Discovery of tofogliflozin, a novel C-arylglucoside with an O-spiroketal ring system, as a highly selective sodium glucose cotransporter 2 (SGLT2) inhibitor for the treatment of type 2 diabetes

Article abstract of DOI:10.1021/jm300884k

Inhibition of sodium glucose cotransporter 2 (SGLT2) has been proposed as a novel therapeutic approach to treat type 2 diabetes. In our efforts to discover novel inhibitors of SGLT2, we first generated a 3D pharmacophore model based on the superposition o

Full text of DOI:10.1021/jm300884k

Article
pubs.acs.org/jmc
Discovery of Tofogliflozin, a Novel C‑Arylglucoside with an
O‑Spiroketal Ring System, as a Highly Selective Sodium Glucose
Cotransporter 2 (SGLT2) Inhibitor for the Treatment of Type 2
Diabetes
Yoshihito Ohtake,*,† Tsutomu Sato,† Takamitsu Kobayashi,† Masahiro Nishimoto,† Naoki Taka,†
Koji Takano,† Keisuke Yamamoto,† Masayuki Ohmori,† Marina Yamaguchi,† Kyoko Takami,†
Sang-Yong Yeu,§ Koo-Hyeon Ahn,‡ Hiroharu Matsuoka,† Kazumi Morikawa,† Masayuki Suzuki,†
Hitoshi Hagita,§ Kazuharu Ozawa,† Koji Yamaguchi,† Motohiro Kato,† and Sachiya Ikeda†
†Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
§Chugai Research Institute for Medical Science, Inc., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
‡C&C Research Laboratories, DRC Natural Sciences Campus, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon,
Gyeonggi-do 440-746, Republic of Korea
S
* Supporting Information
ABSTRACT: Inhibition of sodium glucose cotransporter 2
(SGLT2) has been proposed as a novel therapeutic approach
to treat type 2 diabetes. In our efforts to discover novel
inhibitors of SGLT2, we first generated a 3D pharmacophore
model based on the superposition of known inhibitors. A
search of the Cambridge Structural Database using a series of
pharmacophore queries led to the discovery of an O-spiroketal C-arylglucoside scaffold. Subsequent chemical examination
combined with computational modeling resulted in the identification of the clinical candidate 16d (CSG452, tofogliflozin), which
is currently under phase III clinical trials.
Sodium glucose cotransporters (SGLTs) have recently
attracted considerable attention as new drug targets for the
treatment of diabetes.3 Of several subtypes identified in the
SGLT family, SGLT1 and 2 are expressed in the renal tubules.4
Recent research results indicate that selective inhibition of
SGLT2 could provide a well-tolerated and highly effective
method of glycemic control.5 The expression of SGLT2
appears to be kidney specific. Mutations of the SGLT2 gene
cause familial renal glucosuria,6 but daily activities are not
affected. SGLT1, on the other hand, transports glucose and
galactose not only in the renal tubules but also in the small
intestine.7 Mutations in human SGLT1 lead to glucose−
galactose malabsorption, which is characterized by severe
diarrhea and sometimes mild renal glucosuria.8 In addition,
SGLT1 is expressed in cardiac myocytes and serves as a glucose
transporter in the heart.9 Thus, inhibition of SGLT1 may cause
gastrointestinal symptoms or unexpected adverse effects.
Therefore, highly selective inhibition of SGLT2 versus
SGLT1 is one of the important features to be achieved.
INTRODUCTION
■
Diabetes mellitus is a progressive metabolic disease charac-
terized by chronic hyperglycemia due to a decrease of insulin
secretion from the pancreas and insulin resistance. According to
the World Health Organization, more than 346 million people
worldwide have diabetes, and an estimated 3.4 million people
died from the consequences of high blood sugar in 2004, with
this number expected to double by the year 2030.1 Type 2
diabetes accounts for 90% of all cases of diabetes. Prevention of
diabetes-related microvascular complications such as nephrop-
athy, retinopathy, and neuropathy in these patients requires
tight glycemic control.2 Currently available hypoglycemic
agents can be classified into three categories based on their
mechanism of action: those promoting insulin secretion, those
improving insulin sensitivity, and those delaying glucose
absorption. However, sustained control of blood glucose to
prevent disease progression is difficult to achieve with any
single agent or with stepwise administration of these agents in
combination, with the result that patients eventually require
insulin therapy. In addition, currently available antidiabetic
agents are known to cause undesirable side effects such as
hypoglycemia, body weight gain, gastric symptoms, etc. Thus,
there is a high unmet medical need for novel potent
hypoglycemic agents with a good safety and tolerability profile
that can be used either as a monotherapy or in combination
with other antidiabetic agents.
By targeting renal tubular glucose reabsorption, SGLT2-
selective inhibitors exhibit a novel mechanism of action
resulting in excretion of glucose into the urine. Because this
effect is independent of insulin secretion or insulin sensitivity,
Received: June 22, 2012
Published: August 13, 2012
© 2012 American Chemical Society
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Figure 1. Selected examples of SGLT2 inhibitors.
SGLT2 inhibitors can be used in combination with various
existing oral antihyperglycemia drugs to improve clinical
outcomes in patients with type 2 diabetes. Moreover, SGLT2
inhibitors are also expected to reduce body weight because they
excrete glucose, or “energy”, leading to a lower overall caloric
load. Actually, it has been reported that SGLT2 inhibitors
reduced body weight in clinical trials.10
Phlorizin (1) is a natural nonselective SGLT inhibitor. This
approach to SGLT inhibition was supported by the finding that
chronic subcutaneous administration of 1 reduced the plasma
glucose level of diabetic rodents.11 However, 1 was not
clinically developed as a drug candidate because of its poor
metabolic stability to β-glucosidases.
Over nearly a decade, a lot of different compounds have been
reported as SGLT2 inhibitors.12−15 They are generally divided
into two classes, O-glucosides and C-glucosides. T-1095,16 the
first structural derivative of 1 reported by the Tanabe group,
was followed by sergliflozin17 (2) and remogliflozin18 (3), and
all are representatives of the O-glucosides. However, there have
been no reports that the O-glucoside class of SGLT2 inhibitors
have advanced to phase II clinical trials, probably because, like
phlorizin, they have inherent metabolic instability to β-
glucosidases, and thus relatively large doses are required in
clinic to exert the expected significant efficacy.
Meanwhile, the first of the C-aryl glucoside class of SGLT2
inhibitors has been reported by researchers at Bristol-Myers
Squibb Co. in the previous decade. Their representative,
dapagliflozin19 (4), was found to have more potent hSGLT2
inhibition in vitro and stability in vivo than the O-glucosides
and is currently being developed as an antidiabetic agent. At
present, a number of companies are clinically developing the C-
glucoside class of SGLT2 inhibitors, such as canagliflozin20 (5,
Mitsubishi Tanabe/Johnson & Johnson, phase III) and
empagliflozin21 (Boehringer Ingelheim, phase III), by modify-
ing the aglycon moiety.
clinical candidate 16d as a tofogliflozin,25 according to the
results of both pharmacological and pharmacokinetic studies.
RESULTS AND DISCUSSION
■
New Scaffold Exploration. Three-dimensional structures
of target proteins are very informative for designing new drugs,
but those of hSGLT1 and 2 have not been available so far.26 In
our efforts to identify novel SGLT2 inhibitors, we previously
discovered the carba-glucopyranosides by the ligand-based
approach.27 At nearly the same time, a 3D pharmacophore-
based approach for a lead discovery was applied based on the
accumulated structure−activity relationship information from
the carbasugar derivatives and others. At first, to construct a
hypothetical SGLT2 pharmacophore model, five potent
inhibitors28 (1, 2a, 3, 4, and 5 in Figure 1) and our potent
C-naphthyl glucoside 629 were subjected to superposition with
the Flexible Alignment module in Molecular Operating
Environment (MOE).30 A conformation search by Low-
ModeMD of MOE indicated that the energy difference of
each compound in Figure 2a from the global minimum was
within 5 kcal/mol. The reason why we utilized the additional
in-house compound 6 to construct the pharmacophore model
is that its naphthalene ring served as a template to superpose
two kinds of central aromatic rings such as C-glucosides and O-
glucosides. The superposition model in Figure 2a implies that
having a properly aligned sugar ring, two aromatic moieties, and
special distances between two pairs of pharmacophore points
are important in order to achieve higher activity. The
information obtained from a superposition model in Figure
2a is schematically summarized in Figure 2b. In Figure 2b, the
two aromatic rings are depicted in blue circles and the two
distances by double-headed arrows (Dist. 1, Dist. 2).
Next, we tried many searches in the Cambridge Structural
Database (CSD)31 and other available structure databases using
possible combinations of the pharmacophore points shown in
Figure 2b as a query. When searching databases with all three
pharmacophore points and two distance constraints, no
promising hit was obtained. On the other hand, the search
over CSD using two pharmacophore points (the sugar moiety
and the aromatic group 1 in Figure 2b) and a distance between
them (Dist. 1), followed by careful visual inspection of the hit
lists, provided us with three valuable structures, one of which is
In the course of our efforts to explore potent, selective, and
metabolically stable SGLT2 inhibitors, we successfully dis-
covered a novel class of highly potent and SGLT2-selective
inhibitors incorporating a unique spiroketal structure.22−24
Herein we wish to report the process of producing the O-
spiroketal C-arylglucosides from structure database searches
based on a 3D pharmacophore model, the synthesis of the test
compounds 16a−16n and 21o−21r, and the selection of the
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13a and 13d−13g were synthesized from the aldehyde 12,
which could be obtained by oxidation of 11, followed by
addition of Grignard reagents or lithiated benzenes and
reduction. Compound 16e (R1 = n-Pr) was obtained from
13g (R1 = c-Pr) by the reductive cleavage of the cyclopropane
ring in the final deprotection step described below. On the
other hand, 15b, 15c, and 15h−15n were prepared utilizing the
Suzuki coupling reactions. After debenzylation using boron
trichloride, benzyl alcohol moiety was selectively chlorinated by
treatment of chlorotrimethylsilane with dimethyl sulfoxide.
Four hydroxyl groups of the resulting benzyl chloride were
acetylated to afford 14. Suzuki coupling reactions of 14 with the
corresponding 4-substituted phenylboronic acids under re-
ported conditions34 gave 15b, 15c, and 15h−15n. Finally,
deprotections (debenzylation for 13a and 13d−13g or
deacetylation for 15b, 15c, and 15h−15n) afforded the test
compounds 16a−16n.
The configuration of the spiroketal was determined by single-
crystal X-ray diffraction analysis. Ethyl compound 16d was
converted into monoacetate derivative 17 by treatment with
acetyl chloride in 2,4,6-trimethylpyridine. Single crystal of 17
was obtained by recrystallization from acetonitrile and water as
a monohydrate, and the result of X-ray analysis is shown in
Figure 3 (see Supporting Information for more details).
Compounds 21o−21r with a substitution at position 4 of the
aglycon were synthesized as shown in Scheme 3. Diol 19 was
obtained from commercially available 18 via dibromination,
substitution reaction, and hydrolysis. Compound 19 was
transformed into 20 by a similar method to that in Schemes
1 and 2. Palladium-catalyzed coupling reactions of the chloro-
substituted 20 with boronic acids or trimetylsilylacetylene,35,36
followed by deacetylation under basic conditions, gave 21o−
21r.
In Vitro Activity. The in vitro SGLT inhibitory potential of
the O-spiroketal C-arylglucoside derivatives was assessed by
monitoring the inhibition of methyl-α-D-[U-14C]-
glucopyranoside (AMG) in Chinese hamster ovary cells stably
expressing human or mouse SGLT2 and SGLT1. All the
inhibition activities are shown in Table 1.
Focusing initially on para substitution groups on the distal
benzene ring, methoxy and ethoxy derivatives (16a and 16b)
had similarly high SGLT2 inhibition potential (IC50 = 9.1 and
9.6 nM, respectively). Among the three small straight-chain
alkyl analogues (16c−16e), the ethyl analogue 16d was more
potent and had higher SGLT2 selectivity versus SGLT1
(approximately 3000 times the selectivity) than the two alkoxy
derivatives (16a and 16b). The isopropyl (16f), the cyclopropyl
(16g), and the methylthio (16n) analogues showed similar
inhibition potential to the ethyl analogue 16d, and both 16f and
16g had slightly higher SGLT2 selectivity than 16d. The
Figure 2. Generation of a new O-spiroketal C-arylglucoside scaffold.
(a) Superposition model of six selected SGLT2 inhibitors, (b) a
schematic representation of the pharmacophore model, (c) one of
three hit compounds from CSD search, (d) superposition model of the
newly designed SGLT2 inhibitor 16d (carbon atoms in blue).
Hydrogen atoms are not displayed for clarity. All the figures were
prepared with PyMol (http://www.pymol.org./).
in Figure 2c (and see Supporting Information). They include a
spiroketal moiety in common and are known as synthetic
intermediates of antibiotic papulacandins.32 Finally, a new O-
spiroketal C-arylglucoside class of SGLT inhibitors was
designed by removing presumably unnecessary groups from
the CSD hits and adding p-substituted benzyl group to the
central benzene ring, referring to the structures of sergliflozin
(2) and dapagliflozin (4) (Figure 2d). The conformational
energy of 16d in Figure 2d from its global minimum is within 5
kcal/mol.
Chemistry. The synthetic route was established with
reference to the methods of the synthetic intermediates of
antibiotic papulacandins.33 That is, a key synthetic intermediate
11 for 16a−16n was synthesized as shown in Scheme 1.
Reduction of two carboxyl groups of commercially available 2-
bromoterephtalic acid 7, followed by protection of two
hydroxyl groups with triphenylmethyl, afforded 8. Compound
10 was obtained by adding the tetra benzyl-protected
gluconolactone 9 to the aryl lithium prepared in situ by
treatment of 8 with sec-butyllithium. Deprotection followed by
spirocyclization of 10 using triethylsilane and boron trifluoride-
diethyl etherate (BF3·OEt2) gave the benzyl alcohol 11.
O-Spiroketal C-arylglucosides 16a−16n were prepared from
11 through two pathways as outlined in Scheme 2. Compounds
a
Scheme 1. Preparation of the Benzyl Alcohol 11
a
Reagents and conditions: (a) BH3, THF, 0−40 °C, 2.5 h (90%); (b) Ph3CCl, Et3N, DMAP, DMF−CH2Cl2, 50 °C, 5.5 h (68%); (c) sec-BuLi,
toluene, −78 °C to rt, 30 min (80%); (d) Et3SiH, BF3·OEt2, CH3CN, −40 to 0 °C, 1 h (56%).
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a
Scheme 2. Preparation of Compounds 16a−16n
a
Reagents and conditions: (a) Dess−Martin periodinane, CH2Cl2, rt, 30 min (33%); (b) Grignard reagents, Et2O, 0 °C, 2.5 h (80−92%), or p-R1-
phenyl bromide, n-BuLi, THF or Et2O, −78 °C, 1 h (86−87%); (c) Et3SiH, BF3·OEt2, CH3CN or CH2Cl2, −40 °C, 1 h (83−90%); (d) BCl3, Me5-
benzene, CH2Cl2, −78 °C, 2 h (67%); (e) TMSCl, DMSO, rt, 1.5 h; then Ac2O, NMM, DMAP, 0 °C, 1 h (76%); (f) p-R1-phenylboronic acid,
Pd(OAc)2, Ph3P, K3PO4, toluene, 80 °C, 15 h (50−93%); (g) H2, Pd(OH)2/C, MeOH-AcOEt, 2N HCl aq, rt, 1 h (80−99%), or BCl3, Me5-
benzene, CH2Cl2, −78 °C, 2 h (50%); (h) K2CO3, MeOH, rt, 12 h (29−61%).
We selected 16d and 16f, which had strong hSGLT2
inhibition (IC50 < 5 nM) and high SGLT2 selectivity (around
3000-fold), for further evaluation. Cyclopropyl compound 16g
was not selected, despite having superior in vitro pharmaco-
logical profiles, because it showed high time-dependent
inhibition (TDI) of CYP3A4 (see Supporting Information for
more details).37
In Vivo Efficacy. The effects of these two compounds, 16d
and 16f, were evaluated on renal glucose excretion and blood
glucose level in db/db mice at oral administrations of 1, 3, and
Figure 3. ORTEP representation of the X-ray crystal structure of 17
(monohydrate).
10 mg/kg. Both compounds 16d and 16f showed a significant
and similar degree of increase in renal glucose excretion in the
period 0−6 h after oral dosing at 3 or 10 mg/kg (Figure 4). In
addition, both compounds reduced the blood glucose level in a
dose-dependent manner and to similar levels within the normal
range but not below 100 mg/dL (Figure 5a,b), indicating that
16d and 16f could improve hyperglycemia without inducing
hypoglycemia. Therefore, 16d and 16f were found to be
promising for the treatment of type 2 diabetes.
Pharmacokinetics. To select a clinical candidate, we
compared the pharmacokinetic profiles of the two compounds
(16d and 16f) in normal ICR mice and cynomolgus monkeys.
As shown in Table 2, oral bioavailability (F) of 16d was higher
(75% in mice and 85% in monkeys) than that of 16f (46% in
mice and 21% in monkeys). The large difference in the oral
bioavailability in monkeys is most probably attributable to the
compounds with a hydrophilic group (16h) or electron-
withdrawing groups (16i−16m) showed less potent inhibition
potential than the alkyl or the alkoxy analogues (16a−16g).
We next investigated the effects of substitution on the central
benzene moiety. The introduction of a chloro, methyl, or
ethynyl group in R2 (21o, 21p, and 21q) slightly increased the
SGLT2 inhibition potency compared with 16d while it greatly
increased the SGLT1 inhibition potency; that is, their SGLT2
selectivity was more than 10 times lower than that of 16d.
Interestingly, the substitution with a phenyl group (21r)
resulted in substantially decreased SGLT2 inhibition, suggest-
ing that the large substitution in this position (R2) may be
undesirable.
a
Scheme 3. Preparation of Compounds 21o−21r
a
Reagents and conditions: (a) NBS, AIBN, EtOAc, reflux, 20 min; (b) NaOAc, DMF, 80 °C, 3 h (39% from 17); (c) KOH, THF−EtOH−H2O, 80
°C, 3 h (97%); (d) methylboronic acid, Pd(dba)2, t-Bu3P·BF4, K3PO4, xylene, 145 °C, 15 h (28%); (e) TMS−acetylene, PdCl2(CH3CN)2, Xphos,
Cs2CO3, CH3CN, 90 °C, 2 h (81%); (f) phenylboronic acid, Pd(dba)2, CTC-Q-PHOS, THF, 100 °C, 30 min, microwave (quant); (g) K2CO3,
MeOH, rt, 1.5 h (36−54%).
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Table 1. In Vitro Data for hSGLT Inhibition
a
IC50 SD (nM)
b
compd
R1
R2
hSGLT2
hSGLT1
selectivity
1
16.4 5.2
1.3 0.2
9.1 0.5
9.6 1.6
4.8 1.1
2.9 0.7
7.5 3.8
3.2 1.2
1.6 0.4
29.9 1.1
122.7 42.4
16.8 6.1
117.9 35.3
14.2 3.4
14.5 1.2
3.6 0.8
1.3 0.2
1.4 0.3
1.4 0.3
240.8 56.1
185 70
800 210
8482 2228
11
4
615
932
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
21o
21p
21q
21r
OMe
OEt
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
18745 3791
3086 202
8444 2310
13137 3523
12335 3235
5314 724
4987 1255
18697 449
6281 625
81487 9225
24146 5207
28918 8099
5902 533
158 30
1953
643
2912
1752
3855
3321
167
n-Pr
i-Pr
c-Pr
OH
F
152
Cl
374
CN
CF3
OCF3
SMe
Et
691
1700
1994
1639
122
Et
Me
126 11
90
Et
CCH
388 24
277
Et
Ph
5132 249
21
a
In vitro human SGLT1 or SGLT2 inhibition activities of compounds were determined by evaluating sodium-dependent uptake of methyl-α-D-
[U-14C]glucopyranoside in Chinese hamster ovary (CHO) cells stably expressing SGLT1 or SGLT2. All the values represent the mean of three
determinations except for 1 and 16d (n = 22) and 4 (n = 15). The selectivity values were calculated by IC50 hSGLT1/IC50 hSGLT2.
b
were also compared. We measured the value of the renal
clearance (CLrenal) as a measure of the renal excretion ability
and calculated its ratio to total clearance (CLrenal/CLtotal).
When 1.0 mg/kg of 16d or 16f were administered intra-
venously to monkeys, the urinary excretion ratios (CLrenal
/
CLtotal) were 21.4% for 16d and 8.82% for 16f, respectively,
indicating that the ratio of the hepatic metabolism of 16d is
lower than that of 16f, and consequently the risk of drug−drug
interaction (DDI) is expected to be low. On the basis of the
results described above, compound 16d (tofogliflozin,
CSG452) was selected as a clinical candidate.38
CONCLUSION
■
Figure 4. Effects of single oral administration of 16d and 16f on renal
glucose excretion (0−6 h) in db/db mice. Compounds 16d and 16f
(1, 3, and 10 mg/kg) or vehicle were administered to db/db mice by
oral gavage. The renal glucose excretion (urine glucose concentration/
urine creatinine concentration) was calculated from the urine collected
between 0 and 6 h after drug administration under nonfasting
conditions. Data are expressed as mean SEM (n = 6). *P < 0.05,
**P < 0.01, ***P < 0.001 versus vehicle-treatment group by Dunnett’s
multiple comparison test.
We discovered a novel class of SGLT2 inhibitors characterized
by the O-spiroketal C-arylglucoside scaffold through a structural
database search using a 3D pharmacophore model that was
derived from known inhibitors. We revealed the SAR for
substitution of various groups on the aromatic rings. Regarding
the para-substitution on the distal benzene ring, small alkyl
groups were most favorable for achieving both SGLT2
inhibition activity and SGLT2 selectivity versus SGLT1. As
for the 4-substitution on the central benzene ring, small groups,
such as a chloro, enhanced the SGLT2 inhibition activity but
lowered the SGLT2 selectivity versus SGLT1. Compounds 16d
and 16f, with a well-balanced profile for potency and selective
SGLT2 inhibition, were nominated for further evaluations.
These two compounds almost equally enhanced the renal
glucose excretion and lowered the blood glucose in diabetic db/
db mice. As a result of the pharmacokinetic studies, it was
difference in absorption because CLtotal of both compounds is
almost the same and not as high as the hepatic blood flow of
monkeys, indicating that the hepatic first pass effects of both
the compounds can be considered low. In addition, because the
target organ of SGLT2 inhibitors is the kidney tubule and they
may act from the apical side, a greater amount of inhibitor is
expected to be excreted into the urine. In this regard, urinary
excretion amounts of the two compounds in mice and monkeys
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Figure 5. Effects of single oral administration of 16d and 16f on blood glucose level in db/db mice. (a) blood glucose AUC for 6 h period
(AUC0−6 h), (b) blood glucose level at 6 h after administration. Compounds 16d and 16f (1, 3, and 10 mg/kg) or vehicle were administered to db/
db mice by oral gavage. Blood glucose levels were measured before (0 h) and at 1, 2, 4, and 6 h after drug administration under nonfasting condition.
Data are expressed as mean SEM (n = 6). ***P < 0.001 versus vehicle-treatment group by Dunnett’s multiple comparison test.
Table 2. Pharmacokinetic Parameters of 16d and 16f after Oral and Intravenous Administration to ICR Mice and Cynomolgus
a
Monkeys
mice
monkeys
16d
16f
16d
16f
po
iv
po
iv
po
iv
po
iv
dose (mg/kg)
Cmax (μg/mL)
Tmax (h)
2.8
2.9
2.6
3.1
1.7
1.0
2.8
1.0
1.35
0.63
2.57
3.98
75
0.47
0.44
2.90
1.85
46
0.93
2.00
3.72
4.03
85
0.53
1.75
3.75
1.99
21
T1/2 (h)
2.66
4.75
2.48
4.21
3.54
2.74
4.30
3.37
AUCinf (μg·h/mL)
b
F (%)
Vss (mL/kg)
1416
634
2046
748
1409
356
1364
307
CLtotal (mL/h/kg)
c
CLrenal /CLtotal (%)
7.92
4.06
21.4
8.82
a
Plasma concentrations were measured by LC-MS/MS. PK parameters were calculated by noncompartmental analysis using WinNonlin 5.0. Data
b
c
are expressed as mean (n = 4). Oral bioavailability. CLrenal = {cumulative drug amount excreted into urine (0−8 h for mice, 8−24 h for
monkeys)}/AUC (AUC0−8 h for mice, AUC8−24 h for monkeys).
Classic or Micromass ZQ from Waters (ESI). High resonance mass
spectra (HRMS) were recorded by a Micromass Q-Tof Ultima API
mass spectrometer. Analytical HPLC was measured by a 2690/2996
from Waters or a LC2010A from Shimadzu. The measurement
conditions for HPLC were as follows: (condition A) Column: YMC-
Pack ODS-A 6.0 mm × 150 mm, 5 μm. Mobile phase: elution by
applying a gradient of from 0.1% TFA/MeCN (5%) plus 0.1% TFA/
H2O (95%) to 0.1% TFA/MeCN (100%) for 20 min, and thereafter
under the same condition [0.1% TFA/MeCN (100%)] for 5 min.
Flow Rate: 1.5 mL/min. Column temperature: room temperature.
Detection condition: total plot of the entire wavelength of 230−400
nm. (condition B) Column: YMC-Pack ODS-A 6.0 mm × 150 mm, 5
μm. Mobile phase: elution by applying a gradient of from 0.1% TFA/
MeCN (5%) plus 0.1% TFA/H2O (95%) to 0.1% TFA/MeCN
(100%) for 13 min, and thereafter under the same condition [0.1%
TFA/MeCN (100%)] for 7 min. Flow rate: 1.0 mL/min. Column
temperature: 25 °C. Detection condition: wavelength of 254 nm. All
the test compounds were determined to be >95% pure by HPLC.
(1S,3′R,4′S,5′R,6′R)-3′,4′,5′-Tris(benzyloxy)-6′-((benzyloxy)-
methyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-6-carbaldehyde (Intermediate 12). To a solution of 2-
bromo-terephthalic acid (7) (185.0 g, 755 mmol) in THF (1.85 L), a
THF solution of BH3 (1.1 M, 2.0 L) was added at 0 °C dropwise for
1.5 h and the mixture was stirred for 1 h at 40 °C. After the exothermic
reaction was over, the reaction was cooled by ice−water bath. The
reaction was quenched by dropwise addition of water. The resulting
mixture was extracted with AcOEt. The organic layer was washed with
brine and concentrated under reduced pressure to give (2-bromo-4-
hydroxymethylphenyl)methanol (148 g, 90%) as an off-white solid. 1H
found that 16d had more desirable profiles in oral
bioavailability and renal excretion than 16f. Therefore, 16d
was chosen as a clinical candidate. Compound 16d is currently
undergoing phase III clinical trials for the treatment of type 2
diabetes.
EXPERIMENTAL SECTION
■
All reactions were carried out under an argon or nitrogen atmosphere.
All solvents and reagents were purchased from commercial sources
without further drying. Silica gel 60F254 precoated plates on glass
from Merck KgaA were used for thin layer chromatography (TLC)
and preparative TLC (PTLC). Column chromatography was carried
out on Merck Silica Gel 60 (230−400 mesh) if not otherwise specified.
Preparative HPLC was carried out using a Rainin HPLC employing a
41.4 mm × 300 mm, 8 μm, Dynamax C-18 column at a flow of 49
mL/min and employing a gradient of acetonitrile/water (each
containing 0.75% TFA) typically from 5 to 95% acetonitrile over
35−40 min. Melting points (mp) were determined with a Yanagimoto
1
micro melting point apparatus. H NMR spectra were recorded on
JEOL EX-270, Varian Mercury 300, or JEOL JNM-ECP-400 or Varian
400-MR. Chemical shifts were reported in parts per million (δ)
downfield from tetramethylsilane (δH 0.00) as an internal standard.
Data are presented as follows: chemical shift (δ, ppm), integration,
multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q =
quartet, and m = multiplet), and coupling constant. 13C NMR spectra
were recorded on a Varian 400-MR (100 MHz) spectrometer. The
following internal reference was used (CD3OD, δ 49.0; CDCl3, δ
77.0). Mass spectra (MS) were measured by a Thermo Electron LCQ
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Article
NMR (270 MHz, DMSO-d6) δ: 4.47 (2H, d, J = 5.1 Hz), 4.49 (2H, d,
J = 5.1 Hz), 5.27 (1H, t, J = 6.0 Hz), 5.37 (1H, t, J = 5.7 Hz), 7.31
(1H, d, J = 7.8 Hz), 7.45−7.49 (2H, m). MS (ESI) m/z: 240 [M +
Na]+.
To a solution of the above pentaol (100 mg, 0.34 mmol) in DMSO
(0.19 mL, 2.68 mmol), chlorotrimethylsilane (114 mL, 0.91 mmol)
was added dropwise at rt and the mixture was stirred at the same
temperature for 1.5 h. To the crude product obtained by distilling off
volatile components, N-methylmorpholine (0.74 mL, 6.70 mmol),
DMAP (41 mg, 0.34 mmol), and acetic anhydride (0.32 mL, 3.35
mmol) were added sequentially and the mixture was stirred at 0 °C for
1 h. After addition of brine (1 mL) and water (1 mL), the reaction
mixture was extracted with AcOEt (10 mL). The organic layer was
washed with water (1.5 mL) and brine (1 mL), dried over Na2SO4,
and then concentrated under reduced pressure. The obtained residue
was purified by silica gel column chromatography (AcOEt/hexane =
To a solution of the above obtained diol (148 g, 682 mmol) and
triphenylmethyl chloride (TrCl) (411 g, 1.47 mol) in DMF (300 mL)
and CH2Cl2 (1.0 L), triethylamine (335 mL, 2.40 mol) and DMAP
(20 g, 163.7 mmol) were added and the mixture was stirred for 1.5 h at
50 °C. Triethylamine (38 mL, 273 mmol) and TrCl (76 g, 273 mmol)
were added, and the mixture was stirred for 4 h at 50 °C. The resulting
mixture was cooled, and then white solid was crystallized out. The
solid was filtered off and washed with Et2O−water (2:7) and Et2O and
1
1
dried in vacuo to give 8 (327.0 g, 68%) as a off-white solid. H NMR
2:5) to give 14 as a colorless amorphous (123 mg, 76%). H NMR
(300 MHz, CDCl3) δ: 4.14 (2H, s), 4.20 (2H, s), 7.22−7.34 (21H, m),
7.47−7.53 (12H, m).
(400 MHz, CDCl3) δ: 1.74 (3H, s), 2.01 (3H, s), 2.05 (1H, s), 2.08
(3H, s), 3.99−4.08 (1H, m), 4.24−4.37 (2H, m), 4.61 (2H, s), 5.15−
5.32 (3H, m), 5.57−5.65 (2H, m), 7.22−7.28 (1H, m), 7.38−7.47
(2H, m). 13C NMR (100 MHz, CDCl3) δ: 20.6, 20.6, 20.6, 20.7, 45.6,
61.9, 68.4, 70.0, 70.8, 71.7, 72.9, 108.6, 121.2, 122.9, 130.6, 136.3,
137.6, 140.3, 169.3, 169.5, 170.1, 170.7. MS (ESI) m/z: 485 [M + H]+.
General Procedures for Synthesis of Test Compounds via
Aldehyde (Method A). (1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-
[(4-methoxyphenyl)methyl]-3′,4′,5′,6′-tetrahydro-3H-spiro[2-ben-
zofuran-1,2′-pyran]-3′,4′,5′-triol (16a). To a solution of aldehyde 12
(2.01 g, 3.06 mmol) in Et2O (24 mL), 0.5 M 4-methoxyphenylmag-
nesium bromide in THF solution (12.24 mL, 6.12 mmol) was added
at 0 °C and the mixture was stirred for 2.5 h at rt. After addition of
water, the reaction mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and then evaporated
under reduced pressure to remove the solvent. The obtained residue
was purified by silica gel column chromatography (AcOEt/hexane =
1:4) to give the intermediate (2.15 g, 92%).
To a solution of the above intermediate (270 mg, 0.353 mmol) in
CH2Cl2 (2.7 mL), triethylsilane (281 μL, 1.764 mmol) and BF3·OEt2
(47 μL, 0.37 mmol) were added at −40 °C and the mixture was stirred
at the same temperature for 15 min. After addition of water, the
reaction mixture was extracted with CH2Cl2. The organic layer was
washed with brine, dried over MgSO4, and then evaporated under
reduced pressure to remove the solvent. The obtained residue was
purified by silica gel column chromatography (AcOEt/hexane = 1:10)
to give the intermediate compound (260 mg, 90%).
To a solution of 8 (255.3 mg, 0.36 mmol) in toluene (1.5 mL), 0.99
M sec-BuLi in cyclohexane solution (367 mL, 0.36 mmol) was added
dropwise at rt, and the solution was stirred for 30 min. This solution
was added dropwise at −78 °C to a solution of commercially available
9 (140 mg, 0.26 mmol) in toluene (1.5 mL), and the mixture was
stirred at the same temperature for 30 min. After addition of water, the
reaction mixture was extracted with AcOEt. The organic layer was
washed with brine, dried over MgSO4, and then evaporated under
reduced pressure to remove the solvent. The obtained residue was
purified by silica gel column chromatography (AcOEt/hexane = 1:5)
1
to give 10 (242 mg, 80%) as a mixture of diastereomers. H NMR
(300 MHz, CDCl3) δ: 3.34 (1H, t, J = 9.3 Hz), 3.46−3.51 (3H, m),
3.76 (1H, d, J = 10.8 Hz), 3.92 (1H, t, J = 9.3 Hz), 4.00−4.05 (1H, m),
4.08−4.16 (3H, m), 4.26−4.31 (3H, m), 4.41 (1H, d, J = 12.3 Hz),
4.49−4.58 (2H, m), 4.77−4.84 (3H, m), 6.71−6.76 (2H, m), 6.93−
6.97 (2H, m), 7.02−7.07 (1H, m), 7.11−7.32 (35H, m), 7.47−7.59
(12H, m), 7.69 (1H, d, J = 7.5 Hz). MS (ESI) m/z: 1184 [M + Na]+.
To a solution of the above intermediate 10 (242 mg, 0.21 mmol) in
MeCN (3 mL), triethylsilane (36 mL, 0.23 mmol) and BF3·OEt2 (29
mL, 0.23 mmol) were added at −40 °C and the mixture was stirred at
the same temperature for 1 h. After stirring at 0 °C for an additional 1
h, water was added and the reaction mixture was extracted with
CH2Cl2. The organic layer was washed with brine, dried over MgSO4,
and then evaporated under reduced pressure to remove the solvent.
The obtained residue was purified by silica gel column chromatog-
raphy (AcOEt/hexane = 1:4) to give 11 (77.5 mg, 56%) as a colorless
oil. 1H NMR (300 MHz, CDCl3) δ: 3.66 (1H, d, J = 11.1 Hz), 3.77−
3.93 (4H, m), 4.07−4.18 (4H, m), 4.40−4.63 (5H, m), 4.83−4.95
(3H, m), 5.17 (2H, s), 6.72−6.76 (2H, m), 7.05−7.35 (20H, m),
7.40−7.55 (1H, m). MS (ESI) m/z: 681 [M + Na]+.
To a solution of the above alcohol 11 (77.5 mg, 0.12 mmol) in
CH2Cl2 (1.5 mL), Dess−Martin periodinane (74.8 mg, 0.18 mmol)
was added at rt and the mixture was stirred for 30 min. After addition
of water, the reaction mixture was extracted with CH2Cl2. The organic
layer was washed with brine, dried over MgSO4, and then evaporated
under reduced pressure to remove the solvent. The obtained residue
was purified by silica gel column chromatography (AcOEt/hexane =
1:4) to give 12 as a colorless amorphous compound (25.2 mg, 33%).
1H NMR (300 MHz, CDCl3) δ: 3.61−3.66 (1H, m), 3.76−3.95 (3H,
m), 4.08−4.11 (1H, m), 4.15−4.27 (2H, m), 4.47 (1H, d, J = 12.0
Hz), 4.56 (1H, d, J = 12.0 Hz), 4.63 (2H, d, J = 10.8 Hz), 4.89 (1H, d,
J = 10.8 Hz), 4.95 (2H, s), 5.24 (2H, s), 6.75 (2H, d, J = 6.9 Hz),
7.03−7.15 (3H, m), 7.19−7.41 (16H, m), 7.53 (1H, s), 7.88 (1H, d, J
= 7.8 Hz), 9.85 (1H, s). MS (ESI) m/z: 679 [M + Na]+.
(1S,3′R,4′S,5′R,6′R)-6′-(Acetoxymethyl)-6-(chloromethyl)-
3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triyl Triacetate (Intermediate 14). To a solution of the
prepared 11 (0.59 g, 0.90 mmol) and pentamethylbenzene (1.33 g,
8.95 mmol) in CH2Cl2 (48 mL) was added 1.0 M BCl3 in CH2Cl2
solution (8.95 mL, 8.95 mmol) at −78 °C under a nitrogen stream,
and the mixture was stirred at the same temperature for 2 h. After
addition of MeOH (48 mL), the reaction mixture was warmed to rt
and evaporated under reduced pressure to remove the solvent. The
obtained residue was purified by silica gel column chromatography
(MeOH/CH2Cl2 = 1:6) to give the pentaol compound (0.18 g, 67%).
To a solution of the above intermediate (280 mg, 0.381 mmol) in
MeOH (1 mL) and AcOEt (1 mL), 10% Pd(OH)2 (28.7 mg) was
added and 2 N HCl (15.2 μL) was further added. Under a hydrogen
atmosphere, the reaction mixture was stirred for 45 min at rt and then
filtered to remove the catalyst. After distilling off the solvent under
reduced pressure, the obtained residue was purified by silica gel
column chromatography (MeOH/CH2Cl2 = 1:10) to give 16a (114
mg, 98%) as a colorless amorphous compound; tR = 9.6 min
1
(condition A). H NMR (400 MHz, CD3OD) δ: 3.42−3.49 (1H, m),
3.63−3.67 (1H, m), 3.74 (3H, s), 3.74−3.87 (4H, m), 3.93 (2H, s),
5.07 (1H, d, J = 12.3 Hz), 5.13 (1H, d, J = 12.4 Hz), 6.80−6.83 (2H,
m), 7.12−7.20 (2H, m), 7.21−7.22 (3H, m). 13C NMR (100 MHz,
CD3OD) δ: 41.8, 55.6, 62.8, 71.9, 73.4, 74.9, 76.2, 76.4, 111.6, 114.9,
121.8, 123.5, 130.9, 131.1, 134.5, 139.8, 140.2, 142.8, 159.6. MS (ESI):
389 [M + H]+. HRMS (ESI), m/z calcd for C21H25O7 [M + H]+
389.1595, found 389.1594.
General Procedures for Synthesis of Test Compounds via
Benzyl Chloride (Method B). (1S,3′R,4′S,5′S,6′R)-6-[(4-
Ethoxyphenyl)methyl]-6′-(hydroxymethyl)-3′,4′,5′,6′-tetrahydro-
3H-spiro[2-benzofuran-1,2′-pyran]-3′,4′,5′-triol (16b). To a solution
of benzyl chloride 14 (200 mg, 0.413 mmol) in toluene (2.0 mL),
triphenylphosphine (5.41 mg, 0.021 mmol), palladium acetate (2.36
mg, 0.010 mmol), 4-ethoxyphenylboronic acid (0.103 g, 0.619 mmol),
and potassium phosphate (0.181 g, 0.825 mmol) were added and the
mixture was heated at 80 °C. After the mixture was stirred for 15 h,
water and AcOEt were added, and the mixture was washed with brine.
The organic layer was dried over MgSO4, filtered, and then evaporated
under reduced pressure. The obtained residue was purified by silica gel
column chromatography (AcOEt/hexane = 1:2) to give the titled
1
compound (0.117 g, 50%) as a colorless amorphous compound. H
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NMR (CDCl3) δ 1.39 (3H, t, J = 7.1 Hz), 1.71 (3H, s), 2.00 (3H, s),
2.05 (3H, s), 2.07 (3H, s), 3.88−4.09 (5H, m), 4.21−4.36 (2H, m),
5.07−5.24 (2H, m), 5.25−5.33 (1H, m), 5.55−5.65 (2H, m), 6.79−
6.85 (2H, m), 7.01−7.09 (2H, m), 7.11−7.19 (2H, m), 7.23 (1H, s).
To a solution of the above compound (0.210 g, 0.368 mmol) in
MeOH (5 mL), K2CO3 (100 mg, 0.709 mmol) was added and the
mixture was stirred for 12 h at rt. After addition of water, the reaction
mixture was extracted with AcOEt. The organic layer was washed with
brine and dried over MgSO4. After filtration, the solvent was
concentrated under reduced pressure. The obtained residue was
isopropylphenyl lithium generated by treatment of n-BuLi with 1-
bromo-4-isopropylbenzene following a similar procedure to that
described in Method A (yield 59%, colorless amorphous); tR = 12.1
min (condition A). 1H NMR (400 MHz, CD3OD) δ: 1.21 (6H, d, J =
6.9 Hz), 2.82−2.86 (1H, m), 3.43−3.47 (1H, m), 3.64 (1H, dd, J =
12.1, 5.8 Hz), 3.74−3.81 (4H, m), 3.95 (2H, s), 5.07 (1H, d, J = 12.3
Hz), 5.14 (1H, d, J = 12.3 Hz), 7.09−7.13 (4H, m), 7.17−7.24 (3H,
m). 13C NMR (100 MHz, CD3OD) δ: 24.5, 24.5, 35.0, 42.2, 62.8,
71.8, 73.4, 74.9, 76.2, 76.4, 111.6, 121.8, 123.6, 127.5, 129.9, 131.2,
139.9, 140.2, 142.6, 147.8. MS (ESI): 401 [M + H]+. HRMS (ESI), m/
z calcd for C23H29O6 [M + H]+ 401.1959, found 401.1956.
purified by silica gel column chromatography (MeOH/CH2Cl2
=
1:15) to give the titled compound (42.5 mg, 29%) as a colorless
(1S,3′R,4′S,5′S,6′R)-6-[(4-Cyclopropylphenyl)methyl]-6′-(hydroxy-
methyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16g). Compound 13g was prepared from 12 and 4-
cyclopropylphenyl magnesium bromide generated by treatment of
magnesium with 1-bromo-4-cyclopropylbenznene following a similar
procedure to that described in Method A (yield 73%). Then, to a
solution of 13g (2.55 g, 3.36 mmol) and pentamethylbenzene (4.99 g,
33.7 mmol) in CH2Cl2 (185 mL) was added 1.0 M BCl3 in CH2Cl2
solution (33.3 mL, 33.3 mmol) at −78 °C under a nitrogen stream,
and the mixture was stirred at the same temperature for 2 h. After
addition of MeOH (185 mL), the reaction mixture was warmed to rt
and evaporated under reduced pressure to remove the solvent. The
obtained residue was purified by silica gel column chromatography
(MeOH/CH2Cl2 = 1:10) to give 16g (0.67 g, 50%) as a colorless
1
amorphous compound; tR = 8.1 min (condition B). H NMR (400
MHz, CD3OD) δ: 1.35 (3H, t, J = 6.9 Hz), 3.42−3.48 (1H, m), 3.63−
3.67 (1H, m), 3.74−3.83 (4H, m), 3.92 (2H, s), 3.98 (2H, q, J = 6.9
Hz), 5.07 (1H, d, J = 12.3 Hz), 5.13 (1H, d, J = 12.3 Hz), 6.77−6.83
(2H, m), 7.07−7.14 (2H,m), 7.17−7.22 (3H, m). 13C NMR (100
MHz, CD3OD) δ: 15.2, 41.8, 62.8, 64.4, 71.8, 73.4, 74.9, 76.2, 76.4,
111.6, 115.5, 121.8, 123.5, 130.9, 131.1, 134.5, 139.8, 140.2, 142.8,
158.8. MS (ESI): 403 [M + H]+. HRMS (ESI), m/z calcd for
C22H27O7 [M + H]+ 403.1751, found 403.1748.
(1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-[(4-methylphenyl)-
methyl]-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16c). Compound 16c was prepared from 14 and 4-
methylphenylboronic acid following a similar procedure to that
described in Method B (yield 32%, colorless amorphous); tR = 8.1 min
1
amorphous compound; tR = 11.4 min (condition A). H NMR (400
1
(condition B). H NMR (400 MHz, CD3OD) δ: 2.27 (3H, s), 3.42−
MHz, CD3OD) δ: 0.59−0.63 (2H, m), 0.88−0.92 (2H, m), 1.81−1.87
(1H, m), 3.42−3.47 (1H, m), 3.61 (1H, dd, J = 11.8, 5.6 Hz), 3.74−
3.83 (4H, m), 3.94 (2H, s), 5.06 (1H, d, J = 12.3 Hz), 5.13 (1H, d, J =
12.3 Hz), 6.94−6.97 (2H, m), 7.05−7.08 (2H, m), 7.17−7.22 (3H,
m). 13C NMR (100 MHz, CD3OD) δ: 9.4, 9.4, 15.8, 42.2, 62.8, 71.8,
73.4, 74.9, 76.2, 76.4, 111.6, 121.8, 123.6, 126.7, 129.8, 131.1, 139.4,
139.8, 140.2, 142.6, 143.0. MS (ESI): 399 [M + H]+. HRMS (ESI), m/
z calcd for C23H27O6 [M + H]+ 399.1802, found 399.1800.
3.47 (1H, m), 3.63−3.67 (1H, m), 3.73−3.83 (4H, m), 3.94 (2H, s),
5.07 (1H, d, J = 12.3 Hz), 5.13 (1H, d, J = 12.5 Hz), 7.04−7.10 (4H,
m), 7.17−7.22 (3H, m). 13C NMR (100 MHz, CD3OD) δ: 21.0, 42.2,
62.8, 71.8, 73.4, 74.9, 76.2, 76.4, 111.6, 121.8, 123.6, 129.9, 130.1,
131.1, 136.6, 139.4, 139.9, 140.2, 142.6. MS (ESI): 395 [M + Na]+.
HRMS (ESI), m/z calcd for C21H25O6 [M + H]+ 373.1646, found
373.1647.
(1S,3′R,4′S,5′S,6′R)-6-[(4-Ethylphenyl)methyl]-6′-(hydroxymeth-
yl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16d, CSG452, Tofogliflozin). Compound 16d was
prepared from 12 and 0.5 M 4-ethylphenylmagnesium bromide in
THF solution following a similar procedure to that described in
Method A (yield 66%, colorless amorphous); tR = 11.4 min (condition
(1S,3′R,4′S,5′S,6′R)-6-[(4-Hydroxyphenyl)methyl]-6′-(hydroxy-
methyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16h). Compound 16h was prepared from 14 and 4-
benzyloxyphenylboronic acid following a similar procedure to that
described in Method B (yield 12%, colorless amorphous); tR = 6.3 min
1
(condition B). H NMR (400 MHz, CD3OD) δ: 3.42−3.52 (1H, m),
1
A). H NMR (400 MHz, CD3OD) δ: 1.20 (3H, t, J = 7.6 Hz), 2.58
3.63−3.67 (1H, m), 3.74−3.83 (4H, m), 3.90 (2H, s), 5.07 (1H, d, J =
12.5 Hz), 5.13 (1H, d, J = 12.5 Hz), 6.66−6.70 (2H, m), 6.99−7.02
(2H, m), 7.17−7.22 (3H, m). 13C NMR (100 MHz, CD3OD) δ: 41.8,
62.8, 71.9, 73.4, 74.9, 76.2, 76.4, 111.6, 116.2, 121.8, 123.5, 130.9,
131.1, 133.3, 139.8, 140.1, 143.1, 156.7. MS (ESI): 397 [M + Na]+.
HRMS (ESI), m/z calcd for C20H23O7 [M + H]+ 375.1438, found
375.1439.
(2H, q, J = 7.6 Hz), 3.42−3.47 (1H, m), 3.63−3.67 (1H, m), 3.75−
3.88 (4H, m), 3.95 (2H, s), 5.06 (1H, d, J = 12.3 Hz), 5.12 (1H, d, J =
12.5 Hz), 7.07−7.14 (4H, m), 7.17−7.23 (3H, m). 13C NMR (100
MHz, CD3OD) δ: 16.3, 29.4, 42.3, 62.8, 71.9, 73.4, 74.9, 76.2, 76.4,
111.6, 121.8, 123.6, 128.9, 129.9, 131.1, 139.7, 139.9, 140.2, 142.6,
143.2. MS (ESI): 387 [M + H]+. HRMS (ESI), m/z calcd for
C22H27O6 [M + H]+ 387.1802, found 387.1801.
(1S,3′R,4′S,5′S,6′R)-6-[(4-Fluorophenyl)methyl]-6′-(hydroxymeth-
yl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16i). Compound 16i was prepared from 14 and 4-
fluorophenylboronic acid following a similar procedure to that
described in Method B (yield 26%, colorless amorphous); tR = 7.7
min (condition B). 1H NMR (400 MHz, CD3OD) δ: 3.42−3.47 (1H,
m), 3.63−3.67(1H, m), 3.74−3.83 (4H, m), 3.98 (2H, s), 5.07 (1H, d,
J = 12.5 Hz), 5.13 (1H, d, J = 12.5 Hz), 6.95−7.00 (2H, m), 7.19−7.23
(5H, m). 13C NMR (100 MHz, CD3OD) δ: 41.7, 62.8, 71.8, 73.4,
74.9, 76.2, 76.4, 111.6, 116.0 (d, J = 21.7 Hz), 122.0, 123.6, 131.1,
131.5 (d, J = 8.2 Hz), 138.5 (d, J = 3.0 Hz), 140.1, 140.3, 142.2, 162.9
(d, J = 242.3 Hz). MS (ESI): 399 [M + Na]+. HRMS (ESI), m/z calcd
for C20H22FO6 [M + H]+ 377.1395, found 377.1396.
(1S,3′R,4′S,5′S,6′R)-6-[(4-Chlorophenyl)methyl]-6′-(hydroxy-
methyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16j). Compound 16j was prepared from 14 and 4-
chlorophenylboronic acid following a similar procedure to that
described in Method B (yield 25%, colorless amorphous); tR = 8.3
min (condition B). 1H NMR (400 MHz, CD3OD) δ: 3.43−3.47 (1H,
m), 3.63−3.68 (1H, m), 3.75−3.84 (4H, m), 3.99 (2H,s), 5.07 (1H, d,
J = 12.5 Hz), 5.13 (1H, d, J = 12.5 Hz), 7.17−7.26 (7H, m). 13C NMR
(100 MHz, CD3OD) δ: 41.8, 62.8, 71.8, 73.4, 74.9, 76.2, 76.4, 111.6,
122.0, 123.7, 129.5, 131.1, 131.5, 132.9, 140.2, 140.4, 141.4, 141.8. MS
(1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-[(4-propylphenyl)-
methyl]-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16e). Intermediate 13g (1.68 g, 2.21 mmol) prepared
from 12 and 4-cyclopropylphenylmagnesium bromide generated by
treatment of magnesium with 1-bromo-4-cyclopropylbenznene was
dissolved in MeOH (10 mL) and AcOEt (10 mL). To this solution,
10% Pd(OH)2 (0.21 g) was added. Under a hydrogen atmosphere, the
reaction mixture was stirred for 1.5 h at rt and then filtered to remove
the catalyst. After distilling off the solvent under reduced pressure, the
obtained residue was purified by silica gel column chromatography
(MeOH/CH2Cl2 = 1:10) to give 16e (0.65 g, 73%) as a colorless
1
amorphous compound; tR = 12.3 min (condition A). H NMR (400
MHz, CD3OD) δ: 0.92 (3H, t, J = 7.4 Hz), 1.56−1.65 (2H, m), 2.51−
2.55 (2H, m), 3.42−3.47 (1H, m), 3.63−3.67 (1H, m), 3.74−3.83
(4H, m), 3.96 (2H, s), 5.07 (1H, d, J = 12.3 Hz), 5.13 (1H, d, J = 12.5
Hz), 7.06−7.14 (4H, m), 7.18−7.23 (3H, m). 13C NMR (100 MHz,
CD3OD) δ: 14.1, 25.8, 38.7, 42.3, 62.8, 71.9, 73.4, 74.9, 76.2, 76.4,
111.6, 121.8, 123.6, 129.6, 129.8, 131.2, 139.7, 139.9, 140.2, 141.5,
142.6. MS (ESI): 401 [M + H]+. HRMS (ESI), m/z calcd for
C23H29O6 [M + H]+ 401.1959, found 401.1956.
(1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-[(4-isopropylphenyl)-
methyl]-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-
3′,4′,5′-triol (16f). Compound 16f was prepared from 12 and 4-
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(ESI): 393 [M + H]+. HRMS (ESI), m/z calcd for C20H22ClO6 [M +
H]+ 393.1099, found 393.1102.
Undissolved material was collected by filtration to obtain (4-
acetoxymethyl-2-bromo-5-chloro)benzyl acetate (5.9 g, 39%). 1H
NMR (300 MHz, CDCl3) δ: 2.15 (3H, s), 2.16 (3H, s), 5.14 (2H,
s), 5.16 (2H, s), 7.42 (1H, s), 7.61 (1H, s).
4-(((1S,3′R,4′S,5′S,6′R)-3′,4′,5′-Trihydroxy-6′-(hydroxymethyl)-
3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-6-yl)-
methyl)benzonitrile (16k). Compound 16k was prepared from 14 and
4-cyanophenylboronic acid following a similar procedure to that
described in Method B (yield 43%, colorless amorphous); tR = 7.2 min
To a solution of the above intermediate (28.6 g, 85.2 mmol) in a
mixture of THF (250 mL), EtOH (250 mL), and water (125 mL) was
added potassium hydroxide (14.3 g, 256 mmol), and the resultant
mixture was stirred for 3 h at 80 °C. The reaction mixture was cooled
to rt, and then solvent was removed by distillation under reduced
pressure. To the resulting residue were added water (200 mL) and
AcOEt (100 mL), and this mixture was stirred for 1 h at rt.
Undissolved material was collected by filtration and dried to obtain (2-
bromo-5-chloro-4-hydroxymethylphenyl)methanol (19) (20.7 g,
1
(condition B). H NMR (400 MHz, CD3OD) δ: 3.43−3.47 (1H, m),
3.63−3.68(1H, m), 3.75−3.84 (4H, m), 4.09 (2H, s), 5.08 (1H, d, J =
12.5 Hz), 5.14 (1H, d, J = 12.5 Hz), 7.24−7.25 (3H, m), 7.40 (2H, d, J
= 8.4 Hz), 7.61−7.63 (2H, m). 13C NMR (100 MHz, CD3OD) δ:
42.5, 62.7, 71.8, 73.4, 74.9, 76.2, 76.4, 111.0, 111.6, 119.8, 122.2, 123.9,
131.0, 131.3, 133.4, 140.5, 140.6, 140.8, 148.6. MS (ESI): 406 [M +
Na]+. HRMS (ESI), m/z calcd for C21H22NO6 [M + H]+ 384.1442,
found 384.1446.
1
97%). H NMR (300 MHz, CD3OD) δ: 4.61 (2H, s), 4.66 (2H, s),
7.52 (1H, s), 7.70 (1H, s).
(1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-[(4-(trifluoromethyl)-
phenyl)methyl]-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (16l). Compound 16l was prepared from 14 and
4-trifluoromethylphenylboronic acid following a similar procedure to
that described in Method B (yield 57%, colorless amorphous); tR = 8.7
min (condition B). 1H NMR (400 MHz, CD3OD) δ: 3.43−3.47 (1H,
m), 3.63−3.68 (1H, m), 3.75−3.83 (4H, m), 4.10 (2H,s), 5.08 (1H, d,
J = 12.5 Hz), 5.14 (1H, d, J = 12.5 Hz), 7.22−7.26 (3H, m), 7.39−7.42
(2H, m), 7.55−7.57 (2H, m). 13C NMR (100 MHz, CD3OD) δ: 42.3,
62.8, 71.8, 73.4, 74.9, 76.2, 76.4, 111.6, 122.1, 123.8, 125.8 (q, J =
271.5 Hz), 126.3 (q, J = 3.8 Hz), 129.4 (q, J = 31.5 Hz), 130.5, 131.2,
140.4, 140.5, 141.2, 147.3. MS (ESI): 427 [M + H]+. HRMS (ESI), m/
z calcd for C21H22F3O6 [M + H]+ 427.1363, found 427.1365.
(1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-[(4-(trifluoromethoxy)-
phenyl)methyl]-3″,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (16m). Compound 16m was prepared from 14
and 4-trifluoromethoxyphenylboronic acid following a similar
procedure to that described in Method B (yield 32%, colorless
amorphous); tR = 8.9 min (condition B).; 1H NMR (400 MHz,
CD3OD) δ: 3.43−3.48 (1H, m), 3.64−3.68 (1H, m), 3.75−3.84 (4H,
m), 4.03 (2H, s), 5.08 (1H, d, J = 12.5 Hz), 5.14 (1H, d, J = 12.5 Hz),
7.14−7.32 (7H, m). 13C NMR (100 MHz, CD3OD) δ: 41.8, 62.8,
71.8, 73.4, 74.9, 76.2, 76.4, 111.6, 122.0 (q, J = 256.5 Hz), 122.1, 122.1,
123.7, 131.2, 131.4, 140.2, 140.4, 141.7, 142.0, 148.9 (q, J = 2.2 Hz).
MS (ESI): 443 [M + H]+. HRMS (ESI), m/z calcd for C21H22F3O7
[M + H]+ 443.1312, found 443.1314.
To a solution (500 mL) of 19 (20.7 g, 82.3 mmol) in anhydrous
THF were added 2-methoxypropene (78.8 mL, 823.1 mmol) and
pyridinium p-toluenesulfonate (207 mg, 0.823 mmol) at 0 °C, and the
resultant mixture was stirred for 2 h. Aqueous potassium carbonate
solution (1 M, 200 mL) was added thereto, and then the resultant
mixture was extracted with AcOEt (800 mL) containing Et3N (2.5
mL). The organic layer was washed with water (500 mL) and brine
(500 mL) and then dried over MgSO4. The solvent was then removed
by distillation under reduced pressure to obtain 1-bromo-4-chloro-2,5-
1
bis[(1-methoxy-1-methyl)ethoxymethyl]benzene (33.2 g, 100%). H
NMR (300 MHz, CDCl3) δ: 1.45 (12H, s), 3.22 (6H, s), 4.48 (2H, s),
4.53 (2H, s), 7.51 (1H, s), 7.68 (1H, s).
To a solution (500 mL) of the above intermediate (24.7 g, 62.53
mmol) in anhydrous THF, 1.6 M n-BuLi in hexane solution (39.1 mL,
62.53 mmol) was added dropwise over 5 min at −78 °C under a
nitrogen atmosphere and the resultant mixture was stirred under the
same condition for 20 min. A solution of prepared 2,3,4,6-tetrakis-O-
trimethylsilyl-D-glucono-1,5-lactone39 (26.54 g, 56.85 mmol) in THF
(40 mL) was then added dropwise over 5 min to the resultant mixture.
The reaction mixture was stirred for 1 h, and then water was added
thereto. The resultant mixture was extracted with Et2O. The resultant
organic layer was washed with water and brine and then dried over
MgSO4. The solvent was then removed by distillation under reduced
pressure. The obtained residue (46.97 g) was dissolved in a mixed
solvent of THF (94 mL) and MeOH (47 mL), and p-toluenesulfonic
acid (2.16 g) was added thereto. The mixture was stirred at rt for 15 h
and then cooled with ice. tert-Butyl methyl ether (188 mL) was added
thereto, and the precipitate was collected by filtration. The obtained
solid was dried under reduced pressure to obtain (1S,3′R,4′S,5′S,6′R)-
5-chloro-3′,4′,5′,6′-tetrahydro-6,6′-bis(hydroxymethyl)-spiro[2-benzo-
furan-1(3H),2′-[2H]pyran]-3′,4′,5′-triol (12.7 g, 67%). 1H NMR (300
MHz, CD3OD) δ: 3.44−3.50 (1H, m), 3.63−3.84 (5H, m), 4.71 (2H,
s), 5.07 (1H, d, J = 12.3 Hz), 5.13 (1H, d, J = 12.3 Hz), 7.33 (1H, s),
7.55 (1H, s).
To a solution of the above pentaol (1.0 g, 3.0 mmol) in DMSO (1.7
mL, 24.0 mmol) was added chlorotrimethylsilane (1.1 mL, 8.4 mmol)
at rt, and the resultant mixture was stirred under this condition for 4 h.
The reaction mixture was concentrated under reduced pressure, and
the obtained residue was dried under reduced pressure for 15 h. To a
solution (20 mL) of the obtained residue in THF were added N-
methylmorpholine (3.7 mL, 30 mmol) and DMAP (385 mg, 3.15
mmol) at 0 °C, and Ac2O (1.7 mL, 18 mmol) was then added
dropwise to the resultant mixture. The reaction mixture was stirred
under this condition for 30 min and then stirred for another 30 min at
rt. Excess aqueous phosphoric acid was added thereto, and the
resultant mixture was extracted with AcOEt. The organic layer was
washed with water and brine, and then dried over MgSO4. The solvent
was then removed by distillation under reduced pressure. The
resulting residue was purified by silica gel column chromatography
(AcOEt/hexane = 1:1) to obtain (1S,3′R,4′S,5′R,6′R)-6′-(acetox-
ymethyl)-6-(chloromethyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzo-
furan-1,2′-pyran]-3′,4′,5′-triyl triacetate (840 mg, 54%). 1H NMR
(300 MHz, CDCl3) δ: 1.77 (3H, s), 2.01 (3H, s), 2.05 (3H, s), 2.08
(3H, s), 4.01−4.06 (1H m), 4.25−4.33 (2H, m), 4.70 (2H, d, J = 2.3
(1S,3′R,4′S,5′S,6′R)-6′-(Hydroxymethyl)-6-[(4-(methylthio)-
phenyl)methyl]-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (16n). Compound 16n was prepared from 14 and
4-(methylthio)phenylboronic acid following a similar procedure to
that described in Method B (yield 30%, colorless amorphous); tR = 8.3
1
min (condition B). H NMR (400 MHz, CD3OD) δ: 2.42 (3H, s),
3.42−3.47 (1H, m), 3.63−3.67 (1H, m), 3.74−3.83 (4H, m), 3.96
(2H, s), 5.07 (1H, d, J = 12.5 Hz), 5.13 (1H, d, J = 12.5 Hz), 7.12−
7.23 (7H, m). 13C NMR (100 MHz, CD3OD) δ: 16.0, 42.0, 62.8, 71.8,
73.4, 74.9, 76.2, 76.4, 111.6, 121.9, 123.6, 128.1, 130.5, 131.1, 137.4,
139.5, 140.0, 140.3, 142.2. MS (ESI): 405 [M + H]+. HRMS (ESI), m/
z calcd for C21H25O6S [M + H]+ 405.1366, found 405.1366.
(1S,3′R,4′S,5′S,6′R)-3′,4′,5′-Tri(acetoxy)-6′-acetoxymethyl-5-
chloro-6-[(4-ethylphenyl)methyl]-3′,4′,5′,6′-tetrahydrospiro-
[isobenzofuran-1(3H),2′-[2H]pyran] (20). To a solution (45 mL) of
1-bromo-4-chloro-2,5-dimethylbenzene 18 (10.0 g, 45.5 mmol) in
AcOEt was added N-bromosuccinimide (NBS) (21.0 g, 118.4 mmol)
and 2,2′-azobis(isobutyronitrile) (AIBN) (300 mg), and the resultant
mixture was stirred for 20 min at reflux temperature. The reaction
mixture was cooled to rt, and then AcOEt was added thereto. The
resultant mixture was washed with water and brine. The organic layer
was dried over MgSO4, and the solvent was then removed by
distillation under reduced pressure. The obtained crude product (20.8
g) was dissolved in DMF (100 mL), and AcONa (11.2 g, 136.5 mmol)
was added thereto. The resultant mixture was stirred for 3 h at 80 °C.
The reaction mixture was cooled to rt, and then CH2Cl2 was added
thereto. The mixture was washed with water and brine. The organic
layer was dried over MgSO4, and the solvent was then removed by
distillation under reduced pressure. To the obtained residue was added
AcOEt (100 mL), and this solution was stirred for 15 h at rt.
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Hz), 5.13 (1H, d, J = 13.4 Hz), 5.20 (1H, d, J = 13.4 Hz), 5.25−5.31
(1H, m), 5.55−5.64 (2H, m), 7.32 (1H, s), 7.51 (1H, s).
(3.35 mL) was stirred for 0.5 h at rt. To the mixture was then added
trimethylsilylacetylene (0.26 mL, 1.84 mmol), and the resultant
mixture was stirred for 2 h at 90 °C. The reaction mixture was
concentrated under reduced pressure, and the resulting residue was
purified by silica gel flash column chromatography (AcOEt/hexane =
1:2) to obtain the intermediate (96 mg, 81%). To a solution of the
above compound (101 mg, 0.155 mmol) in MeOH (1 mL) was added
K2CO3 (14 mg), and the resultant mixture was stirred for 1.5 h at rt.
The solvent was then removed by distillation under reduced pressure.
The obtained residue was purified by silica gel column chromatog-
raphy (MeOH/CH2Cl2 = 15:85) to obtain 21q (23 mg, 36%) as a
colorless amorphous compund; tR = 11.6 min (condition A). 1H NMR
(400 MHz, CD3OD) δ: 1.19 (3H, t, J = 7.6 Hz), 2.58 (2H, q, J = 7.6
Hz), 3.40−3.45 (1H, m), 3.62−3.82 (6H, m), 4.12 (1H, d, J = 14.8
Hz), 4.19 (1H, d, J = 14.8 Hz), 5.06 (1H, d, J = 12.7 Hz), 5.12 (1H, d,
J = 12.7 Hz), 7.07 (2H, d, J = 8.0 Hz), 7.14 (2H, d, J = 8.0 Hz), 7.21
(1H, s), 7.42 (1H, s). 13C NMR (100 MHz, CD3OD) δ: 16.2, 29.4,
40.4, 62.7, 71.7, 73.1, 74.8, 76.2, 76.3 76.3, 82.9, 111.5, 124.3, 124.4,
126.3, 128.8, 129.9, 138.9, 140.2, 140.8, 143.2, 144.9. MS (ESI): 411
[M + H]+. HRMS (ESI), m/z calcd for C24H27O6 [M + H]+ 411.1802,
found 411.1802.
(1S,3′R,4′S,5′S,6′R)-6-[(4-Ethylphenyl)methyl]-6′-(hydroxymeth-
yl)-5-phenyl-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (21r). A solution of 20 (67 mg, 0.114 mmol),
bis(dibenzylideneacetone)palladium(0) (0.7 mg, 0.001 mmol),
1,2,3,4,5-pentaphenyl-1′-(di-t-butylphosphino)ferrocene (CTC-Q-
PHOS) (1.7 mg, 0.002 mmol), phenylboronic acid (21 mg, 0.172
mmol), and KF (20 mg, 0.344 mmol) in THF (0.22 mL) was stirred
for 0.5 h at 100 °C under microwave irradiation. The reaction mixture
was concentrated under reduced pressure, and the obtained residue
was purified by silica gel column chromatography (AcOEt/hexane =
1:2), to obtain the intermediate (77 mg, quant). To a solution of the
above compound (77 mg, 0.114 mmol) in MeOH (2 mL) was added
K2CO3 (34 mg, 0.246 mmol), and the resultant mixture was stirred for
1.5 h at rt. The solvent was then removed by distillation under reduced
pressure. The obtained residue was purified by silica gel column
chromatography (MeOH/CH2Cl2 = 1:9) and preparative HPLC to
obtain 21r (24 mg, 45%) as a colorless amorphous compound; tR =
13.5 min (condition A). 1H NMR (400 MHz, CD3OD) δ: 1.17 (3H, t,
J = 7.6 Hz), 2.55 (2H, q, J = 7.6 Hz), 3.43−3.47 (1H, m), 3.65−3.69
(1H, m), 3.76−3.86 (4H, m), 3.90 (2H, d, J = 2.4 Hz), 5.12 (1H, d, J =
12.7 Hz), 5.19 (1H, d, J = 12.7 Hz), 6.82 (2H, d, J = 8.0 Hz), 6.98
(2H, d, J = 8.0 Hz), 7.13 (1H, s), 7.16−7.18 (2H, m), 7.26 (1H, s),
7.32−7.36 (3H, m). 13C NMR (100 MHz, CD3OD) δ: 16.2, 29.4,
39.7, 62.9, 71.9, 73.4, 75.0, 76.3, 76.5, 111.7, 123.4, 125.0, 128.2, 128.7,
129.1, 129.8, 130.3, 139.3, 139.6, 139.7, 140.2, 142.9, 143.0, 145.0. MS
(ESI): 463 [M + H]+. HRMS (ESI), m/z calcd for C28H31O6 [M +
H]+ 463.2115, found 463.2114.
((1S,3′R,4′S,5′S,6′R)-6-[(4-Ethylphenyl)methyl]-3′,4′,5′-trihy-
droxy-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-pyran]-6′-
yl)methyl Acetate (17) for Single Crystal Analysis. To a solution of
16d (CSG452) (100 mg, 0.26 mmol) in 2,4,6-trimethylpyridine (0.68
mL) was added acetyl chloride (74 μL, 1.04 mmol) at −10 °C, and the
resulting mixture was stirred for 2 h at rt. Water was added thereto,
and the mixture was extracted with AcOEt. The organic layer was
washed with brine and then dried over MgSO4. The solvent was then
removed by distillation under reduced pressure. The obtained residue
was purified by silica gel column chromatography (MeOH/CH2Cl2 =
1:10) to obtain monoacetylated compound 17 (81 mg, 73%) as a
colorless solid. Single crystals of monohydrate 17 were obtained by
recrystallization from MeCN−H2O as the solvent; mp 168−169 °C.
1H NMR (400 MHz, CD3OD) δ: 1.19 (3H, t, J = 7.6 Hz), 1.99 (3H,
s), 2.58 (2H, q, J = 7.6 Hz), 3.42−3.47 (1H, m), 3.73−3.76 (2H, m),
3.95−4.00 (3H, m), 4.15 (1H, dd, J = 5.6, 12.0 Hz), 4.31 (1H, dd, J =
2.0, 11.9 Hz), 5.07 (1H, d, J = 12.5 Hz), 5.12 (1H, d, J = 12.5 Hz),
7.08−7.22 (6H, m). 13C NMR (100 MHz, CD3OD) δ: 16.2, 20.7,
29.4, 42.1, 65.1, 71.8, 73.5, 73.5, 74.8, 76.2, 111.6, 121.8, 123.4, 128.9,
129.9, 131.2, 139.5, 139.6, 139.8, 142.7, 143.1, 172.9. MS (ESI): 429
[M + H]+.
To a solution of the above compound (300 mg, 0.58 mmol) in
DMF (2.85 mL) and water (0.15 mL) were added 4-ethyl-
phenylboronic acid (174 mg, 1.16 mmol), tetrakis-
(triphenylphosphine)palladium (34 mg, 0.029 mmol), sodium
carbonate (184 mg, 1.74 mmol), and tetrabutylammonium bromide
(39 mg, 0.116 mmol), and the resultant mixture was stirred for 15 h at
85 °C. The reaction mixture was cooled to rt, and then water (10 mL)
was added thereto. The resultant mixture was extracted with AcOEt
(100 mL). The organic layer was washed with water and brine and
then dried over MgSO4. The solvent was then removed by distillation
under reduced pressure. The obtained residue was purified by silica gel
column chromatography (AcOEt/hexane = 1:3) to obtain 20 (236 mg,
69%) as a colorless amorphous compound. 1H NMR (300 MHz,
CDCl3) δ: 1.22 (3H, t, J = 7.6 Hz), 1.75 (3H, s), 2.00 (3H, s), 2.04
(3H, s), 2.06 (3H, s), 2.62 (2H, q, J = 7.6 Hz), 4.01−4.13 (3H, m),
4.22−4.33 (2H, m), 5.09 (1H, d, J = 12.6 Hz), 5.17 (1H, d, J = 13.1
Hz), 5.23−5.30 (1H, m), 5.52−5.63 (2H, m), 7.07−7.14 (4H, m),
7.26 (2H, s).
(1S,3′R,4′S,5′S,6′R)-5-Chloro-6-[(4-ethylphenyl)methyl]-6′-(hy-
droxymethyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (21o). To a solution of 20 (236 mg, 0.40 mmol)
in MeOH (16 mL) was added potassium carbonate (166 mg, 1.20
mmol), and the resultant mixture was stirred for 1 h at rt. Water (5
mL) was added thereto, and the resultant mixture was extracted with
AcOEt (50 mL). The organic layer was washed with brine and then
dried over MgSO4. The solvent was then removed by distillation under
reduced pressure. The resulting residue was purified by silica gel
column chromatography (MeOH/CH2Cl2 = 1:15) to obtain 21o (110
mg, 65%) as a colorless amorphous compound; tR = 11.2 min
1
(condition B). H NMR (400 MHz, CD3OD) δ: 1.20 (3H, t, J = 7.6
Hz), 2.59 (2H, q, J = 7.6 Hz), 3.38−3.46 (1H, m), 3.63−3.81 (5H, m),
4.04−4.13 (2H, m), 5.07 (1H, d, J = 12.7 Hz), 5.12 (1H, d, J = 12.7
Hz), 7.10 (4H, s), 7.25 (1H, s), 7.36 (1H, s). 13C NMR (100 MHz,
CD3OD) δ: 16.2, 29.5, 39.8, 62.7, 71.7, 72.9, 74.9, 76.3, 76.4, 111.4,
123.1, 125.9, 128.9, 129.9, 136.2, 137.9, 139.2, 139.6, 142.1, 143.4. MS
(ESI): 443 [M + Na]+, 863 [2M + Na]+. HRMS (ESI), m/z calcd for
C22H26ClO6 [M + H]+ 421.1412, found 421.1415.
(1S,3′R,4′S,5′S,6′R)-6-[(4-Ethylphenyl)methyl]-6′-(hydroxymeth-
yl)-5-methyl-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (21p). A solution of 20 (138 mg, 0.23 mmol),
bis(dibenzylideneacetone)palladium(0) (Pd(dba)2) (132 mg, 0.23
mmol), tri-tert-butylphosphonium tetrafluoroborate (t-Bu3P·BF4) (67
mg, 0.23 mmol), methylboronic acid (28 mg, 0.46 mmol), and K3PO4
(146 mg, 0.69 mmol) in xylene (2.8 mL) was stirred for 15 h at 145
°C. The reaction mixture was concentrated under reduced pressure,
and the obtained residue was purified by silica gel column
chromatography (AcOEt/hexane = 2:5) and preparative HPLC to
obtain intermediate (37 mg, 28%). To a solution of the above
compound (37 mg, 0.065 mmol) in MeOH (1 mL) was added K2CO3
(9 mg, 0.065), and the resultant mixture was stirred for 1 h at rt. The
solvent was then removed by distillation under reduced pressure. The
obtained residue was purified by silica gel column chromatography
(MeOH/CH2Cl2 = 15:85) and preparative HPLC to obtain 21p (14
mg, 54%) as a colorless amorphous compound; tR = 11.9 min
1
(condition A). H NMR (400 MHz, CD3OD) δ: 1.19 (3H, t, J = 7.6
Hz), 2.22 (3H, s), 2.58 (2H, q, J = 7.6 Hz), 3.41−3.47 (1H, m), 3.64−
3.68 (1H, m), 3.73−3.84 (4H, m), 3.97 (2H, s), 5.03 (1H, d, J = 12.5
Hz), 5.13 (1H, d, J = 12.4 Hz), 7.01−7.14 (6H, m). 13C NMR (100
MHz, CD3OD) δ: 16.2, 20.1, 29.4, 40.1, 62.8, 71.8, 73.4, 74.9, 76.2,
76.5, 111.7, 123.4, 124.6, 128.8, 129.7, 137.8, 138.7, 139.3, 140.1,
140.4, 143.1. MS (ESI): 401 [M + H]+. HRMS (ESI), m/z calcd for
C23H29O6 [M + H]+ 401.1959, found 401.1956.
(1S,3′R,4′S,5′S,6′R)-6-[(4-Ethylphenyl)methyl]-5-ethynyl-6′-(hy-
droxymethyl)-3′,4′,5′,6′-tetrahydro-3H-spiro[2-benzofuran-1,2′-
pyran]-3′,4′,5′-triol (21q). A solution of 20 (97 mg, 0.165 mmol),
dichlorobis(acetonitrile)palladium(II) (9 mg, 0.035 mmol), 2-
dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (53 mg, 0.111
mmol), and cesium carbonate (141 mg, 0.433 mmol) in MeCN
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Article
Molecular Modeling. All molecular modeling procedures were
performed using Molecular Operating Environment (MOE) version
2010.10.30 Six known SGLT2 inhibitors (1, 2a, 3, 4, 5, 6 in Figure 1)
were constructed and minimized by using a MMFF94 force field in
default condition.40 Superposed models of inhibitors were derived by
the FlexibleAlignment module of MOE in which the H-bond donor
and the aromaticity were assigned for similarity terms. Six atoms in the
pyranose ring moiety of each inhibitor were fixed during the Flexible
Alignment. Although several energetically equivalent models were
generated because of the higher flexibility of inhibitors, the differences
among these models are due to the relative configurations of sugar and
distal aromatic moieties bound to the central aromatic ring. One of
them, which has a similar configuration to the crystal structure of
phlorizin (Cambridge Structural Database ID: CEWWAC01), was
chosen for further discussion. The global minimum energy of each
inhibitor was estimated by the LowModeMD search method.
ASSOCIATED CONTENT
* Supporting Information
The structures of three hit compounds from a CSD search and
CYP3A4 inhibition assay data, X-ray crystallographic data of 17,
and in vitro chemical and metabolic stability data of 16d. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +81(550)87-8644. Fax: +81(550)87-5397. E-mail:
ohtakeysh@chugai-pharm.co.jp.
■
Notes
The authors declare no competing financial interest.
In Vitro SGLT Inhibition Assay. Chinese hamster ovary-
K1(CHO) cells stably expressing human SGLT2 (NM003041) and
human SGLT1 (NM000343) were used for the sodium-dependent
methyl-α-D-glucopyranoside (AMG) uptake inhibition assay. The cells
were incubated in reaction buffer with the compound and 1 mM AMG
containing [14C]AMG for 45 min. AMG uptake activity was
determined by counting the radioactivity of the cell lysates. IC50
values were calculated by curve fitting using a four-parameter logistic
model (XLfit, ID Business Solutions Ltd.).
In Vivo Efficacy in db/db Mice. Male db/db mice were
purchased from CLEA Japan (Tokyo, Japan) and maintained on a
regular diet (CE2, CLEA Japan). All animal care and experiments were
performed in accordance with the guidelines for the care and use of
laboratory animals at Chugai Pharmaceutical Co., Ltd. Compounds
16d, 16f (1, 3, or 10 mg/kg), or a vehicle (0.5% CMC) was orally
administered to nonfasted db/db mice. Blood samples were collected
from the tail vein immediately before administration (0 h) and at 1, 2,
4, and 6 h after administration. Urine samples were collected from
mice metabolic cages for 6 h from drug administration, and the urinary
glucose and creatinine concentrations were measured. Blood and urine
glucose concentrations were measured by the hexokinase method
(Autosera S GLU, Daiichi Pure Chemicals, Japan). Urinary creatinine
concentrations were measured by the enzymatic method (Sikaliquid-N
Cre, Kanto Chemicals, Japan).
Pharmacokinetics Studies. Male Crlj:CD1 (ICR) mice (8-weeks
old; Charles River Laboratories Japan, Inc., Japan) and male
cynomolgus monkeys (body weight: 3.1−3.8 kg) were used for the
pharmacokinetics study. Animals were given a single intravenous or
oral administration of 16d and 16f under fasted conditions. Dosing
vehicles used were 10% (v/v) Cremophor EL aqueous solution (Sigma
Chemical Co., MO, USA) for intravenous and 0.5% (w/v)
carboxymethyl-cellulose sodium salt for oral dosing. Blood samples
were collected at 5 min, 0.25, 0.5, 1, 2, 4, and 8 h after administration
to mice and at 0.25, 0.5, 1, 2, 4, 8, and 24 h after administration to
monkeys. The urine samples were collected during the 8 h just after
administration to mice and collected from 8 to 24 h after
administration to monkeys. The drug concentrations in samples
were determined using an LC-MS/MS system after deproteinization.
LC-MS/MS analysis was performed using a Shimadzu LC-10AD
pump (Shimadzu Co., Japan) and an Applied Biosystems/MDS Sciex
API-300 mass spectrometer (Toronto, Canada) with a TurboIonSpray
source. Chromatographic separation was achieved using a CAPCELL
PAK C18 column (2.0 mm × 150 mm, 5 μm) (Shiseido Co., Japan).
The mobile phase consisted of acetonitrile/10 mM ammonium acetate
solution (4:6, v/v), and the flow rate was set at 0.2 mL/min. The
injection cycle of each sample was set at 10 min. The transitions for
multiple reaction monitoring were 387−267 (16d) and 401−281
(16f). Noncompartmental pharmacokinetic parameters were calcu-
lated based on the plasma concentration−time data using WinNonlin
Professional 5.0 (Pharsight, Mountain View, CA). The cumulative
drug amount excreted into urine was divided by the corresponding
AUC to estimate renal clearance (CLrenal).
ACKNOWLEDGMENTS
■
We thank T. Higuchi, I.-J. Ko, D.-W. Kim, and many chemists
of C&C Research Laboratories for chemical synthesis, H. Suda
for mass spectrometry measurements, M. Aoki and N. Takata
for X-ray structure analysis, A. Higashida and N. Sekiguchi for
CYP inhibition assay, T. Kawai, Y. Hasebe, M. Takeda, and
many biologists of Chugai Research Institute for Medical
Science, Inc., for evaluating the pharmacological profiles in the
animal experiments. We also thank Drs. K. Honda and M.
Fukazawa for useful discussion, and Editing Services at Chugai
Pharmaceutical Co., Ltd for assistance with English usage.
ABBREVIATIONS USED
■
AcOEt, ethyl acetate; AIBN, 2,2′-azobis(2-methylpropioni-
trile); AMG, methyl-α-D-glucopyranoside; AUC, area under
the plasma concentration time curve; CL, clearance; CSD,
Cambridge Structural Database; CYP3A4, cytochrome P450
3A4; 3D, three-dimensional; dba, dibenzylideneacetone; DDI,
drug−drug interaction; DMAP, N,N-dimethylaminopyridine;
DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; F,
oral bioavailability; IC50, half-maximal inhibitory concentration;
MOE, Molecular Operating Environment; mp, melting point;
NBS, N-bromosuccinimide; PK, pharmacokinetics; SD, stand-
ard deviation; SGLT, sodium glucose cotransporter; TDI, time-
dependent inhibition; TFA, trifluoroacetic acid; THF, tetrahy-
drofuran; TMS, trimethylsilyl; Tr, triphenylmethyl; tR, retention
time; Vss, steady-state volume of distribution
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NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published online August 28, 2012, some
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