N-sulfonyl hydroxamate derivatives as inhibitors of class II fructose-1,6-diphosphate aldolase

Article abstract of DOI:10.1016/j.bmcl.2005.09.006

Dihydroxyacetone-phosphate and phosphonate derivatives were synthesized bearing a N-sulfonyl hydroxamate moiety. The phosphate derivatives represent competitive inhibitors for the class II-FBP aldolase catalyzed reaction, while the phosphonate isosteres are comparatively weaker inhibitors.

Full text of DOI:10.1016/j.bmcl.2005.09.006

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5375–5377
N-Sulfonyl hydroxamate derivatives as inhibitors of
class II fructose-1,6-diphosphate aldolase
*
´
Sabine Gavalda, Remi Braga, Chantal Dax, Alain Vigroux and Casimir Blonski
`
´
´ ˆ
Laboratoire de Synthese et de Physico-Chimie de Molecules d’interet Biologique (LSPCMIB),
´
ˆ
Groupe de Chimie Organique Biologique, UMR 5068, Universite Paul Sabatier, Bat IIR1, 118 route de Narbonne,
31062 Toulouse Cedex 4, France
Received 30 June 2005; revised 1 September 2005; accepted 6 September 2005
Abstract—Dihydroxyacetone-phosphate and phosphonate derivatives were synthesized bearing a N-sulfonyl hydroxamate moiety.
The phosphate derivatives represent competitive inhibitors for the class II-FBP aldolase catalyzed reaction, while the phosphonate
isosteres are comparatively weaker inhibitors.
Ó 2005 Elsevier Ltd. All rights reserved.
Aldolases are essential enzymes that catalyze carbon–
carbon bond formation (or cleavage) in living organisms.
Among the more extensively studied aldolases are fruc-
tose bisphosphate aldolases (EC 4.1.2.13) that partici-
pate in the metabolically important pathways of
gluconeogenesis and glycolysis. In gluconeogenesis, these
enzymes catalyze the aldol reaction of two triose-Ps,
dihydroxyacetone-phosphate (DHAP) and D-glyceralde-
hyde 3-phosphate (G3P), to form fructose-1,6-bisphos-
phate (FBP). In glycolysis, FBP aldolases catalyze the
reverse cleavage reaction.1 Aldolases exist in two distinct
classes;2 class I aldolases are found in animals and higher
plants, and catalyze the formation of a Schiff-base inter-
mediate with substrate, whereas class II aldolases are
found in algae, bacteria, and yeasts, and require a biva-
lent metal ion as cofactor. In class I aldolases, the reac-
tion has the following features in the direction of FBP
synthesis: (i) SchiffÕs base formation between DHAP
and a lysyl residue at the active site; (ii) pro-S proton
abstraction at C-3 in the immonium leading to an enam-
ine (so-called carbanion) whose condensation with G3P
yields FBP (new immonium ion intermediate) with an
S configuration at C-3 (Scheme 1). In class II aldolase,
a divalent cation (usually Zn2+) functions as a Lewis acid
to polarize the carbonyl bond of DHAP facilitating pro-
ton abstraction at C-3. The resulting endiolate, equiva-
lent to the enamine intermediate, is stabilized by the
zinc ion.
Class II aldolases are not present in mammals and as
such they represent potential targets for the development
of anti-bacterial and anti-fungal drugs.3 Phospho-
glycolohydroxamic acid (PGH) was, for more than 30
years, the only known potent inhibitor of class II aldolas-
es.4 Recently, two new derivatives of phosphoglycolic
acid were synthesized and shown to be selective inhibi-
tors of this family of enzymes.3d These studies substanti-
ated the feasibility of developing new inhibitors of
therapeutic interest based on the PGH motif while also
affording insight into enzymology of glycolytic aldolases.
Based on these recent results, we prepared a new family
of inhibitors possessing the phosphoglycolate skeleton
bound to a sulfamate group (PGS, Scheme 2). Com-
pounds with a NH moiety between carbonyl and sulfonyl
groups are very acidic; their pKa values are close to 2;5
the presence of a negative charge at physiological pH
at this position of PGS would be expected to improve
the interaction with the active-site metal ion. Additional-
ly, phosphonate isosteres of biologically important phos-
phates are of interest in biological chemistry, due to the
exceptional stability of the phosphorus–carbon bond
toward phosphatases as compared to a phosphorus–
oxygen bond.6 In order to gain understanding of the
influence of such a structural modification on the binding
of the N-sulfonyl hydroxamate inhibitors, we have also
prepared their phosphonate isosteres (p-PGA and
p-PGS, Scheme 2).
Keywords: Fructose bisphosphate aldolase; N-Sulfonyl hydroxamate
derivatives; Inhibitors; Phosphonate.
*
Corresponding author. Tel.: +33 5 61 55 6486; fax: +33 5 61 55
6080; e-mail: blonski@cict.fr
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.006

5376
S. Gavalda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5375–5377
OPO3H-
NH-E
CHO
OPO3H-
O
OPO3H-
NH+-E
OH
OPO3H-
NH-E
OH
E-NH2
class I
OH
OPO3H-
- H+
HO
DHAP
DHAP
OH
OH
OH
OPO3H-
CHO
OPO3H-
OPO3H-
E-NH2
OH
OPO3H-
OPO3H-
- H+
O
O
Zn2+
Zn2+
O
FDP
class II
HO
OH
O
OH
OH
OPO3H-
Scheme 1. Aldolase catalyzed reaction: class I and class II-FBP aldolases.
The interaction of these products with class II aldolase
from Escherichia coli and class I aldolase from rabbit
muscle was analyzed by enzyme kinetics.8 Lineweaver–
Burk plots of enzyme inhibition show clearly that
PGS1 and PGS2 are fully competitive inhibitors for
the reaction catalyzed by the class II aldolase (see
Fig. 1 as an example). This result is an indication that
the two inhibitors act at the active site and chelate the
metal ion.9 No inhibition effect is observed at a concen-
tration up to 3 mM with aldolase from muscle or with
other DHAP-dependent enzymes such as triose phos-
phate isomerase or glyceraldehyde 3-phosphate dehy-
drogenase used in the coupled enzymatic essays.
R
SO2
O
OH
NH
NH
O
O
O
P
OH
OH
OH
X
P
X
OH
PGA (X = O)
p-PGA (X = CH2)
PGS1 (X = O, R = CH3)
PGS2 (X = O, R= Ph)
p-PGS1 (X = CH2 , R= CH3)
p-PGS2 (X = CH2 , R= Ph)
Scheme 2. Structures of inhibitors under study: phosphoglycolo-
hydroxamic acid (PGH) and phosphoglycolosulfamate (PGS) and
their phosphonate derivatives (p-PGH and p-PGS, respectively).
All compounds were synthesized in three steps starting
from dibenzyl ethyl phosphoglycolate or triethyl 3-phos-
phonopropionate according to Scheme 3. In the first
step, compounds are hydrolyzed under basic conditions
to furnish the corresponding sodium salt derivatives.
This is followed by reaction with the hydroxylamine or
sulfonamide in the presence of carbodiimide to yield
compounds 1a,b, 2, and 3a,b. Deprotection of the phos-
phonate or phosphate group yields the expected com-
pounds as the lithium salt.7
It is interesting tonote that the PGS compounds are weak-
er inhibitors than those described previously. Their ac-
tive-site affinity appears to be dependent on the
chemical group used on the sulfonyl hydroxamate of the
inhibitor, PGS2 being more efficient than PGS1 indicat-
ing a specific accommodation of the aromatic moiety by
hydrophobic interaction of the enzyme toward the inhib-
itors (Table 1). This suggests chemical modification at the
sulfamate level (e.g., aromatic ring bearing a reactive elec-
trophile) may improve inhibitor efficiency or transform
O
O
O
O
P
O
P
O
P
a) b)
c) d)
BnO
BnO
O
BnO
BnO
O
I
LiO
LiO
O
OEt
NH SO2 R
R
NH SO2
1a (R = CH3)
1b (R = Ph)
PGS1 (R = CH3)
PGS2 (R= Ph)
O
O
O
O
O
O
P
O
a) b)
c) d)
c) d)
II EtO
LiO
LiO
EtO
OEt
OEt
P
NH OH
NH SO2
P
NH OBn
NH SO2
EtO
EtO
2
p-PGA
O
O
O
P
O
P
O
a) b)
EtO
EtO
EtO
EtO
III
LiO
LiO
R
P
R
3a (R = CH3)
3b (R = Ph)
p-PGS1 (R= CH3)
p-PGS2 (R= Ph)
Scheme 3. Synthesis of inhibitors under study. I: (a) NaOH, H2O, (b) RSO2NH2, DCC, DMAP, CH2Cl2, (c) H2, Pd/C, MeOH, (d) H2O, LiOH; II:
(a) NaOH, H2O, (b) NH2OBn, DCC, DMAP, CH2Cl2, (c) Me3SiBr, (d) H2O, LiOH; III: (a) NaOH, MeOH, (b) RSO2NH2, DMAP, DCC, CH2Cl2,
(c) Me3SiBr, (d) H2O, LiOH.

S. Gavalda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5375–5377
5377
Table 1. Kinetic parameter values (lM)
Aldolase Km
FBP PGS1 PGS2 p-PGS1 p-PGS2 PGH p-PGH
28
26
24
22
20
18
16
14
12
10
8
Ki
[I] = 0 (Km)
[I] = 100 µM
[I] = 250 µM
[I] = 500 µM
Rabbit
E. coli
10
300 350
>104 >104 >104
100
>104
>104
>104
1
>103
0.01 >103
Acknowledgment
6
We are thankful to Professor J. Sygusch for providing
class I FBP aldolase from rabbit muscle and class II
FBP aldolase from E. coli.
4
2
-0,003 -0,002 -0,001 0,000 0,001 0,002 0,003 0,004 0,005
1 / [S] (µM-1)
References and notes
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
1. (a) Horecker, B. L.; Tsolas, O.; Lai, C. Y. Aldolases. In
The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press:
New York, 1972; Vol. 7, pp 213–258; (b) Gefflaut, T.;
Blonski, C.; Perie, J.; Willson, M. Prog. Biophys. Mol.
Biol. 1995, 63, 301.
2. Rutter, W. J. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1964, 23,
1248.
3. (a) Izard, T.; Sygusch, J. J. Biol. Chem. 2004, 279, 11825;
(b) Blom, N.; Sygusch, J. Nat. Struct. Biol. 1996, 3, 856; (c)
Fessner, W. D.; Schneider, A.; Held, H.; Sinerius, G.;
Walter, C.; Hixon, M.; Schloss, J. V. Angew. Chem., Int.
Ed. Engl. 1996, 35, 2219; (d) Fonvielle, M.; Weber, P.;
Dabkowska, K.; Therisod, M. Bioorg. Med. Chem. Lett.
2004, 14, 2923.
4. (a) Lewis, D. J.; Lowe, G. J. Chem. Soc., Chem. Commun.
1973, 713; (b) Collins, K. D. J. Biol. Chem. 1974, 249, 136.
5. Blans, P.; Vigroux, A. Chem. Eur. J. 1999, 5, 1526.
6. (a) Engel, R. Chem. Rev. 1977, 77, 349; (b) Racha, S.; Li,
Z.; El-Subbagh, H.; Abushanab, E. Tetrahedron Lett.
1992, 33, 5491; (c) Page, P.; Blonski, C.; Perie, J.
Tetrahedron Lett. 1995, 36, 8027; (d) Page, P.; Blonski,
C.; Perie, J. Bioorg. Med. Chem. 1999, 7, 1403.
7. All compounds gave satisfactory NMR (300 MHz), ESMS
(negative mode), and CHN data.
-100
0
100
200
300
400
500
[I] (µM)
Ki = 101 µM
Figure 1. Lineweaver–Burk plots of class II-FBP aldolase inhibition
by different concentrations of PGS2.
them into specific irreversible inhibitors by taking advan-
tage of the presence of nucleophilic residues (e.g., Asp-109
or Ser-61) in the active site or in its proximity.10
8. Recombinant enzymes from rabbit muscle and from E.
coli were furnished by Professor J. Sygusch, University of
Montreal, Canada. Enzymatic assays were performed as
described in Blonski, C.; De Moissac, D.; Perie, J.;
Sygusch, J. Biochem. J. 1997, 323, 71.
9. Fortnagel, P.; Freese, E. J. Biol. Chem. 1968, 243, 5289.
10. Claustre, S.; Denier, C.; Lakhdar-Ghazal, F.; Lougare, A.;
Lopez, C.; Chevalier, N.; Michels, P. A. M.; Perie, J.;
Willson, M. Biochemistry 2002, 41, 10183.
Surprisingly, the structural modification of converting a
phosphate into phosphonate significantly weakened
inhibitor binding (Table 1). These results strongly sup-
port the fact that stabilizing interactions made by ac-
tive-site residues with the O1 ester oxygen at the
phosphate binding site are critical for inhibitor binding,
which is not possible with a phosphonate moiety11 and
must be taken into account in the development of stable
intracellular inhibitors against class II FBP aldolases.
11. Arth, H.-L.; Fessner, W.-D. Carbohydrate Res. 1998, 305,
313.