Synthesis of new spiroindolinones incorporating a benzothiazole moiety as antioxidant agents

Article abstract of DOI:10.1016/j.ejmech.2009.12.001

3H-Spiro[1,3-benzothiazole-2,3′-indol]-2′(1′H)-ones 3a-c and 4a-e were synthesized from treating the 5-substituted 1H-indole-2,3-diones with 2-aminothiophenol in ethanol. The structures were confirmed by elemental analyses, spectrometry (IR, ¹H NMR, ¹³C NMR, HSQC-2D and LCMS-APCI) and single crystal X-ray analysis. The new compounds were screened for their antioxidant activities such as the Fe³⁺/ascorbate system induced inhibition of lipid peroxidation (LP) in liposomes, trolox equivalent antioxidant capacity (TEAC), scavenging effect on diphenylpicryl hydrazine (DPPH^{{radical dot}}), and reducing power. These compounds showed potent scavenging activities against DPPH^{{radical dot}} and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS^{{radical dot}+}) radicals, reducing powers, and strong inhibitory capacity on lipid peroxidation. Compound 4a incorporating methyl both at R₁ and R₂ was found to be the most potent antioxidant described in this study. Compounds 3b and 4b were selected as representative compounds by the National Cancer Institute for screening against anticancer activity and these compounds were found to be cytotoxic against CNS cancer cell line SNB-75 in the primary screen.

Full text of DOI:10.1016/j.ejmech.2009.12.001

European Journal of Medicinal Chemistry 45 (2010) 1068–1077
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new spiroindolinones incorporating a benzothiazole moiety as
antioxidant agents
a
a
,
b
c
a
¨
*
¨
¨
Nilg u¨ n Karalı , Ozlen G u¨ zel , Nurten Ozsoy , S u¨ heyla Ozbey , Aydın Salman
a
Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
Istanbul University, Faculty of Pharmacy, Department of Biochemistry, 34116 Beyazıt, Istanbul, Turkey
Hacettepe University, Department of Physics Engineering, 06532 Beytepe, Ankara, Turkey
b
c
a r t i c l e i n f o
a b s t r a c t
0
0
0
Article history:
Received 3 August 2009
Received in revised form
3H-Spiro[1,3-benzothiazole-2,3 -indol]-2 (1 H)-ones 3a–c and 4a–e were synthesized from treating the
-substituted 1H-indole-2,3-diones with 2-aminothiophenol in ethanol. The structures were confirmed
5
1
13
by elemental analyses, spectrometry (IR, H NMR, C NMR, HSQC-2D and LCMS-APCI) and single crystal
X-ray analysis. The new compounds were screened for their antioxidant activities such as the Fe
ascorbate system induced inhibition of lipid peroxidation (LP) in liposomes, trolox equivalent antioxidant
capacity (TEAC), scavenging effect on diphenylpicryl hydrazine (DPPH ), and reducing power. These
compounds showed potent scavenging activities against DPPH and 2,2 -azino-bis(3-ethylbenzthiazo-
line-6-sulphonic acid) (ABTS ) radicals, reducing powers, and strong inhibitory capacity on lipid per-
oxidation. Compound 4a incorporating methyl both at R and R was found to be the most potent
antioxidant described in this study. Compounds 3b and 4b were selected as representative compounds
by the National Cancer Institute for screening against anticancer activity and these compounds were
found to be cytotoxic against CNS cancer cell line SNB-75 in the primary screen.
Ó 2009 Elsevier Masson SAS. All rights reserved.
30 November 2009
3þ
/
Accepted 2 December 2009
Available online 21 December 2009
ꢀ
ꢀ
0
Keywords:
ꢀ
þ
3-Spiroindolinone
Benzothiazole
Synthesis
X-ray analyses
Antioxidant activity
Cytotoxicity
1
2
1. Introduction
led to new medical insight [3,4]. Minimizing oxidative damage may
be an important approach to the primary prevention or treatment of
Antioxidants have gained a lot of importance because of their
potential as prophylactic and therapeutic agents in many diseases.
Free radicals are constantly formed as a result of normal organ
functions or excessive oxidative stress [1]. High levels of free radicals
can cause damage to biomolecules such as lipids, proteins, enzymes
and DNA in cells and tissues, resulting in mutations that can lead to
malignancy. Damage to DNA by oxidative stress has been widely
accepted as a major cause of cancer [2]. DNA mutation is a critical step
in carcinogenesis and elevated levels of oxidative DNA lesions have
been observed in various tumours. The discovery of the role of free
radicals in cancer, diabetes, cardiovascular diseases, autoimmune
diseases, neurodegenerative disorders, aging and other diseases has
these diseases, since antioxidants may stop the free-radical forma-
tion, or interrupt an oxidizing chain reaction. This had attracted
a great deal of research interest in therapeutic antioxidant-based
drugs formulations. The development of synthetic compounds,
capable of scavenging free radicals, has been a great success.
In recent years, many publications have covered the antioxidant
and cytotoxic roles of several indole derivatives. The antioxidant
behaviour of a series of substituted indoline-2-ones and indoline-2-
thiones was investigated using an oxygen radical absorbance capacity
assay. The results indicated that the examined indoline derivatives
had effective activities as radical scavengers [5]. Some indole-3-car-
boxamides were potent inhibitors of superoxide anion formation
[6,7]. Several benzimidazole containing indole derivatives showed
very good in vitro free-radical scavenging properties as shown by
determination of their scavenge superoxide anion formation capacity
0
Abbreviations: ABTS, 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid);
[8]. Phytoalexins are secondary metabolites produced by certain
BHT, butylated hydroxytoluene; CNS, central nervous system; DMF, dime-
thylformamide; DMSO, dimethyl sulfoxide; DPPH, diphenylpicryl-hydrazine; EC50,
effective concentration 50%; GI, growth inhibition; LP, lipid peroxidation; MDA,
malondialdehyde; NSCL, non-small cell lung; ROS, reactive oxygen species; SD,
standard deviation; SRB, sulphorhodamine B; TBA, thiobarbituric acid; TBARS,
thiobarbituric acid reactive substances; TCA, trichloroacetic acid; TEAC, trolox
equivalent antioxidant capacity; TRAP, total radical antioxidant potential.
plantsinresponsetopathogenattackorvariousformsofstress.Indole
phytoalexins from cruciferous plants, such as brassinin, spiro-
brassinin, 1-methoxyspirobrassinol and camalexin demonstrate in
vivo cytostatic/cytotoxic effects against cultured human solid tumour
and leukemia cell lines [9,10]. Synthetic 2-amino analogs of
1-methoxyspirobrassinol were found to be significantly cytotoxic
*
Corresponding author. Tel.: þ90 212 4400000; fax: þ90 212 4400252.
E-mail address: ozlen_guzel@yahoo.com ( O¨ . G u¨ zel).
against human cancer cell lines (Fig. 1) [11,12].
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.001

N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
1069
O
NH
R
N
F
N
N
H
X
N
H
N
H
I
II
III
S
SCH3
N
N
N
S
NH
SCH3
SCH3
S
S
O
OH
N
H
N
H
N
N
H
OCH3
IV
V
VI
VII
Fig. 1. Structures of indoline-2-ones/thiones (I), indole-3-carboxamides (II), indolilbenzimidazoles (III), brassinin (IV), spirobrassinin (V), 1-methoxybrassinol (VI) and
camalexin (VII).
A large number of 2-arylbenzothiazoles have been prepared
because of their wide pharmacological potency, i.e. significant anti-
cancer [13–15] and antioxidant properties [16,17]. 2-Arylbenzothia-
zoles are most commonly synthesized by the condensation of
2. Results and discussion
2.1. Chemistry
0 0 0
3H-Spiro[1,3-benzothiazole-2,3 -indol]-2 (1 H)-ones 3a–c and
2
-aminothiophenolswithsubstitutedaldehydes,carboxylicacids,acyl
0
0
0
0
chlorides, and nitriles [18,19]. The reactivity of 1H-indole-2,3-diones
towards 2-aminothiophenol has been the subject of a number of
reports and some of the products obtained are quite interesting. The
first results reported that 1H-indole-2,3-dione furnished benzothia-
zinone, indolobenzothiazide and spirobenzothiazole when the reac-
tion was carried out in dry xylene in the presence of anhydrous zinc
chloride underreflux. Onthe otherhand,the reaction of1-methyl-1H-
indole-2,3-dione with 2-aminothiophenol under the same conditions
furnished solely the spiro compound (Fig. 2)[20–22].Inaddition, there
is one report on the reaction of 1H-indole-2,3-dione with 2-amino-
thiophenol in ethanol yielding a single spirobenzothiazole [23].
Intrigued by the above observations and in continuation of our
study on indolinone derivatives, we synthesized some new spi-
roindolinone derivatives by incorporating the benzothiazole
moiety. We investigated the antioxidant properties of indole
derivatives in order to evaluate their medicinal value and to point
to an antioxidant agent that could be used in pharmaceutical
1 -methyl-3H-spiro[1,3-benzothiazole-2,3 -indol]-2 (1 H)-ones 4a–
e were synthesized from treatment of 2-aminothiophenol with 5-
substituted 1H-indole-2,3-diones 1 or 5-substituted 1-methyl-1H-
indole-2,3-diones 2 (Fig. 3). The structures of 3 and 4 were
1
13
confirmed by elemental analyses, spectrometry (IR, H NMR,
NMR, HSQC-2D and LCMS-APCI) and single crystal X-ray analysis.
IR spectra of spiroindolinones 3 and 4 showed absorption bands
in the 3354–3299 and 1732–1696 cm regions resulting from the
NH and C]O functions, respectively [23–25]. H NMR spectra of
C
ꢁ
1
1
3a–c displayed the NH protons of the indole (
and benzothiazole ( 7.22–7.33 ppm) rings as two separate signals,
whereas, the benzothiazole NH ( 7.18–7.28 ppm) and the indole N-
CH 3.08–3.20 ppm) signal were observed in the spectra of 4a–e
[23,24,26–28]. The formation of spiroindolinones is evident by both
the presence of a signal assigned to spiro C ( 74.10–74.80 ppm) and
a signal attributed to the C]O function of indolinones ( 174.75–
d 10.01–11.07 ppm)
d
d
3
(d
d
d
13
175.62 ppm) in the C NMR spectra of 4a–e [28]. Further verifi-
cation was obtained from the HSQC spectra of 4a–e, which clearly
3
þ
industry, i.e. antioxidant effect on the Fe /ascorbate-induced lipid
peroxidation in a liposome model system, TEAC, scavenging effect
1
13
show the H– C connections and allow definite assignment of the
ꢀ
1
13
on DPPH radical, and reducing power.
H and C resonances [25,29–31]. LCMS-APCI of 3a, 3b, and 4b
O
S
N
HN
S
N
R1
S
R1
R1
O
N
N
NH2
R2
VIII
IX
X
R = H, 5-F, 6-F
1
R = H, CH3
2
Fig. 2. Structures of benzothiazinone (VIII), indolobenzothiazide (IX) and spirobenzothiazole (X).

1070
N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
Table 2
O
O
Selected bond lengths and angles of compound 4d.
R
R
i
O
O
Bond lengths (Å)
N
CH3
N
H
a-e
Br1–C5
S1–C10
S1–C2
N2–C15
N2–C2
N1–C1
N1–C8
N1–C9
O1–C1
C3–C2
C6–C7
C1–C2
1.890 (4)
1.760 (4)
1.866 (6)
1.407 (6)
1.449 (5)
1.359 (5)
1.406 (5)
1.446 (6)
1.213 (4)
1.504 (5)
1.384 (6)
1.549 (6)
2
a-e
1
NH2
NH2
ii
ii
SH
SH
HN
Bond angles (
C10–S1–C2
C15–N2–C2
C1–N1–C8
C1–N1–C9
C15–C10–S1
C10–C15–N2
C6–C5–Br1
C3–C8–N1
C8–C3–C2
C5–C6–C7
O1–C1–N1
O1–C1–C2
N1–C1–C2
N2–C2–C3
N2–C2–C1
C3–C2–C1
N2–C2–S1
ꢂ
)
HN
R
S
90.1 (2)
112.6 (4)
111.4 (3)
124.7 (3)
112.2 (4)
115.8 (3)
119.4 (3)
110.0 (3)
108.4 (3)
121.0 (4)
125.8 (4)
126.5 (4)
107.6 (3)
115.3 (3)
112.1 (4)
102.4 (3)
107.4 (3)
R
S
O
O
N
N
CH3
H
3
a-c
4a-e
R= CH , Cl, NO
R= CH , CF O, Cl, Br, NO
3 3 2
3
2
Fig. 3. Synthesis of 2, 3 and 4. Reagent and condition: i) NaH, anhyd. DMF, stirred,
.5 h, CH I, reflux, 4 h, ii) EtOH, reflux, 5 h.
0
3
were selected as prototypes and displayed molecular ions with
different intensities.
The X-ray structure of 4d was determined in order to confirm
the assigned structures and to establish conformations of the
molecule. Table 1 summarizes the crystal and experimental data.
Selected bond lengths and angles are listed in Table 2. The mole-
cular structure of 4d is shown in Fig. 4. The molecule is in its spiro
form and it is not planar. The dihedral angle between the indole and
The molecular packing is shown in Fig. 5. The molecules in the
crystal are packed at normal van der Waals distances. There is only
one N–H$$$O intermolecular interaction that is smaller than 3.4 Å
which helps to stabilize the molecules in the crystal, i.e. N2$$$O1
ꢂ
benzothiazole moieties is 84.9(2) . Based on the puckering-
parameters values [32], the benzothiazole ring at C2 is twisted
around the S1–C2 bond. Bond lengths and angles have normal
values.
(
x ꢁ y, x, 1 ꢁ z 1) 3.019(6) Å.
2.2. Antioxidant activity
Table 1
Crystal data and structure refinement parameters for compound 4d.
The antioxidant activities of spiroindolinones were determined
Crystal formula
Formula weight
Crystal dimensions [mm]
Temp [K]
Crystal system
Space group
a [Å]
C
15
H
11
N
2
OSBr
as an index of pharmacological usefulness. Four model systems
3
þ
were used, namely inhibition of the Fe –ascorbate induced
ꢀ ꢀ
347.23
0.71 ꢃ 0.49 ꢃ 0.29
þ
phospholipid degradation, DPPH and ABTS radical scavenging,
and Fe (III) reduction. In the assessment of antioxidant activity, both
293(2)
Trigonal
R-3
26.7410(9)
26.7410(9)
10.1420(3)
90.0
90.0
120.0
6280.7(4)
0.71073
18
b [Å]
c [Å]
ꢂ
a
b
g
[ ]
ꢂ
[ ]
ꢂ
[ ]
3
V [Å ]
l
(MoK
a
) (Å)
Z
D
calc [g cm 3
ꢁ
]
1.652
F(000)
3132
1.52/25.6
3.09
ꢂ
q
range for data collection [ ]
ꢁ
1
m
(MoK
a
)[mm
]
Number of reflections collected
Number of unique reflections
No. of refined parameters
7792 (4892 I > 4s(I))
2728 (1651 used in refinement)
186
R/R
GOF
Final shift
Dr min, (Dr
Measurement
w
values
0.0382/0.0969
1.045
0.001
0.216, ꢁ0.226
max (eÅ 3
ꢁ
)
(
)
)
STOE IPDS 2 diffractometer
Direct methods (SHELXS-97) [37]
Full-matrix least-squares (SHEXL-97) [38]
Structure determination
Refinement
Fig. 4. Molecular structure of 4d showing the atom labelling scheme. Displacement
ellipsoids are drawn as 50% probability level.

N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
1071
Fig. 5. Crystal packing of 4d projected onto ab plane. The dashed lines indicated the intermolecular hydrogen bonds.
synthetic and biologically relevant free radicals were used. The
2.2.1. Antioxidant activity on liposome peroxidation
ꢀ
ꢀ
þ
synthetic nitrogen-centered DPPH and ABTS radicals are not
biologically relevant, but are often used as indicator compounds in
testing hydrogen transfer capacity that are related to the antioxi-
dant activity. The antioxidant properties were expressed as EC50
values (Table 3).
A well-recognized result of oxidant injury is peroxidation of
membrane lipids to organic peroxyl radicals, which initiates a chain
reaction that may explain many membrane-mediated effects of
reactive oxygen species (ROS). The concentration-dependent
inhibitory effects of indole derivatives (3a–c and 4a–e) on LP in
Table 3
EC50 values of 3a–c and 4a–e in antioxidant properties.
HN
R1
S
O
N
R2
EC50 (mM)
Compound
R
1
R
2
Anti-LP
DPPH
ABTS
Reducing Power
a
a
c
d
c
c
d
c
a
b
a
a
b
a
a
a
d
d
a
a
3
3
3
4
4
4
4
4
a
b
c
a
b
c
CH
Cl
NO
CH
3
H
H
H
CH
CH
CH
CH
CH
0.041 ꢄ 0.001
0.049 ꢄ 0.004
0.087 ꢄ 0.001
0.034 ꢄ 0.001
0.084 ꢄ 0.004
0.075 ꢄ 0.002
0.034 ꢄ 0.002
0.092 ꢄ 0.001
–
1.30 ꢄ 0.13
1.78 ꢄ 0.06
1.30 ꢄ 0.10
0.98 ꢄ 0.14
1.62 ꢄ 0.06
1.24 ꢄ 0.11
1.01 ꢄ 0.15
1.27 ꢄ 0.15
0.36 ꢄ 0.04
0.35 ꢄ 0.07
1.02 ꢄ 0.01
1.03 ꢄ 0.05
1.63 ꢄ 0.04
0.98 ꢄ 0.14
1.37 ꢄ 0.05
1.14 ꢄ 0.14
1.04 ꢄ 0.11
1.34 ꢄ 0.04
1.20 ꢄ 0.03
1.20 ꢄ 0.01
1.36 ꢄ 0.13
a
b
0.51 ꢄ 0.11
c
d,e
2
0.80 ꢄ 0.05
a
b,e
3
3
3
3
3
3
0.70 ꢄ 0.17
d
a,e
a
f
CF
Cl
3
O
4.94 ꢄ 0.06
c
4.02 ꢄ 0.15
c
d
e
Br
NO
4.22 ꢄ 0.02
d
e
f
2
5.14 ꢄ 0.38
g
Ascorbic acid
-Tocopherol
0.65 ꢄ 0.10
e
e
a
a
0.153 ꢄ 0.001
1.95 ꢄ 0.04
ꢀ
ꢀ
þ
EC50 value: The effective concentration at which the antioxidant activity was 50%; DPPH and ABTS radicals were scavenged by 50%, respectively, and the absorbance was 0.5
for reducing power. EC50 values were obtained by interpolation from linear regression analysis. Values were the means of three replicates ꢄ SD. Values with different letters in
the same column were significantly (p < 0.05) different.

1072
N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
liposomes were estimated by the amount of thiobarbituric acid
reactive substances (TBARS). -Tocopherol was used as reference
antioxidant. -Tocopherol is the most important lipophilic radical
R
1
-chlorine substituted 4c (1.14 ꢄ 0.14 mM) and
1
R -brome
a
substituted 4d (1.04 ꢄ 0.11 mM) showed similar (p > 0.05) degrees
of efficacy in their scavenging activities against ABTS radical cation,
which were the highest among the tested compounds. The EC50
values of these compounds are lower than the values observed for
a
chain-breaking antioxidant in living organism. As can be seen in
Table 3, the whole of the tested compounds exhibit potent inhibi-
tory effect on the Fe3 /ascorbic acid-induced LP, which is almost
þ
ascorbic acid (1.20 ꢄ 0.03 mM) and
Nevertheless, in R
-nitro substituted 3c (1.63 ꢄ 0.04 mM), R
-nitro
a
-tocopherol (1.20 ꢄ 0.01 mM).
2
–4 times higher than that of
on the EC50 values (effective concentration at which TBARS were
reduced to 50% with respect to controls), R -methyl substituted
compound 4a and R -brome substituted compound 4d incorpo-
rating methyl at R were significantly (p < 0.05) better inhibitors
than other indole derivatives. The EC50 values were 0.034 ꢄ 0.001
and 0.034 ꢄ 0.002 mM, for 4a and 4d, respectively. R -methyl
substituted 3a and R -chlorine substituted 3b were the next most
a
-tocopherol (0.153 ꢄ 0.01 mM). Based
1
1
-tri-
fluoromethoxy substituted 4b (1.37 ꢄ 0.05 mM) and
R
1
1
substituted 4e (1.34 ꢄ 0.04 mM) the activity was decreased some-
1
what when compared with other compounds.
2
In ABTS radical cation scavenging method, the antioxidant
activity of tested compounds was expressed also as the TEAC, the
concentration of Trolox solution having an antioxidant activity
equivalent to 1 mM concentration of the tested compounds. The
TEAC reflects the ability of hydrogen or electron-donating antiox-
idants to scavenge the ABTS radical cation compared with that of
Trolox. TEAC values were found to be 2.01 ꢄ0.010, 2.02 ꢄ 0.020,
2.04 ꢄ 0.045, 1.95 ꢄ 0.005, 2.00 ꢄ 0.027, 2.07 ꢄ 0.022, 2.06 ꢄ 0.070,
2.00 ꢄ 0.027 and 1.94 ꢄ 0.011 mM Trolox for 3a–c and 4a–e,
respectively. These values were not significantly different (p < 0.05)
from that of ascorbic acid (2.13 ꢄ 0.004 mM Trolox) and
1
1
effective indole derivatives with EC50 values of 0.041 ꢄ0.001 and
0
4
0
.049 ꢄ 0.004 mM (p < 0.05), followed by R
c and R -trifluoromethoxy substituted 4b with EC50 values of
.075 ꢄ 0.002 and 0.084 ꢄ 0.004 mM (p < 0.05), respectively.
-nitro substituted compounds 3c and 4e show the lowest
1
-chlorine substituted
1
R
1
inhibitory effect. The decrease in antioxidant activity on LP is
related to their electronic distribution and lipophilicity. EC50 values
of these compounds are 0.087 ꢄ 0.001 and 0.092 ꢄ 0.001 mM
a-tocopherol (2.02 ꢄ 0.011 mM Trolox) at 3.1 mM.
(p < 0.05), respectively. These results suggest that the indole
derivatives may prevent the formation of toxic carbonyl
compounds such as lipid peroxides and their aldehyde decompo-
sition products and may play an important role in protection
against damage to membrane function.
2.2.4. Reducing power
In the ferric to ferrous reduction assay, the electron donation
capacity (reflecting the electron transfer ability) of the compounds
was assessed. From the EC50 (the effective concentration at which
the absorbance was 0.5) it was seen that R
3b (0.51 ꢄ0.11 mM), R -methyl substituted 4a (0.70 ꢄ 0.17 mM),
-nitro substituted 3c (0.80 ꢄ 0.05 mM) and -methyl
1
-chlorine substituted
2
.2.2. DPPH radical scavenging activity
Although the DPPH radical scavenging abilities of all the indole
1
R
1
R
1
derivatives were significantly lower than those of ascorbic acid
substituted 3a (1.36 ꢄ 0.13 mM) showed the highest degree of
electron transfer capacity, indicating the highest antioxidant
potential amongst all the indole derivatives under study. The
capacities of these compounds were generally inferior to ascorbic
acid (0.65 ꢄ 0.10 mM), whereas these values are significantly
(
0.36 ꢄ 0.04 mM) and
a-tocopherol (0.35 ꢄ 0.07 mM), it was
evident that they show reducing ability (possibly by hydrogen atom
transfer) and could serve as free-radical scavengers. Amongst the
3
a (1.30 ꢄ 0.13 mM), 3c (1.30 ꢄ 0.10 mM), 4a (0.98 ꢄ 0.14 mM), 4c
(
1.24 ꢄ 0.11 mM), 4d (1.01 ꢄ0.15 mM) and 4e (1.27 ꢄ 0.15 mM),
higher than the values observed for
a
-tocopherol (1.95 ꢄ 0.04 mM)
there were no significant differences (p > 0.05) in their activities in
scavenging DPPH radicals as shown by the small differences of the
EC50 values (the effective concentration at which the DPPH radicals
were scavenged by 50%). The DPPH radical scavenging activities of
(Table 3). When these data are examined, it is observed that the
substitution of the methyl group at R caused significantly
decrease in reducing power, whereas in 4a incorporating methyl
both at R and R the activity is 2 times higher than R -methyl
2
1
2
1
3
b and 4b were comparable (p > 0.05) with EC50 values of
substituted 3a. 4b–e had the lowest reducing powers of
4.94 ꢄ 0.06, 4.02 ꢄ 0.15, 4.22 ꢄ 0.02 and 5.14 ꢄ 0.38, respectively.
The data from the iron reduction assay suggests that all the indole
derivatives are able to undergo electron transfer to oxidizing
species. Therefore, they should be able to reduce reactive radicals
as well, converting them into more stable and unreactive species.
1
.78 ꢄ 0.06 and 1.62 ꢄ 0.06 mM, respectively. These values are
significantly higher (p < 0.05) than the values observed for the
compounds mentioned above, indicating lower scavenging activity.
2.2.3. Total radical antioxidant potential (TRAP)
From the EC50 values (the effective concentration at which the
ABTS radicals were scavenged by 50%) estimated from the dose–
response curves for the compounds, it can be seen that R -methyl
substituted 3a (1.02 ꢄ 0.01 mM), -chlorine substituted 3b
1.03 ꢄ 0.05 mM), -methyl substituted 4a (0.98 ꢄ 0.14 mM),
2.3. Anticancer activity
1
R
1
Compounds 3b and 4b were selected by the National Cancer
Institute for screening against anticancer activity. A primary
(
R
1
Fig. 6. One dose Mean Graph for CNS cancer cell lines of 3b.

N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
1073
Fig. 7. One dose Mean Graph for CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer cell lines of 4b.
anticancer assay was performed according to the protocol of the
Drug Evaluation Branch of the National Cancer Institute, Bethesda
ring, a growth percentage of 55.80% was obtained for a CNS
cancer cell line SNB-75. The substitution of the methyl group at
position 1 and the trifluoromethoxy group at position 5 of the
indole ring caused an increase in cytotoxicity against renal and
breast cancer cell lines. For compound 4b, growth percentages
were found to be 55.27% for a CNS cancer cell line SNB-75, 58.33%
for a renal cancer cell line UO-31 and 59.99% for a breast cancer
cell line T-47D.
[
33–36]. The cytotoxic and/or growth inhibitory effects of the
ꢁ
5
compounds were tested in vitro at a single dose (10 M) against
the full panel of 60 human tumour cell lines derived from nine
neoplastic diseases. The growth percentage was evaluated spec-
trophotometrically and compared to controls not treated with test
agents. A 48 h continuous drug exposure protocol was followed
and a sulphorhodamine B (SRB) protein assay was used to esti-
mate cell viability or growth. The cell lines used in the National
Cancer Institute screen were leukemia, non-small cell lung cancer
3. Conclusion
(
NSCL), colon cancer, central nervous system (CNS) cancer,
New spiroindolinones incorporating the benzothiazole moiety
were synthesized. The structures were confirmed by elemental
analyses, spectrometry and single crystal X-ray analysis. The new
compounds were evaluated for antioxidant activity. The results
showed that all the indole derivatives had a high degree of
potency in inhibiting lipid peroxidation and demonstrated strong
scavenging activity against the DPPH and ABTS radicals. Hence,
these compounds can be considered as the best antioxidants
amongst all. The antioxidant properties increased due to the
melanoma, ovarian cancer, renal cancer, prostate cancer and
breast cancer cell lines. Mean Graphs were constructed for each
effect, with bars depicting the deviation of individual tumour cell
lines from the overall mean value for all the cells tested. In the
Mean Graph the center point is the mean of all growth inhibition
ꢀ
ꢀ
þ
(
GI) percentages over all cell lines. Bars that point to the right are
cell lines where the inhibition is greater than the average, while
bars that point to the left are cell lines where the inhibition is less
than the average.
substitution of the methyl or halogen at
1
R on the spi-
Single dose Mean Graphs of compound 3b for CNS cancer cell
lines and compound 4b for CNS cancer, melanoma, ovarian
cancer, renal cancer, prostate cancer and breast cancer cell lines
are shown in Figs. 6 and 7, respectively. For compound 3b, it was
observed that incorporating a chlorine at position 5 of the indole
roindolinones. In fact, among the tested compounds, the most
active compounds were 3a, 3b and 4a incorporating methyl or
chlorine at R . Generally, replacement of these groups at R with
1 1
trifluoromethoxy or nitro has been found to yield weak active
compounds. Interestingly, the substitution of the methyl group at

1074
N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
0
0
0
0
R
2
caused significantly decrease in reducing power (except 4a),
whereas in the anti-LP, DPPH and ABTS assays, there were only
minor variations in activities of -methyl substituted
compounds. However, 4a incorporating methyl both at R and R
4.3.2. 5 -Chloro-3H-spiro[1,3-benzothiazole-2,3 -indol]-2 (1 H)-one (3b)
ꢂ
Yellow crystal; yield 60%; m.p. 219 C; IR (KBr):
y
3302 (NH),
(ppm): 6.54 (d, 1H,
–H), 6.63 (t, 1H, J ¼ 7.63 Hz, benzothia. C
H), 6.85 (d, 1H, J ¼ 8.23 Hz, ind. C –H), 6.91 (dt, 1H, J ¼ 7.63, 1.22 Hz,
benzothia. C –H), 7.28 (s,
–H), 7.04 (d, 1H, J ¼ 7.32 Hz, benzothia. C
–H), 7.52
–H), 10.48 (s, 1H, ind. NH). LCMS-APCI (þ)
1
R
2
1714 (C]O); H NMR (DMSO-d
6
, 500 MHz) d
1
2
J ¼ 7.58 Hz, benzothia. C
4
6
–
was found to be the most potent antioxidant described in this
study. Although the indole derivatives under study were found to
be effective antioxidant candidates in these systems, their
potential exploitable beneficial effects and safety in humans need
to be proven in clinical trials.
7
5
7
1H, benzothia. NH), 7.33 (dd, 1H, J ¼ 8.23, 2.13 Hz, ind. C
6
(d, 1H, J ¼ 2.14 Hz, ind. C
4
þ
m/z (%): 289 (MH , 40), 287 (100). Analyses (%) Calcd for
14 9 2
C H ClN OS (288.75): C, 58.23; H, 3.14; N, 9.70. Found: C, 58.34; H,
2.78; N, 9.59.
4
. Experimental section
0
0
0
0
4
.3.3. 5 -Nitro-3H-spiro[1,3-benzothiazole-2,3 -indol]-2 (1 H)-one
4
.1. Chemistry
(3c)
ꢂ
Orange powder; yield 71%; m.p. 212–3 C; IR (KBr):
y
3354 (NH),
(ppm): 6.61 (d, 1H,
–H), 6.69 (t, 1H, J ¼ 7.68 Hz, benzothia. C
H), 6.95 (t,1H, J ¼ 7.67 Hz, benzothia. C –H), 7.07 (d, J ¼ 8.63 Hz, ind.
–H), 7.10 (d, 1H, J ¼ 7.35 Hz, benzothia. C –H), 7.33 (s, 1H, ben-
zothia. NH), 8.25 (dd, 1H, J ¼ 8.63, 2.24 Hz, ind. C –H), 8.27 (d, 1H,
Soybean lecithin (L-
a
-phosphatidylcholine Type IV-S), 2,2-
1
ꢀ
6
1732 (C]O); H NMR (DMSO-d , 500 MHz) d
diphenyl-1-picrylhydrazyl (DPPH ),
5
5
a-tocopherol, ascorbic acid,
J ¼ 7.99 Hz, benzothia. C
4
6
–
-methylisatin, 5-(trifluorometoxy)isatin, 5-chloroisatin and
-nitroisatin were purchased from Sigma–Aldrich Chemical Co. (St.
5
0
C
7
7
Louis, MO, USA). Butylated hydroxytoluene (BHT), 2,2 -azino-bis(3-
ꢀ
þ
6
ethylbenzthiazoline-6-sulphonic acid) (ABTS ) diammonium salt,
-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox),
J ¼ 2.24 Hz, ind. C
4
–H), 11.07 (s, 1H, ind. NH). Analyses (%) Calcd for
S (299.30): C, 56.18; H, 3.03; N, 14.04. Found: C, 56.20; H,
.03; N, 13.63.
6
C
3
14 9 3 3
H N O
sodium hydride (NaH), 5-bromoisatin and 2-aminothiophenol
were purchased from Fluka Chemical Co. (Bushs, Switzerland).
Potassium ferricyanide, thiobarbituric acid (TBA), trichloroacetic
acid (TCA), ferric chloride and iodomethane were purchased from
Merck (Darmstadt, Germany). All other chemicals used were of
analytical grade. Melting points were estimated with a Buchi 540
melting point apparatus in open capillaries and are uncorrected.
Elemental analyses were performed on a Thermo Finnigan Flash EA
0 0
.4. The synthesis of 1 -methyl-3H-spiro[1,3-benzothiazole-2,3 -
4
0
0
indol]-2 (1 H)-ones (4a–e)
To a solution of 5-substituted 1-methyl-1H-indole-2,3-diones 2
3.5 mmol) in ethanol (15 mL) was added 2-aminothiophenol
(3.5 mmol). The mixture was refluxed on a water bath for 5 h. The
product formed after cooling was filtered and recrystallized from
ethanol.
(
1112 elemental analyzer. IR spectra were recorded on KBr discs,
1
using a Perkin–Elmer Model 1600 FT-IR spectrometer. H NMR and
UNITY
HSQC spectra were obtained on Varian
photometers using DMSO-d
AGILENT 1100 MSD instrument.
INOVA 500 spectro-
6
. Mass spectra were determined on an
0
0
0
0
4
.4.1. 1,5 -Dimethyl-3H-spiro[1,3-benzothiazole-2,3 -indol]-2 -one (4a)
ꢂ
Brown powder; yield 41%; m.p. 182–3 C; IR (KBr):
y
3303 (NH),
(ppm): 2.28 (s, 3H,
), 6.52 (d, 1H, J ¼ 7.32 Hz, benzothia.
–H), 6.90 (dt, J ¼ 7.62,
–H), 6.92 (d, J ¼ 7.93 Hz, 1H, ind. C –H),
–H), 7.18 (d, 2H,
–H, benzothia. NH), 7.41 (s, 1H, ind. C –H). HSQC
), 26.91 (N–CH ), 74.80 (spiro
), 109.46 (ind. C ), 119.40 (benzothia. C ),
), 124.77 (ind. C3a), 126.41 (ind. C ), 126.46
), 130.04 (ind. C ), 131.43 (ind. C ), 132.96 (benzothia.
7a), 141.04 (ind. C7a), 147.68 (benzothia. C3a), 175.12 (ind. C ).
Analyses (%) Calcd for C16 OS (282.36): C, 68.06; H, 5.00; N,
9.92. Found: C, 68.14; H, 5.13, N, 9.57.
4
.2. The synthesis of 5-substituted 1-methyl-1H-indole-2,3-diones
1
1
696 (C]O); H NMR (DMSO-d
6
, 500 MHz)
d
(2a–e)
3 3
ind. 5-CH ), 3.08 (s, 3H, N–CH
C
1
7
4
–H), 6.63 (t, 1H, J ¼ 7.32 Hz, benzothia. C
6
A
suspension of 5-substituted 1H-indole-2,3-diones 1a–e
.22 Hz, 1H, benzothia. C
5
7
(
(
5 mmol) and NaH (60% suspension in oil) (0.2 g) in anhydrous DMF
5 mL) was stirred for 0.5 h at room temperature. After addition of
.03 (dd, J ¼ 7.62, 1.22 Hz, 1H, benzothia. C
7
J ¼ 7.80 Hz, ind. C
6
4
iodomethane (15 mmol), the mixture was refluxed for 4 h. The
product was poured onto ice and water, and then filtered.
(
DMSO-d
6
)
d
(ppm): 21.19 (ind. 5-CH
3
3
C), 109.17 (benzothia. C
21.74 (benzothia. C
(benzothia. C
4
7
6
1
7
4
0
4
2
.3. The synthesis of 3H-spiro[1,3-benzothiazole-2,3 -indol]-
5
5
6
0
0
(1 H)-ones (3a–c)
C
2
14 2
H N
To a solution of 5-substituted 1H-indole-2,3-diones 1 (3.5 mmol)
in ethanol (15 mL) was added 2-aminothiophenol (3.5 mmol). The
mixture was refluxed on a water bath for 5 h. The product that was
formed after cooling was filtered and recrystallized from ethanol.
0
0
4.4.2. 1 -Methyl-5 -trifluoromethoxy-3H-spiro[1,3-benzothiazole-
0
0
2,3 -indol]-2 -one (4b)
Yellow crystal; yield 39%; m.p. 187–9 C; IR (KBr):
ꢂ
y
3307 (NH),
(ppm): 3.13 (s, 3H, N–
–H), 6.66 (dt, 1H, J ¼ 7.62,
–H), 6.93 (dt, 1H, J ¼ 7.87, 1.22 Hz, benzothia.
–H), 7.06 (d, 1H, J ¼ 8.54 Hz, ind. C –H), 7.15 (d, J ¼ 7.86 Hz, ben-
zothia. C
–H), 7.28 (s, 1H, benzothia. NH), 7.42 (dd, 1H, J ¼ 8.54,
1.83 Hz, ind. C –H). HSQC
–H), 7.55 (d, 1H, J ¼ 2.44 Hz, ind. C
(DMSO-d (ppm): 27.11 (N–CH ), 74.55 (spiro C), 109.43 (ben-
zothia. C ), 110.97 (benzothia. C ), 119.34 (ind. C ), 119.71 (benzo-
thia. C ), 120.85 (CF O), 121.89 (ind. C ), 124.45 (ind. C3a), 124.58
(ind. C ), 126.59 (benzothia. C ), 131.93 (ind. C7a), 142.64 (benzothia.
7a), 144.77 (ind. C
0
0
0
0
1
4
.3.1. 5 -Methyl-3H-spiro[1,3-benzothiazole-2,3 -indol]-2 (1 H)-
one (3a)
Yellow crystal; yield 74%; m.p. 216 C; IR (KBr):
1703 (C]O); H NMR (DMSO-d
CH
), 6.58 (d, 1H, J ¼ 7.93 Hz, benzothia. C
1.22 Hz, benzothia. C
6
, 500 MHz) d
3
4
ꢂ
y
3299 (NH),
(ppm): 2.25 (s, 3H, ind.
–H), 6.61 (dt, 1H,
6
1
1
5
711 (C]O); H NMR (DMSO-d
-CH
), 6.51 (d, 1H, J ¼ 7.62 Hz, benzothia. C
J ¼ 7.63, 1.22 Hz, benzothia. C –H), 6.71 (d, 1H, J ¼ 7.93 Hz, ind. C
H), 6.89 (dt, 1H, J ¼ 7.62, 1.22 Hz, benzothia. C
6
, 500 MHz)
d
C
5
7
3
4
7
6
7
–
6
4
5
–H), 7.01 (d, 1H,
6
)
d
3
J ¼ 7.62 Hz, benzothia. C
7
–H), 7.08 (dd, 1H, J ¼ 7.93, 1.83 Hz, ind. C
6
–
–H),
4
7
4
H), 7.22 (s, 1H, benzothia. NH), 7.36 (d, 1H, J ¼ 0.61 Hz, ind. C
4
6
3
7
þ
1
(
0.22 (s, 1H, ind. NH). LCMS-APCI (þ) m/z (%): 269 (MH , 6), 267
6
5
100). Analyses (%) Calcd for C15
12
H N
2
OS (268.33): C, 67.14; H, 4.51;
C
5
), 147.38 (benzothia. C3a), 175.18 (ind. C
2
). LCMS-
þ
N, 10.44. Found: C, 67.00; H, 4.35; N, 10.47.
APCI (þ) m/z (%): 353 (MH , 100). Analyses (%) Calcd for

N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
1075
C
16
H
11
F
3
N
2
O
2
S (352.33): C, 54.54; H, 3.15; N, 7.95. Found: C, 54.91;
was corrected for Lorentz-polarisation effects and for absorption
using empirical integration method.
H, 3.08; N, 8.01.
The structure of 4d was solved by direct methods program
SHELXS-97 [37] and refined by the full-matrix least-squares
method based on F using SHELXL-97 [38]. All non-hydrogen atoms
were refined with anisotropic displacement factors. The H atom of
2
N was found in the difference Fourier map, and its position and
isotropic thermal parameters were refined; remaining hydrogen
atoms were positioned geometrically and refined using a riding
model. The geometric calculations were carried out with the Platon
[39] program.
0
0
0
4
2
.4.3. 5 -Chloro-1 -methyl-3H-spiro[1,3-benzothiazole-2,3 -indol]-
0
2
-one (4c)
Dark yellow powder; yield 46%; m.p. 166 C; IR (KBr):
ꢂ
y
3307
(ppm): 3.10 (s,
–H), 6.65 (t, 1H,
–H), 6.92 (dt, 1H, J ¼ 7.62, 1.22 Hz, benzo-
–H), 7.07 (d, 1H, J ¼ 8.23 Hz, ind. C –H), 7.07 (d, 1H,
J ¼ 7.32 Hz, benzothia. C –H), 7.25 (s, 1H, benzothia. NH, D
exchange), 7.45 (dd, 1H, J ¼ 8.23, 2.13 Hz, ind. C –H), 7.57 (d, 1H,
J ¼ 2.12 Hz, ind. C –H). HSQC (DMSO-d (ppm): 27.08 (N–CH ),
4.56 (spiro C), 109.45 (benzothia. C ), 111.43 (ind. C ), 119.71
benzothia. C ), 121.89 (benzothia. C ), 124.54 (ind. C3a), 125.77 (ind.
), 126.57 (benzothia. C ), 127.75 (ind. C ), 131.07 (ind. C ), 132.12
benzothia. C7a), 142.34 (ind. C7a), 147.40 (benzothia. C3a), 174.88
ind. C ). Analyses (%) Calcd for C15 11ClN OS (302.77): C, 59.50; H,
.66; N, 9.25. Found: C, 59.36; H, 4.03; N, 9.19.
1
(
3
NH), 1698 (C]O); H NMR (DMSO-d
H, N–CH
), 6.56 (d, 1H, J ¼ 7.93 Hz, benzothia. C
J ¼ 7.47 Hz, benzothia. C
thia. C
6
, 500 MHz)
d
3
4
6
5
7
7
2
O
6
4
6
)
d
3
4.6. In vitro evaluation of antioxidant activity
7
(
C
(
(
4
7
6
7
4.6.1. Antioxidant activity on liposome peroxidation
4
5
5
6
The antioxidative effects of indole derivatives were quantified
3
þ
by using the inhibition of the Fe /ascorbate induced lipid perox-
idation in liposomes. Phosphatidylcholine (lecithin), a phospho-
lipid believed to be present in high amounts in cell membranes,
was used a liposome-like substrate.
2
H
2
3
0
0
0
4
2
.4.4. 5 -Bromo-1 -methyl-3H-spiro[1,3-benzothiazole-2,3 -indol]-
The lipid peroxidation assay was based on the method described
by Duh et al. [40]. Lecithin (300 mg) was suspended in 30 mL
phosphate buffer (10 mmol/L, pH 7.4). This suspension was soni-
cated with a rod using an ultrasonic homogenizer (Bandelin, Berlin,
Germany) at 30 s intervals for 10 min until an opalescent suspen-
sion was obtained.
0
-one (4d)
ꢂ
Orange crystal; yield 53%; m.p. 244–6 C; IR (KBr):
y
3327 (NH),
(ppm): 3.10 (s, 3H, N–
), 6.56 (d, 1H, J ¼ 7.32 Hz, benzothia. C –H), 6.65 (t, 1H,
–H), 6.92 (t, 1H, J ¼ 7.32 Hz, benzothia. C
H), 7.03 (d, 1H, J ¼ 8.29 Hz, ind. C –H), 7.06 (d, 1H, J ¼ 7.32 Hz,
benzothia. C
–H), 7.25 (s, 1H, benzothia. NH), 7.58 (dd, 1H, J ¼ 8.30,
.95 Hz, ind. C –H). HSQC
–H), 7.67 (d, 1H, J ¼ 1.95 Hz, ind. C
DMSO-d (ppm): 27.07 (N–CH ), 74.51 (spiro C), 109.45 (ben-
zothia. C ), 111.93 (ind. C ), 115.30 (ind. C
21.90 (benzothia. C ), 124.53 (ind. C3a), 126.58 (benzothia. C
1
1
CH
719 (C]O); H NMR (DMSO-d
6
, 500 MHz)
d
3
4
J ¼ 7.32 Hz, benzothia. C
6
5
–
7
The sonicated solution (10 mg/mL), FeCl
indole derivatives (0.025–0.4 mM) or the reference antioxidant
tocopherol (0.1–0.4 mM) were mixed to produce a final concen-
3
, ascorbic acid and the
7
a-
1
(
6
4
6
)
d
3
tration of 3.08 mg liposome/mL, 123.2
m
mol FeCl
3
and 123.2
m
mol
ꢂ
4
7
5
), 119.72 (benzothia. C
6
5
),
),
ascorbic acid. After 1 h incubation at 37 C, the formation of lipid
peroxidation products was assayed by the measurement of
malondialdehyde (MDA) levels on the basis that MDA reacted with
1
1
7
28.46 (ind. C
4
6
), 132.46 (benzothia. C7a), 133.93 (ind. C ), 142.35
(
ind. C7a),147.37 (benzothia. C3a),174.75 (ind. C
2
). Analyses (%) Calcd
TBA at 532 nm according to Buege and Aust [41]. Briefly, 500
this reaction mixture was mixed with 1000 L TCA–TBA reagent
(consisting of 15% w/v TCA and 0.375% TBA in 0.25 N HCl) and 14
mL of
for C15 11BrN OS (347.22): C, 51.89; H, 3.19; N, 8.07. Found: C, 52.12;
H, 3.59; N, 8.06.
H
2
m
mL
butylated hydroxytoluene (BHT) (2% in absolute ethanol). The
mixture was vortexed and heated for 10 min in a boiling water bath.
After cooling, an equal volume of n-butanol was added and the
mixture was shaken vigorously. The n-butanol layer was separated
by centrifugation at 3000 rpm for 5 min. The absorbance of the
sample was measured at 532 nm against a blank, which contained
all reagents except lecithin. The percentage inhibition of lipid per-
oxidation was calculated by comparing the results of the samples
with those of controls not treated with the extract using the
following equation: Inhibition effect (%) ¼ [1 ꢁ (Absorbance of
sample at 532 nm/Absorbance of control at 532 nm)] ꢃ 100.
0
0
0
0
4
.4.5. 1 -Methyl-5 -nitro-3H-spiro[1,3-benzothiazole-2,3 -indol]-2 -
one (4e)
Red powder; yield 75%; m.p. 211 C; IR (KBr):
C]O); H NMR (DMSO-d
ꢂ
y
3348 (NH), 1727
(ppm): 3.20 (s, 3H, N–CH ),
–H), 6.69 (t, 1H, J ¼ 7.32 Hz,
1
(
6
6
, 500 MHz)
d
3
.61 (d, 1H, J ¼ 7.80 Hz, benzothia. C
4
benzothia. C
6
–H), 6.95 (dt, 1H, J ¼ 7.81, 1.47 Hz, benzothia. C
5
–H),
7
7
8
d
.10 (d, 1H, J ¼ 7.81 Hz, benzothia. C
7
–H), 7.27 (s, 1H, benzothia. NH),
.29 (d, J ¼ 8.79 Hz, ind. C
7
–H), 8.29 (d, 1H, J ¼ 2.44 Hz, ind. C
4
–H),
.34 (dd, 1H, J ¼ 8.78, 2.44 Hz, ind. C
6 6
–H). HSQC (DMSO-d )
(ppm): 27.48 (N–CH
10.35 (ind. C ), 120.02 (benzothia. C
benzothia. C ), 124.35 (ind. C3a), 126.74 (benzothia. C
), 131.41 (benzothia. C7a), 143.78 (ind. C ), 147.07 (benzothia. C3a),
7a), 175.62 (ind. ). Analyses (%) Calcd for
S (313.33): C, 57.50; H, 3.54; N, 13.41. Found: C, 57.61; H,
.91; N, 13.60.
3
), 74.10 (spiro C), 109.74 (benzothia. C
), 120.90 (ind. C ), 122.04
), 128.13 (ind.
4
),
1
(
C
1
C
2
7
6
4
7
5
4.6.2. DPPH radical scavenging activity
6
5
The DPPH radical scavenging activities of the indole derivatives
were measured according to the procedure described by Brand-
49.39 (ind.
C
C
2
ꢀ
H
15 11
N
3
O
3
Williams et al. [42]. A purple-colored (DPPH ), is a stable free
radical, which is reduced to DPPH (yellow colored) by reacting with
an antioxidant. A 0.1 mL aliquot of each indole derivatives
4.5. Single crystal X-ray structure determination
(0.2–6.25 mM) in DMSO, ascorbic acid (0.1–0.8 mM) or
a-tocoph-
erol (0.1–0.8 mM) in absolute ethanol was added to 3.9 mL of
ꢁ5
Crystal of 4d of suitable qualityand size, was mounted on the ends
6 ꢃ 10 M methanolic solution of DPPH. The mixture was shaken
vigorously and allowed to stand in the dark at room temperature
for 30 min. The decrease in absorbance of the resulting solution was
then measured spectrophotometrically at 517 nm against meth-
anol. All measurements were made in triplicate and averaged. Two
controls were used for this test, a negative control (containing all
reagents except the test sample) and positive controls (using the
reference antioxidants). The ability to scavenge DPPH radical was
calculated by the following equation: DPPH radical scavenging
of glass and used for the intensity data collection. X-ray crystallo-
graphic measurements were carried out using an STOE IPDS 2 single
crystal diffractometer equipped with image plate detector. Graphite
monochromated MoK
a
radiation (
l
¼ 0.71073 Å) was generated at
5
0 kV and 40 mA. The cell parameters and the orientation matrices
for data collection were obtained from a least-squares refinement
using the setting angles of 10,163 carefully centered reflections in the
ꢂ
range 1.52 <
q
< 26.1 . A total of 7792 reflections were collected. Data

1076
N. Karalı et al. / European Journal of Medicinal Chemistry 45 (2010) 1068–1077
activity (%) ¼ [1 ꢁ (Absorbance of sample at 517 nm/Absorbance of
control at 517 nm)] ꢃ 100.
50% (w/v) TCA (final concentration, 10% TCA) and incubated for
60 min at 4 C. The supernatant was discarded, and the plates were
washed five times with tap water and air-dried. Sulphorhodamine B
ꢂ
4.6.3. Total radical antioxidant potential (TRAP)
(SRB) solution (100 mL) at 0.4% (w/v) in acetic acid was added to each
The total radical antioxidant potential of indole derivatives was
well, and plates were incubated for 10 min at room temperature.
After staining, unbound dye was removed by washing five times
with 1% acetic acid and the plates were air-dried. Bound stain was
subsequently solubilized with 10 mM trizma base, and the absor-
bance was measured on an automated plate reader at a wavelength
of 515 nm. For suspension cells, the methodology was the same
except that the assay was terminated by fixing settled cells at the
measured using TEAC assay as described by Re et al. [43] with
minor modifications.
ꢀ
þ
After addition of 990
derivatives (0.2–6.25 mM), ascorbic acid (0.2–3.1 mM),
pherol (0.2–3.1 mM) or Trolox standards (final concentration
–20 M in ethanol), the decrease in absorbance at 734 nm was
m
L of ABTS solution to 10
m
L of indole
a-toco-
0
m
monitored exactly 6 min after the initial mixing. Appropriate
ethanol blanks were run in each assay. All determinations were
carried out in triplicate. The ability to scavenge ABTS radical was
calculated by the following equation: ABTS radical scavenging
activity (%) ¼ [1 ꢁ (Absorbance of sample at 734 nm/Absorbance of
control at 734 nm)] ꢃ 100.
bottom of the wells by gently adding 50 mL of 80% TCA (final
concentration, 16% TCA). Using the seven absorbance measure-
ments [time zero, (Tz), control growth, (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the
percentage growth was calculated at each of the drug concentra-
tions levels. The percentage growth inhibition was calculated as:
ꢀ
þ
½
ðTi ꢁ TzÞ=ðC ꢁ TzÞꢅ ꢃ 100 for concentrations for which Ti ꢆ Tz
ðTi ꢁ TzÞ=Tzꢅ ꢃ 100 for concentrations for which Ti< Tz
4
.6.4. Reducing power
The reducing powers of the indole derivatives, ascorbic acid and
-tocopherol were determined according to the method described
a
½
by Chung, et al. [44]. A 0.1 mL aliquot of each indole derivative (1.6–
6
6
.25 mM), ascorbic acid (0.4–3.1 mM) or
.25 mM) were mixed with an equal volume of 0.2 M phosphate
a-tocopherol (0.4–
Acknowledgements
buffer (pH 6.6) and 1% potassium ferricyanide, and then incubated
at 50 C for 20 min 0.25 mL of 1% trichloroacetic acid was added to
the mixture to stop the reaction, and then the mixture was
centrifuged at 2790 g for 10 min. The supernatant (0.25 mL) was
3
mixed with 0.25 mL of distilled water and 0.1% of FeCl (0.5 mL) and
ꢂ
We thank the Drug Research and Development, Division of
Cancer Research, National Cancer Institute, Bethesda, Maryland, for
the anticancer activity screening of the compounds. This work was
supported by Istanbul University Research Fund, Projects Number:
BYP-1895.
then the absorbance was measured at 700 nm. The reducing
powers of the tested samples increased with the absorbance values.
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