PYRIMIDOOXADIAZINE AND TRIAZOLOPYRIMIDOOXADIAZINE DERIVATIVES: SYNTHESIS AND CYTOTOXIC EVALUATION IN HUMAN CANCER CELL LINES
© 2015 Seyed-Hadi Mousavi", Hoda Atapour-Mashhad4, Mehdi Bakavolic, Ali Shiric, ^ Marzieh Akbarzadehc, and Zahra Tayarani-Najaran", 1
aPharmacological Research Center of Medicinal Plants, School of Medicine, Mashhad University
of Medical Sciences, Mashhad, Iran bDepartment of Chemistry, Payame Noor University, Mashhad, Iran cDepartment of Chemistry, School of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Received April 23, 2014; in final form, October 20, 2014
In vitro antiproliferative activities of some pyrimido[4,5-e][1,3,4]oxadiazine and [1,2,4]triazo-lo[4',3':1,2]pyrimido[4,5-e][1,3,4]oxadiazine derivatives were examined in human malignant cancer cell lines. All synthesized compounds inhibited the growth of malignant cells in a dose dependent manner, but among them 1,5,7-trimethyl-3-phenyl-1H-[1,2,4]triazolo[4',3':1,2]pyrimido[4,5-e][1,3,4]oxadiazine and [(1,5-dimethyl-3-phenyl-1H-[1,2,4]triazolo[4',3':1,2]pyrimido[4,5-e][1,3,4]oxadiazin-7-yl)sulfa-nyl]acetonitrile, both with triazole moiety, were found to be more effective than other compounds; they also induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to controls, indicating that apoptotic cell death is involved in toxicity they induce. The results showed that compounds with triazole moiety fused to pyrimido[4,5-e][1,3,4]oxadiazine derivatives are more active than those bearing chlorine or pyrrolidine groups at C-7 position.
Keywords: pyrimidooxadiazine, triazolopyrimidooxadiazine, cytotoxicity, tumor cell line, apoptosis.
DOI: 10.7868/S0132342315020074
INTRODUCTION
Oxadiazines bearing heteroatoms at positions 1, 2, and 4 or 1, 3, and 4 are interesting and promising heterocyclic compounds, as they possess diverse biological activities [1]. There are some reports in the literature on their cardiovascular, antibacterial, insecticidal, and anticonvulsive activities [2, 3]. In addition, oxadi-azines are useful intermediates in the synthesis of tenidap prodrugs or P-lactam antibiotics, particularly, the synthesis of carbapenems and penems [4, 5].
On the other hand, pyrimidine-containing compounds have been reported to offer a variety of anticancer potentials including antitumor [6], antineoplastic [7], antiproliferative [8, 9], and cyclin-depen-dent kinase [10], angiogenesis [11] and dihydrofolate reductase [12] inhibitory activities.
The promising therapeutic potential of this class of heterocycles prompted us to synthesize various pyrim-idooxadiazine and triazolopyrimidooxadiazine derivatives and evaluate the antiproliferative profiles of the obtained derivatives against a panel of four human sol-
1 Corresponding authors (e-mail: alishiri@um.ac.ir (A. Shiri); tayaraninz@mums.ac.ir).
id tumor cell lines: adenocarcinomic human alveolar basal epithelial (A549), human cervix carcinoma (HeLa), human liver carcinoma (HepG2), and human breast cancer (MCF-7) cell lines. The most active derivatives were further studied to determine the role of apoptosis in the cytotoxic activity.
RESULTS AND DISCUSSION
Chemical Synthesis
7-Chloro-1,5-dimethyl-3-phenyl-1 H-pyrimido [4,5-e] [1,3,4] oxadiazine (III) was prepared by the reaction of 5-bromo-2-chloro-6-methyl-4-(1-meth-ylhydrazino)pyrimidine (II) with benzoyl chloride in the presence of K2CO3 according to previous published method [13]. Subsequent treatment of compound (III) with pyrrolidine in boiling ethanol led to the replacement ofchorine atom to give 1,5-dimethyl-3-phenyl-7-(pyrrolidin-1-yl)-1H-pyrimido[4,5-e][1,3,4]oxadiaz-ine (IV). On the other hand, compound (III) was treated with hydrazine hydrate and then with triethy-lorthoacetate in boiling acetic acid to give compound (V). Moreover, heating the hydrazino derivative of compound (III) and CS2 in dry pyridine and conversion of
the resulted product into the corresponding alkyl derivatives by reaction with ethylbromide and chloroac-etonitrile provided the desired compounds (Via) and
(VIb), respectively. The structural assignments of compounds (III)—(VI) are based upon the spectroscopic and microanalytical data (Scheme).
O
Cl +
CH3
I
H2N Y Y
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(III)
1) N2H4, EtOH
2) CH3C(OEt)3 AcOH
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(V) Scheme 1.
1) N2H4, EtOH
2) CS2, Pyridine
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CH3 SR
(Via): R (VIb): R
3
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Cytotoxicity of the Synthesized Compounds (III)-(VI)
The cytotoxicities of the synthesized compounds (III)-(VI) were examined in malignant cells. At first, HeLa cells were incubated with various concentrations of compounds (III)-(VI) for 24 h. The results showed that these compounds decreased the viability of cells in a concentration-dependent manner. Among them, compounds (V) and (VIb) were found to be more effective than the other derivatives (P < 0.001) (Fig. 1). The doses of compounds (III)-(V), (VIa), and (VIb) that induced 50% cell growth inhibition (IC50) against HeLa cells were 1092.0, 244.4, 91.06, 122.0, and 49.95 ^mol L-1, respectively (Fig. 1). Among all newly synthesized compounds, compounds (V) and (VIb) were selected to be evaluated via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-um bromide) method for their in-vitro cytotoxic effect
against a panel of four human tumor cell lines including A549, HeLa, Hep-G2, and MCF-7. The IC50 against A549 and HeLa for compound (V) were calculated to be 82.64 and 43.75 ^mol L-1, respectively (Fig. 2), while the IC50 for compound (VIb) against A549, HeLa, HepG2, and MCF-7 cells were 79.85, <25, 135.1, and 156.2 ^mol L-1, respectively, after 48 h (Fig. 3).
The Role of Apoptosis in HeLa Cells Treated with Compounds (III)-(VI)
To determine whether apoptosis is involved in cell toxicity of compounds (V) and (VIb) in A549 and HeLa cell lines, cellular morphological changes and DNA fragmentation were investigated in these cells. HeLa cells were exposed to 60 ^mol L-1 of com-
-O
ce
i>
120 100 80 60 40 20
Via VIb
С 62.5 125 250
Concentration, prnol L-1
C 15 31.2 62.5 125 Concentration, prnol L-1
250
Fig. 1. Dose-dependent growth inhibition of HeLa cells by compounds (III)—(VI) at different concentrations (pmol L-1) after 24 h. Viability was quantitated by MTT assay. IC50 against HeLa cells for compounds (III)-(VI) were 1092.0, 244.4, 91.06, 122.0, and 49.95 ^mol L-1, respectively. Results are mean ± SEM (n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to control (C).
£
-o
a
Via
120 100 80 60 40 20 0
-O
a
Via
120 100 80 60 40 20 0
w HeLa
25 50 75 100 125 150 Concentration, prnol L-1
C 25 50 100 150 200 Concentration, prnol L-1
0
Fig. 2. Dose-dependent growth inhibition of A549 and HeLa cells by compound (V) at different concentration (p,mol L 1) after 48 h. Viability was quantitated by MTT assay. IC50 values against A549 and HeLa cells for compound (V) were 82.64 and 43.75 p,molL-1, respectively. Results are mean ± SEM (n = 3). * P< 0.05, ** P< 0.01, and *** P< 0.001 compared to control (C).
pound (V), incubated for 48 h, stained with DAPI, and examined with fluorescent microscopy. The same protocol was used for compound (VIb) (60 ^mol L-1) on A549 cells. As follows from Figure 4, after treatment with compounds (V) and (VIb), the cells stained with equal intensity of DAPI were condensed and presented strong blue staining under the fluorescent inverted microscope confirming that apoptosis had occurred. After the treatment with compound (VIb) (50 ^mol L-1), apoptosis of A549 cells was measured with PI staining and flow cytometry. The same protocol was used for compounds (V) and (VIb) in HeLa cells to detect
БИООРГАНИЧЕСКАЯ ХИМИЯ том 41 № 2 2015
the sub-G1 peak resulting from DNA fragmentation. Flow cytometry histograms of compounds (V) and (Vlb)-treated cells exhibited a sub-G1 peak in A549 and HeLa cells. This indicates the involvement of an apoptotic process in compounds (V) and (VIb)-in-duced cell death (Figs. 5, 6).
In this study, we investigated the potential antitumor activity of fused heterocyclic compounds of py-rimidooxadiazine and triazolopyrimidooxadiazine derivatives. Different concentrations of compounds (III)-(VI) were tested for their antiproliferative activities. Structural changes on C-7 and a triazole moiety
120 100
^ 80
S 60
-O
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120
100
80
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ilit 60
-О ia
Vi 40
20
0
A549
15.6 31.2 62.5 125 250 500 Concentration, prnol L-1
25 50 75 100 125 150 175 Concentration, prnol L-1
к HepG2
120
100
80
60 ia
Vi 40
20 0
120 100 ^ 80 ilit 60
lo
ia
Vi 40
20 0
HeLa
*** **
25 50 100 150 200 Concentration, prnol L-1
MCF-7
15.6 31.2 62.5 125 250 500 Concentration, prnol L-1
Fig. 3. Dose-dependent growth inhibition of A549, HeLa, HepG2, and MCF-7 cells by compound (VIb) at different concentrations (p,mol L ) after 48 h. Viability was quantitated by MTT assay. IC50 values against A549, HeLa, HepG2, and MCF-7 for compound (VIb) were 79.85, <25, 135.1, and 156.2 p,mol L-1, respectively. Results are mean ± SEM (n = 3). * P < 0.05, ** P< 0.01, and *** P< 0.001 compared to control (C).
fused to pyrimido[4,5-e][1,3,4]oxadiazine heterocyclic ring appear to have considerable effect on the biological activity of the synthesized compounds. The results showed that among compounds (III)-(VI), compounds bearing fused triazole ring are more reactive than those bearing chlorine or pyrrolidine substit-uents on the C-7 position of the compound (III).
Based on these observations, our efforts were focused on compounds (V) and (VIb). They were evaluated for their inhibitory activity on A549, HepG2, and MCF-7 cell lines. We then explored the role of apoptosis in compounds (V) and (VIb)-induced toxicity. In the present study, compounds (V) and (VIb)-induced apoptosis was involved in
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