Derivatives of (2R,3R,4S)-2-aminomethylpyrrolidine-3,4-diol are selective α-mannosidase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00477-2

A collection of (2R,3R,4S)-3,4-dihydroxypyrrolidin-2-yl derivatives have been tested for their inhibitory activities toward 25 glycosidases. Competitive (K_i = 7.4 μM) and selective inhibition of α-mannosidase from jack bean has been found for (

Full text of DOI:10.1016/S0960-894X(01)00477-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2489–2493
Derivatives of (2R,3R,4S)-2-Aminomethylpyrrolidine-3,4-diol are
Selective ꢀ-Mannosidase Inhibitors
Florence Popowycz,a Sandrine Gerber-Lemaire,a Raynald Demange,a
Eliazar Rodriguez-Garcıa,a Ana Teresa Carmona Asenjo,b Inmaculada Robinab
and Pierre Vogela,*
a
´
Section de Chimie de l’Universite de Lausanne, BCH, CH-1015 Lausanne-Dorigny, Switzerland
b
´
Departamento de Quımica Organica, Facultad de Quımica, Universidad de Sevilla, E-41071 Sevilla, Spain
´
´
Received 5 March 2001; revised 29 June 2001; accepted 4 July 2001
Abstract—A collection of (2R,3R,4S)-3,4-dihydroxypyrrolidin-2-yl derivativeshave been tested for their inhibitory activitiestoward
25 glycosidases. Competitive (Ki=7.4 mM) and selective inhibition of a-mannosidase from jack bean has been found for
(2R,3R,4S)-2-[(benzylamino)methyl]pyrrolidine-3,4-diol and other derivatives. # 2001 Elsevier Science Ltd. All rights reserved.
The specific inhibition of N-linked glycoprotein-proces-
sing a-mannosidases may provide a useful anti-cancer
strategy.1 Clinical trials have shown that swainsonine, a
natural a-mannosidase inhibitor that contains a 4-
amino-4-deoxy-mannofuranoside moiety2,3 reduces
solid tumors and hematological malignancies.4 Simpler
synthetic analogues are also potent a-mannosidase
inhibitors.3,5 Mannostatin A and B isolated from the
soil microorganism Streptoverticillum verticillus6 and a
synthetic analogue7 are probably the most potent inhi-
bitorsof a-mannosidases reported thus far.5,8 Often, a-
mannosidase inhibitors that are monosaccharide
mimics9,10 also inhibit other glycosidase types,11 in par-
ticular a-l-fucosidases.9,12 It isbelieved that enzyme
selectivity could be improved if the iminosugar, which
mimicsthe pyranosyl cation intermediate liberated dur-
ing the hydrolytical process, would include some infor-
mation of the glycosidic bond that is cleaved (a vs b, site
of glycosidation of the aglycon) and of the aglycon itself
(shape, electrostatic and/or lyophilic interaction with
the enzyme). Such inhibitorscould be daisccharide
mimicssuch asdideoxy-imino-alditolslinked to mono-
saccharides through non-hydrolyzable linkages.13 For
the moment, the syntheses of these disaccharide mimics
are lengthy and do not offer the necessary flexibility for
drug development. Furthermore, to become a drug, a
good inhibitor must satisfy a number of conditions14
such as membrane permeability, which often requires
the presence of lyophilic groups. We show in this note
that readily prepared (2R,3R,4S)-2-(benzylamino)-
methyl-3,4-dihydroxypyrrolidine and analoguesthat
can be viewed to mimic a transition or intermediate
structure of the hydrolytic process in which the enzyme
15
interveneswith two carboxylic groups(Fig. 1)
good inhibitorsof a-mannosidases.16
can be
17
Following Fleet’smethod,
we converted d-gulono-
lactone into 1 that wasthen transformed into aldehyde
2 (33% overall yield based on d-gulonolactone, seven
steps).18 Aldehyde 2 reacted with one equivalent of pri-
mary amine RNH2 (b–k) in anhydrous1,2-dichloro-
ethane at 20 ꢀC giving the corresponding imines that
were reduced in situ with NaBH(OAc)3 into the corre-
sponding amines 3b–3k (65–90% yield after aqueous
work up with NaHCO3 and purification by flash
chromatography on silica gel).
Deprotection [CF3COOH/H2O (4:1) or HCl 4 M in
H2O, 20 ꢀC, 1–2 h] provided the corresponding diamines
4b–4k nearly quantitatively. Hydrogenolysis [H2/
Pd(OH)2/charcoal, MeOH] of the benzylamino deriva-
tive 3i furnished 3a that wasdeprotected (HCl 4 M) to
give diamine 4a. Acetylation of 3i (Ac2O/pyridine/4-
dimethylaminopyridine, 20 ꢀC) gave 5i (95% yield) that
washydrolyzed with CF COOH/H2O 4:1 (20 ꢀC) pro-
3
*Corresponding author. Tel.: +41-21-692-3971; fax: +41-21-692-
3955; e-mail: pierre.vogel@ico.unil.ch
viding acetamide 6i in good yield (Scheme 1). The
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00477-2

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F. Popowycz et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2489–2493
known cis-pyrrolidine-3,4-diol (7)19 wasalso included in
our enzymatic assays (see below) together with triol 85,20
(derived by NaBH4 reduction of 2 and acidic hydrolysis)
and tetrol 9 (obtained by acidic hydrolysis of 1).17
inhibitory activity. The latter compound isa weak but
selective inhibitor of b-glucosidase (IC50=320 mM) from
almond. In agreement with the hypothesis of Figure 1,
diamine 4a isa better inhibitor of a-mannosidases than
7–9. With a-mannosidase from jack bean, Ki=53mM
wasmeausred [competitive inhibition with p-nitrophe-
nyl a-d-mannopyranoside (Lineweaver–Burk plots)].21
With the hope that alkylation of the primary amines
moiety of 4a might improve inhibitory activity and
selectivity toward a-mannosidases, we tested 4b–4k.
Except for the N-a-thiophenylmethyl 4g and N-benzyl
We have tested compounds 4a–4k, 6i, 7, 8 and 9 for
their inhibitory activitiestoward 25 commercially avail-
able glycosidases. The data are summarized in the Table 1
for five b-galactosidases, five a-glucosidases, two b-glu-
cosidases, and two a-mannosidases. Unless indicated
otherwise, these compounds did not show any inhibitory
activity at 1 mM concentration toward the following
enzymes: a-galactosidases from Aspergillus niger, from
Escherichia coli and from coffee bean, a-l-fucosidase
from bovine epididymis, b-mannosidase from Helix
pomatia, b-xylosidase from A. niger, b-N-acetyl-
glucosaminidase from jack bean, from bovine epididy-
misA and B and a-N-acetylgalactosaminidase from
chicken liver.
The simple cis-pyrrolidine-3,4-diol (7) isa weak inhi-
bitor of several glycosidases including a-mannosidases.
Potency and enzyme selectivity does not change very
much on introducing a (2R)-hydroxymethyl substituent as
in 8 (Table 1). Surprisingly, the introduction of a (2R,10R)-
1,2-dihydroxyethyl substituent as in 9 suppresses most
Figure 1. Design of dicationic mimics of a transition or intermediate
structure of an a-mannosidase-catalyzed hydrolysis of an a-d-manno-
pyranoside.
Scheme 1.

F. Popowycz et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2489–2493
2491
Table 1. Inhibitory activitiesof (2 R,3R,4S)-3,4-dihydroxypyrrolidin-2-yl derivatives. Percentage of inhibitions at 1 mM concentration, IC50 and Ki
in mM, when measured. Optimal pH, 35 ꢀCa,b
Enzyme/inhibitor
4a
4b
4c
4d
4e
4f
4g
4h
4i
ꢁ-Galactosidase
E. coli
Bovine liver
92%
ni
36%
ni
50%
ni
ni
ni
39%
53%
ni
ni
ni
ni
49%
ni
24%
26%
IC50=770
ni
A. niger
24%
47%
61%
IC50=480
32%
26%
39%
ni
41%
ni
A. orizae
Jack bean
60%
76%
ni
39%
ni
21%
ni
26%
ni
34%
ni
ni
24%
36%
ni
ni
46%
ꢀ-Glucosidase
Yeast (maltase)
84%
46%
59%
IC50=410
ni
94%
IC50=70
30%
55% 66%
IC50=610 IC50=280
ni
55%
IC50=610
ni
ni
Rice (maltase)
Baker yeast (isomaltase)
53%
98%
ni
84%
ni
92%
ni
90%
ni
94%
ni
23%
ni
ni
90%
IC50=250
IC50=170 IC50=120
IC50=65
Ki=83 (C) Ki=77 (M) Ki=71 (M) Ki=40 (C) Ki=45 (M)
IC50=120
Ki=40 (C)
ni
A. niger
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
(amyloglucosidase)
Rhizopus mold
(amyloglucosidase)
ni
ꢁ-Glucosidase
Almond
97%
93%
67%
IC50=410
77%
62%
70%
82%
IC50=500 IC50=430 IC50=140
43%
38%
70%
IC50=420
68%
IC50=370
IC50=290
Ki=340 (M)
82%
IC50=180
Ki=170 (M)
Ki=12 (C)
75%
Caldocellum saccharol.
72%
IC50=340
29%
76%
IC50=290 IC50=250
Ki=120 (C) Ki=60 (M)
76%
IC50=290
Ki=120 (C)
ni
ꢀ-Mannosidase
Jack bean
81%
IC50=170
75%
IC50=300
66%
IC50=490
60% 55%
IC50=560 IC50=700
49%
29%
89%
55%
IC50=700
92%
IC50=85
Ki=26 (C)
68%
IC50=350
Ki=98 (C)
IC50=60
Ki=7.4 (C)
69%
IC50=230
Ki=71 (C)
Ki=53 (C) Ki=59 (C) Ki=120 (C)
39%
Almond
51%
IC50=1mM
45%
53%
59%
IC50=570
59%
IC50=570
Enzyme/inhibitor
4j
4k
6i
7
8
9c
1
3
1
4
ꢁ-Galactosidase
E. coli
Bovine liver
25%
84%
34%
ni
ni
ni
41%
25%
ni
51%
ni
32%
ni
ni
48%
32%
IC50=140
Ki=21 (M)
29%
A. niger
ni
ni
39%
ni
ni
ni
91%
IC50=140
Ki=200 (M)
80%
A. orizae
ni
ni
ni
ni
ni
53%
IC50=790
55%
ni
ni
ni
ni
ni
IC50=75 Ki=75 (M)
84%
Jack bean
22%
21%
IC50=720
IC50=110
Ki=25 (M)
ꢀ-Glucosidase
Yeast (maltase)
Rice (maltase)
Baker yeast (isomaltase)
28%
ni
46%
ni
ni
34%
ni
ni
33%
ni
ni
32%
50%
30%
73%
ni
ni
ni
ni
ni
ni
ni
ni
28%
IC50=330
96%
IC50=31
Ki=64 (M)
92%
A. niger
(amyloglucosidase)
ni
ni
ni
ni
ni
ni
24%
47%
ni
ni
ni
ni
21%
40%
Rhizopus mold
(amyloglucosidase)
IC50=20
Ki=44 (N)
ꢁ-Glucosidase
Almond
61%
IC50=550
35%
49%
69%
IC50=430
Ki=220 (M)
43%
60%
88%
73%
IC50=600 IC50=120 IC50=320
ni
ni
91%
IC50=70
Ki=27 (C)
90%
IC50=80
Ki=27 (C)
Ki=53 (M)
90%
IC50=85
Ki=19 (C)
Caldocellum saccharol.
72%
IC50=320
ni
40%
ꢀ-Mannosidase
Jack bean
ni
66%
IC50=440
Ki=170 (C)
ni
ni
70% 54%
IC50=400 IC50=800
ni
ni
33%
ni
81%
IC50=180
Ki=80 (M)
Almond
ni
48%
40%
37%
56%
aFor the conditions of measurements, see ref 21.
b(C), competitive; (M), mixed type; (N), non-competitive inhibition; ni, no inhibition at 1 mM concentration.
cInhibitsalso a-galactosidase from coffee bean (42% at 1 mM) and b-xylosidase from A. niger (42% at 1 mM).

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F. Popowycz et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2489–2493
Scheme 2.
Acknowledgements
derivatives 4i all diamines 4b–4h showed inhibitory
activitiessimilar to that of 4a. The bulky N-(3-hydroxy-
2-methyl-3,3-diphenylpropyl) derivative 4j, apparently,
cannot enter in the active site of the a-mannosidases.
The best inhibitory activity (Ki=7.4 mM, competitive)
wasfound for 4i toward a-mannosidase from jack bean.
We thank the Swiss National Science Foundation, the
Office Federal de l’Enseignement et de la Recherche
(COST D13/0001/99), the FondsHerbette (Lauasnne),
the SOCRATES (Lausanne/Seville) program and the
˜
Direccion General de Ensenanza Superior e Investiga-
cion Cientıfica (Spain) (Grant Number PB 97-0730) for
financial support.
This diamine also presents the best selectivity toward a-
mannosidases. Substitution of the benzyl group of 4i by
a (10R)-a-methylbenzyl group decreases the inhibitory
activity by a factor of 25. Apparently, the diamino
moiety of 4i is necessary for this compound to bind to
a-mannosidases as the acetamide 6i did not inhibit these
enzymes(bulk of the acetamido moiety?). In order to
test the importance of the (2R)-aminomethyl group of 4
in a possible electrostatic interaction with a carboxylic
group of the a-mannosidases, we prepared 13, a struc-
tural isomer of 4i in which the amine moiety of the side
chain isfurther apart from the pyrrolidine base than in
4a–k. In agreement with our working hypothesis (Fig. 1),
13 isa much weaker a-mannosidase inhibitor than 4i
(Table 1).
References and Notes
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several other glycosidases.
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A new lead for the selective inhibition of a-mannosi-
dases has been found with (2R,3R,4S)-2-[(benzyl-
amino)-methyl]pyrrolidine-3,4-diol.23 Work isunderway
to apply parallel synthesis to generate further diamines
4 and to introduce (5R)-hydroxymethyl and other
substitution on their pyrrolidine rings.

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of a-galactosidases, see: Jeong, J. H.; Murray, B. W.;
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Picasso, S.; Vogel, P. J. Org. Chem. 1995, 60, 6806. Picasso, S.;
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23. Selected data for 4i: 1H NMR (400 MHz, MeOD): 7.54
(m, 5H); 4.37 (ddd, 3J=1.6, 4.2, 9.0 Hz, H-4); 4.29 (m, 2H, H-
100); 4.16 (dd, 3J=4.2, 9.0 Hz, H-3); 3.73 (ddd, 3J=4.2, 8.6,
3
2
2J=13.4 Hz, H-2); 3.52 (dd, J=4.2, J=13.1 Hz, H-5); 3.46
(dd, 3J=8.6, 2J=13.4 Hz, H-10); 3.37 (dd, 3J=1.6, 2J=13.1
Hz, H-5). 13C NMR (101 MHz, MeOD): 134.2, 132.1, 131.7,
76.6, 71.7, 59.6, n54.4, 52.8, 49.6. CI-MS (NH3): 223 (100),
222 (43), 211 (4), 120 (22), 91 (25), 77 (29). Selected data for
13: 1H NMR (400 MHz, MeOD): 7.06–7.12 (m, 2H); 6.58–6.66
3
(m, 3H); 4.05 (m, H-4); 3.60 (dd, J=5.3, 7.8 Hz, H-3); 3.18–
3.26 (m, 3H, H-20a, H-20b, H-5a); 3.01 (td, J=7.9 Hz); 2.80
3
(dd, 3J=3.4, 2J=12.2 Hz, H-5b) ; 1.96 (m, H-10a); 1.69 (m, H-
16. 2-(Benzylamino)- and 2-[N-(benzyloxy)amino]-4,5-dihy-
droxy-6-(hydroxymethyl)tetrahydro-1,3-pyrimidine inhibitors
10b).13C NMR (101 MHz, MeOD): 150.1, 130.0, 118.2, 114.3,
25
589
78.3, 72.2, 61.3, 52.4, 42.7, 33.8. [a] =+37 (c 1.2, CH3OH).