Synthesis and mannosidase inhibitory activity of 3-benzyloxymethyl analogs of swainsonine

Article abstract of DOI:10.1016/S0040-4039(01)01777-4

Swainsonine (3), an inhibitor of Golgi α-mannosidase II, is a clinical candidate for cancer treatment. In order to avoid potential problems arising from its co-inhibition of lysosomal mannosidases, we have synthesized 3-benzyloxymethyl analogs of swainsonine (17 and 18). Initial screening of these new compounds is reported.

Full text of DOI:10.1016/S0040-4039(01)01777-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 8267–8271
Synthesis and mannosidase inhibitory activity of
3-benzyloxymethyl analogs of swainsonine
William H. Pearsona,* and Luyi Guob
aDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA
bDepartment of Medicinal Chemistry, School of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA
Received 27 August 2001; revised 19 September 2001; accepted 21 September 2001
Abstract—Swainsonine (3), an inhibitor of Golgi a-mannosidase II, is a clinical candidate for cancer treatment. In order to avoid
potential problems arising from its co-inhibition of lysosomal mannosidases, we have synthesized 3-benzyloxymethyl analogs of
swainsonine (17 and 18). Initial screening of these new compounds is reported. © 2001 Published by Elsevier Science Ltd.
1. Introduction
the surface of cancer cells is associated with metastasis
and disease progression, hence inhibitors of GMII are
potentially useful for cancer treatment.10–13
Golgi a-mannosidase II (GMII), a class II retaining1,2
glycosyl hydrolase of family 38, is a key enzyme in the
biosynthesis of N-linked glycoproteins, catalyzing the
trimming of two mannose residues (M6 and M7) from
the intermediate 1 (GlcNAcMan5GlcNAc2AsnX) to
produce 2 (GlcNAcMan3GlcNAc2AsnX), the precursor
of complex-type glycoproteins (Scheme 1).3–9 The
altered glycosylation patterns of such glycoproteins on
The azasugars14–17 swainsonine (3), deoxymannojir-
imycin (DMNJ, 4), and 1,4-dideoxy-1,4-imino-D-man-
nitol (DIM, 5) are examples of inhibitors of
a-mannosidases, including GMII, and are thought to
mimic the mannosyl cation 6 or the oxonium-like tran-
sition state encountered during hydrolysis (Fig.
1).3,4,14,16,18–23 Swainsonine has been investigated as an
M6
OH
HO
OH
HO
H
α(1→6)
O
HO
HO
8
1
Asn
2
HO
M4
3
OH
OH
O
N
HO
HO
HO
O
O
O
GlcNAc
HO
(-)-swainsonine
G1
GlcNAc
G2
(3)
M7
O
HO
OH
OH
HO
1
Man
M3
H
H
α(1→3)
OH
HO
HO
GlcNAc—Man
HO
NH
NH
G3
M5
DIM
(5)
deoxymannojirimycin
Golgi
α-Mannosidase II
(DMNJ, 4)
2 Man
OH OH
O
(GMII)
HO
HO
D-mannosyl
cation (6)
Man
Man—GlcNAc—GlcNAc—Asn
2
GlcNAc—Man
Scheme 1. Golgi a-mannosidase II (GMII) trims two man-
nose residues from the substrate 1 to produce 2.
Figure 1. Inhibitors of a-mannosidases are proposed to mimic
the mannosyl cation or the oxonium-like transition state
encountered during hydrolysis.
* Corresponding author. E-mail: wpearson@umich.edu
0040-4039/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.
PII: S0040-4039(01)01777-4

8268
W. H. Pearson, L. Guo / Tetrahedron Letters 42 (2001) 8267–8271
anti-cancer agent,10,11,24–29 and in clinical trials, it has
been found to reduce tumor growth and metastasis.10,11
Although swainsonine shows low toxicity and good
oral availability, it also inhibits lysosomal mannosi-
dase,1,4,30,31 a catabolic class II retaining a-mannosi-
dase, causing the accumulation of high-mannose
oligosaccharides in tissues (swainsonine-induced man-
nosidosis).32–34 Synthetic analogs of swainsonine or
other a-mannosidase inhibitors may allow selective
inhibition of the Golgi enzyme over the lysosomal
enzyme. Toward that end, we report herein the synthe-
sis of 3-benzyloxymethyl analogs of swainsonine and
their initial evaluation as inhibitors of jack bean a-man-
nosidase, a commercially available enzyme that is a
useful model for mammalian a-mannosidases.35
be tolerated by GMII, and may lead to the discovery of
analogs with better selectivity for this enzyme over
lysosomal a-mannosidase. It is perhaps unsurprising
that swainsonine cannot discriminate between these two
enzymes, since both are class II retaining a-mannosi-
dases with high sequence homology.1,31 More complex
structures may be more selective; for example, a crucial
requirement for the action of GMII is the presence of a
particular N-acetylglucosamine residue (GlcNAc G3)
(Scheme 1).36
Our initial efforts resulted in the synthesis of (3R)- and
(3S)-3-(hydroxymethyl)swainsonine, 8 and 9, respec-
tively.37,38 Both were found to be inhibitors of a-man-
nosidases, although not as potent as swainsonine (Table
1). Regarding the goal of inhibition of GMII rather
than lysosomal mannosidase, 8 was unselective (as is
swainsonine 3), but 9 was found to show promising
selectivity, an unexpected result, since 9 has the b-
configuration at C-3 (cf expectations based on struc-
tures 1 and 7). Given this promising result, we were
encouraged to study analogs of 8 and 9 wherein the
3-hydroxymethyl group had been modified. We chose
to examine initially 3-benzyloxymethyl derivatives of
swainsonine, not only for synthetic ease, but because
the aryl substituents may serve as a second carbohy-
drate-like group, thus yielding disaccharide mimics of
the M6–M4 or M7–M4 portion of 1 in Scheme 1, where
the aryl group plays the role of M4.
2. Results and discussion
At the time these studies were initiated, there was no
structural information available on GMII to guide
inhibitor design, thus we began by considering the
substrate for the enzyme, molecule 1. Both M6 and M7
are a-linked to M4, thus we proposed that placing a
sugar or sugar-like molecule on swainsonine at C(3),
the position corresponding to the anomeric carbon of
the mannosyl cation 6, i.e. structure 7 in Fig. 2, would
D
-Ribose was transformed into the indolizidinones 10
and 11 using our previously reported procedures.37
Protection of the C-8 hydroxyl group of 10 followed by
desilylation and lactam reduction afforded 12. O-Alky-
lation of 12 with several benzyl halides gave 14, which
could be converted to the desired analogs 16 of (3S)-3-
(hydroxymethyl)swainsonine (9) by acid hydrolysis and
subsequent ion-exchange chromatography. Selective
monosilylation of 11 followed by a similar sequence
provided the analogs 17 of (3R)-3-(hydroxy-
methyl)swainsonine (8) (Scheme 2).
Since GMII and lysosomal mannosidases are not read-
ily available, as a first step toward evaluating these new
analogs, we report their inhibitory action against jack
bean a-mannosidase, a commercially available enzyme
that has proven to be an excellent model for mam-
malian a-mannosidases. It is also a class II, family 38
retaining mannosidase, and shares high sequence
homology with GMII.1,35,42
Figure 2. 3-Substituted swainsonine analogs 7 may mimic the
oxonium-like transition state encountered in the hydrolysis of
1 by GMII. Compounds 8 and 9, for example, have a
hydroxymethyl group at the ‘anomeric’ carbon of swain-
sonine.
Screening of the new analogs 16a–d and 17a–e against
jack bean a-mannosidase was carried out using stan-
Table 1. Concentration of swainsonine and analogs required to inhibit various a-mannosidase by 50% (IC50 in mM)
Jack bean a-mannosidase
Lysosomal a-mannosidase (source)
Golgi a-mannosidase (source)
3
0.4–0.14,19
0.1 (our labs)37
1.237
0.07–0.20 (rat liver)4,30
0.08–0.5 (human)34,39–41
1.7 (human)41
0.08–0.20 (rat liver)4,18,41
0.04 (human)23
8
9
1.3–2.2 (rat liver)41
1.8 (rat liver)41
4537
39 (human)41

W. H. Pearson, L. Guo / Tetrahedron Letters 42 (2001) 8267–8271
8269
OH
O
OMOM
OMOM
OH
HO
O
O
O
H
N
H
N
H
N
H
N
a-c
O
O
O
HO
e
f,g
91%
TBSO
HO
O
O
10
12
14
16
Ar
Ar
D-ribose
(ref. 37)
OMOM
OMOM
OH
OH
O
HO
O
O
O
H
N
H
N
H
N
H
N
d (86%)
O
O
O
HO
e
a-c
91%
f,g
HO
O
O
HO
11
13
15
17
Ar
Ar
Benzylation product
from step e (yield)
Deprotection product
from steps f&g (yield)
Alkylating agent ArCH2X
PhCH2Br
14a (84%)
16a (81%)
16b (70%)
16c (86%)
2-naphthyl-CH2Br
4-Ph-PhCH2Br
4-t-Bu-PhCH2Br
14b (70%) + 12 (28%)
14c (72%) + 12 (21%)
14d (not purified)
16d (24%, 3 steps)
PhCH2Br
15a (89%)
17a (82%)
17b (74%)
17c (100%)
17d (54%)
17e (93%)
2-naphthyl-CH2Br
4-Ph-PhCH2Br
4-t-Bu-PhCH2Br
4-Me-PhCH2Cl
15b (65%) + 13 (23%)
15c (79%) + 13 (5%)
15d (85%)
15e (56%) + 13 (26%)
Scheme 2. Reagents and conditions: (a) i-Pr2NEt, CH3OCH2Cl (MOMCl), −10°C to rt; (b) n-Bu4NF, THF, rt; (c) BH3·SMe2,
THF, rt. (d) t-BuMe2SiCl (TBDMSCl), 2,6-lutidine, CH2Cl2, −15°C; (e) NaH, ArCH2X, n-Bu4NI, THF, rt; (f) 6N HCl, THF, rt;
(g) Dowex 1×8–200.
dard methods.43 The IC50s measured are reported in
Table 2, where they are compared to those of swain-
sonine (3), (3R)-3-(hydroxymethyl)swainsonine (8, a-
3-HMSW) and (3S)-3-(hydroxymethyl)swainsonine (9,
b-3-HMSW), data obtained previously in our labs.37,44
Analogs 17, with an a-oriented 3-benzyloxymethyl
substituent, are considerably more potent inhibitors
than the b-3-benzyloxymethyl analogs 16, which is
consistent with the hypothesis that the C(3) sub-
stituent mimics the a-oriented carbohydrate that is
cleaved by a-mannosidases, e.g. M4 in structure 1
(Scheme 1) and Fig. 2. Another important aspect of
the data in Table 2 is that compounds 17 are as
potent as or more potent than swainsonine in inhibit-
ing this a-mannosidase. Swainsonine is considered to
be one of the best a-mannosidase inhibitors known
and, to date, attempts to make better inhibitors have
met with difficulty. The importance of the a-3-sub-
stituent is further illustrated by the ability of 18 (8-
epihomoaustraline)45 to inhibit this a-mannosidase,
despite the non-manno configuration. We had synthe-
sized this compound earlier in studies aimed at
inhibiting glucosidases, and were surprised to find
that it was an inhibitor of a mannosidase, which led
us to the design and synthesis of 8 and 9. Although
neither of these compounds proved to be as potent as
swainsonine, it is gratifying to find that the a-3-ben-
zyloxymethyl analogs reported herein are excellent a-
mannosidase inhibitors.
In summary, we have synthesized the first analogs of
swainsonine bearing carbohydrate-like substituent at
C(3), the position that has been proposed to be
analogous to the anomeric center of the mannose
substrates of a-mannosidases. Some of the analogs
were shown to exceed swainsonine in the ability to
inhibit jack bean a-mannosidase, with IC50s as low as
50 nM. We will report elsewhere the evaluation of
these and other related analogs against Golgi a-man-
nosidase II and lysosomal mannosidases. Further,
since the first X-ray crystallographic determination of
the structure of an a-mannosidase II has appeared,
including structures with swainsonine or deoxymanno-
jirimycin bound in the active site,46 we will hopefully
be able to design more selective and potent inhibitors
of GMII.
Acknowledgements
We thank the National Institutes of Health
(CA77365) for support of this work. L.G. was sup-
ported in part by National Research Service Award
T32 GM007767.

8270
W. H. Pearson, L. Guo / Tetrahedron Letters 42 (2001) 8267–8271
Table 2. Concentration of swainsonine and analogs
required to inhibit jack bean a-mannosidase by 50%.
Compound 18,45 having a non-manno configuration, is
also shown (see text)
13. Dennis, J. W.; Granovsky, M.; Warren, C. E. Biochim.
Biophys. Acta 1999, 1473, 21–34.
14. Elbein, A. D. FASEB J. 1991, 5, 3055–3063.
15. Ganem, B. Acc. Chem. Res. 1996, 29, 340–347.
16. Iminosugars as Glycosidase Inhibitors; Stu¨tz, A. E., Ed.;
Wiley-VCH: Weinheim, 1999.
OH
OH
OH
HO
HO
HO
H
H
H
17. Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J.
Tetrahedron: Asymmetry 2000, 11, 1645–1680.
18. Elbein, A. D.; Solf, R.; Dorling, P. R.; Vosbeck, K. Proc.
Natl. Acad. Sci. USA 1981, 78, 7393–7397.
19. Elbein, A. D. Annu. Rev. Biochem. 1987, 56, 497–534.
20. Winkler, D. A.; Holan, G. J. Med. Chem. 1989, 32,
2084–2089.
21. Cenci Di Bello, I.; Fleet, G.; Namgoong, S. K.; Tadano,
K.; Winchester, B. Biochem. J. 1989, 259, 855–861.
22. Nishimura, Y. In Studies in Natural Products Chemistry;
Atta-ur-Rahman, Ed.; Elsevier: Amsterdam, 1992; Vol. 10,
pp. 495–583.
HO
HO
HO
N
N
N
HO
HO
3
8
9
OH
OH
OH
HO
HO
HO
H
N
H
N
H
N
HO
HO
HO
O
O
HO
16
17
18
Ar
Compound
Ar
23. Winchester, B.; Al Daher, S.; Carpenter, N. C.; Cenci Di
Bello, I.; Choi, S. S.; Fairbanks, A. J.; Fleet, G. W. J.
Biochem. J. 1993, 290, 743–749.
Ar
IC50 (mM)
3 (swainsonine)
3 (swainsonine)
8 (a-3-HMSW)
9 (b-3-HMSW)
–
–
–
–
0.404
0.1037,42
1.237
24. Newton, S. A.; White, S. L.; Humphries, M. J.; Olden, K.
J. Natl. Cancer Inst. 1989, 81, 1024–1028.
4537
100
17
\200
\200
25. Olden, K.; Breton, P.; Grzegorzewski, K.; Yasuda, Y.;
Gause, B. L.; Oredipe, O. A.; Newton, S. A.; White, S. L.
Pharmacol. Ther. 1991, 50, 285–290.
26. Oredipe, O. A.; White, S. L.; Grzegorzewski, K.; Gause,
B. L.; Cha, J. K.; Miles, V. A.; Olden, K. J. Natl. Cancer
Inst. 1991, 83, 1149–1156.
16a
16b
16c
16d
17a
17b
17c
17d
17e
18
Ph
2-Naphthyl
4-Ph-Ph
4-t-Bu-Ph
Ph
2-Naphthyl
4-Ph-Ph
4-t-Bu-Ph
4-Me-Ph
–
0.39
0.11
0.08
0.05
0.07
27. Goss, P. E.; Baptiste, J.; Fernandes, B.; Baker, M.; Dennis,
J. W. Cancer Res. 1994, 54, 1450–1457.
28. Klein, J. L.; George, M. D.; Roberts, J. D.; Kurtzberg, J.;
Breton, P.; Chermann, J. C.; Olden, K. Proc. Annu. Meet.
Am. Assoc. Cancer Res. 1994, 35, A1909.
15045
29. Oredipe, O. A.; Furbert-Harris, P.; Griffin, W. FASEB J.
References
1995, 9, A95.
30. Dorling, P. R.; Huxtable, C. R.; Colegate, S. M. Biochem.
J. 1980, 191, 649–651.
31. Liao, Y.-F.; Lal, A.; Moremen, K. W. J. Biol. Chem. 1996,
271, 28348–28358.
1. Howard, S.; Braun, C.; McCarter, J.; Moremen, K. W.;
Liao, Y. F.; Withers, S. G. Biochem. Biophys. Res. Com-
mun. 1997, 238, 896–898.
32. Cenci di Bello, I.; Dorling, P.; Winchester, B. Biochem. J.
1983, 215, 693–696.
2. Heightman, T. D.; Vasella, A. T. Angew. Chem., Int. Ed.
1999, 38, 750–770.
33. Dorling, P.; Huxtable, C.; Cenci di Bello, I.; Winchester,
B. Biochem. Soc. Trans. 1983, 11, 717–718.
34. Al Daher, S.; de Gasperi, R.; Daniel, P. H. N.; Warrern,
C. D.; Winchester, B. Biochem. J. 1991, 277, 743–751.
35. Howard, S.; He, S.; Withers, S. G. J. Biol. Chem. 1998, 273,
2067–2072.
36. Harpaz, N.; Schachter, H. J. Biol. Chem. 1980, 255,
4894–4902.
37. Hembre, E. J.; Pearson, W. H. Tetrahedron 1997, 53,
11021–11032.
38. Pearson, W. H.; Hembre, E. J., US Patent 6,262,065, July
17, 2001.
39. de Gasperi, R.; Daniel, P. F.; Warren, C. D. J. Biol. Chem.
1992, 267, 9706–9712.
3. Tulsiani, D. R. P.; Harris, T. M.; Touster, O. J. Biol. Chem.
1982, 257, 7936–7939.
4. Tulsiani, D. R. P.; Broquist, H. P.; Touster, O. Arch.
Biochem. Biophys. 1985, 236, 427–434.
5. Kaushal, G. P.; Szumilo, T.; Pastuszak, I.; Elbein, A. D.
Biochemistry 1990, 29, 2168–2176.
6. Misago, M.; Liao, Y.-F.; Kudo, S.; Eto, S.; Mattei, M.-G.;
Moremen, K. W.; Fukuda, M. N. Proc. Natl. Acad. Sci.
USA 1995, 92, 11766–11770.
7. Moremen, K. W.; Touster, O.; Robbins, P. W. J. Biol.
Chem. 1991, 266, 16876–16885.
8. Moremen, K. W.; Trimble, R. B.; Herscovics, A. Glycobi-
ology 1994, 4, 113–125.
9. Herscovics, A. Biochem. Biophys. Acta 1999, 1473, 96–107.
10. Goss, P. E.; Baker, M. A.; Carver, J. P.; Dennis, J. W. Clin.
Cancer Res. 1995, 1, 935–944.
11. Goss, P. E.; Reid, C. L.; Bailey, D.; Dennis, J. W. Clin.
Cancer Res. 1997, 3, 1077–1086.
40. Dennis, J. W.; White, S. L.; Freer, A. M.; Dime, D.
Biochem. Pharmacol. 1993, 46, 1459–1466.
41. Tests on our compounds, benchmarked against swain-
sonine, were performed at GlycoDesign, Inc. (Toronto)
using enzymes provided by Professor Kelley W. Moremen
(University of Georgia) and reported as compounds EJH-
4-264 [(3R)-3-(hydroxymethyl)swainsonine, 8] and EJH-4-
12. Dennis, J. W.; Granovsky, M.; Warren, C. E. BioEssays
1999, 21, 412–421.

W. H. Pearson, L. Guo / Tetrahedron Letters 42 (2001) 8267–8271
8271
263 [(3S)-3-(hydroxymethyl)swainsonine, 9]. These data
may be found in: Shah, R.; Carver, J.; Marino-Albernas,
J.; Tvaroska, I.; Tropper, F.; Dennis, J., US Patent
6,048,870, 2000.
min at 37°C in a total volume of 450 mL. The p-nitrophenyl
a-mannoside substrate (50 mL of a 12.5 mM solution) was
then added. After 25 min, 2.5 mL of a 2% sodium carbonate
solution was added, and the concentration of p-nitrophe-
nolate was measured at 410 nm. The assays were performed
under conditions where the amount of p-nitrophenol
released was linear with respect to both time and enzyme
concentration.
42. Kang, M. S.; Elbein, A. D. Plant Physiol. 1983, 71, 551–554.
43. Tropea, J. E.; Molyneux, R. J.; Kaushal, G. P.; Pan, Y.
T.; Mitchell, M.; Elbein, A. D. Biochemistry 1989, 28,
2027–2034.
44. Enzyme inhibition was assayed colorimetrically by moni-
toring the release of p-nitrophenol from p-nitrophenyl
a-mannoside (Sigma) at pH 4.5 in 0.1 M sodium acetate
buffer. The enzyme and inhibitor were preincubated for 20
45. Pearson, W. H.; Hembre, E. J. J. Org. Chem. 1996, 61,
5546–5556.
46. van den Elsen, J. M. H.; Kuntz, D. A.; Rose, D. R. EMBO
J. 2001, 20, 3008–3017.