Utility of Hantzsch Ester in Synthesis of Some 3,5-Bis-dihydropyridine Derivatives and Studying Their Biological Evaluation

Article abstract of DOI:10.1002/jhet.1848

The present work involves synthesis of new 3,5-bis-substituted dihydropyridine derivatives 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 starting from dihydropyridine-3,5-dicarboxylate (1) as starting material. Structures of new compounds were establ

Full text of DOI:10.1002/jhet.1848

Month 2015
Utility of Hantzsch Ester in Synthesis of Some 3,5-Bis-dihydropyridine
Derivatives and Studying Their Biological Evaluation
Eman R. Kotb,a Hebat-Allah S. Abbas,a,b Eman M. Flefel, Hayam H. Sayed, and Nayera A. M. Abdelwahed
a
a
c
*
aPhotochemistry Department, National Research Centre, 12622 Dokki, Cairo, Egypt
bChemistry Department, College of Science, King Khalid University, Abha 9004, Saudi Arabia
cNatural and Microbial Product Department, National Research Centre, 12622 Dokki, Cairo, Egypt
*
E-mail: hebanrc@yahoo.com
Received June 26, 2012
DOI 10.1002/jhet.1848
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
HN
N
N
NH
O
O
H2N
NH2
S
O
O
S
N
H
N
H
N
H
N
H
N
S
N
N
N
O
O
HO
O
O
S
N
H
The present work involves synthesis of new 3,5-bis-substituted dihydropyridine derivatives 2–16 starting
from dihydropyridine-3,5-dicarboxylate (1) as starting material. Structures of new compounds were
established by spectral and elemental analyses. Some of the new compounds were evaluated for anticancer
and antimicrobial activity. Screening data of the tested compounds show promising anticancer and
antimicrobial activity. The detail synthesis, spectroscopic data, and pharmacological activities are reported.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
RESULT AND DISCUSSION
In our previous work, we found that pyridine derivatives
showed a broad of biological activity such as antioxidant,
antimicrobial, antitumor, and antiviral [1–7]. In addition,
the literature reports support that pyridine derivatives are
potent antitubercular [8] and anti-inflammatory agents [9].
Also, compounds containing 1,3,4-oxadiazole nucleuses
are associated with diverse pharmacological activities, which
have made them important chemotherapeutic agents [10,11],
analgesic, diuretic, antihypertensive, anti-inflammatory [12],
anticonvulsive [13], antibacterial, antifungal [14], and inhibit
HIV replication [15]. In addition, some derivatives are active
against hepatitis B and HIV-1 [16] viruses.
On the other hand, hydrazones containing azomethine
(–NHN═CH) protons constitute a vital class of compounds
for new drug development [17]. Several heterocyclic
hydrazones were reported to possess various biological
activities, antimicrobial [18], anti-inflammatory, analgesic
[19], anticonvulsant [20], antitubercular, antiplatelet [21],
antiviral, and antimalarial [22] activities. In view of these
observations and in continuation of our work, we synthe-
sized some heterocyclic compounds containing pyridine
moiety and tested their biological activities.
Chemistry. In the course of our investigation, we have
found that diethyl-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-dicarboxylate (1) [23] is an excellent building block for
the synthesis of a numerous heterocyclic ring systems. The
reactivity of 3,5-dicarboxylat 1 towards hydrazine derivatives
was investigated. Thus, 3,5-dicarboxylate 1 was reacted with
different nucleophiles namely hydrazine hydrate or phenyl
hydrazine in ethanol, it afforded 1,4-dihydropyridine-
3,5-dicarbohydrazide derivatives (2a,b) by hydrazinolysis
method (Scheme 1). IR spectra of compound 2a revealed
absorption bands for NH2, NH, and (C═O), and its mass
spectrum showed a peak corresponding to its molecular ion
peak at m/z (%) = 301 (35) [M+] (cf. Experimental).
Further reaction of compound 1 with different amino
acids, namely glycine and anthranilic acid, it afforded the
corresponding compounds 3, 4 respectively (Scheme 1). IR
spectra of compound 3 revealed absorption bands for OH,
NH, and (C═O), and its 1H NMR spectrum showed the pres-
ence of OH protons, NH, and (CH2) signals (cf. Experimental).
1,4-Dihydropyridine-3,5-dicarbohydrazide 2a was proved
chemically via condensation with different acid anhydride
© 2015 HeteroCorporation

E. R. Kotb, H.-A. S. Abbas, E. M. Flefel, H. H. Sayed, and N. A. M. Abdelwahed
Vol 000
Scheme 1
namely, maleic anhydride and phthalic anhydride in
acetic acid gave the corresponding N-amide derivatives 5,
6 respectively (Scheme 2). All the prepared compounds
were provided via elemental analysis and spectral data
(cf. Experimental).
Also, the treatment of hydrazide 2a with p-
chlorobenzaldehyde in ethanol afforded the corresponding
Schiff’s base compound 7 on the basis of its spectral data
(Scheme 2). The IR spectrum showed the disappearance
of the presence of (NH2) stretching vibration in the
spectrum of hydrazide 2a, and their 1H NMR spectra
showed the presence of NH and azo-methine (CH═N)
signals (cf. Experimental).
Bis(4-chlorobenzylidine)-1,4-dihydropyridine-3,5-
dicarbohydrazide (7) was cyclized into the corresponding
1,4-dihydropyridine-bis-1,3,4-oxadiazole derivative 8 via
refluxing in a mixture of acetic anhydride/pyridine (2:1).
The IR spectrum of 8 showed absence of the bands
Scheme 2
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Month 2015
Utility of Hantzsch Ester in Synthesis of Some 3,5-Bis-dihydropyridine Derivatives and
Studying Their Biological Evaluation
corresponding to (C═O) group. Also, the structure of
compound 8 was established chemically upon condensa-
tion of 2a with p-chlorobenzaldehyde in acetic acid
(cf. Experimental).
signals of the alditol protons congregated with the solvent
absorption [40] and the presence of hydroxyl groups
(exchangeable with D2O) (cf. Experimental).
Also, compound 2a was treated with CS2/KOH to give
1,4-dihydropyridine-bis-1,3,4-oxadizole thione derivative
13 (Scheme 4). In the IR spectrum of compound 13, no
signal derived from exocyclic carbonyl function was
observed. Moreover, NHNH2 stretching vibration was
disappeared.
Because of the pharmacological activities of acyclovir
[27], many attempts have been made by nucleoside
chemists to prepare a number of related compounds with
various side chains and glycons [27]. Thus, when the
sodium salt of compound 13 (generated in situ) was treated
with 2-(2-chloroethoxy) ethanol, epichlorohydrine, and
2-chloro ethanol, it afforded the corresponding S-acyclic
nucleosides derivatives 14–16, respectively.
The structure of the aforementioned acyclic nucleosides
was confirmed with spectral data, and 1H NMR spectra re-
vealed hydroxylethoxy ethyl, oxiran ring, and hydroxyethyl
signals. In addition, the IR, 13C NMR spectra revealed that
the side of attack was on the S-atom (cf. Experimental).
The bis-hydrazone derivative 9 (Scheme 3) was pre-
pared via reaction of compound 2a with monosaccharide:
namely, D-glucose in the presence of catalytic amounts of
glacial acetic acid. The products revealed absorption bands
for OH, NH, (C═O), and (C═N) in IR spectra, and their
1H NMR spectra showed the presence of the sugar protons,
NH, and azo-methine (CH═N) signals (cf. Experimental).
Acetylation of bis-hydrazone 9 with acetic anhydride at
room temperature gave the bis-O-acetylated sugar deriva-
tive 10. The IR spectrum of latter compound revealed the
absence of hydroxyl group. The 1H NMR spectrum
showed the absence of OH groups and the presence of
OAc groups (cf. Experimental). Oxidative cyclization of
compounds 10 using bromine/acetic acid [24,25] afforded
the corresponding bis-O-acetylated cyclic C-nucleoside
of 1,3,4-oxadiazoline derivative 11 (Scheme 3). The IR
spectrum of compound 11 showed absorption bands
corresponding to (O–C═O) and (N–C═O) groups. The
1H NMR spectrum showed the absence of azo-methine
(CH═N) and the presence of O-acetyl-methyl protons at d
2.08–2.25 ppm and N-acetyl-methyl protons at 2.41ppm
(cf. Experimental).
Biological evaluation
Anticancer activity. Chemotherapy is a major therapeutic
approach for both localized and metastasized cancers. In the
present work, nine of the newly synthesized compounds 2a,
4, 5, 6, 9, 10, 14, 15, and 16 were selected to evaluate their
in vitro growth inhibitory activities against human cancer
cell line, which is breast cell line (T47D) in comparison
with the known anticancer drug, doxorubicin (DOX) as
a reference drug. The anticancer activity results indicated
that most of the synthesized compounds showed
Deprotection of 11 using ammonium hydroxide solution
in methanol, [26] gave the target free cyclic C-nucleoside
12 (Scheme 3). The structure of the aforementioned
compound was confirmed on the basis of their spectral
data. The IR spectra revealed absorption bands due to
1
(OH) and (C═N); whereas their H NMR spectra showed
Scheme 3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E. R. Kotb, H.-A. S. Abbas, E. M. Flefel, H. H. Sayed, and N. A. M. Abdelwahed
Vol 000
Scheme 4
anticancer activity against breast cell line (T47D) but with
varying intensities in comparison with DOX. Moreover,
compounds 14 and 16 showed the good cytotoxic
activity IC50 22 mg/mL (Table 1), compounds 6 and 15
showed moderate cytotoxic activity IC50 23 mg/mL, and
compounds 5, 9, and 10 showed cytotoxic activity IC50
24–25 mg/mL but compounds 2a and 4 showed no
cytotoxic activity.
The variety of antitumor activities might be explained
because of the difference of side chains. The highest
activity of compounds 14 and 16 was explained because
of the presence of S-acyclic sugar derivatives.
Results were illustrated in Figure 1 for the cytotoxic
activities of the compounds (2a, 4, 5, 6, 9, 10, 14, 15,
and 16) in comparison with DOX.
Antimicrobial activity.
As shown in Table 2, the
antimicrobial effect of the tested compounds was evaluated
by measuring the zone diameters and their results were
compared with those of well-known drugs (standards). It
Table 1
Effect of some selected newly synthesized compounds on breast cancer
cell line (T47D).
1.2
Comp.
IC50
2a
1
4
0.8
0.6
0.4
0.2
0
5
2a
4
5
6
9
10
14
15
16
DOX
==
==
6
9
24 mg mL
23 mg mL
25 mg mL
25 mg mL
22 mg mL
23 mg mL
22 mg/mL
7.8 mg/mL
10
14
15
16
DOX
0
1
12.5
25
50
Conc. ug/ml
Figure 1. Cytotoxic effect of compounds 2a, 4, 5, 6, 9, 10, 14, 15, and 16
on breast cell line (T47D) to (DOX). [Color figure can be viewed in the
online issue, which is available at http://www.interscience.wiley.com.]
IC50: dose of the compounds that reduces survival to 50%.
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Utility of Hantzsch Ester in Synthesis of Some 3,5-Bis-dihydropyridine Derivatives and
Studying Their Biological Evaluation
Table 2
Inhibition zones of the newly synthesized compounds.
Inhibition zone
Gram-negative
Gram-positive bacteria
Bacillus Staphylococcus
bacteria
Fungi
Yeast
Aspergillus
niger
Candida
albicans
Compd. no.
subtilis
aureus
Escherichia coli
DMSO (solvent)
2a
–
++
–
–
–
–
–
–
–
–
2b
++
–
–
+
+
5
6
+++
++
+++
+
–
–
++
+
+++
+
7
9
+++
++
+++
+
–
–
++
+
+++
+
10
12
13
14
++
+
–
++
–
+
+
++++
+++
++
++++
+++
+
+++
++
+
++++
+++
+
–
15
++
+
–
+
+
16
+++
++++
+++
++++
–
++
–
+++
–
Ciprofloxacin
++++
(50 mg mLꢀ1
)
Ketaconazole
–
–
–
++++
++++
(50 mg mLꢀ1
)
–, no antimicrobial effect.
+, low antimicrobial effect (4 mm).
++, moderate antimicrobial effect (8–10 mm).
+++, high antimicrobial effect (15–18 mm).
++++, complete antimicrobial effect (20–22 mm).
Table 3
MIC of the newly synthesized compounds.
MIC (mg/mLꢀ1
)
Gram positive bacteria
Bacillus subtilis Staphylococcus aureus
Gram negative bacteria
Fungi
Yeast
Compd. no.
Escherichia coli
Aspergillus Niger
Candida albicans
DMSO (solvent)
–
–
–
–
–
–
–
–
–
–
–
–
0.45
–
–
–
–
–
2a
2b
5
6
7
0.45
0.45
0.25
0.45
0.45
0.25
0.45
0.075
0.25
0.45
0.45
0.25
0.65
0.45
0.65
0.45
0.65
0.65
0.25
0.45
0.65
0.65
0.45
0.65
0.25
0.65
0.25
0.65
0.65
0.075
0.25
0.65
0.65
0.25
0.25
0.65
0.25
0.45
0.65
0.075
0.25
0.65
0.65
0.25
9
10
12
13
14
15
16
–
–
–
MIC, minimum inhibitory concentration.
is evident that most tested compounds display activity
against Bacillus subtilis and Staphylococcus aureus but
only compound 12 showed moderate activities against
Escherichia coli. While all the tested compounds except
2a, 2b were active against Aspergillus Niger and Candida
albicans, compound 12 was the most active one against all
the listed bacteria, fungi, and yeast. Also, compounds 5, 7,
13, and 16 showed significant antimicrobial activity. The
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E. R. Kotb, H.-A. S. Abbas, E. M. Flefel, H. H. Sayed, and N. A. M. Abdelwahed
Vol 000
(s, 6H, 2CH3), 2.41 (s, 2H, 2NH, exchangeable with D2O), 4.99
(s, 1H, pyridine-H), 6.5–7.3 (m, 15H, Ar–H), 8.06 (s, 2H,
2NH, exchangeable with D2O), 9.08 (s, 1H, NH pyridine,
exchangeable with D2O). 13C NMR (DMSO-d6) (d, ppm): 14.1,
16.6 (2CH3), 50.3 (CH pyridine), 103.6, 104.9, 113.3, 113.9,
121.5, 125.3, 125.9, 129.15, 147.4, 149.5, 150.3, 155.2 (22 C-Ar),
164.9, 165.07 (2C═O). MS: m/z (%) = 453 (29) [M+]. Anal.
Calcd for C27H27N5O2 (453.54): C, 71.5; H, 6.0; N, 15.44.
Found: C, 71.41; H, 6.10; N, 15.52.
minimal inhibitory concentrations (MIC) of these compounds
ranged from 0.075 to 0.65 mgmLꢀ1 (Table 3).
CONCLUSION
In the present work, 16 new 3,5-bis-dihydropyrisine
derivatives were synthesized and characterized using
spectral and elemental analyses. Some of the synthesized
compounds evaluate their in vitro growth inhibitory activ-
ities against human cancer breast (T47D) cell line and
showed good activity because of the presence of S-acyclic
nucleoside derivatives.
Screening data of the prepared compounds show prom-
ising antibacterial and antifungal activities. Compounds
12 showed more significant antibacterial activity because
of the presence of the free cyclic C-nucleoside than com-
pounds 5, 7, 13, and 16.
Synthesis of 2,20[2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-diyl)bis(carbonylimino)]diacetic acid (3).
Glycine (0.15 g,
2 mmol) and Na2CO3 (15 mmol) were dissolved in water (15 mL),
and the pH was adjusted to 9–9.5. Then the compound 1 (3.29 g,
1 mmol) dissolved in ethanol (20 mL) was added, and the reaction
mixture was stirred at 100ꢁC for 8 h at the controlled pH. The
reaction mixture was left overnight at room temperature then
treated with cold formic acid. The solid obtained was filtered
off, washed with H2O, and crystallized from methanol to
yield the corresponding compound 3 as pale white crystals,
mp 158–159ꢁC; 1.89 g (49%); IR (KBr) n: 3412–3341, 3239,
1
3160 (2OH, 3NH), 1689, 1671, 1650 (4C═O) cmꢀ1. H NMR
(DMSO-d6) d 2.34 (s, 6H, 2CH3), 2.42 (s, 2H, 2NH,
exchangeable with D2O), 3.91 (s, 4H, 2CH2), 5.01 (s, 1H,
pyridine-H), 6.89–7.01 (m, 5H, Ar–H), 9.1 (s, 1H, NH
pyridine, exchangeable with D2O), 11.05 (s, 2H, 2OH,
exchangeable with D2O). 13C NMR (DMSO-d6) (d, ppm): 13.9,
14.2 (2CH3), 43.2, 43.9 (2CH2), 48.6 (CH pyridine), 102.4,
103.2, 121.5, 126.3, 126.9, 129.5, 147.4, 149.5 (10 C–Ar),
165.02, 165.12, 171.3, 171.9 (4C═O). MS: m/z (%) = 387 (12)
[M+]. Anal. Calcd for C19H21N3O6 (387.39): C, 58.91; H, 5.46;
N, 10.85. Found: C, 59.01; H, 5.38; N, 10.93.
EXPERIMENTAL
Chemistry. All melting points are uncorrected and measured
using Electro-thermal IA 9100 apparatus, Shimadzu (Japan). IR
spectra were recorded as potassium bromide pellets on a
Perkin-Elmer 1650 spectrophotometer, National Research
Centre, Cairo, Egypt. NMR spectra were determined on a
Jeol-Ex-300 (1H NMR, 13C NMR) spectrometer, and chemical
shifts were expressed as parts per million (ppm) (d values) against
TMS as internal reference (Faculty of Science, Cairo University,
Cairo, Egypt). Mass spectra were recorded on EI+ Q1 MSLMR
UPLR, (Faculty of Science, Cairo University, Cairo, Egypt).
Antitumor screening made by the National Cancer Institute, Cairo
University, Cancer Biology Department, Cairo, Egypt. Follow
up of the reactions and checking the purity of the compounds
was made by TLC on silica gel-coated aluminum sheets
(Type 60F254, Merck, Darmstadt, Germany).
Synthesis of 2,20(2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-diyl)bis-4H-3,1-benzoxazin-4-one (4).
Anthranilic acid
(2.8 g, 2 mmol) and compound 1 (3.29 g, 1 mmol) were fused
in conical flask at 110ꢁC on oil bath for 15 h. The reaction
mixture was cooled and the formed precipitate was filtered off
and recrystallized from methanol to yield the compound 4 as
pale brown powders, mp 182–183ꢁC; 1.99 g (42%). IR (KBr)
n: 3239 (NH), 1689, 1679 (2C═O), 1605 (C═N) cmꢀ1 1H
.
Synthesis of 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-
NMR (CDCl3) d 2.39 (s, 6H, 2CH3), 5.06 (s, 1H, pyridine-H),
6.76–7.32 (m, 13H, Ar–H), 9.08 (s, 1H, NH pyridine,
exchangeable with D2O). 13C NMR (CDCl3) (d, ppm): 16.1, 16.5
(2CH3), 44.6 (CH pyridine), 99.9, 102.8, 103.5, 112.6, 121.6,
122.4, 123.6, 141.1, 142.1, 142.9, 143.4, 150.6, 155.2, 156.4 (24
C–Ar), 161.3, 161.8 (2C═O). MS: m/z (%) = 475 (16) [M+]. Anal.
Calcd for C29H21N3O4 (475.49): C, 73.25; H, 4.45; N, 8.84.
Found: C, 73.32; H, 4.51; N, 8.74.
dicarbohydrazide (2a).
A mixture of compound 1 (3.29 g,
1 mmol) and hydrazine hydrate 99% (3 mL) in absolute ethanol
(30 mL) was refluxed for 8 h. After cooling, the reaction mixture
was poured into cold water, the formed solid was filtered off
and recrystallized from dioxane to give compound 2a as white
powder, mp 137–139ꢁC; 1.5 g (50%); IR (KBr) n: 3398–3350
(2NH2), 3165, 3148 (3NH), 1678 (2C═O) cmꢀ1 1H NMR
.
(CDCl3): d 2.1 (s, 4H, 2NH2, exchangeable with D2O), 2.36
(s, 6H, 2CH3), 4.94 (s, 1H, pyridine-H), 7.0–7.2 (m, 5H, Ar–H),
8.1 (s, 2H, 2NH, exchangeable with D2O), 9.2 (s, 1H, NH
pyridine, exchangeable with D2O). 13C NMR (CDCl3) (d,
ppm): 14.8, 17.9 (2CH3), 49.2 (CH pyridine), 103.2, 104.6,
122.2, 125.8, 126.2, 129.5, 147.4, 149.5, 150.3 (10 C–Ar),
165.15 (2C═O). MS: m/z (%) = 301 (35) [M+]. Anal. Calcd for
C15H19N5O2 (301.34): C, 59.79; H, 6.36; N, 23.24. Found: C,
59.90; H, 6.28; N, 23.35.
Synthesis of compounds (5, 6). General procedure.
A
mixture of compound 2a (3.01 g, 1 mmol) and maleic anhydride
or phthalic anhydride (2 mmol) was refluxed in glacial acetic
acid (30 mL) for 6 h. The reaction mixture was cooled and
poured into ice/water; the solid that formed was filtered off,
dried, and recrystallized from n-butanol.
Synthesis of N3,N5-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-
2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxamide
(5). As white crystals, mp 89–91ꢁC; 2.58 g (56%); IR (KBr)
Synthesis of 2,6-dimethyl-N3,N5,4-triphenyl-1,4-dihydropyridine-
n
3200, 3190, 3160 (3NH), 1710, 1703, 1685, 1676
3,5-dicarbohydrazide (2b).
A mixture of compound 1 (3.29 g,
(6C═O) cmꢀ1). 1H NMR (CDCl3) d 2.35 (s, 6H, 2CH3),
4.93 (s, 1H, pyridine-H), 6.92 (d, 4H, pyrrole ring), 7.06–7.23
(m, 5H, Ar–H), 8.10 (s, 2H, 2NH amide, exchangeable with
D2O), 9.15 (s, 1H, NH pyridine, exchangeable with D2O). 13C
NMR (CDCl3) (d, ppm): 17.6 (2CH3), 49.1 (CH pyridine), 102.5,
1 mmol) and phenyl hydrazine (2.16 g, 2 mmol) in 30 mL absolute
ethanol was refluxed for 6 h. After cooling, the separated solid was
recrystallized from ethanol to give compound 2b as pale reddish
crystals, mp 124–126ꢁC; 3.62 g (80%); IR (KBr) n: 3210, 3160,
3128 (5NH), 1674 (2C═O) cmꢀ1 1H NMR (DMSO-d6) d 2.29
.
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Utility of Hantzsch Ester in Synthesis of Some 3,5-Bis-dihydropyridine Derivatives and
Studying Their Biological Evaluation
104.9, 113.4, 113.9, 1221.3, 123.4, 124.7, 143.2, 144.7, 147.9
(14C-Ar), 163.6, 164.1, 165.3, 165.9 (6C═O). MS: m/z (%) = 461
(27) [M+]. Anal. Calcd for C23H19N5O6 (461.43): C, 59.87; H,
4.15; N, 15.18; Found: C, 59.92; H, 4.08; N, 15.09.
(3.6 g, 2 mmol) in 50 mL ethanol in the presence of 1 mL acetic
acid as catalyst was heated at 70ꢁC for 2 h. The formed
precipitate was filtered off, washed with water, dried, and
recrystallized from ethanol to give 9 pale white crystals, mp
177–179ꢁC; 4.06 g (65%). IR (KBr) n: 3435–3220 (10
Synthesis of N3,N5-bis(1,3-dioxoisoindolin-2-yl)-2,6-dimethyl-
4-phenyl-1,4-dihydropyridine-3,5-dicarboxamide (6). As brown
crystals, mp 261–262ꢁC; 4.65 (83%). IR (KBr) n: 3195,
OH + 3NH), 1673, 1668 (2C═O), 1603 (2C═N) cmꢀ1 1H
.
NMR (CDCl3) d 2.31 (s, 6H, 2CH3), 3.1–3.34 (m, 4H,
2CH2OH), 3.39–4.05 (m, 8H, alditol proton), 4.87–5.02 (m, 6H,
pyridine-H + 5OH, exchangeable with D2O), 5.3–5.74 (m, 5H,
5OH, exchangeable with D2O), 7.08–7.19 (m, 5H, Ar–H), 8.05
(s, 2H, 2NH, exchangeable with D2O), 8.3 (s, 2H, 2 N═CH),
9.16 (s, 1H, NH pyridine, exchangeable with D2O). 13C NMR
(CDCl3) (d, ppm): 15.3, 16.01 (2CH3), 28.23, 28.93 (2CH2),
44.09 (CH pyridine), 61.21, 62.36, 62.98, 71.32, 72.54, 73.15,
73.89 (8 C-alditol), 103.2, 104.6, 122.2, 125.8, 126.2, 129.5,
147.4, 149.5, 150.3 (10 C–Ar), 158,21, 159.01 (2C═N), 165.56
(2C═O). MS: m/z (%) = 625 (19) [M+]. Anal. Calcd for
C27H39N5O12 (625.62): C, 51.83; H, 6.28; N, 11.90; Found: C,
51.75; H, 6.32; N, 12.05.
3182, 3151 (3NH), 1709, 1705, 1679, 1669 (6C═O) cmꢀ1
.
1H NMR (CDCl3) d 2.31 (s, 6H, 2CH3), 5.03 (s, 1H,
pyridine-H), 7.09–7.21 (m, 5H, Ar–H), 7.72–8.06 (m, 10H,
Ar–H + 2NH, exchangeable with D2O), 9.23 (s, 1H, NH
pyridine, exchangeable with D2O). 13C NMR (CDCl3) (d,
ppm): 15.9, 16.3 (2CH3), 48.2 (CH pyridine), 103.9, 104.1,
121.3, 125.4, 126.2, 129.5, 132.5, 133.6, 133.9, 134.2,
144.5, 145.8 147.4, 149.5, 150.3 (22 C–Ar), 164.2, 164.8,
165.09, 165.17, 165.78 (6C═O). MS: m/z (%) = 561 (15)
[M+]. Anal. Calcd for C31H23N5O6 (561.54): C, 66.30; H,
4.13; N, 12.47; Found: C, 66.39; H, 4.23; N, 12.55.
Synthesis of N3,N5-bis(4-chlorobenzylidene)-2,6-dimethyl-4-
Synthesis of 2,6-dimethyl-N3,N5-bis(2,3,4,5,6-penta-acetoxcy-
hexylidene)-4-phenyl-1,4-dihydropyridine-3,5-dicarbohydrazide
phenyl-1,4-dihydropyridine-3,5-dicarbohydrazide (7).
To a
mixture of compound 2a (3.01 g, 1 mmol) in 30 mL ethanol in
the presence of 0.1 mL piperidine, p-chlorobenzaldehyde
(2.82 g, 2 mmol) was added. The reaction mixture was refluxed
for 3 h, the formed solid was filtered off, dried, and
recrystallized from acetic acid to give 7 as pale brown powders,
mp 181–182ꢁC, 5.01 g (92%). IR (KBr) n: 3183, 3148, 3129
(10).
A solution of compound 9 (6.25 g, 1 mmol) in acetic
anhydride 30 mL was stirred at room temperature over night. The
reaction mixture was poured into crushed ice; the precipitate solid
was filtered off, dried, and recrystallized from ethanol to give 10
as pale white crystals, mp 281–283ꢁC; 5.85 g (56%). IR (KBr)
n: 3140, 3127, 3122 (3NH), 1755, 1750, 1742, 1673, 1668
(3NH), 1668, 1661 (2C═O), 1608, 1602 (2C═N) cmꢀ1 1H
.
(12C═O), 1607, 1601 (2C═N) cmꢀ1 1H NMR (DMSO-d6) d
.
NMR (CDCl3) d 2.29 (s, 6H, 2CH3), 4.96 (s, 1H, pyridine-H),
7.11–7.45 (m, 13H, Ar–H), 8.0 (s, 2H, 2NH, exchangeable with
D2O), 8.2 (s, 2H, azomethine proton), 9.06 (s, 1H, NH pyridine,
exchangeable with D2O). 13C NMR (CDCl3) (d, ppm): 19.6
(2CH3), 48.3 (CH pyridine), 104.2, 104.9, 111.9, 113.2, 114.09,
121.8, 122.4, 124.8, 126.95, 130.3, 132.9, 133.04, 144.3, 145.1,
145.33 (22 C–Ar), 148.5 (2C═N), 165.3, 165.9 (2C═O). MS:
m/z (%) = 545 (27) [M+], 547 (14) [M++2]. Anal. Calcd for
C29H25Cl2N5O2 (546.45): C, 63.74; H, 4.61; N, 12.82; Found:
C, 63.68; H, 4.69; N, 12.99.
2.09–2.28 (m, 30H, 10 COCH3), 2.43 (s, 6H, 2CH3), 3.23–3.37
(m, 4H, 2CH2OAc), 4.41–4.81 (m, 4H, 4CHOAc), 5.19–5.23
(m, 5H, pyridine-H + 4CHOAc), 7.18–7.24 (m, 7H, Ar–H+2NH,
exchangeable with D2O), 8.3 (s, 2H, 2N═CH), 9.32 (s, 1H, NH
pyridine, exchangeable with D2O). 13C NMR (DMSO-d6) (d,
ppm): 17.21–19.26 (12CH3), 24.32, 24.50 (2CH2), 44.59
(CH pyridine), 61.21, 62.51, 63.02, 71.48, 72.16, 73.15, 73.94
(8 C-alditol), 103.2, 104.6, 109.26, 121.68, 122.78, 124.26, 126.2,
129.5, 147.4, 149.5 (10 C–Ar), 158,78, 159.11 (2C═N), 165.06–
166.89 (12C═O). Anal. Calcd for C47H59N5O22 (1045.99): C,
53.97; H, 5.69; N, 6.70; Found: C, 54.01; H, 5.80; N, 6.61.
Synthesis of 5,50-(2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-diyl)bis-(2-(4-chlorophenyl)-1,3,4-oxadiazol) (8).
Method
Synthesis of 1,10-(5,50-(2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-diyl)-bis[2-(2,3,4,5,6-pentaacetoxcyhexylidene)-1,3,4-oxadiazole-
A. A solution of compound 7 (5.45g, 1mmol) in 30mL of
acetic anhydride/pyridine (2:1) was refluxed for 2 h. The reaction
mixture poured onto water (100 mL), the formed solid was filtered
off, dried, and recrystallized from acetic acid to give 8.
5,3-(2H)-diyl]diethanone (11).
A solution of compound 10
(1.45 g, 1 mmol) in mixture of glacial acetic acid/bromine
(3:1) 30 mL was stirred at room temperature over night. The
reaction mixture was poured into crushed ice; the separated
solid was filtered off, dried, and recrystallized from ethanol
to give 11 as pale reddish crystals, mp over 300ꢁC; 4.86 g
(43%). IR (KBr) n: 3135 (NH), 1753, 1742, 1750, 1740, 1679,
Method B. To a solution of compound 2a (3.01 g, 1 mmol)
in 30 mL glacial acetic acid, p-chlorobenzaldehyde (2.82 g,
2 mmol) was added. The reaction mixture was refluxed for 3 h,
the formed solid was filtered off, dried, and recrystallized from
acetic acid to give 8 as brown crystals, mp 211–212ꢁC; 3.90 g
1671 (10C═O), 1669 (N–C═O), 1606, 1601 (2C═N) cmꢀ1
.
1
(72%). IR (KBr) n: 3123 (NH), 1608, 1601 (4C═N) cmꢀ1. H
1H NMR (DMSO-d6) d 2.08–2.25 (m, 30H, 10 COCH3),
2.34 (s, 6H, 2CH3), 2.47 (m, 6H, 2N–COCH3), 3.21–3.35
(m, 4H, 2CH2OAc), 4.48–4.69 (m, 4H, 4CHOAc), 5.07–5.26
(m, 4H, pyridine-H + 4CHOAc), 5.49 (s, 2H, 2 oxadiazole-
H), 7.18–7.24 (m, 5H, Ar–H), 9.27 (s, 1H, NH pyridine,
exchangeable with D2O). 13C NMR (DMSO-d6) (d, ppm):
16.29–20.58 (14CH3), 24.12, 24.45 (2CH2), 45.26 (CH
pyridine), 61.89, 62.26, 63.65, 72.03, 72.86, 73.82, 74.14 (8
C-alditol), 103.5, 104.32, 108.56, 121.68, 122.01, 123.27, 126.89,
129.35, 144.4, 147.26 (12C–Ar), 159.23, 159.98 (2C═N),
164.26–166.93 (12C═O). Anal. Calcd for C51H63N5O24
(1130.07): C, 54.20; H, 5.62; N, 6.20; Found: C, 54.11; H,
5.74; N, 6.31.
NMR (DMSO-d6) d 2.33 (s, 6H, 2CH3), 4.99 (s, 1H, pyridine-
H), 7.01–7.43 (m, 13H, Ar–H), 9.16 (s, 1H, NH pyridine,
exchangeable with D2O). 13C NMR (DMSO-d6) (d, ppm): 16.1,
16.86 (2CH3), 48.06 (CH pyridine), 103.2, 104.6, 111.26,
112.08, 121.03, 121.98, 122.2, 125.8, 126.2, 129.5, 133.04,
133.49, 141.64, 144.19, 145.32 (22 C–Ar), 149.21, 149.59
(4C═N). MS: m/z (%) = 541 (40) [M+], 543 (15) [M++2]. Anal.
Calcd for C29H21Cl2N5O2 (542.42): C, 64.21; H, 3.90; N, 12.91;
Found: C, 64.11; H, 3.99; N, 12.79.
Synthesis of 2,6-dimethyl-N3,N5-bis(2,3,4,5,6-pentahydroxy-
hexylidene)-4-phenyl-1,4-dihydropyridine-3,5-dicarbohydrazide
(9). A mixture of compound 2a (3.01 g, 1 mmol) and D-glucose
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E. R. Kotb, H.-A. S. Abbas, E. M. Flefel, H. H. Sayed, and N. A. M. Abdelwahed
Vol 000
Synthesis of 1,10-(5,50-(2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-diyl)-bis[2-(2,3,4,5,6-pentahexylidene)-1,3,4-oxadiazole
[M+]. Anal. Calcd for C25H31N5O6S2 (561.67): C, 53.46; H, 5.56;
N, 12.47; S, 11.42; Found: C, 53.36; H, 5.62; N, 12.36; S, 11.55.
Synthesis of 3,5-bis{5-[(oxiran-2-yl)methylthio]-1,3,4-oxadiazol-
(12).
To a solution of compound 11 (11.3 g, 1 mmol) in
anhydrous methanol (50mL), ammonium hydroxide solution
(5mL, 35%) was added, then the reaction mixtures were stirred at
room temperature for 3 h. The reaction mixtures were evaporated
under reduced pressure at 40ꢁC, and the residues were purified on
silica gel column using chloroform : methanol (4:1) as an eluent to
give products as brown crystals, mp over 288–289ꢁC; 1.42 g
(23%). IR (KBr) n: 3425–3258, 3135 (10OH, NH), 1606, 1601
(4C═N)cmꢀ1. 1H NMR (DMSO-d6) d 2.39 (s, 6H, 2CH3), 3.08–
3.29 (m, 4H, 2CH2OH), 3.31–4.10 (m, 8H, alditol proton), 4.86–
4.93 (m, 5H, 5OH), 5.09–5.26 (m, 6H, pyridine-H + 5OH),
7.05–7.19 (m, 5H, Ar–H), 9.03 (s, 1H, NH pyridine, exchangeable
with D2O). 13C NMR (DMSO-d6) (d, ppm): 17.21, 17.83 (2CH3),
23.02, 23.19 (2CH2), 44.48 (CH pyridine), 61.21, 62.89, 63.24,
71.08, 71.66, 72.35, 73.34 (8 C-alditol), 103.29, 104.32, 109.06,
111.05, 121.68, 122.78, 124.26, 126.32, 129.59, 147.14, 149.09
(10 C–Ar), 159.01, 159.65, 160.82, 161.05 (4C═N). MS: m/z
(%) = 621 (27) [M+]. Anal. Calcd for C27H35N5O12 (621.59): C,
52.17; H, 5.68; N, 11.27; Found: C, 52.23; H, 5.79; N, 11.16.
Synthesis of 5,50-(2,6-dimethyl-4-phenyl-1,4-dihydropyridine-
3,5-diyl)bis-(1,3,4-oxadiazole-2(3H)-thione) (13). To a warmed
solution of KOH (1.12g, 2 mmol in 2 mL water: 30mL ethanol),
compound 2a (3.01 g, 1 mmol) was added. The reaction mixture
was heated for 15 min, and the mixture was cold to room
temperature, and 2 mL carbon disulfide was added. The reaction
mixture was heated under reflux for 10h, and then poured into
crushed ice. The formed solid was filtrated off and recrystallized
from ethanol to give 13 as brown crystals, mp 161–162ꢁC; 2.46 g
(64%). IR (KBr) n: 3145, 3138, 3129 (3NH),1610, 1604 (2C═N)
2-yl}-2,6-dimethyl-4-phenyl-1,4-dihydropyridine (15).
As pale
white powders, mp 189–191ꢁC; 2.68 g (54%). IR (KBr) n: 3131
(NH), 1609, 1601 (4C═N) cmꢀ1. 1H NMR (CDCl3) d 2.33 (s, 6H,
2CH3), 2.62 (d, 4H, 2 oxiranyl-H, J= 4.2 Hz), 2.80 (m, 2H, 2
oxiranyl-H), 3.21 (d, 4H, 2 SCH2, J= 4.6 Hz), 5.01 (s, 1H,
pyridine-H), 7.10–7.26 (m, 5H, Ar–H), 9.20 (s, 1H, NH pyridine,
exchangeable with D2O). MS: m/z (%) = 497 (22) [M+]. Anal.
Calcd for C23H23N5O4S2 (497.59): C, 55.52; H, 4.66; N, 14.07; S,
12.89; Found: C, 55.69; H, 4.59; N, 14.12; S, 12.95.
Synthesis of 3,5-bis(2-hydroxy)ethylthio)-1,3,4-oxadiazol-2-
yl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine (16).
As pale
brown crystals, mp 132–133ꢁC; 1.70 g (36%). IR (KBr) n: 3320
(2OH), 3125 (NH), 1612, 1605 (4C═N) cmꢀ1. 1H NMR (CDCl3)
d 2.21 (d, 2H, 2OH, exchangeable with D2O), 2.38 (s, 6H, 2CH3),
3.2 (t, 4H, 2CH2, J = 4.6 Hz), 3.9 (t, 4H, 2CH2, J = 4.4 Hz), 4.92
(s, 1H, pyridine-H), 7.13–7.29 (m, 5H, Ar–H), 9.26 (s, 1H, NH,
exchangeable with D2O). 13C NMR (CDCl3) (d, ppm): 17.5, 17.9
(2CH3), 43.6 (CH pyridine), 45.2, 62.6 (4 CH2), 102.3, 103.2,
109.32, 112.28, 114.65, 121.6, 122.67, 131.82, 133.74, 140.3
(10 C-Ar), 153.5, 154.1, 155.3, 155.9 (4C═N). MS: m/z
(%) = 473 (48) [M+]. Anal. Calcd for C21H23N5O4S2 (473.57): C,
53.26; H, 4.90; N, 14.79; S, 13.54; Found: C, 53.19; H, 5.01; N,
14.69; S, 13.62.
Biological activity. Antitumor screening. The aim of the
present study was to illustrate the effect of some newly
synthesized compounds on the human breast cancer cell line
(T47D) in comparison with DOX in a trial to get more effective
and less toxic agents.
1228, 1220 (2C═S)cmꢀ1 1H NMR (CDCl3) d 2.37 (s, 6H,
.
2CH3), 5.06 (s, 1H, pyridine-H), 7.08–7.27 (m, 7H, Ar–H + 2NH,
exchangeable with D2O), 9.09 (s, 1H, NH pyridine, exchangeable
with D2O). 13C NMR (CDCl3) (d, ppm): 17.8 (2CH3), 45.6 (CH
pyridine), 103.4, 104.7, 123.2–143.6 (10 C–Ar), 155.1 (2C═N),
157.6, 158.2 (2C═S). MS: m/z (%) = 385 (20) [M+]. Anal. Calcd for
C17H15N5O2S2 (385.46): C, 52.97; H, 3.92; N, 18.17; S, 16.64;
Found: C, 53.07; H, 3.82; N, 18.23; S, 16.73.
MATERIALS AND METHODS
Preliminary experiments used the human tumor cell line
to identify the potential toxicity of some newly synthesized
compounds (2a, 4, 5, 6, 9, 10, 14, 15, and 16) in compari-
son with known anticancer drug DOX by using the method
of Skehan et al. [28].
Alkylation of bis 1,3,4-oxadiazole thione derivatives. Synthesis
of compounds (14–16): General procedure. To a solution of
13 (3.85 g, 1 mmol) in 20 mL DMF, sodium hydride (0.48 g,
2 mmol) was added. The reaction mixture was stirred
at 60ꢁC for 1 h, then the reaction mixture was cooled then
added 2-(2-chloroethoxy) ethanol, epichlorohydrine or 2-chloro
ethanol (2 mmol) was added then stirred at room temperature
over night. The reaction mixture was evaporated under reduced
pressure; the residue was washed with distilled water, filtered
off, dried, and recrystallized from methanol.
• Human tumor cell lines were obtained frozen in liquid
nitrogen (ꢀ180ꢁC) from the American Type Culture
Collection. The tumor cell lines were maintained in the
National Cancer Institute, Cairo, Egypt, by serial sub
culturing. RPMI-1640 medium (Sigma Chemical Co.,
St. Louis, MO, USA).
• The medium was prepared and used for culturing and
maintenance of the human tumor cell lines. The
prepared medium was kept in a refrigerator (4ꢁC) and
checked at regular intervals for contamination.
• Before the use, the medium was warmed at 37ꢁC in
a water bath and supplemented with penicillin/
streptomycin and FBS.
Synthesis of 3,5-bis-(2(2-hydroxyethoxy)ethylthio)-1,3,4-oxzadia-
zol-2-yl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridin (14). As white
crystals, mp 141–143ꢁC; 2.69 g (48%). IR (KBr) n: 3352 (2OH),
3122 (NH), 1610, 1605 (4C═N) cmꢀ1 1H NMR (CDCl3) d 2.1
.
(s, 2H, 2OH, exchangeable with D2O), 2.29 (s, 6H, 2CH3), 3.1 (t,
4H, 2CH2, J= 6.1 Hz), 3.41 (t, 4H, 2CH2, J= 4.2 Hz), 3.64 (t, 4H,
2CH2, J= 6.2 Hz), 3.74 (t, 4H, 2CH2, J= 4.2 Hz), 4.99 (s, 1H,
pyridine-H), 7.01–7.16 (m, 5H, Ar–H), 9.32 (s, 1H, NH pyridine,
exchangeable with D2O). 13C NMR (CDCl3) (d, ppm): 16.32, 16.87
(2CH3), 45.65 (CH pyridine), 46.59–63.2 (8 CH2), 103.87, 105.06,
111.26, 122.68, 122.98, 124.06, 126.32, 129.5, 147.4 (10 C–Ar),
155.26, 156.72, 158,78, 159.11 (4C═N). MS: m/z (%) = 561 (38)
• Different concentrations of the compounds tested (0, 5,
12.5, 25, and 50 mg/mL) were added to the cell
monolayer. Each concentration was evaluated three
times (each dose was incubated with the cells in three
different wells).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Utility of Hantzsch Ester in Synthesis of Some 3,5-Bis-dihydropyridine Derivatives and
Studying Their Biological Evaluation
• Monolayer cells were incubated with the compounds for
48 h at 37ꢁC and atmosphere of 5% CO2.
bacterium/fungus was considered as the MIC of the test
compounds. The MIC values are tabulated in Table 3.
• After 48 h, cells were fixed, washed, and stained with
Sulforhodamine-B stain.
• Excess stain was recovered with Tris EDTA buffer.
• Color intensity was measured in an enzyme-linked
immunosorbent assay reader.
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activity of the synthesized compounds was investigated
against several pathogenic representative Gram-positive
bacteria (B. subtilis) and (S. aureus), Gram-negative bacteria
(E. coli), Fungi (A. niger) and Yeast (C. albicans).
Agar diffusion medium.
Eleven compounds were
screened in vitro for their antimicrobial activity by the agar
diffusion method [29]. A suspension of organisms was
added to a sterile nutrient agar medium at 45ꢁC and the
mixture was transferred to a sterile Petri dish and allowed
to solidify. Holes of 10 mm in diameter were made using a
cork borer and filled with the solution of synthesized
compounds (100 mgmLꢀ1). A hole filled with DMSO was
used as control. The plates were left for 1 h at room
temperature as a period of pre-incubation. The plates were
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Minimum inhibitory concentration.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet