Synthesis, antibacterial and antifungal evaluation of novel 1,4-dihydropyridine derivatives

Article abstract of DOI:10.1016/j.saa.2012.12.069

(Figure Presented) 3,5-Diacetyl-1,4-dihydro-2,4,6-trimethylpyridine (I) has been condensed with aromatic aldehydes to give the corresponding cinnamoyl derivatives IIa-e. the behavior of IIc, d towards thiourea, hydroxylamine hydrochloride, hydrazine hydra

Full text of DOI:10.1016/j.saa.2012.12.069

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, antibacterial and antifungal evaluation of novel
1,4-dihydropyridine derivatives
⇑
Hanaa Abu-Melha
Faculty of Science of Girls, King Khaled University, Abha, Saudi Arabia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Novel 1,4-dihydropyridines were synthesized and evaluated as antimicrobial agents, compounds IIIa and
VIIa, b revealed better activity against Gram-positive bacteria.
"
"
"
"
Novel 1,4-dihydropyridines were
synthesized and characterized by
spectral data.
3,5-Diacetyl-1,4-dihydro-2,4,6-
trimethylpyridine (I) was used as
starting material.
Compounds were screened for their
antibacterial activity and antifungal
activity.
Compounds IIIa and VIIa, b showed
significant activity against Gram-
positive strain.
O
CH3
O
S
S
Ar
Ar
HN
Ar
N
CH3 N NH
H3C
N
H
IIa-e
CH3
Ar
H3C
N
H
CH3
IIa
, Ar = C6H4-NO2-p
, Ar = C6H4-Cl-m
, Ar = C6H4-Cl-p
IId
IIe
, Ar = C6H4-Br-p
IIIa, b
IIb
IIc
IIIa, Ar = C6H4-Cl-p
IIIb, Ar = C6H4-Br-p
, Ar =
O
X
CH3
X
X
X
N
CH3
N
H
3C
CH3
CH3
Ar
Ar
N
H
H3
C
H3C
N
H
CH3
V, X = C(CN)COOEt
VI, X = N-NHPh
VIIa, X = O; Ar = C6H4-Cl-p
VIIb, X = O; Ar = C6H4-Br-p
VIIIa, X = N-Ph; Ar = C6H4-Cl-p
VIIIb, X = N-Ph; Ar = C6H4-Br-p
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 October 2012
Received in revised form 16 December 2012
Accepted 21 December 2012
Available online 24 February 2013
3,5-Diacetyl-1,4-dihydro-2,4,6-trimethylpyridine (I) has been condensed with aromatic aldehydes to
give the corresponding cinnamoyl derivatives IIa–e. the behavior of IIc, d towards thiourea, hydroxyl-
amine hydrochloride, hydrazine hydrate and other bifunctional reagents has been investigated. The
newly synthesized compounds were screened for their antimicrobial activity against Gram-positive bac-
teria, Gram-negative bacteria and as antifungal reagent. Among all the tested compounds, it was found
that compounds IIIa and VIIa, b revealed better activity against the Gram-positive rather than the
Gram-negative bacteria.
Keywords:
1,4-Dihydropyridines
Cinnamoyl derivatives
Antimicrobial activity
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
retention of biological activity and occasionally this moiety is an
essential part of the pharmacophore. Such substitution of @N for
The piperidine ring is contained in the molecules of many syn-
thetic and natural medicaments. One of the synthetic derivatives of
the pyridine is the drug promedol (trimeperidine hydrochloride),
which was developed when searching for analogues of morphine
[1]. Promedol has found wide spread use in medicine as an analge-
sic replacing morphine. It is a pharmacopoeial drug. Substitution of
a pyridine ring for a benzene ring often is compatible with
@CH is an example of the common medicinal chemical strategy
known as bioisomerism. Therefore, pyridine derivatives have a
broad spectrum of biological activity, such as antihypertensive,
bronchodilator, anti-inflammatory and antifungal agents [2–6].
Moreover, a 1,4-dihydropyridine having coronary vasodilatory
activity and, therefore, intended for relief of the intense chest pains
of angina pectoris is nifedipine [7]. Encouraged by these reports
and in continuation of our work in this field from our laboratory
[8–15], we report herein the synthesis and antimicrobial activity
of some new 1,4-dihydropyridine derivatives.
⇑
Tel.: +966 20 506432235; fax: +966 20 502246781.
E-mail address: hanaaabumelha@yahoo.com
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.12.069

116
H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
at 3400 cmꢀ1 (broad NH), 1650, 1630 (C@N) and 1275 (C@S). The
Results and discussion
1H NMR spectrum of IIIa showed signals at d 1.2 ppm of CH3 pro-
tons as doublet, singlet signal at d 1.71 ppm for two methyl group
protons, doublet of doublet at d 1.8 and 2.0 ppm due to CH2 pro-
tons of pyrimidine ring, quartet signal at d 3.3 ppm due to C4AH,
triplet at d 4.01 ppm due to CH of pyrimidine ring, aromatic pro-
tons at d 7.0–7.48 ppm as two doublets (AB system), in addition
to two singlet signals at d 8.2 and 8.75 ppm due to two NH protons.
Fusion of IIc with ethyl cyanoacetate in the presence of ammo-
nium acetate afforded the dicarbonitrile (IV). Compound IV was
confirmed via independent synthesis, by the condensation of com-
pound I with ethyl cyanoacetate to give pyridine diacrylate (V),
which underwent cyclization on treatment with p-chlorobenzalde-
hyde in presence of ammonium acetate to give IV (Fig. 3). Structure
IV was established on the basis of both elemental analysis and
spectral data. The IR spectrum showed absorption bands at
3400–3250, 2220 and 1680 cmꢀ1 corresponding to NH, CN and
CO functions, respectively. The 1H NMR spectrum of IV revealed
signals, in addition to that of pyridine ring, at d 2.33, 2.63 ppm as
doublet of doublet due to CH2 protons of pyridine ring, triplet at
d 4.91 ppm corresponding to CH proton, in addition to aromatic
protons appeared as two doublets at d 7.48–7.58 ppm (AB system).
Treatment of IIc, d with hydroxylamine hydrochloride in boiling
pyridine afforded the corresponding iso-oxazole derivatives VIIa, b,
respectively (Fig. 4). The structures of these compounds were de-
duced from correct analytical results and the IR, 1H NMR and mass
spectra. The IR spectra showed bands at 1630 cmꢀ1 (C@N) and a
broad extending band at 3300 cmꢀ1 (NH). The 1H NMR spectrum
of VIIa showed signals at d 1.26 ppm as doublet of methyl protons
at C4, singlet at d 1.70 ppm due to two methyl protons at positions
2 and 6 of pyridine ring, 1.71, 2.0 doublet of doublet corresponding
to CH2 protons of isoxazole ring, 3.31 ppm as quartet of C4AH of
pyridine ring, finally, the aromatic protons appeared as two dou-
blets at d 6.83 and 7.32 ppm (AB system).
Chemistry
3,5-Diacetyl-1,4-dihydro-2,4,6-trimethylpyridine (I) represents
an adaptable starting material for the introduction of heterocyclic
moieties in its 3- and 5-positions, and for the synthesis of some
new heterocyclic binary systems which have demonstrated
biological activity in different areas of chemotherapy. Compound
I easily undergoes condensation with aromatic aldehydes namely;
p-nitrobenzaldehyde, m- and p-chlorobenzaldehyde, p-bromo-
benzaldehyde and 2-furfuraldehyde to give the corresponding
3,5-dicinnamoyl derivatives (IIa–e), respectively (Fig. 1).
The structures of these compounds were supported from their
correct elemental analysis as well as spectroscopic data. The IR
spectra of IIa–e, in general, showed stretching frequencies at
3300–3150, 1720–1700, 1630, 1600–1590 cmꢀ1 corresponding to
NH, CO, exo C@C and endo C@C, respectively. The 1H NMR spec-
trum of compound IIc revealed signals at d 1.26 and 1.71 ppm
due to the methyl groups at positions 4, 2 and 6 of pyridine ring,
respectively. In addition to quartet signal of C4AH at d 3.31 ppm
and two doublets at d 7.1 and 8.22 ppm corresponding to exo
CH@CH protons, the aromatic protons appeared as two doublets
(AB system) at d 7.56–8.20 ppm. The condensation of IIc, d with
thiourea in boiling ethanolic sodium hydroxide yielded the corre-
sponding thiopyrimidine derivatives IIIa, b, respectively (Fig. 2).
The structures of these compounds have been deduced from cor-
rect analytical data and from their IR spectra which showed bands
O
CH3
O
CH3
CH3
O
CH3
O
Ar
Ar
H3C
H3C
N
H
H3C
N
H
CH3
The reaction of IIc, d with phenyl hydrazine was carried out in a
1:2 M ratio in refluxing acetic acid to give the bis-pyrazoline deriv-
atives VIIIa, b, respectively (Fig. 4), while, refluxing in ethanol
afforded the dianil VI (Fig. 3). Confirmatory evidence for the struc-
tures VIIIa, b was proved by elemental analysis and spectral data.
The IR spectra showed bands at 1680 and 1630 cmꢀ1 (C@N) and a
broad band at 3400–3250 cmꢀ1 (NH). The 1H NMR spectrum of
VIIIa showed a similar picture to that of VIIa.
IIa-e
I
IIa, Ar = C6H4-NO2-p
IIb, Ar = C6H4-Cl-m
IIc, Ar = C6H4-Cl-p
IId, Ar = C6H4-Br-p
IIe, Ar =
O
Fig. 1. Structure of pyridine derivative I and 3,5-dicinnamoyl derivatives IIa–e.
S
S
HN
Ar
N
CH3
N
NH
Ar
X
X
N
CH3
N
Ar
Ar
H3C
N
H
IIIa, b
CH3
H3C
N
H
CH3
VIIa, X = O; Ar = C6H4-Cl-p
VIIb, X = O; Ar = C6H4-Br-p
VIIIa, X = N-Ph; Ar = C6H4-Cl-p
VIIIb, X = N-Ph; Ar = C6H4-Br-p
IIIa
, Ar = C6H4-Cl-p
IIIb
, Ar = C6H4-Br-p
Fig. 4. Structure of iso-oxazole derivatives VIIa, b and bis-pyrazoline derivatives
VIIIa, b.
Fig. 2. Structure of thiopyrimidine derivatives IIIa, b.
O
O
116
21 NC
CH3
X
CH3 X
156
NH
CN
HN
115
114
H3C
H3C
CH3
CH3
129
128
128
133
171
47
29
141
129
38
N
H
136
CH3
17
Cl
Cl
H3C
N
H
V
VI
, X = C(CN)COOEt
, X = N-NHPh
IV
Fig. 3. Structure of dicarbonitrile IV, pyridine diacrylate V and dianil VI.

H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
117
A
O
CH3
O
A
HN
NH CH3
Cl
H3C
N
H
CH3
Cl
Cl
Cl
H3C
N
H
CH3
IXa, A =
X
O
IXb, A = CH(COCH3)COOEt
Fig. 5. Structure of dicyclohexanone IXa, 5-oxo-pentanoate IXb and hexahydroquinoline derivative X.
The bis-chalcone IIa was reacted with cyclohexanone and ethyl
acetoacetate in presence of sodium ethoxide to give the Michael
adducts dicyclohexanone IXa and 5-oxo-pentanoate IXb. Treat-
ment of IXa with ammonium acetate in boiling acetic acid afforded
hexahydroquinoline derivative X (Fig. 5).
Structures IXa, b and X were established on the basis of elemen-
tal and spectral analysis. The IR spectrum of IXb showed stretching
frequencies at 3310 and 1725–1722 cmꢀ1 corresponding to NH
and three carbonyl groups. The IR spectrum of X showed two
bands at 3350–3310 cmꢀ1 corresponding to (NH) stretching vibra-
tion. The 1H NMR of X spectrum showed signals at d 1.27 ppm due
to C4ACH3 protons, multiplet at d 0.8–1.79 due to cyclohexyl ring,
quartet at d 3.32 due to C4ACH3, two triplets and multiplet at d
2.77, 3.42 and 1.80 ppm due to hydroquinoline protons.
N
and ACOANA groups, respectively, and the disappearance of the
band at 1620 cmꢀ1 characteristic for AC@NA group. The 1H NMR
spectrum of XIII showed a characteristic signals at d 3.18 and
3.21 ppm as doublet of doublet due to CH2 protons of oxazine ring,
in addition to the expected picture of 1H NMR spectrum.
The Michael condensation of IIa and ethyl acetoacetate in boil-
ing butanol using piperidine as a basic catalyst gave the corre-
sponding cyclohexne mono carboxylate XIV. The structure of
compound XV was confirmed by hydrolysis and decarboxylation
to give the corresponding cyclohexanone derivative XV. Further-
more, structure of the parent compound XV was confirmed
through independent synthesis, by the condensation of compound
I with p-chlorobenzalacetone to give XV (Scheme 1).
Trials to obtain compound XI by the reduction of IIa with zinc
dust in acetic acid were unsuccessful and compound IIa was recov-
ered unchanged (Fig. 6).
Reaction of compound I with p-toluidine in acetic acid and cat-
alytic amount of freshly fused sodium acetate afforded the bis-anil
XII. The IR spectrum showed the disappearance of CO band and the
appearance of new band at 1615 cmꢀ1 due to the C@N function.
Treatment of XII with malonic acid in presence of acetic anhydride
gave the spiro compound XIII (Fig. 7). The structure was confirmed
by elemental analysis and its spectral data. The IR spectrum
showed absorption bands at 1700 and 1680 cmꢀ1 characteristic for
Pharmacology
Antimicrobial evaluation
Fifteen of the synthesized target compounds were evaluated for
their in vitro antibacterial activity against Gram-positive bacteria
(Bacillus subtilis and Staphylococcus aureus), Gram-negative bacte-
ria (Escherichia coli and Pseudomonas aeruginosa) and fungi (Asper-
gillus niger). Agar-diffusion method 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 zone (IZ) of bacterial and fungal
growth around the disks in mm, the minimum inhibitory concen-
tration (MIC) measurement was determined for compounds
showed significant growth inhibition zones (>14 mm) using two
N
N
CH3
H3C
N
H
CH3
XI
fold serial dilution method [16]. The MIC (lg/mL) and inhibition
zone diameters values are recorded in Table 1. The inhibition zone
Fig. 6. Structure of 2,20-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)diquinoline
(XI).
diameters values cited in Table 1 between brackets are attributed
O
O
H3C
CH3
O
O
CH3
O
O
N
H3C
H3C
N
CH3 N
N
CH3
Ar
Ar
H3C
H3C
CH3
CH3
N
H
CH3
N
H
XIII
XII
Ar = C6H4-CH3-p
Fig. 7. Structure of bis-anil XII and the spiro compound XIII.

118
H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
COOEt
COOEt
O
O
O
O
CH3
CH3
1) OH
2) H
Ar
Ar
Ar
Ar
,
H3C
N
H
CH3
H3C
N
H
CH3
XV
XIV
XIV, XV, Ar = C6H4-Cl-p
O
+
I
Cl
CH3
Scheme 1. Synthesis of 5,500-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis (40-chloro-2,3-dihydro-[1,10-biphenyl]-4(1H)-one) (XV).
to the tested original concentration (1 mg/mL) as preliminary test.
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 fungal strains.
(6.25–100 lg/mL). Among the series of compounds VIIa, b, VIIIa,
b, X, XIII, XIV and XV, compounds VIIa, b and VIIIa, b showed rel-
atively good growth inhibitory profiles against B. subtilis (MIC
3.125–12.5 lg/mL) which were from equipotent to 25% of the
activity of chloramphenicol and 50% of cephalothin against the
Table 1 showed that most of the tested compounds revealed
better activity against the Gram-positive rather than the Gram-
negative bacteria.
same organism (IV).
Moreover, distinctive anti-Gram-positive profile was displayed
by compounds VIIa, b where it proved to be equipotent as chlor-
Regarding the activity of the IIIa–d against Gram-positive bac-
teria, the results revealed that compound IIIa exhibited divergent
antibacterial activity against the tested organisms. In this view,
compound IIIa was equipotent to chloramphenicol in inhibiting
amphenicol against both B. subtilis (MIC 3.125
(MIC 3.125 g/mL). Concerning the antibacterial activity of the
compounds IId, e, IIIa, b and IV revealed weak growth inhibitory
against the tested Gram-negative bacteria (MIC 50 g/mL). On
the other hand, compound VIIa showed equipotent activity as
lg/mL) and S. aureus
l
l
the growth of B. subtilis (MIC 3.125 lg/mL), while its activity was
50% lower than chloramphenicol against S. aureus. Compounds III
chloramphenicol and cephalothin (MIC 6.25 lg/mL) against E. coli
and IV showed 50% of the activity of chloramphenicol (MIC
and P. aeruginosa.
6.25
l
g/mL). On the other hand, compounds I, IIa–e, V, VI, IXa, b
Regarding the activity of I–XV against fungal strains, the results
revealed that compound VIIb was 50% lower than cycloheximide in
and XII exhibited weak to moderate growth inhibitory activity
against Gram-positive bacteria revealed from their MIC values
inhibitory the growth of A. niger (MIC 3.125
lg/mL), while the reac-
Table 1
Minimal inhibitory concentration (MIC,
lg/mL) and inhibition zone (mm) of the newly synthesized compounds.
Compound No.
MICa in
Bacteria
lg/mL and inhibition zone (mm)
Fungi
Gram(+) bacteria
Gram(ꢀ) bacteria
B. subtilis
S. aureus
E. coli
P. aeruginosa
A. niger
I
IIa
IIb
IIc
IId
IIe
IIIa
IIIb
IV
V
VI
VIIa
VIIb
VIIIa
VIIIb
IXa
100 (15)
25 (20)
50 (15)
25 (30)
25 (28)
100 (14)
6.25 (38)
50 (18)
25 (21)
6.25 (34)
50 (30)
6.25 (41)
6.25 (39)
6.25 (40)
6.25 (41)
25 (22)
6.25 (42)
6.25 (44)
6.25 (40)
6.25 (44)
25 (22)
100 (16)
100 (14)
6.25 (38)
50 (24)
50 (23)
50 (18)
50 (19)
50 (16)
50 (18)
12.5 (37)
25 (35)
25 (35)
6.25 (38)
12.5 (35)
12.5 (30)
25 (33)
12.5 (33)
25 (35)
12.5 (33)
6.25 (38)
6.25 (35)
25 (35)
6.25 (39)
6.25 (38)
NT
100 (18)
100 (15)
100 (18)
100 (15)
12.5 (32)
100 (15)
50 (20)
25 (35)
50 (19)
25 (33)
25 (33)
25 (39)
6.25 (38)
25 (33)
25 (35)
25 (30)
25 (35)
50 (20)
50 (30)
25 (33)
50 (25)
25 (35)
6.25 (38)
6.25 (38)
NT
100 (18)
100 (15)
100 (18)
100 (14)
25 (25)
100 (16)
100 (15)
100 (18)
50 (18)
50 (25)
3.125 (38)
6.25 (30)
12.5 (40)
6.25 (44)
50 (15)
3.125 (45)
3.125 (43)
6.25 (38)
3.125 (40)
50 (15)
50 (20)
6.25 (35)
25 (30)
12.5 (35)
12.5 (30)
12.5 (35)
3.125 (42)
6.25 (40)
NT
12.5 (31)
50 (18)
12.5 (35)
6.25 (37)
12.5 (33)
12.5 (30)
50 (20)
12.5 (33)
12.5 (35)
12.5 (33)
50 (18)
25 (25)
12.5 (30)
NT
NT
3.125 (44)
IXb
X
XII
XIII
XIV
XV
50 (15)
6.25 (35)
6.25 (30)
6.25 (30)
12.5 (32)
6.25 (30)
3.125 (44)
6.25 (41)
NT
Chloramphenicol
Cephalothin
Cycloheximide
NT: not tested.
a
MIC: minimal inhibitory concentration values.

H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
119
tivity of compound VIIa was 25% lower than cycloheximide against
A. nieger (MIC 12.5 g/mL).
Synthesis of 3,5-dicinnamoyl-1,4-dihydro-2,4,6-trimethylpyridine
(IIa–e)
l
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 recorded higher antibacterial
activity. (2) The substitution at position 3 and 5 of the pyridine ring
produced a higher antimicrobial activity. (3) Conversion of com-
pounds I and II to their corresponding heterocyclic derivatives
III, IV and VII enhanced also antimicrobial activity. (4) The tested
compounds were more active against Gram-positive than Gram-
negative bacteria, it may be concluded that the antimicrobial activ-
ity of the compounds is related to cell wall structure of the bacte-
ria. 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 piptidoglycan. 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 lipopolysac-
charides and lipoproteins. These differences in cell wall structure
can produce differences in antibacterial susceptibility and some
antibiotics can kill only Gram-positive bacteria and is infective
against Gram-negative bacteria pathogen [17]. (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 inves-
tigation regarding the structural activity relationships, toxicity and
in their biological effects which could be helpful in designing more
potent antibacterial agents for therapeutic use.
General procedure: To a solution of I (0.01 mol) in alcoholic
potassium hydroxide (4%), the appropriate aldehyde (0.02 mol)
was added and the reaction mixture was left to overnight at room
temperature, diluted and acidified with dilute acetic acid. The solid
products thus obtained were filtered off and crystallized from eth-
anol to give compounds IIa–e.
(2E,20E)-1,10-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(3-(4-
nitrophenyl)prop-2-en-1-one) (IIa). Yield (73%); m.p. 151 °C; IR
(KBr):
m
/cmꢀ1 = 3350 (NH), 1720 (CO), 1650 (C@N), 1530, 1350
(NO2); 1H NMR(DMSO-d6) d (ppm): 1.26 (d, 3H, CH3), 1.71 (s, 6H,
2CH3), 3.31 (q, 1H, C4AH), 7.10 (d, 1H, COCH@), 7.56–8.12 (d.d,
4H, ArAH), 8.22 (d, 1H, @CHAAr), 8.71 (s, 1H, NH); 13C
NMR(DMSO-d6) d (ppm): 18 (CH3AC4), 20.5 (2CH3), 23.1 (C4),
117 (C3, C5), 124 (COACH@), 123, 127, 129, 141, 145 (ArAC), 152
(@CHAAr), 157 (C2, C6), 193 (2C@O); MS (EI, 70 eV) m/z (%) = 473
(M+, 100), 474 (M++1, 30), 427 (70), 443 (16). Anal. Calcd. for
C26H23N3O6 (473.48): C, 65.95; H, 4.90; N, 8.87%. Found: C,
65.91; H, 4.80; N, 8.81%.
(2E,20E)-1,10-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(3-(3-
chlorophenyl)prop-2-en-1-one) (IIb). Yield (65%); m.p. 175 °C; IR
(KBr): m
/cmꢀ1 = 3325 (NH), 1715 (CO), 1625 (C@N), 750 (CACl);
1H NMR(DMSO-d6) d (ppm): 1.25 (d, 3H, CH3), 1.70 (s, 6H, 2CH3),
3.33 (q, 1H, C4AH), 7.11 (d, 1H, COCH@), 7.57–8.21 (d.d, 4H, ArAH),
8.22 (d, 1H, @CHAAr), 8.75 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 451
(M+ꢀ1, 100), 452 (M+, 28.6), 453 (M++1, 64.4). Anal. Calcd. for C26-
H23Cl2NO2 (452.37): C, 69.03; H, 5.12; N, 3.10%. Found: C, 68.95; H,
5.01; N, 3.02%.
Conclusion
In conclusion, the objective of the present study was to synthe-
size and investigate the antimicrobial activities of some new func-
tionalized 3- and 5-pyridyl heterocyclic 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 pyridine
moiety attached at 3- and 5-positions. The obtained results clearly
revealed that compounds derived from 3- and 5-pyridyl deriva-
tives exhibited better antimicrobial activity than their unsubstitut-
ed isomers.
(2E,20E)-1,10-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(3-(4-
chlorophenyl)prop-2-en-1-one) (IIc). Yield (73%); m.p. 210 °C; IR
(KBr): m
/cmꢀ1 = 3330 (NH), 1710 (CO), 1640 (C@N), 735 (CACl);
13C NMR(DMSO-d6) d (ppm): 16.3 (C2, C6), 18.2 (CH3AC4), 22.9
(C4), 117 (C3, C5), 124 (COACH@), 123, 127, 129, 130, 145 (ArAC),
152 (@CHAAr), 157 (C2, C6), 195 (2C@O); MS (EI, 70 eV) m/z
(%) = 451 (M+ꢀ1, 100), 452 (M+, 70). Anal. Calcd. for C26H23Cl2NO2
(452.37): C, 69.03; H, 5.12; N, 3.10%. Found: C, 68.89; H, 4.95; N,
3.01%.
(2E,20E)-1,10-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(3-(4-
Experimental
bromophenyl)prop-2-en-1-one) (IId). Yield (81%); m.p. 137oC; IR
(KBr): m
/cmꢀ1 = 3350 (NH), 1720 (2CO), 1630 (C@N), 735 (CABr);
Instruments
1H NMR(DMSO-d6) d (ppm): 1.26 (d, 3H, CH3AC4), 1.69 (s, 6H,
2CH3), 3.3 (q, 1H, C4AH), 7.0 (d, 1H, COCH@), 7.59–8.10 (d.d, 4H,
ArAH), 8.22 (d, 1H, @CHAAr), 8.76 (s, 1H, NH); MS (EI, 70 eV) m/
z (%) = 539 (M+ꢀ2, 50), 541 (M+, 100), 543 (M++2, 52). Anal. Calcd.
for C26H23Br2NO2 (541.27): C, 57.69; H, 4.28; N, 2.59%. Found: C,
57.58; H, 4.17; N, 2.45%.
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 tetramethylsilane (TMS) as an internal standard, and
chemical shifts are expressed as dppm. The mass spectra were deter-
mined on Finnigan Incos 500 (70 eV). Elemental analyses were car-
ried out at the Microanalytical Unit of the Faculty of Science, Cairo
University, Giza, Egypt.
(2E,20E)-1,10-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(3-
(furan-2-yl)prop-2-en-1-one) (IIe). Yield (68%); m.p. 127 °C; IR
(KBr):
m
/cmꢀ1 = 3330 (NH), 1720 (CO), 1630 (C@N); 1H
NMR(DMSO-d6) d (ppm): 1.25 (d, 3H, CH3AC4), 1.73 (s, 6H,
2CH3), 3.35 (q, 1H, C4AH), 6.8 (d, 1H, C4AH furan ring), 7.0 (m,
1H, C3AH furan), 7.1 (d, 1H, COACH@), 7.55–8.2 (d.d, 4H, ArAH),
8.16 (d, 1H, C2AH furan), 8.77 (s, 1H, NH). MS (EI, 70 eV) m/z
(%) = 363 (M+, 100), 364 (M++1, 25), 365 (M++2, 5). Anal. Calcd.
for C22H21NO4 (363.41): C, 72.71; H, 5.82; N, 3.85%. Found: C,
72.65; H, 5.70; N, 3.77%.
Synthesis
Diacetyl-1,4-dihydro-2,4,6-trimethylpyridine (I)
It was prepared according to the reported method [18], m.p.
153 °C.

120
H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
Synthesis of 4,40-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(6-
(4-halo-phenyl)-5,6-dihydropyrimidine-2(1H)-thione) derivatives
(IIIa, b)
(ppm): 1.26 (d, 3H, CH3), 1.3 (t, 6H, 2CH2CH3), 1.70 (s, 6H, 2CH3),
3.30 (q, 1H, C4AH), 4.10 (q, 4H, 2CH2CH3). Anal. Calcd. for
C22H27N3O4 (397.47): C, 66.48; H, 6.85; N, 10.57%. Found: C,
General procedure A mixture of each of IIc, d (0.01 mol), thiourea
(0.02 mol) in sodium ethoxide (0.02 mol sodium, in 50 mL ethanol)
was refluxed for four hours, left to cool, diluted and acidified with
dilute acetic acid. The precipitated solid were filtered and crystal-
lized from ethanol to give compounds IIIa, b, respectively.
66.54; H, 6.93; N, 10.61%.
Condensation of I with phenyl hydrazine; Formation of VI
A
mixture of compound I (0.01 mol) phenyl hydrazine
(0.22 mol) was refluxed in boiling ethanol (30 mL) for 5 h. The
reaction mixture was left to cool and diluted with water to give
the corresponding phenyl hydrazone VI.
4,40-(2,4,6-Trimethyl-1,4-dihydropyridine-3,5-diyl)bis(6-(4-chloro-
phenyl)-5,6-di-hydropyrimidine-2(1H)-thione) (IIIa). Yield (60%);
Yield (65%); m.p. 148 °C; IR (KBr): m
/cmꢀ1 = 3310 (NH), 1635
m.p. 200oC; IR (KBr):
m
/cmꢀ1 = 3400 (NH), 1650, 1630 (C@N),
1275 (C@S), 735 (CACl); 1H NMR(DMSO-d6) d (ppm): 1.21 (d, 3H,
CH3), 1.70 (s, 6H, 2CH3), 1.8:2.0 (d.d, 2H, CH2-pyrimidine), 3.31
(q, 1H, C4AH), 4.02 (t, 1H, CH-pyrimidine), 8.75 (s, 1H, NH), 9.02
(s, 1H, NH); 13C NMR(DMSO-d6) d (ppm): 17 (2CH3), 19.5 (CH3AC4),
25 (C4-pyridine), 36 (2C5-pyrimidine), 54 (2C4-pyrimidine), 100
(C3, C5 pyridine), 128 (benzene ring C), 132 (CACl), 141 (2C, pyrim-
idine ring), 142 (C2, C6, pyridine), 164 (2N@C, pyrimidine), 188
(2C@S); MS (EI, 70 eV) m/z (%) = 567 (M+ꢀ1, 100), 568 (M+, 34),
569 (M++1, 70). Anal. Calcd. for C28H27Cl2N5S2 (568.58): C, 59.15;
H, 4.79; N, 12.32%. Found: C, 59.10; H, 4.61; N, 12.11%.
(C@N). Anal. Calcd. for C24H29N5 (387.52): C, 74.38; H, 7.54; N,
18.07%. Found: C, 74.43; H, 7.57; N, 18.14%.
Condensation of IIc, d with hydroxylamine hydrochloride: Formation
of VIIa, b
A mixture of IIc, d (0.01 mol) and hydroxylamine hydrochloride
(0.02 mol) in pyridine (30 mL) was refluxed for 6 h, left to cool, di-
luted with water and the obtained solid products were crystallized
from ethanol to give compounds VIIa, b, respectively.
4,40-(2,4,6-Trimethyl-1,4-dihydropyridine-3,5-diyl)bis(6-(4-bromo-
3,30-(2,4,6-Trimethyl-1,4-dihydropyridine-3,5-diyl)bis(5-(4-chloro-
phenyl)-5,6-di-hydropyrimidine-2(1H)-thione) (IIIb). Yield (62%);
phenyl)-4,5-di-hydroisoxazole (VIIa). Yield (71%); m.p. 173 °C; IR
m.p. 165oC; IR (KBr):
m
/cmꢀ1 = 3350 (NH), 1650, 1630 (C@N),
(KBr):
m d
/cmꢀ1 = 3310 (NH), 1625 (CO); 1H NMR(DMSO-d6)
1300 (C@S), 750 (CABr); MS (EI, 70 eV) m/z (%) = 655 (M+ꢀ2, 50),
657 (M+, 100), 659 (M++2, 60). Anal. Calcd. for C28H27Br2N5S2
(657.49): C, 51.15; H, 4.14; N, 10.65%. Found: C, 51.10; H, 4.02;
N, 10.51%.
(ppm): 1.25 (d, 3H, CH3), 1.70 (s, 6H, 2CH3), 1.71:1.90 (d.d, 2H,
CH2), 3.33 (q, 1H, CH), 4.5 (t, 1H, CH), 7.2–7.41 (d.d, 4H, ArAH);
MS (EI, 70 eV) m/z (%) = 481 (M+ꢀ1, 100), 482 (M+, 28), 483 (60).
Anal. Calcd. for C26H25Cl2N3O2 (482.40): C, 64.73; H, 5.22; N,
8.71%. Found: C, 64.62; H, 5.10; N, 8.62%.
Synthesis of 4,40-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(6-
(4-chloro-phenyl)-2-oxo-1,2,5,6-tetrahydropyridin-3-carbonitrile (IV)
Method A: A mixture of IIc (0.01 mol) and ethyl cyanoacetate
(0.022 mol) was heated in an oil bath at 150–160 °C, in presence
of ammonium acetate for 1 h. The reaction mixture was left stand
overnight and the crude product washed with water, dried and
recrystallized from ethanol to give IV.
3,30-(2,4,6-Trimethyl-1,4-dihydropyridine-3,5-diyl)bis(5-(4-bromo-
phenyl)-4,5-di-hydroisoxazole (VIIb). Yield (55%); m.p. 205 °C; MS
(EI, 70 eV) m/z (%) = 573 (M++2, 50), 572 (M++1, 51), 571 (M+,
100). Anal. Calcd. for C26H25Br2N3O2 (571.30): C, 54.66; H, 4.41;
N, 7.36%. Found: C, 54.52; H, 4.30; N, 7.25%.
Method B: (a) Condensation of (I) with ethyl cyanoacetate for-
mation of compound (V).
Interaction of IIc, d with phenyl hydrazine: Formation of VIIIa, b
To a mixture of each of IIc, d (0.01 mol), phenyl hydrazine
(0.02 mol) and glacial acetic acid (30 mL) was added. The reaction
mixture was refluxed for 4 h, and left to cool. The solid products
obtained were crystallized from ethanol to give compounds VIIIa,
b, respectively.
Compound (I) (0.01 mol), ethyl cyanoacetate (0.022 mol), and
ammonium acetate (1 g) were dissolved in dry xylene (50 mL)
The solution was heated using a Dean & Stark apparatus and reflux
condenser for 10 h. The solvent was removed under vacuo. The
reaction mixture was diluted with water and the obtained solid
product filtered, dried and recrystallized from ethanol to give V.
(b) A mixture of V (0.01 mol), p-chlorobenzaldehyde (0.22 mol)
and ammonium acetate (0.5 g) was heated in an oil bath at 150–
160 °C for 1.5 h. the reaction mixture was washed with water,
dried and recrystallized from ethanol to give IV.
3,5-Bis(5-(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
2,4,6-trimethyl-1,4-dihydropyridine (VIIIa). Yield (65%); m.p.
180 °C; IR (KBr):
m
/cmꢀ1 = 3310 (2NH), 1630 (C@N); 1H
NMR(DMSO-d6) d (ppm): 1.26 (d, 3H, CH3), 1.70 (s, 6H, 2CH3),
1.71:2.0 (d.d, 2H, CH2), 3.31 (q, 1H, CH), 3.90 (t, 1H, CH), 6.83–
7.32 (d.d, 4H, ArAH); 13C NMR(DMSO-d6) d (ppm): 17 (2CH3), 19
(CH3AC4), 23.5 (C4-pyridine ring), 39.5 (2C4-pyrazole), 55 (2C5-
pyrazole), 106 (C3, C5-pyridine), 113, 117, 128, 129, 132, 143,
141, 149 (ArAC); MS (EI, 70 eV) m/z (%) = 633 (M++1, 60), 631
(M+ꢀ1, 100), 632 (M+, 41). Anal. Calcd. for C38H35Cl2N5 (632.62):
C, 72.15; H, 5.58; N, 11.07%. Found: C, 72.10; H, 5.42; N, 10.95%.
Compound IV. Yield (72%); m.p. 155 °C; IR (KBr): m
/cmꢀ1 = 3400–
3250 (2NH), 2220 (CN), 1685 (CO), 1630 (C@N), 750 (CACl); 1H
NMR(DMSO-d6) d (ppm): 1.29 (d, 3H, CH3), 1.70 (s, 6H, 2CH3),
2.33:2.63 (d.d, 2H, CH2), 3.33 (q, 1H, CH), 4.91 (t, 1H, CH), 7.48–
7.58 (d.d, 4H, ArAH), 8.0 (s, 1H, NH), 8.71 (s, 1H, NH); 13C
NMR(DMSO-d6) d (ppm): 17 (CH3), 21 (CH3AC4), 29 (C4-pyridine),
38 (2C5-pyridine), 47 (2C6), 114 (C3, pyridine), 115 (2CACN), 116
(2CN), 128, 129, 133, 141 (ArAC), 156 (2CO), 171 (2C4-pyridine);
MS (EI, 70 eV) m/z (%) = 583 (M+ꢀ1, 100), 584 (M+, 35), 585
(M++1, 58). Anal. Calcd. for C32H27Cl2N5O2 (584.50): C, 65.76; H,
4.66; N, 11.98%. Found: C, 65.61; H, 4.63; N, 11.82%.
3,5-Bis(5-(4-bromophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-
2,4,6-trimethyl-1,4-dihydropyridine (VIIIb). Yield (70%); m.p.
108 °C; IR (KBr): m
/cmꢀ1 = 3300 (NH), 1630 (C@N); MS (EI, 70 eV)
(2E,20E)-Diethyl 3,30-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)-
m/z (%) = 719 (M+ꢀ2, 51), 721 (M+, 100), 723 (M++2, 50). Anal.
Calcd. for C38H35Br2N5 (721.53): C, 63.26; H, 4.89; N, 9.71%. Found:
C, 63.10; H, 4.72; N, 9.63%.
bis(2-cyanobut-2-eno-ate) (V). Yield (76%); m.p. 135 °C; IR (KBr):
m/
cmꢀ1 = 3330 (NH), 2221 (CN), 1700 (CO); 1H NMR(DMSO-d6) d

H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
121
Synthesis of 2,20-((2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(1-
(4-chloro-phenyl)-3-oxopropane-3,1-diyl))dicyclohexanone (IXa) and
diethyl 5,50-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(2-
acetyl-3-(4-chlorophenyl)-5-oxopentanoate) (IXb)
General procedure A mixture of IIc (0.01 mol), cyclohexanone
and/or ethyl acetoacetate (0.022 mol) was refluxed in absolute eth-
anol for 6 h in the presence of sodium ethoxide (3%). The reaction
mixture was allowed to stand overnight and then acidified by di-
lute HCl. The solid product obtained was crystallized from ethanol
to give the corresponding Michael adducts IXa and IXb,
respectively.
100), 386 (M++1, 28), 387 (4). Anal. Calcd. for C26H31N3 (385.54):
C, 81.00; H, 8.10; N, 10.90%. Found: C, 80.16; H, 7.90; N, 10.75%.
Reaction of XII with malonic acid: Formation of spiro compound XIII
Dissolve (0.05 mol) of compound XII in acetic anhydride
(10 mL) by warming then added (0.12 mol) of malonic acid and stir
to ensure complete dissolving and allowed to stand overnight. The
brown solid precipitate was filtered off and recrystallized from
acetic acid to give the spiro compound XIII.
Synthesis of 2,20-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(2-
Compound IXa. Yield (65%); m.p. 138 °C; IR (KBr): m
/cmꢀ1 = 3315
methyl-3-(p-tolyl)-1,3-oxazinane-4,6-dione) (XIII). Yield (50%); m.p.
(NH), 1720, 1700 (2CO); 1H NMR(DMSO-d6) d (ppm): 1.28 (d, 3H,
C4ACH3), 1.71 (s, 6H, 2 CH3), 1.63:1.87 (m, 12H, 6 CH2), 2.17 (m,
4H, 2 CH2), 2.59 (m, 2H, 2 CH), 3.11:3.36 (m, 6H, 2 COCH2 + 2
CH), 3.41 (q, 1H, C4AH), 7.06–7.19 (d.d, 8H, ArAH); 13C
NMR(DMSO-d6) d (ppm): See Section 2; MS (EI, 70 eV) m/z
(%) = 647 (M+ꢀ1, 100), 648 (M+, 41), 649 (M++1, 63). Anal. Calcd.
for C38H43Cl2NO4 (648.66): C, 70.36; H, 6.68; N, 2.16%. Found: C,
70.25; H, 6.59; N, 2.01%.
135 °C; IR (KBr): m
/cmꢀ1 = 3330 (NH), 1720 (CO); 1H NMR(DMSO-
d6) d (ppm): 1.25 (d, 3H, CH3), 1.70 (s, 6H, 2CH3), 1.77 (s, 6H,
2CH3), 3.18:3.21 (d, 4H, 2CH2 oxazine ring), 3.31 (q, 1H, C4AH pyr-
idine ring), 6.8–7.16 (d.d, 8H, ArAH), 8.71 (s, 1H, NH); MS (EI,
70 eV) m/z (%) = 557 (M++1, 35), 559 (M++2, 7). Anal. Calcd. for
C32H35N3O6 (557.64): C, 68.92; H, 6.33; N, 7.54%. Found: C,
68.81; H, 6.27; N, 7.50%.
Condensation of IIc with ethyl acetoacetate: Formation of compound
XIV
Compound IXb. Yield (71%); m.p. 180 °C; IR (KBr): m
/cmꢀ1 = 3310
(NH), 1725, 1720, 1722 (3CO); 1H NMR (DMSO-d6) d (ppm): 1.29
(d, 3H, C4ACH3), 1.3 (t, 6H, 2 CH2CH3), 1.7 (s, 6H, 2 CH3), 2.09 (s,
6H, 2 COCH3), 3.3 (q, 1H, C4AH), 3.11–3.36 (d.d, 4H, 2 COCH2),
3.56 (d, 2H, 2 CH), 3.85 (m, 2H, 2 CH), 4.12 (q, 4H, 2 COCH2CH3),
7.07–7.19 (dd, 8H, ArAH), 8.75 (s, 1H, NH); MS (EI, 70 eV) m/z
(%) = 713 (M++1, 75), 712 (M+, 44), 711 (M+ꢀ1, 100). Anal. Calcd.
for C38H43Cl2NO8 (712.66): C, 64.04; H, 6.08; N, 1.97%. Found: C,
64.13; H, 6.14; N, 2.06%.
A solution of IIc (0.01 mol), ethyl acetoacetate (0.023 mol), in
50 mL n-butanol, containing piperidine (1 mL) was refluxed for
8 h. The reaction mixture was added to dilute HCl, the formed
product was filtered and crystallized from benzene–ethanol mix-
ture (1:1) to give XIV.
Diethyl
chloro-4-oxo-1,2,3,4-tetrahydro-[1,10-biphenyl]-3-carboxylate) (XIV).
Yield (60%); m.p. 189 °C; IR (KBr):
/cmꢀ1 = 3330 (NH), 1730, 1700
5,500-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(40-
Synthesis of 2,20-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(4-
(4-chlorophenyl) decahydroquinoline) (X)
m
(2CO); 1H NMR(DMSO-d6) d (ppm): 1.26 (d, 3H, CH3), 1.30 (t, 6H,
2CH3), 1.70 (s, 6H, 2CH3), 1.95:2.20 (d.d, 4H, 2CH2), 3.18 (t, 2H,
2CH), 3.31 (q, 1H, CH), 3.45 (m, 2H, 2CH), 4.12 (q, 4H, 2CH2), 7.06
(d, 2H, 2C@CH), 7.19–7.27 (d.d, 8H, ArAH), 8.70 (s, 1H, NH); MS
(EI, 70 eV) m/z (%) = 675 (M+ꢀ1, 100), 676 (M+, 41), 677 (M++1,
63). Anal. Calcd. for C38H39Cl2NO6 (676.63): C, 67.45; H, 5.81; N,
2.07%. Found: C, 67.38; H, 5.77; N, 2.0%.
A mixture of IXa (0.01 mol) and ammonium acetate (0.022 mol)
was refluxed in boiling acetic acid (30 mL) for 4–6 h. The reaction
mixture was left to cool and diluted with water to give compound
X.
Yield (60%); m.p. 175 °C; IR (KBr): m
/cmꢀ1 = 3350–3310 (NH);
1H NMR(DMSO-d6) d (ppm): 0.8–1.79 (m, 20H, 2 cyclohexan rings),
1.27 (d, 3H, CH3), 1.71 (s, 6H, 2CH3), 1.80 (m, 4H, 2CH2), 2.77 (t, 2H,
2CH), 3.32 (q, 1H, C4AH pyridine ring), 3.42 (t, 2H, 2C2AH quino-
line ring), 7.06–7.20 (d.d, 8H, ArAH), 8.3 (s, 1H, NH), 8.76 (s, 1H,
NH); MS (EI, 70 eV) m/z (%) = 617 (M+ꢀ1, 100), 618 (M+, 41), 619
(M++1, 65). Anal. Calcd. for C38H49Cl2N3 (618.72): C, 73.77; H,
7.98; N, 6.79%. Found: C, 73.61; H, 7.80; N, 6.71%.
Synthesis of 5,500-(2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bis(40-
chloro-2,3-dihydro-[1,10-biphenyl]-4(1H)-one) (XV)
Method A: A mixture of I (0.01 mol), p-chlorobenzalideneace-
tone (0.022 mol) was added to sodium methoxide (3%, 55 mL).
The mixture was refluxed for 8 h, cooled, acidified with dilute
HCl. The formed product was filtered off, and crystallized from
methanol to give XV.
Reduction of IIa with zinc dust
To IIa (0.01 mol) in acetic acid (20 mL) was added 2 g zinc dust.
The reaction mixture was refluxed 3 h and IIa was recovered
unchanged.
Method B: Product XIV (0.01 mol) was hydrolyzed by boiling
with aqueous sodium hydroxide solution (5%, 25 mL), for 3 h. After
cooling to room temperature the solution was acidified with dilute
HCl then refluxed for 1 h. After cooling, the obtained product was
filtered off and crystallized from ethanol to give XV. Yield (50%);
Reaction of compound I with p-toluidine: Formation of compound XII
A mixture of I (0.01 mol) and p-toluidine (0.025 mol) was re-
fluxed in glacial acetic acid (30 mL) for 3 h. The reaction mixture
was left to cool to give XII. The crude product was filtered and
recrystallized from ethanol to give yellow crystals of compound
XII.
m.p. 171 °C; IR (KBr):
m
/cmꢀ1 = 3330 (NH), 1700 (CO); 1H
NMR(DMSO-d6) d (ppm): 1.24 (d, 3H, CH3), 1.61–1.86 (m, 4H,
2CH2), 1.71 (s, 6H, 2CH3), 2.89–2.99 (m, 4H, 2CH2), 3.32 (q, 1H,
CH), 3.45 (m, 2H, 2CH), 7.05 (d, 2H, 2C@CH), 7.41–7.44 (d.d, 8H,
ArAH), 8.71 (s, 1H, NH); 1H NMR(DMSO-d6)
d (ppm): 16.6
(2CH3), 19.4 (CH3AC4), 30 (C4-pyridine), 33.8 (2C3-cyclo), 34.8
(2C4), 47.3 (2C5-cyclo), 113.5 (C3, C5, pyridine), 128.9, 129.5, 131,
137 (12CAAr), 132.9 (2C2-ene), 135.7 (2C6-cyclo), 135.9 (C2, C6-
pyridine), 194.4 (2C@O); MS (EI, 70 eV) m/z (%) = 531 (M+ꢀ1,
100), 532 (M++1, 35), 533 (M++1, 61). Anal. Calcd. for C32H31Cl2NO2
(532.50): C, 72.18; H, 5.87; N, 2.63%. Found: C, 72.10; H, 5.73; N,
2.51%.
(N,N0E,N,N0E)-N,N0-((2,4,6-trimethyl-1,4-dihydropyridine-3,5-diyl)bi-
s(ethan-1-yl-1-ylidene))bis(4-methylaniline) (XII). Yield (55%); m.p.
185 °C; IR (KBr):
m
/cmꢀ1 = 3310 (NH), 1615 (C@N); 1H
NMR(DMSO-d6) d (ppm): 0.9 (s, 6H, 2CH3), 1.25 (d, 3H, CH3), 1.71
(s, 6H, 2CH3), 2.34 (s, 6H, 2CH3), 3.31 (q, 1H, CH), 7.1–7.20 (d.d,
8H, ArAH), 8.74 (s, 1H, NH); MS (EI, 70 eV) m/z (%) = 385 (M+,

122
H. Abu-Melha / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 113 (2013) 115–122
Antimicrobial evaluation
corded 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.
The disks of Whatman filter paper were prepared with standard
size (5.0 mm diameter) and kept into 1.0 Oz screw capped wide
mouthed containers for sterilization. These bottles are kept into
hot air oven at a temperature of 150 °C. Then, the standard steril-
ized filter paper disks impregnated with a solution of the test com-
pound in DMF (1 mg/mL) were placed on nutrient agar plate
seeded with 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. Pyr-
ex glass Petri dishes (9 cm in diameter) were used and two disks of
filter paper were inoculated in each plate. The utilized test organ-
isms 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 po-
tential against A. nieger fungal strains. Chloramphenicol, cephalo-
thin and cycloheximide 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 dilution technique, were further evaluated for their
minimal inhibitory concentrations (MICs).
Acknowledgements
To Prof. Dr. Ahmed A. Fadda, Professor of Organic Chemistry,
Chemistry Department, Faculty of Science, Mansoura University,
Mansoura, Egypt for suggesting the research point, his continuous
guidance, precious advice, real support and valuable criticism. Also,
to late Prof. Dr. Salah Ashour, former Professor of Microbiology,
Botany Department, Faculty of Science, Mansoura University for
biological activity evaluation.
References
[1] C. Melentyeva, L. Antonova, Pharmaceutical Chemistry, Mir. Publisher, Moscou,
Russian, 1988, 312
[2] L.A. Walter, W.H. Hunt, R.J. Fosbinder, J. Am. Chem. Soc. 63 (1941) 2771–2773.
[3] K. Shimizu, R. Ushijima, Ger. Pat. (1972), 2,217,084.;
K. Shimizu, R. Ushijima, Chem. Abstr. 78 (1973) 29775f.
[4] W.E. Barth, Ger. Pat. (1972), 2,204,195.;
W.E. Barth, Chem. Abstr. 77 (1972) 151968n.
[5] M.H. Sherlock, US Pat. (1974), 3,839,344.;
M.H. Sherlock, Chem. Abstr. 82 (1975) 16705n.
[6] W. Dittmar, E. Druckrey, H. Urbach, J. Med. Chem. 17 (1974) 753–756.
[7] F. Bossert, W. Vater, South African Pat. 68,01482.;
F. Bossert, W. Vater, Chem. Abstr. 70 (1969) 96641d.
[8] A.A. Fadda, R.E. El-Mekawy, A.I. El-Shafei, H. Freeman, Arch. Pharm. Chem. Life
Sci., in press. doi:10/1002/ardp.201100335 (ardp.201200313.R1).
[9] A.A.-H. Abdel-Rahman, W.A. El-Sayed, E.-S.G. Zaki, A.A. Mohamed, A.A. Fadda, J.
Heterocycl. Chem. 49 (2012) 93–101.
[10] H. Abuo-Melha, A.A. Fadda, Spectrochim. Acta, Part A: Mol. Biomol. Spectrosc.
89 (2012) 123–128.
[11] A.A. Fadda, S. Bondock, W. Khalifa, Eur. J. Med. Chem. 46 (2011) 2555–2561.
[12] A.A. Fadda, F.A. Badria, K.M. El-Attar, Med. Chem. Res. 19 (2010) 413–430.
[13] A.A. Fadda, A. El-Shafei, A.M. Khalil, T.A.E. Ameen, F.A. Badria, Bioorg. Med.
Chem. 17 (2009) 5096–5105.
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, respectively.
Stock solutions of the tested compounds, chloramphenicol, cepha-
lothin and cycloheximide were prepared in DMF at concentration
of 1000
lg/mL. Each stock solution was diluted with standard
[14] A.A. Fadda, E. Abdel-Latif, Rasha E. El-Mekawy, Eur. J. Med. Chem. 44 (2009)
1250–1256.
method broth (Difco) to prepare serial twofold dilutions in the
range of 500–3.125
lg/mL 10 mL of the broth containing about
[15] A.A. Fadda, S. Bondock, R. Rabie, H.A. Etman, Eur. J. Med. Chem. 43 (2008)
2122–2129.
[16] A.H. Sharmroukh, M.E.A. Zaki, E.M.H. Morsy, F.M.E. Abdel-Megeid, Arch.
Pharm. 340 (2007) 345–351.
[17] A.L. Koch, Clin. Microbiol. Rev. 16 (2003) 673–687.
[18] C.A.C. Haley, P. Maitland, J. Chem. Soc. (1951) 3155–3174.
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 re-