Novel synthesis of hydrazide-hydrazone derivatives and their utilization in the synthesis of coumarin, pyridine, thiazole and thiophene derivatives with antitumor activity

Article abstract of DOI:10.3390/molecules16010016

The reaction of cyanoacetyl hydrazine (1) with 3-acetylpyridine (2) gave the hydrazide-hydrazone derivative 3. The latter compound undergoes a series of heterocyclization reactions to give new heterocyclic compounds. The antitumor evaluation of the newly

Full text of DOI:10.3390/molecules16010016

Molecules 2011, 16, 16-27; doi: 10.3390/molecules16010016
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Molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Synthesis of Hydrazide-Hydrazone Derivatives and Their
Utilization in the Synthesis of Coumarin, Pyridine, Thiazole and
Thiophene Derivatives with Antitumor Activity
Rafat M. Mohareb 1,2,*, Daisy H. Fleita 3 and Ola K. Sakka 3
1
Department of Organic Chemistry, Faculty of Pharmacy, October University for Modern Sciences
and Arts, October City, A.R., Egypt
2
Department of Chemistry, Faculty of Science, Cairo University, Giza, A.R., Egypt
Department of Chemistry, Faculty of Science, American University in Cairo, 5th Settlement, A.R.,
3
Egypt
*Author to whom correspondence should be addressed; E-Mail: raafat_mohareb@yahoo.com;
Tel.: +202-37626269 or +202-35676570.
Received: 11 November 2010; in revised form: 5 December 2010 / Accepted: 8 December 2010 /
Published: 23 December 2010
Abstract: The reaction of cyanoacetyl hydrazine (1) with 3-acetylpyridine (2) gave the
hydrazide-hydrazone derivative 3. The latter compound undergoes a series of
heterocyclization reactions to give new heterocyclic compounds. The antitumor evaluation of
the newly synthesized products against three cancer cell lines, namely breast
adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268)
was performed. Most of the synthesized compounds showed high inhibitory effects.
Keywords: hydrazide-hydrazone; 3-acetylpyridine; thiophene; coumarin; antitumor activity
1. Introduction
Hydrazines and their derivatives constitute an important class of compounds that has found wide
utility in organic synthesis [1,2]. While hydrazines have traditionally been employed as reagents for
the derivatization and characterization of carbonyl compounds, in recent years the N-N linkage has
been used as a key structural motif in various bioactive agents. In particular, an increasing number of N-N
bond-containing heterocycles and peptidomimetics have made their way into commercial applications
as pharmaceutical and agricultural agents [3,4]. Recently, hydrazide-hydrazones have gained great

Molecules 2011, 16
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importance due to their diverse biological properties including antibacterial, antifungal, anticonvulsant,
anti-inflammatory, antimalarial and antituberculosis activities [5-17]. With the aim of obtaining novel
hydrazide-hydrazones with a wide spectrum of pharmaceutical applications, we report herein the
synthesis of a series of hydrazide-hydrazones together with their use in a series of heterocyclic
transformations and their evaluation as anti-tumor agents [18-21].
2. Results and Discussion
Recently, our research group became involved with a comprehensive program involving the
synthesis of a series of hydrazide-hydrazone derivatives and their utilization in the synthesis of
heterocyclic compounds with potential pharmaceutical and biological activities [22,23]. In
continuation to this program, we report herein the reaction of cyanoacetylhydrazine (1) with
3-acetylpyridine (2) in 1,4-dioxane to form the hydrazide-hydrazone derivative 3. The structure of
1
compound 3 was confirmed based on its analytical and spectral data. Thus, the H-NMR showed a
singlet at δ 2.28 for the CH3 group, a singlet at δ 4.26 for the CH2 group, a multiplet at δ 7.43-8.99 for
the pyridyl group and a singlet (D2O exchangeable) at δ 10.81 for the NH group. Moreover, the
13C- NMR spectrum showed peaks at δ: 14.2 (CH3), 28.9 (CH2), 116.8 (CN), 122.1, 123.4, 133.7,
150.2, 151.0 (pyridine C), 168.1 (C=N), 169.8 (C=O). Further structure elucidation of compound 3
was obtained through the study of its reactivity towards chemical reagents. Thus, the reaction of 3 with
either benzaldehyde (4a), 4-chlorobenzaldehyde (4b) or 4-methoxybenzaldehyde (4c) gave the
corresponding benzal derivatives 5a-c, respectively (Scheme 1).
Scheme 1. Synthesis of the hydrazide-hydrazones 3 and 5a-c.
O
O
C
CH3
COCH3
CH2
CN
C
NH
N
C
CH2
CN
NHNH2
N
2
N
1
3
O
C
CH3
3
R
CHO
R
HC
NH
N C
C
CN
4a,R=H
b,R=Cl
c,R=OCH3
5a,R=H
b,R=Cl
c,R=OCH3
N
On the other hand, the reaction of compound 3 with salicylaldehyde (6) gave the coumarin
derivative 7 (Scheme 2). Analytical and spectral data of the product are in agreement with the
proposed structure (see Experimental section).
Scheme 2. Synthesis of 7.
O
CH3
CHO
OH
C
NH
N C
3
O
O
N
6
7

Molecules 2011, 16
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Next, we studied the reactivity of the active methylene group present in compound 3 towards
diazonium salts. Thus, the reaction of 3 with either benzenediazonium chloride (8a), 4-chlorobenzene-
diazonium chloride (8b), 4-bromobenzenediazonium chloride (8c) or 4-nitrobenzenediazonium
chloride (8d), gave the hydrazone derivatives 9a-d, respectively (Scheme 3). Analytical and spectral
data of the latter reaction products are all consistent with the proposed structures.
Scheme 3. Synthesis of phenylhydrazo derivatives 9a-d.
O
CH3
R
N
NCl
3
R
NH
N
C
C
NH
N
C
CN
8a, R=H
b,R=Cl
c,R=Br
9a,R=H
b,R=Cl
c,R=Br
N
d,R=NO2
d,R=NO2
Moreover, the reaction of compound 3 with cyclohexanone (10) and elemental sulfur in the presence
of triethylamine was studied as an application of Gewald’s thiophene synthesis. The reaction led to the
formation of 4,5,6,7-tetrahydrobenzo[b]thiophene derivative 11. On the other hand, the reaction of
compound 3 with cyclopentanone (12) and sulfur gave the cyclopentene[b]thiophene derivative 13. The
structures of compounds 11 and 13 were based on analytical and spectral data (see Experimental
section). Further confirmation of structure 11 was obtained through its synthesis via another reaction
route. Thus, the reaction of compound 3 with cyclohexanone in the presence of ammonium acetate in an
oil bath at 140 °C gave the Knoevenagel condensation product 14. The latter reacted with elemental
sulfur in the presence of triethylamine to produce the same tetrahydrobenzo[b]thiophene derivative 11. It
is convenient to notice that the yield (70%) for formation of compound 11 using the latter procedure was
higher than in the synthetic pathway described before (62%) (Scheme 4).
Scheme 4. Synthesis of 11, 13 and 14.
O
O
CH3
N
C NH N C
3
S
NH2
S
10
11
O
O
CH3
N
C NHN C
3
S
NH2
12
S
13
O
O
CH3
C
NH4OAc
C C NHN
CN
3
N
14
10
Et3N
S
11
14

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Next, we studied the reactivity of 3 towards phenyl isothiocyanate in basic dimethylformamide
solution followed by heterocyclization with active methylene reagents like -haloketones with the aim
of synthesizing thiazole derivatives with potential antitumor activity. Thus, compound 3 reacted with
phenylisothiocyanate (15) in DMF/KOH solution at room temperature to give the intermediate
potassium sulphide salt 16. Heterocyclization of 16 with α-haloketones like ethyl bromoacetate (17)
gave the thiophene derivative 18. The structure of 18 was confirmed based on analytical and spectral
1
data. The H-NMR showed a singlet at δ 2.22 for the CH3 group, a singlet at δ 7.46 for the thiazole
hydrogen, a multiplet at δ 7.54-8.95 for the pyridine H and C6H5 and two singlets at 8.80/10.13 for
the NH and OH groups, respectively. Moreover, the 13C-NMR spectrum showed the presence of
peaks at δ 14.3 (CH3), 93.0, 101.2 (C=C), 115.8 (CN), 119.2, 120.5, 121.8, 122.3, 124.0, 124.8, 127.6,
130.5, 133.2, 137.0, 150.7, 152.2 (C6H5, thiazole, pyridine C), 168.4 (C=N), 170.0 (C=O).
In a similar way, the reaction of 16 with ethyl bromocyanoacetate (19) gave the thiazole derivative
20. Furthermore, compound 3 reacted with phenyl isothiocyanate and elemental sulfur in 1,4-dioxane
containing triethylamine to give the thiazole derivative 21 (Scheme 5). The analytical and spectral data
of 21 were in agreement with the proposed structure (see Experimental section).
Scheme 5. Synthesis of 18, 20 and 21
O
CH3
H
CN
C
N
N
C
KOH
DMF
C
C
3
Ph N C S
S-K+
PhHN
CN
N
15
16
CH3
O
N
C
C
NH
C
16
H2C
Br
COOEt
Ph
N
S
N
18
17
HO
CN
O
C
CH3
N
NH
C
16
CH
CN
Br
COOEt
19
Ph N
S
N
EtOOC
20
CN
O
CH3
S
S
C
NH
N C
S
3
Ph N C S
N
Ph
NH2
N
21
Next, we studied the reactivity of the hydrazide-hydrazone derivative 3 towards cinnamonitrile
derivatives. Thus, the reaction of 3 with either 2-benzylidenemalononitrile (22a) or ethyl 2-cyano-3-
phenylacrylate (22b) gave the pyridine derivatives 23a and 23b, respectively (Scheme 6).

Molecules 2011, 16
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Scheme 6. Synthesis of 23a,b.
CH3
N
CN
O
N
C
X
3
Ph CH
C
N
CN
Ph
NH2
CN
22a, X = CN
b, X = COOEt
23a, X = CN
b, X = COOEt
Effect on the Growth of Human Tumor Cell Lines
The effect of compounds 3-25 on the in vitro growth of three human tumor cell lines representing
different tumor types, namely, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460)
and CNS cancer (SF-268) was evaluated after a continuous 48 h exposure. The results are summarized in
Table 1. All of the tested compounds were able to inhibit the growth of the tested human tumor cell lines
in a dose-dependant manner (data not shown). The results indicated in Table 1 revealed that compound 3
showed the highest inhibitory effect against all the three tumor cell lines. In addition compound 5c
showed the best inhibitory effect against CNS cancer (SF-268), while compounds 7 and 23a showed
high inhibitory effects against non-small cell lung cancer (NCI-H460) and breast adenocarcinoma
(MCF-7), respectively. Compounds 5b, 9a, 23b showed the lowest inhibitory effects. The rest of the
compounds showed a moderate growth inhibitory effect. Comparing compound 5b with 5c, it is obvious
thatthe presence of the CH3O group in 5c resulted in a higher inhibitory effect than 5b with the Cl group.
Comparing the pyridine derivatives 23a (with the cyano group) and 23b (with the carboxyethyl group),
the first has a greater inhibitory effect than the second towards the three cell lines.
Table 1. Effect of the newly synthesized product on the growth of three human tumor cell.
GI50 (µM)
Compound
3
5a
5b
5c
7
9a
9b
9c
11
13
14
17
19
21
23a
23b
MCF-7
NCI-H460
0.2 ± 0.001
26.6 ± 1.4
38.9 ± 10.8
3.0 ± 1.6
SF-268
0.1 ± 0.009
20.0 ± 0.2
60.6 ± 16.9
2.0 ± 0.2
66.8 ± 12
74.7 ± 17.5
20 ± 0.4
28.9 ± 0.9
30 ± 0.6
36.0 ± 1.8
50.1 ± 0.7
22 ± 0.4
0.6 ± 0.001
38.4 ± 0.6
28.8 ± 8.6
0.07 ± 0.001
36.8 ± 3.0
62.0 ± 9.01
22.2 ± 0.8
54.8 ± 0.8
22.3 ±1.5
20.5 ± 1.1
18.4 ± 1.8
30 ± 0.8
10 ± 6.2
48.2 ± 12.8
20.3 ± 0.8
40.6 ± 1.8
17.3 ± 1.4
44.0 ± 0.8
23.2 ± 4.8
20.3 ± 0.8
24.1 ± 0.8
24.1 ± 0.8
14.1 ± 0.6
40.6 ± 1.8
22.0 ± 0.2
35.4 ± 10.2
11.9 ± 0.5
70.9 ± 0.9
38.4 ± 0.6
18.9 ± 6.8
20.3 ± 0.5
60.8 ± 0.8
Results are given in concentrations that were able to cause 50 % of cell growth inhibition (GI50).
after a continuous exposure of 48 h and show means ± SEM of three-independent experiments
performed in duplicate. Doxorubicin was used as positive control, GI50: MCF-7 = 42.8 ± 8.2 nM;
NCI-H460 = 94.0 ± 8.7 nM, and SF-280 = 94.0 ± 7.0 nM.

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3. Experimental
3.1. General
Melting points were determined on an Electrothermal melting point apparatus (Electrothermal 9100)
and are uncorrected. IR spectra were recorded for KBr discs on a Pye Unicam SP-1000
1
spectrophotometer. H-NMR and 13C-NMR spectra were measured on a Varian EM-390-200 MHz in
CD3SOCD3 as solvent using TMS as internal standard, and chemical shifts are expressed as δ. Analytical
data were obtained from the Microanalytical Data Unit at Cairo University, Giza, Egypt. Antitumor
evaluation for the newly synthesized products were performed by a research group at the National
Research Center and the National Cancer Institute at Cairo University.
2-Cyano-N'-(1-(pyridine-3-yl)ethylidene)acetohydrazide (3). To a solution of cyanoacetylhydrazine (2,
0.99 g, 0.01 mol) in 1,4-dioxane (20 mL), 3-acetylpyridine (1.21 g, 0.01 mol) was added. The reaction
mixture was heated under reflux for 2 h then poured onto a beaker containing an ice/water mixture.
The formed solid product was collected by filtration and dried to give white crystals (from ethanol).
Yield: 1.50 g, 74%, m.p. 203-205 °C; IR (KBr) υ/cm−1: 3500-3400 (NH), 3066 (CH-aromatic), 2885
(CH3), 2200 (CN), 1680 (C=O); MS m/z (%) 202 [M+, 8%]; 1H-NMR δ: 2.28 (s, 3H, CH3), 4.26 (s, 2H,
13
CH2), 7.43-8.99 (m, 4H, pyridine-H),10.81(s, 1H, NH); C-NMR δ: 14.2 (CH3), 28.9 (CH2), 116.8
(CN), 122.1, 123.4, 133.7, 150.2, 151.0 (pyridine C), 168.1 (C=N), 169.8 (C=O); Anal. Calcd. for
C10H10N4O (202.21): C, 59.40; H, 4.98; N, 27.71%. Found: C, 59.72; H, 5.20; N, 28.01%.
3.2. General Procedure for the Synthesis of 5a, 5b or 5c
To a solution of 3 (2.02 g, 0.01 mol) in 1,4-dioxane (20 mL), either benzaldehyde (1.06 g, 0.01 mol),
p-chlorobenzaldehyde(1.12 g, 0.01 mol) or p-methoxybenzaldehyde (1.08 g, 0.01 mol) was added. The
reaction mixture was heated under reflux for 6 h then poured onto a beaker containing an ice/water
mixture. The formed solid product was collected by filtration and dried.
2-Cyano-3-phenyl-N'-(1-(pyridin-3-yl)ethylidene)acrylohydrazide (5a). White crystals (from acetone).
Yield: 1.32 g, 45.4%, m.p. 115-118 °C; IR (KBr) υ/cm−1: 3460-3380 (NH), 3050 (CH-aromatic), 2890
(CH3), 2220 (CN), 1683 (C=O), 1635 (C=N); MS m/z (%) 290 [M+, 39.7%];1H-NMR δ: 2.28 (s, 3H,
CH3), 3.31 (s, 1H, CH), 7.30-7.91 (m, 9H, C6H5, pyridine-H), 8.71 (s, 1H, NH); 13C-NMR δ: 14.0
(CH3), 98.3 (C=CH), 148.0 (C=CH), 116.8 (CN), 124.3, 125.7, 15.9, 126.2, 128.0, 129.4, 136.5, 150.2,
151.6 (benzene, pyridine C), 165.3 (C=N), 170.0 (C=O); Anal. Calcd. for C17H14N4O (290.32): C,
70.33; H, 4.86; N, 19.30%. Found: C, 70.01; H, 4.92; N, 19.48%.
3-(4-Chlorophenyl)-2-cyano-N'-(1-(pyridin-3-yl)-ethylidene)acrylo-hydrazide (5b). Pale yellow
crystals (from acetone). Yield: 1.95 g, 60.1%, m.p. 100 °C; IR (KBr) υ/cm−1: 3460-3380 (NH), 3050
(CH-aromatic), 2890 (CH3), 2200 (CN), 1683 (C=O), 1623 (C=N); MS m/z (%) 324 [M+, 5.17%];
1H-NMR δ: 2.27 (s, 3H, CH3), 3.38 (s, 1H, CH), 7.17-7.94 (m, 8H, C6H4, pyridine-H), 10.00 (s, 1H,
NH); 13C-NMR δ : 14.1 (CH3), 98.0 (C=CH), 147.8 (C=CH), 116.9 (CN), 122.8, 124.6, 15.9, 126.0,
127.1, 128.9, 136.5, 150.2, 151.6 (benzene, pyridine C), 165.3 (C=N), 170.0 (C=O); Anal. Calcd. for
C17H13ClN4O (324.76): C, 62.87; H, 4.03; N, 17.25%. Found: C, 63.01; H, 4.26; N, 17.32%.

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2-Cyano-3-(4-methoxyphenyl)-N'-(1-(pyridin-3-yl)ethylidene)acrylo-hydrazide (5c). Yellow crystals
(from acetone). Yield: 2.40 g, 75%, m.p. 130-133 °C; IR (KBr) υ/cm−1: 3460-3370 (NH) , 3100 (CH-
aromatic), 2890 (CH3), 2200 (CN), 1683 (C=O), 1640 (C=N); MS m/z (%) 320 [M+, 44.28%]; 1H-
NMR δ: 2.49 (s, 3H, CH3), 3.31(s, 1H, CH), 3.82 (s, 1H, CH3), 7.03-7.82 (m, 8H, C6H4, pyridine-H),
8.62 (s, 1H, NH); 13C-NMR δ: 14.4, 54.6 (2 CH3), 99.9. (C=CH), 148.3 (C=CH), 116.3 (CN), 122.3,
124.6, 125.3, 125.9, 127.8, 128.4, 135.3, 150.0, 151.9 (benzene, pyridine C), 165.6 (C=N), 168.9
(C=O); Anal. Calcd. for C18H16N4O2 (320.35): C, 67.49; H, 5.03; N, 17.49%. Found: C, 67.55; H, 4.89;
N, 17.31%.
2-Oxo-N'-(1-(pyridin-3-yl)ethylidene)-2H-chromene-3-carbohydrazide (7). To a solution of 3 (2.02 g,
0.01 mol) in 1,4-dioxane (20 mL), salicylaldehyde (1.22 g,0.01 mol) was added. The reaction mixture
was heated under reflux for 3 hours then poured onto a beaker containing an ice/water mixture. The
formed solid product was collected by filtration and dried to give white crystals (from ethanol). Yield:
2.10 g, 68%, m.p. 235-238 °C; IR (KBr) υ/cm−1: 3450-3360 (NH), 3200 (CH-aromatic), 2890 (CH3),
2200 (CN), 1670 (C=O), 1650 (C=N); MS m/z (%) 307 [M+, 2.5%]; 1H-NMR δ2.51(s, 3H, CH3), 7.30
(s, 1H, coumarin H-4), 7.48-9.33 (m, C6H4, pyridine-H), 13.69 (s, 1H, NH); 13C-NMR δ: 13.8 (CH3),
121.8, 122.6, 122.9, 124.5, 127.1, 128.0, 130.9, 131.6, 150.2, 152.3 (C6H4, pyridine C), 159.9
(coumarin C=O), 167.9 (C=N), 169.8 (C=O); Anal. Calcd. for C17H13N3O3 (307.30): C, 66.44; H, 4.26;
N, 13.67%. Found: C, 66.38; H, 4.35; N, 13.92%.
3.3. General Procedure for the Synthesis of 9a, 9b, 9c or 9d
To a cold solution of 3 (2.02 g, 0.01 mol) in ethanol (20 mL) containing sodium acetate (3.0 g) was
added with continuous stirring either of the appropriate substituted benzenediazonium salt (0.01 mol)
[prepared by adding sodium nitrite (1.6 g, 0.02 mol) in water (8 mL) to a cold solution of either of the
appropriate substitute aniline 8a-d in the appropriate amount of hydrochloric acid]. The reaction
mixture was stirred for 2 h and the formed solid product, in each case, was collected by filtration.
2-Cyano-2-(2-phenylhydrazinylidene)-N'-[1-(pyridin-4-yl)ethylidene]aceto-hydrazide (9a). Orange
crystals (from ethanol). Yield: 1.92 g, 63%, m.p. 158-161 °C; IR (KBr) υ/cm−1: 3500-3400 (NH), 3050
(CH-aromatic), 2800 (CH3), 2200 (CN), 1696 (C=O); MS m/z (%) 306 [M+, 93.51%]; 1H-NMR δ2.49
(s, 3H, CH3), 7.11-8.22 (C6H5, pyridine H), 10.25, 12.02 (2s, 2H, 2NH); 13C-NMR δ: 14.1 (CH3),
116.0 (CN), 115.9, 116.8, 118.6, 122.3, 123.9, 129.0, 142.8, 150.2, 152.8 (C6H5, pyridine C), 163.2,
166.7 (2 C=N), 168.8 (C=O); Anal. Calcd. for C16H14N6O (306.32): C, 62.74; H, 4.61; N, 27.44%.
Found: C, 62.90; H, 4.51; N, 27.52%.
2-[2-(4-Chlorophenyl)hydrazinylidene]-2-cyano-N'-[1-(pyridin-4-yl)ethylidene] acetohydrazide (9b).
Orange crystals (from ethanol). Yield: 2.32 g, 68%, m.p. 174-177 °C; IR (KBr) υ/cm−1: 3600-3500
1
(NH), 3100 (CH-aromatic), 2890 (CH3), 2200 (CN), 1696 (C=O); MS m/z (%) 340 [M+, 6.17%]; H-
NMR δ2.49 (s, 3H, CH3), 6.95-7.89 (C6H4-pyridine H), 11.737, 12.75 (2s, 2H, 2NH); 13C-NMR δ:
14.0 (CH3), 115.8 (CN), 116.0, 117.3, 118.9, 121.8, 124.5, 125.4, 143.5, 150.0, 151.8 (C6H5, pyridine
C), 163.0, 166.9 (2 C=N), 168.9 (C=O); Anal. Calcd. for C16H13ClN6O (340.77): C, 56.39; H, 3.85; N,
24.66%. Found: C, 56.60; H, 4.01; N, 24.90%.

Molecules 2011, 16
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2-[2-(4-Bromophenyl)hydrazinylidene]-2-cyano-N'-[1-(pyridin-4-yl)ethylidene] acetohydrazide (9c).
Orange crystals (from ethanol). Yield: 2.62 g, 68%, m.p. 183-186 °C; IR (KBr) υ/cm−1: 3550-3400
1
(NH), 3090 (CH-aromatic), 2890 (CH3), 2200 (CN), 1685(C=O); MS m/z (%) 387 [M+, 19 %]; H-
NMR δ 2.49 (s, 3H, CH3), 6.93-7.84 (C6H4, pyridine H), 9.50, 11.75 (2s, 2H, 2NH); 13C-NMR δ: 14.2
(CH3), 116.6 (CN), 116.3, 118.0, 118.6, 122.8, 124.5, 128.0, 138.9, 150.2, 152.5 (C6H5, pyridine C),
163.0, 166.5 (2 C=N), 168.5 (C=O); Anal. Calcd. for C16H13BrN6O (384): C, 49.89; H, 3.40; N,
21.82%. Found: C, 48.93; H, 3.62; N, 22.02%.
2-Cyano-2-[2-(4-nitrophenyl)hydrazinylidene]-N'-[1-(pyridin-4-yl)ethylidene] acetohydrazide (9d).
Orange crystals (from ethanol). Yield: 2.43 g, 69%, m.p. 162-165 °C; IR (KBr) υ/cm−1: 3570-3400
(NH), 3100 (CH-aromatic), 2890 (CH3), 2200 (CN), 1670 (C=O); MS m/z (%) 352 [M+, 20.2 %]; 1H-
NMR δ 2.49 (s, 3H, CH3), 6.57-8.39 (C6H4, pyridine H), 10.48, 12.11 (2s, 2H, 2NH); 13C-NMR δ: 14.1
(CH3), 116.0 (CN), 116.8, 116.9, 118.2, 123.8, 126.3, 128.6, 136.1, 151.0, 152.7 (C6H5, pyridine C),
163.1, 166.4 (2 C=N), 168.9 (C=O); Anal. Calcd. for C16H13N7O3 (351.32): C, 54.70; H, 3.73; N,
27.91%. Found: C, 54.85; H, 3.90; N, 29.11%.
2-Amino-4,5,6,7-tetrahydro-N'-(1-(pyridin-3-yl)ethylidene)benzo[b]thiophene-3-carbohydrazide (11).
Method A: To a solution of 3 (2.02 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1 mL)
and elemental sulfur (0.32 g, 0.01mol), cyclohexanone 10 (0.98 g, 0.01 mol) was added. The reaction
mixture was heated under reflux for 3 hours then poured onto a beaker containing an ice/water
mixture. The formed solid product was collected by filtration and dried obtaining pale yellow crystals
(from ethanol).
Method B: To a solution of compound 14 (2.82 g, 0.01 mol) in 1,4-dioxane (40 mL) containing
triethylamine (0.5 mL), elemental sulfur (0.32 g, 0.01 mol) was added. The reaction mixture was
heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid. The
formed solid product was collected by filtration.
Yield: 1.95g, 62% (method A) and 2.20 g, 70% (method B), m.p. 112 °C; IR (KBr) υ/cm−1: 3400-
3300 (NH2, NH), 3068 (CH-aromatic), 2886 (CH3), 2250 (CN), 1690 (C=O), 1638 (C=C); MS m/z (%)
314 [M+, 2.19 %]; 1H-NMR δ2.29-2.31 (m, 8H, 4CH2), 2.49 (s, 3H, CH3), 4.25 (s, 2H, NH2), 7.41-8.88
(m, 4H, pyridine-H), 10.76 (s, 1H, NH);13C-NMR δ: 13.8 (CH3), 22.8, 23.6, 24.1, 24.9 (4 CH2), 116.5,
122.6, 124.8, 130.2, 150.6, 151.8 (thiophene, pyridine C), 163.6 (C=O), 170.0 (C=N); Anal. Calcd. for
C16H18N4OS (314.41): C, 61.12; H, 5.77; N, 17.82%. Found: C, 60.91; H, 6.01; N, 17.85%.
2-Amino-5,6-dihydro-N'-(1-(pyridin-3-yl)ethylidene)-4H-cyclopenta[b]thiophene-3-carbohydrazide
(13). To a solution of 3 (2.02 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1.0 mL) and
elemental sulfur (0.32 g, 0.01 mol), cyclopentanone 12 (0.98 g, 0.01 mol) was added. The reaction
mixture was heated under reflux for 3 h then poured onto a beaker containing an ice/water mixture.
The formed solid product was collected by filtration and dried obtaining pale yellow crystals (from
ethanol).Yield: 1.82 g, 61%, m.p. 140-144 °C; IR (KBr) υ/cm−1: 3450-3300 (NH2, NH), 3080 (CH-
1
aromatic), 2890 (CH3), 2250 (CN), 1690 (C=O); MS m/z (%) 300 [M+, 0.34 %]; H-NMR δ2.28-2.35
(m, 6H, 3CH2), 2.48 (s, 3H, CH3), 4.25 (s, 2H, NH2), 7.41-8.99 (m, 4H, pyridine-H),10.78 (s, 1H, NH);
13C-NMR δ: 14.0 (CH3), 20.8, 24.6, 26.9 (3 CH2), 116.9, 123.0, 124.6, 133.1, 150.1, 151.4 (thiophene,

Molecules 2011, 16
24
pyridine C), 163.4 (C=O), 169.8 (C=N); Anal. Calcd. for C15H16N4OS (300.3): C, 59.98; H, 5.37; N,
18.65%. Found: C, 59.93; H, 5.39; N, 18.81%
2-Cyano-2-cyclohexylidene-N'-(1-(pyridin-3-yl)ethylidene)acetohydrazide (14). Equimolar amounts of
compound 3 (2.02 g, 0.01 mol) and cyclohexanone 10 (0.98 g, 0.01 mol) were heated in an oil bath at
140 °C for 1 h in presence of ammonium acetate. After cooling the reaction mixture, it was heated in
ethanol, then poured into ice/water mixture and the formed solid product was collected by filtration and
dried to give pale yellow crystals (from ethanol). Yield: 1.52 g, 54% , m.p. 145-146 °C; IR (KBr)
υ/cm−1: 3355-3370 (NH), 3067 (CH-aromatic), 2930 (CH3), 2200 (CN), 1675 (C=O); MS m/z (%) 282
[M+, 0.40 %]; 1H-NMR δ 2.26-2.34 (m, 10H, 5CH2), 2.31(s, 3H, CH3), 7.41-8.99 (m, 4H, pyridine-H),
10.90 (s, 1H, NH); 13C-NMR δ: 13.9 (CH3), 26.8, 27.4, 26.9, 27.0 (cyclohexane C), 93.0 (C=C), 116.8
(CN), 123.4, 126.8, 126.4, 150.5, 151.2 (pyridine C), 168.2 (C=N), 177.3 (C=O); Anal. Calcd. for
C16H18N4O (282.34): C, 68.06; H, 6.43; N, 19.84%. Found: C, 67.85; H, 6.31; N, 20.22%
3.4. General Procedure for the Synthesis of 18 and 20
Compound 3 (2.02 g, 0.01 mol) is dissolved in ethanol and a few sodium hydroxide pellets were
added. Phenylisothiocyanate (15, 1.35, 0.01 mol) is then added and the solution is covered and left
standing overnight. Equimolar amounts of either ethyl 2-bromoacetate (17) or ethyl 2-bromo-2-
cyanoacetate (19) are stirred in the following day, and the solution is covered for another night, after
which the reaction mixture is poured onto ice and the precipitated solid is filtered off.
2-(4-Hydroxy-3-phenylthiazol-2(3H)-ylidene)-2-isocyano-N'-(1-(pyridin-3-yl)ethylidene) acetohydrazide
(18). Orange crystals (from ethanol). Yield: 2.43 g, 64%, m.p. 125-127 °C; IR (KBr) υ/cm−1: 3500-
3370 (NH), 3400 (OH), 3100 (CH-aromatic), 2900 (CH3), 2189 (CN), 1675 (C=O); MS m/z (%) 377
1
[M+, 7.9 %]; H-NMR δ 2.22 (s, 3H, CH3), 7.46 (thiazole H-5),7.54-8.95 (C6H5-pyridine H), 8.80
(NH), 10.13 (OH); 13C-NMR δ: 14.3 (CH3), 93.0, 101.2 (C=C), 115.8 (CN), 119.2, 120.5, 121.8,
122.3, 124.0, 124.8, 127.6, 130.5, 133.2, 137.0, 150.7, 152.2 (C6H5, thiazole, pyridine C), 168.4
(C=N), 170.0 (C=O); Anal. Cald. for C19H15N5O2S (377.42): C, 60.46; H, 4.01; N, 18.56; S, 8.50.
Found: C, 60.50; H, 4.01; N, 18.55; S, 8.48%.
(2Z)-Ethyl-2-((1-(pyridin-3-yl)ethylideneaminocarbamoyl)(cyano)methylene)-4-cyano-3-phenylthiazolidine
-4-carboxylate (20). Orange crystals (from ethanol).Yield: 3.22 g, 70%, m.p. 125-127 °C; IR (KBr)
υ/cm−1: 3577-3370 (NH), 3067 (CH-aromatic), 2930 (CH3), 2200 (CN), 1675 (C=O); MS m/z (%)
1
461.1 [M+, 25.2 %]; H-NMR δ 1.56 (t, 3H, J = 7.02 Hz, CH3), 2.26 (s, 3H, CH3), 4.25 (q, 2H,
J = 7.02 Hz, CH2), 6.47 (s, 2H, thiazole, CH2), 7.08-8.12 (m, 9H, C6H5, pyridine-H), 10.72 (s, 1H,
NH); 13C-NMR δ : 13.9, 14.5 (2 CH3), 40.2 (thiazole CH2), 58.9 (ester CH2), 93.3, 101.6 (C=C), 116.0,
116.7 (2 CN), 119.2, 120.3, 121.2, 121.8, 122.0, 124.7, 125.3, 133.5, 133.9, 150.4, 152.1 (C6H5,
thiazole, pyridine C), 160.2, 164.5 (2 C=O), 168.9 (C=N); Anal. Calcd. for C23H20N6O3S (460.51): C,
59.99; H, 4.38; N, 18.25; S, 6.96%. Found: C, 60.11; H, 4.42; N, 18.13; S, 7.26%.
4-Amino-2,3-dihydro-3-phenyl-N'-(1-(pyridin-3-yl)ethylidene)-2-thioxothiazole-5-carbohydrazide (21).
To a solution of 3 (2.02 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1.0 mL) and
elemental sulfur (0.32 g, 0.01 mol), phenylisothiocyanate (15, 1.35 g, 0.01 mol) was added. The

Molecules 2011, 16
25
reaction mixture was heated under reflux for 3 h then poured onto a beaker containing an ice/water
mixture. The formed solid product was collected by filtration and dried obtaining yellow crystals (from
ethanol). Yield: 2.46 g, 67% ,m.p. 164-167 °C; IR (KBr) υ/cm−1 : 3465-3300 (NH2, NH), 3166 (CH-
aromatic), 2980 (CH3), 1680 (C=O), 1658 (C=N), 1466 (C=C), 1241(C=S); MS m/z (%) 369.9 [M+,
1
13.27%]; H-NMR δ 2.33 (s, 3H, CH3), 3.31(s, 2H, NH2),7.28-7.38 (m, C6H4-pyridine H), 10.67 (s,
1H, NH); 13C-NMR δ: 14.2 (CH3), 120.3, 122.5, 124.1, 127.9, 128.3, 130.1, 133.4, 138.9, 150.0, 152.3
(C6H5, thiazole, pyridine C), 168.2 (C=N), 170.2 (C=O), 180.3 (C=S); Anal. Calcd. for C17H15N5OS2
(369.46): C, 55.26; H, 4.09; N, 18.96; S, 17.36 %. Found: C, 55.40; H, 4.31; N, 19.15; S, 17.60%.
3.5. General Procedure for the Synthesis of 23a or 23b
To a solution of 3 (2.02 g, 0.01 mol) in 1,4-dioxane (20 mL) either 2-benzylidenemalononitrile (22a,
1.54 g, 0.01 mol) or ethyl 2-cyano-3-phenylacrylate (22b, 2.01 g, 0.01 mol) was added. The reaction
mixture was heated under reflux for 3 h then poured onto a beaker containing an ice/water mixture. The
formed solid product was collected by filtration and dried.
1-(1-Phenylethylideneamino)-6-amino-1,2-dihydro-2-hydroxy-4-phenylpyridine-3,5-dicarbonitrile (23a).
White crystals (from ethanol). Yield: 2.40 g, 68%, m.p. >300 °C; IR (KBr) υ/cm−1: 3458-3328 (NH2,
NH), 3215 (CH-aromatic), 2890 (CH3), 2192, 2225 (2 CN), 1688 (C=O), 1640 (C=N); MS m/z (%)
1
355 [M+, 4.9 %]; H-NMR δ 2.301 (s, 3H,CH3), 3.56 (s, 2H, NH2), 7.46-9.22 (m, C6H5, pyridine H);
13C-NMR δ: 18.9 (CH3), 116.9, 118.0 (2 CN), 110.2, 118.9, 120.6, 128.4, 137.2, 150.2, 152.4 (two
pyridine C), 168.9 (C=N), 172.3 (C=O); Anal. Calcd. for C20H14N6O (354.36): C, 67.79; H, 3.98; N,
23.72%. Found: C, 67.80; H, 4.00; N, 23.71%.
Ethyl-1-(1-phenylethylideneamino)-6-amino-5-cyano-1,2-dihydro-2-hydroxy-4-phenylpyridine-3-
carboxylate (23b). White crystals (from ethanol).Yield: 1.96 g, 49%, m.p. 255-259 °C; IR (KBr)
υ/cm−1: 3464-3339 (NH2), 3200 (CH-aromatic), 2890 (CH3), 2180 (CN), 1680 (C=O), 1640 (C=N);
1
MS m/z (%) 401 [M+, 2.8 %]; H-NMR δ: 1.65 (t, 3H, J = 6.83 Hz, CH3), 2.31 (s, 3H, CH3), 3.56 (s,
2H, NH2), 4.18 (q, 2H, J = 6.83 Hz, CH2), 7.27-8.79 (m, C6H5, pyridine H); 13C-NMR δ: 13.7, 19.0 (2
CH3), 59.8 (CH2), 116.9 (CN), 108.0, 117.4, 120.3, 124.6, 125.1, 127.0, 132.0, 138.3, 150.2, 152.4
(C6H5, two pyridine C), 167.3 (C=N), 170.1, 172.6 (2C=O); Anal. Calcd. for C22H19N5O3 (401.42): C,
65.83; H, 4.77; N, 17.45%. Found: C, 66.04; H, 4.83; N, 17.61%.
4. Conclusions
In this work, cyanoacetylhydrazine (1) reacted with 3-acetylpyridine (2) to afford the hydrazide-
hydrazone derivative 3. The latter was reacted with different reagents to give coumarin, pyridine,
thiazole and thiophene derivatives. The antitumor evaluations of the newly synthesized products were
carried out, showing that both the hydrazide-hydrazone derivative 3 and the benzylidene derivative 5c
have the highest inhibitory effects.

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Acknowledgements
R. M. Mohareb would like to express his deepest thanks to the Alexander Von Humboldt Foundation
in Bonn for affording him a fellowship during Summer 2009. Financial support from the American
University in Cairo is greatly acknowledged.
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