Synthesis, cytotoxicity, QSAR, and intercalation study of new diindenopyridine derivatives

Article abstract of DOI:10.1016/j.bmc.2004.03.032

Seven new derivatives of diindenopyridine were synthesized by Hantsch pyridine synthesis. Their biological activity to inhibit cell proliferation was assessed by MTT assay on seven cell lines. 11-(4-Fluoro-phenyl)-diindeno[1,2- b;2′,1′-e]pyridine-10,12-di

Full text of DOI:10.1016/j.bmc.2004.03.032

Bioorganic & Medicinal Chemistry 12 (2004) 2529–2536
Synthesis, cytotoxicity, QSAR, and intercalation
study of new diindenopyridine derivatives
a,b
Ramin Miri,a,b, Katayoun Javidnia, Bahram Hemmateenejad,a Ali Azarpirab
*
and Zahra Amirghofranc
aMedicinal and Natural Products Chemistry Research Centre, Shiraz University of Medical Sciences, PO Box 71345-1149 Shiraz, Iran
bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
cDepartment of Immunology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Received 8 January 2004; revised 15 March 2004; accepted 15 March 2004
Available online 9 April 2004
Abstract—Seven new derivatives of diindenopyridine were synthesized by Hantsch pyridine synthesis. Their biological activity to
inhibit cell proliferation was assessed by MTT assay on seven cell lines. 11-(4-Fluoro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-10,12-
dione and 11-(2-nitro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-10,12-dione were active on K-562 cell line with IC50 values of 79.66 and
78.2 lM, respectively. Effect of structural parameters on the cytotoxicity was evaluated by quantitative structure activity relationship
(QSAR) analysis and a linear relationship was found between the )log IC15 of these compounds and their surface area and molar
refractivity. To model the DNA-intercalator complex, force field molecular mechanic calculation was employed and the binding
energy of the reaction between the intercalating agent and each reasonable double base pairs of DNA was calculated. It was found
that these molecules could intercalate into the DNA. Also, it was observed that 11-(2-nitro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-
10,12-dione, which showed the highest activity in K-562 cell line, produced the most negative binding energy with a moderate
selectivity toward A–G/T–C double base pairs.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
9-hydroxy-2-methyl ellipticinium acetate in clinic has
been reported.5 Derivatives of tetrahydropyrrolo[3,4-a]-
carbazole-1,3-dione and tetrahydropyrido[3,2-b]pyr-
rolo[3,4-g]indole-1,3-dione were screened for cytotoxi-
city, DNA interaction, and topoisomerase II inhibition
potency.6 Some of the N-(alkylamino) alkyl derivatives
of pyrimido[5,6,1-de]acridine-1,3,7-trione have shown
borderline in vivo antitumor activity.7 5,11-Dimethyl-
5H-indole [2,3-b] quinolin displayed a strong antibac-
terial, antimycotic, and cytotoxic activity as well as
significant in vivo antitumor properties.8 New series of
3,5-bis(30-indolyl)pyrazines and 2,4-bis(30-indolyl)pyr-
imidines had a great cytotoxicity against diverse human
cell lines.9 New class of tetracyclic 11-oxo-11H-
indeno[1,2-b]quinoline-6-carboxamide was reported as
potent cytotoxins and potential dual topoisomerase I
and II inhibitors.10 Two common characteristics of these
compounds include the presence of a planar polycyclic
chromophore, able to intercalate into DNA, and at
least one basic side chain, which can increase DNA
binding affinity and in some cases increase solubility
under physiological conditions. Great efforts have
been devoted to modify and determine its optimal
The discovery and development of novel therapeutic
agents for the treatment of malignancy has a vital
importance. The tremendous potential advantages and
challenges associated with the use of a molecular
approach to cancer drugs have been reviewed.1 Efforts
to understand the basis of antitumor activity of anth-
racyclines naturally included a focus on the impact of
drug into DNA double helix structure. Anthracyclines
and DNA intercalators represent one of the most
important classes of anticancer drugs in their overall
utility in clinical oncology.2 Doxorubicin, daunorubicin,
and amsacrin are now being used for treatment in a wide
range of tumors.2 Camptothecin, isolated from a Chi-
nese tree, is a potent topoisomerase I inhibitor.3 Asci-
didemin,
a natural pentacyclic aromatic alkaloid
has toxicity toward topoisomerase II.4 The use of
Keywords: Diindenopyridine; Cytotoxicity; QSAR; Intercalation.
* Corresponding author. Tel.: +98-711-2290091; fax: +98-711-23038-
72; e-mail: mirir@sums.ac.ir
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.032

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R. Miri et al. / Bioorg. Med. Chem. 12 (2004) 2529–2536
characteristics. Results indicate that the planar portion
of the molecule could be tricyclic, tetracyclic, or even
pentacyclic. Although some data indicates a positive
relationship between DNA binding and antitumor
activity, it is normally accepted that DNA intercalation
is a necessary but not sufficient condition for antitumor
activity.11 Also it has been pointed out that DNA
binding, while possibly improve intrinsic activity, can be
even deleterious to normal distribution and penetration
into the cells.11;12
have been found as powerful forces in modern organic
chemistry.27 They are widely used and highly respected,
but, they have also contributed to some of the erroneous
ideas. These methods have been applied to assess the
reaction of intercalating agents such as metal com-
plexes,20;21 indolo[2,3-b]quinoline derivatives,22 Troger
bases,23 quinones,24 anthramycin derivatives,25 acrydine
orange,26 and with DNA. In this paper, we investigated
the intercalation reaction between the synthesized diin-
denopyridine molecules and different reasonable double
base pairs of DNA by the AMBER force field method.
Geita and Vang, in 1962, synthesized four derivatives
of 11-R-diindeno[1,2-b;20,10-e]pyridine-10,12-dione in
which R were methyl, butyl, isopropyl, and isobutyl.13
The structure of these compounds are similar to that of
intercalating agents, which recently synthesized and
evaluated as cytotoxic molecules.3–10;14 According to
these data, we designed and synthesized some new
In addition, a quantitative structure activity relationship
27–32
(QSAR) study
has been conducted to analyze the
effect of various structural parameters of the molecule
on its cytotoxic activity.
derivatives
of
11-R-diindeno[1,2-b;20,10-e]pyridine-
2. Results and discussion
10,12-dione in which R were different substituted phenyl
(linear pentacyclic structures) and their cytotoxicity was
evaluated by MTT assay.15
2.1. Synthesis and antitumor assay
Diindenopyridine analogues (9a–g) were obtained by
photo degradation of related intermediate products,
synthesized by classical Hantsch condensation method,
in which aryl (alkyl) carboxaldehyde (2–7) reacted with
indandione (1) and ammonium acetate (Fig. 1).
There are reports of both experimental techniques such
as spectrophotometry,16 spectroflurimetry,17 NMR,18 X-
ray crystallography,19 and theoretical techniques such as
molecular modeling20–26 for studying the interaction of
intercalators with DNA. Considering the limitations as
well as the difficulty of some experimental techniques,
molecular modeling, and theoretical organic chemistry
The data of cytotoxicity is shown in Table 1. IC50 of all
of the compounds were more than 100 lM, whereas 9d
O
O
R
N
H
8a-8g
O
EtOH
+
RCHO + ACOH CH COO NH
/
3 4
2
O
1
2-7
O
R
N
O
O
R
O
hν
N
H
8a-8g
9a-9g
Figure 1. Synthesis of diindenopyridine derivatives.
Table 1. Cell growth inhibitory activity of compounds 9a–g in vitroa
IC15 (lM)
Compound
R
K-562
Fen
3.7
Vero
Hela
KB
SK-BR-3
U-937
9a
9b
9c
9d
9e
9f
Methyl
Phenyl
21.4
17.1
20.0
3.3
>100.0
>100.0
43.8
>100.0
>100.0
23.4
16.0
>100.0
6.7
>100.0
5.3
100.0
2.1
5.1
2.1
>100.0
28.6
3-Fluoro-phenyl
4-Fluoro-phenyl
2-Nitro-phenyl
4-Nitro-phenyl
<1.0
>100.0
14.3
>100.0
7.5
30.2
36.1
9.0
2.9
3.4
1.5
3.6
35.1
<1
17.7
9.5
1.3
50.3
>100.0
25.1
1.3
46.3
20.4
>100.0
60.0
<1.0
9g
4-Dimethylamino-phenyl 17.2
4.0
<1.0
>100.0
>100.0
<1.0
>100.0
Doxorubicin ––
DMSO ––
<1.0
80.5
1.4
>100.0
28.3
>100.0
>100.0
>100.0
a IC15 is the molar concentration causing 15% growth inhibition of tumor cells. In K-562, IC50 of 9d and 9e was 79.66 and 78.2 lM, respectively.

R. Miri et al. / Bioorg. Med. Chem. 12 (2004) 2529–2536
2531
and 9e in K-562 cell line had an IC50 equivalent to 79.66
and 78.2 lM, respectively. According to the results, the
most cytotoxic effect was seen on K-562 cell line and
among the compounds, 9d and 9e were more active.
Possibly the presence of electrophile moiety at ortho or
para position of the diindenopyridine structure increases
it’s lethal activity on this cell line.
3D structural representations, obtained by AM1 meth-
od, are shown in Figure 2 and some of their electronic
features are summarized in the first five rows of Table 2.
Angle between the phenyl rings at the ends of the di-
indenopyridine plane, (a), is around zero so, almost all
of the compounds have a flat structure. Meanwhile, the
angle between the plane of the C4 substituted phenyl
(9b–g) and diindenopyridine plane, (b), is about 50ꢁ for
ortho substituted phenyl and about 90ꢁ for para. The
dipole moment (DM) of the molecules are also included
in Table 2. Molecule with para-nitro substituent (9f) was
the most polar molecule whereas molecule with para-
dimethylamino substituent (9g) had the least polarity.
The polarity of the most active molecule in K-562 (9e) is
lower than that of 9f but is greater than the polarity of
the others except for 9d.
2.2. QSAR analysis
For QSAR analysis two types of descriptors were used,
which are listed in Table 2. Lipophilicity index (log P),
surface area (SA), volume (V ), hydration energy (HE),
polarizability (Pol), and molar refractivity (MR) are
those descriptors, which were calculated from the whole
structure of the molecule. The rest are substituent con-
stants parameters including electronic (r), hydrophobic
(p), and STERIMOL parameters (L, B1, B5, L=B1, and
B5=B1). These parameters are used to account the
property of the substituent and are derived for an indi-
vidual substituent on a molecule. Among the molecules,
9b–g had an aromatic group on C4 of the pyridine
but 9a contained a methyl group. Therefore, the sub-
stituent constant parameters are not shown for 9a in
Table 2.
Figure 2. The three dimensional representation of diindenopyridine
derivatives.
Second QSAR analysis was performed by using the
substituent constant parameters of the molecules 9b–g
and the following equation was obtained:
The QSAR analysis was performed three times. First, all
of the molecules with the first type of descriptors were
analyzed. The results are shown by Eq. 1:
Log1=IC15 ¼ 0:408ðꢁ0:084Þr þ 4:992ðꢁ0:080Þ
ð2Þ
N ¼ 6; R ¼ 0:925; Se ¼ 0:167; F ¼ 23:7
Among the substituent constants used in this paper,
only the Hammet’s electronic parameter was used by
Eq. 2. This parameter was used to account the electron
withdrawing or donating ability of the substituents by
Hammet. The positive sign of the coefficient of this
parameter describes that by increasing the r, the cyto-
toxicity of the compounds under study is increased.
Referring to the values of r in Table 2 reveals that the
electronic substituent constant of the electron with-
drawing groups such as –F and –NO2 is greater than
that of electron donating groups such as N(CH3)2, and
according to the above discussion, the electron with-
drawing groups increase the activity of the compounds.
Therefore, there is a consistency between the derived
QSAR models.
Log1=IC15 ¼ ꢀ0:160ðꢁ0:040ÞHE þ 4:272ðꢁ0:251Þ;
N ¼ 7; R ¼ 0:850; Se ¼ 0:231; F ¼ 13:0:
ð1Þ
This equation implies that hydration energy of the
molecules affected their antitumor activity. The negative
sign of the coefficient of the HE proposes that more
negative hydration energy are relevant to antitumor
activity. As the polar functional groups produce nega-
tive hydration energy, it can be concluded that presence
of such functional group on the intercalating agents
increase their antitumor activity.

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R. Miri et al. / Bioorg. Med. Chem. 12 (2004) 2529–2536
Table 2. The electronic and structural features of diindenopyridine derivatives
Property
9a
9b
9c
9d
9e
9f
9g
E (kcal/mol)
DHf (kcal/mol)
DM (D)
a (ꢁ)
)4172.0
51.0
)5091.6
90.0
)5101.9
46.5
)5103.8
44.6
)5264.3
97.4
)5268.0
93.7
)5798.7
98.2
3.96
0.02
3.54
3.41
5.31
5.26
9.76
1.80
1.00
53.66
568.90
976.14
)4.70
4.94
0.03
90.00
570.13
974.65
)4.69
5.08
)1.15
52.72
561.74
976.17
)4.59
5.08
)0.10
91.07
587.31
1014.48
)7.29
4.89
)1.04
54.84
586.60
1017.25
)9.17
4.89
)0.10
50.60
633.35
1102.71
)3.23
5.20
b (ꢁ)
––
2
ꢀ
3
SA (A )
484.14
822.03
)3.30
3.72
ꢀ
V (A )
HE (kcal/mol)
log P
Pol
MR
r
p
L
B1
B5
B5=B1
L=B1
33.17
87.50
41.00
107.60
)0.01
1.96
40.91
107.82
0.34
40.95
107.82
0.06
42.84
114.93
1.49
42.88
114.93
0.78
46.02
122.03
)0.83
0.18
––
––
––
––
––
––
––
0.14
2.65
0.14
2.65
)0.28
3.44
1.70
)0.28
3.44
1.70
6.28
1.71
3.53
1.35
1.35
1.35
1.35
1.35
3.11
1.82
2.44
1.44
2.44
1.44
3.08
2.28
1
1.96
1
1.96
3.67
2.02
2.02
2.62
Attempts were also made to use all of the derived
descriptors to perform third QSAR analysis of the 9b–g.
The resulted models were those that obtained previously
(Eqs. 1 and 2) and no new one was obtained. Compar-
ison of the observed relationships from Eqs. 1 and 2
reveal that hydrophile molecules with electron with-
drawing substituent produce higher cytotoxicity in
comparison to other molecules. It should be noted that
the statistical quality of the resulted equation is not high
and therefore they can not be used for the accurate
prediction of the activity. However, according to the
discussion made, they could explain the structure
activity relationships, well.
important. Some of the force field methods did not
result in a reasonable 3D structure for our work but the
AMBER did well. The results showed that these mole-
cules are able to intercalate into the DNA. After finding
the optimum geometry for the intercalation complexes
by the AMBER force field method, the AM1 semi
empirical method was used to calculate the binding
energy (E) and DHf of the resulted complexes. Enthalpy
difference (DH) and binding energy difference (DE)
of the intercalation complex was calculated by sub-
traction of the quantity of the complex from the sum
of the quantities of compounds and DNA ½DE ¼
ðDHf compound þ DHf DNAÞꢂ.33 The results are represented
in Tables 3 and 4. Molecule 9e, with the highest activity
in K-562, produced the strongest complex with DNA
relative to the others. This was measured by the smallest
DH and DE values of intercalation. By comparison of
the results obtained from different DNA double base
pairs, it can be concluded that the compound 9e has a
moderate selectivity toward the A–T/G–C double base
pairs.
Ecomplex ꢀ ðEcompound þ EDNA
Þ
and DH ¼ DHf complex ꢀ
2.3. Intercalation study
We adopted molecular mechanic and after that semi
empirical quantum chemical method to dock the inter-
calators into every reasonable double base pairs of
DNA (A–T/A–T, A–T/T–A, A–T/G–C, A–T/C–G, C–
G/G–C, C–G/C–G). The double base pairs of DNA
were built by the nucleic acid database of Hyperchem
and their 3D geometry were optimized by AM1 semi
empirical method (Fig. 1). The binding energy and DHf
were calculated for each double base pairs. In molecular
modeling studies, finding a suitable force field is
In an attempt to find a relationship between the DH or
DE of intercalation and antitumor activity, the )log IC15
was plotted against different columns of Tables 3 and 4.
The correlation coefficient obtained for each double
Table 3. DE (kcal/mol) of the intercalation reaction between the studied molecules and different double base pairs of DNA
DNA double base pairs
Molecule
A–T/T–A
A–T/C–G
A–T/A–T
A–T/G–C
C–G/G–C
G–G/C–C
9a
9b
9c
)6.6
)19.7
)15.9
)49.9
)42.4
)32.4
)24.6
0.511
)21.4
)23.4
)10.8
)41.3
)35.4
)41.4
)21.6
0.656
)19.1
)42.2
)1.5
)24.6
)11.5
)16.8
)47.6
)13.7
)15.8
)14.7
)44.1
)53.4
)44.2
)12.3
0.928
)17.9
)30.7
)22.7
)50.6
)37.2
)31.0
)21.1
0.364
)15.1
)11.4
)1.5
)32.3
)25.6
)25.6
)10.2
0.685
9d
9e
9f
9g
R2 a
0.230
a R2 is the square of the correlation coefficient for the plot of activity against the DE of intercalation.

R. Miri et al. / Bioorg. Med. Chem. 12 (2004) 2529–2536
2533
Table 4. DH (kcal/mol) of the intercalation reaction between the studied molecules and different double base pairs of DNA
DNA double base pairs
Molecule
A–T/T–A
A–T/C–G
A–T/A–T
A–T/G–C
C–G/G–C
G–G/C–C
9a
9b
9c
)13.5
)19.1
)12.8
)44.6
)33.9
)38.0
)16.3
0.654
)18.2
)32.4
)17.5
)47.3
)43.4
)52.3
)28.7
0.564
)29.7
)12.6
)5.3
)45.5
)35.8
)23.2
)18.9
)22.7
)22.4
)12.4
)48.2
)68.1
)49.9
)12.3
0.934
)15.9
)36.1
)25.9
)53.3
)45.9
)34.0
)29.6
0.331
)8.9
)8.7
)1.0
)42.9
)45.2
)23.1
)3.6
9d
9e
9f
9g
R2 a
0.481
0.792
a R2 is the square of the correlation coefficient for the plot of activity against the DH of intercalation.
base pairs is represented in the last row of Tables 3 and
4. It shows that the DH and DE of the reaction between
intercalators and A–T/G–C double base pairs have more
correlation with the )log IC15 relative to those obtained
from other double base pairs. These observation reveal
that the A–T/G–C has a significant role in interaction
between the studied molecules and DNA.
form new hydrogen bonds with hydrogens of the
guanine.
3. Experimental
3.1. Chemistry
The 3D structure of the intercalation complexes formed
between the molecule 9e and different double base pairs
of DNA (except for A–T/G–C) are shown in Figure 3
and that of A–T/G–C in three different side views are
shown in Figure 4. As we see, the molecule 9e is more or
less intercalated into the DNA. However this molecule
penetrates into the A–T/G–C double base pairs more
than the others. When 9e intercalates into A–T/G–C, the
hydrogen bond between the guanine and cytosine
is broken and the oxygens of the nitro group of 9e
Melting points were determined on a Kofler hot stage
1
apparatus and are uncorrected. H NMR spectra were
run at a Varian Unity Plus 400 MHz spectrometer.
Chemical shifts are reported in parts per million (d)
relative to TMS as an internal standard. The mass
spectra were measured with a HP 6890 spectrometer at
70 eV. The IR spectra were obtained by using a Perkin
Elmer Paragon 1000 spectrophotometer (KBr disks).
Microanalyses were within 0.40% of theoretical values
for C, H, and N. All spectra were consistent with the
Figure 3. Optimized structure of the intercalation complex of 9e with different DNA double base pairs.

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R. Miri et al. / Bioorg. Med. Chem. 12 (2004) 2529–2536
Figure 4. Optimized structure of 9e-(A–T/G–C) intercalation complex in three different views.
assigned structures. All reagents were purchased from
the Aldrich Chemical Co.
(NO2). MS (m=z rel intensity): 404 (Mþ, 100), 372 (95),
358 (80), 303 (21).
3.1.7. 11-(4-Nitro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-
10,12-dione (9f). Yield 6.38%. IR (KBr): m 3022 (CH,
aromatic), 1708 (C@O, ketone), 1536 and 1353 cmꢀ1
(NO2). MS (m=z rel intensity): 404 (Mþ, 100), 374 (8),
358 (48), 301 (20).
3.1.1. General procedure for synthesis. Ammonium ace-
tate (1.54 g, 20 mmol) was added to a stirring solution of
indandione 1 (1.46 g, 10 mmol) and aryl(alkyl)carbox-
aldehydes 2–7, (5 mmol) in 50 mL of absolute acetic acid
and pure ethanol (1:3). The reaction was refluxed at
80 ꢁC for 3 h under exposure of a 100 W tungsten lamp.
After that the reaction mixture was cooled, the precip-
itated sticky solid was removed by filtration. Then it was
washed with pure ethanol and purified by silica gel thin
layer chromatography using chloroform.
3.1.8. 11-(4-Dimethylamino-phenyl)-diindeno[1,2-b;20,10-
e]pyridine-10,12-dione (9g). Yield 15.9%. 1H NMR
(CDCl3): d 7.81 (m, 8H, diindeno), 7.62 (dd, 4H,
phenyl), 3.13 (s, 6H, N(CH3)2). IR (KBr): m 3019 (CH,
aromatic), 1708 cmꢀ1 (C@O, ketone). MS (m=z rel
intensity): 402 (Mþ, 100), 401 (27), 348 (23), 326 (14),
277 (37).
3.1.2. 11-Methyl-diindeno[1,2-b;20,10-e]pyridine-10,12-di-
1
one (9a). Yield 8%. H NMR (CDCl3): d 7.65 (m, 8H,
diindeno), 2.97 (s, 3H, CH3). IR (KBr): m 3019 (CH,
aromatic), 1712 cmꢀ1 (C@O, ketone). MS (m=z rel
intensity): 297 (Mþ, 100), 269 (6), 240 (22).
3.2. Cell culture
Seven cell lines including Vero (monkey kidney primary
cells), Hela (human cervix carcinoma), K-562 (human
chronic myeloid leukemia), SK-BR-3 (human breast
adenocarcinoma), Fen (human bladder epithelial carci-
noma), U-937 (human histiocytic lymphoma) and KB
(human cervix carcinoma, a derivative of Hela) were all
obtained from the Iran Pasteur Institute, Tehran, Iran,
except for the Fen cell line, which was kindly provided
by Dr. A. M. E. Nouri, Department of Medical
Oncology, The Royal London Hospital Trust, London,
UK. Cells were cultured in RPMI-1640 (Sigma, USA)
supplemented with 10% fetal bovine serum (Gibco,
USA), 100 IU/mL penicillin and 100 lg/mL streptomy-
cin. Cells were cultured in 50 cm3 flask (Nunc, Den-
mark) with 5 mL of culture medium in a humified 5%
CO2 incubator at 37 ꢁC.
3.1.3. 11-Phenyl-diindeno[1,2-b;20,10-e ]pyridine-10,12-di-
one (9b). Yield 21.7%. IR (KBr): m 3020 (CH, aromatic),
1716 cmꢀ1 (C@O, ketone). MS (m=z rel intensity): 359
(Mþ, 100), 301 (12), 179 (14), 151 (11).
3.1.4. 11-(3-Fluoro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-
10,12-dione (9c). Yield 9.97%. IR (KBr): m 3018 (CH,
aromatic), 1713 cmꢀ1 (C@O, ketone). MS (m=z rel
intensity): 377 (Mþ, 100), 376 (69), 323 (13), 188 (10).
3.1.5. 11-(4-Fluoro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-
10,12-dione (9d). Yield 16.9%. IR (KBr): m 3021 (CH,
aromatic), 1714 cmꢀ1 (C@O, ketone). MS (m=z rel
intensity): 377 (Mþ, 100), 348 (9), 323 (12), 188 (8).
3.3. Cytotoxicity evaluation
3.1.6. 11-(2-Nitro-phenyl)-diindeno[1,2-b;20,10-e]pyridine-
10,12-dione (9e). Yield 2.9%. IR (KBr): m 3019 (CH,
aromatic), 1712 (C@O, ketone), 1530 and 1344 cmꢀ1
Appropriate amount from each one of the compounds
was mixed with DMSO (dimethyl sulfoxide). Then with

R. Miri et al. / Bioorg. Med. Chem. 12 (2004) 2529–2536
2535
serial dilution in RPMI-1640 four concentrations (10,
References and notes
100, 500, 1000 lM) were made. DMSO, as negative
control, diluted with the same method. For positive
control, doxorubicin was mixed with PBS (phosphated
buffered saline) to make the similar concentrations.
MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetra-
zolium bromide], (Sigma, USA), was dissolved in PBS at
5 mg/mL and then filtered to remove any undissolved
particle. Each well of the microtiter plate was filled with
1–5 · 104 cells (depending on the cell line) in 90 lL cul-
ture medium. Then, 10 lL from the stock solutions of
the compounds, negative and positive controls was
added as triplicate to the wells to reach the final con-
centration of 1, 10, 50, and 100 lM. Three wells con-
taining only the same number of the cells left in each
plate. Plates were kept in a humified incubator for 48 h.
After the incubation period, MTT assay was carried out
by the procedure described by Jabbar et al.15
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ꢀ
less than or equal to 0.01 kcal/mol A.
Acknowledgements
We are thankful to the Research Council of the Shiraz
University of Medical Sciences for financial support and
the Department of Chemistry of Persian Gulf University
for technical help.
28. Hemmateenejad, B.; Akhond, M.; Miri, R.; Shamsipur,
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