New Electronic Analogs of the Sialyl cation: N-Functionalized 4-Acetamido-2,4-dihydroxypiperidines. Inhibition of Bacterial Sialidases

Full text of DOI:10.1021/jo9708497

J . Org. Chem. 1997, 62, 7489-7494
7489
for the design and synthesis of sialidase (or neuramini-
dase) geometric transition state analog inhibitors, par-
ticularly for the influenza enzyme.7 These inhibitors, as
exemplified by 2,3-didehydro-2-deoxy-N-acetylneuramin-
ic acids 2, and more recently reported carbocyclic analogs
37g utilize unsaturation to flatten the pyranosyl ring as
a mimic of the transition state 1a /b. Sialidase electronic
transition state inhibitors reported thus far mimic oxo-
carbenium ion character by replacing the pyranosyl ring
oxygen with a basic nitrogen atom (piperidines 4), which
on protonation would mimic the positive formal charge
placed on oxygen in structure 1b.8
New Electr on ic An a logs of th e Sia lyl
Ca tion : N-F u n ction a lized
4-Aceta m id o-2,4-d ih yd r oxyp ip er id in es.
In h ibition of Ba cter ia l Sia lid a ses
Ian B. Parr and Benjamin A. Horenstein*
Department of Chemistry, University of Florida,
Gainesville, Florida 32611
Received May 13, 1997
In tr od u ction
Sialic acids (N-acetylneuraminic acid, NeuAc) are nine-
carbon ketoses which have the unusual feature of a
highly acidic carboxyl group (pKa ∼ 2.8) immediately
adjacent to the anomeric carbon. Sialic acids are typi-
cally found in terminal glycosidic linkages of cell surface
glycoproteins and glycolipids and are represented in
species as diverse as bacteria, trypanosomes, and higher
members of the animal kingdom, including humans.1,2
A variety of biological phenomena are associated with
recognition of sialosides, including viral replication,
escape of immune detection, and cell adhesion3 providing
considerable interest in the development of inhibitors of
sialyltransferases and sialidases for mechanistic and
clinical applications.4
Kinetic and kinetic isotope effect analyses of viral and
bacterial sialidases have led to the conclusion that as in
solution,5 the enzymatic reaction involves catalysis pro-
ceeding through transition states having oxocarbenium
ion character.6 Sialidase transition states resemble the
resonance pair 1a /b which has development of partial
An alternate design strategy would mimic the elec-
tronic charge distribution of resonance contributor 1a
with inhibitors 5, which place the formal charge at a site
electronically equivalent to the glycosidic carbon of
oxocarbenium ion resonance contributor 1a . Recent
experimental work has shown that placement of the
charge mimic in the position analogous to the anomeric
carbon of a glycosidase substrate can be an effective
strategy for glycosidase inhibition, e.g., isofagomine.9
We are evaluating an approach which employs a
central “core” structure like 5, upon which additional
functionality could be installed to tailor the inhibitor for
its particular target. For example, sialidase inhibitors
might incorporate aglycon functionality to complement
transition state mimicry, or sialyltransferase inhibitors10
might include structural features of the leaving group
(CMP) or acceptor sugar.11a,b At issue in the present work
is synthesis of the core structure; previously reported
inhibitors of the types 2-4 do not readily lend themselves
to these design requirements. Given that hydrophobic
aryl sialosides are good substrates for sialidases,12 we
wished to test the efficacy of inhibitors which included a
positive charge at the C-2 anomeric carbon and an
associated flattening of the pyranosyl ring about atoms
C6-O6-C2-C3. Outstanding progress has been made
* Corresponding author. Phone: 352-392-9859. E-mail: horen@
chem.ufl.edu.
(1) (a) Schauer, R. Adv. Carbohydr. Chem. Biochem. 1982, 40, 131-
234. (b) Varki, A. Glycobiology 1992, 2, 25-40.
(7) (a) Meindl, P.; Tuppy, H. Monatsh Chem. 1969, 100, 1295-1306.
(b) Vasella, A.; Wyler, R. Helv. Chim. Acta 1991, 74, 451-463. (c)
Holzer, C. T.; von Itzstein, M.; J in, B.; Pegg, M. S.; Stewart, W. P.;
Wu, W.-Y. Glycoconjugate J . 1993, 10, 40-44. (d) Singh, S.; J edrzejas,
M. J .; Air, G. M.; Luo, M.; Laver, W. G.; Brouillette, W. J . J . Med.
Chem. 1995, 38, 3217-3225. (e) Bamford, M. J .; Pichel, J . C.; Husman,
W.; Patel, B.; Storer, R.; Weir, N. G. J . Chem. Soc., Perkin Trans. I
1995, 1181-1187. (f) Sollis, S. L.; Smith, P. W.; Howes, P. D.; Cherry,
P. C.; Bethell, R. C. Bioorg. Med. Chem. Lett. 1996, 6, 1805-1808. (g)
Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan,
S.; Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, W.
G.; Stevens, R. C. J . Am. Chem. Soc. 1997, 119, 681-690.
(2) (a) Harduin-Lepers, A.; Recchi, M. A.; Delannoy, P. Glycobiology
1995, 5, 741-758. (b) Broquet, P.; Baubichon-Cortay, H.; George, P.;
Louisot, P. Int. J . Biochem. 1991, 23, 385-389. (c) Ferrero-Garcia, M.
A.; Trombetta, S. E.; Sanchez, D. O.; Reglero, A.; Frasch, A. C. C.;
Parodi, A. J . Eur. J . Biochem. 1993, 213, 765-771. (d) Gotttschalk,
A.; Bhargava, A. S. The Enzymes; Academic Press: New York, 1971;
Vol. 5, pp 321-342.
(3) (a) Schauer, R. Trends Biochem. Sci. 1985, 10, 357-360. (b)
Biology of the Sialic Acids; Rosenberg, A., Ed.; Plenum Press: New
York, 1995.
(4) (a) Taylor, G. Curr. Opin. Struct. Biol. 1996, 6, 830-837. (b)
Colman, P. M. Pure Appl. Chem. 1995, 67, 1683-1688. (c) Bamford,
M. J . J . Enzyme Inhib. 1995, 10, 1-16. (d) Khan, S. H.; Matta, K. L.
In Glycoconjugates. Composition, Structure, and Function; Allen, H.
J ., Kisailus, E. C., Eds.; Marcel Dekker, Inc.: New York, 1992; pp 361-
378.
(5) (a) Ashwell, M.; Guo, X.; Sinnott, M. L. J . Am. Chem. Soc. 1992,
114, 10158-10166. (b) Horenstein, B. A.; Bruner, M. J . Am. Chem.
Soc. 1996, 118, 10371-10379. (c) Horenstein, B. A. J . Am. Chem. Soc.
1997, 119, 1101-1107.
(8) (a) Bernet, B.; Murty, A. R. C. B.; Vasella, A. Helv. Chim. Acta
1990, 73, 940-958. (b) Czollner, L.; Kuszmann, J .; Vasella, A. Helv.
Chim. Acta 1990, 73, 1338-1358. (c) Walliman, K.; Vasella, A. Helv.
Chim. Acta 1990, 73, 1359-1372. (d) Glanzer, B. I.; Gyorgydeak, Z.;
Bernet, B.; Vasella, A. Helv. Chim. Acta 1991, 74, 343-369.
(9) (a) J espersen, T. M.; Dong, W.; Sierks, M. R.; Skrydstrup, T.;
Lundt, I.; Bols, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1778-1779.
(b) Ichikawa, M.; Igarashi, Y.; Ichikawa, Y. Tetrahedron Lett. 1995,
36, 1767-1770. (c) Ichikawa, Y.; Igarashi, Y. Tetrahedron Lett. 1995,
36, 4585-4586. (d) Ichikawa, Y.; Igarashi, Y.; Ichikawa, M. Tetrahe-
dron Lett. 1996, 37, 2707-2708.
(6) (a) Guo, X.; Sinnott, M. L. Biochem. J . 1993, 294, 653-656. (b)
Guo, X.; Laver, W. G.; Vimr, E.; Sinnott, M. L. J . Am. Chem. Soc. 1994,
116, 5572-5578. (c) Tiralongo, J .; Pegg, M. S.; von Itzstein, M. FEBS
Lett. 1995, 372, 148-150. (d) Chong, A. K. J .; Pegg, M. S.; Taylor, N.
R.; von Itzstein, M. Eur. J . Biochem. 1992, 207, 335-343.
(10) Rat liver R(2f6)-sialyltransferase affords â-dideuterium kinetic
isotope effects consistent with a transition state having oxocarbenium
ion character. Horenstein, B. A., Bruner, M. Unpublished results.
S0022-3263(97)00849-9 CCC: $14.00 © 1997 American Chemical Society

7490 J . Org. Chem., Vol. 62, No. 21, 1997
Notes
Sch em e 1. Syn th esis of Azid o Tr ifla te 14
hydrophobic aglycon mimic. Structures 6 and 5b il-
lustrate the correspondence between the transition state
for cleavage of an aryl sialoside and an inhibitor including
functionality which mimics the departing aryl aglycon.
Compounds 5 replace the glycerol C7-C9 side chain
found in NeuAc with a hydroxyl group. This feature
affords the opportunity for preparation of side chain
analogs7g and allows synthesis of the core inhibitor
structure with relative synthetic ease. The sialic acid
carboxylate group is an important component of sialidase
binding interactions;13 this functionality is included in
inhibitors 5 with an intervening methylene linker.14 We
report here the synthesis of trans,trans-4-acetamido-3,5-
dihydroxypiperidines 5a and (()-5b and, to demonstrate
their potential for development of inhibitors of sialic acid
transferring enzymes, show that they are competitive
inhibitors for bacterial sialidases from Salmonella typhi-
murium, Clostridium perfringens, and Vibrio cholerae.
8. The stereochemistry of the secondary alcohol was then
inverted by Swern oxidation16 to the ketone 9 and
stereoselective reduction17 with NaBH4 to ribofuranoside
10.
Resu lts a n d Discu ssion
We anticipated that the required xylo-stereochemistry
for azide 12 could be obtained by displacement of a
suitable derivative of the C-3 hydroxyl in alcohol 10.
Reaction of ribo triflate 11 (obtained from 10 by reaction
with triflic anhydride and pyridine) with sodium azide
afforded the protected azide 12 in 85% yield from 10.
Unlike the reported reaction for the corresponding xylo
triflate, no elimination products were observed.18 The
xylo-configuration of azide 12 was established by X-ray
crystallographic analysis of a diacetyl derivative.19
Desilylation of 12 afforded alcohol 13, which was then
converted to the triflate 14. The hindered base 2,6-di-
tert-butyl-4-methylpyridine had to be used in order to
obtain the triflate in high yield.20 Triflate 14 was
sufficiently stable to purify it by column chromatography
on silica gel, but decomposed slowly at 4 °C, and so was
used immediately after purification. The synthesis of the
key intermediate azido triflate 14 from commercially
available 1,2-isopropylidene-R-D-xylose was achieved in
44% overall yield for seven steps and can be prepared at
the gram scale.
Syn th esis of In h ibitor s. The route to the required
trans,trans-3,5-dihydroxy-4-acetamidopiperidines 5a and
(()-5b was based on the plan that reductive cyclization
of a 5-N-alkylxylose derivative (19, Scheme 2) would
readily afford piperidines with the desired relative ster-
eochemistry; the 5-N-alkylxylose compounds would be
prepared by N-alkylation of primary amines with a
xyloside derivatized at C-5 for nucleophilic displacement,
as outlined in Scheme 1.
The synthesis started from 1,2-isopropylidene-R-D-
xylofuranose15 (7), (Scheme 1), which was selectively
protected in 94% yield as the tert-butyldimethylsilyl ether
The purified triflate 14 was converted to the secondary
amines 15a ,b by displacement with glycine methyl ester
and D/L phenylalanine methyl ester, respectively,21 as
shown in Scheme 2. The displacements proceeded
smoothly at room temperature, with yields of 80% for
(11) (a) We do not intend to imply that an aglycon or nucleophile is
not part of the transition state for the reaction; it is a convenient
artifice of inhibitor design to separate these peripheral transition state
structural features from the core N-acetylneuraminyl unit which serves
as a charge mimic. (b) For a pertinent example of glycosyltransferase
inhibitor design, see: Hashimoto, H.; Endo, T.; Kajihara, Y. J . Org.
Chem. 1997, 62, 1914-1915.
(12) (a) Eschenfelder, V.; Brossmer, R. Carbohydr. Res. 1987, 162,
294-297. (b) Zbiral, E.; Schreiner, E.; Salunkhe, M. M.; Schulz, G.;
Kleineidam, R. G.; Schauer, R. Liebigs Ann. Chem. 1989, 519-526.
(13) (a) Vargese, J . N.; Laver, W. G.; Colman, P. M. Nature 1983,
303, 35-40. (b) Burmeister, W. P.; Ruigrok, R. W. H.; Cusack, S. EMBO
J . 1992, 11, 49-56. (c) Crennell, S. J .; Garman, E. F.; Philippon, C.;
Vasella, A.; Laver, W. G.; Vimr, E. R.; Taylor, G. L. J . Mol. Biol. 1996,
259, 264-280.
(15) A similar transformation of a 1,2-isopropylidene xylofuranoside
to the ribofuranoside has been reported: Ritzmann, G.; Klein, R. S.;
Hollenberg, D. H.; Fox, J . J . Carbohydr. Res. 1975, 39, 227-236.
(16) Mancuso, A. J .; Huang, S.-L.; Swern, D. J . Org. Chem. 1978,
43, 2480-2482.
(17) Sour, W.; Thomas, G. H. S. Can. J . Chem. 1966, 44, 836-837.
(18) (a) Ozols, A. M.; Azhayer, A. V.; Dyatkina, N. B.; Krayezsky,
A. A. Synthesis 1980, 557-559. (b) Ozols, A. M.; Azhayer, A. V.;
Krayezsky, A. A.; Ushakov, A. S.; Gnuchev, N. V.; Cortikh, B. P.
Synthesis 1980, 559-561.
(14) Direct attachment of a carboxylate residue to nitrogen is not
appropriate as the resulting carbamic acid would not be stable. The
methylene bridge between the carboxylate and secondary nitrogen
increases the distance between the carboxylate and anomeric carbon
mimic by ∼1.1 Å relative to the C-COO- bond length for N-
acetylneuraminic acid.
(19) Obtained by reduction of azide 12 to the amine with Raney
nickel, N-acetylation, desilylation, and O-acetylation. X-ray: Abboud,
K.; Parr, I. B.; Horenstein, B. A. Manuscript in prepration.
(20) Binkley, R. W.; Ambrose, M. G. J . Carbohydr. Chem. 1984, 3,
1-49.
(21) Yenakis, D.; Moll, N.; Gross, B. Synthesis 1983, 541-542.

Notes
J . Org. Chem., Vol. 62, No. 21, 1997 7491
Sch em e 2. Syn th esis of In h ibitor s 5a a n d (()-5b
fr om Tr ifla te 14
F igu r e 1. Inhibition of S. typhimurium sialidase by (()-5b.
Double reciprocal plot with 2-O-(p-nitrophenyl)-R-D-N-acetyl-
neuraminic acid at varying concentrations (100-2000 µM)
with fixed concentrations of inhibitor (()-5b: (9) 0, (b); 0.4,
and (4) 2 mM. Reactions were initiated with 3.6 µg of sialidase.
Ta ble 1
Ki, µM
sialidase
5a
(()-5b
Km, µMa
S. typhimurium
C. perfringens
V. cholerae
2500 ( 400
400 ( 100
>2000b
560 ( 30
500 ( 70
1500 ( 200
1200 ( 100
280 ( 30
2700 ( 740
using the substrate 2-O-(p-nitrophenyl)-R-D-N-acetyl-
neuraminic acid (Table 1).12a Figure 1 presents a double-
reciprocal plot for the inhibition of the S. typhimurium
sialidase by (()-5b, from which it is concluded that the
inhibition is competitive; the remaining inhibitor/siali-
dase inhibition kinetics were also well fit to competitive
inhibition patterns. The C. perfringens sialidase is
inhibited to the same extent by 5a or (()-5b, whereas
the S. typhimurium enzyme binds the more hydrophobic
(()-5b almost 5 times more tightly than 5a . Compound
5a did not show inhibition at concentrations below 2 mM,
and (()-5b was a modest inhibitor of the V. cholerae
sialidase, with Ki ) 1.5 mM.
Compounds 5a and (()-5b represent the core structure
of a sialic acid oxocarbenium ion analog, lacking the C6-
C9 glycerol side chain found in the natural sialidase
substrates and p-nitrophenyl glycoside substrate em-
ployed in this work. Despite this, the data presented in
Table 1 show that significant inhibition may be obtained,
particularly for (()-5b. For both the S. typhimurium and
V. cholerae sialidases, (()-5b binds approximately 2 times
tighter than the p-nitrophenyl glycoside substrate (with
the assumption that Km ≈ Kd, based on the observation
of intrinsic kinetic isotope effects6), while for the C.
perfringens sialidase 5a ,b are bound with slightly lower
affinity than substrate. The Ki’s for 5a and (()-5b are
modest, being in the 10-4 - 10-3 M range. The following
observations lead us to suggest that inhibitors 5, which
lack a glycerol side chain, are important leads for further
15a ,b. Amines 15a ,b were then protected as the CBZ
carbamates 16a ,b in 66-89% yields.22
The azides 16a ,b were then reduced23 to the corre-
sponding amines 17a ,b which were acetylated to give the
3-acetamidofuranosides 18a ,b in 60% and 86% overall
yields from 16a ,b. The 1,2-isopropylidene ketal of com-
pounds 18a ,b was hydrolyzed to provide the hemiacetals
19a ,b as a mixture of R- and â-anomers. From earlier
work we found that it was helpful to carry out the
hydrolytic removal of the isopropylidene group with the
secondary amine blocked as the benzyl carbamate.24 The
hemiacetals 19a ,b were then reductively cyclized under
neutral conditions to give the piperidines 20a ,b in 78%
and 57% yields from 18a ,b. To the best of our knowledge,
this is the first report of the synthesis of piperidines with
this substitution pattern. Compounds 20a ,b were con-
verted to the sialidase inhibitors 5a and (()-5b by
alkaline hydrolysis in 70% and 52% respective yields
after chromatography. Presently, preparation of each
antipode of 5b, and side chain analogs, is underway.25a,b
In h ibition Stu d ies. Inhibition of the sialidases from
C. perfringens, S. typhimurium, and V. cholerae by
dihydroxypiperidines 5a and (()-5b was determined
(22) Scwarz, H.; Bumpas, F. M.; Page, I. H. J . Am. Chem. Soc. 1957,
79, 5697-5703.
(23) Kokotos, G.; Constantinou-Kokotou, V. J . Chem. Res., Miniprint
1992, 311.
(24) Acid hydrolysis of the ketal in analogous N-unprotected com-
pounds afforded products which appeared to have aromatized. We
have not fully characterized these reaction products; their aromatic
nature was evident from 1H NMR spectra which showed a multiplet
at δ 7.5-8.0 consisting of four hydrogens. For an example of aroma-
tization of a poly(hydroxypiperidine), see: Nishimura, Y. In Studies
in Natural Products Chemistry; Rahman, A. U., Ed.; Elsevier: Am-
sterdam, 1992; pp 495-578.
(25) (a) Racemic compounds were synthesized since we did not wish
to make initial assumptions regarding which stereoisomer might
ultimately afford the better inhibitor. (b) Synthesis of side chain
analogs raises the synthetic complexity by requiring discrimination
between the two hydroxyl groups of compounds 5 or precursors.

7492 J . Org. Chem., Vol. 62, No. 21, 1997
Notes
further purification. Sialidases from S. typhimurium, C. per-
fringens, and V. cholerae were purchased from Sigma. Solvents
were obtained from Fisher Scientific as ACS reagent grade. THF
was dried immediately before use by distillation from Na/
benzophenone under nitrogen. Moisture sensitive reactions were
performed under an atmosphere of nitrogen using standard
techniques. Analytical TLC was performed on silica gel 60F-
245 plates. Column chromatography was performed with Davi-
sil grade 633, type 60 A, silica gel (200-425 mesh). EI, CI, and
FAB mass spectra were recorded at the University of Florida.
Chemical ionization methods (CI) used either ammonia or
isobutane, and FAB MS used nitrobenzyl alcohol as matrix.
NMR and other spectral data for all compounds are presented
in the Supporting Information.
development of more potent sialidase inhibitors contain-
ing a side chain. Vasella and colleagues8d have measured
Ki’s for a series of piperidine-based inhibitors (structures
4) for the V. cholerae sialidase, in which the side chain
substituent X was varied from the full glycerol function-
ality to hydroxymethyl, methyl, and hydrogen. The
respective Ki’s were 0.029, 6.1, 9.6, and 10 mM, demon-
strating that 102-103 binding affinity is realized for a
full side chain versus truncants. Similarly, a recent
study of NeuAc-2-en analogs 2 in which the side chain X
was varied in length showed a systematic variation in
Ki with chain length against influenza sialidase, consis-
tent with an earlier report.7e,26 Also, side chain recogni-
tion is important for the S. typhimurium and influenza
sialidases, since the X-ray crystal structures show specific
interactions with the glycerol side chain.13
The three sialidases tested show different sensitivity
to the presence of a hydrophobic aglycon mimic in the
inhibitor. The inhibition data suggest that the active site
of the C. perfringens enzyme can accommodate a rela-
tively large hydrophobic group (i.e., the phenyl ring of
(()-5b) but that no net energetic advantage over binding
5a exists. Inhibitors 5a /5b bind weakly to the V. cholerae
sialidase, though the measurable Ki for (()-5b suggests
that it may benefit from a modest hydrophobic binding
effect relative to 5a . This is in contrast to the results
for the S. typhimurium enzyme, which binds (()-5b
almost 5 times more tightly than 5a which demonstrates
that favorable hydrophobic interactions may indeed be
utilized as a component of inhibitor design for the S.
typhimurium sialidase.27 It is also noteworthy that (()-
5b binding (560 ( 30 µM) to the S. typhimurium sialidase
is comparable to that of the geometric transition state
analog 2,3-dehydro-N-acetyl neuraminic acid (380 µM),28
making (()-5b one of the best inhibitors for the S.
typhimurium sialidase. Each antipode of inhibitor (()-
5b is likely to have a different Ki; the observed data
reflect the net inhibition. As mentioned, we are now
preparing each enantiomer of 5b separately to address
this issue.
In conclusion, we have described a synthetic route to
novel N-functionalized 4-acetamido-2,4-dihydroxypiper-
idines which allows for the flexible N-substitution based
on the starting amine employed. This is a new structural
class of sialidase inhibitor which combines transition
state analogy with the ability to include aglycon mimicry.
These compounds competitively inhibit bacterial siali-
dases with Ki’s ranging from ∼10-4-10-3 M, despite the
lack of the glycerol side chain analogous to the C7-C9
tail of N-acetylneuraminic acid. The results indicate that
(1) location of the oxocarbenium ion charge mimic in a
position analogous to the glycosyl carbon and (2) inclusion
of hydrophobic aglycon functionality are viable strategies
for the development of sialidase inhibitors. The inhibi-
tors reported represent the first generation of their type
and have served to demonstrate the potential of the
approach; the next generation of inhibitors will be those
that refine binding interactions for sialidases.
1,2-O-Isop r op ylid en e-5-O-(ter t-bu tyld im eth ylsilyl)-r-D-
xylofu r a n ose (8). 1,2-O-Isopropylidene-D-xylofuranose (7.608
g, 40 mmol) was dissolved in CH2Cl2 (120 mL) and cooled in an
ice bath. Triethylamine (7.8 mL, 56 mmol) and TBDMSCl (7.3
g, 48 mmol) were added. The reaction was stirred at 0 °C for
30 min, then warmed to rt, and stirred for 18 h at which time
additional triethylamine (0.7 mL, 5 mmol) and TBDMSCl (0.7
g, 4.6 mmol) were added to the reaction mixture with stirring
continued for 5 h. The reaction mixture was concentrated in
vacuo, and the residue was suspended in CH2Cl2 (40 mL) and
filtered. CHCl3 (120 mL) was added, and the solution was
washed with 1 M HCl (4 × 30 mL) and saturated aqueous NaCl
(1 × 30 mL) and dried (Na2SO4). Chromatography on silica gel
(1% methanol in CHCl3) afforded compound 8 (10.77 g, 88%) as
an oil. 1H NMR analysis of 8 showed a small amount of
disilylated product which was removed in the next step of the
synthesis: [R]20 -9.3 (c ) 10 g/100 mL, CHCl3); FAB HRMS
D
exact mass calcd for MH+ C14H29SiO5 305.1784, found 305.1784.
1,2-O-Isopr opyliden e-3-oxo-5-O-(ter t-bu tyldim eth ylsilyl)-
D-xylofu r a n ose (9). Oxalyl chloride (3.4 mL, 39 mmol) was
dissolved in dry CH2Cl2 (105 mL) and cooled to -55 °C. DMSO
(5.5 mL, 77.9 mmol) was added such that the temperature
remained below -50 °C, and the mixture was stirred for 2 min.
A solution of 8 (10.77 g, 35.4 mmol) in dry CH2Cl2 (35 mL) was
added slowly over 5 min, and the mixture was stirred at -50
°C for 30 min. Triethylamine (16.3 mL, 117 mmol) in dry CH2Cl2
(30 mL) was added and the mixture stirred at -55 °C for a
further 40 min. The reaction mixture was warmed to rt, stirred
for 2.5 h, then poured into 160 mL of water, and extracted with
CHCl3 (130 mL). The organic layer was then washed with
saturated aqueous NaCl (1 × 80 mL) and dried over Na2SO4.
The crude product was crystallized from hexane to give 9 as a
waxy solid (10.3 g, 96%): mp 32 °C; [R]20 +114 (c ) 10 g/100
D
mL, CHCl3); FAB HRMS exact mass calcd for MH+ C14H27SiO5
303.1627, found 303.1599.
1,2-O-Isop r op ylid en e-5-O-(ter t-bu tyld im eth ylsilyl)-D-r i-
bofu r a n ose (10). Ketone 9 (6.89 g, 22.8 mmol) was dissolved
in ethanol (150 mL) and water (50 mL) and cooled in an ice bath.
NaBH4 (5.61 g, 148.3 mmol) was then added, and stirring was
continued for 3.5 h. The reaction mixture was poured into water
(400 mL) and extracted with ethyl acetate (8 × 150 mL). The
combined organic layers were then dried (Na2SO4) and concen-
trated in vacuo to afford 10 (6.60 g, 95%): [R]20 +25.7 (c ) 4.1
D
g/100 mL, CHCl3).
1,2-O-Isopr opyliden e-3-[(tr iflu or om eth an esu lfon yl)oxy]-
5-O-(ter t-bu tyld im eth ylsilyl)-D-r ibofu r a n ose (11). Triflic
anhydride (1.8 mL, 10.6 mmol) was dissolved in 15 mL of dry
(CH2Cl)2 and added to a solution of pyridine (1.2 mL, 15.6 mmol)
in 10 mL of (CH2Cl)2 at -10 °C. After 3 min a precipitate
formed. A solution of 10 (3.2 g, 10 mmol) in 16 mL of (CH2Cl)2
was added at -10 °C, and the mixture was stirred for 2.25 h at
this temperature. Aqueous 5% NaHCO3 (40 mL) was added;
the reaction mixture was warmed to rt and stirred for 30 min
before being cast into CHCl3 (60 mL). The organic phase was
washed with 5% NaHCO3 (40 mL) and saturated aqueous NaCl
Exp er im en ta l Section
Gen er a l. Starting materials and reagents were purchased
from Sigma, Aldrich, or Fisher Scientific and were used without
(29) (a) Hindsgaul, O.; Kaur, K. J .; Srivastava, G.; Blaszczyk-Thurin,
M.; Crawley, S. C.; Heerze, L. D.; Palic, M. M. J . Biol. Chem. 1991,
266, 17858-17862. (b) Kajihara, Y.; Kodama, H.; Wakabayashi, T.;
Sata, K.; Hashimoto, H. Carbohydr. Res. 1993, 247, 179-193. (c)
Reboul, P.; George, P.; Miquel, D.; Louisot, P.; Broquet, P. Glycocon-
jugate J . 1996, 13, 69-79.
(26) Driguez, P.-A.; Barrere, B.; Quash, G.; Doutheau, A. Carbohydr.
Res. 1994, 262, 297-310.
(27) Angus, D. I., V. Itzstein, M. Carbohydr. Res. 1995, 274, 279-
283.
(28) Hoyer, L. L.; Roggentin, P.; Schauer, R.; Vimr, E. R. J . Biochem.
1991, 110, 462-467.
(30) Gross, H. J .; Bunsch, A.; Paulson, J . C.; Brossmer, R. Eur. J .
Biochem. 1987, 168, 595-602.

Notes
J . Org. Chem., Vol. 62, No. 21, 1997 7493
(30 mL) and then dried (Na2SO4). After being concentrated in
vacuo, the residue was dissolved in toluene (50 mL) and
re-evaporated; this procedure was repeated three times provid-
ing the triflate 11 (4.33 g, 95%) as a red oil.
mL of water, with Na2CO3 (0.826 g, 7.72 mmol) and CbzCl (0.97
mL, 6.79 mmol). Workup and purification as for 16a gave the
product 16b (1.218 g, 77%) as an oil: FAB HRMS exact mass
calcd for MH+ C26H31N4O7 511.2193, found 511.2205.
1,2-O-Isop r op ylid en e-3-d eoxy-3-a zid o-5-O-(ter t-bu tyld i-
m eth ylsilyl)-D-xylofu r a n ose (12). Triflate 11 (4.33 g, 10
mmol) was dissolved in a suspension of NaN3 (3.75 g, 57.7 mmol,
in 50 mL of ethanol) at rt. The reaction mixture was heated to
65-70 °C for 14 h, after which time additional NaN3 (3.75 g,
57.7 mmol) was added followed by reflux for 24 h. The reaction
mixture was then cooled to rt and stirred for 4 days. The
reaction mixture was concentrated and then partitioned between
water (30 mL) and CHCl3 (100 mL). The organic fraction was
then washed with water (2 × 30 mL) and saturated aqueous
NaCl (1 × 30 mL) and dried (Na2SO4). Column chromatography
on silica gel (4:6 hexane/CHCl3) afforded azide 12 (2.912 g, 89%)
as an oil: FAB HRMS exact mass calcd for MH+ C14H28SiN3O4
330.1849, found 330.1824.
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a m in o-5-N-(1′-ca r -
bom eth oxyeth yl)-5-N-[(ben zyloxy)ca r bon yl]-D-xylofu r a n -
ose (17a ). Pd/C (10%, 1 mg per 6-10 mg of azide 16) was
suspended in water, and to it a solution of azide 16a (0.707 g,
1.68 mmol) in 16 mL of methanol was added. NaBH4 (0.191 g,
5.05 mmol) was added in three portions over 15 min at rt. After
stirring for a further 30 min, the reaction mixture was filtered
through Celite. The Celite was washed with methanol (1
reaction volume) and water (10 reaction volumes). The pH of
the combined filtrates was adjusted to pH 7 with 1 M HCl and
the methanol removed under reduced pressure. The aqueous
layer was then adjusted to pH 1, washed with ethyl acetate (2
× 25 mL), basified to pH 10 with 10% Na2CO3, extracted with
CHCl3 (6 × 30 mL), basified to pH 12 with NaOH, and extracted
with ethyl acetate (4 × 20 mL). The organic extracts were
combined, dried (Na2CO3), and then concentrated in vacuo to
yield the crude amine 17a as an oil which was used immediately
without further purification.
1,2-O-Isopr opyliden e-3-deoxy-3-azido-D-xylofu r an ose (13).
The azide 12 (1.84 g, 5.6 mmol) was dissolved in 20 mL of 50%
aqueous THF, and CH3COOH (15 mL) was added. The reaction
mixture was stirred (rt, 18 h); then the reaction was carefully
quenched by the addition of water (60 mL) and sufficient Na2CO3
to raise the pH above 9. The mixture was extracted with CHCl3
(3 × 30 mL), and the combined organic phases were dried
(Na2SO4). Column chromatography on silica gel (1:3 ethyl
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a zid o-5-N-(1′-ca r bo-
m eth oxy-2′-ben zyleth yl)-5-N-[(ben zyloxy)ca r bon yl]-D-xy-
lofu r a n ose (17b). Amine 17b was prepared from azide 16b
(0.844 g, 1.65 mmol) according to the method above for 17a using
methanol (10 mL), water (40 mL), Pd/C (130 mg), and NaBH4
(188 mg, 4.96 mmol). Workup as for 17a afforded 17b as an oil
which was used immediately without further purification.
acetate/hexane) afforded alcohol 13 (1.14 g, 95%) as an oil: [R]20
D
-44 (c ) 3.87 g/100 mL, CHCl3); FAB HRMS exact mass calcd
for MH+ C8H14N3O4 216.0984, found 216.1016.
1,2-O-Isop r op ylid en e-3-d eoxy-3-a zid o-5-[(tr iflu or om eth -
a n esu lfon yl)oxy]-D-xylofu r a n ose (14). 4-Methyl-2,6-di-tert-
butylpyridine (1.28 g, 6.3 mmol) was dissolved in 10 mL of dry
(CH2Cl)2 under N2 and then cooled to -78 °C. Triflic anhydride
(1.05 mL, 6.3 mmol) was added to the solution which was then
stirred for 5 min. A solution of 13 (1.12 g, 5.2 mmol) in 12 mL
of dry (CH2Cl)2 was added to the triflate solution which was then
stirred at -10 °C for 30 min. The reaction mixture was warmed
to rt, 10 mL of hexane was added, and the solution was loaded
onto a silica gel column. Elution with 3:7 hexane/CHCl3 afforded
triflate 14 (1.41 g, 78%): FAB HRMS exact mass calcd for MH+
C9H13SN3F3O6 348.0477, found 348.0477.
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a cet a m id o-5-N-(1′-
ca r bom eth oxyeth yl)-5-N-[(ben zyloxy)ca r bon yl]-D-xylofu r -
a n ose (18a ). The crude amine 17a (663 mg, 1.68 mmol) was
dissolved in 20 mL of 1:1 (v/v) dry pyridine/acetic anhydride and
stirred for 3 h. The reaction mixture was concentrated under
reduced pressure, and the residue was dissolved in ethyl acetate
(50 mL). This was washed with 10% Na2CO3 (3 × 20 mL), 1 M
HCl (3 × 20 mL), and saturated NaCl (1 × 10 mL), and then
dried (Na2SO4) to yield the N-acetate 18a (443 mg, 60% from
16a) as an oil: FAB HRMS exact mass calcd for MH+ C21H29N2O8
437.1924, found 437.1924.
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a cet a m id o-5-N-(1′-
ca r bom eth oxy-2′-ben zyleth yl)-5-N-[(ben zyloxy)ca r bon yl]-
D-xylofu r a n ose (18b). Amine 17b was dissolved in dry pyri-
dine (40 mL) and dry acetic anhydride (50 mL) and stirred for
17 h at rt. Workup and isolation as for 18a and column
chromatography (7:3 ethyl acetate/hexane) afforded N-acetate
18b (747 mg, 86% from 16b) as a white solid, with partial
separation of the diastereoisomers: diastereoisomer A 314 mg,
diastereoisomer A+B 267 mg, diastereoisomer B 166 mg. Isomer
A: mp 54-56 °C; FAB HRMS exact mass calcd for MNa+
C28H34NaN2O8 549.2213, found 549.2227; MH+ C28H35N2O8
527.2393, found 527.2369. Isomer B: mp 60-61 °C; MH+
C28H35N2O8 527.2393, found 527.2379; MNa+ C28H34NaN2O8
549.2213, found 549.2249.
1,2-Dih yd r oxy-3,5-d id eoxy-3-a cet a m id o-5-N-(1′-ca r b o-
m e t h oxye t h yl)-5-N -[(b e n zyloxy)ca r b on yl]-D-xylofu r a n -
ose (19a ). The acetate 18a (200 mg, 0.45 mmol) was dissolved
in TFA (2.27 mL) at 0 °C, and water (0.5 mL) was added
dropwise. The reaction mixture was stirred (0 °C, 18 h), then
warmed to rt, and cautiously neutralized (9 g of Na2CO3 in 80
mL of water). This solution was then extracted with ethyl
acetate (8 × 25 mL). The combined organic fractions were dried
(Na2SO4) and concentrated in vacuo to afford crude 19a as an
oil: 181 mg, quantitative. Hemiacetal 19a was then used
immediately in the next step.
1,2-Dih yd r oxy-3,5-d id eoxy-3-a cet a m id o-5-N-(1′-ca r b o-
m eth oxy-2′-ben zyleth yl)-5-N-[(ben zyloxy)ca r bon yl]-D-xy-
lofu r a n ose (19b). The acetate 18b (267 mg, 0.51 mmol) was
dissolved in TFA (4.5 mL) at 0 °C, and water (0.5 mL) was added
dropwise. The reaction mixture was then stirred between 0 and
4 °C for 21 h before cautiously being added to a solution of
Na2CO3 (6.88 g, 64.9 mmol) in water (60 mL). The solution was
then extracted with ethyl acetate (4 × 40 mL). The combined
organic fractions were dried (Na2SO4) and concentrated in vacuo.
Column chromatography on silica gel (step gradient, 6-10%
methanol in CHCl3) afforded 19b (173 mg, 70%) as an oil which
was used immediately for synthesis of 20b.
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a zid o-5-N-(1′-ca r bo-
m eth oxyeth yl)-D-xylofu r a n ose (15a ). Triflate 14 (1.41 g, 4.05
mmol) was dissolved in 10 mL of dry THF, glycine methyl ester
(1.36 g, 15.3 mmol) in 12 mL of THF was added, and the reaction
mixture was stirred at rt for 14.5 h. The reaction mixture was
concentrated in vacuo, and the crude product was then purified
by column chromatography on silica gel (1:1 hexane/ethyl
acetate) to provide 15a (0.93 g, 80%) as a yellow oil: [R]20 -33
D
(c ) 5 g/100 mL, CHCl3); FAB HRMS exact mass calcd for MH+
C11H19N4O5 287.1355, found 287.1354.
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a zid o-5-N-(1′-ca r bo-
m eth oxy-2′-ben zyleth yl)-D-xylofu r a n ose (15b). 15b was
prepared as for 15a , using 14 (3.94 mmol) in 7.9 mL of THF
and DL-phenylalanine methyl ester (1.41 mL, 7.88 mmol) in THF
(20 mL). Workup as for 16a and column chromatography (1:3
ethyl acetate/hexane) gave 15b (1.19 g, 80%) as an oil: FAB
HRMS exact mass calcd for MH+ C18H25N4O5 377.1825, found
377.1825.
1,2-O-Isopropylidene-3,5-dideoxy-3-azido-5-N-(1′-ca r bom eth -
oxyeth yl)-5-N-[(ben zyloxy)ca r bon yl]-D-xylofu r a n ose (16a ).
The amine 15a (0.92 g, 3.2 mmol) was dissolved in 18 mL of
dioxane and cooled in an ice bath. Na2CO3 (2.19 g, 17.7 mmol)
in 54 mL of water was added to this solution followed by
dropwise addition of benzyl chloroformate (4.57 mL, 32 mmol)
to the rapidly stirred mixture. The reaction mixture was then
stirred at rt for 17 h. The mixture was poured into 10% Na2CO3
(100 mL) and extracted with ethyl acetate (4 × 50 mL). The
combined organic fractions were dried (Na2SO4) and concen-
trated in vacuo. The crude product was then purified twice by
column chromatography on silica gel (8:2 hexane/ethyl acetate,
then 4% methanol in toluene) to afford 16a (0.893 g, 66%) as an
oil: [R]20D -37 (c ) 5 g/100 mL, CHCl3); FAB HRMS exact mass
calcd for MH+ C19H25N4O7 421.1723, found 421.1705.
1,2-O-Isop r op ylid en e-3,5-d id eoxy-3-a zid o-5-N-(1′-ca r bo-
m eth oxy-2′-ben zyleth yl)-5-N-[(ben zyloxy)ca r bon yl]-D-xy-
lofu r a n ose (16b). 16b was prepared as for 16a , using the
amine 15b (1.161 g, 3.095 mmol) in 100 mL of dioxane and 50

7494 J . Org. Chem., Vol. 62, No. 21, 1997
Notes
tr a n s,tr a n s-N-(1′-Ca r bom eth oxyeth yl)-3,5-d ih yd r oxy-4-
a ceta m id op ip er id in e (20a ). The hemiacetal 19a (181 mg,
0.46 mmol) was dissolved in 60 mL of methanol and 10% Pd/C
(200 mg) added. This solution was then hydrogenated using a
Parr hydrogenation apparatus (rt, 48 psi, 17 h). The solution
was filtered through Celite and concentrated in vacuo. Recrys-
tallization once from ethyl acetate/hexane and once again from
ethanol/diethyl ether afforded 20a (87 mg, 78%) as a white
solid: mp 133-136 °C; FAB HRMS exact mass calcd for MH+
C10H19N2O5 247.1294, found 247.1273.
(()-tr a n s,tr a n s-N-(1′-Ca r bom eth oxy-2′-ben zyleth yl)-3,5-
d ih yd r oxy-4-a ceta m id op ip er id in e ((()-20b). The hemiac-
etal 19b (173 mg, 0.35 mmol) was dissolved in methanol (17 mL)
and Pd/C (150 mg) added. This solution was then hydrogenated
using a Parr hydrogenation apparatus at ambient temperature
and 46 psi H2 for 25 h. The solution was filtered through Celite,
washed well with methanol, and concentrated in vacuo. Column
chromatography on silica gel (8% methanol in CHCl3) afforded
20b (95 mg, 81%) as a white solid: mp 156-159 °C; FAB HRMS
exact mass calcd for MH+ C17H25N2O5 337.1763, found 337.1768.
tr a n s,tr a n s-5-N-(1′-Ca r boxyeth yl)-3,5-d ih yd r oxy-4-a cet-
a m id op ip er id in e (5a ). Ester 20a (25 mg, 0.1 mmol) was
dissolved in 1 mL of 80% methanol/water, and LiOH‚H2O (17
mg, 0.4 mmol) was added. The reaction mixture was stirred at
rt for 35 min; then 1 M HCl was added dropwise to lower the
pH to 1. Concentration in vacuo gave the crude product 5a . This
was purified by column chromatography on silica gel (7:2
2-propanol/water) to give the product 5a (19 mg, 70%) as a white
solid: mp 230-234 °C; FAB HRMS exact mass calcd for MH+
C9H17N2O5 233.1137, found 233.1138.
gradient of 2-5% NH4OH. The product-bearing fractions were
combined and concentrated under reduced pressure to give (()-
5b (9.5 mg, 52%) as a white solid: mp 195-197 °C; FAB HRMS
exact mass calcd for MNa+ C16H22N2O5Na 345.1426, found
345.1486; MH+ C16H23N2O5 323.1607, found 323.1607.
In h ibition Stu d ies. Inhibition constants for 5a and (()-5b
were determined for sialidases from S. typhimurium, C. per-
fringens, and V. cholerae using 2-O-(p-nitrophenyl)-R-D-N-acetyl-
neuraminic acid as substrate at 30 ( 0.2 °C. The reaction
buffers employed for the three enzymes were, respectively, 50
mM Na-acetate, 100 mM NaCl, pH 5.5; 25 mM Na-acetate, pH
5.5; and 100 mM Na-acetate, 150 mM NaCl, 4 mM CaCl2, pH
5.5. Reaction mixtures (700 µL) consisted of the appropriate
buffer system containing substrate over a range bracketing Km
and different concentrations of inhibitor 5a or (()-5b. Each
reaction was initiated by addition of 2-5 mU of sialidase
followed by removal of time point aliquots at 5, 10, and 15 min.
Each aliquot was added to 800 µL of 100 mM Na2CO3, pH 10,
and the absorbance at 400 nm due to p-nitrophenolate (ꢀ )
19 200) determined to calculate the initial velocity. Data for
initial velocity were obtained in duplicate measurements of each
substrate/inhibitor concentration. Ki’s were estimated by non-
linear least-squares fit of the initial velocity data to the equation
for competitive inhibition.
Ack n ow led gm en t. We wish to thank the University
of Florida and National Science Foundation (CAREER
Award MCB-9501866 to B.A.H.) for support of this
work.
tr a n s,tr a n s-5-N-(1′-Ca r boxyben zyleth yl)-3,5-d ih yd r oxy-
4-a cet a m id op ip er id in e ((()-5b ). Methyl ester (()-20b (18
mg, 0.05 mmol) was dissolved in water (0.15 mL), and methanol
(0.60 mL) and LiOH‚H2O (2.5 mg, 0.06 mmol) were added. After
stirring at rt for 48 h, the reaction mixture was concentrated to
near dryness in vacuo. The residue was dissolved in 10 mL of
water (pH adjusted to 6) and then purified on Amberlite 120
(H+ form) by initial elution with water followed by a step
Su p p or tin g In for m a tion Ava ila ble: Spectral data for all
compounds (24 pages). This material is contained in libraries
on microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
J O9708497