Synthesis, cytotoxicity, and QSAR study of new aza-cyclopenta[b]fluorene-1,9-dione derivatives

Article abstract of DOI:10.1111/j.1747-0285.2011.01213.x

Thirty novel derivatives of aza-cyclopenta[b]fluorene-1,9-dione were synthesized, and their cytotoxic activities were tested against HeLa, LS180, MCF-7, and Raji cancer cell lines by MTT assay. Two derivatives containing nitrofuryl moiety, including 10-(5-nitro-furan-2-yl)-2,3-dihydro-4-aza-cyclopenta[b]fluorene-1,9-dione (IC₅₀ range: 5.7-13.0μm) and 10-(5-Nitro-furan-2-yl)-2,3,4,10-tetrahydro-4-aza-cyclopenta[b]fluorene-1,9-dione (IC₅₀ range: 3.6-20.2μm), as well as 10-(2-Nitro-phenyl)-2,3,4,10-tetrahydro-4-aza-cyclopenta[b]fluorene-1,9-dione (IC₅₀ range: 3.1-27.1μm) with nitrophenyl moiety on C10 position, were the most effective compounds. Furthermore, the effect of physiochemical descriptors on the cytotoxicity was evaluated by quantitative structure-activity relationship analysis. The quantitative structure-activity relationship results showed that molecular dipole moment, molar refractivity, fragment-based parameters, and some topological indices were influential on the cytotoxic effect. Finally, the good correlation that was found among cytotoxic data obtained from different cell lines may be an implication of a common cytotoxic mechanism in these cell lines. These findings provide useful structural information for the rational design and synthesis of efficient chemotherapeutic agents for treatment for cancer. Thirty novel dihydro and tetrahydro analogous of aza-cyclopenta[b]fluorene-1,9-dione were synthesized and their cytotoxic activities were tested against HeLa, LS180, MCF-7 and Raji cancer cell lines by MTT assay. QSAR analysis determined that molecular dipole moment and fragment-based parameters were important for the activity of these derivatives in the HeLa, MCF-7 and Raji cell lines.

Full text of DOI:10.1111/j.1747-0285.2011.01213.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01213.x
Chem Biol Drug Des 2012; 79: 68–75
Research Article
Synthesis, Cytotoxicity, and QSAR Study of New
Aza-cyclopenta[b]fluorene-1,9-dione Derivatives
Ramin Miri1,2,*, Omidreza Firuzi1, Payam
The condensed polycyclic heteroaromatic compounds are naturally
occurring compounds with widespread biological activities (1). Dif-
ferent polycyclic compounds have demonstrated promising cytotoxic
activities (2,3). One of the common properties of these compounds
Peymani1, Meysam Zamani1, Ahmad Reza
Mehdipour1, Zahra Heydari1, Maryam
Masteri Farahani2,3 and Abbas Shafiee2,4
is the presence of planar polycyclic chromospheres, which can inter-
1Medicinal and Natural Products Chemistry Research Center, Shiraz
calate into the DNA macromolecule and accounts for the cytotoxic
properties of these compounds. They also have at least one basic
side chain, which can improve DNA binding affinity and in some
cases enhance solubility under physiological conditions (4). Different
studies have demonstrated that the planar portion of the molecule
could be tricyclic, tetracyclic, or even pentacyclic. Although some
results have shown a positive relationship between DNA binding
and cytotoxicity, it is generally accepted that DNA intercalation is
necessary but not enough for the antitumoral activity (4,5). There-
fore, the condensed heterocyclic system has been receiving much
attention for further modification as a cytotoxic scaffold and differ-
ent heterocyclic systems such as acridin and acridinone (6–8), diin-
denopyridine derivatives (5) and others have been evaluated for this
purpose.
University of Medical Sciences, Shiraz, Iran
2Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
3Azad University Shahr-e-Rey Branch, Tehran, Iran
4Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran 14174, Iran
*Corresponding author: Ramin Miri, mirir@sums.ac.ir
Thirty novel derivatives of aza-cyclopenta[b]fluo-
rene-1,9-dione were synthesized, and their
cytotoxic activities were tested against HeLa,
LS180, MCF-7, and Raji cancer cell lines by MTT
assay. Two derivatives containing nitrofuryl
moiety,
including
10-(5-nitro-furan-2-yl)-2,3-
dihydro-4-aza-cyclopenta[b]fluorene-1,9-dione (IC50
range: 5.7–13.0 lM) and 10-(5-Nitro-furan-2-yl)-
2,3,4,10-tetrahydro-4-aza-cyclopenta[b]fluorene-1,
9-dione (IC50 range: 3.6–20.2 lM), as well as 10-(2-
Nitro-phenyl)-2,3,4,10-tetrahydro-4-aza-cyclopenta
[b]fluorene-1,9-dione (IC50 range: 3.1–27.1 lM) with
nitrophenyl moiety on C10 position, were the most
effective compounds. Furthermore, the effect of
physiochemical descriptors on the cytotoxicity was
In continuation of our work on condensed heteroaromatic systems
(5,8–10), we have synthesized some novel polycyclic derivatives of
azacyclopenta[b]fluorene-1,9-dione and then evaluated their cyto-
toxic properties on four different human cancer cell lines.
Materials and Methods
evaluated
by
quantitative
structure–activity
Chemistry
relationship analysis. The quantitative structure–
activity relationship results showed that molecular
dipole moment, molar refractivity, fragment-based
parameters, and some topological indices were
influential on the cytotoxic effect. Finally, the good
correlation that was found among cytotoxic data
obtained from different cell lines may be an implica-
tion of a common cytotoxic mechanism in these cell
lines. These findings provide useful structural infor-
mation for the rational design and synthesis of
efficient chemotherapeutic agents for treatment
for cancer.
The synthesis of the tetrahydro-4-aza-cyclopenta[b]fluorene-1,9-di-
one or dihydro-4-aza-cyclopenta[b]fluorene-1,9-dione derivatives was
achieved following the steps outlined in Scheme 1. Reaction of
ammonium acetate 1 with 1,3-cyclopentanedione 2 afforded 3-
imino cyclopentanone 3 (67% yield). The tetrahydro analogues
6a–o were synthesized by molecular condensation of equivalent
amount of 3-imino cyclopentanone 3, corresponding aldehyde 4a–o
and 1,3-indandion 5. Then, the dihydro-4-aza-cyclopenta[b]fluorene-
1,9-dione 7a–o derivatives were achieved by oxidizing the corre-
sponding tetrhydro form using MnO2. The yield and melting point of
final compounds are summarized in Table 1.
Key words: aza-cyclopenta[b]fluorene-1,9-dione, cytotoxicity, MTT
assay, quantitative structure–activity relationship
10-p-Tolyl-2,3,4,10-tetrahydro-4-aza-
cyclopenta[b]fluorene-1,9-dione (6a)
(C22H17NO2) MW = 327 H-NMR (CDCl3): d 2.08 (t, 2H, C3-cyclopen-
Received 13 June 2010, revised 21 July 2011 and accepted for publica-
tion 23 July 2011
1
tyl), 2.21 (s, 3H, tolyl-CH3), 2.48 (t, 2H, C2-cyclopentyl), 4.51 (s, 1H,
68

Synthesis of New Aza-cyclopenta[b]fluorene-1,9-diones
NH
O
Benzene
Reflux
[NH4]+[CH3COO]–
+
O
O
1
2
3
NH
O
O
O
R
O
Ethanol
Reflux
RCH
+
+
N
H
O
O
3
4a–o
5
6a–o
O
O
R
O
O
R
N
MnO2
Dichloromethane
N
H
Scheme 1: General synthetic
pathway for the aza-cyclopenta[b]
fluorene-1,9-dione derivatives.
6a–o
7a–o
C10-H), 7.04–7.60 (m, 4H, tolyl), 7.20–7.60 (m, 4H, phenyl), 11.05 (br
s, 1H, NH)
MS: m ⁄ z (%) 301 (M+, 100), 271 (61), 241 (46), 185 (38), 147 (18),
93 (30), 87 (21)
MS: m ⁄ z (%) 327 (M+, 15), 293 (38), 236 (100), 152 (10), 130 (82),
82 (46), 60 (52)
10-(2-Chloro-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6c)
(C21H14ClNO2) MW = 347 H-NMR (CDCl3): d 2.35 (t, 2H, C3-cyclo-
1
10-p-Tolyl-2,3-dihydro-4-
pentyl), 2.80 (t, 2H, C2-cyclopentyl), 5.00 (s, 1H, C10-H), 7.19–7.23
aza-cyclopenta[b]fluorene-1,9-dione (7a)
(C22H15NO2) MW = 325 1H-NMR (CDCl3): d 2.45 (s, 3H, tolyl-CH3),
2.82 (t, 2H, C3-cyclopentyl), 3.28 (t, 2H, C2-cyclopentyl), 7.25–7.56
(m, 4H, tolyl), 7.66–8.05 (m, 4H, phenyl)
(m, 4H, Cl-phenyl), 7.34–7.49 (m, 4H, phenyl), 11.09 (br s, 1H,
NH)
MS: m ⁄ z (%) 347 (M+, 7), 311 (7), 288 (7), 254 (8), 234 (100), 207
(17), 151 (7), 140 (15), 73 (16)
MS: m ⁄ z (%) 301 (M+, 100), 271 (61), 241 (46), 185 (38), 147 (18),
93 (30), 87 (21)
10-(2-Chloro-phenyl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7c)
1
10-Furan-2-yl-2,3,4,10-tetrahydro-4-
(C21H12ClNO2) MW = 345 H-NMR (CDCl3): d 2.79 (t, 2H, cyclopen-
aza-cyclopenta[b]fluorene-1,9-dione (6b)
(C21H14ClNO2) MW = 303 H-NMR (CDCl3): d 2.07 (t, 2H, C3-cyclo-
tyl), 3.35 (t, 2H, cyclopentyl), 7.26–7.53 (m, 4H, Cl-phenyl), 7.68–
8.03 (m, 4H, phenyl)
1
pentyl), 2.97 (t, 2H, C2-cyclopentyl), 4.71 (s, 1H, C10-H), 5.77 (d, 1H,
C3-furyl), 6.18 (m, 2H, C4-furyl), 7.22 (d, 1H, C5-furyl), 7.3–7.8 (m,
4H, phenyl)
MS: m ⁄ z (%) 345 (M+, 1), 317 (5), 310 (100), 280 (90), 250 (77), 227
(100), 201 (86), 155 (90), 140 (83), 113 (76), 51 (15)
MS: m ⁄ z (%) 303 (M+, 7), 274 (8), 246 (100), 220 (17), 151 (7), 101
(15), 76 (16), 50 (20)
10-(4-Chloro-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6d)
(C21H14ClNO2) MW = 347 H-NMR (CDCl3): d 2.50 (t, 2H, cyclopen-
1
10-Furan-2-yl-2,3-dihydro-4-
tyl), 2.75 (t, 2H, cyclopentyl), 4.59 (s, 1H, C10-H), 7.27 (m, 4H, Cl-
phenyl), 7.49 (m, 4H, H-phenyl), 11.13 (br s, 1H, NH)
aza-cyclopenta[b]fluorene-1,9-dione (7b)
1
(C19H11NO3) MW = 301 H-NMR (CDCl3): d 2.83 (t, 2H, C3-cyclopen-
tyl), 3.22 (t, 2H, C2-cyclopentyl), 6.63 (d, 1H, C5-furyl), 7.58–7.67 (m,
2H, C3,4-furyl), 7.73–7.94 (m, 4H, phenyl)
MS: m ⁄ z (%) 347 (M+, 11), 318 (18), 254 (5), 235 (100), 207 (10),
152 (10), 75 (11)
Chem Biol Drug Des 2012; 79: 68–75
69

Miri et al.
Table 1: Physical characterization and cytotoxic activity data of synthetic compounds assessed by the MTT reduction assay
HeLa cells
LS180 cells
IC16 (lM)
MCF-7 cells
Raji cells
Compound Yield (%) Mp (ꢀC) IC16 (lM)
IC50 (lM)
IC50 (lM)
IC16 (lM)
IC50 (lM)
IC16 (lM)
IC50 (lM)
6a
7a
6b
7b
6c
57
70
40
73
35
40
45
80
55
70
43
73
42
60
40
44
56
67
54
63
84
27
67
42
61
53
56
40
63
52
324–326 25.8 € 5.1
254–255 14.6 € 9.0
278–280 25.3 € 1.3
>100
38.7 € 32.0
>100
>100
76.2 € 42.1
>100
>100
69.1 € 35.5
>100
32.3 € 3.9
8.0 € 1.5
81.5 € 95.8
>100
12.7 € 6.4
10.1 € 5.1
11.5 € 4.5
14.2 € 8.5
9.0 € 2.2
11.1 € 4.5
6.9 € 3.3
5.6 € 1.7
2.9 € 1.6
24.4 € 10.5
28.7 € 18.8
>100
5.3 € 4.3
2.2 € 0.7
7.4 € 7.6
14.4 € 9.3
6.2 € 2.9
19.7 € 17.8
10.8 € 7.4
10.2 € 3.8
6.7 € 2.4
9.0 € 0.4
4.9 € 2.6
18.0 € 9.8
12.1 € 2.3
25.5 € 1.8
5.0 € 3.7
4.3 € 3.3
5.0 € 1.4
8.0 € 5.4
>100
18.6 € 0.7
>100
6.5 € 0.1
4.7 € 3.7
10.0 € 10.6
>100
9.9 € 8.3
2.3 € 2.4
1.5 € 1.8
6.6 € 9.4
30.9 € 11.4
11.6 € 8.3
1.0 € 0.9
1.5 € 1.0
3.0 € 1.5
4.8 € 2.1
3.2 € 0.6
>100
>100
>100
93.4 € 3.0
>100
76.9 € 43.7
>100
>100
33.7 € 20.7
>100
>100
>100
99.7 € 70.8 15.61 € 6.24
53.2 € 12.2
>100
81.3 € 14.6
64.4 € 14.8
>100
37.0 € 0.3
10.2 € 2.9
>100
94.0 € 70.0
54.2 € 36.9
58.8 € 14
72.8 € 18.9
68.9 € 41.1
>100
33.8 € 10.5
26.5 € 16.3
>100
>100
>100
20.2 € 13.1
10.2 € 5.8
>100
26.5 € 6.6
>100
203–204
278–280
5.1 € 1.3
7.3 € 3.1
4.4 € 2.8
14.5 € 11.3
14.6 € 9.9
7.3 € 1.6
0.3 € 0.1
9.89 € 6.6
>100
7c
260–261 32.8 € 1.9
6d
7d
6e
7e
6f
336–337
264–265
269–271
7.9 € 4.6
1.9 € 0.9
1.6 € 1.3
216–217 20.0 € 5.5
292–295 >100
248–250 >100
>100
>100
7f
>100
>100
>100
6g
7g
6h
7h
6i
269–272
259–260
1.0 € 0.3
1.9 € 0.5
3.6 € 1.0
5.7 € 1.1
>100
59.4 € 15.8
8.0 € 1.1
36.7 € 7.0
>100
72.7 € 16.9
14.4 € 5.8
53.7 € 19.2
3.1 € 0.9
>100
69.2 € 9.7
>100
25.7 € 2.4
51.6 € 4.6
48.3 € 23.5
76 € 72.4
5.5 € 3.3
4.9 € 2.5
>100
14.3 € 7.0
13.0 € 4.9
>100
4.4 € 3.8
9.7 € 6.3
60.1 € 70.4
>100
22.4 € 8.7
>100
>100
>100
24.8 € 6.5
>100
13.5 € 9.6
>100
75.3 € 15.0
>100
26.2 € 5.8
>100
>100
>100
207–209 15.7 € 5.7
222–223 18.3 € 10.5
>100
>100
>100
41.0 € 11.4
>100
>100
>100
294–296
224–226
1.8 € 0.3
5.2 € 2.3
1.5 € 0.6
3.1 € 2.6
>100
11.5 € 2.8
24.5 € 9.3
>100
7i
6j
286–288 19.2 € 13.1
226–227 28.3 € 5.2
>100
7j
>100
>100
15.2 € 6.9
3.5 € 2.7
22.3 € 22.8
3.2 € 2.8
>100
16.5 € 0.1
>100
5.5 € 2.6
47.8 € 50.1
>100
6k
7k
6l
286–289
263–265
343–345
3.1 € 1.9
6.4 € 3.1
0.7 € 0.5
2.4 € 0.2
7.8 € 3.0
1.0 € 0.5
32.1 € 4.1
14.9 € 7.7
18.5 € 12.0
3.9 € 1.7
2.0 € 1.8
2.4 € 3.7
11.1 € 10.1
22.8 € 2.8
58.2 € 12.9
6.9 € 3.8
60.4 € 23.2
76.6 € 28.6
>100
31.5 € 15.7
90.6 € 74.2
36.6 € 50.1
>100
>100
54.4 € 22.2
27.1 € 7.7
64.0 € 26.9
>100
>100
49.3 € 9.0
>100
7l
269–270 >100
280–285 30.2 € 5.2
238–240 11.2 € 3.7
6m
7m
6n
7n
6o
7o
DOX
Cisplatin
303–304
258–261
295–297
243–245
–
7.3 € 2.2
9.8 € 4.1
2.8 € 0.2
6.3 € 2.9
>100
>100
>100
–
–
0.054 € 0.024 0.211 € 0.051 0.066 € 0.014 0.365 € 0.140 0.005 € 0.002 0.032 € 0.012 0.049 € 0.066 0.143 € 0.174
0.9 € 0.1 4.7 € 0.6 0.8 € 0.4 6.1 € 2.5 1.7 € 1.0 32.1 € 32.6 2.7 € 0.6 11.3 € 2.5
Values represent the mean € SD of at least three different experiments. Compounds were tested at the maximum final concentration of 100 lM.
10-(4-Chloro-phenyl)-2,3-dihydro-4-
10-(3-Chloro-phenyl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7d)
(C21H12ClNO2) MW = 345 H-NMR (CDCl3): d 2.84 (t, 2H, cyclopen-
tyl), 3.30 (t, 2H, cyclopentyl), 7.26–7.58 (m, 4H, Cl-phenyl), 7.66–
8.04 (m, 4H, phenyl)
aza-cyclopenta[b]fluorene-1,9-dione (7e)
1
1
(C21H12ClNO2) MW = 345 H-NMR (CDCl3): d 2.79 (t, 2H, cyclopen-
tyl), 3.30 (t, 2H, cyclopentyl), 7.25–7.48 (m, 4H, Cl-phenyl), 7.60–
7.95 (m, 4H, phenyl)
MS: m ⁄ z (%) 345 (M+, 100), 309 (15), 252 (15), 200 (12), 125 (12),
75 (25)
MS: m ⁄ z (%) 345 (M+, 16), 301 (7), 196 (46), 151 (4), 105 (100), 77
(7)
10-(3-Chloro-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6e)
(C21H14ClNO2) MW = 347 H-NMR (CDCl3): d 2.43 (t, 2H, cyclopen-
10-Thiophen-2-yl-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6f)
(C19H13NO2S) MW = 319 H-NMR (CDCl3): d 2.54 (t, 2H, cyclopen-
1
1
tyl), 2.75 (t, 2H, cyclopentyl), 4.6 (s, 1H, C10-H), 7.24–7.37 (m, 4H,
Cl-phenyl), 7.45–7.51 (m, 4H, phenyl), 11.15 (br s, 1H, NH)
tyl), 3.27 (t, 2H, cyclopentyl), 4.95 (s, 1H, C10-H), 6.69–7.10 (m, 3H,
thiophene), 7.33–7.6 (m, 4H, phenyl), 10.90 (br s, 1H, NH)
MS: m ⁄ z (%) 347 (M+, 1), 345 (12), 198 (22), 148 (11), 105 (100), 75
(25)
MS: m ⁄ z (%) 319 (M+, 1), 290 (83), 236 (60), 149 (28), 57 (22), 45
(35)
70
Chem Biol Drug Des 2012; 79: 68–75

Synthesis of New Aza-cyclopenta[b]fluorene-1,9-diones
10-Thiophen-2-yl-2,3-dihydro-4-
10-(3-Methoxy-phenyl)-2,3-dihydro-4-aza-
cyclopenta[b]fluorene-1,9-dione (7i)
(C22H15NO3) MW = 341 H-NMR (CDCl3): d 2.82 (t, 2H, cyclopentyl),
aza-cyclopenta[b]fluorene-1,9-dione (7f)
1
1
(C19H11NO2S) MW = 317 H-NMR (CDCl3): d 2.79 (t, 2H, cyclopen-
tyl), 3.23 (t, 2H, cyclopentyl), 7.15–7.30 (m, 3H, thiophene), 7.66–
8.05 (m, 4H, phenyl)
3.33 (t, 2H, cyclopentyl), 3.83 (s, 3H, O-methyl), 6.94–7.25 (m, 4H,
OMe-phenyl), 7.59–8.05 (m, 4H, phenyl)
MS: m ⁄ z (%) 317 (M+, 1000), 310 (92), 153 (13), 105 (10), 77 (4)
MS: m ⁄ z (%) 341 (M+, 100), 310 (62), 239 (29), 170 (18), 115 (9)
10-(5-Nitro-furan-2-yl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6g)
(C19H12N2O5) MW = 348 H-NMR (CDCl3): d 2.42 (t, 2H, cyclopentyl),
2.86 (t, 2H, cyclopentyl), 4.86 (s, 1H, C10-H), 7.34 (d, 1H, C3-furyl),
7.39–7.53 (m, 4H, phenyl), 7.58 (d, 1H, C4-furyl), 11.38 (br s, 1H, NH)
10-(2-Methoxy-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6j)
(C22H17NO3) MW = 343 H-NMR (CDCl3): d 2.38 (t, 2H, cyclopentyl),
2.77 (t, 2H, cyclopentyl), 3.78 (s, 3H, O-methyl), 4.88 (s, 1H, C10-H),
6.66–7.08 (m, 4H, OMe-phenyl), 7.20–7.45 (m, 4H, phenyl), 10.90 (br
s, 1H, NH)
1
1
MS: m ⁄ z (%) 348 (M+, 50), 303 (100), 275 (59), 235 (81), 190 (40),
176 (33), 137 (24), 54 (16)
MS: m ⁄ z (%) 343 (M+, 83), 306 (27), 196 (57), 151 (11), 105 (100),
75 (21)
10-(5-Nitro-furan-2-yl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7g)
(C19H10N2O5) MW = 346 H-NMR (CDCl3): d 2.90 (t, 2H, cyclopentyl),
3.40 (t, 2H, cyclopentyl), 7.34–7.60 (m, 4H, phenyl), 7.73 (d, 1H, fu-
ryl), 7.98 (d, 1H, furyl)
10-(2-Methoxy-phenyl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7j)
(C22H15NO3) MW = 341 H-NMR (CDCl3): d 2.75 (t, 2H, cyclopentyl),
1
1
3.32 (t, 2H, cyclopentyl), 3.72 (s, 3H, O-methyl), 6.95–7.26 (m, 4H,
OMe-phenyl), 7.56–7.96 (m, 4H, phenyl)
MS: m ⁄ z (%) 346 (M+, 100), 316 (32), 288 (48), 243 (22), 214 (33),
187 (33), 165 (15), 110 (6), 87 (33)
MS: m ⁄ z (%) 341 (M+, 45), 310 (100), 292 (15), 240 (7), 213 (14), 55
(4)
10-(4-Methoxy-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6h)
(C22H17NO3) MW = 343 H-NMR (CDCl3): d 2.5 (t, 2H, cyclopentyl),
2.9 (t, 2H, cyclopentyl), 3.66 (s, 3H, O-methyl), 4.5 (s, 1H, C10-H),
6.8–7.1 (m, 4H, OMe-phenyl), 7.21–7.6 (m, 4H, phenyl), 11.05 (br s,
1H, NH)
1
10-(3-Nitro-phenyl)-2,3,4,10-tetrahydro-4-aza-
cyclopenta[b]fluorene-1,9-dione (6k)
(C21H14N2O4) MW = 358 H-NMR (CDCl3): d 2.50 (t, 2H, cyclopentyl),
3.31 (t, 2H, cyclopentyl), 4.80 (s, 1H, C10-H), 7.20–8.10 (m, 7H, aro-
matic), 8.20 (s, 1H, C2-NO2-phenyl), 11.25 (br s, 1H, NH)
1
MS: m ⁄ z (%) 343 (M+, 16), 301 (5), 196 (42), 105 (100), 75 (8)
MS: m ⁄ z (%) 358 (M+, 100), 340 (80), 337 (60), 309 (52), 220 (25),
55 (7)
10-(4-Methoxy-phenyl)-2,3-dihydro-4-aza-
cyclopenta[b]fluorene-1,9-dione (7h)
(C22H15NO3) MW = 341 H-NMR (CDCl3): d 2.85 (t, 2H, cyclopentyl),
3.35 (t, 2H, cyclopentyl), 3.69 (s, 3H, O-methyl), 6.93–7.398 (m, 4H,
OMe-phenyl), 7.50–8.0 (m, 4H, phenyl)
1
10-(3-Nitro-phenyl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7k)
1
(C21H12N2O4) MW = 356 H-NMR (CDCl3): d 2.82 (t, 2H, cyclopentyl),
3.38 (t, 2H, cyclopentyl), 7.30–7.81 (m, 4H, phenyl), 7.90–8.12 (m,
4H, NO2-phenyl), 8.37 (s, 1H, C2-NO2-phenyl)
MS: m ⁄ z (%) 341 (M+, 100), 309 (23), 289 (12), 239 (10), 170 (6),
115 (3)
MS: m ⁄ z (%) 356 (M+, 75), 307 (100), 280 (22), 196 (15), 110 (14)
10-(3-Methoxy-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6i)
(C22H17NO3) MW = 343 H-NMR (CDCl3): d 2.43 (t, 2H, cyclopentyl),
2.74 (t, 2H, cyclopentyl), 3.68 (s, 3H, O-methyl), 4.54 (s, 1H, C10-H),
6.78–7.04 (m, 4H, OMe-phenyl), 7.27–7.48 (m, 4H, phenyl), 11.06 (br
s, 1H, NH)
10-(2-Nitro-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6l)
(C21H14N2O4) MW = 358 H-NMR (CDCl3): d 2.48 (t, 2H, cyclopentyl),
2.79 (t, 2H, cyclopentyl), 5.52 (s, 1H, C10-H), 7.23–7.7 (m, 3H, NO2-
phenyl), 7.81–8.40 (m, 4h, phenyl), 11.23 (br s, 1H, NH)
1
1
MS: m ⁄ z (%) 358 (M+, 5), 341 (39), 311 (100), 309 (27), 236 (91),
208 (27), 152 (13), 74 (4)
MS: m ⁄ z (%) 343 (M+, 83), 311 (21), 297 (15), 235 (100), 155 (14),
75 (17)
Chem Biol Drug Des 2012; 79: 68–75
71

Miri et al.
10-(2-Nitro-phenyl)-2,3-dihydro-4-
MS: m ⁄ z (%) 362 (M+, 42), 345 (14), 261 (14), 236 (100), 208 (14),
aza-cyclopenta[b]fluorene-1,9-dione (7l)
(C21H12N2O4) MW = 356 H-NMR (CDCl3): d 2.80 (t, 2H, cyclopentyl),
152 (6), 54 (7)
1
3.37 (t, 2H, cyclopentyl), 7.25–8.41 (m, 8H, aromatic)
10-(5-Nitro-1-methyl-1H-imidazol-2-yl)-2,3-
dihydro-4-aza-cyclopenta[b]fluorene-1,9-dione
(7o)
MS: m ⁄ z (%) 356 (M+, 30), 310 (12), 283 (14), 236 (100), 208 (38),
151 (15), 75 (8)
1
(C19H12N4O2) MW = 360 H-NMR (CDCl3): d 2.88 (t, 2H, cyclopentyl),
3.43 (t, 2H, cyclopentyl), 3.79 (s, 3H, methyl-imidazole), 7.59–7.77
(m, 4H, H-phenyl), 8.04 (s, 1H, imidazole)
10-(4-Nitro-phenyl)-2,3,4,10-tetrahydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (6m)
(C21H14N2O4) MW = 358 H-NMR (CDCl3): d 2.46 (t, 2H, cyclopentyl),
MS: m ⁄ z (%) 360 (M+, 100), 314 (55), 273 (33), 246 (48), 190 (53),
1
2.76 (t, 2H, cyclopentyl), 4.75 (s, 1H, C10-H), 7.30–7.41 (m, 3H, NO2-
phenyl), 7.51–8.10 (m, 4h, phenyl), 11.23 (br s, 1H, NH)
99 (4), 54 (35)
MS: m ⁄ z (%) 358 (M+, 5), 310 (36), 236 (100), 208 (15), 153 (7), 77
Cytotoxicity section
(9)
Reagents and chemicals
RPMI 1640, fetal bovine serum (FBS), trypsin, and phosphate-buf-
fered saline (PBS) were purchased from Biosera (Ringmer, UK). 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
obtained from Sigma (Saint Louis, MO, USA) and penicillin ⁄ strepto-
mycin was purchased from Invitrogen (San Diego, CA, USA). Doxoru-
bicin and dimethyl sulphoxide were obtained from EBEWE Pharma
(Unterach, Austria) and Merck (Darmstadt, Germany), respectively.
10-(4-Nitro-phenyl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7m)
1
(C21H12N2O4) MW = 356 H-NMR (CDCl3): d 2.84 (t, 2H, cyclopentyl),
3.49 (t, 2H, cyclopentyl), 7.26–7.50 (m, 4H, phenyl), 7.66–8.41 (d,
2H, C2,6-NO2-phenyl), 8.12 (d, 2H, C2,6-NO2-phenyl)
MS: m ⁄ z (%) 356 (M+, 100), 310 (21), 280 (23), 224 (15), 149 (7),
141 (15), 57 (15)
Cell lines and cell culture
HeLa (human cervical adenocarcinoma), LS180 (human colon adeno-
carcinoma), MCF-7 (human breast adenocarcinoma), and Raji (human
B lymphoma) cells were obtained from the National Cell Bank of
Iran, Pasteur Institute, Tehran, Iran. All cell lines were maintained
in RPMI 1640 supplemented with 10% FBS, 100 units ⁄ mL penicillin-
G, and 100 lg ⁄ mL streptomycin. Cells were grown in monolayer
cultures, except for Raji cells, which were grown in suspension, at
37 ꢀC in humidified air containing 5% CO2.
10-(5-Bromo-thiophen-2-yl)-2,3,4,10-tetrahydro-
4-aza-cyclopenta[b]fluorene-1,9-dione (6n)
(C21H14BrO2) MW = 392 H-NMR (CDCl3): d 2.43 (t, 2H, cyclopentyl),
2.81 (t, 2H, cyclopentyl), 4.81 (s, 1H, C10-H), 6.67 (d, 1H, C3-thio-
phene), 6.96 (d, 1H, C4-thiophen), 7.34–7.54 (m, 4H, phenyl), 11.28
(br s, 1H, NH)
1
MS: m ⁄ z (%) 392 (M+, 15), 318 (100), 290 (59), 275 (14), 236 (22),
209 (7), 151 (7)
Cytotoxicity assay
Cell viability following exposure to synthetic compounds was esti-
mated using the MTT reduction assay (11–12). MCF-7 and Raji cells
were plated in 96-well microplates at a density of 5 · 104 cells ⁄ mL
(100 lL ⁄ well). LS180 and HeLa cells were plated at densities of
1 · 105 and 2.5 · 104 cells ⁄ mL, respectively. Control wells con-
tained no drugs and blank wells contained only growth medium for
background correction. After overnight incubation at 37 ꢀC, half of
the growth medium was removed and 50 lL of medium supple-
mented with different concentrations of synthetic compounds dis-
solved in DMSO were added in triplicate. Plates with Raji cells
were centrifuged before this procedure. Maximum concentration of
DMSO in the wells was 0.5%. Cells were further incubated for
72 h, except for HeLa cells, which were incubated for 96 h. At the
end of the incubation time, the medium was removed and MTT
was added to each well at a final concentration of 0.5 mg ⁄ mL, and
plates were incubated for another 4 h at 37 ꢀC. Then, formazan
crystals were solubilized in 200 lL DMSO. The optical density was
measured at 570 nm with background correction at 655 nm using a
Bio-Rad microplate reader (Model 680; Bio-Rad, Hercules, CA, USA).
10-(5-Bromo-thiophen-2-yl)-2,3-dihydro-4-
aza-cyclopenta[b]fluorene-1,9-dione (7n)
(C21H12BrNO2) MW = 390 H-NMR (CDCl3): d 2.83 (t, 2H, cyclopen-
tyl), 3.31 (t, 2H, cyclopentyl), 7.14 (d, 1H, C3-thiophene), 7.26 (d, 1H,
C4-thiophen), 7.53–7.66 (m, 2H, phenyl), 7.72 (d, 1H, phenyl), 8.1 (d,
1H, phenyl)
1
MS: m ⁄ z (%) 390 (M+, 100), 316 (50), 259 (14), 189 (6), 158 (7), 83
(7)
10-(5-Nitro-1-methyl-1H-imidazol-2-yl)-2,3,4,
10-tetrahydro-4-aza-cyclopenta[b]fluorene-1,
9-dione (6o)
1
(C19H14N4O2) MW = 362 H-NMR (CDCl3): d 2.49 (t, 2H, cyclopentyl),
2.82 (t, 2H, cyclopentyl), 4.11 (s, 3H, methyl-imidazole), 4.95 (s, 1H,
C10-H), 7.27–7.55 (m, 4H, H-phenyl), 7.93 (s, 1H, imidazole), 8.31 (br
s, 1H, NH)
72
Chem Biol Drug Des 2012; 79: 68–75

Synthesis of New Aza-cyclopenta[b]fluorene-1,9-diones
The percentage of inhibition of viability compared to control wells
was calculated for each concentration of the compound, and IC16
and IC50 values (13) were calculated with the software CURVEEXPERT
version 1.34 for Windows (Hyams Development, OH, USA). Each
experiment was repeated four times. Data are presented as
mean € SD.
presented for 6a–o and 7a–o have been confirmed by spectro-
scopic data using NMR and MS instruments.
Cytotoxicity
The cytotoxic activities of synthesized compounds were evaluated in
four human cancer cell lines, and IC16 and IC50 values were calcu-
lated for each derivative (Table 1). On the basis of IC16 and IC50 val-
ues, the most and least potent compounds in each cell line were
identified. In HeLa cell line, the most potent compounds in decreas-
ing order of efficiency were 6l > 6g > 7g > 6i, with IC50 values
of 3.1, 3.6, 5.7, and 8.0 lM, respectively. The weakest compounds
were 6f, 7f, and 7l which had IC16 and IC50 values higher than
100 lM. In LS180 cell line, the most potent compounds were
6l > 6e > 6i > 7g > 6g, which had IC50 values of 6.9, 8.0, 11.5,
13.0, and 14.3 lM, respectively. Compounds 6f, 7f, 7b, 6j, 7j, 6h,
and 7h with IC16 and IC50 values of higher than 100 lM were the
weakest compounds. In MCF-7 cells, the order of potency was as
follows; 7g > 6g > 6e > 6l (IC50 values: 10.2, 20.2, 26.5, and
27.1, respectively). Compound 7f, whose IC16 and IC50 values were
both greater than 100 lM, was the weakest compound followed by
6f, 7m, and 7e, which had IC16 values of 28.7, 25.5, and 24.4 lM,
respectively. In Raji cells, the most potent compounds were
6g > 7g > 6l > 6i, with IC50 values of 4.4, 9.7, 13.5, and 22.4 lM,
respectively. Compounds 7a, 7c, 7i, 6j, 7l, 7m, 6o, and 7o had
IC16 and IC50 values of higher than 100 lM.
Computational analysis
The chemical structure of each molecule was drawn with Hyper-
chem software (Version 7; Hypercube Inc., http://www.hyper.com,
USA). The Gaussian98 programa1 was employed to optimize the
molecular structure. The structures were optimized by the ab initio
method at the RHF ⁄ 3-21G level. Some quantum chemical descrip-
tors including the highest occupied molecular orbital (HOMO) and
the lowest unoccupied molecular orbital (LUMO) energies, the dif-
ference between HOMO and LUMO (HLG), and the molecular dipole
moment (MDP) were calculated in Gaussian98. A pool of different
descriptors including physicochemical properties, constitutional
descriptors, geometrical descriptors, and topological indices were
calculated using DRAGON (http://www.talete.mi.it/dragon_exp.htm)
software. The multiple linear regression (MLR) was performed by
the SPSS software (SPSS Inc., Chicago, IL, USA) using the stepwise
selection and elimination method for variable selection. The cross-
validation process was performed by home-made subroutines in
MATLAB environment (Natick, MA, USA).
According to the above data, it can be concluded that the most
potent compounds in all cell lines were 6g, 7g, 6l, and 6i.
Although the potency of these compounds is much lower than doxo-
rubicin, they are comparable and in some instances more potent
than cisplatin. On the other hand, the weakest compounds were
evidently 6f and 7f, whose their IC50 values were greater than
Results and Discussion
Chemistry
In this project, 30 dihydro and tetrahydro analogous of aza-cyclo-
penta[b]fluorene-1,9-dione was synthesized (Figure 1). The structures
CH3
Cl
NO2
Cl
O
S
O
Cl
a
b
c
d
e
f
g
OMe
NO2
OMe
NO2
OMe
NO2
h
i
j
k
l
m
Br
NO2
CH3
S
N
N
n
o
O
O
R
N
O
O
R
Figure 1: Chemical structures of
the aza-cyclopenta[b]fluorene-1,9-di-
one derivatives.
N
H
6a–o
7a–o
Chem Biol Drug Des 2012; 79: 68–75
73

Miri et al.
100 lM in all cell lines and had only one IC16 value of lower than
100 lM (7f, in Raji cell line) and two IC16 values lower than
100 lM (6f, in MCF-7 and Raji cell lines).
In the first step, it was tried to find an appropriate model for cyto-
toxic activity in HeLa cell line. The obtained equation is shown by
eqn 1:
logIC16 ¼ ꢀ0:087ðꢁ0:023Þ MDPz þ 1:722ðꢁ0:358Þ C ꢀ 027
In general, it seems that the derivatives of aza-cyclopenta[b]fluo-
rene-1,9-dione are less potent on Raji and LS180 cell lines and
more potent on the HeLa cell line.
ð1Þ
þ 0:109ðꢁ0:026Þ MR ꢀ 8:689ðꢁ2:298Þ
N ¼ 30; R ¼ 0:770; R2 ¼ 0:592; F ¼ 11:2; RMSE ¼ 0:38; QL2OO
¼ 0:458; QL2MO ¼ 0:520
An interesting finding was obtained by the analysis of the correla-
tion between cytotoxicity in various cell lines demonstrating signifi-
cant associations of cytotoxic properties of these compounds in
various cell lines (Table 2). In fact, in all cases, the correlation
between two cell lines is more than 60% which means that IC50
values of compounds in different cell lines have more than 60%
similarity in their trends. This may indicate that these compounds
probably have similar cytotoxic mechanisms on different cancer cell
lines that we used in this study.
In this equation, the values in the parenthesis represent the SD
of the coefficients. N, R, RMSE, and F are number of components,
correlation coefficient, root mean square error of regression, and
Fisher's F-ratio, respectively. As seen in the eqn 1, this equation
has moderate statistical quality, which can explain and predict
59% and 52% of variances in the cytotoxic activity data in HeLa
cell line, respectively. This model contains the molecular dipole
moment at z co-ordinate (MDPz), a fragment-based parameter (C-
027) defining number of R–CH-X group in the molecule and molar
refractivity.
Some important points were observed in respect for the structure–
activity relationship of these compounds. The dihydro and tetrahy-
dro forms showed similar cytotoxic activity, which indicates that the
oxidation of pyridine ring does not seem to significantly alter the
cytotoxic potency of these derivatives. Furthermore, compounds 6g,
7g, and 6l are among the most potent derivatives and all of them
bear a nitro group on C10 aryl position. This indicates the important
role of this moiety in the cytotoxicity.
The next equation was obtained for cytotoxicity of LS180 cell line:
logIC16 ¼ ꢀ0:087ðꢁ0:023Þ JGI1 þ 1:722ðꢁ0:358Þ J
þ 0:109ðꢁ0:026Þ C ꢀ 026 ꢀ 8:689ðꢁ2:298Þ
N ¼ 30; R ¼ 0:758; R2 ¼ 0:574; F ¼ 10:3; RMSE ¼ 0:57; QL2OO
¼ 0:470; QL2MO ¼ 0:492
QSAR studies
To evaluate the effects of the structural parameters of the investi-
gated derivatives on their cytotoxic activities, quantitative structure–
activity relationship (QSAR) analysis with different types of molecular
descriptors was performed. The activity data were converted to loga-
rithmic scale (i.e., LogIC16 for cytotoxicity in different cell lines).
Because IC50 values could not be calculated for some of the com-
pounds attributable to their low activity, we used IC16 values for
QSAR analysis to have a numerical value for almost all compounds.
It is clear that this equation has a comparable quality with the for-
mer model explaining the 57% of variances in the cytotoxic activity
data in LS180 cell line. The important point, in this equation, is the
presence of two topological parameters, JGI1 as a Galvez topologi-
cal charge index and J the Balaban Index, which indicates the
importance of this type of descriptors. Moreover, the existence of
another fragment-based parameter (C-026) identifying the number
of R–CH–R group is a sign of similarity between these models.
For each set of descriptors, the best linear regression equations
were obtained by the stepwise selection-based MLR subroutine of
SPSS software. The correlation coefficient (R2), standard error (SE) of
regression, correlation coefficient for leave-one-out cross-validation
(QLOO2), and correlation coefficient for leave-ten-out cross-validation
(QL2TO) were employed to judge the validity of regression equation.
Because colinearity degrades the performances of the MLR-based
QSAR equation; first, correlation analysis was performed to detect
the colinear descriptors (R > 0.9).
The next equation was obtained for cytotoxicity of MCF-7 cell line:
logIC16 ¼ 1:233ðꢁ0:187Þ C ꢀ 027 þ 0:061ðꢁ0:014Þ MR
þ 0:014ðꢁ0:006Þ MDPx ꢀ 4:445ðꢁ1:188Þ
N ¼ 30; R ¼ 0:817; R2 ¼ 0:667; F ¼ 15:3; RMSE
¼ 0:20; QL2OO ¼ 0:513; QL2MO ¼ 0:518
The correlation coefficient of this equation shows its better quality
compared to the former equations, although it is an average model.
The interesting point is its high similarity with eqn 1 in which both
models include a molecular dipole moment at z or x co-ordinate
(MDPz for first equation and MDPx for latter one), a fragment-based
parameter (C-027), and molar refractivity. On the other hand, these
activities have a good correlation (intercorrelation between these
cytotoxicity is 0.687) even not so high. These factors may imply that
similarity of trend and mechanism of compounds in these two cell
lines.
Table 2: Correlation coefficients (R2) between IC50 values of syn-
thesized compounds obtained by the MTT assay in various cell lines
HeLa
LS180
MCF-7
Raji
HeLa
LS180
MCF-7
Raji
1
0.903
0.687
0.724
1
0.714
0.698
1
0.647
1
74
Chem Biol Drug Des 2012; 79: 68–75

Synthesis of New Aza-cyclopenta[b]fluorene-1,9-diones
Finally, an equation for cytotoxicity in Raji cell line was obtained as
described below:
2. Sathish N.K., GopKumar P., Rajendra Prasad V.V.S., Shanta Kumar
S.M., Mayur Y.C.S.M. (2010) Synthesis, chemical characterization
of novel 1,3-dimethyl acridones as cytotoxic agents, and their
DNA-binding studies. Med Chem Res;19:674–689.
3. Banik B.K., Becker F.F. (2001) Polycyclic Aromatic compounds as
anticancer agents: synthesis and biological evaluation of chrysen
and pyrene derivatives. Bioorg Med Chem;8:2693–2699.
4. Denny W.A. (1989) DNA-intercalating ligands as anti-cancer drugs:
prospects for future design. Anti-Cancer Drug Des;4:241–263.
5. Miri R., Javidnia K., Hemmateenejad B., Azarpira A., Amirgho-
fran Z. (2004) Synthesis, cytotoxicity, QSAR, and intercalation
study of new diindenopyridine derivatives. Bioorg Med
Chem;12:2529–2536.
logIC16 ¼ ꢀ1:123ðꢁ0:165Þ C ꢀ 029 þ 0:407ðꢁ0:082Þ AMW
þ 0:350ðꢁ0:110Þ nCp ꢀ 0:038ðꢁ0:012Þ MDPz
ꢀ 1:832ðꢁ0:734Þ
N ¼ 30; R ¼ 0:858; R2 ¼ 0:735; F ¼ 14:9; RMSE
¼ 0:21; QL2OO ¼ 0:565; QL2MO ¼ 0:619
This four-parametric QSAR model showed the best statistical quality
among all four cytotoxicity data. Again, in this model, a molecular
dipole moment and a fragment-based parameter (C-029) are pre-
sented indicating the heavy impact of these parameters in overall
cytotoxicity of these compounds.
6. Becker F.F., Mukhopadhyay C., Hackfeld L., Banik I., Banik B.K.
(2000) Polycyclic aromatic compounds as anticancer agents: syn-
thesis and biological evaluation of dibenzofluorene derivatives.
Bioorg Med Chem;8:2693–2699.
7. Bu X., Chen J., Deady L.W., Denny W.A. (2002) Synthesis and
cytotoxicity of potential anticancer derivatives of pyrazolo[3,4,5-
kl]acridine and indolo[2,3-a]acridine. Tetrahedrone;58:175–181.
8. Jamalian A., Shafiee A., Hemmateenejad B., Khoshneviszadeh
M., Madadkar-Sobhani A., Zahra Bathaie S.Z., Akbar Moosavi-
Movahedi A.A. (2011) Novel imidazolyl derivatives of 1, 8-acrid-
inedione as potential DNA-intercalating agents. J Iran Chem
Soc; in press.
9. Miri R., Motamedi R., Rezaei M.R., Firuzi O., Javidnia A., Shafiee
A. (2011) Design, synthesis and evaluation of cytotoxicity of novel
chromeno[4,3-b]quinoline derivatives. Arch Pharm;344:111–118.
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Miri R. (2007) Dihydropyridine derivatives to overcome atypical
multidrug resistance: design, synthesis, QSAR studies, and eval-
uation of their cytotoxic and pharmacological activities. Chem
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Conclusion
Thirty dihydro and tetrahydro analogous of aza-cyclopenta[b]fluo-
rene-1,9-dione synthesized in this study showed weak-to-relatively-
good cytotoxic activities in four human cancer cell lines, which in
some instances were superior to cisplatin cytotoxicity. QSAR analy-
sis found proper relationships between structural characteristics
and the cytotoxic effect. Antitumoral activity in the HeLa, MCF-7,
and Raji cell lines showed that molecular dipole moment and frag-
ment-based parameters (C-027, C-027, and C-029, respectively)
were important for the activity of these derivatives. The presence
of dipole moment may indicate that some transient electrostatic
interactions are involved in the mechanism of action. In LS180 cell
line, in addition to a fragment-based parameter (C-026), two topo-
logical parameters also influenced the cytotoxic activity. Similarity
of the cytotoxic activities of different compounds in various cell
lines indicated the presence of a similar cytotoxic mechanism in all
cell lines. These findings provide helpful information to guide the
rational design and synthesis of more potent cytotoxic molecules
that could be useful chemotherapeutic agents for treatment for can-
cer. According to the condensed and planar structure of these novel
compounds, we speculate that DNA intercalation is a possible
mechanism of cytotoxicity.
12. Jabbar S.A., Twentyman P.R., Watson J.V. (1989) The MTT assay
underestimates the growth inhibitory effects of interferons. Br J
Cancer;60:523–528.
13. Tallarida R.J., Murray R.B. (1986) Manual of Pharmacologic Cal-
culations with Computer Programs. New York: Springer.
Note
Acknowledgments
aFrisch M.J., Trucks G.W., Schlegel H.B., Scuseria G.E., Robb M.A.,
Cheeseman J.R., Zakrzewski V.G., Montgomery Jr. J.A., Stratmann
R.E., Burant J.C., Dapprich S., Millam J.M., Daniels A.D., Kudin
K.N., Strain M.C., Farkas O., Tomasi J., Barone V., Cossi M., Cammi
R., Mennucci B., Pomelli C., Adamo C., Clifford S., Ochterski J., Pet-
ersson G.A., Ayala P.Y., Cui Q., Morokuma K., Salvador P., Dannen-
berg J.J., Malick D.K., Rabuck A.D., Raghavachari K., Foresman J.B.,
Cioslowski J., Ortiz J.V., Baboul A.G., Stefanov B.B., Liu G., Lias-
henko A., Piskorz P., Komaromi I., Gomperts R., Martin R.L., Fox
D.J., Keith T., Al-Laham M.A., Peng C.Y., Nanayakkara A., Challa-
combe M., Gill P.M.W., Johnson B., Chen W., Wong M.W., Andres
J.L., Gonzalez C., Head-Gordon M., Replogle E.S., Pople J.A. (2001)
GAUSSIAN 98 Rev. A11, Pittsburgh, PA, USA: Gaussian Inc.
This work was supported by Research Contract No. 425 ⁄ 1401 from
Tehran University of Medical Sciences, Tehran, Iran. Also, financial
support of the Shiraz University of Medical Sciences, vice-chancellor
of research is acknowledged. The cytotoxicity section of this project
is a part of Pharm. D thesis of P. Peymani (Thesis Number: 390).
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Chem Biol Drug Des 2012; 79: 68–75
75