Synthesis of thieno[2,3-d]pyrimidines, thieno[2,3-d]triazinones and thieno[2,3-e]diazepinones of anticipated anti-cancer activity

Article abstract of DOI:10.3184/174751912X13282020691270

A novel series of 4-aminohexahydrocycloocta[4,5]thieno[2,3-d]pyrimidines, hexahydrocycloocta[4,5]thieno[2,3-d]-1,2,3-triazin-4-one and its N-3 substituted derivatives in addition to 3-aryl hexahydrocycloocta[4,5] thieno[2,3-e]-1,4- diazepin-5-ones were synthesised. Also, 2-(N-ethylcarbamothioylamino) hexahydrocycloocta[b] thiophene-3-carbonitrile and 19-imino tetradecahydrocycloocta[4',5'] thieno[2',3':4,5]pyrimido[1,6-a] cycloocta[4,5]thieno [3,2-e]pyrimidine-9-thione were prepared. Almost all the synthesised compounds exhibited anti-tumour activity against human colon carcinoma (HCT 116) cell line in vitro. Five compounds (IC₅₀: 15.92, 22.59, 25.85, 27.40 and 29.70 μM, respectively) exhibited 2.16 to 1.15 fold more potent antitumour activity than imatinib (IC₅₀: 34.40 μM).

Full text of DOI:10.3184/174751912X13282020691270

JOURNAL OF CHEMICAL RESEARCH 2012
RESEARCH PAPER 105
FEBRUARY, 105–110
Synthesis of thieno[2,3-d]pyrimidines, thieno[2,3-d]triazinones and
thieno[2,3-e]diazepinones of anticipated anti-cancer activity
M.M. Kandeel, Ashraf A. Mounir, Hanan M. Refaat* and Asmaa E. Kassab
Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
A novel series of 4-aminohexahydrocycloocta[4,5]thieno[2,3-d]pyrimidines, hexahydrocycloocta[4,5]thieno[2,3-d]-
1,2,3-triazin-4-one and its N-3 substituted derivatives in addition to 3-aryl hexahydrocycloocta[4,5] thieno[2,3-e]-
1,4-diazepin-5-ones were synthesised. Also, 2-(N-ethylcarbamothioylamino) hexahydrocycloocta[b] thiophene-3-
carbonitrile and 19-imino tetradecahydrocycloocta[4΄,5΄] thieno[2΄,3΄:4,5]pyrimido[1,6-a] cycloocta[4,5]thieno
[3,2-e]pyrimidine-9-thione were prepared. Almost all the synthesised compounds exhibited anti-tumour activity
against human colon carcinoma (HCT 116) cell line in vitro. Five compounds (IC50: 15.92, 22.59, 25.85, 27.40 and
29.70 µM, respectively) exhibited 2.16 to 1.15 fold more potent antitumour activity than imatinib (IC50: 34.40 µM).
Keywords: hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidines, hexahydrocycloocta[4,5]thieno[2,3-d]-1,2,3-triazin-4-ones,
hexahydrocycloocta[4,5] thieno[2,3-e]-1,4-diazepin-5-ones, antitumour activity
The thieno[2,3-d]pyrimidine system is an integral part of
numerous potential cancer chemotherapeutic agents,1,2 express-
ing appreciable anti-cancer activity via inhibition of various
enzymes.3–11 Certain thieno[2,3-d] pyrimidines were originally
prepared as bioisosteres of gefitinib (Iressa®)12 and erlotinib
(Tarceva®)13 (Fig. 1), drugs that have been approved for the
treatment of gastrointestinal stromal tumours and lung cancer.
We have previously synthesised various hexahydrocycloocta
[4,5]thieno[2,3-d]pyrimidine and pyrimidin-4-one derivatives
that could be considered as analogues of gefitinib (M. M.
Kandeel, A. A. Mounir, H. M. Refaat, A. E. Kassab, in
preparation). This synthesis was motivated by the fact that
several reports support the anti-cancer activity for thieno[2,3-
d]pyrimidines fused with various five, six and seven membered
cycloalkyl moieties and / or substituents,14–19 but there are
no reports on the anti-cancer evaluation of thieno[2,3-
d]pyrimidines fused with a hexahydrocycloocta-ring. All the
synthesised compounds exhibited anti-tumour activity against
human colon carcinoma (HCT 116) cell line in vitro and
several compounds exhibited 4.3 to 1.3 fold more potent
anti-tumour activity than imatinib (Fig. 1).
related to gefitinib, we report here the synthesis of a new series
of thieno[2,3-d]pyrimidines, thieno[2,3-d]triazin-4-ones and
thieno[2,3-e]-1,4-diazepin-5-ones. Three elements in the target
compounds are varied while reserving the hexahydrocycloocta-
ring as the cycloalkyl moiety (Fig. 2):
(1) The N-3 substituents in pyrimidine; (2) 4-Amino
moiety; and (3) The size of the annulated ring (pyrimidine,
triazin-4-one and diazepin-5-one).
In the present work, we report the synthesis of the hitherto
unknown title compounds and evaluate their in vitro anticancer
activity.
Results and discussion
The synthetic route to the target compounds is outlined in
Schemes 1 and 2. 2-Amino-4,5,6,7,8,9-hexahydrocycloocta[b]
thiophene-3-carbonitrile (1) and 2-amino-4,5,6,7,8,9,10-hexah
ydrocycloocta[b]thiophene-3-carboxamide (6) were selected
as our primary starting materials for this series of reactions,
and were prepared via reported procedures.20
4-Amino thieno[2,3-d]pyrimidine 2 was obtained by con-
densing the amino cyano derivative 1 with formamide. The IR
spectrum of the title compound indicated the presence of a
forked absorption band at 3394 and 3305 cm−1 corresponding
to NH2 group. While, the 1H NMR spectrum showed an
exchangeable singlet signal at δ 6.83 indicating the two NH2
protons, in addition to a singlet signal at δ 8.18 corresponding
to the C2–H proton.
With the same purpose, our group considered the design and
synthesis of various 2-substituted and 4-substituted hydrazinyl
thieno[2,3-d]pyrimidines as promising anti-tumour agents.
Some products demonstrated 2.89 to 1.07 fold more potent
antitumour activity than imatinib. Moreover, the thienotriazo-
lopyrimidine derivatives were prepared as structure analogues
to 4-hydrazinyl thieno[2,3-d]pyrimidines and were found to be
of moderate activity.
Compounds 5a–d were prepared by reacting the amino-
cyano derivative 1 with the selected isothiocyanate in ethanol
in the presence of a catalytic amount of triethylamine. The IR
spectra of 5a–d showed the presence of two absorption bands
at 3480-3437 and 3460–3440 cm−1 corresponding to the NH2
Encouraged by the above findings and in continuation of
our research programme concerned with the modification
of certain anti-cancer thieno[2,3-d]pyrimidines structurally
Fig. 1 Structures of clinically potent anticancer pyrimidines.
* Correspondent. E-mail: lgchen@tju.edu.cn

106 JOURNAL OF CHEMICAL RESEARCH 2012
Fig. 2 Strategies for structural modifications of gefitinib.
Scheme 2 Synthesis of compounds 7–9.
When 1 was heated with ethyl isothiocyanate in ethanol
without the addition of triethylamine, the thioureido derivative
3 was produced rather than the cyclised form identical to 5a.
Scheme 1 Synthesis of compounds 2–5.
1
The structure of compound 3 was confirmed by IR and H
NMR spectra. The IR spectrum proved useful in tracing the
absence of the absorption band corresponding to an NH2 group
but showed two absorption bands at 3331 and 3265 cm−1
corresponding to the two NH groups, and another two bands
at 2216 and 1247 cm−1 corresponding to CN and C=S groups,
respectively. In addition, the 1H NMR spectrum of the product
revealed the presence of two exchangeable singlet signals at δ
7.38 and 9.23 due to two NH protons instead of NH2 protons.
group, in addition to the C=S group which appeared at 1273–
1
1193 cm−1. While, H NMR spectra revealed the presence of
one exchangeable singlet signal at δ 7.62–8.46 corresponding
to NH2 protons in addition to the characteristic signals of
the N-CH2CH3 (5a) at δ 1.24 and 4.60–5.00 and the signals
corresponding to the N-substituted phenyl groups (5b–d) at
δ 6.72–7.50.

JOURNAL OF CHEMICAL RESEARCH 2012 107
In an attempt to prepare the N-phenyl analogue of com-
pound 5, by reacting 1 with phenyl isothiocyanate in ethanol,
the hexacyclothienopyrimidine 4 was the sole product. The IR
spectrum of compound 4 illustrated the appearance of two
absorption bands at 3288 and 3209 cm−1 due to the two NH
groups, in addition, another absorption band appeared at 1327
cm−1 corresponding to the C=S group. Further evidence was
by elemental analysis and spectral data. The IR spectra of 9a–d
showed the presence of two absorption bands at 3445–3387
and 3248–3109 cm−1 corresponding to two NH groups and
only one band at 1659–1647 cm−1 due to one C=O group,
1
whereas the H NMR spectra of 9a–d revealed the presence
of an C2–H proton signal in the aromatic range. The mass
spectrum of 9d (R1 = 3-NO2) displayed three molecular ion
peaks at m/z 369 (M+), 370 (M+1┐.+) and 371 (M+2┐.+).
1
obtained from the H NMR spectrum which revealed the
absence of signals due to aromatic protons at δ 6–9. In
addition, two exchangeable singlet signals at δ 7.62 and 8.85
appeared corresponding to two NH protons. Moreover, the
mass spectrum of 4 displayed a peak at m/z 454 (M.+). A
literature survey revealed that an analogous compound was
produced under similar reaction conditions.21 A suggested
mechanism for the formation of the title compound is shown
in Fig. 3.
The second approach is concerned with the synthesis of
thieno[2,3-d]triazin-4-ones and thieno[2,3-e]-1,4-diazepin-5-
ones. A literature survey revealed that the thienotriazines22–26
and the thienodiazipinones 27 ring systems were scarcely men-
tioned in the literature. Reaction of o-amino amide derivative 6
with sodium nitrite under acidic conditions yielded the corre-
sponding thienotriazinone derivative 7 which was reacted with
the appropriate halogen compound in dry acetone and in the
presence of anhydrous potassium carbonate to yield 8a–d. The
IR spectrum of 7 revealed the presence of an absorption band
at 3200 cm−1 due to the NH group and a band at 1665 cm−1
corresponding to the C=O group. While the 1H NMR spectrum
showed an exchangeable singlet signal at δ 13.72 due to the
NH proton. Moreover, the mass spectrum displayed three
molecular ion peaks at m/z 235 (M.+), 236 (M+1┐.+) and 237
(M+2┐.+).
Cytotoxic activity
The in vitro growth inhibitory activity of all the prepared com-
pounds was evaluated using colon carcinoma cell line (HCT
116). For comparison purposes, the cytotoxicity of imatinib
(Gleevec®) (Fig. 1), a standard anti-tumour drug used for the
treatment of gasterointestinal tract tumours,28,29 was evaluated
under the same conditions. The IC50 values (dose of the
compound which caused a 50% reduction of survival values)
are shown in Table 1. The results are represented graphically in
Fig. 4. From the analysis of Table 1, it was found that almost
all the compounds showed significant anti-tumour activities.
Interestingly, compounds 4, 3, 8d, 9a and 2 (IC50: 15.92, 22.59,
25.85, 27.40 and 29.70 µM, respectively) exhibited 2.16 to
1.15 fold more potent anti-tumour activity than imatinib (IC50:
34.40 µM) and were the most active among their analogues.
Further, compounds 5a, 9b, 5c and 9d (IC50: 45.32, 45.86,
The IR spectra of 8a–d lacked the presence of an absorption
band due to the NH group and showed two absorption bands at
1755–1668 cm−1 due to two C=O groups. While, the 1H NMR
spectra showed the disappearance of the signal of the NH
proton and displayed the characteristic signals corresponding
to the different NR1 group.
Reacting the amino amide 6 with the selected phenacyl bro-
mide in glacial acetic acid afforded the desired 1,4-diazepino-
nes 9a–d. The structure of these compounds was substantiated
Fig. 4 Cytotoxicity of 2–9d and imatinib against (HCT 116) cell
line.
Table 1 IC50 valuesa of compounds 2–9d and Imatinib against
colon carcinoma cell line (HCT 116)
Compound
a IC50 (µM)
2
29.70
22.59
15.92
45.32
71.74
49.24
63.57
79.90
65.34
143.78
–
25.85
27.40
45.86
64.36
49.26
34.40
3
4
5a
5b
5c
5d
7
8a
8b
8c
8d
9a
9b
9c
9d
Imatinib
a IC50 (µM): dose of the compound which caused 50% reduction
of survival.
Values were calculated from dose–response curves done in
triplicate for each compound.
Fig. 3 Formation of compound 4.

108 JOURNAL OF CHEMICAL RESEARCH 2012
19-Imino-1,2,3,4,5,6,12,13,14,15,16,17,18,19-tetradeca-hydrocycloocta
[4΄,5΄] thieno[2΄,3΄:4,5]pyrimido[1,6-a] cycloocta[4,5]thieno[3,2-e]
pyrimidine-9-thione (4): Phenyl isothiocyanate (0.68 g, 0.005 mol)
was added dropwise to a well stirred solution of 1 (1 g, 0.005 mol) in
absolute ethanol (5 mL). The mixture was then refluxed on a steam
bath for 6 h and left at room temperature overnight. The separated
solid was filtered, washed with ethanol (10 mL) dried and crystallised
from dimethylformamide. M.p. 230–232 °C; yield 50%; IR (KBr)
vmax: 3288, 3209 (2NH), 1629 (C=N) and 1327 (C=S) cm−1; 1H NMR
(DMSO-d6): δ 1.15–1.26 (m, 4H, 2CH2), 1.42–1.50 (m, 4H, 2CH2),
1.60–1.75 (m, 8H, 4CH2), 2.86 (t, 2H, J = 5.7 Hz, CH2), 2.91 (t, 2H,
J = 5.7 Hz, CH2), 3.10 (t, 2H, J = 5.7 Hz, CH2), 3.26 (t, 2H, CH2), 7.62
(s, 1H, NH, D2O exchangeable) and 8.85 (s, 1H, NH, D2O exchange-
able) ppm; MS [m/z,%]: 454 [M.+, 6.49] and 78 [C6H6┐.+, 100]. Anal.
Calcd for C23H26N4S3 (454.66): C, 60.75; H, 5.76; N, 12.32. Found: C,
61.05; H, 5.58; N, 12.15%.
49.24 and 49.26µM, respectively) showed comparable cyto-
toxicity to imatinib. Compounds 5d, 9c, 8a, 5b, 7 and 8b (IC50:
63.57, 64.36, 65.34, 71.74, 79.90 and 143.78 µM) were less
active than imatinib and compound 8c was inactive.
Interestingly, the hexacyclicthienopyrimidine 4 and thio-
phene-3-carbonitrile 3 were the most potent derivatives of
all the synthesised compounds. A literature survey revealed
2,24,30,31
few compounds with structure resemblance to 3 and 4
possessing significant in vitro cytotoxic agents.
Among the 4-amino thieno[2,3-d]pyrimidines 2 and 5a–d,
the unsubstituted analogue 2, was the most active, while
the aliphatic derivative 5a (N-ethyl) manifested comparable
activity to imatinib. Compounds 5b–d, with N-phenyl or
N-substituted phenyl, exhibited moderate potency.
The structure–activity relationship of the thienotriazinones
was studied in view of the N-3 substituents (R1). It was
observed that the derivative with R1 = (3-nitrophenyl)carbonyl
methyl 8d demonstrated privileged activity over imatinib,
whereas 8b, R1 = ethoxycarbonylmethyl and 7, R1 = H exhib-
ited moderate activity. Unexpectedly, the thienotriazinone
8c, R1 = phenylcarbonyl -methyl failed to show significant
inhibition at the concentrations tested while compound 8b,
R1 = benzoyl showed poor activity.
In the thienodiazepinone 9a–d series, the 3-phenyl analogue
(R2 = H) 9a showed more pronounced activity than imatinib.
Whereas the alteration of R2 (when R2 = Cl, Br or NO2) had
little influence on their activity.
The preliminary encouraging results of biological screening
of the thienotriazinones and thienodiazepinones showed that
both new ring systems could offer an excellent framework in
this field and that by proper substitution; potent anti-tumour
agents may be discovered. This may be due to their structure
resemblance to thieno[2,3-d]pyrimidines.
Preparation of 4-amino-3-substituted-3H-5,6,7,8,9,10-hexahydrocy-
cloocta [4,5]thieno[2,3-d]pyrimidine-2-thiones (5a–d); general
procedure
The appropriate isothiocyanate (0.005 mol) was added to 1 (1 g,
0.005 mol) dissolved in absolute ethanol (25 mL) in the presence
of triethylamine (4 drops). The reaction mixture was heated under
reflux for 10 h, cooled and the separated solid was filtered, dried and
crystallised from dimethylformamide.
4-Amino-3-ethyl-3H-5,6,7,8,9,10-hexahydrocycloocta [4,5]thieno[2,3-d]
pyrimidine-2-thione (5a): M.p. 210–212 °C; yield 50%; IR (KBr)
1
vmax: 3437, 3440 (NH2), 1645(C=N) and 1273(C=S) cm−1; H NMR
(DMSO-d6): δ 1.15–1.30 (m, 2H, CH2), 1.24 (t, 3H, CH2CH3), 1.40–
1.50 (m, 2H, CH2), 1.55–1.65 (m, 4H, 2CH2), 2.75–2.80 (m, 2H, CH2),
2.90–3.00 (m, 2H, CH2), 4.60–5.00 (m, 2H, CH2CH3), 7.62 (s, 2H,
NH2, D2O exchangeable) ppm. Anal. Calcd for C14H19N3S2 (293.43):
C, 57.30; H, 6.52; N, 14.32. Found: C, 57.24; H, 6.39; N, 13.97%.
4-Amino-3-phenyl-3H-5,6,7,8,9,10-hexahydrocycloocta [4,5]thieno
[2,3-d]pyrimidine-2-thione (5b): M.p. 234–236 °C; yield 50%; IR
(KBr) vmax: 3452, 3440 (NH2), 1600 (C=N) and 1219 (C=S) cm−1; 1H
NMR (DMSO-d6): δ 1.10–1.15 (m, 2H, CH2), 1.45–1.50 (m, 2H,
CH2), 1.60–1.70 (m, 4H, 2CH2), 2.90–2.95 (m, 2H, CH2), 3.10–3.20
(m, 2H, CH2), 6.86 (t, 1H, J = 7.5 Hz, ArH), 6.97 (t, 2H, J = 7.5 Hz,
ArH), 7.50 (d, 2H, J = 7.5 Hz, ArH) and 8.31 (s, 2H, NH2, D2O
exchangeable) ppm. Anal. Calcd for C18H19N3S2 (341.47): C, 63.30;
H, 5.60; N, 12.30. Found: C, 63.20; H, 5.50; N, 12.00%.
4-Amino-3-(4-bromophenyl)-3H-5,6,7,8,9,10-hexahydrocycloocta
[4,5]thieno[2,3-d]pyrimidine-2-thione (5c): M.p. 208–210 °C; yield
46%; IR (KBr) vmax: 3452, 3450 (NH2), 1597 (C=N), 1222 (C=S), 825
(P-substitution) cm−1; 1H NMR (DMSO-d6): δ 1.00–1.15 (m, 2H,
CH2), 1.40–1.50 (m, 2H, CH2), 1.52–1.55 (m, 2H, CH2), 1.60–1.66
(m, 2H, CH2), 2.85–2.95 (m, 2H, CH2), 3.06–3.20 (m, 2H, CH2), 6.99
(d, 2H, J = 9.0 Hz, ArH), 7.40 (d, 2H, J = 9.0 Hz, ArH) and 8.46 (s,
2H, NH2, D2O exchangeable) ppm. Anal. Calcd for C18H18BrN3S2
(420.36): C, 51.42; H, 4.31; N, 9.99. Found: C, 52.00; H, 4.05; N,
11.49%.
Experimental
Melting points were obtained on a Griffin apparatus and are uncor-
rected. Microanalyses for C, H and N were carried out at the Micro-
analytical Center, Cairo University. IR spectra were recorded on a
Shimadzu 435 spectrometer, using KBr discs. 1H NMR spectra
were performed on a Joel NMR FXQ-200 MHz spectrometer, using
TMS as the internal standard. Mass spectra were recorded on a
GCMP-QP1000 EX mass spectrometer. Progress of the reactions was
monitored by TLC using precoated aluminum sheet silica gel MERCK
60F 254 and was visualised by UV lamp.
4-Amino-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]pyri-
midine (2): A solution of 2-Amino-4,5,6,7,8,9-hexahydrocycloocta
[b]thiophene-3-carbonitrile (1) (0.21 g, 0.001 mol) in formamide
(10 mL) was refluxed for 2 h. The reaction mixture was cooled; the
formed solid was filtered, washed with ethanol (10 mL), dried and
4-Amino-3-(3-methylphenyl)-3H-5,6,7,8,9,10-hexahydrocycloocta
[4,5]thieno[2,3-d]pyrimidine-2-thione (5d): M.p. 209–211 °C; yield
30%; IR (KBr) vmax: 3480, 3460 (NH2), 1610 (C=N) and 1193 (C=S)
cm−1; 1H NMR (DMSO-d6): δ 1.10–1.20 (m, 2H, CH2), 1.45–1.53 (m,
2H, CH2), 1.55–1.65 (m, 4H, 2CH2), 2.14 (s, 3H, CH3), 2.91 (t, 2H,
CH2), 3.16 (t, 2H, CH2), 6.72–6.80 (m, 2H, ArH), 7.35 (d, 1H, ArH),
7.42 (s, 1H, ArH) and 8.20 (s, 2H, NH2, D2O exchangeable) ppm; MS
crystallised from ethanol. M.p. 208–210 °C; yield 65%; IR (KBr) vmax
:
3394, 3305 (NH2) cm−1; H NMR (DMSO-d6): δ 1.13–1.25 (m, 2H,
CH2), 1.40–1.50 (m, 2H, CH2), 1.55–1.65 (m, 4H, 2CH2), 2.85 (t, 2H,
J = 6.0 Hz, CH2), 2.97 (t, 2H, J = 6.0 Hz, CH2), 6.83 (s, 2H, NH2, D2O
exchangeable) and 8.18 (s, 1H, C2-H) ppm; MS [m/z,%]: 235
[M+H+1┐.+ and M+2┐.+, 11.54], 234 [M+H┐.+ and M+1┐.+, 100] and 233
[M.+, 6.09]. Anal. Calcd for C12H15N3S2 (233.32): C, 61.76; H, 6.48; N,
18.00. Found: C, 61.60; H, 6.50; N, 18.19%.
1
[m/z,%]: 356 [M+H┐.+, 15.43] and 340 [M-NH┐.+ and or M-CH3┐.+
,
100]. Anal. Calcd for C19H21N3S2 (355.50): C, 64.18; H, 5.95; N,
11.81. Found: C, 64.30; H, 5.60; N, 12.00%.
2-(N-Ethylcarbamothioylamino)-4,5,6,7,8,9-hexahydrocyclo-octa[b]
thiophene-3-carbonitrile (3): Ethyl isothiocyanate (0.44 g, 0.005 mol)
was added dropwise to a well stirred solution of 1 (1 g, 0.005 mol) in
absolute ethanol (5 mL). The mixture was then refluxed on a steam
bath for 6 h and left overnight at room temperature. The separated
solid was filtered, dried and crystallised from ethanol. M.p. 162–164
°C; yield 50%; IR (KBr) vmax: 3331, 3265 (2NH), 2216 (CN) and 1247
5,6,7,8,9,10-Hexahydrocycloocta[4,5]thieno[2,3-d]-1,2,3-triazin-
4(3H)-one (7): A solution of sodium nitrite (1.12 g, 0.016 mol in 11.2
ml water) was added dropwise with cooling and stirring to a suspen-
sion of 2-amino-4,5,6,7,8,9,10-hexahydrocycloocta[b]thiophene-3-
carboxamide (6) (1 g, 0.004 mol) in hydrochloric acid (6 N, 23 mL)
over a period of 15 minutes. The mixture was further stirred at room
temperature for 30 minutes and left overnight; the formed solid was
filtered, washed with water (20 mL), dried and crystallised from ethyl
acetate. M.p. 120–122 °C; yield 77%; IR (KBr) vmax: 3200 (NH) and
1665 (C=O) cm−1; 1H NMR (CDCl3): δ 1.25–1.40 (m, 2H, CH2), 1.42–
1.55 (m, 2H, CH2), 1.70–1.85 (m, 4H, 2CH2), 2.98 (t, 2H, J = 6.2 Hz,
CH2), 3.16 (t, 2H, J = 6.2 Hz, CH2) and 13.72 (s, 1H, NH, D2O
1
(C=S) cm−1; H NMR (CDCl3): δ 1.31 (t, 3H, J = 7.2 Hz, CH2CH3),
1.26-1.35 (m, 2H, CH2), 1.38-1.50 (m, 2H, CH2), 1.60-1.70 (m, 4H,
2CH2), 2.68 (t, 2H, J = 6.3 Hz, CH2), 2.73 (t, 2H, J = 6.3 Hz, CH2),
3.63 (q, 2H, J = 7.2 Hz, CH2CH3), 7.38 (s, 1H, NH, D2O exchange-
able) and 9.23 (s, 1H, NH, D2O exchangeable) ppm; MS [m/z,%]: 295
[M+H+1┐.+ and M+2┐.+, 51.40], 294 [M+H┐.+ and M+1┐.+, 100] and 293
[M.+, 52.42]. Anal. Calcd for C14H19N3S2 (293.43): C, 57.30; H, 6.52;
N, 14.32. Found: C, 57.58; H, 6.50; N, 14.54.
exchangeable) ppm; MS [m/z,%]: 237 [M+2┐.+, 2.50], 236 [M+1┐.+
,

JOURNAL OF CHEMICAL RESEARCH 2012 109
2.70], 235 [M.+, 18.60], 207 [M-CO┐.+, 37.20] and 164 [C10H12S┐.+
100]. Anal. Calcd for C11H13N3OS (235.29): C, 56.14; H, 5.56; N,
17.85. Found: C, 56.30; H, 5.40; N, 17.39%.
,
1.53–1.66 (m, 4H, 2CH2), 2.85–2.90 (m, 2H, CH2), 3.05–3.10 (m, 2H,
CH2), 7.58 (d, 2H, J = 8.8 Hz, ArH), 7.93 (s, 1H, C2-H), 8.13 (d, 2H,
J = 8.8 Hz, ArH) and 12.55 (s, 1H, NH, D2O exchangeable) ppm.
Anal. Calcd for C19H19ClN2OS (358.87): C, 63.58; H, 5.33; N, 7.80.
Found: C, 63.60; H, 5.40; N, 7.95%.
Preparation of 3-substituted-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno
[2,3-d]-1,2,3-triazin-4(3H)-ones (8a–d); general procedure
A mixture of the triazine 7 (0.28 g, 0.001 mol), anhydrous potassium
carbonate (0.35 g, 0.002 mol) and the selected halogen compound
(0.01 mol) in dry acetone (10 mL) was heated under reflux for 8 h.
The reaction mixture was then cooled, treated with ice cold water
(50 mL) and the separated solid was filtered, washed with water
(20 mL), dried and crystallised from ethanol.
3-Ethoxycarbonylmethyl-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno
[2,3-d]-1,2,3-triazin-4(3H)-one (8a): M.p. 108–110 °C; yield 88%;
IR (KBr) vmax: 1751 and 1678 (2C=O) cm−1; 1H NMR (CDCl3): δ 1.30
(t, 3H, J = 7.0 Hz, CH2CH3), 1.30–1.42 (m, 2H, CH2), 1.45–1.55 (m,
2H, CH2), 1.70–1.82 (m, 4H, 2CH2), 2.99 (t, 2H, J = 5.8 Hz, CH2),
3.14 (t, 2H, J = 5.8 Hz, CH2), 4.26 (q, 2H, J = 7.0 Hz, CH2CH3) and
5.14 (s, 2H, NCH2CO) ppm; Anal. Calcd for C15H19N3O3S (321.38):
C, 56.05; H, 5.95; N, 13.07. Found: C, 55.90; H, 5.80; N, 13.27%.
3-Benzoyl-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]-
1,2,3-triazin-4(3H)-one (8b): M.p. 104–106 °C; yield 50%; IR (KBr)
vmax: 1755 and 1668 (2C=O) cm−1; 1H NMR (DMSO-d6): δ 1.30–1.55
(m, 4H, 2CH2), 1.65–1.75 (m, 4H, 2CH2), 2.90–3.10 (m, 4H, 2CH2),
7.60–7.75 (m, 3H, ArH) and 8.15–8.25 (m, 2H, ArH) ppm. Anal.
Calcd for C18H17N3O2S (339.40): C 63.69; H, 5.05; N, 12.38. Found:
C, 63.80; H, 5.00; N, 12.86%.
3-Phenylcarbonylmethyl-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno
[2,3-d]-1,2,3-triazin-4(3H)-one (8c): M.p. 170–172 °C; yield 50%;
IR (KBr) vmax: 1690 and 1676 (2C=O) cm−1; 1H NMR (CDCl3):
δ 1.25–1.40 (m, 2H, CH2), 1.45–1.55 (m, 2H, CH2), 1.65–1.80 (m, 4H,
2CH2), 2.90–3.05 (m, 2H, CH2), 3.07–3.20 (m, 2H, CH2), 5.85 (s, 2H,
NCH2CO), 7.45–7.70 (m, 3H, ArH) and 8.00–8.13 (m, 2H, ArH) ppm.
Anal. Calcd for C19H19N3O2S (353.42): C, 64.56; H, 5.41; N, 11.88.
Found: C, 64.60; H, 4.71; N, 11.69%.
3-(3-Nitrophenyl)carbonyl-5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno
[2,3-d]-1,2,3-triazin-4(3H)-one (8d): M.p. 98–100 °C; yield 51%; IR
(KBr) vmax: 1668 (2C=O), 1529 and 1348 (NO2) cm−1; 1H NMR
(DMSO-d6): δ 1.25–1.30 (m, 2H, CH2), 1.35–1.45 (m, 2H, CH2),
1.55–1.65 (m, 2H, CH2), 1.66–1.73 (m, 2H, CH2), 3.05–3.10 (m, 4H,
2CH2), 6.14 (s, 2H, NCH2CO), 7.92 (t, 1H, ArH), 8.53–8.58 (m, 2H,
ArH) and 8.79 (s, 1H, ArH) ppm; MS [m/z,%]: 399 [M+1┐.+, 0.04],
398[M.+,0.21]and150[C7H4NO3┐.+,100].Anal.CalcdforC19H18N4O4S
(398.42): C, 57.27; H, 4.55; N, 14.06. Found: C, 57.40; H, 4.60; N,
14.17%.
3-(3-Nitrophenyl)-6,7,8,9,10,11-hexahydro-1H-cycloocta[4,5]thieno
[2,3-e]-1,4-diazepin-5-(4H)-one (9d): M.p. >300 °C; yield 80%; IR
(KBr) vmax: 3387, 3248 (2NH), 1659 (C=O), 1528 and 1346 (NO2)
cm−1; 1H NMR (DMSO-d6): δ 1.25–1.30 (m, 2H, CH2), 1.35–1.46 (m,
2H, CH2), 1.55–1.70 (m, 4H, 2CH2), 2.90 (t, 2H, J = 6.0 Hz, CH2),
3.07 (t, 2H, J = 6.0 Hz, CH2), 7.79–7.85 (m, 2H, ArH and C2-H), 8.38
(d, 1H, J = 8.4 Hz, ArH), 8.54 (d, 1H, J = 8.1 Hz, ArH), 8.94 (s, 1H,
ArH), 12.15 (br s, 1H, NH, D2O exchangeable) and 12.82 (s, 1H, NH,
D2O exchangeable) ppm; MS [m/z,%]: 371 [M+2┐.+, 2.49], 370 [M+1┐.+
and M+H┐.+, 2.07], 369 [M.+, 1.76], 341 [M-CO┐.+, 0.58], 194
[C10H14N2S┐.+, 2.26], 179 [C10H13NS┐.+, 10.80], 164 [C10H12S┐.+, 7.12]
and 41 [C3H5┐.+, 100]. Anal. Calcd for C19H19N3O3S (369.42): C, 61.76;
H, 5.18; N, 11.37. Found: C, 61.79; H, 4.53; N, 11.06%.
Cytotoxic activity studies
Anti-cancer activity studies were carried out at Cairo University,
National Cancer Institute, Cancer Biology Department, Pharmacol-
ogy Unit.
Compounds 2–9d were tested at concentrations between 1 and
10 µg mL−1 using SulfoRhodamine-B (SRB) assay for cytotoxic
activity against human colon tumour cell line (HCT116). Imatinib
which is a 2-substituted aminopyrimidine derivative was chosen as a
reference standard anti-cancer drug because it showed potency against
gasterointestinal tract tumours.28,29
The potential cytotoxicity of the compounds was tested using the
method of Skehan et al.32 Cells were plated in 96 multiwell plates
(104 cells/well) for 24 h before treatment with the compound(s) to
allow attachment to the wall of the plate. Different concentrations of
the compounds (0, 1, 2.5, 5 and 10 µg mL−1) were added to the cell
monolayer and triplicate wells were prepared for each individual dose.
Monolayer cells were incubated with the compound(s) for 48 h at
37 ºc in an atmosphere of 5% CO2. After 48 h, cells were fixed, washed
and stained with SulfoRhodamine-B stain. Excess stain was washed
with acetic acid and the attached stain was recovered with Tris EDTA
buffer. Colour intensity was measured in an ELISA reader. The rela-
tionship between the surviving fraction and the drug concentration
was plotted to obtain the survival curve of each tumour cell line after
the specified compound was administered.
Received 14 December 2011; accepted 16 January 2012
Paper 1101040 doi: 10.3184/174751912X13282020691270
Published online: 23 February 2012
Preparation of 3-aryl-6,7,8,9,10,11-hexahydro-1H-cycloocta[4,5]
thieno[2,3-e]-1,4-diazepin-5-(4H)-ones (9a–d); general procedure
The selected phenacyl bromide (0.004 mol) was added to a solution of
amino amide 6 (1 g, 0.004 mol) in glacial acetic acid (20 mL). The
reaction mixture was heated under reflux for 10 h, and then cooled;
the separated solid was filtered, washed with ethanol (20 mL), dried
and crystallised from dimethylformamide.
3-Phenyl-6,7,8,9,10,11-hexahydro-1H-cycloocta[4,5]thieno[2,3-e]-
1,4-diazepin-5-(4H)-one (9a): M.p. >300 °C; yield 50%; IR (KBr)
vmax: 3445, 3180 (2NH) and 1655 (C=O) cm−1; 1H NMR (DMSO-d6):
δ 1.25–1.35 (m, 2H, CH2), 1.40–1.46 (m, 2H, CH2), 1.60–1.66 (m, 4H,
2CH2), 2.88 (t, 2H, J = 5.7 Hz, CH2), 3.08 (t, 2H, J = 5.7 Hz, CH2),
7.48–7.56 (m, 4H, 3ArH and C2-H), 8.09 (d, 2H, J = 8.1 Hz, ArH) and
12.47 (s, 1H, NH, D2O exchangeable) ppm. Anal. Calcd for
C19H20N2OS (324.43): C, 70.33; H, 6.21; N, 8.63. Found: C, 70.40; H,
6.30; N, 8.89%.
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