Synthesis and in vitro antiproliferative activity of novel (4-chloro- and 4-acyloxy-2-butynyl)thioquinolines

Article abstract of DOI:10.1007/s00044-010-9495-y

The series of new acetylenic thioquinolines containing propargyl, 4-chloro-2-butynyl, and 4-acyloxy-2- butynyl groups have been prepared and tested for antiproliferative activity in vitro against human [SW707 (colorectal adenocarcinoma), CCRF/CEM (leukemia), T47D (breast cancer)] and murine [P388 (leukemia), B16 (melanoma)] cancer lines. Most of the obtained compounds exhibited antiproliferative activity, especially compounds 8, 12, and 21 showed the ID50 values ranging from 0.4 to 3.8 μg/ml comparable to that of cisplatin used as reference compounds. The Author(s) 2010.

Full text of DOI:10.1007/s00044-010-9495-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1402–1410
DOI 10.1007/s00044-010-9495-y
ORIGINAL RESEARCH
Synthesis and in vitro antiproliferative activity of novel
(4-chloro- and 4-acyloxy-2-butynyl)thioquinolines
•
Stanisław Boryczka Wojciech Mol
•
´
•
•
Magdalena Milczarek Joanna Wietrzyk
Ewa Be˛benek
Received: 31 December 2009 / Accepted: 29 October 2010 / Published online: 17 November 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract The series of new acetylenic thioquinolines
containing propargyl, 4-chloro-2-butynyl, and 4-acyloxy-2-
butynyl groups have been prepared and tested for anti-
proliferative activity in vitro against human [SW707
(colorectal adenocarcinoma), CCRF/CEM (leukemia),
T47D (breast cancer)] and murine [P388 (leukemia), B16
(melanoma)] cancer lines. Most of the obtained compounds
exhibited antiproliferative activity, especially compounds
8, 12, and 21 showed the ID50 values ranging from 0.4 to
3.8 lg/ml comparable to that of cisplatin used as reference
compounds.
compounds (Ben-Zvi and Danon, 1994). Among a large
group of synthetic and natural acetylenic compounds the
quinolines possessing an alkynyl moieties are of particular
interest as many of them display important activities,
namely antimicrobiological, anticancer, antiprotozoal, and
antiretroviral (Fuita et al., 1998; Fakhfakh et al., 2003;
Abele et al., 2002). Several acetylenic 2,5-disubstituted
decahydroquinoline alkaloids of wide spectra biological
activities have been isolated from the skins of frogs, toads
and related amphibians (Spande et al., 1999; Michael,
2000). It should be noted that less information is available
concerning the synthesis and biological evaluation of
alkynylthioquinolines (Abele et al., 2002; Makisumi and
Murabayashi, 1969; Boryczka, 1999). It is noteworthy that
no data about the synthesis and cytotoxic activity of
quinolines containing a selenoacetylenic substituent are
available. The chemical and physical properties of sele-
nium are very similar to those of sulfur but the biochem-
istry differs in at least two respects that distinguish them in
biological systems (Aboul-Faddl, 2005). First, in biological
systems selenium compounds are metabolized to more
reduced states, whereas sulfur compounds are metabolized
to more oxidized states; second, selenols are more acidic
than thiols, and they are readily oxidized. In general,
organoselenium compounds are more reactive than their
sulfur analogs due to weaker C–Se bond than the C–S
bond. These properties can be involved in higher activity of
the Se compounds against cancer cells than S derivatives
(Aboul-Faddl, 2005).
Keywords Acetylenic thioquinolines ꢀ
(4-Chloro-2-butynyl)thioquinolines ꢀ
(4-Acyloxy-2-butynyl)thioquinolines ꢀ
Antiproliferative activity
Introduction
The carbon–carbon triple bond is one of the most important
functional groups in organic chemistry and pharmacology.
The structure activity relationship studies suggest that
introduction of alkyne motif may significantly modify the
chemical, physical, and biological properties of acetylenic
´
S. Boryczka (&) ꢀ W. Mol ꢀ E. Be˛benek
Department of Organic Chemistry, Medical University
of Silesia, Sosnowiec PL-41-200, Poland
e-mail: boryczka@sum.edu.pl
The synthetic methods for preparation of acetylenic
compounds are of interest especially with regard to the
synthesis of enediyne antitumor antibiotics or similar
molecules (Nicolaou and Dai, 1991; Grissom et al., 1996;
Joshi et al., 2007; Kumar et al., 2001). Several cyclic and
acyclic models have recently been developed, some of
M. Milczarek ꢀ J. Wietrzyk
Department of Experimental Oncology, Ludwik Hirszfeld
Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, PL-53-114 Wrocław, Poland
123

Med Chem Res (2011) 20:1402–1410
1403
them including pyridine and quinoline units (Rawat et al.,
2001; Knoll et al., 1988; Bhattacharyya et al., 2006).
We have reported a simple and efficient method for the
synthesis of 3,4-disubstituted thioquinolines, which possess
one or two the same or different O, S, Se acetylenic groups.
The new acetylenic thioquinolines exhibited antiprolifera-
tive activity in vitro against a broad panel of human and
murine cancer cell lines comparable to cisplatin (Boryczka
antiproliferative activity, new compounds bearing 4-acyl-
oxy-2-butynyl groups were prepared.
The synthesis of acetylenic thioquinolines 16–25 (Scheme 3)
was accomplished starting with 4-chloro-3-(4-hydroxy-2-
butynylthio)quinoline 5 or 4-(4-hydroxy-2-butynylthio)-3-
propargylthioquinoline13 or 4-(4-hydroxy-2-butynylseleno)-
3-methylthioquinoline 14 or 4-(4-hydroxy-2-butynylthio)-3-
methylthioquinoline 15 which were prepared according to our
´
et al., 2002a, 2002b; Mol et al., 2006, 2008). The struc-
´
previously reported methods (Mol et al., 2008).
ture–activity relationships study show a significant corre-
lation between the antiproliferative activity and the
electronic properties expressed as 13C NMR chemical shift,
lipophilicity, and molecular electrostatic potential (Bor-
yczka et al., 2002b, 2003; Bajda et al., 2007; Boryczka
et al., 2010).
The compounds 5 and 13–15 were converted into esters
16–25 with 42–91% yields by reactions with acylating
agents such as: o-phthalic anhydride, cinnamoyl chloride,
and benzoyl chloride or ethyl chloroformate in dry benzene
in the presence of pyridine.
The crude products were isolated from aqueous sodium
hydroxide by filtration or extraction and separated by col-
umn chromatography.
It is well known that several acetylenic compounds
possessing 2-butynyl motif exhibit specific pharmacologi-
cal activities, although the exact role of the 2-butynyl motif
in the activity of these derivatives is not fully understood
(Ben-Zvi and Danon, 1994).
Antiproliferative activity
As an extension of our work on the development of anti-
cancer drugs, we synthesized derivatives 6–12 and 16–25
with the aim to obtain more information about the influence
of 4-chloro-2-butynyl and 4-acyloxy-2-butynyl groups on
antiproliferative activity in this class of compounds.
The seventeen compounds were tested in SRB or MTT (in
the case of leukemia cells) assay for their antiproliferative
activity in vitro against three human cancer cell lines:
SW707 (colorectal adenocarcinoma), CCRF/CEM (leuke-
mia), T47D (breast cancer) and two murine cancer cell lines:
P388 (leukemia), B16 (melanoma). The results of cytotoxic
activity in vitro were expressed as an ID50 (lg/ml), i.e., the
concentration of compound, which inhibits the proliferation
of 50% of tumor cells as compared to the control untreated
cells. Cisplatin was applied as a referential cytotoxic agent
(positive test control). A value of less than 4 lg/ml was
considered as an antiproliferative activity criterion for syn-
thetic compounds. The results of the cytotoxicity studies are
summarized in Table 1, previously reported data for com-
pounds 4-chloro-3-(4-hydroxy-2-butynylthio)-quinoline 5,
4-(4-hydroxy-2-butynylseleno)-3-methyl-thioquinoline 14
and 4-(4-hydroxy-2-butynylthio)-3-methylthioquinoline 15
Results and discussion
Chemistry
The synthesis of acetylenic thioquinolines 7–12 (Scheme 2)
was accomplished starting with 4-chloro-3-methylthioqui-
noline 3 or 4-chloro-3-propargyl-thioquinoline 4 or 4-chloro-
3-(4-hydroxy-2-butynylthio)quinoline 5.
Compounds 3–5 were prepared according to our previ-
´
ously reported methods (Boryczka et al., 2002b; Mol et al.,
´
2008; Maslankiewicz and Boryczka, 1993). 4-Chloro-
´
were included for comparison (Mol et al., 2008).
quinoline 6 was synthesized as shown in Scheme 1. The
starting 1 was prepared according to our published proce-
´
dure (Maslankiewicz and Boryczka, 1993). Treatment of 1
In general all the compounds 6–12 containing 4-chloro-
2-butynyl substituent exhibited a potent antiproliferative
activity against human and murine cancer lines applied.
4-Chloro-3-(4-chloro-2-butynylthio)quinoline 6 exhibited
high activity against SW707, CCRF/CEM, T47D, B16 and
moderate activity against P388. As reported previously
4-chloro-3-(4-hydroxy-2-butynylthio)quinoline 5 possessed
lower cytotoxic activity than 6 except activity against the
with sodium methoxide in DMSO at 25°C gave sodium
4-chloro-3-quinolinethiolate 1-A and 4-methoxy-3-methyl-
thioquinoline 2, which was removed by extraction. Sodium
salt 1-A after S alkylation using 1-bromo-4-chloro-2-
butyne gave 6 in 65% yield.
Compounds 3–5 were converted into 7–12 in 43–86%
yields by nucleophilic displacement of chlorine atom by
thiourea or selenourea in ethanol, hydrolysis of uronium
salt 3-A and subsequent S or Se alkylation of sodium salt
3-B with 1-bromo-4-chloro-2-butyne (Scheme 2).
´
cells of P388 leukemia (Mol et al., 2008).
In the series of derivatives 7–12, the replacement of
methyl group by propargyl or 4-hydroxy-2-butynyl, com-
pounds 9, 10 and 11, 12, respectively, resulted in decrease
of activity. Among compounds 7–12, the selenium deriv-
atives were more active than sulfur analogs and the
In order to determine whether a acyloxy substituent at
C-4 of 2-butynyl group has any significant influence on the
123

1404
Med Chem Res (2011) 20:1402–1410
Scheme 1 Synthesis of
4-chloro-3-(4-chloro-2-
Cl
N
Cl
N
OMe
butynylthio)quinoline 6.
Reagents and conditions:
a MeONa, DMSO, 25°C,
30 min; b 1-bromo-4-chloro-2-
butyne, NaOHaq, 25°C, 30 min
a
S
N
SNa
SMe
+
N
SMe
1
2
1-A
b
Cl
N
SCH2C CCH2Cl
6
+
than two other cancer cells lines applied with ID50 value of
less than 4 lg/ml, which is considered as an antiprolifer-
ative activity criterion. It is important to note that the
compounds 7–12 exhibited higher cytotoxic activity
against breast cancer (T47D) cells than cisplatin.
NH2
-
Cl
Cl
X C NH2
SR
a
SR
N
The replacement of hydroxy group in 5 by hydroph-
thaloyloxy or cinnamoyloxy groups, compounds 16 and 17,
resulted in decrease of activity.
N
3-6
3-A
3. R = CH3
4. R = CH2C
5. R = CH2C
The substitution of hydroxy group in 4-(4-hydroxy-2-
butynylseleno)-3-methylthioquinoline 14 by hydrophtha-
loyloxy, benzoyloxy, and cinnamoyloxy, compounds 19,
21, and 24, respectively, resulted in decrease of activity
except activity against the cells of B16 melanoma. A
structure–activity relationships observed in compounds 19,
21, and 24 indicated that the rank order of cytotoxic
activity, against all cancer lines applied, according to the
nature of the acyloxy substituent is as follows:
benzoyloxy [ hydrophthaloyloxy [ cinnamoyloxy.
CH
CCH2OH
b
XR'
XNa
SR
N
c
SR
N
3-B
7-12
CCH2Cl, X = S
CCH2Cl, X = Se
7. R = CH3,
8. R = CH3,
R' = CH2C
R' = CH2C
The replacement of hydroxy group in 4-(4-hydroxy-2-
butynylthio)-3-methylthioquinoline 15 by hydrophthaloyl-
oxy, cinnamoyloxy, benzoyloxy, or ethoxycarbonyloxy,
compounds 18, 20, 22, and 23, also resulted in the decrease
of activity. Comparing of compounds 18, 20, 22, and 23
indicated that the cytotoxic activity against SW707, CCRF/
CEM, T47D, and P388 were in the order ethoxycarbonyl-
oxy [ hydrophthaloyloxy [ cinnamoyloxy [ benzoyloxy.
Whereas the activity of these compounds against B16 was
as follows: ethoxycarbonyloxy [ cinnamoyloxy [ ben-
zoyloxy [ hydrophthaloyloxy. It is interesting to note that
the acyloxy compounds 16–25, prepared in this study,
exhibited the most potent cytotoxicity against cancer cell
B16 melanoma. These results may suggest that 4-acyloxy-
2-butynyl function is important for anti-melanoma activity.
CH,
CH,
R' = CH2C CCH2Cl, X = S
R' = CH2C CCH2Cl, X = Se
9. R = CH2C
10. R = CH2C
11. R = CH2C
12. R = CH2C
CCH2OH, R' = CH2C CCH2Cl, X = S
CCH2OH, R' = CH2C CCH2Cl, X = Se
Scheme 2 Synthesis of acetylenic thioquinolines 7–12. Reagents and
conditions: a CS(NH2)2 or CSe(NH2)2, EtOH, 25°C, 1 h; b NaOHaq,
c 1-bromo-4-chloro-2-butyne, NaOHaq, 25°C, 30 min
selenium compound 8 showed the most potent activity with
the ID50 values in the range 0.4–3.5 lg/ml against all
cancer lines applied.
Another noteworthy feature of the obtained compounds
results was the observation that leukemia (CCRF/CEM and
P388) and breast cancer (T47D) cells appear to be more
sensitive to the cytotoxic effects of the compounds 7–12
123

Med Chem Res (2011) 20:1402–1410
1405
Scheme 3 Synthesis of
Cl
N
Cl
N
acetylenic thioquinolines 16–25.
Reagents and conditions:
a o-phthalic anhydride or
cinnamoyl chloride, pyridine,
benzene, 70°C, 1 h; b o-phthalic
anhydride or cinnamoyl
SCH2C CCH2OR'
SCH2C CCH2OH
a
16-17
16. R' = COC6H4COOH
CHC6H5
5
chloride or benzoyl chloride or
ethyl chloroformate, pyridine,
benzene, 70°C, 1 h
17. R' = COCH
XCH2C CCH2OR'
XCH2C CCH2OH
SR
SR
b
N
N
18-25
13-15
CH, X = S
X = Se
X = S
13. R = CH2C
14. R = CH3,
15. R = CH3,
18. R = CH3,
19. R = CH3,
20. R = CH3,
21. R = CH3,
R' = COC6H4COOH, X = S
R' = COC6H4COOH, X = Se
R' = COC6H5,
R' = COC6H5,
R = COOC2H5,
R' = COCH
R' = COCH
X = S
X = Se
X = S
22. R = CH3,
CHC6H5, X =S
23. R = CH3,
24.
CHC6H5, X = Se
R = CH3,
CH, R' = COCH CHC6H5, X = S
25. R = CH2C
Another noteworthy feature of the obtained results was the
observation that acyloxy compounds 19, 21, and 24
exhibited the most potent cytotoxicity with ID50 values
\3.1 lg/ml against B16 cancer cell line, among all the
compounds (5–25) prepared in this study. The replacement
of methyl group by propargyl, compounds 23 and 25,
respectively, resulted in decrease of activity.
compounds. Among the prepared compounds, 8, 12, and 21
were found to be the most active, with ID50 values ranging
from 0.4 to 3.8 lg/ml comparable to that of referential
anticancer drug, cisplatin. It is of interest to note that the
4-acyloxy-2-butynyl function is important for anti-mela-
noma activity. The obtained compounds seem to be good
candidate for further anticancer activity studies in vitro
using a broad panel of human and murine cell lines with the
aim to select compounds for studies in vivo.
Conclusions
Novel acetylenic thioquinolines 6–12 and 16–25, possess-
ing in positions 3 and 4, one or two, propargyl, 4-chloro-2-
butynyl, or 4-acyloxy-2-butynyl groups were synthesized
in good yields using 4-chloro-quinoline derivatives 3–5 and
4-hydroxy-2-butynyl derivatives 13–15 as starting mate-
rial. The obtained compounds were evaluated for antipro-
liferative activity in vitro against three human cancer cell
lines: SW707 (colorectal cancer), CCRF/CEM (leukemia),
T47D (breast cancer) and two murine cancer cell lines:
P388 (leukemia), B16 (melanoma). All the tested com-
pounds showed varied activity against different cancer cell
lines. As a result of the SAR, it was revealed that the nature
of the acetylenic substituent at the C-3 and C-4 positions
and character of the heteroatoms (Se and S) at C-4 criti-
cally influence the anticancer activity in vitro of the studied
Experimental
General techniques
Melting points were determined in open capillary tubes on
1
a Boetius melting point apparatus and are uncorrected. H
NMR (300 MHz) spectra were recorded on a Bruker MSL
300 spectrometer in CDCl3 solvents with tetramethylsilane
as internal standard; chemical shifts are reported in ppm (d)
and J values in Hz. Multiplicity is designated as singlet (s),
doublet (d), triplet (t), quartet (q), and multiplet (m). Mass
spectra were recorded under ?CI conditions on Finnigan
MAT 95 using isobutane as a reagent and temperature of
ion source of 200°C. Elemental C, H, and N analyses were
obtained on a Carlo Erba Model 1108 analyzer. TLC was
123

1406
Med Chem Res (2011) 20:1402–1410
Table 1 Structures of acetylenic thioquinolines 5–12, 14–25 and their antiproliferative activity in vitro and referential cisplatin against the cells
of human and murine cancer cell lines
Compound
Antiproliferative activity ID50 [µg/ml]
Human
Murine
SW707
6.2 1.1
CCRF/CEM
T47D
-
P388
B16
Neg
Cl
N
SCH2C
CCH2OH
CCH2Cl
5*
6
4.1 0.3
3.7 1.5
2.4 0.0
Cl
SCH2C
XR'
3.3 0.3
3.2 2.0
13.4 12.2
4.0 0.5
N
SR
N
R
R’
CCH2Cl
X
S
CH3
CH2C
3.7 0.3
3.2 0.2
2.5 0.4
1.7 0.8
2.9 0.3
1.8 1.5
3.3 1.1
2.6 2.2
0.5 0.3
2.3 0.7
3.9 0.6
0.7 0.4
5.8 1.3
0.4 0.1
5.3 0.7
1.7 1.4
-
2.7 0.8
0.4 0.3
3.0 1.1
0.8 0.7
5.1 3.1
2.0 1.7
2.6 1.1
3.1 0.2
16.9 16.4
3.5 0.1
7
8
CH3
CH2C
CH2C
CH2C
CH2C
CH2C
CH2C
CH2C
CCH2Cl
CCH2Cl
CCH2Cl
CCH2Cl
CCH2Cl
CCH2OH
CCH2OH
Se
S
CH2C
CH2C
CH2C
CH2C
CH3
CH
12.9 8.2
3.2 0.2
52.8 3.8
7.1 3.1
9
CH
Se
S
10
11
12
14*
15*
CCH2OH
CCH2OH
19.8 15.1
3.3 0.7
43.4 12.0
3.8 0.2
Se
Se
S
3.4 0.1
4.2 0.6
CH3
34.9 7.6
-
49.8 0.5
Cl
N
SCH2C CCH2OR'
R’
COC6H4COOH
Neg
Neg
Neg
Neg
53.8 21.6
11.4 9.7
16
17
48.8 4.6
15.4 9.9
22.8 22.3 32.8 23.4
COCH
CHC6H5
XCH2C CCH2OR'
SR
N
R
R’
X
S
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2C
COC6H4COOH
28.1 14.1
3.7 0.5
Neg
14.5 2.3
4.0 0.7
36.1 6.9
3.2 1.4
Neg
35.0 2.0
19.1 11.9
Neg
18.1 10.4
3.1 0.9
17.8 5.4
2.1 2.1
5.5 3.8
9.4 8.2
3.1 1.3
14.7 16.5
4.7 0.2
18
19
COC6H4COOH
COC6H5
Se
S
20
22.2 9.7
1.9 1.4
COC6H5
Se
S
3.6 0.1
33.1 2.6
66.6 2.7
4.7 0.5
Neg
2.2 1.1
34.2 6.4
71.9 16.7
4.3 0.9
Neg
3.5 0.2
34.7 2.2
Neg
21
COOC2H5
13.2 10.3
18.1 9.1
17.8 17.4
50.9 35.1
1.5 0.2
22
23
COCH
COCH
COCH
CHC6H5
S
24
CHC6H5
CHC6H5
Se
S
43.9 18.9
Neg
CH
25
Cisplatin
6.0 0.3
6.7 1.3
1.7 0.2
´
Neg Negative in the concentration used; * See ref. Mol et al., 2008
123

Med Chem Res (2011) 20:1402–1410
1407
performed on silica gel 60 254F plates (Merck) using a
mixture of chloroform and ethanol (15:1, v/v) as an eluent.
UV light and iodine accomplished visualization. Column
chromatography was performed on silica gel 60, \63 lm
(Merck) using a mixture of chloroform and ethanol (30:1,
v/v) as an eluent. Solvents were dried and purified
according to literature procedures.
was poured into 20 ml of 5% aqueous sodium hydroxide.
1-Bromo-4-chloro-2-butyne (0.38 g, 2.3 mmol) was added
dropwise to the aqueous layer, and the mixture was stirred
for 15 min. The resultant solid was filtered off, washed
with water and air-dried to give crude products 7–12 which
were separated by column chromatography using chloro-
form/ethanol (30:1) to give pure products 7–12.
Chemistry
4-(4-Chloro-2-butynylthio)-3-methylthioquinoline (7) Yield
1
86%. Mp: 69–70°C. H NMR (CDCl3, 300 MHz) d: 2.68
The starting compounds: 4-chloro-30-methylthio-3,40-di-
(s, 3H, SCH3), 3.73 (t, J = 2.1 Hz, 2H, CH2), 3.86 (t,
J = 2.1 Hz, 2H, CH2), 7.62–7.72 (m, 2H, H-6 and H-7),
8.11–8.59 (m, 2H, H-5 and H-8), 8.79 (s, 1H, H-2). CI MS m/
z (rel. intensity) 294 (M ? H?, 100), 258 (40). Anal. Calc.
for C14H12ClNS2: C 57.23, H 4.12, N 4.77. Found: C 57.44,
H 4.05, N 4.80.
´
quinolinyl sulfide 1 (Maslankiewicz and Boryczka, 1993),
´
4-chloro-3-(methylthio)quinoline 3 (Maslankiewicz and
´
Boryczka, 1993), 4-chloro-3-propargylthioquinoline 4 (Mol
et al., 2006), 4-chloro-3-(4-hydroxy-2-butynylthio)quino-
´
line 5 (Mol et al., 2008), 1-bromo-4-chloro-2-butyne (Bailey
and Fujiwara, 1955) were obtained according to methods
described previously.
4-(4-Chloro-2-butynylseleno)-3-methylthioquinoline
(8)
1
Yield 56%. Mp: 67–68°C. H NMR (CDCl3, 300 MHz) d:
2.67 (s, 3H, SCH3), 3.62 (t, J = 2.4 Hz, 2H, CH2), 3.88 (t,
J = 2.4 Hz, 2H, CH2), 7.61–7.70 (m, 2H, H-6 and H-7),
8.14–8.52 (m, 2H, H-5 and H-8), 8.76 (s, 1H, H-2). CI MS
m/z (rel. intensity) 342 (M ? H?, 100), 306 (35). Anal.
Calc. for C14H12ClNSSe: C 49.35, H 3.55, N 4.11. Found:
C 49.47, H 3.38, N 4.20.
Synthesis of 4-chloro-3-(4-chloro-2-butynylthio)
quinoline 6
A mixture of 4-chloro-30-methylthio-3,40-diquinolinyl sul-
fide 1 (0.74 g, 2 mmol) and sodium methoxide (0.32 g,
6 mmol) in 8 ml DMSO was stirred at room temperature
for 30 min. The reaction mixture was poured into 20 ml of
5% aqueous sodium hydroxide and extracted with
4 9 5 ml of chloroform. The combined extracts were
washed with water, dried with anhydrous magnesium sul-
fate, and evaporated to give crude 2. To the water layer
1-bromo-4-chloro-2-butyne (0.33 g, 2 mmol) was added
and stirred for 30 min. The mixture was extracted with
4 9 5 ml of chloroform. The combined organic layer was
washed with water and dried with anhydrous magnesium
sulfate. After removal of the solvent the residue was
purified by column chromatography using chloroform/
ethanol (30:1) to give 0.37 g (65%) pure product 6: mp:
4-(4-Chloro-2-butynylthio)-3-(propargylthio)quinoline (9)
Yield 63%. Mp: 109–110°C. 1H NMR (CDCl3, 300 MHz) d:
2.28 (t, J = 2.7 Hz, 1H, CH), 3.74 (t, J = 2,4 Hz, 2H, CH2),
3.84 (d, J = 2.7 Hz, 2H, CH2S), 3.88 (t, J = 2.4 Hz, 2H,
CH2), 7.65–7.72 (m, 2H, H-6 and H-7), 8.10–8.59 (m, 2H, H-
5 and H-8), 9.01 (s, 1H, H-2). CI MS m/z (rel. intensity) 318
(M ? H?, 100), 282 (20), 232 (15). Anal. Calc. for
C16H12ClNS2: C 60.46, H 3.81, N 4.41. Found: C 60.67, H
3.90, N 4.30.
1
4-(4-Chloro-2-butynylseleno)-3-(propargylthio)quinoline
(10) Yield 77%. Mp: 92–93°C. 1H NMR (CDCl3,
300 MHz) d: 2.28 (t, J = 2.7 Hz, 1H, CH), 3.63 (t,
J = 2.4 Hz, 2H, CH2), 3.82 (d, J = 2.7 Hz, 2H, CH2S),
3.89 (t, J = 2.4 Hz, 2H, CH2), 7.66–7.72 (m, 2H, H-6 and
H-7), 8.07–8.53 (m, 2H, H-5 and H-8), 8.99 (s, 1H, H-2).
CI MS m/z (rel. intensity) 366 (M ? H?, 100), 326 (20).
Anal. Calc. for C16H12ClNSSe: C 52.69, H 3.32, N 3.84.
Found: C 52.77, H 3.40, N 3.68.
139–140°C, H NMR (CDCl3) d: 3.82 (t, J = 2.4 Hz, 2H,
SCH2), 4.06 (t, J = 2.4 Hz, 2H, CH2Cl), 7.67–7.80 (m, 2H,
H-6 and H-7), 8.10–8.27 (m, 2H, H-5 and H-8), 8.98 (s, 1H,
H-2). CI MS m/z (rel. intensity) 286 (M ? 4, 10), 284
(M ? 2, 65), 282 (M, 100). Anal. Calc. for C13H9Cl2NS: C
55.33, H 3.21, N 4.96. Found: C 55.50, H 3.11, N 5.08.
General procedure for the synthesis of acetylenic
thioquinolines 7–12
4-(4-Chloro-2-butynylthio)-3-(4-hydroxy-2-butynylthio)
quinoline (11) Yield 58%. Mp: 103–104°C. 1H NMR
(CDCl3, 300 MHz) d: 3.75 (t, J = 2.1 Hz, 2H, CH2),
3.87–3.89 (m, 4H, 29 CH2), 4.24 (t, J = 2.1 Hz, 2H,
CH2), 7.66–7.74 (m, 2H, H-6 and H-7), 8.10–8.58 (m, 2H,
H-5 and H-8), 9.02 (s, 1H, H-2). CI MS m/z (rel. intensity)
A mixture of 4-chloro-3-methylthioquinoline 3 (0.42 g,
2 mmol) or 4-chloro-3-propargylthioquinoline 4 (0.45 g,
2 mmol) or 4-chloro-3-(4-hydroxy-2-butynylthio)quinoline
5 (0.50 g, 2 mmol) and selenourea (0.26 g, 2.1 mmol) or
thiourea (0.16 g, 2.1 mmol) in 99.8% ethanol (8 ml) was
stirred at room temperature for 1 h. The reaction mixture
123

1408
Med Chem Res (2011) 20:1402–1410
348 (M ? H?, 40), 362 (55), 244 (100). Anal. Calc. for
C17H14ClNOS2: C 58.70, H 4.06, N 4.03. Found: C 58.62,
H 4.15, N 3.86.
C6H4 and H-6 and H-7), 8.01–8.48 (m, 2H, H-5 and H-8),
8.85 (s, 1H, H-2). CI MS m/z (rel. intensity) 424 (M ? H?,
10), 276 (100). Anal. Calc. for C22H17NO4S2: C 62.39, H
4.05, N 3.31. Found: C 62.55, H 4.10, N 3.22.
4-(4-Chloro-2-butynylseleno)-3-(4-hydroxy-2-butynylthio)
quinoline (12) Yield 43%. Mp: 99–100°C. 1H NMR
(CDCl3, 300 MHz) d: 3.64 (t, J = 2.4 Hz, 2H, CH2),
3.86–3.89 (m, 4H, 29 CH2), 4.24 (t, J = 2.4 Hz, 2H,
CH2), 7.63–7.72 (m, 2H, H-6 and H-7), 8.06–8.49 (m, 2H,
H-5 and H-8), 8.97 (s, 1H, H-2). CI MS m/z (rel. intensity)
396 (M ? H?, 44), 310 (90), 292 (100). Anal. Calc. for
C17H14ClNOSSe: C 51.72, H 3.57, N 3.55. Found: C 51.90,
H 3.65, N 3.42.
4-(4-Hydrophthaloyloxy-2-butynylseleno)-3-methylthio-
quinoline (19) Yield 52%. Mp: 126–127°C. 1H NMR
(CDCl3, 300 MHz) d: 2.67 (s, 3H, SCH3), 3.51 (t,
J = 2.4 Hz, 2H, CH2), 4.68 (t, J = 2.4 Hz, 2H, CH2),
7.52–7.89 (m, 6H, C6H4 and H-6 and H-7), 8.09–8.40 (m,
2H, H-5 and H-8), 8.78 (s, 1H, H-2). CI MS m/z (rel.
intensity) 472 (M ? H?, 5), 324 (100). Anal. Calc. for
C22H17NO4SSe: C 56.17, H 3.64, N 2.98. Found: C 56.29,
H 3.75, N 3.12.
General procedure for the synthesis of acetylenic
thioquinolines 16–25
4-(4-Benzoyloxy-2-butynylthio)-3-methylthioquinoline (20)
1
Yield 90%. Mp: 88–89°C. H NMR (CDCl3, 300 MHz) d:
A mixture of 4-chloro-3-(4-hydroxy-2-butynylthio)quino-
line 5 (0.53 g, 2 mmol) or 4-(4-hydroxy-2-butynylthio)-
3-propargylthioquinoline 13 or (0.60 g, 2 mmol) 4-(4-
hydroxy-2-butynylseleno)-3-methylthioquinoline 14 (0.64 g,
2 mmol) or 4-(4-hydroxy-2-butynylthio)-3-methylthioquin-
oline 15 (0.55 g, 2 mmol) and pyridine (0.17 g, 2.1 mmol)
and (2.1 mmol) o-phthalic anhydride or cinnamoyl chloride
or benzoyl chloride or ethyl chloroformate in dry benzene
(8 ml) was stirred at 70°C for about 1 h (monitored by TLC
until complete consumption of starting materials) and then
concentrated in vacuo. The residue was separated by column
chromatography using chloroform/ethanol (30:1) to give
pure products 16–25.
2.65 (s, 3H, SCH3), 3.74 (t, J = 2.1 Hz, 2H, CH2), 4.68 (t,
J = 2.1 Hz, 2H, CH2), 7.42–7.61 (m, 7H, C6H5 and H-6
and H-7), 8.15–8.59 (m, 2H, H-5 and H-8), 8.78 (s, 1H, H-
2). CI MS m/z (rel. intensity) 380 (M ? H?, 100). Anal.
Calc. for C21H17NO2S2: C 66.47, H 4.52, N 3.69. Found: C
66.34, H 4.48, N 3.78.
4-(4-Benzoyloxy-2-butynylseleno)-3-methylthioquinoline
(21) Yield 54%. Mp: 92–93°C. 1H NMR (CDCl3,
300 MHz) d: 2.64 (s, 3H, SCH3), 3.63 (t, J = 2.4 Hz, 2H,
CH2), 4.69 (t, J = 2.4 Hz, 2H, CH2), 7.42–7.99 (m, 7H,
C6H5 and H-6 and H-7), 8.05–8.54 (m, 2H, H-5 and H-8),
8.75 (s, 1H, H-2). CI MS m/z (rel. intensity) 428 (M ? H?,
100). Anal. Calc. for C21H17NO2SSe: C 59.15, H 4.02, N
3.28. Found: C 58.94, H 4.15, N 3.34.
4-Chloro-3-(4-hydrophthaloyloxy-2-butynylthio)quinoline
(16) Yield 64%. Mp: 97–98°C. 1H NMR (CDCl3,
300 MHz) d: 3.66 (t, J = 2.1 Hz, 2H, CH2), 4.81 (t,
J = 2.1 Hz, 2H, CH2), 7.31–7.64 (m, 6H, C6H4 and H-6
and H-7), 7.72–8.10 (m, 2H, H-5 and H-8), 8.29 (s, 1H, H-
2). CI MS m/z (rel. intensity) 412 (M ? H?, 10), 246
(100). Anal. Calc. for C21H14ClNO4S: C 61.24, H 3.43, N
3.40. Found: C 61.42, H 3.50, N 3.31.
4-(4-Ethoxycarbonyloxy-2-butynylthio)-3-methylthioquino-
1
line (22) Yield 93%. Mp: 48–49°C. H NMR (CDCl3,
300 MHz) d: 1.30 (t, J = 7.2 Hz, 3H, CH3), 2.68 (s, 3H,
SCH3), 3.70 (t, J = 2.4 Hz, 2H, CH2), 4.18 (q, J = 7.2 Hz,
2H, OCH2), 4.85 (t, J = 2.4 Hz, 2H, CH2), 7.61–7.73 (m,
2H, H-6 and H-7), 8.05–8.59 (m, 2H, H-5 and H-8), 8,79 (s,
1H, H-2). CI MS m/z (rel. intensity) 348 (M ? H?, 100).
Anal. Calc. for C17H17NO3S2: C 58.77, H 4.93, N 4.03.
Found: C 58.98, H 4.85, N 4.19.
4-Chloro-3-(4-cinnamoyloxy-2-butynylthio)quinoline (17)
1
Yield 60%. Mp: 123–124°C. H NMR (CDCl3, 300 MHz)
d: 3.84 (t, J = 2.1 Hz, 2H, CH2), 3.74 (t, J = 2.1 Hz, 2H,
CH2), 6.37 (d, J = 15.9 Hz, 1H, CH), 7.39–7.73 (m, 8H,
CH and C6H5 and H-6 and H-7), 8.07–8.23 (m, 2H, H-5
and H-8), 9.00 (s, 1H, H-2). CI MS m/z (rel. intensity) 394
(M ? H?, 100). Anal. Calc. for C22H16ClNO2S: C 67.09,
H 4.09, N 3.56. Found: C 67.25, H 3.91, N 3.62.
4-(4-Cinnamoyloxy-2-butynylthio)-3-methylthioquinoline
(23) Yield 91%. Mp: 82–83°C. 1H NMR (CDCl3,
300 MHz) d: 2.68 (s, 3H, SCH3), 3.73 (t, J = 2.1 Hz, 2H,
CH2), 4.57 (t, J = 2.1 Hz, 2H, CH2), 6.36 (d, J = 16.2 Hz,
1H, CH), 7.39–7.68 (m, 8H, CH and C6H5 and H-6 and H-
7), 8.04–8.59 (m, 2H, H-5 and H-8), 8.80 (s, 1H, H-2). CI
MS m/z (rel. intensity) 406 (M ? H?, 100). Anal. Calc. for
C23H19NO2S2: C 68.12, H 4.72, N 3.45. Found: C 68.32, H
4.56, N 3.48.
4-(4-Hydrophthaloyloxy-2-butynylthio)-3-metylthioquino-
1
line (18) Yield 50%. Mp: 96–97°C. H NMR (CDCl3,
300 MHz) d: 2.64 (s, 3H, SCH3), 3.61 (t, J = 2,1 Hz, 2H,
CH2), 4.63 (t, J = 2.1 Hz, 2H, CH2), 7.26–7.93 (m, 6H,
123

Med Chem Res (2011) 20:1402–1410
1409
4-(4-Cinnamoyloxy-2-butynylseleno)-3-methylthioquino-
1
reach the required concentrations. DMSO, which was used
as a solvent did not exert any inhibitory effect on cell
proliferation. The cells attached to the plastic were fixed by
gently layering cold 50% TCA (trichloroacetic acid,
Aldrich-Chemie, Germany) on the top of the culture
medium in each well. The plates were incubated at 4°C for
1 h and then washed five times with tap water. The back-
ground optical density was measured in the wells filled
with culture medium, without the cells. The cellular
material fixed with TCA was stained with 0.4% sulforho-
damine B (SRB, Sigma, Germany) dissolved in 1% acetic
acid (POCh, Gliwice, Poland) for 30 min. Unbound dye
was removed by rinsing (49) with 1% acetic acid. The
protein-bound dye was extracted with 10 mM unbuffered
tris base (POCh, Gliwice, Poland) for determination of
optical density (at 540 nm) in a computer-interfaced, 96-
well microtiter plate reader Multiskan RC photometer
(Labsystems, Helsinki, Finland). Each compound in given
concentration was tested in triplicates in each experiment,
which was repeated 3–5 times.
line (24) Yield 42%. Mp: 98–99°C. H NMR (CDCl3,
300 MHz) d: 2.67 (s, 3H, SCH3), 3.63 (t, J = 2.1 Hz, 2H,
CH2), 4.58 (t, J = 2.1 Hz, 2H, CH2), 6.37 (d, J = 15.9 Hz,
1H, CH), 7.39–7.69 (m, 8H, CH and C6H5 and H-6 and H-
7), 8.02–8.53 (m, 2H, H-5 i H-8), 8.77 (s, 1H, H-2). CI MS
m/z (rel. intensity) 453 (M ? H?, 90), 256 (100). Anal.
Calc. for C23H19NO2SSe: C 61.06, H 4.23, N 3.10. Found:
C 60.81, H 4.12, N 3.18.
4-(4-Cinnamoyloxy-2-butynylthio)-3-(propargylthio)quino-
1
line (25) Yield 80%. Mp: 102–103°C. H NMR (CDCl3,
300 MHz) d: 2.27 (t, J = 2,7 Hz, 1H, CH), 3.75 (t,
J = 2,4 Hz, 2H, CH2), 3.84 (d, J = 2.7 Hz, 2H, SCH2),
4.58 (t, J = 2.4 Hz, 2H, CH2), 6.36 (d, J = 15.9 Hz, 1H,
CH), 7.39–7.69 (m, 8H, CH and C6H5 and H-6 and H-7),
8.07–8.60 (m, 2H, H-5 and H-8), 9.01 (s, 1H, H-2). CI MS
m/z (rel. intensity) 430 (M ? H?, 20), 232 (100). Anal.
Calc. for C25H19NO2S2: C 69.90, H 4.46, N 3.26. Found: C
70.12, H 4.52, N 3.38.
Antiproliferative assay in vitro
MTT assay
Cells
This technique was applied for the cytotoxicity screening
against mouse leukemia cells growing in suspension cul-
ture. An assay was performed after 72-h exposure to
varying concentrations (from 0.1 to 100 lg/ml) of the
tested agents. The compounds were dissolved in 10%
DMSO to concentration of 1 mg/ml, and subsequently
diluted in culture medium to reach the required concen-
trations. DMSO, which was used as a solvent did not exert
any inhibitory effect on cell proliferation. For the last
3–4 h of incubation 20 ll of MTT solution were added to
each well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; stock solution: 5 mg/ml). The mito-
chondria of viable cells reduce the pale yellow MTT to a
navy blue formazan: the more viable cells are present in
well, the more MTT will be reduced to formazan. When
incubation time was completed, 80 ll of the lysing mixture
was added to each well (lysing mixture: 225 ml dimeth-
ylformamide, 67.5 g sodium dodecyl sulfate, and 275 ml
of distilled water). After 24 h, when formazan crystals had
been dissolved, the optical densities of the samples were
read on an Multiskan RC photometer at 570 nm
wavelength.
The following established in vitro cancer cell lines were
applied: SW707 (human colorectal adenocarcinoma),
CCRF/CEM (human leukemia), T47D (human breast can-
cer), P388 (mouse leukemia), and B16 (mouse melanoma).
All lines were obtained from the American Type Culture
Collection (Rockville, Maryland, USA) and maintained at
the Cell Culture Collection of the Institute of Immunology
and Experimental Therapy, Wroclaw, Poland.
Twenty-four hours before addition of the tested agents,
the cells were plated in 96-well plate (Sarstedt, USA) at a
density of 104 cells per well in 100 ll of culture medium.
The cells were cultured in the opti-MEM medium supple-
mented with 2 mM glutamine (Gibco, Warsaw, Poland),
streptomycin (50 lg/ml), penicillin (50 U/ml) (both anti-
biotics from Polfa, Tarchomin, Poland), and 5% fetal calf
serum (Gibco, Grand Island, USA). The cell cultures were
maintained at 37°C in humid atmosphere saturated with 5%
CO2.
SRB assay
Each compound in given concentration was tested in
triplicates in each experiment, which was repeated 3–5
times.
The details of this technique were described by Skehan
et al. (1990). The cytotoxicity assay was performed after
72-h exposure of the cultured cells to varying concentra-
tions (from 0.1 to 100 lg/ml) of the tested agents. The
compounds were dissolved in 10% DMSO to concentration
of 1 mg/ml, and subsequently diluted in culture medium to
The results of cytotoxic activity in vitro were expressed
as an ID50—the dose of compound (in lg/ml) that inhibits
proliferation rate of the tumor cells by 50% as compared to
the control untreated cells.
123

1410
Med Chem Res (2011) 20:1402–1410
Acknowledgments This work is supported by Polish Ministry of
Science and Higher Education, Grant No. N405 036 31/2655 and the
Medical University of Silesia, Grant No. KNW-1-029/09.
protozoal and retroviral co-infections. Bioorg Med Chem
11:5013–5023
Fuita M, Chiba K, Nakano J, Tominaga Y, Matsumoto J (1998)
Synthesis and structure-antibacterial activity relationships of 7-
(3-amino-1-propynyl and 3-amino-1-propenyl)quinolones. Chem
Pharm Bull 46:631–638
Grissom JW, Gunawardena GU, Klingberg D, Huang D (1996) The
chemistry of enediyne, enyne allenes and related compounds.
Tetrahedron 52:6453–6518
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
Joshi MC, Bisht GS, Rawat DS (2007) Synthesis and antibacterial
activity of phendioxy substituted cyclic enediynes. Bioorg Med
Chem Lett 17:3226–3230
Knoll J, Budal K, Barenyl E (1988) Quinoline thioethers. GB Patent 2
204 583. Chem Abstr (1989) 110:154170g
References
Abele R, Abele E, Rubina K, Dzenitis O, Arsenyan P, Shestakova I,
Nesterova A, Domracheva I, Popelis J, Grinberga S, Lukevics E
(2002) Synthesis and cytotoxicity of 3-(hetarylothio)-1-propy-
nyl-(trimethyl)silane. Chem Heterocycl Comp 38:867–872
Aboul-Faddl T (2005) Selenium derivatives as cancer preventive
agents. Curr Med Chem Anti Cancer Agents 5:637–652
Bailey WJ, Fujiwara E (1955) Mixed dihalides and halohydrins from
butynediol. J Chem Soc 77:165–166
Kumar D, David WM, Kerwin SM (2001) N-Propargyl-2-alkynylben-
zothiazolium aza-enediynes: role of the 2-alkynylbenzothiazoli-
um functionality in DNA cleavage. Bioorg Med Chem Lett
11:2971–2974
Makisumi Y, Murabayashi A (1969) The thio-Claisen rearrangements
of allyl and propargyl 4-quinolyl sulfides. Tetrahedron Lett
24:1971–1974
´
Maslankiewicz A, Boryczka S (1993) Reactions of 4-methoxy-3-
Bajda M, Boryczka S, Wietrzyk J, Malawska B (2007) Investigation
of lipophilicity of anticancer-active thioquinoline derivatives.
Biomed Chromatogr 21:123–131
quinolinyl and 1, 4-dihydro-4-oxo-3-quinolinyl sulfides aiming
at the synthesis of 4-chloro-3-quinolinyl sulfides. J Heterocycl
Chem 30:1623–1628
Ben-Zvi Z, Danon A (1994) The chemistry of functional groups,
supplement C2. In: Patai S (ed) The chemistry of triple bonded
functional groups. Wiley, New York, pp 739–788
Bhattacharyya S, Pink M, Huffman JC, Zaleski JM (2006) Thioether
coordinated metalloenediynes: syntheses, structures and thermal
reactivity comparison. Polyhedron 5:550–558
Michael JP (2000) Quinoline, quinazoline and acridone alkaloids. Nat
Prod Rep 17:603–620
´
´
Mol W, Naczynski A, Boryczka S, Wietrzyk J, Opolski A (2006)
Synthesis and antiproliferative activity in vitro of diacetylenic
thioquinolines. Pharmazie 61:742–745
´
Mol W, Matyja M, Filip B, Wietrzyk J, Boryczka S (2008) Synthesis
and antiproliferative activity in vitro of novel (2-butynyl)thio-
quinolines. Bioorg Med Chem 16:8136–8141
Boryczka S (1999) Acetylenic derivatives of thioquinolines. Hetero-
cycles 51:631–638
Boryczka S, Wietrzyk J, Opolski A (2002a) Synthesis and antipro-
liferative activity in vitro of new propargyl thioquinolines.
Pharmazie 57:151–154
Nicolaou K, Dai W-M (1991) Chemistry and biology of the enediyne
anticancer antibiotics. Angew Chem Int Ed Engl 30:1387–1416
Rawat DS, Benites PJ, Incarvito CD, Rheingold AL, Zaleski JM
(2001) The contribution of ligand flexibility to metal center
geometry modulated thermal cyclization of conjugated pyridine
and quinoline metalloenediynes of copper (I) and copper (II).
Inorg Chem 40:1846–1857
´
Boryczka S, Wietrzyk J, Nasulewicz A, Pełczynska M, Opolski A
(2002b) New propargyl thioquinolines—synthesis, antiprolifer-
ative activity in vitro and structure–activity relationships.
Pharmazie 57:733–739
Boryczka S, Kulig K, Malawska B (2003) RP TLC determination of
the lipophilicity of anticancer-active propargyl thioquinolines.
J Planar Chromatogr 16:117–120
Boryczka S, Jastrze˛bska M, Nowak M, Kusz J, Wrzalik R, Wietrzyk
J, Matyja M (2010) Synthesis, X-ray structure and antiprolifer-
ative activity of 3-benzylthio-4-propargylselenoquinoline. Med
Chem Res 19:551–564. doi:10.1007/s00044-009-9212-x
Fakhfakh MA, Fournet A, Prina E, Mouscadet J-F, Franck X,
Hocquemiller R, Figadere B (2003) Synthesis and biological
evaluation of substituted quinolines: potential treatment of
Skehan P, Storeng R, Scudiero D, Monks A, Mcmachon J, Vistica D,
Warren JT, Bokesch H, Kenney S, Boyol MR (1990) New
colorimetric cytotoxicity assay for anticancer-drug screening.
J Natl Cancer Inst 82:1107–1112
Spande TF, Jain P, Garraffo HM, Pannell LK, Yeh HJC, Daly JW
(1999) Occurrence and significance of decahydroquinolines from
dendrobatid poison frogs and a myrmicine ant: use of 1H and
13C NMR in their conformational analysis. J Nat Prod 62:5–21
123