Design, synthesis and biological evaluation of novel o-aminobenzamide derivatives as potential anti-gastric cancer agents in vitro and in vivo
Graphical abstract
Introduction
Cancer has been the second leading cause of death for human beings, which has afflicted an estimated 19.3 million people worldwide in 2020 and was predicted an approximately 50% increase between 2020 and 2040 [1]. Among the total cancer burden, gastric carcinoma is the fifth in morbidity and the fourth in mortality. Therefore, gastric cancer has become a major public health problem that seriously threatens the health of global citizens. As for cancer treatment, oral agents are the most preferred manner due to low medical expenses, good patient compliance, non-invasive administration, and improved life quality compared to intravenous injection or infusion [2]. However, oral agents being applied in clinical practice for gastric cancer therapy are quite rare, mainly including capecitabine and tegafur/gimeracil/oteracil, both of which are prodrugs of 5-fluorouracil (5-FU), a cytotoxic drug [3,4]. What's worse, the usage of these two agents above is further limited by a poor complete response rate and considerable toxicities related neutropenia and anemia [5,6]. In this context, developing new oral chemical entities with high efficiency and low toxicity is in great need for gastric cancer therapy.
Histone deacetylases (HDACs) are crucial epigenetic regulators of gene expression by removing an acetyl group from lysine residues of histones and other proteins [7]. Recent evidence has suggested that class I HDAC isoforms (HDAC1, HDAC2, and HDAC3) contribute to gastric cancer progression due to the overexpression and aberrant regulation in gastric carcinomas [[8], [9], [10], [11], [12], [13]]. Besides, knockdown of HDAC 2 induced the growth arrest, apoptosis, and autophagic cell death of gastric cancer cells in vitro and in vivo [14]. Gradually, HDAC inhibitors including Entinostat (MS-275), Trichostatin A (TSA), and Panobinostat (LBH-589), have been verified to inhibit gastric carcinoma alone or in conjunction with other agents [[15], [16], [17], [18]]. Given the evidence above, HDAC inhibitors may be the efficient agent for gastric cancer therapy.
Currently, five HDAC inhibitors were approved and more than twenty inhibitors were in clinical research [[21], [22], [23]] (Fig. 1). The pharmacophore structure of these HDAC inhibitors is summarized as three moieties, including a CAP group that interacts with target protein, a ZBG (Zinc Binding Group) that could chelate with Zn2+, a lipophilic linker with a proper length that connects the two moieties above [22]. Among them, ZBG is a pivotal section to activity, and it is mainly classified as hydroxamic acids and o-phenylenediamine [24,25]. While these two structures are normally used, there are also some drawbacks including toxicity, poor pharmacokinetic properties and low therapeutic index that hamper their use in clinic [[26], [27], [28], [29], [30], [31]]. For instance, hydroxamic acid could generate five-membered ring chelates with Zn2+ that caused a nonspecific chelation ability and toxicity [24,32], o-phenylenediamine could form a seven-membered ring chelate complex with Zn2+ that cause a weak interaction [24,25,32].
As a result, we decide to develop novel HDAC inhibitors bearing a ZBG of o-aminobenzamide instead of o-phenylenediamine via amide reversal based on the non-classical isostere principle (Fig. 2). Since the generation of six-membered ring chelate with Zn2+, the ring strain could be induced leads to an improvement of stability and interaction [27,28]. Besides, the pharmacokinetic stability of o-aminobenzamide could relieve the toxicity.
Herein, we describe a comprehensive medical chemistry study on 41 o-aminobenzamide compounds based on the scaffolds of MS-275 and SAHA, report the structure-activity relationship (SAR) of these novel compounds, and identify the anti-gastric carcinoma potential in vitro and in vivo by suppression of cell growth, induction of apoptosis, arrest of the cell cycle and inhibition of cell migration and invasion. After detection of the HDAC inhibition, we disclose western blotting analysis, IHC analysis, differentially expressed proteins analysis as well as design and synthesis of the CuAAC compatible probe to enrich and identify the probable target protein in order to interpret the mechanism of promising compounds. Collectively, this work offers a potentially oral chemical entity F8 that has high efficiency and low toxicity on gastric cancer therapy.
Section snippets
Chemistry
Herein, we report the syntheses of all the 41 compounds for the first time, as outlined in Scheme 1, Scheme 2, Scheme 3, Scheme 4.
The syntheses of compounds X1-X4 and H1–H5 were started from commercially available 1a-1e. The amino groups in 1a-1e were protected by the Boc group using di-tert-butyl decarbonate under the alkaline condition to generate amines 2a-2e, which then reacted with aniline via condensation followed by Boc deprotection with TFA to produce intermediates 4a-4e. Subsequently,
Conclusions
In summary, we designed and synthesized 41 o-aminobenzamide compounds based on MS-275 and SAHA scaffolds, performed the in-depth SAR study on five human gastric cancer cells, and evaluated the toxicity in two human normal cell lines. The most promising compounds F8 (IC50 = 0.28 μM against HGC-27 cell) and T9 (IC50 = 1.84 μM against HGC-27 cell) displayed remarkable efficacy in inhibition of cell proliferation, induction of cell apoptosis, arrest cell cycle, and suppression of cell migration as
General information
Reagents and solvents were commercially available and used without further purification. Reactions were performed under the argon atmosphere unless otherwise noted. NMR spectra were tested using 400 MHz liquid state NMR spectrometer (Bruker Company, AVANCE III 400, Switzerland) or 300 MHz liquid state NMR spectrometer (VARIAN Company, Mercury plus 300 BB, USA). Proton chemical shifts (δ) are reported relative to a residual solvent peak (CDCl3 at 7.26 ppm, DMSO‑d6 at 2.50 ppm, acetone-d6 at
Cell lines and cell cultures
HGC-27, MGC-803, AGS, GES-1 and WI-38 cell lines were purchased from the China Center for Type Collection (CCTCC, China) and possessed STR identification. Human gastric carcinoma cell lines HGC-27 (undifferentiation), MGC-803 (low differentiation), SGC-7901 (middle differentiation) were developed in RPMI-1640 containing 10% fetal bovine serum (FBS) (Gemini, Bio-Product, CA, USA) and 1% penicillin-streptomycin (Solarbio, Cat.#: P1400, China), while human gastric cancer cell AGS (low
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 was technically supported by Lanzhou University, Ministry of Education of China, and was financially supported by the Recruitment Program of Global Experts (1000 Talents Plan); Gansu Province Science Foundation for Distinguished Young Scholars (20JR5RA304); the Fundamental Research Funds for the Central Universities (lzujbky-2019-ct08). We thank Dr. Na Yan and the Scientific Research and Experiment Center of School of Pharmacy of Lanzhou University for support in instrumentation, and
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2023, European Journal of PharmacologyCitation Excerpt :The complexity of the structure of natural products determines that their anticancer mechanisms are also unique (Ouyang et al., 2014). It is suggested that some alkaloids can cause apoptosis and induce cell cycle arrest (Feng et al., 2022). The continuous activation of carcinogenic signaling pathways occurs at a high frequency in the occurrence of cancer (Jokinen and Koivunen, 2015).
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These authors contributed equally to the work.