Synthesis of pyrrolidine monocyclic analogues of pochonicine and its stereoisomers: Pursuit of simplified structures and potent β-N-acetylhexosaminidase inhibition

Article abstract of DOI:10.3390/molecules25071498

Ten pairs of pyrrolidine analogues of pochonicine and its stereoisomers have been synthesized from four enantiomeric pairs of polyhydroxylated cyclic nitrones. Among the ten N-acetylamino pyrrolidine analogues, only compounds with 2,5-dideoxy-2,5-imino-d-

Full text of DOI:10.3390/molecules25071498

molecules
Article
Synthesis of Pyrrolidine Monocyclic Analogues of
Pochonicine and Its Stereoisomers: Pursuit of
Simplified Structures and Potent
β-N-Acetylhexosaminidase Inhibition
Xin Yan 1,2, Yuna Shimadate 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;
yanxin2012@iccas.ac.cn (X.Y.); 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;
m1961224@ems.u-toyama.ac.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.); tamarali@iccas.ac.cn (Y.-X.L.); yucy@iccas.ac.cn (C.-Y.Y.);
Tel.: +86-010-626-12893 (C.-Y.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Ram
Received: 21 February 2020; Accepted: 23 March 2020; Published: 25 March 2020
ón J. Estévez Cabanas, George Fleet, Atsushi Kato and Robert J. Nash
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Ten pairs of pyrrolidine analogues of pochonicine and its stereoisomers have been
synthesized from four enantiomeric pairs of polyhydroxylated cyclic nitrones. Among the ten
N-acetylamino pyrrolidine analogues, only compounds with 2,5-dideoxy-2,5-imino-d-mannitol
(DMDP) and pochonicine (1) configurations showed potent inhibition of β-N-acetylhexosaminidases
(
β-HexNAcases); while 1-amino analogues lost almost all their inhibitions towards the tested
enzymes. The assay results reveal the importance of the N-acetylamino group and the possible right
configurations of pyrrolidine ring required for this type of inhibitors.
Keywords: Pochonicine; pharmacophore; synthesis;
β-N-acetylhexosaminidase; iminosugars;
structure-activity relationship
1. Introduction
Since its isolation from fungus Pochonia suchlasporia var. suchlasporia TAMA 87 in 2009 [
pochonicine ( ) (Figure 1) has been an attractive synthetic target due to its potent and specific
inhibition of -N-acetylhexosaminidases ( -HexNAcases), including -N-acetylglucosaminidases
-GlcNAcases) and -N-acetylgalactosaminidases ( -GalNAcases) [ ]. -HexNAcases are associated
with many crucial biological processes [ ]. In fungi and insects, the enzymes play important roles in the
metabolism of the polysaccharide chitin [ ]; in mammals, -HexNAcases participate in the regulation
of cell signalling and influence protein expression, degradation and trafficking [ ]. In humans, many
diseases including lysosomal storage disorders [ ], type-II diabetes [ ], insulin resistance [ ] and
Alzheimer’s disease [ 11] can be attributed to abnormality of -HexNAcases. Therefore, study of
β-HexNAcase inhibitors may provide alternate strategies for discovery of therapeutic drugs.
1],
1
β
β
β
(β
β
β
1,2 β
3
4,
5
β
6
7
6
8
9
–
β
Molecules 2020, 25, 1498; doi:10.3390/molecules25071498
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1498
2 of 24
Figure 1. Naturally occurring potent N-acetylhexosaminidases (HexNAcase) inhibitors.
As one of the most potent
pyrrolizidine skeleton, in contrast to the polyhydroxylated piperidine ring present in the other
two naturally occurring iminosugars, nagstatin ( ) [12] and siastatin B ( ) (Figure 1) [13]. The
N-acetylamino group is the common structural feature that distinguishes these natural products from
the other iminosugars [14]. A diverse range of potent synthetic -HexNAcase iminosugar inhibitors
have been reported including pyrrolidines ( 15 17], 18 19], 20 21] and 22]), piperidines
23 24], 25] and 10 26]), azepanes (11 27 29]) and azetidines (12 30] and 13
them contain an N-acetylamino group (Figure 2) [32].
β-HexNAcase inhibitors, pochonicine (1) possesses a polyhydroxylated
2
3
β
4
[
–
5
[
,
6
[
,
7 [
(8
[
,
9
[
[
[
–
[
[31]); almost all of
H
N
·HCl
e
c
NHM
NHA
H
H
N
H
N
H
N
H
O
N
H
O
5
2
c
NHA
c
H
.
H
.
H
H
e
H
O
O
NHA
O
O
O
O
H
O
8
c
H
H
H
H
H
NHA
H
H
O
O
O
O
O
O
O
4
5
6
7
.
.
.
.
.
.
.
15
]
16
]
22
22
[
[
18
]
18
[
20
]
20
24
]
;
7
24
]
0 μ
M[
0 16 μM[
0 30 μM
]
;
15 μM
]
;
]
]
0 03 μM[
0 20 μM
3 4 μM[
0 64 μM[
2 9 μM[
;
;
3 4 μM[
H
N
H
N
H
H
H
N
M HN
O
N
N
H
O
H
O
H
O
c
NHA
H
e
O
H
O
c
c
HO
OH
H
NHA
H
NHA
O
O
c
H
O
NHA
H
O
H
O
H
H
O
O
11
12
13
9
10
.
.
.
30
30
]
=
29
]
n
n 31
[ ]
o
e erm ne
]
;
[
o
o
2 8 μM[
3 3 μM
26
26
x
A Ki 3 6 μM[
;
N t d t
i
d
N i hibiti
]
;
]
41 μM[
65 μM[
H
.
30
]
1 4 μM[
-
.
x
n
w
un
d
n
x
n : r m
n
;
r m um n
n
t
n
e
kid
y
e
case
β H NA
o
n
co
m
o
p
as a e ce
o
o
ac ea
o
a
ace
l
a
o
r m v n
;
f
o
e
11
i hibi
ti
I
ith
p
ti
f
k b
f
h
p
b
i
C50
(
)
j
Figure 2. Examples of synthetic HexNAcase inhibitors.
The novel structure and inhibition properties of pochonicine (
syntheses [ ], together with its enantiomer [33] and analogues [34]. However, syntheses of such
highly substituted bicyclic pyrrolizidines with up to seven contiguous stereogenic centres are long and
complex [35 36]. In contrast, construction of monocyclic pyrrolidine iminosugars with only four chiral
centres are easier to accomplish. The pyrrolidine core of the corresponding iminosugar frequently
appears to be the HexNAcase pharmacophore [37 39], as the pyrrolidine sections are responsible for
mimicking the transition-state of enzyme reaction [40]. Among these monocyclic iminosugars, a series
of pyrrolidines containing acetamide groups which are essential for their -HexNAcase inhibitions
were reported (Figure 2), among which considerable examples were accomplished before the isolation
of pochonicine ( ) [41 42]. Expectedly, quite a number of these five-membered acetamide derivatives
were found to be potent -HexNAcase inhibitors [16 21 25 26]. In continuation of our interests
in structure-activity relationship study of iminosugars [39 43 46], in this work, 20 stereoisomeric
pyrrolidine analogues of pochonicine ( ) were synthesized and systematically assayed as glycosidase
1) led to the rapid report of total
2
,
–
β
1
,
β
, , ,
,
–
1
inhibitors, in order to look for novel molecules with simplified structure and remained potent
inhibitory activities.

Molecules 2020, 25, 1498
3 of 24
2. Results and Discussion
2.1. Synthesis of 1-N-Acetylamino-2,5-Imino-1,2,5-Trideoxy-l-Mannitol hydrochloride (A-10)
Synthesis of acetamide modified pyrrolidines generally rely on asymmetric synthesis from
achiral starting materials or begin with carbohydrate precursors [32]. In the second strategy,
carbohydrate-derived nitrones have significant advantages due to their ready chirality, availability
and versatile chemistry [47,48]. In this work, polyhydroxylated nitrones A–H (Figure 3) were readily
prepared from the enantiomers of xylose, arabinose, lyxose and ribose by literature methods [16,49–51].
O
N
O
N
O
N
O
N
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
OBn
OBn
OBn
OBn
BnO
BnO
OBn
OBn
BnO
BnO
OBn
OBn
B
D
A
C
O
N
O
N
O
N
O
N
G
H
F
E
Figure 3. Polyhydroxylated nitrones used in building pyrrolidine cores.
The d-xylose-derived nitrone
A is a convenient starting material for the synthesis of
1-amino-2,5-imino-1,2,5-trideoxy-l-mannitol hydrochloride (A-11) and its 1-N-acetylamino derivative
(A-10) [16]. Nucleophilic additions with trimethylsilyl cyanide (TMSCN) [16] and nitromethane [52
]
were studied as agents for the introduction of aminomethyl substituents. Reaction of nitrone
A
with TMSCN at room temperature provide hydroxylamine A-2 in 96% yield as the sole product,
and its C-2 configuration was determined as S-configuration through NOESY experiments since a
strong interaction of H-2 and H-4 was observed; in contrast, the aza-Henry reaction gave a pair of
inseparable epimers (A-20a and A-20b) in a 1:1 ratio. Since the corresponding reduction products
were also difficult to purify, the attempt to introduce aminomethyl groups by aza-Henry reaction was
not further investigated. The addition product A-2 was treated with Raney Ni/H2 in the presence of
Boc2O, and then deprotected to afford diamine A-3 in good yield. Final hydrogenation of A-3 gave
1-amino-2,5-imino-1,2,5-trideoxy-l-mannitol dihydrochloride (A-11) in quantitative yield (Scheme 1).
OH
N
O
N
BnO
N
NH2
NH2
BnO
BnO
10% Pd/C,
Raney Ni, H
rt
,
2
HO
H
H
N
1)
N
H2 3 N
HCl
TMSCN, MeOH/THF
Boc
,
2O,
·2 HCl
rt
CH2Cl2
,
yield
two steps
rt
MeOH,
99%
TFA,
85%
for
rt,
0
℃
to 96%
2)
BnO
HO
OH
A-11
BnO
OBn
BnO
OBn
OBn
OBn
A-2
A-3
A
OH
N
OH
N
O
N
BnO
NO2
BnO
NO2
BnO
rt
,
2
Et
CH Cl
3N,
2
+
=
dr 1:1
82%,
BnO
OBn
A-2'b
BnO
OBn
A-2'a
BnO
A
Scheme 1. Synthesis of compound A-11 from l-arabino-nitrone (A).
Treatment of diamine A-3 with acetic anhydride provided compound A-4 in high yield, but the
attempt to release the secondary amine selectively by hydrochloric acid was unsatisfactory, giving
the target product A-5 in only 19% yield together with part of diamine A-3 recovered (Scheme 2).
Selective reduction of the N-O bond of hydroxylamine A-2, can be achieved in the presence of SmI2
according to the reported method in moderate yield [53]. Since excessive amount of SmI2 was needed
in above step, other reduction conditions were also tried. Though the typical Zn-Cu(OAc)2-AcOH
system [16,54] led to complex mixtures, the modified condition with zinc replaced by iron [55] provided

Molecules 2020, 25, 1498
4 of 24
the target amine A-6 in excellent yield with no effect on the nitrile. Subsequent N-protection gave the
carbamate A-7, which was then hydrogenated to convert the cyanide group to the primary amine.
However, the reduction to amine A-8 proceeded in low yield (20–30%). Though the remaining two
steps, N-acetylation and deprotection both can go smoothly to afford their corresponding products, the
unsatisfactory reduction yield of compound A-7 seriously reduced the total yield of the whole route.
Selective acetylation of the primary amine A-3 was also tried by strictly control of the usage of acetic
anhydride and reaction time [15,16], but no target product was obtained. Unexpectedly, acetylation with
acetic acid in dichloromethane provided the monoacetylated compound A-5 in 18% yield (improved
to 24% yield when anhydrous MgSO4 was added). However, further attempts to improve the reaction
were unsuccessful. In contrast, the mild acylation reagent N,N0,N”,N0”-tetraacetylglycoluril [56
]
showed excellent selectivity for primary amine acetylation, giving the monoacetylated A-5 in 92% yield
when refluxing together in dichloromethane. Final hydrogenation of the intermediate then furnished
the target product A-10 quantitatively.
•H
l
C
,
n
c
c
NHA
B
NHA
H
O
O
H
N
H
10 Pd/C
H2 6N H
%
,
N
l
C
-
,
', '', '''
N N N N
,
e
r
t
e raace
co ur
l
M
H
O
T t
C
tyl ly
il
%
g
,
,
n
n
B
O
re ux
fl
99
%
B
H
O
H
O
H
l
92
O
-
2C 2
-
A 10
A 5
c
A
H
O
n
c
n
n
O
n
NH2
B
O
NHA
B
NHR
B
O
B
N
n
O
H
H
N
,
10 Pd/C
%
N
N
.
N
,
c
conc
,
,
s e s
t p
A
DMAP
H
l
2O
C
2
H2 N H
6
O
l
C
,
,
,
e
r
t
r
r
ux
l
e
f
M
H
H
l
t 95
THF
C
2C 2
%
n
n
B
n
n
n
B
O
n
B
n
99
%
B
B
B
O
O
O
O
O
B
B
O
O
-
-
-
-
=
=
A 2
A 4
A 3
A 3
-
R
R
H 32
%
(
)
,
c
A 5
e
u
c
A
A
19
%
( )
F
A
C
H
O
(
)
2
,
,
c
O
r
t 91
%
oc
oc
B
oc
B
N
B
N
n
O
n
c
n
n
B
B
NH2
n
B
NHA
B
N
n
O
n
O
N
O
H
N
,
,
N
,
ane
e
oc
B
R
y Ni H2
c
A
2O
Et3N
,
2O
)
DMAP
(
,
, ,
r
,
,
r
r
t
H
l
t
M
H
t
C
2C 2
99
O
H
l
C
2C 2
93
-
n
B
n
B
O
n
20 30
%
n
n
B
O
%
B
B
B
B
O
O
%
O
O
O
B
O
-
-
-
-
A 7
A 8
A
A 6
9
Scheme 2. Synthesis of monoacetylated compound A-10.
2.2. Synthesis of 1-Amino and 1-N-Acetylamino Modified Pyrrolidine Stereoisomers
According to the strategy in Schemes 1 and 2, other 18 stereoisomeric pyrrolidine analogues of
pochonicine (
) were synthesized from the corresponding nitrones B−H. Reaction of TMSCN with
nitrones gave exclusively trans-addition products in high yields, with nitrone and as exceptions.
Cyanation of nitrone and both afforded a pair of diastereomers with trans/cis ratios as 63:37 and
61:39, respectively. The configurations of the newly constructed chiral centres in compound D-2
F-2b and H-2 were unambiguously confirmed by X-ray crystallographic analysis (See Supplementary
Materials). The structures of their corresponding enantiomers C-2 E-2b and G-2 can also be confirmed
1
E
F
E
F
,
,
since their NMR data were indeed identical. Reduction of the resulting hydroxylamines by Raney
Ni/H2 in the presence of Boc2O and subsequent deprotection afforded diamines B-3−H-3, which were
then acetylated on the primary amine groups to give compounds B-5−H-5. Final hydrogenation
of intermediates B-3−H-3 and B-5−H-5 provided the target products, i.e., 4·HCl
,
C-10−H-10 and
B-11−H-11 (Table 1).
2.3. Glycosidase Inhibition
The synthesized 1-N-acetylamino and 1-amino pyrrolidine analogues were assayed against a
range of enzymes, as shown in Tables 2 and 3.
Compound D-10 which resembles the pyrrolidine ring of pochonicine (
1) exhibited potent
inhibition of
2.8 M, 10
β
-GlcNAcases from various resources including bovine liver, HL60 and Jack bean (IC50
µ
µ
M and 0.12
µM, respectively). While compound 4·HCl, the 1-deoxy-1-N-acetylamino

Molecules 2020, 25, 1498
5 of 24
derivative of 2,5-dideoxy-2,5-imino-d-mannitol (DMDP) [57], also behaved as similar potent inhibitor
of bovine liver and Jack bean -GlcNAcase (IC50 4.7 M and 0.21 M, respectively). Both the
two compounds were found to potent inhibitors of HL60 -GalNAcase (IC50 9.5 M and 8.8 M,
respectively). However, comparing to the natural product pochonicine ( ), the very powerful inhibitor of
-GlcNAcases and -GalNAcases, both the two analogues showed significant decrease in the inhibition
of two enzymes. Unexpectedly, pochonicine ( ) did not exhibit any inhibition of -GalNAcases,
but compound D-10 showed moderate inhibition of chicken liver -GalNAcase (IC50 65.3 M). For
other 1-N-acetylamino compounds tested in Table 2, part of the compounds are only moderate
β
µ
µ
β
µ
µ
1
β
β
1
α
α
µ
or weak inhibition of the tested
inhibitory activities.
β-HexNAcases, and the other completely lost their β-HexNAcase
As shown in Table 3, 1-amino pyrrolidine analogues failed to provide positive assay results. Most
of them showed weak or no inhibition of all the glycosidases tested. As an exception, compound D-11
with the pyrrolidine ring of pochonicine (
(IC50 54 M) and -GlcNAcase (IC50 99
chicken liver. The assay results further indicated the importance of acetamide groups for
inhibitors. However, the more potent inhibition of compound D-11 of -GalNAcase than compound
1
µ
) were found to be moderate inhibitor of
M) from Jack bean, and -GalNAcase (IC50 44
-HexNAcase
α
-mannosidase
µ
β
α
µM) from
β
α
D-10 may indicate the significant role of the scaffold in interaction with active sites of the enzyme
instead of the 1-N-acetylamino group.
Though all the compounds in Tables 2 and 3 can also be regarded as 1-N-acetylamino and 1-amino
derivatives of their corresponding pyrrolidines (for example, compound 4·HCl is the 1-deoxy-1-N-
acetylamino derivative of DMDP), they lost almost all their inhibition towards other tested glycosidases
including glucosidase, galactosidase, mannosidase,
α-l-fucosidase, trehalase, amyloglucosidase,
α
-l-rhamnosidase and -glucuronidase, revealing the importance of C-1 hydroxyl groups in interaction
β
with the corresponding enzymes.
Therefore, both configurations of the pyrrolidine ring and the 1-N-acetylamino group have
significant influences on the inhibition of
pyrrolidine analogues with the same configuration as DMDP and pochonicine (
β
-HexNAcases and
α
-GalNAcase. In detail, 1-N-acetylamino
) showed powerful
1
inhibition of these enzymes, revealing the importance of the right configurations of A ring. Furthermore,
the results indicate that pochonicine analogue with the A ring in DMDP configuration may also turn out
to be potent inhibitors of the above enzymes. The structure-activity relationship reported in this work
may be helpful in pursuing simplified pochonicine (1) analogues and more potent glycosidase inhibitors.

Molecules 2020, 25, 1498
6 of 24
Table 1. 1-Amino and 1-N-acetylamino pyrrolidine analogues synthesized from cyclic nitrones.
Entry
Cyclic Nitrone
Hydroxylamine
Diamine
Monoacetylated Pyrrolidine
1-Amino Product
1-N-Acetylamino Product
OH
N
O
NH2
H
NH2
NHAc
H
H
N
NHAc
H
N
N
N
N
N
5
2
5
2
HO
BnO
BnO
2
HCl
BnO
BnO
HO
HCl
1
BnO
OBn
HO
OH
BnO
OBn
BnO
OBn
HO
BnO
OH
OBn
arabino-nitrone
A)
A-11
A-3
A-5
A-10
-
A-2
L
(88%)c
(99%)d
(99%)e
(85%)b
(96%)a
(
O
N
OH
N
NHAc
NH2
NHAc
H
N
NH2
H
N
H
N
H
N
N
BnO
HO
BnO
BnO
BnO
BnO
HO
HO
HO
HO
HCl
2 HCl
BnO
2
3
4
HO
OH
HCl
BnO
OBn
BnO
OBn
HO
OH
BnO
OBn
BnO
OBn
-
B-5
B-11
B-2
B-3
4
·
D
arabino-nitrone
(91%)
(99%)
B
(94%)
(99%)
(88%)
(
)
O
N
NHAc
OH
N
NH2
2 HCl
NH2
NHAc
H
N
H
N
H
N
H
N
N
.
HO
BnO
BnO
HCl
BnO
HO
OH
HO
OH
BnO
OBn
BnO
OBn
BnO
OBn
-nitrone
BnO
OBn
-
C-3
C-5
C-11
C-2
C-10
L
lyxo
(86%)
(85%)
(99%)
C
(96%)
(99%)
(
)
O
N
OH
N
NH2
2 HCl
NH2
H
N
NHAc
HCl
H
N
NHAc
H
N
N
H
N
HO
BnO
BnO
BnO
HO
OH
BnO
OBn
HO
OH
BnO
OBn
BnO
D-5
OBn
BnO
OBn
-nitrone
D
-
D-3
D-11
D-2
D-10
D
lyxo
(
(89%)
(82%)
(92%)
(99%)
(99%)
)
NHAc
HCl
NH2
NH2
NH2
NHAc
NH2
H
H
N
H
N
H
R
N
R
N
HO
BnO
BnO
BnO
BnO
2
HCl
OH
O
N
HO
OH
BnO
OBn
HO
OH
BnO
OBn
R2
N
E-5a
E-11a
E-10a
E-3a
88%)
R1
BnO
(90%)
(99%)
BnO
(99%)
(
5
BnO
OBn
=
BnO
OBn
-
=
E-2a
E-2b
R1 CN, R2
H
(58%)
(35%)
L
-nitrone
xylo
c
NHA
H
N
NHAc
NH2
H
H
N
H
=
=
CN
R1
R
H,
E
2
N
S
(
)
N
S
HO
BnO
HO
HCl
2 HCl
BnO
OBn
HO
OH
BnO
BnO
BnO
OBn
HO
OH
-
E-11b
E-5b
E-3b
E 10b
99%
(99%)
(82%)
(
)
(90%)
NH2
2 HCl
NHAc
HCl
NHAc
H
H
H
N
H
N
S
R
N
N
HO
HO
BnO
OH
N
HO
OH
BnO
OBn
HO
O
OBn
OH
R1
E-11a
F-3a
F-5a
F-10a
N
R2
(99%)
(87%)
BnO
(83%)
(99%)
BnO
6
BnO
=
OBn
=
BnO
OBn
-
F-2a
F-2b
R1 CN, R2
H
(59%)
(37%)
c
F
NH2
2 HCl
NHAc
NHA
H
H
N
H
D
-ni)trone(
NH2
xylo
H
N
R
=
=
CN
N
R
R1
R
N
H,
2
HO
BnO
H
O
BnO
HCl
HO
OH
OBn
H
O
O
H
BnO
OBn
-
F-5b
(83%)
F-3b
F-11b
F 1
0b
99%
(85%)
(99%)
(
)

Molecules 2020, 25, 1498
7 of 24
Table 1. Cont.
Monoacetylated Pyrrolidine
Entry
Cyclic Nitrone
Hydroxylamine
Diamine
1-Amino Product
1-N-Acetylamino Product
O
OH
NHAc
NH2
H
NH2
H
N
NHAc
H
H
N
N
N
N
N
CN
2 HCl
BnO
HO
BnO
HCl
HO
BnO
BnO
7
HO
OH
BnO
OBn
BnO
OBn
HO
OH
BnO
OBn
BnO
OBn
-
G-11
G-10
G-5
G-2
G-3
L
ribo-nitrone
(99%)
(99%)
(86%)
G
(87%)
(87%)
(
)
O
OH
N
NH2
NH2
2 HCl
NHAc
NHAc
HCl
H
N
H
N
H
N
H
N
N
CN
OBn
BnO
HO
BnO
HO
BnO
BnO
8
BnO
OBn
HO
OH
BnO
OBn
HO
OH
BnO
BnO
-
OBn
H-11
(99%)
H-10
H-3
H-5
H-2
(91%)
D
ribo-nitrone
(99%)
(88%)
H
(81%)
(
)
a Yield of the corresponding hydroxylamines starting from cyclic nitrones; b Total yield in 2 steps starting from hydroxylamines to diamines; c Yield of monoacetylated pyrrolidines from the
corresponding diamines; d Hydrogenation yield of 1-amino products from diamines; e Hydrogenation yield of 1-N-acetylamino products from the corresponding monoacetylated pyrrolidines.
Table 2. Concentrations of 1-N-acetylamino pyrrolidine analogues giving 50% inhibition of various enzymes.
IC50 (µM)
OH
NHAc
HCl
NHAc
HCl
NHAc
HCl
NHAc
NHAc
HCl
NHAc
HCl
NHAc
NHAc
HCl
NHAc
HCl
NHAc
HCl
H
N
H
H
H
H
H
H
H
N
H
H
R
N
R
S
N
N
N
.
N
N
N
N
S
N
5
2
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HCl
HCl
NHAc
Enzyme
H
HO
OH
HO
OH
HO
HO
OH
HO
OH
HO
OH
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO
OH
A-10
4·HCl
C-10
NI (0%)
NI (18.2%)
NI (35.4%)
D-10
E-10a
F-10a
E-10b
F-10b
G-10
H-10
Pochonicine (1)
α-Glucosidase
Yeast
Rice
c
NIa (0.5%)b
450
1000
211
NI (0%)
NI (26.1%)
NI (9.24%)
NI (0%)
NI (15.9%)
NI (12.7%)
NI (6.91%)
NI (12.5%)
NI (14.6%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (12.7%)
NI (0%)
NI (0%)
NI (16.0%)
NI (0%)
NI (5.6%)
NI (13.6%)
—
568
92
—
—
Rat intestinal maltase
β-Glucosidase
Almond
Bovine liver
α-Galactosidase
Coffee beans
β-Galactosidase
Bovine liver
α-Mannosidase
Jack bean
NI (16.1%)
NI (2.0%)
170
NI (0%)
NI (1.0%)
NI (41.0%)
NI (2.4%)
NI (37.9%)
NI (4.1%)
NI (11.6%)
NI (0.3%)
NI (1.2%)
NI (0%)
NI (19.8%)
NI (0%)
NI (15.3%)
NI (0%)
NI (17.1%)
NI (1.7%)
—
—
NI (33.8%)
NI (1.1%)
511
NI (0%)
NI (1.6%)
NI (0%)
NI (0%)
NI (0%)
NI (0.36%)
NI (1.3%)
NI (0.4%)
NI (0%)
380
NI (0.36%)
NI (4.9%)
NI (0.7%)
NI (25.8%)
NI (41.7%)
NI (0.36%)
NI (4.2%)
NI (0%)
NI (0%)
NI (3.9%)
NI (3.8%)
NI (0%)
NI (0%)
NI (4.2%)
NI (1.6%)
NI (0%)
NI (0%)
NI (2.9%)
NI (4.0%)
NI (0%)
NI (0%)
NI (3.6%)
NI (9.5%)
NI (0%)
—
—
—
—
—
NI (3.3%)
205
NI (0%)
296
β-Mannosidase
Snail
α-l-Fucosidase
Bovine kidney
NI (0%)
NI (1.7%)
NI (3.23%)
NI (8.4%)
NI (1.5%)
NI (4.9%)
NI (20.5%)
NI (3.9%)
NI (11.5%)
NI (22.3%)

Molecules 2020, 25, 1498
8 of 24
Table 2. Cont.
IC50 (µM)
OH
OH
NHAc
HCl
NHAc
HCl
NHAc
HCl
NHAc
NHAc
HCl
NHAc
H
NHAc
H
NHAc
HCl
NHAc
HCl
NHAc
HCl
H
N
H
H
H
H
H
N
H
H
R
N
R
S
N
N
N
.
N
N
N
N
S
N
5
2
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HCl
HCl
HCl
NHAc
Enzyme
H
HO
OH
HO
OH
HO
HO
OH
HO
OH
HO
OH
OH
HO
OH
HO
OH
HO
OH
HO
OH
HO
A-10
4·HCl
C-10
NI (0%)
D-10
E-10a
F-10a
E-10b
F-10b
G-10
H-10
Pochonicine (1)
α,α-Trehalase
Porcine kidney
Amyloglucosidase
A. niger
α-l-Rhamnosidase
P. decumbens
NI (4.3%)
NI (5.5%)
NI (10.1%)
NI (20.3%)
NI (40.6%)
NI (6.5%)
NI (0%)
NI (3.8%)
NI (8.3%)
NI (5.6%)
NI (4.0%)
NI (11.8%)
NI (3.0%)
NI (2.8%)
NI (5.3%)
NI (0%)
NI (6.4%)
NI (30.8%)
NI (0%)
NI (5.7%)
NI (3.0%)
NI (5.6%)
NI (7.6%)
NI (9.5%)
NI (40.9%)
NI (8.3%)
NI (7.7%)
—
—
—
NI (1.9%)
NI (3.6%)
β-Glucuronidase
E. coli
Bovine liver
NI (0%)
NI (4.6%)
NI (0.6%)
NI (1.7%)
NI (0.3%)
NI (0%)
NI (0%)
NI (1.3%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (4.3%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
—
—
β-N Acetylhexosaminidase
Bovine liver
HL60
Jack bean
A. oryzae
Human placenta
β-N-Acetylgalactosaminidase
HL60
α-N-Acetylgalactosaminidase
Chicken liver
943
NI (12.7%)
202
4.7
34
0.21
—
NI (36.3%)
NI (3.9%)
2.8
10
0.12
—
95
591
10
—
—
NI (23.2%)
NI (0%)
NI (42%)
—
NI (38.4%)
NI (4.5%)
652
NI (16.6%)
299
NI (18.8%)
NI (33.7%)
NI (0.3%)
0.021 [2]
0.018 [2]
0.0016 [2]
0.33 [2]
129
—
—
115
—
—
98
—
—
26
—
—
241
—
—
—
—
—
—
—
0.012 [2]
NI (13.0%)
NI (0%)
9.5
NI (1.4%)
8.8
490
NI (0.9%)
NI (10.1%)
NI (36.1%)
NI (15.9%)
NI (2.3%)
NI (5.3%)
NI (6.1%)
0.049 [2]
NI (3.3%)
NI (6.1%)
65.3
NI (1.4%)
NI (0%)
NI (0.5%)
NI (2.3%)
NI (9.0%) [2]
a
NI: No Inhibition (less than 50% at 1000 µM); b ( ): Inhibition % at 1000 µM; c —: Not determined.
Table 3. Concentrations of 1-amino pyrrolidine analogues giving 50% inhibition of various enzymes.
IC50 (µM)
NH2
NH2
NH2
NH2
2
NH2
H
NH2
H
NH2
NH2
NH2
H
H
NH2
H
H
N
S
H
R
N
H
H
N
R
H
N
S
N
N
N
N
N
N
2 HCl
HO
HO
2 HCl
HO
HO
2 HCl
HO
HO
2 HCl
HO
HO
HO
HO
2 HCl
HCl
2 HCl
2 HCl
2 HCl
2 HCl
Enzyme
HO
OH
HO
OH
HO
OH
HO
HO
OH
HO
OH
HO
OH
OH
HO
OH
HO
OH
HO
OH
A-11
B-11
C-11
D-11
E-11
F-11a
E-11b
NI (0%)
NI (12.9%)
NI (10.9%)
F-11b
G-11
H-11
α-Glucosidase
Yeast
Rice
Rat intestinal maltase
β-Glucosidase
NIa (0%)b
286
NI (42.7%)
725
NI (9.55%)
NI (0%)
NI (37.1%)
NI (4.52%)
NI (8.4%)
NI (4.02%)
NI (9.55%)
NI (15.7%)
NI (9.95%)
NI (10.1%)
NI (31.5%)
NI (48.0%)
NI (0%)
NI (16.9%)
NI (20.8%)
NI (0%)
NI (11.8%)
NI (%2.64)
NI (0%)
NI (3.4%)
NI (10.1%)
68
251

Molecules 2020, 25, 1498
9 of 24
Table 3. Cont.
IC50 (µM)
NH2
2 HCl
NH2
2 HCl
NH2
2 HCl
NH2
2
NH2
2 HCl
NH2
2 HCl
NH2
2 HCl
H
NH2
2 HCl
NH2
H
NH2
2 HCl
H
H
N
S
H
R
N
H
N
H
N
H
H
N
R
H
N
S
N
N
N
N
HO
HO
HO
HO
HO
HO
HO
HCl
HO
HO
HO
2 HCl
Enzyme
HO
OH
HO
OH
HO
OH
HO
HO
OH
HO
OH
HO
OH
OH
HO
OH
HO
OH
HO
OH
A-11
B-11
C-11
D-11
E-11
F-11a
E-11b
F-11b
G-11
H-11
Almond
Bovine liver
α-Galactosidase
Coffee beans
β-Galactosidase
Bovine liver
α-Mannosidase
Jack bean
β-Mannosidase
Snail
α-l-Fucosidase
Bovine kidney
α,α-Trehalase
Porcine kidney
Amyloglucosidase
A. niger
NI (0%)
NI (0%)
NI (44.4%)
NI (16.2%)
NI (13.0%)
NI (0.7%)
419
NI (12.4%)
NI (0%)
NI (6.0%)
NI (0%)
NI (4.8%)
NI (0%)
NI (13.5%)
NI (0%)
NI (2.3%)
NI (0%)
NI (18.8%)
NI (3.8%)
NI (3.1%)
NI (42.2%)
NI (6.5%)
54
NI (0%)
NI (0%)
NI (36.3%)
NI (0%)
NI (0%)
NI (0%)
NI (4.7%)
589
NI (1.9%)
NI (2.6%)
NI (0%)
NI (0%)
NI (2.0%)
NI (0.32%)
NI (2.6%)
NI (21.2%)
NI (4.7%)
NI (9.1%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (2.6%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (4.2%)
NI (3.81%)
NI (1.2%)
NI (16.2%)
NI (0%)
NI (1.0%)
NI (0%)
NI (0.97%)
NI (1.9%)
NI (46.4%)
NI (0%)
NI (12.1%)
NI (0%)
NI (5.19%)
NI (0%)
NI (0%)
NI (3.35%)
NI (39.9%)
NI (0.9%)
NI (0%)
NI (2.23%)
NI (8.0%)
NI (0%)
NI (0%)
NI (13.1%)
NI (0%)
NI (19.9%)
NI (2.2%)
NI (0%)
NI (16.2%)
NI (0.6%)
NI (0%)
NI (16.2%)
NI (3.8%)
NI (1.1%)
NI (1.6%)
NI (0.9%)
NI (0%)
NI (0.7%)
NI (0%)
NI (0%)
NI (2.1%)
NI (1.3%)
α-l-Rhamnosidase
P. decumbens
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (1.6%)
β-Glucuronidase
E. coli
Bovine liver
NI (0%)
NI (0.8%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (5.7%)
NI (0%)
NI (4.1%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (0%)
NI (4.9%)
β-N-Acetylhexosaminidase
Bovine liver
HL60
NI (2.1%)
NI (0%)
NI (21.1%)
NI (0%)
NI (0%)
NI (14.5%)
NI (0%)
NI (0%)
NI (14.5%)
NI (0%)
NI (3.1%)
99
NI (0%)
NI (1.3%)
NI (19.3%)
NI (0%)
NI (2.7%)
NI (30.7%)
NI (0%)
NI (0%)
NI (23.5%)
NI (1.6%)
NI (0%)
264
NI (1.6%)
NI (0%)
NI (29.5%)
NI (1.3%)
NI (0%)
NI (7.2%)
Jack bean
β-N-Acetylgalactosaminidase
HL60
α-N-Acetylgalactosaminidase
Chicken liver
NI (5.2%)
NI (4.7%)
NI (3.8%)
NI (2.9%)
NI (14.8%)
NI (4.8%)
NI (6.0%)
NI (11.9%)
NI (8.1%)
NI (0%)
NI (1.9%)
NI (0%)
NI (7.5%)
NI (0.5%)
44
NI (0%)
NI (5.1%)
NI (0%)
NI (13.6%)
NI (2.8%)
a
NI: No Inhibition (less than 50% at 1000 µM); b ( ): Inhibition % at 1000 µM.

Molecules 2020, 25, 1498
10 of 24
3. Materials and Methods
3.1. General Methods
All reagents were used as received without any further purification or prepared as described in
the literature. 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,
Inno-chem, Beijing, China). NMR spectra were measured in CDCl3 (with TMS as internal standard)
or D2O (with H2O as internal standard) on a Bruker AV300, AV400 or AV500 magnetic resonance
spectrometer (Bruker, Ettlingen, Germany) (1H NMR at 300 MHz, 400 MHz or 500 MHz, 13C NMR
at 125 MHz). High-resolution mass spectra (HRMS) were performed on a Thermo Fisher Exactive
Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Polarimetry was determined using an
Optical Activity AA-10R polarimeter with concentrations (c) given in gram per 100 mL. Infrared spectra
were recorded as films on KBr plates on a Nicolet-6700 FT-IR spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA).
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. [58] 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 [59].
3.3. Chemistry
3.3.1. General Procedure for Synthesis of Hydroxylamines A-2, B-2, C-2, D-2, E-2a, E-2b, F-2a, F-2b,
G-2 and H-2 with A-2 as an Example
To a solution of nitrone
A
(1.25 g, 3.00 mmol) in THF (5 mL) and me◦thanol (25 mL) was
added dropwise TMSCN (0.45 mL, 3.60 mmol) under Ar atmosphere at 0 C. After stirring at
room temperature for 6–8 h, TLC showed completion of the reaction. The resulting solution was
concentrated in vacuo, and the residue was purified by flash column chromatography (silica gel,
petroleum ether/EtOAc = 6/1) to give hydroxylamine A-2 (colourless syrup, 1.28 g, 96% yield).
Data for (2S,3S,4S,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine (A-2)
23
(Ref. [16]): [
α
]
D
−
9.5 (c 1.1 in CH2Cl2);
ν
max/cm−1: 3366 (s), 3088 (m), 3063 (m), 3030 (s), 2920 (s), 2867
1
(vs), 2237 (w), 1497 (s), 1454 (vs), 1362 (s), 1207 (m), 1100 (vs), 1028 (s), 738 (vs), 697 (vs); H NMR
(500 MHz, CDCl3)
1H, H-2), 4.13 (t, J = 2.1 Hz, 1H, H-3), 3.93 (dd, J = 6.5 and 2.2 Hz, 1H, H-4), 3.72 (dd, J = 10.5 and 3.3 Hz,
1H, H-6), 3.54 (dd, J = 10.5 and 4.0 Hz, 1H, H-60), 3.28–3.25 (m, 1H, H-5); 13C NMR (125 MHz, CDCl3)
δ (ppm): 7.36–7.20 (m, 15H), 6.52 (s, 1H, OH), 4.54–4.37 (m, 6H), 4.20 (d, J = 1.5 Hz,
δ
(ppm): 137.4, 137.3, 136.4, 128.7, 128.4, 128.3, 128.0, 127.97, 127.93, 127.88, 127.82, 115.7, 83.6 (C-3), 81.2
(C-4), 73.3 (PhCH2), 72.3 (PhCH2), 72.0 (PhCH2), 69.4 (C-5), 66.4 (C-6), 61.2 (C-2); HRMS (ESI): calcd for
C27H28O4N2Na+ [M + Na+] 467.1941, found 467.1941.
Data for (2R,3R,4R,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine
22
(B-2): Colourless syrup, 1.26 g, 94% yield from nitrone
in CH2Cl2);
1097 (vs), 1027 (m), 737 (s), 697 (vs); 1H NMR (500 MHz, CDCl3)
B
(1.26 g, 3.02 mmol); [
α
]
D
+8.7 (c 1.0
ν
max/cm−1: 3359 (m), 3031 (m), 2868 (m), 2239 (w), 1496 (m), 1454 (s), 1362 (m), 1207 (m),
δ
(ppm): 7.36–7.20 (m, 15H), 6.46
(s, 1H, OH), 4.55–4.37 (m, 6H), 4.20 (d, d, J = 1.5 Hz, 1H, H-2), 4.14 (t, J = 2.1 Hz, 1H, H-3), 3.94 (dd,
J = 6.5 and 2.2 Hz, 1H, H-4), 3.72 (dd, J = 10.5 and 3.3 Hz, 1H, H-6), 3.54 (dd, J = 10.5 and 4.0 Hz, 1H,

Molecules 2020, 25, 1498
11 of 24
H-60), 3.28–3.26 (m, 1H, H-5); 13C NMR (125 MHz, CDCl3)
δ
(ppm): 137.4, 137.3, 136.4, 128.7, 128.4,
128.3, 128.0, 127.96, 127.93, 127.87, 127.82, 115.7, 83.6 (C-3), 81.2 (C-4), 73.3 (PhCH2), 72.3 (PhCH2), 72.0
(PhCH2), 69.4 (C-5), 66.4 (C-6), 61.1 (C-2); HRMS (ESI): calcd for C27H28O4N2Na+ [M + Na+] 467.1941,
found 467.1937.
Data for (2S,3S,4R,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine (C-2)
23
(Ref. [60]): Colourless syrup, 1.32 g, 96% yield from nitrone
CH2Cl2);
(m), 1144 (s), 1102 (s), 1027 (s), 736 (s), 697 (vs); 1H NMR (500 MHz, CDCl3)
C
(1.30 g, 3.12 mmol); [
α
]
+8.1 (c 1.5 in
D
ν
max/cm−1: 3306 (m), 3030 (m), 2925 (s), 2855 (s), 2251 (w), 1497 (m), 1454 (s), 1362 (m), 1209
δ
(ppm.): 7.34–7.23 (m,
15H), 6.72 (s, 1H, OH), 4.69–4.44 (m, 6H), 4.33 (t, J = 5.5 Hz, 1H, H-3), 4.28 (d, J = 5.6 Hz, 1H. H-2), 4.22
(t, J = 5.7 Hz, 1H, H-4), 3.72 (dd, J = 9.6 Hz and 6.9 Hz, 1H, H-6), 3.66 (dd, J = 9.3 Hz and 6.7 Hz, 1H,
H-60), 3.55–3.51 (m, 1H, H-5); 13C NMR (125 MHz, CDCl3)
δ (ppm): 137.79, 137.70, 136.7, 128.6, 128.45,
128.43, 128.2, 128.0, 127.9, 127.89, 127.81, 116.4, 80.2 (C-3), 75.8 (C-4), 73.8 (PhCH2), 73.4 (PhCH2), 73.1
(PhCH2), 68.9 (C-5), 67.4 (C-6), 60.4 (C-2); HRMS (ESI): calcd for C27H28O4N2Na+ [M + Na+] 467.1941,
found 467.1939.
Data for (2R,3R,4S,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine
23
(D-2) (Ref. [61]): Colourless syrup, 1.19 g, 92% yield from nitrone
1.0 in CH2Cl2);
1209 (m), 1145 (s), 1102 (s), 1027 (s), 736 (s), 697 (vs); 1H NMR (500 MHz, CDCl3)
D
(1.22 g, 2.93 mmol); [
α
]
D
−
10.2 (c
ν
max/cm−1: 3328 (m), 3030 (m), 2925 (s), 2870 (s), 2251 (w), 1497 (m), 1454 (s), 1362 (m),
δ
(ppm): 7.36–7.22 (m,
15H), 6.78 (s, 1H, OH), 4.69–4.43 (m, 6H), 4.33 (t, J = 5.5 Hz, 1H, H-3), 4.28 (d, J = 5.6 Hz, 1H. H-2),
4.22 (t, J = 5.7 Hz, 1H, H-4), 3.72 (dd, J = 9.6 Hz and 6.9 Hz, 1H, H-6), 3.65 (dd, J = 9.3 Hz and 6.7 Hz,
1H, H-60), 3.55–3.51 (m, 1H, H-5); 13C NMR (125 MHz, CDCl3)
δ (ppm): 137.79, 137.71, 136.7, 128.6,
128.46, 128.43, 128.2, 128.0, 127.89, 127.81, 116.5, 80.2 (C-3), 75.8 (C-4), 73.8 (PhCH2), 73.4 (PhCH2), 73.1
(PhCH2), 69.0 (C-5), 67.4 (C-6), 60.4 (C-2); HRMS (ESI): calcd for C27H28O4N2Na+ [M + Na+] 467.1941,
found 467.1938.
(2R,3R,4R,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine (E-2a) and
(2S,3R,4R,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine (E-2b): 92%
total yield from nitrone E (1.34 g, 3.21 mmol).
Data for E-2a: White solid, 821 mg, 58% yield; m.p. 117–119 ◦C; [
3308 (m), 3028 (m), 2870 (m), 2245 (w), 1497 (m), 1453 (m), 1361 (m), 1144 (m), 1109 (s), 1043 (m),
1028 (m), 736 (vs), 693 (vs); 1HNMR (500 MHz, CDCl3)
(ppm):7.37–7.23 (m,15H), 5.75 (s, 1H, OH),
4.56–4.45 (m, 6H), 4.15 (dd, J = 5.6 and 1.4 Hz, 1H, H-2), 4.00 (dd, J = 6.1 and 1.4 Hz, 1H, H-4), 3.83
(t, J = 9.1 Hz,1H), 3.73 (m, 2H, H-6), 3.39–3.35 (m,1H, H-5); 13C NMR (125 MHz, CDCl3)
(ppm):
α] ν :
23 +7.8 (c 0.7 in CH2Cl2); max/cm−1
D
δ
δ
137.8, 137.2, 136.4, 128.6, 128.5, 128.4, 128.3, 128.0, 127.91, 127.90, 127.83, 127.81, 118.35, 83.9 (C-3), 80.0
(C-4), 73.5 (PhCH2), 72.4 (PhCH2), 72.3 (PhCH2), 69.1 (C-6), 67.1 (C-5), 63.2 (C-2); HRMS (ESI): calcd for
C27H28O4N2Na+ [M + Na+] 467.1941, found 467.1938.
◦
23
Data for E-2b: White solid, 491 mg, 35% yield; m.p. 111–114 C; [
α
]
−
38.1 (c 1.0 in CH2Cl2);
D
ν
max/cm−1: 3359 (m), 3031 (m), 2868 (m), 2237 (w), 1496 (m), 1454 (m), 1362 (m), 1207 (m), 1097 (s),
1027 (m), 737 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
δ
(ppm):7.35–7.21 (m, 15H), 5.98 (s, 1H, OH),
4.59–4.48 (m, 6H), 4.37 (d, J = 6.4 Hz, 1H, H-2), 4.18 (dd, J = 7.7 and 4.3 Hz, 1H, H-4), 4.07 (dd, J = 6.3
and 4.4 Hz, 1H, H-3), 3.80 (dd, J = 9.7 and 5.7 Hz, 1H, H-6), 3.68 (dd, J = 9.6 and 5.7 Hz, 1H, H-60),
3.63 δ (ppm): 137.7, 137.5, 136.6, 128.6, 128.4, 128.3,
3.59 (m, 1H, H-5); 13C NMR (125 MHz, CDCl3)
−
128.2, 127.9, 127.8, 127.78, 127.73, 114.6, 81.1 (C-3), 80.8 (C-4), 73.5 (PhCH2), 73.0 (PhCH2), 72.9 (PhCH2),
67.4 (C-6), 66.7 (C-5), 60.6 (C-2); HRMS (ESI): calcd for C27H28O4N2Na+ [M + Na+] 467.1941, found
467.1939.
(2S,3S,4S,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine (F-2a) and
(2R,3S,4S,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine (F-2b): 96%
total yield from nitrone F (1.50 g, 3.60 mmol).

Molecules 2020, 25, 1498
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23
Data for F-2a: White solid, 985 mg, 59% yield; m.p. 118–119 ◦C; [
α
]
D
−
5.5 (c 1.0 in CH2Cl2); ν :
max/cm−1
3326 (m), 3028 (m), 2911 (m), 2870 (m), 2245 (w), 1497 (m), 1454 (s), 1241 (m), 1216 (m), 1144 (s), 1110 (vs),
736 (vs), 693 (vs); 1H NMR (500 MHz, CDCl3)
(ppm):7.37–7.23 (m,15H), 5.70 (s, 1H, OH), 4.56–4.45
(m, 6H), 4.15 (dd, J = 5.7 and 1.4 Hz, 1H, H-2), 4.00 (dd, J = 6.1 and 1.4 Hz, 1H, H-4), 3.83 (dd, J = 9.2
and 7.7Hz,1H, H-3), 3.75–3.71 (m, 2H, H-6), 3.39–3.35 (m,1H); 13C NMR (125 MHz, CDCl3)
(ppm):
δ
δ
137.8, 137.2, 136.4, 128.6, 128.5, 128.4, 128.3, 128.0, 127.91, 127.90, 127.83, 127.81, 118.3, 83.9 (C-3), 80.0
(C-4), 73.5 (PhCH2), 72.4 (PhCH2), 72.3 (PhCH2), 69.1 (C-6), 67.1 (C-5), 63.2 (C-2); HRMS (ESI): calcd for
C27H28O4N2Na+ [M + Na+] 467.1941, found 467.1938.
◦
23
Data for F-2b: White solid, 612 mg, 37% yield; m.p. 113–115 C; [
α
]
+35.3 (c 1.0 in CH2Cl2);
D
ν
max/cm−1: 3359 (m), 3031 (m), 2868 (m), 2239 (w), 1496 (m), 1454 (m), 1362 (m), 1207 (s), 1097 (s), 1027
(m), 737 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
δ
(ppm):7.35–7.21 (m, 15H), 6.04 (s, 1H, OH), 4.59
–4.47 (m, 6H), 4.37 (d, J = 6.3 Hz, 1H, H-2), 4.18 (dd, J = 7.7 and 4.3 Hz, 1H, H-4), 4.07 (dd, J = 6.3 and
4.4 Hz, 1H, H-3), 3.80 (dd, J = 9.7 and 5.7 Hz, 1H, H-6), 3.68 (dd, J = 9.6 and 5.7 Hz, 1H, H-60), 3.63
3.59
(m, 1H, H-5); 13C NMR (125 MHz, CDCl3)
(ppm): 137.7, 137.5, 136.6, 128.6, 128.4, 128.3, 128.2, 127.9,
−
δ
127.8, 127.78, 127.74, 114.6, 81.1 (C-3), 80.8 (C-4), 73.5 (PhCH2), 73.0 (PhCH2), 72.9 (PhCH2), 67.4 (C-6),
66.7 (C-5), 60.6 (C-2); HRMS (ESI): calcd for C27H28O4N2Na+ [M + Na+] 467.1941, found 467.1939.
Data for (2R,3R,4S,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine
(G-2) (Ref. [61]): White solid, 1.39 g, 87% yield from nitrone
G
(1.51 g, 3.62 mmol); m.p. 106–109 ◦C;
23
[
α
]
+0.6 (c 1.0 in CH2Cl2); ν
max/cm−1: 3403 (m), 3029 (m), 2922 (m), 2871 (m), 2242 (w), 1497 (m),
D
1453 (m), 1352 (m), 1224 (m), 1145 (s), 1101 (s), 1027 (s), 742 (s), 694 (vs); 1HNMR (500 MHz, CDCl3)
(ppm):7.35–7.24 (m,15H), 6.05 (s, 1H, OH), 4.70–4.43 (m, 6H), 4.10–4.05 (m, 2H, H-3, H-2), 3.83 (t,
J = 6.1 Hz, 1H, H-4), 3.53–3.51 (m, 2H, H-6, H-60), 3.31 3.30 (m,1H, H-5); 13C NMR (125 MHz, CDCl3)
(ppm): 137.6, 137.4, 136.7, 128.6, 128.48, 128.44, 128.2, 128.13, 128.11, 127.98, 127.90, 127.88, 127.86,
δ
−
δ
118.5, 77.3 (C-3), 75.2 (C-4), 73.3 ((PhCH2)), 72.8 (PhCH2), 72.2 (PhCH2), 72.0 (C-5), 68.0 (C-6), 61.8 (C-2);
HRMS (ESI): calcd for C27H28O4N2Na+ [M + Na+] 467.1941, found 467.1936.
Data for (2S,3S,4R,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-1-hydroxypyrrolidine
23
(H-2): White solid, 1.45 g, 91% yield from nitrone
(c 1.1 in CH2Cl2);
1225 (m), 1149 (s), 1102 (s), 1035 (s), 1028 (s), 743 (vs), 694 (vs); 1H NMR (500 MHz, CDCl3)
7.34–7.23 (m,15H), 6.11 (s, 1H, OH), 4.68–4.43 (m, 6H), 4.10–4.05 (m, 2H, H-3, H-2), 3.81 (t, J= 5.1 Hz,
1H, H-4), 3.53–3.48 (m, 2H, H-6, H-60), 3.31–3.28 (m,1H, H-5); 13C NMR (125 MHz, CDCl3)
(ppm):
H
(1.50 g, 3.60 mmol); m.p. 109–111 ◦C; [
α
]
−
1.4
D
ν
max/cm−1: 3407 (m), 3029 (m), 2873 (m), 2242 (w), 1497 (m), 1453 (m), 1352 (m),
δ
(ppm):
δ
137.6, 137.4, 136.7, 128.6, 128.49, 128.45, 128.2, 128.13, 128.11, 127.98, 127.91, 127.8, 118.6, 77.3 (C-3), 75.2
(C-4), 73.3 (PhCH2), 72.8 (PhCH2), 72.2 (PhCH2), 72.0 (C-5), 68.0 (C-6), 61.8 (C-2); HRMS (ESI): calcd for
C27H28O4N2Na+ [M + Na+] 467.1941, found 467.1933.
3.3.2. General Procedure for Synthesis of Hydroxylamines A-3, B-3, C-3, D-3, E-3a, E-3b, F-3a, F-3b,
G-3 and H-3 with A-3 as an Example
Hydroxylamine A-2 (1.28 g, 2.88 mmol) was dissolved in methanol (10 mL), followed by Boc2O
(1.46 mL, 6.30 mmol) and Raney Ni (1.0 g, 60%). The suspension was stirred under hydrogen
atmosphere for 4 h when TLC showed completion of the reaction. Hydrogen was replaced by
nitrogen, and catalyst was removed from the reaction mixture by filtration. The filtrate was
concentrated in vacuo to afford the crude produc◦t as a colourless oil. The intermediate was dissolved
in dichloromethane (10 mL) and cooled to 0 C, trifluoroacetic acid (0.54 mL, 7.20 mmol) was
added dropwise. After stirring at room temperature for 3 h, TLC showed completion of the
reaction. The mixture was neutralized by aqueous NaHCO3 and extracted with dichloromethane
(3
×
10 mL). The organic phases were combined, washed with brine and dried over MgSO4. After
concentrated in vacuo, the crude product was purified by flash column chromatography (silica gel,
dichloromethane/methanol = 25:1) to give diamine A-3 (colourless syrup, 1.05 g, 85% yield for two
steps). Data for (2S,3S,4S,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine

Molecules 2020, 25, 1498
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23
(
A-3): [
α
]
−
16.3 (c 1.0 in CH2Cl2);
ν
max/cm−1: 3031 (m), 2923 (s), 2865 (s), 1682 (vs), 1453 (m), 1203
D
(vs), 1130 (vs), 1027 (m), 737 (s), 721 (s), 697 (s); 1H NMR (500 MHz, CDCl3)
δ
(ppm): 7.34–7.24 (m, 15H),
3.50 (m, 2H), 3.32 (q, J = 5.0 Hz,
1H), 3.15–3.12 (m, 1H), 2.79–2.71 (m, 2H), 1.77 (s, 3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
(ppm):
4.55–4.49 (m, 6H), 3.91 (t, J = 3.8 Hz, 1H), 3.76 (t, J = 3.8 Hz, 1H), 3.56
−
δ
138.2, 138.16, 138.15, 128.44, 128.41, 127.8, 127.78, 127.75, 127.71, 127.6, 87.3, 86.1, 73.2, 71.9, 71.7, 70.3,
64.1, 61.6, 44.3; HRMS (ESI): calcd for C27H33O3N2+ [M + H+] 433.2486, found 433.2478.
Data for (2R,3R,4R,5R)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (B-3)
:
23
Colourless syrup, 380 mg, 88% yield from hydroxylamine B-2 (450 mg, 1.01 mmol); [
in CH2Cl2);
736 (s), 720 (s), 697 (s); 1H NMR (500 MHz, CDCl3)
(t, J = 3.8 Hz, 1H), 3.76 (t, J = 3.8 Hz, 1H), 3.56–3.50 (m, 2H), 3.32 (q, J = 5.0 Hz, 1H), 3.16–3.12 (m, 1H),
2.79–2.71 (m, 2H), 1.76 (s, 3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
(ppm): 138.2, 138.16, 138.15,
α
]
+11.8 (c 0.8
D
ν
max/cm−1: 3030 (m), 2866 (m), 1682 (vs), 1496 (m), 1453 (m), 1204 (vs), 1130 (vs), 1027 (m),
δ
(ppm): 7.34–7.24 (m, 15H), 4.55–4.49 (m, 6H), 3.91
δ
128.44, 128.41, 127.8, 127.78, 127.75, 127.71, 127.6, 87.3, 86.1, 73.2, 71.9, 71.7, 70.3, 64.1, 61.6, 44.3; HRMS
(ESI): calcd for C27H33O3N2+ [M + H+] 433.2486, found 433.2478.
Data for (2S,3S,4R,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (C-3)
:
22
Colourless syrup, 372 mg, 86% yield from hydroxylamine C-2 (450 mg, 1.01 mmol); [
in CH2Cl2);
736 (s), 696 (vs); 1H NMR (500 MHz, CDCl3)
J = 4.1 Hz, 1H), 3.70–3.61 (m, 3H), 3.42 (q, J = 6.6 Hz, 1H), 3.30 (q, J = 6.3 Hz, 1H), 2.77 (dd, J = 12.6 and
4.2 Hz, 1H), 2.59 (dd, J = 12.6 and 6.5 Hz, 1H), 1.50 (s, 3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
α
]
D
+13.3 (c 1.0
ν
max/cm−1: 3328 (m), 3030 (m), 2864 (m), 1495 (m), 1453 (s), 1143 (s), 1094 (s), 1027 (m),
δ
(ppm): 7.32–7.25 (m, 15H), 4.73–4.43 (m, 6H), 4.04 (t,
δ
(ppm): 138.6, 138.2, 138.1, 128.44, 128.41, 128.3, 127.8, 127.78, 127.76, 127.6, 127.5, 81.9, 78.0, 73.3, 73.2,
72.3, 69.7, 61.9, 59.0, 45.2; HRMS (ESI): calcd for C27H33O3N2+ [M + H+] 433.2486, found 433.2479.
Data for (2R,3R,4S,5R)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (D-3)
:
23
Colourless syrup, 372 mg, 86% yield from hydroxylamine D-2 (450 mg, 1.01 mmol); [
in CH2Cl2);
(m), 734 (s), 696 (vs); 1H NMR (500 MHz, CDCl3)
α
]
D
−
19.6 (c 1.2
ν
max/cm−1: 3292 (w), 3029 (m), 2859 (m), 1585 (m), 1495 (m), 1453 (s), 1143 (s), 1094 (s), 1027
δ
(ppm): 7.32–7.25 (m, 15H), 4.73–4.43 (m, 6H), 4.04
(t, J = 4.1 Hz, 1H), 3.70–3.61 (m, 3H), 3.42 (q, J = 6.6 Hz, 1H), 3.30 (q, J = 6.3 Hz, 1H), 2.77 (dd, J = 12.6
and 4.2 Hz, 1H), 2.59 (dd, J = 12.6 and 4.3 Hz, 1H), 1.50 (s, 3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
δ
(ppm): 138.6, 138.2, 138.1, 128.44, 128.41, 128.3, 127.8, 127.78, 127.76, 127.6, 127.5, 81.9, 78.0, 73.3, 73.2,
72.3, 69.7, 61.9, 59.0, 45.2; HRMS (ESI): calcd for C27H33O3N2+ [M + H+] 433.2486, found 433.2478.
Data for (2R,3R,4R,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (E-3a)
:
Colourless syrup, 379 mg, 88% yield from hydroxylamine E-2a (450 mg, 1.01 mmol); [
in CH2Cl2);
max/cm−1: 3062 (w), 3029 (m), 2923 (s), 2860 (s), 1688 (m), 1496 (m), 1453 (s), 1201 (m), 1093
(vs), 735 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
(ppm): 7.35–7.24 (m, 15H), 4.57–4.44 (m, 6H), 3.92
α]
D
23 +12.1 (c 1.0
ν
δ
(dd, J = 4.6 and 1.1 Hz, 1H), 3.73 (dd, J = 9.2 and 6.0 Hz, 1H), 3.65 (dd, J = 4.5 and 1.1 Hz,1H), 3.62 (dd,
J = 9.2 and 6.8 Hz, 1H), 3.52 (q, J = 5.95 Hz, 1H), 3.12–3.08 (m,1H), 2.85 (dd,12.6 and 4.9 Hz,1H), 2.76
(dd, J = 12.6 and 7.1Hz, 1H), 1.61 (s, 3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
δ (ppm): 138.3, 138.2,
138.0, 128.48, 128.41, 128.3, 127.8, 127.79, 127.75, 127.72, 127.69, 127.63, 127.60, 85.8, 82.9, 73.4, 71.67,
71.64, 69.7, 66.0, 60.6, 45.5; HRMS (ESI): calcd for C27H33O3N2+ [M + H+] 433.2486, found 433.2477.
Data for (2S,3R,4R,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (E-3b)
:
23
Colourless syrup, 387 mg, 90% yield from hydroxylamine E-2b (450 mg, 1.01 mmol); [
0.9 in CH2Cl2);
1027 (m), 735 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
α
]
−
27.1 (c
D
ν
max/cm−1: 3062 (w), 3029 (m), 2920 (s), 2861 (s), 1495 (m), 1453 (s), 1362 (m), 1094 (vs),
δ
(ppm): 7.34–7.25 (m, 15H), 4.57–4.38 (m, 6H),
4.04 (dd, J = 4.6 and 2.3 Hz, 1H), 3.96 (dd, J = 5.3 and 2.3 Hz,1H), 3.67–3.60 (m, 2H), 3.55 (dd, J = 8.6
and 6.3 Hz,1H), 3.36 (q, J = 6.1 Hz,1H), 2.86–2.78 (m, 2H), 1.63 (s, 3H, NH, NH2); 13C NMR (125 MHz,
CDCl3) δ (ppm): 138.3, 138.2, 138.1, 128.4, 128.39, 128.35, 127.78, 127.74, 127.71, 127.6, 127.57, 127.56,
+
82.9, 82.5, 73.3, 72.2, 71.9, 69.5, 61.1, 58.5, 42.1; HRMS (ESI): calcd for C27H33O3N2 [M + H+] 433.2486,
found 433.2479.

Molecules 2020, 25, 1498
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Data for (2S,3S,4S,5R)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (F-3a):
23
Colourless syrup, 359 mg, 83% yield from hydroxylamine F-2a (450 mg, 1.01 mmol); [
in CH2Cl2);
1201 (s), 1094 (vs), 736 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
α
]
−
9.3 (c 1.0
D
ν
max/cm−1: 3087 (w), 3029 (m), 2922 (s), 2859 (s), 1688 (m), 1496 (m), 1453 (s), 1363 (m),
δ
(ppm):7.35–7.24 (m, 15H), 4.58–4.44
(m, 6H), 3.92 (dd, J = 4.6 and 1.2 Hz, 1H), 3.73 (dd, J = 9.2 and 6.0 Hz, 1H), 3.65 (dd, J = 4.1 and 1.2 Hz,),
3.62 (dd, J = 9.2 and 6.8 Hz, 1H), 3.54–3.50 (m, 1H), 3.12–3.08 (m, 1H), 2.84 (dd, J = 12.6 and 4.9 Hz,1H),
2.76 (dd, J = 12.6 and 7.1Hz, 1H), 1.60 (s, 3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
δ (ppm): 138.3,
138.2, 138.0, 128.48, 128.41, 128.3, 127.8, 127.79, 127.72, 127.69, 127.63, 127.60, 85.8, 82.9, 73.4, 71.69,
71.64, 69.7, 66.0, 60.6, 45.5; HRMS (ESI): calcd for C27H33O3N2+ [M + H+] 433.2486, found 433.2478.
Data for (2R,3S,4S,5R)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (F-3b)
Colourless syrup, 367 mg, 85% yield from hydroxylamine F-2b (450 mg, 1.01 mmol); [
22 +23.8 (c 1.0
in CH2Cl2);
max/cm−1: 3062 (w), 3029 (m), 2922 (s), 2858 (s), 1495 (m), 1453 (s), 1362 (m), 1093 (vs),
1027 (m), 734 (s), 696 (vs); 1H NMR (500 MHz, CDCl3)
(ppm): 7.34–7.25 (m, 15H), 4.57–4.38 (m, 6H),
:
α
]
D
ν
δ
4.04 (dd, J = 4.6 and 2.3 Hz, 1H), 3.96 (dd, J = 5.3 and 2.3 Hz, 1H), 3.67–3.60 (m, 2H), 3.55 (dd, J = 8.6
and 6.3 Hz, 1H), 3.36 (q, J = 6.1Hz, 1H), 2.86–2.78 (m, 2H), 1.62 (s, 3H, NH, NH2); 13C NMR (125 MHz,
CDCl3)
δ (ppm): 138.3, 138.2, 138.1, 128.47, 128.42, 128.3, 127.8, 127.77, 127.74, 127.69, 127.60, 127.5,
+
82.9, 82.5, 73.4, 72.3, 71.9, 69.6, 61.1, 58.6, 42.1; HRMS (ESI): calcd for C27H33O3N2 [M + H+] 433.2486,
found 433.2478.
Data for (2R,3R,4S,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (G-3)
:
22
Colourless syrup, 375 mg, 87% yield from hydroxylamine G-2 (450 mg, 1.01 mmol); [
in CH2Cl2);
1027 (m), 735 (vs), 697 (vs); 1H NMR (400 MHz, CDCl3)
α
]
+9.2 (c 1.1
D
ν
max/cm−1: 3362 (w), 3061 (m), 3029 (m), 2859 (s), 1496 (m), 1453 (s), 1363 (m), 1094 (vs),
δ
(ppm): 7.34–7.22 (m, 15H), 4.60–4.42 (m,
6H), 3.75 (t, J = 5.0 Hz, 1H), 3.55 (t, J = 5.9 Hz,1H), 3.49 (q, J = 5.0 Hz, 1H), 3.46–3.34 (m, 2H), 3.30 (dd,
J = 10.8 and 5.9 Hz,1H), 2.78 (dd, J = 12.8 and 4.4 Hz, 1H), 2.60 (dd, J = 12.8 and 6.1 Hz,1H), 1.45 (s,
3H, NH, NH2); 13C NMR (125 MHz, CDCl3):
127.7, 127.69, 127.66, 79.4, 78.6, 73.2, 72.1, 71.7, 71.5, 62.9, 61.1, 44.9; HRMS (ESI): calcd for C27H33O3N2
[M + H+] 433.2486, found 433.2479.
δ
(ppm): 138.31, 138.30, 138.27, 128.4, 128.3, 128.1, 127.9,
+
Data for (2S,3S,4R,5R)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)pyrrolidine (H-3)
:
23
Colourless syrup, 380 mg, 88% yield from hydroxylamine H-2 (450 mg, 1.01 mmol); [
in CH2Cl2);
(m), 736 (s), 697 (vs); 1H NMR (400 MHz, CDCl3)
α
]
D
−
15.4 (c 1.0
ν
max/cm−1: 3061 (m), 3029 (m), 2861 (s), 1496 (m), 1453 (s), 1362 (m), 1122 (s), 1094 (s), 1027
δ
(ppm): 7.34–7.23 (m, 15H), 4.60–4.43 (m, 6H), 3.75
(t, J = 5.0 Hz, 1H), 3.55 (t, J = 5.9 Hz,1H), 3.49 (dd, J = 9.9 and 5.0 Hz, 1H), 3.46–3.34 (m, 2H), 3.30 (dd,
J = 10.8 and 5.9 Hz,1H), 2.78 (dd, J = 12.8 and 4.4 Hz, 1H), 2.60 (dd, J = 12.8 and 6.1 Hz, 1H), 1.43 (s,
3H, NH, NH2); 13C NMR (125 MHz, CDCl3)
δ (ppm): 138.3, 138.29, 138.26, 128.4, 128.3, 128.1, 127.9,
+
127.7, 127.69, 127.65, 79.4, 78.6, 73.2, 72.1, 71.7, 71.5, 62.9, 61.1, 44.9; HRMS (ESI): calcd for C27H33O3N2
[M + H+] 433.2486, found 433.2479.
3.3.3. Synthesis of (2S,3S,4S,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-2-cyano-pyrrolidine (A-6)
To a suspension of iron powder (560 mg, 10.00 mmol, used as received) in acetic acid was added
Copper (II) acetate (20 mg, 0.10 mmol), and the mixture was stirred at room temperature for 5–10 min
until the bluish green suspension turned into reddish brown. The solution of hydroxylamine A-2
(450 mg, 1.01 mmol) in acetic acid (10 mL) was added, and the reaction mixture was stirred at room
temperature overnight. Solvent was removed in vacuo, the residue was neutralized by aqueous
NaHCO3 and filtered. The resulting filtrate was extracted with EtOAc (3
phases were combined, dried over MgSO4 and concentrated under reduced pressure. Purification
×
50 mL), then organic
by flash chromatography on silica gel (petroleum ether/EtOAc = 3/1) afforded pyrrolidine A-6 (light
yellow syrup, 389 mg, 91% yield). Data for (2S,3S,4S,5S)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)
23
-2-cyano-pyrrolidine (A-6): [
α
]
−
21.2 (c 1.0 in CH2Cl2); ν
max/cm−1: 3293 (w), 3030 (w), 2926 (m),
D
2869 (m), 2246 (w), 1717 (m), 1661 (s), 1453 (s), 1397(m), 1149 (s), 1102 (s), 1027 (m), 736 (s), 697 (vs); 1H

Molecules 2020, 25, 1498
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NMR (500 MHz, CDCl3)
δ
(ppm): 7.36–7.25 (m, 15H), 4.59–4.45 (m, 6H), 4.27 (t, J = 3.1 Hz, 1H), 3.76 (d,
J = 2.9 Hz, 1H), 3.83 (dd, J = 5.5 and 3.3 Hz, 1H), 3.59 (q, J = 6.5 Hz, 1H), 3.49–3.45 (m, 2H), 2.45 (br, 1H,
NH); 13C NMR (125 MHz, CDCl3)
127.78, 127.74, 119.3, 87.4, 84.1, 73.3, 72.5, 72.2, 69.7, 62.1, 51.9; HRMS (ESI): calcd for C27H29O3N2
[M + H+] 429.2173, found 429.2181.
δ
(ppm): 137.7, 1377.5, 136.7, 128.6, 128.4, 128.2, 128.0, 127.87, 127.82,
+
3.3.4. Synthesis of tert-butyl-(2S,3S,4S,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5-(benzyloxy
methyl)pyrrolidine-1-carboxylate (A-8)
The mixture of compound A-6 (389 mg, 0.91 mmol) and Et3N (190
µL, 1.37 mmol) in
dichloromethane (5 mL) was cooled by an ice-water bath, and Boc2O (298 mg, 1.37 mmol) was
added. After stirring overnight at room temperature, the reaction was quenched by aqueous NaHCO3.
The solution was then extracted with dichloromethane (3
×
10 mL), then organic phases were combined,
dried over MgSO4 and concentrated in vacuo to give intermediate A-7 as a light yellow syrup (479 mg,
99% yield). The crude A-7 was directly dissolved in methanol (5 mL), and Raney Ni (500 mg, 60%)
was added. The suspension was stirred under hydrogen atmosphere for 24 h when TLC showed
part of intermediate A-7 remained unreacted. Longer reaction time did not lead to any further
change. Hydrogen was then replaced by nitrogen, and catalyst was removed from the reaction
mixture. The filtrate was concentrated in vacuo to afford a colourless oil, which was purified by flash
chromatography (silica gel, dichloromethane/methanol = 50:1) to give compound A-8 (colourless
syrup, 126 mg, 26% yield). Data for tert-butyl-(2S,3S,4S,5S)-2-(aminomethyl)-3,4-bis(benzyloxy)-5
23
-(benzyloxymethyl)pyrrolidine-1-carboxylate (A-8): [
α
]
D
−
12.7 (c 0.6 in CH2Cl2); ν
max/cm−1: 3029
(w), 2973 (m), 2929 (m), 1688 (s), 1453 (m), 1391 (vs), 1367 (s), 1160 (s), 1108 (s), 1027 (m), 734 (s), 697 (s);
1H NMR (500 MHz, CDCl3)
(ppm): 7.37–7.23 (m, 15H), 4.77–4.41 (m, 6H), 4.18 (d, J = 7.7 Hz, 1H),
4.15–4.10 (m, 2H), 3.99–3.94 (m, 1H), 3.94–3.87 (m, 1H), 3.57 (d, J = 6.5 Hz,1H), 3.24 (d, J = 13.0 Hz, 1H),
2.87 (dd, J = 12.9 and 8.5 Hz, 1H), 1.39 (s, 9H); 13C NMR (125 MHz, CDCl3)
(ppm): 156.7, 138.3, 137.8,
δ
δ
137.7, 128.38, 128.33, 128.2, 127.89, 127.84, 127.69, 127.60, 127.5, 127.4, 82.1, 80.3, 77.2, 73.2, 72.4, 72.1,
69.5, 60.3, 59.2, 44.4, 28.2; HRMS (ESI): calcd for C32H40O5N2+ [M + H+] 533.3010, found 533.3013.
3.3.5. Synthesis of tert-butyl-(2S,3S,4S,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-
(benzyloxymethyl)pyrrolidine-1-carboxylate (A-9)
Compound A-8 (126 mg, 0.24 mmol) was dissolved in dichloromethane (5 mL), followed by Ac2O
(28
for 3-4 h, when TLC showed completion of the reaction. The solution was quenched by aqueous
NaHCO3, and extracted with dichloromethane (3 10 mL). The organic phases were combined,
µL, 0.29 mmol) and catalytic amount of DMAP. The solution was stirred at room temperature
×
dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography
(silica gel, petroleum ether/EtOAc = 1/1) to afford compound A-9 (colourless syrup, 124 mg, 93%
yield). Data for tert-butyl-(2S,3S,4S,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxy
23
methyl)pyrrolidine-1-carboxylate (A-9): [
α
]
−
19.1 (c 1.0 in CH2Cl2); ν
max/cm−1: 3299 (w), 3029
D
(w), 2927 (m), 1690 (vs), 1682 (m), 1453 (m), 1390 (s), 1366 (s), 1274 (m), 1174 (m), 1096 (s), 1027 (m), 735
(m), 697 (m); 1H NMR (500 MHz, CDCl3)
(ppm): 7.33–7.25 (m, 15H), 6.64 (br, 1H, NHCO), 4.75–4.44
(m, 6H), 4.18 (dd, J = 8.8 and 6.1 Hz, 1H), 4.15–4.09 (m, 2H), 3.95–3.85 (m, 2H), 3.57 (d, J = 6.5 Hz,1H),
3.46–3.37 (m, 1H), 3.19–3.10 (m, 1H), 1.87 (s, 1H), 1.39 (s, 9H); 13C NMR (125 MHz, CDCl3)
(ppm):
δ
δ
170.3, 155.5, 138.4, 138.1, 128.3, 128.2, 127.8, 127.7, 127.68, 127.62, 127.4, 80.7, 79.8, 77.8, 73.2, 72.5, 72.4,
70.0, 61.7, 58.6, 43.0, 28.3, 23.2; HRMS (ESI): calcd for C34H43O6N2+ [M + H+] 575.3116, found 575.3118.
3.3.6. General Procedure for Synthesis of Compounds A-5, B-5, C-5, D-5, E-5a, E-5b, F-5a, F-5b, G-5
and H-5 with A-5 as an Example
To the solution of compound A-3 (215 mg, 0.50 mmol) in dichloromethane (10 mL) was added
N,N’,N”,N”’-tetraacetylglycoluril (170 mg, 0.55 mmol). The solution was refluxed for 3–5 h, when TLC
showed disapearance of the starting material. The reaction was quenched by aqueous NaHCO3, and

Molecules 2020, 25, 1498
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extracted with dichloromethane (3
concentrated under reduced pressure. The crude product was purified by flash chromatography (silica
×
10 mL). The organic phases were combined, dried over MgSO4 and
gel, dichloromethane/methanol = 50:1) to give compound A-5 (colourless syrup, 208 mg, 88% yield).
Data for (2S,3S,4S,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5- (benzyloxymethyl)pyrrolidine
23
(A-5): [
α
]
−
7.7 (c 0.9 in CH2Cl2);
ν
max/cm−1: 3292 (w), 3030 (m), 2860 (m), 1656 (s), 1453 (m), 1363
D
1
(m), 1094 (s), 1027 (m), 737 (s), 697 (vs); H NMR (500 MHz, CDCl3)
δ (ppm): 7.34–7.25 (m, 15H),
6.08 (br, 1H, NHCO), 4.55–4.49 (m, 6H), 3.88 (t, J = 3.5 Hz, 1H), 3.76 (t, J = 3.4 Hz, 1H), 3.54–3.48
(m, 2H), 3.43–3.38 (m, 1H), 3.37–3.31 (m, 2H), 3.28–3.23 (m, 1H), 2.1 (br, 1H, NH), 1.88 (s, 3H); 13
NMR (125 MHz, CDCl3) (ppm): 170.2, 137.9, 137.83, 137.81, 128.46, 128.44, 127.87, 127.84, 127.81,
C
δ
+
127.77, 127.73, 86.8, 85.5, 73.2, 72.0, 71.8, 69.8, 61.9, 61.0, 41.4, 23.2; HRMS (ESI): calcd for C29H35O4N2
[M + H+] 475.2591, found 475.2588.
Data for (2R,3R,4R,5R)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
23
(B-5) (Ref. [62]): Colourless syrup, 216 mg, 91% yield from diamine B-3 (215 mg, 0.50 mmol); [
+11.9 (c 1.0 in CH2Cl2);
(s), 1028 (m), 737 (s), 697 (vs); 1H NMR (500 MHz, CDCl3)
α]
D
ν
max/cm−1: 3283 (w), 3030 (m), 2859 (m), 1656 (s), 1454 (m), 1363 (m), 1093
(ppm): 7.34–7.25 (m, 15H), 6.08 (br, 1H,
δ
NHCO), 4.55–4.49 (m, 6H), 3.88 (t, J = 3.5 Hz, 1H), 3.76 (t, J = 3.4 Hz, 1H), 3.54–3.48 (m, 2H), 3.43–3.38
(m, 1H), 3.37–3.31 (m, 2H), 3.28–3.23 (m, 1H), 2.16 (br, 1H, NH), 1.88 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ (ppm): 170.2, 137.9, 137.83, 137.81, 128.46, 128.44, 127.87, 127.84, 127.81, 127.77, 127.73, 86.8,
+
85.5, 73.2, 72.0, 71.8, 69.8, 61.9, 61.0, 41.4, 23.2; HRMS (ESI): calcd for C29H35O4N2 [M + H+] 475.2591,
found 475.2586.
Data for (2S,3S,4R,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
23
(C-5): Colourless syrup, 201 mg, 85% yield from diamine C-3 (215 mg, 0.50 mmol); [
in CH2Cl2);
1027 (m), 736 (s), 697 (s); 1H NMR (500 MHz, CDCl3)
4.73–4.49 (m, 6H), 4.00 (t, J = 4.0 Hz, 1H), 3.68 3.62 (m, 2H), 3.58 (t, J = 7.5 Hz, 1H), 3.44–3.38 (m, 2H),
3.35–3.25 (m, 2H), 2.16 (br, 2H, NH2), 1.89 (s, 3H); 13C NMR (125 MHz, CDCl3)
(ppm): 170.3, 138.3,
α
]
+11.9 (c 1.0
D
ν
max/cm−1: 3288 (w), 3029 (w), 2924 (m), 2855 (m), 1652 (s), 1453 (m), 1366 (m), 1089 (s),
δ
(ppm): 7.35–7.25 (m, 15H), 5.87 (br, 1H, NH),
−
δ
138.0, 137.8, 128.47, 128.42, 128.3, 127.89, 127.88, 127.86, 127.80, 127.7, 127.6, 82.4, 77.6, 73.4, 73.2, 72.5,
69.5, 59.07, 59.04, 42.3, 23.2; HRMS (ESI): calcd for C29H35O4N2 [M + H+] 475.2591, found 475.2588.
+
Data for (2R,3R,4S,5R)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
23
(D-5): Colourless syrup, 194 mg, 82% yield from diamine D-3 (215 mg, 0.50 mmol); [
CH2Cl2);
(s), 1027 (m), 736 (s), 696 (s); 1H NMR (500 MHz, CDCl3)
NHCO), 4.73–4.49 (m, 6H), 4.00 (t, J = 4.1 Hz, 1H), 3.68–3.62 (m, 2H), 3.58 (t, J = 7.5 Hz, 1H), 3.44–3.38
(m, 2H), 3.35–3.25 (m, 2H), 2.16 (br, 1H, NH), 1.89 (s, 3H); 13C NMR (125 MHz, CDCl3)
(ppm): 170.3,
α
]
−
2.5 (c 1.0 in
D
ν
max/cm−1: 3296 (w), 3028 (w), 2921 (m), 2855 (m), 1652 (s), 1554 (m), 1453 (s), 1365(m), 1091
δ
(ppm): 7.35–7.25 (m, 15H), 5.87 (br, 1H,
δ
138.4, 138.0, 137.9, 128.49, 128.44, 128.3, 127.9, 127.8, 127.7, 127.6, 82.4, 77.7, 73.4, 73.3, 72.6, 69.5, 59.09,
59.06, 42.3, 23.2; HRMS (ESI): calcd for C29H35O4N2+ [M + H+] 475.2591, found 475.2588.
Data for (2R,3R,4R,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
23
(E-5a): Colourless syrup, 213 mg, 90% yield from diamine E-3a (215 mg, 0.50 mmol); [
in CH2Cl2);
1091 (s), 1027 (m), 736 (s), 696 (s); 1H NMR (500 MHz, CDCl3)
α
]
D
+9.2 (c 1.0
ν
max/cm−1: 3295 (w), 3030 (w), 2924 (m), 2855 (m), 1652 (s), 1554 (m), 1493 (s), 1365 (m),
δ
(ppm): 7.34–7.24 (m, 15H), 6.81 (br, 1H,
NHCO), 4.58–4.42 (m, 6H), 4.19 (br, 1H, NH), 3.90 (d, J = 3.9 Hz, 1H), 3.77 (s, 1H), 3.73 (dd, J = 9.3
and 5.8 Hz, 1H), 3.68 (dd, J = 9.5 and 6.7 Hz, 2H), 3.62 (dd, J = 10.3 and 6.0 Hz, 1H), 3.48–3.43 (m, 1H),
3.39–3.34 (m, 1H), 1.76 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ (ppm): 170.6, 138.1, 137.7, 137.6, 128.54,
128.52, 128.4, 127.99, 127.91, 127.8, 127.79, 127.75, 127.6, 85.0, 82.2, 73.5, 71.9, 71.8, 69.6, 62.0, 60.4, 42.4,
22.8; HRMS (ESI): calcd for C29H35O4N2+ [M + H+] 475.2591, found 475.2585.
Data for (2S,3R,4R,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
23
(E-5b): Colourless syrup, 194 mg, 82% yield from diamine E-3b (215 mg, 0.50 mmol); [
α
]
D
−
17.2 (c
1.2 in CH2Cl2);
ν
max/cm−1: 3290 (m), 3063 (m), 3030 (m), 2925 (s), 2861 (s), 1651 (vs), 1549 (m), 1453 (s),

Molecules 2020, 25, 1498
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1367 (s), 1092 (vs), 1027 (m), 736 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
δ
(ppm): 7.36–7.25 (m, 15H),
6.11 (br, 1H, NHCO), 4.56–4.35 (m, 6H), 3.99 (d, J = 1.6 Hz, 1H), 3.93 (d, J = 3.1 Hz, 1H), 3.63–3.61 (m,
2H), 3.57–3.52 (m, 3H), 3.30–3.25 (m, 1H), 2.36 (br, 1H, NH), 1.85 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ
(ppm): 170.4, 138.1, 137.9, 137.7, 128.6, 128.4, 128.0, 127.89, 127.86, 127.84, 127.7, 82.6, 82.0, 73.4, 72.3,
72.1, 68.8, 58.9, 58.2, 39.6, 23.2; HRMS (ESI): calcd for C29H35O4N2+ [M + H+] 475.2591, found 475.2584.
Data for (2S,3S,4S,5R)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
22
(F-5a): Colourless syrup, 206 mg, 87% yield from diamine F-3a (215 mg, 0.50 mmol); [
1.0 in CH2Cl2);
(m), 1365 (m), 1091 (s), 1027 (m), 736 (s), 696 (s); 1H NMR (500 MHz, CDCl3)
α
]
−
4.1 (c
D
ν
max/cm−1: 3295 (w), 3064 (m), 3030 (m), 2924 (m), 2855 (m), 1652 (s), 1554 (m), 1493
δ
(ppm): 7.34–7.23 (m,
15H) 6.54 (br, 1H, NHCO), 4.58–4.42 (m, 6H), 3.89 (dd, J = 4.1 and 0.6 Hz, 1H), 3.74–3.71 (m, 2H), 3.65
(dd, J = 8.9 and 6.9 Hz, 1H), 3.59 (dd, J = 10.0 and 5.4 Hz, 1H), 3.44–3.40 (m, 2H), 3.35–3.31 (m, 1H),
3.19 (br, 1H, NH), 1.73 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ (ppm): 170.6, 138.1, 137.7, 137.6, 128.54,
128.52, 128.4, 127.99, 127.9, 127.8, 127.79, 127.75, 127.6, 85.0, 82.2, 73.5, 71.9, 71.8, 69.6, 62.0, 60.4, 42.4,
22.8; HRMS (ESI): calcd for C29H35O4N2+ [M + H+] 475.2591, found 475.2587.
Data for (2R,3S,4S,5R)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
(F-5b): Colourless syrup, 197 mg, 83% yield from diamine F-3b (215 mg, 0.50 mmol); [
1.0 in CH2Cl2);
max/cm−1: 3293 (m), 3063 (m), 3030 (m), 2924 (s), 2861 (s), 1651 (vs), 1549 (m), 1453
(s), 1367 (s), 1092 (vs), 1027 (m), 736 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
(ppm): 7.35–7.24 (m,
15H) 6.24 (br, 1H, NHCO), 4.55–4.35 (m, 6H), 3.98 (dd, J = 6.0 and 1.9 Hz, 1H), 3.92 (dd, J = 4.9 and
1.9 Hz, 1H), 3.63 (m, 2H), 3.57–3.52 (m, 3H), 3.29–3.23 (m, 1H), 3.12 (br, 1H, NH), 1.84 (s, 3H); 13
NMR (125 MHz, CDCl3) (ppm): 170.4, 138.1, 137.9, 137.7, 128.6, 128.48, 128.42, 128.0, 127.89, 127.86,
α]
22 +20.5 (c
D
ν
δ
C
δ
+
127.85, 127.7, 82.5, 82.0, 73.4, 72.3, 72.1, 68.9, 58.9, 58.2, 39.6, 23.2; HRMS (ESI): calcd for C29H35O4N2
[M + H+] 475.2591, found 475.2585.
Data for (2R,3R,4S,5S)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
(G-5): Colourless syrup, 204 mg, 86% yield from diamine G-3 (215 mg, 0.50 mmol); [
23 +7.5 (c 1.0 in
CH2Cl2);
max/cm−1: 3293 (m), 3062 (m), 3029 (m), 2924 (s), 2861 (s), 1653 (vs), 1539 (m), 1453 (s), 1365
(m), 1100 (vs), 1027 (m), 736 (s), 697 (vs); 1H NMR (500 MHz, CDCl3)
(ppm): 7.34–7.24 (m, 15H), 6.12
(br, 1H, NHCO), 4.58–4.45 (m, 6H), 3.77 (t, J = 4.7 Hz, 1H), 3.60 (t, J = 5.5 Hz, 1H), 3.50 (q, J = 4.4 Hz,
1H), 3.47–3.38 (m, 4H), 3.18–3.13 (m, 1H), 2.14 (br, 1H, NH), 1.74 (s, 3H); 13C NMR (125 MHz, CDCl3)
α
]
D
ν
δ
δ
(ppm): 170.2, 138.0, 137.9, 128.47, 128.40, 128.1, 128.0, 127.86, 127.80, 127.7, 80.0, 78.2, 73.3, 72.0, 71.6,
61.0, 59.6, 42.3, 23.0; HRMS (ESI): calcd for C29H35O4N2+ [M + H+] 475.2591, found 475.2587.
Data for (2S,3S,4R,5R)-2-N-acetylaminomethyl-3,4-bis(benzyloxy)-5-(benzyloxymethyl) pyrrolidine
23
(H-5): Colourless syrup, 192 mg, 81% yield from diamine H-3 (215 mg, 0.50 mmol); [
CH2Cl2);
(vs), 1027 (m), 736 (vs), 697 (vs); 1H NMR (500 MHz, CDCl3)
α
]
D
−
1.6 (c 1.2 in
ν
max/cm−1: 3304 (m), 3062 (m), 3030 (m), 2865 (s), 1651 (vs), 1549 (m), 1453 (s), 1365 (m), 1099
δ
(ppm): 7.33–7.24 (m, 15H), 6.11 (br, 1H,
NHCO), 4.57–4.43 (m, 6H), 3.76 (t, J = 4.8 Hz, 1H), 3.59 (t, J = 5.6 Hz, 1H), 3.49 (dd, J = 8.9 and 4.4 Hz,
1H), 3.47–3.37 (m, 4H), 3.18–3.13 (m, 1H), 2.14 (br, 1H, NH), 1.74 (s, 3H); 13C NMR (125 MHz, CDCl3):
δ
(ppm): 170.2, 138.0, 137.9, 128.47, 128.40, 128.1, 128.0, 127.86, 127.80, 127.7, 80.0, 78.2, 73.3, 72.0, 71.6,
61.0, 59.6, 42.3, 23.0; HRMS (ESI): calcd for C29H35O4N2+ [M + H+] 475.2591, found 475.2588.
3.3.7. General Procedure for Synthesis of 1-N-Acetylamino Derivatives (A-10, 4·HCl, C-10, D-10,
E-10a, E-10b, F-10a, F-10b, G-10 and H-10) and 1-Amino Derivatives (A-11, B-11, C-11, D-11, E-11a,
E-11b, F-11a, F-11b, G-11 and H-11) with A-10 as an Example
To a stirred solution of A-5 (95 mg, 0.20 mmol) and 3 N HCl (0.5 mL) in MeOH (10 mL)
was added Pd/C (10 wt%, 30 mg) under Ar atmosphere and the reaction mixture was stirred
under H2 atmosphere for 8 h. Then the catalyst was filtered and the solvent was removed
under reduced pressure to afford compound A-10 (colourless syrup, 47 mg, 99% yield). Data
for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-l-mannitol hydrochloride (A-10) (Ref. [16], reported

Molecules 2020, 25, 1498
18 of 24
23
as free base): [
α
]
−
30.4 (c 0.5 in MeOH);
ν
max/cm−1: 3290 (vs), 2932 (s), 1646 (m), 1550 (m), 1369
D
(m), 1132 (m), 1041 (m); 1H NMR (500 MHz, D2O)
δ
(ppm): 4.25 (s, 1H,), 4.11 (s, 1H), 3.98 (dd, J = 12.0
Hz and 4.6 Hz, 1H), 3.95–3.91 (m, 1H), 3.86 (dd, J = 11.8 Hz and 8.7 Hz, 1H), 3.66 (d, J = 6.8 Hz, 2H),
3.63–3.61 (m, 1H), 2.04 (s, 3H, COCH3); 13C NMR (125 MHz, D2O):
δ (ppm): 175.2, 75.7, 74.6, 67.5, 61.5,
59.2, 35.8, 21.7; HRMS (ESI): calcd for C8H17O4N2+ [M + H+] 205.1183, found 205.1181.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-d-mannitol hydrochloride (4·HCl) (Ref. [16
,62],
reported as free base): Colourless syrup, 50 mg, 99% yield from compound B-5 (101 mg, 0.21 mmol);
25
[
α
]
+34.6 (c 0.5 in MeOH); ν
max/cm−1: 3297 (vs), 2937 (s), 1644 (m), 1550 (m), 1369 (m), 1132 (m),
D
1042 (m); 1H NMR (500 MHz, D2O)
δ (ppm): 4.22 (d, J = 2.0 Hz, 1H), 4.09 (t, J = 1.6 Hz, 1H), 3.95 (dd,
J = 12.1 Hz and 4.8 Hz, 1H), 3.91 (dt, J = 6.9 Hz and 3.7 Hz, 1H), 3.83 (dd, J = 12.1 Hz and 8.6 Hz, 1H),
3.66 (d, J = 6.8 Hz, 2H), 3.61–3.58 (m, 1H), 2.01 (s, 3H, COCH3); 13C NMR (125 MHz, D2O)
δ
(ppm):
+
175.2, 75.7, 74.6, 67.5, 61.5, 59.2, 35.8, 21.7; HRMS (ESI): calcd for C8H17O4N2 [M + H+] 205.1183,
found 205.1183.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-l-altritol hydrochloride (C-10) (Ref. [17], reported
22
as free base): Colourless syrup, 45 mg, 99% yield from compound C-5 (90 mg, 0.19 mmol); [
α
]
D
−
18.0
(c 0.7 in MeOH);
(m); H NMR (500 MHz, D2O)
ν
max/cm−1: 3291 (vs), 2936 (s), 1635 (m), 1550 (m), 1419 (m),1369 (m), 1136 (m), 1042
1
δ
(ppm): 4.33(s, 1H), 4.23 (dd, J = 9.3 Hz and 3.7 Hz, 1H), 3.99 (dd,
J = 12.0 Hz and 4.8 Hz, 1H), 3.90 (dd, J = 11.6 Hz and 8.4 Hz, 1H), 3.82–3.80 (m, 1H), 3.72–3.66 (m, 2H),
3.59 (dd, J = 15.3 Hz and 7.7 Hz, 1H), 2.04 (s, 3H, COCH3); 13C NMR (125 MHz, D2O)
δ
(ppm): 176.0,
+
72.5, 69.8, 62.0, 60.5, 57.4, 38.9, 21.6; HRMS (ESI): calcd for C8H17O4N2 [M + H+] 205.1183, found
205.1186.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-d-altritol hydrochloride (D-10) (Ref. [16,61],
reported as free base): Colourless syrup, 41 mg, 99% yield from compound D-5 (82 mg, 0.17 mmol);
22
[
α
]
+21.3 (c 0.4 in MeOH); ν
max/cm−1: 3296 (vs), 2936 (s), 1635 (m), 1550 (m), 1419 (m),1371 (m),
D
1136 (m), 1042 (m); 1H NMR (500 MHz, D2O)
δ (ppm) 4.33 (t, J = 3.2 Hz, 1H), 4.23 (dd, J = 9.3 Hz and
3.7 Hz, 1H), 3.99 (dd, J = 12.0 Hz and 4.8 Hz, 1H), 3.90 (dd, J = 11.6Hz and 8.4Hz, 1H), 3.82–3.80 (m,
1H), 3.72–3.66 (m, 2H), 3.59 (dd, J = 15.3 Hz and 7.75 Hz, 1H), 2.04 (s, 3H, COCH3); 13C NMR (125 MHz,
D2O) δ
(ppm): 176.0, 72.5, 69.8, 62.0, 60.5, 57.4, 38.9, 21.6; HRMS (ESI): calcd for C8H17O4N2+ [M + H+]
205.1183, found 205.1185.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-d-glucitol hydrochloride (E-10a) (Ref. [63],
reported as free base): Colourless syrup, 46 mg, 99% yield from compound E-5a (91 mg, 0.19 mmol);
[α] ν
25 +36.9 (c 0.85 in MeOH); max/cm−1: 3292 (vs), 2935 (s), 1645 (m), 1550 (m), 1369 (m), 1132 (m),
D
1045 (m); 1H NMR (400 MHz, D2O)
δ (ppm): 4.27 (s, 1H), 4.12 (s, 1H), 3.98 (d, J = 9.4 Hz, 1H), 3.94–3.88
(m, 2H), 3.68–3.57 (m, 3H), 2.01 (s, 3H, COCH3); 13C NMR (125 MHz, D2O)
δ (ppm):175.8, 76.8, 74.5,
65.1, 63.0, 57.0, 39.4, 21.6; HRMS (ESI): calcd for C8H17O4N2+ [M + H+] 205.1183, found 205.1182.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-l-iditol hydrochloride (E-10b) (Ref. [64], reported
25
as free base): Colourless syrup, 52 mg, 99% yield from compound E-5b (104 mg, 0.22 mmol); [α]
D
−
9.6 (c 1.2 in MeOH); ν
max/cm−1: 3292 (vs), 2936 (s), 1651 (m), 1553 (m), 1370 (m), 1136 (m), 1042 (m);
1H NMR (500 MHz, D2O) δ (ppm): 4.35 (d, J = 3.3 Hz, 1H), 4.28 (d, J = 2.0 Hz, 1H), 4.01–3.95 (m, 3H),
3.87 (dd, J = 11.9 and 8.3 Hz, 1H), 3.60 (d, J = 6.8 Hz, 2H), 2.01 (s, 3H, COCH3); 13C NMR (125 MHz,
D2O) δ
(ppm): 175.8, 75.5, 74.1, 62.6, 61.3, 57.9, 38.6, 21.6; HRMS (ESI): calcd for C8H17O4N2+ [M + H+]
205.1183, found 205.1180.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-l-glucitol hydrochloride (F-10a)
: Colourless
25
syrup, 44 mg, 99% yield from compound F-5a (88 mg, 0.19 mmol); [
α
]
−
29.2 (c 1.05 in MeOH);
D
ν
max/cm−1: 3285 (vs), 2931 (s), 1645 (m), 1551 (m), 1370 (m), 1090 (m), 1063 (m); 1H NMR (400 MHz,
D2O)
δ
(ppm): 4.30 (t, J = 1.6 Hz, 1H), 4.16 (t, J = 1.6 Hz, 1H), 4.01 (dd, J = 8.8 and 2.8 Hz, 1H), 3.95–3.88
(ppm):175.8, 76.8, 74.6,
65.2, 63.1, 57.0, 39.4, 21.6; HRMS (ESI): calcd for C8H17O4N2+ [M + H+] 205.1183, found 205.1183.
(m, 2H), 3.69–3.59 (m, 3H), 2.04 (s, 3H, COCH3); 13C NMR (125 MHz, D2O)
δ

Molecules 2020, 25, 1498
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Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-d-iditol hydrochloride (F-10b): Colourless syrup,
42 mg, 99% yield from compound F-5b (83 mg, 0.17 mmol); [
25 +7.7 (c 1.0 in MeOH); max/cm−1
3296 (vs), 2936 (s), 1651 (m), 1553 (m), 1371 (m), 1136 (m), 1041 (m); 1H NMR (500 MHz, D2O)
(ppm):
4.34 (d, J = 3.4 Hz, 1H), 4.27 (t, J = 1.8 Hz, 1H), 4.00–3.94 (m, 3H), 3.86 (dd, J = 11.6 and 8.3 Hz, 1H), 3.60
α
]
ν
:
D
δ
(d, J = 6.9 Hz, 2H), 2.01 (s, 3H, COCH3); 13C NMR (125 MHz, D2O)
δ (ppm): 175.8, 75.5, 74.1, 62.6, 61.3,
57.9, 38.6, 21.7; HRMS (ESI): calcd for C8H17O4N2+ [M + H+] 205.1183, found 205.1183.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-d-allitol hydrochloride (G-10): Colourless syrup,
23
46 mg, 99% yield from compound G-5 (92 mg, 0.19 mmol); [
α
]
−
23.1 (c 1.0 in MeOH); ν :
max/cm−1
D
3306 (vs), 2928 (s), 1635 (s), 1551 (s), 1419 (m),1370 (m), 1089 (m), 1063 (m); 1H NMR (500 MHz, D2O)
(ppm): 4.22–4.19 (m, 2H), 3.93 (dd, J = 12.6 Hz and 3.8 Hz, 1H), 3.83 (dd, J = 12.6 Hz and 3.8 Hz,
1H), 3.78–3.74 (m, 1H), 3.73–3.66 (m, 2H), 3.57 (dd, J = 15.1 Hz and 7.4 Hz, 1H), 2.04 (s, 1H, COCH3);
δ
13C NMR (125 MHz, D2O):
δ(ppm): 176.1, 71.1, 70.2, 64.2, 62.8, 57.9, 38.5, 21.6; HRMS (ESI): calcd for
C8H17O4N2+ [M + H+] 205.1183, found 205.1182.
Data for 1-N-acetylamino-2,5-imino-1,2,5-trideoxy-l-allitol hydrochloride (H-10): Colourless syrup,
41 mg, 99% yield from compound H-5 (81 mg, 0.17 mmol); [
3296 (vs), 2936 (s), 1651 (s), 1546 (s), 1419 (m), 1372 (m), 1091 (m), 1061 (m); 1H NMR (500 MHz, D2O)
(ppm): 4.22–4.18 (m, 2H), 3.93 (dd, J = 12.5 Hz and 3.6 Hz, 1H), 3.82 (dd, J = 12.6 Hz and 3.6 Hz,
1H), 3.78 3.74 (m, 1H), 3.71–3.66 (m, 2H), 3.57 (dd, J = 15.1 Hz and 7.4 Hz, 1H), 2.04 (s, 1H, COCH3);
α] ν :
23 +27.3 (c 0.95 in MeOH); max/cm−1
D
δ
−
13C NMR (125 MHz, D2O)
δ(ppm): 176.1, 71.1, 70.2, 64.2, 62.8, 57.9, 38.5, 21.5; HRMS (ESI): calcd for
C8H17O4N2+ [M + H+] 205.1183, found 205.1184.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-l-mannitol dihydrochloride (A-11) (Ref. [61], reported as
23
free base): Colourless syrup, 46 mg, 99% yield from compound A-3 (86 mg, 0.2 mmol); [
1.0 in MeOH);
D2O) (ppm): 4.20–4.12 (m, 2H), 3.98 (dd, J = 12.5 Hz and 3.5 Hz, 1H), 3.91–3.81 (m, 2H), 3.74–3.69
α
]
−
66.7 (c
D
ν
max/cm−1: 3313 (s), 2939 (s), 1115 (m), 1058 (m), 1033 (m), 1014 (m); 1H NMR (400 MHz,
δ
(m, 1H), 3.57 (d, J = 10.5 Hz, 2H); 13C NMR (125 MHz, D2O)
δ (ppm): 76.5, 73.9, 63.4, 58.4, 58.1, 38.7;
HRMS (ESI): calcd for C6H15O3N2+ [M + H+] 163.1077, found 163.1077.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-d-mannitol dihydrochloride (B-11) (Ref. [17]): Colourless
23
syrup, 35 mg, 99% yield from compound B-3 (66 mg, 0.15 mmol); [
ν
α
]
−
66.7 (c 1.0 in MeOH);
(ppm):
4.19–4.12 (m, 2H), 3.98 (dd, J = 12.6 Hz and 3.6 Hz, 1H), 3.88 (dd, J = 12.5 Hz and 3.6 Hz, 1H), 3.83 (q, J
D
max/cm−1: 3314 (s), 2942 (s), 1113 (m), 1063 (m), 1033 (m), 1014 (m); 1H NMR (400 MHz, D2O)
δ
= 7.2 Hz, 1H), 3.73–3.69 (m, 1H), 3.56 (d, J = 10.5 Hz, 2H); 13C NMR (125 MHz, D2O)
δ (ppm): 76.5,
73.9, 63.4, 58.4, 58.1, 38.7; HRMS (ESI): calcd for C6H15O3N2+ [M + H+] 163.1077, found 163.1077.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-l-altritol dihydrochloride (C-11) (Ref. [17]): Colourless
23
syrup, 39 mg, 99% yield from compound C-3 (73 mg, 0.17 mmol); [
ν
α
]
−
42.0 (c 1.0 in MeOH);
(ppm): 4.38 (t,
J = 3.2 Hz, 1H), 4.34 (dd, J = 9.5 Hz and 3.7 Hz, 1H), 4.04–3.97 (m, 1H), 3.97–3.92 (m, 2H), 3.85 (ddd,
D
max/cm−1: 3314 (s), 2923 (s), 1132 (m), 1033 (m), 1014 (m); 1H NMR (500 MHz, D2O)
δ
J = 13.8 Hz and 7.8 Hz and 5.9 Hz, 1H), 3.62–3.53 (m, 2H); 13C NMR (125 MHz, D2O)
δ (ppm): 74.0,
69.3, 62.9, 57.3, 56.9, 39.0; HRMS (ESI): calcd for C6H15O3N2+ [M + H+] 163.1077, found 163.1078.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-d-altritol dihydrochloride (D-11) (Ref. [61], reported as
23
free base): Colourless syrup, 37 mg, 99% yield from compound D-3 (69 mg, 0.2 mmol); [
0.8 in MeOH);
(ppm): 4.38 (t, J = 2.5 Hz, 1H), 4.34 (dd, J = 9.5 Hz and 3.7 Hz, 1H), 4.06–4.01 (m, 1H), 3.98–3.93 (m,
α
]
+39.0 (c
D
ν
max/cm−1: 3318 (s), 2923 (s), 1131 (m), 1033 (m), 983 (m); 1H NMR (500 MHz, D2O)
δ
2H), 3.86 (dd, J = 14.9 Hz and 7.9 Hz, 1H), 3.62–3.53 (m, 2H); 13C NMR (125 MHz, D2O)
δ (ppm): 74.0,
69.3, 62.9, 57.3, 56.9, 39.0; HRMS (ESI): calcd for C6H15O3N2+ [M + H+] 163.1077, found 163.1076.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-d-glucitol dihydrochloride (E-11a) (Ref. [17]): Colourless
23
syrup, 38 mg, 99% yield from compound E-3a (71 mg, 0.16 mmol); [
ν
α
]
+21.6 (c 0.5 in MeOH);
(ppm): 4.32–4.29
D
max/cm−1: 3313 (s), 2935 (s), 1118 (m), 1063 (m), 1033 (m); 1H NMR (300 MHz, D2O)
δ

Molecules 2020, 25, 1498
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(m, 1H), 4.26 (t, J = 2.8 Hz, 1H), 4.02–3.90 (m, 3H), 3.80 (dt, J = 6.9 and 2.9 Hz, 1H), 3.54 (d, J = 6.8 Hz, 2H);
+
13C NMR (125 MHz, D2O)
δ (ppm): 77.5, 74.1, 64.1, 62.4, 56.9, 39.2; HRMS (ESI): calcd for C6H15O3N2
[M + H+] 163.1077, found 163.1077.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-l-iditol dihydrochloride (E-11b) (Ref. [17]): Colourless
23
syrup, 44 mg, 99% yield from compound E-3b (81 mg, 0.19 mmol); [
ν
α
]
+5.5 (c 1.0 in MeOH);
(ppm): 4.46 (d,
D
max/cm−1: 3322 (s), 2924 (s), 1131 (m), 1039 (m), 984 (m); 1H NMR (500 MHz, D2O)
δ
J = 5.1 Hz, 1H), 4.44 (d, J = 5.1Hz, 1H), 4.22 (dt, J = 6.8 Hz and 3.5 Hz, 1H), 4.12-4.09 (m, 1H), 4.03 (dd,
J = 12.2 Hz and 4.6Hz, 1H), 3.94 (dd, J = 12.2 Hz and 8.8 Hz, 1H), 3.62 (dd, J = 13.7 Hz and 6.8 Hz, 1H),
3.55–3.51(dd, J = 13.7 Hz and 6.8 Hz, 1H); 13C NMR (125 MHz, D2O)
δ(ppm): 74.6, 74.2, 63.8, 58.3, 57.3,
35.9; HRMS (ESI): calcd for C6H15O3N2+ [M + H+] 163.1077, found 163.1076.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-l-glucitol dihydrochloride (F-11a) (Ref. [61], reported as
23
free base): Colourless syrup, 31 mg, 99% yield from compound F-3a (57 mg, 0.13 mmol); [
(c 0.65 in MeOH);
(ppm): 4.35 (s, 1H), 4.31 (s, 1H), 4.05–3.96 (m, 3H), 3.88–3.84 (m, 1H), 3.59 (d, J = 6.6 Hz, 2H); 13
NMR (125 MHz, D2O)
[M + H+] 163.1077, found 163.1076.
α
]
−
27.5
D
ν
max/cm−1: 3313 (s), 2935 (s), 1118 (m), 1063 (m), 1033 (m); 1H NMR (500 MHz, D2O)
δ
C
+
δ(ppm): 77.4, 74.0, 64.1, 62.5, 56.8, 39.2; HRMS (ESI): calcd for C6H15O3N2
Data for 1-amino-2,5-imino-1,2,5-trideoxy-d-iditol dihydrochloride (F-11b) (Ref. [61], reported as
23
free base): Colourless syrup, 34 mg, 99% yield from compound F-3b (62 mg, 0.14 mmol); [
0.3 in MeOH);
α
]
−
7.3 (c
D
ν
max/cm−1: 3310 (s), 2936 (s), 1131 (m), 1076 (m), 1031 (m); 1H NMR (500 MHz, D2O)
δ
(ppm): 4.41–4.39 (m, 2H), 4.22 (dt, J = 6.7 Hz and 3.5 Hz, 1H), 4.05–3.99 (m, 2H), 3.91 (dd, J = 11.8 Hz
and 8.5 Hz, 1H), 3.57 (dd, J = 13.7 Hz and 6.8 Hz, 1H), 3.47(dd, J = 13.7 Hz and 6.8 Hz, 1H); 13C NMR
(ppm): 74.7, 74.4, 63.6, 58.1, 57.4, 36.0; HRMS (ESI): calcd for C6H15O3N2 [M + H+]
+
(125 MHz, D2O)
δ
163.1077, found 163.1076.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-d-allitol dihydrochloride (G-11) (Ref. [61], reported as free
base): Colourless syrup, 35 mg, 99% yield from compound G-3 (65 mg, 0.15 mmol); [
in MeOH);
max/cm−1: 3322 (s), 2947 (s), 1122 (m), 1078 (m), 1034 (m), 979 (m); 1H NMR (500 MHz,
D2O) (ppm): 4.33 (dd, J = 7.6 Hz and 5.0 Hz, 1H), 4.29 (dd, J = 4.7 Hz and 4.0 Hz, 1H), 3.96 (dd,
α]
23 +8.1 (c 1.0
D
ν
δ
J = 12.2 Hz and 3.5 Hz 1H), 3.92–3.84 (m, 3H) 3.61–3.53 (m, 2H); 13C NMR (125 MHz, D2O)
δ (ppm):
72.5, 70.0, 66.0, 58.6, 58.1, 38.4; HRMS (ESI): calcd for C6H15O3N2+ [M + H+] 163.1077, found 163.1077.
Data for 1-amino-2,5-imino-1,2,5-trideoxy-l-allitol dihydrochloride (H-11) (Ref. [61], reported as free
22
base): Colourless syrup, 36 mg, 99% yield from compound H-3 (67 mg, 0.16 mmol); [
in MeOH);
D2O) (ppm):4.34–4.27 (m, 2H), 3.96 (dd, J = 12.0 Hz and 3.4 Hz, 1H), 3.92–3.84 (m, 3H), 3.62–3.52
α
]
−
7.2 (c 1.0
D
ν
max/cm−1: 3313 (s), 2951 (s), 1124 (m), 1079 (m), 1034 (m), 980 (m); 1H NMR (400 MHz,
δ
(m, 2H); 13C NMR (125 MHz, D2O)
δ (ppm): 72.5, 70.1, 66.1, 58.7, 58.1, 38.4; HRMS (ESI): calcd for
C6H15O3N2+ [M + H+] 163.1077, found 163.1077.
4. Conclusions
In summary, a general and efficient synthetic strategy has been developed for the synthesis
of 1-N-acetylamino and 1-amino pyrrolidine analogues of pochonicine (
1
) with l-arabino-nitrone
), l-xylo-nitrone ( ), d-xylo-nitrone ( ),
) as the starting materials 4 and 5 steps, respectively. Glycosidase
(A
), d-arabino-nitrone (
B), l-lyxo-nitrone (
C
), d-lyxo-nitrone (
D
E
F
l-ribo-nitrone (
G
) and d-ribo-nitrone (
H
inhibition assays on a range of enzymes showed that 1-N-acetylamino pyrrolidine analogues with
the same configuration as DMDP and pochonicine (1) showed powerful inhibition of β-HexNAcases
and moderate inhibition of α-GalNAcase, while the other compounds showed weak or no inhibition
of the tested glycosidases. This work has further examined the glycosidase inhibition of pyrrolidine
analogues of pochonicine and its stereoisomers, and would be helpful for the study of potent and
selective β-HexNAcase inhibitors.

Molecules 2020, 25, 1498
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Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/7/1498/s1:
Copies of 1H NMR, 13C NMR and 2D NMR spectra, and data of crystal structures.
Author Contributions: X.Y. performed the chemical syntheses, analysed the data and wrote the original draft;
Y.S. and A.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. All authors have read and agreed to the published version of
the manuscript.
Funding: Financial support from the National Natural Science Foundation of China (No. 21772206) and National
Engineering Research Center for Carbohydrate Synthesis of Jiangxi Normal University is gratefully acknowledged.
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the
Promotion of Science (JSPS KAKENHI Grant Number JP17K08362) (AK).
Conflicts of Interest: The authors declare no conflict of interest.
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