Synthesis of Some 1,4,6-Trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles and Their Biological Evaluation as Cytotoxic and Antimicrobial Agents

Article abstract of DOI:10.1002/ardp.201500175

A series of novel 1,4,6-trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles supported with some functionalities reported to contribute to significant chemotherapeutic potential were synthesized and evaluated for their antimicrobial and/or cytotoxic activities. Thirteen compounds exhibited cytotoxic potential against a panel of three human tumor cell lines. Compounds 15, 23, and 24 proved to be the most active agents with a broad spectrum of cytotoxic activity. Analog 24 was considered as the most active cytotoxic agent, being 2.5 times more active than doxorubicin against the colon HT29 carcinoma cell line. Seventeen compounds were able to exert a variable antimicrobial profile, among which analogs 15, 20, 21, 23, and 24 were prominently active. The highest antimicrobial potential was displayed by analog 24, being equipotent to ampicillin against Staphylococcus aureus and Escherichia coli, together with a considerable antifungal activity comparable with clotrimazole. Collectively, compounds 15, 23, and 24 could be considered as possible dual antimicrobial-anticancer candidates. Novel substituted 2-oxo-1,2-dihydropyridine-3-carbonitriles (A, B) and some derived 1,2,4-triazolo[4,3-a]-pyridinones (C) were synthesized and evaluated for their antimicrobial and cytotoxic activities. Three analogs are possible dual antimicrobial-anticancer candidates.

Full text of DOI:10.1002/ardp.201500175

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Full Paper
Synthesis of Some 1,4,6-Trisubstituted-2-oxo-1,2-dihydropyridine-
3-carbonitriles and Their Biological Evaluation as Cytotoxic and
Antimicrobial Agents
Hassan M. Faidallah1, Sherif A. F. Rostom2,3, Mona H. Badr2,3, Azza E. Ismail3,
and Ameen M. Almohammadi4
1
Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah,
2
Saudi Arabia
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria,
Egypt
4
Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
A series of novel 1,4,6-trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles supported with some
functionalities reported to contribute to significant chemotherapeutic potential were synthesized and
evaluated for their antimicrobial and/or cytotoxic activities. Thirteen compounds exhibited cytotoxic
potential against a panel of three human tumor cell lines. Compounds 15, 23, and 24 proved to be
the most active agents with a broad spectrum of cytotoxic activity. Analog 24 was considered as the
most active cytotoxic agent, being 2.5 times more active than doxorubicin against the colon HT29
carcinoma cell line. Seventeen compounds were able to exert a variable antimicrobial profile, among
which analogs 15, 20, 21, 23, and 24 were prominently active. The highest antimicrobial potential
was displayed by analog 24, being equipotent to ampicillin against Staphylococcus aureus and
Escherichia coli, together with a considerable antifungal activity comparable with clotrimazole.
Collectively, compounds 15, 23, and 24 could be considered as possible dual antimicrobial-anticancer
candidates.
Keywords: 1,3-Benzodioxole / Antimicrobial / Cytotoxic / Pyridines / Synthesis
Received: May 21, 2015; Revised: September 12, 2015; Accepted: September 14, 2015
DOI 10.1002/ardp.201500175
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
several complications among which toxicity and cellular drug
resistance are the most prevalent [2]. Therefore, there is
Introduction
Cancer is a multifaceted disease that continues to be the
second most common cause of death worldwide [1]. Although
chemotherapy is the mainstay for cancer treatment, the use of
available chemotherapeutic agents is usually associated with
still an urgent need to search for new lead structures that
might be beneficial in designing novel anticancer agents with
higher selectivity toward tumor cells and minimum side
effects [3]. Meanwhile, it is well documented that patients
undergoing anticancer chemotherapy are immune-suppressed
and are strongly susceptible to life-threatening systemic
microbial infections. However, the emergence of multi-drug
resistant bacteria and fungi constitutes a serious medical
problem that persists to provide impetus for the search and
discovery of new non-conventional antimicrobial agents
active against these pathogens [4]. Based on these facts, it
might be advantageous for patients suffering from cancer
Correspondence: Dr. Sherif A. F. Rostom, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz
University, P.O. Box 80260, Jeddah 21589, Saudi Arabia.
E-mail: sherifrostom@yahoo.com
Fax: þ9662-6400000-22327
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1,4,6-Trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles as Antimicrobial Agents
Archiv der Pharmazie
diseases accompanied with microbial infections to receive a
single drug entity which would possess a dual anticancer-
antimicrobial activity, especially in patients with malfunc-
tions in the liver and/or kidney. Such dually acting agent
would be beneficial in minimizing the frequency of drug
administration, and subsequently reducing the costs of the
treatment course. Besides, it could circumvent or delay the
development of multiple drug resistance.
attempt to enhance the biological activity of the pyridine
motif, the newly synthesized compounds were designed so as
to reserve the C3-carbonitrile function and comprise a C4-1,3-
benzodioxole moiety as essential counterparts at the main
pyridine ring. In addition, the target compounds were
substituted at the N1 by several functionalities such as the
nitroso, benzenesulfonyl, alkyl, thiocarbamoyl, formyl, and
acetyl groups (A and B; Fig. 1), that would represent different
electronic, lipophilic and steric environment, which would
assist the aimed biological actions. Moreover, owing to the
reported role of the triazole ring in inducing many
anticancer [31–33] and/or antimicrobial [34, 35] activities,
the pyridine ring was annulated in a triazolopyridine ring
system (C; Fig. 1), aiming to potentiate the targeted
anticancer and/or antimicrobial activities.
Comprehensive literature survey revealed that a wide range
of chemotherapeutic activities have been ascribed to pyridine
carbonitriles including antimicrobial [5–7], antifungal [8],
antitubercular [9], antiviral [10], and anticancer [10–12]
activities. Moreover, several pyridine-containing compounds
were documented to possess dual anticancer and antimicro-
bial activities [13–16]. Consequently, pyridine carbonitriles
and their derived fused rings would constitute a promising
template toward the design of a new category of chemother-
apeutic agents. On the other hand, the 1,3-benzodioxole
counterpart was found to be incorporated in a variety of
anticancer drugs with excellent bioavailability and low
toxicity [17–19]. Xia et al. [20] have documented that the
introduction of methylenedioxy moiety for 2-amino-chalcone
led to enhanced activity against multidrug resistant KB
carcinoma cells, compared with the unsubstituted analog.
Moreover, several 1,3-benzodioxole derivatives were found
to possess cytotoxic activity against a wide range of human
tumor cell lines including multidrug-resistant nasopharyngeal
carcinoma cells [20–23], in addition to remarkable anti-
bacterial [24] and antifungal [25] activities.
Over the past few years, we have been principally
engrossed in the synthesis and biological evaluation of
several pyridine-incorporating compounds [26–30] as novel
chemotherapeutic agents, among which those comprising
the substituted pyridine-3-carbonitrile scaffold have received
much concern due to their promising broad spectrum
antitumor and/or antimicrobial potentials.
Inspired by the afore-mentioned finding, and with the
hope to develop novel chemotherapeutic pyridine deriva-
tives, we report herein the synthesis, in vitro antimicrobial and
anticancer evaluation of some novel 1,4,6-trisubstituted-2-
oxo-1,2-dihydropyridine-3-carbonitrile derivatives. In an
Results and discussion
Chemistry
The synthetic strategies employed to obtain the intermediate
and target compounds are illustrated in Schemes 1 and 2.
In Scheme 1, two synthetic strategies were adopted to
synthesize the key intermediates 6-aryl-4-(benzo[1,3]dioxol-
5-yl)-2-oxo-1,2-dihydropyridine-3-carbonitriles 7–9. The first
method involved the formation of 1-aryl-3-(benzo[1,3]dioxol-
5-yl)-prop-2-en-1-ones 4–6 (chalcones) via Claisen-Schmidt
condensation of the appropriate aromatic ketones 1–3 with
benzo[1,3]dioxole-5-carbaldehyde (piperonal) in ethanolic
potassium hydroxide [36]. These chalcones 4–6 were subjected
to cycloaddition condensation reaction with ethyl cyanoace-
tate and excess ammonium acetate as a nitrogen source to
obtain the requested intermediates 7–9. On the other hand,
the same compounds 7–9 were directly prepared by one-pot
multicomponent reaction (MCR) of the appropriate aromatic
ketone 1–3, piperonal, ethyl cyanoacetate, and an excess of
ammonium acetate in boiling ethanol. The latter method
gave better results (higher yields and less time consumption)
when compared with the two-stepped procedure. Reacting
7–9 with sodium nitrite in the presence of cold acetic acid
yielded the corresponding N-nitroso derivatives 10–12.
Figure 1. General structures of the newly synthesized compounds A, B, and C.
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Scheme 1. Synthesis of compounds 4–24.
Moreover, refluxing compounds 4–6 with benzenesulfonyl
achieved by heating 7–9 with trifluoroacetic acid in tetrahy-
drofuran. Finally, condensing the N-acylated analogs 26 and
30–32 with hydrazine hydrate afforded the bicyclic substituted
1,2,4-triazolo[4,3-a]pyridinones 29 and 33–35, respectively.
chloride in pyridine as
a basic medium afforded the
corresponding N-benzenesulfonyl derivatives 13–15. Further-
more, alkylation of compounds 7–9 with the appropriate alkyl
iodide in pyridine favored the production of the N-alkyl
derivatives 16–21. Their IR spectra were characterized by
the existence of the carbonyl absorption bands at 1654–
1645 cmꢀ1, while they lacked the NH bands. Analogously, the
N-phenylthiocarbamoyl analogs 22–24 were prepared by
heating 7–9 with phenyl isothiocyanate in pyridine. Their IR
spectra showed a new absorption band at 1236–1220 cmꢀ1
Biology
In vitro MTT cytotoxicity assay
All the newly synthesized 29 compounds were investigated
for their in vitro cytotoxic effect against a panel of three
human tumor cell lines, namely, Caucasian breast adenocar-
cinoma MCF7, hepatocellular carcinoma Hep-G2, and colon
carcinoma HT29, via the standard [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] (MTT) method [37, 38].
The results are presented in Table 1 as LC50 (mg/mL) which
is the lethal concentration of the compound which causes
death of 50% of the cells in 24 h.
The obtained data revealed that 13 analogs, 8, 9, 14, 15, 17,
18, 23, 24, 25, 31, 32, 34, and 35, exhibited variable degrees of
inhibitory activity toward the three tested human tumor cell
lines, whereas the rest of the compounds were totally
inactive. In this respect, the human colon carcinoma HT29
was proved to be the most sensitive cell line as its growth was
–
–
due to the C S function in addition to the C O and CN
–
–
absorptions in the regions 1654–1648 and 2228–2220 cmꢀ1
,
respectively.
In Scheme 2, the same key intermediates 7–9 served as
precursors for the synthesis of the target compounds 25–35. In
this view, heating compound 8 with formic acid furnished the
N-formyl derivative 25. Whereas, reacting compounds 7–9
with acetic anhydride in the presence of anhydrous sodium
acetate resulted in the formation of the corresponding
N-acetyl derivatives 26–28. In an analogous fashion, the
formation of the N-trifluoroacetyl derivatives 30–32 was
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1,4,6-Trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles as Antimicrobial Agents
Archiv der Pharmazie
Scheme 2. Synthesis of compounds 25–35.
inhibited by the 13 active compounds, particularly compounds
23 and 24 (LC50 14.9 and 8.4 mg/mL, respectively), which were
remarkably more active than doxorubicin (LC50 21.1 mg/mL),
the reference standard cytotoxic agent utilized in this
study. Moreover, compounds 14, 15, and 32 displayed a
remarkable cytotoxic potential (LC50 34.2, 29.6, and
31.2 mg/mL), while the remaining compounds showed mod-
erate to mild activity (LC50 41.5–72.1 mg/mL) against the same
cell line. Regarding activity against the human hepatocellular
carcinoma Hep-G2 cell line, compounds 15, 23, and 24
revealed the highest cytotoxicity profiles (LC50 18.5, 11.6,
and 7.2 mg/mL, respectively) as compared to doxorubicin
Table 1. Cytotoxic effects (LC50; mg/mL)a) of the active
compounds on some human tumor cell lines using the
MTT assay.
Compound
HT29b)
Hep-G2c)
MCF7d)
8
9
14
15
17
18
23
24
25
31
32
34
65.8
47.1
34.2
29.6
54.6
41.5
14.9
8.4
72.1
43.6
31.2
51.3
42.8
21.1
57.2
26.3
22.4
18.5
46.3
34.1
24.8
25.2
–
e)
–
–
–
(LC50 1.69 mg/mL). However,
a weak growth inhibitory
11.6
7.2
12.1
9.7
–
efficiency was recorded for the rest of the active compounds
against the same cell line (LC50 21.3–57.2 mg/mL). Although
the human breast cancer MCF7 was proved to be the least
sensitive cell line as it was affected by only eight of the
active compounds, yet a noticeable activity was displayed by
compounds 23 and 24 (LC50 12.1 and 9.7 mg/mL, respectively),
when compared with doxorubicin (LC50 2.14 mg/mL).
–
38.5
29.2
26.6
21.3
1.69
–
–
46.2
40.6
2.14
35
Doxorubicinf)
With regard to the broad spectrum of cytotoxic activity,
eight analogs, namely 8, 9, 14, 15, 23, 24, 34, and 35,
demonstrated considerable activity against the three tested
human tumor cell lines, among which the analogs 15, 23,
and 24 were considered to be the most active with a broad
spectrum of cytotoxic activity. In particular, the analog 24 has
stemmed as the most active member in the present study
a) LC50: Lethal concentration of the compound which causes
death of 50% of cells in 24 h.
b) HT29 (human colon carcinoma cell line).
c) Hep-G2 (human hepatocellular carcinoma cell line).
d) MCF7 (human breast cancer cell line).
e) Totally inactive against this cell line.
f) Positive control cytotoxic agent.
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being 2.5 times more active than doxorubicin against the
colon HT29 carcinoma cell line, besides showing about 25% of
the activity of the same standard against the Hep-G2 and
MCF7 carcinoma cell lines (Table 1).
into the [1,2,4]triazolo[4,3-a]pyridine derivative 29 resulted
in a complete abolishment of activity, yet the introduction
of a trifluoroacetyl moiety at position-1 of the pyridine ring
gave rise to compounds 31 and 32 with moderate cytotoxicity
against the HT29 and Hep-G2 carcinoma cell lines. Finally,
cyclization of the latter analogs 31 and 32 into the
corresponding [1,2,4]triazolo[4,3-a]pyridine derivatives 34
and 35 resulted in relative improvement in the spectrum of
cytotoxic activity, however, their potency was comparable
with the starting analogs 8 and 9 against the three tested
carcinoma cell lines.
The obtained cytotoxic potential of the active compounds
could be correlated to the performed structure variations
and modifications. In general, the cytotoxic efficiency
was connected with derivatives encountering bromo- and
methoxyphenyl moieties (R ¼ Br or OCH3) at position-6 of
the pyridine ring, whereas those with unsubstituted phenyl
ring (R ¼ H), were totally inactive. In this context, the key
precursors 8 (R ¼ Br) and 9 (R ¼ OCH3) exhibited moderate to
weak cytotoxic profiles toward the three tested tumor cell
lines. Nitrosation as in compounds 10–12 resulted in a
complete abolishment of activity. However, introduction of
a benzenesulfonyl moiety at position-1 (14 and 15) led to an
obvious improvement in the cytotoxic potential, especially 15
(R ¼ OCH3) which was nearly equiactive with doxorubicin
against the colon HT29 carcinoma cell line. Whereas,
N-alkylation with a methyl or ethyl groups led to a complete
loss of activity, except compounds 17 (R ¼ Br; R1 ¼ CH3) and 18
(R ¼ OCH3; R1 ¼ CH3), which showed about 40–50% of the
activity of doxorubicin against the colon HT29 carcinoma cell
line. On the other hand, a significant enhancement in both
cytotoxic potency and spectrum of activity was exemplified
by the incorporation of a phenylthiocarbamoyl moiety at
position-1 of the pyridine scaffold (23; R ¼ Br) and (24;
R ¼ OCH3). Among these, the anlog 24 was proved to be
the most active member in the present study when compared
with doxorubicin. Furthermore, formylation represented
by the analog 25 led to a dramatic reduction in activity.
Although acetylation (26–28) and subsequent cyclization
In vitro antibacterial and antifungal activity
All the newly synthesized compounds were evaluated for
their in vitro antibacterial activity against Staphylococcus
aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), and
Micrococcus luteus (ATCC 21881) as examples of Gram-
positive bacteria and Escherichia coli (ATCC 25922), Pseudo-
monas aeruginosa (ATCC 27853), and Klebsiella pneumonia
(clinical isolate) as examples of Gram-negative bacteria. They
were also evaluated for their in vitro antifungal potential
against Candida albicans (ATCC 10231) and Aspergillus niger
(recultured) fungal strains. Their inhibition zones using agar
cup diffusion technique were measured [39]. The minimum
inhibitory concentration (MIC, mg/mL) was also determined
using the twofold serial dilution method [40]. Ampicillin was
employed as a standard antibacterial agent, while clotrimazole
was used as antifungal reference drug. Dimethylsulfoxide
(DMSO) was used as a blank and showed no antimicrobial
activity. The MIC (mg/mL) values are recorded in Table 2.
The results depicted in Table 2 revealed that 17 out of
the newly 35 synthesized compounds displayed variable
Table 2. Minimal inhibitory concentrations (MIC, mg/mL) of the active compounds.
Compound no.
S. aureus
B. subtilis
E. coli
P. aeruginosa
C. albicans
a)
7
8
9
13
14
15
17
20
21
22
23
24
25
31
32
34
100
50
25
50
25
12.5
50
25
12.5
25
12.5
6.25
100
100
25
200
100
6.25
–
100
100
50
100
100
25
100
100
50
50
50
25
200
100
100
–
100
50
25
100
25
6.25
100
12.5
6.25
25
12.5
ꢀ
–
–
100
50
100
50
25
100
25
25
100
50
25
200
50
50
–
–
–
100
25
–
100
50
50
25
25
–
6.25
100
50
25
–
100
–
–
35
–
200
6.25
–
–
Ampicillin
Clotrimazole
12.5
–
12.5
–
–
–
6.25
a) Inactive: MIC > 200 mg/mL.
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inhibitory effects on the growth of the tested Gram-positive
and Gram-negative bacterial strains. In general, the Gram-
positive S. aureus and the Gram-negative E. coli were found to
be the most sensitive microorganisms. On the contrary, the
tested compounds lacked any antibacterial potential against
Gram-positive M. luteus and Gram-negative K. pneumonia.
Moreover, some members showed moderate antifungal
activity against C. albicans, while all the tested compounds
were totally inactive against Asp. niger.
Among the tested Gram-positive bacteria, S. aureus,
showed relative high sensitivity toward the tested com-
pounds. In particular, compound 24 was equipotent to
ampicillin (MIC 6.25 mg/mL), whereas the analogs 15, 21,
and 23 (MIC 12.5 mg/mL) exhibited half the activity of
ampicillin against S. aureus. Furthermore, compounds 9, 14,
20, 22, and 32 (MIC 25 mg/mL) showed 25% of the activity of
ampicillin against the same strain. Regarding the activity
against B. subtilis, compounds 15 and 24 showed 50% of the
activity of ampicillin. On the other hand, investigation of the
antibacterial potential of the active compounds against
the Gram-negative strains, two analogs namely 15 and 24
were able to display a distinctive growth inhibitory activity
against E. coli (MIC 6.25 mg/mL) being equiactive with
ampicillin. Compounds 20 and 23 (MIC 12.5 mg/mL) showed
half the activity of ampicillin, whereas, 9, 14, 22, and 32
(MIC 25 mg/mL) exhibited moderate activity against the
same organism. On the contrary, the tested P. aeruginosa
strain revealed the least sensitivity toward most of the active
compounds, except 15, 20, 21, and 24 (MIC 25 mg/mL), which
displayed 50% of the activity of ampicillin.
especially against E. coli (equipotent to ampicillin), S. aureus,
B. subtilis, and P. aeruginosa (half the activity of ampicillin),
together with an appreciable antifungal activity against
C. albicans. Alkylation with a methyl or ethyl group gave rise
to compounds 16–21 with variable degrees of antimicrobial
activity. Among these, compounds 20 (R ¼ Br; R1 ¼ C2H5) and
21 (R ¼ OCH3; R1 ¼ C2H5) showed an obvious improvement
in the antimicrobial activity against S. aureus, E. coli, and
P. aeruginosa when compared to their precursors 8 and 9;
respectively, in addition to a moderate antifungal activity.
Furthermore, introduction of a phenylthiocarbamoyl moiety
as in compounds 22–24 resulted in a significant enhancement
in the overall antimicrobial potential, among which com-
pound 24 (R ¼ OCH3) was the most active member. When
compared with the parent compound 9, the analog 24
displayed a fourfold increase in the antibacterial activity
against S. aureus and E. coli, together with a moderate
antifungal activity. Introduction of a formyl group, as in
compound 25, led to an obvious reduction of the overall
activity, when compared with the parent compound 8,
whereas, the N-acetyl derivatives 26–28 were completely
devoid of any antimicrobial activity. Moreover, incorporating
a trifluoromethyl moiety at position-1 of the pyridine ring as
in compounds 30–32 did not offer a significant advantage to
the antimicrobial efficiency. Finally, cyclization of the N-acetyl
or N-trifluoroacetyl derivatives into the corresponding [1,2,4]-
triazolo[4,3-a]pyridines 29 and 33–35 resulted in either a
dramatic reduction in the antimicrobial spectrum and activity
(34 and 35) or a complete loss of activity (29 and 33).
Concerning the antifungal activity, compounds 15, 23, and
24 (MIC 25 mg/mL) were the most active members against
C. albicans, as they displayed 25% of the activity of
clotrimazole (MIC 6.25 mg/mL), whereas the other tested
compounds showed very mild or completely devoid of activity
(MIC 50 to >200 mg/mL).
Conclusion
The aim of this study was to synthesize novel 1,4,6-
trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles sup-
ported with some functionalities that are reported to exert
potential chemotherapeutic activities to be evaluated for
their cytotoxic and/or antimicrobial activities. The results
revealed that 13 compounds were able to exhibit remarkable
cytotoxic potential against human colon carcinoma HT29,
hepatocellular carcinoma Hep-G2, and Caucasian breast
adenocarcinoma MCF7 cell lines. The obtained results
indicated that compounds 15, 23, and 24 were the most
active with a broad spectrum of cytotoxic activity, especially
the analog 24 which is considered the most active cytotoxic
member being 2.5 times more active than doxorubicin against
the colon HT29 carcinoma cell line, together with showing
about 25% of the activity of the same standard against the
Hep-G2 and MCF7 carcinoma cell lines. Meanwhile, 17
compounds were able to exert antibacterial activity against
S. aureus and E. coli strains, in addition to a moderate
antifungal activity against C. albicans. Among these, five
analogs namely 15, 20, 21, 23, and 24 could be considered as
the most active broad spectrum antimicrobial members
identified in this study. Compound 24 proved to be the most
active candidate as it showed equipotency with ampicillin
A deep insight in the structures of the tested compounds
revealed that their antimicrobial activity seems to be
modulated by the nature of the substituent at position-1,
together with the type of substituent at the C6 phenyl group
of the pyridine scaffold. In general, it could be deduced that
significant antibacterial activity with broad spectrum of
activity was associated with compounds incorporating either
a methoxy or a bromo substituent at position-4 of the
C6 phenyl ring. In this respect, the key intermediates 4,6-
disubstituted-2-oxo-1,2-dihydropyridine carbonitriles 7–9
showed moderate to weak antibacterial activity, among
which the brominated (8, R ¼ Br) and the methoxylated
analogs (9, R ¼ OCH3) were the most active members,
particularly against S. aureus and E. coli. Introduction of a
nitroso group at position-1 as in compounds 10–12 led to
a complete abolishment of activity. On the other hand,
incorporating a benzenesulfonyl moiety at position-1 fur-
nished three active compounds 13–15, in which the methoxy-
lated derivative 15 displayed a significant enhancement in
both the potency and spectrum of the antibacterial activity
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against S. aureus and E. coli, together with a considerable
antifungal activity comparable with clotrimazole. Collective-
ly, compounds 15, 23, and 24 could be considered as possible
dual antimicrobial-anticancer candidates that makes such
pyridine scaffold the appropriate subject for future derivati-
zation with the hope of discovering more potent and selective
cytotoxic and/or antimicrobial agents.
1H NMR (d ppm) of compound 9: 3.80 (s, 3H, OCH3), 6.14 (s, 2H,
CH2), 7.18–7.72 (m, 8H, Ar H, and pyridine C5-H), 8.14 (s, 1H,
NH). 13C NMR (d ppm) of 9: 56.0 (OCH3), 92.9 (CH2), 117.9 (CN),
100.1, 101.6, 114.0, 120.3, 124. 5, 127.1, 127.2, 127.3, 127.8,
130.4, 136.6, 136.8, 161.2, 162.5 (Ar C), 162.9 (CO).
General procedure for the synthesis of 6-aryl-4-(benzo[1,3]-
dioxol-5-yl)-1-nitroso-2-oxo-1,2-dihydropyridine-3-
carbonitriles 10–12
To an ice-cooled stirred solution of 7–9 (10 mmol) in acetic acid
(15 mL) was added drop-wise a solution of sodium nitrite
(1.05 g, 15 mmol) in water (5 mL) over a period of 1 h. Stirring
was maintained for further 2 h, and then the reaction mixture
was left at room temperature overnight. The resulting solid
product was filtered, washed with water, dried, and recrystal-
lized from DMF. Physicochemical and analytical data are
recorded in Table 3.
Experimental
Chemistry
Melting points were determined on a Gallenkamp melting
point apparatus and are uncorrected. The infrared (IR) spectra
were recorded on Shimadzu FT-IR 8400S infrared spectro-
photometer using the KBr pellet technique. H and 13C NMR
1
spectra were recorded on
a Bruker WM-600 FT NMR
spectrometer using tetramethylsilane as the internal standard
and dimethyl sulfoxide-d6 (DMSO-d6) as a solvent (chemical
shifts in d, ppm). Splitting patterns were designated as
follows: s: singlet; d: doublet; t: triplet; q: quartet and m:
multiplet. Elemental analyses were performed on a 2400
Perkin Elmer Series 2 analyzer and the found values were
within ꢁ0.4% of the theoretical values. Follow up of the
reactions and checking the homogeneity of the compounds
were made by thin layer chromatography on silica gel-
protected aluminum sheets (Type 60 F254, Merck) and the
spots were detected by exposure to UV-lamp at l 254.
IR (cmꢀ1) of compounds 10–12: 2220–2215 (CN), 1652–1645
–
(C O).
–
1H NMR (d ppm) of compound 10: 6.04 (s, 2H, CH2), 7.14–
7.88 (m, 9H, Ar H, and pyridine C5-H). 13C NMR (d ppm) of 10:
92.4 (CH2), 117.5 (CN), 101.4, 114.3, 119.8, 121.9, 124.2, 126.8,
127.3, 128.0, 129.1, 130.7, 131.3, 139.7, 148.4, 164.8 (Ar C),
163.3 (CO).
1H NMR (d ppm) of compound 11: 6.12 (s, 2H, CH2), 7.15–
7.55 (m, 8H, Ar H, and pyridine C5-H). 13C NMR (d ppm) of 11:
91.8 (CH2), 117.8 (CN), 104.7, 108.6, 122.3, 123.8, 126.3, 127.8,
128.3, 129.1, 130.2, 130.8, 131.9, 132.2, 148.5, 153.6 (Ar C),
162.8 (CO).
General procedure for the synthesis of 6-aryl-4-(benzo[1,3]-
dioxol-5-yl)-2-oxo-1,2-dihydropyridine-3-carbonitriles 7–9
Method A: A mixture of 4–6 (10 mmol), ethyl cyanoacetate
(1.1 g, 10mmol), and ammonium acetate (6.2 g, 80 mmol) in
absolute ethanol (30 mL) was heated under reflux for 4 h. After
being cooled to R.T., the solid product formed was filtered,
washed with water, dried, and recrystallized from ethanol.
1H NMR (d ppm) of compound 12: 3.81 (s, 3H, OCH3), 6.15 (s,
2H, CH2), 7.19–7.76 (m, 8H, Ar H, and pyridine C5-H). 13C NMR
(d ppm) of 12: 56.1 (OCH3), 91.6 (CH2), 117.3 (CN), 100.2, 101.4,
108.0, 114.2, 126.5, 127.3, 127.4, 127.5, 128.0, 130.3, 136.6,
136.7, 161.1, 163.9 (Ar C), 162.8 (CO).
General procedure for the synthesis of 6-aryl-1-
Method B:
A
mixture of piperonal (1.5 g, 10 mmol),
benzenesulfonyl-4-(benzo[1,3]dioxol-5-yl)-2-oxo-1,2-
dihydropyridine-3-carbonitriles 13–15
arylaldehyde 1–3 (10 mmol), ethyl cyanoacetate (1.1 g,
10 mmol), and ammonium acetate (6.2 g, 80 mmol) in abso-
lute ethanol (50 mL) was heated under reflux for 6 h. The
reaction mixture was allowed to cool, and the formed
precipitate was filtered, washed with water, dried, and
recrystallized from ethanol. Physicochemical and analytical
data are recorded in Table 3.
A mixture of 7–9 (10 mmol) and benzenesulfonyl chloride
(1.77 g, 10 mmol,) in pyridine (10 mL) was heated under reflux
for 5 h. After cooling, the reaction mixture was poured onto
crushed ice and the separated solid product was filtered,
washed with water, dried, and recrystallized from ethanol.
Physicochemical and analytical data are recorded in Table 3.
IR (cmꢀ1) of compounds 13–15: 2232–2224 (CN), 1652–1640
IR (cmꢀ1) of compounds 7–9: 3314–3278 (NH), 2226–2218
–
–
(CN), 1650–1648 (C O).
(C O), 1382–1370, 1168–1159 (SO ).
–
–
2
1H NMR (d ppm) of compound 7: 6.04 (s, 2H, CH2), 7.10–7.98
(m, 9H, Ar H, and pyridine C5-H), 8.14 (s, 1H, NH). 13C NMR
(d ppm) of 7: 93.6 (CH2), 117.1 (CN), 101.9, 114.5, 120.0, 122.2,
124.6, 127.0, 127.2, 128.2, 129.4, 130.9, 131.6, 139.9, 148.2,
164.6 (Ar C), 162.7 (CO).
1H NMR (d ppm) of compound 13: 6.15 (s, 2H, CH2), 6.98–
7.87 (m, 14H, Ar H, and pyridine C5-H). 13C NMR (d ppm) of 13:
92.3 (CH2), 117.2 (CN), 102.2, 125.2, 126.3, 127.1, 127.3, 128.2,
129.2, 131.6, 132.4, 134.5, 136.6, 136.9, 144.2, 148.0, 154.5,
160.4 (Ar C), 162.7 (CO).
1H NMR (d ppm) of compound 8: 6.07 (s, 2H, CH2), 7.06–7.92
(m, 8H, Ar H, and pyridine C5-H), 8.11 (s, 1H, NH). 13C NMR
(d ppm) of 8: 93.4 (CH2), 117.3 (CN), 102.1, 111.6, 120.0, 122.4,
124.3, 127.1, 127.3, 128.1, 129.2, 130.8, 131.5, 139.8, 148.4,
164.3 (Ar C), 163.2 (CO).
1H NMR (d ppm) of compound 14: 6.05 (s, 2H, CH2), 6.79–
7.98 (m, 13H, Ar H, and pyridine C5-H). 13C NMR (d ppm) of 14:
92.1 (CH2), 117.1 (CN), 102.0, 125.4, 126.3, 127.1, 127.2, 128.3,
129.1, 131.7, 132.4, 134.6, 136.2, 136.9, 144.4, 148.1, 154.6,
160.6 (Ar C), 162.8 (CO).
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1,4,6-Trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles as Antimicrobial Agents
Archiv der Pharmazie
Table 3. Physicochemical and analytical data for compounds 7–35.
Mol. formulaa)
(mol. weight)
Compd. no.
7
R
R1
Yield (%)
M.p. (°C)
H
–
65 (A)
86 (B)
59 (A)
87 (B)
56 (A)
80 (B)
72
74
71
72
74
72
86
84
85
86
84
85
76
84
79
70
84
86
86
73
72
72
70
73
72
292–4
C19H12N2O3 (316.31)
8
9
Br
–
–
340–2
264–6
C19H11 BrN2O3 (395.21)
C20H14N2O4 (346.34)
OCH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
H
Br
OCH3
H
Br
OCH3
H
Br
OCH3
H
Br
OCH3
H
Br
OCH3
Br
H
Br
OCH3
H
–
328–30
288–90
262–4
278–80
268–70
238–40
276–8
328–30
258–60
267–9
258–60
142–3
220–2
282–3
C19H11N3O4 (345.31)
–
C19H10BrN3O4 (424.2)
C20H13N3O5 (375.33)
C25H16N2O5S (456.47)
C25H15BrN2O5S (535.37)
C26H18N2O6S (486.5)
C20H14N2O3 (330.1)
C20H13BrN2O3 (409.23)
C21H16N2O4 (360.36)
C21H16N2O3 (344.36)
C21H15BrN2O3 (423.26)
C22H18N2O4 (374.39)
–
–
–
–
CH3
CH3
CH3
C2H5
C2H5
C2H5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
C26H17N3O3S (451.5)
C26H16BrN3O3S (530.39)
C27H19N3O4S (481.52)
C20H11BrN2O4 (423.22)
C21H14N2O4 (358.35)
262–3
291-–3
318–20
286–8
270-–2
298-–9
318-–20
325–7
268–70
265–7
269–71
264–6
C21H13BrN2O4 (437.24)
C22H16N2O5 (388.37)
C21H14N4O2 (354.11)
C21H11F3N2O4 (412.32)
C21H10BrF3N2O4(489.98)
C22H13F3N2O5 (442.34)
C21H11F3N4O2 (408.33)
C21H10BrF3N4O2 (487.23)
C22H13F3N4O3 (438.36)
H
Br
OCH3
H
Br
OCH3
75
a) The found values are within ꢁ0.4% of the calculated values.
1H NMR (d ppm) of compound 15: 3.81 (s, 3H, OCH3), 5.92 (s,
2H, CH2), 6.86–8.10 (m, 13H, Ar H, and pyridine C5-H). 13C NMR
(d ppm) of 15: 56.0 (OCH3), 92.4 (CH2), 117.0 (CN), 100.6, 101.3,
114.3, 125.4, 127.0, 127.2, 128.0, 129.2, 131.3, 132.2, 134.3,
136.4, 136.7, 144.2, 154.8, 161.3 (Ar C), 163.0 (CO).
(d ppm) of 16: 31.0 (CH3), 93.6 (CH2), 117.1 (CN), 101.9, 114.7,
120.0, 122.2, 124.6, 127.0, 127.1, 128.0, 129.2, 130.7, 131.3,
138.9, 148.1, 164.5 (Ar C), 162.8 (CO).
1H NMR (d ppm) of compound 17: 3.34 (s, 3H, NCH3), 6.09 (s,
2H, CH2), 7.02–7.97 (m, 8H, Ar H, and pyridine C5-H). 13C NMR
(d ppm) of 17: 31.2 (CH3), 93.4 (CH2), 117.5 (CN), 102.9, 111.6,
120.1, 122.3, 124.3, 127.1, 127.3, 128.5, 129.7, 130.5, 131.3,
139.8, 148.1, 164.3 (Ar C), 163.6 (CO).
General procedure for the synthesis of 1-alkyl-6-aryl-4-
(benzo[1,3]dioxol-5-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitriles 16–21
1H NMR (d ppm) of compound 18: 3.36 (s, 3H, NCH3), 3.82 (s,
3H, OCH3), 6.04 (s, 2H, CH2), 7.18–7.72 (m, 8H, Ar H and
pyridine C5-H). 13C NMR (d ppm) of 18: 31.3 (NCH3), 56.0
(OCH3), 91.9 (CH2), 117.6 (CN), 100.1, 101.4, 114.0, 120.5,
126.3, 127.1, 127.3, 127.4, 127.7, 130.2, 136.6, 136.9, 161.1,
162.9 (Ar C), 163.6 (CO).
To a solution of 7–9 (10 mmol) in pyridine (10 mL), alkyl iodide
(10 mmol) was added and the mixture was heated under
reflux for 4 h. The reaction mixture was allowed to attain
room temperature, poured onto ice cold water, and the
separated solid product was filtered, washed with water,
dried, and recrystallized from ethanol. Physicochemical and
analytical data are recorded in Table 3.
1H NMR (d ppm) of compound 19: 1.22 (t, J ¼ 6 Hz, 3H, CH3),
3.03 (q, J ¼ 6 Hz, 2H, CH2), 6.04 (s, 2H, CH2), 6.59–8.05 (m, 9H,
Ar H and pyridine C5-H). 13C NMR (d ppm) of 19: 13.9 (CH3),
37.2 (CH2), 93.7 (CH2), 117.9 (CN), 100.1, 101.9, 120.3, 122.1,
124.8, 127.0, 127.1, 128.2, 129.3, 130.6, 131.5, 138.5, 148.0,
164.7 (Ar C), 161.4 (CO).
IR (cmꢀ1) of compounds 16–21: 2232–2229 (CN), 1654–1645
–
(C O).
–
1H NMR (d ppm) of compound 16: 3.33 (s, 3H, NCH3), 5.97 (s,
2H, CH2), 7.10–8.01 (m, 9H, Ar H, and pyridine C5-H). 13C NMR
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H. M. Faidallah et al.
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1H NMR (d ppm) of compound 20: 1.18 (t, J ¼ 6 Hz, 3H, CH3),
3.09 (q, J ¼ 6 Hz, 2H, CH2), 6.12 (s, 2H, CH2), 6.63–7.92 (m, 8H,
Ar H and pyridine C5-H). 13C NMR (d ppm) of 20: 14.0 (CH3),
37.8 (CH2), 93.4 (CH2), 117.5 (CN), 101.8, 111.6, 120.1, 122.3,
124.3, 127.1, 127.3, 128.5, 129.7, 130.5, 131.3, 139.8, 148.1,
164.3 (Ar C), 163.6 (CO).
General procedure for the synthesis of 1-acetyl-6-aryl-4-
(benzo[1,3]dioxol-5-yl)-2-oxo-1,2-dihydropyridine-3-
carbonitriles 26–28
To a solution of 7–9 (10 mmol) in acetic anhydride (10 mL),
anhydrous sodium acetate (1.2 g, 15 mmol) was added. The
reaction mixture was heated under reflux for 4 h, allowed to
cool then poured onto crushed ice with vigorous stirring. The
formed solid product was filtered, thoroughly washed with
water, dried, and recrystallized from ethanol. Physicochemical
and analytical data are recorded in Table 3.
1H NMR (d ppm) of compound 21: 1.24 (t, J ¼ 6 Hz, 3H, CH3),
3.05 (q, J ¼ 6 Hz, 2H, CH2), 3.80 (s, 3H, OCH3), 6.10 (s, 2H, CH2),
6.67–7.83 (m, 8H, Ar H and pyridine C5-H). 13C NMR (d ppm) of
21: 13.9 (CH3), 37.1(CH2), 56.1 (OCH3), 91.8 (CH2), 118.1 (CN),
100.1, 101.8, 114.0, 120.4, 126.2, 127.0, 127.2, 127.3, 127.8,
130.4, 136.7, 137.1, 161.2, 162.9 (Ar C), 161.6 (CO).
IR (cmꢀ1) of compounds 26–28: 2230–2228 (CN), 1708–1702
–
(CO acetyl), 1646–1642 (C O pyridone).
–
1H NMR (d ppm) of compound 26: 2.47 (s, 3H, COCH3), 5.95
(s, 2H, CH2), 6.81–8.11 (m, 9H, Ar H, and pyridine C5-H).
13C NMR (d ppm) of 26: 17.5 (CH3), 92.3 (CH2), 118.1 (CN),
101.2, 104.7, 120.2, 122.1, 124.4, 127.0, 127.2, 128.1, 129.0,
130.8, 131.2, 139.2, 148.1, 164.4 (Ar C), 160.9 (CO), 169.2 (CO).
1H NMR (d ppm) of compound 27: 2.48 (s, 3H, COCH3), 6.159
(s, 2H, CH2), 6.69–7.93 (m, 8H, Ar H, and pyridine C5-H).
13C NMR (d ppm) of 27: 17.8 (CH3), 93.2 (CH2), 118.2 (CN),
100.2, 111.4, 120.2, 122.5, 124.6, 127.0, 127.2, 128.4, 129.5,
130.4, 131.2, 139.7, 148.0, 164.4 (Ar C), 161.0 (CO), 169.6 (CO).
1H NMR (d ppm) of compound 28: 2.50 (s, 3H, COCH3); 3.81
(s, 3H, OCH3), 6.10 (s, 2H, CH2), 6.58–7.79 (m, 8H, Ar H, and
pyridine C5-H). 13C NMR (d ppm) of 28: 18.0 (CH3), 56.1 (OCH3),
92.3 (CH2), 117.9 (CN), 100.2, 101.4, 120.4, 114.1, 126.3, 127.0,
127.3, 127.5, 127.7, 130.2, 136.6, 136.8, 161.1, 161.6 (Ar C),
164.0 (CO), 168.9 (CO).
General procedure for the synthesis of 6-aryl-4-(benzo[1,3]-
dioxol-5-yl)-3-cyano-2-oxo-N-phenylpyridine-1(2H)-
carbothioamides 22–24
A mixture of 7–9 (10 mmol) and the phenyl isothiocyanate
(1.35 g, 10 mmol) in pyridine (10 mL) was heated under reflux
for 6 h. After being cooled to room temperature, the reaction
mixture was poured onto ice cold water and the separated
solid product was filtered, washed with water, dried, and
recrystallized from ethanol. Physicochemical and analytical
data are recorded in Table 3.
IR (cmꢀ1) of compounds 22–24: 1236–1220 (CS), 2228–2220
–
(CN), 1654–1648 (C O).
–
1H NMR (d ppm) of compound 22: 6.13 (s, 2H, CH2), 6.78–
7.89 (m, 15H, Ar H, pyridine C5-H and NH). 13C NMR (d ppm) of
22: 92.3 (CH2), 117.2 (CN), 102.2, 125.2, 126.3, 127.1, 127.3,
128.2, 129.2, 131.6, 132.4, 134.5, 136.6, 136.9, 144.2, 148.0,
154.5, 160.4 (Ar C), 162.7 (CO), 179.8 (CS).
General procedure for the synthesis of 6-aryl-4-(benzo[1,3]-
dioxol-5-yl)-1-trifluoroacetyl)-2-oxo-6-phenyl-1,2-
dihydropyridine-3-carbonitriles (30–32)
A solution of 7–9 (10 mmol) and trifluoroacetic anhydride
(2.1 g, 10 mmol) in THF (25 mL) was heated under reflux for
4 h. The reaction mixture was then cooled, poured onto ice
cold water, and the precipitated product was filtered, washed
with water, dried, and recrystallized from ethanol. Physico-
chemical and analytical data are recorded in Table 3.
IR (cmꢀ1) of compounds 30–32: 2228–2225 (CN), 1710–1706
1H NMR (d ppm) of compound 23: 6.05 (s, 2H, CH2), 6.79–
7.88 (m, 14H, Ar H, pyridine C5-H and NH). 13C NMR (d ppm) of
23: 92.4 (CH2), 117.4 (CN), 102.0, 125.2, 126.5, 127.0, 127.1,
128.3, 129.1, 131.7, 132.6, 134.7, 136.4, 137.1, 144.2, 148.1,
154.7, 161.3 (Ar C), 162.8 (CO) 179.9 (CS).
1H NMR (d ppm) of compound 24: 3.81 (s, 3H, OCH3), 6.02 (s,
2H, CH2), 6.86–8.10 (m, 14H, Ar H, pyridine C5-H, and NH).
13C NMR (d ppm) of 24: 56.0 (OCH3), 92.3 (CH2), 117.2 (CN),
100.2, 101.5, 114.0, 125.2, 127.1, 127.3, 128.1, 129.3, 131.2,
132.1, 134.1, 136.4, 136.8, 144.4, 154.4, 161.2 (Ar C), 163.1
(CO), 180.1 (CS).
–
(CO trifluoroacetyl), 1648–1644 (C O pyridone).
–
1H NMR (d ppm) of compound 30: 6.03 (s, 2H, CH2); 6.71–
8.01 (m, 9H, Ar H, and pyridine C5-H). 13C NMR (d ppm) of 30:
92.1 (CH2), 117.2 (CN), 100.0, 101.1, 114.3, 120.3, 122.4, 124.3,
127.1, 127.2, 128.2, 129.2, 130.6, 131.4, 139.0, 148.0, 164.3
(Ar C, CF3), 160.8 (CO), 168.8 (CO).
General procedure for the synthesis of 4-(benzo[1,3]-
dioxol-5-yl)-6-(4-bromophenyl)-1-formyl-2-oxo-1,2-
dihydropyridine-3-carbonitrile 25
A solution of 8 (3.95 g, 10 mmol) in formic acid (5 mL) was
heated under reflux for 4 h. The reaction mixture was poured
onto crushed ice and the separated solid product was filtered,
washed with water, dried, and recrystallized from ethanol.
Physicochemical and analytical data are recorded in Table 3.
1H NMR (d ppm) of compound 31: 6.12 (s, 2H, CH2), 6.72–
7.85 (m, 8H, Ar H, and pyridine C5-H). 13C NMR (d ppm) of 31:
90.6 (CH2), 117.3 (CN), 100.2, 101.7, 114.3, 115.6, 120.6, 126.7,
127.2, 129.4, 129.8, 132.5, 134.8, 138.0, 139.3, 147.5, 153.8
(Ar C, CF3), 162.5 (CO), 169.2 (CO).
IR (cmꢀ1): 2222 (CN), 1672 (CHO), 1648 (C O pyridone).
1H NMR (d ppm) of compound 32: 3.82 (s, 3H, OCH3); 6.12
(s, 2H, CH2); 6.55–7.76 (m, 8H, Ar H, and pyridine C5-H).
13C NMR (d ppm) of 32: 56.0 (OCH3), 92.1 (CH2), 118.0 (CN),
100.1, 101.6, 114.0, 120.1, 122.5, 126.4, 127.0, 127.4, 127.6,
127.8, 130.2, 136.8, 136.9, 161.0, 161.6 (Ar C, CF3), 161.3 (CO),
167.9 (CO).
–
–
1H NMR (d ppm): 6.07 (s, 2H, CH2), 7.11–7.98 (m, 8H, Ar H, and
pyridine C5-H), 8.23 (s, 1H, CHO). 13C NMR (d ppm): 93.1 (CH2),
117.3 (CN), 101.6, 111.6, 120.2, 122.5, 125.1, 127.0, 127.2,
128.2, 129.0, 131.1, 131.8, 139.9, 147.6, 164.2 (Ar C), 157.7
(CO), 163.0 (CO).
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Archiv der Pharmazie
General procedure for the synthesis of 7-(benzo[1,3]-
dioxol-5-yl)-5-phenyl-3-substituted-[1,2,4]triazolo[4,3-a]-
pyridine-8-carbonitriles 29 and 33–35
A mixture of 26 or 30–32 (10 mmol) and hydrazine hydrate
(0.9 g, 15 mmol) in ethanol (15 mL) was heated under reflux
for 8 h. The reaction mixture was allowed to attain room
temperature, poured on crushed ice and the precipitated solid
product was filtered, washed with water, dried, and recrystal-
lized from DMF. Physicochemical and analytical data are
recorded in Table 3.
mixture (10000 IU/mL penicillin potassium, 10000 mg/mL
streptomycin sulphate, and 25 mg/mL amphotericin B), and
1% L-glutamine in 96-well flat bottom microplate at 37°C
under 5% CO2. After 24 h of incubation, the medium was
aspirated, 40 mL of MTT salt (2.5 mg/mL) were added to each
well, and incubated for further 4 h at 37°C under 5% CO2.
To stop the reaction and dissolve the formed crystals, 200 mL
of 10% sodium dodecyl sulphate (SDS) in deionized water
was added to each well and incubated overnight at 37°C.
The absorbance was then measured using a microplate
multi-well reader (Bio-Rad Laboratories Inc., model 3350,
Hercules, California, USA) at l 595 nm and a reference
wavelength of l 620 nm. A statistical significance was tested
between samples and negative control (cells with vehicle)
using independent t-test by SPSS 11 program. The results are
presented in Table 1 as LC50 (mg/mL) which is the lethal
concentration of the compound which causes death of 50%
of the cells in 24 h.
IR (cmꢀ1) of compounds 10 and 33–35: 2229–2224 (CN).
1H NMR (d ppm) of compound 29: 2.35 (s, 3H, CH3), 6.14 (s,
2H, CH2), 6.82–7.59 (m, 9H, Ar H). 13C NMR (d-pm) of 29: 14.0
(CH3), 93.0 (CH2), 117.1 (CN), 101.8, 111.9, 113.3, 115.4, 118.7,
120.3, 127.3, 127.4, 129.5, 131.4, 139.3, 142.0, 147.4, 151.5,
161.8, 164.2 (Ar C).
1H NMR (d ppm) of compound 33: 6.12 (s, 2H, CH2), 6.82–
7.59 (m, 9H, Ar H). 13C NMR (d ppm) of 33: 93.2 (CH2), 117.2
(CN), 118.9 (CF3), 101.5, 104.7, 111.7, 113.3, 115.1, 120.2,
127.4, 127.3, 129.4, 131.3, 139.2, 142.1, 147.5, 151.3, 160.8,
164.0 (Ar C).
In vitro antibacterial and antifungal activities
Inhibition-zone (IZ) measurements: All the newly synthe-
sized compounds were evaluated for their in vitro anti-
bacterial activity by the agar cup diffusion technique [39]
using a 1 mg/mL solution in DMSO. The test organisms
were Staphylococcus aureus (ATCC 6538), Bacillus subtilis
(ATCC 6633), and Micrococcus luteus (ATCC 21881) as
examples of Gram-positive bacteria and Escherichia coli
(ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and
Klebsiella pneumonia (clinical isolate) as examples of
Gram-negative bacteria. They were also evaluated for
their in vitro antifungal potential against Candida albicans
(ATCC 10231) and Aspergillus niger (recultured) fungal
strains. Each 100 mL of sterile molten agar (at 45°C)
received 1 mL of 6 h-broth culture and then the seeded
agar was poured into sterile Petri dishes. Cups (8 mm
in diameter) were cut in the agar. Each cup received
0.1 mL of the 1 mg/mL solution of the test compounds.
The plates were then incubated at 37°C for 24 h or, in case
of C. albicans, for 48 h. A control using DMSO without
the test compound was included for each organism.
Ampicillin trihydrate and clotrimazole were used as
reference drugs. The results were recorded for each tested
compound as the average of three measurements of the
diameter of inhibition zones (IZ) of bacterial or fungal
growth around the discs in mm.
Minimal inhibitory concentration (MIC) measurements: The
minimal inhibitory concentrations (MIC) of the most active
compounds were measured using the twofold serial broth
dilution method [40]. The test organisms were grown in
their suitable broth: 24 h for bacteria and 48 h for fungi at
37°C. Twofold serial dilutions of solutions of the test
compounds were prepared using 200, 100, 50, 25, 12.5,
6.25, and 3.12 mg/mL. The tubes were then inoculated with
the test organisms; each 5 mL received 0.1 mL of the above
inoculum and were incubated at 37°C for 48 h. Then, the tubes
were observed for the presence or absence of microbial
1HNMR (d ppm) of compound 34: 6.11(s, 2H, CH2), 6.72–7.77
(m, 8H, Ar H). 13CNMR (d ppm) of 34: 93.2 (CH2), 117.6 (CN),
119.8 (CF3), 101.9, 104.5, 112.2, 113.8, 115.7, 120.0, 127.2,
127.4, 129.2, 131.3, 139.7, 142.0, 147.4, 151.8, 161.7, 164.4
(Ar C).
1H NMR (d ppm) of compound 35: 3.80 (s, 3H, OCH3), 6.11 (s,
2H, CH2), 6.63–7.86 (m, 8H, Ar H). 13C NMR (d ppm) of 35: 58.2
(OCH3), 93.0 (CH2), 117.3 (CN), 119.9 (CF3), 101.2, 105.1, 112.5,
114.3, 115.8, 120.3, 127.4, 127.5, 129.3, 131.5, 139.6, 142.1,
147.6, 151.5, 161.6, 164.3 (Ar C).
Biology
In vitro MTT cytotoxicity assay
The synthesized compounds were investigated for their
in vitro cytotoxic effect via the standard [3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide] method (MTT) [37,
38] against a panel of three human tumor cell lines, namely
Caucasian breast adenocarcinoma MCF7, hepatocellular
carcinoma HepG2, and colon carcinoma HT29. The proce-
dures were done in a sterile area using a laminar flow cabinet
biosafety class II level (Baker, SG403INT, Stanford, ME, USA).
Cells were batch cultured for 10 days, then seeded at
concentration of 10 ꢂ 103 cells/well in fresh complete growth
medium in 96-well microtiter plastic plates at 37°C for 24 h
under 5% CO2 using a water jacketed carbon dioxide
incubator (Sheldon, TC2323, Cornelius, OR, USA). Media
was aspirated, fresh medium (without serum) was added,
and cells were incubated either alone (negative control) or
with different concentrations of the test compounds to give
a final concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.56,
and 0.78 mg/mL. DMSO was employed as a vehicle for
dissolution of the tested compounds and its final concentra-
tion on the cells was less than 0.2%. Cells were suspended
in RPMI 1640 medium (for Hep-G2 and HT29 cell lines)
and DMEM (for MCF7 cell line), 1% antibiotic–antimycotic
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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833

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 824–834
H. M. Faidallah et al.
Archiv der Pharmazie
growth. The MIC values in mg/mL of the prepared compounds
are listed in Table 2.
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