Synthesis and antimicrobial activity of some new benzo and naphthonitrile derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.11.017

The reaction of 2-(cyanomethyl)benzonitrile (1) with different diazonium salts gave the corresponding aryldiazenyl derivatives 2a-e which reacted with sodium hydroxide and ethyl chloroacetate and hydroxylamine hydrochloride to afford the corresponding acetamides 4a-e, pyrazoloisoquinolines 6a-e and triazoloisoquinoline derivatives 8a-e, respectively. Moreover, the reaction of 1 with arylidene malononitriles 9a-c afforded dihydronaphthalenes 11a, b and naphthalene derivative 12, respectively. The newly synthesized compounds were characterized by analytical and spectral data. The investigated compounds were screened for their antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and antifungal activity. Among the synthesized compounds, (E)-2-(cyano((4-nitrophenyl)diazenyl)methyl)benzonitrile (2e) exhibited a significant activity toward both Gram-positive, Gram-negative bacteria and exhibit the most potent in vitro antifungal with MIC's (6.25 μg/mL) against Botrytis fabae.

Full text of DOI:10.1016/j.ejmech.2012.11.017

European Journal of Medicinal Chemistry 60 (2013) 421e430
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activity of some new benzo and naphthonitrile
derivatives
*
Ahmed A. Fadda , El-Sayed M. Afsah, Radwa S. Awad
Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhoria Street, 35516 Mansoura, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of 2-(cyanomethyl)benzonitrile (1) with different diazonium salts gave the corresponding
aryldiazenyl derivatives 2aee which reacted with sodium hydroxide and ethyl chloroacetate and
hydroxylamine hydrochloride to afford the corresponding acetamides 4aee, pyrazoloisoquinolines 6aee
and triazoloisoquinoline derivatives 8aee, respectively. Moreover, the reaction of 1 with arylidene
malononitriles 9aec afforded dihydronaphthalenes 11a, b and naphthalene derivative 12, respectively.
The newly synthesized compounds were characterized by analytical and spectral data. The investigated
compounds were screened for their antibacterial activity against Gram-positive bacteria, Gram-negative
bacteria and antifungal activity. Among the synthesized compounds, (E)-2-(cyano((4-nitrophenyl)dia-
zenyl)methyl)benzonitrile (2e) exhibited a significant activity toward both Gram-positive, Gram-negative
Received 12 October 2012
Received in revised form
9 November 2012
Accepted 13 November 2012
Available online 20 November 2012
Keywords:
2-(Cyanomethyl)benzonitrile
Pyrazoloisoquinolines
1,2,3-Triazoloisquinolines
Dihydronaphthalenes
Naphthalene
bacteria and exhibit the most potent in vitro antifungal with MIC’s (6.25 mg/mL) against Botrytis fabae.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antimicrobial activity
1. Introduction
In a sequel of continuation, the present work is undertaken to
study the structural chemistry of these aryl azo derivatives through
discussing IR, 1H NMR, mass and UV spectra to support the tauto-
meric behavior of these compounds.
Aroyl acetonitriles (u-cyanoacetophenone and its derivatives)
are versatile reagents reported to have many applications in
heterocyclic synthesis. Many problems are encountered with the
synthesis of such compounds. We report herein a novel synthesis
of new intermediates for the synthesis of such compounds.
We have carried out intensive studies with activated nitriles as
potential intermediates for the preparation of variety of heterocy-
clic systems for biological screening program in our laboratory and
as extension of our recently reported procedures for the prepara-
tion of active cyanomethylene group [1e9].
The rich chemistry of azo compounds is also associated with
several important biological reactions such as protein synthesis,
carcinogenesis, azo reduction monoamine oxidase inhibition
mutagenic, immunochemical affinity labeling, nitrogen fixation,
important medical and industrial uses [10,11]. Fadda and
2. Results and discussion
2.1. Chemistry
Encouraged by these results, we have tried the coupling reaction
of compound 1 with some diazotized aromatic amines under the
similar reported conditions. Thus, in sodium acetate buffered
solution of ethanol, 2-(cyanomethyl)benzonitrile (1) reacted with
diazotized aryl amines to form the corresponding (E)-2-(cya-
no(aryldiazenyl)methyl)benzonitrile derivatives 2aee in overall
good yields (Fig. 1). However, no details of the dying behavior,
synthesis or their antimicrobial activity were reported.
coworkers [2,12e22] have published
a series of papers to
The possible tautomeric forms of arylazo derivatives could be
set out as outlined in Fig. 2.
throw the light on the chemistry of azo dyes. In our laboratory,
some (E)-2-(cyano(aryldiazenyl)methyl) benzonitrile derivatives
have been prepared and characterized by elemental analysis and
spectral data.
There are referred to as the CH azo form (A), the NH azo form (B)
and the cyanohydrazone form (C). Therefore, it was considered
worthwhile preparing arylazo compounds containing the benzo-
nitrile ring in order to evaluate their biological activities. In this
investigation, five (E)-2-(cyano(aryldiazenyl)methyl)benzonitrile
or (Z)-2-cyano-N0-aryl benzohydrazonyl cyanide derivatives 2aee
* Corresponding author. Tel.: þ20 506432235; fax: þ20 502246781.
E-mail address: afadda50@yahoo.com (A.A. Fadda).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.017

422
A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
Fig. 1. Structures of the investigated (E)-2-(cyano(aryldiazenyl)methyl) benzonitrile derivatives 2aee.
were prepared by coupling reaction of 1 with the appropriate
diazonium salt in ethanol containing a catalytic amount of freshly
fused sodium acetate. The newly prepared dyes were characterized
by elemental analysis, as well as by spectral data.
the polarity change of the absorbing system caused by solvent
interactions because of the general solvent effect [25a].
The azo compounds generally showed two absorption bands at
400e410 and 290e300 nm, corresponding to nep* and
pe
p*
On the basis of the IR spectrum of compound 2, it was possible
to assign the absorption bands of the diazonium coupling prod-
ucts 2. Each of the investigated compounds possessed a weak and
broad band in the region 3452e3255 cmꢀ1. This was assigned to
NH stretching of the hydrazone moiety. The large shift and
broadening of this band, as reported by Ramirez and Kerby [23]
for simple hydrazones, can be only from intramolecular
hydrogen bonding in (C). The fact that the compound 2 showed
evidence for intramolecular hydrogen bonding is in favor of the
hydrazone structure. The IR spectra of all compounds showed
two absorption bands in the region 2250e2203 cmꢀ1. These
bands were assigned to C^N vibration. So, on the basis of these
data, structure (B) can be ruled out. The IR spectra of most of
transitions, respectively [25b]. On the other hand, monoaryl
hydrazones showed three intense bands in the region of 220e230,
250e280, and 330e390 nm [25a].
It is clear that these dyes exhibited three bands; of these, the
medium and high wavelength bands seem to be affected by the
nature of the polar substituent in the arylazo group, but the low
wavelength band is unaffected. The 250 nm band of the parent
benzonitrile exhibited a profound hypsochromic shift, often dis-
appearing from the measurable region, which is conceivably caused
by the cis arrangement around N]N bond.
Table 1 showed that both electron withdrawing and electron
donating groups cause the absorption to occur at higher wave-
lengths. A “C]N” linkage was reported to have properties espe-
cially analogous to those of an ethylenic linkage [26]. Hence, an
electronic effect of a substituent on the aromatic nucleus of the
arylazo moiety will be transmitted to the whole conjugate system
compounds showed absorption in the region 1580e1550 cmꢀ1
.
These bands, however, can be assigned to N]N group might be
taken as an indication that structure (A) is present in the studied
series.
through pep conjugation, exerting a considerable influence upon
Since, it has been deduced that compounds 2aee existed as
tautomeric forms azo-hydrazone (A) and (C) (Fig. 3), a band due to
C]N would be expected in the double bond region. Bellamy [24]
the conjugation bands (A) and (B). Table 1 showed also that the
presence of electron donating or electron withdrawing groups has
not brought about any marked increase or decrease in lmax in the
quoted
compounds. The IR spectral data revealed that none of the
compounds exhibited absorption bands above 1600 cmꢀ1
a
range of 1630e1625 cmꢀ1 for
a
,
b
-unsaturated
visible region and log
3
has nearly remained constant. This does
point toward the hydrazone structure (C) where the resonance in
the diazo components is minimal, owing to steric factors.
.
However, each compound showed strong absorption band between
the aromatic 1500 cmꢀ1 band and NH deformation band near
1550 cmꢀ1, this band is relatively strong and in some cases, it could
not be resolved from the 1500 cmꢀ1 band. Such a band was not
observed in the spectra of the starting compound. This new band
may be due to the C]N vibration. Fadda suggested that the
downward shift of the C]N band of the newly prepared
compounds may be attributed to its conjugation with the nitrile
group.
The UV spectra of the diazonium coupling products of 1 provide
additional evidence that such compounds have a tautomeric rela-
tionship with arylazo-monohydrazones (A) and (C). Most of the
dyes showed three main absorption bands in the region 210e
390 nm. The relatively small difference in lmax may be a result of
The 1H NMR spectra of arylazo derivatives 2aee provided
a more confirmation for the tautomeric azo-hdrazo forms which
showed in general a singlet signal of an NH proton at
8.38 ppm and showed also a singlet signal at 3.80e4.20 ppm
due to the methine proton. Also, 1H NMR spectra revealed multi-
plet signals at 7.18e8.10 ppm corresponding to the aromatic
d 8.00e
d
d
protons. The mass spectra of arylazo derivatives 2aee, in general,
confirm the proposed formula by detecting the following peaks. For
compound 2a, the observed peak at m/z ¼ 246 (C15H10N4 calculated
atomic mass 246.27) represents the molecular ion peak with 75%
abundance. In general, from the obtained data for all compounds
we conclude that the molecular weight was in good agreement
with the calculated molecular weight of the investigated
compounds.
A
B
C
A
C
Fig. 2. The possible tautomeric forms of compounds 2aee.
Fig. 3. The tautomeric forms azo-hydrazone (A) and (C) of compounds 2aee.

A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
423
stretching frequency of two CN groups around 2220 cmꢀ1 and
showed stretching vibration bands at 1700 and 1630 cmꢀ1 due to
CO and C]N, respectively. Also, the 1H NMR spectrum exhibited
Table 1
UV absorption bands of the newly synthesized compounds 2aee.
Dye no.
R
Color of
crystals
lmax (log 3 )
triplet signals at
4.29 ppm for CH2 protons, two singlet signals at
7.10 ppm attributable to 2NH protons, in addition to the expected
aromatic protons at 7.20e7.57 ppm as multiplet signals. The mass
d
1.30 ppm for CH2CH3 protons, quartet signal at
2a
2b
2c
2d
C6H5
C6H4eCl(p)
CH3(p)
Orange
Yellow
Brown
Orange
299 (4.42), 399 (4.21)
418 (3.95), 448 (3.96), 331 (4.01)
406 (4.05), 430 (4.04)
253 (4.21), 271 (4.22), 304 (4.13),
419 (4.05), 430 (4.05)
297 (3.93), 444 (3.89)
d
d
4.00 and
d
OCH3(p)
spectrum revealed the molecular ion peak at m/z ¼ 332 (Mþ, 100%),
such data was in accordance with the proposed structure 6a.
Compound 8a was established its structure also on the basis of
elemental analysis and spectral data, the IR spectrum showed
absorption bands at 3440 cmꢀ1 due to NH group, C]N at
1628 cmꢀ1 and the disappearance of stretching frequency of two
CN groups around 2200 cmꢀ1. The 1H NMR spectrum revealed
2e
NO2(p)
Brown
Cyclization of azo derivatives 2aee by refluxing in ethanolic
sodium hydroxide solution to afford the corresponding pyridazine
carbonitrile derivatives 3aee was failed, and instead it afforded (E)-
2-(2-cyanophenyl)-2-(p-aryldiazenyl)acetamide derivatives 4aee,
respectively (Scheme 1). Assignment of structures 4aee was
made on the basis of analytical and spectral data. Thus, their IR
spectra showed in general an NH2 absorption band at 3442e
3221 cmꢀ1, cyano stretching vibration and amidic carbonyl group
appeared at 2214e2201 and 1658e1621 cmꢀ1, respectively, also its
1H NMR spectra exhibited, beside the expected signals, a singlet
signals at
at
d 4.00 and 7.10 ppm for 2NH protons and multiplet signals
d
7.40e8.09 ppm corresponding to aromatic protons. While, the
mass spectrum showed the molecular ion peak at m/z ¼ 261 (Mþ,
30%).
As a continuation of previous work to investigate the behavior
of some activated nitriles toward arylidene malononitriles, we
report herein the behavior of 2-cyanomethylbenzonitrile (1)
toward some arylidene malononitriles. The work gave rise to the
development of convenient approaches to the synthesis of
a variety of polyfunctionally substituted 4-aminonaphthalene
derivatives in good yield. Thus, compound 1 reacted with equi-
molar amounts of arylidene malononitriles 9aec in absolute
ethanol containing a catalytic amount of triethylamine under
reflux to yield the corresponding 1:1 adducts 11a, b in acceptable
yield. The nonisolable intermediates 10a, b are believed to be
formed via Michael type addition of active methylene in
compound 1 to the double bond in compound 9 to yield the
nonisolable intermediate 10 that cyclized directly yielding
compound 11a, b as final product in cases of 10a, b while in case of
10c the reaction directly afforded the aromatized compound 12 by
loss one molecule of HCN from the nonisolable intermediate 11c
derivative which aromatized under the reaction conditions [27].
Both elemental analysis and spectral data of 11a, b and 12 are
consistent with the assigned structures. Thus, the IR spectra of 11a,
b showed bands at 3226, 3343 and 2277e2177 cmꢀ1 due to
stretching vibration of NH functions as well as three CN stretching
absorption peaks. The 1H NMR spectrum of 11a revealed two
signal at
d 4.10e4.30 ppm attributed to CH proton and at d 7.20e
7.30 ppm due to NH2 protons. The mass spectrum of 4a showed
the molecular ion peak at m/z 264 (Mþ, 100%). Such data were in
accordance with the proposed structures 4aee.
Unexpectedly, the azo derivatives 2aee underwent cyclization
upon refluxing with ethyl chloroacetate in N,N-dimethyformamide
in presence of catalytic amount of dry potassium carbonate affor-
ded the corresponding pyrazoloisoquinoline derivatives 6aee
instead of the nonisolable intermediate b-aminoester 5aee, while
refluxing of 2aee with hydroxylamine hydrochloride in N,N-
dimethyformamide in presence of catalytic amount of glacial acetic
acid and freshly fused sodium acetate afforded the corresponding
triazoloisoquinoline derivatives 8aee via the nonisolable inter-
mediates 7aee, respectively (Scheme 2).
Compounds 6aee were assumed to be formed via the elimina-
tion of a molecule of hydrochloric acid which followed by nucleo-
philic attack of NH2 to C^N group according to the proposed
mechanism in Scheme 3.
Similarly, compounds 8aee were assumed to be formed via the
elimination of a water molecule followed by double nucleophilic
addition of NH2 to the C^N group (Scheme 4).
doublets at
d 3.20 and 4.15 ppm corresponding to two CH protons
Assignments of structures 6aee and 8aee were made on the
basis of analytical and spectral data. Thus, the IR spectrum of 6a
showed an NH absorption band at 3450 cmꢀ1, disappearance of
besides singlet signal due to exchangeable proton at d 5.10 ppm for
NH proton, while the 1H NMR spectrum of 11b showed a similar
picture to that of 11a in addition to the expected OCH3 signal at
Scheme 1. Synthesis of (E)-2-(2-cyanophenyl)-2-(p-aryldiazenyl)acetamide derivatives 4aee.

424
A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
Scheme 2. Synthesis of pyrazolo[4,3-c]isoquinolines 6aee and triazolo[4,5-c]isoquinolines 8aee.
d
3.80 ppm. The mass spectra of 11a, b showed the molecular ion
2.2. Antimicrobial evaluation
peaks at m/z 330 (Mþ, 50%) and 324 (Mþ ꢀ 2, 100%) corresponding
to the molecular formula (C19H11ClN4) and (C20H14N4O), respec-
tively. The 1H NMR spectrum of compound 12 revealed a singlet
Twenty seven of the newly synthesized targeted compounds
were evaluated for their in vitro antibacterial activity against
Bacillus subtilis and Bacillus thuringiensis as example 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 method [28] was used for the determination of
the preliminary antibacterial and antifungal activity. Chloram-
phenicol, Cephalothin and Cycloheximide were used as reference
drugs. The results were recorded for each tested compound as the
average diameter of inhibition zones (IZ) of bacterial or fungal
growth around the disks in mm. The minimum inhibitory
concentration (MIC) measurement was determined for compounds
signal at
3.90 ppm for three methoxy protons, in addition to multiplet
signals at 6.80e8.80 ppm for aromatic protons.
All attempts to aromatize 11a, b to obtain the corresponding
aminonaphthalene 12a, b by refluxing in boiling nitrobenzene were
failed. Structures 11a, b and 12 were more confirmed by alternative
synthesis via condensation of 1 with appropriate aldehydes 13aec
to yield the corresponding arylidene 14aec, respectively. Heating of
d 10.10 ppm for NH2 protons, multiplet signals at d 3.70e
d
14a,
b with malononitrile afforded the corresponding dihy-
dronaphthalene derivatives 11a, b while heating of 14c with
malononitrile under the same previous conditions afforded product
which identified as compound 12 (Scheme 5).
Scheme 3. The proposed mechanism for the synthesis of pyrazolo[4,3-c]isoquinolines 6aee.

A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
425
Scheme 4. The proposed mechanism for the synthesis of triazolo[4,5-c]isoquinolines 8aee.
showed significant growth inhibition zones (>14 mm) using two
fold serial dilution method [29].
organisms. Compounds with electron withdrawing groups such as
C6H4eNO2-p, Cl, C6H5, CO and N]N recorded higher activity. In this
view, compounds of azo derivatives showed higher activity in order
of 2e > 2b > 2a > 2d. Also, compounds 2e, 2b were equipotent to
Chloramphenicol and Ampicillin in inhibiting the growth of
The MIC (mg/mL) and inhibition zone diameters values are
recorded in Table 2. The results depicted in Table 2 revealed that the
most of tested compounds displayed variable inhibitory effects on
the growth of the tested Gram-positive and Gram-negative bacte-
rial strains, and also against antifungal strain.
In general, most of the tested compounds revealed better
activity against the Gram-positive rather than the Gram-negative
bacteria. It would be also noticed that compounds belonging to
the azo and acetamide derivatives (Scheme 1) exhibited better
antibacterial potentials than parent 1 and members of the pyrazole
and triazole series (Scheme 2). Regarding the structure activity
relationship (SAR) of the azo derivatives against Gram-positive
bacteria, the results revealed that compounds 2a, 2b, 2d and 2e
exhibited broad spectrum antibacterial profile against the tested
B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than
of Chloramphenicol against B. thuringiensis. Moreover, compounds
of acetamide derivatives showed higher activity in order of
4e > 4b > 4a > 4d, while compounds 4e and 4b were about 25% of
the activity Chloramphenicol and Ampicillin and 50% of Cephalo-
thin against B. subtilis, and its activity was 50% lower than of
Chloramphenicol and 25% lower than Cephalothin against
B. thuringiensis.
On the other hand, compounds 6aee and 8aee exhibited
moderate growth inhibitory activity against Gram-positive bacteria
as revealed from their MIC values (25e50 mg/mL). Compounds of
Scheme 5. Reactions of 2-(cyanomethyl)benzonitrile (1) with arylidene malononitrile derivatives 9aec.

426
A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
Table 2
Minimum inhibitory concentration (MIC,
m
g/mL) and inhibition zone (mm) of some new synthesized compounds.
Compound No.
MICa in
Bacteria
mg/mL, and inhibition zone (mm)
Fungi
Gram-positive bacteria
Gram-negative bacteria
B. subtilis
B. thuringiensis
E. coli
P. aeruginosa
F. oxysporum
B. fabae
1
2a
2b
2c
2d
2e
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
8a
8b
8c
8d
8e
11a
11b
12
100(15)
12.5(37)
3.125(42)
12.5(35)
6.25(39)
3.125(43)
12.5(37)
6.25(38)
25(31)
25(32)
6.25(33)
25(33)
25(33)
25(32)
50(40)
25(38)
50(33)
25(32)
50(30)
50(33)
50(38)
50(20)
50(15)
25(30)
100(16)
25(33)
6.25(37)
25(30)
12.5(35)
6.25(36)
25(15)
12.5(27)
50(15)
12.5(30)
25(15)
50(20)
50(25)
50(15)
50(30)
50(15)
50(20)
50(15)
50(15)
50(19)
25(26)
100(15)
12.5(30)
12.5(35)
25(16)
25(30)
6.25(35)
25(31)
25(15)
12.5(16)
25(32)
25(16)
50(19)
25(33)
50(15)
50(20)
50(16)
50(19)
50(19)
50(15)
50(15)
50(15)
100(31)
100(26)
100(32)
100(20)
100(26)
100(18)
6.25(38)
50(14)
12.5(38)
6.25(38)
12.5(33)
12.5(37)
6.25(41)
12.5(27)
25(16)
25(38)
25(16)
12.5(35)
50(33)
25(16)
50(25)
25(38)
25(19)
25(20)
25(15)
50(19)
25(19)
25(15)
50(20)
50(19)
50(33)
100(15)
50(20)
100(15)
12.5(40)
6.25(39)
25(35)
12.5(38)
6.25(40)
25(15)
12.5(16)
25(20)
50(26)
12.5(38)
25(20)
50(27)
50(16)
50(19)
50(20)
25(16)
50(15)
50(20)
25(15)
25(16)
50(26)
100(20)
50(28)
100(26)
100(25)
6.25(38)
12.5(33)
25(36)
6.25(37)
12.5(37)
12.5(33)
50(36)
12.5(19)
12.5(35)
25(35)
50(38)
50(20)
25(37)
25(37)
25(37)
25(38)
50(26)
50(35)
50(38)
50(33)
100(18)
50(35)
50(20)
100(18)
100(15)
3.125(44)
100(33)
100(31)
100(35)
100(15)
100(19)
100(16)
6.25(37)
100(15)
50(26)
50(21)
100(26)
100(20)
3.125(44)
14a
14b
14c
100(19)
100(20)
b
b
Chloramphenicol
Cephalothin
Cycloheximide
Ampicillin
b
b
6.25(36)
6.25(37)
6.25(38)
6.25(37)
b
b
b
b
3.125(43)
3.125(42)
b
b
b
b
3.125 (40)
6.25(38)
a
MIC: Minimum inhibitory concentration values with SEM ¼ 0.02 (The lowest concentration that inhibited the bacterial growth).
b
NT: Not tested.
pyrazole derivatives showed relatively good growth inhibitory
3. Experimental
profiles against B. subtilis in order of 6e > 6b > 6a > 6d, while 6e, 6b
were about 25% of the activity Chloramphenicol and Ampicillin and
50% of Cephalothin against the same organism. Also, compounds
11a, 11b, 12 and 14aec showed weak growth inhibitory against
3.1. Instruments
All melting points are recorded on Gallenkamp electric melting
point apparatus and are uncorrected. The IR spectra
/cmꢀ1 (KBr)
B. subtilis in order of 12 > 11a > 11b (MIC 50e100
mg/mL). Con-
n
cerning the antibacterial activity of the compounds 11a, 11b, 12 and
14aec revealed weak growth inhibitory against the tested Gram-
negative bacteria (MIC 50e100 mg/mL).
Regarding to the activity of azo derivatives, against antifungal
strains, the results revealed that compounds 2e and 2b were 50%
lower than Cycloheximide in inhibitory the growth of B. fabae and
were recorded on Perkin Elmer Infrared Spectrophotometer Model
157, Grating. The 1H NMR and 13C NMR spectra were run on Varian
Spectrophotometer at 300 and 75 MHz, respectively, using tetra-
methylsilane (TMS) as an internal reference and DMSO-d6 as
solvent. The mass spectra (EI) were recorded on 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 micro analytical center
of Cairo University, Giza, Egypt, the results were found to be in good
agreement (ꢁ0.3%) with the calculated values.
F. oxysporum (MIC 6.25
and 2d was 25% lower than Cycloheximidine against F. oxysporum
(MIC 12.5 g/mL). The substituted pattern was also crucial. It is
mg/mL), while the activity of compounds 2a
m
worth mentioning that incorporation of arylazo group to triazole
nucleus decreased the antimicrobial activity. On the other hand,
conversion of compound 1 to arylazo derivatives 2aee enhanced
the antimicrobial activity. High biological activity can be correlated
with low electron density of ring systems.
In conclusion, the objective of the present study was to synthesize
and investigate the antimicrobial activities of some new functional-
ized arylazo derivatives, acetamide derivatives and arylazotriazole
derivatives with the hope of discovering new structure leads serving
as antimicrobial agents. Our aim has been verified by the synthesis of
three different groups of structure hybrids comprising basically the
arylazo moiety. The obtained resultsclearly revealed thatcompounds
of arylazo derivatives exhibited better antimicrobial activity than
compounds containing triazole moiety.
3.1.1. General procedure for the synthesis of (E)-2-
(cyano(aryldiazenyl)methyl)benzonitrile derivatives 2aee
A well stirred solution of the base aromatic amine (0.01 mol) in
2 N hydrochloric acid (15 mL) was cooled in an ice-salt bath and
diazotized with sodium nitrite (0.01 mol) in water (5 mL). The
mixture was then tested for complete diazotization using starch
iodide paper which gives a weak blue test. If the mixture does not
give the test, more sodium nitrite was added dropwise until
a positive test is obtained and the color is stable for few minutes. If,
on the other hand, strong test for nitrite is obtained, a few drops of
a dilute solution of the base hydrochloride are added until the
nitrite test is nearly negative. The above cold diazonium solution
was added slowly to a well stirred solution of compound 1 (1.42 g,

A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
427
0.01 mol) in ethanol (20 mL) containing sodium acetate (1 g) and
3.1.2. General procedure for the synthesis of (E)-2-(2-cyanophenyl)-
the mixture was cooled in an ice-salt bath. After the addition of the
diazonium salt solution, the reaction was tested for coupling
reaction. A drop of the reaction mixture was placed on a filter paper
and the colorless ring surrounding the spot dye was treated with
a drop of an alkaline solution of a reactive coupler, such as a sodium
salt of 3-hydroxy-2-naphthanilide. If unreacted diazonium salt is
present, a dye is formed. The presence of unreacted coupler can be
determined in a similar manner using a diazonium salt solution to
test the colorless ring. After the coupling reaction was complete,
the reaction mixture was stirred for 15 min at room temperature to
coagulate the dye particles. The precipitated deep-colored product
formed was filtered off, washed with water several times, dried and
crystallized from dioxane to give compounds 2aee.
2-(p-aryldiazenyl)acetamide derivatives 4aee
To a solution of each of 2aee (0.01 mol) in ethanol (20 mL),
NaOH (20%, 10 mL) solution was added. The reaction mixture was
refluxed in each case for 3e4 h, and then left to cool overnight. The
solid products formed were collected by filtration, washed with
cold water, dried and recrystallized from DMF.
3.1.2.1. (E)-2-(2-cyanophenyl)-2-(phenyldiazenyl)acetamide
(4a).
Brown powder; yield (60%); m.p. > 300 ꢂC; IR (KBr):
n
/cmꢀ1 ¼3442,
3420 (NH2), 2201 (CN), 1657 (CO), 1614 (C]N); 1H NMR (DMSO-d6)
d
(ppm): 4.21 (s, 1H, CH), 7.20 (s, 2H, NH2), 7.40e7.61 (m, 9H, Ar-H);
13C NMR (DMSO-d6)
d
(ppm): 172.7, 150.9, 139.4, 133.5, 132.7, 130.3,
129.2, 128.3, 125.7, 122.6, 115.8, 113.9, 71.7; MS (EI, 70 eV): m/z
(%) ¼ 264 (Mþ, 100), 142 (12), 128 (16), 114 (17), 102 (9). Anal. Calcd.
for C15H12N4O (264.28): C, 68.17; H, 4.58; N, 21.20%. Found: C,
68.07; H, 4.38; N, 21.05%.
3.1.1.1. (E)-2-(Cyano(phenyldiazenyl)methyl)benzonitrile
(2a).
Orange powder; yield (70%); m.p. 80 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 3255
(NH), 2250, 2225 (2CN),1629 (C]N),1537 (N]N); 1H NMR (DMSO-
d6)
d
(ppm): 3.80 (s, 1H, CH), 7.20e7.61 (m, 9H, Ar-H), 8.0 (s, 1H,
(ppm): 150.9, 133.6, 132.2, 131.4, 129.5,
3.1.2.2. (E)-2-((4-chlorophenyl)diazenyl)-2-(2-cyanophenyl)acet-
NH); 13C NMR (DMSO-d6)
d
amide (4b). Brown powder; yield (50%); m.p. 200 ꢂC; IR (KBr):
n/
129.2, 128.7, 125.7, 122.6, 115.8, 114.9, 113.1, 53.5; MS (EI, 70 eV): m/z
(%) ¼ 245 (Mþ ꢀ 1, 75), 204 (15), 177 (15), 149 (28), 142 (25), 116
(100). Anal. Calcd. for C15H10N4 (246.27): C, 73.16; H, 4.09; N,
22.75%. Found: C, 73.02; H, 4.24; N, 22.55%.
cmꢀ1 ¼3348, 3224 (NH2), 2207 (CN),1655 (CO),1614 (C]N); 1H NMR
(DMSO-d6) d (ppm): 4.23 (s, 1H, CH), 7.21 (s, 2H, NH2), 7.30e7.60 (m,
8H, Ar-H); 13C NMR (DMSO-d6)
d (ppm): 172.7,149,139.4,133.5,132.7,
131.3, 130.3, 129.3, 128.3, 124, 115.8, 113.9, 71.7; MS (EI, 70 eV): m/z
(%) ¼ 300 (Mþþ2, 4), 298 (Mþ, 6.1), 284 (100), 267 (11), 242 (12), 142
(22),128(17),114 (28). Anal. Calcd. for C15H11ClN4O (298.37): C, 60.31;
H, 3.71; N, 18.76%. Found: C, 60.16; H, 3.51; N, 18.66%.
3.1.1.2. (E)-2-(((4-Chlorophenyl)diazenyl)(cyano)methyl)benzonitrile
(2b). Yellow powder; yield (63%); m.p. 88 ꢂC; IR (KBr):
n/
cmꢀ1 ¼ 3452 (NH), 2250, 2226 (2CN), 1630 (C]N), 1550 (N]N); 1H
NMR (DMSO-d6)
d
(ppm): 3.80 (s, 1H, CH), 7.27e7.61 (m, 8H, Ar-H),
3.1.2.3. (E)-2-(2-cyanophenyl)-2-(p-tolyldiazenyl)acetamide
(4c).
8.10 (s, 1H, NH); 13C NMR (DMSO-d6)
d
(ppm): 149, 133.6, 132.2,
Brown powder; yield (70%); m.p. 169 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 3390,
131.4, 131.3, 129.5, 129.3, 128.7, 124, 115.8, 114.9, 113.1, 53.5; MS (EI,
70 eV): m/z (%) ¼ 282 (Mþþ2, 6), 280 (Mþ, 11), 257 (19), 149 (11),
142 (30), 116 (100). Anal. Calcd. for C15H9ClN4 (280.71): C, 64.18; H,
3.23; N, 19.96%. Found: C, 64.04; H, 3.08; N, 19.76%.
3336 (NH2), 2214 (CN), 1644 (CO), 1608 (C]N); 1H NMR (DMSO-d6)
d
(ppm): 2.37 (s, 3H, CH3), 4.30 (s,1H, CH), 7.20 (s, 2H, NH2), 7.30e8.50
(m, 8H, Ar-H); 13C NMR (DMSO-d6)
d
(ppm): 147.9,135.4,133.6,132.2,
131.4, 129.5, 128.7, 122.5, 115.8, 114.9, 113.1, 53.5, 21.3; MS (EI, 70 eV):
m/z (%) ¼ 278 (Mþ, 100), 260 (46), 243 (42), 232 (30), 208 (27), 156
(33), 132 (46), 103 (85). Anal. Calcd. for C16H14N4O (278.31): C, 69.05;
H, 5.07; N, 20.13%. Found: C, 69.20; H, 5.27; N, 20.03%.
3.1.1.3. (E)-2-(Cyano(p-tolyldiazenyl)methyl)benzonitrile
(2c).
Brown powder; yield (70%); m.p. 73e76 ꢂC; IR (KBr):
n
/cmꢀ1 ¼3258
(NH), 2225, 2203 (2CN),1625 (C]N),1535 (N]N); 1H NMR (DMSO-
d6)
d
(ppm): 2.38 (s, 3H, CH3), 4.20 (s, 1H, CH), 7.50e7.90 (m, 8H, Ar-
(ppm): 147.9, 135.4,
3.1.2.4. (E)-2-(2-cyanophenyl)-2-((4-methoxyphenyl)diazenyl)acet-
H), 8.38 (s, 1H, NH); 13C NMR (DMSO-d6)
d
amide (4d). Brown powder; yield (50%); m.p. > 300 ꢂC; IR (KBr):
n/
133.6, 132.2, 131.4, 129.5, 128.7, 122.5, 115.8, 114.9, 113.1, 53.5, 21.3;
MS (EI, 70 eV): m/z (%) ¼ 261 (Mþþ1, 7), 260 (Mþ, 31), 259 (48), 142
(8), 129 (11),116 (93),105 (62). Anal. Calcd. for C16H12N4 (260.29): C,
73.83; H, 4.65; N, 21.52%. Found: C, 73.62; H, 4.45; N, 21.42%.
cmꢀ1 ¼ 3390, 3329 (NH2), 2204 (CN), 1658 (CO), 1612 (C]N); 1H
NMR (DMSO-d6) d (ppm): 4.20 (s, 1H, CH), 3.90 (s, 3H, CH3), 7.30 (s,
2H, NH2), 7.50e8.50 (m, 8H, Ar-H); 13C NMR (DMSO-d6)
d (ppm):
172.7,157.6,143.2,139.4,133.5,132.7,130.3,128.3,123.6,115.8,114.8,
113.9, 71.7, 55.8; MS (EI, 70 eV): m/z (%) ¼ 294 (Mþ,10), 284 (100),142
(14),128 (15),115 (17),101 (12). Anal. Calcd. for C16H14N4O2 (294.31):
C, 65.30; H, 4.79; N, 19.04%. Found: C, 65.20; H, 4.59; N, 19.19%.
3.1.1.4. (E)-2-(Cyano((4-methoxyphenyl)diazenyl)methyl)benzoni-
trile (2d). Orange powder; yield (70%); m.p. 73e76 ꢂC; IR (KBr):
n/
cmꢀ1: 3432 (NH), 2250, 2226 (2CN), 1628 (C]N), 1550 (N]N); 1H
NMR (DMSO-d6) (ppm): 3.50 (s, 3H, OCH3), 4.20 (s, 1H, CH), 7.40e
7.90 (m, 8H, Ar-H), 8.15 (s, 1H, NH); 13C NMR (DMSO-d6)
(ppm):
d
3.1.2.5. (E)-2-(2-cyanophenyl)-2-((4-nitrophenyl)diazenyl)acet-
d
amide (4e). Brown powder; yield (60%); m.p. 133 ꢂC; IR (KBr):
n/
157.6,143.2, 133.6,132.2,131.4,129.5,128.7,123.6,115.8,114.9,114.8,
55.8, 53.5; MS (EI, 70 eV): m/z (%) ¼ 276 (Mþ, 11), 257 (14), 229 (2),
204 (3), 139 (2), 116 (100). Anal. Calcd. for C16H12N4O (276.29): C,
69.55; H, 4.38; N, 20.28%. Found: C, 69.35; H, 4.23; N, 20.18%.
cmꢀ1 ¼ 3351, 3221 (NH2), 2208 (CN), 1621 (CO), 1621 (C]N); 1H
NMR (DMSO-d6)
d (ppm): 4.10 (s, 1H, CH), 7.20 (s, 2H, NH2), 7.40e
8.50 (m, 8H, Ar-H); 13C NMR (DMSO-d6)
d (ppm): 172.7, 157,
144.9, 139.4, 133.5, 132.7, 130.3, 128.3, 124.4, 120.5, 115.8, 113.9, 71.7;
MS (EI, 70 eV): m/z (%) ¼ 309 (Mþ, 7), 297 (3), 284 (100), 270 (13),
256 (16), 229 (17), 142 (33), 128 (21), 114 (26), 102 (22). Anal. Calcd.
for C15H11N5O3 (309.28): C, 58.25; H, 3.58; N, 22.64%. Found: C,
58.20; H, 3.43; N, 22.42%.
3.1.1.5. (E)-2-(Cyano((4-nitrophenyl)diazenyl)methyl)benzonitrile
(2e). Brown powder; yield (70%); m.p. 66 ꢂC; IR (KBr):
n/
cmꢀ1 ¼ 3430 (NH), 2250, 2226 (2CN), 1630 (C]N), 1517 (N]N); 1H
NMR (DMSO-d6)
d (ppm): 4.18 (s, 1H, CH), 7.18e8.10 (m, 8H, Ar-H),
8.30 (s, 1H, NH); 13C NMR (DMSO-d6)
d
(ppm): 157, 144.9, 133.6,
3.1.3. General procedure for the synthesis of ethyl-2-aryl-5-imino-
4,5-dihydro-2H-pyrazolo[4,3-c]isoquinoline-3-carboxylate
derivatives (6aee)
A solution of each of 2aee (0.01 mol), in DMF (30 mL), and ethyl
chloroacetate (1.22 g, 0.01 mol) in presence of dry K2CO3 (1.38 g,
0.01 mol), was refluxed for 8 h. The reaction mixture was allowed to
132.2, 131.4, 129.5, 128.7, 124.4, 120.5, 115.8, 114.9, 113.1, 53.5; MS
(EI, 70 eV): m/z (%) ¼ 292 (Mþ þ 1, 2), 291 (Mþ, 10), 257 (17), 149
(13), 142 (11), 134 (13), 116 (100). Anal. Calcd. for C15H9N5O2
(291.26): C, 61.85; H, 3.11; N, 24.04%. Found: C, 61.65; H, 3.01; N,
24.19%.

428
A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
cool, poured onto crushed ice and neutralized by dilute HCl. The
obtained solid product was collected by filtration, washed, dried and
crystallized from ethanol to give compounds 6aee, respectively.
presence of anhydrous sodium acetate (0.82 g, 0.01 mol), and few
drops of acetic acid. The reaction mixture was refluxed in each case
for 8 h and left to cool and poured onto crushed ice. The obtained
solid product was collected by filtration, washed, dried and crys-
tallized from ethanol to give compounds 8aee, respectively.
3.1.3.1. Ethyl-5-imino-2-phenyl-4,5-dihydro-2H-pyrazolo[4,3-c]iso-
quinoline-3-carboxylate (6a). Brown powder; yield (50%); m.p.
200 ꢂC; IR (KBr):
n
d
/cmꢀ1 ¼ 3450 (2NH), 1700 (CO), 1630 (C]N); 1H
3.1.4.1. 2-Phenyl-2H-[1,2,3]triazolo[4,5-c]isoquinolin-5(4H)-imine
NMR (DMSO-d6)
(ppm): 1.30 (t, 3H, CH3), 4.00 (s, 1H, NH), 4.29 (q,
(8a). Brown crystals; yield (50%); m.p. > 300 ꢂC; IR (KBr):
n/
2H, CH2), 7.10 (s,1H, NH), 7.20e7.57 (m, 9H, Ar-H); 13C NMR (DMSO-
cmꢀ1 ¼ 3440 (2NH), 1628 (C]N); 1H NMR (DMSO-d6)
d (ppm): 4.00
d6)
d
(ppm): 160.6, 156.2, 141.1, 139.7, 134.6, 131.4, 130.6, 129.3,
(s,1H, NH), 7.10 (s,1H, NH), 7.40e8.09 (m, 9H, Ar-H); 13C NMR (DMSO-
128.9, 128.8, 126.6, 126.3, 126.2, 124.3, 123.9, 60.9, 14.1; MS (EI,
70 eV): m/z (%) ¼ 332 (Mþ, 100), 298 (26), 246 (100), 215 (19), 196
(28), 140 (30), 102 (38). Anal. Calcd. for C19H16N4O2 (332.36): C,
68.66; H, 4.85; N, 16.86%. Found: C, 68.46; H, 4.75; N, 16.71%.
d6) d (ppm): 156.2, 138.8, 132.8, 131.4, 130.6, 128.9, 128.8, 128.7, 126.6,
124.6,123.9,119.0; MS (EI, 70 eV): m/z (%) ¼ 261 (Mþ, 30), 246 (22), 97
(22), 85 (33),73 (22). Anal. Calcd. for C15H11N5 (261.28): C, 68.95; H,
4.24; N, 26.80%. Found: C, 68.72; H, 4.14; N, 26.65%.
3.1.3.2. Ethyl-2-(4-chlorophenyl)-5-imino-4,5-dihydro-2H-pyrazolo
3.1.4.2. 2-(4-Chlorophenyl)-2H-[1,2,3]triazolo[4,5-c]isoquinolin-
[4,3-c]isoquinoline-3-carboxylate (6b). Brown powder; yield (40%);
5(4H)-imine (8b). Brown crystals; yield (40%); m.p. > 300 ꢂC; IR
m.p. 180 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 3439 (2NH), 1705 (CO), 1628 (C]N);
(KBr):
(ppm): 4.10 (s, 1H, NH), 7.00 (s, 1H, NH), 7.40e7.60 (m, 8H, Ar-H);
13C NMR (DMSO-d6)
(ppm): 156.2, 136.9, 134.3, 132.8, 131.4, 130.6,
n
/cmꢀ1: 3439 (2NH), 1630 (C]N); 1H NMR (DMSO-d6)
1H NMR (DMSO-d6)
d
(ppm):1.25 (t, 3H, CH3), 4.00 (s,1H, NH), 4.20(q,
d
2H, CH2), 7.20 (s, 1H, NH), 7.40e7.80 (m, 8H, Ar-H); 13C NMR (DMSO-
d6) (ppm): 160.6, 156.2, 141.1, 137.8, 134.6, 131.8, 131.4, 130.6, 129.4,
d
d
128.9, 128.8, 126.6, 124.6, 123.9, 122.9; MS (EI, 70 eV): m/z (%) ¼ 297
(Mþþ2, 6), 295 (Mþ, 14), 235 (12), 210 (12), 144 (12), 122 (18), 90
(20). Anal. Calcd. for C15H10ClN5 (295.73): C, 60.92; H, 3.41; N,
23.68%. Found: C, 60.72; H, 3.31; N, 23.48%.
128.9, 128.8, 126.6, 124.3, 123.9, 119.8, 60.9, 14.1; MS (EI, 70 eV): m/z
(%) ¼ 368 (Mþ þ 2, 6), 366 (Mþ, 13), 348 (5), 272 (18), 246 (100), 190
(22), 140 (14), 109 (17). Anal. Calcd. for C19H15ClN4O2 (366.80): C,
62.21; H, 4.12; N, 15.27%. Found: C, 62.01; H, 4.02; N, 15.17%.
3.1.4.3. 2-p-Tolyl-2H-[1,2,3]triazolo[4,5-c]isoquinolin-5(4H)-imine
3.1.3.3. Ethyl-5-imino-2-p-tolyl-4,5-dihydro-2H-pyrazolo[4,3-c]iso-
(8c). Brown crystals; yield (60%); m.p. 100 ꢂC; IR (KBr):
n/
quinoline-3-carboxylate (6c). Brown powder; yield (50%); m.p.
cmꢀ1 ¼3450 (2NH), 1625 (C]N); 1H NMR (DMSO-d6)
d (ppm): 2.30
194 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 3435 (2NH), 2215 (CN), 1715 (CO), 1625
(s, 3H, CH3), 4.20 (s, 1H, NH), 7.10 (s, 1H, NH), 7.40e7.80 (m, 8H, Ar-
(C]N); 1H NMR (DMSO-d6)
d
(ppm): 1.30 (t, 3H, CH3), 2.34 (s, 3H,
H); 13C NMR (DMSO-d6)
d (ppm): 156.2, 138.4, 135.8, 132.8, 131.8,
CH3), 4.10 (q, 2H, CH2), 4.20 (s, 1H, NH), 7.10 (s, 1H, NH), 7.40e7.90
131.4, 130.6, 128.9, 128.8, 126.6, 124.6, 123.9, 121.3, 21.3; MS (EI,
70 eV): m/z (%) ¼ 275 (Mþ, 14), 260 (31), 247 (16), 231 (26),180 (22),
116 (18). Anal. Calcd. for C16H13N5 (275.31): C, 69.80; H, 4.76; N,
25.44. Found: C, 69.60; H, 4.65; N, 25.34.
(m, 8H, Ar-H); 13C NMR (DMSO-d6)
d
(ppm): 160.6, 156.2, 141.1,
136.7, 135.9, 134.6, 131.4, 130.6, 129.6, 128.8, 128.9, 126.6, 125.1,
124.3, 123.9, 60.9, 21.3, 14.1; MS (EI, 70 eV): m/z (%) ¼ 346 (Mþ, 19),
298 (50), 276 (34), 246 (100), 212 (31), 194 (56), 170 (44), 113 (56).
Anal. Calcd. for C20H18N4O2 (346.38): C, 69.35; H, 5.24; N, 16.17%.
Found: C, 69.20; H, 5.04; N, 16.07%.
3.1.4.4. 2-(4-Methoxyphenyl)-2H-[1,2,3]triazolo[4,5-c]isoquinolin-
5(4H)-imine (8d). Brown crystals; yield (50%); m.p. > 300 ꢂC; IR
(KBr):
n
/cmꢀ1 ¼ 3430 (2NH), 1628 (C]N); 1H NMR (DMSO-d6)
3.1.3.4. Ethyl-5-imino-2-(4-methoxyphenyl)-4,5-dihydro-2H-pyr-
d (ppm): 3.90 (s, 3H, OCH3), 4.12 (s, 1H, NH), 7.10 (s, 1H, NH), 7.50e
azolo[4,3-c]isoquinoline-3-carboxylate (6d). Brown powder; yield
8.00 (m, 8H, Ar-H); 13C NMR (DMSO-d6)
d (ppm): 160.6,156.2,132.8,
(60%); m.p. 178 ꢂC; IR (KBr):
n
d
/cmꢀ1 ¼ 3340 (2NH), 1710 (CO), 1629
131.4, 131.1, 130.6, 128.9, 128.8, 126.6, 124.6, 123.9, 114.3, 55.8; MS
(EI, 70 eV): m/z (%) ¼ 291 (Mþ, 19), 284 (34), 246 (72), 130 (56), 116
(44), 102 (69). Anal. Calcd. for C16H13N5O (291.31): C, 65.97; H, 4.50;
N, 24.04%. Found: C, 65.77; H, 4.40; N, 24.19%.
(C]N); 1H NMR (DMSO-d6)
(ppm): 1.20 (t, 3H, CH3), 3.80 (s, 3H,
OCH3), 4.00 (s, 1H, NH), 4.20 (q, 2H, CH2), 7.00 (s, 1H, NH), 7.20e7.90
(m, 8H, Ar-H); 13C NMR (DMSO-d6)
d
(ppm): 160.6, 158.1, 156.2,
141.1, 134.6, 132.0, 131.4, 130.6, 128.9, 128.8, 126.6, 124.3, 123.9,
114.9, 112.6, 60.9, 55.8, 14.1; MS (EI, 70 eV): m/z (%) ¼ 362 (Mþ, 11),
246 (100), 215 (15), 190 (28), 142 (19), 109 (31). Anal. Calcd. for
C20H18N4O3 (362.38): C, 66.29; H, 5.01; N, 15.46%. Found: C, 66.19;
H, 5.16; N, 15.26%.
3.1.4.5. 2-(4-Nitrophenyl)-2H-[1,2,3]triazolo[4,5-c]isoquinolin-
5(4H)-imine (8e). Brown crystals; yield (60%); m.p. > 300 ꢂC; IR
(KBr):
n
/cmꢀ1 ¼ 3444 (2NH), 1630 (C]N); 1H NMR (DMSO-d6)
d
(ppm): 4.20 (s, 1H, NH), 7.10 (s, 1H, NH), 7.50e8.00 (m, 8H, Ar-H);
13C NMR (DMSO-d6)
d (ppm): 156.2, 147.9, 144.9, 132.8, 131.4, 130.6,
3.1.3.5. Ethyl-5-imino-2-(4-nitrophenyl)-4,5-dihydro-2H-pyrazolo
128.9, 128.8, 126.6, 124.6, 123.9, 120.9; MS (EI, 70 eV): m/z (%) ¼ 306
(Mþ, 10), 278 (40), 246 (40), 198 (30), 170 (35), 150 (50), 114 (50).
Anal. Calcd. for C15H10N6O2 (306.28): C, 58.82; H, 3.29; N, 27.44%.
Found: C, 58.67; H, 3.19; N, 27.24%.
[4,3-c]isoquinoline-3-carboxylate (6e). Brown powder; yield (40%);
m.p. 173 ꢂC; IR (KBr): /cmꢀ1: 3439 (2NH), 1700 (CO), 1630 (C]N);
n
1H NMR (DMSO-d6)
(q, 2H, CH2), 7.00 (s, 1H, NH), 7.40e8.40 (m, 8H, Ar-H); 13C NMR
(DMSO-d6) (ppm): 160.6, 156.2, 145.8, 145.4, 141.1, 134.6, 131.4,
d (ppm): 1.30 (t, 3H, CH3), 4.10 (s, 1H, NH), 4.30
d
3.1.5. General procedure for the synthesis of 2-aryl-4-imino-1,2-
dihydronaphthalene-1,3,3(4H)-tricarbonitrile derivatives (11a, b)
and 4-amino-2-(3,4,5-trimethoxyphenyl)naphthalene-1,3-
dicarbonitrile (12)
3.1.5.1. Method A. A suspension of 1 (1.42 g, 0.01 mol) in ethanol
(30 mL) containing few drops of piperidine was treated with the
130.6, 128.9, 128.8, 126.6, 124.5, 124.3, 123.9, 118.4, 60.9, 14.1; MS
(EI, 70 eV): m/z (%) ¼ 377 (Mþ, 19), 277 (49), 247 (14), 212 (13), 142
(19),115 (14). Anal. Calcd. for C19H15N5O4 (377.35): C, 60.47; H, 4.01;
N, 18.56%. Found: C, 60.27; H, 4.11; N, 18.36%.
3.1.4. General procedure for the synthesis of 2-aryl-2H-[1,2,3]
triazolo[4,5-c]isoquinolin-5(4H)-imine derivatives 8aee
To a solution of each of 2aee (0.01 mol) in DMF (30 mL),
hydroxylamine hydrochloride (0.69 g, 0.01 mol) was added in
appropriate a-cinnamonitrile derivatives 9aec (0.01 mol). The
reaction mixture was refluxed for 6e8 h. The excess of solvent was
evaporated in vacuo then left to cool. The solid products formed was
filtered off, washed, dried and crystallized from ethanol.

A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
429
3.1.5.2. Method B. A suspension of 14aec (0.01 mol) in ethanol
(30 mL) was treated with malononitrile (0.66 g, 0.01 mol) and few
drops of triethylamine. The reaction mixture was refluxed for 3e4 h
and then evaporated and allowed to cool. The solid product was
collected by filtration, washed, dried and crystallized from ethanol.
The samples obtained by methods A and B were confirmed by
m.p., mixed m.p. and by comparison of their spectra.
118.8, 115.8, 114.2, 109.1, 99.5, 55.8; MS (EI, 70 eV): m/z (%) ¼ 260
(Mþ, 100), 245 (23), 216 (11), 190 (23), 163 (17), 113 (11). Anal. Calcd.
for C17H12N2O (260.29): C, 78.44; H, 4.65; N, 10.76%. Found: C,
78.34; H, 4.45; N, 10.56%.
3.1.6.3. 2-(1-Cyano-2-(3,4,5-trimethoxyphenyl)vinyl)benzonitrile
(14c). Brown crystals; yield (56%); m.p. 120 ꢂC; IR (KBr): /cmꢀ1
n :
2220, 2225 (2CN), 1610 (C]C); 1H NMR (DMSO-d6)
3.90 (m, 9H, 3OCH3), 7.00e7.50 (m, 8H, Ar-H), 8.00 (s, 1H, CH); 13
NMR (DMSO-d6) (ppm): 153.0, 147.5, 138.4, 132.9, 132.1, 129.5,
d (ppm): 3.70e
3.1.5.3. 2-(4-Chlorophenyl)-4-imino-1,2-dihydronaphthalene-
C
1,3,3(4H)-tricarbonitrile (11a). Yellow crystals; yield (70%); m.p.
d
140 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 3226 (NH), 2220, 2210, 2177 (3CN), 1613
128.6, 127.1, 118.8, 115.8, 109.1, 103.8, 99.5, 60.8, 56.1; MS (EI, 70 eV):
m/z (%) ¼ 320 (Mþ, 100), 306 (27), 277 (11), 234 (15), 191 (32), 164
(31). Anal. Calcd. for C19H16N2O3 (320.34): C, 71.24; H, 5.03; N,
8.74%. Found: C, 71.14; H, 5.18; N, 8.54%.
(C]C); 1H NMR (DMSO-d6)
d (ppm): 3.20 (d, 1H, CH), 4.15 (d, 1H,
CH), 5.10 (s, 1H, NH), 7.00e7.50 (m, 8H, Ar-H); 13C NMR (DMSO-d6)
(ppm): 164.6, 146.5, 139.3, 131.5, 131.1, 128.7, 128.5, 128.2, 127.5,
d
126.1, 124.6, 120.3, 116.2, 35.0, 33.0, 28.1; MS (EI, 70 eV): m/z
(%) ¼ 332 (Mþþ2, 22), 330 (Mþ, 50), 303 (28), 164 (28), 149 (100),
105 (86). Anal. Calcd. for C19H11ClN4 (330.77): C, 68.99; H, 3.35; N,
16.94%. Found: C, 68.79; H, 3.25; N, 16.74%.
3.2. Antimicrobial evaluation
Standard sterilized filter paper disks (5 mm diameter) impreg-
nated with a solution of the tested compound in DMF (1 mg/mL)
was placed on an agar plate seeded with the appropriate test
organism in triplicates. 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. oxy-
sporum and B. fabae fungal strains. Chloramphenicol, Cephalothin
and Cycloheximide were used as standard antibacterial and anti-
fungal agents, respectively [2,28]. DMF alone was used as control at
the same above-mentioned concentration. The plates were
3.1.5.4. 4-Imino-2-(4-methoxyphenyl)-1,2-dihydronaphthalene-
1,3,3(4H)-tricarbonitrile (11b). Brown crystals; yield (49%); m.p.
124e126 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 3343 (NH), 2277, 2250, 2226 (3CN),
1606 (C]C); 1H NMR (DMSO-d6)
d (ppm): 3.30 (d, 1H, CH), 3.80 (s,
3H, OCH3), 4.20 (d, 1H, CH), 5.10 (s, 1H, NH), 7.50e7.90 (m, 8H, Ar-
H); 13C NMR (DMSO-d6)
d
(ppm): 164.6, 157.8, 140.7, 139.3, 131.1,
128.7, 128.2, 127.1, 126.1, 124.6, 120.3, 116.2, 114.0, 55.8, 35.0, 33.0,
28.1; MS (EI, 70 eV): m/z (%) ¼ 324 (Mþ ꢀ 2,100), 299 (30), 284 (40),
102 (40), 101 (20), 98 (75). Anal. Calcd. for C20H14N4O (326.35): C,
73.61; H, 4.32; N, 17.17%. Found: C, 73.41; H, 4.22; N, 17.02%.
Table 3
3.1.5.5. 4-Amino-2-(3,4,5-trimethoxyphenyl)naphthalene-1,3-
Minimum bactericidal concentration (MBC,
compounds.
mg/mL) of some new synthesized
dicarbonitrile (12). Brown crystals; yield (56%); m.p. 124 ꢂC; IR
(KBr):
n
/cmꢀ1 ¼ 3343, 3300 (NH2), 2188, 2207 (2CN), 1610 (C]C);
Compound MBCa in
mg/mL
No.
1H NMR (DMSO-d6)
d (ppm): 3.70e3.90 (m, 9H, 3OCH3), 6.80e8.80
(m, 6H, Ar-H), 10.10 (s, 2H, NH2); 13C NMR (DMSO-d6)
d (ppm):
Bacteria
Fungi
153.1,152.1,139.5,138.1,133.5,130.8,130.2,126.8,123.9,120.5,119.8,
117.3, 113.9, 106.3, 99.4, 89.2, 60.8, 56.1; MS (EI, 70 eV): m/z
(%) ¼ 359 (Mþ, 24), 331 (24), 261 (39), 245 (27), 154 (67), 125 (52).
Anal. Calcd. for C21H17N3O3 (359.38): C, 70.18; H, 4.77; N, 11.69%.
Found: C, 70.08; H, 4.57; N, 11.59%.
Gram-positive
bacteria
Gram-negative
bacteria
B.
B.
E.
P.
F.
B.
subtilis thuringiensis coli
aeruginosa oxysporum fabae
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
1
2a
2b
2c
2d
2e
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
200
100
50
100
100
50
100
50
100
100
50
200
200
200
200
200
200
200
200
200
200
200
200
200
200
100
200
50
200
200
200
200
50
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
3.1.6. General procedure for the synthesis of (Z)-2-(2-aryl-1-
cyanovinyl)benzonitrile derivatives (14aec)
A suspension of 1 (1.42 g, 0.01 mol) in dioxan (30 mL) containing
a few drops of piperidine was treated with the appropriate
aromatic aldehydes 13aec (0.01 mol). The reaction mixture was
refluxed for 4e6 h. The solid product formed after addition of cold
water (20e30 mL) was collected by filtration, washed, dried and
recrystallized from ethanol to give compounds 14aec, respectively.
100
200
100
200
100
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
200
25
3.1.6.1. 2-(2-(4-Chlorophenyl)-1-cyanovinyl)benzonitrile
(14a).
Yellow crystals; yield (70%); m.p. 137 ꢂC; IR (KBr):
n
/cmꢀ1 ¼ 2210,
2220 (2CN), 1614 (C]C), 785 (CeCl); 1H NMR (DMSO-d6)
d (ppm):
8a
8b
8c
8d
7.50 (s, 1H, CH), 7.60e7.90 (m, 8H, Ar-H); 13C NMR (DMSO-d6)
d
(ppm): 147.5, 138.4, 133.5, 133.3, 132.9, 132.1, 129.0, 128.7, 128.6,
127.1, 118.8, 115.8, 109.1, 99.5; MS (EI, 70 eV): m/z (%) ¼ 266 (Mþþ2,
40), 264 (Mþ, 100), 229 (96), 201 (20), 175 (14), 139 (16), 116 (29),
100 (32). Anal. Calcd. for C16H9ClN2 (264.71): C, 72.60; H, 3.43; N,
10.58%. Found: C, 72.40; H, 3.33; N, 10.48%.
8e
11a
11b
12
14a
14b
14c
Ampicillin
3.1.6.2. 2-(1-Cyano-2-(4-methoxyphenyl)vinyl)benzonitrile
Yellow crystals; yield (42%); m.p. 128 ꢂC; IR (KBr): /cmꢀ1: 2225,
2227 (2CN), 1612 (C]C); 1H NMR (DMSO-d6)
(ppm): 3.80 (s, 3H,
OCH3), 7.00e7.50 (m, 8H, Ar-H), 8.00 (s, 1H, CH); 13C NMR (DMSO-
d6) (ppm): 159.8, 147.5, 138.4,132.9, 132.1, 130.2, 128.6, 127.5, 127.1,
(14b).
n
d
a
MBC: Minimum bactericidal concentration (the lowest concentration at which
no bacterial growth was observed).
b
d
NT: Not tested.

430
A.A. Fadda et al. / European Journal of Medicinal Chemistry 60 (2013) 421e430
incubated at 37 ꢂC for 24 h for bacteria and 48 days for fungi.
Compounds that showed significant growth inhibition zones
(>14 mm) using the twofold serial dilution technique, were further
evaluated for their minimum inhibitory concentration (MICs) [2,29].
[6] A.A. Fadda, S. Bondock, A.-G. Tarhoni, Arkivoc ii (2011) 272e279.
[7] A.A. Fadda, F.M. Abdelrazek, K.S. Mohamed, H.M. Gieth, H.A. Etman, Eur. J.
Chem. 1 (2010) 90e95.
[8] F. Abdelrazek, A.A. Fadda, A.N. Elsayed, Synth. Commun. 41 (2011) 1119e
1126.
[9] A.A. Fadda, M. Hammouda, E.M. Afsah, A.A. Hanash, Phosphorus Sulfur Silicon
Relat. Elem. 185 (2010) 433e446.
[10] E. Lohse, Pharmazie 41 (1986) 815. C.A. 106 (1987) 95471y.
[11] S. Pati, The Chemistry of the Hydrazo and Azoxy Groups. Part 1, John Wiley,
New York, 1975.
3.3. Minimum inhibitory concentration (MIC) and minimum
bacterial concentration (MBC) measurements [2]
[12] A.A. Fadda, S.A. Elagizy, Indian J. Text. Res. 14 (1989) 177.
[13] A.A. Fadda, F.A. Amer, A.M. El-Said, S.A. Elagizy, Indian J. Fib. Text. Res. 16
(1991) 226e231.
[14] A.A. Fadda, A.M. El-said, S.S. Elmorsy, E.M. Kandeel, S.A. Elagizy, Indian J. Fib.
Text. Res. 16 (1991) 159e165.
[15] M.A. Hanna, M.M. Girges, A.A. Fadda, J. Chem. Technol. Biotechnol. 55 (1992)
9e16.
[16] A.A. Fadda, H.A. Etman, F.A. Amer, K.S. Mohamed, J. Chem. Technol. Bio-
technol. 61 (1994) 343e349.
[17] A.A. Fadda, H.A. Etman, M.M. Ali, A. Fouda, Indian J. Fibre Text. Res. 20 (1995)
34e38.
[18] A.A. Fadda, H.A. Etman, M.M. Ali, A. Fouda, Indian J. Fibre Text. Res. 20 (1995)
108.
[19] A.A. Fadda, H.A. Etman, F.A. Amer, K.S. Mohamed, J. Chem. Technol. Bio-
technol. 62 (1995) 165e169.
[20] A.A. Fadda, H.A. Etman, S.I. El-Desoky, S. Bondock, Boliet. Chim. Farmac. 137
(1998) 191e194.
The minimum inhibitory and bactericidal concentrations (MICs
and MBCs) were determined using 96-well microtitre plates. The
microdilution susceptibility test in MüllereHinton Broth (Oxoid)
and Sabouraud Liquid Medium (Oxoid) was used for the determi-
nation of antibacterial and antifungal activity, respectively. Stock
solutions of the tested compounds, Chloramphenicol, Cephalothin,
Cycloheximide and Ampicillin were prepared in DMF at concen-
tration of 1000
tions of (500, 250, 3.125
m
g/mL followed by twofold dilution at concentra-
g/mL). The microorganism suspensions at
m
106 CFU/mL (Colony Forming U/mL) concentrations were inocu-
lated to the corresponding wells. Plates were incubated at 36 ꢂC for
24e48 h and the minimum inhibitory concentration (MICs) were
determined. Control experiments were also done. The MIC and
[21] A.A. Fadda, H.M. Refat, M.E.A. Zaki, E. Mounier, Heterocycl. Commun. 12
(2006) 47e52.
MBC values were expressed in mg/mL as shown in Tables 2 and 3.
[22] A.A. Fadda, M. Hammouda, M. Mashaly, Arch. Pharm. Res. 18 (1995) 213e214.
[23] F. Ramirez, A.F. Kirby, J. Am. Chem. Soc. 76 (1954) 1037e1044.
[24] L.J. Bellamy, Infrared Spectra of Complex Molecules, Methuen, London, 1954,
pp. 263e272.
[25] (a) A.E. Gilman, E.S. Stern, An Introduction to Electronic Absorption Spec-
troscopy in Organic Chemistry, second ed., Edward Arnold Publisher Ltd,
London, 1957, p. 302;
Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.11.017.
(b) A.E. Gilman, E.S. Stern, An Introduction to Electronic Absorption Spec-
troscopy in Organic Chemistry, second ed., Edward Arnold Publisher Ltd,
London, 1960, p. 271.
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