Synthesis and antioxidant potential of novel synthetic benzophenone analogues

Article abstract of DOI:10.1016/j.ejmech.2008.09.010

Considering that oxidative stress is strongly implicated in the toxicity of chemotherapy, much effort is focused on the research of diverse antioxidants as protective agents. An efficient synthesis of three novel benzophenones containing 1,3-thiazol moiety (6a-c) is described. Their antioxidant power was evaluated in vitro and in three cell lines (the cancerous MCF7 and the non-cancerous hTERT-HME1 mammary cells, and the H9c2 cardiomyoblastic cells). One analogue 5-(2,5-dihydroxybenzoyl)-2(3H)-benzothiazolone (6c), displayed an important antioxidant activity, a low cytotoxicity, and could decrease reactive oxygen species production generated by tert-butyl hydroperoxide (tBHP) in all three cell lines. Interestingly, 6c was able to protect the non-cancerous cells against tBHP-induced death. Further studies are underway to determine its relevance as an adjuvant in oxidative stress inducing chemotherapy.

Full text of DOI:10.1016/j.ejmech.2008.09.010

European Journal of Medicinal Chemistry 44 (2009) 2724–2730
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and antioxidant potential of novel synthetic benzophenone analogues
,
,
*
Tzvetomira Tzanova a c, Mariana Gerova b, Ognyan Petrov b, Margarita Karaivanova a, Denyse Bagrel c
a Medical University of Sofia, Faculty of Pharmacy, Department of Pharmacology and Toxicology, 2 Dunav Street, Sofia 1000, Bulgaria
b University of Sofia St. Kliment Ohridski, Faculty of Chemistry, Department of Applied Organic Chemistry, 1 James Bourchier Avenue, 1164 Sofia, Bulgaria
c Laboratoire d’Inge´nierie Mole´culaire et Biochimie Pharmacologique, Universite´ Paul Verlaine – Metz, FR CNRS 2843, UFR SciFA, Campus Bridoux, rue du Ge´ne´ral Delestraint,
57070 Metz, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 May 2008
Received in revised form 6 August 2008
Accepted 4 September 2008
Available online 19 September 2008
Considering that oxidative stress is strongly implicated in the toxicity of chemotherapy, much effort is
focused on the research of diverse antioxidants as protective agents. An efficient synthesis of three novel
benzophenones containing 1,3-thiazol moiety (6a–c) is described. Their antioxidant power was evalu-
ated in vitro and in three cell lines (the cancerous MCF7 and the non-cancerous hTERT-HME1 mammary
cells, and the H9c2 cardiomyoblastic cells). One analogue 5-(2,5-dihydroxybenzoyl)-2(3H)-benzothia-
zolone (6c), displayed an important antioxidant activity, a low cytotoxicity, and could decrease reactive
oxygen species production generated by tert-butyl hydroperoxide (tBHP) in all three cell lines. Inter-
estingly, 6c was able to protect the non-cancerous cells against tBHP-induced death. Further studies are
underway to determine its relevance as an adjuvant in oxidative stress inducing chemotherapy.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
2(3H)-Benzothiazolones
Benzophenones
Oxidative stress
Cytoprotection
1. Introduction
natural hydroxybenzophenones are biologically active metabolites
present in plants and especially in Guttiferae family [12,13]. Radical
Toxicity, as well as the development of resistance, is a major
obstacle for successful chemotherapy and radiotherapy. Multiple
organ systems can be affected, with both acute and chronic side
effects. Cytoprotection, or protection of normal cells, is a strategy
now being investigated in preclinical and clinical models [1]. The
ideal protective agent would possess selectivity (only healthy tissue
protection), broad spectrum activity (protection of numerous tissues
against a wide variety of cytotoxic agents), and a favourable side
effect profile (well tolerated) [2]. It would prevent all toxic side
effects, from non-life threatening (alopecia) to potentially fatal
(severe cardiomyopathy, severe thrombocytopenia), without
diminishing the antitumor efficacy of the cancer therapy [3].
Considering the fact that oxidative stress is strongly implicated in
the toxicity of chemotherapy, much effort has been focused on
the research of diverse antioxidants as potential chemoprotective
agents [4,5]. Thus, the phenolic antioxidants which are effective
agents against oxidative stress-mediated disorders (cardiovascular
dysfunctions, inflammatory diseases, neurodegenerative diseases,
diabetes.) [6–8] are also protectors in cancer therapy [9,10]. Their
pharmacological action is mostly ascribed to free radical scavenging,
chelation of redox active metal ions, modulation of gene expression
and interaction with cell signalling pathways [11]. Among phenolics,
scavenging activity represents one of their most relevant functions.
For example, garcinol, a polyisoprenylated benzophenone isolated
from Garcinia indica suppressed hydroxyl radical more strongly than
DL-a-tocopherol in the Fenton reaction system [14]. Some of the
benzophenone glycosides isolated from Hypericum annulatum
showed a free radical scavenging activity and exerted substantial
protection of bone-marrow cells against anticlonogenic effects
induced by epirubicine [15]. Based upon these natural models,
different benzophenone derivatives were synthesized. For example,
the antioxidant properties of synthetic benzophenone and its
trihydroxy-derivative were reported to have similar characteristics
to those of phenol and resorcinol [16]. Polyhydroxybenzophenones
displayed significant scavenging effects on the superoxide anion and
prevented cell death in menadione-treated myoblasts [17].
These interesting biological effects led us to design and
synthesize novel benzophenones containing a benzothiazolone
moiety which could adjoin important pharmacological activities.
Indeed, 2(3H)-benzothiazolone derivatives are pharmacophores
largely used in medicinal chemistry [18]. In particular, they display
anti-inflammatory properties [19–21] and can inhibit Cox pathway
[22]. These two tightly linked properties are strongly related to
their antioxidant activity.
In order to develop antioxidant adjuvants for chemotherapy,
this preliminary study is devoted to the design and synthesis of
three novel benzophenone derivatives, i.e. 5-(hydroxybenzoyl)-
2(3H)-benzothiazolones. Their antioxidant activity was assessed in
* Corresponding author. Tel.: þ33 3 87 37 84 04; fax: þ33 3 87 37 84 23.
E-mail address: bagrel@univ-metz.fr (D. Bagrel).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.010

T. Tzanova et al. / European Journal of Medicinal Chemistry 44 (2009) 2724–2730
2725
vitro, and their toxicity was verified in cell cultures. These two
analyses allowed the choice of the most effective of these
analogues, named 6c, whose effect on cell redox status and efficacy
against oxidative stress induced by tert-butyl hydroperoxide (tBHP)
was explored in two human mammary cell lines (a cancerous one,
MCF7, and a non-cancerous one, hTERT-HME1) and in a car-
diomyoblast cell line, H9c2. We suggest that this compound could
be a potential candidate as a chemoprotective agent.
groups on compounds 5a–c by treatment in refluxing 48%
aqueous HBr.
2.2. Pharmacology
2.2.1. In vitro antioxidant capacity
The ferric reducing antioxidant power (FRAP) method [28] was
used for assessing the antioxidant potential of 6a–c derivatives.
The highest antioxidant activity was observed for 6b (6.1 nmol
Fe2þ/nmol compound), while 6a lacked any ability to reduce Fe3þ
(Fig. 1). The antioxidant capacity of 6b and 6c was, respectively, 2
and 1.5 times higher than that of Trolox (6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid) a water soluble derivative of
vitamin E, used as a reference antioxidant.
2. Results and discussion
2.1. Chemistry
The general synthetic method shown in Scheme
1 was
employed for the preparation of the new benzophenones con-
taining 1,3-thiazol moiety. The preparation involved a straightfor-
ward reaction sequence: (i) Friedel–Crafts acylation of
methoxybenzenes 1a–c, (ii) reduction of the nitro group, (iii)
formation of thiazol ring, (iv) replacement of the thione group by
a carbonyl group and (v) demethylation of aryl methyl ethers to
afford the desired hydroxyl substituted benzophenones 6a–c.
As the Friedel–Crafts acylation of 2(3H)-benzothiazolone was
previously studied and was found to proceed with high regiose-
lectivity in position 6 [23–25], the access to the 5-benzoyl deriva-
tives of 2(3H)-benzothiazolone was realized by us using an
alternative route (Scheme 1).
2.2.2. Cytotoxicity
The cytotoxicity of benzophenone derivatives was tested on the
cancerous MCF7 and the non-cancerous hTERT-HME1 mammary
cell lines, following 48 or 72 h exposure, using the 3-(4,5-dime-
thylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay
[29]. MTT was used here as a first intention screening method
in order to eliminate compounds that could present any cell
toxicity.
As evident from IC50 values summarized in Table 1, the most
cytotoxic compound was 6b, while 6c did not show noteworthy
cytotoxic effects below 100 mM on both cell lines. Thus, 6c which
In the first step the Friedel–Crafts acylation of activated
benzenes such as anisole (1a), veratrole (1b), and 1,4-dimethox-
ybenzene (1c) was realized. Acylation was carried out with
freshly prepared 4-chloro-3-nitrobenzoyl chloride in dichloro-
methane in the presence of AlCl3 as a catalyst [26]. Selective
reduction of the nitro group in compounds 2a–c was accom-
plished with tin(II) chloride according to a published procedure
[27] to obtain the o-chloroanilines 3a–c in high yields. These
compounds underwent smooth cyclization with ethyl potassium
xanthate in dimethylformamide (DMF) as solvent to afford 5-
aroyl-2(3H)-benzothiazolethiones (4a–c). Thiones 4a–c were
oxidized with KMnO4 under basic conditions to the correspond-
ing benzothiazole-2-sulfonic acids, which were used in the next
stage without isolation. After acidic hydrolysis 5-aroyl-2(3H)-
benzothiazolones (5a–c) were obtained. The preparation of target
compounds 6a–c was achieved by demethylation of the methoxy
displayed a high in vitro antioxidant potential, without significant
toxicity in a non-cancerous cell line was selected for further studies.
The concentration of 100
according to IC20 values.
mM was chosen for further experiments
2.2.3. Effect on cellular redox status
In order to verify whether 6c could play a functional role in
biological systems, the levels of intracellular reduced glutathione
(GSH) were quantified by high-performance liquid chromatog-
raphy (HPLC) using a post-column reaction with ortho-phtha-
laldehyde [30].
In both cell lines, a significant increase of GSH levels was
observed after a 48 h-incubation (88.9 ꢀ 7.2 vs 71.3 ꢀ 2.5 for MCF7,
p < 0.05 vs control and 59.4 ꢀ1.9 vs 43.3 ꢀ1.1 for hTERT-HME1,
p < 0.001 vs control) (Fig. 2). In non-cancerous cells, a significant
increase was also detected after 72 h of incubation.
R1
R1
R4
O
R1
R4
O
R2
R3
R2
R3
R2
R3
NO2
Cl
NH2
Cl
i
ii
iii
R4
1a-c
2a-c
3a-c
R1
R4
O
R1
R4
O
R1
R4
O
H
N
H
N
H
N
R2
R3
R2
R3
R2
R3
iv
v
S
O
O
S
S
S
6a-c
4a-c
5a-c
1 - 5a R1 = R2 = R4 = H; R3 = OCH3
1 - 5b R1 = R4 = H; R2 = R3 = OCH3
1 - 5c R1 = R4 = OCH3; R2 = R3 = H
6a R1 = R2 = R4 = H; R3 = OH
6b R1 = R4 = H; R2 = R3 = OH
6c R1 = R4 = OH; R2 = R3 = H
Scheme 1. Schematic representation of the synthesis and chemical structures of the tested 5-(hydroxybenzoyl)-2(3H)-benzothiazolones (6a–c). Reagents and conditions: (i) 4-
chloro-3-nitrobenzoyl chloride, AlCl3, CH2Cl2, 45 ꢁC; (ii) SnCl2$2H2O, 37% HCl, C2H5OH; (iii) C2H5OC(S)SK, DMF, reflux; (iv) KMnO4, KOH, 60–70 ꢁC; (v) 48% HBr, CH3COOH, n-
Bu4NþBrꢂ, reflux.

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T. Tzanova et al. / European Journal of Medicinal Chemistry 44 (2009) 2724–2730
hTERT-HME1 cells were pretreated with 100 mM 6c for 4 h (a time
treatment compatible to an antioxidant activity as shown by Fig. 3),
and thereafter exposed to 0.5 mM tBHP.
The most significant production of reactive oxygen species
(ROS) in cells was measured after 0.5 and 1.5 h of tBHP treatment.
During this length of time, a pre-treatment with 6c, at the
7
6
5
4
3
2
1
0
concentration of 100 mM, significantly lowered the ROS levels down
to levels for controls not exposed to tBHP in both cell lines (Fig. 4A).
In parallel, cell survival was evaluated using a flow cytometric
analysis with H2DCFDA and PI (propidium iodide). H2DCFDA enters
viable cells freely where it is converted to the green fluorescent
dichlorofluoresceine, whereas the red fluorophore PI only enters
altered membrane-cells. Therefore, viable cells are identified with
a high green and a low red fluorescence [33]. tBHP dramatically
induced cell death in hTERT-HME1, while this effect was discrete in
MCF7. Compound 6c efficiently protected the non-cancerous cell
line against cell death provoked by tBHP, as shown by the increase
in cell survival (43–59%) observed in the co-treated (6c þ tBHP)
cells (Fig. 4B).
Since the sensitivity of cardiac cells to oxidative stress limits
anthracyclin-based chemotherapy, we evaluated whether 6c
could also protect the cardiomyoblastic cells, H9c2 exposed to
tBHP.
6a
6b
6c
Trolox
Fig. 1. Antioxidant capacity of benzophenone derivatives (6a–c) (FRAP method).
Values are expressed as antioxidant ratio (nmol Fe2þ/nmol compound). Data are
means ꢀ SD (n ¼ 3), ***p < 0.001 with respect to Trolox.
Fig. 5A shows that H9c2 cells are extremely sensitive to tBHP.
Indeed, a 0.5 h treatment by tBHP 0.5 mM induced cell mortality of
89% vs 6% in the control group (DMSO). To avoid such an over-
whelming mortality, and allow realistic evaluation of 6c to protect
from oxidative stress, we decreased the dose of tBHP. H9c2 cells
were treated by very low doses of tBHP (0.05 mM). Fig. 5B and C
shows that 6c induced an important decrease of ROS levels (up to 7-
fold) produced by tBHP and saved cell viability, indicating that the
benzophenone was able to efficiently protect these cells against
tBHP-induced toxicity.
Moreover, ROS levels were detected by flow cytometric analysis
using H2DCFDA (20,70-dichlorodihydrofluorescein diacetate) as
described previously [31]. H2DCFDA is capable of crossing the
plasma membrane to enter a cell interior, where esterases hydro-
lyze its acetyl moiety to produce 20,70-dichlorodihydrofluorescein
(H2DCF). The de-acetylated form of the probe is then susceptible to
oxidation, generating
fluorescein (DCF).
a
fluorescent product, 20,70-dichloro-
In MCF7 cells, during the first 24 h of incubation, 6c was
a more powerful reducer of H2O2 production than Trolox (Fig. 3).
After 24 h, 6c did not act as a radical scavenger, while Trolox
continuously inhibited ROS production by 20–30% in both cell
lines.
3. Conclusion
Many adverse effects of chemotherapy agents are related to the
rise of oxidative stress levels in multiple organ systems. In order
to develop novel antioxidant molecules as chemoprotectants for
non-cancerous cells, we designed new molecules associating two
chemical structures that could be efficient against oxidative stress
(a benzophenone and a benzothiazolone moiety). This paper
describes the successful synthesis of three derivatives, 5-
(hydroxybenzoyl)-2(3H)-benzothiazolones (6a–c), and the evalu-
ation of the pharmacological properties of the lead candidate, i.e.
(6c) [5-(2,5-dihydroxybenzoyl)-2(3H)-benzothiazolone]. Comp-
ound 6c exhibited evident antioxidant properties in vitro. A
screening on human mammary cell lines – the cancerous MCF7
and the non-cancerous hTERT-HME1 showed that 6c did not exert
any cytotoxic effect. Moreover, this benzophenone derivative
displayed a potent free radical scavenging activity and was able to
efficiently protect cells against oxidative stress provoked by tert-
butylhydroperoxide. These properties could be due to the signif-
icant increase of GSH level observed in 6c treatments. This study
must now be extended by comparing the antioxidant properties of
the two moieties of the molecule with those of the bifunctional
resulting compound.
2.2.4. Cytoprotective action
Many biological functions of polyphenols have been attributed
to their free radical scavenging and antioxidant properties [6]. The
new benzophenone 6c could be particularly efficacious due to its
high antioxidant in vitro efficacy (FRAP assay). Thus, it was inter-
esting to test the protective potential of this compound against
oxidative-induced stress in cells. For that, tert-butyl hydroperoxide
(tBHP) was used as a membrane-permeant pro-oxidant agent
[32]. To evaluate protection against oxidative stress, MCF7 and
Table 1
Cytotoxic activity of benzophenone analogues (6a–c) on MCF7 and hTERT-HME1
breast cell lines after 48 and 72 h of incubation (MTT assay)
Compound
Breast cancer cell line, MCF7
Non-cancer breast cell line,
hTERT-HME1
IC20
(m
M)
IC50
(
m
M)
IC20
(
m
M)
IC50 (mM)
48 h
6a
6b
Moreover, the protective action of 6c was also evident in
H9c2, a cardiomyoblast cell line. These results are of particular
relevance considering that the cardiotoxicity of doxorubicin,
a drug often used in breast cancer treatment, is due to oxidative
stress generation. Thus, the protective effect of 6c against doxo-
rubicin toxicity has now to be tested in cancer cells and in
cardiac cells in order to appreciate its eventual relevance in
therapeutics.
44.5 ꢀ 4.7
12.1 ꢀ 3.9
93.5 ꢀ 6.8
120.9 ꢀ 2.1
43.6 ꢀ 4.7
167.6 ꢀ 2.4
73.1 ꢀ 3.7
43.5 ꢀ 4.4
149.9 ꢀ 4.3
109.2 ꢀ 3.4
155.3 ꢀ 4.9
182.2 ꢀ 6.2
6c
72 h
6a
6b
37.8 ꢀ 4.5
8.3 ꢀ 3.2
95,9 ꢀ 5,1
28.1 ꢀ 2.7
173.2 ꢀ 4.1
78.4 ꢀ 2.1
33.8 ꢀ 3.6
144.6 ꢀ 4.7
110.6 ꢀ 4.8
116.3 ꢀ 4.7
167.4 ꢀ 3.8
6c
126.0 ꢀ 2.3
IC values are the mean of 8 assays ꢀ SD.

T. Tzanova et al. / European Journal of Medicinal Chemistry 44 (2009) 2724–2730
2727
Breast cancer cell line
Non-cancer breast cell line
120
100
80
60
40
20
0
MCF7
120
100
80
60
40
20
0
hTERT-HME1
48
72
48
72
Treatment time (h)
Fig. 2. Intracellular GSH levels in MCF7 and hTERT-HME1 breast cells after 48 and 72 h of incubation with 100
compared to respective control cells (,), which were performed with DMSO.
mM 6c ( ). Data are means ꢀ SD (n ¼ 3), *p < 0.05 and ***p < 0.001
4. Experimental protocol
diisopropyl ether. Yield 4.88 g (67%). M.p. 103 ꢁC. IR (nujol, cmꢂ1):
1640, 1540, 1340, 1270. Anal. Calcd. for C14H10ClNO4: C 57.65; H
3.46; N 4.80. Found: C 57.84; H 3.49; N 4.82.
4.1. Chemistry
Melting points were determined on a Boetius hot-stage micro-
scope and are uncorrected. IR spectra were recorded in nujol on
a Specord 71 spectrometer. 1H NMR spectra were obtained with
a Bruker DRX 300 spectrometer operating at 300 MHz in DMSO-d6.
4.1.2. 4-Chloro-30,40-dimethoxy-3-nitrobenzophenone (2b)
Compound 2b was synthesized as for 2a starting from veratrole.
Recrystallization from ethanol gave 2.70 g (34%). M.p. 157–158 ꢁC.
IR (nujol, cmꢂ1): 1650, 1510, 1370, 1270. Anal. Calcd. for
C15H12ClNO5: C 56.00; H 3.76; N 4.35. Found: C 56.35; H 3.78; N
4.35.
Chemical shifts were recorded in parts per million (ppm,
d)
downfield from TMS as an internal standard. Elemental analyses
were performed on a Vario EL III microanalyzer (Elementar Ana-
lysensysteme GmbH, Germany). Obtained results were within 0.4%
of theoretical values. Thin layer chromatography (TLC) was carried
out on silica gel plates (Merck 60 F254) using toluene–chloroform–
ethylacetate (3:1:1) as eluent; zones were detected visually under
ultraviolet irradiation (254 nm).
4.1.3. 4-Chloro-20,50-dimethoxy-3-nitrobenzophenone (2c)
Compound 2c was synthesized analogously as for 2a starting
from 1,4-dimethoxybenzene. Recrystallization from ethanol gave
4.80 g (60%). M.p. 112–113 ꢁC. IR (nujol, cmꢂ1): 1670, 1540, 1350,
1270. Anal. Calcd. for C15H12ClNO5: C 56.00; H 3.76; N 4.35. Found:
C 56.37; H 3.79; N 4.31.
4.1.1. 4-Chloro-40-methoxy-3-nitrobenzophenone (2a)
Anisol (2.7 ml, 25 mmol) was added to a solution of freshly
prepared 4-chloro-3-nitrobenzoyl chloride (6.05 g, 27.5 mmol) in
dichloromethane (25 ml). The resulting solution was cooled and
then aluminum chloride (3.67 g, 27.5 mmol) was slowly added at
0–5 ꢁC. After complete addition, the mixture was allowed to stir at
room temperature for 1 h, and then was heated up to 45 ꢁC for 4 h.
The reaction mixture was poured onto ice (250 g) and conc. HCl
(15 ml). The crude product was isolated by extraction with
dichloromethane. The combined organic layers were washed with
10% aqueous NaHCO3, water, dried over Na2SO4 and evaporated in
vacuum. The residue was purified by recrystallization from
4.1.4. 5-(4-Methoxybenzoyl)-2(3H)-benzothiazolethione (4a)
To a stirred suspension of 2a (2.33 g, 8 mmol) in ethanol (20 ml),
a hot solution of SnCl2$2H2O (7.24 g, 32 mmol) in conc. HCl (16 ml)
was slowly added. The stirring was continued for 30 min at room
temperature. The obtained clear solution was treated with 30%
aqueous NaOH until pH was strongly basic and the amino deriva-
tive 3a was isolated in 85% yield. Then, a mixture of 3a (2.61 g,
10 mmol) and ethyl potassium xanthate (3.59 g, 22 mmol) in DMF
(20 ml) was refluxed for 3 h. After heating, the reaction mixture
was poured onto water (50 ml) and then was acidified with 10%
HCl. The obtained precipitate was filtered, washed with water and
Breast cancer cell line
Non-cancer breast cell line
hTERT-HME1
MCF7
1,2
1,2
1
1
0,8
0,6
0,4
0,2
0
0,8
0,6
0,4
0,2
0
4
8
12
4
8
12
24
48
24
48
Treatment time (h)
Fig. 3. Effect of 6c on redox status of MCF7 and hTERT-HME1 cells in culture. Cells were treated for 4, 8, 12, 24 and 48 h with 6c (
) and Trolox (
) (100 mM). ROS levels
were detected by flow cytometry as dichlorofluoresceine (DCF) fluorescence ratio between treatment and DMSO control (d). Data are means ꢀ SD (n ¼ 3).

2728
T. Tzanova et al. / European Journal of Medicinal Chemistry 44 (2009) 2724–2730
Breast cancer cell line
MCF7
Non-cancer breast cell line
hTERT-HME1
A
5
4
3
2
1
0
5
4
3
2
1
0
0.5
1.5
2.5
3.5
0.5
1.5
2.5
3.5
120
100
80
60
40
20
0
B 120
100
80
60
40
20
0
0.5
1.5
2.5
3.5
0.5
1.5
2.5
3.5
Duration of incubation with tBHP(h)
Fig. 4. Evaluation of the protective potential of 6c against oxidative stress induced by tert-butyl hydroperoxide (tBHP) in MCF7 and hTERT-HME1 cells. (A) ROS production was
assessed by flow cytometric analysis as DCF fluorescence ratio between treatment and control. Co-treated cells ( ) were pre-incubated with 6c for 4 h, followed by
a subsequent incubation (0.5–3.5 h) with tBHP (0.5 mM). Three controls were performed for testing the effects of a 4 h pre-incubation with DMSO (d) or 6c ( ), and for
testing the effects of a 0.5–3.5 h incubation with tBHP alone ( ). (B) Cytotoxicity was assessed by flow cytometric analysis with PI and H2DCFDA. Co-treated cells ( ) were pre-
incubated with 6c for 4 h, followed by a subsequent incubation (0.5–3.5 h) with tBHP (0.5 mM). Three controls were performed for testing the effects of a 4 h pre-incubation with
DMSO (,) or 6c ( ), and for testing the effects of a 0.5–3.5 h incubation with tBHP alone (-). Results are the mean of triplicate assays ꢀ SD.
Myoblastic cell line H9c2
0.5 mM tBHP
0.05 mM tBHP
A
B5
120
100
80
60
40
20
0
4
3
2
1
0
0.5
1.5
2.5
3.5
0.5
1.5
2.5
3.5
Duration of incubation with tBHP(h)
C
0.05 mM tBHP
120
100
80
60
40
20
0
0.5
1.5
2.5
3.5
Duration of incubation with tBHP(h)
Fig. 5. Evaluation of the protective potential of 6c against oxidative stress induced by tert-butyl hydroperoxide (tBHP) in cardiomyoblastic cells H9c2. (A,C) cytotoxicity was assessed
by flow cytometric analysis with PI and H2DCFDA. Co-treated cells ( ) were pre-incubated with 6c for 4 h, followed by a subsequent incubation (0.5–3.5 h) with tBHP (0.5 mM (A)
or 0,05 mM (C)). Three controls were performed for testing the effects of a 4 h pre-incubation with DMSO (,) or 6c ( ), and for testing the effects of a 0.5–3.5 h incubation with
tBHP alone (-). (B) ROS production was assessed by flow cytometric analysis as DCF fluorescence ratio between treatment and control. Co-treated cells (
incubated with 6c for 4 h, followed by a subsequent incubation (0.5–3.5 h) with tBHP (0.05 mM). Three controls were performed for testing the effects of a 4 h pre-incubation with
DMSO (d) or 6c ( ), and for testing the effects of a 0.5–3.5 h incubation with tBHP alone (
). Results are the mean of triplicate assays ꢀ SD.
) were pre-

T. Tzanova et al. / European Journal of Medicinal Chemistry 44 (2009) 2724–2730
2729
dried to yield 2.85 g (95%) of 4a. Recrystallization from 2-methox-
yethanol gave 2.38 g (79%). M.p. 225–228 ꢁC. IR (nujol, cmꢂ1):
3000–3100, 1640, 1250. Anal. Calcd. for C15H11NO2S2: C 59.78; H
3.68; N 4.63; S 21.28. Found: C 60.12; H 3.58; N 4.33; S 21.43.
J ¼ 8.1 Hz), 7.66 (d, 2H, ArH, J ¼ 8.7 Hz), 7.73 (d, 1H, ArH, J ¼ 8.1 Hz),
10.42 (br s,1H, OH),12. 04 (br s,1H, NH). Anal. Calcd. for C14H9NO3S:
C 61.98; H 3.34; N 5.16; S 11. 82. Found: C 61.78; H 3.20; N 5.01; S
11.47.
4.1.5. 5-(3,4-Dimethoxybenzoyl)-2(3H)-benzothiazolethione (4b)
Compound 4b was synthesized as for 4a starting from 2b.
Recrystallization from 2-methoxyethanol gave 1.06 g (32%). M.p.
243–244 ꢁC. IR (nujol, cmꢂ1): 3200, 1640, 1250. Anal. Calcd. for
C16H13NO3S2: C 57.99; H 3.95; N 4.23; S 19.35. Found: C 58.15; H
3.91; N 4.21; S 19.65.
4.1.11. 5-(3,4-Dihydroxybenzoyl)-2(3H)-benzothiazolone (6b)
Compound 6b was synthesized as for 6a starting from 5b.
Recrystallization from acetone gave 0.12 g (43%). M.p. 309–310 ꢁC.
IR (nujol, cmꢂ1): 3050–3350,1690,1640. 1H NMR (300 MHz, DMSO-
d6): 6.84 (d, 1H, ArH, J ¼ 8.2 Hz), 7.10 (dd, 1H, ArH, J ¼ 2.1 Hz,
J ¼ 8.2 Hz), 7.22 (d, 1H, ArH, J ¼ 2.1 Hz), 7.35 (d, 1H, ArH, J ¼ 1.3 Hz),
7.41 (dd, 1H, ArH, J ¼ 1.4 Hz, J ¼ 8.1 Hz), 7.72 (d, 1H, ArH, J ¼ 8.1 Hz),
9.48 (br s, 1H, OH), 9.83 (br s, 1H, OH), 12.05 (br s, 1H, NH). Anal.
Calcd. for C14H9NO4S: C 58.53; H 3.16; N 4.88; S 11.16. Found: C
58.18; H 3.46; N 4.85; S 10.81.
4.1.6. 5-(2,5-Dimethoxybenzoyl)-2(3H)-benzothiazolethione (4c)
Compound 4c was synthesized as for 4a starting from 2c.
Recrystallization from ethanol gave 1.35 g (41%). M.p.176–179 ꢁC. IR
(nujol, cmꢂ1): 3000–3100, 1640, 1280. Anal. Calcd. for C16H13NO3S2:
C 57.99; H 3.95; N 4.23; S 19.35. Found: C 58.13; H 4.09; N 4.35; S
19.60.
4.1.12. 5-(2,5-Dihydroxybenzoyl)-2(3H)-benzothiazolone (6c)
Compound 6c was synthesized as for 6a starting from 5c.
Recrystallization from ethanol gave 0.12 g (42%). M.p. 240–242 ꢁC.
IR (nujol, cmꢂ1): 3400, 3000–3150, 1710, 1660. 1H NMR (300 MHz,
DMSO-d6): 6.71 (d, 1H, ArH, J ¼ 2.9 Hz), 6.80 (d, 1H, ArH, J ¼ 8.8 Hz),
6.86 (dd, 1H, ArH, J ¼ 2.9 Hz, J ¼ 8.8 Hz), 7.40 (d, 1H, ArH, J ¼ 1.1 Hz),
7.46 (dd, 1H, ArH, J ¼ 1.1 Hz, J ¼ 8.1 Hz), 7.72 (d, 1H, ArH, J ¼ 8.2 Hz),
9.08 (s, 1H, OH), 9.64 (s,1H, OH),12.03 (br s,1H, NH). Anal. Calcd. for
C14H9NO4S: C 58.53; H 3.16; N 4.88; S 11.16. Found: C 58.88; H 3.23;
N 4.84; S 10.79.
4.1.7. 5-(4-Methoxybenzoyl)-2(3H)-benzothiazolone (5a)
To a stirred solution of 4a (1.50 g, 5 mmol) in 10% aqueous KOH,
a hot solution of 10% KMnO4 was slowly added for 20–30 min. The
end of the oxidation reaction was determined by the excess of
KMnO4 in reaction mixture. The precipitate of MnO2 was filtered
and washed with hot water. The filtrate was acidified to pH ¼ 2 with
conc. HCl and refluxed until the evolving of SO2 finished. The
obtained precipitate of 5a was collected by filtration, washed with
water and dried. Recrystallization from dioxane gave 0.93 g (65%).
M.p. 214–215 ꢁC. IR (nujol, cmꢂ1): 3000–3100, 1680, 1640, 1270. 1H
NMR (300 MHz, DMSO-d6): 3.86 (s, 3H, OCH3), 7.09 (d, 2H, ArH,
J ¼ 8.8 Hz), 7.39 (d, 1H, ArH, J ¼ 1.5 Hz), 7.44 (dd, 1H, ArH, J ¼ 1.5 Hz,
J ¼ 8 Hz), 7.74–7.78 (m, 3H, ArH),12.10 (br s,1H, NH). Anal. Calcd. for
C15H11NO3S: C 63.14; H 3.89; N 4.91; S 11.24. Found: C 63.47; H 3.68;
N 4.66; S 11.55.
4.2. Pharmacology
4.2.1. Ferric reducing antioxidant power (FRAP)
The FRAP assay [28] depends upon the reduction of a ferric
tripyridyltriazine (Fe3þ–TPTZ) complex to the ferrous tripyridyl-
triazine (Fe2þ–TPTZ) by a reductant at low pH. Fe2þ–TPTZ has an
intensive blue color and was monitored at 593 nm. Trolox was used
as standard antioxidant. Working FRAP reagent was prepared as
required by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution, and
2.5 ml FeCl3$6H2O solution. Freshly prepared FRAP reagent was
warmed at 37 ꢁC for 30 min and added to the samples every 30 s.
Absorbance readings were taken after 30 min every 30 s.
4.1.8. 5-(3,4-Dimethoxybenzoyl)-2(3H)-benzothiazolone (5b)
Compound 5b was synthesized as for 5a starting from 4b.
Recrystallization from ethanol gave 0.50 g (32%). M.p. 232–235 ꢁC.
IR (nujol, cmꢂ1): 3000–3100, 1680, 1640, 1250. 1H NMR (300 MHz,
DMSO-d6): 3.83 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 7.11 (d, 1H, ArH,
J ¼ 8.4 Hz), 7.35 (dd, 1H, ArH, J ¼ 1.8 Hz, J ¼ 8.3 Hz), 7.38 (d, 1H, ArH,
J ¼ 1.8 Hz), 7.54 (d, 1H, ArH, J ¼ 1.3 Hz), 7.60 (dd, 1H, ArH, J ¼ 1.3 Hz,
J ¼ 8.2 Hz), 7.83 (d, 1H, ArH, J ¼ 8.4 Hz), 12.25 (br s, 1H, NH). Anal.
Calcd. for C16H13NO4S: C 60.94; H 4.16; N 4.44; S 10.17. Found: C
60.68; H 4.30; N 4.33; S 9.87.
4.2.2. Cell lines and cell culture
Human breast adenocarcinoma cell line MCF7 (ATCC) was
grown in phenol red-free RPMI-1640 medium, supplemented with
10% heat inactivated fetal bovine serum, 10,000 U/ml penicillin and
10,000 mg/ml streptomycin, 2 mM L-glutamine, 1.25 mM sodium
pyruvate, amino acids and vitamins (Eurobio, France). Human
breast epithelial cell line hTERT-HME1 (ATCC) was grown in
a mixture of RPMI-1640 (90%) and Mammary Epithelial Growth
Medium (10%) (Cambrex, USA). The cell lines were cultivated at
37 ꢁC in a humidified atmosphere containing 5% CO2. Rat myoblast
embryonic cell line H9c2 (ATCC) was cultured in standard DMEM
medium containing 3.7 g/L sodium bicarbonate, 4.5 g/L glucose
4.1.9. 5-(2,5-Dimethoxybenzoyl)-2(3H)-benzothiazolone (5c)
Compound 5c was synthesized as for 5a starting from 4c.
Recrystallization from ethanol gave 1.06 g (67%). M.p. 210–214 ꢁC.
IR (nujol, cmꢂ1): 3000–3100, 1680, 1640, 1270. 1H NMR (300 MHz,
DMSO-d6): 3.61 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 6.89 (d, 1H, ArH,
J ¼ 2.8 Hz), 7.09 (dd, 1H, ArH, J ¼ 2.8 Hz, J ¼ 8.8 Hz), 7.13 (d, 1H, ArH,
J ¼ 8.8 Hz), 7.40 (d, 1H, ArH, J ¼ 1.5 Hz), 7.45 (dd, 1H, ArH, J ¼ 1.5 Hz,
J ¼ 8.2 Hz), 7.71 (d, 1H, ArH, J ¼ 8.2 Hz). Anal. Calcd. for C16H13NO4S:
C 60.94; H 4.16; N 4.44; S 10.17. Found: C 60.59; H 4.12; N 4.48; S
10.05.
supplemented with 10% fetal bovine serum and 4 mM
at 37 ꢁC in a humidified atmosphere of 10% CO2.
L-Glutamine
4.2.3. Cell viability determination (MTT assay)
Cell survival was detected by MTT reduction as described by
Mosmann [29]. Cells were seeded in 96-well plates at a density of
1 ꢃ104 cells/well and were incubated 24 h. After 48 or 72 h expo-
sure to benzophenones, the medium with tested compounds was
removed and fresh media with MTT (final concentration of 0.5 mg/
ml) was added. The incubation continued for 3 h at 37 ꢁC. The
formazan crystals were dissolved in dimethyl sulfoxide (DMSO)
and the spectrophotometric determinations were done at 550 nm.
Determination of inhibitory concentrations was performed using
generalized linear models as described previously [34].
4.1.10. 5-(4-Hydroxybenzoyl)-2(3H)-benzothiazolone (6a)
To a suspension of 5a (0.29 g, 1 mmol) in acetic acid (2 ml), an
aqueous solution of 48% HBr (7 ml) and tetrabutylammonium
bromide (5 mg) was added. The mixture was refluxed for 4 h and
after cooling was poured onto water (50 ml). The precipitate was
collected, washed with water and dried. Recrystallization from
ethanol gave 0.14 g (52%). M.p. 297–299 ꢁC. IR (nujol, cmꢂ1): 3000–
3250, 1680, 1640. 1H NMR (300 MHz, DMSO-d6): 6.89 (d, 2H, ArH,
J ¼ 8.7 Hz), 7.36 (d, 1H, ArH, J ¼ 1.5 Hz), 7.42 (dd, 1H, ArH, J ¼ 1.5 Hz,

2730
T. Tzanova et al. / European Journal of Medicinal Chemistry 44 (2009) 2724–2730
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The authors thank Gilbert Kirsch for helpful discussions and
Alexandra Tomova for critical reading of the manuscript. The work
´
was funded by grants from the ‘‘Contrat de Plan Etat-Region de
´
Lorraine’’ and the ‘‘Ligue contre le Cancer (Comite de Moselle)’’. T.
Tzanova was recipient of a fellowship provided by the French
Government (Ambassade de France en Bulgarie).
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