Synthesis, antioxidant and antitumor activities of some of new cyclobutane containing triazoles derivatives

Article abstract of DOI:10.1080/10426507.2019.1597363

Thiosemicarbazides (2a–e) were obtained by the interaction of furan-2-carboxylic acid hydrazide (1) with five different isothiocyanate (RNCS) derivatives. By addition of KOH to the reaction medium, ethyl, allyl, phenyl and benzyl, p-tolyl substituted 1,2,4-triazoles (3a–e) were obtained. 3a–e were dissolved in dry acetone containing K₂CO₃ in the presence of 2-chloro-1-(3-methyl-3-mesitylcyclobutyl) ethanone (4) to give 3,4,5-trisubstituted 1,2,4-triazole sulfanyl compounds containing a cyclobutane ring (5a–e). The structures of the final compounds were confirmed by elemental analyses, FT-IR, ¹H-NMR and ¹³C-NMR. The antioxidant and antitumor properties of the synthesized compounds were also investigated. Three of the triazole derivatives with p-tolyl, benzyl and phenyl substituents (5c–e) displayed good antioxidant and antitumor activity in comparison to the standards.

Full text of DOI:10.1080/10426507.2019.1597363

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: https://www.tandfonline.com/loi/gpss20
Synthesis, antioxidant and antitumor activities
of some of new cyclobutane containing triazoles
derivatives
Pelin Koparir
To cite this article: Pelin Koparir (2019): Synthesis, antioxidant and antitumor activities of some of
new cyclobutane containing triazoles derivatives, Phosphorus, Sulfur, and Silicon and the Related
Elements, DOI: 10.1080/10426507.2019.1597363
To link to this article: https://doi.org/10.1080/10426507.2019.1597363
View supplementary material
Published online: 02 Apr 2019.
Submit your article to this journal
Article views: 25
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=gpss20

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2019.1597363
Synthesis, antioxidant and antitumor activities of some of new cyclobutane
containing triazoles derivatives
Pelin Koparir
Department of Chemistry, Forensic Medicine Institute, Malatya, Turkey
ABSTRACT
ARTICLE HISTORY
Received 7 November 2018
Accepted 17 March 2019
Thiosemicarbazides (2a–e) were obtained by the interaction of furan-2-carboxylic acid hydrazide
(1) with five different isothiocyanate (RNCS) derivatives. By addition of KOH to the reaction
medium, ethyl, allyl, phenyl and benzyl, p-tolyl substituted 1,2,4-triazoles (3a–e) were obtained.
3a–e were dissolved in dry acetone containing K2CO3 in the presence of 2-chloro-1-(3-methyl-3-
mesitylcyclobutyl) ethanone (4) to give 3,4,5-trisubstituted 1,2,4-triazole sulfanyl compounds con-
taining a cyclobutane ring (5a–e). The structures of the final compounds were confirmed by elem-
ental analyses, FT-IR, 1H-NMR and 13C-NMR. The antioxidant and antitumor properties of the
synthesized compounds were also investigated. Three of the triazole derivatives with p-tolyl, ben-
zyl and phenyl substituents (5c–e) displayed good antioxidant and antitumor activity in compari-
son to the standards.
KEYWORDS
1,2,4-Triazole; antioxidant
activity; antitumor activity
GRAPHICAL ABSTRACT
1. Introduction
triazoles derivatives (5a–e) as anticancer and antioxi-
dant agents.
Compounds containing five membered heterocyclic rings
such as triazoles, oxadiazoles, thiadiazoles and imidazoles
have been synthesized in recent years due to their differ- 2. Results and discussion
ent biological activities such as antimicrobial, anti-inflam-
The 3,4,5-trisubstituted 1,2,4-triazoles obtained in the first
matory, analgesic, antitumor, antihypertensive or antiviral
properties. In addition, some current drugs such as
Rizatriptan (antimigraine agent), Itraconazole and
Fluconazole (antifungal agents), Ribavirin (antiviral agent),
Alprazolam (anxiolytic agent) are some potent molecules
containing a triazole ring.[1–8] It is well known that cyclo-
butane derivatives have anti-oxidant and anti-cancer activ-
ities.[9,10] Compounds containing a furan unit have been
reported to exhibit broad spectrum bioactivities.[11–13] For
this purpose, heterocyclic compounds such as cyclobutane
derivatives, 1,2,4-triazoles, and furan rings have shown
satisfactory antioxidant and cytotoxicity. It seems that
hybridization of these skeletons would also create more
effective agents. In this work, we focused on the design,
step in this work are reacted with 2-chloro-1-(3-methyl-3-
mesityl-cyclobutyl) ethanone in dry acetone containing
K2CO3 to obtain the 4,5-trisubstituted 1,2,4-triazole sulfanyl
compounds. 3,4,5-trisubstituted 1,2,4-triazoles reacted with
2-chloro-1-(3-methyl-3-mesityl-cyclobutyl)-ethanone in the
presence of K2CO3 via the SN2 reaction mechanism to yield
the sulfanyl compounds containing the triazole and cyclobu-
tane ring. Efficient yields range from 62% to 81%. When the
IR spectra of 5a–e in the 3,4,5-tri substituted 1,2,4-triazole
sulfanyl compounds are examined, the most characteristic
peak is the C ¼ O peak. The C ¼ O stretching vibration
occurs in the range of 1700–1710 cmꢀ1. The C-S-C strain
peculiar to the structure is around 720 cmꢀ1. Around
2950–3100 cmꢀ1 Ar-H, around 2860–2920 cmꢀ1 C-H,
synthesis, and evaluation of new cyclobutane containing around 1160 cmꢀ1 C-C peaks for the cyclobutane ring and
CONTACT Pelin Koparir
mpelin23@hotmail.com
Department of Chemistry, Forensic Medicine Institute, Malatya, TR-44000, Turkey.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
Supplemental data for this article can be accessed on the publisher website.
ß 2019 Taylor & Francis Group, LLC

2
P. KOPARIR
C ¼ C bands for the mesityl ring at 1600 cmꢀ1 derived compounds have been reported to reduce MDA lev-
els.[25] In another study, some Schiff base derivatives except
were obtained.
triazole were found to reduce MDA.[26]
When the proton NMR spectrum is examined, one of the
Table S4 shows changes in vitamin A and E levels of
Saccharomyces cerevisiae cells treated with the substances.
As it can be seen from the Tables, it appears that there is
a statistically reduced level of vitamins A and E. The free
radical scavenging activities of the compounds containing
furan and cyclobutane groups were examined by the
DPPH reduction method. The reduction ability of DPPH
radicals was determined by the decrease in absorbance at
517 nm induced by antioxidants and calculated percent
inhibition values. The findings are given in Table S5.
From the data presented in the Table, it was determined
that 5d and 5e of the test compounds exhibited the high-
est radical scavenging activities which were better than the
reference antioxidant compound. On the other hand, the
compound 5c showed good activities which were similar
to reference antioxidant compound. Of all the synthesized
derivatives compounds 5c, 5d and 5e with phenyl, benzyl
and p-tolyl group showed the best DPPH radical scaveng-
ing activity. Antioxidants are thought to be due to their
hydrogen donor ability.[27] DPPH is a stable free radical
and accepts an electron or hydrogen radical to become a
stable diamagnetic molecule.[28] A similar correlation was
observed for antitumor activity. All compounds greatly
improved their activity compared to precursors. Therefore,
the significant antioxidant and antitumor activity of com-
pounds may be due to the presence of 1,2,4-triazole rings
with cyclobutane and furan moiety in addition to phenyl,
benzyl and p-tolyl group. This biological activity results
agree with the results reported in literature, take into
characteristic peaks in the 3,4,5-trisubstituted 1,2,4-triazole
sulfanyl is signaled by nine -CH3 protons in the mesityl
group. This signal appeared as a singlet at approximately
2.14 ppm and was found to be equivalent to nine hydrogen
signals. The signal of -CH3 in the cyclobutane ring appeared
as a singlet at about 1.50 ppm. Four hydrogens in the cyclo-
butane ring were distinctly resonant as -cis and -trans.
These four protons occurred as
a
multiplet at
2.45–2.55 ppm. Data for all compounds are given in the
Experimental section.
Organic compounds containing five-membered aromatic
and heterocyclic rings are found in many natural struc-
tures, but play a major role in biochemical events. As a
result of these, heterocyclic rings have created a very
important and new field of study for chemists. Now a
days, the heterocyclic chemistry brings reagents and syn-
thetic methods of its own usual activity in synthesis of
drugs, antioxidant and anticancer agents as well as into
the correlated fields such as biochemistry, polymers and
material sciences.[14–18]
In this study, it was determined that antitumor activity of
furan and cyclobutane groups was changed in duration and
dose dependent. Statistically significant differences were
observed between the control group and the other groups at
30 and 60 lM concentrations and at 24 and 48 hours (Table
S1 and Table S2 Supplemental Materials). According to the
results obtained in the One-way ANOVA multi variance
Tukeyꢀs test in SPSS statistical program, it can be said that
the compounds containing furan and cyclobutane groups
have antitumor activity.
1,2,4-triazole,
phenyl,
benzyl
and
p-tolyl
the
groups.[21,29–31]
When the studies in the literature are examined, it is
seen that there are studies consistent with our results. For
example, in one study, some anti-cancer activities of 1,2,4
triazole compounds have been investigated and these com-
pounds have been reported to show anticancer activity
against kidney, leukemia, colon and breast cancers.[19]
Some studies in the literature[20–23] results are also con-
sistent with our study. As a result of our study, it is import-
ant that 5c–e of test compounds show activity close
to cisplatin.
This is because these electron donating substituents
may have increased the electron density of the ring, result-
ing in a more potent activity, resulting in more antitumor
and antioxidant activities than the other compounds. In
the literature, the methods used to determine the antioxi-
dant or antitumor properties are different, so comparison
may not give accurate information. In addition, the com-
pounds examined in the literature are different; it is not
possible to make a complete comparison. On the other
hand, in the literature, there are many studies showing
that the compounds in the 1,2,4 triazole structure that
support this study have antioxidant and antitumor activ-
ities.[21,29–31] In this regard, the necessity of building-activ-
ity studies has also emerged. This field will be planned for
our future work.
In this study, it was also found that the effects of com-
pounds containing furan and cyclobutane groups on the
MDA (Malondialdehyde) levels applied to Saccharomyces
cerevisiae cells had different effects in comparison with the
control group. Statistical differences in MDA levels were
observed between the control group and the test com-
pounds. Table S3 shows there is a statistically significant dif-
ference in MDA levels among all groups of substances. It
was found that 5c–e of the test compounds decreased the
MDA level compared to the control. According to the litera-
ture, studies on lipid peroxidation partially support the
results of this study. For example, in one study, inhibitory
activity of two types of diphenyl alkyl piperazine derivative
compounds against lipid peroxidation after oxidative dam-
3. Experimental
3.1. Materials
The hydrogen nuclear magnetic resonance (1H-NMR) and
carbon-13 nuclear magnetic resonance (13C-NMR) spectra
were taken on Bruker Ascend 400 NMR spectrometer oper-
1
ating at 400 MHz for H and 100 MHz for 13C NMR com-
age has been reported.[24] In another study, 1,2,4 triazole- pound were dissolved in deuterated dimethyl sulfoxide

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Figure 1. The synthesis route of the compounds 1-5.
(DMSO-d6). Elemental analyses were done on a LECO- C24H29N3O2S Calculated: C, 68.05; H, 6.90; N, 9.92; S, 7.57.
CHNS-938 and chemicals were purchased from Merck. The
reactions for the synthesis are shown in Figure 1.
Found: C, 68.15; H, 6.71; N, 9.85; S, 7.65.
Thiosemicarbazides compounds (2a–e), 4-(aryl-alkyl)-5-
3.2.2. 1-(3-Methyl-3-mesityl)-cyclobutyl-2–f[5-(furan-2-yl)-
4-allyl-4H-[1,2,4]triazol-3-yl]sulfanylg-ethanone (5b)
(2-furanyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(3a–e)
and 2-chloro-1-(3-Methyl-3-mesityl-cyclobutyl)-ethanone (4)
were synthesized according to the general procedure
described earlier.[32,33] The Supplemental Materials file con-
White solid (77%) mp. 115–117 ꢁC; FT-IR (KBr, cmꢀ1) t:
2950–3035 (Ar-H), 2862–2914 (C-H), 1709 (C ¼ O), 1475
(C ¼ N), 1132 (N-N), 1014 (C-S), 760 (C-S-C); 1H NMR
(400 MHz, DMSO-d6, d, ppm): 1.49 (s, 3H, -CH3 (cyclobu-
tane)), 2.14 (s, 9H, -CH3 (mesityl)), 2.38–2.53 (m, 4H,
-CH2- (cyclobutane)), 3.56 (p, 1H, -CH- (cyclobutane),
J ¼ 8.78 Hz) , 4.30 (s, 2H, S-CH2-), 4.78–4.86 (m, 2H, -N-
CH2-CH-CH2), ve 1H, -N-CH2-CH-CH2(trans)), 5.24 (d,
1H, N-CH2-CH-CH2, Jcis ¼ 10.6 Hz), 5.97–6.06 (m, 1H, N-
CH2-CH-CH2), 6.70 (s, 2H, Ar-H (mesityl)), 6.73 (dd, 1H,
Ar-H, J ¼ 3.5 Hz, J ¼ 1.8 Hz), 7.07 (d, 1H, Ar-H, J ¼ 3.5 Hz)
7.96 (d, 1H, Ar-H, J ¼ 1.8 Hz), 13C NMR (100 MHz, DMSO-
d6, d, ppm): 20.5, 21.4, 25.2, 39.1, 41.4, 46.9, 117.4, 127.9,
130.5, 132.5, 134.9, 143.7, 150.5, 151.2, 205.3. Elemental ana-
lysis: C25H29N3O2S Calculated: C, 68.93; H, 6.71; N, 9.65; S,
7.36. Found: C, 68.90; H, 6.75; N, 9.60; S, 7.30.
1
tains sample H and 13C NMR spectra for products 5a – 53
(Figures S1–S10).
3.2. General synthesis of 1-(3-Methyl-3-mesityl)-
cyclobutyl-2-(5-furan-2yl-4-(aryl-alkyl)-4H-[1,2,4]triazol-
3-ylsulfanyl)-ethanone (5a-e)
A solution of 0.445 g (2 mmol) of 2-chloro-1-(3-Methyl-3-
mesityl-cyclobutyl)-ethanone (4) was dissolved in dry acet-
one (30 mL) containing (2 mmol) K2CO3. To this solution,
(2 mmol) of 4-(aryl-alkyl)-5-(2-furanyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (3a–e) in dry acetone (20 mL) was
added dropwise over a 1 h period at room temperature with
stirring. The resulting solid was collected by filtration, dried
and recrystallized from ethyl alcohol.
3.2.3. 1-(3-Methyl-3-mesityl)-cyclobutyl-2–f[5-(furan-2-yl)-
4-phenyl-4H-[1,2,4]triazol-3-yl]sulfanylg-ethanone (5c)
White solid (65%) mp. 172 ꢁC; FT-IR (KBr, cmꢀ1) t:
2953–3117 (Ar-H), 2862–2937 (C-H), 1703 (C ¼ O), 1499
(C ¼ N), 1125 (N-N), 1072 (C-S), 773 (C-O-C); 1H NMR
(400 MHz, DMSO-d6, d, ppm): 1.50 (s, 3H, -CH3 (cyclobu-
tane)), 2.14 (s, 9H, -CH3 (mesityl)), 2.47–2.52 (m, 4H,
-CH2- (cyclobutane)), 3.56 (p, 1H, -CH- (cyclobutane),
J ¼ 9.10 Hz) , 4.26 (s, 2H, S-CH2-), 6.14 (d, 1H, Ar-H,
J ¼ 3.2 Hz), 6.51 (dd, 1H, Ar-H, J ¼ 3.2 Hz J ¼ 1.5 Hz), 6.71
(s, 2H, Ar-H, (mesityl)), 7.47–7.49 (m, 2H, Ar-H), 7.59–7.63
(m, 3H, Ar-H), 7.76 (d, 1H, Ar-H, J ¼ 1.5 Hz) 13C NMR
(100 MHz, DMSO-d6, d, ppm): 20.5, 21.4, 25.2, 39.0, 40.9,
111.6, 112.1, 128.0, 130.5, 131.0, 133.8, 134.4, 134.9, 141.4,
143.7, 145.3, 147.7, 151.6, 205.3. Elemental analysis:
C28H29N3O2S Calculated: C, 71.31; H, 6.20; N, 8.91; S, 6.80.
3.2.1. 1-(3-Methyl-3-mesityl)-cyclobutyl-2-f[5-(furan-2-yl)-
4-
ethyl-4H-[1,2,4]triazol-3-yl]sulfanylg-ethanone (5a)
White solid (62%) mp. 108–110 ꢁC; FT-IR (KBr, cmꢀ1) t:
2975–3145 (Ar-H), 2911–2956 (C-H), 1705 (C ¼ O), 1488
1
(C ¼ N), 1226 (C-O-C), 1129 (N-N), 1001 (C-S); H NMR
(400 MHz, DMSO-d6, d, ppm): 1.29 (t, 3H, N-CH2-CH3,
J ¼ 7.2 Hz), 1.49 (s, 3H, -CH3 (cyclobutane)), 2.14 (s, 9H,
-CH3 (mesityl)), 2.47–2.51 (m, 4H, -CH2- (cyclobutane)),
3.57 (p, 1H, -CH- (cyclobutane), J ¼ 9.10 Hz) , 4.16 (q, 2H,
N-CH2-CH3, J ¼ 7.1 Hz), 4.31 (s, 2H, S-CH2-), 6.70 (s, 2H,
Ar-H (mesityl)), 6.73 (dd, 1H, Ar-H, J ¼ 3.3 Hz, J ¼ 1.5 Hz),
7.07 (d, 1H, Ar-H, J ¼ 3.3 Hz) 7.96 (d, 1H, Ar-H, J ¼ 1.5 Hz),
13C NMR (100 MHz, DMSO-d6, d, ppm): 15.5, 20.5, 21.4,
25.2, 39.1, 41.4, 111.9, 112.4, 130.5, 134.4, 134.9, 141.8,
143.7, 145.5, 147.3, 150.6, 205.3. Elemental analysis: Found: C, 71.51; H, 6.45; N, 8.75; S, 6.89.

4
P. KOPARIR
3.2.4. 1-(3-Methyl-3-mesityl)-cyclobutyl-2–f[5-(furan-2-yl)-
4-benzyl-4H-[1,2,4]triazol-3-yl]sulfanylg-ethanone (5d)
White solid (81%) mp. 152–154 ꢁC; FT-IR (KBr, cmꢀ1) t:
2950–3100 (Ar-H), 2860–2910 (C-H), 1705 (C ¼ O), 1465
(C ¼ N), 1125 (N-N), 1024 (C-S), 777 (C-O-C); 1H NMR
(400 MHz, DMSO-d6, d, ppm): 1.48 (s, 3H, -CH3 (cyclobu-
tane)), 2.13 (s, 9H, -CH3 (mesityl)), 2.45–2.55 (m, 4H,
-CH2- (cyclobutane)), 3.54 (p, 1H, -CH- (cyclobutane),
J ¼ 9.05 Hz) , 4.28 (s, 2H, S-CH2-), 5.43 (s, 2H, N-CH2-
C6H5), 6.12 (d, 1H, Ar-H, J ¼ 3.5 Hz), 6.51 (dd, 1H, Ar-H,
J ¼ 3.5 Hz J ¼ 1.8 Hz), 6.70 (s, 2H, Ar-H), 7.00–7.04 (m, 2H,
Ar-H), 7.26–7.34 (m, 3H, Ar-H), 7.77 (d, 1H, Ar-H,
J ¼ 1.8 Hz) 13C NMR (100 MHz, DMSO-d6, d, ppm): 20.5,
21.4, 25.2, 39.0, 41.4, 47.9, 126.5, 127.9, 128.3, 128.6, 129.4,
129.6, 130.5, 134.4, 134.9, 135.6, 143.7, 150.4, 151.7, 205.3.
Elemental analysis: C29H31N3O2S Calculated: C, 71.72; H,
6.43; N, 8.65; S, 6.60. Found: C, 71.60; H, 6.50; N, 8.70;
S, 6.65.
was replaced with newly-prepared DC5. This process was
repeated at intervals of three days. The number of the cells
increased and began to form layers on top of each other by
completely covering the bottom of the flask. At the end of
the 15th day, the medium in the flask was removed and
replaced with 3 mL of trypsin. The flasks were shaken
slightly in 2-3 min intervals to allow the cells to separate
from the adhered surface. After all cells were separated from
the surface of the flask, 12 mL of DC5 was added to the
flask and trituration, dissociation by drawing the suspension
into the pipette, was carefully carried out to separate the
cells into the solution homogeneously. Cells were counted
using a hemocytometer. A cell suspension with 1x105 cells
was added to each flask and DC5 was added (total volume
25 mL), and then all flasks were placed in the incubator.
Cultivation, feeding and experiments of the cells were per-
formed in
a
sterile Class II Laminair Flow
(Biolaf, Ankara).[34,35]
3.5. The determination of cell numbers and drug dosage
3.2.5. 1-(3-Methyl-3-mesityl)-cyclobutyl-2–f[5-(furan-2-yl)-
4-p-tolyl-4H-[1,2,4]triazol-3-yl]sulfanylg-ethanone (5e)
White solid (73%) m. p. 168–170 ꢁC; FT-IR (KBr, cmꢀ1) t:
2961–3147 (Ar-H), 2863–2931 (C-H), 1709 (C ¼ O), 1476
(C ¼ N), 1125 (N-N), 1019 (C-S), 742 (C-O-C); 1H NMR
(400 MHz, DMSO-d6, d, ppm): 1.49 (s, 3H, -CH3 (cyclobu-
tane)), 2.14 (s, 9H, -CH3 (mesityl)), 2.42 (s, 3H, C6H5-CH3),
2.47–2.53 (m, 4H, -CH2- (cyclobutane)), 3.55 (p, 1H, -CH-
(cyclobutane), J ¼ 9.20 Hz) , 4.26 (s, 2H, S-CH2-), 6.13 (d,
1H, Ar-H, J ¼ 3.4 Hz), 6.51 (dd, 1H, Ar-H, J ¼ 3.2 Hz
J ¼ 1.6 Hz) 6.70 (s, 2H, Ar-H), 7.34 (d, 2H, Ar-H, J ¼ 8.2 Hz,
(p-tolyl)), 7.41 (d, 2H, Ar-H, J ¼ 8.2 Hz, (p-tolyl)), 7.77 (d,
1H, Ar-H, J ¼ 1.6 Hz). 13C NMR (100 MHz, DMSO-d6, d,
ppm): 20.5, 21.3, 21.4, 25.2, 39.0, 40.9, 111.6, 112.1, 127.5,
130.5, 131.1, 134.4, 134.9, 140.8, 141.4, 143.7, 145.3, 147.8,
151.8, 205.3. Elemental analysis: C29H31N3O2S Calculated: C,
71.72; H, 6.43; N, 8.65; S, 6.60. Found: C, 71.80; H, 6.40; N,
8.61; S, 6.53.
The L 1210 leukemia cell suspension was centrifuged at
2000 rpm for 5 min. Cells were counted using the hemocyt-
ometer and the number of cells was adjusted to 1x105 cells/
mL for L 1210 cell assays. Preliminary experiments were done
to determine the doses and the more or less method was used.
One mL of cell suspension was transferred to each test tube
and the test agents (test groups) were added at concentrations
of 30, 60 lM. Same amount of serum physiological was added
to the negative control tubes and same amount of DMSO (for
control group) was added to the Vehicle tubes and then all
tubes were placed in the incubator. The amount of DMSO in
cell suspensions was not more than 1%. After 24 h, the tube
was removed from the incubator and triturated and then the
cell suspension was counted in 100 cell hemocytometer ran-
domly selected at 1:1 (v/v) with 0.4% tryphan blue. Cell viabil-
ity was expressed as a percentage. GI50 is the concentration
for 50% of maximal inhibition of cell proliferation, GI50 value
is calculated. The same procedure was repeated after 48 h and
the experiment was ended.[36]
3.3. Bioactivity of Synthesized Compounds
3.3.1. Antitumor Activity
3.6. In vitro antioxidant activity measurements
3.3.1.1. Cells used in the experiment. L 1210 cellular cul-
ture (rodent leukemia cell) was used to determine antitu-
mor activity.
3.6.1. Measurement of Saccharomyces cerevisiae
specimens
Saccharomyces cerevisiae yeast cells used for identification of
antioxidant activity is a single-celled microorganism belongs
to the fungi kingdom. Saccharomyces cerevisiae is asymmet-
ric yeast. The high vitamin content of the yeast increases its
value as a nutrient. Many Saccharomyces cerevisiae species
synthesize other vitamins, especially B-vitamin.[37]
Saccharomyces cerevisiae was inoculated in malt extract
broth and incubated at 25 ꢁC. Experimental studies were
performed by taking 106 cells/mL in the exponential phase.
These cells are often used as a model for oxidative stress
response studies of molecular metabolism.[38]
3.4. Thawing, flasking, feeding and division
of L 1210 cells
Frozen L 1210 rodent leukemia cells obtained from the cell
culture bank (Sigma, USA) were dissolved at room tempera-
ture and transferred into a 75 mL flask. Previously prepared
DC5f3-([9-oxo-9H-fluorene-1-carbonyl]-amino)-benzoic
acidg (25 mL) was added to the flask and flasks were placed
in a Nuaire Mark, 5% CO2 medium incubator (Plymouth,
MN, USA). The state of the cells was daily controlled using
an inverted microscope (Soif Mark, Soif Optical Inc.,
HPLC chromatographic method was used to determine
China), and at the end of the third day, the DC5 in the flask the antioxidant properties of the ingredients. For the

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
measurements of the antioxidant vitamins (A and E) and 3.7. Statistical evaluation
MDA, a solution containing 2 mL of cells was placed in
each test tube. The materials were dissolved in DMSO and
the solutes were prepared at specific concentrations and final
concentrations were added to the tubes as 50 lM and
100 lM. For all experiments, additives were added so that
the amount of DMSO in the tubes would be equal. Tubes
with an equal volume of DMSO added were used as the
control group. After the addition of the substance, the rele-
vant amount of time was waited, in accordance to the rele-
vant literature.[39–41] The doses were determined by
considering the cell number at mL to be 106 cells/mL and
taking into account the solubility of the substances.
The statistical analyses were carried out by SPSS15.0 (SPSS
Inc., Chicago, IL). One-way ANOVA multi variance Tukeyꢀs
test for the antitumor feature and LSD test for vitamins (A
and E) and MDA were used for the data obtained from the
experimental studies. In the evaluation of the test results,
the P values expressed in the tables are meaningful when
the significance value is less than 0.05. (n) values indicate
the number of samples.
4. Conclusions
The fact that triazoles are biologically active compounds has
led to a wide field of application of these compounds in
pharmaceutical chemistry. In this study, bioactivities of
newly synthesized compounds containing cyclobutane ring
in biologically active furan and triazole structure were inves-
tigated. The antioxidant activities of the synthesized com-
pounds (5a-e) were investigated by two different methods.
In addition their antitumor activities were also investigated.
According to the results some of the compounds were found
to have good antioxidant and antitumor potentials.
3.6.2. MDA analysis
For MDA analysis, the cells treated with chemicals were
shaken by adding 250 lL of 15% trichloroacetic acid and
750 lL of 0.5 M HClO4. The cells were separated into small
pieces and the lysate was centrifuged at 4500 rpm for 5 min
and then clear part was taken and analyzed by HPLC.[42]
3.6.3. Vitamins A and E analysis
For the Vitamins A and E analysis, the cells treated with
chemicals were shaken by adding 250 lL of 15% trichloro-
acetic acid and 750 lL of 0.5 M HClO4. So the cells were
lysed. Proteins were then precipitated by the addition of
2 mL of ethyl alcohol containing 1% H2SO4. The mixture
was thoroughly mixed with the vortex and then centrifuged
at 4500 rpm for 5 min, and then 0.3 mL of n-hexane was
added to the samples. The fat-soluble vitamins were
extracted to the hexane phase by the addition of hexane.
After the addition of the hexane, it was vortexed again
and the tube was centrifuged. At the end of the centrifuga-
tion, the hexane phase was carefully removed and trans-
ferred in the glass tubes. The sample was mixed again by
adding 0.3 mL of n-hexane, centrifuged, and the n-hexane
phase was combined with the hexane phase in the glass
tube. The extracted hexane was carefully removed under dry
nitrogen. The residue was dissolved in 100 lL of methanol
and analyzed by HPLC.[43]
References
[1] Holla, B. S.; Akberali, P. M.; Shivananda, M. K. Studies on
Nitrophenylfuran
Characterization, Antibacterial and Antiviral Activities of Some
Nitrophenylfurfurylidene-1,2,4-Triazolo[3,4-b]-1,3,4-
Thiadiazines. Farmaco 2001, 56, 919–927. DOI: 10.1016/S0014-
827X(01)01124-7.
Derivatives:
Part
XII.
Synthesis,
[2] Kakefuda, A.; Suzuki, T.; Tobe, T.; Tsukada, J.; Tahara, A.;
Sakamoto, S.; Tsukamoto, S. I., Synthesis and Pharmacological
Evaluation of 5-(4-Biphenyl)-3-Methyl-4-Phenyl-1,2,4-Triazole
Derivatives as a Novel Class of Selective Antagonists for the
Human Vasopressin V1a Receptor. J. Med. Chem. 2002, 45,
2589–2598. DOI: 10.1021/jm010544r.
[3] Kojima, N.; Fushimi, T.; Tatsukawa, T.; Tanaka, T.; Okamura,
M.; Akatsuka, A.; Yamori, T.; Dan, S.; Iwasaki, H.; Yamashita,
M. Thiophene-3-Carboxamide Analogue of Annonaceous
Acetogenins as Antitumor Drug Lead. Eur. J. Med. Chem. 2014,
86, 684–689. DOI: 10.1016/j.ejmech.2014.09.026.
[4] Zhang, B.; Li, Y.-H.; Liu, Y.; Chen, Y.-R.; Pan, E.-S.; You, W.-
W.; Zhao, P.-L. Design, Synthesis and Biological Evaluation of
Novel 1,2,4-Triazolo [3,4-b][1,3,4] Thiadiazines Bearing Furan
and Thiophene Nucleus. Eur. J. Med. Chem. 2015, 103,
335–342. DOI: 10.1016/j.ejmech.2015.08.053.
[5] Saito, Y.; Kishimoto, M.; Yoshizawa, Y.; Kawaii, S. Synthesis
and Structure–Activity Relationship Studies of Furan-Ring
Fused Chalcones as Antiproliferative Agents. Anticancer Res.
2015, 35, 811–817.
3.6.4. Measurement with DPPH radical reduction method
The DPPH radical reduction method, which is another
method of determining antioxidant activity, was used.[44]
4 mL of the solution prepared in the form of 5 mg/L DPPH
in methanol was taken into each tube individually. The final
concentrations of 100, 250, 500 and 1000 lM were prepared
by adding to each group from the test substances dissolved
in DMSO at a concentration of 4000 lM. The reaction mix-
ture was allowed to stand in the dark at room temperature
for 30 min to form the reaction. It was decided whether to
be read in spectrophotometer at 517 nm by looking at the
color opening. After it was color expansion, reading was car-
ried out on the spectrophotometer. To compare DPPH
activity, BHT (Butylhydroxytoluene) was used at the same
concentration as the standard antioxidant.
[6] Catarzi, D.; Varano, F.; Poli, D.; Squarcialupi, L.; Betti, M.;
Trincavelli, L.; Martini, C.; Dal Ben, D.; Thomas, A.; Volpini,
R. 1,2,4-Triazolo[1,5-a]Quinoxaline Derivatives and Their
Simplified Analogues as Adenosine A3 Receptor Antagonists.
Synthesis, Structure–Affinity Relationships and Molecular
Modeling Studies. Bioorg. Med. Chem. 2015, 23, 9–21. DOI:
10.1016/j.bmc.2014.11.033.
€
[7] Aytac¸, S. P.; Tozkoparan, B.; Kaynak, F. B.; Aktay, G.; Gokta¸s,
€
€
O.; Unu€var, S. Synthesis of 3,6-Disubstituted 7H-1,2,4-
Triazolo[3,4-b]-1,3,4-Thiadiazines as Novel Analgesic/anti-
Inflammatory Compounds. Eur. J. Med. Chem. 2009, 63,
340–346. DOI: 10.1016/j.ejmech.2009.06.026.

6
P. KOPARIR
[8] Cansiz, A.; Etin, A.; Kutulay, P.; Koparir, M. Synthesis of [24] Yao, K.; Ina, Y.; Nagashima, K.; Ohmori, K.; Ohno, T.
Antioxidant Effects of Calcium Antagonists in Rat Brain
Homogenates. Biol. Pharm. Bull. 2000, 23, 766–769.
Tautomeric Forms of 5-(2-Hydroxyphenyl)-4-Substituted-3H-1,
2, 4-Triazole-3-Thione. Asian J. Chem. 2009, 21, 617.
[25] Nagaraja, G. K.; Reshma, K.; Manjunath, B.; Peethambar, S. K.;
Arulmoli, T. Antioxidant and Metal Chelating Activities of
Some Novel Imidazoquinolie Incorporated [1,2,4]-Triazolo
Heterocycles. J. Pharma Res. 2014, 3, 23–25.
[26] Genc¸, Z. K.; Selc¸uk, S.; Sandal, S.; C¸olak, N.; Keser, S.; S¸ekerci,
M.; Karatepe, M. Spectroscopic, Antiproliferative and
Antiradical Properties of Cu(II), Ni(II), and Zn(II) Complexes
with Amino Acid Based Schiff Bases. Med. Chem. Res. Res.
2014, 23, 2476–2485. DOI: 10.1007/s00044-013-0826-7.
[9] Jiang, J.; Lei, J.; Chen, X. Z.; Lou, L. G.; Ye, Q. S.; Chang,
Q. W.; Xu, Y. P.; Liu, P. W. Synthesis and Anticancer Activity
of
a New Water-Soluble Derivative of Heptaplatin, Cis-
fPt(II)[(4R,5R)-4,5-Bis(Aminomethyl)-2-Isopropyl-1,3-
Dioxolane](3-Acetoxyl-Cyclobutane-1,1-Dicarboxylato)g.
Chem. Intermed. 2015, 41, 6149–6155.
Res.
[10] Karatepe, M.; Karatas, F. Antioxidant, Pro-Oxidant Effect of the
Thiosemicarbazone Derivative Schiff Base (4-(1-
Phenylmethylcyclobutane-3-yl)-2-(2-
[27] Kedare, S. B.; Singh, R. P. Genesis and Development of DPPH
Method of Antioxidant Assay. J. Food Sci. Technol. 2011, 48,
412–422. DOI: 10.1007/s13197-011-0251-1.
[28] Soare, J. R.; Dinis, T. C. P.; Cunha, A. P.; Almeida, L.
Antioxidant Activities of Some Extracts of Thymus Zygis. Free
Hydroxybenzylidenehydrazino) Thiazole) and Its Metal
Complexes on Rats. Cell Biochem. Funct. 2006, 24, 547–554.
DOI: 10.1002/cbf.1266.
[11] Koparir, M.; Orek, C.; Parlak, A. E.; So€ylemez, A.; Koparir, P.;
Karatepe, M.; Dastan, S. D. Synthesis and Biological Activities
of Some Novel Aminomethyl Derivatives of 4-Substituted-5-(2-
Thienyl)-2,4-Dihydro-3H-1,2,4-Triazole-3-Thiones. Eur. J. Med.
Chem. 2013, 63, 340–346. DOI: 10.1016/j.ejmech.2013.02.025.
[12] Madhu Sekhar, M.; Nagarjuna, U.; Padmavathi, V.; Padmaja,
A.; Reddy, N. V.; Vijaya, T. Synthesis and Antimicrobial
Activity of Pyrimidinyl 1,3,4-Oxadiazoles, 1,3,4-Thiadiazoles
and 1,2,4-Triazoles. Eur. J. Med. Chem. 2018, 145, 1. DOI:
10.1016/j.ejmech.2017.12.067.
[13] V. Dehmlow, E.; Schmidt, S. Synthese Von Stereoisomeren 3-
Substituierten Cyclobutancarbons€aure-Derivaten. Liebigs Ann.
Chem. 1990, 1990, 411–414. DOI: 10.1002/jlac.199019900179.
[14] Koparir, P.; Karaarslan, M.; Orek, C.; Koparir, M. Synthesis and
in-Vitro Antimicrobial Activity of Novel Aminophosphinic
Acids Containing Cyclobutane and 1,3-Thiazole. Phosphorus.
Sulfur. Silicon Relat. Elem. 2011, 186, 2368–2376. DOI: 10.1080/
10426507.2011.604660.
[15] Kandeel, M. M.; Refaat, H. M.; Kassab, A. E.; Shahin, I. G.;
Abdelghany, T. M. Synthesis, Anticancer Activity and Effects
on Cell Cycle Profile and Apoptosis of Novel Thieno[2,3-
d]Pyrimidine and Thieno[3,2-e] Triazolo[4,3-c]Pyrimidine
Derivatives. Eur. J. Med. Chem. 2015, 90, 620–632. DOI:
10.1016/j.ejmech.2014.12.009.
[16] Mohamed, M. S.; Abd El-Hameed, R. H.; Sayed, A. I.; Soror,
S. H. Novel Antiviral Compounds Against Gastroenteric Viral
Infections. Arch. Pharm. Chem. Life. Sci. 2015, 348, 194–205.
DOI: 10.1002/ardp.201400387.
Radic.
Res.
1997,
26,
469–478.
DOI:
10.3109/
10715769709084484.
[29] Kharb, R.; Sharma, P. C.; Yar, M. S. Pharmacological
Significance of Triazole Scaffold. J. Enzyme Inhib. Med. Chem.
2011, 26, 1–21. DOI: 10.3109/14756360903524304.
[30] Zhang, H.-Z.; Damu, G.; Cai, G.-X.; Zhou, C.-H. Current
Developments in the Syntheses of 1,2,4-Triazole Compounds.
COC 2014, 18, 359–406. DOI: 10.2174/13852728113179990025.
[31] Kaur, R.; Dwivedi, A. R.; Kumar, B.; Kumar, V. Recent
Developments on 1,2,4-Triazole Nucleus in Anticancer
Compounds: A Review. Anticancer Agents Med. Chem. 2016,
16, 465–489. DOI: 10.2174/1871520615666150819121106.
ꢂ
[32] Koparır, M.; Cansız, A.; Demirdag, A. Synthesis of Some New
4,5-Substituted-4H-1,2,4-Triazole-3-Thiol Derivatives. Molecules
2004, 9, 204–212. DOI: 10.3390/90400204.
[33] Koparır, M.; Cansız, A.; Ahmedzade, M.; C¸etin, A.
Cyclobutane-Substituted Diacetamido Sulfides and 2,5-
Diacylthiophenes. Heteroatom Chem. 2004, 15, 26–31. DOI:
10.1002/hc.10207.
[34] Offiong, O. E.; Martelli, S. Stereochemistry and Antitumour
Activity of Platinum Metal Complexes of 2-Acetylpyridine
Thiosemicarbazones. Transit. Met. Chem. 1997, 22, 263–269.
DOI: 10.1023/A:1018416624951.
[35] Belicchi Ferrari, M.; Capacchi, S.; Pelosi, G.; Reffo, G.;
Tarasconi, P.; Albertini, R.; Pinelli, S.; Lunghi, P. Synthesis,
Structural Characterization and Biological Activity of Helicin
Thiosemicarbazone Monohydrate and a Copper(II) Complex of
Salicylaldehyde Thiosemicarbazone. Inorganica Chim. Acta.
1999, 286, 134–141. DOI: 10.1016/S0020-1693(98)00383-1.
[36] Kumamoto, T.; Toyooka, K.; Nishida, M.; Kuwahara, H.;
Yoshimura, Y.; Kawada, J.; Kubota, S. Effect of 2,4-Dihydro-
3H-1,2,4-Triazole-3-Thiones and Thiosemicarbazones on
Lodide Uptake by the Mouse Thyroid: The Relationship
Between Their Structure and Anti-Thyroid Activity. Chem.
Pharm. Bull. 1990, 38, 2595–2596. DOI: 10.1248/cpb.38.2595.
[37] Orek, C.; Sarac, K.; Parlak, A. E.; Koparir, P.; Turkoglu, S.
Koparır, M. Synthesis, Structure Investigation, Spectral
Properties and in Vitro Antioxidant Evaluation of New 1-(3-
Methyl-3-Mesityl)-Cyclobutyl-2-(5-Thiophen-4-Ethyl-2h-
[1,2,4]Triazol-3-Ylsulfanyl)- Ethanone. Fresenius Environ. Bull.
2018, 27, 2992–3005.
[17] Rombouts, F. J. R.; Tresadern, G.; Buijnsters, P.; Langlois, X.;
ꢁ
Tovar, F.; Steinbrecher, T. B.; Vanhoof, G.; Somers, M.; Andres,
J.-I.; Trabanco, A. A. Pyrido[4,3- e] [1,2,4]Triazolo[4,3- a]
Pyrazines as Selective, Brain Penetrant Phosphodiesterase 2
(PDE2) Inhibitors. ACS Med. Chem. Lett. 2015, 6, 282–286.
DOI: 10.1021/ml500463t.
[18] Zhu, X.; Zhang, M.; Liu, J.; Ge, J.; Yang, G. Ametoctradin Is a
Potent Qo Site Inhibitor of the Mitochondrial Respiration
Complex III. J. Agric. Food Chem. 2015, 63, 3377–3386. DOI:
10.1021/acs.jafc.5b00228.
[19] Lesyk, R.; Vladzimirska, O.; Holota, S.; Zaprutko, L.; Gzella, A.
New 5-Substituted Thiazolo [3,2-b][1,2,4] Triazol-6-Ones:
Synthesis and Anticancer Evaluation. Eur. J. Med. Chem. 2007,
42, 641–648. DOI: 10.1016/j.ejmech.2006.12.006.
[20] Khursheed, A.; Alimuddin, A. S. A Short Review on 1, 2, 4-
Triazole with Various Pharmacological Activity. SD Int. J.
Pharmaceut. Sci. 2018, 1, 14–22.
[38] Herdeiro, R. S.; Pereira, M. D.; Panek, A. D.; Eleutherio,
E. C. A. Trehalose Protects Saccharomyces Cerevisiae from
Lipid Peroxidation During Oxidative Stress. Biochim. Biophys.
[21] Ku€c¸u€kgu€zel, S¸. G.; C¸ıkla-Su€zgu€n, P. Recent Advances Bioactive
1,2,4-Triazole-3-Thiones. Eur. J. Med. Chem. 2015, 97, 830–870.
DOI: 10.1016/j.ejmech.2014.11.033.
[22] Kumudha, D.; Reddy, R. R.; Kalavathi, T. 1,2,4-Triazoles: As
Biologically Important Agents. Int. J. Pharm. Sci. Res. 2012, 3,
4562–4572. DOI: 10.13040/IJPSR.0975-8232.3(12).4562-72.
[23] Singhal, N.; Sharma, P. K.; Dudhe, R.; Kumar, N. Recent
Advancement of Triazol Derivatives and Their Biological
Significance. J. Chem. Pharm. Res. 2011, 3, 126–133.
Acta
- Gen. Subj. 2006, 1760, 340–346. DOI: 10.1016/
j.bbagen.2006.01.010.
[39] Yeh, S.-L.; Hu, M.-L. Antioxidant and Pro-Oxidant Effects of
Lycopene in Comparison with b-Carotene on Oxidant-Induced
Damage in Hs68 Cells. J. Nutr. Biochem. 2000, 11, 548–554.
DOI: 10.1016/S0955-2863(00)00117-0.
[40] Abid-Essefi, S.; Ouanes, Z.; Hassen, W.; Baudrimont, I.; Creppy,
E.; Bacha, H. Cytotoxicity, Inhibition of DNA and Protein
Syntheses and Oxidative Damage in Cultured Cells Exposed to

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
7
€
ꢁ
Zearalenone. Toxicol. Vitr. 2004, 18, 467–474. DOI: 10.1016/ [43] Cetınkaya, N.; Ozcan, H. Investigation of Seasonal Variations in
j.tiv.2003.12.011.
Cow Serum Retinol and b -Carotene by High Performance
Liquid Chromatographic Method. Comp. Biochem. Physiol. Part
Physiol. 1991, 100, 1003–1008. DOI: 10.1016/0300-
9629(91)90328-A.
[41] Buss, J. L.; Neuzil, J.; Ponka, P. Oxidative Stress Mediates
Toxicity of Pyridoxal Isonicotinoyl Hydrazone Analogs. Arch.
Biochem. Biophys. 2004, 421, 1–9. DOI: 10.1016/
j.abb.2003.09.044.
A
[44] Liyanapathirana, C.; Shahidi, F. Optimization of Extraction of
Phenolic Compounds from Wheat Using Response Surface
Methodology. Food Chem. 2005, 93, 47–56. DOI: 10.1016/
j.foodchem.2004.08.050.
[42] Karatepe, M. Simultaneous Determination of Ascorbic Acid and
Free Malondialdehyde in Human Serum by HPLC-UV. LC GC
North Am. 2004, 22, 362–365.