Synthesis, spectral characterization and in vitro antimicrobial activity of some new azopyridine derivatives

Article abstract of DOI:10.1016/j.saa.2011.12.054

A series of arylpicolino and/or isonicotinohydrazonyl cyanide 2a-d and 4a-f were prepared by coupling the approprite aryl diazonium salt with 2-cyanomethyl and/or 4-cyanomethyl-pyridine, respectively. These compounds were characterized by analytical and spectral analyses and screened for their antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and antifungal activity. Among the synthesized compounds, N′-(4-phenyldiazenyl) phenylisonicotinohydrazonyl cyanide 4f showed a significant activity toward both Gram-positive, Gram-negative bacteria and exhibit the most potent in vitro antifungal with MIC's (625 μg/mL) against Aspergillus nieger.

Full text of DOI:10.1016/j.saa.2011.12.054

Spectrochimica Acta Part A 89 (2012) 123–128
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, spectral characterization and in vitro antimicrobial activity of some
new azopyridine derivatives
Hanaa Abuo-Melhaa, A.A. Faddab,∗
a Faculty of Education of Girls, King Khaled University, Abha, Saudi Arabia
b Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhoria Street, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2011
Received in revised form 8 December 2011
Accepted 21 December 2011
A series of arylpicolino and/or isonicotinohydrazonyl cyanide 2a–d and 4a–f were prepared by coupling
the approprite aryl diazonium salt with 2-cyanomethyl and/or 4-cyanomethyl-pyridine, respectively.
These compounds were characterized by analytical and spectral analyses and screened for their antibac-
terial activity against Gram-positive bacteria, Gram-negative bacteria and antifungal activity. Among the
synthesized compounds, Nꢀ-(4-phenyldiazenyl)phenylisonicotinohydrazonyl cyanide 4f showed a sig-
nificant activity toward both Gram-positive, Gram-negative bacteria and exhibit the most potent in vitro
antifungal with MIC’s (625 ␮g/mL) against Aspergillus nieger.
Keywords:
Arylpicoline
Isonicotinohydrazonyl cyanide
Gram-positive and Gram-negative bacteria
Antifungal activity
© 2011 Elsevier B.V. All rights reserved.
Minimum inhibitory concentration (MIC)
1. Introduction
to form the corresponding (Z)-Nꢀ-arylpicolinohydrazonyl cyanide
(2) and its isonicotino-hydrazonyl derivatives 4 in overall good
yields (Fig. 1). However, no details of the dyeing behavior or their
antimicrobial activity are reported.
The possible tautomeric forms of arylazo derivatives could be
set out as outlined in Fig. 2.
There are referred to as the CH azo form (A), the NH azo form (B)
and the cyanohydrazone form (C). The presently available data indi-
cate that the tautomeric structure 2 or 4 is the chelated hydrazone
form (C). It was therefore considered worthwhile preparing arylazo
compounds containing the pyridine ring to evaluate their biologi-
cal activities. In this investigation, ten Nꢀ-arylpicolinohydrazonyl
cyanides 2a–d and Nꢀ-arylisonicotinohydrazonyl cyanides 4a–f
were prepared by coupling of 2 and/or 4-cyanomethylpyridines 1
and/or 3 with the appropriate diazonium salt in ethanol containing
sodium acetate. The newly prepared dyes were characterized by
elemental analyses, as well as by spectral analysis.
The rich chemistry of azo compounds is associated with
several important biological reactions such as protein synthe-
sis, carcinogenesis, azo reduction monoamine oxidase inhibition
mutagenic, immunochemical affinity labeling, nitrogen fixa-
tion, important medical and industrial uses [1,2]. Fadda et al.
[3–19] published
a series of papers to throw light on the
chemistry of azo dyes. In our laboratory, some (E)-2-(aryl
diazenyl)-2-(pyridine-2-yl)acetonitrile and its isomer 2-(pyridine-
4-yl)acetonitrile derivatives have been prepared and characterized
by elemental and spectral analyses. The electronic absorption spec-
tra of these aryl azo derivatives were studied.
In a sequel of continuation, the present work is undertaken to
study the structural chemistry of these aryl azo derivatives through
(i) discussing IR, 1H NMR, mass and UV spectra to support the tau-
tomeric behavior of these compounds. (ii) These compounds were
assayed against different Gram-positive bacteria, Gram-negative
bacteria and antifungal activity in order to test their biological
activity.
On the basis of the IR spectra of arylhydrazones 2 and 4, it
was possible to assign the absorption bands of the diazonium
coupling products 2 and 4. Each of the compounds investigated
possessed a weak and broad band in the region 3100–3300 cm−1
.
2. Results and discussion
This was assigned to NH stretching of the hydrazone moiety. The
large shift and broadening of this band, as reported by Ramirez and
Kirby [15], for simple hydrazones, can result only from intramolec-
ular hydrogen bonding as in (C). The fact that the compounds
2 and 4 show evidence for intramolecular hydrogen bonding is
in favor of the hydrazone structure. The IR spectra of all com-
2.1. Chemistry
In a sodium acetate buffered solution of ethanol 2- and 4-
cyanomethylpyridine (1) and (3) reacts with diazotized aryl amines
pounds showed absorption bands in the region 2220–2222 cm−1
.
∗
These bands assigned to C N vibration. So, on the basis of these
data, structure (B) can be ruled out. The IR spectra of almost all
Corresponding author. Tel.: +20 506432235; fax: +20 502246781.
E-mail address: afadda2@yahoo.com (A.A. Fadda).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.12.054

124
H. Abuo-Melha, A.A. Fadda / Spectrochimica Acta Part A 89 (2012) 123–128
Ar
HN
N
NC
NC
CN
N
CN
N
N
N
NH
Ar
1
3
N
4a-f
2a-d
2a, Ar= C6H5
4a, Ar= C6H5
2b, Ar= C6H4-CH3-p
2c, Ar= C6H4-OCH3-p
2d, Ar= C6H4-NO2-p
4b, Ar= C6H4-CH3-p
4c, Ar= C6H4-OCH3-p
4d, Ar= C6H4-NO2-p
4e, Ar= C6H4-Cl-p
4f, Ar= C6H4-N=N-C6H5 -p
Fig. 1. Structures of the investigated arylpicolino and isonicotinohydrazonyl cyanide derivatives 2a–d and 4a–f.
compounds showed absorption in the region 1400–1450 cm−1
These bands, however, cannot be assigned to N N vibration accord-
ing to LeFèvre [16]. Thus, the absence of bands characteristic for the
.
compounds generally show two absorption bands at 410–400 and
290–300 nm corresponding to n–␲* and ␲–␲* transitions, respec-
tively [17]. The monophenylhydrazones, on the other hand, show
three intense bands in the 220–228, 250–280 and 330–390 nm
regions [18].
N
N group might be taken as an indication that structure (A) is not
present in the series studied.
The UV spectra of compounds 2 and 4 investigated cannot be
interpreted in terms of the azo structure of the types (A and B)
but evidently they appear compatible with the indicated hydra-
zone structure (C). The presence of diazonium coupling products
of 1 and 3, apparently exclusively, in the hydrazone form can be
explained by a smaller degree of resonance stabilization of the
azo forms (A and B). The six-membered hydrogen-bonded ring
structure in the hydrazone form, as in (C), would also undoubt-
edly enhance its relative stability [21], since the color of an azodye
depends on the structure of the diazotizable amines. It is clear that,
these dyes exhibit three absorption bands, of these, the medium
and high wavelength bands seem to be affected by the nature of
the polar substituent in the arylazo group and the low wavelength
band is unaffected. Table 1 shows that, both electron withdraw-
ing and electron donating groups cause the absorption to occur at
higher wavelengths A “C N” linkage was reported to have prop-
erties especially analogous to those of an ethylenic linkage [22].
Hence, an electronic effect of a substituent on the aromatic nucleus
of the arylazo moiety will be transmitted to the whole conjugate
system through ␲–P conjugation, exerting a considerable influence
upon the conjugation bands A and B. Table 1 also shows that, the
presence of electron donating or electron withdrawing groups has
not brought about any marked increase or decrease in ꢀmax in the
visible region and log ε has nearly remained constant. This does
point towards the hydrazone structure (C) where the resonance
in the diazo components is minimal, owing to steric factors. The
1H NMR spectra of arylazo derivatives 2a–d and 4a–f showed a
Since, it has been deduced that compounds 2a–d and 4a–f
exist as arylhydrazone (C), a band due to C N would be expected
in the double bond region. Bellamy [20], quoted a range of
1690–1640 cm−1 for the C N stretching vibration falling slightly
to 1680–1630 cm−1 for ␣,␤-unsaturated compounds. The IR spec-
tral data revealed that none of the compounds under investigation
exhibited absorption bands above 1600 cm−1. However, each com-
pound showed strong absorption between the aromatic 1500 cm−1
band and NH deformation band near 1550 cm−1, this band is rela-
tively strong and in some cases, it could not be resolved from the
1500 cm−1 band. Such a band was not observed in the spectra of
the corresponding 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 nearly prepared compounds may be attributed
to its conjugation with the nitrile group.
The UV spectra of the diazonium coupling products of 1 and
3 provide additional evidence that such compounds have the
hydrazone structure (C) rather than (A) or (B). Most of the dyes
show three main absorption bands in the region 240–390 nm
(2445–2455 nm “␲–␲*”, 2710–2950 “n–␲*” related to pyridine
moiety and 3445–390 “n–␲*” assigned to hydrazo center). These
bands are referred to as A, B and C, starting from the long wave-
length side. The data indicate that absorption maxima in the
solvents examined almost coincide. This suggests that such a com-
pound is not in a tautomeric equilibrium, but exists in one form.
The relatively small differences in ꢀmax may be due to the general
solvent effect [18].
It has been reported that, the UV spectra of monophenylazo
compounds differ from those of monophenylhydrazones The azo
Table 1
UV absorption bands of the newly synthesized compounds 2a–d and 4a–f.
Dye
ꢀmax (nm)a
C
2a
2b
2c
2d
4a
4b
4c
4d
4e
4f
395 (45)
385 (45)
390 (46)
390 (46)
390 (46)
395 (47)
385 (46)
390 (47)
385 (46)
390 (46)
275 (41)
280 (41)
275 (42)
285 (40)
285 (42)
275 (42)
275 (41)
285 (41)
280 (41)
280 (41)
248 (43)
248 (43)
248 (42)
245 (41)
245 (42)
248 (41)
248 (42)
245 (42)
248 (42)
245 (41)
C
C
N
N
H
N
H
N
N
N
N
N
N
Ar
N
N
N
Ar
Ar
(A)
(B)
(C)
Fig. 2. The possible tautomeric forms of 2-, 4-[substituted-diazenyl]-2-[pyridin-2-
yl](acetonitrile/ethenimine) and (Z)-Nꢀ-substituted-(picolino/nicotino)hydrazonoyl
cyanide derivatives 2 and 4.
aValues in parentheses show log ε.

H. Abuo-Melha, A.A. Fadda / Spectrochimica Acta Part A 89 (2012) 123–128
125
Table 2
Minimal inhibitory concentration (MIC, ␮g/mL) and inhibition zone (mm) of the newly synthesized compounds.
Compound no
MICa in ␮g/mL and inhibition zone (mm)
Bacteria
Fungi
Gram(+) bacteria
Gram(−) bacteria
B. subtilis
S. aureus
E. coli
P. aeruginosa
A. nieger
1
2a
2b
2c
2d
3
4a
4b
4c
4d
4e
4f
100 (15)
25 (20)
50 (15)
25 (30)
3125 (38)
50 (25)
100 (14)
625 (38)
50 (18)
25 (21)
625 (34)
50 (30)
625 (41)
625 (39)
625 (40)
625 (41)
625 (42)
625 (44)
3125 (44)
625 (41)
NT
100 (16)
100 (14)
625 (38)
50 (24)
50 (23)
50 (18)
50 (19)
50 (16)
50 (18)
125 (37)
25 (35)
625 (38)
625 (39)
625 (38)
NT
100 (18)
100 (15)
100 (18)
100 (15)
125 (32)
100 (15)
50 (20)
25 (35)
50 (19)
25 (33)
25 (39)
625 (38)
625 (38)
625 (38)
NT
100 (18)
100 (15)
100 (18)
100 (14)
25 (25)
100 (16)
100 (15)
100 (18)
50 (18)
125 (31)
125 (35)
625 (37)
NT
125 (38)
25 (30)
125 (40)
625 (44)
3125 (45)
3125 (43)
3125 (42)
625 (40)
NT
Chloramphenicol
Cephalothin
Cycloheximide
NT
3125 (44)
MIC, minimal inhibitory concentration values; NT, not tested.
singlet signal at ı 88 ppm corresponding to NH proton. The down-
field shift of the former occurs because the nearby azo group has
a deshielding effect [23]. Compound 2a–d showed a doublet at ı
865 ppm corresponding to ␣-proton (C-6) in pyridine ring, while
compounds 4a–f showed two doublets (AB system) due to ␣- and
␤-protons of the pyridine ring at ␦ 855 and 721 ppm, respectively.
Also, the 1H NMR spectra of arylazo derivatives revealed a multiplet
at ı 691–745 ppm corresponding to the aromatic protons.
Mass spectra of the newly synthesized arylazo derivatives, in
general, confirm the proposed formula by detecting the following
peaks. For compound 2a, the observed peak at m/z 222 (C13H10N4,
calculated atomic mass 22,209) represents the molecular ion peak
with 25% abundance, respectively The other molecular ion peaks
(221, 195, 92, 91, 90, and 78) appeared in the mass spectra are
attributed to the fragmentation of molecule obtained from the rup-
ture of different bonds inside the molecule.
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 4-cyanomethylpyridine exhibited better antibacterial poten-
tials than members of the 2-cyanomethylpyridine.
Regarding the activity of the 2a–d against Gram-positive bac-
teria, the results revealed that compound 2d exhibited divergent
antibacterial activity against the tested organisms. In this view,
compound 2d was equipotent to chloramphenicol in inhibiting the
growth of B. subtilis (MIC 3125 ␮g/mL), while its activity was 50%
lower than chloramphenicol against S. aureus. Compounds 2a and
2d showed 50% of the activity of chloramphenicol (MIC 625 ␮g/mL).
On the other hand, compounds 1, 2b and 2c exhibited weak to
moderate growth inhibitory activity against Gram-positive bac-
teria revealed from their MIC values (25–100 ␮g/mL). Among the
series of compounds 4a–f, compounds 4a, 4c, 4d, 4e and 4f showed
relatively good growth inhibitory profiles against B. subtilis (MIC
3125–125 ␮g/mL) which were from equipotent to 25% of the activ-
ity of chloramphenicol and 50% of cephalothin against the same
organism (4c).
In general, from the data obtained for all compounds we con-
clude that the molecular weight was in good agreement with the
calculated molecular weight of the investigated compounds.
Moreover, distinctive anti-Gram positive profile was displayed
by compounds 4e, f where it proved to be equipotent as chloram-
phenicol against both B. subtilis (MIC 3125 ␮g/mL) and S. aureus
(MIC 3125 ␮g/mL). Concerning the antibacterial activity of the com-
pounds 2c, 3 and 4a–c revealed weak growth inhibitory against the
tested Gram-positive bacteria (MIC 50 ␮g/mL). On the other hand,
compound 4f showed equipotent activity as chloramphenicol and
cephalothin (MIC 625 ␮g/mL) against E. coli and P. aeruginosa.
Regarding the activity of 2a–d and 4a–f against fungal strains,
the results revealed that compound 4f was 50% lower than cyclo-
heximide in inhibitory the growth of A. nieger (MIC 3125 ␮g/mL),
while the reactivity of compounds 4d and 4e was 25% lower than
cycloheximide against A. nieger (MIC 125 ␮g/mL).
The results of antimicrobial screening demonstrated the follow-
ing assumptions about the structural activity relationships (SAR’s):
(1) it is interesting to point out that all compounds having electron
withdrawing groups such as NO2, Cl, aminoazobenzene recorded
higher antibacterial activity. (2) The substitution at position 4 of
the pyridine ring produced a higher antimicrobial activity. (3)
Conversion of compounds 1 and 3 to their corresponding arylhy-
drazone derivatives 2a–d and 4a–f enhanced also antimicrobial
activity. (4) The tested compounds were more active against Gram-
positive than Gram-negative bacteria, it may be concluded that the
2.2. Pharmacology
2.2.1. Antimicrobial evaluation
Ten of the newly synthesized target compounds besides the
starting compounds were evaluated for their in vitro antibacte-
rial activity against Gram positive bacteria (Bacillus subtilis and
Staphylococcus aureus), Gram negative bacteria (Escherichia coli
and Pseudomonas aeruginosa) and fungi (Aspergillus nieger). Agar-
diffusion method 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 if inhi-
bition zone (IZ) of bacterial and fungal growth around the disks in
mm, the minimum inhibitory concentration (MIC) measurement
was determined for compounds showed significant growth inhi-
bition zones (>14 mm) using two fold serial dilution method [24].
The MIC (␮g/mL) and inhibition zone diameters values are recorded
in Table 2. The inhibition zone diameters values cited in Table 2
between brackets are attributed to the tested original concentra-
tion (1 mg/mL) as preliminary test. The results depicted in Table 2
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 fungal strains.

126
H. Abuo-Melha, A.A. Fadda / Spectrochimica Acta Part A 89 (2012) 123–128
antimicrobial activity of the compounds is related to cell wall struc-
ture of the bacteria. It is possible because the cell wall is essential
to the survival of bacteria and some antibiotics are able to kill
bacteria by inhibiting a step in the synthesis of peptidoglycan.
Gram-positive bacteria possess a thick cell wall containing many
layers of peptidoglycan and teichoic acid, but in contrast, Gram
negative bacteria have a relatively thin cell wall consisting of a few
layers of peptidoglycan surrounded by a second lipid membrane
containing lipopolysaccharides and lipoproteins. These differences
in cell wall structure can produce differences in antibacterial sus-
ceptibility and some antibiotics can kill only Gram positive bacteria
and is infective against Gram negative bacteria pathogen [25]. (5)
The importance of such work lies in the possibility that the new
compounds might be more effective drugs against bacteria for
which a through investigation regarding the structural activity rela-
tionships, toxicity and in their biological effects which could be
helpful in designing more potent antibacterial agents for therapeu-
tic use.
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 crude product was filtered, dried and recrystallized
from ethanol to give N-arylpicolino and/or isonicotinohydrazonyl
cyanide derivatives 2a–d and 4a–f (Table 2).
4.1.1. (Z)-Nꢀ-Phenylpicolinohydrazonyl cyanide (2a)
This compound was prepared from coupling of benzene diazo-
nium chloride with 2-cyanomethylpyridine.
Yield (70%); brown crystals; mp 160 ◦C; IR (KBr): ꢁ/cm−1 = 3310
(NH), 1550 (C N); 1H NMR(DMSO-d6) ı (ppm): 68–73 (m, 5H,
Ar–H), 76–796 (m, 3H, Py–H), 86 (d, 1H, C-6,-Py), 88 (s, 1H, NH); MS
(EI, 70 eV) m/z (%) = 222 (M+, 286), 195 (96), 92 (44), 91 (100), 90
(40), 78 (19), 76 (38), 65 (34), 57 (30), 50 (57), 30 (51), 49 (30). Anal
Calcd for C13H10N4 (22,225): C, 70.26; H, 454; N, 2521%. Found: C,
70.11; H, 441; N, 2510%.
4.1.2. (Z)-Nꢀ-p-Tolylpicolinohydrazonyl cyanide (2b)
This compound was prepared from coupling of p-tolyl diazo-
nium chloride with 2-cyanomethylpyridine.
3. Conclusion
In conclusion, the objective of the present study was to syn-
thesize and investigate the antimicrobial activities of some new
functionalized 2- and 4-pyridyl arylhydrazone derivatives with
the hope of discovering new structure leads serving as potent
antimicrobial agents. Our aim has been verified by the synthesis of
different groups of structure hybrids comprising basically the pyri-
dine moiety attached at 2- and 4-positions. The obtained results
clearly revealed that compounds derived from 4-pyridyl deriva-
tives exhibited better antimicrobial activity than their 2-isomers.
Yield (75%); brown crystals; mp 139 ◦C; IR (KBr): ꢁ/cm−1 = 3300
(NH), 1552 (C N); 1H NMR(DMSO-d6) ı (ppm): 234 (s, 3H, CH3), 65
(d, 2H, Ar–H), 696 (d, 2H, Ar–H), 764–796 (m, 3H, Py–H), 861 (d, 1H,
C-6,-Py), 88 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 236 (M+, 557), 207
(18), 182 (4), 180 (5), 117 (23), 90 (100), 78 (17), 77 (21), 76 (24),
65 (37), 63 (16), 51 (20), 50 (12). Anal Calcd for C14H12N4 (23,627):
C, 7117; H, 512; N, 2371%. Found: C, 710; H, 510; N, 2368%.
4.1.3. (Z)-Nꢀ-(4-Methoxyphenyl)picolinohydrazonyl cyanide (2c)
This compound was prepared from coupling of 4-
methoxyphenyl diazonium chloride with 2-cyanomethylpyridine.
Yield (80%); brown crystals; mp 154 ◦C; IR (KBr): ꢁ/cm−1 = 3320
(NH), 1552 (C N); 1H NMR(DMSO-d6) ı (ppm): 284 (s, 3H, OCH3),
703 (d, 2H, Ar–H), 753 (d, 2H, Ar–H), 761–793 (m, 3H, Py–H), 866
(d, 1H, C-6,-Py); MS (EI, 70 eV) m/z (%) = 252 (M+, 50), 236 (52), 235
(26), 221 (18), 220 (20), 207 (20), 149 (23), 121 (20), 118 (21), 117
(21), 106 (36), 104 (23), 92 (23), 91 (100), 89 (32), 80 (25). Anal
Calcd for C14H12N4O (25,227): C, 6665; H, 479; N, 2221%. Found: C,
6661; H, 473; N, 2220%.
4. Experimental
All melting points are recorded on Gallenkamp electrothermal
melting point apparatus. The IR spectra were recorded for KBr disc
on a Mattson 5000 FTIR spectrophotometer. The 1H NMR spectra
were measured on a Bruker AC 300 (300 MHz) in DMSO-d6 as sol-
vent, using TMS as an internal standard, and chemical shifts are
expressed as ıppm. The mass spectra were determined on Finnigan
Incos 500 (70 ev). Elemental analyses were carried out at the Micro-
analytical Unit of the Faculty of Science, Cairo University, Giza,
Egypt.
4.1.4. (Z)-Nꢀ-(4-Nitrophenyl)picolinohydrazonyl cyanide (2d)
This compound was prepared from coupling of 4-nitrophenyl
diazonium chloride with 2-cyanomethylpyridine.
4.1. Synthesis of arylhydrazonyl cyanide 2a–d and 4a–f
General procedure: a well stirred solution of the base aromatic
amine (0.1 mol) in 2 N hydrochloric acid (125 mL) was cooled in
an ice-salt bath and diazotized with 1 N sodium nitrite solution
(100 mL). The mixture was then tested for complete diazotiza-
tion 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 hydrochlo-
ride are added until the nitrite test is nearly negative. The above
cold diazonium solution was added slowly to a well stirred solution
to cyanomethyl pyridine (0.1 mol) in ethanol (150 mL) containing
10% sodium acetate solution (50 mL) and the mixture was cooled
in an ice-salt bath. After the addition of the diazonium salt solu-
tion, 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
Yield (85%); yellowish brown crystals; mp 180 ◦C; IR
(KBr): ꢁ/cm−1 = 3310 (NH), 1555 (C N), 1530, 1350 (NO2); 1H
NMR(DMSO-d6) ı (ppm): 72 (d, 2H, Ar–H), 764–795 (m, 3H, Py–H),
80 (d, 2H, Ar–H), 861 (d, 1H, C-6-Py), 882 (s, 1H, NH); MS (EI, 70 eV)
m/z (%) = 267 (M+, 91), 266 (72), 241 (22), 240 (27), 136 (45), 123
(18), 122 (22), 117 (18), 104 (45), 91 (60), 90 (77), 89 (31), 80 (45),
78 (41), 65 (36), 64 (91), 63 (100), 62 (45), 58 (36), 57 (54), 51 (60),
50 (54). Anal Calcd for C13H9N5O2 (26,724): C, 5843; H, 339; N,
2621%. Found: C, 5840; H, 336; N, 2620%.
4.1.5. (E)-Nꢀ-Phenylisopicolinohydrazonyl cyanide (4a)
This compound was prepared from coupling of benzene diazo-
nium chloride with 4-cyanomethylpyridine.
Yield (76%); yellow crystals; mp 179 ◦C; IR (KBr): ꢁ/cm−1 = 3300
(NH), 1551 (C N); 1H NMR(DMSO-d6) ı (ppm): 681–735 (m, 5H,
Ar–H), 798 (d, 2H, ␤-Py–H), 866 (d, 2H, ␣-Py–H), 882 (s, 1H, NH);
MS (EI, 70 eV) m/z (%) = 222 (M+, 54), 92 (78), 91 (100), 89 (76), 65
(31). Anal Calcd for C13H10N4 (22,225): C, 70.26; H, 454; N, 2521%.
Found: C, 70.19; H, 449; N, 2520%.

H. Abuo-Melha, A.A. Fadda / Spectrochimica Acta Part A 89 (2012) 123–128
127
4.1.6. (E)-Nꢀ-(p-Tolyl)isopicolinohydrazonyl cyanide (4b)
This compound was prepared from coupling of p-tolyl diazo-
nium chloride with 4-cyanomethylpyridine.
the appropriate test organism in triplicates. Standard conditions
of 106 CFU/mL (Colony Forming U/mL) and 104 CFU/mL were used
for antibacterial and antifungal assay, respectively Pyrex glass petri
dishes (9 cm in diameter) were used and two disks of filter paper
were inoculated in each plate The utilized test organisms were B.
subtilis and S. aureus 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 A.
nieger fungal strains chloramphenicol, cephalothin and cyclohex-
imide were used as standard antibacterial and antifungal agents,
respectively. DMF alone was used as control at the same above
mentioned concentration and due this there was no visible 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 twofold serial dilu-
tion technique, were further evaluated for their minimal inhibitory
concentrations (MICs).
Yield (76%); yellow crystals; mp 167 ◦C; IR (KBr): ꢁ/cm−1 = 3280
(NH), 1553 (C N); 1H NMR(DMSO-d6) ı (ppm): 231 (s, 3H, CH3),
651 (d, 2H, Ar–H), 699 (d, 2H, Ar–H), 796 (d, 2H, ␤-Py–H), 865 (d, 2H,
␣-Py–H), 89 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 236 (M+, 78), 207
(40), 117 (38), 91 (100), 90 (50), 65 (51). Anal Calcd for C14H12N4
(23,627): C, 7117; H, 512; N, 2371%. Found: C, 710; H, 510; N, 2369%.
4.1.7. (E)-Nꢀ-(p-Tolyl)isopicolinohydrazonyl cyanide (4c)
This compound was prepared from coupling of p-
methoxyphenyl diazonium chloride with 4-cyanomethylpyridine.
Yield (81%); yellow crystals; mp 155 ◦C; IR (KBr): ꢁ/cm−1 = 3310
(NH), 1556 (C N); 1H NMR(DMSO-d6) ı (ppm): 391 (s, 3H, OCH3),
701 (d, 2H, Ar–H), 753 (d, 2H, Ar–H), 795 (d, 2H, ␤-Py–H), 863 (d,
2H, ␣-Py–H), 879 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 252 (M+, 50),
236 (65), 207 (38), 149 (30), 107 (32), 91 (100), 52 (50). Anal Calcd
for C14H12N4O (25,227): C, 6665; H, 479; N, 2221%. Found: C, 6662;
H, 477; N, 2220%.
4.3. Minimal inhibitory concentration (MIC) measurement
The microdilution susceptibility test in Muller–Hinton Broth
(Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for
the determination of antibacterial and antifungal activity, respec-
tively. Stock solutions of the tested compounds, chloramphenicol,
cephalothin and cycloheximide were prepared in DMF at con-
centration of 1000 ␮g/mL. Each stock solution was diluted with
standard method broth (Difco) to prepare serial twofold dilutions in
the range of 500–3125 ␮g/mL 10 mL of the broth containing about
106 CFU/mL of test bacteria was added to each well of 96-well
microtiter plate. 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 minimal inhibitory concentrations (MICs) 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.
4.1.8. (E)-Nꢀ-(p-Nitrophenyl)isopicolinohydrazonyl cyanide (4d)
This compound was prepared from coupling of p-nitrophenyl
diazonium chloride with 4-cyanomethylpyridine.
Yield (80%); yellowish brown crystals; mp 289 ◦C; IR (KBr):
ꢁ/cm−1 = 3315 (NH), 1550 (C N); 1H NMR(DMSO-d6) ı (ppm): 791
(d, 2H, Ar–H), 798–701 (m, 4H, Ar–H + 2␤-Py–H), 865 (d, 2H, ␣-
Py–H), 885 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 267 (M+, 54), 266
(50), 122 (30), 117 (20), 104 (50), 91 (66), 90 (80), 65 (100). Anal
Calcd for C13H9N5O2 (26,724): C, 5843; H, 339; N, 2621%. Found: C,
5839; H, 335; N, 2620%.
4.1.9. (E)-Nꢀ-(p-Chlorophenyl)isopicolinohydrazonyl cyanide (4e)
This compound was prepared from coupling of p-chlorophenyl
diazonium chloride with 4-cyanomethylpyridine.
Yield (70%); pale yellow crystals; mp 212 ◦C; IR (KBr):
ꢁ/cm−1 = 3310 (NH), 1555 (C N); 1H NMR(DMSO-d6) ı (ppm):
710–726 (m, 4H, Ar–H), 801 (d, 2H, ␤-Py–H), 869 (d, 2H, ␣-Py–H),
889 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 258 (M++2, 15), 257 (M++1,
13), 256 (M+, 78), 220 (60), 126 (413), 125 (100), 124 (13), 111 (17),
101 (13), 100 (7), 99 (45), 75 (21), 63 (27), 51 (33), 50 (15). Anal
Calcd for C13H9ClN4 (25,669): C, 60.83; H, 353; N, 1381%. Found: C,
60.77; H, 351; N, 1379%.
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(E)-Nꢀ-(4-((E)-Phenyldiazenyl)phenyl)isonicotinohydrazonoyl
cyanide (4f)
This compound was prepared from coupling of p-amino-
azobenzene diazonium chloride with 4-cyanomethylpyridine.
Yield (85%); brown crystals; mp 223 ◦C; IR (KBr): ꢁ/cm−1 = 3315
(NH), 1580 (N N), 1555 (C N); 1H NMR(DMSO-d6) ı (ppm):
714–798 (m, 11H, 9Ar–H + 2␤-Py–H), 861 (d, 2H, ␣-Py–H), 889 (s,
1H, NH); MS (EI, 70 eV) m/z (%) = 328 (M++2, 15), 327 (M++1, 25),
326 (M+, 70), 249 (25), 221 (78), 166 (50), 125 (51), 124 (30), 106
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128
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