Synthesis and evaluation of 9-anilinothiazolo[5,4-b]quinoline derivatives as potential antitumorals

Article abstract of DOI:10.1016/j.ejmech.2003.05.002

Five new 9-anilinothiazolo[5,4-b]quinoline derivatives (compounds 5, 7, 9, 10, 11) have been prepared. Some of the compounds were prepared by coupling properly substituted anilines to the novel compound 9-chloro-2-(methylthio) thiazolo[5,4-b]quinoline. Of

Full text of DOI:10.1016/j.ejmech.2003.05.002

European Journal of Medicinal Chemistry 39 (2004) 5–10
www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of 9-anilinothiazolo[5,4-b]quinoline derivatives
as potential antitumorals
Pilar Rodríguez-Loaiza a, Angelina Quintero b, Rogelio Rodríguez-Sotres c,
José D. Solano b, Alfonso Lira-Rocha a,*
a Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, 04510, México
b Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, 04510, México
c Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, 04510, México
Received 7 March 2003; received in revised form 26 May 2003; accepted 26 May 2003
Abstract
Five new 9-anilinothiazolo[5,4-b]quinoline derivatives (compounds 5, 7, 9, 10, 11) have been prepared. Some of the compounds were
prepared by coupling properly substituted anilines to the novel compound 9-chloro-2-(methylthio)thiazolo[5,4-b]quinoline. Of these,
compound 7 (9-anilino-2-[[2-(N,N-diethylamino)ethyl]amino]thiazolo[5,4-b]quinoline) showed the best cytotoxic activity in several cell
lines. All compounds demonstrated DNA binding in nanomolar range. Compound 7 inhibited the 14C-thymidine incorporation into DNA.
Results indicate that these derivatives deserve more considerations as potential antitumoral drugs.
© 2003 Elsevier SAS. All rights reserved.
Keywords: 9-anilinothiazolo[5,4-b]quinoline derivatives; Antitumoral; DNA intercalation; Cytotoxic activity
1. Introduction
H3CO
HN
NHSO2CH3
Several tricyclic compounds have been studied in the
search of better antitumoral agents. Particularly,
9-aminoacridines, 9-arylacridines, 9-alkylaminoacridines or
9-arylaminoacridines, with or without substituents at posi-
tions 3, 4, or/and 5, have been characterized extensively
[1–4]. The activity of these compounds seems to be due to
their interaction with DNA. One example is amsacrine 1
(N-[4-(9-acridylamino)-3-methoxy-phenyl]methane-
sulphonamide, m-AMSA), a potent antileukemic drug and a
moderate DNA-intercalating agent [5,6] (Fig. 1).
Recently, a new approach to this kind of compounds was
reported byAlvarez-Ibarra et al. [7] where benzene moiety in
these acridines was isosterically replaced with thiazole. Sev-
N
Fig. 1. m-AMSA structure 1.
eral 9-hydroxy or 9-alkylaminothiazolo[5,4-b]quinoline de-
rivatives were synthesized and evaluated in three cell lines
(P-388, A-549, Ht-29) demonstrating good cytotoxic activ-
ity. This resulted in the formation of a new class of potential
antitumor agents.
The present study describes the synthesis, the preliminary
antitumoral evaluation, and the intercalating properties of
thiazolo[5,4-b]quinoline derivatives that combine the gen-
eral substitution pattern of acridine derivatives with
thiazolo[5,4-b]quinoline template.
Abbreviations: Ac2O, acetic anhydride; AcOH, acetic acid; DMSO,
dimethyl sulfoxide; EtOH, ethanol; H2O2, hydrogen peroxide; KMnO4,
potassium permanganate; KOH, potassium hydroxide; MeOH, methanol;
Na2SO4, sodium sulfate; NaHCO3, sodium bicarbonate; POCl3, phosphorus
oxychloride; PPA, polyphosphoric acid; Py, pyridine; SOCl2, thionyl chlo-
ride.
2. Chemistry
The synthesis of the novel compounds is outlined in
* Corresponding author.
E-mail address: lira@servidor.unam.mx (A. Lira-Rocha).
Fig. 2. The preparation of 7 is based on the methodology used
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.05.002

6
P. Rodríguez-Loaiza et al. / European Journal of Medicinal Chemistry 39 (2004) 5–10
O
O
HN
EtO
O
N
R
O
N
c
a
N
S
SCH3
R
N
H
SCH3
S
N
H
S
N
2
3 R = OH
5 R = SCH3
b
d
e
4 R = NH-C6H5
6 R = SO2CH3
7 R = HNCH2CH2N(Et)2
c
R
Cl
N
f
HN
SCH3
S
N
S
N
SCH3
8
N
h
9 R = NH2
10 R = NHAc
g
NH2
OH
HN
N
SCH3
S
N
11
Fig. 2. (a) KOH/EtOH; (b) (1) SOCl2/Py/C6H6, (2) aniline/C6H6; (c) POCl3/PPA; (d) H2O2/AcOH; (e) H2NCH2CH2N(Et)2; (f) HCl/MeOH/
m-phenylendiamine; (g)Ac2O/Py; (h) HCl/MeOH/3,5-diaminobenzyl alcohol.All compounds were characterized by mp, spectroscopic data (IR, 1H NMR, MS)
and elemental analysis.
by Alvarez-Ibarra et al. [7], with some modifications. The
sequence of reactions started from compound 2. Basic hy-
drolysis of 2 afforded the carboxylic acid derivative 3, which
was treated with SOCl2 and the acid chloride formed was
treated in situ with aniline to give compound 4. Cyclization
of the amide 4 to thiazolo[5,4-b]quinoline derivative 5 was
achieved by the treatment of 4 with POCl3/PPA. When we
tried to oxidize compound 5 with KMnO4/AcOH, according
to a reported procedure, we obtained the starting material.
The desired compound was obtained in good yield by making
react 5 with H2O2 in AcOH. The incorporation of the N,N-
diethylethylendiamine group at position 2 of thiazolo[5,4-
b]quinoline ring was achieved in a moderate yield. All at-
tempts to increase this yield were unsuccessful. Alvarez-
Ibarra et al. [7] reported quantitative yields for the same
reaction. We presume that the anilino group at position 9 ex-
erts some influence on this reaction. On the other hand, the
preparation of the other 9-anilinothiazolo[5,4-b]quinoline
derivatives was carried out by using a different approach.
Thus, to prepare 9, novel compound 8 (prepared by treating 2
with POCl3/PPA at 136 °C for 4 h) and m-phenylendiamine
were heated under reflux for 8 h, under acid catalysis. Several
attempts under neutral or basic conditions were unsuccess-
ful. Following this procedure, but using 3,5-diaminobenzyl
alcohol instead, compound 11 was prepared. The compound
10 was obtained by treating 9 withAc2O/Py mixture at 25 °C.
3. Biological activity
3.1. Cells and cytotoxic assay
Cytotoxic assay and 14C-thymidine incorporation into
DNA were carried out using a reported procedure [8]. The
results are shown in Tables 1 and 2, respectively.
3.2. Ethidium displacement
DNA intercalation was determined from the displacement
of ethidium bromide from DNA [9]. The results are shown in
Table 3.
4. Results and discussion
We selected compounds 12 and 13 as templates for this
study. 9-Anilinoacridine 12 is a DNA intercalating agent [10]
but it does not have antitumor activity. Compound 13
(AHMA) exhibits potent antitumor activity against L-1210
and HL-60 and various murine tumors both in vitro and in
vivo [11] as well as intercalating properties (Fig. 3).
Compounds 14 and 15, also used as templates, are re-
ported byAlvarez-Ibarra et al. [7] as good antitumoral agents
against P-388, A-549 and HT-29 cell lines. The intercalating

P. Rodríguez-Loaiza et al. / European Journal of Medicinal Chemistry 39 (2004) 5–10
7
Table 1
Cytotoxic activity of compounds 5, 7, 9–11 and the reference compound m-AMSA in human cancer cell lines a,b
Compounds
MCF-7
HeLa
Calo
C-33
SW480
>200
SW620
CHO
K-562
5
7
9
10
>200
>200
198.3 (15.8)
29.9 (1.5)
128.1 (25.4)
>200
153.7 (7.7)
22.4 (4.2)
138.8 (23.9)
>200
>200
>200
>200
16.6 (2.7)
74.3 (19.2)
132.3 (28.5)
138.5 (23.4)
4.1 (1.2)
15.96 (0.3)
176.5 (30.3)
>200
37.7 (0.8)
>200
21.6 (1.8)
>200
30.6 (11.1)
136.8 (18.2)
58.8 (2.7)
111.4 (6.5)
9.3 (2.7)
16.8 (0.5)
143.4 (11.5)
85.3 (10.2)
143.4 (6.9)
19.9 (0.8)
>200
153.9 (28.6)
183.9 (69.9)
16.7 (2.8)
11
>200
106.4 (2.1)
18.3 (1.5)
>200
>200
m-AMSA
9.5 (0.6)
8.8 (0.5)
27.7 (2.0)
a IC50, µM, compound concentration inhibiting 50% of cellular growth assessed by MTT assay.
b Values are means of three experiments, standard deviation is given in parentheses.
properties of these compounds have not been reported yet
(Fig. 4).
compound 9 is accompanied by a higher affinity for these
sites. In this case, it appears that the presence of the amino
group increases the affinity while the acyl derivative, com-
pound 7, has lower affinity suggesting that the amino group
favors binding.
On the basis of our hypothesis given above, we propose
that compounds 5, 7, 9–11 (Fig. 2) should differ in their
cytotoxic and intercalating properties, because the anilino
group at position 9 enhances DNA intercalation, as it inserts
itself on to the minor groove. The various substituents were
chosen to modify the intercalation and the antitumoral prop-
erties. The effect of a diethylethylendiamine substituent at
position 2 was also studied, because compound 15 is 12-fold
more active than compound 14.
At the present time, we have evaluated the cytotoxic activ-
ity of compounds 5, 7, 9–11. The results, listed in the Table 1,
show that compound 7 was more potent than compounds 5,
9–11 in all the cell lines.
5. Conclusions
The results indicated that compound 9 formed the tightest
intercalation complex, but this did not correlate with its
cytotoxic activity. On the other hand, compound 7 formed the
weakest and most extensive complexes with DNA being
highly cytotoxic.A similar trend has been observed in several
studies. For instance, trying to establish a correlation be-
tween DNA-drug binding and cytotoxicity, it was observed
that m-AMSA showed the weakest intercalation complexes,
but its cytotoxic activity was higher than the other com-
pounds. On the contrary, o-AMSA showed a higher affinity
for DNA than m-AMSA but the former was inactive as a
cytotoxic agent [12–14]. We know that the real target of
many tricyclic compounds is the topoisomerase II and the
intercalating ability of many compounds is not reflective of
good antitumoral property. Further studies are required to
propose a plausible action mechanism. Our current efforts
are directed toward improving the antitumoral properties. We
propose that 9-anilinothiazolo[5,4-b]quinoline derivatives
are potential antitumorals.
We assayed 14C-thymidine incorporation into DNA in the
cancer cell lines to evaluate whether there is inhibition of cell
growth (at the DNA synthesis level) by compound 7. The
results (Table 2) indicate that compound 7 inhibits DNA
replication.
Comparison of cytotoxic activity data between compound
5 and 9 showed that the incorporation of an amino group to
the anilino ring increased the activity and, in some cases, the
additional incorporation of hydroxymethyl group showed
enhanced activity. On the other hand, the acyl derivative of
compound 9, compound 10, showed lower activity on some
cell lines. It appears that the amino group plays significant
role in enhancing the cytotoxic properties of the compounds.
The ethidium bromide displacement assay was also car-
ried out to evaluate the DNA-binding affinity of compounds
5, 7, 9–11 (Table 3). All compounds were able to displace
ethidium bromide from DNA.
6. Experimental protocols
6.1. Chemistry
The results are consistent with compound 7 binding to
more sites than compound 9, which suggests that compound
9 binds to a specific subset of DNA sites, while compound 7
may have a broader specificity. The higher specificity of
Melting points were determined on a Fisher-Jones appa-
ratus and are uncorrected. Infrared spectra were recorded on
a Nicolet FT-5SX spectrophotometer model. 1H NMR spec-
Table 2
Inhibition of 14C-thymidine incorporation into DNA by compound 7 and m-AMSA in human cancer cell lines
Cell line
MCF-7
HeLa
Calo
C-33
SW-480
SW-620
CHO
80.44 (9.38) 73.28 (14.58)
73.7 (6.81) 76.8 (9.38)
K-562
Compound 7
m-AMSA
83.14 (3.24) 78.84 (0.48) 45.64 (5.25) 44.43 (9.04)
84.91 (3.13) 73.58 (6.70) 62.67 (7.39) 74.56 (11.36)
55.63 (2.96)
81.49 (5.20)
59.35 (9.30)
60.93 (16.79)
a Concentration of each compound (20 µM) in each experiment. The results are expressed as % of the inhibition of 14C-thymidine incorporation in DNA in
relation to the control (without compound).
b Values are means of three experiments, standard deviation is given in parentheses.

8
P. Rodríguez-Loaiza et al. / European Journal of Medicinal Chemistry 39 (2004) 5–10
Table 3
The reaction mixture was heated under reflux for 30 min.
Normal work up after acidification, isolation and crystalliza-
tion from EtOH afforded 3 as pink crystals. Yield: 3.51 g
(90.23%). m.p.: 142–144 °C; IR (KBr): 3428, 3258, 3018,
1638, 1550, 1232 cm–1; 1H NMR (CDCl3): d 10.7 (br s; 1H);
9.6 (s; 1H, D2O exchangeable); 7.3 (dd; 2H, J = 7.5, 7.8 Hz);
7.2 (d; 2H; J = 8 Hz); 7.1 (dd; 1H, J = 7.5, 7.2 Hz); 2.6 (s;
3H); MS (EI): m/z 267 (M++1, 21%); 266 (M+, 56%); 221
(M+-45, 100%).
Apparent constant of ethidium bromide displacement from DNA by com-
pounds 5, 7, 9–11
b
Compounds
Q a
Qmax
5
7
9
10
11
13.62 (4.64)
52.46 (14.08)
6.96 (2.095)
18.447 (8.97)
22.64 (6.207)
0.2987 (0.03)
1.7428 (0.1769)
0.5861 (0.045)
1.0781 (0.1982)
1.2804 (0.1418)
a Concentration to give 50% quenching of fluorescence of bound ethidium
(nM). Values are means of three experiments, standard deviation is given in
parentheses.
6.1.3. 4-(Phenylcarbamoyl)-2-(methylthio)-5-(phenylamino)
thiazole 4
b Maximum quenching.
Obtained as greenish crystals from 3 (3.03 g, 11.4 mmol)
according to a procedure already described [7].Yield: 2.98 g
(76.8%). m.p.: 99–102 °C.; IR (KBr): 3364, 1646, 1594,
1560, 1526 cm–1; 1H NMR (CDCl3): d 10.3 (s; 1H); 8.8 (s;
1H); 7.65 (dd; 2H, J = 9.0, 1.2 Hz); 7.3 (m; 4H); 7.2 (dd; 2H,
J = 8.7, 1.0 Hz); 7.1 (t, 2H, J = 5 Hz); 2.7 (s; 3H); MS (EI):
m/z 341 (M+,100%); 248 (M+-93, 25%); 215 (M+-126, 58%).
tra were recorded on a Varian VxR (300 MHz). Chemical
shifts are reported in ppm (d) and the signals are described as
singlet (s), doublet (d), triplet (t), quartet (q) broad (br), broad
singlet (br s) and multiplet (m). Coupling constants are re-
ported in Hz. EI-MS was done on a JEOL JMS-AX505-HA
apparatus. CI- and FAB-MS were done on a JEOL Sx102
apparatus. Silica gel plates (Merck254) were used for analyti-
cal chromatography. Solvents and reagents were purchased
from the commercial vendors in the appropriate grade and
were used without further purification.
6.1.4. 9-Anilino-2-(methylthio)thiazolo[5,4-b]quinoline 5
To compound 4 (1 g, 2.9 mmol) were added PPA (213 mg)
and POCl3 (0.9 ml). The reaction mixture was vigorously
stirred at 130–135 °C for 4 h. The reaction mixture was
cooled at room temperature, 1 ml of EtOH was carefully
added and the mixture was concentrated at reduced pressure.
The suspension was poured into water (20 ml) and neutral-
ized with a saturated solution of NaHCO3. The yellow–
orange precipitate formed was collected and dried by
vacuum filtration. The crude was washed several times with
acetone to afford 5 as a dark yellow solid.Yield: 1 g (98.9%).
m.p.: 226–229 °C; IR (KBr): 3353, 3243-2711, 1593, 1549,
1415 cm–1; 1H NMR (DMSO-d6): d 8.4 (d; 1H, J = 8.4 Hz);
7.9 (dd; 1H, J = 8.4, 0.9 Hz); 7.7 (dd; 1H, J = 8.4, 1.5 Hz); 7.5
(td; 1H, J = 7.8,1.5 Hz); 2.3 (s; 3H) thiazolo[5,4-b]quinoline
protons; 10.5 (s,1H); 7.3 (dd; 1H, J = 7.8, 7.2 Hz); 7.1 (d; 2H,
J = 7.2 Hz); 7.0 (dd; 1H, J = 7.5, 7.2 Hz) anilino ring protons;
MS (CI): m/z 324 (M++1, 100%); 323 (M+, 83%).
6.1.1. 4-(Ethoxycarbonyl)-2-(methylthio)-5-(phenylamino)
thiazole 2
The compound 2 was prepared according to a procedure
already described [7].
6.1.2. 4-Carboxy-2-(methylthio)-5-(phenylamino)thiazole 3
To a solution of compound 2 (4.3 g, 14.62 mmol) in EtOH
(43 ml) was added an KOH aqueous solution (2.45 g, 3.5 ml).
R1
HN
R2
N
6.1.5. 9-Anilino-2-(methylsulphonyl)thiazolo[5,4-b]quino-
line 6
12 R1 = R2 = H
13 R1 = NH2 R2 = CH2OH
To a solution of 5 (200 mg) in glacial AcOH (6 ml) was
slowly added 30% H2O2 solution (6 ml). The suspension was
stirred at 30 °C for 3 h. After this time the reaction mixture
was stirred overnight at 25 °C. The yellow solution was then
poured into stirred cold water (50 ml). The yellow precipitate
was collected, washed with cold water and dried by vacuum
filtration to afford 6 as a light yellow solid. Yield: 180 mg
(82.2%); m.p.: 212–214 °C; IR (KBr): 3373, 2921, 1577,
Fig. 3. 9-Anilinoacridine structure 12, AHMA structure 13.
OH
N
R
S
N
1
1313, 1140 cm–1; H NMR (Me2CO-d6): d 8.0 (dd; 1H,
14 R = SCH3
15 R = NHCH2CH2N(CH2CH3)2
J = 8.4, 0.6 Hz); 8.3 (dd; 1H, J = 8.7, 0.9 Hz); 7.8 (ddd; 1H,
J = 9.0, 8.4, 1.2 Hz); 7.5 (ddd; 1H, J = 9.0, 8.1, 1.2 Hz)
thiazolo[5,4-b]quinoline ring protons; 9.2 (br s; 1H); 7.3 (m;
3H); 7.2 (t; 2H, J = 6.9 Hz) anilino ring protons; 3.3 (s; 3H);
MS (EI): m/z 355 (M+, 100%).
Fig. 4. 9-hydroxy-2-(methylthio)thiazolo[5,4-b]quinoline structure 14,
2-[[2-(N,N-diethylamino)ethyl]amino]-9-hydroxy-thiazolo[5,4-b]quinoline
structure 15.

P. Rodríguez-Loaiza et al. / European Journal of Medicinal Chemistry 39 (2004) 5–10
9
6.1.6. 9-Anilino-2-[[2-(N,N-diethylamino)ethyl]amino]thia-
zolo[5,4-b]quinoline 7
7.6 (dd; 1H, J = 7.8, 7.5 Hz); 7.3 (dd, 1H, J = 9.0, 7.5 Hz); 7.0
(dd, 1H, J = 9.0, 7.2 Hz); 6.4 (m; 3H); 4.2 (br s; 2H); 2.6 (s;
3H); MS (EI): m/z 338 (M+, 100); 322 (M+-16, 16%); 305
(M+-33, 53%).
To compound 6 (0.177 g, 0.5 mmol) was added N,N-
diethylethylendiamine (0.214 ml, 3 mmol). The suspension
was stirred at 140 °C for 20 min. The reaction mixture was
then cooled to room temperature. CHCl3 (30 ml) was then
added. The CHCl3 solution was successively extracted with
1 N NaOH solution (3 × 10 ml), NH4Cl saturated solution
(3 × 10 ml) and brine (3 × 10 ml), and dried over Na2SO4. The
organic phase, after filtration, was evaporated under reduced
pressure to a small volume. The precipitate formed was
collected and dried under vacuum filtration to afford 7 as a
white solid. Yield: 215 mg (55%); m.p.: 253–256 °C (dec);
IR (KBr): 3211, 3003-2596, 1588-1519 cm–1; 1H NMR
(DMSO-d6): d 8.5 (s; 1H); 8.2 (s; 1H); 8.0 (d; 1H, J = 9 Hz);
7.8 (d; 1H, J = 9.3 Hz); 7.5 (dd; 1H, J = 9.0, 6.0 Hz); 7.4 (dd;
1H, J = 9.0, 6.0 Hz); 7.1 (dd; 1H, J = 8.4, 7.2 Hz); 6.8 (m;
3H); 3.2 (m; 2H); 2.5 (dd, 2H, J = 7.2, 6.3 Hz); 2.3 (q; 4H;
J = 7.2 Hz); 0.8 (t; 6H, J = 7.0 Hz); MS (EI): m/z 391 (M+,
92 %); 389 (M+-2, 100%).
6.1.9. 9-[(3-Acetamidephenyl)amino]-2-(methylthio)thiazo-
lo[5,4-b]quinoline 10
To compound 9 (200 mg, 0.6 mmol) were successively
added Ac2O (2 ml) and pyridine (2 ml). The reaction mixture
was stirred at room temperature for 14 h. The mixture was
then poured into stirred water (30 ml). The solid formed was
collected by vacuum filtration. The residue was purified by
crystallization from MeOH to afford 10 as a yellow solid.
Yield: 160 mg (71.4%); m.p.: 207–210 °C; IR (KBr): 3283,
2923, 1667, 1586, 1551 cm–1; 1H NMR (DMSO-d6): d 8.3 (d;
1H, J = 12 Hz); 7.9 (d; 1H, J = 12 Hz); 7.7 (ddd; 1H, J = 12.0,
10.0, 2.1 Hz); 7.5 (ddd; 1H, J = 12.6, 10.5, 2.1 Hz); 7.3-7.1
(m; 3H); 6.7 (d; 1H, J = 1.2 Hz); 2.4 (s; 3H); 1.2 (s; 3H); MS
(EI): m/z 381 (M++1, 21%); 380 (M+, 100%); 365 (M+-15,
9%); 337 (M+-43, 11%).
6.1.7. 9-Chloro-2-(methylthio)thiazolo[5,4-b]quinoline 8
To compound 2 (2.94 g, 10 mmol) were added PPA
(710 mg) and POCl3 (3 ml). The reaction mixture was vigor-
ously stirred at 136 °C for 4 h. The mixture was cooled to
room temperature and EtOH (10 ml) was carefully added.
The suspension was poured into water (20 ml) and neutral-
ized with a NaHCO3 saturated solution. The yellow precipi-
tate formed was collected and dried by vacuum filtration. The
crude was suspended in MeOH. The solid was collected and
dried by vacuum filtration to afford 8 as a yellow solid.Yield:
1.2 g (60.3%); m.p.: 160 °C; IR (KBr): 3447, 1583, 1543,
6.1.10. 9-[((3-Amino-5-(hydroxymethyl)phenyl)amino]-2-
(methylthio)thiazolo[5,4-b]quinoline 11
To compound 8 (0.133 g, 0.5 mmol) were successively
added MeOH (7 ml), CHCl3 (3.5 ml) and three drops of HCl
(36%). The mixture was stirred for ten minutes. Meanwhile,
to a solution of 3,5-diaminobenzyl alcohol dihydrochloride
(315 mg, 1.5 mmol) in MeOH anhydrous (3 ml) was added
NaHCO3 anhydrous (200 mg). The suspension was stirred
for ten minutes and the free base solution was transferred to
the initial solution. The mixture reaction was heated under
reflux for 48 h. After this time, the reaction mixture was
concentrated under reduced pressure. The solid residue was
suspended in water (10 ml) and a 10% NaHCO3 solution was
added to pH 8. The solid was collected and dried by vacuum
filtration. The residue (150 mg) was dissolved in MeOH and
coevaporated with silica gel (200 mg) in vacuo to dryness.
The residue was put on the top of a silica gel column (3 ×
30 cm), which was washed with CHCl3/MeOH (99:1 v/v) to
afford compound 11 as a light yellow solid. Yield: 105 mg
(42.9%), m.p.: 220 °C (dec.); IR (KBr): 3384, 3357, 3328,
2922, 1607, 1584 cm–1; 1H NMR (DMSO-d6): d 8.9 (s; 1H,
exchangeable with D2O); 8.23 (d; 1H, J = 8.1 Hz); 7.87 (d;
1H, J = 7.5 Hz), 7.67 (ddd; 1H, J = 8.4, 6.8, 0.9); 7.43 (ddd;
1H, J = 7.7, 6.8, 1.2 Hz); 6.27 (s; 1H); 6.23 (s; 1H); 6.12
(s;1H), 5.0 (s; 1H, exchangeable with D2O); 4.9 (s;1H, ex-
changeable with D2O); 4.3 (s; 2H); 2.58 (s;3H); MS (EI): m/z
368 (M+,100%); 335 (M+-33,15%).
1
1371, 1014, 751 cm–1; H NMR (CDCl3): d 8.3 (dd; 1H,
J = 8.7, 0.9 Hz); 8.0 (dd; 1H, J = 7.2, 0.6 Hz); 7.7 (ddd; 1H,
J = 9.0, 6.6, 1.8 Hz); 7.6 (ddd; 1H, J = 7.6, 1.5, 1.2 Hz); 2.88
(s, 3H); MS (EI): m/z 268 (M++2, 41%); 266 (M+, 100%);
MS (FAB+, High resolution): m/z 267 (M++1); Anal. for
C11H8N2ClS2: C, 49.53; H, 2.64; N, 10.50; S, 24.04. Found:
C, 49.67; H, 2.46; N, 10.50; S, 23.52.
6.1.8.
9-[(3-Aminophenyl)amino]-2-(methylthio)thiazolo
[5,4-b]quinoline 9
To compound 8 (0.266 g, 0.1 mmol) were added succes-
sively MeOH (15 ml) and two drops of HCl (36%). The
mixture was stirred for ten minutes and m-phenylendiamine
(80 mg) was then added. The stirred mixture was heated
under reflux for 8 h.After cooling, the MeOH was evaporated
under reduced pressure. The residue was suspended in
EtOAc (25 ml), stirred several minutes and collected by
vacuum filtration. The solid was again suspended in a 10%
NaHCO3 solution, stirred for 15 min, collected and dried by
vacuum filtration. The solid residue was purified by crystal-
lization from MeOH to afford 9 as yellow crystals. Yield:
200 mg (59.2%). m.p.: 178–183 °C, IR (KBr) 3447, 3347,
3301, 3186, 1593, 1550, 1490 cm–1; 1H NMR (DMSO-d6): d
8.1 (s; 1H); 8.0 (d; 1H, J = 9.0 Hz); 7.9 (d; 1H, J = 8.4 Hz);
6.2. Cells and cytotoxic assay
Cells lines. The cell lines used here were: two human
colorectal cancer cell lines (SW-480 and SW-620); three
cervical cell lines (HeLa, C-33, Calo); one breast cancer cell
line (MCF-7), one ovarian cell line (CHO), and one myelog-
enous leukemia human cell line (K-562) [9].

10
P. Rodríguez-Loaiza et al. / European Journal of Medicinal Chemistry 39 (2004) 5–10
6.3. Ethidium displacement
Acknowledgements
We are grateful to Maricela Guitérrez, Rosa Isela del
Villar, Georgina Duarte and Margarita Guzmán for the deter-
mination of all spectra. We thank Dr. David J. Kroll from
Duke University Medical Center for supplying a sample of
amsacrine.
DNA intercalation was determined from the displacement
of ethidium bromide from DNA [10]. Sterile solutions of
high molecular weight DNA from calf thymus (Gibco, BRL,
New York, USA) in 0.1 M Tris–HCl buffer, 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% dimethylsul-
phoxide (DMSO), and the solution fluorescence intensity
was recorded at 584 nm with an excitation 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 to that deter-
mined in 100% aqueous buffers. The recorded fluorescence
change was corrected for the dilution caused by serial addi-
tions of this solvent.
References
[1] G.J. Atwell, B.F. Cain, R.N. Seelye, J. Med. Chem 15 (1972) 611–
615.
[2] B.F. Cain, R.N. Seelye, G.J. Atwell, J. Med. Chem 17 (1974) 922–
930.
[3] B.F. Cain, G.J. Atwell, W.A. Denny, J. Med. Chem 18 (1975) 1110–
1117.
[4] G.W. Rewcastle, G.J. Atwell, D. Chambers, B.C. Gaguley,
W.A. Denny, J. Med. Chem 29 (1986) 472–477.
[5] B.F. Cain, G.J. Atwell, Eur. J. Cancer 10 (1974) 539–549.
[6] Z. Arlin, Cancer Treat. Rep 67 (1983) 967–970.
[7] C.Alvarez-Ibarra, R. Fernández-Granda, M.L. Quiroga,A. Carbonell,
F. Cárdenas, E. Giralt, J. Med. Chem 40 (1997) 668–676.
[8] A. Quintero, A. Pelcastre, J. Dolores, A. Guzmán, E. Díaz, J. Pharm.
Pharmaceut. Sci. 2 (1999) 108–112.
[9] A.W. McConnaughie, T.C. Jenkins, J. Med. Chem 38 (1995) 3488–
3501.
[10] T.D. Sakore, B.S. Reddy, H.M. Sobell, J. Mol. Biol 135 (1979)
763–785.
[11] T.L. Su, J.T. Huang, K.A. Watanabe, G.M. Otter, F.M. Sirotnak,
X.-B. Kong, T.-C. Chou, Proc. Am. Assoc. Cancer Res 34 (1993)
2241.
[12] R.C. Baguley, W.A. Denny, G.H. Atwell, B.F. Cain, J. Med. Chem 24
(1981) 170–177.
[13] W.A. Denny, P.G. Wakelin, Cancer Res 46 (1986) 1717–1721.
[14] K.-X. Chen, N. Gresh, B. Pullman, Nucleic Acids Res 16 (1988)
3061–3073.
The compounds were tested in the 1–100 µM range, ex-
cept when, at lower concentration, precipitation was detected
as light dispersal in the solution (at 600 nm and 90°). The
displacement curves were fitted by non-linear regression
analysis to a rectangular hyperbola. Evidence of the direct
interaction of the compounds with DNA was obtained from
the differential fluorescence spectra of high molecular
weight DNA, excited at 260 nm and recorded from 400 to
500 nm, in the absence and presence of the ligand. Two new
peaks appeared at 420 and 440 nm and their intensity in-
creased in a concentration dependent manner. Those peaks
were not present in the spectra of the pure compound or pure
DNA solutions.