Stereoselective total synthesis of aminoiminohexitols via carbamate annulation

Article abstract of DOI:10.1021/jo201151b

New methodology for the preparation of a variety of aminoiminohextitols is described. Key in the synthesis is the application of a diastereoselective Strecker reaction and the extension of our carbamate annulation methodology to protected and functionaliz

Full text of DOI:10.1021/jo201151b

Article
pubs.acs.org/joc
Stereoselective Total Synthesis of Aminoiminohexitols via
Carbamate Annulation
Anna L. Win-Mason,†,‡ Seino A. K. Jongkees,§ Stephen G. Withers,§ Peter C. Tyler,∥
Mattie S. M. Timmer,*,† and Bridget L. Stocker*,‡
†School of Chemical and Physical Sciences, Victoria University of Wellington, P.O. Box 600, Wellington 6140, New Zealand
‡Malaghan Institute of Medical Research, P.O. Box 7060, Wellington 6242, New Zealand
§Chemistry Department, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
∥Carbohydrate Chemistry Team, Industrial Research Limited, P.O. Box 31-310, Lower Hutt 5040, New Zealand
S
* Supporting Information
ABSTRACT: New methodology for the preparation of a
variety of aminoiminohextitols is described. Key in the
synthesis is the application of a diastereoselective Strecker
reaction and the extension of our carbamate annulation
methodology to protected and functionalized alkenylamines.
Insight into the effects that the substitution patterns of the alkenylamines have on the diastereoselectivity of the iodocyclization
and carbamate annulation is discussed. An evaluation of the glycosidase inhibitory activity of the aminoiminohexitols and
derivatives is also presented, with the previously undisclosed D-talo isomer showing good selective inhibition of β-D-glucosidase.
nidase.4 The subsequent synthesis of a library of N-
INTRODUCTION
■
functionalized DMDP analogues5−8 then led to the identi-
The ability of azasugars to mimic the oxocarbenium transition
fication of potential drug candidates for osteoarthritis9,10 and
state of carbohydrate-processing enzymes1 has imparted on
bacterial infection.11 Similarly, the groups of Stutz and
̈
them much potential in the treatment of diseases such as
diabetes, cancer, and lysosomal storage disorders.2 Azasugars
Wrodnigg developed a series of glycosidase inhibitors via the
one-step N-derivatization of 1-amino-1,2,5-trideoxy-2,5-imino-
used as clinical drugs include the N-alkylated piperidine Glyset
D-mannitol (4),12−15 while more recently, Ramesh and co-
(1a, Figure 1), for noninsulin dependent diabetes, and Zavesca
workers prepared several selective glycosidase inhibitors based
on modifications to the L-ido azasugar 5.16 An N-acyl derivative
of imino-D-glucitol 6 was also found to exhibit potent and
selective activity against β-glucosidase while the parent
compound was inactive.17
Given the potential of pyrrolidines as glycosidase inhib-
itors,18 we were interested in developing efficient methodology
for the preparation of aminoiminohexitols. To this end, we
demonstrated that the combination of a diastereoselective
Strecker reaction and our recently developed I2-promoted
carbamate annulation methodology19 allowed for the synthesis
of the L-ido-derived aminoiminohexitol 5 in 39% overall yield
from D-arabinose (Scheme 1).20 Here, we illustrated that
benzyl-protected aldehyde 7, readily prepared from D-arabinose,
underwent a highly diastereoselective Strecker21 reaction to
Figure 1. Representative azasugars.
give the syn- and anti-alkenylamines 8 (dr = 9:1), which were
(1b), for the control of Gaucher’s disease. 1-Deoxygalactonojir-
imycin (DGJ, 2), currently in clinical trials, has shown great
promise in the treatment of Fabry’s disease.3 Five-membered
azasugars, or pyrrolidines, also exhibit much promise as drug
candidates. Notably, Wong et al. observed that derivatization of
the naturally occurring 2,5-bis(hydroxymethyl)-3,4-dihydrox-
ypyrrolidine (DMDP, 3a) to the 1-amino-1-deoxy-DMDP
analogue 3b resulted in the generation of a compound with
pronounced inhibitory activity against N-acetyl-β-D-glucosami-
readily separated by flash column chromatography. The major
syn-isomer was subjected to I2 and excess NaHCO3 in THF/
H2O to give, by way of intermediate iodide 9, the 4,5-cis
carbamate 10 in excellent yield and diastereoselectivity (dr >
95:5). Treatment of carbamate 10 with Pd(OH)2/C in the
presence of 2 M HCl then produced deprotected amine 11,
Received: June 4, 2011
Published: October 24, 2011
© 2011 American Chemical Society
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Scheme 1. Preparation of 5 from D-Arabinose
which upon hydrolysis gave the known L-ido-aminoiminohexitol
5.16
selectivity of the Strecker reaction does not appear to be
affected by the relative configuration between C-2 and C-3 of
the linear aldehyde, with similar diastereoselectivities being
obtained for both the arabinose (cf. Scheme 1)20 and ribose
derived aldehydes. The diastereomers, syn-17 and anti-17, were
readily separated via flash column chromatography (hexanes/
EtOAc, 2:1; Rf = 0.27 for α-aminonitrile syn-17, Rf = 0.16 for α-
aminonitrile anti-17) and their relative configurations con-
firmed following subsequent cyclization (vide infra).
To further explore this methodology, we herein report on the
application of our carbamate annulation for the synthesis of
other aminoiminohexitols including the D-manno 4 and D-gluco
6 derived azasugars (Figure 1) and the previously undisclosed
stereoisomers with the D-galacto 12, D-talo 13, and L-altro 14
configurations (Figure 2). Factors influencing the diastereose-
The major Strecker product syn-aminonitrile 17 was then
subjected to a carbamate annulation via treatment with
NaHCO3 and I2 (Scheme 3). Surprisingly, in contrast to the
Scheme 3. Carbamate Annulations with D-Ribose-Derived α-
Aminonitriles
Figure 2. Novel aminoiminohexitols.
lectivity of the iodocyclization and carbamate annulation will be
presented, as well as an evaluation of the inhibitory activity of
the aminoiminohexitols.
RESULTS AND DISCUSSION
■
To gain access to the 3,4-cis-series of hydroxylated azasugars,
we required the 3,4-anti-substituted α-aminonitrile precursors
and, consequently, D-ribose as the starting material (Scheme 2).
Scheme 2. Preparation of α-Aminonitriles Commencing with
D-Ribose
usual high selectivity of this reaction,19,20 two diastereomeric
carbamates 18 and 19 (dr = 4:1) were formed. Here, the major
carbamate was the 4,5-cis-isomer 18, as confirmed by X-ray
analysis (Figure 3A). Cyclization of the minor Strecker product,
anti-aminonitrile 17, however, gave only one diastereomer 20,
in 53% yield. This was also crystallized and a crystal structure
obtained (Figure 3B). Though the annulation yield for anti-
1
aminonitrile 17 was modest, there was no evidence by H
NMR of the other carbamate diastereomer or the intermediate
primary iodide. 1H NMR of the crude reaction mixture,
however, did indicate that retro-Strecker products (e.g.,
aldehydes) were formed in addition to the desired product. It
is also interesting that both ribose-derived alkenylamines took
longer to cyclize (6 days) when compared to the cyclization of
arabinose-derived syn-8 (20 h). This suggests that the activation
energies for the formation of 18−20 are higher than for the
formation of 10, which could be due to steric constraints
encountered by the benzyloxy groups at C-3 and C-4 when
transforming from the linear to cyclized structures. In previous
studies by Yoshida and co-workers,24 it was also observed that
Methyl iodoglycoside 15 was conveniently prepared according
to literature precedent22 and subjected to a Vasella reaction23 to
give aldehyde 16 in excellent (97%) yield. Aldehyde 16 was
then treated with NH4OAc and TMSCN to give α-aminonitrile
syn-17, the chelation-controlled Cram product, as the major
diastereomer (syn-17:anti-17, 8:1) and in excellent (92%)
overall yield. It is interesting to note that the syn-stereo-
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Figure 3. Crystal structures of carbamates 18 (A) and 20 (B).
cyclization of 1,2-alkoxy-substituted pentenylic alcohols pro-
ceeded much more slowly when forming the 2,5-trans, as
compared to the 2,5-cis, products.
To further tease out the effects that the substituents have on
the diastereoselectivity of the annulation, the minor arabinose-
derived aminonitrile anti-8 was also subjected to the carbamate
annulation conditions (Scheme 4). Here, cyclization resulted in
To explain the diastereoselectivities observed in the
carbamate annulation reaction, the transition states for the
formation of the observed carbamates need to be considered.
We previously established that the primary iodide (9, Scheme
1) is a key intermediate during the annulation20 and as the
formation of the iodide is irreversible, it is likely that this step
determines the diastereoselectivity of the reaction. Building on
seminal work by Chamberlin et al.25−27 and the more recent
theoretical studies presented by Gouverneur and co-workers,28
we propose that the attack of the internal nucleophile on the
I2−alkene complex takes place via a 5-membered ring transition
state in which the nitrogen approaches the double bond in an
Scheme 4. I2-Mediated Carbamate Annulation Using D-
Arabinose-Derived anti-8
envelope conformation and follows a Burgi−Dunitz-like
̈
trajectory. In the case of the cyclization of the D-ribose-derived
Strecker product syn-17 (Figure 4), the benzyloxy substituent
on the ring (depicted in blue) can be positioned either in the
plane of the double bond (A, OBn-in-plane) or almost
perpendicular to that plane (B, H-in-plane). Of these two
transition states, A, which ultimately leads to the major
carbamate 18, has minimal overlap between the electron-
withdrawing σ*C−O and reacting πCC orbitals, thereby
resulting in a lower energy transition state. In contrast,
the generation of two carbamate products (21:22, dr = 6:1)
following stirring at room temperature for 48 h, with the
configuration of each diastereomer being determined at
subsequent stages in the synthesis (vide infra).
Figure 4. Transition states leading to the formation of carbamate 18 (A, major product) and 19 (B, minor product) from alkenylamine syn-17 and
the formation of carbamate 20 (C) from alkenylamine anti-17.
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transition state B has overlapping benzyloxy σ*C−O and double
bond πCC orbitals, which results in the removal of electron
density from the π-bond and a higher energy transition state. At
this point, however, the influence of the nitrile substituent on
the transition state also needs to be considered. In A, the
dihedral angle between the plane of the nitrile dipole and the
lone pair of electrons on the amine is approximately 180°
(depicted in green). This results in electron density being
withdrawn from the nitrogen nucleophile, thus raising the
transition state energy of conformer A. In contrast, the dihedral
angle between the nitrogen dipole and the lone pair of
electrons of the amine in transition state B is approximately 90°
and thus has less of an influence on the nucleophilicity of the
amine. Accordingly, the transition states for A and B become
closer in energy and some of the trans-carbamate product 19 is
also formed in the cyclization.
When considering the formation of carbamate 20 from the
anti-α-aminonitrile 17 (Figure 4), similar transition states can
be drawn with the benzyloxy substituent in the plane of the
double bond (C, OBn-in-plane, lower energy TS) or almost
perpendicular to that plane (D, H-in-plane, higher energy TS).
In addition, the nitrile group in transition state C does not
reduce the nucleophilicity of the amine, while in transition state
D the nitrile dipole leads to electron density being withdrawn
from the amine through n−σ*CCN overlap. For this reason,
there is a large energy difference between the two transition
states, and carbamate 20 (from C) is the only product
observed. Similar analyses can also be used when explaining the
observed diastereoselectivity of the carbamate annulation for
the D-arabinose-derived alkenylamines syn-8 and anti-8.
carbonyl peak at δ 164.0 ppm in the 13C NMR spectra, which
correlated by HMBC to two protons at δ 3.59 and 3.49 ppm.
Further analysis by 2D NMR (COSY, HMBC, HSQC)
revealed that, rather than the desired amino sugar, urea
derivative 23 was formed in 30% yield (unoptimized),
presumably via the ring closure of the intermediate amine on
the carbamate in 24. An attempt was made to prevent urea
formation by changing the deprotection order through the
hydrolysis of carbamate 18 with LiOH; however, this led to
epimerization of the proton α to the nitrile and carbamate 20
was isolated in quantitative yield. While not effecting the
desired transformation, this epimerization to favor the
thermodynamically more stable trans relationship between the
carbamate and the nitrile provides a high-yielding route for the
preparation of carbamate 20. Previously, carbamate 20 could
only be prepared from the minor ribose-derived Strecker
product. The hydrogenation of carbamate 18 using Pd(OH)2/
C was then repeated, and during workup it was observed that if
the amine remained protonated, by keeping the conditions
acidic throughout, the desired amine functionalized carbamate
24 could be obtained in excellent (99%) yield and without the
need for further purification. Attempts to hydrolyze carbamate
24 then followed, but surprisingly, the use of 6 M HCl gave
chloroaminoiminohexitol 25 as the major product and only
minor amounts of the desired D-galacto aminoiminohexitol 12.
Though a useful synthetic intermediate, chloride 25 decom-
posed upon purification by silica gel chromatography and thus
could not be isolated in sufficient purity for subsequent
biological assessment. Subjecting carbamate 24 to 3 M H2SO4
at reflux, however, followed by neutralization with basic
DOWEX allowed for aminoiminohexitol 12 to be obtained in
quantitative yield as the free base. Urea derivative 23 could also
be quantitatively converted to the desired aminoiminohexitol
12 as the hydrochloride salt following treatment with refluxing
6 M HCl. Using the optimized route (18 → 24 → 12), the
target aminoiminohexitol 12 can now be prepared in 36%
overall yield from D-ribose.
Having determined the key influences on the diastereose-
lectivity of the carbamate annulation, we were then interested
in using the said carbamates as intermediates for the synthesis
of aminoiminohexitols. To this end, carbamate 18 was first
hydrogenated using Pd(OH)2/C in the presence of 2 M HCl
with the objective of simultaneously removing the benzyl
groups and reducing the nitrile to the amine (Scheme 5). Via
Armed with this knowledge, the remaining carbamates were
then transformed into their corresponding aminoiminohexitols
(Scheme 6). Treatment of carbamate 19 with Pd(OH)2/C in
the presence of 2 M HCl led to the smooth conversion into
amine 26 (98% yield). The carbamate in 26 was then
hydrolyzed using a solution of NaOH in EtOH followed by
neutralization with DOWEX-H+ to complete the synthesis and
generate novel D-talo aminoiminohexitol 13. Here, it is
important to note that when there is a 2,5-trans relationship
between the aminomethyl and carbamate moieties, the
corresponding urea derivative is not formed and base-mediated
hydrolysis can be used. Carbamate 20 was then hydrogenated
to give the corresponding amine 27 (99% yield), and
subsequent hydrolysis proceeded uneventfully to yield the
novel L-altro derived aminoiminohexitol 14 in 37% overall yield
from D-ribose. Though we had already assigned the stereo-
chemistry of 14 from the crystal structure of the precursor
carbamate 20, the 1H NMR and 13C NMR data were consistent
with those of the known D-altro enantiomer and the optical
rotation of similar magnitude but opposite sign.29,30 Carbamate
21, with the cis-relationship between the aminomethyl and
carbamate functionalities, was then hydrogenated and the
ammonium salt 28 obtained in excellent (99%) yield. The acid-
catalyzed hydrolysis of 28 using 3 M H2SO4 then gave the D-
glucitol-derived aminoiminohexitol 6, again in excellent yield.
Spectral data for 6 matched that previously reported.12 Finally,
Scheme 5. Deprotection of Carbamate 18
TLC analysis, this reaction appeared to proceed smoothly, and
following silica gel chromatography (DCM/EtOH/MeOH/
30% aq NH3, 5/2/2/1, v/v/v/v) only one product was
observed. HRMS also gave a molecular ion of 189.0869 [M +
H]+ corresponding to the expected [C7H12N2O4 + H]+. Close
analysis of the spectral data, however, revealed the presence of a
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Scheme 6. Carbamate Deprotection and Hydrolysis
Figure 5. Structures of acetylated aminoiminohexitols 30−33.
a
Table 1. Glycosidase Inhibition Studies
amines
carbamates
24 26
n.i. n.i.
acetamides
enzyme
5
12
13
n.i.
14
11
27
n.i.
30
31
32
33
GCase
SpHEX
ABG
9
n.i.*
n.i.
n.i.§
n.i.*
n.i.§
1.1
n.i.*
n.i.
n.i.§
n.i.*
0.5
n.i.*
n.i.§
n.i.§
n.i.*
n.i.§
0.07
n.i.*
0.5
n.i.
n.i.§
n.i.
n.i.§
n.i.§
n.i.§
n.i.§
0.006
0.06
2.3
0.8
1.3
a
Key: Ki values (mM) of compounds with GCase = β-D-glucocerebrosidase from human lysosome, SpHex = N-acetyl-β-D-hexosaminidase from
Streptomyces plicatus, ABG = β-D-glucosidase from Agrobacterium sp., n.i. = no inhibition was observed up to 10 mM inhibitor concentration, n.i.* =
no inhibition was observed up to 2 mM inhibitor concentration, and n.i.§ = no inhibition was observed up to 1 mM inhibitor concentration.
carbamate 22, derived from D-arabinose, was hydrogenated in
90% yield to generate the deprotected amine 29, which was
subsequently hydrolyzed under basic conditions to give the
known D-mannitol-derived aminoiminohexitol 4.12,31
Acetylated derivatives of the novel stereoisomers D-galacto
12, D-talo 13 and L-altro 14 were then prepared as literature
precedence suggests that N-acylation improves glycosidase
activity.5−8,12−17 Subjection of D-galacto-hexitol 12 to acetic
anhydride in MeOH15 gave acetamide 30 along with the bis-
acetamide 31 in 57% combined yield. Selective acetylation of D-
talo 13 and L-altro 14, however, proceeded smoothly to give the
corresponding acetamides 32 and 33 in 77% and 65% yield,
respectively (Figure 5).
D-hexosaminidase from Streptomyces plicatus (SpHEX), and β-D-
glucosidase from Agrobacterium sp. (ABG) (Table 1). Only
weak inhibition of GCase, the enzyme involved in Gaucher’s
disease, was observed with a single compound (L-ido amine 5)
at levels lower than 10 mM, and this level of inhibition is likely
to be ineffective for any therapeutic effect.32 In the case of
SpHEX, two inhibitors were identified with activity in the
submillimolar range. Both the D-galacto and L-altro isomers of
the acylated aminoiminohexitols 30 and 33 had inhibitory
activities of 0.5 and 0.5 mM, respectively. Given that the D-
manno isomer of these compounds (3b, Figure 1) has been
shown to inhibit Jack bean N-acetyl-β-D-glucosaminidase (Ki =
1.9 μm),4 it appears as though the functional group pattern
around the pyrrolidine scaffold is more important than
stereochemistry for the inhibition of this enzyme.
The novel aminoiminohexitols (12−14), their carbamate
precursors (24−27) and acetamides (30−33), and the
previously reported L-ido derivative (5)16 and carbamate
precursor (11) were then tested for their ability to inhibit
human lysosomal β-D-glucocerebrosidase (GCase), N-acetyl-β-
Five compounds were found to be selective ABG inhibitors.
The L-altro carbamate 27 and amine 14 had moderate Ki values
of 2.3 and 1.1 mM, respectively, while the D-talo acetamide 32
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vacuo to provide the aldehyde 16 as a colorless oil (1.03 g, 3.48 mmol,
and carbamate 26 had more potent inhibitory values of 0.07
and 0.06 mM. D-Talo amine 13 had the most potent inhibitory
activity (Ki = 0.006 mM). This is surprising as generally N-
acylated derivatives of iminohexitols are better inhibitors than
their parent compounds.5−8,12−17 Here, it should also be noted
that more lipophilic N-substituted derivatives generally exhibit
improved inhibitory activity. For example, D-manno-acetamide
3b and amine 4 (Figure 1) both exhibited a Ki of 0.025 mM
against ABG,15 whereas N-functionalized lipophilic D-manno
derivatives showed potent inhibition in the nanomolar scale.13
In view of this, the preparation of lipophilic derivatives of the D-
talo scaffold has the potential to produce noteworthy
glycosidase inhibitors.
97%): Rf = 0.57 (hexanes/EtOAc, 2/1, v/v); [α]17.6 = −29 (c = 1.0,
D
CHCl3); IR (film) 3064, 3031, 2925, 2868, 1729, 1455, 1207, 1069,
1027, 933, 735, 696 cm−1; 1H NMR (500 MHz, CDCl3) δ 9.64 (d, J1,2
= 2.0 Hz, 1H, H-1), 7.38−7.27 (m, 10H, H−Ar), 5.88 (ddd, J4,5b
=
=
17.7, J4,5a = 10.4, J3,4 = 7.6 Hz, 1H, H-4), 5.38 (dt, J4,5a = 10.4, 2J5a,5b
4J3,5a = 1.2 Hz, 1H, H-5a), 5.35 (dt, J4,5b = 17.7, 2J5a,5b = 4J3,5b = 1.2 Hz,
1H, H-5b), 4.71 (d, 2Ja,b = 12.0 Hz, 1H, CHa 2-O-Bn), 4.66 (d, 2Ja,b
=
2
12.0 Hz, 1H, CHb 2-O-Bn), 4.65 (d, Ja,b = 12.0 Hz, 1H, CHa 3-O-
2
Bn), 4.42 (d, Ja,b = 12.0 Hz, 1H, CHb 3-O-Bn), 4.17 (ddt, J3,4 = 7.6,
J
2,3 = 4.7 Hz, 4J3,5 = 1.2 Hz, 1H, H-3), 3.90 (dd, J2,3 = 4.7, J1,2 = 2 Hz,
1H, H-2); 13C NMR (125 MHz, CDCl3) δ 201.8 (C-1), 137.9 (C-i 3-
O-Bn), 137.3 (C-i 2-O-Bn), 134.2 (C-4), 128.6 (C-Ar), 128.5 (C-Ar),
128.2 (C-Ar), 128.2 (C-Ar), 127.9 (C-Ar), 128.8 (C-Ar), 120.2 (C-5),
85.0 (C-2), 80.3 (C-3), 73.1 (CH2 2-O-Bn), 70.6 (CH2 3-O-Bn);
HRMS(ESI) m/z calcd for [C19H20O3 + Na]+ 319.1305, obsd
319.1311.
CONCLUSION
■
In summary, an efficient route for the preparation of a number
of aminoiminohexitols has been presented. In particular, the
two novel aminoiminohexitols 12 and 14 were efficiently
prepared in 36% and 37% overall yield from D-ribose,
respectively. During the course of this work, the power of a
highly diastereoselective Strecker reaction without the need for
chiral Lewis acids or catalysts has been demonstrated. The
diastereoselectivity of the reaction can be explained by the
Cram-chelation control model for both D-arabinose- and D-
ribose-derived aldehydes. Moreover, the potential of our novel
carbamate annulation for the cyclization of protected and
functionalized alkenylamine precursors has been highlighted,
and the effects that the substituent pattern of the α-aminonitrile
has on the diastereoselectivity of the annulation reaction have
been explained. The applicability of the carbamate annulation
to a variety of alkenylamines and an understanding of the
factors controlling the diastereoselectivity of the reaction
should make this methodology a valuable addition to the
synthetic chemists toolbox. Furthermore, several amino-
iminohexitol derivatives have shown promising selective
glycosidase activity (e.g., 13, 14, 26, and 32), with the D-talo
configuration exhibiting good selective inhibition of β-D-
glucosidase. Further derivatization of the D-talo scaffold has
the potential to provide powerful glycosidase inhibitors, and we
are currently exploring this avenue of research.
(2S,3S,4R)-2-Amino-3,4-dibenzyloxyhex-5-enenitrile (syn-
17) and (2S,3S,4R)-2-Amino-3,4-dibenzyloxyhex-5-enenitrile
(anti-17). To a solution of aldehyde 16 (166 mg, 0.56 mmol) in
EtOH (11.2 mL) was added NH4OAc (471 mg, 5.60 mmol). The
mixture was stirred at room temperature for 15 min, at which point
TMSCN (147 μL, 1.12 mmol) was added dropwise. The clear
colorless solution was stirred at room temperature for 48 h. The
resulting solution was diluted with EtOAc, washed with satd aq
NaHCO3, H2O, and brine, dried (MgSO4), filtered, and concentrated
in vacuo. Purification of the α-aminonitriles using gradient flash
chromatography (hexanes/EtOAc, 10/1 → 1/1, v/v) gave first α-
aminonitrile syn-17 as a pale yellow oil (147 mg, 0.46 mmol, 82%) and
then α-aminonitrile anti-17 as a pale yellow oil (18.9 mg, 0.06 mmol,
10%) (dr = 8:1, syn-17:anti-17). syn-17: Rf = 0.27 (hexanes/EtOAc, 2/
1, v/v); [α]19D = −22.4 (c = 1.0, CHCl3); IR (film) 3397, 3327, 3088,
3064, 3032, 2871, 2372, 1710, 1497, 1455, 1392, 1214, 1088, 1072,
1
935, 741, 698 cm−1; H NMR (500 MHz, CDCl3) δ 7.37−7.30 (m,
10H, H−Ar), 5.84 (ddd, J5,6a = 17.2, J5,6b = 10.5, J4,5 = 7.9 Hz, 1H, H-
5), 5.46 (ddd, J5,6a = 17.2, 2J6a,6b = 1.7, 4J4,6a = 0.7 Hz, 1H, H-6a), 5.45
(ddd, J5,6b = 10.5, 2J6a,6b = 1.7, 4J4,6a = 0.7 Hz, 1H, H-6b) 4.76 (d, 2Ja,b
=
2
10.8 Hz, 1H, CHa 3-O-Bn), 4.68 (d, Ja,b = 10.8 Hz, 1H, CHb 3-O-
2
2
Bn), 4.62 (d, Ja,b = 11.4 Hz, 1H, CHa 4-O-Bn), 4.36 (d, Ja,b = 11.4
Hz, 1H, CHb 4-O-Bn), 4.08 (d, J2,3 = 2.0 Hz, 1H, H-2), 4.06 (tt, J4,5
=
J
3,4 = 7.9, 4J4,6 = 0.7 Hz, 1H, H-4), 3.66 (dd, J3,4 = 7.9, J2,3 = 2.0 Hz, 1H,
H-3), 1.82 (br s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ 137.7 (C-
i 4-O-Bn), 137.2 (C-i 3-O-Bn), 135.3 (C-5), 128.7 (C-Ar), 128.6 (C-
Ar), 128.6 (C-Ar), 128.5 (C-Ar), 128.5 (C-Ar), 128.4 (C-Ar), 128.2
(C-Ar), 128.2 (C-Ar), 128.0 (C-Ar), 121.3 (C-1), 120.8 (C-6), 81.5
(C-3), 79.0 (C-4), 74.8 (CH2 3-O-Bn), 70.8 (CH2 4-O-Bn), 44.4 (C-
2); HRMS (ESI) m/z calcd for [C20H22N2O2 + H]+ 323.1754, obsd
EXPERIMENTAL SECTION
■
323.1762. anti-17: Rf = 0.16 (hexanes/EtOAc, 2/1, v/v); [α]19
=
Unless otherwise stated, all reactions were performed under
atmospheric air. THF was distilled from LiAlH4 prior to use. All
chemicals obtained from commercial suppliers were used without
further purification. Zn dust was activated by the careful addition of
concd H2SO4, followed by decantation and washing with EtOH (3×)
and hexanes (3×) and storage under dry hexanes. All solvents were
removed by evaporation under reduced pressure. Reactions were
monitored by TLC analysis on silica gel coated plastic sheets with
detection by coating with 20% H2SO4 in EtOH followed by charring at
ca. 150 °C, by coating with a solution of ninhydrin in EtOH followed
by charring at ca. 150 °C, by coating with Hanessian’s stain followed
by charring at ca. 150 °C, or by coating with a solution of 5% KMnO4
and 1% NaIO4 in H2O followed by heating. 1H and 13C chemical shifts
(δ) were internally referenced to the residual solvent peak. NMR peak
assignments are based on 2D NMR experiments (COSY, HSQC, and
HMBC).
D
−2.8 (c = 1.0, CHCl3); IR (film) 3388, 3325, 3088, 3065, 3031, 2869,
2231, 1643, 1497, 1455, 1392, 1210, 1086, 1070, 1028, 936, 738, 698
1
cm−1; H NMR (500 MHz, CDCl3) δ 7.38−7.30 (m, 10H, H−Ar),
5.90 (ddd, J5,6a = 17.3, J5,46b = 10.3, J4,5 = 7.8 Hz, 1H, H-5), 5.51 (ddd,
2
J5,6a = 17.3, J6a,6b = 1.7, J4,6a = 1.0 Hz, 1H, H-6a), 5.47 (ddd, J5,6b
=
2
4
2
10.3, J6a,6b = 1.7, J4,6a = 1.0 Hz, 1H, H-6b) 4.81 (d, Ja,b = 11.2 Hz,
1H, CHa 3-O-Bn), 4.64 (d, 2Ja,b = 11.3 Hz, 1H, CHa 4-O-Bn), 4.62 (d,
2Ja,b = 11.2 Hz, 1H, CHb 3-O-Bn), 4.39 (d, 2Ja,b = 11.3 Hz, 1H, CHb 4-
O-Bn), 4.08 (d, J2,3 = 4.2 Hz, 1H, H-2), 4.05 (tt, J4,5 = J3,4 = 7.8, 4J4,6
=
0.7 Hz, 1H, H-4), 3.64 (dd, J3,4 = 7.8, J2,3 = 4.2 Hz, 1H, H-3), 1.76 (br
s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ 137.8 (C-i 4-O-Bn),
137.6 (C-i 3-O-Bn), 135.2 (C-5), 128.6 (C-Ar), 128.1 (C-Ar), 128.1
(C-Ar), 128.0 (C-Ar), 128.0 (C-Ar), 121.2 (C-1), 120.5 (C-6), 82.6
(C-3), 81.0 (C-4), 75.2 (CH2 3-O-Bn), 70.9 (CH2 4-O-Bn), 45.9 (C-
2); HRMS(ESI) m/z calcd for [C20H22N2O2 + H]+ 323.1754, obsd
323.1763.
General Procedure for the Carbamate Annulation. To a
solution of α-aminonitrile (1 mmol) in THF (3.5 mL) were added I2
(634 mg, 2.5 mmol), H2O (3.5 mL), and NaHCO3 (1.68 g, 20 mmol).
The reaction mixture was stirred at room temperature for varying
lengths of time (20 h−6 d), quenched with satd aq Na2S2O3, and
extracted with EtOAc. The organic layer was washed with H2O and
(2R,3R)-2,3-Dibenzyloxypent-4-enal (16). To a solution of
methyl iodoriboside 1522 (1.63 g, 3.59 mmol) in EtOH/H2O/
AcOH (40/2/1, v/v/v, 54 mL) was added Zn (1.17 g, 18.0 mmol).
The mixture was stirred at reflux for 3 h, cooled to room temperature,
and filtered through a Celite plug with EtOAc. The resulting mixture
was further diluted with EtOAc, washed twice with satd aq NaHCO3
then H2O and brine, dried (MgSO4), filtered, and concentrated in
9616
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Article
brine, dried (MgSO4), filtered, and concentrated in vacuo. Purification
was achieved using gradient flash chromatography (hexanes/EtOAc,
v/v) or (DCM/EtOAc, v/v).
0.3 mL). The reaction mixture was stirred at room temperature for 4 h,
quenched with satd aq NH4Cl, and extracted with EtOAc. The organic
layer was washed with H2O and brine, dried (MgSO4), and
concentrated in vacuo to provide carbamate 20 as a colorless oil
(11.0 mg, 0.03 mmol, quantitative). Spectral data as above.
(5R,6S,7R,7aS)-6,7-Dibenzyloxy-3-oxotetrahydropyrrolo[1,2-
c]oxazole-5-carbonitrile (18). By subjecting α-aminonitrile syn-17
(492 mg, 1.53 mmol) to the general procedure for the carbamate
annulation for 6 d, carbamate 18 was obtained as a white solid (378
mg, 1.04 mmol, 68%, dr = 4:1, 18:19). Crystallization (DCM) yielded
fine white needles for which a single-crystal X-ray diffraction was
obtained and then solved and refined using Olex2:33 Rf = 0.19 (DCM/
(5R,6R,7R,7aS)-6,7-Dibenzyloxy-3-oxotetrahydropyrrolo-
[1,2-c]oxazole-5-carbonitrile (21). By subjecting α-aminonitrile
anti-820 (140 mg, 0.43 mmol) to the general procedure for the
carbamate annulation for 48 h, carbamate 21 was obtained as a
colorless oil (129 mg, 0.35 mmol, 81%, dr = 6:1, 21:22): Rf = 0.29
EtOAc, 50/1, v/v); mp = 198.9 − 199.9 °C; [α]20 = +34.5 (c = 1.0,
(hexanes/EtOAc, 1/1, v/v); [α]24 = +25.7 (c = 1.0, CHCl3); IR
D
D
CHCl3); IR (film) 3066, 3028, 2953, 2921, 2887, 2251, 1737, 1395,
(film) 3064, 3032, 2919, 2872, 2247, 1752, 1471, 1395, 1246, 1096,
1
1269, 1256, 1150, 1101, 1046, 1002, 702, 694 cm−1; H NMR (500
1077, 1007, 740, 698 cm−1; 1H NMR (500 MHz, CDCl3) δ 7.42−7.27
2
2
MHz, CDCl3) δ 7.43−7.30 (m, 10H, H−Ar), 5.18 (d, Ja,b = 11.8 Hz,
(m, 10H, H−Ar), 4.73 (d, Ja,b = 12 Hz, 1H, CHa 7-O-Bn), 4.60 (d,
1H, CHa 7-O-Bn), 4.78 (d, 2Ja,b = 11.9 Hz, 1H, CHa 6-O-Bn), 4.71 (d,
2Ja,b = 11.9 Hz, 1H, CHb 6-O-Bn) 4.68 (d, 2Ja,b = 11.8 Hz, 1H, CHb 7-
2
2Ja,b = 11.9 Hz, 1H, CHa 6-O-Bn), 4.55 (s, 1H, H-6), 4.53 (d, Ja,b
=
11.9 Hz, 1H, CHb 6-O-Bn), 4.46 (dd, 2J1a,1b = 8.6, J1a,7a = 5.6 Hz, 1H,
O-Bn), 4.51 (dd, 2J1a,1b = 8.8, J1a,7a = 6.8, Hz, 1H, H-1a), 4.42 (d, J5,6
=
H-1a), 4.44 (d, 2Ja,b = 12 Hz, 1H, CHb 7-O-Bn), 4.42 (t, J1a,1b = J1b,7a
=
7.1 Hz, 1H, H-5), 4.35 (t, J1a,1b = J1b,7a = 8.8 Hz, 1H, H-1b), 4.37 (dd,
5,6 = 7.1, J6,7 = 2.7 Hz, 1H, H-6), 4.09 (ddd, J7a,1b = 8.8, J1a,7a = 6.8, J7,7a
8.6 Hz, 1H, H-1b), 4.36 (ddd, J1b,7a = 8.6, J1a,7a = 5.6, J7,7a = 3.2 Hz, 1H,
H-7a), 4.22 (d, J5,6 = 0.5 Hz, 1H, H-5), 3.79 (dd, J7,7a = 3.2, J6,7 = 1.0
Hz, 1H, H-7); 13C NMR (125 MHz, CDCl3) δ 157.5 (C-3), 136.3 (C-
i 7-O-Bn), 135.9 (C-i 6-O-Bn), 129.0 (C-Ar), 128.9 (C-Ar), 128.9 (C-
Ar), 128.6 (C-Ar), 128.4 (C-Ar), 128.2 (C-Ar), 113.5 (CN), 87.0 (C-
6), 78.2 (C-7), 73.2 (CH2 6-O-Bn), 71.9 (CH2 7-O-Bn), 62.6 (C-1),
62.0 (C-7a), 50.6 (C-5); HRMS(ESI) m/z calcd for [C21H20N2O4 +
Na]+ 387.1315, obsd 387.1324.
J
= 2.7 Hz, 1H, H-7a), 3.87 (t, J7,7a = J6,7 = 2.7 Hz, 1H, H-7); 13C NMR
(125 MHz, CDCl3) δ 157.4 (C-3), 137.4 (C-i 7-O-Bn), 136.1 (C-i 6-
O-Bn), 129.0 (C-Ar), 128.9 (C-Ar), 128.7 (C-Ar), 128.3 (C-Ar), 128.3
(C-Ar), 128.2 (C-Ar), 112.9 (CN), 83.7 (C-6), 74.5 (C-7), 73.8 (CH2
6-O-Bn), 73.6 (CH2 7-O-Bn), 64.1 (C-1), 58.8 (C-7a), 47.2 (C-5);
HRMS(ESI) m/z calcd for [C21H20N2O4 + Na]+ 387.1315, obsd
387.1321.
(5R,6R,7R,7aR)-6,7-Dibenzyloxy-3-oxotetrahydropyrrolo-
[1,2-c]oxazole-5-carbonitrile (22). By subjecting α-aminonitrile
anti-820 (140 mg, 0.43 mmol) to the general procedure for the
carbamate annulation for 48 h, carbamate 22 was obtained as a
colorless oil (21.4 mg, 0.06 mmol, 13.6%, dr = 6:1, 21:22). Rf = 0.50
(hexanes/EtOAc, 1/1, v/v): [α]24D = +5.7 (c = 1.0, CHCl3); IR (film)
3064, 3032, 2919, 2872, 2250, 1762, 1455, 1391, 1211, 1099, 1072,
(5R,6S,7R,7aR)-6,7-Dibenzyloxy-3-oxotetrahydropyrrolo-
[1,2-c]oxazole-5-carbonitrile (19). By subjecting α-aminonitrile
syn-17 (492 mg, 1.53 mmol) to the general procedure for the
carbamate annulation for 6 d, carbamate 19 was obtained as a colorless
oil (94.5 mg, 0.26 mmol, 17%, dr = 4:1, 18:19): Rf = 0.31 (DCM/
EtOAc, 20/1, v/v); [α]20 = +45.2 (c = 1.0, CHCl3); IR (film) 3064,
D
3032, 2919, 2872, 1761, 1455, 1393, 1306, 1214, 1136, 1027, 1010,
1
740, 698 cm−1; H NMR (500 MHz, CDCl3) δ 7.41−7.28 (m, 10H,
1
737, 699 cm−1. H NMR (500 MHz, CDCl3) δ 7.45−7.27 (m, 10H,
2
H−Ar), 4.80 (d, J5,6 = 2.1 Hz, 1H, H-5), 4.65 (d, Ja,b = 12.0 Hz, 1H,
2
H-Ar), 4.96 (d, Ja,b = 11.5 Hz, 1H, CHa 6-O-Bn), 4.77 (d, J2,3 = 5.0
2
CHa 7-O-Bn), 4.63 (d, Ja,b = 11.5 Hz, 1H, CHa 6-O-Bn), 4.51 (ddd,
Hz, 1H, H-5), 4.73 (d, 2Ja,b = 11.5 Hz, 1H, CHb 6-O-Bn), 4.64 (d, 2Ja,b
2
J
1a,7a = 8.7, 2J1a,1b = 7.5, 4J1a,7 = 4.0 Hz, 1H, H-1a), 4.50 (d, Ja,b = 12.0
2
= 11.9 Hz, 1H, CHa 7-O-Bn), 4.46 (dd, J1a,1b = 9.6, J1a,7a = 8.3 Hz,
2
Hz, 1H, CHb 7-O-Bn), 4.46 (d, Ja,b = 11.5 Hz, 1H, CHb 6-O-Bn),
2
1H, H-1a), 4.36 (d, Ja,b = 11.9 Hz, 1H, CHb 7-O-Bn), 4.29 (t, J6,7
=
4.37 (dd, J5,6 = 2.1, J6,7 = 2.7 Hz, 1H, H-6), 4.12 (dd, J1a,1b = 7.5, J1b,7a
=
J
5,6 = 5.0 Hz, 1H, H-3), 4.23 (td, J1a,7a = J7,7a = 8.3, J1b,7a = 2.7 Hz, H-
4.4 Hz, 1H, H-1b), 4.09 (ddd, J1a,7a = 8.7, J1b,7a = 4.4, J7,7a = 0.5 Hz, 1H,
2
7a), 4.08 (dd, J1a,1b = 9.6, J1b,7a = 2.7 Hz, 1H, H-1b), 3.56 (dd, J7,7a
=
4
H-7a), 3.87 (ddd, J1a,7 = 4.0, J6,7 = 2.7, J7,7a = 0.5 Hz, 1H, H-7); 13C
8.3, J6,7 = 5.0 Hz, 1H, H-7); 13C NMR (125 MHz, CDCl3) δ 160.2 (C-
3), 136.6 (C-i 7-O-Bn), 136.5 (C-i 6-O-Bn), 129.0 (C-Ar), 128.8 (C-
Ar), 128.8 (C-Ar), 128.6 (C-Ar), 128.6 (C-Ar), 128.0 (C-Ar), 115.0
(CN), 80.7 (C-7), 77.1 (C-6), 74.5 (CH2 6-O-Bn), 73.2 (CH2 7-O-
Bn), 66.3 (C-1), 60.0 (C-7a), 53.1 (C-5); HRMS(ESI) m/z calcd for
[C21H20N2O4 + Na]+ 387.1315, obsd 387.1328.
NMR (125 MHz, CDCl3) δ 160.0 (C-3), 136.6 (C-i 7-O-Bn), 135.7
(C-i 6-O-Bn), 129.1 (C-Ar), 129.0 (C-Ar), 128.9 (C-Ar), 128.8 (C-
Ar), 128.7 (C-Ar), 128.6 (C-Ar), 128.3 (C-Ar), 128.1 (C-Ar), 115.9
(CN), 86.8 (C-7), 86.4 (C-6), 73.0 (CH2 7-O-Bn), 72.8 (CH2 6-O-
Bn), 67.4 (C-1), 62.7 (C-7a), 52.3 (C-5); HRMS(ESI) m/z calcd for
[C21H20N2O4 + Na]+ 387.1315, obsd 387.1321.
(5S,6S,7R,7aS)-6,7-Dibenzyloxy-3-oxotetrahydropyrrolo[1,2-
c]oxazole-5-carbonitrile (20). By subjecting α-aminonitrile anti-17
(25.0 mg, 0.08 mmol) to the general procedure for the carbamate
annulation for 6 d, carbamate 20 was obtained as a white solid (14.9
mg, 0.04 mmol, 53%). Crystallization (DCM) yielded colorless plates
for which a single-crystal X-ray diffraction was obtained then solved
(5S,6R,7S,7aR)-6,7-Dihydroxy-5-(hydroxymethyl)tetrahydro-
1H-pyrrolo[1,2-c]imidazol-3(2H)-one (23). Carbamate 18 (147
mg, 0.40 mmol) was dissolved in CHCl3 (3.0 mL) and transferred into
a Fischer−Porter bottle, EtOH (5.0 mL), HCl in ether (1.0 mL, 2 M),
and Pd(OH2)/C (110 mg) were then added, and the vessel was
charged with 7 bar of pressure of H2. The reaction mixture was stirred
vigorously at room temperature for 3 d. After H2 pressure was
released, the reaction mixture was filtered through a plug of Celite,
washed with EtOH and DCM/MeOH/EtOH/30% aq NH3 (5/2/2/1,
v/v/v/v), and concentrated in vacuo. Purification was achieved by dry-
loading using gradient flash chromatography (DCM/MeOH/EtOH/
30% aq NH3, 50/2/2/1 → 5/2/2/1, v/v/v/v) to provide the urea 23
as a colorless oil (23.0 mg, 0.12 mmol, 30%): Rf = 0.48 (DCM/
MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); [α]21D = −22.0 (c =
1.0, H2O); IR (film) 3288, 2927, 1658, 1492, 1451, 1372, 1130, 1025,
978, 757, 700 cm−1; 1H NMR (500 MHz, D2O) δ 4.61 (dd, J5,6 = 8.1,
and refined using Olex2: Rf = 0.45 (DCM/EtOAc, 20/1, v/v); [α]20
D
̊
= +13.6 (c = 0.5, CHCl3); mp = 97.4 − 97.6C; IR (film) 3034, 2953,
2923, 2852, 1757, 1456, 1395, 1207, 1141, 1075, 1005, 772, 735, 696
1
cm−1; H NMR (500 MHz, CDCl3) δ 7.43−7.25 (m, 10H, H−Ar),
4.93 (d, 2Ja,b = 11.9 Hz, 1H, CHa 7-O-Bn), 4.79 (d, 2Ja,b = 11.9 Hz, 1H,
2
CHa 6-O-Bn), 4.69 (d, Ja,b = 11.9 Hz, 1H, CHb 6-O-Bn), 4.59 (d,
2Ja,b = 11.9 Hz, 1H, CHb 7-O-Bn), 4.57 (d, J5,6 = 6.8 Hz, 1H, H-5),
4.46 (dd, J6,7 = 3.4, J5,6 = 6.8 Hz, 1H, H-6), 4.40 (dd, 2J1a,1b = 8.6, J7a,1a
2
= 3.7 Hz, 1H, H-1a), 4.34 (t, J1a,1b = J1a,7a = 8.6 Hz, 1H, H-1b), 4.03
(td, J1a,7a = J1b,7a = 8.6, J7,7a = 3.4 Hz, 1H, H-7a), 3.95 (t, J7,7a = J6,7 = 3.4
Hz, 1H, H-7); 13C NMR (125 MHz, CDCl3) δ 160.8 (C-3), 137.2 (C-
i 7-O-Bn), 136.1 (C-i 6-O-Bn), 129.0 (C-Ar), 128.8 (C-Ar), 128.8 (C-
Ar), 128.4 (C-Ar), 128.1 (C-Ar), 128.0 (C-Ar), 117.9 (CN), 86.0 (C-
6), 76.2 (C-7), 73.8 (CH2 7-O-Bn), 73.8 (CH2 6-O-Bn), 63.7 (C-1),
59.5 (C-7a), 50.9 (C-5); HRMS(ESI) m/z calcd for [C21H20N2O4 +
Na]+ 387.1315, obsd 387.1320.
J
6,7 = 4.0 Hz, 1H, H-6), 4.13 (dd, 2Ja,b = 12.0, J5,CH2Oa = 3.2, Hz, 1H, H-
CH2Oa) 4.12 (ddd, J1b,7a = 9.5, J1a,7a = 8.1, J7,7a = 4.0 Hz, 1H, H-7a),
3.99 (t, J6,7 = J7,7a = 4.0 Hz, 1H, H-7), 3.76 (dt, J5,6 = 8.1, J5,CH2Oa
=
2
J5,CH2Ob = 3.2 Hz, 1H, H-5), 3.72 (dd, Ja,b = 12.0, J5,CH2Ob = 3.2 Hz,
2
H−CH2Ob) 3.59 (dd, J1a,1b = 9.5, J1a,7a = 8.1 Hz, 1H, H-1a), 3.49 (t,
2J1a,1b = J1b,7a = 9.5 Hz, 1H, H-1b); 13C NMR (125 MHz, D2O) δ 164.0
Synthesis of 20 via Epimerization. To a solution of carbamate
18 (11.0 mg, 0.03 mmol) in THF (0.6 mL) was added LiOH aq (2 M,
(C-3), 74.2 (C-6), 69.1 (C-7), 60.1 (C-7a), 58.5 (C-5), 55.3 (CH2O),
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Article
38.7 (C-1); HRMS(ESI) m/z calcd for [C7H12N2O4 + H]+ 189.0870,
obsd 189.0869.
MHz, D2O) δ 4.63−4.59 (m, 1H, H-1a), 4.52−4.47 (m, 2H, H-7a, and
H-1b), 4.15 (t, J6,7 = J5,6 = 1.9 Hz, 1H, H-6), 4.01 (dd, J7,7a = 2.8, J6,7
=
1.9 Hz, 1H, H-7), 3.72 (ddd, J5,CH2Nb = 10, J5,CH2Na = 3.2, J5,6 = 1.9 Hz,
General Procedure for Hydrogenation of Carbamates.
Protected carbamate (0.27 mmol) was dissolved in CHCl3 (4.5 mL)
and transferred into a Fischer−Porter bottle, EtOH (7.5 mL), HCl in
ether (1.4 mL, 2 M), and Pd(OH2)/C (230 mg) were then added, and
the vessel was charged with 7 bar of pressure of H2. The reaction
mixture was stirred vigorously at room temperature for 3 d. After H2
pressure was released, the reaction mixture was filtered through a plug
of Celite, washed with EtOH and H2O, and concentrated in vacuo to
provide the amino carbamate as the hydrochloride salt.
2
1H, H-5), 3.59 (dd, Ja,b = 13.9, J5,CH2Nb = 3.2 Hz, 1H, CH2Na), 3.53
(dd, 2Ja,b = 13.9, J5,CH2Nb = 10 Hz, 1H, CH2Nb); 13C NMR (125 MHz,
D2O) δ 160.2 (C-3), 82.2 (C-6), 73.3 (C-7), 63.9 (C-1), 63.2 (C-7a),
62.5 (C-5), 39.3 (CH2N); HRMS(ESI) m/z calcd for [C7H12N2O4 +
H]+ 189.0870, obsd 189.0871.
( 5 R , 6 R , 7 R , 7 a R ) - 5 - ( A m i n o m e t h y l ) - 6 , 7 -
dihydroxytetrahydropyrrolo[1,2-c]oxazol-3(1H)-one Hydro-
chloride (29). By subjecting carbamate 22 (16 mg, 0.04 mmol) to
the general procedure for the hydrogenation of carbamates,
ammonium salt 29 was obtained as a colorless oil (7.0 mg, 0.04
mmol, 90%): Rf = 0.41 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1,
v/v/v/v); [α]24.8D = +10.0 (c = 0.4, H2O); IR (film) 3330, 3247, 2924,
( 5 R , 6 S , 7 R , 7 a S ) - 5 - ( A m i n o m e t h y l ) - 6 , 7 -
dihydroxytetrahydropyrrolo[1,2-c]oxazole-3(1H)-one Hydro-
chloride (24). By subjecting carbamate 18 (60 mg, 0.16 mmol) to
the general procedure for the hydrogenation of carbamates,
ammonium salt 24 was obtained as a white solid (30.8 mg, 0.16
mmol, 99%) and further crystallized from MeOH: Rf = 0.29 (DCM/
MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); [α]26.1D = +30.0 (c =
1.0, H2O); mp = 230.0−230.5 °C; IR (film) 3362, 2960, 2930, 1724,
1
1731, 1641, 1404, 1235, 1084, 1012, 918, 773 cm−1. H NMR (500
2
MHz, D2O) δ 4.70 (dd, J1a,1b = 9.6, J1a,7a = 8.1 Hz, 1H, H-1a), 4.48
(dd, 2J1a,1b = 9.6, J1b,7a = 4 Hz, 1H, H-1b), 4.07−4.02 (m, 3H, H-6, H-7
and H-7a), 3.84 (ddd, J5,CH2Nb = 11, J5,6 = 4.9, J5,CH2Na = 3.3 Hz, 1H, H-
5), 3.37 (dd, 2Ja,b = 13.1, J5,CH2Na = 3.3 Hz, 1H, CH2Na), 3.19 (dd, 2Ja,b
= 13.1, J5,CH2Nb = 11, 1H, CH2Nb); 13C NMR (125 MHz, D2O) δ
163.6 (C-3), 79.2 (C-6), 78.4 (C-7), 68.4 (C-1), 61.6 (C-5), 60.9 (C-
7a), 40.8 (CH2N); HRMS(ESI) m/z calcd for [C7H12N2O4 + H]+
189.0870, obsd 189.0874.
1
1633, 1433, 1261, 1127, 1075, 949, 897 cm−1; H NMR (500 MHz,
2
D2O) δ 4.66 (dd, J5,6 = 7.6, J6,7 = 3.4 Hz, 1H, H-6), 4.56 (t, J1a,1b
J
=
1a,7a = 9.3 Hz, 1H, H-1a), 4.49 (dd, 2J1a,1b = 9.3, J1a,7a = 7.1 Hz, 1H, H-
1b), 4.25 (ddd, J1a,7a = 9.3, J1b,7a = 7.1, J7,7a = 3.4 Hz, 1H, H-7a), 4.13 (t,
7,7a = J6,7 = 3.4 Hz, 1H, H-7), 4.04 (td, J5,6 = J5,CH2Nb = 7.6, J5,CH2Na
J
=
2
1-Amino-6-chloro-1,2,5,6-tetradeoxy-2,5-imino-D-galactitol
Hihydrochloride (25). Amino carbamate 24 (4 mg, 0.07 mmol) was
dissolved in HCl (1.2 mL, concd). The solution was stirred at reflux
for 3 h, cooled, and concentrated in vacuo. Purification was attempted
using gradient flash chromatography (DCM/MeOH/EtOH/30% aq
NH3, 15/2/2/1 → 5/2/2/1, v/v/v/v) to provide chloroiminogalacti-
tol 25 as a white solid with an inseparable decomposition product also
observed: Rf = 0.37 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1, v/
3.4 Hz, 1H, H-5), 3.59 (dd, Ja,b = 14.2, J5,CH2Na = 3.4 Hz, 1H,
CH2Na), 3.38 (dd, Ja,b = 14.2, J5,CH2Nb = 7.6 Hz, 1H, CH2Nb); 13C
2
NMR (125 MHz, D2O) δ 159.7 (C-3), 74.3 (C-6), 69.1 (C-7), 64.5
(C-1), 60.3 (C-7a), 55.0 (C-5), 36.7 (CH2N); HRMS(ESI) m/z calcd
for [C7H12N2O4 + H]+ 189.0870, obsd 189.0869.
( 5 R , 6 S , 7 R , 7 a R ) - 5 - ( A m i n o m e t h y l ) - 6 , 7 -
dihydroxytetrahydropyrrolo[1,2-c]oxazol-3(1H)-one Hydro-
chloride (26). By subjecting carbamate 19 (4 mg, 0.01 mmol) to
the general procedure for the hydrogenation of carbamates,
ammonium salt 26 was obtained as a colorless oil (2.0 mg, 0.01
mmol, 98%): Rf = 0.25 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1,
v/v/v/v); [α]25.7D = −10.0 (c = 0.7, H2O); IR (film) 3382, 3284, 2927,
1
v/v/v); H NMR (500 MHz, 2% DCl in D2O) δ 4.65 (dd, J2,3 = 7.1,
J
J
3,4 = 4.4 Hz, 1H, H-3), 4.47 (t, J4,5 = J3,4 = 4.4 Hz, 1H, H-4), 4.08 (td,
2,3 = J1a,2 = 7.1, J1b,2 = 5.4 Hz, 1H, H-2), 4.02 (dd, 2J6a,6b = 13.2, J5,6a
=
4.4 Hz, 1H, H-6a), 4.01 (td, J5,6b = 11.5, J5,6a = J4,5 = 4.4, Hz, 1H, H-5),
2
2
1
2855, 1735, 1630, 1402, 1243, 1221, 1114, 999, 768 cm−1; H NMR
3.90 (dd, J6a,6b = 13.2, J5,6b = 11.5 Hz, 1H, H-6b), 3.61 (dd, J1a,1b
=
13.7, J1a,2 = 7.1 Hz, 1H, H-1a), 3.44 (dd, J1a,1b = 13.7, J1b,2 = 5.4 Hz,
1H, H-1b); 13C NMR (125 MHz, 2% DCl in D2O) δ 70.2 (C-3), 70.2
(C-4), 62.4 (C-5), 55.8 (C-2), 39.5 (C-6), 36.9 (C-1); HRMS(ESI)
m/z calcd for [C6H1335ClN2O2 + H]+ 181.0738, obsd 181.0741; m/z
calcd for [C6H1337ClN2O2 + H]+ 183.0709, obsd 183.0713.
General Procedure for Acidic Hydrolysis of Carbamates.
Amino carbamate (0.05 mmol) was dissolved in H2SO4 aq (1 mL, 3
M). The solution was refluxed for 12−18 h, cooled, and neutralized by
the addition of Dowex-OH− ion-exchange resin. The resin mixture was
filtered with H2O and the filtrate concentrated in vacuo to provide the
pure aminoiminohexitols as the free base. Iminohexitols were also
characterized as the dihydrochloride salt with the addition of
deuterium chloride.
(500 MHz, D2O) δ 4.59−4.56 (m, 1H, H-1a), 4.36−4.34 (m, 2H, H-6
2
and H-1b), 4.00−3.94 (m, 3H, H-5, H-7 and H-7a), 3.17 (dd, Ja,b
=
=
2
13.2, J5,CH2Na = 3.6 Hz, 1H, CH2Na), 3.12 (dd, Ja,b = 13.2, J5,CH2Nb
7.8 Hz, 1H, CH2Nb); 13C NMR (125 MHz, D2O) δ 163.7 (C-3), 74.3
(C-6), 73.3 (C-7), 67.9 (C-1), 60.2 (C-7a), 58.7 (C-5), 39.6 (CH2N);
HRMS(ESI) m/z calcd for [C7H12N2O4 + H]+ 189.0870, obsd
189.0873.
( 5 S , 6 S , 7 R , 7 a S ) - 5 - ( A m i n o m e t h y l ) - 6 , 7 -
dihydroxytetrahydropyrrolo[1,2-c]oxazol-3(1H)-one Hydro-
chloride (27). By subjecting carbamate 20 (104 mg, 0.29 mmol)
to the general procedure for the hydrogenation of carbamates,
ammonium salt 27 was obtained as a colorless oil (53.0 mg, 0.28
mmol, 99%): Rf = 0.22 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1,
v/v/v/v); [α]25D = +14.8 (c = 1.0, H2O); IR (film) 3233, 2978, 2903,
1-Amino-1,2,5-trideoxy-2,5-imino-D-galactitol (12). By sub-
jecting amino carbamate 24 (9.7 mg, 0.05 mmol) to the general
procedure for the acidic hydrolysis of carbamates for 18 h, imino-D-
galactitol 12 was obtained as a white solid (8.4 mg, 0.05 mmol,
quantitative): Rf = 0.01 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1,
v/v/v/v); (HCl salt) [α]19.6D = −2.0 (c = 0.47, H2O); IR (film) 3316,
2837, 1736, 1622, 1506, 1403, 1236, 1089, 1013, 916, 823, 775 cm−1
;
2
1H NMR (500 MHz, D2O) δ 4.57 (t, J1a,1b = J1a,7a = 9.3 Hz, 1H, H-
2
1a), 4.54 (dd, J1a,1b = 9.3, J1b,7a = 3.7 Hz, 1H, H-1b), 4.24 (dd, J5,6
=
7.8, J6,7 = Hz, 1H, H-6), 4.21−4.20 (m, 1H, H-7a) 4.06 (br s, 1H, H-
7), 3.62 (td, J5,6 = J5,CH2Nb = 7.8, J5,CH2Na = 3.4 Hz, 1H, H-5), 3.28 (dd,
1
2965, 1644, 1364, 1315, 1081, 1019, 835 cm−1; H NMR (500 MHz,
2
2Ja,b = 13.2, J5,CH2Na = 3.4 Hz, 1H, CH2Na), 3.01 (dd, Ja,b = 13.2,
J5,CH2Nb = 7.8 Hz, 1H, CH2Nb); 13C NMR (125 MHz, D2O) δ 164.4
(C-3), 76.6, (C-6), 71.4 (C-7), 64.6 (C-1), 60.7 (C-5), 60.4 (C-7a),
42.0 (CH2N); HRMS(ESI) m/z calcd for [C7H12N2O4 + H]+
189.0870, obsd 189.0872.
D2O, 2HCl salt) δ 4.59 (dd, J2,3 = 7.2, J3,4 = 4.7 Hz, 1H, H-3), 4.42 (t,
J
4,5 = J3,4 = 4.7 Hz, 1H, H-4), 4.03 (td, J2,3 = J1a,2 = 7.2, J1b,2 = 5.7 Hz,
2
1H, H-2), 3.97 (dd, J6a,6b = 12.5, J5,6a = 4.7 Hz, 1H, H-6a), 3.88 (dd,
2J6a,6b = 12.5, J5,6b = 9.0 Hz, 1H, H-6b), 3.75 (dt, J5,6b = 9.0, J5,6a = J4,5
=
2
4.7 Hz, 1H, H-5), 3.57 (dd, J1a,1b = 13.9, J1a,2 = 7.2 Hz, 1H, H-1a),
( 5 R , 6 R , 7 R , 7 a S ) - 5 - ( A m i n o m e t h y l ) - 6 , 7 -
dihydroxytetrahydropyrrolo[1,2-c]oxazol-3(1H)-one Hydro-
chloride (28). By subjecting carbamate 21 (100 mg, 0.27 mmol)
to the general procedure for the hydrogenation of carbamates,
ammonium salt 28 was obtained as a white solid (51.4 mg, 0.27 mmol,
99%) and further crystallized from EtOH: Rf = 0.62 (DCM/MeOH/
3.42 (dd, J1a,1b = 13.9, J1b,2 = 5.7 Hz, 1H, H-1b); 13C NMR (125
2
MHz, D2O, 2HCl salt) δ 70.1 (C-3), 69.7 (C-4), 62.1 (C-5), 57.3 (C-
6), 55.7 (C-2), 36.8 (C-1); HRMS(ESI) m/z calcd for [C6H14N2O3 +
1
H]+ 163.1077, obsd 163.1084; H NMR (500 MHz, 2% NaOD in
D2O) δ 4.21 (dd, J4,5 = 6.2, J3,24 = 5.1 Hz, 1H, H-4), 4.03 (t, J3,4 = J2,3
=
EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); [α]25.8 = +60.2 (c = 0.83,
5.1 Hz, 1H, H-3), 3.63 (dd, J6a,6b = 11.4, J5,6a = 5.1 Hz, 1H, H-6a),
D
2
H2O); mp = 183.3−184.0 °C; IR (film) 3356, 2961, 2929, 1716, 1633,
3.58 (dd, J6a,6b = 11.4, J5,6b = 5.1 Hz, 1H, H-6b), 3.07 (dt, J4,5 = 6.2,
1
1428, 1262, 1143, 1094, 1063, 1007, 949, 893 cm−1; H NMR (500
J
5,6a = J5,6b = 5.1 Hz, 1H, H-5), 2.92 (td, J1a,2 = J1b,2 = 6.5, J2,3 = 5.1 Hz,
9618
dx.doi.org/10.1021/jo201151b|J. Org. Chem. 2011, 76, 9611−9621

The Journal of Organic Chemistry
Article
2
1H, H-2), 2.76 (dd, J1a,1b = 13, J1a,2 = 6.5 Hz, 1H, H-1a), 2.61 (dd,
J5,6a = J5,6b = 8.7, J4,5 = 2.9 Hz, 1H, H-5), 3.66 (ddd, J2,3 = 9.7, J1a,2 =
2J1a,1b = 13, J1b,2 = 6.5 Hz, 1H, H-1b); 13C NMR (125 MHz, 2% NaOD
in D2O) δ 73.0 (C-4), 71.6 (C-3), 61.3 (C-2), 60.4 (C-6), 60.3 (C-5),
40.2 (C-1).
7.9, J1b,2 = 5.9 Hz, 1H, H-2), 3.41 (dd, 2J1a,1b = 14.0, J1a,2 = 7.9 Hz, 1H,
2
H-1a), 3.36 (dd, J1a,1b = 14.0, J1b,2 = 7.9 Hz, 1H, H-1b); 13C NMR
(125 MHz, 2% DCl in D2O) δ 74.0 (C-4), 69.3 (C-3), 62.8 (C-5),
57.3 (C-6), 56.9 (C-2), 39.0 (C-1); HRMS(ESI) m/z calcd for
1-Amino-1,2,5-trideoxy-2,5-imino-D-glucitol (6).12 By sub-
jecting amino carbamate 28 (4.2 mg, 0.02 mmol) to the general
procedure for the acidic hydrolysis of carbamates for 12 h, imino-D-
glucitol 6 was obtained as a white solid (3.6 mg, 0.02 mmol,
quantitative): Rf = 0.01 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1,
v/v/v/v); (HCl salt) [α]20.1D = +8.0 (c = 0.38, H2O); IR (film) 3121,
1
[C6H14N2O3 + H]+ 163.1077, obsd 163.1079; H NMR (500 MHz,
2% NaOD in D2O) δ 4.05 (t, J4,5 = J3,4 = 4.1 Hz, 1H, H-4), 3.79 (dd,
J
2,3 = 8.1, J3,4 = 4.1 Hz, 1H, H-3), 3.70 (dd, 2J6a,6b = 11.2, J5,6a = 6.3 Hz,
1H, H-6a), 3.58 (dd, 2J6a,6b = 11.2, J5,6b = 6.3 Hz, 1H, H-6b), 3.17 (td,
5,6a = J5,6b = 6.3, J4,5 = 4.1 Hz, 1H, H-5), 2.93 (td, J2,3 = J1b,2 = 8.1, J1a,2
J
2
3050, 2930, 2799, 1633, 1401, 1082, 964, 879 cm−1; H NMR (500
1
= 4.4 Hz, 1H, H-2), 2.76 (dd, J1a,1b = 13.2, J1a,2 = 4.4 Hz, 1H, H-1a),
2
MHz, D2O, 2HCl salt) δ 4.38 (t, J4,5 = J3,4 = 3.0 Hz, 1H, H-4), 4.29 (t,
2.57 (dd, J1a,1b = 13.2, J1b,2 = 8.1 Hz, 1H, H-1b); 13C NMR (125
J
3,4 = J2,3 = 3.0 Hz, 1H, H-3), 4.03 (dd, 2J6a,6b = 9.8, J5,6a = 3.0 Hz, 1H,
MHz, 2% NaOD in D2O) δ 75.6 (C-3), 72.6 (C-4), 62.7 (C-2), 60.6
H-6a), 3.99 (dt, J5,6b = 7.3, J5,6a = J4,5 = 3.0 Hz, 1H, H-5), 3.96 (dd,
2J6a,6b = 9.8, J5,6b = 7.3 Hz, 1H, H-6b), 3.84 (td, J1,2 = 6.9, J2,3 = 3 Hz,
1H, H-2), 3.57 (d, J1,2 = 6.9 Hz, 2H, H-1); 13C NMR (125 MHz, D2O,
2HCl salt) δ 77.5 (C-3), 74.0 (C-4), 64.1 (C-5), 62.5 (C-2), 56.9 (C-
6), 39.2 (C-1); HRMS(ESI) m/z calcd for [C6H14N2O3 + H]+
(C-6), 59.6 (C-5), 43.8 (C-1).
1-Amino-1,2,5-trideoxy-2,5-imino-D-mannitol (4).12,31 By
subjecting amino carbamate 29 (4.0 mg, 0.02 mmol) to the general
procedure for the basic hydrolysis of carbamates, iminomannitol 4 was
obtained as a colorless oil (3.2 mg, 0.02 mmol, 91%): Rf = 0.01
(DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); (HCl salt)
1
163.1077, obsd 163.1081; H NMR (500 MHz, 2% NaOD in D2O)
[α]27.4 = +40.0 (c = 0.10, H2O); IR (film) 3209, 2925, 1603, 1503,
2
δ 3.97 (dd, J4,5 = 5.5, J3,4 = 2.7 Hz, 1H, H-4), 3.67 (dd, J6a,6b = 11.5,
D
1406, 1123, 1065, 1034, 813 cm−1. H NMR (500 MHz, 2% DCl in
1
J5,6a = 6.2 Hz, 1H, H-6a), 3.65 (t, J3,4 = J2,3 = 2.7 Hz, 1H, H-3), 3.55
(dd, 2J6a,6b = 11.5, J5,6b = 6.2 Hz, 1H, H-6b), 3.16 (td, J5,6a = J5,6b = 6.2,
D2O) δ 4.08 (t, J3,4 = J2,3 = 7.0 Hz, 1H, H-3), 4.04 (t, J4,5 = J3,4 = 7.0
2
Hz, 1H, H-4), 3.89 (dd, J6a,6b = 12.7, J5,6a = 3.9 Hz, 1H, H-6a), 3.79
J
4,5 = 5.5 Hz, 1H, H-5), 2.78 (ddd, J1b,2 = 7.0, J1a,2 = 5.1, J2,3 = 2.7 Hz,
2
2
(dd, J6a,6b = 12.7, J5,6b = 7.0 Hz, 1H, H-6b), 3.73 (td, J1,2 = 7.2, J2,3
=
1H, H-2), 2.74 (dd, J1a,1b = 12.8, J1a,2 = 5.1 Hz, 1H, H-1a), 2.60 (dd,
2J1a,1b = 12.8, J1b,2 = 7.0 Hz, 1H, H-1b); 13C NMR (125 MHz, 2%
NaOD in D2O) δ 80.7 (C-3), 77.6 (C-4), 65.5 (C-2), 60.7 (C-5), 60.3
(C-6), 43.5 (C-1).
7.0 Hz, 1H, H-2), 3.61 (td, J4,5 = J5,6b = 7.0, J5,6a = 3.9 Hz, 1H, H-5),
3.36 (d, J1,2 = 7.2 Hz, 2H, H-1); 13C NMR (125 MHz, 2% DCl in
D2O) δ 76.4 (C-3), 73.9 (C-4), 63.4 (C-5), 58.4 (C-6), 58.0 (C-2),
38.6 (C-1); HRMS(ESI) m/z calcd for [C6H14N2O3 + H]+: 163.1077,
obsd 163.1084; 1H NMR (500 MHz, 2% NaOD in D2O) δ 3.83 (t, J4,5
= J3,4 = 7.6 Hz, 1H, H-4), 3.75 (t, J3,4 = J2,3 = 7.6 Hz, 1H, H-3), 3.72
(dd, 2J6a,6b = 11.7, J5,6a = 4.4 Hz, 1H, H-6a), 3.64 (dd, 2J6a,6b = 11.7, J5,6b
= 6.1 Hz, 1H, H-6b), 2.99 (ddd, J4,5 = 7.6, J5,6b = 6.1, J5,6a = 4.4 Hz, 1H,
H-5) 2.95 (td, J2,3 = J1b,2 = 7.6, J1a,2 = 4.4 Hz, 1H, H-2), 2.83 (dd, 2J1a,1b
= 13.2, J1a,2 = 4.4 Hz, 1H, H-1a), 2.67 (dd, 2J1a,1b = 13.2, J1b,2 = 7.6 Hz,
1H, H-1b); 13C NMR (125 MHz, 2% NaOD in D2O) δ 79.4 (C-3),
77.7 (C-4), 62.0 (C-2), 61.9 (C-6), 61.5 (C-5), 43.5 (C-1).
General Procedure for the Acetylation of Aminoiminohex-
itols.15 Aminoiminohexitol (0.05 mmol) was coevaporated with dry
toluene (0.5 mL) three times, placed under argon, and dissolved in dry
MeOH (500 μL). Acetic anhydride (0.05 mmol) was added to the
solution at −15 °C. The solution was concentrated in vacuo when
TLC analysis (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/v)
revealed the reaction was complete. Purification was achieved using
flash chromatography to provide the pure acetamides (DCM/MeOH,
5/1, v/v containing 1% of 30% aq NH3).
1-Acetamido-1,2,5-trideoxy-2,5-imino-D-galactitol (30). By
subjecting imino-D-galactitol 12 (8.0 mg, 0.05 mmol) to the general
procedure for the acetylation of aminoiminohexitols for 1 h at −15 °C,
acetamide 30 was obtained as a colorless oil (4.0 mg, 0.02 mmol,
40%): Rf = 0.29 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/
v); 1H NMR (500 MHz, D2O) δ 4.48 (dd, J3,4= 4.7, J2,3 = 6.3 Hz, 1H,
H-3), 4.41 (t, J3,4 = J4,5 = 4.7 Hz, 1H, H-4), 3.94 (dd, J6a,6b = 12.2, J5,6a
= 4.5 Hz, 1H, H-6a), 3.87 (dd, J6a,6b = 12.2, J5,6b = 8.6 Hz, 1H, H-6b),
3.77−3.74 (m, 2H, H-2, H-5), 3.64 (dd, J1a,1b = 14.5, J1a,2 = 5.4 Hz, 1H,
H-1a), 3.59 (dd, J1a,1b = 14.5, J1b,2 = 7.8 Hz, 1H, H-1b), 2.00 (s, 3H,
COCH3); 13C NMR (125 MHz, D2O) δ 175.2 (CO), 69.9, 69.7 (C-
3, C-4), 61.2, 59.4, 57.8 (C-2, C-5, C-6), 36.8 (C-1), 21.6 (COCH3);
HRMS(ESI) m/z calcd for [C8H17N2O4 + H]+ 205.1183, obsd
205.1185.
General Procedure for Basic Hydrolysis of Carbamates. A
solution of NaOH in EtOH (0.5 mL, 2 M) was added to the amino
carbamate (0.08 mmol) and the mixture stirred under reflux for 2 h.
The resulting reaction mixture was loaded directly on to a Dowex-H+
ion-exchange resin column and washed with H2O to remove excess
salt. The amine product was then eluted with 30% aq NH3 and
concentrated in vacuo to provide the pure aminoiminohexitols as a
mixture of protonation states, which were characterized as both the
free base by adding sodium deuteroxide and the dihydrochloride salt
with the addition of deuterium chloride
1-Amino-1,2,5-trideoxy-2,5-imino-D-talitol (13). By subjecting
amino carbamate 26 (5 mg, 0.03 mmol) to the general procedure for
the basic hydrolysis of carbamates, iminotalitol 13 was obtained as a
colorless oil (4.1 mg, 0.03 mmol, 95%): Rf = 0.01 (DCM/MeOH/
EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); (HCl salt) [α]27.4D = +11.1 (c
= 0.27 H2O); IR (film) 3322, 2927, 1622, 1497, 1403, 1278, 1127,
1
1074, 1035, 981 cm−1; H NMR (500 MHz, 2% DCl in D2O) δ 4.42
(t, J3,4 = J2,3 = 3.6 Hz, 1H, H-3), 4.31 (dd, J4,5 = 8.6, J3,4 = 3.6 Hz, 1H,
2
H-4), 3.99−3.97 (m, 1H, H-2), 3.97 (dd, J6a,6b = 12.7, J5,6a = 3.4 Hz,
2
1H, H-6a), 3.83 (dd, J6a,6b = 12.7, J5,6b = 5.8 Hz, 1H, H-6b), 3.70
2
(ddd, J4,5 = 8.6, J5,6b = 5.8, J5,6a = 3.4 Hz, 1H, H-5), 3.60 (dd, J1a,1b
=
2
13.9, J1a,2 = 7.1 Hz, 1H, H-1a), 3.43 (dd, J1a,1b = 13.9, J1b,2 = 6.1 Hz,
1H, H-1b); 13C NMR (125 MHz, 2% DCl in D2O) δ 71.2 (C-4), 70.0
(C-3), 62.4 (C-5), 57.9 (C-6), 57.6 (C-2), 35.9 (C-1); HRMS(ESI)
m/z calcd for [C6H14N2O3 + H]+ 163.1077, obsd 163.1084; 1H NMR
(500 MHz, 2% NaOD in D2O) δ 4.03 (t, J3,4 = J2,3 = 4.1 Hz, 1H, H-3),
3.89 (dd, J4,5 = 8.3, J3,4 = 4.1 Hz, 1H, H-4), 3.68 (dd, 2J6a,6b = 11.7, J5,6a
= 3.9 hz, 1H, H-6a), 3.56 (dd, 2J6a,6b = 11.7, J5,6b = 6.3 Hz, 1H, H-6b),
3.09 (td, J1a,2 = J1b,2 = 7.1, J2,3 = 4.1 Hz, 1H, H-2), 2.99 (ddd, J4,5 = 8.3,
2
J5,6b = 6.3, J5,6a = 3.9 Hz, 1H, H-5), 2.77 (dd, J1a,1b = 13.0, J1a,2 = 7.1
2
Hz, 1H, H-1a), 2.62 (dd, J1a,1b = 13.0, J1b,2 = 7.1 Hz, 1H, H-1b); 13C
NMR (125 MHz, 2% NaOD in D2O) δ 74.1 (C-4), 72.4 (C-3), 62.4
(C-6), 62.0 (C-5), 60.6 (C-2), 40.4 (C-1).
1-Acetamido-1,2,5-trideoxy-2,5-acetimido-D-galactitol
(31). By subjecting imino-D-galactitol 12 (8.0 mg, 0.05 mmol) to the
general procedure for the acetylation of aminoiminohexitols for 1 h at
−15 °C, bis-acetamide 31 was obtained as a colorless oil and appeared
as a 1:1 mixture of rotamers in the NMR spectra (2.0 mg, 0.008 mmol,
17%): Rf = 0.50 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/
1-Amino-1,2,5-trideoxy-2,5-imino-L-altritol (14). By subject-
ing amino carbamate 27 (12.3 mg, 0.06 mmol) to the general
procedure for the hydrolysis of carbamates, iminohexitol 14 was
obtained as a colorless oil (10.6 mg, 0.06 mmol, quantitative): Rf =
0.01 (DCM/MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); (HCl
1
salt) [α]27.4 = −20.0 (c = 0.45 H2O); IR (film) 3302, 2928, 1622,
v); H NMR (500 MHz, D2O) δ 4.38 (dd, J4′,5′ = 6.3, J3′,4′ = 5.6 Hz,
D
1
1402, 1342, 1221, 1124, 1039, 1027 cm−1; H NMR (500 MHz, 2%
1H, H-4′), 4.34 (dd, J4,5 = 7.6, J3,4 = 5.4 Hz, 1H, H-4), 4.27 (dd, J2,3
=
DCl in D2O) δ 4.18 (dd, J3,4 = 3.7, J4,5 = 2.9 Hz, 1H, H-4), 4.14 (dd,
7.0, J3,4 = 5.4 Hz, 1H, H-3), 4.24 (dd, J2′,3′ = 3.0, J3′,4′ = 5.6 Hz, 1H, H-
3′), 4.18−4.12 (m, 4H, H-2, H-2′, H-5, H-5′), 3.89 (dd, J6a,6b = 12.2,
J
2,3 = 9.7, J2,3 = 3.7 Hz, 1H, H-3), 3.84 (dd, 2J6a,6b = 16.1, J5,6a = 8.7 Hz,
2
1H, H-6a), 3.75 (dd, J6a,6b = 16.1, J5,6b = 8.7 Hz, 1H, H-6b), 3.74 (td,
J
5,6a = 5.0 Hz, 1H, H-6a), 3.87 (dd, J6a′,6b′ = 11.5, J5′,6a′ = 6.0 Hz, 1H, H-
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Article
6a′), 3.81 (dd, J6a′,6b′ = 11.5, J5′,6b′ = 4.5 Hz, 1H, H-6b′), 3.79 (dd, J6a,6b
=
described. Kinetic studies were performed at 37 °C in pH 5.0 sodium
citrate/sodium phosphate buffer (25/50 mM) and NaCl (75 mM)
with enzyme at 25 nM. Plate screening was as above for ABG, with 4-
nitrophenyl N-acetyl-β-D-glucosaminide (0.33 mM; 1.5 × KM)
monitored at 340 nm. Following initial screening, the highest inhibitor
concentrations able to be tested were limited to 8 mM for 11, 14, and
5 and to 14 mM for 12, due to a limited amount of inhibitor. For 30, a
full Ki determination was carried out as described above for 14 with
ABG, with substrate at 0.03−1 mM and inhibitor at 0.16−4 mM.
Recombinant Human β-D-Glucocerebrosidase (GCase) (Gen-
zyme). The enzyme was assayed as previously described.32 Kinetic
studies were performed at 37 °C in pH 5.5 sodium citrate/sodium
phosphate buffer (20/50 mM), EDTA (1 mM), Triton X-100 (0.25%
v/v), and taurocholic acid (0.25% w/v) with enzyme at 4.0 nM. Plate
screening was as above for ABG, with 2,4-dinitrophenyl β-D-glucoside
(2.4 mM; 1.5 × KM) monitored at 405 nm. No clear hits were detected
in the initial screen, so all compounds were tested at four higher
inhibitor concentrations (2−0.2 mM) to verify those compounds
showing weak inhibition, with only compounds that gave repeatable
concentration dependent inhibition with a determined Ki below 10
mM being reported.
12.2, J5,6b = 5.5, 1H, H-6b), 3.60 (dd, J1a,1b = 14.1, J1a,2 = 6.3 Hz, 1H,
H-1a), 3.57 (dd, J1a′,1b′ = 13.4, J1a′,2′ = 5.8 Hz, 1H, H-1a′), 3.50 (dd, J1a,1b
= 14.1, J1b,2 = 6.3 Hz, 1H, H-1b), 3.48 (dd, J1a′,1b′ = 13.4, J1b′,2′ = 7,7 Hz,
1H, H-1b), 2.12 (s, 3H, NCOCH3), 2.11 (s, 3H, NCOCH3′), 1.97 (s,
3H, NHCOCH3), 1.94 (s, 3H, NHCOCH3′); 13C NMR (125 MHz,
D2O) δ 175.1 (NCO), 175.0 (NCO′), 174.3 (NHCO), 174.1
(NHCO′), 70.3 (C-3), 70.2 (C-4), 70.1 (C-4′), 69.6 (C-3′), 62.2
(C-5), 61.1 (C-5′), 60.0 (C-2′), 59.3 (C-6), 59.0 (C-6′), 58.7 (C-2),
38.9 (C-1), 38.1 (C-1′), 21.8 (NHCOCH3), 21.8 (NHCOCH3′), 21.7
(NCOCH3), 21.2 (NCOCH3′); HRMS(ESI) m/z calcd for
[C10H18N2O5 + Na]+ 269.1108, obsd 269.1115.
1-Acetamido-1,2,5-trideoxy-2,5-imino-D-talitol (32). By sub-
jecting imino-D-talitol dihydrochloride 13 (4.0 mg, 0.017 mmol) to the
general procedure for the acetylation of aminoiminohexitols for 3 h at
−15 °C, then 1 h at room temperature, acetamide 32 was obtained as a
colorless oil (2.0 mg, 0.009 mmol, 77%): Rf = 0.30 (DCM/MeOH/
EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); 1H NMR (500 MHz, D2O) δ
4.17 (t, J2,3 = J3,4 = 3.8 Hz, 1H, H-3), 4.11 (dd, J4,5 = 8.8, J3,4 = 3.8 Hz,
1H, H-4), 3.83 (dd, J6a,6b = 12.2, J5,6a = 3.5 Hz, 1H, H-6a), 3.71 (dd,
J6a,6b = 12.2, J5,6b = 5.8 Hz, 1H, H-6b), 3.55−3.49 (m, 1H, H-1a),
3.45−3.36 (m, 3H, H-1b, H-2, H-5), 1.97 (s, 3H, COCH3); 13C NMR
(125 MHz, D2O) δ 174.7 (CO), 72.2, 71.0 (C-3, C-4), 61.7, 59.9,
59.1 (C-2, C-5, C-6), 37.8 (C-1), 21.7 (COCH3); HRMS(ESI) m/z
calcd for [C8H17N2O4 + H]+ 205.1183, obsd 205.1184.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra (1H and 13C) for all new compounds and X-ray
structures and CIFs of 18 and 20. This material is available free
of charge via the Internet at http://pubs.acs.org.
1-Acetamido-1,2,5-trideoxy-2,5-imino-L-altritol (33). By sub-
jecting imino-L-altritol dihydrochloride 13 (5.0 mg, 0.021 mmol) to
the general procedure for the acetylation of aminoiminohexitols for 2 h
at −15 °C, then 3 h at room temperature, acetamide 33 was obtained
as a colorless oil (2.6 mg, 0.013 mmol, 65%): Rf = 0.31 (DCM/
MeOH/EtOH/30% aq NH3, 5/2/2/1, v/v/v/v); 1H NMR (500 MHz,
AUTHOR INFORMATION
■
Corresponding Author
D2O) δ 4.18 (t, J4,5 = J3,4 = 3.9 Hz, 1H, H-4), 3.98 (dd, J2,3 = 8.5, J3,4
=
*Email: bstocker@malaghan.org.nz; mattie.timmer@vuw.ac.nz.
3.9 Hz, 1H, H-3), 3.80 (dd, J6a,6b = 11.4, J5,6a = 6.8 Hz, 1H, H-6a), 3.66
(dd, J6a,6b = 11.4, J5,6b = 6.8 Hz, 1H, H-6b), 3.47 (dd, J1a,1b = 14.1, J1a,2
= 4.4 Hz, 1H, H-1a), 3.41 (td, J5,6a = J5,6b = 6.8, J4,5 = 3.9 Hz, 1H, H-5),
3.32 (dd, J1a,1b = 14.1, J1b,2 = 7.4 Hz, 1H, H-1b), 3.22−3.25 (m, 1H, H-
2), 2.00 (s, 3H, COCH3), 174.9 (CO), 74.5 (C-3), 71.3 (C-4), 60.0
(C-5), 59.7 (C-2), 59.6 (C-6), 41.1 (C-1), 21.7 (COCH3);
HRMS(ESI) m/z calcd for [C8H17N2O4 + H]+ 205.1183, obsd
205.1185.
ACKNOWLEDGMENTS
■
We acknowledge the financial contributions of Victoria
University of Wellington (Postgraduate Scholarship, Curtis−
Gordon Research Scholarship, A.L.W.-M.) and the Lotteries
Health Research Grant (B.L.S. and M.S.M.T.).
Kinetic Studies. Agrobacterium sp. β-D-Glucosidase/-Gal-
actosidase (ABG). The enzyme was purified and assayed as
previously described.34 Kinetic studies were performed at 37 °C in
pH 7.0 sodium phosphate buffer (50 mM) containing 0.1% bovine
serum albumin with enzyme at 3 × 10−3 mg/mL for plate assays and
7.2 × 10−5 mg/mL for full Ki determination. Approximate values of Ki
were determined by a screen of four inhibitor concentrations ranging
from 0.01 to 1 mM at a fixed concentration of 4-nitrophenyl β-D-
glucoside (0.11 mM; 1.5 × KM) in a 96-well plate. Initial rates were
monitored at 405 nm. The intersection of a linear fit to a plot of 1/V
against [I] with a horizontal line through 1/Vmax gave −Ki. Vmax was
determined by measuring initial rates with six substrate concentrations
ranging from 0.2 to 5 × KM on the same plate and fit to the
Michaelis−Menten equation. Compounds giving apparent inhibition
in this screen were further tested at a range of five inhibitor
concentrations ranging from 0.2 to 5 times the apparent initial Ki to
verify the results and further refine this value. Full Ki determination
was carried out on the best inhibitor from the plate screens (14) by
measuring rates at five inhibitor concentrations (0.2−5 × Ki
determined in the plate assays; 0.6−15 μM) while also varying the
substrate concentration from 0.2 to 5 × KM (14 − 350 μM). Initial
rates were determined by monitoring at 405 nm in matched quartz
cuvettes. Data were fit directly to the Michaelis−Menten equation
describing reaction in the presence of a competitive inhibitor, with
competitive inhibition mode being confirmed by a Dixon plot. Initial
rate fitting was performed in Microsoft Excel for the plate assays and
proprietary software for the Cary 4000, while analyses of these initial
rates were performed in GraFit 5.
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