Design of benzimidazole- and benzoxazole-2-thione derivatives as inhibitors of bacterial hyaluronan lyase

Article abstract of DOI:10.1016/j.ejmech.2011.07.014

Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC₅₀ values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch.

Full text of DOI:10.1016/j.ejmech.2011.07.014

European Journal of Medicinal Chemistry 46 (2011) 4419e4429
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design of benzimidazole- and benzoxazole-2-thione derivatives as inhibitors of
bacterial hyaluronan lyase
*
Stephan Braun, Alexander Botzki, Sunnhild Salmen, Christian Textor, Günther Bernhardt, Stefan Dove ,
Armin Buschauer
Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular
matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the
enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme
inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of
Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors,
1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the
inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-
phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC50
Received 21 March 2011
Received in revised form
29 June 2011
Accepted 8 July 2011
Available online 19 July 2011
Keywords:
Hyaluronan lyase inhibitors
Streptococcus agalactiae
Structure-based design
Benzimidazole-2-thiones
Benzoxazole-2-thiones
values: 24 mM at pH 7.4, 15 mM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles
and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode
characterized by interactions with residues in the catalytic site and with a hydrophobic patch.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
HA is degraded by hyaluronidases, a class of enzymes with two
main families, the eukaryotic hyaluronoglucosaminidases (EC
Hyaluronan, the polyanionic form of hyaluronic acid (HA), is
a linear polyglycosaminoglycan consisting of repeating disaccha-
3.2.1.35) and the prokaryotic hyaluronan lyases (EC 4.2.2.1) [6,7].
The bacterial enzymes cleave HA at the -1,4-glycosidic linkage
between -glucuronic acid and N-acetyl- -glucosamine. The
product of this elimination reaction is the unsaturated disaccharide
2-acetamido-2-deoxy-3-O-( -gluco-4-enepyranosyluronic acid)-
-glucose [6,8]. The best characterized hyaluronan lyases are from
Streptococcus pneumoniae (SpnHyal) and Streptococcus agalactiae
(SagHyal) [9,10]. The 3D structures of both enzymes were eluci-
dated by X-ray analyses in the native form as well as in complex
with substrates, products and in the case of SpnHyal also with
inhibitors [9e16]. Fig. 1 presents an overview of the SpnHyal
structure complexed with a hexasaccharide substrate. It consists of
b
ride units of (
b
-1,4)-
D
-glucuronic acid (
b
-1,3)-N-acetyl-
D
-glucos-
D
D
amine. It was first isolated from the vitreous humor of bovine eyes
[1] and is a major component of the extracellular matrix (ECM) of
animal tissues. HA is present in the capsule of some bacteria and in
essentially all vertebrates where it mainly occurs in the skin and in
the CNS [2]. HA interacts with various receptors (e.g., CD44) and
hyaluronan binding proteins (hyaladherins like aggrecan, versican,
neurocan and brevican) on the surface of cells [3,4]. As main
structural element in the matrix HA plays an important role for
tissue architecture. Moreover, HA is involved in many biological
processes including fertilization, embryonic development, cell
migration and differentiation, wound healing, inflammation as well
as growth and metastasis of tumor cells [2,4,5].
b-D
D
an
a-helical and a b-sheet domain, both linked by a loop of 10
amino acids. The active site is located in a long cleft and comprises
four main parts: a positive patch, a catalytic triad (Asn349, His399
and Tyr408) responsible for degradation, a hydrophobic/aromatic
patch (Trp291, Trp292 and Phe343) responsible for correct positioning
of the substrate chains, and a negative patch implicated in the
release of the product (Glu388, Asp398, and Thr400) [12].
The S. pneumoniae enzyme shows 53% sequence identity with
the nearly (98%) identical hyaluronan lyases from two S. agalactiae
strains, 4755 (SagHyal4755) and 3502 (SagHyal3502) [17]. However,
the amino acids of the active sites of the three enzymes are entirely
the same. In contrast to the structure of SpnHyal, a fully active two-
Abbreviations: BSA, bovine serum albumin; CTAB, cetyltrimethylammonium
bromide; DMAP, 4-(dimethylamino)pyridine; DMF, dimethylformamide; DMSO,
dimethyl sulfoxide; ECM, extracellular matrix; HA, hyaluronan; Hyal, hyaluronan
lyase; SagHyal4755, Streptococcus agalactiae strain 4755 hyaluronan lyase; SpnHyal,
Streptococcus pneumoniae hyaluronan lyase; TCDI, 1,10-thiocarbonyl diimidazole;
THF, tetrahydrofuran; Vc, L-ascorbic acid; Vcpal, L-ascorbic acid-6-hexadecanoate.
* Corresponding author. Tel.: þ49 941 943 4673; fax: þ49 941 943 4820.
E-mail address: Stefan.Dove@chemie.uni-regensburg.de (S. Dove).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.014

4420
S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
contacts with Trp292 and Tyr408 are important. Higher activity was
predicted for derivatives which additionally fit to the hydrophobic
patch. Indeed, -ascorbic acid-6-hexadecanoate (Vc palmitate,
Vcpal, 2, Fig. 2), a highly effective antioxidant [22] and glutathione-
S-transferase inhibitor [23], showed an IC50 value of 4.2 M at
L
m
SagHyal4755 [15]. The X-ray structure of the SpnHyaleVcpal
complex [15], determined at 1.65 Å resolution, confirmed the
hypothesis that additional hydrophobic interactions with Phe343
,
His399 and Thr400 lead to increased inhibition. Within a congeneric
series of 6-O-acylated Vc derivatives, -ascorbic acid-6-
octadecanoate was the most potent SagHyal4755 inhibitor (IC50
0.9 M) [24].
Recent work on hyaluronan lyase inhibitors in our laboratory
L
m
was focused on the generation of new leads. The program LUDI [25]
identified numerous hits by docking of molecules from databases
into a SagHyal4755 model [26]. 13 of 19 synthesized compounds
proved to be active against SagHyal4755. 1,3-Diacetylbenzimidazole-
Fig. 1. Schematic representation of the SpnHyal structure in complex with a hex-
asaccharide substrate (black sticks). Cylinders - a-helices, ribbons - b-sheets. Amino
2-thione (3, Fig. 2) was the most potent inhibitor (IC50
5 mM at pH
acids of the catalytic triad (1 e His399, 2 e Tyr408, 3 e Asn349) are shown as graysticks,
residues of the hydrophobic patch (4 e Trp291, 5 e Trp292, 6 e Phe343) as space fill
model.
7.4). Another hit from these investigations, indole-2-carboxylic acid
(4, Fig. 2), was active only at concentrations above 1 mM, but
initiated the rational design of a series of inhibitors based on the 2-
phenylindole scaffold [27,28]. The most active compound, 1-decyl-
2-(4-sulfamoyloxyphenyl)-1H-indol-6-yl sulfamate (5, Fig. 2, IC50
domain truncated version [12], the SagHyal3502 X-ray structure
reveals an additional b-sheet domain at the N-terminus and is not
on SagHyal4755 11 mM at pH 5, 16 mM at pH 7.4) was crystallized in
catalytically active because of an w7.3 Å wider binding cleft [13].
HA degradation by hyaluronan lyases consistently results in
unsaturated disaccharides, which can be utilized by bacteria as an
additional carbon source [18], and leads to reduced viscoelasticity
of the ground substance in the ECM of the host. Thus, the spread of
the pathogens and of their associated toxins into tissues may be
facilitated [18]. Streptococcal hyaluronan lyases can therefore be
regarded as significant virulence factors involved in human infec-
tions which are major causes of meningitis, pneumonia, septicemia,
as well as many other serious diseases that lead to neonatal
mortality. Hyaluronan lyase inhibitors may serve as useful phar-
macological tools to study the precise role of the enzyme, its
substrates and products in the course of bacterial infection and
spread and are of potential interest as candidates for antibacterial
treatment in combination with antibiotics. However, the detection
and the design of inhibitors are still at the beginning (for recent
review, see [19]).
complex with SpnHyal [16], indicating a similar interaction pattern
as in the case of Vcpal. In particular, hydrophobic chains at the
indole nitrogen were shown to mimic the hexadecanoyl moiety of
Vcpal.
The present study is dealing with the derivation and optimiza-
tion of two other leads, namely benzimidazole- and benzoxazole-2-
thiones, based on comparison of SpnHyal-inhibitor complexes and
exploration of possible interaction sites. The synthesis and the
structureeactivity relationships of these novel SagHyal4755 inhibi-
tors will be described and discussed in the light of a common
binding mechanism.
2. Results and discussion
2.1. Lead generation
4-(3-Phenyl-1H-indol-2-yl)phenol inhibited bovine testes
hyaluronidase (IC50 107 mM) [29]. Based on indole-2-carboxylic acid
Various unsaturated fatty acids inhibited the enzyme from
Streptococcus dysgalactiae with IC50 values ranging from 17 to
(4, Fig. 2), a hit from structure-based LUDI searches [26], a series of
2-phenylindole derivatives was synthesized and tested for inhi-
bition of SagHyal4755 [27] (to be published elsewhere). For
minimum activity, hydroxy, methoxy or ester groups in 5- or 6-
position of the indole nucleus and in 4-position of the phenyl
ring seem to be necessary. The binding mechanism of 2-
phenylindoles could be analyzed by the crystal structure of
SpnHyal in complex with 1-decyl-2-(4-sulfamoyloxyphenyl)-1H-
indol-6-yl sulfamate (5, Fig. 2) [16]. Fig. 3 shows that the inhibitor 5
172
m
M [20]. Glycyrrhetinic acid, a triterpene, was active against
SagHyal (IC50 60
m
M) [21]. -Ascorbic acid (Vc, 1, Fig. 2) was
L
described as another, but weak competitive inhibitor (IC50 w6 mM)
[14]. The crystal structure of the SpnHyal-Vc complex [14] indicated
that Vc binds in the active site of the enzyme and that hydrophobic
binds in the cleft formed by the a-domain in the area of the cata-
lytic triad (Asn349, His399 and Tyr408) and the hydrophobic patch
(Trp291, Trp292 and Phe343) and, therefore, in the same site in which
the HA disaccharide product [17], Vc [14] as well as Vcpal [15] were
located in our earlier studies. Hydrophobic interactions of the rings
and the aliphatic tail as well as hydrogen bonds of the 6-
sulfamoyloxy group contribute to the binding affinity. The indole
plane of the inhibitor is in parallel with that of Trp292. The opposite
side of the indole moiety interacts with the guanidino group of
Arg462. The phenyl nucleus of the inhibitor is sandwiched between
Trp291 (nearly perpendicular ring arrangement) and Asn580. The
decyl chain fits into a surface crevice lined mainly by hydrophobic
residues e Trp291, Phe343, Met579 e but also by Arg336, Glu388 and
His399. The 6-sulfamoyloxy group forms a network of hydrogen
Fig. 2. Chemical structures of hyaluronan lyase inhibitors. 1: Vitamin C, L-ascorbic acid,
Vc; 2: L-ascorbic acid-6-hexadecanoate, Vcpal; 3: 1,3-diacetylbenzimidazole-2-thione;
4: indole-2-carboxylic acid; 5: 1-decyl-2-(4-sulfamoyloxyphenyl)-1H-indol-6-yl sul-
famate, 6: N-substituted 2-(4-hydroxyphenyl)-3-methyl-5-hydroxyindoles.

S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
4421
implies that an unsubstituted nitrogen in 3-position may form
a hydrogen bond with the amide oxygen of Asn290 (cf. Fig. 3A).
Replacing this nitrogen by oxygen leads to benzoxazole-2-thiones.
In this case, a hydrogen bond of the benzoxazole oxygen with the
amide group of Asn290 may be suggested. The acetylation of the N1
nitrogen in compound 5 could enable additional interactions with
His399, Tyr408 and/or Arg462. Fig. 3B shows the lipophilic potential
mapped on the surface of the binding site of SpnHyal together with
the phenylindole 5 and a hexasaccharide substrate, both extracted
from the respective aligned crystal structures [11,16]. A continuous
hydrophobic region ranges from Trp292 outwards to Phe343
,
accommodating parts of the indole ring and nearly the complete
decyl chain as well as the HA disaccharide unit to be released. Based
on this comparison, the idea was to mimic the terminal N-acetyl-
glucosamine moiety by an aromatic or alicyclic ring, i.e., to replace
1-alkanoyl or 1-alkyl substituents by, e.g.,
u-phenylalkanoyl
groups. 3-Phenyl substituted 1-propionyl derivatives were pre-
dicted to be optimal because of possible overlap of the N-acetyl-
glucosamine residue with the terminal phenyl moiety.
Fig. 3B indicates that parts of the phenyl moiety and the 40-
substituent project into the solvent. Therefore, shorter groups may
be more appropriate. Benzimidazole-2-thiones and benzoxazole-2-
thiones are new leads bearing a sulfur atom instead of the 40-
substituted phenyl ring present in compounds 5 and 6.
2.2. Chemistry
The benzimidazole-2-thione derivatives were synthesized as
shown in Fig. 4. The preparation of 1-acetyl-benzimidazole-2-
thione (10) was performed according to the procedure described
by Saxena et al. [30], whereas the other 1-monoacylated benzimi-
dazol-2-thione derivatives 11e15 were accessible by a slightly
modified protocol using benzimidazol-2-thione
7 as starting
material and acid chlorides or acid anhydrides as acylating
reagents. Pyridine was used as solvent and base.
1,3-Diacetyl-benzimidazol-2-thione (3) was obtained by acety-
lation of benzimidazole-2-thione with acetyl chloride in the pres-
ence of triethylamine as base and N,N-dimethylaminopyridine
(DMAP) as catalyst at room temperature. The 1-hexanoyl homolog
18 was synthesized by treating 13 with acetyl chloride and trie-
thylamine in dry THF (modified protocol according to [31]).
The oxo analog of compound 3, 1,3-diacetyl-benzimidazol-2-
one (16), was prepared according to previously described
methods [32,33]. From the chemical point of view, 16 is an inter-
esting reagent, but probably, its use as an enzyme inhibitor is
limited due to its high reactivity toward nucleophiles, although it is
more stable than the corresponding thio analogs.
Fig. 3. SpnHyal binding site of the phenylindole inhibitor 5 (view into the substrate
binding cleft with the -domain in the foreground). A) Detailed representation of
interactions. Colors of C atoms: inhibitor gray, residues with hydrophobic contacts to
the inhibitor orange, catalytic triad cyan, other residues green. Magenta balls: oxygen
of water molecules participating in the SpnHyal-inhibitor H-bond network. B) Lipo-
philic potential of the binding site mapped onto a MOLCAD surface. Cpd. 5 (C atoms
a
white) and
a hexasaccharide substrate (C atoms black) are aligned according to
a backbone rms fit of the complexes PDB 2BRP and 1LOH. Hydrophobic regions are
colored in brown, polar regions in green and green-blue. The 4-sulfamoyloxy
substituent is exposed to the solvent. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
The alkylated derivatives 9 and 17 were synthesized by modi-
fication of published methods [31,34] via N,N0-dialkylation of o-
phenylenediamine followed by ring closure to obtain the corre-
sponding benzimidazol-2-thiones. o-Phenylenediamine was alky-
lated with bromoethane and sodium hydride as base in DMF
yielding both the diethylated (21%) and the monoethylated
bonds with the protein and water, including charge assisted
bidentate H bonds between Arg466 and two sulfamoyl oxygen
atoms. Both oxygen atoms and the nitrogen are additionally linked
to the protein by through-water interactions.
In a homologous series of N-substituted 2-(40-hydroxyphenyl)-
3-methyl-5-hydroxyindoles (6, Fig. 2), SagHyal4755 inhibition at pH
7.4 increases in the order R ¼ H (IC50 280
n-propyl (IC50 160 M), n-pentyl (IC50 36
12
m
M), methyl (IC50 220
mM),
m
mM), and n-heptyl (IC50
m
M) [27]. Thus, the fit of the aliphatic chains to the hydrophobic
SagHyal4755 pocket corresponding to SpnHyal Trp291, Phe343 and
Met579 significantly contributes to the binding affinity.
An appropriate strategy to derive new leads from the phenyl-
indole inhibitors is to retain a bicyclic scaffold. The high activity of
1,3-diacetylbenzimidazole-2-thione (3, Fig. 2), a hit from LUDI
searches [26], provides first clues to possible variations. The
benzimidazole moiety may be docked into SpnHyal in superposi-
tion with the indole scaffold of compound 5. This docking mode
Fig. 4. Synthesis of N-mono and diacylated benzimidazole-2-(thi)ones. Reagents and
conditions: (a) R2Cl or (R2)2O, pyridine (not used for 16); (b) R2Cl, Et3N, THF.

4422
S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
Table 1
substance (38%). The cyclization was performed with TCDI in
anhydrous THF. Overnight stirring at ambient temperature resulted
in high yields of 9 (93%) which was converted to 17 by acylation
with acetyl chloride in anhydrous THF in the presence of
triethylamine.
Inhibitory activities (IC50 values) of benzimidazole and benzoxazole derivatives
determined on SagHyal4755 at pH 5.0 and pH 7.4.
The preparation of the N-acylated benzoxazole-2-(thi)ones
22e36 was performed using modified protocols (22e34 [35], 35
and 36 [31]) by treating benzoxazole-2-thiol (19) and benzoxazol-
2-one (20), respectively, with either appropriate acid chloride or
acid anhydride in anhydrous THF as shown in Fig. 5. The N-alky-
lated benzoxazole-2-thione 38 was obtained via a ring closing
reaction of 2-(ethylamino)phenol (37) with TCDI (synthesis modi-
fied according to [36]).
Compound R1
R2
X
Y
hylB4755, IC50 [m
M] or %a
pH 5.0
160
pH 7.4
5
3b
H
H
H
H
COCH3
COCH3
CH2CH3
NCOCH3
NCOCH3
NCOCH3
S
O
S
S
16b
17b
18
Inact. (200) 10% (200)
2.3. Structureeactivity relationships
10% (380)
50% (49)
19 ꢀ 1
CO(CH2)4CH3 NCOCH3
12 ꢀ 1
The inhibitory activities (IC50 values) of the benzimidazole and
benzoxazole derivatives determined by a turbidimetric assay [37]
on SagHyal4755 at pH 5.0, the pH optimum of the enzyme, and pH
7.4 are reported in Table 1. Strikingly, all compounds except 28 are
more potent at pH 7.4 than at pH 5. It is unlikely that this behavior is
simply due to the effect of Km on the relationship between IC50 and
Ki since the HA concentrations used in the assays were not signif-
icantly greater than estimated Km values and since the
2-phenylindole 5 and some of its derivatives are equiactive at both
pH values, too [27].
Generallythe displacementof the thione sulfurbyoxygen(16, 35,
36) leads to inactivity. Further common considerations are that the
potencies of the benzimidazoles at pH 5 are much lower than those
of the benzoxazoles, and that at least one N-acyl substituent must be
present since the unsubstituted compounds 7 and 19 as well as the
N-ethyl derivatives 9 and 38 are nearly inactive in contrast to the
7b
H
H
H
H
H
H
H
H
H
NH
NH
NH
NH
S
S
S
S
S
S
S
S
1148 ꢀ 50
19% (4200)
20% (460)
4% (180)
7% (190)
14% (170)
5% (100)
1862 ꢀ 117
28% (4200)
17 ꢀ 1
9b
CH2CH3
COCH3
COCH2CH3
CO(CH2)2CH3 NH
CO(CH2)4CH3 NH
10b
11b
12
13
14
15
26 ꢀ 3
20 ꢀ 2
16 ꢀ 1
CO(CH2)2Ph
CO-m-ClC6H4 NH
NH
29 ꢀ 5
16% (400)
70 ꢀ 10
19b
21b
22b
23b
24
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
S
42% (3800)
2542 ꢀ 125
CO2CH3
1299 ꢀ 223 27% (2000)
H
H
H
H
H
H
H
H
H
H
H
H
COCH3
125 ꢀ 5
42 ꢀ 3
48 ꢀ 2
29 ꢀ 1
25 ꢀ 4
17 ꢀ 1
69 ꢀ 2
24 ꢀ 1
19 ꢀ 1
36% (200)
20 ꢀ 1
62 ꢀ 2
COCH2CH3
CO(CH2)4CH3
CO(CH2)8CH3
CO(CH2)14CH3
COCH2Ph
CO(CH2)2Ph
CO(CH2)3Ph
COCH]CHPh
CO(CH2)2Cy
COCH2OPh
CO2CH2Ph
128 ꢀ 5
51% (130)
41% (100)
23% (63)
260 ꢀ 41
15 ꢀ 1
25
26
27
28b
29b
30b
31
47% (100)
9% (80)
1-acetyl-3-ethyl benzimidazole 17 (IC50 19 mM at pH 7.4).
227 ꢀ 122
The diacylated benzimidazole-2-thiones 3 and 18 show the
highest inhibitory potency of the whole series at pH 7.4. At pH 5
they are still moderately active in contrast to the almost inactive
monoacylated derivatives 10e15. At pH 7.4, the IC50 values of 10e15
approach those of 3 and 18, but there is no distinct effect of the
chain length since all homologs except the m-Cl substituted ben-
zoylated compound 15 are nearly equiactive. This result was rather
disappointing with regard to the original intention to fit the acyl
chains to the hydrophobic patch like in the case of Vcpal (2) and the
phenylindole 5.
By contrast, the SAR of the acylated benzoxazole-2-thiones
22e33 are more pronounced. At pH 7.4, the higher open-chained
homologs 24e26 are by a factor of w2 more active than the
acetyl and propionyl derivatives 22 and 23, respectively. With
a terminal phenyl moiety, optimal potency was observed on
3-phenylpropionyl (28) and 4-phenylbutanoyl (29) substitution.
Having in mind that only compound 28 inhibits SagHyal4755 also at
pH 5 with an IC50 value in the range of the best results at pH 7.4
32b
33b
34
213 ꢀ 30
Inact. (100) 556 ꢀ 20
CO2CH3 CO(CH2)2Ph
56% (200)
21 ꢀ 1
35b
36
H
H
H
COCH3
CO(CH2)4CH3
CH2CH3
Inact. (2000) 1466 ꢀ 76
Inact. (150) Inact. (300)
38b
46% (400)
17% (1000)
Reference compoundsc
1 (Vc)
2 (Vcpal)
5
6100 ꢀ 100
4.2 ꢀ 0.13
11
16
a
Inhibition was expressed as IC50 ꢀ SEM in
mM or as % inhibition at the
concentration of inhibitor given in parentheses, highest tested concentrations were
dependent on the solubility of the compounds.
b
The following compounds are not original: 3 [38e40], 7 (purchased), 9 [38], 10
[38,39,41], 11 [39], 16 [33,41], 17 [41], 19 (purchased), 21 [42,43], 22 [35,44], 23
[45], 28 [44,46], 29 [46], 30 [47], 32 [44], 33 [44,48], 35 [31,49], 38 [36,50].
c
Data of 1 and 2 from Ref. [15], data of 5 from Ref. [16].
3-phenylpropionyl group for fit to the hydrophobic patch is
confirmed in the case of benzoxazole-2-thiones. Retaining the
chain length of 28, a terminal cyclohexyl moiety (31) is still favor-
able, whereas a phenoxyacetyl group (32) reduces the potency by
a factor of 3. A benzyloxycarbonyl substituent (33) and even more
the rigidisation of the chain by introduction of a double bond (30)
drastically decreases activity.
(15 mM), it may be concluded that the predicted optimum of the
An additional methoxycarbonyl substituent in 5-position of the
benzoxazole ring does not increase SagHyal4755 inhibition (cf. cpd.
19 with 21 and 28 with 34). Compared to 28, the activity of cpd. 34
at pH 5 is even more than one order of magnitude lower.
All of the compounds in Table 1 were inactive as inhibitors of
bovine testicular hyaluronidase at the highest concentration tested
at pH 5.
Fig. 5. Synthesis of N-substituted benzoxazole-2-(thi)ones. Reagents and conditions:
(a) R2Cl or (R2)2O, Et3N, THF; (b) TCDI, THF.

S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
4423
2.4. Discussion
His399, Tyr408 and Arg462, respectively, indicates hydrogen bonds
and/or ionedipole interactions (see Fig. 6A). The phenylpropionyl
moiety of compound 28 mainly interacts with Trp291 and Phe343
The structureeactivity relationships of SagHyal4755 inhibition
indicate that the predicted binding mode, based on the fit of the
bicyclic scaffolds to the indole moiety of the phenylindole 5, is
reasonable at least in the case of the benzoxazole-2-thiones. Fig. 6A
shows the most active inhibitor 28 docked into the SpnHyal -
compound 5 complex. The benzoxazole oxygen is suggested to
form hydrogen bonds with the amide NH2 groups of Asn290 and
(hydrophobic patch), but also with Arg336 (catione
p interactions)
and His399. Because of the similar potency of compounds 5 and 28,
it may be speculated that some interactions of 5, in particular of the
6-sulfamoyloxy group and of the 2-phenyl substituent, are
compensated in the case of acylated benzoxazole-2-thiones by
interactions of the acyl function, the oxazole oxygen and the thione
sulfur.
Asn580
. Because of the inactivity of the benzoxazole-2-one
compounds 35 and 36, hydrophobic interactions of the thione
sulfur with Trp291 and Trp292 are predicted. These interactions
partially replace those of the 2-phenyl group in compound 5 (see
Fig. 3A). The remarkable increase in potency observed for
1-acylated vs. 1-alkylated derivatives (compare e.g., cpds. 22 and
38) suggests that the acyl oxygen contributes to binding. Its short
distance (<3Å) to the imidazolyl, hydroxy and guanidino groups of
In Fig. 6B, the docking pose of compound 28 is compared with
the binding modes of a hexasaccharide substrate [11], of Vcpal (2)
[15] and of the phenylindole 5 [16]. The more or less polar cores and
the hydrophobic chains of the inhibitors are largely aligned. The
heterocyclic scaffolds of compounds 5 and 28 as well as the ring-
opened [15] vitamin C moiety of Vcpal occupy the region around
the scissile bond of the substrate, whereas the alkyl or arylalkanoyl
chains correspond to the disaccharide unit to be released. Obvi-
ously, the 3-phenylpropionyl group of the benzoxazole-2-thione 28
optimally aligns with this HA unit since the 1,3-glycosidic linkage is
mimicked by the two methylene groups and since the N-acetyl-
glucosamine residue overlaps with the terminal phenyl ring.
Fig. 6B additionally contains the binding modes predicted for
1,3-diacetylbenzimidazole-2-thione (3). Using a SagHyal4755 model,
two possible poses resulted from LUDI runs [25] with different
search spheres (radii of 5 and 8 Å, respectively) [26]. The two poses
adopt nearly mirror-inverted positions. In each pose, the acetyl
groups interact with the guanidine moieties of two arginines
(SpnHyal: Arg243 and Arg466) bridging the walls at the narrowest
site of the HA binding cleft, and the thione sulfur is less than 3.5 Å
distant from a Trp side chain corresponding to SpnHyal Trp292
.
However, the predicted poses are rather questionable in the light of
the SAR. The high activity of the hexanoyl derivative 18 is still
conceivable since the alkanoyl chain might align with the long HA
binding cleft on both sides, and also the lower potency of the
1-ethyl-substituted benzimidazole 17 is not surprising due to the
presence of only one acetyl group. But there is no reasonable
explanation for the inactivity of the benzimidazole-2-one 16. It may
be speculated that the binding mode of compound 3 corresponds to
that of the phenylindole 5, but a close superposition of both
heterocyclic scaffolds on docking to the SpnHyal structure is
impossible because of steric clashes of one of the acetyl groups with
Asn290 and Asn580. However, considering the specific topology of
the substrate binding site e a rather long cleft instead of a deep
pocket like in many other enzymes e multiple and overlapping
inhibitor binding modes may exist, especially in the case of small
molecules like 3.
The binding mode of the benzimidazole-2-thiones 10e15 may
be similar to that of the benzoxazole-2-thiones 22e33. The NH
function in 3-position, however, suggests interaction with the
amide oxygens of Asn290 and/or Asn580. The temperature factors of
the ligand-free SpnHyal structure [12] indicate high flexibility of the
Asn290 side chain unlike that of Asn580. As a consequence, the
benzimidazole-2-thiones may dock in a position which slightly
differs from the binding modes in Fig. 3A and Fig. 6A and which is
characterized by the same type of interactions as in the case of the
benzoxazole derivatives. This would explain why the optimal
members of both series are equipotent at pH 7.4 and why both N1-
ethylated compounds 9 and 38 are inactive, but also why the effects
of the chain lengths are different.
Fig. 6. Putative SpnHyal binding sites of the benzoxazole-2-thione 28 and 1,3-
diacetylbenzimidazole-2-thione 3. (A) Detailed representation of the suggested inter-
actions of cpd. 28, docked into the crystal structure PDB 2BRP (view corresponding to
Fig. 3A). Colors of C atoms: inhibitor gray, residues with hydrophobic contacts to the
inhibitor orange, catalytic triad cyan, other residues green. Pink lines: suggested key
interactions. (B) Comparison of the putative binding modes of cpds. 28 (blue) and 3
(predicted by LUDI [25] using sphere radii of 5 Å e pose 1, magenta, and 8 Å e pose 2,
green) with binding modes of a hexasaccharide substrate (gray, only disaccharide units
1 and 2 around the scissile bond drawn), of Vcpal (2, red) and the phenylindole 5
(yellow). The alignment is based on backbone rms fits of the PDB complexes 2BRP (cpd.
5), 1LOH (hexasaccharide), 1W3Y (Vcpal), and the SagHyal4755 model used for LUDI
searches (cpd. 3). For better orientation, some important SpnHyal residues are shown
(cyan). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
According to the binding mode of the phenylindole 5 (see Figs. 3
and 6B), an additional methoxycarbonyl substituent in 5-position
of the benzoxazole ring should actually improve SagHyal4755 inhi-
bition since in the model in Fig. 6 the oxygen atoms of this polar
group would fit to appropriate amino acids (Tyr408, Arg462, Arg466
)

4424
S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
like the corresponding 6-sulfamoyloxy group of compound 5.
However, no increase in potency was observed for compounds 21
and 34. Possible reasons are slight differences between the
SagHyal4755 and SpnHyal binding sites or a nearly unchanged free
antibacterial properties in vivo are intended, since inhibition of the
spreading of pathogenic Streptococci in such infections as bacter-
emia and meningitis may reduce or even avoid the application of
classical antibiotics.
energy balance if
interactions.
H bonds are replaced by through-water
The pH influence on the inhibitory activity is difficult to inter-
pret. Generally greater IC50 values of the inhibitors at the pH
optimum of 5 could be due to higher affinity of the substrate HA.
However, a number of SagHyal4755 inhibitors (e.g., 28, some phe-
nylindoles [27] and LUDI hits [26]) prefer pH 5. Therefore the
protonation state of some active site residues (glutamates, aspar-
tates and the histidine of the catalytic triad) may directly affect the
binding of individual compounds. But why are just benzimidazole-
2-thiones inactive at pH 5? It is surely too simple to explain this
result by the different states of a single amino acid like the histidine
corresponding to SpnHyal His599. The analysis of two new SpnHyal
structures revealed the great flexibility of the substrate binding site,
allowing open and closed conformations of the cleft and, by this,
the controlled sequence of HA binding, catalysis, disaccharide
release and translocation of the substrate by one disaccharide unit
to the next cleavage position [51]. The influence of the flexibility
and the different conformations on inhibitor association and
dissociation is hardly to interpret, all the more as all SpnHyal
structures were analyzed at pH 6e6.5 (close to the optimum of this
enzyme). The lower pH optimum of SagHyal4755 may point to
slightly different energetic and conformational properties which
are not sufficiently reflected by flexible docking of the inhibitors to
SpnHyal.
4. Experimental protocols
4.1. Chemical syntheses
Starting materials and solvents were purchased from Acros
Organics (Belgium), Lancaster Synthesis GmbH (Germany), Sig-
maeAldrich Chemie GmbH (Germany) or Merck (Germany). THF
was distilled from NaH. Commercially available petroleum ether
(60e80 ꢁC) was distilled before use and dichloromethane was
either dried over CaCl2 or distilled from P2O5. All other solvents
used were of analytical grade.
Nuclear Magnetic Resonance (1H NMR and 13C NMR) spectra
were recorded on a Bruker ARX-300 NMR spectrometer with per-
deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloro-
form (CDCl3). The chemical shift
(ppm) with reference to the chemical shift of the residual protic
solvent compared to tetramethylsilane (TMS,
¼ 0 ppm). Coupling
d is given in parts per million
d
constants (J) are reported in Hz throughout. 2D-NMR (HeH and
CeH) COSY techniques were used in a few cases to support inter-
pretation of 1D spectra. Mass spectrometry analysis (MS) was
performed on a Finnigan MAT 95 (PI-EIMS, 70 eV) or a Finnigan SSQ
710A (PI-EIMS, 70 eV, CI-MS (NH3)) spectrometer. Melting points
(Mp) were measured on a BÜCHI 530 electrically heated copper
block apparatus using an open capillary and are uncorrected.
Column chromatography was carried out using Merck silica gel SI
60 (0.063e0.200). Elemental analyses were performed by the
Department of Microanalysis, University of Regensburg.
Compounds were dried in vacuo (0.1e1 Torr) at room temperature
or with heating up to 50 ꢁC for at least 24 h prior to submission for
elemental analysis. Analyses indicated by the symbols of the
elements or functions were within ꢀ0.4% of the theoretical values.
Reactions were routinely monitored by thin layer chromatography
(TLC) on Merck silica gel 60 F254 aluminum sheets, and spots were
visualized with UV light at 254 nm.
Also the 5- to 25-fold reduced inhibitory potency of Vc, Vcpal
and the phenylindole 5 at SpnHyal compared to SagHyal4755 [16]
indicates that both enzymes with identical active site residues
differ in conformational details. Since the Vmax values are similar
and since a reduction of the HA concentration did not significantly
lower IC50, the weaker SpnHyal activity should be attributed to the
inhibitors themselves, e.g., to slower association or higher disso-
ciation rates due to the specific topology around the binding cleft or
to the flexibility of the enzymeeinhibitor complexes.
3. Conclusions
Based on crystal structures of SpnHyaleligand complexes with
a hexasaccharide substrate and with the inhibitors 2 and 5,
respectively, 1-acylated benzimidazole-2-thiones and benzox-
azole-2-thiones were derived as new leads for the inhibition of
streptococcal hyaluronan lyases. In the benzoxazole series,
structure-based optimization of the 1-substituents led to N-(3-
phenylpropionyl)benzoxazole-2-thione (28) with IC50 values of
4.1.1. 1,3-Diacetylbenzimidazole-2-thione (3)
A mixture of benzimidazole-2-thione (7) (1.5 g, 10 mmol), trie-
thylamine (2.88 g, 28.5 mmol), a catalytic amount (100 mg) of
DMAP and anhydrous dichloromethane was stirred at room
temperature for 10 min. A solution of acetyl chloride (2.83 g) and
anhydrous dichloromethane was added dropwise and stirring was
continued for 16 h. After diluting with water (100 ml) and
dichloromethane (50 ml), the organic layer was separated, washed
three times with an aqueous solution of potassium hydrogen
sulfate (1 M, 50 ml) and water (50 ml) and dried over sodium
sulfate. The solvent was removed under reduced pressure, and the
product was purified by column chromatography on silica gel
eluting with a 1:8 (v/v) mixture containing ethyl acetate and
petroleum ether at 60e80 ꢁC to give a white solid. The product
decomposes at ambient temperature and moisture to 1-
24 mM (pH 7.4) and 15 mM (pH 5) on SagHyal4755. The high potency
of 28 at the pH optimum (pH 5) is unique among all analyzed
derivatives; this compound is among the three most potent
SagHyal4755 inhibitors known so far (IC50 values at pH 5 for Vcpal
4.2 mM, for the phenylindole 5 11 mM). At physiological pH also
some other benzimidazole-2-thione and benzoxazole-2-thione
derivatives like 17, 18, 10e14, 24e26, 29 and 31, respectively,
inhibit SagHyal4755 with potencies close to that of the most active
compound 3. Especially in the case of the benzoxazoles, the
structureeactivity relationships are in agreement with the
proposed binding mode characterized by polar interactions of the
oxazole and the acyl oxygens with asparagine, arginine, tyrosine
and histidine residues in the active site and by fitting of the thione
sulfur and the alkanoyl or arylalkanoyl chain to the hydrophobic
patch of the enzyme. The results provide a basis for further
structure-based inhibitor design to optimize potency, selectivity
and pharmacokinetic properties. Additional investigations toward
acetylbenzimidazole-2-thione. Yield 1.3
g (5.55 mmol), 56%,
white solid. Mp: 100e102 ꢁC (Mp: 102e103 ꢁC [40]). 1H NMR
(CDCl3)
d
3.05 (s, 6H, OCH3), 7.31 (dd, 2H, 4J ¼ 3.4 Hz, 3J ¼ 6.3 Hz, H-5
and H-6), 7.97 (dd, 2H, 4J ¼ 3.4 Hz, 3J ¼ 6.3 Hz, H-4 and H-7). MS (EI)
m/z 234 (Mꢂþ). Anal. (C11H10N2O2S) C, H, N, S.
4.1.2. Synthesis of the monoacylated benzimidazoles 10e15
The following procedures are modifications of the protocol
described in Ref. [30].

S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
4425
General method: Benzimidazole-2-thiol (7) (1 eq) and pertinent
acid chloride or acid anhydride (1.1 eq) were dissolved in pyridine
(4.4 eq) under a nitrogen atmosphere. The reaction mixture was
stirred overnight at room temperature. After quenching with water
(10e25 ml), ethyl acetate (30e45 ml) was added. The organic phase
was extracted with 3M hydrochloric acid (3 ꢃ 25 ml), dried over
magnesium sulfate and concentrated under reduced pressure. The
crude product was purified as stated below.
161e162.5 ꢁC. 1H NMR (DMSO-d6):
d
3.05 (t, 2H, 3J ¼ 7.8 Hz,
COCH2CH2Ph), 3.90 (t, 2H, 3J ¼ 7.8 Hz, COCH2CH2Ph), 7.16e7.34 (m,
8H, AreH), 7.96e7.99 (m, 1H, AreH), 13.33 (br, 1H, NH). 13C NMR
(DMSO-d6):
d
30.0 (ꢄ, CH2), 40.7 (ꢄ, CH2), 109.5 (þ, AreC), 115.1 (þ,
AreC), 123.3 (þ, AreC), 125.3 (þ, AreC), 126.0 (þ, AreC), 128.3 (þ,
4AreC),130.9 (Cquart, AreC),131.1 (Cquart, AreC),140.5 (Cquart, AreC),
169.4 (Cquart, CS), 174.4 (Cquart, CO). MS (EI): m/z (%) 282 ([Mꢂþ], 6),
249 ([M ꢄ SH]þ, 19), 150 ([M ꢄ C6H5CH2CHCO]þ, 100), 91 ([C7H7]þ,
28). Anal. (C16H14N2OS) C, H, N, S.
4.1.2.1. 1-(2-Thioxo-1H-benzo[d]imidazol-1-yl)ethanone (10). Yield:
3.80 g (19.77 mmol, 74%, white solid). Mp: 193e194 ꢁC (Mp:
4.1.2.6. (3-Chlorophenyl)(2-thioxo-1H-benzo[d]imidazol-1-yl)meth-
anone (15). Reaction of 7 (0.30 g, 2.00 mmol) with m-chlor-
obenzoyl chloride (0.26 ml, 2.00 mmol) and pyridine (3.50 ml,
42.92 mmol); purification by flash column chromatography on
silica gel, elution with dichloromethane. Yield: 0.13 g (0.45 mmol,
23%, pale yellow solid). Mp: 170e172 ꢁC. 1H NMR (DMSO-d6):
198e200 ꢁC [30]). 1H NMR (DMSO-d6):
d 3.00 (s, 3H, CH3), 7.16e7.33
(m, 3H, AreH), 7.97e8.02 (m, 1H, AreH), 13.31 (br, 1H, NH). MS (EI):
m/z (%) 192 ([Mꢂþ], 20), 150 ([M ꢄ CH2CO]þ, 100). Anal. (C9H8N2OS)
C, H, N.
4.1.2.2. 1-(2-Thioxo-1H-benzo[d]imidazol-1-yl)propan-1one (11).
Reaction of 7 (1.03 g, 6.86 mmol) with propanoyl chloride (0.64 ml,
7.33mmol)andpyridine(2.40ml,29.73mmol);recrystallizationfrom
ethyl acetate. Yield: 0.78 g (3.79 mmol, 55%, white flakes), Mp:
d 7.21e7.35 (m, 3H, benzimidazoleeH), 7.45e7.48 (m, 1H,
benzimidazoleeH), 7.55 (ddd,1H, 3J ¼ 8.1 Hz, 3J ¼ 7.7 Hz, 5J ¼ 0.4 Hz,
H-5), 7.75 (ddd, 1H, 3J ¼ 8.1 Hz, 4J ¼ 2.1 Hz, 5J ¼ 1.1 Hz, H-4/6), 7.80
(ddd, 1H, 3J ¼ 7.7 Hz, 4J ¼ 1.7 Hz, 5J ¼ 1.1 Hz, H-4/6), 7.91 (ddd, 1H,
4J ¼ 2.1 Hz, 4J ¼ 1.7 Hz, 5J ¼ 0.4 Hz, H-2), 13.29 (br, 1H, NH). 13C NMR
176e177.5 ꢁC, 1H NMR (DMSO-d6):
d
1.19 (t, 3H, 3J ¼ 7.1 Hz, CH2CH3),
3.56 (q, 2H, 3J ¼ 7.1 Hz, CH2CH3), 7.17e7.34 (m, 3H, AreH), 7.97e8.02
(m, 1H, AreH), 13.29 (br, 1H, NH). MS (EI): m/z (%) 206 ([Mꢂþ], 6), 150
([M ꢄ CH3CHCO]þ, 100). Anal. (C10H10N2OS) C, H, N, S.
(DMSO-d6):
d
110.0 (þ, AreC), 112.1 (þ, AreC), 123.2 (þ, AreC),
124.9 (þ, AreC), 128.8 (þ, AreC), 129.7 (þ, AreC), 130.5 (þ, AreC),
131.3 (Cquart, AreC), 131.9 (Cquart, AreC), 133.1 (Cquart, AreC), 133.5
(þ, AreC), 134.8 (Cquart, AreC), 168.5 (Cquart, CO), 169.5 (Cquart, CS).
MS (EI): m/z (%) 288 ([Mꢂþ], 41), 139 ([ClC6H4CO]þ, 100), 111
([ClC6H4]þ, 47) Anal. (C14H9ClN2OS) C, H, N, S.
4.1.2.3. 1-(2-Thioxo-1H-benzo[d]imidazol-1-yl)butan-1-one (12).
Reaction of 7 (1.00 g, 6.66 mmol) with butyric anhydride (1.20 ml,
7.32 mmol) and pyridine (2.40 ml, 29.73 mmol); crude product
was treated with a 2:1 (v/v) mixture of petroleum ether (boiling
point range 60e80 ꢁC) and ethyl acetate to separate pure
product; analytically pure product was obtained by column
chromatography on silica gel eluting with a 4:1 (v/v) mixture of
petroleum ether (60e80 ꢁC) and ethyl acetate. Yield: 0.98 g
(4.45 mmol, 67%, white solid). Mp: 152e153 ꢁC. 1H NMR (DMSO-
4.1.3. 1,3-Diacetyl-1H-benzo[d]imidazol-2(3H)-one (16)
The protocol described in Ref. [33] was modified.
2-Hydroxybenzimidazole (8) (0.50 g, 3.73 mmol) was suspended in
acetic anhydride (15 ml) and heated under reflux for 6 h. Water
(20 ml) and ethyl acetate (30 ml) were added and the separated
organic phase was extracted with a solution of saturated sodium
carbonate (3 ꢃ 20 ml). The organic layer was washed with water
(20 ml), dried over magnesium sulfate and concentrated under
reduced pressure. The crude product was recrystallized from a 1:1
(v/v) mixture of petroleum ether (60e80 ꢁC) and ethyl acetate.
Yield: 0.59 g (2.70 mmol, 73%, white needles). Mp: 145.5e146.5 ꢁC
d6):
d
0.98 (t, 3H, 3J ¼ 7.4 Hz, (CH2)2CH3), 1.67e1.81 (m, 2H,
CH2CH2CH3), 3.56 (t, 2H, 3J ¼ 7.3 Hz, COCH2e), 7.17e7.34 (m, 3H,
AreH), 7.95e8.00 (m, 1H, AreH), 13.29 (br, 1H, NH). 13C NMR
(DMSO-d6):
d
¼ 13.3 (þ, CH3), 17.6 (ꢄ, CH2), 40.7 (ꢄ, CH2), 109.4
(þ, AreC), 115.0 (þ, AreC), 123.3 (þ, AreC), 125.2 (þ, AreC), 130.9
(Cquart, AreC), 131.0 (Cquart, AreC), 169.4 (Cquart, CS), 175.1 (Cquart
,
(Mp: 149e151 ꢁC [33]). 1H NMR (DMSO-d6):
7.21e7.37 (m, 2H, AreH), 8.03e8.18 (m, 2H, AreH). 13C NMR
(DMSO-d6):
d 2.65 (s, 6H, 2COCH3),
CO). MS (EI): m/z (%) 220 ([Mꢂþ], 13), 150 ([M ꢄ C4H9CHCO]þ, 100).
Anal. (C11H12N2OS) C, H, N, S.
d
25.8 (þ, 2CH3), 114.4 (þ, 2AreC), 124.8 (þ, 2AreC),
126.5 (Cquart, 2AreC), 150.6 (Cquart, CO), 170.1 (Cquart, 2COCH3). MS
(EI): m/z (%) 218 ([Mꢂþ], 15), 176 ([M ꢄ CH2CO]þ, 18), 134
([M ꢄ 2CH2CO]þ, 100). Anal. (C11H10N2O3) C, H, N.
4.1.2.4. 1-(2-Thioxo-1H-benzo[d]imidazol-1-yl)hexan-1-one (13).
Reaction of 7 (1.02 g, 6.79 mmol) with hexanoic anhydride (1.80 ml,
7.81 mmol) and pyridine (2.40 ml, 29.73 mmol); purification by
column chromatography on silica gel, elution with a 4:1 (v/v)
mixture of petroleum ether (60e80 ꢁC) and ethyl acetate. Yield:
0.58 g (2.34 mmol, 34%, white solid). Mp: 132.5e133 ꢁC. 1H NMR
4.1.4. 1-(3-Ethyl-1,2-dihydro-2-thioxobenzo[d]imidazole-1-yl)
ethanone (17)
The protocol described in Ref. [31] was modified. A mixture of 1-
ethyl-1H-benzo[d]imidazole-2(3H)-thione (9) (0.14 g, 0.79 mmol),
dissolved in absolute THF (5 ml), triethylamine (0.11 ml, 0.79 mmol)
and acetyl chloride (0.07 ml, 0.98 mmol) was stirred at room
temperature for 1 h under an inert atmosphere. The reaction
mixture was concentrated under reduced pressure, and water
(5 ml) was added. After extraction with ethyl acetate (3 ꢃ 10 ml),
the combined organic layers were dried over magnesium sulfate
and concentrated in vacuo. The crude product was purified by
column chromatography on silica gel eluting with a 4:1 (v/v)
mixture of petroleum ether (60e80 ꢁC) and ethyl acetate. Yield:
0.07 g (0.32 mmol, 40%, white solid). Mp: 81.5e82.5 ꢁC. 1H NMR
(DMSO-d6):
d
0.89 (t, 3H, 3J ¼ 7.0 Hz, (CH2)4CH3), 1.28e1.40 (m, 4H,
(CH2)2CH3), 1.67e1.78 (m, 2H, COCH2CH2e), 3.57 (t, 2H, 3J ¼ 7.3 Hz,
COCH2e), 7.17e7.33 (m, 3H, AreH), 7.94e7.99 (m, 1H, AreH), 13.29
(br,1H, NH). 13C NMR (DMSO-d6):
d
13.7 (þ, CH3), 21.8 (ꢄ, CH2), 23.7
(ꢄ, CH2), 30.5 (ꢄ, CH2), 38.7 (ꢄ, CH2), 109.5 (þ, AreC), 115.0 (þ,
AreC), 123.3 (þ, AreC), 125.2 (þ, AreC), 130.9 (Cquart, AreC), 131.1
(Cquart, AreC), 169.4 (Cquart, CS), 175.2 (Cquart, CO). MS (EI): m/z (%)
248 ([Mꢂþ], 9), 150 ([M ꢄ C4H9 ꢄ CHCO]þ, 100). Anal. (C13H16N2OS)
C, H, N, S.
4.1.2.5. 1-(2-Thioxo-1H-benzo[d]imidazol-1-yl)-3-phenylpropan-1-
one (14). Reaction of 7 (0.50 g, 3.33 mmol) with 3-phenylpropanoyl
chloride (0.50 ml, 3.33 mmol) and pyridine (2.20 ml, 26.98 mmol);
purification by flash column chromatography on silica gel, elution
with a 5:2 (v/v) mixture of petroleum ether (60e80 ꢁC) and ethyl
(DMSO-d6):
d
1.27 (t, 3H, 3J ¼ 7.1 Hz, CH2CH3), 3.03 (s, 3H, COCH3),
4.37 (q, 2H, 3J ¼ 7.1 Hz, CH2CH3), 7.28e7.43 (m, 2H, AreH),
7.52e7.57 (m, 1H, AreH), 8.03e8.08 (m, 1H, AreH). MS (EI): m/z (%)
220 ([Mꢂþ], 42),178 ([M ꢄ CH2CO]þ, 97),150 ([M ꢄ C2H4 ꢄ CH2CO]þ,
100), 43 ([H3C ꢄ CO]þ, 24). Anal. (C11H12N2OS) C, H, N.
acetate. Yield: 0.46
g (1.63 mmol, 49%, white solid). Mp:

4426
S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
4.1.5. 1-(3-Acetyl-1,2-dihydro-2-thioxobenzo[d]imidazol-1-yl)
hexan-1-one (18)
[35]). 1H NMR (DMSO-d6):
AreH), 7.55e7.60 (m, 1H, AreH), 7.96e8.00 (m, 1H, AreH). 13C NMR
(DMSO-d6):
(þ, AreC), 126.0 (þ, AreC), 129.5 (Cquart, AreC), 145.9 (Cquart, AreC),
170.9 (Cquart, CO), 178.8 (Cquart, CS). MS (EI): m/z (%) 193 ([Mꢂþ], 17),
151 ([M ꢄ CH2CO]þ, 100). Anal. (C9H7NO2S) C, H, N, S.
d 2.94 (s, 3H, COCH3), 7.37e7.44 (m, 2H,
The protocol described in Ref. [31] was modified. Acetyl chloride
(0.10 ml, 1.40 mmol) was added to a mixture of 1-(2-thioxo-1H-
benzo[d]imidazol-1-yl)hexan-1-one (13) (0.29 g, 1.17 mmol), trie-
thylamine (0.19 ml, 1.37 mmol) and absolute THF (10 ml). After
stirring for 1 h at room temperature, the reaction mixture was
concentrated in vacuo. Water (10 ml) was added and the aqueous
phase was extracted with ethyl acetate (3 ꢃ 10 ml). The combined
organic layers were washed with a solution of saturated sodium
carbonate (10 ml), dried over magnesium sulfate and concentrated
under reduced pressure. The crude product was purified by column
chromatography on silica gel eluting with an 8:1 (v/v) mixture of
petroleum ether (60e80 ꢁC) and ethyl acetate. Yield: 20 mg
d
27.3 (þ, CH3), 109.7 (þ, AreC), 115.8 (þ, AreC), 125.5
4.1.7.2. 1-(2-Thioxobenzo[d]oxazol-3(2H)-yl)propan-1-one (23).
Method A: Reaction of 19 (0.50 g, 3.31 mmol) with propanoyl
chloride (0.30 ml, 3.44 mmol); recrystallization from ethanol.
Yield: 0.30 g (1.45 mmol, 44%, white needles). Mp: 81e82 ꢁC. 1H
NMR (DMSO-d6):
d
1.18 (t, 3H, 3J ¼ 7.1 Hz, CH3), 3.43 (q, 2H,
3J ¼ 7.1 Hz, CH2), 7.38e7.43 (m, 2H, AreH), 7.55e7.60 (m,1H, AreH),
7.99e8.04 (m, 1H, AreH). 13C NMR (DMSO-d6):
d
8.2 (þ, CH3), 32.3
(0.07 mmol, 6%, white solid). 1H NMR (DMSO-d6):
d
0.88 (t, 3H,
(ꢄ, CH2), 109.7 (þ, AreC), 115.9 (þ, AreC), 125.3 (þ, AreC), 126.0 (þ,
AreC), 129.7 (Cquart, AreC), 146.0 (Cquart, AreC), 174.6 (Cquart, CO),
179.5 (Cquart, CS). MS (EI): m/z (%) 207 ([Mꢂþ], 22), 179 ([M ꢄ C2H4]þ,
14), 151 ([M ꢄ CH3CHCO]þ, 100). Anal. (C10H9NO2S) C, H, N.
3J ¼ 7.1 Hz, (CH2)4CH3), 1.29e1.40 (m, 4H, (CH2)2CH3), 1.70e1.80 (m,
2H, COCH2CH2e), 2.98 (s, 3H, COCH3), 3.45 (t, 2H, 3J ¼ 7.3 Hz,
COCH2e), 7.34e7.40 (m, 2H, AreH), 7.69e7.74 (m, 1H, AreH),
7.86e7.90 (m, 1H, AreH). Anal. (C15H18N2O2S) C, H, N.
4.1.7.3. 1-(2-Thioxobenzo[d]oxazol-3(2H)-yl)hexan-1-one
(24).
4.1.6. 1-Ethyl-1H-benzo[d]imidazole-2(3H)-thione (9)
Method B: Reaction of 19 (0.50 g, 3.31 mmol) with hexanoic anhy-
dride (0.76 ml, 3.30 mmol); purification by chromatography on
a silica gel column, elution with chloroform; analytically pure
product was obtained by recrystallization from methanol. Yield:
0.30 g (1.20 mmol, 36%, white solid). Mp: 46e47.5 ꢁC. 1H NMR
The protocol described in Ref. [34] was modified. N1-Ethyl-
benzene-1,2-diamine (0.23 g, 1.69 mmol) was dissolved in absolute
THF (10 ml). This solution was treated with TCDI (0.33 g,1.85 mmol)
under a nitrogen atmosphere. After stirring at room temperature
for 22 h, the mixture was concentrated in vacuo. Ethyl acetate
(15 ml) and water (15 ml) were added to the residue and the organic
phase was separated. The aqueous phase was extracted three times
with ethyl acetate (15 ml). The combined organic phases were dried
over magnesium sulfate and concentrated in vacuo. The crude
product was purified by column chromatography on silica gel
eluting with a 1:1 (v/v) mixture of petroleum ether (60e80 ꢁC) and
ethyl acetate. Yield: 0.28 g (1.57 mmol, 93%, white needles). Mp:
(CDCl3):
d
0.94 (t, 3H, 3J ¼ 7.1 Hz, (CH2)4CH3), 1.32e1.48 (m, 4H,
(CH2)2CH3), 1.77e1.88 (m, 2H, COCH2CH2e), 3.51 (t, 2H, 3J ¼ 7.3 Hz,
COCH2e), 7.27e7.35 (m, 3H, AreH), 8.06e8.12 (m, 1H, AreH). 13C
NMR (CDCl3):
d
14.0 (þ, CH3), 22.5 (ꢄ, CH2), 24.0 (ꢄ, CH2), 31.1 (ꢄ,
CH2), 39.2 (ꢄ, CH2),109.7 (þ, AreC), 116.5 (þ, AreC),125.5 (þ, AreC),
126.1 (þ, AreC),130.0 (Cquart, AreC),146.6 (Cquart, AreC),174.3 (Cquart
,
CO), 178.8 (Cquart, CS). MS (EI): m/z (%) 249 ([Mꢂþ], 8), 221 ([M ꢄ CO]þ,
10), 151 ([M ꢄ C4H9CHCO]þ, 100). Anal. (C13H15NO2S) C, H, N.
161.5e162.5 ꢁC (Mp: 158e161 ꢁC [38]). 1H NMR (DMSO-d6):
d 1.23
(t, 3H, 3J ¼ 7.1 Hz, CH2CH3), 4.26 (q, 2H, 3J ¼ 7.1 Hz, CH2CH3),
4.1.7.4. 1-(2-Thioxobenzo[d]oxazol-3(2H)-yl)decan-1-one
(25).
7.08e7.28 (m, 3H, AreH), 7.31e7.47 (m,1H, AreH), 12.74 (s, 1H, NH).
Method A: Reaction of 19 (0.50 g, 3.31 mmol) with decanoyl chloride
(0.69ml, 3.35 mmol); stirring for 3 h;recrystallization from methanol.
Yield: 0.51 g (1.67 mmol, 51%, white solid). Mp: 64.5e65 ꢁC. 1H NMR
13C NMR (DMSO-d6):
d
12.8 (þ, CH3), 37.9 (ꢄ, CH2), 109.3 (þ, AreC),
109.6 (þ, AreC), 122.1 (þ, AreC), 122.7 (þ, AreC), 130.7 (Cquart
,
AreC), 131.9 (Cquart, AreC), 167.6 (Cquart, CS). MS (EI): m/z (%) 178
(CDCl3):
d
0.88 (t, 3H, 3J ¼ 6.9 Hz, (CH2)8CH3), 1.25e1.42 (m, 12H,
([Mꢂþ], 100), 150 ([M ꢄ C2H4]þ, 91). Anal.: (C9H10N2S) C, H, N.
(CH2)6CH3), 1.76e1.87 (m, 2H, COCH2CH2e), 3.51 (t, 2H, J ¼ 7.3 Hz,
3
COCH2e), 7.29e7.36(m, 3H, AreH), 8.05e8.12(m,1H, AreH).13CNMR
4.1.7. Synthesis of the benzoxazoles 22e34
The following procedures are modifications of the protocol
described in Ref. [35].
(CDCl3):
d
14.1 (þ, CH3), 22.7(ꢄ, CH2), 24.3 (ꢄ, CH2), 29.0(ꢄ, CH2), 29.3
(ꢄ, CH2), 29.4 (ꢄ, 2CH2), 31.9 (ꢄ, CH2), 39.2 (ꢄ, CH2), 109.7 (þ, AreC),
116.5 (þ, AreC),125.5 (þ, AreC),126.1 (þ, AreC),130.0 (Cquart, AreC),
146.6 (Cquart, AreC),174.3 (Cquart, CO),178.8 (Cquart, CS). MS (EI, 70 eV):
m/z (%) 305 ([Mꢂþ], 2), 277 ([M ꢄ CO]þ, 2), 151 ([M ꢄ C8H17CHCO]þ,
100). Anal. (C17H23NO2S) C, H, N, S.
Method A. Benzoxazole-2-thiol (19) (1 eq) was dissolved in
absolute THF (5 ml) under an inert atmosphere. Triethylamine
(1.1 eq) was added and the solution was cooled to 0 ꢁC. The particular
acid chloride (1 eq), dissolved in 5 ml absolute THF, was added
dropwise. After stirring for 30 min at room temperature, the reaction
mixture was poured into ice water (35 ml). The precipitated product
was collected, washed with water, dried in vacuo and recrystallized.
Method B. Benzoxazole-2-thiol (19) (1 eq) was dissolved in
absolute THF (5 ml) and mixed with triethylamine (1.1 eq) under
a nitrogen atmosphere. Alkanoic anhydride (1 eq) was added drop-
wise and the mixture was heated under reflux (7 h). After diluting
with water (30 ml), the mixture was extracted three times with ethyl
acetate (10 ml). The combined organic phases were washed with
a saturated solution of sodium carbonate (10 ml), dried over magne-
sium sulfate and the solvent was removed under reduced pressure.
The product was purified by column chromatography on silica gel.
4.1.7.5. 1-(2-Thioxobenzo[d]oxazol-3(2H)-yl)hexadecan-1-one (26).
Method A: Reaction of 19 (0.51 g, 3.37 mmol) with hexadecanoyl
chloride (1.00 ml, 3.31 mmol); stirring for 3 h; recrystallization from
methanol. Yield: 0.65 g (1.67 mmol, 50%, white solid). Mp:
81e81.5 ꢁC. 1H NMR (CDCl3):
d
0.88 (t, 3H, 3J ¼ 6.7 Hz, (CH2)14CH3),
1.23e1.50 (m, 24H, CH2(CH2)12CH3),1.76e1.87 (m, 2H, COCH2CH2e),
3.51 (t, 2H, 3J ¼ 7.3 Hz, COCH2e), 7.28e7.37 (m, 3H, AreH), 8.05e8.11
(m, 1H, AreH). 13C NMR (CDCl3):
d
14.1 (þ, CH3), 22.7 (ꢄ, CH2), 24.3
(ꢄ, CH2), 28.9 (ꢄ, CH2), 29.4 (ꢄ, 2CH2), 29.5 (ꢄ, CH2), 29.6 (ꢄ, CH2),
29.7 (ꢄ, 5CH2), 31.9 (ꢄ, CH2), 39.2 (ꢄ, CH2),109.6 (þ, AreC),116.5 (þ,
AreC), 125.5 (þ, AreC), 126.0 (þ, AreC), 130.0 (Cquart, AreC), 146.5
(Cquart, AreC),174.3 (Cquart, CO),178.8 (Cquart, CS). MS (EI, 70 eV): m/z
(%) 389 ([Mꢂþ], 25), 361 ([M ꢄ CO]þ, 83), 151 ([M ꢄ C14H29CHCO]þ,
100). Anal. (C23H35NO2S) C, N. H: calcd, 9.06; found 9.54.
4.1.7.1. 1-(2-Thioxobenzo[d]oxazol-3(2H)-yl)ethanone (22). Method
A: Reaction of 19 (0.50 g, 3.31 mmol) with acetyl chloride (0.24 ml,
3.36 mmol); recrystallization from cyclohexane. Yield: 0.46 g
(2.38 mmol, 72%, beige solid). Mp: 113e114 ꢁC (Mp: 120e121 ꢁC
4.1.7.6. 2-Phenyl-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)ethanone (27).
Method A: Reaction of 19 (0.50 g, 3.31 mmol) with phenylacetyl

S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
4427
chloride (0.44 ml, 3.33 mmol) in 10 ml anhydrous acetone;
recrystallization from ethanol. Yield: 0.28 g (1.04 mmol, 31%, white
from ethanol. Yield: 0.54 g (1.87 mmol, 56%, white solid). Mp:
76e77.5 ꢁC.1H NMR (CDCl3):
d 0.89e1.44 (m, 5H, 2CH2CH),1.62e1.83
solid). Mp: 132.5e134 ꢁC. 1H NMR (DMSO-d6):
d
3.56 (s, 2H, CH2),
7.23e7.32 (m, 8H, AreH), 7.49e7.54 (m, 1H, AreH). 13C NMR
(DMSO-d6):
40.6 (ꢄ, CH2), 109.9 (þ, AreC), 110.3 (þ, AreC), 123.7
(þ, AreC), 125.1 (þ, AreC), 126.5 (þ, AreC), 128.1 (þ, 2AreC), 129.3
(m, 8H, 4CH2), 3.53 (t, 2H, 3J ¼ 7.7 Hz, COCH2e), 7.27e7.36 (m, 3H,
AreH), 8.05e8.10 (m, 1H, AreH). 13C NMR (CDCl3):
d
26.2 (ꢄ, 2CH2),
d
26.5 (ꢄ, CH2), 31.6 (ꢄ, CH2), 33.1 (ꢄ, 2CH2), 36.9 (ꢄ, CH2), 37.1 (þ, CH),
109.6 (þ, AreC), 116.5 (þ, AreC), 125.5 (þ, AreC), 126.0 (þ, AreC),
130.0 (Cquart, AreC), 146.5 (Cquart, AreC), 174.6 (Cquart, CO), 178.8
(þ, 2AreC), 131.1 (Cquart, AreC), 134.9 (Cquart, AreC), 148.1 (Cquart
,
AreC), 172.6 (Cquart, CO), 178.9 (Cquart, CS). MS (EI): m/z (%) 269
([Mꢂþ], 26), 151 ([M ꢄ C6H5CHCO]þ, 28), 118 ([C6H5 ꢄ CHCO]þ, 85),
91 ([C6H5CH2]þ, 100). Anal. (C15H11NO2S) C, H, N.
(Cquart
,
CS). MS (EI): m/z (%) 289 ([Mꢂþ], 5), 151
([M
ꢄ
C6H11CH2CHCO]þ, 100), 121 (benzoxazole, 56). Anal.
(C16H19NO2S) H, N. C: calcd, 66.40; found 65.86.
4.1.7.7. 3-Phenyl-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)propan-1-one
(28). Method A: Reaction of 19 (0.50 g, 3.31 mmol) with
3-phenylpropionyl chloride (0.66 g, 3.91 mmol); recrystallization
from ethanol. Yield: 0.33 g (1.16 mmol, 35%, yellow solid). Mp:
4.1.7.11. 2-Phenoxy-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)ethanone
(32). Method A: Reaction of 19 (0.50 g, 3.31 mmol) with phenox-
yacetyl chloride (0.46 ml, 3.33 mmol); recrystallization from
ethanol. Yield: 0.32 g (1.12 mmol, 34%, light-brown solid). Mp:
73e75 ꢁC. 1H NMR (DMSO-d6):
d
3.06 (t, 2H, 3J ¼ 7.4 Hz,
138e139 ꢁC. 1H NMR (DMSO-d6):
d 4.66 (s, 2H, CH2), 6.84e6.99 (m,
COCH2CH2Ph), 3.87 (t, 2H, 3J ¼ 7.4 Hz, COCH2CH2Ph), 7.16e7.26 (m,
4H, AreH), 7.39e7.44 (m, 2H, AreH), 7.56e7.61 (m, 1H, AreH),
7.74e7.79 (m, 1H, AreH), 7.98e8.03 (m, 1H, AreH). 13C NMR
3H, AreH), 7.21e7.33 (m, 2H, AreH), 7.36e7.53 (m, 2H, AreH), 7.77
(d, 2H, 3J ¼ 6.8 Hz, AreH). 13C NMR (DMSO-d6):
64.2 (ꢄ, CH2),110.9
d
(þ, AreC), 114.3 (þ, 2AreC), 119.5 (þ, AreC), 120.8 (þ, AreC), 125.2
(þ, AreC), 125.9 (þ, AreC), 129.3 (þ, 2AreC), 141.1 (Cquart, AreC),
151.7 (Cquart, AreC), 157.6 (Cquart, AreC), 160.0 (Cquart, CO), 170.1
(Cquart, CS). MS (EI): m/z (%) 285 ([Mꢂþ], 36), 192 ([M ꢄ OC6H5]þ, 46),
164 ([M ꢄ OC6H5CO]þ, 100), 151 ([M ꢄ C6H5OCHCO]þ, 34), 77
([C6H5]þ, 71). Anal. (C15H11NO3S) C, H, N.
(DMSO-d6):
d
29.5 (ꢄ, CH2), 40.1 (ꢄ, CH2), 109.8 (þ, AreC), 110.9 (þ,
AreC), 115.8 (þ, AreC), 119.5 (þ, AreC), 125.2 (þ, AreC), 125.5 (þ,
AreC), 125.9 (þ, AreC), 128.2 (þ, 2AreC), 129.6 (Cquart, AreC), 140.1
(Cquart, AreC), 146.0 (Cquart, AreC), 173.1 (Cquart, CO), 179.3 (Cquart
,
CS). MS (EI): m/z (%) 283 ([Mꢂþ], 15), 250 ([M ꢄ SH]þ, 64), 151
([M
ꢄ
C6H5CH2CHCO]þ, 100), 105 ([C6H5CH2CH2]þ, 69), 91
([C6H5CH2]þ, 78). Anal. (C16H13NO2S) C, H, N.
4.1.7.12. Benzyl 2-thioxobenzo[d]oxazol-3(2H)-carboxylate (33).
Method A: Reaction of 19 (0.50 g, 3.31 mmol) with benzyl chlor-
oformate (0.48 ml, 3.36 mmol); different workup: reaction mixture
was concentrated under reduced pressure and ethyl acetate (15 ml)
and water (10 ml) were added; the organic phase was dried over
magnesium sulfate and the solvent was removed under reduced
pressure; crystallization from petroleum ether (60e80 ꢁC) and
4.1.7.8. 4-Phenyl-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)butan-1-one
(29). Method A: Reaction of 19 (0.50 g, 3.31 mmol) with
4-phenylbutanoyl chloride (0.87 g, 4.76 mmol); the reaction
mixture was extracted two times with chloroform (15 ml); the
combined organic phases were dried over magnesium sulfate and
the solvent was removed under reduced pressure; purification by
column chromatography on silica gel, elution with chloroform.
Yield: 0.13 g (0.44 mmol, 13%, brown solid). Mp: 145e146.5 ꢁC. 1H
subsequent recrystallization from methanol. Yield: 0.30
(1.05 mmol, 32%, white needles). Mp: 86e87 ꢁC (Mp: 89e92 ꢁC
[48]). 1H NMR (DMSO-d6):
5.55 (s, 2H, CH2), 7.31e7.51 (m, 5H,
AreH), 7.52e7.62 (m, 3H, AreH), 7.66e7.73 (m 1H, AreH). 13C NMR
(DMSO-d6):
g
d
NMR (DMSO-d6): d 1.71e1.89 (m, 2H, COCH2CH2CH2Ph), 2.21 (t, 2H,
3J ¼ 7.4 Hz, COCH2e), 2.56 (t, 2H, 3J ¼ 7.4 Hz, CO(CH2)2CH2Ph),
d
69.7 (ꢄ, CH2), 110.0 (þ, AreC), 115.0 (þ, AreC), 125.3
7.14e7.20 (m, 3H, AreH), 7.21e7.34 (m, 5H, AreH), 7.42e7.60 (m,
(þ, AreC), 125.9 (þ, 2AreC), 128.6 (þ, 4AreC), 128.8 (Cquart, AreC),
134.3 (Cquart, AreC), 145.6 (Cquart, AreC), 148.8 (Cquart, CO), 176.4
(Cquart, CS). MS (EI): m/z (%) 285 ([Mꢂþ], 5), 91 ([C6H5CH2]þ, 100).
Anal. (C15H11NO3S) C, H, N.
1H, AreH). 13C NMR (DMSO-d6):
d
26.2 (ꢄ, CH2), 32.9 (ꢄ, CH2), 34.3
(ꢄ, CH2), 109.9 (þ, AreC), 110.4 (þ, AreC), 123.7 (þ, AreC), 125.1 (þ,
AreC), 125.7 (þ, AreC), 128.2 (þ, AreC), 128.2 (þ, 3AreC), 131.1
(Cquart, AreC), 141.4 (Cquart, AreC), 148.0 (Cquart, AreC), 174.2 (Cquart
,
CO), 180.0 (Cquart, CS). MS (EI): m/z (%) 264 ([M ꢄ SH]þ, 55), 151
([M ꢄ C6H5(CH2)2CHCO]þ, 58), 147 ([C6H5CH2CH2CH2CO]þ, 65), 91
([C6H5CH2]þ, 100). Anal. (C17H15NO2S) C, H, N.
4.1.7.13. Methyl-3-(3-phenylpropanoyl)-2,3-dihydro-2-thioxobenzo
[d]oxazole-5-carboxylate (34)
4.1.7.13.1. Synthesis of methyl 2-sulfanylbenzo[d]oxazole-5-
carboxylate (21). 3-Amino-4-hydroxybenzoic acid (0.40 g,
2.61 mmol) was dissolved in anhydrous methanol (10 ml) and
treated with TMSCl (0.75 ml, 5.94 mmol). The mixture was stirred
at 55 ꢁC for 2 days. After evaporation of the solvent, the obtained
residue was purified by column chromatography on silica gel
eluting with ethyl acetate (yield: 0.25 g, 1.50 mmol, 57%, white
solid). 0.20 g of the obtained methyl 3-amino-4-hydroxybenzoate,
dissolved in anhydrous THF (10 ml), was mixed with TCDI (0.26 g,
1.46 mmol) under an inert atmosphere. The solution was stirred
overnight at room temperature. After evaporation of the solvent the
residue was treated with water (15 ml) and extracted with ethyl
acetate (3 ꢃ 15 ml). The combined organic layers were dried over
magnesium sulfate and concentrated under reduced pressure. The
remaining crude product was recrystallized from ethanol yielding
0.22 g (1.05 mmol, 88%) of methyl 2-sulfanylbenzo[d]oxazole-5-
carboxylate (21) as a brown solid. Mp: 203e205 ꢁC.
4.1.7.9. (E)-3-Phenyl-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)prop-2-
en-1-one (30). Method A: Reaction of 19 (0.50 g, 3.31 mmol) with
cinnamoyl chloride (0.55 g, 3.30 mmol); recrystallization from
ethanol. Yield: 0.53 g (1.88 mmol, 57%, yellow powder). Mp:
150.5e151.5 ꢁC (Mp: 148e149 ꢁC [47]). 1H NMR (DMSO-d6):
d
7.41e7.46 (m, 2H, AreH), 7.49e7.53 (m, 3H, AreH), 7.61e7.66 (m,
1H, AreH), 7.73e7.81 (m, 2H, AreH), 7.83e7.89 (m, 1H, AreH), 7.93
(d, 1H, 3J ¼ 15.8 Hz, ]CH), 8.04 (d, 1H, 3J ¼ 15.8 Hz, ]CH). 13C NMR
(DMSO-d6):
d
110.1 (þ, AreC),114.6 (þ, AreC),119.7 (þ, ]CH),125.6
(þ, AreC), 126.0 (þ, AreC), 128.7 (þ, AreC), 129.2 (þ, 3AreC), 129.6
(Cquart, AreC), 131.2 (þ, AreC), 134.0 (Cquart, AreC), 145.8 (þ, ]CH),
146.5 (Cquart, AreC), 165.5 (Cquart, CO), 178.4 (Cquart, CS). MS (EI): m/z
(%) 281 ([Mꢂþ], 16), 253 ([M ꢄ CO]þ, 3), 151 ([M ꢄ C6H5CHCHCO]þ,
9), 131 ([C6H5CHCHCO]þ, 100), 103 ([C6H5eCH]CH]þ, 34), 77
([C6H5]þ, 18). Anal. (C16H11NO2S) C, H, N.
Method A: Reaction of methyl 2-sulfanylbenzo[d]oxazole-5-
carboxylate (21) (0.20 g, 0.96 mmol) with 3-phenylpropanoyl
chloride (0.15 ml, 0.96 mmol); recrystallization from ethanol.
Yield: 0.17 g (0.50 mmol, 52%, beige solid). Mp: 112.5e115.5 ꢁC. 1H
4.1.7.10. 3-Cyclohexyl-1-(2-thioxobenzo[d]oxazol-3(2H)-yl)propan-
1-one (31). Method A: Reaction of 19 (0.5 g, 3.31 mmol) with
3-cyclohexylpropanoyl chloride (0.80 g, 4.58 mmol); recrystallization

4428
S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
NMR (CDCl3):
d
3.17 (t, 2H, 3J ¼ 7.5 Hz, COCH2CH2Ph), 3.85 (t, 2H,
96e98 ꢁC [36]). 1H NMR (DMSO-d6):
d
1.30 (t, 3H, 3J ¼ 7.2 Hz, CH3),
3J ¼ 7.5 Hz, COCH2CH2Ph), 3.95 (s, 3H, CO2CH3), 7.22e7.36 (m, 6H,
AreH), 8.09 (dd, 1H, 3J ¼ 8.5 Hz, 4J ¼ 1.7 Hz, H-6), 8.74 (dd, 1H,
4.24 (q, 2H, 3J ¼ 7.2 Hz, CH2), 7.30e7.43 (m, 2H, AreH), 7.48e7.63 (m,
2H, AreH). 13C NMR (CDCl3):
d
11.7 (þ, CH3), 40.2 (ꢄ, CH2), 110.1 (þ,
4J ¼ 1.7 Hz, 5J ¼ 0.6 Hz, H-4). 13C NMR (CDCl3):
d
30.1 (ꢄ, CH2), 40.7
AreC),110.5 (þ, AreC),124.3 (þ, AreC),125.1 (þ, AreC),131.0 (Cquart
,
(ꢄ, CH2), 52.6 (þ, CH3), 109.4 (þ, AreC), 117.9 (þ, AreC), 126.5 (þ,
AreC), 127.9 (Cquart, AreC), 128.4 (þ, AreC), 128.5 (þ, 2AreC), 128.7
AreC), 146.4 (Cquart, AreC), 178.6 (Cquart, CS). MS (EI): m/z (%) 179
([Mꢂþ], 100), 151 ([M ꢄ C2H4]þ, 93). Anal. (C9H9NOS) C, H, N.
(þ, 2AreC), 130.1 (Cquart, AreC), 139.7 (Cquart, AreC), 149.4 (Cquart
,
AreC), 165.9 (Cquart, CO2Me), 173.0 (Cquart, CO), 178.6 (Cquart, CS). MS
4.2. Hyaluronan lyase assay
(EI): m/z (%) 341 ([Mꢂþ], 8), 308 ([M
ꢄ
SH]þ, 36), 209
([M ꢄ C6H5CH2CHCO]þ, 87), 178 ([M ꢄ C6H5CH2CHCOOCH3]þ, 35),
105 ([C6H5CH2CH2]þ, 100), 91 ([C6H5CH2]þ, 89). Anal. (C18H15NO4S)
C, H, N.
Hyaluronan (hyaluronic acid) from Streptococcus zooepidemicus
was purchased from Aqua Biochem (Dessau, Germany). Bovine
serum albumin (BSA) was obtained from Serva (Heidelberg,
Germany). Stabilized hyaluronan lyase, i.e., 200,000 units (0.572 mg
from S. agalactiae strain 4755 plus 2.2 mg of BSA and 37 mg of
TriseHCl per vial) of lyophilized SagHyal4755, was kindly provided
by id-Pharma (Jena, Germany). All other chemicals were of
analytical grade and were received from Merck or Sigma.
4.1.8. 3-Acetylbenzo[d]oxazol-2(3H)-one (35)
The protocol described in Ref. [31] was modified. Benzoxazol-2-
one (20) (0.67 g, 4.96 mmol), dissolved in anhydrous THF (10 ml),
and triethylamine (2.0 ml,14.43 mmol) weremixed under a nitrogen
atmosphere. After cooling in an ice bath, acetyl chloride (0.43 ml,
6.03 mmol) was added dropwise. The reaction mixture was heated
under reflux for 2 h and then poured into ice-cold water (100 ml).
After stirring for 1 h, the precipitate was collected, washed with
water and recrystallized from ethanol. Yield: 0.28 g (1.58 mmol, 32%,
white solid). Mp: 90.5e91.5 ꢁC (Mp: 95e96 ꢁC [31]).1H NMR (DMSO-
To determine the solubility of the inhibitors, a sample containing
600
300
m
m
l of citrateephosphate buffer (pH 5 and pH 7.4), 396
l of BSA solution (0.2 mg/ml in water) and a solution of 54
ml of water,
ml of
the respective test compound (dissolved in DMSO) of various
concentrations was measured at 600 nm. A cuvette filled with water
served as reference. Compounds were tested for hyaluronan lyase
inhibition at concentrations, where no turbidity was measured.
The inhibitory effects of the compounds on SagHyal4755 were
measured using a turbidimetric assay [37]. Enzyme activity was
quantified by determining the turbidity caused by the residual high
d6):
d 2.61 (s, 3H, COCH3), 7.22e7.37 (m, 2H, AreH), 7.38e7.48 (m,1H,
AreH), 7.89e7.99 (m, 1H, AreH). 13C NMR (CDCl3):
d
24.6 (þ, CH3),
109.7 (þ, AreC), 115.0 (þ, AreC), 124.4 (þ, AreC), 124.9 (þ, AreC),
127.7 (Cquart, AreC), 141.7 (Cquart, AreC), 150.9 (Cquart, CO), 169.6
(Cquart, CO). MS (EI): m/z (%) 177 ([Mꢂþ], 26),135 ([M ꢄ CH2CO]þ,100),
43 ([CH3CO]þ, 84). Anal. (C9H7NO3) C, H, N.
molecular weight substrate (molecular weight
precipitated with cetyltrimethylammonium bromide (CTAB). The
incubation mixture contained 120 l of citrateephosphate buffer
> 6e8 kDa)
m
4.1.9. 3-Hexanoylbenzo[d]oxazol-2(3H)-one (36)
(solution A: 0.1 M Na2HPO4/0.1 M NaCl, solution B: 0.1 M citric acid/
0.1 M NaCl, solution A and B were mixed in appropriate proportions
The protocol described in Ref. [31] was modified. Benzoxazol-2-
one (20) (0.50 g, 3.70 mmol) was dissolved in dry THF (10 ml) under
an inert atmosphere, triethylamine (0.63 ml, 4.54 mmol) and
hexanoic anhydride (1.03 ml, 4.47 mmol) were added, and the
mixture was heated under reflux for 4 h. Then water (15 ml) was
added and the mixture was extracted three times with ethyl acetate
(15 ml). The combined organic layers were washed with a saturated
solution of sodium carbonate (15 ml), dried over magnesium
sulfate and concentrated in vacuo. The crude product was purified
by column chromatography on silica gel eluting with chloroform.
Yield: 0.60 g (2.57 mmol, 70%, pale light-brown solid). Mp:
to reach pH 5.0 and 7.4, respectively), 30
ml in water), 30 l of HA substrate solution (2 mg/ml in water), 50
H2O, 10 l dimethyl sulfoxide (DMSO) and 30 l enzyme solution
(2.9 ng of SagHyal4755 in 30 l BSA solution). To determine the
enzyme activities in the presence of the test compounds, instead of
10 l DMSO, 10 l of varying concentrations of inhibitors were used.
ml of BSA solution (0.2 mg/
m
ml
m
m
m
m
m
The final DMSO concentration was 3.8% (v/v).
After incubation of the assay mixture for 30 min (pH 5) or for 3 h
(pH 7.4) at 37 ꢁC, 720
ml of a 2.5% (m/v) cetyltrimethylammonium
bromide solution (2.5 g CTAB dissolved in 100 ml 0.5 M sodium
hydroxide solution, pH 12.5) were added to precipitate the residual
high molecular weight substrate and to stop the enzyme reaction.
This mixture was again incubated at 25 ꢁC for 20 min, and the
turbidity of each sample was determined at 600 nm with an Uvikon
930 UV spectrophotometer (Kontron, Eching, Germany).
The turbidity of the sample without inhibitor was taken as
reference for 100% enzyme activity, whereas the turbidity of the
sample without enzyme (30 ml BSA) was taken as reference for 0%
enzyme activity. The activities were plotted against the logarithm
of the inhibitor concentration. 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 exper-
iments performed in duplicate. To exclude factors affecting
turbidity (interactions of the test compound with the substrate,
absorption by the test compound etc.), controls containing the
72.5e73.5 ꢁC. 1H NMR (DMSO-d6):
d 0.80e0.99 (m, 3H, CH3),
1.24e1.43 (m, 4H, (CH2)2CH3), 1.60e1.71 (m, 2H, COCH2CH2e), 3.02
(t, 2H, 3J ¼ 7.4 Hz, COCH2e), 7.23e7.34 (m, 2H, AreH), 7.36e7.50 (m,
1H, AreH), 7.89e8.05 (m, 1H, AreH). 13C NMR (DMSO-d6):
d
13.7 (þ,
CH3), 21.8 (ꢄ, CH2), 22.8 (ꢄ, COCH2CH2), 30.5 (ꢄ, CO(CH2)2CH2),
35.7 (ꢄ, COCH2e), 109.7 (þ, AreC), 115.1 (þ, AreC), 124.3 (þ, AreC),
124.8 (þ, AreC), 127.8 (Cquart, AreC), 141.8 (Cquart, AreC), 150.8
(Cquart, CO), 172.4 (Cquart, CO). MS (EI): m/z (%) 233 ([Mꢂþ], 29), 135
([M ꢄ C4H9CHCO]þ, 100), 99 ([C4H9CHCO]þ, 60), 71 ([C4H9 ꢄ CH2]þ,
44). Anal. (C13H15NO3) C, H, N.
4.1.10. 3-Ethylbenzo[d]oxazole-2(3H)-thione (38)
The protocol described in Ref. [36] was modified. 2-(Ethylamino)
phenol (37) [34] (0.15 g, 1.09 mmol) was dissolved in dry THF (5 ml)
under an inert atmosphere. Subsequently, TCDI (0.21 g, 1.20 mmol)
was added in small portions and the mixture was stirred for 3 h at
ambient temperature. The reaction mixture was concentrated under
reducedpressure, andwater (15 ml)was added. Afterextractionwith
ethyl acetate (3 ꢃ 15 ml), the combined organic layers were dried
over magnesium sulfate and evaporated in vacuo. The crude product
was purified by column chromatography on silica gel eluting with
a 3:1 (v/v) mixture of petroleum ether (60e80 ꢁC) and ethyl acetate.
Yield: 0.07 g (0.39 mmol, 36%, beige solid). Mp: 105e107 ꢁC (Mp.
incubation mixture without enzyme (30
instead) were run in parallel.
ml of BSA solution was used
4.3. Molecular models of SpnHyal in complex with ligands
The SpnHyal crystal structures PDB ID 1EGU [12] (free), 2BRP
[16] (cpd. 5), 1LOH [11] (hexasaccharide) and 1W3Y [15] (Vcpal)
were taken from the Brookhaven Protein Databank [52]. Modeling

S. Braun et al. / European Journal of Medicinal Chemistry 46 (2011) 4419e4429
4429
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software package SYBYL 7.3 (Tripos, L.P., St. Louis, USA). Energy
minimizations were based on the AMBER FF99 force field [53] with
AMBER FF99 charges (distance dependent dielectric constant of 4)
up to a root mean square gradient of 0.05 kcal/mol/Å (Powell
conjugate gradient method). Surfaces and lipophilic potentials
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(MOLCAD GmbH, Darmstadt, Germany) contained within SYBYL.
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Acknowledgment
This study was supported by the Graduate Research Training
Program of the Deutsche Forschungsgemeinschaft (Gra-
duiertenkolleg 760 “Medicinal Chemistry”), by the Bayerische For-
schungsstiftung (AZ-829-08) and by the Studienstiftung des
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