Novel macrocyclic C-aryl glucoside SGLT2 inhibitors as potential antidiabetic agents

Article abstract of DOI:10.1016/j.bmc.2011.07.045

Novel macrocyclic C-aryl glucoside SGLT2 inhibitors were designed and synthesized. Two different synthetic routes of macrocyclization were adopted to prepare novel ansa SGLT2 inhibitors. Among the compounds tested, [1,7]dioxacyclopentadecine macrocycles p

Full text of DOI:10.1016/j.bmc.2011.07.045

Bioorganic & Medicinal Chemistry 19 (2011) 5468–5479
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel macrocyclic C-aryl glucoside SGLT2 inhibitors as potential
antidiabetic agents
Min Ju Kim, Suk Ho Lee, So Ok Park, Hyunku Kang, Jun Sung Lee, Ki Nam Lee, Myung Eun Jung,
⇑
Jeongmin Kim, Jinhwa Lee
Research Center, Green Cross Corporation, 303 Bojeong-Dong, Giheung-Gu, Yongin, Gyeonggi-Do 446-770, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel macrocyclic C-aryl glucoside SGLT2 inhibitors were designed and synthesized. Two different syn-
thetic routes of macrocyclization were adopted to prepare novel ansa SGLT2 inhibitors. Among the com-
pounds tested, [1,7]dioxacyclopentadecine macrocycles possessing methylthiophenyl at the distal ring
40 or ethoxyphenyl at the distal ring 23 showed the best in vitro inhibitory activity in this series to date
(40, IC50 = 0.778 nM and 23, IC50 = 0.899 nM) against hSGLT2.
Received 5 July 2011
Revised 22 July 2011
Accepted 22 July 2011
Available online 28 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
This paper is dedicated to Professor Eun Lee
on the occasion of his retirement from Seoul
National University in August 2011.
Keywords:
SGLT
Dapagliflozin
Ring-closing metathesis
Glucoside
Macrocycle
1. Introduction
SGLT1 and SGLT2.4 SGLT1 is typically in the GI tract of humans
and the GI tract represents a major locus of action of SGLT1 in glu-
Diabetes has become an augmenting concern to the world’s
population. In 2007, approximately 246 million people were influ-
enced by the disease, with an additional 7 million people catching
the disease every year.1 The ADA states, ‘‘Diabetes mellitus is a
group of metabolic diseases characterized by hyperglycemia
resulting from defects in insulin secretion, insulin action, or both.
The chronic hyperglycemia of diabetes is associated with long-
term damage, dysfunction, and failure of various organs, especially
the eyes, kidneys, nerves, heart, and blood vessels.’’19. There are
two identified form of diabetes: Type 1 diabetes is distinguished
as an autoimmune disease involving pancreatic b-cells, while type
2 diabetes is associated with b-cell dysfunction2 and is defined by a
defect in glucose regulation and metabolism. Type 2 diabetes is the
most dominant disorder of glucose homeostasis, accounting for
nearly 90–95% of all cases of diabetes. While diabetes is often a re-
sult of impared insulin secretion or action, the actual measure of
diabetes relates to glucose levels.
cose trafficking. Also, SGLT1 is a high-affinity, low-capacity trans-
porter and therefore accounts for only a small fraction of renal
glucose reabsorption.6 In contrast, SGLT2 is a low-affinity, high-
capacity transporter located exclusively at the apical domain of
the epithelial cells in the early proximal convoluted tubule. It is
estimated that 90% of renal glucose reabsorption is facilitated by
SGLT2; the remaining 10% is likely mediated by SGLT1 in the late
proximal straight tubule.7 Since SGLT2 appears to be responsible
for the majority of renal glucose reabsorption based on human
mutation studies,8 it has become a target of therapeutic interest.
Cl
OEt
Me
O
O
HO
HO
F
S
HO
HO
OH
OH
O
OH
OH
canagliflozin
dapagliflozin
Sodium-dependent glucose cotransporters (SGLTs) couple the
transport of glucose against a concentration gradient with the
simultaneous transport of Na+ down a concentration gradient.3
Two important SGLT isoforms have been cloned and identified,
Cl
OEt
Cl
OEt
O
MeS
HO
O
HO
HO
OH
PF-04971729
OH
OH
OH
Lexicon's
⇑
Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9020.
E-mail address: jinhwalee@greencross.com (J. Lee).
Figure 1. Structures of C-aryl glucoside SGLT2 inhibitors.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.045

M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
5469
Bristol–Myers Squibb has identified dapagliflozin (Fig. 1), a
potent, selective SGLT2 inhibitor for the treatment of type 2
diabetes.9–11 At present, dapagliflozin is the most advanced SGLT2
inhibitor in clinical trials. On the other hand, Mitsubishi Tanabe, in
collaboration with Johnson & Johnson, is developing canagliflozin
2, another novel C-glucoside-derived SGLT2 inhibitor.12 In addi-
tion, Boehringer Ingelheim (BI 10773), Lexicon (LX4211), Astellas
(ASP1941), and Pfizer (PF-04971729) are reported to be in various
phase of clinical trials.13 Our efforts on identifying inhibitors that
target SGLT2 have been previously described.14 One of our most
recent efforts involved exploration of ansa-structure A of C-aryl
glucoside SGLT2 inhibitors (Fig. 2). We performed synthesis of
C-glucosides associated with cyclic diarylpolynoid utilizing versa-
tile organozinc chemistry and subsequent ring-closing olefin
metathesis using 2nd generation Grubbs catalyst. The synthesized
ansa-analogs showed the modest in vitro inhibitory activity
against hSGLT2 (B, IC50 = 59.5 nM).15
In the present study, novel macrocyclic C-glucosides connecting
carbohydrate and the proximal ring were exploited in order to de-
velop novel SGLT2 targeting antidiabetic agents. Theracos has
shown that the O-spiroketal C-aryl glucosides and C-spiro C-aryl
glucosids are potent SGLT2 inhibitors and introduction of a non-
cyclized substituent at 6-position of the proximal aryl ring not only
improved potency but also provided high selectivity toward
SGLT2.18 Adopting these results and ring closing strategy used in
a variety of medicinal chemistry practices, we envisioned that con-
joining the proximal ring of dapagliflozin with a handle of glucose
ring as shown in F (Fig. 3) would be a valuable approach for mod-
ulating physicochemical property, and possibly improving biologi-
cal activity. Thus, we decided to test this hypothesis.
O
HO
HO
OH
Cl
OEt
To be best of our knowledge, this would be the first example of
macrocyclic SGLT2 inhibitor formed by connecting glucose and
proximal phenyl ring. For this purpose, structure of dapagliflozin
was modified into G forming macrocycle between C-6 position of
carbohydrate and ortho-position at the proximal ring as shown in
Figure 3. Subsequently, we envisioned that macrocyclic ring could
be obtained by ring-closing olefin metathesis using Grubbs catalyst
or simple intramolecular alkylation using hydroxyl group as a
nucleophile and alkyl halide as an electrophile at the other side
of the molecule. Herein, we report design, synthesis and biological
evaluation of novel C-glucoside macrocyclic congeners.
OH
A
O
HO
HO
OH
dapagliflozin
O
0
OH
O
HO
HO
OH
B: IC50 = 59.5 nM
OH
Figure 2. Glucosides with cyclic diarylpolynoid as SGLT2 inhibitor.
2. Results and discussion
2.1. Chemistry
O
R1
R2
E
O
m
O
n
C
D
O
As shown in Scheme 1, preparation of the target compounds
commenced with preparation of aglycone 8. Thus, 2-chloro-4-
hydroxybenzonitrile (1) was brominated selectively to produce
structure of 2 with NBS and trifluoromethanesulfonic acid. The
structure of 2 was methylated with dimethylsulfate and LiOH in
a suitable solvent such as THF to provide 3. Treatment of 3 with
NaOH in aqueous EtOH, followed by thionyl chloride produced
HO
OH
HO
OH
OH
G
OH
F
Figure 3. Exploration of C-glucoside conjoining the proximal ring with a handle of
glucose ring.
HO
Br
Cl
HO
Cl
O
Cl
TfOH, NBS
DMS, LiOH.H2O
CN
-40 oC->RT
86 %
THF, 75 o
85 %
C
CN
Br
CN
1
3
2
O
O
Cl
NaOH
thionyl chloride
OH
Br
EtOH/H2O
98 %
AlCl3, DCM
94 % (2 steps)
toluene, cat.DMF
90 oC, 4h
O
4
O
Cl
O
NaSEt
DMF
Et3SiH, BF3.OEt2
O
Cl
O
DCM/ACN
79 %
Br
Br
O
6
5
Br
O
Cl
O
HO
Br
Cl
O
K2CO3
Br
acetone, 65 o
C
8
95 % (2 steps)
7
Scheme 1. Preparation of intermediate 8. TfOH: trifluoromethanesulfonic acid; DMS: dimethylsulfate; DCM: dichloromethane; ACN:acetonitile; DMF : N,N-dimethylamide.

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M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
O
Cl
O
O
O
1) BuLi,
O
Cl
O
TMSO
O
THF/Toluene
HO
HO
+
OMe
OH
TMSO
OTMS
Br
2) MeOH,
MeSO3H
OTMS
OH
8
9
10
O
Cl
O
O
Cl
O
.
1) Ac2O, DMAP, pyridine, DCM
2) EtOH (crystallization)
O
O
Et3SiH, BF3 OEt2,
HO
HO
AcO
DCM/ACN
OH
AcO
OAc
OH
OAc
37 % (4 steps, beta-form)
11
12
O
Cl
O
O
Cl
O
O
BnBr, NaH
DMF
BnO
BnO
O
NaOMe
MeOH
HO
OBn
HO
OH
OBn
OH
96 % (2 steps)
14
13
O
Cl
O
O
Cl
O
O
NaOMe
MeOH
TMSOTf
O
HO
BnO
AcO
Ac2O/DCM
OBn
BnO
OBn
OBn
OBn
97 % (2 steps)
16
15
Scheme 2. Preparation of advanced intermediate 16.
the corresponding acyl chloride, which was directly reacted with
ethoxybenzene and aluminum chloride in dichloromethane to
generate ketone 5. Subsequent reduction of ketone 5 with triethyl-
silane in the presence of boron trifluoride diethyl etherate pro-
BCl3 in methylene chloride or hydrogenolysis on Pd/C in MeOH
and THF to produce the target compound 23 (Scheme 3).
Another approachtoward macrocyclization involves ring-closing
olefin metathesis (RCM) as described in Scheme 4. Thus, compound
13 was treated with 3,3-dimethoxyprop-1-ene in the presence of
CSA (10-camphorsulfonic acid) in DMF to provide the 2-vinyl-1,3-
dioxane24. Selective and reductive ring opening using TfOH (trifluo-
romethanesulfonic acid) and sodium cyanoborohydride produced
compound 25. Ring-closing olefin metathesis was undergone with
divinyl intermediate 26 using Grubbs 2nd generation catalyst in
moderate yield. Finally, removal of acetyl groups was accomplished
using sodium methoxide in methanol to generate the target com-
pound 28.
Another way performing RCM is described in Scheme 5. Thus,
compound 16 prepared in Scheme 2, was allylated (allyl bromide,
sodium hydride, DMF) to produce diene 29. Ring-closing metathe-
sis (RCM) using Grubbs 2nd generation catalyst afforded macro-
cycle 30 along with recovered 29. Finally, subjection of 30 to
hydrogen atmosphere on Pd/C in a mixture of MeOH and THF
produced the target compound 31 in 22% yields.
duced diphenylmethane 6. The requisite aglycone
8 was
produced with demethylation by sodium ethanethiolate in DMF
and the subsequent allylation with allyl bromide and potassium
carbonate in acetone.
Preparation of the key intermediate 16 was accomplished as
shown in Scheme 2. Thus, lithiation and subsequent etherification
produced anomeric mixture 10 with concomitant desilylation
(Scheme 2). Reduction of 10 with triethylsilane in the presence
of boron trifluoride diethyl etherate, and subsequent acetylation
of the resulting compound, followed by crystallization in EtOH
generated beta-anomeric tetraacetate 12. Switching its acetyl
groups to benzyl groups was accomplished by hydrolysis with so-
dium methoxide in MeOH, and the subsequent benzylation. The
requisite alcohol 16 was obtained in 97% yield overall by (i) the
selective acetylation of 14 into acetate 15 with TMSOTf and acetic
anhydride, and (ii) the subsequent hydrolysis.
Alkylation of alcohol 16 with (5-bromopentyloxy)(tert-butyl)-
diphenylsilane (17) in the presence of sodium hydride in DMF
produced 18 in 42% yield. Desilylation of 18 with TBAF gave alcohol
19 in 93% yield. Removal of the allyl group was carried out
smoothly using NaBH4 in the presence of tetrakis[triphenylphos-
phine]palladium(0) to give phenol 20 in quantitative yield. The pri-
mary alcohol of 20 was transformed into the corresponding iodide
21 by action of iodine, triphenylphosphine, and imidazole in ben-
zene. The iodide underwent macrocyclization under conditions of
potassium carbonate and 18-crown-6 in DMF. Removal of the
benzyl groups on the carbohydrate moiety proceeded with either
2.2. Structure–activity relationship studies
The cell-based SGLT2 AMG (Methyl-a-D-glucopyranoside) inhi-
bition assay was performed to evaluate the inhibitory effects of all
prepared compounds on hSGLT2 activities.16,17 Exploration of the
SAR began by connecting ortho-position on the proximal phenyl
ring of dapagliflozin with C-6 position of glucose moiety. These
results are shown in Table 1. Synthesis of the series started from
macrocyclic ring with m = 2 (in CmHn) and R1 = OEt, since macro-
cyclic ring with m = 1 can be considered to be chemically labile.

M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
5471
OTBDPS
Br
OTBDPS
O
Cl
O
O
Cl
O
17
O
O
HO
BnO
, NaH
O
TBAF
THF
BnO
OBn
OBn
DMF
42 %
OBn
OBn
93 %
16
18
OH
OH
HO
Cl
O
O
O
Cl
O
NaBH4, Pd(PPh3)4
THF
O
I2, PPh3, imidazole
Benzene
O
BnO
OBn
O
OBn
BnO
OBn
100 %
OBn
93 %
20
19
I
O
Cl
O
O
HO
Cl
O
O
K2CO3, 18-crown-6
O
O
BnO
OBn
DMF
OBn
BnO
OBn
OBn
64 %
22
21
O
Cl
O
Pd/C, H2
O
BCl3
O
or
MeOH/THF
DCM
HO
OH
OH
20 %
23
Scheme 3. Synthesis of precursor 21 and subsequent key intramolecular cyclization.
To our delight, the first compound we prepared in this series
turned out to be as good as dapagliflozin in terms of inhibitory
activity against hSGLT2 (32, IC50 = 1.34 nM vs dapagliflozin,
IC50 = 1.35 nM), suggesting that C-6 OH on glucose is not necessar-
ily critical for inhibitory activity against hSGLT2 as long as the mol-
ecule is poised to adopt favorable conformation for optimal
inhibitory activity. As we tested macrocycle homologated by one
more carbon (m = 3) in this series, the compound proved to be two-
fold less active (33, IC50 = 2.95 nM). One more homologation 31
slightly improved in vitro inhibitory activity, but failed to reach
the same level of activity as that of compound 32 (31,
IC50 = 2.27 nM). However, one more homologation into [1,7]dioxa-
cyclopentadecine 23 appears to provide better inhibitory activity
against hSGLT2 (IC50 = 0.899 nM). The higher homolog (m = 6) be-
comes less potent, the compound become less inhibitory against
hSGLT2. The observations are more clearly described in Figure 4.
This is a kind of combination of bell-shaped curve and zig–zag
curve depending on the number of the bridging carbon atoms.
Interestingly, the olefinic macrocycle 28 demonstrated subnanom-
olar inhibitory activity against hSGLT2, showing the molecule can
adopt appropriate conformation with the olefinic character in the
macrocycle. Next, the compounds with R1 = Et on the distal ring
were tested to examine if the particular modification would influ-
ence the inhibitory activity against hSGLT2. As shown in Table 1,
regardless of macrocycle ring size (m = 3–6), the compounds in
the series show consistently good activity, displaying IC50 = 1.38–
1.59 nM (35–38). Further investigation was carried out to other
substituents such as R1 = SMe or R1 and R2 connected by 1,4-diox-
ane bridge. For both cases, the similar observations were made as
described previously in 4-OEt or 4-Et series. Thus, [1,7]dioxacycl-
opentadecine compounds with m = 5 showed the excellent activity,
exhibiting IC50 = 0.778 nM (40) and IC50 = 1.38 nM (42), respec-
tively. On the basis of the excellent in vitro inhibitory data against
hSGLT2, compound 23 and 37 were evaluated briefly for its
potential to induce UGE (urine glucose excretion) in vivo. UGE
values of dapagliflozin, 23, and 37 in normal SD rats were
1648 228 mg/200 g body weight, 226 97 mg/200 g body
weight, and 116 50 mg/200 g body weight at a single oral dose
of 1 mg/kg, respectively. Thus, it is concluded that the decreased
in vivo efficacy of the current macrocycle SGLT2 inhibitors 23
and 37 compared with dapagliflozin, should be attributed to the
poor pharmacokinetic properties, since there appears to be no
significant difference in inherent in vitro potencies.
3. Conclusion
In the present study, novel macrocyclic C-aryl glucoside SGLT2
inhibitors were designed and synthesized. We envisioned that
conjoining the proximal ring of dapagliflozin with a handle of

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M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
O
Cl
O
O
Cl
O
OMe
TfOH, NaCNBH3,
Molecular sieves
O
, CSA
O
HO
HO
OMe
DMF
O
THF
OH
O
OH
OH
OH
13
24
O
Cl
O
O
Cl
O
O
Grubbs 2nd gen.
Ac2O, DMAP, pyridine
O
O
O
DCM
DCM,60 o
23 %
C
AcO
OAc
HO
OH
61 % (3 steps)
OAc
OH
25
26
O
Cl
O
O
Cl
O
O
O
O
NaOMe
MeOH
O
+
26
HO
OH
AcO
OAc
OH
OAc
56 %
28
27
Scheme 4. Preparation of diene precursor 26 and Grubbs catalyst-mediated macrocyclization.
O
Cl
O
O
Cl
O
Br
O
O
NaH
HO
BnO
O
OBn
BnO
OBn
DMF, 0 o
71 %
C
OBn
OBn
29
16
O
Cl
O
O
Cl
O
O
O
Grubbs 2nd gen.
DCM, 60oC
26 %
Pd/C, H2
O
O
+
22
MeOH/THF
HO
OH
BnO
OBn
OH
OBn
22 %
31
30
Scheme 5. Preparation of diene precursor 29 and Grubbs catalyst-mediated macrocyclization.
glucose ring would be a valuable approach for modulating physico-
chemical property, and possibly improving biological activity. We
trust that this would be the first example of macrocyclic SGLT2
inhibitor formed by connecting glucose and proximal phenyl ring.
For this purpose, structure of dapagliflozin was modified into mac-
rocycles between C-6 position of carbohydrate and ortho-position
at the proximal ring. Two different synthetic routes of macrocycli-
zation such as ring-closing olefin metathesis using Grubbs catalyst
and simple intramolecular alkylation using hydroxyl group as a
nucleophile and alkyl halide as an electrophile at the other side
of the molecule, were adopted to prepare novel macrocyclic SGLT2
inhibitors. Among the compounds tested, [1,7]dioxacyclopentade-
cine macrocycles possessing ethylphenyl at the distal ring 40 or
ethoxyphenyl at the distal ring 23 showed the best in vitro inhib-
itory activity in this series to date (40, IC50 = 0.778 nM and 23,
IC50 = 0.899 nM) against hSGLT2. The promising profile of 23
prompted evaluation of its potential to induce UGE in vivo, but
23 turned out to show only moderate in vivo UGE value.
4. Experimental
4.1. General methods
All reactions are conducted under an inert atmosphere at room
temperature, unless otherwise noted. All reagents were purchased
at the highest commercial quality and used without further purifi-
cation, unless otherwise indicated. Microwave reaction was con-
ducted with a Biotage Initiator microwave reactor. NMR spectra
were obtained on a Varian 400-MR (400 MHz 1H, 100 MHz 13C)
spectrometer. NMR spectra were recorded in ppm (d) relative to

M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
5473
Table 1
In vitro inhibitory activity against hSGLT2. Ref. Dapagliflozin (1.35 0.15)a
Hn
Cm
O
Cl
O
R1
R2
O
HO
OH
OH
b
b
Compd
R1, R2
CmHn
hSGLT2 IC50 (nM)
Compd
R1, R2
CmHn
hSGLT2 IC50 (nM)
32c
33c
28
OEt, H
OEt, H
OEt, H
OEt, H
OEt, H
OEt, H
Et, H
C2H4
C3H6
C4H6
C4H8
C5H10
C6H12
C3H6
1.34
2.95
0.974
2.27
0.899
3.63
1.59
36c
37c
38c
39c
40c
41c
42c
Et, H
Et, H
Et, H
SMe, H
SMe, H
1,4-Dioxane
1,4-Dioxane
C4H8
1.40
1.38
1.40
1.35
0.778
3.65
1.38
C5H10
C6H12
C4H8
C5H10
C4H8
31
23c
34c
35c
C5H10
a
b
c
This data was obtained by multiple determinations.
These data were obtained by single determinations.
These compounds were synthesized by the same procedure shown in Scheme 3.
Figure 4. In vitro inhibitory activity against hSGLT2 in homologous series of macrocycles. aThis data was obtained by multiple determinations. bThese data were obtained by
single determinations.
tetramethylsilane (d = 0.00) as an internal standard unless stated
otherwise and are reported as follows: chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet,
sext = sextet, m = multiplet, and br = broad), coupling constant,
and integration. 13C NMR spectra were referenced to the residual
chloroform-d1 (d = 77.0) or DMSO-d6 (d = 39.7). Mass spectra were
obtained with an Agilent 6110 quadruple LC-MSD (ESI+). High res-
olution mass spectra were obtained on a Jeol JMS-700 Mstation
(10 kV, HFAB). Optical rotations were obtained on a Rudolph Auto-
pol III digital polarimeter. Preparative HPLC purifications were per-
formed on a Gilson purification system. For preparative HPLC, ca.
100 mg of a product was injected in 1 mL of methanol onto a Sun-
10 min at À30 °C, before adding of N-bromosuccinimide (16.2 g,
91 mmol). After 18 h stirring at ambient temperature, the solution
was quenched with aqueous saturated sodium hydrogen carbon-
ate. The organic layers was extracted with ethyl acetate two times,
washed with brine, dried over magnesium sulfate, filtered and con-
centrated in vacuo. The resulting crude residue was purified on a
BiotageÒ purification apparatus (silica gel, 5–40% ethyl acetate in
hexanes gradient) to yield the title compound (13 g, 56 mmol,
86%) as a white solid. 1H NMR (400 MHz, CD3OD) d 7.84 (s, 1H),
6.99 (s, 1H); MH+ 232.
4.2.1.2. 5-Bromo-2-chloro-4-methoxybenzonitrile (3). To
a
Fire Prep C18 OBD 5
l
m 30 Â 100 mm Column with a 30 min gra-
solution 5-bromo-2-chloro-4-hydroxybenzonitrile (2, 29 g,
124 mmol) in tetrahydrofuran (500 mL) was added lithium
hydroxide monohydrate (6.7 g, 161 mmol) and dimethyl sulfite
(15.2 mL, 161 mmol). The resulting mixture was heated at 75 °C
for 5 h, re-cooled to room temperature, and quenched with water.
The mixture was extracted with ethyl acetate and the organic layer
was washed with brine, dried over magnesium sulfate, filtered and
evaporated in vacuo. The crude residue was purified on BiotageÒ
purification apparatus (silica gel, 3–10% ethyl acetate in hexanes)
to yield the title compound (26 g, 104 mmol, 85%) as a white
solid. 1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H), 7.00 (s, 1H), 3.98
(s, 3H).
dient from 5% to 90% acetonitrile in water and a 45 mL/min flow
rate. Biotage SP1 and Isolera purification systems were used for
normal phase column chromatography with ethyl acetate and
hexane. Flash chromatography was performed using E. Merck
230–400 mesh silica gel according to the procedure of Still et al.
Reactions were monitored by either thin-layer chromatography
(TLC) on 0.25 mm E. Merck silica gel plates (60F-254) using UV
light and p-anisaldehyde solution as visualizing agents or HPLC
analysis on an Agilent 1200 series system.
4.2. Chemistry
4.2.1. Preparation of intermediate 8
4.2.1.3. 5-Bromo-2-chloro-4-methoxybenzoic acid (4). To a
4.2.1.1. 5-Bromo-2-chloro-4-hydroxybenzonitrile (2). To
a
solution of 5-bromo-2-chloro-4-methoxybenzonitrile (3, 30.6 g,
124 mmol) in ethanol (450 mL)/H2O (225 mL) was added sodium
hydroxide (124 g, 3.1 mol). The solution was refluxed at 100 °C
overnight, cooled to room temperature and evaporated ethanol.
solution of 2-chloro-4-hydroxybenzonitrile (1, 10 g, 65 mmol) in
acetonitrile (200 mL) at À30 °C was added dropwise trifluorometh-
anesulfonic acid (10 mL, 71 mmol). The solution is stirred for

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M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
The aqueous layer was cooled to 0 °C, acidified with concentrated
hydrogen chloride (190 mL). The generated white solid was fil-
tered, washed with water and dried in vacuo to yield the title com-
pound (32 g, 122 mmol, 98%) as a white solid.
1H), 6.83 (d, J = 8.0 Hz, 2H), 6.09–5.99 (m, 1H), 5.47 (d,
J = 17.2 Hz, 1H), 5.33 (d, J = 10.4 Hz, 2H), 4.57 (d, J = 4.8 Hz, 2H),
4.01 (q, J = 6.8 Hz, 2H), 3.93 (s, 2H), 1.40 (t, J = 6.8, 3H).
4.2.2. Preparation of intermediate 21
4.2.1.4. (5-Bromo-2-chloro-4-methoxyphenyl)(4-ethoxyphenyl)-
methanone (5). To a solution of 5-bromo-2-chloro-4-methoxy-
benzoic acid (4, 15 g, 56.5 mmol) in toluene (72 mL) was added
thionyl chloride (8.24 mL, 113 mmol) and N,N-dimethylformamide
(0.1 mL). The solution was refluxed at 90 °C for 4 h, cooled to room
temperature and evaporated toluene and residual reagent. The ob-
tained acyl chloride was diluted with dichloromethane (240 mL)
and added portionwise aluminum chloride (8.3 g, 62.2 mmol)
and phenetole (7.2 mL, 56.5 mmol) at 0 °C. The reaction mixture
was stirred at room temperature overnight, quenched with 1 N
HCl (15 mL) and H2O (15 mL). The organic layer was extracted with
dichloromethane two times, washed with 1 N HCl and brine, dried
over magnesium sulfate, filtered and concentrated in vacuo to yield
the title compound as a yellowish solid which was used without
4.2.2.1.
(2R,3R,4R,5S,6S)-2-(Acetoxymethyl)-6-(2-(allyloxy)-4-
chloro-5-(4-ethoxybenzyl)phenyl)tetrahydro-2H-pyran-3,4,5-
triyl triacetate (12). To a solution of (8, 10.2 g, 27 mmol) in
tetrahydrofuran (25 mL)/toluene (50 mL) at À78 °C under an atmo-
sphere of nitrogen was added dropwise n-butyllithium (2.5 M in
hexanes, 12 mL, 30 mmol), and the mixture was stirred for 1 h at
the same temperature. Then a solution of 2,3,4,6-tetra-O-trimetyl-
silyl-b-D-glucolatone (9, 13.8 g, 30 mmol) in tetrahydrofuran
(30 mL) was added dropwise, and the mixture was stirred for
1.5 h at the same temperature. The reaction mixture was quenched
by addition of saturated ammonium chloride solution. After com-
plete addition, the solution was gradually raised to room temper-
ature. The organic layer was separated and the aqueous layer
was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered
and concentrated in vacuo to yield the title compound (10) as a
yellowish solid which was used without further purification.
To a stirred À25 °C solution of O-methylglucoside (10) in
dichloromethane (50 mL)/acetonitile (50 mL) was added triethylsi-
lane (8.7 mL, 54 mmol) followed by boron trifluoride diethyl ether-
ate (5.2 mL, 41 mmol) at À25 °C. The solution was allowed to
warm to 0 °C over 3 h prior to quenching with saturated sodium
carbonate solution. After removal of organic volatiles under re-
duced pressure, the residue was partitioned between ethyl acetate
and water. Following extraction of the aqueous layer with ethyl
acetate, the combined organic layers were washed with water
prior to drying over magnesium sulfate. Filtration and removal of
volatiles under reduced pressure yield desired tetraol (11) as a yel-
lowish solid.
The obtained tetraol (11) was diluted dichloromethane (60 mL)
and added acetic anhydride (22.2 mL, 235 mmol), DMAP (165 mg,
1.35 mmol) and pyridine (19 mL, 235 mmol). After 18 h, the reac-
tion was quenched by addition of H2O, whereupon the resulting
mixture was extracted with dichloromethane (2Â). The combined
organic layers were washed with 1 N HCl (2Â) and brine (2Â) prior
to drying over magnesium sulfate. After filtration and concentra-
tion under reduced pressure, residue was slurried in ethanol
(80 mL) and heated to reflux with stirring. The reaction mixture
was held at reflux for 1 h to ensure that all of solution had homog-
enized; it was then cooled evenly at 15 °C/h to ambient tempera-
ture and stirred overnight at this temperature. The resulting solid
was isolated by filtration and dried in vacuo to yield the title com-
pound (12, 7.0 g, 11 mmol, 41%, ca. 60% separation yield) as a white
solid. MNa+ 655.
further purification. 1H NMR (400 MHz, CDCl3)
d 7.77 (d,
J = 9.2 Hz, 2H), 7.57 (s, 1H), 6.94 (s, 1H), 6.92 (d, J = 9.6 Hz, 2H),
4.11 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 1.43 (t, J = 6.4 Hz, 3H); MH+
368.
4.2.1.5. 1-Bromo-4-chloro-5-(4-ethoxybenzyl)-2-methoxyben-
zene (6). To a stirred À10 °C solution of (5-bromo-2-chloro-4-
methoxyphenyl)(4-ethoxyphenyl)methanone (54.3 mmol) from
Step 4 in dichloromethane (150 mL)/acetonitile (150 mL) was
added triethylsilane (20 mL, 109 mmol) followed by boron trifluor-
ide diethyl etherate (16 mL, 109 mmol) at À10 °C. The solution was
allowed to warm to 0 °C over 2 h prior to quenching with saturated
sodium carbonate solution. After removal of organic volatiles un-
der reduced pressure, the residue was partitioned between ethyl
acetate and water. Following extraction of the aqueous layer with
ethyl acetate, the combined organic layers were washed with
water prior to drying over magnesium sulfate. Filtration and con-
centration under reduced pressure yield the title compound as a
yellowish solid which was used without further purification. 1H
NMR (400 MHz, CDCl3) d 7.29 (s, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.90
(s, 1H), 6.83 (d, J = 8.8 Hz, 2H), 4.00 (q, J = 6.8 Hz, 2H), 3.93 (s,
2H), 3.87 (s, 3H), 1.40 (t, J = 6.8, 3H).
4.2.1.6. 2-Bromo-5-chloro-4-(4-ethoxybenzyl)phenol (7). To a
solution of 1-bromo-4-chloro-5-(4-ethoxybenzyl)-2-methoxyben-
zene (6, 11.3 mmol) in N,N-dimethylformamide (50 mL) was added
sodium ethane thiolate (3.17 g, 34 mmol) and heated at 90 °C for
3 h. The reaction mixture was cooled to 0 °C, neutralized with
1 N HCl, and the organic layer was separated and the aqueous layer
was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered
and concentrated in vacuo to yield the title compound as a yellow
oil which was used without further purification. 1H NMR
(400 MHz, CDCl3) d 7.26 (s, 1H), 7.07 (d, J = 8.4 Hz, 2H), 7.06 (s,
1H), 6.83 (d, J = 8.4 Hz, 2H), 4.01 (q, J = 6.8 Hz, 2H), 3.93 (s, 2H),
1.40 (t, J = 6.8 Hz, 3H).
4.2.2.2. (2S,3R,4R,5S,6R )-2-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-6-(hydroxyl-methyl)tetrahydro-2H-pyran-3,4,5-
triol (13). To a solution (2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-(2-
(allyloxy)-4-chloro-5-(4-ethoxybenzyl)phenyl)tetrahydro-2H-pyr-
an-3,4,5-triyl triacetate (12, 4 g, 6.32 mmol) from Step
1 in
methanol (90 mL) was added sodium methoxide (25% in methanol,
9 mL) and the reaction mixture stirred at ambient temperature for
5 h. The solution was cooled to 0 °C prior to neutralizing with ace-
tic acid (4.5 mL). After removal of organic volatiles under reduced
pressure, the residue was diluted with ethyl acetate and water. The
organic layer was separated and the aqueous layer was extracted
with ethyl acetate. The combined organic layers were washed with
sodium hydrogen carbonate, brine, dried over magnesium sulfate,
filtered and concentrated in vacuo to yield the title compound
(13) as a white solid which was used without further purification.
1H NMR (400 MHz, CDCl3) d 7.19 (s, 1H), 7.05 (d, J = 8.4 Hz, 2H),
4.2.1.7. 1-(Allyloxy)-2-bromo-5-chloro-4-(4-ethoxybenzyl)ben-
zene (8). To a solution of 2-bromo-5-chloro-4-(4-ethoxyben-
zyl)phenol (7, 55 mmol) in acetone (180 mL) was added
potassium carbonate (15.2 g, 110 mmol) and allyl bromide (7 mL,
83 mmol) and heated at 65 °C for 3 h. The insoluble material was
removed by filter and the filtrate was evaporated under reduced
pressure. The crude residue was purified on BiotageÒ purification
apparatus (silica gel, 3–10% ethyl acetate in hexanes) to yield the
title compound (21 g, 52 mmol, 95%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d 7.29 (s, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.90 (s,

M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
5475
6.92 (s, 1H), 6.79 (d, J = 8.8 Hz, 2H), 6.06–5.96 (m, 1H), 5.40 (dd,
J = 1.6, 17.6 Hz, 1H), 5.30 (dd, J = 1.6, 10.4 Hz, 1H), 4.66 (d,
J = 9.6 Hz, 2H), 4.58–4.49 (m, 2H), 4.01–3.96 (m, 4H), 3.89–3.86
(m, 1H), 3.80–3.75 (m, 1H), 3.66–3.62 (m, 2H), 3.55–3.47 (m,2H),
3.22 (m, 1H), 3.05 (m, 1H), 2.42 (m, 1H), 2.13–2.11 (m, 1H), 1.38
(t, J = 6.8 Hz, 3H), MNa+ 487.
ica gel, 5–30% tetrahydrofuran in hexanes) to yield the title com-
pound (16, 7.2 g, 9.8 mmol, 98%) as a white solid. MNa+ 757.
4.2.3. Synthesis of precursor 31 and subsequent key
intramolecular cyclization
4.2.3.1. (5-Bromopentyloxy)(tert-butyl)diphenylsilane (17). To
a solution of 5-bromopentan-1-ol (0.43 mL, 3.00 mmol) in tetrahy-
drofuran (15 mL) at 0 °C under an atmosphere of nitrogen was
added imidazole (210 mg, 3.00 mmol) and t-butylchlorodiphenyl-
silane (0.78 mL, 3.00 mmol) at 0 °C. After 5 h at room temperature,
the reaction mixture was quenched by addition of water (5 mL)
and the mixture was extracted with diethyl ether. The organic
layer was washed with brine, dried over magnesium sulfate, fil-
tered and evaporated in vacuo. The resulting crude residue was
purified on a BiotageÒ purification apparatus (silica gel, 0–10%
ethyl acetate in hexanes gradient) to yield the title compound
4.2.2.3. (2S,3S,4R,5R,6R )-2-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetra-
hydro-2H-pyran (14). To
a solution of tetraol (13, 8.4 g,
18 mmol) from Step 2 in N,N-dimethylformamide (100 mL) at
0 °C under an atmosphere of nitrogen was added sodium hydride
(60% dispersion in mineral oil, 10.1 g, 252 mmol), and the mixture
was stirred for 30 min at the same temperature. Then benzyl bro-
mide (19.5 mL, 162 mmol) was added dropwise, and the mixture
was stirred with gradual warming to ambient temperature over
5 h. After re-cooling to 0 °C, the reaction mixture was quenched
by addition of water (100 mL). The mixture was diluted with water
and extracted with ethyl acetate. The organic layer was washed
with brine, dried over magnesium sulfate, filtered and evaporated
in vacuo. The resulting crude residue was purified on a BiotageÒ
purification apparatus (silica gel, 5–20% tetrahydrofuran in hex-
anes gradient) to yield the title compound (14, 14.8 g, 18 mmol,
100%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.43 (br,
2H), 7.33–7.25 (m, 12H), 7.21–7.13 (m, 5H), 7.06–7.04 (m, 3H),
6.83 (d, J = 6.4 Hz, 1H), 6.71 (d, J = 8.8 Hz, 1H), 6.00–5.93 (m, 1H),
5.38 (d, J = 17.2 Hz, 1H), 5.18 (dd, J = 1.6, 10.8 Hz, 1H), 4.80 (s,
2H), 4.76 (d, J = 11.2 Hz, 1H), 4.59–4.40 (m, 6H), 3.94–3.86 (m,
5H), 3.75 (m, 2H), 3.63–3.58 (m, 4H), 1.26 (t, J = 6.8 Hz, 3H);
MNa+ 847.
(17, 980 mg, 2.42 mmol, 81%) as
a
colorless oil. 1H NMR
(400 MHz, CDCl3) d 7.72–7.69 (m, 4H), 7.48–7.39 (m, 6H), 3.70 (t,
J = 6.4 Hz, 1H), 3.42 (t, J = 6.8 Hz, 1H), 1.91–1.84 (m, 2H), 1.62–
1.58 (m, 2H), 1.47–1.42 (m, 4H), 1.09 (s, 9H).
4.2.3.2. (5-(((2R,3R,4R,5S,6S)-6-(2-(Allyloxy)-4-chloro-5-(4-eth-
oxybenzyl)phenyl)-3,4,5-tris-(benzyloxy)tetrahydro-2H -pyran-
2-yl)methoxy)pentyloxy)(tert-butyl)diphenylsilane (18). To
a
solution of ((2R,3R,4R,5S,6S)-6-(2-(allyloxy)-4-chloro-5-(4-
ethoxybenzyl)phenyl)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-
2-yl)methanol (26, 1.1 g, 1.51 mmol) in N,N-dimethylformamide
(25 mL) was added sodium hydride (60% dispersion in mineral
oil, 120.4 mg, 3.01 mmol) at 0 °C, and the reaction mixture stirred
at ambient temperature for 0.5 h. After 1 h at room temperature,
the reaction mixture was re-cooled to 0 °C and (5-bromopentyl-
oxy)(tert-butyl)diphenylsilane (17, 980 mg, 2.42 mmol) was added
dropwise, and the mixture was stirred with gradual warming to
ambient temperature over 5 h. After re-cooling to 0 °C, the reaction
mixture was quenched by addition of water (25 mL). The mixture
was diluted with water and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over magnesium sulfate,
filtered and evaporated in vacuo. The resulting crude residue was
purified on a BiotageÒ purification apparatus (silica gel, 5–20% tet-
rahydrofuran in hexanes gradient) to yield the title compound (18,
664 mg, 0.63 mmol, 42%) as a yellowish oil. 1H NMR (400 MHz,
CDCl3) d 7.69–7.67 (m, 4H), 7.45–7.40 (m, 6H), 7.35–7.32 (m,
10H), 7.26–7.14 (m, 6H), 7.06–7.02 (m, 1H), 6.92–6.86 (m, 3H),
6.77–6.75 (m, 1H), 6.04–5.94 (m,1H), 5.43–5.39 (m, 1H),
5.27–5.24 (m, 1H), 4.96–4.89 (m, 3H), 4.72 (d, J = 11.2 Hz, 1H),
4.49–4.43 (m, 3H), 4.05–3.89 (m, 5H), 3.85–3.77 (m, 2H),
3.75–3.68 (m, 2H), 3.67–3.64 (m, 3H), 3.56–3.47 (m, 2H), 3.42–
3.34 (m, 1H), 1.63–1.53 (m, 2H), 1.40 (t, J = 6.8 Hz, 3H), 1.06 (s,
9H).
4.2.2.4. ((2R,3R,4R,5S,6S)-6-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)-
methyl acetate (15). To a stirred À55 °C solution of (2S,3S,4R,5R,
6R)-2-(2-(allyloxy)-4-chloro-5-(4-ethoxybenzyl)phenyl)-3,4,5-
tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-2H-pyran
(14,
8.54 g, 10 mmol) from Step 3 in acetic anhydride (50 mL)/dichloro-
methane (50 mL) was dropwise added trimethylsilyl trifluoro-
methanesulfonate (4.7 mL, 26 mmol) at a rate such that the
reaction temperature was maintained between À50 and À55 °C.
The solution was allowed to warm to À45 °C over 1.5 h prior to
quenching with saturated sodium hydrogen carbonate solution.
The reaction mixture was extracted with dichloromethane and
the combined organic layers were washed with saturated sodium
hydrogen carbonate solution prior to drying over magnesium sul-
fate. Filtration and concentration under reduced pressure and the
crude residue was purified on a BiotageÒ purification apparatus
(silica gel, 5–20% tetrahydrofuran in hexanes gradient) to yield
the title compound (15, 7.8 g, 10 mmol, 100%) as a white solid.
MNa+ 799.
4.2.3.3. 5-(((2R,3R,4R,5S,6S)-6-(2-(Allyloxy)-4-chloro-5-(4-eth-
oxybenzyl)phenyl)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-
2-yl)methoxy)pentan-1-ol (19). To a solution of (5-(((2R,3R,
4R,5S,6S)-6-(2-(allyloxy)-4-chloro-5-(4-ethoxybenzyl)phenyl)-3,4,
5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methoxy)pentyloxy)(-
tert-butyl)diphenylsilane (18, 664 mg, 0.626 mmol) from Step 1 in
tetrahydrofuran (8 mL) was added tetrabutylammonium fluoride
(1.0 M in tetrahydrofuran, 1.9 mL, 1.88 mmol) and the reaction
mixture stirred at ambient temperature for 2 h. After removal of
organic volatiles under reduced pressure, the residue was parti-
tioned between ethyl acetate and saturated ammonium chloride
solution. The organic layer was separated and the aqueous layer
was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered
and concentrated in vacuo. The crude residue was purified on
BiotageÒ purification apparatus (silica gel, 10–60% tetrahydrofuran
4.2.2.5. ((2R,3R,4R,5S,6S)-6-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)-
methanol (16). To a solution ((2R,3R,4R,5S,6S)-6-(2-(allyloxy)-4-
chloro-5-(4-ethoxybenzyl)phenyl)-3,4,5-tris(benzyloxy)tetrahy-
dro-2H-pyran-2-yl)methyl acetate (15, 7.8 g, 10 mmol) from Step 4
in methanol (130 mL) was added sodium methoxide (25% in meth-
anol, 13 mL) and the reaction mixture vigorously stirred at ambi-
ent temperature for 5 h. The solution was cooled to 0 °C prior to
neutralizing with acetic acid (6.5 mL). After removal of organic vol-
atiles under reduced pressure, the residue was diluted ethyl ace-
tate and water. The organic layer was separated and the aqueous
layer was extracted with ethyl acetate. The combined organic lay-
ers were washed with sodium hydrogen carbonate, brine, dried
over magnesium sulfate, filtered and concentrated in vacuo. The
crude residue was purified on BiotageÒ purification apparatus (sil-

5476
M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
in hexanes) to yield the title compound (19, 479 mg, 0.583 mmol,
(m, 10H), 7.26–7.12 (m, 7H), 7.10–7.07 (m, 1H), 6.89–6.82 (m, 2H),
6.78–6.71 (m, 1H), 4.81–4.77 (m, 3H), 4.67 (d, J = 10.8 Hz, 1H),
4.48–4.43 (m, 1H), 4.26–4.23 (m, 2H), 4.10–4.01 (m, 2H), 3.98–
3.88 (m, 6H), 3.80–3.60 (m, 4H), 3.48–3.41 (m, 3H), 1.95–1.86 (m,
2H), 1.64–1.61 (m, 2H), 1.31 (t, J = 6.8 Hz, 3H); MNa+ 785.
93%) as a white. MNa+ 843.
4.2.3.4.
5-Chloro-4-(4-ethoxybenzyl)-2-((2S,3S,4R,5R,6R)-3,4,
5-tris(benzyl-oxy)-6-((5-hydr-oxypentyloxy)methyl)tetrahydro-
2H-pyran-2-yl)phenol (20). To a solution of 5-(((2R,3R,4R,5S,6S)-
6-(2-(allyloxy)-4-chloro-5-(4-ethoxybenzyl)phenyl)-3,4,5-tris-
(benzyloxy)tetrahydro-2H-pyran-2-yl)methoxy)pentan-1-ol (19,
479 mg, 0.584 mmol) from Step 2 in tetrahydrofuran (6 mL) was
added sodium borohydride (177 mg, 4.668 mmol) and tetrakis(tri-
phenylphosphine)palladium (67.5 mg, 0.058 mmol). The reaction
mixture was stirred at room temperature overnight. The mixture
was diluted with ethyl acetate and quenched with saturated
ammonium chloride, extracted with ethyl acetate. The organic
layer was washed with brine, dried over magnesium sulfate, fil-
tered and evaporated in vacuo. Flash chromatography on a Bio-
tageÒ apparatus (silica gel, 10–50% tetrahydrofuran in hexanes
gradient) gave the title compound (20, 459 mg, 0.58 mmol, 100%)
as a colorless oil. . 1H NMR (400 MHz, DMSO-d6) d 9.84 (s, 1H),
7.38–7.27 (m, 10H), 7.23–7.10 (m, 5H), 7.07–7.02 (m, 2H), 6.93–
6.86 (m, 3H), 6.76–6.74 (m, 1H), 4.81–4.78 (m, 3H), 4.65–4.60
(m, 2H), 4.42–4.37 (m, 1H), 4.00–3.85 (m, 4H), 3.76–3.67 (m,
2H), 3.65–3.51 (m, 4H), 3.47–3.40 (m, 1H), 3.35–3.28 (m, 2H),
3.25–3.20 (m, 2H), 1.53–1.33 (m, 4H), 1.36–1.21 (m, 2H), 1.29 (t,
J = 6.8 Hz, 3H); MNa+ 803.
4.2.3.7.
3,4,5,6,8,9,10,11,12,13-decah-ydro-2H-9,13-epoxybenzo[h][1,7]-
dioxacyclopentadecine-10,11,12-triol (23). To solution of
(9R,10S,11R,12R,13S)-16-Chloro-15-(4-ethoxybenzyl)-
a
(2S,3R,4R,5S,6R)-2-(4-chloro-3-((5-(pyrazin-2-yl)-1,3,4-thiadiazol-
2-yl)methyl)phenyl)-6-(hydroxymethyl)-tetrahydro-2H-pyran-
3,4,5-triol (22, 261 mg, 0.34 mmol) in dichloromethane (12 mL) at
0 °C was added boron trichloride (1.0 M in dichloromethane,
1.8 mL, 5.3 mmol). The reaction mixture was stirred at 0 °C for
0.5 h and quenched with methanol (3 mL), then the solution was
concentrated in vacuo. Purification by reverse phase preparative
HPLC (WatersÒ, SunFire™ Prep, 5–50% acetonitrile in water gradi-
ent) provided the title compound (23, 33 mg, 0.067 mmol, 20%) as
a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.26 (s, 1H), 7.11 (d,
J = 8.4 Hz, 2H), 7.08 (s, 1H), 6.84 (d, J = 8.8 Hz, 2H), 5.08 (m, 1H),
5.01 (m, 1H), 4.83–4.82 (m, 1H), 4.21 (d, J = 10.0 Hz, 1H), 4.04 (m,
2H), 3.98 (q, J = 7.2 Hz, 2H), 3.91 (d, J = 12.4 Hz, 2H), 3.71–3.67
(m, 1H), 3.59 (d, J = 11.6 Hz, 1H), 3.48–3.39 (m, 4H), 3.31–3.26
(m, 2H), 3.16–3.3.14 (m, 1H), 1.70–1.68 (m, 3H), 1.54–1.42 (m,
3H), 1.31 (t, J = 6.8 Hz, 3H); M+–H2O 475, M+–NH4 457.
+
4.2.3.7.1. (6R,7S,8R,9R,10S)-13-Chloro-12-(4-ethoxybenzyl)-2,3,5,
6,7,8,9,10-octahydro-6,10-epoxybenzo[e][1,4]dioxacyclododecine-7,8,
9-triol (32). 1H NMR (400 MHz, DMSO-d6) d 7.12 (s, 1H), 7.05 (d,
J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 6.52 (s, 1H), 4.97 (br, 2H),
4.41 (d, J = 9.2 Hz, 1H), 3.97 (q, J = 6.8 Hz, 2H), 3.81 (d, J = 3.6 Hz,
2H), 3.69 (d, J = 10.8 Hz, 1H), 3.48–3.25 (m, 8H), 3.18–3.12 (m,
3H), 1.31 (t, J = 7.2 Hz, 3H); MH+–4H+ 447.
4.2.3.7.2. (7R,8S,9R,10R,11S)-14-Chloro-13-(4-ethoxybenzyl)-3,4,
6,7,8,9,10,11-octahydro-2H-7,11-epoxybenzo[f][1,5]dioxacyclotride-
cine-8,9,10-triol (33). 1H NMR (400 MHz, DMSO-d6) d 7.47 (s, 1H),
7.27 (s, 1H), 7.11 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 5.33–
4.79 (br, 2H), 4.25 (d, J = 10.0 Hz, 1H), 4.18–4.14 (m, 1H), 4.01–
3.95 (m, 3H), 3.91–3.80 (m, 3H), 3.74–3.67 (m, 2H), 3.61 (t,
J = 9.6 Hz, 1H), 3.46–3.40 (m, 2H), 3.38–3.34 (m, 3H), 3.15–3.11
(m, 1H), 1.93–1.84 (m, 1H), 1.79–1.73 (m, 1H), 1.31 (t, J = 6.8 Hz,
3H); MH+–H2O 447.
4.2.3.7.3. (10R,11S,12R,13R,14S)-17-Chloro-16-(4-ethoxybenzyl)-
2,3,4,5,6,7,9,10,11,12,13,14 -undecahydro-10,14-epoxybenzo[h][1,8]-
dioxacyclohexadecine-11,12,13-triol (34). 1H NMR (400 MHz,
DMSO-d6) d 7.29 (s, 1H), 7.10 (d, J = 8.8 Hz, 2H), 7.03 (s, 1H), 6.83
(d, J = 8.8 Hz, 2H), 5.11 (br, 1H), 5.02 (br, 1H), 4.83 (br, 1H), 4.39
(d, J = 10.0 Hz, 1H), 4.15–4.13 (m, 1H), 4.01–3.96 (m, 3H), 3.89–
3.86 (m, 2H), 3.66 (t, J = 9.2 Hz, 1H), 3.53 (d, J = 12.0 Hz, 1H), 3.46
(t, J = 8.0 Hz, 2H), 3.38–3.30 (m, 2H), 2.99–2.97 (m, 1H), 1.82 (m,
1H), 1.68–1.66 (m, 2H), 1.54–1.39 (m, 5H), 1.31 (t, J = 6.8 Hz, 3H);
MNa+ 529, MH+–H2O 489, MH+–NH4+ 471, IR (neat, cmÀ1) 3429,
2917, 1608, 1509, 1242, 1077, 1040, 989, 833.
4.2.3.5.
5-Chloro-4-(4-ethoxybenzyl)-2-((2S,3S,4R,5R,6R)-3,4,
5-tris(benzyloxy)-6-((5-iodo-pentyloxy)methyl)tetrahydro-2H-
pyran-2-yl)phenol (21). To a solution of 5-chloro-4-(4-ethoxy-
benzyl)-2-((2S,3S,4R,5R,6R)-3,4,5-tris(benzyloxy)-6-((5-hydroxy-
pentyloxy)methyl)tetrahydro-2H-pyran-2-yl)phenol (20, 459 mg,
0.58 mmol) in benzene (3 mL) under an atmosphere of nitrogen
was added imidazole (448 mg, 1.76 mmol) and triphenylphosphine
(467 mg,1.76 mmol). After 5 min, iodine (448 mg, 1.76 mmol) in
benzene (2 mL) was added dropwide to the reaction mixture at
room temperature. The reaction solution was stirred for 1 h, di-
luted with diethyl ether, quenched with saturated sodium hydro-
gen carbonate, extracted with diethyl ether (2Â). The organic
layer was washed with brine, dried over magnesium sulfate, fil-
tered and evaporated in vacuo. The resulting crude residue was
purified on a BiotageÒ purification apparatus (silica gel, 5–25% tet-
rahydrofuran in hexanes gradient) to yield the title compound (21,
485 mg, 0.54 mmol, 93%) as a yellow foam. 1H NMR (400 MHz,
DMSO-d6) d 9.83 (s, 1H), 7.38–7.28 (m, 10H), 7.24–7.10 (m, 5H),
7.06–7.04 (m, 2H), 6.92–6.86 (m, 3H), 6.76–6.74 (m, 1H), 4.82–
4.79 (m, 3H), 4.66–4.60 (m, 2H), 4.42–4.35 (m, 1H), 4.00–3.85
(m, 4H), 3.76–3.67 (m, 2H), 3.65–3.51 (m, 4H), 3.47–3.40 (m,
1H), 3.35–3.28 (m, 2H), 3.25–3.20 (m, 2H), 1.80–1.70 (m, 2H),
1.53–1.43 (m, 2H), 1.41–1.32 (m, 2H), 1.28 (t, J = 6.8 Hz, 3H);
MNa+ 913.
4.2.3.6. (9R,10S,11R,12R,13S)-10,11,12-Tris(benzyloxy)-16-chloro-
15-(4-ethoxybenz-yl)-3,4-,5,6,8,9,10,11,12,13-decahydro-2H-9,
13-epoxybenzo[h]-[1,7]dioxacyclopentadecine (32). To a solu-
tion of 5-chloro-4-(4-ethoxybenzyl)-2-((2S,3S,4R,5R,6R)-3,4,5-
tris(benzyloxy)-6-((5-iodopentyloxy)methyl)tetrahydro-2H-pyran-2-
yl)phenol (21, 480 mg, 0.54 mmol) from Step 4 in toluene (54 mL)
was added potassium carbonate (15 mg, 1.08 mmol) and 18-
crown-6 (288 mg, 1.08 mmol). The resulting mixture was stirred
at room temperature overnight and quenched with H2O. After dilu-
tion with ethyl acetate, the organic layer was washed with water and
brine prior to drying over magnesium sulfate. Filtration and removal
of volatiles under reduced pressure, then flash chromatography on a
BiotageÒ apparatus (silica gel, 10–50% tetrahydrofuran in hexanes
gradient) gave the macrocyclic compound (22, 261 mg, 0.34 mmol,
64%) as a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.36–7.27
4.2.3.7.4.
(7R,8S,9R,10R,11S)-14-Chloro-13-(4-ethylbenzyl)-3,4,
6,7,8,9,10,11-octahydro-2H-7,11-epoxybenzo[f][1,5]dioxacyclotride-
cine-8,9,10-triol (35). 1H NMR (400 MHz, DMSO-d6) d 7.50 (s, 1H),
7.29 (s, 1H), 7.12 (s, 4H), 5.17–5.14 (m, 2H), 5.03 (d, J = 5.6 Hz, 1H),
4.25 (d, J = 10.0 Hz, 1H), 4.17–4.15 (m, 1H), 4.01 (d, J = 14.8 Hz, 1H),
3.92 (d, J = 14.4 Hz, 1H), 3.90–3.80 (m, 2H), 3.74–3.68 (m, 2H),
3.64–3.58 (m, 1H), 3.46–3.38 (m, 2H), 3.15–3.09 (m, 1H), 2.55 (q,
J = 7.6 Hz, 2H), 1.90–1.84 (m, 1H), 1.78–1.72 (m, 1H), 1.16 (t,
J = 7.6 Hz, 1H),3H); MH+–H2O 431, IR (neat, cmÀ1) 3374, 2962,
2927, 2872, 1607, 1488, 1381, 1251, 1087, 1039, 987, 846.
4.2.3.7.5. (8R,9S,10R,11R,12S)-15-Chloro-14-(4-ethylbenzyl)-2,3,4,
5,7,8,9,10,11,12-decahyd-ro-8,12-epoxybenzo[g][1,6]dioxacyclotetrade-
cine-9,10,11-triol (36). 1H NMR (400 MHz, DMSO-d6) d 7.32 (s,
1H), 7.13 (s, 4H), 7.07 (s, 1H), 5.05 (d, J = 4.8 Hz, 1H), 5.00 (d,

M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
5477
J = 5.2 Hz, 1H), 4.92 (d, J = 6.4 Hz, 1H), 4.17 (d, J = 10.0 Hz, 1H), 3.98
(d, J = 14.8 Hz, 1H), 3.91 (d, J = 14.8 Hz, 1H), 3.71–3.61 (m, 3H),
3.56–3.52 (m, 1H), 3.40–3.37 (m, 4H), 3.31–3.25 (m, 1H), 3.19–
3.15 (m, 1H), 2.56 (q, J = 7.6 Hz, 2H), 1.79 (m, 2H), 1.73–1.69 (m,
1H), 1.61–1.60 (m, 1H), 1.16 (t, J = 7.6 Hz, 1H); MNa+ 485, MH+–
3380, 2917, 1506, 1463, 1284, 1255, 1122, 1087, 1067, 1045,
1000, 982, 952, 919, 885.
4.2.3.7.11.
benzo[b ][1,4]dioxin-6-yl)-methyl)-3,4,5,6,8,9,10,11,12,13-decahydro-
2H-9,13-epoxybenzo[h ][1,7]diox-acyclopentadecine-10,11,12-triol
(9R,10S,11R,12R,13S)-16-Chloro-15-((2,3-dihydro-
H2O 445, MH+–NH4 427, IR (neat, cmÀ1) 3375, 2964, 2860, 1616,
(42). 1H NMR (400 MHz, DMSO-d6) d 7.25 (s, 1H), 7.06 (s, 1H),
6.77–6.71 (m, 1H), 6.68–6.63 (m, 2H), 5.05 (d, J = 4.4 Hz, 1H),
5.00 (d, J = 5.5 Hz, 1H), 4.82 (d, J = 6.1 Hz, 1H), 4.23–4.16 (m, 5H),
4.08–3.97 (m, 2H), 3.84 (quartet, J = 14.8 Hz, 2H), 3.71–3.63 (m,
1H), 3.58 (d, J = 11.9 Hz, 1H), 3.51–3.37 (m, 4H), 3.32–3.23 (m,
1H), 3.18–3.09 (m, 1H), 1.78–1.59 (m, 3H), 1.57–1.38 (m, 3H);
MNa+ 529; IR (neat, cmÀ1) 3375, 2912, 1591, 1507, 1495, 1285,
1256, 1126, 1069, 1034, 1003, 920, 882.
+
1495, 1329, 1253, 1086, 1048, 983, 832.
4.2.3.7.6. (9R,10S,11R,12R,13S)-16-Chloro-15-(4-ethylbenzyl)-3,4,
5,6,8,9,10,11,12,13-decah-ydro-2H-9,13-epoxybenzo[h][1,7]dioxa-
cyclopentadecine-10,11,12-triol (37).
O
Cl
O
O
4.2.4. Preparation of diene precursor and macrocyclization
4.2.4.1. (4aR,6S,7R,8R,8aS)-6-(2-(Allyloxy)-4-chloro-5-(4-eth-
oxybenzyl)phenyl)-2-vinylhexahydropyrano[3,2-d][1,3]diox-
ine-7,8-diol (24). To a solution of intermediate tetraol (13,
500 mg, 1.08 mmol) in N,N-dimethylformamide (3 mL) under an
atmosphere of nitrogen was added acrolein diethyl acetal
(0.51 mL, 3.23 mmol) and camphorsulfonic acid (64 mg,
0.26 mmol), and the mixture was stirred at the same temperature
overnight. The reaction mixture was quenched by addition of tri-
ehylamine (0.045 mL, 0.32 mmol) and the removal of volatiles un-
der reduced pressure yielded desired acetal compound (24) as a
white-off solid which was used without further purification.
MNa+ 525.
HO
OH
OH
1H NMR (400 MHz, DMSO-d6) d 7.29 (s, 1H), 7.13 (s, 4H), 7.09 (s,
1H), 5.06 (br, 1H), 5.00 (br, 1H), 4.84 (br, 1H), 4.21 (d, J = 10.0 Hz,
1H), 4.04 (m, 2H), 4.99 (d, J = 14.8 Hz, 1H), 3.91 (d, J = 14.8 Hz,
1H), 3.72–3.68 (m, 1H), 3.60 (d, J = 12.0 Hz, 1H), 3.46–3.39 (m,
4H), 3.30–3.26 (m, 1H), 3.16–3.14 (m, 1H), 2.56 (q, J = 7.6 Hz,
1H), 1.68 (m, 3H), 1.50–1.42 (m, 3H), 1.16 (t, J = 7.6 Hz, 1H);
MNa+ 499, MH+–H2O 459, MH+–NH4 441, IR (neat, cmÀ1) 3358,
+
2918, 1608, 1568, 1493, 1466, 1257, 1212, 1062, 991, 915, 829.
4.2.3.7.7. (10R,11S,12R,13R,14S )-17-Chloro-16-(4-ethylbenzyl)-
2,3,4,5,6,7,9,10,11,12,13,14-undecahydro-10,14-epoxybenzo[h][1,8]-
dioxacyclohexadecine-11,12,13-triol (38). 1H NMR (400 MHz,
DMSO-d6) d 7.31 (s, 1H), 7.12 (s, 4H), 7.03 (s, 1H), 5.08 (d,
J = 4.4 Hz, 1H), 5.01 (d, J = 4.4 Hz, 1H), 4.82 (d, J = 5.2 Hz, 1H),
4.39 (d, J = 10.0 Hz, 1H), 4.16–4.13 (m, 1H), 4.01 (d, J = 14.8 Hz,
1H), 3.93–3.86 (m, 2H), 3.70–3.65 (m, 1H), 3.53 (d, J = 12.0 Hz,
1H), 3.48–3.44 (m, 2H), 3.38–3.29 (m, 2H), 2.97 (m, 1H), 2.56 (q,
J = 7.2 Hz, 1H), 1.82 (m, 1H), 1.68–1.66 (m, 2H), 1.54–1.49 (m,
1H), 1.46–1.39 (m, 4H), 1.16 (t, J = 7.6 Hz, 1H); MNa+ 513, MH+–
4.2.4.2. (2S,3R,4R,5S,6R)-2-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-6-(allyloxyme
thyl)tetrahydro-2H-pyran-3,4,5-
triol (25). To
a
solution (4aR,6S,7R,8R,8aS)-6-(2-(allyloxy)-4-
chloro-5-(4-ethoxybenzyl)phenyl)-2-vinylhexahydropyrano[3,2-d]-
[1,3]dioxine-7,8-diol (24, 669 mg, 1.33 mmol) from Step 1 in tetra-
hydrofuran (13 mL) was added sodium cyanoborohydride(642 mg,
9.71 mmol), molecular sieves (407 mg) and was added cautiously
trifluoromethanesulfonic acid. After addition, the mixture was stir-
red for another 0.5 h, before being poured into H2O. The aqueous
phase was extracted with dichloromethane and the combined
organic fractions were washed with brine. The organic layer was
dried over magnesium sulfate and filtered through a pad of silica
gel. The filtrate was evaporated and went to next step. MNa+ 527.
+
H2O 473, MH–NH4 455; IR (neat, cmÀ1) 3396, 2917, 1610, 1497,
1323, 1255, 1134, 1059, 1003, 944, 827.
4.2.3.7.8.
(8R,9S,10R,11R,12S)-15-Chloro-14-(4-(methylthio)-
benzyl)-2,3,4,5,7,8,9,10,11,12-d ecahydro-8,12-epoxybenzo[g][1,6]-
dioxacyclotetradecine-9,10,11-triol (39). 1H NMR (400 MHz,
DMSO-d6) d 7.30 (s, 1H), 7.23–7.09 (m, 4H), 7.06 (s, 1H), 5.07–
4.89 (m, 3H), 4.15 (d, J = 9.8 Hz, 1H), 4.05–3.93 (m, 4H), 3.74–
3.58 (m, 3H), 3.56–3.61 (m, 1H), 3.41–3.31 (m, 2H), 3.29–3.21
(m, 1H), 3.19–3.09 (m, 1H), 2.42 (s, 3H), 1.85–1.55 (m, 4H);
MNa+ 503; IR (neat, cmÀ1) 3387, 2917, 1492, 1254, 1081, 1052,
1002, 914, 834.
4.2.4.3. (2S,3S,4R,5R,6R)-2-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-6-(allyloxyme
thyl)tetrahydro-2H-pyran-3,4,5-
triyl triacetate (26). The obtained triol (25) was diluted dichloro-
methane (15 mL) and added acetic anhydride (1.1 mL, 12.1 mmol),
DMAP (9.2 mg, 0.076 mmol) and pyridine (1.0 mL, 12.11 mmol).
After 18 h, the reaction was quenched by addition of H2O, where-
upon the resulting mixture was extracted with dichloromethane
(2Â). The combined organic layers were washed with 1 N HCl
(2Â) and brine (2Â) prior to drying over magnesium sulfate. After
filtration and concentration under reduced pressure , the resulting
oil was purified on BiotageÒ purification apparatus (silica gel, 5–
30% tetrahydrofuran in hexanes) to yield the title compound (26,
580 mg, 0.92 mmol, 61%; three steps) as a white solid. 1H NMR
(400 MHz, CDCl3) d 7.20 (s, 1H), 7.04 (d, J = 8.8 Hz, 2H), 6.84 (s,
1H), 6.79 (d, J = 8.4 Hz, 2H), 6.07–5.99 (m, 1H), 5.85–5.78 (m,
1H), 5.44 (dd, J = 1.6, 17.6 Hz, 1H), 5.34–5.23 (m, 3H), 5.19–50.13
(m, 2H), 4.82 (d, J = 9.2 Hz, 1H), 4.50 (d, J = 4.0 Hz, 1H), 4.02–3.90
(m, 6H), 3.79–3.74 (m, 1H), 3.53 (d, J = 4.4 Hz, 1H), 2.04 (s, 3H),
2.00 (s, 3H), 1.74 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H); MNa+ 653.
4.2.3.7.9.
(9R,10S,11R,12R,13S)-16-Chloro-15-(4-(methylthio)-
benzyl)-3,4,5,6,8,9,10,11,12,13-decahydro-2H-9,13-epoxybenzo[h][1,7]-
dioxacyclopentadecine-10,11,12-triol (40). 1H NMR (400 MHz,
DMSO-d6) d 7.27 (s, 1H), 7.21–7.10 (m, 4H), 7.07 (s, 1H), 5.05 (d,
J = 4.4 Hz, 1H), 4.99 (d, J = 5.3 Hz, 1H), 4.81 (d, J = 5.8 Hz, 1H), 4.19
(d, J = 10.0 Hz, 1H), 4.06–3.88 (m, 4H), 3.71–3.62 (m, 1H), 3.58 (d,
J = 11.6 Hz, 1H), 3.52–3.37 (m, 4H), 3.27–3.21 (m, 1H), 3.18–3.08
(m, 1H), 1.77–1.58 (m, 3H), 1.56–1.39 (m, 3H); MNa+ 517; IR (neat,
cmÀ1) 3378, 2918, 1609, 1492, 1255, 1034, 997, 833, 774.
4.2.3.7.10.
(8R,9S,10R,11R,12S)-15-Chloro-14-((2,3-dihydro-
benzo[b][1,4]dioxin-6-yl)-methyl)-2,3,4,5,7,8,9,10,11,12-decahydro-
8,12-epoxybenzo[g][1,6]dioxacyc-lotetradecine-9,10,11-triol
(41). 1H NMR (400 MHz, DMSO-d6) d 7.28 (s, 1H), 7.05 (s, 1H),
6.77–6.71 (m, 1H), 6.69–6.62 (m, 2H), 5.04 (d, J = 4.0 Hz, 1H),
4.99 (d, J = 4.4 Hz, 1H), 4.91 (d, J = 5.4 Hz, 1H), 4.22–4.11 (m, 5H),
4.09–3.96 (m, 2H), 3.84 (quartet, J = 14.8 Hz, 2H), 3.70–3.58 (m,
3H), 3.57–3.44 (m, 1H), 3.39–3.32 (m, 2H), 3.29–3.22 (m, 1H),
4.2.4.4. (8R,9S,10R,11R,12S)-9,10,11-Tris(acetyloxy)-15-chloro-
14-(4-ethoxybenzyl)-5,7,8,9, 10,11,12-hexahydro-2H-8,12-epox-
ybenzo[g ][1,6]dioxacyclotetradecine (27). To
a solution of
3.17–3.09 (m, 1H), 1.85–1.52 (m, 4H); MNa+ 515; IR (neat, cmÀ1
)
(2S,3S,4R,5R,6R)-2-(2-(allyloxy)-4-chloro-5-(4-ethoxybenzyl)phenyl)-

5478
M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
6-(allyloxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (26,
580 mg, 0.92 mmol) from Step 3 in dichloromethane (184 mL,
0.005 M) under an atmosphere of nitrogen was added Grubbs
2nd generation catalyst (156 mg, 0.184 mmol), and the mixture
was heated at 60 °C for three days. After re-cooling to room tem-
perature, the reaction mixture was filtered through a pad of Cel-
iteÒ, washed with ethyl acetate (20 mL) and the filtrated was
evaporated under reduced pressure. The resulting crude residue
was purified on a BiotageÒ purification apparatus (silica gel, 5–
20% tetrahydrofuran in hexanes gradient) to yield the title com-
pound (27, (Z)-form, 125 mg, 0.208 mmol, 23%) as a white solid.
1H NMR (400 MHz, CDCl3) d 7.22 (s, 1H), 7.18 (s, 1H), 7.11 (d,
J = 8.4 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 5.95–5.87 (m, 1H), 5.72
(dt, J = 4.0, 15.2 Hz, 1H), 5.56 (t, J = 9.2 Hz, 1H), 5.35–5.28 (m,
1H), 4.92 (t, J = 10.0 Hz, 1H), 4.77–4.67 (m, 2H), 4.41–4.36 (m,
1H), 4.14–4.11 (m, 1H), 4.05–3.94 (m, 5H), 3.72 (t, J = 8.8 Hz, 1H),
3.65 (d, J = 13.2 Hz, 1H), 3.54–3.48 (m, 1H), 2.07 (s, 3H), 2.02 (s,
3H), 1.75 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H); MNa+ 625.
Grubbs 2nd generation catalyst (100 mg, 0.16 mmol), and the mix-
ture was heated at 60 °C overnight. After re-cooling to room tem-
perature, the reaction mixture was filtered through a pad of
CeliteÒ, washed with ethyl acetate (100 mL) and the filtrated was
evaporated under reduced pressure. The resulting crude residue
was purified on a BiotageÒ purification apparatus (silica gel, 5–
20% tetrahydrofuran in hexanes gradient) to yield the title com-
pound (30, (Z)-form, 279 mg, 0.374 mmol, 26%) as a white solid.
1H NMR (400 MHz, DMSO-d6) d 7.29–7.25 (m, 10H), 7.22–7.13
(m, 5H), 7.12–7.02 (m, 3H), 6.89 (s, 1H), 6.86–6.82 (m, 2H), 6.79–
6.69 (m, 2H), 5.96–5.92 (m, 2H), 4.79–4 (d, J = 5.6 Hz, 1H), 4.65
(d, J = 6.0 Hz, 1H), 4.15 (d, J = 10.0 Hz, 1H), 4.02.77 (m, 3H), 4.68–
4.65 (m, 1H), 4.56–4.53 (m, 1H), 4.49–4.43 (m, 1H), 4.41–4.34
(m, 2H), 4.22–4.13 (m, 1H), 4.12–4.02 (m, 2H), 3.99–3.81 (m,
5H), 3.76–3.71 (m, 1H), 3.68–3.63 (m, 2H), 3.47–3.45 (m, 1H),
1.32 (t, J = 6.8 Hz, 3H); MNa+ 769.
4.2.4.8. (8R,9S,10R,11R,12S)-15-Chloro-14-(4-ethoxybenzyl)-2,3,
4,5,7,8,9,10,11,12-decahyd
dioxacyclotetradecine-9,10,11-triol (31). To
ro-2H-8,12-epoxybenzo[g][1,6]-
solution of
4.2.4.5. (8R,9S,10R,11R,12S)-15-Chloro-14-(4-ethoxybenzyl)-5,7,
8,9,10,11,12-hexahydro-2H-8,12-epoxybenzo[g][1,6]dioxacycl-
otetradecine-9,10,11-triol (28). To a solution (2S,3S,4R,5R,6R)-2-
(2-(allyloxy)-4-chloro-5-(4-ethoxybenzyl)phenyl)-6-(allyloxym-
ethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (27, 125 mg,
0.208 mmol) from Step 4 in methanol (4 mL) was added sodium
methoxide (25% in methanol, 0.6 mL) and the reaction mixture
stirred at ambient temperature for 1 h. The solution was cooled
to 0 °C prior to neutralizing with acetic acid (0.3 mL). After removal
of organic volatiles under reduced pressure, the residue was diluted
with methanol. Purification by reverse phase preparative HPLC
(WatersÒ, SunFire™ Prep, 5–50% acetonitrile in water gradient) pro-
vided the title compound (28, 56 mg, 0.117 mmol, 56%) as a white
solid. 1H NMR (400 MHz, DMSO-d6) d 7.33 (s, 1H), 7.30 (s, 1H), 7.29
(d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.8 Hz, 2H), 5.86–5.80 (m, 1H), 5.72
(dt, J = 7.6, 15.2 Hz, 1H), 5.07 (d, J = 4.4 Hz, 1H), 5.01 (d, J = 5.6 Hz,
1H), 4.79 (d, J = 6.0 Hz, 1H), 4.62 (dd, J = 8.8, 12.0 Hz, 1H), 4.38 (dd,
J = 5.6, 11.6 Hz, 1H), 4.23 (d, J = 10.0 Hz, 1H), 4.00–3.88 (m, 6H),
3.83 (d, J = 11.6 Hz, 1H), 3.66–3.60 (m, 1H), 3.28–3.24 (m, 3H),
a
(2S,3R,4R,5S,6R)-2-(4-chloro-3-((5-(pyrazin-2-yl)-1,3,4-thiadiazol-
2-yl)methyl)phenyl)-6-(hydroxymethyl)-tetrahydro-2H-pyran-3,
4,5-triol (30, 279 mg, 0.374 mmol) in methanol (3 mL)/tetrahydro-
furan (3 mL) was added 10% palladium on charcoal (22 mg). The
reaction mixture was stirred under hydrogen gas overnight. The
reaction solution was filtered through syringe filter and the filtrate
was evaporated under reduced pressure. The crude compound was
diluted with methanol and purified by reverse phase preparative
HPLC (WatersÒ, SunFire™ Prep, 5–50% acetonitrile in water gradi-
ent) provided the title compound (31, 38 mg, 0.08 mmol, 22%) as a
white solid. 1H NMR (400 MHz, DMSO-d6) d 7.27 (s, 1H), 7.09 (d,
J = 8.8 Hz, 1H), 7.05 (s, 1H), 6.82 (d, J = 8.4 Hz, 1H), 5.01 (d,
J = 4.4 Hz, 1H), 4.96 (d, J = 5.6 Hz, 1H), 4.65 (d, J = 6.0 Hz, 1H),
4.15 (d, J = 10.0 Hz, 1H), 4.02–3.94 (m, 2H), 3.96 (q, J = 7.2 Hz,
2H), 3.89 (d, J = 9.2 Hz, 1H), 3.67–3.59 (m, 2H), 3.54–3.50 (m,
1H), 3.38–3.36 (m, 1H), 3.29–3.23 (m, 1H), 3.15–3.14 (m, 1H),
1.77–1.58 (m, 4H), 1.29 (t, J = 7.2 Hz, 3H); MH+–H2O 461, MH+–
NH4+ 443, IR (neat, cmÀ1) 3444, 3288, 2977, 2931, 1608, 1512,
1476, 1392, 1349, 1238, 1176, 1012, 959, 842.
2.98–2.93 (m, 1H), 1.30 (t, J = 7.2 Hz, 3H); MH+ 499, IR (neat, cmÀ1
)
3425, 2922, 1607, 1511, 1485, 1245, 1079, 1028, 974, 826.
4.3. In vitro assay
4.2.4.6. (2S,3S,4R,5R,6R )-2-(2-(Allyloxy)-4-chloro-5-(4-ethoxyb-
enzyl)phenyl)-6-(allyloxyme thyl)-3,4,5-tris(benzyloxy)tetrahy-
dro-2H-pyran (29). To a solution of primary alcohol (16, 1.5 g,
2.04 mmol) from Step 1 in N,N-dimethylformamide (10 mL) at
0 °C under an atmosphere of nitrogen was added sodium hydride
(60% dispersion in mineral oil, 123 mg, 3.06 mmol), and the mix-
ture was stirred for 30 min at the same temperature. Then allyl
bromide (0.26 mL, 3.06 mmol) was added dropwise, and the mix-
ture was stirred with gradual warming to ambient temperature
over 5 h. After re-cooling to 0 °C, the reaction mixture was
quenched by addition of water (10 mL). The mixture was diluted
with saturated ammonium chloride and extracted with ethyl ace-
tate. The organic layer was washed with brine, dried over magne-
sium sulfate, filtered and evaporated in vacuo. The resulting crude
residue was purified on a BiotageÒ purification apparatus (silica
gel, 5–20% tetrahydrofuran in hexanes gradient) to yield the title
compound (29, 1.12 g, 1.44 mmol, 71%) as a white solid. MNa+ 797.
4.3.1. Cloning and cell line construction for human SGLT2
Human SGLT2 (hSGLT2) gene was amplified by PCR from cDNA-
Human Adult Normal Tissue Kidney (Invitrogen, Carlsbad, CA). The
hSGLT2 sequence was cloned into pcDNA3.1(+) for mammalian
expression and were stably transfected into Chinese hamster ovary
(CHO) cells. SGLT2-expressing clones were selected based on resis-
tance to G418 antibiotic (GeneticinÒ, Invitrogen, Carlsbad, CA) and
activity in the 14C-
a-methyl-D
-glucopyranoside (14C-AMG) uptake
assay.
4.3.2. Inhibitory effects on human SGLT2 activities
For sodium-dependent glucose transport assay, cells expressing
hSGLT2 were seeded into a 96-well culture plate at a density of
5 Â 104 cells/well in RPMI medium 1640 containing 10% fetal bo-
vine serum. The cells were used one day after plating. They were
incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris,
140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2,
pH 7.4) at 37 °C for 10 min. They were then incubated in uptake
buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) con-
4.2.4.7.
(Z)-((8R,9S,10R,11R,12S)-9,10,11-Tris(benzyloxy)-15-
7,8,9,10,11,12-hexahydro-2H-
chloro-14-(4-ethoxybenzyl)-5,
8,12-epoxybenzo[g][1,6]dioxacyclotetradecine (30). To a solu-
tion of (2S,3S,4R,5R,6R)-2-(2-(allyloxy)-4-chloro-5-(4-ethoxyben-
zyl)phenyl)-6-(allyloxymethyl)-3,4,5-tris(benzyloxy)tetrahydro-2H-
pyran (29, 1.12 g, 1.44 mmol) from Step 3 in dichloromethane
(150 mL, 0.01 M) under an atmosphere of nitrogen was added
taining 14C-labeled (8
lM) and inhibitor or dimethyl sulfoxide
(DMSO) vehicle at 37 °C for 2 h. Cells were washed twice with
washing buffer (pretreatment buffer containing 10 mM AMG at
room temperature) and then the radioactivity was measured using

M. J. Kim et al. / Bioorg. Med. Chem. 19 (2011) 5468–5479
5479
Presented at the 238th American Chemical Society National Meeting and
Exposition, Washington, DC, August 16–20, 2009; Medi-151.; (b) Nomura, S.;
Sakamaki, S.; Hongu, M.; Kawanshi, E.; Koga, Y.; Sakamoto, T.; Yamamoto, Y.;
Ueta, K.; Kimata, H.; Nakayama, K.; Tsuda-Tsukimoto, M. J. Med. Chem. 2010,
53, 6355.
a liquid scintillation counter. IC50 was determined by nonlinear
regression analysis using GraphPad PRISM.16,17
Acknowledgment
13. (a)
Boehringer
Ingelheim
Pharmaceuticals,
www.clinicaltrials.gov,
NCT01164501, 2010.; (b) Lexicon Pharmaceuticals, www.clinicaltrials.gov,
NCT00962065, 2009.; (c) Astellas Pharma Inc., www.clinicaltrials.gov,
NCT01135433, 2010.; (d) Mascitti, V. Presented at the 240th American
Chemical Society National Meeting and Exposition, Boston, MA, Aug 22–26,
2010; Medi-151.; (e) Mascitti, V.; Maurer, T. S.; Robinson, R. P.; Bian, J.;
Boustany-Kari, C. M.; Brandt, T.; Collman, B. M.; Kalgutkar, A. S.; Klenotic, M. K.;
Leininger, M. T.; Lowe, A.; Maguire, R. J.; Masteson, V. M.; Miao, Z.; Mukaiyama,
E.; Patel, J. D.; Pettersen, J. C.; Preville, C.; Samas, B.; She, L.; Sobol, Z.; Steppan,
C. M.; Stevens, B. D.; Thuma, B. A.; Tugnait, M.; Zeng, D.; Zhu, T. J. Med. Chem.
2011, 54, 2952.
We appreciate Dr. Eun Chul Huh for his leadership and supports
as Head of R&D at Green Cross Corporation.
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Further reading
5. Zhou, L.; Cryan, E. V.; D’Andrea, M. R.; Belkowski, S.; Conway, B. R.; Demarest, K.
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Yamamoto, Y.; Ueta, K.; Kimata, H.; Nakayama, K.; Tsuda-Tsukimoto, M.
T. J. Cell. Biochem. 2003, 90, 339.