Novel 6-O-acylated vitamin C derivatives as hyaluronidase inhibitors with selectivity for bacterial lyases

Article abstract of DOI:10.1016/j.bmcl.2006.07.087

Previously, we identified ascorbic acid 6-O-hexadecanoate as an up to 1500 times more potent inhibitor of bacterial and bovine hyaluronidases than the parent compound, vitamin C, and determined a crystal structure of hyaluronidase from Streptococcus pneumoniae in complex with the inhibitor. As the alkanoyl chain interacts with a hydrophobic patch of the enzyme we synthesized other 6-O-acylated vitamin C derivatives bearing various lipophilic residues and investigated the inhibition of Streptococcus agalactiae strain 4755 hyaluronidase (SagHyal₄₇₅₅) and of bovine testicular hyaluronidases (BTH) in a turbidimetric assay. All compounds showed selectivity for the bacterial enzyme. Whereas vitamin C 6-O-hexanoate only weakly inhibited SagHyal₄₇₅₅, the inhibition of both enzymes increased with the length of the aliphatic chain. In the case of the 6-O-octadecanoate, IC₅₀ values of 0.9 and 39 μM for SagHyal₄₇₅₅ and BTH, respectively, were determined. Partial replacement of the aliphatic chain with a phenyl, p-phenylene or p-biphenylyl group resulted in inhibitors with activity in the lower micromolar range, too. The title compounds are among the most potent inhibitors of both enzymes known to date.

Full text of DOI:10.1016/j.bmcl.2006.07.087

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5313–5316
Novel 6-O-acylated vitamin C derivatives as hyaluronidase
inhibitors with selectivity for bacterial lyases
Martin Spickenreither, Stephan Braun, Gunther Bernhardt,
¨
Stefan Dove and Armin Buschauer*
Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry II, University of Regensburg,
Universita¨tsstr. 31, D-93040 Regensburg, Germany
Received 20 July 2006; revised 27 July 2006; accepted 27 July 2006
Abstract—Previously, we identified ascorbic acid 6-O-hexadecanoate as an up to 1500 times more potent inhibitor of bacterial and
bovine hyaluronidases than the parent compound, vitamin C, and determined a crystal structure of hyaluronidase from Streptococ-
cus pneumoniae in complex with the inhibitor. As the alkanoyl chain interacts with a hydrophobic patch of the enzyme we synthe-
sized other 6-O-acylated vitamin C derivatives bearing various lipophilic residues and investigated the inhibition of Streptococcus
agalactiae strain 4755 hyaluronidase (SagHyal4755) and of bovine testicular hyaluronidases (BTH) in a turbidimetric assay. All com-
pounds showed selectivity for the bacterial enzyme. Whereas vitamin C 6-O-hexanoate only weakly inhibited SagHyal4755, the inhi-
bition of both enzymes increased with the length of the aliphatic chain. In the case of the 6-O-octadecanoate, IC50 values of 0.9 and
39 lM for SagHyal4755 and BTH, respectively, were determined. Partial replacement of the aliphatic chain with a phenyl, p-pheny-
lene or p-biphenylyl group resulted in inhibitors with activity in the lower micromolar range, too. The title compounds are among
the most potent inhibitors of both enzymes known to date.
Ó 2006 Elsevier Ltd. All rights reserved.
Hyaluronidases are enzymes degrading hyaluronic acid
(hyaluronan), an important component of the extracel-
lular matrix. Hyaluronan consists of 1 ! 4-linked
b-D-glucuronic acid-(1 ! 3)-b-N-acetyl-D-glucosamine
disaccharide units. According to their catalytic mecha-
nism, the hyaluronidases are divided into three main
families. In contrast to hyaluronidases from other sourc-
es, bacterial hyaluronan lyases (EC 4.2.2.1) degrade the
substrate into unsaturated disaccharide units, cleaving
the b-1,4-glycosidic bond between N-acetyl-b-D-glucosa-
mine and D-glucuronic acid residues by an elimination
reaction.1 As the role of hyaluronidases is far from being
understood, potent and selective inhibitors are required
as pharmacological tools. Moreover, inhibitors of the
bacterial and mammalian enzymes might be useful
agents in the treatment of various diseases, for example,
bacterial infections,2 cancer,3 or as contraceptives.4
Vitamin C, reported to inhibit the bovine testicular hyal-
uronidase (BTH),5 was found to be a weak inhibitor of
the hyaluronan lyase of Streptococcus pneumoniae (IC50
value: 5.8 mM). X-ray analysis verifies that ascorbic acid
binds to the active site6 and suggests that additional
interactions with a hydrophobic patch might increase
the potency. Therefore, we investigated L-ascorbic
acid-6-O-hexadecanoate (vitamin C palmitate, Vcpal,
cpd. 13i in Table 1), a highly effective antioxidant, for
the inhibition of hyaluronan lyases from S. pneumoniae
(SpnHyal) and Streptococcus agalactiae strain 4755
(SagHyal4755) and of BTH. Furthermore, SpnHyal was
crystallized in complex with Vcpal and the X-ray struc-
ture was determined.7 Compared to vitamin C, Vcpal is
an up to 1500 times more potent inhibitor of bacterial
and bovine hyaluronidases.
As shown in Figure 1, Vcpal binds exactly in the same
region of the catalytic cleft of SpnHyal as a hexasaccha-
ride substrate.8 In addition to hydrogen bonds of the
carboxylate group of the inhibitor (the ring-opened
form of ascorbic acid was found in the crystal structure)
with Tyr408 and Arg462, and of the hydroxy groups at
C-4 and C-5 with Asn290, extensive interactions with
the hydrophobic patch (Trp291, Trp292, Phe343) are
Keywords: Ascorbic acid derivatives; Hyaluronidase inhibitors; Hya-
luronan; Hyaluronan lyases; Bovine testicular hyaluronidase.
*
Corresponding author. Tel.: +49 941 943 4827; fax: +49 941 943
4820; e-mail: armin.buschauer@chemie.uni-regensburg.de
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.087

5314
M. Spickenreither et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5313–5316
Table 1. Inhibitory effects of L-ascorbic acid derivatives on hyaluronan lyase from S. agalactiae strain 4755 (SagHyal4755) and bovine testicular
hyaluronidase (BTH; NeopermeaseÒ) at pH 5.0
Compound
R1
R2
R3
SagHyal4755
BTH
IC50 [lM] or % inhibitiona, pH 5.0
1
H
H
H
6100 100
Inactivec
355 59
Inactiveb
Inactivec
Inactivec
Inactivec
Inactived
Inactived
Inactived
Inactivee
Inactive
Inactive
1380 49
580 21
6
Me
Bn
Me
Bn
H
7
H
8
CH2CH2
CH2CH2
H
24% (2000)
5% (160)
Inactived
32% (190)
43% (1100)
475 16
9
Me
Bn
Me
Bn
CO(CH2)14CH3
CO(CH2)14CH3
CO(CH2)14CH3
COC(CH3)3
10
11
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
13r
13s
13t
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CO(CH2)4CH3
CO(CH2)6CH3
CO(CH2)8CH3
CO(CH2)9CH3
CO(CH2)10CH3
CO(CH2)11CH3
CO(CH2)12CH3
CO(CH2)14CH3
CO(CH2)16CH3
COPh
772 19
102
72
5
2
2
47
208
96
71
57
39
4
14.3 0.3
8.4 0.2
4.2 0.1
0.9 0.1
5
1
1
1
132
6
33% (1430)
2006 40
Inactive
COCH2-p-C6H4-Ph
CO(CH2)5OPh
CO(CH2)5OCH2Ph
CO(CH2)5O-p-C6H4-Ph
CO(CH2)5OCH2-p-C6H4-Ph
CO(CH2)5O-p-C6H4-C2H5
CO(CH2)5O-p-C6H4-OCH2Ph
CO(CH2)10O-Ph
CO(CH2)10O-p-C6H4-Ph
358 15
717 28
437 77
Inactive
3
61
1
188
543 15
Inactive
102
280
76
2
9
1
210
105
3
1
31 0.4
7.5 0.2
37 0.4
a Inhibition of enzyme expressed as IC50 (lM), mean values SEM (N = 2, experiments performed in duplicate), or as % inhibition at inhibitor
concentration given in parentheses; substances were tested up to a concentration of 1.5 mM unless otherwise indicated.
b At concentrations 6100 mM.
c At concentrations 6 13 mM.
d At concentrations 6200 lM.
e At concentrations 6 1.1 mM.
evident. The lipophilic face of the vitamin C portion lies
flat on the side chain of Trp292, and the palmitoyl group
interacts with Trp291 and Phe343, also with His399 and
Thr400. This prompted us to synthesize various ascorbic
acid derivatives with increased lipophilicity as potential
hyaluronidase inhibitors.
dure was used (Scheme 2, Method B): ascorbic acid was
treated with the corresponding methyl esters in the pres-
ence of lipase from Candida antarctica immobilized on
acrylic resin (Novozyme 435Ò) at 60 °C under reduced
pressure (200 mbar).10,11
The synthesized compounds were investigated for inhi-
bition of SagHyal4755 and BTH at pH 5.0 in a modified
turbidimetric assay16 based on the method of Di Ferran-
te.17 Under these conditions both enzymes show compa-
rably high activity.18 The results are summarized in
Table 1 and exemplarily depicted in Figure 2.
The ascorbic acid derivatives 9–11 were synthesized
according to a modified four-step reaction sequence by
analogy with the procedure described by Sanders9
(Scheme 1). First the acetal 2 was formed, which was
alkylated to obtain the 2,3-di-O-protected derivatives
3–5. After cleavage of the isopropylidene-protecting
group, acylation was carried out using palmitoyl chlo-
ride to obtain compounds 9–11. The 6-O-acylated ascor-
bic acid derivatives 13a, b, i–k were accessible by
acylation of benzyl-protected ascorbic acid 7 followed
by hydrogenolysis of the benzyl ether groups (Scheme
2, Method A). For the synthesis of the 6-O-acylated
ascorbic acid derivatives 13c–h, l–t an alternative proce-
Ascorbic acid (1) is only a very weak inhibitor of the
bacterial enzyme. Structural modifications of the enediol
system as in 6 and 8 are not tolerated, whereas the 2-O-,
3-O-dibenzylated analog 7 is more active than 1. This is
in agreement with the hypothesis that the potency of
hyaluronidase inhibitors derived from vitamin C may
be increased by additional hydrophobic interactions

M. Spickenreither et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5313–5316
5315
Scheme 2. Synthesis of the 6-O-acylated ascorbic acid derivatives 13a–
t, Reagents and conditions: (i) X = Cl: pyridine/DCM, DMAP (cat.);
X = OH: 1,10-carbonyl-diimidazole (CDI) or dicyclohexyl carbodii-
mide (DCC), DCM, DMAP (cat.); (ii) 10% Pd/C (cat.), H2, 4 bar,
EtOH. (iii) Lipase acrylic resin from Candida antarctica, tert-amyl
alcohol, 60 °C, 200 mbar, 16–24 h. The alkanoic acids and acid
chlorides used for the preparation of 12a, b, i–k are commercially
available. The building blocks 14c–h, l–t were prepared according to
standard procedures starting from the corresponding commercially
available alkanoic acids, x-bromo-alkanoic acids, methyl x-bromoalk-
anoates or from e-caprolacton.12–15
Figure 1. Binding modes of Vcpal and a hyaluronan hexasaccharide.
(A) Binding mode of Vcpal (13i) in the active site of SpnHyal (PDB
code 1w3y). The inhibitor was found to bind in a ring-opened form.
Missing electron density did not permit modeling of the last three
carbon atoms of the palmitoyl chain. (B) SpnHyal in complex with a
hyaluronic acid hexasaccharide (PDB code 11oh, for clearity only the
tetrasaccharide is shown).
Figure 2. Enzymatic activity of hyaluronan lyase from S. agalactiae
strain 4755 in the presence of compounds 13c, f, j, o, t at pH 5.0.
with amino acids in the active site. Regarding com-
pounds 13b–j, the inhibition of both enzymes increases
with the length of the 6-O-acyl residue, hence the octa-
decanoate 13j inhibits SagHyal4755 and BTH with the
lowest IC50 values of 0.9 and 39 lM, respectively. Com-
Scheme 1. Synthesis of the 6-O-palmitoyl ascorbic acid derivatives 9–
11. Reagents and conditions: (i) abs. acetone, AcCl (cat.); (ii) K2CO3,
MeI or BnBr or 1,2-dibromoethane, DMF, 40–60 °C; (iii) 50% HOAc,
MeOH, 80 °C; (iv) CH3(CH2)14COCl, pyridine, dichloromethane
(DCM), 4-dimethylaminopyridine (DMAP) (catalytic amount).

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.
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