Enaminonitriles in heterocyclic synthesis: Synthesis and biological evaluation of novel indeno[2,1-b]thiophene derivatives

Article abstract of DOI:10.3906/kim-1204-53

A series of 1,3-indanedione derivatives were synthesized and evaluated as antimicrobial agents using 2- amino-8-oxo-8H-indeno[2,1-b]thiophene-3- carbonitrile (2) as a starting material. Compound 2 reacted with various types of reagents under different conditions to yield thienopyrazole 3, thienopyrimidines (6, 8, 9), thienopyridines (10, 11), and thiazolylidines (15, 17, 19, 21, 23). These compounds were screened for their antibacterial activity against gram-positive bacteria (Bacillus subtilis and Bacillus thuringiensis) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and antifungal activity against Fusarium oxysporum and Botrytis fabae strains. 2-((3,4-Diphenylthiazol-2(3H)-ylidene)amino)-8-oxo-8H-indeno[2,1-b] thiophene-3-carbonitrile (15) and 8-oxo-2-((4-oxo- 3-phenylthiazolidin-2- ylidene)amino)-8H-indeno[2,1-b] thiophene-3-carbonitrile (17) were found to exhibit equal activity compared with chloramphenicol against B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than that of chloramphenicol against B. thuringiensis. TUeBITAK.

Full text of DOI:10.3906/kim-1204-53

Turk J Chem
Turkish Journal of Chemistry
(2013) 37: 134 – 148
¨
http://journals.tubitak.gov.tr/chem/
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1204-53
Research Article
Enaminonitriles in heterocyclic synthesis: synthesis and biological evaluation of
novel indeno[2,1-b]thiophene derivatives
Ahmed A. FADDA∗, Abd El-Galil M. KHALIL and Eman H. TAWFIK
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, 35516, Egypt
Received: 19.04.2012
•
Accepted: 04.12.2012
•
Published Online: 24.01.2013
•
Printed: 25.02.2013
Abstract: A series of 1,3-indanedione derivatives were synthesized and evaluated as antimicrobial agents using 2-
amino-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (2) as a starting material. Compound 2 reacted with various
types of reagents under different conditions to yield thienopyrazole 3, thienopyrimidines (6, 8, 9), thienopyridines
(10, 11), and thiazolylidines (15, 17, 19, 21, 23). These compounds were screened for their antibacterial activity
against gram-positive bacteria (Bacillus subtilis and Bacillus thuringiensis) and gram-negative bacteria (Escherichia
coli and Pseudomonas aeruginosa) and antifungal activity against Fusarium oxysporum and Botrytis fabae strains.
2-((3,4-Diphenylthiazol-2(3H)-ylidene)amino)-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (15) and 8-oxo-2-((4-oxo-
3-phenylthiazolidin-2-ylidene)amino)-8H-indeno[2,1-b] thiophene-3-carbonitrile (17) were found to exhibit equal activity
compared with chloramphenicol against B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than that of
chloramphenicol against B. thuringiensis.
Key words: Indeno[2,1-b]thiophenes, thienopyrazole, thienopyrimidines, thienopyridines, thiazolylidines, antimicrobial
activity, MIC
1. Introduction
Indeno-fused heterocycles have attracted considerable attention due to their medicinal and synthetic importance
in recent years and broad range of biological activity.1−3 Thus, indeno[1,2-c]pyrazole-4-ones have recently been
identified as potent and selective cyclin dependent kinase (CDK),4 and check point kinase (CHK1) inhibitors,5,6
showing excellent activity against various tumor cell linings and in vivo activity in human xenograph models. A
few of these compounds were reported to exhibit antidepressive activity.7 Similarly, a series of indeno derivatives
were reported to display marked cytotoxic activity8 and were potent DNA topoisomerase inhibitors; in fact,
indenoquinoline has undergone advanced clinical trials.8a A series of indeno-fused pyrimidines, pyridazines, and
1,2,4-triazines were reported to exhibit inhibitory potency towards monoamine oxidase (MAO),9 whereas an
indeno-fused pyridone has been found to exhibit cardiotonic activity (PDE 3 inhibitor).10a Some indeno-fused
pyrimidines were identified as a novel class of potent A2 A receptor antagonists.10b Similarly, indeno-fused
thiophene derivatives have been extensively explored in the field of organometallic chemistry because their
transition metal complexes have been found to be promising catalysts for olefin polymerization.11 However,
despite their biological activities and other applications, syntheses of these indeno-fused heterocycles have not
received much attention, involving diverse methods with many limitations and lacking generality.
∗Correspondence: afadda50@yahoo.com
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FADDA et al./Turk J Chem
We therefore extended our efforts directed towards the development of convenient synthetic approaches
for the synthesis of thiophenes,12−16 with an expected broad spectrum of biological activity. We wished to
broaden the scope of the Gewald reaction utilizing 1,3-indandione (1) as a key precursor for the synthesis of
some hitherto unreported polyfunctionally substituted 2-amino-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile
(2). The latter, in turn, could be successfully annulated to polyfunctionally substituted thieno[2,3-d]pyrimidine,
thieno[2,3-c]pyrazole, thieno[2,3-b]pyridine, and thiazolidine amino indeno[2,1-b]thiophene derivatives. The
importance of these compounds is due to their potential biological and physiological activities.17−19
2. Results and discussion
2.1. Chemistry
Gewald reaction of 1,3-indanedione (1) with elemental sulfur and malononitrile in absolute ethanol containing a
catalytic amount of morpholine furnished upon heating exclusively and in reasonable good yield a product that
could be formulated as 2-amino-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (2) (Scheme 1). The elemental
analysis and spectroscopic data of 2 are consistent with the assigned structure. The 1 H NMR spectrum revealed
the presence of a D2 O exchangeable broad singlet at δ 5.65 ppm (2H) attributable to the NH2 protons and
multiplet signals at δ 7.12–7.65 ppm (4H) corresponding to aromatic protons. The synthetic potency of β-
enaminonitriles as versatile reagents in heterocyclic synthesis is well documented.20,21 On the other hand,
β-enaminonitrile moiety in 2 proved to be highly reactive towards nitrogen nucleophiles. Thus, compound 2
reacted with hydroxylamine hydrochloride in refluxing glacial acetic acid containing anhydrous sodium acetate
to afford 3-aminoindeno[1ꢀ ,2ꢀ :4,5]thieno[2,3-c]pyrazol-8-(1H)-one (3). The 1 H NMR spectrum of 3 revealed
singlet signals of 2 types of exchangeable protons at δ 5.0 (2H) and 8.53 ppm (1H) attributable to NH2 and
NH protons, respectively.
Acylation of compound 2 using acetic anhydride under reflux conditions gave N-(3-cyano-1-oxo-1H-inden-
Scheme 1. Synthesis and reactions of 2-amino-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (2).
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FADDA et al./Turk J Chem
2-yl)acetamide (4). Structure 4 was established based on both elemental analysis and spectral data The 1 H
NMR showed singlet signals at δ 2.88 ppm (3H) for acetyl protons, at 5.12 ppm (1H) corresponding to NH
proton, and a multiplet signal at δ 7.12-8.22 ppm (4H) for aromatic protons. Compound 2 reacted readily with
phenylisothiocyanate to yield 4-amino-3-phenyl-2-thioxo-2H-indeno[1ꢀ,2ꢀ :4,5]thieno[2,3-d]pyrimidin-9(3H)-one
(6) via the thiourea intermediate (5) (Scheme 1). Structure 6 was ascertained by spectroscopic data and
elemental analysis. The IR spectrum showed absorption bands at 3380 and 3322 cm−1 for 2NH and at 1683
cm−1 for α, β-unsaturated carbonyl group. Its 1 H NMR spectrum showed singlet signals at δ 5.11 ppm (H)
for NH, at 9.4 ppm (1H) for NH proton, and a multiplet signal at δ 6.49–7.52 ppm (9H) for aromatic protons.
In addition, refluxing of compound 2 in boiling formic acid or formamide afforded thienopyrimidinone
(7). Refluxing of compound 7 with phosphorus oxychloride yielded the corresponding 4-chloro thienopyrimidine
(8). Reaction of compound 8 with p-chloroaniline gave the thieno[3,2-d]pyrimidine (9) (Scheme 2). Structure
7 was proved by both elemental and spectral analysis. Its 1 H NMR spectrum revealed 2 singlet signals at δ
8.09 and 11.85 ppm attributable to the C-H and NH protons, respectively, and multiplet signals at δ 7.23–8.15
ppm corresponding to aromatic protons. In addition, the mass spectrum showed a molecular ion peak at m/z
254 as a base peak. The 1 H NMR spectrum of 8 showed a singlet signal at δ 9.01 ppm for C-H and multiplet
signals at δ 7.33–8.42 ppm due to aromatic protons. The mass spectrum showed a molecular ion peak at m/z
272. Moreover, the 1 H NMR spectrum of 9 showed 2 singlet signals at δ 10.3 and 8.43 ppm corresponding
to the NH and C-H proton, respectively, a doublet of doublets at δ 6.43–7.06 ppm due to the AB system of
the p-Cl-phenyl group, and a multiplet at δ 7.50–7.89 ppm due to aromatic protons. Moreover, the reaction of
enaminonitrile 2 with malononitrile in ethanolic sodium ethoxide solution afforded the corresponding pyridine
derivatives 10 (Scheme 2). The structure of 10 was confirmed by elemental analysis and spectral data. The
IR spectrum showed absorption frequencies at 3444, 3421, 3420, and 3340 cm−1 due to stretching vibrations
of 2 NH2 groups at 2204 cm−1 due to CN function and the α, β-unsaturated carbonyl group at 1720 cm−1
.
Moreover, its mass spectrum showed a molecular ion peak at m/z 292.
In a similar manner, compound 2 reacted with 2-(benzo[d]thiazol-2-yl)acetonitrile in dimethylformamide
catalyzed by triethylamine to afford compound 11 (Scheme 2). The 1 H NMR spectrum of 11 showed 2 singlet
signals at δ 8.65 and 8.85 ppm corresponding to 2 amino protons and multiplet signals at δ 7.27–8.30 ppm due
to aromatic protons.
Previously, we investigated the reaction of phenyl isothiocyanate with enaminonitrile compounds in
alkaline medium, which has been proved to be a convenient route for the synthesis of thiazole, pyrazole,
oxazine, and pyrimidine ring systems.12−16 We have extended our synthetic program to the synthesis of
otherwise inaccessible heterocyclic ring systems utilizing phenyl isothiocyanate as a key starting material. It
is known that a great variety of reactants bearing the (N=C=S) fragment undergo cyclization on reaction
with α-halocarbonyl compounds to afford thiazoles, 2,3-dihydrothiazoles, and thiazolidines,22 which have been
shown to exhibit local anesthetic,23 antiprotozoal,24 and fungicidal properties.25 In this paper, we describe
a generally applicable extension of this synthetic approach, first reported by Hantzsch and Weber.26 Thus,
the base-prompted reaction of enaminonitrile derivative 2 with phenyl isothiocyanate in dry DMF at room
temperature yielded the nonisolable intermediate 12, which gave compound 13 upon treatment with dil. HCl
(Scheme 3). The 1 H NMR spectrum of 13 revealed 2 singlet signals at δ 1.72 and 4.5 ppm due to SH and NH
protons, respectively, besides multiplet signals at δ 6.48–8.62 ppm for aromatic protons. The mass spectrum
showed a molecular ion peak at m/z 361, which is in agreement with the molecular formula C19 H11 N3 OS2 .
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FADDA et al./Turk J Chem
Scheme 2. Syntheses of pyrimidines 7–9 and pyridine derivatives 10 and 11.
Scheme 3. Synthesis of Nꢀ -(3-cyano-8-oxo-8H-indeno[2,1-b]thiophen-2-yl)-N-phenyl carbamimidothioic acid (13).
Furthermore, the intermediate 12 undergoes in situ cyclization upon the reaction with equimolar amounts
of phenacyl bromide in boiling DMF containing a catalytic amount of TEA to give thiazole derivative 15. The
structure of 15 was inferred from its spectral data and elemental analysis. Thus, the IR spectrum showed
absorption bands at υ 2230, 1690, and 1610 cm−1 corresponding to CN, C=O, and C=C functions and the
disappearance of the NH2 stretching band. It was found that treatment of 12 with phenacyl bromide in
stirring DMF afforded acyclic intermediate 14. Structure 14 was suggested for the reaction product on the
basis of both elemental and spectral analysis. Refluxing 14 in DMF with a few drops of triethylamine led to the
formation of a product identical in all respects (m.p., IR, mass) to 15. The intermediate 12 reacted readily with
equimolar amounts of chloroacetyl chloride in DMF at room temperature to give a product, 16, that analyzed as
C21 H12 ClN3 O2 S2 , which was isolated in each case in good yield. The acyclic structure of 16 was established
based on its IR spectrum, which showed bands at υ 3279, 2220, 1730, 1688, and 675 cm−1 due to stretching
vibrations of NH, CN, 2 C=O, and C-Cl groups, respectively. Refluxing of 16 in DMF containing a catalytic
amount of TEA afforded the corresponding thiazole derivative 17. Compound 17 was confirmed by spectral
data and elemental analysis. The IR spectrum that showed absorption bands at υ 2225, 1723, and 1680 cm−1
related to CN and 2 CO function groups, respectively. Moreover, its mass spectrum showed a molecular ion
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FADDA et al./Turk J Chem
Scheme 4. Reactions of the nonisolable intermediate 12 with different α-halo carbonyl compounds.
peak at m/z 401. Similarly, when the intermediate 12 was treated with chloroacetonitrile in stirring DMF, the
corresponding acyclic intermediate 18 was exclusively isolated in good yield. The mass spectrum of 18 showed
a molecular ion peak at m/z. Furthermore, heating of the intermediate 18 in DMF containing a catalytic
amount of TEA afforded the thiazole derivative 19. The structure of 19 was established based on its IR
spectrum, which showed bands due to stretching vibrations of NH2 , CN, and CO functions at 3400, 3390, 2227,
and 1690 cm−1 , respectively. Compound 12 reacted readily with chloroacetone in stirring DMF to afford the
acyclic intermediate 20 by NaCl elimination. The structure of 20 was confirmed by its IR spectrum, which
showed bands at υ 3218, 2218, 1700, and 1685 cm−1 due to stretching vibrations of NH, CN, and 2 C=O
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FADDA et al./Turk J Chem
functions. Its mass spectrum showed a molecular ion peak at m/z 417. Refluxing 20 in DMF containing a
catalytic amount of TEA gave the thiazole derivative 21, whose structure was confirmed by elemental analysis
and spectral data. The IR spectrum showed absorption frequencies at υ 2230, 1670, and 1550 cm−1 due to
stretching vibrations of CN, C=O, and phenyl groups, respectively. Its mass spectrum showed a molecular ion
peak at m/z 399. Moreover, intermediate 12 reacted with 2-bromoethyl acetate in stirring DMF to give the
acyclic intermediate derivative 22. The IR spectrum of 22 showed absorption frequencies at υ 3320, 2230,
1720, 1670, and 1550 cm−1 due to stretching vibrations of NH, CN, 2 C=O, and phenyl groups, respectively.
Its mass spectrum showed a molecular ion peak at m/z 447. Moreover, refluxing of compound 22 in boiling
DMF containing a catalytic amount of TEA afforded compound 23 (Scheme 4). The IR spectrum of 23 showed
absorption frequencies at υ 2235, 1680, and 1550 cm−1 due to stretching vibrations of CN, CO, and phenyl
groups, respectively. Its mass spectrum showed a molecular ion peak at m/z 387.
2.2. Pharmacology
2.2.1. Antimicrobial evaluation
Twenty of the newly synthesized target compounds were evaluated for their in vitro antibacterial activity against
Bacillus subtilis and Bacillus thuringiensis (as examples of gram-positive bacteria) and Escherichia coli and
Pseudomonas aeruginosa (as examples of gram-negative bacteria). They were also evaluated for their in vitro
antifungal potential against Fusarium oxysporum and Botrytis fabae fungal strains.
Agar diffusion was used for the determination of the preliminary antibacterial and antifungal activity.
Chloramphenicol, cephalothin, and cycloheximide were used as reference drugs. The results were recorded for
each tested compound as the average diameter of inhibition zones of bacterial or fungal growth around the disks
in millimeters. The minimum inhibitory concentration (MIN) measurement was determined for compounds
that showed significant growth inhibition zones (>14 mm) using 2-fold serial dilution.27 The MIC (μg/mL)
and inhibition zone diameters values are recorded in Table 1.
The results depicted in Table 1 revealed that most of the tested compounds displayed variable inhibitory
effects on the growth of the tested gram-positive and gram-negative bacterial strains, and also against the
antifungal strains.
In general, most of the tested compounds revealed better activity against the gram-positive rather than
the gram-negative bacteria. It should be also noted that compounds belonging to the thienopyrimidine and
thiazolylidine series exhibited better antibacterial potentials than members of the thienopyridine, thienopyrazole,
and indenothiophene derivatives.
Regarding the structure–activity relationship of the thiazolylidines against gram-positive bacteria, the
results revealed that compounds 15, 17, 21, and 23 exhibited broad spectrum antibacterial profiles against the
tested organisms. Thiazolylidines with electron withdrawing groups such as CO and Ph exist in compounds 15
and 17, respectively, and recorded higher activity than compound 21 and 23. In this view, compounds 15 and
17 were found to be equipotent to chloramphenicol in inhibiting the growth of B. subtilis (MIC 3.125 μg/mL),
while its activity was 50% lower than that of chloramphenicol against B. thuringiensis. Compounds 21 and 23
showed 50% of the activity of chloramphenicol (MIC 6.25 μg/mL) but they were equipotent to cephalothin in
inhibiting the growth of B. subtilis and B. thuringiensis (MIC 6.25 μg/mL).
On the other hand, compounds 2, 3, 4, 10, 11, 13, 14, 16, 18, 20, and 22 exhibited moderate growth
inhibitory activity against gram-positive bacteria as revealed by their MIC values (25–50 μg/mL). Among
these, compounds 3, 10, 11, 14, 16, 18, 20, and 22 showed relatively good growth inhibitory profiles against
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FADDA et al./Turk J Chem
B. subtilis (MIC 12.5 μg/mL) that were about 25% of the activity of chloramphenicol and 50% of cephalothin
against the same organism. Moreover, a distinctive anti-gram-positive profile was displayed by compound 7
where it proved to be equipotent to chloramphenicol against B. subtilis (MIC 3.125 μg/mL) together with
significant activity against B. thuringiensis (MIC 6.25 μg/mL).
Table 1. Minimal inhibitory concentration (MIC, μg/mL) and inhibition zone (mm) of the investigated compounds.
a
MIC in μg/mL, and inhibition (mm)
Gram-positive bacteria
Gram-negative bacteria
Fungi
Compound
B. subtilis B. thuringiensis
E. coli
100(15)
6.25(37)
50(19)
50(20)
50(19)
100(15)
25(27)
100(15)
100(14)
100(15)
50(20)
P. aeruginosa F. oxysporum
B. fabae
50(18)
6.25(38)
100(14)
50(19)
12.5(32)
100(15)
100(16)
50(20)
25(26)
100(16)
50(20)
25(27)
100(16)
2
3
4
6
7
8
9
10
11
13
14
15
16
17
18
19
20
21
22
23
50(18)
12.5(33)
25(27)
6.25(39)
3.125(43)
6.25(37)
6.25(38)
12.5(31)
12.5(32)
25(27)
12.5(33)
3.125(45)
12.5(32)
3.125(44)
12.5(32)
12.5(33)
12.5(32)
6.25(37)
12.5(33)
6.25(38)
100(14)
50(20)
50(19)
100(15)
100(16)
50(20)
100(14)
12.5(31)
100(15)
100(16)
6.25(38)
100(16)
50(19)
12.5(33)
100(15)
50(20)
100(16)
100(15)
50(19)
100(16)
100(15)
50(19)
100(15)
50(20)
100(15)
6.25(39)
6.25(37)
6.25(37)
6.25(38)
6.25(36)
50(19)
100(15)
100(14)
6.25(38)
50(20)
25(27)
100(16)
6.25(38)
100(16)
50(19)
12.5(33)
100(16)
25(25)
6.25(38)
50(19)
100(16)
100(15)
12.5(33)
50(19)
25(25)
100(15)
100(14)
100(15)
100(14)
100(15)
100(15)
50(19)
6.25(37)
100(15)
50(20)
50(19)
50(20)
100(16)
100(15)
12.5(32)
100(15)
100 (16)
6.25(38)
6.25(37)
6.25(38)
6.25(38)
3.125(44)
6.25(37)
100(15)
50(20)
25 (26)
6.25(37)
6.25(38)
100(15)
6.25(38)
6.25(37)
100(15)
b
b
Chloramphenicol 3.125(44)
NT
NT
NT
NT
b
b
Cephalothin
Cycloheximide
6.25(36)
b
b
b
b
NT
NT
NT
NT
3.125(43)
3.125(42)
aMIC: Minimal inhibitory concentration values with SEM = 0.02.
bNT: Not tested
Antibacterial activities of compounds 4, 6, 7, 14, and 22 revealed weak growth inhibitory against the
tested gram-negative bacteria (MIC 50 μg/mL). On the other hand, compounds 3 and 11 showed equipotent
activity to chloroamphenicol and cephalothin against E. coli and P. aeruginosa.
Regarding the activity of thienopyrdine, thienopyrimidine, thienopyrazole, and thiazolylidine against
antifungal strains, the results revealed that compounds 3, 10, and 17 were 50% lower than cycloheximide in
inhibiting the growth of B. fabae and F. oxysporum (MIC 6.25 μg/mL), while the activity of compound 14
and 21 was 25% lower than that of cycloheximidine against F. oxysporum (MIC 6.25 μg/mL).
The substituted pattern was also crucial. It is worth mentioning that formation of fused thienopyrimidine,
thienopyridine, thienopyrazole, and thiazolylidine produced high antimicrobial activity.28,29 On the other hand,
conversion of 1,3-indanedione to indeno[2,1-b]thiophene 2 unfortunately produced weak antimicrobial activity.
High biological activity can be correlated with low electron density of ring systems.30,31
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FADDA et al./Turk J Chem
In conclusion, the objective of the present study was to synthesize and investigate the antimicrobial
activities of some new functionalized thienopyrimidines and thiazolylidines with the hope of discovering new
structure leads serving as antimicrobial agents. Our aim has been verified by the synthesis of 4 different
rings. The obtained results clearly revealed that compounds derived from thienopyrimidine and thiazolylidine
exhibited better antimicrobial activity than their indenothiophene derivatives.
3. Experimental
3.1. Instruments
All melting points are recorded on a Gallenkamp electric melting point apparatus and are uncorrected. The IR
spectra υ cm−1 (KBr) were recorded on a PerkinElmer Infrared Spectrophotometer Model 157, Grating. The
1 H and 13 C NMR spectra were run on a Varian Spectrophotometer at 400 and 75 MHz, respectively, using TMS
as an internal reference and DMSO-d6 as solvent. The mass spectra (EI) were recorded at 70 eV with Kratos
MS equipment and/or a Varian MAT 311 A Spectrometer. Elemental analyses (C, H, and N) were carried out
at the microanalytical center of Cairo University, Giza, Egypt; the results were found to be in good agreement
with the calculated values. Biological activity screening was carried out at the Department of Botany, Faculty
of Science, Mansoura University.
3.1.1. Synthesis of 2-amino-8-oxo-8H-indeno[2,1-b]thiophene-3-carbonitrile (2)
To a solution of 1,3-indanedione (1) (1.46 g, 0.01 mol) in absolute ethanol (25 mL) containing morpholine (0.2
mL), malononitrile (0.66 g, 0.01 mol) and elemental sulfur (0.32 g, 0.01 mol) were added. The reaction mixture
was refluxed for 4 h, then cooled, poured onto ice cold water and neutralized by dilute HCl. The solid so formed
was collected by filtration and crystallized from ethanol to give compound 2 (Tables 2 and 3).
3.1.2. Synthesis of 3-aminoindeno[1ꢀ ,2ꢀ :4,5]thieno[2,3-c]pyrazol-8-(1H)-one (3)
A mixture of 2 (2.26 g, 0.01 mol), hydroxylamine hydrochloride (0.695 g, 0.01 mol), and anhydrous sodium
acetate (0.82 g, 0.01 mol) in glacial acetic acid (20 mL) was refluxed for 7 h; then the reaction mixture was
left to cool. The solid product formed after pouring onto ice cold water was filtered and recrystallized from
ethanol/DMF (3:1) mixture to give product 3 (Tables 2 and 3).
3.1.3. Synthesis of N-(3-cyano-1-oxo-1H-inden-2-yl) acetamide (4)
Compound 2 (2.26 g, 0.01 mol) in acetic anhydride (15 mL) was refluxed for 5 h. The reaction mixture was left
to cool and the formed precipitate was filtered off, dried, and crystallized from ethanol/DMF (2:1) mixture to
give compound 4 (Tables 2 and 3).
3.1.4. Synthesis of 4-amino-3-phenyl-2-thioxo-2H-indeno[1ꢀ ,2ꢀ :4,5]thieno[2,3-d] pyrimidin-9(3H)-
one (6)
A solution of 2 (2.26 g, 0.01 mol) in DMF (20 mL) was treated with phenylisothiocyanate (1.35 g, 0.01 mol)
and a few drops of triethylamine (0.4 mL) were added. The reaction mixture was refluxed for 8–10 h and left
to cool at room temperature. The formed solid product was filtered off, dried, and crystallized from a mixture
of DMF/ethanol to give 6 (Tables 2 and 3).
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FADDA et al./Turk J Chem
Table 2. Physical data of the newly synthesized compounds.
Yield
(%)
mp
◦C
Calcd. (Found)
Compound
Color
Fw. (M.Wt.)
C %
63.70
H %
2.67
N %
12.38
C12H6N2OS
(226.25)
2
97
reddish brown powder
dark brown powder
dark gray crystals
black powder
270
220
(63.50) (2.54) (12.21)
59.74 2.92 17.42
(59.57) (2.77) (17.26)
62.67 3.01 10.44
(62.48) (3.11) (10.64)
63.14 3.07 11.63
(63.21) (3.12) (11.69)
61.41 2.38 11.02
(61.33) (2.27) (10.96)
57.25 1.85 10.27
(57.17) (1.76) (9.93)
62.72 2.77 11.55
(62.67) (2.81) (11.62)
61.63 2.76 19.17
(61.76) (2.69) (19.29)
62.98 3.02 13.99
(62.90) (3.11) (13.87)
63.14 3.07 11.63
(62.95) (2.97) (11.57)
67.62 3.57 8.76
(67.71) (3.62) (8.68)
70.26 3.28 9.10
(70.31) (3.21) (9.17)
57.60 2.76 9.60
(57.67) (2.83) (9.68)
62.83 2.76 10.47
(62.76) (2.81) (10.41)
62.98 3.02 13.99
(62.91) (3.14) (13.94)
62.98 3.02 13.99
(63.10) (3.08) (14.03)
63.29 3.62 10.06
(63.35) (3.67) (10.14)
66.14 3.28 10.52
(66.23) (3.35) (10.59)
61.73 3.83 9.39
(61.66) (3.76) (9.32)
65.09 3.38 10.84
(65.14) (3.43) (10.93)
C12H7N3OS
(241.27)
3
80
83
90
86
79
87
78
88
98
85
97
90
99
82
87
94
87
81
87
C14H8N2O2S
(268.29)
C19H11N3OS2
(361.44)
C13H6N2O2S
(254.26)
C13H5ClN2OS
(272.71)
4
254
6
168
7
pale brown powder
black powder
>300
>300
>300
>300
180
8
C19H10ClN3OS
(363.82)
9
black powder
C15H8N4OS
(292.32)
C21H12N4OS2
(400.48)
C19H11N3OS2
(361.44)
10
11
13
14
15
16
17
18
19
20
21
22
23
dark green crystals
green powder
reddish brown powder
pale brown powder
gray crystals
130
C27H17N3O2S2
(479.57)
110
C27H15N3OS2
(461.56)
C21H12ClN3O2S2
(437.92)
C21H11N3O2S2
(401.46)
C21H12N4OS2
(400.48)
160
brown powder
135
dark brown crystals
yellow powder
153
156
C21H12N4OS2
(400.48)
faint brown powder
faint brown powder
dark brown crystals
pale yellow powder
deep brown powder
195
C22H15N3O2S2
(417.50)
C22H13N3OS2
(399.49)
C23H17N3O3S2
(447.53)
C21H13N3OS2
(387.48)
178
195
152
195
142

FADDA et al./Turk J Chem
Table 3. Spectroscopic data of the newly synthesized compounds.
1H NMR (400 MHz,
13C NMR (75 MHz,
Compound
2
IR
MS m/z (%)
DMSO-d6) δ (ppm)
DMSO-d6) δ (ppm)
3450–3312 (NH2), 2215
(CN), 1675 (CO), 1601
(C=C), 1486 (Ph)
5.65 (s, 2H, NH2),
7.12–7.65 (m, 4H,
Ar-H)
182.8, 165.4, 148.2, 146.8, 136.2,
132.8, 129.4, 127.3, 126.9, 118.5,
116.3, 82.5
226 (M+, 100), 199 (8.5), 170
(10), 127 (11)
241 (M+, 80), 226 (56), 221
(35), 198 (48), 138 (30), 127
(43), 111 (54), 76 (38), 75
(76), 73 (100), 50 (50), 45
(95)
5.00 (s, 2H, NH2),
8.53 (s, H, NH),
7.30–7.91 (m, 4H,
Ar-H).
3430–3334 (NH2), 3195
(NH), 1685 (CO), 1617
(C=C), 1494 (Ph).
181.5, 153.9, 150.2, 146.3, 133.6,
132.8. 130.3, 129.5, 125.2, 121.9,
119.4, 93.5.
3
4
3408 (NH), 1729 (amidic
carbonyl group), 1690 (α,β-
unsaturated ketone), 2228
(CN), 1609 (C=C), 1509
(Ph)
2.88 (s, 3H, CH3),
5.12 (s, H, NH),
7.21–7.98 (m, 4H,
Ar-H)
267 (M+, 20), 254 (40), 226
(69), 198 (100), 170 (30), 154
(28), 127 (26), 126 (25), 69
(23).
182.9, 170.4, 165.7, 153.2, 142.9,
133.4, 132.9, 129.7, 127.2, 125.8,
124.6, 117.2, 110.6, 33.4
329 (M+-S, 15), 313 (25), 249
(24), 187 (27), 93 (24), 92
(25), 79(29), 77 (64), 76 (61),
70 (33), 68 (48), 54 (42), 51
(48), 48 (100), 45 (70)
254 (M+, 100), 226 (40),
199(32), 171 (25), 170 (24),
154 (23), 144 (21), 127 (39),
69 (22)
273 (M++ 1, 33), 272 (M+,
100), 254 (23), 237 (21), 217
(22), 209 (28), 181 (26), 160
(29), 137 (30), 105 (25), 92
(31), 75 (33), 62 (31)
182.4, 180.1, 174.4, 155.7, 148.1,
142.9, 137.2, 135.3, 131.4, 130.1,
129.2, 127.9, 126.2, 124.6, 123.5,
118.6, 115.9
3380, 3322 (2NH), 1683
(α,β-unsaturated ketone),
1600 (C=C), 1490 (Ph)
5.11 (s, H, NH), 9.4
(s, H, NH), 6.49–7.52
(m, 9H, Ar-H)
6
7
8
3446 (NH), 1700 (amidic
carbonyl), 1693 (α,β-
unsaturated ketone), 1600
(C=C), 1496 (Ph)
8.09 (s, H, CH),
11.85 (s, H, NH),
7.23–8.15 (m, 4H,
Ar-H)
183.4, 173.2, 162.9, 144.2, 143.2,
142.4, 135.6, 132.3, 130.2, 129.6,
125.4, 123.5, 118.6
1693 (α,β-unsaturated
ketone), 1603 (C=C), 1500
(Ph), 650 (C-Cl)
9.01 (s, H, CH),
7.33–8.42 (m, 4H,
Ar-H)
179.3, 163.9, 159.4, 154.6, 148.9,
142.5, 136.2, 132.7, 127.4, 125.3,
124.6, 121.8, 119.6
363 (M+, 15), 242 (30), 215
(26), 187 (43), 143 (25), 90
(27), 89 (26), 79 (80), 78 (33),
76 (89), 64 (34), 51 (86), 50
(100), 48 (53)
3222 (NH), 1660 (α,β-
unsaturated ketone), 1606
(C=C), 1500 (Ph), 757 (C-
Cl)
8.43 (s, H, CH), 10.3
(s, H, NH), 6.43–7.06
(dd, 4H, Ar-H), 7.50-
7.89 (m, 4H, Ar-H)
178.6, 162.3, 157.7, 151.9, 150.6,
145.3, 141.6, 133.4, 131.9, 129.8,
127.2, 126.3, 125.5, 124.6, 123.7,
120.2, 121.6
9
3444, 3421, 3420, 3340
(2NH2), 2204 (CN), 1720
(α,β-unsaturated ketone),
1602 (C=C), 1494 (Ph)
292 (M+, 12), 194 (75), 165
(30), 154 (27), 104 (23), 88
(20), 76 (29), 64 (21), 46 (45),
45(100)
183.4, 164.3, 163.2, 154.4, 155.1,
145.4, 133.8, 132.6, 129.2, 127.1,
123.7, 121.8, 118.7, 116.6, 78.4
10
---
400 (M+, 17), 381(25), 367
(19), 280 (29), 272 (25), 266
(27), 252 (34), 227 (55), 198
(100), 177 (26), 173 (23), 170
(61), 125 (30), 74 (26), 50
(33)
179.7, 162.6, 159.3, 158.2, 154.7,
153.6, 151.7, 146.1, 136.6, 134.9,
132.3, 129.9, 127.8, 124.6, 123.4,
122.2, 120.7, 120.0, 119.4, 118.3,
106.5
3439, 3429, 3415, 3316
(2NH2), 1700 (α,β-
unsaturated ketone), 1617
8.65 (s, 2H, NH2),
8.85 (s, 2H, NH2),
7.27–8.30 (m, 8H,
Ar-H)
11
(C=C), 1511 (Ph)
361 (M+, 10), 135 (62), 119
(52), 93 (91), 91 (35), 77 (79),
76 (26), 73 (100), 66 (64), 65
(52), 64 (60), 63 (29), 52 (29),
51 (53), 50 (36), 48 (25), 45
(38)
3250 (NH), 2200 (CN),
1690 (α,β-unsaturated
ketone), 1600 (C=C), 1545
(Ph)
1.72 (s, 1H, SH),
4.50 (s, 1H, NH),
6.48–8.62 (m, 9H,
Ar-H)
183.3, 174.2, 160.8, 151.6, 144.9,
140.4, 136.3, 132.4, 129.8, 128.1,
127.4, 126.1, 123.5, 121.4, 120.2,
116.9, 82.4
13
143

FADDA et al./Turk J Chem
Table 3. Continued.
1H NMR (400 MHz,
13C NMR (75 MHz,
Compound
IR
MS m/z (%)
DMSO-d6) δ (ppm)
DMSO-d6) δ (ppm)
195.6, 183.4, 174.5, 162.2, 152.5,
146.3, 141.4, 136.9, 135.1, 134.6,
132.5, 130.5, 129.3, 128.3, 127.4,
126.9, 125.4, 123.2, 122.5, 121.3,
116.5, 82.8, 37.4
180.9, 175.1, 159.2, 147.9, 146.1,
142.7, 141.9, 136.2, 134.3, 131.2,
129.9, 129.1, 128.6, 127.9, 126.3,
125.3, 124.6, 123.2, 122.5, 120.8,
114.2, 108.3, 80.6
479 (M+, 8), 164 (25), 135 (23),
119 (42), 105 (95), 93 (65), 77
(100), 72 (73), 65 (25), 64(26), 51
(28), 45 (19)
3330 (NH), 2224 (CN), 1700
(CO), 1685 (α,β-unsaturated
ketone), 1600 (C=C), 1500 (Ph)
4.32 (s, 1H, NH), 5.11
(s, 2H, CH2), 6.85–8.73
(m, 14H, Ar-H)
14
461 (M+, 15), 408 (13), 372 (16),
234 (17), 343 (16), 312 (14), 251
(13), 104 (80), 92(31), 90 (20), 76
(100), 64 (47), 69 (15), 52 (17), 50
(68), 45 (44)
438 (M+, 12), 343 (25), 312 (28),
198 (21), 135 (47), 93 (89), 92
(25), 91 (30), 77 (98), 76 (70), 75
(29), 66 (49), 50 (100), 48 (77), 45
(48)
401 (M+, 17), 313 (18), 286 (25),
226 (22), 210 (21), 198 (29), 195
(49), 193 (100), 179 (23), 147
(32), 77 (38), 65 (29), 51 (37), 50
(23), 45 (16)
3320 (NH), 2230 (CN), 1690
(α,β-unsaturated ketone), 1610
(C=C), 1500 (Ph)
7.11 (s, 1H, CH), 6.83–
8.67 (m, 14H, Ar-H)
15
16
17
3279 (NH), 2220 (CN), 1730
(CO), 1688 (α,β-unsaturated
ketone), 1607 (C=C), 1490
(Ph), 675 (C-Cl)
184.2, 174.3, 171.5, 158.2, 150.4,
145.3, 140.4, 135.6, 134.7, 131.2,
129.5, 127.8, 126.2, 123.4, 121.4,
120.7, 116.5, 80.3, 42.6
4.23 (s, 1H, NH), 4.52
(s, 2H, CH2), 6.52–8.40
(m, 9H, Ar-H)
184.6, 174.7, 173.6, 157.3, 150.2,
145.3, 138.2, 136.3, 132.7, 131.2,
129.4, 127.7, 126.1, 125.4, 124.6,
122.0, 116.5, 83.6, 35.7
2225 (CN), 1723 (CO), 1680
(α,β-unsaturated ketone), 1600
(C=C), 1510 (Ph)
4.33 (s, 2H, CH2), 7.11–
8.65 (m, 9H, Ar-H)
400 (M+, 7), 356 (20), 321 (23),
297 (42), 296 (100), 254 (32), 226
(60), 223 (29), 194(23), 119 (47),
93 (84), 91 (22), 77 (27), 73 (46),
66 (43), 65 (22), 50 (18), 45 (20)
400 (M+, 10), 297 (39), 296 (100),
226 (65), 223 (26), 77 (26), 73
(46), 66 (46), 65 (22), 50 (18), 45
(30)
417 (M+, 23), 399 (25), 367 (90),
313 (33), 296 (34), 226 (54), 198
(40), 93 (80), 78 (24), 77 (100), 75
(25), 52 (20), 51 (66), 50 (30), 45
(26)
399 (M+, 32), 368 (30), 367 (100),
342 (18), 321 (17), 312 (59), 77
(98), 76 (41), 75 (27), 66 (47), 64
(33), 51 (68), 45 (29)
447 (M+, 35), 416 (31), 387 (10),
234 (17), 218 (15), 167 (14), 105
(23), 92 (27), 90 (40), 77 (32),
73(70), 64 (47), 54 (30), 51 (23),
47 (100), 45 (30)
182.0, 175.6, 160.7, 153.4, 144.5,
140.8, 133.6, 132.7, 128.9, 127.2,
126.3, 125.8, 123.1, 122.7, 121.6,
119.6, 117.4, 80.5, 15.6
3200 (NH), 2220, 2195 (2CN),
1690 (α,β-unsaturated ketone),
1601 (C=C), 1497 (Ph)
4.15 (s, 1H, NH), 4.35
(s, 2H, CH2), 6.75–8.39
(m, 9H, Ar-H)
18
19
20
21
22
23
180.3, 176.2, 160.9, 154.6, 150.2,
145.7, 143.2, 135.8, 132.9, 130.8,
127.6, 125.3, 124.6, 121.7, 120.5,
118.7, 116.4, 83.8, 78.2
3400, 3390 (NH2), 2227 (CN),
1690 (α,β-unsaturated ketone),
1612 (C=C), 1520 (Ph)
8.65 (s, 2H, NH2), 5.78
(s, 1H, CH), 6.64–8.33
(m, 9H, Ar-H)
2.42 (s, 3H, CH3), 4.20
(s, 1H, NH), 4.28 (s,
2H, CH2), 6.85–8.45
(m, 9H, Ar-H)
203.4, 181.9, 175.6, 161.5, 151.3,
143.5, 141.5, 136.7, 134.5, 130.2,
128.2, 127.3, 125.9, 123.4, 121.6,
120.5, 114.3, 180.5, 40.2, 25.4
3218 (NH), 2218 (CN), 1700
(CO), 1685 (α,β-unsaturated
ketone), 1612 (C=C), 1510 (Ph)
181.6, 175.3, 159.2, 151.3, 148.4,
146.3, 139.2, 133.6, 132.6, 129.1,
128.3, 127.9, 125.6, 124.7, 121.3,
119.4, 114.6, 100.5, 83.4, 19.3
2230 (CN), 1670 (α,β-
unsaturated ketone), 1614
(C=C), 1550 (Ph)
1.89 (s, 3H, CH3), 6.08
(s, 1H, CH), 7.25–8.48
(m, 9H, Ar-H)
2.97 (s, 2H, CH2), 3.40
(s, 2H, CH2), 3.72 (s,
3H, CH3), 6.75–8.54
(m, 9H, Ar-H)
181.9, 173.8, 171.2, 158.3, 151.7,
144.5, 140.4, 136.6, 135.3, 131.8,
129.2, 125.2, 124.7, 123.6, 120.8,
119.4, 116.5, 81.9, 53.5, 34.3, 27.7
3320 (NH), 2230 (CN), 1720
(CO), 1670 (α,β-unsaturated
ketone), 1610 (C=C), 1550 (Ph)
180.1, 172.4, 158.9, 150.8, 148.4,
142.9, 134.5, 132.3, 128.8, 127.9,
125.8, 124.6, 122.4, 121.0, 120.7,
115.7, 80.5, 36.7, 26.4
387 (M+, 12), 328 (13), 234 (15),
235 (17), 218 (20), 219 (23), 203
(17), 267 (20), 51 (24), 47 (100),
46 (30), 45 (44)
2235 (CN), 1680 (α,β-
unsaturated ketone), 1600
(C=C), 1550 (Ph)
3.86 (t, 2H, CH2), 3.64
(t, 2H, CH2), 6.96–8.54
(m, 9H, Ar-H)
144

FADDA et al./Turk J Chem
3.1.5. Synthesis of 3H-indeno[1ꢀ ,2ꢀ :4,5]thieno[2,3-d]pyrimidine-4,9-dione (7)
A mixture of compound 2 (2.26 g, 0.01 mol) and formic acid (15 mL) was refluxed for 12 h and then left to cool
at room temperature. The reaction mixture was poured onto crushed ice, neutralized with sodium hydroxide
solution (2 N), filtered, dried, and crystallized from a mixture of EtOH/DMF (2:1) to give compound 7 (Tables
2 and 3).
3.1.6. Synthesis of 4-chloro-9H-indeno[1ꢀ,2ꢀ :4,5]thieno[2,3-d]pyrimidin-9-one (8)
A mixture of 7 (2.54 g, 0.01 mol) and phosphorus oxychloride (25 mL) was refluxed for 15 h. After completion
of the reaction, the reaction mixture was left to cool and was poured onto crushed ice; the resulting solid was
filtered off, dried, and crystallized from a mixture of ethanol/DMF (4:1) to give compound 8 (Tables 2 and 3).
3.1.7. Synthesis of 4-((4-chlorophenyl)amino)-9H-indeno[1ꢀ,2ꢀ :4,5]thieno[2,3-d] pyrimidin-9-one
(9)
To a solution of compound 8 (2.72 g, 0.01 mol) in DMF (25 mL) was added p-chloroaniline (1.275 g, 0.01 mol)
in the presence of a few drops of TEA. The reaction mixture was refluxed for 13 h. The reaction mixture was
left to cool, poured onto crushed ice, and neutralized by dilute HCl. The precipitated solid product was filtered
and crystallized from a mixture of ethanol/DMF (2:1) to give compound 9 (Tables 2 and 3).
3.1.8. Synthesis of 2,4-diamino-9-oxo-9H-indeno[1ꢀ,2ꢀ :4,5]thieno[2,3-b]pyridine-3-carbonitrile (10)
A mixture of 2 (2.26 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) was added to freshly prepared sodium
ethoxide solution [prepared by adding 0.23 g of sodium metal to ethanol (20 mL)]; the reaction mixture was
refluxed for 8 h and left to cool overnight. The obtained solid product was collected by filtration, washed, and
crystallized from ethanol to afford 10 (Tables 2 and 3).
3.1.9. Synthesis of 2,4-diamino-3-(benzo[d]thiazol-2-yl)-9H-indeno[1ꢀ,2ꢀ :4,5]thieno [2,3-b]pyridine-
9-one (11)
A solution of 2 (2.26 g, 0.01 mol) in DMF (30 mL) and 2-(benzo[d]thiazol-2-yl)acetonitrile (1.74 g, 0.01 mol)
containing TEA (0.5 mL) was refluxed for 7 h, left to cool, and poured into crushed ice. The obtained solid
product was collected by filtration, washed, and crystallized from a mixture of ethanol/DMF to give compound
11 (Tables 2 and 3).
3.1.10. Preparation of sodium Nꢀ -(3-cyano-8-oxo-8H-indeno[2,1-b]thiophen-2-yl)-N-phenylcarba-
mimidothioate (12)
To a vigorously stirred solution of DMF (20 mL) containing an equimolar amount of sodium hydroxide (0.4 g,
0.01 mol), enaminonitrile 2 (2.26 g, 0.01 mol) and phenyl isothiocyanate (1.35 g, 0.01 mol) were added. The
reaction mixture was stirred for a further 24 h at room temperature.
145

FADDA et al./Turk J Chem
3.1.11. Synthesis of Nꢀ -(3-cyano-8-oxo-8H-indeno[2,1-b]thiophen-2-yl)-N-phenyl carbamimidoth-
ioic acid (13)
To a vigorously stirred solution of DMF (20 mL) containing an equimolar amount of sodium hydroxide (0.4 g,
0.01 mol), enaminonitrile 2 (2.26 g, 0.01 mol) and phenyl isothiocyanate (1.35 g, 0.01 mol) were added. The
reaction mixture was stirred for 24 h at room temperature. The reaction mixture was poured onto crushed ice
and neutralized by dil. HCl. The obtained solid product was filtered, dried, and crystallized from ethanol to
give compound 13 (Tables 2 and 3).
3.1.12. General procedure for the synthesis of substituted Nꢀ -(3-cyano-8-oxo-8H-indeno[2,1-b]
thiophen-2-yl)-N-phenylcarbamimidothioates 14, 16, 18, 20, and 22
Equimolar amounts of 12 (0.01 mol) in DMF and α-haloketones, namely phenacyl bromide (1.99 g, 0.01 mol),
chloroacetyl chloride (1.13 g, 0.01 mol), chloroacetonitrile (0.76 g, 0.01 mol), chloroacetone (0.93 g, 0.01 mol),
or 2-bromoethyl acetate (1.67 g, 0.01 mol), were stirred for 10 h at room temperature, then left to stand at the
same temperature for 24 h, and then poured into ice water. The separated solid material was filtered off, washed
with water, dried, and crystallized from an ethanol/DMF mixture to give 14, 16, 18, 20, and 22, respectively
(Tables 2 and 3).
General procedure for the synthesis of thiazolylidene derivatives 15, 17, 19, 21, and 23
Method A: A mixture of equimolar amounts of 12 (0.01 mol) and α-haloketones, namely phenacyl bromide
(1.99 g, 0.01 mol) and/or [chloroacetyl chloride (1.13 g, 0.01 mol) and/or chloroacetic acid (0.945 g, 0.01
mol) and/or ethyl bromocetate (1.67 g, 0.01 mol), and/or ethyl chloroacetate (1.225 g, 0.01 mol)] and/or
chloroacetonitrile (0.755 g, 0.01 mol) and/or chloroacetone (0.925 g, 0.01 mol) and/or 2-bromoethyl acetate (1.67
g, 0.01 mol), was refluxed in DMF (20 mL) for 9 h. The reaction mixture was left to cool at room temperature,
filtered off, dried, and recrystallized from a mixture of ethanol/DMF to give thiazolylidene derivatives 15, 17,
19, 21, and 23, respectively.
Method B: Refluxing the acyclic intermediate (14, 16, 18, 20, and/or 22) in DMF (20 mL) containing
a catalytic amount of TEA (0.5 mL) for 5 h afforded the corresponding substituted thiophene derivatives 15,
17, 19, 21, and 23, respectively (Tables 2 and 3).
3.2. Antimicrobial evaluation
Disks of Whatman filter paper of standard size (5.0 mm diameter) were prepared and put into 30-mL screw-
capped wide-mouthed containers for sterilization. These bottles were kept in a hot air oven at a temperature
of 150 ◦ C. Then the standard sterilized filter paper disks impregnated with a solution of the test compound in
DMF (1 mg/mL) were placed on a nutrient agar plate seeded with the appropriate test organism in triplicate.
Standard concentrations of 106 CFU/mL and 104 CFU/mL were used for antibacterial and antifungal assay,
respectively. Pyrex glass petri dishes (9 cm in diameter) were used and 2 disks of filter paper were inoculated
in each plate. The utilized test organisms were B. subtilis and B. thuringiensis (as examples of gram-positive
bacteria) and E. coli and P. aeruginosa (as examples of gram-negative bacteria). They were also evaluated
for their in vitro antifungal potential against F. oxysporum and B. fabae fungal strains. Chloramphenicol,
cephalothin, and cycloheximide were used as standard antibacterial and antifungal agents, respectively. DMF
alone was used as the control in the same above-mentioned concentration and due to this there was no visible
146

FADDA et al./Turk J Chem
change in bacterial growth. The plates were incubated at 37 ◦ C for 24 h for bacteria and for 48 h for fungi.
Compounds that showed significant growth inhibition zones (>14 mm) using the 2-fold serial dilution technique
were further evaluated for their minimal inhibitory concentrations (MICs).
3.3. MIC measurement
The microdilution susceptibility test in Mueller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid)
was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the
tested compounds, chloramphenicol, cephalothin, and cycloheximide, were prepared in DMF at a concentration
of 1000 mg/mL. Each stock solution was diluted with standard method broth (Difco) to prepare serial 2-fold
dilutions in the range of 500–3125 mg/mL and 10 mL of the broth containing about 106 CFU/mL of test
bacteria was added to each well of 96-well microtiter plates. The sealed microplates were incubated at 37 ◦ C
for 24 h for antibacterial activity and at 37 ◦ C for 48 h for antifungal activity in a humid chamber. At the
end of the incubation period, the MIC values were recorded as the lowest concentrations of the substance that
had no visible turbidity. Control experiments with DMF and uninoculated media were run parallel to the test
compounds under the same conditions.
Acknowledgments
The authors are very grateful to Dr Ehab Ali, Department of Botany, Faculty of Science, Mansoura University,
for performing the antimicrobial evaluation.
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