Novel cyano- and N-isopropylamidino-substituted derivatives of benzo[b]thiophene-2-carboxanilides and benzo[b]thieno[2,3-c]quinolones: Synthesis, photochemical synthesis, crystal structure determination, and antitumor evaluation. 2

Article abstract of DOI:10.1021/jm049541f

Derivatives of 3-chlorobenzo[b]thiophene-2-carboxanilides and their "cyclic" analogues benzo-[b]thieno[2,3-c]quinolones were synthesized. Spectroscopic study of the interactions of some representatives of "cyclic" derivatives and their "acyclic" precursor

Full text of DOI:10.1021/jm049541f

2346
J. Med. Chem. 2005, 48, 2346-2360
Novel Cyano- and N-Isopropylamidino-Substituted Derivatives of
Benzo[b]thiophene-2-carboxanilides and Benzo[b]thieno[2,3-c]quinolones:
Synthesis, Photochemical Synthesis, Crystal Structure Determination, and
Antitumor Evaluation. 2
Ivana Jarak,† Marijeta Kralj,‡ Lidija Sˇuman,‡ Gordana Pavlovic´,§ Jasna Dogan,†,| Ivo Piantanida,
Mladen Zˇinic´, Kresˇimir Pavelic´,‡ and Grace Karminski-Zamola*,†
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulic´ev trg 20,
-
P.O. Box 177, HR-10000 Zagreb, Croatia, Division of Molecular Medicine, “Ruder Bosˇkovic´” Institute, Bijenicˇka cesta 54,
P.O. Box 180, HR-10000 Zagreb, Croatia, Faculty of Textile Technology, University of Zagreb, Pierottieva 6,
HR-10000, Zagreb, Croatia, and Division of Organic Chemistry & Biochemistry, Laboratory of Supramolecular and
Nucleoside Chemistry, “Rud-er Bosˇkovic´” Institute, P.O. Box 180, HR-10002 Zagreb, Croatia
Received June 11, 2004
Derivatives of 3-chlorobenzo[b]thiophene-2-carboxanilides and their “cyclic” analogues benzo-
[b]thieno[2,3-c]quinolones were synthesized. Spectroscopic study of the interactions of some
representatives of “cyclic” derivatives and their “acyclic” precursors with ds-DNA/RNA supported
strong intercalative binding of the former and weak nonintercalative binding of the latter group
of compounds. All tested compounds showed a certain antiproliferative effect on a series of
human tumor cells and on a normal cell line. Among the compounds, those with one amidino-
substituent have shown the best effect. The most active benzo[b]thieno[2,3-c]quinolones induced
apparent S and G2/M arrests of the cell cycle, which resulted in apoptosis. These results strongly
suggest that the compounds may act as topoisimerase “poisons”, which is in good agreement
with their intercalative mode of binding to ds-DNA/RNA, in contrast to the studied “acyclic”
group of derivatives. 6a and 6d showed the best selectivity by inhibiting the growth of tumor
cells but not of normal fibroblasts.
Introduction
naphthyridine-3-carboxylic acids as antitumor agents
have been described recently.14
There are not many recent literature data about the
synthesis and antitumor evaluation of heterocyclic
condensed quinolones. Novel quinoline-quinone, e.g.
streptonigrin (SN), is an antitumor antibiotic that has
activity against a broad range of tumors.1-3 SN was
studied clinically as an antitumor agent, but its use was
limited by reports of delayed myelotoxicity.4,5 Neverthe-
less, positive results were reported for SN both as a
single agent6,7 and in combination chemotherapy.8,9 SN
is an excellent substrate for oxidoreductase (NQ01).10
Pyranoquinoline-2-ones were synthesized and evalu-
ated for their in vitro cytotoxicity against a panel of
human tumor cell lines,11 while 2-arylquinazolinones
displayed significant growth inhibitory action against
tumor cell lines, some of them being potent inhibitors
of tubulin polymerization. Some displayed selective
activity against P-gp-expressing epidermoid carcinoma
of the nasopharynx.12 Besides, trifluoromethyl-substi-
tuted pyranoquinolinone was prepared and tested for
the ability to modulate the transcriptional activity of
the human androgen receptor (HAR).13
The authors investigated the structure-activity re-
lationships in this series of compounds by changing N-1
and C-7 positions and the core ring structure itself and
evaluated the synthesized compounds against several
murine and human tumor cell lines. These modifications
led to the following findings. The 2-thiazolyl group at
the N-1 position of the naphthyridine structure is the
best substituent for antitumor activity. Regarding the
core ring structure, the naphthyridine derivative is the
most active, followed by the pyridopyrimidine analogue.
At the C-7 position, aminopyrrolidine derivatives are
more effective than other amines or thioether deriva-
tives. Finally, the trans-3-amino-4-methoxypyrrolidinyl
derivative and the 3-amino-3-methylpyrrolidinyl deriva-
tive as well as 3-aminopyrrolidinyl derivative were
found to be effective in in vitro and in vivo antitumor
assays, and their activity was comparable to that of
etoposide.
Indolo-, pyrrolo-, and benzofuroquinolones and anili-
noindoloquinolone derivatives were synthesized and
evaluated in vitro against a three-cell line panel consist-
ing of MCF7, NCI-H460, and SF268. The results have
shown that cytotoxicity decreases in the order: anili-
noindoloquinolones > indoloquinolones > pyrroloqui-
nolones > benzofuroquinolones.15 New synthesized hy-
droxymethyl- and methoxymethylfuro[2,3-h]quinolin-
2(1H)-ones inhibited topoisomerase II, leading to a
moderate antiproliferative activity in mammalian cells.
The antiproliferative activity was also tested upon UVA
Synthesis and structure-activity relationships of
novel 7-substituted 1,4-dihydro-4-oxo-1-(2-thiazolyl)-1,8-
* To whom correspondence should be addressed. Phone:
++38514597215. Fax: ++38514597250. E-mail: gzamola@pierre.fkit.hr.
† Department of Organic Chemistry, University of Zagreb.
-
‡ Division of Molecular Medicine, “Ruder Bosˇkovic´” Institute.
§ Faculty of Textile Technology, University of Zagreb.
|| Present address: PLIVA d.d., Research and Development, Prilaz
baruna Filipovic´a 25, HR-10000 Zagreb, Croatia.
-
Laboratory of Supramolecular and Nucleoside Chemistry, Ruder
Bosˇkovic´” Institute.
10.1021/jm049541f CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/17/2005

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2347
irradiation in mammalian cells: all compounds showed
higher activity than 8-MOP, without mutagenicity and
skin phototoxicity.16
Recent data describe the quinolone-metal complexes
as antraquinone intercalators investigated on leukaemia
L1210 cells. A new series of quinolone-platinum(II)
conjugates, [Pt(Q′-NH2)(dmso)X2] and cis-[Pt(Q′′-en)X2],
where Q′ and Q′′ are quinolones (flumequine, nalidixic
acid, or oxolinic acid) linked to monodentate and biden-
tate amine ligands, respectively, and X2 is Cl2 or 1,1-
cyclobutanedicarboxylate, have been synthesized with
the aim of examining the synergetic antitumor activity
of quinolone intercalation and platinum(II) chelation.17
Searching for compounds related to heterocyclic qui-
nolones as antitumor active compounds, we have pre-
pared new derivatives of thieno[3′,2′:4,5]thieno[2,3-
c]quinolones and examined their antitumor activity.18
Later on, a class of substituted benzo[b]thieno[2,3-c]-
quinolones containing a 3-dimethylaminopropyl sub-
stituent on the quinolone nitrogen or a 3-dimethylami-
nopropyl substituent in the amide part of the molecule
was prepared. All prepared compounds were tested on
several human tumor cell lines and exhibited strong
inhibitory activities in vitro against all the cell lines
tested. The compounds that bear a dimethylaminopropyl
substituent on the quinolone nitrogen showed higher
antitumor activity than compounds bearing the same
substituent on the amidic nitrogen.19
Figure 1. Substituted 3-chlorobenzo[b]thiophene-2-carbox-
amides and benzo[b]thieno[2,3-c]qinolones (2a, 2b, 4a, 4b, 5a-
g, 7a-e, 8a-e, and 3a, 3b, 6a-g, 9a-d).
[2,3-c]quinolones 3a, 3b, 6a-g, and 9a-d were pre-
pared by the photochemical dehydrohalogenation reaction
in very good yields.
In this work, we have prepared a new group of
substituted derivatives of benzo[b]thieno-2-carboxanil-
ides (4a, 4b, 5a-g, 7a-e, and 8a-e) and benzo[b]-
thieno[2,3-c]quinolones bearing a cyano or a substituted
amidino group in different positions (6a-g and 9a-d)
of the condensed ring and evaluated their antitumor
activity.
Crystal Structure of 9a. The molecular and crystal
structures of compound 9a were determined by single-
crystal X-ray diffractometry. The ORTEP view (Figure
2; Cl- ions have been omitted for clarity) of the molec-
ular structure clearly shows that the asymmetric unit
contains two crystallographically independent mol-
ecules, which differ only in the conformation of the
isopropyl fragment. The π-electron delocalization through
the molecules includes planar benzo[b]thieno[2,3-c]-
quinolone moieties, while the N-isopropylamidino frag-
ment does not lie in the plane of the remaining mol-
ecules. Protonation of the amidine group is confirmed
by the equal bond distance values of C11-N13 and
C11-N12 bonds (of the first molecule) and C21-N23
and C21-N22 bonds (of the second molecule).
All hydrogen atoms, belonging either to the amidino
nitrogen atoms or to the nitrogen atoms of the quino-
lonic part, participate in hydrogen-bond formations of
the N-H‚‚‚Cl- or N-H‚‚‚O type. The geometry of the
N-H‚‚‚Cl- hydrogen bonds is characterized by the
N‚‚‚Cl- distances being in the range 3.183(7)-3.264(7)
Å and the angles in the range 154(7)°-172(5)°. Nitrogen
atoms N12 and N22 form intermolecular hydrogen
bonds with O1 and O2 atoms (N22-H7‚‚‚O2 and N12-
H8‚‚‚O1 distances are 2.814(8) and 2.852(8) Å, respec-
tively).
Chemistry
All compounds (2a-9d) shown in Figure 1 were
prepared according to Scheme 1, starting from 3-chlo-
robenzo[b]thiophene-2-carbonyl chloride 1a or 6-substi-
tuted 3-chlorobenzo[b]thiophene-2-carbonyl chlorides
1b-f, which were already known from the literature.20
2- and 9-methoxycarbonyl-substituted benzo[b]thieno-
[2,3-c]quinolones 3a and 3b were prepared from the
corresponding carboxanilides 2a and 2b. Carboxanilides
2a and 2b served as reference compounds in the
biological examination, while quinolones 3a and 3b
could not be examined for their antitumor activity
because of insolubility. Novel derivatives of cyano-
substituted 3-chlorobenzo[b]thiophene-2-carboxanilides
4a, 4b, and 7a-e were prepared in the reaction of the
corresponding carbonyl chlorides with p-cyanoaniline or
with 6-cyano-substituted 3-chlorobenzo[b]thiophene-2-
carbonyl chloride 1f with the corresponding p-substi-
tuted anilines. Novel N-isopropylamidino-substituted
3-chlorobenzo[b]thiophene-2-carboxanilides 5a, 5f, and
8a-e were prepared in the Pinner21 reaction from cyano
derivatives (4a, 4b, and 7a-e) while carboxanilides
5b-e were prepared by direct condensation of 3-chlo-
robenzo[b]thiophene-2-carbonyl chlorides 1b-e with
4-(N-isopropylamidino)aniline, as well as diamidino-
substituted carboxanilide 5g. All prepared 6-substituted
3-chlorobenzo[b]thiophene-2-carboxanilides were ob-
tained in 15-81% yields. All substituted benzo[b]thieno-
Molecular Structure of 9a and Its Correlation
with Binding to DNA/RNA. Most aromatic com-
pounds built of three or more condensed aromatic units
base their biological activity on intercalation.22 The
determined crystal structure of compound 9a shows that
the benzo[b]thieno[2,3-c]quinolone moiety is planar and
aromatic, while the N-isopropylamidino substituent is
in extended conformation and lies only slightly out of

2348 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Jarak et al.
Scheme 1
the plane (Figure 3a). Although the aromatic surface of
9a is more than large enough for intercalation,22 it
should be stressed that the longer axis of 9a (13.6 Å)
significantly surpasses longer axes of some classical
intercalators (ethidium bromide 9.8 Å, proflavine 9.5 Å).
The question therefore arises whether insertion of 9a
and its analogues between DNA/RNA basepairs is
possible.
To study if the sterical properties of 9a allow inter-
calation into the double stranded helix, we have chosen
the intercalative ds-5′-d(CpGpCpG)-3′/Flexi-Di complex,
whose crystal structure was previously determined.23
Flexi-Di (an analogue of ditercalinium)24 consists of two
linked 7H-pyridocarbazole units (Figure 3b), which are
actually close structural analogues of the here studied
benzo[b]thieno[2,3-c]quinolones (e.g. 9a, Figure 3a).
Flexi-Di was converted into two separate molecules of
9a by deleting the aliphatic linker and modifying the
structure of 7H-pyridocarbazole into 9a by introducing
parameters of atoms taken from the crystal structure
of the latter compound.
Structures represented in Figure 4 indicate that the
curved shape of 9a allowed its intercalation into ds-DNA
without steric clashes with the polynucleotide backbone.

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2349
Both shown positions of the amidinium substituent
allow formation of additional hydrogen bonds (Figure
4a).
Interactions of Acyclic Derivatives (5d, 8d) and
Their Cyclic Analogues (6d, 9c) with Double-
Stranded DNA and RNA. Compounds studied in this
paper can be divided into two major groups, “acyclic”
derivatives (Figure 1; compounds 2, 4, 5, 7, 8) and their
“cyclic” analogues (Figure 1; compounds 3, 6, 9). Cyclic
derivatives consist of a large planar moiety (see X-ray
data, Figure 3a), which should form stable intercalative
complexes with double-stranded (ds) DNA and RNA (as
shown in Figure 4). On the other hand, acyclic deriva-
tives are much more flexible and possess no condensed
aromatic moiety large enough for stable intercalation
into ds-polynucleotides.22 Owing to the mentioned struc-
tural differences, acyclic and cyclic derivatives can differ
in the modes of their biological action.
To shed more light on the structure-biological activ-
ity relationship, we have studied interactions of the close
structural analogues 5d, 8d, 6d, and 9c (Figure 1) with
ds-polynucleotides, namely natural calf thymus (ct)
DNA and synthetic RNA homo-polynucleotides. Since
application of spectrophotometric methods was neces-
sary for these studies, we have characterized aqueous
solutions of the compounds by means of electronic
absorption (UV/vis) and fluorescence emission spectra.
Figure 2. (a and b) Molecular structure of 9a showing two
crystallographically independent molecular ions. Chloride ions
have been omitted.
Absorbencies of aqueous solutions of 5d, 8d, 6d, and
9c are proportional to their concentrations up to 1 ×
10-4 mol dm-3, indicating that there is no significant
intermolecular stacking that could give rise to hypo-
chromicity effects. The pH dependence of the absorption
spectra of 6d and 9c under neutral and basic conditions
can be attributed to the protonation of amidine substit-
uents, commonly having pK ) 8-9.25 To have predomi-
nantly protonated amidines under the experimental
conditions used (more than 99%), we performed our
studies at pH ) 6.2.
Figure 3. Single molecule of 9a isolated from its crystal
structure (A). 7H-Pyridocarbazole unit taken from the crystal
structure of 5′-d(CpGpCpG)-3′/Flexi-Di complex23 (B).
Figure 4. Single molecule of 9a taken from its crystal structure and inserted into ds-5′-d(CpGpCpG)-3′ on positions of the bis-
intercalator Flexi-Di.23 Green dotted lines on structure A mark possible hydrogen bonds. Views B and C show the overlapping of
basepairs with the aromatic system of 9a (green).

2350 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Table 1. Electronic Absorption Data of 5d, 8d, 6d and 9ca
Jarak et al.
Table 2. Stability Constants (log Ks) and Ratios n ([bound
compound]/[polynucleotide]) Calculated According to UV/Vis
Titrations of 5d and 8d and Fluorimetric Titrations of 6d and
9c with Polynucleotidesa,b
λmax
/
ꢀ × 103/
λmax
/
ꢀ × 103/
nm
dm3 mol-1 cm-1
nm
dm3 mol-1 cm-1
5d
8d
250
304
250
304
30.2
35.8
24.0
25.0
6d
9c
264
354
370
260
353
368
43.5
9.0
ct-DNA
log Ks
polyA-polyU
polyG-polyC
8.2
n
log Ks
n
log Ks
n
36.9
6.6
6.1
5d
8d
6d
9c
5.07
5.05
5.96
6.21
0.60
0.37
0.25
0.25
5.07
c
5.74
5.79
0.86
c
0.10
0.20
c
c
c
c
5.25
5.91
0.08
0.03
a Sodium cacodilate/HCl buffer, I ) 0.05 mol dm-3, pH ) 6.2.
a UV/vis titrations were performed at pH ) 6.2 (sodium caco-
dilate/HCl buffer, I ) 0.05 mol dm-3). b Accuracy of n ( 30%,
consequently varying values log Ks ( 0.5. c To small changes (total
less than ∆Abs ) 0.05) for evaluation of binding.
Cyclization of 5d and 8d into 6d and 9c, respectively,
resulted in pronounced bathochromic shifts of the
absorption maxima of acyclic compounds (Table 1).
Comparison of ꢀ values revealed that all ꢀ(5d) > ꢀ(8d)
and also ꢀ(6d) > ꢀ(9c), strongly suggesting that the
positioning of amidine and carbmethoxy substituents
has a systematic impact on the electron absorption
properties of studied compounds and the likely electron
distribution in the studied systems. Aqueous solutions
of all studied compounds were shown to be stable over
longer periods and also at increased temperature during
shorter periods (a few hours at up to 100 °C).
All studied compounds exhibit characteristic fluores-
cence emission with maxima at about 400 nm, their
excitation spectra being in good agreement with the
absorption spectra. Fluorescence intensities were found
to be linearly concentration dependent up to 4 × 10-6
mol dm-3. Under the same conditions, cyclic compounds
6d and 9c exhibit 14 and 80 times stronger emission
than acyclic analogues 5d and 8d, respectively.
Addition of ct-DNA to cyclic compounds (c6d,9c ) 6 ×
10-5 mol dm-3) resulted in strong hypochromic effects
(27% for 6d and 43% for 9c) and pronounced broadening
of UV/vis spectra, which resulted in the merging of two
maxima (354 and 370 nm) into one at 361 nm. The
observed changes in the UV/vis spectra of 6d and 9c
strongly suggest intercalation as the dominant binding
mode.26 For both compounds, titration with ct-DNA
yielded measurable changes only in excess of the studied
compounds; consequently, titration ended at an equimo-
lar ratio of c(6d, 9c)/c(ct-DNA phosphates). This obser-
vation suggested high values of the binding constants,
Ks > 105 M-1. Since aqueous solutions of 6d and 9c
exhibit strong fluorescence, it was possible to perform
fluorimetric titrations at up to 100 times lower concen-
trations than used in UV/vis experiments. Addition of
any ds-polynucleotide strongly quenched emission of 6d
and 9c. Processing the titration data by means of the
Scatchard equation27 gave the binding constants and
ratios n ([bound compound]/[polynucleotide phosphate])
presented in Table 2.
Unlike 6d and 9c, titration with ct-DNA induced only
minor changes in the UV/vis spectra of their acyclic
analogues 5d and 8d (19% hypochromic effect and
negligible bathochromic shift of the maximum at 304
nm). Titration with ds-RNA polynucleotides in most
cases yielded even smaller changes, some of them close
to the error of the instrument. Due to the low fluores-
cence of the aqueous solutions of 5d and 8d, fluorimetric
titrations were not possible. Therefore, UV/vis titration
data (when more than 10 data points could be collected)
were processed by means of the Scatchard equation27
to give the binding constants and ratios n (Table 2).
Table 3. ∆Tm Valuesa (°C) of Different ds-Polynucleotides with
5d, 8d, 6d, and 9c at pH ) 6.2 (sodium cacodilate/HCl buffer, I
) 0.05 mol dm-3
)
rb
5d
0.8
1.9
2.3
3.0
>0.5
>0.5
>0.5
>0.5
8d
6d
9c
ct-DNA
0.1
0.2
0.3
0.5
0.1
0.2
0.3
0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
2.6
5.2
5.9
7.7
1.2
1.9
2.3
2.6
3.6
4.2
6.4
1.9
3.1
polyA-polyU
3.5/11.8c
3.9/16.9c
3.1/23.8c
a Error in ∆Tm, (0.5 °C. b r ) [compound]/[polynucleotide].
c Biphasic curves.
Results of the titrations reveal significantly higher
affinity of cyclic 6d and 9c toward ds-DNA and RNA
than found for their acyclic analogues 5d and 8d. This
finding can be correlated with the size of the aromatic
surface, since cyclic analogues are characterized by a
planar system more than large enough for intercalation
into ds-DNA/RNA.22 Also, the obtained ratios n vary
considerably; only those found for cyclic 6d and 9c agree
well with the concentration of the intercalative binding
sites.28
Interactions of 5d, 8d, 6d, and 9c with ds-polynucle-
otides were studied also by thermal melting experiments
(Table 3). Addition of cyclic 6d and 9c significantly
stabilized the double helices of ct-DNA and polyA-
polyU, revealing pronounced DNA selectivity; both
observations are characteristic of the intercalative bind-
ing mode.22,28 Biphasic curves observed for polyA-polyU
at higher ratios r can be explained by two coexistent
modes of binding. The first transition (∆Tm value almost
identical to ∆Tm values at lower ratios r) can be
attributed to intercalation of the studied compound into
the double helix, while the second transition is very
likely a consequence of additional nonintercalative
binding (electrostatic binding of a positively charged
compound to the negatively charged phosphate back-
bone) of the compound in excess over the intercalation
binding sites (r > 0.25).29 Acyclic derivatives 5d and 8d
do not stabilize double helices in most cases, with the
exception of the weak stabilization of ct-DNA observed
upon addition of 5d (Table 3).
As a general conclusion, studies of interactions of ds-
polynucleotides with acyclic derivatives 5d and 8d and
their cyclic analogues 6d and 9c strongly support weak,
nonintercalative binding of the former and strong,
intercalative binding of the latter compounds.

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2351
Table 4. In Vitro Inhibition of the Compounds on the Growth
of Tumor Cells and Normal Human Fibroblasts (WI 38)
Biological Results and Discussion
Compounds 2a-9d were tested for their potential
antiproliferative effect on a panel of six human cell lines,
five of which derived from five cancer types: HeLa
(cervical carcinoma), MCF-7 (breast carcinoma), SW 620
(colon carcinoma), MiaPaCa-2 (pancreatic carcinoma),
Hep-2 (laryngeal carcinoma), and WI 38 (diploid fibro-
blasts).
All tested compounds showed a certain antiprolifera-
tive effect (Table 4). 2a and 2b produced little or no
growth inhibition except at the highest concentration
tested, but when the cyano group was introduced into
the molecule as a R1 or R2 substituent, slightly more
pronounced activity was obtained (4a, 7a, 7c, 7d; Figure
5E). In contrast, carboxanilides that bear an isopropy-
lamidino substituent on the quinolone part of the acyclic
molecule (R2) were much more active, in general show-
ing mostly “nondifferential” cytotoxicity at the highest
concentrations tested (5b, 5c, 5d, 5e; Figure 5A), except
for analogues bearing strongly polarizable groups 5f (R1
) CN) and 5g [R1 ) isopropylamidino (iso-pr-am)]. A
comparable, but less pronounced effect was obtained
with the compounds that bear isopropylamidino as R1
substituent (8a-e; Figure 5B).
IC50 (µM)a
compd HeLa
MCF-7 MiaPaCa-2 Hep-2 SW 620 WI 38
2a
2b
4a
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
95.46
44.34
53.62
8.12
36.4
3.3
4.01
2.85
2.99
25.1
86.9
0.86 g100
6.50 >100
15.3 27.8
12.9
1.967
6.99
3.0
3.49
3.7
4.44
4.67
3.49
3.51
8.72
75.9
7.3
3.94
2.55
5.37
2.37
3.53
3.86
3.67
3.93
8.02
2.7
2.24
2.15
1.23
2.54
22.2
23.9
15.6
1.36
5.4
38
65.1
5.78
21
25.6
25.3
4.3
67.3 >100
16.6
32.2 g100
5.98
6.73
5.64
3.71
8.59
7.57
20.6
18.66
40.6
2.34
4.79
1.7
34.7
45.9
48.8 >100
6.95
6.84
>100
g100
19
g100
g100
g100
6.14 >100
42.1
86.9
>100
>100
g100
>100
6g
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
31.6
0.35
>100
40.35
24.10 >100
>100 >100
1.27
78.5
84.93
>100
17.56
0.69 g100
>100
>100
0.75
90.87
48.38 >100
23.43
2.71
5.99
4.7
>100
>100
59.06
4.62
>100
23.8
2.96
>100
4.26
2.35
5.83
5.65
3.05
7.23
4.57
7.26
17.37
2.36
2.62
21.3
5.58
5.56
6.28
5.11
22.49
7.10
2.54
18
3.54
8.24
2.03
29.81
6.90
3.64
37.4
19.07
5.33
3.88
9.70
8.16
3.14
3.60
4.18
49.2
20.9
3.76
6.43
15.76
8.92
6.67
6.93
7.99
8.51
97.02
9.79
22.67
8.11
To check whether, due to structural differences, the
“cyclic” analogues (compounds 6 and 9) will have
significantly different activity compared to the above-
discussed “acyclic” ones (compounds 2, 5, 7, 8), the
13.16
a IC50, the concentration that causes a 50% reduction of the cell
growth.
Figure 5. Dose-response profiles for compounds 5b (A), 8b (B), 6a (C), 6d (D), 7d (E), and 9c (F). PG ) percentage of growth.

2352 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Jarak et al.
antiproliferative effect on the same cell lines was also
studied. It was definitely shown that condensed quino-
lones with an isopropylamidino substituent at the R2
position have distinct antiproliferative and rather dif-
ferential effects (Figure 5C,D), again except for those
with strongly polarizable cyano (6f) and isopropylami-
dino groups (6g) as R1 substituents. Similar, but some-
what less obvious, effects were achieved with condensed
quinolones that bear the isopropylamidino group as R1
substituent (9a-d; Figure 5F). Interestingly, compari-
son of the IC50 values of all compounds indicates that
compounds 6a and 6d showed the best selective effect;
they selectively inhibited the growth of tumor cells but
not of normal fibroblasts (WI38) (Figure 5C,D).
induce any substantial changes in the cell cycle phases
distribution in the MiaPaCa-2 cells, it induced massive
apoptosis in the SW 620 and HeLa cells (Figure 8), but
only at the higher tested concentration (10 µM), cor-
relating again with the growth inhibition results IC50
(SW 620) ) 4.6 ( 2.9 µM, while IC50 (MiaPaCa-2) >
100 µM and IC50 (HeLa) ) 24.1 ( 44.7 µM.
Induction of apoptosis was further confirmed by the
Annexin-V assay, an immunofluorescence technique for
detection and quantification of apoptotic cells at the
single cell level (Table 5).
Both the mechanism of cell death and the overall
cytotoxic effect seem to depend on at least three differ-
ent parameters, including: (1) pharmacological factors
(e.g. dose, exposure time), (2) cell type, and (3) expres-
sion of certain oncogenes and oncogene suppressors.30
For a given cell type, different mechanisms of cell death
may be triggered off as a function of dose, which was
obviously the case after cell treatment with 6a, 6b, 6d,
9c, and 9d. Lower doses resulted in a mixture of
cytostatic and delayed cytotoxic effects, and the cells
ultimately died of delayed apoptosis. However, different
cell types responded differently to the same compound,
so they have diverse tolerance toward drug-induced
lesions. One of the most important factors that influence
the response to chemotherapeutic drug treatment is the
endogenous status of the p53 suppressor gene. In most
cases, a functional p53 is required for drug-induced
apoptosis. However, as HeLa, MiaPaCa-2, and SW 620
have all either mutated or inactivated p53, another
mechanism of partial resistance of colon carcinoma cells
SW 620 to even higher doses of compound 6d is
relevant. One of the possible explanations is that colon
carcinoma cells have a high level of natural resistance,
which could be due to the expression of different
resistance mechanisms.30,33
Compounds that manifest “differential” growth inhi-
bition and/or cytotoxicity (especially without an inhibi-
tory effect on normal fibroblasts) are mostly of particular
interest as the basis for additional research applications,
as well as for the selection and prioritization of com-
pounds for in vivo evaluation. We therefore selected
several compounds that manifested the most interesting
differential effect and subjected them to further testing
to obtain a more precise insight into the possible
mechanism of their antiproliferative effect. First of all,
to establish whether the cell growth inhibition was
caused by specific perturbation of cell cycle-related
events, flow cytometric analyses were performed for
compounds 6a, 6b, 6d, 7d, 9c, and 9d, at concentrations
of 5 × 10-6 and 10-5 mol/L after 24, 48, and 72 h. DNA
contents of MiaPaCa-2, HeLa, SW 620, and WI 38 cells
were measured. The results revealed that all tested
compounds noticeably influenced the distribution of cell
cycle phases; however, the results vary among the cell
lines. For instance, no significant changes in the cell
cycle population were observed in WI 38 after treatment
with compounds 6a, 6b, 6d, and 9c for as long as 72 h,
except for a slight reduction in the percentage of the
cells in the S and G2/M phases and a slight increase in
the G0/G1 phase (Figures 6J-L and 7D-F), which is
in agreement with the cell proliferation assay. On the
other hand, the most distinct changes of DNA histo-
grams were observed after the treatment of MiaPaCa-2
and HeLa cells with compounds 6d and 9c (Figures
6D-I and 7A-C). This caused a decrease in the G0/G1
fraction and accumulation of cells in the S and G2/M
phases after 24 h. This initial S and G2/M arrest
persisted for the following 48 h, when an increase in
the subG1 fraction (apoptotic cells) was detected, but
only in cells treated with the higher concentration (10
µM). Parallel with these results, morphological changes
of the treated cells were noticed, such as the presence
of giant cells, characteristic of G2/M-arrested cells.
Similar results (G2/M arrest) were obtained with the
MiaPaCa-2 cells treated with compounds 6a, 6b, and
9d (data not shown). Interestingly, after treatment of
the SW 620 cells with compound 6d, the percentage of
arrested cells in the G2/M phase was much lower than
in MiaPaCa-2 and HeLa cells, which correlates with the
most pronounced growth inhibition of these two cell
lines: IC50 (SW 620) ) 48.8 ( 17.5 µM, while IC50
(MiaPaCa-2) ) 5.6 ( 1.7 µM and IC50 (HeLa) ) 4.8 (
1.5 µM (Figure 5D).
Conclusions
Among the studied compounds, those with one ami-
dino group substituent have shown the best antiprolif-
erative effect on the cell lines tested. This could be
explained by the permanent positive charge placed on
the amidino group, which may start a number of
additional interactions. Unexpectedly, however, the
introduction of two amidino groups diminished the
biological activity, possibly due to the steric effects.
The presented cell-cycle distribution related results
strongly suggest that compounds 6a, 6b, 6d, 9c, and
9d may act as topoisimerase “poisons”. In addition,
studies on interactions of compounds 6d and 9c with
ds-DNA and RNA have shown that these compounds
efficiently bind to double-stranded polynucleotides by
intercalation. Since it is well-known that intercalators
act as topoisimerase inhibitors and/or “poisons”, it is
reasonable to propose intercalation of 6d and 9c but also
of other cyclic analogues (groups 6 and 9) as the main
mode of action in the treated cell lines. For final
confirmation, detailed mechanistic studies based on
topoisomerase assays should be additionally performed.
Representatives of the “acyclic” group of derivatives
(5d and 8d) either do not bind to ds-DNA and RNA
(Tables 2, 3) or exhibit weak nonintercalative interac-
tions. Therefore, the mode of their action in the cell lines
is not likely to be targeted at nucleic acids.
On the other hand, compound 7d induced completely
different effects on the tested cell lines. While it did not

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2353
Figure 6. Effect of the compound 6d on the cell cycle of SW 620 (A-C), HeLa (D-F), MiaPaCa-2 (G-I), and WI 38 (J-L) cells.
The cells were untreated (A, D, G, and J) or treated with 5 µM (B, E, H, and K) or 10 µM (C, F, I, and L) concentration of
compound 6d for 72 h, fixed, and stained with propidium iodide to determine the DNA content. Percentage of the cells in each
phase of the cell cycle was obtained by flow cytometric analysis.
Comparison of the IC50 values of all compounds
indicates that compounds 6a (R1 ) H, R2 ) iso-
propyamidino) and 6d (R1 ) COOCH3, R2 ) isopropya-
midino) showed the best selective effect; they selectively
inhibited the growth of tumor cells but not of normal
fibroblasts (WI38).
On the basis of the presented results (the superior
properties of derivatives bearing a positively charged
substituent, amidine), future prospects in the here
presented area should be focused on the benzo[b]thieno-
[2,3-c]quinolone system substituted by groups bearing
more permanent positive charges (e.g. spermine deriva-
tives) or stronger positive charges (e.g. guanidin-
ium).
Experimental Section
Chemistry. Melting points were determined on a Kofler
hot stage microscope and are uncorrected. IR spectra were
recorded on a Nicolet Magna 760 spectrophotometer with KBr
disks. 1H and 13C NMR spectra were recorded on either a Var-
ian Gemini 300 or a Bruker Avance DPX 300 spectrometer
using TMS as an internal standard in DMSO-d6. Elemental
analysis for carbon, hydrogen, and nitrogen were performed
on a Perkin-Elmer 2400 elemental analyzer. Where analyses
are indicated only as symbols of elements, analytical results
obtained are within 0.4% of the theoretical value. Irradiation
was performed at room temperature with a water-cooled im-
mersion well with an “Original Hanau” 400-W high-pressure
mercury arc lamp using Pyrex filter. All compounds were rou-
tinely checked by TLC with Merck silica gel 60F-254 glass
plates.

2354 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Jarak et al.
Figure 7. Effect of the compound 9c on the cell cycle of MiaPaCa-2 (A-C) and WI 38 (D-F) cells. The cells were untreated (A
and D) or treated with 5 µM (B and E) or 10 µM (C and F) concentration of compound 9c for 72 h, fixed, and stained with
propidium iodide to determine the DNA content. Percentage of the cells in each phase of the cell cycle was obtained by flow
cytometric analysis.
Figure 8. Effect of the compound 7d on the cell cycle of HeLa cells. The cells were untreated (A) and treated with 10 µM (B)
concentration of compound 7d for 72 h, fixed, and stained with propidium iodide to determine the DNA content. Percentage of
the cells in each phase of the cell cycle was obtained by flow cytometric analysis.
Table 5. Percentage of Apoptosis Induced by Compounds 6a, 6b, 6d, 7d, 9c, and 9d in MiaPaCa-2, HeLa, and SW 620 Cells after 72
h
apoptotic cells (%)
control
6a
6b
6d
7d
9c
9d
cell line
0 µM
5 µM
10 µM
5 µM
10 µM
5 µM
10 µM
5 µM
10 µM
5 µM
10 µM
5 µM
10 µM
MiaPaCa-2
HeLa
SW 620
2
2
4
4
9
9
11
11
13
3
6
15
17
8
16
4
12
7
13
17
9
3
6
6
9
52
71
6
13
6
14
15
7
3
10
9
11
15
13
4′-Carbmethoxy-N-phenyl-3-chlorobenzo[b]thiophene-
2-carboxamide (2a). To a solution of 1a (1.00 g, 3.05 mmol)
in dry toluene (45 mL) was added a solution of methyl
4-aminobenzoate (1.02 g, 10.9 mmol) in dry toluene (5 mL)
dropwise, followed by the addition of Et3N (0.6 mL, 4.3 mmol).
The mixture was refluxed for 2.5 h. After cooling, precipitated
crystals were filtered off and washed with diluted HCl and
water. After recrystallization from acetone, 1.23 g (81.8%) of
6.09 Hz, Harom.), 3.84 (s, 3H, COOCH3); 13C NMR (DMSO-d6)
(δ ppm) 166.2, 159.9, 143.0, 137.3, 136.1, 132.1, 130.8 (2C),
128.2, 126.7, 125.6, 124.0, 123.1, 120.6, 120.1 (2C), 52.4. Anal.
(C17H12ClNO3S) C, H, N.
Methyl 2-(N-Phenylcarbamoyl)-3-chlorobenzo[2,3-c]-
thiophene-6-carboxylate (2b). Compound 2b was prepared
using the method described for the preparation of 2a, from
1d (1.00 g, 4.30 mmol) in dry THF (23 mL) and aniline (1.31
g, 8.70 mmol) in dry THF (5 mL) after refluxing for 3 h; 0.34
g (33%) of ivory crystals was obtained: mp 192-194 °C (lit.19
mp 197-198 °C).
white crystals was obtained: mp 189-190 °C; IR (KBr) (νmax
/
cm-1) 3420, 1710, 1640, 1590; 1H NMR (DMSO-d6) (δ ppm)
10.91 (s, 1H, NHamide), 8.17 (dd, 1H, J1 ) 6.25 Hz, J2 ) 2.89
Hz, Harom.), 8.99 (d, 2H, J ) 8.72 Hz, Hanilide), 7.95 (dd, 1H, J1
) 5.89 Hz, J2 ) 3.39 Hz, Harom.), 7.87 (d, 2H, J ) 8.73 Hz,
Methyl 6-Oxo-5,6-dehydro[1]benzothieno[2,3-c]quino-
lin-2-carboxylate (3a). A solution of 2a (0.1 g, 0.29 mmol)
in 10% methanol/toluene solution (18.7 mL) was irradieted at
Hanilide), 7.635 (d, 1H, J ) 6.09 Hz, Harom.), 7.63 (d, 1H, J )

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2355
room temperature with 400-W high-pressure mercury lamp
using a Pyrex filter for 1.5 h. The air was bubbled through
the solution. The solution was concentrated and the thus
obtained solid was filtered off and washed with acetone. After
recrystallization from DMF, 0.083 g (93.7%) of white crystals
was obtained: mp >300 °C; IR (KBr) (νmax/cm-1) 3000, 2960,
126.6, 124.4, 123.5, 122.6, 120.3, 119.7 (2C), 44.9, 21.2 (2C).
Anal. (C19H19Cl2N3OS) C, H, N.
6-Methyl-N-[4′-(N′-isopropylamidino)phenyl]-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (5b).
To a solution of 6-methyl-3-chlorobenzo[b]thiophene-2-carbonyl
chloride (1.15 g, 4.7 mmol) in dry toluene (40 mL) was added
dropwise a solution of p-(N-isopropyl)amidinoaniline (1 g, 4.7
mmol) in dry DMF (70 mL), followed by the addition of Et3N
(1 mL, 7 mmol). The mixture was refluxed for 6 h. After
cooling, precipitated crystals were filtered off, washed with hot
chloroform, and recrystallized from DMF to produce 0.54 g
1
1720, 1655, 1610; H NMR (DMSO-d6) (δ ppm) 12.46 (s, 1H,
NHquinolone), 9.13 (s, 1H, Harom.), 8.65 (d, 1H, J ) 7.81 Hz, Harom.),
8.29 (d, 1H, J ) 7.55 Hz, Harom.), 8.09 (d, 1H, J ) 8.21 Hz,
H
arom.), 7.77-7.67 (m, 2H, Harom.), 7.59 (d, 1H, J ) 8.65 Hz,
H
arom.), 3.94 (s, 3H, COOCH3); 13C NMR (DMSO-d6) (δ ppm)
165.9, 158.1, 141.4, 140.9, 135.3, 135.1, 133.2, 129.1, 127.7,
126.3, 124.9, 124.7, 124.4, 123.6, 117.0, 116.9, 52.3. Anal.
(C17H11NO3S) C, H, N.
Methyl 6-Oxo-5,6-dehydro[1]benzothieno[2,3-c]quino-
lin-9-carboxylate (3b). Compound 3b was prepared using
the method described for the preparation of 3a, from 2b (0.095
g, 0.27 mmol) in 10% methanol/toluene solution (16.5 mL) and
2 h of irradiation. After recrystallization from DMF, 0.076 g
(89.6%) of white crystals was obtained: mp >300 °C (lit.19 mp
>300 °C).
N-(4′-Cyanophenyl)-3-chlorobenzo[b]thiophene-2-car-
boxamide (4a). To a solution of 1a (2.08 g, 9.0 mmol) in dry
toluene (40 mL) was added dropwise a solution of 4-aminoben-
zonitrile (1.063 g, 9.0 mmol) in dry toluene (90 mL), followed
by the addition of Et3N (2 mL, 14 mmol). The mixture was
refluxed for 4 h. After cooling, precipitated crystals were
filtered off and washed with diluted HCl and water to yield
(27%) of white crystals: mp >300 °C; IR (KBr) (νmax/cm-1
)
3360, 3200, 3000, 1660, 1600; 1H NMR (DMSO-d6) (δ ppm)
10.88 (s, 1H, NHamide), 9.51 (1H, J ) 8.18 Hz, NHamidine), 9.47
(s, 1H, NHamidine), 9.01 (s, 1H, NHamidine), 7.995 (d, 2H, J ) 6.41
Hz, Hanilide), 7.91 (s, 1H, Harom.), 7.85 (d, 1H, J ) 8.23 Hz, Harom.),
7.78 (d, 2H, J ) 8.77 Hz, Hanilide), 7,47 (dd, 1H, J1 ) 8.35 Hz,
J2 ) 0.96 Hz, Harom.), 4.097-4.007 (m, 1H, J ) 6.76 Hz, CHi-pr),
3.3 (s, 3H, CH3), 1.29 (d, 6H, J ) 6.39 Hz, 2CH3 i-pr); 13C NMR
(DMSO-d6) (δ ppm) 161.2, 159.6, 142.5, 138.0, 137.0, 133.6,
132.0, 131.0, 129.3, 127.9, 124.3, 122.9, 122.3, 119.6 (2C), 44.9,
21.2 (2C). Anal. (C20H21Cl2N3OS) C, H, N.
General Method for the Synthesis of 6-Substituted-
N-[4′-(N′-isopropylamidino)phenyl]-3-chlorobenzo[b]-
thiophene-2-carboxamide hydrochloride (5c-f). To a
solution of p-(N-isopropyl)amidinoaniline in dry DMF was
added dropwise a solution of 6-substituted-3-chlorobenzo[b]-
thiophene-2-carbonyl chloride (1c-f) in dry THF (10 mL),
followed by the addition of Et3N. The mixture was refluxed
for 4-24 h. After cooling, precipitated crystals were filtered
off, washed with hot chloroform, and recrystallized.
2.34 g (83%) of white crystals: mp 211-213 °C; IR (KBr) (νmax
/
cm-1) 3880, 2220, 1660, 1590; 1H NMR (DMSO-d6) (δ ppm)
11.00 (s, 1H, NHamide), 8.19 (m, 1H, Harom.), 7.98 (m, 1H, Harom.),
7.92 (d, 2H, J ) 9.07 Hz, Hanilide), 7.87 (d, 1H, J ) 8.98 Hz,
6-Methoxy-N-[4′-(N′-isopropylamidino)phenyl]-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (5c):
from p-(N-isopropyl)amidinoaniline (0.41 g, 1.92 mmol) in dry
DMF (30 mL), 6-methoxy 3-chlorobenzo[b]thiophene-2-carbo-
nyl chloride (0.5 g, 1.91 mmol) in dry THF (10 mL), and Et3N
(2.27 mL, 1.94 mmol) after refluxing for 24 h: 0.49 g (58%) of
white crystals; mp 292-295 °C; IR (KBr) (νmax/cm-1) 3450,
3200, 2900, 2660, 1660, 1600; 1H NMR (DMSO-d6) (δ ppm)
10.92 (s, 1H, Hamide), 9.52 (d, 1H, J ) 7.86 Hz, Hamidine), 9.39
(s, 1H, Hamidine), 9.02 (s, 1H, Hamidine), 7.91 (d, 2H, J ) 8.64 Hz,
H
anilide), 7.65 (dd, 2H, J1 ) 5.88 Hz, J2 ) 6.26 Hz, Harom.); 13C
NMR (DMSO-d6) (δ ppm) 159.6, 142.3, 136.8, 135.3, 133.3 (2C),
131.3, 127.7, 126.2, 123.5, 122.6, 120.3, 120.17 (2C), 118.8,
106.2. Anal. (C16H9ClN2OS) C, H, N.
6-Cyano-N-(4′-cyanophenyl)-3-chlorobenzo[b]thio-
phene-2-carboxamide (4b). To a solution of 4-aminoben-
zonitrile (1.18 g, 10.0 mmol) in dry toluene (90 mL) was added
dropwise a suspension of 1f (1.78 g, 8.7 mmol) in dry toluene
(50 mL), followed by the addition of Et3N (3.5 mL, 25 mmol).
The mixture was refluxed for 14 h. After cooling, precipitated
crystals were filtered off and washed with diluted HCl, water,
and acetone to yield 0.81 g (38.6%) of white crystals: mp 290-
293 °C; IR (KBr) (νmax/cm-1) 3400, 2220, 1680, 1600; 1H NMR
(DMSO-d6) (δ ppm) 11.17 (s, 1H, NHamide), 8.84 (dd, 1H, J1 )
1.35 Hz, J2 ) 0.63 Hz, Harom.), 8.13 (dd, 1H, J1 ) 8.44 Hz, J2
) 0.61 Hz, Harom.), 8.01 (dd, 1H, J1 ) 8.39 Hz, J2 ) 1.42 Hz,
Hanilide), 7.83 (d, 1H, J ) 8.87 Hz, Harom.), 7.76 (d, 2H, J ) 8.95
Hz, Hanilide), 7.758 (s, 1H, Harom.), 7.23 (dd, 1H, J1 ) 8.87 Hz,
J2 ) 2.19 Hz, Harom.), 4.07-4.03 (m, 1H, J ) 6.76 Hz, CHi-pr),
3.88 (s, 3H, OCH3), 1.27 (d, 6H, J ) 6.47 Hz, CH3 i-pr); 13C NMR
(DMSO-d6) (δ ppm) 161.2, 159.5, 142.6, 138.8, 129.5, 129.3
(2C), 128.5, 124.2, 123.6, 120.3, 119.6 (2C), 116.6, 105.6, 55.8,
44.9, 21.2 (2C). Anal. (C20H21Cl2N3O2S) C, H, N.
Methyl 2-[N-4′-(N′-isopropylamidino)phenylcarbam-
oyl]-3-chlorobenzo[b]thiophene-6-carboxylate hydro-
chloride (5d): from 1d (0.8 g, 2.8 mmol) in dry THF (15 mL),
p-(N-isopropyl)amidinoaniline (0.534 g, 2.5 mmol) in dry DMF
(30 mL), and Et3N (0.56 mL, 4 mmol), after refluxing for 21 h
and recrystallization from the mixture of methanol and DMF
(1:1): 0.19 g (16.3%) of white powder; mp 286-289 °C; IR (KBr)
(νmax/cm-1) 3360, 3200, 3000, 1720, 1670, 1610; 1H NMR
(DMSO-d6) (δ ppm) 11.1 (s, 1H, NHamide), 9.52 (d, 1H, J ) 7.51
Hz, NHamidine), 9.37 (s, 1H, NHamidine), 8.99 (s, 1H, NHamidine),
8.89 (s, 1H, Harom.), 8.15 (d, 1H, J ) 8.57 Hz, Harom.), 8.075 (d,
1H, J ) 8.54 Hz, Harom.), 7.93 (d, 2H, J ) 8.53 Hz, Hanilide),
7.78 (d, 2H, J ) 8.29 Hz, Hanilide), 4.04-4.01 (m, 1H, J ) 6.45
Hz, CHi-pr), 3.93 (s, 3H, COOCH3), 1.28 (d, 6H, J ) 6.11 Hz,
2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.3, 159.2, 142.3,
138.7, 136.7, 129.3 (2C), 128.5, 126.3, 125.5, 124.6, 122.9, 119.7
(3C). Anal. (C21H21Cl2N3O3S) C, H, N.
6-Bromo-N-[4′-(N′-isopropylamidino)phenyl]-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (5e):
from 1e (0.62 g, 2.0 mmol) in dry THF (10 mL), p-(N-isopropyl)-
amidinoaniline (0.43 g, 2.0 mmol) in dry DMF (30 mL), and
Et3N (0.42 mL, 3 mmol), after refluxing for 20 h and recrys-
tallization from DMF: 0.19 g (15.2%) of white powder; mp
>300 °C; IR (KBr) (νmax/cm-1) 3320, 3000, 1670, 1650, 1610;
1H NMR (DMSO-d6) (δ ppm) 10.99 (s, 1H, NHamide), 9.51 (d,
1H, J ) 7.65 Hz, NHamidine), 9.37 (s, 1H, NHamidine), 9.02 (s,
H
arom.), 7.92 (d, 2H, J ) 9.22 Hz, Hanilide), 7.88 (d, 2H, J ) 9.19
Hz, Hanilide); 13C NMR (DMSO-d6) (δ ppm) 159.0, 142.1, 138.3,
136.7, 135.9, 133.4 (2C), 128.9, 128.6, 123.6, 120.2 (2C), 118.7,
118.4, 109.7, 106.4. Anal. (C17H8ClN3OS) C, H, N.
N-[4′-(N′-Isopropylamidino)phenyl]-3-chlorobenzo[b]-
thiophene-2-carboxamide hydrochloride (5a). Dry HCl
was bubbled for 4 h into the suspension of 4a (2 g, 0.106 mol)
in absolute ethanol (100 mL). The suspension was stirred at
room temperature until the -CN band was undetectable (IR).
After anhydrous diethyl ether was added, the corresponding
iminoether was collected by filtration. The product was
suspended in absolute ethanol (10 mL), and isopropylamine
(11 mL, 0.13 mol) was added. The mixture was refluxed for
17 h. The volume was reduced by evaporation and the product
was filtered off and washed with hot acetone. After recrystal-
lization from ethanol, 0.8 g (83%) of white crystals was
obtained: mp >300 °C; IR (KBr) (νmax/cm-1) 3360, 3200, 3000,
1650, 1605; 1H NMR (DMSO-d6) (δ ppm) 10.91 (bs, 1H,
NHamide), 9.44 (bs, 1H, NHamidine), 9.29 (bs, 1H, NHamidine), 8.93
(bs, 1H, NHamidine), 8.12 (dd, 1H, J1 ) 5.2 Hz, J2 ) 3.9 Hz,
H
arom.), 7.90 (dd, 1H, J1 ) 6.38 Hz, J2 ) 2.89 Hz, Harom.), 7.707
(d, 2H, J ) 8.60 Hz, Hanilide), 7.705 (d, 2H, J ) 8.61 Hz, Hanilide),
7.58 (m, 2H, Harom.), 3.98-3.95 (m, 1H, J ) 6.32 Hz, CHi-pr),
1.22 (d, 6H, J ) 6.38 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ
ppm) 161.1, 159.6, 142.5, 136.8, 135.6, 131.5, 129.4 (2C), 127.7,

2356 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
1H, NHamidine), 8.51 (s, 1H, Harom.), 7.90 (d, 2H, J ) 8.64 Hz,
Jarak et al.
0.074 g (81%); mp >300 °C; IR (KBr) (νmax/cm-1) 3220, 3000,
1650, 1610; 1H NMR (DMSO-d6) (δ ppm) 12.58 (s, 1H,
NHquinolone), 9.86 (bs, 1H, NHamidine), 9.65 (bs, 1H, NHamidine),
9.11 (bs, 1H, NHamidine), 8.95 (s, 1H, Harom.), 8.85 (d, 1H, J )
8.44 Hz, Harom.), 8.11 (s, 1H, Harom.), 7.87 (d, 1H, J ) 8.60 Hz,
Hanilide), 7.87 (d, 1H, J ) 9.31 Hz, Harom.), 7.76 (d, 3H, J ) 8.65
Hz, 2Hanilide, 1Harom.), 4.077-4.007 (m, 1H, J ) 6.64 Hz, CHi-pr),
1.26 (d, 6H, J ) 6.32 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ
ppm) 161.3, 159.3, 142.5, 138.4, 134.8, 132.3, 129.6, 129.4 (2C),
126.2, 124.5, 124.4, 121.0, 120.7, 120.2, 119.7 (2C), 45.0, 21.3
(2C). Anal. (C19H18BrCl2N3OS) C, H, N.
6-Cyano-N-[4′-(N′-isopropylamidino)phenyl]-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (5f):
from 1f (0.7 g, 2.7 mmol) in dry toluene (40 mL), p-(N-
isopropyl)amidinoaniline (0.49 g, 2.3 mmol) in dry DMF (30
mL), and Et3N (0.56 mL, 4 mmol), after refluxing for 4 h and
recrystallization from a mixture of DMF and methanol (1:1):
H
arom.), 7.69 (d, 1H, J ) 8.61 Hz, Harom.), 7.59 (d, 1H, J ) 8.65
Hz, Harom.), 4.11-4.07 (m, 1H, J ) 6.39 Hz, CHi-pr), 3.39 (s,
3H, CH3), 1.35 (d, 6H, J ) 6.35 Hz, 2CH3 i-pr); 13C NMR
(DMSO-d6) (δ ppm) 161.6, 157.9, 141.7, 140.7, 138.0, 125.3,
132.7, 132.4, 128.3, 127.6, 125.7, 124.2, 123.8, 123.0, 116.75,
116.73, 45.1, 21.3 (2C), 21.08. Anal. (C20H20ClN3OS) C, H, N.
2-(N′-Isopropylamidino)-9-methoxy-6-oxo-5,6-dihydro-
[1]benzothieno[2,3-c]quinoline hydrochloride (6c): yield
0.074 g (81%); mp >300 °C; IR(KBr) (νmax/cm-1) 3400, 3100,
2920, 2840, 1670, 1640, 1600; 1H NMR (DMSO-d6) (δ ppm)
12.56 (s, 1H, NHquinolone), 9.71 (bs, 2H, NHamidine), 9.18 (bs, 1H,
NHamidine), 8.91 (s, 1H, Harom.), 8.85 (d, 1H, J ) 9.19 Hz, Harom.),
7.89 (s, 1H, Harom.), 7.875 (d, 1H, J ) 7.86 Hz, Harom.), 7.69 (d,
1H, J ) 8.47 Hz, Harom.), 7.325 (d, 1H, J ) 8.70 Hz, Harom.),
4.13-4.11 (m, 1H, J ) 5.04 Hz, CHi-pr), 4.08 (s, 3H, OCH3),
1.35 (d, 6H, J ) 5.98 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ
ppm) 162.0, 159.7, 144.3, 141.2, 135.8, 131.5, 130.0, 128.9,
127.4, 124.6, 123.5, 117.2, 117.1, 116.5, 106.8, 56.2, 45.6, 21.8
(2C). Anal. (C20H20ClN3O2S) C, H, N.
0.39 g (39.2%) of white powder; mp 292-295 °C; IR (KBr) (νmax
/
cm-1) 3340, 3200, 3000, 2200, 1670, 1600; H NMR (DMSO-
d6) (δ ppm) 11.0 (s, 1H, NHamide), 9.51 (d, 1H, J ) 7.81 Hz,
NHamidine), 9.37 (bs, 1H, NHamidine), 9.00 (bs, 1H, NHamidine), 8.84
(s, 1H, Harom.), 8.13 (d, 1H, J ) 8.48 Hz, Harom.), 8.01 (dd, 1H,
J1 ) 8.46 Hz, J2 ) 1.32 Hz, Harom.), 7.93 (d, 2H, J ) 8.82 Hz,
Hanilide), 7.79 (d, 2H, J ) 8.81 Hz, Hanilide), 4.09-4.02 (m, 1H, J
) 6.58 Hz, CHi-pr), 1.29 (d, 6H, J ) 6.0 Hz, 2CH3 i-pr); 13C NMR
(DMSO-d6) (δ ppm) 161.1, 159.0, 142.3, 128.4, 136.7, 136.1,
129.4 (2C), 128.9, 128.6, 124.6, 123.7, 120.0, 199.7 (2C), 118.5,
109.7, 44.9, 21.3 (2C). Anal. (C20H18Cl2N4OS) C, H, N.
1
6-N′′-Isopropylamidino-N-[4′-(N′-isopropylamidino)-
phenyl]-3-chlorobenzo[b]thiophene-2-carboxamide di-
hydrochloride (5g). Compound 5g was prepared using the
method described for the preparation of 5a; from 4b (0.75 g,
2.5 mmol) in dry methoxyethanol (80 mL). The obtained imino
ether was collected by filtration after 7 days and suspended
in absolute ethanol (40 mL). Isopropylamine (1.8 mL, 21 mmol)
was added and the mixture was stirred at room temperature
for 3 days. Crude product was recrystallized from methanol
and then from water to yield 0.25 g (24.4%) of white crystals:
Methyl 2-(N′-isopropylamidino)-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinolin-9-carboxylate hydrochloride
(6d): yield 0.083 g (83.5%); mp 280-283 °C; IR (KBr) (νmax
/
cm-1) 3200, 3020, 1710, 1670, 1650; 1H NMR (DMSO-d6) (δ
ppm) 12.70 (bs, 1H, NHquinolone), 9.72 (bs, 2H, NHamidine), 9.16
(bs, 1H, NHamidine), 9.11 (d, 1H, J ) 8.61 Hz, Harom.), 8.98 (s,
1H, Harom.), 8.97 (s, 1H, Harom.), 8.23 (dd, 1H, J1 ) 8.72 Hz, J2
) 1.61 Hz, Harom.), 7.90 (d, 1H, J ) 8.46 Hz, Harom.), 7.72 (d,
1H, J ) 8.64 Hz, Harom.), 4.12-4.08 (m, 1H, J ) 6.38 Hz,
CHi-pr), 3.96 (s, 3H, COOCH3), 1.36 (d, 6H, J ) 6.33 Hz,
2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 165.7, 161.5, 157.7,
141.3, 140.7, 138.3, 136.6, 134.6, 128.7, 128.4, 126.3, 125.8,
125.7, 124.2, 123.3, 116.9, 116.6, 52.6, 45.2, 21.3 (2C). Anal.
(C21H20ClN3O3S) C, H, N.
2-(N′-Isopropylamidino)-9-bromo-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinoline hydrochloride (6e): yield
0.094 g (90%); mp >300 °C; IR (KBr) (νmax/cm-1) 3200, 3000,
2820, 2780, 1650, 1610; 1H NMR (DMSO-d6) (δ ppm) 12.70 (s,
1H, NHquinolone), 9.73 (s, 1H, Hamidine), 9.64 (s, 1H, NHamidine),
9.12 (s, 1H, NHamidine), 9.82 (d, 2H, J ) 8.56 Hz, Harom.), 8.66
(d, 1H, J ) 1.49 Hz, Harom.), 7.91 (d, 1H, J ) 6.99 Hz, Harom.),
7.89 (d, 1H, J ) 5.42 Hz, Harom.), 7.70 (d, 1H, J ) 8.63 Hz,
mp 306-308 °C; IR (KBr) (νmax/cm-1) 3320, 3180, 3000, 1670,
1
1610; H NMR (DMSO-d6) (δ ppm) 9.75-9.49 (bs, 6H, NHami
-
dine), 8.64 (s, 1H, Harom.), 8.15 (d, 1H, J ) 8.44 Hz, Harom.), 7.95
(d, 2H, J ) 8.83 Hz, Hanilid), 7.92 (d, 2H, J ) 8.76 Hz, Harom.),
7.80 (d, 2H, J ) 8.61 Hz, Hanilide), 4.06-4.02 (m, 2H, CHi-pr),
1.35 (d, 6H, J ) 6.62 Hz, 2CH3 i-pr), 1.29 (d, 6H, J ) 6.50 Hz,
2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.5, 161.3, 159.2,
142.4, 138.6, 136.5, 135.3, 129.5 (2C), 128.4, 125.9, 124.7,
122.9, 120.0, 119.9 (2C), 45.4, 45.1, 21.4 (2C), 21.3 (2C). Anal.
(C23H28Cl3N5OS) C, H, N.
General Method for the Synthesis of 6-Oxo-5,6-dihydro-
[1]benzothieno[2,3-c]quinoline (6a-g). A solution of 6-sub-
stituted N-[4′-(N′-isopropylamidino)phenyl]-3-chlorobenzo[2,3-
c]thiophene-2-carboxamide (5a-g) (0.24 mmol) in methanol
(10 mL) was irradiated at room temperature with 400-W high-
pressure mercury lamp using a Pyrex filter for 1.5 h. The air
was bubbled through the solution. The solution was concen-
trated and the obtained solid was filtered off and washed with
acetone. After recrystallization from methanol, white crystals
were obtained.
2-(N′-Isopropylamidino)-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinoline hydrochloride (6a). Com-
pound 6a was prepared using the general method described
for the preparation of 6a-g; a solution of 5a (0.106 g, 0.25
mmol) in methanol (10 mL) was irradiated for 1 h. The formed
precipitate was filtered off and recrystallized from ethanol to
give 0.061 g (64%) of white crystals: mp >300 °C; IR (KBr)
(νmax/cm-1) 3060, 2940, 1650, 1620; 1H NMR (DMSO-d6) (δ
ppm) 12.62 (bs, 1H, NHquinolone), 9.72 (bs, 1H, NHamidine), 9.62
(bs, 1H, NHamidine), 9.18 (bs, 1H, NHamidine), 8.92 (d, 1H, J )
8.32 Hz, Harom.), 8.90 (d, 1H, J ) 1.69 Hz, Harom.), 8.23 (dd, 1H,
J1 ) 7.64 Hz, J2 ) 1.21 Hz, Harom.), 7.83 (dd, 1H, J1 ) 8.60 Hz,
J2 ) 1.74 Hz, Harom.), 7.69 (dd, 1H, J1 ) 7.02 Hz, J2 ) 1.06 Hz,
H
arom.), 4.12-4.09 (m, 1H, J ) 7.59 Hz, CHi-pr), 1.35 (d, 6H, J
) 6.29 Hz, CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.9, 158.3,
143.6, 141.2, 134.8, 134.2, 134.0, 129.5, 129.1, 128.1, 127.3,
124.6, 123.7, 121.7, 117.3, 116.9, 45.6, 21.8 (2C). Anal. (C19H17-
BrClN3OS) C, H, N.
2-(N′-Isopropylamidino)-9-cyano-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinoline hydrochloride (6f): yield
0.059 g (65%); mp >300 °C; IR (KBr) (νmax/cm-1) 3400, 3000,
2220, 1650, 1610; 1H NMR (DMSO-d6) (δ ppm) 12.47 (bs, 1H,
NHquinolone), 9.68 (bs, 3H, NHamidine), 9.13 (d, 1H, J ) 8.65 Hz,
Harom.), 8.91 (s, 2H, Harom.), 8.09 (d, 1H, J ) 8.64 Hz, Harom.),
7.89 (d, 1H, J ) 8.64, Harom.), 7.70 (d, 1H, J ) 8.64, Harom.),
4.15-4.11 (m, 1H, J ) 6.43 Hz, CHi-pr), 1.34 (d, 6H, J ) 6.32
Hz, CH3 i-pr); 13C NMR (δ ppm) (DMSO-d6) 161.7, 140.9, 138.2,
135.5, 131.8, 129.4, 129.1, 128.5, 127.3, 124.4, 123.6, 119.0,
117.2, 117.0, 110.1, 45.4 (2C), 21.4. Anal. (C20H17ClN4OS) C,
H, N.
2-(N′-Isopropylamidino)-9-(N′′-isopropylamidino)-6-
oxo-5,6-dihydro[1]benzothieno[2,3-c]quinoline dihydro-
chloride (6g). Compound 6g was prepared using the general
method described for the preparation of 6a-g; a solution of
5g (0.10 g, 0.19 mmol) in methanol (12 mL) was irradiated
for 1.5 h. The formed precipitate was filtered off and recrystal-
lized from methanol to yield 0.06 g (64%) of white crystals:
H
arom.), 7.67-6.55 (m, 2H, Harom.), 4.12-4.07 (m, 1H, CHi-pr),
1.28 (d, 6H, J ) 6.41 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ
ppm) 161.1, 158.0, 141.4, 140.7, 135.3, 135.1, 133.4, 128.5,
127.9, 126.2, 126.0, 124.28, 124.24, 123.2, 116.8 (2C), 45.2, 21.4
(2C). Anal. (C19H18ClN3OS) C, H, N.
1
mp >300 °C; IR (KBr) (νmax/cm-1) 3040, 1650, 1640, 1610; H
NMR (DMSO-d6) (δ ppm) 12.82 (s, 1H, NHquinolone), 9.93 (d, 1H,
J ) 9.35 Hz, NHamidine), 9.20 (d, 1H, J ) 9.46 Hz, NHamidine),
9.80 (s, 1H, NHamidine), 9.78 (s, 1H, NHamidine), 9.39
2-(N′-Isopropylamidino)-9-methyl-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinoline hydrochloride (6b): yield

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2357
(s, 1H, NHamidine), 9.26 (s, 1H, NHamidine), 9.18 (d, 1H, J ) 8.81
Hz, Harom.), 8.97 (s, 1H, Harom.), 8.75 (d, 1H, J ) 1.63 Hz, Harom.),
8.01 (dd, 1H, J1 ) 8.67 Hz, J2 ) 1.70 Hz, Harom.), 7.93 (dd, 1H,
J1 ) 8.64 Hz, J2 ) 1.74 Hz, Harom.), 7.74 (d, 1H, J ) 8.66 Hz,
Harom.), 4.19-4.14 (m, 2H, J ) 6.55 Hz, CHi-pr), 1.35 (d, 6H, J
of light yellow crystals; mp 252-255 °C; IR (KBr) (νmax/cm-1
)
3480, 2220, 1710, 1680, 1620; 1H NMR (DMSO-d6) (δ ppm)
10.85 (s, 1H, NHamide), 8.82 (s, 1H, Harom.), 8.10 (d, 1H, J )
8.43 Hz, Harom.), 7.99 (d, 1H, J ) 8.44 Hz, Harom.), 7.695 (d, 1H,
J ) 8.84 Hz, Hanilide), 7.585 (d, 2H, J ) 8.42 Hz, Hanilide); 13C
NMR (DMSO-d6) (δ ppm) 158.9, 138.9, 137.9, 137.1, 136.9,
132.3 (2C), 129.4, 129.1, 124.1, 122.6 (2C), 119.0, 116.9, 110.1.
Anal. (C16H8BrClN2OS) C, H, N.
) 6.41 Hz, 2CH3 i-pr), 1.33 (d, 6H, J ) 6.41 Hz, 2CH3 i-pr); 13
C
NMR (DMSO-d6) (δ ppm) 161.4, 161.3, 157.7, 140.9, 140.8,
138.0, 136.0, 134.8, 128.9, 128.1, 126.4, 125.3, 124.8, 124.4,
123.3, 116.9, 116.5, 45.3, 45.2, 21.3 (2C), 21.2 (2C). Anal.
(C23H27Cl2N5OS) C, H, N.
General Method for the Synthesis of 4′-Substituted-
6-cyano-N-phenyl-3-chlorobenzo[b]thiophene-2-carbox-
amide (7a-e). To a solution of 6-cyano-3-chlorobenzo[2,3-
c]thiophene-2-carbonyl chloride (1f) was added dropwise a
solution of 4-substituted aniline. The mixture was refluxed for
1-2.5 h. After cooling, precipitated crystals were filterd off,
washed with diluted HCl and water, and recrystallized from
acetone.
6-Cyano-N-phenyl-3-chlorobenzo[b]thiophene-2-car-
boxamide (7a): from 1f (2.3 g, 9.0 mmol) in dry toluene (70
mL) and aniline (1.68 g, 18.0 mmol) after refluxing for 1 h:
2.16 g (76%) of light yellow crystals; mp 216-218 °C; IR (KBr)
(νmax/cm-1) 3320, 3040, 2220, 1640, 1600; 1H NMR (DMSO-d6)
(δ ppm) 10.73 (s, 1H, NHamide), 8.30 (s, 1H, Harom.), 8.11 (d, 1H,
J ) 8.43 Hz, Harom.), 7.91 (dd, 1H, J1 ) 8.43 Hz, J2 ) 1.38 Hz,
General Method for the Synthesis of 4′-Substituted-
6-N′′-isopropylamidino-N-phenyl-3-chlorobenzo[b]-
thiophene-2-carboxamide hydrochloride (8a-e). Dry HCl
was bubbled for 4 h into a suspension of 4′-substituted-6-N′′-
cyano N-phenyl-3-chlorobenzo[b]thiophene-2-carboxamide (7a-
e) in absolute ethanol. The suspension was stirred at room
temperature until the -CN band was undetectable (IR). After
anhydrous diethyl ether was added, the corresponding imino
ether was collected by filtration. The product was suspended
in absolute ethanol (20 mL), isopropylamine was added, and
the mixture was refluxed for 17-27 h. The volume was reduced
by evaporation, and the product was filtered off and washed
with hot acetone.
6-N′′-Isopropylamidino-N-phenyl-3-chlorobenzo[b]-
thiophene-2-carboxamide hydrochloride (8a): from 7a
(1.00 g, 3.20 mmol) in absolute EtOH (65 mL), which gave the
corresponding imino ether after 14 days. After addition of
isopropylamine (1.3 mL, 15.30 mmol) the mixture was refluxed
for 20 h. The crude product was recrystallized from ethanol
to yield 0.61 g (49.6%) of white crystals: mp 260-264 °C; IR
(KBr) (νmax/cm-1) 3420, 3200, 3020, 1660, 1590; UV (methanol)
(λmax/nm) 262, 350, 366; 1H NMR (DMSO-d6) (δ ppm) 10.74 (s,
1H, NHamide), 9.50 (bs, 3H, NHamidine), 8.62 (s, 1H, Harom.), 8.14
(d, 1H, J ) 8.45 Hz, Harom.), 7.89 (d, 1H, J ) 8.47 Hz, Harom.),
7.73 (d, 2H, J ) 7.90 Hz, H2′,6′), 7.41 (t, 2H, J ) 7.53 Hz, H3′,5′),
7.18 (t, 1H, J ) 7.28 Hz, H4′), 4.11-4.08 (m, 1H, J ) 5.99 Hz,
CH i-pr), 1.31 (d, 6H, J ) 6.09 Hz, 2CH3 i-pr); 13C NMR (DMSO-
d6) (δ ppm) 165.2, 157.6, 141.3, 138.7, 138.1, 137.6, 135.3,
129.1, 128.8, 128.3, 125.9, 125.4, 123.5, 122.8, 120.2, 117.1,
116.8, 45.7, 21.6 (2C). Anal. (C19H19Cl2N3OS) C, H, N.
H
arom.), 7.72 (d, 2H, J ) 7.68 Hz, H2′,6′), 7.40 (t, 2H, J ) 7.89
Hz, H3′,5′), 7.18 (t, 1H, J ) 7.37 Hz, H4′); 13C NMR (DMSO-d6)
(δ ppm) 158.3, 157.5, 138.4, 137.9, 136.8, 136.6, 128.8 (2C),
128.5, 124.6, 123.5, 120.2 (2C), 119.4, 118.5, 109.5. Anal.
(C16H9ClN2OS) C, H, N.
6-Cyano-N-(4′-methylphenyl)-3-chlorobenzo[b]thio-
phene-2-carboxamide (7b): from 1f (1.0 g, 3.9 mmol) in dry
toluene (40 mL) and p-methylaniline (1.25 g, 11.7 mmol) in
dry toluene (40 mL) after refluxing for 2.5 h: 0.92 g (72.4%)
of light brown crystals; mp 238-240 °C; IR (KBr) (νmax/cm-1
)
3400, 2220, 1650, 1600; 1H NMR (DMSO-d6) (δ ppm) 10.63 (s,
1H, NHamide), 8.81 (d, 1H, J ) 0.625 Hz, Harom.), 8.10 (d, 1H, J
) 8.42 Hz, Harom.), 7.98 (dd, 1H, J1 ) 8.45 Hz, J2 ) 1.33 Hz,
Harom.), 7.60 (d, 2H, J ) 8.30 Hz, Hanilide), 7.20 (d, 2H, J ) 8.35
6-N′′-Isopropylamidino-N-(4′-methylphenyl)-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (8b):
from 7b (0.35 g, 1.10 mmol) in absolute EtOH (70 mL), which
gave the corresponding imino ether after 12 days. After
addition of isopropylamine (4.6 mL, 53.60 mmol) the mixture
was refluxed for 17 h. The crude product was recrystallized
from methanol to yield 0.38 g (81.9%) of yellow crystals: mp
286-290 °C; IR (KBr) (νmax/cm-1) 3400, 3160, 3000, 1660, 1600;
1H NMR (DMSO-d6) (δ ppm) 10.55 (s, 1H, NHamide), 9.55 (bs,
3H, NHamidine), 8.60 (s, 1H, Harom.), 8.12 (d, 1H, J ) 8.50 Hz,
Hz, Hanilide), 2.29 (s, 3H, CH3); 13C NMR (DMSO-d6) (δ ppm)
158.3, 138.6, 137.0, 135.7, 133.9, 129.4 (2C), 128.9, 128.7,
123.6, 120.3 (2C), 119.5, 118.7, 109.6, 20.6. Anal. (C17H11ClN2-
OS) C, H, N.
6-Cyano
N-(4′-methoxyphenyl)-3-chlorobenzo[b]-
thiophene-2-carboxamide (7c): from 1f (1.2 g, 4.7 mmol)
in dry toluene (50 mL) and p-methoxyaniline (1.46 g, 11.9
mmol) in dry toluene (20 mL) after refluxing for 1.5 h: 0.95 g
(58.7%) of yellow crystals; mp 211 °C; IR (KBr) (νmax/cm-1
)
3400, 3040, 1650, 1600; 1H NMR (DMSO-d6) (δ ppm) 10.58 (s,
1H, NHamide), 8.81 (d, 1H, J ) 1.24 Hz, Harom.), 8.10 (d, 1H, J
) 8.43 Hz, Harom.), 8.09 (dd, 1H, J1 ) 8.46 Hz, J2 ) 1.34 Hz,
Harom.), 7.90 (d, 1H, J ) 5.56 Hz, Harom.), 7.615 (d, 2H, J ) 8.21
Hz, Hanilide), 7.21 (d, 2H, J ) 8.37 Hz, Hanilide), 4.16-4.11 (m,
1H, J ) 6.36 Hz, CHi-pr), 2.31 (s, 3H, CH3), 1.32 (d, 6H, J )
6.34 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.4, 158.4,
138.6, 136.2, 136.1, 135.7, 133.7, 129.3 (2C), 128.0, 125.7,
124.6, 122.7, 120.3 (2C), 119.3, 45.3, 21.3 (2C), 20.6. Anal.
(C20H21Cl2N3OS) C, H, N.
Harom.), 7.46 (d, 2H, J ) 8.99 Hz, Hanilide), 6.97 (d, 2H, J ) 9.02
Hz, Hanilide), 3.76 (s, 3H, OCH3); 13C NMR (DMSO-d6) (δ ppm)
158.4, 156.7, 139.0, 137.5, 137.0, 131.6, 129.3, 129.0, 124.0,
122.9 (2C), 119.8, 119.0, 114.5 (2C), 109.9, 55.7. Anal. (C17H11-
ClN2O2S) C, H, N.
6-N′′-Isopropylamidino N-(4′-methoxyphenyl)-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (8c):
from 7c (0.45 g, 1.30 mmol) in absolute EtOH (80 mL), which
gave the corresponding imino ether after 10 days. After
addition of isopropylamine (4.5 mL, 52.50 mmol) the mixture
was refluxed for 20 h. The crude product was recrystallized
from methanol to yield 0.39 g (67.5%) of yellow crystals: mp
264-267 °C; IR (KBr) (νmax/cm-1) 3400, 3200, 3000, 1650, 1600;
1H NMR (DMSO-d6) (δ ppm) 10.57 (s, 1H, NHamide), 9.73-9.40
(bs, 3H, NHamidine), 8.61 (s, 1H, Harom.), 8.135 (d, 1H, J ) 8.50
Hz, Harom.), 7.89 (d, 1H, J ) 8.51 Hz, Harom.), 7.65 (d, 2H, J )
8.66 Hz, Hanilide), 6.98 (d, 2H, J ) 8.71 Hz, Hanilide), 4.12-4.03
(m, 1H, J ) 6.22 Hz, CHi-pr), 3.76 (s, 3H, OCH3), 1.31 (d, 6H,
J ) 6.26 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.4,
158.1, 138.6, 136.2 (2C), 131.1, 128.1, 125.7, 124.5, 122.7, 121.9
(2C), 119.1, 114.0 (2C), 55.2, 45.3, 21.3 (2C). Anal. (C20H21-
Cl2N3O2S) C, H, N.
6-Cyano-N-(4′-carbmethoxyphenyl)-3-chlorobenzo[b]-
thiophene-2-carboxamide (7d): from 1f (0.86 g, 3.36 mmol)
in dry THF (10 mL), methyl 4-aminobenzoate (1.12 g, 6.7
mmol) in dry THF (30 mL), and Et3N (0.47 mL, 3.4 mmol) after
refluxing for 2 h: 0.81 g (64.6%) of white crystals; mp 193-
196 °C; IR (KBr) (νmax/cm-1) 3387, 2987, 2229, 1714, 1669,
1597; 1H NMR (DMSO-d6) (δ ppm) 11.05 (s, 1H, NHamide), 8.84
(d, 1H, J ) 0.62 Hz, Harom.), 8.12 (d, 1H, J ) 8.46 Hz, Harom.),
8.01 (d, 1H, J ) 7.65 Hz, Harom.), 7.99 (d, 2H, J ) 8.46 Hz,
H
anilide), 7.87 (d, 2H, J ) 8.82 Hz, Hanilide), 3.85 (s, 2H,
COOCH3); 13C NMR (DMSO-d6) (δ ppm) 166.2, 159.4, 142.8,
138.9, 137.2, 136.7, 130.8 (2C), 129.4, 129.1, 125.8, 124.1,
120.5, 120.1 (2C), 119.0, 110.4, 52.5. Anal. (C18H11ClN2O3S)
C, H, N.
6-Cyano-N-(4′-bromophenyl)-3-chlorobenzo[b]-
thiophene-2-carboxamide (7e): from 1f (1.0 g, 3.9 mmol)
in dry toluene (50 mL) and p-bromoaniline (2.01 g, 11.7 mmol)
in dry toluene (40 mL) after refluxing for 1.5 h: 1.05 g (68.8%)
6-N′′-Isopropylamidino-N-(4′-carbmethoxyphenyl)-3-
chlorobenzo[b]thiophene-2-carboxamide hydrochloride

2358 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Jarak et al.
(8d): from 7d (0.49 g, 1.3 mmol) in absolute ethanol (70 mL),
which gave the corresponding imino ether after 30 days. After
addition of isopropylamine (4 mL, 0.047 mol), the mixture was
refluxed for 3 h. The crude product was recrystallized from
methanol to yield 0.23 g (38%) of white crystals: mp 193-
196 °C; IR (KBr) (νmax/cm-1) 3460, 3000, 1710, 1660, 1590; 1H
NMR (DMSO-d6) (δ ppm) 11.06 (s, 1H, NHamide), 9.78 (d, 1H,
J ) 7.76 Hz, NHamidine), 9.58 (s, 1H, NHamidine), 9.21 (s, 1H,
NHamidine), 8.56 (s, 1H, J ) 0.91 Hz, Harom.), 8.08 (d, 1H, J )
8.45 Hz, Harom.), 7.94 (dd, 2H, J1 ) 8.95 Hz, J2 ) 2.29 Hz,
7.51 (d, 1H, J ) 8.36 Hz, Harom.), 7.45 (d, 1H, J ) 8.42 Hz,
Harom.), 4.16-4.12 (m, 1H, J ) 6.09 Hz, CHi-pr), 2.55 (s, 3H,
CH3), 1.34 (d, 6H, J ) 6.21 Hz, 2CH3 i-pr); 13C NMR (DMSO-
d6) (δ ppm) 161.6, 157.5, 140.9, 138.6, 135.8, 135.3, 135.1,
132.4, 130.6, 127.9, 126.3, 125.4, 124.8, 123.1, 117.1, 116.9,
45.4, 21.3 (2C), 20.9. Anal. (C20H20ClN3OS) C, H, N.
2-Carbmethoxy-9-(N′-isopropylamidino)-6-oxo-5,6-
dihydro[1]benzothieno[ 2,3-c]quinoline hydrochloride
(9c). A solution of 8d (0.10 g, 0.22 mmol) in methanol (14 mL)
was irradiated for 2 h and 10 min. The formed precipitate was
filtered off and recrystallized from methanol to yield 0.059 g
Hanilide), 7.83 (dd, 1H, J1 ) 8.33 Hz, J2 ) 1.58 Hz, Harom.), 7.82
(63%) of white crystals: mp 280-283 °C; IR (KBr) (νmax/cm-1
)
(d, 2H, J ) 8.68 Hz, 2Hanilide), 4.04-4.02 (m, 1H, J ) 6.27 Hz,
CHi-pr), 3.78 (s, 3H, COOCH3), 1.28 (d, 6H, J ) 6.41 Hz,
2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 165.6, 161.4, 158.9,
142.3, 138.4, 136.3, 135.4, 130.2 (2C), 128.1, 125.6, 125.2,
124.5, 122.7, 119.9, 119.6 (2C), 51.9, 45.2, 21.2 (2C). Anal.
(C21H21Cl2N3O3S) C, H, N.
3260, 3040, 3960, 1710, 1620; 1H NMR (DMSO-d6) (δ ppm)
12.78 (s, 1H, NHquinolone), 9.68 (bs, 3H, NHamidine), 9.16 (s, 1H,
Harom.), 8.83 (d, 1H, J ) 8.77 Hz, Harom.), 8.75 (s, 1H, Harom.),
8.14 (dd, 1H, J1 ) 8.57 Hz, J2 ) 0.96 Hz, Harom.), 8.06 (d, 1H,
J ) 8.47 Hz, Harom.), 7.67 (d, 1H, J ) 8.64 Hz, Harom.), 4.16-
4.12 (m, 1H, J ) 6.30 Hz, CHi-pr), 3.95 (s, 3H, COOCH3), 1.34
(d, 6H, J ) 6.28 Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm)
167.0, 162.6, 159.1, 142.3, 142.2, 139.3, 137.0, 136.0, 130.7,
129.4, 127.0, 126.4, 126.3, 125.9, 125.0, 118.5, 117.9, 53.7, 46.6,
22.5 (2C). Anal. (C21H20ClN3O3S) C, H, N.
6-N′′-Isopropylamidino-N-(4′-bromophenyl)-3-chlo-
robenzo[b]thiophene-2-carboxamide hydrochloride (8e):
from 7e (0.43 g, 1.10 mmol) in absolute EtOH (50 mL), which
gave the corresponding imino ether after 17 days. After
addition of isopropylamine (4.6 mL, 53.60 mmol), the mixture
was refluxed for 27 h. The crude product was recrystallized
from methanol to yield 0.33 g (61%) of yellow crystals: mp
275-278 °C; IR (KBr) (νmax/cm-1) 3380, 3040, 1670, 1630, 1590;
1H NMR (DMSO-d6) (δ ppm) 10.88 (s, 1H, NHamide), 9.57 (bs,
3H, NHamidine), 8.62 (s, 1H, Harom.), 8.14 (d, 1H, J ) 8.52 Hz,
2-Bromo-9-(N′-isopropylamidino)-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinoline hydrochloride (9d). A solu-
tion of 8e (0.10 g, 0.21 mmol) in methanol (13 mL) was
irradiated for 1.5 h. The formed precipitate was filtered off
and recrystallized from ethanol to yield 0.059 g (62.5%) of
white crystals: mp >300 °C; IR (KBr) (νmax/cm-1) 3000, 1630;
1H NMR (DMSO-d6) (δ ppm) 12.57 (bs, 1H, NHquinolone), 9.71
(bs, 3H, NHamidine), 9.06 (d, 1H, J ) 8.71 Hz, Harom.), 8.79 (d,
1H, J ) 1.50 Hz, Harom.), 8.71 (s, 1H, Harom.) 7.95 (d, 1H, J )
8.60 Hz, Harom.), 7.80 (dd, 1H, J1 ) 8.83 Hz, J2 ) 1.31 Hz,
Harom.), 7.90 (dd, 1H, J1 ) 7.97 Hz, J2 ) 1.30 Hz, Harom.), 7.20
(d, 2H, J ) 8.83 Hz, Hanilide), 7.60 (d, 2H, J ) 8.88 Hz, Hanilide),
4.12-4.06 (m, 1H, J ) 6.17 Hz, CH i-pr), 1.31 (d, 6H, J ) 6.36
Hz, 2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.9, 159.1, 138.9,
137.9, 136.8, 136.1, 132.2 (2C), 128.7, 126.2, 125.0, 123.3, 122.7
(2C), 120.2, 116.9, 45.7, 21.7 (2C). Anal. (C19H18BrCl2N3OS)
C, H, N.
H
arom.), 7.56 (d, 1H, J ) 8.81 Hz, Harom.), 4.15-4.11 (m, 1H, J
) 6.36 Hz, CHi-pr), 1.36 (d, 6H, J ) 6.31 Hz, 2CH3 i-pr). 13C
NMR (DMSO-d6) (δ ppm) 161.3, 157.3, 140.7, 137.9, 136.8,
136.0, 133.9, 131.9, 127.9, 125.8, 125.4, 125.2, 124.7, 118.9,
118.5, 114.9, 45.3, 21.2 (2C). Anal. (C19H17BrClN3OS) C, H,
N.
General Method for the Synthesis of 2-Substituted-9-
(N′-isopropylamidino)-6-oxo-5,6-dihydro[1]benzothieno-
[2,3-c]quinoline hydrochloride (9a-d). A solution of 4′-
substituted-6-N′′-isopropylamidino-N-phenyl-3-chloro-
benzo[b]thiophene-2-carboxamide hydrochloride (8a,b,d,e) in
methanol or water was irradiated at room temperature with
a 400-W high-pressure mercury lamp using a Pyrex filter for
1-14 h. The air was bubbled through the solution. The solution
was concentrated and the obtained solid was filtered off and
washed with acetone.
Interactions with DNA and RNA. The electronic absorp-
tion spectra were obtained on Varian Cary 100 Bio spectrom-
eter, and fluorescence emission spectra were recorded on
Varian Eclipse fluorimeter, in both cases using quartz cuvettes
(1 cm).
Polynucleotides were purchased from Sigma and Aldrich
and used without further purification. Polynucleotides were
dissolved in the sodium cacodylate buffer, 0.05 mol dm-3, pH
) 7, and their concentration was determined spectroscopically
as the concentration of phosphates. The measurements were
performed in aqueous buffer solution (pH ) 6.2; sodium
cacodilate/HCl buffer, I ) 0.05 mol dm-3). Under the experi-
mental conditions used, the absorbance and fluorescence
intensities of studied compounds were proportional to their
concentrations. In fluorimetric titrations, excitation wave-
lengths of 310 nm (5d, 8d) and 370 nm (6d, 9c) were used to
avoid inner filter effects caused by absorption of excitation light
of added polynucleotides, and changes of emission were
monitored at 400 nm. The stability constants (Ks) and [bound
compound]/[polynucleotide phosphate] ratio (n) were calculated
according to the Scatchard equation28 by nonlinear least-
squares fitting.29 Values for Ks and n given in Table 2 all have
satisfying correlation coefficients (>0.999). Thermal melting
curves for DNA, RNA, and their complexes were determined
as previously described34 by following the absorption change
at 260 nm as a function of temperature. The absorbance of
the ligands was subtracted from every curve, and the absor-
bance scale was normalized. Tm values are the midpoints of
the transition curves, determined from the maximum of the
first derivative or graphically by a tangent method.34 ∆Tm
values were calculated by subtracting Tm of the free nucleic
acid from Tm of the complex. Every ∆Tm value here reported
was the average of at least two measurements, the error in
∆Tm is ( 0.5 °C.
9-(N′-Isopropylamidino)-6-oxo-5,6-dihydro[1]ben-
zothieno[2,3-c]quinoline hydrochloride (9a). Method a.
A solution of 8a (0.12 g, 0.29 mmol) in methanol (10 mL) was
irradiated for 1 h. The formed precipitate was filtered off and
recrystallized from ethanol to yield 0.082 g (74.5%) of white
crystals.
Method b. A solution of 8a (0.10 g, 0.25 mmol) in distilled
water (13 mL) was irradiated for 2 h. The formed precipitate
was filtered off and recrystallized from ethanol to yield 0.085
g (93%) of white crystals: mp >300 °C; IR (KBr) (νmax/cm-1
)
3040, 1630; 1H NMR (DMSO-d6) (δ ppm) 12.46 (s, 1H,
NHquinolone), 9.87 (d, 1H, J ) 6.06 Hz, NHamidine), 9.67 (s, 1H,
NHamidine), 9.32 (s, 1H, NHamidine), 9.14 (d, 1H, J ) 8.53 Hz,
Harom.), 8.82 (d, 1H, J ) 8.08 Hz, Harom.), 8.72 (d, 1H, J ) 1.51
Hz, Harom.), 7.95 (dd, 1H, J1 ) 8.60 Hz, J2 ) 1.52 Hz, Harom.),
7.47-7.43 (m, 1H, Harom.), 6.64-6.62 (m, 2H, Harom.), 4.15-4.12
(m, 1H, J ) 6.98 Hz, CH i-pr), 1.33 (d, 6H, J ) 6.22 Hz,
2CH3 i-pr); 13C NMR (DMSO-d6) (δ ppm) 161.5, 157.6, 140.9,
138.5, 137.7, 135.4, 134.9, 129.3, 127.9, 126.0, 125.3, 124.8,
123.7, 122.9, 117.1, 116.9, 45.3, 21.2 (2C). Anal. (C19H18ClN3-
OS) C, H, N.
2-Methyl-9-(N′-isopropylamidino)-6-oxo-5,6-dihydro[1]-
benzothieno[2,3-c]quinoline hydrochloride (9b). A solu-
tion of 8b (0.10 g, 0.24 mmol) in methanol (11 mL) was
irradiated for 14 h. The formed precipitate was filtered off and
recrystallized from methanol to yield 0.035 g (38%) of yellow
crystals: mp >300 °C; IR (KBr) (νmax/cm-1) 3000, 1620; 1H
NMR (DMSO-d6) (δ ppm) 12.37 (bs, 1H, NHquinolone), 9.62 (bs,
3H, NHamidine), 9.18 (d, 1H, J ) 8.67 Hz, Harom.), 8.97 (s, 1H,
Antitumor Activity Assays. The HeLa (cervical carci-
noma), MCF-7 (breast carcinoma), SW 620 (colon carcinoma),
Harom.), 8.56 (s, 1H, Harom.), 7.945 (d, 1H, J ) 8.51 Hz, Harom.),

Antitumor Heterocyclic Condensed Quinolones
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2359
MiaPaCa-2 (pancreatic carcinoma), Hep-2 (laryngeal carci-
noma), and WI 38 (diploid fibroblasts) cells were cultured as
monolayers and maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/
mL streptomycin in a humidified atmosphere with 5% CO2 at
37 °C.
washed with PBS, pelleted, and resuspended in staining-
solution (Annexin-V-fluorescein labeling reagent and pro-
pidium iodide (PI) in Hepes buffer). The cells were then
analyzed under a fluorescence microscope. Annexin-V (green
fluorescent) cells were determined to be apoptotic and An-
nexin-V and PI cells were determined to be necrotic. The
percentage of apoptotic cells was expressed as the number of
fluorescent cells in relation to the total cell number (fluorescent
and nonfluorescent cells), which was expressed as 100%.
The growth inhibition activity was assessed according to the
slightly modified procedure performed at the National Cancer
Institute, Developmental Therapeutics Program.31 The cells
were inoculated onto standard 96-well microtiter plates on day
0. The cell concentrations were adjusted according to the cell
population doubling time (PDT): 1 × 104/mL for HeLa, Hep-
2, MiaPaCa-2, and SW 620 cell lines (PDT ) 20-24 h); 2 ×
104/mL for MCF-7 cell lines (PDT ) 33 h); and 3 × 104/ml for
WI 38 (PDT ) 47 h). Test agents were then added in five 10-
fold dilutions (10-8 to 10-4 mol/L) and incubated for a further
72 h. Working dilutions were freshly prepared on the day of
testing. The solvent was also tested for eventual inhibitory
activity by adjusting its concentration to be the same as in
working concentrations. After 72 h of incubation, the cell
growth rate was evaluated by performing the MTT assay,32
which detects dehydrogenase activity in viable cells. The
absorbency (OD, optical density) was measured on a microplate
reader at 570 nm. The percentage of growth (PG) of the cell
lines was calculated according to one or the other of the
following two expressions:
Acknowledgment. Support for this study by the
Ministry of Science, Education and Sport of Croatia is
gratefully acknowledged (Projects 0125005, 00981104,
0119633, 0098053).
Supporting Information Available: Elemental analysis
of novel compounds, X-ray crystal structure analysis, and
selected crystallographic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Oleson, J. J.; Calderella, L. A.; Mjos, K. J.; Reith, A. R.; Thie, R.
S.; Toplin, I. The Effects of Streptonigrin on Experimental
Tumors. Antibiot. Chemother. 1961, 11, 158-164.
(2) Teller, M. N.; Wagshul, S. F.; Wolley, G. W. Transplantable
Human Tumors in Experimental Chemotherapy. Effects of
Streptonigrin on HS-1 and Hep-3 in the Rat. Antibiot. Chemoth-
er. 1961, 11, 165-173.
(3) Reilly, H. C.; Sigura, K. An Antitumor Spectrum of Streptoni-
grin. Antibiot. Chemother. 1961, 11, 174-177.
(4) Hackethal, C. A.; Golbey, R. B.; Tan, C. T. C.; Karnofsky, D. A.;
Burchenal, J. H. Clinical Observation on the Effects of Strep-
tonigrin in Patients with Neoplastic Disease. Antibiot. Chemoth-
er. 1961, 11, 178-183.
(5) Rivers, S. L.; Whittington, R. M.; Medrek, T. J. Treatment of
Malignant Lymphomas with Methyl Ester of Streptonigrin (NSC
45384). Cancer 1966, 19(10), 1377-1385.
(6) Kaung, D. T.; Whittington, R. M.; Spencer, H. H.; Patno, M. E.
Comparison of Chlorambucil and Streptonigrin (NSC-45383) in
the Treatment of Chronic Lymphocytic Leukemia. Cancer 1969,
23(3), 597-600.
(7) Kaung, D. T.; Whittington, R. M.; Spencer, H. H.; Patno, M. E.
Comparison of Chlorambucil and Streptonigrin (NSC-45383) in
the Treatment of Malignant Lymphomas. Cancer 1969, 23(6),
1280-1283.
(8) Banzet, P.; Jacquillat, C.; Civatte, J.; Puissant, A.; Maral, J.;
Chastang, C.; Israel, L.; Belaich, S.; Jourdain, J.-C.; Weil, M.;
Auclerc, G. Adjuvant Chemotherapy in the Management of
Primary Malignant Melanoma. Cancer 1978, 41(4), 1240-1248.
(9) Forcier, R. J.; McIntyre, O. R.; Nissen, N. I.; Pajak, T. F.;
Glidewell, O.; Holland, J. F. Combination Chemotherapy of non-
Hodgkin Lymphoma. Med. Pediatr. Oncol. 1978, 4(4), 351-362.
(10) Fryatt, T.; Goroski, D. T.; Nilson, Z. D.; Moody, C. J.; Beall, H.
D. Novel Quinolinequinone Antitumor Agents: Structure-
Metabolism Studies with NAD(P)H:Quinone Oxidoreductase
(NQO1). Bioorg. Med. Chem. Lett. 1999, 9, 2195-2198.
(11) Yang, Z.-Y.; Xia, Y.; Xia, P.; Tachibana, Y.; Bastow, K. F.; Lee,
K.-H. Antitumor Agents. 187. Synthesis and Cytotoxicity of
Substituted 8,8-Dimethyl-2H,8H-pyrano[6,5-h]quinoline-2-one
and Related Compounds. Bioorg. Med. Chem. Lett. 1999, 9, 713-
716.
If (mean ODtest - mean ODtzero) ) 0, then
PG ) 100(mean ODtest - mean ODtzero)/
(mean ODctrl - meanODtzero
If (mean ODtest - mean ODtzero) < 0, then
PG ) 100(mean ODtest - mean ODtzero)/ODtzero
)
where mean ODtzero ) the average of optical density measure-
ments before exposure of cells to the test compound, mean
ODtest ) the average of optical density measurements after
the desired period of time, and mean ODctrl ) the average of
optical density measurements after the desired period of time
with no exposure of cells to the test compound.
Each test point was performed in quadruplicate in three
individual experiments. The results are expressed as IC50,
which is the concentration necessary for 50% inhibition. The
IC50 values for each compound are calculated from dose-
response curves using linear regression analysis by fitting the
test concentrations that give PG values above and below the
reference value (i.e. 50%). If, however, for a given cell line all
of the tested concentrations produce PGs exceeding the respec-
tive reference level of effect (e.g. PG value of 50), then the
highest tested concentration is assigned as the default value,
which is preceded by a “>” sign. Each result is a mean value
from three separate experiments.
(12) Xia, Y.; Yang, Z.-Y.; Hour, M.-J.; Kuo, S.-C.; Xia, P.; Bastow, K.
F.; Nakanishi, Y.; Nampoothiri, P.; Hackl, T.; Hamel, E.; Lee,
K.-H. Antitumor Agents. Part 204: Synthesis and Biological
Evaluation of Substituted 2-Aryl Quinazolinones. Bioorg. Med.
Chem. Lett. 2001, 11, 1193-1196.
Cell Cycle Analysis. A total of 1 × 106 cells were seeded
per 100 mm plate. After 24 h the tested compounds were added
at concentrations of 5 × 10-6 and 10-5 mol/L. After the desired
length of time, the attached cells were trypsinized, combined
with floating cells, washed with phosphate buffer saline (PBS),
and fixed with 70% ethanol. Immediately before the analysis,
the cells were washed with PBS and stained with 1 µg/mL of
propidium iodide (PI) with the addition of 0.2 µg/µL of RNAse
A. The stained cells were then analyzed with a Becton
Dickinson FACScalibur flow cytometer (20 000 counts were
measured). The percentage of the cells in each cell cycle phase
was determined using the WinMDI software based on the DNA
histograms. Statistical analysis was performed in SigmaStat
2.0 by using the one-way ANOVA test.
(13) Edwards, J. P.; Higuchi, R. I.; Winn, D. T.; Pooley, C. L. F.;
Caferro, T. R.; Hamann, L. G.; Zhi, L.; Marschke, K. B.;
Goldman, M. E.; Jones, T. K. Nonsteroidal Androgen Receptor
Agonists Based on 4-(Trifluoromethyl)-2H-pyrano[3,2-g]quinolin-
2-one. Bioorg. Med. Chem. Lett. 1999, 9, 1003-1008.
(14) Tomita, K.; Tsuzuki, Y.; Shibamori, K.; Tashima, M.; Kajikawa,
F.; Sato, Y.; Kashimoto, S.; Chiba, K.; Hino, K. Synthesis and
Structure-Activity Relationships of Novel 7-Substituted 1,4-
dihydro-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylic ac-
ids as Antitumor Agents. J. Med. Chem. 2002, 45, 5564-5575.
(15) Chen, Y.-L.; Chung, C.-H.; Chen, I.-L.; Chen, P.-H.; Jeng, H.-
Y. Synthesis and Cytotoxic activity evaluation of Indolo-, Pyrrolo-
and Benzofuro-Quinoli-2(1H)-Ones and 6-Anilinoindoloquinoline
Derivatives. Bioorg., & Med. Chem. 2002, 10, 2705-2712.
(16) Chilin, A.; Marzano, C.; Baccichetti, F.; Simonato, M.; Guiotto,
A. 4-Hydroxymethyl- and 4-Methoxymethylfuro[2,3-h]-quinolin-
2(1H)-ones: Synthesis and Biological Properties. Bioorg. Med.
Chem. 2003, 11, 1311-1318.
Annexin-V Assay. Detection and quantification of apoptotic
cells at the single-cell level was performed using an Annexin-
V-FLUOS staining kit (Roche), according to the manufactur-
er’s recommendations. After the desired length of time, both
floating and attached cells were collected. The cells were then

2360 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Jarak et al.
(17) Kim, Y. S.; Kim, K. M.; Song, R.; Jun, M. J.; Sohn, Y. S.
“Synthesis, Characterization and Antitumor Activity of Qui-
nolone-platinum(II) conjugates. J. Inorg. Biochem. 2001, 87,
157-163.
(27) McGhee, J. D.; von Hippel, P. H. Theoretical Aspects of DNA-
Protein Interactions: Cooperative and Non-Cooperative Binding
of Large Ligands to a One-Dimensional Homogeneous Lattice.
J. Mol. Biol. 1976, 103, 679-684.
(28) Zimmermann, H. W. Physicochemical and Cytochemical Inves-
tigations of the Binding of Ethidium and Acridine Dyes to DNA
and to Organelles in Living Cells. Angew. Chem., Int. Ed. Engl.
1986, 25, 115-196.
(29) Piantanida, I.; Palm, B. S.; Zˇinic´, M.; Schneider, H.-J. A New
4,9-Diazapyrenium Intercalator for Single and Double Stranded
Nucleic Acids: Distinct Differences to Related Diazapyrenium
Compounds and Ethidium Bromide. J. Chem. Soc., Perkin Trans.
2 2001, 1808-1816.
(30) Larsen, A. K.; Skladanowski, A. Cellular Resistance to Topoi-
somerase-targeted Drugs: From Drug Uptake to Cell Death.
Bioch. Bioph. Acta 1998, 257-274.
(31) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute In Vitro Anticancer
Drug Discovery Screen. Drug Dev. Res. 1995, 34, 91-109.
(32) Mossman, T. J. Rapid Colorimetric Assay for Cellular Growth
and Survival: Application to Proliferation and Cytotoxicity
Assays. J. Immunol. Methods 1983, 65, 55-61.
(33) Szakacs, G.; Annereau, J.-P.; Lababidi, S.; Shankavaram, U.;
Arciello, A.; Bussey, K. J. n; Reinhold, W.; Guo, Y.; Kruh, G. D.;
Reimers, M.; Weinstein, J. N.; Gottesman, M. M. Predicting drug
sensitivity and resistance: Profiling ABC transporter genes in
cancer cells. Cancer Cell 2004, 6, 129-137.
(34) Wilson, W. D.; Tanious, F. A.; Fernandez-Saiz, M.; Rigl, C. T.
Evaluation of Drug/Nucleic acid Interactions by Thermal Melt-
ing Curves, In Methods in Molecular Biology, Vol. 90: Drug-
DNA Interaction Protocols; Fox, K. R., Ed.; Humana Press Inc.,
Totowa, N. Y., 1998.
(18) Dogan Koruznjak, J.; Slade, N.; Zamola, B.; Pavelic´, K.; Karmin-
ski-Zamola, G. Synthesis, photochemical synthesis and antitu-
mor evaluation of novel derivatives of thieno[3′,2′:4,5]thieno[2,3-
c]quinolones. Chem. Pharm. Bull. 2002, 50, 656-660.
(19) Dogan Koruznjak, J.; Grdis´a, M.; Slade, N.; Zamola, B.; Pavelic´,
K.; Karminski-Zamola, G. Novel Derivatives of Benzo[b]thieno-
[2,3-c]Quinolones: Synthesis, Photochemical Synthesis and
Antitumor Evaluation. J. Med. Chem. 2003, 45, 4516-4524.
(20) Ried, W.; Oremek, G.; Ocakcioglu, B. Synthese von Substitu-
ierten Benzo[b]thiophenen. Liebigs Ann. Chem. 1980, 1424-
1427.
(21) Fairley, T. A.; Tidwell, R. R.; Donkor, I.; Naiman, N. A.;
Ohemeng, K. A.; Lombardy, R. J.; Bentley, J. A.; Cory, M.Struc-
ture, DNA minor-Groove Binding and Base-Pair Specificity of
Alkyl-Linked and Aryl-Linked Bis(amidinobenzimidazoles) and
Bis(amidinoindoles). J. Med. Chem. 1993, 36, 1746-1753.
(22) Demeunynck, M.; Bailly, C.; Wilson, W. D. In DNA and RNA
Binders; Wiley-VCH: Weinheim, 2002.
(23) Peek, M. E.; Lipscomb, L. A.; Bertrand, J. A.; Gao, Q.; Roques,
B. P.; Garbay-Jaureguiberry, C.; Williams, L. D. DNA Distortion
in Bis-Intercalated Complexes. Biochemistry 1994, 33, 3794-
3800.
(24) Gao, Q.; Williams, L. D.; Egli, M.; Rabinovich, D.; Chen, S.-L.;
Quigley, G. J.; Rich, A. Drug-Induced DNA Repair: X-ray
Structure of a DNA-Ditercalinium Complex. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 2422-2426.
(25) Oszczapowicz, In The Chemistry of Amidines and Imidates;
Patai, S., Rappoport, Z., Eds.; John Wiley and Sons: London,
New York, 1991; Vol. 2, p 623.
(26) Dougherty, G.; Pilbrow, J. R. Physico-Chemical Probes of
Intercalation. Int. J. Biochem. 1984, 16, 1179.
JM049541F