An efficient synthesis of new substituted spiro pyrazolethieno, pyrimidinethieno, and benzodiazepinethieno pyridazine derivatives with biological activities

Article abstract of DOI:10.1080/10426500903095531

3-Ethyl 2-amino-4-methyl-5-phenyl thiophene carboxylate 1 was used as a starting material to synthesize 2a,b via coupling with malononitrile or acetyl acetone, respectively. When heated, under reflux in sodium ethoxide solution, 2a,b give 3a,b. On the other hand, when compounds 3a,b were heated under reflux in ethanol with hydrazine hydrate, thiourea, or 1,1-phenylenediamine hydrochloride and a catalytic amount of piperidine 4a,b, 5a,b and 6a,b, were produced, respectively. The new compounds were tested for their antimicrobial activity. Compounds 2a-6b showed antibacterial activities, and 2a,2b and 4b showed antifungal activities. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file. Copyright Taylor & Francis Group, LLC.

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An Efficient Synthesis of New
Substituted Spiro Pyrazolethieno,
Pyrimidinethieno, and
Benzodiazepinethieno Pyridazine
Derivatives with Biological Activities
Rasha A. M. Faty a , Hoda A. R. Hussein b & Ayman M. S. Youssef c
a Cairo University, Faculty of Science, Giza, Egypt
b Department of Photochemistry (Heterocyclic Unit), National
Research Centre, Dokki, Egypt
c Fayoum University, Faculty of Science, Fayoum, Egypt
Published online: 03 Jul 2010.
To cite this article: Rasha A. M. Faty , Hoda A. R. Hussein & Ayman M. S. Youssef (2010): An Efficient
Synthesis of New Substituted Spiro Pyrazolethieno, Pyrimidinethieno, and Benzodiazepinethieno
Pyridazine Derivatives with Biological Activities, Phosphorus, Sulfur, and Silicon and the Related
Elements, 185:7, 1484-1490
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Phosphorus, Sulfur, and Silicon, 185:1484–1490, 2010
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500903095531
AN EFFICIENT SYNTHESIS OF NEW SUBSTITUTED
SPIRO PYRAZOLETHIENO, PYRIMIDINETHIENO, AND
BENZODIAZEPINETHIENO PYRIDAZINE DERIVATIVES
WITH BIOLOGICAL ACTIVITIES
Rasha A. M. Faty,1 Hoda A. R. Hussein,2
and Ayman M. S. Youssef3
1Cairo University, Faculty of Science, Giza, Egypt
2Department of Photochemistry (Heterocyclic Unit), National Research Centre,
Dokki, Egypt
3Fayoum University, Faculty of Science, Fayoum, Egypt
3-Ethyl 2-amino-4-methyl-5-phenyl thiophene carboxylate 1 was used as a starting material
to synthesize 2a,b via coupling with malononitrile or acetyl acetone, respectively. When
heated, under reflux in sodium ethoxide solution, 2a,b give 3a,b. On the other hand, when
compounds 3a,b were heated under reflux in ethanol with hydrazine hydrate, thiourea, or 1,1-
phenylenediamine hydrochloride and a catalytic amount of piperidine 4a,b, 5a,b and 6a,b,
were produced, respectively. The new compounds were tested for their antimicrobial activity.
Compounds 2a–6b showed antibacterial activities, and 2a,2b and 4b showed antifungal
activities.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental
file.
Keywords Antibacterial; antifungal; benzodiazepinethieno-pyridazine; biological activity;
pyrimidinethieno; spiro pyrazolethieno
INTRODUCTION
From a chemical structure point of view, various natural products or synthetic
molecules containing the rigid conformations of spiro cyclic skeletons show a wide range of
biological and pharmacological properties.1–3 For example, spirohydantoins have been re-
ported as glycogen and phosphorylase inhibitors, herbicides, and anti-inflammatory agents,4
and spiro-isoxazolines and spiro-dioxazoles exhibit antitumor and anti-HIV activities.5–8 In
spite of the immense biological activities of many spiro heterocyclic compounds, no report
is yet available on the synthesis of pyrazolothienopyridazine spiro derivatives. Prompted
by this observation and in continuation of our earlier interest on chemical synthesis of bio-
dynamic heterocyclic compounds,9 we have made an attempt to synthesize novel tricyclic
spiro derivatives.
Received 7 January 2009; accepted 3 June 2009.
Address correspondence to Rasha A. M. Faty, Cairo University, Faculty of Science, Giza, Egypt. E-mail:
rashafaty@yahoo.com
1484

NEW SUBSTITUTED SPIRO DERIVATIVES
1485
RESULTS AND DISCUSSION
Chemistry
For synthesis of novel spiro derivatives, at first, we attempted to obtain dinitrile or
diacetyl derivatives by oxidative cyclization of diazo thieno derivatives. Thus, by using
3-ethyl 2-amino-4-methyl-5-phenylthiophene carboxylate10 1 as a starting material, it was
coupled with some active methylene groups namely, malononitrile or acetylacetone, re-
spectively, to obtain 3-Ethoxycarbonyl-4-methyl-5-phenyl(thien-2-ylazo)malononitrile 2a
or 3-ethoxycarbonyl-4-methyl-5-phenyl(thien-2-ylazo)acetylacetone 2b. Elemental analy-
ses as well as spectral data of compounds 2a,b are in agreement with the proposed structures
(see the Experimental section).
Heating 2a,b under reflux with ethanolic sodium ethoxide solution for 3 h yielded
3,3-disubstituted thienopyridazine derivatives 3a,b. Compounds 3a,b give expected values
in elemental analyses and spectral data. The IR spectrum (KBr) cm−1 of 3b, for example,
showed absorption bands at: 1719 (2CO), 1673 (CO); 1H NMR (DMSO-d6) δ ppm showed
signals at: 2.31 (s, 3H, CH3), 3.80 (s, 6H, 2CH3), 7.43–7.46 (m, 5H, aromatic protons); MS
(m/z): 326 (M+) 80%.
Compounds 3a,b underwent oxidative cyclization upon heating under reflux in
ethanol with hydrazine hydrate in the presence of a catalytic amount of piperidine to
produce 3,5-diamino(diacetyl)-5ꢀ-methyl-6ꢀ-phenyl-4ꢀH-spiro-(pyrazole-4,3ꢀ-thieno[2,3-
c]pyridazine)-4ꢀ-one 4a,b with new ring systems (Scheme 1). Compounds 4a,b gave an
expected elemental analyses (see the Experimental section). The IR spectrum (KBr) cm−1
1
of 4a, for example, showed: 3313, 3180(NH2), and 1660(CO); H NMR (DMSO-d6)
δ ppm: 2.22 (s, 3H, CH3), 3.81 (br., 2H, NH2, D2O exchangeable), 7.40–7.57 (m, 5H,
aromatic protons), and 9.44 (br., 2H, NH2, D2O exchangeable); 13C NMR (75 MHz,
CDCl3) of 4a for example showed signals at δ189 (S) ppm, 160 (S, 2C), 145 (S), 132 (S),
130 (S, 3C), 129 (d, 2C), 127 (d, 2C), 126 (d), 79 (S), 5 (q).
On the other hand, compounds 3a,b also reacted with thiourea in boiling ethanol
containing a catalytic amount of piperidine to produce other novel spiro- compounds
4,6-diamino(diacetyl)-5ꢀ-methyl-6ꢀ-phenyl-2-thioxo-2H,4ꢀH-spiro-[pyrimidine-5,3ꢀ-
thieno[2,3-c]pyridazin]-4ꢀ-one 5a,b, which showed expected spectral data and elemental
analyses (see the Experimental section and Scheme 1).
Also, heating 3a,b under reflux with o-phenylenediamine in boiling ethanol
containing a catalytic amount of piperidine produced other novel spiro compounds
2,4-diamino(diacetyl)-5ꢀ-methyl-6ꢀ-phenyl-4ꢀH-spiro[1,5-benzodiazepine-3,3ꢀ-thieno[2,3-
1
c]pyridazin]-4ꢀ-one 6a,b. Besides the expected values of elemental analyses, the IR, H
NMR, and 13C NMR spectra of 6a,b are in agreement with the assigned structure (see the
Experimental section and Scheme 1).
Biology
All new compounds were evaluated for their antimicrobial properties. Compound
2a showed inhibition zones and therefore antibacterial activity against staphylococcus
aureus (G+) more than the reference compound tetracycline antibacterial agent. (See the
Supplemental Materials available online, Table S1.)
CONCLUSION
This work is concerned with the synthesis and reactions of 3-ethyl-2-amino-4-methyl-
5-phenyl thiophene carboxylate with functional and bifunctional groups leading to spiro

1486
R. A. M. FATY ET AL.
H C
3
COOC H
2 5
X
H C
+
2
X
Ph
S
NH
2
a , X = CN
b , X = COCH
1
3
H C
3
COOC H
2 5
H C
3
COOC H
2 5
X
X
X
X
CH
N
Ph
S
N
Ph
S
N
H
N
C
2a,b
R
O
N
O
N
R
S
O
R
N
H C
3
H C
3
H C
3
X
X
N
N
N
N
N
R
Ph
S
Ph
S
N
Ph
S
N
3a,b
5a,b
4a,b
R
N
O
H C
3
N
N
R
Ph
S
N
6a,b
a , R = NH
2
b , R =CH
3
Scheme 1
pyrazolethieno, pyrimidinethieno and benzodiazepinethieno pyridazin derivatives. Nine of
the synthesized compounds were found to be highly active against Gram positive bacteria
(Staphylococcus aureus G+), Gram negative bacteria (Pseudomon asaeruginosa G−), and
fungi (Aspergillus flavus and Candida albicans).
EXPERIMENTAL
All melting points are uncorrected and were taken in open capillaries on a Gallen
Kamp Apparatus. Microanalyses were carried out at the Microanalytical Unit, National
Research Center and Faculty of Science, Cairo University. IR spectra were recorded on a
FT/IR-300 E Jasco using KBr discs. 1H NMR spectra were measured in DMSO or CDCl3,
using a JEOL-JNM-Ex270 NMR spectrometer. Signals were measured with reference to
TMS as an internal standard. The mass spectra were recorded on Finnigan SSQ 7000
spectrometer. Microbiological analyses were carried out by the Microanalytical Center,

NEW SUBSTITUTED SPIRO DERIVATIVES
1487
Table I Physical data for the products 2a–6b
Elemental analyses
(calcd./found)
Compound No.
Mp ◦C
Yield %
80
Molecular formala (M. wt.)
%C
%H
%N
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
151–153
170–172
212–214
180–182
190–192
200–202
195–197
205–207
185–187
202–204
C17H14N4O2S
338.39
C19H20N2O4S
372.44
60.34
60.30
61.27
61.22
61.63
61.60
62.56
62.54
55.54
55.50
63.33
63.30
52.15
52.10
58.99
58.97
62.98
62.97
69.32
69.32
4.17
4.00
5.41
5.40
2.75
2.74
4.32
4.30
3.72
3.70
4.37
4.35
3.28
3.25
3.85
3.85
4.02
4.00
4.55
4.55
16.55
16.50
7.52
75
7.50
80
C
15H8N4OS
292.32
19.16
19.12
8.58
75
C
17H14N2O3S
326.37
8.55
73
C
15H12N6OS
324.36
25.90
25.90
17.37
17.35
22.80
22.80
15.28
15.26
20.98
20.97
14.06
14.00
70
C
17H14N4OS
322.39
75
C
C
16H12N6OS2
368.44
18H14N4OS2
366.47
65
75
C
21H16N6OS
400.46
60
C
23H18N4OS
398.49
Faculty of Science, Cairo University, Giza, Egypt. All solid compounds were recrystallized
to produce constant melting points. See Table I.
3-Ethyl-2-amino-4-methyl-5-phenyl thiophene carboxylate (1)
Compound 1 was prepared according to the Gewald method.10
General Method for Preparation of 2a,b
To an ice-cold solution of the appropriate amine 1 (0.01 mol), glacial acetic acid (30
mL), and phosphoric acid (10 mL), a solution of sodium nitrite (1.03 g, 0.01 mol) dissolved
in the minimum amount of water was added dropwise, in an ice bath at −5◦C. This
previously prepared diazonium salt was added dropwise to a mixture of active methylene
malononitrile or acetyl acetone (0.01 mol) and anhydrous sodium acetate in ethanol. The
reaction mixture was allowed to stand overnight at room temperature, then it was poured
into water. The formed solid was filtered off, washed with water, dried, and recrystallized
from the appropriate solvent to produce 2a,b.
3-Ethoxycarbonyl-4-methyl-5-phenyl(thien-2-ylazo)malononitrile (2a).
Compound 2a was obtained by the reaction mixture of 1 (2.60 g, 0.01 mol) and malononi-
trile (0.66 g, 0.01 mol). The compound was recrystallized from ethanol to produce 2a as
deep orange crystals; IR spectrum (KBr) cm−1: 3318 (br, NH), 2232, 2211 (2CN), and
1
1668 (CO); H NMR (DMSO-d6) δ ppm (J, Hz): 1.29 (t, 3H, CH3, J 4.2), 2.24(s, 3H,

1488
R. A. M. FATY ET AL.
CH3), 3.12 (s, 1H, NH, D2O exchangeable), 4.26 (q, 2H, CH2, J 4.2) and 7.34–7.47 (m,
5H, aromatic protons); MS (m/z): 338 (M+) 80%.
3-Ethoxycarbonyl-4-methyl-5-phenyl(thien-2-ylazo)acetylacetone (2b).
Compound 2b was obtained by the reaction mixture of 1 (2.60 g, 0.01 mol) and acetyl
acetone (1.00 g, 0.01 mol). The compound was recrystallized from ethanol to produce 2b
as orange crystals; IR spectrum (KBr) cm−1: 3324(br, NH), 1671, 1645 (3CO); 1H NMR
(DMSO-d6) δ ppm (J, Hz): 1.31 (t, 3H,CH3, J
4.7), 2.21 (s, 3H, CH3), 4.00 (s, 6H,
2CH3), 4.10 (s, 1H,NH, D2O exchangeable), 4.40 (q, 2H, CH2, J 4.7) and 7.34–7.45 (m,
5H, aromatic protons); MS(m/z): 372(M+) 100%.
3,3-Dicyano(diacetyl)-5-methyl-6-phenylthieno[2,3-c]pyridazine-4-one
(3a,b)
Compound 2a,b (0.01 mol) was refluxed in sodium ethoxide solution [(prepared by
dissolving sodium metal (0.23 g, 0.01 mol) in absolute ethanol (30 mL)] was heated under
reflux for 5 h. The reaction mixture was allowed to cool to room temperature, poured into
water, and neutralized by dilute acetic acid solution. The solid product that precipitated was
filtered off, dried, and recrystallized from the proper solvent to produce 3a,b.
3,3-Dicyano -5-methyl-6-phenylthieno[2,3-c]pyridazine-4-one (3a). Com-
pound 3a was obtained by refluxing 2a (3.38 g, 0.01 mol) in sodium ethoxide solution.
The solid product was recrystallized from dioxane to produce 3a as deep orange crystals;
IR spectrum (KBr) cm−1: 2219 (2CN) and 1708 (CO); 1H NMR (DMSO-d6) δ ppm: 2.33
(s, 3H, CH3), 7.34–7.47 (m, 5H, aromatic protons); 13C NMR (75 MHz, CDCl3): δ 190 (s)
ppm, 148 (s), 135 (s), 134 (s, 3C), 129 (d, 3C), 128 (d, 2C), 117(s, 2C), 58 (s), 5 (q); MS
(m/z): 292(M+) 70%.
3,3-Diacetyl-5-methyl-6-phenylthieno[2,3-c]pyridazin-4-one (3b). Com-
pound 3b was obtained by refluxing 2b (3.72 g, 0.01 mol) in sodium ethoxide solution.
The solid product was recrystallized from dioxane to produce 3b as orange crystals; IR
spectrum (KBr) cm−1: 1719 (2CO), 1673 (CO); 1H NMR (DMSO-d6) δ ppm: 2.31(s, 3H,
CH3), 3.80 (s, 6H, 2CH3), 7.43–7.46 (m, 5H, aromatic protons); 13C NMR (75 MHz,
CDCl3): δ 204 (s, 2C) ppm, 192 (s), 147 (s), 135 (s), 133 (s, 3C), 129 (d, 3C), 128 (d, 2C),
103 (s), 24 (q, 2C), 5 (q); MS(m/z): 326 (M+) 80%.
3,5-Diamino(diacetyl)-5ꢁ-methyl-6ꢁ-phenyl-4ꢁH-spiro[pyrazole-4,3ꢁ-
thieno[2,3-c]pyridazin]-4ꢁ-one (4a,b)
A mixture of 3a,b (0.01 mol) and hydrazine hydrate (99–100%) (7 mL, 0.03 mol)
was refluxed in ethanol (30mol) containing a catalytic amount of piperidine for 5 h. The
reaction mixture was cooled, filtered off, and recrystallized from the proper solvent to
produce 4a,b.
3,5-Diamino-5ꢁ-methyl-6ꢁ-phenyl-4ꢁH-spiro[pyrazole-4,3ꢁ-thieno[2,3-c]-
pyridazin]-4ꢁ-one (4a). Compound 4a was obtained by refluxing 3a (2.92 g, 0.01 mol)
and hydrazine hydrate in ethanol containing a catalytic amount of piperidine. The solid
product recrystallized from dioxane to produce 4a as deep red crystals; IR spectrum (KBr)
cm−1: 3313, 3180 (NH2) and 1660 (CO); 1H NMR (DMSO-d6) δ ppm: 2.22 (s, 3H, CH3),
3.81 (br.,2H, NH2, D2O exchangeable), 7.40–7.57 (m, 5H, aromatic protons) and 9.44
(br.,2H, NH2, D2O exchangeable); MS (m/z): 324 (M+) 14.80%.

NEW SUBSTITUTED SPIRO DERIVATIVES
1489
3,5-Diacetyl-5ꢁ-methyl-6ꢁ-phenyl-4ꢁH-spiro[pyrazole-4,3ꢁ-thieno[2,3-
c]pyridazin]-4ꢁ-one (4b). Compound 4b was obtained by refluxing 3b (3.26 g, 0.01
mol) and hydrazine hydrate in ethanol containing a catalytic amount of piperidine. The
solid product recrystallized from dioxane to produce 4b as deep red crystals; IR spectrum
1
(KBr) cm−1: 1670 (CO); H NMR (DMSO-d6) δ ppm: 2.23 (s, 6H, 2CH3), 3.72 (s, 3H,
CH3) and 7.32–7.44 (m, 5H, aromatic protons); 13C NMR (75 MHz, CDCl3): δ190 (s)
ppm, 165 (s, 2C), 147 (s), 136 (s), 134 (s, 3C), 129 (d, 3C), 128(d, 2C), 80 (s), 19 (q, 2C),
5 (s); MS (m/z): 322 (M+) 65%.
4,6-Diamino(diacetyl)-5ꢁ-methyl-6ꢁ-phenyl-2-thioxo-2H-4ꢁH-spiro-
[pyrimidine-5,3ꢁ-thieno[2,3-c]pyridazin]-4ꢁ-one (5a,b)
A mixture of 3a,b (0.01 mol) and thiourea (0.01, mol) was refluxed in ethanol
containing a catalytic amount of piperidine for 5 h. The reaction mixture was cooled
and poured into water. The solid product that precipitated was filtered off, dried, and
recrystallized from the proper solvent to produce 5a,b.
4,6-Diamino-5ꢁ-methyl-6ꢁ-phenyl-2-thioxo-2H-4ꢁH-spiro[pyrimidine-5,3ꢁ-
thieno[2,3-c]pyridazin]-4ꢁ-one (5a). Compound 5a was obtained by refluxing of 3a
(2.92 g, 0.01 mol) and thiourea (0.76 g, 0.015 mol). The solid product recrystallized from
dioxane to produce 5a as yellow crystals; IR spectrum (KBr) cm−1: 3310, 3150 (NH2)
1
and 1654 (CO); H NMR (DMSO-d6) δ ppm: 2.33 (s, 3H, CH3), 3.88 (br., 2H, NH2,
D2O exchangeable), 7.22–7.47 (m, 5H, aromatic protons) and 9.44 (br., 2H, NH2, D2O
exchangeable); 13C NMR (75 MHz, CDCl3): δ229(s) ppm, 191 (s), 165 (s, 2C), 147 (s),
135 (s), 134 (s, 3C), 129 (d, 3C), 128 (d, 2C), 72 (s), 5 (q); MS (m/z): 368(M+) 96%.
4,6-Diacetyl-5ꢁ-methyl-6ꢁ-phenyl-2-thioxo-2H-4ꢁH-spiro[pyrimidine-5,3ꢁ-
thieno[2,3-c]pyridazin]-4ꢁ-one (5b). Compound 5b was obtained by refluxing 3b
(3.26g, 0.01 mol) and thiourea (0.76 g, 0.015 mol). The solid product recrystallized from
dioxane to produce 5b as orange crystals; IR spectrum (KBr) cm−1: 1673 (CO); 1H NMR
(DMSO-d6) δ ppm: 2.21 (s, 3H, CH3), 3.20 (s, 6H, 2CH3) and 7.22–7.48 (m, 5H, aromatic
protons); 13C NMR (75 MHz, CDCl3): δ 229(s) ppm, 192 (s), 165 (s, 2C), 147 (s), 135 (s),
134 (s, 3C), 129 (d, 3C), 128 (d, 2C), 72 (s), 15 (q, 2c), 5 (q); MS (m/z): 366(M+) 70%.
2,4-Diamino(diacetyl)-5ꢁ-methyl-6ꢁ-phenyl-4ꢁH-spiro[1,5-
benzodiazepine-3,3ꢁ-thieno[2,3-c]pyridazin]-4ꢁ-one (6a,b)
A mixture of 6a,b (0.01 mol) and o-phenylenediamine hydrochloride (0.01 mol) was
refluxed in boiling ethanol containing a catalytic amount of piperidine for 5 h. The reaction
mixture was cooled and poured into water. The formed solid was filtered off, dried, and
recrystallized from the appropriate solvent to produce 6a,b.
2,4-Diamino-5ꢁ-methyl-6ꢁ-phenyl-4ꢁH-spiro(1,5-benzodiazepine-3,3ꢁ-thieno
[2,3-c]pyridazin)-4ꢁ-one (6a). Compound 6a was obtained by refluxing 3a (2.92 g,
0.01 mol) and o-phenylenediamine hydrochloride. The solid product recrystallized from
dilute dimethyl formamide (80%) to produce 6a as yellow crystals; IR spectrum (KBr)
cm−1: 3430–3360 (br, 2NH2), 1680 (CO); 1H NMR (DMSO-d6) δ ppm: 2.13 (s, 3H, CH3),
2.74 (br, 4H, 2NH2, D2O exchangeable), 7.01–7.41 (m, 9H, aromatic protons). 13C NMR
(75 MHz, CDCl3): δ190 (s) ppm, 160 (s, 2C), 145 (s), 140 (s, 2C), 136 (s), 134 (s,2C),

1490
R. A. M. FATY ET AL.
132 (s), 130 (d, 2C), 129 (d), 128 (d, 2C), 127 (d, 2C), 122 (d, 2C), 75 (s), 50 (q). MS
(m/z): 400 (M+) 70%.
2,4-Diacetyl-5ꢁ-methyl-6ꢁ-phenyl-4ꢁH-spiro(1,5-benzodiazepine-3,3ꢁ-thieno
[2,3-c]pyridazin)-4ꢁ-one (6b). Compound 6b was obtained by refluxing 3b (3.26g,
0.01 mol) and o-phenylenediamine. The solid product recrystallized from dilute dimethyl
1
formamide (80%) to produce 6b; 1710 (2CO), 1670 (CO); H NMR (DMSO-d6) δ ppm:
2.13 (s, 3H, CH3), 2.24 (s, 6H, 2CH3), 7.01–7.41 (m, 9H, aromatic protons); MS (m/z):
398 (M-86) 20%.
Biology
Measurement of Antimicrobial Activity Using Diffusion Disc Method.
The prepared compounds were tested against one strain of Gram positive bacteria (Staphy-
lococcus aureus G+), Gram-negative bacteria (Esherichia coli G−), and fungi (Aspergillus
flavus and Candida albicans).11,12
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