Synthesis and chemistry of noeuromycin and isofagomine analogues

Article abstract of DOI:10.1081/CAR-200030070

Several N-substituted analogues of noeuromycin ((2RS,3S,4R,5R)-2,3,4- trihydroxy-5-hydroxymethylpiperidine) and isofagomine ((3R,4R,5R)-3,4-dihydroxy- 5-hydroxy-methylpiperidine) were synthesised. The isofagomine analogues (3RS,4RS,5RS)-N-(2-phosphonoethyl)-3,4-dihydroxy-5-hydroxymethyl-piperidine, (3SR,4SR,5RS)-N-(2-phosphonoethyl)-3,4-dihydroxy-5-hydroxy-methylpiperidine, and (3R,4R,5R)-N-(10-chloro-9-anthracenemethyl)-3,4-dihydroxy-5-hydroxy- methylpiperidine were synthesised by direct alkylation of the corresponding azasugar. N-Substituted noeuromycin derivatives could not be made in this straightforward manner, but were made by modification of a synthesis intermediate. By this method (2RS,3S,4R,5R)-N-(4-methoxyphenyl)-2,3,4- trihydroxy-5-hydroxymethylpiperidine and (2RS,3S,4R,5R)-N-nonyl-2,3,4- trihydroxy-5-hydroxymethylpiperidine were synthesised. The stability of noeuromycin was studied and was found to depend on stereochemistry and pH. The L-fuco isomer ((2RS,3R,4R,5R)-2,3,4-trihydroxy-5-methylpiperidine) was observed to undergo a particularly facile Amadori rearrangement at neutral pH to the 3-ketopiperidine. A noeuromycin analogue, that could not undergo the Amadori rearrangement, was synthesised. Copyright

Full text of DOI:10.1081/CAR-200030070

This article was downloaded by: [State University NY Binghamton]
On: 16 April 2013, At: 13:41
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
Synthesis and Chemistry of Noeuromycin
and Isofagomine Analogues
Huizhen Liu a , Vinni H. Lillelund a , Jens Andersch a , Xifu Liang a &
Mikael Bols a
a Department of Chemistry, University of Aarhus, Langelandsgade
140, DK‐8000, Aarhus, Denmark
Version of record first published: 17 Aug 2006.
To cite this article: Huizhen Liu , Vinni H. Lillelund , Jens Andersch , Xifu Liang & Mikael Bols (2004):
Synthesis and Chemistry of Noeuromycin and Isofagomine Analogues, Journal of Carbohydrate
Chemistry, 23:4, 223-238
To link to this article: http://dx.doi.org/10.1081/CAR-200030070
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation
that the contents will be complete or accurate or up to date. The accuracy of any
instructions, formulae, and drug doses should be independently verified with primary
sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
demand, or costs or damages whatsoever or howsoever caused arising directly or
indirectly in connection with or arising out of the use of this material.

JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 4, pp. 223–238, 2004
Synthesis and Chemistry of Noeuromycin and
Isofagomine Analogues
Huizhen Liu, Vinni H. Lillelund, Jens Andersch, Xifu Liang,
*
and Mikael Bols
Department of Chemistry, University of Aarhus, Aarhus, Denmark
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 233
A. N-Substitution of 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
B. N-Substitution of 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
C. Stability Study with 1 . . . . . . . . . . . . . . . . . . . . . . . . . . 235
D. Synthesis of 31; a 3-C-Methyl Analogue of 1 . . . . . . . . . . . . 236
E. Glycosidase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . 238
III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
A. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
B. Ki Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
ACKNOWLEDGMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
*
Correspondence: Mikael Bols, Department of Chemistry, University of Aarhus, Langelandsgade
140, DK-8000, Aarhus, Denmark; Fax: þ4586196199; E-mail: mb@chem.au.dk.
223
DOI: 10.1081/CAR-200030070
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

224
Liu et al.
ABSTRACT
Several N-substituted analogues of noeuromycin ((2RS,3S,4R,5R)-2,3,4-trihydroxy-
5-hydroxymethylpiperidine) and isofagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxy-
methylpiperidine) were synthesised. The isofagomine analogues (3RS,4RS,5RS)-
N-(2-phosphonoethyl)-3,4-dihydroxy-5-hydroxymethyl-piperidine, (3SR,4SR,5RS)-N-
(2-phosphonoethyl)-3,4-dihydroxy-5-hydroxy-methylpiperidine, and (3R,4R,5R)-N-
(10-chloro-9-anthracenemethyl)-3,4-dihydroxy-5-hydroxy-methylpiperidine were syn-
thesised by direct alkylation of the corresponding azasugar. N-Substituted noeuro-
mycin derivatives could not be made in this straightforward manner, but were made
by modification of a synthesis intermediate. By this method (2RS,3S,4R,5R)-N-(4-
methoxyphenyl)-2,3,4-trihydroxy-5-hydroxymethylpiperidine and (2RS,3S,4R,5R)-N-
nonyl-2,3,4-trihydroxy-5-hydroxymethylpiperidine were synthesised. The stability of
noeuromycin was studied and was found to depend on stereochemistry and pH. The
L-fuco isomer ((2RS,3R,4R,5R)-2,3,4-trihydroxy-5-methylpiperidine) was observed to
undergo a particularly facile Amadori rearrangement at neutral pH to the 3-ketopiper-
idine. A noeuromycin analogue, that could not undergo the Amadori rearrangement,
was synthesised.
Key Words: Noeuromycin; Isofagomine; Glycoside metabolism; Gaucher’s disease;
Glycosidase inhibitor; 1-N-Iminosugar; 1-Azasugar; N-Alkyl.
INTRODUCTION
Selective inhibition of glycoside metabolism has very large potential applications.
Not only are several glycosidase inhibitors currently used in treatment of diabetes,
but also a number of others are under clinical investigation to treat a variety of diseases
and disorders such as HIV, Gaucher’s disease, hepatitis, and cancer.[1] Success in this
area have been difficult however because most of the potent glycosidase inhibitors are aza-
sugars (imino- or aminosugars) of various kinds[2] that are very polar and difficult to
absorb making them unsuitable drugs. This problem is frequently overcome by attaching
lipophilic groups, such as a butyl group to the nitrogen of 1-deoxynojirimycin,[3,4] though
these modifications may compromise enzyme binding.
Noeuromycin (1)[5] and isofagomine (2)[6] are members of a new type of azasugars
with high biological activity (Fig. 1). However in order to use these potent enzyme
ligands as drugs it may be necessary to improve lipophilicity, stability, or specificity.
Figure 1. Structure of noeuromycin and isofagomine.

Noeuromycin and Isofagomine Analogues
225
In this paper, we have investigated various structural modifications that improve some of
these properties in 1 or 2.
RESULTS AND DISCUSSION
N-Substitution of 2
Isofagomine (2) and noeuromycin (1) are potent glycogen phosphorylase inhibitors.[1]
Binding of the inhibitors to the enzyme is presumably associated with simultaneous
binding of phosphate since the crystal structure of 1 shows the presence of a phosphate
molecule coordinated to N1.[1] Joining the phosphate and azasugar together, possibly
through a ethylphosphonate, might improve binding and specificity towards this
enzyme. For this reason, we investigated the incorporation of this group at N1 in 2.
Reaction of (+)-2 with diethyl 2-bromoethylphosphonate in EtOH in the presence of
anhydrous K2CO3 gave the alkylation product 3 in 68% yield (Sch. 1). By the same
Scheme 1.

226
Liu et al.
procedure the stereoisomer (+)-5 was converted to 6 in 40% yield. The two esters 3 and 6
were hydrolysed by hydrochloric acid to form the phosphonic acids 4 and 7 in 85% and
96% yield, respectively. A similar modification of 2 was made in the fluorescence labelled
inhibitor 8 (Sch. 1). Reductive amination of 2 with 10-chloro-9-anthralaldehyde and
sodium cyanoborohydride gave the product 8 in modest yield.
N-Substitution of 1
Derivatives of 1 with lipophilic groups at nitrogen could convey better absorption
characteristics to this very polar compound. We have synthesised two such derivatives.
Modification of the nitrogen of 1 could not be carried out on 1 for obvious reasons. Reduc-
tive amination reaction similar to the synthesis of 8 results in destruction of the hemiami-
nal function, while an alkylation reaction such as that used in the synthesis of 3 and 6 is
problematic due to the poor nucleophilicity of the nitrogen atom. Substitution of the nitro-
gen therefore had to be carried out at an earlier stage in the synthesis, and we have used
two different protocols. The synthesis of the N-p-methoxybenzyl modified compound 14
was carried out from 9 (Sch. 2), which is obtained from levoglucosan.[7] Treatment of 9
with p-methoxybenzylamine and NaCNBH3 gave the crude secondary amine 10, which
Scheme 2.

Noeuromycin and Isofagomine Analogues
227
was protected with a Boc group to provide 11 in 44% yield. Cleavage of the diol in 11 with
sodium periodate in MeOH/H2O gave the pyranose 12 in 58% yield. Hydrogenolysis of
the benzyl group gave 13 in 52% yield, which after deprotection of the Boc group with
TFA gave 14 in 85% yield.
The N-nonyl analogue 17 was made in a different manner (Sch. 3). The nitro deriva-
tive 15, which is obtained from D-arabinose,[8] was reduced to the amine by catalytic
hydrogenation. Reductive amination with pelargonaldehyde and NaCNBH3 followed by
Boc protection afforded the Boc derivative 16 in 44% yield. Hydrogenolysis of the
benzyl group followed by treatment with hydrochloric acid gave 17 as a hydrochloride
in 67% yield.
Stability Study with 1
In his classical work Paulsen found that nojirimycin and similar compounds[9] under-
went pH dependant Amadori rearrangement. Similar reactions could be expected to occur
in compound 1 and analogues, but pH dependence might be different due to the different
base strength of the amine in 1 and nojirimycin. Noeuromycin (1) was found to be stable
for a month when kept as the hydrochloride. However at neutral pH the compound began
to rearrange: in D2O at pD 6.8, as maintained by a buffer, the compound was converted to
the hydrated ketone 18 over 7 hr (Sch. 4). The incorporation of deuterium is stereospecific,
but its configuration not determined. The rearrangement must occur by elimination of
water from 1 to imine 21, rearrangement to 22, and addition of D2O to 22 to reach 18.
Even faster, the fuco isomer 19 rearranged in D2O to ketone hydrate 20 again with stereo-
specific incorporation of deuterium. The same rearrangement has recently been observed
by Nishimura’s group starting from various 2-amino derivatives that presumably hydro-
lyse to 19.[10]
Scheme 3.

228
Liu et al.
Scheme 4.
Synthesis of 31; a 3-C-Methyl Analogue of 1
The instability of 1 is a possible problem that limits its suitability as a drug. We there-
fore considered ways to prevent the rearrangement of 1. A C-3 methyl group would
prevent the rearrangement of 21 to 22 and might not affect inhibition significantly. We
therefore synthesised a C-3 methyl analogue starting from the known D-arabinose deriva-
tive 23. Compound 23 as well as its C-2 epimer is readily obtained from D-arabinose
by stereoselective addition of organometallic reagents to the corresponding ketone.[11]
Removal of the acetonide group in 23 was accomplished with aqueous TFA to give the
triol 24 in 98% yield (Sch. 5). Oxidation of 24 with dibutyltin oxide and bromine gave
a crude product containing mainly 25. The reaction is not as regioselective as the corres-
ponding oxidation of benzyl b-D-arabinoside[8] and oxidation at C-3 is also occurring. The
product is however difficult to analyse and separate due to the presence of intermolecular
hemiacetal formation. Treatment of this product with nitromethane and triethylamine gave
a mixture of three different isomers 26, 27, and 28 in a combined yield of 56%. By chrom-
atography the individual compounds were isolated in 11%, 18%, and 10% yield, respect-
ively. The absolute stereochemistries at the newly formed tertiary centers were not
determined. Compound 26 was acetylated by treatment with Ac2O and scandium triflate,
and then treated with NaBH4 which gave b-elimination and conjugate reduction of the

Noeuromycin and Isofagomine Analogues
229
Scheme 5.
resulting nitroalkene (Sch. 6). Complete deacetylation with NaOMe in methanol gave diol
29 in 37% yield. Catalytic hydrogenation of the nitro group followed by Boc protection
gave the Boc protected amine 30 in 81% yield. Finally hydrogenolysis of the benzyl
group and removal of the Boc group with hydrochloric acid gave the 3-C methyl analogue
of noeuromycin 31 in 61% yield.
Scheme 6.

230
Liu et al.
Glycosidase Inhibition
The new analogues were investigated for inhibition of a- and b-glucosidases
(Table 1). (Solutions of 18 were prepared by incubating 1 in phosphate buffer overnight.)
The compounds 14, 18, and 31 were found to be 100–1000 times weaker than 1. In the
case of 14, it is clear that the bulky p-methoxybenzyl group disrupts the efficient
binding of the azasugar moiety. The weaker binding of 18 and 31 suggests that there is
no space for an axial substituent in the 3-position of the azasugar. The isofagomine ana-
logue 8 inhibited almond b-glucosidase with a Ki of 24 mM. The phosphonates 4 and 7
were found to be very poor inhibitors of glycogen phophorylase.
In summary we have established a number of synthetic methods that can be used to
prepare analogues of 1 and 2, which will be important for obtaining pharmaceutically
acceptable derivatives of these compounds. The stability of 1 has also been investigated
and it has been found to undergo Amadori rearrangement at neutral pH.
EXPERIMENTAL
General
1H (200 or 400 MHz) and 13C NMR (50 MHz) spectra were recorded on a Varian
Gemini 200 or on a Varian 400 spectrometer using the signal of the solvent as reference.
Table 1. Ki values, in mM, of some noeuromycin analogues towards various glycosidases.
Yeast
a-glu’ase
Almond
b-glu’ase
Yeast
isomaltase
Compound
Structure
1
0.022
3.0
0.069
45.0
0.025
—
14
18
31
8.5
7.5
3.0
5.3
110.0
—

Noeuromycin and Isofagomine Analogues
231
Chemical shifts were expressed in d values (ppm). Where a compound is present as a
mixture of anomers NMR signals may be assigned to integrate to a fraction of a proton
reflecting the fraction present of that anomer. Optical rotations were measured with a
Perkin–Elmer Model 241 polarimeter. The mass spectra and the high-resolution mass
spectra were taken by a Micromass LC-TOF. For Ki determination a Milton Roy
Genesys 5 spectrophotometer was used. Unless otherwise stated, all reactions were
performed under a normal atmosphere and pressure. THF was distilled prior to use
from sodium and benzophenone, and CH2Cl2 was distilled with CaH2. The acetone
˚
used was HPLC grade, and DMF was dried using 4 A molecular sieves. Other solvents
and reagents from stock or purchased were used as received. When dry glassware was
necessary it was heated in an oven at a temperature above 1258C for atleast 6 hr, then
quickly assembled and cooled under a stream of dry inert gas. Flash column chromato-
graphy was performed on silica gel (Merck silica gel 60, 230–400 mesh). TLC plates
were viewed under an ultraviolet lamp or treated with one of the reagents listed here:
(i) 3 g of KMnO4, 20 g of K2CO3, 5 mL of NaOH (5%, aq.) and 300 mL of H2O; (ii)
10 g of Ce(IV)sulphate, 15 g of (NH4)2MoO4 in 1 L 10% H2SO4. Ion-exchange chromato-
graphy was performed with Amberlite IR-120, Hþ and eluted with 2.5% NH3 (aq.). Rotary
evaporation was performed under reduced pressure (minimum 25 mbar) and maximum
temperature 408C.
(+)-2-[3,4-trans-4,5-trans)-5-Hydroxymethyl-3,4-dihydroxypiperidine-1-yl]ethyl-
phosphono acid diethylester (3). A mixture of (+)-isofagomine (2, 30 mg, 0.2 mmol),
diethyl 2-bromoethylphosphonate (0.12 mL, 0.6 mmol), and K2CO3 (138 mg, 1.0 mmol)
in 3 mL of EtOH was heated reflux for 20 hr. After the reaction, the solid was filtered
off and the obtained solution was concentrated. The residue was purified by chromato-
1
graphy (CHCl3/EtOH, 3 : 1) to provide product 3 (43 mg, 68%) as an oil. H NMR
0
0
0
(200 MHz, CD3OD): d 3.97 (q, 4H, J 7.5 Hz, CH2CH3), 3.70 (dd, 1H, J5 a,5 b11.3, J5,5 a
3.80, H-50a), 3.57–3.26 (m, 3H, H-3, H-4, H-50b), 3.04–2.80 (m, 2H, H-2eq, H-6eq),
2.64–2.40 (m, 2H, H-2ax, H-6ax), 2.00–1.70 (m, 4H, H-10a, H-10b, H-100a, H-1b00),
1.60 (m, 1H, H-5), 1.20 (t, 6H, CH3). 13C NMR (50 MHz, CD3OD): d 74.1 (C-4), 71.3
(C-3), 61.4 (d, 2C, CH2CH3), 60.8 (C-50), 57.4 (C-2), 54.0 (C-6), 50.3 (NCH2), 43.3
(C-5), 22.0 (d, PCH2), 14.9 (d, 2C, CH3). HRMS (ES): calcd for C12H26NO6P þ Naþ
334.1395, found 334.1389.
(+)-2-[(3,4-trans-4,5-cis)-5-Hydroxymethyl-3,4-dihydroxypiperidine-1-yl]-ethyl-
phosphono acid diethylester (6). Compound 5 (25 mg, 0.17 mmol) was processed as
described above for 2. Chromatography (CHCl3/EtOH, 3 : 1) provided 6 (20 mg, 40%)
1
as an oil. H NMR (200 MHz, CD3OD): d 4.22–3.90 (m, 4H, CH2CH3), 3.70–3.45
(m, 4H, H-3, H-4, H-50a, H-50b), 2.6–2.27 (m, 6H, H-2ax, H-2eq, H-6ax, H-6eq, H-100a,
H-100b), 2.10–1.85 (m, 3H, H-10a, H-10b, H-5), 1,23 (t, 6H, CH2CH3, J ¼ 7.5 Hz).
13C NMR (50 MHz, CD3OD): d 69.6 (C-4), 68.4 (C-3), 61.4 (d, 2C, CH2 of Et), 60.7
(C-50), 60.0 (C-2), 54.1 (C-6), 50.4 (NCH2CH2), 39.1 (C-5), 22.0 (d, PCH2), 14.8 (d, 2C,
CH3). HRMS (ES): calcd for C12H26NO6P þ Hþ 312.1576, found 312.1579.
(+)-2-[(3,4-trans-4,5-trans)-5-Hydroxymethyl-3,4-dihydroxypiperidine-1-yl]-ethyl-
phosphono acid (4). A solution of 3 (36 mg, 0.12 mmol) in 2 mL of 6 N HCl was heated
reflux for 8 hr. Concentration of the reaction solution provided 4 (25 mg, 85%) as a colour-
less oil. 1H NMR (400 MHz, D2O): d 3.80–3.50 (m, 5H, H-2eq, H-3, H-50a, H-50b, H-6eq),
3.50–3.23 (m, 3H, H-4, H-2ax, H-6ax), 3.04–2.70 (m, 2H, H-10a, H-10b), 2.26-1.97
(m, 2H, H-100a, H-100b), 1.88 (m, 1H, H-5). 13C NMR(50 MHz, D2O): d 70.3 (C-4), 68.1

232
Liu et al.
(C-3), 58.3 (C-50), 53.9 (C-2), 52.7 (NCH2CH2), 51.8 (C-6), 40.5 (C-5), 22.4 (d, PCH2).
HRMS (ES): calcd for C8H18NO6P þ Hþ 256.0949, found 256.0946.
(+)-2-[(3,4-trans-4,5-cis)-5-Hydroxymethyl-3,4-dihydroxypiperidine-1-yl]-ethyl-
phosphono acid diethylester (7). Compound 6 (20 mg, 0.064 mmol) was processed as
described above for 3 to provide 7 as a colourless oil (18 mg, 96%). 1H NMR
(200 MHz, D2O): d 4.08 (bs, 1H, H-3), 3.95 (bs, 1H, H-4), 3.75–3.20 (m, 7H, H-10a,
H-10b, H-2ax, H-2eq, H-50a, H-50b, H-6eq), 2.97 (t, 1H, H-6ax), 2.42 (m, 1H, H-5),
2.28–1.96 (m, 2H, H-100a, H-100b). 13C NMR (50 MHz, D2O): d 65.7 (C-4), 64.1 (C-3),
59.6 (C-50), 52.2 (C-2), 51.9 (NCH2), 48.6 (C-6), 37.4 (C-5), 22.3 (d, PCH2). HRMS
(ES): calcd for C8H18NO6P þ Hþ 256.0949, found 256.0948.
N-(10-Chloro-9-anthracenemethyl] isofagomine (8). A mixture of isofagomine
(2, 22 mg, 0.15 mmol) and 10-chloro-9-anthralaldehyde (36 mg, 0.15 mmol) in methanol
(15 mL) and CHCl3 (1 mL) was heated to reflux until a homogeneous solution was
formed. Then NaCNBH3 (18 mg, 0.29 mmol) was added. After 10 min HOAc (0.1 mL)
was added, and the mixture heated to about 608C for 4 hr and furthermore stirred at
room temperature for 24 hr. The solution was evaporated and the residue chromatographed
with CHCl3 until the lipophilic compounds were eluted (7 mg 20% 10-chloro-9-anthral-
aldehyde and 16 mg 44% 10-chloro-9-anthracenemethanol) and then changed to CHCl3/
MeOH (8 : 1) giving 20 mg (31%) of N-(10-chloro-9-anthracenemethyl)isofagomine (8) as
yellow acetic acid salt (RF 0.35, CHCl3/MeOH 8 : 1). Evaporation of a solution of the
1
acetic acid salt in HCl (1 mL, 1.0 M) provided the corresponding hydrochloride. H
NMR (200 MHz, D2O): d 8.08 (d, 2H, J 8.8 Hz), 7.90 (d, 2H, J 8.8 Hz), 7.46 (m, 4H),
4.95 (s, 2H, ArCH2), 3.33–3.71 (m, 6H, H-2eq, H-6eq, H-3, H-4, H-50a, H-50b), 3.07
(m, 2H, H-2ax, H-6ax), 1.74 (m, 1H, H-5). UV (nm): 399 (3.93), 378 (3.96), 360
(3.76), 344ꢀ (3.46), 327ꢀ (3.14), 251 (.4.32), 220 (4.13). HRMS (ES): calcd for
M þ Hþ 372.1366, found 372.1364.
2-[(N-tert-Butylcarbonyl)-(p-methoxyphenyl)amino]methyl-4-O-benzyl-2-deoxy-
D-mannitol (11). 9 (0.23 g, 0.81 mmol) and p-methoxybenzylamine (0.2 mL, 1.5 mmol)
in H2O (0.3 mL) was heated to 608C. After the mixture became homogeneous, the
heating was continued for 3 hr at the same temperature. Then methanol (1 mL) was
added, and the obtained solution cooled to room temperature. Sodium borohydride
(0.06 g, 1.6 mmol) was added slowly in portion so that the temperature did not exceed
308C. After stirring for 18 hr at room temperature, the reaction solution was concentrated
in vacuo, redissolved in methanol (2 mL) and acidified to pH 2 with 3 M HCl followed by
concentration twice with methanol (2 ꢁ 2 mL). The residue was taken up to 2 mL of
methanol, and the obtained mixture was filtered. Concentration of the filtrate provided a
crude product of 10. (1H NMR (200 MHz, CD3OD): d 7.33–7.25 (m, 5H, Ph), 7.16
(d, 2H, Ar, J 8.5 Hz), 6.82 (d, 2H, Ar), 4.67 (d, 1H, PhCH2, J 11.0 Hz), 4.46 (d, 1H,
PhCH2), 3.93–3.53 (m, 9H, H-20a, H-20b, H-3, H-4, H-5, H-6a, H-6b, NCH2Ar), 3.65
(s, 3H, OCH3), 3.16 (dd, 1H, J1a,2 7.0 Hz, J1a,1b 12.5 Hz, H-1a), 3.01 (dd, 1H, J1b,2
7.0 Hz, H-1b), 2.28 (m, 1H, H-2). 13C NMR (50 MHz, CD3OD): d 161.5, 138.7, 131.4,
132.6, 131.6, 129.5, 129.0 (Ph, Ar), 126.1, 124.0, 115.3 (Ar), 80.2 (C-4), 73.7, 73.2,
71.5 (C-3, C-5, PhCH2), 64.0, 62.0 (C-6, C-20), 55.8 (OCH3), 51.7 (PhCH2N), 43.8
(C-1), 42.3 (C-2). MS (ES): m/z 406.1 (M þ H)). To the crude 10 (0.45 g, 0.81 mmol)
in a solution of 1,4-dioxane/H2O (1 : 1, 30 mL) was added Na2CO3 (0.26 g, 2.5 mmol).
Subsequently a solution of (Boc)2O (0.47 g, 2.2 mmol) was added dropwise to the
mixture during 2 min at room temperature. After stirring for 18 hr at the same temperature,

Noeuromycin and Isofagomine Analogues
233
the reaction mixture was concentrated in vacuo. The residue was taken up into ethyl
acetate and washed twice with H2O (30 mL). The organic phase was dried over MgSO4,
concentrated in vacuo and purified by chromatography firstly with ethyl acetate, and
1
then with ethyl acetate/MeOH 99 : 1 to give 11 (0.18 g, 44% over two steps). H NMR
(200 MHz, CDCl3): d 7.34–7.25 (m, 5H, Ph), 7.00 (d, 2H, J 7.5 Hz, Ar), 6.74 (d, 2H,
Ar), 4.64 (d, 1H, J 11.5 Hz, PhCH2), 4.51 (d, 1H, PhCH2), 4.36 (d, 1H, J 15.5 Hz,
ArCH2), 4.07 (d, 1H, ArCH2), 3.86–3.75 (m, 3H, H-3, H-6a, H-6b), 3.71 (s, 3H,
CH3O), 3.65–3.41 (m, 5H, H-1a, H-20a, H-20b, H-4, H-5), 3.01 (dd, 1H, J1b,2 5.0 Hz,
J1a,1b 14.5 Hz, H-1b), 1.80 (m, 1H, H-2), 1.40 (s, 9H, (CH3)3C). 13C NMR (50 MHz,
CDCl3): d 158.9 (Ar), 157.2 (CO), 138.0, 129.8, 128.4–127.8 (Ph, Ar), 114.0 (Ar), 80.9
((CH3)3CO), 79.1 (C-4), 73.2 (PhCH2), 71.7, 70.4 (C-3, C-5), 63.6, 59.5 (C-20, C-6),
55.3 (OCH3), 50.5 (PhCH2N), 44.7 (C-5), 41.2 (C-2), 28.4 ((CH3)3C). HRMS (ES):
calcd for C27H39NO8 þ Na 528.2573, found 528.2587.
3-O-Benzyl-4-[N-(tert-butyloxycarbonyl)-N-(p-methoxybenzyl)amino]methyl-4-
deoxy-a/b-D-arabino-pyranose (12). To a solution of 11 (101 mg, 0.20 mmol) in H2O
(0.6 mL) was added dropwise a solution of NaIO4 (214 mg, 1.0 mmol) in H2O (2 mL)
over 10 min and subsequently MeOH (1 mL) was added. The obtained mixture was
stirred at 458C for 3 hr and then filtered. The solution was concentrated in vacuo. The
residue was taken up into ethyl acetate/EtOH (1 : 1, 3 mL), and undissolved solid filtered
off. The obtained solution was concentrated in vacuo and purified by chromatography
(ethyl acetate/CHCl3, 1 : 5) to give 12 as a yellow oil (54 mg, 58%). 1H NMR (200 MHz,
CDCl3): d 7.42–7.16 (m, 5H, Ph), 7.08 (d, 2H, J 8.5 Hz, Ar), 6.80 (d, 2H, Ar), 5.37 (bs,
1H, H-1), 5.12–4.79 (m, 1H, OH), 4.70 (m, 4H, CH2Ph, CH2Ar), 4.00–3.20 (m, 6H, H-2,
H-3, H-5a, H-5b, H-40a, H-40b), 3.70 (s, 3H, CH3), 2.53 (m, 1H, H-4), 1.41 (s, 9H,
(CH3)3C). HRMS (ES): calcd for C26H35NO7 þ Naþ 496.2311, found 496.2307.
4-[N-(tert-Butyloxycarbonyl)-N-(p-methoxybenzyl)amino]methyl-4-deoxy-a/b-
D-arabino-pyranose (13). The mixture of 12 (50 mg, 0.11 mmol) and Pd/C (10%, 20 mg)
in CH3COOH (2 mL) was hydrogenolysed for 18 hr at room temperature. After the reac-
tion, the mixture was filtered and the solution was concentrated in vacuo. The residue was
purified by chromatography (CHCl3/CH3OH, 20 : 1) to give 13 (21 mg, 52%) as an oil. 1H
NMR (200 MHz, CDCl3): d 7.08 (d, 2H, J 8.5 Hz, Ar), 6.80 (d, 2H, Ar), 5.48 (d, 0.5 H, J1,2
8.0 Hz, H-1a), 5.42 (d, 0.5H, J1,2 1.5 Hz, H-1b), 4.93 (s, 0.5H, OH), 4.85 (d, 0.5H, OH),
4.70 (s, 0.5H, OH), 4.56 (d, 1H, J 15.0 Hz, CH2Ar), 3.98 (d, 1H, J 15.0 Hz, CH2Ar), 3.80–
3.64 (m, 2H, H-3, H-4), 3.71 (s, 3H, OCH3), 3.60–3.40 (m, 4.5H, H-3, H-5a, H-5b, H-40a,
H-40ba), 3.22 (dd, 0.5H, J4 a,4 b12.5, J4,4 b 3.5 Hz, H-40bb), 2.54 (m, 1H, H-4), 1.41 (s, 9H,
(CH3)3C). 13C NMR (50 MHz, CDCl3): d 128.9–114.3 (Ar), 95.2 (C-1a), 92.7 (C-1b),
82.0, 81.5 ((CH3)3C), 70.0, 67.5, 67.0 (C-3, C-4), 63.4 (C-5a), 56.8 (C-5b), 55.5
(OCH3), 50.8 (CH2Ar), 43.5, 42.7 (C-40), 34.6, 33.7 (C-4), 28.5 ((CH3)3C). HRMS
(ES): calcd for C19H29NO7 þ Naþ 406.1842, found 406.1845.
0
0
0
(2R/S,3S,4R,5R)-1-(p-Methoxybenzyl)-5-(hydroxymethyl)-2,3,4-trihydroxy-
piperidine tri-fluoroacetate (14). Compound 13 (10 mg, 0.026 mmol) was dissolved in
CF3COOH (0.5 mL). The obtained solution was stirred for 5 min and concentrated in
1
vacuo to furnish 14 (8.8 mg, 85%) as a yellow oil. H NMR (200 MHz, D2O): d 7.40–
7.04 (d, 4H, J 8.6 Hz, Ar), 5.08 (d, 0.33H, J2,3 2.9 Hz, H-2b), 4.88 (d, 0.67H, J2,3
7.7 Hz, H-2a), 4.20 (bs, 2H, CH2Ar), 4.10–3.48 (m, 3H, H-3, H-4, H-6a), 3.83 (s, 3H,
CH3O), 3.40–3.00 (m, 3H, H-50a, H-50b, H-6b), 2.37 (m, 1H, H-5). HRMS (ES): calcd
for (C14H21NO5 2 H2O) þ Hþ 266.1392, found 266.1395.

234
Liu et al.
Benzyl 4-(N-tert-butyloxycarbonyl-N-nonyl)methyl-4-deoxy-b-D-arabino-pyra-
noside (16). A mixture of 15 (110 mg, 0.39 mmol), Et3N and Pd/C (10%, 20 mg) in
EtOH (10 mL) was hydrogenated under 1 atm at room temperature for 2 hr. The catalyst
was filtered off and the solution was concentrated in vacuo. The residue was dissolved
in CH3OH (2.5 mL). The pH value of the solution was adjusted to about 6 by using CH3-
CO2H. Then, to the solution were added pelargonaldehyde (78 mg, 0.55 mmol) in CH3OH
(1.5 mL) and NaCNBH3 (27 mg, 0.43 mmol) at room temperature. The obtained mixture
was stirred at room temperature for 18 hr and then concentrated in vacuo. The residue
was taken up to acetone/H2O (1 : 1, 5 mL). To this mixture was added NaHCO3 (0.2 g,
2.38 mmol) and (Boc)2O (174 mg, 0.80 mmol). The reaction mixture was stirred at
room temperature for 2hr, and the acetone was removed in vacuo. To this aqueous
residue was added H2O (5 mL). The aqueous mixture was extracted three times with
ethyl acetate. The combined organic phases were dried over MgSO4 and concentrated
in vacuo. The residue was purified by chromatography (pentane/ethyl acetate 3 : 1) to
1
furnish 16 (82 mg, 44%) as an oil. H NMR (200 MHz, CDCl3): d 7.40–7.13 (m, 5H,
Ph), 4.80 (d, 1H, J 11.6HZ, CH2Ph), 4.79 (bs, 1H, H-1), 4.55 (d, 1H, J 11.6, CH2Ph),
4.55 (m, 1H, H-3), 3.73 (bs, 1H, H-2), 3.60–3.40 (m, 3H, H-40a, H-5a, H-5b), 3.23–
2.86 (m, 2H, CH2CH2N), 2.64 (dd, 1H, H-40b), 2.35 (bs, 1H, OH), 2.20 (m, 1H, H-4),
1.60–1.10 (m, 14H, 7CH2), 1.40 (s, 9H, (CH3)3C), 0.90 (t, 3H, CH2CH3). 13C NMR
(50 MHz, CDCl3): d 137.5, 128.6, 128.2, 121.1 (Ph), 97.8 (C-1), 80.6 ((CH3)3C), 70.6
(CH2Ph), 69.8, 67.4 (C-2, C-3), 62.9 (C-5), 47.9 (C-40), 43.5 (CH2CH2N), 36.0 (C-4),
32.1, 29.7, 29.4, 27.0, 22.9 (7CH2), 28.6 ((CH3)3C), 14.3 (CH2CH3).
(2R/S,3S,4R, 5R)-5-(Hydroxymethyl)-1-nonyl-2,3,4-trihydroxy-piperidine
hydrochloride (17). The mixture of 16 (57 mg, 0.12 mmol) and Pd/C (10%, 20 mg) in
EtOH (22 mL) was hydrogenolysed under 1 atm of H2 pressure at room temperature for
20 hr. After reaction, the catalyst was filtered off, and the solution was concentrated in
vacuo. The residue was purified by chromatography (CHCl3/MeOH 10 : 1) to give an
oil. The oil was treated with aqueous HCl (2 M, 1.5 mL) at room temperature for 5 min
1
and concentrated in vacuo to furnish 17 (30 mg, 67%) as a brownish oil. H NMR
(200 MHz, D2O): d 5.10 (s, 0.33 H, H-2b), 4.50 (d, 0.67H, J2,3 8.5 Hz, H-2a), 4.1–2.9
(m, 8H, H-3, H-4, H-6a, H-6b, H-50a, H-50b, CH2CH2N), 2.43–2.26 (m, 1H, H-5),
1.90–1.00 (m, 14H, 7CH2), 0.9 (bs, 3H, CH3). 13C NMR (50 MHz, D2O): d 96.5 (C-
2b), 91.5 (C-2a), 71.6, 71.1, 68.5, 68.0 (C-3a, C-3b, C-4a, C-4b), 62.4 (C-50b), 60.0
(C-50a), 47.9 (C-6), 45.1 (CH2CH2N), 37.2 (C-5), 30.7, 28.0, 27.8, 25.3, 24.9, 21.7
(7CH2), 13.0 (CH3). HRMS (ES): calcd for (C15H31NO4 2 H2O) þ Hþ: 272.2226,
found 272.2220.
Hydrolysis of 1. A 10 mg of 1 was dissolved in pD 6.8 phosphate buffer (0.2 M,
ꢀ0.3 mL). After standing for 7 hr at room temperature, 1 had been completely converted
1
to the rearrangement product 18 according to H- and 13C NMR. The stereochemistry at
C-2 was not determined. 1H NMR (200 MHz, D2O): d 3.83–3.66 (m, 2H, H-50a, H-50b),
3.62 (d, 1H, J4,5 10.5 Hz, H-4), 3.44 (dd, 1H, J5,6eq 4.1, J6,6 12.5 Hz, H-6eq), 3.34 (s, 1H,
H-2), 2.96 (t, 1H, J5,6ax 12.5 Hz, H-6ax), 2.10 (m, 1H, H-5).13C NMR (50 MHz, D2O):
d 90.6 (C-3), 70.7 (C-4), 58.8 (C-50), 49.9 (t, C-3), 44.4 (C-6), 39.3 (C-5).
Hydrolysis of 19. A 10 mg of 19 was dissolved in pD 6.8 phosphate buffer (0.2 M,
ꢀ0.3 mL). After standing for 40 min at room temperature, 19 had been completely con-
1
verted to the rearranged product 20 according to H- and 13C NMR spectra. The stereo-
1
chemistry of C-2 was not determined. H NMR (200 MHz, D2O): d 3.52 (d, 1H, J4,5

Noeuromycin and Isofagomine Analogues
235
2.0 Hz, H-4), 3.00 (s, 1H, H-2), 2.98 (dd, 1H, J5,6eq 4.2, J6,6 12.1 Hz, H-6eq), 2.70 (t, 1H,
0
J5,6ax 12.1 Hz, H-6ax), 2.20 (m, 1H, H-5), 0.89 (d, 3H, J5,5 6.0 Hz, CH3).13C NMR
(50 MHz, D2O): d 91.6 (C-3), 71.8 (C-4), 45.8 (t, C-3), 43.3 (C-6), 30.1 (C-5), 13.3
(CH3). These NMR data are identical to those given in Ref.[10]
.
Benzyl 2-C-methyl-b-D-arabinopyranoside (24). To a solution of CF3CO2H/H2O
(2/1, 3 mL) was added benzyl 3,4-O-isopropylidene-2-C-methyl-b-D-arabinopyranoside
(1.0 g, 3.54 mmol, 23) at room temperature. The obtained solution was stirred at the
same temperature for 10 min and concentrated in vacuo to provide 24 (0.88 g, 98%) as
a yellowish oil. 1H NMR (200 MHz, CDCl3/D2O): d 7.40–7.20 (bs, 5H, Ph), 4.73
(d, 1H, J 11.4 Hz, CH2Ph), 4.46 (d, 1H, CH2Ph), 4.60 (s, 1H, H-1), 3.98 (ddd, 1H, J3,4
3.6, J4,5eq 2.8, J4,5ax 4.2 Hz, H-4), 3.80 (dd, 1H, J5ax,5eq 12.4 Hz, H-5eq), 3.78 (d, 1H,
H-3), 3.63 (dd, 1H, H-5ax), 1.29 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3): d
136.9, 128.8, 128.3 (Ph), 101.7 (C-1), 73.1 (CH2Ph), 72.6 (C-2), 70.4, 67.9 (C-3, C-4),
63.0 (C-5), 20.3 (CH3). HRMS (ES): calcd for C13H18O5 þ Naþ 277.1052, found
277.1053.
Benzyl 4-C-nitromethyl-2-C-methylpentopyranoside (26 and 27, and benzyl 3-C-
nitromethyl-2-C-methylpentopyranoside (28). To a mixture of 24 (2.0 g, 7.86 mmol)
˚
and molecular sieves (3 A, 3.5 g) in CH2Cl2 (40 mL) was added (Bu3Sn)2O (9.4 g,
15.7 mmol) at room temperature. The mixture was refluxed for 3.5 hr and then cooled
to 08C. To this mixture was added bromine (0.9 mL, 17.9 mmol) dropwise until the solu-
tion was faintly coloured. The mixture was poured on a column with silica and washed
thoroughly with CHCl3 in order to remove tin compounds. The product 25 was washed
down with ethyl acetate and was obtained after concentration as a mixture (1.5 g, 79%)
of several different forms that could not be separated. These were not characterised but
used directly in the next step.
To a suspension of 25 (1.0 g, 4.3 mmol) in dry CH3NO2 (30 mL) was added Et3N
(0.45 g, 4.45 mmol). The solution was stirred at room temperature for 18 hr and then
concentrated in vacuo. The residue was chromatographed on silica gel by using
pentane/ethylacetate 3 : 2 to furnish 27 (220 mg, 18%), 28 (120 mg, 10%), 26 (140 mg,
1
11%), and a mixture of 27 and 28 (200 mg). 26: H NMR (200 MHz, CDCl3): d 7.35–
7.12 (m, 5H, Ph), 4.70–4.39 (m, 5H, H-1, H-40a, H-40b, CH2Ph), 3.90 (d, 1H, J5ax,5eq
12.3 Hz, H-5eq), 3.60 (d, 1H, H-5ax), 3.48 (s, 1H, H-3), 1.20 (s, CH3). 13C NMR
(50 MHz, CDCl3): d 136.9, 127.6, 127.4, 127.0 (Ph), 101.5 (C-1), 79.5 (C-40), 71.7,
71.6, 71.2 (C-2, C-3, C-4), 69.1 (CH2Ph), 63.5 (C-5), 19.1 (CH3). HRMS (ES): calcd
for C14H19NO7 þ Naþ 336.1059, found 336.1057. 27: 1H NMR (200 MHz, CDCl3):
d 7.40–7.24 (m, 5H, Ph), 4.84 (dd, 1H, J4 a,5eq 1.7, J4 a,4 b 10.9 Hz, H-40a), 4.77 (d, 1H,
J 11.3 Hz, CH2Ph), 4.71 (d, 1H, H-40b), 4.68 (s, 1H, H-1), 4.52 (d, 1H, CH2Ph), 4.17
(s, 1H, H-3), 4.14 (dd, 1H, J5ax,5eq 13.5 Hz, H-5eq), 4.0 (bs, 1H, OH), 3.13 (bs, 1H,
OH), 2.80 (bs, 1H, OH), 3.72 (d, 1H, H-5ax), 1.32 (s, 3H, CH3). 13C NMR (50 MHz,
CDCl3): d 135.9, 129.0, 128.8, 128.4 (Ph), 103.6 (C-1), 76.1 (C-40), 75.6 (C-4), 71.1
(C-3), 70.1 (C-2), 68.9 (CH2Ph), 60.7 (C-5), 21.3 (CH3). HRMS (ES): calcd for
0
0
0
1
C14H19NO7 þ Naþ 336.1059, found 336.1057. 28: H NMR (200 MHz, CDCl3/D2O):
d 7.40–7.20 (m, 5H, Ph), 4.86 (d, 1H, J 12.0 Hz, CH2Ph), 4.76 (s, 2H, H-30a, H-30b),
4.60 (s, 1H, H-1), 4.57 (d, 1H, CH2Ph), 4.20 (dd, 1H, J4,5aq 10.1, J4,5eq 5.6 Hz, H-4),
3.88 (dd, 1H, J5ax,5eq 11.4 Hz, H-5eq), 3.52 (dd, 1H, H-5ax), 1.24 (s, 3H, CH3). 13C
NMR (50 MHz, CDCl3): d 137.0, 128.7, 128.3 (Ph), 99.5 (C-1), 75.4 (C-30), 74.5 (C-2),
71.0 (C-4), 66.3 (CH2Ph), 63.5 (C-5), 18.3 (CH3).

236
Liu et al.
Benzyl 4-nitromethyl-4-deoxy-2-methyl-b-D-arabinopyranoside (29). To a solu-
tion of 26 (110 mg, 0.35 mmol) and Ac2O (0.16 mL) in CH3CN (4 mL) was added
Sc(OTf)3 (8 mg, 0.016 mmol) at room temperature. The obtained mixture was stirred at
the same temperature for 2 hr and then quenched with saturated aqueous solution of
NaHCO3. The mixture was extracted three times with CH2Cl2. The combined organic
phases were dried over MgSO4 and concentrated in vacuo to afford a crude triacetate
(150 mg). This product was dissolved in EtOH (6 mL), and to this solution was added
NaBH4 (20 mg, 0.53 mmol) at room temperature. The reaction mixture was stirred at
the same temperature for 1 hr and then concentrated in vacuo. The residue was dissolved
in MeOH (6 mL) and NaOMe (20 mL) was added. The mixture was stirred at room temp-
erature for 0.5 hr, and Amberlite IR 120 Hþ was added until pH reached 6. The resin was
removed by filtration. The filtrate was concentrated in vacuo. The residue was purified by
chromatography (pentane/ethyl acetate 2 : 1) to furnish 29 (39 mg, 37%) as a colourless
1
solid. H NMR (200 MHz, CDCl3/D2O): d 7.35–7.17 (m, 5H, Ph), 4.78–4.50 (d, 2H, J
12.5 Hz, CH2Ph), 4.56 (bs, 1H, H-1), 4.47 (dd, 1H, J4,4 a 6.5, J4 a,4 b 12.5 Hz, H-40a),
0
0
0
4.28 (dd, 1H, J4,4 b 8.7 Hz, H-40b), 3.80 (dd, 1H, J4,5eq 4.2, J5ax,5eq 12.9 Hz, H-5eq),
0
3.76 (d, 1H, J3,4 4.0 Hz, H-3), 3.60 (dd, 1H, J4,5ax 8.4 Hz, H-5ax), 2.88 (m, 1H, H-4),
1.15 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3): d 137.0, 128.7, 128.3 (Ph), 100.4 (C-1),
73.7 (C-40), 72.0 (C-3), 71.6 (C-2), 70.7 (CH2Ph), 61.4 (C-5), 36.8 (C-4), 20.8 (CH3).
HRMS (ES): calcd for C14H19NO6 þ Naþ 320.1110, found 320.1110.
Benzyl 4-(N-tert-butyloxycarbonyl)methyl-4-deoxy-2-methyl-b-D-arabinopyra-
noside (30). A mixture of 29 (20 mg, 0.07 mmol), Pd/C (10%, 15 mg) and Et3N
(0.03 mL) in MeOH (1.5 mL) was hydrogenated under 1 atm at room temperature for
2 hr. The catalyst was filtered off and the solution was concentrated in vacuo. The
residue was dissolved in CH2Cl2. To this solution was added Et3N (0.10 mL) and
(Boc)2O (30 mg, 0.14 mmol). The obtained solution was stirred at room temperature for
2 hr. Afterwards, the reaction solution was concentrated in vacuo. The residue was purified
by chromatography (pentane/ethyl acetate 2 : 1) giving 30 (20 mg, 81%) as a white solid.
1H NMR (400 MHz, CDCl3): d 7.36–7.20 (m, 5H, Ph), 4.82–4.52 (d, 2H, J 11.6 Hz,
0
0
CH2Ph), 4.76 (dd, 1H, JNH,4 a 8.8, JNH,4 b 5.2 Hz, NH), 4.52 (bs, 1H, H-1), 3.60–3.40
0
(m, 3H, H-3, H-5a, H-5b), 3.25 (ddd, 1H, J4,4 a 10.8 Hz, J4 a,4 b 14.8 Hz, H-40a), 2.69
(ddd, 1H, J4,4 b 5.2 Hz, H-40b), 2.18 (m, 1H, H-4), 1.38 (s, 9H, C(CH3)3), 1.25 (s, 3H,
0
0
0
CH3). 13C NMR (50 MHz, CDCl3): d 137.9, 128.6, 128.2, 128.0 (Ph), 100.4 (C-1), 80.6
(C(CH3)3), 71.5 (C-2), 71.1 (C-3), 70.9 (CH2Ph), 63.0 (C-5), 38.5 (C-40), 37.6 (C-4),
28.5 (C(CH3)3), 22.3 (CH3). HRMS (ES): calcd for C19H29NO6 þ Naþ 390.1893, found
390.1892.
(2R/S,3S,4R,5R)-5-(Hydroxymethyl)-3-methyl-2,3,4-trihydroxy-piperidine tri-
fluoroacetate (31). A mixture of 30 (20 mg, 0.054 mmol), Pd/C (10%, 10 mg) in
ethanol (10 mL) was hydrogenated under 1 atm at room temperature for 18 hr. The catalyst
was filtered off, and the solution was concentrated in vacuo. The residue was treated
with aqueous HCl (0.2 M, 1 mL) for 5 min, and the reaction solution was concentrated
1
in vacuo to give 31 (7 mg, 61%) as a brownish oil. H NMR (200 MHz, D2O): d 4.90
(s, 0.5H, H-2b), 4.30 (s, 0.5H, H-2a), 3.8–3.2 (m, 4H, H-4, H-50a, H-50b, H-6eq),
2.95 (t, 1H, J5,6ax ¼ J6ax,6eq 12.2 Hz, H-6ax), 1.95 (m, 1H, H-5), 1.20 (s, 3H, CH3).
13C NMR (50 MHz, D2O): d 82.5 (C-2b), 81.4 (C-2a), 74.3 (C-4b), 71.9 (C-3a), 71.8
(C-4a), 69.3 (C-3b), 59.3 (C-50b), 58.9 (C-50a), 40.9 (C-6b), 39.5 (C-6a), 38.7 (C-5),
17.1 (CH3).

Noeuromycin and Isofagomine Analogues
Ki Determination
237
Each glycosidase assay was performed by preparing fourteen 2 mL samples in cuv-
ettes, containing 1 mL sodium phosphate buffer (0.1 M) of pH 6.8 or acetate buffer of
pH 4.0, along with 0.05–0.70 mL of different substrate. The concentration of the substrate
was in the range of 0.25Km–5Km. The substrates used were 4-nitrophenyl b-D-glucopyr-
anoside or 4-nitrophenyl a-D-glucopyranoside. Also added was 0.025–0.1 mL of a sol-
ution of either the inhibitor or water, and finally each cuvette was filled up to a total
volume of 1.9 mL with distilled water. Seven of the samples contained the inhibitor at a
fixed concentration but with varying concentrations of nitrophenyl glycoside. The other
seven samples contained no inhibitor, but also varying concentrations of nitrophenyl gly-
coside. Finally the reaction was started by adding 0.1 mL of a diluted solution of enzyme
solution. The formation of 4- or 2-nitrophenol was monitored for 2 min at 258C by
measurement of the absorbance at 400 nm. Initial velocities were calculated from the
slopes from each reaction and used to construct two Hanes plots ([S]/v vs. [S]), one
wit0h and one without inhibitor. From the two Michaelis–Menten constants, Km and
Km , thus obtained, the inhibition constant, Ki, was calculated.
ACKNOWLEDGMENT
We thank the National Danish Research council for financial support.
REFERENCES
1. Lillelund, V.H.; Jensen, H.H.; Liang, X.; Bols, M. Recent developments of transition
state analogue glycosidase inhibitors of non-natural product origin. Chem. Rev. 2002,
102, 515–553 (and references herein).
2. Stu¨tz, A. Iminosugars as Glycosidase Inhibitors; Wiley-VCH: Weinheim, 1999.
3. Baxter, E.W.; Reitz, A.B. Expeditious synthesis of aza sugars by the double reductive
amination of dicarbonyl sugars. J. Org. Chem. 1994, 59, 3175–3185.
4. Matos, C.R.R.; Lopes, R.S.C.; Lopes, C.C. Synthesis of 1-deoxynojirimycinand
N-butyl-1-deoxynojirimycin. Synthesis 1999, 571–573.
5. Liu, H.; Liang, X.; Søhoel, H.; Bu¨low, A.; Bols, M. Noeuromycin, a glycosyl cation
mimic that strongly inhibit glycosidases. J. Am. Chem. Soc. 2001, 123, 5116–5117.
6. Bols, M. 1-Azasugars, apparent transition state analogs of equatorial glycoside
formation/cleavage. Acc. Chem. Res. 1998, 31, 1–8.
7. Jespersen, T.M.; Dong, W.; Skrydstrup, T.; Sierks, M.R.; Lundt, I.; Bols, M. Isofago-
mine, a potent new glycosidase inhibitor. Angew. Chem. Int. Ed. Engl. 1994, 33,
1778–1779.
8. Andersch, J.; Bols, M. Efficient synthesis of isofagomine and noeuromycin. Chem.
Eur. J. 2001, 7, 3744–3747.
9. Paulsen, H. Monosaccaride mit Stickstoffhaltigem Ring. V. Amadori–Umlagerung
von 5-Amino-5-desoxy-D-xylopiperidinose zu 1.5-Didesoxy–1.5-imino-D-threopen-
tulose. Liebigs Ann. Chem. 1965, 683, 187–198.

238
Liu et al.
10. Kondo, K.-I.; Adachi, H.; Shitara, E.; Kojima, F.; Nishimura, Y. Glycosidase
inhibitors of gem-diamine 1-N-iminosugars: structures in media of enzyme assays.
Bioorg. Med. Chem. 2001, 9, 1091–1095.
11. Ireland, R.E.; Courtney, L.; Fitzsimmons, B.J. Total synthesis of polyether antibiotics.
Synthesis of the enantiomer of lasalocid A (X–537A). J. Org. Chem. 1983, 48,
5186–5198.
Accepted August 2, 2003