Synthesis and biological evaluation of N-glucosyl indole derivatives as sodium-dependent glucose co-transporter 2 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.11.006

Sodium-dependent glucose co-transporter 2 (SGLT2) inhibition has been demonstrated to efficiently control hyperglycemia via an insulin secretion-independent pathway. The unique mode of action eliminates the risk of hypoglycemia and makes SGLT2 inhibitors

Full text of DOI:10.1016/j.bioorg.2018.11.006

Accepted Manuscript
Synthesis and biological evaluation of N-glucosyl indole derivatives as sodium-
dependent glucose co-transporter 2 inhibitors
Kuang-Feng Chu, Jen-Shin Song, Chiung-Tong Chen, Teng-Kuang Yeh,
Tsung-Chih Hsieh, Chung-Yu Huang, Min-Hsien Wang, Szu-Huei Wu, Chun-
Hsu Yao, Yu-Sheng Chao, Jinq-Chyi Lee
PII:
S0045-2068(18)30789-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.006
YBIOO 2614
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 July 2018
2 November 2018
5 November 2018
Please cite this article as: K-F. Chu, J-S. Song, C-T. Chen, T-K. Yeh, T-C. Hsieh, C-Y. Huang, M-H. Wang, S-H.
Wu, C-H. Yao, Y-S. Chao, J-C. Lee, Synthesis and biological evaluation of N-glucosyl indole derivatives as sodium-
dependent glucose co-transporter 2 inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.006
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Synthesis and biological evaluation of N-glucosyl indole derivatives as
sodium-dependent glucose co-transporter 2 inhibitors
Kuang-Feng Chu, Jen-Shin Song, Chiung-Tong Chen, Teng-Kuang Yeh, Tsung-Chih Hsieh,
Chung-Yu 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.
Abstract
Sodium-dependent glucose co-transporter 2 (SGLT2) inhibition has been demonstrated to efficiently
control hyperglycemia via an insulin secretion-independent pathway. The unique mode of action
eliminates the risk of hypoglycemia and makes SGLT2 inhibitors an attractive option for the
treatment of type 2 diabetes. In a continuation of our previous studies on SGLT2 inhibitors bearing
different sugar moieties, sixteen new N-glucosyl indole derivatives were designed, synthesized, and
evaluated for their inhibitory activity against hSGLT2. Of these sixteen, acethydrazide-containing
N-glucosyl indole 9d was found to be the most potent SGLT2 inhibitor, and caused a significant
elevation in urine glucose excretion in rats at 50 mg/kg, relative to the vehicle control.
1

1. Introduction
Sodium-dependent glucose co-transporter 2 (SGLT2) regulates glucose homeostasis by
reabsorbing glucose from the glomerular filtrate, and is therefore an attractive target for the
treatment of type 2 diabetes. SGLT2, a member of the solute carrier 5A (SLC5A) gene family, is a
high-capacity and low-affinity glucose transporter located on the S1 segment of the proximal tubule
in the kidney, which mediates the reabsorption of the majority (>90%) of renal glucose [1–4].
Inhibition of SGLT2 has been demonstrated to control hyperglycemia via an insulin
secretion-independent pathway by suppressing the reabsorption of glucose in the kidney and
inducing urinary glucose excretion [5]; and SGLT2 inhibitors such as dapagliflozin, canagliflozin,
ipragliflozin, tofogliflozin, luseogliflozin, empagliflozin, and ertugliflozin have been successfully
used in the treatment of type 2 diabetes [6–12]. In addition to lowering blood sugar, SGLT2
inhibitors have also been found to show pleiotropic effects on other disorders, including
cardiovascular protection, hepatoprotective potential, renoprotection, and so on [13,14]. Their
unique mode of action eliminates the risk of hypoglycemia and makes SGLT2 inhibitors more
attractive than conventional therapeutic agents currently used in the treatment of type 2 diabetes.
SGLT1, which belongs to the same solute carrier family as SGLT2, is responsible for the
reuptake of the remaining renal glucose that is not reabsorbed by SGLT2. SGLT1 is mainly
distributed in the small intestine as well as the kidney; and its inhibition gives rise to
gastrointestinal side effects [15,16]. Accordingly, most studies recommend the use of selective
SGLT2 inhibitors; but SGLT2 inhibitors with the ability to partially inhibit SGLT1 have been
proposed based on the results of clinical efficacy evaluations [17,18]. Additionally, a promising
dual SGLT1/SGLT2 inhibitor sotagliflozin (LX4211) has also been developed (phase III) [19].
Whether or not good selectivity for SGLT2 over SGLT1 is an essential prerequisite for the
development of a practical drug remains to be seen.
Numerous effective and structurally diverse SGLT2 inhibitors, including selective and dual
SGLT1/SGLT2 inhibitors, have been identified [20,21]. When our discovery program was initiated,
2

we found that most SGLT2 inhibitor development focused on the modification of the aglycone
moieties. Instead, we sought to explore the role of the sugar moiety in SGLT inhibition; to help
researchers better understand the associated structure-activity relationships (SARs); and to discover
novel and potent SGLT2 inhibitors [22-25]. As shown in Figure 1, we have identified several
potential SGLT2 inhibitors with hSGLT1/hSGLT2 selectivities of 1.3- to 78-fold, and bearing a
variety of C- and N-sugar moieties. Comparisons between our results and those reported in the
literature [26-28] revealed that the C6 position of the sugar moiety plays a critical role in SGLT2
inhibitory potency and selectivity. For N-glycosyl indoles, the SGLT2 inhibitory potency of a
selection of compounds with a glucose moiety was better than those bearing xylose (1) or
6-amido-6-deoxyglucose (3) units when the same aglycone moiety was installed at the C1 position;
however, the latter imparted better hSGLT1/hSGLT2 selectivity [24]. In the series of C-glycosyl
compounds, 6-oxime-containing C-glucosylarene (4) was found to exhibit inferior SGLT2
inhibition and selectivity than glucose-based dapagliflozin, but comparable in vivo efficacy in
glucosuria and anti-hyperglycemic studies [25]. Accordingly, whether N-glucosyl indoles bearing
oxime, hydrazone, hydroxylamine, or hydrazide at the C6-position of glucose show the same trend
is of interest to us, too.
Cl
Cl
R
Y
N
OEt
X
H
OH
OH
OH
OH
OH
OH
O
O
O
H
N
N
HO
O
OH
OH
OH
1: X= N, Y = C, R = 4-Cl
2: X= C, Y = N, R = 7-F
3
4
Figure 1. Our previously-reported SGLT2 inhibitors, bearing differing sugar moieties.
In this study, we further explored the role of the sugar moiety of the synthesized N-glucosyl
indoles
on
inhibitory
potency,
wherein
the
aglycone
unit
is
fixed
as
4-chloro-3-(4-cyclopropylbenzyl)-1H-indole. Detailed descriptions of syntheses, SAR,
pharmacokinetics, and animal studies are all presented.
3

2. Results and discussion
2.1 Synthesis
The synthesis of test compounds is depicted in Scheme 1, starting from N-glucosyl indole 5.
As reported previously [25], the key intermediate 6-aldehyde 7 could be obtained via a
straightforward reaction sequence: (1) selective protection of the primary alcohol with
tert-butylchlorodimethylsilane (TBDMSCl) in pyridine at 70 °C, followed by immediate
peracetylation by addition of acetic anhydride to give the fully protected N-glucoside (83% yield
over both steps); (2) desilylation under acidic conditions to avoid the exchange with the O4-acetyl
group with boron trifluoride etherate (BF3．Et2O) yielded the free primary alcohol at the 6-position
(91%); (3) Dess-Martin Periodinane (DMP) oxidation of the generated 6-OH 6 to give the desired
aldehyde 7, which was used without further purification. Condensation of 7 with a small library of
hydroxylamines and hydrazines followed by deacetylation gave oxime ethers 8a-8c and hydrazones
Cl
O
Cl
O
Cl
O
N
N
N
a
c
b
OH
OH
OAc
OAc
OAc
OAc
HO
HO
H
OH
OAc
O
OAc
5
6
7
Cl
O
Cl
a: R = OH
b: R = OCH
3
c: R = OCH CH
2
3
N
N
O
d: R = NHC(O)CH
d
3
OH
OH
OH
OH
e: R = NHC(O)OCH
3
H
f: R = NHC(O)OCH CH
2
3
RN
N
t
R
g: R = NHC(O)O Bu
OH
OH
9a-9h
h: R = NHC(O)Ph
8a-8h
Scheme 1. The synthetic route to compounds 8a-8h and 9a-9h. Reagents and conditions: (a) (i)
TBDMSCl, DMAP, pyridine, 70 °C, 18 h; then Ac2O, rt, 2 h, 83%; (ii) BF3·OEt2, CH2Cl2, 0 °C, 20
min, 91%; (b) Dess-Martinperiodinane, CH2Cl2, rt, 2 h; (c) For 8a-8c: (i) H2NOR1, pyridine, rt, 2 h;
(ii) NaOMe, MeOH/CH2Cl2 = 2/1, 0 °C to rt, 2 h, 44% (3 steps) (8a), 33% (3 steps) (8b), 66% (3
steps) (8c); For 8d-8h: (i) H2NNHR2, EtOH, rt, 2 h; (ii) 1 N LiOH(aq), THF/MeOH/CH2Cl2 = 2/3/1,
0 °C, 2 h, 53% (3 steps) (8d), 78% (3 steps) (8e), 43% (3 steps) (8f), 45% (3 steps) (8g), 55% (3
steps) (8h); (d) NaBH3CN, 6 N HCl in MeOH, MeOH, 0 °C to rt, 1 h, 31% (9a), 27% (9b), 62%
(9c), 80% (9d), 84% (9e), 76% (9f), 79% (9g), 87% (9h).
4

8d-8h, respectively. Their corresponding reduction product hydroxylamine N-glucosyl indoles
9a-9c and hydrazide N-glucosyl indoles 9d-9h were also successfully synthesized, using sodium
cyanoborohydride (NaBH3CN) under acidic conditions.
2.2 Inhibitory activity against hSGLT2
The inhibitory activities (EC50) of all synthesized N-glucosyl indole derivatives 8a-8h and
9a-9h were determined by measuring the inhibition of the uptake of [14C]-labeled
α-methyl-D-glucopyranoside into Chinese hamster ovary (CHO) cells stably expressing human
SGLT2 (hSGLT2) or hSGLT1 [29,30]; using phlorizin (hSGLT2: EC50 = 96 ± 21 nM; hSGLT1:
EC50 = 179 ± 42 nM) and dapagliflozin (hSGLT2: EC50 = 2.8 ± 0.7 nM; hSGLT1: EC50 = 501 ± 147
nM) as positive controls. The effect of oxime and N-acylhydrazone moieties on activity was tested
first, using eight N-glucosyl indoles, as shown in Table 1. In the case of oxime ethers 8a-8c, those
bearing small substituents (8a: EC50 = 212 nM; 8b: EC50 = 286 nM) possessed better inhibitory
activity against hSGLT2, as discovered for C-glycosyl compounds [25]. Replacing the oxime ether
functionality with N-acylhydrazone moiety gave 8d-8h, which were of similar potency to
compounds 8a and 8b, except 8f (EC50 = 1162 nM) and 8g (EC50 = 867 nM). Of these, compound
8h (EC50 = 258 nM) bearing a phenyl group was a more potent inhibitor of SGLT2.
We then examined hydroxylamines 9a-9c and hydrazides 9d-9h, the reduction products of
oximes 8a-8c and N-acylhydrazones 8d-8h, respectively. Reduction products 9a-9c showed
improved inhibitory activity against hSGLT2 compared to oximes 8a-8c, with EC50 values ranging
from of 45 to 294 nM. Among them, compound 9b (EC50 = 45 nM) bearing a methoxyamine group
was found to be the most potent SGLT2 inhibitor; in contrast, substitution with a simple
hydroxylamine (9a, EC50 = 160 nM) and ethoxyamine group (9c, EC50 = 294 nM) weakened the
hSGLT2 inhibition 3.5- and 6.5-fold, respectively. Among the series of hydrazides 9d-9h,
acethydrazide 9d was found to exhibit the greatest hSGLT2 inhibition with an EC50 value of 33 nM.
5

Table 1
In vitro inhibitory activity of N-glucosyl indole derivatives 8a-8h and 9a-9h on hSGLT2.
hSGLT2
hSGLT2
Compd
R =
Compd
R =
EC50 (nM)a
212 ± 17
286 ± 36
1222 ± 212
355 ± 56
316 ± 69
1162 ± 129
867 ± 258
258 ± 72
96 ± 21
EC50 (nM)a
HO
CH3O
HO
CH3O
160 ± 14
45 ± 1
8a
8b
9a
9b
Cl
O
CH3CH2O
CH3CH2O
294 ± 45
33 ± 6
8c
9c
Cl
O
CH3C(O)HN
CH3OC(O)HN
CH3CH2OC(O)HN
tBuOC(O)HN
PhC(O)HN
CH3C(O)HN
CH3OC(O)HN
CH3CH2OC(O)HN
tBuOC(O)HN
PhC(O)HN
8d
9d
N
N
OH
OH
OH
OH
H
N
63 ± 1
8e
9e
R
RN
OH
OH
134 ± 11
1761 ± 228
156 ± 3
2.8 ± 0.7
8f
9f
8g
9g
8h
9h
PZNb
Dapac
a Data obtained by at least two independent experiments, each experiment performed in triplicate.
b PZN: phlorizin.
c Dapa: dapagliflozin.
Similar potency was observed when the carbomethoxyhydrazino group (9e, EC50 = 63 nM)
occupied the glucosyl C6 position. Replacement of the methoxy unit with ethoxy or tert-butoxy
gave 9f (EC50 = 134 nM) and 9g (EC50 = 1761 nM), both of which were less potent than 9e,
suggesting that larger alkyl groups may not be tolerated. Decreased potency was also observed
when the methoxy moiety was substituted with a phenyl group (9h, EC50 = 156 nM).
2.3 Selectivity and in vivo studies
The selectivity of the most active acethydrazide-containing N-glucosyl indole 9d for SGLT2
over SGLT1 was evaluated. The obtained data indicated that 9d lacked selectivity for SGLT1, with
an EC50 value of 37 nM for hSGLT1. In the course of our discovery program, compounds with good
hSGLT2 inhibition were evaluated for their in vivo ability to induce urinary glucose excretion, to
assess their potential for further development. Thus, 9d was administered orally in single doses of 1,
10 and 50 mg/kg to normal Sprague-Dawley (SD) rats; the results are presented in Figure 2.
6

Unfortunately, increased urinary glucose excretion could only be observed at a relatively high dose
of 50 mg/kg (825 mg glucose per 200g body weight (BW) over 24 h); which indicated 9d exhibited
inferior efficacy compared to dapagliflozin (2762 mg glucose/200g BW at a single dose of 1 mg/kg).
This pharmacokinetic data suggests 9d to be a metabolically stable compound with low total body
clearance (15.4 mL/min/kg) after intravenous administration of a single dose of 1.1 mg/kg to rats;
however, the very low plasma concentration (below the limit of quantification) observed after oral
administration (1.1 mg/kg) to rats indicated the absorption of 9d to be very poor – this is believed to
be the cause of the unfavorable in vivo results.
Figure 2. Effect of acethydrazide-containing N-glucosyl indole 9d and dapagliflozin on urine
glucose excretion over 24 h in normal Sprague–Dawley rats. Data are expressed as the mean ± SEM
(n = 5/group): *p < 0.05 vs vehicle.
3. Conclusion
Sixteen new glucose-modified N-glucosyl indoles have been synthesized and evaluated for
their hSGLT2 inhibitory activity. Of these, hydrazide 9d with an EC50 value of 33 nM for hSGLT2
was identified and advanced into selectivity, pharmacokinetic, and in vivo glucosuria studies;
unfortunately, it was found to have poor pharmacokinetic properties and increased glucosuria in rats
only at high doses. Compared to our earlier reports, regardless of the amide or hydrazide moiety at
the C6-position of glucose, all the sugar-modified N-glucosyl indole SGLT2 inhibitors synthesized
herein showed unsatisfactory in vivo profiles. Although prospects for their further development are
7

limited, the in vitro/in vivo outcomes obtained indeed improve our understanding of N-glucosyl
indoles.
4. Experimental
4.1. General
All chemicals and solvents were used as received without further purification (unless stated
otherwise). Chemical reactions were monitored by analytical thin layer chromatography (TLC) on
glass-backed plates pre-coated with SiO2 60 F254. Column chromatography was performed using
1
13
SiliaFlash P60 SiO2 of 230−400 mesh size. H and C NMR spectra were recorded on Varian
Mrcury-300 or Mercury-400 spectrometers; chemical shifts are reported in δ (ppm) relative to the
13
internal standard signal of CD3OD (1H, δ = 3.31 ppm; C, δ = 49.15 ppm). High resolution mass
spectra were measured with an electrospray ionization (ESI) source, and spectral data were recorded
as m/z values.
4.2. Test compounds 8a-8h and 9a-9h
All test compounds were synthesized using our previously reported procedures for sugar-modified
C-glycosyl compounds [25].
4.2.1.
(2R,3R,4S,5S,6R)-2-[4-Chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-6-[(hydroxyimino)
methyl]-tetra-hydro-2H-pyran-3,4,5-triol (8a). According to the literature, the proton signals of
CH=N and Glc H5 revealed the configuration of the major isomer was (E) [31]. The NMR data of
1
8a specified is for the major isomer only. H NMR (400 MHz, CD3OD) δ 7.44 (d, J = 8.4 Hz, 1H,
ArH), 7.31 (d, J = 7.2 Hz, 1H, CH=N), 7.10-7.05 (m, 3H, ArH), 7.01-6.95 (m, 4H, ArH), 5.46 (d, J
= 8.8 Hz, 1H, Glc H1), 4.28 (s, 2H, CH2), 4.09 (dd, J = 9.2, 7.2 Hz, 1H, Glc H5), 3.83 (t, J = 8.8 Hz,
1H, Glc H2), 3.58 (t, J = 8.8 Hz, 1H, Glc H3), 3.53 (t, J = 8.8 Hz, 1H, Glc H4) 1.88-1.81 (m, 1H,
13
cyclopropyl CH), 0.92-0.86 (m, 2H, cyclopropyl CH2), 0.64-0.60 (m, 2H, cyclopropyl CH2); C
NMR (100 MHz, CD3OD) δ 148.81, 142.75, 140.28, 139.91, 129.88, 127.48, 126.64, 126.61,
8

123.67, 121.91, 117.64, 110.53, 86.52, 78.60, 77.54, 73.40, 73.26, 33.23, 15.95, 9.55; HRMS (ESI)
m/z: [M + H]+ calcd for C24H26ClN2O5 457.152476, found 457.152044.
4.2.2.
(2R,3R,4S,5S,6R)-2-[4-Chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-6-[(methoxyimino)
methyl]tetrahydro-2H-pyran-3,4,5-triol (8b). According to the literature, the proton signals of
CH=N and Glc H5 revealed the configuration of the major isomer was (E) [31]. The NMR data of
1
8b specified is for the major isomer only. H NMR (400 MHz, CD3OD) δ 7.43 (d, J = 8.0 Hz, 1H,
ArH), 7.31 (d, J = 6.8 Hz, 1H, CH=N), 7.10-7.05 (m, 3H, ArH), 7.01-6.93 (m, 4H, ArH), 5.46 (d, J
= 9.2 Hz, 1H, Glc H1), 4.27 (s, 2H, CH2), 4.08 (dd, J = 9.2, 7.2 Hz, 1H, Glc H5), 3.84 (t, J = 9.2 Hz,
1H, Glc H2), 3.80 (s, 3H, OCH3), 3.58 (t, J = 9.2 Hz, 1H, Glc H3), 3.52 (t, J = 9.2 Hz, 1H, Glc H4),
1.87-1.80 (m, 1H, cyclopropyl CH), 0.91-0.85 (m, 2H, cyclopropyl CH2), 0.63-0.59 (m, 2H,
cyclopropyl CH2); 13C NMR (100 MHz, CD3OD) δ 148.66, 142.75, 140.29, 139.88, 129.88, 127.49,
126.61, 123.70, 121.94, 117.70, 110.50, 86.49, 78.58, 77.25, 73.33, 73.15, 62.28, 33.23, 15.94, 9.55;
HRMS (ESI) m/z: [M + H]+ calcd for C25H28ClN2O5 471.168126, found 471.166869.
4.2.3.
(2R,3R,4S,5S,6R)-2-[4-Chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-6-[(ethoxyimino)
methyl]tetrahydro-2H-pyran-3,4,5-triol (8c). According to the literature, the proton signals of
CH=N and Glc H5 revealed the configuration of the major isomer was (E) [31]. The NMR data of
1
8c specified is for the major isomer only. H NMR (400 MHz, CD3OD) δ 7.44 (dd, J = 8.4, 0.8 Hz,
1H, ArH), 7.32 (d, J = 7.2 Hz, 1H, CH=N), 7.11-7.06 (m, 3H, ArH), 7.02-6.95 (m, 4H, ArH), 5.47
(d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H, CH2), 4.12-4.05 (m, 3H, CH2, Glc H5), 3.85 (dd, J = 9.2,
8.8 Hz, 1H, Glc H2), 3.60 (t, J = 8.8 Hz, 1H, Glc H3), 3.54 (dd, J = 9.6, 8.8 Hz, 1H, Glc H4),
1.87-1.81 (m, 1H, cyclopropyl CH), 1.20 (t, J = 6.8 Hz, 3H, CH3), 0.92-0.88 (m, 2H, cyclopropyl
CH2), 0.64-0.60 (m, 2H, cyclopropyl CH2); 13C NMR (100 MHz, CD3OD) δ 148.41, 142.74, 140.27,
139.88, 129.88, 127.49, 126.61, 123.69, 121.93, 117.69, 110.51, 86.51, 78.59, 77.38, 73.36, 73.16,
70.70, 33.24, 15.95, 14.87, 9.55; HRMS (ESI) m/z: [M + H]+ calcd for C26H30ClN2O5 485.183776,
found 485.183010.
9

4.2.4.
N'-({(2R,3S,4S,5R,6R)-6-[4-Chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-3,4,5-tri-
hydroxytetrahydro-2H-pyran-2-yl}methylidene)acetohydrazide (8d). The ratio of the obtained
isomers was ~1.4:1. The proton chemical shifts of CH=N groups of the isomers are at 7.33 (d, J =
5.2 Hz, 1H) and 7.18 (d, J = 6.4 Hz, 1H) ppm, respectively; HRMS (ESI) m/z: [M + H]+ calcd for
C26H29ClN3O5 498.179025, found 498.178590.
4.2.5.
Methyl 2-({(2R,3S,4S,5R,6R)-6-[4-chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-3,4,5-
1
trihydroxytetrahydro-2H-pyran-2-yl}methylidene)hydrazinecarboxylate (8e). H NMR (400 MHz,
CD3OD) δ 7.45 (dd, J = 8.4, 0.8 Hz, 1H, ArH), 7.20 (d, J = 6.0 Hz, 1H, CH=N), 7.11-7.06 (m, 3H,
ArH), 7.01-6.95 (m, 4H, ArH), 5.49 (d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H, CH2), 4.13 (dd, J =
9.2, 6.0 Hz, 1H, Glc H5), 3.85 (t, J = 9.2 Hz, 1H, Glc H2), 3.74 (s, 3H, OCH3), 3.64-3.57 (m, 2H,
Glc H3, H4), 1.89-1.82 (m, 1H, cyclopropyl CH), 0.93-0.88 (m, 2H, cyclopropyl CH2), 0.65-0.61
(m, 2H, cyclopropyl CH2); 13C NMR (100 MHz, CD3OD) δ 156.99, 145.46, 142.76, 140.32, 130.89,
129.88, 127.49, 126.59, 123.70, 121.95, 117.72, 110.53, 86.45, 78.86, 78.61, 73.34, 73.02, 53.21,
33.23, 15.95, 9.55; HRMS (ESI) m/z: [M + H]+ calcd for C26H29ClN3O6 514.173940, found
514.173493.
4.2.6.
Ethyl 2-({(2R,3S,4S,5R,6R)-6-[4-chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-3,4,5-
1
trihydroxytetrahydro-2H-pyran-2-yl}methylidene)hydrazinecarboxylate (8f). H NMR (400 MHz,
CD3OD) δ 7.45 (dd, J = 8.4, 0.8 Hz, 1H, ArH), 7.19 (d, J = 6.0 Hz, 1H, CH=N), 7.09-7.05 (m, 3H,
ArH), 7.01-6.94 (m, 4H, ArH), 5.49 (d, J = 9.2 Hz, 1H, Glc H1), 4.27 (s, 2H, CH2), 4.17 (q, J = 7.2
Hz, 2H, CH2), 4.13 (dd, J = 9.2, 6.0 Hz, 1H, Glc H5), 3.86 (t, J = 9.2 Hz, 1H, Glc H2), 3.65-3.58 (m,
2H, Glc H3, H4), 1.86-1.80 (m, 1H, cyclopropyl CH), 1.25 (t, J = 7.2 Hz, 3H, CH3), 0.92-0.87 (m,
2H, cyclopropyl CH2), 0.63-0.60 (m, 2H, cyclopropyl CH2); 13C NMR (100 MHz, CD3OD) δ
156.54, 145.35, 142.74, 140.30, 139.88, 129.88, 127.48, 126.60, 123.70, 121.94, 117.71, 110.53,
86.45, 78.85, 78.59, 73.33, 73.00, 62.80, 33.23, 15.95, 14.95, 9.56; HRMS (ESI) m/z: [M + H]+
calcd for C27H31ClN3O6 528.189590, found 528.188070.
10

4.2.7.
tert-Butyl 2-({(2R,3S,4S,5R,6R)-6-[4-chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-
1
3,4,5-trihydroxytetrahydro-2H-pyran-2-yl}methylidene)hydrazinecarboxylate (8g). H NMR (400
MHz, CD3OD) δ 7.45 (dd, J = 8.4, 0.8 Hz, 1H, ArH), 7.15 (d, J = 5.6 Hz, 1H, CH=N), 7.10-7.05
(m, 3H, ArH), 7.01-6.93 (m, 4H, ArH), 5.48 (d, J = 9.2 Hz, 1H, Glc H1), 4.27 (s, 2H, CH2), 4.12
(dd, J = 9.2, 5.6 Hz, 1H, Glc H5), 3.86 (t, J = 9.2 Hz, 1H, Glc H2), 3.65-3.58 (m, 2H, Glc H3, H4),
1.87-1.81 (m, 1H, cyclopropyl CH), 1.47 (s, 9H, 3CH3), 0.92-0.87 (m, 2H, cyclopropyl CH2),
0.64-0.60 (m, 2H, cyclopropyl CH2); 13C NMR (75 MHz, CD3OD) δ 155.59, 144.51, 142.74,
140.29, 139.87, 129.87, 127.47, 126.60, 123.69, 121.93, 117.68, 110.54, 86.45, 82.20, 78.86, 78.59,
73.33, 72.98, 33.23, 28.68, 15.95, 9.56; HRMS (ESI) m/z: [M + H]+ calcd for C29H35ClN3O6
556.220890, found 556.219917.
4.2.8.
N'-({(2R,3S,4S,5R,6R)-6-[4-Chloro-3-(4-cyclopropylbenzyl)-1H-indol-1-yl]-3,4,5-tri-
1
hydroxytetrahydro-2H-pyran-2-yl}methylidene)benzohydrazide (8h). H NMR (400 MHz, CD3OD)
δ 7.85 (d, J = 7.2 Hz, 2H, ArH), 7.61-7.55 (m, 2H, ArH, CH=N), 7.49-7.45 (m, 3H, ArH), 7.10-7.06
(m, 3H, ArH), 7.01-6.94 (m, 4H, ArH), 5.54 (d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H, CH2), 4.26
(dd, J = 9.2, 5.6 Hz, 1H, Glc H5), 3.89 (t, J = 9.2 Hz, 1H, Glc H2), 3.72-3.64 (m, 2H, Glc H3, H4),
1.88-1.81 (m, 1H, cyclopropyl CH), 0.92-0.87 (m, 2H, cyclopropyl CH2), 0.64-0.60 (m, 2H,
cyclopropyl CH2);13C NMR (100 MHz, CD3OD) δ 167.39, 149.84, 142.76, 140.34, 139.88, 133.89,
133.61, 129.87, 128.93, 127.49, 126.61, 123.73, 121.97, 117.77, 110.55, 86.52, 78.83, 78.64, 73.33,
72.93, 33.23, 15.95, 9.55;HRMS (ESI) m/z: [M + H]+ calcd for C31H31ClN3O5 560.194675, found
560.193241.
4.2.9.
4-Chloro-3-(4-cyclopropylbenzyl)-1-[6-deoxy-6-(hydroxyamino)-β-D-glucopyranosyl]-
1
1H-indole (9a). H NMR(400 MHz, CD3OD) δ 7.46 (dd, J = 8.4, 0.8 Hz, 1H, ArH), 7.11-7.06 (m,
3H, ArH), 7.02-6.95 (m, 4H, ArH), 5.39 (d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H, CH2), 3.85-3.79
(m, 2H, Glc H2, H5), 3.55 (t, J = 9.2 Hz, 1H, Glc H3), 3.36-3.30 (m, 2H, Glc H4, H6a), 2.91 (dd, J
= 13.6, 8.4 Hz, 1H, Glc H6b), 1.89-1.82 (m, 1H, cyclopropyl CH), 0.93-0.89 (m, 2H, cyclopropyl
11

CH2), 0.65-0.61 (m, 2H, cyclopropyl CH2); 13C NMR (75 MHz, CD3OD) δ 142.76, 140.19, 139.97,
129.88, 127.49, 126.82, 126.61, 123.65, 121.87, 117.46, 110.69, 86.78, 79.12, 75.73, 73.93, 73.63,
56.57, 33.21, 15.95, 9.55; HRMS (ESI) m/z: [M + H]+ calcd for C24H28ClN2O5 459.168126, found
459.171187.
4.2.10. 4-Chloro-3-(4-cyclopropylbenzyl)-1-[6-deoxy-6-(methoxyamino)-β-D-glucopyranosyl]-1H
-indole (9b). 1H NMR (400 MHz, CD3OD) δ 7.44 (d, J = 8.4 Hz, 1H, ArH) 7.10-7.05 (m, 3H, ArH),
7.02-6.94 (m, 4H, ArH), 5.38 (d, J = 8.8 Hz, 1H, Glc H1), 4.28 (s, 2H, CH2), 3.81 (t, J = 8.8 Hz, 1H,
Glc H2), 3.76 (ddd, J = 9.6, 8.4, 2.8 Hz, 1H, Glc H5), 3.54 (t, J = 8.8 Hz, 1H, Glc H3), 3.45 (s, 3H,
OCH3), 3.41 (dd, J = 13.6, 2.8 Hz, 1H, Glc H6a), 3.34-3.27 (m, 1H, Glc H4), 2.86 (dd, J = 13.6, 8.4
Hz, 1H, Glc H6b), 1.88-1.81 (m, 1H, cyclopropyl CH), 0.92-0.87 (m, 2H, cyclopropyl CH2),
13
0.64-0.60 (m, 2H, cyclopropyl CH2); C NMR (100 MHz, CD3OD) δ 142.74, 140.21, 139.93,
129.88, 127.51, 126.75, 126.60, 123.65, 121.89, 117.50, 110.58, 86.67, 79.11, 75.93, 73.86, 73.54,
61.41, 53.73, 33.20, 15.94, 9.56; HRMS (ESI) m/z: [M + H]+ calcd for C25H30ClN2O5 473.183776,
found 473.182591.
4.2.11. 4-Chloro-3-(4-cyclopropylbenzyl)-1-[6-deoxy-6-(ethoxyamino)-β-D-glucopyranosyl]-1H-
indole (9c). 1H NMR (400 MHz, CD3OD) δ 7.44 (d, J = 8.0 Hz, 1H, ArH), 7.10-7.06 (m, 3H, ArH),
7.01 (d, J = 8.0 Hz, 2H, ArH), 6.95 (d, J = 8.0 Hz, 2H, ArH), 5.38 (d, J = 9.2 Hz, 1H, Glc H1), 4.28
(s, 2H, CH2), 3.81 (t, J = 9.2 Hz, 1H, Glc H2), 3.80-3.75 (m, 1H, Glc H5), 3.68-3.62 (m, 2H, CH2),
3.55 (t, J = 9.2 Hz, 1H, Glc H3), 3.39 (dd, J = 14.0, 2.8 Hz, 1H, Glc H6a), 3.34-3.30 (m, 1H, Glc
H4), 2.87 (dd, J = 14.0, 8.8 Hz, 1H, Glc H6b), 1.88-1.81 (m, 1H, cyclopropyl CH), 1.09 (t, J = 7.2
Hz, 3H, CH3), 0.92-0.88 (m, 2H, cyclopropyl CH2), 0.64-0.60 (m, 2H, cyclopropyl CH2); 13C NMR
(100 MHz, CD3OD) δ 142.74, 140.22, 139.94, 129.88, 127.52, 126.75, 126.60, 123.62, 121.89,
117.50, 110.59, 86.67, 79.13, 76.04, 73.91, 73.54, 69.76, 54.24, 33.20, 15.94, 14.59, 9.55; HRMS
(ESI) m/z: [M + H]+ calcd for C26H32ClN2O5 487.199426, found 487.199100.
4.2.12. 1-[6-(2-Acetylhydrazinyl)-6-deoxy-β-D-glucopyranosyl]-4-chloro-3-(4-cyclopropylbenzyl)
12

-1H-indole (9d). 1H NMR (400 MHz, CD3OD) δ 7.50 (dd, J = 8.4, 0.8 Hz, 1H, ArH), 7.12-7.07 (m,
3H, ArH), 7.04-6.95 (m, 4H, ArH), 5.36 (d, J = 8.8 Hz, 1H, Glc H1), 4.29 (s, 2H, CH2), 3.81 (dd, J
= 9.2, 8.8 Hz, 1H, Glc H2), 3.65 (ddd, J = 10.0, 7.6, 2.4 Hz, 1H, Glc H5), 3.53 (dd, J = 9.2, 8.8 Hz,
1H, Glc H3), 3.36 (dd, J = 9.6, 9.2 Hz, 1H, Glc H4), 3.18 (dd, J = 12.8, 2.4 Hz, 1H, Glc H6a), 2.94
(dd, J = 12.8, 7.6 Hz, 1H, Glc H6b), 1.89-1.82 (m, 1H, cyclopropyl CH), 1.74 (s, 3H, CH3),
13
0.93-0.88 (m, 2H, cyclopropyl CH2), 0.65-0.61 (m, 2H, cyclopropyl CH2); C NMR (75 MHz,
CD3OD) δ 171.70, 142.75, 140.20, 140.01, 129.88, 127.47, 126.85, 126.63, 123.66, 121.86, 117.43,
110.79, 86.81, 79.06, 78.40, 73.59, 73.06, 53.85, 33.20, 20.74, 15.95, 9.54; HRMS (ESI) m/z: [M +
H]+ calcd for C26H31ClN3O5 500.194675, found 500.193341.
4.2.13. 4-Chloro-3-(4-cyclopropylbenzyl)-1-{6-deoxy-6-[2-(methoxycarbonyl)hydrazinyl]-β-D-
1
glucopyranosyl}-1H-indole (9e). H NMR (400 MHz, CD3OD) δ 7.48 (d, J = 7.6 Hz, 1H, ArH),
7.11-7.06 (m, 3H, ArH), 7.01-6.95 (m, 4H, ArH), 5.37 (d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H,
CH2), 3.80 (t, J = 9.2 Hz, 1H, Glc H2), 3.67 (ddd, J = 10.0, 8.0, 2.4 Hz, 1H, Glc H5), 3.55-3.51 (m,
4H, Glc H3, OCH3), 3.36 (dd, J = 9.6, 9.2 Hz, 1H, Glc H4), 3.18 (dd, J = 12.8, 2.4 Hz, 1H, Glc
H6a), 2.94 (dd, J = 12.8, 8.0 Hz, 1H, Glc H6b), 1.89-1.82 (m, 1H, cyclopropyl CH), 0.93-0.89 (m,
2H, cyclopropyl CH2), 0.65-0.61 (m, 2H, cyclopropyl CH2); 13C NMR (75 MHz, CD3OD) δ 160.14,
142.74, 140.17, 139.99, 129.88, 127.43, 126.86, 126.60, 123.61, 121.81, 117.38, 110.78, 86.78,
79.02, 78.16, 73.67, 73.22, 54.18, 52.73, 33.20, 15.94, 9.55; HRMS (ESI) m/z: [M + H]+ calcd for
C26H31ClN3O6 516.189590, found 516.188604.
4.2.14. 4-Chloro-3-(4-cyclopropylbenzyl)-1-{6-deoxy-6-[2-(ethoxycarbonyl)hydrazinyl]-β-D-
1
glucopyranosyl}-1H-indole (9f). H NMR (400 MHz, CD3OD) δ 7.48 (d, J = 8.0 Hz, 1H, ArH),
7.11-7.05 (m, 3H, ArH), 7.02-6.94 (m, 4H, ArH), 5.36 (d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H,
CH2), 3.97-3.95 (m, 2H, CH2), 3.81 (t, J = 9.2 Hz, 1H, Glc H2), 3.67 (ddd, J = 10.0, 8.0, 2.8 Hz, 1H,
Glc H5), 3.54 (t, J = 9.2 Hz, 1H, Glc H3), 3.37 (dd, J = 9.6, 9.2 Hz, 1H, Glc H4), 3.19 (dd, J = 12.8,
2.8 Hz, 1H, Glc H6a), 2.95 (dd, J = 12.8, 8.0 Hz, 1H, Glc H6b), 1.88-1.81 (m, 1H, cyclopropyl CH),
13

1.12 (t, J = 7.2 Hz, 3H, CH3), 0.92-0.88 (m, 2H, cyclopropyl CH2), 0.64-0.60 (m, 2H, cyclopropyl
13
CH2); C NMR (75 MHz, CD3OD) δ 159.69, 142.73, 140.15, 139.96, 129.89, 127.44, 126.87,
126.59, 123.60, 121.81, 117.38, 110.79, 86.80, 79.00, 78.22, 73.65, 73.22, 62.17, 54.17, 33.21,
15.94, 14.99, 9.56; HRMS (ESI) m/z: [M + H]+ calcd for C27H33ClN3O6 530.205240, found
530.203954.
4.2.15. 1-{6-[2-(tert-Butoxycarbonyl)hydrazinyl]-6-deoxy-β-D-glucopyranosyl}-4-chloro-3-(4-
cyclopropylbenzyl)-1H-indole (9g). 1H NMR (400 MHz, CD3OD) δ 7.49 (d, J = 8.0 Hz, 1H, ArH),
7.11-7.06 (m, 3H, ArH), 7.02-6.94 (m, 4H, ArH), 5.37 (d, J = 9.2 Hz, 1H, Glc H1), 4.28 (s, 2H,
CH2), 3.82 (t, J = 9.2 Hz, 1H, Glc H2), 3.65 (ddd, J = 10.0, 8.0, 2.8 Hz, 1H, Glc H5), 3.54 (t, J =
8.8 Hz, 1H, Glc H3), 3.37 (dd, J = 9.6, 9.2 Hz, 1H, Glc H4), 3.16 (dd, J = 12.8, 2.8 Hz, 1H, Glc
H6a), 2.93 (dd, J = 12.8, 8.0 Hz, 1H, Glc H6b), 1.88-1.81 (m, 1H, cyclopropyl CH), 1.37 (s, 9H,
13
3CH3), 0.92-0.88 (m, 2H, cyclopropyl CH2), 0.64-0.60 (m, 2H, cyclopropyl CH2); C NMR (75
MHz, CD3OD) δ 158.83, 142.72, 140.20, 139.96, 129.88, 127.45, 126.84, 126.61, 123.62, 121.85,
117.41, 110.77, 86.75, 81.07, 79.03, 78.02, 73.58, 73.21, 54.20, 33.21, 28.80, 15.95, 9.53; HRMS
(ESI) m/z: [M + H]+ calcd for C29H37ClN3O6 558.236540, found 558.234633.
4.2.16. 1-[6-(2-Benzoylhydrazinyl)-6-deoxy-β-D-glucopyranosyl]-4-chloro-3-(4-cyclopropyl-
benzyl)-1H-indole (9h). 1H NMR (400 MHz, CD3OD) δ 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.42-7.38 (m,
3H, ArH), 7.21 (t, J = 7.6 Hz, 2H, ArH), 7.07-6.98 (m, 5H, ArH), 6.93 (d, J = 8.0 Hz, 2H, ArH),
5.42 (d, J = 9.2 Hz, 1H, Glc H1), 4.26 (s, 2H, CH2), 3.85 (t, J = 9.2 Hz, 1H, Glc H2), 3.79-3.74 (m,
1H, Glc H5), 3.56 (t, J = 9.2 Hz, 1H, Glc H3), 3.41 (dd, J = 9.6, 9.2 Hz, 1H, Glc H4), 3.36-3.31 (m,
1H, Glc H6a), 3.05 (dd, J = 13.6, 8.0 Hz, 1H, Glc H6b), 1.88-1.81 (m, 1H, cyclopropyl CH),
13
0.92-0.87 (m, 2H, cyclopropyl CH2), 0.63-0.59 (m, 2H, cyclopropyl CH2); C NMR (75 MHz,
CD3OD) δ 168.57, 142.73, 140.12, 139.90, 134.15, 132.70, 129.90, 129.61, 128.01, 127.54, 126.89,
126.69, 126.61, 123.75, 121.93, 117.46, 110.74, 86.81, 79.67, 79.05, 73.47, 72.98, 53.58, 33.23,
15.94, 9.54; HRMS (ESI) m/z: [M + H]+ calcd for C31H33ClN3O5 562.210325, found 562.208968.
14

4.3. In vitro human SGLT inhibition assays
The transporter assays were performed according to the protocols of Castaneda and Kinne
[29,30], with necessary modification [22].
4.4. Pharmacokinetics and urine glucose excretion studies
Pharmacokinetics and urine glucose excretion effect of acethydrazide-containing N-glucosyl
indole 9d were studied using our previously reported protocols [22].
Conflicts of interest
The authors declare no competing interests.
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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A,
and
6’-N-hydroxydibekacin, Carbohydr. Res. 136 (1985) 195–206.
18

Highlights
․ A series of N-glucosyl indole derivatives bearing modifications at the C6-position of glucose
was designed and synthesized.
․ All the synthesized derivatives were tested in vitro for their potential to inhibit the activity of
hSGLT2.
․ Acethydrazide-containing N-glucosyl indole 9d was found to be the most active compound.
․ The selectivity, pharmacokinetic, and glucosuria potential of 9d were also studied.
19

Graphical Abstract
Cl
OEt
H
OH
OH
Cl
O
O
RN
OH
N
OH
OH
O
H
Cl
O
N
N
H
OH
9d
N
OH
OH
Z
OH
20