Design, Synthesis and Inhibitory Properties against Coxsackie B3/b6 of Some Novel Triazole Derivatives

Copyright © 2013 Dongping Shao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT A series of 1,2,4-triazole derivatives were synthesized, and their abilities to inhibit the in vitro replication of Coxsackie B3/B6 were evaluated. Among the 1,2,4-triazole derivatives, compound 3 g displayed potent activity, with a high anti-viral potency (IC 50 = 1.71 μM (against CVB3), 1.43 μM (against CVB6)). The structures of all the new synthesized compounds were confirmed by 1 H-NMR spectra, mass spectra and elemental analyses.


Introduction
In recent years heterocyclic compounds analogues and derivatives have gained significant interest in various fields of drug discovery due to their wide range of biological activities, such as, anti-microbial, anti-tumor, anthelmintic, anti-leishmanial, anti-convulsant and antiinflammatory [1].And triazoles as important aromatic nitrogen-containing heterocycle possess a lot of desirable features and have been incorporated into a wide variety of potent therapeutic agents [2].Introduction of a triazole ring into nucleosides to improve bioactivity in antivirus agents has become widespread in drug design practices since the first synthetic nucleoside drug, ribavirin, showed a broad spectrum of antiviral activity against many RNA and DNA viruses.
Human enteroviruses are the most common cause for the majority of human respiratory diseases along with rhinoviruses.Moreover, enteroviruses may cause aseptic meningitis, encephalitis, febrile illness, hand-foot-mouth disease, and myocarditis [3].It is estimated that worldwide enteroviruses cause 10 -15 billion (or more) enteroviral infections annually, and many outbreaks have been reported from Asian countries [4].With more than 200 enterovirus serotypes existing, there is no effective anti-viral drug for clinical use of enteroviral infections so far [5].This highlights the urgency and significance for developing anti-enterovirus agents.
Enteroviruses are non-enveloped, single-stranded (+) RNA viruses belonging to the picornavirus family, which associated with several human and mammalian diseases [6].Conventionally, enteroviruses were classified into polioviruses, coxsackie A viruses (CVA), coxsackie B viruses (CVB), echoviruses, and enteroviruses 68 -71 (EV 68 -71).Coxsackie viruses, and in particular CVB 3, have often been associated with the development of myocarditis, which may lead to sudden death in young adults or progress to dilated cardiomyopathy [7,8].
In our previous work [9,10], a series of 2-pyridyl-1H-benzimidazole-4-carboxamide derivatives were synthesized, screened and indentified as modest inhibitors of CVB3.In view of its novel structural template, which differed from those of all reported anti-enterovirus agents, we were interested to introduce 1,2,4-triazoles moiety.A series of amide derivatives containing 1,2,4-triazole ring were synthesized, screened and indentified as modest inhibitors of coxsackie viruses.It was believed that these compounds could be found effective against picornavirus.The inhibitory activities of these triazole derivatives were tested against CVB3, CVB6.No active clinical drugs against enterovirus employed by now, a relatively effective drug, ribavirin (RBV), was selected as the positive control drug.These triazole derivatives were found to exhibit good inhibitory activities against the two kinds of enteroviruses.
The virus strains of CVB3, CVB6 were provided by American Type Culture Collection (ATCC).The positive control drug, RBV was produced by Hubei Keyi Pharmaceutical Factory.The newly synthesized triazole compounds (3a-4a) were dissolved in DMSO and diluted with the culture medium.Vero cells were planted in 96-well culture plates.After 24 h the plates were placed in the corresponding virus bulk for 2 h.Then the solutions of triazole compounds and RBV were added in the plates and cell wells and virus wells were set simultaneously.When the cytopathic effect (CPE) of virus wells was over 4, the CPE of cell wells was observed.The concentration required to inhibit virus growth by 50% (IC 50 ) was determined by the Reed-Muench method [15].

Results and Discussion
The cytotoxicity and antiviral potency of these synthesized 1,2,4-triazole derivatives were evaluated in vero cells against CVB3/B6.No active clinical drugs against CVB3/B6 employed by now, RBV was the recommended clinical antiviral drug in China, the IC 50 values of RVB were provided as comparable data.
The results were summarized in Table 1.The antienterovirus activity of each compound was expressed as the concentration of compound that achieved 50% inhibition (IC 50 ) of enterovirus growth.The cytotoxicity of each compound was expressed as the concentration of compound required to kill 50% (TC 50 ) of the vero cells.As a major pharmaceutical parameter for possible future clinical development, the selectivity index (SI) was de- termined as the ratio of TC 50 to IC 50 .The bioactivity of each compound was evaluated by the combination of its IC 50 and SI.
As shown in Table 1, the 1,2,4-triazole derivatives (3a-4a) were evaluated against CVB3.It is found that most of the synthesized compounds had good antiviral activity, far more active than RVB with IC 50 value of 384.90μM.Each of the bioactive compounds possessed an IC 50 value within the range from 1.71 μM to 66.67 μM, and a TC 50 value ranging from 4.14 μM to more than 200 μM.Analyzing the activities of the synthesized compounds, the following structure-activity relationship (SAR) was observed.
Compared with compound 3g (IC 50 = 1.71 μM), all other derivatives showed equivalent or decreased antiviral activities.Due to the introduction of dinitro phenyl in the R 2 position, compounds 3a-h exhibited good antiviral potency against CVB3.The introduction of methyl in the R 2 position resulted in approximately no biological activities, but their cytotoxicities were significantly decreased with TC 50 values of more than 200 μM.Compounds 3j-k (IC 50 > 66.67 μM) with methyl substituent at the R 2 position caused significantly decrease in antiviral activity, but showed lower cytotoxicity with TC 50 values of more than 200 μM.
Compound 3c (IC 50 = 22.22 μM) with moiety of isoxazole at the R 3 position displayed lower activity than compounds 3a and 3b (IC 50 = 2.47 and 2.47 μM, respectively), but was less cytotoxic with TC 50 value of 96.15 μM.Compound 3d with methyl at the R 3 position showed higher IC 50 (IC 50 = 22.22 μM) than compound 3e with phenyl at the R 3 position (IC 50 = 38.49μM).Compound 4a proved inactive against CVB3/B6 in vero cells which was the oxidation product of compound 3a, but the cytotoxicity to vero cells was decreased.
These structure-activity relationship analyses indicated that the introduction of dinitro phenyl in the R 2 position was critical for the high antiviral activity, such as 3a, 3b, 3g.The bioactive compounds 3a, 3b, 3g expressed better IC 50 s (IC 50 = 2.47, 2.47, 1.71μM).They could be considered as promising candidates for the development of new derivatives with anti-enterovirus activity and for additional studies concerning the antiviral activity of this group of compounds.The other bioactive compounds 3cf, 3h showed higher IC 50 s (IC 50 > 20 μM), but they appeared to be less toxic (TC 50 = 96.15,66.67, 138.67, 46.22, 138.67 μM, respectively).

Conclusion
In summary, a novel series of 1,2,4-triazole derivatives (3a-4a) have been synthesized and evaluated for their antiviral activities against CVB3, CVB6 replication in cell culture.Most of the 1,2,4-triazole derivatives pronounced good anti-CVB3 activities and some pronounced good anti-CVB6 activities.Moreover, some of the synthesized compounds had excellent antiviral activity, but they were cytotoxic to cells, such as compounds 3a, 3b and 3g (IC 50 < 3 μM, TC 50 < 10 μM).And some of the synthesized compounds had low cytotoxicities, such as 3h (IC 50 > 20 μM, TC 50 > 100 μM), but their antiviral activites were not eminent.Further studies are in progress in our laboratories to increase the antiviral potency and decrease cytotoxicity, most importantly, to improve the TC 50 /IC 50 ratio.The modifications on 1,2,4triazoles moiety display valuable biological activities, and these modifications can be utilized as potent therapeutic agents in future.

Table 1 . Activity of the triazole derivatives against coxsackie virus B3/B6 in vero cells.
a Cytotoxic concentration required to inhibit vero cell growth by 50%; b Concentration required to inhibit CVB3 growth by 50%; c Selectivity index values equaled to TC 50 /IC 50 ; d Not active in the largest concentration which were not toxic to vero cells.