ortho-Substituted C-aryl glucosides as highly potent and selective renal sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.04.088

A series of 2-substituted C-aryl glucosides have been synthesized and evaluated for inhibition of hSGLT1 and hSGLT2. Introduction of an appropriate ortho substituent at the proximal phenyl ring adjacent to the glycosidic bond was found to improve SGLT2 in

Full text of DOI:10.1016/j.bmc.2010.04.088

Bioorganic & Medicinal Chemistry 18 (2010) 4422–4432
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
ortho-Substituted C-aryl glucosides as highly potent and selective renal
sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors
b
Baihua Xu a,b,c, , Yan Feng , Binhua Lv a,b,c, Ge Xu b, Lili Zhang b, Jiyan Du b, Kun Peng b, Min Xu b,
*
Jiajia Dong b, Wenbin Zhang b, Ting Zhang b, Liangcheng Zhu b, Haifeng Ding b, Zelin Sheng b,
Ajith Welihinda d, Brian Seed d, Yuanwei Chen a,b,
*
a Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu, Sichuan 610041, PR China
b Egret Pharma (Shanghai) Limited, Shanghai 201203, PR China
c Graduate School of Chinese Academy of Sciences, Beijing 100049, PR China
d Theracos, Inc., Sunnyvale, CA 94085, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-substituted C-aryl glucosides have been synthesized and evaluated for inhibition of hSGLT1
and hSGLT2. Introduction of an appropriate ortho substituent at the proximal phenyl ring adjacent to the
glycosidic bond was found to improve SGLT2 inhibitory activity and dramatically increase selectivity for
hSGLT2 over hSGLT1. Selected compounds were investigated for in vivo efficacy.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 17 March 2010
Revised 23 April 2010
Accepted 24 April 2010
Available online 29 April 2010
Keywords:
Diabetes
ortho-Substituted C-aryl glucosides
SGLT
Sodium-dependent glucose co-transporter
Urinary glucose
Blood glucose
1. Introduction
opened ortho-substituted compounds (Fig. 1), based on compounds
1, 2 and 3, as SGLT2 inhibitors. The synthetic strategy is centered
Inhibition of renal sodium-dependent glucose co-transporter
(SGLT) proteins is an attractive strategy for the mitigation of
hyperglycemia in type 2 diabetes.1 It has been reported that 90%
of renal glucose re-absorption is mediated by SGLT2, which is
localized to the S1 and S2 segments of the proximal tubule.2 The
remaining renal glucose re-absorption is thought to be principally
mediated by SGLT1, which is localized to the more distal S3 seg-
ment of the proximal tubule.2 Multiple classes of metabolically
instable O-glucosides1a,3 and metabolically more robust C-gluco-
sides, like dapagliflozin,4 have been reported as SGLT2 inhibitors
to date.
We have previously discussed the activity of O-spiroketal C-aryl
glucosides (formula II, Fig. 1), as SGLT2 inhibitors.5 Compared to
dapagliflozin 1, O-spiroketal glucoside 3 exhibited similar in vitro
hSGLT2 inhibitory activity and slightly higher selectivity toward
hSGLT2 than SGLT1, but had less in vivo activity and displayed
unfavorable gastrointestinal side effects (diarrhea and cecal
dilation due to gas accumulation).5 In this study we identify ring-
on the introduction of a substituent at the 6-position of the prox-
imal phenyl ring of formula I to obtain 2-substituted C-aryl gluco-
side SGLT2 inhibitors (formula III).6
2. Results and discussion
2.1. Chemistry
Scheme 1 describes the formation of the core aglycones, a ben-
zyl ether series, and a phenethyl ether series, that were diversified
in this study from intermediates 4a and 4b, the synthetic method
of which was described in details in the patent WO2008122014.6
Phenylethanols 7a and 7b were constructed using Brown hydrob-
oration reaction of styrenes 6a and 6b, which were synthesized
by the oxidation of benzylalcohols 4a and 4b5 followed by Wittig
olefination of 5a and 5b. Alkylation of benzyl alcohol 4a and
phenylethanols 7a–b gave aglycones 8a–b (Scheme 1, top) and
9a–b (Scheme 1, bottom) respectively.
As illustrated in Scheme 2, the b-C-aryl glucosides 11a–b and
12a–b were synthesized via a previously reported method.7 Lithi-
ation of bromodiarylmethanes 8a–b and 9a–b followed by their
* Corresponding authors. Tel.: +86 28 85232730; fax: +86 28 85259387.
E-mail addresses: xubaihua2002@126.com, baihuaxu@egretpharma.com (B. Xu),
chenyw@cioc.ac.cn (Y. Chen).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.088

B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
4423
anol under Mitsunobu reaction conditions and finally by hydrogen-
olysis of the benzyl groups.
Cl
R
6
O
R1
HO
HO
2
Cl
R2
The synthesis of analogs 23–26 is shown in Scheme 4. A meth-
ylene extended 3-propoxy alcohol 23 was obtained directly by
Brown hydroboration of alkene 11b (path a). Wacker–Tsuji
oxidation13 of the alkene 11b (path b) provided ketone 24, which
was subjected to reduction with sodium borohydride and to reduc-
tive amination with sodium cyanoborohydride and methylamine
to provide the corresponding secondary alcohol 25 and secondary
amine 26 (as a mixture of diastereomers).
Scheme 5 depicts the synthesis of fluorides 29 and 30. Wacker–
Tsuji oxidation13 of alkene 14 generated ketone 27 which was
reduced by sodium borohydride to obtain alcohol 28. Fluorination
of alcohol 28 and ketone 27 with DAST11 followed by hydrogenol-
ysis produced the corresponding monofluoro and difluoro-substi-
O
n
OH
O
I
HO
HO
OH
1 R = OEt
OH
2 R = Et
III
OH
2
Cl
R
O
O
HO
HO
OH
II
OH
3 R = OEt
Figure 1.
R2
Cl
R1
8a R1 = Et, R2 = Me
8b R1 = Et, R2 =
O
tuted analogs 29 (as
a mixture of diastereomers) and 30
Br
respectively.
d
As shown in Scheme 6, peracetylation of 11b yielded tetraace-
tate 31 that was subjected to deallylation12 to give benzyl alcohol
32, which subsequently underwent Mitsunobu reaction followed
by hydrolysis to generate phenyl-substituted analog 33.
Alkyne derivatives 34 and 35 described in Scheme 7 were ob-
tained from the corresponding alkenes 11b and 12b by electro-
philic addition of bromine to the double bond followed by
elimination under strong alkaline conditions.
O
Cl
R1
a
Cl
R1
HO
Br
H
Br
4a R1 = Et
5a R1 = Et
5b R1 = OEt
b
4b R1 = OEt
Cl
R1
Cl
R1
2
c
HO
Br
Br
6a R1 = Et
7a R1 = Et
2.2. Biological activity evaluation
6b R1 = OEt
7b R1 = OEt
d
The target compounds were screened in cell-based SGLT func-
tional assays, in which 293 cells expressing hSGLT2 and COS-7 cells
expressing hSGLT1 were used. The results were presented in Table 1.
In the benzylic ether series, 2-methoxymethyl-substituted gluco-
side 11a was 7-fold more potent and 11-fold more selective for inhi-
bition of hSGLT2 compared to dapagliflozin 114 and O-spiroketal
glucoside 3. In general the benzylic ethers tolerated a variety of sub-
stitutions and resulted in higher selectivity for hSGLT2 compared to
hSGLT1. Some notably less potent outliers were the allyl ether 11b,
which was 57-fold less active than the methyl ether, the trifluoro-
ethyl ether 22, which was 190-fold less active, and the hydroxypro-
pyl ether 23, which was 125-fold less active. The reduced activity of
the trifluoroethyl ether is striking when considered in the context of
the di- and monofluoroethyl ethers, both of which exhibited excel-
lent potency and selectivity. A related steep SAR can be noted among
the fluorinated propyl ethers, for example comparing the activities
of the 2-fluoro and the 2,2-difluoro analogs 29 and 30. The
1-hydroxypropyl ether 23 was significantly less active than the
2-hydroxypropyl ether 25, and introduction of a cationic center into
the side chain (compound 26) was poorly tolerated. Similarly
disruptive was the introduction of a bulky phenyl group, 33. Surpris-
ingly, the propargyl ether 34 displayed an exceptional combination
of potency and specificity, with sub-nanomolar activity and more
than 3300-fold selectivity for SGLT2.
9a R1 = OEt, R2 = Me
9b R1 = Et, R2 =
Cl
R1
R2O
2
Br
Scheme 1. Construction of the aglycones. Reagents and conditions: (a) Dess–
Martin periodinane, DCM, 0 °C to rt, (96% for 5a, 89% for 5b); (b) Ph3P+MeIꢁ,
KHMDS, toluene, rt (81% for 6a, 94% for 6b); (c) 9-BBN, THF, 0 °C to rt, then MeOH,
NaOH, 30% H2O2 (70% for 7a, 64% for 7b); (d) NaH, TBAI, DMF or THF, 0 °C or rt, then
MeI or allylbromide (78–91%).
addition to 2,3,4,6-tetra-O-trimethylsilyl-D-gluconolactone 10 and
finally treatment with methanesulfonic acid provided desilylated
O-methyl lactols, which were reduced using triethylsilane and
BF3ꢀOEt2 to give the desired ortho-substituted C-aryl glucosides.8
Schemes 3–7 illustrate the synthetic routes to modify the allyl
moiety of glucoside 11b. The synthesis of compounds 17–20 and
22 is shown in Scheme 3. Bromodiarylmethane 8b was lithiated
and added to 2,3,4,6-tetra-O-benzyl-D
-gluconolactone 13.9 Subse-
quent reduction of the lactol gave predominantly b-linked C-aryl
glucoside 1410 followed by ozonolysis to provide aldehyde 15.
Reduction of this aldehyde afforded alcohol 16 which was depro-
tected by hydrogenolysis to give the hydroxyl-substituted analog
17. Methylation of alcohol 16 followed by deprotection yielded
methoxy-substituted compound 18. Fluorination of alcohol 16
and aldehyde 15 with DAST11 followed by hydrogenolysis
produced mono and difluoro-substituted analogs 19 and 20,
respectively. Trifluoroethoxy compound 22 was constructed by
deallylation12 of 14 followed by alkylation with 2,2,2-trifluoroeth-
Limited comparisons between the benzyl and phenethyl ether
series showed that short side chains were well tolerated in the
phenethyl context, and that the allyl ether was more effective
and more selective than in the benzyl context. The propargyl ether
11a n = 1, R1 = Et, R2 = Me
O
O
TMSO
TMSO
1) a, b
R2
11b n = 1, R1 = Et, R2
=
Cl
R1
OTMS
O
n
OTMS
O
8a-b
9a-b
HO
HO
10
12a n = 2, R1 = OEt, R2 = Me
12b n = 2, R1 = OEt, R2
OH
=
2) c
OH
Scheme 2. General route employed in the synthesis of C-arylglucosides. Reagents and conditions: (a) n-BuLi, THF/toluene (1:2), ꢁ78 °C, then 10; (b) MeSO3H, MeOH, ꢁ78 °C
to rt; (c) Et3SiH, BF3ꢀOEt2, MeCN/DCM (1:1), ꢁ30 to ꢁ10 °C (22–58% three steps).

4424
B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
O
O
BnO
BnO
Cl
Et
Cl
Et
Cl
Et
F3C
HO
O
O
HO
O
O
O
OBn
g
1) a
h, i
OBn
13
BnO
BnO
BnO
HO
OH
OBn
Et
BnO
OBn
d
8b
OH
OBn
21
OBn
14
c
2) b
22
HO
HO
Cl
Et
Cl
Et
HO
Cl
i
O
OHC
BnO
O
O
O
O
O
BnO
HO
OH
BnO
OBn
BnO
OBn
OH
OBn
OBn
15
16
17
e, i
Et
f, i
f, i
O
Cl
Et
F
F
HO
F
Cl
Cl
Et
O
O
O
O
O
O
HO
HO
HO
OH
HO
OH
HO
OH
OH
OH
OH
18
19
20
Scheme 3. Reagents and conditions: (a) n-BuLi, THF, ꢁ78 °C, then 13; (b) BF3ꢀOEt2, Et3SiH, MeCN, ꢁ40 °C (28% two steps); (c) O3, ꢁ78 °C, then PPh3 (75%); (d) NaBH4, THF, 0 °C
(96%); (e) NaH, THF, 0 °C, then TBAI and MeI; (f) DAST, DCM, ꢁ78 °C; (g) PdCl2, AcONa, AcOH/H2O (9:1), 70 °C (73%); (h) ADDP, PBu3, CF3CH2OH, toluene, rt; (i) H2, 10% pd/C,
THF/MeOH (2:1), 1,2-dichlorobenzene (71% for 17, two steps: 45% for 18, 48% for 19, 39% for 20, 10% for 22).
Cl
Et
Cl
Cl
Et
HO
HO
HO
O
O
O
O
a
b
O
11b
HO
HO
OH
OH
OH
23
OH
24
d
c
H
N
HO
Et
Cl
Et
O
O
O
O
HO
HO
HO
HO
OH
OH
OH
25
OH
26
Scheme 4. Reagents and conditions: (a) BH3ꢀTHF, 0 °C to rt, then 30% NaOH, 30% H2O2 (22%); (b) PdCl2, CuCl, O2, DMF/H2O (10:1), rt (91%); (c) NaBH4, THF, rt (67%); (d)
MeNH2, NaBH3CN, cat. AcOH, EtOH, 0 °C to rt (9%).
Cl
Et
Cl
Et
O
O
O
n
O
a, b
O
a
11b
12b
HO
HO
BnO
14
OH
BnO
OBn
OH
OBn
34 n = 1
35 n = 2
27
b
c, d
R2
Cl
Et
Scheme 7. Reagents and conditions: (a) Br2, CCl4, ꢁ5 °C; (b) KOH, EtOH, reflux (two
steps: 47% for 34, 45% for 35).
Cl
Et
O
O
O
O
F
HO
F
R1O
R1O
OR1
OR1
28 R1 = Bn, R2 = OH
29
HO
OH
OH
30
35 had high activity and selectivity in the phenethyl context, but
both attributes were diminished compared to those of the benzylic
ether 34. Both the benzyl and phenethyl ether series exhibited
higher selectivity for SGLT2 over SGLT1 compared to compounds
1, 2 and 3.
c, d
R
1 = H, R2 = F
Scheme 5. Reagents and conditions: (a) PdCl2, CuCl, O2, DMF, rt (58%); (b) NaBH4,
THF, rt (98%); (c) DAST, DCM, ꢁ78 °C to rt; (d) H2, 10% Pd/C, 1,2-dichlorobenzene,
THF/MeOH (2:1) (two steps: 23% for 29, 20% for 30).
Cl
Et
Cl
Et
O
O
HO
O
Cl
Et
c, d
O
O
b
R1O
R1O
AcO
AcO
HO
HO
OR1
OAc
OAc
OR1
OH
OH
33
1
11b
R
= H
a
32
31
R
1 = Ac
Scheme 6. Reagents and conditions: (a) Ac2O, pyridine, DMAP, CH2Cl2, rt (67%); (b) PdCl2, AcONa, AcOH/H2O (9:1), 70 °C (91%); (c) phenol, DIAD, PPh3, ꢁ5 °C to rt (30%); (d)
LiOHꢀH2O, THF/MeOH/H2O, rt (2:3:1), rt (65%).

B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
4425
Table 1
In vitro data for hSGLT inhibitory activity and selectivity
Cl
R1
R2
O
n
O
HO
HO
OH
OH
Compds
n
R1
R2
hSGLT2 IC50 (nM)
hSGLT1 IC50
(l
M)
Selectivity versus hSGLT1
114
2
3
NA
NA
NA
1
OEt
Et
OEt
Et
NA
NA
NA
Me
6.7
1.9
6.5
0.9
0.89
0.72
1.3
130
378
200
>2200
11a
>2
11b
17
18
19
20
22
23
24
25
26
29
30
1
1
1
1
1
1
1
1
1
1
1
1
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
52
91
4.2
>10
>5
1700
3300
>2700
>3100
>3300
780
HOCH2CH2
MeOCH2CH2
FCH2CH2
F2CHCH2
F3CCH2
HOCH2CH2CH2
MeCOCH2
MeCH(OH)CH2
MeNHCH(Me)CH2
MeCHFCH2
MeCF2CH2
1.2
3.6
1.6
3
>10
134
103
>10
60
170
110
6.0
16
930
>1600
3700
ND
ND
1000
2%a
<10
130
36%b
10%c
140
33
1
Et
12%a
0%b
ND
34
1
2
Et
0.3
0.9
>1
>2
>3300
>2200
12a
OEt
Me
12b
35
2
2
Et
Et
12
31
2500
6.7
>20
>2900
a
b
c
Inhibition at a screening concentration of 0.1
Inhibition at a screening concentration of 100
Inhibition at a screening concentration of 1 M.
l
l
M.
M.
l
In the mouse diarrhea test (CD1 mice), no diarrhea was ob-
4. Experimental
4.1. Chemistry
4.1.1. General procedures
served after 6 h following oral administration of a single dose of
25 mg/kg of benzyl ether analogs, such as 11a–b and 20 whereas
severe diarrhea was noted following oral administration of an
equivalent dose of O-spiroketal C-aryl glucosides.5b Compounds
11b and 12b were evaluated for their ability to induce urinary glu-
cose excretion in normal male Sprague-Dawley rats compared to
compound 2. When a single dose of 1.0 mg/kg of compound was
administered orally, compound 11b induced excretion of 830 mg
of urinary glucose per 200 g body weight over 24 h, 34% of glucose
excretion relative to reference compound 2. However, compound
12b induced excretion of 1170 mg of urinary glucose at the same
dose, 56% of glucose excretion relative to reference compound 2.
In fact, compounds 1 and 2 had the similar in vivo activity as that
the urinary glucose induced by compound 2 was 88% of that in-
duced by compound 1 when tested in the same experiment. In
the same experiment, a 31% and 38% AUC reduction in blood glu-
cose level versus vehicle controls was observed at the 2 h time
point after a single 1 mg/kg oral dose of 11b and reference com-
pound 2, respectively, to normal male C57BL/6 J mice compared
with a pre-dose levels of 84–115 mg/dL of blood glucose.
All reagents and solvents were commercially available. Com-
mercial grade anhydrous solvents were purchased from SK Chem-
ical. Silica gel (Qingdao Haiyang) was used for analytical TLC (F254
plates) and flash chromatography (200–300 mesh). 1H, 13C and 19
F
NMR spectra were recorded on a Varian Mercury Plus 300 MHz or
Bruker 400 MHz using CDCl3, CD3OD or DMSO-d6 as solvent and
TMS as internal reference. Chemical shifts values are expressed
in d ppm. The data were processed using the program NUTS
4.9.9.9. Abbreviations used for signal patterns are as follows: s, sin-
glet; bs, broad singlet; d, doublet; t, triplet; q, quadruplet; m, mul-
tiplet. LC–MS system was a Waters ZQ2000 mass spectrometer,
interfaced with a Waters series 2695 liquid chromatography and
a Waters 2996 photodiode array detector. Electrospray (ESI+) was
used as the ionization technique. Ion polarity was positive and neg-
ative. Chromatographic separation was achieved using a Waters
XTerra C18 column (5
lm, 50 ꢂ 2.1 mm column). The mobile
phases A and B were 0.045% ammonium formate in acetonitrile
and 0.1% ammonium formate or formic acid in Milli-Q water,
respectively. A gradient elution was performed from 10% to 95%
A in 6 min, followed by a 2 min isocratic step at 95% A, at a flow
rate of 1.0 mL/min. The photodiode array detector used wave-
lengths between 190 and 400 nm. Preparative LC–MS system was
a Waters ZQ2000 mass spectrometer, interfaced with a Waters ser-
ies 2525 liquid chromatography and a Waters 2487 UV absorbance
detector. The mobile phases A and B were 100% acetonitrile and
0.1% ammonium formate or formic acid in Milli-Q water, respec-
tively. Chromatographic separation was achieved using a Waters
3. Conclusion
In summary, we have demonstrated that introduction of an
appropriate ortho substituent adjacent to the glycosidic bond at
the proximal phenyl ring resulted in improved SGLT2 inhibitory
activity consistent with an analysis presented by Washburn,3b
and also increased selectivity toward SGLT2 as compared to dapa-
gliflozin 1, 2 and O-spiroketal glucoside 3. Further in vitro and
in vivo SAR investigation of this series will be reported in due
course.

4426
B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
XTerra Prep MS C18 OBD column (5
l
m, 19 ꢂ 100 mm column). A
stirring for 3 h at room temperature, the mixture was extracted
3ꢂ with ethyl acetate. The combined organic extracts were washed
with brine and dried over sodium sulfate. After filtration and re-
moval of the volatiles under reduced pressure, the residue was
purified by flash column using 100% PE to 5:1 PE/ethyl acetate as
eluent to give 7a (336 mg, 70%) as a yellow solid. 1H NMR
(300 MHz, CDCl3) d 7.34 (s, 1H), 7.30 (s, 1H), 7.13 (q, 4H), 4.00 (s,
2H), 3.87 (t, J = 6.6 Hz, 2H), 2.96 (t, J = 6.6 Hz, 2H), 2.63 (q,
J = 7.6 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H).
gradient elution was performed from 33% to 95% A in 15 min,
followed by a 5 min isocratic step at 95% A, at a flow rate of
17 mL/min. UV absorbance detector at wavelength 225 nm was
used to monitor the chromatography.
4.1.2. Synthesis of 2-bromo-5-chloro-4-(4-ethylbenzyl)benzal-
dehyde (5a)
Toastirred, cold(ꢁ5 °C)solutionof 4a(9.7 g, 29.4 mmol)inanhy-
drous DCM (100 mL)under argon was added a solutionof Dess–Mar-
tin’s reagent (18.7 g, 44 mmol) in DCM (100 mL) dropwise. After
stirring for 2 h, the reaction was then quenched with 1 M aqueous
sodium hydroxide (10 mL). After separation, the organic layer was
washed with saturated aqueous sodium bisulfite (1ꢂ), saturated
aqueous sodium bicarbonate (2ꢂ) and brine (3ꢂ), and dried over
anhydrous sodium sulfate. After filtration and removal of the vola-
tiles under reduced pressure, 5a was obtained as a white solid
(9.3 g, 96%). 1H NMR (300 MHz, CDCl3) d 10.24 (s, 1H), 7.91 (s, 1H),
7.40 (s, 1H), 7.17 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 4.08 (s,
2H), 2.64 (q, J = 7.8 Hz, 2H), 1.23 (t, J = 7.8 Hz, 3H).
4.1.7. Synthesis of 2-(2-bromo-5-chloro-4-(4-ethoxybenzyl)-
phenyl)ethanol (7b)
Compound 7b was prepared from 6b in 64% yield using a sim-
ilar method as used for the preparation of 6a. 1H NMR (300 MHz,
CDCl3) d 7.30 (s, 1H), 7.29 (s, 1H), 7.09 (d, J = 8.0 Hz, 2H), 6.83 (d,
J = 8.0 Hz, 2H), 4.01 (t, J = 6.9 Hz, 2H), 3.96 (s, 2H), 3.87 (t,
J = 6.6 Hz, 2H), 2.95 (t, J = 6.6 Hz, 2H), 1.40 (t, J = 6.9 Hz, 3H).
4.1.8. Synthesis of 1-bromo-4-chloro-5-(4-ethylbenzyl)-2-(meth-
oxymethyl)benzene (8a)
To a stirred solution of 4a (0.5 g, 1.5 mmol) and tetrabutylam-
monium iodide (42 mg) in anhydrous THF (5 mL) under argon
was added sodium hydride (60% suspension in mineral oil,
0.11 g, 2.8 mmol) in portions. After stirring for 20 min, iodometh-
ane (0.43 g, 3 mmol) was added dropwise to the reaction mixture.
After stirring overnight at room temperature, the reaction was
quenched with ice water and extracted 3ꢂ with ethyl acetate.
The combined organic extracts were washed with brine and dried
over anhydrous sodium sulfate. After filtration and removal of the
volatiles under reduced pressure, the residue was purified by flash
column using 10:1 PE/ethyl acetate as eluent to give 8a (404 mg,
78%). 1H NMR (400 MHz, CDCl3) d 7.47 (s, 1H), 7.35 (s, 1H), 7.18
(d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 4.55 (s, 2H), 4.02 (s,
2H), 3.43 (s, 3H), 2.65 (q, J = 7.8 Hz, 2H), 1.25 (t, J = 7.8 Hz, 3H).
4.1.3. Synthesis of 2-bromo-5-chloro-4-(4-ethoxybenzyl)benz-
aldehyde (5b)
Compound 5b was prepared from 4b in 89% yield using a sim-
ilar method as used for the preparation of 5a. 1H NMR (300 MHz,
CDCl3) d 10.23 (s, 1H), 7.90 (s, 1H), 7.36 (s, 1H), 7.09 (d,
J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.08 (s, 2H), 4.03 (q,
J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H).
4.1.4. Synthesis of 1-bromo-4-chloro-5-(4-ethylbenzyl)-2-
vinylbenzene (6a)
To a stirred solution of Ph3P+CH3Iꢁ (14.4 g, 35.7 mmol) in anhy-
drous toluene (100 mL) under argon was added potassium hexam-
ethyldisilazane (15% in toluene, 47.5 g, 35.7 mmol) dropwise. After
stirring for 15 min, a solution of 5a (10 g, 29.8 mmol) in toluene
(100 mL) was added dropwise to the reaction mixture. After stir-
ring for 2.5 h at room temperature, the reaction was quenched
with saturated aqueous sodium bicarbonate (15 mL). After separa-
tion, the organic layer was washed with water (2ꢂ) and brine (2ꢂ),
and dried over anhydrous sodium sulfate. After filtration and re-
moval of the volatiles under reduced pressure, the residue was
purified by flash column using petroleum ether (PE) as eluent to
give 6a as a colorless oil (8.1 g, 81%). 1H NMR (300 MHz, CDCl3) d
7.54 (s, 1H), 7.33 (s, 1H), 7.15 (d, J = 8.1 Hz, 2H), 7.10 (d,
J = 8.1 Hz, 2H), 6.94 (dd, J = 17.4, 10.8 Hz, 1H), 5.69 (d, J = 17.4 Hz,
1H), 5.37 (d, J = 10.8 Hz, 1H), 4.02 (s, 2H), 2.63 (q, J = 7.5 Hz, 2H),
1.23 (t, J = 7.5 Hz, 3H).
4.1.9. Synthesis of 1-(allyloxymethyl)-2-bromo-5-chloro-4-(4-
ethylbenzyl)benzene (8b)
To a stirred, cold (ꢁ5 °C) solution of 4a (3.4 g, 10 mmol) in
anhydrous DMF (50 mL) under argon was added sodium hydride
(60% suspension in mineral oil, 0.8 g, 20 mmol) in portions. After
stirring for 1 h, tetrabutylammonium iodide (0.37 g, 1 mmol) was
added followed by addition of a solution of allyl bromide (1.45 g,
12 mmol) in DMF (10 mL). After stirring for 1 h at the same tem-
perature, the reaction was quenched with ice water and extracted
3ꢂ with ethyl acetate. The combined organic extracts were washed
with brine and dried over anhydrous sodium sulfate. After filtra-
tion and removal of the volatiles under reduced pressure, the res-
idue was purified by flash column using 10:1 PE/ethyl acetate as
eluent to give 8b (3.22 g, 85%). 1H NMR (400 MHz, CDCl3) d 7.56
(s, 1H), 7.35 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz,
2H), 6.07–5.96 (m, 1H), 5.42–5.36 (m, 1H), 5.29 (dd, J = 17.0,
10.2 Hz, 1H), 4.54 (s, 2H), 4.16–4.14 (m, 2H), 4.06 (s, 2H), 2.67 (q,
J = 7.6 Hz, 2H), 1.27 (t, J = 7.8 Hz, 3H).
4.1.5. Synthesis of 1-bromo-4-chloro-5-(4-ethoxybenzyl)-2-
vinylbenzene (6b)
Compound 6b was prepared from 5b in 94% yield as a yellow
solid using a similar method as used for the preparation of 6a. 1H
NMR (300 MHz, CDCl3) d 7.53 (s, 1H), 7.29 (s, 1H), 7.09 (d,
J = 8.7 Hz, 2H), 6.93 (dd, J = 17.7, 11.1 Hz, 1H), 6.83 (d, J = 8.7 Hz,
2H), 5.68 (d, J = 17.7 Hz, 1H), 5.37 (d, J = 11.1 Hz, 1H), 4.01 (q,
J = 7.5 Hz, 2H), 3.97 (s, 2H), 1.40 (t, J = 6.9 Hz, 3H).
4.1.10. Synthesis of 1-bromo-4-chloro-5-(4-ethoxybenzyl)-2-(2-
methoxyethyl)benzene (9a)
Compound 9a was prepared from 7a in 82% yield using a similar
method as used for the preparation of 8a. 1H NMR (300 MHz,
CDCl3) d 7.32 (s, 1H), 7.0 (s, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.78 (d,
J = 8.4 Hz, 2H), 4.01 (q, J = 7.2 Hz, 2H), 3.35 (s, 3H), 3.66 (t,
J = 7.0 Hz, 2H), 2.98 (t, J = 7.0 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H).
4.1.6. Synthesis of 2-(2-bromo-5-chloro-4-(4-ethylbenzyl)-
phenyl)ethanol (7a)
To a stirred 0 °C solution of 6a (453 mg, 1.3 mmol) in THF
(5 mL) was added 9-BBN (0.5 M in THF, 3.2 mL, 1.6 mmol) drop-
wise over 30 min. After the addition was complete, the ice bath
was removed and the reaction mixture was stirred at room tem-
perature overnight. After cooling to 0 °C, methanol (2.2 mL), 2 M
aqueous sodium hydroxide (5.6 mL) and 30% hydrogen peroxide
(1.2 mL) were added to the reaction mixture successively. After
4.1.11. Synthesis of 1-(2-(allyloxy)ethyl)-2-bromo-5-chloro-4-
(4-ethylbenzyl)benzene (9b)
Compound 9b was prepared from 7b in 91% yield using a sim-
ilar method as used for the preparation of 8b. 1H NMR (300 MHz,

B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
4427
CDCl3) d 7.31 (s, 1H), 7.30 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 7.10 (d,
J = 8.4 Hz, 2H), 5.97–5.84 (m, 1H), 5.31–5.23 (m, 1H), 5.21–5.15
(m, 1H), 4.02–3.98 (m, 4H), 3.64 (t, J = 7.0 Hz, 2H), 2.98 (t,
J = 7.0 Hz, 2H), 2.63 (q, J = 7.8 Hz, 2H), 1.24 (t, J = 7.8 Hz, 3H).
4.1.15. Synthesis of (2S,3R,4R,5S,6R)-2-(2-(2-(allyloxy)ethyl)-4-
chloro-5-(4-ethylbenzyl)phenyl)-6-(hydroxymethyl)tetrahydro-
2H-pyran-3,4,5-triol (12b)
Compound 12b was prepared from 9b in 58% yield as a white
solid using a similar method as used for the preparation of 11a.
1H NMR (300 MHz, CD3OD) d 7.39 (s, 1H), 7.26 (s, 1H), 7.08 (s,
4H), 5.97–5.84 (m, 1H)?5.29–5.12 (m, 2H)?4.44 (d, J = 9.0 Hz,
1H), 4.03–3.97 (m, 4H), 3.85 (d, J = 11.1 Hz, 1H), 3.70–3.62 (m,
3H), 3.48–3.37 (m, 4H), 3.12–3.03 (m, 1H), 2.95–2.86 (m, 1H),
2.58 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H); 13C NMR
(100 MHz, CD3OD) d 140.2, 136.5, 135.4, 135.1, 135.0, 133.1,
131.3, 128.8, 128.7, 126.8 (2C), 125.8 (2C), 144.3, 79.5, 77.0, 76.1,
73.1, 69.9, 68.9 (2C), 60.1, 36.5, 30.5, 26.5, 13.3; ESI MS (m/z):
477 [M+H]+, 521 [M+HCO2]ꢁ, calcd 476.
4.1.12. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-(methoxymethyl)phenyl)-6-(hydroxymethyl)tetrahydro-
2H-pyran-3,4,5-triol (11a)
To a stirred ꢁ78 °C solution of 8a (54 mg, 0.15 mmol) in 2:1
anhydrous toluene/THF (0.8 mL) was added n-BuLi (2.5 M in hex-
ane, 0.07 mL, 0.18 mmol) dropwise, keeping the internal tempera-
ture below ꢁ60 °C. After stirring for 40 min, a solution of 10
(86 mg, 0.18 mmol) in toluene (0.8 mL) was added to the above
reaction solution. After stirring for another 1.5 h, the reaction
was quenched with saturated aqueous ammonium chloride
(5 mL). The mixture was extracted 3ꢂ with ethyl acetate. The com-
bined organic extracts were washed with brine and dried over
anhydrous sodium sulfate. After filtration and removal of the vol-
atiles, the resulting residue was redissolved in 1:1 DCM/acetoni-
4.1.16. Synthesis of (2S,3S,4R,5R,6R)-2-(2-(allyloxymethyl)-4-
chloro-5-(4-ethylbenzyl)phenyl)-3,4,5-tris(benzyloxy)-6-(ben-
zyloxymethyl)tetrahydro-2H-pyran (14)
To a stirred ꢁ78 °C solution of 8b (3.2 g, 8.5 mmol) in anhy-
drous THF (30 mL) was added n-BuLi (2.5 M in hexane, 3.7 mL,
9.4 mmol) dropwise, keeping the internal temperature below
ꢁ60 °C. After stirring for 2 h, a solution of 13 (6.9 g, 12.8 mmol)
in THF (20 mL) was added to the above reaction solution. After stir-
ring for another 2 h, the reaction was quenched with saturated
aqueous ammonium chloride (10 mL). The mixture was extracted
3ꢂ with ethyl acetate. The combined organic extracts were washed
with brine and dried over anhydrous sodium sulfate. After filtra-
tion and removal of the volatiles, the resulting residue was redis-
solved in acetonitrile (30 mL) and cooled to ꢁ40 °C. Et3SiH (1.5 g,
13 mmol) was added followed by addition of BF3ꢀOEt2 (1.8 g,
13 mmol) while keeping the internal temperature below ꢁ25 °C.
After the addition was complete, the reaction mixture was allowed
to warm to ꢁ15 °C and stirred for 2 h. Saturated aqueous sodium
bicarbonate was then added to quench the reaction. After concen-
tration, the resulting residue was extracted 3ꢂ with ethyl acetate.
The combined organic extracts were washed 1ꢂ with water and
brine, respectively, and dried over anhydrous sodium sulfate prior
to filtration and concentration under reduced pressure. The result-
ing residue was purified by flash column using 40:1 PE/ethyl
acetate as eluent to give 14 (2.0 g, 28%). ESI MS (m/z): 840
[M+NH4]+, calcd 822.
trile (0.8 mL) and cooled to ꢁ15 to ꢁ10 °C. Et3SiH (48
l
l
L,
L,
0.3 mmol) was added followed by addition of BF3ꢀOEt2 (29
0.23 mmol) while keeping the internal temperature below
ꢁ25 °C. After the addition was complete, the reaction mixture
was allowed to warm to ꢁ15 °C and stirred for 5 h. Saturated aque-
ous sodium bicarbonate was then added to quench the reaction.
After concentration, the resulting residue was extracted 3ꢂ with
ethyl acetate. The combined organic extracts were washed 1ꢂ with
water and brine respectively, and dried over anhydrous sodium
sulfate prior to filtration and concentration under reduced pres-
sure. The resulting residue was purified by preparative LC–MS to
give 11a (15 mg, 22%) as a white solid. 1H NMR (400 MHz, CD3OD)
d 7.47 (s, 1H), 7.40 (s, 1H), 7.09 (s, 4H), 4.69 (d, J = 12.4 Hz, 1H),
4.49 (d, J = 12.4 Hz, 1H), 4.42 (d, J = 8.8 Hz, 1H), 4.08 (d,
J = 15.0 Hz, 1H), 4.03 (d, J = 15.0 Hz, 1H), 3.86 (d, J = 12.4 Hz, 1H),
3.70–3.63 (m, 1H), 3.50–3.37 (m, 7H), 2.58 (q, J = 7.8 Hz, 2H),
1.19 (t, J = 7.8 Hz, 3H); ESI MS (m/z): 437 [M+H]+, 481 [M+HCO2]ꢁ,
calcd 436.
4.1.13. Synthesis of (2S,3R,4R,5S,6R)-2-(2-(allyloxymethyl)-4-
chloro-5-(4-ethylbenzyl)phenyl)-6-(hydroxymethyl)tetrahydro-
2H-pyran-3,4,5-triol (11b)
Compound 11b was prepared from 8b in 35% yield as a white
solid using a similar method as used for the preparation of 11a.
1H NMR (400 MHz, CD3OD) d 7.48 (s, 1H), 7.43 (s, 1H), 7.11 (s,
4H), 6.04–5.98 (m, 1H), 5.35 (dd, J = 16.4, 1.6 Hz, 1H), 5.23 (dd,
J = 10.0, 1.6 Hz, 1H), 4.76 (d, J = 12.2 Hz, 1H), 4.68 (d, J = 12.2 Hz,
1H), 4.47 (d, J = 9.2 Hz, 1H), 4.13–4.02 (m, 4H), 3.87 (dd, J = 12.4,
2.4 Hz, 1H), 3.71–3.66 (m, 1H), 3.50–2.74 (m, 4H), 2.60 (q,
J = 7.2 Hz, 2H), 1.13 (d, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d 140.3, 136.4, 135.5, 135.2, 135.1, 132.9, 131.5, 128.8, 127.2, 126.8
(2C), 125.9 (2C), 114.8, 79.5, 77.0, 75.9, 73.5, 69.5, 68.9, 66.9, 60.1,
36.5, 26.5, 13.4; ESI MS (m/z): 463 [M+H]+, 480 [M+NH4]+, 507
[M+HCO2]ꢁ, calcd 462.
4.1.17. Synthesis of 2-(5-chloro-4-(4-ethylbenzyl)-2-((2S,3S,4R,
5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-
2H-pyran-2-yl)benzyloxy)acetaldehyde (15)
Ozone was bubbled through a stirred ꢁ78 °C solution of 14
(0.2 g, 0.24 mmol) in DCM (100 mL) until the color of solution
turned to blue. At this point, argon was bubbled through the reac-
tion solution until blue disappeared. A solution of triphenylphos-
phine (0.19 g, 0.72 mmol) in DCM (2 mL) was then added. The
reaction solution was allowed to warm to room temperature and
stirred for 30 min. After removal of the volatiles under reduced
pressure, the residue was purified by flash column using 5:1
PE/ethyl acetate as eluent to give 15 (152 mg, 75%). 1H NMR
(300 MHz, CD3OD) d 9.57 (s, 1H), 7.29 (m, 13 H), 7.22 (m, 6H),
7.04 (m, 4H), 6.84 (m, 2H), 4.87 (m, 4H), 4.52 (m, 1H), 4.43 (m,
5H), 4.05 (dd, J = 18.0, 15.0 Hz, 2H), 3.96 (m, 5H), 3.77 (m, 5H),
3.71 (m, 2H), 2.56 (q, J = 7.5 Hz, 2H), 1.76 (t, J = 7.5 Hz, 3H); ESI
MS (m/z): 842 [M+NH4]+, calcd 824.
4.1.14. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethoxyb-
enzyl)-2-(2-methoxyethyl)phenyl)-6-(hydroxymethyl)tetrahy-
dro-2H-pyran-3,4,5-triol (12a)
Compound 12a was prepared from 9a in 40% yield as a white
solid using a similar method as used for the preparation of 11a.
1H NMR (300 MHz, CD3OD) d 7.37 (s, 1H), 7.24 (s, 1H), 7.08 (d,
J = 8.7 Hz, 2H), 6.78 (d, J = 8.7 Hz, 2H), 4.42 (d, J = 9.0 Hz, 1H),
4.02–3.94 (m, 4H), 3.85 (d, J = 12.3 Hz, 1H), 3.67–3.58 (m, 3H),
3.49–3.43 (m, 2H), 3.40–3.36 (m, 2H), 3.34 (s, 3H), 3.10–2.99 (m,
1H), 2.95–2.85 (m, 1H), 1.35 (t, J = 7.2 Hz, 3H); ESI MS (m/z): 467
[M+H]+, 511 [M+HCO2]ꢁ, calcd 466.
4.1.18. Synthesis of 2-(5-chloro-4-(4-ethylbenzyl)-2-((2S,3S,4R,
5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-
2H-pyran-2-yl)benzyloxy)ethanol (16)
To a stirred 0 °C solution of 15 (0.14 g, 0.17 mmol) in THF
(10 mL) was added solid sodium borohydride (13 mg, 0.34 mmol).
After stirring for 1.5 h at room temperature, the reaction was

4428
B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
quenched with methanol (2 mL). After removal of the volatiles un-
der reduced pressure, the residue was purified by flash column
using 5:1 PE/ethyl acetate as eluent to give 16 (135 mg, 96%). ESI
MS (m/z): 844 [M+NH4]+, calcd 826.
4.08 (d, J = 15.0 Hz, 1H), 4.02 (d, J = 15.0 Hz, 1H), 3.84–3.78 (m,
2H), 3.71–3.61 (m, 2H), 3.45–3.31 (m, 4H), 2.58 (q, J = 7.8 Hz,
2H), 1.18 (t, J = 7.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 140.3,
136.5, 135.3, 135.2, 135.1, 131.5, 128.8, 127.2, 126.8 (2C), 125.9
(2C), 81.1 (d, J = 167 Hz), 79.4, 77.0, 75.8, 73.5, 68.9, 67.9, 67.8,
60.1, 36.6, 26.5, 13.4; 19F NMR (376 MHz, CD3OD) d ꢁ224.2 to
ꢁ224.5 (m, 1F); ESI MS (m/z): 469 [M+H]+, 486 [M+NH4]+, 513
[M+HCO2]ꢁ, calcd 468.
4.1.19. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2-hydroxyethoxy)methyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (17)
The mixture of 16 (50 mg, 0.06 mmol), 10% Pd/C (50 mg),
1,2-dichlorobenzene (45 mg, 0.3 mmol) and 2:1 THF/methanol
(3 mL) was stirred for 3 h under hydrogen atmosphere (1 atm.).
After filtration of the solid and removal of the volatiles under
reduced pressure, the residue was purified by preparative LC–MS
to give 17 (20 mg, 71%) as a white solid. 1H NMR (300 MHz,
CD3OD) d 7.46 (s, 1H), 7.45 (s, 1H), 7.07 (s, 4H), 4.74 (d,
J = 12.0 Hz, 1H), 4.60 (d, J = 12.0 Hz, 1H), 4.47 (d, J = 9.3 Hz, 1H),
4.08 (d, J = 14.7 Hz, 1H), 4.02 (d, J = 15.0 Hz, 1H), 3.86 (d,
J = 11.8 Hz, 1H), 3.72–3.58 (m, 5H), 3.51–3.37 (m, 4H), 2.57 (q,
J = 7.5 Hz, 2H), 1.18 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d 141.9, 138.0, 137.1, 136.8 (2C), 133.0, 130.4, 129.0, 128.3 (2C),
127.4 (2C), 81.0, 78.5, 77.3, 75.1, 71.6, 70.5, 69.6, 61.7, 60.8, 38.1,
28.1, 14.9; ESI MS (m/z): 467 [M+H]+, 511 [M+HCO2]ꢁ, calcd 466.
4.1.22. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-2-((2,2-diflu-
oroethoxy)methyl)-5-(4-ethylbenzyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (20)
Compound 20 was prepared from 15 in 39% yield as a white so-
lid using a similar method as used for the preparation of 19. 1H
NMR (300 MHz, CD3OD) d 7.46 (s,1H) 7.41 (s, 1H), 7.07 (s, 4H),
5.99 (tt, J = 55.2, 3.9 Hz, 1H), 4.86 (d, J = 12.6 Hz, 1H), 4.65 (d,
J = 12.6 Hz, 1H), 4.44 (d, J = 9.0 Hz, 1H), 4.08 (d, J = 15.0 Hz, 1H),
4.02 (d, J = 15.0 Hz, 1H), 3.85 (d, J = 11.4 Hz, 1H), 3.78–3.62 (m,
3H), 3.50–3.35 (m, 4H), 2.58 (q, J = 7.5 Hz, 2H), 1.19 (t, J = 7.5 Hz,
3H); 13C NMR (100 MHz, CD3OD) d 140.3, 136.7, 135.2, 135.1,
134.8, 131.5, 128.9, 127.1, 126.8 (2C), 125.9 (2C), 113.1 (t,
J = 238 Hz), 79.4, 76.9, 75.8, 73.4, 68.8, 68.5, 67.5 (t, J = 27 Hz),
36.5, 26.5, 13.3; 19F NMR (376 MHz, CD3OD)
d -126.9 (t,
4.1.20. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2-methoxyethoxy)methyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (18)
J = 14.3 Hz, 1F), -127.1 (t, J = 14.3 Hz, 1F); ESI MS (m/z): 504
[M+NH4]+, 531 [M+HCO2]ꢁ, calcd 486.
To a stirred 0 °C solution of 16 (60 mg, 0.072 mmol) in anhy-
drous THF (2 mL) under argon was added 60% NaH (suspended in
mineral oil, 35 mg, 0.86 mmol). After stirring for 1 h at the same
temperature, tetrabutylammonium iodide (5 mg) and iodometh-
ane (15 mg, 0.11 mmol) was added to the reaction mixture. After
stirring overnight at room temperature, the reaction was quenched
with ice water and extracted 3ꢂ with ethyl acetate. The combined
organic extracts were washed with brine and dried over anhydrous
sodium sulfate. Removal of the volatiles under reduced pressure
gave benzyl-protected product (60 mg). Debenzylation of the
above intermediate using a similar method as used for the prepa-
ration of 17 gave 18 (16 mg, 45% two steps) as a white solid. 1H
NMR (300 MHz, CD3OD) d 7.46 (s, 1H), 7.42 (s, 1H), 7.07 (s, 4H),
4.74 (d, J = 12.0 Hz, 1H), 4.58 (d, J = 12.0 Hz, 1H), 4.46 (d,
J = 9.3 Hz, 1H), 4.08 (d, J = 15.0 Hz, 1H), 4.02 (d, J = 15.0 Hz, 1H),
3.85 (d, J = 12.0 Hz, 1H), 3.68–3.62 (m, 3H), 3.60–3.56 (m, 2H),
3.47–3.33 (m, 7H), 2.58 (q, J = 7.5 Hz, 2H), 1.19 (t, J = 7.5 Hz, 3H);
13C NMR (100 MHz, CD3OD) d 140.3, 136.5, 135.4, 135.3, 135.2,
131.4, 128.8, 127.3, 126.8 (2C), 125.9 (2C), 79.5, 77.0, 75.8, 73.6,
69.9, 68.9, 68.0, 67.7, 60.2, 56.2, 36.5, 26.5, 13.3; ESI MS (m/z)
481 [M+H]+, 498 [M+NH4]+, 525 [M+HCO2]ꢁ, calcd 480.
4.1.23. Synthesis of (5-chloro-4-(4-ethylbenzyl)-2-((2S,3S,4R,5R,
6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-2H-
pyran-2-yl)phenyl)methanol (21)
The mixture of 14 (50 mg, 0.061 mmol), PdCl2 (24 mg,
0.13 mmol), sodium acetate (76 mg, 0.29 mmol) and 9:1 acetic
acid/H2O (1 mL) was stirred at 70 °C for 1 h. Upon cooling to room
temperature, water and ethyl acetate was added. After separation
of the layers, the aqueous layer was extracted 2ꢂ with ethyl ace-
tate. The combined extracts were washed 1ꢂ with water and brine,
respectively, and dried over sodium sulfate. After filtration and re-
moval of the volatiles under reduced pressure, the residue was
purified by preparative TLC using 3:1 PE/ethyl acetate as eluent
to give 21 (30 mg, 63%). ESI MS (m/z): 800 [M+NH4]+, calcd 782.
4.1.24. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2,2,2-trifluoroethoxy)methyl)phenyl)-6-(hydroxy-
methyl)tetrahydro-2H-pyran-3,4,5-triol (22)
To a stirred solution of 21 (0.78 g, 1 mmol) in toluene (2 mL)
was added ADDP (0.5 g, 2 mmol) at room temperature. The
reaction mixture was then stirred for 10 min and tributylphos-
phine (0.5 mL, 2 mmol) was added. After stirring for another 1 h,
2,2,2-trifluoroethanol (0.72 mL, 10 mmol) was added. The reaction
mixture was stirred overnight. After removal of the volatiles, the
residue was purified by flash column using 10:1 to 3:1 PE/ethyl
acetate as eluent to give benzyl-protected product. Debenzylation
of the above intermediate using a similar method as used for the
preparation of 17 gave 22 (50 mg, 10% two steps) as a white solid.
1H NMR (300 MHz, CD3OD) d 7.47(s, 1H), 7.41 (s, 1H), 7.08 (s, 4H),
4.94 (d, J = 12.0 Hz, 1H), 4.71 (d, J = 12.0 Hz, 1H), 4.42 (d, J = 9.3 Hz,
1H), 4.06 (d, J = 6.6 Hz, 2H), 3.98 (t, J = 8.7 Hz, 2H), 3.85 (d,
J = 1.2 Hz, 1H), 3.69–3.63 (m, 1H), 3.46–3.36 (m, 4H), 2.58 (q,
J = 7.5 Hz, 2H), 1.18 (t, J = 7.8 Hz, 3H); ESI MS (m/z): 505 [M+H]+,
549 [M+HCO2]ꢁ, calcd 504.
4.1.21. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2-fluoroethoxy)methyl)phenyl)-6-(hydroxymethyl)tet-
rahydro-2H-pyran-3,4,5-triol (19)
To a stirred ꢁ78 °C solution of 16 (150 mg, 0.18 mmol) in anhy-
drous DCM (2 mL) under argon was added DAST (36 lL,
0.27 mmol). The reaction solution was allowed to slowly warm
to room temperature and stirred for 3 h. The reaction was then
quenched with methanol and the mixture was evaporated in vac-
uum. The residue was taken up with water and ethyl acetate. After
separation of the layers, the organic layer was washed with brine
and dried over anhydrous sodium sulfate. After filtration and re-
moval of the volatiles under reduced pressure, the residue was
purified by preparative TLC using 5:1 PE/ethyl acetate as eluent
to give benzyl-protected product (100 mg). Debenzylation of the
above intermediate using a similar method as used for the prepa-
ration of 17 gave 19 (41 mg, 48% two steps) as a white solid. 1H
NMR (300 MHz, CD3OD) d 7.47 (s, 1H), 7.42 (s, 1H), 7.07 (s, 4H),
4.80 (d, J = 12.3 Hz, 1H), 4.66–4.59 (m, 2H), 4.50–4.44 (m, 2H),
4.1.25. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((3-hydroxypropoxy)methyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (23)
To a stirred 0 °C solution of 11b (22 mg, 0.048 mmol) in THF
(0.6 mL) was added BH3ꢀTHF (1 M in THF, 0.5 mL, 0.5 mmol)
dropwise. After stirring for 2 h at room temperature, 30%

B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
4429
aqueous sodium hydroxide (1 mL) was added followed by addi-
tion of 30% aqueous hydrogen peroxide (1 mL). After stirring
for another 1.5 h, the mixture was extracted 3ꢂ with ethyl ace-
tate. The combined extracts were washed 1ꢂ with water and
brine respectively, and dried over sodium sulfate. After filtration
and removal of the volatiles under reduced pressure, the residue
was purified by preparative TLC using 10:1 ethyl acetate/metha-
nol as eluent to give desired product 23 (5 mg, 22%) and isomer
25 (4 mg, 17%) as white solid. 1H NMR (300 MHz, CD3OD) d 7.46
(s, 1H), 7.40 (s, 1H), 7.08 (s, 4H), 4.73 (d, J = 12.2 Hz, 1H), 4.53 (d,
J = 12.2 Hz, 1H), 4.45 (d, J = 9.0 Hz, 1H), 4.08 (d, J = 15.0 Hz, 1H),
4.02 (d, J = 15.0 Hz, 1H), 3.86 (d, J = 12.0 Hz, 1H), 3.68–3.60 (m,
4H), 3.44–3.29 (m, 5H), 2.58 (q, J = 7.6 Hz, 2H), 1.86–1.79 (m,
4.1.28. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2-(methylamino)propoxy)methyl)phenyl)-6-(hydroxy-
methyl)tetrahydro-2H-pyran-3,4,5-triol (26)
To a stirred 0 °C solution of 24 (0.48 g, 1 mmol) in ethanol
(5 mL) was added a solution of methylamine in ethanol (10 mmol)
and followed by addition of catalytic amount of acetic acid. After
stirring for 1 h, sodium cyanoborohydride (0.19 g, 3 mmol) was
added. The reaction was monitored by LC–MS. Once the reaction
was complete, the reaction mixture was evaporated prior to adding
water and ethyl acetate. The organic layer was separated, washed
with brine, and dried over sodium sulfate. After filtration and re-
moval of the volatiles under reduced pressure, the residue was
purified by preparative LC–MS to give 26 (44 mg, 9%) as white so-
lid. 1H NMR (300 MHz, CD3OD) d 7.45 (s, 1H), 7.41 (s, 1H), 7.06 (s,
4H), 4.90–4.77 (m, 2H), 4.62–4.57 (m, 1H), 4.49 (d, J = 9.0 Hz, 1H),
4.04 (s, 2H), 3.88 (d, J = 11.7 Hz, 1H), 3.75–3.59 (m, 2H), 3.55–3.38
(m, 5H), 2.65–2.53 (m, 6H), 1.27 (d, J = 6.9 Hz, 3H), 1.19 (t,
J = 7.5 Hz, 3H); ESI MS (m/z): 494 [M+H]+, 538 [M+HCO2]ꢁ, calcd
493.
2H), 1.19 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d
140.3, 136.4, 135.6, 135.2, 135.1, 131.4, 128.8, 127.3, 126.8
(2C), 125.8 (2C), 79.5, 77.0, 75.9, 73.5, 68.9, 67.8, 65.6, 60.1,
57.0, 36.5, 30.7, 26.5, 13.3; ESI MS (m/z): 481 [M+H]+, 525
[M+HCO2]ꢁ, calcd 480.
4.1.26. Synthesis of 1-(5-chloro-4-(4-ethylbenzyl)-2-((2S,3R,4R,
5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)benzyloxy)propan-2-one (24)
4.1.29. Synthesis of 1-(5-chloro-4-(4-ethylbenzyl)-2-((2S,3S,4R,
5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-
2H-pyran-2-yl)benzyloxy)propan-2-one (27)
The mixture of PdCl2 (5.3 mg, 0.03 mmol), CuCl (15 mg,
0.15 mmol) and 10:1 DMF/H2O (2 mL) was stirred for 2.5 h under
oxygen atmosphere (1 atm.). Compound 11b (0.13 g, 0.28 mmol)
was then added. After stirring overnight at room temperature,
water was added and the mixture was extracted 3ꢂ with ethyl ace-
tate. The combined extracts were washed 1ꢂ with water and brine
respectively, and dried over sodium sulfate. After filtration and re-
moval of the volatiles under reduced pressure, the residue was
purified by preparative TLC using 10:1 ethyl acetate/methanol as
eluent to give 24 (122 mg, 91%) as gel. 1H NMR (300 MHz, CD3OD)
d 7.47 (s, 1H), 7.44 (s, 1H), 7.08 (s, 4H), 4.79 (d, J = 12.0 Hz, 1H), 4.60
(d, J = 12.0 Hz, 1H), 4.55 (d, J = 9.6 Hz, 1H), 4.22 (s, 2H), 4.10 (d,
J = 14.8 Hz, 1H), 4.05 (d, J = 14.8 Hz, 1H), 3.85 (dd, J = 11.7, 2.1 Hz,
1H), 3.68–3.63 (m, 1H), 3.50–3.36 (m, 4H), 2.58 (q, J = 7.8 Hz,
2H), 2.12 (s, 3H), 1.19 (t, J = 7.8 Hz, 3H); 13C NMR (100 MHz,
CD3OD) d 205.8, 140.3, 136.8, 135.5, 135.2, 134.7, 131.5, 128.9,
127.6, 126.8 (2C), 125.9 (2C), 79.4, 76.9, 75.7, 73.6, 73.2, 68.9,
68.1, 60.1, 36.5, 26.5, 23.3, 13.3; ESI MS (m/z): 479 [M+H]+, 523
[M+HCO2]ꢁ, calcd 478.
Compound 27 was prepared from 14 in 58% yield using a similar
method as used for the preparation of 24. 1H NMR (300 MHz,
DMSO-d6) d 7.46 (s, 2H), 7.32–7.27 (m, 13H), 7.20–7.10 (m, 5H),
7.04 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 8.1 Hz, 2H), 6.79 (dd, J = 7.8,
1.8 Hz, 2H), 4.79 (s, 2H), 4.76–4.71 (m, 2H), 4.64–4.32 (m, 6H),
4.13 (s, 2H), 4.00 (s, 2H), 3.82–3.76 (m, 2H), 3.63–3.54 (m, 5H),
2.45 (q, J = 8.1 Hz, 2H), 1.99 (s, 3H), 1.06 (t, J = 8.1 Hz, 3H); ESI
MS (m/z): 839 [M+H]+, calcd 838.
4.1.30. Synthesis of 1-(5-chloro-4-(4-ethylbenzyl)-2-((2S,3S,4R,
5R,6R)-3,4,5-tris(benzyloxy)-6-(benzyloxymethyl)tetrahydro-
2H-pyran-2-yl)benzyloxy)propan-2-ol (28)
Compound 28 was prepared from 27 in 98% yield using a similar
method as used for the preparation of 16. ESI MS (m/z): 860
[M+NH4]+, calcd 842.
4.1.31. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2-fluoropropoxy)methyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (29)
Compound 29 was prepared from 28 in 23% yield as a white so-
lid using a similar method as used for the preparation of 19. 1H
NMR (300 MHz, CD3OD) d 7.46 (s, 1H), 7.41 (s, 1H), 7.08 (s, 4H),
4.80 (d, J = 12.6 Hz, 1H), 4.59 (d, J = 12.3 Hz, 1H), 4.46 (dd, J = 9.3,
2.4 Hz, 1H), 4.08 (d, J = 15.0 Hz, 1H), 4.02 (d, J = 15.0 Hz, 1H), 3.85
(d, J = 12.0 Hz, 1H), 3.68–3.61 (m, 2H), 3.55 (d, J = 4.8 Hz, 1H),
3.46–3.35 (m, 4H), 2.58 (q, J = 7.5 Hz, 2H), 1.31 (ddd, J = 23.4, 6.6,
0.6 Hz, 3H), 1.19 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CD3OD) d
140.3, 136.5, 135.3, 135.2, 135.1, 131.4, 128.8, 127.2, 126.8 (2C),
125.9 (2C), 87.5 (d, J = 167 Hz), 79.4, 77.0, 75.8, 73.5, 71.6 (d,
J = 22 Hz), 70.7, 68.6, 59.7, 36.5, 26.5, 14.7 (d, J = 22 Hz), 13.3; 19F
NMR (376 MHz, CD3OD) d ꢁ180.2 to ꢁ180.7 (m, 1F); ESI MS
(m/z): 483 [M+H]+, 500 [M+NH4]+, 527 [M+HCO2]ꢁ, calcd 482.
4.1.27. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((2-hydroxypropoxy)methyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (25)
To a stirred solution of 24 (3.7 mg, 0.008 mmol) in anhydrous
THF (0.5 mL) under argon was added sodium borohydride
(1.8 mg, 0.046 mmol) at room temperature. After stirring for
2 h, the reaction was quenched with saturated aqueous ammo-
nium chloride. The mixture was extracted 2ꢂ with ethyl acetate.
The combined organic extracts were washed with water and
brine, dried over sodium sulfate. After filtration and removal of
the volatiles under reduced pressure, the residue was purified
by preparative TLC using 10:1 ethyl acetate/methanol as eluent
to give 25 (2.5 mg, 67%) as white solid. 1H NMR (300 MHz,
CD3OD) d 7.45 (s, 1H), 7.43 (s, 1H), 7.08 (s, 4H), 4.85–4.71 (m,
2H), 4.58 (dd, J = 12.0, 3.9 Hz, 1H), 4.47 (dd, J = 9.3, 3.3 Hz, 1H),
4.10 (d, J = 14.8 Hz, 1H), 4.05 (d, J = 14.8 Hz, 1H), 3.95–3.83 (m,
1H), 3.85 (dd, J = 11.7, 1.5 Hz, 1H), 3.68–3.62 (m, 1H), 3.47–3.33
(m, 5H), 2.60 (q, J = 7.8 Hz, 2H), 1.19 (t, J = 7.8 Hz, 3H), 1.15
(dd, J = 6.6, 1.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 140.3,
136.5, 135.5, 135.3, 135.2, 131.4, 128.8, 127.4, 126.8 (2C), 125.9
(2C), 79.5, 77.0, 75.7, 74.2, 73.5, 68.9, 68.2, 64.5, 60.1, 36.5,
26.5, 16.8, 13.3; ESI MS (m/z): 481 [M+H]+, 525 [M+HCO2]ꢁ, calcd
480.
4.1.32. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-2-((2,2-diflu-
oropropoxy)methyl)-5-(4-ethylbenzyl)phenyl)-6-(hydroxy-
methyl)tetrahydro-2H-pyran-3,4,5-triol (30)
Compound 30 was prepared from 27 in 20% yield as a white so-
lid using a similar method as used for the preparation of 19. 1H
NMR (300 MHz, CD3OD) d 7.47 (s, 1H), 7.41 (s, 1H), 7.08 (s, 4H),
4.87 (d, J = 12.0 Hz, 1H), 4.64 (d, J = 12.0 Hz, 1H), 4.45 (d,
J = 9.0 Hz, 1H), 4.08 (d, J = 15.0 Hz, 1H), 4.03 (d, J = 15.0 Hz, 1H),
3.85 (d, J = 12.0 Hz, 1H), 3.72–3.63 (m, 3H), 3.50–3.35 (m, 4H),
2.58 (q, J = 7.8 Hz, 2H), 1.64 (t, J = 18.6 Hz, 3H), 1.18 (t, J = 7.8 Hz,

4430
B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
3H); 13C NMR (100 MHz, CD3OD) d 140.3, 136.6, 135.2 (2C), 134.9,
131.5, 128.8, 127.1, 126.8 (2C), 125.9 (2C), 120.6 (t, J = 237 Hz),
79.4, 76.9, 75.8, 73.5, 70.0 (t, J = 32 Hz), 68.8, 68.6, 60.1, 36.5,
26.5, 18.2 (t, J = 25 Hz), 13.4; 19F NMR (376 MHz, CD3OD) d ꢁ99.3
to ꢁ99.5 (m, 2F); ESI MS (m/z): 518 [M+NH4]+, 545 [M+HCO2]ꢁ,
calcd 500.
1H), 3.47–3.35 (m, 4H), 2.58 (q, J = 7.5 Hz, 2H), 1.19 (t, J = 7.5 Hz,
3H); 13C NMR (100 MHz, CD3OD) d 157.0, 140.3, 136.4, 135.2,
134.7, 134.6, 131.6, 128.9, 127.6 (2C), 126.8 (2C), 126.6, 125.9
(2C), 119.1, 113.0 (2C), 79.5, 76.9, 76.2, 73.4, 68.8, 64.9, 60.1,
36.5, 26.5, 13.3; ESI MS (m/z): 516 [M+NH4]+, 543 [M+HCO2]ꢁ,
calcd 498.
4.1.33. Synthesis of (2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-(2-
(allyloxymethyl)-4-chloro-5-(4-ethylbenzyl)phenyl)tetrahydro-
2H-pyran-3,4,5-triyl triacetate (31)
4.1.36. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-((prop-2-ynyloxy)methyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (34)
To a stirred solution of 11b (1.2 g, 2.6 mmol) in DCM (18 mL)
was added pyridine (2.1 mL, 26 mmol), acetic anhydride (2.7 mL,
29 mmol) and DMAP (32 mg) successively at room temperature.
After stirring overnight, the reaction was quenched with water
(10 mL). The mixture was extracted 2ꢂ with DCM. The combined
organic extracts were washed 2ꢂ with 1 N HCl and brine, and dried
over sodium sulfate. After filtration and removal of the volatiles
under reduced pressure, the residue was purified by flash column
using 3:1 PE/ethyl acetate as eluent to give 31 (1.1 g, 67%) as a
white solid. 1H NMR (300 MHz, CD3OD) d 7.61 (s, 1H), 7.26 (s,
1H), 7.10 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 6.04–5.93 (m,
1H), 5.52 (t, J = 9.6 Hz, 1H), 5.43–5.36 (m, 1H), 5.27–5.20 (m, 3H),
4.85 (d, J = 13.8 Hz, 1H), 4.58 (d, J = 13.5 Hz, 1H), 4.36 (dd,
J = 12.3, 4.8 Hz, 1H), 4.16–4.10 (m, 2H), 4.08–3.95 (m, 5H), 2.59
(q, J = 7.8 Hz, 2H), 2.10 (s, 3H), 2.07–2.04 (m, 6H), 1.94 (s, 3H),
1.19 (t, J = 7.8 Hz, 3H); ESI MS (m/z): 648 [M+NH4]+, 675
[M+HCO2]ꢁ, calcd 630.
To a stirred, cold (ꢁ5 °C) solution of 11b (306 mg, 0.66 mmol) in
chloroform (5.0 mL) was added a solution of bromine (0.1 g,
0.66 mmol) in chloroform (1 mL) dropwise until the color of reac-
tion solution turned from colorless to yellow. After stirring for
30 min, water (10 mL) was added to the reaction solution. The
resulting mixture was extracted 2ꢂ with ethyl acetate. The com-
bined organic layer was washed 2ꢂ with brine, dried over sodium
sulfate, and finally evaporated to give 368 mg of dibromide as a
yellow solid. A mixture of above obtained dibromide (53 mg,
0.086 mmol), solid potassium hydroxide (82%, 11 mg) and ethanol
(1 mL) was refluxed for 6 h. After concentration of the solution, the
residue was taken up with water and ethyl acetate. After separa-
tion of the layers, the organic layer was washed with 1 N HCl
(2ꢂ) and brine (3ꢂ), dried over sodium sulfate. After filtration
and removal of the volatiles under reduced pressure, the residue
was purified by preparative LC–MS to give 34 (21 mg, 47% two
steps) as a white solid. 1H NMR (400 MHz, CD3OD) d 7.48 (s, 1H),
7.42 (s, 1H), 7.11 (s, 4H), 4.85 (d, J = 12.0 Hz, 1H), 4.62 (d,
J = 12.0 Hz, 1H), 4.51 (d, J = 9.2 Hz, 1H), 4.23 (s, 2H), 4.10 (d,
J = 14.8 Hz, 1H), 4.05 (d, J = 14.8 Hz, 1H), 3.88 (d, J = 12.4 Hz, 1H),
3.71–3.67 (m, 1H), 3.52–3.41 (m, 4H), 2.94 (s, 1H), 2.60 (q,
J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d 141.9, 138.3, 136.9, 136.7, 136.3, 133.0, 130.4, 129.4, 128.4 (2C),
127.5 (2C), 81.0, 79.2, 78.5, 77.4, 75.2, 74.9, 70.4, 67.9, 61.7, 56.9,
38.2, 28.1, 15.0; ESI MS (m/z): 461 [M+H]+, 478 [M+NH4]+, 505
[M+HCO2]ꢁ, calcd 460.
4.1.34. Synthesis of (2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-(4-
chloro-5-(4-ethylbenzyl)-2-(hydroxymethyl)phenyl)tetrahydro-
2H-pyran-3,4,5-triyl triacetate (32)
Compound 32 was prepared from 31 in 91% yield as a white so-
lid using a similar method as used for the preparation of 21. 1H
NMR (400 MHz, CDCl3) d 7.44 (s, 1H), 7.12–7.05 (m, 5H), 5.36–
5.27 (m, 2H), 5.20 (t, J = 9.6 Hz, 1H), 4.69 (s, 2H), 4.57 (d,
J = 10.0 Hz, 1H), 4.23–4.12 (m, 2H), 4.06 (d, J = 15.2 Hz, 1H), 3.99
(d, J = 15.2 Hz, 1H), 3.85–3.81 (m, 1H), 2.60 (q, J = 7.6 Hz, 2H),
2.07 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.63 (s, 3H), 1.19 (t,
J = 9.6 Hz, 3H); ESI MS (m/z): 608 [M+NH4]+, 635 [M+HCO2]ꢁ, calcd
590.
4.1.37. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-(2-(prop-2-ynyloxy)ethyl)phenyl)-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3,4,5-triol (35)
Compound 35 was prepared from 11b in 45% yield as a white
solid using a similar method as used for the preparation of 34.
1H NMR (300 MHz, CD3OD) d 7.38 (s, 1H), 7.26 (s, 1H), 7.10 (s,
4H), 4.45–4.42 (m, 1H), 4.15 (d, J = 2.4 Hz, 2H), 4.05 (d,
J = 15.6 Hz, 1H), 3.99 (d, J = 15.6 Hz, 1H), 3.85 (dd, J = 12.3, 1.5 Hz,
1H), 3.74 (t, J = 7.1 Hz, 2H), 3.67–3.62 (m, 1H), 3.48–3.45 (m, 2H),
3.40–3.37 (m, 2H), 3.13–3.04 (m, 1H), 2.96–2.89 (m, 1H), 2.83 (t,
J = 2.4 Hz, 1H), 2.57 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H); 13C
NMR (100 MHz, CD3OD) d 140.3, 136.4, 135.4, 135.2, 135.1,
131.5, 128.9, 128.7, 126.8 (2C), 125.9 (2C), 79.5, 77.0, 76.2, 73.1,
73.0, 69.0, 68.4, 60.2, 55.9 (2C), 36.5, 30.3, 26.5, 13.3; ESI MS
(m/z): 475 [M+H]+, 492 [M+NH4]+, 519 [M+HCO2]ꢁ, calcd 474.
4.1.35. Synthesis of (2S,3R,4R,5S,6R)-2-(4-chloro-5-(4-ethylben-
zyl)-2-(phenoxymethyl)phenyl)-6-(hydroxymethyl)tetrahydro-
2H-pyran-3,4,5-triol (33)
A solution of phenol (22 mg, 0.23 mmol) and triphenylphos-
phine (60 mg, 0.23 mmol) in anhydrous THF (0.5 mL) under argon
was stirred for 1 h at ꢁ5 °C. Then DIAD (45
lL, 0.23 mmol) was
added dropwise to the above solution. After stirring for 1 h below
10 °C, a solution of 32 (68 mg, 0.12 mmol) in THF (0.5 mL) was
added dropwise. The reaction mixture was stirred for another 5 h
prior to concentration in vaccum. The resulting residue was puri-
fied by preparative TLC using 4:1 PE/ethyl acetate as eluent to give
23 mg of product as a white solid, which was redissolved in 2:3:1
THF/methanol/H2O (0.6 mL). Solid LiOHꢀH2O (1.8 mg, 0.043 mmol)
was added to the above solution. After stirring overnight at room
temperature, the reaction mixture was evaporated and the result-
ing residue was taken up with ethyl acetate and water. After sep-
aration, the organic layer was washed with brine, dried over
sodium sulfate. After filtration and removal of the volatiles under
reduced pressure, the residue was purified by preparative TLC
using 8:80:1 PE/ethyl acetate/methanol as eluent to give 33
(11 mg, 65%) as a colorless gel. 1H NMR (300 MHz, CD3OD) d 7.49
(s, 1H), 7.48 (m, 1H), 7.30–7.23 (m, 2H), 7.09 (s, 4H), 7.04–6.98
(m, 2H), 6.93 (t, J = 7.5 Hz, 1H), 5.31 (d, J = 12.6 Hz, 1H), 5.16 (d,
J = 12.9 Hz, 1H), 4.44 (d, J = 9.3 Hz, 1H), 4.09 (d, J = 15.0 Hz, 1H),
4.03 (d, J = 15.0 Hz, 1H), 3.86 (d, J = 11.7 Hz, 1H), 3.70–3.62 (m,
4.2. In vitro test
4.2.1. Preparation of human SGLT2 expression vector
A full-length cDNA clone expressing human SGLT2 (GenScript
Corporation) was subcloned into Hind III and Not I sites of the
pEAK15 expression vector. Clones harboring the cDNA inserts were
identified by restriction analysis.
4.2.2. Preparation of a cell line stably expressing human SGLT2
A plasmid containing human SGLT2 was linearized with Nsi I
and purified by agarose gel electrophoresis. Using Lipofectamine
2000 Transfection Reagent (Invitrogen Corporation), DNA was
transfected into HEK293.ETN cells and cultured in Dulbecco’s

B. Xu et al. / Bioorg. Med. Chem. 18 (2010) 4422–4432
4431
Modified Eagle Medium (DMEM) containing 10% fetal bovine ser-
um (FBS) at 37 °C under 5% carbon dioxide for 24 h. Transfectants
were selected in the same growth medium supplemented with
puromycin (Invitrogen Corporation) for two weeks. Puromycin-
resistant cells were recovered and seeded on a fresh 96-well plate
(single cell per well) and cultured in the presence of puromycin un-
til cells became confluent. Puromycin-resistant clones were evalu-
4.3.2. Urinary glucose excretion in SD rats
Reference compound 2 was used as a positive control in every
test. Each test compound was dissolved in 30% PEG400 and admin-
istered orally to overnight-fasted normal male SD rats by gavage at
the dose level of 1 mg/kg. Control rats were given 30% PEG400
only. One hour post dosing, glucose solution (2 g/kg, 10 mL/kg)
was administered by oral gavage. Urine was collected within met-
abolic cages from 0 to 24 h post-dosing for urine volume and glu-
cose measurement. Food was removed 16 h before dosing and then
provided 4 h after dosing. Water was supplied ad libitum. The con-
centration of urinary glucose was determined at 24 h post-dose on
the Integra 400 Plus Automatic Biochemistry Analyzer (Roche).
ated for SGLT2 activity in the methyl-a-D-[U-14C]glucopyranoside
uptake assay described below. The clone that exhibited the highest
signal-to-background ratio was used for the methyl-
a-D-
[U-14C]glucopyranoside uptake assay.
4.2.3. Preparation of human SGLT1 expressing cells
Full-length human SGLT1 cDNA in the pDream 2.1 expression
vector was obtained from GenScript Corporation and propagated
in Escherichia coli strain DH5 using Luria–Bertani (LB) medium con-
taining ampicillin. Plasmid DNA was isolated using the QIAGEN
Plasmid Midi Kit (QIAGEN Inc.). Human SGLT1 expression plasmid
DNA was transfected into COS-7 cells (American Type Culture Col-
lection) using Lipofectamine 2000 Transfection Reagent according
to a manufacturer suggested protocol. Transfected cells were
stored in DMEM containing 10% dimethyl sulfoxide (DMSO) at
ꢁ80 °C.
4.3.3. AUC reduction in blood glucose level test
Reference compound 2 was used as a positive control in the
test. Compounds are dissolved in 30% PEG400 and administered or-
ally to overnight fasted C57BL/6 J mice by gavage at different
doses. Control mice are given vehicle only. After 1 h the adminis-
tration of the drugs or vehicle, glucose solution (2 g/kg) is given or-
ally. Blood samples are collected from tail tips just before and 5, 15,
30, 60, and 120 min after glucose loading for determination of glu-
cose. The blood glucose was determined by a glucose meter (John-
son & Johnson). The AUC was calculated using trapezoidal rule.
4.2.4. Methyl-
a-
D-[U-14C]glucopyranoside uptake assay
Acknowledgements
Cells expressing SGLT1 or SGLT2 were seeded on 96-well Scin-
tiPlates (PerkinElmer, Inc.) in DMEM containing 10% FBS
lL medium) incubated at 37 °C under
5% carbon dioxide for 48 h prior to the assay. Cells were washed
We gratefully acknowledge Ying Chen for in vitro activity test-
ing, the analytical group of Egret Pharma (Shanghai) Limited for
analytical service and Dr. Jacques Y. Roberge for thoughtful review
of this manuscript.
(1 ꢂ 105 cells per well in 100
twice with 150 lL of either sodium buffer (137 mM NaCl, 5.4 mM
KCl, 2.8 mM CaCl2, 1.2 mM MgCl2, 10 mM tris(hydroxymeth-
yl)aminomethane/N-2-hydroxyethylpiperazine-N0-ethanesulfonic
acid [Tris/Hepes], pH 7.2) or sodium-free buffer (137 mM
N-methyl-glucamine, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgCl2,
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lM respectively.
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4.3.1. Diarrhea test
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methods used in this paper.