Synthesis and biological evaluation of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamides as pyruvate dehydrogenase kinase 1 (PDK1) inhibitors to reduce the growth of cancer cells

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Abstract

Most cancer cells exhibit a high rate of glycolysis and reduced capacity in mitochondrial oxidative phosphorylation. The expression of pyruvate dehydrogenase kinases (PDKs) was found to be increased in many cancer cells. Inhibition of PDKs increases the oxidative phosphorylation of glucose, which may disrupt the balance between the demand and supply of oxygen in cancer cell, thus leading to cell death. Several reports suggested that compounds containing (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide group could inhibit PDKs in pyruvate dehydrogenase primary enzymatic assay. However, none of them were capable of reducing the growth of cancer cells. Herein, we report the synthesis and biological evaluation of some novel PDK1 inhibitors containing the (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide warhead. Excitingly, these novel PDK1 inhibitors exhibited good potency to reduce the growth of cancer cells. We have demonstrated that these compounds could physically associate with PDK1 and activate pyruvate dehydrogenase in low micromolar levels.

Introduction

Despite significant progress in cancer therapy over the last decades, cancer remains one of the most serious threats to human life. According to current estimates, 8.9 million people worldwide died from cancer in 2015, and the numbers are likely to increase even further (Sautier et al., 2016). Therefore, development of effective anticancer therapeutics is urgently needed.

Unlike the normal cells, most cancer cells tend to metabolize glucose by means of the cytoplasm-based glycolysis rather than mitochondria-based oxidative phosphorylation, even under normoxia (Vander Heiden et al., 2009). This altered cellular metabolic pathway meets the survival and proliferation needs during tumor progression (Vander Heiden, 2012). Disruption this metabolic aberration represents therapeutic opportunities in anticancer strategy (Zhang et al., 2016a).

Pyruvate dehydrogenase complex (PDC) is responsible for converting pyruvate to acetyl-CoA and plays an essential role in glucose metabolism (Zhang et al., 2015). Structurally, PDC consists four major components like pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), dihydrolipoamide dehydrogenase (E3), and E3-binding protein (E3BP), as well as the two kinds of dedicated regulatory enzymes PDKs and pyruvate dehydrogenase phosphatase (PDP) (Yu et al., 2008). On average, PDC contains only two or three kinase molecules per complex, and is organized around a structural core comprising 30 copies of E1, 60 copies of E2 and 6 copies of E3. This entire complex is assembled around the 60-mer of E2 with other components being attached to the E2 cores (Patel et al., 2009). Human E2 protein consists of two lipoyl domains (L1 and L2) and a small E1-binding domain. PDKs are an integral part of PDC and attached to the L2 domain. This attachment could facilitate the movement of PDKs around the E2 domain to reach copies of E1 cores (Hiromasa et al., 2006). Phosphorylation of PDC occurs to multiple serine residues (S264, S271 and S203) in E1.

It has been suggested that the attenuation of mitochondrial function may be partly due to the inhibition of PDC via an elevated expression of PDKs (Saunier et al., 2016). To date, four PDK isoforms (PDK1, PDK2, PDK3, and PDK4) in mitochondria have been characterized in terms of their differences in activities, tissue distribution and regulations (Kato et al., 2005, Devedjiev et al., 2007). Among the isoforms, PDK1 is considered to be mostly related to cancer malignancy (Wigfield et al., 2008). It had been reported that PDK1 was remarkably overexpressed in multiple human tumors such as lung cancer (Koukourakis et al., 2007), head squamous cancer (Wigfield et al., 2008), myeloma (Fujiwara et al., 2013), and gastric cancer (Hur et al., 2013). Moreover, the expression of the PDK1 gene in cancer cells is upregulated by the oncogenes c-Myc and hypoxia-inducible factor-1α (HIF-1α) to control the metabolic and malignant phenotypes (Kaplon et al., 2013). In particular, PDK1 can be regulated by the post-translational modification, which leads to the promotion of the Warburg effect and tumor growth (Hitosugi et al., 2011). Collectively, these pieces of evidence suggest that the inhibition of PDKs, especially PDK1, may offer a new therapeutic option for the treatment of cancer.

In 1999 and 2000, researchers from Novartis and AstraZeneca reported that (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamides (Fig. 1) inhibited PDKs in primary PDC enzymatic assay (Aicher et al., 1999, Aicher et al., 2000, Bebernitz et al., 2000, Butlin et al., 1999). However, the use of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamides as PDKs inhibitor to reduce the growth of cancer cells has not been reported in scientific literature. Herein, for the first time we report the synthesis and biological evaluation of some novel PDK1 inhibitors containing (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide warhead, which reduce the growth of cancer cells.

Section snippets

Cell Culture

Glioblastoma cell SF188 and glioma cell U251 were a gift from Prof. Gang Li, Faculty of Health Sciences, University of Macau, and the colon cancer cell RKO was from Prof. Hang Fai KWOK, Faculty of Health Sciences, University of Macau. The cancer cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2 at 37 °C. Cells at exponentially growing stage were used for all experiments.

Cell Viability Assay

Cancer cell viability upon treatment by the prepared

Chemistry

Lipoamide-binding site is located at the end of N-terminal R domain of PDKs, which is crucial to regulate the activity of PDKs (Zhang et al., 2015). The warhead of Nov 3r or AZD7545, trifluoromethylpropanamide, projects into the lipoamide-binding site of PDK. This interaction results in the inhibition of PDKs activities by blocking kinase binding to the PDC scaffold (Kato et al., 2007). We thus retained the (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide warhead in our target compounds

Conclusion

In this paper, we described the synthesis and biological evaluation of some novel PDK1 inhibitors containing the (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide warhead. Their structures were confirmed by 1H NMR, 13C NMR, and HRMS. Antiproliferative assay indicated that most of the compounds inactivated against SF188, RKO, and U251 cancer cell lines at 40 μM, similar to AZD7545. Interestingly, three of these compounds, namely 6a, 6e, and 6s, strongly inhibited the growth of RKO cancer cells,

Acknowledgements

We thank the financial support from the Science and Technology Development Fund, Macao S.A.R. (FDCT) (project reference no. 086/2014/A2).

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