissolved in

dichloromethane (1,200 mL), and to the mixture was added aluminum chloride (30.3 g, 227.2 mmol) at 0 �C (internal temperature). After being stirred at the same temperature for 30 min, the mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was poured into ice-water (1,200 mL). The organic layer was separated, and the aqueous layer was extracted with chloroform (500 mL) three times. The organic layers were combined and dried over potas- sium carbonate. The solvent was concentrated under reduced pressure, and to the mixture was added n-hexane to produce a precipitate. The precipitate was collected by filtration, washed with n-hexane, and dried to give (5-bromo-2-methylphenyl)(5-(4- fluorophenyl)-2-thienyl)methanone (66.44 g, 85.7%) as yellow crys- tals: mp 121-122 �C. IR (Nujol) 1668, 1627, 1597, 1585, 1556, 1532, 1505 cm

-1 . APCI-MS m/z 375/377 (M þ H). 1H NMR

(DMSO-d6) δ 2.24 (3H, s), 7.31-7.37 (3H, m), 7.46 (1H, d, J = 4.0 Hz, thio), 7.63-7.68 (3H, m), 7.87 (2H, m). Anal. Calcd for C18H12BrFOS: C, 57.61; H, 3.22; Br, 21.29; F, 5.06; S, 8.54. Found: C, 57.61; H, 3.14; Br, 21.07; F, 4.96; S, 8.58.

A solution of the above obtained (5-bromo-2-methylphenyl)- (5-(4-fluorophenyl)-2-thienyl)methanone (62.23 g, 165.8 mmol) and triethylsilane (76.8 mL, 481 mmol) in dichloromethane (620 mL)-acetonitrile (620 mL) was cooled to 0 �C (internal temperature), and to the mixture was added dropwise boron trifluoride-ethyl ether complex (58.8 mL, 464 mmol) over 15 min under nitrogen atmosphere. Subsequently, the mixture was stirred at room temperature for 4 h and was cooled again under ice-water. To the mixture was added slowly a saturated aqueous sodiumhydrogencarbonatesolution(1,200mL) understirring.The organic layer was separated, and the aqueous layer was extracted with chloroform (500 mL) three times. The combined organic layer was washed with brine and dried over magnesium sulfate. The solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (1,200 mL)-methanol (600 mL), and the mixture was treatedwithactivatedcarbon(3.0 g). The insoluble was filtered off, and the filtrate was concentrated under reduced pres- sure. To the mixture was added methanol to produce a precipitate. Theprecipitatewascollectedbyfiltration,washedwithethylacetate- methanol(1:3),anddriedtogive2-(5-bromo-2-methylbenzyl)-5-(4- fluorophenyl)thiophene 5b-3 (46.83 g, 78.2%) as pale-yellow crystals: mp 101-103 �C. IR (Nujol) 1879, 1746, 1592, 1511

Figure 2. Effects of single oral dosing of 4b-3 on blood glucose levels and food intake in high-fat diet fed KK (HF-KK) (A) and normal (B) mice. Data are expressed as the mean ( SEM (n = 5): * P < 0.05, ** P < 0.01 vs vehicle.

Article Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6359

cm -1 . GC-MS m/z 362 (M

þ ).

1 H NMR (DMSO-d6) δ 2.25 (3H, s),

4.15 (2H, s, Ph-CH2-thio), 6.85 (1H, d, J = 3.5 Hz, thio), 7.17 (1H, d, J = 8.0 Hz), 7.21 (2H, quasi-t), 7.31 (1H, d, J = 3.5 Hz, thio), 7.36 (1H, dd, J = 8.0, 1.9 Hz), 7.44 (1H, d, J = 1.9 Hz), 7.60 (2H, m). Anal. Calcd for C18H14BrFS: C, 59.84; H, 3.91; Br, 22.12; F, 5.26; S, 8.87. Found: C, 59.89; H, 3.86; Br, 21.93; F, 5.17; S, 8.85.

To a solution of the above obtained 2-(5-bromo-2-methyl- benzyl)-5-(4-fluorophenyl)thiophene 5b-3 (28.9 g, 80.0 mmol) in tetrahydrofuran (480 mL) and toluene (480 mL) was added n-butyllithium (1.6 M n-hexane solution, 50.0 mL, 80.0 mmol) dropwise over 10 min at -67 to -70 �C (internal temperature) under argon atmosphere, and the mixture was stirred for 20 min at the same temperature (dark-blue solution). To the mixture was added a solution of 2,3,4,6-tetra-O-trimethylsilyl-β-D-glu- conolactone

22 (34.0 g, 72.8 mmol) in toluene (240 mL) dropwise

over 30 min at -67 to -70 �C (internal temperature), and the mixture was further stirred for 1 h at the same temperature (slightly brown solution). Subsequently, to the mixture was added a solution of methanesulfonic acid (21.0 g, 219 mmol) in methanol (480 mL) dropwise over 15 min, and the resulting mixture was allowed to warm to room temperature and stirred for 17 h. The mixture was again cooled in ice-water, and to it was added a saturated aqueous sodium hydrogen carbonate solution (1,000 mL). The mixture was extracted with ethyl acetate (1,000 mL) twice, and the combined organic layer was washed with brine (1,000 mL) and dried over magnesium sul- fate. The insoluble was filtered off, and the solvent was evapo- rated under reduced pressure. The residue was triturated with toluene (100 mL)-n-hexane (400 mL) to give 1-(1-meth- oxyglucopyranosyl)-4-methyl-3-(5-(4-fluorophenyl)-2-thienyl- methyl)benzene 6b-3 (31.6 g, 91.3%) as a pale-yellow powder: HPLC 88.5% (tR = 8.1 min, L-column ODS (5 μm particle size, 4.6 � 150 mm), CH3CN/20 mM phosphate buffer (pH 6.5) (45/ 55)). APCI-MS m/z 492 (M þ NH4), 460 (M þ NH4 - MeOH), 443 (M þ H - MeOH). 1H NMR (DMSO-d6) δ 2.26 (3H, s, Me), 2.91 (1H, m, sugar), 2.95 (3H, s, OMe), 3.21 (1H, m, sugar), 3.37 (1H, m, sugar), 3.51-3.61 (2H, m, sugar), 3.75 (1H, m, sugar), 4.09, 4.18 (each 1H, d, J = 15.9 Hz, Ph-CH2-thio), 4.51 (1H, t, J = 6.0 Hz, OH), 4.65 (1H, d, J = 7.2 Hz, OH), 4.69 (1H,d,J=5.1Hz,OH),4.94(1H,d,J=5.5Hz,OH),6.77(1H,d, J = 3.5 Hz, Thio), 7.14 (1H, d, J = 8.0 Hz, Ph), 7.20 (2H, quasi-t, J = 8.8 Hz, Ph), 7.26 (1H, d, J = 3.5 Hz, thio), 7.32 (1H, dd, J = 8.0, 1.5 Hz, Ph), 7.42 (1H, d, J = 1.5 Hz, Ph), 7.57 (2H, m, Ph).

A solution of the above obtained 1-(1-methoxyglucopy- ranosyl)-4-methyl-3-(5-(4-fluorophenyl)-2-thienylmethyl)benzene 6b-3 (63.1 g, 132 mmol) and triethylsilane (46.4 g, 399 mmol) in dichloromethane (660 mL) was cooled in a dry ice-acetone bath under argon atmosphere, and to the mixture was added dropwise boron trifluoride-ethyl ether complex (50.0 mL, 395 mmol) over 5 min. The mixture was stirred at the same temperature. The mix- ture was allowed to warm to 0 �C and stirred under ice-water for 2 h. At the same temperature, a saturated aqueous sodium hydro- gen carbonate solution (800 mL) was added, and the mixture was stirred for 30 min. The organic solvent was evaporated under reduced pressure, and the residue was poured into water (1,500 mL) and extracted with ethyl acetate (1,000 mL) twice. The com- bined organic layer was washed with water (500 mL) twice, dried over magnesium sulfate, and treated with activated carbon. The insoluble was filtered off and the solvent was evaporated under reduced pressure. The residue was crystallized from ethyl acetate (300 mL)-diethyl ether (600 mL)-water (6 mL) to give desired 1-(β-D-glucopyranosyl)-4-methyl-3-(5-(4-fluorophenyl)-2-thienyl- methyl)benzene 4b-3 (33.5 g, 56.7%) as colorless crystals: mp 98-100 �C. IR (Nujol) 1626, 1600, 1549, 1507 cm-1. HPLC 99.5% (tR = 11.6 min, L-column ODS (5 μm particle size, 4.6 � 150 mm), CH3CN/20 mM phosphate buffer (pH 6.5) (40/60)). APCI-MS m/z 462 (M þ NH4). 1H NMR (DMSO-d6) δ 2.26 (3H, s, Me),3.13-3.28 (4H, m, sugar),3.44 (1H, m, sugar), 3.69(1H, m, sugar), 3.96 (1H, d, J = 9.3 Hz, sugar), 4.10, 4.15 (each 1H, d, J = 16.0 Hz, Ph-CH2-thio), 4.43 (1H, t, J = 5.8 Hz, OH), 4.72 (1H, d,

J = 5.6 Hz, OH), 4.92 (2H, d, J = 4.8 Hz, OH), 6.80 (1H, d, J = 3.5 Hz, thio), 7.11-7.15 (2H, m, Ph), 7.18-7.25 (3H, m, Ph), 7.28 (1H, d, J = 3.5 Hz, thio), 7.59 (2H, dd, J = 8.8, 5.4 Hz, Ph). Anal. Calcd for C24H25FO5S 3 0.5H2O: C, 63.56; H, 5.78; F, 4.19; S, 7.07. Found: C, 63.52; H, 5.72; F, 4.08; S, 7.00.

Supporting Information Available: Description of in vitro hSGLT1, hSGLT2 and GLUT1 assays, and in vivo urinary glucose excretion and blood glucose-lowering studies; detailed synthetic procedures for 4a-1, 4b-1, 4b-2, 4c-1, 4d-1 and 4e-1; HPLC analysis of 4b-3. This material is available free of charge via the Internet at http://pubs.acs.org.

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(14) Washburn, W. N. Evolution of Sodium Glucose Co-Transporter 2 Inhibitors as Anti-Diabetic Agents. Expert Opin. Ther. Patents 2009, 19, 1485–1499.

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S1

Supporting Information

Discovery of Canagliflozin, a Novel C-Glucoside with Thiophene Ring, as

Sodium-Dependent Glucose Cotransporter 2 Inhibitor for the Treatment of Type 2

Diabetes Mellitus

Sumihiro Nomura, Shigeki Sakamaki, Mitsuya Hongu, Eiji Kawanishi, Yuichi Koga,

Toshiaki Sakamoto, Yasuo Yamamoto, Kiichiro Ueta, Hirotaka Kimata, Keiko

Nakayama, Minoru Tsuda-Tsukimoto

Mitsubishi Tanabe Pharma Corporation, 2-2-50 Kawagishi, Toda, Saitama, Japan

Contents:

Pharmacology ................................................................................................................. S2

Chemistry........................................................................................................................ S6

HPLC Analysis of 4b-3 ................................................................................................ S19

References. ................................................................................................................... S20

S2

Pharmacology

Sodium-Dependent Glucose Uptake in CHO cells Expressing human SGLT1

and SGLT2.

Parental Chinese hamster ovary-K (CHOK) cells expressing human SGLT1 and

SGLT21 were used in these experiments. For the uptake assay, cells were seeded into

24-well plates, and were post-confluent on the day of assay.

Cells were rinsed one time with 400 µL Assay Buffer (137 mM NaCl, 5 mM KCl,

1 mM CaCl2, 1 mM MgCl2, 50 mM HEPES, 20 mM Tris Base, pH 7.4), and were

pre-incubated with the solutions of compounds (250 µL) for 10 min at 37 °C. The

transport reaction was initiated by addition of 50 µL alpha methyl-D-glucopyranoside

(AMG) / 14C-AMG solution (16.7 µCi; final concentration, 0.3 mM for CHOK-SGLT1

and 0.5 mM for CHOK-SGLT2, respectively) and incubated for 120 min at 37 °C.

After the incubation, the AMG uptake was halted by aspiration of the incubation

mixture followed by immediate washing three times with PBS. The cells were

solubilized in 0.3 N NaOH of 300 µL and the radioactivity associated with the cells was

monitored by a liquid scintillation counter (Quantasmart™ (Packard, Boston, MA,

USA)). Inhibitory concentration of 50% (IC50) was calculated by nonlinear least

squares analysis using a four-parameter logistic model (Prism version 4; GraphPad

Software, San Diego, CA, USA).

S3

2-Deoxy-Glucose (2-DG) Uptake in L6 Myoblast Cells.

The rat skeletal muscle cell line, L6 (JCRB9081), was obtained from Health

Science Research Resources Bank (HSRRB, Osaka, Japan). L6 myoblast cells were

maintained in Dulbecco’s modified Eagle’s medium containing 5.6 mM glucose

supplemented with 10% FBS. Cells were seeded in 24-well plates at a density of 3.0 x

105 cells / well and cultured for 24 hours in an atmosphere of 5% CO2 at 37 ºC before

the experiment. Prior to the transport experiment, cells were rinsed twice with KRPH

buffer (pH 7.4, 150 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.25 mM CaCl2, 10 mM

HEPES, 2.9 mM Na2HPO4), and were pre-incubated with compounds (250 µ l) for 5 min

at room temperature. The transport reaction was initiated by addition of 50 µ l

3H-2-deoxy-glucose solution (0.625 µ Ci; final concentration, 750 µ M) and incubated

for 15 min at room temperature. After the incubation, the 2-DG uptake was halted by

aspiration of the incubation mixture. Cells were immediately washed three times with

ice-cold PBS and were solubilized in 300 µL of 0.3 N NaOH. The radioactivity

associated with the cells was determined by a liquid scintillation counter

(Quantasmart™ (Packard, Boston, MA, USA)).

S4

Urinary Glucose Excretion (UGE) Study.

Male Sprague-Dawley (SD) rats aged 4-5 weeks were obtained from Japan SLC

(Shizuoka, Japan) and were used for experiments at 6 weeks of age after acclimation

period. The animals were divided into experimental groups matched for body weight

(n = 2-3). The compounds were prepared in vehicles as suspension or solution. UGE

studies were performed after two-day acclimation period in metabolic cages. The

compounds or vehicle were orally administered at a dose of 30 mg/kg in 0.2%

CMC/0.2% Tween 80. Urine samples were collected for 24 hours using metabolic

cages to measure urinary glucose excretion. Urine glucose contents were determined

by an enzymatic assay kit (UGLU-L, Serotec, Hokkaido, Japan). All animals were

allowed free access to a standard pellet diet (CRF1; Oriental Yeast Co., Ltd., Tokyo,

Japan) and tap water.

Single Oral Dosing Study.

Male KK/Ta Jcl mice aged 9 weeks were obtained from CLEA Japan Inc. (Tokyo,

Japan) and kept on a standard diet (CRF-1; 5.7% (w/w) fat, 3.59 kcal/g, Oriental Yeast

Co., Ltd., Tokyo, Japan), 20-week-old mice were fed with a high-fat diet (60 kcal%,

Research Diets, Inc., New Brunswick, NJ) for 4 weeks. The experiment was carried

out at the age of 24 weeks. Male C57BL/6N mice aged 11 weeks were obtained from

S5

Charles River Laboratories Japan, INC. (Yokohama, Japan) and were also used in this

study. The animals were divided into experimental groups matched for body weight

and blood glucose levels, which were measured in the fed state on the day of the

experiment.

The compounds (3 mg/kg) or vehicle (0.2% CMC/0.2% Tween 80) were orally

administered at a volume of 10 mL/kg. The blood samples were collected from the tail

vein before and at 1, 2, 4, 6 and 24 hr after the administration.

The blood glucose level was determined using commercially available kits based

on the glucose oxidase method (Glucose CII-Test Wako; Wako Pure Chemical

Industries, Osaka, Japan). Data are expressed as means ± SEM. Area under the

curve for blood glucose levels (AUCglucose 0-6 hr) was calculated by the trapezoidal rule.

Differences between groups were analyzed by repeated measurement ANOVA followed

by Student's t-test (EXSAS, Arm Systex Co. Ltd.). Probabilities less than 5% (P<0.05)

were considered to be statistically significant.

S6

Chemistry

All reactions were carried out under inert gas or with CaCl2 tube and reaction

mixtures were stirred magnetically. All reagents and solvents were purchased from

commercial suppliers and used without further purification unless otherwise noted.

Reaction products were monitored by TLC using 0.25 mm E. Merck silica gel plates (60

F254) and were visualized using UV light or 5% phosphomolybdic acid in 95% EtOH.

Silica gel column chromatography was performed on Silica gel BW-300 (Fuji Silicia) or

NH-silica gel Chromatorex (Fuji Silicia), using the solvent systems (volume ratios)

indicated below. NMR spectra were collected on JEOL JNM-ECX400P and Varian

UNITY INOVA500 spectrometers. Chemical shifts were given in parts per million

(ppm) downfield from internal reference tetramethylsilane standard; coupling constants

(J value) were given in hertz (Hz). Melting points were measured by BÜCHI Melting

Point B-545 and were uncollected. APCI-MS spectra were obtained on Finnigan MAT

SSQ7000C or ThermoQuest LCQ Advantage eluting with 10 mM AcONH4/MeOH.

GC-MS spectra were measured on Shimadzu GCMS-QP2010. Analytical HPLC

spectra were reported using Agilent 1100 with a UV detector measuring absorbance at

210 nm. All compounds were found to be >95% pure by HPLC analysis unless

otherwise noted.

S7

1-(ββββ-D-Glucopyranosyl)-3-(5-ethyl-2-furylmethyl)benzene (4a-1). A solution

of 1,3-dibromobenzene (4.45 g, 18.84 mmol) in diethyl ether (100 ml) was cooled to

–70 °C under argon atmosphere, and thereto was added dropwise n-butyllithium (2.44

M n-hexane solution, 7.41 ml, 18.08 mmol). The reaction mixture was stirred at the

same temperature for 30 minutes, and thereto was added dropwise a solution of

5-ethyl-2-furaldehyde (2.34 g, 18.84 mmol) in diethyl ether (20 ml). The mixture was

stirred at the same temperature for 15 minutes, and thereto was added a saturated

ammonium chloride solution, and the reaction mixture was warmed to room

temperature. The mixture was extracted with diethyl ether, and the extract was dried

over magnesium sulfate, and the solvent was evaporated under reduced pressure. The

residue was purified by aminosilane-treated silica gel column chromatography

(n-hexane : ethyl acetate = 5 : 1) to give (3-bromophenyl)(5-ethyl-2-furyl)methanol

(4.30 g, 81.2%) as a yellow oil: APCI-Mass m/Z 263/265 (M+H-H2O). 1H-NMR

(CDCl3) δ 1.21 (3H, t, J = 7.6 Hz), 2.35 (1H, d, J = 4.4 Hz), 2.62 (2H, q, J = 7.6 Hz),

5.75 (1H, d, J = 4.4 Hz), 5.90 (1H, d, J = 3.4 Hz), 5.97 (1H, d, J = 3.4 Hz), 7.23 (1H, t,

J = 8.7 Hz), 7.33-7.38 (1H, m), 7.43 (1H, ddd, J = 7.9, 1.7, 1.3 Hz), 7.61-7.62 (1H, m).

A solution of the above obtained (3-bromophenyl)(5-ethyl-2-furyl)methanol

(3.09 g, 11.0 mmol) in acetonitrile (11 ml) was added dropwise to a mixture of

chlorotrimethylsilane (6.48 ml, 51.0 mmol) and sodium iodide (7.65 g, 51.0 mmol) at

S8

0 °C over 1 hour. Thereto was added a 10% aqueous sodium hydroxide solution to be

basified. The mixture was extracted with diethyl ether, and the extract was washed

with an aqueous sodium thiosulfate solution and dried over magnesium sulfate. The

solvent was evaporated under reduced pressure, and the residue was purified by silica

gel column chromatography (n-hexane : ethyl acetate = 100 : 0 – 97 : 3) to give

2-(3-bromobenzyl)-5-ethylfuran 5a-1 (1.87 g, 64.1%) as a pale orange syrup: GC-Mass

m/Z 264/266 (M+). 1H-NMR (CDCl3) δ 1.20 (3H, t, J = 7.6 Hz), 2.59 (2H, q, J = 7.6

Hz), 3.88 (2H, s), 5.87 (1H, d, J = 3.0 Hz), 5.89 (1H, d, J = 3.0 Hz), 7.12-7.19 (2H, m),

7.35 (1H, m), 7.38 (1H, m).

The above obtained 2-(3-bromobenzyl)-5-ethylfuran 5a-1 was treated in a manner

similar to 4b-3 to give 1-(β-D-glucopyranosyl)-3-(5-ethyl-2-furylmethyl)benzene 4a-1

as a colorless foam: HPLC 99.6% (RT 3.36 min., L-column ODS (3 µm particle size,

4.6 x 50 mm), CH3CN/20mM-phosphate buffer (pH6.5) (30/70)). APCI-Mass m/Z

366 (M+NH4). 1H-NMR (DMSO-d6) δ 1.13 (3H, t, J = 7.5 Hz), 2.54 (2H, q, J = 7.5

Hz), 3.13-3.30 (4H, m), 3.44 (1H, dd, J = 11.9, 6.0 Hz), 3.69 (1H, d, J = 10.1 Hz), 3.89

(2H, s), 3.98 (1H, d, J = 9.5 Hz), 4.43 (1H, br), 4.74 (1H, br), 4.92 (2H, br), 5.94 (1H, d,

J = 2.9 Hz), 5.97 (1H, d, J = 2.9 Hz), 7.09-7.12 (1H, m), 7.17-7.20 (1H, m), 7.21-7.26

(2H, m).

S9

1-(ββββ-D-Glucopyranosyl)-3-(5-ethyl-2-thienylmethyl)benzene (4b-1).

1-(ββββ-D-Glucopyranosyl)-4-chloro-3-(5-chloro-2-thienylmethyl)benzene (4b-2).

5b-1, 4b-1, 5b-2 and 4b-2 were prepared in a manner similar to 4b-3 from the

corresponding benzoic acid and thiophene.

5b-1: APCI-Mass m/Z 281/283 (M+H). 1H-NMR (CDCl3) δ 1.27 (3H, t, J = 7.5 Hz),

2.80 (2H, q, J = 7.5 Hz), 4.04 (2H, s), 6.56-6.62 (2H, m), 7.14-7.18 (2H, m), 7.31-7.37

(1H, m), 7.37-7.40 (1H, m).

4b-1: HPLC 99.2% (RT 2.80 min., L-column ODS (3 µm particle size, 4.6 x 50 mm),

CH3CN/20mM-phosphate buffer (pH6.5) (35/65)). APCI-Mass m/Z 382 (M+NH4).

1H-NMR (DMSO-d6) δ 1.20 (3H, t, J = 7.5 Hz), 2.71 (2H, q, J = 7.5 Hz), 3.11-3.29 (4H,

m), 3.38-3.48 (1H, m), 3.89 (1H, ddd, J = 11.6, 5.4, 1.6 Hz), 3.98 (1H, d, J = 9.2 Hz) ,

4.04 (2H, s), 4.44 (1H, d, J = 5.8 Hz), 4.75 (1H, d, J = 5.6 Hz), 4.92 (2H, d, J = 4.6 Hz),

6.60-6.63 (1H, m), 6.65-6.68 (1H, m), 7.10-7.28 (4H, m).

5b-2: GC-Mass m/Z 320/322/324 (M+). 1H-NMR (DMSO-d6) δ 4.19 (2H, s), 6.79 (1H,

d, J = 3.7 Hz), 6.95 (1H, d, J = 3.7 Hz), 7.43 (1H, d, J = 8.5 Hz), 7.50 (1H, dd, J = 8.6,

2.2 Hz), 7.67 (1H, d, J = 2.4 Hz).

4b-2: HPLC 99.6% (RT 10.21 min., L-column ODS (5 µm particle size, 4.6 x 150 mm),

CH3CN/20mM-phosphate buffer (pH6.5) (35/65)). APCI-Mass m/Z 422/424

(M+NH4). 1H-NMR (DMSO-d6) δ 3.13-3.28 (4H, m), 3.42-3.50 (1H, m), 3.68-3.72

S10

(1H, m), 4.01 (1H, d, J = 9.4 Hz), 4.16, 4.20 (each 1H, d, J = 15.7 Hz), 4.44 (1H, t, J =

5.8 Hz), 4.84 (1H, d, J = 5.8 Hz), 4.94 (1H, d, J = 3.8 Hz), 4.96 (1H, d, J = 3.3 Hz), 6.74

(1H, d, J = 3.7 Hz), 6.93 (1H, d, J = 3.7 Hz), 7.27 (1H, dd, J = 8.3, 1.8 Hz), 7.40 (1H, d,

J = 5.3 Hz), 7.41 (1H, br s).

1-(ββββ-D-Glucopyranosyl)-4-chloro-3-(1-phenyl-1H-pyrazol-4-ylmethyl)benzene

(4c-1). A solution of 4-bromo-1-phenyl-1H-pyrazole2 (2.23 g, 10.0 mmol) in diethyl

ether (30 ml) was cooled to –78 °C under argon atmosphere, and added dropwise

thereto was n-butyllithium (1.59 M n-hexane solution, 6.9 ml, 11.0 mmol). The

mixture was stirred at –20 °C to –10 °C for 5 hours, and added dropwise thereto at the

same temperature was a solution of 5-bromo-2-chlorobenzaldehyde (2.19 g, 10.0 mmol)

in diethyl ether (30 ml). The mixture was stirred at the same temperature for 30

minutes, and added thereto was tetrahydrofuran (30 ml), and the mixture was stirred at

0 °C for further 30 minutes. A saturated aqueous ammonium chloride solution was

added thereto, and the mixture was extracted with ethyl acetate. The extract was

washed with brine and dried over sodium sulfate. The solvent was evaporated under

reduced pressure, and the residue was purified by silica gel column chromatography

(n-hexane : ethyl acetate = 83 : 17 – 80 : 20) to give (5-bromo-2-chlorophenyl)-

(1-phenyl-1H-pyrazol-4-yl)methanol (831 mg, 23%) as a yellow caramel: APCI-Mass

S11

m/Z 363/365 (M+H). 1H-NMR (DMSO-d6) δ 5.97 (1H, d, J = 4.8 Hz), 6.14 (1H, d, J =

4.7 Hz), 7.28 (1H, t, J = 7.4 Hz), 7.38 (1H, d, J = 8.4 Hz), 7.46 (2H, t, J = 8.0 Hz), 7.51

(1H, dd, J = 8.5, 2.6 Hz), 7.60 (1H, s), 7.80 (2H, d, J = 7.9 Hz), 7.89 (1H, d, J = 2.4 Hz),

8.34 (1H, s).

A solution of the above obtained (5-bromo-2-chlorophenyl)-

(1-phenyl-1H-pyrazol-4-yl)methanol (790 mg, 2.17 mmol) in trifluoroacetic acid (8 ml)

was cooled to 0 °C, and thereto was added portionwise sodium triacetoxyborohydride

(3.30 g, 10.9 mmol). The mixture was stirred at room temperature overnight, and

cooled again to 0 °C. Thereto was added a 10% aqueous sodium hydroxide solution to

be basified. The mixture was extracted with chloroform, and the extract was washed

with brine, and dried over sodium sulfate. The solvent was evaporated under reduced

pressure and the residue was purified by silica gel column chromatography (n-hexane :

ethyl acetate = 100 : 0 – 95 : 5) to give 4-(5-bromo-2-chlorobenzyl)-

1-phenyl-1H-pyrazole 5c-1 (548 mg, 72.6%) as a colorless powder: APCI-Mass m/Z

347/349 (M+H). 1H-NMR (DMSO-d6) δ 3.95 (2H, s), 7.28 (1H, m), 7.42 (1H, d, J =

8.5 Hz), 7.44-7.51 (3H, m), 7.60 (1H, d, J = 2.2 Hz), 7.63 (1H, s), 7.79 (2H, d, J = 7.9

Hz), 8.35 (1H, s).

The above obtained 4-(5-bromo-2-chlorobenzyl)-1-phenyl-1H-pyrazole 5c-1 was

treated in a manner similar to 4b-3 to give 1-(β-D-glucopyranosyl)-4-chloro-

S12

3-(1-phenyl-1H-pyrazol-4-ylmethyl)benzene 4c-1 as a colorless foam: HPLC 99.6%

(RT 4.19 min., L-column ODS (3 µm particle size, 4.6 x 50 mm),

CH3CN/20mM-phosphate buffer (pH6.5) (30/70)). APCI-Mass m/Z 431/433 (M+H).

1H-NMR (DMSO-d6) δ 3.08-3.28 (4H, m), 3.41-3.47 (1H, m), 3.66-3.72 (1H, m), 3.92,

3.97 (each 1H , d, J = 15.4 Hz), 4.00 (1H, d, J = 9.5 Hz), 4.44 (1H, d, J = 5.9 Hz), 4.84

(1H, d, J = 5.8 Hz), 4.95 (2H, t, J = 5.3 Hz), 7.25 (1H, dd, J = 8.4, 1.9 Hz), 7.27 (1H, t,

J = 7.4 Hz), 7.38 (1H, m), 7.40 (1H, d, J = 4.8 Hz), 7.47 (2H, t, J = 8.0 Hz), 7.58 (1H,

br s), 7.77 (2H, d, J = 7.7 Hz), 8.28 (1H, s).

1-(ββββ-D-Glucopyranosyl)-4-chloro-3-(2-ethyl-5-pyridylmethyl)benzene (4d-1).

A solution of 2,5-dibromopyridine (5.00 g, 21.1 mmol) in diethyl ether (300 ml) was

cooled to –78 °C under argon atmosphere, and thereto was added dropwise

n-butyllithium (2.59 M n-hexane solution, 8.31 ml, 21.52 mmol) over 15 minutes. The

reaction mixture was further stirred at the same temperature for 40 minutes, and thereto

was added dropwise a solution of 5-bromo-2-chloro-N-methoxy-N-methylbenzamide

(7.05 g, 25.32 mmol) in diethyl ether (200 ml) and tetrahydrofuran (15 ml) over 5

minutes. After being stirred at the same temperature for 30 minutes, thereto was added

a saturated ammonium chloride solution. The mixture was extracted with ethyl acetate,

and the extract was washed with brine and dried over magnesium sulfate. The solvent

S13

was evaporated under reduced pressure. The residue was purified by silica gel column

chromatography (n-hexane : ethyl acetate = 98 : 2 – 92 : 8) to give

(5-bromo-2-chlorophenyl)(6-bromopyridin-3-yl)methanone (2.46 g, 31.1%) as colorless

crystals: APCI-Mass m/Z 374/376 (M+H). 1H-NMR (DMSO-d6) δ 7.60 (1H, d, J = 8.7

Hz), 7.83 (1H, dd, J = 8.7, 2.4 Hz), 7.87 (1H, s), 7.88 (1H, d, J = 6.1 Hz), 8.04 (1H, dd,

J = 8.4, 2.4 Hz), 8.69 (1H, d, J = 2.6 Hz).

The above obtained (5-bromo-2-chlorophenyl)(6-bromopyridin-3-yl)methanone

(3.20 g, 9.86 mmol) was dissolved in tetrahydrofuran (80 ml), and added thereto were

successively triethylaluminum (1.0 M n-hexane solution, 9.9 ml, 9.86 mmol),

tetrakis(triphenylphosphine)palladium (0) (570 mg, 0.49 mmol) and cerium (III)

chloride (7.30 g, 29.6 mmol), and the mixture was stirred at 30 °C for 1.5 hours. The

reaction mixture was diluted with methanol, and the solution was basified with a

saturated aqueous sodium hydrogen carbonate solution. The insoluble materials were

filtered off, and the filtrate was extracted with ethyl acetate. After being dried over

magnesium sulfate, the solvent was evaporated under reduced pressure. The residue

was purified by silica gel column chromatography (n-hexane : ethyl acetate = 99 : 1 –

85 : 15) to give (5-bromo-2-chlorophenyl)(6-ethylpyridin-3-yl)methanone (1.98 g,

61.9%) as colorless crystals: APCI-Mass m/Z 324/326 (M+H). 1H-NMR (DMSO-d6)

δ 1.25 (3H, t, J = 7.5 Hz), 2.86 (2H, q, J = 7.5 Hz), 7.49 (1H, d, J = 8.2 Hz), 7.59 (1H,

S14

d, J = 8.5 Hz), 7.81 (1H, dd, J = 8.5, 2.2 Hz), 7.84 (1H, d, J = 2.2 Hz), 8.03 (1H, dd, J =

8.2, 2.2 Hz), 8.77 (1H, d, J = 1.9 Hz).

To a solution of the above obtained (5-bromo-2-chlorophenyl)-

(6-ethylpyridin-3-yl)methanone (1.97 g, 6.07 mmol) in ethylene glycol (25 ml) were

added hydrazine hydrate (0.97 ml, 20.0 mmol) and potassium hydroxide (1.23 g, 21.9

mmol), and the mixture was stirred at 190 °C for 1.5 hours. After being cooled to

room temperature, the reaction mixture was poured into water, and the mixture was

extracted with diethyl ether. The extract was washed with water twice and dried over

magnesium sulfate. The solvent was evaporated under reduced pressure, and the

residue was purified by silica gel column chromatography (n-hexane : ethyl acetate =

98 : 2 – 85 : 15) to give 5-(5-bromo-2-chlorobenzyl)-2-ethylpyridine 5d-1 (1.21 g,

64.2%) as a colorless syrup: APCI-Mass m/Z 310/312 (M+H). 1H-NMR (DMSO-d6)

δ 1.18 (3H, t, J = 7.6 Hz), 2.69 (2H, q, J = 7.6 Hz), 4.03 (2H, s), 7.18 (1H, d, J = 8.0

Hz), 7.40 (1H, d, J = 8.5 Hz), 7.45-7.51 (2H, m), 7.63 (1H, d, J = 2.2 Hz), 8.37 (1H, d, J

= 1.4 Hz).

The above obtained 5-(5-bromo-2-chlorobenzyl)-2-ethylpyridine 5d-1 was treated

in a manner similar to 4b-3 to give 1-(β-D-glucopyranosyl)-4-chloro-

3-(2-ethyl-5-pyridylmethyl)benzene 4d-1 as a colorless foam: HPLC 99.6% (RT 3.94

min., L-column ODS (3 µm particle size, 4.6 x 50 mm), CH3CN/20mM-phosphate

S15

buffer (pH6.5) (25/75)). APCI-Mass m/Z 394/396 (M+H). 1H-NMR (DMSO-d6)

δ 1.19 (3H, t, J = 7.5 Hz), 2.69 (2H, q, J = 7.5 Hz), 3.07-3.28 (4H, m), 3.44 (1H, m),

3.69 (1H, m), 4.00 (1H, d, J = 9.5 Hz), 4.01, 4.06 (each 1H , d, J = 15.1 Hz), 4.44 (1H,

t, J = 5.9 Hz), 4.84 (1H, d, J = 5.8 Hz), 4.95 (2H, t, J = 4.7 Hz), 7.15 (1H, d, J = 8.0

Hz), 7.24 (1H, dd, J = 8.3, 2.0 Hz), 7.37 (1H, d, J = 8.2 Hz), 7.38 (1H, d, J = 1.9 Hz),

7.45 (1H, dd, J = 8.0, 2.3 Hz), 8.34 (1H, d, J = 1.9 Hz).

1-(ββββ-D-Glucopyranosyl)-4-methyl-3-(5-(4-fluorophenyl)-2-thiazolylmethyl)-

benzene (4e-1). To a solution of 5-bromo-2-methylphenylacetic acid (1.15 g, 5.02

mmol) and 2-amino-4´-fluoroacetophenone hydrochloride (1.14 g, 6.01 mmol) were

successively added 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (1.31 ml, 6.01

mmol), 1-hydroxybenzotriazole (0.811 g, 6.01 mmol) and triethylamine (0.836 ml, 6.01

mmol), and the mixture was stirred at room temperature overnight. The reaction

mixture was poured into water, and the resulting mixture was extracted with chloroform.

The extract was successively washed with a 10% aqueous hydrochloric acid solution, a

saturated aqueous sodium hydrogen carbonate solution and brine, and dried over sodium

sulfate. The solvent was evaporated under reduced pressure, and the residue was

purified by silica gel column chromatography (n-hexane : ethyl acetate = 9 : 1 – 3 : 1) to

give N-(2-oxo-2-(4-fluorophenyl)ethyl)-2-(5-bromo-2-methylphenyl)acetamide (1.14 g,

S16

62.4%) as a colorless foam: APCI-Mass m/Z 364/366 (M+H). 1H-NMR (DMSO-d6)

δ 2.24 (3H, s), 3.57 (2H, s), 4.61 (2H, d, J = 5.6 Hz), 7.12 (1H, d, J = 8.2 Hz), 7.32 (1H,

dd, J = 8.0, 2.1 Hz), 7.36 (2H, t, J = 8.8 Hz), 7.44 (1H, d, J = 2.1 Hz), 8.06 (2H, dd, J =

8.8, 5.5 Hz), 8.39 (1H, t, J = 5.5 Hz).

To a solution of the above obtained N-(2-oxo-2-(4-fluorophenyl)ethyl)-

2-(5-bromo-2-methylphenyl)acetamide (1.14 g, 3.13 mmol) in 1,4-dioxane (50 ml) was

added Lawesson’s reagent (1.90 g, 7.05 mmol), and the mixture was heated at reflux for

2 hours. The solvent was evaporated under reduced pressure, and the residue was

purified by silica gel column chromatography (n-hexane : ethyl acetate = 100 : 0 – 75 :

25) to give 2-(5-bromo-2-methylbenzyl)-5-(4-fluorophenyl)thiazole 5e-1 (1.00 g,

88.2%) as a yellow oil: APCI-Mass m/Z 362/364 (M+H). 1H-NMR (DMSO-d6) δ 2.25

(3H, s), 4.36 (2H, s), 7.18 (1H, d, J = 8.0 Hz), 7.25 (2H, t, J = 8.8 Hz), 7.39 (1H, dd, J =

8.0, 1.9 Hz), 7.53 (1H, d, J = 1.9 Hz), 7.66 (2H, dd, J = 8.6, 5.4 Hz), 8.06 (1H, s).

To a solution of the above obtained 2-(5-bromo-2-methylbenzyl)-

5-(4-fluorophenyl)thiazole 5e-1 (362 mg, 1.00 mmol) in tetrahydrofuran (5 ml) and

toluene (10 ml) was added n-butyllithium (2.67 M n-hexane solution, 0.41 ml, 1.10

mmol) dropwise at –78 °C under argon atmosphere, and the mixture was stirred for 10

minutes at the same temperature. Thereto was added tert-butyllithium (1.49 M

n-pentane solution, 1.41 ml, 2.10 mmol) dropwise at –78 °C, and the mixture was

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further stirred for 15 minutes at the same temperature. To the resultant mixture was

added a solution of 2,3,4,6-tetra-O-trimethylsilyl-β-D-gluconolactone3 (1.40 g, 3.00

mmol) in toluene (5 ml) dropwise at –78 °C, and the mixture was further stirred for 1

hour at the same temperature. Subsequently, thereto was added a solution of

methanesulfonic acid (0.324 ml, 4.99 mmol) in methanol (15 ml) dropwise at –78 °C,

and the resultant mixture was allowed to warm to room temperature and stirred

overnight. The mixture was cooled to 0 °C, and thereto was added a saturated aqueous

sodium hydrogen carbonate solution. The resultant mixture was extracted with ethyl

acetate, and the extract was washed with brine and dried over sodium sulfate. The

solvent was evaporated under reduced pressure to give crude

1-(1-methoxyglucopyranosyl)-4-methyl-3-(5-(4-fluorophenyl)-2-thiazolylmethyl)-

benzene 6e-1.

To a solution of the above obtained 1-(1-methoxyglucopyranosyl)-4-methyl-

3-(5-(4-fluorophenyl)-2-thiazolylmethyl)benzene 6e-1 in dichloromethane (7 ml) and

acetonitrile (7 ml) were added successively triethylsilane (0.798 ml, 5.00 mmol) and

boron trifluoride · ethyl ether complex (0.634 ml, 5.00 mmol) at 0 °C, and the mixture

was stirred at the same temperature for 4 hours. The resultant mixture was basified

with a saturated aqueous sodium hydrogen carbonate solution, and the organic solvent

was evaporated under reduced pressure. The residual mixture was extracted with ethyl

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acetate, and the extract was washed with brine and dried over sodium sulfate. The

solvent was evaporated under reduced pressure, and the residue was purified by silica

gel column chromatography (chloroform : methanol = 100 : 0 – 90 : 10) and

crystallization from methanol – ethyl acetate to give 1-(β-D-glucopyranosyl)-4-methyl-

3-(5-(4-fluorophenyl)-2-thiazolylmethyl)benzene 4e-1 (159 mg, 35.7%) as colorless

crystals: mp 187-188 °C. HPLC 99.6% (RT 2.67 min., L-column ODS (3 µm particle

size, 4.6 x 50 mm), CH3CN/20mM-phosphate buffer (pH6.5) (35/65)). APCI-Mass

m/Z 446 (M+H). 1H-NMR (DMSO-d6) δ 2.27 (3H, s), 3.12-3.29 (4H, m), 3.41-3.48

(1H, m), 3.68-3.73 (1H, m), 3.98 (1H, d, J = 9.3 Hz), 4.30, 4.33 (each 1H , d, J = 16.0

Hz), 4.44 (1H, t, J = 4.7 Hz), 4.74 (1H, d, J = 5.6 Hz), 4.93 (2H, d, J = 4.2 Hz),

7.13-7.20 (2H, m), 7.24 (2H, t, J = 8.7 Hz), 7.29 (1H, s), 7.63 (1H, d, J = 8.7 Hz), 7.65

(1H, d, J = 8.7 Hz), 8.04 (1H, s).

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HPLC Analysis of 4b-3

HPLC analysis shows compound 4b-3 is 99.54% purity.

Column: L-column ODS (5 µm particle size, 4.6 x 150 mm). Mobile phase: CH3CN/20 mM-phosphate

buffer (pH6.5) (40/60), flux rate: 1 ml/min, column temperature: 40 degree, sample: 1 mg/ml MeOH.

S20

References

1 Dudash, J., Jr.; Zhang, X.; Zeck, R. E., Johnson, S. G.; Cox, G. G.; Conway, B. R.;

Rybczynski, P. J.; Demarest, K. T. Glycosylated Dihydrochalcones as Potent and

Selective Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors. Bioorg. Med. Chem.

Lett. 2004, 14, 5121-5125.

2 Khan, M. A.; Lynch, B. M.; Hung, Y.-Y. Reactions of Phenyl-Substituted

Heterocyclic Compounds II. Nitrations and Brominations of 1-Phenylpyrazole

Derivatives. Can. J. Chem. 1963, 41, 1540-1547.

3 Horton, D.; Priebe, W. Synthetic Routes to Higher-Carbon Sugars. Reaction of

Lactones with 2-Lithio-1,3-Dithiane. Carbohydr. Res. 1981, 94, 27-41.