Design, synthesis, and cytotoxicity of pyridine, pyrazole, and thiazole derivatives derived from N-alkyl-4,5,6,7-tetrahydro-1-benzothiophene

Article abstract of DOI:10.1007/s10593-013-1259-4

The reaction of 4,5,6,7-tetrahydro-1-benzothiophene derivatives with ethyl acetoacetate gave oxobutanamide derivatives. The reactivity of these products towards some chemical reagents was studied to afford new heterocyclic derivatives. The cytotoxicity of the newly synthesized compounds was evaluated against three human tumor cells lines and three normal cell lines. The results showed that some of these compounds exhibit high inhibitory effects towards the three tumor cell lines and the normal cell lines.

Full text of DOI:10.1007/s10593-013-1259-4

Chemistry of Heterocyclic Compounds, Vol. 49, No. 3, June, 2013 (Russian Original Vol. 49, No. 3, March, 2013)
DESIGN, SYNTHESIS, AND CYTOTOXICITY OF PYRIDINE,
PYRAZOLE, AND THIAZOLE DERIVATIVES DERIVED
FROM N-ALKYL-4,5,6,7-TETRAHYDRO-1-BENZOTHIOPHENE
M. M. Kamel1*, A. K. El-Ansary1, Y. R. Milad2
The reaction of 4,5,6,7-tetrahydro-1-benzothiophene derivatives with ethyl acetoacetate gave
oxobutanamide derivatives. The reactivity of these products towards some chemical reagents was
studied to afford new heterocyclic derivatives. The cytotoxicity of the newly synthesized compounds was
evaluated against three human tumor cells lines and three normal cell lines. The results showed that
some of these compounds exhibit high inhibitory effects towards the three tumor cell lines and the
normal cell lines.
Keywords: pyridine, tetrahydro-1-benzothiophene, thiazole, thiophene, cytotoxocity.
Aromatic thiophenes are the most promising small-molecule selective protein kinase inhibitors [1-4].
Recently [5], a tricyclic compound, tetrahydro-1-benzothieno[2,3-d]pyrimidine, was identified as an initial hit
with an enzyme inhibition IC50 2.6 M.
In recent years, thiophenes and their fused derivatives showed promising results as anticancer agents
[6-9]. Moreover, Mohareb et al. [10] reported that 2-acylamino-4,5,6,7-tetrahydro-1-benzothiophene derivatives
were tested against three human tumor cells lines, namely, breast adenocarcinoma (MCF-7), non-small cell lung
cancer (NCI-H460), and human glioblastoma (SF-268). The results showed that most of the tested compounds
exhibit high inhibitory effects towards all three tumor cell lines. To our knowledge, some research has been
done on the variation of substitution at positions 2 and 3 of the tetrahydro-1-benzothiophene nucleus
_______
*To whom correspondence should be addressed, e-mail: mona_mounir50@hotmail.com.
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo
11562, Egypt.
2Department of Organic Chemistry, Faculty of Pharmacy, October University for Modern sciences & Arts
(MSA), El-Wahaat Road, Giza, Egypt; e-mail: yara.sihaya@googlemail.com.
________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 3, pp. 423-435, March, 2013. Original
article submitted July 23, 2012.
392
0009-3122/13/4903-0392©2013 Springer Science+Business Media New York

[8, 11-13]. In light of these observations, our efforts towards drug discovery prompted us to design, synthesize,
and evaluate the cytotoxocity of some new tetrahydro-1-benzothiophene derivatives. In the present work, a new
series of thiophene derivatives has been synthesized by incorporation of a variety of ring systems such as
pyridine, thiazole, and thiophene at position 2 of the benzothiophene ring and either cyano or ethyl carboxylate
group at position 3. The newly synthesized products have been evaluated as to their antitumor activity against
the three cancer cell lines mentioned above. Some of them were tested for activity towards human normal cell
lines. The comparison between the activity of the newly synthesized thiophene derivatives towards tumor and
normal cell lines will direct future research towards the synthesis of good anticancer agents.
In an attempt to obtain an antitumor agent with high activity, the substitution pattern at positions 2 and 3
of the thiophene pharmacophore was selected in order to alter the electronic environment and thus affect the
lipophilicity of the target molecules. Our aim was the acylation of the 2-amino groups in the known tetrahydro-
1-benzothiophene nucleus 1a,b and the introduction of 1,3-dicarbonyl [14], -unsaturated carbonyl groups
[15], and a nitrogen- or sulfur-containing heterocyclic ring [16] that are known to contribute to the enhancement
of antitumor activity. The incorporation of a heterocyclic ring at position 2 of the thiophene pharmacofore was
of great importance in order to increase the lipophilicity and thus radically modify the bioavailability and
efficacy of the synthesized compounds. Therefore, in the present work we want to provide a comparison
between some previously reported [17-20] tetrahydro-1-benzothiophenes 1a-e and our newly synthesized
compounds derived from the synthons 1a,b.
In the present work, we are considering the reported thiophene derivatives 1a-e where antitumor
reactivity will be compared to the newly synthesized compounds derived from the acylated derivatives of
synthons 1a,b.
R
NH2
S
1a–e
a R = CN, b R = CO2Et, c R = CONH2,
d R = CONHPh, e R = CONH(4-ClC6H4)
Thus, the reaction of compounds 1a,b with ethyl acetoacetate at 140°C afforded the N-acyl derivatives
2a,b, respectively. The structures of these and other synthesized compounds were assigned on the basis of their
1
analytical and spectral data (Tables 1 and 2). Thus, the H NMR spectrum of compound 2a showed signals for
all of the aliphatic groups and a singlet (D2O exchangeable) at 11.57 ppm corresponding to the NH group.
The reactivity of compounds 2a,b towards different electrophilic reagents to form benzylidene, hydrazo,
pyridine, and thiophene derivatives was studied with a view of investigating their biological activity. Thus, the
reaction with benzaldehyde or salicylaldehyde gave the corresponding arylidene derivatives 3a,b and 4,
respectively.
393

The reaction of compounds 2a or 2b with aromatic diazonium salts 5a-c gave arylhydrazo derivatives
6a-f. The analytical and spectral data of the latter products were consistent with the proposed structures.
The reactivity of compounds 2a,b towards formation of hydrazide-hydrazone derivatives was studied on
the example of the reaction with cyanoacetylhydrazine which gave hydrazones 7a,b. The 1H NMR spectra were
the basis of the structure elucidation of hydrazones 7a,b. Thus, the 1H NMR spectrum of compound 7a showed
signals of all expected aliphatic groups and two singlets (D2O exchangeable) at 10.18 and 11.01 ppm for the two
NH groups.
Next, we moved towards studying the reactivity of compounds 2a,b towards active methylene reagents
under different conditions. Thus, the reaction of compound 2a with either malononitrile (8a) or ethyl cyanoacetate
(8b) in 1,4-dioxane in the presence of triethylamine afforded pyridine derivatives 9a,b, respectively [21]. The
1H NMR spectrum of compound 9a showed, besides the expected signals of the cyclohexene moiety, a singlet at
2.77 ppm corresponding to the CH3 group, a singlet at 3.34 ppm (D2O exchangeable) for one NH2 group, and a
singlet at 6.01 ppm corresponding to the pyridine H-3. Similarly, the reaction between compounds 2b and 8a in
1,4-dioxane/Et3N solution gave the pyridine derivative 9c. On the other hand, carrying out the same reactions of
compound 2a with compounds 8a,b but in the presence of ammonium acetate in an oil bath at 120°C gave the
Knoevenagel condensation products 10a,b. The analytical and spectral data of the latter products were in
agreement with their respective structures (Tables 1, 2).
The reaction of either compound 2a or 2b with acetylacetone gave the pyridine derivatives 11a and 11b,
1
respectively. The H NMR spectrum of compound 11a showedthree singlets at 2.41, 2.63, and 3.59 ppm
corresponding to the three CH3 groups and a singlet at 6.91 ppm indicating the presence of the pyridine H-5
atom.
394

TABLE 1. Physicochemical Characteristics of the Synthesized Compounds
Found, %
Calculated, %
——————
Com-
pound
Empirical
formula
Yield,
%
Mp*, °C
C
H
N
S
78
C13H14N2O2S
C15H19NO4S
C20H18N2O2S
C22H23NO4S
C20H18N2O3S
C19H18N4O2S
C20H20N4O2S
C19H17ClN4O2S
C21H23N3O4S
C22H25N3O4S
C21H22ClN3O4S
C16H17N5O2S
C18H22N4O4S
C16H14N4OS
C18H19N3O3S
C18H19N3O3S
C16H14N4OS
C18H19N3O3S
C18H18N2O2S
C20H23NO4S
C22H19N3O3S2
C23H21N3O2S2
C26H28N2O5S2
C30H28N2O4S2
C25H26N2O4S2
59.60
59.52
58.02
58.23
68.38
68.55
66.44
66.48
65.37
65.55
61.99
62.28
63.09
63.14
57.23
56.93
60.95
61.00
61.74
61.81
56.49
56.31
56.21
55.96
55.64
55.37
61.69
61.92
60.34
60.49
60.72
60.49
61.44
61.92
60.34
60.49
66.02
66.23
64.01
64.32
59.28
60.39
62.02
63.42
60.73
60.92
65.88
66.15
5.60
5.38
5.35
6.19
5.44
5.18
6.07
5.83
5.17
4.95
5.12
4.95
5.18
5.30
4.52
4.27
5.72
5.61
6.01
5.89
4.60
4.95
5.12
4.99
5.89
5.68
4.67
4.55
5.38
5.36
5.66
5.36
4.83
4.55
5.38
5.36
5.84
5.55
6.49
6.21
5.19
4.38
5.08
4.86
5.29
5.51
5.15
5.18
10.59
10.68
4.73
4.53
8.28
7.99
3.82
3.52
7.93
7.64
14.84
15.29
15.07
14.73
14.11
13.98
9.84
10.16
10.02
9.83
9.66
9.38
19.92
20.39
14.15
14.35
17.83
18.05
12.04
11.76
12.06
11.76
17.88
18.05
12.04
11.76
8.69
8.58
3.49
3.75
9.52
9.60
9.92
9.65
5.05
5.46
11.89
12.22
10.14
10.36
8.84
9.15
7.71
8.07
9.16
8.75
9.05
8.75
8.18
8.43
7.83
8.00
7.69
7.75
7.09
7.50
6.80
7.16
9.26
9.34
8.52
8.21
10.11
10.33
8.68
8.97
8.62
8.97
10.01
10.33
9.36
8.97
10.27
9.82
8.82
8.59
14.52
14.66
14.41
14.72
12.92
12.51
12.04
11.77
217-219
210-212
144-145
138-139
189-190
170-171
251-252
240-241
183-184
100-102
139-140
222-223
120-121
268-269
110-111
133-134
280-283
184-185
216-217
180-181
200-201
277-278
170-171
220-221
160-161
2a
66
70
83
62
77
80
71
64
60
88
87
79
58
70
76
75
59
62
70
77
80
80
60
74
2b
3a
3b
4
6a
6b
6c
6d
6e
6f
7a
7b
9a
9b
9c
10a
10b
11a
11b
15
16a
16b
16c
16d
4.99
5.14
6.29
5.80
62.16
62.22
5.67
5.43
12.93
13.29
_______
* Recrystallization solvents: ethanol (compounds 2a,b, 3a,b, 4, 6a-f, 7a,b, 11a,b, 15, and 16a-d)
395

396

397

It has been reported [22-24] that the reaction of active methylene reagents with phenylisothiocyanate in
basic dimethyl formamide gives intermediate potassium sulfide salts that undergo heterocyclization with
-halocarbonyl compounds to give either thiophene or thiazole derivatives, depending on the reaction
conditions or the nature of the -halocarbonyl compound. In a similar way, we expected the reaction of either
compound 2a or 2b with phenylisothiocyanate in DMF/KOH solution to give the salts 12a,b that would further
react with the -halocarbonyl derivatives 13a-c to yield intermediate thioether derivatives of the type 14a-e
[23]. The reaction of compound 2a with phenylisothiocyanate, and further with ester 13a, produced the thiazole
derivative 15. The 1H NMR spectrum of compound 15 showed, besides the expected CH2 and Ph group signals, a
singlet at 2.72 ppm for the CH3 group, a singlet at 5.61 ppm indicating the presence of the thiazole H-5 proton,
and two singlets at 10.22 and 11.30 ppm corresponding to the NH and OH groups, respectively. Formation of
the thiazole ring of compound 15 most likely took place via ethanol elimination from the intermediate 14a due
to the presence of the good ethoxy leaving group. On the other hand, the reaction of either nitrile 2a with
chloroacetone (13c) or ester 2b with -halocarbonyl compounds 13a-c afforded the thiophene derivatives
16a-d, which were formed via water elimination from the intermediates 14b-e.
A concurrent formation of thiazoles of the type 15 and thiophenes of the type 16a-d has been also reported
in the literature [25]. It is important to note that in such reaction, although the same reagent ethyl chloroacetate
(12a) was used, thiazole derivative 15 and thiophene derivative 16a were alternatively formed. Such findings are
complementary to those reported earlier [26].
All synthesized compounds 2-16 were evaluated for their capacity to inhibit the in vitro growth of breast
adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460), and human glioblastoma (SF-268) cell lines,
after 48 h continuous exposure (Table 3). The In vitro Anticancer Drug Discovery Screen, a procedure adopted
by the National Cancer Institute (NCI, USA), was employed, which uses the protein-binding dye sulforhodamine
B to assess cell growth [27]. The dose–response curves were obtained and, in each case, the concentration of the
compound that inhibits 50% of the net cell growth (IC50) was calculated [27]. The above-mentioned three cancer
cell lines were selected as our compounds are electron-rich systems substituted with electronegative groups and
many reports from previous work [28-31] used such cell lines together with the use of doxorubicin, which was
shown to be the best positive control against these three cell lines.
398

TABLE 3. Effect of the Synthesized Compounds on the Growth of
Human Tumor Cell Lines
Inhibitory concentration IC50, 
Compound
MCF-7
20.0 ± 2.2
NCI-H460
26.3 ± 2.4
SF-268
24 ± 4.6
1b
1c
26.2 ± 2.9
34.2 ± 12.6
22.2 ± 4.5
10.0 ± 0.6
8.0 ± 4.2
24.8 ± 4.6
33.7 ± 6.6
22.8 ± 4.2
8.4 ± 2.4
28.2 ± 12.6
44.2 ± 8.2
18.0 ± 4.4
8.8 ± 4.8
1d
1e
2a
6.3 ± 2.6
8.0 ± 1.8
2b
0.2 ± 0.09
30.2 ± 10.9
0.02 ± 0.01
2.2 ± 0.8
0.8 ± 0.08
22.7 ± 2.8
0.08 ± 0.01
4.6 ± 0.4
0.2 ± 0.06
40.2 ± 6.0
0.06 ± 0.02
1.2 ± 0.8
3a
3b
4
6a
30.0 ± 2.5
10.0 ± 0.8
30.4 ± 2.8
0.01 ± 0.008
77.8 ± 10.0
0.4 ± 0.1
22.0 ± 4.6
8.3 ± 2.8
18.5 ± 2.8
16.5 ± 4.0
36.3 ± 4.5
0.08 ± 0.08
70.2 ± 12.6
0.1 ± 0.02
10.2 ± 4.8
4.2 ± 1.8
6b
6c
20.1 ± 4.6
0.01 ± 0.006
64.2 ± 8.4
0.2 ± 0.01
10.0 ± 2.6
2.6 ± 0.4
6d
6e
6f
7a
14.2 ± 8.2
4.2 ± 0.8
7b
9a
32.0 ± 2.5
0.01 ± 0.008
10.0 ± 0.8
44.4 ± 6.8
70.8 ± 10.0
10.2 ± 8.2
0.4 ± 0.1
24.0 ± 4.6
0.01 ± 0.006
8.3 ± 2.8
26.5 ± 2.8
0.08 ± 0.08
16.5 ± 4.0
34.3 ± 2.5
74.2 ± 12.6
6.2 ± 2.8
9b
9c
10a
10b
11a
11b
15
26.1 ± 2.6
66.2 ± 8.4
8.0 ± 2.6
0.2 ± 0.01
20.1 ± 0.6
0.02 ± 0.006
22.2 ± 8.4
6.0 ± 1.6
0.1 ± 0.02
36.3 ± 0.5
0.06 ± 0.08
12.2 ± 4.6
8.2 ± 2.8
20.4 ± 2.8
0.04 ± 0.008
30.8 ± 10.0
8.2 ± 2.2
16a
16b
16c
16d
Doxorubicin
0.04 ± 0.008
0.09 ± 0.008
0.09 ± 0.007
Some selected compounds were also evaluated against three normal cell lines: human fibroblast (WI-
38), normal prostate epithelial cells (PrEC), and normal colon mucosal (NCM 460) cells. To further characterize
the possible differential effects of the obtained compounds on tumor and normal cells, we compared cell
viability (scored as membrane integrity by the trypan blue exclusion assay) [32] after treatment with the
synthesized compounds.
The normal PrEC cells showed minimal loss of viability up to at least 25 M of the tested compound
(i.e., about 75IC50) even after a 24 h continuous treatment (Table 4). Other normal cell lines showed similar
marginal decreases in cell viability.
The results show that all compounds were able to inhibit the growth of both human tumor and normal
cell lines in a dose-dependent manner (Table 3, 4). Although in most cases the growth inhibiting effect was
moderate, a more pronounced effect was found with compounds 3a, 4, 6e, 7a, 9c, 15, and 16b where such
399

TABLE 4. Effect of the Obtained Compounds on the Growth of Normal
Cell Lines
Com-
Inhibitory concentration IC50, 
pound
WI-38
3.11 ± 0.08
PrEC
NCM 460
2.81 ± 0.70
0.67 ± 0.02
0.03 ± 0.002
4.42 ± 0.9
4.21 ± 1.12
2a
2b
3a
3b
4
0.18 ± 0.08
0.14 ± 0.006
6.33 ± 1.13
0.06 ± 0.003
0.36 ± 0.02
0.18 ± 0.03
0.88 ± 0.01
0.18 ± 0.04
0.21 ± 0.01
3.47 ± 0.28
0.18 ± 0.03
0.24 ± 0.02
0.04 ± 0.006
2.28 ± 0.4
0.08 ± 0.005
0.22 ± 0.02
0.29 ± 0.01
1.33 ± 0.09
0.13 ± 0.02
0.38 ± 0.05
5.45 ± 2.03
0.17 ± 0.04
0.01 ± 0.004
0.42 ± 0.01
0.49 ± 0.004
1.68 ± 0.03
0.28 ± 0.01
0.49 ± 0.08
5.09 ± 1.74
0.28 ± 0.06
6a
6e
7a
9c
15
16a
16b
compounds showed the best results, exhibiting submicromolar inhibitory effect against the three tumors, but
also against normal cell lines. Compounds 4, 6e and 9c showed higher inhibitory effect towards the tumor cell
lines than the reference control doxorubicin. However, only compound 7a showed selective inhibition of the
tumor cell lines.
In summary, a series of new 2-substituted 4,5,6,7-tetrahydro-1-benzothiophene derivatives was
synthesized, an in vitro cell viability assay was employed to investigate the inhibition effect of 31 compounds
against three tumor cell lines, and eleven selected compounds were also evaluated against normal cell lines. It
was found that some of the compounds achieved promising cytotoxicity with IC50 values lower than 5 M
against some cancer cell lines.
EXPERIMENTAL
IR spectra were recorded in KBr pellets on a Pye Unicam SP-1000 spectrophotometer. 1H NMR spectra
were recorded on a Varian EM-390 spectrometer at 200 MHz in DMSO-d6 using TMS as internal standard.
Mass spectra were recorded with a Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu
instruments. Melting points were determined on an Electrothermal 9100 melting point apparatus and are
uncorrected. Elemental analysis data were obtained from the Microanalytical Data Unit at Cairo University,
Giza, Egypt. Antitumor activity evaluation of the newly synthesized products was performed by a research
group at the National Research Center (Medicinal Section) and the National Cancer Institute at Cairo
University. Fetal bovine serum and L-glutamine were from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640
medium was from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, penicillin,
streptomycin, and sulforhodamine B were from Sigma Chemical Co. (Saint Louis, MO, USA). Stock solutions
of all compounds were prepared in DMSO and kept at -20C. Appropriate dilutions of the compounds were
freshly prepared just prior to assays. The final concentrations of DMSO did not interfere with the cell growth.
MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), and NCI-H460
and SF-268 were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). WI-38 and PrEC cells
were purchased from the American Type Culture, and NCM 460 cells were obtained from In Cell Corporation
LLC. Gen-Probe kits were obtained from Clonetics.
N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-3-oxobutanamide (2a) and Ethyl 2-[(3-oxo-
butanoyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (2b) (General Method). Ethyl
acetoacetate (13.0 g, 0.1 mol) was heated to 140°C, then compound 1a (17.8 g, 0.1 mol) or 1b (22.5 g, 0.1 mol)
400

was added with continuous heating until the temperature reached 125°C, and the whole reaction mixture was
heated under reflux for 20 min, then left to cool. The formed solid product was triturated with ethanol and
collected by filtration.
2-Benzylidene-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-3-oxobutanamide (3a) and
Ethyl
2-(2-Benzylidene-3-oxobutanamido)-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate
(3b)
(General Method). Benzaldehyde (1.06 g, 0.01 mol) was added to a solution of compound 2a (2.62 g, 0.01 mol)
or 2b (3.09 g, 0.01 mol) in 1,4-dioxane (40 ml) containing piperidine (0.5 ml). The reaction mixture was heated
under reflux for 2 h, then poured onto ice/water containing a few drops of hydrochloric acid to reach pH 6. The
solid product was collected by filtration.
N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-2-(2-hydroxybenzylidene)-3-oxobutanamide
(4). Salicylaldehyde (1.22 g, 0.01 mol) was added to a solution of compound 2a (2.62 g, 0.01 mol) in
1,4-dioxane (40 ml) containing piperidine (0.5 ml). The reaction mixture was heated under reflux for 3 h, then
worked up as above.
2-(2-Arylhydrazinylidene)-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-3-oxobutanamides
6a-c and Ethyl 2-[2-(2-Arylhydrazinylidene)-3-oxobutanamido]-4,5,6,7-tetrahydro-1-benzothiophene-3-carb-
oxylates 6d-f (General Method). A solution of the corresponding diazonium salt 5a-c (0.01 mol), prepared by
adding sodium nitrite solution (0.70 g, 0.01 mol) to a stirred cold solution of the corresponding aniline
(0.01 mol) in concentrated hydrochloric acid (20 ml), was added with stirring to a cold (0-5°C) solution of
compound 2a (2.62 g, 0.01 mol) or 2b (3.09 g, 0.01 mol) in ethanol (40 ml) containing 5% sodium hydroxide
(5 ml). The reaction mixture was maintained at room temperature for 1 h, and the formed solid product was
collected by filtration.
3-[2-(Cyanoacetyl)hydrazinylidene)]-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)butan-
amide (7a) and Ethyl 2-{3-[2-(Cyanoacetyl)hydrazinylidene]butanamido}-4,5,6,7-tetrahydro-1-benzo-
thiophene-3-carboxylate (7b) (General Method). Cyanoacetylhydrazine (1.0 g, 0.01 mol) was added to a
solution of either compound 2a (2.62 g, 0.01 mol) or 2b (3.09 g, 0.01 mol) in 1,4-dioxane (40 ml). The reaction
mixture was heated under reflux for 2 h, then concentrated under vacuum. The residue was triturated with
ethanol, and the formed solid product was collected by filtration.
2-Amino-1-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-4-methyl-6-oxo-1,6-dihydropyridine-
3-carbonitrile (9a) and Ethyl 2-Amino-1-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-4-methyl-
6-oxo-1,6-dihydropyridine-3-carboxylate (9b) (General Method). Either malononitrile (0.66 g, 0.01 mol) or
ethyl cyanoacetate (1.13 g, 0.01 mol) was added to a solution of compound 2a (2.62 g, 0.01 mol) in 1,4-dioxane
(40 ml) containing triethylamine (0.50 ml). The reaction mixture was heated under reflux for 4 h, then poured
onto ice/water containing 18 M hydrochloric acid (0.50 ml) to reach pH 6, and the formed solid product was
collected by filtration.
Ethyl 2-(6-Amino-5-cyano-4-methyl-2-oxopyridin-1(2H)-yl)-4,5,6,7-tetrahydro-1-benzothiophene-
3-carboxylate (9c). Malononitrile (0.66 g, 0.01 mol) was added to a solution of compound 2b (3.09 g, 0.01 mol)
in 1,4-dioxane (40 ml) containing triethylamine (0.50 ml). The reaction mixture was heated under reflux for 6 h,
then poured onto ice/water containing hydrochloric acid (0.5 ml), and the formed solid product was collected by
filtration.
4,4-Dicyano-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-3-methylbut-3-enamide (10a) and
Ethyl 2-Cyano-5-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-ylamino)-3-methyl-5-oxopent-2-enoate
(10b) (General Method). Either malononitrile (0.66 g, 0.01 mol) or ethyl cycanoacetate (1.13 g, 0.01 mol) was
added to a dry solid compound 2a (2.62 g, 0.01 mol). Ammonium acetate (0.50 g) was added to the reaction
mixture. The reaction mixture was heated in an oil bath at 120C for 15 min, then left to cool. The residue was
triturated with ethanol, and the formed solid product was collected by filtration.
2-(3-Acetyl-4,6-dimethyl-2-oxopyridin-1(2H)-yl)-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile
(11a) and Ethyl 2-(3-Acetyl-4,6-dimethyl-2-oxopyridin-1(2H)-yl)-4,5,6,7-tetrahydro-1-benzothiophene-
3-carboxylate (11b) (General Method). Acetylacetone (1.0 g, 0.01 mol) was added to a solution of compound
401

2a (2.62 g, 0.01 mol) or 2b (3.09 g,0.01 mol) in 1,4-dioxane (40 ml). The whole reaction mixture was heated
under reflux for 8 h, then concentrated under vacuum. The residue was triturated with ethanol, and the formed
solid product was collected by filtration.
N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-2-(4-hydroxy-3-phenylthiazol-2(3H)-ylidene)-3-
oxobutanamide (15), 5-Acetyl-N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-4-methyl-2-(phenyl-
amino)thiophene-3-carboxamide (16a), and Ethyl 2-[5-Acyl-4-methyl-2-(phenylamino)thiophene-3-carb-
oxamido]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylates 16b-d (General Method). Potassium
hydroxide (0.56 g, 0.01 mol) and then phenylisothiocyanate (1.30 g, 0.01 mol) were added to a solution of
compound 2a (2.62 g, 0.01 mol) or 2b (3.09 g, 0.01 mol) in dimethyl formamide (30 ml). The reaction mixture
was stirred at room temperature overnight. On the next day, the corresponding -halocarbonyl compound 13a,
13b (with 2b only), or 13c (0.01 mol) was added with continuous stirring overnight at room temperature. The
reaction mixture was poured onto ice/water containing a few drops of 18 M hydrochloric acid (0.50 ml) to reach
pH 6. The solid product formed in each case was collected by filtration.
Tumor Cell Cultures. The human tumor cells grow as a monolayer and were routinely maintained in
RPMI-1640 medium supplemented with 5% heat-inactivated FBS, 2 M glutamine, and antibiotics (penicillin
100 U/ml, streptomycin 100 g/ml) at 37°C in a humidified atmosphere containing 5% CO2. Exponentially
growing cells were obtained by plating 1.5105 cells/ml for MCF-7 and SF-268 and 0.75104 cells/ml for
NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these
cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum
concentration (0.5%) of DMSO used in each assay.
Normal Cell Cultures. All cell lines were tested regularly for Mycoplasma contamination by the DNA
hybridization method using a Gen-Probe kit [33].
Cell Growth Assay. Exponentially growing cells in 96-well plates were exposed for 48 h to five serial
concentrations of each compound, starting from a maximum concentration of 150 M. Following this exposure
period, adherent cells were fixed, washed, and stained with sulforhodamine B [33]. The bound stain was
solubilized, and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc.,
Powerwave XS, Winooski, Vermont, USA).
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