Novel derivatives of benzo[b]thieno[2,3-c]quinolones: Synthesis, photochemical synthesis, and antitumor evaluation

Article abstract of DOI:10.1021/jm0210966

Novel derivatives of benzo[b]thieno[2,3-c]quinolones 3a-j were synthesized in a multistep synthesis starting from substituted benzo[b]thiophene-2-carbonyl chlorides, to their corresponding benzo[b]thiophene-2-carboxamides, which were photochemically dehyd

Full text of DOI:10.1021/jm0210966

4516
J . Med. Chem. 2003, 46, 4516-4524
Novel Der iva tives of Ben zo[b]th ien o[2,3-c]qu in olon es: Syn th esis, P h otoch em ica l
Syn th esis, a n d An titu m or Eva lu a tion
J asna Dogan Koruzˇnjak,†,‡ Mira Grdisˇa,§ Neda Slade,§ Branimir Zamola,| 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, Rudjer Boskovic Institute, Bijenicˇka cesta 54,
P.O. Box 1016, HR-10000 Zagreb, Croatia, and Department of Biochemical Engineering, Faculty of Food and Biotechnology,
University of Zagreb, Pierrottieva 6, HR-10000 Zagreb, Croatia
Received November 19, 2002
Novel derivatives of benzo[b]thieno[2,3-c]quinolones 3a -j were synthesized in a multistep
synthesis starting from substituted benzo[b]thiophene-2-carbonyl chlorides, to their corre-
sponding benzo[b]thiophene-2-carboxamides, which were photochemically dehydrohalogenated
to their corresponding substituted benzo[b]thieno[2,3-c]quinolones. Compound 4 was prepared
from 3i by alkylation with 3-dimethylaminopropyl chloride in the presence of NaH. Compounds
7a ,b were prepared from 3g in the multistep synthesis from compounds 5 and 6. Compounds
3b, 3c-f, 3h , 7a , and 7b were found to exert cytostatic activity against malignant cell lines:
pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa),
laryngeal carcinoma (Hep2), colon carcinoma (CaCo-2), melanoma (HBL), human fibroblast
cell lines (WI-38). The compounds that bear a 3-dimethylaminopropyl substituent on the
quinolone nitrogen (3b, 3c-f, 3h ) showed higher antitumor activity than compounds bearing
the same substituent on the amidic nitrogen (7a and 7b). The compound 3h , which has a
3-dimethylaminopropyl substituent on the quinolone nitrogen and a methoxycarbonyl sub-
stituent at position 9, had marked antitumor activity. Because of strong cytotoxic effect of
compound 4 on melanoma cells (HBL, ME 67.3, and ME 67.1), a potential mechanism of action
was examined. Analysis of DNA and Annexin-V-FLUOS staining indicated that compound 4
causes cell death by apoptosis.
In tr od u ction
Few data have been publish describing the antitumor
activity of heterocyclic condensed quinolones. The novel
quinoline-quinone streptonigrin (SN) is an antitumor
antibiotic that has activity against a broad range of
tumors.12-14 Although SN has been studied clinically as
an antitumor agent, its use has been limited by reports
of delayed myelotoxicity.15,16 Nevertheless, positive
results were reported for SN either as a single agent17,18
or in combination chemotherapy.19,20 SN is an excellent
substrate for oxidoreductase (NQ01).21
A lot of 2- and 4-quinolones substituted with different
substituents showed in vitro inhibition of human tumor
cell lines and also tubulin polymerization. This class of
compounds are 2-phenyl-4-quinolones substituted with
different substituents on the benzene part of the mol-
ecule. In these compounds a good correlation was found
between cytotoxicity and inhibition of tubulin polymeri-
zation.1-6 The same group of compounds displayed
significant growth inhibitory action against a panel of
tumor cell lines including human ileocecal carcinoma
(HCT-8), murine leukemia (P-388), human melanoma
(RPMI), and human central nervous system tumor (TE
671) cells. Some of these compounds displayed DNA
topoisomerase I inhibitory activity in vitro and DNA
topoisomerase II inhibitory activity in vitro.7-9 Some
new analogues of 4-quinolinones and 1,7-naphthyridin-
4-ones have been synthesized and tested in vitro for
their antitumor activity against CNS (SNB-75), breast
(T-47D), and lung (NCI-H 522) cancer cell lines.10
Substituted 3-hydroxy-2-quinolone has also been syn-
thesized and evaluated by [3H]-glycine and [3H]-AMPA
binding to rat cortical membranes.11
Pyranoquinolin-2-ones were synthesized and evalu-
ated for their in vitro cytotoxicity against a panel of
human tumor cell lines,22 and 2-arylquinazolinones had
significant growth inhibitory action against tumor cell
lines, some of them being potent inhibitors of tubulin
polymerization. Some of them displayed selective activ-
ity against P-gp-expressing epidermoid carcinoma of the
nasopharrynx.23 Recently trifluoromethyl-substituted
pyranoquinolinone was prepared and tested for its
ability to modulate the transcriptional activity of the
human androgen receptor (HAR).24
Searching for compounds related to these classes of
biologically and pharmacologically active compounds, we
have prepared new derivatives of thieno[3′,2′:4,5]thieno-
[2,3-c]quinolones and examined their antitumor activ-
ity.25
* To whom the correspondence should be addressed. Phone
++38514597215; fax ++38514597250; e-mail: gzamola@pierre.fkit.hr.
† Department of Organic Chemistry, University of Zagreb.
‡ Present address: PLIVA d.d., Research and Development, Prilaz
baruna Filipovic´a 25, HR-10000 Zagreb, Croatia.
In this work we prepared benzo[b]thieno[2,3-c]qino-
lones containing a 3-dimethylaminopropyl substituent
on the quinolone nitrogen (3b-f, 3h , and 4) or a
§ Rudjer Boskovic Institute.
| Department of Biochemical Engineering, University of Zagreb.
10.1021/jm0210966 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/28/2003

Antitumor Benzo[b]thieno[2,3-c]quinolones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4517
F igu r e 1. Benzo[b]thieno[2,3-c]qinolones (3b-f, 3h , 4, 7a ,
and 7b).
3-dimethyaminopropyl substituent in the amide part of
the molecule (7a , 7b) and evaluated their antitumor
activity.
Ch em istr y
All compounds 3a -j shown in Figure 1 were prepared
in multistep synthesis according to Scheme 1. Starting
from different 6-substituted 3-chlorobenzo[b]thiophene-
2-carbonyl chlorides 1a -g, which already were known
from the literature.26 The yield varied from 50 to 80%
by refluxing of chlorides 1a , 1b, and 1c with aniline in
toluene. Corresponding 3-chlorobenzo[b]thiophene-2-
carboxamides were obtained (2a 27 in 98% yield, 2g28 in
88.9% yield, and 2i29 in 35.4% yield).
On the other hand, N-[3-(dimethyamino)propyl]-
substituted 3-chlorobenzo[b]thiophene-2-carboxamides
2b -f, 2h , and 2j were obtained in yields of 40-70%
from chlorides 1a-g by the Shotten-Baumann method30
with N-[3-(dimethyamino)propyl]aniline at 0 °C. The
anilides 2a -j were converted by the photochemical
dehydrohalogenation reaction into their corresponding
quinolones 3a -j in 50-90% yields.29 Better yield was
achieved in cases where the N-atom on the anilide
moiety of a molecule was not substituted with an N-[3-
(dimethyamino)propyl] substituent.
Hydrolysis of ester group of compound 3g and conver-
sion of corresponding carboxylic acid 5 into carbonyl
chloride 6 gave the intermediate which was used for
preparation of both quinolones: N-[3-(dimethyamino)-
propyl]-6-oxo-5,6-dihydro[1]benzothieno[2,3-c]quinoline-
9-carboxamide hydrochloride (7a ) (reflux with 3-(di-
methylamino)propylamine) and N-[3-(dimethyamino)-
propyl]-6-oxo-5,6-dihydro[1]benzothieno[2,3-c]quinoline-
9-carboxanilide hydrochloride (7b).28
F igu r e 2. 1H NMR spectra of compounds 2i, 3i, and 4.
1
Figure 2 shows H NMR spectra of compounds 2i, 3i,
and 4 from which it is evident that a monoalkylation
reaction had taken place and that the reaction occurred
at the quinolone part of the molecule. The structures of
the novel compounds were determined on the basis of
analysis of chemical shifts and H-H coupling constants
1
in H NMR spectra and 13C NMR spectra as well as by
elemental analysis.
Biologica l Resu lts a n d Discu ssion
An titu m or Activity. The effect of all compounds on
cell proliferation was tested on the following malignant
human tumor cell lines: cervical carcinoma (HeLa),
breast carcinoma (MCF-7), colon carcinoma (CaCo-2),
pancreatic carcinoma (MIA PaCa-2), melanoma (HBL),
and laryngeal carcinoma (Hep-2), as well as on a human
fibroblast cell line (WI-38). Compound 4 was tested also
on additional melanoma cell lines, ME 67.1 and ME
67.3. The results are presented in Table 1 and Figure 3.
N-Alkylation of compound 3i with 3-dimethylamino-
propyl chloride hydrochloride in the presence of NaH,
after stirring at room temperature, gave the expected
quinolone derivative 4.

4518 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Koru et al.
Sch em e 1
All tested compounds exhibited strong antitumor
activity with IC50 ranging from 0.01 to 34.7 µM. Com-
pounds 3f, 3h , and 4 had the strongest inhibition.
Compound 4 was the strongest inhibitor of growth for
melanoma cell lines, HBL (IC50 ) 0.01 µM), ME 67.3
(IC50 ) 0.19 µM), and ME 67.1 (IC50 ) 0.79 µM) and for
CaCo-2 cells (IC50 ) 0.49 µM). Compound 3h also mostly
inhibited HBL cells (IC50 ) 0.35 µM), as well as MCF-
7, CaCo-2, and Hep-2 cell lines (IC50 ) 0.47-0.65 µM).
Compound 3f was not as toxic for HBL cells (IC50 ) 1.58
µM) as for CaCo-2 (IC50 ) 0.47 µM) and MIA PaCa-2
cells (IC50 ) 0.51 µM). Compound 7b had the most
pronounced selectivity in cytostatic activity: HBL cells
were inhibited the most (IC50 ) 0.67 µM), HeLa (IC50
)
34.7 µM) and MCF-7 cells (IC50 ) 19.5 µM) the least.
No other compound showed so big a difference in
inhibition. In comparison with antitumor activity of
similar quinolones,3-6,9,11 the substances tested in these
experiments were very strong inhibitors of tumor cell
proliferation at very low concentrations.
From results in Table 1, we noticed a very strong
inhibitory effect of compound 4 on melanoma cell line
HBL, and we speculated that this compound could act
as a specific inhibitor for melanoma cells. For this

Antitumor Benzo[b]thieno[2,3-c]quinolones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4519
Ta ble 1. Inhibitory Effect of Compounds on the Growth of
Tumor Cell Lines and Normal Fibroblasts (WI-38)
Ta ble 3. Percent of Apoptotic Cells after Treatment of HBL
Cells with Compound 4 for 5 h
IC50 (µM)a
concentration (M)
apoptotic cells (%)
compd HeLa MCF-7 CaCo-2 MIAPaCa-2 HBL Hep-2 WI-38
control
1
9
29
35
1 × 10-6
5 × 10-6
1 × 10-5
3b
3c
3d
3e
3f
3h
4
2.5
0.65
1.0
1.0
0.85
1.3
2.8
6.3
34.7
4.27
4.6
5.8
4.5
2.2
0.59
2.63
10.0
19.5
0.62
0.98
0.79
1.7
0.47
0.47
0.49
3.8
1.4
2.5
1.25
1.1
0.51
1.0
1.0
6.6
4.79
0.58
3.98
1.2
3.63
3.16
3.54
3.63
2.96
0.65
1.77
6.6
4.5
2.9
2.4
2.45
2.3
1.6
2.3
4.4
1.1
1.58
0.35
0.01
1.0
comparison with control cells, fragmentation of DNA
increased with concentration of compound 4. The pres-
ence of DNA fragments in treated cells pointed to
apoptosis as a mechanism of cell death induced by
compound 4. Induction of apoptosis by compound 4 was
confirmed with Annexine-V-FLUOS staining. The num-
ber of apoptotic cells (green fluorescence) increased with
concentration of 4 (Table 3).
7a
7b
4.79
0.67
3.16
10.0
a
50% inhibitory concentration or compound concentration
required to inhibit cell proliferation by 50%.
Apoptosis is characterized by cell shrinkage, conden-
sation of the nucleus, and DNA fragmentation.31-35
A
variety of cancer chemotherapeutic agents induce cell
death by apoptosis. From the above results it is very
difficult to say whether compound 4 acts as a specific
inhibitor for melanoma cells, but we can say that
compound 4 induced apoptosis in melanoma cells and
influenced their adhesion. Whether induction of apop-
tosis caused detachment of the cells or the converse is
not clear. Whether compound 4 exerts its effect directly
on the cell cycle or indirectly through interaction with
the cell membrane has not been determined. Even
though compound 4 induced apoptosis in melanoma
cells, other mechanisms responsible for cell death have
not been excluded (e.g., inhibition of tubulin polymeri-
zation36-39).
F igu r e 3. The inhibitory effect of different concentration of
the most active substance (3h ) on the growth of human
malignant tumor cell lines (HeLa, MCF-7, CaCo-2, MIA PaCa-
2, HBL, Hep-2) and normal human fibroblasts (WI-38).
Ta ble 2. Influence of Compound 4 on Adhesion of HBL Cells
Con clu sion s
time of incubation (h)
All tested compounds exhibited strong inhibitory
activities against all cell lines tested. The most sensitive
were HBL cells, although cell sensitivity varied from
compound to compound. The most potent antitumor
effect against all cells lines was found for compounds
3f, 3h , and 4. Compound 4 induced apoptosis in HBL
cells and altered cell adhesion. Future study will be
focused on more profound examination of the mecha-
nisms of action for these compounds, as well as their in
vivo effect.
concentration (M)
1
2
3
24
1 × 10-5
87%
90%
87%
98%
81%
95%
28%
62%
5 × 10-6
reason, inhibition of growth of additional melanoma cell
lines (ME 67.1 and ME 67.3) was investigated. Both of
these cell lines were sensitive to compound 4 (ME 67.3,
IC50 ) 0.19 µM; ME 67.1, IC50 ) 0.79 µM). A potential
mechanism of action was also examined for compound
4. The influence of compound 4 on adhesion of HBL cells
was examined. By MTT test we determined the cells
attached on plastic substratum, after treatment with 4
at different time point. The results (Table 2) are
expressed as a ratio (%) between treated and control
cells. One of three parallel experiments is shown.
The influence of compound 4 on cell cycle was also
examined. Melanoma cell line (HBL) was treated with
compound 4, and after staining with propidium iodide
(PI), a flow cytometry analysis was performed (Figure
4). In comparison with control cells (G0/G1 ) 60.19%;
G2/M ) 4.26%; S ) 37.06%), the number of cells in G2/M
phase increased about 2.25 times (G2/M ) 9.6) and in
S phase decreased about 1.33 times (S ) 27.78), without
significant changes in G0/G1 phase (62.62%) (the results
are average from two independent experiments).
To better understand the mechanism of action, DNA
from HBL cells was isolated after treatment with
compound 4. DNA was separated on an agarose gel, and
fragmentation of DNA was observed (Figure 5). In
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Kofler
hot stage microscope and are uncorrected. IR spectra were
recorded on a Nicolet Magna 760 spectrophotometer between
sodium chloride plates. UV spectra were recorded on either a
Perkin-Elmer 124 or a Hewlett-Packard 8452A spectropho-
tometer in methanol or ethanol. 1H and 13C NMR spectra were
recorded on either a Varian Gemini 300 or a Bruker Avance
DPX 300 spectrometer using TMS as an internal standard in
DMSO-d6, CDCl3, or D2O. Mass spectra were recorded on
either a Varian-MAT 311A (electron impact and fast atom
bombardment) or a Micromass Platform LCZ (Electrospray).
Elemental analysis for carbon, hydrogen, and nitrogen was
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 immersion well fitted with an “Hanovia” 450 W medium
pressure and with an “Original Hanau” 125 or 400 W high
pressure mercury arc lamp using quartz or Pyrex filter. All
compounds were routinely checked by TLC with Merck silica

4520 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Koru et al.
F igu r e 4. Flow cytometry analysis of HBL cells treated with compound 4 and stained with PI. The cells were grown under
confluences and treated with compound 4 for 5 h.
N-[3-(Dim et h yla m in o)p r op yl]-N-p h en yl-3-ch lor ob en -
zo[b]th iop h en e-2-ca r boxa m id e (2b): from 1a by method
B, brown oily product after purification by column chroma-
tography (10% ethanol/toluene), yield 62.7%; IR (NaCl)(νmax
/
/
cm-1) 2766-2942 (CH3, CH2), 1651 (CdO); UV (methanol)(λmax
nm) 230, 284; 1H NMR (CDCl3)(δ/ppm) 7.77-7.67 (m, 2H, J )
9.03, 7.70, 2.23 Hz), 7.43-7.39 (m, 2H, J ) 8.79, 7.64, 2.21
Hz), 7.32-7.18 (m, 5H), 4.03 (t, 2H, J ) 7.45 Hz), 2.49 (t, 2H,
J ) 7.37 Hz), 2.30 (s, 6H), 1.93 (tt, 2H, J ) 7.40 Hz).; 13C NMR
(CDCl3)(δ/ppm) 162.6 (s), 141.4 (s), 137.6 (s), 137.6 (s), 135.4
(s), 130.9 (s), 129.0 (d), 129.0 (d), 127.6 (d), 127.4 (d), 127.4
(d), 126.3 (d), 125.0 (d), 122.5 (d), 122.4 (d), 56.7 (t), 48.5 (t),
45.1 (q), 45.1 (q), 22.5 (t); ΜS m/z 373 (M + 1). Anal. (C20H21
-
F igu r e 5. DNA analysis after treatment of HBL cells with
compound 4. Lane 1, control cells; lane 2, cells treated with
compound 4 at concentration of 5 × 10-6 M; and lane 3, cells
treated with compound 4 at concentration of 10-5 M.
ClN2OS) C, H, N.
N-[3-(Dim eth ylam in o)pr opyl]-N-ph en yl-6-flu or o-3-ch lo-
r oben zo[b]th iop h en e-2-ca r boxa m id e (2c): from 1d by
method B, white crystals after recrystallization from chloro-
form:ethyl acetate (1:3), yield 65.6%; mp 203-204 °C; IR (KBr)-
(νmax/cm-1) 3417-2469 (CH3, CH2), 1640 (CdO); UV (Methanol)-
(λmax/nm) 206, 232, 286; 1H NMR (DMSO-d6)(δ/ppm) 7.94 (dd,
1H, J ) 9.08, 2.28 Hz), 7.72 (dd, 1H, J ) 8.90, 5.07 Hz), 7.39-
7.22 (m, 6H), 3.95 (t, 2H, J ) 7.10 Hz), 3.13 (t, 2H, J ) 8.06
Hz), 2.73 (s, 6H), 1.99 (tt, 2H, J ) 7.45, 7.75 Hz); 13C NMR
(DMSO-d6)(δ/ppm) 161.8 (s), 161.4 (s), 159.9 (s), 140.5 (s), 138.2
(s), 131.1 (s), 129.1 (d), 129.1 (d), 127.9 (d), 127.6 (d), 127.6
(d), 123.7 (d), 118.4 (s), 114.9 (d), 109.6 (d), 53.9 (t), 46.7 (t),
gel 60F-254 glass plates or Merck aluminum oxide plates in
KBr disks or as a liquid film.
Gen er a l Meth od for th e Syn th esis of 6-Su bstitu ted
3-Ch lor ob en zo[b]t h iop h en e-2-ca r b oxa m id es
(2a -j).
Meth od A. A solution of 3-chlorobenzo[b]thiophene-2-carbonyl
chloride (1a -c) (0.02 mol) and aniline (0.03 or 0.07 mol) in
toluene (80 mL) was refluxed for 1 h. After cooling, precipitated
crystals were filtered off and recrystallized. Meth od B. A
solution of 3-chlorobenzo[b]thiophene-2-carbonyl chloride (1a -
g) (0.02 mol) in chloroform (400 mL) was added dropwise to a
cold mixture of N-[3-(dimethylamino)propyl]aniline (0.02 or
0.04 mol) and 5% NaOH (9 or 18 mL) at 2-5 °C. The resulting
mixture was stirred at the same temperature for 30 min and
then 1 h at room temperature. The organic layer was separated
and washed first with 3% HCl and then with water and dried
over anhydrous Na2SO4. After evaporation of the solvent, the
crude product was purified by column chromatography (neu-
tral aluminum oxide) or recrystallization.
41.8 (q), 41.8 (q), 22.3 (t); ΜS m/z 391 (M + 1). Anal. (C20H20
ClFN2OS) C, H, N.
-
N-[3-(Dim eth ylam in o)pr opyl]-N-ph en yl-6-flu or o-3-ch lo-
r oben zo[b]th iop h en e-2-ca r boxa m id e (2d ): from 1e by
method B, yellow crystals after recrystallization from chloro-
form:ethyl acetate (1:3), yield 51.5%; mp 227-228 °C; IR (KBr)-
(νmax/cm-1) 3505-2467 (CH3, CH2), 1634 (CdO); UV (methanol)-
1
(λmax/nm) 206, 226, 308; H NMR (DMSO-d6)(δ/ppm) 9.11 (s,
1H), 8.25 (dd, 1H, J ) 8.91, 1.94 Hz), 7.90 (d, 1H, J ) 8.90
Hz), 7.44-7.22 (m, 5H), 3.97 (t, 2H, J ) 6.98 Hz), 3.15 (t, 2H,
N -P h e n y l-3-c h lo r o b e n z o [b ]t h i o p h e n e -2-c a r b o x -
a m id e (2a ): from 1a by method A, white crystals after
recrystallization from chloroform:cyclohexane (1:2), yield 98.6%;
mp 170 °C (lit.27 mp 169 °C).
J ) 7.87 Hz), 2.74 (s, 6H), 1.99 (tt, 2H, J ) 7.54, 7.79 Hz); 13
C
NMR (DMSO-d6)(δ/ppm) 161.0 (s), 145.6 (s), 140.0 (s), 138.5
(s), 138.1 (s), 137.0 (s), 129.2 (d), 129.2 (d), 128.2 (d), 127.6

Antitumor Benzo[b]thieno[2,3-c]quinolones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4521
(d), 127.6 (d), 122.7 (d), 120.5 (d), 120.2 (d), 118.2 (s), 53.9 (t),
46.6 (t), 41.9 (q), 41.9 (q), 22.3 (t); ΜS m/z 418 (M + 1). Anal.
(C20H20ClN3O3S) C, H, N.
methanol/toluene, light brown crystals after recrystallization
from methanol, yield 65.6%; mp > 300 °C (lit.40 mp > 310 °C).
5-[3-(Dim e t h yla m in o)p r op yl]-6-oxo-5,6-d ih yd r o [1]-
ben zoth ien o[2,3-c]qu in olin -6-on e h ydr och lor ide (3b): from
2b after 30 min of irradiation with 400 W high-pressure
mercury arc lamp using Pyrex filter in 9% methanol/benzene.
After evaporation of the solvent, the crude product was purified
by column chromatography (20% ethanol/toluene) and then
converted into hydrochloride salt; light brown crystals, yield
50.3%; mp 260-263 °C; IR (KBr)(νmax/cm-1) 2475-2948 (CH3,
CH2), 1615 (CdO); UV (ethanol)(λmax/nm) 234, 256, 302, 318,
N -[3-(Dim e t h yla m in o)p r op yl]-N -p h e n yl-6-m e t h yl-
3-ch lor oben zo[b]th iop h en e-2-ca r boxa m id e (2e): from 1f
by method B, white crystals after recrystallization from
chloroform:ethyl acetate (1:3), yield 91.6%; mp 201-203 °C;
IR (KBr)(νmax/cm-1) 3555-2476 (CH3, CH2), 1641 (CdO); UV
(Methanol)(λmax/nm) 208, 232, 292; 1H NMR (DMSO-d6)(δ/
ppm): 7.75 (s, 1H), 7.58 (d, 1H, J ) 8.23 Hz), 7.38-7.19 (m,
6H), 3.95 (t, 2H, J ) 6.94 Hz), 3.13 (t, 2H, J ) 7.99 Hz), 2.73
(s, 6H), 2.39 (s, 3H), 1.98 (tt, 2H, J ) 7.50, 7.62 Hz); 13C NMR
(DMSO-d6)(δ/ppm) 162.0 (s), 140.7 (s), 137.3 (s), 137.1 (s), 132.4
(s), 130.1 (s), 129.2 (d), 129.2 (d), 128.0 (d), 127.7 (d), 127.7
(d), 127.5 (d), 122.8 (d), 121.7 (d), 118.8 (s), 54.1 (t), 46.8 (t),
42.0 (q), 42.0 (q), 22.5 (t), 21.2 (q); ΜS m/z 387 (M+1). Anal.
(C21H23ClN2OS) C, H, N.
1
342, 360; H NMR (DMSO-d6)(δ/ppm) 10.92 (s, 1H), 8.94 (dd,
1H, J ) 9.31 Hz), 8.88 (d, 1H, J ) 7.67 Hz), 8.27 (dd, 1H, J )
9.28 Hz), 7.90 (d, 1H, J ) 8.42 Hz), 7.76-7.67 (m, 3H), 7.53
(dd, 1H, J ) 7.62 Hz), 4.53 (t, 2H, J ) 7.13 Hz), 3.25 (dt, 2H,
J ) 5.05 Hz), 2.76 (s, 3H), 2.75 (s, 3H), 2.18 (tt, 2H, J ) 7.27
Hz); 13C NMR (DMSO-d6)(δ/ppm) 157.7 (s), 141.7 (s), 137.4 (s),
135.4 (s), 135.2 (s), 131.8 (s), 129.5 (d), 127.8 (d), 126.2 (d),
126.2 (d), 124.6 (d), 124.3 (d), 123.2 (d), 118.9 (s), 116.3 (d),
54.2 (t), 42.2 (q), 42.2 (q), 39.1 (t), 22.8 (t); ΜS m/z 337 (free
base M + 1). Anal. (C20H21ClN2OS) C, H, N.
N-[3-(Dim eth yla m in o)p r op yl]-N-p h en yl-6-m eth oxy-3-
ch lor oben zo[b]th iop h en e-2-ca r boxa m id e (2f): from 1g by
method B, white crystals after recrystallization from chloro-
form:ethyl acetate (1:3), yield 29.9%; mp 188-190 °C; IR (KBr)-
(νmax/cm-1) 3436-2474 (CH3, CH2), 1642 (CdO); UV (Ethanol)-
An attempt was made to synthesize compound 3b by
N-alkylation41 of 3a , but the reaction was unsuccessful and
only starting material was isolated.
1
(λmax/nm) 197, 233, 301; H NMR (DMSO-d6)(δ/ppm) 7.58 (d,
1H, J ) 8.92 Hz), 7.55 (d, 1H, J ) 2.12 Hz), 5.37-5.23 (m,
5H), 7.07 (dd, 1H, J ) 8.88, 2.28 Hz), 3.94 (t, 2H, J ) 7.11
Hz), 3.80 (s, 3H), 3.13 (t, 2H, J ) 8.13 Hz), 2.74 (s, 6H), 1.96
(tt, 2H, J ) 7.92, 8.34 Hz); 13C NMR (DMSO-d6)(δ/ppm): 162.0
(s), 158.9 (s), 140.9 (s), 139.0 (s), 133.4 (s), 129.3 (d), 129.3 (d),
128.3 (s), 128.0 (d), 127.8 (d), 127.8 (d), 122.9 (d), 119.1 (s),
116.1 (d), 105.5 (d), 55.8 (q), 54.2 (t), 46.9 (t), 42.2 (q), 42.2 (q),
22.5 (t); ΜS m/z 403 (M + 1). Anal. (C21H23ClN2O2S) C, H, N.
Meth yl 2-(N-p h en ylca r ba m oyl)-3-ch lor oben zo[b]th io-
p h en e-6-ca r boxyla te (2 g): from 1b by method A, white
crystals after recrystallization from acetone, yield 88.8%; mp
197-198 °C (lit.28 mp 197-198 °C).
5-[3-(D i m e t h y la m i n o )p r o p y l]-9-flu o r o -6-o x o -5,6-
d ih yd r o[1]ben zoth ien o[2,3-c]qu in olin -6-on e h yd r och lo-
r id e (3c): from 2c after 20 min of irradiation with 400 W high-
pressure mercury arc lamp using Pyrex filter in 9% methanol/
toluene. After evaporation of the solvent, the crude product
was purified by recrystallization from toluene:methanol (1:1)
and then converted into hydrochloride salt; white crystals,
yield 62.5%; mp 252 °C; IR (KBr)(νmax/cm-1) 3501-2483 (CH3,
CH2), 1620 (CdO); UV (methanol)(λmax/nm) 204, 232, 254, 302,
340, 356; 1H NMR (DMSO-d6)(δ/ppm) 10.39, (s, 1H), 8.97 (dd,
1H, J ) 9.22, 5.05 Hz), 8.83 (d, 1H, J ) 7.53 Hz), 8.21 (dd,
1H, J ) 9.02, 2.54 Hz), 7.89 (d, 1H, J ) 8.33 Hz), 7.74 (dd,
1H, J ) 7.49, 7.45 Hz), 7.58-7.49 (m, 2H), 4.52 (t, 2H, J )
7.11 Hz), 3.24 (t, 2H, J ) 7.90 Hz), 2.75 (s, 6H), 2.16 (tt, 2H,
J ) 7.22, 7.68 Hz); 13C NMR (DMSO-d6)(δ/ppm) 162.8 (s), 159.5
(s), 157.2 (s), 143.1 (s), 137.2 (s), 134.5 (s), 132.0 (s), 129.5 (d),
127.7 (d), 124.2 (d), 123.0 (d), 118.3 (s), 116.0 (d), 114.5 (d),
110.1 (d), 54.0 (t), 42.0 (q), 42.0 (q), 39.7 (t), 22.6 (t); ΜS m/z
355 (free base M+1). Anal. (C20H20ClFN2OS) C, H, N.
Met h yl
2-{N-[3-(d im et h yla m in o)p r op yl]-N-p h en yl-
ca r b a m oyl}-3-ch lor ob en zo[b]t h iop h en e-6-ca r b oxyla t e
(2h ): from 1b by method B, yellow crystals after recrystalli-
zation from chloroform:ethyl acetate (1:2), yield 65.6%; mp
191-195 °C (lit.28 mp 191-195 °C).
N,N′-Dip h en yl-3-ch lor ob en zo[b]t h iop h en e-2,6-d ica r -
boxa m id e (2i): from 1c by method A, light yellow crystals
after recrystallization from DMF:water (1:2), yield 85.6%; mp
278 °C (lit.29 mp 278 °C).
5-[3-(D i m e t h y la m i n o )p r o p y l]-9-n i t r o -6-o x o -5,6-
d ih yd r o[1]ben zoth ien o[2,3-c]qu in olin -6-on e h yd r och lo-
r id e (3d ): from 2d after 20 min of irradiation with 400 W
high-pressure mercury arc lamp using Pyrex filter in 9%
methanol/toluene. After evaporation of the solvent, the crude
product was purified by recrystallization from toluene:metha-
nol (1:1) and then converted into hydrochloride salt; yellow
crystals, yield 43.6%; mp 270 °C; IR (KBr)(νmax/cm-1) 3417-
2360 (CH3, CH2), 1634 (CdO); UV (methanol)(λmax/nm) 224,
278, 299, 370; 1H NMR (DMSO-d6)(δ/ppm) 10.79 (s, 1H), 9.07
(d, 1H, J ) 2.15 Hz), 8.84 (d, 1H, J ) 9.25 Hz), 8.55 (d, 1H, J
) 7.96 Hz), 8.20 (dd, 1H, J ) 8.94, 2.11 Hz), 7.82 (d, 1H, J )
8.39 Hz), 7.67 (dd, 1H, J ) 7.25, 7.83 Hz), 7.40 (dd, 1H, J )
7.40, 7.19 Hz), 4.23 (t, 2H, J ) 6.78 Hz), 3.27 (t, 2H, J ) 7.68
Hz), 2.78 (s, 6H), 2.17 (tt, 2H, J ) 6.90, 7.50 Hz); 13C NMR
(DMSO-d6)(δ/ppm) 157.0 (s), 145.5 (s), 141.6 (s), 139.4 (s), 137.1
(s), 136.1 (s), 133.7 (s), 129.8 (d), 126.6 (d), 124.1 (d), 123.2
(d), 120.1 (d), 120.1 (d), 118.0 (s), 116.2 (d), 54.1 (t), 42.1 (q),
N,N′-Dip h en yl-N,N′-b is[3-(d im et h yla m in o)p r op yl]-3-
ch lor oben zo[b]th iop h en e-2,6-d ica r boxa m id e (2j): from
1c by method B, brown oily product after purification by
column chromatography (5% ethanol/benzene), yield 35.4%; IR
(NaCl)(νmax/cm-1) 2740-2920 (CH3, CH2), 1635 (CdO); UV
(Methanol)(λmax/nm) 220, 290; 1H NMR (CDCl3)(δ/ppm) 7.68
(s, 1H), 7.43 (d, 1H, J ) 8.46 Hz), 7.20-7.08 (m, 9H), 7.00-
6.98 (m, 2H), 3.99-3.94 (m, 4H), 2.37-2.30 (m, 4H), 2.17 (s,
12H), 1.83-1.79 (m, 4H); 13C NMR (CDCl3)(δ/ppm): 169.3 (s),
162.1 (s), 142.9 (s), 141.1 (s), 136.9 (s), 135.8 (s), 134.3 (s), 132.9
(s), 129.1 (d), 129.1 (d), 129.0 (d), 129.0 (d), 127.6 (d), 127.5
(d), 127.5 (d), 127.3 (d), 127.3 (d), 126.7 (d), 125.2 (d), 123.2
(d), 121.5 (d), 119.9 (s), 56.7 (t), 56.6 (t), 48.6 (t), 48.3 (t), 45.1
(q), 45.1 (q), 45.1 (q), 45.1 (q), 25.5 (t), 25.4 (t); ΜS m/z 577 (M
+ 1). Anal. (C32H37ClN4O2S) C, H, N.
Gen er a l Meth od for th e Syn th esis of [1]Ben zoth ien o-
[2,3-c]qu in olin -6-on es (3a -j). A solution of 6-substituted
3-chlorobenzo[b]thiophene-2-carboxamides (2a -j) (1.6 mmol)
and triethylamine (1.6 mmol) in 9% methanol/toluene (ben-
zene) or dioxane (550 mL) was irradiated with 450 W medium
pressure or 125 or 400 W high-pressure mercury arc lamp
using quartz or Pyrex filter at room temperature for 10 min
up to 2 h. The air was bubbled through the solution. After
evaporation of the solvent, the crude product was purified by
column chromatography (neutral aluminum oxide) or recrys-
tallization. Free base was then converted into the hydrochlo-
ride salt by stirring it with saturated ethanolic HCl.
39.6 (t), 22.7 (t); ΜS m/z 382 (free base M+1). Anal. (C20H20
ClN3O3S) C, H, N.
-
5-[3-(D im e t h y la m in o )p r o p y l]-9-m e t h y l-6-o x o -5,6-
d ih yd r o[1]ben zoth ien o[2,3-c]qu in olin -6-on e h yd r och lo-
r id e (3e): from 2e after 20 min of irradiation with 400 W
high-pressure mercury arc lamp using Pyrex filter in 9%
methanol/toluene. After evaporation of the solvent, the crude
product was purified by recrystallization from toluene:metha-
nol (1:1) and then converted into hydrochloride salt; white
crystals, yield 60.4%; mp 261-263 °C; IR (KBr)(νmax/cm-1
)
6-Oxo-5,6-d ih yd r o[1]b e n zot h ie n o[2,3-c]q u in olin -6-
on e (3a ): from 2a after 20 min of irradiation with 450 W
medium-pressure mercury arc lamp using Pyrex filter in 9%
3444-2479 (CH3, CH2), 1639 (CdO); UV (methanol)(λmax/nm)
204, 234, 256, 308, 342, 358; 1H NMR (DMSO-d6)(δ/ppm) 10.31
(s, 1H), 8.85 (d, 1H, J ) 7.27 Hz), 8.81 (d, 1H, J ) 8.62 Hz),

4522 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Koru et al.
8.05 (s, 1H), 7.88 (d, 1H, J ) 8.40 Hz), 7.72 (dd, 1H, J ) 7.29,
7.42 Hz), 7.54-7.50 (m, 2H), 4.52 (t, 2H, J ) 7.02 Hz), 3.24 (t,
2H, J ) 7.93 Hz), 2.75 (s, 6H), 2.53 (s, 3H), 2.16 (tt, 2H, J )
7.73, 7.44 Hz); 13C NMR (DMSO-d6)(δ/ppm) 157.7 (s), 142.0
(s), 137.9 (s), 137.3 (s), 135.2 (s), 133.0 (s), 131.0 (s), 129.4 (d),
127.8 (d), 125.7 (d), 124.6 (d), 123.8 (d), 123.1 (d), 118.8 (s),
116.1 (d), 54.2 (t), 42.2 (q), 42.2 (q), 39.6 (t), 22.8 (t), 21.2 (q);
ΜS m/z 351 (free base M + 1). Anal. (C21H23ClN2OS) C, H, N.
5-[3-(Dim e t h yla m in o)p r op yl]-9-m e t h oxy-6-oxo-5,6-
d ih yd r o[1]ben zoth ien o[2,3-c]qu in olin -6-on e h yd r och lo-
r id e (3f): from 2f after 20 min of irradiation with 400 W high-
pressure mercury arc lamp using Pyrex filter in 9% methanol/
toluene. After evaporation of the solvent, the crude product
was purified by recrystallization from toluene:methanol (1:1)
and then converted into hydrochloride salt; white crystals,
yield 76.7%; mp 254-255 °C; IR (KBr)(νmax/cm-1) 3439-2681
(CH3, CH2), 1634 (CdO); UV (methanol)(λmax/nm) 205, 234,
256, 164, 324, 336, 353; 1H NMR (DMSO-d6)(δ/ppm) 10.47 (s,
1H), 8.79 (d, 2H, J ) 9.38 Hz), 7.88-7.83 (m, 2H), 7.71 (dd,
1H, J ) 7.51 Hz), 7.50 (dd, 1H, J ) 7.58 Hz), 7.24 (dd, 1H, J
) 8.14, 2.04 Hz), 4.50 (t, 2H, J ) 7.10 Hz), 3.92 (s, 3H), 3.24
(t, 2H, J ) 7.95 Hz), 2.75 (s, 6H), 2.16 (tt, 2H, J ) 7.92, 7.12
Hz); 13C NMR (DMSO-d6)(δ/ppm) 158.9 (s), 157.3 (s), 143.8 (s),
137.1 (s), 135.0 (s), 133.0 (s), 129.2 (d), 128.6 (s), 126.6 (d),
124.3 (d), 122.8 (d), 118.4 (s), 115.9 (d), 115.7 (d), 106.3 (d),
55.6 (q), 53.9 (t), 41.9 (q), 41.9 (q), 39.6 (t), 22.6 (t); ΜS m/z
367 (free base M + 1). Anal. (C21H23ClN2O2S) C, H, N.
130.9 (d), 130.9 (d), 130.4 (d), 129.4 (d), 129.4 (d), 129.2 (d),
125.9 (d), 125.2 (d), 124.6 (d), 124.1 (d), 123.7 (d), 118.4 (s),
116.2 (d), 56.1 (t), 55.6 (t), 48.1 (t), 43.6 (q), 43.6 (q), 43.6 (q),
43.6 (q), 40.6 (t), 23.4 (t), 23.1 (t); ΜS m/z 541 (free base M +
1). Anal. (C32H38Cl2N4O2S) C, H, N.
5-[3-(Dim e t h yla m in o)p r op yl]-6-oxo-5,6-d ih yd r o[1]-
ben zoth ien o[2,3-c]qu in olin e-9-ca r boxa n ilid e Hyd r och lo-
r id e (4). To a cold solution of 3i (2.00 g, 5.4 mmol) in
anhydrous DMF (100 mL) was added sodium hydride (1.00 g,
26.0 mmol) in three portions.42 After the solution was stirred
under nitrogen for 15 min, a solution of 3-(dimethylamino)-
propyl chloride hydrochloride (2.23 g, 14.0 mmol) in DMF (50
mL) was added, and the reaction mixture was stirred for 2 h
at 0 °C and 60 h at room temperature. The solid (sodium
chloride) was separated by filtration, and the solvent was
removed under reduced pressure. The crude product was
purified by recrystallization from chloroform:cyclohexane (1:
2), and free base was then converted into the hydrochloride
salt by stirring it with saturated ethanolic HCl. Resulting
precipitate was filtered off, washed with absolute ethanol, and
dried in vacuo at 50 °C for 5 h to give yellow crystals (1.26 g);
yield 47.4%; mp 173-175 °C; IR (KBr)(νmax/cm-1) 2670-3028
(CH3, CH2), 1629 (CdO); UV (ethanol)(λmax/nm) 234, 264, 282,
310, 350, 368; 1H NMR (DMSO-d6)(δ/ppm) 10.62 (s, 1H), 10.59
(s, 1H), 9.04 (d, 1H, J ) 8.83 Hz), 8.90 (d, 1H, J ) 7.45 Hz),
8.88 (d, 1H, J ) 1.62 Hz), 8.22 (dd, 1H, J ) 8.72, 1.62 Hz),
7.91 (d, 1H, J ) 8.65 Hz), 7.87 (d, 2H, J ) 7.64 Hz), 7.75 (dd,
1H, J ) 7.33, 8.38 Hz), 7.55 (dd, 1H, J ) 7.49, 7.74 Hz), 7.40
(dd, 2H, J ) 7.68, 8.14 Hz), 7.15 (dd, 1H, J ) 7.37, 8.34 Hz),
4.53 (t, 2H, J ) 7.19 Hz), 3.27 (dt, 2H, J ) 5.32 Hz), 2.76 (s,
3H), 2.75 (s, 3H), 2.19 (tt, 2H, J ) 7.66 Hz); 13C NMR (DMSO-
d6)(δ/ppm) 164.8 (s), 157.5 (s), 141.4 (s), 139.2 (s), 137.3 (s),
137.3 (s), 134.7 (s), 133.7 (s), 133.6 (s), 129.7 (d), 128.7 (d),
128.7 (d), 125.9 (d), 125.4 (d), 124.6 (d), 124.0 (d), 123.7 (d),
123.2 (d), 120.6 (d), 120.6 (d), 118.7 (s), 116.2 (d), 56.1 (t), 42.1
(q), 42.1 (q), 39.4 (t), 22.7 (t); ΜS m/z 456 (free base M + 1).
Anal. (C27H26ClN3O2S) C, H, N.
6-Oxo-5,6-d ih ydr o[1]ben zoth ien o[2,3-c]qu in olin e-9-ca r -
boxylic Acid (5). A solution of NaOH (0.60 g, 15.0 mmol) in
water (300 mL) was added to the solution of 3g (1.50 g, 5.0
mmol) in ethanol (250 mL). The resulting solution was refluxed
for 1 h. Ethanol was removed under reduced pressure, and
the residue was dissolved in water and acidified with concen-
trated HCl. The precipitate was filtered off, washed with
water, and recrystallized from DMSO to give 6 (1.35 g) as
white crystals; yield 95.1%; mp > 300 °C (lit.28 mp > 300 °C).
6-Oxo-5,6-d ih ydr o[1]ben zoth ien o[2,3-c]qu in olin e-9-ca r -
bon yl Ch lor id e (6). A mixture of 5 (0.40 g, 1.4 mmol) and
thionyl chloride (10 mL, 137.7 mmol) was refluxed for 5 h.
Excess of thionyl chloride was removed under reduced pressure
to give 7 (0.45 g) as yellow crystals in quantitative yield; mp
> 300 °C (lit.28 mp > 300 °C).
Meth yl 6-oxo-5,6-d ih yd r o[1]ben zoth ien o[2,3-c]qu in o-
lin -9-ca r boxyla te (3g): from 2g after 1 h of irradiation with
450 W medium-pressure mercury arc lamp using quartz filter
in 9% methanol/benzene; white crystals after recrystallization
from DMF, yield 87.0%; mp > 300 °C (lit.28 mp > 310 °C).
Methyl5-[3-(dimethylamino)propyl]-6-oxo-5,6-dihydro[1]-
b en zot h ien o[2,3-c]q u in olin e-9-ca r b oxyla t e h yd r och lo-
r id e (3h ): from 2h after 10 min of irradiation with 450 W
medium-pressure mercury arc lamp using quartz filter in 9%
methanol/benzene. After evaporation of the solvent, the crude
product was purified by recrystallization from ethanol and
then converted into hydrochloride salt; white crystals, yield
78.9%; mp 245-247 °C; IR (KBr)(νmax/cm-1) 2683-2922 (CH3,
CH2), 1729, 1629 (CdO); UV (ethanol)(λmax/nm) 232, 266, 282,
294, 306, 328, 354, 370; 1H NMR (DMSO-d6)(δ/ppm) 10.25 (s,
1H), 9.01 (d, 1H, J ) 8.80 Hz), 8.86 (d, 1H, J ) 1.55 Hz), 8.84
(d, 1H, J ) 8.25 Hz), 8.14 (dd, 1H, J ) 8.73, 1.63 Hz), 7.89 (d,
1H, J ) 8.50 Hz), 7.74 (dd, 1H, J ) 7.80, 7.88, 1.03 Hz), 7.53
(dd, 1H, J ) 7.80, 7.45 Hz), 4.52 (t, 2H, J ) 7.17 Hz), 3.95 (s,
3H), 3.25 (tt, 2H, J ) 5.25 Hz), 2.76 (s, 3H), 2.75 (s, 3H), 2.16
(tt, 2H, J ) 7.59 Hz); 13C NMR (DMSO-d6)(δ/ppm): 165.7 (s),
157.4 (s), 141.5 (s), 138.5 (s), 137.2 (s), 134.5 (s), 134.4 (s), 129.6
(d), 128.1 (s), 126.1 (d), 125.9 (d), 125.6 (d), 124.4 (d), 123.2
(d), 118.5 (s), 116.2 (d), 54.1 (t), 52.5 (q), 42.1 (q), 42.1 (q), 39.3
(t), 22.6 (t); ΜS m/z 395 (free base M + 1). Anal. (C22H23
ClN2O3S) C, H, N.
-
N -[3-(Dim e t h yla m in o)p r op yl]-6-oxo-5,6-d ih yd r o[1]-
ben zoth ien o[2,3-c]qu in olin e-9-ca r boxa m id e Hyd r och lo-
r id e (7a ). A solution of 6 (0.82 g, 2.6 mmol) and [3-(dimeth-
ylamino)propyl]amine (12 mL, 95.4 mmol) in toluene (35 mL)
was refluxed for 30 min. After cooling, the precipitate was
filtered off and washed with 20% HCl and then with water.
Recrystallization from DMSO, followed by conversion into the
hydrochloride salt by stirring it with saturated ethanolic HCl,
gave light brown crystals (0.346 g); yield 62.0%; mp 230-235
°C; IR (KBr)(νmax/cm-1) 2711-2963 (CH3, CH2) 1661 (CdO);
UV (methanol)(λmax/nm) 234, 264, 306, 326, 350, 366; 1H NMR
(DMSO-d6)(δ/ppm) 12.37 (s, 1H), 10.51 (s, 1H), 9.04 (t, 1H, J
) 5.71 Hz), 9.01 (d, 1H, J ) 8.80 Hz), 8.83 (d, 1H, J ) 8.24
Hz), 8.77 (d, 1H, J ) 1.50 Hz), 8.15 (dd, 1H, J ) 8.69, 1.50
Hz), 7.65-7.57 (m, 2H), 7.45-7.42 (m, 1H), 3.43 (dt, 2H, J )
6.46, 6.03 Hz), 3.14 (t, 2H, J ) 7.87 Hz), 2.77 (s, 6H), 2.00 (tt,
2H, J ) 6.77, 7.81 Hz); 13C NMR (DMSO-d6)(δ/ppm) 165.6 (s),
157.6 (s), 141.0 (s), 137.6 (s), 137.2 (s), 135.5 (s), 134.0 (s), 132.8
(s), 129.0 (d), 125.5 (d), 124.7 (d), 123.6 (d), 123.1 (d), 122.7
(d), 117.1 (s), 116.7 (d), 54.4 (t), 41.9 (q), 41.9 (q), 36.5 (t), 24.1
(t); ΜS m/z 380 (free base M + 1). Anal. (C21H22ClN3O2S) C,
H, N.
6-Oxo-5,6-d ih yd r o[1]ben zoth ien o[2,3-c]qu in olin -9-ca r -
boxa n ilid e (3i): from 2i after 2 h of irradiation with 450 W
medium-pressure mercury arc lamp using quartz filter in
dioxane; yellow crystals after recrystallization from DMF:
water (1:2), yield 85.7%; mp > 300 °C (lit.29 mp > 300 °C).
N,N′-Bis[3-(dim eth ylam in o)pr opyl]-6-oxo-5,6-dih ydr o[1]-
b en zot h ien o[2,3-c]q u in olin e-9-ca r b oxa n ilid e d ih yd r o-
ch lor id e (3j): from 2j after 2 h of irradiation with 400 W
high-pressure mercury arc lamp using Pyrex filter in 9%
methanol/benzene. After evaporation of the solvent, the crude
product was purified by column chromatography (10% ethanol/
benzene) and then converted into dihydrochloride salt; white
crystals, yield 28.7%; mp 238-242 °C; IR (KBr)(νmax/cm-1
)
2682-2957 (CH3, CH2), 1642, 1634 (CdO); UV (methanol)(λmax
/
nm) 232, 260, 308, 348, 364; 1H NMR (D2O)(δ/ppm) 7.33-7.18
(m, 7H), 7.01 (d, 1H, J ) 8.51 Hz), 6.66-6.61 (m, 2H), 6.34-
6.31 (m, 2H), 4.06 (t, 2H, J ) 7.57 Hz), 3.60 (t, 2H, J ) 7.57
Hz), 3.22 (t, 2H, J ) 7.48 Hz), 3.07 (t, 2H, J ) 7.48 Hz), 2.87
(s, 6H), 2.79 (s, 6H), 2.05 (tt, 2H, J ) 7.38 Hz), 1.80 (tt, 2H, J
) 7.38 Hz); 13C NMR (D2O)(δ/ppm) 172.5 (s), 158.3 (s), 142.8
(s), 142.0 (s), 136.3 (s), 135.5 (s), 135.0 (s), 134.7 (s), 131.5 (s),

Antitumor Benzo[b]thieno[2,3-c]quinolones
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4523
N -[3-(Dim e t h yla m in o)p r op yl]-6-oxo-5,6-d ih yd r o[1]-
ben zoth ien o[2,3-c]qu in olin e-9-ca r boxa n ilid e Hyd r och lo-
r id e (7b). The chloride 6 was converted into free base of 7b
by the method B described earlier for the synthesis of
6-substituted 3-chlorobenzo[b]thiophene-2-carboxamides 2a -
j. Free base was then converted into the hydrochloride salt by
stirring it with saturated ethanolic HCl. Resulting precipitate
was filtered off, washed with absolute ethanol, and dried in
vacuo at 50 °C for 5 h to give light yellow crystals; yield 47.7%;
mp 235-240 °C; IR (KBr)(νmax/cm-1) 2865-3123 (CH3, CH2),
1660, 1651 (CdO); UV (ethanol)(λmax/nm) 230, 260, 304, 350,
in 20 µL of staining solution and incubated for 15 min at
ambient temperature. The evaluation was performed by
fluorescence microscopy. The results are expressed as a ratio
between apoptotic (green) and total number of the cells.
Ack n ow led gm en t. Support for this study by the
Ministry of Science (Project No 0125005 and 00981104)
and Ministry of small and medium Enterprises of
Croatia is gratefully acknowledged.
Refer en ces
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364; H NMR (DMSO-d6)(δ/ppm) 12.35 (s, 1H), 10.98 (s, 1H),
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9.02 (d, 1H, J ) 8.97 Hz), 8.85 (d, 1H, J ) 1.43 Hz), 8.77 (d,
1H, J ) 8.14 Hz), 8.17 (dd, 1H, J ) 8.75, 1.65 Hz), 7.65-7.15
(m, 8H), 4.00 (t, 2H, J ) 6.83 Hz), 3.20 (dt, 2H, J ) 5.99 Hz),
2.75 (s, 3H), 2.73 (s, 3H), 2.01 (tt, 2H, J ) 5.17 Hz); 13C NMR
(DMSO-d6)(δ/ppm) 168.9 (s), 157.7 (s), 142.5 (s), 140.6 (s), 137.7
(s), 135.8 (s), 135.6 (s), 135.3 (s), 133.6 (s), 129.4 (d), 129.4 (d),
129.1 (d), 128.2 (d), 128.2 (d), 127.1 (d), 125.6 (d), 125.0 (d),
124.3 (d), 123.6 (d), 122.8 (d), 117.2 (s), 116.8 (d), 55.0 (t), 47.4
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Anal. (C27H26ClN3O2S) C, H, N.
Cell Lin es a n d Cu ltu r in g. Human tumor cell lines (HeLa,
cervical carcinoma; MCF-7, breast carcinoma; CaCo-2, colon
carcinoma; MIA PaCa-2, pancreatic carcinoma; HBL, ME 67.3
and ME 67.1, melanoma; Hep-2, laryngeal carcinoma) and
normal human fibroblasts (WI-38) were tested for sensitivity
in vitro. All cell lines were grown in DMEM medium (supple-
mented with 10% heat inactivated fetal bovine serum, 2 mM
L-glutamine, 100 U/mL penicillin, and 100 µg/mL strepto-
mycin) at 37 °C in a humidified atmosphere with 5% CO2. For
the purpose of the experiment, the cells were plated in
quadruplicates in 96-microwell flat bottom plates at 2 × 104
cells/mL (all tumor cell lines) and 3 × 104 cells/mL (WI-38).
The next day (24 h later) compounds were added to the cells
at different concentrations (5 × 10-4 M, 10-4 M, and 10-5 M).
Compounds were dissolved in DMSO at a concentration of 10-1
M and diluted with DMEM medium into working concentra-
tions. The concentration of DMSO was too small to affect the
growth. Control cells (without any compound) were growing
under the same conditions.
Cell viability was measured immediately before (day 0) and
72 h after addition of compounds, using MTT assay, which
detects dehydrogenase activity in viable cells.43,44 For this
purpose the medium was discarded and 40 µL of MTT (1%)
was added to each well. After 4 h of incubation at 37 °C, the
precipitates were dissolved in 160 µL of DMSO. The absor-
bance was measured on an ELISA reader at 570 nm, and the
percentage of growth was calculated. Each number was the
mean of three individual experiments done in quadruplicate.
The cytotoxic effects of each compound were obtained as IC50
values, which represents the molar drug concentrations re-
quired to cause 50% inhibition.
Isola tion of DNA-Ap op totic F r a gm en ts. Melanoma
cells (HBL) were treated with compound 4, at concentration
of 5 × 10-6 M and 10-5 M for 24 h. The apoptotic fragments of
DNA were isolated according to a method described by
Herrmann et al.45
F low Cytom etr y. Melanoma cells (HBL) were grown under
confluence and treated with compound 4 (10-5 M) for 5 h.
Fixation and DNA staining for cell cycle analysis was per-
formed (ICBR Flow Cytometry Core Laboratory, http://
www.biotech.ufl.edu). PI stained cells were analyzed by FACS
CALIBUR (Becton Dickinson), using ModFitLT V2.0 (PMac)
software.
Detection a n d Qu a n tifica tion of Ap op tosis by An -
n exin -V-Sta in in g. Melanoma cells (HBL) were plated at 2
× 105 cells/mL in a multiwell plate (2 mL). After 48 h, the
cells were treated with 10-6 M, 5 × 10-6 M, and 10-5 M of
compound 4 5 h. Staining with Annexin-V-FLUOS (Staining
kit, Roche Diagnostics GmbH, Mannheim, Germany) was
performed according to the manufacture procedure, with slight
modification. The cells were collected, washed with PBS, and
centrifuged at 200g for 5 min. The cell pellets were suspended
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