Identification of an oxime-containing C-glucosylarene as a potential inhibitor of sodium-dependent glucose co-transporter 2

Article abstract of DOI:10.1016/j.ejmech.2017.11.019

Treatment of hyperglycemia with drugs that block renal glucose reabsorption via inhibition of sodium-dependent glucose cotransporter 2 (SGLT2) is a novel approach to diabetes management. In this study, twenty-seven aryl C-glycosides bearing a C=N/C?N linkage at the glucosyl C6 position were designed, synthesized and evaluated for their inhibitory activity against human SGLT2 (hSGLT2). Compounds with good hSGLT2 inhibition were further investigated to determine their selectivity over hSGLT1. Of these, five representative aryl C-glycosides were chosen for pharmacokinetic analysis. Oxime 2a was determined to have the most promising pharmacokinetic properties and was selected for in vivo glucosuria and plasma glucose level studies, which found it to exhibit comparable efficacy to dapagliflozin (1). Furthermore, 2a was not found to exhibit either significant cytotoxicity (CC₅₀ > 50 μM) or human ether-a-go-go related gene (hERG) inhibition (2% inhibition at 10 μM). Taken together, these efforts culminated in the discovery of oxime 2a as a potential SGLT2 inhibitor.

Full text of DOI:10.1016/j.ejmech.2017.11.019

Accepted Manuscript
Identification of an oxime-containing C-glucosylarene as a potential inhibitor of
sodium-dependent glucose co-transporter 2
Mao-Chia Yuan, Teng-Kuang Yeh, Chiung-Tong Chen, Jen-Shin Song, Yu-Chen
Huang, Tsung-Chih Hsieh, Chung-Yu Huang, Yu-Ling Huang, Min-Hsien Wang, Szu-
Huei Wu, Chun-Hsu Yao, Yu-Sheng Chao, Jinq-Chyi Lee
PII:
S0223-5234(17)30914-5
10.1016/j.ejmech.2017.11.019
EJMECH 9894
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 August 2017
Revised Date: 28 September 2017
Accepted Date: 7 November 2017
Please cite this article as: M.-C. Yuan, T.-K. Yeh, C.-T. Chen, J.-S. Song, Y.-C. Huang, T.-C. Hsieh, C.-
Y. Huang, Y.-L. Huang, M.-H. Wang, S.-H. Wu, C.-H. Yao, Y.-S. Chao, J.-C. Lee, Identification of an
oxime-containing C-glucosylarene as a potential inhibitor of sodium-dependent glucose co-transporter 2,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.019.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Cl
OEt
O
RN
HO
H
OH
OH
Twenty-seven
aryl
C-glycosides,
whose
glycone
is
a
6-amino-/6-imino-6-deoxy-β-
D-glucosyl group, were synthesized. Biological,
pharmacokinetic and efficacy studies culminated in the identification of oxime 2a as a
potential SGLT2 inhibitor.

ACCEPTED MANUSCRIPT
Identification of an oxime-containing C-glucosylarene as a potential inhibitor of
sodium-dependent glucose co-transporter 2
Mao-Chia Yuan1, Teng-Kuang Yeh1, Chiung-Tong Chen, Jen-Shin Song, Yu-Chen Huang, Tsung-Chih
Hsieh, Chung-Yu Huang, Yu-Ling Huang, Min-Hsien Wang, Szu-Huei Wu, Chun-Hsu Yao, Yu-Sheng
Chao, Jinq-Chyi Lee*
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, 35 Keyan
Road, Zhunan Town, Miaoli County 35053, Taiwan
* Corresponding author: E-mail address: jinqchyi@nhri.org.tw (J.-C. Lee);
Tel.: +886 37 246166x35781; fax: +886 37 586456.
1 These authors contributed equally to this work.
Author contributions: T.-K.Y., C.-T.C., J.-S.S., Y.-S.C., and J.-C.L. designed research; M.-C.Y., Y.-C.H.,
T.-C.H., C.-Y.H., Y.-L.H., M.-H.W., S.-H.W., and C.-H.Y. performed research; M.-C.Y., T.-K.Y.,
C.-T.C., J.-S.S., and J.-C.L. analyzed data; and J.-C.L. wrote the paper. The authors declare no conflict
of interest.

ACCEPTED MANUSCRIPT
Abstract: Treatment of hyperglycemia with drugs that block renal glucose reabsorption via inhibition of
sodium-dependent glucose cotransporter 2 (SGLT2) is a novel approach to diabetes management. In this
study, twenty-seven aryl C-glycosides bearing a C=N/C−N linkage at the glucosyl C6 position were
designed, synthesized and evaluated for their inhibitory activity against human SGLT2 (hSGLT2).
Compounds with good hSGLT2 inhibition were further investigated to determine their selectivity over
hSGLT1. Of these, five representative aryl C-glycosides were chosen for pharmacokinetic analysis.
Oxime 2a was determined to have the most promising pharmacokinetic properties and was selected for
in vivo glucosuria and plasma glucose level studies, which found it to exhibit comparable efficacy to
dapagliflozin (1). Furthermore, 2a was not found to exhibit either significant cytotoxicity (CC50 >50 ꢀM)
or human ether-a-go-go related gene (hERG) inhibition (2% inhibition at 10 ꢀM). Taken together, these
efforts culminated in the discovery of oxime 2a as a potential SGLT2 inhibitor.
Keywords: oxime, sodium-dependent glucose co-transporter, aryl C-glycosides, diabetes management
Abbreviations used
Ac2O, acetic anhydride; AMG, α-methyl- -glucopyranoside; BF3·OEt2, boron trifluoride etherate; CHO,
D
Chinese hamster ovary; DMAP, 4-N,N-dimethylaminopyridine; hERG, low human ether-a-go-go related
gene; NaBH3CN, sodium cyanoborohydride; SAR, structure-activity relationship; SD, Sprague-Dawley;
SGLT2,
sodium-dependent
glucose
co-transporter
2;
STZ,
streptozotocin;
TBSCl,
tert-butylchlorodimethylsilane; T2DM, type 2 diabetes mellitus; TBAF, tetra-n-butylammonium
fluoride; TLC, thin layer chromatography.

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1. Introduction
Due to the increasing prevalence of type 2 diabetes mellitus (T2DM) and the adverse effects
associated with traditional anti-diabetic drugs, novel treatments for this disease are urgently required.
Since the kidneys of diabetic patients are known to reabsorb greater amounts of glucose back into the
bloodstream compared to healthy individuals [1], one possible approach entails the use of
sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors, which inhibit reabsorption of glucose in
the kidney and induce urinary glucose excretion, thereby lowering blood sugar without a significant risk
of hypoglycemia (because of their insulin-independent mechanism of action) [2]. SGLT2 inhibitors are
also associated with a reduced risk of cardiovascular disease, lower blood pressure, and modest weight
loss [3]; and can be administered in combination with other oral, anti-diabetic drugs as well as insulin
[4,5]. Although adverse effects of SGLT2 inhibitors include ketoacidosis [6], currently the benefits of
SGLT2 inhibitors are considered to outweigh the risks.
SGLT2 is a high-capacity and low-affinity glucose transporter located on the S1 segment of the
proximal tubule in the kidney, and mediates the reabsorption of the majority (>90%) of renal glucose
filtered by the kidney glomeruli; the remainder is performed by SGLT1, a low-capacity and high-affinity
glucose transporter mainly expressed in the small intestine, but also presented in the S2/S3 segment of
the proximal tubule [7-10]. Some studies recommend the use of selective SGLT2 inhibitors, in order to
avoid the gastrointestinal side effects involved in SGLT1 inhibition [11,12]. On the contrary, dual
SGLT1/SGLT2 or selective SGLT1 inhibitors have also been proposed; of these, sotagliflozin (LX4211)
is the most highly developed (Phase III) [13,14]. The trial data acquired so far indicates that
sotagliflozin is well tolerated: adverse events were mild, and equally distributed across treatment and
placebo groups; and no dose-limiting toxicities have been reported. Additionally, and based on the
results of clinical trials [15,16], SGLT2 inhibitors with the ability to partially inhibit SGLT1 have also
been proposed. Taken together, these findings cast doubt on the notion that good selectivity over SGLT1
is an essential prerequisite for the development of a practical drug.
The first known SGLT inhibitor, phlorizin, was isolated from the bark of the apple tree in 1835

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[17], but found to be metabolically unstable due to the presence of an O-glycosidic bond in its structure.
Structural modifications of phlorizin have given rise to several, selective, SGLT2 inhibitors; including
prodrugs and N-/C-linked glycosides [18]. Of these, the β-C-glycosides were found to exhibit
particularly high metabolic stability, oral bioavailability, and plasma exposure; and six have been
approved for the treatment of type 2 diabetes: dapagliflozin, canagliflozin, ipragliflozin, tofogliflozin,
luseogliflozin, and empagliflozin [19-24]. Also, phlorizin C-glucoside and analogues were recently
reported as potent and selective SGLT2 inhibitors [25]. Regarding the role of the sugar, we have shown
that N-xylosylindole is less well tolerated than N-glucosylindole within the family of N-glycosylindoles
reported as SGLT2 inhibitors [26]. We also found that inhibitors bearing 6-amido-6-deoxyglucosyl
groups imparted good bioactivities and better selectivity than unmodified N-glucosides. Herein, we
build on these results and explore C-glycosides bearing modifications at the glucosyl C6 position,
whose C1 is C-C-linked to position 4 of 1-chloro-2-(4-ethoxybenzyl)benzene (Fig. 1). Dapagliflozin (1)
was used as a template; and all compounds were designed to be structural modifications of it. Synthesis
and structure-activity relationship (SAR) studies of the newly developed aryl C-glycosides,
incorporating the C=N/C−N linkage at the glucosyl C6 position, are presented. Further pharmacokinetic
and animal studies of selected compounds are also discussed.
Cl
OEt
Cl
OEt
O
R
O
HO
HO
H
H
OH
HO
OH
OH
OH
R1O
R3O
R4N
Dapagliflozin (1)
2: R =
3: R =
4: R =
5: R =
N
N
N
H
R2N
N
H
Fig. 1. Design and general structures of desired molecules.

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2. Results and Discussion
2.1.Chemistry
Scheme 1 depicts our synthesis of the novel compounds described herein. Starting from
5-bromo-2-chloro-benzoic acid, a mixture of α- and β-C-glucosides 6 was obtained [27]. A sequential
one-pot process, involving regioselective 6-O-silylation and per-O-acetylation of 6, was carried out to
synthesize the fully protected β-C-glucoside 7. Selective O-silylation at 6-OH of the glucose moiety was
performed with tert-butylchlorodimethylsilane (TBSCl) / pyridine in the presence of a catalytic amount
of 4-N,N-dimethylaminopyridine (DMAP), to provide the 6-OTBS glucoside. Per-O-acetylation was
then accomplished by immediate addition of acetic anhydride (Ac2O), and the fully protected 7 could be
purified by column chromatography on silica gel and isolated in 62% yield. The next step was to
introduce the aldehyde at C6 position, for further coupling with various hydrazines or hydroxylamines.
In order to avoid the exchange with the O4-acetyl group, the TBS group was removed under acidic
condition by treatment of 7 with boron trifluoride etherate (BF3·OEt2) in CH2Cl2, instead of the more
widely used desilylation reagent tetra-n-butylammonium fluoride (TBAF). Subsequent oxidation of the
resulting primary alcohol 8 using Dess-Martin periodinane gave the key intermediate aldehyde 9.
Without further purification, condensation of 9 with a diverse range of hydroxylamines in pyridine
followed by deacetylation under Zemplén conditions afforded the desired oxime ether derivatives 2. In a
similar fashion, the corresponding hydrazones 3 were also successfully synthesized by the sequential
coupling of 9 with various hydrazines in EtOH, and LiOH/MeOH-mediated deacetylation. Further
reduction of 2 and 3 with sodium cyanoborohydride (NaBH3CN) under acidic condition provided the
hydroxylamines 4 and hydrazines/hydrazides 5, respectively.

ACCEPTED MANUSCRIPT
Cl
OEt
Cl
OEt
Cl
OEt
Cl
O
O
O
ref. 27
a
b
HO
HO
HO
AcO
TBSO
AcO
H
H
H
Br
CO H
2
OH
OAc
OAc
OH
OAc
OAc
6
8
7
Cl
OEt
Cl
OEt
Cl
OEt
H
O
O
R
O
R
O
c
d
e
f
N
N
H
H
H
AcO
OAc
AcO
OAc
HO
OH
OAc
OAc
OH
1
1
9
10: R = OR
2: R = OR
2
2
11: R = NR
3: R = NR
Cl
OEt
R
O
N
H
H
HO
OH
OH
1
4: R = OR
2
5: R = NR
Scheme 1. Reagents and conditions: (a) TBSCl, DMAP, pyridine, 0 °C to rt, 18 h; then Ac2O, rt, 3 h; (b)
BF3·OEt2, CH2Cl2, 0 °C, 1 h; (c) Dess-Martinperiodinane, CH2Cl2, rt, 3 h; (d) For 10: H2NOR1, pyridine,
rt, 2 h; For 11: H2NNHR2, EtOH, rt, 2 h; (e) For 2: NaOMe, MeOH/CH2Cl2, 0 °C, 1-2 h; For 3: 1 N
LiOH(aq), MeOH/THF/CH2Cl2, 0 °C, 2 h; (f) NaBH3CN, 6 N HCl in MeOH, 0 °C, 2 h.
2.2.Biological evaluations
The inhibitory activities of all synthesized oximes 2, hydrazones 3, and their reduction products 4
and 5 against human SGLT2 (hSGLT2) or hSGLT1 were ascertained by measuring the inhibition of the
sodium-dependent uptake of [14C]-labeled α-methyl-
-glucopyranoside (AMG) into Chinese hamster
D
ovary (CHO) cells stably expressing hSGLT [28,29]; the data are presented as EC50 values. Phlorizin
and dapagliflozin (1) were used as standards in this in vitro activity evaluation system. The results are
compiled in Table 1, and the results of related SAR studies are discussed below.
In this study, SAR exploration was initiated by investigating the effect of oxime/oxime
ether-containing C-glycosides (2a-2j), as shown in Table 1. Of these, compound 2a bearing an oxime
group at the C6 position of the sugar was found to be the most potent SGLT2 inhibitor, with an EC50
value of 46 nM. Replacing the hydrogen with a methyl group (2b, EC50 = 92 nM) or an ethyl group (2c,
EC50 = 335 nM) led to a 2-fold and 7-fold loss in hSGLT2 inhibitory activity, respectively. Decreased

ACCEPTED MANUSCRIPT
potency was also observed when the ethyl moiety was modified to a 2-hydroxyethyl (2d, EC50 = 194
nM), 2-methoxyethyl (2e, EC50 = 678 nM), or 3,3-dimethylbutyl (2f, EC50 = 8541 nM) group, compared
to 2a. Furthermore, potency was not recovered by substitution with an alkynyl linker (2g, EC50 = 431
nM; 2h, EC50 = 396 nM), a cyclopropylmethyl group (2i, EC50 = 1953 nM), or a benzyl group (2j, EC50
= 2251 nM). Accordingly, the O-substituent on the oxime ethers was restricted to hydrogen and methyl
only.
In an effort to improve the potency of the glucose-C6-substituted C-glycosides, we built
functionality into hydrazone derivatives 3a-3m. In contrast, compound 3a with a 2-hydroxyethyl group
was found to exhibit greater potency (EC50 = 88 nM) than 2d, with an oxime ether (EC50 = 194 nM).
The same trend was observed when the benzyl group was incorporated (3b, EC50 = 38 nM; 2j, EC50 =
2251 nM); suggesting that hydrazone functionality is a potential liability when seeking to enhance
inhibitory activity against hSGLT2.
More analogues were synthesized and explored. When those bearing a nitro substituent on the
ortho- (3c) or para-position (3d) of the phenyl group were constructed, decreased potency was noted
with EC50 values of 457 nM and 370 nM, respectively. In addition, there was no improvement in
hSGLT2 inhibition using imidazoline (3e, EC50 = 429 nM) or benzothiazole (3f, EC50 = 204 nM)
substitution, indicating that aryl or heteroaryl rings bound to nitrogen without a spacer are not well
tolerated at this site. As the project progressed, the N-acylhydrazone moiety was also included to study
the corresponding hSGLT2 inhibition, being a privileged structure associated with diverse
pharmacological activities [30,31]. In general, this subset (3g-3m) of compounds exhibited moderate to
good hSGLT2 inhibitory activity, with EC50 values ranging from 65 to 395 nM. Among them,
compound 3i bearing a 4-chlorophenyl group was found to be the most potent inhibitor of hSGLT2
(EC50 = 65 nM); in contrast, the unsubstituted phenyl group made the resulting 3h (EC50 = 130 nM) two
times less potent than 3i. Decreased potency was also observed when a small methyl group was
introduced (3g, EC50 = 395 nM), which weakened the hSGLT2 inhibition 6-fold. In addition,
replacement of the phenyl group with heteroaryl rings, pyridine, thiophene or furan, gave rise to

ACCEPTED MANUSCRIPT
compounds 3j-3m; no improvements in inhibitory potency compared to 3h resulted (EC50 = 173 – 301
nM).
We next investigated the contribution of the hydroxylamine 4a and methoxyamine 4b, the
reduction products of 2a and 2b, respectively. Interestingly, the reverse trend in potency was observed:
the hydroxylamine 4a (EC50 = 95 nM) was an inferior inhibitor of hSGLT2 compared with 4b (EC50 =
63 nM). The methoxyamine 4b possessed slightly improved potency compared to oxime ether 2b,
whereas the reduction product of 2a, the hydroxylamine 4a, exhibited a 2-fold loss in hSGLT2
inhibitory activity. In the case of reduction of hydrazone 3d and N-acylhydrazone 3m, the resulting
p-nitrophenyl hydrazine 5a and 2-furoic hydrazide 5b were found to show somewhat improved
inhibitory activity against hSGLT2, with EC50 values of 211 nM and 151 nM, respectively.
Subsequently, the selectivity profile of the most active glucose-C6-substituted C-glycosides from
each series was evaluated. The ratios of these eight selected compounds ranged from 63 for 3i to 143 for
3a. Taken together, from the SAR studies of the SGLT2 inhibitors, we have shown (1) good tolerance
for small substituents on the oxime ether moiety; and (2) the moderate to good hSGLT2 inhibitory
activity of hydrazone-based glycosides.

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Table 1
Effect of aryl C-glycosides with the C=N/C−N linkage at glucose C6-position on hSGLT inhibitory activity and selectivity.
Sel.b
hSGLT2
hSGLT1
Compound
Structures
EC50 (nM)a
EC50 (nM)a
2a
2b
2c
2d
2e
2f
46 ± 7
92 ± 14
3576 ± 1206
78
64
－
－
－
－
H
Me
Et
5933 ± 527
－
－
－
－
335 ± 111
194 ± 7
HO
MeO
tBu
Cl
OEt
678 ± 85
8541 ± 3245
O
O
N
H
－
－
431 ± 67
2g
2h
2i
HO
OH
OH
－
－
－
－
396 ± 78
1953 ± 376
－
－
2251 ± 1159
88 ± 36
2j
3a
3b
12572 ± 4095
4931 ± 420
143
130
HO
38 ± 4
NO2
－
－
457 ± 111
3c
－
－
－
－
－
－
－
－
370 ± 133
429 ± 71
204 ± 64
395 ± 79
3d
3e
3f
O2N
N
NH
N
S
Cl
OEt
3g
O
H
N
O
N
H
130 ± 42
65 ± 5
18080 ± 6739
4091 ± 30
139
63
3h
3i
HO
OH
O
O
OH
Cl
N
－
－
－
－
－
－
－
－
173 ± 58
175 ± 31
208 ± 45
301 ± 111
3j
3k
3l
O
O
S
S
O
O
3m
O
H
Cl
Cl
OEt
OEt
95 ± 15
63 ± 17
8167 ± 1822
5390 ± 772
86
86
4a
4b
O
O
N
H
HO
H
OH
Me
OH
O
－
－
－
－
211 ± 98
151 ± 25
5a
5b
H
N
O2N
N
H
HO
H
O
OH
OH
O
2 ± 1
860 ± 162
240 ± 10
430
3
1
phlorizin
79 ± 10
a Data obtained by at least two independent experiments, each experiment performed in triplicate. b Sel: Selectivity values were calculated by EC50
hSGLT1/EC50 hSGLT2.

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2.3.Pharmacokinetic and animal studies
To assess the potential of the designed molecules as SGLT2 inhibitors, representative aryl
C-glycosides from each series, 2a, 2b, 3a, 3h, and 4b, were chosen for pharmacokinetic analysis using
an established model [32]; the results are presented in Table 2. Among them, oxime 2a has the most
favorable pharmacokinetic properties, with the low clearance (5.0 mL/min/kg) and good oral
bioavailability (64%) in Sprague-Dawley (SD) rats. The elimination half-life was 9.0 hr. Methyloxime
2b and its reduction product 4b possessed similar pharmacokinetic profiles with moderate
bioavailability (F% = 40.5 and 32.8%, respectively). On the contrary, neither hydrazone 3a nor
N-acylhydrazone 3h showed druggable properties in rats. Even though the N-acylhydrazone scaffold is a
privileged structure in medicinal chemistry, its vulnerability to hydrolysis is a major consideration for
drug discovery. N-Acylhydrazone 3h was found to convert into dapagliflozin (1) after administration to
rats.
Table 2
Pharmacokinetic properties of selected compounds after oral and intravenous administration to rats
Compound
2a
2b
3a
3h
4b
IV
3
PO
3
IV
3
PO
3
IV
3
PO
3
IV
3
PO
3
IV
3
PO
3
Parameter
N
Dose (mg/kg)
T1/2 (hr)
1.0
4.7
5.0
1.8
1.1
9.0
1.0
3.0
12.6
2.9
1.0
2.5
1.1
0.7
17.6
0.8
1.1
1.2
0.9
0.36
75.7
1.4
0.9
－
1.0
1.9
16.5
1.8
1.0
3.0
Clearance (mL/min/kg)
Vss (L/kg)
－
－
－
－
Cmax (ng/mL)
Tmax (hr)
238.7
1.0
115.9
1.0
34.5
0.7
73
67.2
1.3
AUC(0-inf.) (ng/mL*hr)
F (%)
3267
2273
64.0
1316
540
1026
206
1067
352
32.8
40.5
7.1
To advance the development of oxime 2a for hyperglycemia management via inhibition of SGLT2,
animal studies including glucosuria evaluation and an anti-hyperglycemic assessment were carried out
[32]. As shown in Figure 2A, single oral administrations of 2a of 0.1, 1, 10, and 50 mg/kg to SD rats
induced urine glucose excretions of 275, 2215, 1935, and 2732 mg of glucose per 200 g of body weight

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over 24 h, respectively; resulting in a 476- to 4730-fold elevation in glucosuria relative to the vehicle
control. The efficacy profile of oxime 2a at various doses in this experiment was similar to that of 1.
Compound 2a was further evaluated for anti-hyperglycemic effects in streptozotocin (STZ)-induced
diabetic SD rats (blood glucose of >450 mg/dL). After a single oral administration of 2a (0.1 mg/kg or
10 mg/kg), dapagliflozin (1) (10 mg/kg), or vehicle, blood samples were collected from the tail vein at 0
(predose), 0.5, 1, 2, 3, 4, and 5 h for blood glucose analysis. As shown in Figure 2B, a gradual decrease
in blood glucose level was observed during the period of 5 h; it was found that 2a caused a 44%
reduction in blood glucose level compared with the control, and showed comparable efficacy with 1 at
an oral dose of 10 mg/kg. Additional results of low inhibitory activity in the human ether-a-go-go
related gene (hERG) binding assay (2% inhibition at 10 µM), and a lack of significant cytotoxicity
(CC50 >50 µM), emphasize the potential of 2a as a promising SGLT2 inhibitor.
(A)
(B)
Fig. 2. (A) Effect of oral administration of oxime 2a and dapagliflozin (1) on urine glucose excretion
over 24 h in normal Sprague-Dawley rats. Data are expressed as the mean ± SEM (n = 4/group; vehicle:
n = 8): *p < 0.05 vs vehicle. (B) Antihyperglycemic effect of oxime 2a (0.1 mg/kg or 10 mg/kg) and
dapagliflozin (1) (10 mg/kg) in STZ-induced diabetic Sprague-Dawley rats. Data are expressed as the
mean ± SEM (n = 6/group): *p < 0.05 vs vehicle.

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3. Conclusion
The rational design of aryl
C
-glycoside SGLT2 inhibitors with the C=N/C−N linkage at the
glucosyl C6 position led to the identification of oxime 2a as a potential lead compound. Oxime 2a
showed good in vitro inhibitory activity against hSGLT2, 78-fold selectivity over SGLT1, no significant
cytotoxicity, and low hERG inhibition. Further in vivo studies indicated that dapagliflozin (1) and 2a
could induce glucosuria in normal SD rats and lower plasma glucose levels in STZ-induced diabetic rats
to a comparable extent. The favorable pharmacokinetic profile exhibited by 2a establishes it as a
promising SGLT2 inhibitor for further development. Finally, since 2a is a SGLT2 inhibitor that also
partially inhibits SGLT1, it may inform discussions regarding the importance of selectivity towards
SGLT1 and should be of utility for investigative studies.
4. Experimental section
4.1. Chemistry
Unless otherwise stated, all reagents and solvents were used as received without further
purification. Reaction progress was monitored by analytical thin layer chromatography (TLC), carried
out on glass-backed plates pre-coated with SiO2 60 F254. Column chromatography was carried out
using SiliaFlash P60 SiO2 of 230−400 mesh size. Chromatograms were visualized with UV irradiation
at 254 nm, followed by staining with an aqueous solution of Ce(NH4)2(NO3)6, (NH4)6Mo7O24, and
1
13
H2SO4, and heating on a hot plate. H and C NMR spectra were recorded on Varian Mercury-300 or
Mercury-400 spectrometers. Chemical shifts are reported relative to the internal standard signal of
13
CD3OD (1H,
CDCl3 (1H,
δ
= 3.31 ppm; 13C,
= 7.26 ppm; 13C,
δ
= 49.00 ppm), DMSO-d6 (1H,
δ
= 2.50 ppm; C,
δ
= 39.50 ppm) or
δ
δ = 77.00 ppm), and splitting patterns are recorded as s, singlet; d,
doublet; t, triplet; q, quartet; and m, multiplet. LC/MS data are recorded as m/z values obtained with an
Agilent MSD-1100 mass spectrometer using electrospray ionization (ESI) source. High-resolution mass
spectra were obtained on MAT-95XL or JEOL JMS-700 high resolution mass spectrometers in electron
impact (EI) or fast atom bombardment (FAB) ionization modes. The analysis of test compounds was

ACCEPTED MANUSCRIPT
performed by a Hitachi 2000 series HPLC system with an Agilent ZORBAX Eclipse XDB-C18
reverse-phase column (5 ꢀm, 4.6 mm × 150 mm) in gradient conditions starting from mobile phase A
(MeCN) / mobile phase B (10 mM NH4OAc aqueous solution containing 0.1% formic acid) = 10/90%
to A/B = 90/10% in 45 min at a flow rate of 0.5 mL/min.
4.1.1.
(1S)-2,3,4-Tri-O-acetyl-1,5-anhydro-6-O-[tert-butyl(dimethyl)silyl]-1-[4-chloro-3-(4-ethoxy-
-glucitol (7)
benzyl)phenyl]-
D
A solution of TBSCl (724 mg, 4.8 mmol) in pyridine (3.7 mL) was added to a mixture of 6 (1.51 g,
o
3.7 mmol) and DMAP (207 mg, 1.8 mmol) in pyridine (3.7 mL) at 0 C under N2(g). The reaction
mixture was allowed to warm to room temperature and left overnight, with stirring. After the reaction
was complete, Ac2O (3.5 mL, 36.9 mmol) was added and the solution stirred at room temperature for
another 3 h. The reaction was quenched with H2O at 0 °C, and the resulting mixture was extracted with
CH2Cl2. The organic layer was sequentially washed with 1 N HCl(aq), H2O, and saturated NaHCO3(aq)
,
dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column
1
chromatography (EtOAc/n-Hexane = 1/3) to provide 7 (1.48 g, 62%) as a white solid. H NMR (400
MHz, CDCl3)
1H, ArH), 7.07−7.03 (m, 2H, ArH), 6.82−6.79 (m, 2H, ArH), 5.28 (t,
= 9.6 Hz, 1H, Glc H4), 4.99 (t, = 9.6 Hz, 1H, Glc H2), 4.28 (d, = 10.0 Hz, 1H, Glc H1), 4.08−3.93
(m, 4H, 2 x CH2), 3.78−3.68 (m, 2H, Glc H6a, H6b), 3.63 (ddd, = 9.6, 4.8, 2.4 Hz, 1H, Glc H5), 2.04
= 7.2 Hz, 3H, CH3), 0.86 (s, 9H, 3 x CH3),
170.51, 169.33, 168.80, 157.39,
δ
7.33 (d,
J
= 8.4 Hz, 1H, ArH), 7.16 (dd,
J
= 8.0, 2.0 Hz, 1H, ArH), 7.10 (d,
J = 2.0 Hz,
J
= 9.6 Hz, 1H, Glc H3), 5.20 (t,
J
J
J
J
(s, 3H, CH3), 1.99 (s, 3H, CH3), 1.71 (s, 3H, CH3), 1.39 (t,
J
13
0.01 (s, 3H, CH3), -0.03 (s, 3H, CH3); C NMR (100 MHz, CDCl3)
δ
138.90, 135.58, 134.30, 131.08, 129.73, 129.56, 126.03, 114.41, 79.31, 78.92, 74.46, 72.85, 68.81,
63.30, 62.44, 38.25, 25.74, 20.69, 20.68, 20.31, 18.26, 14.83, -5.37; MS (ESI) m/z: 671 (MNa+).
4.1.2 General procedure for the synthesis of compound 2
BF3·OEt2 (2.7 equiv) was added to a stirred solution of 7 (1.0 equiv) in CH2Cl2 at 0 °C under Ar(g).
After stirring at 0 °C for 30 min, the reaction was quenched by the addition of saturated NaHCO3(aq)
,

ACCEPTED MANUSCRIPT
and the resulting mixture was extracted with CH2Cl2. The organic layer was dried over MgSO4, filtered,
and concentrated under reduced pressure to afford a crude extract of 8, which was used for oxidation
directly without further purification. 1H NMR (400 MHz, CDCl3)
= 8.4, 2.0 Hz, 1H), 7.08−7.03 (m, 3H), 6.83−6.80 (m, 2H), 5.33 (dd,
9.6 Hz, 1H), 5.04 (dd, = 10.0, 9.6 Hz, 1H), 4.28 (d,
δ
7.35 (d,
J
= 8.0 Hz, 1H), 7.19 (dd,
J
J
= 9.6, 9.2 Hz, 1H), 5.13 (t,
J
=
J
J
= 9.6 Hz, 1H), 4.07−3.95 (m, 4H), 3.77−3.70 (m,
1H), 3.65-3.58 (m, 2H), 2.25 (dd,
J = 8.8, 5.2 Hz, 1H), 2.04 (s, 3H), 2.00 (s, 3H), 1.71 (s, 3H), 1.40 (t, J
= 7.2 Hz, 3H); MS (ESI) m/z: 557 (MNa+).
Dess-Martin periodinane (1.5 equiv) was added to a stirred solution of crude 8 (1.0 equiv) in
CH2Cl2 at room temperature under Ar(g). After stirring for 3 h, the solution was diluted with CH2Cl2,
saturated NaHCO3(aq) and saturated Na2S2O3(aq) were then added to the reaction sequentially. The
resulting mixture was stirred for another 30 min at room temperature. The organic layer was separated,
and the aqueous layer was extracted with EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure to afford a crude extract of the aldehyde 9, which was used for the
1
next reaction without further purification. H NMR (400 MHz, CDCl3)
δ
9.58 (s, 1H), 7.38 (d,
J = 8.4
Hz, 1H), 7.21 (dd,
J
= 8.0, 2.0 Hz, 1H), 7.10−7.04 (m, 3H), 6.84−6.80 (m, 2H), 5.34 (t,
J
= 9.2 Hz, 1H),
5.27 (dd, = 10.0, 9.6 Hz, 1H), 5.08 (dd,
J
J
= 9.6, 9.2 Hz, 1H), 4.39 (d,
J = 9.6 Hz, 1H), 4.09−3.91 (m,
5H), 2.07 (s, 3H), 2.01 (s, 3H), 1.71 (s, 3H), 1.40 (t,
J
= 6.8 Hz, 3H); MS (ESI) m/z: 555 (MNa+).
Hydroxylamine (1.5 equiv) was added to a stirred solution of aldehyde 9 (1.0 equiv) in pyridine at
room temperature. After stirring at the same temperature for 2 h, the solvent was removed under
reduced pressure. Water was added to the residue, and the mixture was extracted with CH2Cl2. The
organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by column chromatography (EtOAc/ n-Hexane) to provide compound 10 as a mixture of
E/Z-isomers.
A 30% solution of NaOMe in MeOH (1.1 equiv) was added to a stirred solution of compound 10
(1.0 equiv) in mixed solvents (MeOH/CH2Cl2, v/v: 1/1) in an ice bath. The reaction was warmed up to
room temperature and stirred for 1~2 h. The reaction was neutralized with Amberlite-120 acidic resin,

ACCEPTED MANUSCRIPT
and the mixture was filtered to remove the resin followed by washing with MeOH. The filtrate was
concentrated in vacuo and the residue was purified by column chromatography (MeOH/CH2Cl2) to give
compound 2 as a mixture of E/Z isomers. The major isomer was determined to be present in at least
80% for all samples by HPLC analysis. The NMR data specified is for the major isomer only.
4.1.2.1 (2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-[(hydroxyimino)methyl]tetrahydro-
2H-pyran-3,4,5-triol (2a)
The title compound was obtained from 7 according to the general procedure in 40% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ
7.34 (d,
J
= 8.0 Hz, 1H, ArH), 7.33 (d,
J
= 7.2 Hz, 1H, CH=N),
= 9.2 Hz, 1H, Glc
7.26−7.21 (m, 2H, ArH), 7.10−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.13 (d,
J
H1), 4.05−3.87 (m, 5H, 2 x CH2, Glc H5), 3.50−3.46 (m, 2H, Glc H3, H4), 3.34−3.30 (m, 1H, Glc H2),
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
1.35 (t,
J
δ 158.85, 149.31, 140.04, 139.56, 134.60,
132.78, 131.76, 130.86, 130.19, 128.06, 115.43, 82.93, 79.12, 79.08, 76.19, 73.51, 64.41, 39.23, 15.20;
MS (ESI) m/z: 422 (MH+), 444 (MNa+); HRMS (FAB) for C21H25ClNO6: calcd, 422.1370; found,
422.1375.
4.1.2.2 (2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-[(methoxyimino)methyl]tetrahydro-
2H-pyran-3,4,5-triol (2b)
The title compound was obtained from 7 according to the general procedure in 35% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ
7.34 (d,
J
= 8.0 Hz, 1H, ArH), 7.33 (d,
J
= 7.2 Hz, 1H, CH=N),
= 9.6 Hz, 1H, Glc
7.26−7.21 (m, 2H, ArH), 7.09−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.13 (d,
J
H1), 4.05−3.86 (m, 5H, 2 x CH2, Glc H5), 3.82 (s, 3H, OCH3), 3.49−3.44 (m, 2H, Glc H3, H4),
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
δ 158.86,
3.33−3.28 (m, 1H, Glc H2), 1.35 (t,
J
149.13, 140.07, 139.49, 134.63, 132.77, 131.75, 130.86, 130.20, 128.07, 115.43, 82.96, 79.12, 78.83,
76.14, 73.40, 64.41, 62.08, 39.22, 15.20; MS (ESI) m/z: 436 (MH+), 458 (MNa+); HRMS (EI) for
C22H26ClNO6: calcd, 435.1449; found, 435.1456.
4.1.2.3 (2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-[(ethoxyimino)methyl]tetrahydro-
2H-pyran-3,4,5-triol (2c)

ACCEPTED MANUSCRIPT
The title compound was obtained from 7 according to the general procedure in 17% yield over four
steps. 1H NMR (300 MHz, CD3OD)
δ
7.34 (d,
J
= 8.1 Hz, 1H, ArH), 7.33 (d,
J
= 6.9 Hz, 1H, CH=N),
= 9.3 Hz, 1H, Glc
7.26−7.20 (m, 2H, ArH), 7.10−7.05 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.13 (d,
J
H1), 4.08 (q,
J
= 7.2 Hz, 2H, CH2), 4.01−3.86 (m, 5H, 2 x CH2, Glc H5), 3.50−3.43 (m, 2H, Glc H3,
= 7.2 Hz, 3H, CH3); 13C NMR
H4), 3.42−3.28 (m, 1H, Glc H2), 1.35 (t, J = 7.2 Hz, 3H, CH3), 1.21 (t, J
(100 MHz, CD3OD)
δ 158.86, 148.89, 140.07, 139.51, 134.62, 132.77, 131.75, 130.86, 130.20, 128.07,
115.43, 82.95, 79.13, 78.96, 76.16, 73.42, 70.49, 64.40, 39.23, 15.20, 14.77; MS (ESI) m/z: 450 (MH+),
472 (MNa+); HRMS (EI) for C23H28ClNO6: calcd, 449.1605; found, 449.1603.
4.1.2.4 (2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[(2-hydroxyethoxy)imino]methyl}-
tetrahydro-2H-pyran-3,4,5-triol (2d)
The title compound was obtained from 7 according to the general procedure in 23% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ 7.39 (d, J = 6.8 Hz, 1H, CH=N), 7.34 (d, J = 8.4 Hz, 1H, ArH),
7.26−7.21 (m, 2H, ArH), 7.09−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.15−4.09 (m, 3H, CH2,
Glc H1), 4.05−3.88 (m, 5H, 2 x CH2, Glc H5), 3.74−3.71 (m, 2H, CH2), 3.51−3.44 (m, 2H, Glc H3, H4),
3.34−3.28 (m, 1H, Glc H2), 1.35 (t,
J δ 158.83,
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
149.56, 140.06, 139.45, 134.63, 132.75, 131.73, 130.84, 130.20, 128.07, 115.43, 82.93, 79.06, 78.90,
76.31, 76.11, 73.38, 64.41, 61.40, 39.21, 15.20; MS (ESI) m/z: 466 (MH+), 488 (MNa+); HRMS (EI) for
C23H28ClNO7: calcd, 465.1554; found, 465.1552.
4.1.2.5 (2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{ [(2-methoxyethoxy)imino]methyl}-
tetrahydro-2H-pyran-3,4,5-triol (2e)
The title compound was obtained from 7 according to the general procedure in 20% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ 7.37 (d, J = 6.8 Hz, 1H, CH=N), 7.34 (d, J = 8.4 Hz, 1H, ArH),
7.26−7.21 (m, 2H, ArH), 7.09−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.19−4.12 (m, 3H, CH2,
Glc H1), 4.05−3.88 (m, 5H, 2 x CH2, Glc H5), 3.62−3.59 (m, 2H, CH2), 3.48−3.46 (m, 2H, Glc H3, H4),
3.33−3.29 (m, 4H, OCH3, Glc H2), 1.35 (t,
J δ
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
158.85, 149.58, 140.06, 139.50, 134.62, 132.75, 131.74, 130.86, 130.19, 128.07, 115.42, 82.95, 79.08,

ACCEPTED MANUSCRIPT
78.90, 76.15, 74.10, 73.37, 71.92, 64.39, 59.11, 39.21, 15.20; MS (ESI) m/z: 480 (MH+), 502 (MNa+);
HRMS (EI) for C24H30ClNO7: calcd, 479.1711; found, 479.1713.
4.1.2.6
(2S,3R,4R,5S,6R)-2-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-{[(3,3-dimethylbutoxy)imino]-
methyl}tetrahydro-2 H-pyran-3,4,5-triol (2f)
The title compound was obtained from 7 according to the general procedure in 74% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ 7.33 (d, J = 8.4 Hz, 1H, ArH), 7.32 (d, J = 7.2 Hz, 1H, CH=N),
7.26−7.21 (m, 2H, ArH), 7.09−7.05 (m, 2H, ArH), 6.80−6.76 (m, 2H, ArH), 4.15−4.08 (m, 3H, CH2,
Glc H1), 4.05−3.87 (m , 5H, 2 x CH2, Glc H5), 3.50−3.44 (m, 2H, Glc H3, H4), 3.34−3.28 (m, 1H, Glc
H2), 1.55 (t,
J
= 7.2 Hz, 2H, CH2), 1.34 (t,
158.82, 148.83, 140.05, 139.47, 134.62, 132.76, 131.75, 130.85, 130.20, 128.06,
J
= 7.2 Hz, 3H, CH3), 0.92 (s ,9H, 3 x CH3); 13C NMR (100
MHz, CD3OD)
δ
115.44, 82.92, 79.13, 78.91, 76.13, 73.42, 72.57, 64.40, 43.10, 39.22, 30.43, 30.17, 15.21; MS (ESI) m/z
:
506 (MH+), 528 (MNa+); HRMS (EI) for C27H36ClNO6: calcd, 505.2231; found, 505.2224.
4.1.2.7
(2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[(prop-2-yn-1-yloxy)imino]-
methyl}tetrahydro-2H-pyran-3,4,5-triol (2g)
The title compound was obtained from 7 according to the general procedure in 39% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ
7.39 (d, J = 7.2 Hz, 1H, CH=N), 7.34 (d, J = 8.0 Hz, 1H, ArH),
7.26−7.21 (m, 2H, ArH), 7.10−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.63 (d,
J = 2.4 Hz, 2H,
CH2), 4.14 (d,
J
= 9.6 Hz, 1H, Glc H1), 4.06−3.90 (m, 5H, 2 x CH2, Glc H5), 3.49−3.46 (m, 2H, Glc H3,
= 7.2 Hz, 3H, CH3); 13C NMR
H4), 3.34−3.29 (m, 1H, Glc H2), 2.84 (t,
J = 2.4 Hz, 1H, CH), 1.35 (t, J
(100 MHz, CD3OD)
δ 158.85, 150.44, 140.07, 139.45, 134.64, 132.77, 131.75, 130.87, 130.20, 128.07,
115.43, 82.97, 80.27, 79.09, 78.75, 76.13, 76.06, 73.35, 64.41, 62.38, 39.22, 15.20; MS (ESI) m/z: 460
(MH+), 482 (MNa+); HRMS (EI) for C24H26ClNO6: calcd, 459.1449; found, 459.1440.
4.1.2.8
(2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[(pent-2-yn-1-yloxy)imino]-
methyl}tetrahydro-2H-pyran-3,4,5-triol (2h)
The title compound was obtained from 7 according to the general procedure in 20% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ 7.37 (d, J = 6.8 Hz, 1H, CH=N), 7.34 (d, J = 8.4 Hz, 1H, ArH),

ACCEPTED MANUSCRIPT
7.26−7.21 (m, 2H, ArH), 7.09−7.07 (m, 2H, ArH), 6.80−6.78 (m, 2H, ArH), 4.59 (t,
J = 2.4 Hz, 2H,
CH2), 4.14 (d, J = 9.6 Hz, 1H, Glc H1), 4.05−3.89 (m, 5H, 2 x CH2, Glc H5), 3.50−3.44 (m, 2H, Glc H3,
H4), 3.34−3.27 (m, 1H, Glc H2), 2.17 (qt, = 7.2 Hz, 3H, CH3), 1.09
J
= 7.6, 2.4 Hz, 2H, CH2), 1.35 (t,
J
(t,
J
= 7.6 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
δ 158.85, 150.03, 140.06, 139.46, 134.63,
132.77, 131.76, 130.86, 130.20, 128.07, 115.43, 89.29, 82.96, 79.08, 78.80, 76.13, 75.92, 73.37, 64.40,
62.97, 39.22, 15.20, 14.15, 13.04; MS (ESI) m/z: 510 (MNa+); HRMS (EI) for C26H30ClNO6: calcd,
487.1762; found, 487.1765.
4.1.2.9
(2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[(cyclopropylmethoxy)imino]-
methyl}tetrahydro-2H-pyran-3,4,5-triol (2i)
The title compound was obtained from 7 according to the general procedure in 49 % yield over four
steps. 1H NMR (400 MHz, CD3OD)
7.26−7.21 (m, 2H, ArH), 7.10−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.13 (d,
H1), 4.05−3.95 (m, 4H, 2 x CH2), 3.91−3.83 (m, 3H, CH2, Glc H5), 3.50−3.44 (m, 2H, Glc H3, H4),
δ
7.35 (d,
J
= 7.2 Hz, 1H, CH=N), 7.34 (d,
J
= 8.0 Hz, 1H, ArH),
= 9.6 Hz, 1H, Glc
J
3.34−3.28 (m, 1H, Glc H2), 1.35 (t,
J = 7.2 Hz, 3H, CH3), 1.15−1.06 (m, 1H, CH), 0.53−0.48 (m, 2H,
CH2), 0.26−0.22 (m, 2H, CH2); 13C NMR (100 MHz, CD3OD)
δ
158.85, 148.83, 140.06, 139.50, 134.63,
132.80, 131.74, 130.86, 130.19, 128.06, 115.47, 82.94, 79.83, 79.14, 78.93, 76.16, 73.46, 64.44, 39.23,
15.20, 10.98, 3.43, 3.37; MS (ESI) m/z: 476 (MH+), 498 (MNa+); HRMS (EI) for C25H30ClNO6: calcd,
475.1762; found, 475.1763.
4.1.2.10 (2R,3S,4R,5R,6S)-2-{[(Benzyloxy)imino]methyl}-6-[4-chloro-3-(4-ethoxybenzyl)phenyl]tetra-
hydro-2H-pyran-3,4,5-triol (2j)
The title compound was obtained from 7 according to the general procedure in 79% yield over four
1
steps. H NMR (400 MHz, CD3OD)
δ
7.42 (d,
J
= 7.2 Hz, 1H, CH=N), 7.35−7.20 (m, 8H, ArH),
= 9.6 Hz, 1H, Glc H1),
7.09−7.06 (m, 2H, ArH), 6.80−6.75 (m, 2H, ArH), 5.06 (s, 2H, CH2), 4.13 (d,
J
4.04−3.88 (m, 5H, 2 x CH2, Glc H5), 3.48−3.46 (m, 2H, Glc H3, H4), 3.34−3.31 (m, 1H, Glc H2), 1.34
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
(t,
J
δ 158.83, 149.65, 140.06, 139.45, 138.87,
134.62, 132.75, 131.75, 130.86, 130.19, 129.35, 129.28, 128.87, 128.07, 115.42, 82.92, 79.07, 78.85,

ACCEPTED MANUSCRIPT
77.07, 76.12, 73.40, 64.39, 39.21, 15.20; MS (ESI) m/z: 534 (MNa+); HRMS (EI) for C28H30ClNO6:
calcd, 511.1762; found, 511.1755.
4.1.3 General procedure for the synthesis of compound 3
As described above, key intermediate aldehyde 9 for the synthesis of hydrazone/N-acylhydrazone 3
could be obtained from 7 via a 2-step procedure. Without purification, 9 (1.0 equiv.) was reacted with
various hydrazines (1.0 equiv.) in EtOH at room temperature for 2 h. Water was added to the reaction,
and the resulting mixture was extracted with CH2Cl2 or EtOAc. The organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography
(EtOAc/ n-Hexane) to provide compound 11.
A 1 M solution of LiOH in H2O (1.17 equiv) was added to a stirred solution of compound 11 (1.0
o
equiv) in mixed solvents (MeOH/THF/CH2Cl2, v/v: 2/2/1) in an ice bath. After stirring at 0 C for 2 h,
the reaction was neutralized with CH3COOH. The reaction mixture was extracted with EtOAc, and the
organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by
column chromatography (MeOH/CH2Cl2) to provide compound 3 as see above E/Z isomers. The major
isomer was determined to be present in at least 90% for all samples by HPLC analysis except compound
3g (The ratio of the isomers was ~1.4:1 presented in 1H-NMR spectrum). The NMR data specified is for
the major isomer only.
4.1.3.1
(2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[2-(2-hydroxyethyl)hydrazinyl-
idene]methyl}tetrahydro-2H-pyran-3,4,5-triol (3a)
The title compound was obtained from 7 according to the general procedure in 51% yield over four
1
steps. H NMR (400 MHz, CD3OD)
δ
7.33 (d,
= 6.0 Hz, 1H, CH=N), 6.80−6.77 (m, 2H, ArH), 4.12 (d,
1H, Glc H1), 4.05−3.95 (m, 4H, 2 x CH2), 3.83 (dd, = 9.2, 6.0 Hz, 1H, Glc H5), 3.65 (t,
J = 8.0 Hz, 1H, ArH), 7.26−7.20 (m, 2H, ArH),
7.09−7.05 (m, 2H, ArH), 6.94 (d,
J
J
= 9.6 Hz,
J
J = 5.6 Hz,
2H, CH2), 3.52−3.45 (m, 2H, Glc H3, H4), 3.34−3.27 (m, 1H, Glc H2), 3.17 (t,
J = 5.6 Hz, 2H, CH2),
1.35 (t,
J
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
δ 158.86, 140.06, 139.71, 138.84, 134.58,
132.78, 131.81, 130.85, 130.18, 128.14, 115.43, 82.89, 81.07, 79.17, 76.25, 73.84, 64.41, 60.91, 51.68,

ACCEPTED MANUSCRIPT
39.21, 15.20; MS (ESI) m/z: 465 (MH+), 487 (MNa+); HRMS (EI) for C23H29ClN2O6: calcd, 464.1714;
found, 464.1707.
4.1.3.2 (2R,3S,4R,5R,6S)-2-[ (2-Benzylhydrazinylidene)methyl]-6-[4-chloro-3-(4-ethoxybenzyl)phenyl]-
tetrahydro-2H-pyran-3,4,5-triol (3b)
The title compound was obtained from 7 according to the general procedure in 11% yield over four
steps. 1H NMR (400 MHz, DMSO-d6)
7.09−7.05 (m, 2H, ArH), 6.82−6.79 (m, 2H, ArH), 6.75 (d,
1H, OH), 4.89−4.86 (m, 2H, 2 x OH), 4.12 (d, = 5.2 Hz, 2H, CH2), 4.03 (d,
δ
7.35 (d,
J
= 8.0 Hz, 1H, ArH), 7.29−7.15 (m, 8H, ArH, NH),
= 6.8 Hz, 1H, CH=N), 5.03 (d, = 4.0 Hz,
= 9.6 Hz, 1H, Glc H1),
J
J
J
J
4.00−3.92 (m, 4H, 2 x CH2), 3.67 (dd,
J = 9.2, 6.8 Hz, 1H, Glc H5), 3.39−3.12 (m, 3H, Glc H2, H3, H4),
1.28 (t,
J
= 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3)
δ 157.37, 139.13, 136.96, 136.87, 136.37,
134.26, 131.15, 130.35, 129.78, 129.69, 128.73, 128.23, 127.73, 126.48, 114.43, 81.13, 77.98, 77.20,
74.50, 72.80, 63.34, 52.44, 38.37, 14.84; MS (ESI) m/z: 511 (MH+), 533 (MNa+); HRMS (FAB) for
C28H31ClN2O5: calcd, 510.1921; found, 510.1922.
4.1.3.3
(2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[2-(2-nitrophenyl)hydrazinyl-
idene] methyl}tetrahydro-2H-pyran-3,4,5-triol (3c)
The title compound was obtained from 7 according to the general procedure in 43% yield over four
steps. 1H NMR (400 MHz, CD3OD)
ArH), 7.49 (ddd, = 8.8, 7.2, 1.6 Hz, 1H, ArH), 7.44 (d,
ArH), 7.29−7.23 (m, 2H, ArH), 7.07−7.03 (m, 2H, ArH), 6.82 (ddd,
6.77−6.73 (m, 2H, ArH), 4.20 (d, = 9.2 Hz, 1H, Glc H1), 4.06−3.90 (m, 5H, 2 x CH2, Glc H5), 3.61
δ
8.09 (dd,
J
= 8.8, 1.6 Hz, 1H, ArH), 7.81 (dd,
= 6.0 Hz, 1H, CH=N), 7.33 (d,
= 8.8, 7.2, 1.6 Hz, 1H, ArH),
J
= 8.8, 1.6 Hz, 1H,
J
J
J
= 8.4 Hz, 1H,
J
J
(dd,
J
= 9.6, 8.8 Hz, 1H, Glc H4), 3.53 (dd, = 9.6, 8.8 Hz, 1H,
J
= 9.2, 8.8 Hz, 1H, Glc H3), 3.37 (dd,
J
Glc H2), 1.32 (t,
J
= 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
δ 158.82, 145.03, 142.95, 140.11,
139.59, 136.99, 134.63, 132.74, 132.45, 131.74, 130.84, 130.20, 128.14, 126.61, 119.30, 117.20, 115.42,
82.94, 81.03, 79.22, 76.21, 73.48, 64.38, 39.21, 15.19; MS (ESI) m/z: 564 (MNa+); HRMS (FAB) for
C27H28ClN3O7: calcd, 541.1616; found, 541.1619.
4.1.3.4
(2S,3R,4R,5S,6R)-2-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-6-{[2-(4-nitrophenyl)hydrazinyl-

ACCEPTED MANUSCRIPT
idene]methyl}tetrahydro-2H-pyran-3,4,5-triol (3d)
The title compound was obtained from 7 according to the general procedure in 26% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ
8.07 (d, J = 9.2 Hz, 2H, ArH), 7.33 (d, J = 8.4 Hz, 1H, ArH),
7.28−7.20 (m, 3H, ArH, CH=N), 7.07−7.02 (m, 4H, ArH), 6.78−6.75 (m, 2H, ArH), 4.19 (d,
1H, Glc H1), 4.04−3.92 (m, 5H, 2 x CH2, Glc H5), 3.58 (t, = 8.8 Hz, 1H, Glc H4), 3.53 (t,
1H, Glc H3), 3.37 (t, = 8.8 Hz, 1H, Glc H2), 1.32 (t,
CD3OD) 158.82, 152.15, 142.09, 140.72, 140.09, 139.57, 134.64, 132.74, 131.77, 130.84, 130.21,
J
= 9.6 Hz,
J
J
= 8.8 Hz,
J
J
= 6.8 Hz, 3H, CH3); 13C NMR (100 MHz,
δ
128.13, 126.90, 115.43, 112.28, 82.94, 81.02, 79.28, 76.20, 73.59, 64.40, 39.22, 15.19; MS (ESI) m/z
:
542 (MH+), 564 (MNa+); HRMS (FAB) for C27H29ClN3O7: calcd, 542.1694; found, 542.1687.
4.1.3.5 (2S,3R,4R,5S,6R)-2-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-{[2-(4,5-dihydro-1H-imidazol-2-yl)-
hydrazinylidene]methyl}tetrahydro-2H-pyran-3,4,5-triol (3e)
The title compound was obtained from 7 according to the general procedure in 17% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ
7.39 (d,
J
= 5.6 Hz, 1H, CH=N), 7.35 (d,
J
= 8.4 Hz, 1H, ArH),
= 9.6 Hz, 1H, Glc
7.25−7.21 (m, 2H, ArH), 7.09−7.05 (m, 2H, ArH), 6.80−6.76 (m, 2H, ArH), 4.16 (d,
J
H1), 4.05−3.94 (m, 5H, 2 x CH2, Glc H5), 3.69 (s, 4H, 2 x CH2), 3.56 (t,
(t, = 8.8 Hz, 1H, Glc H3), 3.36 (dd, = 9.6, 8.8 Hz, 1H, Glc H2), 1.34 (t,
NMR (100 MHz, CD3OD)
J
= 9.2 Hz, 1H, Glc H4), 3.50
13
J
J
J
= 6.8 Hz, 3H, CH3); C
δ
158.85 149.71, 140.14, 139.37, 134.72, 132.74, 131.82, 130.85, 130.26,
128.13, 115.44, 83.07, 80.43, 79.18, 76.01, 73.25, 64.42, 44.02, 39.18, 15.20; MS (ESI) m/z: 489 (MH+),
511 (MNa+); HRMS (FAB) for C24H29ClN4O5: calcd, 488.1826; found, 488.1833.
4.1.3.6
(2R,3S,4R,5R,6S)-2-{[2-(1,3-benzothiazol-2-yl)hydrazinylidene]methyl}-6-[4-chloro-3-(4-
ethoxybenzyl)phenyl]tetrahydro-2H-pyran-3,4,5-triol (3f)
The title compound was obtained from 7 according to the general procedure in 28% yield over four
steps. 1H NMR (400 MHz, CD3OD)
(d, = 6.4 Hz, 1H, CH=N), 7.32 (d,
ArH), 6.77−6.73 (m, 2H, ArH), 4.19 (d,
(q, = 7.2 Hz, 2H, CH2), 3.59 (dd, = 9.2, 8.8 Hz, 1H, Glc H4), 3.54 (t, J
δ
7.60 (d,
= 8.4 Hz, 1H, ArH), 7.29−7.22 (m, 3H, ArH), 7.11−7.03 (m, 3H,
= 9.6 Hz, 1H, Glc H1), 4.02−3.98 (m, 3H, CH2, Glc H5), 3.92
= 8.8 Hz, 1H, Glc H3), 3.37
J = 7.6 Hz, 1H, ArH), 7.42 (d, J = 8.0 Hz, 1H, ArH), 7.35
J
J
J
J
J

ACCEPTED MANUSCRIPT
(dd, J = 9.2, 8.8 Hz, 1H, Glc H2), 1.31 (t, J δ 169.86,
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
158.83, 140.09, 139.56, 134.65, 132.75, 131.75, 130.86, 130.20, 128.14, 127.09, 123.28, 122.25, 115.42,
82.92, 80.79, 79.19, 76.20, 73.55, 64.39, 39.24, 15.19; MS (ESI) m/z: 554 (MH+), 576 (MNa+); HRMS
(FAB) for C28H29ClN3O5S: calcd, 554.1516; found, 554.1509.
4.1.3.7 N'-[{(2R,3S,4R,5R,6S)-6-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl}methylidene]acetohydrazide (3g)
The title compound was obtained from 7 according to the general procedure in 23% yield over four
steps. The ratio of the isomers was ~1.4:1. The proton chemical shifts of CH3 groups of the isomers are
at 2.19 (s) and 2.00 (s) ppm, respectively; MS (ESI) m/z: 463 (MH+); HRMS (EI) for C23H27ClN2O6:
calcd, 462.1558; found, 462.1557.
4.1.3.8 N'-[{(2R,3S,4R,5R,6S)-6-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl}methylidene]benzohydrazide (3h)
The title compound was obtained from 7 according to the general procedure in 34% yield over four
steps. 1H NMR (400 MHz, CD3OD)
CH=N), 7.63−7.56 (m, 1H, ArH), 7.51−7.47 (m, 2H, ArH), 7.34 (d,
2H, ArH), 7.09−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.20 (d,
δ
7.87 (dd,
J
= 7.2, 1.6 Hz, 2H, ArH), 7.63 (d,
= 8.4 Hz, 1H, ArH), 7.27−7.23 (m,
= 9.6 Hz, 1H, Glc H1),
J = 6.0 Hz, 1H,
J
J
4.07−3.94 (m, 5H, 2 x CH2, Glc H5), 3.62 (t,
3.36 (t, = 9.2 Hz, 1H, Glc H2), 1.34 (t,
J
= 9.2 Hz, 1H, Glc H4), 3.53 (t,
J = 8.8 Hz, 1H, Glc H3),
J
J
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
δ
163.18, 156.91, 148.55, 139.08, 138.06, 133.23, 132.20, 131.81, 131.14, 130.80, 129.59, 128.84, 128.50,
127.58, 127.36, 114.29, 80.96, 79.45, 77.83, 74.33, 72.02, 62.87, 37.64, 14.70; MS (ESI) m/z: 525
(MH+), 547 (MNa+); HRMS (FAB) for C28H29ClN2O6: calcd, 524.1714; found, 524.1711.
4.1.3.9 4-Chloro-N'-[{(2R,3S,4R,5R,6S)-6-[4-chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetra-
hydro-2H-pyran-2-yl}methylidene]benzohydrazide (3i)
The title compound was obtained from 7 according to the general procedure in 34% yield over four
steps. 1H NMR (400 MHz, CD3OD)
δ
7.87 (d,
J
= 8.4 Hz, 2H, ArH), 7.62 (d,
J
= 5.6 Hz, 1H, CH=N),
= 8.8
7.51 (d, = 8.8 Hz, 2H, ArH), 7.35 (d,
J
J
= 8.0 Hz, 1H, ArH), 7.26−7.23 (m, 2H, ArH), 7.07 (d, J

ACCEPTED MANUSCRIPT
Hz, 2H, ArH), 6.80−6.78 (m, 2H, ArH), 4.20 (d,
J
= 10.0 Hz, 1H, Glc H1), 4.07−3.95 (m, 5H, 2 x CH2,
Glc H5), 3.62 (dd,
Glc H2), 1.27 (t,
J
= 9.6, 9.2 Hz, 1H, Glc H4), 3.53 (t,
J
= 8.8 Hz, 1H, Glc H3), 3.36 (t,
J = 9.2 Hz, 1H,
J
= 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
δ 162.09, 156.90, 148.96,
139.08, 138.05, 136.65, 132.20, 131.90, 131.14, 130.78, 129.58, 129.53, 128.84, 128.61, 127.35, 114.29,
80.95, 79.38, 77.82, 74.33, 71.99, 62.87, 37.64, 14.70; MS (ESI) m/z: 559 (MH+), 581 (MNa+); HRMS
(FAB) for C28H28Cl2N2O6: calcd, 558.1324; found, 558.1325.
4.1.3.10 N'-[{(2R,3S,4R,5R,6S)-6-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl}methylidene]pyridine-4-carbohydrazide (3j)
The title compound was obtained from 7 according to the general procedure in 8% yield over four steps.
1H NMR (400 MHz, CD3OD)
1H, CH=N), 7.35 (d, = 8.0 Hz, 1H, ArH), 7.27−7.23 (m, 2H, ArH), 7.10−7.06 (m, 2H, ArH),
6.81−6.78 (m, 2H, ArH), 4.20 (d, = 9.6 Hz, 1H, Glc H1), 4.08−3.94 (m, 5H, 2 x CH2, Glc H5), 3.62
δ 8.73−8.70 (m, 2H, ArH), 7.84−7.80 (m, 2H, ArH), 7.67 (d, J = 5.6 Hz,
J
J
(dd,
J
= 9.6, 9.2 Hz, 1H, Glc H4), 3.53 (t,
J
= 8.8 Hz, 1H, Glc H3), 3.67 (t,
J
= 9.2 Hz, 1H, Glc H2),
1.34 (t,
J
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
δ 161.66, 156.88, 150.32, 149.92,
140.27, 139.04, 138.02, 132.18, 131.12, 130.75, 129.55, 128.81, 127.32, 121.47, 114.28, 80.92, 79.26,
77.80, 74.29, 71.95, 62.85, 37.61, 14.67; MS (ESI) m/z: 548 (MNa+); HRMS (FAB) for C27H28ClN3O6:
calcd, 525.1667; found, 525.1664.
4.1.3.11 N'-[{(2R,3S,4R,5R,6S)-6-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl}methylidene]thiophene-2-carbohydrazide (3k)
The title compound was obtained from 7 according to the general procedure in 51% yield over four
steps. 1H NMR (400 MHz, CD3OD)
(d, = 5.6 Hz, 1H, CH=N), 7.36−7.15 (m, 4H, ArH), 7.07 (d,
2H, ArH), 4.19 (d, = 9.2 Hz, 1H, Glc H1), 4.06−3.93 (m, 5H, 2 x CH2, Glc H5), 3.61 (dd,
δ
7.80 (d,
J
= 2.8 Hz, 1H, ArH), 7.74 (d,
J = 4.4 Hz, 1H, ArH), 7.61
J
J
= 8.8 Hz, 2H, ArH), 6.78 (d,
J
= 8.4 Hz,
J
J = 9.6, 8.8
Hz, 1H, Glc H4), 3.53 (t,
J
= 8.8 Hz, 1H, Glc H3), 3.36 (dd,
J
= 9.2, 8.8 Hz, 1H, Glc H2), 1.34 (t,
J =
7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD/CDCl3)
δ
160.81, 157.88, 148.90, 139.37, 138.13,
137.06, 134.38, 132.35, 132.04, 131.08, 130.29, 130.13, 129.85, 128.41, 127.25, 114.95, 82.23, 79.35,

ACCEPTED MANUSCRIPT
78.34, 75.12, 72.11, 63.98, 38.80, 15.02; MS (ESI) m/z: 531 (MH+), 553 (MNa+); HRMS (FAB) for
C26H27ClN2O6S: calcd, 530.1278; found, 530.1282.
4.1.3.12 N'-[{(2R,3S,4R,5R,6S)-6-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl}methylidene]thiophene-3-carbohydrazide (3l)
The title compound was obtained from 7 according to the general procedure in 41% yield over four
steps. 1H NMR (400 MHz, CD3OD)
7.57−7.50 (m, 2H, ArH), 7.34 (d,
ArH), 6.80-6.77 (m, 2H, ArH), 4.19 (d,
δ
8.19 (d,
= 8.0 Hz, 1H, ArH), 7.27−7.23 (m, 2H, ArH), 7.08-7.05 (m, 2H,
= 9.2 Hz, 1H, Glc H1), 4.06−3.94 (m, 5H, 2 x CH2, Glc H5),
J = 1.6 Hz, 1H, ArH), 7.61 (d, J = 5.6 Hz, 1H, CH=N),
J
J
3.62 (dd,
1.34 (t,
J
= 9.6, 9.2 Hz, 1H, Glc H4), 3.53 (t,
J
= 8.8 Hz, 1H, Glc H3), 3.36 (t,
J
= 9.2 Hz, 1H, Glc H2),
J
= 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6)
δ 158.67, 156.94, 148.13, 139.14,
138.08, 135.89, 132.24, 131.16, 130.79, 129.93, 129.62, 128.87, 127.37, 127.20, 126.97, 114.31, 80.98,
79.44, 77.86, 74.42, 72.07, 62.90, 37.69, 14.73; MS (ESI) m/z: 531 (MH+), 553 (MNa+); HRMS (FAB)
for C26H27ClN2O6S: calcd, 530.1278; found, 530.1274.
4.1.3.13 N'-[{(2R,3S,4R,5R,6S)-6-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-3,4,5-trihydroxytetrahydro-2H-
pyran-2-yl}methylidene]furan-2-carbohydrazide (3m)
The title compound was obtained from 7 according to the general procedure in 39% yield over four
steps. 1H NMR (400 MHz, CD3OD)
7.34 (d, = 8.4 Hz, 1H, ArH), 7.26−7.22 (m, 3H, ArH), 7.08−7.05 (m, 2H, ArH), 6.79−6.76 (m, 2H,
ArH), 6.62 (dd, = 3.6, 1.6 Hz, 1H, ArH), 4.19 (d, = 9.6 Hz, 1H, Glc H1), 4.06−3.93 (m, 5H, 2 x CH2,
δ 7.72 (d, J = 1.2 Hz, 1H, ArH), 7.63 (d, J = 5.6 Hz, 1H, CH=N),
J
J
J
Glc H5), 3.61 (t, = 9.2 Hz, 1H, Glc
J
= 9.2 Hz, 1H, Glc H4), 3.52 (t,
J
= 8.8 Hz, 1H, Glc H3), 3.36 (t,
J
H2), 1.33 (t,
J
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
δ 158.84, 157.62, 150.68, 147.40,
147.23, 140.09, 139.48, 134.65, 132.74, 131.79, 130.85, 130.21, 128.10, 117.36, 115.43, 113.27, 83.05,
80.42, 79.14, 76.12, 73.19, 64.40, 39.20, 15.20; MS (ESI) m/z: 515 (MH+), 537 (MNa+); HRMS (EI) for
C26H27ClN2O7: calcd, 514.1507; found, 514.1504.
4.1.4 General procedure for the synthesis of compounds 4 and 5
A 6 N solution of HCl in MeOH was added dropwise to a solution of oxime / hydrazone (1.0 equiv)

ACCEPTED MANUSCRIPT
and sodium cyanoborohydride (NaBH3CN, 2.0 equiv.) in MeOH at 0 oC until pH 1~3. After stirring at 0
°C for 2 h, the reaction was quenched by the addition of saturated NaHCO3(aq), and the resulting mixture
was extracted with EtOAc. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo
The residue was purified by column chromatography (MeOH/CH2Cl2) to provide 4 or 5.
.
4.1.4.1 (1S)-1,5-Anhydro-1-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-deoxy-6-(hydroxyamino)-
D
-glucitol
(4a). Yield 41%; 1H NMR (400 MHz, CD3OD)
δ
7.34 (d, J = 8.4 Hz, 1H, ArH), 7.26−7.22 (m, 2H, ArH),
7.10−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.08 (d,
2 x CH2), 3.62 (ddd, = 9.6, 8.4, 2.8 Hz, 1H, Glc H5), 3.44 (t,
3H, Glc H2, H4, H6a), 2.91 (dd,
NMR (100 MHz, CD3OD)
J = 9.6 Hz, 1H, Glc H1), 4.06−3.95 (m, 4H,
J
J
= 8.8 Hz, 1H, Glc H3), 3.41−3.24 (m,
J
= 13.2, 8.4 Hz, 1H, Glc H6b), 1.35 (t, J
= 7.2 Hz, 3H, CH3); 13C
δ
158.86, 140.02, 139.91, 134.47, 132.82, 131.78, 130.86, 130.17, 128.06,
115.42, 82.83, 79.58, 77.24, 76.50, 74.27, 64.41, 56.80, 39.19, 15.20; MS (ESI) m/z: 424 (MH+), 446
(MNa+); HRMS (FAB) for C21H27ClNO6: calcd, 424.1527; found, 424.1523.
4.1.4.2 (1S)-1,5-Anhydro-1-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-deoxy-6-(methoxyamino)-
D-glucitol
(4b). Yield 68%; 1H NMR(400 MHz, CD3OD)
δ
7.34 (d, J = 8.0 Hz, 1H, ArH), 7.25−7.21 (m, 2H, ArH),
7.10−7.06 (m, 2H, ArH), 6.81−6.77 (m, 2H, ArH), 4.06 (d,
2 x CH2), 3.55 (ddd, = 9.6, 8.4, 2.4 Hz, 1H, Glc H5), 3.45 (s, 3H, OCH3), 3.42 (t,
H3), 3.38 (dd,
J
= 9.6 Hz, 1H, Glc H1), 4.05−3.95 (m, 4H,
= 9.2 Hz, 1H, Glc
= 13.6, 8.4 Hz,
J
J
J
= 13.6, 2.4 Hz, 1H, Glc H6a), 3.29-3.23 (m, 2H, Glc H2, H4), 2.87 (dd,
J
1H, Glc H6b), 1.35 (t,
J
= 7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
δ 158.86, 140.04, 139.87,
134.47, 132.80, 131.75, 130.87, 130.16, 128.06, 115.42, 82.82, 79.56, 77.49, 76.46, 74.17, 64.39, 61.21,
53.93, 39.18, 15.20; MS (ESI) m/z: 438 (MH+), 460 (MNa+); HRMS (EI) for C22H28ClNO6: calcd,
437.1605; found, 437.1601.
4.1.4.3
(1S)-1,5-Anhydro-1-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-deoxy-6-[2-(4-nitrophenyl)-
hydrazinyl]-D-glucitol (5a). Yield 12%; 1H NMR (400 MHz, CD3OD)
δ
7.91 (d, J = 9.6 Hz, 2H, ArH),
7.31 (d,
ArH), 4.00−3.92 (m, 5H, 2 x CH2, Glc H1), 3.50 (ddd,
4H, Glc H2, H3, H4, H6a), 2.95 (dd,
J = 8.0 Hz, 1H, ArH), 7.19−7.14 (m, 2H, ArH), 7.09−7.06 (m, 2H, ArH), 6.80−6.74 (m, 4H,
J
= 9.2, 8.0, 2.4 Hz, 1H, Glc H5), 3.43−3.23 (m,
J
= 13.2, 8.0 Hz, 1H, Glc H6b), 1.33 (t, J
= 7.2 Hz, 3H, CH3); 13C

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NMR (100 MHz, CD3OD)
δ
158.87, 157.34, 139.99, 139.91, 138.61, 134.47, 132.79, 131.62, 130.85,
130.08, 128.03, 127.02, 115.44, 111.19, 82.88, 79.89, 79.68, 76.45, 73.48, 64.40, 53.56, 39.18, 15.19;
MS (ESI) m/z: 544 (MH+), 566 (MNa+); HRMS (FAB) for C27H31ClN3O7: calcd, 544.1851; found,
544.1844.
4.1.4.4
hydrazinyl]-
ArH), 7.31 (d,
0.8 Hz, 1H, ArH), 6.80−6.76 (m, 2H, ArH), 6.50 (dd,
Glc H1), 4.05−3.93 (m, 4H, 2 x CH2), 3.54 (ddd, = 10.0, 7.6, 2.4 Hz, 1H, Glc H5), 3.43 (t,
= 12.8, 7.6 Hz, 1H, Glc H6b), 1.33 (t,
(1S)-1,5-Anhydro-1-[4-chloro-3-(4-ethoxybenzyl)phenyl]-6-deoxy-6-[2-(furan-2-ylcarbonyl)-
1
D
-glucitol (5b). Yield 67%; H NMR (400 MHz, CD3OD)
δ
7.50 (dd,
J
= 2.0, 0.8 Hz, 1H,
= 3.6,
= 9.6 Hz, 1H,
= 8.8 Hz,
J
= 8.4 Hz, 1H, ArH), 7.27−7.22 (m, 2H, ArH), 7.10−7.06 (m, 2H, ArH), 6.99 (dd,
= 3.6, 2.0 Hz, 1H, ArH), 4.09 (d,
J
J
J
J
J
1H, Glc H3), 3.37−3.27 (m, 3H, Glc H2, H4, H6a), 3.02 (dd,
7.2 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD)
J
J
=
δ
159.37, 158.85, 147.85, 146.22, 139.96, 139.91,
134.40, 132.79, 131.82, 130.89, 130.15, 128.03, 115.42, 115.33, 112.77, 82.79, 80.19, 79.56, 76.35,
73.34, 64.40, 53.92, 39.24, 15.19; MS (ESI) m/z: 517 (MH+), 539 (MNa+); HRMS (EI) for
C26H29ClN2O7: calcd, 516.1663; found, 516.1659.
4.2. In vitro human SGLT inhibition assays
In vitro transporter assays were carried out according to the methods of Castaneda and Kinne, with
necessary modification [28,29,32].
4.3. hERG potassium channel assay
The radioligand binding assay was performed by Ricerca Biosciences, LLC.
4.4. Pharmacokinetics, glucosuria, and anti-hyperglycemia evaluations
Studies of pharmacokinetics, glucosuria, and the anti-hyperglycemic effect of selected aryl
C
-glycoside(s) were performed as previously reported [27,32].
Acknowledgements
We are grateful to the National Health Research Institutes and the Ministry of Science and
Technology of Taiwan (NSC 99-2323-B-400-003) for financial support.

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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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