Synthesis and in vitro anticancer activity of some novel cyclohepta[b]thiophene-3-carboxamides bearing pyrazole moiety

Article abstract of DOI:10.1002/jhet.4041

2-Aminothiophene 3 was achieved through the one-pot multicomponent reaction of cycloheptanone, cyanoacetamide, elemental sulfur, and morpholine in ethanol. Diazotization of 2-aminothiophene 3 with NaNO₂/HCl gave the corresponding diazonium salt

Full text of DOI:10.1002/jhet.4041

Received: 11 April 2020
DOI: 10.1002/jhet.4041
Revised: 12 May 2020
Accepted: 14 May 2020
A R T I C L E
Synthesis and in vitro anticancer activity of some novel
cyclohepta[b]thiophene-3-carboxamides bearing pyrazole
moiety
Mohammed Al-Ghorbani1,2
| Moustafa A. Gouda1,3
1Department of Chemistry, Faculty of
Science and Arts, Ulla, Taibah University,
Medina, Saudi Arabia
2Department of Chemistry, Faculty of
Education, Thamar University, Dhamar,
Yemen
3Department of Chemistry, Faculty of
Science, Mansoura University, Mansoura,
Egypt
Abstract
2-Aminothiophene 3 was achieved through the one-pot multicomponent reaction
of cycloheptanone, cyanoacetamide, elemental sulfur, and morpholine in ethanol.
Diazotization of 2-aminothiophene 3 with NaNO2/HCl gave the corresponding
diazonium salt 4, that combined with the appropriate active methylene compo-
nents; 5a, 5b, 7, 11, 13, 16, 18, 21, 9, 19, 22a, and 22b in pyridine (AcONa/EtOH)
to form the corresponding hydrazones 6a, 6b, 8, 10, 14, 15, 17, 20, 23, 24, 25a, and
25b, respectively. Heating of compound 8 with malononitrile 9 in ethanol gave
the thiazole 10. Treatment of compound 10, 25a, and 25b with hydrazine hydrate
achieve the pyrazoles 12, 27a, and 27b, respectively. Hydrazinolysis of compound
14 with hydrazine hydrate, followed by condensation of the obtained hydrazide
15 with acetylacetone 19 gave the pyrazole 20. The recently orchestrated thio-
phenes were assessed for their cytotoxic action. The result revealed that com-
pound 12 indicated comparable and better action towards HePG2, HCT-116,
MCF-7, and PC3 cancer cell lines than Doxorubicin.
Correspondence
Moustafa A. Gouda, Department of
Chemistry, Faculty of Science and Arts,
Ulla, Taibah University, Medina, Saudi
Arabia.
Email: dr_mostafa_chem@yahoo.com
1 | INTRODUCTION
agents.[18–21] AT9283 is a powerful inhibitor of Aurora A
kinase and of different cancers-associated kinases, PHA-
The chemistry of 2-aminothiophenes has received much
attention; due to their convenient availability through the
versatile synthesis introduced by Gewalled.[1] Furthermore,
they were discovered to have diverse organic programs,
which include, N-(4-methoxyphenyl)-N,2,5-trimethylthieno
[2,3-days]pyrimidin-4-amine (I), a potent apoptosis indu
cer[2]; N-(4-chlorophenethyl)-2,5-dimethylthieno[2,3-days]
pyrimidin-4-amine (II), a potential anti-inflammatory and
anti-osteoporosis agent[3]; (2-amino-4,5-dimethylthiophen-
3-yl)(3-[trifluoromethyl]phenyl)methanone (III), an agonist
of allosteric enhancers (AE)[4] (Figure 1).
Moreover, cyclohepta[b]thiophene possesses a huge
spectrum of pharmacological activities, including:
antimicrobial,[5–8] antiviral,[9,10] anti-inflammatory,[11,12]
and antitumor activities.[7,13–17] Moreover, pyrazole deriv-
atives are a distinctive lead nucleus that represents a
basic framework of a large number of anticancer
739358 is likewise by and by in Stage II of clinical prelim-
inary, basically for the cure of leukemia.[22,23] The
2-aminothiophene bearing pyrazole nucleus was hardly
ever acknowledged.[24–30] Therefore, our research deals
with the effective use of 2-amino-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide in the synthesis
of novel pyrazoles incorporating thiophene nucleus to
assess their antitumor activity.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The aminopyrazoles 6a,b had been prepared in multi-step
reaction sequences as shown in Scheme 1. Treatment of
cycloheptanone 1 with cyanoacetamide 2 in ethanol and
J Heterocyclic Chem. 2020;1–9.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals LLC.
1

2
AL-GHORBANI AND GOUDA
20. Compound 15 may be also prepared through heating
of compound 14 with hydrazine hydrate. Cyclization of
compound 15 under basic condition gave also, the
aminopyrazole 17. Compound 15 was further converted
into 3,5-dimethylpyrazole 20 via the reaction with
acetylacetone in EtOH/DMF (Scheme 3).
Finally, the intermediate
4 was reacted with
FIGURE 1 The biological applications of some
malononitrile 9, acetylacetone 19, or 3-methyl-1-H-
pyrazol-5(4-H)-one 20a,b[35,36] in ethanol/sodium acetate
(pyridine) to afford the corresponding hydrazone 23, 24,
25a, and 25a,b, respectively. Compounds 23 and 24
cyclized with hydrazine hydrate in ethanol to give the
3,5-diaminopyrazole 26 and 3,5-dimethylpyrazole 27,
respectively (Scheme 4).
2-aminothiophene derivatives
in the presence of morpholine and sulfur, resulted the
corresponding 2-aminothiophene-3-carboxamide 3. Com-
pounds 3 was ultimately subjected to diazotization with
sodium nitrite in AcOH/HCl to afford the corresponding
diazonium salt 4, that combined with 3-amino-5-(cyano
methyl)-1H-pyrazole-4-carbonitriles 5a,b[31] in pyridine to
give the preferred cyclohepta[b]thiophene-3-carboxamides
6a,b (Scheme 1).
2.2 | In vitro anticancer activity
Moreover, the intermediate 4 was coupled with
1-phenyl-2-thiocyanatoethanone 7[32] in pyridine giving
the corresponding hydrazo 8 which, underwent ring clo-
sure with malononitrile 9 in DMF in the presence of
piperidine to achieve the thiazole 10. Furthermore, com-
pound 10 was prepared through interaction of 4 with
substituted thiazole 11[33] in pyridine. Cyclization of
malononitrile derivative 10 with hydrazine hydrate in
DMF achieved the 3,5-diaminopyrazole 12 (Scheme 2).
In addition, the diazonium salt 4 was coupled with var-
ious compounds specifically: ethyl 2-cyanoacetate 13 or
2-cyanoacetohydrazide 14[34] or 5-amino-1H-pyrazol-3-ol
16, or 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile
19[34] in ethanol in the presence sodium acetate or pyri-
dine to gain the corresponding hydrazones 14, 15, 17, and
The anticancer activity of 15 novel pyrazoles were evalu-
ated in vitro against a liver (HePG2), breast (MCF-7),
colon (HCT-116), and prostate (PC3 human) cancer cell
lines, using the standard MTT method.[37] All tested com-
pounds were screened for cytotoxic activity with imply
50% inhibition concentration (IC50). MTT assay is mea-
suring cell growth by colorimetric changes. It is utilized
to decide cytotoxicity of capacity therapeutic specialists
and other dangerous materials. In summary, yellow MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro
mide) is decreased to purple formazan by mitochondrial
dehydrogenases of living cells. An appropriate solvent is
used to dissolve purple formazan product. The absor-
bance of this colored solution might be evaluated at a def-
inite wavelength. At the point when the measure of
SCHEME 1 Synthesis of thiophene-3-carboxamides 6a,b

AL-GHORBANI AND GOUDA
3
SCHEME 2 Synthesis of
diaminopyrazoles 12
SCHEME 3 Synthesis of pyrazoles 17 and 20
purple formazan created by cells treated with an agent is
compared with that produced by unreacted control cells,
the viability of the agent in causing death of cells can be
deduced, through the production of a dose response
curve.[38]
The cytotoxicity consequences Table 1, found out that
compounds, 12 was the first-rate inhibitory activity in oppo-
sition to HCT-116, HePG2, PC3, and MCF-7 cancer cell
lines. Whereas, compounds 8 and 25a are the vulnerable
inhibitory activity towards the selected cancer cell lines. As

4
AL-GHORBANI AND GOUDA
CN
SCHEME 4 Synthesis of
pyrazoles 25-27
NC
CONH
2 H2N
CN
CN
H2NOC
H
9
H
N
NH2NH2.H2O
N
N
N
N
N
EtOH
reflux, 13h.
S
EtOH
/AcONa
S
stirr,3h, 0-5 0C
26
NH2
23
COCH3
CONH
2 H3C
COCH3
COCH3
NH2NH2.H2O
H2NOC
H
H
N
19
N
N
4
N
COCH3
EtOH
N
N
S
EtOH
/AcONa
stirr,3h, 0-5 0C
CH3
S
reflux, 13h.
CH3
27
24
N
O
N
CONH2
R
22
H3C
H
N
N
N
pyridine
stirr,3h, 0-5 0C
S
N R
O
25
25a ;R=H
25b ;R=Ph
TABLE 1
pyrazole derivatives against four human
tumor cells
Cytotoxic activity of
In vitro cytotoxicity IC50 (μg/mL)a
Compound no.
HePG2
HCT-116
MCF-7
PC3
Doxorubicin
4.50 0.2
20.36 1.8
59.50 3.8
81.29 4.3
8.94 0.7
3.81 0.2
45.49 3.2
15.40 1.4
6.14 0.4
47.23 3.4
48.13 3.6
7.58 0.6
92.65 5.1
25.08 2.0
14.32 1.3
5.23 0.3
38.52 3.2
76.60 4.6
78.63 4.6
20.13 1.8
5.85 0.4
62.41 4.1
35.76 2.9
16.84 1.4
71.42 4.5
66.05 4.1
13.76 1.2
85.35 4.8
43.78 3.5
31.12 2.7
4.17 0.2
32.73 2.5
63.84 4.2
69.93 4.5
11.65 1.0
4.92 0.3
53.41 3.8
24.55 2.0
7.82 0.6
56.90 3.8
58.76 3.9
9.39 0.8
73.18 4.7
35.18 2.7
22.69 1.9
8.87 0.6
32.73 2.5
82.33 4.9
93.82 5.3
28.31 2.8
9.70 0.8
79.39 4.8
45.94 3.7
27.39 2.4
89.43 5.1
76.29 4.8
20.78 1.7
>100
5
6
8
10
12
14
15
17
20
23
24
25a
25b
27
59.04 3.9
41.45 3.6
aIC50 (μg/mL): 1-10 (very strong); 11-20 (strong); 21-50 (moderate); 51-100 (weak); and above
100 (non-cytotoxic).
for hobby in the competition to HepG2 cell line, the very
highest cytotoxic activity has become showed with the aid
of compound 10, 17, and 24, that displayed the proportion
viability IC50 at 8.94, 6.14, and 7.58 mg/mL, respectively,
while, the best cytotoxic activity was hobby changed into
displayed by compound 5, 15, and 27 which, confirmed the
proportion viability IC50 at 20.36, 15.40, and 14.32 mg/mL
respectively. The MCF-7, and HCT-116 cell lines showed
moderate activity towards compounds 5, 15, 25b, and 27
which have IC50 at 38.52 (32.73), 35.76 (24.55), 31.12
(22.69), and 43.18 (35.18) mg/mL, respectively. However,
compound 5 and 15 showed slight activity, while, com-
pounds 6, 12, 22, and 23 displayed weak cytotoxic activities
against MCF-7, PC3, and HCT-116 cancer cell lines.

AL-GHORBANI AND GOUDA
5
Compound 12 showed comparable and better activity in
opposition to HCT-116, MCF-7, PC3, and HePG2 cancer
cell lines than Doxorubicin.
3.3.1 | (Z)-2-(2-((3-amino-4-cyano-1H-
pyrazol-5-yl)(cyano)methylene)hydrazinyl)
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (6a)
3 | EXPERIMENTAL
Yield 83%; reddish brown powder; mp > 320ꢀC. IR (KBr)
νmax/cm−1 = 3424, 3314, 3214, 3171 (2NH2, 2NH), 2923,
2853 (C-aliph), 2215 (2CN), 1649 (CO), 1596 (C C), 1526
3.1 | Materials and instrumentation
(C N). 1H-NMR (DMSO-d6)
δ
1.50-1.68 (m, 4H,
All melting points were decided on Gallenkamp elec-
tronic melting point equipment. The infrared (IR) spectra
were recorded on a Mattson 5000 Fourier transform-IR
(FT-IR) spectrophotometer (λ, cm−1). The 1H-NMR
(DMSO-d) spectra were determined on a Bruker AV
300 MHz, using TMS as internal standard. Mass spectra
were obtained on GCMS/QP1000 Ex mass spectrometer
at 70 eV. Elemental analyses (C, H, and N) had been
done at the Microanalytical Center of Cairo University,
Giza, Egypt. The biological activities were carried in
Pharmacognosy Department, Faculty of Pharmacy, Man-
soura University, Mansoura, Egypt.
cycloheptane, CH2), 1.75-1.81 (m, 2H, cycloheptane, CH2),
2.66-2.78 (m, 4H, cycloheptane, CH2), 7.17 (br, 2H, NH2),
7.34 (br, 2H, CONH2), 7.65 (br, 1H, NHpyrazole), 12.54 (br, 1H,
NHhydrazo); ms: (m/z, %): 368 (M+, 22.23), 329.27 (61.16),
317.94 (100), 282.34 (76.65), 272.01 (67.84), 254.61 (97.11),
227.27 (40.08), 194.30 (46.51), 131.51 (38.68), 94.82 (51.91),
69.86 (44.49). Anal. Calcd for C16H16N8OS: C, 52.16; H,
4.38; N, 30.41. Found: C, 51.99; H, 4.15; N, 30.22%.
3.3.2 | (Z)-2-(2-((3-amino-4-cyano-
1-phenyl-1H-pyrazol-5-yl)(cyano)
methylene) hydrazin-yl)-5,6,7,8-tetrahydro-
4H-cyclohepta[b]thiophene-
3.2 | Chemistry
3-carboxamide (6b)
Coupling of the diazonium salt of 2-aminothiophene
derivative 4 with 3-amino-5-(cyanomethyl)-1-H-pyrazole-
4-carbonitrile 5a or 3-amino-5-(cyanomethyl)-1-phenyl-
1-H-pyrazole-4-carbonitrile 5b or 1-phenyl-2-thiocyanato
ethanone 7 or 2-(4-phenylthiazol-2[3H]-ylidene) malono-
nitrile 10 or 3-methyl-1H-pyrazol-5(4H)-one 22a, or 3-
methyl-1-phenyl-1H-pyrazol-5(4H)-one 22b.
Yield 69%; reddish brown powder; mp > 320ꢀC. IR (KBr)
νmax/cm−1 = 3428 (br), 3332, 3224, 3185 (2NH2, NH),
2924, 2854 (C-aliph), 2208 (2CN), 1634 (CO), 1628
1
(C C), 1528 (N N). H-NMR (DMSO-d6) δ 1.50-1.73 (m,
4H, cycloheptane, CH2), 1.75-1.81 (m, 2H, cycloheptane,
CH2), 2.63-2.80 (m, 4H, cycloheptane, CH2), 6.85 (br, 2H,
NH2), 7.20-7.67 (m, 9H, ArH, CONH2), 7.93 (br, 1H,
NHpyrazol), 12.50 (br, 1H, NHhydrazo). Anal. Calcd for
C22H20N8OS; C, 59.44; H, 4.54; N, 25.21. Found: C,
59.26; H, 4.29; N, 25.17%.
3.3 | General procedure
A solution of 2-aminothiophene derivative 3 (10 mM;
2.10 g) in 5 mL acetic acid, and 3 mL conc. HCl was stirred
and cooled in ice bath. Then, a solution of sodium nitrite
(0.8 g, 1.15 mM) in 8 mL water was brought dropwise
for 10 min. The cold diazonium solution become delivered
3.3.3 | (Z)-2-(2-[2-oxo-2-phenyl-
1-thiocyanatoethylidene] hydrazinyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (10)
slowly to
a properly stirred solution of 3-amino-
5-(cyanomethyl)-1H-pyrazole-4-carbonitrile (1.47 g, 10
mM) 5a, 3-amino-5-(cyanomethyl)-1-phenyl-1H-pyrazole-
4-carbonitrile (2.23 g, 10 mM) 5b, 1-phenyl-2-thiocyanato
ethanone 7 (1.77 g, 10 mM), 2-(4-phenylthiazol-2-[3H]-
ylidene)malononitrile 10 (2.25 g, 10 mM), 3-methyl-1H-
pyrazol-5(4H)-one 22a (0.98 g, 10 mM), or 3-methyl-1-phe-
nyl-1H-pyrazol-5(4H)-one 22b (1.74 g, 10 mM) in pyridine
(40 mL). Then, the reaction mixture was stirred for
3 hours, and the formed precipitate was filtered off, dried,
and recrystallized from ethanol/benzene to afford 6a,b, 10,
12, and 25a,b.
Yield 90%; pale yellow powder; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3433 (br), 3300, 3185 (NH2, NH), 2924, 2854
(C-aliph), 2203 (CN), 1662 (2CO), 1599 (C C), 1571
(N N). 1H-NMR (DMSO-d6)
δ 1.50-1.62 (m, 4H,
cycloheptane CH2), 1.81-1.89 (m, 2H, cycloheptane, CH2),
2.59-2.92 (m, 4H, cycloheptane, CH2), 7.18-7.84 (m, 7H,
ArH, CONH2,), 13.05 (br, 1H, NH); ms: (m/z, %): 400 (M
+ 2, 22.23), 398.76 (M+, 7.61), 368.82 (100), 351.08 (36.97),
229.14 (49.30), 228 (58.63), 200.01 (40.08), 144.93 (54.73).
Anal. Calcd for C19H18N4O2S2; C, 57.27; H, 4.55; N, 14.06.
Found: C, 57.09; H, 4.26; N, 14.13%.

6
AL-GHORBANI AND GOUDA
3.3.4 | (E)-2-(2-[dicyanomethylene]-
4-phenyl-2,3-dihydrothiazol-5-yl) diazenyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (10)
3.4.1 | (E)-2-(2-(3-methyl-5-oxo-1H-
pyrazol-4[5H]-ylidene) hydrazinyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (25a)
Yield 83%; green powder; mp > 320ꢀC. IR (KBr)
νmax/cm−1 = 3445, 3363, 3170 (NH2, NH), 2920, 2847
(C-aliph), 2214, 2201 (2CN), 1649 (CO), 1593 (C C),
Yield 93%; scarlet red crystal; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3423, 3357, 3195 (NH2, NH), 2924, 2854 (C-
1
aliph), 1690, 1659 (2CO), 1587 (N N); H NMR (DMSO-
1
1543 (N N). H-NMR (DMSO-d6) δ 1.50-1.69 (m, 4H,
d6) (δ/ppm): 1.50-1.68 (m, 4H, cycloheptane, CH2),
1.73-1.84 (m, 2H, cycloheptane, CH2), 2.62-2.83 (m, 4H,
cycloheptane, CH2), 2.91 (s, 3H, CH3), 7.58 (br, 2H, NH2),
8.55 (br,1H, NHpyrazolone), 12.50 (br, 1H, NHhydrazo); ms:
(m/z, %): 320 (M + 1, 22.44), 319 (M+, 32.12), 304.23
(64.13), 276.98 (69.42), 250.35 (74.27), 231.07 (78.16),
204.47 (67.68), 190.35 (69.76), 150.27 (67.25), 122.04
(100), 67.55 (60.34); Anal. for C14H17N5O2S; C, 52.65; H,
5.37; N, 21.93. Found: C, 52.33; H, 5.13; N, 22.05%.
cycloheptane, CH2), 1.75-1.81 (m, 2H, cycloheptane,
CH2), 2.63-2.75 (m, 4H, cycloheptane CH2), 7.33-7.84
(m, 8H, Ar-H, NH2, NH); ms: (m/z, %): 447 (M + 1,
22.33), 446 (M+, 19.49), 400.79 (40.58), 373.31
(34.33), 303.48 (56.07), 271.96 (32.54), 175.35 (31.06),
140.17 (40.89), 126.96 (55.51), 92.39 (100), 73.42
(59.09), 53.30 (43.02). Anal. Calcd for C22H18N6
OS2; C, 59.17; H, 4.06; N, 18.82. Found: C, 58.94; H,
4.13; N, 18.66%.
Compound 10 was additionally, prepared via
heating of (Z)-2-(2-[2-oxo-2-phenyl-1-thiocyanatoethyli
dene] hydrazinyl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thi
ophene-3-carbox-amide 8 (1.78 g, 10 mM), malononitrile
9 (0.66 g, 10 mM) and piperidine (0.85 g, 10 mM) in etha-
nol 50 mL for 10 hours. The reaction mixture became
cooled and the fashioned precipitate became filtered to
give compound 9. Yield 80%; green powder.
3.4.2 | (E)-2-(2-(3-methyl-5-oxo-1-phenyl-
1H-pyrazol-4[5H]-ylidene)hydrazinyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (25b)
Yield 90%; scarlet red crystal; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3422, 3313, 3195 (NH2, NH), 2919, 2849 (C-
1
aliph), 1671, 1631 (2CO), 1542 (N N). H-NMR (DMSO-
d6) δ 1.50-1.66 (m, 4H, cycloheptane, CH2), 1.73-1.84 (m,
2H, cycloheptane, CH2), 2.57 (s, 3H, CH3), 2.65-2.81 (m,
4H, cycloheptane CH2), 6.80-7.65 (m, 7H, ArH, NH2), 9.12
(br, 1H, NHhydrazo); ms: (m/z, %): 395(M+, 14.97), 235.17
(100), 217.30 (71.30), 177.28 (18.28), 126.93 (46.29), 89.16
(60.17), 74.59 (56.19), 64.18 (67.33), 62.91 (64.25), 55.40
(46). Anal. Calcd for C20H21N5O2S; C, 60.74; H, 5.35; N,
17.71. Found: C, 60.63; H, 5.42; N, 17.49%.
Reaction of the diazonium salt of 2-aminothiophene
derivative 4 with ethyl 2-cyanoacetate, 2-cyanoaceto
hydrazide 16, 5-amino-1H-pyrazol-3-ol 18, 3-(3,5-
dimethyl-1-H-pyrazol-1-yl)-3-oxopropanenitrile 21, mal-
ononitrile 9, or acetylacetone 19.
3.4 | Synthesis of (E)-2-([2-3,5-diamino-
4H-pyrazol-4-ylidene]-4-phenyl-
2,3-dihydrothia-zol-5-yl)diazenyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (12)
A suspension of 10 (0.446 g, 1 mM), and hydrazine
hydrate (0.26 g, 4 mM mol) in ethanol (40 mL) was
refluxed for 11 hours. The formed precipitate after
cooling was filtered off and crystallized from
DMF/EtOH to yield compound 12. Yield 66%; brown
crystals; mp > 320ꢀC. IR (KBr) νmax/cm−1 = 3431,
3376, 3328, 3271 (3NH2, NH), 2922, 2851 (C-aliph),
1
2198 (CN), 1648 (CO), 1592 (C C), 1546 (N N). H-
NMR (DMSO-d6) δ 1.50-1.69 (m, 4H, cycloheptane,
CH2), 1.75-1.81 (m, 2H, cycloheptane, CH2),
2.63-2.75 (m, 4H, cycloheptane, CH2), 7.34-7.84 (m,
12H, Ar-H, 3NH2, NH); ms: (m/z, %): 479 (M + 1,
0.81), 478 (M+, 9.47), 386.58 (100), 341.28 (54.64),
216.28 (16.02), 187.11 (54.61), 156.08 (36.07), 129.25
3.5 | General procedure
A solution of 2-aminothiophene 3 (2.10 g, 10 mM) in 5 mL
AcOH, and 3 mL conc. HCl was stirred and cooled in ice
bath. Then, a solution of sodium nitrite (0.8 g, 1.15
10 mM) in 8 mL water was added dropwise for 10 minutes.
The cold diazonium salt was added slowly to a nicely
stirred cold solution of ethyl 2-cyanoacetate (1.13 g,
10 mM), 2-cyanoacetohydrazide 16 (1.00 g, 10 mM),
(36.56),
68.22
(19.26).
Anal.
Calcd
for
C22H22N8OS2; C, 55.21; H, 4.63; N, 23.41. Found: C,
55.13; H, 4.55; N, 23.53%.

AL-GHORBANI AND GOUDA
7
5-amino-1-H-pyrazol-3-ol 18 (1.00 g10 mM), 3-(3,5-
dimethyl-1-H-pyrazol-1-yl)-3-oxopropanenitrile 21 (1.63 g,
10 mM), malononitrile 9 (0.66 g, 10 mM), or acetylacetone
19 (1.00 g, 10 mM) in EtOH (60 mL) containing AcONa
(4 g, 50 mM). The reaction mixture was stirred for 3 hours.
The formed precipitate was filtered off, dried, and rec-
rystallized from EtOH/benzene to give 14, 15, 17, 20, 23,
and 24.
cooled and the formed precipitate was filtered to offer
compound 15; Yield 79%; mp >320ꢀC.
3.5.3 | (E)-2-([5-amino-3-hydroxy-1H-
pyrazol-4-yl] diazenyl)-5,6,7,8-tetrahydro-
4H-cyclohepta[b]thiophene-
3-carboxamide (17)
Yield 80%; black powder; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3421, 3414, 3357, 3285, 3185 (2NH2, NH,
OH), 2923, 2852 (C-aliph), 2205 (CN), 1635 (CO), 1513
3.5.1 | (E)-Ethyl 2-(2-(3-carbamoyl-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophen-2-yl) hydrazono)-
2-cyanoacetate (14)
(N N). 1H-NMR (DMSO-d6)
δ 1.50-1.62 (m, 4H,
cycloheptane, CH2), 1.81-1.89 (m, 2H, cycloheptane,
CH2), 2.59-2.92 (m, 4H, cycloheptane, CH2), 6.95 (br, 2H,
NH2), 7.43 (br, 2H, CONH2), 10.35 (br, 1H, NH), 11.25
(br, 1H, OH); MS (rel.int.%) (70 ev, %): 322 (M + 2, 6.11),
320 (M+, 15.46), 294.72 (13.45), 227.53 (11.25), 209.40
(17.17), 134.60 (14.12), 109.48 (16.05), 76.13 (100), 59.17
(35.53). Anal. Calcd for C13H16N6O2S; C, 48.74; H,
5.03; N, 26.23. Found: C, 48.53; H, 4.89; N, 26.11%.
Compound 17 may be prepared via heating a suspen-
sion of acetohydrazide 15 (0.32 g, 1 mM) in ethanol
(20 mL) containing pyridine (5 mL) for 12 hours. The
reaction mixture was cooled and the formed precipitate
after neutralization the reaction mixture with dilute
HCl, was filtered to form compound 17. Yield 82%;
mp > 320ꢀC.
Yield 81%; reddish brown powder; mp = 315ꢀC. IR(KBr)
νmax/cm−1 = 3428, 3385, 3185 (NH2, NH), 2924, 2853 (C-
1
aliph), 2191 (CN), 1661 (br, 2CO), 1542 (N N). H-NMR
(DMSO-d6) δ 1.14 (t, 3H, CH3, J = 6.9), 1.50-1.69 (m, 4H,
cycloheptane, CH2), 1.75-1.81 (m, 2H, cycloheptane,
CH2), 2.63-2.75 (m, 4H, cycloheptane, CH2), 4.12 (q, 2H,
CH2, J = 6.9), 7.44 (br, 2H, NH2), 12.52 (br, 1H,
NHhydrazo) ms: (m/z, %): 334(M+, 7.47), 331.37 (33.37),
309.60 (56.89), 268.28 (41.99), 249.99 (26.59), 193.20
(21.13), 152.31 (49.89), 94.56 (30.07), 69.29 (100), 53.39
(52.31). Anal. Calcd for C15H18N4O3S; C, 53.88; H,
5.43; N, 16.75. Found: C, 53.53; H, 5.29; N, 16.68%.
3.5.2 | (E)-2-(2-(3-carbamoyl-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophen-2-yl) hydrazono)-
3.5.4 | (E)-2-(2-(1-cyano-2-[3,5-dimethyl-
1H-pyrazol-1-yl]-2-oxoethylidene)
hydrazinyl)-5, 6, 7, 8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (20)
2-cyanoacetohydrazide (15)
Yield 79%; reddish brown powder; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3385, 3333, 3242, 3185 (2NH2, 2NH), 2923,
2851 (C-aliph), 2221 (CN), 1649 (br, 2CO), 1519 (N N ms:
(m/z, %): 320 (M+, 6.93), 311.27 (34.89), 278.28 (15.73),
268.08 (16.67), 229.37 (16.10), 165.21 (30.68), 120.15 (39.59),
85.17 (63.57), 76.18 (57.55), 52.22 (34.97) .1H-NMR (DMSO-
d6) δ 1.55-1.72 (m, 4H, cycloheptane, CH2), 1.75-1.81 (m,
2H, cycloheptane, CH2), 2.63-2.75 (m, 4H, cycloheptane,
CH2), 3.85 (br, 2H, NH2), 7.29 (br, 2H, CONH2), 9.23 (br,
1H, NHhydrazon) 12.15 (br, 1H, NHCO) ms: (m/z, %):
322 (M + 2, 2.15), 320 (M+, 4.38), 242.30 (100), 226.49
(67.77), 197.96 (46.80), 184.80 (69), 146.40 (86.99), 130.16
(54.77), 104.19 (71.05), 84.27 (68.02), 70.37 (59.72), 52.21
(30.18). Anal. Calcd for C13H16N6O2S; C, 48.74; H, 5.03; N,
26.23. Found: C, 48.54; H, 4.87; N, 26.08%.
Yield 67%; red powder; mp > 320ꢀC. IR (KBr)
νmax/cm−1 = 3431, 3342, 3185 (NH2, NH), 2926, 2855 (C-
1
aliph), 2207 (CN), 1671 (br, 2CO), 1515 (N N). H-NMR
(DMSO-d6) δ 1.50-1.69 (m, 4H, cycloheptane, CH2),
1.75-1.81 (m, 2H, cycloheptane, CH2), 2.63-2.75 (m, 4H,
cycloheptane, CH2), 3.05 (s, 3H, CH3), 3.15 (s, 3H, CH3),
6.66 (s, 1H, CH, pyrazole), 7.44 (br, 2H, NH2), 12.52 (br,
1H, NHhydrazo) ms: (m/z, %): 386 (M + 2, 17.87), 384 (M+,
21.24), 347.34 (49.38), 328.44 (23.19), 250.85 (37.77), 202.23
34.83), 169.33 (35.34), 160.19 (39.82), 115.78 (55.06), 106.13
(87.86), 104.26 (100), 93.09 (67.95), 74.72 (41.90), 66.13
(12.80). Anal. Calcd for C18H20N6O2S; C, 56.23; H, 5.24; N,
21.86. Found: C, 56.17; H, 5.33; N, 21.75%.
Compound 20 can be also, prepared via refluxing a
mixture of acetohydrazide 15 (0.32 g, 1 mM) and
acetylacetone 19 (0.1 g, 1 mM) in ethanol (20 mL) con-
taining dimethylformamide (5 mL) was refluxed for
10 hours. The reaction mixture was cooled and the
obtained precipitate after pouring the reaction mixture in
Also, compound 15 can be prepared via refluxing a sus-
pension of (E)-ethyl 2-(2-(3-carbamoyl-5,6,7,8-tetrahydro-
4H-cyclohepta[b]thiophen-2-yl)hydrazono)-2-cyanoacetate
14 (0.344 g, 1 mM) and hydrazine hydrate (0.26 g, 4 mM)
in ethanol (40 mL) for 8 hours. The response mixture was

8
AL-GHORBANI AND GOUDA
ice cooled water was filtered to achieve compound 20.
4H-cyclohepta[b]thioph-ene-3-carboxamide 24 (0.321 g,
1 mM) and hydrazine hydrate (0. 26 g, 4 mM) in
EtOH (45 mL) was refluxed for 13 hours. The reac-
tion mixture was cooled and the formed precipitate
was filtered to afford compounds 26 and 27.
Yield 67%; mp > 320ꢀC.
3.5.5 | 2-(2-hydrazonoemalonyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (23)
3.6.2 | Compound 26
Yield 70%; reddish brown powder; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3434, 3342, 3192 (NH2, NH), 2927, 2855 (C-
Yield 78%; black powder; mp > 320ꢀC. IR(KBr)
νmax/cm−1 = 3424, 3357, 3192 (3NH2, NH), 2922, 2852
(C-aliph), 1641 (CO), 1528 (N N). 1H-NMR (DMSO-d6)
δ 1.50-1.73 (m, 4H, cycloheptane, CH2), 1.75-1.81 (m,
2H, cycloheptane, CH2), 2.63-2.80 (m, 4H,
cycloheptane, CH2), 5.75 (br, 4H, 2NH2), 7.17 (br, 2H,
CONH2), 12.54 (br, 1H, NHhydrazo); ms: (m/z, %):
321 (M + 2, 26.63), 319 (M+, 5.54), 245.19 (24.15),
236.33 (21.83), 220.41 (26.64), 190.23 (18.70), 164.99
(19.67), 126.28 (22.59), 105.20 (4.26), 96.23 (46.19),
77.19 (100), 64.10 (55.78), 51.15 (27.70); Anal. for
C13H17N7OS; C, 48.89; H, 5.36; N, 30.70. Found: C,
48.77; H, 5.23; N, 30.55%.
1
aliph), 2201 (br, 2CN), 1659 (CO), 1588 (N N). H-NMR
(DMSO-d6) δ 1.50-1.69 (m, 4H, cycloheptane, CH2),
1.75-1.91 (m, 2H, cycloheptane, CH2), 2.60-2.90 (m, 4H,
cycloheptane, CH2), 7.48 (br, 2H, CONH2,), 13.23 (br, 2H,
NH N) ms: (m/z, %): 287 (M+, 26.63), 266.05 (47.26),
225.61 (39.26), 170.24 (95.05), 139.64 (44.85), 133.61 (100),
113.82 (50.15), 105.13 (92.61), 101.63 (73.71), 95.42 (32.62),
65.02 (40.54). Anal. Calcd for C13H13N5OS; C, 54.34; H,
4.56; N, 24.37. Found: C, 54.12; H, 4.37; N, 24.39%.
3.5.6 | 2-(2-[2, 4-Dioxopentan-3-ylidene]
hydrazinyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carboxamide (24)
3.6.3 | Compound 27
Yield 73%; pale yellow powder; mp > 320ꢀC. IR (KBr)
νmax/cm−1 = 3373, 3342, 3190 (2NH2, NH), 2923, 2852 (C-
aliph), 2208 (CN), 1670 (br, 2CO), 1638 (CONH2), 1551
Yield 75%; black powder; m.p 320ꢀC. IR (KBr)
νmax/cm−1 = 3402, 3314, 3214, 3192 (NH2, NH), 2922,
2851 (C-aliph), 1655 (CO), 1585 (N N). 1H-NMR
(DMSO-d6) δ 1.50-1.74 (m, 4H, cycloheptane, CH2),
1.81-1.89 (m, 2H, cycloheptane, CH2), 2.59-2.92 (m, 4H,
cycloheptane, CH2), 3.02 (s, 3H, CH3), 3.05 (s, 3H,
CH3), 7.42 (br, 2H, CONH2,), 13.23 (br, 1H, NH N);
ms: (m/z, %): 333 (M+, 14.19), 304.23 (64.13), 250.35
(74.27), 217.07 (30.78), 204.47 (67.68), 150.27 (67.25),
122.04 (100), 113.95 (26.90), 78.36 (35.61), 67.55
(60.34), 58.42 (31.30); Anal. for C15H19N5OS; C,
56.76; H, 6.03; N, 22.06. Found: C, 56.34; H,
6.12; N, 22.02%.
(N N). 1H-NMR (DMSO-d6)
δ
1.50-1.62 (m, 4H,
cycloheptane, CH2), 1.81-1.89 (m, 2H, cycloheptane, CH2),
2.34 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.59-2.81 (m, 4H,
cycloheptane, CH2), 7.60 (br, 2H, CONH2), 15.02 (br, 1H,
NHhydrazo) ms: (m/z, %): 321.00 (M+, 2.8), 245.19 (24.15),
220.41 (26.64), 126.28 (22.59), 115.48 (24.45), 105.75 (39.89),
96.23 (46.19), 88.31 (32.17), 77.19 (100), 74.28 (48.80), 69.19
(33.55), 63.17 (44.55). Anal. Calcd for C15H19N3O3S; C,
56.06; H, 5.96; N, 13.07. Found: C, 56.19; H, 6.03; N, 13.34%.
3.6 | Synthesis of 2-(2-[3,5-diamino-4H-
pyrazol-4-ylidene]hydrazinyl)-
5,6,7,8-tetrahydro-4H-cyclohepta[b]
thiophene-3-carboxamide (26) and
2-(2-[3,5-dimethyl-4H-pyrazol-4-ylidene]
hydrazinyl)-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thioph-ene-
3.7 | Biological activity
The reagents RPMI-1640 medium (Sigma Co.,
St. Louis) Fetal Bovine serum (GIBCO, UK) and the
human cell lines HepG2, HCT-116, MCF-7, and PC3
were obtained from ATCC. The cell lines cited above
have been used to decide the inhibitory effects of com-
pounds on cell growth using the MTT assay. Extra
information about the MTT assay, cytotoxicity activity
and IC50 for each compound was presented in litera-
ture [39].
3-carboxamide (27)
3.6.1 | General procedure
A mixture of dicyanide 23 (0.287 g, 1 mM) or 2-(2-
[2, 4-dioxopentan-3-ylidene]hydrazinyl)-5,6,7,8-tetrahydro-

AL-GHORBANI AND GOUDA
9
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Mohammed Al-Ghorbani https://orcid.org/0000-0002-
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How to cite this article: Al-Ghorbani M,
Gouda MA. Synthesis and in vitro anticancer
activity of some novel cyclohepta[b]thiophene-3-
carboxamides bearing pyrazole moiety.
J Heterocyclic Chem. 2020;1–9. https://doi.org/10.
1002/jhet.4041
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S. M. El-Ashry, J. Am. Sci. 2016, 12, 55.