Biomedical Research

Abstract

We herein report the anti-cancer activities of some synthesized pyridine derivatives substituted with thiazole, pyrazole and triazole moieties. A series of these derivatives 1-12 were synthesized and evaluated as anti-inflammatory, analgesic, anticonvulsant and antiparkinsonian agents before. Twelve compounds were conveniently screened for their in vitro cytoyoxicity against a wide rannge of cell lines and they are also showed potent activities against renal and prostate cancer cell lines. The in vivo antirenal cancer and antiprostate cancer of the most active in vitro compounds was estimated and founded highly potent. In search for the mechanism of action of anticancer activities it was foundeded that these compounds exert its action via histone decarboylase inhibition and inhibition of p53 ubiquitination.

Keywords

2,4,6-Trisubstituted pyridine, Heterocyclic derivatives, Anticancer activities.

Abbreviations

ARF: Alternative Reading Frame; DMEM: Dulbecco's Modified Eagle's Medium; DMSO: Dimethyl Sulfoxide; HLI98: HDM2 Ligase Inhibitor 98 Class.

Introduction

Thieno[2,3-b]pyridine candidates which substituted with pyridine, cyclopentyl, tetrahydroquinoline, pyrimidine, 1,6- naphthiridin, benzofuro[2,3-b]pyridine, imidazo[1,2- c]pyrimidine, triazolo[1,5-a]pyrimidine were synthesized and used a new agents for their in vitro antitumor activities against liver HepG-2 and breast MCF-7 cell lines [1-3]. Many pyridine derivatives were synthesised and foundeded to have cytotoxic activities in vitro against a wide range of cell lines especially renal and prostate types [4]. Imidazopyridine derivatives have potent antitumor activities [5]. In addition, two series of 4,6- diaryl-2-imino-1,2-dihydropyridine-3-carbonitriles and imidazo[2,1-b]pyridine/ pyrimidine chalcones were also synthesized and evaluated for their in vitro capacity to inhibit PDE3A and the growth of the human HT-29 colon adenocarcinoma tumor cell line [6-8]. In view of these observations and in continuation of our previous work [9-16] in biological and pharmacological studies for heterocyclic candidates, we herein reported the anti-cancer activities of some heterocyclic substituted pyridine derivatives.

Experimental

Chemistry

All the tested compounds 1-12 were confirmed by physical and spectroscopic evidences according to the previously reported procedure [16].

Cytotoxic activities

In vitro determination of IC50 of tested compounds against differrent cancer cell line via using the MTT Assay

The cytotoxicity of the newly synthesized compounds against cancer cell lines in vitro was performed with the MTT assay according to the previous reported method [17].

In vivo determination of the cytotoxic activities

Effects of compounds tested compounds on prostate cancer in vivo

To investigate the effect of tested compounds on prostate cancer cells in vivo use the adopted methods [18,19].

In vivo antirenal cancer

Cell lines: OUR-10 cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum (FCS). OUR-10 and DU145 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated FCS.

Proliferation assay: The experimental method which was used in proliferation assay has been adopted from Oka et al. [20].

Xenograft model: The experimental method which was used in xenograft model has been adopted from Liang et al. [21].

In vitro inhibition of histone deacetylase: The experimental method which was used in vitro inhibition of histone deacetylase has been adopted from Yoshid et al., [22] and Farooq et al., [23].

Biological assay in vitro ubiquitination assay: The experimental method which was used for biological assay in vitro ubiquitination assay has been adopted from Roxburgh et al., [24].

Non-fluorescent in vitro ubiquitination assays: The experimental method which was used for non-fluorescent in vitro ubiquitination assays has been adopted from Roxburgh et al., [24].

Statistical analysis

The data were expressed as means plus; standard deviation (s.d.). Statistical analysis was performed by Student's t-test (two-tailed). The criterion for statistical significance was taken as P<0.05.

Results and Discussion

In continuation of our previous work, a series of substituted pyridine, pyrazole, triazole and thiazolotriazole derivatives 1-12 (Figure 1) were synthesized before and screened as anti-inflammatory, analgesic, anticonvulsant and antiparkinsonian agents [16]. Herein, we report the activities of these compounds for evaluation as anticancer agents.

Figure 1: Chemical structure for tested compounds 1-12.

Anti-cancer activities

The cytotoxicity of the newly synthesized compounds against cancer cell lines in vitro was performed with the MTT assay according to the previous reported [17].

All the tested compounds showed potent cytotoxic activities in vitro against aKB6, SKOV-3, SF-268, NCI H 460, RKOP27, PC3, OUR-10, HL60, U937, K561, G361, SK-MEL-28, GOTO, NB-1, HeLa, MCF-7, HT1080 and HepG2 at micro molar level for all cell except for PC3, OUR-10 the potent activities lies on nanomolar level (Table 1).

Table 1: In vitro cytotoxicity activities of the tested compounds 1-12 against several cancer cell lines using MTT assay.

In vivo antiprostate cancer

The in vivo anti-prostate cancer activities of the tested compounds were estimated and culminated on the tested compounds exerts potent anti-prostate carcinoma in the following descending order 12, 11, 9, 10, 8, 7, 6, 5, 4, 3, 2 and 1 (Table 2).

Table 2: In vivo antiprostate carcinoma for tested compounds 1-12.

In vivo anti-renal cancer

In this study, from table 3 compounds 1-12 were used to test the effect on renal cancer cell lines. As shown in table 3, treatment with tested compounds reduced cell viability of renal cancer cells (OUR-10) in a time-dependent manner [18].

Table 3: Cytotoxic effect of treatment with tested compounds 1-12 against OUR-10.

Xenograft model

We examined whether tested compounds could inhibit the growth of OUR-10 tumors in mice. We selected an injection dose of 7 μM/Kg 3 times per week because this dose effectively inhibited in vivo OUR-10 tumor growth without adverse effects in a preliminary study. About 6 weeks after the start of treatment with compounds 12, 11, 9, 10, 8, 7, 6, 5, 4, 3, 2 and 1, the increase in OUR-10 tumor volume was measured and given in table 4. The in vivo antirenal cancer activities of the tested compounds were estimated and culminated on the tested compounds exerts potent anti prostate carcinoma in the following descending order 12, 11, 9, 10, 8, 7, 6, 5, 4, 3, 2 and 1.

Table 4: In vivo antirenal carcinoma for tested compounds 1-12.

Mechanism of anticancer activities

In search for the mechanism of action of the anticancer activities of the tested compounds the author's resorts to many in vitro assays for many targeted enzymes and cancer modulation molecules, these experiments revealed on the following two mechanistic pathways.

In vitro Inhibition of histone deacetylase

All the tested compounds inhibited the enzyme histone decarboxylase in the following descending order 12, 11, 9, 10, 8, 7, 6, 5, 4, 3, 2 and 1 (Table 5).

Table 5: IC50 of histone decarboxylate inhibitor activities of the tested compounds1-12.

Ubiquitination assay

All the testes compounds showed inhibition of p53 ubiquitination, the most active ones were 12, 11, 9, 10, 8, 7, 6, 5, 4, 3, 2 and 1 (Table 6) [18].

Table 6: IC50 of p53 ubiquitination of the newly synthesized compounds 1-12 (in vitro).

Conclusion

All tested compounds inhibited in vivo prostate and renal carcinoma via inhibition of the histone decaroxylase and inhibition of p53 ubiquitination also inhibited the EGFR and VEGFR-2 kinase.

Structural Activity Relationships

• Chloropyridine and pyrazoles essential for potent activities but the methyl pyrazole exerts more potent activities than the oxo-ones, while sulfones showed the least potent activities.

• Hydroxy pyridines provide less potent activities than the chloropyridines.

• Fusion of another heterocyclic ring system into the triazole ring greatly reduces the activities.

• Macrocyclic moieties and polychloro ones greatly reduces the activities.

Acknowledgement

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group project no. RGP -172.

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