Mono, di and tri-mannopyranosyl phosphates as mannose-1-phosphate prodrugs for potential CDG-Ia therapy

Article abstract of DOI:10.1016/j.bmcl.2006.09.074

An efficient and convergent method for the synthesis of mannose-1-phosphate prodrugs is described as a potential therapy for congenital disorders of glycosylation-Ia (CDG-Ia). The key feature of the proposed approach is the silver assisted nucleophilic substitution of 2,3,4,6-tetra-O-protected-α-d-mannopyranosyl bromides with various silver phosphate salts to afford mono, di, and tri-mannopyranosyl phosphates. A preliminary biological evaluation of the synthesized phosphate prodrugs has been carried out.

Full text of DOI:10.1016/j.bmcl.2006.09.074

Bioorganic & Medicinal Chemistry Letters 17 (2007) 152–155
Mono, di and tri-mannopyranosyl phosphates
as mannose-1-phosphate prodrugs for potential CDG-Ia therapy
a
a
b
b
b
´
´
Renaud Hardre, Amira Khaled, Alexandra Willemetz, Thierry Dupre, Stuart Moore,
Christine Gravier-Pelletiera,* and Yves Le Merrera,*
aUniversite´ Rene´ Descartes, UMR 8601 CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
`
45 rue des Saints-Peres, 75006 Paris, France
bCentre de Recherche Biome´dicale Bichat Beaujon—CRB3 INSERM U773, Faculte´ de Me´decine Xavier Bichat,16 rue Henri Huchard,
BP 416, 75018 Paris, France
Received 20 July 2006; revised 21 September 2006; accepted 22 September 2006
Available online 17 October 2006
Abstract—An efficient and convergent method for the synthesis of mannose-1-phosphate prodrugs is described as a potential ther-
apy for congenital disorders of glycosylation-Ia (CDG-Ia). The key feature of the proposed approach is the silver assisted nucleo-
philic substitution of 2,3,4,6-tetra-O-protected-a-D-mannopyranosyl bromides with various silver phosphate salts to afford mono,
di, and tri-mannopyranosyl phosphates. A preliminary biological evaluation of the synthesized phosphate prodrugs has been carried
out.
Ó 2006 Elsevier Ltd. All rights reserved.
The Congenital Disorders of Glycosylation1 (CDG)
Therapeutic treatments
F
CDG-Ib :
CDG-Ia :
Prodrugs
Extracellular
medium
belong to a recently recognized group of inherited
multisystem metabolic disorders characterized by the
abnormal glycosylation of a number of serum glycopro-
teins.2 The most common clinical case associates an
encephalomyopathy3 and a peripheral neuropathy.4
Among the CDG, CDG-Ia5 and CDG-Ib6 are the most
frequently encountered forms and are related to a defi-
ciency in the production of GDP-mannose (guanosine-
diphosphate-mannose). GDP-mannose is generated
from mannose-6-phosphate (Fig. 1) which is derived
from either glucose, fructose or mannose metabolism.
Mannose-6-phosphate can be generated by transfor-
mation of fructose-6-phosphate by phosphomannose
isomerase (PMI), or by phosphorylation of the free
intracellular mannose by an hexokinase. The mannose-
6-phosphate (M6P) is then isomerised by the action of
phosphomannose mutase (PMM) into mannose-1-phos-
phate (M1P). This one is subsequently converted to
exogeneous
mannose
G
M
M
Cytosol
Prodrugs
G
F
Hexokinase
Hexokinase
Hexokinase
Esterases
PMI
M
G
P
6
P
F
6
6
P
CDG-Ib
PMM
CDG-Ia
M
1
P
G
: glucose
: fructose
: mannose
F
Guanosine monophosphate
transferase
M
M
GDP
Figure 1. GDP-mannose biosynthesis.
Keywords: Mannose-1-phosphate; Prodrugs; Silver phosphate; Con-
genital disorders of glycosylation.
GDP-mannose by the action of guanosine-monophos-
phate transferase.
*
Corresponding authors. Tel.: +33 142862181 (C.G.P.); +33
142862176 (Y.L.M.); fax: +33 142868387; e-mail addresses:
Enzymatic deficiencies of PMM and PMI are, respec-
tively, responsible for CDG-Ia and Ib. CDG-Ib patients
christine.gravier-pelletier@univ-paris5.fr;
paris5.fr
yves.le-merrer@univ-
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.074

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R. Hardre et al. / Bioorg. Med. Chem. Lett. 17 (2007) 152–155
153
can be treated by oral supplementation with free man-
nose in order to offset the lack of M6P.7
OAc
OAc
OAc
O
O
OAc
b
O
AcO
AcO
AcO
AcO
AgO P
OBn
+
O
OBn
However, there is currently no treatment for patients
suffering from CDG-Ia which accounts for 70% of type
I observed cases. Due to good recovery in patients
affected with CDG-Ib obtained by supplementation with
mannose, one could speculate treating CDG-Ia patients
with M1P supplementation. However, due to its high
polarity limiting passive diffusion through membranes
and its relative instability in blood, it seems more appro-
priate to use prodrugs8 of M1P. Such drugs should be
neutral lipophilic phosphotriesters.
Br
O P OR
3
1
OR
4 : R = Bn
5 : R = H
O
c
a
HO P OBn
OBn
d
e
2
OH
OH
O
OAc
OAc
O
O
HO
AcO
AcO
HO
O
O P OBn
OBn
O
O P O
O
The goal of this work was to synthesize various phos-
phoester prodrugs containing at least one O-protected-
1-mannosyl moiety (Fig. 2).
O
O
7
6
O
Scheme 1. Reagents and conditions: (a) Na2CO3, H2O then AgNO3,
˚
H2O, rt, 77%; (b) toluene, MS 4 A, rt, 18 h, 94%; (c) H2, Pd/C 10%,
Indeed, expected behaviour from such prodrugs would
be: (i) an easy diffusion from extracellular medium to
cytosol due to its high lipophilicity, (ii) release of M1P
in cytosol by esterases and (iii) possible transphospho-
rylation catalyzed by the guanosine-monophosphate
transferase to directly produce GDP-mannose.
2 h, 100%; (d) Ag2CO3, CH3CN, chloromethyl pivalate (7 equiv), 24 h,
52%; (e) MeONa, MeOH, then AmberliteÒ resin IR120 H+.
The synthesis of mono-a-D-mannosyl phosphate pro-
drugs is outlined in Scheme 1 from 2,3,4,6-tetra-O-
acetyl-a-D-mannopyranosyl bromide11 1 and silver
dibenzyl phosphate 3. The peracetylated a-D-mannosyl
bromide 1 was easily obtained by acetylation of mannose
(Ac2O, DMAP, Et3N, CH2Cl2, rt) followed by bromina-
tion (HBr-AcOH)12 in 86% overall yield. The phosphate
3 resulted from treatment of the commercially available
dibenzyl phosphate 2 with sodium carbonate and precip-
itation with silver nitrate (77% yield).13 The silver assisted
nucleophilic substitution of bromomannose 1 by silver
dibenzyl phosphate 3 was carried out in toluene in the
In a recent work, Marquadt et al. described the synthesis
of protected derivatives of mono-1-mannopyranosyl
phosphate.9 Their strategy involved reaction between a
conveniently 2,3,4,6-O-protected mannose as a nucleo-
phile with either a dibenzyl di-iso-propylphosphorami-
dite or chloridophosphites as an electrophile, followed
by subsequent oxidation at the phosphorus atom. Alter-
natively, the preparation of bis-pivaloyloxymethyl
(POM) peracetylated M1P was also described in better
yield by nucleophilic substitution of chlorophosphoryl-
bisPOM by peracetylated mannose.10 Our work focused
on a complementary and more convergent approach
which involves a silver assisted nucleophilic substitution
of a conveniently O-protected 1-mannopyranosyl bro-
mide, as an electrophile, by an anionic phosphate ester.
˚
presence of an excess of molecular sieves 4 A and efficient-
ly led to the dibenzyl peracetylated a-D-mannopyranosyl
phosphate 4.14 Indeed, this compound was isolated in
94% yield as a single a-anomer. Hydrogenolysis of both
benzyl esters with dihydrogen in the presence of 10%
Pd/C quantitatively led to the phosphate 5 which could
be then converted into its POM derivative9,10 6 (52%
yield). The acetyl and POM15 protecting groups are
expected to facilitate cell penetration and to be further
cleaved into M1P by intracellular esterases and
phosphoesterases. Alternatively, methanolysis of the
acetate protecting groups of 4 led to the phosphate 7 as
a substrate for further O-chemoselective protection of
the primary alcohol function.
According to that general approach, the synthesis of
mono, di, and tri-a-D-mannopyranosyl phosphates is
straightforward. It has to be pointed out that, among
these M1P prodrugs, the symmetrical trimannopyrano-
syl phosphate would have the advantage of leading to
M1P and consequently, to GDP-mannose, whatever
the phosphorus–oxygen bond to be disconnected. So,
we herein report an efficient and practical method for
the large-scale synthesis of mono, di, and tri-a-D-
mannopyranosyl phosphates from commercially avail-
able D-mannose.
We next turned to the preparation of di-a-D-mannopyr-
anosyl phosphate prodrugs according to this strategy
(Scheme 2). It involved the disilver phenyl phosphate
10 which could be prepared by silver nitrate treat-
ment of the commercially available disodium phenyl-
phosphate 8. Its condensation with the peracetylated
mannopyranosyl bromide 1 in toluene formed the corre-
sponding phenyl mannopyranosyl phosphate 12 in 78%
yield. Then, hydrogenolysis of phenyl moiety in the
presence of PtO2 in methanol gave the phosphate 14 in
moderate yield (27%).
OR1
H2O
ROH
OH
OH
OR1
O
R1O
R1O
O
HO
HO
O
P
O
P
OR2
esterases
O
O
O
OR2
O
Mannose-1-phosphate
Prodrugs
R1 = H, acyl
R2 = H, mannose, ...
To improve the yield of the hydrogenolysis reaction, we
observed that it was more efficient to use the monoben-
Figure 2. Structure of the targeted prodrugs.

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R. Hardre et al. / Bioorg. Med. Chem. Lett. 17 (2007) 152–155
154
O
OAc
O
OAc
1
a
NaO P OPh
OAc
O
O
AO
P
OR
AcO
AcO
P(OH)3
a
b
OAc
b or c
1
ONa
O
OA
O
8
AgO P OR
OAg
AcO
AcO
9
c
O
OR
P
2
O
A = Ag or H
P(OH)3
9
O P OR
2
12 : R = Ph
19a : R = Et
20a : R = Et
20b : R = 1-glyceryl [
20c : R = pNO2-C6H4
10 : R = Ph
11 : R = Bn
19b : R = 1-glyceryl [
19c : R = pNO2-C6H4
or S]
or S]
+
( )
+
( )
-
-
d e
13 : R = Bn
14 : R = H
A = Ag
A = H
19d : R = 1-naphthyl
19e : R = 2-naphthyl
19f : R = isopentenyl
20d : R = 1-naphthyl
20e : R = 2-naphthyl
20f : R = isopentenyl
Scheme 2. Reagents and conditions: (a) AgNO3, H2O, rt, 91%; (b) i—
BnOH, Et3N, I2, ii—C6H11NH2 excess, resin 50X8-100 Na+ form then
˚
AgNO3, H2O, rt, 62%; (c) toluene, MS 4A, 18 h, rt, 78 and 70% for 12
Scheme 4. Reagents and conditions: (a) i—EtOH for 19a (or isopen-
tenol for 19f), Et3N, I2, ii—C6H11NH2 excess, resin 50X8-100 Na+
˚
form then AgNO3, H2O, rt; (b) toluene, MS 4 A, 18 h, rt; (c) Ag2CO3,
and 13, respectively; (d) H2 (1 atm), PtO2 10%, 2 h, 27%; (e) H2, Pd/C
10%, THF–MeOH, 60%.
˚
CH2Cl2, MS 4 A, 18 h, rt.
zyl phosphate analogue 13. Its preparation required
condensation of the corresponding disilver benzyl phos-
phate 11. This reagent was prepared by treatment of
phosphorous acid with a solution of benzyl alcohol
and triethylamine containing iodine.16 Precipitation as
its dicyclohexyl ammonium salt, followed by cation ex-
change (DowexÒ resin 50X8-100 Na+ form), afforded
the disilver benzylphosphate 11 which was isolated by
silver nitrate treatment as previously. Then, the nucleo-
philic substitution of the peracetylated 1-mannopyrano-
could be used as nucleophile towards the peracetylated
a-D-mannosyl bromide (Scheme 4). Thus, treatment of
phosphorous acid with ethanol in the same conditions
as for the preparation of the benzyl phosphate 11 sup-
plied the corresponding disilver phosphate 19a in 90%
yield. In addition, glyceryl, p-nitrophenyl, 1-naphthyl
and 2-naphthyl phosphates, commercially available as
their dicyclohexyl ammonium, disodium and monosodi-
um salts, respectively, were turned into their corre-
sponding disilver phosphates 19b–e in quantitative
overall yield. Nucleophilic substitution of the manno-
pyranosyl bromide 1 in toluene with derivatives 19a–e
led to the expected dimannopyranosyl phosphates
20a–e in good to excellent yield. In the specific case of
isopentenyl moiety (3-methyl-but-3-en-1-yl), the suitable
conditions involved direct condensation of the diacid
19f, in the presence of a stoichiometric amount of silver
carbonate, affording 20f in moderate yield (25%). Inter-
mediate 19f was readily obtained from the correspond-
ing dicyclohexyl ammonium precursor through H+
resin treatment. Among these derivatives, the p-nitro-
phenyl and naphthyl phosphate esters have the advan-
tage of exhibiting a chromophore which facilitates the
kinetic studies during the hydrolysis into MIP; while,
the isopentenyl and glyceryl phosphate esters provide
leaving groups expected to be non-toxic.
syl bromide
1
as above yielded the benzyl
dimannopyranosyl phosphate 13 (70%). As expected,
subsequent hydrogenolysis in the presence of 10% Pd/
C afforded the deprotected phosphate 14 in good yield.
Interestingly, this strategy could be extended, on
one hand, to other O-protected a-D-mannosyl bro-
mides which can be easily prepared from D-mannose
(Scheme 3).
Thus, treatment of D-mannose with i- or n-butyric anhy-
dride (DMAP, Et3N, CH2Cl2, rt) followed by bromina-
tion (HBr, AcOH) gave the corresponding mannosyl
bromide 15 or 16 in 61% and 70% overall yield, respec-
tively. Then, nucleophilic substitution was carried out,
as above, with disilver phosphate compound 10 or 11
yielding, respectively, the lipophilic phenyl or benzyl
di-mannosyl phosphate 17a,b or 18a,b. On the other
hand, diversity could be introduced on the phosphate re-
agent, since alkyl, alkenyl, benzyl or aryl phosphate
Finely, the same strategy was also suitable to obtain the
tri-a-D-mannosyl phosphate 2217 (Scheme 5).
Thus, treatment of the commercially available trisilver
phosphate 21 with peracetylated a-D-mannosyl bromide
1 afforded the prodrug 22 in 31% yield.
OP
OP
OP
OP
a, b
O
c
PO
PO
O
PO
PO
D-mannose
O
O
OR
P
2
OAc
Br
O
OAc
a
O
15 : P = CO-iPr
16 : P = CO-nPr
17a : P = CO-iPr, R = Ph
+
AgO P OAg
AgO
AcO
AcO
1
O
17b : P = CO-iPr, R = Bn
18a : P = CO-nPr, R = Ph
O P
3
18b : P = CO-nPr, R = Bn
22
21
Scheme 3. Reagents and conditions: (a) (i-PrCO)2O or (n-PrCO)2O,
DMAP, Et3N, CH2Cl2, rt; (b) HBr, AcOH, 61% and 70% overall yield
˚
from D-mannose; (c) 10 or 11, toluene, MS 4 A, 18 h, rt.
˚
Scheme 5. Reagents and conditions: (a) toluene, MS 4 A, 3 weeks, rt,
31%.

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R. Hardre et al. / Bioorg. Med. Chem. Lett. 17 (2007) 152–155
155
2. (a) De Praeter, C. M.; Gerwig, G. J.; Bause, E.; Nuytinck,
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Figura, K.; Freeze, H. H.; Harms, H. K.; Marquardt, T.
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A preliminary biological evaluation of the synthesized
di-mannosyl phosphate prodrugs 12, 14, 17a, 18b and
20d has been carried out. These prodrugs have been
screened, on the one hand, for their toxicity, and on
the other, for their ability to interfere with 2[3H]man-
nose incorporation into cellular glycoconjugates. In fact,
if mannose 1-phosphate can be generated from the pro-
drugs intracellularly, we would expect it to compete with
2[3H]mannose 1-phosphate for entry into the glycopro-
tein biosynthetic pathway.18,19 Lymphoblast cells de-
rived from both a control subject and a CDG-Ia
patient were preincubated for 30 min with prodrugs
(300 lM) in 500 lL glucose-free RPMI 1640 medium
supplemented with 0.5 mM glucose and 10% dialysed
foetal calf serum prior to addition of 50 lCi 2[3H]man-
nose. The incubations were continued for a further
30 min before extracting cellular glycoconjugates as pre-
viously described. 18 Results revealed that 12 (26%), 17a
(78%), 18b (71%) and 20d (56%) inhibited 2[3H]mannose
incorporation into glycoconjugates. The toxicity of pro-
drugs was evaluated by measuring the release of cellular
lactate dehydrogenase into the cell culture medium after
treating the cells with 300 lM prodrugs for 16 h, and in
no case was the toxicity greater than 25% higher than
that observed with the drug carrier methanol alone.
The mechanism of action of the di-mannosyl phosphate
prodrugs is presently under investigation.
7. (a) Panneerselvan, K.; Freeze, H. H. J. Clin. Invest. 1996,
97, 1478; (b) Alton, G.; Kjaergaard, S.; Etchinson, J. R.;
Skovby, F.; Freeze, H. H. Biochem. Mol. Med. 1997, 60,
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Beaune, G.; Castelnau, P.; Kretz, M.; Durand, G.;
Saudubray, J. M.; Seta, N. J. Pediatr. 1999, 135, 379.
8. Schultz, C. Bioorg. Med. Chem. 2003, 11, 885, and
references cited therein.
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Bioorg. Med. Chem. 2002, 10, 4043; (b) Muus, U.; Kranz,
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13. This reagent was prepared according to a slight modifi-
cation of Lynen’s method: Lynen, F. Berichte 1940, 73B,
367.
In summary, we have described the synthesis of various
mannose-1-phosphate prodrugs by nucleophilic substi-
tution of 2,3,4,6-tetra-O-protected-a-D-mannopyranosyl
bromides with silver phosphate salts. The versatility of
this convergent and efficient strategy has been demon-
strated by the synthesis of mono, di and tri-a-D-manno-
pyranosyl phosphate prodrugs for which the phosphate
and carbohydrate protecting groups can be chosen to
adjust the pharmacomodulation in the correction of
the hypoglycosylation pattern. According to the de-
scribed method, the synthesis of a family of M1P pro-
drugs leading to various nontoxic leaving groups is
under progress. The preliminary in vitro biological eval-
uation of the synthesized prodrugs showed that di-
mannosyl prodrugs are promising membrane-permeant
derivatives of mannose-1-phosphate. Further work con-
cerning such prodrugs is currently in progress.
14. During the course of our work, a similar approach to
mono 1-mannopyranosyl phosphate was reported Eklund,
E. A.; Merbouh, N.; Ichikawa, M.; Nishikawa, A.; Clima,
J. M.; Dorman, J. A.; Norberg, T.; Freeze, H. H.
Glycobiology 2005, 15, 1084.
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16. Farquhar, D.; Khan, S.; Srivastva, D. N.; Saunders, P. P.
J. Med. Chem. 1994, 37, 3902.
17. Colowick, S. P. J. Biol. Chem. 1938, 124, 557.
Acknowledgments
We thank Orphan Europe for financial support and for
providing post-doctoral research fellowships to R.H.
ˆ
and A.K. We are also grateful to Dr P. de Lonlay, Hopi-
tal Necker-Enfants malades, for helpful discussions.
´
18. Chantret, I.; Dupre, T.; Delenda, C.; Bucher, S.; Dan-
court, J.; Barnier, A.; Charollais, A.; Heron, D.; Bader-
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