5644
Y. Yamamoto et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5641–5645
Table 2
Pharmacokinetic parameters of 8a and 9d in SD rats
Compound
8a
9d
Dose (mg/kg; iv)
3
3
t1/2 (h)
AUC0Àinf (lg h/mL)
CLtot (L/h/kg)
Vdss (L/kg)
0.58
0.41
7.4
4.5
3.0
8.7
0.35
1.3
10. Ellsworth, B.; Washburn, W. N.; Sher, P. M.; Wu, G.; Meng, W. PCT Int. Appl.
WO200127128, 2001; Chem. Abstr. 2001, 134, 281069.
Dose (mg/kg; po)
t1/2 (h)
tmax (h)
10
10
0.43
0.25
0.43
0.23
17
2.9
0.5
2.0
17
59
Cmax (lg/mL)
AUC0Àinf (lgÁh/mL)
F (%)
13. Reactions without any additives in refluxing MeOH were sluggish and finally
led to a complex mixture. There is a patent literature, in which ammonium
sulfate is used as an additive; see Tam, T. F.; Karimian, K.; Leung-Toung, R. C. S.
H.; Zhao, Y.; Wodzinska, J. M.; Li, W.; Lowrie, J. N. Eur. Pat. Appl. EP1348710,
2003; Chem. Abstr. 2003, 139, 276904.
14. The anomeric stereochemistry in 8a is b-configuration, which was confirmed
by the coupling constant (J = 8.4 Hz) between anomeric C–H and adjacent C–H
in 1H NMR studies (D2O exchange in DMSO-d6).
15. (a) Sodium-dependent glucose uptake in CHO cells expressing hSGLT1 and
hSGLT2; parental CHOK cells expressing hSGLT2 and hSGLT1 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
tion between the glucose moiety and the distal phenyl group.
The N-glucoside 9d possessing an indole core structure showed
good hSGLT2 inhibitory activity (IC50 = 7.1 nM). The hSGLT1
inhibitory activity of 9d was also evaluated, and IC50 was
1956 nM (hSGLT1/hSGLT2 ratio = 275). Thus, 9d was found to
be a potent and selective hSGLT2 inhibitor. As for indazoles,
the 2-glucosylated isomer 9e exhibited very weak activity
(IC50 = 1098 nM), whereas its 1-isomer 9f was moderately active
(IC50 = 69 nM). Similar to the case of the C-glucoside hSGLT2
inhibitors,7b 1,3-orientation of the N-glucose moiety relative to
the distal phenyl ring is considered to be preferable rather than
1,2-substitution. Next, the effect of indole-N-glucoside 9d on
UGE in SD rats was evaluated. As expected, 9d demonstrated im-
proved oral activity (UGE = 1830 mg/day), which is 20-fold as po-
tent as that of 8a (UGE = 93 mg/day), and furthermore,
comparable to that of aryl-C-glucoside 3 (UGE = 1485 mg/day).
As shown in Table 2, this large enhancement of in vivo potency
of 9d relative to 8a was clearly supported by the pharmacoki-
netic results, wherein 9d showed favorable profiles such as low-
er clearance, higher AUC, and better bioavailability. We attribute
these enhanced in vivo profiles of 9d in part to the inherent sta-
bility of the C–N glucosidic bond against hydrolysis, because 9d
was stable under acidic aqueous condition (0.5 N HCl, 37 °C) in
our preliminary experiments (data not shown), and no aglycon
13d was observed in the pharmacokinetic studies of 9d in rats.
In summary, we synthesized a series of N-glucosides and eval-
uated their hSGLT2 inhibitory activities. The key concept of hetero-
aromatic-N-glucoside 9 was the combination of aryl-C-glucoside 7
and aniline-N-glucoside 8 to improve the stability of the C–N glu-
cosidic bond. As a result, indole-N-glucoside 9d was identified as a
novel class of selective hSGLT2 inhibitor with favorable both
in vitro and in vivo potencies. Encouraged by these findings, we
started the more detailed SAR studies around 9d. Further explora-
tion will be reported in due course.
400
50 mM HEPES, 20 mM Tris Base, pH 7.4), and were pre-incubated with the
solutions of compounds (250 L) for 10 min at 37 °C. The transport reaction
was initiated by addition of 50 Ci; final
L AMG/14C-AMG solution (16.7
lL Assay Buffer (137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
l
l
l
concentration, 0.3 mM for CHOK-hSGLT1 and 0.5 mM for CHOK-hSGLT2,
respectively), and incubated for 120 min at 37 °C. After the incubation, the
AMG uptake was halted by aspiration of the incubation mixture followed by
immediate washing three times with PBS. The cells were solubilized in 0.3 N
NaOH of 300
by a liquid scintillation counter (Quantasmart™ (Packard, Boston, MA, USA)).
Inhibitory concentration of 50% (IC50 was calculated by nonlinear least
squares analysis using four-parameter logistic model (Prism version 4;
lL and the radioactivity associated with the cells was monitored
)
a
GraphPad Software, San Diego, CA, USA); see Dudash, J., Jr.; Zhang, X.; Zeck, R.
E., Johnson, S. G.; Cox, G. G.; Conway, B. R.; Rybczynski, P. J.; Demarest, K. T.
Bioorg. Med. Chem. Lett. 2004, 14, 5121; (b) Rat UGE Study; male SD rats aged 4-
5 weeks were obtained from Japan SLC (Shizuoka, Japan) and were used for
experiments at 6 weeks of age after acclimation period. The animals were
divided into experimental groups matched for body weight (n = 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 h 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..
condition of hSGLT inhibitory activities (pH 7.4; see Ref. 15a).
17. The stereochemistry of 9a–9f at the anomeric center was determined as
b-configuration by the coupling constant between anomeric C–H and adjacent
C–H in 1H NMR studies (J = 8.8–9.3 Hz in DMSO-d6).
18. Neyts, J.; Das, A. R.; Hung, S. C.; Hwu, J. R.; Singha, R. PCT Int. Appl.
WO2007128086, 2007; Chem. Abstr. 2007, 147, 522523.
19. In the cases of pyridones 15b and 15c, N-glucosylated products were obtained.
These N-glucosides were confirmed by the presence of carbonyl absorptions in
the IR spectrum of the corresponding deacetylated compounds 9b and 9c
(1667 cmÀ1 and 1650 cmÀ1, respectively).
References and notes
20. Synthesis of 3-(4-ethylphenylmethyl)-1-(b-D-Glucopyranosyl)indole 9d from 13d:
To a solution of 13d (920 mg, 3.9 mmol) in MeCN (30 mL) was added BSA
(>75%, 1.6 mL, ca. 4.7 mmol) and the mixture was stirred at 60 °C for 3 h under
argon atmosphere. After being cooled to ambient temperature, the solvent was
removed by evaporation and the resulting residue was dissolved in
dichloroethane (35 mL). Then, 14 (2.5 g, 6.4 mmol) and TMSOTf (1.1 mL,
6.1 mmol) were added to the solution, and the reaction mixture was stirred at
80 °C for an hour followed by at 60 °C for 12 h under argon atmosphere. After
being cooled to ambient temperature, the reaction mixture was poured into a
saturated aqueous NaHCO3 solution and extracted with AcOEt. The organic
layer was dried over Na2SO4, concentrated under reduced pressure, and
purified by silica gel column chromatography (hexane: AcOEt = 98: 2–50:50)
to give 15d (315 mg, 17%) as a powder. NaOMe (28% MeOH solution, 5 drops)
was added to a stirred solution of 15d (300 mg, 0.53 mmol) in MeOH (10 mL)–
THF (5 mL) at room temperature. After 30 min, the reaction mixture was
concentrated under reduced pressure, and purified by silica gel column
chromatography (CHCl3: MeOH = 100: 0–86: 14) to give 9d (145 mg, 48%) as a
Yamamoto, Yasuo
