Synthesis of novel heterocyclic compounds incorporate 4,5,6,7-tetrahydrobenzo[b]thiophene together with their cytotoxic evaluations

Article abstract of DOI:10.1248/cpb.c16-00925

The 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene was the key starting compound used to synthesize new thiazole, pyrimidine, pyran, pyridine and thiazine derivatives. The cytotoxicity of the synthesized compounds was studied towards the three cancer cell lines namely MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and SF-268 (central nervous system (CNS) cancer) in addition to the normal cell line (WI-38) using doxorubicin as the reference drug. The study showed that compounds 5, 9a, 15b, 17c, 18 and 21b were the most potent compounds.

Full text of DOI:10.1248/cpb.c16-00925

Vol. 65, No. 5
Chem. Pharm. Bull. 65, 469–477 (2017)
469
Regular Article
Synthesis of Novel Heterocyclic Compounds Incorporate
4,5,6,7-Tetrahydrobenzo[b]thiophene Together with Their Cytotoxic
Evaluations
,a
Amira Elsayed Mahmoud Abdallah,* Rafat Milad Mohareb,b Eid Metwally Khalil,a and
Menna Alla Mohamed Abd Elaleem Elshamya
a Department of Chemistry, Faculty of Science, Helwan University; Ain Helwan, Cairo 11795, A. R. Egypt: and
b Department of Chemistry, Faculty of Science, Cairo University; Giza 12614, A. R. Egypt.
Received November 28, 2016; accepted February 19, 2017
The 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene was the key starting compound used to syn-
thesize new thiazole, pyrimidine, pyran, pyridine and thiazine derivatives. The cytotoxicity of the synthe-
sized compounds was studied towards the three cancer cell lines namely MCF-7 (breast adenocarcinoma),
NCI-H460 (non-small cell lung cancer) and SF-268 (central nervous system (CNS) cancer) in addition to the
normal cell line (WI-38) using doxorubicin as the reference drug. The study showed that compounds 5, 9a,
15b, 17c, 18 and 21b were the most potent compounds.
Key words tetrahydrobenzo[b]thiophene; cytotoxic evaluation; multicompounant reaction; pyrimidine; thiazole
The reactions of the 2-amino-tetrahydrobenzo[b]thiophene 3 were in agreement with its structure (see Experimental
derivatives with different chemical organic reagents were section). The reaction of compound 2-amino-3-cyano-4,5,6,7-
found to be of great interest in heterocyclic chemistry lead- tetrahydrobenzo[b]thiophene with phenylisothiocyanate and
ing to the formation of this ring system incorporated in many chloroacetone gave the thiazole derivative 5. Formation of the
heterocyclic compounds including thiazole,1) pyrimidine,2) thia- latter product took place through the intermediate formation
zine,3) pyridine4) and pyran.5) On the other side, the derivatives of 4 followed by water elimination (Chart 1).
of tetrahydrobenzo[b]thiophene exist in many pharmaceutical
Next we studied the possibility of compound 2-amino-
applications such as anti-tumor,6) antimicrobial,7–9) antiviral,10) 3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene to form thio-
anti-leishmanial agents,11) antioxidant activity,12) anti-arrhyth- urea derivative. Thus, compound 2-amino-3-cyano-4,5,6,7-
mic, serotonin antagonist and anti-anxiety activities.13)
tetrahydrobenzo[b]thiophene
reacted
with
phenyl
Recently we were involved through the synthesis
of polyfunctional heterocyclic compounds, where the
2-cyanomethylbenzo[c]imidazole was used as the key start-
ing compound.14) In addition, we were involved through
the reaction of ethyl acetoacetate with elemental sulphur
and either malononitrile or ethyl cyanoacetate gave thio-
phene derivatives.15) In the present work we were concerned
through exploring the reactivity of the amino group present
in the 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene16)
towards different chemical reagents to afford a vast number
of heterocyclic compounds involving tetrahydrobenzo[b]-
thiophene moiety and evaluating their cytotoxicity.
Results and Discussion
Chemistry The reaction of the 2-amino-3-cyano-4,5,6,7-
tetrahydrobenzo[b]thiophene with triethyl orthoformate in ace-
tic acid gave the ethyl N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]-
thiophen-2-yl)formimidate 1. The analytical and spectral data
of compound 1 were the tools of its structural elucidation.
Compound 1 reacted with aniline to give the N-phenyl for-
mamidine derivative 2. The elemental and spectral data of
compound 2 were consistent with its proposed structure. Thus,
1H-NMR spectrum of compound 2 showed, beside the expect-
ed signals, a singlet at 6.95ppm δ for CH moiety, a muliplet
at δ 7.27–7.90ppm for phenyl ring and a singlet at δ 8.36ppm
for NH group. On the other hand, the reaction of compound
1 with malononitrile gave the imino-methyl malononitrile
derivative 3. The analytical and spectral data of compound
Chart 1.
*To whom correspondence should be addressed. e-mail: amiraelsayed135@yahoo.com
© 2017 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 65, No. 5 (2017)
Chart 3.
Chart 2.
isothiocyanayte in ethanol containing a catalytic amount of
triethylamine to give the N-phenylthioura derivative 6. The
latter reacted with ethyl 2-chloroacetate in ethanol to give
the thiazole derivative 7. The structure of compound 7 was
confirmed on the basis of analytical and spectral data. Thus,
1
the H-NMR showed, beside the expected signals, a singlet at
δ 4.50ppm for CH thiazole ring, a multiplet at δ 6.92–7.70ppm
indicating phenyl group and a singlet at δ 11.10ppm (D2O
exchangeable) for OH group. In addition, the 13C-NMR spec-
trum showed beside the expected signals, a signal at δ 180.0
corresponding to the excocyclic C=N group and a signal at δ
60.1 corresponding to the thiazole C-5. Similarly, the reaction
of compound 6 with 2-bromo-1-(4-chlorophenyl)ethanone gave
the thiazole derivative 8 the structure of which structure was
based on its analytical and spectral data (see Experimental
section).
Chart 4.
Compound 6 was used to synthesize pyrimidine deriva-
tives with potential biological activities. Thus, the reaction of
compound 6 with either malononitrile or ethyl cyanoacetate was based on the reported literature which showed that
gave the 4,6-diaminodihydropyrimidine derivatives 9a and 9b, this structure is the favored one.17) Compound 6 underwent
respectively. On the other hand, the reaction of compound 6 ready cyclization in 1,4-dioxane and piperidine to afford the
with ethyl acetoacetate gave the 4-methyl-6-oxodihydropyrim- 4-imino-3-phenyl-3,4,5,6,7,8-hexa-hydrobenzo[4,5]thieno[2,3-
idine derivative 10. The reaction took place through ethanol d]pyrimidine-2-thiol 13 (Chart 3). The analytical and spectral
and water elimination. The analytical and spectral data of data of compound 13 were the basis of its structural elucida-
compounds 9a, 9b and 10 are consistent with their respective tion (see Experimental section).
structures (Chart 2).
In addition compound 6 underwent multi-component reac-
However, the reaction of compound 6 with acetylac- tions through its reaction with malononitrile and any of benz-
etone in 1,4-dioxane containing a catalytic amount of pi- aldehyde, 4-chlorobenzaldehyde or 4-methoxy-benzaldehyde
peridine gave the 4,6-dimethyl-6-hydroxydihydropyrimidine to produce the thiazine derivatives 15a–c. The reaction took
derivative 11. The latter compound, due to the presence of place through the intermediates formation of 14a–c followed
the acidic pyrimidine H-5 coupled with benzenediazonium by cyclization (Chart 4).
chloride gave the phenyl azo derivative 12. It is worthy to
note that through the products 11 and 12 the OH group thiophene underwent
The compound 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]-
multi-component reaction when
a
being at the carbon adjacent to the N-ph not at the carbon reacted with phenyl isothiocyanate, malononitrile and any
adjacent to the tetahydrobenzo[b]thiophene moiety this of benzaldehyde, 4-chlorobenzaldehyde or 4-methoxybenz-

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
471
Chart 6.
the benzaldehyde react with compound 19 in 1,4-dioxane/N,N-
dimethylformamide (DMF) solvent to produce first the
benzylidine derivative this was followed by its reaction of
malononitrile in the second step to produce the 2-pyridone
Chart 5.
aldehyde to give the tetrahydropyrimidine derivatives 17a–c, derivatives. However, in this work the reaction is considered
respectively. The reaction took place through the intermedi- as a multi-component reaction occurred in one step where
ates formation of 16a–c. The analytical and spectral data of similar multi-component reactions producing pyran deriva-
17a–c was in agreement with their respective structures. Thus tives were reported before.29–31) The produced pyran deriva-
1H-NMR spectrum of 17a (as an example) showed beside tives 20a–c were found to be stable especially when heated
the expected signals, a singlet at δ 7.52ppm indicating the in 1,4-dioxane/DMF solvent during our trials to convert them
pyrimidine H-6, multiplet at δ 7.53–7.99ppm for two phenyl to the 2-pyridone derivatives. On carrying the same multi-
moieties and singlet at δ 8.66ppm (D2O exchangeable) for component reaction but in the presence of ammonium acetate
NH2 group. Similarly, the multi-component reaction of com- instead of triethylamine gave the pyridine derivatives 21a–c,
1
pound 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene respectively (Chart 6). Thus, the H-NMR and mass spectrum
with phenyl isothiocyanate, malononitrile and furfural in of compound 21c (as an example) showed that singlet at δ
ethanol and triethylamine produced the tetrahydropyrimidine 1.30ppm for CH3 group, multiplet at δ 1.69–2.68ppm for the
derivative 18 (Chart 5).
four CH2 groups of cyclohexene ring, singlet at 6.95ppm for
The analytical and spectral data of the latter compound pyridine ring, multiplet for [C6H4] moiety at δ 7.09–7.99 ppm,
were consistent with its proposed structure (see Experimental singlet at δ 8.30ppm for NH2 group and two singlets at δ 8.39
section).
and 8.54ppm for two 2NH moieties. Also, the mass spectrum
It has been reported that the compound 2-amino-3-cyano- of 21c exhibited a molecular ion peak [M+] (m/z 428) corre-
4,5,6,7-tetrahydrobenzo[b]thiophene reacted with ethyl cya- sponding to molecular formula C23H20N6OS.
noacetate in dimethylformamide gave the N-cyanoacetamido
Biological Activity Evaluations
In Vitro Cytotoxicity Evaluations of the Newly Synthesized
derivative 19.18,19) It has been reported that multi-component
reactions (MCRs) can combine three or more components Compounds
together in a single reaction vessel and produce final products
The cytotoxicity of the newly synthesized compounds was
with a minimum of synthetic time and effort20) because they evaluated in the presence of three cancer cell lines and the
are no need to separate any reaction intermediate.21) Such results were demonstrated (Table 1). The three cancer cell
MCRs often result in high atomeconomy and high selectiv- lines were [MCF-7 (breast adenocarcinoma), NCI-H460 (non-
ity products.22) They are also applicable to the synthesis of small cell lung cancer), SF-268 (central nervous system (CNS)
heterocyclic systems.23) It is quite remarkable that many top- cancer)] and normal fibroblasts cells (WI38). The data showed
selling pharmaceuticals containing 4-H pyran derivatives24–28) that some compounds showed promising results, and the re-
which encouraged us to synthesize the 4-H pyran and 4-H sults were compared to the anti-proliferative affects of the
pyridine derivatives through the multi-component reactions reference control doxorubicin.32) All the compounds were dis-
of compound 19. Thus, compound 19 underwent the MCR of solved in dimethyl sulfoxide (DMSO) at 1mg/mL immediately
malononitrile and any of benzaldehyde, 4-chlorobenzaldehyde before use and diluted just before addition to the cell culture.
or 4-methoxybenzaldehyde in ethanolic triethylamine solution
The data (Table 1) represents the mean standard error
to give the pyran derivatives 20a–c, respectively. It should be of the mean (S.E.M.) of three independent experiments per-
useful to mention that similar reaction was carried before by formed in duplicate. The results indicated that most com-
our research group18) but it was in two steps reaction, firstly pounds demonstrated substantial growth inhibitory effects

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against the human tumor cells at the concentrations tested.
Structure Activity Relationship
more potency with GI50’s 0.01, 0.04 and 0.70µM against the
three cancer cell lines MCF-7, NCI-H460 and SF-268, re-
It is of great value to notice that the newly synthesized prod- spectively than compound 9b with the 6-oxo moiety. The
ucts 5, 9a, 15b, 17c, 18 and 21b exhibited optimal cytotoxic hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine derivative 13
effect against the three cancer cell lines than the reference showed high potency against the three cancer cell lines and
control doxorubicin, with GI50’s (50% of growth inhibition) this was attributed due to its annulated structure. Comparing
in the µM range. The cytotoxic effect of compound 5 showed the cytotoxicity of the thiazine derivatives 15a–c, it is obvious
that high potency due to the presence of the cyclohexene moi- that the cytotoxicity of 15b the highest cytotoxicity among the
ety together with the 3-cyano group of the thiophene moiety. three compounds. The presence of the chlorine (Cl) and the
Considering the dihydropyrimidine derivatives 9a and 9b it thiophene groups were responsible for its high potency.
is obvious that compound 9a with the 6-imino group showed
On the other hand, for the tetrahydropyrimidine derivatives
Fig. 1. The Optimal Cytotoxic Effect of the Six Newly Synthesized Compounds against the Three Cancer Cell Lines
Table 1. Cytotoxic Evaluations of the Newly Synthesized Compounds in GI50 (µM) on the Growth of Three Human Tumor Cell Lines and Normal
Human Cell Line
GI50 (µM)a)
Compound
MCF-7
NCI-H460
SF-268
WI-38
2-Amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene
11.20 2.23
30.8 7.21
16.29 4.06
30.02 3.38
0.01 0.001
30.0 3.22
28.22 6.15
0.01 0.003
22.8 6.18
18.29 3.61
12.80 2.39
28.75 6.16
0.60 0.01
2.84 2.04
0.03 0.002
1.29 0.04
36.20 6.24
0.91 0.29
0.02 0.004
0.02 0.005
2.84 0.14
31.41 2.83
0.60 0.07
18.31 8.22
0.08 0.002
18.61 6.22
0.0428 0.0082
16.29 2.26
22.8 3.70
8.01 2.39
12.31 1.89
18.29 6.39
18.21 6.29
0.06 0.002
21.30 2.82
16.20 2.93
0.40 0.09
>100
>100
>100
1
2
20.81 8.29
28.19 8.24
0.02 0.004
20.81 4.21
44.19 4.67
0.07 0.008
21.05 5.02
14.11 3.36
18.02 4.11
24.58 16.07
0.4 0.02
3
58.70 10.20
5
7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8
9a
9b
20.39 2.29
10.18 3.53
30.30 4.12
18.41 4.22
0.02 0.006
6.08 2.16
10
11
12
13
15a
15b
15c
17a
17b
17c
18
3.62 1.04
0.02 0.003
5.29 1.84
0.05 0.002
3.14 1.06
26.19 6.22
0.80 0.41
22.40 3.21
0.38 0.06
0.06 0.003
0.06 0.004
4.62 1.04
0.08 0.005
0.02 0.003
6.08 1.16
20a
20b
20c
21a
21b
21c
*Doxorubicin
8.21 1.61
28.2 6.2
>100
>100
>100
20.80 4.33
0.80 0.14
18.21 3.70
0.018 0.002
10.39 4.23
0.02 0.002
17.21 4.51
0.0940 0.0070
32.21 6.30
0.08 0.003
20.22 10.11
0.0940 0.0087
>100
>100
a) Drug concentration required to inhibit tumor cell proliferation by 50% after continuous exposure of 48h; data are expressed as means S.E.M. of three independent ex-
periments performed in duplicates; *Doxorubicin was used as positive control.

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
473
17a–c, it is clear that both of compounds 17b and 17c were 0.01mol) was added. The reaction mixture was heated under
more cytotoxic than compound 17a this was attributed to reflux for 2h, then cooled and neutralized by pouring onto
the presence of the Cl and OCH3 groups in both compounds, ice/water mixture. The solid product formed was collected by
respectively. However, compound 17c showed higher potency filtration and crystallized from acetic acid.
than 17b. It is clear from Table 1 that compound 18 indicated
Brown crystals, mp 102–105°C, IR (KBr) cm−1: 2995, 2928,
1
high cytotoxicity and this is attributed to the presence of the 2214, 1621, 1435, 1554. H-NMR (DMSO-d6) δ: 1.20 (t, 3H,
pyrimidine, furan and the thione moieties. Considering com- CH3), 1.70–1.99 (m, 4H, 2CH2), 2.13–2.99 (m, 4H, 2CH2),
pounds 21a–c where compound 21b showed higher cytotoxic- 4.10 (q, 2H, CH2), 6.96 (s, 1H, CH). 13C-NMR (DMSO-d6) δ:
ity than 21a and 21c due to the presence of Cl moiety (Fig. 1). 162.7, 158.9, 146.6, 145.0, 114.2, 101.0, 62.0, 24.8, 24.0, 23.4,
It is very clear from our present finding that the newly syn- 21.7, 21.6. MS m/z (%): 236 [M++2] (0.36), 235 [M++1] (0.12),
thesized products with the electronegative moieties such as Cl, 234 [M+] (0.11), 233 [M+−1] (0.07), 232 [M+−2] (0.05), 150
cyano (CN) and methoxy (OCH3) groups in the pyrimidine (100.00). Anal. Calcd for C12H14N2OS (234.32): C, 61.51; H,
and thiazine derivatives show greater cytotoxic property and 6.02; N, 11.96; S, 13.68. Found: C, 61.73; H, 6.30; N, 12.20;
might play a very important role in enhancing the cytotoxic S, 13.32.
effect. It is important to mention that our previous reports
Preparation of N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]-
showed the cytotoxicity of other unsaturated benzothiophene, thiophen-2-yl)-N-phenyl Formamidine (2) To a mixture
pyran and other N-containing heterocyclic derivatives.33–35)
of equimolar amount of 2-amino-4,5,6,7-tetrahydrobenzo[b]-
thiophene-3-carbonitrile (2.34g, 0.01mol) in acetic acid
(20mL), aniline (0.93g, 0.01mol) was added. The reaction
Conclusion
The objective for the present study was to synthesize a mixture were heated under reflux for 2h, and then cooled by
series of tetrahydrobenzo[b]thiophene derivatives based on pouring onto ice/water mixture. The solid product formed col-
2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile. lected by filtration and crystallized from acetic acid.
Study the reactivity of the latter towards different chemical
Off white crystals, mp 179–182°C, IR (KBr) cm−1: 3426,
reagents and establish the structure of the newly synthesized 3210, 3080, 2997, 2840, 2217, 1622, 1438, 1555. 1H-NMR
compounds based on elemental analysis and spectral data. (DMSO-d6) δ: 1.70–1.91 (m, 4H, 2CH2), 2.04–2.77 (m, 4H,
On the other extreme, the cytotoxic activity of some of the 2CH2), 6.95 (s, 1H, CH), 7.27–7.90 (m, 5H, C6H5), 8.36 (s, 1H,
newly synthesized compounds (twenty six compounds) was NH). 13C-NMR (DMSO-d6) δ: 162.7, 159.0, 146.6, 146.0, 145.0,
evaluated on three human cancer cell lines and normal human 126.9, 126.9, 120.5, 116.8, 116.1, 114.2, 101.0, 24.6, 24.0, 23.7,
cell line. Moreover, the results showed that compounds 5, 9a, 21.7. MS m/z (%): 282 [M++1] (8.51), 281 [M+] (43.35), 280
15b, 17c, 18 and 21b revealed higher effect than the reference [M+−1] (4.26), 80 (100.00), 77 [C6H5]+ (38.30). Anal. Calcd
doxorubicin when screened in vitro against the three human for C16H15N3 (281.38): C, 68.30, H, 5.37, N, 14.93, S, 11.40.
cancer cell lines tested such as MCF-7 (breast adenocarcino- Found: C, 67.90; H, 5.02; N, 14.69; S, 11.13.
ma), NCI-H460 (non-small cell lung cancer) and SF-268 (CNS
cancer) and normal fibroblasts human cell line (WI-38).
Preparation of 2-(2,2-Dicyanoethylideneamino)-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carbonitrile (3) This com-
pound was prepared according to the same procedure as that
applied for 2 using malononitrile (0.66g, 0.01mol).
Experimental
General All melting points were determined on an Elec-
Brown crystals, mp 152–155°C, IR (KBr) cm−1: 2996, 2841,
1
tro-thermal digital meting point apparatus and are uncorrect- 2260, 2212, 2195, 1623, 1436, 1555. H-NMR (DMSO-d6) δ:
ed. IR Spectra (KBr discs) were recorded on a FITR plus 460 1.70–1.91 (m, 4H, 2CH2), 2.17–2.57 (m, 4H, 2CH2), 6.80, 6.95
1
or Pye Unicam SP-1000 spectrophotometer. H-NMR spectra (2s, 2H, 2CH). 13C-NMR (DMSO-d6) δ: 162.7, 158.1, 146.6,
were recorded with Varian Gemini-200 (200 MHZ) (Cairo 144.1, 116.1, 114.2, 114.2, 101.1, 24.4, 23.9, 23.4, 22.2, 21.7.
University) and Jeol AS 500MHz (National Research Center) MS m/z (%): 256 [M++2] (1.13), 255 [M++1] (1.23), 254 [M+]
instruments in DMSO-d6 as solvent using tetramethylsilane (0.30), 253 [M+−1] (0.34), 252 [M+−2] (0.38), 69 (100.00).
(TMS) as internal standard and chemical shifts are expressed Anal. Calcd for C13H10N4S (254.31): C, 61.40; H, 3.96; N,
as δ ppm. The mass spectra were recorded with Hewlett Pack- 22.03; S, 12.61. Found: C, 61.10; H, 3.69; N, 21.80; S, 9.64.
ard 5988 AGC/MS system and GCMS-QP1000 Ex shimadzu
Preparation of (2Z)-2-(4-Methyl-3-phenylthiazole-2(3H)-
instruments. Analytical data were obtained from the Micro ylideneamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-car-
analytical data unit at Cairo University and were performed bonitrile (5) Equimolar amount of 2-amino-4,5,6,7-
on Vario El III Elemental CHNS analyzer.
tetrahydrobenzo[b]-thiophene-3-carbonitrile (1.78g, 0.01mol)
Chemistry
in absolute ethanol (25mL) containing a catalytic amount of
Preparation of the 2-Amino-3-cyano-4,5,6,7-tetrahydro- triethylamine (0.50mL), α-chloroacetone (0.92g, 0.01mol)
benzo[b]thiophene16) To a solution of cyclohexanone (9.30g, and phenyl isothiocyanates (1.35g, 0.01mol) were added. The
0.10mol) in ethanol (50mL) containing triethylamine (1.50mL) reaction mixture was heated under reflux for 3h and cooled
both of elemental sulfur (3.20g, 0.10mol) and malononitrile by pouring onto acidified ice/water mixture the solid product
(6.60g, 0.10mol) were added. The whole reaction mixture was formed in each case was collected by filtration, washed with
heated under reflux for 30min. then left to cool. The formed water and crystallized from absolute ethanol.
crystals were collected by filtration.
Off white crystals, mp 142–145°C, IR (KBr) cm−1: 3100,
Preparation of Ethyl N-(3-Cyano-4,5,6,7-tetrahydrobenzo- 2954, 2836, 2195, 1521, 1619, 1431. 1H-NMR (DMSO-d6)
[b]thiophen-2-yl)formimidate (1) To a solution of 2-amino- δ: 1.20 (s, 3H, CH3), 1.69–1.90 (m, 4H, 2CH2), 2.32–2.51
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile
(1.78g, (m, 4H, 2CH2), 6.94 (s, 1H, thiazole C5), 6.94–7.80 (m, 5H,
0.01mol) in acetic acid (20mL), triethyl orthoformate (1.48g, C6H5). 13C-NMR (DMSO-d6) δ: 162.7, 158.1, 149.1, 144.0,

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Vol. 65, No. 5 (2017)
141.3, 141.1, 120.7, 120.7, 116.8, 116.1, 116.1, 115.0, 101.1, 83.2, tetrahydrobenzo[b]thiophene-3-carbonitrile (9a and b)
24.5, 24.0, 23.4, 21.8, 18.5. MS m/z (%): 353 [M+2] (2.16), Derivatives To a mixture of equimolar amounts of 6 (3.13g,
352 [M++1] (0.43), 351 [M+] (32.03), 55 (100.00). Anal. Calcd 0.01mol), in absolute ethanol (25mL) containing triethylamine
for C19H17N3S2 (351.49): C, 64.92; H, 4.87; N, 11.95; S, 18.25. (0.50mL), malononitrile (0.66g, 0.01mol) or ethyl cyanoac-
Found: C, 64.58; H, 4.98; N, 11.63; S, 17.95.
etate (1.13g, 0.01mol) was added. The reaction mixture, in
Preparation of 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]- each case, was heated under reflux for 3h then poured onto
thiophene-2-yl)-3-phenylthiourea (6) Equimolar amount a beaker containing acidified ice/water mixture. The formed
of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile solid products were collected by filtration and crystallized
(1.78g, 0.01mol) in absolute ethanol (25mL) containing a cata- from absolute ethanol.
lytic amount of triethylamine (0.50mL) and phenyl isothiocya-
2-(6-Amino-3,4-dihydro-4-amino-3-phenyl-2-thioxo-
nate (1.35g, 0.01mol) were added. The reaction mixture was pyrimidin-1(2H)-yl)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-
heated under reflux for 2h and cooled by pouring onto acidi- carbonitrile (9a)
fied ice/water mixture the solid product formed in each case
was collected by filtration, washed with water and crystallized 3209, 3100, 2934, 2836, 2195, 1619, 1435, 1334, 1282. 1H-NMR
from absolute ethanol.
(DMSO-d6) δ: 1.50–1.71 (m, 4H, 2CH2), 2.33–2.51 (m, 4H,
Off white crystals; mp 97–100°C, IR (KBr) cm−1: 3438,
Brown crystals, mp 97–100°C, IR (KBr) cm−1: 3445, 3204, 2CH2), 6.93 (s, 1H, pyrimidine C5), 7.20–7.70 (m, 5H, C6H5),
3022, 2932–2835, 2195, 1617, 1499, 1332, 1281. 1H-NMR 8.80 (s, 2H, NH2), 11.10 (s, 1H, NH). 13C-NMR (DMSO-d6)
(DMSO-d6) δ: 1.32–1.79 (m, 4H, 2CH2), 2.33–2.50 (m, 4H, δ: 180.2, 164.0, 162.8, 141.0, 135.2, 134.1, 131.1, 129.1, 129.1,
2CH2), 6.91 −7.57 (m, 5H, C6H5), 9.80, 11.08 (2s, 2H, 2NH). 126.5, 126.5, 124.2, 116.1, 83.3, 67.0, 24.5, 24.0, 23.4, 21.8. MS
13C-NMR (DMSO-d6) δ: 178.0, 154.3, 141.1, 138.5, 132.0, m/z (%): 380 [M++1] (18.87), 379 [M+] (21.85), 378 [M+−1]
128.1, 128.1, 126.3, 126.3, 124.4, 114.5, 83.3, 24.3, 24.0, 23.4, (20.53), 77 [C6 H4]+ (100.00). Anal. Calcd for C19H17N5S2
21.8. MS m/z (%): 315 [M++2] (10.75), 314 [M++ 1] (24.45), (379.50): C, 60.13; H, 4.52; N, 18.45; S, 16.90. Found: C, 59.89;
313 [M+] (92.59), 312 [M+−1] (45.27), 311 [M+−2] (2.02), H, 4.31; N, 18.31; S, 16.54.
77 [C6H5]+ (100.00). Anal. Calcd for C16H15N3S2 (313.44): C,
61.31; H, 4.82; N, 13.41; S, 20.46. Found: C, 61.28; H, 5.07; N, thioxopyrimidin-1(2H)-yl)-4,5,6,7-tetrahydro-benzo[b]-
2 - (6 - A m i n o -3 , 4 - d i h y d r o - 4 - o x o -3 - p h e n y l - 2 -
13.06; S, 20.18.
Preparation of (2E)-2-(4-Hydroxy-3-phenylthiazol-2(3H)-
thiophene-3-carbonitrile (9b)
Off white crystals, mp 127–130°C, IR (KBr) cm−1: 3429,
ylideneamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-car- 3333, 3100, 2938–2835, 2196, 1660, 1620, 1435, 1334, 1282.
bonitrile (7) Equimolar amount of 6 (3.13g, 0.01mol) in 1H-NMR (DMSO-d6) δ: 1.69–1.70 (m, 4H, 2CH2), 2.32–251
absolute ethanol (25mL) containing a catalytic amount of (m, 4H, 2CH2), 6.95 (s, 1H, pyrimidine C5), 7.18–7.60 (m,
triethyleamine (0.50mL), ethyl chloroacetate (1.22g, 0.01mol) 5H, C6H5), 8.20 (s, 2H, NH2). Anal. Calcd for C19H16N4OS2
was added. The reaction mixture was heated under reflux for (380.49): C, 59.98; H, 4.24; N, 14.73; S, 16.85. Found: C, 59.58;
2h. The formed solid products, in each case, upon pouring H, 3.97; N, 14.35; S, 16.45.
onto ice/water mixture containing few drops of hydrochloric
acid were collected by filtration and crystallized from absolute methyl- 4-oxo-3-phenyl-2-thioxopyrimidin-1-(6H)-yl)-
ethanol.
benzo[b]thiophene-3-carbonitrile (10) To a mixture of
Preparation of 4,5,6,7-Tetrahydro-2-(3,4-dihydro-6-
Pale brown crystals, mp 127–130°C, IR (KBr) cm−1: 3442, equimolar amounts of 6 (3.13g. 0.01mol) in absolute ethanol
3203, 3019, 2931, 2839, 2192, 1618, 1439, 1521. 1H-NMR (25mL) containing a catalytic amount of piperidine (0.50mL),
(DMSO-d6) δ: 1.69–1.80 (m, 4H, 2CH2), 2.33–2.50 (m, 4H, ethyl acetoacetate (1.30g, 0.01mol) was added. The reaction
2CH2), 4.50 (s, 1H, thiazole C5), 6.92–7.70 (m, 5H, C6H5), mixture was heated under reflux for 3h then poured onto
11.10 (s, 1H, OH). 13C-NMR (DMSO-d6) δ: 180.0, 162.7, 155.1, acidified ice/water mixture. The solid products were collected
145.0, 141.0, 131.1, 129.6, 129.6, 118.1, 116.8, 116.1, 115.0, by filtration and crystallized from absolute ethanol.
101.0, 60.1, 24.4, 24.0, 23.4, 21.8. MS m/z (%): 354 [M++1]
Pale brown crystals, mp 267–270°C, IR (KBr) cm−1: 3219,
(29.32), 353 [M+] (23.56), 352 [M+−1] (41.36), 351 [M+−2] 2935, 2836, 2196, 1617, 1555, 1438, 1329, 1280. 1H-NMR
(2.62), 127 (100.00). Anal. Calcd for C18H15N3OS2 (353.46): C, (DMSO-d6) δ: 1.10 (s, 3H, CH3), 1.70–1.80 (m, 4H, 2CH2),
61.16; H, 4.28; N, 11.89; S, 18.14. Found: C, 61.46; H, 4.63; N, 2.40–2.79 (m, 4H, CH2), 6.74 (s, 1H, pyrimidine C5),
11.54; S, 17.89.
6.93–7.73 (m, 5H, C6H5). MS m/z (%): 379 [M+] (25.45), 378
Preparation of 2-((4-(4-Chlorophenyl)-3-phenylthiazol- [M+−1] (19.71), 92 (100.00), 77 [C6H5]+ (16.85). Anal. Calcd
2(3H)-ylidene)amino)-4,5,6,7-tetrahydro-benzo[b]thio- for C20H17N3OS2 (379.50): C, 63.30; H, 4.52; N, 11.07; S, 16.90.
phene-3-carbonitrile (8) Prepared according to the method Found: C, 62.93; H, 4.34; N, 11.47; S, 17.29.
applied for 7, using p-chlorobromo acetate (2.33g, 0.01mol).
Preparation of 4,5,6,7-Tetrahydro-2-(2,3-dihydro-4-
Pale brown crystals, mp 97–100°C, IR (KBr) cm−1: 3050, hydroxy-4,6-dimethyl-3-phenyl-2-thioxo-pyrimidine-1(2H-
1
2931–2840, 2194, 1620, 1436, 1518. H-NMR (DMSO-d6) δ: yl)benzo[b]thiophene-3-carbonitrile (11) To a mixture of
1.70–1.80 (m, 4H, 2CH2), 2.33–2.50 (m, 4H, 2CH2), 4.76 (s, equimolar amounts of 6 (3.13g. 0.01mol) in absolute ethanol
1H, thiazole C3), 6.93–8.03 (m, 9H, C6H4, C6H5). MS m/z (%): (25mL) containing a catalytic amount of piperidine (0.50mL),
448 [M+] (0.46), 447 [M+−1] (3.60), 76 [C6H4]+ (10.11), 150 acetylacetone (1.00g, 0.01mol) was added. The reaction mix-
(100.00). Anal. Calcd for C24H18N3S2Cl (448.00): C, 64.34; H, ture was heated under reflux for 3h then poured onto acidified
4.05; N, 9.38; S, 14.31. Found: C, 64.43; H, 4.34; N, 9.60; S, ice/water mixture. The solid products were collected by filtra-
14.61.
General Procedure for Preparation of 2-(6-Amino-3,4-
tion and crystallized from absolute ethanol.
Off white crystals, mp 147–150°C, IR (KBr) cm−1: 3443,
dihydro-3-phenyl-2-thioxopyrimidin-1(2H)-yl)-4,5,6,7- 3198, 3026, 2929, 2844, 2195, 1586, 1440, 1335, 1290.

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
475
1H-NMR (DMSO-d6) δ: 1.20 (s, 3H, CH3), 1.50 (s, 3H, CH3), (DMSO-d6) δ: 1.70–1.91 (m, 4H, 2CH2), 2.32–2.79 (m, 4H,
1.71–1.79 (m, 4H, 2CH2), 2.33–2.50 (m, 4H, 2CH2), 6.92 (s, 2CH2), 6.93 (s, 1H, thiazine C4), 7.07–7.98 (m, 10H, 2C6H5),
1H, pyrimidine C5), 7.32–7.57 (m, 5H, C6H5), 11.10 (s, 1H, 8.64 (s, 2H, NH2). MS m/z (%): 467 [M+−1] (21.98), 466
OH). MS m/z (%): 397 [M++2] (0.51), 396 [M++1] (0.46), [M+−2] (14.66), 55 (100.00). Anal. Calcd for C26H21N5S2
395 [M+] (0.68), 394 [M+−1] (0.63), 206 (100.00), 77 [C6H5]+ (467.61): C, 66.78; H, 4.53; N, 14.98; S, 13.71. Found: C, 66.41;
(43.79). Anal. Calcd for C21H21N3OS2 (395.54): C, 63.77; H, H, 4.28; N, 15.09; S, 13.88.
5.35; N, 10.62; S, 16.21. Found: 63.48; H, 4.99; N, 10.92; S,
16.59.
(2E)-2-(3-Cyano-4,5,6,7-tetrahydro[b]thiophen-2-
ylimino)-6-amino-4-(4-chloro-phenyl)-3,4-dihydro-3-
Preparation of 2-(5E)-5-(2-Phenyldiazenyl)-3,4-dihydro- phenyl-2H-1,3-thiazine-5-carbonitrile (15b)
4-hydroxy-4,6-dimethyl-3-phenyl-2-thioxo-pyrimidin-
Brown crystals, mp 67–70°C, IR (KBr) cm−1: 3445, 3327,
1-(2H)-yl)-5,6,7-tetrahydrobenzo[b]thiophene-3-carboni- 3206, 2932, 2837, 2260, 2196, 1624, 1491, 1520. 1H-NMR
trile (12) To a cold solution (0–5°C) of 11 (3.95g, 0.01mol) (DMSO-d6) δ: 1.69–1.79 (m, 4H, 2CH2), 2.33–2.79 (m, 4H,
containing sodium hydroxide (1.00g), an equimolar amount 2CH2), 6.76 (s, 1H, thiazine C4), 6.79–7.99 (m, 9H, C6H4,
of diazotized aniline (0.93mL, 0.01mol) was gradually added C6H5), 8.64 (s, 2H, NH2). MS m/z (%): 502 [M+] (6.98), 501
while stirring. The solid product formed upon cooling in an [M+−1] (8.90), 500 [M+−2] (7.09), 76 [C6H4]+ (2.82), 64
ice-path was collected by filtration, washed with water and (100.00). Anal. Calcd for C26H20N5S2Cl (502.05): C, 62.20; H,
crystallized from absolute ethanol.
4.02; N, 13.95; S, 12.77. Found: C, 62.18; H, 4.09; N, 14.35; S,
Dark brown crystals, mp 92–95°C, IR (KBr) cm−1: 3428, 12.49.
3058, 2933, 2206, 1595, 1445, 1494, 1329, 1250. 1H-NMR
(2E)-2-(3-Cyano-4,5,6,7-tetrahydro[b]thiophen-2-
(DMSO-d6) δ: 1.06 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.51–1.99 ylimino)-6-amin-3,4-dihydro-4-(4-methoxyphenyl)-3-
(m, 4H, 2CH2), 2.17–2.65 (m, 4H, 2CH2), 6.75–8.02 (m, 10H, phenyl-2H-1,3-thiazine-5-carbonitrile (15c)
2C6H5), 11.10 (s, 1H, OH). MS m/z (%): 500 [M+] (0.06), 499
Yellowish brown crystals, mp 72–75°C, IR (KBr) cm−1:
[M+−1] (0.09), 498 [M+−2] (0.07), 80 (100.00), 77 [C6H5]+ 3429, 3332, 3026, 2930, 2837, 2217, 2195, 1602, 1439, 1562.
(5.62). Anal. Calcd for C27H25N5OS2 (499.65): C, 64.90; H, 1H-NMR (DMSO-d6) δ: 1.31 (s, 3H, CH3), 1.70–191 (m, 4H,
5.04; N, 14.02; S, 12.83. Found: C, 64.69; H, 4.89; N, 14.08; 2CH2), 2.16–2.68 (m, 4H, 2CH2), 6.94 (s, 1H, thiazine C4),
S, 12.89.
7.08–7.98 (m, 9H, C6H4, C6H5), 8.54 (s, 2H, NH2). MS m/z
Preparation of 4-Imino-3-phenyl-3,4,5,6,7,8-hexa-hydro- (%): 497 [M+−1] (13.17), 496 [M+−2] (2.83), 268 (100.00), 76
benzo[4,5]thieno[2,3-d]pyrimidine-2-thiol (13) To a solu- [C6H4]+ (7.00). Anal. Calcd for C27H23N5OS2 (497.63): C, 65.17;
tion of compound 6 (3.13g, 0.01) in 1,4-dioxane (25mL) a cat- H, 4.66; N, 14.07; S, 12.89. Found: C, 65.07; H, 5.06; N, 14.39;
alytic amount of piperidine (0.50mL) was added the reaction S, 12.68.
mixture was heated under reflux for 3h and the formed solid
General Procedure for Preparation of 6-Amino-3-(3-
product pouring onto acidified ice/water mixture was collected cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-1,2,3,4-
by filtration and crystallized from 1,4-dioxane.
tetrahydro-1-phenyl-2-thioxopyrimidine-5-carbonitrile
Pale yellow crystals, mp 117–120°C, IR (KBr) cm−1: 3444, Derivatives (17a–c) Equimolar amounts of 2-amino-4,5,6,7-
1
3206, 3100, 2933–2835, 1618, 1432, 1520. H-NMR (DMSO- tetrahydrobenzo[b]thiophene-3-carbonitrile (1.78g, 0.01mol)
d6) δ: 1.69–1.91 (m, 4H, 2CH2), 2.31–2.79 (m, 4H, 2CH2), in absolute ethanol (25mL) containing a catalytic amount
6.95–7.58 (m, 5H, C6H5), 8.60 (s, 1H, SH), 9.00 (s, 1H, NH). of triethylamine (0.50mL), benzaldehyde (1.06g, 0.01mol),
13C-NMR (DMSO-d6) δ: 162.7, 161.0, 143.4, 141.0, 131.1, 129.6, 4-chlorobenzaldehyde (1.12g, 0.01mol) or 4-methoxybenzal-
129.6, 129.0, 127.2, 118.8, 116.8, 116.8, 25.0, 24.0, 23.4, 21.8. dehyde (1.08g, 0.01mol) was added with malononitrile (0.66g,
MS m/z (%): 315 [M++2] (19.16), 314 [M++1] (8.35), 313 [M+] 0.01mol) and phenyl isothiocynate (1.35g, 0.01mol). The reac-
(7.62), 121 (100.00). Anal. Calcd for C16H15N3S2 (313.44): C, tion mixture, in each case, was heated under reflux for 3h, the
61.31; H, 4.82; N, 13.41; S, 20.46. Found: C, 60.93; H, 4.49; N; formed solid products pouring onto ice/water mixture contain-
13.06; S, 20.06.
ing few drops of hydrochloric acid were collected by filtration
General Procedure for Preparation of (2E)-2-(3-Cyano- and crystallized from absolute ethanol.
4,5,6,7-tetrahydro[b]thiophen-2-ylimino)-6-amino-3,4-
6-Amino-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
dihydro-3-phenyl-2H-1,3-thiazine-5-carbonitrile Deriva- yl)-1,2,3,4-tetrahydro-1,4-diphenyl-2-thioxopyrimidine-5-
tives (15a–c) To a solution of compound 6 (3.13g, 0.01mol) carbonitrile (17a)
in absolute ethanol (25mL), benzaldehyde (1.06g, 0.01mol),
Yellow crystals, mp over 300°C, IR (KBr) cm−1: 3421,
4-chlorobenzaldehyde (1.12g, 0.01mol) or 4-methoxybenz- 3251, 3067, 2926–2840, 2210, 2195, 1603, 1441, 1328, 1249.
aldehyde (1.08g, 0.01mol) was added with malononitrile 1H-NMR (DMSO-d6) δ: 1.80–1.81 (m, 4H, 2CH2), 2.50–2.72
(0.66g, 0.01mol) in the presence of a catalytic amount of (m, 4H, 2CH2), 7.52 (s, 1H, pyrimidine C6), 7.53–7.99 (m,
triethylamine (0.50mL). The reaction mixture was heated 10H, 2C6H5), 8.66 (s, 2H, NH2). MS m/z (%): 467 [M+−1]
under reflux for 3h. The solid products formed, in each case, (25.83), 466 [M+−2] (17.22), 124 (100.00), 77 [C6H5]+ (31.79).
upon pouring onto ice/water mixture containing few drops of Anal. Calcd for C26H21N5S2 (467.61): C, 66.78; H, 4.53; N,
hydrochloric acid were collected by filtration and crystallized 14.98; S, 13.71. Found: C, 66.38; H, 4.19; N, 15.07; S, 13.46.
from absolute ethanol.
6 -A mino- 4(4 -chlorophenyl)-3-(3-cyano- 4,5,6,7-
(2E)-2-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- tetrahydrobenzo[b]thiophen-2-yl)-1,2,3,4-tetrahydro-1-
ylimino)-6-amino-3,4-dihydro-3,4-diphenyl-2H-1,3-thiazine-5- phenyl-2-thioxopyrimidine-5-carbonitrile (17b)
carbonitrile (15a)
Pale brown crystals, mp 82–85°C, IR (KBr) cm−1: 3444,
Dark brown crystals, mp 87–90°C, IR (KBr) cm−1: 3423, 3326, 3027, 2931, 2843, 2220, 2195, 1619, 1440, 1335, 1289.
3249, 3057, 2927, 2835, 2213, 2195, 1598, 1447, 1556. H-NMR 1H-NMR (DMSO-d6) δ: 1.69–1.80 (m, 4H, 2CH2), 2.32–2.72
1

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Vol. 65, No. 5 (2017)
(m, 4H, 2CH2), 6.90 (s, 1H, pyrimidine C6), 7.34–8.00 (m, 9H, C22H17N5OS (399.47): C, 66.15; H, 4.29; N, 17.53; S, 8.03.
C6H4, C6H5), 8.55 (s, 2H, NH2). MS m/z (%): 502 [M+] (10.01), Found: C, 65.79; H, 4.62; N, 17.13; S, 7.98.
501 [M+−1] (13.45), 77 [C6H5]+ (36.48), 69 (100.00). Anal.
2-(3-Cyano–4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6-
Calcd for C26H20N5S2Cl (502.05): C, 62.20; H, 4.02; N, 13.95; amino-4-(4-chlorophenyl)-4H-pyran-3,5-dicarbonitrile (20b)
S, 12.77. Found: C, 61.95; H, 4.11; N, 13.79; S, 12.63.
Brown crystals, mp over 300°C, IR (KBr) cm−1: 3462, 3225,
6-Amino-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2- 3100, 2936, 2861, 2260, 2214, 2195, 1598, 1482. 1H-NMR
yl)-1,2,3,4-tetrahydro-4-(4-methoxyphenyl)-1-phenyl-2- (DMSO-d6) δ: 1.82–1.91 (m, 4H, 2CH2), 2.50–2.96 (m, 4H,
thioxopyrimidine-5-carbonitrile (17c)
2CH2), 6.90 (s, 1H, pyran C4), 7.59–7.75 (m, 4H, C6H4), 8.10
Reddish brown crystals, mp 67–70°CC, IR (KBr) cm−1: (s, 2H, NH2), 9.24 (s, 1H, NH). MS m/z (%): 433 [M+−1]
3415, 3181, 3063, 3021, 2936, 2843, 2216, 2146, 1599, 1445, (21.39), 432 [M+−2] (16.76), 118 (100.00), 76 [C6H4]+ (10.12).
1318, 1271. 1H-NMR (DMSO-d6) δ: 1.20 (s, 3H, CH3), Anal. Calcd for C22H16N5OSCl (433.91): C, 60.90; H, 3.72; N,
1.79–1.80 (m, 4H, 2CH2), 2.50–2.68 (m, 4H, 2CH2), 7.09 (s, 16.14; S, 7.39. Found: C, 60.58; H, 4.09; N, 16.20; S, 7.39.
1H, pyrimidine C6), 7.11–7.99 (m, 9H, C6H4, C6H5), 8.53 (s,
2H, NH2). MS m/z (%): 498 [M+] (7.95), 497 [M+−1] (10.18), amino-4-(4-methoxy-phenyl)-4H-pyran-3,5-dicarbonitrile (20c)
496 [M+−2] (7.95), 63 (100.00). Anal. Calcd for C27H23N5OS2 Pale brown crystals, mp over 300°C, IR (KBr) cm−1:
(497.63): C, 65.17; H, 4.66; N, 14.07; S, 12.89. Found: C, 65.28; 3465, 3220, 3065, 2936, 2836, 2220, 2210, 2195, 1611, 1440.
H, 4.29; N, 13.79; S, 12.59.
1H-NMR (DMSO-d6) δ: 1.20 (s, 3H, CH3), 1.60–1.82 (m, 4H,
2-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl-amino)-6-
Preparation of 6-Amino-3-(3-cyano-4,5,6,7-tetrahydro- 2CH2), 2.50–2.96 (m, 4H, 2CH2), 6.90 (s, 1H, pyran C4),
benzo[b]thiophen-2-yl)-4-(furan-3-yl)-1,2,3,4-tetrahydro- 7.12–7.69 (m, 4H, C6H4), 8.10 (s, 2H, NH2), 9.18 (s, 1H, NH).
1-phenyl-2-thioxopyrimidine-5-carbonitrile (18) To
solution of compound 2-amino-4,5,6,7-tetrahydrobenzo[b]- Anal. Calcd for C23H19N5O2S (429.49): C, 64.32; H, 4.46; N,
a
MS m/z (%): 429 [M+] (86.41), 428 [M+−1] (53.40), 87(100.00).
thiophene-3-carbonitrile (1.78g, 0.01mol) in absolute etha- 16.31; S, 7.47. Found: C, 63.93; H, 4.06; N, 15.93; S, 7.09.
nol (25mL) containing a catalytic amount of triethylamine
General Procedure for Preparation of 2-(3-Cyano-
(0.50mL), malononitrile (0.66g, 0.01mol), phenyl isothio- 4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6-amino-
cynate (1.35g, 0.01mol) and furfural (0.96g, 0.01mol) were 1,4-dihydro-pyridine-3,5-dicarbonitrile Derivatives (21a–c)
added the reaction mixture was heated under reflux for 2h. Equimolar amount of 19 (2.45g, 0.01mol) in absolute ethanol
The solid products formed upon pouring onto acidified ice/ (25mL), containing a catalytic amount of ammonium acetate,
water mixture were collected by filtrations and crystallized benzaldehyde (1.06g, 0.01mol), 4-chlorobenzaldehyde (1.12g,
from absolute ethanol.
0.01mol) or 4-methoxybenzaldehyde (1.08g, 0.01mol) was
Dark brown crystals, mp 132–135°CC, IR (KBr) cm−1: added with malononitrile (0.66g, 0.01mol). The reaction mix-
3426, 3300, 3113, 2936, 2861, 2218, 2195, 1608, 1486, 1333, ture, in each case, was heated under reflux for 3h. The solid
1287. 1H-NMR (DMSO-d6) δ: 1.78–19.1 (m, 4H, 2CH2), products formed, in each case, upon pouring onto ice/water
2.50–2.68 (m, 4H, 2CH2), 6.76 (s, 1H, pyrimidine C6), mixture were collected by filtration and crystallized from ab-
6.77–8.06 (m, 8H, C6H5, furan C), 8.40 (s, 2H, NH2). MS m/z solute ethanol.
(%): 458 [M+] (16.14), 457 [M+−1] (18.92), 64 (100.00). Anal.
Calcd for C24H19N5OS2 (457.57): C, 63.00; H, 4.19; N, 15.31; S, amino-1,4-dihydro-4-phenylpyridine-3,5-dicarbonitrile (21a)
14.02. Found: C, 62.72; H, 3.89; N, 15.29; S, 13.79.
Yellowish brown crystals, mp over 300°C, IR (KBr) cm−1:
2-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6-
Preparation of 2-Cyano-N-(3-cyano-4,5,6,7-tetrahydro- 3445, 3326, 3205, 3032, 2933, 2836, 2240, 2223, 2195, 1620,
benzo[b]thiophen-2-yl)acetamide (19) The data for com- 1520. 1H-NMR (DMSO-d6) δ: 1.69–1.79 (m, 4H, 2CH2),
pound 19 has been published earlier.18)
2.32–2.66 (m, 4H, 2CH2), 6.95 (s, 1H, pyridine C4), 7.09–7.99
General Procedure for Preparation of 2-(3-Cyano- (m, 5H, C6H5), 8.10 (s, 2H, NH2), 8.39, 8.54 (2s, 2H, 2NH).
4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6-amino- MS m/z (%): 399 [M++1] (13.08), 398 [M+] (11.32), 396
4H-pyran-3,5-dicarbonitrile Derivatives (20a–c) Equimo- [M+−2] (6.86), 177 (100.00), 77 [C6H5]+ (24.56). Anal. Calcd
lar amount of 19 (2.45g, 0.01mol) in absolute ethanol (25mL), for C22H18N6S (398.48): C, 66.31; H, 4.55; N, 21.09; S, 8.05.
containing a catalytic amount of triethylamine (0.50mL), Found: C, 66.66; H, 3.99; N, 21.48; S, 8.45.
benzaldehyde (1.06g, 0.01mol), 4-chlorobenzaldehyde (1.12g,
2-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6-
0.01mol) or 4-methoxybenzaldehyde (1.08g, 0.01mol) was amino,4-(4-chloro-phenyl)-1,4-dihydro-pyridine-3,5-
added with malononitrile (0.66g, 0.01mol). The reaction mix- dicarbonitrile (21b)
ture, in each case, was heated under reflux for 3h. The solid
Pale brown crystals, mp 97–100°C, IR (KBr) cm−1: 3444,
products formed, in each case, upon pouring onto acidified 3327, 3205, 3033, 2932, 2836, 2240, 2225, 2196, 1622, 1520.
ice/water mixture were collected by filtration and crystallized 1H-NMR (DMSO-d6) δ: 1.69–1.80 (m, 4H, 2CH2), 2.33–2.72
from absolute ethanol.
(m, 4H, 2CH2), 6.95 (s, 1H, pyridine C4), 7.27–7.98 (m, 4H,
2-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6- C6H5), 8.01 (s, 2H, NH2), 8.56, 8.67 (2s, 2H, 2NH). MS m/z
amino-4-phenyl-4H-pyran-3,5-dicarbonitrile (20a)
(%): 433 [M+] (2.09), 432 [M+−1] (2.86), 431 [M+−2] (2.44),
Brown crystals, mp over 300°C, IR (KBr) cm−1: 3462, 153 (100.00), 76 [C6H4]+ (34.93). Anal. Calcd for C22H17N6SCl
3225, 3100, 2933, 2861, 2220, 2210, 2195, 1628, 1450. 1H-NMR (432.93): C, 61.03; H, 3.96; N, 19.41; S, 7.41. Found: C, 60.66;
(DMSO-d6) δ: 1.82–1.91 (m, 4H, 2CH2), 2.50–2.96 (m, 4H, H, 4.36; N, 19.02; S, 7.81.
2CH2), 6.90 (s, 1H, pyran C4), 7.58–7.72 (m, 5H, C6H5), 8.10
2-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylamino)-6-
(s, 2H, NH2), 9.21 (s, 1H, NH). MS m/z (%): 400 [M++1] amino-1,4-dihydro-4-(4-methoxyphenyl)pyridine-3,5-
(55.74), 399 [M+] (72.95), 398 [M+−1] (15.57), 397 [M+−2] dicarbonitrile (21c)
(58.20), 225 (100.00), 77 [C6H5]+ (60.66). Anal. Calcd for
Yellow crystals, mp 72–75°C, IR (KBr) cm−1: 3444, 3326,

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
477
3206, 3026, 2931, 2840, 2260, 2217, 2193, 1610, 1515. 1H-NMR
(DMSO-d6) δ: 1.30 (s, 3H, CH3), 1.69–1.79 (m, 4H, 2CH2),
2.32–2.68 (m, 4H, 2CH2), 6.95 (s, 1H, pyridine C4), 7.09–7.99
(m, 4H, C6H4), 8.30 (s, 2H, NH2), 8.39, 8.54 (2s, 2H, 2NH).
MS m/z (%): 429 [M++1] (3.33), 428 [M+] (3.72), 107 (100.00).
Anal. Calcd for C23H20N6OS (428.51): C, 64.47; H, 4.70; N,
19.61; S, 7.48. Found: C, 64.17; H, 4.38; N, 19.32; S, 7.84.
5) Mohareb R. M., Schatz J., Bioorg. Med. Chem., 19, 2707–2713
(2011).
6) El-Sharkawy K. A., El-Sehrawi H. M., Ibrahim R. A., Int. J. Org.
Chem., 2, 126–134 (2012).
7) Khalil A. M., Berghot M. A., Abd El-Ghani G. E., Gouda M. A.,
Synth. Commun., 40, 1658–1669 (2010).
8) Srivastava S., Das B., Der Pharma Chem., 3, 103–111 (2011).
9) Mohareb R. M., Abdallah A. E. M., Helal M. H. E., Mohammed H.
N. A., Eur. Chem. Bull., 2, 618–628 (2013).
Biology
10) Kapustina M. V., Kharizomenova I. A., Shvedov V. I., Fomina A.
N., Nikolaeva I. S., Golovanova E. A., Bogdanova G. A., Alekseeva
L. M. Khim.-Farm. Zh., 24, 39–40 (1990).
11) Mohareb R. M., Fahmy A. A., Eur. Chem. Bull., 2, 545–553 (2013).
12) Gouda M. A., Abu-Hashem A. A., Arch. Pharm. Chem. Life Sci.,
344, 170–177 (2011).
13) Amr A. G., Sherif M. H., Assy M. G., Al-Omar M. A., Ragab I.,
Eur. J. Med. Chem., 45, 5935–5942 (2010).
14) Mohareb R. M., Mohamed A. A., Abdallah A. E. M., Acta Chim.
Slov., 63, 227–240 (2016).
Materials and Methods Fetal bovine serum (FBS) and
L-glutamine, were obtained from Gibco Invitrogen Com-
pany (Scotland, U.K.). RPMI-1640 medium was provided from
Cambrex (NJ, U.S.A.). Dimethyl sulfoxide (DMSO), doxoru-
bicin, penicillin, streptomycin and sulforhodamine B (SRB)
were obtained from Sigma Chemical Company. (Saint Louis,
MO, U.S.A.).
Samples Stock solutions of the newly compounds were
prepared in DMSO and kept at −20°C. Appropriate dilutions
of the compounds were freshly prepared just prior the assays.
15) Mohareb R. M., Abdallah A. E. M., Helal M. H., Shaloof S. M. H.,
Acta Pharm., 66, 53–68 (2016).
Final concentrations of DMSO did not interfere with the cell 16) Ramanathan J. D., Namboothiri D. G., Shah G. F., Radhakrishnan
A. V., Mehta H. J., Padhya A. C., J. Indian Chem. Soc., 55, 822–825
(1978).
17) Singh H., Singh P., Deep K., Tetrahedron, 39, 1655–1660 (1983).
18) Shams H. Z., Mohareb R. M., Helal M. H. E., Mahmoud A. E., Mol-
ecules, 16, 52–73 (2010).
19) Shams H. Z., Mohareb R. M., Helal M. H., Mahmoud A. E.-S., Mol-
ecules, 16, 6271–6305 (2011).
20) Xu X., Shi W., Zhou Y., Wang Y., Zhang M., Song L., Deng H., J.
growth.
Cell Cultures Three human tumor cell lines, MCF-7
(breast adenocarcinoma), NCI-H460 (non-small cell lung
cancer), and SF-268 (CNS cancer) were used. MCF-7 was
obtained from the European Collection of Cell Cultures
(ECACC, Salisbury, U.K.) and NCI-H460 and SF-268 were
kindly provided by the National Cancer Institute (NCI, Cairo,
Egypt). They grow as monolayer and routinely maintained in
RPMI-1640 medium supplemented with 5% heat inactivated
FBS, 2mM glutamine and antibiotics (penicillin 100U/mL,
streptomycin 100µg/mL), at 37°C in a humidified atmosphere
containing 5% CO2. Exponentially growing cells were ob-
tained by plating 1.5×105 cells/mL for MCF-7 and SF-268 and
0.75×104 cells/mL for NCI-H460, followed by 24h of incuba-
tion. The effect of the vehicle solvent (DMSO) on the growth
of these cell lines was evaluated in all the experiments by ex-
posing untreated control cells to the maximum concentration
(0.5%) of DMSO used in each assay.
Fluor. Chem., 176, 127–133 (2015).
21) Penta S., Gadidasu K. K., Basavoju S., Rajeswar V. R., Tetrahedron
Lett., 54, 5663–5666 (2013).
22) Vereshchagin A. N., Elinson M. N., Ryzhkov F. V., Nasybullin R.
F., Bobrovsky S. I., Egorov M. P., Goloveshkin A. S., C. R. Chim.,
18, 1344–1349 (2015).
23) Hazeri N., Maghsoodlou M. T., Mir F., Kangani M., Saravani H.,
Molashahi E., Chin. J. Catal., 35, 391–395 (2014).
24) Wagh Y. B., Tayade Y. A., Padvi S. A., Patil B. S., Patil N. B., Dalal
D. S., Chin. Chem. Lett., 26, 1273–1277 (2015).
25) Goli-Jolodar O., Shirini F., Seddighi M., J. Mol. Liq., 224, 1092–
1101 (2016).
26) Zakeri M., Nasef M. M., Abouzari-Lotf E., Moharami A., Heravi
M. M., J. Ind. Eng. Chem., 29, 273–281 (2015).
27) Tabassum S., Govindaraju S., Khan R. U., Pasha M. A., Ultrason.
Sonochem., 24, 1–7 (2015).
28) El-Sayed N. N., Abdelaziz M. A., Wardakhan W. W., Mohareb R.
M., Steroids, 107, 98–111 (2016).
29) Khaligh N. G., Hamid S. B. A., Chin. J. Catal., 36, 728–733 (2015).
30) Udhaya Kumar C., Sethukumar A., Arul Prakasam B., J. Mol.
Struct., 1036, 257–266 (2013).
31) Nemouchi S., Boulcina R., Carboni B., Debache A., C. R. Chim.,
15, 394–397 (2012).
32) Li L. H., Yu F. H., Biochem. Mol. Biol. Int., 31, 879–887 (1993).
33) Mohareb R. M., Abbas N. S., Abdelaziz M. A., Steroids, 86, 45–55
(2014).
34) Mohareb R. M., Wardakhan W. W., Hamed F. I., Med. Chem. Res.,
24, 2043–2054 (2015).
35) Mohareb R. M., Wardakhan W. W., Ibrahim R. A., Med. Chem.
Res., 25, 2187–2204 (2016).
Acknowledgment The authors thank the research group
at the National Cancer Institute in Egypt for recording the
cytotoxic activity.
Conflict of Interest The authors declare no conflict of
interest.
References
1) Gouda M. A., Berghot M. A., Abd El-Ghani G. E., Khalil A. M.,
Eur. J. Med. Chem., 45, 1338–1345 (2010).
2) Abdelaziz M. A., El-Sehrawi H. M., Mohareb R. M., Med. Chem.
Res., 24, 3932–3948 (2015).
3) Hemdan M. M., Abd El-Mawgoude H. K., Chem. Pharm. Bull., 63,
450–456 (2015).
4) Mohareb R. M., Mohamed M. H., Heteroatom Chem., 12, 518–527
(2001).