Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular modeling and structure-activity relationship of 9-anilinothiazolo[5,4-b]quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2009.03.052

Some novel 9-anilinothiazolo[5,4-b]quinoline derivatives were synthesized and their cytotoxic activities were examined. The inhibition of some of the most active compounds over human topoisomerase II (Topo II) activity was assessed with the kDNA decatenat

Full text of DOI:10.1016/j.bmc.2009.03.052

Bioorganic & Medicinal Chemistry 17 (2009) 3266–3277
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cytotoxic activity, DNA topoisomerase-II inhibition, molecular
modeling and structure–activity relationship of
9-anilinothiazolo[5,4-b]quinoline derivatives
Marco A. Loza-Mejía a, , Susana Olvera-Vázquez a, Karina Maldonado-Hernández a,
Teresita Guadarrama-Salgado a, Ignacio González-Sánchez b, Fernando Rodríguez-Hernández c,
José D. Solano b, Rogelio Rodríguez-Sotres c, Alfonso Lira-Rocha a,
*
a Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, México 04510, Mexico
b Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, México 04510, Mexico
c Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, México 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Some novel 9-anilinothiazolo[5,4-b]quinoline derivatives were synthesized and their cytotoxic activities
were examined. The inhibition of some of the most active compounds over human topoisomerase II (Topo
II) activity was assessed with the kDNA decatenation assay. The novel compounds differ in the substitu-
ents attached to the anilino ring, a dialkylamino alkylamino group, a saturated heterocyclic moiety, a
methylthio group at position 2 and a fluorine atom present or absent at 7-position. According to the data,
compounds with a diethylaminopropylamino group and a chlorine atom at 40-position of the anilino ring
were the most cytotoxic. The molecular models of all compounds indicated a correlation between hydro-
phobicity and cytotoxic activity although the direction and magnitude of the dipole moment also had a
significant influence on its cytotoxicity. The 2-dialkylaminoalkylamino substituent is flexible and is
known to facilitate the crossing of cell membranes; thus, this last barrier may be a limiting step in the
mechanisms mediating the cytotoxicity. On the other hand, the activity of 2-methylthio derivatives
seems to rely more on the electronic effects brought about by the substitution of the aniline ring. The syn-
thesis, cytotoxicity against cancer cell lines, in vitro inhibition of human topoisomerase II, molecular
modeling and the preliminary analysis of structure–activity relationships are presented.
Received 21 February 2009
Revised 20 March 2009
Accepted 25 March 2009
Available online 1 April 2009
Keywords:
Thiazolo[5,4-b]quinoline
Molecular modeling
Cytotoxic activity
DNA topoisomerase-II inhibition
Structure–activity relationship
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Several compounds based on tricyclic templates have been
studied in the search for better anticancer agents. Among these,
9-anilinoacridines have been extensively studied as potential ant-
itumorals, and their capability of interacting with DNA and inhib-
iting the DNA topoisomerase II has been proposed as a mechanism
of action. The stabilization of a enzyme-drug-DNA ternary complex
covalent-intermediate has been shown to lead to DNA lesions, the
interaction anilino ring-enzyme being one of the principal stabiliz-
ing factors.1–3 One of these derivatives, amsacrine (Fig. 1, m-AMSA,
1a), has been clinically used for leukemia and lymphoma treat-
ment.4,5 Structural modifications in the acridine nucleus have led
to a novel agent, asulacrine 1b,6 which was capable of inhibiting
the growth of some solid tumors. On the other hand, the isosteric
replacement of a benzene moiety in the acridine template has
* Corresponding author. Tel.: + 52 55 56 22 52 86; fax: +52 55 56 22 53 29.
E-mail address: lira@servidor.unam.mx (A. Lira-Rocha).
Taken in part from the PhD dissertation of Marco A. Loza-Mejía.
Figure 1. Chemical structures of amsacrine 1a, asulacrine 1b and some aniline-
substituted tricyclic templates 2–4.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.052

M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
3267
the tricyclic ring to the nucleophilic attack, apparently by steric
hindrance. Compounds 10a–10f, 11a–11e and 8d–8f were ob-
tained by the oxidation of the methylthio derivative to the respec-
tive sulfone, and subsequent condensation with the appropriate
amine in moderate to good yields. We also tried to obtain com-
pounds 8d–8f by condensation of aniline with 8a–8c by using dif-
ferent conditions, but the starting material was recovered each
time. Compounds of series 7 were obtained by a method previously
reported,7 but in the case of fluorinated derivatives it was neces-
sary to extend reaction time. All compounds were characterized
by IR, 1H NMR, MS and elemental analysis. Unequivocal assign-
ment of all 1H NMR signals was possible by NOESY experiments
carried out with compound 10e.
Figure 2. 9-Anilinothiazolo[5,4-b]quinoline template and the substituents consid-
ered in the present study.
3. Biological
led to compounds such as 9-anilinothiazolo[5,4-b]quinoline 2,7,8 4-
anilinofuro[2,3-b]quinoline 39 and 4-anilinopyrazolo[3,4-b]quino-
line 410 derivatives which have shown good cytotoxic activity.
The incorporation of a five- or six-membered heterocyclic ring
fused to an anthracenedione or acridine nucleus usually increases
the cytotoxic activity, thus enabling the compounds to overcome
multidrug resistance of tumor cells.11,12
3.1. In vitro cytotoxicity
The results of the evaluation of cytotoxic activity of the newly
prepared and some previously reported compounds7,8 are shown
in Table 1. 9-Anilino-7-fluoro-susbtituted derivatives were not as
active as their non-fluorinated analogues (compare 7a–e and 7i–
m), except for compound 7c, which was more active than the
non-fluorinated analogue 7k. In general, the trend differs from
the one found by Alvarez-Ibarra et al. for 9-hydroxythiazoloquino-
line derivatives,13 where a substitution with a fluorine atom in po-
sition 7 favored cytotoxicity.
Since, according to our previous studies, the presence of an elec-
tron withdrawing group at 30-position favored cytotoxicity, addi-
tional substituents were incorporated at this position, that is,
compounds 7f–7h. As discussed in that preceding paper,8 a puta-
tive hydrogen bond acceptor atom seems to be relevant to cytotox-
icity of such a compound series. In the new compounds, only the
oxime-substituted compound was cytotoxic. Thus, the cytotoxic
activity may be influenced by the distance from the anilino ring
to the putative hydrogen bond acceptor atom (distance to HBA,
Figure 3), because the cyano derivative 7j was cytotoxic, but a
shorter or longer distance to HBA, as in 7f and 7g, eliminated the
activity. In addition, the alignment of the cyano group possibly
contributes to a better hydrogen bond interaction. Moro, Capranico
et al. have proposed a model for the interaction of some intercala-
tors with DNA and DNA topoisomerase II, based on docking stud-
ies.14 Strong interactions were found between a hydroxyl group
of Thr744 of topoisomerase II (hydrogen bond donor atom, HBD)
and the sulfonamide moiety (HBA) of the m-amsacrine. A similar
interaction may mediate anilinothiazolo[5,4-b]quinolines antitu-
mor activity, and if so, the HBA group could be involved.
Compounds of series 8 that carry a six-member saturated nitro-
gen-containing heterocyclic substituent at 2-position showed low
activity. A similar result was observed for 9-hydroxy-2-saturated
thiazoloquinoline substituted compounds.13 Thus, flexibility of the
alkylamino chain at 2-position seems to have a positive influence
on cytotoxic activity. In addition, compounds of series 10 and 11
showed similar cytotoxic activity, 11e being the most active. Since
the ‘propylenediamine’ substituted compounds in these series were
more active than ‘ethylenediamine’ substituted compounds, the
length of the alkylamino chain was relevant but not critical for cyto-
toxic activity. On the other hand, attachment of the substituents at
any position of the anilino ring in these derivatives did not improve
activity and in some cases it even reduced cytotoxicity.
In our previous paper, the importance of the substitution pat-
tern in the anilino ring of 2-methylthio derivatives of 2 was
explored by introducing different substituents at positions 30 and
40 of the anilino ring as well as a diethylaminoethylamino chain
at positions 2 and 9 of the tricyclic nucleus. Electron withdrawing
groups (EWG) and hydrogen bond acceptors (HBA) attached to
position 30 of the anilino ring improved cytotoxic activity. Besides,
the position of the diethylaminoethylamino chain had less influ-
ence on cytotoxicity than it did in the DNA binding affinity.7,8
In the present study, the effect of additional substituents on the
anilino ring of the 9-anilinothiazoloquinoline system (Fig. 2, Scheme
1, series 7f–7h) is presented. In those compounds previously identi-
fied as good to fair cytotoxics (with substituents at positions 30 or 40
of the anilino ring), the influence of a dialkylaminoalkylamino
(diethylaminoethylamino or diethylaminopropylamino) chain at
position 2 was studied (Scheme 1, series 10 and 11). In particular,
the chain length and flexibility of the dialkylaminoalkylamino chain
was modified by incorporating a saturated heterocycle substituent
at position 2, while keeping a nitrogen atom attached to the C-2 of
the tricyclic system (Scheme 1, series 8). Finally, fluorination of posi-
tion 7 of the thiazoloquinoline system was included in some com-
pounds (Scheme 1, series 7a–7e) since it was reported to increase
cytotoxic effects.13 Cytotoxic activity as well as the inhibition of hu-
man topoisomerase II activity of the novel and some previously re-
ported compounds were evaluated, and a preliminary study
structure–activity relationship was carried out. The information ob-
tained has rendered important clues to the understanding of the
cytotoxic profile for these types of compounds.
2. Chemistry
The synthesis of the proposed compounds is depicted in Scheme
1. Preparation of intermediates for compounds 5a–5b has already
been described.7,13 Compounds 6a and 6b were prepared by cycli-
zation of 5a and 5b with POCl3/PPA,7 but lower yields were ob-
tained for the fluorinated analogue (60% for 6a, 35% for 6b).
Attempts to increase the yield (higher temperature and/or incre-
ment of POCl3/PPA) were unsuccessful. Although compounds 8a–
8c were obtained from 6a by direct condensation with the hetero-
cyclic amine in good yields (over 80%), this method was not useful
for preparing anilino-substituted compounds 8d–8f or those of ser-
ies 10 and 11. The anilino group diminishes the susceptibility of
3.2. Inhibition of human DNA topoisomerase II activity
As reported in our previous paper8, the cytotoxicity of several
thiazolo[5,4-b]quinoline derivatives did not correlate with the

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M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
Scheme 1. Reactions and conditions (a) POCl3/PPA, 130 °C, 4 h; (b) H2N–C6H4–R, MeOH, reflux, 6 h; (c) H2O2/AcOH, rt, overnight; (d) method A amine, reflux 30 min; method
B amine, DMF, rt, 3 h; (e) POCl3/PPA, 150 °C, 4 h; (f) H2N–C6H4–R, EtOH, reflux, 20 h; (g) amine, rt, 24 h; (h) NH2OH, MeOH, reflux, 2 h.
in vitro DNA-intercalation ability of the compounds. In the case of
acridines the mechanism of action seems to be related to the
stabilization of the ternary complex between DNA, Topoisomerase
II and the drug.15 The decatenation of kDNA molecules was used to
assay human Topoisomerase II (Topo II) activity. Only topoisomer-
ase type II can resolve individual DNA molecules from the inter-
locked network of covalently closed minicircles composing the
kDNA. The resulting individual molecules were analyzed by aga-
rose-gel electrophoresis, and the activity was calculated from the
percentage of kDNA converted to individual DNA molecules, as
judged from densitometric analysis of ethidium bromide stained
gels. In control lanes no residual kDNA was detected, this activity
being considered 100%. In the initial screening, the most cytotoxic
compounds were tested at a final concentration of 160 M in the
assay. For those showing significant activity the IC50 was calculated
from dose–response curves in the range of concentration of
1–160 lM. The resulting curves were sigmoidal in most cases with
Hill numbers ranging from 2 to 8. Apparent cooperativity binding
may indicate the participation of more than one molecule of the
compound in the enzyme–DNA–inhibitor complex or the existence
of high and low affinity DNA subsites where the compound binds,
yet not all of them lead to the inhibition of Topo II. The concentra-
tion values giving 50% inhibition (IC50) were calculated by non-lin-
ear regression fits of the data to the following equation:
l

M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
3269
Table 1
Table 2
In vitro cytotoxic activity of novel and already reported thiazolo[5,4-b]quinoline
Inhibitory activity of thiazolo[5,4-b]derivatives and m-amsacrine, as reference,
against on topoisomerase II (IC50 M)
derivatives
, l
R3
Compound
Activity
se
Hill
se
R4
7l
7o
7p
7r
7s
7t
7u
7v
21.20
20.49
12.27
15.42
64.34
16.74
18.04
29.56
168.90
40.95
9.94
0.61
0.24
0.28
0.01
5.13
0.14
1.66
6.21
6.07
8.66
0.51
8
7
3
6
7
4
2
2
1
1
4
1.52
0.57
0.15
0.01
1.86
0.10
0.48
0.40
0.06
0.05
0.56
HN
R1
N
S
R2
N
R1
R2
R3
R4
HeLa
SW480
SW620
K-562
10g
11c
m-AMSA
7a
7b
7c
7d
7e
7f
7g
F
F
F
F
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
H
CN
OMe
H
H
CO2Et
COMe
OXM
H
CN
OMe
Cl
H
H
H
CN
OMe
H
H
H
H
H
H
H
CN
OMe
Cl
H
H
H
H
>80
>80
15.8
>80
>80
>80
>80
13.7
>80
>80
>80
>80
37.3
>80
28.68
66.65
>80
>80
>80
>80
>80
13.6
65.13
>80
>80
>80
>80
>80
>80
12.2
>80
>80
>80
55.36
n.t
n.t
54.91
>80
>80
32.8
>80
8.01
22.17
80.26
>80
77.2
79.45
46.85
12.54
67.06
>80
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>80
>80
>80
>80
100ICn5H0
Activity ð%Þ ¼
nH
ICn5H0 þ ½Iꢀ
7h
34.2
>80
7.75
25.34
69.37
>80
>80
>80
46.22
21.69
43.34
>80
39.4
>80
43.75
26.58
>80
>80
>80
>80
>80
19.72
62.28
>80
>80
>80
7ic
7jd
7kd
7ld
7md
7nd
7od
7pd
7qd
7rd
7sd
7tc
7uc
7vc
where nH is the cooperativity index and I the concentration of added
compound. Non-linear regression was performed with the Sigmas-
tat 2.0 statistical package (Jendel Sci. Inc. San Rafael, Ca.).
H
H
H
There was no correlation between in vitro Topo II inhibition
(Table 2) and the cytotoxicity of the compounds (Table 1). Com-
pound 7p was as nearly a good Topo II inhibitor as m-AMSA, but
it exhibited low cytotoxicity, while compound 11c was highly
cytotoxic, but, as an inhibitor of Topo II, it was four times less
potent than m-AMSA. Thus, the thiazolo[5,4-b]quinoline deriva-
tives may not be acting by the same mechanism as m-AMSA,
may be differentially modified by cell metabolism, or may present
large differences in their ability to penetrate the cell.
NMe
(CO)E
CF3
OH
NH2
NH2
NHAc
a
H
>80
>80
>80
>80
>80
>80
b
H
>80
a
a
a
8a
8b
8c
8d
8e
8f
H
H
H
H
H
H
PIP
>80
>80
>80
>80
60.7
>80
>80
>80
>80
>80
41.8
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
73.01
>80
a
MPZ
MOR
PIP
MPZ
MOR
a
4. Molecular modeling
H
H
H
H
H
H
The cytotoxic activity of these compounds must be related to
their molecular structure, and molecular modeling of the isolated
molecules may reveal some of the factors involved. Molecular
models were obtained with SPARTAN00416 software using the semi-
empirical AM1 method. A systematic search protocol (torsion
10a
10b
10c
10d
10e
10f
H
H
H
H
H
H
H
E
E
E
E
E
E
E
CN
Cl
OMe
H
H
H
H
H
H
CN
Cl
13.33
9.12
19.2
15.18
10.16
13.60
15.96
13.10
14.33
11.5
14.18
12.56
12.04
37.7
14.62
17.78
20.0
16.49
12.20
16.38
21.6
12.48
12.19
23.5
8.36
7.26
angles sa and sb) was used to obtain the minimal energy conform-
OMe
H
10.87
16.8
10gc
H
ers. Table 3 shows some of the calculated molecular parameters.
Optimized geometries of selected compounds are shown in Figure
4. Substituents attached to the phenyl ring linked to the amino
group had a greater effect on the phenyl conformation than those
attached to 2-position of the tricyclic nucleus system. The results
of the conformational analysis show an oblique orientation of the
anilino ring in relation to the tricyclic nucleus. In accordance with
the models, the analysis of the NOESY spectrum of 10e showed a
long-range interaction between the protons of the anilino ring
and those of alkylamino chain.
Because DNA–intercalator complex can be stabilized through
frontier molecular orbital interactions, LUMO energy values of
the compounds were calculated. All compounds appear to behave
as electron acceptors since their LUMO energies were negative. In
contrast, the DNA base pairs have been shown to act as electron
donors.17 This type of interaction should be very important for
fluorinated compounds 7a–7e because their LUMO energy values
were the lowest. In the case of alkylamino derivatives, a cationic
species can be formed due to the basic properties of the side chain,
which can interact the phosphate backbone, facilitating DNA inter-
calation. This last interaction has been shown to occur in similar
compounds18 and was suggested by us based on previous results.8
The dipole moment describes the charge distribution in a mol-
ecule and somehow summarizes electronic effects involved in
molecular recognition. With DNA intercalators, a direct correlation
between their dipole moment values and cytotoxicity has been
11a
11b
11c
11d
11e
H
H
H
H
H
P
P
P
P
P
H
CN
Cl
H
H
H
H
CN
Cl
6.27
19.21
7.46
24.18
8.82
9.5
6.90
16.56
19.65
10.17
29.15
7.48
7.52
12.88
9.84
12.88
3.36
19.9
11.53
7.91
22.70
4.92
27.7
H
Amsacrinec
16.7
IC50, lM, compound concentration inhibiting 50% of cellular growth assessed by the
MTT assay.
E = NH(CH2)2NEt2 P = NH(CH2)3NEt2, PIP = -1-piperidinyl, MPZ = -4-methylpiperaz-
inyl, MOR = 1-morpholinyl, OXM = C(NOH)Me, n.t. not tested.
a
Chlorine atom in position 9 instead of aniline ring.
50-CH2OH substituted.
Taken from Ref. 7.
Taken from Ref. 8.
b
c
d
Figure 3. Distance from aniline ring to hydrogen bond acceptor atom (distance to
HBA).

3270
M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
Table 3
Selected parameters calculated for thiazolo[5,4-b]quinoline derivatives
R3
R4
R2
τb
τa
HN
1
9
R1
N
S
N
R1
R2
R3
R4
E HOMO (eV)
E LUMO (eV)
Dipole (Debye)
PSA (Å2)
LogP
sa (°)
7a
7b
7c
7d
7e
7f
7g
7h
7i
F
F
F
F
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
H
H
H
H
CN
OMe
H
H
H
H
H
H
H
CN
OMe
Cl
H
H
H
ꢁ8.2812
ꢁ8.5381
ꢁ8.2981
ꢁ8.5720
ꢁ8.1733
ꢁ8.3488
ꢁ8.3493
ꢁ8.2219
ꢁ8.1929
ꢁ8.4384
ꢁ8.2095
ꢁ8.3269
ꢁ8.4712
ꢁ8.1371
ꢁ8.3010
ꢁ8.4467
ꢁ8.6759
ꢁ8.8215
ꢁ8.5419
ꢁ8.4487
ꢁ8.4069
ꢁ8.5198
ꢁ1.2490
ꢁ1.4596
ꢁ1.2551
ꢁ1.5062
ꢁ1.2143
ꢁ1.2317
ꢁ1.2319
ꢁ1.1237
ꢁ1.0978
ꢁ1.3072
ꢁ1.1038
ꢁ1.2085
ꢁ1.3560
ꢁ1.0973
ꢁ1.2130
ꢁ1.3666
ꢁ1.5141
ꢁ1.5828
ꢁ1.3882
ꢁ1.3750
ꢁ1.1691
ꢁ1.4433
2.7750
2.9112
2.9116
4.3856
2.5061
1.5972
1.8153
2.2472
2.6069
1.4718
3.0656
1.0670
3.0940
1.8501
1.7710
3.9320
1.9465
4.1788
4.2182
3.4153
5.0351
1.045
25.4798
40.94
32.6213
40.9403
32.6746
44.009
5.33
5.37
5.21
5.37
5.21
5.33
4.49
4.88
5.18
5.21
5.05
5.73
5.21
5.05
5.73
4.67
4.99
6.10
4.79
4.37
3.8
ꢁ60.9
ꢁ66.0
ꢁ62.4
ꢁ67.2
ꢁ58.4
ꢁ64.5
ꢁ63.4
ꢁ62.6
ꢁ61.8
ꢁ66.3
ꢁ63.2
ꢁ64.6
ꢁ67.1
ꢁ60.0
ꢁ64.3
ꢁ67.9
ꢁ70.9
ꢁ79.0
ꢁ74.0
ꢁ70.0
ꢁ70.0
ꢁ68.0
CN
OMe
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CO2Et
COMe
OXM
H
CN
OMe
Cl
H
H
H
NMe
(CO)E
CF3
OH
NH2
NH2
NHAc
a
38.628
59.969
25.4955
40.9621
32.6417
25.5319
40.9473
32.6991
25.5309
37.5510
53.1684
25.8454
45.2594
50.388
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
H
H
b
7u
7v
67.5379
50.2311
H
4.08
a
a
a
8a
8b
8c
8d
8e
8f
H
H
H
H
H
H
PIP
ꢁ8.5277
ꢁ8.5574
ꢁ8.6512
ꢁ8.1366
ꢁ8.1507
ꢁ8.2160
ꢁ0.9740
ꢁ1.0003
ꢁ1.0832
ꢁ0.8559
ꢁ0.8753
ꢁ0.9483
2.1853
1.9366
0.4513
2.8936
2.4780
1.1818
16.5785
19.1469
24.4935
27.3391
29.895
6.13
4.82
4.66
6.52
5.21
5.06
n/a
n/a
n/a
ꢁ67.0
ꢁ66.3
ꢁ65.2
a
MPZ
MOR
PIP
MPZ
MOR
a
H
H
H
H
H
H
35.2189
10a
10b
10c
10d
10e
10f
H
H
H
H
H
H
H
E
E
E
E
E
E
E
CN
Cl
OMe
H
H
H
H
H
H
CN
Cl
ꢁ8.4021
ꢁ8.2583
ꢁ8.1415
ꢁ8.3940
ꢁ8.2344
ꢁ8.0092
ꢁ8.1317
ꢁ1.0062
ꢁ0.9071
ꢁ0.8290
ꢁ1.0392
ꢁ0.9171
ꢁ0.7954
ꢁ0.8131
3.7587
3.2760
4.4488
6.0452
3.8532
3.4852
3.7866
54.4904
39.0601
46.2024
54.4736
39.0872
46.2707
39.0819
5.38
5.9
5.22
5.38
5.9
ꢁ75.1
ꢁ71.1
ꢁ68.7
ꢁ72.7
ꢁ71.3
ꢁ67.1
ꢁ69.3
OMe
H
5.22
5.35
10g
H
11a
11b
11c
11d
11e
H
H
H
H
H
P
P
P
P
H
H
H
H
CN
Cl
ꢁ8.0845
ꢁ8.3473
ꢁ8.2280
ꢁ8.3434
ꢁ8.1920
ꢁ8.2450
ꢁ0.7612
ꢁ0.9520
ꢁ0.8615
ꢁ0.9862
ꢁ0.8659
ꢁ1.3367
4.1858
4.6902
4.0702
6.9274
4.3978
1.7783
36.7885
52.1346
34.2395
52.1784
36.7761
5.63
5.66
6.18
5.66
6.18
3.11
ꢁ69.9
ꢁ75.2
ꢁ72.9
ꢁ73.1
ꢁ72.4
ꢁ114
CN
Cl
H
P
H
Amsacrine
E = NH(CH2)2NEt2 P = NH(CH2)3NEt2, PIP = -1-piperidinyl, MPZ = -4-methylpiperazinyl, MOR = 1-morpholinyl, OXM = C(NOH)Me, n/a = not applicable.
a
Chlorine atom in position 9 instead of aniline ring.
50-CH2OH substituted.
b
found.19–21 In the present study, the dipole moment vector and
magnitude were calculated.
5. From the data, the presence of EWG in the anilino ring and incor-
poration of a fluorine atom at position 7 cause changes in the
dipole moment direction. It is worthy of note that 7g and 7f lack
EWG at position 40 but belong to group D, since their dipole vectors
resemble the 7m dipole instead of the one of 7j.
A cyano group or chlorine atom increased the dipole moment
magnitude more when placed at 40-position than when placed at
30-position. The opposite trend is observed for the methoxy group.
For compounds with a dialkylaminoalkylamino substituent, the
incorporation of an electron withdrawing group (EWG) notably
increased the dipole moment magnitude. The replacement of the
methylthio group at 2-position by a dialkylaminoalkylamino resi-
due also resulted in a higher dipole moment, and the incorporation
of a saturated heterocycle reduced its magnitude slightly. How-
ever, the variations in the direction of the dipole moment may be
of greater importance, and based on the dipole moment direction,
the compounds can be grouped as indicated in Table 4 and Figure
5. Structure–activity relationship
5.1. Cytotoxic activity
In Table 4, the compounds with a dialkylaminoalkyl group,
regardless of their dipole orientation, are the most active; in the
case of 2-methyltio derivatives (series 7), however, only the

M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
3271
Figure 4. Optimized geometries for selected compounds.
Table 4
Analyzed compounds grouped according to direction of dipole moment vector and avg IC50
(l
M) is included (HeLa cell line)
+y
3'
5'
+x
NH
N
7
+z
S
N
10
Group A (position 10)
Avg IC50
>80a
Group B (position 7)
Avg IC50
Group C (position 30)
Avg IC50
24.2
Group D (position 40)
Avg IC50
7h, 7i, 7k, 7l, 7n, 7o, 7s, 7t, 7u, 7v
7a, 7c, 7e
>80b
>80c
13.6
7.52
7j, 7q, 7r
7b, 7d, 7f, 7g, 7m
8c
10a, 10d
11b, 11d
>80
>80
14.2
21.7
8a, 8b, 8d, 8e, 8f
10b, 10c, 10e, 10f, 10g
11a, 11c, 11e
a
Except 7h IC50 = 34.2, 7k, IC50 = 25.34.
Except 7c IC50 = 15.8.
Except 8e IC50 = 60.7.
b
c
compounds with the dipole orientation through 30-position and an
EWG are active. The comparison of compound 7j with 10a and 11d
indicates a greater influence of the alkyl chain on the dipole orien-
tation, and while compounds 10a, 10d, 11b and 11d show no cell
line selectivity at all, compound 7j is very active only against HeLa
and K-562 cell lines. Moreover, the incorporation of a fluorine atom
in 7j severely modifies its dipole orientation (relative to compound
7b) and diminishes its cytotoxic activity. There is possibly a low
density region in the putative interaction site, which is only
accessed through a substituent with a high charge density. This
proposal would explain the low activity of compound 7l, which,
in spite of its chlorine atom at 30-position, has a different dipole
orientation relative to 7j.
The influence of the dipole moment on cytotoxicity is clear
because the 2-methylthio compounds of group D (series 7) have
no cytotoxic activity while the same kind of compounds of groups
A and C are cytotoxic. Nevertheless, other factors ought to be con-
sidered since in 30-substituted compounds of group A, a higher
dipole moment magnitude is associated with higher cytotoxicity
(7h, 7k), but the opposite trend was found for compounds in group
C (7j,7q) (Fig. 6). Furthermore, compounds of group D (10d and
11d) with higher dipole moment are among the less active

3272
M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
Figure 6. Relationships between dipole moments and cytotoxic activity in HeLa cell
line. Groups are those included in Table 4.
for K-562 cell line was: log(1/IC50) = 1.14576 * LUMO + 0.13084 * -
dipole moment ꢁ0.59538 (n = 19; r2 = 0.850; F = 45.583). Interest-
ingly, with this subset of compounds, two subgroups become
evident, one including alkylamino substituted compounds and the
other comprising methylthio substituted compounds (Fig. 7). A sim-
ilar tendency was observed with data from the other cell lines.
For the compounds with a dialkylaminoalkylamino group, the
hydrophobic factors appear to be important since compounds with
a higher logP were among the most active. To develop a 2D-QSAR,
several descriptors representative of size (polar surface area, polar
volume, etc.), electronic and lipophilic characteristics were used to
characterize the compounds, but only one satisfactory QSAR
equation was found for compounds of series 10 and 11 (HeLa cell
line): log(1/IC50) = ꢁ0.01964 PSA ꢁ0.19409 (n = 10; r2 = 0.740;
F = 24.541), 10c and 10g as outliers. Where PSA is the Polar Surface
Area descriptor. Thus, one important factor for the activity of these
compounds lies in their ability to pass through biological mem-
branes because this parameter strongly correlates with membrane
permeability.22 Therefore, the better activity of series 11 could be
Figure 5. Analyzed compounds grouped according to the direction of the dipole
vector. Red arrow indicates dipole orientation.
compounds of the alkylamino series, and hence, other electronic
factors influence cytotoxicity. In any case, hydrophobic factors
remain among the most relevant.
Some QSAR equations were derived, but in this case compounds
with IC50 above 80 lM were excluded from the SAR analysis. Com-
pounds7j, 7q, 8e and 11edid notfit into thefirst model. Thiscan pos-
sibly be explained by the different direction of the dipole moment
toward the nitrogen atom of the central ring instead of pointing
towards the anilino ring as in the remaining compounds (see
Fig. 4). When these compounds were excluded, the equation found
Figure 7. Predicted activities, calculated by equation A, versus observed activities. Lower subset includes alkylamino substituted compounds, upper, methylthio substituted
compounds.

M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
3273
attributed to higher ability to cross cell membranes and not neces-
sarily to the better binding to DNA.
a JEOL Sx102 apparatus. Compounds 5a–b and 6a–b were prepared
according to procedures already described.7,8,13
5.2. Topoisomerase II inhibition
7.1. General preparation of 2-(methylthio)-9-anilinothiazolo[5,4-
b]quinolines (7a–h)
For the inhibition of human Topo II, the IC50 value correlated only
with the LUMO parameter [log(1/IC50) = ꢁ0.99025 * LUMO ꢁ
2.46625; n = 07; r2 = 0.810; F = 21.233, 8r, 8s and 8t as outliers]. As
discussed before, the LUMO parameter is relevant to the DNA inter-
calation and appears to be important in the formation of the Topoi-
somerase II-DNA-inhibitor ternary complex, at least for these
compounds. An unusual feature of the Topo II inhibition curves is
the presence of positive cooperativity, with Hill numbers approach-
ing 4. Therefore, four or more molecules of the compound seem to be
required to produce the inhibition. This result does not necessarily
reflect the presence of 4 inhibitor molecules at the ternary complex
but may result from high-affinity-intercalation of the inhibitor to
DNA sites, which are poor substrates of the Topoisomerase II.
Taking all data together, the 2-alkylamino compounds (series
10 and 11) have different structural requirements for cytotoxic
activity as compared with 2-methylthio derivatives (series 7). For
the first group of compounds the capacity to pass through biolog-
ical membranes is more important and for the latter the nature of
the substituent in position 30 becomes predominant. Possibly this
last factor affects the binding of these compounds to a putative
receptor. Even in series 7, two different behaviors were observed,
one class of compounds with dipole moment oriented to the anili-
no ring and the other class with dipole moment oriented to the tri-
cyclic template. Perhaps compounds of series 10 and 11 have a
different mode of binding to DNA or to topoisomerase II, from
those of series 7. Studies on the determination of mechanism of
action of both classes of compounds are in course.
This method has already been described.7,8 Briefly, to the com-
pound 6a or 6b (0.5 mmol) were successively added methanol or
ethanol for fluorinated analogues (5 mL) and two drops of HCl
(36%). The cream-colored suspension formed was stirred for ten
minutes. Meanwhile, a mixture of the aniline with the desired sub-
stitution pattern (0.7 mmol) in 5 mL of methanol or ethanol was
prepared. This mixture was added to the suspension of 6a or 6b
and heated to reflux for 6–18 h. After cooling, the solvent was
evaporated under reduced pressure; the residue was suspended
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.
7.1.1. 7-Fluoro-9-(phenylamino)-2-(methylthio)-9-
anilinothiazolo[5,4-b]quinoline (7a)
Yellow solid; 138 mg (81.5%); mp 187–190 °C. IR (KBr, cmꢁ1):
3423 (NH), 1624, 1597, 1545, 1525, 1493, (aromatic); 1H NMR
(DMSO-d6, d): 2.33 (s, 3H) SCH3; 7.15 (d, J = 7.6 Hz, 2H), H-20, H-
60; 7.32 (t, J = 8.0 Hz, 2H), H-30, H-50; 7.09 (t, J = 7.6 Hz, 1H) H-40;
7.99 (dd, J = 9.2, 5.2 Hz, 1H) H-5; 7.71 (ddd, J = 10.8, 9.6, 2.8 Hz,
1H) H-6; 8.27 (dd, J = 11.2, 2.1 Hz, 1H) H-8; 9.70 (br), NHAr. MS
(FAB, m/z): 342 (M++1, 100%), 341 (M+, 41%) Anal. Calcd for
C17H12FN3S2: C, 59.80; H, 3.54; N, 12.31; S, 18.78. Found: C,
59.65; H, 3.79; N, 12.16; S, 18.93.
7.1.2. 9-[[(3-Cyano)phenyl]amino]-7-fluoro-2-(methylthio)-9-
anilinothiazolo[5,4-b]quinoline (7b)
Yellow solid; 153 mg (81%); mp 252–253 °C. IR (KBr, cmꢁ1):
3430 (NH), 2222 (CN), 1603, 1586, 1543, 1511, 1479, (aromatic);
1H NMR (DMSO-d6, d): 2.31 (s, 3H) SCH3; 7.65 (s, 1H), H-20; 7.53
(m, 3H), H-40, H-50, H-60; 8.01 (dd, J = 9.6, 5.6 Hz, 1H) H-5; 7.57
(dd, J = 9.2, 2.0 Hz, 1H) H-6; 8.49 (dd, J = 10.4, 2.1 Hz, 1H) H-8;
10.42 (br), NHAr. MS (FAB, m/z): 367 (M++1, 100%), 366 (M+, 17%)
Anal. Calcd for C18H11FN4S2: C, 59.00; H, 3.03; N, 15.29; S, 17.50.
Found: C, 59.25; H, 3.18; N, 15.04; S, 17.35.
6. Conclusions
Novel 9-anilinothiazolo[5,4-b]quinoline derivatives with a dif-
ferent substitution pattern in the anilino ring and in the tricyclic
template were synthesized and their cytotoxic activity, tested
in vitro. Incorporation of a saturated heterocyclic ring and of a fluo-
rine atom in positions 2 and 7 of the tricyclic template, respectively,
resulted in a depletion of activity. A flexible dialkylaminoalkylamino
residue in position 2 improves activity; its length is important but
not critical for cytotoxicity. For 2-methylthio derivatives, the nature
of the substituent in position 30 is relevant for cytotoxicity, appar-
ently through the formation of hydrogen bonds with its in vivo
molecular target, the direction of dipole moment being likely to ori-
ent the compound into the target molecule, thus enhancing or weak-
ening the binding.
7.1.3. 9-[[(3-Methoxy)phenyl]amino]-7-fluoro-2-(methylthio)-
9-anilinothiazolo[5,4-b]quinoline (7c)
Yellow solid; 131 mg (70%); mp 193–195 °C. IR (KBr, cmꢁ1):
3424 (NH), 3028 (OMe), 1599, 1578, 1549, 1510, 1486, (aromatic);
1H NMR (DMSO-d6, d): 2.35 (s, 3H) SCH3; 3.61 (s, 3H) OCH3, 6.74
(m, 3H), H-20, H-40, H-60; 7.25 (t, J = 8.7 Hz, 1H), H-50; 7.75 (ddd,
J = 10.5, 7.8, 2.7 Hz, 1H) H-6; 7.97 (dd, J =9.2, 5.1 Hz, 1H) H-5;
8.27 (dd, J = 10.4, 2.1 Hz, 1H) H-8; 10.13 (br), NHAr. MS (FAB, m/
z): 372 (M++1, 100%), 371 (M+, 17%)%) Anal. Calcd for
C18H14FN3OS2: C, 58.20; H, 3.80; N, 11.31; S, 17.26. Found: C,
58.38; H, 3.87; N, 11.23; S, 17.09.
7. Experimental
All starting materials were commercially available research-
grade chemicals and used without further purification. Reactions
were monitored by analytical TLC on precoated silica gel 60 F254
plates (Aldrich). Column chromatography was carried out on Silica
Gel 60 (70–230 mesh, Merck). Melting points were determined on
a Fisher–Jones apparatus and are uncorrected. Infrared spectra
were recorded on a Nicolet FT-5SX spectrophotometer model. 1H
and 13C NMR spectra were recorded on a Varian VxR-300S spec-
trometer (300 and 75.5 MHz, respectively). Chemical shifts are
reported in ppm (d) and the signals are described as singlet (s),
doublet (d), triplet (t), quartet (q), broad (br) and multiplet (m);
coupling constants are reported in Hertz. EI-MS were carried out
on a JEOL JMS-AX505-HA apparatus. FAB-MS were carried out on
7.1.4. 9-[[(4-Cyano)phenyl]amino]-7-fluoro-2-(methylthio)-9-
anilinothiazolo[5,4-b]quinoline (7d)
Yellow solid; 111 mg (75%); mp 235–238 °C. IR (KBr, cmꢁ1):
3422 (NH), 2220 (CN), 1600, 1575, 1534, 1511, 1496, (aromatic);
1H NMR (DMSO-d6, d): 2.49 (s, 3H) SCH3; 7.2 (d, J = 8.1 Hz, 2H),
H-20, H-60; 7.66 (d, J = 8.1 Hz, 2H), H-30, H-50; 7.72 (dd, J = 9.6,
7.5 Hz, 1H) H-6; 8.07 (d, J = 9.2, 1H) H-5; 8.15 (d, J = 9.0 Hz, 1H)
H-8; 9.90 (br), NHAr. MS (FAB, m/z): 367 (M++1, 100%), 366 (M+,
28%) Anal. Calcd for C18H11FN4S2: C, 59.00; H, 3.03; N, 15.29; S,
17.50. Found: C, 59.13; H, 3.10; N, 15.16; S, 17.43.

3274
M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
7.1.5. 9-[[(4-Methoxy)phenyl]amino]-7-fluoro-2-(methylthio)-
9-anilinothiazolo[5,4-b]quinoline (7e)
yellow crude product was purified by crystallization from metha-
nol or hexane.
Yellow solid; (72%); mp 203–205 °C. IR (KBr, cmꢁ1): 3444 (NH),
3026 (OCH3), 1602, 1577, 1545, 1504, 1444, (aromatic); 1H NMR
(DMSO-d6, d): 2.30 (s, 3H) SCH3; 3.77 (s, 3H) OCH3, 6.96 (d,
J = 8.8 Hz, 2H), H-30, H-50; 7.22 (d, J = 8.1, 2H), H-20, H-60; 7.78
(ddd, J = 9.6, 8.0, 2.8 Hz, 1H) H-6; 7.98 (dd, J = 9.2, 5.6 Hz, 1H) H-
5; 8.41 (dd, J = 11.2, 2.4 Hz, 1H) H-8; 10.11 (br), NHAr. MS (FAB,
m/z): 372 (M++1, 100%), 371 (M+, 28%) Anal. Calcd for
C18H14FN3OS2: C, 58.20; H, 3.80; N, 11.31; S, 17.26. Found: C,
58.41; H, 3.91; N, 11.20; S, 17.05.
7.2.1. 9-Chloro-2-(1-piperidinyl)thiazolo[5,4-b]quinoline (8a)
Pale yellow solid; 130 mg (80%); mp 165–168 °C (hexane). IR
(KBr, cmꢁ1): 2937, 2850 (CH), 1601, 1543, (aromatic); 1H NMR
(CDCl3, d): 1.76 (s, 6H) H-3a, H-4a, H-6a; 3.76 (s, 4H) H-2a, H-5a,
7.60 (m, 2H), H-6, H-7; 8.0 (dd, J = 7.2,. 1.8 Hz, 1H), H-5; 8.25 (dd,
J = 7.5, 1.8 Hz, 1H) H-8. MS (EI, m/z): 305 (M++2, 37%), 303 (M+,
100%) Anal. Calcd for C15H14ClN3S: C, 59.30; H, 4.64; N, 13.83; S,
10.55. Found: C, 59.45; H, 4.89; N, 13.70; S, 10.28.
7.1.6. 9-[[(3-Ethoxycarbonyl)phenyl]amino]-2-(methylthio)-
thiazolo[5,4-b]quinoline (7f)
7.2.2. 9-Chloro-2-[1-[(4-methyl)piperazinyl]]thiazolo[5,4-b]-
quinoline (8b)
Yellow solid; (70%); mp 173–5 °C. IR (KBr, cmꢁ1): 3411 (NH),
3129, 3056, 2971 (CH), 1713 (C@O), 1621, 1570, 1545, 1507 (aro-
matic); 1H NMR (DMSO-d6, d): 1.30 (t, J = 6.9 Hz, 3H) CH3; 2.21
(s, 3H) SCH3; 4.30 (q, J = 6.9 Hz, 2H) OCH2; 7.52 (t, J = 6.9 Hz, 1H)
H-50; 7.55 (dt, J = 6.9, 1.5 Hz, 1H) H-60; 7.68 (ddd, J = 8.7, 6.3, 1.2,
1H) H-7; 7.70 (dt, J = 6.9, 1.5 Hz, 1H) H-40; 7.89 (t, J = 1.5 Hz, 1H)
H-20; 7.92 (ddd, J = 8.7, 6.3, 1.2 Hz, 1H) H-6; 7.99 (dd, J = 8.7, 1.2,
1H) H-5; 8.70 (d, J = 8.4 Hz, 1H) H-8; 10.7 (br, 1H) NH; MS (FAB,
m/z): 396 (M++1, 100%) Anal. Calcd for C20H17N3O2S2: C, 60.74; H,
4.33; N, 10.62; S, 16.22. Found: C, 60.52; H, 4.54; N, 10.46; S, 16.39.
Pale yellow solid; 130 mg (80%); mp 165–168 °C (methanol). IR
(KBr, cmꢁ1): 2932, 2841, 2789 (CH), 1601, 1548, (aromatic); 1H
NMR (CDCl3, d): 2.42 (s, 3H) NCH3; 2.64 (t, J = 4.8 Hz, 4H) H-3a,
H-5a; 3.86 (t, J = 4.5 Hz, 4H), H-2a, H-6a; 7.61 (m, 2H), H-6, H-7;
8.0 (dd, J = 7.8,. 1.8 Hz, 1H), H-5; 8.25 (dd, J = 7.5, 1.8 Hz, 1H) H-8.
MS (EI, m/z): 320 (M++2, 10%), 318 (M+, 30%), 70 (100%) Anal. Calcd
for C15H15ClN4S: C, 56.51; H, 4.74; N, 17.57; S, 10.06. Found: C,
56.51; H, 4.74; N, 17.57; S, 10.06.
7.2.3. 9-Chloro-2-(1-morphonyl)thiazolo[5,4-b]quinoline (8c)
Pale yellow solid; 130 mg (80%); mp 165–168 °C (hexane). IR
(KBr, cmꢁ1): 2967, 2937, 2880 (CH), 1600, 1548, (aromatic); 1H
NMR (CDCl3, d): 3.80 (s, 4H) H-2a, H-6a; 3.87 (s, 4H) H-3a, H-5a;
7.64 (m, 2H), H-6, H-7; 8.04 (d, J = 7.2, 1H), H-5; 8.27 (d, J = 7.5,
1H) H-8. MS (EI, m/z): 307 (M++2, 37%), 305 (M+, 100%) Anal. Calcd
for C14H12ClN3OS: C, 54.99; H, 3.96; N, 13.74; S, 10.49. Found: C,
54.84; H, 4.10; N, 13.61; S, 10.66.
7.1.7. 1-(3-{[2-(Methylthio)thiazolo[5,4-b]quinolin-9-yl]-
amino}phenyl)ethanone (7g)
Yellow solid; (70%); mp 185–7 °C. IR (KBr, cmꢁ1): 3411 (NH),
3129, 3122, 3059 (CH), 1681 (C@O), 1621, 1570, 1550, 1504 (aro-
matic); 1H NMR (DMSO-d6, d): 2.24 (s, 3H) COCH3; 2.55 (s, 3H)
SCH3; 7.52 (t, J = 6.9 Hz, 1H) H-50; 7.55 (dt, J = 6.9, 1.5 Hz, 1H) H-
60; 7.67 (ddd, J = 8.7, 6.3, 1.2, 1H) H-7; 7.78 (dt, J = 6.9, 1.5 Hz,
1H) H-40; 7.86 (t, J = 1.5 Hz, 1H) H-20; 7.91 (ddd, J = 8.7, 6.3,
1.2 Hz, 1H) H-6; 7.99 (dd, J = 8.7, 1.2, 1H) H-5; 8.58 (d, J = 8.4 Hz,
1H) H-8; 10.66 (br, 1H) NH; MS (FAB, m/z): 366 (M++1, 100%) Anal.
Calcd for C19H15N3OS2: C, 62.44; H, 4.14; N, 11.50; S, 17.55. Found:
C, 62.63; H, 4.33; N, 11.42; S, 17.23.
7.3. General preparation of 2-methylsulphanyl derivatives 9a–f
A suspension of 0.5 mmol of the 2-(methylthio)-9-anilinothiaz-
olo[5,4-b]quinoline derivative with the desired substitution
pattern in the aniline ring, in 10 mL of a mixture of H2O2 30%-acetic
acid (1:1) was stirred overnight at room temperature. 20 mL of
cold water were then added and the orange precipitate of the 2-
methylsulphanyl derivative was collected by filtration, dried by
suction and used without further purification.
7.1.8. (1E)-1-(3-{[2-(Methylthio)thiazolo[5,4-b]quinolin-9-
yl]amino}phenyl)ethanone oxime (7h)
To a suspension of 110 mg (0.3 mmol) of compound 7f in 10 mL
of methanol 50 mg of hydroxylamine chlorhydrate in 5 mL of
methanol were added and heated to reflux for 1 h. After cooling,
the solvent was evaporated under reduced pressure; the residue
was suspended in 10 mL of water and a 10% NaHCO3 solution
was added to render pH 8. The solid was collected by vacuum fil-
tration and washed with cold acetone, giving 100 mg (87%) of title
compound as a yellow solid, mp 201–3 °C. IR (KBr, cmꢁ1): 3411
(NH), 3227, 3053 (CH), 1617 (C@N), 1585, 1551, 1496, (aromatic);
1H NMR (DMSO-d6, d): 2.10 (s, 3H) C(NOH)CH3; 2.41 (s, 3H) SCH3;
7.08 (m, 1H) H-50; 7.28 (m, 2H) H-40, H-60; 7.39 (t, J = 1.5 Hz, 1H) H-
20; 7.51 (ddd, J = 8.7, 6.3, 1.2, 1H) H-7; 7.71 (ddd, J = 8.7, 6.3, 1.2 Hz,
1H) H-6; 7.92 (dd, J = 8.7, 1.2, 1H) H-5; 8.37 (d, J = 8.4 Hz, 1H) H-8;
9.36 (br, 1H) NH; 11.12 (s, 1H) OH; MS (FAB, m/z): 366 (M++1,
100%) Anal. Calcd for C19H16N4OS2: C, 59.98; H, 4.24; N, 14.73; S,
16.85. Found: C, 59.81; H, 4.37; N, 14.70; S, 16.95.
7.4. General preparation of compounds10a–10f, 11a–11e and8d–8f
Method A: A solution of the 2-methylsulphanyl derivative
(0.4 mmol) in 0.5 mL of the corresponding amine was heated to
reflux for 30 min. After that time, 30 mL of dichloromethane were
added and the yellowish solution was washed with a NaOH 2 N
(3 ꢂ 5 mL), saturated NH4Cl solution (3 ꢂ 5 mL) and water
(3 ꢂ 5 mL) and dried over Na2SO4. After concentration under
reduced pressure, the pale yellow crude was purified by column
chromatography (silica gel, mixture of CH2Cl2/MeOH/NH4OH
98:2:0.1 as eluent) or crystallization from methanol for com-
pounds 8d–f. Method B: An alternative procedure for compounds
10a–f and 11a–e was as follows: To a solution of the 2-methylsul-
phanyl derivative in 1 mL of DMF 1.5 equiv of the aliphatic amine
were added and stirred overnight at room temperature; the dark
solution was poured into a 10 mL of a well stirred brine solution
and left at room temperature overnight. The precipitate formed
was filtered, dried by suction and treated with hot hexane. This
procedure gave similar yields as method A.
7.2. Preparation of compounds 8a–c
A solution of 6a (133 mg, 0.5 mmol) in 2 mL of the correspond-
ing amine was stirred for 24 h at room temperature. After that
time, 10 mL of a saturated aqueous solution of NH4Cl was added,
the greenish suspension was extracted with dichloromethane
(3 ꢂ 5 mL) and the organic extracts were collected, washed with
a NaHCO3 solution (3 ꢂ 5 mL) and water (3 ꢂ 5 mL) and dried over
Na2SO4. After concentration under reduced pressure, the pale
7.4.1. 9-[[(3-Cyano)phenyl]amino]-2-[2-(N,N-diethylamino)]
ethylaminothiazolo[5,4-b]quinoline (10a)
Pale yellow solid; (69%); mp 110–111 °C. IR (KBr, cmꢁ1) 3311
(NH), 2966, 2810 (CH); 2227 (CN); 1600, 1560, 1476 (aromatic);

M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
3275
1H NMR (CDCl3, d): 1.02 (t, J = 7.2 Hz, 6H) 2-CH3; 2.55 (q, J = 7.2 Hz,
4H) CH2; 2.68 (t, J = 6.9 Hz, 2H) CH2; 3.48 (q, J = 6.9 Hz, 2H) NHCH2;
6.44 (br, 1H) -NH-; 7.00 (s, 1H) –NHAr; 7.04 (d, J = 8.1 Hz, 1H) H-60;
7.08 (s, 1H) H-20; 7.18 (d, J = 8.1 Hz, 1H) H-4; 07.26 (t, J = 8.1, 1H) H-
50; 7.37 (ddd; J = 8.7, 6.9, 1.2 Hz; 1H) H-7; 7.60 (ddd; J = 8.7, 6.9,
1.5 Hz; 1H) H-6; 7.67 (d, J = 8.7 Hz, 1H) H-5; 8.03 (d, J = 8.7 Hz,
1H) H-8; MS (FAB, m/z): 417 (M++1, 100%) Anal. Calcd for
C23H24N6S: C, 66.32; H, 5.81; N, 20.18; S, 7.70. Found: C, 66.17;
H, 6.00; N, 20.96; S, 7.51.
7.4.6. 9-[[(4-Methoxy)phenyl]amino]-2-[2-(N,N-diethylamino)]
ethylaminothiazolo[5,4-b]quinoline (10f)
Yellow solid; (65%); mp 110–112 °C. IR (KBr, cmꢁ1) 3179 (NH)
2966, 2827, (CH); 1599, 1563, 1508, 1495, 1469 (aromatic). 1236
(OCH3); 1H NMR (DMSO-d6, d): 0.88 (t, J = 7.2 Hz, 6H) 2-CH3; 2.41
(q, J = 7.2 Hz, 4H) CH2; 2.45 (t, J = 6.9 Hz, 2H) CH2; 3.23 (q,
J = 6.9 Hz, 2H) NHCH2; 3.68 (s, 3H) OCH3; 6.80 (m, 4H) H-2’, H-60,
H-30, H-50; 7.36 (ddd, J = 8.7, 6.9, 0.9 Hz, 1H) H-7; 7.52 (ddd,
J = 8.7, 6.9, 0.9 Hz, 1H) H-6; 7.79 (dd, J = 8.7, 0.9 Hz, 1H) H-5;
8.06 (dd, J = 8.7, 0.9 Hz, 1H) H-8; 8.13 (br, t, J = 6.9, 1H) –NH–;
8.29 (s, 1H) –NHAr–; MS (EI, m/z): 421 (M+, 15%), 322 (M-99,
80%), 86 (M-335, 100%) Anal. Calcd for C23H27N5OS: C, 65.53; H,
6.46; N, 16.61; S, 7.61. Found: C, 65.66; H, 6.49; N, 16.59; S, 7.47.
7.4.2. 9-[[(3-Chloro)phenyl]amino]-2-[2-(N,N-diethylamino)]
ethylaminothiazolo[5,4-b]quinoline (10b)
Yellow solid; (70%); mp 109–110 °C. IR (KBr, cmꢁ1) 3411
(NH), 2966, 2919, 2849 (CH); 1549, 1561, 1475 (aromatic); 1H
NMR (DMSO-d6, d): 1.10 (t, J = 7.2 Hz, 6H) 2-CH3; 2.97 (q,
J = 7.2 Hz, 4H) CH2; 3.10 (t, J = 6.9 Hz, 2H) CH2; 3.59 (q,
J = 6.9 Hz, 2H) NHCH2; 6.78 (d, J = 8.1 Hz, 1H) H-60; 6.90 (d,
J = 8.1 Hz, 1H) H-40; 7.20 (t, J = 8.1, 1H) H-50; 7.30 (s, 1H) H-20;
7.51 (ddd; J = 8.7, 6.9, 1.2 Hz, 1H) H-7; 7.64 (ddd, J = 8.7, 6.9,
1.5 Hz, 1H) H-6; 7.90 (d, J = 8.7 Hz, 1H) H-5; 8.22 (d, J = 8.7 Hz,
1H) H-8; 8.97 (br, t, J = 6.9, 1H) –NH–; 9.22 (s, 1H) –NHAr–;
MS (FAB, m/z): 426 (M++1, 40%), 86 (M-339, 100%) Anal. Calcd
for C22H24ClN5S: C, 62.03; H, 5.68; N, 20.69; S, 7.49. Found: C,
62.17; H, 5.81; N, 20.52; S, 7.35.
7.4.7. 9-(Phenylamino)-2-[3-(N,N-diethylamino)]propylamino-
thiazolo[5,4-b]quinoline (11a)
Yellow solid; 62 mg (59%); mp 72–75 °C. IR (KBr, cmꢁ1) 3233,
1562, 1493 (–NH–); 2967,2818 (CH); 1597 (aromatic). 1H NMR
(DMSO-d6, d): 0.9 (t, J = 7.2 Hz, 6H) 2-CH3; 1.6 (q, J = 7.0 Hz, 2H) –
CH2–; 2.4 (m, 6H) 3-CH2–; 3.2 (sa, 2H) –CH2–; 6.8 (d, J = 7.2 Hz,
3H) H-20, H-40, H-6’; 7.1 (ddd, J = 8.4,6.9,1.8 Hz, 2H) H-30, H-50;
7.4 (ddd, J = 8.4,6.9,1.5 Hz, 1H) H-7; 7.5 (ddd, J = 8.4,6.9,1.5 Hz,
1H) H-6; 7.9 (dd, J = 8.7,0.9 Hz, 1H) H-5; 8.0 (dd, J = 8.7,0.9 Hz,
1H) H-8; 8.3 (t, J = 5.4 Hz, 1H) –NH–; 8.5(s, 1H) –NH– MS (EI, m/
z) 405 (M+ 100%); 376 (M+-29, 15%); 319 (M+-89, 56%); 306 (M+-
99, 53%); Anal. Calcd for C23H27N5S: C, 68.11; H, 6.71; N, 17.27;
S, 7.91. Found: C, 68.15; H, 6.67; N, 17.31; S, 7.87.
7.4.3. 9-[[(3-Methoxy)phenyl]amino]-2-[2-(N,N-diethylamino)]
ethylaminothiazolo[5,4-b]quinoline (10c)
Brownish solid; (50%); mp 118–120 °C. IR (KBr, cmꢁ1): 3120
(NH), 3053, 2966 2850 (CH), 1595, 1571, 1543, 1503, 1476, (aro-
matic), 1H NMR (DMSO-d6, d): 0.88 (t, J = 7.2 Hz, 6H) 2-CH3; 2.41
(q, J = 7.2 Hz, 4H) CH2; 2.45 (t, J = 6.9 Hz, 2H) CH2; 3.23 (q,
J = 6.9 Hz, 2H) NHCH2; 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, 1H) H-5; 8.53 (d,
J = 8.4 Hz) H-8; 8.13 (br, t, J = 6.9, 1H) –NH–; 8.29 (s, 1H) –NHAr–
; MS (EI, m/z): 421 (M+, 15%), 322 (M-99, 80%), 86 (M-335, 100%)
Anal. Calcd for C23H27N5OS: C, 65.53; H, 6.46; N, 16.61; S, 7.61.
Found: C, 65.41; H, 6.52; N, 16.67; S, 7.76.
7.4.8. 9-[[(3-Cyano)phenyl]amino]-2-[3-(N,N-diethylamino)]
propylaminothiazolo[5,4-b]quinoline (11b)
Yellow solid; 68 mg (57%); mp 73–76 °C. IR (KBr, cmꢁ1) 3209 (–
NH–); 2960, 2815 (CH), 2277 (CN); 1599, 1562,1494 (aromatic); 1H
NMR (DMSO-d6, d): 0.9 (t, J = 7.2 Hz, 3H) 2-CH3; 1.6 (q, J = 7.2 Hz;
2H) –CH2–; 2.4 (m, 6H) 3-CH2–; 3.2 (q, J = 7.2 Hz, 2H) –CH2–; 7.1
(d, J = 7.5 Hz, 2H) H-20, H-60; 7.2 (d, J = 7.5 Hz, 1H) H-40; 7.3 (ddd,
J = 8.4, 7.8, 0.9 Hz, 1H) H-50; 7.5 (ddd, J = 8.1,6.9,1.2 Hz, 1H) H-7;
7.6 (ddd, J = 8.4,6.9,1.2 Hz, 1H) H-6; 7.9 (d, J = 7.5 Hz, 1H) H-5;
8.1 (d, J = 7.8 Hz, 1H) H-8; 8.5 (br) –NH–; 9.0 (br) –NH– MS (EI,
m/z) 430 (M+ 100%); 401 (M+-29, 12%); 344(M+-86, 30%); 330
(M+-100, 27%). Anal. Calcd for C24H26N6S: C, 66.95; H, 6.09; N,
19.52; S, 7.45. Found: C, 66.74; H, 6.39; N, 19.33; S, 7.54.
7.4.4. 9-[[(4-Cyano)phenyl]amino]-2-[2-(N,N-diethylamino)]
ethylaminothiazolo[5,4-b]quinoline (10d)
Pale yellow solid; (61%); mp 114–115 °C. IR (KBr, cmꢁ1) 3320
(NH); 2967, (CH) 2216 (CN), 1602, 1580, 1560, 1511 (aromatic);
1H NMR (DMSO-d6, d):1.10 (t, J = 7.2 Hz, 6H) 2-CH3; 2.97 (q,
J = 7.2 Hz, 4H) CH2; 3.10 (t, J = 6.9 Hz, 2H) CH2; 3.59 (q, J = 6.9 Hz,
2H) NHCH2; 6.84 (d, J = 8.4 Hz, 2H) H-20, H-60; 7.55 (m, 3H) H-30,
H-50, H-7; 7.64 (ddd, J = 7.2, 6.9, 1.2 Hz, 1H) H-6; 7.91 (d,
J = 7.8 Hz, 1H) H-5; 8.04 (d, J = 8.4 Hz, 1H) H-8; 8.50 (br, 1H) –
NH– 9.3 (s, 1H) –NH– MS (FAB, m/z) 430 (M++1, 50%) 86 (M-344,
100%) Anal. Calcd for C23H24N6S: C, 66.32; H, 5.81; N, 20.18; S,
7.70. Found: C, 66.47; H, 5.93; N, 20.10; S, 7.50.
7.4.9. 9-[[(3-Chloro)phenyl]amino]-2-[3-(N,N-diethylamino)]
propylaminothiazolo[5,4-b]quinoline (11c)
Yellow solid; 45 mg (55.0%); mp 55–58 °C. IR (KBr, cmꢁ1) 2966,
1375 (–CH); 1594, 1561, 1497 (aromatic); 1H NMR (DMSO-d6, d):
0.9 (t, J = 7.2 Hz, 6H) 3-CH3; 1.6 (q, J = 6.9 Hz, 2H) –CH2–; 2.4 (m,
6H) 3-CH2–; 3.3 (c, J = 6.6 Hz, 2H) –CH2–; 6.7 (d, J = 9.0 Hz, 1H)
H-60; 6.8 (d, J = 6.3 Hz, 2H) H-20, H-40; 7.1 (t, J = 8.1 Hz, 1H) H-50;
7.5 (t, J = 7.5 Hz, 1H) H-7; 7.6 (t, J = 7.5 Hz, 1H) H-6; 7.9 (t,
J = 8.4 Hz, 1H) H-5; 8.1 (d, J = 8.4 Hz, 1H) H-8; 8.4 (br) –NH–; 8.8
(s, 1H) –NH– MS (EI, m/z) 438 (M+, 100%); 410 (M+-29, 8%); 353
(M+-57, 28%). Anal. Calcd for C23H26ClN5S: C, 62.78; H, 5.96; N,
15.92; S, 7.29. Found: C, 62.78; H, 5.85; N, 15.81; S, 7.29.
7.4.5. 9-[[(4-Chloro)phenyl]amino]-2-[2-(N,N-diethylamino)]
ethylaminothiazolo[5,4-b]quinoline (10e)
Yellow solid; (63%); mp 113–114 °C. IR (KBr, cmꢁ1) 3410 (NH),
2965, 2809, (CH); 1600, 1586, 1566 (aromatic); 1H NMR (DMSO-d6,
d): 1.08 (t, J = 7.2 Hz, 6H) 2-CH3; 2.94 (q, J = 7.2 Hz, 4H) CH2; 3.08 (t,
J = 6.9 Hz, 2H) CH2; 3.58 (q, J = 6.9 Hz, 2H) NHCH2; 6.82 (d,
J = 8.7 Hz, 2H) H-20, H-60; 7.21 (d, J = 8.7 Hz, 2H) H-30, H-50; 7.48
(ddd, J = 8.7, 6.9, 1.2 Hz, 1H) H-7; 7.60 (ddd, J = 8.7, 6.9, 1.5 Hz,
1H) H-6; 7.88 (d, J = 8.7 Hz, 1H) H-5; 8.16 (d, J = 8.7 Hz, 1H) H-8;
8.4 (br, t, J = 6.9, 1H) –NH–; 8.91 (s, 1H) –NHAr–; MS (FAB, m/z):
426 (M++1, 40%), 86 (M-339, 100%) Anal. Calcd for C22H24ClN5S:
C, 62.03; H, 5.68; N, 20.69; S, 7.49. Found: C, 62.21; H, 5.73; N,
20.64; S, 7.30.
7.4.10. 9-[[(4-Cyano)phenyl]amino]-2-[3-(N,N-diethylamino)]
propylaminothiazolo[5,4-b]quinoline (11d)
Yellow solid; 92 mg (71%); mp 75–78 °C IR (KBr, cmꢁ1) 3316 (R–
NH–R); 2960, 1466, 1378 (CH); 2217 (CN); 1603, 1561, 1512, 1466
(aromatic); 1H NMR (DMSO-d6, d):0.9 (t, J = 7.2 Hz, 6H) 2-CH3; 1.6
(q, J = 6.9 Hz, 2H) –CH2–; 2.4 (m, 6H) 3-CH2–; 3.2 (sa, 2H) –CH2–;
6.8 (d, J = 8.4 Hz, 2H) H-2’, H-60; 7.5 (d, J = 8.7 Hz, 3H) H-30, H-50,
H-7; 7.6 (ddd; J = 7.2, 6.9, 1.2 Hz, 1H) H-6; 7.9 (d, J = 7.8 Hz, 1H)
H-5; 8.0 (d, J = 8.4 Hz, 1H) H-8; 8.5 (br, 1H) –NH– 9.3 (s, 1H) –
NH– MS (EI, m/z) 430 (M+, 71%) 401 (M+-29, 9%) 344 (M+-57,

3276
M. A. Loza-Mejía et al. / Bioorg. Med. Chem. 17 (2009) 3266–3277
31%); 330 (M+-14, 31%) Anal. Calcd for C24H26N6S: C, 66.95; H, 6.09;
N, 19.52; S, 7.45. Found: C, 66.84; H, 6.28; N, 19.44; S, 7.44.
Beckman photometer model DUR-64. Cell growth inhibition was
determined by the formula% cell growth inhibition = (1 ꢁ absor-
bance of treated cells/absorbance of untreated cells) ꢂ 100. The
assays were carried out in three independent experiments in
quadruplicate.
7.4.11. 9-[[(4-Chloro)phenyl]amino]-2-[3-(N,N-diethylamino)]
propylaminothiazolo[5,4-b]quinoline (11e)
Yellow solid; 75 mg (61%); mp 120–123 °C IR (KBr, cmꢁ1) 2965,
2809, (CH); 1600, 1586, 1566 (aromatic); 1H NMR (DMSO-d6, d):
0.92 (t, J = 7.2 Hz, 6H) 2-CH3; 1.6 (q, J = 6.9 Hz, 2H) –CH2–; 2.4
(m, 6H) 3-CH2–; 3.2 (c, J = 6.6 Hz, 2H) –CH2–; 6.8 (d, J = 8.7 Hz,
2H) H-2’, H-60; 7.1 (d, J = 8.7 Hz, 2H) H-30, H-50; 7.4 (ddd, J = 8.1,
6.9, 1.2 Hz, 1H) H-7; 7.6 (ddd, J = 8.1, 6.9, 1.5 Hz, 1H) H-6; 7.9 (d,
J = 7.5 Hz, 1H) H-5; 8.1 (d, J = 7.8 Hz, 1H) H-8; 8.4 (br, 1H) –NH–;
8.7 (s, 1H) –NH–; MS (EI, m/z): 439 (M+, 75%) 410 (M+-29, 11%);
367 (M+-43, 13%); 353 (M+-14, 40%). Anal. Calcd for C23H26ClN5S:
C, 62.78; H, 5.96; N, 15.92; S, 7.29. Found: C, 62.73; H, 5.90; N,
15.86; S, 7.34.
7.6. Decatenation assay for topoisomerase II activity24
DNA topoisomerase activity was monitored with the
jDNA
decatenation assay using human DNA topoisomerase II, and the
TM
DNA topoisomerase II determination kit from Topogen . Briefly,
jDNA from Crithidia fasciculata, at a concentration of 5 lg/mL, was
incubated in 30 mM Tris–HCl, pH 7.6 containing 60 mM NaCl,
8 mM MgCl2, 15 mM 2-mercaptoethanol, 3 mM ATP, 4 units of
human DNA topoisomerase II, 8% (v/v) dimethyl sulphoxide, and
with or without the indicated concentration of the inhibitor in a
final volume of 20
30 min at 37 °C, terminated by the addition of 2
treated with proteinase K for 15 min at 37 °C. The samples were
mixed with 2.2 L of loading dye (0.025% w/v bromophenol blue in
50% glycerol) and extracted with 25 L of chloroform/isoamyl alco-
hol24:1. Phase separation waspromoted by rapidcentrifugationin a
microfuge, and the DNA in the aqueous layer was analyzed by elec-
trophoresis in a standard agarose gel with 0.5 lg/mL of ethidium
l
L. Reactions were allowed to proceed for
7.4.12. 9-Phenylamino-2-(1-piperidinyl)thiazolo[5,4-b]quinoline
l
L of 10% SDS and
(8d)
Yellow solid; (84%); mp 200–203 °C (methanol). IR (KBr, cmꢁ1
)
l
3440 (NH), 3223, 2953, 2851 (C–H); 1600, 1494 (aromatic); 1H
NMR (CDCl3, d): 1.71 (s, 6H) H-3a, H-4a, H-6a; 3.64 (s, 4H) H-2a,
H-5a, 7.02 (d, J = 8 Hz, 2H) H-20, H-60; 7.07 (t, J = 7.2 Hz, 1H) H-7;
7.24 (m, 3H) H-30, H-4’, H-50; 7.54 (td, J = 7.6, 1.2 Hz, 1H) H-6;
7.68 (dd, J = 8.4, 0.8 Hz, 1H) H-5; 8.04 (d, J = 8.4 Hz, 1H) H-8; MS
(EI, m/z): 390 (M+, 100%) Anal. Calcd for C21H20N4S: C, 69.97; H,
5.59; N, 15.54; S, 8.90. Found: C, 69.78; H, 5.53; N, 15.76; S, 8.93.
l
bromide. The gel was destained for 30 min and photographed under
TM
UV light using a Bio-Rad FlourS imaging system. The relative
amounts of decatenated jDNA were measured using the Bio-Rad’s
Quantity-one software or the ImageJ software.25 Both programs
essentially rendered the same results. The activity was calculated
as the density of the circular-decatenated plus linear DNA bands as
a percentage of the total DNA. Activity in a control reaction, in the
absence of inhibitors, was taken as 100%.
TM
7.4.13. 9-Phenylamino-2-[1-[(4-methyl)piperazinyl]]thiazolo[5,4-
b]quinoline (8e)
Pale yellow solid; (97%); mp 150–152 °C (methanol). IR (KBr,
cmꢁ1): 3374 (NH), 2936, 2847, 2799 (CH), 1599, 1494, (aromatic);
1H NMR (CDCl3, d): 2.37 (s, 3H) NCH3; 2.55 (t, J = 4.8 Hz, 4H) H-3a,
H-5a; 3.71 (t, J = 5.2 Hz, 4H), H-2a, H-6a; 6.94 (t, J = 7.6, 2H) H-20,
H-60; 6.99 (t, J = 7.2 Hz, 1H) H-7; 7.24 (m, 3H) H-3’, H-40, H-50;
7.53 (td, J = 7.6, 1.6 Hz, 1H) H-6; 7.71 (dd, J = 8.8, 1.8 Hz, 1H) H-5;
7.97 (dd, J = 8.4, 0.8 Hz, 1H) H-8, MS (EI, m/z): 375 (M+, 30%), 70
(100%) Anal. Calcd for C21H21N5S: C, 67.17; H, 5.64; N, 18.65; S,
8.54. Found: C, 67.34; H, 5.87; N, 18.42; S, 8.31.
7.7. Molecular modeling procedure
All calculations were performed with SPARTAN’04 software.16 The
molecules were built by assembling standard fragments, and the
resulting geometries were optimized by molecular mechanics.
Conformational analysis of the compounds by Systematic Search
protocol around rotable bonds was performed using the MMFF94
force field. The most frequent conformer for each compound was
selected and geometry optimization and calculation of their elec-
tronic properties were carried out with semiempirical AM1
method.
7.4.14. 9-Phenylamino-2-(1-morphonyl)thiazolo[5,4-b]quinoline
(8f)
Pale yellow solid; (80%); mp 205–208 °C (methanol–water). IR
(KBr, cmꢁ1): 3330(NH) 2956, 2904, 2859 (CH), 1600, 1548, (aro-
matic); 1318 (C–O) 1H NMR (CDCl3, d): 3.66 (t, J = 4.4 Hz, 4H) H-
2a, H-6a; 3.83 (t, J = 4.4 Hz, 4H) H-3a, H-5a; 7.05 (d, J = 8 Hz, 2H)
H-20, H-6’; 7.11 (t, J = 7.6, 1H) H-7; 7.28 (m, 3H) H-30, H-40, H-50;
7.40 (sa, 1H) NH; 7.57 (td, J = 7.6, 1.2 Hz, 1H) H-6; 7.71 (dd,
J = 8.8, 1.2 Hz, 1H) H-5; 8.06 (d, J = 8.4 Hz, 1H) H-8. MS (EI, m/z):
362 (M+, 100%) Anal. Calcd for C20H18N4OS: C, 66.28; H, 5.01; N,
15.46; S, 8.85. Found: C, 66.53; H, 5.23; N, 15.24; S, 8.60.
Acknowledgments
We thank Maricela Gutiérrez, Rosa Isela del Villar, Victor M.
Arroyo, Georgina Duarte and Margarita Guzmán for determina-
tion of all spectra and Nayeli López for elemental analysis. M.
A. L.-M. wishes to thank Consejo Nacional de Ciencia
y
Tecnología (CONACYT-Mexico) for a scholarship for his doctorate
studies. We also thank DGAPA-UNAM for financing project PAP-
IIT IN202805 as well as Facultad de Química for financial sup-
port (PAIP 6390-10, 6290-09).
7.5. Cytotoxic assay23
The effects of the compounds were determined in one cervical
cell line (HeLa), two human colorectal cancer cell lines (SW480
and SW620) and one myelogenous leukemia human cell line (K-
562). The cytotoxic assays were carried out according to the micro-
culture MTT method. Cells were harvested at 4.5 to 5.0 ꢂ 104 cells/
mL/well and inoculated in 24 well microtiter plates. The culture
cells were then inoculated free and with the compounds (which
were dissolved in DMSO and added in a volume maximum of
2 mL/mL/well). After 72 h incubation, 100 mg/mL of MTT (in PBS,
pH 7.2) were added. 1 mL of DMSO was added to each well, fol-
lowed by gentle shaking, dissolved the formazan dye. After centri-
fugation the extinction coefficient was measured at 540 nm using a
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.03.052.
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