Synthesis of novel 1,2,4-triazoles and triazolo-thiadiazines as anticancer agents

Article abstract of DOI:10.3906/kim-1504-13

A new series of 7-arylazo-5H-3-(tri uoromethyl)-6-methyl-1,2,4-triazolo-[3,4- b ]-1,3,4-thiadiazines was pre- pared by reaction of 4-amino-3-tri uoromethyl-5-mercapto-1,2,4-triazoles with N -aryl-2-oxo-propane hydrazonoyl chlo- ride in dioxane under re ux

Full text of DOI:10.3906/kim-1504-13

Turk J Chem
Turkish Journal of Chemistry
(2015) 39: 955 – 969
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1504-13
Research Article
Synthesis of novel 1,2,4-triazoles and triazolo-thiadiazines as anticancer agents
Thoraya Abd El-Reheem FARGHALY1,2,∗, Magda Ahmad ABDALLAH1, Huda Kamel MAHMOUD1
1Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
2Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah Almukkarramah,
Saudi Arabia
Received: 06.04.2015
•
Accepted/Published Online: 15.05.2015
•
Printed: 30.10.2015
Abstract: A new series of 7-arylazo-5H -3-(trifluoromethyl)-6-methyl-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazines was pre-
pared by reaction of 4-amino-3-trifluoromethyl-5-mercapto-1,2,4-triazoles with N -aryl-2-oxo-propane hydrazonoyl chlo-
ride in dioxane under reflux in the presence of triethylamine. Furthermore, Schiff bases of 4-amino-5-mercapto-1,2,4-
triazole derivatives were reacted with a variety of hydrazonoyl chlorides and gave the respective hydrazonothioates. In
addition, the novel bis-(1,2,4-triazole-3-thione) was reacted with the appropriate hydrazonoyl chloride in dioxane under
reflux in the presence of triethylamine to give the corresponding bis-(1,2,4-triazolethiohydrazonoate). The structures of
the new compounds were established based on elemental and spectral data. The mechanism of the studied reaction was
also discussed. Moreover, some of the new products were screened for their anticancer activity and the results obtained
are promising and indicate that compounds 4a and 4i are the most active inhibitors against HEPG-2 and compounds
4a and 13b are active against HCT cell lines.
Key words: 4-Amino-3-trifluoromethyl-5-mercapto-1,2,4-triazole, hydrazonoyl halides, anticancer activity, triazolo[3,4-
b][1,3,4]thiadiazines
1. Introduction
4-Amino-5-mercapto-1,2,4-triazole derivative is a readymade building block for construction of various organic
heterocycles since they contain two nucleophilic groups, amino and thiol groups. On the other hand, Schiff
bases of 1,2,4-triazoles find diverse applications and extensive biological activity. For example, Schiff bases
derived from 3-substituted-4-amino-5-mercapto-1,2,4-triazoles show analgesic, antimicrobial, anti-inflammatory,
antiproliferative, and antidepressant activities.1,2
On the other hand, 4-amino-5-mercapto-1,2,4-triazoles react with different types of hydrazonoyl halides to
give a wide variety of fused 1,2,4-triazoles.3−5 Moreover, the fluorine substituent in the heterocyclic compounds
improves their biological activities.6,7 From all the above findings and in continuation of our previous work on
hydrazonoyl halides,8−15 we were interested in studying the reaction of hydrazonoyl halides with 4-amino-3-
trifluoromethyl-5-mercapto-1,2,4-triazoles and the respective Schiff bases. Our objective after such a study was
to shed some light on the site-selectivity of such reactions and also to test the biological activity of the products
against HPG-2 and HCT cancer cell lines.
∗Correspondence: thoraya-f@hotmail.com
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FARGHALY et al./Turk J Chem
2. Results and discussion
2.1. Chemistry
The required starting material, namely 4-amino-3-trifluoromethyl-5-mercapto-1,2,4-triazole 1, was prepared as
previously reported.6 Thus, the reaction of trifluoroacetic acid hydrazide with carbon disulfide in alcoholic
solution of potassium hydroxide gave potassium trifluoroacetyldithiocarbazate, which upon refluxing with
hydrazine hydrate produced 4-amino-5-trifluoromethyl-4H -1,2,4-triazole-3-thiol 1.6 Reaction of 1 with N -
aryl-2-oxopropanehydrazonoyl chloride 2 in dioxane under reflux in the presence of triethylamine afforded in
each case one isolable product as evidenced by TLC analysis (Figure 1). The products were assigned structure
4 or 7 rather than structure 6 since they were free of sulfur. Moreover, elemental analysis and spectral data are
in agreement with structure 4 rather than structure 7. For example, IR spectra of the products revealed in each
case the absence of the NH2 group, the acetyl carbonyl group, and the C=S group and at the same time showed
only absorption bands at 3439–3317 cm−1 , which were assigned to the hydrazone NH group. The 1 H NMR
spectra revealed a singlet signal at δ 11.02–10.29 ppm assigned to the hydrazone NH proton, in addition to the
expected signals for the methyl and aromatic protons. The mass spectra revealed in each case a molecular ion
peak corresponding to elimination of water molecule from intermediate 3, and in agreement with the molecular
formula of the products 4. In addition, the electronic absorption spectra of compounds 4a–j in dioxane revealed
in each case two characteristic absorption bands in the regions λmax 386–357 and 277–244 nm (Table 1). Such
an absorption pattern is similar to that of typical hydrazone chromophore 4A.9
Table 1. UV spectral data of compounds 4a–j in dioxane.
Compd. no. λmax (log ε)
4a
4b
4c
4d*
4e
4f
4g
4h
4i
385 (4.56), 275 (5.09)
357 (6.45), 244 (6.17)
377(5.34), 251 (5.75)
374 (6.35), 246 (6.36)
370 (5.57), 256 (5.83)
370 (6.07), 246 (6.06)
363 (5.45), 253 (5.91)
386 (5.30), 244 (5.40)
378 (5.39), 277 (5.33)
375 (5.70), 277 (5.60)
4j
∗ Solvent λmax (log ε): chloroform: 376 (6.09), 244 (6.23); ethanol: 383 (5.55), 225 (5.52); DMF 359 (4.17), 304 (4.60).
Our study was extended to examine the chemical reactivity of Schiff bases of 4-amino-5-mercapto-1,2,4-
triazole derivative 1 towards a variety of hydrazonoyl halides 2. The Schiff bases 8 were prepared as previously
reported by reaction of triazole derivative 1 with the appropriate aromatic aldehyde16,17 under reflux for 5 h
in glacial acetic acid. Products 8 in turn reacted with hydrazonoyl halides 2 in dioxane under reflux to give
the intermediate thiohydrazonoate ester 9 (Figure 2). The other isomeric structures N -triazolyltriazoles 10
and 11 were discarded based on IR and 1 H NMR spectral data. For example, the IR spectra of products 9
revealed the absence of the absorption band of the SH group at 2500–2600 cm−1 and revealed the presence
of an absorption band near 3430 cm−1 assigned to the NH group.18 The 1 H NMR spectra of the products 9
exhibited in each case a singlet signal at δ 8.07–9.48 ppm characteristic for the azomethine–N=CH– protons,
which is similar to compound I2,19,20 and differ from the CH triazoles types II and III21−24 (Figure 3). More
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FARGHALY et al./Turk J Chem
N
N
Cl
H
N
+
F3C
SH
H3COC
N
Ar
N
2
NH2
1
Dioxane/ Et3N / reflux
- HCl
N N
COCH3
S
F3C
N
N
NH
NH2
3
Ar
- H2O
N N
N N
Ar
F3C
N
SH
F3C
N
S
N
N
H
N
N
NH2
N
COCH3
Ar
5
4A
CH3
-H2S
- H2O
N
N
N
N
F3C
Ar
N
N
S
N
N
Ar
F3C
N
HN
F3C
Ar
N
N
N
N
N
N
NH
HN
CH3
S
COCH3
4B
6
H3C
7
Ar = XC6H4
X : a, 4-OCH3 ; b, 4-CH3; c, 3-CH3; d, H; e, 3-Cl; f, 4-Cl; g, 3-NO2;
h, 4-NO2; i, 4-COCH3; j, 4-COOEt
Figure 1. Synthesis of compounds 4a–j.
evidence for the elucidation of the structure 9 rather than 10 or 11 is the 13 C NMR of the isolated products.
As shown in Figure 3, the 13 C NMR of compounds 9a and 9k revealed the absence of the signals of the C-3 or
C-4 for the triazoles II or III,23−25 which are similar to compounds 10 and 11 in Scheme 2. Instead the 13 C
NMR of compounds 9a and 9k showed signals at 150.4 ppm and 150.1 ppm, respectively. Furthermore, the
13 C NMR spectra of the products 9a and 9k supported the assigned structure 9A for the isolated products,
which revealed the absence of a thiohydrazide carbon (N–C=S) signal near δ 170–180 ppm and the presence
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FARGHALY et al./Turk J Chem
of a signal near δ 141–142 ppm due to the carbon atom of the –S–C=N–NH– group.26−28 All the above data
proved that the isolated products have structure 9A.
N
N
Ar'CHO +
F3C
SH
Cl
N
NH2
1
AcOH/ reflux
H
N
N
N
F3C
SH
+
N
N
N
Ar
R
2
C Ar'
H
8
Dioxane/ Et3N / reflux
- HCl
N
N
N
N
N
N
N
R
H
F3C
Ar
SH
Ar'
F3C
R
SH
Ar'
F3C
N
N
N
N
S
N
N
Ar
N
N
CH
Ar'
N
9A
N
N
Ar
N
R
HS
N
N
Ar
10A
11A
F3C
R
N
N
N
N
NH
N
NH
F3C
Ar
S
CH
9B
F3C
R
S
N
N
N
N
Ar'
N
N
Ar'
Ar
Ar'
S
N
N
N
N
N
R
F3C
R
N
H
10B
N
Ar
11B
N
9C
CH
Ar'
Comp. No.
Ar
R
Ar'
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
MeCO
MeCO
MeCO
MeCO
EtOCO
EtOCO
EtOCO
EtOCO
MeCO
EtOCO
Ph
Ph
9a
9b
9c
9d
9e
9f
9g
9h
9i
4-MeC6H4
4-ClC6H4
4-NO2C6H4
Ph
4-MeC6H4
4-ClC6H4
4-NO2C6H4
Ph
3,4-(MeO)2C6H3
3,4-(MeO)2C6H3
3,4-(MeO)2C6H3
Ph
9j
9k
9l
Ph
Ph
EtOCO
Me
O
Figure 2. Synthesis of compounds 9a–l.
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FARGHALY et al./Turk J Chem
N N
R'
H
N
R
R'
N
N
S
Ar
N
Ar'
1H NMR 4.90-5.70 ppm
13C NMR 60-67ppm
N
CH
R
1H NMR 8.0-10.16 ppm
13C NMR 152.3-159.0 ppm
Ar
II
I
R'
N
H
N
N
R
H
N
R
Ar'
1H NMR 5.97-6.66 ppm
13C NMR 74-87 ppm
F3C
N
N
N
S
Ar
N
Ar
N
III
CH
1H NMR 8.07-9.48 ppm
9 Ar'
13C NMR 150.4, 150.1 ppm
Figure 3. 1 H and 13 C NMR of compounds I–III and 9.
In order to increase the biological activity of triazole derivatives, we also synthesized a new symmetric
dimeric derivative, namely 1,4-bis(iminomethyl-3-trifluoromethyl-5-mercapto-1,2,4-triazole-4-benzene 12. Thus,
reaction of 2 moles of 4-amino-5-mercapto-3-trifluoromethyl-1,2,4-triazole 1 with terephthaldehyde in acetic acid
under reflux for 5 h furnished the corresponding bis-Schiff base 12, which was obtained in good yield (90%)
(Figure 4). Reaction of the latter with 2 mole equivalents of the appropriate hydrazonoyl chloride 2 in dioxane
under reflux in the presence of triethylamine afforded in each case the corresponding bis-thiohydrazonoate
esters 13 rather than the bis(N -triazolyltriazole) 14 or its regioisomer 15. Elemental analyses and spectral
data are in support of structure 13 for the products. For example, the IR spectrum of compound 13a showed
a characteristic absorption band at 3431 and 1629 cm−1 for the hydrazone –NH and the azomethine groups,
respectively. Moreover, the 1 H NMR spectrum displayed three singlet signals at δ 10.69, 8.27, and 2.49 ppm
for two –NH, two –CH=N–, and two methyl protons, in addition to the other expected signals assigned for the
aromatic protons in the structural formula.
2.2. Antitumor activity
The compounds 4a, 4i, 4j, 9a, 9b, 9e, 9i, 9j, 13a, and 13b were assayed for their cytotoxicity against HEPG 2
cell line and HCT cell line. The results depicted in Table 2 indicated that the most active ones were compounds
9b and 9i since their inhibitory effect was 82.3%. The other compounds, 4 and 13, are also active but relatively
less than compounds 9b and 9i (HEPG-2 cell line). On the other hand, compounds 4a and 9i are the most
active HCT cell line as depicted in Table 3. Compounds 4, 9, and 13a have moderate activity in comparison
with compounds 4a and 9i.
From the above data, we recorded the IC50 of the most reactive compounds against the two cancer cell
lines, HepG2 (hepatic cancer) and HCT (colon carcinoma).
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FARGHALY et al./Turk J Chem
Table 2. Single dose experiment on HPG2 cell line (100
µg/mL).
Table 3. Single dose experiment on HCT cell line (100
µg/mL).
Sample no. Surviving % Inhibition %
Sample no. Surviving % Inhibition %
4a
4i
4j
9a
9b
9e
9i
9j
13a
13b
22
25
27
30
17.7
42
17.7
26
78
75
73
70
82.3
58
82.3
74
4a
4i
4j
9a
9b
9e
9i
9j
13a
13b
25
75
36.4
32.7
45.5
36.4
51.4
19.3
37.5
31
63.6
67.3
54.5
63.6
48.6
80.7
62.5
69
30
29
70
71
28
72
N
N
OHC
CHO
+
F3C
SH
2
N
NH2
AcOH / reflux
1
H
N
N
Cl
H
N
F3C
NH
S
N
N
N
N
+
2
R
N
Ar
H
C
S
F3C
N
C
H
2
12
Dioxane / Et3N / reflux
- HCl
R
N
N
Ar
S
N
N
N
H
N
CF3
Ar
N
R
Ar
N
N
N
N
N
CF3
N
N
N
F3C
CH
R
N
N
HS
N
SH
SH
N
R
HS
N
HC
N
S
N
N
N
N
NH
N
N
N
N
R
Ar
N
F3C
F3C
N
N
Ar
N
N
N
R
Ar
CF3
13
15
14
R / Ar: a, COCH3 / Ph; b, COOEt / Ph
Figure 4. Synthesis of compounds 13.
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FARGHALY et al./Turk J Chem
Doxorobucin was used as the reference drug for screening, exerting IC50 3.6 and 4.28 µg/L against
HepG2 and HCT, respectively.
HepG-2 was the cell line that showed highest sensitivity towards four tested compounds, 4a, 4i, 9b, and
9i. Compounds 4i and 4a displayed the highest activity against HepG2 exerting the following IC50 5.63 and
8.63 µg/mL. On the other hand, 9i and 9b showed mild anti-HepG2 activity representing IC50 of 18.7 and
22.7 µg/mL respectively, compared to the reference drug (Table 4). For colon carcinoma cancer cell line HCT,
compounds 4a and 13b were the most prominent anti-HCT among the test compounds. They exhibited IC50
of 8.33 and 8.45 µg/mL compared to doxorubicin. In addition, the activity of 9i cannot be ignored: IC50 14.9
µg/mL compared to the reference drug (Table 5). The dose response profile was illustrated for the most sensitive
cell lines HepG2 and HCT of the tested compounds compared to the reference drug doxorubicin (Figures 5 and
6).
Table 4. IC50 of compounds 4a, 4i, 9b, and 9i against
Table 5. IC50 of compounds 4a, 9i, and 13b against
HEPG-2.
HCT.
Comp. no. IC50µg/mL
Comp. no. IC50µg/mL
4a
4i
8.63
5.63
22.7
19.7
3.6
4a
9i
8.33
14.9
8.45
4.28
9b
9i
13b
DOX
DOX
Figure 5. Dose-response profiles of compounds 4a, 4i,
9b, and 9i against HEPG2 cell line.
Figure 6. Dose-response profiles of compounds 4a, 9i,
and 13b against HCT cell line.
3. Conclusion
In summary, we described a facile one-pot synthesis of a novel series of 3,6,7-trisubstituted-1,2,4-triazolo-[3,4-
b][1,3,4]thiadiazines (4a–j) using 3-trifluoromethyl-4-amino-5-mercapto-1,2,4-triazole (1) as the key synthon
for their preparation. Moreover, 3-substituted-4-arylideneamino-1,2,4-triazol-5-yl)thiohydrazonate esters (9a–
l) were also synthesized by reaction of Schiff bases (8) each with a variety of hydrazonoyl halides (2). A new
symmetric dimeric Schiff base (12) was also synthesized and reacted with hydrazonoyl halides (2) to give the
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FARGHALY et al./Turk J Chem
respective bis-(1,2,4-triazol-5-yl-thiohydrazonoate) (13). The structure of the newly synthesized compounds was
elucidated on the basis of elemental analysis and spectral data (IR, 1 H NMR, and MS spectra). Moreover, some
substituted 1,2,4-triazole derivatives were assayed for anticancer activity. The results showed that compounds
4a, 4i, and 13b are the most active inhibitors against HEGP-2 cell line and/or HCT cell line, which provides
a good lead for the design and discovery of new high potent drugs by structure-based molecular modification.
4. Experimental
4.1. General
Melting points were determined on a Gallenkamp apparatus. IR spectra were recorded in potassium bromide
using PerkinElmer FTIR 1650 and Pye-Unicam SP300 infrared spectrophotometers. 1 H NMR spectra were
recorded in deuterated dimethyl sulfoxide using a Varian Gemini 300 NMR spectrometer. Mass spectra were
recorded on a GCMS-QP 1000 EX Shimadzu and a GCMS 5988-A HP spectrometer. Electronic absorption
spectra were recorded on a PerkinElmer Lambda 40 spectrophotometer. Elemental analyses were carried
out using a German made Elementar vario LIII CHNS analyzer at the Microanalytical Laboratory of Cairo
University, Giza, Egypt. Antitumor activities were recorded at J-Natl-Cancer Inst., Cairo, Egypt. Hydrazonoyl
chlorides 229 were prepared as previously described.
4.2. Reaction of compound 1 with hydrazonoyl chloride 2
To a mixture of 1 (0.46 g, 2.5 mmol) and the hydrazonoyl chloride 2 (2.5 mmol) in dioxane (30 mL) was added
triethylamine (0.35 mL), and the mixture was heated under reflux for 5 h. The reaction mixture was then
poured on ice water and acidified with HCl. The solid produced was collected by filtration and crystallized
from the appropriate solvent to give the corresponding compounds 4a–j. The products 4a–j together with their
physical constants are listed below.
4.2.1. 6-Methyl-3-trifluoromethyl-7-(4-methoxyphenylhydrazono)[1,2,4]triazolo [3,4-b][1,3,4]thia-
diazine (4a)
Yellow solid, yield (0.53 g, 60%), mp 226–228 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3434 (NH), 3050,
2951, 1597, 1536, 1505, 1459, 142, 1393, 1356, 1299, 1234, 1164, 1033 cm−1
.
1 H NMR (DMSO-d6) 2.46 (s,
3H, CH3), 3.73 (s, 3H, OCH3), 6.92 (d, J = 9.0 Hz, 2H, Ar-H), 7.29 (d, J = 9.0 Hz, 2H, Ar-H), 10.29 (s,
1H, NH). MS m/z (%) 357 (M+ +1, 4), 356 (M+ , 24), 122 (100), 107 (11), 95 (17), 77 (7). Anal. Calcd. for
C13 H11 N6 SF3 O (356.32) Calcd: C, 43.81; H, 3.11; N, 23.58. Found: C, 43.57; H, 3.29; N, 23.29%.
4.2.2. 6-Methyl-3-trifluoromethyl-7-(4-methylphenylhydrazono)-[1,2,4]triazolo [3,4-b][1,3,4]thia-
diazine (4b)
Yellow solid, yield (0.62 g, 73%), mp 214–216 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3317 (NH), 3208,
3031, 1652, 1612, 1525, 1491, 1360, 1277, 1230, 1179, 1156, 1004 cm−1
.
1 H NMR (DMSO-d6) 1.76 (s, 3H,
CH3), 2.23 (s, 3H, CH3), 7.06–7.39 (m, 4H, Ar H), 10.87 (s, 1H, NH). MS m/z (%) 341 (M+ +1, 10), 340 (M+ ,
52), 185 (5), 174 (9), 119 (6), 106 (100), 91 (71), 79 (51), 77 (56), 65 (15). Anal. Calcd. For C13 H11 N6 SF3
(340.33). Calcd: C, 45.87; H, 3.25; N, 24.69. Found: C, 45.57; H, 3.47; N, 24.89%.
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FARGHALY et al./Turk J Chem
4.2.3. 6-Methyl-3-trifluoromethyl-7-(3-methylphenylhydrazono)-[1,2,4]triazolo [3,4-b][1,3,4]thia-
diazine (4c)
Yellow solid, yield (0.65 g, 76%), mp 286–288 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3436 (NH), 3208,
3052, 1609, 1540, 1507, 1459, 1390, 1318, 1261, 1190, 1159, 1040 cm−1
.
1 H NMR (DMSO-d6) 2.31 (s, 3H,
CH3), 2.48 (s, 3H, CH3), 6.8–7.18 (m, 4H, Ar H), 10.32 (s, 1H, NH). MS m/z (%) 341 (M+ +1, 11), 340 (M+ ,
59), 180 (7), 106 (68), 91 (100), 79 (60), 77 (60), 65 (17). Anal. Calcd. forC13 H11 N6 SF3 (340.33) Calcd: C,
45.87; H, 3.25; N, 24.69. Found: C, 45.59; H, 3.46; N, 24.82%.
4.2.4. 6-Methyl-3-trifluoromethyl-7-phenylhydrazono-[1,2,4]triazolo[3,4-b][1,3,4]- thiadiazine (4d)
Yellow solid, yield (0.75 g, 70%), mp 276–278 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3432 (NH), 3050,
3010, 1598, 1536, 1498, 1459, 1393, 1360, 1242, 1162, 1077, 1041 cm−1
.
1 H NMR (DMSO-d6) 2.47 (s, 3H,
CH3), 6.99 (d, J = 8.1 Hz, 2H, Ar-H), 7.34 (d, J = 8.1 Hz, 2H, Ar-H), 10.31 (s, 1H, NH). MS m/z (%)
327 (M+ +1, 10), 326 (M+ , 60), 105 (13), 92 (91), 91 (46), 77 (100), 70 (11), 65 (77). Anal. Calcd. for
C12 H9 N6 SF3 (326.31) Calcd: C, 44.17; H, 2.78; N, 25.75. Found: C, 44.38; H, 3.01; N, 25.99%.
4.2.5. 7-(3-Chlorophenylhydrazono)-3-trifluoromethyl-6-methyl-[1,2,4]triazolo-[3,4-b][1,3,4] thiadi-
azine (4e)
Yellow solid, yield (0.63 g, 70%), mp 270–272 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3430 (NH), 1595,
1535, 1467, 1430, 1396, 1357, 1264, 1241, 1193, 1157, 1073, 1042, 1003 cm−1
.
1 H NMR (DMSO-d6) 2.41 (s,
3H, CH3), 7.0–7.36 (m, 4H, Ar-H), 10.56 (s, 1H, NH). MS m/z (%) 362 (M+ +2, 20), 361 (M+ +1, 10), 360
(M+ , 56), 180 (27), 139 (12), 128 (19), 126 (56), 111 (100), 101 (26), 99 (80), 90 (31), 75 (23), 70 (26), 63 (28).
Anal. Calcd. for C12 H8 N6 SF3 Cl (360.75) Calcd: C, 39.95; H, 2.23; N, 23.29. Found: C, 40.25; H, 2.48; N,
23.40%.
4.2.6. 7-(4-Chlorophenylhydrazono)-3-trifluoromethyl-6-methyl-[1,2,4]triazolo-[3,4-b][1,3,4]thia-
diazine (4f)
Yellow solid, yield (0.68 g, 75%), mp 260–262 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3437 (NH), 1653,
1599, 1533, 1489, 1398, 1360, 1239, 1161, 1095, 1005 cm−1 1 H NMR (DMSO-d6) 1.77 (s, 3H, CH3), 7.16–7.49
.
(m, 4H, Ar-H), 10.42 (s, 1H, NH). MS m/z (%) 363 (M+ +2, 4), 362 (M+ +1, 19), 361 (M+ , 10), 360 (M+ –1,
48), 180 (17), 139 (13), 126 (100), 111 (72), 101 (25), 99 (74), 90 (27), 80 (89), 70 (26), 63 (98), 63 (28). Anal.
Calcd. for C12 H8 N6 SF3 Cl (360.75) Calcd: C, 39.95; H, 2.23; N, 23.29. Found: C, 40.11; H, 2.0; N, 23.57%.
4.2.7. 6-Methyl-3-trifluoromethyl-7-(3-nitrophenylhydrazono)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zine (4g)
Buff solid, yield (0.74 g, 80%), mp 256–258 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3428 (NH), 3085,
3043, 3018, 1617, 1543, 1531, 1478, 1457, 1397, 1347, 1271, 1242, 1185, 1154, 1079, 1039, 1003 cm−1 1 H NMR
.
(DMSO-d6) 2.49 (s, 3H, CH3), 7.58–8.13 (m, 4H, Ar-H), 10.76 (s, 1H, NH). MS m/z (%) 372 (M+ +1, 16),
371(M+ , 84), 180 (41), 137 (16), 122 (64), 91 (59), 80 (67), 79 (20), 70 (34), 64 (100), 63 (50). Anal. Calcd.
for C12 H8 N7 SF3 O2 (371.30) Calcd: C, 38.81; H, 2.17; N, 26.40. Found: C, 39.09; H, 2.45; N, 26.17%.
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4.2.8. 6-Methyl-3-trifluoromethyl-7-(4-nitrophenylhydrazono)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zine (4h)
Buff solid, yield (0.65 g, 70%), mp 282–284 ◦ C, dioxane, IR (KBr, cm−1 ): 3390 (NH), 1596, 1505, 1405, 1330,
1249, 1154, 1112, 1033 cm−1 1 H NMR (DMSO-d6) 2.49 (s, 3H, CH3), 7.49 (d, J = 9.1 Hz, 2H, Ar-H), 8.21
.
(d, J = 9.2 Hz, 2H, Ar-H), 11.02 (s, 1H, NH). MS m/z (%) 372 (M+ +1, 17), 371 (M+ , 100), 180 (42), 169
(7), 153 (13), 35 (17), 122 (83), 111 (12), 107 (31), 98 (10), 92 (20), 91 (37), 80 (52), 76 (22), 70 (38), 65 (75),
64 (16), 63 (49). Anal. Calcd. for C12 H8 N7 SF3 O2 (371.30) Calcd: C, 38.81; H, 2.17; N, 26.40. Found: C,
38.56; H, 2.47; N, 26.64%.
4.2.9. 7-(4-Acetylphenylhydrazono)-3-trifluoromethyl-6-methyl-[1,2,4]triazolo-[3,4-b][1,3,4]thia-
diazine (4i)
Yellow solid, yield (0.65 g, 71%), mp 278–280 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3435 (NH), 1669
(CO), 1596, 1534, 1459, 1422, 1358, 1249, 1157, 1039 cm−1 1 H NMR (DMSO-d6) 2.49 (s, 3H, CH3), 2.51 (s,
.
3H, CH3) 7.42 (d,J = 9.0 Hz, 2H, Ar-H), 7.93 (d, J = 9.0 Hz, 2H, Ar-H), 10.73 (s, 1H, NH). MS m/z (%) 369
(M+ +1, 2), 368 (M+ , 9), 106 (4), 91 (7), 80 (100), 64 (47). Anal. Calcd. for C14 H11 N6 SF3 O (368.34) Calcd:
C, 45.65; H, 3.01; N, 22.81. Found: C, 45.92; H, 3.29; N, 23.06%.
4.2.10. 7-(4-Ethoxycarbonylphenylhydrazono)-3-trifluoromethyl-6-methyl-[1,2,4]-triazolo[3,4-b]
[1,3,4]thiadiazine (4j)
Yellow solid, yield (0.65 g, 65%), mp 268–270 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3439 (NH), 1703
(CO), 1603, 1532, 1458, 1392, 1243, 1157, 1013 cm−1
.
1 H NMR (DMSO-d6) 1.3 (t, J = 7.1 Hz, 3H, CH3),
2.4 (s, 3H, CH3), 4.25 (q, J = 7.1 Hz, 2H, CH2), 7.37 (d, J = 8.9 Hz, 2H, Ar-H), 7.87 (d, J = 8.9 Hz, 2H,
Ar-H), 10.79 (s, 1H, NH). MS m/z (%) 399 (M+ +1, 20), 398 (M+ , 100), 370 (16), 179 (18), 149 (64), 135 (24),
121 (46), 119 (42), 108 (88), 103 (42), 91 (54), 81 (23), 76 (14), 65 (60). Anal. Calcd. for C15 H13 N6 SF3 O2
(398.37) Calcd: C, 45.22; H, 3.28; N, 21.09. Found C, 45.49; H, 3.40; N, 21.32%.
4.3. Synthesis of compounds 8
A mixture of compound 1 (2.5 mmol) and the appropriate aromatic aldehyde (2.5 mmol) was refluxed in glacial
acetic acid (20 mL) for 5 h. The solution was then cooled and the solid produced was filtered and recrystallized
from the appropriate solvent.
Compounds 8a and b were prepared by the same method described in the literature.16,17
4.3.1. 4-[(5-Methyl-furan-2-ylmethylene)-amino]-5-trifluoromethyl-4H-[1,2,4]-triazole-3-thiol (8c)
White solid, yield (0.54 g, 80%), mp 208–210 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3431 (NH), 2912,
2742, 1612, 1590, 1569, 1552, 1522, 1497, 1451, 1369, 1348, 1293, 1268, 1195, 1165, 1142, 1104, 1027 cm−1
.
1 H NMR (DMSO-d6) 2.44 (s, 3H, CH3), 6.46 (d, J = 4.3, 1H, CH), 7.33 (d, J = 4.3, 1H, CH), 9.48 (s, 1H,
N=CH), 14.78 (s, 1H, NH). MS m/z (%) 276 (M+ , 2), 274 (13), 169 (43), 118 (13), 111 (20), 106 (100), 70
(12), 80 (67), 69 (56), 64 (83). Anal. Calcd. for C9 H7 N4 SF3 O (276.24) Calcd: C, 39.13; H, 2.55; N, 20.28.
Found: C, 39.31; H, 2.83; N, 20.01%.
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FARGHALY et al./Turk J Chem
4.4. Reaction of compounds 8 with hydrazonoyl chlorides 2
To a mixture of 8 (2.5 mmol of each) and the hydrazonoyl chloride 2 (2.5 mmol) in dioxane (30 mL) was
added triethylamine (0.35 mL), and the mixture was heated to reflux for 5 h, and then the reaction mixture
was poured on ice water and acidified with HCl. The solid produced was collected by filtration and crystallized
from the appropriate solvent to give the corresponding compounds 9a–l. The products 9a–l together with their
physical constants are listed below.
4.4.1. 4-[((E)-Benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl (E)-2-oxo-N-phenylp-
ropanehydrazonothioate (9a)
Orange solid, yield (0.76 g, 70%), mp 200–202 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3436 (NH), 1682
(CO), 1631, 1578, 1507, 1442, 1407, 1362, 1293, 1193, 1141, 1062, 1016 cm−1 1 H NMR (DMSO-d6): 2.61 (s,
.
3H, COCH3), 7.40–8.13 (m, 10H, Ar-H), 8.26 (s, 1H, =CH), 10.61 (s, 1H, NH). 13 C NMR (DMSO-d6): 24.9,
116.8, 122.0, 126.4, 127.0, 128.6, 129.0, 129.4, 133.9, 138.3, 141.4, 144.2, 150.3, 164.3, 189.2. MS m/z (%) 433
(M+ +1, 3), 432 (M+ , 11), 273 (7), 228 (10), 135 (26), 118 (92), 104 (60), 91 (53), 77 (100). Anal. Calcd. for
C19 H15 N6 SF3 O (432.43) Calcd: C, 52.77; H, 3.49; N, 19.43. Found: C, 52.56; H, 3.76; N, 19.70%.
4.4.2. 4-[((E)-Benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl (E)-2-oxo-N-(4-methy-
lphenyl)propanehydrazonothioate (9b)
Orange solid, yield (0.76 g, 68%), mp 208–210 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3434 (NH), 1678
(CO), 1631, 1574, 1510, 1441, 1403, 1292, 1161, 1063, 1005 cm−1
.
1 H NMR (DMSO-d6) 2.40 (s, 3H, CH3),
2.60 (s, 3H, COCH3), 7.37–7.46 (m, 5H, Ar-H), 7.61 (d, J = 8.2 Hz, 2H, Ar-H), 7.95 (d, J = 8.2 Hz, 2H,
Ar-H), 8.26 (s, 1H, =CH), 10.56 (s, 1H, NH). MS m/z (%) 447 (M+ +1, 3), 446 (M+ , 9), 149 (13), 132 (100),
118 (26), 105 (67), 91 (69), 77 (45). Anal. Calcd. for C20 H17 N6 SF3 O (446.46) Calcd: C, 53.80; H, 3.83; N,
18.82. Found: C, 54.0; H, 3.54; N, 19.06%.
4.4.3. 4-[((E)-Benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl (E)-N-(4-chlorophenyl)-
2-oxopropanehydrazonothioate (9c)
Orange solid, yield (0.93 g, 85%), mp 230–232 ◦ C dioxane, IR (KBr, cm−1 ): 3428 (NH), 1687 (CO), 1632,
1574, 1531, 1487, 1440, 1402, 1366, 1286, 1193, 1152, 1059, 1011 cm−1
.
1 H NMR (DMSO-d6) 2.49 (s, 3H,
COCH3), 7.40–7.46 (m, 5H, Ar-H), 7.61 (d, J = 12.2 Hz, 2H, Ar-H), 8.18 (d, J = 12.2 Hz, 2H, Ar-H), 8.28 (s,
1H, =CH), 10.63 (s, 1H, NH). MS m/z (%) 467 (M+ +1, 20), 466 (M+ , 32), 295 (20), 262 (22), 214 (23), 169
(30), 152 (60), 125 (38), 104 (37), 103 (25), 96 (64), 77 (100). Anal. Calcd. for C19 H14 N6 SF3 OCl (466.88)
Calcd: C, 48.88; H, 3.02; N, 18.00. Found: C, 48.59; H, 3.28; N, 18.24%.
4.4.4. 4-[((E)-benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl (E)-N-(4-nitrophenyl)-
2-oxopropanehydrazonothioate (9d)
Reddish brown solid, yield (0.72 g, 60%), mp 206–208 ◦ C, dioxane, IR (KBr, cm−1 ): 3428(NH), 1689 (CO),
1631, 1589, 1511, 1442, 1405, 1336, 1282, 1187, 1152, 1111, 1011 cm−1
.
1 H NMR (DMSO-d6) 2.65 (s, 3H,
COCH3), 7.44–7.69 (m, 5H, Ar-H), 8.23 (d, J = 7.1 Hz, 2H, Ar H), 8.40 (d,J = 7.1 Hz, 2H, Ar-H), 9.14 (s,
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FARGHALY et al./Turk J Chem
1H, =CH), 10.76 (s, 1H, NH). MS m/z (%) 478 (M+ +1, 15), 477 (M+ , 21), 313 (16), 243 (23), 192 (14), 163
(19), 104 (59), 90 (53), 77 (100). Anal. Calcd. for C19 H14 N7 SF3 O3 (477.43) Calcd: C, 47.79; H, 2.95; N,
20.52. Found: C, 47.99; H, 2.73; N, 20.27%.
4.4.5. Ethyl (E)-2-[(4-(((E)-benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(2-phenylhydrazono)acetate (9e)
Orange solid, yield (0.98 g, 85%), mp 218–220 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3431 (NH), 1711
(CO), 1622, 1580, 1528, 1495, 1443, 1405, 1314, 1193, 1145, 1105, 1009 cm−1
.
1 H NMR (DMSO-d6) 1.33 (t,
J = 7.3 Hz, 3H, CH3), 4.38 (q, J = 7.3 Hz, 2H, CH2), 7.37–8.06 (m, 10H, Ar-H), 8.26 (s, 1H, =CH), 10.61 (s,
1H, NH). MS m/z (%) 463 (M+ +1, 4), 462 (M+ , 16), 135 (23), 118 (18), 104 (43), 96 (37), 91 (77), 77 (100),
69 (18). Anal. Calcd. for C20 H17 N6 SF3 O2 (462.46) Calcd: C, 51.94; H, 3.70; N, 18.17. C, 52.20; H, 3.99; N,
18.38%.
4.4.6. Ethyl (E)-2-[(4-(((E)-benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(2-(p-tolyl)hydrazono)acetate (9f)
Yellow solid, yield (0.89 g, 75%), mp 196–198 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ):, 3451 (NH), 1740
(CO), 1626, 1583, 1533, 1441, 1404, 1328, 1284, 1186, 1143, 1105, 1014 cm−1
.
1 H NMR (DMSO-d6) 1.30
(t, J = 7 Hz, 3H, CH3), 4.38 (q, J = 7 Hz, 2H, CH2), 7.35–7.44 (m, 5H, Ar-H), 7.61 (d, J = 8.0 Hz, 2H,
Ar-H), 7.88 (d, J = 8.0 Hz, 2H, Ar-H), 8.26 (s, 1H, =CH), 10.55 (s, 1H, NH). MS m/z (%) Anal. Calcd: for
C21 H19 N6 SF3 O2 (476.48) Calcd: C, 52.93; H, 4.01; N, 17.63. Found: C, 52.64; H, 4.31; N, 17.42%.
4.4.7. Ethyl (E)-2-[(4-(((E)-benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(2-(4-chlorophenyl)hydrazono)acetate (9g)
Yellow solid, yield (0.74 g, 60%), mp 164–266 ◦ C, dioxane/ethanol (3:1), IR (KBr, cm−1 ):, 3437 (NH), 1732
(C=O), 1630, 1584, 1536, 1486, 1442, 1402, 1283, 1190, 1145, 1095, 1010 cm−1 1 H NMR (DMSO-d6) 1.31 (t,
.
J = 7.2 Hz, 3H, CH3), 4.38 (q, J = 7.2 Hz, 2H, CH2), 7.40 (d, J = 8.0 Hz, 2H, Ar-H), 7.56–7.64 (m, 5H,
Ar-H), 8.08 (d, J = 8.0 Hz, 2H, Ar-H), 8.28 (s, 1H, =CH), 10.60 (s, 1H, NH). MS m/z (%) 498 (M+ +2, 7),
497 (M+ +1, 4), 496 (M+ , 18), 241 (5), 172 (11), 169 (29), 152 (19), 138 (19), 125 (100), 111 (34), 104 (47),
96 (93), 90 (61), 77 (99). Anal. Calcd. for C20 H16 N6 SF3 O2 Cl (496.90) Calcd: C, 48.34; H, 3.24; N, 16.91.
Found: C, 48.60; H, 3.44; N, 16.70%.
4.4.8. Ethyl (E)-2-[(4-(((E)-benzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl)thio)-2-
(2-(4-nitrophenyl)hydrazono)acetate (9h)
Reddish brown solid, yield (1.08 g, 86%), mp 150–152 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3412 (NH),
1698 (CO), 1602, 1511, 1476, 1341, 1269, 1205, 1159, 1084, 1015 cm−1
.
1 H NMR (DMSO-d6) 1.35 (t, J =
6.9 Hz, 3H, CH3), 4.16 (q, J = 6.9 Hz, 2H, CH2), 7.41 (d, J = 9.0 Hz, 2H, Ar-H), 7.60 (d, J = 9.0 Hz,
2H, Ar-H), 7.65–8.20 (m, 5H, Ar-H), 9.13 (s, 1H, =CH), 11.42 (s, 1H, NH). MS m/z (%) 508 (M+ +1, 2), 507
(M+ , 7), 250 (10), 136 (12), 103 (42), 96 (14), 90 (31), 77 (100), 69 (22). Anal. Calcd. for C20 H16 N7 SF3 O4
(507.45) Calcd: C, 47.33; H, 3.17; N, 19.32. Found: C, 47.60; H, 3.29; N, 19.58%.
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FARGHALY et al./Turk J Chem
4.4.9. 4-[((E)-3,4-Dimethoxybenzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl (E)-2-
oxo-N-phenylpropanehydrazonothioate (9i)
Orange solid, yield (0.98 g, 80%), mp 174–176 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3435 (NH), 1691
(CO), 1631, 1579, 1513, 1438, 1409, 1368, 1268, 1191, 1145, 1017 cm−1
.
1 H NMR (DMSO-d6) 2.49 (s, 3H,
COCH3), 3.76 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 6.99–8.12 (m, 8H, Ar-H), 8.16 (s, 1H, =CH), 10.47 (s, 1H,
NH). MS m/z (%) 493 (M+ +1, 0.3), 492 (M+ , 1), 273 (6), 163 (100), 148 (32), 137(10), 120 (21), 118 (11),
102 (11), 79 (26), 77 (44), 65 (18). Anal. Calcd. for C21 H19 N6 SF3 O3 (492.48) Calcd: C, 51.21; H, 3.88; N,
17.06. Found: C, 51.39; H, 4.02; N, 17.33%.
4.4.10. Ethyl (E)-2-[(4-(((E)-3,4-dimethoxybenzylidene)amino)-5-(trifluoromethyl)-4H-1,2,4-tri-
azol-3-yl)thio]-2-(2-phenylhydrazono)acetate (9j)
Yellow solid, yield (1.1 g, 85%), mp 170–172 ◦ C, dioxane/ethanol (3:1), IR (KBr, cm−1 ): 3432 (NH), 1733
(CO), 1636, 1581, 1518, 1439, 1406, 1273, 1190, 1136, 1018 cm−1
.
1 H NMR (DMSO-d6) 1.30 (t,J = 7.2 Hz,
3H, CH3), 3.76 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 4.35 (q, J = 7.2 Hz, 2H, CH2 ), 6.99–8.05 (m, 10H, Ar-H),
8.16 (s, 1H, =CH), 10.48 (s, 1H, NH). MS m/z (%) Anal. Calcd. for C22 H21 N6 SF3 O4 (522.51) Calcd: C,
50.57; H, 4.05; N, 16.08. Found: C, 50.34; H, 4.33; N, 16.30%.
4.4.11. 4-[((E)-3,4-dimethoxybenzylidene)amino]-5-(trifluoromethyl)-4H-1,2,4-triazol-3-yl (E)-N-
phenylbenzohydrazonothioate (9k)
Orange solid, yield (1.0 g, 78%), mp 192–194 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3436 (NH), 1622,
1591, 1511, 1440, 1411, 1329, 1264, 1197, 1134, 1020 cm−1
.
1 H NMR (DMSO-d6): 3.77 (s, 3H, OCH3), 3.78
(s, 3H, OCH3), 6.99–8.20 (m, 13H, Ar-H), 8.22 (s, 1H, =CH), 10.44 (s, 1H, NH). 13 C NMR (DMSO-d6): 55.2,
55.5, 108.4, 111.5, 112.0, 120.5, 121.3, 125.8, 126.0, 126.8, 128.8, 129.1, 129.2, 130.9, 139.0, 140.8, 143.8, 148.7,
150.0, 150.1, 163.4. MS m/z (%) 527 (M+ +1, 2), 526 (M+ , 70, 362 (8), 194 (53), 91 (100), 77 (33), 64 (15).
Anal. Calcd. for C25 H21 N6 SF3 O2 (526.54) Calcd: C, 57.02; H, 4.02; N, 15.96. Found: C, 57.32; H, 4.22; N,
15.67%.
4.4.12. Ethyl (E)-2-[(4-(((E)-(5-methylfuran-2-yl)methylene)amino)-5-(trifluoro-methyl)-4H-1,2,4-
triazol-3-yl)thio]-2-(2-phenylhydrazono)acetate (9l)
Orange solid, yield (0.94 g, 81%), mp 150–152 ◦ C, dioxane/ethanol (2:1), IR (KBr, cm−1 ): 3409 (NH), 1711
(CO), 1620, 1530, 1491, 1437, 1407, 1313, 1186, 1099, 1013 cm−1
.
1 H NMR (DMSO-d6) 1.30 (t,J = 7.0 Hz,
3H, CH3), 2.31 (s, 3H, CH3), 4.37 (q, J = 7.0 Hz, 2H, CH2), 6.21 (d, J = 2.3 Hz, 1H, CH), 6.69 (d, J = 2.3
Hz, 1H, CH), 7.40–8.04 (m, 5H, Ar-H), 8.07 (s, 1H, =CH), 10.40 (s, 1H, NH). MS m/z (%) 467 (M+ +1, 4), 466
(M+ , 18), 217 (22), 135 (16), 108 (23), 96 (15), 91 (100), 79 (19), 77 (31). Anal. Calcd. for C19 H17 N6 SF3 O3
(466.44) Calcd: C, 48.92; H, 3.67; N, 18.02. Found: C, 48.64; H, 3.95; N, 18.31%.
4.5. Synthesis of compound 12
A mixture of compound 1 (0.92 g, 5 mmol) and terephthaldehyde (2.5 mmol) was refluxed in glacial acetic
acid (30 mL) for 5 h. The solution was then cooled and the solid produced was filtered and recrystallized from
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FARGHALY et al./Turk J Chem
dioxane to give compound 12 as yellowish white solid, yield (1.29 g, 90%), mp 250 ◦ C, dioxane, IR (KBr,
cm−1 ): cm−1 1658 (2C=N), 1 H NMR (DMSO-d6): 7.97 (d, J = 8.0 Hz, 2H, Ar H), 8.06 (d, J = 8.0 Hz, 2H,
Ar H), 8.1 (s, 2H, 2=CH), 10.69 (s, 2H, 2SH). MS m/z (%) 467 (M+ +1, 4), 466 (M+ , 4), 300 (11), 170 (82),
132 (49), 130 (100), 104 (31), 102 (44), 89 (33), 77 (69), 69 (45). Anal. Calcd. for C14 H8 N8 S2 F6 (466.39)
Calcd: C, 36.05; H, 1.72; N, 24.02. Found: C, 36.25; H, 1.45; N, 24.32%.
4.6. Reaction of compound 12 with hydrazonoyl chlorides 2
To a mixture of 12 (1.17 g, 2.5 mmol) and the hydrazonoyl chloride 2 (5 mmol) in dioxane (40 mL) was added
triethylamine (0.7 mL), and the mixture was heated to reflux for 5 h, and then the reaction mixture was poured
on ice water and acidified with HCl. The solid produced was collected by filtration and crystallized from the
appropriate solvent to give the corresponding compounds 13a and b.
4.6.1. (13a)
Yellow solid, yield (2.3 g, 85%), mp 240–242 ◦ C, dioxane, IR (KBr, cm−1 ): 3431 (2NH), 1688 (2C=O) 1629
(2C=N), 1 H NMR (DMSO-d6): 2.49 (s, 6H, 2COCH3), 7.44–7.56 (m, 10H, Ar H), 7.59 (d, J = 7.8 Hz, 2H, Ar
H), 8.1 (d, J = 7.8 Hz, 2H, Ar H), 8.27 (s, 2H, 2=CH), 10.69 (s, 2H, 2NH). Anal. Calcd. for C32 H24 N12 S2 F6 O2
(786.75) Calcd: C, 48.85; H, 3.07; N, 21.36. Found: C, 48.65; H, 3.29; N, 21.25%.
4.6.2. (13b)
Orange solid, yield (2.4 g, 87%), mp 262–264 ◦ C, dioxane/ethanol (1:1), IR (KBr, cm−1 ): 3428 (NH), 1711
(C=O), 1623 (C=N), 1 H NMR (DMSO-d6) 1.32 (t, J = 7.0 Hz, 6H, 2CH3), 4.38 (q, J = 7.0 Hz, 2H, CH2),
7.42–8.05 (m, 14H, Ar H), 8.27 (s, 2H, 2=CH), 10.68 (s, 2H, 2NH). Anal. Calcd. for C34 H28 N12 S2 F6 O4
(846.80) Calcd: C, 48.22; H, 3.33; N, 19.85. Found: C, 48.40; H, 3.07; N, 20.01%.
4.7. Biological activity
4.7.1. Measurement of cytotoxicity by SRB assay
Potential cytotoxicity of the compounds was tested using the method of Skehan et al.30 Cells were plated in
a 96-multiwell plate (104 cells/well) for 24 h before treatment with the compounds to allow attachment of
cells to the wall of the plate. A single concentration of the compound under test (100 µg/mL) was added to
the cell monolayer triplicate wells prepared for each individual dose. Monolayer cells were incubated with the
compounds for 48 h at 37 ◦ C and in atmosphere of 5% CO2 .
After 48 h, cells were fixed, washed, and stained with sulfo-Rhodamine-B stain. Excess stain was washed
with acetic acid and attached stain was recovered with Tris EDTA buffer. Color intensity was measured in an
ELISA reader. The relation between inhibition % and drug concentration was calculated.30
968

FARGHALY et al./Turk J Chem
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