Synthesis and biological evaluation of a 2-aryl polyhydroxylated pyrrolidine alkaloid-based library

Article abstract of DOI:10.1016/j.bmc.2008.10.063

Inspired by polyhydroxylated pyrrolidine alkaloid natural products, a 18-membered library of 2-aryl polyhydroxylated pyrrolidines has been efficiently prepared in two or three synthetic steps from the known chiral cyclic nitrones with high yield and purity and excellent stereoselectivity. The inhibitory activity of all these compounds against various glycosidase enzymes was evaluated. Interestingly, 15 and 19 show better inhibitory activities than radicamine A (20) and B (18) against α-glucosidases. The IC₅₀ values of 15 and 19 are 1.1 and 0.5 μM, respectively. In this study, we also discovered the substituent(s) on the aryl ring could affect the inhibition potency and selectivity against glycosidases.

Full text of DOI:10.1016/j.bmc.2008.10.063

Bioorganic & Medicinal Chemistry 16 (2008) 10198–10204
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a 2-aryl polyhydroxylated pyrrolidine
alkaloid-based library
*
*
En-Lun Tsou, Sih-Yu Chen, Ming-Hsun Yang, Shih-Chi Wang, Ting-Ren Rachel Cheng , Wei-Chieh Cheng
The Genomics Research Center, Academia Sinica, No. 128, Academia Road Sec. 2, Nankang District, Taipei 11529, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2008
Revised 27 October 2008
Accepted 27 October 2008
Available online 5 November 2008
Inspired by polyhydroxylated pyrrolidine alkaloid natural products, a 18-membered library of 2-aryl
polyhydroxylated pyrrolidines has been efficiently prepared in two or three synthetic steps from the
known chiral cyclic nitrones with high yield and purity and excellent stereoselectivity. The inhibitory
activity of all these compounds against various glycosidase enzymes was evaluated. Interestingly, 15
and 19 show better inhibitory activities than radicamine A (20) and B (18) against
a-glucosidases. The
IC50 values of 15 and 19 are 1.1 and 0.5 M, respectively. In this study, we also discovered the substitu-
ent(s) on the aryl ring could affect the inhibition potency and selectivity against glycosidases.
l
Keywords:
Polyhydroxylated pyrrolidine alkaloid
Natural product-like molecule
Chiral cyclic nitrone
Ó 2008 Elsevier Ltd. All rights reserved.
Glycosidase inhibitors
Structural diversity
1. Introduction
and B, revision of their absolute configurations of these nature
products.8,9
Naturally occurring polyhydroxylated pyrrolidines impart a
variety of biological activities including glycosidase inhibition,
antiviral activity, and antidiabetic activity.1,2 A new class of poly-
hydroxylated pyrrolidines was recently discovered, in which an
aryl moiety was found to be directly attached to the C-2 position
of the pyrrolidine ring (Fig. 1).3–5 Members of this class include
(ꢀ)-codonopsinine and (ꢀ)-codonopsine, both isolated from
Codonopsis clematidea (a herb used in folk medicine to improve
hepatic function), and were found to exhibit antibiotic activity
and hypotensive activity without affecting the central nervous sys-
tem.3 Recently, another new alkaloid, codonopsinol, was reported
by Ishida et al.,4 and in 2001, Kusano and co-workers first isolated
radicamines A and B from Lobelia chinensis Lour, an important
herb prescribed in traditional Chinese medicine as a diuretic, anti-
dote, and carcinostatic agent. Radicamines A and B were subse-
Our research interests include the design and synthesis of nat-
ural product-based small molecule libraries via a combinatorial ap-
proach.10 Clearly, the variety of biological activities exhibited by
molecules containing the 2-aryl pyrrolidine skeleton suggests this
motif to be a privileged scaffold of potential use in combinatorial
chemistry for drug discovery.11 Meanwhile, recent reports show
that the enantiomers of natural products or synthetic bioactive
molecules, such as
L-nucleoside analogues or L-iminocyclitols,
may possess interesting or unexpected biological activity.12,13 To
OH
OH
OMe
OMe
OMe
CH3
N
OH
HO
OH
HO
OH
HO
H
N
H
N
quently shown to be new glycosidase inhibitors.5 From
a
OH
OH
OH
structural point of view, the absolute configurations of these mol-
ecules are (2R,3R,4R,5R) or (2S,3S,4S,5S) at the four stereogenic
centers and the relative stereochemistry of the substituents at
C2, C3 is trans (Fig. 1). The interesting structures of these molecules
and their potentially useful biological activity have inspired the to-
tal synthesis of specific targets6,7 and, in the cases of radicamines A
radicamine B
revised
radicamine A
revised
codonopsinol
H
OMe
OMe
OMe
CH3
CH3
N
H3C
HO
N
H3C
OH
HO
OH
* Corresponding authors. Tel.: +886 2 2789 1262 (W.-C.C.); fax: +886 2 27899924
(T.-R.R.C.).
(-)-codonopsinine
(-)-codonopsine
E-mail addresses: tingjenc@gate.sinica.edu.tw (T.-R.R. Cheng), wcheng@gate.
sinica.edu.tw (W.-C. Cheng).
Figure 1. Natural polyhydroxylated pyrrolidine alkaloids containing aryl moieties.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.063

E.-L. Tsou et al. / Bioorg. Med. Chem. 16 (2008) 10198–10204
10199
their preparation. Eight diverse un-, mono-, or di-substituted aryl
Grignard reagents 3a–h (Fig. 3) will be prepared. It is noteworthy
that the naturally occurring alkaloids—radicamine A (20) and B
(18) could be synthesized from cyclic nitrone 4 with Grignard re-
agents 3b and 3d, respectively.6,8,15
R1
R1
R2
N
R2
N
OH
OH
HO
HO
OH
OH
2.2. Synthesis
2
1
Mirror
The requisite aryl Grignard reagents 3a–g (Fig. 3) were synthe-
sized starting from the corresponding commercially available
mono- or dihydroxybenzyl bromide. Although the preparation of
3d has been reported using bromine,8 we adapted the procedure
(Scheme 2) for use with N-bromosuccinimide (NBS) since it is eas-
ier and safer to handle.17 Guaiacol 6 was reacted with MsCl under
basic conditions to protect the hydroxyl group and assist the regio-
selective bromination of the aromatic ring with NBS to give the 5-
brominated compound 8.18 Deprotection of the mesyl group of 8
with lithium diisopropylamide (LDA)19 followed by benzylation
afforded the aryl bromide 10 in a yield of 83% over four steps, with-
out the need for column chromatography. Treatment of 10 with
magnesium metal in THF at 70 °C gave the desired reagent 3d as
a pale yellow solution in THF.20 All other Grignard reagents were
similarly and freshly prepared by addition of magnesium powder
to the appropriate aryl halides.
Figure 2. Desired small molecule libraries 1 and 2.
the best of our knowledge, however, use of the 2-aryl pyrrolidine
skeleton as a privileged scaffold in combinatorial chemistry for
the synthesis of both enantiomers of various derivatives bearing
substituents on the aryl ring and the nitrogen atom of the pyrrol-
idine ring has not been completely explored.
Herein, we describe the preparation of a small molecule library
based on a general 2-aryl polyhydroxylated pyrrolidine core struc-
ture with two points of substitution diversity (R1 and R2 in 1, see
Fig. 2). In addition, the enantiomeric library 2 was also prepared
(structural diversity).14 The inhibitory activity of these molecules
against various glycosidases was also examined.
The enantiopure tri-O-benzyl cyclic nitrone 4 (Scheme 3) was
smoothly prepared from 2,3,5-tri-O-benzyl-D-arabinofuranose
(11) in a yield of 71% over five steps (oximation, TBDPS protection,
iodolization, deprotection, and in situ cyclization).16 Similarly, tri-
O-benzyl cyclic nitrone 5 was also prepared in 74% yield from
2,3,5-tri-O-benzyl-D-xylofuranose (13).16
2. Results and discussion
2.1. Design and strategy
The stereoselective addition of a nucleophile to a chiral cyclic
nitrone to give a polyhydroxylated pyrrolidine has been reported
by Goti, Gurjar, Yu, and others.6,8,15 In this approach, a Grignard re-
agent attacks a cyclic nitrone16 on the less hindered face opposite
to that of the neighboring benzyl ether group to give a product
having a 2,3-trans configuration (Scheme 1). A common structural
feature of alkaloid libraries 1 and 2 is the 2,3-trans configuration
(Fig. 2), and therefore this chemistry is potentially applicable to
OMe
OMe
OMe
OMs
(c)
OH
OH
(a)
(d)
(b)
(e)
OMs
OBn
Br
8
6
7
OMe
OMe
OMe
R1
OBn
O
N
O
N
OBn
BnO
OBn
BnO
Br
9
Br
10
MgBr
3d
MgX
3
OBn
OBn
5
4
steps
Scheme 2. Preparation of Grignard reagent 3d. Reagents and conditions: (a) MsCl,
NEt3, CH2Cl2, 0 °C, 0.5 h, 96%; (b) NBS, DMF, rt, 24 h, 96%; (c) LDA, THF, 0 °C, 5 min,
91%; (d) BnBr, NaH, THF, 0 °C ? rt, 15 h, 99%; (e) Mg, THF, reflux.
steps
2
1
Scheme 1. General synthetic route towards libraries 1 and 2.
OBn
OMe
OMe
OBn
MgCl
MgBr
3b
MgBr
3c
MgBr
3d
3a
OMe
OMe
OMe
MeO
OMe
OMe
OMe
Scheme 3. Preparation of chiral cyclic nitrones 4 and 5. Reagents and conditions:
(a) i—NH2OHꢁHCl, NaOMe MeOH, rt; ii—TBDPSCl, imidazole, CH2Cl2, rt; (b) i—I2,
PPh3, imidazole, toluene, reflux; ii—TBAF, toluene, reflux, 71% yield from 11;
(c) i—MsCl, Et3N, CH2Cl2, rt; ii—TBAF, THF, 0 °C; then NH2OH HCl, NaHCO3, MeOH/
H2O (3/1), 60 °C, 74% yield from 13.
MgBr
3e
MgBr
3g
MgBr
3f
MgBr
3h
Figure 3. Set of Gringard reagents 3a–h for the library.

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E.-L. Tsou et al. / Bioorg. Med. Chem. 16 (2008) 10198–10204
Table 1
OH
N
OMe
OMe
Solution-phase synthesis of 2-arylpolyhydroxylated pyrrolidine-based library.
(b)
(a)
4
BnO
Nucleophilic addition
(nitrone/Grignard reagent)
Deprotection
Product (yield %)
N-alkylation
product (yield %)
deprotection
nucleophilic
addition
BnO
OBn
14
4/3a
4/3b
4/3c
4/3d
4/3d
4/3e
4/3f
4/3g
5/3c
5/3d
5/3e
4/3h
17 (88)
18 (89)
19 (90)
20 (92)
20 (92)
15 (87)
21 (90)
22 (73)
23 (86)
24 (90)
25 (88)
32 (92)
Me
N
26 (84)
27 (86)
28a (80)
16 (95)
29 (77)
30 (90)
31 (82)
OMe
OMe
OMe
OMe
H
N
Ha
(c)
HO
HO
N-alkylation
OH
HO
OH
HO
16
15
Scheme 4. Preparation of 15 and its N-methylated analogue 16. Reagents and
conditions: (a) 3e, THF, 0 °C, 85%; (b) Pd(OH)2, H2(g), HCl(cat.), THF, 87%; (c)
formaldehyde, Pd(OH)2, H2(g), MeOH, 95%.
a
N-butylated product.
Preparation of the 2-aryl polyhydroxylated pyrrolidine library
could then commence, and a variety of reaction conditions or puri-
fication procedures were examined. For example, the reaction was
found to take 10–12 h to complete when only 1 or 2 equiv. of Grig-
nard reagent was used. Use of elevated temperatures (0 °C ? rt)
resulted in multiple products. In our optimized procedure (Scheme
4), chiral cyclic nitrone 4 (0.25 g, 0.60 mmol) was reacted with
freshly prepared Grignard reagent 3e (4 equiv.) at 0 °C for 1 h. Solu-
tion-phase extraction with CH2Cl2 and simple filtration through a
silica-gel cartridge were carried out to afford the only single isomer
14 in good yield (85%) and high purity (>95%). Impurities, derived
from the excess of Grignard reagent, were easily removed using a
1:20 mixture of ethyl acetate: hexanes as the elutant (30 mL).
The product 14 was directly collected by eluting a 1:5 mixture of
ethyl acetate: hexanes (50 mL) without any fractionation. Subse-
quent hydrogenolysis of 14 with Pd(OH)2 under acidic conditions
for 15 h gave alkaloid 15 (87%) without the need for purification.20
N-Methylation of 5 was performed to give the desired product 16
in good yield (95%).21
3.8 ppm and the coupling constant was 9.0 Hz, confirming the
structural feature of the 2,3-trans configuration. As expected, the
same chemical shift and coupling constant but opposite optical
rotation data {½a D20
= ꢀ36.4 (c = 0.44, H2O)} were observed for com-
ꢂ
pound 25.
This procedure was used to synthesize a total of 18 distinct alka-
loids (Fig. 4), including radicamine A (20) and B (18) and -radic-
L
amine A (24) in high yields and high purities (Table 1). Next,
biological evaluation of this focused library against different glycosi-
dases was conducted.
2.3. Biological evaluation
The inhibitory activities (Table 2) of all library members against
a
-glucosidase (bacillus and yeast), b-glucosidase (almonds), and b-
mannosidase (helix pomatia) were measured.22,23 All compounds
with a -gluco configuration including the natural alkaloids 18
and 20 showed better inhibitory activities against -glucosidases
D
a
Alkaloid 25, the enantiomer of 15, was also prepared from the
cyclic nitrone 5 in 91% yield over two steps using the same proce-
dure. In the 1H NMR spectrum of 15, the chemical shift of Ha was
than b-glucosidase or b-mannosidase. Comparing the molecules
with a mono-substituent on the aryl ring, the inhibitory activities
OH
HO
OH
HO
OH
HO
OH
OH
HO
H
N
H
N
OH
HO
H
N
OMe
OMe
OMe
OH
OMe
OMe
H
N
H
N
OH
OH
OH
OH
OH
OH
OH
20
)
Radicamine A(
15
19
17
Radicamine B(18)
OMe
OH
H
OH
HO
OH
H
OH
HO
OMe
OH
24
OH
HO
OMe
OMe
H
N
OMe
OMe
H
N
H
N
N
N
OMe
OMe
HO
OH
HO
OH
OH
OH
23
L-Radicamine A(
)
25
21
22
Me
OH
Me
N
Bu
Me
N
Me
N
OMe
OMe
OH
HO
OMe
OMe
OH
OH
HO
OH
HO
OH
HO
OMe
OH
N
N
OMe
OH
HO
OH
OH
OH
OH
OH
16
28
29
27
26
OMe
Me
N
Me
N
OH
HO
OH
HO
OH
HO
OMe
H
N
OMe
OMe
OH
31
32
30
Figure 4. Chemical structures of the 2-aryl polyhydroxylated pyrrolidine library.

E.-L. Tsou et al. / Bioorg. Med. Chem. 16 (2008) 10198–10204
10201
Table 2
6.0
lM and no inhibition against other enzymes, even bacillus a-
glucosidase.24
Inhibition activities of 15–32 against glycosidases.
Although we attempted to introduce a methyl or butyl group
into the imino group in 26–31 via reductive amination to increase
the hydrophobic interactions,25–27 these modifications failed to en-
hance the binding affinity and instead resulted in a dramatic de-
crease or complete loss of inhibitory activities.
Compound
IC50
(lM)
a
-Glucosidasea
a
-Glucosidaseb b-Glucosidasec b-Mannosidased
17
18
19
20
15
21
22
23
24
25
26
27
28
16
29
30
31
32
36
10
8.8
12
1.8
8.4
4.0
—
—
—
9.2
—
—
10
24
93
197
—
122
—
34
96
169
—
—
—
—
—
—
—
—
—
—
ND
e
—
0.5
4.2
1.1(0.7)g
7.8
2.6
21
—
6.0
38
28
—
72
—
21
66
52
172
—
—
113
—
61
184
—
—
86
—
3. Conclusion
A natural product-based 2-aryl polyhydroxylated pyrrolidine
alkaloid library with three diversity elements (one structural diver-
sity; two substituent diversities) has been efficiently prepared
using the chiral cyclic nitrones 4 and 5 as starting materials. Based
on this solution-phase combinatorial approach, our small molecule
library is obtained in high yield and purity and in excellent stere-
oselectivity. This synthetic protocol is expected to be suitable for
a large library preparation with assistance of automated equip-
ments such as synthesizers and liquid handlers. The inhibitory
activities of all the compounds synthesized against various glyco-
sidases enzymes were measured and compounds 15 and 19 were
found to show better inhibitory activities than radicamine B (18)
24
—
—
—
31
—
—
2.4
—
NDf
a
From Bacillus stearothermophilus lyoph.
From yeast.
From almonds.
From Helix Pomatia.
IC50 more than 200
Not determined.
b
c
d
e
f
and A (20) against yeast
a-glucosidase. The IC50 values of 15 and
19 are 1.1 and 0.5 M, respectively. In this study, we also discov-
l
lM.
ered the substituent(s) on the aryl ring could affect the inhibition
potency and selectivity against glycosidases.
g
Ki (lM).
4. Experimental
4.1. Chemistry
of 19 (with a para methoxy moiety) and 32 (with a meta-methoxy
moiety) exhibited a higher potency than that of natural product 18
(having a hydroxyl group at the para position) specifically against
yeast
2.4 M and 24
Comparing the positions of dimethoxy groups on the aryl ring,
the 3,4-dimethoxy substituted 15 was more potent than 21 and
22, having 2,4- or 2,5-dimethoxy groups, respectively. Compound
a
-glucosidase. The IC50 values of 19, 32, and 18 were 0.5
lM,
Mass spectra were measured with a Bruker BioTOF III (ESI-MS).
Optical rotations were measured with a PerkinElmer Model 341
polarimeter. NMR spectra (1H at 400 or 600 MHz, 13C at 100 or
150 MHz) were recorded in CDCl3, CD3OD, or D2O solvents. Silica
l
lM, respectively.
gel was used Merck Kieselgel Si60 (40–63 lm) and CC refers to col-
15 showed potent inhibitory activities against
(bacillus and yeast) and the IC50 values were 1.8
respectively. Presumably, because the conformation, electron dis-
tribution, and orientation of hydroxyl groups of -azasugar 15
may resemble the transition state of enzymatic glycosidic hydroly-
sis, it could act as a transition state mimic to inhibit -glucosi-
daes (bacillus and yeast) at the active site.22,23 The Lineweaver–
Burk plot of yeast -glucosidae kinetics was shown in Figure 5.
The kinetic results demonstrated that the inhibition pattern of 15
was competitive with a Ki value of 0.7 M.
Compound 24, the -enantiomer of natural product 20, was
inactive towards various glycosidase enzymes. Interestingly,
though 25 was the -enantiomer of 15, it still showed some inhib-
a-D-glucosidases
umn chromatography. Thin layer chromatography (TLC) was per-
formed on glass plates coated to a thickness of 1 mm with Merck
Kieselgel 60F254. All reagents, enzymes, substrates, and solvents
were purchased from commercial suppliers, and used without fur-
ther purification. Concentration refers to rotary evaporation. Prep-
aration of 10 is described in the supporting material. Cyclic
nitrones 4 and 5 were prepared as previously described.16
l
M and 1.1 M,
l
D
a-D
a
4.1.1. Typical procedure for the preparation of Grignard reagent
3e
l
L
A mixture of 4-bromo-1,2-dimethoxybenzene (0.52 g, 2.4 mmol)
andmagnesiumpowder(0.129 g, 5.37 mmol)inTHF(5 mL)wasstir-
redunderrefluxfor30 minandcooledto roomtemperaturetoafford
the freshly prepared 3e (0.25 M, 5 mL) as a yellow solution. Other
Grignard reagents 3a–d, f, g were prepared using a similar method.
L
itory activity towards yeast
a-glucosidase with IC50 value of
2
0.18
4.1.2. Typical procedure for the synthesis of (2R,3R,4R,5R)-2-
(3,4-dimethoxyphenyl)-5-(hydroxymethyl)pyrrolidine-3,4-diol
(15)
1
0.14
6.6 μM
The freshly prepared Grignard reagent 3e (4 equiv., 0.25 M,
5 mL) was added dropwise into a solution of cyclic nitrone 4
(0.25 g, 0.6 mmol) in THF (5 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 1 h, quenched with sat. NH4Cl(aq) (5 mL), and ex-
tracted with DCM (2ꢃ 10 mL). The combined organic extracts were
dried and concentrated. The residue was loaded onto the top of a
silica gel cartridge (12 g, silica gel; 2ꢃ 6 cm), and eluted with ethyl
acetate-hexanes (1: 20, 30 mL, first fraction) to remove impurities,
followed by eluting with ethyl acetate–hexanes (1:5, 50 mL, second
fraction) to directly give the intermediate 14 (0.28 g, 85%) as a
-2
2
4
6
0.10
[ I ] (μM)
Ki = 0.7 μM
3.3 μM
2.2 μM
1.1 μM
0.06
0.02
-3
-1
1
3
5
)
7
9
-0.02
1/[S] (μM-1
white solid: ½a 2D0
ꢂ
= ꢀ6.8 (c 0.6, CHCl3); 1H NMR (600 MHz, CDCl3)
Figure 5. Lineweaver-Burk double reciprocal plots of alkaloid 15.

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E.-L. Tsou et al. / Bioorg. Med. Chem. 16 (2008) 10198–10204
d 3.66–3.67 (m, 1H), 3.75 (q, 1H, J = 7.0, 7.1, 9.3 Hz), 3.77 (s, 3H),
3.81 (dd, 1H, J = 4.3, 9.5 Hz), 3.85 (s, 3H), 4.05 (m, 1H), 4.10 (t, 1H,
J = 3.1 Hz), 4.17 (d, 1H, J = 7.4 Hz), 4.34 (dd, 2H, J = 11.9, 18.8 Hz),
4.45–4.58 (m, 4H), 6.80 (d, 1H, J = 7.9 Hz), 6.93 (d, 2H, J = 8.8 Hz),
7.07 (t, 2H, J = 2.6, 4.8 Hz), 7.22-7.32 (m, 13H); 13C NMR
(150 MHz, CDCl3) d 148.7, 148.5, 138.0, 137.8, 137.6, 128.3, 128.3,
128.2, 128.0, 127.7, 127.6, 127.6, 121.0, 110.8, 110.5, 86.6, 83.2,
73.6, 73.3, 71.9, 71.6, 68.8, 66.6, 55.7, 55.7; HRMS calcd for
[C34H37NO6+H]+ 556.2694, found 556.2697.
132.0, 119.6, 114.3, 112.4, 81.6, 77.0, 63.3, 62.1, 61.5, 55.8; HRMS
calcd for [ C12H17NO5+H ]+ 256.1179, found 256.1167.
4.1.7. (2R,3R,4R,5R)-2-(2,4-Dimethoxyphenyl)-5-(hydroxy-
methyl)pyrrolidine-3,4-diol (21)
Compound 21 was prepared from 4 and 3f followed by hydrogen-
olysis according to the procedure previously described for the syn-
thesis of compound 15. The title compound was isolated (90%
yield over two steps) as a colorless solid: ½a D20
ꢂ
= +27.0 (c 1.9, H2O);
A
mixture of 14 (0.1 g, 0.18 mmol), palladium hydroxide
1H NMR (600 MHz, D2O) d 3.26 (m, 1H), 3.70 (dd, 1H, J = 6.4,
11.5 Hz), 3.76 (dd, 1H, J = 4.4, 11.5 Hz), 3.85 (S, 3H), 3.86 (s, 3H),
3.95 (t, 1H, J = 7.4, 7.5 Hz), 4.11 (d, 1H, J = 8.9 Hz), 4.35 (dd, 1H,
J = 7.4, 8.7 Hz), 6.63 (dd, 1H, J = 2.3, 8.3 Hz), 6.67 (d, 1H, J = 2.3 Hz),
7.28 (d, 1H, J = 8.4 Hz); 13C NMR (150 MHz, D2O) d 160.0, 158.8,
129.5, 119.5, 105.1, 99.0, 79.9, 77.6, 62.3, 61.5, 60.0, 55.4, 55.4;
HRMS calcd for [C13H19NO5+H]+ 270.1336, found 270.1331.
(0.09 g), and 37% HCl(aq) (0.1 mL) in THF (5 mL) was stirred for
15 h under a hydrogen atmosphere. The reaction mixture was fil-
tered through Celite and then the solution was neutralized with
DOWEX(OHꢀ) and concentrated to give 15 (42.2 mg, 0.16 mmol,
87%) as a white solid: ½a D20
ꢂ
= +35 (c 0.43, H2O); 1H NMR (600 MHz,
D2O) d 3.26 (m, 1H), 3.70 (dd, 1H, J = 6.4, 11.5 Hz), 3.76 (dd, 1H,
J = 4.5, 11.5 Hz), 3.81 (S, 3H), 3.85 (s, 3H), 3.90 (d, 1H, J = 9.0 Hz),
3.96 (t, 1H, J = 7.3, 7.3 Hz), 4.09 (dd, 1H, J = 7.4, 8.7 Hz), 6.99 (s,
2H), 7.04 (s, 1H); 13C NMR (150 MHz, D2O) d 148.2, 147.7, 132.6,
120.1, 111.7, 110.5, 82.0, 77.3, 63.7, 62.4, 61.6, 55.6; HRMS calcd
for [C13H19NO5+H]+ 270.1336, found 270.1337.
4.1.8. (2R,3R,4R,5R)-2-(2,5-Dimethoxyphenyl)-5-(hydroxy-
methyl)pyrrolidine-3,4-diol (22)
Compound 22 was prepared from 4 and 3g followed by hydrog-
enolysis according to the procedure previously described for the
synthesis of compound 15. The title compound was isolated (73%
4.1.3. (2R,3R,4R,5R)-2-(Hydroxymethyl)-5-phenylpyrrolidine-
3,4-diol (17)
Compound 17 was prepared from 4 and 3a followed by hydrog-
enolysis according to the procedure previously described for the
synthesis of compound 15. The title compound was isolated (88%
yield over two steps) as a yellow syrup: ½a D20
ꢂ
= +44.1 (c 0.8, H2O);
1H NMR (600 MHz, D2O) d 3.30 (m, 1H), 3.71 (dd, 1H, J = 6.2,
11.6 Hz), 3.76 (dd, 1H, J = 4.5, 11.6 Hz), 3.79 (S, 3H), 3.81 (s, 3H),
3.95 (t, 1H, J = 7.4, 7.3 Hz), 4.17 (d, 1H, J = 8.7 Hz), 4.30 (dd, 1H,
J = 7.3, 8.6 Hz), 6.96 (m, 2H), 7.02 (d, 1H, J = 8.6 Hz); 13C NMR
(150 MHz, D2O) d 152.8, 152.1, 128.1, 114.7, 113.9, 113.1, 80.3,
77.4, 61.9,61.7, 60.1, 56.1, 55.8; HRMS calcd for [C13H19NO5+H]+
270.1336, found 270.1332.
yield for two steps) as a colorless syrup: ½a D20
ꢂ
= 45 (c 0.12, H2O);
1H NMR (600 MHz, D2O) d 3.29 (m, 1H), 3.72 (dd, 1H, J = 6.5,
11.6 Hz), 3.78 (dd, 1H, J = 4.6, 11.6 Hz), 3.98 (m, 2H), 4.15 (dd,
1H, J = 7.6, 9.1 Hz), 7.40–7.47 (m, 5H); 13C NMR (150 MHz, D2O) d
139.5, 128.9, 128.2, 127.3, 82.1, 77.4, 63.9, 62.3, 61.7; HRMS calcd
for [C11H15NO3+H]+ 210.1125, found 210.1131.
4.1.9. (2R,3R,4R,5R)-2-(Hydroxymethyl)-5-(3-methoxy-
phenyl)pyrrolidine-3,4-diol (32)
Compound 32 was prepared from 4 and 3h followed by hydrog-
enolysis according to the procedure previously described for the
synthesis of compound 15. The title compound was isolated (92%
4.1.4. (2R,3R,4R,5R)-2-(Hydroxymethyl)-5-(4-hydroxyphenyl)
pyrrolidine-3,4-diol (18)
Compound 18 was prepared from 4 and 3b followed by hydrog-
enolysis according to the procedure previously described for the
synthesis of compound 15. The title compound was isolated (89%
yield over two steps) as a colorless syrup: ½a D20
ꢂ
= +37.1 (c 0.93,
H2O); 1H NMR (600 MHz, CD3OD) d 3.24 (m, 1H), 3.66 (m, 1H,
J = 6.2, 11.5 Hz), 3.74 (dd, 1H, J = 4.1, 11.5 Hz), 3.79 (s, 3H), 3.91 (t,
1H, J = 6.2, 6.6 Hz), 3.98 (m, 2H), 6.85 (m, 1H), 7.02 (t, 2H, J = 2.5,
8.7 Hz), 7.26 (t, 1H, J = 7.8, 7.9 Hz); 13C NMR (150 MHz, CD3OD) d
161.5, 144.1, 130.7, 120.7, 114.3, 113.9, 84.8, 79.3, 66.7, 64.8, 63.4,
55.8; HRMS calcd for [C12H17NO4+H]+ 240.1230, found 240.1232.
yield over two steps) as a colorless syrup: ½a D20
ꢂ
= +69 (c 0.86, H2O);
1H NMR and 13C NMR was described as ref report about radicamine
B; HRMS calcd for [C11H15NO4+H]+ 226.1074, found 226.1075.
4.1.5. (2R,3R,4R,5R)-2-(Hydroxymethyl)-5-(4-methoxyphenyl)
pyrrolidine-3,4-diol (19)
Compound 19 was prepared from 4 and 3c followed by hydrog-
enolysis according to the procedure previously described for the
synthesis of compound 15. The title compound was isolated (90%
4.1.10. Typical procedure for the synthesis of (2R,3R,4R,5R)-2-
(3,4-dimethoxyphenyl)-5-(hydroxymethyl)-1-methyl-
pyrrolidine-3,4-diol (16)
A mixture of 15 (22.4 mg, 0.083 mmol) and formaldehyde solu-
tion (37% in H2O, 0.1 mL) in MeOH (5 mL) was stirred at room tem-
perature for 10 min. Pd(OH)2 (10 mg) was added to the reaction
mixture and the reaction was stirred for overnight under a hydro-
gen atmosphere. The mixture was filtered and the solution was
yield over two steps) as a colorless syrup: ½a D20
ꢂ
= +42.5 (c 0.38,
H2O); 1H NMR (600 MHz, D2O) d 3.24 (m, 1H), 3.69 (dd, 1H, J = 6.5,
11.5 Hz), 3.75 (dd, 1H, J = 4.6, 11.5 Hz), 3.81 (S, 3H), 3.89 (d, 1H,
J = 9.1 Hz), 3.95 (t, 1H, J = 7.3, 7.3 Hz), 4.08 (dd, 1H, J = 7.6, 9.1 Hz),
7.00 (d, 2H, J = 8.7 Hz), 7.36 (d, 2H, J = 8.7 Hz); 13C NMR (150 MHz,
D2O) d 158.5, 132.2, 128.6, 114.2, 82.1, 77.4, 63.3, 62.5, 61.6, 55.3;
HRMS calcd for [C12H17NO4+H]+ 240.1230, found 240.1246.
concentrated to give 16 (22.3 mg, 95%): ½a D20
= ꢀ15 (c 0.22, MeOH);
ꢂ
1H NMR (600 MHz, CD3OD) d 2.20 (s, 3H), 3.10 (m, 1H), 3.66 (d, 1H,
J = 6.5 Hz), 3.81-3.87 (m, 8H), 3.94 (dd, 1H, J = 5.0, 6.4 Hz), 4.03 (t,
1H, J = 4.4, 4.4 Hz), 6.90 (s, 2H), 7.03 (s, 1H); 13C NMR (150 MHz,
CD3OD) d 150.7, 150.1, 134.5, 122.5, 112.8, 112.8, 85.8, 80.1,
75.1, 71.2, 60.9, 56.6, 56.5, 35.1; HRMS calcd for [C14H21NO5+H]+
284.1492, found 284.1492.
4.1.6. (2R,3R,4R,5R)-2-(3-Hydroxy-4-methoxyphenyl)-5-
(hydroxymethyl)pyrrolidine-3,4-diol (20)
Compound 20 was prepared from 4 and 3d followed by hydrog-
enolysis according to the procedure previously described for the
synthesis of compound 15. The title compound was isolated (92%
4.1.11. (2R,3R,4R,5R)-2-(Hydroxymethyl)-5-(4-methoxy-
phenyl)-1-methylpyrrolidine-3,4-diol (26)
Compound 26 was prepared from 19 following the procedure
previously described for the synthesis of compound 16. The title
yield for two steps) as a white solid: ½a D20
ꢂ
= +36 (c 0.11, H2O); 1H
3.26 (m, 1H), 3.69 (dd, 1H, J = 6.4,
NMR (600 MHz, D2O)
d
11.6 Hz), 3.75 (dd, 1H, J = 4.4, 11.6 Hz), 3.81 (S, 3H), 3.85 (d, 1H,
J = 9.0 Hz), 3.94 (t, 1H, J = 7.4 Hz), 4.07 (dd, 1H, J = 7.6, 9.0 Hz),
6.89-6.97 (m, 3H); 13C NMR (150 MHz, D2O) d 147.3, 145.2,
compound was obtained (84% yield) as
a colorless solid:
½
a 2D0
ꢂ
= ꢀ7 (c 0.16, MeOH);1H NMR (600 MHz, CD3OD) d 2.20 (s,

E.-L. Tsou et al. / Bioorg. Med. Chem. 16 (2008) 10198–10204
10203
3H), 3.10 (dd, 1H, J = 4.3, 8.6 Hz), 3.71 (d, 1H, J = 6.7 Hz), 3.78 (s,
3H), 3.82(dd, 1H, J = 4.3, 11.6 Hz), 3.86 (dd, 1H, J = 4.1, 11.6 Hz),
3.96 (dd, 1H, J = 5.0, 6.7 Hz), 4.02 (t, 1H, J = 4.6, 4.7 Hz), 6.90 (d,
2H, J = 8.6 Hz), 7.28 (d, 2H, J = 8.6 Hz); 13C NMR(150 MHz, CD3OD)
d 160.9, 130.8, 114.9, 85.4, 79.8, 75.4, 71.2, 60.8, 55.8, 49.9, 35.2;
HRMS calcd for [C13H19NO4+H]+ 254.1387, found 254.1388.
nitrophenyl-glycopyranoside (40 mM, 20 mM, 10 mM, 5 mM,
2.5 mM, 1.25 mM, 0.625 mM, 0 mM) at 405 nm using multi-detec-
tion reader (SpectraMax M5, Molecular Dectce). The data obtained
were fitted to the Michaelis–Menten equation using the GraphPad
to determine the Km values and Vmax values. For example, the Km
values for bacillus
a-glucosidase and yeast a-glucosidase are
1.5 mM and 0.19 mM, respectively. The substrate concentrations
were used at two fold Km values for evaluation of the inhibitory ef-
fect against various glycosidases. Enzymes at 0.25–1 units per mL
were used to provide an ideal progression curve, and inhibitors
4.1.12. (2R,3R,4R,5R)-2-(3-Hydroxy-4-methoxyphenyl)-5-
(hydroxymethyl)-1-methylpyrrolidine-3,4-diol (27)
Compound 27 was prepared from 20 following the procedure
previously described for the synthesis of compound 16. The title
were tested initially at 500 lM. The compounds that showed activ-
compound was obtained (86%) as a colorless solid: ½a D20
ꢂ
= +2 (c
ities were selected and further tested at lower concentration to
determine IC50. The assays performed in wells of a 96-well micro-
titer plate containing either sodium phosphate buffer (100 mM, pH
0.69, MeOH); 1H NMR(600 MHz, CD3OD) d 2.17 (s, 3H), 3.05 (m,
1H), 3.57 (d, 1H, J = 6.6 Hz), 3.78–3.84 (m, 5H), 3.93 (t, 1H, J = 6.4,
5.0 Hz), 3.98 (t, 1H, J = 4.7, 4.9 Hz), 6.73 (dd, 1H, J = 1.9, 8.2 Hz),
6.84 (m, 2H); 13C NMR (150 MHz, CD3OD) d 148.8, 134.7, 126.3,
121.0, 116.4, 112.6, 85.7, 80.0, 75.5, 71.0, 61.0, 56.6, 49.9, 35.1;
HRMS calcd for [C13H19NO5+H]+ 270.1336, found 270.1338.
6.8, for
a-glucosidase (E.C. 3.2.1.20) and b-glucosidase (E.C.
3.2.1.21), or sodium citrate buffer (100 mM, pH 6.4, for
a- and b-
mannosidase (E.C. 3.2.1.25).
Acknowledgment
4.1.13. (2R,3R,4R,5R)-1-Butyl-2-(3-hydroxy-4-methoxyphenyl)-
5-(hydroxymethyl)pyrrolidine-3,4-diol (28)
Following the same procedure as described for the preparation
of 16, instead of butraldehyde from formaldehyde, the title com-
This work was supported by the National Science Council and
Academia Sinica.
pound 28 was obtained as
a colorless syrup in 80% yield:
Supplementary data
½
a 2D0
ꢂ
= ꢀ35.1 (c 0.54, MeOH); 1H NMR (600 MHz, CD3OD) d 0.81
(t, 3H, J = 7.4, 7.3 Hz), 1.21 (m, 1H), 1.29 (m, 1H), 1.38 (m, 2H),
2.36 (m, 1H), 2.52 (m, 1H), 3.21 (dd, 1H, J = 3.5, 8.2 Hz), 3.66 (t,
1H, J = 6.8, 6,9 Hz), 3.78 (dd, 1H, J = 3.6, 11.3 Hz), 3.82–3.86 (m,
5H), 4.05 (t, 1H, J = 3.8, 3.7 Hz), 6.79 (dd, 1H, J = 1.7, 8.2 Hz), 6.85
(d, 1H, J = 8.2 Hz), 6.88 (d, 1H, J = 1.8 Hz); 13C NMR (150 MHz,
CD3OD) d 148.5, 147.6, 135.9, 121.0, 116.1, 112.5, 86.6, 80.5,
74.9, 68.4, 60.3, 56.5, 47.2, 31.3, 21.5, 14.4; HRMS calcd for
[C16H21NO5+H]+ 312.1805, found 312.1806.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.10.063.
References and notes
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methyl)-1-methylpyrrolidine-3,4-diol (29)
Compound 29 was prepared from 21 following the procedure
previously described for the synthesis of compound 16. The title
compound was obtained (77%) as a colorless syrup in 77% yield:
½
a 2D0 = +16.1 (c 1, MeOH); 1H NMR (600 MHz, CD3OD) d 2.32 (s,
ꢂ
3H), 3.21 (br, 1H), 3.81 (s, 3H), 3.85 (m, 4H), 3.90 (dd, 1H, J = 3.2,
11.9 Hz), 4.05 (t, 1H, J = 5.9, 5.9 Hz), 4.30 (t, 1H, J = 5.3,
6.0 Hz),4.39 (br, 1H), 6.59 (dd, 2H, J = 1.7, 8.4 Hz), 7.30 (d, 1H,
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106.3, 99.7, 81.6, 78.4, 71.6, 59.7, 56.1, 56.0, 49.9, 35.9; HRMS calcd
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compound was obtained (90%) as a colorless solid: ½a D20
= ꢀ9
ꢂ
(c 0.08, MeOH); 1H NMR (600 MHz, CD3OD) d 2.19 (s, 3H), 3.08
(dd, 1H, J = 4.3, 9.1 Hz), 3.8175 (s, 3H), 3.78–3.81 (m, 4H), 3.85
(dd, 1H, J = 4.1, 11.6 Hz), 4.01 (t, 1H, J = 4.9, 4.9 Hz), 4.10 (t, 1H,
J = 4.8, 5.1 Hz), 4.29 (d, 1H, J = 5.6 Hz), 6.81 (dd, 1H, J = 3.1,
8.9 Hz), 6.91 (d, 1H, J = 8.9 Hz), 7.01 (d, 1H, J = 3.0 Hz); 13C NMR
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71.4, 60.9, 56.7, 56.2, 50.0, 35.2; HRMS calcd for [C14H21NO5+H]+
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