The reaction of cyanoacetylhydrazine with ω-bromo(4-methyl) acetophenone: Synthesis of heterocyclic derivatives with antitumor activity

Article abstract of DOI:10.3390/molecules15053602

New approaches for the synthesis of hydrazide-hydrazone derivatives were demonstrated as well as some heterocyclizations of such derivatives to afford 1,3,4-triazine, pyridine and 1,3,4-oxadiazine derivatives. The antitumor evaluation of the newly synthes

Full text of DOI:10.3390/molecules15053602

Molecules 2010, 15, 3602-3617; doi:10.3390/molecules15053602
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Reaction of Cyanoacetylhydrazine with ω-Bromo(4-
methyl)acetophenone: Synthesis of Heterocyclic Derivatives
with Antitumor Activity
Rafat M. Mohareb 1,2,* and Abeer A. Mohamed 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
3
National Organization for Drug Control & Research (NODCAR), P.O. 29, Cairo, Egypt;
E-Mail: abeer_aam@yahoo.com (A.A.M.)
* Author to whom correspondence should be addressed; E-Mail: raafat_mohareb@yahoo.com;
Tel: +202-37626269 or +202-35676570.
Received: 22 March 2010; in revised form: 14 April 2010 / Accepted: 21 April 2010 /
Published: 17 May 2010
Abstract: New approaches for the synthesis of hydrazide-hydrazone derivatives were
demonstrated as well as some heterocyclizations of such derivatives to afford 1,3,4-
triazine, pyridine and 1,3,4-oxadiazine derivatives. 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) were recorded. Most of
the synthesized compounds showed high inhibitory effects.
Keywords: hydrazide-hydrazone; 1,3,4-triazine; pyridine; pyridazine; antitumor
1. Introduction
Hydrazide-hydrazones are an important class of compounds that has gained much importance in
recent years due to their diverse biological activities [1–10]. The therapeutic prominence of hydrazide-
hydrazone derivatives is well established [11–12]. Hydrazide-hydrazones were also reported to elicit
anticancer [13–20] and anti-HIV properties [21] and hence they have gained an important place in

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Molecules 2010, 15
medicinal chemistry. The discovery of the antineoplastic activity of the naturally occurring Schiff’s
bases has stimulated considerable research efforts in the field of condensed systems [22]. With the aim
of constructing such condensed systems with the hydrazide-hydrazone nucleus, we turned our attention
to using such compounds as synthons for heterocyclic derivatives and their anitumor evaluation [23,24].
2. Results and Discussion
In this work we report the reaction of cyanoacetylhydrazine (1) with ω-bromo-(4-methyl-
acetophenone) (2) in 1,4-dioxane which gave the condensed product 3. The structure of compound 3
was confirmed based on analytical and spectral data. Thus, the 1H-NMR showed a singlet at δ 2.51 for
the CH3, two singlets at δ 4.31, 4.72 for the two CH2 groups, a multiplet at δ 6.50–7.76 for the C6H4
group and a singlet at δ 11.46 (D2O exchangeable) for the NH group. The reactivity of compound 3
towards different chemical reagents was studied. The reaction of 3 with either potassium cyanide or
potassium thiocyanate gave the corresponding cyanide or the thiocyanate derivatives 4a and 4b,
respectively (Scheme 1).
Scheme 1. Synthesis of the hydrazide-hydrazone 3 and 4a, b.
CH2Br
O
C
O
C
H2C
CN
NHN C
H3C
COCH2Br
H2C
CN
NHNH2
+
1
2
3
CH3
CH2X
O
C
H2C
CN
NHN C
K
X
3
+
a, X =CN
b, X =SCN
CH3
4a, X =CN
b, X = SCN
The reaction of compound 3 with either hydrazine hydrate (5a) or phenylhydrazine (5b) gave the
hydrazine derivative 6a or 6b, respectively. Analytical and spectral data of the reaction products are in
agreement with the proposed structures (see Experimental section). The reaction of either 6a or 6b
with benzaldehyde (7) gave the benzal derivative 8a or 8b, respectively (Scheme 2).
On the other hand, the reaction of either 6a or 6b with either benzenediazonium chloride (9a), 3-
cyano-4,5,6,7-tetrahydrobenzo[b]thiophene-2-diazonium chloride (9b) or ethyl 3-cyano-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate-2-diazonium chloride (9c) gave the 3-(α-hydrazo-
acetonitrilo)-1,2,4-triazine derivatives 10a–f, respectively (Scheme 3). The analytical and spectral data
of the latter reaction products are all consistent with the proposed structures.

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Molecules 2010, 15
Scheme 2. Synthesis of the hydrazine derivatives 6a, b and 8a, b.
CH2NHNHR
O
C
H2C
CN
NHN
C
3
R
NHNH2
+
5a, R =H
b, R = Ph
CH3
6a, R = H
b, R = Ph
CH2NHNHR
O
C
C
NHN C
PhHC
6a, R = H
b, R = Ph
PhCHO
+
CN
7
CH3
8a, R = H
b, R = Ph
Scheme 3. Synthesis of the 1,2,4-triazines 10a–f.
CH2NHNHR
O
NHR
N
H2C C NHN
C
H
R1 N N C
-H2O
R1
N
NCl
+
CN
N
CN
N
CH3
9a, R` = Ph
CN
COOEt
6a, R = H
b, R = Ph
CH3
10a-f
b, R` =
S
R1
R
10
a
c, R` =
S
Ph
H
CN
b
c
H
S
COOEt
H
Ph
Ph
S
d
e
Ph
CN
S
S
COOEt
f
Ph
Next, we moved towards studying the reactivity of 6a and 6b with active methylene reagents. Thus,
their reactions with either malononitrile (11a) or ethyl cyanoacetate (11b) gave the pyridine
derivatives 12a–d, respectively (Scheme 4). The structures of the latter products were established on
the basis of their analytical and spectral data. Thus, the 1H-NMR spectrum of 12c showed a singlet at
δ 2.51 for the CH3 group, a singlet at δ 3.38 for the CH2 group, two singlets at δ 4.38, 4.79 for the two
NH2 groups and a multiplet at δ 6.48–8.19 for the pyridine H-3, C6H5 and C6H4, two singlets at
δ 10.82, 11.20 for the two NH groups, respectively.

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Molecules 2010, 15
Scheme 4. Synthesis of 2-oxopyridines 12a–d.
O
CH2NHNHR
NC
CN
X
N
N
X
C
6a, R = H
b, R = Ph
H2C
+
H2N
11a, X = CN
CH3
b, X = COOEt
12a-d
X
R
H
12
a
NH2
OH
NH2
OH
H
b
c
Ph
Ph
d
On the other hand, the reaction of either 6a or 6b with either acetylacetone (13a) or ethyl
acetoacetate (13b) gave the pyridine derivatives 14a–d, respectively (Scheme 5). The structures of the
latter products were confirmed by their analytical and spectral data (see Experimental section).
Scheme 5. Synthesis of 2-oxopyridines 14a–d.
CH2NHNHR
O
O
CH2NHNHR
H2C C NHN
C
O
NC
O
N N
X
C
CN
+
X
H3C
13a, X= CH3
b, X= OEt
6a, R = H
b, R = Ph
CH3
CH3
14a-d
X
R
H
14
CH3
a
OH
H
b
c
CH3
Ph
Ph OH
d
Compound 3 underwent ready cyclization when heated in sodium ethoxide solution to give the
1,3,4-oxadiazine derivative 15, whose structure was established from its analytical and spectral data
(see Experimental section). The oxadiazine derivatives 15 seemed to be an intermediate for many
reactions between 3 and many chemical reagents. Thus, the reaction of 3 with benzaldehyde (7) gave
the 2-(α-benzalacetonitrilo)-1,3,4-oxadiazine derivative 16. The analytical and spectral data of 16 were

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Molecules 2010, 15
in agreement with the proposed structure. Thus, the 1H-NMR spectrum showed a singlet at δ 2.51 for
the CH3 group, a singlet at δ 4.22 for the CH2 group, a singlet at δ 5.16 for the (=CH) group and a
multiplet at δ 7.35–8.02 for the C6H5 and C6H4 groups. The same product 16 was obtained through the
reaction of compound 15 with benzaldehyde (7) (confirmed by m.p., mixed m.p. and fingerprint IR
spectrum). On the other hand, the reaction of 15 with benzenediazonium chloride (9) gave the 2-(α-
phenylhydrazo)-1,3,4-oxadiazine derivative 17 (Scheme 6).
Scheme 6. Synthesis of 1,3,4-oxadiazines 15, 16 and 17.
CH2Br
O
O
N
H2C
H2C C NHN C
CN
NaOEt
Heat
N
CN
CH3
15
3
CH3
O
C
PhHC
Ethanol
Piperidine
3
+ PhCHO
N
CN
N
CH3
7
16
16
Ethanol
15
PhCHO
+
Piperidine
7
O
O
N
H2C
PhHN N
C
PhN
+
NCl
N
N
CN
NC
N
CH3
CH3
9
17
15
Effect on the Growth of Human Tumor Cell Lines
The effect of compounds 4–17 was evaluated 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), after a continuous exposure of 48 h. The results are
summarized in Table 1. All the compounds were able to inhibit the growth of the tested human tumor
cell lines in a dose-dependent manner (data not shown). The results showed that compound 14b, with
its 2-hydroxypyridine group, showed the highest inhibitory effect against all the three tumor cell lines.
In addition compounds 10a, 10f, 12a, 12b and 14c showed high inhibitory effects. On the other hand,
compounds 4b, 6a, 8b, 10b, 10d, 12c, 12d, 14a, 14c, 16 and 17 showed the lowest inhibitory effect

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Molecules 2010, 15
towards adenocarcinoma (MCF-7). The rest of compounds showed the moderate growth inhibitory
effects. Comparing compound 4a with 4b, it is obvious that the presence of the α-SCN present in 4b
showed lower inhibitory effect than 4a with it’s α-CN group. Comparing 1,2,4-triazine derivatives 10a
(with the 4-phenylamino group) and 10b (with the 4-thiophenoamino group), the first has a greater
inhibitory effect than the second towards the three cell lines.
Table 1. Effect of the newly synthesized products on the growth of three human tumor cell lines.
GI50 (μΜ)
Compound
MCF-7
NCI-H460
12.9 ± 1.6
38.1 ± 10.8
20.2 ± 12.6
22.4 ± 8.6
77.9 ± 5.0
70.9 ± 6.1
11.7 ± 8.8
55.6 ± 5.8
8.4 ± 1.8
SF-268
15 ± 1.1
4a
4b
6a
6b
8a
8b
10a
10b
10c
10d
10e
10f
12a
12b
12c
12d
14a
14b
14c
16
10.0 ± 0.6
70.6 ± 15.3
50.8 ± 18.5
25.8 ± 12.5
24.6*
48.0 ± 9.1
50.0 ± 8.7
20.3 ± 8.6
35.0*
50.2 ± 2.2
14.8 ± 1.6
42.6 ± 8.6
10.4 ± 8.4
40.9 ± 2.6
8.3 ± 9.2
14 ± 1.1
80.9 ± 10.2
8.0 ± 0.6
70.6 ± 8.6
24.2 ± 0.6
88.4 ± 4.3
12.4 ± 2.6
10.0 ± 0.4
12.4 ± 1.6
8.6 ± 2.4
86.8 ± 6.0
90.8 ± 2.4
66.4 ± 6.0
4.2 ± 2.8
66.8 ± 12.0
6.8 ± 1.2
14.9 ± 1.6
10.8 ± 4.0
10.4 ± 6.2
48.5 ± 4.0
78.2 ± 2.2
42.4 ± 6.0
10.2 ± 6.8
64.8 ± 6.4
32.6 ± 4.0
77.8 ± 3.1
16 ± 6.0
12.0 ± 4.2
38.4 ± 2.6
86.2 ± 1.8
62.3 ± 6.0
10.8 ± 5.0
50.8 ± 6.4
26.8 ± 8.4
47.0 ± 6.4
80.3 ± 4.0
40.5 ± 2.6
60.4 ± 6.0
17
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. *Results from two-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-268 = 94.0 ± 7.0 nM.
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
13
spectrophotometer. H-NMR & C-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.

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Molecules 2010, 15
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 & the National Cancer Institute at Cairo University.
4-Methyl-ω-bromoacetophenone cyanoacetylhydrazone (3). To a solution of cyanoacetylhydrazine (1,
2.44 g, 0.02 mol) in 1,4-dioxane (20 mL), ω-bromo-(4-methylacetophenone) (5.24 g, 0.02 mol) was
added. The reaction mixture was stirred at room temperature for 1 hr then poured onto a beaker
containing an ice/water mixture. The solid product formed was collected by filtration and dried
obtaining pale yellow crystals (from ethanol).Yield: 5.02 g (71%), m.p. 148 °C; IR (KBr) υ/cm−1:
3400–3378 (NH), 3105 (CH aromatic), 2956 (CH3), 2259 (CN), 1681 (C=O), 1610 (C=C); 1H-NMR δ:
2.51 (s, 3H, CH3), 4.31, 4.72 (2s, 4H, CH2), 6.50–7.76 (m, 4H, C6H4), 11.46 (s, 1H, NH). MS: (m/z)
293 (M.+) (6%), 200 (28%), 117 (100%), 68 (69.9%); 13C-NMR: 22.6 (CH3), 27.0, 58.7 (2CH2), 118.5
(CN), 126.8, 128.4, 129.5, 138.8 (C6H5), 156.3 (C=N), 173.8 (C=O); Anal. Calcd. for C12H12N3OBr
(294.24): C, 48.00; H, 4.11; N, 14.29%. Found: C, 49.27; H, 4.45; N, 14.35%.
3.2. General Procedure for the Synthesis of 4a or 4b
To a solution of 3 (0.54 g, 1.83 × 10−3 mol) in ethanol (25 mL) in a water bath at 60 °C, either
potassium cyanide (0.11 g, 1.83 × 10−3 mol) or potassium thiocyanate (0.17 g, 1.83 × 10−3 mol) was
added with continuous stirring. The reaction mixture was left in the water bath for 30 min at 60 °C
then poured onto a beaker containing ice/water mixture and few drops of hydrochloric acid. The
formed solid product was collected by filtration and dried.
4-Methyl-ω-cyanoacetophenonecyanoacetylhydrazone (4a). Pale brown crystals (from ethanol). Yield:
0.20 g (62%), m.p. 160 °C; IR (KBr) υ/cm−1: 3450–3205 (NH), 3031 (CH aromatic), 2260, 2209
1
(2CN), 1680 (C=O); H NMR δ: 2.39 (s, 3H, CH3), 4.46, 5.06 (2s, 4H, 2CH2), 7.18–7.84 (m, 4H,
C6H4), 10.82 (s, 1H, NH); 13C NMR: 23.8 (CH3), 27.3, 29.5 (2CH2), 116.9, 117.0 (2CN), 127.3, 128.0,
129.8, 138.9 (C6H4), 155.8 (C=N), 173.8 (C=O); Anal. Calcd. for C13H12N4O (240.26): C, 64.99;
H, 5.03; N, 23.32%. Found: C, 64.79; H, 5.11; N, 22.99%.
4-Methyl-ω-thiocyanoacetophenoecyanoacetylhydrazone (4b). Buff crystals (from ethanol). Yield:
0.33 g (67%), m.p. 130 °C; IR (KBr) υ/cm−1: 3445–3225 (NH), 3099 (CH aromatic), 3034 (CH3),
2969, 2925 (2CH2), 2264, 2225 (2CN), 1666 (C=O), 1606 (C=C); 1H-NMR δ: 2.49 (s, 3H, CH3), 4.50,
13
5.08 (2s, 4H, 2CH2), 7.06–7.93 (m, 4H, C6H4), 11.52 (s, 1H, NH); C-NMR: 23.8 (CH3), 27.3, 29.4
(2CH2), 116.7, 117.8 (2CN), 127.0, 128.3, 129.6, 138.6 (C6H4), 155.5 C=N), 173.9 (C=O); Anal. Calcd
for C13H12N4OS (272.32): C, 57.33; H, 4.44; N, 20.57; S, 11.77%. Found: C, 57.38; H, 4.64; N, 20.68;
S, 11.57%.
3.3. General Procedure for the Synthesis of 6a and 6b
To a solution of compound 3 (1.50 g, 5.09 × 10−3 mol) in ethanol (35 mL) either hydrazine hydrate
(0.25 g, 5.09 × 10−3 mol) or phenylhydrazine (0.55 g, 5.09 × 10−3 mol) was added. The reaction

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Molecules 2010, 15
mixture was heated under reflux for 3 hrs then poured onto a beaker containing an ice/water mixture
and a few drops of hydrochloric acid. The formed solid product was collected by filtration and dried.
4-Methyl-ω-hydrazinoacetophenonecyanoacetylhydrazone (6a). Greenish brown crystals (from
ethanol). Yield: 0.65 g (52%), m.p. 120 °C; IR (KBr) υ/cm−1: 3400–3204 (NH2, 2NH), 3027 (CH
1
aromatic), 2918 (CH3), 2203 (CN), 1688 (C=O), 1607 (C=C); H-NMR δ: 2.51 (s, 3H, CH3), 3.39,
4.23 (2s, 4H, 2CH2), 4.82 (s, 2H, NH2), 6.87–8.40 (m, 4H, C6H4), 9.26, 11.41 (2s, 2H, 2NH);
13C-NMR: 23.6 (CH3), 27.0, 29.6 (2CH2), 127.8, 128.3, 129.5, 138.6 (C6H4), 155.6, 164.2 (2C=N),
173.8 (C=O); Anal. Calcd. for C12H15N5O (245.28): C, 58.76; H, 6.16; N, 28.55%. Found: C, 58.55; H,
5.96; N, 28.32%.
4-Methyl-ω-phenylhydrazinoacetophenonecyanoacetylhydrazone (6b). Brown crystals (from ethanol).
Yield: 0.95 g (63%), m.p. 72 °C; IR (KBr) υ/cm−1: 3500–3174 (3NH), 3026 (CH aromatic), 2969
(CH3), 2205 (CN), 1687 (C=O), 1600 (C=C); 1H-NMR δ: 2.51 (s, 3H, CH3), 3.39, 4.35 (2s, 4H, 2CH2),
13
6.79–8.40 (m, 9H, C6H5, C6H4), 10.82, 11.19, 12.63 (3s, 3H, 3NH); C-NMR: 23.6 (CH3), 27.0, 29.9
(2CH2), 120.3, 122.5, 127.3, 128.4, 129.3, 138.5, 140.1 (C6H4, C6H5), 155.6, 164.2 (2C=N), 173.8
(C=O); Anal. Calcd. for C18H19N5O (321.38): C, 67.27; H, 5.96; N, 21.79%. Found: C, 67.37; H, 5.84;
N, 22.61%.
α-Benzal-4-methyl-ω-hydrazinoacetophenonecyanoacetylhydrazone (8a): To a solution of compound
6a (0.29 g, 1.18 × 10−3 mol) in ethanol (25 mL) containing piperidine (0.5 mL), benzaldehyde (0.11 g,
1.48 × 10−3 mol) was added. The reaction mixture was heated under reflux for 3hrs then poured onto a
beaker containing an ice/water mixture and few drops of hydrochloric acid. The formed solid product
was collected by filtration and dried obtaining deep yellowish brown crystals (from ethanol).Yield:
0.26 g (66%), m.p. 90 °C; IR (KBr) υ/cm−1: 3500–3194 (NH2, 2NH), 3027 (CH aromatic), 2919 (CH3),
1
2218 (CN), 1680 (C=O), 1608 (C=C); H-NMR δ: 2.51 (s, 3H, CH3), 3.36 (s, 2H, CH2), 4.77 (s, 2H,
13
NH2), 5.99 (s, 1H, =CH), 7.27–8.40 (m, 9H, C6H5, C6H4), 8.81, 9.15 (2s, 2H, 2NH); C-NMR: 23.2
(CH3), 27.4, 29.2 (2CH2), 122.3, 122.8, 122.5, 126.0 128.3, 129.9, 138.3 (C6H5, C6H4) 155.0, 164.3
(2C=N), 174.3 (C=O); Anal. Calcd. for C19H19N5O (333.39): C, 68.45; H, 5.74; N, 21.01%. Found: C,
68.55; H, 5.54; N, 21.31%.
α-Benzal-4-methyl-ω-phenylhydrazinoacetophenonecyanoacetyl-hydrazone (8b). To a solution of
compound 6b (0.36 g, 1.12 × 10−3 mol) in ethanol (25 mL) containing piperidine (0.5 mL),
benzaldehyde (0.11 g, 1.12 × 10−3 mol) was added. The reaction mixture was heated under reflux for
3hrs then poured onto a beaker containing an ice/water mixture and a few drops of hydrochloric acid.
The formed solid product was collected by filtration and dried obtaining yellowish brown crystals
(from ethanol). Yield: 0.33 g (73%), m.p. 102 °C; IR (KBr) υ/cm−1: 3300-3179 (3NH), 3026 (CH
1
aromatic), 2920 (CH3), 2210 (CN), 1688 (C=O), 1600 (C=C); H NMR δ: 2.88 (s, 3H, CH3), 3.37 (s,
2H, CH2), 6.90–8.40 (m, 15H, =CH, 2C6H5, C6H4), 9.20, 10.83, 12.62 (3s, 3H, 3NH); 13C-NMR: 23.2
(CH3), 27.4, 29.2 (2CH2), 120.9, 121.1, 122.3, 122.8, 123.4, 123.9, 122.5, 126.0 128.3, 129.5, 138.6
(2C6H5, C6H4) 155.3, 164.0 (2C=N), 174.6 (C=O); Anal. Calcd. for C25H23N5O (409.48): C, 73.33; H,
5.66; N, 17.10%. Found: C, 73.52; H, 5.97; N, 17.33%.

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Molecules 2010, 15
1-Amino-6-(4-methylphenyl)-3-(α-phenylhydrazoacetonitrilo)-1,2,4-triazine (10a). To a cold solution
(0–5 °C) of compound 6a (0.40 g, 1.63 × 10−3 mol) in ethanol (50 mL) containing sodium hydroxide
(10 mL, 10%) and a solution of benzenediazonium chloride (1.63 × 10−3 mol) [which was prepared by
dissolving sodium nitrite (0.16 g, 2.44 × 10−3 mol) in water, 2 mL was added to a cold solution of
aniline (0.15 g, 1.63 × 10−3 mol) containing the appropriate amount of hydrochloric acid and with
continuous stirring] was added with continuous stirring. The formed solid product was collected by
filtration and dried to give reddish brown crystals (from ethanol and few drops of dimethylformamide).
Yield: 0.58 g (63%), m.p. 170 °C; IR (KBr) υ/cm−1: 3400–3307 (NH2, NH), 3027 (CH aromatic),
1
2919, 2862 (CH3, CH2), 2211 (CN), 1600 (C=C); H-NMR δ: 2.51 (s, 3H, CH3), 3.37 (s, 2H, CH2),
13
4.22 (s, 2H, NH2), 6.50–8.20 (m, 9H, C6H5, C6H4), 11.64 (s, 1H, NH); C-NMR: 23.8 (CH3), 51.9
(triazine CH2), 115.8 (CN), 118.3, 118.9, 119.2, 121.6, 124.8, 133.0, 139.6 (C6H5, C6H4), 156.9, 163.8
164.1 (3 C=N); Anal. Calcd. for C18H17N7 (331.38): C, 65.24; H, 5.17; N, 29.58%. Found: C, 65.42; H,
5.27; N, 29.36%.
3.4. General Procedure for the Synthesis of 10b and 10c
To a cold solution (0–5 °C) of compound 6a (0.49 g, 1.99 × 10−3 mol) in ethanol (50 mL)
containing sodium hydroxide solution (10 mL, 10%) was added with continuous stirring a solution of
either 3-cyano-4,5,6,7-terahydrobenzo[b]thiophene-2-diazonium chloride (9b) (1.99 × 10−3 mol) or
ethyl 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-2-diazonium chloride (9c) (1.99 × 10−3 mol)
[which was prepared by dissolving sodium nitrite (0.20 g, 2.99 × 10−3 mol) in water, 2 mL was added
to a cold solution of either the 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene (0.35 g,
1.99 × 10−3 mol) or ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (0.45 g,
1.99 × 10−3 mol) dissolved in acetic acid (50 mL) containing the appropriate amount of hydrochloric
acid and with continuous stirring]. The solid product formed was collected by filtration and dried.
1-Amino-6-(4-methylphenyl)-3-(α-(3-cyano-2-hydrazo-4,5,6,7-tetra-hydrobenzo[b]thiophene)aceto-
nitrilo)-1,2,4-triazine (10b). Deep brown crystals (from ethanol). Yield: 0.67 g (75%), m.p. 210–214 °C;
IR (KBr) υ/cm−1: 3600–3425 (NH2, NH), 3030 (CH aromatic), 2930 (CH3), 2250, 2217 (2CN), 1606
(C=C); 1H-NMR δ: 1.63–2.34 (m, 8H, cyclohexene 4CH2), 2.51 (s, 3H, CH3), 3.60 (s, 2H, CH2), 4.78
(s, 2H, NH2), 6.97–8.32 (m, 4H, C6H4), 9.84 (s, 1H, NH); 13C-NMR: 19.4, 23.0, 25.1, 27.8
(cyclohexene 4CH2), 24.8 (CH3), 51.9 (triazine CH2), 115.8, 116.4 (2CN), 118.3, 118.9, 119.2, 121.6,
124.6, 133.2, 136.5, 136.9 139.6 (thiophene C, C6H5, C6H4), 156.0, 163.5 164.3 (3 C=N); Anal. Calcd.
for C21H20N8S (416.50): C, 60.56; H, 4.84; N, 26.90; S, 7.70%. Found: C, 60.28; H, 5.03; N, 26.61;
S, 7.88%.
Ethyl 1-amino-6-(4-methylphenyl)-3-(α(2-hydrazo-4,5,6,7-tetra-hydrobenzo[b]thiophene-3-carboxyl-
ate)acetonitrilo)-1,2,4-triazine (10c). Brown crystals (from ethanol) Yield: 0.53 g (57%), m.p. 90 °C;
IR (KBr) υ/cm−1: 3400–3287 (NH2, NH), 2976, 2934, 2860 (2CH3, CH2), 2213 (CN), 1711 (C=O);
1H- NMR δ: 1.07–1.92 (m, 8H, cyclohexene 4CH2), 2.51 (s, 3H, CH3), 2.77 (t, 3H, J = 7.04 Hz, CH3),
3.36 (s, 2H, CH2), 4.24 (q, 2H, J = 7.04 Hz, CH2), 4.99 (s, 2H, NH2), 6.50–8.08 (m, 4H, C6H4), 9.88
13
(s, 1H, NH); C-NMR: 14.5, 24.6 (2 CH3), 23.2, 23.6, 25.1, 27.6 (cyclohexene 4CH2), 55.4 (triazine

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Molecules 2010, 15
CH2), 60.8 (ester CH2), 128.7, 129.0, 129.7, 132.1, 136.5, 138.9, 140.8 (C6H5, thiophene C), 115.9,
116.7 (2CN), 155.2, 158.0, 163.1 (3 C=N), 165.6 (CO); Anal. Calcd. for C23H25N7O2S (463.56):
C, 59.59; H, 5.44; N, 21.15; S, 6.93%. Found: C, 59.83; H, 5.46; N, 20.91; S, 7.05%.
1-Phenylamino-6-(4-methylphenyl)-3-(α-phenylhydrazoacetonitrilo)-1,2,4-triazine (10d). To a cold
solution (0–5 °C) of compound 6b (0.53 g, 1.64 × 10−3 mol) in ethanol (50 mL) containing sodium
hydroxide (10 mL, 10%) was added with continuous stirring a solution of benzenediazonium chloride
(1.64 × 10−3 mol) [which was prepared by dissolving sodium nitrite (0.17 g, 2.47 × 10−3 mol) in water,
2 mL was added to a cold solution of aniline (0.15 g, 1.64 × 10−3 mol) containing the appropriate
amount of hydrochloric acid and with continuous stirring]. The formed solid product was collected by
filtration to give reddish brown crystals (from ethanol and few drops of dimethylformamide). Yield:
0.58 g (86%), m.p. 120 °C; IR (KBr) υ/cm−1: 3500–3422 (2NH), 3057 (CH aromatic), 3028,
2921(CH3, CH2), 2211 (CN), 1600 (C=C); 1H-NMR δ: 2.50 (s, 3H, CH3), 3.31 (s, 2H, CH2), 6.60–7.65
13
(m, 14H, 2C6H5, C6H4), 8.16, 9.05 (s, 2H, 2NH); C-NMR: 23.6 (CH3), 51.9 (triazine CH2), 115.6
(CN), 118.6, 118.7, 119.2, 120.1, 121.6, 124.8, 133.6, 134.8, 139.9 (2C6H5, C6H4), 156.6, 163.7, 164.0
(3 C=N); Anal. Calcd. for C24H21N7 (407.47): C, 70.74; H, 5.19; N, 24.06%. Found: C, 71.05; H, 5.38;
N, 23.87%.
3.5. General Procedure for the Synthesis of 10e and 10f
To a cold solution (0–5 °C) of compound 6b (0.40 g, 1.24 × 10−3 mol) in ethanol (50 mL)
containing sodium hydroxide solution (10 mL, 10%) was added with continuous stirring a solution of
either 3-cyano-4,5,6,7-terahydrobenzo[b]thiophene-2-diazonium chloride (9b) (1.24 × 10−3 mol) or
ethyl 4,5,6,7-tetrahydrobenzo[b]thiophen-3-carboxylate-2-diazonium chloride (9c) (1.24 × 10−3 mol)
[which was prepared by dissolving sodium nitrite (0.12 g, 1.86 × 10−3 mol) in water, 2 mL was added
to a cold solution of either the 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene (0.22 g,
1.99 × 10−3 mol) or ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (0.28 g,
1.99 × 10−3 mol) dissolved in acetic acid (50 mL) containing the appropriate amount of hydrochloric
acid and with continuous stirring]. The solid product formed was collected by filtration and dried.
1-Phenylamino-6-(4-methylphenyl)-3-(α-(3-cyano-2-hydrazo-4,5,6,7-tetrahydrobenzo[b]thiophene)-
acetonitrilo)-1,2,4-triazine (10e). Reddish brown crystals (from ethanol), yield: 0.34 g (55%), m.p. 190 °C;
IR (KBr) υ/cm−1: 3500–3425 (2NH), 3030 (CH aromatic), 2930 (CH3), 2250, 2217 (2CN), 1606
1
(C=C); H-NMR δ: 1.63–2.34 (m, 8H, cyclohexene), 2.51 (s, 3H, CH3), 3.60 (s, 2H, CH2), 6.97–8.32
(m, 9H, C6H5, C6H4), 9.84, 10.00 (s, 2H, 2NH); 13C-NMR: 19.1, 23.3, 25.4, 27.4 (cyclohexene 4CH2),
24.9 (CH3), 51.7 (triazine CH2), 115.6, 116.3 (2CN), 118.3, 118.9, 119.2, 121.6, 124.6, 133.2, 136.5,
136.9 139.4 (thiophene C, C6H5, C6H4), 156.1, 163. 3, 164.6 (3 C=N). Anal. Calcd. for C27H24N8S
(492.60): C, 65.83; H, 4.91; N, 22.75; S, 6.51%. Found: C, 65.96; H, 5.24; N, 22.55; S, 6.79%.
Ethyl
1-phenylamino-6-(4-methylphenyl)-3-(α(2-hydrazo-4,5,6,7-tetrahydrobenzo-[b]thiophen-3-
carboxylate)acetonitrilo)-1,2,4-triazine (10f). Pale reddish brown crystals (from ethanol).
Yield: 0.40 g (59%), m.p. 150–160 °C; IR (KBr) υ/cm−1: 3400–3287 (2NH), 2976, 2934, 2860 (2CH3,

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Molecules 2010, 15
1
CH2), 2213 (CN), 1711 (C=O); H-NMR δ: 1.07–1.92 (m, 8H, cyclohexene), 2.51 (s, 3H, CH3), 2.77
(t, 3H, J = 6.89 Hz, CH3), 3.36 (s, 2H, CH2), 4.24 (q, 2H, J = 6.89 Hz, CH2), 6.50–8.08 (m, 9H, C6H5,
13
C6H4), 9.88, 10.30 (2s, 2H, 2NH); C-NMR: 14.3, 24.8 (2 CH3), 23.0, 23.3, 25.1, 27.8 (cyclohexene
4CH2), 55.54 (triazine CH2), 60.8 (ester CH2), 128.7, 129.2, 129.7, 132.0, 136.5, 138.9, 140.8 (C6H5,
thiophene C), 115.8, 116.5 (2CN), 155.2, 158.0, 163.3 (3 C=N), 165.6 (CO); Anal. Calcd. for
C29H29N7O2S (539.65): C, 64.54; H, 5.42; N, 18.17; S, 5.94%. Found: C, 64.68; H, 5.23; N, 17.97;
S, 6.20%.
3.6. General Procedure for the Synthesis of 12a and 12b
To a solution of compound 6a (0.47 g, 1.91 × 10−3 mol) in ethanol (20 mL) containing triethylamine
(0.5 mL), either malononitrile (0.12 g, 1.91 × 10−3 mol) or ethyl cyanoacetate (0.21 g, 1.91 × 10−3 mol)
was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker
containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was
collected by filtration and dried.
3-Cyano-4,6-diamino-2-oxo-1-imino-(4-methyl-ω-hydrazinoaceto-phenonylidieno)pyridine
(12a).
Brown crystals (from ethanol). Yield: 0.38 g (64%), m.p. 158 °C; IR (KBr) υ/cm−1: 3500–3227 (3NH2,
NH), 3028 (CH aromatic), 2974 (CH3), 2919 (CH2), 2201 (CN), 1685 (C=O), 1600 (C=C); 1H-NMR δ:
2.51 (s, 3H, CH3), 3.38 (s, 2H, CH2), 4.38, 4.79, 5.30 (3s, 6H, 3NH2), 7.05–8.19 (m, 5H, pyridine H-3,
13
C6H4), 10.82 (s, 1H, NH); C-NMR: 23.8 (CH3), 51.7 (CH2), 80.9, 89.5, 114.8, 125.6 (pyridine C),
117.9 (CN), 126.2, 128.0, 129.3, 129.6 (C6H5), 163.5 (C=O), 170.9 (C=N); Anal. Calcd. for
C15H17N7O (311.34): C, 57.87; H, 5.50; N, 31.49%. Found: C, 57.96; H, 5.48; N, 31.68%.
4-Amino-3-cyano-6-hydroxy-2-oxo-1-imino(4-methyl-ω-hydrazino-acetophenonylidieno)pyridine
(12b). Pale brown crystals (from ethanol).Yield: 0.66 g (111%), m.p. 140 °C; IR (KBr) υ/cm−1:
3600–3227 (OH, 2NH2, NH), 3028 (CH aromatic), 2974 (CH3), 2919 (CH2), 2201 (CN), 1685 (C=O),
1
1600 (C=C); H-NMR δ: 2.50 (s, 3H, CH3), 3.39 (s, 2H, CH2), 4.34, 4.77, (2s, 4H, 2NH2), 7.05–8.19
(m, 5H, pyridine H-3, C6H4), 10.84 (s, 1H, NH), 12.64 (s, 1H, OH); 13C-NMR: 23.6 (CH3), 51.4 (CH2),
82.9, 89.5, 116.8, 125.9 (pyridine C), 117.9 (CN), 126.4, 128.0, 129.3, 129.6 (C6H5), 170.9, 176.5
(2C=N), 164.3 (2C=O); Anal. Calcd. for C15H16N6O2 (312.33): C, 57.68; H, 5.16; N, 26.90%. Found:
C, 57.42; H, 5.09; N, 26.76%.
3.7. General Procedure for the Synthesis of 12c and 12d
To a solution of compound 6b (0.60 g, 1.86 × 10−3 mol) in ethanol (20 mL) containing
triethylamine (0.5 mL), either malononitrile (0.12 g, 1.86 × 10−3 mol) or ethyl cyanoacetate (0.21 g,
1.86 × 10−3 mol) was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a
beaker containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed
was collected by filtration and dried.

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Molecules 2010, 15
3-Cyano-4,6-diamino-2-oxo-1-imino-(4-methyl-ω-phenylhydrazino-acetophenonylidieno)pyridine (12c)
Green crystals (from ethanol). Yield: 0.51 g (70%), m.p. 80 °C; IR (KBr) υ/cm−1: 3480–3227 (2NH2,
2NH), 3028 (CH aromatic), 2974 (CH3), 2919 (CH2), 2201 (CN), 1685 (C=O), 1600 (C=C); 1H-NMR
δ: 2.51 (s, 3H, CH3), 3.38 (s, 2H, CH2), 4.38, 4.79, (2s, 4H, 2NH2), 6.48–8.19 (m, 10H, pyridine H-3,
C6H5, C6H4), 10.82, 11.20 (2s, 2H, 2NH); 13C-NMR: 23.7 (CH3), 51.9 (CH2), 80.7, 89.3, 114.9, 125.3
(pyridine C), 118.3 (CN), 126.4, 128.0, 129.3, 129.6 (C6H5), 170.9 (C=N), 162.5 (C=O); Anal. Calcd.
for C21H21N7O (387.44): C, 65.10; H, 5.46; N, 25.30%. Found: C, 64.21; H, 5.36; N, 25.38%.
4-Amino-3-cyano-6-hydroxy-2-oxo-1-imino(4-methyl-ω-phenyl-hydrazinoacetophenonylidieno) pyridine
(12d). Yellowish green crystals (from ethanol). Yield: 0.58 g (80%). Mp 84–100 °C; IR (KBr) υ/cm−1:
3600–3185 (OH, NH2, 2NH), 3027 (CH aromatic), 2975 (CH3), 2919 (CH2), 2206 (CN), 1684 (C=O),
1600 (C=C); 1H-NMR δ: 2.50 (s, 3H, CH3), 3.39 (s, 2H, CH2), 4.34 (s, 2H, NH2), 6.21 (s, 1H, pyridine
H-3), 7.33–8.70 (m, 9H, C6H5, C6H4), 10.84, 11.22 (2s, 2H, 2NH), 12.64 (s, 1H, OH); 13C-NMR: 23.6
(CH3), 51.6 (CH2), 80.2, 89.5, 114.5, 125.0 (pyridine C), 118.1 (CN), 126.2, 128.2, 129.5, 129.6
(C6H5), 164.8 (C=O), 170.9 (C=N); Anal. Calcd. for C21H20N6O2 (388.43): C, 64.93; H, 5.19; N,
21.63%. Found: C, 65.42; H, 5.69; N, 21.13%.
3.8. General Procedure for the Synthesis of 14a and 14b
To a solution of compound 6a (0.52 g, 2.12 × 10−3 mol) in ethanol (20 mL) containing piperidine
(0.5 mL), either acetylacetone (0.21 g, 2.21 × 10−3 mol) or ethyl acetoacetate (0.27 g, 2.21 × 10−3 mol)
was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker
containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was
collected by filtration and dried.
3-Cyano-4,6-diamethyl-2-oxo-1-imino-(4-methyl-ω-hydrazinoaceto-phenonylidieno)pyridine
(14a)
Brown crystals (from ethanol). Yield: 0.37 g (56%), m.p. 144 °C; IR (KBr) υ/cm−1: 3433–3229 (NH2,
NH,), 3027 (CH aromatic), 2221 (CN), 1685 (C=O), 1600 (C=C); 1H-NMR δ: 2.34, 2.54, 3.04 (3s, 9H,
3CH3), 3.39 (s, 2H, CH2), 6.21 (s, 1H, pyridine H-3), 6.72–7.45 (m, 4H, C6H4), 10.84, 11.63 (s, 2H,
13
2NH); C-NMR: 16.0, 19.2, 24.3 (3CH3), 51.2 (CH2), 80.0, 88.3, 115.3, 123.9 (pyridine C), 116.8
(CN), 126.0, 127.9, 128.3, 129.1 (C6H5), 164.9 (C=O), 173.6 (C=N); Anal. Calcd. for C17H19N5O
(309.37): C, 66.00; H, 6.19; N, 22.63%; found: C, 66.29; H, 6.23; N, 23.53%.
3-Cyano-6-hydroxy-4-methyl-2-oxo-1-imino(4-methyl-ω-hydrazino-acetophenonylidieno)pyridine
(14b). Brown crystals (from ethanol). Yield: 0.38 g (57%), m.p. 136 °C; IR (KBr) υ/cm−1: 3549–3321
1
(OH, NH2, NH), 3027 (CH aromatic), 2213 (CN), 1680 (C=O), 1600 (C=C); H-NMR δ: 2.53, 3.13
(3s, 9H, 3CH3), 3.39 (s, 2H, CH2), 6.22 (s, 1H, pyridine H-3), 6.73–7.39 (m, 4H, C6H4), 10.85, 11.33
13
(s, 2H, 2NH), 12.21 (s, 1H, OH); C-NMR: 16.1, 19.0, 24.3 (3CH3), 51.3 (CH2), 80.2, 88.3, 115.3,
152.1 (pyridine C), 116.6 (CN), 126.3, 127.6, 128.3, 129.1 (C6H5), 165.3 (C=O), 173.8 (C=N); Anal.
Calcd. for C16H17N5O2 (311.34): C, 61.44; H, 5.50; N, 22.49%; found: C, 61.23; H, 5.67; N, 22.30%.

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Molecules 2010, 15
3.9. General Procedure for the Synthesis of 14c and 14d
To a solution of compound 6b (0.60 g, 1.86 × 10−3 mol) in ethanol (20 mL) containing piperidine
(0.5 mL), either acetylacetone (0.18 g, 1.86 × 10−3 mol) or ethyl acetoacetate (0.24 g, 1.86 × 10−3 mol)
was added. The reaction mixture was heated under reflux for 3 hrs then poured onto a beaker
containing an ice/water mixture and a few drops of hydrochloric acid. The solid product formed was
collected by filtration and dried.
3-Cyano-4,6-diamethyl-2-oxo-1-imino-(4-methyl-ω-phenylhydrazino-acetophenonylidieno)pyridine
(14c) Yellowish brown crystals (from ethanol). Yield: 0.61 g (85%), m.p. 100 °C; IR (KBr) υ/cm−1:
3450–3229 (2NH), 3027 (CH aromatic), 2213 (CN), 1680 (C=O), 1600 (C=C); 1H-NMR δ: 2.34, 2.51,
3.01 (3s, 9H, 3CH3), 3.37 (s, 2H, CH2), 6.21 (s, 1H, pyridine H-3), 6.50–8.41 (m, 9H, C6H5, C6H4),
13
10.82, 12.63 (s, 2H, 2NH); C-NMR: 16.3, 19.1, 24.3 (3CH3), 51.5 (CH2), 80.3, 88.6, 115.0, 127.7,
155.8 (pyridine C), 116.5 (CN), 118.7, 119.0, 120.8, 122.5, 126.0, 127.8, 128.7, 129.8 (C6H5, C6H4), ,
166.2 (C=O), 173.6 (C=N); Anal. Calcd. for C23H23N5O (385.47): C, 71.66; H, 6.01; N, 18.16%;
found: C, 71.86; H, 5.98; N, 17.99%.
3-Cyano-6-hydroxy-4-methyl-2-oxo-1-imino(4-methyl-ω-phenyl-hydrazinoacetophenonylidieno)-
pyridine (14d). Yellowish brown crystals (from ethanol). Yield: 0.48 g (66%). m.p. 96 °C;
IR (KBr) υ/cm−1: 3600–3220 (OH, 2NH), 3027 (CH aromatic), 2211 (CN), 1682 (C=O), 1600 (C=C);
1H-NMR δ: 2.51, 3.00 (2s, 6H, 2CH3), 3.40 (s, 2H, CH2), 6.54 (s, 1H, pyridine H-3), 7.34–8.40 (m,
13
9H, C6H5, C6H4), 9.62, 10.82 (2s, 2H, 2NH), 12.65 (s, 1H, OH); C-NMR: 16.3, 19.4, 24.6 (3CH3),
51.5 (CH2), 80.8, 88.4, 116.5, 155.4 (pyridine C), 116.5 (CN), 118.8, 119.0, 120.3, 121.7, 126.0,
127.8, 128.9, 129.0 (C6H5, C6H4), , 166.4 (C=O), 173.9 (C=N); Anal. Calcd. for C22H21N5O2 (387.44):
C, 68.20; H, 5.46; N, 18.07%; Found: C, 68.49; H, 5.84; N, 17.79%.
2-Acetonitrilo-5-(4-methylphenyl)-1,3,4-oxadiazine (15). A solution of compound 3 (1.00 g,
3.39 × 10−3 mol) in sodium ethoxide (50 mL) was heated under reflux for 3 hrs then poured onto a
beaker containing an ice/water mixture and a few drops of hydrochloric acid. The formed solid product
was collected by filtration and dried to give pale brown crystals (from ethanol). Yield 0.39 g (54%),
m.p. 190–193 °C; IR (KBr) υ/cm−1: 3028 (CH aromatic), 2998 (CH3), 2922, 2854 (2CH2), 2210 (CN),
1673 (C=N), 1609 (C=C); 1H-NMR δ: 2.51 (s, 3H, CH3), 3.35, 4.42 (2s, 4H, 2CH2), 7.00–8.40 (m, 4H,
13
C6H4); C-NMR: 24.4 (CH3), 20.3 (CH2), 64.3 (oxadiazine CH2), 116.9 (CN), 126.0, 127.8, 128.9,
129.0, 133.1 (C6H5), 170.5 (C=N); Anal. Calcd. for C12H11N3O (213.24): C, 67.59; H, 5.19; N,
19.70%; found: C, 67.70; H, 5.38; N, 19.88%.
2-(α-Benzalacetonitrilo)-5-(4-methylphenyl)-1,3,4-oxadiazine hydrochloride (16). To a solution of
compound 3 (2.00 g, 6.79 × 10−3 mol) in ethanol (30 mL) containing piperidine (0.5 mL),
benzaldehyde (0.72 g, 6.79 × 10−3 mol) was added. The reaction mixture was heated under reflux for
3 hrs then poured onto a beaker containing an ice/water mixture with a few drops of hydrochloric acid.
The formed solid product was collected by filtration and dried to afford brown crystals (from ethanol).
Yield: 1.35 g (58%), m.p. 100 °C; IR (KBr) υ/cm−1: 3050 (CH aromatic), 3029 (CH3), 2921 (CH2),

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1
2830 (CH), 2216 (CN), 1604 (C=C); H-NMR δ: 2.51 (s, 3H, CH3), 4.22 (s, 2H, CH2), 5.16 (s, 1H,
13
=CH), 7.35–8.02 (m, 9H, C6H5, C6H4); C-NMR: 24.6 (CH3), 63.8 (oxadiazine CH2), 116.7 (CN),
118.2 (CH=C), 120.4, 121.9, 124.5, 126.2, 127.4, 127.9, 129.2, 133.9 (C6H4, C6H5), 144.2 (CH=C),
171.5, 173.9 (2C=N); Anal. Calcd. for C19H15N3O (301.34): C, 75.73; H, 5.02; N, 13.94%; found: C,
75.52; H, 4.91; N, 14.31%.
2-(α-Phenylhydrazoacetonitrilo)-5-(4-methylphenyl)1,3,4-oxadiazine (17). To a cold solution (0–5 °C)
of compound 15 (1.00 g, 3.39 × 10−3 mol), in ethanol (50 mL) was added with continuous stirring
benzenediazonium chloride (3.39 × 10−3 mol) [which was prepared by dissolving sodium nitrite
(0.35 g, 5.09 × 10−3 mol) in water, 2 mL was added to a cold solution of aniline (0.31 g, 3.39 × 10−3 mol)
containing the appropriate amount of hydrochloric acid and with continuous stirring]. The solid
product formed after adjusting the pH 6 and stirring was collected by filtration and dried to give
reddish brown crystals (from ethanol and dimethylformamide). Yield: 0.96 g (89%), m.p. 130 °C; IR
(KBr) υ/cm−1: 3300–3215 (NH), 3057 (CH aromatic), 3030 (CH3), 2920 (CH2), 2219 (CN), 1679
(C=N), 1603 (C=C); 1H-NMR δ: 2.52 (s, 3H, CH3), 3.38 (s, 2H, CH2), 7.21–8.82 (m, 9H, C6H5, C6H4),
13
10.22 (s, 1H, NH). ). C-NMR: 24.3 (CH3), 63.0 (oxadiazine CH2), 116.5 (CN), 121.5, 121.9, 124.0,
123.5, 126.7, 127.0, 127.6, 129.2, 133.0 (C6H4, C6H5), 166.9, 170.5, 173.9 (3C=N). Anal. Calcd. for
C18H15N5O (317.35): C, 68.12; H, 4.76; N, 22.06%; found: C, 67.92; H, 4.91; N, 21.25%.
4. Conclusions
In this work cyanoacetyl hydrazine (1) reacted with α-haloketone 2 to afford the
α-bromohydrazide-hydrazone derivative 3. The latter compound was used in a series of heterocyclic
transformations to give compounds that were tested as antitumor agents. The 2-hydroxypyridine
derivatives 14b showed the highest inhibitory activity.
Acknowledgments
R. M. Mohareb expressed his deepest thank to the Alexander von Humboldt Foundation in Bonn for
its financial support through a fellowship in Erlangen University during Summer 2009 and for the
completion of this work.
References and Notes
1. Bharti, S.K.; Nath, G.; Tilak, R.; Singh, S.K. Synthesis, anti-bacterial and anti-fungal activities of
some novel Schiff bases containing 2,4-disubstituted thiazole ring. Eur. J. Med. Chem. 2010, 45,
651–660.
2. Loncle, C.; Brunel, J.M.; Vidal, N.; Dherbomez, M.; Letourneux, Y. Synthesis and antifungal
activity of cholesterol-hydrazone derivatives. Eur. J. Med. Chem. 2004, 39, 1067–1071.
3. Garoufalias, S.P.; Pouli, N.; Marakos, P.; Ladas, A.C. Synthesis antimicrobial and antifungal
activity of some new 3-substituted derivatives of 4-(2,4-dichlorophenyl)-5-adamantyl-1H-1,2,4-
triazole. Farmaco 2002, 57, 973–977.

3616
Molecules 2010, 15
4. Vicini, P.; Zani, F.; Cozzini P.; Doytchinova, I. Hydrazones of 1,2-benzisothiazole hydrazides:
synthesis, antimicrobial activity and QSAR investigations. Eur. J. Med. Chem. 2002, 37,
553–567.
5. Popp, F.D. Potential anticonvulsant. XII. anticonvulsant activity of some aldehyde derivatives.
Eur. J. Med. Chem. 1989, 24, 313–316.
6. Sridhar, S.K.; Pandeya, S.N.; Stables J.P.; Atmakuru, R. Anticonvulsant activity of hydrazones,
Schiff and Mannich bases of isatin derivatives Eur. J. Pharm. Sci. 2002, 16, 129–132.
7. Küçükgüzel, S.G.; Mazi, A.; Sahin, F.; Öztürk, S.; Stables, J.P. Synthesis and biological activities
of diflunisal hydrazide–hydrazones. Eur. J. Med. Chem. 2003, 38, 1005–1013.
8. Todeschini, A.R.; Miranda, A.L.P.; Silva, K.C.M.; Parrini, S.C.; Barreiro, E. Synthesis and
evaluation of analgesic, antiinflammatory and antiplatelet properties of new
2-pyridylarylhydrazone derivatives. Eur. J. Med. Chem. 1998, 33, 189–199.
9. Gaston, M.A.; Dias, L.R.S.; Freitas, A.C.C.; Miranda, A.L.P.; Barreiro, E. Synthesis and
Analgesic Properties of New 4-Arylhydrazone 1H-Pyrazolo[3,4-b] Pyridine Derivatives.
J. Pharm. Acta Helv. 1996, 71, 213–219.
10. Melnyk, P.; Leroux, V.; Sergheraert, C.; Grellier, P. Design, synthesis and in vitro antimalarial of
an acylhydrazone library. Bioorg. Med. Chem. Lett. 2006, 16, 31–35.
11. Küçükgüzel, S.G.; Rollas, S., Küçükgüzel, I.; Kiraz, M. Synthesis and antimycobacterial activity
of some coupling products from 4-aminobenzoic acid hydrazones. Eur. J. Med. Chem. 1999, 34,
1093–1100.
12. Kaymakçıoğlu, B.K.; Rollas, S. Synthesis, characterization and evaluation of antituberculosis
activity of some hydrazones. Farmaco 2002, 57, 595–599.
13. Terzioglu, N.; Gursoy, A. Synthesis and anticancer evaluation of some new hydrazone derivatives
of 2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazide. Eur. J. Med. Chem. 2003, 38,
781–786.
14. Sondhi, S.M.; Dinodia, M., Kumar, A. Synthesis, anti-inflammatory and analgesic activity
evaluation of some amidine and hydrazone derivatives Bioorg. Med. Chem. 2006, 14, 4657–4663.
15. Boga, C.; Fiume, L.; Baglioni, M.; Bertucci, C.; Farina, C.; Kratz, F.; Manerba, M.; Naldi, M.;
Stefano, G. Characterisation of the conjugate of the (6-maleimidocaproyl)hydrazone derivative of
13
doxorubicin with lactosaminated human albumin by C NMR spectroscopy. Eur. J. Pharm. Sci.
2009, 38, 262–269.
16. Garnett, M.C. Targeted drug conjugates: principles and progress. Adv. Drug Deliv. Rev. 2001, 53,
171–216.
17. Rodrigues, P.C.; Scheuermann, K.; Stockmar, C.; Maier, G.; Fiebig, H.; Unger, C.; Mülhaupt, R.;
Kratz, F. Synthesis and In vitro efficacy of acid-Sensitive poly(ethylene glycol) paclitaxel
conjugates. Bioorg. Med. Chem. Lett. 2003, 13, 355–360.
18. El-Sabbagh, O.I.; Rady, H.M. Synthesis of new acridines and hydrazones derived from cyclic
β-diketone for cytotoxic and antiviral evaluation. Eur. J. Med. Chem. 2009, 44, 3680–3686.
19. Krakovicova, H.; Etrych, T.; Ulbrich, K. HPMA-based polymer conjugates with drug
combination. Eur. J. Pharm. Sci. 2009, 37, 405–412.

3617
Molecules 2010, 15
20. Zhang, H.Z.; Drewe, J.; Tseng, B.; Kasibhatla, S.; Cai, S.X. Discovery and SAR of indole-2-
carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-
based HTS assay. Bioorg. Med. Chem. 2004, 12, 3649–3655.
21. Vicini, P.; Incerti, M.I.; Colla, P.L.; Loddo, R. Anti-HIV evaluation of benzo[d]isothiazole
hydrazones. Eur. J. Med. Chem. 2009, 44, 1801–1807.
22. Gürsoy, E.; Güzeldemirci, N.U. Synthesis and primary cytotoxicity evaluation of new
imidazo[2,1-b]thiazole derivatives. Eur. J. Med. Chem. 2007, 42, 320–326.
23. Hao, J.J.; Xu, Y.; Geng,C.; Liu, W.Y.; Wang, E.; Gong, Z.; Ulbrich, N. Purification of α-Sarcin
and an Antifungal Protein from Aspergillus giganteusby Blue Sepharose CL-6B Affinity
Chromatography. Protein Expr. Purif. 1998, 14, 295–301.
24. Ke, S.Y.; Qian, X.H.; Liu, F.Y.; Wang, N.; Yang, Q.; Zhong, L. Novel 4H-1,3,4-oxadiazin-5(6H)-
ones with hydrophobic and long alkyl chains: Design, synthesis, and bioactive diversity on
inhibition of monoamine oxidase, chitin biosynthesis and tumor cell. Eur. J. Med. Chem. 2009,
44, 2113–2121.
Sample Availability: Samples of the compounds 4a–17 are available from the authors.
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