Synthesis, cytotoxic evaluation, and DNA binding of novel thiazolo[5,4-b]quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2007.10.084

A series of novel alkylamino and 9-anilinothiazolo[5,4-b]quinolines were synthesized as potential antitumoral agents. The in vitro cytotoxicity of these compounds was evaluated on several cell lines. The inclusion of electron-withdrawn/acceptor hydrogen-b

Full text of DOI:10.1016/j.bmc.2007.10.084

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1142–1149
Synthesis, cytotoxic evaluation, and DNA binding
of novel thiazolo[5,4-b]quinoline derivatives
a
a
´
´
Marco A. Loza-Mejıa, Karina Maldonado-Hernandez,
b
b
´
´
´
´
Fernando Rodrıguez-Hernandez, Rogelio Rodrıguez-Sotres,
c
c
´
Ignacio Gonzalez-Sanchez, Angelina Quintero,
c
a,
*
´
Jose D. Solano and Alfonso Lira-Rocha
aDepartamento de Farmacia, Facultad de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Cd. Universitaria, Coyoaca´n,
Me´xico 04510, Mexico
bDepartamento de Bioqu´ımica, Facultad de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Cd. Universitaria, Coyoaca´n,
Me´xico 04510, Mexico
cDepartamento de Biolog´ıa, Facultad de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Cd. Universitaria, Coyoaca´n,
Me´xico 04510, Mexico
Received 3 August 2007; revised 19 October 2007; accepted 23 October 2007
Available online 30 October 2007
Abstract—A series of novel alkylamino and 9-anilinothiazolo[5,4-b]quinolines were synthesized as potential antitumoral agents. The
in vitro cytotoxicity of these compounds was evaluated on several cell lines. The inclusion of electron-withdrawn/acceptor hydrogen-
bond groups at position 30 of the anilino ring and the presence of an alkylamino chain on the tricyclic framework (regardless of its
position) seem to be structural features relevant to cytotoxic activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
oloquinoline template 1 (Fig. 1). Our results revealed
an increase in cytotoxic activity with the presence of
a diethylethylenediamino group at the 2 position of
a thiazoloquinoline nucleus 2, but noteworthy, the
AHMA 7 analogue, compound 3, showed low activity.
In order to gain a better understanding of the rela-
tionship between the substitution pattern and cytotox-
icity and to acquire insight into the contribution of
the diethylethylendiamino group, additional com-
pounds with different substituents were prepared.
Here, the synthesis, in vitro cytotoxicity, and DNA
intercalating properties of novel thiazoloquinoline
derivatives are reported.
The acridine derivatives are a group of compounds
believed to express their antitumor activity through
an interaction with DNA.1–4 The acridine intercalation
properties are responsible for their high affinity for
DNA. However, intercalation ability does not ensure
antitumor activity, and a requirement for specific
interaction with other targets, such as DNA topoiso-
merase,5 has frequently been proposed. Besides acri-
dine derivatives, other fused compounds, such as
thiazolo[5,4-b]quinolines,6 furo[2,3-b]quinolines,7 and
pyrazolo[3,4-d]pyrimidines,8
have
been
studied.
Recently, the cytotoxic activity and intercalation prop-
erties of some derivatives of 9-anilinothiazolo[5,
4-b]quinoline have been reported.9 The chemical struc-
ture of these compounds accommodates the general
substitution pattern of acridine derivatives on a thiaz-
2. Chemistry
The synthesis of the compounds is illustrated in Scheme
1. The preparation of compounds 8 and 9 has already
been described.6,9 From compound 9, the synthesis of
compounds 10a–11c was achieved in good yield under
acidic conditions. Attempts, however, under neutral
and basic conditions were unsuccessful and resulted in
low yield or decomposition.
Keywords: Thiazolo[5,4-b]quinoline derivatives; Antitumoral; Cyto-
toxic activity; Structure–activity relationship; DNA binding.
*
Corresponding author. Tel.: +52 56 22 52 86; fax: +52 56 22 53 29;
e-mail: lira@servidor.unam.mx
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.084

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M. A. Loza-Mejıa et al. / Bioorg. Med. Chem. 16 (2008) 1142–1149
1143
(MS-FAB), and the preservation of the thiazoloquino-
line ring (13C NMR). Therefore, it was concluded that
novel compound 13 and not 15 was formed during the
reaction. The yield of this reaction was increased by di-
rect condensation of the amine with 9 at room tempera-
ture (25 ꢁC). The preparation of 15 was carried out by
basic hydrolysis of compound 8, the coupling of the car-
bonyldiimidazole derivative of 8 with 2-(N,N-diethyleth-
ylenediamino)ethylamine, compound 14, and the
cyclization of the last compound with POCl3/PPA to af-
ford 15 in low yield.
HN
N
S
N
1
R1
HN
N
R2
R3
N
S
Notably, aromatic amines react at position 9 of the thiaz-
oloquinoline system while the aliphatic amines do so at
position 2. This behavior may be explained with the
HSAB principle10 since aliphatic amines are hard bases
which should react with the harder region of the molecule
(thiazole ring), while aromatic amines are medium-soft
bases and should react with the softer region (the central
ring of tricyclic system), as illustrated in Fig. 2. An alter-
native explanation could be a steric hindrance of the sul-
fur atom at position 2 to react with aromatic amines; such
hindrance does not exist with aliphatic amines.
2 R1 = R2 = H R3=NH(CH2)2N(CH2CH3)2
3 R1 = NH2 R2 = CH2OH R3=SCH3
4 R1 = R2 = H R3=SCH3
5 R1 = NH2 R2 = H R3=SCH3
6 R1 = NHAc R2 = H R3=SCH3
NH2
OH
HN
All thiazolo[5,4-b]quinoline derivatives 10–15 were char-
N
1
acterized by H and 13C NMR and elemental analysis.
7
NOESY experiments of compounds 10a, 10b and 10f
led to the unequivocal assignment of the aromatic protons
for all compounds. The assignment was based on the long
range interaction between the H-8 proton and the one
present at the amino group linking both aromatic rings
(Fig. 3). The data are summarized in Table 1.
Figure 1. Structures of tricyclic compounds.
Preparation of 15 was attempted by a direct condensa-
tion of 9 with 2-(N,N-diethyl)ethylenediamine in MeOH
as solvent. Though one principal product was obtained,
the spectral data showed the absence of the methylthio
group (1H NMR), the presence of one chlorine atom
An interesting interaction between the H-20 and H-60
protons and the methylthio group protons was observed
R
4'
O
HN
Cl
N
N
S
EtO
HN
N
a
b
SCH3
SCH3
SCH3
S
N
S
N
9
10a R = 3' -Cl
10b R = 3' -CN
10c R = 3' -OMe
10d R = 3' -CF3
10e R = 3' -NO2
10f R = 3' -OH
c
Cl
N
8
N
S
H
N
N
10g R= 3' -NHMe
d
11a R= 4' -Cl
11b R= 4' -OMe
13
11c R= 4' -CN
12 R= 3' -(CO)NH(CH2)2N(CH2CH3)2
N
O
HN
N
N
e
N
S
N
H
N
SCH3
SCH3
HN
S
15
14
Scheme 1. Reactions and conditions: (a) POCl3/PPA, 130 ꢁC, 4 h; (b) NH2–C6H4–R in MeOH/HCl, reflux, 6 h; (c) H2NCH2CH2N(CH2CH3)2, rt,
24 h., (d) 1— KOH/EtOH; 2— 1,1-carbonyldiimidazole in CH2Cl2, reflux, 1 h; 3— H2NCH2CH2N(CH2CH3)2 in CH2Cl2, reflux, 4 h; (e) POCl3/PPA,
120 ꢁC, 30 min.

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M. A. Loza-Mejıa et al. / Bioorg. Med. Chem. 16 (2008) 1142–1149
1144
at least two. The arrangements are illustrated in Figure
4.
Cl
N
N
S
SCH3
SCH3
Clearly, the substituents attached to the anilino ring
influence the spatial arrangement of this ring, possibly
through an electronic repulsion between the anilino ring
and the nitrogen thiazole atom because an electron-
releasing group (compounds 10a, 10f, and 10g) increases
the electron density on the anilino ring while an elec-
tron-withdrawing group (compound 10b) diminishes it.
The different chemical shifts for the methylthio group
found in the compounds support this proposal. This
phenomenon was observed only in the 30-substituted
compounds.
soft region
Cl
N
S
N
hard region
Figure 2. Hard and soft regions in the thiazolo[5,4-b]quinoline ring.
R
The assignment of the 13C NMR signals (Table 2) was
carried out by HETCOR and HMBC experiments. All
10a R = 3' -Cl
10b R = 3' -CN
10f R = 3' -OH
H
N
N
H
N
S
OH
SCH3
H
N
H
N
H
H
OH
Figure 3. Spatial interaction expected between H-8 and NH.
N
S
N
S
X
SCH3
SCH3
in the same experiment. This fact suggests that the ani-
lino ring is not orthogonal to the tricyclic skeleton,
but oblique. A similar spatial arrangement has been ob-
served for several 9-anilinoacridines and has been as-
cribed to a high degree of conjugation between C9 and
N-anilino atoms.11,12 Another interesting interaction
was observed for protons H-20 and H-60 and the anilino
proton Ar–NH–Ar. In the case of compounds 10a, 10f,
and 10g a strong interaction between H-20 and Ar–NH–
Ar was observed but not with H-60, while protons H-20
and H-60 in compound 10b interacted with Ar–NH–Ar.
Therefore, compounds 10a, 10f, and 10g present one
preferred conformation, while compound 10b exists in
N
N
10f
CN
H
N
H
N
H
H
CN
N
S
N
S
SCH3
SCH3
N
N
10b
Figure 4. Possible conformers in 30-substituted 9-anilinothiazolo[5,4-
b]quinolines.
Table 1. 1H NMR chemical shifts (ppm) of all novel thiazolo[5,4-b]quinoline derivatives synthesizeda
10a
10b
2.30
10c
2.38
10d
2.33
10e
2.36
10f
2.38
10g
11a
2.32
11b
2.28
11c
2.44
12
13
15
SMe
H-5
2.42
7.94
7.79
7.58
8.45
7.18
2.52
7.89
7.70
7.47
8.28
6.37
2.68
7.81
7.62
7.29
7.99
7.70
2.77
7.73
7.61
7.41
8.26
8.00
7.89
7.68
8.64
7.68
7.97
7.84
7.60
8.53
6.74
8.00
7.87
7.65
8.61
7.55
7.97
7.84
7.65
8.47
7.96
7.97
7.90
7.63
8.59
6.71
7.94
7.91
7.67
8.68
7.31
7.43
7.95
7.90
7.64
8.66
7.28
6.98
8.01
7.84
7.64
8.50
7.21
7.70
8.00
7.63
7.58
8.25
H-6
H-7
H-8
H-20
H-30
H-40
7.09
7.30
7.06
9.87
7.56
7.56
7.56
6.74
7.25
6.74
7.51
7.58
7.43
7.88
7.58
6.72
7.17
6.67
6.32
7.02
6.37
9.22
7.64
7.31
7.07
5.20
8.06
H-50
7.43
7.31
6.98
7.28
7.70
7.21
H-60
7.53
10.23
NHAr
–CONH–
NH
10.5
10.2
10.2
10.5
10.66
10.6
10.4
6.78
3.58
2.78
2.61
1.07
7.51
4.52
2.52
2.50
0.92
–NHCH2–
–CH2N–
–NCH2–
–CH3
Others
3.69
2.97
2.88
1.21
OCH3
3.71
OH
9.59
NHCH3
2.62
OCH3
3.77
NHCH3
3.39
a All spectra were carried out in DMSO-d6, except for 12, 13, and 15, which were carried out in CDCl3.

´
M. A. Loza-Mejıa et al. / Bioorg. Med. Chem. 16 (2008) 1142–1149
1145
Table 3. Apparent constants for ethidium bromide displacement from
DNA by thiazolo[5,4-b]quinoline derivatives and AHMA, as reference
methine carbon signals were assigned by HETCOR
experiments, and quaternary carbon atom signals were
assigned by HMBC experiments. These experimental
data are in agreement with the theoretical assignment
a
b
Q50
Compound
Qmax
Qmax/Q50
10a
10b
10c
10d
10f
10g
11a
11b
11c
12
2.60 (0.32)
7.84 (0.45)
6.65 (0.58)
1.62 (0.2)
1.29 (0.36)
11.54 (3.6)
3.71 (1.8)
0.53 (0.22)
7.64 (1.7)
4.18 (1.9)
2.19 (2.3)
9.48 (5.7)
14.26 (8.2)
16.57 (5.4)
11.1 (3.3)
9.44 (2.5)
7.36 (2.01)
52.46 (14.1)
22.64 (6.2)
13.62 (4.6)
6.96 (2.1)
18.45 (8.9)
2.0
6
´
carried out by Alvarez-Ibarra et al.
0.68
1.79
3.08
2.15
2.37
0.84
0.39
0.90
1.39
2.36
4.87
6.49
0.46
0.79
0.31
1.17
0.82
16.45 (1.5)
9.89 (1.8)
1.84 (0.5)
3. Biological
3.1. DNA binding (ethidium bromide displacement)
3.73 (0.69)
12.81 (2.4)
23.08 (1.4)
26.24 (1.03)
46.00 (1.12)
47.79 (1.36)
24.31 (0.18)
17.86 (0.14)
4.17 (0.03)
8.18 (0.05)
15.04 (0.19)
Apparent DNA intercalation constants were determined
by a conventional method based on quenching of the
fluorescence of the ethidium bromide–DNA complex,
as previously described.9 According to the data pre-
sented in Table 3, the compounds carrying an ethylene-
diamino group (12, 13, and 15) displace ethidium
bromide from more sites (higher Qmax). Comparison
of the binding affinity of 13 and 15 with 2 indicates that
the presence of the anilino group is unfavorable. In
addition, the position of the ethylenediamino group al-
ters the maximum quenching and binding affinity, which
was higher for 15 than for 12 and 13. On the other hand,
the compounds with a 30-anilino substituted group
(10b,10c) have higher binding affinity than compounds
40-anilino substituted (11b,11c); this roughly correlates
with the cytotoxic data. The ratio Qmax/Q50 can be con-
sidered as an apparent efficiency index because com-
pounds with higher ratios present a better combination
of binding strength and displacement ability. Accord-
ingly, the well-known DNA intercalating compound
AHMA, used here as a reference, performed better than
any other of the compounds studied, and only com-
pound 15 was nearly as good. On the opposite site, com-
pounds 11b and 10b showed efficiencies almost as low as
the compounds reported in a previous study;9 neverthe-
less, most of the substituent patterns tested here fa-
voured DNA intercalation.
13
15
AHMA
2c
3c
4c
5c
6c
a Maximum quenching.
b Concentration to give 50 % quenching of fluorescence of bound
ethidium bromide (mM). Values are means of three experiments;
standard deviation given in parentheses.
c Data taken from Ref. 9.
3.2. In vitro cytotoxicity
The results of evaluation of cytotoxic activity of thiazol-
o[5,4-b]quinolines 10a–f, 11a–c, 12, 13, and 15 as well as
2–69 against the proliferation of two cervical cell lines
(HeLa, C-33), two colorectal cancer cell lines (SW480,
SW620), and one leukemic cell line (K-562) in vitro
are shown in Table 4.
In general, the most active compounds were 2, 10b, 12,
13, and 15, four of which (2, 12, 13, and 15) carry the
Table 2. 13C NMR chemical shifts (ppm) of novel thiazolo[5,4-b]quinoline derivatives synthesized
No. C
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
14.8
12
15.2
13
15
15.4
SMe
C-2
C-3a
C-4a
C-5
14.5
14.6
14.5
14.3
14.6
163.8
143
14.8
14.6
14.6
15.3
162.9
138.4
143.5
125.9
130.2
124.8
123.9
119
163.8
138.6
142.7
125.4
130.6
125.1
124.0
119.0
159.0
131.7
143.4
124.7
110.9
126.2
129.4
126.3
162.5
137.5
144.0
128.1
129.1
124.1
123.9
119.6
161.6
132.1
146.4
106.5
159.3
107.6
128.8
113.2
163.2
140.7
141.0
129.2
131.4
125.3
124.5
118.5
142.2
131.5
141.7
119.4
129.4
120.1
126.7
123.6
161.6
141.3
142.0
123.1
131.4
124.9
124.5
117.6
157.5
130.9
150.1
111.9
156.4
114.6
128.9
110.8
162.1
143.3
146
162
161.3
133.7
139.7
127.8
133.1
125.9
125.1
117.1
158.3
130.2
145
165
167.2
137
166
162.8
140.8
146.5
128.1
129.2
124.4
122.3
117.2
160.9
146.5
134.9
142.5
126.1
132.1
125.4
124.5
122
137.6
143.5
130.7
132
142.8
144.8
127.7
128.1
126.4
123.7
125.4
158.7
125.4
143.2
127.6
130.7
125.3
124.1
119.1
158.8
131.9
147.7
115.5
138.5
117.1
129.2
115.5
143.3
124.5
129.1
124.1
128.8
118.9
166.4
134.8
147.2
119.4
135
127.5
129.4
124.2
124
C-6
C-7
125.5
124.4
120
C-8
C-8a
C-9
119.2
161
132
159.5
131.7
144
155.4
130.6
139.6
117.4
128.1
129.1
128.1
117.4
159
C-9a
C-10
133.5
146.7
120.3
132.4
103
149
106
C-20
119.9
132.4
121.1
129.5
122.3
121.8
114.4
155
C-30
138.6
110.6
128.7
107.6
C-40
121.5
129
123
C-50
114.4
121.8
132.4
120.3
C-60
–NHCH2
–CH2N
–NCH2
–CH3
Others
36.2
52.3
41.3
51.1
46.8
10.7
41.2
55.0
49.3
47.7
10.0
9.24
CN
118.8
OMe
54.9
CF3
124.3
NHCH3
30.4
OCH3
56.1
CN
119.6
C@O
161.1

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M. A. Loza-Mejıa et al. / Bioorg. Med. Chem. 16 (2008) 1142–1149
1146
Table 4. In vitro cytotoxic activity of compounds 2–6 and 10a–15a,b
Compound
HeLa
C-33
SW480
SW620
K-562
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
12
69.37 (20.92)
7.75 (1.13)
25.34 (10.86)
43.34 (11.80)
152.21 (29.77)
>200
129.33 (16.59)
15.08 (10.36)
75.41 (2.30)
59.73 (3.46)
>200
110.69 (14.64)
28.68 (16.88)
66.65 (19.93)
65.13 (5.29)
>200
129.73 (24.04)
43.75 (15.47)
26.58 (1.60)
62.28 (5.43)
160.82 (1.02)
146.34 (5.17)
118.69 (25.70)
145.95 (14.07)
110.8 (15.51)
>200
80.26 (8.36)
8.01 (0.65)
22.17 (11.33)
67.06 (22.08)
>200
146.12 (10.35)
133.98 (56.67)
>200
146.95 (59.51)
101.38 (0.04)
>200
173.34 (3.98)
46.85 (16.93)
79.45 (4.10)
77.2 (25.16)
120.01 (28.70)
12.54 (3.03)
9.28 (0.65)
7.85 (1.85)
16.8 (0.50)
143.4 (6.90)
>200
46.22 (11.55)
123.86 (78.19)
>200
>200
>200
>200
>200
140.02 (51.82)
21.69 (3.35)
12.86 (2.34)
12.97 (3.83)
15.9 (0.30)
>200
19.95 (5.59)
9.35 (1.96)
9.06 (1.86)
22.4 (4.20)
>200
13.6 (3.44)
19.48 (9.81)
27.97 (9.22)
37.7 (0.80)
>200
19.72 (0.48)
15.22 (2.34)
16.87 (2.19)
21.6 (1.80)
183.9 (69.90)
>200
13
15
2c
3c
4c
>200
153.7 (7.70)
138.8 (23.90)
>200
>200
>200
5c
176.5 (30.30)
>200
9.50 (0.60)
>200
153.9 (28.60)
16.70 (2.80)
143.4 (11.50)
85.3 (10.20)
19.90 (0.80)
6c
>200
27.70 (2.00)
Amsacrinec
8.80 (0.50)
a IC50, lM, compound concentration inhibiting 50% of cellular growth assessed by the MTT assay.
b Values are means of three experiments, standard deviation given in parentheses.
c Data taken from Ref. 9.
2-(N,N-diethyl)ethylenediamino moiety, although the
group is attached to different positions and in 2 position
is present twice. To detect if part of the cytotoxicity can
be explained by the intercalating properties, a Log Log
multiple linear correlation analysis of the ꢀLogIC50 in
Table 4 was carried out for every cell line, against the
LogQmax and the ꢀLogQ50 in Table 4. A significant
correlation (p < 0.05) was found for only Qmax and the
C33 cells, but even in this case, less than 25% of the cyto-
toxicity can be explained by the maximal intercalation
ability (adjusted R squared was 0.21) with a negative
correlation. Therefore, if the intercalation of these com-
pounds into DNA is related to their cytotoxicity, only a
subset of the DNA binding sites seems to be relevant.
It is also important to notice that compounds 10a (30-
Cl), 10b (30-CN), and 10c (30-OMe) were more potent
than compounds 11a (40-Cl), 11b (40-OMe), and 11c
(40-CN); thus, the substitution pattern in the anilino ring
is critical for biological activity.
Compound 10b is as potent as the reference compound
amsacrine despite the lack of an alkylamino residue.
Perhaps, in addition to its electron-withdrawn proper-
ties, the nitrile group acts as an acceptor in the forma-
tion of a hydrogen bond. The great difference in
activity between compounds 10c (30-OMe) and 10f (30-
OH) is in agreement with this last proposal since, in
the first case, the oxygen atom can act only as a hydro-
gen-bond acceptor, while in the second case it acts as a
donor.
As already mentioned the presence of an alkylamine res-
idue seems to improve cytotoxic activity, since com-
pounds 12, 13, and 15 were as potent as the reference
compound, amsacrine. It is interesting to note that the
change of position of the alkylamino residue has no sig-
nificant effect on cytotoxic activity.
In light of these results, we propose the following
requirements for good cytotoxic activity of 9-anilino-
thiazolo[5,4-b]quinolines: cytotoxic activity is improved
by the incorporation of an alkylamino residue, but the
location of the alkylamino residue is not critical. Incor-
poration of electron-withdrawing groups into the anili-
no ring enhances cytotoxic activity in comparison with
that of electron-releasing groups. The substitution pat-
tern of the anilino ring is critical for good cytotoxic
activity, position 30 being the most favorable. The pres-
ence of hydrogen-bond acceptors may also be relevant
to this activity.
The 9-anilinothiazolo[5,4-b]quinoline derivatives previ-
ously reported had only electron-releasing groups and
showed poor cytotoxic activity.9 Then in this study the
effect of electron-withdrawn groups was analyzed. It
was observed, in a general way, that the incorporation
of electron-withdrawn groups improves cytotoxic activ-
ity. This is in opposition to the 9-anilinoacridine deriva-
tives, where the incorporation of electron-releasing
groups is critical for good cytotoxic activity. The only
exception was compound 10e (30-nitro derivative), but
this compound had solubility problems, this possibly
being the cause of its poor activity. Therefore, if the 9-
anilinothiazolo[5,4-b]quinoline derivatives share a com-
mon mechanism of action with the 9-anilinoacridines,
at least their binding modes should differ significantly
because the structural requirements for cytotoxic activ-
ity are not the same.
4. Conclusions
A new series of 9-anilinothiazolo[5,4-b]quinoline deriva-
tives was synthesized and characterized. The preliminary
antitumor studies showed that some derivatives exhib-
ited significant cytotoxic activity in vitro. In line with
the data reported here, the presence of electron-with-
drawing or a hydrogen-bond acceptor in the anilino ring

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M. A. Loza-Mejıa et al. / Bioorg. Med. Chem. 16 (2008) 1142–1149
1147
and the incorporation of an alkylamino substituents im-
prove the cytotoxic activity of thiazolo[5,4-b]quinolines.
J = 8.4, 6.9, 1.2 Hz) H-6; 8.00 (dd, J = 8.7, 0.9 Hz) H-
5; 8.64 (d, J = 8.4 Hz) H-8; 10.5 (br) NHAr; 13C NMR
(DMSO-d6): 14.6, 110.9, 118.8, 119.0, 124.0, 124.6,
125.1, 125.4, 126.2, 126.3, 129.4, 130.6, 131.7, 138.5,
142.7, 143.3, 159.0, 163.3; MS (FAB, m/z): 349 (M++1,
100%), 348 (M+, 55%), 315 (M-33, 5.8%). Anal. Calcd
for C18H12N4S2: C, 62.05; H, 3.47; N, 16.08; S, 18.40.
Found: C, 62.40; H, 3.45; N, 16.01; S, 18.31.
5. Experimental
All starting materials were commercially available re-
search-grade chemicals and used without further purifi-
cation. Reactions were monitored by analytical TLC on
precoated silica gel 60 F254 plates (Aldrich). Column
chromatography was carried out on silica gel 60
(Merck). Melting points were determined on a Fisher–
Johns apparatus and are uncorrected. Infrared spectra
were recorded on a Nicolet FT-5SX spectrophotometer
5.1.3. 9-[[(3-Methoxy)phenyl]amino]-2-(methylthio)thiaz-
olo[5,4-b]quinoline (10c). Yellow solid; 132 mg (75%);
mp 175–179 ꢁC. IR (KBr, cmꢀ1): 3120 (NH), 3053,
2966 (CH), 2850 (O–CH), 1595, 1571, 1543, 1503,
1476, (aromatic), 1300 (C–S), 1266 (ArO); 1H NMR
(DMSO-d6, d): 2.38 (s, 3H) SCH3; 3.71 (s, 3H) OCH3;
6.74 (m, 3H) H-20, H-40, H-60; 7.25 (t, J = 8.7 Hz) H-
50; 7.60 (ddd, J = 8.4, 6.6, 1.2 Hz) H-7; 7.84 (ddd,
J = 8.7, 6.6, 1.2 Hz) H-6; 7.97 (dd, J = 8.7, 0.9 Hz) H-
5; 8.53 (d, J = 8.4 Hz) H-8; 10.2 (br) NHAr. MS (EI,
m/z): 354 (M++1, 23%), 353 (M+, 100%), 352 (M+-1,
11%), 338 (M+-15, 6%), 320 (M+-33, 21%); Anal. Calcd
for C18H15N3OS2: C, 61.16; H, 4.28; N, 11.89; S, 18.14.
Found: C, 61.23; H.4.22; N, 11.69; S, 18.03.
1
model. H and 13C NMR spectra were recorded on a
Varian VxR-300S spectrometer (300 and 75.5 MHz).
Chemical shifts are reported in ppm (d) and the signals
are described as singlet (s), doublet (d), triplet (t), quar-
tet (q), broad (br), and multiplet (m), coupling constants
are reported in Hz. EI-MS were carried out on a JEOL
JMS-AX505-HA apparatus. FAB-MS were carried out
on a JEOL Sx102 apparatus. Compounds 9 and 10 were
prepared according to procedures already described.10,11
5.1. General preparation of 2-(methylthio)-9-anilinothiaz-
olo[5,4-b]quinolines (10a–f, 11a–c, 12)
5.1.4.
9-[[(3-Trifluoromethyl)phenyl]amino]-2-(methyl-
thio)thiazolo[5,4-b]quinoline (10d). Yellow solid; 130 mg
(67%); mp 168–170 ꢁC. IR (KBr, cmꢀ1): 3124 (NH),
3054 (C–H), 1598, 1522, 1503, 1472, 1451 (aromatic),
1290 (C–S); 1H NMR (DMSO-d6, d): 2.33 (s, 3H)
SCH3; 7.43 (d J = 7.8 Hz, 1H), H-60; 7.51 (dd J = 8.4,
2.1 Hz, 1H), H-40; 7.55 (dd, J = 2.1, 2.1 Hz, 1H) H-20;
7.58 (t, J = 7.8 Hz, 1H) H-50; 7.65 (ddd J = 8.4, 6.9,
0.9 Hz, 1H) H-7, 7.87 (ddd, J = 8.1, 6.9, 0.9 Hz, 1H)
H-6; 8.00 (dd, J = 8.1, 0.9 Hz, 1H) H-5; 8.61 (dd,
J = 8.1, 0.9 Hz, 1H) H-8; 10.2 (br), NHAr. MS (FAB,
m/z): 392 (M++1, 100%), 391 (M+, 15%), 376 (M+-15,
2%), 358 (M+-33, 2%). Anal. Calcd for C18H12F3N3S2:
C, 55.23; H, 3.09; F, 14.56; N, 10.73; S, 16.38. Found:
C, 54.74; H, 2.95; N, 10.84; S, 16.62.
To compound 9 (133 mg, 0.5 mmol) were added succes-
sively methanol (5 ml) and two drops of HCl (36%). The
mixture was stirred for 10 min. Meanwhile, a mixture of
the aniline with the desired substitution pattern
(0.7 mmol) in 5 ml of methanol was prepared. This mix-
ture was added to the mixture of 9 in methanol/HCl and
heated to reflux for 6 h. After cooling, methanol was
evaporated under reduced pressure, the residue was sus-
pended in 10 ml of water and a 10% NaHCO3 solution
was added to render pH 8. The solid was collected by
vacuum filtration and washed with cold acetone. In
the case of compound 12 crystallization from ethanol–
water was used for purification.
5.1.5. 9-[[(3-Nitro)phenyl]amino]-2-(methylthio)thiazol-
o[5,4-b]quinoline (10e). Yellow solid; 140 mg (76%); mp
201–204 ꢁC. IR (KBr, cmꢀ1): 3000 (NH), 2990 (C–H),
1595, 1571, 1543, 1503, 1476, (aromatic), 1518 (NO2),
1348 (N–O), 1300 (C–S); 1H NMR (DMSO-d6, d):
2.36 (s, 3H) SCH3; 7.53 (ddd, J = 8.1, 2.1, 2.1 Hz, 1H),
H-60; 7.58 (t, J = 8.1 Hz, 1H), H-50; 7.65 (ddd, J = 8.4,
6.9, 1.2 Hz, 1H) H-7; 7.84 (ddd, J = 8.4, 6.9, 1.2 Hz,
1H) H-6; 7.88 (ddd J = 8.1, 2.1, 2.1 Hz, 1H) H-40, 7.96
(t J = 2.1 Hz, 1H) H-40; 7.97 (d, J = 8.1 Hz, 1H) H-5;
8.47 (d, J = 8.1 Hz, 1H) H-8; 10.23 (br), NHAr. MS
(EI, m/z): 369 (M++1, 23%), 368 (M+, 100%), 367(M+-
1, 2.5%), 353 (M+-15, 2.8%), 338 (M+-30, 32%), 335
(M+-33, 7.5%); Anal. Calcd for C17H12N4O2S2: C,
55.42; H, 3.28; N, 15.21; O, 8.68; S, 17.41.Found: C,
55.70; H, 3.38; N, 15.10; O, 8.73; S, 17.09.
5.1.1. 9-[[(3-Chloro)phenyl]amino]-2-(methylthio)thiazol-
o[5,4-b]quinoline (10a). Yellow solid; 133 mg (74.5%);
mp 185–187 ꢁC. IR (KBr, cmꢀ1): 3108 (NH), 3050 (C–
H), 1572, 1548, 1516, 1477, 1430, (aromatic), 1266 (C–
1
S); H NMR (DMSO-d6, d): 2.42 (s, 3H) SCH3; 7.06
(m, 1H), H-60; 7.09 (m, 1H), H-40; 7.18 (t, J = 2.1 Hz,
1H) H-20; 7.30 (t, J = 8.1 Hz, 1H) H-50; 7.58 (ddd,
J = 8.1, 6.6, 2.1 Hz, 1H) H-7, 7.79 (ddd, J = 8.1, 6.6,
1.2 Hz, 1H) H-6; 7.94 (dd, J = 8.4, 0.9 Hz 1H) H-5;
8.45 (d, J = 8.4 Hz, 1H) H-8; 9.87 (br), NHAr; MS
(FAB, m/z): 360 (M++3, 43.5%), 358 (M++1, 100%),
357(M+, 25%), 324 (M+ꢀ33, 2.8%). Anal. Calcd for
C17H12ClN3S2: C, 57.05; H, 3.38; N, 11.74; S, 17.92.
Found: C, 56.97; H, 3.39; N 11.59; S, 17.55.
5.1.2. 9-[[(3-Cyano)phenyl]amino]-2-(methylthio)thiazol-
o[5,4-b]quinoline (10b). Yellow solid; 136 mg (72%); mp
210–212 ꢁC. IR (KBr, cmꢀ1): 3245 (NH), 2231 (CN),
1624, 1573, 1550, 1498, 1473, 1433 (aromatic), 1286
(C–S); 1H NMR (DMSO-d6, d): 2.30 (s, 3H) SCH3;
7.56 (m, 3H) H-40, H-50, H-60; 7.68 (ddd, J = 8.4, 6.9,
1.2 Hz) H-7; 7.86 (t, J = 1.5 Hz) H-20; 7.89 (ddd,
5.1.6. 9-[[(3-Hydroxy)phenyl]amino]-2-(methylthio)thiaz-
olo[5,4-b]quinoline (10f). Yellow solid; 124 mg (73%); mp
227–230 ꢁC. IR (KBr, cmꢀ1): 3147 (NH), 2775 (O–CH),
1607, 1573, 1549, 1497, 1453, (aromatic), 1268 (C–S),
1222 (ArO); 1H NMR (DMSO-d6, d): 2.38 (s, 3H)
SCH3; 6.67 (ddd, J = 8.1, 1.5, 1.5 Hz, 1H), H-40; 6.71

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1148
(t, J = 1.2 Hz, 1H), H-20; 6.72 (m, 1H) H-60; 7.17 (t,
J = 8.7 Hz) H-50; 7.63 (ddd, J = 8.4, 6.6, 1.2 Hz) H-7;
7.90 (ddd, J = 8.7, 6.9, 0.9 Hz) H-6; 7.97 (dd, J = 8.4,
1.2 Hz) H-5; 8.59 (d, J = 8.4 Hz) H-8; 9.59 (s) OH;
10.2 (br) NHAr. MS (EI, m/z): 340 (M++1, 23%), 339
(M+, 100%), 338 (M+-1, 11%), 338 (M+-15, 5%), 320
(M+-33, 32%); Anal. Calcd for C17H13N3OS2: C,
60.15; H, 3.86; N, 12.38; 0, 4.71; S, 18.89. Found: C,
60.31; H, 3.56; N, 12.18; S, 18.49.
8.50 (d, J = 8.4 Hz, 1H) H-8; 10.41 (br, 1H) NH. MS
(FAB, m/z): 349 (M++1, 100%), 348 (M+, 72%); Anal.
Calcd for C18H12N4S2: C, 62.05; H, 3.47; N, 16.08; S,
18.40. Found: C, 61.97; H, 3.49; N, 15.98; S, 18.29.
5.1.11. 9-[[[3-[2-(N,N-Diethylamino)ethyl]carbamoyl]phe-
nyl]amino]-2-(methylthio)thiazolo[5,4-b]quinoline
(12).
White solid; 118 mg (68%); mp 178–180 ꢁC. IR (KBr,
cmꢀ1): 3250 (NH), 3057, 2964, 2926, 2812 (C–H), 1581
(C@N), 1550, 1521, 1467 (aromatic), 1288 (C–S); 1H
NMR (CDCl3,, d): 1.21 (t, J = 7.2 Hz, 6H) CH3; 2.68 (s,
3H), SCH3, 2.88 (c, J = 7.2 Hz, 4H) –NCH2–; 2.97 (t,
J = 5.1 Hz, 2H) –CH2N–; 3.69 (td, J = 5.7, 5.1 Hz, 2H)
NHCH2–; 5.20 (br, 1H), NH; 7.07 (dd, J = 7.8, 2.1 Hz,
1H) H-60; 7.29 (ddd, 8.7, 6.9, 1.2 Hz,1H) H-7; 7.31 (t,
J = 7.8 Hz, 1H) H-50; 7.62 (ddd, J = 8.7, 6.6, 1.2 Hz,
1H) H-6; 7.64 (dd, J = 7.2, 1.8 Hz), H-40; 7.70 (t,
J = 1.8 Hz) H-20; 7.81 (dd, J = 8.4, 0.9 Hz) H-5; 7.99
(dd, J = 8.4, 0.9 Hz, 1H) H-8; 8.07 (br, 1H) CONH. MS
(EI, m/z): 466 (M++1, 3%), 465 (M+, 8%), 450 (M+-15,
4%), 393 (M+-72, 9%), 366 (M+-99, 23%), 350 (M+-115,
10%), 100 (M+-365, 10%), 86 (M+-379, 100%); Anal.
Calcd for C24H27N5OS2: C, 61.91; H, 5.84; N, 15.04; S,
13.77. Found. C, 61.74; H, 5.97; N, 14.97; S, 13.91.
5.1.7. 9-[[(3-N-Methylamino)phenyl]amino]-2-(methylthio)-
thiazolo[5,4-b]quinoline (10g). Yellow solid; 131 mg
(74.5%); mp 173–175 ꢁC. IR (KBr, cmꢀ1): 3414 (NH),
2921 (C–H), 1588, 1549, 1494, 1464 (aromatic), 1298
(C–S); 1H NMR (DMSO-d6, d): 2.52 (s, 3H) SCH3;
2.62 (s, 3H) NHCH3; 6.32 (d, J = 8.4 Hz) H-40; 6.37
(m, 2H) H-20, H-60; 7.02 (t, J = 8.4 Hz, 1H) H-50; 7.47
(ddd, J = 8.4, 6.9, 1.2 Hz) H-7; 7.70 (ddd, J = 8.1, 6.9,
1.2 Hz) H-6; 7.89 (dd, J = 8.4, 1.2 Hz) H-5; 8.28 (dd,
J = 8.7, 0.9 Hz) H-8; 9.22 (s) NH. MS (FAB, m/z): 353
(M++1, 100%), 352 (M+, 61%), 351 (M+-1, 12.8%),
337 (M+-33, 2%), 319 (M+-33, 2%); Anal. Calcd for
C18H16N4S2 C,61.34; H, 4.58: N, 15.90; S, 18.19. Found:
C, 61.54; H, 4.62; N, 15.79; S, 18.05.
5.1.8. 9-[[(4-Chloro)phenyl]amino]-2-(methylthio)thiazol-
o[5,4-b]quinoline (11a). Yellow solid; 125 mg (70%); mp
186–187 ꢁC. IR (KBr, cmꢀ1): 3196 (NH), 3104, 2989
(C–H), 1627, 1573, 1540, 1486, 1467 (aromatic) 1295
(C–S); 1H NMR (DMSO-d6, d): 2.32 (s, 3H) SCH3;
7.31 (d, J = 8.4 Hz, 2H) H-20, H-60; 7.43 (d,
J = 8.8 Hz, 2H) H-30, H-50; 7.67 (t, J = 7.6 Hz, 1H) H-
7; 7.91 (t, J = 7.6 Hz, 1H) H-6; 7.94 (d, J = 8.4, 1H)
H-5; 8.68 (d, J = 8.4 Hz, 1H) H-8; 10.66 (s,1H) NH.
MS (FAB, m/z): 360 (M++3, 43.5 %), 358 ((M++1),
100%), 357 (M+, 25%), 342 (M+-15, 5%), 324 (M+-33,
2%); Anal. Calcd for C17H12ClN3S: C, 57.05; H, 3.38;
N, 11.74; S, 17.92. Found: C,57.01; H, 3.37; N, 11.62;
S, 17.57.
5.1.12. 9-Chloro-2-[[2-(N,N-diethylamino)ethyl]amino]-
thiazolo[5,4-b]quinoline (13). A solution of 9 (500 mg,
1.88 mol) in 0.3 ml of N,N-diethylethylenediamine was
stirred for 48 h at room temperature. To the brownish
solution, dichloromethane (30 ml) was added and the
resulting solution was successively extracted with a sat-
urated aqueous solution of NH4Cl (3 · 10 ml), water
(3 · 10 ml) and dried over Na2SO4. After concentration
under reduced pressure, the pale yellow crude product
was purified by column chromatography (dichlorometh-
ane/methanol/NH4OH 99:1:0.1) to give 13 as white crys-
tals. 13: 475 mg (75%), IR (KBr, cmꢀ1): 3202 (N–H),
2968, 2968, 2815(CH2, CH3); 1H NMR (CDCl3, d):
1.07 (t, J = 7.2, 6H) 2CH3; 2.61 (c, J = 7.2, 4H) 2CH2;
2.78 (t, J = 6.0, 2H) CH2; 3.58 (ta, 2H) CH2; 6.78 (br,
1H) NH; 7.63 (ddd, J = 8.1, 6.9, 1.8, 1H) H-6; 7.58
(ddd J = 7.8, 6.6, 1.5, 1H, H-7); 8.00 (dd, J = 8.1, 1.5,
1H, H-5); 8.25 (dd, J = 8.1, 1.2, 1H) H-8. MS (FAB,
m/z): 337(M++3, 35%), 335 (M++1, 100%), 333 (M+-1,
24%), 262 (M+-72, 44%), 100 (M+-234, 33.5%), 86
(M+-248, 67%); Anal. Calcd for C16H19ClN4S: C,
57.39; H 5.72; N, 16.73; S, 9.58. Found: C, 57.73; H,
5.67; N, 16.63; S, 9.68.
5.1.9. 9-[[(4-Methoxy)phenyl]amino]-2-(methylthio)thiaz-
olo[5,4-b]quinoline (11b). Yellow solid; 120 mg (68%);
mp 205–207 ꢁC. IR (KBr, cmꢀ1): 3196 (NH), 3200
(NH), 3062, 3028 (C–H), 2835 (O–CH), 1608, 1573,
1546, 1510, 1475 (aromatic), 1295 (C–S); 1H NMR
(DMSO-d6, d): 2.28 (s, 3H) SCH3; 3.77 (s, 3H) OCH3;
6.98 (d, J = 8.4 Hz, 2H) H-30, H-50; 7.28 (d,
J = 8.4 Hz, 2H) H-20, H-60; 7.64 (t, J = 7.6 Hz, 1H) H-
7; 7.90 (t, J = 7.6 Hz, 1H) H-6; 7.95 (d, J = 8.4, 1H)
H-5; 8.66 (d, J = 8.4 Hz, 1H) H-8; 10.68 (s,1H) NH;
MS (FAB, m/z): 355 (M++2, 32%), 354 (M++1, 100%),
353 (M+, 23%), 338 (M+-15, 7%); Anal. Calcd for
C18H15N3OS2: C, 61.16; H, 4.28; N, 11.89; S, 18.14.
Found: C, 61.25; H, 4.24; N, 11.66; S, 18.02.
5.1.13. 5-(Phenylamino)-4-[[2-(N,N-diethylamino)ethyl]car-
bamoyl]-2-(methylthio)thiazole (14). The carboxylic acid
used as precursor of amide 14 was obtained with high
yields (90%) by saponification of ester 8, by a procedure
already described.10,11 To a suspension of the 1,10-carbon-
yldimidazole (2.5 mmol) in 5 ml of dichloromethane,
500 mg (1.87 mmol) of the carboxylic acid was added.
The mixture was stirred and heated to reflux for 1 h. After
reflux, a solution of0.2 ml of N,N-diethylethylenediamine
in 5 ml of dichloromethane was added to the reaction mix-
ture and heated to reflux for 4 h. After cooling, 20 ml of
dichloromethane was added. The solution was washed
with a 10% NaHCO3 solution (3 · 10 ml), a saturated
aqueous solution of NH4Cl (3 · 10 ml), water
5.1.10. 9-[[(4-Cyano)phenyl]amino]-2-(methylthio)thiazol-
o[5,4-b]quinoline (11c). Yellow solid; 118 mg (68%); mp
178–180 ꢁC. IR (KBr, cmꢀ1): 3423 (NH), 2222 (CN),
1622, 1573, 1532, 1497, 1424 (aromatic), 1274 (C–S);
1H NMR (DMSO-d6, d): 2.44 (s, 3H), SCH3; 7.21 (d,
J = 8.8 Hz, 2H) H-20, H-60; 7.64 (t, J = 7.2 Hz, 1H) H-
7; 7.70 (d, J = 8.8 Hz, 2H) H-30, H-50; 7.84 (t,
J = 7.2 Hz, 1H) H-6; 8.01 (d, J = 8.4 Hz, 1H) H-5;

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1149
(3 · 10 ml), and dried over Na2SO4. After concentration
of the solution under reduced pressure, the pale yellow
crude oil product was purified by column chromatogra-
phy (dichloromethane/methanol/NH4OH 99:1:0.1) to
give 14 as a colorless oil. 14: 515 mg (75%); IR (KBr,
cmꢀ1): 3401(N–H), 2966, 2926, 2811 (CH2, CH3), 1633
(C@O), 1589, 1533, 1526, 1496 (aromatic); 1H NMR
(CDCl3,, d): 1.07 (t, J = 7.1, 6H) 2CH3; 2.60 (t, J = 7.2,
4H), 2CH2; 2.63 (s, 3H) SCH3; 2.70 (c, J = 7.2, 2H)
CH2; 3.5 (dt, 2H) CH2; 7.1 (dd, J = 7.2, 1.2, 1H) H-40;
7.32 (ddd, J = 8.7, 7.5, 1.8, 2H) H-30, H-50; 7.15 (dd,
J = 8.7, 1.2, 2H) H-20, H-60; 7.45 (t, 1H) CONH; 10.29
(s, 1H) NH; MS (FAB, m/z): 365 (M++1, 100%), 364
(M+, 47.5%), 292 (M+-72, 35%), 249 (M-115, 22.5%).
Tris–HCl buffer at pH 7.4, 0.15 M NaCl, and 5 mM
ethidium bromide (ultrapure from Gibco, BRL, New
York, USA) were mixed with serial additions of the
compounds to be tested dissolved in 100% dimethyl-
sulfoxide (DMSO), and the fluorescence intensity of
the solution was recorded at 584 nm with an excita-
tion light of 546 nm. The DMSO concentration never
exceeded 8%. The effect of this amount of DMSO was
small and had no effect on the shape of emission or
excitation fluorescence spectra of a DNA–ethidium
bromide complex as compared with that determined
in 100% aqueous buffers. The recorded fluorescence
change was corrected for the dilution caused by serial
additions of this solvent.
5.1.14. 9-[[2-(N,N-diethylamino)]ethyl]amino]-2-(methyl-
thio)-thiazolo[5,4-b]quinoline (15). Compound 15 was
prepared from 14 with a procedure already described.6,9
The crude product was purified by column chromatog-
raphy (CH2Cl2/CH3OH/NH4OH 99:1:0.1) to render a
yellowish oil, which was treated with hexane to give 15
as white crystals (30%). 15: mp 94–98 ꢁC, IR (KBr,
cmꢀ1): 3308 (N–H), 2970 (C–H), 1554, 1531, 1497,
1470(aromatic), 1267; 1H NMR (CDCl3,, d): 0.92 (t,
J = 7.2, 6H) 2CH3; 2.50 (c, 4H) 2CH2; 2.52 (t, J = 7.2,
2H) CH2; 2.77 (s, 3H) SCH3; 2.70 (t, J = 7.2, 2H)
CH2; 4.58 (dt, 2H) CH2; 7.51 (t, J = 6.3, 1H) NH; 7.41
(ddd, J = 8.4, 6.9, 1.5, 1H) H-7; 7.61 (ddd J = 6.9, 6.0,
1.5, 1H); H-6 7.73 (dd, J = 8.4, 0.9 Hz, 1H) H-5; 8.26
(dd, J = 8.7, 0.6, 1H) H-8; MS (CI, m/z): 347 (M++1,
39%), 346 (M+, 24.5%) 274 (M+-72, 4.5%), 260 (M+-
86, 4.5%), 100 (M+-247, 10%), 86 (M+-261, 100%). Anal.
Calcd for C17H22N4S2: C, 57.39; H, 5.72; N, 16.73; S,
9.58. Found: C, 57.33; H, 5.77; N, 16.70; S, 9.49.
The concentration of the compounds tested varied in the
range of 1–100 lM depending on their respective solubil-
ity. The precipitation of the compound from the solution
was detected from an increase in 600 nm light dispersal at
90 degrees in a Shimadzu RF5000U fluorescence spectro-
photometer. The displacement curves were fitted by non-
linear regression analysis to a rectangular hyperbola.
Acknowledgments
´
We thank Maricela Gutierrez, Rosa Isela del Villar, Vic-
tor M. Arroyo, Georgina Duarte, and Margarita Guz-
´
´
man for determination of all spectra and Nayeli Lopez
for elemental analysis. We also thank DGAPA-UNAM
for financing project PAPIIT IN202805 as well as Facul-
tad de Qu´ımica, UNAM for financial support (PAIP
6390-10, 6290-9).
5.2. Cytotoxic assay
References and notes
The effects of the compounds were determined in two cer-
vical cell lines (HeLa, C-33), two human colorectal cancer
cell lines (SW480 and SW620), and one myelogenous leu-
kemia human cell line (K-562). The cytotoxic assays were
performed according to the microculture MTT method.
Cells were harvested at 4.5–5.0 · 104 cells/ml/well and
inoculated in 24 well microtiter plates. Then the culture
cells were inoculated free and with the compounds (which
were dissolved in DMSO and added in a volume maxi-
mum of 2 ml/ml/well). After 72 h incubation, 100 mg/ml
of MTT (in PBS, pH 7.2) was added. Adding 1 ml of
DMSO to each well, followed by gentle shaking, dissolved
the formazan dye. After centrifugation the extinction
coefficient was measured at 540 nm using a Beckman
photometer model DUR-64. Cell growth inhibition
was determined by the formula % cell growth inhibi-
tion = (1 ꢀ absorbance of treated cells/ absorbance of
untreated cells) · 100. The assays were carried out in three
independent experiments in quadruplicate.
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5.3. DNA affinity and intercalation
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