Design, synthesis, and structure-activity relationships of a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl β-d-glycopyranosides substituted with novel hydrophilic groups as highly potent inhibitors of sodium glucose co-transporter 1 (SGLT1)

Article abstract of DOI:10.1016/j.bmc.2012.11.041

Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of therapeutic options for postprandial hyperglycemia. Previously, we reported potent and selective SGLT1 inhibitors 1 and 2 showing efficacy in oral carbohydrate tolerance tests in diabetic rat models. In a pharmacokinetic (PK) study of 2, excessive systemic exposure to metabolites of 2 was observed, presumably due to the high permeability of its aglycone (2a). To further improve SGLT1 inhibitory activity and reduce aglycone permeability, a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl β-d-glycopyranoside derivatives bearing novel hydrophilic substitution groups on the phenyl ring were synthesized and their inhibitory activity toward SGLTs was evaluated. Optimized compound 14c showed an improved profile satisfying both higher activity and lower permeability of its aglycone (22f) compared with initial leads 1 and 2. Moreover, the superior efficacy of 14c in various carbohydrate tolerance tests in diabetic rat models was confirmed compared with acarbose, an α-glucosidase inhibitor (α-GI) widely used in the clinic.

Full text of DOI:10.1016/j.bmc.2012.11.041

Accepted Manuscript
Design, synthesis, and structure–activity relationships of a series of 4-benzyl-5-
isopropyl-1H-pyrazol-3-yl β-D-glycopyranosides substituted with novel hydro‐
philic groups as highly potent inhibitors of sodium glucose co-transporter 1
(
SGLT1)
Nobuhiko Fushimi, Hirotaka Teranishi, Kazuo Shimizu, Shigeru Yonekubo,
Kohsuke Ohno, Takashi Miyagi, Fumiaki Itoh, Toshihide Shibazaki, Masaki
Tomae, Yukiko Ishikawa-Takemura, Takeshi Nakabayashi, Noboru Kamada,
Yuji Yamauchi, Susumu Kobayashi, Masayuki Isaji
PII:
S0968-0896(12)00934-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2012.11.041
BMC 10448
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 October 2012
15 November 2012
18 November 2012
Please cite this article as: Fushimi, N., Teranishi, H., Shimizu, K., Yonekubo, S., Ohno, K., Miyagi, T., Itoh, F.,
Shibazaki, T., Tomae, M., Ishikawa-Takemura, Y., Nakabayashi, T., Kamada, N., Yamauchi, Y., Kobayashi, S.,
Isaji, M., Design, synthesis, and structure–activity relationships of a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-
yl β-D-glycopyranosides substituted with novel hydrophilic groups as highly potent inhibitors of sodium glucose
co-transporter 1 (SGLT1), Bioorganic & Medicinal Chemistry (2012), doi: http://dx.doi.org/10.1016/j.bmc.
2
012.11.041
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Design, synthesis, and structure–activity relationships of a series of
-benzyl-5-isopropyl-1H-pyrazol-3-yl -D-glycopyranosides substituted
4
with novel hydrophilic groups as highly potent inhibitors of sodium
glucose co-transporter 1 (SGLT1)
a,
a
a
a
Nobuhiko Fushimi *, Hirotaka Teranishi , Kazuo Shimizu , Shigeru Yonekubo ,
a
a
a
a
Kohsuke Ohno , Takashi Miyagi , Fumiaki Itoh , Toshihide Shibazaki , Masaki Tomae
a
a
a
a
,
Yukiko Ishikawa-Takemura , Takeshi Nakabayashi , Noboru Kamada , Yuji
a
b
a
Yamauchi , Susumu Kobayashi , and Masayuki Isaji
a
Central Research Laboratory, Kissei Pharmaceutical Company, 4365-1 Kashiwabara,
Hotaka, Azumino, Nagano Prefecture 399-8304, Japan
b
Faculty of Pharmaceutical Sciences, Tokyo University of Science (RIKADAI), 2641
Yamazaki, Noda-shi, Chiba Prefecture 278-8510, Japan
*
Corresponding author.
Tel: +81-263-82-8820; Fax: +81-263-82-8827.
E-mail: nobuhiko_fushimi@pharm.kissei.co.jp
-
Page 1 of 87 -

Keywords: Diabetes, Postprandial hyperglycemia, SGLT1, SGLT1 inhibitor
Abstract
Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of
glucose in the gut and is considered a promising target in the development of
therapeutic options for postprandial hyperglycemia. Previously, we reported potent and
selective SGLT1 inhibitors 1 and 2 showing efficacy in oral carbohydrate tolerance tests
in diabetic rat models. In a pharmacokinetic (PK) study of 2, excessive systemic
exposure to metabolites of 2 was observed, presumably due to the high permeability of
its aglycone (2a). To further improve SGLT1 inhibitory activity and reduce aglycone
permeability, a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl -D-glycopyranoside
derivatives bearing novel hydrophilic substitution groups on the phenyl ring were
synthesized and their inhibitory activity toward SGLTs was evaluated. Optimized
compound 14c showed an improved profile satisfying both higher activity and lower
permeability of its aglycone (22f) compared with initial leads 1 and 2. Moreover, the
superior efficacy of 14c in various carbohydrate tolerance tests in diabetic rat models
was confirmed compared with acarbose, an -glucosidase inhibitor (-GI) widely used
-
Page 2 of 87 -

in the clinic.
1
. Introduction
In individuals with prediabetes, the metabolic abnormalities are frequently found
as elevations in postprandial plasma glucose. Epidemiological studies have shown that
postprandial glycemia has a strong association with cardiovascular (CV) risks, and
addressing postprandial hyperglycemia is now recommended by International Diabetes
1
Federation (IDF). In a clinical trial to prevent progression to non-insulin-dependent
diabetes mellitus (NIDDM) in patients with impaired glucose tolerance (IGT)
(STOP-NIDDM), an α-glucosidase inhibitor (α-GI), acarbose, that delays the digestion
of carbohydrates, not only reduced progression to diabetes mellitus (DM) but also
2
protected patients against CV disease.
Digested monosaccharides derived from carbohydrates in the diet are absorbed
3
via several types of transporters in the small intestine. Sodium glucose co-transporter 1
(SGLT1), expressed in the brush border membrane of the enterocytes, plays a central
4
role in the absorption of glucose and galactose. Therefore, inhibition of SGLT1 can be
an efficacious approach to improve postprandial hyperglycemia in DM and IGT
5
patients.
-
Page 3 of 87 -

Currently, high-fructose corn syrup (typically comprising 45% glucose and 55%
fructose) and other forms of sugar that are rich in glucose are routinely added to
6
processed food and many types of beverages. Thus, patients are frequently exposed to
carbohydrates in the form of glucose. Therefore, the ability to suppress postprandial
hyperglycemia after the intake of glucose may be an advantage of SGLT1 inhibitors
over -GI as an antidiabetic medication. We have been exploring SGLT1 inhibitors as
novel agents for the treatment of postprandial hyperglycemia.
SGLT2 (the isoform of SGLT1) is distributed predominantly in the proximal
tubules of the kidney, and plays a dominant role in the reabsorption of
7
glomerular-filtered glucose. Because compounds that inhibit both SGLTs could induce
urinary glucose excretion and normalize blood glucose levels by inhibiting renal SGLT2
if they were absorbed, we targeted synthesis of an SGLT1-selective molecule to
investigate efficacy based on the inhibition of SGLT1 alone.
8
In a previous paper, we reported a structure–activity relationship (SAR) study
of 4-benzyl-1H-pyrazol-3-yl -D-glucopyranoside derivatives having inhibitory activity
on SGLT1. Among the analogs explored, compounds 1 and 2 (Figure 1) were potent
and selective SGLT1 inhibitors in vitro and demonstrated robust efficacy in oral mixed
9
carbohydrate tolerance tests in streptozotocin-nicotinamide-induced diabetic rats
-
Page 4 of 87 -

1
0
(
NA-STZ rats). In a pharmacokinetic (PK) study of compound 2 (10 mg/kg, p.o.) in
rats, very low levels of 2 were detected in the plasma and were eliminated immediately,
indicating that systemic exposure to 2 was low (Cmax: 8.2 ng/mL, AUC(0–6 h): 67
min·ng/mL). However, in the same plasma, relatively high levels of the aglycone of 2
(2a) were also observed (Cmax: 201 ng/mL, AUC(0–6 h): 8600 min·ng/mL). Furthermore,
levels of 2a were further increased by enzymatic hydrolysis with -glucuronidase (an
enzyme with hydrolytic activity for conjugates including glycosides, glucuronides, and
sulfates) before quantification analysis (Cmax: 1087 ng/mL, AUC(0–6 h): 81,227
min·ng/mL), suggesting that systemic exposure to metabolites derived from 2 was high.
The Caco-2 cell permeability of 2 and 2a indicated that the higher permeability of the
aglycone, formed by hydrolysis of 2 in the gut, meant that 2a would be easily absorbed
1
1
(
Table 5). Because these metabolites would have no impact on efficacy, we considered
that the permeability of the aglycone should be reduced to avoid unnecessary systemic
exposure and ensure the safety of the administered compounds. Accordingly, the
additional objective of reducing the permeability of the aglycones as well as further
enhancement of the inhibitory activity of SGLT1 was included in this study.
It is known that some physicochemical parameters of compounds show
1
2
correlation with membrane permeability, and hydrophilic compounds having a larger
-
Page 5 of 87 -

polar surface area (PSA) are preferable to reduce absorption. In our previous study, we
8
focused on investigations into relatively simple substitution groups. Therefore, in this
study, we instead explored analogs bearing higher functionalized hydrophilic groups in
order to concurrently achieve improvement of both pharmacological and
physicochemical parameters.
In the present paper, we report the design, synthesis, and SAR of a series of
4
-benzyl-5-isopropyl-1H-pyrazol-3-yl -D-glycopyranoside derivatives bearing novel
hydrophilic functional groups on the phenyl ring. These compounds were investigated
in order to identify further improved SGLT1 inhibitors. We also describe the Caco-2
cell permeability of the aglycones of several derivatives as well as in vivo
pharmacological profiles of selected compounds.
2
. Chemistry
-Benzyl-5-isopropyl-1H-pyrazol-3-yl -D-glycopyranoside derivatives bearing
4
various functional groups on the phenyl ring were prepared as illustrated in Scheme 1–4.
As shown in Scheme 1, 4-benzyl-1,2-dihydro-5-isopropyl-3H-pyrazol-3-one derivatives
(5a–g) were synthesized by the cyclization of substituted 2-benzyl ketoesters (4a–g),
prepared by either Knoevenagel condensation of 4-nitrobenzaldehyde (3a) and methyl
-
Page 6 of 87 -

4
-methyl-3-oxopentanoate followed by Pd–C-catalyzed hydrogenation of both the
double bond and nitro group of the benzylidene intermediate or by alkylation of the
same ketoester with substituted benzyl mesylates prepared by methanesulfonylation of
the hydroxyl groups of the corresponding benzyl alcohols (3b–g). The pyrazolone
derivatives obtained (5a–g) were glycosidated to react with
tetra-O-acetyl--D-glycopyranosyl bromides in two-phase solvents consisting of
CH Cl and aqueous NaOH solution in the presence of the phase transfer catalyst
2
2
13
BnN(n-Bu) Cl. The yields of these glycosylation reactions were low to moderate
3
(11-38%, see experiment) due to decomposition of the sugar donors and remaining of
the unreacted aglycones. Subsequent removal of the benzyl (Bn) protective groups of
the ester derivatives, except for aniline (6a), was performed by Pd–C-catalyzed
hydrogenation, in which the double bonds of the alkene-containing derivatives were
simultaneously reduced to afford carboxylates (6b–h). Within these intermediates, the
acetyl (Ac) groups on the sugar of the aniline (6a) were removed by hydrolysis using
NaOMe in MeOH to give deprotected glucopyranoside (7).
Substituted aniline derivatives (8a–f) were synthesized via reaction of aniline
(6a) with acid anhydrides or acid chlorides in the presence of pyridine (method A) or by
reaction of carboxylic acids with condensing agents,
-
Page 7 of 87 -

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDCI) and 1-hydroxybenzotriazole
HOBt) (method B), following hydrolysis of the Ac groups as described above. In the
(
synthesis of amines (9a, b), benzyloxycarbonyl (Cbz) groups of 8e and 8f were
removed by Pd–C-catalyzed hydrogenation (Scheme 2).
Various amide derivatives (10a–e, 11a–f) were prepared via similar reaction
sequences of condensation and hydrolysis as described for the synthesis of 8e and 8f,
but using carboxylic acids (6b–e, h) as precursors (Scheme 3).
For the synthesis of 2-acylamino-2-methylpropanamide derivatives (14a–i),
2
-acylamino-2-methylpropanoic acids (12a–c) were prepared initially via condensation
of butanoic acids (6e–g) and benzyl 2-amino-2-methylpropanoate followed by removal
of Bn groups by Pd–C-catalyzed hydrogenation. Subsequent conversion of 12a–c into
amides (14a–i) was performed using similar reaction sequences as described above. In
1
4a–c, Cbz groups were removed by Pd–C-catalyzed hydrogenation before hydrolysis
of the Ac groups (Scheme 4).
Starting materials (3b–g) were synthesized by either benzylation of carboxylic
acids (20a, b) using BnBr and K CO or by reduction of benzaldehydes (17a, b, 19, 21)
2
3
by NaBH , as illustrated in Scheme 5. Benzyl butanoates (17a, b) were prepared by
4
conversion of ethyl esters (16a, b), which were obtained by the
-
Page 8 of 87 -

TM
14
PEPPSI –IPr-catalyzed Negishi cross-coupling reaction of aryl bromides (15a, b)
with an alkylzinc bromide reagent, into benzyl esters via hydrolysis. Pent-4-enoate (19)
was synthesized by benzylation of pent-4-enoic acid, prepared by Wittig olefination of
mono-protected terephthalaldehyde (18), in which the diethyl acetal group was
deprotected under acidic work-up of the Wittig reaction.
In the above PK study of compound 2, hydrolysis of the glucosidic linkage of 2
was observed by treatment with β-glucuronidase. Therefore, synthesis of the aglycones
of the key compounds was carried out by enzymatic hydrolysis of the parent glycosides
using -glucuronidase in a 0.2 M acetate buffer (pH 5.0). For the amine (22c), the Cbz
group was removed by following Pd–C-catalyzed hydrogenation of the N-protected
aglycone (22b) (Scheme 6).
3
. Results and Discussion
All compounds were screened for their inhibitory effects on the uptake of
1
4
radiolabeled -methyl-D-glucopyranoside ([ C]-AMG) into COS-7 cells transiently
expressing human SGLT1 (hSGLT1) or SGLT2 (hSGLT2).
It is known that some aniline derivatives can mimic the phenol structure and
1
5
function as biologically equivalent groups. Focusing on the para-hydroxyl group of
-
Page 9 of 87 -

the phenyl ring of our lead compound 2, we first examined the effects of the
replacement with phenol bioisoster groups. For synthetic ease, novel designed groups
were assessed in the absence of the methyl group at the ortho-position to the
pyrazolylmethyl group, and the results are summarized in Table 1.
Compared with phenol (23), aniline (7) showed reduced SGLT1 inhibitory
activity. However, it was slightly more potent than unsubstituted compound 24,
suggesting that the amino group had some enhancing effect on activity. Conversion of
aniline (7) into acetoanilide (8a) was well tolerated and its inhibitory activity was
maintained, with a slight improvement in selectivity versus SGLT2 also observed. In
contrast, elongation of the acyl group was found to be an unfavorable conversion, and
the resulting propanamide (8b) and butanamide (8c) showed diminished activity
according to chain length. In addition, methanesulfonamide (8d) showed decreased
activity and reduced selectivity for SGLT1 compared with every aniline derivative
described above, except for compound 8c. To reduce the lipophilic parameter,
introduction of an amino group onto the acyl groups was next examined. Although,
glycinamide (9a) showed weaker activity than acetoanilide (8a), recovery of activity
with improved selectivity for SGLT1 was observed in -alaninamide (9b) compared
with glycinamide (9a) or propanamide (8b). Butanamide (8c), possessing a methyl
-
Page 10 of 87 -

group instead of the terminal amino group of 9b, showed weaker activity than 9b,
indicating that the hydrogen-bond donor character and/or basic functionality of the
amino group at this position might contribute to the interaction with SGLT1. By the
introduction of these phenol bioisosteric groups, a reduction in calculated LogD values
and/or larger topological PSA (TPSA) values were observed and reduced permeability
of their aglycones was expected. Indeed, in the Caco-2 cell permeability test, the
aglycones (22a, c) of selected compounds (8a, 9b) were found to be less permeable than
the aglycones of lead compounds 1 and 2 (1a, 2a) (Table 5).
Because these amide derivatives incorporate an aniline structure, the formation
16
of aniline metabolites was of concern owing to the known mutagenicity of anilines.
Therefore, a PK study of a representative amide (8a) was performed in rats (10 mg/kg,
p.o.) before further optimization of this series of compounds. The plasma concentration
of the aglycone of compound 8a (22a) was found to be lower, even after hydrolysis by

-glucronidase (Cmax: 78 ng/mL, AUC(0–6 h): 16,107 min·ng/mL), than that of the
aglycone of compound 2 (2a), as described above. However, a trace amount of the
particular aniline metabolite (7) and its aglycone (5a) was detected in the same plasma,
and the level of 5a increased after treatment with -glucronidase (Cmax: 38 ng/mL,
AUC(0–6 h): 7458 min·ng/mL), indicating that the amide bond of 8a is not stable in vivo
-
Page 11 of 87 -

and that aniline metabolites could be formed easily. Moreover, one of the aniline
metabolites (5a) was unfortunately found to be positive in the Ames test (7: not tested).
Based on these disappointing results, we discontinued further optimization of this series
of compounds and turned our attention instead to nonaniline derivatives to ensure
greater safety.
As we wished to incorporate some characteristics of the above aniline amide
derivatives in order to improve physicochemical parameters and the potential to
introduce further substitution groups, we next investigated the effects of introducing
1
7
inverse amide-containing groups. The results are summarized in Table 2. Initial use of
benzamide (10a) or derivatives having relatively short linking alkylene groups (10b, c)
afforded a disappointing reduction in SGLT1 inhibitory activity compared with phenol
(23). However, a marked recovery of activity was observed by elongation of the linking
group to propyrene (10d). Good consistency observed in the number of tethering atoms
between the phenyl group and the terminal –NH group with a previous -alaninamide
2
derivative (9b) suggested that at least the hydrogen-bond donor character at this
position might be efficacious to interact with SGLT1. Because further elongation into
pentanamide (10e) led to a decrease in activity, we selected butanamide (10d) as a lead
compound for further optimization investigations.
-
Page 12 of 87 -

With a nonaniline lead compound in hand, we next examined the effects of
introducing functional groups on the amide-nitrogen atom of butanamide (10d).
Because the presence of substitution groups should improve not only SGLT1 inhibitory
activity but also physicochemical parameters, alkyl groups possessing polar groups such
as an alcohol or amide were used for investigation and the results are illustrated in Table
3
. Substitution with a 2-hydroxylethyl group on the amide-nitrogen atom was well
tolerated and resulting compound 11a showed a slightly increased inhibitory activity on
both SGLTs compared with the N-unsubstituted amide (10d). In contrast, further
substitution with a methyl group on the nitrogen atom of 2-hydroxyethyl amide (11a)
resulted in a decrease in activity, indicating that at least one of the hydrogen atoms on
the nitrogen of the amide group should be left for hydrogen-bond donation (11b). In
addition, 3-hydroxypropyl amide (11c) showed comparable inhibitory activity on
SGLT1 with 2-hydroxyethyl amide (11a), but a slight decrease in selectivity for SGLT1
was observed. Although lipophilicity was increased and not limited to SGLT1, a notable
enhancement of inhibitory activity was achieved by disubstitution with methyl groups
on the methylene bonded to the amide-nitrogen atom of compound 11a, suggesting that
a degree of bulkiness at this position might be critical for the expression of higher
activities (11d). These enhancement effects on the inhibitory activities on both SGLTs
-
Page 13 of 87 -

of the dimethyl groups were also observed in subsequent glycinamide derivatives
investigated. Glycinamide (11e) was less potent than basal N-unsubstituted amide (10d),
but substitution with dimethyl groups at the corresponding position to 11d led to a
marked increase of inhibitory activities on both SGLTs (11f). In addition to the strong
potency, the permeability of the aglycone of compound 11f (22e) was distinctly lowered
relative to that of the aglycones of initial lead compounds 1 and 2 (1a, 2a) and was
further reduced even compared with those of the aglycones of the above aniline
derivatives (22a, c), possibly owing to changes in physicochemical properties (Table 5).
On the other hand, the aglycone of the most potent compound 11d (22d) showed higher
permeability than that of 22e. Of these two types of potent compounds, 11d and 11f, we
focused on the terminal carbamoyl group of 11f. The effects of further modification of
this group were finally investigated (Table 4).
Based on the same objective to explore substituents on the butanamide nitrogen
atom, 2-hydroxyethyl amide (14g) and glycinamide (14h) were designed but no
improvement in activity or selectivity was observed compared with the N-unsubstituted
amide (11f). In contrast, a further increase of inhibitory activity could be achieved by
conversion of the carbamoyl group into amine-containing amide groups such as
2
-(dimethylamino)ethylamide (14i) and 4-methylpiperazin-1-ylamide (14d). Because
-
Page 14 of 87 -

cyclized amine (14d) was more potent than alkylamine (14i), fixation of the amine
position might be advantageous to the interaction with SGLTs. To reduce the lipophilic
parameter, a methyl group on the piperazine ring of compound 14d was eliminated and
the resulting unsubstituted piperazine (14a) showed a slightly decreased but still potent
inhibitory activity on SGLT1 along with potent inhibition of SGLT2. Conversion of this
piperazine group into either piperidine (14e) or morpholine (14f) caused a decrease of
activity, suggesting that basic functionality at this position might be efficacious in
achieving strong inhibitory activities on both SGLTs. The above conversion of the
N-unsubstituted amide into piperazinamide was also found to be efficacious in
maintaining the low permeability of the aglycone. The aglycone of 14a (22f) showed a
corresponding low permeability compared with that of the aglycone of 11f (22e) (Table
5
).
Of all derivatives investigated in this study, we selected 14a as the most
promising compound and examined whether its inhibitory activity toward SGLT2 could
be reduced. In the previous study, we discovered that the introduction of a methyl group
at the ortho-position of the pyrazolylmethyl group on the phenyl ring could improve
8
selectivity toward SGLT1; therefore, an analog of 14a bearing the methyl group at the
corresponding position was synthesized. Obtained compound 14b actually exhibited
-
Page 15 of 87 -

weaker inhibitory activity on SGLT2 compared with 14a (Table 4). Moreover, unlike
lead compounds 1 and 2, the potency on SGLT1 of 14b was not diminished even by the
introduction of this methyl group. This conversion was not so efficient to maintain the
8
potency on SGLT1 in the lead compound analogs (for example, 2 vs. 23). As another
approach to improve selectivity for SGLT1, conversion of the sugar portion into
galactoside was also investigated. Unlike glucose, which is a substrate of both SGLT1
7
and SGLT2, galactose is a substrate of SGLT1 only. As expected, the inhibitory
activity of galactoside (14c) on SGLT2 was notably reduced compared with glucoside
(
14a), and this conversion of sugar was more effective than the introduction of the
methyl group on the phenyl ring with respect to decreasing activity on SGLT2 (14c:
7-fold reduction vs. 14b: 19-fold reduction). Galactoside (14c) also showed reduced
8
inhibitory activity on SGLT1 compared with glucoside (14a), but the superior activity
of 14a ensured that 14c was a still potent compound. A slight difference in the
recognition of the natural substrates, glucose and galactose, might be a possible
1
8
explanation of this reduction of the inhibitory activity on SGLT1. Selectivity toward
SGLT1 might not be important for the compounds that would be poorly absorbed such
as 14b and 14c. However, simplicity of the structure modifications, installation of
methyl group or conversion of glucoside into galactoside, led us to pursue the
-
Page 16 of 87 -

selectivity to exclude possibility of increasing urinary glucose excretion, if they were
absorbed unexpectedly.
As shown in Table 6, these potent and selective SGLT1 inhibitors (14b, c)
showed greater efficacy than initial lead compound 2 in oral liquid meal tolerance tests
1
9
(
OMTT) in NA-STZ rats. The higher metabolic stability of compound 14c might
contribute to the higher efficacy compared with compound 14b, despite the relatively
weaker inhibitory activity on rat SGLT1 of compound 14c. On the basis of these in vivo
efficacies of the two compounds, we selected 14c and a comparison of its efficacy with
acarbose, an -GI that is widely used in the clinic, was conducted.
In tolerance tests in streptzotocin-induced-diabetic rats (STZ rats), 14c
suppressed the escalation of plasma glucose levels induced by oral loading of various
carbohydrates and carbohydrate-containing meals in a dose-dependent manner, as
shown in Figure 2. Furthermore, its efficacy was greater than that of acarbose. An
ability to respond with glucose would be an advantage of SGLT1 inhibitors over -GI,
because they have no effects on monosaccaride absorption. While acarbose showed no
efficacy, even at a high dose (30 mg/kg), distinct suppressing effects on glucose
absorption were observed with 14c in this study.
-
Page 17 of 87 -

In in vivo experiments, the abdominal symptoms such as soft feces or diarrhea,
2
which were common adverse effects with α-GI, were not observed in every treatment
groups, except for the higher dose (1.0 mg/kg) of 14c in the comparative study among
SGLT1 inhibitors (Table 6). Continuous administration for 2 days with liquid meal
(t.i.d., see experiment) before OMTT and the greater efficacy of 14c (Table 6), might be
possible factors that induced those symptoms of 14c.
4
. Conclusions
To improve potency of SGLT1 inhibition and reduce the permeability of the
aglycones of initial lead compounds 1 and 2, novel hydrophilic substitution groups on
the phenyl ring were investigated. SAR studies identified that N-substituted butanamide
derivatives exhibited strong inhibitory activities on both SGLTs, whereas the
introduction of a methyl group into the meta-position of the butanamide group on the
phenyl ring or conversion of glucoside into galactoside was found to improve selectivity
toward SGLT1. Finally, reduction of lipophilic parameters and/or enlargement of PSA
were effective to lower the Caco-2 cell permeability of the aglycones.
We achieved both enhancement of inhibitory activity on SGLT1 and a marked
reduction in the permeability of its aglycone in 14c compared with initial lead
-
Page 18 of 87 -

compounds 1 and 2. The improved profile of 14c might contribute to a lower required
dose and reduced risks of unnecessary systemic exposure to compounds, including
metabolites. Compound 14c showed superior efficacy over acarbose in various
carbohydrate tolerance tests in STZ rats and could suppress absorption of glucose itself.
These findings indicate the potential of this compound instead of -GI as a novel
therapeutic option for the treatment of postprandial hyperglycemia in DM or IGT
patients.
5
5
. Experimental
.1. Chemistry
1
H nuclear magnetic resonance (NMR) spectra were recorded using Bruker
+
13
+
Avance II 400 instruments. C NMR spectra were recorded using Bruker Avance II
00 or Avance III 600 instruments and chemical shifts were reported in parts per
million (ppm) (), downfield from tetramethylsilane as the internal standard. In
4
experiments using D O as the solvent, chemical shifts were referenced to dioxane as the
2
internal standard ( = 66.5 ppm). Peak patterns are shown using the following
c
abbreviations: br, broad; s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet.
Mass spectra (MS) were measured using an Agilent Technologies 6140 (electrospray
-
Page 19 of 87 -

ionization; ESI) or Shimadzu LCMS-2010EV (ESI) spectrometer. High-resolution mass
spectra (HRMS) were recorded using an Agilent Technologies 6520 Accurate-Mass
Q-TOF instrument. Silica gel 60F -precoated glass plates from Merck KgaA or
2
54
aminopropyl silica gel (APS)-precoated NH plates from Fuji Silysia Chemical Ltd. were
used for thin-layer chromatography (TLC). Flash or medium-pressure liquid
chromatography was performed on silica gel BW-350 from Fuji Silysia Chemical Ltd.
or APS Daisogel IR-60 (particle size, 25–40 M) from Daiso Co., Ltd. or SNAP
®
cartridge KP-Sil or KP-NH from Biotage . All reagents and solvents were
commercially available unless otherwise indicated.
5
.1.1. 4-(4-Aminophenylmethyl)-1,2-dihydro-5-isopropyl-3H-pyrazol-3-one (5a)
A mixture of 3a (5.00 g, 33.1 mmol), methyl 4-methyl-3-oxopentanoate (4.77 g,
3.1 mmol), piperidine (196 L, 1.98 mmol), and AcOH (133 L, 2.32 mmol) in iPrOH
3
(15 mL) was stirred at room temperature for 4 days. After dilution with water, the
resulting mixture was extracted with Et O. The extract was washed with water and brine,
2
dried over anhydrous MgSO , and then, concentrated under reduced pressure to give
4
crude methyl 4-methyl-2-(4-nitrobenzylidene)-3-oxopentanoate. This intermediate was
dissolved in MeOH (90 mL), and 10% Pd–C (50% wet with water, 3.00 g) was added to
-
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the solution. The mixture was stirred under a hydrogen atmosphere at room temperature
overnight. The insoluble material was removed by filtration and washed with MeOH.
The filtrate and washing were concentrated under reduced pressure to give crude 4a.
This intermediate was dissolved in toluene (50 mL) and N H ·H O (3.21 mL, 66.2
2
4
2
mmol) was added to the solution. After refluxing for 2 h, the reaction mixture was
cooled to room temperature. The precipitates were collected by filtration, washed with
hexane and water, and then, dried under reduced pressure to give 5a (5.28 g, 69%) as a
white solid.
1
H NMR (DMSO-d ) δ: 1.06 (6H, d, J = 7.0 Hz), 2.80 (1H, heptet, J = 7.0 Hz),
6
3
.39 (2H, s), 4.73 (2H, s), 6.40-6.45 (2H, m), 6.79 (2H, d, J = 8.3 Hz), 8.40-12.20 (2H,
+
br); MS m/z: 232 (M+H) .
5
.1.2. Benzyl 4-(1,2-dihydro-3-oxo-5-isopropyl-3H-pyrazol-4-ylmethyl)benzoate
(5b)
Methanesulfonyl chloride (MsCl) (1.74 mL, 22.4 mmol) was added to a solution
of 3b (5.18 g, 21.4 mmol) and Et N (3.28 mL, 23.5 mmol) in tetrahydrofuran (THF) (20
3
mL) under ice cooling, and the mixture was stirred for 20 min. The insoluble material
was removed by filtration and washed with THF (4 mL). The filtrate and washing were
-
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added to a mixture prepared by the addition of NaH (855 mg, 21.4 mmol, 60% oil
dispersion) into a solution of methyl 4-methyl-3-oxopentanoate (3.08 g, 21.4 mmol) in
THF (40 mL) under ice cooling and stirring for 5 min. The mixture was then refluxed
for 10 h. After the mixture was cooled to room temperature, 1 N HCl (30 mL) was
added and then the resulting mixture was extracted with EtOAc. The extract was
washed with brine, dried over anhydrous MgSO , and then, concentrated under reduced
4
pressure to give crude 4b. This intermediate was dissolved in iPrOH (15 mL), and
N H ·H O (1.56 mL, 32.0 mmol) was added to the solution. After the mixture was
2
4
2
stirred at room temperature overnight, it was diluted with water and then the resulting
mixture was extracted with EtOAc. The extract was washed with brine, dried over
anhydrous MgSO and then concentrated under reduced pressure. The residue was
4
purified by column chromatography on silica gel (eluent: CH Cl /MeOH =
2
2
1
:0–19:1–9:1) to give 5b (4.14 g, 55%) as an oil with a slight yellow color.
1
H NMR (DMSO-d ) δ: 1.06 (6H, d, J = 7.0 Hz), 2.83 (1H, heptet, J = 7.0 Hz),
6
3
.66 (2H, s), 5.33 (2H, s), 7.27-7.48 (7H, m), 7.85-7.91 (2H, m), 8.60-12.20 (2H, br);
+
MS m/z: 351 (M+H) .
5
.1.3. Benzyl
-
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2
-[4-(1,2-dihydro-3-oxo-5-isopropyl-3H-pyrazol-4-ylmethyl)phenyl]acetate (5c)
The title compound was prepared from 3c, as described for the synthesis of 5b,
in 23% yield.
1
H NMR (DMSO-d ) δ: 1.07 (6H, d, J = 7.0 Hz), 2.83 (1H, heptet, J = 7.0 Hz),
6
3
.55 (2H, s), 3.65 (2H, s), 5.09 (2H, s), 7.09 (2H, d, J = 8.2 Hz), 7.13 (2H, d, J = 8.2
+
Hz), 7.27-7.38 (5H, m), 8.80-9.80 (1H, br), 10.70-11.50 (1H, br); MS m/z: 365 (M+H) .
5
.1.4. Benzyl
(E)-3-[4-(1,2-dihydro-3-oxo-5-isopropyl-3H-pyrazol-4-ylmethyl)phenyl]acrylate
(5d)
The title compound was prepared from 3d, as described for the synthesis of 5b,
in 47% yield.
1
H NMR (DMSO-d ) δ: 1.07 (6H, d, J = 7.0 Hz), 2.84 (1H, heptet, J = 7.0 Hz),
6
3
.60 (2H, s), 5.21 (2H, s), 6.62 (1H, d, J = 15.9 Hz), 7.19 (2H, d, J = 8.3 Hz), 7.30-7.45
(5H, m), 7.61 (2H, d, J = 8.3 Hz), 7.64 (1H, d, J = 15.9 Hz), 9.00-10.00 (1H, br),
+
1
0.70-11.60 (1H, br); MS m/z: 377 (M+H) .
5
.1.5. Benzyl
-
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4
-[4-(1,2-dihydro-3-oxo-5-isopropyl-3H-pyrazol-4-ylmethyl)phenyl]butanoate (5e)
The title compound was prepared from 3e, as described for the synthesis of 5b,
in 62% yield.
1
H NMR (DMSO-d ) δ: 1.06 (6H, d, J = 7.0 Hz), 1.74-1.84 (2H, m), 2.32 (2H, t,
6
J = 7.4 Hz), 2.48-2.54 (2H, m), 2.82 (1H, heptet, J = 7.0 Hz), 3.53 (2H, s), 5.07 (2H, s),
7
.01 (2H, d, J = 8.4 Hz), 7.05 (2H, d, J = 8.4 Hz), 7.28-7.40 (5H, m), 8.90-10.00 (1H,
+
br), 10.60-11.50 (1H, br); MS m/z: 393 (M+H) .
5
4
.1.6. Benzyl
-[4-(1,2-dihydro-3-oxo-5-isopropyl-3H-pyrazol-4-ylmethyl)-3-methylphenyl]butan
oate (5f)
The title compound was prepared from 3f, as described for the synthesis of 5b,
in 64% yield.
1
H NMR (DMSO-d ) δ: 1.03 (6H, d, J = 7.0 Hz), 1.74-1.84 (2H, m), 2.24 (3H, s),
6
2
.32 (2H, t, J = 7.3 Hz), 2.48 (2H, t, J = 7.8 Hz), 2.72 (1H, heptet, J = 7.0 Hz), 3.47 (2H,
s), 5.07 (2H, s), 6.79 (1H, d, J = 7.9 Hz), 6.83 (1H, d, J = 7.9 Hz), 6.90 (1H, s),
.29-7.41 (5H, m), 8.80-9.80 (1H, br), 10.60-11.60 (1H, br); MS m/z: 407 (M+H) .
+
7
-
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5
.1.7. Benzyl
E)-5-[4-(1,2-dihydro-3-oxo-5-isopropyl-3H-pyrazol-4-ylmethyl)phenyl]pent-4-eno
ate (5g)
The title compound was prepared from 3g, as described for the synthesis of 5b,
(
in 29% yield.
1
H NMR (DMSO-d ) δ: 1.06 (6H, d, J = 6.9 Hz), 2.39-2.56 (4H, m), 2.82 (1H,
6
heptet, J = 6.9 Hz), 3.55 (2H, s), 5.10 (2H, s), 6.17 (1H, dt, J = 16.1, 6.7 Hz), 6.36 (1H,
d, J = 16.1 Hz), 7.08 (2H, d, J = 8.3 Hz), 7.21 (2H, d, J = 8.3 Hz), 7.28-7.37 (5H, m),
+
9
.00-9.80 (1H, br), 10.70-11.45 (1H, br); MS m/z: 405 (M+H) .
5
2
.1.8. 4-(4-Aminophenylmethyl)-5-isopropyl-1H-pyrazol-3-yl
,3,4,6-tetra-O-acetyl--D-glucopyranoside (6a)
To a mixture of 5a (5.50 g, 23.8 mmol), acetobromoglucose (9.79 g, 23.8 mmol),
and BnN(n-Bu) Cl (7.42 g, 23.8 mmol) in CH Cl (100 mL) was added 5 N NaOH
3
2
2
(19.0 mL, 95.0 mmol), and the mixture was stirred at room temperature overnight. The
reaction mixture was extracted with EtOAc, and the extract was washed with brine and
concentrated under reduced pressure. The residue was purified by column
-
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chromatography on aminopropyl silica gel (eluent: hexane/EtOAc = 3:2–2:3–3:7–1:4)
to give 6a (1.41 g, 11%) as a white solid.
1
H NMR (DMSO-d ) δ: 1.06 (3H, d, J = 7.0 Hz), 1.07 (3H, d, J = 7.0 Hz), 1.92
6
(
3H, s), 1.95 (3H, s), 1.989 (3H, s), 1.992 (3H, s), 2.86 (1H, heptet, J = 7.0 Hz), 3.33
1H, d, J = 15.6 Hz), 3.38 (1H, d, J = 15.6 Hz), 3.97 (1H, dd, J = 12.3, 2.0 Hz),
(
4
5
8
.09-4.16 (1H, m), 4.20 (1H, dd, J = 12.3, 4.5 Hz), 4.80 (2H, br s), 4.95-5.03 (2H, m),
.42 (1H, t, J = 9.5 Hz), 5.72 (1H, d, J = 8.3 Hz), 6.39-6.44 (2H, m), 6.74 (2H, d, J =
+
.3 Hz), 11.63 (1H, s); MS m/z: 562 (M+H) .
5
4
.1.9.
-[3-(Tetra-O-acetyl--D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethyl]b
enzoic acid (6b)
To a mixture of 5b (2.00 g, 5.71 mmol), acetobromoglucose (2.35 g, 5.71 mmol),
and BnN(n-Bu) Cl (1.78 g, 5.71 mmol) in CH Cl (25 mL) was added 5 N NaOH (4.57
3
2
2
mL, 22.9 mmol), and the mixture was stirred at room temperature overnight. After the
addition of 1 N HCl (40 mL), the resulting mixture was extracted with EtOAc. The
extract was washed with brine and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel (eluent: hexane/EtOAc =
-
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7
4
:3–2:3–3:7–1:4) to give benzyl
-[3-(tetra-O-acetyl--D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethyl]benzo
ate (1.46 g, 38%). This intermediate (1.39 g, 2.04 mmol) was dissolved in MeOH (14
mL), and 10% Pd–C (50% wet with water, 278 mg) was added to the solution. The
mixture was stirred under a hydrogen atmosphere at room temperature overnight. The
insoluble material was removed by filtration and washed with MeOH. The filtrate and
washing were concentrated under reduced pressure, and the residue was purified by
column chromatography on silica gel (eluent: hexane/EtOAc = 1:4–0:1) to give 6b (1.07
g, 88%) as a white solid.
1
H NMR (DMSO-d ) δ: 1.076 (3H, d, J = 7.0 Hz), 1.077 (3H, d, J = 7.0 Hz),
6
1
.88 (3H, s), 1.95 (3H, s), 1.98 (3H, s), 1.99 (3H, s), 2.89 (1H, heptet, J = 7.0 Hz), 3.63
2H, s), 3.97 (1H, dd, J = 12.1, 2.0 Hz), 4.10-4.16 (1H, m), 4.18 (1H, dd, J = 12.1, 4.5
Hz), 4.94-5.01 (2H, m), 5.42 (1H, t, J = 9.5 Hz), 5.75 (1H, d, J = 8.0 Hz), 7.21 (2H, d, J
(
=
8.3 Hz), 7.80 (2H, d, J = 8.3 Hz), 11.78 (1H, br s), 12.76 (1H, br s); MS m/z: 591
+
(
M+H) .
5
2
.1.10.
-{4-[3-(Tetra-O-acetyl--D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethy
-
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l]phenyl}acetic acid (6c)
The title compound was prepared from 5c, as described for the synthesis of 6b,
in 34% yield.
1
H NMR (DMSO-d ) δ: 1.08 (3H, d, J = 6.9 Hz), 1.09 (3H, d, J = 6.9 Hz), 1.88
6
(3H, s), 1.95 (3H, s), 1.985 (3H, s), 1.987 (3H, s), 2.90 (1H, heptet, J = 6.9 Hz), 3.47
(2H, s), 3.50 (1H, d, J = 15.8 Hz), 3.55 (1H, d, J = 15.8 Hz), 3.97 (1H, dd, J = 12.2, 2.0
Hz), 4.09-4.15 (1H, m), 4.19 (1H, dd, J = 12.2, 4.5 Hz), 4.95-5.04 (2H, m), 5.42 (1H, t,
J = 9.5 Hz), 5.74 (1H, d, J = 8.3 Hz), 7.03 (2H, d, J = 8.0 Hz), 7.10 (2H, d, J = 8.0 Hz),
+
1
1.71 (1H, br s), 12.22 (1H, br s); MS m/z: 605 (M+H) .
5
3
.1.11.
-{4-[3-(Tetra-O-acetyl--D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethy
l]phenyl}propanoic acid (6d)
The title compound was prepared from 5d, as described for the synthesis of 6b,
in 32% yield.
1
H NMR (DMSO-d ) δ: 1.07 (3H, d, J = 6.9 Hz), 1.08 (3H, d, J = 6.9 Hz), 1.88
6
(
3H, s), 1.95 (3H, s), 1.99 (6H, s), 2.45 (2H, t, J = 7.7 Hz), 2.73 (2H, t, J = 7.7 Hz), 2.89
1H, heptet, J = 6.9 Hz), 3.48 (1H, d, J = 15.8 Hz), 3.53 (1H, d, J = 15.8 Hz), 3.97 (1H,
(
-
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dd, J = 12.2, 2.0 Hz), 4.09-4.15 (1H, m), 4.19 (1H, dd, J = 12.2, 4.6 Hz), 4.94-5.03 (2H,
m), 5.42 (1H, t, J = 9.5 Hz), 5.72 (1H, d, J = 8.0 Hz), 6.99 (2H, d, J = 8.0 Hz), 7.06 (2H,
+
d, J = 8.0 Hz), 11.71 (1H, br s), 11.73-12.50 (1H, br); MS m/z: 619 (M+H) .
5
4
.1.12.
-{4-[3-(Tetra-O-acetyl--D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethy
l]phenyl}butanoic acid (6e)
The title compound was prepared from 5e, as described for the synthesis of 6b,
in 31% yield.
1
H NMR (DMSO-d ) δ: 1.06 (3H, d, J = 7.0 Hz), 1.07 (3H, d, J = 7.0 Hz),
6
1
7
3
.69-1.79 (2H, m), 1.88 (3H, s), 1.95 (3H, s), 1.98 (3H, s), 1.99 (3H, s), 2.17 (2H, t, J =
.3 Hz), 2.48-2.54 (2H, m), 2.88 (1H, heptet, J = 7.0 Hz), 3.49 (1H, d, J = 15.8 Hz),
.54 (1H, d, J = 15.8 Hz), 3.97 (1H, dd, J = 12.2, 2.1 Hz), 4.09-4.15 (1H, m), 4.19 (1H,
dd, J = 12.2, 4.5 Hz), 4.95-5.03 (2H, m), 5.42 (1H, t, J = 9.7 Hz), 5.73 (1H, d, J = 8.3
+
Hz), 6.98-7.05 (4H, m), 11.71 (1H, br s), 12.02 (1H, br s); MS m/z: 633 (M+H) .
5
4
.1.13.
-{4-[3-(Tetra-O-acetyl--D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethy
-
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