Synthesis of novel 3-allylseleno-6-alkylthiopyridazines: Their anticancer activity against MCF-7 cells

Article abstract of DOI:10.1007/s12272-013-0244-x

A new series of 3-allylseleno-6-alkylthiopyridazines 6a-6g was synthesized by two synthetic routes from 3,6-dichloropyridazine to develop new anticancer agents. These new compounds showed antiproliferative activities against breast cancer (MCF-7) cells in CCK-8 assays, and could be promising candidates for chemotherapy of carcinomas. Compound 6e (3-allylseleno-6-pentylthiopyridazine) showed higher potency than 5FU for inhibiting the growth of these cell lines. This suggests the potential anticancer activity of compound 6e.

Full text of DOI:10.1007/s12272-013-0244-x

Arch. Pharm. Res. (2014) 37:452–458
DOI 10.1007/s12272-013-0244-x
RESEARCH ARTICLE
Synthesis of novel 3-allylseleno-6-alkylthiopyridazines:
their anticancer activity against MCF-7 cells
•
Chaewon Kim Saet-Byeul Kim Myung-Sook Park
•
Received: 22 July 2013 / Accepted: 13 September 2013 / Published online: 26 September 2013
Ó The Pharmaceutical Society of Korea 2013
Abstract A new series of 3-allylseleno-6-alkylthiopyr-
idazines 6a–6g was synthesized by two synthetic routes
from 3,6-dichloropyridazine to develop new anticancer
agents. These new compounds showed antiproliferative
activities against breast cancer (MCF-7) cells in CCK-8
assays, and could be promising candidates for chemother-
apy of carcinomas. Compound 6e (3-allylseleno-6-pen-
tylthiopyridazine) showed higher potency than 5FU for
inhibiting the growth of these cell lines. This suggests the
potential anticancer activity of compound 6e.
were tested (Jung et al. 2001; Kwon and Moon 2005)
(Fig. 1). Allylthioheterocyclo(or aryl)alkylaminopyrida-
zines and allylthioaminopyridazines showed especially
good anticancer activities (Lee et al. 2009; Won and Park
2010).
The pyridazine group is an important moiety present in
many drugs acting at various pharmacological targets
(Kleemann and Engel 2001; Song et al. 2008; Cha et al.
2012). The pyridazine moiety has been combined with the
allylthio group (Lee et al. 2003; Shin and Kwon 2003).
We recently reported the synthesis of allylthioheter-
ocyclopyridazines (Park and Park 2005, 2007). The synthesis
of allylthioheterocyclo(or aryl)alkylaminopyridazines and
their antitumor activities against SK-Hep-1 human livercancer
cells were also reported (Kwon and Lee 2005). More recently,
the synthesis of 3-(6-arylamino)pyridazinylamino benzoic
acids and their antitumor activities against HT-29 colon cancer
cells were investigated (Abouzid et al. 2013). The synthesis of
isoxazolo (or thiazolo) [4,5-d]pyridazines and their antimi-
crobial activities were also reported (Faidallah et al. 2013).
The isosteric replacement of the sulfur of allylthiopy-
ridazines by a selenium atom yields the allylselenopyrid-
azines (Fig. 1). We designed new 3-allylseleno-6-
alkylthiopyridazine derivatives 6a–6g in order to discover
potential anti-tumor candidates. We tested the ability of
these synthetic compounds to inhibit the growth of breast
cancer MCF-7 cell lines.
Keywords Allylselenopyridazines ꢀ Pyridazines ꢀ
Organoseleniums ꢀ Anticancer activities ꢀ MCF-7 cells
Introduction
The allylthio group of allicin and other organosulfur com-
pounds that are isolated from garlic is considered an important
pharmacopore (Cavallito et al. 1944; Block et al. 1986;
Yamasaki et al. 1991; Agarwal 1996), i.e., a key structural
component of the molecules that is responsible for their anti-
tumor activities. Garlic is said to contain selenoproteins (Wang
et al. 1989) and a selenopolysaccharide (Yang et al. 1992).
Selenium plays an important role in biology. It is required for
the correct functioning of the immune system and for cellular
defense against oxidative damage, and may play a role in the
prevention of cancer and premature aging (Block 2010).
In previous studies, various allylthioalkoxypyridazines
and allylthioalkylthiopyridazines were synthesized (Lee
et al. 2001; Kwon 2002a, b) and their biological activities
Materials and methods
Chemicals
C. Kim ꢀ S.-B. Kim ꢀ M.-S. Park (&)
College of Pharmacy, Duksung Women’s University,
Seoul 132-714, Korea
Chemicals were supplied by Aldrich, Sigma, Merck, and
Tokyo Kasei. Melting points were determined in open
e-mail: mspark@duksung.ac.kr
123

Synthesis of anticancer 3-allylseleno-6-alkylthiopyridazines
453
Fig. 1 Allicin,
organoseleniums, reported
allylthiopyridazines and
allylselenopyridazines as target
compounds
O
S
=
-
-
CH2 CH CH2 Se CH3
=
-
-
-
CH2 CH=CH2
CH2 CH CH2 S
Volatile organoselenium compounds
Allicin from garlic
from genus Allium plants
N
N
N
N
=
-
-
R
CH2 CH CH2 Se
S
=
-
R
CH2 CH-CH2 S
O
Allylthiopyridazines
Allylselenopyridazines
Dipropylthiopyridazinyl diselenide (5c)
capillary tubes on a Bu¨chi 535 melting point apparatus and
were uncorrected. NMR spectra were recorded using a Bruker
300 MHz NMR spectrometer. Chemical shifts are reported in
parts per million and were recorded in chloroform-d or
dimethyl-d6 sulfoxide with tetramethylsilane as the internal
standard. NMR spin multiplicities are indicated by the sym-
bols: s (singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). IR spectra were recorded on a Perkin-Elmer 16F
PC FT-IR spectrometer using NaCl discs and pellets.
Yield: 51 %; mp 137–140 °C; 1H NMR (CDCl3) d 7.73 (d,
J = 9.0 Hz, 2H, CH, pyridazine), 7.18 (d, J = 9.0 Hz, 2H,
CH, pyridazine), 3.28 (t, J = 7.2 Hz, 4H, SCH2, propyl),
1.84–1.72 (m, 4H, CH2, propyl), 1.04 (t, J = 7.5 Hz, 6H,
CH3, propyl); 13C NMR (CDCl3) d 161.86, 154.36, 127.38,
127.27 (pyridazine), 32.23, 22.40, 13.35 (propyl).
Dibutylthiopyridazinyl diselenide (5d)
General synthetic procedure
Yield: 38 %; mp 128–133 °C; 1H NMR (CDCl3) d 7.74 (d,
J = 9.0 Hz, 2H, CH, pyridazine), 7.19 (d, J = 9.0 Hz, 2H,
CH, pyridazine), 3.32 (t, J = 7.5 Hz, 4H, SCH2, butyl),
1.80–1.73 (m, 4H, CH2, butyl), 1.55–1.42 (m, 4H, CH2,
butyl), 0.95 (t, J = 7.2 Hz, 6H, CH3, butyl); 13C NMR
(CDCl3) d 161.91, 154.36, 127.36, 127.26 (pyridazine),
31.02, 30.04, 21.99, 13.53 (butyl).
for the dialkylthiopyridazinyl diselenides 5a–5e
To a vigorously stirred mixture of powdered sodium
hydroxide (0.3 g, 7.4 mmol), elemental selenium (0.39 g,
4.93 mmol) and dimethylformamide (DMF) (20 mL),
100 % hydrazine hydrate (0.16 mL, 4.93 mmol) was added
slowly. After stirring for nearly 6 h at rt, 6-alkylthiopy-
ridazinyl chloride 4a–4e (4.93 mmol) was added slowly.
The mixture was refluxed for 1 h. When the solution turned
to red black from green black, it was cooled to rt. After the
excess selenium was filtered, the solution was diluted with
cold water (40 mL). The mixture was left for 48 h at 4 °C.
The yellow solid was formed in aqueous solution. The
crude product was filtered to afford 5a–5e.
Dipentylthiopyridazinyl diselenide (5e)
Yield: 60 %; mp 148–152 °C; 1H NMR (CDCl3) d 7.72 (d,
J = 9.0 Hz, 2H, CH, pyridazine), 7.17 (d, J = 9.0 Hz, 2H,
CH, pyridazine), 3.29 (t, J = 7.5 Hz, 4H, SCH2, pentyl),
1.80–1.70 (m, 4H, CH2, pentyl), 1.47–1.28 (m, 8H, CH2,
pentyl), 0.89 (t, J = 7.2 Hz, 6H, CH3, pentyl); 13C NMR
(CDCl3) d 161.91, 154.35, 127.35, 127.26 (pyridazine),
31.02, 30.32, 28.64, 22.18, 13.84 (pentyl).
Dimethylthiopyridazinyl diselenide (5a)
1
Yield: 42 %; mp 181 °C; H NMR (CDCl3) d 7.76 (d,
General synthetic procedure for the 3-allylseleno-6-
J = 9.0 Hz, 2H, CH, pyridazine), 7.23 (d, J = 9.0 Hz, 2H,
CH, pyridazine), 2.70 (s, 6H, SCH3, methyl); 13C NMR
(CDCl3) d 161.89, 154.46, 127.23, 127.07 (pyridazine),
13.35 (methyl).
alkylthiopyridazines 6a–6g
Method A
To a stirred mixture of powdered sodium hydroxide (89 mg,
2.23 mmol), water and absolute methanol (30 mL),
3-allylseleno-6-chloropyridazine 3 (0.5 g, 2.23 mmol) was
added at rt. Sodium hydroxide and related thiols were useful
reagents for generating the alkylthioxide in the case of long
chain thiols such as ethanethiol, propanethiol, butanethiol,
pentanethiol, hexanethiol and heptanethiol as shown in
Diethylthiopyridazinyl diselenide (5b)
1
Yield: 55 %; mp 159–162 °C; H NMR (CDCl3) d 7.74 (d,
J = 9.0 Hz, 2H, CH, pyridazine), 7.18 (d, J = 9.0 Hz, 2H,
CH, pyridazine), 3.31 (q, J = 7.2 Hz, 4H, SCH2, ethyl), 1.41
(t, J = 7.2 Hz, 6H, CH3, ethyl); 13C NMR (CDCl3) d 161.70,
154.39, 127.33, 127.32 (pyridazine), 24.72, 14.22 (ethyl).
123

454
C. Kim et al.
Table 1. Related thiol (2.23 mmol) was dropwised to reac-
tion mixture at 0 °C. The mixture was stirred for 1 * 17 h
at 0 °C. Upon completion, the reaction was stopped and the
excess thiol was evaporated under reduced pressure. The
residue was dissolved in ethyl acetate (50 mL). The organic
layer was washed with water (50 mL 9 2) and then dried
over anhydrous Na2SO4. After solvent evaporation, the
residue was purified by column chromatography (hexanes:
EtOAc) on silica gel to afford 6a–6g.
allyl), 5.31 (d, J = 16.3 Hz, 1H, =CH2, allyl), 5.11 (d,
J = 10.0 Hz, 1H, =CH2, allyl), 4.02 (d, J = 7.5 Hz, 2H,
SeCH2), 2.72 (s, 3H, SCH3, methyl); 13C NMR (CDCl3) d
160.21, 154.60, 127.90, 125.24 (pyridazine), 134.00,
117.79, 28.70 (allyl), 13.24 (SCH3).
3-Allylseleno-6-ethylthiopyridazine (6b)
1
Yield: 28 % (method A), 19 % (method B); oil; H NMR
(CDCl3) d 7.19 (d, J = 9.0 Hz, 1H, CH, pyridazine), 7.04
(d, J = 9.0 Hz, 1H, CH, pyridazine), 6.12–6.03 (m, 1H,
CH, allyl), 5.48 (d, J = 16.9 Hz, 1H, =CH2, allyl), 5.06 (d,
J = 10.0 Hz, 1H, =CH2, allyl), 3.99 (d, J = 7.5 Hz, 2H,
SeCH2), 3.33 (q, J = 7.4 Hz, 2H, SCH2, ethyl), 1.44 (t,
J = 7.4 Hz, 3H, CH3, ethyl); 13C NMR (CDCl3) d 159.98,
154.57, 128.07, 125.65 (pyridazine), 134.02, 117.81, 28.37
(allyl), 24.51 (SCH3), 14.36 (ethyl); FT-IR (NaCl) cm-1
3081 (aromatic), 2926 (aromatic), 1562 (N=N), 1396
(CH2), 1129 (CH3), 988, 916 (allyl double band),
821 (CSe); GC–MS m/z (%) 260 (M?), 245.0 (100.0),
260.0 (76.0), 243.0 (49.7), 258.0 (37.6), 118.1 (36.7).
Method B
To a stirred mixture of dipyridazyinyl diselenide 5a–5e
(5.2 mmol), powdered sodium hydroxide (1.03 g, 26 mmol)
and tetrabutylammonium bromide (0.34 g, 1.04 mmol) in
absolute THF (30 mL), was added a solution of hydrazine
monohydrate (0.054 mL, 1.73 mmol) at rt. After 1 h of
stirring, the color of the reaction mixture turned to orange red
from pale yellow. The reaction mixture was then cooled to
0 °C, allyl bromide (0.86 mL, 10.4 mmol) was added slowly
dropwise and stirring continued for 1 h at 0 °C until the color
of the reaction mixture turned to yellow from orange red.
After the reaction was complete, the solvent THF was
evaporated under reduced pressure. The residue was dis-
solved in ethyl acetate (50 mL). The organic layer was
washed with water (50 mL 9 4) and then dried over anhy-
drous Na2SO4. After solvent evaporation, the residue was
purified by column chromatography (hexanes: EtOAc) on
silica gel to afford 6a–6e.
3-Allylseleno-6-propylthiopyridazine (6c)
1
Yield: 10 % (method A), 22 % (method B); mp 58 °C; H
NMR (CDCl3) d 7.26 (d, J = 9.0 Hz, 1H, CH, pyridazine),
7.05 (d, J = 9.0 Hz, 1H, CH, pyridazine), 6.11–6.02 (m,
1H, CH, allyl), 5.25 (d, J = 17.1 Hz, 1H, =CH2, allyl),
5.05 (d, J = 10.1 Hz, 1H, =CH2, allyl), 3.99 (d,
J = 7.5 Hz, 2H, SeCH2), 3.30 (t, J = 7.2 Hz, 2H, SCH2,
propyl), 1.86–1.73 (m, 2H, CH2, propyl), 1.05 (t,
J = 7.3 Hz, 3H, CH3, propyl); 13C NMR d 160.56, 154.92,
128.41, 126.07 (pyridazine), 134.40, 118.20, 28.76 (allyl),
32.40, 22.78, 13.89 (propyl); FT-IR (NaCl) cm-1 3034
(aromatic), 2925 (aromatic), 1566 (N=N), 1393 (CH2),
1150 (CH3), 1014, 896 (allyl double band), 849 (CSe);
GC–MS m/z (%) 274 (M?), 259.0 (100.0), 274.1 (78.1),
257.0 (50.5), 272.1 (38.7), 118.1 (34.3).
3-Allylseleno-6-methylthiopyridazine (6a)
Yield: 19 % (method B); mp 31–32 °C; 1H NMR (CDCl3)
d 7.23 (d, J = 9.1 Hz, 1H, CH, pyridazine), 7.12 (d,
J = 9.1 Hz, 1H, CH, pyridazine), 6.18–6.02 (m, 1H, CH,
Table 1 Totals yields of two synthetic routes for target compounds
6a–6e and their antiproliferative activity against breast cancer cell
lines (MCF-7) in CCK-8 assays
3-Allylseleno-6-butylthiopyridazine (6d)
Yield: 24 % (method A), 24 % (method A); mp 34–36 °C;
1H NMR (CDCl3) d 7.19 (d, J = 9 Hz, 1H, pyridazine),
7.05 (d, J = 9 Hz, 1H, pyridazine), 6.14–6.00 (m, 1H, CH,
allyl), 5.25 (d, J = 16.4 Hz, 1H, =CH2, allyl), 5.05 (d,
J = 9.9 Hz, 1H, =CH2, allyl), 3.98 (d, J = 7.4 Hz, 2H,
SeCH2), 3.32 (t, J = 7.2 Hz, 2H, SCH2), 1.79–1.69 (m,
2H, CH2, butyl), 1.55–1.45 (m, 2H, CH2, butyl), 0.95 (t,
J = 7.5 Hz, 3H, CH3, butyl); 13C NMR (CDCl3) d 160.64,
154.91, 128.39, 126.03 (pyridazine), 134.39, 118.18, 28.76
(allyl), 31.35, 30.25, 22.45, 14.06 (butyl); FT-IR (NaCl)
cm-1 3080 (aromatic), 2957 (aromatic), 1561 (N=N), 1384
(CH2), 1130 (CH3), 987, 916 (allyl double band), 823
Compound
R
Total yieldsa Total yieldsb Mean IC50
(%)
(%)
(lM)
6a
6b
6c
6d
6e
5FU
a
Methyl 1.19
Ethyl 9.13
Propyl 3.26
Butyl 7.83
Pentyl 1.30
5.77
7.11
7.85
6.38
5.98
842
766
1,512
1,122
207
6,112
Total yields referred to isolated product (method A)
b
Total yields referred to isolated product (method B)
123

Synthesis of anticancer 3-allylseleno-6-alkylthiopyridazines
455
(CSe); GC–MS m/z (%) 288 (M?), 272.9 (100.0), 288.0
315.0 (100.0), 313.0 (50.8), 330.0 (44.4), 119.0 (26.4),
118.0 (24.8).
(68.7), 271.0 (48.7), 118.0 (46.0), 119.0 (43.1).
Materials and methods for bioassays: cell line culture
conditions
3-Allylseleno-6-pentylthiopyridazine (6e)
Yield: 4 % (method A), 16 % (method B); mp 52–54 °C;
1H NMR (CDCl3) 7.18 (d, J = 8.9 Hz, 1H, pyridazine),
7.04 (d, J = 8.9 Hz, 1H, pyridazine), 6.11–6.02 (m, 1H,
CH, allyl), 5.25 (d, J = 17.5 Hz, 1H, =CH2, allyl), 5.05 (d,
J = 9.9 Hz, 1H,=CH2, allyl), 3.98 (d, J = 7.0 Hz, 2H,
SeCH2), 3.31 (t, J = 7.3 Hz, 2H, SCH2), 1.79–1.74 (m,
2H, CH2, pentyl), 1.46–1.31 (m, 4H, CH2, pentyl), 0.91 (t,
J = 7.1 Hz, 3H, CH3, pentyl); 13C NMR (CDCl3) d
160.28, 154.53, 128.03, 125.66 (pyridazine), 134.02,
117.81, 28.64 (allyl), 31.08, 30.16, 28.40, 22.28, 13.97
(pentyl); FT-IR (NaCl) cm-1 3036 (aromatic), 2925 (aro-
matic), 1565 (N=N), 1034, 1018 (allyl double band),
845 (CSe). GC–MS m/z (%) 302 (M?), 146.0 (100.0),
148.0 (59.7), 147.0.0 (50.4), 160.0 (41.0), 173.0 (31.3).
MCF-7 breast cancer cells were purchased from the ATCC
(Manassas, USA) and maintained at 37 °C in a humidified
atmosphere, with 5 % CO2, in MEM (Gibco-BRL Inc.)
medium supplemented with 10 % fetal bovine serum
(Gibco-BRL Inc., Korea).
Antiproliferative CCK-8 (cell counting kit-8) assays (Jung
et al. 2012)
The cytotoxic activity of the compounds was determined
in vitro using the CCK-8 assay kit (Dojindo, Korea). The
human breast cancer cells were seeded in 96-well plates at
densities of 5000 cells/well with five replicates for each
drug concentration and maintained at 37 °C in a 5 % CO2
humidified incubator for 24 h. Control cells were treated
with dimethyl sulfoxide (DMSO) equal to the highest
percentage of solvent used in the experimental condi-
tions. 5FU was used as a positive control. Then, the MCF-7
cells were treated with various concentrations of synthetic
compounds (the final concentrations of 6a–6e were 6.25,
25, 100, and 400 lg/mL) for 24 h. 10 lL of Cell Counting
Kit-8 solution were added into each well (containing
100 lL), and the plates were further incubated for 2.5 h.
The absorbance at 450 nm was measured by a micro
ELISA reader (ASYS Biotech, Cambridge, BK). The cell
3-Allylseleno-6-hexylthiopyridazine (6f)
Yield: 4 % (method A); mp 42–44 °C; 1H NMR (CDCl3) d
7.18 (d, J = 9.0 Hz, 1H, pyridazine), 7.04 (d, J = 9.0 Hz,
1H, pyridazine), 6.14–6.00 (m, 1H, CH, allyl), 5.25 (d,
J = 17.1 Hz, 1H, =CH2, allyl), 5.05 (d, J = 9.9 Hz, 1H,
=CH2, allyl), 3.98 (d, J = 7.2 Hz, 2H, SeCH2), 3.31 (t,
J = 7.3 Hz, 2H, OCH2), 1.80–1.70 (m, 2H, CH2, hexyl),
1.50–1.40 (m, 2H, CH2, hexyl), 1.35–1.25 (m, 4H, CH2,
hexyl), 0.89 (t, J = 7.0 Hz, 3H, CH3, hexyl); 13C NMR
(CDCl3) d 160.27, 154.51, 128.01, 125.64 (pyridazine),
134.03, 117.81, 28.87 (allyl), 31.38, 30.18, 28.61, 28.39,
22.55, 14.03 (hexyl); FT-IR (NaCl) cm-1 3046 (aromatic),
2920 (aromatic), 1549 (N=N), 988, 915 (allyl double band),
830 (CSe); GC–MS m/z (%) 316 (M?), 234.0 (100.0),
150.1 (66.4), 85.2 (54.7), 117.1 (40.0), 55.1 (23.0).
viability ratio was calculated as follows: (test group A450
/
control group A450) 9 100 %. IC50 values were determined
from three independent experiments.
Results and discussion
3-Allylseleno-6-heptylthiopyridazine (6g)
Activated aryl halides react well with thiols to give the
corresponding thioethers. The SN reaction of an aryl halide
with a thiol is not only important for the synthesis of
thioethers, but is also essential for the preparation of
pharmaceuticals. Many reports have been published on the
nucleophilic thiolation of aryl halides. Even though a
synthetic pathway for 3-allylthiopyridazines has been
developed (Kwon 2002a; Kwon and Lee 2005; Kwon and
Moon 2005), the synthesis of 3-allylselenopyridazines has
not been reported until now. We applied a general method
of preparing allylthiopyridazines from pyridazinyl halides
and thiols (Kwon 2002b; Lee et al. 2003) such as synthetic
method A. Also, a method of splitting the selenium–sele-
nium bond, allylation of dipyridazinyl diselenide and allyl
bromide such as synthetic method B was applied (Bhasin
Yield: 3 % (method A); mp 37–38 °C; 1H NMR (CDCl3) d
7.18 (d, J = 9.0 Hz, 1H, pyridazine), 7.04 (d, J = 9.0 Hz,
1H, pyridazine), 6.11–6.01 (m, 1H, CH, allyl), 5.25 (d,
J = 17.0 Hz, 1H, =CH2, allyl), 5.05 (d, J = 9.9 Hz, 1H,
=CH2, allyl), 3.98 (d, J = 7.2 Hz, 2H, SeCH2), 3.31 (t,
J = 7.2 Hz, 2H, SCH2), 1.80–1.70 (m, 4H, CH2, heptyl),
1.48–1.40 (m,2H, CH2, heptyl), 1.37–1.27 (m, 4H, CH2,
heptyl), 0.90 (t, J = 4.7 Hz, 3H, CH3, heptyl); 13C NMR
(CDCl3) d 160.69, 154.92, 128.41, 126.04 (pyridazine),
134.41, 118.19, 28.79 (allyl), 32.11, 30.56, 29.31, 29.29,
29.25, 22.99, 14.46 (heptyl); FT-IR (NaCl) cm-1 3100
(aromatic), 2920 (aromatic), 1556 (N=N), 987, 915 (allyl
double band), 830 (CSe); GC–MS m/z (%) 330 (M?),
123

456
C. Kim et al.
Scheme 1 Synthetic routes for
target 3-allylseleno-6-
alkylthiopyridazines 6a–6g
through method A and
method B
Method B
N N
N N
R-SH, NaOH, MeOH
rt, 1h, 60~91%
RS
l
C
Cl
Cl
1
4a-4e
A
Na2Se2, DMF
rt, 60h,48%
Na2Se2, DMF
d
o
R
h
reflux, 1h, 38~60%
t
e
M
a
b
c
d
e
f
methyl
ethyl
propyl
butyl
pentyl
hexyl
heptyl
N N
N N
N N
N N
RS
Se Se
SR
l
C
Cl
Se Se
2
5a-5e
g
NH2NH2,
allylbromide,
NH2NH2
allylbromide,
,
A
d
o
h
t
NaOH, TBAB, THF,
0oC, 1h, 16~24%
NaOH, TBAB,
THF, 0oC, 1h, 68%
e
M
N N
N N
NaOH/R-SH
0oC,1~17h, 3~28%
Method A
R
S
Se CH2 CH
CH2
l
C
Se CH2 CH CH2
6a-6g
3
and Singh 2002; Bhasin et al. 2004; Scianowski et al.,
Scianowski 2006; Lee and Park 2011; Lee et al. 2011).
A series of 3-allylseleno-6-alkylthiopyridazines 6a–6g
was prepared by diselenylation, allylation and alkylthiola-
tion as shown in method A (Scheme 1). The allylseleno
group, a pharmacologically active group, was introduced
on one side of the pyridazine ring. Also, an alkylthio
moiety such as methylthio, ethylthio, propylthio, butylthio,
pentylthio, hexylthio and heptylthio was introduced into
the para-position of pyridazines 3. The key intermediates
in these preparations were dichloropyridazinyl diselenide
2, which could be obtained from the corresponding 3,6-
dichloropyridazine 1 by reaction with sodium selenide.
Dichloropyridazinyl diselenide 2 was converted to all-
ylselenopyridazinyl chloride 3 by allylation using hydra-
zine hydrate and allyl bromide. Condensation of the
allylselenopyridazinyl chloride 3 with various thiols gave
the final target products 6a–6g (Table 1). On the other
hand, 6a–6e were also prepared by alkylthiolation, disele-
nylation and allylation as shown in method B. The allylthio
group was introduced on one side of the 3,6-dichloropyr-
idazine ring. An alkylthio moiety such as methylthio, eth-
ylthio, propylthio, butylthio and pentylthio was introduced
into the C6-position of pyridazines for alkylthiopyridazinyl
chloride 4a–4e. Condensation of the 3,6-dichloropyrid-
azine 1 with various thiols gave 4a–4e. The key interme-
diates in these synthetic routes were dipyridazinyl
diselenides 5a–5e, which could be readily obtained from
the corresponding 4a–4e by diselenylation with sodium
selenide. Diselenides 5a–5e was converted to final all-
ylselenopyridazines 6a–6e by allylation using hydrazine
hydrate and allyl bromide.
Here, we present the synthesis of new 3-allylseleno-6-
alkylthiopyridazine 6a–6g by two synthetic routes. In
Table 1, we summarize the total yield of two method and
the antiproliferative activity for compounds 6a–6e.
3-Allylseleno-6-chloropyridazine 3 was converted to final
6a–6g by nucleophilic aromatic substitution with thiols in
the presence of sodium hydroxide (Scheme 1). The
nucleophilic displacement of chlorine in 3-allylseleno-6-
chloropyridazine 3 requires prolonged the reaction time at
0 °C. A typical reaction consisted of a mixture of thiols
(2.23 mmol), 6-allylthio-3-chloropyridazine (2.23 mmol),
and sodium hydroxide (2.23 mmol) in absolute methanol
stirred at 0 °C for 1–17 h. The reaction was usually carried
out using 1: 1 equivalents of 3-allylseleno-6-chloropyr-
idazine 3: thiols. 3 were converted into the corresponding
pyridazines 6a–6g in low yields (3–28 %).
Thus we designed synthetic method B to improve the
low yield. Method B differed from method A in the order
of reactions. The mono-alkylthiolation from 3,6-dichloro-
pyridazines 1 to 4a–4e produced high yields (60–91 %).
We previously reported the synthesis of 3-allylthio-6-
chloropyridazine in 95 % yield through allylthiolation
(Park and Park 2007). 3-Alkylthio-6-chloropyridazine 4a–
4e and sodium selenide were reacted in DMF to form the
corresponding diselenides 5a–5e in 38–60 % yield
(Scheme 1). Diselenides 5a–5e were converted into the
123

Synthesis of anticancer 3-allylseleno-6-alkylthiopyridazines
457
corresponding compounds 6a–6e in lower yields
(16–24 %). But, the total average yield of method B was
better than that of method A.
compound 6e markedly inhibited MCF-7 cell growth at
IC50 (207 lM) in a dose-dependent manner at a low con-
centration (6.25 lg/mL). Five compounds (6a–6e) showed
higher potencies than 5FU at IC50 (6,112 lM) in inhibiting
the growth of MCF-7 cells, suggesting the potential anti-
cancer activity of these compounds (Table 1.). The results
revealed that compound 6e had high activity towards MCF-
7 cells.
The pyridazine NMR peak of 5a–5e appeared at
7.17–7.23 and 7.72–7.76 ppm. The alkyl (methyl, ethyl,
propyl, butyl and pentyl) NMR peak of 5a–5e appeared at
0.89–3.33 ppm as splitting signal. The pyridazine 13C
NMR peak appeared at 127.07–127.32, 127.23–127.38,
154.35–154.46 and 161.70–161.91 ppm, and the alkyl peak
appeared at 13.75–32.23 ppm.
Finally, we synthesized new dipyridazinyl diselenides
5a–5e and 3-allylseleno-6-alkylthiopyridazines 6a–6g in
order to discover potential anticancer candidates. Com-
pound 6e (3-allylseleno-6-pentylthiopyridazine) showed
higher potency than 5-FU for inhibiting the growth of these
MCF-7 cell lines.
The pyridazine NMR peak of 6a–6g appeared at 7.04–7.12
and 7.18–7.23 ppm, and the allyl peak appeared at 3.98–4.02,
5.05–5.11, 5.25–5.48, and 6.00–6.18 ppm. The alkyl (methyl,
ethyl, propyl, butyl, pentyl, hexyl and heptyl) NMR peak of
6a–6g appeared at 0.89–3.33 ppm as splitting signal. The
pyridazine 13C NMR peak appeared at 125.24–126.07,
127.9–128.41, 154.51–154.92 and 159.98–160.69 ppm, and
the allyl peak appeared at 28.37–28.87, 117.79–118.20, and
134.00–134.41 ppm.
Acknowledgments This research was supported by Basic Science
Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Science, ICT and future
Planning (No 2012050022). We acknowledge to the professor Joohee
Jung (College of Pharmacy, Duksung Women’s University) for the
CCK-8 assays of antiproliferative activities.
In order to investigate the potential anti-cancer activity
of the seven synthetic compounds, the growth-inhibitory
effect of the synthetic compounds was examined against
breast cancer (MCF-7) cells. CCK-8 assays were con-
ducted on cells treated with various concentrations of the
compounds. 5-Fluorouracil (5FU), which produces partial
responses in 10–20 % of patients with metastatic colon
carcinomas, upper gastrointestinal tract carcinomas, and
breast carcinomas (Brunton et al. 2006), was used as a
positive control. The IC50 values for these compounds were
determined from the concentration range used in this study.
Of the seven target compounds, five compounds (6a–6e)
inhibited the growth of breast (MCF-7) cells at standard
concentrations (6.25, 25, 100, 400 lg/mL) (Fig. 2). 5FU, a
positive control, showed inhibitory effects on the growth of
MCF-7 cells at standard concentrations (6.25, 25, 100,
400 lg/mL). We further investigated the antiproliferative
activity of compound 6e, which caused greater inhibition of
cell growth than the other compounds. As shown in Fig. 2,
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