Comparative studies on conventional and microwave synthesis of some benzimidazole, benzothiazole and indole derivatives and testing on inhibition of hyaluronidase

Article abstract of DOI:10.3390/molecules13040736

We have synthesized twelve 2-substituted benzimidazole, benzothiazole and indole derivatives using on both microwave irradiation and conventional heating methods. The microwave method was observed to be more beneficial as it provides an increase of yield from 3% to 113% and a 95 to 98 % reduction in time. All compounds were tested by a stains-all assay at pH 7 and by a Morgan-Elson assay at pH 3.5 for hyaluronidase inhibitory activity at a concentration of 100 μM. The most potent compound was 2-(4-hydroxyphenyl)-3-phenylindole (12) with an IC₅₀ value of 107 μM at both pH 7 and 3.5.

Full text of DOI:10.3390/molecules13040736

Molecules 2008, 13, 736-748
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules
Full Paper
Comparative Studies on Conventional and Microwave Synthesis
of Some Benzimidazole, Benzothiazole and Indole Derivatives
and Testing on Inhibition of Hyaluronidase
Oztekin Algul 1,*, Andre Kaessler 2, Yagmur Apcin 1, Akin Yilmaz 1 and Joachim Jose 2
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin 33169,
Turkey; Tel: (+90) 532 414 01 99; Fax: (+90) 324 341 30 22; E-mails: utku_eczanesi@hotmail.com
(Akin Yilmaz), yagmurapcin@hotmail.com (Yagmur Apcin)
2
Bioanalytics, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University,
Düsseldorf 40225, Germany; E-mails: Andre.Kaessler@uni-duesseldorf.de (Andre Kaessler),
joachim.jose@uni-duesseldorf.de (Joachim Jose)
* Author to whom correspondence should be addressed; E-mail: oztekinalgul@mersin.edu.tr
Received: 12 February 2008; in revised form: 20 March 2008 / Accepted: 20 March 2008 / Published:
27 March 2008
Abstract: We have synthesized twelve 2-substituted benzimidazole, benzothiazole and
indole derivatives using on both microwave irradiation and conventional heating methods.
The microwave method was observed to be more beneficial as it provides an increase of
yield from 3% to 113% and a 95 to 98 % reduction in time. All compounds were tested by
a stains-all assay at pH 7 and by a Morgan-Elson assay at pH 3.5 for hyaluronidase
inhibitory activity at a concentration of 100 µM. The most potent compound was 2-(4-
hydroxyphenyl)-3-phenylindole (12) with an IC50 value of 107 µM at both pH 7 and 3.5.
Keywords: Hyaluronidase, benzimidazole, benzothiazole, indole, Morgan-Elson assay,
stains-all assay and microwave irradiation.
Introduction
Tumor metastasis is a complex phenomenon involving a sequence of events that still remain poorly
understood. However, it is known that tumor cells must be able to avoid intercellular adhesion, detach

Molecules 2008, 13
737
from the tumor mass and overcome physical barriers imposed by the extracellular matrix. Hyaluronic
acid (HA), one of the most important components of the extracellular matrix, plays an important role
in growth, development and repair of tissues. HA also correlates to endothelial cell motility,
proliferation, differentiation and migration, and finally leads to angiogenesis [1]. Higher HA
concentrations have been found in several human cancer cells than in normal tissues. HA may support
tumor growth by stimulation and proliferation of tumor cells [2,3]. On the other hand, hyaluronidases
(HAases) degrade HA into small angiogenic fragments and play essential roles in ovum fertilization,
angiogenesis, cell adhesion and proliferation, and also in the progression of severe diseases like
arthrosis or bladder, breast and prostate cancer. Moreover, it has been shown that both tumor-
associated HA and tumor-derived HAase possibly play a role in tumor progression [4, 5].
Consequently, potent inhibitors of hyaluronidases could promise a new concept of antitumor drugs.
Due to the lack of potent and selective inhibitors, several compounds were studied as
hyaluronidase inhibitors. Several natural and synthetic inhibitors such as apigenin [6], TNP-470 [7],
marimastat [8], cis-hinokiresinol [9] and SU6668 [10] have been developed for targeting endothelial
cell proliferation, invasion, the VEGF receptor (VEGFR) signaling pathway, angiogenesis and
inhibitory activity on HAases [11]. Buschauer and collaborators identified 1,3-diacetylbenzimidazole-
2-thione, 1-decyl-2-(4-sulfamoyloxyphenyl)-1H-indol-6-yl sulfamate [12] and N-substituted
benzoxazole-2-thione derivatives as inhibitors of streptococcal Hyal. Several of them showed high
hyaluronidase inhibitor activity especially against hyaluronidase derived from Streptococcus
agalactiae [13,14]. Structures and IC50 values of these compounds are shown in Table 1.
Table 1. Inhibitors of bacterial hyaluron lyase as synthesized, tested and published
recently by Buschauer and colleagues [13,14].
A
R1
N
R3
R2
Sag Hyal IC50
A
R1
R2
R3
Compound
[µM], pH 5
-N-COCH3
I
=S
-COCH3
-C10H21
-H
160
11
II
-CH2-
-O-
-Ph (OSO2NH2)
-OSO2NH2
III
IV
V
=S
=S
=S
-COCH2Ph
-CO(CH2)2Ph
-CO(CH2)3Ph
-H
-H
-H
260
15
-O-
-O-
110
Olgen et al. found that the inhibition hyaluronidase activity of indole-2- and 3-carboxamide
derivatives was found to be slightly different. While N-benzyl mono-halogenated benzamide-
containing compounds substituted at position 2 had good activity, their position 3 congeners did not
show any enzymatic inhibition [15]. Recently, we reported the synthesis and characterization of some
benzimidazole, bis-benzimidazole and benzoxazole derivatives and observed that the presence of aryl
groups at the 2 position of all compounds resulted in more inhibitory activity than alkyl groups [16].

Molecules 2008, 13
738
The most active compound was di(1H-benzo[d]imidazol-2-yl)methane, which showed hyaluronidase
inhibitory activity of 67 % at pH 7 and 63 % at pH 3.5 at a concentration of 100 µM. Hence, we also
made a substitution in the C-2 position to increase lipophilicity of the molecule and the insertion of
such a group enhanced the hyaluronidase inhibitory activities.
However, some of our compounds and other natural and synthetic group containing compounds
showed prominent or potent activity as hyaluronidase inhibitors. Therefore, these structures can be
used as the starting point for the development of hyaluronidase inhibitors. Selective inhibitors are
needed to study the relationship between the enzyme activity and the physiological and
pathophysiological effects of HAases. The lack of potent and selective inhibitors of human HAases
prompt us to synthesize and test some benzimidazole, benzoxazole and indole derivatives as inhibitors
of hyaluronidase in order to obtain the compounds with better inhibitory activity. In this study, all
compounds were synthesized utilizing both microwave and conventional methods. The performance of
two methods was compared and hyaluronidase inhibitory activity for such compounds was
investigated.
Results and Discussion
With the goal of investigating the structure–activity relationships of benzimidazole, benzoxazole
and indole-based molecules, twelve analogs were synthesized (Scheme 1, Table 2). Our initial efforts
of optimizing the benzimidazole structures were focused on either replacing the 4-substituted aryl
groups or 3(4)-substituted benzyl groups and on different substituents at different positions of the
indole groups. We recently determined the significance of the 2 position of the benzimidazole ring for
inhibitory activity. We also investigated the possible effect of any change in the linker between both
aromatic rings upon the bioactivity.
Therefore, we synthesized benzimidazole compounds with 2-aryl substituted groups (compounds
1-3) and 3(4)-substituted benzyl groups (compounds 6-8). Besides the compounds containing
benzimidazole as the main ring, compounds bearing indole rings with methyl, phenyl, 4-
hydroxyphenyl groups in the 1,2,3 or 5 position (compounds 9-12) and 2-substituted benzothiazole
compounds (compounds 4, 5) were also synthesized. All compounds were synthesized using
conventional and microwave assisted methods. The structures of the synthesized compounds were
confirmed using IR, NMR and elemental analysis methods. The comparative data of the synthesized
compounds are provided in Table 2. The reaction time for the synthesis of all compounds by
conventional methods was 2 to 8 h, in comparison with the microwave heating one (3-10 min), an
obvious many-fold time reduction. Overall approximately a 95 to 98 % decrease in reaction times and
a 3% to 113% increase in the yields was obtained.
We tested these compounds for inhibitory activity using microtiter plate assays. We modified new
in vitro enzymatic assay methods at pH 7 based on stains-all assay and at pH 3.5 using the Morgan-
Elson assay [26]. The assays have been carried out using bovine testes hyaluronidase (BTH) because
of the limited availability of purified human enzyme. The bovine enzyme exhibits a homology of about
65% identical amino acid residues to human PH-20 and 40% to human Hyal-1.

Molecules 2008, 13
739
Scheme 1. The syntheses of compounds 1-12.
HOOC
R
R1
A, E
NH2
N
D
OHC
R
(CH2)n
R
N
H
R1
NH2
A, G
HOOC
R
A:PPA/MW, D:Na2S2O5/DMF, E:PPA, G: HCl
HOOC
O
R
B
N
SH
R
S
NH2
R
F
Cl
B: Et2O/Silica gel/MW, F: Pyridine
NHNH2
O
CH2CH3
R1
C, H
R2
R
CH3
R2
N
H
NNH2
C, H
R
O
NHNH2
CH2
C, H
OH
N
HO
H
C:CCl3COOH/MW, H:CCl3COOH
It is known that hyaluronidases are active at different pH values. BTH and human PH-20 show
high activity over a broader range, starting at neutral pH down to pH 3 than human Hyal-1, which is
much more active at acidic pH than at neutral pH [27]. We tested the inhibition profile of the
compounds at neutral (pH 7) as well as the acidic pH (pH 3.5). It is known that lower pH values are
common for cancer tissues and inflamed tissues. Therefore, the inhibition of hyaluronidases at lower
pH values is important, especially for cancer treatment.
The principle behind the stains-all assay is based on the measurement of absorbance values of blue
colored complexes formed upon the reaction of longer chains of HA with stain-all reagent (1-ethyl-2-
[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtha[1,2-d] thiazolium bromide)
at 620 nm. Small fragments formed by degradation of HA do not form these blue colored complexes.

Molecules 2008, 13
740
However, this assay method is not suitable for low pH values, because of the instability of the stains-
all complexes. Therefore, another method is required to measure enzyme activity at pH 3.5. The
Morgan-Elson reaction can be used for the assay at this pH. When incubated with hyaluronidase, long
chains of HA are decomposed to shorter chains with N-acetyl-D-glucosamine at the reducing end. At
least one of the resultant chromogens, 3-acetamido-5-(1,2-dihydroxy-ethyl)furane, reacts with
dimethylamino-benzaldehyde (DMAB) in acidic environment leading to the formation of a red colored
product. The intensity of the red color is measured at 590 nm.
Table 2. Chemical structures of all synthetic compounds and their synthetic methods
R1
R2
A
(CH2)n
R
B
Microwave heating
Conventional heating
Comp
No
Time
Power
(watt)
Yield
Temp.
Time
(h)
Yield
(%)
Mp.
A
B
n
R
R1
R2
Method*
(min)
(%)
(˚C)
(˚C)
N
N
NH
NH
NH
S
0
0
0
0
0
1
1
1
0
0
0
0
H
F
H
H
H
H
A, D
A, E
A, E
B, F
B, F
A, G
A, G
C, H
C, H
C, H
C, H
C, H
8
10
8
100
100
100
50
88
90
74
90
94
85
80
75
80
78
70
75
180
200
5
5
5
2
2
7
7
8
2
2
3
4
83
87
70
70
80
50
40
35
70
70
50
60
293 (17)
250 (18)
225 (17-19)
115 (20)
123 (21)
182 (22)
165 (23)
152-155
88 ( 24)
1
2
N
OCH3
H
H
H
180
3
N
H
H
5
30
4
N
S
OCH3
H
H
H
6
50
30
5
N
NH
NH
NH
NH
NH
N-CH3
NH
H
H
5
150
150
150
50
Reflux
Reflux
Reflux
100
6
N
OCH3
H
H
H
5
7
N
OCH3
H
H
7
8
C-CH3
C-CH3
C-CH3
C-Ph
H
H
3
9
H
H
CH3
H
3
50
100
105-108
69 (25)
10
11
12
H
H
4
50
100
OH
H
H
5
50
100
147
* Methods are described in detail in the Experimental section.
The IC50 values of all compounds exhibiting an inhibition of more than 40 % at a concentration of
100 µM, were determined. All results are summarized in Table 3. Eight inhibitors (compounds 5-12)
showed inhibition on the enzyme, the most potent compound being 12, with an IC50 value of 107 μM.
Examination of substituent effects of benzimidazole and benzothiazole compounds on activity
showed that position 2 needs to be substituted with a large compound such as a phenyl or benzyl
group. All benzimidazole or benzothiazole compounds carrying a phenyl ring (compounds 1-4) were
inactive and only compound 5 showed higher activity than the corresponding phenyl ring subsituted
benzimidazole compounds (compounds 2,3). Interestingly, compound 5 lost all inhibitory activity at
pH 3.5, which might be explained with a higher polarity due to protonation.

Molecules 2008, 13
741
If the phenyl ring is changed into a benzimidazole with a benzyl ring, an increase in inhibitory
activity was obtained at pH 7 (compounds 6,7). As expected, the inhibition at pH 3.5 was again much
lower. Presumably, the protonation of the benzimidazole ring and the associated higher polarity of the
molecule at a low pH like 3.5 decreased enzyme-inhibitor interaction within the active site. Moreover,
an additional substituent at meta or para position of the benzyl ring had no effect (compounds 7, 8).
Despite the fact that the indole ring based derivatives did not bear a benzyl ring at position 2, they
showed a higher activity, between 16-48 % at pH 7 and 8-42 % at pH 3.5, than similar benzimidazole
analogues. The missing basic nitrogen in position 3 seems to have a positive effect on inhibitory
activity as the compound is now acidic (except for compound 11), less polar and no longer amphoteric.
Methyl group substituents at the positions 1, 3 or 5 had only weak influence (compounds 9-11). A
substitution of the phenyl ring at position 3 resulted in an extreme increase in the activity at pH 7
(compound 12). This compound was also substituted with a hydroxy at the para position of the phenyl
ring. It is difficult to say whether the higher activity is due to the phenyl or the hydroxy group. On the
other hand, Olgen et al. [15] recently reported about the importance of lipophilicity and potency of
indol compounds for higher hyaluronidase inhibitory activity. So it might be assumed that phenyl or
benzyl substitutions have positive effects that were combined in this compound and can explain the
much higher inhibitory activity than all other tested derivatives.
Table 3. The effects of the synthesized compounds on hyaluronidase activities at different pH values.
Inhibition [%]
at 100 μM
IC50 [μM]
Compound No
pH
7
pH7 3.5
3.5
1
6
0
0
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
n.d n.d
107 107
2
2
3
3
0
4
2
2
5
10
13
15
16
17
16
16
48
0
6
5
7
5
8
11
14
8
9
10
11
9
12
42
Ascorbic acid
99%
99%
18
8
palmitate
n.d: not determined
In our experiments Vcpal (6-palmitoyl-L-ascorbic acid), a known hyaluronidase inhibitor [28], was
used as a control substance with an IC50 value of about 8 µM. Although all compounds 1-12 were not
as active as Vcpal, they can be used as lead structures. Our further investigations aim at adding large
lipophilic groups with hydrophilic substituents of different positions on the core indole backbone.

Molecules 2008, 13
742
Moreover, it might be considered to keep hydrophilic substituents on phenyl ring or replace it with
benzyl groups which help in the interaction with the active site of the enzyme.
Experimental
General
All chemicals and solvents were supplied by Aldrich, Merck, Sigma and were used without further
purification. HAases from bovine testes was purchased from Serva (Heidelberg, Germany), hyaluronic
acid was purchased from Sigma Aldrich (Steinheim, Germany). Water was purified using a Milli-Q
Biocel system. All other chemicals were of analytical or HPLC grade. Microwave reaction was carried
out in focused mono-mode microwave oven (Discover by CEM). Melting points were measured on an
Electrothermal 9200 melting point apparatus and were uncorrected. Proton magnetic resonance (1H-
NMR) spectra were recorded on Bruker 400 MHz NMR spectrometer using DMSO-d6 as solvent.
Chemical shifts were reported in parts per million relative to internal standard tetramethylsilane. The
room temperature attenuated total reflection Fourier transform infrared (FT-IR ATR) spectrum of the
compounds were recorded using Varian FTS1000 FT-IR spectrometer with Diamond/ZnSe prism
(4000–525 cm−1; number of scans: 250; resolution: 1 cm−1) in the solid. Thin-layer chromotography
(TLC) was perfomed on pre-coated aluminium plates (Silica gel 60 F254, Merck). Plates were
visualized by UV light, Dragendorff reagent, and iodine vapour.
Chemistry
In recent years, the use of microwave irradiation to simplify and improve classical organic
reactions has become a very popular method [20] because it often leads to high yields, clean reactions,
and shorter reaction times. We carried out the reaction of aromatic aldehydes/aromatic acid with 2-
amino-thiophenol/1,2-phenylendiamine in the presence of silica gel/ polyphosphoric acid (PPA) under
microwave irradiation. The compounds were obtained in good yield. In this study, we found an
oppurtunity to compare the compounds synthesized by conventional methods to those obtained
employing microwave irradiation.
General method for the synthesis of 2-aryl-1H-benzimidazoles, 2-arylbenzoxazole and 2-arylindole
derivatives
Microwave irradiation conditions:
Method A. In the presence of PPA (5 mL), the mixture of 4-substituted benzoic acid (15 mmol) and
1,2-phenylendiamine (10 mmol) was stirred and irridated in MW (5-10 min, 100-150 W). After the
reaction was complete, the mixture was allowed to cool to room temperature and then poured into cold
water (50 mL). Stirring was continued for several minutes and the mixture was neutralized with
NaHCO3. The resulting precipitate was filtered off, washed several times with water and purified by
recrystallization.

Molecules 2008, 13
743
Method B. To a solution of 4-substituted benzaldehyde (3 mmol) and 2-aminothiophenol (6 mmol) in
diethylether (10 mL) silica gel (3 g) was added. The slurry was mixed thoroughly and the solvent was
removed by rotary evaporation. The solid obtained was subjected to microwave irradiation using
microwave oven operated at 50W for 5-6 min. After cooling, the product was extracted with ethyl
acetate. The extract was filtered and the filtrate was evaporated under reduced pressure to yield the
crude product. The product was purified by recrystallization in MeOH/H2O.
Method C. The solution of phenylhydrazine (1 mmol), ketones (1 mmol) and trichloroacetic acid (3
mmol) was stirred and irridated in MW (3-5 min) (50W). After the reaction was completed, the
mixture was cooled with cold water. The precipitate was collected by filtration, washed with water and
recrystallized from MeOH/H2O.
Classical conditions:
Method D. A mixture of 1,2-phenylenediamine (0.313 mmol), benzaldehyde (1.01 equiv.) and sodium
metabisulfite (1.01 equiv.) in DMF (10 mL) was heated to reflux for 5 h. After cooling, water (20 mL)
was added and the mixture was extracted with AcOEt (3x15 mL). The organic layer was dried over
magnesium sulfate and removed under vacuum. Purification was done by chromatography on silica gel
eluting with chloroform and recrystallization from adequate solvent [17].
Method E. The 4-substituted benzoic acid (15 mmol), 1,2-phenylendiamine (10 mmol) and PPA (5
mL) were placed in a round bottomed flask. Then the mixture was heated and stirred at 180-200 ˚C for
5 h. After the reaction was complete, the mixture was allowed to cool to room temperature and then
poured into cold water (50 mL). The mixture was neutralized with NaHCO3. The resulting precipitate
was filtered off, washed several times with water and purified by recrystallization.
Method F. 2-Aminothiophenol (0.04 mol) was added to a stirred solution of 4-substituted benzoyl
chloride (0.04 mol) in pyridine (50 mL). The mixture was stirred at room temperature for 2 h and
poured into water (200 mL). The product was washed with water and crystallized from MeOH/H2O.
Method G. Equimolar amounts of 1,2-phenylenediamine and the corresponding phenylacetic acid in
5N hydrochloric acid were refluxed for 7-8 h. The solution was cooled in an ice bath and neutralized
with NaHCO3. The resulting precipitate was filtered off, washed several times with water and purified
by recrystallization.
Method H. A mixture of phenylhydrazine (1 mmol), ketones (1 mmol) and trichloroacetic acid (3
mmol) was heated at 100 °C for 2-4 h. Water was added to the cooled mixture which was filtered. The
retentate was washed with water, recrystallized from MeOH/H2O and finally dried in vacuum to give
corresponding analytically pure indoles.
Characterization data

Molecules 2008, 13
744
2-Phenyl-1H-benzo[d]midazole (1): M.p. 293°C (lit. [17] 289-291°C); yield 88 % (Method A); IR
(cm-1): 3247, 1648, 1621, 1541; 1H-NMR: δ = 6.95 (m, 2H), 7.20-7.21 (d, 2H), 7.48 (m, 1H), 8.16-8.18
(m, 4H) 12.89 (br, s, 1H) ppm; Anal. calc. for C13H10N2 (194.08): C, 80.39; H, 5.19; N, 14.42. Found:
C, 80.6; H, 5.3; N, 14.7 %.
2-(4-Fluorophenyl)-1H-benzo[d]midazole (2): M.p. 250˚C (lit. [18] 247-248˚C); yield 87% (Method
1
E); IR (cm-1): 3445, 1622; H-NMR: δ = 7.13-7.28 (m, 2H), 7.30-7.72 (m, 4H), 8.13-8.32 (m, 2H),
12.91 (br, s, 1H) ppm; Anal. calc. for C13H9FN2 (212.07): C, 73.57; H, 4.27; N, 13.20. Found: C, 73.6;
H, 4.3; N, 13.7 %.
2-(4-Methoxyphenyl)-1H-benzo[d]imidazole (3): M.p. 225 °C (lit. [17-19] 224-226 °C); yield 74 %
(Method A); IR (cm-1): 3451, 1625; 1H-NMR: δ = 12.88 (br, s, 1H), 8.28 (d, 2H), 7.74 (d, 1H), 7.48 (d,
1H), 7.24 (m, 4H), 3.84 (s, 3H) ppm; Anal. calc. for C14H12N2O (224.09) Calc. C, 74.9; H. 5.3; N,
12.4. Found C, 75.1; H, 5.4; N, 12.4 %.
2-Phenylbenzo[d]thiazole (4): M.p. 115 ˚C (lit. [20] 113-114 °C ); yield 90 % (Method B); IR (cm-1):
3387, 1643, 1587; 1H-NMR: δ = 8.15 (m, 3H), 7.91 (d, 1H), 7.46 (m, 4H), 7.40 (t, 1H); Anal. calc. for
C13H9NS (211.05) C, 73.9; H, 4.3; N, 6.6. Found: C, 73.9; H, 4.7; N, 6.3 %.
2-(4-Methoxyphenyl)benzo[d]thiazole (5): M.p. 123 ˚C (lit. [21] 121-122); yield 80 % (Method F); IR
1
(cm-1): 1603, 1521; H-NMR: δ = 8.04 (d, 1H), 8.02 (s, 1H), 7.87 (d, 1H), 7.46 (t, 1H), 7.35 (t, 1H),
7.00 (d, 1H), 6.90 (2H, d), 3.87 (3H, s) ppm; Anal. calc. for C14H11NSO (241.06) C, 69.7; H, 4.6; N,
5.8. Found: C, 69.5; H, 4.5; N, 5.8 %.
2-Benzyl-1H-benzo[d]imidazole (6): M.p. 182 °C (lit. [22] 179-180°C); yield: 85% (Method A); IR
(cm-1): 1665, 1597, 1535, 745; 1H-NMR: δ = 4.60 (s, 2H), 7.58–7.20 (m, 9H, ArH), 12.34 (s, 1H) ppm;
Anal. calc. for C14H12N2 (208.10) C, 80.7; H, 5.8; N, 13.5. Found: C, 80.5; H, 5.6; N, 13.5 %.
2-(4-Methoxybenzyl)-1H-benzo[d]imidazole (7): M.p. 165 °C (lit. [23] 165°C); yield: 80 % (Method
A); IR (cm-1): 3664, 1610, 1535. 1H-NMR: δ = 3,80 (s, 3H), 4.60 (s, 2H), 6.85-6.90 (m, 2H), 7.10-7.15
(m, 2H), (7.35 (d, 2H), (7.36-7.52 (m, 2H), 12.27 (s, 1H) ppm. Anal. calc. for C15H14N2O (238.11) C,
75.6; H, 5.9; N, 11.8. Found: C, 75.4; H, 5.7; N, 11.8 %.
2-(3-Methoxybenzyl)-1H-benzo[d]imidazole (8): m.p.152-155 °C; yield: 75 % (Method C); IR (cm-1):
1
1613, 1586. H-NMR: δ = 3.36 (s, 3H), 7.10-7.14 (m, 5H), 7.19-7.29 (m, 5H), 12.29 (brs, 1H) ppm;
Anal. calc. for C15H14N2O (238.11) C, 75.6; H, 5.9; N, 11.8. Found: C, 75.4; H, 5.6; N, 11.7 %.
3-Methyl-2-phenyl-1H-indole (9): M.p. 88 °C (lit. [24] [91-92.5°C); yield: 70 % (Method H); IR(cm-1):
3420, 3000, 1600, 1460 cm-1; 1H-NMR: δ = 2.39 (s, 3H), 6.97-7.14 (m, 2H), 7.36-7.38 (m, 3H), 7.47-
7.54 (7, 2H), 7.67 (d, 2H), 11.16 (bs, 1H) ppm; Anal. calcd. for C15H13N (207.10) C, 86.92; H, 6.32; N,
6.76. Found: C, 86.8; H, 6.4; N, 6.8 %.

Molecules 2008, 13
745
3,5-Dimethyl-2-phenyl-1H-indole (10): M.p. 105-108 °C; yield: 78 % (Method C); IR (cm-1): 3395,
1
3049, 1612, 1454, 1246, 718 cm-1; H-NMR: δ = 2.39 (s, 3H), 2.50 (s, 3H), 6.95-6.92 (dd, 1H), 7.24-
7.57 (m, 3H), 7.49 (t, 2H), 7.66 (d, 2H) 10.91 (s, 1H) ppm; Anal. calc. for C16H15N (221.12) C, 86.8;
H, 6.8; N, 6.3; Found: C, 86.8; H, 6.9; N, 6.4 %.
1,3-Dimethyl-2-phenyl-1H-indole (11): M.p. 69 °C. (lit. [25] [66-67.5°C); yield: 70% (Method C); IR
(cm-1): 3409, 3050, 1660, 1445, 1210, 739 cm-1; 1H-NMR: δ = 2.21 (s, 3H), 3.59 (s, 3H), 7.03-7.21 (m,
3H), 7.42-7.60 (m, 6H) ppm; Anal. required for C16H15N (221.12) C, 86.8; H, 6.8; N, 6.3; Found: C,
86.6; H, 6.9; N, 6.4 %.
4-(3-Phenyl-1H-indol-2-yl)phenol (12): M.p. 147°C; yield: 75 % (Method C); IR (cm-1): 3395, 3049,
1612, 1454, 1246, 718 cm-1; 1H-NMR: δ = 6.74 (d, 2H), 7.28-7.47 (m, 9H), 6.97-7.15 (m, 2H), 9.67 (s,
1H), 11.38 (s, 1H) ppm; Anal. calc. for C20H15NO (285.12) C, 84.2; H, 5.3; N, 4.9. Found: C, 84.3; H,
5.6; N, 4.9 %.
Assays for the measurement of hyaluronidase activity
Stains-all-assay
0.2 M and 50 mM phosphate buffer solutions were prepared and the pH was adjusted to 7.0 with
HCl. Pure hyaluronidase powder (3110 U/mg) was dissolved in phosphate buffer (50 mM) to give
hyaluronidase solutions with an activity 100 U/mL. The inhibitor substances were dissolved in DMSO
to a concentration of 10 mM. An aqueous stock solution of hyaluronic acid (2 mg/mL) was prepared,
then the inhibitor solutions were added to the enzyme solutions to give 50 µM, 75 µM and 100 µM
inhibitor concentrations. These inhibitor/enzyme solutions were incubated for one hour.
Stains-all (22.4 mg), ascorbic acid (35.2 mg), glacial acetic acid (23 µL) and butylated
hydroxytoluene (BHT, 1.3 mg) were mixed and dissolved by adding dioxane (100 mL) and water (100
mL). This staining solution was stored protected from light.
The substrate solution was prepared by mixing 0.2 M phosphate buffer (390 µL), 2 mg/mL HA
solution (110 µL) and water (500 µL). Inhibitor/enzyme solution (12.5 µL) and substrate solution
(12.5 µL) were mixed directly onto a microplate. The first value at zero minutes was measured after
immediate adding staining solution (112.5 µL) and water (62.5 µL). The absorption was read at 650
nm using a Berthold LB940 Mithras Multilabel microplate reader. The microplate was then incubated
at 37 °C for one hour. After incubation, staining solution (112.5 µL) and water (62.5 µL) were added
to the other wells and the absorption at 650 nm was read again using a microtiter plate reader. The
activity of the positive controls with pure DMSO instead of inhibitor solution was set to 100 %. The
activity was calculated depending on the change in absorption using the following formula:
ΔAInhibitor
ΔAPositive Control
Activity[%]=
×100
(1)

Molecules 2008, 13
746
Morgan-Elson Assay
A formate buffer containing 0.1 M sodium formate and 0.1 M NaCl was prepared as incubation
buffer and adjusted to a pH of 3.5 with formic acid. Bovine serum albumine (BSA) was dissolved in
water to get a solution with a concentration of 0.2 mg/mL. Boric acid solution was prepared by
dissolving boric acid (4.94 g) and KOH (1.98 g) in water (100 mL). A solution of dimethylamino-
benzaldehyde (DMAB, 5 g) in 10 N HCl (6.25 mL) was made up to 50 mL with glacial acetic acid.
This staining solution was used as a stock solution and stored under light protection.
Hyaluronidase powder (3110 U/mg) was dissolved in incubation buffer to prepare 800 U/mL
hyaluronidase solutions. The inhibitor substances were dissolved and diluted in DMSO to a final
concentration of 10 mM/L. The stock solution of hyaluronic acid (5 mg/mL) was prepared by
dissolving hyaluronic acid in water. Then the inhibitor solutions were added to the enzyme solution for
preparing inhibitor/enzyme solutions with 25 µM, 75 µM and 100 µM inhibitor concentration and
incubated for one hour. After incubation BSA solution (100 µL), incubation buffer (100 µL), water
(150 µL) and the inhibitor/enzyme solution (50 µL) were mixed in a microfuge tube. The assay was
initiated by adding HA solution (50 µL) to this mixture. To get the values at zero time, 45 µL were
pipetted into a microfuge tube and boric acid solution (10 µL) was added. The microfuge tube was
then heated at 100 °C for 4.5 min. A microplate was placed onto ice and the heated solutions were
transferred completely into the wells. The tubes containing the mixture were incubated for one hour at
37 °C. While incubating, the staining solution was diluted with glacial acetic acid by nine folds. After
incubation, the tubes were centrifugated. Each solution (45 µL) was put into a new microfuge tube and
boric acid solution (10 µL) was added. The microfuge tube was then heated at 100°C for 4.5 min. The
heated solutions were also transferred completely onto the microplate. To initiate the staining process,
diluted staining solution (300 µL) was added to each well and the microplate was incubated at 37 °C
for 20 min. The absorption at 590 nm was read using a Berthold LB940 Mithras Multilabel microplate
reader. The activity of the positive controls with pure DMSO instead of inhibitor solution was set to
100%. The activity was calculated depending on the change in absorption using equation (1). For both
assays a negative control lacking hyaluronidase enzyme was used. 6-Palmitoyl-L-ascorbic acid was
used as a control compound in the same way as the other inhibitors.
Acknowledgements
This work was financially supported by The Scientific and Technological Research Council of
Turkey (TÜBİTAK) and JULICH [Grants: SBAG-JULICH-4 (104S413)] and by the Mersin
University Research Fund (Project no: BAP-SBE MB(YA) 2006-3 YL and BAP-SBE MB(AY) 2006-
3 YL).
References and Notes
1. West, D. C.; Kumar, S. The Effect of Hyaluronate and its oligosaccharides on endothelial cell
proliferation and monolayer integrity. Exp. Cell Res. 1989, 183, 179-196.

Molecules 2008, 13
747
2. Auvinen, P.; Tammi, R.; Parkkinen, J.; Tammi, M.; Ågren, U.; Johansson, R.; Hirvikoski, P.;
Ekselinen, M.; Kosma, V.M. Hyaluronan in peritumoral stroma and malignant cells associates
with breast cancer spreading and predicts survival. Am. J. Pathol. 2000, 156, 342-347.
3. Girish, S.; Kemparaju, K. The magic glue hyaluronan and its eraser hyaluronidase: A biological
overview. Life Sci. 2007, 80, 1921-1943.
4. Lokeshwar, V. B.; Rubinowicz, D.; Schroeder, G. L.; Forgacs, E.; Minna, J. D.; Block, N. L.;
Nadji, M.; Lokeshwar, B. L. Stromal and epithelial expression of tumor markers hyaluronic acid
and HYAL1 hyaluronidase in prostate cancer. J. Biol. Chem. 2001, 275, 11922-11932.
5. Liu, D.; Pearlman, E.; Diaconu, E.; Guo, K.; Mori, H.; Haqqi, T.; Markowitz, S.; Wilson, J.; Sy,
M.-S. Expression of hyaluronidase by tumor cells induces angiogenesis in vivo. Proc. Natl.
Acad. Sci. USA 1996, 93, 7832-7837.
6. Trochon, V.; Blot, E.; Cymbalista, F.; Engelmann, C.; Tang, R. -P.; Thomdis, A.; Vasse, M.;
Soria, J.; Lu, H.; Soria, C. Apigenin inhibits endothelial-cell proliferation in g2/m phase whereas
it stimulates smooth-muscle cells by inhibiting p21 and p27 expression. Int. J. Cancer 2000, 85,
691–696.
7. Herbst, R. S.; Madden, T. L.; Tran, H. T.; Blumenschein, G. R.; Meyers, Jr. C. A.; Seabrooke, L.
F.; Khuri, F. R.; Puduvalli, V. K.; Allgood, V.; Fritsche, H. A.; Hinton, Jr. L.; Newman, R.A.;
Crane, E. A.; Fossella, F. A.; Dordal, M.; Goodin, T.; Hong W. K. Safety and pharmacokinetic
effects of TNP-470, an angiogenesis inhibitor, combined with paclitaxel in patients with solid
tumors: evidence for activity in non–small-cell lung cancer. J. Clin. Oncol. 2002, 20, 4440-4447.
8. Sridhar, S. S.; Shepherd, F. A. Targeting angiogenesis: a review of angiogenesis inhibitors in the
treatment of lung cancer. Lung Cancer 2003, 42, 581-591.
9. Jeong, S. -J.; Higuchi, R.; Ono, M.; Kuwano, M.; Kim, Y. -C.; Miyamoto, T. cis-Hinokresinol, a
norlignan from anmemarrhena asphodeloides, inhibits angiogenic responce in vitro an in vivo.
Biol. Pharm. Bull. 2003, 26, 1721-1724.
10. Laird, A. D.; Vajkoczy, P.; Shawver, L. K.; Thurnher, A.; Liang, C.; Mohammadi, M.;
Schlessinger, J.; Ullrich, A.; Hubbard, S. R.; Blake, R. A.; Fong, A. T.; Strawn, L. M.; Sun, L.;
Tang, C.; Hawtin, R.; Tang, F.; Shenoy, N.; Hirth, K. P.; McMahon, G.; Cherrington, J.M.
SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established
tumors. Cancer Res. 2000, 60, 4152-4160.
11. Jeong, S. J.; Ahn, N. H.; Kim, Y. C.; Inagaki, M.; Miyamoto, T.; Higuchi, R. Norlignans with
hyaluronidase inhibitory activity from Anemarrhena asphodeloides. Planta Medica 1999, 65,
367-368.
12. Salmen, S. Inhibitors of Bacterial and Mammalian Hyaluronidases: Synthesis and Structure-
Activity Relationships. Ph.D. Thesis, University of Regensburg Press: Regensburg, Germany,
2003.
13. Botzki, A.; Salmen, S.; Bernhardt, G.; Buschauer, A.; Dove, S. Structure-based design of
bacterial hyaluronan lyase inhibitors. QSAR Comb. Sci. 2005, 24, 458–469.
14. Rigden, D. J.; Botzki, A.; Nukui, M.; Mewbourne, R. B.; Lamani, E.; Braun, S.; von Angerer, E.;
Bernhardt, G.; Dove, S.; Buschauer, A.; Jedrzejas, M. J. Design of new benzoxazole-2-thione-
derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a
2-phenylindole. Glycobiology 2006, 16, 757–765.

Molecules 2008, 13
748
15. Olgen, S.; Kaeßler, A.; Nebioglu, D.; Jose, J. New potent indole derivatives as hyaluronidase
inhibitors. Chem. Biol. Drug. Des. 2007, 70, 547–551.
16. Kaessler, A.; Algul, O.; Jose, J. A microplate based screening of benzimidazole derivatives on
hyaluronidase inhibition at pH 7 and 3.5. Lett. Drug Des. Discov. 2007, 4, 562-569.
17. Du, L. -H; Wang, Y. -G. A rapid and efficient synthesis of benzimidazoles using hypervalent
iodine as oxidant. Synthesis 2007, 5, 675-678.
18. Getvoldsen, G.; Fredriksson, A.; Elander, N.; Stone-Elander, S. Microwave-assisted
cyclocondersation of 1,2-diaminobenzene with [4-18F]fluorobenzoic acid:microwave synthesis
of 2-([4-18F]fluorophenyl)benzimidazole. J. Label. Compd. Radiopharm. 2004, 47, 139-145.
19. Arslan, H.; Algul, O. Vibrational spectrum and assignments of 2-(4-methoxyphenyl)-1H-
benzo[d]imidazole by ab initio Hartree–Fock and density functional methods. Spectrochim. Acta
A: Mol. Biomol. Spectrosc. doi:10.1016/j.saa.2007.07.027.
20. Kodomari, M.; Tamaru, Y.; Aoyama, T. Solvent-free synthesis of 2-aryl and 2-
alkylbenzothiazoles on silica gel under microwave irradiation. Synth. Commun. 2004, 3029-3036.
21. Arslan, H.; Algul, O. Synthesis and Ab initio/DFT studies on 2-(4-methoxyphenyl)benzo-
[d]thiazole. Int. J. Mol. Sci. 2007, 8, 1-18.
22. Charton, J.; Girault-Mizzi, S.; Debreu-Fontaine, M.-A.; Foufelle, F.; Hainault, I.; Bizot-Espiard,
J.-G.; Caignard, D.-H.; Sergheraert, C. Conversion of sterically hindered diacylated 1,2-
phenylenediamines into 2-substituted benzimidazoles. Chem. Pharm. Bull. 2005, 53, 492-497.
23. Gumus, F.; Demirci, A.B.; Özden, T.; Eroglu, H.; Diril, N. Synthesis, characterization and
mutagenicity of new cis-[Pt(2-substituted-benzimidazole)2Cl2]complexes. Pharmazie 2003, 58,
303-307.
24. Matsumoto, K.; Tanaka, A.; Yukio, I.; Hayashi, N.; Toda, M.; Bulman, R. A. Fischeer Indole
Synthesis in the Absence of a Solvent. Heterocycl. Commun. 2003, 9, 9-12.
25. Katritzky, A. R.; Fan, W-Q.; Akutagawa, K.; Wang, J. Carbon dioxide: A reagemt for the
simultaneous protection of nucleophilic and the activation of alternative locations to electrophilic
attack. 16. A novel synthetic method for the side-chain functionalization of N-methyl-o-toluidine
and for the preparation of 2-substituted N-methylindoles. Heterocycles 1990, 30, 407-414.
26. Benchetrit, L.C; Pahuja, S.L.; Gray, E.D.; Edstrom, R.D. A sensitive method for the assay of
hyaluronidase activity. Anal. Biochem. 1977, 79, 431-37.
27. Stern, R.; Jedrzejas, M. Hyaluronidases: their genomics, structures, and mechanisms of action. J.
Chem. Rev. 2006, 106, 818–839.
28. Botzki, A.; Rigden, D. J.; Braun, S.; Nukui, M.; Salmen, S.; Hoechstetter, J.; Bernhardt, G.;
Dove, S.; Jedrzejas, M. J.; and Buschauer, A. L-Ascorbic acid 6-hexadecanoate, a potent
hyaluronidase inhibitor: X-Ray structure and molecular modeling of enzyme-inhibitor
complexes. J. Biol. Chem. 2004, 279, 45990–45997.
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