Synthesis and high-throughput screening of N-acetyl-β-hexosaminidase inhibitor libraries targeting osteoarthritis

Article abstract of DOI:10.1021/jo049355h

C1 Nitrogen iminocyclitols are potent inhibitors of N-acetyl-β- hexosaminidases. Given hexosaminidases' important roles in osteoarthritis, we developed two straightforward and efficient syntheses of C1 nitrogen iminocyclitols from two readily available starting materials, D-mannosamine hydrochloride and the microbial oxidation product of fructose. A diversity-oriented synthetic strategy was then performed by coupling these core structures with various aldehydes, carboxylic acids, and alkynes to generate three separate libraries. High-throughput screening of the generated libraries with human N-acetyl-β-hexosaminidases produced only moderate inhibitory activities. However, the synthetic approach and screening strategy for these compounds will be applied to develop new potent inhibitors of human N-acetyl-β-hexosaminidases, particularly when combined with the structural information of these enzymes.

Full text of DOI:10.1021/jo049355h

Syn th esis a n d High -Th r ou gh p u t Scr een in g of
N-Acetyl-â-h exosa m in id a se In h ibitor Libr a r ies Ta r getin g
Osteoa r th r itis
J unjie Liu,† Mehdi M. D. Numa,† Haitian Liu, Shi-J ung Huang, Pamela Sears,
Alexander R. Shikhman,‡ and Chi-Huey Wong*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La J olla, California 92037
wong@scripps.edu
Received April 16, 2004
C1 Nitrogen iminocyclitols are potent inhibitors of N-acetyl-â-hexosaminidases. Given hexosamini-
dases’ important roles in osteoarthritis, we developed two straightforward and efficient syntheses
of C1 nitrogen iminocyclitols from two readily available starting materials, D-mannosamine
hydrochloride and the microbial oxidation product of fructose. A diversity-oriented synthetic strategy
was then performed by coupling these core structures with various aldehydes, carboxylic acids,
and alkynes to generate three separate libraries. High-throughput screening of the generated
libraries with human N-acetyl-â-hexosaminidases produced only moderate inhibitory activities.
However, the synthetic approach and screening strategy for these compounds will be applied to
develop new potent inhibitors of human N-acetyl-â-hexosaminidases, particularly when combined
with the structural information of these enzymes.
In tr od u ction
N-Acetyl-â-hexosaminidases (EC 3.2.1.52) belong to the
group of lysosomal hydrolases. They catalyze the hy-
drolysis of terminal, nonreducing N-acetyl-â-D-glucosamine
and N-acetyl-â-D-galactosamine residues in glycoproteins,
G
M2-gangliosides, and glycosaminoglycans.1 Human N-
acetyl-â-hexosaminidases are dimeric enzymes composed
of R and â subunits and hence have three isoforms:
N-acetyl-â-hexosaminidase A (Râ, HexA), N-acetyl-â-
hexosaminidase B (RR, HexB), and N-acetyl-â-hexosamini-
dase S (ââ, HexS). The â subunit hydrolyzes mainly
neutral substrates, whereas the R subunit is able to
hydrolyze charged substrates, although dimerization of
the two units is a prerequisite for the enzyme’s activity.2
N-Acetyl-â-hexosaminidases are the dominant glycosami-
noglycan-degrading glycosidases released by chondro-
cytes into the extracellular compartment, and the domi-
nant glycosidases in synovial fluid of patients with
osteoarthritis. Stimulation of chondrocytes with proin-
flammatory cytokine interleukin-1â results in a selective
secretion of N-acetyl-â-hexosaminidases.3 Of particular
interest is the enzyme’s direct involvement in the carti-
lage matrix degradation.4
F IGURE 1. Postulated mechanism of hexosaminidases ca-
talysis and an iminocyclitol structure.
generally believed to exhibit a flattened half-chair con-
formation with substantial oxocarbenium ion character
at the anomeric position, which is stabilized by a depro-
tonated carboxyl group from the enzyme (Figure 1).5 The
mechanism involving the participation of the neighboring
C2 acetamido group of the substrate was also suggested.6
Iminocyclitols, whose ring nitrogen would be protonated
under physiological conditions, are transition state ana-
logues for glycosidase catalysis and therefore good inhibi-
tors for glycosidases. The inhibition constants of imi-
nocyclitols toward various glycosidases are usually in the
nanomolar range.7 In our previous study, C1 N-acetyl
iminocyclitols 1, 2, 3, and 4 were found to show very
potent inhibition activity against N-acetyl-â-hexosamini-
dases from human placenta (Figure 2). In particular,
During the course of hexosaminidase catalysis, the
terminal N-acetyl-â-hexosamine of polysaccharides is
† These authors contributed equally.
(4) Liu, J .; Shikhman, A. R.; Lotz, M. K.; Wong, C.-H. Chem. Biol.
2001, 8, 701.
(5) (a) Rye, C. S.; Withers, S. G. Curr. Opin. Chem. Biol. 2000, 4,
573. (b) Sinnott, M. Chem. Rev. 1990, 90, 1171.
(6) (a) Vocadlo, D. J .; Davies, G. J .; Laine, R.; Withers, S. G. Nature
2001, 412, 835. (b) Knapp, S.; Vocadlo, D. J .; Gao, Z.; Kirk, B.; Lou, J .;
Withers, S. G. J . Am. Chem. Soc. 1996, 118, 6804.
(7) (a) Bols, M. Acc. Chem. Res. 1998, 32, 1. (b) Takayama, S.;
Martin, R.; Wu, J .; Laslo, K.; Siuzdak, G.; Wong, C.-H. J . Am. Chem.
Soc. 1997, 119, 8145.
‡ Division of Arthritis Research, The Scripps Research Institute.
(1) (a) Winchester, B. G. Subcell. Biochem. 1996, 27, 191. (b)
Watanabe, K. J . Biochem. (Tokyo) 1936, 24, 297.
(2) (a) Mahuran, D. J .; Lowden, J . A. Can. J . Biochem. 1980, 58,
287. (b) Hou, Y.; Tse, R.; Mahuran, D. J . Biochemistry 1996, 35, 3963.
(c) Meier, E. M.; Schwarzmann, G.; Fu¨rst, W.; Sandhoff, K. J . Biol.
Chem. 1991, 266, 1897.
(3) Shikhman, A. R.; Brinson, D. C.; Lotz, M. K. Arthritis Rheum.
2000, 43, 1307.
10.1021/jo049355h CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/07/2004
J . Org. Chem. 2004, 69, 6273-6283
6273

Liu et al.
SCHEME 2. Retr osyn th etic An a lysis
F IGURE 2. Structures of N-acetyl iminocyclitol inhibitors of
N-acetyl-â-hexosaminidases.
SCHEME 1a
SCHEME 3a
a
Reagents and conditions: (a) (i) FDP aldolase; (ii) acid
phosphatase; (b) H2, Pd-C.
incubation of human chondrosarcoma cells with imino-
cyclitol 2 resulted in an accumulation of glycosaminogly-
cans in the cell-associated fraction and a decrease in the
release of glycosaminoglycans into the culture superna-
tant.4 The discovery of iminocyclitols as potential chon-
droprotective agents suggests a new avenue for the
development of drugs to treat osteoarthritis.
In view of their interesting biological properties, con-
siderable effort has been devoted to the development of
efficient syntheses of various iminocyclitols.8 Our group
has developed a chemoenzymatic method to generate
numerous iminocyclitols, which generally involves an
aldol condensation catalyzed by fructose 1,6-diphosphate
(FDP) aldolase (EC 4.1.1.13). Various glucose-type imi-
nocyclitols can be easily prepared by a three-step proce-
dure: condensation of an aldehyde and DHAP catalyzed
by FDP aldolase, followed by dephosphorylation with acid
phosphatase and then intramolecular reductive amina-
tion (Scheme 1).9 FDP aldolase’s high stereospecificity
and broad substrate tolerance allow the straightforward
preparation of a large number of iminocyclitols.10 How-
ever, the high cost of removing the phosphate group of
the aldol condensation products with acid phosphatase
(EC 3.1.3.2) ($440/2,500 U) combined with FDP aldolase’s
low activity to certain unnatural substrates make large-
scale chemoenzymatic iminocyclitol synthesis unfavor-
able. As an extension of our research on the effect of
iminocyclitols on N-acetyl-â-hexosaminidases for the
possible treatment of osteoarthritis, we report here two
novel routes to synthesize C1 nitrogen iminocyclitol
a
Reagents and conditions: (a) (i) TfN3 (5 equiv), K2CO3, MeOH,
H2O, CuSO4 (cat.); (ii) Ac2O, Pyr, 0 °C, 89% for two steps; (b)
p-toluene sulfide (4 equiv), BF3‚Et2O (6 equiv), 0 °C, 90%; (c) (i)
MeONa (2 equiv), MeOH, 0 °C, (ii) NaH, BnBr, DMF, 0 °C, 87%;
(d) NIS (3 equiv), acetone, H2O, 65%; (e) NaBH4 (excess), MeOH,
83%; (f) TBDMSCl (1.1 equiv), Pyr, 0 °C, 71%; (g) Dess-Martin
reagent (1.1 equiv), 91%, CH2Cl2.
structures: one starting from D-mannosamine hydro-
chloride and another starting with the microbial oxida-
tion product of fructose. Reported also are the rapid
generation of three libraries from three different imi-
nocyclitol core structures and their high-throughput
screening with human N-acetyl-â-hexosaminidases.
Resu lts a n d Discu ssion
(8) (a) Ganem, B. Acc. Chem. Res. 1996, 29, 340. (b) Koumbis, A.
E.; Gallos, J . K. Curr. Org. Chem. 2003, 7, 771. (c) Legler, G. In
Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond; Stu¨tz,
A. E., Ed.; Wiley-VCH: Weinheim, 1999; pp 49-56.
(9) (a) Gijsen, H. J . M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. Rev.
1996, 96, 443. (b) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic
Organic Chemistry; Elsevier: NewYork, 1994; pp 195-210. (c) Wong,
C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, Y. Angew. Chem., Int.
Ed. Engl. 1995, 34, 412.
New Rou te to Syn th esize N-Acetyl Im in ocyclitols
5 a n d 6. The retrosynthetic pathway of compounds 5 and
6 is represented in Scheme 2. The key step is the
preparation of pyrrolidine 7 by cyclization of the azido-
ketone 8 through intramolecular reductive amination.
Compound 8 is accessible from commercially available
D-mannosamine hydrochloride 9.
(10) (a) Takaoka, Y.; Kajimoto, T.; Wong, C.-H. J . Org. Chem. 1993,
58, 4809. (b) Hung, R. R.; Straub, J . A.; Whitesides, G. M. J . Org. Chem.
1991, 56, 3849.
As shown in Scheme 3, the primary amine of D-
mannosamine hydrochloride 9 was first converted into
6274 J . Org. Chem., Vol. 69, No. 19, 2004

N-Acetyl-â-hexosaminidase Inhibitor Libraries
SCHEME 4a
F IGURE 3. Reduction of imines.
ration.14 The same reductive amination of the hemi-
aminal with NaBH(OAc)3 and p-TsOH in dichloromethane
afforded 7 and the undesired 16 in a 1:3 ratio.15 When
ketone 8 was reduced with 100 psi H2 in the presence of
Pd-C in methanol, the major product was still 16 as
shown by TLC after a short period of reaction time
(Scheme 4b). Interestingly, reductive amination with
NaBH3CN or NaBH(OAc)3 performed on 17 gave the C4-
C5 trans isomer 18 as the predominant product in 72%
yield (Scheme 4c). After removal of the TBDMS of 8,
reductive amination of 19 under similar conditions
produced a 1:1 mixture of the two epimers (Scheme 4d).
Previous studies suggest that the intramolecular re-
ductive amination of unprotected glucose-type azido-
ketone substrates usually favors C4-C5 trans configu-
ration as a result of torsional strain.16 Our current
research suggests that the steric effect caused by the
bulky C1 group (TBDMS) plays a critical role in the
intramolecular reductive amination of azido ketones.
Figure 3 shows how the C1 TBDMS blocks the R face of
the imine intermediate to favor the â face hydrogen
addition.
As a result of the inability to obtain the desired isomer
7 in high stereoselectivity by imine reduction, an alterna-
tive approach was sought. Ketone 8 was reduced using
NaBH4 in MeOH (Scheme 5), where a Felkin-Ahn-type
addition afforded predominantly the secondary alcohol
20 with the (5S) configuration.17 The hydroxyl group of
20 was activated as mesylate, and after reduction of the
azido group, there followed an immediate cyclization to
give the desired isomer 7 as the major product (7/16 )
61:26).18
a
Reagents and conditions: (a) H2, Pd-C, Pyr, THF; (b)
NaBH3CN, p-TsOH, MeOH or NaBH(OAc)3, p-TsOH, CH2Cl2; (c)
H2 (100 psi), Pd-C, MeOH; (d) TBDMSCl, Pyr, 0 °C.
an azido functionality using a diazo transfer reaction.11
The crude product was peracetylated using acetic anhy-
dride in pyridine to give 10 as an anomeric mixture in
89% yield for two steps. Treatment of this anomeric
mixture with p-toluene sulfide and BF3‚Et2O provided the
hemi-thioketal 11 in 90% yield with traces of â-anomer.
After removal of the acetate groups using sodium meth-
oxide, benzylation of the crude product with NaH and
BnBr in DMF provided compound 12 in 87% yield for
two steps. Hydrolysis of the hemi-thioketal functionality
in the presence of N-iodo succinimide gave 13 in 65%
yield. This C2-azido-pyranose was reduced to the corre-
sponding alcohol 14 using NaBH4. After selective protec-
tion of the primary hydroxyl group using TBDMSCl in
pyridine, the secondary hydroxyl group of 15 was oxidized
with the Dess-Martin periodinane to provide the azido-
ketone 8.12
(14) The stereochemistry of 16 was ascertained by reacting 15 with
MsCl and then reducing the azido group using H2 and Pd-C in
pyridine under hydrogen atmosphere (1 atm).
Several reductive amination conditions were tried in
order to cyclize 8 to give products with the desired
stereochemistry. The azido group was first partially
reduced using Pd-C in the presence of pyridine under 1
atm of H2 (Scheme 4a).13 The hemi-aminal intermediate
formed was reduced with NaBH3CN and p-TsOH in
methanol to give the undesired epimer 16 as the pre-
dominant product, where C4 and C5 are in cis configu-
(15) (a) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff,
C. A.; Shah, R. D. J . Org. Chem. 1996, 61, 3849. (b) For a review on
reductive amination, see: Baxter, E.; Reitz, A. B. Org. React. 2002,
59, 1.
(16) (a) Liu, K. K.-C.; Kajimoto, T.; Chen, L.; Zhong, Z.; Ichikawa,
Y.; Wong, C.-H. J . Org. Chem. 1991, 56, 6280. (b) Kajimoto, T.; Liu,
K. K.-C.; Pederson, R. L.; Zhong, Z.; Ichikawa, Y.; Porco, J . A., J r.;
Wong, C.-H. J . Am. Chem. Soc. 1991, 113, 6187. (c) Wang, Y.-F.;
Dumas, D. P.; Wong, C.-H. Tetrahedron Lett. 1993, 34, 403.
(17) Mengel, A.; Reiser, O. Chem. Rev. 1999, 99, 1191.
(11) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029.
(12) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155.
(13) Medgyes, A.; Bajza, I.; Farkas, E.; Pozsgay, V.; Liptak, A. J .
Carbohydr. Chem. 2000, 3, 285.
(18) Liu, J .; Wong, C.-H. Angew. Chem., Int. Ed. 2002, 41, 1404.
J . Org. Chem, Vol. 69, No. 19, 2004 6275

Liu et al.
SCHEME 5a
SCHEME 7a
a
Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) (i) MsCl,
Pyr, 0 °C, (ii) H2 (1 atm), Pd-C, Pyr, THF, 61% for 3 steps.
SCHEME 6a
a
Reagents and conditions: (a) MsCl, Pyr; (b) NaN3, NaI, 87%
from 26; (c) Ph3P, THF, 50 °C; (d) Ac2O, Pyr, 87% from 28; (e) H2
(50 psi), Pd-C, 89%.
F IGURE 4. Derivatives of iminocyclitol.
was treated directly with NaN3 in pyridine to afford azido
compound 28. Reduction of the azido group followed by
acetylation of the resulting amine generated 30, which
was subjected to hydrogenation to give the final N-meth-
yl-â-acetamido-iminocyclitol 4 in high yield.
Ar om a tic Der iva tives of th e Rin g Nitr ogen of
-
a
Im in ocyclitol 1. We have previously shown that 6-SO3
Reagents and conditions: (a) TBAF, THF, 0 °C, 97%; (b) Boc2O,
iminocyclitol (R1 ) SO3-, R2 ) Me, R3 ) Ac, Figure 4)
was able to selectively inhibit N-acetyl-â-hexosaminidase
A, which would cause fewer side effects.4 In our current
research, we investigated the substituent effects on the
ring nitrogen (R2) and C1 nitrogen (R3).
It has long been recognized that aromatic moieties are
major players in molecular recognition as a result of their
capability to interact with both hydrophobic residues and
polar substituents.21 Accordingly, we synthesized a series
of aromatic derivatives from the core iminocyclitol struc-
ture 1 (Scheme 8). The couplings of different aromatic
aldehydes with 1 proceeded smoothly via reductive
amination with NaBH3CN to give various aromatic
derivatives in 60-70% yields.
The IC50’s of aromatic derivatives 31a -e against
human N-acetyl-â-hexosaminidases in the presence of
4-methylumbellyferyl-N-acetyl-â-D-glucosamine were all
in the low micromolar range (Table 1). Compared with
iminocyclitol 2’s 24 nM, the aromatic moieties decrease
the iminocyclitol’s activity against N-acetyl-â-hexosamini-
dases. Compound 31b is the best inhibitor in this
Et3N, 0 °C, 83%; (c) (i) MsCl, Et3N, 0 °C, (ii) NaN3, DMF, 75 °C,
53%; (d) TFA, 0 °C, 97%; (e) H2 (70 psi), Pd-C, MeOH, aq HCl
(pH 4), (quant); (f) TfN3, K2CO3, MeOH, H2O, CuSO4, 85%.
The silyl protecting group of 7 was removed using
TBAF to give the alcohol 21, whose secondary amine was
then protected with the treatment of Boc2O to afford 22
in 83% yield, which existed as a mixture of atropoisomers
(Scheme 6). The hydroxyl group of 22 was converted to a
mesylate, followed by displacement with NaN3 at 75 °C
to give the azido compound 23 in 53% yield for two steps.
The Boc group was removed using trifluoroacetic acid to
provide 24 in 97% yield. Reduction of 24 using Pd-C in
methanol and aqueous HCl (pH 4) under H2 afforded 5
in quantitative yield. Amine 5 was treated with a solution
of TfN3 to afford azido iminocyclitol 6 in 85% yield.
New Rou te to N-Acetyl Im in iocyclitol 4. We also
developed a new method to synthesize N-methyl-â-isomer
iminocyclitol 4 from 2,5-anhydro-2,5-imino-D-glucitol 25,
which can be produced in large quantities by the micro-
bial oxidation of fructose (Scheme 7).19 Following the
procedure described in the literature, 25 was transformed
to N-methyl iminocyclitol 26 in four steps.20 The hydroxyl
group in 26 was activated with MsCl to afford 27, which
(20) Wong, C.-H.; Provencher, L.; Porco, J . A.; J ung, S.-H.; Wang,
Y.-F.; Chen, L.; Wang, R.; Steensma, D. H. J . Org. Chem. 1995, 60,
1492.
(21) Hajduk, P. J .; Bures, M.; Praestgaard, J .; Fesik, S. W. J . Med.
Chem. 2000, 43, 3443.
(19) Baxter, E. W.; Reitz, A. B. J . Org. Chem. 1994, 59, 3175.
6276 J . Org. Chem., Vol. 69, No. 19, 2004

N-Acetyl-â-hexosaminidase Inhibitor Libraries
SCHEME 8a
CHART 1. Am id e Libr a r y Bioa ssa y Resu lts
from the core structures 5 and 6 (Scheme 9): one is based
on amide formation, where the primary amine of 5
reacted with various carboxylic acids, a strategy that has
been successfully used in the discovery of HIV protease
inhibitors,22 and the other involves triazole formation,
where the azido iminocyclitol 6 was coupled with different
alkynes to generate a triazole library. This azide-alkyne
coupling has been the focus of recent publications because
the azido group is orthogonal to most functional groups
in biological systems and the azide-alkyne coupling is
an extremely efficient reaction.23 Along with the triazole
formation in situ in microtiter plates, the triazole library
was used to screen various lectins.24
a
Reagents and conditions: Ar-CHO, NaBH3CN, MeOH, rt,
60-70%.
TABLE 1. In h ibition Activities of Ar om a tic Der iva tives
of Im in ocyclitol 31a -c
31a
31b
31c
31d
31e
IC50(µM)
9.5
4.1
38
10
18
SCHEME 9a
The coupling reactions were carried out in 96-well
microtiter plates and the crude reaction products used
directly for the enzymatic assay to make the high-
throughput synthesis and screening more efficient. This
method entails that compound synthesis will be scaled
up for characterization, once a potent inhibitor is identi-
fied.
The mixture of iminocyclitol 5, carboxylic acid (Figure
5), diisopropyl ethylamine (DIEA), and HBTU in metha-
nol was shaken in a 96-well microtiter plate overnight.
After the starting material was consumed, the mixture
was diluted and transferred to another 96-well microtiter
plate for the N-acetyl-â-hexosaminidases inhibition as-
say, where the final inhibitor concentration was 25 µM
(based on 100% conversion of starting materials to
amides). The inhibition percentage relative to the control
with no inhibitor was calculated on the basis of the
a
Reagents and conditions: (a) HBTU, DIEA, rt; (b) 80 °C,
overnight.
category, where it appears that the hydrophilic group
may help to improve inhibitory activity.
Ra p id Gen er a tion a n d High -Th r ou gh p u t Scr een -
in g of Im in ocyclitol Libr a r ies. To investigate the
effect of a substituent on the C1 nitrogen position, a rapid
generation and high-throughput screening program was
initiated. Two strategies were used to generate libraries
F IGURE 5. Carboxylic acid building blocks.
J . Org. Chem, Vol. 69, No. 19, 2004 6277

Liu et al.
6278 J . Org. Chem., Vol. 69, No. 19, 2004

N-Acetyl-â-hexosaminidase Inhibitor Libraries
F IGURE 6. Alkyne building blocks and bioassay results (% inhibition shown in parentheses).
fluorescence readings of the released 4-methylumbelli-
ferone from the substrate 4-methylumbellyferyl-N-acetyl-
â-D-glucosamine (Chart 1). Acetic acid derivative (a li-2,
Figure 5) is the best inhibitor (92% inhibition at 25 µM).
It is interesting to note the large activity difference
between the acetic acid derivative and other carboxylic
acids derivatives, which indicates that the acetamido
group is critical for the inhibitor’s activity against human
N-acetyl-â-hexosaminidases. There is no apparent activ-
ity difference between aromatic and aliphatic inhibitors,
although none is as potent as the iminocyclitol 2.
Figure 6 shows the 97 alkynes containing different
functional groups that were coupled to 6 by heating at
80 °C for 24 h generating both the 1,4- and 1,5-regiois-
mers for greater diversity, although 1,4-triazoles can be
generated exclusively using copper salts.25 In cases where
two nonequivalent alkyne groups existed (a lk -62 and
a lk -65, Figure 6), four isomers could be generated. At
25 µM, the inhibitors were tested against human N-acetyl-
â-hexosaminiadses, and the inhibition percentage was
calculated as described above for the amide library. No
inhibitor as potent as iminocyclitol 2 (Figure 2) has been
yet discovered.
Con clu sion
In summary, two new methods to prepare iminocycli-
tols were described, starting from D-mannosamine hy-
drochloride and a modification of fructose’s microbial
oxidation product. The substituent effects on the ring
nitrogen and C1 nitrogen of R-iminocyclitol were inves-
tigated through a rapid synthesis of diversity-oriented
libraries through reductive amination, amide coupling,
and triazole formation reactions. The later two reactions
could be carried out in 96-well microtiter plates, where
the crude products were screened directly against human
N-acetyl-â-hexosaminidases. The high efficiency of the
coupling reactions meant that many iminocyclitol deriva-
tives could be easily generated and screened in a short
time. Although no significant improvement in inhibition
was found, our results indicate that modifying both the
ring nitrogen and C1 nitrogen causes significant activity
losses; possibly these groups are directly involved in
interactions within the enzymes active site. With the
availability of the structure of human N-acetyl-â-hexos-
aminidase B,26 it is possible to rationally design potent
N-acetyl-â-hexosaminidase inhibitors using similar high-
throughput approaches. Work is in progress to synthesize
(22) Brik, A.; Lin, Y.-C.; Elder, J .; Wong, C.-H. Chem. Biol. 2002, 9,
891.
(23) Breinbauer, R.; Ko¨hn, M. ChemBioChem. 2003, 4, 1147.
(24) Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J . C.; Wong, C.-H.
J . Am. Chem. Soc. 2002, 124, 14397.
(25) (a) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier,
P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 1053. (b) Tornoe, C. W.; Christensen, C.; Meldal, M. J . Org.
Chem. 2002, 67, 3057.
J . Org. Chem, Vol. 69, No. 19, 2004 6279

Liu et al.
1.5 h at 0 °C and 1 h at room temperature, the solvent was
removed in vacuo. The residue was dissolved in dry DMF (80
mL) and cooled to 0 °C. NaH (1.7 g, 4.7 equiv) and BnBr (8
mL, 67.1 mmol, 7.4 equiv) were successively added, and the
solution was stirred under argon overnight. After the solvent
was removed in vacuo, the residue was purified by flash
chromatography (silica, 1:8 EtOAc/hexane) to yield compound
12 (4.61 g, 87%) as a pale-yellow oil: 1H NMR (600 MHz,
CDCl3) δ 7.36-7.16 (m, 17H), 6.98 (d, J ) 7.8 Hz, 2H), 5.39
(s, 1H, H-1), 4.82 (d of AB, J ) 10.9 Hz, 1H, PhCH2), 4.69 (d
of AB, J ) 10.9 Hz, 1H, PhCH2), 4.60 (d of AB, J ) 12.2 Hz,
1H, PhCH2), 4.56 (d of AB, J ) 11.0 Hz, 1H, PhCH2), 4.49 (d
of AB, J ) 10.5 Hz, 1H, PhCH2), 4.40 (d of AB, J ) 12.2 Hz,
1H, PhCH2), 4.30 (dd, J ) 4.4, 9.6 Hz, 1H, H-6), 4.04 (m, 1H,
H-4), 4.01 (dd, J ) 3.5, 9.2 Hz, 1H, H-6′), 3.92 (t, J ) 9.6 Hz,
1H, H-5), 3.75 (dd, J ) 4.0, 10.9 Hz, 1H, H-3), 3.65 (d, J )
10.9 Hz, 1H, H-2), 2.22 (s, 3H, ArCH3); 13C NMR (150 MHz,
CDCl3) δ 137.9, 137.8, 137.7, 137.2, 132.1, 129.6, 129.2, 128.2,
128.07, 128.05, 127.7, 127.6, 127.5, 127.42, 127.40, 127.2, 86.2,
79.7, 74.9, 74.4, 73.0, 72.4, 72.3, 68.5, 62.3, 20.8; MALDI-TOF
m/z 604 (M + Na+).
2-Deoxy-2-a zid o-3,4,6-tr i-O-ben zyl-D-m a n n itol (14). To
a solution of compound 12 (433 mg, 0.74 mmol) in acetone (72
mL) were successively added water (0.8 mL) and NIS (1.3 g,
5.7 mmol, 7.8 equiv). After 3 h at room temperature, the
reaction was quenched by addition of aqueous Na2S2O3 (1 M).
The aqueous phase was extracted with CH2Cl2 (150 mL). The
organic phase was then dried over anhydrous Na2SO4, filtered,
and evaporated in vacuo. The residue was purified by flash
chromatography (silica, 1:3 EtOAc/hexane) to yield 3,4,6-tri-
O-benzyl-2-deoxy-2-azido-â-D-mannopyranose 13 (544 mg, 65%)
as a pale-yellow oil: HRMS calcd for C27H29N3O5 [M + Na]+•
498.1999, found 498.1992.
To a solution of compound 13 (495 mg, 1.04 mmol) in dry
MeOH (18 mL) was added one portion of NaBH4 (79 mg, 2.08
mmol, 2 equiv) at 0 °C under argon. The solution was stirred
at 0 °C for 5 min and then at room temperature for 2 h.
Portions of NaBH4 were added under the same conditions until
total disappearance of the starting material. The reaction
mixture was then cooled to 0 °C and quenched by addition of
glacial AcOH (1 mL). After the solvent was removed in vacuo,
the residue was purified by flash chromatography (silica, 1:2
EtOAc/hexane) to yield compound 14 (410 mg, 83%) as a pale-
yellow oil: 1H NMR (600 MHz, CDCl3) δ 7.34-7.23 (m, 15H),
4.70 (d of AB, J ) 11.4 Hz, 1H, PhCH2), 4.61 (d of AB, J )
11.4 Hz, 1H, PhCH2), 4.59 (d of AB, J ) 11.4 Hz, 1H, PhCH2),
4.54 (d of AB, J ) 11.4 Hz, 1H, PhCH2), 4.51 (d of AB, J )
11.8 Hz, 1H, PhCH2), 4.48 (d of AB, J ) 11.4 Hz, 1H, PHCH2),
4.10 (dd, J ) 7.0, 14.0 Hz, 1H, H-3), 3.98 (ddd, J ) 4.4, 8.3,
11.8 Hz, 1H, H-5), 3.89-3.86 (m, 2H, H-4, OH), 3.77-3.72 (m,
2H, H-6, OH), 3.69-3.65 (m, 2H, H-6′, OH), 3.61 (dd, J ) 4.9,
14.4 Hz, 1H, H-2); 13C (150 MHz, CDCl3) δ 137.7, 137.6, 137.5,
128.4, 128.36, 128.34, 128.29, 128.27, 128.1, 127.96, 127.93,
127.86, 127.83, 127.7, 78.4, 77.7, 74.4, 74.1, 73.3, 70.9, 69.8,
62.9, 62.0; HRMS calcd for C27H31N3O5 [M + Na]+• 500.2156,
found 500.2163.
1-(ter t-Bu tyl-dim eth yl-sylan yloxy)-2-deoxy-2-azido-3,4,6-
tr i-O-ben zyl-D-m a n n itol (15). To a solution of compound 14
(1.4 g, 2.93 mmol) in dry pyridine (46 mL) was added
TBDMSCl (484 mg, 3.22 mmol, 1.1 equiv) at 0 °C under argon.
The reaction mixture was stirred overnight. After the solvent
was removed in vacuo, the residue was purified by flash
chromatography (silica, 1:5 EtOAc/hexane) to yield compound
15 (1.23 g, 71%) as a pale-yellow oil: 1H NMR (600 MHz,
CDCl3) δ 7.35-7.23 (m, 15H), 4.71 (d of AB, J ) 11.4 Hz, 1H,
PhCH2), 4.61 (d of AB, J ) 11.4 Hz, 2H, PhCH2), 4.54 (d of
AB, J ) 11.4 Hz, 1H, PhCH2), 4.53 (d of AB, J ) 11.8 Hz, 1H,
PhCH2), 4.50 (d of AB, J ) 11.8 Hz, 1H, PhCH2), 4.01-3.97
(m, 2H, H-3, 4), 3.89 (dd, J ) 2.2, 8.7 Hz, 1H, H-6), 3.81 (dd,
J ) 6.1, 10.6 Hz, 1H, H-5), 3.76 (dd, J ) 2.2, 8.7 Hz, 1H, H-6′),
3.68 (dd, J ) 3.5, 9.6 Hz, 1H, H-1), 3.63 (dd, J ) 4.8, 9.6 Hz,
1H, H-1′), 3.60-3.58 (m, 1H, H-2), 2.55 (d, J ) 6.6 Hz, 1H),
and screen novel iminocyclitols following the methodology
described above and will be reported in due course.
Exp er im en ta l Section
Gen er a l P r oced u r es. All reactions were carried out under
an argon atmosphere with dry, freshly distilled solvents under
anhydrous conditions, unless otherwise noted. Tetrahydro-
furan (THF) was distilled from sodium-benzophenone and
dichloromethane (CH2Cl2) from calcium hydride. All reagents
were purchased at highest commercial quality and used
without further purification unless otherwise stated.
2-Deoxy-2-a zid o-1,3,4,6-t et r a -O-a cet yl-D-m a n n op yr a -
n osid e (10). To a mixture of NaN3 (46 g, 0.71 mole) in water
(93 mL) and CH2Cl2 (93 mL) was added dropwise Tf2O (22 mL,
5.5 equiv) over 30 min under vigorous stirring at 0 °C. After
2 h, the organic phase was separated, and the aqueous phase
was extracted twice with CH2Cl2 (46 mL). The organic layers
were combined and washed successively with aqueous satu-
rated NaHCO3 (150 mL) and water (150 mL). The solution of
TfN3 was used immediately without drying.
D-Mannosamine hydrochloride (5 g, 23.2 mmol), K2CO3 (4.25
g, 30.7 mmol, 1.32 equiv), and CuSO4 (53 mg, 0.33 mmol) were
dissolved in water (67 mL) and MeOH (130 mL). The solution
of TfN3 previously obtained (186 mL) was added dropwise
under vigorous stirring at room temperature. After the green
biphasic mixture was stirred at room temperature for 2 days,
the solvent was removed in vacuo. To the mixture of the crude
solid in dry pyridine (150 mL) was added dropwise a solution
of acetic anhydride (40 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 1 h and then at room temperature for 5 h.
The solvent was removed in vacuo, and the residue was
purified by flash chromatography (silica, 1:1 EtOAc/hexane)
to yield compound 10 (7.18 g, 89%) as a pale-yellow oil: 1H
NMR (600 MHz, CDCl3) δ 6.11 (s, 1H, H-1 R), 5.89 (s, 0.5H,
H-1 â), 5.38 (t, J ) 6.1 Hz, 2H), 5.27 (t, J ) 9.6 Hz, 0.5H),
5.14 (dd, J ) 3.1, 9.6 Hz, 0.5H), 4.28-4.23 (m, 1.5H), 4.18 (d,
J ) 3.5 Hz, 0.5H), 4.11-4.03 (m, 3.5H), 3.80 (dd, J ) 3.5, 6.1
Hz, 0.5H), 2.19-2.05 (8s, 18H, CH3); 13C NMR (150 MHz,
CDCl3) δ 170.3, 169.65, 169.64, 169.60, 169.0, 168.0, 167.87,
167.85, 72.7, 71.4, 70.3, 70.1, 64.9, 64.6, 61.4, 60.7, 60.1, 20.4,
20.3, 20.29, 20.21, 20.1, 20.09; HRMS calcd for C14H19N3O9 [M
+ Na]+• 396.1017, found 396.1013.
p-Meth ylp h en yl 2-Deoxy-2-a zid o-3,4,6-tr i-O-a cetyl-1-
th io-D-m a n n op yr a n osid e (11). Compound 10 (3.8 g, 10.2
mmol) and p-toluene sulfide (2.5 g, 20.2 mmol, 1.9 equiv) in
dry CH2Cl2 (68 mL) were cooled to 0 °C. BF3‚Et2O (2.5 mL, 20
mmol, 1.9 equiv) was added dropwise under argon and the
reaction mixture was stirred for 6 h. After the solvent was
removed in vacuo, the residue was purified by flash chroma-
tography (silica, 2:7 EtOAc/hexane) to yield compound 11 (4.0
g, 90%) as a pale-yellow oil: 1H NMR (500 MHz, CDCl3) δ 7.41
(d, J ) 8.0 Hz, 1H, â), 7.37 (d, J ) 8.1 Hz, 2H), 7.13 (d, J )
8.1 Hz, 2H), 5.46 (s, 1H), 5.36-5.33 (m, 2H and 1H, â), 5.13
(ddd, J ) 2.2, 4.0, 9.9 Hz, 1H, â), 4.80 (s, 1H, â), 4.50 (m, 1H),
4.29 (t, J ) 1.5 Hz, 1H), 4.26 (dd, J ) 5.5, 12.1 Hz, 1H), 4.13-
4.06 (m, 2H), 2.33 (s, 3H), 2.13-2.02 (7s, 12H); 13C NMR (125
MHz, CDCl3) δ 170.28, 170.24, 169.7, 169.6, 169.2, 169.1, 138.3,
138.1, 132.3, 132.2, 129.8, 129.6, 129.5, 128.3, 85.9, 85.8, 76.0,
73.6, 70.8, 69.2, 65.8, 65.5, 63.2, 62.3, 62.2, 61.9, 20.8, 20.4,
20.38, 20.37, 20.34, 20.2, 20.1; ESI+ m/z 460 (M + Na+), ESI-
m/z 472 (M + Cl-).
p-Meth ylp h en yl 3,4,6-Tr i-O-ben zyl-2-d eoxy-2-a zid o-1-
th io-â-D-m a n n op yr a n osid e (12). To a solution of compound
11 (3.99 g, 9.1 mmol) in dry MeOH (200 mL) was added
NaOMe (3.6 mL, 1.8 mmol) dropwise at 0 °C under argon. After
(26) (a) Maier, T.; Strater, N.; Schuette, C. G.; Klingenstein, R.;
Sandhoff, K.; Saenger, W. J . Mol. Biol. 2003, 328, 669. (b) Mark, B.
L.; Mahuran, D. J .; Cherney, M. M.; Zhao, D.; Knapp, S.; J ames, M.
N. G. J . Mol. Biol. 2003, 327, 1003.
6280 J . Org. Chem., Vol. 69, No. 19, 2004

N-Acetyl-â-hexosaminidase Inhibitor Libraries
0.91 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H); 13C NMR (150 MHz,
CDCl3) δ 137.97, 137.96, 137.7, 128.5, 128.4, 128.3, 128.12,
128.10, 128.0, 127.9, 127.8, 127.7, 78.6, 76.9, 74.5, 74.3, 73.4,
70.9, 69.6, 63.6, 62.4, 25.8, 18.1, -5.6; HRMS calcd for
Data for compound 16: 1H NMR (500 MHz, CDCl3) δ 7.35-
7.25 (m, 15H), 4.57-4.34 (m, 6H, PhCH2), 3.93 (dd, J ) 1.1,
4.4 Hz, 1H, H-3), 3.86 (dd, J ) 1.1, 4.0 Hz, 1H, H-4), 3.74 (dd,
J ) 6.2, 9.1 Hz, 1H, H-6), 3.70-3.65 (m, 2H, H-1, 1′), 3.62 (dd,
J ) 7.0, 9.1 Hz, 1H, H-6′), 3.52 (dd, J ) 6.2, 11.0 Hz, 1H, H-2),
3.18 (dd, J ) 4.7, 9.1 Hz, 1H, H-5), 1.91 (bs, 2H), 0.80 (s, 9H,
C(CH3)3), 0.01 (s, 6H, CH3); 13C NMR (125 MHz, CDCl3) δ
138.36, 138.34, 138.33, 128.4, 128.3, 128.29, 128.28, 127.7,
127.6, 127.58, 127.56, 127.54, 84.4, 83.5, 73.4, 71.5, 71.3, 69.5,
65.5, 63.9, 60.8, 25.9, 18.2, -5.4; ESI+ m/z 548 (M + H+), 570
(M + Na+).
1-(ter t-Bu tyl-dim eth yl-sylan yloxy)-2-deoxy-2-azido-3,4,6-
tr i-O-ben zyl-5-oxo-D-glu citol (17). Compound 17 was pre-
pared from D-glucosammine hydrochloride following the same
route as the synthesis of 8: 1H NMR (400 MHz, CDCl3) δ 7.34-
7.24 (m, 15H), 4.41-4.63 (m, 6H, PhCH2), 4.28-4.25 (m, 2H,
H-6, 6′), 4.17 (d, J ) 4.4 Hz, 1H, H-3), 3.90 (t, J ) 4.4 Hz, 1H,
H-4), 3.67-3.58 (m, 3H, H-1, 1′, 2), 0.87 (s, 9H, C(CH3)3), 0.17
(s, 6H, CH3); 13C NMR (100 MHz, CDCl3) δ 137.2, 137.1, 136.6,
128.6, 128.5, 128.3, 128.2, 128.0, 127.9, 81.9, 78.5, 74.5, 74.3,
73.8, 73.3, 63.2, 62.9, 25.8, 18.7, -5.5; ESI+ m/z 590 (M +
H+), 612 (M + Na+).
C
33H45N3O5Si [M + Na]+• 614.3021, found 614.3029.
1-(ter t-Bu tyl-dim eth yl-sylan yloxy)-2-deoxy-2-azido-3,4,6-
tr i-O-ben zyl-5-oxo-D-m a n n itol (8). To a solution of com-
pound 15 (149 mg, 0.25 mmol) in dry CH2Cl2 (2.4 mL) was
added Dess-Martin reagent (110 mg, 0.26 mmol, 1.04 equiv)
at 0 °C under argon. The solution was stirred for 30 min at 0
°C and then overnight at room temperature. After the solvent
was removed in vacuo, the residue was purified by flash
chromatography (silica, 1:6 EtOAc/hexane) to yield compound
8 (135 mg, 91%) as a pale-yellow oil: 1H NMR (500 MHz,
CDCl3) δ 7.34-7.19 (m, 15H), 4.58-4.48 (m, 4H, PhCH2, H-6,
6′), 4.45-4.39 (m, 2H, PhCH2), 4.32 (d of AB, J ) 18.3 Hz,
1H, PhCH2), 4.27 (d, J ) 2.5 Hz, 1H, H-4), 4.22 (d of AB, J )
18.7 Hz, 1H, PhCH2), 3.99 (dd, J ) 2.5, 5.1 Hz, 1H, H-3), 3.97
(t, J ) 2.9 Hz, 1H, H-1), 3.80 (dd, J ) 5.5, 11.0 Hz, 1H, H-1′),
3.62 (ddd, J ) 2.9, 5.5, 8.8 Hz, 1H, H-2), 0.91 (s, 9H, C(CH3)3),
0.08 (s, 6H, CH3); 13C NMR (125 MHz, CDCl3) δ 208.8, 137.08,
137.07, 136.5, 128.57, 128.52, 128.46, 128.43, 128.3, 128.2,
128.0, 127.98, 127.95, 83.7, 78.4, 74.8, 74.6, 73.3, 62.9, 61.9,
25.8, 18.1, -5.6; HRMS calcd for C33H44N3O5Si [M + Na]+•
612.2864, found 612.2864.
(2R,3R,4R,5R)-2-(ter t-Bu tyl-d im eth yl-syla n yloxy-m eth -
yl)-3,4-d i-O-ben zyl-5-(O-ben zyl-m eth yl)-p yr r olid in e (7)
a n d (2R,3R,4R,5S)-2-(ter t-Bu t yl-d im et h yl-syla n yloxy-
m e t h yl)-3,4-d i-O-b e n zyl-5-(O-b e n zyl-m e t h yl)-p yr r oli-
d in e (16). Meth od A. Compound 8 (110 mg, 0.18 mmol) was
stirred in THF/pyridine (1:1, 4 mL) in the presence of Pd-C
(200 mg) under hydrogen atmosphere (1 atm) for 4 h. After
the catalyst was filtered off using Celite, the solvent was
evaporated in vacuo. To the solution of the crude product in
dry MeOH (1.5 mL) were added NaBH3CN (17 mg, 0.27 mmol,
1.5 equiv) and p-TsOH (38 mg, 0.2 mmol, 1.1 equiv). The
reaction mixture was stirred overnight at room temperature.
After the solvent was removed in vacuo, the residue was
purified by flash chromatography (silica, 2:5 EtOAc/hexane)
to yield compounds 7 (7 mg, 7%) and 16 (70 mg, 69%).
(2S,3R,4R,5R)-2-(ter t-Bu tyl-d im eth yl-syla n yloxy-m eth -
yl)-3,4-d i-O-ben zyl-5-(O-ben zyl-m eth yl)-p yr r olid in e (18).
A mixture of compound 17 (18 mg, 0.03 mmol) and Lindlar
catalyst (10 mg) in toluene (0.1 mL) was stirred under
hydrogen atmosphere (1 atm) for 1 h. After the catalyst was
filtered off using Celite, the solvent was evaporated in vacuo.
To a solution of the crude product in dry MeOH (0.25 mL) were
added NaBH3CN (3 mg, 0.05 mmol, 1.5 equiv) and p-TsOH
(6.4 mg, 0.033 mmol, 1.1 equiv) at room temperature. The
reaction mixture was stirred at room temperature for 3 h. After
the solvent was removed in vacuo, the residue was purified
by flash chromatography (silica, 5:2 EtOAc/hexane) to afford
18 (12 mg, 72% yield): 1H NMR (600 MHz, CDCl3) δ 7.36-
7.24 (m, 15H), 4.55-4.44 (m, 6H, PhCH2), 4.01-3.99 (m, 2H,
H-3, 4), 3.75 (dd, J ) 6.6, 9.6 Hz, 1H, H-6), 3.68-3.64 (m, 3H,
H-6′, 1, 1′), 3.54 (t, J ) 10.2 Hz, 1H, H-2), 3.49 (dd, J ) 6.1,
11.8 Hz, 1H, H-5), 0.88 (s, 9H, C(CH3)3), 0.04 (s, 6H, CH3); 13
C
NMR (150 MHz, CDCl3) δ 138.42, 138.40, 138.3, 128.33,
128.31, 127.7, 127.59, 127.56, 127.55, 127.50, 82.7, 82.3, 73.3,
72.3, 72.2, 69.9, 62.0, 60.3, 50.0, 25.9, 18.3, -5.3; ESI+ m/z
548 (M + H+).
Meth od B. Following the similar procedure described in
method A, 7 and 16 were isolated in a 1:3 ratio when NaBH-
(OAc)3 was used instead of NaBH3CN.
(2R,3R,4R,5R)-2-(H yd r oxy-m et h yl)-3,4-d i-O-b en zyl-5-
(O-ben zyl-m eth yl)-p yr r olid in e (21). To a solution of com-
pound 7 (197 mg, 0.36 mmol) in dry THF (4.5 mL) was added
TBAF (0.9 mL, 0.9 mmol, 2.5 equiv) at 0 °C. The reaction
mixture was stirred at room temperature for 4 h. After the
solvent was removed in vacuo, the residue was purified by
flash chromatography (silica, 5:0.3 CHCl3/MeOH) to yield
compound 21 (125 mg, 97%) as a white crystalline solid: 1H
NMR (600 MHz, CDCl3) δ 7.35-7.25 (m, 15H), 4.67-4.55 (m,
6H, PhCH2), 3.94 (dd, J ) 3.9, 5.0 Hz, 1H, H-3), 3.86 (t, J )
3.9 Hz, 1H, H-4), 3.60 (dd, J ) 4.4, 11.0 Hz, 1H, H-6), 3.57-
3.54 (m, 2H, H-6′, 1), 3.51 (dd, J ) 5.7, 9.7 Hz, 1H, H-1′), 3.36
(m, 2H, H-2, 5), 3.06 (bs, 2H); 13C NMR (150 MHz, CDCl3) δ
137.85, 137.83, 137.79, 128.45, 128.41, 128.40, 128.84, 127.80,
127.77, 127.73, 85.5, 85.1, 73.2, 72.0, 71.2, 69.1, 63.1, 61.9, 61.4;
HRMS calcd for C27H31NO4 [M + H]+• 434.2326, found 434.2323.
N-ter t-Bu t yloxyca r b on yl-(2R,3R,4R,5R)-2-(h yd r oxy-
m e t h yl)-3,4-d i-O-b e n zyl-5-(O-b e n zyl-m e t h yl)-p yr r oli-
d in e (22). To a solution of compound 21 (151 mg, 0.35 mmol)
in CH2Cl2 (1.75 mL) in the presence of Et3N (74 µL) was added
Boc2O (108 mg, 0.50 mmol, 1.4 equiv) at 0 °C under argon.
The reaction mixture was stirred at room temperature over-
night. After the solvent was removed in vacuo, the residue was
purified by flash chromatography (silica, 1:3 EtOAc/hexane)
to yield compound 22 (155 mg, 83%) as a pale-yellow oil. The
spectra are consistent with those reported.27
Meth od C. To a solution of compound 8 (400 mg, 0.82 mmol)
in dry MeOH (8.5 mL) was added NaBH4 (31 mg, 0.82 mmol,
1 equiv) at 0 °C under argon. After 1 h, the reaction was
quenched by addition of glacial AcOH (0.17 mL). After the
solvent was removed in vacuo, the crude product was passed
through a short silica column (1:5 EtOAc/hexane) to yield 20
(393 mg, 98%), which was then dissolved in dry pyridine (2.7
mL). To the solution was added dropwise MsCl (0.1 mL) at 0
°C under argon. The reaction mixture was stirred at 0 °C for
2 h. After the solvent was removed in vacuo, the residue was
filtered through a pack of silica using EtOAc as eluent. After
the solvent was removed in vacuo, the oily residue was
dissolved in pyridine/THF (1:1 16 mL) and stirred under
hydrogen atmosphere (1 atm) in the presence of Pd-C (900
mg) for 24 h. The reaction mixture was then filtered through
Celite. After the solvent was removed in vacuo, the residue
was purified by flash chromatography (silica, 2:5 to 5:5 EtOAc/
hexane) to yield compound 7 (189 mg, 62%) and compound 16
(82 mg, 27%) as pale-yellow oils.
Data for compound 7: 1H NMR (500 MHz, CDCl3) δ 7.33-
7.25 (m, 15H), 4.57-4.50 (m, 6H, PhCH2), 3.93 (m, 2H, H-3,
4), 3.66 (d, J ) 5.8 Hz, 2H, H-6, 6′), 3.55 (dd, J ) 5.8, 9.5 Hz,
1H, H-1), 3.51 (dd, J ) 6.2, 9.5 Hz, 1H, H-1′), 3.36 (dd, J )
5.1, 10.3 Hz, 1H, H-2), 3.21 (dd, J ) 5.1, 10.3 Hz, 1H, H-5),
2.0 (bs, 2H), 0.9 (s, 9H, C(CH3)3), 0.05 (s, 6H, CH3); 13C NMR
(125 MHz, CDCl3) δ 138.4, 138.3, 138.2, 128.37, 128.32, 128.2,
127.74, 127.70, 127.67, 127.65, 127.63, 127.5, 86.7, 86.0, 73.2,
71.8, 71.7, 71.0, 63.3, 63.1, 61.6, 25.9, 18.3, -5.4; HRMS calcd
for C33H45N1O4Si [M + H]+• 548.3190, found 548.3185.
(27) Takebayashi, M.; Hiranuma, S.; Kanie, Y.; Kajimoto, T.; Kanie,
O.; Wong, C.-H. J . Org. Chem. 1999, 64, 5280.
J . Org. Chem, Vol. 69, No. 19, 2004 6281

Liu et al.
N-ter t-Bu tyloxyca r bon yl-(2R,3R,4R,5R)-2-(a zid o-m eth -
yl)-3,4-d i-O-ben zyl-5-(O-ben zyl-m eth yl)-p yr r olid in e (23).
To a solution of compound 22 (154 mg, 0.35 mmol) and Et3N
(56 µL) in dry CH2Cl2 (1.8 mL) was added dropwise MsCl (30
µL, 0.39 mmol, 1.1 equiv) at 0 °C under argon. The reaction
mixture was stirred at 0 °C for 2 h, diluted with EtOAc (6
mL), and washed successively with aqueous saturated NaHSO4
(1.5 mL), aqueous saturated NaHCO3 (1.5 mL), and water (1.5
mL). The organic phase was dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo to yield a yellow oil. The
crude product was taken up in dry DMF (3 mL) in the presence
of NaN3 (190 mg, 2.92 mmol), and the reaction mixture was
stirred at 75 °C under argon for 24 h. After the solvent was
removed in vacuo, the residue was purified by flash chroma-
tography (silica, 1:9 EtOAc/hexane) to yield compound 23
(mixture of atropoisomers) (85 mg, 53%) as a pale-yellow oil:
1H NMR (500 MHz, CDCl3) δ 7.35-7.20 (m, 15H), 4.65-4.39
(m, 6H, PhCH2), 4.15-4.19 (m, 1.5H), 4.00-3.93 (m, 3H), 3.85
(dd, J ) 3.7, 11.0 Hz, 0.5H), 3.74 (dd, J ) 4.0, 8.8 Hz, 0.5H
atropoisomer b), 3.69 (dd, J ) 4.0 Hz, 11.4 Hz, 0.5H atropo-
isomer a), 3.51-3.45 (m, 1H), 3.28-3.35 (m, 1H), 1.47 (s, 4.5H),
1.4 (s, 4.5H); 13C NMR (125 MHz, CDCl3) δ 154.4, 138.1,
137.54, 137.51, 137.4, 128.48, 128.42, 128.41, 128.3, 128.2,
127.8, 127.79, 127.72, 127.6, 127.59, 127.58, 127.4, 83.4, 82.7,
82.0, 81.2, 73.03, 73.00, 71.3, 71.26, 71.22, 71.0, 68.3, 67.5, 63.0,
62.9, 62.7, 62.6, 50.9, 49.6, 28.4, 28.3; HRMS calcd for
2H, H-1, 6), 3.80 (dd, J ) 5.8, 12.4 Hz, 1H, H-6′), 3.73 (dd, J
) 6.6, 13.5 Hz, 1H, H-1′), 3.50-3.46 (m, 1H, H-5), 3.44-3.40
(m, 1H, H-2); 13C NMR (150 MHz, D2O) δ 86.3, 85.3, 72.6, 70.2,
69.2, 60.2; HRMS calcd for C6H12N4O3 [M + H]+• 189.0982,
found 189.0984.
(2S,3R,4R,5R)-2-(Azid om et h yl)-3,4-b is(b en zyloxy)-5-
((ben zyloxy)m eth yl)-1-m eth ylp yr r olid in e (28). To a solu-
tion of compound 26 (170 mg, 0.4 mmol) and pyridine (122
µL, 1.5 mmol) in CH2Cl2 (5 mL) was added MsCl (110 µL, 1.4
mmol) at 0 °C. The mixture was stirred at room temperature
for 2 h. The solvent was removed under high vacuum. Without
purification, the residue was dissolved in DMF (20 mL). NaN3
(200 mg, 3.1 mmol) and NaI (60.5 mg, 0.40 mmol) were added
to this solution. After 5 h, the reaction was quenched with H2O
and extracted with CH2Cl2. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (silica, 1:2
EtOAc/hexane) to afford azido compound 28 (156 mg, 87%):
1H NMR (500 MHz, CDCl3) δ 7.36-7.24 (m, 15H, Ph), 4.54 (d
of AB, J ) 11.7 Hz, 1H, PhCH2), 4.53 (d of AB, J ) 12.1 Hz,
1H, PhCH2), 4.50 (d of AB, J ) 12.1 Hz, 1H, PhCH2), 4.46 (d
of AB, J ) 12.1 Hz, 1H, PhCH2), 4.45 (d of AB, J ) 12.1 Hz,
1H, PhCH2), 4.39 (d of AB, J ) 11.7 Hz, 1H, PhCH2), 3.90 (d,
J ) 4.5 Hz, 1H, H-4), 3.81 (m, 1H, H-3), 3.59 (dd, J ) 8.5,
11.8 Hz, 1H, H-1), 3.54 (dd, J ) 5.2, 8.6 Hz, 1H, H-6), 3.42
(dd, J ) 8.2, 8.6 Hz, 1H, H-6′), 3.32 (dd, J ) 4.8, 11.8 Hz, 1H,
H-1′), 2.85 (dt, J ) 4.8, 8.5 Hz, 1H, H-2), 2.76 (ddd, J ) 2.5,
5.2, 7.7 Hz, 1H, H-5), 2.43 (s, 3H, NCH3); 13C NMR (125 MHz,
CDCl3) δ 138.2, 138.0, 137.7, 128.4, 128.3, 127.9, 127.7, 127.7,
127.6, 127.6, 127.5, 82.5, 82.0, 73.1, 71.9, 71.6, 71.0, 67.5, 49.4,
41.3; HRMS calcd for C28H32N4O3 [M + H]+• 473.2553, found
473.2528.
N-(((2S,3R,4R,5R)-3,4-Bis(b en zyloxy)-5-((b en zyloxy)-
m eth yl)-1-m eth ylp yr r olid in -2-yl)m eth yl)a ceta m id e (30).
To a solution of 28 (104 mg, 0.22 mmol) in dry THF (10 mL)
was added PPh3 (118 mg, 0.45 mmol). The mixture was heated
to 50 °C for 4 h and concentrated in vacuo. The residue was
dissolved in pyridine (8 mL). Acetic anhydride (91 µL, 0.86
mmol) was added to the reaction mixture at 0 °C. After 12 h,
the reaction was quenched with water (50 mL) and extracted
with CH2Cl2. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. Purification of the
residue by flash chromatography (silica, 60:1 CH2Cl2/MeOH)
afforded 30 (91 mg, 87% yield from 28): 1H NMR (500 MHz,
CDCl3) δ 7.37-7.25 (m, 15H, Ph), 4.61 (d of AB, J ) 11.9 Hz,
1H, PhCH2), 4.57 (d of AB, J ) 12.1 Hz, 1H, PhCH2), 4.55 (d
of AB, J ) 12.1 Hz, 1H, PhCH2), 4.53 (d of AB, J ) 11.9 Hz,
1H, PhCH2), 4.52 (d of AB, J ) 11.9 Hz, 1H, PhCH2), 4.26 (d
of AB, J ) 11.9 Hz, 1H, PhCH2), 3.84 (d, J ) 3.3 Hz, 1H, H-4),
3.81 (d, J ) 4.9 Hz, 1H, H-3), 3.60 (dd, J ) 4.6, 9.7 Hz, 1H,
H-6), 3.54 (ddd, J ) 3.0, 5.3, 13.4 Hz, 1H, H-1), 3.48 (dd, J )
7.3, 9.7 Hz, 1H, H-6′), 3.34 (ddd, J ) 5.3, 7.6, 13.4 Hz, 1H,
H-1′), 2.77 (ddd, J ) 3.3, 4.9, 7.6 Hz, 1H, H-2), 2.72 (dt, J )
4.3, 7.6 Hz, 1H, H-5), 2.37 (s, 3H, NCH3), 1.69 (s, 3H, COCH3);
13C NMR (125 MHz, CDCl3) δ 169.9, 138.2, 138.1, 137.7, 128.7,
128.4, 128.3, 128.2, 128.1, 127.7, 127.6, 127.6, 83.0, 82.7, 71.4,
71.2, 71.2, 70.6, 40.2, 37.1, 23.1; HRMS calcd for C30H36N2O4
[M + H]+• 489.2753, found 489.2738.
C
32H38N4O5 [M + Na]+• 581.2734, found 581.2742.
(2R,3R,4R,5R)-2-(Azid o-m et h yl)-3,4-d i-O-b en zyl-5-(O-
ben zyl-m eth yl)-p yr r olid in e (24). To a solution of compound
23 (59 mg, 0.10 mmol) in dry CH2Cl2 (0.6 mL) was added
dropwise TFA (0.5 mL) at 0 °C under argon. The reaction
mixture was stirred at this temperature for 2 h. After the
solvent was removed in vacuo, the residue was purified by
flash chromatography (silica, 1:1 EtOAc/hexane) to yield
compound 24 (47 mg, 97%) as a pale-yellow oil: 1H NMR (500
MHz, CDCl3) δ 7.34-7.26 (m, 15H), 4.55-4.49 (m, 6H, PhCH2),
3.91 (t, J ) 3.7 Hz, 1H, H-3), 3.83 (t, J ) 4.4 Hz, 1H, H-4),
3.53 (dd, J ) 5.5, 9.5 Hz, 1H, H-6), 3.50 (dd, J ) 5.8, 9.5 Hz,
1H, H-6′), 3.41-3.30 (m, 4H, H-1, 1′, 2, 5); 13C NMR (125 MHz,
CDCl3) δ 138.0, 137.9, 137.8, 128.44, 128.41, 127.8, 127.79,
127.76, 127.72, 86.5, 85.7, 73.2, 72.00, 71.97, 70.1, 61.8, 61.4,
53.5; HRMS calcd for C27H30N4O3 [M + H]+• 459.2391, found
459.2394.
(2R,3R,4R,5R)-2-(Am in o-m eth yl)-3,4-d ih yd r oxy-5-(h y-
d r oxy-m eth yl)-p yr r olid in e (5). To a solution of compound
24 (32 mg, 70 µmol) in MeOH (0.6 mL) and aqueous HCl (0.15
mL, pH 4) was added Pd-C (20 mg). The reaction mixture
was stirred under hydrogen atmosphere (70 psi) for 24 h before
being filtered through Celite. After the solvent was removed
in vacuo, the dihydrochloride of compound 5 was obtained
(quant) as an oily residue: 1H NMR (600 MHz, D2O) δ 4.00-
3.95 (m, 2H, H-3, 4), 3.84 (dd, J ) 4.8, 14.5 Hz, 1H, H-6), 3.75
(dd, J ) 7.5, 14.5 Hz, 1H, H-6′), 3.52-3.47 (m, 1H, H-5), 3.36
(dd, J ) 6.0, 15.8 Hz, 1H, H-1), 3.34-3.28 (m, 1H, H-2), 3.27
(dd, J ) 10.0, 15.8 Hz, 1H, H-1′); 13C NMR (150 MHz, D2O) δ
78.4, 76.1, 62.9, 60.2, 58.5, 40.9; ESI+ m/z 163 (M + H+), 185
(M + Na+).
(2R,3R,4R,5R)-2-(Azid o-m et h yl)-3,4-d ih yd r oxy-5-(h y-
d r oxy-m eth yl)-p yr r olid in e (6). To a solution of compound
24 (40 mg, 87 µmol) in MeOH (0.8 mL) and aqueous HCl (0.2
mL, pH 4) was added Pd-C (25 mg). The reaction mixture
was stirred under hydrogen atmosphere (70 psi) for 24 h and
filtered through Celite, and the solvent was removed in vacuo.
The crude product obtained, CuSO4 (1 mg) and K2CO3 (35 mg,
0.25 mmol, 3 equiv), were dissolved in MeOH (0.8 mL) and
H2O (0.2 mL). TfN3 in CH2Cl2 (0.7 mL) was added dropwise
over 1 h at room temperature, and the reaction mixture was
vigorously stirred at room temperature for 24 h. After the
solvent was removed in vacuo, the residue was purified by
flash chromatography (silica, 8:2:0.2 CHCl3/MeOH/H2O) to
yield compound 6 (14 mg, 85% yield) as a white solid: 1H NMR
(500 MHz, D2O) δ 4.03-4.01 (m, 2H, H-3, 4), 3.88-3.83 (m,
N-(((2S,3R,4R,5R)-3,4-Dih yd r oxy-5-(h yd r oxym eth yl)-1-
m eth ylp yr r olid in -2-yl)m eth yl)a ceta m id e (4). Compound
30 (34 mg, 0.07 mmol) was dissolved in AcOH/THF/H2O (4:2:
1, 6 mL) and treated with Pd-C (10%) (10 mg). The reaction
mixture was hydrogenated under 50 psi pressure. After 1 d,
the catalyst was removed by filtration, and the filtrate was
concentrated and purified by flash chromatography (silica, 6:4:
0.7 CHCl3/MeOH/H2O) to yield 4 (16.6 mg, 89%) as an oil: 1H
NMR (500 MHz, D2O) δ 4.06 (dd, J ) 3.0, 5.5 Hz, 1H, H-4),
3.77 (dd, J ) 3.0, 4.4 Hz, 1H, H-3), 3.75 (dd, J ) 7.3, 11.7 Hz,
1H, H-6), 3.68 (dd, J ) 4.4, 11.7 Hz, 1H, H-6′), 3.46 (dd, J )
4.1, 14.0 Hz, 1H, H-1), 3.22 (dd, J ) 7.0, 14.0 Hz, 1H, H-1′),
2.73 (m, 1H, H-2), 2.45 (m, 1H, H-5), 2.35 (s, 3H, NCH3), 1.96
(s, 3H, COCH3); 13C NMR (125 MHz, D2O) δ 175.2, 79.4, 76.7,
6282 J . Org. Chem., Vol. 69, No. 19, 2004

N-Acetyl-â-hexosaminidase Inhibitor Libraries
71.8, 69.3, 59.3, 40.7, 40.4, 22.6; HRMS calcd for C9H18N2O4
[M + Na]+• 241.1164, found 241.1172.
7.23 (d, J ) 8.8 Hz, 2H), 6.74 (d, J ) 8.8 Hz, 2H), 3.98 (t, J )
2.2 Hz, 1H, H-4), 3.85 (d of AB, J ) 14.0 Hz, 1H, ArCH2), 3.82
(d of AB, J ) 14.0 Hz, 1H, ArCH2), 3.79 (t, J ) 2.6 Hz, 1H,
H-3), 3.75 (dd, J ) 5.3, 11.4 Hz, 1H, H-6), 3.63 (dd, J ) 3.1,
11.4 Hz, 1H, H-6′), 3.40 (dd, J ) 3.1, 13.6 Hz, 1H, H-1), 3.25
(dd, J ) 5.7, 13.6 Hz, 1H, H-1′), 3.08 (m, 1H, H-2), 3.01 (m,
1H, H-5), 2.88 (s, 6H, N(CH3)2), 1.91 (s, 3H, COCH3); 13C NMR
(150 MHz, CD3OD) δ 177.3, 151.0, 129.6, 113.8, 80.7, 80.1,
69.4, 69.1, 60.4, 51.4, 40.7, 39.6, 22.1; HRMS calcd for
Gen er a l P r oced u r e To P r ep a r e Ar om a tic Der iva tives
of Im in ocyclitol 4. To the mixture of 4 (11 mg, 0.053 mmol)
and aldehyde (0.11 mmol) in MeOH (0.7 mL) was added
NaBH3CN (6.7 mg, 0.11 mmol). After the mixture was stirred
at room temperature overnight, the solvent was removed in
vacuo. Purification of the residue with flash chromatography
(silica, 9:1 CHCl3/MeOH) afforded the product.
N-(((2R,3R,4R,5R)-1-(4-(P yr id in -2-yl)ben zyl)-3,4-d ih y-
d r oxy-5-(h yd r oxym et h yl)p yr r olid in -2-yl)m et h yl)a cet a -
C
17H27N3O4 [M + Na]+• 360.1899, found 360.1896.
Assa y for Hexosa m in id a se Activity. 4-Methylumbelly-
1
m id e (31a ). Yield 62%; H NMR (500 MHz, CD3OD) δ 8.58
feryl-N-acetyl-â-D-glucosamine at a final concentration of 0.1
mM in sodium citrate buffer (0.1 M, pH 4.5) was used as a
substrate for hexosaminidase activity assay. Human placental
hexosaminidases were diluted in sodium citrate buffer (0.1 M,
pH 4.5) and incubated with the substrate and inhibitor at 37
°C for 2 h. The reaction was then stopped by the addition of
sodium glycine buffer (0.5 M, pH 10.5). Hexosaminidase
activity was measured by the release of 4-methylumbelliferone,
utilizing fluorometry with the excitation wavelength of 360
nm and the emission wavelength of 460 nm.
Gen er a l P r oced u r e for Am id e Cou p lin g Rea ction s. To
each 200-µL well of a microtiter plate containing diisopropyl
ethylamine (10 µL, 80 mM solution in MeOH), HBTU (6 µL,
106 mM solution in MeOH), and carboxylic acid (2 µL, 400
mM solution in MeOH) was added 5 (2 µL, 400 mM solution
in MeOH). The mixture was kept at room temperature
overnight before being diluted with H2O and transferred to a
second 96-well microtiter plate to make a 0.25 mM solution of
the inhibitor (based on 100% conversion of 5) for the enzymatic
assay.
Gen er a l P r oced u r e for Tr ia zole F or m a tion Rea ction s.
To each 200-µL well of a microtiter plate containing alkynes
(10 µL, 400 mM solution in toluene) was added 2 µL of 6 (400
mM solution in MeOH), except when the starting material was
solid and 50 µL of toluene was used as solvent. The mixture
was heated to 80 °C for 24 h before diluted with H2O and
transferred to a second 96-well microtiter plate to make a 0.25
mM solution of the inhibitor (based on 100% conversion of 6)
for the enzymatic assay.
Biologica l Assa ys of Micr otiter P la te Libr a r y. The
microtiter plate assay was performed on a fluorescence spec-
trophotometer at room temperature. Assays were run in citrate
buffer (pH 4.5). Human placenta hexosaminidases stock (10
µL, 2.38 U/mL), inhibitor (10 µL, 0.25 mM), and 4-methylum-
bellyferyl-N-acetyl-â-D-glucosamine (10 µL, 0.2 mM in DMF)
were used in the assay (total volume was 100 µL). The
fluorescence signal was monitored by λ excitation at 335 nM
and λ emission at 460 nM.
(d, J ) 4.0 Hz, 1H, CHdN-C), 7.91-7.81 (m, 4H), 7.54 (d, J
) 8.0 Hz, 2H), 7.34 (ddd, J ) 1.1, 5.1, 7.3 Hz, 1H, CHCHdN),
4.02 (m, 3H, ArCH2, H-4), 3.83 (t, J ) 2.7, 1H, H-3), 3.77 (dd,
J ) 4.8, 11.4 Hz, 1H, H-6), 3.64 (dd, J ) 3.0, 11.4 Hz, 1H,
H-6′), 3.44 (dd, J ) 3.3, 14.0 Hz, 1H, H-1), 3.31 (dd, J ) 5.9,
14.0 Hz, 1H, H-1′), 3.13 (m, 1H, H-2), 3.06 (m, 1H, H-5), 1.93
(s, 3H, COCH3); 13C NMR (125 MHz, CD3OD) δ 173.5, 158.9,
150.2, 142.4, 139.1, 138.9, 129.7, 128.1, 123.6, 122.5, 81.1, 80.7,
69.9, 69.7, 61.1, 51.9, 40.2, 22.7; HRMS calcd for C20H25N3O4
[M + H]+• 372.1923, found 372.1911.
N-(((2R,3R,4R,5R)-1-(4-Hyd r oxyben zyl)-3,4-d ih yd r oxy-
5-(h ydr oxym eth yl)pyr r olidin -2-yl)m eth yl)acetam ide (31b).
Yield 62%; 1H NMR (600 MHz, CD3OD) δ 7.20 (d, J ) 8.8 Hz,
2H), 6.71 (d, J ) 8.8 Hz, 2H), 3.98 (t, J ) 2.2 Hz, 1H, H-4),
3.87 (d of AB, J ) 13.8 Hz, 1H, ArCH2), 3.82 (d of AB, J )
13.8 Hz, 1H, ArCH2), 3.79 (t, J ) 2.6 Hz, 1H, H-3), 3.75 (dd,
J ) 5.3, 11.4 Hz, 1H, H-6), 3.62 (dd, J ) 3.1, 11.4 Hz, 1H,
H-6′), 3.40 (dd, J ) 3.5, 13.8 Hz, 1H, H-1), 3.25 (dd, J ) 6.2,
13.8 Hz, 1H, H-1′), 3.07 (dt, J ) 3.0, 6.2 Hz, 1H, H-2), 3.00
(dt, J ) 2.2, 4.4 Hz, 1H, H-5), 1.92 (s, 3H, COCH3); 13C NMR
(150 MHz, CD3OD) δ 173.5, 157.4, 131.3, 130.5, 116.1, 81.3,
80.7, 69.9, 69.6, 61.0, 51.8, 40.2, 22.7; HRMS calcd for
C
15H22N2O5 [M + H]+• 311.1607, found 311.1598.
N-(((2R,3R,4R,5R)-1-((Ben zo[d ][1,3]d ioxol-4-yl)m eth yl)-
3,4-d ih yd r oxy-5-(h yd r oxym et h yl)p yr r olid in -2-yl)m et h -
yl)a ceta m id e (31c). Yield 67%; 1H NMR (600 MHz, CD3OD)
δ 6.86 (d, J ) 7.9 Hz, 1H), 6.72 (t, J ) 7.9 Hz, 1H), 6.64 (d, J
) 7.9 Hz, 1H), 5.88 (d, J ) 1.3 Hz, 1H, OCH2O), 5.86 (d, J )
1.3 Hz, 1H, OCH2O), 3.92 (t, J ) 2.6 Hz, 1H, H-4), 3.87 (d of
AB, J ) 14.0 Hz, 1H, ArCH2), 3.78 (d of AB, J ) 14.0 Hz, 1H,
ArCH2), 3.75 (dd, J ) 4.8, 11.4 Hz, 1H, H-6), 3.70 (t, J ) 3.1
Hz, 1H, H-3), 3.59 (dd, J ) 2.6, 11.4 Hz, 1H, H-6′), 3.45 (dd, J
) 2.6, 14.0 Hz, 1H, H-1), 3.19 (dd, J ) 6.0, 14.0 Hz, 1H, H-1′),
3.02 (dt, J ) 3.6, 6.0 Hz, 1H, H-2), 2.90 (dt, J ) 2.6, 4.8 Hz,
1H, H-5), 1.86 (s, 3H, COCH3); 13C NMR (150 MHz, CD3OD)
δ 173.5, 148.8, 146.8, 123.6, 122.6, 122.4, 108.2, 102.0, 81.2,
80.6, 69.6, 69.4, 60.8, 45.7, 39.7, 22.7; HRMS calcd for
C
16H22N2O6 [M + H]+• 339.1556, found 339.1552.
N-(((2R,3R,4R,5R)-1-((5-(4-Ch lor op h en yl)fu r a n -2-yl)-
Ack n ow led gm en t. We thank the labs of Professors
K. B. Sharpless and V. Fokin for sharing the samples
of alkynes and azides for these studies. This research
was supported by Optimer Pharmaceuticals, Co.
m eth yl)-3,4-dih ydr oxy-5-(h ydr oxym eth yl)pyr r olidin -2-yl)-
m eth yl)a ceta m id e (31d ). Yield 60%; 1H NMR (500 MHz,
CD3OD) δ 7.64 (m, 2H), 7.35 (m, 2H), 6.71 (d, J ) 3.3 Hz, 1H),
6.38 (d, J ) 3.3 Hz, 1H), 4.08 (d of AB, J ) 15.0 Hz, 1H,
ArCH2), 3.96 (d of AB, J ) 15.0 Hz, 1H, ArCH2), 3.94 (dd, J )
4.0, 11.4 Hz, 1H, H-6), 3.86 (dd, J ) 4.8, 11.4 Hz, 1H, H-6′),
3.76 (m, 2H, H-3, 4), 3.51 (dd, J ) 3.0, 13.8 Hz, 1H, H-1), 3.22
(dd, J ) 6.2, 13.8 Hz, 1H, H-1′), 3.14 (m, 2H, H-2, 5), 1.89 (s,
3H, COCH3); 13C NMR (125 MHz, CD3OD) δ 173.5, 154.8,
153.3, 133.7, 131.0, 129.9, 126.0, 111.1, 107.6, 81.1, 80.5, 70.5,
69.6, 61.1, 45.1, 40.2, 22.7; HRMS calcd for C19H23N2O5Cl [M
+ Na]+• 417.1193, found 417.1189.
Note Ad d ed a fter ASAP P ostin g. Several structures
were missing from Figure 6 in the version posted ASAP
August 7, 2004; the corrected version was posted August
19, 2004.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra for
compounds 4-8, 10-12, 14-18, 21, 23, 24, 26, 28, 30, and
31a -e. This material is available free of charge via the
Internet at http://pubs.acs.org.
N-(((2R,3R,4R,5R)-1-(4-(Dim et h yla m in o)b en zyl)-3,4-
d ih yd r oxy-5-(h yd r oxym et h yl)p yr r olid in -2-yl)m et h yl)-
1
a ceta m id e (31e). Yield 70%; H NMR (600 MHz, CD3OD) δ
J O049355H
J . Org. Chem, Vol. 69, No. 19, 2004 6283