New sugar-mimic alkaloids from the pods of Angylocalyx pynaertii

Article abstract of DOI:10.1021/np010360f

Chromatographic separation of the pod extract of Angylocalyx pynaertii resulted in the isolation of 13 sugar-mimic alkaloids (1-13). The structures of the new alkaloids were elucidated by spectroscopic methods as the 6-O-β-D-glucoside (10) and N-hydroxyethyl derivative (11) of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) (1), 1,6-dideoxynojirimycin (12), and 1,3,4-trideoxynojirimycin (13). 2,5-Imino-1,2,5-trideoxy-L-glucitol (7), 2,5-dideoxy-2,5-imino-D-fucitol (8), and β-homofuconojifimycin (9), isolated from the pods as well as the bark, were very specific inhibitors of α-L-fucosidase with no significant inhibitory activity toward other glycosidases. In this work, 1,4-dideoxy-1,4-imino-D-ribitol (6) was found to be a better inhibitor of lysosomal β-mannosidase than 2,5-imino-1,2,5-trideoxy-D-mannitol (2). N-Hydroxyethyl-1-deoxynojirimycin (miglitol), which is commercially available for the treatment of diabetes, retained its inhibitory potential toward rat intestinal maltase and sucrase, whereas 11 and the synthetic N-hydroxyethyl derivative of 2,5-dideoxy-2,5-imino-D-mannitol markedly lowered or abolished their inhibition toward all enzymes tested.

Full text of DOI:10.1021/np010360f

1
98
J . Nat. Prod. 2002, 65, 198-202
New Su ga r -Mim ic Alk a loid s fr om th e P od s of An gyloca lyx pyn a er tii
†
†
†
†
‡
#
Kayo Yasuda, Haruhisa Kizu, Toru Yamashita, Yukihiko Kameda, Atsushi Kato, Robert J . Nash,
|
§
,†
George W. J . Fleet, Russell J . Molyneux, and Naoki Asano*
Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-1181, J apan, Department of
Hospital Pharmacy, Toyama Medical and Pharmaceutical University, Toyama 930-0194, J apan, Institute of Grassland and
Environmental Research, Aberystwyth, Cardiganshire SY23 3EB, U.K., Dyson Perrins Laboratory, Oxford University, Oxford
OX1 3QY, U.K., and Western Regional Research Center, Agricultural Research Service, USDA, Albany, California 94710
Received J uly 20, 2001
Chromatographic separation of the pod extract of Angylocalyx pynaertii resulted in the isolation of 13
sugar-mimic alkaloids (1-13). The structures of the new alkaloids were elucidated by spectroscopic
methods as the 6-O-â-D-glucoside (10) and N-hydroxyethyl derivative (11) of 1,4-dideoxy-1,4-imino-D-
arabinitol (DAB) (1), 1,6-dideoxynojirimycin (12), and 1,3,4-trideoxynojirimycin (13). 2,5-Imino-1,2,5-
trideoxy-L-glucitol (7), 2,5-dideoxy-2,5-imino-D-fucitol (8), and â-homofuconojirimycin (9), isolated from
the pods as well as the bark, were very specific inhibitors of R-L-fucosidase with no significant inhibitory
activity toward other glycosidases. In this work, 1,4-dideoxy-1,4-imino-D-ribitol (6) was found to be a
better inhibitor of lysosomal â-mannosidase than 2,5-imino-1,2,5-trideoxy-D-mannitol (2). N-Hydroxyethyl-
1
-deoxynojirimycin (miglitol), which is commercially available for the treatment of diabetes, retained its
inhibitory potential toward rat intestinal maltase and sucrase, whereas 11 and the synthetic N-
hydroxyethyl derivative of 2,5-dideoxy-2,5-imino-D-mannitol markedly lowered or abolished their inhibition
toward all enzymes tested.
The genus Angylocalyx (Leguminosae) consists of seven
species of shrubs and trees growing in tropical African
1
forests. Six of them have been examined with regard to
alkaloidal composition, and 1,4-dideoxy-1,4-imino-D-ara-
binitol (DAB) (1) has been detected or isolated from all
examined, including Angylocalyx pynaertii DeWild.2 In
1
993, Molyneux et al. isolated 2,5-imino-1,2,5-trideoxy-D-
mannitol (6-deoxy-DMDP) (2), 1-deoxymannojirimycin (DMJ)
3), and fagomine (4), in addition to 1, from the seeds of A.
(
pynaertii and found that 2 is a weak competitive inhibitor
3
of â-mannosidase. In the course of a search for specific
lysosomal glycosidase inhibitors from plants, we found that
the extract of the bark of A. pynaertii shows a potent
inhibitory activity toward rat R-L-fucosidase (IC50 ) 3.1 µg/
mL) and reported that a thorough examination of the bark
extract resulted in the isolation of 15 sugar-mimic alka-
loids, including the known alkaloids 1, 2, 3, 2-O-â-D-
glucopyranosyl-DAB (5), N-methyl-DAB, 1,4-dideoxy-1,4-
imino-D-ribitol (DRB) (6), 1,4-dideoxy-1,4-imino-D-xylitol,
2
,5-imino-1,2,5-trideoxy-L-glucitol (7), 2,5-dideoxy-2,5-imino-
D-fucitol (8), 2,5-imino-1,2,5-trideoxy-D-altritol, N-methyl-
DMJ , 6-O-R-L-rhamnopyranosyl-DMJ (R-Rha-DMJ ), â-L-
homofuconojirimycin (â-HFJ ) (9), 1-deoxyaltronojirimycin,
4
and 1-deoxygulonojirimycin. We previously reported that
7
, R-Rha-DMJ , and 9 are very potent competitive inhibitors
of bovine epididymis R-L-fucosidase, with K values of 0.49,
i
4
0
.06, and 0.0053 µM, respectively.
In this paper, we describe the isolation of 13 sugar-mimic
alkaloids (1-13) from the pods of A. pynaertii, structural
determination of new alkaloids 10-13, and inhibitory
activities of the alkaloids obtained in this work and
chemically synthetic ones toward a variety of glycosidases.
Resu lts a n d Discu ssion
We previously reported that the bark extract treated
with Amberlite IR-120B (H form) and Dowex1-X2 (OH
*
To whom correspondence should be addressed. Fax: +81 76 229 2781.
+
-
E-mail: naoki22@po.incl.ne.jp.
†
form) showed a potent inhibitory activity toward rat R-L-
Hokuriku University.
Toyama Medical and Pharmaceutical University.
Institute of Grassland and Environmental Research.
Oxford University.
‡
4
fucosidase (IC50 ) 3.1 µg/mL). The 50% aqueous EtOH
#
|
extract of the pods was also similarly treated with ion-
exchange resins and tested for inhibitory activity of rat
§
Western Regional Research Center.
1
0.1021/np010360f CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/06/2002

Sugar-Mimic Alkaloids from Angylocalyx
J ournal of Natural Products, 2002, Vol. 65, No. 2 199
epididymis glycosidases. The pod extract showed a more
potent inhibitory activity (IC50 ) 0.23 µg/mL) than did the
bark extract toward rat epididymis R-L-fucosidase.
The pods (2.2 kg) of A. pynaertii were extracted with 50%
aqueous EtOH. Chromatographic separation of the extract
afforded 13 alkaloids (1-13). Alkaloids 1-9, other than 4,
were isolated from the pods as well. Alkaloid 4 was
determined to be fagomine from the optical rotation and
1
13
H NMR and C NMR spectral data. The structures of new
alkaloids 10-13 isolated from the pods were determined
from HRFABMS and various 1D and 2D NMR spectral
data. We report here the structural determination of new
alkaloids 10-13 and present full details on the structural
determination of 7 and 8, which were not reported in our
previous paper.
6 3
Alkaloid 7 showed the molecular formula C H13NO by
1
3
HRFABMS. The C NMR spectral data revealed the
presence of a single methyl (δ 15.4), a single methylene (δ
6
4.6), and four methine (δ 58.7, 68.5, 81.7, 82.3) carbon
atoms, suggesting that 7 was an isomer of 6-deoxy-DMDP
2). Irradiation of H-5 enhanced the NOE intensity of H-2
and H-3, and a definite NOE between H-4 and C-6 (CH
(
2
-
OH) protons was also observed. These results suggest that
H-2, H-3, H-4, and H-5 are in the R, R, â, and R orienta-
tions, respectively. Thus, alkaloid 7 was determined to be
2
,5-imino-1,2,5-trideoxy-L-glucitol or its enantiomer. In the
NMR spectra of 7, minor signals were always observed, and
no separations were observed using several chromato-
graphic techniques. Since the intensity of the minor signals
changed appreciably with different solvents and temper-
ature in the NMR measurement, these minor signals were
suggested to be due to those of the conformational isomer.
F igu r e 1. NOE interactions of 2,5-dideoxy-2,5-imino-D-fucitol (8) (a)
and 1,4-dideoxy-1,4-imino-(hydroxyethyliminiumyl)-D-arabinitol (11)
(b).
droxymethyl. The methine carbons at δ 78.3 and 81.7 were
assigned to C-2 and C-3 bearing OH groups, and the
methylene carbons at δ 59.1 and 62.2 to the N-hydroxyethyl
group. The C-4 methine at δ 74.9 and C-1 methylene at δ
61.6 carbons must be bonded to the nitrogen of the
pyrrolidine ring. Irradiation of H-4 enhanced the NOE
signal intensity of H-2 and H-1R, and the NOE effect
Alkaloid 8 showed the molecular formula C
HRFABMS. The 13C NMR spectral data (δ 15.2, 58.1, 62.8,
3.4, 79.3, 80.9) suggest that 8 is also an isomer of 6-deoxy-
6 3
H13NO by
6
DMDP. The definite NOEs between H-2 and H-3, between
H-3 and H-4, and between H-4 and H-5 indicate that H-2,
H-3, H-4, and H-5 are all in the R orientation (Figure 1a).
Thus, alkaloid 8 was determined to be 2,5-dideoxy-2,5-
imino-D-fucitol or its enantiomer.
between H-3 and the C-5 (CH OH) protons were also
2
observed (Figure 1b). These results suggest that H-2, H-3,
Alkaloid 10 showed the molecular formula C11
by HRFABMS. The response to the naphthoresorcinol-
sulfuric acid reagent and the characteristic anomeric proton
H
21NO
8
and H-4 are in the R, â, and R orientations, respectively.
Thus, alkaloid 11 was determined to be N-hydroxyethyl-
DAB. Furthermore, the NMR spectral data and specific
rotation of the N-hydroxyethyl derivative prepared by
heating DAB (1) with 2-bromoethanol in the presence of
anhydrous potassium carbonate in DMF were completely
in accord with those of the natural product. The 2-deoxy
derivative of 11, N-(hydroxyethyl)-2-(hydroxymethyl)-3-
hydroxypyrrolidine, has been isolated from the seeds of
Castanospermum australe.7
(
H-1′, δ 4.51, J ) 7.8 Hz) and carbon (C-1′, δ 105.6) signals
in the NMR suggested that 10 was a glycoside of an
alkaloid. Six signals (δ 63.7, 72.6, 76.1, 78.6, 78.9, 105.6)
in the 13C NMR spectrum were completely in accord with
those of the â-D-glucopyranosyl group in 6-O-â-D-glucopy-
5
ranosyl-1-deoxynojirimycin and 7-O-â-D-glucopyranosyl-
6
R-homonojirimycin. The strong NOEs from the anomeric
proton to the C-5 hydroxymethyl protons (H-5a and H-5b)
of the aglycone indicated that the â-glucosidic linkage site
is C-5. The aglycone part was determined to be DAB from
the decoupling and COSY experiments, the NOE effect
6 3
Alkaloid 12 showed the molecular formula C H13NO by
13
HRFABMS. C NMR spectral data revealed the presence
of a single methyl (δ 19.8), a single methylene (δ 51.5), and
four methine (δ 57.9, 73.9, 79.2, 80.9) carbon atoms.
Decoupling experiments and COSY spectra elucidated a
between H-3 and H-5b (δ 4.13) or H-1â (δ 3.26), and 13
C
NMR chemical shifts. Therefore, 10 was shown to be 5-O-
â-D-glucopyranosyl-DAB. The 7.8 ppm downfield shift for
C-5 and 1.3 ppm upfield shift for C-4 produced by the
â-glucoside formation also supported this structure.
CH
2
-CH-CH-CH-CH-CH
3
moiety, showing a linear
sequence from the C-1 methylene group to the C-6 methyl.
In the H NMR spectrum, the large J values (J 2,3 ) J 3,4
1
)
J
4,5 ) 9.3 Hz) seen in the signals of H-3 and H-4 indicated
Alkaloid 11 showed the molecular formula C
7
H
15NO
4
by
an all trans-axial orientation of H-2, H-3, H-4, and H-5.
HRFABMS. The 13C NMR spectra showed the presence of
three methine and four methylene carbon signals. The
complete connectivity of the carbon and hydrogen atoms
was defined from analysis of decoupling experiments and
COSY and HMBC spectra. These experiments elucidated
An NOE effect between H-1ax and H-3 or H-5 indicated
4
that the six-membered ring was in a C
1
chair conforma-
tion. Thus, alkaloid 12 was determined to be 1,6-dideoxy-
nojirimycin. Although an N,O-tribenzyl derivative of 12 has
been chemically synthesized as a structural unit of a
pseudooligosaccharide type of inhibitor, its free base has
not previously been characterized.8
a CH
2
-CH-CH-CH-CH
2
OH fragment, showing a linear
sequence from the C-1 methylene group to the C-5 hy-

2
00 J ournal of Natural Products, 2002, Vol. 65, No. 2
Yasuda et al.
Ta ble 1. Concentration of Sugar-Mimic Furanoses and the Glucosides Giving 50% Inhibition of Various Glycosidases
IC50 (µM)
enzyme
1
2
5
6
7
8
10
11
R-glucosidase
rice
120
1.2
55
16
5.8
-
-
-
-
-
-
1000
230
-
-
-
-
-
-
-
-
-
-
-
-
-
290
-
yeast
270
-
570
-
rat intestinal maltase
rat intestinal sucrase
rat intestinal isomaltase
R-mannosidase
rat epididymis
â-mannosidase
rat epididymis
R-galactosidase
coffee bean
â-galactosidase
bovine liver
R-L-fucosidase
bovine epididymis
amyloglucosidase
Aspergillus niger
-
-
95
-
-
250
84
17
150
290
-a
-
93
-
-
-
-
-
-
-
18
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
700
-
-
30
380
-
420
1
-
-
150
160
35
-
400
1000
510
-
620
a
-
indicates no inhibition (less than 50% inhibition at 1000 µM).
Ta ble 2. Concentration of 1,4-Dideoxy-1,4-imino-D-arabinitol (DAB), 2,5-Dideoxy-2,5-imino-D-mannitol (DMDP), 1-Deoxynojirimycin
(DNJ ), and Their N-Hydroxyethyl (N-HE) Derivatives Giving 50% Inhibition of Various Glycosidases
IC50 (µM)
enzyme
DAB (1)
N-HE-DAB (11)
DMDP
N-HE-DMDP
DNJ
N-HE-DNJ
R-glucosidase
rice
120
0.84
0.11
55
16
5.8
-b
78
12
-
250
84
300
2.0
2.0
290
-
1000
260
240
-
0.05
110
42
0.36
0.21
0.30
0.15
-
a
yeast maltase
yeast isomaltase
a
-
rat intestinal maltase
rat intestinal sucrase
rat intestinal isomaltase
R-mannosidase
rat epididymis
â-mannosidase
rat epididymis
â-galactosidase
bovine liver
trehalase
0.49
0.19
1.10
-
91
-
150
290
-
-
-
-
-
-
14
-
-
-
-
320
-
-
-
-
3.3
200
150
700
porcine kidney
2.5
41
a
Activities of yeast maltase and isomaltase were determined using maltose and isomaltose, respectively, as substrate at pH 6.8. The
released D-glucose was determined colorimetrically using Glucose B-test Wako. b - indicates no inhibition (less than 50% inhibition at
000 µM).
1
Alkaloid 13 showed the molecular formula C
6
H
13NO
2
by
tase.10,11 Recently, DAB has been found to be a potent
HRFABMS. 13C NMR spectral data revealed the presence
inhibitor of glycogen phosphorylase both in vitro and in
vivo.1
2,13
The N-methyl derivative of DAB was isolated from
of four methylene (δ 29.0, 34.8, 54.1, 67.7) and two methine
(
δ 59.1, 70.0) carbon atoms. Decoupling experiments,
COSY, and HMBC spectra revealed a CH -CH-CH
CH -CH-CH OH moiety. The coupling pattern of H-1ax
1ax,2 ) 10.2, J 1ax,1eq ) 11.5 Hz) indicated axial orientation
of H-2. An NOE effect between H-1ax and H-3 or H-5
the bark, while N-hydroxyethyl-DAB (11) was found in the
pods. The N-hydroxyethyl derivative of 1-deoxynojirimycin
2
2
-
2
2
(DNJ ), miglitol, is commercially available for the treatment
(J
of diabetes in several countries. We previously reported
that the introduction of the N-alkyl residues to furanose-
mimic alkaloids significantly lowered its inhibition toward
4
indicated that the six-membered ring was in a
conformation. Thus, alkaloid 13 was determined to be 1,3,4-
trideoxynojirimycin.
We previously reported that alkaloids 2, 3, 7, 8, and 9
are inhibitors of various R-L-fucosidases.4 Among these
alkaloids, 7 and 9 were very potent inhibitors of bovine
epididymis R-L-fucosidase, with IC50 values of 1 and 0.01
µM, respectively. The IC50 values of sugar-mimic furanoses
(
glycosidases are shown in Table 1. Although 6-deoxy-
DMDP (2) has been reported to be a weak inhibitor of snail
â-mannosidase, DRB (6) was a better inhibitor of lysoso-
mal â-mannosidase than 6-deoxy-DMDP. The isomers of
6
R-L-fucosidase with no significant inhibitory activity toward
other glycosidases. DAB (1) is known to be a potent
inhibitor of yeast R-glucosidase and mammalian isomal-
1
C chair
14
glycosidases. To investigate the change in potency and
specificity of bioactivity by the introduction of a polar
substituent to iminosugars, we prepared the N-hydroxy-
ethyl derivatives of DMDP and DNJ . As shown in Table 2,
the N-hydroxyethylation of DAB and DMDP markedly
lowered or abolished its inhibition toward all enzymes
tested. The derivatization of DNJ showed no change for
rat intestinal maltase and sucrase but slightly lowered its
potential toward rat isomaltase.
4
polyhydroxylated pyrrolidine alkaloids) toward various
3
Although DNJ is a very potent inhibitor of various
R-glucosidases,11 the deoxygenation at C-6 to form 12
completely abolished its inhibition toward all R-glucosi-
dases tested. This suggests a favorable interaction between
the C-6 hydroxyl group and the active site of R-glucosidase.
The 3,4-dideoxy derivative (13) of DNJ also showed no
-deoxy-DMDP, 7 and 8, were very specific inhibitors of
9

Sugar-Mimic Alkaloids from Angylocalyx
J ournal of Natural Products, 2002, Vol. 65, No. 2 201
significant inhibitory activity toward any glycosidases
tested.
dd, J ) 4.4, 10.7 Hz, H-5b), 4.21 (1H, ddd, J ) 3.9, 3.9, 5.6
1
3
Hz, H-2), 4.51 (1H, d, J ) 7.8 Hz, H-1′); C NMR (100 MHz,
D
2
O) δ 53.4 (C-1), 63.7 (C-6′), 66.7 (C-4), 72.6 (C-5), 72.7 (C-
4
1
′), 76.2 (C-2′), 78.6 (C-3′), 78.9 (C-5′), 79.7 (C-2), 81.3 (C-3),
05.6 (C-1′); HRFABMS m/z 296. 1342 [M + H] (C11H22NO
8
Exp er im en ta l Section
+
Gen er al Exper im en tal P r ocedu r es. The purity of samples
was checked by HPTLC on silica gel 60F254 (E. Merck) using
requires 296.1346).
1,4-Did eoxy-1,4-im in o-(h yd r oxyeth ylim in iu m yl)-D-a r a -
1
the solvent system PrOH-AcOH-H
2
O (4:1:1), and a chlorine-
bin itol (11): [R]D +54.7° (c 0.38, H O); H NMR (400 MHz,
2
o-tolidine reagent was used for detection. Optical rotations
D
2
O) δ 2.58 (1H, m, N-CH -), 2.61 (1H, m, H-4), 2.82 (1H, dd,
2
1
were measured with a J asco DIP-370 digital polarimeter. H
J ) 5.9, 11.2 Hz, H-1a), 3.02 (1H, m, N-CH
dd, J ) 2.2, 11.2 Hz, H-1b), 3.69-3.73 (4H, 2 × CH
(1H, br dd, J ) 2.9, 5.2 Hz, H-3), 4.13 (1H, ddd, J ) 2.2, 2.9,
.9 Hz, H-2); 13C NMR (100 MHz, D
C-1), 62.2 (N-CH -CH OH), 63.7 (C-5), 74.9 (C-4), 78.3 (C-2),
1.7 (C-3); HRFABMS m/z 178.1079 [M + H] (C
2
-), 3.08 (1H, br
1
3
NMR (400 MHz) and C NMR (100 MHz) spectra were
recorded on a J EOL J NM-GX 400 spectrometer. Chemical
shifts are expressed in ppm downfield from sodium 3-(trim-
2
OH), 3.95
5
2
O) δ 59.1 (N-CH -), 61.6
2
(
8
2
2
2
ethylsilyl)propionate (TSP) in D O as an internal standard.
+
7 4
H16NO
The assignment of proton and carbon signals in NMR was
determined from extensive homonuclear decoupling experi-
requires 178.1079).
N-Hyd r oxyeth yla tion of Im in osu ga r s. 2-Bromoethanol
0.2 mL) and anhydrous K CO (200 mg) were added to a
1
13
ments, DEPT, H- C COSY, and HMBC spectral data.
FABMS were measured using glycerol as a matrix on a J EOL
J MS-SX 102A spectrometer.
(
2
3
solution of DAB (1) (100 mg) in DMF. The reaction mixture
was heated overnight at 60 °C and diluted with MeOH. The
diluted solution was applied to an Amberlyst 15 (20 mL)
P la n t Ma ter ia ls. The pods of A. pynaertii were collected
3
on J une 8, 1988, in the Republic of Cameroon. The voucher
column, washed with MeOH, eluted with 1 M NH
4
OH, and
specimen (No. 3738415) is deposited in the Missouri Botanical
Garden Herbarium. The dried plant materials were pulverized
with a ball mill to produce powder.
concentrated. The N-hydroxyethylated derivative was purified
-
+
4
form)
by Dowex 1-X2 (OH form) and Amberlite CG-50 (NH
chromatography with water as eluant to give 5 (95 mg) as a
colorless oil. The N-hydroxyethylated derivatives of DMDP and
DNJ were also synthesized in a similar manner.
Extr a ction a n d Isola tion . The pod powder (2.2 kg) of A.
pynaertii was extracted three times with 6 L of 50% aqueous
EtOH. The extract was applied to a column of Amberlite IR-
2
,5-Dideoxy-2,5-im in o-(h ydr oxyeth ylim in iu m yl)-D-m an -
n itol (N-h yd r oxyeth yl-DMDP ): [R] -39.4° (c 0.89, H O);
-), 62.1 (C-1,6), 62.3
OH), 70.8 (C-2,5), 81.1 (C-3,4); HRFABMS m/z
1
20B (500 mL, H+ form). The 0.5 M NH
4
OH eluate was
D
2
concentrated to give a brown syrup (46 g), which was chro-
13
C NMR (100 MHz, D
N-CH -CH
08.1183 [M + H] (C
,5-Did eoxy-1,5-im in o-(h yd r oxyeth ylim in iu m yl)-D-glu -
citol (N-h yd r oxyeth yl-1-d eoxyn ojir im ycin , m iglitol): [R]
2
O) δ 51.0 (N-CH
2
matographed over an Amberlite CG-50 column (3.8 × 95 cm,
(
2
2
2
+
NH
4
form) with H
2
O as eluant (fraction size 15 mL). Fractions
+
8 5
H18NO requires 208.1185).
2
8-63 and 64-90 were collected and concentrated to give 350
1
mg (pool I) and 580 mg (pool II), respectively. The 0.5 M NH
4
-
D
OH eluate from the same column was concentrated to give a
brown syrup of 3.45 g (pool III). Each pool was further
13
-
)
(
11.0° (c 0.55, H
, 58.9 (C-1), 60.4 (C-6), 60.7 (N-CH
C-2), 72.8 (C-4), 81.1 (C-3); HRFABMS m/z 208.1183 [M +
2
O); C NMR (100 MHz, D
2
O) δ 55.7 (N-CH -
2
2
-CH OH), 68.5 (C-5), 71.6
2
-
chromatographed with Dowex 1-X2 (OH form) and/or CM-
+
Sephadex C-25 (NH
4
form) columns to give alkaloids 11 (6
+
H] (C
8
H
18NO
5
requires 208.1185).
,6-Did eoxyn ojir im ycin (12): [R]
H NMR (400 MHz, D O) δ 1.16 (3H, d, J ) 6.4 Hz, CH
1H, dd, J ) 10.7, 12.5 Hz, H-1ax), 2.52 (1H, dq, J ) 6.4, 9.3
mg) from pool I, 3 (565 mg) and 12 (4 mg) from pool II, and 1
1.03 g), 2 (51 mg), 4 (10 mg), 5 (22 mg), 6 (5 mg), 7 (729 mg),
1
D
+37.8° (c 0.26, H
2
O);
), 2.46
(
1
2
3
8
(6 mg), 9 (36 mg), 10 (3 mg), and 13 (5 mg) from pool III.
,5-Im in o-1,2,5-tr id eoxy-D-glu citol (7): [R] +23.3° (c
O) the major conformer, δ
), 3.01 (1H, ddd, J ) 4.9, 4.9, 6.4
(
2
D
Hz, H-5), 3.01 (1H, dd, J ) 9.3, 9.3 Hz, H-4), 3.07 (1H, dd, J
5.1, 12.5 Hz, H-1eq), 3.28 (1H, dd, J ) 9.3, 9.3 Hz, H-3),
1
0
1
.64, H
2 2
O); H NMR (400 MHz, D
)
3
D
8
.16 (3H, d, J ) 6.8 Hz, CH
3
13
.51 (1H, ddd, J ) 5.1, 9.3, 10.7 Hz, H-2); C NMR (100 MHz,
O) δ 19.8 (C-6), 51.5 (C-1), 57.9 (C-5), 73.9 (C-2), 79.2 (C-4),
Hz, H-5), 3.32 (1H, dq, J ) 4.4, 6.8 Hz, H-2), 3.68 (1H, dd, J
6.4, 11.5 Hz, H-6a), 3.76 (1H, dd, J ) 4.9, 11.5 Hz, H-6b),
.88 (1H, dd, J ) 2.2, 4.9 Hz, H-4), 3.96 (1H, dd, J ) 2.2, 4.4
),
.54 (1H, ddd, J ) 3.4, 5.9, 6.8 Hz, H-5), 3.65 (1H, dd, J ) 5.9,
1.5 Hz, H-6a), 3.87 (1H, dd, J ) 3.4, 11.5 Hz, H-6b), 3.94 (1H,
2
)
3
+
0.9 (C-3); HRFABMS m/z 148.0973 [M + H] (C
6
H
14NO
3
requires 148.0974).
Hz, H-3); the minor conformer, δ 1.07 (3H, d, J ) 6.6 Hz, CH
3
1
3
1
,3,4-Tr id eoxyn ojir im ycin (13): [R]
D
+36.6° (c 0.23, H
2
O);
1
H NMR (400 MHz, D O) δ 1.18 (1H, m, H-4ax), 1.35 (1H, m,
2
H-3ax), 1.76 (1H, m, H-4eq), 2.07 (1H, m, H-3eq), 2.40 (1H,
dd, J ) 10.2, 11.5 Hz, H-1ax), 2.63 (1H, m, H-5), 3.16 (1H,
ddd, J ) 2.2, 4.6, 11.5 Hz, H-1eq), 3.43 (1H, dd, J ) 7.3, 11.3
Hz, H-6a), 3.55 (1H, dd, J ) 4.9, 11.3 Hz, H-6b), 3.65 (1H, m,
dd, J ) 6.8, 8.1 Hz, H-4), 4.03 (1H, dq, J ) 6.6, 7.3 Hz, H-2),
.05 (1H, dd, J ) 7.3, 8.1 Hz, H-3); 13C NMR (100 MHz, D
O)
the major conformer, δ 15.4 (C-1), 58.7 (C-2), 64.6 (C-6), 68.5
C-5), 81.7 (C-3), 82.3 (C-4); the minor conformer, δ 17.9 (C-
), 57.3 (C-2), 65.9 (C-5), 66.6 (C-6), 77.6 (C-3), 78.3 (C-4);
4
2
(
13
H-2); C NMR (100 MHz, D
2
O) δ 29.0 (C-4), 34.8 (C-3), 54.1
1
(
1
C-1), 59.1 (C-5), 67.7 (C-6), 70.0 (C-2); HRFABMS m/z
32.1024 [M + H] (C
Glycosid a se In h ibitor y Activities. The enzymes R-glu-
+
HRFABMS m/z 148.0972 [M + H] (C
1
6
H
14NO
3
requires
+
6 2
H14NO requires 132.1025).
48.0974).
2
,5-Did eoxy-2,5-im in o-D-fu citol (8): [R]
D
-11.1° (c 0.28,
O) δ 1.19 (3H, d, J ) 6.8 Hz, CH ),
cosidases (from rice and yeast), R-galactosidase (from coffee
beans), â-galactosidase (from bovine liver), amyloglucosidase
(from Aspergillus niger), p-nitrophenyl glycosides, and disac-
charides were purchased from Sigma Chemical Co. The rat
epididymal fluid was purified from epididymis according to the
1
H
3
7
2
O); H NMR (400 MHz, D
2
3
.53 (1H, dq, J ) 3.7, 6.8 Hz, H-2), 3.60 (1H, ddd, J ) 4.6, 6.4,
.1 Hz, H-5), 3.69 (1H, dd, J ) 7.1, 11.2 Hz, H-6a), 3.81 (1H,
dd, J ) 6.4, 11.2 Hz, H-6b), 4.02 (1H, dd, J ) 2.0, 3.7 Hz, H-3),
.27 (1H, dd, J ) 2.0, 4.6 Hz, H-4); 13C NMR (100 MHz, D
δ 15.2 (C-1), 58.1 (C-2), 62.8 (C-6), 63.4 (C-5), 79.3 (C-4), 80.9
O)
15
4
2
method of Skudlarek et al. Brush border membranes pre-
pared from rat small intestine according to the method of
+
15
(
1
C-3); HRFABMS m/z 148.0973 [M + H] (C
48.0974).
,4-Did eoxy-1,4-im in o-(5-O-â-D-glu cop yr a n osyl)-D-a r a -
6
H
14NO
3
requires
Kessler et al. were used as the enzyme source of rat intestinal
maltase, sucrase, and isomaltase. The activity of rice R-glu-
cosidase, rat intestinal glycosidases, and amyloglucosidase was
determined using the appropriate disaccharides as substrates
at the optimum pH of each enzyme. The released D-glucose
was determined colorimetrically using the Glucose B-test
Wako (Wako Pure Chemical Ind.). Other glycosidase activities
were determined using an appropriate p-nitrophenyl glycoside
as a substrate at the optimum pH of each enzyme. The reaction
1
1
bin itol (10): [R] +40.1° (c 0.16, H2O); H NMR (400 MHz,
D
D O) δ 2.97 (1H, br dd, J ) 3.9, 12.5 Hz, H-1a), 3.26 (1H, dd,
2
J ) 5.6, 12.5 Hz, H-1b), 3.26-3.30 (1H, m, H-4), 3.33 (1H, dd,
J ) 7.8, 9.3 Hz, H-2′), 3.40 (1H, dd, J ) 9.0, 9.8 Hz, H-4′),
3
9
(
.49 (1H, ddd, J ) 2.2, 5.9, 9.8 Hz, H-5′), 3.52 (1H, dd, J )
.0, 9.3 Hz, H-3′), 3.74 (1H, dd, J ) 5.9, 12.5 Hz, H-6′a), 3.82
1H, dd, J ) 7.6, 10.7 Hz, H-5a), 3.93 (1H, dd, J ) 2.2, 12.5
was stopped by adding 400 mM Na
2 3
CO . The released p-
Hz, H-6′b), 3.97 (1H, br, dd, J ) 3.9, 4.9 Hz, H-3), 4.13 (1H,
nitrophenol was measured spectrometrically at 400 nm.

2
02 J ournal of Natural Products, 2002, Vol. 65, No. 2
Yasuda et al.
Ack n ow led gm en t. This work was supported in part by
the Special Research Fund of Hokuriku University (N.A.).
(8) Liotta, L. J .; Bernotas, R. C.; Wilson, D. B.; Ganem, B. J . Am. Chem.
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L. E.; Nash, R. J . Tetrahedron Lett. 1985, 26, 3127-3130.
10) Scofield, A. M.; Fellows, L. E.; Nash, R. J .; Fleet, G. W. Life Sci. 1986,
Refer en ces a n d Notes
(
(
(
(
(
(
(
(
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(
(
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9
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NP010360F