Synthesis and biological evaluation of C-glucosides with azulene rings as selective SGLT2 inhibitors for the treatment of type 2 diabetes mellitus: Discovery of YM543

Article abstract of DOI:10.1016/j.bmc.2013.03.067

Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had relatively good SGLT2 inhibitory activity, comp

Full text of DOI:10.1016/j.bmc.2013.03.067

Bioorganic & Medicinal Chemistry 21 (2013) 3934–3948
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of C-glucosides with azulene
rings as selective SGLT2 inhibitors for the treatment of type 2
diabetes mellitus: Discovery of YM543
a,
a
a
a
Kazuhiro Ikegai a, , Masakazu Imamura , Takayuki Suzuki , Keita Nakanishi , Takeshi Murakami ,
Eiji Kurosaki a, Atsushi Noda b, Yoshinori Kobayashi b, Masayuki Yokota b, Tomokazu Koide b,
Kazuhiro Kosakai b, Yasufumi Ohkura b, Makoto Takeuchi a, Hiroshi Tomiyama b, Mitsuaki Ohta a
⇑
⇑
a Drug Discovery Research, Astellas Pharmaceutical Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Research Laboratories, Kotobuki Pharmaceutical Co., Ltd, 6351 Sakaki-machi, Hanishina-gun, Nagano 389-0697, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory
activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had
relatively good SGLT2 inhibitory activity, compound 8a which has a 3-[(azulen-2-yl)methyl]phenyl group
was identified as a lead compound for further optimization. Introduction of a phenolic hydroxyl group
onto the central benzene ring afforded a potent and selective SGLT2 inhibitor 8e, which reduced blood
glucose levels in a dose-dependent manner in rodent diabetic models. A mono choline salt of 8e
(YM543) was selected as a clinical candidate for use in treating type 2 diabetes mellitus.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 1 March 2013
Revised 26 March 2013
Accepted 27 March 2013
Available online 10 April 2013
Keywords:
Type 2 diabetes mellitus
SGLT2 inhibitor
Azulene
YM543
1. Introduction
dependent glucose cotransporters (SGLTs), and of the six known
SGLT isoforms, the physiological and pathophysiological roles of
Type 2 diabetes mellitus (T2DM) is a metabolic disorder charac-
terized by persistent hyperglycemia and insulin resistance.1 Long-
term lack of glycemic control in T2DM patients increases risk of
macro- and microvascular complications, resulting in significant
morbidity and early mortality.2 As such, the principal management
strategy for T2DM involves control of blood glucose levels. To date,
various oral drugs that enhance insulin secretion and/or improve
insulin sensitivity have been used for the treatment of T2DM. How-
ever, due to their limited efficacy and adverse side effects (e.g.,
weight gain and hypoglycemia), it is difficult to maintain good gly-
cemic control in most diabetic patients. Thus, additional agents,
especially those with insulin-independent mechanisms of action,
are needed to control hyperglycemia at all stages of the disease
whether for monotherapy or for use in combination therapy.
The kidney plays an important role for the body’s glucose
homeostasis. Plasma glucose is filtered through the glomerulus
and then reabsorbed in the proximal tubules of the nephron. Renal
glucose reabsorption is known to be mediated by sodium-
the two major isoforms, SGLT1 and SGLT2 are well-documented.3
SGLT1 is a low-capacity, high-affinity transporter that is found
mainly in the brush border in the small intestine but also present
in the S3 segment of the proximal tubule.4,5 In contrast, SGLT2 is
a high-capacity, low-affinity transporter expressed selectively in
the S1 and S2 segments of the proximal tubule. SGLT2 is responsi-
ble for the reuptake of approximately 90% of filtered glucose.6,7 In-
fants with SGLT1 gene mutations experience glucose–galactose
malabsorption, resulting in frequent, watery diarrhea and dehydra-
tion when on a diet that does not exclude glucose and milk prod-
ucts.8,9 In contrast, in humans with SGLT2 gene mutations,
persistent renal glucosuria is the only reported finding.10,11 These
observations suggest that selective SGLT2 inhibitors could, by
inhibiting glucose reabsorption in the proximal tubule, induce uri-
nary glucose excretion (UGE) and reduce plasma glucose concen-
trations without adverse gastrointestinal side effects associated
with SGLT1 inhibition. Indeed, a recent study confirmed that SGLT2
inhibitors can increase UGE and reduce hyperglycemia in patients
with T2DM.12
Optimization of the natural O-glucoside phlorizin (1), a nonse-
lective SGLT inhibitor,13 led to the identification of O-aryl glucoside
selective SGLT2 inhibitors such as T-1095A (2)14 as shown in
Figure 1. These derivatives were of limited usefulness in vivo,
⇑
Corresponding authors. Tel.: +81 332442687 (K. Ikegai), +81 298296653 (M.
Imamura)
E-mail addresses: kazuhiro.ikegai@astellas.com (K. Ikegai), masakazu.imamur-
a@astellas.com (M. Imamura).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.067

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3935
O
-Glucosides
OH
OH
O
OH
O
HO
O
O
O
O
O
HO
HO
RO
HO
OH
OH
2 : T-1095A (R = H)
3 : T-1095 (R = COOMe)
OH
OH
1 : Phlorizin
C
-Glucosides
OH
O
O
O
HO
HO
HO
HO
O
OH
OH
4 (Kotobuki)
5 (Bristol-Myers Squibb)
OH
OH
F
O
S
HO
HO
6 : Ipragliflozin
(Astellas-Kotobuki)
OH
OH
Figure 1. Structure of SGLT2 inhibitors.
R1
R2
O
O
HO
HO
7
HO
OH
HO
OH
OH
OH
hSGLT2 IC50 = 99 nM
Figure 2. Design of novel azulene derived SGLT2 inhibitors.
however, due to their degradation by glucosidase enzymes in the
gut, suggesting that O-glucosides were better administered as pro-
drugs (e.g., 3).15 The problem has been successfully addressed via
the discovery of potent and selective C-aryl glucoside SGLT2 inhib-
itors, that are not subject to GI tract degradation. In 2001, scientists
at Kotobuki Pharmaceutical Co., Ltd and Bristol–Myers Squibb
independently submitted patent applications describing C-aryl
glucosides such as 416 and 5,17 which are resistant to degradation
than O-glucosides. Pursuing these findings, researchers at Bristol–
Myers Squibb identified dapagliflozin,18 which has been approved
in the European Union (EU) last November to be the first medicine
in the new SGLT2 class. Our group reported the discovery of
ipragliflozin (6; ASP1941),19 a potent and selective SGLT2 inhibitor
that is currently being investigated in clinical trials for the treat-
ment of T2DM.
derivatives have been found to inhibit gastric acid secretion and
prevent the development of duodenal ulcers,22 stimulate the repair
of gastric mucosa,23 increase gastric mucosal blood flow,24 act as
antioxidants,25 and block TXA2/PGH2 receptors in the platelet
membrane.21e,26 Additionally, azulene derivatives are also known
to have antiallergenic,21i anticancer,21b,21f,27,28 local anesthetic,29
and cardioprotective30 properties. To the best of our knowledge,
no reports have commented on the use of azulene derivatives for
the treatment of T2DM. We therefore initiated the optimization
study of 7 based on the synthetic plan depicted in Figure 2.
Here, we describe the synthesis and structure–activity relation-
ships of azulene-derived SGLT2 inhibitors, which led to the discov-
ery of YM543 (Fig. 5), a promising candidate for use in the
treatment of T2DM.
Our investigation of other C-glucoside SGLT2 inhibitors has
found azulene derivative 7 (Fig. 2) to have relatively good SGLT2
inhibitory activity (IC50 = 99 nM) and high selectivity versus SGLT1
(140-fold). Azulene, an aromatic isomer of naphthalene, has an
electrically positive seven-membered ring and an electrically neg-
ative five-membered ring, and is considered an arene bioisostere of
benzene and naphthalene.20 Although the use of azulene motifs in
the field of medicinal chemistry21 is uncommon, a search of the lit-
erature reveals that certain azulene derivatives possess significant
pharmacological and therapeutic actions. For example, azulene
2. Chemistry
In general, b-C-glucoside linkages are synthesized from glucon-
olactone 13 (Scheme 1) in a stereoselective manner that has been
previously reported.19,31
Compounds 7, 8a, and 11 were prepared from b-C-glucoside
14,19 as shown in Scheme 1. Treatment of 14 with PPh3 and CBr4
produced benzyl bromide 15, which was transformed into the cor-
responding zinc reagent in situ and subjected to Negishi coupling
with 6-bromoazulene (16),32 producing the coupling product 17.

3936
K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
O
O
a
BnO
BnO
O
OH
BnO
BnO
OBn
OBn
(Ref. 19)
OBn
OBn
13
14
O
Br
b
BnO
BnO
O
RO
RO
OBn
OR
OBn
OR
15
Br
17: R = Bn
7: R = H
16
c
R
b
c
O
O
HO
HO
BnO
BnO
CO2Me
CO2Me
OH
OBn
OH
OBn
19
20: R = H
21: R = Bn
Cl
CO2Me
18
R
R
O
O
d
e
HO
HO
HO
HO
CO2H
OH
OH
OH
OH
8a: R = H
11: R = Bn
22: R = H
23: R = Bn
Scheme 1. Reagents and conditions: (a) PPh3, CBr4, CH2Cl2, rt, quant.; (b) Zn, BrCH2CH2Br, TMSCl, THF, reflux to rt, then 16 or 18, Pd(PPh3)4, reflux, 28% for 17, 62% for 19; (c)
BCl3 or BBr3, CH2Cl2, À78 °C, 47% for 7, 49 and 18% for 20 and 21; (d) aq NaOH, MeOH, reflux, 80% for 22, 78% for 23; (d) p-TsOHÁH2O, benzene, reflux, 76% for 8a, 62% for 11.
b
a
Br
Br
O
25
26
24
e
O
c, d
O
BnO
BnO
HO
HO
OBn
OH
OBn
OH
27
9
Scheme 2. Reagents and conditions: (a) 3-bromobenzoyl chloride, AlCl3, CH2Cl2, 0 °C, 26%; (b) BF3ÁEt2O, NaBH4, diglyme–diethyl ether, 0 °C to rt, 46%; (c) n-BuLi/hexane, THF,
À78 °C then 13, 59%; (d) iPr3SiH, BF3ÁEt2O, CH2Cl2, À40 °C, 65%; (e) BCl3, CH2Cl2, À78 °C, 17%.
Deprotection of benzyl groups of 17 with BCl3 at À78 °C yielded the
desired b-C-glucoside 7. Similarly, a coupling reaction of the zinc
reagent generated from 15 with methyl 2-chloroazulene-1-carbox-
ylate (18)33 smoothly proceeded to produce a coupling product 19.
Deprotection of benzyl groups of 19 in the presence of BBr3 gave 20
along with the minor product 21. Saponification of the methyl es-
ters of 20 and 21, followed by decarboxylation in the presence of p-
TsOHÁH2O, afforded the b-C-glucosides 8a and 11, respectively.
The synthesis of compound 9 is depicted in Scheme 2. Friedel–
Crafts acylation of 1-methylazulene (24) with 3-bromobenzoyl
chloride produced ketone 25, which was then reduced by using
NaBH4 and BF3ÁOEt2 to afford biarylmethane 26. The addition of
the aryl lithium reagent generated by lithium halogen exchange
of aglycon 26–13, followed by the reduction of lactol yielded the
corresponding b-C-glucoside 27 stereoselectively. Finally, removal
of the benzyl groups with BCl3 resulted in the desired product 9.
The preparation of derivatives 10 and 12 which have substitu-
ents on the azulene moiety is described in Scheme 3. Acetylation
of 8a followed by Friedel–Crafts acylation of 28 in the presence
of Ac2O and AlCl3 produced 29. The ketone moiety was reduced

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3937
d
a
O
O
AcO
AcO
HO
HO
8a
R
R
OAc
OH
OAc
OH
10: R = Et
12: R = Ac
28: R = H
29: R = Ac
30: R = Et
b
c
Scheme 3. Reagents and conditions: (a) Ac2O, pyridine, rt, quant.; (b) Ac2O, AlCl3, CH2Cl2, 0 °C to rt, 42%; (c) BF3ÁEt2O, NaBH4, diglyme–diethyl ether, 0 °C to rt, quant.; (d)
NaOH, THF–MeOH, 0 °C, 77% for 10, 89% for 12.
R1
R2
R1
R2
R1
Br
R2
a, b
c
O
O
Br
BnO
BnO
BnO
BnO
OH
OBn
OBn
OTBS
OBn
33: R1 = F, R2 = H
OBn
35: R1 = F, R2 = H
36: R1 = H, R2 = OMe
31: R1 = F, R2 = H
34: R1 = H, R2 = OMe
32: R1 = H, R2 = OMe
R2
R1
R2
R1
d
e, f, g
O
O
BnO
HO
HO
CO2Me
BnO
OBn
OH
OBn
OH
37: R1 = F, R2 = H
8b: R1 = F, R2 = H
38: R1 = H, R2 = OMe
8h: R1 = H, R2 = OMe
Scheme 4. Reagents and conditions: (a) n-BuLi/hexane, THF, À78 °C then 13; (b) iPr3SiH, BF3ÁEt2O, CH2Cl2, -40 °C, 58% for 33 (two steps), 31% for 34 (two steps); (c) PPh3, CBr4,
CH2Cl2, rt, 78% for 35, 86% for 36; (d) Zn, BrCH2CH2Br, TMSCl, THF, reflux to rt, then 18, Pd(PPh3)4, reflux, 72% for 37, 34% for 38; (e) BBr3, CH2Cl2, À78 °C; (f) 10% aq NaOH,
MeOH, reflux; (g) p-TsOHÁH2O, benzene, reflux, 62% for 8b (three steps), 5% for 8h (three steps).
by treatment with NaBH4 and BF3ÁOEt2 resulting in 30. Removal of
the acetyl groups of 29 and 30 under basic conditions yielded 10
and 12.
59 and 60, respectively. Deprotection of the benzyl groups using
BCl3 in the presence of pentamethylbenzene yielded the desired
C-glucosides 8f and 8g.
In Scheme 4, the synthetic strategy for 8a, which utilized Negi-
shi coupling was also used to prepare azulene derivatives 8b and
8h. Addition of organolithium reagents generated from aryl bro-
mides 31 and 32 to lactone 13 followed by stereoselective reduc-
tion of lactols produced b-C-glucosides 33 and 34. Bromination of
a hydroxyl group and subsequent Negishi coupling produced 37
and 38, which were converted to the final products 8b and 8h in
three steps.
3. Results and discussion
The in vitro SGLT inhibitory potential (IC50) of all synthesized
compounds was assessed by monitoring the inhibition of accumu-
lating [14C]methyl-
a-D-glucopyranoside (AMG) in Chinese hamster
ovary (CHO) cells expressing human SGLT2 or SGLT1.37
Compounds 8c, 8d, and 8e were also prepared via Negishi cou-
pling as shown in Scheme 5. b-C-glucosides 41 and 42 were ob-
tained from lactone 13 based on the abovementioned procedure.
Exchange of protecting groups produced acetyl-protected C-gluco-
We previously described the synthesis and preclinical evalua-
tion of a series of C-glucoside SGLT2 inhibitors bearing heteroaro-
matic rings in the aglycon moieties.19a Replacement of the terminal
benzene ring of 5 with various heteroaromatic rings was found to
improve inhibitory activities. Thus, we initiated this study with the
modification of the terminal azulene ring of 7 (Table 1). Azulen-2-
yl derivative 8a was found to be much more potent (IC50 = 22 nM)
and selective for SGLT2 (590-fold compared to SGLT1) than azulen-
6-yl derivative 7, while azulen-1-yl derivative 9 was not toler-
ated.38 Introduction of alkyl and acetyl substituents at the 1-posi-
tion of azulene in 8a was also tested (compounds 10–12), induced
in a dramatic loss of SGLT2 inhibitory activity. We next confirmed
the potent antihyperglycemic effect of 8a in KK/Ay type 2 diabetic
mice.19b A single oral administration of 8a (3 mg/kg) in diabetic
mice reduced blood glucose levels significantly (46% reduction
compared to vehicle). Following this encouraging result, we next
focused our attention on the optimization of 8a.
sides 43 and 44. Conversely, b-C-glucoside 46 was obtained from D-
glucose pentaacetate (45) and 1-methoxy-4-methyltoluene by
SnCl4/AgOTf-promoted C-glycosylation according to Satoh’s proce-
dure.34 Bromination of 43, 44 and 46 using NBS and a catalytic
amount of AIBN in CCl4 produced benzyl bromides 47–49, which
were transformed into coupling products 50–52. Finally, removal
of acetyl groups and decarboxylation yielded desired C-glucosides
8c, 8d and 8e.
To reduce the number of reaction steps, we then discovered a
Suzuki coupling reaction with azulenyl boronate 58,35 which was
applied to the synthesis of compounds 8f and 8g as shown in
Scheme 6. Benzyl bromides 56 and 57,36 reacted with 58 in the
presence of a Pd catalyst and K3PO4, produced coupling products

3938
K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
R
R
c, e (for 43)
d, e (for44)
R
a, b
O
O
BnO
BnO
AcO
AcO
Br
OBn
OAc
OBn
OAc
39: R = Cl
40: R = OBn
41: R = Cl
42: R = OBn
43: R = Cl
44: R = OAc
g
MeO
O
R
O
O
OAc
OAc
g
f
AcO
AcO
Br
AcO
AcO
AcO
AcO
OAc
OAc
OAc
OAc
OAc
45
46
47: R = Cl
48: R = OMe
49: R = OAc
R
R
i, j, k
O
O
h
AcO
AcO
HO
HO
CO2Me
OAc
OH
OAc
OH
8c: R = Cl
8d: R = OMe
8e: R = OH
50: R = Cl
51: R = OMe
52: R = OAc
Scheme 5. Reagents and conditions: (a) n-BuLi/hexane, THF, À78 °C then 13; (b) iPr3SiH, BF3ÁEt2O, CH2Cl2, -40 °C, 63% for 41 (two steps), 37% for 42 (two steps); (c) BBr3,
CH2Cl2, À78 °C; (d) H2, Pd(OH)2/C, MeOH–EtOAc, rt; (e) Ac2O, Py, rt, 31% for 43 (two steps), 82% for 44 (two steps); (f) 1-methoxy-4-methylbenzene, SnCl4, AgOTf, CH2Cl2, 0 °C,
41%; (g) NBS, AIBN, CCl4, reflux, 96% for 47, 54% for 48, 48% for 49; (h) Zn, BrCH2CH2Br, TMSCl, THF, reflux to rt, then 18, Pd(PPh3)4, reflux, 44% for 51, 47% for 52; (i) NaOMe,
MeOH, 0 °C; (j) 10% aq NaOH, MeOH, reflux; (k) p-TsOHÁH2O, benzene, reflux, 10% for 8c (four steps), 41% for 8d (three steps), 53% for 8e (three steps).
Table 1
Optimization of the azulene moiety
Ar
O
HO
HO
OH
OH
Compd
Ar
hSGLT2 IC50 (nM)
99
Selectivity versus hSGLT1a (fold)
140
Compd
Ar
hSGLT2 IC50 (nM)
5200
Selectivity versus hSGLT1a (fold)
>1.9
7
10
Ph
8a
9
22
590
230
11
12
970
>10
O
44,000
12,000
>0.8
a
Data were obtained from at least two independent experiments.
Next, we investigated substituent effects on the central benzene
8.9 nM, respectively) with sufficient selectivity on SGLT1 (2100-
and 280-folds, respectively). Halogen substituents (8b and 8c) at
this position slightly decreased the potency compared with 8a. Ta-
ken together, these results indicate that the introduction of oxy-
ring of 8a (Table 2). Introduction of methoxy (8d) or hydroxyl (8e)
groups at the ortho position to the glucoside (6-position; R1) was
found to increase inhibitory activity against SGLT2 (IC50 = 16 and

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3939
F
F
F
a, b
c, d, e
O
f
BnO
H
Br
Br
OH
BnO
OBn
O
OTBDPS
OBn
53
54
55
R
R
O
Br
g
h
O
BnO
BnO
BnO
BnO
OBn
OBn
OBn
OBn
59: R = F
60: R = Cl
56: R = F
57: R = Cl
(pin)B
58
R
O
HO
HO
OH
OH
8f: R = F
8g: R = Cl
Scheme 6. Reagents and conditions: (a) NaBH4, EtOH, 0 °C,%; (b) TBDPSCl, imidazole, DMF, rt, 72% (two steps); (c) n-BuLi/hexane, THF, À78 °C then 13; (d) iPr3SiH, BF3ÁEt2O,
CH2Cl2, À40 °C; (e) TBAF, THF, rt, 36% (three steps); (f) PPh3, CBr4, CH2Cl2, rt, 81%; (g) 58, Pd2dba3, P(2-fulyl)3, K3PO4, THF, reflux; (h) BCl3, pentamethylbenzene, CH2Cl2,
À40 °C, 26% for 8f (two steps), 36% for 8g (two steps).
Table 2
functional groups at this position is beneficial for improving
inhibitory activity against SGLT2.19a Additionally, substitution with
Optimization of the central benzene ring
fluorine (8f) or chlorine (8g) atoms at the para position to the glu-
coside (4-position; R2) improved potency (IC50 = 14 and 7.9 nM,
R1
R2
respectively) with good selectivity for SGLT2 over SGLT1 (610-
and 140-fold, respectively). Introduction of a methoxy group at this
position (8h) also increased inhibitory activity against SGLT2
(IC50 = 17 nM) but resulted in a consequent loss of selectivity for
SGLT2 over SGLT1 (35-fold).
O
HO
HO
OH
OH
Among the aforementioned azulene derivatives, one of the
most potent SGLT2 inhibitors, 8e, was chosen for further phar-
macokinetic studies in normal rats (Table 3). Administration of
a single 1.0 mg/kg dose intravenously or a single 3 mg/kg dose
orally revealed that 8e is notable for rapid oral absorption (Tmax
0.5 h) and metabolism (Cltot 2483 L/h/kg) with a bioavailability
of 29%. We also confirmed that the renal drug concentration
was 23 times that in plasma, suggesting robust extraction by
the nephron, the site of the target transporter (data not
shown).
a
Compd R1
R2
hSGLT2 IC50
(nM)
Selectivity versus hSGLT1a
(fold)
8a
8b
8c
8d
8e
8f
H
F
Cl
OMe
OH
H
H
H
H
H
H
F
22
39
70
16
8.9
14
7.9
590
123
200
2100
280
610
140
35
8g
8h
H
H
Cl
OMe 17
We next examined the in vivo pharmacological profile of 8e.39
A
a
Data were obtained from at least two independent experiments.
single oral dose of 8e (0.1–3.0 mg/kg) resulted in a dose-dependent
and sustained increase in urinary glucose excretion (UGE) that
lasted over 12 h at doses of 0.3 mg/kg or higher (Fig. 3). 8e also
showed potent and long-lasting antihyperglycemic activity in both
KK/Ay mice and streptozotocin-induced diabetic rats (STZ rats, a
type 1 diabetes mellitus model), without decreases into the hypo-
glycemic range (Fig. 4). The reduction of glucose AUC reached 56%
versus vehicle in KK/Ay mice by the administration of 8e (3 mg/kg),
which was stronger than that of 8a at the same dose (46% vs vehi-
cle). We therefore concluded that 8e is an orally active and selec-
tive SGLT2 inhibitor with the best pharmacological profile in the
azulene series. After salt-screening experiments, a mono choline
salt of 8e (YM543; see Fig. 5) was selected for further clinical
development.
Table 3
Pharmacokinetic properties of 8e after oral and intravenous administration to rats
Parameter
Unit
SD rats
iv
po
Dose
T1/2
CLtot
Vdss
Cmax
Tmax
AUC(0–inf)
F
mg/kg
h
L/h/kg
L/kg
ng/mL
h
1.0
0.9
2483
3360
3.0
1.3
101
0.50
ng h/mL
%
403
29

3940
K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
Vehicle
500
400
300
200
100
0
1000
0.1 mg/kg
0.3 mg/kg
1 mg/kg
800
600
400
200
0
3 mg/kg
0-4 h
4-8 h
8-12 h
12-24 h
0-24 h
Figure 3. Effects of 8e on urinary glucose excretion in KK/Ay type 2 diabetic mice. 8e was orally administered to diabetic mice and spontaneously voided urine was collected
for 24 h. Values are mean SEM for 6–7 animals in each group. ⁄p <0.05 versus vehicle using the Dunnett’s multiple range test.
Figure 4. Antihyperglycemic effect of 8e in KK/Ay mice and STZ rats. (A) Change in blood glucose levels. (B) Suppression of glucose AUC. Values are expressed as mean SEM
for 6 animals in each group. ⁄p <0.05 versus vehicle using the Dunnett’s multiple range test.
4. Conclusion
N+
HO
O
The synthesis and structure–activity relationships of azulene-
derived C-glucoside SGLT2 inhibitors were explored. Structural
O
HO
HO
modification of the azulene moiety of compound 7 led to the iden-
tification of compound 8a. Incorporation of a phenolic hydroxyl
group at the central benzene ring resulted in the more potent
SGLT2 inhibitor 8e, which exerted a strong and sustained antihy-
perglycemic effect in both KK/Ay type 2 diabetic mice and STZ type
OH
OH
Figure 5. The structure of YM543.

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3941
1 diabetic rats after oral administration. Given these encouraging
preclinical findings, a mono choline salt of 8e (YM543) was se-
lected as a clinical candidate.
1H NMR (CD3OD) d: 3.34–3.89 (6H, m), 4.12 (1H, d, J = 9.3 Hz),
4.17 (2H, s), 7.18 (2H, d, J = Hz), 7.19–7.38 (6H, m), 7.77 (1H, t,
J = 3.7 Hz), 8.25 (2H, d, J = 10.3 Hz). MS (ESI) m/z: 381 (M++H), calcd
for C23H24O5: 380.
5.1.4. (1S)-2,3,4,6-Tetra-O-benzyl-1,5-anhydro-1-(3-{[1-
(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-D-glucitol (19)
5. Experimental
5.1. Chemistry
To a mixture of zinc dust (370 mg, 5.66 mmol) and THF (3 mL)
was added 1,2-dibromoethane (2 drops) and the mixture was
warmed at 60 °C for 3 min, then cooled to room temperature. To
the mixture was added trimethylsilyl chloride (2 drops) and the
mixture was stirred at room temperature for 15 min. To the mix-
ture was added a solution of 15 (2.00 g, 2.88 mmol) in THF
(5 mL) and the mixture was stirred at 80 °C for 30 min to form
the corresponding benzyl zinc bromide in situ. To the mixture were
added methyl 2-chloroazulene-1-carboxylate (18)33 (450 mg,
2.04 mmol) and Pd(PPh3)4 (230 mg, 0.20 mmol) and the mixture
was stirred at 80 °C for 12 h. After cooled to room temperature,
the mixture was filtered through a pad of CeliteÒ (washed with
EtOAc). After evaporation, the residue was purified by column
chromatography on silica gel (hexane/EtOAc = 10/1) to give the ti-
tle compound (1.0 g, 62% yield) as a red–purple oil.
1NMR spectra were obtained on a JEOL JNM-EX400 spectrome-
ter and the chemical shifts are expressed in d (ppm) values with
tetramethylsilane as an internal standard. Abbreviations 1H NMR
signal patterns are as follows: s, singlet; d, doublet; dt, double trip-
let; t, triplet; q, quartet; m, multiplet; br, broad. Mass spectra were
obtained on a JEOL JMS-DX300 or HITACHI M-80 spectrometer.
Column chromatography on silica gel was performed with Kiesel-
gel 60 (E. Merck).
5.1.1. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-
(bromomethyl)phenyl]-D-glucitol (15)
To a solution of 14 (3.88 g, 6.15 mmol) in CH2Cl2 (70 mL) were
added CBr4 (4.08 g, 12.3 mmol) and PPh3 (1.94 g, 7.40 mmol) and
the mixture was stirred at rt for 2 h. Additional CBr4 (4.08 g,
12.3 mmol) and PPh3 (1.94 g, 7.40 mmol) were added and the mix-
ture was stirred at for additional 1 h. The reaction was quenched
with by adding satd aq NaHCO3. The separated organic layer was
dried over Na2SO4, filtered and evaporated in vacuo. The residue
was purified by column chromatography on silica gel (eluent: hex-
ane/EtOAc = 20/1–10/1) to give the title compound (4.27 g, quant.)
as a colorless oil.
1H NMR (CDCl3) d: 3.52–3.82 (7H, m), 3.93 (3H, s), 4.22–4.95
(10H, m), 6.86–7.68 (28H, m), 8.04 (1H, d, J = 9.8 Hz), 9.53 (1H, d,
J = 9.8 Hz). MS (ESI) m/z: 799 (M++H), calcd for C53H50O7: 798.
5.1.5. (1S)-1,5-Anhydro-1-(3-{[1-(methoxycarbonyl)azulen-2-
yl]methyl}phenyl)-D-glucitol (20) and (1S)-1,5-anhydro-1-(3-
{[1-benzyl-3-(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-
glucitol (21)
D-
1H NMR (CDCl3) d: 3.46–3.63 (2H, m), 3.72–3.84 (5H, m), 4.24
(1H, d, J = 6.9 Hz), 4.38–4.68 (6H, m), 4.83–4.99 (3H, m), 6.88–
6.96 (2H, m), 7.16–7.50 (22H, m).
To a cold (À78 °C) solution of 19 (1.0 g, 1.25 mmol) in CH2Cl2
(20 mL) was added BBr3 (1.0 M in CH2Cl2; 6.3 mL, 6.3 mmol) and
the mixture was stirred at À78 °C for 1 h. The reaction was
quenched by adding 10% aq HCl. The mixture was extracted with
EtOAc (Â3). The combined organic layer was washed with satd
aq NaHCO3 and brine, dried over Na2SO4, and evaporated in vacuo.
The residue was purified by column chromatography on silica gel
(CHCl3/MeOH = 10/1) to give the title compounds 20 (270 mg,
49% yield) and 21 (120 mg, 18% yield) as a red–purple solid.
Compound 20: 1H NMR (CD3OD) d: 3.42–3.93 (m, 6H), 3.93 (3H,
s), 4.18 (1H, d, J = 9.3 Hz), 4.63 (2H, s), 7.02 (1H, s), 7.02–7.33 (4H,
m), 7.42 (1H, t, J = 9.6 Hz), 7.53 (1H, t, J = 10.3 Hz), 7.73 (1H, t,
J = 9.8 Hz), 8.30 (1H, d, J = 9.8 Hz), 9.48 (1H, d, J = 9.8 Hz). MS
(ESI) m/z: 439 (M++H), calcd for C25H26O7: 438.
5.1.2. (1S)-2,3,4,6-Tetra-O-benzyl-1,5-anhydro-1-[3-(azulen-6-
ylmethyl)phenyl]-D-glucitol (17)
To a mixture of zinc dust (40 mg, 0.61 mmol) and THF (4.0 mL)
was added 1,2-dibromoethane (1 drop) and the mixture was
warmed at 60 °C for 3 min, then cooled to room temperature. To
the mixture was added trimethylsilyl chloride (1 drop) and the
mixture was stirred at room temperature for 15 min. To the mix-
ture was added 15 (230 mg, 0.33 mmol) and the mixture was stir-
red at 80 °C for 30 min to form the corresponding benzyl zinc
bromide in situ. To the mixture were added 6-bromoazulene
(16)32 (50 mg, 0.24 mmol) and Pd(PPh3)4 (30 mg, 0.026 mmol)
and the mixture was stirred at 80 °C for 3 h. After cooled to room
temperature, the mixture was filtered through a pad of CeliteÒ
(washed with EtOAc). After evaporation, the residue was purified
by column chromatography on silica gel (hexane/EtOAc = 10/1) to
give the title compound (50 mg, 28% yield) as a blue oil.
Compound 21: 1H NMR (CD3OD) d: 3.29–3.42 (5H, m), 3.65 (1H,
m), 3.82 (3H, s), 3.99 (1H, d, J = 8.0 Hz), 4.41 (2H, s), 4.59 (2H, s),
6.87 (1H, d, J = 7.8 Hz), 7.00 (1H, d, J = 8.0 Hz), 7.10–7.19 (7H, m),
7.44 (1H, t, J = 8.0 Hz), 7.53 (1H, t, J = 8.0 Hz), 7.78 (1H, t,
J = 8.0 Hz), 8.43 (d, 1H, J = 8.0 Hz), 9.43 (1H, d, J = 8.0 Hz). MS
(ESI) m/z: 529 (M++H), calcd for C32H32O7: 528.
1H NMR (CDCl3) d: 3.47–3.81 (7H, m), 4.15 (2H, s), 4.21–4.93
(8H, m), 6.85–7.36 (28H, m), 7.81 (1H, t, J = 3.9 Hz), 8.16 (d, 2H,
J = 10.3 Hz). MS (ESI) m/z: 741 (M++H), calcd for C51H48O5: 740.
5.1.6. (1S)-1,5-Anhydro-1-{3-[(1-carboxyazulen-2-
yl)methyl]phenyl}-D-glucitol (22)
To a solution of 20 (270 mg, 0.62 mmol) in MeOH (8.0 mL) was
added 10% aq NaOH (4.0 mL) and the mixture was refluxed for 2 h.
After cooling, the mixture was neutralized with 10% aq HCl (ꢀpH
4) and extracted with EtOAc (Â3). The combined organic layer
was washed with brine, dried over Na2SO4, filtered, and evaporated
in vacuo. The resulting residue was purified by column chromatog-
raphy on silica gel (eluent: CHCl3/MeOH = 10/1) to give the title
compound (210 mg, 80% yield) as a red–purple solid.
5.1.3. (1S)-1,5-Anhydro-1-[3-(azulen-6-ylmethyl)phenyl]-D-
glucitol (7)
To a cold (À78 °C) solution of 17 (50 mg, 0.067 mmol) in CH2Cl2
(6.0 mL) was added BCl3 (1.0 M in CH2Cl2; 0.27 mL, 0.27 mmol) and
the mixture was stirred at À78 °C for 1 h. The reaction was
quenched by adding 10% aq HCl. The mixture was extracted with
EtOAc (Â3). The combined organic layer was washed with satd
aq NaHCO3 and brine, dried over Na2SO4, and evaporated in vacuo.
The residue was purified by column chromatography on silica gel
(CHCl3/MeOH = 10/1) to give the title compound (12 mg, 47% yield)
as a blue-purple solid.
1H NMR (CD3OD) d: 3.34–3.52 (4H, m), 3.70–3.93 (2H, m), 4.15
(1H, d, J = 9.3 Hz), 4.69 (2H, s), 7.06 (1H, s), 7.24–7.84 (7H, m), 8.38
(1H, d, J = 9.8 Hz), 9.57 (1H, d, J = 9.8 Hz). MS (ESI) m/z: 423 (M--H),
calcd for C24H24O7: 424.

3942
K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
5.1.7. (1S)-1,5-Anhydro-1-[3-(azulen-2-ylmethyl)phenyl]-
glucitol (8a)
D
-
5.1.12. (1S)-2,3,4,6-Tetra-O-benzyl-1,5-anhydro-1-{3-[(3-
methylazulen-1-yl)methyl]phenyl}- -glucitol (27)
D
To a mixture of 22 (0.19 g, 0.45 mmol) and benzene (10 mL) and
p-TsOHÁH2O (20 mg, mmol) at room temperatue. The mixture was
refluxed for 2 h. After cooling, the mixture was poured into ice-
water and extracted with EtOAc (Â3). The combine organic layer
was washed with brine, dried over Na2SO4, and evaporated in va-
cuo. The purple residue was purified by column chromatography
on silica gel (eluent: CHCl3/MeOH = 10/1) to give the title com-
pound (130 mg, 76%) as a purple solid.
Step 1: To a cold (À78 °C) solution of 26 (1.19 g, 3.81 mmol) in
THF (8.0 mL) was added n-BuLi (1.56 M in hexane; 2.44 mL,
3.81 mmol) and the mixture was stirred at À78 °C for 1 h. To the
reaction mixture was added a solution of 14 (2.80 g, 5.20 mmol)
in THF (8.0 mL) and the mixture was stirred at À78 °C for 1 h.
The reaction was quenched by satd aq NH4Cl and extracted with
EtOAc. The organic layer was washed with brine, dried over
Na2SO4, filtered, and evaporated in vacuo. The resulting residue
was purified by column chromatography on silica gel (hexane/
EtOAc = 20/1) to give the corresponding lactol (2,3,4,6-tetra-O-
benzyl-1-C-{3-[(3-methylazulen-1-yl)methyl]phenyl}glucopyra-
nose) as a blue oil (1.74 g, 59%).
1H NMR (CD3OD) d: 3.34–3.93 (6H, m), 4.16 (1H, d, J = 9.3 Hz),
4.37 (2H, s), 7.15–7.58 (9H, m), 8.24 (2H, d, J = 9.8 Hz). MS (ESI)
m/z: 381 (M++H), calcd for C23H24O5: 380.
1H NMR (CDCl3) d: 2.56 (3H, s), 2.93 (1H, s), 3.54–4.36 (8H, m),
4.40 (2H, s), 4.50–4.91 (6H, m), 6.87–7.56 (28H, m), 8.13 (2H, dd,
J = 9.8, 14.2 Hz). MS (ESI) m/z: 793 (M++Na), calcd for C52H50O6:
770.
5.1.8. (1S)-1,5-Anhydro-1-{3-[(1-benzyl-3-carboxyazulen-2-
yl)methyl]phenyl}-D-glucitol (23)
The title compound was prepared from 21 in the same manner
as described for 22 (78% yield, a blue solid).
1H NMR (CD3OD) d: 3.31–3.87 (6H, m), 4.05 (1H, d, J = 9.8 Hz),
4.17 (2H, s), 4.42 (2H, s), 6.92–7.19 (9H, m), 7.40 (1H, t,
J = 9.8 Hz), 7.51 (1H, t, J = 10.3 Hz), 7.73 (1H, t, J = 9.8 Hz), 8.40
(1H, d, J = 9.3 Hz), 9.51 (1H, d, J = 9.3 Hz). MS (ESI) m/z: 513
(MÀÀH), calcd for C31H30O7: 514.
Step 2: To a cold (À40 °C) solution of the lactol obtained above
(2.30 g, 2.98 mmol) in CH3CN (40 mL) were added iPr3SiH
(1.23 mL, 5.99 mmol) and BF3ÁOEt2 (0.39 mL, 3.08 mmol) and the
mixture was stirred at À40 °C for 2 h. The reaction was quenched
by adding satd aq Na2CO3 and extracted with EtOAc (Â2). The com-
bined organic layer was dried over Na2SO4, filtered, and evaporated
in vacuo. The resulting residue was purified by column chromatog-
raphy on silica gel (eluent: hexane/EtOAc = 10/1) to give the title
compound 27 (1.47 g, 65% yield) as a blue oil.
5.1.9. (1S)-1,5-Anhydro-1-{3-[(1-benzylazulen-2-
yl)methyl]phenyl}-D-glucitol (11)
The title compound was prepared from 23 in the same manner
as described for 8a (62% yield, a purple solid).
1H NMR (CDCl3) d: 2.55 (3H, s), 3.47–3.81 (7H, m), 4.11–4.32
(2H, m), 4.40 (2H, s), 4.51–4.93 (6H, m), 6.86–7.46 (27H, m), 7.49
(1H, s), 8.13 (2H, dd, J = 9.5, 14.7 Hz). MS (ESI) m/z: 754 (M++H),
calcd for C52H50O5: 753.
1H NMR (CD3OD) d: 3.31–3.88 (6H, m), 4.06 (1H, d, J = 9.8 Hz),
4.17 (2H, s), 4.42 (2H, s), 7.02–7.26 (12H, m), 7.48 (1H, t,
J = 9.3 Hz), 8.14 (1H, d, J = 9.3 Hz), 8.24 (1H, d, J = 9.8 Hz). MS
(ESI) m/z: 471 (M++H), calcd for C30H30O5: 470.
5.1.13. (1S)-1,5-Anhydro-1-{3-[(3-methylazulen-1-
yl)methyl]phenyl}-D-glucitol (9)
5.1.10. 3-Bromobenzoyl-1-methylazulene (25)
To a cold (À78 °C) solution of 27 (760 mg, 1.01 mmol) in CH2Cl2
(20 mL) was added BCl3 (1.0 M in heptane; 20.0 mL, 20.0 mmol)
and the mixture was stirred at À78 °C for 0.5 h. The mixture was
diluted by adding a mixed solvent of CH2Cl2 (40 mL) and toluene
(20 mL). The reaction was quenched by adding MeOH at À78 °C.
The mixture was evaporated in vacuo. The residue was purified
by column chromatography on silica gel (CHCl3/MeOH = 20/1) to
give the title compound (68 mg, 17% yield) as a blue–green solid.
1H NMR (DMSO-d6) d: 2.57 (3H, s), 3.14–3.43 (5H, m), 3.66–3.70
(1H, m), 3.96 (1H, d, J = 8.9 Hz), 4.35 (2H, s), 4.41–4.94 (4H, m),
6.99–7.21 (6H, m), 7.54 (1H, t, J = 10.0 Hz), 7.61 (1H, s), 8.20 (1H,
d, J = 9.8 Hz), 8.34 (1H, d, J = 9.8 Hz). MS (DI) m/z: 394 (M+), calcd
for C24H26O5: 394.
To a solution of 1-methylazulene (2.00 g, 14.1 mmol) in CH2Cl2
(20 mL) at 0 °C was added AlCl3 (1.87 g, 14.1 mmol) in one-potion.
After being stirred for 15 min at 0 °C, a CH2Cl2 (5 mL) solution of
3-bromobenzoyl chloride (1.86 mL, 14.1 mmol) was dropped to
the reaction mixture, which was further stirred for 1 h at the
same temperature. The mixture was poured into ice-cooled 10%
aq HCl and extracted with ethyl acetate. The organic layer was
washed with brine and dried over Na2SO4. After the evaporation
of the solvent, the residue was purified by silica gel column chro-
matography (eluent: hexane/EtOAc) to afford the title compound
(1.20 g, 26%).
1H NMR (CDCl3) d: 2.63 (3H, s), 7.35–7.39 (1H, m), 7.50 (1H, t,
J = 9.6 Hz), 7.59 (1H, t, J = 9.6 Hz), 7.67–7.86 (4H, m), 7.96 (1H, s),
8.43 (1H, d, J = 9.6 Hz), 9.67 (1H, d, J = 9.6 Hz). MS (DI) m/z: 326
(M++H), calcd for C18H13BrO: 325.
5.1.14. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-1-[3-(azulen-2-
ylmethyl)phenyl]-D-glucitol (28)
5.1.11. 3-Bromobenzyl-1-methylazulene (26)
To a solution of 8a (236 mg, 0.620 mmol) in pyridine (5.0 mL)
was added acetic anhydride (0.30 mL, 3.2 mmol) at room temper-
ature. The mixture was stirred at room temperature for 15 h. The
mixture was poured into aq HCl and extracted with EtOAc (Â2).
The combined organic layer was washed with satd aq NaHCO3
and brine, then dried over Na2SO4. After the removal of the solvent
in vacuo, the residue was purified by silica gel column chromatog-
raphy (eluent: hexane/EtOAc = 2/1) to afford the title compound
(0.34 g, quant.) as a blue solid.
To
a solution of 3-bromobenzoyl-1-methylazulene (0.48 g,
1.48 mmol) in diglyme–diethyl ether (20 mL, v/v = 1/1) were added
BF3ÁEt2O (1.17 mL, 9.2 mmol) and NaBH4 (0.68 g, 1.8 mmol) at 0 °C.
After the addition, the reaction mixture was stirred at room tem-
perature for 1 h. The mixture was poured into ice-water, extracted
with ethyl acetate, and extracted with ethyl acetate. The organic
layer was washed with brine and dried over Na2SO4. After the
evaporation of the solvent, the residue was purified by silica gel
column chromatography (eluent: hexane/EtOAc) to afford the title
compound (0.21 g, 46%).
1H NMR (CDCl3) d: 1.63 (3H, s), 1.98 (3H, s), 2.05 (3H, s), 2.06
(3H, s), 3.80–4.38 (4H, m), 4.17 (2H, s), 5.12 (1H, t, J = 9.5 Hz),
5.22 (1H, t, J = 9.8 Hz), 5.31 (1H, t, J = Hz), 7.11–7.32 (8H, m), 7.51
(1H, t, J = 10.0 Hz), 8.20 (2H, d, J = 9.3 Hz). MS (ESI) m/z: 549
(M++H), calcd for C31H32O9: 548.
1H NMR (CDCl3) d: 2.63 (3H, s), 4.37 (2H, s), 6.95–7.53 (8H, m),
8.14–8.17 (2H, m). MS (DI) m/z: 312 (M++H), calcd for C18H15Br:
311.

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3943
5.1.15. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-1-{3-[(1-
acetylazulen-2-yl)methyl]phenyl}- -glucitol (29)
the reaction mixture was added
a
solution of 14 (10.0 g,
D
18.6 mmol) in THF (40 mL) and the mixture was stirred at À78 °C
for 1 h. The reaction was quenched with satd aq NH4Cl, extracted
with EtOAc. The organic layer was washed with brine, dried over
MgSO4, filtered, and evaporated in vacuo. The residue was purified
by column chromatography on silica gel (eluent: hexane/
EtOAc = 5/1) to give the corresponding lactol (2,3,4,6-tetra-O-ben-
zyl-1-C-[5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-fluoro-
phenyl]glucopyranose) (14.8 g, quant.).
To a solution of 28 (200 mg, 0.37 mmol) in CH2Cl2 (20 mL) at
0 °C was added AlCl3 (240 mg, 1.8 mmol) in one-potion. After being
stirred for 30 min at 0 °C, acetic anhydride (0.17 mL, 1.8 mmol)
was dropped to the reaction mixture, which was then refluxed
for 16 h. After cooling, the mixture was poured into ice-cooled
10% aq HCl and extracted with ethyl acetate. The organic layer
was washed with brine and dried over Na2SO4. After the evapora-
tion of the solvent, the residue was purified by silica gel column
chromatography (eluent: hexane/EtOAc = 5/1) to afford the title
compound (90 mg, 42%).
1H NMR (CDCl3) d: 0.10 (6H, s), 0.94 (9H, s), 3.56–4.89 (17H, m),
6.92–7.60 (23H, m). MS (ESI) m/z: 796 (M++NH4), calcd for
C47H55FO7Si: 778.
1H NMR (CDCl3) d: 1.71 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 2.09
(3H, s), 2.70 (3H, s), 3.80–4.38 (4H, m), 4.57 (2H, s), 5.11 (1H, t,
J = 9.5 Hz), 5.22 (1H, t, J = 9.5 Hz), 5.30 (1H, t, J = 9.3 Hz), 6.84
(1H, s), 7.17–7.55 (6H, m), 7.72 (1H, t, J = 9.5 Hz), 8.28 (1H, d,
J = 9.8 Hz), 9.35 (1H, d, J = 10.3 Hz). MS (ESI) m/z: 589 (M-H)-, calcd
for C33H34O10: 590.
Step 2: To a cold (À40 °C) solution of the lactol obtained above
(14.8 g, 19.0 mmol) in CH3CN (100 mL) were added iPr3SiH
(7.8 mL, 38.0 mmol) and BF3ÁOEt2 (2.4 mL, 18.9 mmol) and the
mixture was stirred at À40 °C for 2 h. The reaction was quenched
with satd aq Na2CO3 and extracted with EtOAc (Â2). The organic
layer was washed with brine, dried over MgSO4, filtered, and evap-
orated in vacuo. The residue was purified by column chromatogra-
phy on silica gel (eluent: hexane/EtOAc = 5/1) to give the title
compound 33 (7.10 g, 58% yield) as a colorless solid.
5.1.16. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-1-{3-[(1-
ethylazulen-2-yl)methyl]phenyl}-
To a solution of 29 (90 mg, 0.15 mmol) in diglyme (3.0 mL) and
diethyl ether (3.0 mL) at 0 °C was added BF3ÁOEt2 (60 L,
D-glucitol (30)
1H NMR (CDCl3) d: 3.59–3.63 (2H, m), 3.73–3.84 (4H, m), 4.01
(1H, d, J = 10.7 Hz), 4.47–4.56 (2H, m), 4.60–4.65 (5H, m), 4.85–
4.96 (3H, m), 6.89–6.92 (2H, m), 7.05 (1H, t, J = 9.3 Hz), 7.14–7.35
(19H, m), 7.43 (1H, dd, J = 2.2, 6.6 Hz). MS (ESI) m/z: 666
(M++NH4), calcd for C41H41FO6: 648.
l
0.47 mmol). The mixture was stirred at 0 °C for 0.5 h, then
NaBH3CN (30 mg, 0.47 mmol) was added. The mixture was then re-
fluxed for 15 min. After cooling, the mixture was poured into ice-
water, and extracted with EtOAc (Â2). The combined organic layer
was washed with brine, and dried over Na2SO4. After evaporation
of the solvent, the residue was purified by silica gel column chro-
matography (eluent: hexane/EtOAc = 3/1) to afford the title com-
pound (88 mg, quant.) as a blue oil.
5.1.20. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[5-
(bromomethyl)-2-fluorophenyl]-D-glucitol (35)
The title compound was prepared from 33 in the same manner
as described for 15 (78% yield, a colorless solid).
1H NMR (CDCl3) d: 3.60–4.00 (7H, m), 4.41–4.95 (10H, m), 6.90–
7.51 (23H, m). MS (FAB) m/z: 733 (M++Na), calcd for C41H40BrFO5:
710.
1H NMR (CDCl3) d: 1.81 (3H, t, J = 7.8 Hz), 1.67 (3H, s), 1.98 (3H,
s), 2.05 (3H, s), 2.08 (3H, s), 3.04 (2H, dd, J = 7.8 Hz), 3.60–4.36 (6H,
m), 5.08–5.31 (3H, m), 6.99–7.51 (8H, m), 8.09 (1H, d, J = 9.8 Hz),
8.20 (1H, d, J = 9.3 Hz). MS (ESI) m/z: 577 (M++H), calcd for
C33H36O9: 576.
5.1.21. (1S)-2,3,4,6-Tetra-O-benzyl-1,5-anhydro-1-(2-fluoro-5-
{[1-(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-
(37)
D-glucitol
5.1.17. (1S)-1,5-Anhydro-1-{3-[(1-ethylazulen-2-
yl)methyl]phenyl}- -glucitol (10)
D
The title compound was prepared from 35 in the same manner
as described for 19 (72% yield, a red–purple oil).
To a solution of 30 (88 mg, 0.15 mmol) in THF (2 mL) and MeOH
(2.0 mL) at 0 °C was added NaOMe (4.0 mg, 0.074 mmol). The mix-
ture was stirred at 0 °C for 2 h, and then neutralized with acidic
ion-exchange resin (ꢀpH 6). After filtration and evaporation, the
residue was purified by silica gel column chromatography (eluent:
CHCl3/MeOH = 10/1) to afford the title compound (48 mg, 77%) as a
blue solid.
1H NMR (CDCl3) d: 3.60–3.99 (7H, m), 3.91 (3H, s), 4.43–4.93
(10H, m), 6.85–7.44 (25H, m), 7.50 (1H, t, J = 10.3 Hz), 7.68 (1H, t,
J = 9.8 Hz), 8.05 (1H, d, J = 9.3 Hz), 9.51 (1H, d, J = 10.3 Hz). MS
(ESI) m/z: 834 (M++NH4), calcd for C53H49FO7: 816.
1H NMR (CD3OD) d: 1.20 (3H, t, J = 7.5 Hz), 3.07 (2H, dd, J = 7.6,
14.9 Hz), 3.30–3.88 (6H, m), 4.10 (1H, d, J = Hz), 4.31 (2H, s), 7.00–
7.45 (8H, m), 8.08 (1H, d, J = 9.3 Hz), 8.22 (1H, d, J = 9.8 Hz). MS
(ESI) m/z: 409 (M++H), calcd for C25H28O5: 408.
5.1.22. (1S)-1,5-Anhydro-1-[5-(azulen-2-ylmethyl)-2-
fluorophenyl]-
Step 1:
D
-glucitol (8b)
(1S)-1,5-Anhydro-1-(2-fluoro-5-{[1-(methoxycar-
bonyl)azulen-2-yl]methyl}phenyl)-D-glucitol was prepared from
37 in the same manner as described for 20 (72% yield, a red–purple
oil).
5.1.18. (1S)-1,5-Anhydro-1-{3-[(1-acetylazulen-2-
yl)methyl]phenyl}-D-glucitol (12)
1H NMR (CD3OD) d: 3.38–3.88 (6H, m), 3.93 (3H, s), 4.48–4.51
(1H, m), 4.57 (2H, s), 6.97 (1H, dd, J = 8.3, 9.8 Hz), 7.06 (1H, s),
7.14–7.18 (1H, m), 7.44–7.51 (2H, m), 7.56 (1H, t, J = 9.8 Hz), 7.80
(1H, m), 8.38 (1H, d, J = 9.3 Hz), 9.46 (1H, d, J = 9.8 Hz). MS (ESI)
m/z: 457 (M++H), calcd for C25H25FO7: 456.
The title compound was prepared from 29 in the same manner
as described for 10 (89% yield, a red–purple foam).
1H NMR (CD3OD) d: 2.69 (3H, s), 3.30–3.87 (6H, m), 4.06 (2H, s),
4.10 (1H, d, J = 9.3 Hz), 7.03 (1H, s), 7.12–7.46 (4H, m), 7.49 (1H, t,
J = 9.8 Hz), 7.57 (1H, t, J = 9.5 Hz), 7.79 (1H, t, J = 9.7 Hz), 8.37 (1H,
d, J = 9.8 Hz), 9.30 (1H, d, J = 9.8 Hz). MS (ESI) m/z: 423 (M++H),
calcd for C25H26O6: 422.
Step 2: (1S)-1,5-Anhydro-1-{5-[(1-carboxyazulen-2-yl)methyl]-
2-fluorophenyl}-D-glucitol was prepared in the same manner as
described for 22 (92% yield, a red–purple solid).
1H NMR (CD3OD) d: 3.38–3.87 (6H, m), 4.49 (1H, m), 4.61 (2H,
s), 6.96–7.56 (6H, m), 7.77 (1H, t, J = 9.3 Hz), 8.34 (1H, d,
J = 9.8 Hz), 9.52 (1H, d, J = 9.8 Hz). MS (ESI) m/z: 441 (MÀÀH), calcd
for C24H23FO7: 442.
5.1.19. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[2-fluoro-5-
(hydroxymethyl)phenyl]-D-glucitol (33)
Step 1: To a cold (À78 °C) solution of 31 (12.1 g, 37.9 mmol) in
THF (100 mL) was added n-BuLi (1.6 M in hexane; 24.0 mL,
38.4 mmol) and the mixture was stirred at À78 °C for 0.5 h. To
Step 3: The title compound 8b was prepared in the same man-
ner as described for 8a (93% yield, a blue solid).

3944
K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
1H NMR (CD3OD) d: 3.34–3.88 (6H, m), 4.31 (2H, s), 4.49–4.83
tion was washed with satd aq Na2CO3 and brine. The organic layer
was dried over Na2SO4, filtered, and evaporated in vacuo. To the
obtained residue were added pyridine (20 mL) and acetic anhy-
dride (10 mL) and the mixture was stirred at room temperature
overnight. The reaction was quenched by adding MeOH and the
mixture was evaporated in vacuo (co-evaporated with toluene
2 times) and dried to give the title compound (0.86 mg, 31% yield)
as a pale yellow solid.
(1H, m), 6.97–7.54 (8H, m), 8.20 (2H, d, J = 9.7 Hz). MS (ESI) m/z:
399 (M++H), calcd for C23H23FO5: 398.
5.1.23. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-
(hydroxymethyl)-4-methoxyphenyl]-D-glucitol (34)
The title compound was prepared from 32 in the same manner
as described for 33 (31% yield over two steps, a colorless solid).
1H NMR (CDCl3) d: 2.14 (1H, t, J = 6.6 Hz), 3.46–3.84 (7H, m), 3.89
(3H, s), 4.19–4.97 (m, 10H), 6.88 (1H, d, J = 7.8 Hz), 6.93–7.38 (m,
22H). MS (ESI) m/z: 678 (M++NH4), calcd for C42H44O7: 660.
1H NMR (CDCl3) d: 1.84 (3H, s), 2.01 (3H, s), 2.06 (3H, s), 2.10
(3H, s), 2.33 (3H, s), 3.88 (1H, ddd, J = 2.2, 4.8, 9.9 Hz), 4.16 (1H,
dd, J = 2.2, 12.5 Hz), 4.29 (1H, dd, J = 4.8, 12.5 Hz), 4.97 (1H, d,
J = 9.9 Hz), 5.16–5.43 (3H, m), 7.05 (1H, dd, J = 2.2, 8.1 Hz), 7.21
(1H, d, J = 8.1 Hz), 7.30 (1H, d, J = 2.2 Hz). MS (FAB) m/z: 457
(M++H), calcd for C21H25ClO9: 456.
5.1.24. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-
(bromomethyl)-4-methoxyphenyl]-D-glucitol (36)
The title compound was prepared from 34 in the same manner
as described for 15 (86% yield, a colorless oil).
5.1.29. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-(5-
(bromomethyl)-2-chlorophenyl)-D-glucitol (47)
1H NMR (CDCl3) d: 3.49–3.88 (7H, m), 3.91 (3H, s), 4.17–4.96
(10H, m), 6.85–7.41 (23H, m).
To a solution of 2 (850 mg, 1.86 mmol) in CCl4 (20 mL) was
added N-bromosuccinimide (397 mg, 2.23 mmol) at room temper-
ature. After the mixture was warmed up to reflux, azobisisobutyro-
nitrile (76 mg, 0.46 mmol) was added in one portion and the
mixture was refluxed for additional 1.5 h. The mixture was poured
into satd aq Na2CO3 and extracted with CHCl3 (Â2). The organic
layer was washed with brine, dried over Na2SO4, filtered, and evap-
orated in vacuo. The resulting residue was purified by column
chromatography on silica gel (hexane/EtOAc = 75/25) to give the ti-
tle compound (960 mg, 96% yield) as a colorless foam.
5.1.25. (1S)-2,3,4,6-Tetra-O-benzyl-1,5-anhydro-1-(4-methoxy-
3-{[1-(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-
(38)
D-glucitol
The title compound was prepared from 33 in the same manner
as described for 37 (34% yield, a colorless solid).
1H NMR (CDCl3) d: 3.49–3.89 (10H, m), 3.94 (3H, s), 4.09–4.95
(10H, m), 6.74–7.68 (27H, m), 7.94 (1H, d, J = 9.8 Hz), 9.52 (1H, d,
J = 9.8 Hz). MS (ESI) m/z: 851 (M++Na), calcd for C54H52O8: 828.
1H NMR (CDCl3) d: 1.85 (3H, s), 2.01 (3H, s), 2.07 (3H, s), 2.11
(3H, s), 3.89 (1H, ddd, J = 2.2, 4.8, 9.9 Hz), 4.17 (1H, dd, J = 2.2,
12.3 Hz), 4.29 (1H, dd, J = 4.7, 12.3 Hz), 4.40–4.50 (2H, m), 4.96
(1H, d, J = 9.9 Hz), 5.18–5.41 (3H, m), 7.27–7.64 (3H, m). MS (EI)
m/z: 535 (M+), calcd for C21H24BrClO9: 535.
5.1.26. (1S)-1,5-Anhydro-1-[3-(azulen-2-ylmethyl)-4-
methoxyphenyl]-
Step 1: (1S)-1,5-Anhydro-1-(4-methoxy-3-{[1-(methoxycar-
bonyl)azulen-2-yl]methyl}phenyl)- -glucitol was prepared from
D-glucitol (8h)
D
38 in the same manner as described for 20 (34% yield, a red–purple
oil).
1H NMR (CDCl3) d: 3.29–4.56 (m, 15H), 6.79–7.18 (4H, m), 7.41
(1H, t, J = 9.8 Hz), 7.52 (1H, t, J = 9.8 Hz), 7.74 (1H, t, J = 9.8 Hz), 8.28
(1H, d, J = 9.8 Hz), 9.42 (1H, d, J = 9.8 Hz). MS (ESI) m/z: 469 (M++H),
calcd for C26H28O8: 468.
5.1.30. (1S)-1,5-Anhydro-1-[5-(azulen-2-ylmethyl)-2-
chlorophenyl]-D-glucitol (8c)
To a mixture of zinc dust (83 mg, 1.26 mmol) and THF (4 mL)
was added 1,2-dibromoethane (2 drops) and the mixture was
warmed at 60 °C for 2 min, then cooled to room temperature.
To the mixture was added trimethylsilyl chloride (2 drops) and
the mixture was stirred at room temperature for 10 min. To the
mixture was added 47 (450 mg, 0.84 mmol) and the mixture
was stirred at 80 °C for 1 h to form the corresponding benzyl zinc
bromide in situ. To the mixture were added methyl 2-chloroazu-
lene-1-carboxylate (220 mg, 1.01 mmol) and Pd(PPh3)4 (115 mg,
0.10 mmol) and the mixture was stirred at 80 °C for 5 h. After
cooled to room temperature, the mixture was filtered through a
pad of CeliteÒ (washed with EtOAc). The filtrate was concentrated
in vacuo to give crude 50. To the obtained crude product were
added MeOH and a catalytic amount of NaOMe and the mixture
was stirred at room temperature overnight (the formation of
the corresponding tetraol was confirmed by TLC monitoring).
The mixture was neutralized with Dowex 50 W-X8, filtered, and
evaporated in vacuo. The crude residue was mixed with MeOH
and 10% aq NaOH (excess) and the mixture was stirred at 90 °C
overnight. After cooling, the mixture was neutralized with 1 M
aq HCl and extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, filtered, and evaporated in vacuo
to give the crude carboxylic acid. To the residue were added
CH3CN and 1 M HCl/EtOAc (excess) and the mixture was stirred
at 90 °C for 30 min. After evaporation, the residue was purified
by column chromatography on silica gel (eluent: CHCl3/
MeOH = 95/5) to give the title compound (35 mg, 10% yield over
four steps) as a purple solid.
Step 2: (1S)-1,5-Anhydro-1-{3-[(1-carboxyazulen-2-yl)methyl]-
4-methoxyphenyl}-D-glucitol was prepared in the same manner as
described for 22 (77% yield, a red–purple solid).
1H NMR (CD3OD) d: 3.30–4.50 (7H, m), 3.80 (3H, s), 4.28 (2H, s),
6.92–7.31 (6H, m), 7.48 (1H, t, J = 9.3 Hz), 8.16 (2H, d, J = 9.3 Hz).
MS (ESI) m/z: 477 (M++Na), calcd for C25H26O8: 454.
Step 3: The title compound 8h was prepared in the same man-
ner as described for 8a (18% yield, a blue–purple solid).
1H NMR (CD3OD) d: 3.30–4.29 (7H, m), 3.83 (3H, s), 4.30 (2H, s),
6.94–7.29 (7H, m), 7.46 (1H, t, J = 9.8 Hz), 8.16 (2H, d, J = 9.3 Hz).
MS (ESI) m/z: 411 (M++H), calcd for C24H26O6: 410.
5.1.27. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-(2-chloro-5-
methylphenyl)-D-glucitol (41)
The title compound was prepared from 39 in the same manner
as described for 33 (63% yield over two steps, a colorless oil).
1H NMR (CDCl3) d: 2.31 (3H, s), 3.58–3.90 (6H, m), 3.96 (1H, d,
J = 10.6 Hz), 4.38 (1H, d, J = 10.4 Hz), 4.50–4.68 (3H, m), 4.78–4.98
(4H, m), 6.91–7.08 (3H, m), 7.12–7.38 (20H, m). MS (FAB) m/z: 649
(M++H), calcd for C41H41ClO5: 648.
5.1.28. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-(2-chloro-5-
methylphenyl)-D-glucitol (43)
To a cold (À78 °C) solution of 41 (4.00 g, 6.16 mmol) in CH2Cl2
(120 mL) was added BBr3 (1.0 M in CH2Cl2; 28.0 mL, 28.0 mmol)
and the mixture was stirred at À78 °C for 1 h. The reaction was
quenched by adding MeOH at À78 °C. The mixture was evaporated
in vacuo. The residue was diluted with EtOAc and the organic solu-
1H NMR (CD3OD) d: 3.35–3.56 (4H, m), 3.67–3.71 (1H, m), 3.81
(1H, d, J = 12.2 Hz), 4.32 (2H, s), 4.69–4.75 (1H, m), 7.06–7.21 (5H,

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3945
m), 7.30 (1H, d, J = 8.3 Hz), 7.46–7.58 (2H, m), 8.20 (2H, d,
J = 9.2 Hz). MS (FAB) m/z: 415 (M++H), calcd for C23H23ClO5: 414.
Step 2: (1S)-1,5-Anhydro-1-{5-[(1-carboxyazulen-2-yl)methyl]-
2-methoxyphenyl}- -glucitol was prepared in the same manner as
D
described for 22 (98% yield, a red–purple solid).
5.1.31. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-(2-methoxy-5-
1H NMR (CD3OD) d: 3.29–3.66 (5H, m), 3.78 (3H, s), 3.84 (1H,
m), 4.55 (2H, s), 4.70 (1H, d, J = 9.3 Hz), 6.88 (1H, d, J = 8.3 Hz),
6.98 (1H, s), 7.15 (1H, dd, J = 2.2, 8.5 Hz), 7.37–7.42 (2H, m), 7.49
(1H, t, J = 9.8 Hz), 7.72 (1H, t, J = 9.8 Hz), 8.28 (1H, d, J = 9.3 Hz),
9.48 (1H, d, J = 9.8 Hz). MS (ESI) m/z: 453 (MÀÀH), calcd for
methylphenyl)-
D-glucitol (46)
To a cooled (0 °C) solution of 1,2,3,4,6-penta-O-acetyl-
D-gluco-
pyranose (45) (10.0 g, 25.6 mmol) and 1-methoxy-4-methylben-
zene (6.50 mL, 51.5 mmol) in CH2Cl2 (125 mL) were added AgOTf
(8.50 g, 33.1 mmol) and SnCl4 (1 M solution in CH2Cl2; 91 mL,
91 mmol) dropwise. The mixture was stirred for 4 h at 0 °C and
quenched with satd aq NaHCO3. Insoluble materials were filtered
off, and the filtrate was extracted with CH2Cl2. The organic layer
was washed with brine, dried over Na2SO4, filtered, and evaporated
in vacuo. The resulting residue was purified by column chromatog-
raphy on silica gel (hexane/EtOAc = 2/1) to give the title compound
(4.75 g, 41% yield) as a pale yellow oil.
C25H26O8: 454.
Step 3: The title compound 8d was prepared from the above-
mentioned carboxylic acid in the same manner as described for
8a (61% yield, a blue solid).
1H NMR (CD3OD) d: 3.36–3.90 (6H, m), 3.86 (3H, s), 4.31 (2H, s),
4.74 (1H, d, J = 9.3 Hz), 6.97 (1H, d, J = 8.3 Hz), 7.15–7.43 (6H, m),
7.54 (1H, t, J = 9.8 Hz), 8.23 (2H, d, J = 9.3 Hz). MS (ESI) m/z: 411
(M++H), calcd for C24H26O6: 410.
1H NMR (CDCl3) d: 1.77 (3H, s), 2.01 (3H, s), 2.05 (3H, s), 2.08
(3H, s), 2.28 (3H, s), 3.80 (3H, s), 3.84 (1H, m), 4.13 (1H, m), 4.27
(1H, d, J = 4.9, 12.2 Hz), 4.92 (1H, d, J = 9.8 Hz), 5.21–5.38 (3H,
m), 6.74 (1H, d, J = 8.3 Hz), 7.06 (1H, d, J = 2.0, 8.3 Hz), 7.18
(1H, d, J = 2.0 Hz). MS (ESI) m/z: 453 (M++H), calcd for
5.1.35. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[2-
(benzyloxy)-5-methylphenyl]-D-glucitol (42)
Step 1: To a cold (À78 °C) solution of 1-benzyloxy-2-bromo-4-
methylbenzene (40)40 (20.0 g, 72.2 mmol) in THF (250 mL) was
added n-BuLi (1.56 M in hexane; 50.0 mL, 78.0 mmol) and the mix-
ture was stirred at À78 °C for 1 h. To the reaction mixture was
added a solution of 13 (35.0 g, 65.0 mmol) in THF (200 mL) and
the mixture was stirred at À78 °C for 1.5 h. The reaction was
quenched with 1 N aq HCl (10 mL) and dried over MgSO4. After fil-
tration (washed with EtOAc), the filtrate was evaporated in vacuo
to give a brown residue. The crude product was purified by column
chromatography on silica gel (eluent: hexane/EtOAc = 85/15) to
give the corresponding lactol (2,3,4,6-tetra-O-benzyl-1-C-[2-(ben-
C22H28O10: 452.
5.1.32. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-[5-
(bromomethyl)-2-methoxyphenyl]- -glucitol (48)
D
To a solution of 46 (4.75 g, 10.5 mmol) in CCl4 (30 mL) were
added NBS (2.24 g, 12.6 mmol) and a catalytic amount of AIBN.
The mixture was refluxed for 30 min. After cooling, the organic
solution was washed with satd aq NaHCO3 and brine, dried over
Na2SO4, filtered, and evaporated in vacuo. The resulting residue
was passed through short column on silica gel (hexane/
EtOAc = 2/1) to give the title compound (3.00 g, 54% yield) as a col-
orless solid.
zyloxy)-5-methylphenyl)glucopyranose) as
(37.6 g, 79%).
a
colorless solid
1H NMR (CDCl3) d: 2.29 (3H, s), 3.66–3.86 (3H, m), 3.47–4.22
(4H, m), 4.47–4.64 (4H, m), 4.77–5.00 (6H, m), 6.80–7.45 (28H,
m). MS (FAB) m/z: 719 (M+ÀOH), calcd for C48H48O7: 736.
1H NMR (CDCl3) d: 1.77 (3H, s), 2.01 (3H, s), 2.06 (3H, s), 2.09
(3H, s), 3.85 (3H, s), 3.87 (1H, m), 4.14 (1H, dd, J = 2.2, 12.2 Hz),
4.27 (1H, dd, J = 4.9, 12.2 Hz), 4.46 (1H, ABq, J = 10.3 Hz), 4.49
(1H, ABq, J = 10.3 Hz), 4.88 (1H, d, J = 9.8 Hz), 5.23–5.36 (3H, m),
6.82 (1H, d, J = 8.3 Hz), 7.32 (1H, dd, J = 2.4, 8.3 Hz), 7.39 (1H, d,
J = 2.4 Hz).
Step 2: To a ice-cold solution of the lactol obtained above
(37.0 g, 50.2 mmol) in CH2Cl2 (100 mL) and CH3CN (300 mL) were
added iPr3SiH (30.9 mL, 151 mmol) and BF3ÁOEt2 (12.6 mL,
100 mmol) and the mixture was stirred in a ice-water bath for
1 h (the internal temperature raised up to 9 °C after the addition
of BF3ÁOEt2, then kept below 5 °C). The reaction mixture was
poured into satd aq Na2CO3 (800 mL) and extracted with CHCl3
(Â2). The organic layer was dried over MgSO4, filtered, and evapo-
rated in vacuo. The residue was purified by column chromatogra-
phy on silica gel (hexane/EtOAc = 90/10) to give the title
compound 42 (16.9 g, 47% yield) as a pale yellow oil.
5.1.33. (1S)-2,3,4,6-Tetra-O-acetyl-1,5-anhydro-1-(2-methoxy-5-
{[1-(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-
D-glucitol
(51)
The title compound was prepared from 48 in the same manner
as described for 19 (44% yield, a red–purple oil).
1H NMR (CDCl3) d: 1.74–2.09 (12H, m), 3.82 (3H, s), 3.96 (3H, s),
4.11–4.27 (2H, m), 4.55 (2H, s), 4.91–5.36 (5H, m), 6.79–7.69 (7H,
m), 8.29 (1H, d, J = 9.8 Hz), 9.53 (1H, d, J = 10.4 Hz). MS (ESI) m/z:
637 (M++H), calcd for C34H36O12: 636.
1H NMR (CDCl3) d: 2.35 (3H, s), 3.54–3.87 (6H, m), 3.99 (1H, d,
J = 10.7 Hz), 4.43 (1H, d, J = 10.8 Hz), 4.53 (1H, d, J = 12.2 Hz), 4.58–
4.67 (2H, m), 4.74–5.02 (6H, m), 6.81 (1H, d, J = 8.3 Hz), 6.86–6.93
(2H, m), 7.01–7.41 (25H, m). MS (FAB) m/z: 719 (MÀÀH), calcd for
C48H48O6: 720.
5.1.34. (1S)-1,5-Anhydro-1-[5-(azulen-2-ylmethyl)-2-
methoxylphenyl]-D-glucitol (8d)
5.1.36. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-[2-(acetyloxy)-
5-methylphenyl]- -glucitol (44)
Step 1: To a cold (0 °C) solution of 51 (0.19 g, 0.30 mmol) in
MeOH (5.0 mL) was added NaOMe (8.0 mg, 0.10 mmol). The mix-
ture was stirred at 0 °C for 1 h and neutralized with acidic ion-ex-
change resin (ꢀpH 6). After filtration and evaporation in vacuo, the
residue was purified by column chromatography on silica gel
(CHCl3/MeOH = 10/1) to give (1S)-2,3,4,6-1,5-anhydro-1-(2-meth-
D
To a solution of 42 (16.8 g, 23.3 mmol) in EtOAc (150 mL) and
MeOH (75 mL) was added Pd(OH)2/C (20 wt %; 168 mg) and the
mixture was stirred under H2 atmosphere (1 atm) at room temper-
ature overnight. The catalyst was filtered off and the filtrate was
evaporated in vacuo. The solid residue was washed with EtOAc
to give the crude product as a colorless solid (6.57 g). The obtained
crude product was mixed with pyridine (30 mL) and acetic anhy-
dride (15 mL), then the mixture was stirred at room temperature
overnight. The reaction was quenched by adding MeOH and the
mixture was evaporated in vacuo (co-evaporated with toluene 2
times). Crystallization in ethanol gave the title compound (9.13 g,
82% yield) as a colorless solid.
oxy-5-{[1-(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-
tol (0.095 g, 68% yield) as a red–purple oil.
D-gluci-
1H NMR (CD3OD) d: 3.30–3.66 (5H, m), 3.78 (3H, s), 3.86 (1H, d,
J = 11.7 Hz), 3.91 (3H, s), 4.49 (2H, s), 4.70 (1H, d, J = 8.8 Hz), 6.88
(1H, d, J = 8.3 Hz), 7.00 (1H, s), 7.09–7.72 (5H, m), 8.28 (1H, d,
J = 8.8 Hz), 9.40 (1H, d, J = 10.2 Hz). MS (ESI) m/z: 469 (M++H), calcd
for C26H28O8: 468.

3946
K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
1H NMR (CDCl3) d: 1.79 (3H, s), 2.01 (3H, s), 2.05 (3H, s),
tered through a pad of Celite to remove inorganic salts (washed
with CHCl3–MeOH). After the evaporation of the filtrate, the purple
residue was purified by column chromatography on silica gel (elu-
ent: CHCl3/MeOH = 20/1–13/1) to give the title compound
(759 mg, 53%) as a blue–purple solid.
2.09 (3H, s), 2.34 (3H, s), 2.34 (3H, s), 3.81 (1H, ddd, J = 2.4,
4.8, 9.9 Hz), 4.10 (1H, dd, J = 1.9, 12.2 Hz), 4.31 (1H, dd, J = 4.8,
12.2 Hz), 4.61 (1H, d, J = 9.7 Hz), 5.17–5.35 (3H, m), 6.94 (1H,
d, J = 8.3 Hz), 7.13 (1H, dd, J = 2.5, 8.3 Hz), 7.25 (1H, d, J =
2.5 Hz). MS (FAB) m/z: 481 (M++H), calcd for C23H28O11: 480.
1H NMR (CD3OD) d: 3.37–3.59 (4H, m), 3.70 (1H, dd, J = 5.4,
12.2 Hz), 3.86 (1H, dd, J = 2.0, 11.7 Hz), 4.23 (2H, s), 4.56 (1H, d,
J = 9.8 Hz), 6.76 (1H, d, J = 8.3 Hz), 7.02–7.16 (5H, m), 7.29 (1H, d,
J = 2.4 Hz), 7.49 (1H, t, J = 10.0 Hz), 8.17 (2H, d, J = 9.3 Hz). MS
(FAB) m/z: 397 (M++H), calcd for C23H24O6: 396. Anal. Calcd for
5.1.37. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-[2-acetyloxy-
5-(bromomethyl)phenyl]-D-glucitol (49)
To a refluxed solution of 44 (10.0 g, 210.9 mmol) and N-bromo-
succinimide (7.43 g, 41.7 mmol) in EtOAc (20 mL) was added azob-
isisobutyronitrile (1.03 g, 6.26 mmol) in one portion and the
mixture was refluxed for 1 h. After cooling, the mixture was
washed with water (150 mL Â 3 times) and brine (100 mL). The or-
ganic layer was dried over Na2SO4, filtered, and evaporated in va-
cuo. The obtained residue was dissolved in hot ethanol (35 mL,
temperature maintained below 45 °C), then cooled to room tem-
perature. After the addition of hexane–Et2O (v/v = 5/1; 100 mL),
the precipitate was collected by filtration and dried to give the title
compound (5.56 g, 48% yield) as a colorless solid.
C23H24O6Á0.5 H2O: C, 68.14; H, 6.21. Found: C, 68.05; H, 6.26.
5.1.40. [(5-Bromo-2-fluorobenzyl)oxy](tert-
butyl)diphenylsilane (54)
To a cold (0 °C) solution of 5-bromo-2-fluorobenzaldehyde
(10.0 g, 49.2 mmol) in EtOH (100 mL) was added NaBH4 (2.00 g,
54.2 mmol) and the mixture was stirred at 0 °C for 1 h. The reac-
tion was quenched with 1 M aq HCl, and extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4, fil-
tered, and evaporated in vacuo to give the crude material. To a
ice-cold solution of the crude alcohol in DMF (150 mL) were added
imidazole (5.00 mL, 73.8 mmol) and tert-butyl(chloro)diphenylsi-
lane (13.9 mL, 54.1 mmol) and the mixture was stirred at room
temperature for 1 h. The reaction mixture was poured into water
and extracted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4, filtered, and evaporated in va-
cuo. The residue was purified by column chromatography on silica
gel (hexane/EtOAc = 19/1) to give the title compound (15.7 g, 72%
yield) as a pale yellow oil.
1H NMR (CDCl3) d: 1.80 (3H, s), 2.01 (3H, s), 2.06 (3H, s), 2.10
(3H, s), 2.36 (3H, s), 3.82 (1H, ddd, J = 2.0, 4.8, 9.7 Hz), 4.10 (1H,
dd, J = 2.0, 12.5 Hz), 4.33 (1H, dd, J = 4.8, 12.3 Hz), 4.43–4.51 (2H,
m), 4.62 (1H, d, J = 9.3 Hz), 5.17–5.37 (3H, m), 7.06 (1H, d,
J = 8.4 Hz), 7.38 (1H, dd, J = 2.2, 8.3 Hz), 7.46 (1H, d, J = 2.2 Hz).
MS (FAB) m/z: 559 (M++H), calcd for C23H27BrO11: 558.
5.1.38. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-acetyl-1-(2-acetoxy-5-
{[1-(methoxycarbonyl)azulen-2-yl]methyl}phenyl)-
D-glucitol
(52)
1H NMR (CDCl3) d: 1.11 (9H, s), 4.77 (2H, s), 6.85 (1H, t,
J = 9.0 Hz), 7.29–7.49 (7H, m), 7.65–7.74 (5H, m). MS (FAB) m/z:
443 (M++H), calcd for C23H24BrFOSi: 442.
To a mixture of zinc dust (200 mg, 3.06 mmol) and THF (6.0 mL)
was added 1,2-dibromoethane (4 drops) and the mixture was
warmed at 60 °C for 2 min, then cooled to room temperature. To
the mixture was added trimethylsilyl chloride (4 drops) and the
mixture was stirred at room temperature for 15 min. To the mix-
ture was added 49 (856 mg, 1.53 mmol) and the mixture was stir-
red at 80 °C for 30 min to form the corresponding benzyl zinc
bromide in situ. To the mixture were added methyl 2-chloroazu-
lene-1-carboxylate (18)33 (307 mg, 1.39 mmol) and Pd(PPh3)4
(162 mg, 0.14 mmol) and the mixture was refluxed for 2 h. After
cooled to room temperature, the mixture was filtered through a
pad of CeliteÒ (washed with EtOAc). After evaporation, the residue
was purified by column chromatography on silica gel (hexane/
EtOAc = 4/1–2/1) to give the title compound (437 mg, 47% yield)
as a purple foam.
5.1.41. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[4-fluoro-3-
(hydroxymethyl)phenyl]-D-glucitol (55)
Step 1: To a cold (À78 °C) solution of 54 (15.6 g, 35.2 mmol) in
THF (75 mL) and toluene (75 mL) was added n-BuLi (1.59 M in
hexane; 24.5 mL, 38.7 mmol) and the mixture was stirred at
À78 °C for 0.5 h. To the reaction mixture was added a solution
of 13 (17.0 g, 31.7 mmol) in a mixed solvent of THF–toluene (v/
v = 1/1, 170 mL) and the mixture was stirred at À40 °C for 2 h.
The reaction was quenched with satd aq NH4Cl, extracted with
EtOAc. The organic layer was washed with brine, dried over
MgSO4, filtered, and evaporated in vacuo to give the crude lactol
(20.3 g).
Step 2: To a cold (À20 °C) solution of the above-obtained lac-
tol (20.3 g) in CH3CN (300 mL) were added iPr3SiH (9.2 mL,
45.0 mmol) and BF3ÁOEt2 (3.4 mL, 27.0 mmol) and the mixture
was stirred at the same temperature for 2 h. The reaction mix-
ture was poured into satd aq Na2CO3 and extracted with EtOAc
(Â2). The organic layer was washed with brine, dried over
MgSO4, filtered, and evaporated in vacuo to give the crude
material.
1H NMR (CDCl3) d: 1.75 (3H, s), 1.99 (3H, s), 2.02–2.06 (6H, m),
2.36 (3H, s), 3.77–3.85 (1H, m), 3.95 (3H, m), 4.05–4.12 (1H, m),
4.30 (1H, dd, J = 4.6, 12.4 Hz), 4.57–4.65 (3H, m), 5.14–5.35 (3H,
m), 6.92 (1H, s), 6.98–7.04 (1H, m), 7.22 (1H, dd, J = 2.0, 8.3 Hz),
7.35–7.58 (3H, m), 7.72 (1H, t, J = 9.9 Hz), 8.30 (1H, d, J = 9.5 Hz),
9.54 (1H, d, J = 9.9 Hz). MS (FAB) m/z: 665 (M++H), calcd for
C
35H36O13: 664.
5.1.39. (1S)-1,5-Anhydro-1-[5-(azulen-2-ylmethyl)-2-
hydroxyphenyl]- -glucitol (8e)
Step 3: The above-obtained crude material was dissolved in THF
(300 mL) and then mixed with tetra-n-butylammonium fluoride
(1 M in THF; 50 mL, 50 mmol). The mixture was stirred at room
temperature for 1 h and concentrated in vacuo. The residue was di-
luted with EtOAc. The organic solution was washed with water and
brine, dried over MgSO4, filtered, and evaporated in vacuo. The res-
idue was purified by column chromatography on silica gel (hex-
ane/EtOAc = 80/20–75/25) to give the title compound 55 (8.2 g,
36% yield) as a colorless solid.
D
To a solution of 52 (1.74 g, 3.65 mmol) in MeOH (3.0 mL) was
added aq NaOH (292 mg, 7.29 mmol in H2O 5.0 mL). The mixture
was refluxed (bath temp. 110 °C) for 1 h. Additional aq NaOH
(1.46 g, 36.5 mmol in H2O 10 mL) and MeOH (12 mL) were added
and the mixture was refluxed for 2 h. After being cooled to room
temperature, the solvents were evaporated in vacuo to give a dark
purple residue (4.84 g). This residue was mixed with CH3CN
(40 mL) and 4 M HCl in EtOAc (20 mL) at room temperatue. The
mixture was refluxed (bath temp. 93 °C) for 40 min. Additional
CH3CN (20 mL) and 4 M HCl in EtOAc (10 mL) were added and
the blue–purple mixture was refluxed for 2 h. The mixture was fil-
1H NMR (CDCl3) d: 3.40–3.63 (2H, m), 3.71–3.87 (5H, m), 4.23
(1H, d, J = 9.6 Hz), 4.44–4.72 (6H, m), 4.83–4.98 (3H, m), 6.90–
7.48 (23H, m). MS (FAB) m/z: 649 (M++H), calcd for C41H41FO6: 648.

K. Ikegai et al. / Bioorg. Med. Chem. 21 (2013) 3934–3948
3947
5.1.42. (1S)-1,5-Anhydro-2,3,4,6-tetra-O-benzyl-1-[3-
(bromomethyl)-4-fluorophenyl]- -glucitol (56)
stirred. The solid was collected with filtration and dried to give
the title compound (1.68 g, 60% yield) as a blue–purple solid.
1H NMR (CD3OD) d: 3.34–3.44 (4H, m), 3.67 (1H, dd, J = 12.0,
5.2 Hz), 3.86 (1H, m), 4.09 (1H, d, J = 9.2 Hz), 4.45 (2H, d,
J = 5.6 Hz), 7.10–7.15 (4H, m), 7.29 (1H, dd, J = 8.4, 2.4 Hz), 7.37
(1H, d, J = 8.4 Hz), 7.42 (1H, d, J = 2.0 Hz), 7.50 (1H, t, J = 10.0 Hz),
8.16 (1H, s), 8.19 (1H, s). MS (DI) m/z: 415 (M++H), calcd for
D
The title compound was prepared from 55 in the same manner
as described for 15 (81% yield, a pale yellow oil).
1H NMR (CDCl3) d: 3.41 (1H, m), 3.60 (1H, m), 3.77 (5H, m),
4.59–4.94 (10H, m), 6.90–7.51 (23H, m).
C23H23ClO5: 414.
5.1.43. (1S)-1,5-Anhydro-1-[3-(azulen-2-ylmethyl)-4-
fluorophenyl]-D-glucitol (8f)
5.2. In vivo pharmacology
Step 1: To a solution of 56 (0.31 g, 0.44 mmol) in THF (10 mL)
were added 58 (0.11 g, 0.44 mmol), Pd2dba3 (12 mg, 0.013 mmol),
tri(2-furyl)phosphine (9.0 mg, 0.057 mmol), K3PO4 (0.14 g,
0.66 mmol), and water (0.1 mL). The mixture was refluxed for
2 h. After cooling, the mixture was filtered through CeliteÒ. The fil-
trate was diluted with EtOAc and washed with water, satd aq NaH-
CO3, and brine. The organic layer was dried over Na2SO4, filtered,
and evaporated in vacuo. The residual solid was collected by filtra-
tion (washed with MeOH) to give crude (1S)-2,3,4,6-tetra-O-ben-
5.2.1. Animals
Male Sprague–Dawley rats were purchased from Charles River
Laboratories Japan (Kanagawa, Japan) at age 5–7 weeks. STZ was
dissolved in 50 mM citric acid buffer before intravenous adminis-
tration at 50 mg/kg. Normal control rats were given physiological
saline. Blood glucose levels were measured in the diabetic rats
1 week later, after which the rats were grouped such that the blood
glucose levels were similar in each group. Male Institute of Cancer
Research normal and KK/Ay type 2 diabetic mice, which exhibit
hyperglycemia, insulin resistance, hyperinsulinemia, hyperlipid-
emia, and obesity, were purchased from Japan SLC, Inc. (Shizuoka,
Japan) and CLEA Japan (Kanagawa, Japan), respectively, at the age
of 5–6 weeks. The diabetic mice were also grouped such that each
group had similar blood glucose levels. All animals were housed
under conventional conditions with controlled temperature,
humidity, and light (12-h light–dark cycle) and were provided with
a standard commercial diet and water (ad libitum). Animals were
handled and cared for in accordance with the Guide for the Care
and Use of Laboratory Animals, and all procedures were approved
by the Animal Ethical Committee of Astellas Pharma Inc.
zyl-1,5-anhydro-1-[3-(azulen-2-ylmethyl)-4-fluorophenyl]-D-
glucitol (59) (0.21 g, 64% yield) as a blue–purple solid.
1H NMR (CDCl3) d: 3.42–3.45 (1H, m), 3.59–3.61 (1H, m), 3.68–
3.90 (5H, m), 4.17 (1H, d, J = 9.6 Hz), 4.42 (1H, d, J = 9.6 Hz), 4.58–
4.63 (4H, m), 4.83–4.95 (4H, m), 6.87–7.36 (27H, m), 7.44 (1H, t,
J = 9.8 Hz), 8.01 (2H, d, J = 9.8 Hz).
Step 2: To a cold (À78 °C) solution of 59 (0.23 g, 0.30 mmol) and
pentamethylbenzene (0.45 g, 3.0 mmol) in CH2Cl2 (5 mL) was
added BCl3 (1 M in CH2Cl2; 1.8 mL, 1.8 mmol). The mixture was
stirred at the same temperature for 1 h. The reaction was quenched
by adding MeOH (5 mL). The mixture was diluted with EtOAc
(20 mL), washed with satd aq NaHCO3 and brine. The organic layer
was dried over Na2SO4, and evaporated in vacuo. The residue
was purified by column chromatography on silica gel (eluent:
CHCl3/MeOH = 7/1) to give the title compound (50 mg, 41% yield)
as a blue–purple solid.
5.2.2. Effects of 8e on urinary glucose excretion in KK/Ay type 2
diabetic mice
1H NMR (CD3OD) d: 3.29–3.44 (4H, m), 3.66 (1H, dd, J = 5.2,
12.0 Hz), 3.85 (1H, d, J = 12.0 Hz), 4.08 (1H, d, J = 10.4 Hz), 4.33
(2H, d, J = 4.4 Hz), 7.03–7.16 (5H, m), 7.30 (1H, m), 7.37 (1H, d,
J = 7.2 Hz), 7.51 (1H, t, J = 9.6 Hz), 8.17 (2H, d, J = 9.2 Hz). MS (DI)
m/z: 399 (M++H), calcd for C23H23FO5: 398.
Compound 8e (0.1–3 mg/kg) was administered to non-fasted
normal mice, and spontaneously voided urine was collected for
24 h after administration while the animals were kept in metabolic
cages. After the urine volume had been measured, the glucose con-
centration in the urine was measured using the Glucose CII test re-
agent (Wako, Osaka, Japan).
5.1.44. (1S)-1,5-Anhydro-1-[3-(azulen-2-ylmethyl)-4-
chlorophenyl]-D-glucitol (8g)
5.2.3. Effects of single administration of 8e in diabetic animals
To investigate its antihyperglycemic effect, 8e (0.1–3 mg/kg)
was administered to STZ-induced type 1 diabetic rats and KK/Ay
type 2 diabetic mice in the fed condition. Blood glucose levels were
then measured for 8 h under fasting conditions, in order to elimi-
nate the influence of feeding during the experiment.
Step 1: To a solution of 5736 (7.4 g, 10.2 mmol) in THF (30 mL)
was added 58 (2.6 g, 10.2 mmol), Pd2dba3 (280 mg, 0.31 mmol),
tri(2-furyl)phosphine (210 mg, 0.91 mmol), K3PO4 (3.2 g,
15 mmol), and water (0.4 mL). The mixture was refluxed for 2 h.
After cooling, the mixture was filtered through CeliteÒ. The filtrate
was diluted with EtOAc and washed with water, satd aq NaHCO3,
and brine. The organic layer was dried over Na2SO4, filtered, and
evaporated in vacuo. The residual solid was collected by filtration
(washed with MeOH) to give crude (1S)-2,3,4,6-tetra-O-benzyl-
Acknowledgments
We are grateful to Dr. Atsuo Tahara, Mr. Masanori Yokono, Mr.
Daisuke Yamajuku, Ms. Rumi Kihara, Ms. Takako Furukawa and Dr.
Fumihiko Ushigome for performing the biological experiments,
and members of the Division of Analytical Science Laboratories of
Astellas Pharma Inc. for elemental analysis and spectral
measurements.
1,5-anhydro-1-[3-(azulen-2-ylmethyl)-4-chlorophenyl]-D-glucitol
(60) (4.8 g, 60%) as a blue-purple solid.
1H NMR (CDCl3) d: 3.46 (1H, m), 3.55 (1H, m), 3.71–3.76 (4H,
m), 3.85 (1H, d, J = 10.4 Hz), 4.17 (1H, d, J = 9.6 Hz), 4.36–4.62
(6H, m), 4.82–4.91 (3H, m), 6.87 (2H, d, J = 6.8 Hz), 7.07–7.41
(25H, m), 7.48 (1H, t, J = 9.8 Hz), 8.09 (2H, d, J = 9.2 Hz)
References and notes
Step 2: To a cold (À40 °C) solution of 60 (4.8 g, 6.2 mmol) and
pentamethylbenzene (4.6 g, 31 mmol) in CH2Cl2 (30 mL) was
added BCl3 (1 M in CH2Cl2; 31 mL, 31 mmol). The mixture was stir-
red at the same temperature for 1 h. The reaction was quenched by
adding MeOH (10 mL). The mixture was diluted with EtOAc
(100 mL), washed with satd aq NaHCO3 and brine. The organic
layer was dried over Na2SO4, and evaporated in vacuo. The purple
residue was mixed with hexane–EtOAc (v/v = 1/1, 10 mL) and
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