Large-scale synthesis of the glucosylceramide synthase inhibitor N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojirimycin

Article abstract of DOI:10.1021/op700295x

A synthetic route for the preparation of glucosylceramide synthase inhibitor N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojirimycin methanesulfonic acid salt (AMP-DNM) has been developed. Herein we report the development and optimization of this synthetic route from its initial version in an academic research laboratory at milligram-scale to the final optimized route that was implemented in a cGMP miniplant on kilogram-scale. The definitive route starts with the separate synthesis of building blocks 2,3,4,6-tetra-O- benzyl-1-deoxynojirimycin and 5-(adamantan-1-ylmethoxy)-pentanal. The aldehyde was synthesized from 1,5-pentanediol in five steps and 45% overall yield. Protected 1-deoxynojirimycin was prepared by a successive hemiacetal reduction/Swern oxidation/double reductive amination sequence of 2,3,4,5-tetra-O-benzyl-D-glucopyranose in 52% overall yield. Reductive amination of the two building blocks produced the benzylprotected penultimate that was isolated as its crystalline (+)DTTA salt in 68% yield. Hydrogenolysis of the penultimate and crystallization of the end product as its methanesulfonic acid salt produced AMP-DNM in 76% yield with a purity of >99.5%. The described route enables the production of multikilogram amounts of inhibitor AMP-DNM as a stable crystalline solid with high purity under cGMP control.

Full text of DOI:10.1021/op700295x

Organic Process Research & Development 2008, 12, 414–423
Large-Scale Synthesis of the Glucosylceramide Synthase Inhibitor
N-[5-(Adamantan-1-yl-methoxy)-pentyl]-1-deoxynojirimycin
Tom Wennekes,† Bernhard Lang,§ Michel Leeman, Gijsbert A. van der Marel,† Elly Smits, Matthias Weber,§
Jim van Wiltenburg, Michael Wolberg,§ Johannes M.F.G. Aerts,*,‡ and Herman S. Overkleeft*,†
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden UniVersity, PO Box 9502, 2300 RA Leiden, The Netherlands,
Department of Medical Biochemistry, Academic Medical Center, UniVersity of Amsterdam, 1105 AZ Amsterdam, The
Netherlands, DSM Pharma Chemicals - ResCom, Donaustaufer Strasse 378, 93055 Regensburg, Germany, and Syncom BV,
Kadijk 3, PO Box 2253, 9704 CE Groningen, The Netherlands
Abstract:
A synthetic route for the preparation of glucosylceramide synthase
inhibitor N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojiri-
mycin methanesulfonic acid salt (AMP-DNM) has been developed.
Herein we report the development and optimization of this
synthetic route from its initial version in an academic research
laboratory at milligram-scale to the final optimized route that was
implemented in a cGMP miniplant on kilogram-scale. The defini-
tive route starts with the separate synthesis of building blocks
2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin and 5-(adamantan-1-yl-
methoxy)-pentanal. The aldehyde was synthesized from 1,5-
pentanediol in five steps and 45% overall yield. Protected 1-deox-
ynojirimycin was prepared by a successive hemiacetal reduction/
Swern oxidation/double reductive amination sequence of 2,3,4,5-
Figure 1. Structures of 1-deoxynojirimycin (1), Miglitol (2),
Zavesca (3), glucosylceramide (4), and N-[5-(adamantan-1-yl-
methoxy)-pentyl]-1-deoxynojirimycin (5).
tetra-O-benzyl-D-glucopyranose in 52% overall yield. Reductive
in 1966,3,4 preceded its discovery as a natural product in 1976,
when it was isolated from the leaves of mulberry trees5 and
certain species of bacteria.6 Since then numerous processes for
the preparation of 1 have been reported that include synthetic
approaches based on both carbohydrate and non-carbohydrate
precursors7–9 as well as chemoenzymatic syntheses.10 Further
research into the synthesis and biological activity of 1-deox-
ynojirimycin derivatives has already spawned two registered
drugs. Miglitol (2)11 is an oral drug for the treatment of non-
insulin-dependent diabetes and Zavesca (3)12,13 is an oral drug
for the treatment of Gaucher’s disease. In the latter case drug
action takes place by inhibition of the enzyme glucosylceramide
synthase (GCS). The product of GCS action is glucosylceramide
(4), which is a member of the glycosphingolipid family and
the crucial metabolic precursor in the biosynthesis of almost
all complex glycosphingolipids. Glycosphingolipids are com-
amination of the two building blocks produced the benzyl-
protected penultimate that was isolated as its crystalline (+)DTTA
salt in 68% yield. Hydrogenolysis of the penultimate and crystal-
lization of the end product as its methanesulfonic acid salt
produced AMP-DNM in 76% yield with a purity of >99.5%. The
described route enables the production of multikilogram amounts
of inhibitor AMP-DNM as a stable crystalline solid with high
purity under cGMP control.
Introduction
Ever since the discovery of iminosugars during the 1960s
and the unearthing of their ability to inhibit glycosidases1,2 in
the 1970s, they have been subject of extensive studies in both
organic chemistry and biochemistry. Iminosugars (also known
as azasugars) are polyhydroxylated alkaloids that can be
regarded as monosaccharide analogues with nitrogen replacing
the ring oxygen. From this extensive family of compounds, the
best known member is 1-deoxynojirimycin (1; Figure 1). Its
chemical synthesis from L-sorbose, by Paulsen and co-workers
(3) Paulsen, H. Angew. Chem., Int. Ed. 1966, 5, 495–510.
(4) Paulsen, H.; Sangster, I.; Heyns, K. Chem. Ber. 1967, 100, 802–815.
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(7) Afarinkia, K.; Bahar, A. Tetrahedron: Asymmetry 2005, 16, 1239–
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* To whom correspondence should be addressed. E-mail: h.s.overkleeft@
chem.leidenuniv.nl.
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Eur. J. Org. Chem. 2005, 2159–2191.
† Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University.
‡ Department of Medical Biochemistry, Academic Medical Center, University
of Amsterdam.
(9) Behling, J.; Farid, P.; Medich, J. R.; Scaros, M. G.; Prunier, M.; Weier,
R. M.; Khanna, I. Synth. Commun. 1991, 21, 1383–1386.
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§ DSM Pharma Chemicals - ResCom.
Syncom BV.
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4696.
(1) Stütz, A. E., Ed. Iminosugars as Glycosidase Inhibitors: Nojirimycin
and Beyond ; 1999, Wiley-VCH: New York.
(2) Compain, P.; Martin, O. R. Iminosugars: From Synthesis to Thera-
peutic Applications; 2007, Wiley-VCH: New York.
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10.1021/op700295x CCC: $40.75
2008 American Chemical Society
Published on Web 04/29/2008

ponents of the outer plasma membrane and as such are involved
in many (patho)physiological processes, including intercellular
recognition, signaling processes, and interactions with path-
ogens.14–28 The biosynthesis of 4 occurs at the outer membrane
of the Golgi apparatus where GCS is membrane bound.
Catabolism of 4 is effected in the lysosomes where the enzyme
glucocerebrosidase (GBA1) assisted by activator protein saposin
C, cleaves the glycosidic bond in 4.29 There is also a second
enzymatic activity that is capable of cleaving this glycosidic
bond about which we reported in 1993.30,31 Although the exact
function of this membrane-bound enzyme, located at the plasma
membrane, is still unknown, it has recently been identified by
us and Yildiz and co-workers as ꢀ-glucosidase 2 (GBA2).32,33
During our research focussing on the development of
selective and potent inhibitors of the three enzymes involved
in glucosylceramide metabolism (GCS, GBA1, and GBA2)30,32,34
we found N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojir-
imycin (5) to be a remarkably potent inhibitor of glucosylce-
ramide biosynthesis (100-fold more potent than 3 in inhibiting
GCS; 3: IC50 ) 25–50 µM; 5: IC50 ) 200 nM in our assay).
Besides a potential application of 5 in the treatment of Gaucher’s
disease and related sphingolipidoses,35 the role of glycosphin-
golipids in many other (patho)biological processes points
towards a wider range of applications. Recently, we showed
that inhibition of GCS through oral dosage of compound 5 to
ob/ob mice, which is a type II diabetes model, downregulates
glycosphingolipid biosynthesis and restores insulin receptor
sensitivity.36 We have also found that administration of 5 to
mice with hapten-induced ulcerative colitis resulted in beneficial
antiinflammatory responses.23 The crucial role of GCS at the
root of glycosphingolipid biosynthesis and its role in these
pathological processes make it an interesting drug target and
thereby GCS inhibitor 5 a promising therapeutic lead.
For potential clinical development we needed access to a
large supply of compound 5. Consequently, a study was started
to develop an efficient chemical synthesis of 5, suitable for
preparation of kilogram amounts in a miniplant. In this report
we describe the development and optimization of the synthetic
route for compound 5 from its initial synthesis in an academic
research laboratory to the successfully implemented final
synthetic route in a cGMP miniplant.
Results and Discussion
The first synthesis of compound 5 was reported by us in
1998, where it was part of a library of lipophilic iminosugars
generated to produce a specific inhibitor for GBA2.30 The
strategy for its synthesis then was to first prepare two building
blocks, 1-deoxynojirimycin (1) and 5-(adamantan-1-yl-meth-
oxy)-pentanal (17) and condense these via a reductive amination
to provide 5. In this synthesis, 1 was derived from commercially
available 2,3,4,5-tetra-O-benzyl-D-glucopyranose (6) by trans-
formation of its lactone 7 to lactam intermediate 11, which could
be further reduced and deprotected to provide 1 in 29% yield
over seven steps (Scheme 1).30,37,38 Aldehyde 17 was obtained
from commercially available glutaric dialdehyde39 in five steps
and 2% overall yield. Finally, reductive amination of 1 and 17
provided 60 mg of 5 in 50% yield. Although this route
successfully produced 5, we deemed it unsuitable for larger-
scale synthesis of 5. The main objections to this route were the
low overall yield in the synthesis of 5 and the need for several
column chromatography purifications. The larger quantities
(∼100 g) of 5 that were needed at that time for investigations
into its biological applications23,32,36,40 prompted us to search
for alternate procedures for the production of 5.
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van Assche, G.; Geboes, K.; Rutgeerts, P.; Ceuppens, J. L. Int.
Immunopharmacol. 2004, 4, 939–951.
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F.; Uemura, S.; Ogawa, C.; Ishii, A.; Saito, M.; Ohtsuka, Y.; Sakaue,
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Development of an alternative route commenced with
changing the starting material for the synthesis of 17 to 1,5-
pentanediol (18; Scheme 2). Transformation of 18 into 17 now
started according to a literature procedure41 with successive
monotosylation (19), Swern oxidation (20) and protection of
the resulting aldehyde (20) as the 1,3-dioxolane acetal to
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M.; Dubbelhuis, P. F.; Aten, J.; Kuipers, F.; Serlie, M. J.; Wennekes,
T.; Sethi, J. K.; O’Rahilly, S.; Overkleeft, H. S. Diabetes 2007, 56,
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415

Scheme 1
Reagents and conditions: (a) DMSO, Ac2O, 12 h, used crude. (b) NH3 in MeOH, 1.5 h, 86% 2 steps. (c) DMSO, Ac2O, 12 h, used crude. (d) NH3 in MeOH, 1.5 h,
10a:10b; 1.8:1 92% 2 steps. (e) NaBH3CN, HCOOH/CH3CN, reflux, 2 h, 79%. (f) LiAlH4, THF, 70 °C, 3 h, 63%. (g) Pd(OH)2/C, 5 bar H2, MeOH/EtOH, HCl, 48 h,
74%. (h) NaBH4, EtOH, 3 h, 41%. (i) MsCl, Et3N, CH2Cl2, 1 h, used crude. (j) 1: adamantanemethanol, NaH, DMF, 1 h; 2: addition 15, 70 °C, 4 h, 34%. (k) 5% aq
HCl, acetone, 1 h, quantitative. (l) 1*HCl, NaBH3CN, AcOH, MeOH, 20 h, 50%.
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) TsCl, DMAP, Et3N, CH2Cl2, 16 h, 70%. (b)
1: DMSO, (COCl)2, CH2Cl2, -75 °C, 2 h; 2: addition 19, 1.5 h; 3: Et3N, -75
°C to rt, 2 h, 91%. (c) Ethylene glycol, pTsOH, benzene, reflux, 95%. (d) 1:
adamantane-methanol, NaH, DMF, 1 h; 2: addition 21, 70 °C, 4 h, 71%. (e) 6
M aq HCl, acetone, 74 °C, 15 min, quantitative.
produce 21 in 61% yield over the three steps. Substitution of
the tosylate (21) with adamantanemethanol proved more
productive than that of mesylate 15 in the previous route and
yielded 22 in 71% yield after purification by distillation.
Subsequent acidic hydrolysis of the acetal in 22 provided
building block 17 in a yield of 43% over five steps. Compound
17 was used without further purification and proved stable when
stored at -20 °C.
a Reagents and conditions: (a) 2,2-dimethoxypropane, SnCl4, reflux, 4 h, used
crude. (b) 1: NaH, DMSO/THF, 1.5 h; 2: addition BnCl, 16 h, used crude. (c)
0.24% aq H2SO4, acetone, 48 h, 24% 3 steps. (d) Pd/C, 1 bar H2, EtOAc, 16 h,
quantitative. (e) TsCl, Et3N, pyridine, 16 h, used crude. (f) NaN3, DMF, 100 °C,
20 h, 50% 2 steps. (g) PPh3, H2O/THF, 16 h, used crude. (h) 12 M aq HCl, 0
C, 87% 2 steps. (i) PtO2, 5 bar H2, 16 h, 70%. (j) 17, Pd/C, 5 bar H2, NaOAc,
AcOH, EtOH, 67%.
For larger-scale synthesis of the second building block (1)
we selected a route reported by Behlings and co-workers9 and
also found in patent literature.42 The route uses L-sorbose (23)
as an economical starting material and is claimed to be suitable
for kg-scale preparation of 1. In the first step 23 is protected as
1,2;4,6 diacetonide 24, followed by selective hydrolysis of the
4,6 acetonide. Installation of an amine functionality at the C6
position and cyclization via a reductive amination after first
hydrolyzing the 4,6 acetonide produces 1 (Scheme 3). However,
when the first two steps were attempted at a scale of 500 g,
several problems arose that prevented the isolation of reasonable
amounts of 27. Diacetonide 24, produced after the first step,
turned out not to be the thermodynamically more stable isomer
under most reaction conditions. Also, during workup of the
second step, the acidic aqueous hydrolysis of 24, the 1,2
acetonide in product 27, isomerized considerably. An optimized
procedure for the first step was found in treating 100-g batches
of 23 with SnCl4 in dimethoxypropane.43 Unfortunately, the
isomerization side reaction of the second step could only be
circumvented by first protecting the 3-hydroxyl as a benzyl
ether, adding two additional steps to the route. Hydrolysis of
the 1,2 acetonide in benzylated 25 with dilute sulfuric acid
produced 26 in 24% yield over the three steps. After quantitative
debenzylation with Pd/C catalyzed hydrogenolysis and 6-O-
tosylation,44 azide 28 was obtained in 50% yield over two steps
via substitution of the crude tosylate. Purification of 28 by
column chromatography proved to be an unavoidable necessity.
Staudinger reduction of 28 and hydrolysis of the 1,2-acetonide
(43) Chen, C. C.; Whistler, R. L. Carbohydr. Res. 1988, 175, 265–271.
(44) Instead of the tri-isopropylbenzenesulfonyl group used in the original
procedure by Behling and co-workers, we found that the tosyl group
can successfully be used instead.
(42) Stoltefuss, J. Preparation of 1-deoxynojirimycin and N-substituted
derivatives. U.S. Patent 4,220,782, 1979.
416
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Scheme 4a
reductive amination Matos and co-workers were also able, after
hydrogenolysis, to produce 3. Recently, we successfully em-
ployed a modified version of this procedure for the synthesis
of derivatives of 5.34 Application of the original protocol, which
uses a Pfitzner-Moffat oxidation and a double reductive
amination at room temperature to produce 12, led to irrepro-
ducible and low yields in our hands. We found that the
procedure could be optimized by using a Swern oxidation to
give 33 and most importantly to execute the double reductive
amination of 33 at 0 °C in the presence of a larger excess of
ammonium salt. First, 6 was reduced to glucitol 32 with LiAlH4
in THF. Crude 32 was subjected to a Swern oxidation, which
after completion was concentrated under reduced pressure with
moderate heating to minimize degradation of the unstable
intermediate 33. The reductive amination was carried out on
crude 33 with an excess of ammonium formate in methanol at
0 °C under the agency of NaBH3CN and in the presence of 3Å
molecular sieves. These conditions could reproducibly generate
multigram amounts of 12 in yields of 60–65% over the three
steps.
a Reagents and conditions: (a) LiAlH4, THF, 20 h, used crude. (b) 1. DMSO,
(COCl)2, CH2Cl2, -75 °C, 2 h; 2. Et3N, -75 to 0 °C, 2 h, reaction mixture used
neat. (c) NaBH3CN, excess NH4HCO2, 3Å mol sieves, MeOH, 0 °C to rt, 20 h,
65% 3 steps. (d) 1.1 equiv of 17, Pd/C, 5 bar H2, AcOH, EtOH, 20 h, used
crude. (e) Pd/C, 1 bar H2, HCl, EtOH, 20 h, 89% 2 steps.
However, when the scale of this process was increased, the
Swern reaction mixture took an increasing amount of time to
concentrate. This extended exposure to heat resulted in marked
degradation of intermediate 33 and, as a result, significantly
lower yields of 12. Because degradation of 33 increases at
elevated temperature, we reasoned that omitting the concentra-
tion step and adding the crude Swern reaction mixture to the
mixture of reductive amination components might improve the
overall yield. This adaptation indeed efficiently produced 10 g
of 12 in 73% yield over the three steps. The next reaction would
be deprotection of 12 to 1, but because of the persistent
moderate yields obtained in the previous large-cale reactions
of 1 with aldehyde 17, we chose to first explore the reductive
amination of 12 with 17. When 12 and 17 were exposed to
Pd/C-catalyzed hydrogenolysis at 5 bar in an ethanol/acetic acid
mixture, the sole product was 34. After filtration and concentra-
tion, a second hydrogenolysis of crude 34, now in the presence
of hydrochloric acid, produced 2.8 g of target compound 5 in
89% over the two steps.
With this tandem reductive amination/deprotection method
and the optimized synthesis of building blocks 12 and 17 in
hand, the stage was set for translating the improved synthesis
of 5 to a kg-scale miniplant process. Process development of
the route for building block 17 focussed on optimizing the purity
of all intermediates and 17 itself without using column purifica-
tion. This was quite a challenge as all intermediates are oily
liquids and only 22 is stable enough for distillation. Suitable
in-process control by HPLC (up to 21) and GC (22 and 17)
was developed, which enabled the reactions to be monitored
and controlled in an efficient way to ensure complete conver-
sions and effective workup procedures. The synthesis of 17
started with monotosylation of 1,5-pentanediol (18). The
formation of ditosylate could be minimized to <5% by using
0.5 equiv of tosylchloride to produce 19. Swern oxidation of
19 was replaced by a TEMPO/bleach oxidation in order to
prevent formation of dimethylsulfide and the difficult handling
of all reaction phases thereof. Protection of aldehyde 20 with
ethylene glycol and catalytic p-toluenesulfonic acid resulted in
produced labile 29 in 87% yield over two steps, which when
isolated as its HCl salt proved stable when stored at 4 °C. The
final reductive amination was carried out on 20-g batches of
29 via a Pt-catalyzed hydrogenolysis at 5 bar to produce the
HCl salt of 1 in an average yield of 70%. The next stage was
the optimization of the reductive amination between building
blocks 1 and 17. Initially, the best reproducible conditions were
the use of sodium triacetoxyborohydride in ethanol, with
additional sodium acetate to liberate the HCl salt of 1. These
conditions, however, produced 5 on a 1-g scale in an unim-
proved yield of 50%. Furthermore, workup of the reaction
would be complicated on a larger scale. Instead we found that
Pd/C-catalyzed hydrogenolysis at 5 bar of a suspension of 1
and NaOAc in an ethanol/acetic acid mixture with 17 was more
efficient and produced 17 g of 5 in an average yield of 67%.
However, column purification of 5 proved necessary to remove
a side product, 6-deoxy derivative 31. This side product
originated from 6-deoxy 30 that was formed by a side reaction
during the Pt-catalyzed hydrogenolysis of 29. Overall, this route
produced 64 g of 5 in 8% yield over nine steps, and the five-
step synthesis of building block 17 was optimized from 2% to
43% yield.
The route for building block 17 was now set for translation
to kg-scale synthesis. On the other hand, we deemed the route
explored for the second building block, 1-deoxynojirimycin (1)
unsuitable for this next stage, mainly because of the low overall
yield and the requirement for column chromatography purifica-
tion of intermediate 28 and target compound 5. In search of a
shorter and more efficient route for the large-scale synthesis of
1 we evaluated a procedure reported by Matos and co-workers
that transforms 6 into 1 in four steps.45 The key reaction in this
synthesis is the cyclization of hexosulose 33 via a double
reductive amination with ammonium formate to produce 12
(Scheme 4). By switching to N-butylammonium formate in the
(45) Matos, C. R. R.; Lopes, R. S. C.; Lopes, C. C. Synth.-Stuttgart 1999,
571–573.
Vol. 12, No. 3, 2008 / Organic Process Research & Development
•
417

Scheme 5. Overview of cGMP miniplant process for the
synthesis of building block 12 and target compound 5
resulting from the Swern oxidation was kept below -60 °C
and directly transferred to a 0 °C suspension of NaBH3CN,
NH4OAc, and Na2SO4 in methanol. Laboratory development
had shown that the order of addition, the ammonium source,
the temperature, and the methanol dilution (>0.1 M) are critical
for this process. Molecular sieves could be replaced by Na2SO4
and instead of 20 equiv of NH4HCO2 10 equiv of NH4OAc
were used. The resulting reaction mixture, containing 12, is
contaminated with dimethylsulfide, which has to be completely
removed to prevent poisoning of the palladium catalyst used
in the final two steps. Treatment of crude 12 with an aqueous
solution of sodium hypochlorite during workup accomplished
this. During process development we observed that 12 is an oil
upon isolation, which in purified form only slowly solidifies
over time. In order to facilitate purification and isolation, the
hydrochloric acid salt of 12 was generated that could be
precipitated from acetone at 0 °C and isolated via centrifugation
to provide 12*HCl as an off-white solid in 52% yield over the
three reactions. Minor impurities were removed by means of
an additional reslurrying step in acetone that produced 12*HCl
with 93% recovery in high purity as a white solid (6.7 kg; 98.7
area % by HPLC).
The synthesis of 34 was accomplished in the miniplant via
the earlier described selective Pd/C-catalyzed hydrogenation of
the intermediate imine of 12 and 17 in the presence of acetic
acid (Scheme 5). Aldehyde 17 was now applied in a larger
excess (1.5 equiv) to ensure complete consumption of 12.
Afterwards, excess 17 was removed by formation of the HCl
salt of 34 in methanol/water and repeated washing with heptane.
As a minor side reaction partial debenzylation was detected
(∼10%), but this did not effect further processing to 5.
Penultimate 34 was chosen to be the cGMP starting material
and was therefore required to be of defined composition and
high in purity. As 34*HCl is a noncrystalline hygroscopic solid,
a salt screening was performed in order to identify a stable
nonhygroscopic salt that allows efficient purification by crystal-
lization. The (+)-di-p-toluoyl-L-tartaric acid ((+)DTTA) salt
provided a stable crystalline solid that proved most convenient
for purification and isolation. In the production run, 6.5 kg of
12 furnished 10.0 kg (75% yield) of 34*(+)DTTA salt, which
could be isolated in high purity (98.4 area % by HPLC including
the debenzylated side products).
The miniplant procedure for debenzylation of the penultimate
34*(+)DTTA was identical to the earlier described catalytic
hydrogenation in the presence of hydrochloric acid. However,
just before the scheduled start of the production run, HPLC
analysis of a laboratory test batch of 5 indicated the presence
of previously undetected 6-O-benzoylated 35 as a minor side
product (∼1%). Byproduct 35 was most probably generated
by oxidation of the 6-O-benzyl ether of 12 during workup of
the double reductive amination reaction mixture with sodium
hypochlorite. Laboratory experiments showed that removal of
this side product was best accomplished before the catalytic
hydrogenation of 34 by basic saponification of the benzoyl ester.
Correspondingly, deprotection during miniplant production
commenced with the generation of free base 34 in MTBE, after
which the solvent was exchanged from MTBE to ethanol, and
6 M sodium hydroxide was added. When HPLC analysis
the 1,3-dioxolane acetal 21 in 96% yield. Instead of benzene,
MTBE was used as reaction solvent because the lower reflux
temperature prevented the onset of decomposition of both the
starting material (20) and product (21). As fractional distillation
is not feasible on kg-scale, 0.85 equiv of adamantanemethanol
was used in the SN2 substitution of 21 to minimize the amount
of unreacted adamantanemethanol. Remaining traces of ada-
mantanemethanol could be removed with an extractive purifica-
tion in which a 21-containing heptane phase was washed
repeatedly with a methanol/water mixture. Finally, short-path
distillation provided 22 (92.3 area % by GC) as a colourless
oil in 92% yield related to adamantanemethanol or 68% related
to 21. During process development for the acidic hydrolysis of
22 we observed that an equilibrium is reached at 8% remaining
starting material and that prolonged reaction times only lead to
degradation of product 17. A solution for this problem was
found in performing the reaction two consecutive times with
extractive workup in between. This diminishes the remaining
starting material to <2% and yielded 5.1 kg of crude 17 in
45% yield over the five steps. The obtained purity of 17 (86.8
area % by GC) allowed implementing the crude product in the
subsequent reductive amination of 17 with 12.
Process development of the route for the second building
block 12 concentrated on adapting the challenging tandem
oxidation/double reductive amination sequence to the miniplant
and finding a suitable purification procedure for 12. In the final
production run two 8.5-kg batches of 6 (Scheme 5) were
reduced quantitatively with NaBH4 in refluxing dichloromethane/
methanol (Scheme 5). After extractive workup, a 12.5 kg portion
of crude 32 was oxidized to hexosulose 33 using the Swern
protocol. As isolation of the unstable 33 is not desirable, a
telescoped procedure was developed for the consecutive double
reductive amination of crude 33. Thus, the reaction mixture
418
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Vol. 12, No. 3, 2008 / Organic Process Research & Development

indicated complete saponification of 36 to 37, the reaction
mixture was acidified with hydrochloric acid and subjected to
Pd/C hydrogenolysis at atmospheric hydrogen pressure. After
removal of the catalyst by filtration, residual Pd was reduced
to a level of <20 ppm by a treatment with Ecosorb C-941.
The good solubility of 5*HCl in various solvents including
water made it difficult to remove the inorganic salts from the
reaction mixture. This problem was solved by treating crude
5*HCl with ammonia in methanol and subsequently exchanging
the solvent for dichloromethane from which all inorganic salts
precipitated and in which 5 remained dissolved.
Previous biological studies were performed with 5*HCl, but
it was evident that an alternative for this highly hygroscopic
and noncrystalline HCl salt had to be found. Free amine 5 also
showed the same hygroscopic property and also proved to be
unstable after prolonged storage at room temperature. Therefore,
a salt screening was initiated to identify a suitable counterion
that forms a stable, crystalline and nonhygroscopic salt of
defined composition. From a number of possible pharmaceuti-
cally accepted counterions the sulfonic acid salts of methansul-
fonic acid (MSA), ethanesulfonic acid, and p-toluenesulfonic
acid showed the desired properties. A brief toxicological study
showed identical results for the 5*MSA salt when compared
to the previously evaluated 5*HCl salt. On production scale
the MSA salt of 5 was prepared by addition of a slight excess
of methanesulfonic acid to a hot solution of 5 in isopropanol.
Slow cooling to ambient temperature and seeding with crystals
of 5*MSA resulted in crystallization, which after filtration
yielded 5*MSA in 69.5% with a purity of >99.5 area % by
HPLC.
are currently investigating the clinical development of 5*MSA
as an inhibitor for GCS.
Experimental Section
General. All solvents and reagents were obtained com-
mercially and used as received unless stated otherwise. Ada-
mantanemethanol was obtained from Inter-Chemical Ltd.
(Shenzhen, China) and 2,3,4,6-tetra-O-benzyl-D-glucose from
Farmak (Olomouc, Czech Republic). Reactions were executed
at ambient temperatures and under inert atmosphere unless
stated otherwise. Reaction progress was monitored by HPLC
and GC analysis (details in Supporting Information). 1H NMR
spectra were recorded on a Bruker DRX-400, except spectra
for 5*MSA were recorded on a Bruker AV-500. Chemical shifts
are given in ppm (δ) relative to the signal of the internal standard
tetramethylsilane for CDCl3 or the deuterated solvent signal for
CD3OD and DMSO-d6. Coupling constants (J) are given in Hz.
DSC measurements were conducted on a Mettler Toledo
DSC822e (temperature program 50 to 300 °C at 10 °C/min).
Optical rotation for 5*MSA was measured on an automatic
Propol polarimeter (Sodium D-line, λ ) 589 nm).
5-(Toluene-4-sulfonyloxy)-1-pentanol (19). To a cooled
(0 °C) solution of pentane-1,5-diol (18, 9.76 kg, 93.71 mol),
DMAP (390 g, 2.92 mol) and triethyl amine (4.88 kg, 51.72
mol) in MTBE (68.30 L) was added a cooled (0 °C) solution
of TsCl (8.78 kg, 46.84 mol) in CH2Cl2 (9.76 L) over a 2 h
period. The reaction mixture was kept at 0 °C for 2 h after
which it was warmed to 20 °C within a 1 h period and stirred
for an additional 18 h. Water (39.04 L) was added to the reaction
mixture over a 30 min period, followed by 2 M aqueous HCl
(19.52 L). After stirring the mixture for 30 min, the organic
layer was isolated and washed with saturated aqueous NaCl (2
× 19.52 L). The organic layer was concentrated using moderate
heating (Tmax < 40 °C: 19 slowly decomposes when heated) to
produce a yellow oil. A solution of crude 19 in 2-propanol (24.4
L) was stirred for 1 h at rt during which the ditosylate byproduct
precipitated as a white solid. The mixture was cooled at -5 °C
for 2 h after which the precipitate was removed by centrifugation
and washed with precooled 2-propanol (2 × 0.97 L; T ) 0
°C). The mother liquor and washings were concentrated using
moderate heating (Tmax < 40 °C) and degassed at 30 °C under
full vacuum for 2 h to provide 19 (8.82 kg, 34.14 mol, 84.8
area % by HPLC) as a light-yellow oil in 72.9% yield, which
was stable when stored under inert atmosphere, below 5 °C in
the dark. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J ) 8.3, 2H),
7.29 (d, J ) 8.1, 2H), 3.96 (t, J ) 6.4, 2H), 3.80 (s, 1H), 3.52
(t, J ) 6.5, 2H), 2.38 (s, 3H), 1.65-1.56 (m, 2H), 1.49-1.40
(m, 2H), 1.37-1.27 (m, 2H).
Conclusion
In summary, we have developed and successfully imple-
mented a synthetic route for the preparation of kilogram
amounts of GCS inhibitor 5*MSA in a cGMP miniplant. This
large-scale synthetic preparation of N-[5-(adamantan-1-yl-
methoxy)-pentyl]-1-deoxynojirimycin (5) complements the
chemoenzymatic synthesis of N-butyl-1-deoxynojirimycin (3)
reported in 2002 by Landis and co-workers, in which the key
step is a selective oxidation of N-butylglucamine on C-5 by
Gluconobacter oxidans.46 The synthesis of 5 started with the
preparation of the two building blocks 12 and 17. Benzyl-
protected 1-deoxynojirimycin (12) was synthesized from com-
mercially available 6 in three steps in an overall yield of 52%.
The second building block (17) was prepared from 1,5-
pentandiol (18) in five steps and 45% overall yield. Aldehyde
17 was used crude in a selective reductive amination with the
free base of 12 to provide 34, which was purified by crystal-
lization as its (+)DTTA salt in 75% yield. The production batch
of penultimate 34*(+)DTTA was split up and deprotected to
provide a batch for toxicological studies and a second clinical
batch under full cGMP control. Crystallization of 5 as its MSA
salt resulted in two lots of 1.4 kg of 5*MSA (average yield
68.4%) as an air-stable, nonhygroscopic, crystalline white solid
in a purity of >99.5 area % by HPLC. With the well-defined
batches of 5*MSA, prepared via the here reported route, we
5-(Toluene-4-sulfonyloxy)-1-pentanal (20). To a solution
of 19 (7.93 kg, 30.69 mol) and TEMPO (52.4 g, 0.33 mol) in
CH2Cl2 (43.65 L) was added a solution of KBr (0.43 kg) in
water (1.74 L) and the mixture was cooled to 5 °C. Separately,
sodium hypochlorite (∼20.07 L; 12–15% in water; 30.69 mol)
was diluted with enough aqueous NaHCO3 (∼20.07 L; 9%) to
reach a pH between 8.5 and 9.5 and then cooled to 5 °C.
Controlled addition of an equimolar amount of NaOCl was
essential to prevent overoxidation of 19. Therefore, the exact
concentration of the commercial NaOCl solution has to be
determined. A portion (30.55 L) of the NaOCl/NaHCO3 solution
(46) Landis, B. H.; McLaughlin, J. K.; Heeren, R.; Grabner, R. W.; Wang,
P. T. Org. Process. Res. DeV. 2002, 6, 547–552.
Vol. 12, No. 3, 2008 / Organic Process Research & Development
•
419

was added over a period of 2 h at 5 °C to the mixture containing
19, which caused an initial orange coloration of the reaction
mixture that slowly disappeared. The reaction mixture was
stirred for an additional 30 min at 5 °C, after which the reaction
progress was checked with HPLC by sampling the organic layer.
Additional portions (1–2 L) of the NaOCl/NaHCO3 solution
were added over 15 min periods with 30 min of additional
stirring at 5 °C and intermittent HPLC analysis in between until
less than 1% of 19 remained. The reaction mixture was warmed
to 20 °C, and the aqueous layer was separated and extracted
with CH2Cl2 (11.34 L). Aqueous 2.87 M HCl (34.92 L)
containing KI (87 g) was slowly added to the combined organic
layers. The aqueous layer was removed, and the organic layer
was successively extracted with aqueous Na2S2O3 (34.9 L;
10%), saturated aqueous NaHCO3 (34.9 L), and water (34.9
L). The organic layer was isolated, concentrated using moderate
heating (Tmax < 40 °C: 20 slowly decomposes when heated),
and degassed at 30 °C under full vacuum for 2 h to afford 20
(7.93 kg, 30.93 mol, 90.5 area % by HPLC) as a yellow oil in
91.5% yield, which was stable when stored under inert
saturated aqueous NaCl (2 × 43.5 L) and concentrated using
moderate heating (Tmax < 40 °C). The residue was dissolved
in heptane (76.5 L) and extracted with a methanol/water mixture
(4 × 84.2 L, 8/3 methanol/water, v/v). The heptane layer was
isolated and concentrated (Tmax < 40 °C) to afford a light-yellow
oil. This residue was further purified by short-path distillation
at a temperature of 120 °C and a pressure below 0.1 mbar to
afford 22 (5.84 kg, 19.83 mol; 92.3 area % by GC) in 80.5%
yield as a colorless oil, which was stable when stored under
inert atmosphere, below 5 °C in the dark. 1H NMR (400 MHz,
CDCl3) δ 4.82 (t, J ) 4.8, 1H), 3.98-3.88 (m, 2H), 3.87-3.77
(m, 2H), 3.35 (t, J ) 6.5, 2H), 2.92 (s, 2H), 1.92 (s, 3H),
1.71-1.53 (m, 11H), 1.52–1.40 (m, 9H).
5-(Adamantan-1-yl-methoxy)-1-pentanal (17). At 30 °C
and under rapid stirring 6 M aqueous HCl (113.6 L) was added
to a solution of 22 (5.68 kg, 19.29 mol) in acetone (56.9 L).
The turbid reaction mixture was heated to 40 °C and stirred
for 30 min. Stirring was stopped, and the organic layer was
analyzed with GC. After stirring for an additional 30 min at 40
°C, the reaction mixture was quenched by transfer to a 0 °C
mixture of 3 M aqueous NaOH (227.2 L) and MTBE (113.6
L) with the temperature being kept below 25 °C (additional 3
M NaOH was added if pH was not >7). The layers were
separated, and the aqueous layer was extracted with MTBE
(56.8 L). The combined organic layers were isolated, washed
with water (56.8 L), and concentrated at 40 °C to a volume of
∼5 L. The light-yellow residue was dissolved in acetone (56.8
L), and under rapid stirring 6 M aqueous HCl (56.8 L) was
added with the temperature being kept below 40 °C. The
reaction mixture was stirred for 1 h to 40 °C with midway
analysis by GC. The reaction mixture was quenched by rapid
transfer to a 0 °C mixture of 3 M aqueous NaOH (113.6 L)
and MTBE (56.8 L) with the temperature being kept below 25
°C (additional 3 M NaOH was added if pH was not >7). The
layers were separated, and the aqueous layer was extracted with
MTBE (28.4 L). The combined organic layers were successively
washed with water (28.4 L) and saturated aqueous NaCl (28.4
L). The organic layer was isolated, concentrated at 40 °C (17
slowly decomposes when heated above 40 °C for prolonged
time), and degassed at 30 °C under full vacuum for 2 h to afford
17 (5.10 kg, ∼16 mol, 86.8 area % by GC) as a light-yellow
oil in ∼91% yield, which still contained residual MTBE and
was stable when stored under inert atmosphere, at -20 °C in
the dark.1H NMR (400 MHz, CDCl3) δ 9.72 (t, J ) 1.6, 1H),
3.34 (t, J ) 6.1, 2H), 2.90 (s, 2H), 2.42 (td, J ) 1.6, 7.2, 2H),
1.90 (s, 3H), 1.72–1.50 (m, 10H), 1.47 (d, J ) 2.3, 6H).
2,3,4,6-Tetra-O-benzyl-D-glucitol (32). A solution of 6 (8.50
kg, 15.72 mol) in CH2Cl2 (42.5 L + 1.7 L for rinsing) was
added to a suspension of NaBH4 (1.61 kg, 42.45 mol) in CH2Cl2
(11.1 L). The resulting suspension was vigorously stirred and
heated to reflux (36–40 °C), during which methanol (11.1 L)
was carefully added over a 6 h period. Following the addition
of methanol, the reaction mixture was heated for an additional
hour, after which it was cooled to 20 °C, and remaining
hydrogen gas was evacuated with a nitrogen flow. The reaction
mixture was quenched by careful addition of 2 M aqueous
H3PO4 (21.3 L) under vigorous stirring over a 2 h period,
cooling the reaction mixture to keep the temperature below 30
1
atmosphere, below 5 °C in the dark. H NMR (400 MHz,
CDCl3) δ 9.68 (t, J ) 1.3, 1H), 7.74 (d, J ) 8.3, 2H), 7.31 (d,
J ) 8.0, 2H), 3.99 (t, J ) 5.8, 2H), 2.44-2.37 (m, 5H),
1.68-1.58 (m, 4H).
2-(4-[Toluene-4-sulfonyloxy]-butyl)-1,3-dioxolane (21). To
an emulsion of ethylene glycol (2.8 kg, 5.9 mol) and TsOH
(171 g; 0.1 mol) in MTBE (27.27 L) was added a solution of
20 in MTBE (27.27 L) over a period of 30 min. The water
liberated up to now was removed, and the reaction mixture was
refluxed (∼56 °C) for 3 h or until HPLC analysis indicated
complete conversion. The reaction mixture was cooled to 20
°C, and saturated aqueous NaHCO3 (35.06 L) was added over
a 30 min period. The organic layer was washed with saturated
aqueous NaCl (35.06 L), concentrated using moderate heating
(Tmax < 40 °C: 21 slowly decomposes when heated) and
degassed at 30 °C under full vacuum for 2 h. Compound 21
(8.81 kg, 29.33 mol, 89.5 area % by HPLC) was obtained as a
light-yellow oil in 96.5% yield, which was stable when stored
under inert atmosphere, below 5 °C in the dark. 1H NMR (400
MHz, CDCl3) δ 7.75 (d, J ) 8.3, 2H), 7.31 (d, J ) 8.0, 2H),
4.76 (t, J ) 4.6, 1H), 3.99 (t, J ) 6.5, 2H), 3.95-3.84 (m,
2H), 3.84-3.75 (m, 2H), 2.41 (s, 3H), 1.71-1.61 (m, 2H),
1.61-1.54 (m, 2H), 1.46-1.36 (m, 2H).
2-(4-[Adamantan-1-yl-methoxy]-butyl)-1,3-dioxolane (22).
Water content of commercial adamantanemethanol (4.09 kg,
24.62 mol) was removed by azeotropic distillation with toluene
(17.4 L), and the residue was dissolved in DMF (26.1 L) at 40
°C. Sodium hydride (1.97 kg, 60% in mineral oil, 49.23 mol)
was suspended in DMF (26.1 L) and heated to 40 °C after which
the adamantanemethanol solution was carefully added over a
period of 1 h. After additional stirring for 30 min a 40 °C
solution of 21 (7.79 kg, 28.96 mol) in DMF (17.4 L) was added
over a 1 h period. The reaction mixture was stirred for 2 h after
which it was cooled to 20 °C, and methanol (3.48 L) was
carefully added over a 1 h period, keeping the temperature under
30 °C. Water (87 L) was added to the reaction mixture, and
after cooling to 20 °C the mixture was extracted with MTBE
(2 × 87 L). The combined organic layers were washed with
420
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Vol. 12, No. 3, 2008 / Organic Process Research & Development

C°. After the addition, the mixture was vigorously stirred for
30 min, whilst evacuating the remaining hydrogen gas with a
nitrogen flow. After the two-phase mixture had settled for 1 h,
the organic phase was isolated, and the turbid aqueous phase
was back-extracted once with CH2Cl2 (11.1 L). The combined
organic layers were washed with water (2 × 11.1 L), concen-
trated, and degassed at 30 °C under full vacuum for 2 h to
produce 32 (8.58 kg, 15.72 mol, 97.6 area % by HPLC) as a
colorless oil in quantitative yield. 1H NMR (400 MHz, CDCl3)
δ 7.37–7.19 (m, 20H), 4.71 (d, J ) 11.3, 1H), 4.68–4.47 (m,
7H), 4.07–3.99 (m, 1H), 3.89 (dd, J ) 3.7, 6.4, 1H), 3.82–3.68
(m, 3H), 3.68–3.60 (m, 2H), 3.60–3.52 (m, 1H), 2.98 (d, J )
4.9, 1H), 2.17 (s, 1H).
and another hour whilst cooling at 0–5 °C. The product was
isolated by centrifugation, washed twice with precooled acetone
(T ) 0–5 °C; portion 1 ) 12.7 L; portion 2 ) 6.5 L), and
dried under vacuum at 35–40 °C to afford 12*HCl (7.27 kg;
12.97 mol) as an off-white solid in 56% yield. Remaining
impurities were removed through a reslurrying step in which a
suspension of 12*HCl (7.15 kg; 12.76) was vigorously stirred
in acetone (46.3 L) for 3 h at 20–25 °C and another hour at
0–5 °C. The solvent was removed by centrifugation, and the
product was washed with precooled acetone (T ) 0–5 °C; 17.9
L) to afford, after drying under vacuum at 35–40 °C, 12*HCl
(6.66 kg; 11.89 mol; 98.7 area % by HPLC) as a white solid
1
with 93% recovery.*** H NMR (400 MHz, DMSO-d6) δ
2,3,4,6-Tetra-O-benzyl-1-deoxynojirimycin Hydrochloric
Acid Salt (12*HCl). A solution (water content was verified to
be KF < 0.05%) of DMSO (9.54 kg; 122.10 mol) in CH2Cl2
(8.68 L) was slowly added to a cooled (-75 °C) solution of
oxalylchloride (12.56 kg; 9.91 mol) in CH2Cl2 (45.5 L; water
content was verified to be KF < 0.03%) so the internal tem-
perature of the reaction mixture did not exceed -65 °C. The
resulting mixture was stirred for 30 min at -75 °C, whereupon
a solution of 32 (12.5 kg; 23.03 mol) in CH2Cl2 (14.47 L) was
slowly added so the internal temperature of the reaction mixture
did not exceed -65 °C (32 was dried by azeotropic distillation
with CH2Cl2 until water content was KF < 0.03%). After
addition of 32, the reaction mixture was stirred for 2 h at -75
°C after which Et3N (25.17 kg; 248.7 mol) was slowly added
so the internal temperature of the reaction mixture did not
exceed -65 °C. The resulting suspension was stirred for 4 h
at -75 °C and then transferred to a cooled (0–5 °C) mixture*
of NH4OAc (17.76 kg, 230.40 mol), Na2SO4 (9.81 kg, 69.06
mol) and NaBH3CN (5.79 kg, 92.10 mol) in methanol (207.3
L + 23.0 L for rinsing). The reaction mixture was stirred for
18 h and allowed to warm to 20–25 °C. The reaction mixture
was cooled to 5–10 °C, and water (46.06 L) was slowly added
over a 30 min period so the internal temperature of the mixture
did not exceed 35 °C.* An aqueous NaOH (18.3 L, 50 wt %)
solution was added to the mixture** followed by addition of
water (414.6 L) over a 1 h period (T < 35 °C). The two-phase
mixture was stirred for 1 h at 18–25 °C after which the organic
phase was isolated. The aqueous phase was back-extracted once
with CH2Cl2 (102.5 L). The combined organic phases were
cooled to 5–10 °C, and under vigorous stirring an aqueous
NaOCl solution (131.1 L, 12 wt %) was added over a 1 h period
(T < 35 °C). The two-phase reaction mixture was vigorously
stirred for 1 h at 18–25 °C and then the organic phase was
isolated and cooled to 5–10 °C. Aqueous 2 M HCl (115.2 L)
was added to the organic phase over a 1 h period (T < 35 °C)
after which the mixture was vigorously stirred for another hour
at 18–25 °C. Isolation of 12 through precipitation was achieved
by solvent exchange from CH2Cl2 to acetone. The organic phase
was isolated, and CH2Cl2 (∼150 L) was evaporated until the
residue reached 54–55 °C. Acetone (151.5 L) was added to
the residue, and the mixture was vigorously stirred (T < 35
°C) for 30 min during which 12*HCl precipitated as white
floccules. Residual CH2Cl2 was removed by successively
evaporating ∼50 L of solvent and adding acetone (23.1 L). The
suspension was vigorously stirred for 1 h at ambient temperature
10.18 (s, 1H), 9.51 (s, 1H), 7.42–7.25 (m, 18H), 7.17–7.10 (m,
2H), 4.86 (d, J ) 11.2, 1H), 4.77–4.67 (m, 3H), 4.62 (d, J )
11.7, 1H), 4.58 (d, J ) 12.2, 1H), 4.50 (d, J ) 12.2, 1H), 4.45
(d, J ) 10.8, 1H), 3.91 (ddd, J ) 5.0, 8.9, 11.0, 1H), 3.82 (dd,
J ) 2.3, 10.5, 1H), 3.79–3.62 (m, 3H), 3.41 (dd, J ) 5.0, 12.1,
2H), 2.89 (dd, J ) 11.6, 1H). Mp (DSC) 174 °C. *: Precautions
should be taken for possible liberation of hydrogen cyanide gas
from the mixture. **: The pH of the aqueous layer was checked
to be above 10 to ensure fixation of hydrogen cyanide. ***:
Solubility of 12*HCl in acetone at ambient temperature was
determined to be ∼6 g L-1.
2,3,4,6-Tetra-O-benzyl-N-[5-(adamantan-1-yl-methoxy)-
pentyl]-1-deoxynojirimycin DTTA Salt (34*(+)DTTA). Aque-
ous 1 M NaOH (65.0 L) was added to a suspension of 12*HCl
(6.50 kg, 11.60 mol) in EtOAc (65.0 L) over a 15 min period.
After stirring the two-phase mixture for 10 min, the organic
phase was isolated and successively washed with aqueous 1 M
NaOH (19.5 L) and a saturated aqueous NaCl solution (19.5
L). The organic phase was concentrated (T < 40 °C) and
coevaporated twice with ethanol (2 × 13.0 L) to produce free-
base 12 as a light-yellow oil. Crude 17 (4.50 kg, 17.97 mol)
and Pd/C catalyst (650 g, slurry in ethanol) were successively
added to a solution of 12 in ethanol (65.0 L)* and acetic acid
(6.7 L). The reaction mixture was purged of oxygen by flushing
it with nitrogen for 15 min. Hydrogen was bubbled through
the vigorously stirred reaction mixture for 20 h (initially with
cooling to keep T < 30 °C). The reaction mixture was purged
of hydrogen by flushing with nitrogen for 15 min, and reaction
progress was determined with HPLC. If the amount of remain-
ing 12 was still above 0.5%, the reaction mixture was placed
under hydrogen for a further 20 h. Celite (1.3 kg) was added to
the nitrogen-flushed reaction mixture when HPLC analysis
indicated reaction completion. The reaction mixture was filtered,
the filter cake was washed with ethanol (3 × 12.5 L), and the
combined filtrate was concentrated (T < 40 °C). A methanolic
hydrochloric acid solution was prepared separately by adding
acetylchloride (3.7 L, 52.16 mol) over a 30 min period to
methanol (32.5 L) at 5–10 °C and stirring for an additional 30
min. The residue resulting from the concentrated filtrate was
dissolved in methanol (32.5 L) and added to the methanolic
hydrochloric acid solution over a 15 min period at 20–25 °C.
The combined methanolic solutions were washed with heptane
(3 × 32.5 L) and subsequently concentrated (T < 40 °C). The
residue was dissolved in MTBE (65.0 L) and washed succes-
sively with aqueous 1 M NaOH (2 × 32.5 L) and a saturated
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aqueous NaCl solution (32.5 L). The organic phase was isolated
and concentrated (T < 40 °C). The residue was dissolved in
heptane (16.7 L) and added to a solution of (+)-di-p-toluoyl-
L-tartaric acid (4.29 kg; 11.10 mol; exact amount should be
equimolar to HPLC-determined amount of 34 in the heptane
solution) in ethanol (8.4 L) over a 15 min period. The stirred
solution was seeded with 34*(+)DTTA that resulted in
precipitation of 34*(+)DTTA as a white solid. Heptane (25.1
L) was added, and the mixture was stirred for 30 min at ambient
temperature followed by 1 h at 0–5 °C. The precipitate was
isolated by centrifugation and washed with a heptane/ethanol
mixture (2 × 8.4 L; 5/1 v/v). After drying for 20 h under
vacuum (T < 40 °C), 34*(+)DTTA (10.01 kg, 8.74 mol; 98.4
area % by HPLC) was obtained as a white solid in 75% yield.
1H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J ) 8.2, 4H), 7.37
(d, J ) 8.2, 4H), 7.35-7.22 (m, 18H), 7.19-7.14 (m, 2H),
5.79 (s, 2H), 4.85 (d, J ) 11.3, 1H), 4.76 (d, J ) 10.9, 1H),
4.72 (d, J ) 11.3, 1H), 4.65 (d, J ) 11.9, 1H), 4.58 (d, J )
11.8, 1H), 4.47-4.36 (m, 3H), 3.67-3.57 (m, 2H), 3.57-3.50
(m, 1H), 3.40 (dd, J ) 8.2, 16.3, 2H), 3.27 (t, J ) 6.2, 2H),
3.18 (dd, J ) 4.5, 11.2, 1H), 2.89 (s, 2H), 2.79-2.69 (m, 1H),
2.56-2.48 (m, 2H), 2.38 (s, 6H), 2.25 (dd, J ) 9.9, 1H), 1.90
(s, 3H), 1.62 (dd, J ) 11.8, 30.6, 6H), 1.48 (d, J ) 1.9, 6H),
1.46-1.33 (m, 4H), 1.23-1.10 (m, 2H). Mp (DSC) 106 °C. *:
Absence of acetaldehyde in the used batch of ethanol should
be verified beforehand; otherwise significant formation of a
2,3,4,6-tetra-O-benzyl-N-ethyl-1-deoxynojirimycin byproduct is
possible.
N-[5-(Adamantan-1-yl-methoxy)-pentyl]-1-deoxynojiri-
mycin Methanesulfonic Acid Salt (5*MSA). A solution of
34*(+)DTTA (4.90 kg, 4.28 mol) in MTBE (49.0 L) was
washed successively with aqueous 1 M NaOH (1 × 24.5 L;
then 1 × 12.3 L) and saturated aqueous NaCl (12.3 L). The
organic phase was isolated, concentrated (T < 40 °C) to 5–10
L, and coevaporated with ethanol (3 × 25 L; or until MTBE
level <2%) to quantitatively produce 34 (3.24 kg; 4.28 mol)
as a colorless oil. The byproduct 36 contaminating 34 was
debenzoylated by adding aqueous 6 M NaOH (3.3 L) to a
cooled (0–10 °C) solution of 34 (3.24 kg; 4.28 mol) in ethanol
(63.7 L) over a 10 min period (T < 10 °C) and stirring the
resulting turbid reaction mixture for 2 h at 20–25 °C. The
reaction mixture was cooled (10 °C), aqueous 10.17 M HCl
(1.9 L) was added over a 10 min period (altering the pH to
5–8), and subsequently aqueous 2 M HCl (12.3 L) was added
at 10–30 °C to produce a clear solution with acidic pH.
Palladium on carbon (321 g; slurry in ethanol) was added to
the solution, and oxygen was purged by flushing the mixture
for 15 min with nitrogen. Hydrogen was bubbled through the
vigorously stirred reaction mixture for 6–12 h (initially with
cooling to keep T < 30 °C). The reaction mixture was purged
of hydrogen by flushing with nitrogen for 15 min, and reaction
progress was determined by HPLC analysis. If the amount of
34 and partially debenzylated intermediates was higher than 1
area % compared to generated toluene, the reaction mixture
was placed under hydrogen for further 2–4 h periods until
complete. When HPLC analysis indicated reaction completion,
the mixture was filtered, and the Pd/C filter cake was washed
with ethanol (3 × 7.3 L). Ecosorb (172 g) was added to the
combined filtrate, whereupon the mixture was stirred for 1 h at
ambient temperature. The mixture was filtered, and the Ecosorb
filter cake was washed with ethanol (3 × 7.3 L). The combined
filtrate was concentrated (T < 50 °C) to a volume of 5–10 L
and coevaporated with ethanol (3 × 32 L). The remaining
mixture was diluted to a volume of 34.3 L with ethanol (water
content was verified to be KF < 2%), and 7 M methanolic
NH3 (3.9–6.9 L) was added to the solution until the pH was
adjusted to 8–9.5. The mixture was concentrated (T < 45 °C)
to 5–10 L and coevaporated with CH2Cl2 (5 × 122.5 L; or until
ethanol level <2%). The remaining mixture was diluted to a
total volume of 24.5 L with CH2Cl2, and the precipitated salts
were removed by filtration. The precipitate was washed with
CH2Cl2 (3 × 2.5 L) and the combined filtrate concentrated (T
< 45 °C) to 5–10 L. (The residue could at this stage be further
concentrated and degassed under vacuum for 4 h at T < 45 °C
to provide 5 as an off-white hygroscopic foam.) 1H NMR (400
MHz, CD3OD) δ 3.96 (d, J ) 12.0, 1H), 3.88 (dd, J ) 2.6,
12.2, 1H), 3.64-3.53 (m, 1H), 3.47 (dd, J ) 9.5, 1H), 3.40 (t,
J ) 6.2, 2H), 3.28-3.14 (m, 2H), 3.04 (s, 1H), 2.97 (s, 2H),
2.87 (s, 1H), 2.54 (s, 2H), 1.95 (s, 3H), 1.76 (d, J ) 12.1, 3H),
1.72-1.58 (m, 7H), 1.56 (d, J ) 2.1, 6H), 1.46-1.35 (m, 2H).
The solution of 5 in CH2Cl2 was coevaporated with isopropanol
(3 × 24.5 L; or until CH2Cl2 level <1%). Any remaining
particulate was removed by a polish filtration step followed by
additional rinsing with isopropanol (3 × 2.4 L) of the reaction
vessel and filter. At ambient temperature a solution of meth-
anesulfonic acid (473 g, 4.92 mol) in isopropanol (3.9 L + 2.45
rinse) was added to the isopropanol solution of 5. The solution
was heated to 70 °C and slowly cooled to ambient temperature
over a 4–8 h period during which at ∼50 °C seed crystals of
5*MSA (5 g) were added. The solution slowly turned turbid,
and 5*MSA precipitated as a coarse white solid. The mixture
was stirred for 16 h at ambient temperature and filtered, and
the collected solids were washed with isopropanol (3 × 2.4
L). Drying of the product under vacuum (T < 40 °C) provided
5*MSA (1.38 kg, 2.80 mol; 99.9 area % by HPLC) as a stable
white solid in 65% yield. 1H NMR (500 MHz, D2O, COSY) δ
4.06 (d, J ) 13.1, 1H, H-6a), 3.92 (dd, J ) 2.5, 13.2, 1H, H-6b),
3.78 (td, J ) 4.9, 11.0, 1H, H-2), 3.63 (dd, J ) 9.9, 1H, H-4),
3.52 (dd, J ) 4.9, 12.2, 1H H-1a), 3.47 (dd, J ) 4.8, 14.2, 1H,
H-3), 3.44 (t, J ) 6.5, 2H, OCH2-5′ pentyl), 3.37-3.28 (m,
1H, NCHH-1′ pentyl), 3.24-3.16 (m, 1H, NCHH-1′ pentyl),
3.14 (m, 1H, H-5), 3.04 (m, 1H, H-1b), 3.02 (s, 2H, OCH2-
adamantane), 2.75 (s, 3H, CH3 MsOH), 1.92 (br s, 3H, 3 ×
CH adamantane), 1.81-1.56 (m, 10H, CH2-4′ pentyl, 3 × CH2
adamantane, CH2-2′ pentyl), 1.50 (d, J ) 1.5, 6H, 3 × CH2
adamantane), 1.46-1.33 (m, 2H, CH2-3′ pentyl).13C NMR (126
MHz, D2O, HSQC) δ 81.68 (OCH2-adamantane), 75.90 (C-3),
71.42 (OCH2-5′ pentyl), 67.05 (C-4), 65.96 (C-2), 65.33 (C-
5), 53.94 (C-6), 53.00 (C-1), 52.69 (NCH2-1′ pentyl), 39.31 (3
× CH2 adamantane), 38.50 (CH3 MsOH), 36.91 (3 × CH2
adamantane), 33.72 (Cq adamantane), 28.23 (CH2-4′ pentyl),
28.09 (3 × CH adamantane), 22.67(CH2-3′ pentyl), 22.33(CH2-
2′ pentyl). Mp (DSC) 178 °C. [R]20 ) –3.2 (c 1.02, H2O).
D
Found C: 55.9; H: 8.9; N: 2.8, calculated for C23H43NO8S )
C: 56.0; H: 8.8; N: 2.8.
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Acknowledgment
and HSQC for 5*MSA) and DSC (for 12*HCl, 34*(+)DTTA,
5, and 5*MSA) spectra for all reported compounds and purity
information for 5*MSA. This material is available free of charge
via the Internet at http://pubs.acs.org.
We thank Edward van Wezel for assistance in the coordina-
tion on this project and Macrozyme BV for funding of the
project.
Supporting Information Available
HPLC and GC in-process control details and chromatograms
for all reactions; 1H NMR (for all compounds; 13C-APT, COSY
Received for review December 21, 2007.
OP700295X
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