Synthesis and antiproliferative assessments of new derivatives of isothiazolo[3,4-d]pyrimidine

Article abstract of DOI:10.1080/17415993.2020.1841192

Various derivatives of 5-aryl-4-imino-3-(phenylamino)-4,5-dihydroisothiazolo[3,4-d]pyrimidines (3a-f) were synthesized. The synthesis has been done through treatment of 3-amino-4-cyano-5-(phenylamino)isothiazole with various aryl isothiocyanates. The isothiazole skeleton was obtained by the reaction of malononitrile and phenyl isothiocyanate followed by chloramine treatment. Some of the synthesized dihydroisothiazolo[3,4-d]pyrimidines were tested against different cancer cell lines, including ACHN, HeLa, HL-60, MCF-7, and PC3. Malignant cells were cultured in RPMI medium and incubated with different concentrations of the mentioned compounds. Cell viability was assessed using the MTS assay. The cytotoxicities of the synthesized compounds are not significant and are probably safe for other biological use.

Full text of DOI:10.1080/17415993.2020.1841192

Journal of Sulfur Chemistry
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Synthesis and antiproliferative assessments of
new derivatives of isothiazolo[3,4-d]pyrimidine
Hamideh Khoshniazi , Hossein Eshghi , Mona Tayarani-Najjaran , Zahra
Tayarani-Najaran , Kayvan Saadat & Ali Shiri
To cite this article: Hamideh Khoshniazi , Hossein Eshghi , Mona Tayarani-Najjaran ,
Zahra Tayarani-Najaran , Kayvan Saadat & Ali Shiri (2020): Synthesis and antiproliferative
assessments of new derivatives of isothiazolo[3,4-d]pyrimidine, Journal of Sulfur Chemistry, DOI:
10.1080/17415993.2020.1841192
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JOURNAL OF SULFUR CHEMISTRY
https://doi.org/10.1080/17415993.2020.1841192
Synthesis and antiproliferative assessments of new
derivatives of isothiazolo[3,4-d]pyrimidine
a
a
b
Hamideh Khoshniazi , Hossein Eshghi , Mona Tayarani-Najjaran ,
c
a
a
Zahra Tayarani-Najaran , Kayvan Saadat
and Ali Shiri
a
b
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Department
c
of Chemistry, The Graduate Center of the City University of New York, New York, USA; Medical Toxicology
Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
ABSTRACT
ARTICLE HISTORY
Various derivatives of 5-aryl-4-imino-3-(phenylamino)-4,5-dihydroi
sothiazolo[3,4-d]pyrimidines (3a-f) were synthesized. The synthesis
has been done through treatment of 3-amino-4-cyano-5-(phenyla
mino)isothiazole with various aryl isothiocyanates. The isothiazole
skeleton was obtained by the reaction of malononitrile and phenyl
isothiocyanate followed by chloramine treatment. Some of the syn-
thesized dihydroisothiazolo[3,4-d]pyrimidines were tested against
different cancer cell lines, including ACHN, HeLa, HL-60, MCF-7, and
PC3. Malignant cells were cultured in RPMI medium and incubated
with different concentrations of the mentioned compounds. Cell via-
bility was assessed using the MTS assay. The cytotoxicities of the
synthesized compounds are not significant and are probably safe for
other biological use.
Received 21 March 2020
Accepted 14 October 2020
KEYWORDS
Isothiazolo[4,3-d]pyrimidine;
heterocyclization; aryl
isothiocyanate; cytotoxicity;
MTS assay
Introduction
Pyrimidines and isothiazolopyrimidines are two promising compounds that have been
used for the synthesis of various compounds with a broad spectrum of biological activ-
ities [1,2]. Various synthetic methods for these compounds have been reported in the
literature for use in studying such biological properties. For example, the condensation
of aroyl halides with malononitrile yields an intermediate which is converted to isothia-
zolopyrimidine via treatment with PCl , diethyl dithiophosphate, and H O , respectively
5
2
2
CONTACT Ali Shiri
alishiri@um.ac.ir
This paper is dedicated to Prof. Mehdi Bakavoli, who passed away in 2018.
©
2020 Informa UK Limited, trading as Taylor & Francis Group

2
H. KHOSHNIAZI ET AL.
Scheme 1. Common synthetic routes to Isothiazolines and Isothiazolopyrimidines.
[
5
3]. Other examples are the sulfonation and intramolecular cyclization of 4-mercapto-
-acetylpyrimidine derivatives[4], cyclization of 3-amino-3-mercaptoacrylonitriles [5],
oxidative cyclization of β-aminothioamide [6], self-condensation reaction of 1,3-
disubstituted 5-amino-6-methyluracil in bromobenzene [7], treatment of 6-amino-1,3-
dialkyluracils with 4,5-dichloro-5H-1,2,3-dithiazolium chloride [8], and cyclization of
arylmethylene-malononitriles with thionyl chloride in pyridine [9] (Scheme 1).
Biological applications of fused isothiazolopyrimidine are reported in the literature. For
instance, they have been shown to have antimicrobial [10] and anti-HIV activity [11], and
used as a tyrosine kinase receptor [3], and PDE7 inhibitors [12]. Such characteristics are
well-known for pyrimidines and fused-pyrimidines [13–19]. It is noteworthy that isothia-
zole chemistry also has interesting biological implications [20] and new rational synthesis
routes for the production of isothiazolopyrmidine moieties can are needed.
In continuation of our interests in the synthesis of potentially biological active hetero-
cyclic compounds derived from pyrimidine and isothiazole scaffolds [21–24] we report in
this study an efficient protocol for the synthesis of some new isothiazolo[3,4-d]pyrimidine
derivatives. We also investigated the reaction with various aryl isothiocyanates. This report
further describes an investigation of the possible cytotoxic effects of some of the synthe-
sized isothiazoloprymidine moieties on five different malignant cancer cell lines including
human breast cancer (MCF-7), human epithelial (HeLa), human prostatic cancer (PC3),
human promyelocytic leukaemia (HL-60), and human kidney adenocarcinoma cell line
(
ACHN).

JOURNAL OF SULFUR CHEMISTRY
3
Scheme 2. Synthesis of 5-aryl-4-imino-3-(phenylamino)-4,5-dihydroisothiazolo[3,4-d]pyrimidine-
(7H)-thiones.
6
Results and discussion
Chemistry
The general synthetic strategy for the preparation of isothiazolopyrimidines is described in
Scheme 2. The condensation of phenylisothiocyanate with malononitrile in the presence
of EtONa/EtOH led to the formation of sodium 2,2-dicyano-1-(phenylamino)ethene-1-
thiolate (1). Treatment of compound (1) with the mixture of NH and HOCl (Chlo-
3
ramine) subsequently converted it to 3-amino-4-cyano-5-(phenylamino)isothiazole (2)
[
5]. The reaction of compound (2) with various aryl isothiocyanates in the presence of
3
Et N and subsequent refluxing in t-BuOK/t-BuOH yielded the desired compounds (3a-f)
(
Scheme 2).
The proposed heterocyclization mechanism of the aryl isothiocyanates and 3-amino-
4
-cyano-5-(phenylamino)isothiazole (2) is shown in Scheme 3. The reaction starts with
nucleophilic attack of NH moiety of 3-amino-4-cyano-5-(phenylamino)isothiazole to
2
arylisothiocyanate, followed by a proton exchange. Further cyclization completes via nucle-
ophilic attack of NHAr to the nitrile substitution of isothiazole precursor. The structures of
isothiazolo[3,4-d]pyrimidine 3(a-f) were confirmed from their spectral and microanalyti-
1
cal data. For example, the H NMR spectrum of compound (3d) did not show the signal at
δ = 5.82 and 9.51 ppm, belonging to NH and NH moiety of the precursor (2), respectively,
2
but instead showed three singlet peaks at δ = 10.66 (the NHPh group attached to isoth-
iazole), 10.75 (the NH of imine group attached to pyrimidine ring), and 12.86 ppm (the
NH of thioamide moiety on pyrimidine ring) in compound (3d) which disappeared when
adding D O. Furthermore, the spectrum also showed the peaks in the aromatic region,
2
confirming the presence of phenyl and p-Br-phenyl rings and the occurrence of heterocy-
clization. The aromatic proton nuclei of (3d) can be classified into two groups for each
existing aromatic ring. The protons of phenyl group showed two multiplet signals at δ
7
.03–7.25 ppm. The other two signals at δ 7.49 and 7.54 ppm with coupling constants of
1
J = 6.3 Hz could be observed in the H NMR spectrum for p-bromophenyl moiety. The

4
H. KHOSHNIAZI ET AL.
Scheme 3. The proposed mechanism of the synthesis of 5-aryl-4-imino-3-(phenylamino)-4,5-
dihydroisothiazolo[3,4-d]pyrimidine-6(7H)-thiones.
IR spectrum was also devoid of the stretching vibration bands at 3446 and 3349 cm−1, for
−
1
the NH moiety of the precursor. Instead, it converted to one band at 3394 cm for the
2
+
NH moiety. The molecular ion peaks of compound (3d) were observed at 429 (M ) and
+
4
31 (M + 2) corresponding to the molecular formula of C H BrN S .
1
7
12
5 2
Cell culture and cytotoxic assessments
ACHN, HL-60, MCF-7, and PC3 cancer cell lines were obtained from Pasteur Institute
Tehran, Iran) and maintained at 37°C in a humidified atmosphere (90%) containing 5%
(
CO . All cell lines were cultured in Roswell Park Memorial Institute medium (RPMI)
2
except ACHN which were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with
5
% (v/v) Fetal Calf Serum (FCS), 100 units/mL penicillin, and 100 μM streptomycin.
The protocol was the same as previously reported [25]. 5,000 cell/well (MCF-7, Hela,
PC3, HL-60, and ACHN) were seeded in each well of 96-microwell plates overnight and
then treated for 48 h with various concentrations (60, 125, and 250 μM) of some of the
synthesized isothiazoloprymidine compounds. There was a control sample for each con-
centration and time course study, which remained untreated and received an equal volume
of the medium. After incubation for 48 h, the MTS assay for cell viability measurement was
®
done. Cell Titer 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA), which
is composed of the novel tetrazolium compound MTS and an electron coupling reagent
phenazine methosulfate (PES, a redox intermediary) was added to each well according to
the manufacturer’s instructions. After two hours, cell viability was determined by measur-
ing the absorbance at 490 nm using an ELISA microplate reader (Awareness, Palm City,
FL, USA).
The results are presented in Table 1. Compounds (3a) and (3f) were insoluble in
the medium. Hence, their antiproliferative characteristic measurement was difficult. The
antiproliferative assessment of the other compounds, namely (3c), (3d), and (3e), demon-
strated that the presence of halogens on the phenyl group has moderate effects on the
toxicity of the mentioned compounds. The cytotoxic effects of the newly synthesized com-
pounds on cell lines were not statistically significant. It could be because of their low
solubility in the medium or low cytotoxic effect of the compounds.
Conclusion
This study reports a simple and efficient protocol for the synthesis of new derivatives
of isothiazolo[3,4-d]pyrimidine through the intermediates formed by the reaction of 3-
amino-4-cyano-5-(phenylamino)isothiazole (2) with various aryl isothiocyanates. The

JOURNAL OF SULFUR CHEMISTRY
5
Table 1. The dose-dependent growth inhibition of ACHN, PC3, MCF-7, and HL-60 cells by compounds
3c), (3d), and (3e) with various concentrations (60–250 µM) after 48 h. Viability was quantified by
(
MTS assay. Results are mean ± SEM. Values are from at least three independent experiments, each in
triplicates.a
Concentration (µM)
Cell line
ACHN
Compound
3c
C
60
125
250
100.0 ± 0.88
100.0 ± 0.88
100.0 ± 0.88
100.0 ± 2.038
100.0 ± 2.038
100.0 ± 2.038
100 ± 0.26
90.89 ± 1.27
89.78 ± 3.82
78.91 ± 4.66
110.67 ± 1.74
110.97 ± 4.36
98.68 ± 9.75
104.92 ± 6.71
105.38 ± 5.57
96.81 ± 5.57
96.24 ± 3.04
111.30 ± 4.26
94.07 ± 6.56
92.78 ± 3.38
82.90 ± 0.11
76.80 ± 6.32
106.84 ± 1.60
104.85 ± 6.01
88.39 ± 1.31
98.21 ± 3.56
97.68 ± 2.27
118.01 ± 3.33
101.5 ± 5.53
109.44 ± 0.59
107.53 ± 3.91
87.12 ± 1.49
83.40 ± 0.55
78.80 ± 3.32
96.99 ± 0.97
95.92 ± 4.75
79.22 ± 3.10
102.88 ± 6.33
97.15 ± 3.03
109.36 ± 4.17
120.31 ± 4.189
126.20 ± 3.28
113.5 ± 3.63
3d
3e
pC3
3c
3d
3e
MCF7
HL60
3c
3d
100 ± 0.26
3e
100 ± 0.26
3c
100 ± 0.71
3d
100 ± 0.71
3e
100 ± 0.71
aBonferroni’s Multiple comparisons.
results demonstrate that the cytotoxicity of newly synthesized halogenated compounds
3c), (3d), and (3e) on MCF-7, HeLa, PC3, HL-60, and ACHN cancer cell lines were mod-
(
erate and these results can be of value for researchers who screen chemicals for potential
cytotoxic activities or even other biological effects due to its low cytotoxicity.
Experimental
Instrumental
Melting points were measured by an Electrothermal type-9100 melting point apartus
(
Staffordshire, U.K.). The FT-IR spectra was recorded with Avatar 370 FT-IR Thermo
1
Nicolet (Madison, WI) and only well-known absorptions were highlighted. H-NMR
(
1
3
400 MHz) and C-NMR (100 MHz) spectra obtained via Bruker Avance DRX-400
Fourier transformer spectrometer (Rheinstetten, Germany) and reported as downfield
of tetramethylsilane internal reference with parts per million units. Varian Mat CH-7
(
Palo Alto, CA) and Thermo Finnigan Flash EA microanalyzer (Milan, Italy) were utilized
respectively to scanning mass spectra and elemental analysis.
Synthesis of 3-amino-4-cyano-5-(phenylamino)isothiazole (2)
Malononitrile (25 mmol, 1.65 g) was added to a stirred solution of (0.6 g Na in 25 ml abso-
lute ethanol) and then placed in an ice bath. The solution was stirred for 10 min. Then,
phenylisothiocyanate (25 mmol, 2.98 g) was added and stirred for 12 h at room temper-
ature. Then, a solution of ClNH , which was prepared from the dropwise addition of
2
NaOCl(aq) (2%, 300 ml) to NH (25%, 50 ml) was added gradually and stirred at room
3
temperature for 24 h. The mixture was neutralized by 10% HCl solution, and the resulting
solid was filtered off and recrystallized in EtOH. The spectral data of the product was in
accordance with the product which was prepared with a little modification following the
previously published method [5].

6
H. KHOSHNIAZI ET AL.
General procedure for the synthesis of isothiazolo[3,4-d]pyrimidine (3a-f)
The appropriate isothiocyanate (1 mmol) was added to a stirred solution of compound
(
2) (1 mmol, 0.216 g) and Et N (1.5 mmol, 0.15 g) in CH CN (10 ml). The solution then
3 3
refluxed for 12 h. The progress of the reaction was monitored by TLC using CHCl : MeOH
3
(
15:1). The solvent was evaporated under reduced pressure with a rotary evaporator and
the resulting solid was filtered off, washed with water, and dried. The resulting solid was
dissolved in a solution of potassium (1.5 mmol, 0.058 g) in t-BuOH (20 ml) and refluxed
for 12 h. The solvent was removed under reduced pressure and neutralized with 10% HCl
solution. The resulting solid was filtered off and recrystallized in DMF-water.
4
,5-Dihydro-4-imino-5-phenyl-3-(phenylamino)isothiazolo[3,4-d]pyrimidine-6(7H)
-thione (3a)
Yield = 68% (0.238 g, insoluble in water and chloroform, soluble in DMSO), mp = 308–
1
3
10°C (decomposed), H NMR (DMSO-d , ppm): δ 7.02–7.43 (m, 5H, PhH), 7.36–7.75
6
(
m, 5H, PhH), 7.53 (br s, 1H, NHPh, D O exchangeable), 9.71 (br s, 1H, NH(thiouracil,
2
HN=C−N), D O exchangeable), 13.35 (br s, 1H, NH(thiouracil, HN−C=S), D O
2
2
exchangeable); 1 C NMR (DMSO-d , ppm): δ 177.5, 155.3, 157.2, 147.6, 136.9, 135.5,
3
6
1
28.5, 129.2, 128.7, 127.4, 123.4, 122.2, 117.4; IR (KBr disc) ν 3392 (N-H stretching), 3174
−
1
+
(
N-H stretching), 3112, 1638 (C=N stretching), 1455, 1266 cm ; MS: m/z: 351 [M ],
+
+
2
74 [M - (C H )], 260 [M - NHPh]. Anal. Calcd. for C H N S (%): C, 58.10; H,
6 5 17 13 5 2
3.73; N,19.93; S, 18.25. Found: C, 58.01; H, 3.81; N.19.89; S, 18.47.
5
-(3-Chlorophenyl)-4-imino-3-(phenylamino)-4,5-dihydroisothiazolo
[3,4-d]pyrimidine-6(7H)-thione (3b)
Yield = 54% (0.208 g, insoluble in water and chloroform, soluble in DMSO), mp = 297–
1
2
4
1
99°C (decomposed), H NMR (DMSO-d , ppm) δ 7.03–7.44 (m, 5H, PhH), 7.19–7.68 (m,
6
H, ArH), 10.70 (br s, 2H, NHPh and NH(thiouracil, HN=C−N), D O exchangeable),
2
1
3
2.89 (br s, 1H, NH(thiouracil, HN−C=S), D O exchangeable); C NMR (DMSO-d ,
2
6
ppm): δ 179.6, 157.0, 156.2, 147.8, 139.9, 138.1, 135.3, 134.6, 131.1, 130.4, 129.5, 128.3,
1
23.4, 122.4, 117.8; IR (KBr disc) ν 3378 (N-H stretching), 3182 (N-H stretching), 3108,
−
1
+
+
+
1
640 (C=N stretching), 1457, 1268 cm ; MS: m/z: 385 [M ], 387 [M + 2], 294 [M -
+
NHPh], 274 [M - (3-Cl-C H )]. Anal. Calcd. For C H ClN S (%) C, 52.91; H, 3.13;
6
4
17 12
5 2
N, 18.15; S, 16.62. Found: C, 52.88; H, 3.10; N, 18.11; S, 16.59.
5
-(4-Chlorophenyl)-4-imino-3-(phenylamino)-4,5-dihydroisothiazolo
[3,4-d]pyrimidine-6(7H)-thione (3c)
Yield = 62% (0.239 g, insoluble in water and chloroform, soluble in DMSO), mp = 303–
1
3
05°C (decomposed), H NMR (DMSO-d , ppm) δ 7.02–7.44 (m, 5H, PhH), 7.24 (d, 2H,
6
J = 6.7 Hz, ArH), 7.63 (d, 2H, J = 6.7 Hz, ArH), 10.71 (br s, 2H, NHPh and NH(thiouracil,
HN=C−N), D O exchangeable), 12.88 (br s, 1H, NH(thiouracil, HN−C=S), D O
2
2
exchangeable); 1 C NMR (DMSO-d , ppm): δ 176.3, 167.2, 154.5, 146.0, 140.2, 132.5,
3
6
130.1, 124.3, 123.8, 123.0, 122.2, 118.1, 116.2; IR (KBr disc) ν 3394 (N-H stretching), 3186

JOURNAL OF SULFUR CHEMISTRY
7
−
1
+
(
[
(
N-H stretching), 3100, 1627 (C=N stretching), 1457, 1267 cm ; MS: m/z: 385 [M ], 387
+
+
+
M + 2], 294 [M - NHPh], 274 [M - (4-Cl-C H )]. Anal. Calcd. For C H ClN S
6
4
17 12
5 2
%) C, 52.91; H, 3.13; N, 18.15; S, 16.62. Found: C, 52.85; H, 3.10; N, 18.15; S, 16.60.
5
-(4-Bromophenyl)-4-imino-3-(phenylamino)-4,5-dihydroisothiazolo
[3,4-d]pyrimidine-6(7H)-thione (3d)
Yield = 64% (0.275 g, insoluble in water and chloroform, soluble in DMSO), mp = 309–
1
3
11°C (decomposed), H NMR (DMSO-d , ppm) δ 7.03–7.42 (m, 5H, PhH), 7.22 (d, 2H,
6
J = 6.3 Hz, ArH), 7.63 (d, 2H, J = 6.3 Hz, ArH), 10.66 (br s, 1H, NHPh, D O exchange-
2
able), 10.75 (br s, 1H, NH(thiouracil, HN=C−N), D O exchangeable), 12.86 (br s, 1H,
2
1
3
NH(thiouracil, HN−C=S), D O exchangeable); C NMR (DMSO-d , ppm): δ 176.6,
2
6
1
67.6, 153.9, 146.3, 139.7, 132.5, 130.4, 124.9, 124.6, 123.4, 122.7, 118.5, 116.3; IR (KBr
disc) ν 3394 (N-H stretching), 3182 (N-H stretching), 3108, 1630 (C=N stretching), 1457,
−1
+
+
+
+
1
266 cm ; MS: m/z: 429 [M ], 431 [M + 2], 338 [M - NHPh], 274 [M - (4-Br-C H )].
6
4
Anal. Calcd. For C H BrN S (%) C, 47.45; H, 2.81; N, 16.27; S, 14.90. Found: C, 47.55;
1
7
12
5 2
H, 2.86; N, 16.66; S, 14.96.
5
-(4-Fluorophenyl)-4-imino-3-(phenylamino)-4,5-dihydroisothiazolo
[3,4-d]pyrimidine-6(7H)-thione (3e)
Yield = 43% (0.159 g, insoluble in water and chloroform, soluble in DMSO), mp = 311–
1
3
13°C (decomposed), H NMR (DMSO-d , ppm) δ 6.97–7.39 (m, 5H, PhH), 7.26–7.58
6
(
m, 4H, ArH), 9.35 (br s, 1H, NHPh, D O exchangeable), 10.22 (br s, 1H, NH(thiouracil,
2
HN=C−N), D O exchangeable), 12.88 (br s, NH(thiouracil, HN−C=S), D O exchange-
2
2
1
3
able); C NMR (DMSO-d , ppm): δ 175.6, 161.3, 157.0, 156.0, 147.7, 139.2, 136.0, 130.3,
6
1
29.3, 129.1, 123.5, 122.6, 117.7, 116.8; IR (KBr disc) ν 3390 (N-H stretching), 3186 (N-H
−
1
+
+
stretching), 3112, 1640 (C=N stretching), 1489, 1266 cm ; MS: m/z: 369 [M ], 278 [M
+
-
NHPh], 274 [M - (4-F-C H )]. Anal. Calcd. For C H FN S (%) C, 55.27; H, 3.27; N,
6
4
17 12
5 2
18.96; S, 17.36. Found: C, 55.14; H, 3.24; N, 18.93; S, 17.36.
4
,5-Dihydro-4-imino-5-(4-nitrophenyl)-3-(phenylamino)isothiazolo
[3,4-d]pyrimidine-6(7H)-thione (3f)
Yield = 67% (0.266 g, insoluble in water and chloroform, soluble in DMSO), mp = 310–
1
3
12°C (decomposed), H NMR (DMSO-d , ppm) δ 7.10–7.59 (m, 5H, PhH), 8.14 (d,
6
2
H, J = 8.2 Hz, ArH), 8.33 (d, 2H, J = 8.2 Hz, ArH), 10.65 (br s, 1H, NHPh, D O
2
exchangeable), 11.02 (br s, 1H, NH(thiouracil, HN=C−N), D O exchangeable), 12.92 (br
2
1
3
s, NH(thiouracil, HN−C=S), D O exchangeable); C NMR (DMSO-d , ppm): δ 177.6,
2
6
1
68.6, 154.9, 147.3, 139.7, 133.5, 131.4, 125.9, 126.6, 124.4, 123.7, 119.5, 117.3; IR (KBr
disc) ν 3390 (N-H stretching), 3178 (N-H stretching), 3105, 1637 (C=N stretching), 1335
−
1
+
+
+
cm ; MS: m/z: 396 [M ], 305 [M - NHPh], 274 [M - (4-NO -C H )]. Anal. Calcd.
2
6
4
For C H N S O (%) C, 51.50; H, 3.05; N, 21.20; S, 16.18. Found: C, 51.29; H, 3.00; N,
1
7
12 6 2
2
2
0.93; S, 15.87.

8
H. KHOSHNIAZI ET AL.
Aknowledgement
The authors gratefully acknowledge Research Council of Ferdowsi University of Mashhad for finan-
cial support of this project. This paper has been derived from the related conference paper that was
presented at 12th Iranian Congress of Toxicology.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The authors gratefully acknowledge Research Council of Ferdowsi University of Mashhad for finan-
cial support of this project. This paper has been derived from the related conference paper that was
presented at 12th Iranian Congress of Toxicology.
ORCID
Hossein Eshghi http://orcid.org/0000-0002-8417-220X
Kayvan Saadat http://orcid.org/0000-0003-1912-8875
Ali Shiri http://orcid.org/0000-0002-5736-6287
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