Discovery of novel chloropyramine-cinnamic acid hybrids as potential FAK inhibitors for intervention of metastatic triple-negative breast cancer

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Highlights

Abstract

To search for novel focal adhesion kinase (FAK) inhibitors for intervention of metastatic triple-negative breast cancer (TNBC), a series of hybrids 7a-s from chloropyramine and cinnamic acid analogs were designed, synthesized and biologically evaluated. The most active compound 7d could potently inhibit the proliferation, invasion and migration of TNBC cells in vitro. The docking analysis of 7d was performed to elucidate its possible binding modes to focal adhesion targeting (FAT) domain of FAK scaffold. Further mechanism studies indicated the ability of 7d in disrupting Y925 autophosphorylation of FAK, reducing formation of focal adhesions (FAs) and stress fibers (SFs) as well as inducing apoptosis of TNBC cells. Together, 7d is a novel FAK inhibitor to inhibit the essential nonkinase scaffolding function of FAK via binding FAT domain and may be worth studying further for intervention of TNBC.

Introduction

Tumor metastasis accounts for the vast majority of deaths in patients with cancer, especially for triple-negative breast cancer (TNBC).1 TNBC is the most aggressive subtype of breast cancer characterized by the absence of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor-2 (HER2) expression.2 Patients with TNBC tend to a poor survival and prognosis due to rapid metastatic recurrence and the lack of efficient targeted therapies.3

Focal adhesion kinase (FAK) refers to a non-receptor intracellular tyrosine kinase that stimulates tumor progression and metastasis formation by regulating adhesion, proliferation, migration, invasion, and angiogenesis through both its kinase-dependent functions and kinase-independent functions.4, 5 Notably, overexpression of FAK has been detected in many human cancer cells including TNBC cells, and enhanced FAK expression is associated with invasion and metastasis of TNBC.5, 6, 7, 8 Thus, FAK inhibitors have been emerging as one of effective targeted therapy strategies for the intervention of TNBC. Accordingly, several ATP-competitive kinase inhibitors of FAK have been developed, and some of them have reached preclinical or clinical trials for varieties of malignant cancers.6 However, existing kinase inhibitors can only act on the ATP binding site of protein kinase domain to inhibit FAK kinase catalytic function, while most of which cannot block the essential nonenzymatic functions of FAK. Extensive research has indicated that small molecules targeting the FAT (focal adhesion targeting) domain of FAK scaffold represent a unique class of FAK inhibitors that provide a powerful approach to restrain FAK signaling transduction in a nonkinase-dependent manner via disturbing the phosphorylation or protein–protein interactions of FAT domain.9, 10, 11 Additionally, FAK-FAT domain inhibitors with ATP-noncompetitive are able to enhance pharmacological potency and selectivity of FAK inhibitors.

Chloropyramine (1), a typical FAK inhibitor targeting the interaction between FAK and vascular endothelial growth factor receptor 3 (VEGFR-3) in FAT domain, was identified via virtual screening and exerted antitumor activity at relatively high micromolar concentrations.6, 12 To improve its potency, many synthetic chloropyramine analogues have been prepared, such as compounds 2, 3, 4 (Fig. 1), and others.6, 10, 11, 13 These compounds have strong antitumor activity, and compound 4 exhibited the potential for intervention of metastatic TNBC.10 Thus, the structural modification of chloropyramine may be valuable for the development of novel FAK inhibitors to fight metastatic TNBC.

Bioactive moiety/fragment derived from natural products (NPs) have been one of the most important sources for the drug design strategy of molecular hybridization in order to improve the biological activity of known bioactive molecules. Natural cinnamic acid and its analogs, a class of aromatic fatty acid widely presented in citric fruits, coffee, propolis and wine, were frequently used as versatile pharmacophores in anticancer drug design because of their antiproliferative and antitumorigenic potency.14, 15 Importantly, previous studies have shown that cinnamic acid and its analogs can effectively suppress proliferation and metastasis of TNBC.16, 17

Accordingly, the above investigations led us to hypothesize that hybridization of chloropyramine scaffold with types of cinnamoyl moiety would provide a new class of FAK inhibitors with inhibitory effect on nonkinase scaffolding function via binding to FAT domain of FAK against metastatic TNBC. To test the hypothesis, we designed, synthesized the hybrids 7a-s by substitution of the p-chlorobenzyl group of chloropyramine with various cinnamoyl moiety through construction of amide, and evaluated the anti-metastasis activity of these compounds against TNBC (Fig. 2).

Section snippets

Chemistry

The synthesis of the target compounds is depicted in Scheme 1. The condensation of 2-chloropyridine 5 with N, N-dimethyl ethylenediamine at 140 °C in a sealed tube gave the desired key intermediate 6.13 Next, the generated 6 was reacted with indicated substituted cinnamic acid, respectively, in the presence of dicyclohexylcarbodiimide (DCC) to afford the corresponding amide 7a − e. In addition, different substituted cinnamic acid was treated with thionyl chloride respectively and the resulting

Conclusion

In conclusion, nineteen hybrids containing chloropyramine scaffold and cinnamoyl moiety were designed, synthesized and identified as novel FAK inhibitors targeting the FAT domain for the intervention of metastatic TNBC. Among them, the most active compound 7d exhibited low micromolar antiproliferative activity against three TNBC cells (MDA-MB-231, MDA-MB-157 and MDA-MB-453), superior to positive control chloropyramine. In contrast, 7d had approximately 5-fold less effect on non-cancer MCF10A

Chemical analysis

All commercially available compounds were used without further purification, unless otherwise noted. Anhydrous solvents (CH2Cl2) were used as commercially available. Analytical and preparative TLC was performed on silica gel (200–300 mesh) GF/UV 254 plates, and the chromatograms were visualized under UV light at 254 and 365 nm. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 5001H, 500 MHz; 13C, 126 MHz) spectrometer at 303 K, using TMS as an internal standard. High resolution mass

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study received financial support from the National Natural Science Foundation of China (NO. 81903470), Natural Science Foundation of Hunan Province (2020JJ5786), Central South University Postgraduate Independent Exploration and Innovation Project (No. 160171018), Central South University College Students Innovation and Entrepreneurship Project (S2021105330834) and Open Sharing Fund for the Large-scale Instruments and Equipments of Central South University.

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  • Cited by (5)

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    These authors contributed equally to this work.

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