Synthesis and glycosidase inhibition of broussonetine M and its analogues

Article abstract of DOI:10.3390/molecules24203712

Cross-metathesis (CM) and Keck asymmetric allylation, which allows access to defined stereochemistry of a remote side chain hydroxyl group, are the key steps in a versatile synthesis of broussonetine M (3) from the D-arabinose-derived cyclic nitrone 14. By a similar strategy, ent-broussonetine M (ent-3) and six other stereoisomers have been synthesized, respectively, starting from L-arabino-nitrone (ent-14), L-lyxo-nitrone (ent-3-epi-14), and L-xylo-nitrone (2-epi-14) in five steps, in 26%–31% overall yield. The natural product broussonetine M (3) and 10’-epi-3 were potent inhibitors of β-glucosidase (IC50 = 6.3 μM and 0.8 μM, respectively) and β-galactosidase (IC50 = 2.3 μM and 0.2 μM, respectively); while their enantiomers, ent-3 and ent-10’-epi-3, were selective and potent inhibitors of rice α-glucosidase (IC50 = 1.2 μM and 1.3 μM, respectively) and rat intestinal maltase (IC50 = 0.29 μM and 18 μM, respectively). Both the configuration of the polyhydroxylated pyrrolidine ring and C-10’ hydroxyl on the alkyl side chain affect the specificity and potency of glycosidase inhibition.

Full text of DOI:10.3390/molecules24203712

molecules
Article
Synthesis and Glycosidase Inhibition of Broussonetine M
and Its Analogues
Qing-Kun Wu 1,2, Kyoko Kinami 3, Atsushi Kato 3,
* ,
, Yi-Xian Li 1,2, Yue-Mei Jia 1,2
George W. J. Fleet 4,5 and Chu-Yi Yu 1,2,5,
*
1
Beijing National Laboratory for Molecular Science (BNLMS), CAS Key Laboratory of Molecular Recognition
and Function, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China;
lsqkwu@iccas.ac.cn (Q.-K.W.); tamarali@iccas.ac.cn (Y.-X.L.); jiayuemei@iccas.ac.cn (Y.-M.J.)
University of Chinese Academy of Sciences, Beijing 100049, China
Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930–0194, Japan;
kimagure1242@yahoo.co.jp
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road,
Oxford OX13TA, UK; george.fleet@chem.ox.ac.uk
National Engineering Research Center for Carbohydrate Synthesis, Jiangxi Normal University,
Nanchang 330022, China
2
3
4
5
*
Correspondence: kato@med.u-toyama.ac.jp (A.K.); yucy@iccas.ac.cn (C.-Y.Y.); Tel.: +86-010-626-12893 (C.-Y.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Ramón J. Estévez Cabanas
Received: 17 September 2019; Accepted: 14 October 2019; Published: 15 October 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Cross-metathesis (CM) and Keck asymmetric allylation, which allows access to defined
stereochemistry of a remote side chain hydroxyl group, are the key steps in a versatile synthesis of
broussonetine M (
M (ent-3) and six other stereoisomers have been synthesized, respectively, starting from l-arabino-nitrone
ent-14), l-lyxo-nitrone (ent-3-epi-14), and l-xylo-nitrone (2-epi-14) in five steps, in 26%–31% overall
yield. The natural product broussonetine M ( ) and 10’-epi-3 were potent inhibitors of -glucosidase
(IC50 = 6.3 µM and 0.8 M, respectively) and -galactosidase (IC50 = 2.3 µM and 0.2 M, respectively);
while their enantiomers, ent-3 and ent-10’-epi-3, were selective and potent inhibitors of rice -glucosidase
(IC50 =1.2 Mand1.3 M, respectively)andratintestinalmaltase(IC50 = 0.29 µMand18 M, respectively).
3) from the d-arabinose-derived cyclic nitrone 14. By a similar strategy, ent-broussonetine
(
3
β
µ
β
µ
α
µ
µ
µ
Both the configuration of the polyhydroxylated pyrrolidine ring and C-10’ hydroxyl on the alkyl side
chain affect the specificity and potency of glycosidase inhibition.
Keywords: broussonetine M; analogue; synthesis; glycosidase inhibition; structure-activity relationship
1. Introduction
In the past several decades, numerous polyhydroxylated pyrrolidine iminosugars have been isolated
and synthesized, which has enriched the extended family of iminosugars [
together with their synthetic analogues, have exhibited a variety of important biological activities and
shown great potential as chemotherapeutic agents for an ever widening number of diseases [ ]. DAB
(1,4-dideoxy-1,4-imino-d-arabinitol, ) [ ] and DMDP (2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine,
) [10] are among the most widespread and studied iminosugars (Figure 1). DAB ( ), isolated in 1985,
is a powerful -glucosidase inhibitor [11 15], whereas DMDP ( ) is a potent inhibitor of bovine liver
-glucosidase and -galactosidase (IC50 = 9.7 M and 3.3 M, respectively) [16]. DAB ( ) was also
1–7]; these natural products,
6,8
1
9
2
1
α
–
2
β
β
µ
µ
1
found to be a potent inhibitor of glycogen phosphorylase, which made it a potential therapeutic agent
for the treatment of diabetes [17]. In contrast, the enantiomers LAB (1,4-dideoxy-1,4-imino-l-arabinitol)
(ent-1) [15] and l-DMDP (ent-2) are both potent and specific α-glucosidase inhibitors [16,18].
Molecules 2019, 24, 3712; doi:10.3390/molecules24203712
www.mdpi.com/journal/molecules

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Figure 1. Structures of DAB, DMDP, and their related derivatives.
Many efforts have been devoted to the synthesis and glycosidase inhibition studies of DAB
) [19 20] and various analogues [13 21 28]. Among these studies, the biological evaluation results of
-C-1-alkyl-DABs and their l-enantiomers [29] attracted our attention. While -C-1-alkyl-DABs shifted
to be -glucosidase inhibitors with improved potency as the length of the C-1 alkyl chain increased,
-C-1-alkyl-LABs upheld similar -glucosidase inhibitory activities to the parent compound. The above
(1
,
, –
α
α
β
α
α
trend in changes of glycosidase inhibition reminded us of a special class of polyhydroxylated pyrrolidine
alkaloids, broussonetines, which are a subgroup of natural iminosugars with more than 30 members
(Figure 2). Broussonetines were isolated from the branches of the deciduous tree Broussonetia Kazinoki
SIEB, which is widely distributed in several Asian countries and has been used as a folk medicine [30–37].
Most broussonetines share a common (2R, 3R, 4R, 5R)-pyrrolidine moiety with a 13-carbon chain
containing various functional groups on the C-5 position. Therefore, they can be structurally regarded as
DAB-related iminosugars or
activities, broussonetines are better to be considered as analogues of DMDP (
found to be good -glucosidase and -galactosidase inhibitors. For example, broussonetine E (
F ( ), and G ( ) show potent -glycosidase inhibition and have therapeutic potential as antitumor and
anti-HIV agents [38]. Broussonetine M ( ), isolated in 2000 by Kusano’s group, was a potent inhibitor of
α
-1-C-alkylated-DABs. However, with respect to glycosidase inhibitory
), of which most were
),
2
β
β
4
5
6
β
3
β-galactosidase from bovine liver (IC50 = 8.1 µM) [36].
According to our previous study on the synthesis and glycosidase inhibition of broussonetines [39,40],
enantiomers of the above broussonetines would probably demonstrate a similar inhibitory profile by
analogy with LAB (ent-1) and l-DMDP (ent-2), and as such [22
potential in the treatment of type II diabetes, cancers, and viral infections [43
broussonetine M ( ) was selected as the research objective; synthesis and glycosidase inhibition of the
,
26
,41
,
42], these compounds may have
–
46]. Hence, in this work,
3
natural product and its analogues, including l-enantiomers and pyrrolidine core stereoisomers, were
finished, aiming for a better understanding of structure–activity relationship (SAR) of this interesting
class of pyrrolidine iminosugars.
Figure 2. Some representative broussonetines.

Molecules 2019, 24, 3712
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Though the first synthesis of broussonetine M (3) was accomplished with d-serine as the starting
material [47], we have shown that most broussonetines can be efficiently constructed via a general synthetic
strategy employing sugar-derived cyclic nitrones [39 40]. The pyrrolidine core of this class of iminosugars
can be derived from cyclic sugar nitrones [44 48 50] with the corresponding stereochemistry in the
hydroxylated pyrrolidine ring, while the various side chains could be installed via cross-metathesis (CM)
reactions [46 47 51 52]. This general strategy is capable of synthesizing a number of natural broussonetines,
as well as a variety of broussonetine analogues for SAR study; it has been successful in the synthesis of
broussonetine I ( ), J2 (10), and W (11) [39 40]. Therefore, this strategy was applied in the construction of
,
,
–
,
, ,
7
,
broussonetine M (3) and its analogues.
2. Results and Discussion
2.1. Synthesis of Broussonetine M
Our retrosynthesis for broussonetine M (
M was obtained by the CM reaction between the pyrrolidine 13 and the alcohol 15. The pyrrolidine 13 was
conveniently prepared from d-arabinose-derived cyclic nitrone 14 53 57]. The alcohol 15, which contains
3) is presented in Scheme 1. The precursor 12 of broussonetine
[
–
one stereocenter, was synthesized through asymmetric Keck allylation of aldehyde 16. In this synthetic
route, only the stereocenter in alcohol 15 is constructed by virtue of an asymmetric reaction; of the four
stereocenters on the pyrrolidine ring, three were determined by sugar-derived cyclic starting nitrone 14
and the fourth was formed by the high diastereoselectivity of organometallic addition to the nitrone 14.
Scheme 1. Retrosynthesis of broussonetine M (3) from d-arabino-nitrone (14).
For the synthesis of broussonetine M (3), addition of Grignard reagent 18, prepared from
8-bromo-1-octene, to d-arabino-nitrone (14) afforded the hydroxylamine 19 in high yield and excellent
diastereoselectivity; none of the other diastereomers were formed (Scheme 2) [54]. Due to its chemical
instability, the hydroxylamine 19 was directly used in the next step of reaction without further purification.
SuccessivezincreductionandN-Cbzprotectionofthecrudehydroxylamine19providedthepyrrolidine13
,
as the required CM reaction precursor, in 64% overall yield in three steps. The configuration of the newly
formed C-5 chiral center was determined as R by nuclear Overhauser effects experiment on 19, with the
observation of the strong correlation between H-5 and H-6a, H-6b (Scheme 2).
Scheme 2. Synthesis of the pyrrolidine core 13.

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The key step in the synthesis of the chiral alkyl alcohol 15 was the asymmetric Keck allylation
(Scheme 3) [58]. Treatment of butyl glycol 20 with BnBr/NaH/TBAI in DMF-THF gave the mono-O-benzylated
alcohol 21 in 91% yield. Swern oxidation of alcohol 21 afforded aldehyde 16 (89%), which on enantioselective
Keck allylation using (S)-BINOL produced alcohol 15 (93% ee) [59]. Using (R)-BINOL as the ligand, alcohol
ent-15 was also prepared by the same method (See the Supplementary Materials for HPLC analysis of
compounds 15 and ent-15).
Scheme 3. Synthesis of the side chain.
CM reaction between pyrrolidine 13 and alcohol 15 completed the synthesis of broussonetine M (3).
In this case, the free hydroxyl group of the alcohol 15 was tolerated for the CM reaction with no need for
O-protection. CM reaction of pyrrolidine 13 and alcohol 15 promoted by Grubbs II catalyst produced olefin
12 as an inseparable Z/E mixture in moderate yield (43%). Pd/C-catalyzed hydrogenation of compound
12 in acidic methanol afforded the target product broussonetine M (
Thus, broussonetine M ( ) was synthesized in five linear steps starting from d-arabino-nitrone (14) in
28% total yield. The 1H- and 13C-NMR spectra and the specific rotation of the synthetic broussonetine
M ( ) were all consistent with those reported for the synthetic broussonetine M (3) 47], but had some
differences with those of natural products [36] (See the Supplementary Materials for comparison of NMR
data). Since the structure and configuration of product were ensured by all the synthetic materials and
3) in quantitative yield (Scheme 4).
3
3
[
3
procedures, as in the work of Alberto Marco et al. [47], the NMR spectra differences may be explained by
minute pH variation or metal impurities [60,61].
Scheme 4. Completion of the synthesis of broussonetine M (3).
2.2. Synthesis of Analogues of Broussonetine M
Glycosidase inhibition by pyrrolidine iminosugars are among those that are most difficult to
predict [16]; minor modification of the iminosugar can lead to a distinct change of the inhibition
profile. In order to explore the preliminary structure–activity relationship of this type of iminosugar,
seven analogues of broussonetine M (
then prepared. By the same strategy as that for pyrrolidine 13, synthesis of ent-13
2-epi-13 were accomplished from the corresponding sugar-derived cyclic nitrones ent-14
and 2-epi-14. The configurations of the newly constructed chiral centers in these compounds were
all confirmed by NOE experiments. With these pyrrolidines in hand, CM reaction with alcohol 15
and subsequent hydrogenation, provided the target products, i.e., ent-10’-epi-3 ent-3,10’-di-epi-3, and
2-epi-3, of which, the C10’-hydroxyls retained S configuration as that of the natural product (Table 1).
3
), including its l-enantiomer and other six stereoisomers, were
ent-3-epi-13, and
ent-3-epi-14
,
,
,
,
,

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In order to evaluate the influence of the C10’-hydroxyl on glycosidase inhibition, the C10’-epimers
of broussonetine M ( ), including 10’-epi-3 ent-3 ent-3-epi-3, and 2,10’-di-epi-3, were also synthesized
3
,
,
from the above four pyrrolidines and alcohol ent-15 by the same strategy.
Table 1. Broussonetine M (3) and analogues synthesized from different cyclic nitrones and alcohols.
Cyclic
Nitrone
Yield b
Entry
Pyrrolidine
Yield a
Alcohol
Product
1
43%
64%
2
3
4
5
6
7
8
43%
41%
45%
40%
41%
43%
43%
65%
64%
71%
a
b
Total yield in 3 steps starting from cyclic nitrones to the corresponding pyrrolidine. Total yield in 2 steps starting from
pyrrolidine cores to broussonetine M or its analogues.
2.3. Glycosidase Inhibition
The synthetic broussonetine M (3) and its analogues were all assayed as potential glycosidase
inhibitors of a range of enzymes, as shown in Tables 2 and 3.
According to the results, all the compounds showed moderate to potent inhibition of β-galactosidase
from bovine liver; 10’-epi-3 was the most potent inhibitor (IC50 = 0.2
M (3) and 10’-epi-3 showed potent inhibition toward -glucosidase (IC50 = 6.3 µM and 0.8
and -galactosidase (IC50 = 2.3 M and 0.2 M, respectively) from bovine liver, which were consistent
with DMDP ( ) and other DAB or DMDP derivatives with a long alkyl side chain on C-1 position [29].
However, different from DMDP ( ), compounds and 10’-epi-3 lost their inhibitions against -glucosidase,
-mannosidase, and trehalase, whereas they showed moderate -glucuronidase inhibition. Furthermore,
µM). The natural product broussonetine
β
µM, respectively)
β
µ
µ
2
2
3
α
α
β
10’-epi-3 with C-10’ having R configuration was about 4 times better as an inhibitor than broussonetine M
(3) with C-10’ having S configuration.

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Table 2. Concentrations of Broussonetine M (3) and its DMDP-related analogs giving 50% inhibition of various enzymes.
IC50 (µM)
Enzyme
DAB (1) [15]
DMDP (2) [16]
3
10’-epi-3
LAB (ent-1) [15]
l-DMDP (ent-2) [16]
ent-10’-epi-3
ent-3
α-Glucosidase
Yeast
NI a (15.6%) b
214
0.15
250
NI (2.6%)
NI (0%)
NI (4.6%)
NI (0%)
70
3.2
NI (34.9%)
5.8
NI (1.9%)
1.3
NI (1.9%)
1.2
Rice
Rat intestinal
maltase
55
290
NI (0%)
NI (26.4%)
0.93
1.2
0.18
0.29
β-Glucosidase
Almond
250
638
10
9.7
NI (10.0%)
6.3
NI (29.1%)
0.8
NI
NI (13%)
NI (12.1%)
NI (4.6%)
NI (8.0%)
51
NI (9.6%)
NI (46.5%)
Bovine liver
α-Galactosidase
Coffee beans
β-Galactosidase
Bovine liver
α-Mannosidase
Jack bean
NI (7%)
NI (37%)
320
NI (10.5%)
3.3
NI (0%)
2.3
NI (0%)
0.2
NI (2%)
NI (16%)
NI (0%)
NI (1%)
NI (0%)
75
NI (13.8%)
NI (0%)
NI (17.3%)
NI (12.6%)
NI (0%)
48
NI (12.2%)
9.0
NI (0.3%)
50
NI (31.5%)
721
NI (2.5%)
NI (1.7%)
NI (0%)
NI (0%)
NI (3.8%)
NI (6.1%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (36.5%)
NI (4.7%)
NI (1.8%)
NI (2.0%)
NI (6.1%)
NI (0%)
NI (2.2%)
NI (0%)
NI (0%)
NI (2.1%)
NI (0%)
NI (13.3%)
β-Mannosidase
Snail
NI (10%)
NI (11%)
61
α-l-Fucosidase
Bovine kidney
α,α-Trehalase
Porcine kidney
Amyloglucosidase
A. niger
α-l-Rhamnosidase
P. decumbens
β-Glucuronidase
E.coli
NI (37.2%)
200
362
—
NI (41%)
803
—
NI (6.1%)
NI (1.8%)
NI (5%)
NI (24.1%)
NI (45.6%)
c
—
NI (0.4%)
—
86
20
—
—
NI (0.8%)
—
NI (36.2%)
NI (14.9%)
NI (44.3%)
NI (2.3%)
Bovine liver
—
NI (18.4%)
NI (17.2%)
a
NI: No inhibition (less than 50% at 100 µM for broussonetine M and its analogs; less than 50% at 1000 µ µM for broussonetine M
M for DAB, DMDP, LAB, and l-DMDP); b ( ): inhibition % at 100
and its analogs; inhibition % at 1000 µM for DMDP and l-DMDP; c —: not determined.

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Table 3. Concentrations of Broussonetine M’s analogs related with l-altro-DMDP and d-gluco-DMDP giving 50% inhibition of various enzymes.
IC50 (µM)
Enzyme
l-altro-DMDP [16]
ent-3,10-di-epi-3
ent-3-epi-3
d-gluco-DMDP [16]
2-epi-3
2,10-di-epi-3
α-Glucosidase
Yeast
NI a (35.1%) b
NI (9.5%)
754
NI (6.6%)
NI (9.5%)
84
NI (0%)
NI (23.0%)
42
167
131
138
NI (1.6%)
NI (5.6%)
NI (16.8%)
NI (3.2%)
Rice
12
1.4
Rat intestinal maltase
β-Glucosidase
Almond
Bovine liver
α-Galactosidase
Coffee beans
β-Galactosidase
Bovine liver
α-Mannosidase
Jack bean
NI (15.9%)
NI (10.3%)
NI (7.3%)
68
NI (4.6%)
71
256
523
NI (8.6%)
23
NI (10.9%)
33.9
120
NI (5.9%)
NI (37.6%)
NI (3.7%)
205
NI (0%)
31
NI (0%)
41
NI (38.4%)
361
NI (0%)
7.4
NI (0%)
13
NI (0%)
NI (2.3%)
98
NI (1.3%)
NI (0%)
50
NI (13.3%)
NI (14.6%)
NI (37.2%)
379
NI (0%)
NI (0%)
NI (4.3%)
NI (0%)
NI (3.8%)
NI (3.3%)
NI (0.32%)
NI (0%)
NI (10.8%)
NI (0%)
NI (6.8%)
NI (4.7%)
β-Mannosidase
Snail
α- l-Fucosidase
Bovine kidney
α,α-Trehalase
Porcine kidney
Amyloglucosidase
A. niger
α-l-Rhamnosidase
P. decumbens
β-Glucuronidase
E.coli
1000
NI (3.8%)
NI (0%)
NI (11.4%)
NI (0%)
NI (0%)
NI (15.2%)
c
—
—
91
NI (24.1%)
NI (2.0%)
—
42
NI (28.7%)
NI (0%)
NI (5.1%)
—
73
80
Bovine liver
NI (14.9%)
NI (5.2%)
NI (1.7%)
a
NI: No inhibition (less than 50% at 100 µM for broussonetine M’s analogs; less than 50% at 1000 µ µM for broussonetine M’s analogs;
M for l-altro-DMDP and d-gluco-DMDP); b ( ): inhibition % at 100
inhibition % at 1000 µM for l-altro-DMDP and d-gluco-DMDP; c —: not determined.

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Ent-broussonetine M (ent-3) and ent-10’-epi-3 exhibited potent and more selective inhibition of
-glucosidase from rice (IC50 = 1.2 M and 1.3 M) and rat intestinal maltase (IC50 = 0.29 M and
18 M), analogous to the inhibition by l-DMDP [16 18], ent-broussonetine I, J2 [39], and other LAB or
l-DMDP derivatives with a long alkyl chain on the C1 position [29 62]. Ent-3-epi-3 and ent-3,10-di-epi-3
showed moderate and broad inhibition against -glucosidase, -glucosidase, -galactosidase, and
-l-fucosidase, while l-altro-DMDP was a selective moderate -glycosidase inhibitor. d-gluco-DMDP
-glucosidase. For d-gluco-DMDP-related broussonetine M
-glucosidase,
-glucosidase, while
α
µ
µ
µ
µ
,
,
α
β
β
α
α
exhibited moderate inhibition against
α
analogs, compounds 2-epi-3 and 2,10-di-epi-3 both showed moderate inhibition against
-galactosidase, and -glucuronidase; 2,10’-di-epi-3 was a potent inhibitor of
β
β
β
α
2-epi-3 did not show such inhibition. The results indicate that the correct configuration at C-10’ is
essential for α-glucosidase inhibition.
Therefore, configurations of both the pyrrolidine ring and C-10’ have significant influences on
glycosidase inhibition of broussonetine M and its analogs. In detail, comparison of the inhibitory
profiles of ent-broussonetine M (ent-3) and ent-10’-epi-3 with those of broussonetine M (3) and
10’-epi-3 showed high similarity to those of DAB and LAB derivatives (or DMDP and l-DMDP
derivatives), of which we have previously reported opposite inhibitions toward
α- and β-glycosidases
for enantiomers [16 18 42]. In contrast, inhibitory activities of other broussonetine analogues with
,
,
l-altro-DMDP and d-gluco-DMDP pyrrolidine cores are more difficult to predict, but the presence of
the 13-carbon chains basically narrowed down the inhibitory profiles.
The structure–activity relationship uncovered in this work would be helpful in the research and
development of new glycosidase inhibitors.
3. Materials and Methods
3.1. General Methods
NMR spectra was recorded at 300 MHz, 400 MHz, or 500 MHz (1H-NMR) and 75 MHz, 100 MHz,
or 125 MHz (13C-NMR) in CDCl3 (with TMS as internal standard), C5D5N, or CD3OD (with solvent
signal as internal standard). High-resolution mass spectra (HRMS) were performed on a LTQ/FT linear
ion trap mass spectrometer. All reagents were used as received without any further purification or
prepared, as described in the literature. CH2Cl2 was freshly distilled from CaH2. Tetrahydrofuran was
distilled from sodium benzophenone. TLC plates were visualized by ultraviolet light or by treatment
with a spray of Pancaldi reagent ((NH4)6MoO4, Ce(SO4)2, H2SO4, H2O) or a 0.5% solution of KMnO4
in acetone. Column chromatography was performed on a flash column chromatography with silica
gel (200–300 mesh). Polarimetry was determined using an Optical Activity AA-10R polarimeter with
concentrations (c) given in grams per 100 mL. IR data were measured as films on KBr plates and are
given only when relevant functions are present. Chiral HPLC analyses were performed on an Agilen
1100 Series using a Daicel Chiralpak (OD-H) column with hexanes/i-PrOH as the eluent.
3.2. Material and Methods for the Enzyme Inhibition Assay
With rat intestinal maltase as an exception, other enzymes were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, Mo. USA). Brush border membranes prepared from rat small intestine
according to the method of Kessler et al. [63] were assayed at pH 6.8 for rat intestinal maltase using
maltose. The released d-glucose was determined colorimetrically using the Glucose CII-test Wako
(Wako Pure Chemical Ind.; Osaka, Japan). Other glycosidase activities were determined using an
appropriate p-nitrophenyl glycoside as substrate in a buffer solution at the optimal pH value of each
enzyme. The reaction was stopped by adding 400 mM Na2CO3. The released p-nitrophenol was
measured spectrometrically at 400 nm [16].

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3.3. Chemistry
3.3.1. Synthesis of 4-(benzyloxy)butan-1-ol (21)
NaH (60%, 11.52 g, 0.48 mol) was added slowly to a solution of butane-1,4-diol 20 (36.05 g, 0.4 mol)
in dry THF (300 mL) at 0 ◦C. After stirring at room temperature for 0.5 h, tetrabutylammonium iodide
(0.74 g, 8.0 mmol) was added, followed by a dropwise solution of benzyl bromide (68.41 g, 0.4 mol) in
THF while the temperature was kept between 45 ◦C and 50 ◦C. The reaction mixture was stirred for 2 h
and then quenched by aq. NH4Cl solution (20 mL). Water (200 mL) was added and the organic layer
separated; the aqueous layer was extracted with EtOAc (200 mL
were dried over MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash
×
3). The combined organic phases
column chromatography (silica gel, petroleum ether/EtOAc = 3/1) to give 4-(benzyloxy)butan-1-ol
1
21 (light yellow oil, 69.9 g, 97% yield). H-NMR (300 MHz, CDCl3)
δ
7.35–7.28 (m, 1H), 4.52 (s, 2H),
3.68–3.63 (d, J = 5.8 Hz, 2H), 3.54–3.50 (d, J = 5.9 Hz, 2H), 1.81–1.60 (m, 4H); 13C-NMR (75 MHz, CDCl3)
δ 138.2, 128.4, 127.7, 127.7, 73.1, 70.4, 62.7, 30.1, 26.7.
3.3.2. Synthesis of 4-(benzyloxy)butanal (16)
A solution of DMSO (8.74 mL, 0.26 mol) in dry CH2Cl2 (20 mL) was added dropwise to a solution
of (COCl)2 (24.57 mL, 0.29 mol) in dry CH2Cl2 (100 mL) at –78 ◦C. The mixture was stirred for 5 min.
A solution of 4-(benzyloxy)butan-1-ol 21 (43.3 g, 0.24 mol) in dry CH2Cl2 (50 mL) was then added
◦
dropwise while the temperature was kept below
was added dropwise. After stirring for 10 min at
−
65 C. After 15 min, NEt3 (166.94 mL, 1.2 mol)
−
78 ◦C, the reaction mixture was allowed to warm
to room temperature and diluted with CH2Cl2 (200 mL). The organic layer was washed with brine
(2 × 100 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated under
reduced pressure. Purification by flash chromatography on silica gel (petroleum ether/EtOAc = 10/1)
1
afforded 4-(benzyloxy)butanal 16 (39.8 g, 93% yield) as a yellow oil. H-NMR (300 MHz, CDCl3)
δ 9.77
(t, J = 1.6 Hz, 1H), 7.35–7.30 (m, 5H), 4.48 (s, 2H), 3.50 (t, J = 5.9 Hz, 2H), 2.54 (dt, J = 7.2, 1.6 Hz, 1H),
1.98–1.91 (m, 2H); 13C-NMR (75 MHz, CDCl3) δ 202.4, 138.3, 128.4, 127.7, 73.0, 69.1, 41.0, 22.6.
3.3.3. General Procedure for Synthesis of (R)-7-(benzyloxy)hept-1-en-4-ol (15) and (S)-7-(benzyloxy)hept-
1-en-4-ol (ent-15), with Alcohol 15 as an Example
Under Ar atmosphere, (S)-BINOL (52 mg, 0.2 mmol) and Ti(OiPr)4 (45 mg, 0.2 mmol) were added
to a solution of dried 4 Å molecular sieves (2.2 g) in CH2Cl2 (20 mL). The reaction mixture was heated at
reflux for 1 h, and then allowed to cool to room temperature. A solution of aldehyde 16 (356 mg, 2 mmol)
in CH2Cl2 (15 mL) was added to the reaction mixture. After stirring for 0.5 h at room temperature, the
solution was cooled to
mixture was stirred for an additional 20 min at
mixture was filtered through a pad of celite into a 200 mL flask that contained a stirred sat. aq. NaHCO3
solution (50 mL); the resulting reaction mixture was stirred for 1 h. Then, the layers were separated and
−
78 ◦C and allyltributyltin (993 mg, 3 mmol) was added dropwise. The reaction
78 ◦C, then kept at 20 ◦C. After 12 h, the reaction
−
−
the aqueous layer was extracted with CH2Cl2 (3
×
50 mL). The combined organic extracts were dried
with MgSO4; subsequent removal of all volatiles under reduced pressure and column chromatography
of the residue on silica gel (petroleum ether/EtOAc = 20/1) afforded homoallylic alcohol 15 (729.7 mg,
83% yield) as colorless oil.
20
Data for 15: [
α
]
+4.14 (c 3.85 in CH2Cl2); HPLC analysis: 92.6% ee [Daicel CHIRALPAK OD-H
max/cm−1
3401 (s), 3068 (w), 2924 (s), 1679 (w), 1453 (m), 1096 (s), 1021 (m), 697 (m); 1H-NMR (300 MHz, CDCl3)
D
column, 20 ◦C, 220 nm, hexane/i-PrOH = 95:5, 1 mL/min, 20.8 min (major), 23.9 min (minor)];
ν
:
δ
7.39–7.23 (m, 5H), 5.93–5.75 (m, 1H), 5.15–5.10 (m, 1H), 5.08 (t, J = 1.2 Hz, 1H), 4.50 (s, 2H), 3.64 (tt, J = 8.1,
4.5 Hz, 1H), 3.50 (t, J = 6.0 Hz, 2H), 2.52 (br, 1H), 2.26–2.15 (m, 2H), 1.77–1.60 (m, 3H), 1.54–1.45 (m, 1H);
13C-NMR (75 MHz, CDCl3)
δ
138.3, 135.1, 128.4, 127.7, 127.7, 117.7, 73.0, 70.6, 70.5, 42.0, 34.0, 26.2;
HRMS(ESI) calcd for C14H21O2+ [M + H]+ 243.13555, found 243.13564.

Molecules 2019, 24, 3712
10 of 19
20
Data for ent-15: colorless oil; yield: 86%; [
α
]
+4.08 (c 4.45 in CH2Cl2); HPLC analysis: 92.8% ee
D
[Daicel CHIRALPAK OD-H column, 20 ◦C, 220 nm, hexane/i-PrOH = 95:5, 1 mL/min, 21.3 min (minor),
23.1 min (major)];
ν
max/cm−1: 3401 (s), 3068 (w), 2924 (s), 1679 (w), 1453 (m), 1096 (s), 1021 (m), 697 (m);
7.38–7.31 (m, 4H), 7.31–7.26 (m, 1H), 5.90–5.76 (m, 1H), 5.15–5.12 (m, 1H),
1H-NMR (400 MHz, CDCl3)
δ
5.10 (s, 1H), 4.50 (s, 2H), 3.7–3.61 (m, 1H), 3.51 (t, J = 6.0 Hz, 2H), 2.36 (d, J = 3.2 Hz, 1H), 2.32–2.24
(m, 1H), 2.23–2.14 (m, 1H), 1.82–1.70 (m, 2H), 1.70–1.60 (m, 1H), 1.50 (m, 1H); 13C-NMR (100 MHz,
CDCl3) δ 138.3, 135.1, 128.4, 127.7, 127.7, 117.7, 73.0, 70.6, 70.5, 42.0, 34.0, 26.2; HRMS(ESI) calcd for
C14H21O2Na+ [M + Na]+ 243.13555, found 243.13544.
3.3.4. General Procedure for Synthesis of Compounds 19, ent-19, ent-3-epi-19, and 2-epi-19, with 19 as
an Example
Part of the solution of 8-bromo-1-octene (573.3 mg, 3.0 mmol) in THF (2 mL) was quickly added via
syringe to a stirred solution of Mg (1.16 g, 5.0 mmol) and I2 (cat.) in THF (5 mL) under Ar atmosphere.
The mixture was heated until the color disappeared; then, the remaining 8-bromo-1-octene was added
dropwise. After the addition was completed, the resulting reaction mixture was heated to reflux for
1 h and then was allowed to cool to room temperature. The prepared Grignard reagent was added
slowly to a solution of d-arabino-nitrone (14) (417.5 mg, 1.0 mmol) in THF (10 mL) via syringe at 0 ◦C
under Ar atmosphere. The reaction mixture was stirred for 0.5 h; then sat. aq. NH4Cl was added
to quench the reaction. The organic layer was separated and the aqueous layer was extracted with
EtOAc (3 × 20 mL). The combined organic phases were dried over MgSO4 and filtered; the solvent was
removed under reduced pressure to give the crude product hydroxylamine 19, which was used without
further purification because of its instability. The sample for structure characterization was purified by
flash column chromatography on silica gel (petroleum ether/EtOAc = 5/1) as a colorless syrup.
Data for (2R,3R,4R,5R)-3,4-bis(benzyloxy)-2- ((benzyloxy)methyl)-1-hydroxyl-5-(oct-7-en-1-yl)pyrrolidine
(19): [
735 (m), 697 (s); 1H-NMR (400 MHz, CDCl3)
α] ν
20 -8.6 (c 1.2 in CH2Cl2); max/cm−1: 3030 (w), 2926 (s), 2855 (s), 1454 (m), 1362 (w), 1097 (s),
D
δ
7.32–7.24 (m, 15H), 5.80 (ddt, J = 16.9, 10.2, 6.6 Hz, 1H),
5.01–4.91 (m, 2H), 4.56–4.42 (m, 6H), 3.95–3.92 (m, 1H), 3.80–3.76 (m, 2H), 3.58 (dd, J = 9.2, 6.9 Hz, 1H),
3.54–3.50 (m, 1H), 3.17 (dt, J = 7.5, 5.4 Hz, 1H), 2.04–1.99 (m, 2H), 1.88–1.83 (m, 1H), 1.50–1.43 (m, 1H),
1.42–1.28 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 139.3, 138.3, 138.2, 138.2, 128.5, 128.5, 128.1, 128.0,
127.8, 127.8, 127.7, 114.3, 86.8, 84.7, 73.5, 71.8, 71.8, 70.2, 70.1, 68.4, 33.9, 29.8, 29.2, 29.0, 26.7; HRMS(ESI)
calcd for C34H44O4N+ [M + H]+ 530.32649, found 530.32565.
Data for (2S,3S,4S,5S)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-1-hydroxyl-5-(oct-7-en-1-yl) pyrrolidine
(ent-19): colorless syrup; [
α] ν
20 + 7.8 (c 2.8 in CH2Cl2); max/cm−1: 3030 (w), 2926 (s), 2856 (s), 1454 (m),
D
1362 (w), 1099 (s), 735 (m), 697 (s); 1H-NMR (400 MHz, CDCl3)
δ
7.31–7.25 (m, 15H), 6.60 (br, 1H), 5.80 (ddt,
J = 16.9, 10.2, 6.7 Hz, 1H), 5.00–4.91 (m, 2H), 4.56–4.42 (m, 6H), 3.94 (t, J = 3.2 Hz, 1H), 3.80–3.77 (m, 2H),
3.60–3,56 (dd, J = 9.2, 6.9 Hz, 1H), 3.54–3.50 (m, 1H), 3.16 (dt, J = 7.2, 5.4 Hz, 1H), 2.02 (q, J = 6.9 Hz, 2H),
1.89–1.84 (m, 1H), 1.59–1.43 (m, 1H), 1.37–1.22 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 139.3, 138.3, 138.2,
138.2, 128.5, 128.4, 128.4, 128.1, 128.0, 127.8, 127.8, 127.7, 114.3, 86.8, 84.7, 73.4, 71.8, 71.7, 70.2, 70.0, 68.4,
33.9, 29.8, 29.2, 29.0, 26.6; HRMS(ESI) calcd for C34H44O4N+ [M + H]+ 530.32649, found 530.32550.
Data for (2S,3R,4S,5S)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-1-hydroxyl–5-(oct-7-en-1-yl) pyrrolidine
20
(
ent-3-epi-19
):
colorless syrup; [
α
]
−
24.5 (c 4.5 in CH2Cl2); ν
max/cm−1: 3030 (w), 2926 (s), 2855 (s),
D
1454 (m), 1363 (w), 1098 (br), 734 (m), 697 (s); 1H-NMR (400 MHz, CDCl3)
δ 7.30–7.24 (m, 15H), 6.64
(br, 1H), 5.84–5.74 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.03–4.88 (m, 2H), 4.68–4.43 (m, 6H), 4.19 (t, J = 5.1 Hz,
1H), 3.86–3,76 (m, 2H), 3.68 (dd, J = 6.8, 5.3 Hz, 1H), 3.56–3.51 (m, 1H), 3.29–3.24 (m, 1H), 2.04–1.99
(m, 2H), 1.75–1.69 (m, 1H), 1.49–1.28 (m, 9H); 13C-NMR (100 MHz, CDCl3)
δ 139.2, 138.5, 138.4, 138.2,
128.6, 128.4, 128.4, 128.2, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.0, 114.3, 82.9, 76.8, 73.6, 73.5, 72.7,
70.2, 69.3, 67.4, 33.9, 30.1, 29.8, 29.2, 29.0, 26.9; HRMS(ESI) calcd for C34H44O4N+ [M + H]+ 530.32649,
found 530.32573.

Molecules 2019, 24, 3712
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Data for (2S,3R,4R,5R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-1-hydroxyl-5-(oct-7-en-1-yl) pyrrolidine
2-epi-19 colorless syrup; [
20 + 22.8 (c 2.5 in CH2Cl2); max/cm−1: 3290 (br), 3028 (w), 2925 (s), 2856
(s), 1453 (m), 1353 (w), 1112 (s), 729 (s), 693 (s); 1H-NMR (400 MHz, CDCl3)
7.36–7.23 (m, 15H), 5.85–5.75
(
):
α]
D
ν
δ
(ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.32 (s, 1H), 5.01–4.91 (m, 2H), 4.57–4.50 (m, 4H), 4.42–4.32 (m, 2H),
3.94–3.87 (m, 2H), 3,81–3.78 (m, 1H), 3,53–3.52 (m, 1H), 3.36–3.31 (dt, J = 8.0, 5.3 Hz, 1H), 2.85–2.80
(dt, J = 8.0, 5.3 Hz, 1H), 2.05–2.00 (dd, J = 7.1, 6.6 Hz, 1H), 1.81–1.75 (m, 1H), 1.57–1.50 (m, 1H), 1.42–1.28
(m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 139.3, 138.3, 138.2, 138.0, 128.5, 128.0, 127.9, 127.9, 127.8, 127.8,
114.3, 85.4, 79.8, 73.6, 73.1, 72.3, 71.6, 69.7, 68.0, 33.9, 32.8, 29.8, 29.2, 29.0, 26.2; HRMS(ESI) calcd for
C34H44O4N+ [M + H]+ 530.32649, found 530.32577.
3.3.5. General Procedure for Synthesis of Compounds 13, ent-13, 3-epi-ent-13, and 2-epi-13, with 13 as
an Example
Zinc powder (653.8 mg, 10 mmol) was added to a suspension of Cu(OAc)2 (18.2 mg, 0.1 mmol)
in AcOH (10 mL) and the reaction mixture was stirred for 0.5 h. Then, a solution of the crude
hydroxylamine 19 in AcOH (5 mL) was added and the reaction mixture was stirred for 10 h. The solid
was removed by filtration and all volatiles were removed under reduced pressure. The residue was
dissolved in EtOAc (20 mL) and sat. aq. NaHCO3 was added to neutralize the solution. The resulting
precipitate was removed by filtration and the organic layers were collected; the aqueous layer was
extracted with EtOAc (3
×
10 mL). The combined organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure to give the crude amine, which was used in the next step without
further purification. NaHCO3 (252.0 mg, 3.0 mmol) and CbzCl (255.9 mg, 1.5 mmol) were added
slowly to a stirred solution of the crude amine in methanol (10 mL) and the reaction mixture was
stirred at room temperature for 6 h. Then sat. aq. NaHCO3 (20 mL) was added to quench the reaction
and EtOAc (20 mL) was added. The organic layer was separated and the aqueous layer was extracted
by EtOAc (3
×
10 mL). The combined organic layers were dried over MgSO4, filtered, and the solvent
was removed under reduced pressure. Purification by flash chromatography on silica gel (petroleum
ether/EtOAc = 15/1) afforded the carbamate 13 as light yellow syrup (423 mg, yield: 64% for 3 steps).
Data for (2R, 3R, 4R, 5R)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-2-((benzyloxy)methyl)-5-(oct-7-en-1-
36.4 (c 2.25 in CH2Cl2); ν
max/cm−1: 2926 (m), 2855 (w), 1700 (vs), 1407
20
yl)pyrrolidine (13): [
α
]
−
D
(m), 1347 (w), 1093 (m), 696 (m); 1H-NMR (400 MHz, CDCl3)
δ 7.38–7.23 (m, 17H), 7.23–7.15 (m, 3H),
5.86–5.72 (m, 1H), 5.22–5.15 (m, 1H), 5.05 (s, 0.5H), 5.02–5.01 (m, 1H), 4.96–4.92 (m, 1.5H), 4.65–4.57
(m, 1.5H), 4.47–4.32 (m, 4.5H), 4.27–4.23 (m, 0.5H),4.15–4.12 (m, 1.5H), 4.07–4.03 (m, 0.5H), 3.86–3.71
(m, 2.5H), 3.50–3.44 (m, 1H), 2.09–1.94 (m, 2.5H), 1.78– 1.48 (m, 1.5H), 1.29 (m, 4.5H), 1.16 (m, 3.5H);
13C-NMR (100 MHz, CDCl3)
δ 153.6, 153.2, 138.1, 138.1, 137.5, 137.3, 136.9, 136.9, 136.7, 136.6, 135.6,
135.6, 127.5, 127.4, 127.3, 127.3, 127.1, 126.99, 126.95, 126.69, 126.65, 126.60, 126.57, 126.52, 126.46, 113.2,
113.1, 83.4, 82.3, 82.1, 81.0, 72.0, 71.9, 70.1, 70.0, 69.8, 67.7, 66.8, 65.8, 65.7, 64.0, 63.6, 61.8, 61.5, 32.7,
32.7, 30.4, 29.1, 28.2, 28.0, 27.8, 27.8, 27.7, 25.5, 25.5; HRMS(ESI) calcd for C42H49O5NNa+ [M + Na]+
670.35029, found 670.34943.
Data for (2S,3S,4S,5S)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-2-((benzyloxy)methyl)-5-(oct-7-en-
1-yl)pyrrolidine (ent-13): light yellow syrup; yield for 3 steps: 65%; [α]
20 + 35.0 (c 6.95 in CH2Cl2);
D
1
ν
δ
max/cm−1: 3031 (w), 2926 (m), 1701 (vs), 1408 (m), 1095 (vs), 697 (m); H-NMR (400 MHz, CDCl3)
7.37–7.14 (m, 20H), 5.80–5.73 (m, 1H), 5.22–5.14 (m, 1H), 5.02–4.92 (m, 3H), 4.62–4.52 (m, 1.5H),
4.44–4.29 (m, 5H), 4.18 (d, J = 8.4 Hz, 1H), 4.11–4.08 (m, 1H), 3.93–3.72 (m, 2.5H), 3.51–3.46 (m, 1H),
2.18–1.84 (m, 2.5H), 1.81–1.48 (m, 1.5H), 1.41–1.05 (m, 8H); 13C-NMR (100 MHz, CDCl3)
154.8, 154.4,
δ
139.2, 139.2, 138.8, 138.5, 138.2, 138.1, 137.9, 137.9, 136.9, 136.8, 128.7, 128.62, 128.59, 128.54, 128.51,
128.33, 128.25, 128.2, 128.1, 127.94, 127.88, 127.82, 127.76, 127.7, 127.6, 114.54, 114.49, 84.6, 83.5, 83.3, 82.1,
73.2, 73.1, 71.3, 71.2, 71.0, 69.0, 68.0, 67.0, 66.9, 65.3, 64.9, 63.2, 62.8, 34.0, 33.9, 31.6, 30.3, 29.5, 29.2, 29.1,
28.99, 28.95, 26.69, 26.65; HRMS(ESI) calcd for C42H49O5NNa+ [M + Na]+ 670.35029, found 670.34998.

Molecules 2019, 24, 3712
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Data for (2S,3R,4S,5S)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-2-((benzyloxy)methyl)-5-(oct-7-en-
20
1-yl)pyrrolidine (ent-3-epi-13): light yellow syrup; yield for 3 steps: 64%; [
CH2Cl2);
CDCl3)
4.13 (br, 1H), 3.87–3.67 (m, 2.5H), 3.60 (m, 0.5H), 2.08–1.86 (m, 2.5H), 1.64 (s, 0.5H), 1.41–0.92 (m, 9H);
13C-NMR (100 MHz, CDCl3)
154.7, 154.6, 139.2, 138.8, 138.5, 138.4, 136.8, 128.6, 128.5, 128.3, 128.2,
α
]
+ 5.1 (c 3.45 in
D
ν
max/cm−1: 3030 (w), 2926 (m), 1701 (vs), 1406 (m), 1094 (s), 697 (m); 1H-NMR (400 MHz,
δ
7.35–7.19 (m, 20H), 5.82–5.75 (m, 1H), 5.23–4.92 (m, 4H), 4.82–4.51 (m, 5H), 4.40–4.26 (m, 3H),
δ
128.1, 128.0, 127.82, 127.75, 127.6, 127.5, 127.4, 127.3, 114.5, 81.1, 79.9, 78.1, 77.7, 77.4, 77.1, 73.1, 73.00,
72.8, 72.3, 72.3, 72.0, 70.3, 69.2, 67.0, 66.9, 62.8, 62.40, 58.0, 57.9, 33.9, 33.3, 32.0, 29.4, 29.1, 29.0, 28.9, 26.7,
26.5; HRMS(ESI) calcd for C42H49O5NNa+ [M + Na]+ 670.35029, found 670.35004.
Data for (2S,3R,4R,5R)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-2-((benzyloxy)methyl)-5-(oct-7- en-1-
20
yl)pyrrolidine (2-epi-13): light yellow syrup; yield for 3 steps: 71%; [
ν
α
1
]
−
4.2 (c 1.95 in CH2Cl2);
D
max/cm−1: 3031 (w), 2927 (m), 1702 (vs), 1406 (m), 1095 (s), 697 (m); H-NMR (400 MHz, CDCl3)
δ
7.35–7.17 (m, 20H), 5.83–5.73 (m, 1H), 5.10 (s, 2H), 5.00–4.91 (m, 2H), 4.61–4.59 (m, 2H), 4.62–4.33 (m, 5H),
4.15–4.12 (m, 1H), 3.92–3.90 (m, 1H), 3.80–3.65 (m, 3H), 2.00–1.98 (m, 2H), 1.78 (m, 1H), 1.62–1.54 (m, 1H),
1.31–1.105 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 155.8, 139.3, 138.6, 138.2, 138.1, 136.9, 128.6, 128.5,
128.4, 128.0, 127.9, 127.83, 127.79, 127.7, 127.6, 114.3, 84.9, 82.5, 73.4, 73.0, 71.7, 68.2, 67.0, 62.3, 59.1, 33.9,
33.5, 29.5, 29.1, 29.0, 26.0; HRMS(ESI) calcd for C42H49O5NNa+ [M + Na]+ 670.35029, found 670.34973.
3.3.6. General Procedure for Synthesis of Compounds 12, 10’-epi-12, ent-10’-epi-12, ent-12,
ent-3,10’-di-epi-12, ent-3-epi-12, 2-epi-12, and 2,10’-di-epi-12, with 12 as an Example
Grubbs II catalyst (6.8 mg, 0.008 mmol) was added to a solution of 13 (50 mg, 0.077 mmol) and
15 (25.4 mg, 0.116 mmol) in dry CH2Cl2 and the resulting reaction mixture was heated to reflux for
4 h. Then the solvent was removed under reduced pressure and purification of the residue by flash
chromatography on silica gel (petroleum ether/EtOAc = 5/1) afforded 12 (Z/E mixture) as a yellow
syrup (28 mg, 43% yield).
Data for (2R,3R,4R,5R)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5- ((R)-13-(benzyloxy)-10-hydroxydec-
20
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (12): [
α
]
−
20.0 (c 1.50 in CH2Cl2);
ν
max/cm−1: 3079
7.45–7.13
D
(w), 2925 (s), 2854 (m), 1699 (s), 1409 (m), 1094 (s), 697 (m); 1H-NMR (400 MHz, CDCl3)
δ
(m, 25H), 5.49–5.39 (m, 2H), 5.22–5.15 (dd, J = 14.8, 12.6 Hz, 1H), 5.05–5.02 (d, J = 12.3 Hz, 1H), 4.65–4.57
(m, 1.5H), 4.50–4.32 (m, 6.5H), 4.2–4.23 (dd, J = 10.5, 4.0 Hz, 0.5H), 4.18–4.12 (m, 1.5H), 4.07–4.03
(dd, J = 8.7, 4.1 Hz, 0.5H), 3.85–3.71 (m, 1.5H), 3.62–3.58 (d, J = 3.8 Hz, 1H), 3.52–3.44 (m, 3H), 2.26–1.87
(m, 5H), 1.79–1.40 (m, 5H), 1.38–1.14 (m, 8H); 13C-NMR (100 MHz, CDCl3) δ 153.6, 153.2, 137.5, 137.3,
136.93, 136.88, 136.63, 135.58, 133.32, 133.25, 131.9, 127.5, 127.4, 127.32, 127.28, 127.1, 126.98, 126.95,
126.64, 126.56, 126.5, 125.0, 83.4, 82.3, 82.1, 81.0, 76.3, 76.0, 75.7, 72.0, 71.9, 71.7, 70.1, 70.0, 69.8, 69.5, 69.4,
67.7, 66.8, 65.6, 64.0, 63.6, 61.9, 61.6, 39.8, 34.4, 32.8, 31.6, 31.1, 30.4, 29.1, 28.7, 28.4, 28.2, 28.1, 28.0, 26.4,
25.5, 25.2, 25.0; HRMS(ESI) calcd for C54H65O7NNa+ [M + Na]+ 862.46532, found 862.46423.
Data for (2R,3R,4R,5R)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((S)-13-(benzyloxy)-10- hydroxydec-
20
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (10’-epi-12): yellow syrup; yield: 43%; [
CH2Cl2);
CDCl3)
α
]
−
20.0 (c 1.50 in
D
ν
max/cm−1: 3080 (w), 2927 (s), 2855 (m), 1699 (s), 1409 (m), 1095 (s), 697 (m); 1H-NMR (400 MHz,
7.33–7.18(m, 25H), 5.54–5.38 (m, 2H), 5.22–5.15(m, 1H), 5.05–5.02 (m, 1H), 4.65–4.57 (m, 1.5H),
δ
4.51–4.32 (m, 6.5H), 4.26–4.22 (m, 0.5H), 4.16–4.11 (m, 1.5H), 4.07–4.03 (m, 0.5H), 3.86–3.71 (m, 2.5H),
3.62–3.58 (m, 1H), 3.53–3.44 (m, 3H), 2.24–1.86 (m, 5H), 1.74–1.61(m, 4H), 1.52–1.38 (m, 1H), 1.36–1.16
(m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 154.7, 154.3, 138.61, 138.36, 138.03, 137.98, 137.76, 136.67, 134.30,
134.23, 132.98, 130.02, 128.6, 128.49, 128.46, 128.4, 128.2, 128.10, 128.06, 127.8, 127.74, 127.69, 127.65, 127.6,
126.1, 84.5, 83.4, 83.2, 82.0, 73.1, 73.0, 72.7, 71.2, 71.1, 70.9, 70.6, 70.5, 68.8, 67.9, 66.91, 66.87, 65.1, 64.8, 63.0,
62.7, 40.9, 33.9, 32.7, 32.6, 31.5, 30.2, 29.5, 29.2, 29.1, 27.5, 26.6, 26.3; HRMS(ESI) calcd for C54H65O7NNa+
[M + Na]+ 862.46532, found 862.46411.

Molecules 2019, 24, 3712
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Data for (2S,3S,4S,5S)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((R)-13-(benzyloxy)-10- hydroxydec-
20
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (ent-10’-epi-12): yellow syrup; yield: 41%; [
α
]
+ 28.6
D
(c 1.85 in CH2Cl2);
ν
max/cm−1: 3470 (m), 3031 (w), 2925 (s), 1699 (vs), 1409 (m), 1096 (vs), 697 (m); 1H-NMR
(400 MHz, CDCl3)
δ 7.35–7.17 (m, 25H), 5.54–5.34 (m, 2H), 5.22–5.15 (m, 1H),5.05–5.02 (m, 1H), 4.65–4.56
(m, 1.5H), 4.52–4.32 (m, 6.5H), 4.26–4.22 (dd, J = 4.0 Hz, 0.5H), 4.15–4.10 (m, 1.5H), 4.06–4.03 (dd, J = 8.7,
4.2 Hz, 0.5H), 3.87–3.72 (m, 2.5H), 3.64–3.56 (m, 1H), 3.52–3.44 (m, 3H), 2.24–1.91 (m, 5H), 1.78–1.58
(m, 4H), 1.52–1.48 (m, 1H), 1.35–1.11 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 154.7, 154.3, 138.6, 138.4,
138.0, 138.0, 137.7, 137.6, 136.7, 134.3, 134.2, 129.7, 129.6, 128.6, 128.50, 128.47, 128.4, 128.2, 128.10, 128.07,
127.74, 127.65, 127.6, 127.5, 126.1, 84.5, 83.4, 83.2, 82.0, 73.1, 73.0, 71.2, 71.1, 70.9, 70.6, 70.5, 68.8, 67.8,
66.9, 65.1, 64.8, 63.0, 62.7, 40.9, 33.9, 32.7, 31.5, 30.2, 29.5, 29.2, 29.1, 26.6, 26.3, 26.1; HRMS(ESI) calcd for
C54H65O7NNa+ [M + Na]+ 862.46532, found 862.46407.
Data for (2S,3S,4S,5S)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((S)-13-(benzyloxy)-10- hydroxydec-
20
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (ent-12): yellow syrup; yield: 45%; [
in CH2Cl2);
(400 MHz, CDCl3)
α
]
+ 27.8 (c 1.39
D
ν
max/cm−1: 3462 (m), 3031 (w), 2925 (s), 1699 (vs), 1409 (m), 1096 (vs), 697 (m); 1H-NMR
δ
7.41–7.16 (m, 25H), 5.54–5.33 (m, 2H), 5.22–5.14 (m, 1H), 5.04–5.01 (m, 1H), 4.65–4.55
(m, 1.5H), 4.51–4.32 (m, 6.5H), 4.27–4.23 (m, 0.5H), 4.15–4.12 (m, 1.5H), 4.06–4.03 (dd, J = 8.2, 3.9 Hz,
0.5H), 3.85–3.69 (m, 2.5H), 3.59–3.54(m, 1H), 3.51–3.47 (m, 3H), 2.22–1.90 (m, 5H), 1.77–1.52 (m, 5H),
1.38–1.12 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 154.7, 154.3, 138.6, 138.3, 138.00, 137.95, 137.7, 136.6,
134.34, 134.28, 128.5, 128.44, 128.41, 128.37, 128.2, 128.1, 127.73, 127.69, 127.65, 127.6, 126.1, 84.5, 83.4,
83.2, 82.03, 77.5, 77.2, 76.8, 73.1, 73.0, 71.2, 71.1, 70.9, 70.54, 70.45, 68.79, 67.84, 66.9, 65.1, 64.7, 62.973.0,
62.6, 40.8, 35.4, 33.9, 32.7, 32.2, 31.5, 30.1, 29.4, 29.3, 29.2, 29.1, 27.5, 26.6, 26.3, 26.0; HRMS(ESI) calcd for
C54H65O7NNa+ [M + Na]+ 862.46532, found 862.46418.
Data for (2S,3R,4S,5S)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((R)-13-(benzyloxy)-10- hydroxydec-
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (ent-3,10’-di-epi-12): yellow syrup; yield: 40%; [α]D 20 + 5.1
(c 1.05 in CH2Cl2);
1H-NMR (400 MHz, CDCl3)
4.39–4.22 (m, 3H), 4.12–4.17 (m, 1H), 3.81–3.74 (m, 2H), 3.71–3.68 (m, 0.5H), 3.65–3.57 (m, 1.5H), 3.52–3.46
(t, J = 6.1 Hz, 2H), 2.26–1.88 (m, 5H), 1.79–1.46 (m, 5H), 1.32–1.01 (m, 8H); 13C-NMR (100 MHz, CDCl3)
ν
max/cm−1: 3461 (m), 3030 (w), 2926 (s), 1700 (vs), 1408 (m), 1096 (vs), 697 (m);
δ
7.38–7.17 (m, 25H), 5.53–5.38 (s, 2H), 5.25–5.00 (m, 2H), 4.85–4.45 (m, 7H),
δ
154.7, 154.5, 138.7, 138.4, 136.7, 134.2, 128.5, 128.5, 128.4, 128.2, 128.0, 127.72, 127.65, 127.5, 127.4, 126.30,
126.2, 81.1, 79.9, 78., 77.5, 77.2, 76.9, 73.0, 72.7, 72.5, 72.2, 71.9, 70.9, 70.6, 69.1, 66.9, 62.7, 62.3, 57.9, 40.9,
37.3, 33.9, 33.3, 32.7, 31.9, 29.4, 29.1, 26.6, 26.3; HRMS(ESI) calcd for C54H65O7NNa+ [M + Na]+ 862.46532,
found 862.46412.
Data for (2S,3R,4S,5S)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((S)-13-(benzyloxy)-10-hydroxydec-
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (ent-3-epi-12): yellow syrup; yield: 41%; [
in CH2Cl2);
max/cm−1: 3461 (m), 3030 (w), 2926 (s), 1701 (vs), 1408 (m), 1096 (vs), 697 (m); 1H-NMR
(500 MHz, CDCl3) 7.38–7.17 (m, 25H), 5.53–5.36 (m, 2H), 5.24–5.01 (m, 2H), 4.82–4.75 (m, 1H), 4.71–4.46
α]
20+ 5.9 (c 3.3
D
ν
δ
(m, 6H), 4.42–4.34 (m, 1H), 4.31–4.23 (m, 2H), 4.15–4.14 (d, J = 3.7 Hz, 1H), 3.81–3.77 (m, 2H), 3.70–3.68
(d, J = 8.1 Hz, 0.5H), 3.60–3.57 (m, 1.5H), 3.52–3.46 (m, 2H), 2.22–1.89 (m, 5H), 1.78–1.58 (m, 5H), 1.34–1.01
(m, 8H); 13C-NMR (125 MHz, CDCl3)
δ 154.7, 154.5, 1390, 138.7, 138.4, 138.3, 136.7, 136.6, 134.24, 134.17,
132.9, 129.6, 128.50, 128.45, 128.4, 128.2, 128.1, 127.99, 127.95, 127.73, 127.65, 127.5, 127.4, 127.3, 127.2,
126.3, 126.1, 81.0, 79.8, 78.0, 77.6, 77.4, 77.2, 76.9, 73.0, 72.9, 72.7, 72.4, 72.2, 71.9, 71.3, 70.9, 70.5, 70.5, 69.1,
66.9, 62.6, 62.3, 57.9, 57.8, 40.8, 35.5, 34.1, 33.9, 33.3, 32.8, 32.7, 32.6, 32.2, 31.8, 29.4, 29.3, 29.1, 29.0, 27.4,
26.6, 26.5, 26.4, 26.3, 26.0; HRMS(ESI) calcd for C54H65O7NNa+ [M + Na]+ 862.46532, found 862.46437.
Data for (2S,3R,4R,5R)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((R)-13-(benzyloxy)-10- hydroxydec-
20
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (2-epi-12): yellow syrup; yield: 43%; [
in CH2Cl2);
(400 MHz, CDCl3)
(m, 6H), 4.36–4.32 (m, 1H), 4.15–4.12 (dd, J = 6.8, 4.6 Hz, 1H), 3.91–3.89 (t, J = 4.0 Hz, 1H), 3.85–3.66
α
]
−
3.7 (c 0.88
D
ν
max/cm−1: 3461 (m), 3030 (w), 2926 (s), 1700 (vs), 1409 (m), 1097 (vs), 697 (m); 1H-NMR
δ
7.34–7.24 (m, 25H), 5.52–5.38 (m, 2H), 5.11 (s, 2H), 4.64–4.58 (m, 2H), 4.55–4.46

Molecules 2019, 24, 3712
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(m, 3H), 3.61–3.57 (m, 1H), 3.52–3.46 (t, J = 6.1 Hz, 2H), 2.25–1.92 (m, 4H), 1.81–1.60 (m, 4H), 1.52–1.42
(m, 1H), 1.35–1.12 (m, 8H); 13C-NMR (100 MHz, CDCl3)
155.6, 138.5, 138.4, 138.2, 138.0, 136.8, 134.4,
δ
132.9, 128.5, 128.48, 128.45, 128.4, 128.3, 128.0, 127.8, 127.8, 127.7, 127.6, 127.5, 126.0, 89.6, 82.5, 80.7, 73.4,
73.0, 73.0, 72.7, 71.6, 71.3, 70.9, 70.6, 70.5, 67.0, 62.3, 59.0, 40.9, 35.5, 34.1, 33.9, 32.7, 29.5, 29.4, 29.1, 27.5,
26.4, 26.3, 26.0, 25.9; HRMS(ESI) calcd for C54H65O7NNa+ [M + Na]+ 862.46532, found 862.46422.
Data for (2S,3R,4R,5R)-3,4-bis(benzyloxy)-1-benzyloxycarbonyl-5-((S)-13-(benzyloxy)-10- hydroxydec-
20
7-en-1-yl)-2-((benzyloxy)methyl)pyrrolidine (2,10’-di-epi-12): yellow syrup; yield: 43%; [
(c 1.43 in CH2Cl2);
1H-NMR (400 MHz, CDCl3)
α
]
D
−
3.8
ν
max/cm−1: 3470 (m), 3030 (w), 2926 (s), 1700 (vs), 1409 (m), 1097 (vs), 697 (m);
δ
7.36–7.24 (m, 25H), 5.52–5.34 (m, 2H), 5.13 (s, 2H), 4.65–4.48 (m, 8H),
4.36–4.32 (m, 1H), 4.15–4.12 (dd, J = 6.8, 4.7 Hz, 1H), 3.92–3.90 (t, J = 3.9 Hz, 1H), 3.83 –3.67 (m, 3H),
3.52–3.45 (m, 2H), 2.26–1.85 (m, 5H), 1.77–1.41 (m, 5H), 1.35–1.13 (m, 8H); 13C-NMR (100 MHz, CDCl3)
δ 153.0, 138.51, 138.45, 138.2, 138.1, 136.8, 134.3, 130.0, 128.53, 128.51, 128.47, 128.46, 128.4, 128.0, 1279,
127.79, 127.76, 127.69, 127.66, 127.5, 126.1, 83.9, 79.9, 73.4, 73.1, 73.0, 72.9, 71.7, 71.3, 71.1, 70.9, 70.64,
70.57, 69.4, 68.1, 67.0, 62.3, 59.1, 40.9, 37.4, 34.8, 34.7, 33.9, 32.8, 29.5, 29.4, 29.2, 26.4, 26.3, 25.9, 25.8;
HRMS(ESI) calcd for C54H65O7NNa+ [M + Na]+ 862.46532, found 862.46448.
3.3.7. General Procedure for Synthesis of Compounds 3, 10’-epi-3, ent-10’-epi-3, ent-3,
ent-3,10’-di-epi-3, ent-3-epi-3, 2-epi-3, 2, and 10’-di-epi-3, with Broussonetine M (3) as an Example
Pd/C (10 wt%) was added to a stirred solution of 9 (43 mg, 0.055 mmol) and 3 N HCl (1 mL) in
MeOH (10 mL) under Ar atmosphere and the reaction mixture was stirred under H2 atmosphere for
12 h. Then, the catalyst was filtered and the solvent was removed under reduced pressure. Purification
of the residue by flash chromatography on silica gel (CHCl3/MeOH/NH3 H2O (2 N) = 90/9/1) afforded
·
(2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxyl-5-((10R)-10,13-(dihydroxyl)dec- 1-yl)pyrrolidine (3)
20
as white solid (14.4 mg, 0.055 mmol, quantative yield). Data for broussonetine M (
3): [α
]
+ 4.0
D
20
(c 0.7 in CH3OH) [lit. [36] [
(vs); H-NMR (500 MHz, C5D5N)
α
]
+ 5.9 (c 0.3 in CH3OH)];
6.41 (br, 7.15–5.54, 4H), 4.92 (t, J = 6.7 Hz, 1H), 4.67 (t, J = 7.1
ν
max/cm−1: 3104 (vs), 2926 (s), 2857 (w),1402
D
1
δ
Hz, 1H), 4.49–4.42 (m, 2H), 4.31–4.28 (m, 1H), 4.09–4.05 (m, 1H), 3.96 (t, J = 6.4 Hz, 2H), 3.92–3.87
(m 1H), 2.34–2.29 (m, 2H), 2.15–2.09 (m, 1H), 2.03–1.97 (m, 1H), 1.90–1.79 (m, 3H), 1.74–1.56 (m, 4H),
1.48–1.45 (m, 1H), 1.33–1.07 (m, 10H); 13C-NMR (125 MHz, C5D5N)
δ
80.3, 76.2, 70.8, 65.0, 62.7, 62.3,
59.2, 38.3, 35.1, 31.5, 30.2, 29.9, 29.7, 29.5, 29.4, 29.3, 26.6, 26.1; 1H-NMR (400 MHz, CD3OD)
4.00–3.97
(t, J = 6.1 Hz, 1H), 3.90–3.80 (m, 3H), 3.58–3.48 (m, 4H), 3.34 (m, 1H), 1.98–1.28 (m, 22H); 13C-NMR
δ
(100 MHz, CD3OD) δ 80.3, 76.6, 72.3, 65.5, 64.3, 63.1, 59.5, 38.4, 34.7, 32.0, 30.8, 30.7, 30.6, 30.39, 30.35,
29.9, 27.2, 26.8; HRMS(ESI) calcd for C18H38O5N+ [M + H]+ 348.27445, found 348.27432.
Data for (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxyl-5-((10S)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
(
10’-epi-3): white solid, quantative yield; [
2861 (w), 1402 (vs); 1H-NMR (400 MHz, CD3OD)
(m, 3H), 3.49–3.45 (m, 1H), 3.37–3.35 (m, 1H), 1.96–1.84 (m, 1H), 1.80–1.64 (m, 2H), 1.63–1.28 (m, 19H);
α] ν
20 + 4.0 (c 0.4 in CH3OH); max/cm−1: 3104 (vs), 2927 (s),
D
δ
3.99–3.96 (t, J = 5.7 Hz, 1H), 3.85 (m, 3H), 3.62–3.51
13C-NMR (100 MHz, CD3OD)
δ 80.3, 76.6, 72.3, 65.5, 64.3, 63.2, 59.6, 38.4, 34.8, 32.2, 30.8, 30.7, 30.6, 30.4,
29.9, 27.2, 26.8; HRMS(ESI) calcd for C18H38O5N+ [M + H]+ 348.27445, found 348.27411.
Data for (2S,3S,4S,5S)-2-hydroxymethyl-3,4-dihydroxyl-5-((10R)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
20
(
ent-10’-epi-3): white solid, quantative yield; [
α
]
−
16.8 (c 1.5 in CH3OH);
4.01–3.98 (t, J = 6.2 Hz, 1H), 3.91–3.80 (m, 3H),
3.59–3.47 (m, 4H), 3.36–3.33 (m, 1H), 1.95–1.30 (m, 22H); 13C-NMR (100 MHz, CD3OD)
80.3, 76.6, 72.3,
ν
max/cm−1: 3134 (vs), 2928
D
(s), 2854 (w), 1402 (vs); 1H-NMR (400 MHz, CD3OD)
δ
δ
65.5, 64.3, 63.1, 59.5, 38.4, 34.8, 32.0, 30.8, 30.7, 30.6, 30.39, 30.35, 29.9, 27.2, 26.8; HRMS(ESI) calcd for
C18H38O5N+ [M + H]+ 348.27445, found 348.27430.

Molecules 2019, 24, 3712
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Data for (2S,3S,4S,5S)-2-hydroxymethyl-3,4-dihydroxyl-5-((10S)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
20
(
ent-3): white solid, quantative yield; [
α
]
−
20.4 (c 1.55 in CH3OH);
δ 4.01–3.98 (t, J = 6.1 Hz, 1H), 3.90–3.80 (m, 3H), 3.58–3.45
ν
max/cm−1: 3134 (vs), 2928 (s),
D
2854 (w), 1403 (s); 1H-NMR (400 MHz, CD3OD)
(m, 4H), 3.36–3.33 (m, 1H), 1.94–1.29 (m, 22H); 13C-NMR (100 MHz, CD3OD)
δ 80.3, 76.6, 72.3, 65.5, 64.3,
63.1, 59.5, 38.4, 34.8, 32.0, 30.8, 30.7, 30.6, 30.39, 30.35, 29.9, 27.2, 26.8; HRMS(ESI) calcd for C18H38O5N+
[M + H]+ 348.27445, found 348.27416.
Data for (2S,3R,4S,5S)-2-hydroxymethyl-3,4-dihydroxyl-5-((10R)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
20
(
ent-3,10’-di-epi-3): white solid, quantative yield; [
α
]
−
19.4 (c 0.5 in CH3OH);
ν
max/cm−1: 3124 (vs),
D
1
2926 (s), 2853 (w), 1402 (s); H-NMR (500 MHz, CD3OD)
δ
4.16 (s, 1H), 3.97–3.92 (m, 2H), 3.90–3.85
(m, 1H), 3.70–3.68 (dd, J = 8.0, 3.9 Hz, 1H), 3.59–3.54 (m, 3H), 3.46–3.41 (td, J = 9.1, 5.2 Hz, 1H), 1.89–1.29
(m, 22H); 13C-NMR (125 MHz, CD3OD)
δ 77.4, 72.3, 71.7, 63.5, 63.1, 62.0, 59.4, 38.5, 34.8, 32.0, 30.8, 30.7,
30.6, 30.46, 30.38, 29.9, 27.6, 26.8; HRMS(ESI) calcd for C18H38O5N+ [M + H]+ 348.27445, found 348.27424.
Data for (2S,3R,4S,5S)-2-hydroxymethyl-3,4-dihydroxyl-5-((10S)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
20
(
ent-3-epi-3): white solid, quantative yield; [
α
]
−
33.4 (c 0.65 in CH3OH);
4.16 (t, J = 3.4 Hz, 1H), 3.97–3.85 (m, 3H), 3.68–3.63
(m, 1H), 3.56 (t, J = 6.4 Hz, 2H), 3.53 (m, 1H), 3.42 (td, J = 9.2, 5.2 Hz, 1H), 1.90–1.28 (m, 22H); 13C-NMR
ν
max/cm−1: 3364 (vs), 2925
D
(vs), 2852 (m), 1402 (s); 1H-NMR (400 MHz, CD3OD)
δ
(100 MHz, CD3OD) δ 77.4, 72.3, 71.7, 63.5, 63.1, 62.0, 59.4, 38.5, 34.8, 32.08, 30.8, 30.7, 30.6, 30.5, 30.4, 29.9,
27.6, 26.8; HRMS(ESI) calcd for C18H38O5N+ [M + H]+ 348.27445, found 348.27427.
Data for (2S,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxyl-5-((10R)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
20
(
2-epi-3): white solid, quantative yield; [
α
]
+ 3.1 (c 0.45 in CH3OH);
4.07 (s, 1H), 3.98–3.89 (m, 3H), 3.72–3.68 (m, 1H), 3.56
(t, J = 6.3 Hz, 2H), 3.54–3.52 (m, 1H), 3.30–3.26 (m, 1H), 1.88–1.29 (m, 22H); 13C-NMR (125 MHz, CD3OD)
ν
max/cm−1: 3124 (s), 2923 (s),
D
2852 (w), 1402 (s); 1H-NMR (500 MHz, CD3OD)
δ
δ
80.8, 76.5, 72.3, 68.7, 65.6, 63.1, 58.6, 38.5, 34.8, 33.1, 30.8, 30.7, 30.6, 30.5, 30.3, 29.9, 27.8, 26.8; HRMS(ESI)
calcd for C18H38O5N+ [M + H]+ 348.27445, found 348.27439.
Data for (2S,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxyl-5-((10S)-10,13-(dihydroxyl)dec-1-yl) pyrrolidine
(
2,10’-di-epi-3): white solid, quantative yield; [
(s), 2853 (m), 1403 (s); 1H-NMR (400 MHz, CD3OD)
(m, 1H), 3.60–3.50 (m, 3H), 3.31–3.27 (m, 1H), 1.93–1.76 (m, 2H), 1.71–1.28 (m, 20H); 13C-NMR (100 MHz,
α] ν
20 + 0.12 (c 1.2 in CH3OH); max/cm−1: 3127 (s), 2923
D
δ
4.08 (d, J = 2.0 Hz, 1H), 3.99–3.88 (m, 3H), 3.71
CD3OD) δ 80.8, 76.5, 72.3, 68.7, 65.5, 63.1, 58.6, 38.4, 34.8, 33.1, 30.8, 30.7, 30.6, 30.4, 30.3, 29.9, 27.8, 26.8;
HRMS(ESI) calcd for C18H38O5N+ [M + H]+ 348.27445, found 348.27429.
4. Conclusions
In summary, a general and versatile synthetic strategy has been developed for the synthesis of
broussonetine M (3), ent-broussonetine M (ent-3), and six other stereoisomers with d-arabino-nitrone
(14), l-arabino-nitrone (ent-14), l-lyxo-nitrone (ent-3-epi-14), and l-xylo-nitrone (2-epi-14) as the starting
materials in 26%–31% total yield for five linear steps. Glycosidase inhibition assays on a range of
enzymes showed that natural product broussonetine M (
of -glucosidase from bovine liver, while ent-3 and ent-10’-epi-3 were potent and selective inhibitors
of rice -glucosidase and rat intestinal maltase. It was also found that the configuration at C-3 was
essential for -glucosidase inhibition. This work has further explored the spectrum of glycosidase
3) and 10’-epi-3 showed potent inhibition
β
α
α
inhibition by DAB- and LAB-related iminosugars and will be helpful for the future design of potent
and selective glycosidase inhibitors.
1
Supplementary Materials: The following are available online: copies of H-NMR, 13C-NMR, and 2D NMR
spectra, and HPLC analysis methods.
Author Contributions: Q.-K.W. performed the chemical syntheses, analyzed the data, and wrote the original
draft; K.K. performed the glycosidase inhibition assay; Y.-X.L. and Y.-M.J. validated all the experimental data
and revised the manuscript; A.K., G.W.J.F., and C.-Y.Y. designed the experiments and reviewed the manuscript.
All authors approved the final manuscript.

Molecules 2019, 24, 3712
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Funding: Financial support from the National Natural Science Foundation of China (No. 21272240 and No.
21772206) and National Engineering Research Center for Carbohydrate Synthesis of Jiangxi Normal University is
gratefully acknowledged. This work was supported in part by an Emeritus Leverhulme Research Fellowship
(G.W.J.F.) and a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science
(JSPS KAKENHI Grant Number JP17K08362) (A.K.).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Compain, P.; Martin, O.R. Iminosugars: From Synthesis to Therapeutic Applications, 1st ed.; Wiley-VCH:
Chichester, UK, 2007; pp. 328–446.
Wrodnigg, T.M. From Lianas to Glycobiology Tools: Twenty-Five Years of 2,5-dideoxy-2,5-imino-d-mannitol.
Mon. Chem. 2002, 133, 393–426. [CrossRef]
Asano, N.; Nash, R.J.; Molyneux, R.J.; Fleet, G.W.J. Sugar-mimic glycosidase inhibitors: Natural occurrence,
biological activity and prospects for therapeutic application. Tetrahedron Asymmetry 2000, 11, 1645–1680.
[CrossRef]
4.
5.
6.
7.
8.
9.
Watson, A.A.; Fleet, G.W.J.; Asano, N.; Molyneux, R.J.; Nash, R.J. Polyhydroxylated alkaloids—Natural
occurrence and therapeutic applications. Phytochemistry 2001, 56, 265–295. [CrossRef]
Horne, G.; Wilson, F.X.; Tinsley, J.; Williams, D.H.; Storer, R. Iminosugars past, present and future: Medicines
for tomorrow. Drug Discov. Today 2010, 16, 107–118. [CrossRef]
Nash, R.J.; Kato, A.; Yu, C.-Y.; Fleet, G.W.J. Iminosugars as Therapeutic Agents: Recent Advances and
Promising Trends. Future Med. Chem. 2011, 3, 1513–1521.
Davis, B.G. A silver-lined anniversary of Fleet iminosugars: 1984–2009, from DIM to DRAM to LABNAc.
Tetrahedron Asymmetry 2009, 20, 652–671. [CrossRef]
Butters, T.D.; Dwek, R.A.; Platt, F.M. Inhibition of Glycosphingolipid Biosynthesis: Application to Lysosomal
Storage Disorders. Chem. Rev. 2000, 100, 4683–4696. [CrossRef]
Nash, R.J.; Bell, E.A.; Williams, J.M. 2-Hydroxymethyl-3,4-dihydroxypyrrolidine in fruits of angylocalyx
boutiqueanus. Phytochemistry 1985, 24, 1620–1622. [CrossRef]
10. Welter, A.; Jadot, J.; Dardenne, G.; Marlier, M.; Casimir, J. 2,5-Dihydroxymethyl 3,4-dihydroxypyrrolidine
dans les feuilles de Derris elliptica. Phytochemistry 1976, 15, 747–749. [CrossRef]
11. Jones, D.W.C.; Nash, R.J.; Bell, E.A.; Williams, J.M. Identification of the 2-hydroxymethyl-3,4-
dihydroxypyrrolidine (or 1,4-dideoxy-1,4-iminopentitol) from angylocalyx boutiqueanus and from
arachniodes standishii as the (2R, 3R, 4S)-isomer by the synthesis of its enantiomer. Tetrahedron Lett.
1985, 26, 3125–3126. [CrossRef]
12. Fleet, G.W.J.; Nicholas, S.J.; Smith, P.W.; Evans, S.V.; Fellows, L.E.; Nash, R.J. Potent competitive
inhibition of
α-galactosidase and α-glucosidase activity by 1,4-dideoxy-1,4-iminopentitols: Syntheses
of 1,4-dideoxy-1,4-imino-d-lyxitol and of both enantiomers of 1,4-dideoxy-1,4-iminoarabinitol. Tetrahedron
Lett. 1985, 26, 3127–3130. [CrossRef]
13. Asano, N.; Kizu, H.; Oseki, K.; Tomioka, E.; Matsui, K.; Okamoto, M.; Baba, M. N-Alkylated
Nitrogen-in-the-Ring Sugars: Conformational Basis of Inhibition of Glycosidases and HIV-1 Replication.
J. Med. Chem. 1995, 38, 2349–2356. [CrossRef] [PubMed]
14. Merino, P.; Delso, I.; Tejero, T.; Cardona, F.; Marradi, M.; Faggi, E.; Parmeggiani, C.; Goti, A. Nucleophilic
additions to cyclic nitrones en route to iminocyclitols - Total syntheses of DMDP, 6-deoxy-DMDP, DAB-1,
CYB-3, nectrisine, and radicamine B. Eur. J. Org. Chem. 2008, 2929–2947. [CrossRef]
15. Cruz, F.P.d.; Newberry, S.; Jenkinson, S.F.; Wormald, M.R.; Butters, T.D.; Alonzi, D.S.; Nakagawa, S.; Becq, F.;
Norez, C.; Nash, R.J.; et al. 4-C-Me-DAB and 4-C-Me-LAB—enantiomeric alkyl-branched pyrrolidine
iminosugars—are specific and potent
Tetrahedron Lett. 2011, 52, 219–223. [CrossRef] [PubMed]
16. Ayers, B.J.; Ngo, N.; Jenkinson, S.F.; Mart nez, R.F.; Shimada, Y.; Adachi, I.; Weymouth-Wilson, A.C.; Kato, A.;
α-glucosidase inhibitors; acetone as the sole protecting group.
í
Fleet, G.W.J. Glycosidase Inhibition by All 10 Stereoisomeric 2,5-Dideoxy-2,5-iminohexitols Prepared from
the Enantiomers of Glucuronolactone. J. Org. Chem. 2012, 77, 7777–7792. [CrossRef] [PubMed]
17. Fosgerau, K.; Westergaard, N.; Quistorff, B.; Grunnet, N.; Kristiansen, M.; Lundgren, K. Kinetic and Functional
Characterization of 1,4-Dideoxy-1,4-imino-d-arabinitol: A Potent Inhibitor of Glycogen Phosphorylase with
Anti-hyperglyceamic Effect in ob/ob Mice. Arch. Biochem. Biophys. 2000, 380, 274–284. [CrossRef]

Molecules 2019, 24, 3712
17 of 19
18. Yu, C.-Y.; Asano, N.; Ikeda, K.; Wang, M.-X.; Butters, T.D.; Wormald, M.R.; Dwek, R.A.; Winters, A.L.; Nash, R.J.;
Fleet, G.W.J. Looking glass inhibitors: l-DMDP, a more potent and specific inhibitor of alpha-glucosidases
than the enantiomeric natural product DMDP. Chem. Commun. 2004, 1936–1937. [CrossRef]
19. Wang, G.-N.; Yang, L.; Zhang, L.-H.; Ye, X.-S. A Versatile Approach to N-Alkylated 1,4-Dideoxy-
1,4-imino-d-arabinitols and 1,4-Dideoxy-1,4-imino-l-xylitols. J. Org. Chem. 2011, 76, 2001–2009. [CrossRef]
20. Bouillon, M.E.; Pyne, S.G. Diastereoselective concise syntheses of the polyhydroxylated alkaloids DMDP
and DAB. Tetrahedron Lett. 2014, 55, 475–478. [CrossRef]
21. Li, Y.-X.; Kinami, K.; Hirokami, Y.; Kato, A.; Su, J.-K.; Jia, Y.-M.; Fleet, G.W.J.; Yu, C.-Y. Gem-difluoromethylated
and trifluoromethylated derivatives of DMDP-related iminosugars: Synthesis and glycosidase inhibition.
Org. Biomol. Chem. 2016, 14, 2249–2263. [CrossRef]
22. Jenkinson, S.F.; Best, D.; Saville, A.W.; Mui, J.; Mart
í
nez, R.F.; Nakagawa, S.; Kunimatsu, T.; Alonzi, D.S.;
-Glucosidase Inhibition by Enantiomers of
Butters, T.D.; Norez, C.; et al. C-Branched Iminosugars:
α
isoDMDP, isoDGDP, and isoDAB–l-isoDMDP Compared to Miglitol and Miglustat. J. Org. Chem. 2013, 78,
7380–7397. [CrossRef] [PubMed]
23. Best, D.; Jenkinson, S.F.; Saville, A.W.; Alonzi, D.S.; Wormald, M.R.; Butters, T.D.; Norez, C.; Becq, F.;
Blériot, Y.; Adachi, I.; et al. Cystic fibrosis and diabetes: isoLAB and isoDAB, enantiomeric carbon-branched
pyrrolidine iminosugars. Tetrahedron Lett. 2010, 51, 4170–4174. [CrossRef]
24. Matassini, C.; Vanni, C.; Goti, A.; Morrone, A.; Marradi, M.; Cardona, F. Multimerization of DAB-1 onto Au
GNPs affords new potent and selective N-acetylgalactosamine-6-sulfatase (GALNS) inhibitors. Org. Biomol.
Chem. 2018, 16, 8604–8612. [CrossRef] [PubMed]
25. D’Adamio, G.; Forcella, M.; Fusi, P.; Parenti, P.; Matassini, C.; Ferhati, X.; Vanni, C.; Cardona, F. Probing the
Influence of Linker Length and Flexibility in the Design and Synthesis of New Trehalase Inhibitors. Molecules
2018, 23, 436. [CrossRef] [PubMed]
26. Liu, Z.; Ma, S. Recent Advances in Synthetic -Glucosidase Inhibitors. Chemmedchem 2017, 12, 819–829.
[CrossRef]
27. Li, Y.-X.; Shimada, Y.; Adachi, I.; Kato, A.; Jia, Y.-M.; Fleet, G.W.J.; Xiao, M.; Yu, C.-Y. Fluorinated and
Conformationally Fixed Derivatives of l-HomoDMDP: Synthesis and Glycosidase Inhibition. J. Org. Chem.
2015, 80, 5151–5158. [CrossRef]
28. Ghavami, A.; Johnston, B.D.; Jensen, M.T.; Svensson, B.; Pinto, B.M. Synthesis of Nitrogen Analogues of
Salacinol and Their Evaluation as Glycosidase Inhibitors. J. Am. Chem. Soc. 2001, 123, 6268–6271. [CrossRef]
29. Kato, A.; Nakagome, I.; Sato, K.; Yamamoto, A.; Adachi, I.; Nash, R.J.; Fleet, G.W.J.; Natori, Y.;
Watanabe, Y.; Imahori, T.; et al. Docking study and biological evaluation of pyrrolidine-based iminosugars
as pharmacological chaperones for Gaucher disease. Org. Biomol. Chem. 2016, 14, 1039–1048. [CrossRef]
30. Shibano, M.; Kitagawa, S.; Kusano, G. Studies on the constituents of Broussonetia species. 1. Two new
pyrrolidine alkaloids, broussonetines C and D, as beta-galactosidase and beta-mannosidase inhibitors from
Broussonetia kazinoki SIEB. Chem. Pharm. Bull. 1997, 45, 505–508. [CrossRef]
31. Shibano, M.; Kitagawa, S.; Nakamura, S.; Akazawa, N.; Kusano, G. Studies on the Constituents of Broussonetia
Species. II. Six New Pyrrolidine Alkaloids, Broussonetine A, B, E, F and Broussonetinine A and B, as Inhibitors
of Glycosidases from Broussonetia kazinoki SIEB. Chem. Pharm. Bull. 1997, 45, 700–705. [CrossRef]
32. Shibano, M.; Nakamura, S.; Akazawa, N.; Kusano, G. Studies on the Constituents of Broussonetia Species. III.
Two New Pyrrolidine Alkaloids, Broussonetines G and H, as Inhibitors of Glycosidase, from Broussonetia
kazinoki SIEB. Chem. Pharm. Bull. 1998, 46, 1048–1050. [CrossRef] [PubMed]
33. Shibano, M.; Nakamura, S.; Kubori, M.; Minoura, K.; Kusano, G. Studies on the constituents of Broussonetia
species. IV. Two new pyrrolidinyl piperidine alkaloids, broussonetines I and J, from Broussonetia kazinoki
SIEB. Chem. Pharm. Bull. 1998, 46, 1416–1420. [CrossRef]
34. Shibano, M.; Nakamura, S.; Motoya, N.; Kusano, G. Studies on the constituents of Broussonetia species. V.
Two new pyrrolidine alkaloids, broussonetines K and L, as inhibitors of glycosidase, from Broussonetia
kazinoki SIEB. Chem. Pharm. Bull. 1999, 47, 472–476. [CrossRef]
35. Shibano, M.; Tsukamoto, D.; Kusano, G. A new pyrrolizidine alkaloid, Broussonetine N, as an inhibitor of
glycosidase, from Broussonetia kazinoki Sieb. and absolute stereostructures of Broussonetines A and B.
Chem. Pharm. Bull. 1999, 47, 907–908. [CrossRef]

Molecules 2019, 24, 3712
18 of 19
36. Shibano, M.; Tsukamoto, D.; Fujimoto, R.; Masui, Y.; Sugimoto, H.; Kusano, G. Studies on the Constituents of
Broussonetia Species. VII. Four New Pyrrolidine Alkaloids, Broussonetines M, O, P, and Q, as Inhibitors of
Glycosidase, from Broussonetia kazinoki SIEB. Chem. Pharm. Bull. 2000, 48, 1281–1285. [CrossRef]
37. Tsukamoto, D.; Shibano, M.; Okamoto, R.; Kusano, G. Studies on the constituents of Broussonetia species VIII.
Four new pyrrolidine alkaloids, broussonetines R, S, T, and V and a new pyrroline alkaloid, broussonetine U,
from Broussonetia kazinoki Sieb. Chem. Pharm. Bull. 2001, 49, 492–496. [CrossRef]
38. Shibano, M.; Tsukamoto, D.; Kusano, G. Polyhydroxylated Alkaloids with Lipophilic Moieties as Glycosidase
Inhibitors from Higher Plants. Heterocycles 2002, 57, 1539–1553.
39. Zhao, H.; Kato, A.; Sato, K.; Jia, Y.-M.; Yu, C.-Y. Total synthesis and glycosidase inhibition of broussonetine I
and J2. J. Org. Chem. 2013, 78, 7896–7902. [CrossRef]
40. Song, Y.-Y.; Kinami, K.; Kato, A.; Jia, Y.-M.; Li, Y.-X.; Fleet, G.W.J.; Yu, C.-Y. First total synthesis of
(+)-broussonetine W: Glycosidase inhibition of natural product & analogs. Org. Biomol. Chem. 2016, 14,
5157–5174.
41. D’Alonzo, D.; Guaragna, A.; Palumbo, G. Glycomimetics at the mirror: Medicinal chemistry of l-iminosugars.
Curr. Med. Chem. 2009, 16, 473–505. [CrossRef]
42. Best, D.; Wang, C.; Weymouth-Wilson, A.C.; Clarkson, R.A.; Wilson, F.X.; Nash, R.J.; Miyauchi, S.; Kato, A.;
Fleet, G.W.J. Looking glass inhibitors: Scalable syntheses of DNJ, DMDP, and (3R)-3-hydroxy- l-bulgecinine
from d-glucuronolactone and of l-DNJ, l-DMDP, and (3S)-3-hydroxy-d-bulgecinine from l-glucuronolactone.
DMDP inhibits β-glucosidases and β-galactosidases whereas l-DMDP is a potent and specific inhibitor of
α-glucosidases. Tetrahedron Asymmetry 2010, 21, 311–319.
43. Mohan, S.; Eskandari, R.; Pinto, B.M. Naturally Occurring Sulfonium-Ion Glucosidase Inhibitors and Their
Derivatives: A Promising Class of Potential Antidiabetic Agents. Acc. Chem. Res. 2014, 47, 211–225.
[CrossRef] [PubMed]
44. Boisson, J.; Thomasset, A.; Racine, E.; Cividino, P.; Sainte-Luce, T.B.; Poisson, J.-F.; Behr, J.-B.; Py, S.
Hydroxymethyl-Branched Polyhydroxylated Indolizidines: Novel Selective alpha-Glucosidase Inhibitors.
Org. Lett. 2015, 17, 3662–3665. [CrossRef] [PubMed]
45. Kato, A.; Zhang, Z.-L.; Wang, H.-Y.; Jia, Y.-M.; Yu, C.-Y.; Kinami, K.; Hirokami, Y.; Tsuji, Y.; Adachi, I.;
Nash, R.J.; et al. Design and Synthesis of Labystegines, Hybrid Iminosugars from LAB and Calystegine, as
Inhibitors of Intestinal
α-Glucosidases: Binding Conformation and Interaction for ntSI. J. Org. Chem. 2015,
80, 4501–4515. [CrossRef] [PubMed]
46. Jackova, D.; Martinkova, M.; Gonda, J.; Vilkova, M.; Pilatova, M.B.; Takac, P. Stereoselective synthesis and
anticancer activity of broussonetine analogues. Tetrahedron Asymmetry 2017, 28, 1175–1182. [CrossRef]
47. Ribes, C.; Falomir, E.; Murga, J.; Carda, M.; Alberto Marco, J. Convergent, stereoselective syntheses of the
glycosidase inhibitors broussonetines D and M. Org. Biomol. Chem. 2009, 7, 1355–1360. [CrossRef]
48. D’Adamio, G.; Parmeggiani, C.; Goti, A.; Moreno-Vargas, A.J.; Moreno-Clavijo, E.; Robina, I.; Cardona, F.
6-Azido hyacinthacine A(2) gives a straightforward access to the first multivalent pyrrolizidine architectures.
Org. Biomol. Chem. 2014, 12, 6250–6266. [CrossRef]
49. Lahiri, R.; Ansari, A.A.; Vankar, Y.D. Recent developments in design and synthesis of bicyclic azasugars,
carbasugars and related molecules as glycosidase inhibitors. Chem. Soc. Rev. 2013, 42, 5102–5118. [CrossRef]
50. Lahiri, R.; Palanivel, A.; Kulkarni, S.A.; Vankar, Y.D. Synthesis of Isofagomine-Pyrrolidine Hybrid Sugars
and Analogues of (-)-Steviamine and (+)-Hyacinthacine C-5 Using 1,3-Dipolar Cycloaddition Reactions.
J. Org. Chem. 2014, 79, 10786–10800. [CrossRef]
51. Chatterjee, A.K.; Choi, T.L.; Sanders, D.P.; Grubbs, R.H. A general model for selectivity in olefin cross
metathesis. J. Am. Chem. Soc. 2003, 125, 11360–11370. [CrossRef]
52. Wennekes, T.; van den Berg, R.J.B.H.N.; Boltje, T.J.; Donker-Koopman, W.E.; Kuijper, B.; van der Marel, G.A.;
Strijland, A.; Verhagen, C.P.; Aerts, J.M.F.G.; Overkleeft, H.S. Synthesis and Evaluation of Lipophilic
Aza-C-glycosides as Inhibitors of Glucosylceramide Metabolism. Eur. J. Org. Chem. 2010, 1258–1283.
[CrossRef]
53. Wang, W.-B.; Huang, M.-H.; Li, Y.-X.; Rui, P.-X.; Hu, X.-G.; Zhang, W.; Su, J.-K.; Zhang, Z.-L.; Zhu, J.-S.;
Xu, W.-H.; et al. A Practical Synthesis of Sugar-derived Cyclic Nitrones: Powerful Synthons for the Synthesis
of Iminosugars. Synlett 2010, 3, 488–492.

Molecules 2019, 24, 3712
19 of 19
54. Cardona, F.; Faggi, E.; Liguori, F.; Cacciarini, M.; Goti, A. Total syntheses of hyacinthacine A2 and
7-deoxycasuarine by cycloaddition to a carbohydrate derived nitrone. Tetrahedron Lett. 2003, 44, 2315–2318.
[CrossRef]
55. Carmona, A.T.; Whightman, R.H.; Robina, I.; Vogel, P. Synthesis and glycosidase inhibitory activity of
7-deoxycasuarine. Helv. Chim. Acta. 2003, 86, 3066–3073. [CrossRef]
56. Tsou, E.-L.; Chen, S.-Y.; Yang, M.-H.; Wang, S.-C.; Cheng, T.-R.-R.; Cheng, W.-C. Synthesis and biological
evaluation of a 2-aryl polyhydroxylated pyrrolidine alkaloid-based library. Bioorg. Med. Chem. 2008, 16,
10198–10204. [CrossRef]
57. Tsou, E.-L.; Yeh, Y.-T.; Liang, P.-H.; Cheng, W.-C. A convenient approach toward the synthesis of enantiopure
isomers of DMDP and ADMDP. Tetrahedron 2009, 65, 93–100. [CrossRef]
58. Keck, G.E.; Geraci, L.S. Catalytic asymmetric allylation (CAA) reactions. II. A new enantioselective allylation
procedure. Tetrahedron Lett. 1993, 34, 7827–7828. [CrossRef]
59. Bhoite, S.P.; Kamble, R.B.; Suryavanshi, G.M. An enantioselective synthesis of (+)-hygroline and
(+)-pseudohygroline via Keck allylation and CBS reduction. Tetrahedron Lett. 2015, 56, 4704–4705. [CrossRef]
60. Wormald, M.R.; Nash, R.J.; Hrnciar, P.; White, J.D.; Molyneux, R.J.; Fleet, G.W.J. Configurational and
conformational analysis of highly oxygenated pyrrolizidines: Definitive identification of some naturally
occurring 7a-epi-alexines. Tetrahedron Asymmetry 1998, 9, 2549–2558. [CrossRef]
61. Donohoe, T.J.; Sintim, H.O.; Hollinshead, J. A Noncarbohydrate Based Approach to Polyhydroxylated
Pyrrolidizines: Total Syntheses of the Natural Products Hyacinthacine A1 and 1-Epiaustraline. J. Org. Chem.
2005, 70, 7297–7304. [CrossRef]
62. Calveras, J.; Egido-Gabás, M.; Gómez, L.; Casas, J.; Parella, T.; Joglar, J.; Bujons, J.; Clapés, P. Dihydroxyacetone
Phosphate Aldolase Catalyzed Synthesis of Structurally Diverse Polyhydroxylated Pyrrolidine Derivatives
and Evaluation of their Glycosidase Inhibitory Properties. Chem. Eur. J. 2009, 15, 7310–7328. [CrossRef]
[PubMed]
63. Kessler, M.; Acuto, O.; Storelli, C.; Murer, H.; Semenza, G.A. A modified procedure for the rapid preparation
of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating
some properties of d-glucose and choline transport systems. Biochim. Biophys. Acta 1978, 506, 136–154.
[CrossRef]
Sample Availability: Not available.
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