New Bis(dihydropyridine-3,5-dicarbonitrile) Derivatives: Green Synthesis and Cytotoxic Activity Evaluation

Article abstract of DOI:10.1002/jhet.2867

A synthesis of bis(dihydropyridine-3,5-dicarbonitrile) by a three-component reaction of one equivalent of bis-cyanoacetamides with two equivalents of both arylaldehydes and malononitrile in ethanol-containing piperidine is reported. Bis-cyanopyridones could also be obtained by the condensation of bis-cyanoacetamides with substituted arylidenemalononitriles in the presence of piperidine, chitosan, or montmorillonite as basic catalysts. The cytotoxicity of the synthesized products against the heterogeneous human epithelial colorectal adenocarcinoma cell line (Caco-2) was assessed by WST-1 assay with concentration dependent cellular growth inhibitory effect especially for compounds 5l, 5h, and 5d. The dose response curves indicate that IC50 were 87?±?3.11?μg/mL, 104?±?4.78?μg/mL, and 108?±?5.12?μg/mL, respectively.

Full text of DOI:10.1002/jhet.2867

Month 2017 New Bis(dihydropyridine-3,5-dicarbonitrile) Derivatives: Green Synthesis
and Cytotoxic Activity Evaluation
a
b
a
a
*
*
and Ahmed H. M. Elwahy
Amr M. Abdelmoniem, Taher A. Salaheldin, Ismail A. Abdelhamid,
aChemistry Department, Faculty of Science, Cairo University, Giza, Egypt
bNanotechnology & Advanced Materials Central Lab, Agriculture Research Centre, Cairo 12631, Egypt
*
E-mail: ismail_shafy@yahoo.com; aelwahy@hotmail.com
Received October 1, 2016
DOI 10.1002/jhet.2867
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
A synthesis of bis(dihydropyridine-3,5-dicarbonitrile) by a three-component reaction of one equivalent of
bis-cyanoacetamides with two equivalents of both arylaldehydes and malononitrile in ethanol-containing
piperidine is reported. Bis-cyanopyridones could also be obtained by the condensation of bis-
cyanoacetamides with substituted arylidenemalononitriles in the presence of piperidine, chitosan, or mont-
morillonite as basic catalysts. The cytotoxicity of the synthesized products against the heterogeneous hu-
man epithelial colorectal adenocarcinoma cell line (Caco-2) was assessed by WST-1 assay with
concentration dependent cellular growth inhibitory effect especially for compounds 5l, 5h, and 5d. The
dose response curves indicate that IC50 were 87 3.11 μg/mL, 104 4.78 μg/mL, and 108 5.12 μg/mL,
respectively.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
RESULTS AND DISCUSSION
Substituted cyanoacetamides are important intermediates
in the synthesis of a variety of agrochemicals, dyes, and
pharmacologically active compounds [1]. They were
also found to exhibit significant biological activities as
antifungal and antibacterial agents [2]. In addition, they
are versatile and convenient intermediates for the synthesis
of wide variety of various nitrogen-containing heterocyclic
compounds [3–6].
The bis-cyanoacetamides 3 were chosen as key
intermediates to a variety of novel bis-cyanopyridone
derivatives. They can be prepared by cyanoacetylation of
the bisamines 1 with ethyl cyanoacetate 2 according to
recently reported procedure (Scheme 1) [38].
To find the optimal reaction conditions for the
synthesis of bis-pyridine derivatives 5, we examined the
reaction between the bis-cyanoacetamide 3a and
Moreover, pyridine derivatives have attracted much
attention as they exhibit important biological activities
including antiviral [7,8], antitumor [9,10], and anti-
inflammatory [11].
Furthermore, bis-heterocyclic compounds have been
reported to possess interesting biological properties [12–14]
including antihypertensive [15,16], antiallergenic [17],
and antitumor activities [15,18].
As a part of an ongoing research program on
bis-heterocycles [19–29], Michael addition reaction to
acrylonitrile derivatives [30–37], as well as the utility of
green benign approaches in organic synthesis [32,35], we
report the results of our investigations concerning the
different reactivity patterns of bis-cyanoacetamides
toward cinnamonitrile derivatives.
benzylidenemalononitrile derivative 4a as a simple
model system in the presence of a wide range of bases
including piperidine, chitosan, DABCO, DBU, and
montmorillonite (Scheme 2). The % yields in all cases
are cited in Table 1.
As seen in this table, it was noticed that, although the
reaction worked out best in refluxing ethanol in all
catalysts, the reactions using piperidine, chitosan, and
montmorillonite were found to achieve the best yields.
Encouraged by this success and in a trial to develop the
scope of these reactions, a variety of bis-cyanoacetamides
3a–c undergo nucleophilic addition reaction to the double
bond of arylidenmalononitrile 4a–d via a Michael-type
addition reaction under the optimized conditions in the
presence of piperidine (Method A), chitosan (Method B),
© 2017 Wiley Periodicals, Inc.

A. M. Abdelmoniem, T. A. Salaheldin, I. A. Abdelhamid, and A. H. M. Elwahy
Vol 000
Scheme 1. Synthesis of bis(2-cyanoacetamide) compounds 3a–c.
Scheme 2. Synthesis of bispyridine compound 5a.
Table 1
Table 2
Optimizing the yield of compound 5a.
The % yields of 5b–l obtained using piperidine, chitosan, and
montmorillonite.
Entry
Time (h)
Solvent
Catalyst
Piperidine
DABCO
Chitosan
Montmorillonite
DBU
Yield (%)
Yield (%)
1
2
3
4
5
3
3
3
3
3
EtOH
EtOH
EtOH
EtOH
EtOH
92
84
90
88
83
Compound
Piperidine
Chitosan
Montmorillonite
5b
5c
5d
5e
5f
5g
5h
5i
93
89
90
91
89
87
85
93
91
88
87
92
87
91
90
88
88
83
90
89
86
88
90
90
89
89
90
85
85
86
85
87
85
and montmorillonite (Method C) as catalysts. The results
are summarized in Scheme 3 and Table 2.
It is honestly to mention that during preparation of this
paper we read the articles of Kheder et al. [39,40] in
which they have also reported the formation of the
5j
5k
5l
Scheme 3. Synthesis of bispyridine compounds 5b–l.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
New Bis(dihydropyridine-3,5-dicarbonitrile) Derivatives: Green Synthesis and
Cytotoxic Activity Evaluation
bis-pyridine derivatives 5a upon heating 3a with 4a in
ethanol at reflux in the presence of piperidine as a
catalyst.
reaction of bis(2-cyano-3-arylacrylamide) 13e–g with
malononitrile 14 in refluxing dioxane-containing few
drops of piperidine (Scheme 5, Method D).
Taking into account the results obtained, one can
suggest the following mechanistic pathway. The pyridine
structures 5 are formed through the initial addition of
the active methylene in the cyanoacetamides 3 to the
Compounds 5 were also obtained in good to
excellent yields by a three-component reaction of two
equivalents of both arylaldehyde 12 and malononitrile
14 with one equivalent of bis-cyanoacetamides 3 in
double bond of cinnamonitriles
4
followed by
refluxing ethanol with piperidine as
a
catalyst
cyclization involving NH of the amide and then
elimination of two molecules of hydrogen (pathway A).
On the other hand, the cyclization step that involves the
carbonyl group leads to pyran structure 6 (pathway B).
(Scheme 5, Method E).
Cytotoxic activity against Caco-2 cell line. The cytotoxic
effect of synthesized products group 1 [5a, 5b, 5c, 5d, 5i,
5j] and group 2 [5k, 5l, 5e, 5f, 5g, 5h] were evaluated
against heterogeneous human epithelial colorectal
adenocarcinoma cell line (Caco-2), compared with
doxorubicin as a reference drug, using the WST-1 cell
proliferation assay for quantification of cytotoxic effect in
form of cell proliferation and viability. Basically, WST-1
assay is based on the cleavage of the tetrazolium salt
WST-1 to formazan by cellular mitochondrial
dehydrogenases. The increase in the number of viable
cells results in an increase in the overall mitochondrial
dehydrogenases activity. The augmentation in enzyme
activity leads to the increase in the amount of formazan
dye formed. The formazan dye produced by viable cells
can be quantified by a multiwell microplate reader by
measuring the absorbance of the dye solution at 450 nm.
Tables 3 and 4 represents the cytotoxic effect of the
1
Structures 6 were readily excluded because the H NMR
spectra of the products lacked the pyran H-4 signal,
which should appear at approximately δ ≈ 4–5 ppm
(Scheme 4).
It is worth mentioning that the reaction of malononitrile
14 with a preheated mixture of aldehydes 12 and
bis-cyanoacetamides 3 resulted in the formation of the
corresponding bis-pyridine derivatives. In support of this
viewpoint, we could manage to isolate the Knoevenagel
condensation products, N,N0-(alkane-1,3-diyl)bis(2-cyano-
3-arylacrylamide) 13 in some cases. Thus, compounds
13e–g were obtained by Knoevenagel condensation of
bis-cyanoacetamides 3b with the appropriate aromatic
aldehyde 12a–c according to reported procedure [41,42].
The bis-pyridine derivatives 5e–g were obtained by the
Scheme 4. Proposed mechanism.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. M. Abdelmoniem, T. A. Salaheldin, I. A. Abdelhamid, and A. H. M. Elwahy
Vol 000
Scheme 5. Synthesis of bis-pyridine compounds 5e–g through the reaction of cyanoacrylonitriles with malononitrile and through one pot three-component
reaction.
Table 3
Cytotoxic effect (%) of group 1 compounds against Caco-2.
Cytotoxic effect %
Conc. (μg/mL)
5a
5b
5c
5d
5i
5j
0
0
0
0
0
0
0
10
20
30
50
100
150
200
8.4218
12.9865
17.0549
25.6566
37.8847
57.1156
71.3804
7.3477
13.5362
16.9272
24.3542
40.5667
59.95122
75.3305
6.86664
9.3097
9.9268
15.6091
22.4538
32.592
50.8879
65.1504
82.5903
5.05131
7.67915
10.6434
15.7704
27.4308
45.087
2.56258
5.2207
9.7604
17.5443
30.7843
46.2823
62.3462
14.8764
20.5581
32.2284
52.6709
65.8664
57.0844
Table 4
Cytotoxic effect (%) of group 2 compounds against Caco-2.
Cytotoxic effect %
Conc.(μg/mL)
5k
5l
5e
5f
5g
5h
Reference
0
0
0
0
0
0
0
0
10
20
30
50
100
150
200
1.7654
3.8809
7.3372
12.8897
23.987
39.7074
53.0448
14.3402
22.5643
30.5569
40.5337
60.8975
79.459
4.9863
8.5497
6.2387
11.3249
16.4533
22.4353
36.0826
51.9768
65.3709
2.7849
7.4932
11.638
18.1895
23.8392
34.9765
51.6649
68.4604
82.3978
15.548
25.476
32.652
43.438
64.622
82.541
97.338
12.4887
20.3385
37.5573
50.3453
67.1552
12.4599
19.5508
30.4326
43.4665
55.4398
90.6579
tested compounds in response to their concentration as
illustrate in Figures 1 and 2 (data generated were used to
plot a dose response curve of which the concentration of
test compounds required to kill 50% of cell population
(IC50) was estimated).
Cytotoxic activity was expressed as the mean IC50 of
three independent experiments. The results are
represented in Table 5 and Figure 3.
The results revealed that compounds 5l, 5h, and 5d
containing benzo[d][1,3]dioxole substituent (IC50 were
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
New Bis(dihydropyridine-3,5-dicarbonitrile) Derivatives: Green Synthesis and
Cytotoxic Activity Evaluation
Figure 1. Cytotoxic effect chart of group 1compounds against Caco-2 cell line.
Figure 2. Cytotoxic effect chart of group 2 compounds against Caco-2 cell line.
Table 5
87 3.11 μg/mL, 104 4.78 μg/mL, and 108 5.12 μg/mL,
respectively) have the highest cytotoxic effect
against heterogeneous human epithelial colorectal
adenocarcinoma cell line (Caco-2), while 5b, 5a, 5f,
5c, and 5e have moderate cytotoxic activity (IC50
IC50 values of tested compounds and standard deviation against Caco-2.
Compound No. IC50 (μg/mL) Compound No. IC50 (μg/mL)
5a
5b
5c
5d
5i
5j
132 6.08
125 5.54
147 6.98
108 5.12
173 8.67
160 7.84
80 5.65
5 k
5 l
5e
5f
5 g
5 h
180 9.26
87 3.11
148 5.37
145 5.79
174 9.17
104 4.78
were 125
5.54 μg/mL, 132
6.08 μg/mL,
145 5.79 μg/mL, 147
6.98 μg/mL, and
148 5.37 μg/mL). On the other hand, 5j, 5i, 5g, and 5k
have lower cytotoxic activity against the tested cell
Reference
line (IC50 = 160
7.84 μg/mL, 173
8.67 μg/mL,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. M. Abdelmoniem, T. A. Salaheldin, I. A. Abdelhamid, and A. H. M. Elwahy
Vol 000
Figure 3. Estimated IC50 values of tested compounds against Caco-2 cell line.
174 9.17 μg/mL, and 180 9.26 μg/mL, respectively).
IC50 of Doxorubicin as a reference drug was found to be
Varian Gemini NMR spectrometer at 400 MHz using
TMS as internal standard. Chemical shifts are reported as
δ values in ppm. Mass spectra were recorded with a
Shimadzu GCMS–QP–1000 EX mass spectrometer in EI
(70 eV) model. The elemental analyses were performed
at the Micro analytical center, Cairo University.
General procedure of synthesis of compound 5a–l is as
follows:
80
5.65 μg/mL, the smaller values of IC50 for the
higher cytotoxic effect. It is clear that the presence of
benzo[d][1,3]dioxole substituent has shown to play an
important role on the observed growth inhibition. These
results are in agreement with that previously reported
[43–47].
• Methods A–C: A mixture of bisamides 3a–c (1 mmol)
and activated cinnamonitriles 4a–d (2.2 mmol) in abso-
lute ethanol (15 mL) was heated at reflux in the presence
of piperidine (0.2 mL, method A), chitosan (0.02 g, method
B) or montmorillonite (0.02 g, method C) for 3 h. The
crude solid was isolated and recrystallized from the proper
solvent whereby chitosan and montmorillonite can be
recovered and washed with acetone, dried, and reused
in another reaction.
• Method D: To a mixture of bis(2-cyanoacrylamides),
13a–l (1 mmol) and malononitrile 14 (2.2 mmol) in
dioxane (10 mL) was added piperidine (0.2 mL), and
the mixture was set at reflux for 3 h. The crude solid
was isolated and recrystallized from the proper solvent.
• Method E: To a mixture of bisamides 3a–c, aromatic
aldehydes 12a–d (2.2 mmol) and malononitrile 14
(2.2 mmol) in absolute ethanol (15 mL) was added pi-
peridine (0.2 mL) for 3 h, and the mixture was heated
at reflux for 3 h. The crude solid was isolated and re-
crystallized from the proper solvent.
CONCLUSIONS
The bis-cyanoacetamide moiety, behaving like C-
nucleophiles, affects simple and facile Michael addition
reactions with various arylidenemalononitriles, and
regioselectively yielding different 1,2-dihydropyridine-
3,5-dicarbonitrile derivatives. Moreover, piperidine,
montmorillonite, and chitosan were found to be the best
catalysts for this type of reactions. Compounds 5l, 5h,
and 5d may have significant and promising growth
inhibatory efficiency against heterogeneous human
epithelial colorectal adenocarcinoma cell line (Caco-2).
The anticancer effect might be due to such inhibitory
effect to the inner mitochondrial membrane
dehydrogenase enzyme, which in turn decreases the
cellular activity including the rate of cell division.
EXPERIMENTAL
1,10-(Ethane-1,2-diyl)bis(6-amino-2-oxo-4-phenyl-1,2-dihydr
Materials and methods. Melting points were measured
with a Stuart melting point apparatus and are uncorrected.
The IR spectra were recorded using an FTIR Bruker–
vector 22 spectrophotometer as KBr pellets. The 1H
NMR spectra were recorded in DMSO–d6 as solvent on
opyridine-3,5-dicarbonitrile) (5a) [39].
Yellowish white
crystals (ethanol/dioxane (1:1)), Mp > 300°C, IR (KBr):
ν = 3360, 3282 (br, NH2), 2218 (CN), 1681 (CO) cmꢀ1
,
1H NMR (400 MHz, DMSO-d6): δ = 4.42 (s, 4H, 2CH2),
7.42–7.53 (m, 10H, Ar─H), 8.57 (br s, 4H, 2NH2) ppm,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
New Bis(dihydropyridine-3,5-dicarbonitrile) Derivatives: Green Synthesis and
Cytotoxic Activity Evaluation
MS (EI, 70 eV): m/z (%) = 498 [M+], Anal. Calcd for
C28H18N8O2: C, 67.46; H, 3.64; N, 22.48. Found: C,
67.32; H, 3.55; N, 22.39.
(m, 2H, CH2), 3.82 (s, 6H, 2OCH3), 4.09 (m, 4H,
2CH2), 7.05–7.47 (dd, 8H, Ar─H), 8.38 (br s, 4H,
2NH2) ppm, MS (EI, 70 eV): m/z (%) = 572 [M+],
Anal. Calcd for C31H24N8O4: C, 65.03; H, 4.23; N,
19.57. Found: C, 64.91; H, 4.16; N, 19.51.
1,10-(Ethane-1,2-diyl)bis(6-amino-4-(4-chlorophenyl)-2-oxo-
1,2-dihydropyridine-3,5-dicarbonitrile) (5b).
Pale yellow
1,10-(Propane-1,3-diyl)bis(6-amino-4-(benzo[d][1,3]dioxol-5-
crystals (ethanol/dioxane (1:1)), Mp > 300°C, IR (KBr):
IR (KBr): ν = 3416, 3362 (br, NH2), 2215 (CN), 1661,
yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile)
(5h).
1630 (CO) cmꢀ1
,
1H NMR (400 MHz, DMSO-d6):
Yellow crystals (ethanol/dioxane (3:1)), Mp = 230–
232°C, IR (KBr): IR (KBr): ν = 3343, 3186 (br, NH2),
δ = 4.39 (s, 4H, 2CH2), 7.43–7.64 (dd, 8H, Ar─H), 8.61
(br s, 4H, 2NH2) ppm, MS (EI, 70 eV): m/z (%) = 567
[M+], Anal. Calcd for C28H16Cl2N8O2: C, 59.27; H, 2.84;
N, 19.75. Found: C, 59.18; H, 2.69; N, 19.59.
2215 (CN), 1648 (CO) cmꢀ1 1H NMR (400 MHz,
,
DMSO-d6): δ = 1.70 (m, 2H, CH2), 3.99 (m, 4H, 2CH2),
6.14 (s, 4H, 2OCH2O), 6.97–7.10 (m, 6H, Ar─H), 8.41
(br s, 4H, 2NH2) ppm, MS (EI, 70 eV): m/z (%) = 600
[M+], Anal. Calcd for C31H20N8O6: C, 62.00; H, 3.36; N,
18.66. Found: C, 61.85; H, 3.19; N, 18.57.
1,10-(Ethane-1,2-diyl)bis(6-amino-4-(4-methoxyphenyl)-2-
oxo-1,2-dihydropyridine-3,5-dicarbonitrile)
(5c).
Pale
yellow crystals (ethanol/dioxane (1:1)), Mp > 300°C, IR
(KBr): IR (KBr): ν = 3331, 3133 (br, NH2), 2214 (CN),
1,10-(Butane-1,4-diyl)bis(6-amino-2-oxo-4-phenyl-1,2-dihydr
1645 (CO) cmꢀ1
,
1H NMR (400 MHz, DMSO-d6):
opyridine-3,5-dicarbonitrile) (5i).
Pale yellow crystals
δ = 3.83 (s, 6H, 2OCH3), 4.38 (s, 4H, 2CH2), 7.06–7.41
(dd, 8H, Ar─H), 8.49 (br s, 4H, 2NH2) ppm, MS (EI,
70 eV): m/z (%) = 558 [M+], Anal. Calcd for
C30H22N8O4: C, 64.51; H, 3.97; N, 20.06. Found: C,
(ethanol/dioxane (3:1)), Mp > 300°C, IR (KBr): IR
(KBr): ν = 3425, 3331 (br, NH2), 2213 (CN), 1619
1
(CO) cmꢀ1, H NMR (400 MHz, DMSO-d6): δ = 1.62
(m, 4H, 2CH2), 4.05 (m, 4H, 2CH2), 7.48–7.56 (m, 8H,
Ar─H), 8.42 (br s, 4H, 2NH2) ppm, MS (EI, 70 eV):
m/z (%) = 526 [M+], Anal. Calcd for C30H22N8O2: C,
68.43; H, 4.21; N, 21.28. Found: C, 68.31; H, 4.14; N,
21.16.
64.46; H, 3.82; N, 20.19.
1,10-(Ethane-1,2-diyl)bis(6-amino-4-(benzo[d][1,3]dioxol-5-
yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile) (5d). Yellow
crystals (ethanol/dioxane (1:1)), Mp > 300°C, IR (KBr):
IR (KBr): ν = 3309, 3277 (br, NH2), 2206 (CN), 1646
1,10-(Butane-1,4-diyl)bis(6-amino-4-(4-chlorophenyl)-2-oxo-
1
(CO) cmꢀ1, H NMR (400 MHz, DMSO-d6): δ = 4.16 (s,
1,2-dihydropyridine-3,5-dicarbonitrile) (5j).
Pale yellow
4H, 2CH2), 6.12 (s, 4H, 2OCH2O), 6.93–7.06 (m, 6H,
Ar─H), 8.31 (br s, 4H, 2NH2) ppm, MS (EI, 70 eV): m/z
(%) = 586 [M+], Anal. Calcd for C30H18N8O6: C, 61.43;
H, 3.09; N, 19.11. Found: C, 61.31; H, 3.15; N, 18.98.
1,10-(Propane-1,3-diyl)bis(6-amino-2-oxo-4-phenyl-1,2-dihyd
crystals (ethanol/dioxane (3:1)), Mp > 300°C, IR (KBr):
IR (KBr): ν = 3373, 3331, 3212 (br, NH2), 2217 (CN),
1651 (CO) cmꢀ1 1H NMR (400 MHz, DMSO-d6):
,
δ = 1.62 (m, 4H, 2CH2), 4.04 (m, 4H, 2CH2), 7.51–7.65
(dd, 8H, Ar─H), 8.45 (br s, 4H, 2NH2) ppm, MS (EI,
70 eV): m/z (%) = 595 [M+], Anal. Calcd for
C30H20Cl2N8O2: C, 60.51; H, 3.39; N, 18.82. Found: C,
60.44; H, 3.18; N, 18.77.
ropyridine-3,5-dicarbonitrile) (5e).
Yellow crystals
(ethanol), Mp = 218–220°C, IR (KBr): ν = IR (KBr):
ν = 3436, 3348 (br, NH2), 2216 (CN), 1643 (CO) cmꢀ1
,
1H NMR (400 MHz, DMSO-d6): δ = 1.56 (m, 2H, CH2),
4.09 (m, 4H, 2CH2), 7.46–7.55 (m, 10H, Ar─H), 8.36
(br s, 4H, 2NH2), MS (EI, 70 eV): m/z (%) = 512 [M+],
Anal. Calcd for C29H20N8O2: C, 67.96; H, 3.93; N,
1,10-(Butane-1,4-diyl)bis(6-amino-4-(4-methoxyphenyl)-2-
oxo-1,2-dihydropyridine-3,5-dicarbonitrile)
(5k).
Pale
yellow crystals (ethanol/dioxane (3:1)), Mp > 300°C, IR
(KBr): IR (KBr): ν = 3331, 3168 (br, NH2), 2212 (CN),
1610 (CO) cmꢀ1 1H NMR (400 MHz, DMSO-d6):
,
21.86. Found: C, 67.82; H, 3.88; N, 21.79.
1,10-(Propane-1,3-diyl)bis(6-amino-4-(4-chlorophenyl)-2-
oxo-1,2-dihydropyridine-3,5-dicarbonitrile) (5f). Pale yellow
δ = 1.62 (m, 4H, 2CH2), 3.84 (s, 6H, 2OCH3), 4.03 (m,
4H, 2CH2), 7.08–7.48 (dd, 8H, Ar─H), 8.34 (br s, 4H,
2NH2) ppm, MS (EI, 70 eV): m/z (%) = 586 [M+], Anal.
Calcd for C32H26N8O4: C, 65.52; H, 4.47; N, 19.10.
crystals (ethanol), Mp = 224–226°C, IR (KBr): ν = IR
(KBr): ν = 3429, 3361 (br, NH2), 2214 (CN), 1645 (CO)
1
cmꢀ1, H NMR (400 MHz, DMSO-d6): δ = 1.55 (m, 2H,
Found: C, 65.44; H, 4.32; N, 19.03.
1,10-(Butane-1,4-diyl)bis(6-amino-4-(benzo[d][1,3]dioxol-5-
yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile) (5l). Yellow
CH2), 4.07 (m, 4H, 2CH2), 7.50–7.63 (dd, 8H, Ar─H),
8.42 (br s, 4H, 2NH2) ppm, MS (EI, 70 eV): m/z
(%) = 581 [M+], Anal. Calcd for C29H18Cl2N8O2: C,
59.91; H, 3.12; N, 19.27. Found: C, 59.86; H, 3.16; N,
19.19.
crystals (ethanol/dioxane (3:1)), Mp > 300°C, IR (KBr):
IR (KBr): ν = 3378, 3215 (br, NH2), 2214 (CN), 1652
(CO) cmꢀ1, 1H NMR (400 MHz, DMSO-d6): δ = 1.61 (m,
4H, 2CH2), 4.03 (m, 4H, 2CH2), 6.14 (s, 4H, 2OCH2O),
6.98–7.09 (m, 6H, Ar─H), 8.38 (br s, 4H, 2NH2) ppm,
MS (EI, 70 eV): m/z (%) = 614 [M+], Anal. Calcd for
C32H22N8O6: C, 62.54; H, 3.61; N, 18.23. Found: C,
62.49; H, 3.52; N, 18.14.
1,10-(Propane-1,3-diyl)bis(6-amino-4-(4-methoxyphenyl)-2-
oxo-1,2-dihydropyridine-3,5-dicarbonitrile) (5g).
Yellow
crystals (ethanol), Mp = 220–222°C, IR (KBr): IR
(KBr): ν = 3434, 3222 (br, NH2), 2215 (CN), 1643
(CO) cmꢀ1 1H NMR (400 MHz, DMSO-d6): δ = 1.61
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. M. Abdelmoniem, T. A. Salaheldin, I. A. Abdelhamid, and A. H. M. Elwahy
Vol 000
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Cytotoxic activity. The heterogeneous human epithelial
colorectal adenocarcinoma cell line (Caco-2) were cultured
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DMEM with 10% FBS at 37°C humidified with 5% CO2.
Various concentrations of the compound under test and
doxorubicin that was used as a reference drug (0.0, 10,
20, 30, 50, 100, 150, and 200 μg/mL) were dissolved in
mixture of 50% DMSO, 40% ethanol, and 10% deionized
water, then added to the cell monolayer in triplicate wells
individual dose, and its cytotoxicity was tested using a
standard WST-1 cell proliferation assay as a fast and sensi-
tive quantification of cell proliferation and viability, in a
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gratefully acknowledge the Alexander von Humboldt Foundation
for a research fellowship and the Nanotechnology and Advanced
Materials Central Lab, Agriculture Research Center for
bioactivity screening.
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Journal of Heterocyclic Chemistry
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