Synthesis of novel triazoles, tetrazine, thiadiazoles and their biological activities

Article abstract of DOI:10.3390/molecules20022591

An expedient synthesis of novel triazoles, tetrazine and thiadiazoles, using conveniently accessible and commercially available starting materials has been achieved. The synthesized compounds were characterized by spectroscopic and elemental analyses, and screened for their antibacterial activities against four different strains, namely E. coli, P. aeruginosa, S. aureus and B. megaterium. In particular, the compounds 5, 24 and 26h exhibited excellent antibacterial activities compared to the reference antibiotic. To get further insight about their behavior, these compounds were tested for their antioxidant activities via SOD-like activity, DPPH free radical scavenging activity, ABST and NO, which showed promising results. Furthermore, these compounds effectively promoted the cleavage of genomic DNA as well, in the absence of any external additives.

Full text of DOI:10.3390/molecules20022591

Molecules 2015, 20, 2591-2610; doi:10.3390/molecules20022591
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Novel Triazoles, Tetrazine, Thiadiazoles and Their
Biological Activities
Mohammed A. Al-Omair 1, Abdelwahed R. Sayed 1,2,* and Magdy M. Youssef 1,3
1
Department of Chemistry, Faculty of Science, King Faisal University, Hofuf 31982, Saudi Arabia;
E-Mails: alomair@kfu.edu.sa (M.A.A.-O.); mmm_youssef@yahoo.com (M.M.Y.)
2
Department of Chemistry, Faculty of Science, University of Beni Suef, Beni Suef 62511, Egypt
3
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
* Author to whom correspondence should be addressed; E-Mail: arsayed@kfu.edu.sa or
arashad_2003@yahoo.com; Tel.: +966-561-009-338.
Academic Editor: Roman Dembinski
Received: 1 December 2014 / Accepted: 23 January 2015 / Published: 2 February 2015
Abstract: An expedient synthesis of novel triazoles, tetrazine and thiadiazoles, using
conveniently accessible and commercially available starting materials has been achieved.
The synthesized compounds were characterized by spectroscopic and elemental analyses,
and screened for their antibacterial activities against four different strains, namely E. coli,
P. aeruginosa, S. aureus and B. megaterium. In particular, the compounds 5, 24 and 26h
exhibited excellent antibacterial activities compared to the reference antibiotic. To get
further insight about their behavior, these compounds were tested for their antioxidant
activities via SOD-like activity, DPPH free radical scavenging activity, ABST and NO,
which showed promising results. Furthermore, these compounds effectively promoted the
cleavage of genomic DNA as well, in the absence of any external additives.
Keywords: triazoles; tetrazine; thiadiazoles; antibacterial; antioxidant; DNA
1. Introduction
In recent years, heterocyclic compounds have received considerable attention due to their
significant importance in pharmacological and agricultural fields [1–7]. Notably, nitrogen-containing
heterocycles exhibit excellent biological activities [8]. Free radicals and reactive oxygen species such

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as hydroxyl radical, hydrogen peroxide and superoxide radical anion are frequently synthesized
through many biological processes and may be considered as indicators of biological inadequacy.
Reactive oxygen species are also capable of damaging essential biomolecules such as nucleic acids,
proteins, lipids, and carbohydrates and may cause DNA damage that can lead to mutations. Biological
systems use antioxidant molecules such as nitrogen-containing heterocycles to neutralize the excessive
levels of reactive oxygen and nitrogen species [9].
1,2,4-Triazole rings are typically planar 6π-electron aromatic systems and extensive research has
been carried out in this domain [10,11]. These represent one of the most bioactive classes of
compounds, which display diverse biological activities in the medicinal and agrochemical fields,
including anti-inflammatory [12,13], antifungal [14,15], herbicidal [16], antimicrobial [16,17],
antiparasitic [18], cytostatic [19], and brassinosteroid biosynthesis inhibitory activities [20].
Triazolopyrimidines possess a range of biological activities, including activities against Aspergillus
and Pencicillium species [21–26], and have been evaluated as bioregulator agents [27]. Tetrazines are
of considerable interest not only because of their inherent biological potential [28], but also because of
their value as building blocks in synthetic transformations. 1,2,3-Thiadiazoles have also been reported
to have significant agricultural applications [29,30] and antiviral activities [31].
Hydrazones are of great interest to researchers because of their diverse biological and
clinical applications. They have been reported to exhibit antimicrobial, anticonvulsant, analgesic,
anti-inflammatory, antiplatelet, antitubercular and antitumor activities [32–34]. Small heterocyclic
molecules that interact with DNA through recognition, binding, crosslinking or cleaving have gained
significant attention and are considered as promising area of research in the fields of chemistry,
biology and medicine. Such heterocyclic compounds are capable of hydrolyzing and manipulating
DNA, and potentially can be used as chemotherapeutic agents [35]. In the present study, our synthetic
investigations towards novel heterocyclic compounds, their biological evaluations and genomic DNA
degradation activities are described.
2. Results and Discussion
2.1. Chemistry
In continuation of our active research in an area of hydrazonoyl halides and their reactions [36–40],
a novel compound 4 was prepared by the reaction of salicylaldehyde (1) with phenylhydrazine (2) in
boiling ethanol to afford salicylaldehydephenylhydrazone (3, Scheme 1) [41]. The hydrazone 3 was
then treated with N-bromosuccinimide to give the desired 2-hydroxy-N-phenylbenzohydrazonoyl
bromide (4). Spectroscopic data and microanalytical analyses confirmed the structure of bromide 4.
Scheme 1. Synthesis of 2-hydroxy-N-phenylbenzohydrazonoyl bromide 4.

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Pleasingly, treatment of hydrazonoyl bromide 4 or 2-(2-phenylhydrazono)-2-bromo-1-(naphthalene-
2-yl)ethanone (6) with 2-cyano-3-methylthio-3-phenylaminoacrylonitrile (5) in the presence of
trimethylamine under reflux conditions proceeded smoothly to afford 1,2,4-triazole derivatives 9 and
10, respectively (Scheme 2). It is proposed that the reactions involve the loss of hydrogen bromide and
methanethiol during the course of reaction to provide the desired products 9 and 10. The compounds
1
were characterized by elemental analysis, spectral data (IR, MS, H-NMR spectra), which confirmed
the absence of sulfur atoms.
Scheme 2. Synthesis of triazoline derivatives 9 and 10.
After successful accomplishment of this reaction, a series of novel compounds 12–15 were prepared
via the reaction of hydrazonoyl bromide 4 with 2-thioxo or 2-methylthio derivatives 11a–d in boiling
chloroform, in the presence of triethylamine as a base catalyst until hydrogen sulfide or methanethiol
gas ceased to evolve (Scheme 3). After standard work-up, the crude reaction mixture showed single
spots on TLC for each reaction mixrture, from which the desired products 12–15 were isolated in
excellent yields and characterized.

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O
Ph
N
H
N
O
Ph
OH
H3CH2C
H
O
H3CH2C
N
O
NH
N
H3C
H3C
N
11a
SR
12
O
N
O
OH
NH
N
N
N
H3C
SR
H3C
N
11b
13
Br
H
N
N
OH
H
N
OH
4
SR
N
N
N
N
11c
N
14
O
NH2
SR
N
O
H
N
H3C
N
N
11d
N
OH
H3C
Where R = H or CH3
N
N
Ph
15
Scheme 3. Synthesis of triazolines 12–14 and tetrazine 15.
Treatment of hydrazonoyl bromide 4 with potassium thiocyanate (16) in hot ethanol provided
3-phenyl-5-(2-hydroxyphenyl)-1,3,4-thiadiazol-2(3H) imine (20) as sole isolated product via
elimination of hydrogen bromide, followed by cyclization (Scheme 4). In similar manner, the reaction
of hydrazonoyl bromide 4 with methyl hydrazinecarbodithioate (17) afforded the desired product 21
via elimination of hydrogen bromide and methanethiol.
Scheme 4. Synthesis of thiadiazoles 20 and 21.

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Also, the reaction of hydrazonoyl bromide 4 with methyl hydrogenphenylcarbonimidodithioate (22)
in EtOH/TEA gave thiadiazole 24 (Scheme 5), which was characterized by its elemental analysis and
spectral data.
Scheme 5. Synthesis of thiadiazole 24.
In addition to this, 1,5-diarylidenethiocarbonohydrazide derivatives 26a–h were prepared according
to the literature methods [42–44], which involve the treatment of thiocarbohydrazide 25 with
aldehydes in hot ethanol to afford the desired dihydrazones 26a–h (Scheme 6).
S
S
H
O
H2N
NH2
H
N
N
R
EtOH
N
N
N
N
2
R
H
H
H
H
R
H
25
26
R : a, 4-CH3OC6H4; b, 4-CH3C6H4; c, 2-OHC6H4; d, C6H5; e, 4-ClC6H4; f, 4-BrC6H4; g, 4-NO2C6H4;h, 2-Furanyl
Scheme 6. Synthesis of dihydrazones 26.
2.2. Biology
2.2.1. Antibacterial Activities
The antibacterial activity of the synthesized heterocyclic triazoles, tetrazine and thiadiazoles
compounds were tested against both Gram-negative and Gram-positive bacteria using their single
concentration in DMSO as a control. The inhibition zone against the growth of the verified bacteria for
the compounds is given in Table 1. From the results, it is evident that compounds 5, 24 and 26h
showed excellent antibacterial activity towards all the investigated bacterial strains compared to the
tetracycline used as a reference antibiotic. The other compounds 4, 14, 26a, 26b, 26f and 26c revealed
good antimicrobial activity towards the examined bacteria. The compounds 9, 12, 15, 20, 21, 26d and
26e displayed moderate levels of antimicrobial activity, while compound 26g offered the lowest
antibacterial activity. From the antibacterial assay data presented in Table 1, it seems that enhanced
biological activity for the compound 24 is due to its electron donating group (hydroxyl) and the
poly-conjugated nature of the compound. For compound 26h, the furan motif acts as an electron
donating group as well as a diene in the Diels-Alder reaction and delocalization of electrons through
the furan ring facilitates the electron density on the hydrazone structure [45]. The ortho-, meta- or
para-position of the electron donating groups in the aromatic system plays a significant role in the
bioactivities. In addition to this, the presence of heteroatoms such as oxygen, sulfur, and nitrogen also
play a vital role in the observed antibacterial activity. It is also suggested that the sulfur-containing

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compounds might inhibit enzyme synthesis, since enzymes need specific groups for their activity and
are especially susceptible to deactivation by the compounds. The presence of sulfur and nitrogen atoms
in the structure of the active compounds facilitates their diffusion through the lipid layer of the
microorganism membranes to the site of action, eventually killing them by linking with essential
groups of certain cell enzymes [46].
Table 1. Effect of compounds on Gram-negative and Gram-positive microorganisms.
Results expressed as inhibition zone diameter in mm.
Gram-Negative
E. coli P. aeruginosa
Gram-Positive
S. aureus B. megaterium
Compound No.
4
15
17
13
12
14
12
13
12
18
13
12
13
12
13
14
9
13
14
12
11
13
11
11
12
16
12
14
14
11
12
13
11
20
18
16
19
14
13
15
14
14
13
18
13
12
12
13
14
15
11
20
22
18
20
15
14
16
14
16
15
21
15
16
17
14
13
17
10
21
24
5
9
12
14
15
20
21
24
26a
26b
26c
26d
26e
26f
26g
26h
Tetracycline
21
20
2.2.2. Minimum Inhibitory Concentrations
The minimum concentration at which no growth was detected was taken as the MIC value. The
MIC of the compounds against Gram-negative and Gram-positive bacterial strains was evaluated.
Comparison of the MICs (in μg/mL) of the synthesized compounds and tetracycline used as a standard
drug against susceptible organisms is presented in Table 2. It was found that compounds 4, 5, 14, 24
and 26h showed the highest inhibitory activity against all measured strains (MIC values in the range of
15–35 μg/mL). Compounds 9, 12, 15, 20, 21, 26a, 26b, 26c, 26f and 26d had good inhibitory activity
against all tested strains (MIC range: 30–55 μg/mL). A plausible explanation for these results is that
the antibacterial activity of compounds may result from the basic skeleton of the molecules as well as
from the nature of the nitrogen and sulfur atoms and the presence of substituents such as NO2, Br, Cl,
OH, CH3 and OCH3 groups.

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Table 2. Minimum inhibitory concentration (μg/mL) of compounds against Gram-negative
and Gram-positive microorganisms.
Gram-Negative
Gram-Positive
Compound No.
E. coli
25
15
35
40
20
45
40
35
15
30
30
25
40
45
35
55
15
10
P. aeruginosa
S. aureus
B. megaterium
4
30
15
40
45
30
40
45
40
20
35
30
35
45
45
40
50
20
15
30
20
35
50
35
45
50
50
20
35
35
40
45
55
40
70
20
15
35
20
45
50
35
50
40
45
20
35
35
35
55
50
35
50
20
15
5
9
12
14
15
20
21
24
26a
26b
26c
26d
26e
26f
26g
26h
Tetracycline
2.2.3. DNA Cleavage Assay
The ability of the compounds to cleave genomic DNA was studied and compared to the controls
using the agarose gel electrophoresis technique. In the current study, the compounds displayed DNA
degradation effect which proves their binding aptitude to the genomic DNA (Figure 1). When DNA
was allowed to interact with compounds at 4 μg, DNA cleavage was noticed, as shown in Figure 1.
The result indicates that the compounds 5, 24 and 26h are able to perform an efficient cleavage of
DNA at 4 μg. Furthermore, the compounds 4, 14, 26a, 26b, 26f and 26c also demonstrated an efficient
cleavage of the genomic DNA as demonstrated by agarose gel electrophoresis. The investigational
observations revealed that the compounds have promising capability to degrade the genomic DNA
in vitro. The obtained results also indicate that the examined compounds at 4 μg can effectively digest
DNA. The weak DNA cleavage ability of the compounds 12, 15, 21, 26d, 26e and 26g may be
attributed to the difference in the pharmacokinetic properties of the substituents in these compounds.
These results suggest that the DNA cleavage activity depends on the particular structures of the studied
compounds. It was observed that replacing one or more of the electron donating groups such as
hydroxyl or furan ring results in an increase in the DNA cleavage affinity. The compounds thus may
have use as endonuclease mimics due to their interesting structural features. The present study
demonstrates that the compounds 5, 24 and 26h have a significant nuclease activity toward the
cleavage of genomic DNA in the absence of any external additives. The DNA-binding evaluations are
important for the rational strategy and construction of new and extra-efficient drugs targeting DNA.

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The DNA cleavage activity without any additive is an appreciated feature for these compounds, which
provides a potential application of these compounds as chemotherapeutic agents in anticancer treatments.
Figure 1. A figure showing the degradation effect of 4 μg of series (Lanes 3–19) on the
genomic DNA isolated from E. coli, Lane1 E. coli DNA and lane 2 E. coli DNA + DMSO.
2.2.4. Superoxide Dismutase Mimetic Catalytic Activity
As some of the synthesized compounds demonstrated good DNA binding affinity, we envisaged
•−
studying their antioxidant activity too. Superoxide anions (O2 ) are originators for dynamic free
radicals that have the potential to react with biological macromolecules thus inducing cell damage.
Superoxide anions have a short half-life and consequently, these are being constantly produced. In this
colorimetric based assay, the studies of O2•− dismutation were accomplished by a catalyst and PMS as
•−
O2 photogenerator at room temperature. Inhibition of the reduction of NBT to formazan by the
synthesized compounds was employed for recognition of the SOD-mimetic catalytic activity of these
compounds in a phosphate buffer comparable to a biological environment. As the reaction proceeds,
the formazan color is established and then it changes from colorless to blue, which is related with an
increase in the absorbance at 560 nm. SOD decreases the superoxide ion concentration and thereby
lowers the rate of formazan formation. In the SOD-like activity test, the compounds compete with
NBT for oxidation of the generated superoxide ions. The more efficient the compound, the lower the
concentration that corresponds to 50% inhibition of NBT reduction; this concentration is called the
IC50. The data in Table 3 present the scavenging efficiency of each compound, giving its final
concentration that produces efficient quenching of the superoxide anion radical. The compounds 26h,
5, 14 and 24 displayed a significant SOD-like activity and caused percent inhibitions of 53.3, 51.8,
50.4 and 50.2, respectively. In addition to this, compounds 26f, 4, 26a, 26c, 26b and 9 exhibited a
moderate SOD-like activity with the inhibition percentages of 46.4, 45.8, 44.3, 43.8, 42.7 and 40.5,
respectively. However, the compounds 20, 21, 26e, 12, 15, 26d and 26g displayed a weak SOD-like
activity with inhibition percentages of 39.4, 38.5, 34.7, 33.6, 30.5, 30.3, and 29.6, respectively.

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Table 3. Superoxide dismutase mimetic catalytic activity of compounds as antioxidant enzyme.
Compound No.
Δ through 4 min
0.548
% Inhibition
—
Control
Horseradish peroxidase
0.169
69.2
45.8
51.8
40.5
33.6
50.4
30.5
39.4
38.5
50.2
44.3
42.7
43.8
30.3
34.7
46.4
29.6
53.3
4
0.297
5
0.264
9
0.326
12
0.364
14
0.272
15
0.381
20
0.332
21
0.337
24
0.273
26a
26b
26c
26d
26e
26f
26g
26h
0.305
0.314
0.308
0.382
0.358
0.294
0.386
0.256
2.2.5. DPPH Free Radical Scavenging Activity
The DPPH• radical scavenging activity (RSA) evaluation is a typical assay used in antioxidant
activity studies that offers a rapid technique for screening the RSA of specific compounds. The
interaction of synthesized compounds with stable DPPH free radical indicates their free radical
scavenging ability. The compounds showed antiradical activity by inhibiting DPPH radical (Figure 2).
Majority of the tested compounds in the series revealed high to moderate interactions with the DPPH
radical at 2 μg/mL concentration. The maximum antioxidant activity was observed in compounds in
the following order 26h > 24 > 5, which displayed more than 50% inhibition, which is comparable to
that of the standard (vitamin C) at a similar concentration, as shown in Figure 2. The presence of either
electron-withdrawing or electron-donating groups in the aromatic rings may play a functional role in
the activity. When the electron-donating groups such as furan ring is introduced in the compounds, the
antioxidant activity is increasedm as reflected by compound 26h, but electron-withdrawing groups
such as NO2 or Cl decrease the activity, as observed for 26g or 26e, respectively. The other compounds
exhibited moderate RSA in the order 14 > 4 > 26f > 26a > 26c > 9 > 26b > 12 (Figure 2). The
antioxidant activity of these compounds is related with their electron delocalization or hydrogen
radical donating ability to DPPH radical.

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Figure 2. Antioxidant activities of compounds using DPPH.
2.2.6. ABTS Radical Cation Decolorization Assay
The antioxidant activity evaluation employed here is one of the numerous assays that depends on
determining the consumption of stable free radicals and used to assess the free radical scavenging
activity of the examined compound. The reduction in colour intensity of the free radical solution due to
scavenging of the free radical by the antioxidant material is determined colorimetrically at 734 nm.
The test employs the radical cation formed from ABTS as a stable free radical. The benefit of the
ABTS-derived free radical process over other approaches is that the produced colour remains stable for
more than 1 h and the reaction is stoichiometric. The antioxidant activities of seventeen newly
synthesized compounds were subjected to ABTS assessment (Figure 3). From these results, it is
concluded that compounds 26h > 24 > 5 > 4 exhibited more than 50% inhibition of the ABTS radical
cation. On the other hand, the compounds 14 > 21 > 26a > 26b > 20 > 26c > 26f > 9 > 15 showed
weak scavenging activities with lower than 50% inhibition of the ABTS (Figure 3).
Figure 3. Antioxidant activities of compounds using ABTS.

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2.2.7. Nitric Oxide (NO) Scavenging Assay
The compounds were also examined for antioxidant properties by the nitric oxide scavenging activity
assay (Figure 4). The compounds displayed nitric oxide radical scavenging activity in the following
order: 5 > 24 > 26h > 4 > 14 > 26b > 26f > 26a. The other evaluted compounds 26c, 21, 9, 12, 15 and
26d showed moderate antioxidant activity, whereas the compounds 26e and 26g displayed weak nitric
oxide scavenging properties compared to the control. Compounds that can neutralize the effects of NO
formation may be of interest for avoiding the severe effects of extreme NO generation in living
organisms. This study shows that the compounds are potent nitric oxide scavengers. Nitric oxide
generated from sodium nitroprusside reacts with oxygen to form nitrite, therefore, these compounds
may inhibit nitrite formation by competing with oxygen and reacting with nitric oxide. Nitric oxide is a
gassy signaling molecule known to have an important function in a variety of biological systems.
Nitric oxide free radicals are formed non-enzymatically and have a short lifetime, but cause harm to
most biomolecules, including proteins and DNA. Nitric oxide has a role in many of human diseases
such as inflammation and tumors.
Figure 4. Antioxidant activities of compounds using NO.
3. Experimental Section
3.1. General Information
All melting points were determined on an Electrothermal apparatus and are uncorrected. IR spectra
1
were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer. H-NMR spectra were
recorded in CDCl3 and (CD3)2SO solutions on a Varian Gemini 300 MHz spectrometer and chemical
shifts are expressed in δ units using TMS as an internal reference. Mass spectra were recorded on a
Shimadzu GC-MS QP 1000 EX instrument. Elemental analyses were carried out at the Microanalytical
Canter of Cairo University. The starting reagents 3 [41], 5 [47], 6 [48], 11a [49], 11b [50], 11c [51],
11d [52], 17 [53], 22 [54] and 27 [42–46] were prepared as previously described in the
respective references.

Molecules 2015, 20
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3.1.1. Synthesis of 2-Hydroxy-N-phenylbenzohydrazonoyl Bromide (4)
To a solution of N-bromosuccinimide (10 mmol) dissolved in CH2Cl2 (70 mL) at 0 °C was added
dimethyl sulfide (1.12 g, 18 mmol) and the reaction mixture was stirred for 15 min at 0 °C and then
kept in a deep freezer (−20 °C) overnight. To this solution was added the hydrazone (9 mmol) in
CH2Cl2 (20 mL) and the reaction mixture was left in the deep freezer overnight and then allowed to
warm to room temperature for 2 h. The progress of the reaction was monitored by TLC. The reaction
mixture was quenched with cold water. The contents were extracted with CH2Cl2 (40 mL), washed with
water (2 × 40 mL) and dried (MgSO4). The solvent was removed under reduced pressure and
crystallization from MeOH afforded the final product 2-hydroxy-N-phenylbenzohydrazonoyl bromide
(4). Green solid; yield (86%); mp 175 °C; IR: 3433 (OH), 3317 (NH) and 1623 (C=N) cm−1. 1H-NMR
(DMSO-d6): 6.94–8.17 (m, 9H, ArH), 10.25 (s, H, NH) and 10.46 (s, 1H, OH) ppm. MS m/z (%) = 291
(M+, 72); Anal. Found for C13H11BrN2O (291.14): C, 53.68; H, 3.84; N, 9.57; requires: C, 53.63; H,
3.81; N, 9.62%.
3.1.2. Synthesis of 2-(5-(2-Hydroxyphenyl)-2,4-diphenyl-2H-1,2,4-triazol-3(4H)-ylidene)malononitrile
(9) and 2-(3-(2-Naphthoyl-1,4-diphenyl-1H-1,2,4-triazol-5(4H)-ylidene)malononitrile (10)
To a mixture of 2-[(methylthio)-(phenylamino)methylene]malononitrile (5, 1.075 g, 0.005 mol) and
hydrazonoyl chloride 4 or 6 (0.005 mol) in ethanol (20 mL) was added triethylamine (0.7 mL, 0.005 mol)
and the mixture was refluxed till methane thiol ceased to evolve (6 h). The precipitate that was formed
was filtered off and crystallized from DMF/MeOH mixture to give the 1,2,4-triazole derivatives 9 and
10, respectively. The physical constants of the compounds prepared are listed below.
2-(5-(2-Hydroxyphenyl)-2,4-diphenyl-2H-1,2,4-triazol-3(4H)-ylidene)malononitrile (9). Pale green
1
solid (71%), mp 171 °C. IR (KBr) ν 3433 (OH) and 2258, 2206 (CN) cm−1; H-NMR (DMSO-d6):
7.01–8.32 (m, 14H, ArH) and 10.45 (s, 1H, OH) ppm. MS m/z (%) 377 (M+, 18), Anal. Found: C,
73.16; H, 3.98; N, 18.64%; C23H15N5O (377.13) requires: C, 73.20; H, 4.01; N, 18.56.
1,4-Diphenyl-3-naphthoyl-5-dicyanomethylene-1,2,4-triazole carboxylate (10). Yellow solid (50%),
1
mp 288 °C. IR (KBr) ν 2199, 2188 (CN) and 1690 (C=O) cm−1; H-NMR (DMSO-d6): 7.32–8.04
(m, 17H, ArH); MS m/z (%) 439 (M+, 10), Anal. Found: C, 76.49; H, 3.95; N, 15.91; C28H17N5O
(439.47) requires: C, 76.52; H, 3.90; N, 15.94%.
3.1.3. General Procedure for the Preparation of Bis([1,2,4]triazolo[4,3-a]pyrimidine) Derivatives
12,13), Bis([1,2,4]triazolo[4,5-a]benzimidazole) Derivative 14 and 3-(2-Hydroxyphenyl)-8-methyl-1-
phenyl-1H-pyrimido[1,2-b][1,2,4,5]tetrazine-6-(4H)-one (15)
A mixture of the thioxo or 2-methylthio derivatives 11a–d (10 mmol) and 2-hydroxy-N-
phenylbenzohydrazonoyl bromide (4, 1.455 g, 5 mmol) were refluxed in chloroform (20 mL) in the
presence of triethylamine (1.01 g, 1.39 mL, 10 mmol) till hydrogen sulfide or methane thiol ceased to
evolve (4–6 h) and then cooled. The cold reaction mixture was then poured onto cold petroleum ether
with stirring. The solid that precipitated was collected, washed with water several times, dried and
recrystallized from MeOH to give the final products 12–15.

Molecules 2015, 20
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Ethyl-1,5-dihydro-3-(2-hydroxyphenyl)-7-methyl-1,5-diphenyl-[1,2,4]triazolo[4,3-a]pyrimidine-6-
crboxylate (12). Pale yellow solid (82%), mp 206 °C. IR (KBr) ν 3425 (OH) and 1666 (C=O) cm−1;
1H-NMR (DMSO-d6): 1.23 (t, 3H, OCH2CH3), 2.41 (s, 3H, CH3), 4.14 (q, 2H, OCH2CH3); 5.37 (s,1H,
pyrimidine-1H); 6.99–8.12 (m, 14H, ArH) ppm; 10.44 (s, 1H, OH); MS m/z (%) 452 (M+, 17), Anal.
Found: C, 71.69; H, 5.29; N, 12.42; C27H24N4O3 (452.5) requires: C, 71.67; H, 5.35; N, 12.38%.
3-(2-Hydroxyphenyl)-7-methyl-1-phenyl-[1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (13). Yellow brown
solid (74%), mp 184 °C. IR (KBr) ν 3433 (OH), and 1680 (C=O) cm−1; 1H-NMR (DMSO-d6): 2.35 (s, 3H,
CH3), 6.16 (s, 1H, pyrimidine-H), 6.97–8.02 (m, 9H, ArH), 10.41 (s, 1H, OH); MS m/z (%) 318 (M+,
31), Anal. Found: C, 67.89; H, 4.48; N, 17.57; C18H14N4O2 (319.12) requires: C, 67.91; H, 4.43; N,
17.60%.
3-(2-Hydroxyphenyl)-1-phenyl-1,2,4]triazolo[4,5-a]benzimidazole (14). Brown solid (80%), mp
163 °C. IR (KBr) ν 3425 (OH) and 1605 (C=N) cm−1; 1H-NMR (DMSO-d6): 6.97–8.34 (m, 13H, ArH)
and 10.48 (s, 1H, OH) ppm; MS m/z (%) 326 (M+, 25), Anal. Found: C, 73.58; H, 4.38; N, 17.23;
C20H14N4O (326.35) requires: C, 73.61; H, 4.32; N, 17.17%.
3-(2-Hydroxyphenyl)-8-methyl-1-phenyl-1H-pyrimido[1,2-b][1,2,4]tetrazine-6(4H)-one (15). Pale green
solid (63%), mp 170 °C. IR (KBr) ν 3433 (OH), 3317 (NH), and 1690 (C=O) cm−1; 1H-NMR (DMSO-d6):
6.93–8.13 (m, 13H, Ar’s), 9.51 (s, 1H, NH) and 10.48 (s, 1H, OH); MS m/z (%) 333 (M+, 32), Anal.
Found: C, 64.81; H, 4.49; N, 21.06; C18H15N5O2 (333.34) requires: C, 64.86; H, 4.54; N, 21.01%.
3.1.4. Synthesis of 2-(4,5-Dihydro-5-imino-4-phenyl-1,3,4-thiadiazol-2-yl)phenol (20)
A mixture of 2-hydroxy-N-phenylbenzohydrazonoyl bromide (4, 2.90 g, 10 mmol) and the appropriate
of potassium thiocynate (16) in EtOH (20 mL) was boiled under reflux for 6 h. The cold reaction
mixture was then poured onto ice-cold hydrochloric acid with stirring. The solid that precipitated was
collected. The resulting solids filtered, washed with water several times and crystallized from MeOH
to give the final product 2-(4,5-dihydro-5-imino-4-phenyl-1,3,4-thiadiazol-2-yl)phenol (20). Yellow
1
brown solid (74%) mp 176 °C. IR (KBr) ν 3433 (OH) and 3115 (NH) cm−1; H-NMR (DMSO-d6):
7.03–8.69 (m, 10H, ArH and =NH) and 10.49 (s, 1H, OH); MS m/z (%) 269 (M+, 16), Anal. Found: C,
62.39; H, 4.14; N, 15.63; C14H11N3OS (269.32) requires: C, 62.43; H, 4.12; N, 15.60%.
3.1.5. Synthesis of 1-(2-Hydroxyphenyl-3-phenyl-1,3,4-thiadiazol-4,(3H)-ylidene)hydrazine (21) and
4,5-Dihydro-4-phenyl-5-(phenylimino)-1,3,4-thiadiazol-2-yl)phenol (24)
A mixture of 2-hydroxy-N-phenylbenzohydrazonoyl bromide (4, 2.90 g, 10 mmol) and methyl
hydrazinecarbodithioate (17, 10 mmol) or methyl hydrogenphenylcarbonimidodithioate (22, 1.83 g,
10 mmol) in EtOH (20 mL) was stirred in the presence of triethylamine (1.01 g, 1.39 mL, 10 mmol). The
resulting mixture was refluxed for 4 h, and then cooled. The cold reaction mixture was then poured onto
ice-cold water with stirring. The solid that precipitated was collected. The resulting solids were filtered,
washed with water several times and recrystallized from MeOH to give the final products 21 and 24.
1-(2-Hydroxyphenyl-3-phenyl-1,3,4-thiadiazol-4,(3H)-ylidene)hydrazine (21). Green solid (69%) mp
185 °C. IR (KBr) ν 3433 (OH) and 3190, 3171 (NH2) cm−1; 1H-NMR (DMSO-d6): 5.83 (s, 2H, NH2),

Molecules 2015, 20
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7.32–8.04 (m, 11H, ArH) and 10.43 (s, 1H, OH); MS m/z (%) 284 (M+, 29), Anal. Found: C, 59.23; H,
4.21; N, 19.64; C14H12N4OS (284.34) requires: C, 59.14; H, 4.25; N, 19.70%.
4,5-Dihydro-4-phenyl-5-(phenylimino)-1,3,4-thiadiazol-2-yl)phenol (24). Pale green solid (75%) mp
150 °C. IR (KBr) ν 3379 (OH) and 1617 (C=N) cm−1; 1H-NMR (DMSO-d6): 7.01–8.15 (m, 14H, ArH)
and 10.41 (s, 1H, OH); MS m/z (%) 345 (M+, 41), Anal. Found: C, 69.58; H, 4.34; N, 12.14;
C20H15N3OS (345.42) requires: C, 69.54; H, 4.38; N, 12.17%.
3.2. Biology
3.2.1. Antibacterial Activities
Bacterial strains utilized in the present work were obtained from the American Type Culture
Collection (ATCC; Manassas, VA, USA). The antimicrobial evaluation of triazoles, tetrazine and
thiadiazoles compounds were carried out via the cup diffusion procedure [55]. The antibacterial
activity was tested against the Gram-positive bacterial strains Staphylococcus aureus (S. aureus)
ATCC 25923 and Bacillus megaterium (B. megaterium) ATCC 14591 and the Gram-negative ones
Escherichia coli (E. coli) ATCC 25922 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853.
The studied compounds were dissolved in dimethylsulfoxide (DMSO) at a concentration 1 mg/mL.
The Luria-Bertani Agar (LBA) Medium (5 g yeast extract, 10 g bacto-tryptone, 10 g NaCl, and 20 g
agar in 1 Liter de-ionized water) was used for inoculation and bacterial growth. An aliquot of the
solution of the compounds equivalent to 100 μg was placed separately in cups cut in the agar. The
LBA plates were incubated at 37 °C for 24 h and the resulting inhibition zones were measured. From
the inhibition zone diameter data analysis, the antimicrobial activities of compounds against
Gram-positive and Gram-negative bacteria were estimated.
3.2.2. Minimum Inhibitory Concentrations (MIC)
Determination of MIC of compounds was completed according to the standard method summarized
by the CLSI/NCCLS norms [56]. In brief, to sterilized LB broth solution, several concentrations
(0.5–50 μg/mL) of the compounds in DMSO were added individually. The test cultures of Gram-negative
E. coli, P. aeruginosa and Gram-positive S. aureus, B. megaterium bacterial strains were selected for
the present study. All bacterial strains were grown in LB broth separately with 50 μL LB containing
approximately 5 × 104 colony forming units (CFU) of 18 h grown cultures of each microorganism to
be tested. Microorganisms were inoculated and the final optical density (OD) of the test solution was
kept as ≈0.5 and the compounds were incubated for 24 h at 37 °C. Respective blanks (culture and broth
alone) were conserved, accordingly. Following incubation, the turbidity of the growth medium was
measured at 600 nm. The concentration at which the solution (OD is 0.5) turns turbid, was measured as
the MIC of the test samples. Experiments were carried out in duplicate.
3.2.3. DNA Cleavage Assay
The DNA cleavage study of the compounds was performed as described by Youssef et al. [57]. In brief,
the compounds were dissolved in DMSO (0.5 mg/mL) and 4 μg was added to 2 µg of E. coli DNA. DNA

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only and DNA with DMSO were used as a control. The reactions were done for one hour at 37 °C. A
solution of 10% w/v ficol 400, 0.06% w/v bromophenol blue, and 0.5% w/v SDS was added to the reaction
mixtures before running the horizontal gel. The DNA was assessed via agarose gel electrophoresis
done using 1.0% agarose gel at a constant voltage of 80 V for 60 min in TEA (40 mM Tris-HCl pH
7.9, 5 mM sodium acetate, 1 mM EDTA) buffer. The agarose gels were stained with ethidium bromide
(0.5 µg/mL) and visualized through a UV transilluminator and photographed using a digital camera.
3.2.4. Superoxide Dismutase (SOD) Mimetic Catalytic Activity Assay
The SOD mimetic catalytic activity of compounds was evaluated by nitroblue tetrazolium/phenazine
methosulphate/reduced nicotinamide (NBT/PMS/NADH) to photogenerate a reproducible and
•−
continuous flux of O2 at pH = 8.3 (phosphate buffer). Reduction of NBT to blue formazan was
•−
employed as an indicator of O2 production and observed spectrophotometrically at 560 nm. PMS
(0.93 mM) was added to an aqueous solution of NBT (0.03 mM), NADH (0.47 mM), and phosphate
buffer (final volume of 2 mL). The reaction in blank samples and in the presence of compounds was
measured for 5 min. For comparison purposes, the activity of native horseradish superoxide dismutase
(HR SOD) was also measured. The percent of inhibition of free radical production from SOD mimetic
catalytic activity was considered by the following equation:
I% = [(ΔAcontrol − ΔAsample)/ΔAcontrol] × 100
where Acontrol is the absorbance of the control reaction (containing all reagents except the test
compound) and Asample is the absorbance of the test compound. Tests were carried at in triplicate.
3.2.5. 2,2,-Diphenyl-1-Picrylhydrazyl (DPPH) Free Radical Scavenging Activity Assay
The electron donation ability of compounds was evaluated from the reduction of a purple-colored stable
free radical DPPH into the yellow-colored diphenylpicryl hydrazine. When odd electrons in DPPH• are
removed the absorbance at 517 nm is reduced steadily due to the increase of non-radical DPPH forms.
As this electron becomes paired off in the presence of a free radical scavenger, the absorption
disappears and the resulting decolorization is stoichiometric with respect to the number of electrons
taken up. Briefly, 1 mL of compounds (1 μg) in DMSO/ ethanol (1:1 v:v) or standard (vitamin C) were
added to 4 mL of 0.004% (w/v) ethanol solution of DPPH and vortexes thoroughly. After a 30 min
incubation period at 30 °C, the absorbance was read against blank (DMSO/methanol 1:1 v:v) at 517 nm.
The percent of inhibition of free radical production from DPPH was considered by the following equation:
I% = [(Acontrol − Asample)/ Acontrol] × 100
where Acontrol is the absorbance of the control reaction (containing all reagents except the test
compound) and Asample is the absorbance of the test compound. Tests were carried out in triplicate.
3.2.6. 2,2'-Azino-bis-3-ethylbenzthiazoline-6-sulphonic Acid (ABTS) Radical Cation Assay
ABTS forms a relatively steady free radical, which decolorizes to its non-radical form. The analysis
of ABTS free radical scavenging activity was assessed spectrophotometrically according to the method
reported in [58]. A mixture of ABTS (1 mL, 0.1 g/100 mL) and MnO2 (1.5 mL, 25 mg/mL), prepared in

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phosphate buffer (PB: pH 7.4, 100 mM) was vortexed, centrifuged and decanted. Prior to the
evaluation, the resulting green-blue solution was diluted in PB to give an absorbance (Acontrol of ABTS
radical solution) at 734 nm of 0.600 in a 1 cm quartz cuvette and equilibrated to 30 °C, the temperature
at which all the estimates were done. Antioxidant activities of the verified compounds were assessed
using the radical cations of ABTS. The absorbance (Asample) was determined upon the addition of 2 g of
the compounds in DMSO/PB (1:1 v/v) to the ABTS mixture. Ascorbic acid was used as a standard
antioxidant (positive control). Blank samples were run by solvent without ABTS. The decrease in
absorbance is expressed as % inhibition which is calculated from the following formula:
(%) Inhibition = [(Acontrol − Asample)/Acontrol] × 100
where Acontrol is the absorbance of the control reaction (containing all reagents except the test
compound) and Asample is the absorbance of the test compound. All total antioxidant activity data are
the average of triplicate analyses.
3.2.7. Nitric Oxide (NO) Scavenging Assay
The nitric oxide scavenging activity procedure was carried out according to [59], whereby sodium
nitroprusside generates NO radicals, which interact with oxygen to produce nitrite ions. These can be
assessed using the Greiss reagent (0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, 2% H3PO4
and 1% sulfanilamide). Scavengers of nitric oxide participate with oxygen leading to less production of
nitrite ions. For the assessment, sodium nitroprusside (10 mM) in PB saline was mixed with
compounds (2 μg) and standard. The reaction mixtures were incubated at 25 °C for 150 min and after
that, 0.5 mL of Griess reagent [60] was added. The absorbance of the pink colour formed during
diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylenediamine
was read at 546 nm and referred to the absorbance of standard solutions of sodium nitrite treated in the
same way with Griess reagent. Nitric oxide scavenging activity was calculated by the following equation:
% Nitric oxide scavenging activity = [(Acontrol − Asample)/Acontrol] × 100
where Acontrol is the absorbance of the control reaction (containing all reagents except the test
compound) and Asample is the absorbance of the test compound. All total antioxidant activity data are
the average of triplicate analyses.
4. Conclusions
In conclusion, a facile synthesis of novel bioactive heterocycles such as triazoles, tetrazine and
thiadiazoles is reported. The compounds were evaluated for their biological properties and gratifyingly,
the compounds 5, 24 and 26h exhibited excellent antibacterial and antioxidant activities. These compounds
also effectively promoted the cleavage of genomic DNA in the absence of external additives, which
suggests a potential application of these compounds as chemotherapeutic agents in anticancer treatment.
Acknowledgments
The financial support by the Deanship of Scientific Research (Project Number 130158) King Faisal
University, Saudi Arabia is gratefully acknowledged.

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Author Contributions
Abdelwahed Sayed planned to synthesized the Novel Triazoles, Tetrazine, Thiadiazoles derivatives.
Magdy Youssef planned and carried out all biological experiments. Abdelwahed Sayed and
Magdy Youssef performed the statistical analyses and drafted the manuscript with assistance from
Mohammed Al-Omair.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3,4,9,10, 12–15, 20 and 24 are available from the authors.
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