Synthesis of an α-amylase inhibitor: Highly stereoselective glycosidation and regioselective manipulations of hydroxyl groups in carbohydrate derivatives

Article abstract of DOI:10.1021/op500306p

Here, we describe the efficient synthesis of α-amylase inhibitor 1. To introduce the most expensive C ring unit at a late stage in the synthesis, we developed 1,2-cis-O-glycosidation of AB and C ring intermediates. Taking advantage of the effect of non-neighboring protecting groups, reaction solvents, and temperature for the glycosidation led to high stereoselectivity and high yield of the 1,2-cis-glycoside product bearing the API skeleton. We also explored protection and deprotection methods for regioselective manipulation of hydroxyl groups in A and B ring intermediates. One-pot benzylation of the 2,3-hydroxyl groups of d-glucose under phase-transfer conditions and regioselective anomeric deacetylation with N-methylpiperazine were developed for the syntheses of A, B, and AB ring intermediates. Thus, the efficiency of the process was dramatically improved. The raw material cost of API was reduced to approximately one-third that of the original route, and the total process was decreased by six steps.

Full text of DOI:10.1021/op500306p

Article
pubs.acs.org/OPRD
Synthesis of an α‑Amylase Inhibitor: Highly Stereoselective
Glycosidation and Regioselective Manipulations of Hydroxyl Groups
in Carbohydrate Derivatives
Tsuyoshi Ueda,* Masaki Hayashi, Yutaka Ikeuchi, Takumi Nakajima, Eiji Numagami,
and Satoshi Kobayashi
Process Technology Research Laboratories, Pharmaceutical Technology Division, Daiichi Sankyo Co., Ltd., 1-12-1 Shinomiya,
Hiratsuka, Kanagawa 254-0014, Japan
S
* Supporting Information
ABSTRACT: Here, we describe the efficient synthesis of α-amylase inhibitor 1. To introduce the most expensive C ring unit at a
late stage in the synthesis, we developed 1,2-cis-O-glycosidation of AB and C ring intermediates. Taking advantage of the effect of
non-neighboring protecting groups, reaction solvents, and temperature for the glycosidation led to high stereoselectivity and high
yield of the 1,2-cis-glycoside product bearing the API skeleton. We also explored protection and deprotection methods for
regioselective manipulation of hydroxyl groups in A and B ring intermediates. One-pot benzylation of the 2,3-hydroxyl groups of
D-glucose under phase-transfer conditions and regioselective anomeric deacetylation with N-methylpiperazine were developed for
the syntheses of A, B, and AB ring intermediates. Thus, the efficiency of the process was dramatically improved. The raw material
cost of API was reduced to approximately one-third that of the original route, and the total process was decreased by six steps.
ful general method for 1,2-cis glycosidation has been developed
despite considerable progress and extensive efforts toward
glycosidation techniques.5−7 Second, methods for the regiose-
lective manipulation of hydroxyl groups in carbohydrate
derivatives are required for the efficient synthesis of API.8
Differentially and/or partially protected intermediates are key
feedstocks for the glycosidation reaction. The synthesis of these
compounds is complicated by the several steps required
conventional synthetic strategies. In addition, neighboring and
even non-neighboring protecting groups often play important
roles in modulating the reactivity of glycosyl donors and
acceptors as well as directing the stereochemistry of
glycosidation reactions. Therefore, selecting suitable protecting
groups is critical for success.5−7
INTRODUCTION
■
(2R,3R,4R)-4-Hydroxy-2-(hydroxymethyl)pyrrolidine-3-yl 4-O-
(6-deoxy-α-D-glucopyranosyl)-α-D-glucopyranoside (1) is an α-
amylase inhibitor designed by Daiichi Sankyo to suppress
postprandial hyperglycemia and excessive insulin secretion for
type II diabetes (Figure 1).1,2 In the same therapeutic category,
Figure 1. Structure of α-amylase inhibitor 1.
First-Generation Method. A practical method applicable
to the multikilogram synthesis of 1 is required to support
preclinical and early clinical studies. At this stage, a medicinal
chemistry route is expected to be sufficiently reliable for the
bulk supply. Therefore, we initially focused on modification of
the original method. The first-generation method for 1 based
on a medicinal chemistry route is shown in Scheme 1. 1,2,3,4-
Tetrabenzoyl-6-deoxy-β-D-glucopyranose (2)9−11 and 2,3-O-
dibenzyl-4,6-O-benzylidene-D-glucopyranose (3)12 as A and B
units were prepared from D-glucose (5) over five and six steps,
respectively, by modifying reported procedures. C ring units,
N,5-O-carbonyl-2-O-benzoyl-1,4-deoxyimino-D-arabinitol (4),
were synthesized from diacetone-D-glucose (6) over 15 steps
as described previously.13
α-glycosidase inhibitors3 such as voglibose and acarbose are
already widely used in clinical practice. However, they often
induce gastrointestinal tract-related symptoms such as diarrhea
and flatulence as side effects.4 It is preferable to inhibit α-
amylase specifically at an early step of sugar metabolism.
Accordingly, there is considerable interest in developing α-
amylase inhibitors that do not cause side effects in order to
enhance patient satisfaction.
To support the development and potential commercial
production of α-amylase inhibitor 1, a scalable cost-effective
synthesis that allows timely and cost-effective large-scale
production is required. This represents a significant challenge
because of the complexity of the trisaccharide structure
comprising 6-deoxy-D-glucose (A ring), D-glucose (B ring),
and 1,4-deoxyimino-D-arabinitol (C ring). The stereoselective
construction of the 1,2-cis-O-glycoside bond is expected to be
especially challenging. In contrast to a reliable methodology for
1,2-trans-O-glycoside formation based on neighboring-group
participation in 2-O-acyl-protected glycosyl donor, no success-
Schmidt glycosidation,14 which is a key step of this synthesis,
was performed by using trichloroacetimidate 7 and C unit 4 in
Received: September 21, 2014
© XXXX American Chemical Society
A
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Scheme 1. First-generation method for synthesizing 1
the presence of a catalytic amount of trimethylsilyl trifluor-
omethanesulfonate (TMSOTf) to afford 8 with moderate
selectivity (α/β = 92:8). Deprotection of benzylidene acetal
and the following toluoylation gave a BC unit 9 for which
crystallization was found to be effective for removing the
undesired β-isomer. 1,2-trans-Glycosidation of A unit 2 and BC
unit 9 with a stoichiometric amount of TMSOTf proceeded
selectively because of the neighboring-group participation by
benzoyl group. Hydrolysis of benzoyl groups and cyclic
carbamate afforded the final intermediate 11 with a yield of
71% from 9. Finally, deprotection of benzyl ether in
hydrogenolysis conditions afforded 1 with a yield of 93%.
Although this first-generation process proved suitable for the
manufacturing of medium-sized lots of 1 (up to 45 kg), several
drawbacks would be encountered when running this process at
a commercial production scale, including (1) the large number
of steps (total 33 steps), (2) linear route with low overall yield
(22%) in the synthesis of C unit 4, (3) moderate selectivity of
1,2-cis-glycosidation, (4) introduction of the most expensive C
unit 4 at an early stage of the process, resulting in poor cost-
efficiency, (5) the use of a stoichiometric amount of expensive
TMSOTf in the formation of the 1,2-trans-glycoside bond, and
(6) tedious protection and deprotection steps in the syntheses
of each units. These issues more than tripled the manufacturing
cost of API compared to the target cost. Herein, we will detail
the development of a stereoselective synthesis of 1 to overcome
above issues.
Scheme 2. New synthesis strategy for α-amylase inhibitor 1
trichloroacetimidate, which is easily activated by a catalytic
amount of Lewis acid such as TMSOTf, is the most popular
and reliable.5−7 Therefore, we applied this methodology to the
1,2-cis-glycoside-bond formation as well as the first-generation
method. The ether protecting group at the 2-O position of the
donor is well-known to favor the formation of 1,2-cis-glycoside
by virtue of the anomeric effect. Therefore, benzyl ether was
selected as the protecting groups at the 2 and 3 positions of the
B unit.
In this approach, the particularly high stereoselectivity in the
reaction is especially desirable, because the undesired 1,2-trans-
glycoside product formed at a late stage in the synthesis would
complicate the purification and quality control of API.
1,2-cis-Glycosidation of AB and C Units: Selection of
the Protecting Groups. Non-neighboring protecting groups
RESULTS AND DISCUSSION
■
Our synthetic strategy is shown in Scheme 2. In this strategy,
the most expensive C ring unit is introduced at a late stage in
the synthesis, in which 1,2-cis-glycosidation of AB and C units
are a key step. Among the various glycoside-bond formation
developed to date, Schmidt glycosidation based on O-glycosyl
B
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Table 1. Effects of protecting groups on the selectivity of cis-glycosidation
a
b
entry
donor
acceptor
temp. (°C)
α/β ratio
conv. (%)
1
2
3
4
5
6
7
8
9
12a
12b
12c
12d
12c
12c
12c
12c
12c
4
−20
−20
−20
−20
−40
−40
−40
−40
−40
84:16
84:16
89:11
88:12
94:6
83
64
86
87
4
4
4
c
4
97 (76)
13a
13b
14a
14b
93:7
76
85
92
89:11
90:10
93:7
c
99 (84)
a
b
c
Determined by HPLC. Ratio of the product to the remaining donor in HPLC analysis. HPLC yield.
Table 2. Solvent study of cis-glycosidation
a
a
entry
solvent
α/β ratio
yield (%)
1
CH2Cl2/i-Pr2O (3:2)
toluene
93:7
94:6
96:4
93:7
91:9
96:4
97:3
96:4
94:6
89:11
96:4
84
82
70
85
75
75
63
76
34
71
86
2
3
xylene
4
chlorobenzene
anisole
5
6
CPME
7
TBME
8
DME
9
toluene/Bu2O (3:2)
toluene/dioxane (9:1)
toluene/DME (9:1)
10
11
a
Determined by HPLC.
of intermediates are crucial, because they could affect not only
the reactivity and selectivity of the glycosidation reaction, but
also the synthetic difficulty of each unit. Therefore, we initially
tested the glycosidations of acceptor 4 and donors 12a−d with
different protecting groups in order to determine the best
structure for each unit (Table 1). Interestingly, the acetyl group
at the 6-position of the B unit (entries 3 and 4) significantly
enhanced the selectivity compared to benzoyl and toluoyl
groups (entries 1 and 2). It can be hypothesized that an acetyl
group at the 6-position of glucosyl donor shields the β face of
the sugar ring, thus promoting high selectivity in glycosidation
reaction; however, this long-range assistance is not well-
established in glycosidation chemistry.5−7 Reducing the
reaction temperature to −40 °C dramatically improved
conversion and selectivity to afford the corresponding 1,2-cis-
glycoside product with an α/β ratio of 94:6 (entry 5). On the
basis of these results, 12c was selected as a suitable donor. Next,
acceptors 13a,b and 14a,b were tested for the glycosidation
with 12c (entries 6−9). Unexpectedly, the protecting groups of
the acceptor also significantly affected the reaction. Only N-
C
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Scheme 3. Retrosynthesis of AB and C units
a
Scheme 4. Synthesis of B unit 17a
a
Compounds in brackets were used for the next step without isolation.
methoxylcarbonyl-2,5-O-dibenzoyl-1,4-deoxyimino-D-arabinitol
(14b) demonstrated excellent reactivity comparable to 4.
Meanwhile, 14b was easier to prepare than 4 bearing an
oxazolidinone ring, which allows 1 step to be saved in the
preparation of the C unit. Therefore, it was selected as a
suitable C ring intermediate.
comprising toluene and an ether-type solvent (entries 9−11).
The optimal solvent was toluene/DME (9:1 v/v); the yield
increased significantly to 86% as the α/β ratio remained high
(96:4) (entry 11).
Development of the Synthesis of AB and C Units. We
subsequently explored the efficient synthesis of the AB and C
units (12c and14b) in which the protecting groups must be
manipulated such that each can be selectively introduced or
removed during the synthetic route. Unlike the first-generation
method using a stoichiometric amount of expensive TMSOTf,
catalytic Schmidt glycosidation was utilized to form the 1,2-
trans-glycoside bond in order to reduce the required amount of
expensive TMSOTf.
AB ring unit 12c was synthesized from the corresponding A
unit 16 and B unit 17a, both of which can be prepared from
inexpensive methyl-α-glucopyranose (18) by modifying the
precedent procedures. The C unit 14b can prepared from 19,
the synthesis of which from diacetone-glucose (6) has been
reported and applied in the first-generation method.13 The
synthesis of 19 from D-xylose (20) was attempted in pursuit of
a more efficient method for the C unit (Scheme 3).
1,2-cis-Glycosidation of 12c and 14b: Solvent Study.
The reaction solvent in 1,2-cis-glycosidation is well-known to be
one of the most important factors affecting anomeric
stereoselectivity. In particular, ether-type solvents generally
participate in the glycosidation processes; as a result, the
equatorial carbocation is preferentially formed, leading to axial
glycosidic bond formation.7 Therefore, we subsequently
examined the effect of solvent on the glycosidation of 12c
and 14b at −40 °C (Table 2). As expected, ether-type solvents
such as CPME, TBME, and DME demonstrated high
stereoselectivity (α/β ratio = 96:4 to 97:3). However, a
significant amount of unreacted acceptor 14b was observed
after the reactions, and the yields were moderate (entries 6−8);
this might be because 12c or its carbocation would be less
stable in those solvents than in nonpolar solvents such as
toluene. Therefore, we subsequently tried using mixed solvents
D
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Synthesis of B Unit 17a. Field and Mukhopadhyay15
report the synthesis of 17a via the formation of cyclic
orthoesters and subsequent benzylation of the other alcohols.
However, their method suffers from low efficiency because of
the requirement of excess amounts of BnBr and NaH, which is
a safety issue owing to the rapid hydrogen evolution. Therefore,
we improved upon this method by applying the phase-transfer
conditions using BnCl, tetrabutylammonium bromide (TBAB),
and NaOH (aq) to afford a mixture of 17a and 17b with good
yield (HPLC assay yield: 85%). Fortunately, the acetyl group at
the 4-position in 17b could be transferred to the 6-position in
the presence of NEt3 in i-PrOH, which gave B unit 17a (57%)
from 18 (Scheme 4, Method I).16 As an another high-yield
access, the mixture of 17a and 17b was hydrolyzed to give 22
(82%), which was subsequently acetylated regioselectively to
afford 17a (95%) (Scheme 4, Method II).
Table 3. Regioselective anomeric deacetylation of 25
a
a
entry
amine
BnNH2
conversion (%)
yield (%)
1
2
100
100
72
68
n-PrNH2
i-PrNH2
75
3
76
b
4
Me2NH
100
1
85
5
Et2NH
trace
trace
78
6
n-Pr2NH
2
7
MeBnNH
72
8
morpholine
Me2N−C2H4−NH2
N-Me-piperazine
100
100
100
96
9
73
10
87
Synthesis of A Unit 25. 23 was prepared via selective
iodination and the subsequent one-pot acetylation using
Garegg and Samuelsson’s procedure.17 The crystallization of
23 from i-PrOH was effective for removal of triphenylphos-
phine oxide to afford 23 with a yield of 85%. Deiodination
under hydrogenolysis conditions and subsequent acetolysis
using Ac2O and H2SO4 in AcOH gave the desired A ring
intermediate 25 (91%) (Scheme 5).
a
b
Determined by HPLC. THF solution.
than the basicity of amine significantly affects the reaction. In
addition, secondary amines, especially cyclic amines, exhibited
excellent reactivities while suppressing the overreaction of the
product. Next, N-Me-piperazine and N,N-dimethylethylenedi-
amine were tested, because the corresponding acetamide can be
removed by simple extraction using acidic aqueous solution.
Consistent with the above-mentioned findings, the reaction
using N-methylpiperazine, which is a cyclic secondary amine,
resulted in the complete conversion of 25 and a good yield of
26. Furthermore, as expected, the corresponding acetamide was
easily removed from the reaction mixture by washing with
diluted aqueous HCl (entry 10).
a
Scheme 5. Synthesis of A unit 25
Synthesis of AB Unit 29. Having obtained required
intermediates, 1,2-trans-glycosidation of trichloroacetimidate
16, which was prepared from 25 over 2 steps without isolation,
and B unit 17a in the presence of a catalytic amount of
TMSOTf was attempted. The reaction proceeded selectively,
and the subsequent acetolysis using Ac2O and H2SO4 in AcOH
afforded the desired product 28 with an isolated yield of 80%.
Regioselective deacetylation with N-methylpiperazine, which
was developed for the synthesis of 26, was also effective for the
reaction of 28 to give AB unit 29 in excellent yield (Scheme 6).
Synthesis of C Unit 14b from 20. Fleet and Witty report
the synthesis of 19 from diacetone-glucose (6) over nine
steps,13 which was successfully applied to the synthesis of C
unit 4 in the first-generation method. However, this route
requires one carbon degradation by Malaprade glycol oxidative
cleavage with NaIO4 and the subsequent reduction to afford the
protected D-xylose derivative 35. To reduce the number of
steps, we subsequently explored the synthesis of 14b from D-
xylose (20) itself (Scheme 7). Regioselective protection of 20
and the following tosylation afforded 30 with a yield of 64%.29
The introduction of the azide function at C-5 and the following
benzylation gave 31. Treatment with HCl in MeOH and
mesylation at the 3-O position gave 32 as an anomeric mixture.
Hydrogenolysis caused the reduction of azide to give the
corresponding amine, which underwent cyclization to afford
bicyclic 19; 19 was then protected by methylcarbamate and
hydrolyzed with HCl to afford 33. Reduction with NaBH4 gave
the diol 34 with a yield of 59% over nine steps. Protection by
BzCl at the 2,5-O positions and the deprotection of benzyl
ether afforded the desired 14b with a yield of 86% (33% overall
yield from 20). Compared to the first-generation method, this
a
Compounds in brackets were used for the next step without isolation,
Regioselective Anomeric Deacetylation of 25. Selective
deacylation on C-1 of the carbohydrate residue often plays an
important role in anomeric functional group transformations.
Various existing methods for regioselective anomeric deacety-
lation are used for oligosaccharide synthesis.18−27 Among them,
aminolysis is the most effective owing to the low cost and ease
of handling of amine. Benzylamine is frequently used for this
conversion,28 but an excess amount of amine is sometimes
required, and the removal of benzylacetamide, a byproduct of
the reaction, poses a barrier to scaling-up. The reactivity of
other amines for this conversion is not well-understood.
Therefore, we examined the regioselective deacetylation of 25
with various amines (Table 3). Complete conversions of 25
were observed when using primary amines such as BnNH2 and
n-PrNH2. However, the yields were moderate, and several
unknown impurities that were assumed to arise from the
overreaction of 26 were detected (entries 1 and 2). While
secondary amines such as Et2NH and n-Pr2NH afforded only
trace amounts of 26 (entries 5 and 6), Me2NH exhibited the
highest reactivity, achieving complete conversion and good
yield of 26 (entries 4). Surprisingly, morpholine, which has
weak basicity (pKa = 8.36), afforded the highest product yield
and suppressed the overreaction of 26 (entry 8), although it
was difficult to remove the corresponding acetamide from the
reaction mixture. These results suggest the steric effect rather
E
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a
Scheme 6. Synthesis of AB unit 29
a
Compounds in brackets were used for the next step without isolation.
a
Scheme 7. Synthesis of C unit 14b from 20
a
Compounds in brackets were used for the next step without isolation.
route has three fewer steps and increased the total yield from
and 14b (Scheme 8). The donor 12c was prepared from 29 in
the presence of 1.5 mol % DBU. 12c could be utilized for the
next glycosidation without any purification. Slowly adding the
reaction mixture of 12c to a solution containing C unit 14b and
22% to 33%.
End-Game Synthesis. Finally, we examined the synthesis
of α-amylase inhibitor 1 via the glycosidation reaction of 12c
F
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a
Scheme 8. Synthesis of α-amylase inhibitor 1 via glycosidation of AB and C units
a
Compounds in brackets were used for the next step without isolation.
Scheme 9. Outline of second-generation method
TMSOTf in toluene-DME allowed the reaction to proceed well
to afford 15 with an α/β ratio of 96/4. The hydrolysis of the O-
acetyl, benzoyl, and N-methoxycarbonyl groups afforded 11,
which is the same final intermediate as that in the first-
generation method. Fortunately, the undesired β-isomer was
removed effectively by crystallization of 11. Finally, depro-
tection of benzyl ether in hydrogenolysis conditions afforded
the final product with a yield of 93%. Compared to the first-
generation method, this route dramatically reduced the
consumption rate of the expensive C unit in the synthesis of
API.
Aminolysis with N-methylpiperazine was effective for regiose-
lective anomeric deacetylation and was successfully applied to
the reaction of 25 and 28 to afford the corresponding A and AB
units with good yields.
EXPERIMENTAL SECTION
■
General. Commercially available chemicals including
solvents were purchased from commercial supplier and used
as received. NMR spectra were measured on a JEOL JNM-
1
ECA500 NMR spectrometer at 500 MHz for H spectra and
125 MHz for 13C spectra. Reactions were monitored by HPLC
analysis that was performed using a Agilent 1100 or Shimazu
LC 10 series. HPLC methods are described below. Gradient
condition of each methods was set in consideration of retention
time of a measuring compound.
CONCLUSION
■
The outline of the second-generation synthetic method for α-
amylase inhibitor 1 is shown in Scheme 9. This method has
dramatically better efficiency than the medicinal chemistry
route (i.e., the first-generation method). The raw material costs
are around one-third those of the original route, and the total
number of steps was reduced from 33 to 27. In this work, the
key to success was the development of highly stereoselective
1,2-cis-glycosidation of the AB and C ring intermediates, which
made it possible to introduce the most expensive C unit at a
late stage in the synthesis. Furthermore, the efficient synthesis
of AB units was achieved by manipulating hydroxyl groups,
which also reduced costs. In the synthesis of the AB units, one-
pot regioselective benzylation of 18 in phase-transfer conditions
was developed, and the B unit 17a was obtained in three steps.
HPLC Method A. Column: SPELCO Ascentis RP-amide 4.6
× 250 mm (5 μm), eluents: MeCN/2−10 mM aq. NH4OAc
(25/75−90/10), detector: UV (210 or 220 nm), or Corona
CAD, flow rate: 1.0 mL/min, column temp.: 40 °C, injection:
10 μL.
HPLC Method B. Column: Imtakt Unison UK-C8 4.6 × 150
mm (3 μm), eluents: MeCN/2−10 mM aq. NH4OAc (25/75−
80/20), detector: UV (210 or 220 nm), flow rate: 1.0 mL/min,
column temp.: 40 °C, injection: 10 μL.
HPLC Method C. Column: L-column 4.6 × 250 mm (5 μm),
eluents: MeCN/2 mM aq. NH4OAc (25/75−85/15), detector:
G
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UV (210 or 220 nm), flow rate: 1.0 mL/min, column temp.: 40
°C, injection: 10 μL.
HPLC Method D. Column: SUPELCO Discovery HSF5 4.6
× 250 mm (5 μm), eluents: MeCN/0.01% aq. TFA (40/60,
isocratic), detector: ELSD, flow rate: 1.0 mL/min, column
temp.: 40 °C, injection: 10 μL.
1H NMR (500 Hz, CDCl3) β-anomer: δ: 5.69 (d, 1H, J = 8.5
Hz), 5.21 (t, 1H, J = 9.5 Hz), 5.11 (dd, 1H, J = 8.5, 9.5 Hz),
4.86 (t, 1H, J = 10.0 Hz), 3.75−3.69 (m, 1H), 2.11 (s, 3H),
2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.25 (d, 3H, J = 6.0
Hz); α-anomer: δ: 6.27 (d, 1H, J = 4.0 Hz), 5.43 (t, 1H, J =
10.0 Hz), 5.07 (dd, 1H, J = 6.0, 10.5 Hz), 4.86 (t, 1H, J = 10.0
Hz), 4.04−4.00 (m, 1H), 2.11 (s, 3H), 2.06 (s, 3H), 2.02 (s,
3H), 2.00 (s, 3H), 1.21 (d, 3H, J = 6.0 Hz); 13C NMR (125 Hz,
CDCl3) δ: 170.2, 170.1, 169.6, 169.3, 169.1, 169.0, 91.6, 73.2,
72.9, 72.7, 70.9, 70.6, 69.7, 69.5, 20.8, 20.6, 20.54, 20.52, 20.4,
17.3, and 17.2; IR (KBr pellet) 1755, 1377, 1228, 1150, 1076,
and 1039 cm−1; HRMS (ESI+) [M + Na]+ calcd for
Synthesis of A Unit 25 (Scheme 5). Methyl-2,3,4-tri-O-
acetyl-6-deoxy-6-iodo-α-D-glucopyranoside (23). PPh3
(89.15 g, 339.89 mmol) and imidazole (29.45 g, 432.39
mmol) were added to a mixture of methyl-α-D-glucopyranoside
(18) (60.00 g, 308.99 mmol) in THF (300 mL) at rt. The
reaction mixture was warmed to 70 °C, and then a solution of I2
(86.26 g, 339.89 mmol) in THF (150 mL) was added dropwise
over 3 h. After completion of the reaction (confirmed by TLC),
the mixture was cooled to room temperature, and then pyridine
(146.65 g, 1853.94 mmol) and Ac2O (157.72g, 1544.95 mmol)
were added to the mixture. The mixture was warmed to 35 °C
and stirred for 17 h. After completion of the reaction
(confirmed by TLC), EtOAc (720 mL) and 10% aq. NaCl
(480 mL) were added, and the organic layer was separated. The
organic layer was washed with 10% aq. NaCl (360 mL)
containing c-HCl (150 mL) and 10% aq. NaCl (480 mL)
containing Na2S2O3 (24 g). The obtained organic layer was
concentrated to 420 mL, i-PrOH (900 mL) was added, and the
mixture was concentrated to 600 mL. The precipitation of 23
was observed during the concentration. After i-PrOH (120 mL)
was added to the slurry, the mixture was cooled to 0 °C, stirred
for 1 h, and filtered. The product was washed with chilled i-
PrOH (180 mL) and dried in vacuo at 40 °C to afford 23
(111.41 g, 83.8%) as a white solid.
20
C14H20NaO9: 355.1005; found 355.1013; [α]D = +119.70 (c
= 1.0, CH2Cl2).
Synthesis of B Unit 17a (Scheme 4, Method I). Methyl
2,3-di-O-benzyl-6-O-acetyl-α-D-glucopyranoside (17a). To a
mixture of methyl-α-D-glucopyranoside (18) (15.00 g, 77.24
mmol) in MeCN (75 mL), CH3C(OMe)3 (13.92 g, 115.86
mmol) and TsOH·H2O (0.294 g, 1.54 mmol) were added. The
mixture was warmed to 50 °C and stirred while MeOH was
removed at 50 °C in vacuo and MeCN was added to maintain
the volume of the mixture. After completion of the reaction
(confirmed by HPLC Method A), NEt3 (0.391 g, 3.62 mmol)
was added, and the mixture was concentrated to 37.5 mL.
Tetrabutylammonium bromide (1.25 g, 3.86 mmol), BnCl
(39.12 g, 308.96 mmol), and 48% aq. NaOH (30 mL) were
added, and the mixture was stirred at 40 °C for 4 h. After
completion of the reaction (confirmed by HPLC Method A),
toluene (150 mL) and H2O (75 mL) were added, and the
organic layer was separated, washed with 1 M HCl aq. (81 mL),
and concentrated to 48 mL. i-PrOH (75 mL) and NEt3 (15.63
g, 154.48 mmol) were added, and the mixture was stirred at rt
for 55 h. After completion of the reaction (confirmed by HPLC
Method A), toluene (117 mL) and 1 M aq. HCl (170 mL,
169.93 mmol) were added, and then the pH of the mixture was
adjusted to ca. 7.0 with 48% aq. NaOH. The organic layer was
separated and concentrated to 45 mL. i-PrOH (22 mL) and
ethylcyclohexane (113 mL) was added dropwise. The
precipitation of 17a was observed, and the slurry was stirred
at 0 °C and filtered. The product was washed with chilled
ethylcyclohexane (69 mL) and dried in vacuo to afford 17a
(18.46 g, 57.4%) as a white solid.
1H NMR (500 Hz, CDCl3) δ: 5.47 (t, 1H, J = 10.0 Hz), 4.96
(d, 1H, J = 3.5 Hz), 4.90−4.85 (m, 2H), 3.82−3.78 (m, 1H),
3.48 (s, 3H), 3.30 (dd, 1H, J = 11.0, 2.5 Hz), 3.14 (dd, 1H, J =
8.5, 11.0 Hz), 2.08 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H); 13C
NMR (125 Hz, CDCl3) δ: 170.1, 170.0, 169.6, 96.6, 72.4, 70.8,
69.6, 68.6, 55.7, 20.7, 20.6, and 3.6; IR (KBr pellet) 1740, 1264,
1233, and 1036 cm−1; HRMS (ESI+) [M + Na]+ calcd for
20
C13H19INaO8: 453.0022; found 453.0007; [α]D = +117.20 (c
= 1.0, CH2Cl2).
1,2,3,4-Tetra-O-acetyl-6-deoxy-D-glucopyranose (α,β-Mix-
ture) (25). To a mixture of 23 (50.00 g, 116.23 mmol) and
AcONa (19.07g, 232.46 mmol) in EtOAc (500 mL) and H2O
(100 mL), 5% Pd/C (moisture content: 54.5%) (16.48 g) was
added. The mixture was stirred for 5.5 h at 35 °C under H2
pressure (3 bar). After completion of the reaction (confirmed
by HPLC Method A), the mixture was filtered and washed with
EtOAc (250 mL). The filtrate was washed with 10% aq. NaCl
(400 mL) containing Na2S2O3 (22.05 g, 139.48 mmol) and
10% aq. NaCl (250 mL). The organic layer was concentrated to
50 mL, and AcOH (100 mL) was added. The mixture was
concentrated to 50 mL of Ac2O (15.77g, 232.46 mmol); H2SO4
(5.70 g, 58.12 mmol) was added, and the mixture was stirred at
25 °C for 5 h. After completion of the reaction (confirmed by
HPLC Method A), AcONa (9.53 g, 116.23 mmol) was added,
and the mixture was stirred for 15 min. EtOAc (500 mL) and
5% aq. NaHCO3 (500 mL) were added. The organic layer was
separated, washed with 5% aq. NaHCO3 (500 mL) 2 times and
concentrated to 100 mL. i-PrOH (400 mL) was added, and the
mixture was concentrated to 175 mL. The precipitation of 25
was observed, and then H2O (800 mL) was added dropwise.
The slurry was stirred at 0 °C, filterd, washed with chilled i-
PrOH/H2O (16/84 mL), and dried in vacuo to afford 25
(34.80 g, 90.1%) as a white solid.
1H NMR (500 Hz, CDCl3) δ: 7.38−7.29 (m, 10H), 5.00 (d,
1H, J = 11.5 Hz), 4.77 (d, 1H, J = 11.5 Hz), 4.74 (d, 1H, J =
11.5 Hz), 4.66 (d, 1H, J = 12.0 Hz), 4.62 (d, 1H, J = 3.5 Hz),
4.40 (dd, 1H, J = 11.5, 4.0 Hz), 4.21 (dd, 1H, J = 12.0, 2.5 Hz),
3.79 (t, 1H, J = 9.5 Hz), 3.75−3.72 (m, 1H), 3.51 (dd, 1H, J =
3.5, 9.5 Hz), 3.42 (t, 1H, J = 9.0 Hz), 3.38 (s, 3H), 2.51 (d, 1H,
J = 2.5 Hz), 2.07 (s, 3H); 13C NMR (125 Hz, CDCl3) δ: 171.3,
138.6, 137.9, 128.6, 128.5, 128.1, 128.0, 127.96, 127.88, 98.2,
81.1, 79.4, 75.5, 73.2, 69.8, 69.2, 63.2, 55.2, and 20.8.; IR (KBr
pellet) 3524, 2917, 1727, 1264, 1116, 1061, 1035, and 744
cm−1; HRMS (ESI+) [M + Na]+ calcd for C23H28NaO7:
439.1733; found 439.1745; [α]D20 = +41.50 (c = 1.0, CH2Cl2).
Synthesis of 22 (Scheme 4, Method II). Methyl-2,3-di-
O-benzyl-α-D-glucopyranoside (22). To a mixture of methyl-
α-D-glucopyranoside (18) (100.00 g, 514.99 mmol) in MeCN
(500 mL), CH3C(OEt)3 (125.32 g, 772.49 mmol), and TsOH·
H2O (1.96 g, 10.30 mmol) was added. The mixture was stirred
at rt for 5 h. After completion of the reaction (confirmed by
HPLC Method A), NEt3 (2.60 g, 25.75 mmol) was added and
the mixture was concentrated to 250 mL. Tetrabutylammonium
bromide (8.55 g, 25.75 mmol), BnCl (260.77 g, 2059.96
H
dx.doi.org/10.1021/op500306p | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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Article
mmol), and 48% aq. NaOH (200 mL) were added and the
mixture was stirred at 40 °C for 4 h. After completion of the
reaction (confirmed by HPLC Method A), toluene (1000 mL)
and H2O (500 mL) were added, and the organic layer was
separated and washed with 2 M HCl aq. (500 mL). 2 M aq.
NaOH (450 mL) and MeOH (50 mL) were added, and the
mixture was stirred at 55 °C for 3 h. The organic layer was
separated, washed with H2O (300 mL), and concentrated to
500 mL. Toluene (400 mL) was added, and then heptane (700
mL) was added dropwise at 45 °C. The mixture was stirred for
1 h, and the precipitation of 22 was observed. Heptane (700
mL) was added dropwise at 45 °C, and the slurry was stirred at
0 °C and filtered. The product was washed with chilled mixed
solution of toluene (100 mL) and heptane (200 mL) and dried
in vacuo to afford 22 (157.21 g, 81.5%) as a white solid.
1H NMR (500 Hz, CDCl3) δ: 7.38−7.28 (m, 10H), 5.01 (d,
1H, J = 11.0 Hz), 4.76 (d, 1H, J = 12.0 Hz), 4.71 (d, 1H, J =
12.0 Hz), 4.65 (d, 1H, J = 11.5 Hz), 4.59 (d, 1H, J = 3.5 Hz),
3.81−3.71 (m, 3H), 3.62−3.58 (m, 1H), 3.52 (d, 1H, J = 9.0
Hz), 3.49 (dd, 1H, J = 3.5, 9.5 Hz), 3.37 (s, 3H), 2.52 (brs,
1H), 2.09 (brs, 1H); 13C NMR (125 Hz, CDCl3) δ: 138.7,
137.9, 128.6, 128.5, 128.1, 128.0, 127.9, 127.88, 98.1, 81.3, 79.7,
75.4, 73.1, 70.7, 70.3, 62.3, and 55.2; IR (KBr pellet) 3286,
2922, 1739, 1453, 1120, 1061, 1028, 756, 737, and 698 cm−1;
HRMS (ESI+) [M + Na]+ calcd for C21H26NaO6: 397.1627;
(7.0 mL, 39.12 mmol) in toluene (1950 mL) at −30 °C over
2.5 h and stirred for 1 h. After the reaction (confirmed by
HPLC Method A), NEt3 (8.24 mL, 58.68 mmol) was added,
and the mixture was concentrated to 1300 mL. The residue was
washed with 25% aq. MeOH (1300 mL) three times, and the
organic layer was concentrated to 260 mL. AcOH (650 mL)
was added, and the mixture was concentrated to 260 mL. After
addition of AcOH (390 mL), H2SO4 (9.1 mL, 170.71 mmol)
and Ac2O (91 mL, 962.68 mmol) were added at 20 °C, and the
mixture was stirred at 20 °C for 4 h. After completion of the
reaction (confirmed by HPLC Method A), AcONa (45.5 g,
554.67 mmol) was added, and the mixture was stirred for 30
min. H2O (520 mL) and i-PrOH (910 mL) was added, and the
precipitation of 28 was observed. After stirring for 17 h at rt,
H2O (1300 mL) was added dropwise. The slurry was cooled to
0 °C, stirred for 2 h, and filtered. The product was washed with
chilled 40% aq. i-PrOH (650 mL) and dried in vacuo to afford
28 (156.12 g, 79.5%, α/β = 4:1) as a white solid.
1H NMR (500 Hz, CDCl3) α-anomer: δ: 7.37−7.21 (m,
10H), 6.27 (d, 1H, J = 3.5 Hz), 5.07 (d, 1H, J = 9.5 Hz), 4.98−
4.90 (m, 3H), 4.81−4.75 (m, 1H), 4.68−4.64 (m, 2H), 4.61−
4.57 (m, 1H), 4.40 (dd, 1H, J = 2.5, 12.5 Hz), 4.10 (dd, 1H, J =
4.5, 12.0 Hz), 3.91−3.86 (m, 2H), 3.78−3.75 (m, 1H), 3.62
(dd, 1H, J = 3.5, 9.5 Hz), 3.37−3.32 (m, 1H), 2.17 (s, 3H),
2.08 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.01 (d,
3H, J = 6.0 Hz); β-anomer: δ: 7.37−7.21 (m, 10H), 5.58 (d,
1H, J = 8.0 Hz), 5.10−5.07 (m, 1H), 4.98−4.90 (m, 3H),
4.81−4.75 (m, 2H), 4.72−4.70 (m, 1H), 4.61−4.57 (m, 1H),
4.42 (dd, 1H, J = 2.0, 12.0 Hz), 4.12 (dd, 1H, J = 5.5, 12.5 Hz),
3.80 (t, 1H, J = 8.5 Hz), 3.69 (t, 1H, J = 8.5 Hz), 3.64−3.62 (m,
1H), 3.54 (t, 1H, J = 8.5 Hz), 3.42−3.36 (m, 1H), 2.10 (s, 3H),
2.05 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.04 (d, 3H, J = 6.0
Hz); 13C NMR (125 Hz, CDCl3) δ: 170.4, 170.2, 169.6, 169.4,
169.2, 138.8, 137.3, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9,
127.75, 127.70, 127.2, 126.9, 100.7, 93.5, 89.3, 79.3, 78.3, 77.7,
75.0, 73.2, 72.98, 72.97, 72.4, 70.5, 70.0, 62.1, 21.0, 20.8, 20.6,
20.5, and 17.1; IR (KBr pellet) 1745, 1374, 1247, 1220, 1072,
and 1039 cm−1; HRMS (ESI+) [M + Na]+ calcd for
20
found 397.1613; [α]D = +49.25 (c = 1.0, CH2Cl2).
Synthesis of B Unit 17a (Scheme 4, Method II). Methyl-
2,3-di-O-benzyl-6-O-acetyl-α-D-glucopyranoside (17a). To a
solution of methyl 2,3-di-O-benzyl-α-D-glucopyranoside (22)
(150.00 g, 400.61 mmol) in EtOAc (450 mL), NEt3 (46.62 g,
460.70 mmol), and Ac2O (47.03 g, 460.70 mmol) was added at
20 °C. The mixture was stirred for 16 h. After completion of
the reaction (confirmed by HPLC Method A), H2O (450 mL)
was added, and the organic layer was separated. i-PrOH (900
mL) was added, and the mixture was concentrated to 450 mL.
After addition of i-PrOH (300 mL), the mixture was stirred for
1 h, and the precipitation of 17a was observed. H2O (1200 mL)
was added dropwise, and the slurry was cooled to 0 °C and
filtered. The product was washed with chilled mixed solution of
i-PrOH (150 mL) and H2O (300 mL) and dried in vacuo to
afford 17a (156.21 g, 93.6%) as a white solid.
Synthesis of 28 (Scheme 6). 1,6-Di-O-acetyl-2,3-di-O-
benzyl-4-O-(2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosyl)-
D-glucopyranose (28). To a mixture of 1,2,3,4-tetra-O-acetyl-6-
deoxy-D-glucopyranose (α,β-mixture) (25) (130.00 g, 391.21
mmol) in THF (195 mL), N-methylpiperazine (58.77 g, 586.82
mmol) was added dropwise at 15 °C. The mixture was stirred
at 15 °C for 14 h. After completion of the reaction (confirmed
by HPLC Method A), the mixture was cooled to 0 °C, and c-
HCl (65.21 g, 625.94 mmol) was added dropwise. EtOAc
(1300 mL) and H2O (390 mL) were added, and the organic
layer was separated and washed with H2O (390 mL) three
times. The organic layer was concentrated to 260 mL, and then
toluene (1300 mL) was added. The solution was concentrated
to 520 mL. The addtion of toluene (1300 mL) and the
following concentration to 520 mL were repeated to control
the water content less than 100 ppm. To the solution, Cl3CCN
(73.43 g, 508.57 mmol) and DBU (0.89 g, 5.87 mmol) was
added. The mixture was stirred at 25 °C for 14 h. After
completion of the reaction (confirmed by HPLC Method B),
toluene (900 mL) was added. The solution was added dropwise
to the mixture of methyl 2,3-di-O-benzyl-6-O-acetyl-α-D-
glucopyranoside (17a) (114.05 g, 273.85 mmol) and TMSOTf
20
C36H44NaO15: 739.2578; found 739.2558; [α]D = +44.20 (c
= 1.0, CH2Cl2).
Synthesis of 29 (Scheme 6). 6-O-Acetyl-2,3-di-O-benzyl-
4-O-(2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosyl)-D-gluco-
pyranose (29). To a solution of 1,6-di-O-acetyl-2,3-di-O-
benzyl-4-O-(2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosyl)-D-
glucopyranose (28) (60.00 g, 83.71 mmol) in THF (120 mL),
N-methylpiperazine (33.54 g, 334.85 mmol) was added at 15
°C. The mixture was stirred at 15 °C for 48 h. After completion
of the reaction (confirmed by HPLC Method A), the mixture
was cooled to 0 °C. EtOAc (600 mL) and 1 M aq. HCl (600
mL) were added, and the organic layer was separated, washed
with H2O (600 mL) and 1% aq. NaHCO3 (600 mL), and
concentrated to 180 mL. i-PrOH (360 mL) was added, and the
mixture was concentrated to 180 mL. i-PrOH (240 mL) was
added, and then H2O (1260 mL) was added dropwise over 2 h.
During the addtion, the precipitation of 29 was observed. The
slurry was stirred at rt for 1 h and filtered. The product was
washed with 25% aq. i-PrOH (300 mL) and dried in vacuo to
afford 29 (52.68 g, 93.3%) as a white solid.
1H NMR (500 Hz, CDCl3) α/β-mixture: δ: 7.38−7.24 (m,
20H), 5.15−5.14 (m, 1H), 5.11−5.06 (m, 2H), 4.98−4.92 (m,
4H), 4.89−4.84 (m, 3H), 4.81−4.75 (m, 2H), 4.73−4.69 (m,
3H), 4.65−4.61 (m, 3H), 4.49−4.43 (m, 2H), 4.13−4.03 (m,
3H), 3.90 (t, 1H, J = 9.0 Hz), 3.76−3.61 (m, 4H), 3.55−3.51
I
dx.doi.org/10.1021/op500306p | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development
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(m, 2H), 3.41−3.35 (m, 3H), 3.23 (brs, 1H), 2.10 (s, 3H), 2.09
(s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H),
1.99 (s, 3H), 1.98 (s, 3H), 1.71 (brs, 2H), 1.03 (d, 3H, J = 6.5
Hz), 1.01 (d, 3H, J = 6.0 Hz); 13C NMR (125 Hz, CDCl3) δ:
170.5, 170.4, 170.11, 170.08, 169.5, 169.3, 169.2, 138.7, 138.5,
137.9, 137.5, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4,
127.0, 126.9, 126.8, 100.33, 100.31, 96.9, 90.4, 82.4, 82.2, 79.12,
79.07, 76.7, 74.7, 74.5, 72.9, 72.84, 72.79, 72.3, 72.1, 69.78,
69.76, 67.9, 62.4, 62.2, 20.62, 20.60, 20.4, 20.3, 16.9, and 16.8;
IR (KBr pellet) 3466, 1745, 1248, 1218, 1069, and 1039 cm−1;
HRMS (ESI+) [M + Na]+ calcd for C34H42NaO14: 697.2472;
783.99 mmol), the mixture was stirred at 100 °C for 3 h. After
completion of the reaction (confirmed by HPLC Method C),
EtOAc (900 mL) was added. ClCO2Me (29.64 g, 313.60
mmol) was added dropwise to the mixture at 0 °C. After
completion of the reaction (confirmed by HPLC Method C),
10% aq. NaCl (720 mL) was added, and the organic layer was
separated (HPLC purity of the product: 69.4%). H2O (270
mL) and c-HCl (54 mL) were added, and the mixture was
stirred at 30 °C for 1 h. After completion of the reaction
(confirmed by HPLC Method C), the organic layer was
separated and washed with 5% aq. NaHCO3 (360 mL) (HPLC
purity of 33: 80.4%). To the solution, NaBH4 (7.91 g, 209.06
mmol) was added portionwise at 0 °C. The mixture was stirred
at 0 °C for 20 min. After completion of the reaction (confirmed
by HPLC Method C), 10% aq. NH4Cl (270 mL) was added,
and the organic layer was separated and washed with 10% aq.
NaCl (270 mL). Activated carbon (13.5 g) was added, and the
mixture was stirred at 30 °C for 30 min, filtered, and washed
with EtOAc (270 mL). The filtrate was concentrated to 360 mL
and warmed to 50 °C, and heptane (450 mL) was added
dropwise. During the addtion, the precipitation of 34 was
observed. The slurry was stirred at rt for 1 h and filterd. The
product was washed with EtOAc/heptane (1:3, 360 mL) and
dried in vacuo to afford 34 (58.67 g, 56.1% from 30) as a white
solid.
20
found 697.2474; [α]D = +34.60 (c = 1.0, CH2Cl2).
Synthesis of 34 (Scheme 7). 3-O-Benzyl-N-methoxycar-
bonyl-1,4-dideoxy-1,4-imino-D-arabinitol (34). To a solution
of 5-O-tosylate-1,2-O-isopropylidene-α-D-xylofuranose (30)15
(90.00 g, 261.33 mmol) in DMSO (360 mL), NaN3 (20.39 g,
313.60 mmol) was added. The mixture was stirred at 110 °C
for 5 h. After completion of the reaction (confirmed by HPLC
Method C), the mixture was cooled to rt, and 20% aq. NaCl
(540 mL) and toluene (450 mL) were added. The organic layer
was separated, and the aqueous layer was extracted with toluene
(450 mL) two times. The combined organic layer was
concentrated to 360 mL (HPLC purity of the product:
96.0%). The solution was added dropwise to a solution of 60%
NaH in oil (12.54 g, 313.60 mmol) in THF (495 mL) at 0 °C.
BnCl (34.73 g, 274.40 mmol) and tetrabutylammonium
bromide (8.42 g, 26.13 mmol) were added, and the mixture
was stirred at 25 °C for 4 h. After completion of the reaction
(confirmed by HPLC Method C), 20% aq. NaCl (450 mL) was
added dropwise, and the organic layer was separated and
concentrated to 360 mL. MeOH (900 mL) was added, and the
mixture was concentrated to 360 mL. MeOH (900 mL) was
added to the residue, and the mixture was concentrated to 360
mL. MeOH (270 mL) was added (HPLC purity of 31: 88.7%).
To the solution of 31, 2 M HCl in MeOH (270 mL) was
added, and the mixture was warmed to 55 °C and stirred for 2
h. After completion of the reaction (confirmed by HPLC
Method C), the pH of the mixture was adjusted to 7−8 with
20% aq. NaOH. Then heptane (180 mL) was added, and the
aqueous layer was separated and concentrated to 360 mL.
EtOAc (900 mL) was added to the residue, and the mixture
was concentrated to 360 mL. EtOAc (360 mL) and 20% aq.
NaCl (450 mL) were added, and the organic layer was
separated and concentrated to 360 mL. EtOAc (900 mL) was
added to the residue, and the mixture was concentrated to 360
mL. EtOAc (270 mL) was added (HPLC purity of the product:
88.3%). To the solution, NEt3 (39.66 g, 392.00 mmol) and
MsCl (32.93 g, 287.46 mmol) were added dropwise at 0 °C,
and the mixture was stirred. After completion of the reaction
(confirmed by HPLC Method C), 5% aq. NaHCO3 (900 mL)
was added dropwise, and the mixture was stirred for 1 h. The
mixture was separated, and the obtained aqueous layer was
extracted by EtOAc (1350 mL). The combined organic layer
was washed with 5% aq. NaHCO3 (450 mL), 1 M aq. HCl (450
mL), 5% aq. NaHCO3 (450 mL), and 20% aq. NaCl (450 mL)
and concentrated to 270 mL (HPLC purity of 32: 91.0%). To
the solution of 32, MeOH (450 mL) and 5% Pd/C containing
54.5% of water (9.89 g) were added, and the mixture was
stirred under H2 pressure (3 bar) at 50 °C for 9 h. After
completion of the reaction (confirmed by HPLC Method C),
the mixture was filtered and washed with MeOH (360 mL).
The filtrate was concentrated to 450 mL, and DMSO (450 mL)
was added to the residue. After addition of NEt3 (79.33 g,
1H NMR (500 MHz, CDCl3) δ 7.37−7.28 (m, 5H), 5.00 (d,
0.3H (a rotamer), J = 8.5 Hz), 4.71 (d, 0.7H (a rotamer), J =
9.0 Hz), 4.59 (d, 2H, J = 2.0 Hz), 4.20−4.15 (m, 2H), 4.05−
3.87 (m, 3H), 3.72−3.65 (m, 1H), 3.68 (s, 3H), 3.54−3.47 (m,
1H) ; 13C NMR (125 MHz, CDCl3) δ 156.4, 156.0, 137.3,
128.3, 127.8, 127.5, 127.4, 87.1, 86.0, 72.3, 71.4, 71.3, 64.9,
64.5, 61.5, 61.4, 55.0, 54.3, and 52.5 (rotamers were observed);
IR (KBr pellet) 3357, 1728, 1395, 1318, 1223, 1180, 1100, and
1057 cm−1; HRMS (ESI+) [M + H]+ calcd for C14H20NO5:
282.1341; found 282.1338; [α]D20 = −28.23 (c = 1.0, DMSO).
Synthesis of C Unit 14b (Scheme 7). 2,5-O-Dibenzoyl-N-
methoxycarbonyl-1,4-dideoxy-1,4-imino-D-arabinitol (14b).
To a mixture of 3-O-benzyl-N-methoxycarbonyl-1,4-dideoxy-
1,4-imino-D-arabinitol (34) (30.00 g, 106.65 mmol) and
pyridine (42.18 g, 533.25 mmol) in EtOAc (120 mL), BzCl
(37.48 g, 266.63 mmol) was added dropwise at 25 °C. The
mixture was warmed to 70 °C and stirred for 5 h. After that, the
mixture was cooled to 25 °C and stirred for 15 h. After
completion of the reaction (confirmed by HPLC Method C),
EtOAc (180 mL) and 2 M aq. HCl (300 mL) were added, and
the organic layer was separated and washed with 2 M aq. HCl
(150 mL), 5% aq. NaHCO3 (150 mL), and 10% aq. NaCl (150
mL). The obtained organic layer was concentrated to 90 mL
(HPLC purity of the product: 90.4%). To the solution, MeOH
(210 mL), c-HCl (9 mL), and 5% Pd/C containing 54.5% of
water (13.19 g) were added, and the mixture was stirred at 60
°C under H2 pressure (3 bar) for 1 h. After completion of the
reaction (confirmed by HPLC Method C), the mixture was
filtered and washed with EtOAc (120 mL). The filtrate was
concentrated to 120 mL. EtOAc (180 mL) and 1 M aq. HCl
(150 mL) were added, and the organic layer was separated and
washed with 5% aq. NaHCO3 (150 mL) 2 times and 10% aq.
NaCl (150 mL). To the obtained organic layer, activated
carbon (4.5 g) was added, and the mixture was stirred for 30
min, filtered, and washed with EtOAc (120 mL). The filtrate
was concentrated to 120 mL. Heptane (120 mL) was added
dropwise to the solution at 50 °C, and the mixture was stirred
at 50 °C for 17 h (the precipitation of 14b was observed). After
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that, heptane (360 mL) was added dropwise at 50 °C, and the
mixture was cooled to 20 °C, stirred for 18 h, and filtered. The
product was washed with EtOAc/heptane (1:4, 150 mL) and
dried in vacuo to afford 14b (36.67 g, 86.1%) as a white solid.
1H NMR (500 MHz, CDCl3) δ 8.03−8.00 (m, 4H), 7.60−
7.52 (m, 2H), 7.46−7.38 (m, 4H), 5.35−5.32 (m, 1H), 4.60
(brs, 2H), 4.53 (brs, 1H), 4.32−4.10 (m, 2H), 3.70 (brs, 3H),
3.69 (brs, 1H), 3.42−3.34 (m, 1H); 13C NMR (125 MHz,
CDCl3) δ 166.0, 165.7, 155.7, 133.3, 132.9, 132.8, 129.41,
129.36, 128.8, 128.3, 128.1, 78.1, 77.3, 75.9, 75.0, 63.7, 63.2,
63.0, 62.4, 52.6, 50.8, and 50.5 (rotamers were observed); IR
(KBr pellet) 3436, 1725, 1686, 1460, 1397, 1282, 1115, and
710 cm−1; Elementary analysis. Found: C, 63.24; H, 5.32; N,
3.67%. Calcd for C21H21NO7: C, 63.15; H, 5.30; N, 3.51%;
12.0 Hz), 1.15 (d, 3H, J = 6.0 Hz); 13C NMR (125 Hz,
CD3OD) δ: 140.6, 139.5, 129.4, 129.2, 129.0, 128.9, 128.7,
128.3, 104.4, 97.0, 85.5, 81.5, 80.6, 78.1, 77.7, 77.3, 77.0, 76.2,
76.0, 74.2, 73.4, 72.8, 66.8, 63.0, 61.4, 53.9, and 18.2; IR (KBr
pellet) 3381, 2908, 1108, 1066, 1039, 734, and 697 cm−1;
HRMS (ESI+) [M + H]+ calcd for C31H44NO12: 622.2864;
20
found 622.2859; [α]D = +113.00 (c = 1.0, 1 M aq. HCl).
Synthesis of 1 (Scheme 8). (2R,3R,4R)-4-Hydroxy-2-
(hydroxymethyl)pyrrolidine-3-yl 4-O-(6-deoxy-α-D-glucopyr-
anosyl)-α-D-glucopyranoside (1). To a mixture of 11 (11.78 g,
content rate 67.9%, net amount 8.00 g, 12.87 mmol) in EtOH
(64 mL), c-HCl (1.68 g, 16.09 mmol) was added, and the
mixture was stirred at 40 °C for 1 h. NaCl was removed by
filtration and washed with EtOH (16 mL). To the filtrate, 7.5%
Pd/C containing 52.8% of water (0.85 g) and H2O (8 mL)
were added. The mixture was stirred at 40 °C under H2
pressure (3 bar) for 2 h. After completion of the reaction
(confirmed by HPLC Method D), the mixture was filtered and
washed with 90% aq. EtOH (40 mL). The pH of the filtrate was
adjusted to ca. 12 with 2 M aq. LiOH, and then activated
carbon (0.2 g) was added. The mixture was stirred for 1 h,
filtered, washed with 90% aq. EtOH (40 mL), and concentrated
to 24 mL. EtOH (160 mL) was added, and the mixture was
concentrated to 80 mL. The precipitation of 1 was observed.
EtOH (80 mL) was added, and the mixture was concentrated
to 80 mL. EtOH (40 mL) was added, and the mixture was
stirred for 16 h and filtered. The product was washed with
EtOH (80 mL) and dried in vacuo to afford 1 (5.42 g, 95.4%
from 11) as a white solid.
LRMS (FAB) [M + H]+ 400; [α]D = −23.20 (c = 1.0,
20
DMSO).
Synthesis of ABC Unit 11 (Scheme 8). (2R,3R,4R)-4-
Hydroxy-2-(hydroxymethyl)pyrrolidine-3-yl 2,5-O-dibenzyl-4-
O-(6-deoxy-α-D-glucopyranosyl)-α-D-glucopyranoside (11).
To a mixture of 6-O-acetyl-2,3-di-O-benzyl-4-O-(2,3,4-tri-O-
acetyl-6-deoxy-β-D-glucopyranosyl)-D-glucopyranose (29)
(21.96 g, 32.55 mmol) and trichloroacetonitrile (7.05 g,
48.83 mmol) in toluene (7 mL), DBU (99 mg, 0.65 mmol)
was added at 25 °C, and the mixture was stirred for 24 h. The
completion of the reaction was confirmed by HPLC Method A
(solution of 12c).
To a solution of 2,5-O-dibenzoyl-N-methoxycarbonyl-1,4-
dideoxy-1,4-imino-D-arabinitol (14b) (10.00 g, 25.04 mmol) in
toluene (215 mL) and DME (35 mL) at −40 °C, TMSOTf
(0.68 mL, 3.76 mmol) was added dropwise. To the mixture, the
solution of 12c was added dropwise at −40 °C over 10 h, and
the mixture was stirred for 30 min. After completion of the
reaction (confirmed by HPLC Method A), NEt3 (1.05 mL, 7.51
mmol) was added. The mixture was washed with 20% aq. NaCl
(100 mL) and concentrated to 70 mL. i-PrOH (60 mL), H2O
(30 mL), and 5 M aq. NaOH (120 mL) were added, and the
mixture was stirred at 70 °C for 7 h. After completion of the
reaction (confirmed by HPLC Method A), the mixture was
cooled to 25 °C, and c-HCl (55 mL) was added (pH: <1.0).
Toluene was added, and the aqueous layer was separated. The
pH of the aqueous layer was adjusted to ca. 11.0 with 5 M aq.
NaOH (36 mL). 25% aq. NaCl (150 mL) was added, and
crystal of 11 (5 mg) was seeded. The mixture was stirred at 30
°C for 16 h and then concentrated to 330 mL. The mixture was
warmed to 50 °C, stirred for 1 h, cooled to 25 °C, and filtered.
The product was washed with 25% aq. NaCl (50 mL) and dried
in vacuo to afford crude 11 (21.63 g) as a white solid.
The obtained crude solid was added to the solution of
MeCN (126 mL) and THF (84 mL). The mixture was warmed
to 70 °C, stirred for 1.5 h, gradually cooled to 0 °C over 3 h,
stirred for 2 h, and filtered. The product was washed with
chilled mixed solvent of MeCN (63 mL)/THF (42 mL) and
dried in vacuo to afford 11 (16.61 g, content rate: 67.9%, net
yield 75% from 14b) as a white solid. This solid contained ca.
30% of NaCl.
1H NMR (500 MHz, D2O) δ 4.85 (d, 1H, J = 3.5 Hz), 4.21
(d, 1H, J = 8.0 Hz), 4.06−4.03 (m, 1H), 3.70−3.68 (m, 1H),
3.66−3.59 (m, 2H), 3.57−3.51 (m, 2H), 3.45 (dd, 2H, J = 1.0,
5.5 Hz), 3.37−3.33 (m, 2H), 3.28−3.23 (m, 1H), 3.20 (t, 1H, J
= 9.5 Hz), 3.06 (t, 1H, J = 8.0 Hz), 2.93 (t, 1H, J = 9.5 Hz),
2.90−2.86 (m, 1H), 2.84 (d, 1H, J = 5.0 Hz), 2.63 (dd, 1H, J =
3.0, 12.0 Hz), 1.07 (d, 3H, J = 5.5 Hz); 13C NMR (125 MHz,
D2O) δ 103.1, 97.7, 84.9, 79.6, 76.5, 75.8, 75.3, 73.9, 72.6, 71.9,
71.5, 71.3, 65.0, 62.3, 60.3, 52.1, and 17.4; IR (KBr pellet)
3338, 2909, 1442, 1378, 1181, 1161, 1042, and 651 cm−1;
Elementary analysis. Found: C, 45.97; H, 7.23; O, 43.68%; N,
3.12%. Calcd for C17H31NO12: C, 46.25; H, 7.08; O, 43.49; N,
3.17%; HRMS (ESI+) [M + H]+ calcd for C17H32NO12:
20
442.1925; found 442.1910; [α]D = +91.8 (c = 1.0, H2O).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR and 13C NMR spectra for all isolated compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel.: +81-463-31-6404. Fax: +81-463-31-6525. E-mail: ueda.
tsuyoshi.aj@daiichisankyo.co.jp.
1H NMR (500 Hz, CD3OD) δ: 7.39 (d, 2H, J = 7.0 Hz),
7.33−7.22 (m, 8H), 5.11 (d, 1H, J = 3.5 Hz), 5.00 (d, 1H, J =
11.0 Hz), 4.73 (d, 1H, J = 11.0 Hz), 4.66 (s, 2H), 4.49 (d, 1H, J
= 8.0 Hz), 4.15−4.13 (m, 1H), 3.99−3.98 (m, 1H), 3.94 (dd,
1H, J = 3.0, 12.0 Hz), 3.86−3.79 (m, 3H), 3.76−3.73 (m, 1H),
3.68 (d, 2H, J = 11.0 Hz), 3.51 (dd, 1H, J = 4.0, 9.5 Hz), 3.32−
3.28 (m, 1H), 3.22−3.19 (m, 2H), 3.11−3.07 (m, 1H), 3.06 (d,
1H, J = 5.0 Hz), 2.99 (t, 1H, J = 9.0 Hz), 2.87 (dd, 1H, J = 2.0,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dr. Makoto Michida, Mr. Shunsuke
Koiwa, Dr. Ryo Itooka, and Ms. Ayako Saito for their important
contributions to the development of first-generation method in
Process Development Laboratories. We also gratefully acknowl-
K
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edge Dr. Takeshi Honda and Dr. Shigeo Yamanoi for the early
medicinal chemistry work in Medicinal Chemistry Research
Laboratories.
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