5316
M. Spickenreither et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5313–5316
9. Sanders, J. Doctoral thesis, Aachen, 1984.
10. Gruning, B.; Hills, G. Eur. Patent Appl. EP 924301, 1999;
Chem. Abstr. 1999, 131, 57865.
pound 13k, bearing only a relatively small benzoyl resi-
due, is more active than the alkanoyl analogs 13b and
13c on the bacterial enzyme. Structural modifications
at the hydroxy groups of the enediol system of the
strong hyaluronidase inhibitor 13i (see compounds 9–
11) led to significantly lower potency on the bacterial
enzyme and to inactivity on the BTH, respectively.
Comparing compounds 13m with 13o and 13s with
13t, a remarkably increased inhibitory potency on both
enzymes results when the phenyl groups are replaced
with p-biphenylyl residues. The synthetic approach
using an ether linkage to introduce aromatic residues
makes the desired compounds easily accessible. More-
over, the ether group is capable of forming H-bonds in
the active site by analogy with the substrate hyaluronan.
The more rigid phenyl and biphenylyl residues are
suitable alternatives to the flexible alkyl chains, as the
inhibitory activities are in the same range as those of
the corresponding aliphatic compounds.
11. General procedure for the preparation of 6-O-acylated
ascorbic acid derivatives using immobilized lipase from
Candida antarctica (Novozyme 435Ò): Vitamin C (1
equiv), the pertinent methyl alkanoate (1.5–4 equiv), and
the immobilized enzyme (50 mg/mmol ascorbic acid) were
suspended in tert-amyl alcohol and submitted to rotary
evaporation at 60 °C bath temperature and 200 mbar
vacuum for 14–24 h. After the major part of ascorbic acid
was consumed, the reaction mixture was cooled to room
temperature, solids were removed by filtration, the solvent
was removed under reduced pressure, and the resulting
material was taken up in EtOAc. After washing with brine
and water, the organic phase was dried over MgSO4.
Removal of the solvent under reduced pressure and
recrystallization from tert-butyl methyl ether/hexane yield-
ed the target compounds as white solids.
12. Hocke, H.; Uozumi, Y. Tetrahedron 2004, 60, 9297.
13. Jacobi, P. A.; Li, Y. Org. Lett. 2003, 5, 701.
14. Parang, K.; Knaus, E. E.; Wiebe, L. I.; Sardari, S.;
Daneshtalab, M.; Csizmadia, F. Arch. Pharm. Pharm.
Med. Chem. 1996, 329, 475.
15. Wolfe, S.; Wilson, M.-C.; Cheng, M.-H.; Shustov, G. V.;
Akuche, C. I. Can. J. Chem. 2003, 81, 937.
In summary, the presented 6-O-acyl derivatives of
L-ascorbic acid are among the most potent inhibitors
of hyaluronidases known so far. The title compounds
are rather selective for the bacterial enzyme and repre-
sent promising lead structures for rational optimization
in ongoing work.
16. Determination of enzyme inhibition: 10 lL of
a
0.2 lM–2 mM solution of the test compound in
DMSO was incubated at 37 °C in a mixture composed
of 120 lL of McIlvaine’s buffer (solution A: 0.2 M
Na2HPO4, 0.1 M NaCl, solution B: 0.1 M citric acid,
0.1 M NaCl; solution A and B were mixed in the
appropriate proportions to reach pH 5.0), 30 lL BSA
solution (0.2 mg/mL in water), 50 lL water, 30 lL
hyaluronan solution (2 mg/mL in water) and 30 lL of
either bacterial or bovine hyaluronidase solution
(SagHyal4755 (id-Pharma, Jena, Germany) was dis-
solved in BSA solution (0.2 mg/mL in water); BTH
(Sanabo, Vienna, Austria) was dissolved in 1 mL H2O
and further diluted with BSA solution (0.2 mg/mL in
water)). Equiactive concentrations of BTH (54 ng) or
SagHyal4755 (2.9 ng) were incubated for 30 min. The
final DMSO concentration was 3.7% (v/v). After
incubation of the reaction mixture, the residual high
molecular weight hyaluronan was precipitated by
addition of 700 lL of a 2.5% (w/v) cetyltrimethylam-
monium bromide (CTAB) solution (2.5 g CTAB dis-
solved in 100 mL of 0.5 M NaOH solution, pH 12.5).
The turbidity of each sample was quantified at 600 nm
with a Uvikon 930 UV spectrophotometer (Kontron,
Eching, Germany) after an incubation period of
20 min at 25 °C. Samples without inhibitor, and
enzyme were taken as references. The activities were
plotted against the logarithm of the inhibitor concen-
tration, and IC50 SEM values were calculated by
curve fitting of the experimental data with Sigma Plot
8.0 (SPSS Inc., Chicago, IL) and are the means of at
least two independent experiments performed in
duplicate.
Acknowledgments
We thank Mrs. L. Schneider for technical assistance.
Support to this work by the Graduate Training Program
(Graduiertenkolleg) GRK 760 ‘‘Medicinal Chemistry:
Molecular Recognition – Ligand-Receptor Interac-
tions’’ of the Deutsche Forschungsgemeinschaft is grate-
fully acknowledged.
References and notes
1. Glycoforum.
2. Hynes, W. L.; Walton, S. L. FEMS Microbiol. Lett. 2000,
183, 201.
3. Isoyama, T.; Thwaites, D.; Selzer, M. G.; Carey, R. I.;
Barbucci, R.; Lokeshwar, V. B. Glycobiology 2006, 16,
11.
4. Zaneveld, L. J.; Waller, D. P.; Anderson, R. A.; Chany, C.
n.; Rencher, W. F.; Feathergill, K.; Diao, X. H.; Doncel,
G. F.; Herold, B.; Cooper, M. Biol. Reprod. 2002, 66, 886.
5. Menzel, E. J.; Farr, C. Cancer Lett. 1998, 131, 3.
6. Li, S.; Taylor, K. B.; Kelly, S. J.; Jedrzejas, M. J. J. Biol.
Chem. 2001, 276, 15125.
7. Botzki, A.; Rigden, D. J.; Braun, S.; Nukui, M.; Salmen,
S.; Hoechstetter, J.; Bernhardt, G.; Dove, S.; Jedrzejas, M.
J.; Buschauer, A. J. Biol. Chem. 2004, 279, 45990.
8. Jedrzejas, M. J.; Mello, L. V.; De Groot, B. L.; Li, S. J.
Biol. Chem. 2002, 277, 28287.
17. Di Ferrante, N. J. Biol. Chem. 1956, 220, 303.
18. Hoechstetter, J. Doctoral thesis, Regensburg, 2005.