Discovery of remogliflozin etabonate: A potent and highly selective SGLT2 inhibitor

Article abstract of DOI:10.1016/j.bmc.2021.116033

We optimized the structure of an active metabolite (1) of WAY-123783, which was obtained from mouse urine after oral administration, to improve selectivity for SGLT2 and oral bioavailability. O-glucoside derivative 24 (remogliflozin etabonate) was subsequently identified as a potent, highly selective, and orally available SGLT2 inhibitor.

Full text of DOI:10.1016/j.bmc.2021.116033

Bioorg. Med. Chem. 34 (2021) 116033
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of remogliflozin etabonate: A potent and highly selective
SGLT2 inhibitor
,b,
Kazuo Shimizua *, Hideki Fujikuraa, Nobuhiko Fushimia, Toshihiro Nishimuraa,
Kazuya Tatania, Kenji Katsunoa, Yoshikazu Fujimoria, Shinjiro Watanabea,
Masahiro Hiratochia, Takeshi Nakabayashia, Noboru Kamadaa, Koichi Arakawaa,
,
Hidemasa Hikawab, Isao Azumayab *, Masayuki Isajia
a Central Research Laboratory, Kissei Pharmaceutical Co., Ltd., 4365-1,Hotakakashiwabara, Azumino, Nagano 399-8304, Japan
b Faculty of Pharmaceutical Sciences, Toho University, 2-2-1, Miyama, Funabashi, Chiba 274-8510, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
We optimized the structure of an active metabolite (1) of WAY-123783, which was obtained from mouse urine
after oral administration, to improve selectivity for SGLT2 and oral bioavailability. O-glucoside derivative 24
(remogliflozin etabonate) was subsequently identified as a potent, highly selective, and orally available SGLT2
inhibitor.
Type 2 diabetes mellitus
SGLT2 inhibitor
O-glucoside
Remogliflozin etabonate
1. Introduction
diabetic drugs in US, Europe, and/or Japan9, and more recently sota-
gliflozin was approved as a T1DM medication.10 All approved SGLT2
The International Diabetes Federation has reported that the global
prevalence of diabetes was estimated to be 9.3% (463 million people) in
2019 and is expected to increase to 10.2% (578 million) by 2030 and
10.9% (700 million) by 2045 without sufficient action to address this
pandemic.1 Diabetes has been classified into type-1 (T1DM) and type-2
(T2DM) diabetes mellitus. Prolonged exposure to high glucose levels in
the blood can result in neurological and cardiovascular complications.2,3
Although a number of anti-diabetic drugs have been developed and
marketed, adverse effects of oral anti-diabetic drugs, including hypo-
glycemia and weight gain, increase the demand for safer and newer
classes of anti-diabetic drugs.3
inhibitors are C-aryl glucosides and their analogs.
SGLT2 inhibitors originated from the natural product phlorizin
(Figure 1), which was demonstrated to induce urinary glucose excretion
and was successfully used to lower serum glucose levels.11 However,
phlorizin was not developed for clinical use because of limitations such
as low oral bioavailability resulting from rapid hydrolysis of O-glucoside
linkage in the intestine and low selectivity for SGLT2.11–13 Phlorizin also
inhibits SGLT1, which is responsible for the absorption of dietary
glucose and galactose transport in the small intestine. Loss-of-function
mutations in SGLT1 cause severe diarrhea and dehydration by
glucose/galactose malabsorption.14 Therefore, SGLT2-selective non-O-
glucoside derivatives have been intensively explored. Alternatively, O-
glucoside SGLT2 inhibitors with greater intestinal stability and selec-
tivity represent another development route. T-1095 (Figure 1), a de-
rivative of phlorizin, was identified as the first orally active SGLT
inhibitor by Tanabe Seiyaku Co., Ltd.15 T-1095 overcame some disad-
vantages of phlorizin in development and progressed to clinical trials.5
We independently performed lead identification and optimization
studies focusing on the non-phlorizin type benzylpyrazole O-glucoside
derivatives, which culminated in the discovery of remogliflozin eta-
bonate (24) (Figure 2, RE) and its subsequent launch in India in 2019.
A number of sodium/glucose cotransporter 2 (SGLT2) inhibitors,
such as dapagliflozin (Figure 1), have been developed and in clinical use
as a relatively new class of anti-diabetic drugs. SGLT2 is primarily
expressed in the proximal tubule in the kidney, and approximately 90%
of filtered glucose is reabsorbed by SGLT2. Hence, the inhibition of
SGLT2 increases urinary glucose excretion and effectively reduces
circulating plasma glucose levels.4,5 Furthermore, SGLT2 inhibitors
have been shown to exert cardiorenal protective effects and are now
recommended as second-line pharmacotherapy in patients with
T2DM.6–8 To date, seven SGLT2 inhibitors have been approved as anti-
* Corresponding authors at: Central Research Laboratory, Kissei Pharmaceutical Co., Ltd., 4365-1,Hotakakashiwabara, Azumino, Nagano 399-8304, Japan (K.
Shimizu); Faculty of Pharmaceutical Sciences, Toho University, 2-2-1, Miyama, Funabashi, Chiba 274-8510, Japan (I. Azumaya).
E-mail addresses: kazuo_shimizu@pharm.kissei.co.jp (K. Shimizu), isao.azumaya@phar.toho-u.ac.jp (I. Azumaya).
https://doi.org/10.1016/j.bmc.2021.116033
Received 7 December 2020; Received in revised form 14 January 2021; Accepted 16 January 2021
Available online 22 January 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
Here, we describe the structure–activity relationships (SARs) and the
discovery of RE as the first marketed O-glucoside-type SGLT2 inhibitor.
phlorizin-type compounds such as T-1095, we focused on the non-
glycoside pyrazole compound WAY-123783, which has shown an anti-
hyperglycemic effect in db/db mice.19 Given that WAY-123783 had a
robust glycosuric effect when administered to normal mice, and did not
block intestinal glucose absorption, it was proposed that renal SGLT2
might represent the molecular target of this compound. To test this
hypothesis, we evaluated the inhibitory activity of WAY-123783 on
human SGLT2 expressed in COS-7 cells; an apparent effect was not
observed (IC50 value: >10ꢀ 4 M), suggesting the presence of an active
metabolite. Next, we analyzed the major metabolites of WAY-123783
and identified an atypical urinary glucose conjugate in mice. Inspired
by the structure of phlorizin, we synthesized corresponding O-glucoside
of WAY-123783 and confirmed that its retention time was consistent
with that of the metabolite in HPLC (data not shown). Furthermore, the
synthesized compound 1 exerted excellent inhibitory activity on SGLT2
(Table 1), indicating that this compound was an active metabolite of
WAY-123783.
2. Chemistry
4-Benzylpyrazole-O-glucoside derivatives 1–7 and 9–17 were pre-
pared as illustrated in Scheme 1. Benzyl halides or benzyl alcohols, in
which hydroxyl groups of benzyl alcohols were methanesulfonylated
using methanesulfonyl chloride (MsCl) with Et3N before condensation,
were allowed to react with ketoesters in the presence of NaH to afford 2-
substituted ketoesters. Subsequent cyclization with hydrazine mono-
hydrate gave aglycones 2a–7a and 9a–17a. Reaction of aglycones with
acetobromoglucose was carried out in two-phase solvents consisting of
CH2Cl2 and aqueous NaOH solution in the presence of the phase-transfer
catalyst BnN(nBu)3Cl or in CH3CN (for 1b) using K2CO3 as a base to
afford glucoside 1b–7b and 9b–17b. Finally, the acetyl groups pro-
tecting the hydroxyl groups on the sugars were removed by solvolysis
using NaOMe in MeOH to give 1–7 and 9–17.16
Resistance to the enzymatic hydrolysis of the glucosidic linkage
should differ from that of the phlorizin-type dihydrochalcone com-
pounds because an alkylated pyrazole ring instead of the acylated
phenol in phlorizin is attached to the glucose in 1. The character of this
novel scaffold prompted us to clarify the unknown SARs of 1 for later
optimization of orally available selective SGLT2 inhibitors.
4-(2-Phenylethyl)indazole-O-glucoside derivative (8) was prepared
as illustrated in Scheme 2. 4-Bromo-1,2-dihydro-3H-indazol-3-one was
glycosylated using 2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide
with K2CO3. Subsequent introduction of the phenethyl group was car-
ried out by Mizoroki–Heck reaction using styrene, followed by catalytic
hydrogenation of the double bond. The structure of the product 8b was
confirmed by X-ray crystal structure analysis.17 Finally, the pivaloyl
groups on the sugar were removed by solvolysis using LiOH in MeOH to
give 8.
Although the synthesis, SGLT inhibitory activity in brush border
membrane vehicles of rat kidney and urinary glucose excretion in rats by
intravenous injection of two hypothetical conjugates of WAY-123783
analogue have been reported previously,20 we considered it valuable
to report the detailed SAR of the first marketed orally available O-
glycoside SGLT2 inhibitor RE herein.
Preparation of 18 and N-alkylated-4-benzylpyrazole-O-glucoside
derivatives (19–22) is shown in Scheme 3. 18 was prepared by solvolysis
using NaOMe in MeOH of 18b, which was synthesized according to the
method reported by Kobayashi et al.18 The synthesis of N-alkylated-4-
benzylpyrazole-O-glucoside derivatives (19b–22b) was achieved by
alkylation of 18b with corresponding alkyl iodides in the presence of
NaH. 19b was isolated through column chromatography as a minor
product. The pivaloyl groups on the sugars were removed by solvolysis
using NaOMe in MeOH to give 19–22.18
The lead compound 1 showed potent inhibitory activity on SGLT2
but poor selectivity for SGLT2 (Table 1) and low oral bioavailability in
rats was observed (data not shown). In subsequent metabolic stability
testing using rat intestinal S9, 1 was rapidly hydrolyzed to WAY-123783
as with phlorizin, indicating that stability of the glucosidic linkage of 1
was low. Therefore, we initiated the optimization of 1 to improve
selectivity for SGLT2 and oral bioavailability.
All compounds were screened for their inhibitory effects on the up-
It has been reported that the hydrolysis of phenyl β-ᴅ-glucosides by
β-glucosidase is facilitated by the introduction of electron-withdrawing
groups into the benzene ring.21 Given that both the carbonyl group in
phlorizin and the CF3 group in 1 attenuate the stability of their gluco-
sides, we initially converted the CF3 group of 1 into electron-donating
groups (Table 1).
take of radiolabeled
α
-methyl-ᴅ-glucopyranoside ([14C]-AMG) into COS-
7 cells transiently expressing human SGLT1 or SGLT2.13
3. Results and discussion
To identify a novel scaffold SGLT2 inhibitor that differs from
As expected, the metabolic stability in rat intestinal S9 increased
Fig. 1. Structure of SGLT inhibitors and WAY-123783.
2

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
following the replacement of the CF3 group with a methyl group (2).
Moreover, methyl derivative (2) exhibited three-fold more potent SGLT2
inhibitory activity with four-fold reduced inhibition of SGLT1, indi-
cating that this structural conversion was favorable for SGLT2 selec-
tivity. Elongation of the methyl group (2) into ethyl group (3) or
isopropyl group (4) markedly decreased the inhibitory activity and
selectivity for SGLT2; the methyl group was thus considered optimal. In
a recent report on the predicted binding mode of phlorizin to SGLTs, a
hydrophobic cage (H80, F98, and H268 on EL5c) around the central ring
of the aglycone tail of phlorizin was shown to be formed in SGLT2,
whereas a salt bridge (R267 and D268) was created in SGLT1 because
268 is aspartic acid.22 The subtype selectivity related to the bulkiness of
the pyrazole substituent may be attributed to differences in the amino
acid environment surrounding the pyrazole ring.
Compound
n
1
1
1
1
1
2
3
1
1
1
1
1
1
1
1
1
R1
CF3
Me
Et
R2
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
R3
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
R4
SMe
SMe
SMe
SMe
H
Next, to establish an optimal scaffold, the linker length between the
pyrazole ring and benzene ring was verified using aglycone without a
methylsulfanyl group as shown in Table 2. In unsubstituted phenyl de-
rivative (5), elongation of methylene into ethylene (6) or trimethylene
(7) led to remarkable loss of activity, suggesting that the conformational
difference in each scaffold would cause a differentiated SAR. Because
phlorizin has a carbonyl group in the linker chain and ortho-hydroxyl
group on the benzene ring, an intramolecular hydrogen bond between
these groups might contribute to fixing the active conformation of this
compound.23 To mimic the pseudo 6-membered ring of phlorizin, we
designed and synthesized indazole derivative (8) by cyclizing the 4- and
5-substituents of the pyrazole ring of 7 and found that this conversion
was highly effective in enhancing the inhibitory activity on SGLT2.
However, we did not further explore this new scaffold, because 8 was
equipotent on SGLT1. In previous reports, indole compounds derived
from phlorizin also showed reduced selectivity for SGLT2.24 Thus, in
obtaining a highly SGLT2-selective inhibitor, presence of the fused ring
scaffolds derived from phlorizin was disadvantageous.
1
2
3
iPr
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
4
5
H
6
H
7
H
9
H
10
11
12
13
14
15
16
17
Me
Et
Because urinary glucose excretion is affected by substituents on the
distal benzene ring of phlorizin derivatives25, those groups should
impact SGLT2 inhibition. In fact, 2 was more potent than 5 in our study,
and we next optimized the benzene ring substituents. As shown in
Table 3, our initial attempt at methyl scanning demonstrated that only
4-methyl derivative (11) enhanced SGLT2 inhibitory activity. In benzyl
phenyl C-glucoside type SGLT2 inhibitors, including dapagliflozin,
substituents at the 4-position of the distal benzene ring have been
intensively explored,26 meaning that this position is an established site
for potential optimization, regardless of the scaffolds present.
nPr
iPr
OMe
OEt
OnPr
Scheme 1. Reagents and conditions: (a) MsCl, Et3N, THF, 0 ◦C (b) NaH,
R1COCH2CO2Et, THF or DME, 0 ◦C to reflux; (c) K2CO3, methyl acetoacetate,
toluene, 75 ◦C; (d) N2H4-H2O, toluene, rt to reflux, (2 or 3 steps, 16%–70%); (e)
K2CO3, acetobromoglucose, CH3CN, rt, 57%; (f) 5 M NaOH aq., BnN(nBu)3Cl,
acetobromoglucose, CH2Cl2, rt, (6%–25%); (g) NaOMe, MeOH, rt, (43%–74%).
Subsequent elongation of the 4-methyl group into ethyl group (12)
improved both SGLT2 inhibitory activity and selectivity, and isopropyl
derivative (14) exerted higher selectivity compared with 12 despite
diminished activity (Table 4). Alkoxy groups were also examined, and
SGLT2 inhibitory activities of derivatives (15–18) were found to be
comparable to that of 11. However, elongation of the alkoxy group
decreased SGLT1 inhibitory activity with increasing carbon number,
resulting in improved selectivity for SGLT2. Because the highest selec-
tivity was achieved by introducing an isopropoxy group, we selected
compound 18 for further evaluation. In the marketed C-glucoside type
SGLT2 inhibitors, the 4-ethyl or 4-ethoxy group is primarily selected as
the optimal substituent.9,26 Our SAR studies with pyrazole O-glucoside
Fig. 2. Structure of remogliflozin (23) and remogliflozin etabonate (24).
3

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
Scheme 2. Reagents and conditions: (a) 2,3,4,6-tetra-O-pivaloyl-
α
-D-glucopyranosyl bromide, K2CO3, CH3CN, rt, 30%; (b) styrene, Pd(OAc)2, Tri(o-tolyl)phosphine,
Et3N, CH3CN, reflux, 41%; (c) H2, Pd-C, THF, rt, 94%; (d) LiOH-H2O, MeOH, rt, 60%.
Scheme 3. Reagents and conditions: (a) R5I, NaH, 1,3-dimethyl-2-imidazolidinone, rt, 6.9% (19b), 51% (20b), 71% (21b), 78% (22b); (b) NaOMe, MeOH, rt,
(59%–67%).
Table 1
Table 3
In vitro inhibitory activity, selectivity, and metabolic stability of compounds
In vitro inhibitory activity and selectivity for compounds 9–11.
1–4.
Compound
Compound
R2
R3
R4
IC50 (nM)a
Selectivityb
R1
IC50 (nM)a
hSGLT2
Selectivityb
Metabolic
stabilityc
CLint (µL/min/
mg)
hSGLT2
hSGLT1
hSGLT1
9
Me
H
H
H
914 ± 211
757 ± 82
12.2 ± 1.2
1630 ± 349
8710 ± 1630
321 ± 29
1.8
12
26
10
Me
H
11
H
H
Me
1
2
3
CF3
Me
Et
16.3 ± 0.7
4.9 ± 0.5
51.2 ±
109 ± 18
405 ± 29
378 ± 18
6.7
83
73.2
3.2
a
Concentration of each compound required to inhibit [14C]-
α
-methyl-ᴅ-glu-
7.4
4.8
copyranoside by 50%. Data represent the mean ± SEM of at least three inde-
14.9
pendent experiments unless otherwise stated.
4
iPr
154 ± 23
468 ± 37
3.0
4.6
b
Selectivity values were calculated as IC50 hSGLT1/IC50 hSGLT2.
a
Concentration of each compound required to inhibit [14C]-
α-methyl-ᴅ-glu-
copyranoside by 50%. Data represent the mean ± standard error of the mean
(SEM) of at least three independent experiments unless otherwise stated.
b
Selectivity values were calculated as IC50 hSGLT1/IC50 hSGLT2.
c
Incubated with rat intestinal S9.
Table 4
In vitro inhibitory activity and selectivity of compounds 12–18.
Compound
R4
IC50 (nM)a
Selectivityb
hSGLT2
hSGLT1
Table 2
12
13
14
15
16
17
18
Et
3.9 ± 0.6
8.8 ± 1.2
16.2 ± 0.9
12.3 ± 1.6
10.8 ± 0.4
15.4 ± 2.3
10.0 ± 1.2
233 ± 48
60
In vitro inhibitory activity and selectivity of compounds 5–8.
nPr
345 ± 38
39
Compound
n
IC50 (nM)a
Selectivityb
iPr
1690 ± 416
626 ± 133
1030 ± 64
1630 ± 143
2780 ± 148
104
51
OMe
OEt
OnPr
OiPr
hSGLT2
hSGLT1
95
5
6
7
8
1
2
3
–
181 ± 12
1320 ± 69
7.3
6.0
6.0
1.7
106
278
5290 ± 939
3950 ± 651
86.0 ± 2.9
31500 ± 7610
23700 ± 1700
150 ± 15
Concentration of each compound required to inhibit [14C]-
α
-methyl-ᴅ-glu-
a
copyranoside by 50%. Data represent the mean ± SEM of at least three inde-
a
Concentration of each compound required to inhibit [14C]-
α
-methyl-ᴅ-glu-
pendent experiments unless otherwise stated.
copyranoside by 50%. Data represent the mean ± SEM of at least three inde-
b
Selectivity values were calculated as IC50 hSGLT1/IC50 hSGLT2.
pendent experiments unless otherwise stated.
b
Selectivity values were calculated as IC50 hSGLT1/IC50 hSGLT2.
4

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
demonstrated that the 4-isopropoxy group was potentially comparable
with those groups.
Table 6
ADME profiles and pharmacokinetics data in rats for compounds 18, 23 and 24.
b
Unfortunately, compound 18 did not show a sufficient improvement
in oral bioavailability (Table 6), despite having robust metabolic sta-
bility in rat intestinal S9. This suboptimal result may be attributable to
low membrane permeability resulting from 18 having multiple
hydrogen bond donors and therefore being less lipophilic. Next, we
introduced the substituents onto the nitrogen atoms of the pyrazole ring
to modulate the physical parameters of 18.
Compound
Metabolic
Caco-2 Papp
AUC0-tz (ng⋅min/
F (%)
stabilitya
mL)c
CLint (μL/
18
23
24
18d
23e
min/mg)
4.1
18
23
24
1.02
N/A
N/A
N.A.
3.16
10.11
N.A.
N.A.
4.29
385 ±
130
N.A.
0.43f
3.23g
5.79h
3.9
3.4
1833
± 568
4088
±
1677
± 504
3005
±
As shown in Table 5, compound 20 with a 1-methyl group exhibited
weakened but partly retained SGLT2 inhibitory activity while remaining
highly selective for SGLT2, whereas unacceptable decreased activity was
observed in 2-methylated compound 19. In the 1H NMR spectra of both
compounds, changes in the chemical shift of the signals of the sugar
moiety of 19 by N-methylation were larger than those of 20, suggesting
that substituents introduced more closely to the glucopyranosyloxy
group disrupted the active conformation.
1597
1147
a
Incubated with rat intestinal S9.
b
c
d
Papp (10-6 cm/s) = mL/(
μ
M)⋅1/cm2⋅(
μ
M/s)⋅10-6.
3 mg/kg oral administration, 0.5% MC (mean ± standard deviation),
Plasma concentrations of 18 after single oral administration of 18, 23, and
24.
e
Plasma concentrations of 23 after single oral administration of 23 and 24.
F value of 18 was calculated as a ratio between the AUC of 18 after oral
Subsequent elongation of the 1-methyl group into ethyl and n-propyl
groups (21, 22) recovered the SGLT2 inhibitory activity, and this posi-
tion was thus considered to accept the lipophilic groups. Furthermore,
introduction of an isopropyl group furnished compound 23, which
showed comparable activity with 18 and excellent selectivity for SGLT2.
As expected, compared with unsubstituted 18, compound 23 was more
lipophilic and showed better Caco-2 permeability (Table 6). Among
these derivatives, oxidative cleavage of the N-alkyl groups was a concern
in terms of metabolic instabilty,27 but 23 showed the highest metabolic
stability when incubated with human liver microsome because its
branched alkyl group might be less reactive (Table 5). An improvement
in oral absorption was also observed, and the bioavailability in rats of N-
alkylated 23 was higher than that of unsubstituted 18 (Table 6). In the
pharmacokinetic (PK) study of 23, the active metabolite 18 was also
detected, suggesting that the N-substituent plays a potential role as a
prodrug group.
f
administration and the AUC of 18 after iv administration (1 mg/kg, 20% DMA /
80% saline) of 18.
g
F value of 23 was calculated as a ratio between the AUC of 23 after oral
administration of 23 and the AUC of 23 after iv administration of 23.
h
F value of 24 was calculated as a ratio between the AUC of 23 after oral
administration of 24 and the AUC of 23 after iv administration of 23.AUC0-tz: the
area under the plasma concentration–time curve up to the last observation point,
F: bioavailability, N.D.: not detected, N.A.: not applicable.
prodrug group also improved the absorption of 23 in vivo. As a result of
this study, we determined that compound 24, remogliflozin etabonate
(RE), had potent and highly selective SGLT2 inhibitory activity and
favorable oral absorption, leading to its selection as a clinical candidate
for further development. The biological evaluation of RE has previously
been reported.13
Finally, to further increase blood exposure of compound 23, we
attempted a prodrug formation focusing on esterification of the sugar
moiety. In oral administration of the phlorizin derivatives in rats, some
prodrug groups of the hydroxyl groups enhanced excretion of the uri-
nary glucose.15 Based on those findings, ethyl carbonate of the 6-hydrox-
yl group of the glucose residue of 23 was designed and synthesized
(Figure 2). Fortunately, the resultant compound 24 showed higher Caco-
2 permeability compared with its parent 23, likely because of its greater
lipophilicity (Table 6). In this permeation study, a significant amount of
23 was detected in the basolateral side, indicating that 24 had excellent
permeability.
4. Conclusion
In the present study, a series of pyrazole-O-glucoside derivatives of
the active metabolite of WAY-123783 was designed and synthesized to
identify novel orally available selective SGLT2 inhibitors. SAR studies
revealed that the substituent at 5-position of the pyrazole ring could
affect the intestinal stability of the glucosidic linkage, and that a 5-meth-
ylpyrazole bearing 4-isopropoxybenzyl group was the optimal structure
of this scaffold. To improve oral absorption, alkylation of the 1-nitrogen
atom of the pyrazole ring and prodrug formation of the sugar moiety
were effective; therefore, isopropyl and ethoxycarbonyl groups were
respectively introduced.
In the subsequent PK study of orally administered 24 in rats, the
active metabolite 18 was also detected in addition to the parent 23
(Table 6), but 24 was not observed (<1.0 ng/mL) probably due to rapid
metabolism in vivo. The total area under the curve (AUC) of these
compounds was larger than that of the parent 23, indicating that the
Optimized remogliflozin etabonate (RE) has been shown to be clin-
ically useful and in 2019 achieved its first approval for the treatment of
type 2 diabetes in India.28 Multiple reports of clinical evaluations of this
Table 5
In vitro activity, selectivity, ClogP analysis, and metabolic stability of compounds 18–23.
Compound
R5
IC50 (nM)a
Selectivityb
ClogPc
Metabolic stabilityd
CLint (µL/min/mg)
hSGLT2
hSGLT1
18
19
20
21
22
23
H
10.0 ± 1.2
> 1000
2780 ± 148
278
–
124
306
156
902
1.21
–
1.33
1.69
2.21
2.10
1.44
N.T.
3.73
4.15
6.75
0.85
Me
Me
Et
> 100 µM
110 ± 21
43.4 ± 16.0
66.1 ± 29.6
18.4 ± 1.6
13600 ± 2270
13300 ± 2080
10300 ± 1190
16600 ± 1810
nPr
iPr
a
Concentration of each compound required to inhibit [14C]-
α-methyl-ᴅ-glucopyranoside by 50%. Data represent the mean ± SEM of at least three independent
experiments unless otherwise stated.
b
Selectivity values were calculated as IC50 hSGLT1/IC50 hSGLT2.
c
d
ClogP was calculated by Chemaxon’s Marvin ver. 19.21.
Incubated with human liver microsome.N.T., not tested.
5

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
agent are available.29 In recent years, beneficial extra-glycemic effects of
SGLT2 inhibitors, such as cardiorenal protection, have been reported
and attracting significant attention in this field.30 As the first marketed
O-glucoside-type SGLT2 inhibitor, we expect RE to address a current
unmet need.31
5.1.4. 4-Benzyl-5-methyl-1,2-dihydro-3H-pyrazol-3-one (5a)
NaH (323 mg, 8.07 mmol, 60% oil dispersion), under ice cooling,
was added to a solution of ethyl 3-oxobutanoate (1.00 g, 7.68 mmol) in
THF (10 mL), and the mixture was stirred for 30 min. Benzyl chloride
(973 mg, 7.68 mmol) was added, and the mixture was refluxed over-
night. After cooling to room temperature, saturated NH4Cl aq. was
added, and the resulting mixture was extracted with EtOAc. The extract
was washed with water and brine, and dried over anhydrous MgSO4.
The solvent was removed in vacuo to give crude ethyl 2-benzyl-3-oxobu-
tanoate. This material was dissolved in toluene (10 mL), and hydrazine
monohydrate (1.15 g, 23.1 mmol) was added, and the reaction mixture
was stirred at 80 ◦C overnight. The reaction mixture was cooled to room
temperature, and subjected to phase separation after the addition of
water (10 mL) and EtOAc (10 mL). The organic layer was washed with
brine, dried over MgSO4 and then concentrated in vacuo. The precipitate
was washed with Et2O to give 5a as a white solid (828 mg, 57%). 1H
NMR (DMSO‑d6) δ: 2.04 (3H, s), 3.60 (2H, s), 7.10–7.19 (3H, m),
7.20–7.29 (2H, m), 9.00–13.3 (2H, br); MS m/z: 189 (M+H)+.
5. Experimental
5.1. Chemistry
All reagents and solvents were purchased from commercial sources
and used without further purification. All moisture and air sensitive
reactions were carried out under an argon atmosphere. Reactions were
monitored by thin layer chromatography (TLC) using Merck Kieselgel 60
F254 plates. Flash column chromatography was performed on YAMA-
ZEN amino packed inject column and Biotage prepacked columns using
an automated flash chromatography system (Biotage, Isolera One). 1
H
NMR spectra were recorded on a Bruker AV400 M spectrometer at 400.1
MHz. Chemical shifts (δ) are reported in parts per million (ppm) units
using tetramethylsilane as an internal standard. Data are presented as
follows; chemical shift, integration, multiplicity (s, singlet; d, doublet; t,
5.1.5. 5-Methyl-4-(2-phenylethyl)-1,2-dihydro-3H-pyrazol-3-one (6a)
To a mixture of (2-bromoethyl)benzene (3.00 g, 16.2 mmol), ethyl 3-
oxo-butanoate (2.32 g, 17.8 mmol) and toluene (5 mL) were added
K2CO3 (5.68 g, 40.7 mmol) and water (0.1 mL). The mixture was heated
to 75 ◦C and stirred at the same temperature overnight. The reaction
mixture was cooled to room temperature, and subjected to phase sepa-
ration after the addition of water (10 mL) and toluene (2 mL). The
organic layer was washed twice with water, dried over MgSO4 and then
concentrated in vacuo to give the crude product of ethyl 3-oxo-2-(2-phe-
nylethyl)butanoate. This material was dissolved in toluene (15 mL), and
hydrazine monohydrate (2.43 g, 48.6 mmol) was added. The reaction
mixture was stirred at 90 ◦C for 12 h then cooled to room temperature.
The resulting slurry was stirred at 0 ◦C for 1 h. The precipitate was
filtered off, washed with cooled toluene and water and dried in vacuo at
60 ◦C to give 6a (2.20 g, 67%) as a white solid. 1H NMR (DMSO‑d6) δ:
1.84 (3H, s), 2.40–2.50 (2H, m), 2.60–2.72 (2H, m), 7.10–7.20 (3H, m),
7.21–7.30 (2H, m), 8.70–11.5 (2H, br); MS m/z: 203 (M + H)+.
triplet; q, quartet; m, multiplet; br, broad), and coupling constant. 13
C
NMR spectra were recorded on a Bruker AV400M spectrometer at 100.6
MHz with complete proton decoupling. Chemical shifts (δ) are reported
in parts per million (ppm) with the solvent as the internal reference (δ
20.00 in AcOH-d4, 49.00 in CD3OD).32,33 Mass spectra (MS) were
measured using an Agilent Technologies 6150 (ESI). High resolution
mass spectra (HRMS) were recorded on an Agilent Technologies 6520
Accurate-Mass Q-TOF instrument using electrospray ionization (ESI,
positive or negative ion mode).
5.1.1. 5-Methyl-4-[4-(methylsulfanyl)benzyl]-1,2-dihydro-3H-pyrazol-3-
one (2a)
NaH (161 mg, 4.03 mmol, 60% oil dispersion), under ice cooling,
was added to a solution of ethyl 3-oxobutanoate (0.50 g, 3.84 mmol) in
1,2-dimethoxyethane (5 mL), and the mixture was stirred for 5 min. 4-
(Methylsulfanyl)benzyl chloride (663 mg, 3.84 mmol) was added, and
the mixture was refluxed for 12 h. After cooling to room temperature,
saturated NH4Cl aq. was added, and the resulting mixture was extracted
with EtOAc. The extract was washed with brine and dried over anhy-
drous MgSO4. The solvent was removed in vacuo to give crude ethyl 2-
[4-(methylsulfanyl)benzyl]-3-oxobutanoate. This material was dis-
solved in toluene (5 mL), and hydrazine monohydrate (192 mg, 3.84
mmol) was added. The reaction mixture was stirred at 90 ◦C for 10 h and
then cooled to room temperature. The precipitate was filtered off,
washed with toluene and water and dried in vacuo to give 2a (350 mg,
39%) as a white solid. 1H NMR (DMSO-d6) δ: 2.00 (3H, s), 2.42 (3H, s),
3.50 (2H, s), 7.07–7.18 (4H, m), 8.80–11.6 (2H, br); MS m/z: 235 (M +
H)+.
5.1.6. 5-Methyl-4-(3-phenylpropyl)-1,2-dihydro-3H-pyrazol-3-one (7a)
The title compound was prepared from (3-bromopropyl)benzene, as
described for the synthesis of 2a, in 49% yield. 1H NMR (DMSO‑d6) δ:
1.62–1.76 (2H, m), 2.01 (3H, s), 2.21 (2H, t, J = 7.4 Hz), 2.54 (2H, t, J =
7.8 Hz), 7.12–7.21 (3H, m), 7.23–7.31 (2H, m), 8.70–11.5 (2H, br); MS
m/z: 217 (M + H)+.
5.1.7. 5-Methyl-4-(2-methylbenzyl)-1,2-dihydro-3H-pyrazol-3-one (9a)
NaH (299 mg, 7.47 mmol, 60% oil dispersion), under ice cooling,
was added to a solution of ethyl 3-oxobutanoate (0.93 g, 7.11 mmol) in
THF (10 mL), and the mixture was stirred for 30 min. 1-(chloromethyl)-
2-methylbenzene (1.00 g, 7.11 mmol) was added, and the mixture was
refluxed overnight. After cooling to room temperature, saturated NH4Cl
aq. was added, and the resulting mixture was extracted with EtOAc. The
extract was washed with brine and dried over anhydrous MgSO4. The
solvent was removed in vacuo to give crude ethyl 2-(2-methylbenzyl)-3-
oxobutanoate. This material was dissolved in toluene (10 mL), and hy-
drazine monohydrate (1.07 g, 21.3 mmol) was added. The reaction
mixture was stirred at 80 ◦C overnight and then stirred under ice cooling
for 1 h. The precipitate was filtered off, washed with toluene and water
and dried in vacuo to give 9a (834 mg, 58%) as a white solid. 1H NMR
(DMSO‑d6) δ: 1.92 (3H, s), 2.28 (3H, s), 3.49 (2H, s), 6.90–6.99 (1H, m),
7.00–7.14 (3H, m), 8.70–11.8 (2H, m); MS m/z: 203 (M + H)+.
5.1.2. 5-Ethyl-4-[4-(methylsulfanyl)benzyl]-1,2-dihydro-3H-pyrazol-3-
one (3a)
The title compound was prepared from ethyl 3-oxopentanoate, as
described for the synthesis of 2a, in 65% yield. 1H NMR (DMSO‑d6) δ:
1.02 (3H, t, J = 7.6 Hz), 2.39 (2H, q, J = 7.6 Hz), 2.42 (3H, s), 3.51 (2H,
s), 7.05–7.20 (4H, m), 8.80–11.6 (2H, br); MS m/z: 249 (M + H)+.
5.1.3. 5-Isopropyl-4-[4-(methylsulfanyl)benzyl]-1,2-dihydro-3H-pyrazol-
3-one (4a)
The title compound was prepared from ethyl 4-methyl-3-oxopenta-
noate, as described for the synthesis of 2a, in 34% yield. 1H NMR
(DMSO‑d6) δ: 1.07 (6H, d, J = 7.1 Hz), 2.42 (3H, s), 2.83(1H, heptet, J =
7.1 Hz), 3.53 (2H, s), 7.06–7.17 (4H, m), 8.95–9.70 (1H, br), 10.9–11.5
(1H, br); MS m/z: 263 (M + H)+.
5.1.8. 5-Methyl-4-(3-methylbenzyl)-1,2-dihydro-3H-pyrazol-3-one (10a)
The title compound was prepared from 1-(chloromethyl)-3-methyl-
benzene, as described for the synthesis of 9a, in 43% yield. 1H NMR
(DMSO‑d6) δ: 2.00 (3H, s), 2.24 (3H, s), 3.50 (2H, s), 6.87–6.99 (3H, m),
7.06–7.15 (1H, m), 8.60–11.8 (2H, br); MS m/z: 203 (M + H)+.
6

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
5.1.9. 5-Methyl-4-(4-methylbenzyl)-1,2-dihydro-3H-pyrazol-3-one (11a)
The title compound was prepared from 1-(chloromethyl)-4-methyl-
benzene, as described for the synthesis of 9a, in 70% yield. 1H NMR
(DMSO‑d6) δ: 1.98 (3H, s), 2.23 (3H, s), 3.48 (2H,s), 7.03 (4H, s),
8.60–11.8 (2H, br); MS m/z: 203 (M + H)+.
5.1.16. 4-[4-(Methylsulfanyl)benzyl]-5-trifluoromethyl-1H-pyrazol-3-yl
2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (1b)
A mixture of 4-[4-(methylthio)benzyl]-5-trifluoromethyl-1,2-dihy-
19
dro-3H-pyrazol-3-one
(WAY-123783) (830 mg, 2.88 mmol), aceto-
bromoglucose (1.18 g, 2.88 mmol), K2CO3 (438 mg, 3.17 mmol) and
CH3CN (10 mL) was stirred at room temperature for 22 h. To the mixture
was added water (10 mL) and extracted with EtOAc (30 mL). The
organic layer was washed with brine and dried over anhydrous MgSO4.
The solvent was removed in vacuo, and the residue was purified by flash
column chromatography on silica gel (eluent: hexane/EtOAc = 2:1–1:1)
to give 1b (1.01 g, 57%) as a white solid. 1H NMR (CDCl3) δ: 1.91 (3H,
s), 2.03 (3H, s), 2.04 (3H, s), 2.08 (3H, s), 2.44 (3H, s), 3.73 (2H, s),
3.78–3.88 (1H, m), 4.20 (1H, dd, J = 12.5 Hz, 2.0 Hz), 4.27 (1H, dd, J =
12.5 Hz, 4.2 Hz), 5.15–5.30 (3H, m), 5.32–5.52 (1H, br), 7.05–7.11 (2H,
m), 7.12–7.20 (2H, m), 9.20–10.8 (1H, br); MS m/z: 617(Mꢀ H)ꢀ .
5.1.10. 4-(4-Ethylbenzyl)-5-methyl-1,2-dihydro-3H-pyrazol-3-one (12a)
The title compound was prepared from 1-(chloromethyl)-4-ethyl-
benzene, as described for the synthesis of 9a, in 43% yield. 1H NMR
(DMSO‑d6) δ: 1.13 (3H, t, J = 7.6 Hz), 1.99 (3H, s), 2.52 (2H, q, J = 7.6
Hz), 3.49 (2H, s), 7.06 (4H, s), 9.00–9.80 (1H, br), 10.5–11.5 (1H, br);
MS m/z: 217 (M + H)+.
5.1.11. 5-Methyl-4-(4-propylbenzyl)-1,2-dihydro-3H-pyrazol-3-one (13a)
MsCl (4.06 g, 35.4 mmol), under ice cooling, was added to a solution
of (4-propylphenyl)methanol (5.1 g, 33.7 mmol) and Et3N (3.76 g, 37.1
mmol) in THF (30 mL), and the mixture was stirred for 10 min. The
insoluble material was removed by filtration and washed with THF (20
mL). The filtrate and washing were added to the mixture prepared by the
addition of NaH (1.62 g, 37.1 mmol, 55% oil dispersion) into a solution
of ethyl 3-oxobutanoate (4.39 g, 33.7 mmol) in THF (50 mL) under ice
cooling and stirring for 5 min, then the mixture was refluxed for 8 h.
After cooling to room temperature, and to the resulting mixture was
added water and extracted with EtOAc. The aqueous layer was washed
with EtOAc, and combined organic layers were washed with water and
brine and dried over anhydrous MgSO4. The solvent was removed in
vacuo to give crude ethyl 3-oxo-2-(4-propylbenzyl) butanoate. This
material was dissolved in toluene (40 mL), and hydrazine monohydrate
(2.94 g, 58.8 mmol) was added to the solution. The reaction mixture was
stirred at 100 ◦C for 5 h and then cooled to room temperature. The
precipitate was filtered off, washed with toluene and water and dried in
vacuo to give 13a (2.30 g, 34%) as a white solid. 1H NMR (DMSO‑d6) δ:
0.86 (3H, t, J = 7.3 Hz), 1.46–1.60 (2H, m), 1.99 (3H, s), 2.46–2.50 (2H,
m), 3.49 (2H, s), 7.06 (4H, s), 8.50–12.0 (2H, br); MS m/z: 231 (M +
H)+.
5.1.17. 5-Methyl-4-[4-(methylsulfanyl)benzyl]-1H-pyrazol-3-yl 2,3,4,6-
tetra-O-acetyl-β-D-glucopyranoside (2b)
Aqueous 5 M NaOH (0.84 mL, 4.27 mmol) was added to a mixture of
2a (250 mg, 1.07 mmol), acetobromoglucose (439 mg, 1.07 mmol), and
benzyltributylammonium chloride (333 mg, 1.07 mmol) in CH2Cl2 (5
mL), and the mixture was stirred at room temperature overnight. 1 M
HCl (5 mL) was added, and the resulting mixture was extracted with
EtOAc. The extract was washed with brine and dried over anhydrous
MgSO4. The solvent was removed in vacuo, and the residue was purified
by flash column chromatography on silica gel (eluent: hexane/EtOAc =
2:1–1:4) to give 2b (70 mg, 12%) as a white solid. 1H NMR (CDCl3) δ:
1.88 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.07 (3H, s), 2.12 (3H, s), 2.44
(3H, s), 3.55 (1H, d, J = 15.8 Hz), 3.62 (1H, d, J = 15.8 Hz), 3.82–3.89
(1H, m), 4.13 (1H, dd, J = 12.4 Hz, 2.4 Hz), 4.31 (1H, dd, J = 12.4 Hz,
4.1 Hz), 5.17–5.30 (3H, m), 5.55–5.63 (1H, m), 7.03–7.09 (2H, m),
7.12–7.17 (2H, m), 8.65–8.85 (1H, br); MS m/z: 565 (M + H)+.
5.1.18. 5-Ethyl-4-[4-(methylsulfanyl)benzyl]-1H-pyrazol-3-yl 2,3,4,6-
tetra-O-acetyl-β-D-glucopyranoside (3b)
The title compound was prepared from 3a, as described for the
synthesis of 2b, in 12% yield. 1H NMR (CDCl3) δ: 1.13 (3H, t, J = 7.6
Hz), 1.88 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.07 (3H, s), 2.44 (3H, s),
2.51 (2H, q, J = 7.6 Hz), 3.56 (1H, d, J = 16.0 Hz), 3.63 (1H, d, J = 16.0
Hz), 3.83–3.89 (1H, m), 4.14 (1H, dd, J = 12.4 Hz, 2.3 Hz), 4.31 (1H, dd,
J = 12.4 Hz, 4.0 Hz), 5.17–5.30 (3H, m), 5.56–5.62 (1H, m), 7.03–7.09
(2H, m), 7.11–7.16 (2H, m), 8.80–9.20 (1H, br); MS m/z: 579 (M + H)+.
5.1.12. 4-(4-Isopropylbenzyl)-5-methyl-1,2-dihydro-3H-pyrazol-3-one
(14a)
The title compound was prepared from 1-(chloromethyl)-4-iso-
propylbenzene, as described for the synthesis of 9a, in 47% yield. 1H
NMR (DMSO‑d6) δ: 1.16 (6H, d, J = 6.9 Hz), 2.01 (3H, s), 2.81 (1H,
heptet, J = 6.9 Hz), 3.49 (2H, s), 7.02–7.13 (4H, m), 9.00–9.80 (1H, br),
10.6–11.4 (1H, br); MS m/z: 231 (M + H)+.
5.1.19. 5-Isopropyl-4-[4-(methylsulfanyl)benzyl]-1H-pyrazol-3-yl 2,3,4,6-
tetra-O-acetyl-β-D-glucopyranoside (4b)
5.1.13. 4-(4-Methoxybenzyl)-5-methyl-1,2-dihydro-3H-pyrazol-3-one
(15a)
The title compound was prepared from 4a, as described for the
synthesis of 2b, in 17% yield. 1H NMR (CDCl3) δ: 1.17 (6H, dd, J = 7.0
Hz, 1.0 Hz), 1.86 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.07 (3H, s), 2.44
(3H, s), 2.91 (1H, heptet, J = 7.0 Hz), 3.58 (1H, d, J = 16.2 Hz), 3.65
(1H, d, J = 16.2 Hz), 3.83–3.90 (1H, m), 4.14 (1H, dd, J = 12.4 Hz, 2.2
Hz), 4.32 (1H, dd, J = 12.4 Hz, 4.0 Hz), 5.17–5.30 (3H, m), 5.56–5.59
(1H, m), 7.03–7.08 (2H, m), 7.11–7.16 (2H, m); MS m/z: 593 (M + H)+.
The title compound was prepared from 1-(chloromethyl)-4-
methoxybenzene, as described for the synthesis of 9a, in 66% yield. 1H
NMR (DMSO‑d6) δ: 1.99 (3H, s), 3.45 (2H, s), 3.69 (3H, s), 6.75–6.83
(2H, m), 7.02–7.10 (2H, m), 8.50–12.0 (2H, m); MS m/z: 219 (M + H)+.
5.1.14. 4-(4-Ethoxybenzyl)-5-methyl-1,2-dihydro-3H-pyrazol-3-one
(16a)
The title compound was prepared from (4-ethoxyphenyl)methanol,
as described for the synthesis of 13a, in 16% yield. 1H NMR (DMSO‑d6)
δ: 1.29 (3H, t, J = 7.0 Hz), 1.98 (3H, s), 3.46 (2H, s), 3.95 (2H, q, J = 7.0
Hz), 6,75–6.81 (2H, m), 7.00–7.08 (2H, m), 9.00–9.70 (1H, br),
10.7–11.4 (1H, br); MS m/z: 233 (M + H)+.
5.1.20. 4-Benzyl-5-methyl-1H-pyrazol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-
glucopyranoside (5b)
The title compound was prepared from 5a, as described for the
synthesis of 2b, in 20% yield. 1H NMR (CDCl3) δ: 1.86 (3H, s), 2.01 (3H,
s), 2.03 (3H, s), 2.07 (3H, s), 2.12 (3H, s), 3.59 (1H, d, J = 16.0 Hz), 3.66
(1H, d, J = 16.0 Hz), 3.81–3.90 (1H, m), 4.13 (1H, dd, J = 12.5 Hz, 2.2
Hz), 4.31 (1H, dd, J = 12.5 Hz, 4.0 Hz), 5.17–5.30 (3H, m), 5.54–5.60
(1H, m), 7.09–7.18 (3H, m), 7.19–7.30 (2H, m); MS m/z: 519 (M + H)+.
5.1.15. 5-Methyl-4-(4-propoxybenzyl)-1,2-dihydro-3H-pyrazol-3-one
(17a)
The title compound was prepared from (4-propoxyphenyl)methanol,
as described for the synthesis of 13a, in 55% yield. 1H NMR (DMSO‑d6)
δ: 0.95 (3H, t, J = 7.4), 1.63–1.75 (2H, m), 1.98 (3H, s), 3.46 (2H, s),
3.85 (2H, t, J = 6.5 Hz), 6.75–6.82 (2H, m), 7.00–7.07 (2H, m),
8.60–11.8 (2H, m); MS m/z: 247 (M + H)+.
5.1.21. 5-Methyl-4-(2-phenylethyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (6b)
The title compound was prepared from 6a, as described for the
synthesis of 2b, in 6.0% yield. 1H NMR (CDCl3) δ: 1.85 (3H, s), 2.02 (3H,
7

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
s), 2.036 (3H, s), 2.042 (3H, s), 2.06 (3H, s), 2.41–2.81 (4H, m),
3.86–3.92 (1H, m), 4.16 (1H, dd, J = 12.4 Hz, 2.4 Hz), 4.34 (1H, dd, J =
12.4 Hz, 4.1 Hz), 5.20–5.37 (3H, m), 5.56–5.63 (1H, m), 7.08–7.13 (2H,
m), 7.15–7.20 (1H, m), 7.21–7.26 (2H, m), 8.40–9.10 (1H, br); MS m/z:
533 (M + H)+.
(M + H)+.
5.1.25. 5-Methyl-4-(3-methylbenzyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (10b)
The title compound was prepared from 10a, as described for the
synthesis of 2b, in 13% yield.1H NMR (CDCl3) δ: 1.86 (3H, s), 2.01 (3H,
s), 2.03 (3H, s), 2.07 (3H, s), 2.12 (3H, s), 2.30 (3H, s), 3.56 (1H, d, J =
15.9 Hz), 3.63 (1H, d, J = 15.9 Hz), 3.81–3.89 (1H, m), 4.13 (1H, dd, J
= 12.5 Hz, 2.3 Hz), 4.32 (1H, dd, J = 12.5 Hz, 4.1 Hz), 5.16–5.30 (3H,
m), 5.54–5.61 (1H, m), 6.90–6.99 (3H, m), 7.08–7.16 (1H, m),
8.50–9.10 (1H, br); MS m/z: 533 (M + H)+.
5.1.22. 5-Methyl-4-(3-phenylpropyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (7b)
The title compound was prepared from 7a, as described for the
synthesis of 2b, in 17% yield. 1H NMR (CDCl3) δ: 1.70–1.82 (2H, m),
1.90 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.04 (3H, s). 2.13 (3H, s),
2.21–2.43 (2H, m), 2.57 (2H, t, J = 8.0 Hz), 3.84–3.91 (1H, m), 4.15
(1H, dd, J = 12.4 Hz, 2.2 Hz), 4.32 (1H, dd, J = 12.4 Hz, 4.0 Hz),
5.18–5.33 (3H, m), 5.55–5.64 (1H, m), 7.11–7.32 (5H, m), 8.50–9.40
(1H, br); MS m/z: 547 (M + H)+.
5.1.26. 5-Methyl-4-(4-methylbenzyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (11b)
The title compound was prepared from 11a, as described for the
synthesis of 2b, in 20% yield. 1H NMR (CDCl3) δ: 1.87 (3H, s), 2.02 (3H,
s), 2.03 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 2.28 (3H, s), 3.55 (1H, d, J =
15.8 Hz), 3.63 (1H, d, J = 15.8 Hz), 3.80–3.89 (1H, m), 4.08–4.17 (1H,
m), 4.13 (1H, dd, J = 12.5 Hz, 4.0 Hz), 5.15–5.31 (3H, m), 5.50–5.60
(1H, m), 6.89–7.15 (5H, m); MS m/z: 533 (M + H)+.
5.1.23. 4-(2-Phenylethyl)-1H-indazol-3-yl 2,3,4,6-tetra-O-pivaloyl-β-D-
glucopyranoside (8b)
Step 1: A mixture of 4-bromo-1,2-dihydro-3H-indazol-3-one (2 g,
9.39 mmol), 2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide (6.53
g, 11.3 mmol), K2CO3 (2.60 g, 18.8 mmol) and CH3CN (30 mL) was
stirred at room temperature overnight. The mixture was poured into
water and extracted with EtOAc. The organic layer was washed with
water and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (eluent: n-hexane/EtOAc =
10:1–2:1) to give 4-bromo-1H-indazol-3-yl 2,3,4,6-tetra-O-pivaloyl-ß-D-
glucopyranoside 8a (2.02 g, 30%) as a pale yellow solid. 1H NMR
(CDCl3) δ: 1.09 (9H, s), 1.14 (9H, s), 1.17 (9H, s), 1.20 (9H, s), 3.96–4.04
(1H, m), 4.16 (1H, dd, J = 12.4 Hz, 4.9 Hz), 4.26 (1H, dd, J = 12.4 Hz,
1.8 Hz), 5.28–5.35 (1H, m), 5.40–5.52 (2H, m), 5.88 (1H, d, J = 7.6 Hz),
7.14–7.20 (1H, m), 7.21–7.27 (2H, m), 8.59–9.15 (1H, br); MS m/z: 710,
712 (Mꢀ H)ꢀ .
5.1.27. 4-(4-Ethylbenzyl)-5-methyl-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (12b)
The title compound was prepared from 12a, as described for the
synthesis of 2b, in 12% yield. 1H NMR (CDCl3) δ: 1.19 (3H, t, J = 7.6
Hz), 1.86 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.06 (3H, s), 2.12 (3H, s),
2.58 (2H, q, J = 7.6 Hz), 3.55 (1H, d, J = 15.8 Hz), 3.64 (1H, d, J = 15.8
Hz), 3.81–3.89 (1H, m), 4.13 (1H, dd, J = 12.5 Hz, 2.3 Hz), 4.31 (1H, dd,
J = 12.5 Hz, 3.9 Hz), 5.16–5.34 (3H, m), 5.51–5.61 (1H, m), 6.98–7.18
(4H, m); MS m/z: 547 (M + H)+.
5.1.28. 5-Methyl-4-(4-propylbenzyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (13b)
Step 2: A mixture of 8a (2.00 g, 2.81 mmol), styrene (0.877 g, 8.42
mmol), Pd(OAc)2 (0.0637 g, 0.284 mmol), tri(o-tolyl)phosphine (0.171
g, 0.562 mmol), Et3N (1.42 g, 14.0 mmol) and CH3CN (60 mL) was
refluxed for 8 h. After volatile components were removed in vacuo, the
residue was purified by flash column chromatography on silica gel
(eluent: hexane/EtOAc = 10:1–2:1) . The obtained material (1.61 g) was
recrystallized from hexane–EtOAc to afford 4-[(E)-styryl]-1H-indazol-3-
yl 2,3,4,6-tetra-O- pivaloyl-β-D-glucopyranoside (0.841 g, 41%) as a
white solid. 1H NMR (CDCl3) δ: 0.98 (9H, s), 1.16 (9H, s), 1.18 (9H, s),
1.19 (9H, s), 3.97–4.05 (1H, m), 4.10–4.30 (2H, m), 5.25–5.35 (1H, m),
5.45–5.63 (2H, m), 5.96 (1H, d, J = 8.1 Hz), 7.10–7.40 (4H, m),
7.41–7.51 (3H, m), 7.67 (2H, d, J = 7.7 Hz), 7.78 (1H, d, J = 16.4 Hz),
8.89 (1H, br); MS m/z: 733 (Mꢀ H)ꢀ .
The title compound was prepared from 13a, as described for the
synthesis of 2b, in 25% yield. 1H NMR (CDCl3) δ: 0.91 (3H, t, J = 7.3
Hz), 1.50–1.65 (2H, m), 1.86 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.07 (3H,
s), 2.12 (3H, s), 2.47–2.55 (2H, m), 3.56 (1H, d, J = 15.8 Hz), 3.64 (1H,
d, J = 15.8 Hz), 3.81–3.88 (1H, m), 4.13 (1H, dd, J = 12.4 Hz, 2.2 Hz),
4.31 (1H, dd, J = 12.4 Hz, 4.0 Hz), 5.16–5.32 (3H, m), 5.52–5.62 (1H,
m), 7.00–7.11 (4H, m), 8.45–9.45 (1H, br); MS m/z: 561 (M + H)+.
5.1.29. 4-(4-Isopropylbenzyl)-5-methyl-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (14b)
The title compound was prepared from 14a, as described for the
synthesis of 2b, in 16% yield. 1H NMR (CDCl3) δ: 1.20 (6H, d, J = 6.9
Hz), 1.84 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.07 (3H, s), 2.13 (3H, s),
2.84 (1H, heptet, J = 6.9 Hz), 3.56 (1H, d, J = 15. 8 Hz), 3.63 (1H, d, J =
15.8 Hz), 3.80–3.89 (1H, m), 4.13 (1H, dd, J = 12.5 Hz, 2.3 Hz), 4.31
(1H, dd, J = 12.4 Hz, 4.0 Hz), 5.15–5.35 (3H, m), 5.50–5.60 (1H, m),
7.01–7.15 (4H, m), 8.70–9.30 (1H, br); MS m/z: 561 (M + H)+.
Step 3: A mixture of the 4-[(E)-styryl]-1H-indazol-3-yl 2,3,4,6-tetra-
O-pivaloyl-β-D-glucopyranoside (0.600 g, 0.817 mmol) and 10% Pd/C
(60 mg) in THF (12 mL) was stirred under an atmosphere of H2 at room
temperature for 3 h. The mixture was filtered through celite and the
filtrate was concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel to provide 8b (0.564 g, 94%) as a
white solid. 1H NMR (CDCl3) δ: 1.04 (9H, s), 1.13 (9H, s), 1.15 (9H, s),
1.17 (9H, s), 2.88–3.22 (3H, m), 3.26–3.36 (1H, m), 3.93–4.02 (1H, m),
4.13 (1H, dd, J = 12.4 Hz, 4.8 Hz), 4.21 (1H, dd, J = 12.4 Hz, 1.9 Hz),
5.25–5.33 (1H, m), 5.40–5.52 (2H, m), 6.05 (1H, d, J = 7.6 Hz),
6.68–6.74 (1H, m), 7.10–7.24 (5H, m), 7.27–7.32 (2H, m), 8.94 (1H, s);
MS m/z: 735 (Mꢀ H)ꢀ .
5.1.30. 4-(4-Methoxylbenzyl)-5-methyl-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (15b)
The title compound was prepared from 15a, as described for the
synthesis of 2b, in 9.9% yield. 1H NMR (CDCl3) δ: 1.89 (3H, s), 2.02 (3H,
s), 2.03 (3H, s), 2.07 (3H, s), 2.11 (3H, s), 3.53 (1H, d, J = 15.9 Hz), 3.60
(1H, d, J = 15.9 Hz), 3.76 (3H, s), 3.81–3.90 (1H, m), 4.13 (1H, dd, J =
12.6 Hz, 2.3 Hz), 4.31 (1H, dd, J = 12.6 Hz, 4.0 Hz), 5.16–5.31 (3H, m),
5.54–5.60 (1H, m), 6.74–6.82 (2H, m), 7.01–7.10 (2H, m), 8.20–9.20
(1H, br); MS m/z: 549 (M + H)+.
5.1.24. 5-Methyl-4-(2-methylbenzyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (9b)
The title compound was prepared from 9a, as described for the
synthesis of 2b, in 12% yield. 1H NMR (CDCl3) δ: 1.80 (3H, s), 2.00 (3H,
s), 2.025 (3H, s), 2.032 (3H, s), 2.07 (3H, s), 2.30 (3H, s), 3.54 (1H, d, J
= 16.5 Hz), 3.65 (1H, d, J = 16.5 Hz), 3.79–3.88 (1H, m), 4.15–4.17
(1H,m), 4.30 (1H, dd, J = 12.4 Hz, 3.9 Hz), 5.15–5.28 (3H, m),
5.52–5.57 (1H, m), 6.90–6.97 (1H, m), 7.02–7.15 (3H, m); MS m/z: 533
5.1.31. 4-(4-Ethoxybenzyl)-5-methyl-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (16b)
The title compound was prepared from 16a, as described for the
synthesis of 2b, in 23% yield. 1H NMR (CDCl3) δ: 1.38 (3H, t, J = 7.0
Hz), 1.89 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.06 (3H, s), 2.10 (3H, s),
8

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
3.53 (1H, d, J = 15.8 Hz), 3.59 (1H, d, J = 15.8 Hz), 3.81–3.90 (1H, m),
3.98 (2H, q, J = 7.0 Hz), 4.13 (1H, dd, J = 12.4 Hz, 2.3 Hz), 4.31 (1H, dd,
J = 12.4 Hz, 4.0 Hz), 5.15–5.30 (3H, m), 5.52–5.60 (1H, m), 6.72–6.80
(2H, m), 6.99–7.08 (2H, m), 8.50–9.80 (1H, br); MS m/z: 563 (M + H)+.
(1H, d, J = 8.1 Hz), 6.70–6.78 (2H, m), 6.98–7.08 (2H, m); MS m/z: 787
(M + H)+.
5.1.36. 4-[4-(Methylsulfanyl)benzyl]-5-trifluoromethyl-1H-pyrazol-3-yl
β-D-glucopyranoside (1)
5.1.32. 5-Methyl-4-(4-propoxybenzyl)-1H-pyrazol-3-yl 2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside (17b)
A mixture of 1b (900 mg, 1.46 mmol), NaOMe (28% MeOH solution,
0.56 mL, 2.91 mmol), and MeOH (6 mL) was stirred at room tempera-
ture for 20 h. The solvent was removed in vacuo, and the residue was
purified by flash column chromatography on silica gel (eluent: CH2Cl2/
MeOH = 19:1–9:1–4:1) to give 1 (398 mg, 61%) as an amorphous solid.
1H NMR (AcOH-d4) δ: 2.42 (3H, s), 3.54–3.62 (1H, m), 3.64–3.80 (3H,
m), 3.85 (2H, s), 3.89 (1H, dd, J = 12.5 Hz, 4.5 Hz), 4.00 (1H, dd, J =
12.5 Hz, 2.3 Hz), 5.15 (1H, d, J = 7.7 Hz), 7.16 (4H, s); 13C NMR (AcOH-
d4) δ: 15.82, 27.10, 61.71, 70.18, 74.14, 76.92, 77.34, 103.27, 106.43,
121.85 (q, J = 263 Hz), 127.67, 129.78, 136.17 (q, J = 37.9 Hz), 137.39,
137.62, 155.58 ; HRMS-ESI (m/z): [M + H]+ calcd for C18H22F3N2O6S,
451.1145; found 451.1145.
The title compound was prepared from 17a, as described for the
synthesis of 2b, in 18% yield. 1H NMR (CDCl3) δ: 1.01 (3H, t, J = 7.4
Hz), 1.70–1.83 (2H, m), 1.89 (3H,s), 2.02 (3H, s) 2.03 (3H,s), 2.07 (3H,
s), 2.11 (3H, s), 3.53 (1H, d, J = 15.7 Hz), 3.59 (1H, d, J = 15.7 Hz),
3.81–3.92 (3H, m), 4.14 (1H, dd, J = 12.4 Hz 2.3 Hz), 4.31 (1H, dd, 12.4
Hz, 4.0 Hz), 5.15–5.32 (3H, m), 5.52–5.60 (1H m), 6.73–6.80 (2H, m),
7.00–7.07 (2H, m), 8.50–9.50 (1H, br); MS m/z: 577 (M + H)+.
5.1.33. 4-(4-Isopropoxybenzyl)-2,5-dimethyl-1H-pyrazol-3-yl 2,3,4,6-
tetra-O-pivaloyl-β-D-glucopyranoside (19b)
4-(4-Isopropoxybenzyl)-1,5-dimethyl-1H-pyrazol-3-yl 2,3,4,6-tetra-
O-pivaloyl-β-D-glucopyranoside (20b)
5.1.37. 5-Methyl-4-[4-(methylsulfanyl)benzyl]-1H-pyrazol-3-yl β-D-
glucopyranoside (2)
A solution of 4-[(4-isopropoxyphenyl)methyl]-5-methyl-1H-pyrazol-
3-yl 2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranoside 18b18 (500 mg, 0.671
mmol) and iodomethane (381 mg, 2.69 mmol) in 1,3-dimethyl-2-imida-
zolidinone (DMI) (2 mL) was added dropwise to a suspension of NaH
(81 mg, 2.01 mmol, 60% oil dispersion) in DMI (3 mL) while main-
taining the internal temperature between 10 and 20 ◦C. The reaction
mixture was stirred at this temperature for 2 h. The reaction mixture was
added dropwise to a mixture of H2O (10 mL), glacial acetic acid (80.6
mg, 1.34 mmol) and the resulting mixture was extracted with EtOAc.
The extract was washed with water and brine, and dried over anhydrous
MgSO4. The solvent was removed in vacuo, and the residue was purified
by flash column chromatography on silica gel (gradient: 10–30% EtOAc
in hexane) to give 19b (35 mg, 6.9%) as a white solid and 20b (262 mg,
51%) as a white solid. 19b: 1H NMR (CDCl3) δ: 1.11 (9H, s), 1.12 (9H, s),
1.147 (9H, s), 1.152 (9H, s), 1.32 (3H, d, J = 6.1 Hz), 1.33 (3H, d, J =
6.1 Hz), 2.03 (3H, s), 3.22–3.34 (1H, m), 3.60 (1H, d, J = 16.7 Hz), 3.62
(3H, s), 3.68 (1H, d, J = 16.7 Hz), 3.97 (1H, dd, J = 12.5 Hz, 2.2 Hz),
4.04 (1H, dd, J = 12.5 Hz, 5.2 Hz), 4.52 (1H, heptet, J = 6.1 Hz), 4.71
(1H, d, J = 7.9 Hz), 5.08–5.19 (2H, m), 5.20–5.29 (1H, m), 6.77–6.85
(2H, m), 6.92–7.01 (2H, m); MS m/z: 759 (M + H)+; 20b: 1H NMR
(CDCl3) δ: 1.05 (9H, s), 1.12 (9H, s), 1.15 (9H, s), 1.18 (9H, s), 1.292
(3H, d, J = 6.1 Hz), 1.295 (3H, d, J = 6.1 Hz), 2.02 (3H, s), 3.51 (2H, s),
3.56 (3H, s), 3.82–3.92 (1H, m), 4.10 (1H, dd, J = 12.4 Hz, 5.1 Hz), 4.20
(1H, dd, J = 12.4 Hz, 1.7 Hz), 4.46 (1H, heptet, J = 6.1 Hz), 5.16–5.44
(3H, m), 5.65 (1H, d, J = 8.1 Hz), 6.70–6.79 (2H, m), 6.99–7.08 (2H, m);
MS m/z: 759 (M + H)+.
NaOMe (28% MeOH solution, 0.020 mL, 0.104 mmol) was added to a
solution of 2b (59 mg, 0.104 mmol) in MeOH (2 mL), and the mixture
was stirred at room temperature for 20 h. Acetic acid (6.3 mg, 0.105
mmol) was added to this reaction mixture, and the solvent was removed
in vacuo. The residue was loaded on amino packed inject column and
purified by flash column chromatography on silica gel (gradient: 0–20%
MeOH in CH2Cl2) to give 2 (21 mg, 51%) as an amorphous solid.1H NMR
(CD3OD) δ: 2.06 (3H, s), 2.42 (3H, s), 3.20–3.45 (4H, m), 3.63–3.70 (2H,
m), 3.72 (1H, d, J = 16.1 Hz), 3.80–3.88 (1H, m), 5.00–5.10 (1H, m),
7.10–7.20 (4H, m); 13C NMR (CD3OD) δ: 10.08, 16.19, 27.65, 62.68,
71.23, 74.93, 77.99, 78.39, 102.59, 104.06, 128.05, 129.96, 136.86,
139.67, 139.85, 161.61; HRMS-ESI (m/z): [M
18H25N2O6S, 397.1428; found 397.1430.
+
H]+ calcd for
C
5.1.38. 5-Ethyl-4-[4-(methylsulfanyl)benzyl]-1H-pyrazol-3-yl β-D-
glucopyranoside (3)
The title compound was prepared from 3b, as described for the
synthesis of 2, in 54% yield. 1H NMR (CD3OD) δ: 1.06 (3H, t, J = 7.6 Hz),
2.42 (3H, s), 2.47 (2H, q, J = 7.6 Hz), 3.32–3.44 (4H, m), 3.64–3.77 (2H,
m), 3.74 (1H, d, J = 16.2 Hz), 3.81–3.88 (1H, m), 5.04–5.10 (1H, m),
7.12–7.17 (4H, m); 13C NMR (CD3OD) δ: 13.65, 16.16, 19.20, 27.61,
62.70, 71.24, 74.95, 78.01, 78.40, 102.58, 103.25, 128.00, 129.96,
136.89, 139.88, 145.65, 161.52; HRMS-ESI (m/z): [M + H]+ calcd for
C19H27N2O6S, 411.1584; found 411.1586.
5.1.39. 5-Isopropyl-4-[4-(methylsulfanyl)benzyl]-1H-pyrazol-3-yl β-D-
glucopyranoside (4)
5.1.34. Ethyl-4-(4-isopropoxybenzyl)-5-methyl-1H-pyrazol-3-yl 2,3,4,6-
tetra-O-pivaloyl-β-D-glucopyranoside (21b)
The title compound was prepared from 4b, as described for the
synthesis of 2, in 43% yield. 1H NMR (CD3OD) δ: 1.125 (3H, d, J = 7.1
Hz), 1.132 (3H, d, J = 7.1 Hz), 2.42 (3H, s), 2.89 (1H, heptet, J = 7.1
Hz), 3.32–3.42 (4H, m), 3.61–3.73 (2H, m), 3.76 (1H, d, J = 15.8 Hz),
3.84 (1H, dd, J = 12.2 Hz, 1.7 Hz), 5.05–5.15 (1H, m), 7.15–7.20 (4H,
m); 13C NMR (CD3OD) δ: 16.16, 21.98, 21.99, 26.60, 27.60, 62.73,
71.26, 74.96, 78.02, 78.40, 102.46, 102.53, 127.97, 129.94, 136.87,
The title compound was prepared using iodoethane instead of
iodomethane, as described for the synthesis of 20b, in 71% yield. 1H
NMR (CDCl3) δ: 1.05 (9H, s), 1.12 (9H, s), 1.15 (9H, s), 1.18 (9H, s),
1.25–1.35 (9H, m), 2.03 (3H, s), 3.52 (2H, s), 3.82–3.92 (3H, m), 4.11
(1H, dd, J = 12.4 Hz, 4.8 Hz), 4.18 (1H, dd, J = 12.4 Hz, 1.9 Hz), 4.47
(1H, heptet, J = 6.0 Hz), 5.18–5.32 (2H, m), 5.34–5.44 (1H, m), 5.69
(1H, d, J = 8.1 Hz), 6.70–6.78 (2H, m), 7.00–7.08 (2H, m); MS m/z: 773
(M + H)+.
140.04, 149.44, 161.69; HRMS-ESI (m/z): [M
+
H]+ calcd for
C20H29N2O6S, 425.1741; found 425.1742.
5.1.35. 4-(4-Isopropoxybenzyl)-5-methyl-1-propyl-1H-pyrazol-3-yl
2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranoside (22b)
5.1.40. 4-Benzyl-5-methyl-1H-pyrazol-3-yl β-D-glucopyranoside (5)
The title compound was prepared from 5b, as described for the
synthesis of 2, in 54% yield. 1H NMR (CD3OD) δ: 2.05 (3H, s), 3.32–3.45
(4H, m), 3.63–3.74 (2H, m), 3.77 (1H, d, J = 16.0 Hz), 3.80–3.87 (1H,
m), 5.01–5.10 (1H m), 7.05–7.25 (5H, m); 13C NMR (CD3OD) δ: 10.05,
28.20, 62.68, 71.22, 74.94, 77.99, 78.38, 102.55, 104.17, 126.77,
129.27, 129.37, 139.85, 142.52, 161.68; HRMS-ESI (m/z): [M + H]+
calcd for C17H23N2O6, 351.1551; found 351.1551.
The title compound was prepared using iodopropane instead of
iodomethane, as described for the synthesis of 20b, in 78% yield. 1H
NMR (CDCl3) δ: 0.86 (3H, t, J = 7.5 Hz), 1.04 (9H, s), 1.12 (9H, s), 1.15
(9H, s), 1.18 (9H, s), 1.293 (3H, d, J = 6.1 Hz), 1.296 (3H, d, J = 6.1 Hz),
1.64–1.82 (2H, m), 2.03 (3H, s), 3.52 (2H, s), 3.77 (2H, t, J = 7.2 Hz),
3.81–3.90 (1H, m), 4.11 (1H, dd, J = 12.5 Hz, 4.7 Hz), 4.18 (1H, dd, J =
12.5 Hz, 1.7 Hz), 4.47 (1H, heptet, J = 6.1 Hz), 5.18–5.43 (3H, ms), 5.70
9

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Bioorganic & Medicinal Chemistry 34 (2021) 116033
5.1.41. 5-Methyl-4-(2-phenylethyl)-1H-pyrazol-3-yl β-D-glucopyranoside
(6)
5.00–5.07 (1H, m), 7.00–7.10 (4H, m); 13C NMR (CD3OD) δ: 10.10,
21.02, 27.76, 62.68, 71.22, 74.94, 77.97, 78.37, 102.59, 104.33,
129.26, 129.87, 136.22, 139.39, 139.96, 161.53; HRMS-ESI (m/z): [M
+ H]+ calcd for C18H25N2O6, 365.1707; found 365.1709.
The title compound was prepared from 6b, as described for the
synthesis of 2, in 62% yield. 1H NMR (CD3OD) δ: 1.80 (3H, s), 2.55–2.70
(2H, m), 2.71–2.86 (2H, m), 3.32–3.47 (4H, m), 3.69 (1H, dd, J = 12.1
Hz, 5.2 Hz), 3.87 (1H, dd, J = 12.1 Hz, 1.8 Hz), 4.99–5.07 (1H, m),
7.08–7.16 (3H, m), 7.17–7.24 (2H m); 13C NMR (CD3OD) δ: 9.53, 24.89,
37.25, 62.80, 71.34, 75.00, 78.05, 78.42, 102.55, 104.14, 126.74,
129.16, 129.92 ,139.70, 143.48, 161.73; HRMS-ESI (m/z): [M + H]+
calcd for C18H25N2O6, 365.1707; found 365.1710.
5.1.47. 4-(4-Ethylbenzyl)-5-methyl-1H-pyrazol-3-yl β-D-glucopyranoside
(12)
The title compound was prepared from 12b, as described for the
synthesis of 2, in 52% yield. 1H NMR (CD3OD) δ: 1.18 (3H, t, J = 7.6 Hz),
2.05 (3H, s), 2.57 (2H, q, J = 7.6 Hz), 3.32–3.43 (4H, m), 3.62–3.76 (3H,
m), 3.83 (1H, d, J = 12.1 Hz), 5.01–5.07 (1H, m), 7.03–7.13 (4H, m); 13
C
5.1.42. 5-Methyl-4-(3-phenylpropyl)-1H-pyrazol-3-yl β-D-glucopyranoside
(7)
NMR (CD3OD) δ: 10.10, 16.32, 27.77, 29.44, 62.67, 71.21, 74.94, 77.97,
78.37, 102.58, 104.33, 128.71, 129.31, 139.68, 139.96, 142.84, 161.49;
HRMS-ESI (m/z): [M + H]+ calcd for C19H27N2O6, 379.1864; found
379.1866.
The title compound was prepared from 7b, as described for the
synthesis of 2, in 65% yield. 1H NMR (CD3OD) δ: 1.75–1.88 (2H, m),
2.11 (3H, s), 2.32–2.46 (2H, m), 2.56–2.65 (2H, m), 3.32–3.47 (4H, m),
3.67 (1H, dd, J = 12.0 Hz, 5.3 Hz), 3.85 (1H, dd, J = 12.0 Hz, 1.9 Hz),
5.03–5.11 (1H, m), 7.09–7.27 (5H, m); 13C NMR (CD3OD) δ: 9.91, 22.11,
32.98, 36.54, 62.76, 71.31, 74.97, 78.07, 78.40, 102.29, 104.71,
126.63, 129.26, 129.47, 139.32, 143.84, 161.55; HRMS-ESI (m/z): [M
+ H]+ calcd for C19H27N2O6, 379.1864; found 379.1865.
5.1.48. 5-Methyl-4-(4-propylbenzyl)-1H-pyrazol-3-yl β-D-glucopyranoside
(13)
The title compound was prepared from 13b, as described for the
synthesis of 2, in 43% yield. 1H NMR (CD3OD) δ: 0.91 (3H, t, J = 7.5 Hz),
1.54–1.65 (2H, m), 2.05 (3H, s), 2.48–2.56 (2H, m), 3.32–3.44 (4H, m),
3.60–3.75 (3H, m), 3.83 (1H, d, J = 11.9 Hz), 5.02–5.06 (1H, m),
7.01–7.06 (2H, m), 7.07–7.12 (2H, m) ; 13C NMR (CD3OD) δ: 10.11,
14.09, 25.85, 27.79, 38.65, 62.67, 71.21, 74.94, 77.97, 78.37, 102.59,
104.33, 129.22, 129.36, 139.71, 139.99, 141.15, 161.48; HRMS-ESI (m/
z): [M + H]+ calcd for C20H29N2O6, 393.2020; found 393.2023.
5.1.43. 4-(2-Phenylethyl)-1H-indazol-3-yl β-D-glucopyranoside (8)
LiOH-H2O (525 mg, 12.5 mmol) and water (1 mL) were added to a
solution of 8b (461 mg, 0.626 mmol) in MeOH (10 mL), and the mixture
was stirred at room temperature overnight. The reaction mixture was
concentrated in vacuo. The residue was dissolved in water, and acetic
acid (939 mg, 16.6 mmol) was added to the solution. The mixture was
extracted by solid-phase extraction on a C18 Bond Elute cartridge
(eluent: MeOH) and the extracts were concentrated in vacuo. The res-
idue was purified by flash column chromatography on silica gel
(gradient: 10–20% MeOH in CH2Cl2) to give 8 (151 mg, 60%) as an
amorphous solid.1H NMR (CD3OD) δ: 2.90–3.08 (2H, m), 3.10–3.24
(1H, m), 3.35–3.65 (5H, m), 3.71 (1H, dd, J = 12.2 Hz, 5.5 Hz), 3.89
(1H, dd, J = 12.2 Hz, 2.2 Hz), 5.66 (1H, d, J = 7.9 Hz), 6.76 (1H, d, J =
6.9 Hz), 7.10–7.30 (7H, m); 13C NMR (CD3OD) δ: 36.44, 39.10, 62.60,
71.27, 75.14, 78.40, 78.55, 101.49, 108.86, 111.87, 120.99, 126.75,
129.02, 129.26, 129.63, 137.64, 143.65, 144.05, 156.80; HRMS-ESI (m/
z): [M + H]+ calcd for C21H25N2O6, 401.1707; found 401.1706.
5.1.49. 4-(4-Isopropylbenzyl)-5-methyl-1H-pyrazol-3-yl β-D-
glucopyranoside (14)
The title compound was prepared from 14b, as described for the
synthesis of 2, in 41% yield. 1H NMR (CD3OD) δ: 1.20 (6H, d, J = 7.0
Hz), 2.05 (3H, s), 2.83 (1H, heptet, J = 7.0 Hz), 3.32–3.43 (4H, m),
3.58–3.86 (4H, m), 5.00–5.08 (1H, m), 7.06–7.13 (4H, m) ; 13C NMR
(CD3OD) δ: 10.10, 24.53, 27.75, 34.99, 62.67, 71.21, 74.94, 77.98,
78.37, 102.56, 104.32, 127.23, 129.28, 139.84, 140.04, 147.47, 161.66;
HRMS-ESI (m/z): [M + H]+ calcd for C20H29N2O6, 393.2020; found
393.2021.
5.1.50. 4-(4-Methoxybenzyl)-5-methyl-1H-pyrazol-3-yl β-D-
glucopyranoside (15)
5.1.44. 5-Methyl-4-(2-methylbenzyl)-1H-pyrazol-3-yl β-D-glucopyranoside
(9)
The title compound was prepared from 15b, as described for the
synthesis of 2, in 74% yield. 1H NMR (CD3OD) δ: 2.05 (3H, s), 3.32–3.42
(4H, m), 3.60–3.72 (3H, m), 3.73 (3H, s), 3.84 (1H, d, J = 12.0 Hz),
5.00–5.09 (1H, m), 6.73–6.81 (2H, m), 7.07–7.14 (2H, m) ; 13C NMR
(CD3OD) δ: 10.08, 27.30, 55.61, 62.69, 71.23, 74.95, 77.99, 78.38,
102.58, 104.54, 114.66, 130.26, 134.51, 139.83, 159.30, 161.57;
HRMS-ESI (m/z): [M + H]+ calcd for C18H25N2O7, 381.1656; found
381.1657.
The title compound was prepared from 9b, as described for the
synthesis of 1, in 65% yield. 1H NMR (CD3OD) δ: 1.96 (3H, s), 2.31 (3H,
s), 3.32–3.38 (4H, m), 3.60–3.86 (4H, m), 4.97–5.05 (1H, m), 6.94–7.15
(4H, m); 13C NMR (CD3OD) δ: 10.15, 19.78, 26.00, 62.67, 71.20, 74.89,
77.94, 78.35, 102.61, 102.94, 126.77, 127.02, 129.44, 130.90, 137.38,
139.82, 140.16, 161.66; HRMS-ESI (m/z): [M
18H25N2O6, 365.1707; found 365.1710.
+
H]+ calcd for
C
5.1.51. 4-(4-Ethoxybenzyl)-5-methyl-1H-pyrazol-3-yl β-D-glucopyranoside
(16)
5.1.45. 5-Methyl-4-(3-methylbenzyl)-1H-pyrazol-3-yl β-D-glucopyranoside
(10)
The title compound was prepared from 16b, as described for the
synthesis of 2, in 63% yield. 1H NMR (CD3OD) δ: 1.34 (3H, t, J = 7.0 Hz),
2.05 (3H, s), 3.32–3.44 (4H, m), 3.60–3.72 (3H, m), 3.84 (1H, d, J =
12.1 Hz), 3.97 (2H, q, J = 7.0 Hz), 5.00–5.08 (1H, m), 6.72–6.84 (2H,
m), 7.04–7.15 (2H, m) ; 13C NMR (CD3OD) δ: 10.10, 15.21, 27.31, 62.69,
64.40, 71.23, 74.95, 77.98, 78.37, 102.58, 104.55, 115.30, 130.24,
134.45, 139.93, 158.55, 161.45; HRMS-ESI (m/z): [M + H]+ calcd for
The title compound was prepared from 10b, as described for the
synthesis of 1, in 53% yield. 1H NMR (CD3OD) δ: 2.05 (3H, s), 2.27 (3H,
s), 3.32–3.43 (4H, m), 3.61–3.75 (3H, m), 3.80–3.87 (1H, m), 5.00–5.08
(1H, m), 6.91–7.04 (3H, m), 7.06–7.13 (1H, m); 13C NMR (CD3OD) δ:
10.07, 21.47, 28.12, 62.70, 71.23, 74.95, 77.99, 78.40, 102.55, 104.20,
126.43, 127.45, 129.17, 130.02, 138.90, 139.85, 142.39, 161.69;
HRMS-ESI (m/z): [M + H]+ calcd for C18H25N2O6, 365.1707; found
365.1709.
C19H27N2O7, 395.1813; found 395.1814.
5.1.52. 5-Methyl-4-(4-propoxybenzyl)-1H-pyrazol-3-yl β-D-
glucopyranoside (17)
5.1.46. 5-Methyl-4-(4-methylbenzyl)-1H-pyrazol-3-yl β-D-glucopyranoside
(11)
The title compound was prepared from 17b, as described for the
synthesis of 2, in 48% yield. 1H NMR (CD3OD) δ: 1.02 (3H, t, J = 7.4 Hz),
1.70–1.80 (2H, m), 2.05 (3H, s), 3.32–3.43 (4H, m), 3.60–3.73 (3H, m),
3.80–3.86 (1H, m), 3.87 (2H, t, J = 6.5 Hz), 5.00–5.08 (1H, m),
The title compound was prepared from 11b, as described for the
synthesis of 2, in 61% yield. 1H NMR (CD3OD) δ: 2.04 (3H, s), 2.26 (3H,
s), 3.32–3.43 (4H, m), 3.61–3.76 (3H, m), 3.83 (1H, d, J = 12.1 Hz),
10

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
6.74–6.85 (2H, m), 7.06–7.12 (2H, m) ; 13C NMR (CD3OD) δ: 10.06,
10.85, 23.71, 27.31, 62.69, 70.53, 71.23, 74.95, 77.99, 78.37, 102.55,
104.55, 115.32, 130.24, 134.41, 139.77, 158.74, 161.62; HRMS-ESI (m/
z): [M + H]+ calcd for C20H29N2O7, 409.1969; found 409.1970.
5.2. Biology
5.2.1. SGLT1 and SGLT2 inhibition assay
Human SGLT expression plasmids were constructed and cell culture,
transfection, and [14C]-AMG uptake experiments were performed as
reported previously.13 In the experiments, 0.3 mM AMG concentration
in the uptake buffer was used. The concentration of test compounds
required to inhibit a 50% uptake of [14C]-AMG (IC50) was calculated
using a logit plot. Phlorizin was included in all experiments as a refer-
ence standard.
5.1.53. 4-(4-Isopropoxybenzyl)-5-methyl-1H-pyrazol-3-yl β-D-
glucopyranoside (18)
The title compound was prepared from 18b18, as described for the
synthesis of 2, in 59% yield. 1H NMR (CD3OD) δ: 1.26 (6H, d, J = 6.1
Hz), 2.05 (3H, s), 3.30–3.43 (4H, m), 3.60–3.73 (3H, m), 3.80–3.87 (1H,
m), 4.51 (1H, heptet, J = 6.1 Hz), 5.00–5.08 (1H, m), 6.73–6.80 (2H, m),
7.04–7.14 (2H, m) ; 13C NMR (CD3OD) δ: 10.10, 22.38, 27.32, 62.69,
71.04, 71.23, 74.95, 77.99, 78.38, 102.59, 104.53, 116.96, 130.28,
134.59, 139.93, 157.35, 161.45; HRMS-ESI (m/z): [M + H]+ calcd for
5.2.2. Human liver microsome stability
Each test compound (30 μM) was incubated with human liver mi-
crosomes (Thermo Fisher Scientific) at a protein concentration of 1 mg/
mL in phosphate buffer (pH 7.4, 100 mmol/L) containing MgCl2⋅6H2O
(10 mM) and NADPH (1 mg/mL). An aliquot (50 µL) of each reaction
mixture was collected at 0, 15, 30, and 60 min. Reactions were quenched
with 2.5 volumes of ice-cold acetonitrile and centrifuged at 2280 × g for
20 min. Supernatants were analyzed using LC-MS/MS (QTRAP 6500, AB
SCIEX, Framingham, MA). The half-life (t1/2) and intrinsic clearance (CL
int) calculated from the remaining test compound at 15, 30, and 60 min
were estimated as metabolic stability.
C
20H29N2O7, 409.1969; found 409.1969.
5.1.54. 4-(4-Isopropoxybenzyl)-2,5-dimethyl-1H-pyrazol-3-yl β-D-
glucopyranoside (19)
The title compound was prepared from 19b, as described for the
synthesis of 1, in 67% yield. 1H NMR (CD3OD) δ: 1.27 (6H, d, J = 6.1
Hz), 1.97 (3H, s), 2.95–3.05 (1H, m), 3.23–3.32 (1H, m), 3.33–3.44 (2H,
m), 3.58–3.66 (2H, m), 3.68 (3H, s), 3.74 (1H, d, J = 16.4 Hz), 3.80 (1H,
d, J = 16.4 Hz), 4.52 (1H, heptet, J = 6.1 Hz), 4.58 (1H, d, J = 7.8 Hz),
6.74–6.82 (2H, m), 6.98–7.07 (2H, m) ; 13C NMR (CD3OD) δ: 12.69,
22.38, 27.69, 34.48, 62.08, 70.80, 71.05, 74.87, 77.80, 78.31, 104.71,
106.58, 117.03, 130.06, 134.16, 147.53, 151.21, 157.47; HRMS-ESI (m/
z): [M + H]+ calcd for C21H31N2O7, 423.2126; found 423.2123.
5.2.3. Rat intestinal S9 stability
Each test compound (10 μM) was incubated with rat intestinal S9
(Sekisui XenoTech, Kansas City, KS) at a protein concentration of 0.5
mg/mL in Tris HCl buffer (pH 7.4, 50 mM) containing MgCl2⋅6H2O (10
mM) and NADPH (1 mg/mL). Reaction mixtures (50 μL) were incubated
5.1.55. 4-(4-Isopropoxybenzyl)-1,5-dimethyl-1H-pyrazol-3-yl β-D-
glucopyranoside (20)
at 37 ◦C for 0, 15, 30, and 60 min and subsequently quenched with cold
acetonitrile before centrifugation at 2280 × g for 20 min. Supernatants
were analyzed using LC-MS/MS (API-3000, AB SCIEX). The half-life (t1/
2) and intrinsic clearance (CL int) calculated from the remaining test
compound at 15, 30, and 60 min were estimated as metabolic stability.
The title compound was prepared from 20b, as described for the
synthesis of 2, in 67% yield. 1H NMR (CD3OD) δ: 1.26 (6H, d, J = 6.1
Hz), 2.07 (3H, s), 3.30–3.42 (4H, m), 3.58–3.72 (3H, m), 3.62 (3H, s),
3.79–3.86 (1H, m), 4.51 (1H, heptet, J = 6.1 Hz), 5.01–5.06 (1H, m),
6.73–6.84 (2H, m), 7.04–7.12 (2H, m) ; 13C NMR (CD3OD) δ: 9.80,
22.38, 27.61, 35.70, 62.64, 71.02, 71.19, 74.95, 77.98, 78.35, 102.69,
105.46, 116.95, 130.22, 134.58, 139.93, 157.36, 159.97; HRMS-ESI (m/
z): [M + H]+ calcd for C21H31N2O7, 423.2126; found 423.2126.
5.2.4. Rat PK study
Test compounds (1, 18, 23, or 24) were dissolved in dimethylace-
tamide (DMA) and diluted five-fold with saline to prepare intravenous
dosing formulations (1 mg/kg/mL). For the oral formulations (3 mg/kg/
5 mL), test compounds were suspended in 0.5 w/v% methyl cellulose
400 solution (MC). Rat blood was collected from the jugular vein at 5,
10, 15, 30, 60, 120, 240, and 360 min after single oral or intravenous
administration of the test compound to rats. Plasma concentrations were
measured using LC-MS/MS (API-3000, AB SCIEX).
5.1.56. 1-Ethyl-4-(4-isopropoxybenzyl)-5-methyl-1H-pyrazol-3-yl β-D-
glucopyranoside (21)
The title compound was prepared from 21b, as described for the
synthesis of 2, in 67% yield. 1H NMR (CD3OD) δ: 1.26 (6H, d, J = 6.1
Hz), 1.29 (3H, t, J = 7.2 Hz), 2.09 (3H, s), 3.30–3.45 (4H, m), 3.58–3.72
(3H, m), 3.78–3.85 (1H, m), 3.96 (2H, q, J = 7.2 Hz), 4.51 (1H, heptet, J
= 6.1 Hz), 5.01–5.07 (1H, m), 6.70–6.80 (2H, m), 7.02–7.10 (2H, m) ;
13C NMR (CD3OD) δ: 9.67, 15.70, 22.38, 27.60, 44.35, 62.65, 71.02,
71.22, 75.00, 78.00, 78.31, 102.72, 105.52, 116.96, 130.21, 134.62,
5.2.5. Permeability of test compounds across Caco-2 cell monolayers
Caco-2 cells were plated at a density of 1.0 × 105 cells/well on
polycarbonate membranes (BD Falcon 24 multiwell insert systems, 0.3
cm2 growth area, 1
μm pores, Corning, Corning, NY). Cell confluence
138.89, 157.36, 160.13; HRMS-ESI (m/z): [M
+
H]+ calcd for
was monitored by transepithelial electrical resistance (TEER) measure-
ments. Transport experiments were performed on monolayers at 21 days
post-seeding. Bi-directional transport of the compounds was investi-
gated using Caco-2 cell monolayers. HBSS with pH adjusted to 7.4 using
10 mM HEPES was used as the transport buffer (TB).
C22H33N2O7, 437.2282; found 437.2283.
5.1.57. 4-(4-Isopropoxybenzyl)-5-methyl-1-propyl-1H-pyrazol-3-yl β-D-
glucopyranoside (22)
The title compound was prepared from 22b, as described for the
synthesis of 2, in 60% yield. 1H NMR (CD3OD) δ: 0.87 (3H, t, J = 7.4 Hz),
1.26 (6H, d, J = 6.1 Hz), 1.68–1.80 (2H, m), 2.07 (3H, s), 3.30–3.42 (4H,
m), 3.59–3.73 (3H, m), 3.81 (1H, dd, J = 12.1 Hz, 2.0 Hz), 3.88 (2H, t, J
= 7.2 Hz), 4.51 (1H, heptet, J = 6.1 Hz), 5.00–5.07 (1H, m), 6.72–6.80
(2H, m), 7.03–7.10 (2H, m) ; 13C NMR (CD3OD) δ: 9.88, 11.28, 22.38,
24.59, 27.58, 50.91, 62.63, 71.02, 71.21, 75.00, 78.00, 78.28, 102.75,
105.33, 116.96, 130.18, 134.60, 139.45, 157.36, 160.14; HRMS-ESI (m/
z): [M + H]+ calcd for C23H35N2O7, 451.2439; found 451.2439.
23 (Remogliflozin) and 24 (Remogliflozin etabonate) were prepared
as reported previously.18
Following pre-incubation (30 min) of both sides of the monolayers
with compound-free TB, the TB was removed from the apical (A) side
while TB containing each compound was added to the removal side.
Following incubation in a CO2 incubator (37 ◦C, 5% CO2) for 60 min at
37 ◦C, a portion (50
μL) of buffer was collected from the opposite side.
The concentrations of test compounds were measured using LC-MS/MS
(API-3000, AB SCIEX).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
11

K. Shimizu et al.
Bioorganic & Medicinal Chemistry 34 (2021) 116033
sodium glucose co-transporter 1 (SGLT1) inhibitors with therapeutic activity on
postprandial hyperglycemia. Bioorg Med Chem. 2012;20(22):6598–6612.
17 The crystal structures of 8b has been deposited at the Cambridge Crystallographic
Data Centre and the CCDC deposition number are 2034277.
Acknowledgements
We thank Dr. Noritaka Furuya for X-ray crystal analysis of compound
8b, Dr. Hideyuki Muranaka for HRMS measurements of the compounds,
Dr. Minoru Fujioka and Kazuma Hirata for metabolite identification of
WAY-123783, and Dr. Takuro Endo and Dr. Hideki Takeuchi for
providing valuable suggestions during the preparation of this manu-
script. We thank Dr. William O Wilkison from Avolynt, Inc for valuable
comments on this manuscript.
18 Kobayashi M, Isawa H, Sonehara J, Kubota M, Ozawa T. O-Glycosylation of 4-
(substituted benzyl)-1,2-dihydro-3H-pyrazol-3-one derivatives with 2,3,4,6-tetra-O-
acyl-α-D-glucopyranosyl bromide via N1-acetylation of the pyrazole ring. Chem
Pharm Bull (Tokyo). 2016;64:1009–1018. https://doi.org/10.1248/cpb.c15-00982.
19 Kees KL, Fitzgerald Jr JJ, Steiner KE, et al. New potent antihyperglycemic agents in
db/db mice: synthesis and structure-activity relationship studies of (4-substituted
benzyl) (trifluoromethyl)pyrazoles and -pyrazolones. J Med Chem. 1996;39:
3920–3928. https://doi.org/10.1021/jm960444z.
20 Ohsumi K, Matsueda H, Hatanaka T, Hirama R, Umemura T, Oonuki A, et al.
Pyrazole-O-glucosides as novel Na+-glucose cotransporter (SGLT) inhibitors. Bioorg
Med Chem Lett. 2003;13(14):2269–2272.
Appendix A. Supplementary material
21 Nath RL, Rydon HN. The influence of structure on the hydrolysis of substituted
phenyl β-D-glucosides by emulsin. Biochem J. 1954;57:1–10. https://doi.org/
10.1042/bj0570001.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116033.
22 Bisignano P, Ghezzi C, Jo H, Polizzi NF, Althoff T, Kalyanaraman C, et al. Inhibitor
binding mode and allosteric regulation of Na+-glucose symporters. Nat Commun.
2018;9(1). https://doi.org/10.1038/s41467-018-07700-1.
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