Diarylamine-Guided Carboxamide Derivatives: Synthesis, Biological Evaluation, and Potential Mechanism of Action

Diarylamines are a class of important skeleton widely existing in drugs or natural products. To discover novel diarylamine analogues as potential drugs, two series of diamide and carboxamide derivatives containing diarylamine scaffold were designed, synthesized and evaluated for their potential cytotoxic activities. The bioassay results indicated that some of the obtained compounds (C5, C6, C7, C11) exhibited good cytotoxic effect on cancer cell lines (SGC-7901, A875, HepG2), especially, compound C11 present significantly selective proliferation inhibition activity on cancer and normal cell lines (MARC145). In addition, the possible apoptosis induction for highly potential molecules was investigated, which present compound C11 could be used as novel lead compound for discovery of promising anticancer agents.


INTRODUCTION
According to the progress report published by American Association for Cancer Research in 2019, cancer has become the leading cause of morbidity and mortality around the world, accounting for about 16 percent of deaths worldwide (American Cancer Society, 2018; AACR, 2019). Annual cancer cases and deaths have been increasing since 2000, and in 2018, 24% of estimated new cancer cases and 30% of cancer-related deaths globally occurred in China . It is reported that the most common causes of cancer death were lung cancer, liver cancer and stomach cancer in China . The traditional treatment for cancer includes surgical resection combined with radiotherapy and chemotherapy; however, there are adverse drug reactions or therapeutic resistance. The devastating impact of cancer is predicated to grow significantly in the coming decades unless new and more effective anticancer drugs are developed. From 2009 to 2018, the number of cancer drug trails in China showed remarkable growth, with an average annual growth rate of 33% . The China cancer drugs market size is projected to reach USD 30 billion by the year 2024. Thus, there is an urgent need for developing new anticancer agents with safer toxicological profiles and novel modes of action.
Diarylamines are a class of important scaffold widely existing in drugs and natural products, which can construct diverse molecular structures with extensive pharmacological activity (Kumar and Mishra, 2018;Abduelkarem et al., 2020). Diarylamines have also been used as typical synthons for construction of novel molecules due to their special multifunctional features, and many diarylamines analogues have been demonstrated to exhibit broad range of biological functions (Guan et al., 2013;Wang et al., 2013;Chai et al., 2014;Soussi et al., 2014;Wild et al., 2016;Wang et al., 2017;Zhang et al., 2017). Especially, some important diarylamine derivatives such as selumetinib, imatinib, dasatinib, niflumic acid, and flufenamic acid have been discovered and developed as commercial drugs (Figure 1), which further identify that this special scaffold is an attractive pharmacophore in the discovery of highly potential molecules, and will lead to some promising drug candidates. Therefore, diarylamines as easily available substrates have proven a convenient choice for construction of structural diversity molecules with promising bioactivity. In addition, carboxamides are also a useful class of molecules with potential pharmacological (Odingo et al., 2017;Tang et al., 2018;Barker et al., 2019;Ling et al., 2019;Sethy et al., 2019;Sun et al., 2019;Yang et al., 2019) and agroactive activities (Sartori et al., 2018;Tsikolia et al., 2018;Yu et al., 2018;Liu et al., 2019;Siber et al., 2019;Tsikolia et al., 2019;Liu et al., 2020), and many carboxamides have been developed as clinical drugs or agrochemicals, which demonstrate that amide units will play an important role in the development of drugs and pesticides.
Recently, during the course of our research on discovery of functional molecules, a series of diamides derivatives bearing nicotinamide unit were obtained (Peng et al., 2017), and an interesting phenomenon was found based on the preliminary results. All of the similar diamides without nicotinamide moiety lost the inhibitory activity except the compound bearing diarylamine unit (A2, R = Me), and this compound exhibited good cytotoxic activity against the tested lung cancer cell lines (NCI-H460, A549, and NCI-H1975) with IC 50 values of 14.66-46.42 μM ( Figure 2). However, the corresponding N-unsubstituted diarylamine-amide derivative A1 (R = H) almost lost activity (IC 50 > 80 μM). These interesting findings may provide some useful information for developing diarylamine derivatives as potential anticancer agents, which also urge us to investigate the possible structure and activity relationships (SARs) for these diarylamine derivatives bearing diamide scaffold.
So, a series of extended diarylamine-guided diamides analogues based on the lead compound A2 was constructed for exploring prospective SARs with good cytotoxic activity as shown in Figure 3, and a series of diarylamine-guided carboxamide derivatives have also been designed for comparison with that of diarylamine-guided diamides. We report herein the synthesis and characterization of two series of diarylamine derivatives that have flexible substituent patterns, and their in vitro cytotoxic activities against SGC-7901 (human gastric cancer cell line), A875 (human melanoma cell line), HepG2 (human hepatocellular liver carcinoma cell line) and MARC145 (A subclone of African green monkey kidney cell line MA-104) cell lines were fully investigated. The results demonstrated that compound C11 may be a highly potential selective cytotoxic agent between cancer and normal cell lines. Scheme 1 | General synthetic route for target molecules B1-13 and C1-11. 2 RESULTS AND DISCUSSION

Chemistry
According to the structure of lead molecule A2, various orthoamino aryl acid 2 and diverse substituted aryl amines 5 were utilized as the building blocks to construct these diarylamine guided carboxamide or diamide derivatives. The target molecules B1-13 and C1-11 were conveniently prepared as described in Scheme 1. First, the easily available flufenamic acid 1 (X = CH) was used as starting material for construction of diarylamine-guided diamides B1-13, which were reacted with various ortho-amino aryl acid 2 to obtain the corresponding oxazinone heterocyclic intermediates. Then these oxazinones 3 were treated with various amines resulting in the target diamides derivatives B1-13 via nucleophilic substitution reaction. Subsequently, diverse substituted arylamines 5 were directly adopted to construction diarylamine guided carboxamides C1-11 via nucleophilic substitution reaction of acids 1 (X = CH or N). The structures of all obtained molecules were characterized on the basis of satisfied spectral analysis, and the substituents of all the compounds were present in Scheme 1. All of the newly prepared diamides B1-13 and carboxamide derivatives C1-11 were confirmed by IR, 1 H NMR, 13 C NMR and ESI-MS analyses, and their chemical structures and basic physicochemical properties were summarized in Experimental. The 1 H NMR of these compounds indicated typical proton signal peaks for different groups, and the ESI-MS spectrum indicated all these diarylamineguided diamide derivatives B1-13 displayed an obvious [M + Na] + addition ion fragmentation peak, however, the MS for diarylaminecontaining carboxamide derivatives C1-11 exhibited [M + H] + or [M−H] + ion fragmentation peak.
First, the lead molecule A2 exhibit good in vitro cytotoxic activity against lung cancer cell lines including NCI-H460, A549, and NCI-H1975 based on our previous investigation (Peng et al., 2017). As far as we know, the substituents attached to aryl ring usually have obvious influence on the activity. So, in order to investigate the possible effect of this unit on the activity of final molecules, a series of diarylamine guided diamide derivatives B1-13 were extended synthesized, and their cytotoxic activity was tested against SGC-7901, A875, HepG2, and MARC145 cell lines. Unfortunately, all newly prepared diarylamine-guided diamide derivatives B1-13 present poor effects on these tested cell lines as indicated in the Table 1, however, the lead compound A2 still exhibit very good cytotoxicity with IC 50 values of 7.59-12.43 μM. These results demonstrated that the change of an appropriate substituents on the aryl ring could dramatically change the biological activity.
With these results in hand, we think that the part of orthoamino aryl acid may be important for the bioactivity, and so the molecular simplification strategy was adopted to investigate the potential active molecules. As shown in Scheme 1, a series of   Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 953523 diarylamine guided carboxamides C1-11 were further constructed using diverse substituted aryl amines, and the preliminary screening for these molecules were treated at the concentration of 100 µM ( Table 2). What's interesting is that some of the compounds (C5, C6, C7, C10, and C11) exhibit good inhibition activity, and especially the inhibition rate is even higher than that of the control 5-FU ( Figure 4). We also can find from Figure 4 that compound C11 indicated good inhibition on all three cancer cell lines (SGC-7901, A875, and HepG2), however, which has low inhibition on normal cell lines (MARC145). Base on the aforementioned results, some of the diarylamineguided carboxamide derivatives have been demonstrated to present good inhibitory activities against all tested cancer cell lines, so in order to further investigate the potential activities, the IC 50 values were tested based on the above cell-based method. The in vitro activities described as IC 50 values for these compounds were present in Table 3.
As shown in Table 3, the results further confirmed that some of these diarylamine-guided carboxamide derivatives (C5, C6, C7, C10, and C11) indicated higher inhibition activities compared to the control 5-FU. Generally, the compounds bearing p-isopropylaniline and o-trifluormethylaniline present poor inhibition activities against all tested cell lines (Entries 1-4). The compounds C6 containing m-trifluormethylaniline exhibited significant cytotoxic activities with the IC 50 values of 5.53-12.77 μM (Entry 6), respectively. However, for the compounds containing p-trifluormethylaniline, the compound bearing flufenamic acid (X = CH) present the highest potential activities (Entry 7) than that of the compound containing niflumic acid (X = N). Especially, compound C11 bearing 6chloropyridin-3-amine had a stronger inhibition on cancer cell lines (SGC-7901 IC 50 = 9.13 μM; A875 IC 50 = 12.34 μM; HepG2 IC 50 = 10.15 μM) than the normal cells line MARC145 (IC 50 > 102 μM), and it displayed a significantly selective proliferation inhibition activity on cancer and normal cell lines, and the selectivity index up to 8.26 ( Figure 5). These interesting finds may provide some useful information for developing potential cytotoxicity agents.
In addition, the dose-response analysis of cell growth inhibition for highly potential molecules C5, C6, C7, C11, and 5-FU have been displayed in Figure 6, which identified that these compounds exhibited obvious cytotoxic effects on SGC-7901, A875, HepG2, and MARC145 cell lines with a significant concentration dependence.

Analysis for Cell Apoptosis
Apoptosis induction is one of the important modes of action for antitumor drugs. So, the potential mechanism of highly effective compounds (C6 and C11) on A875 cells were investigated by flow cytometry. The apoptotic effect of compounds C6 and C11 was evaluated and analyzed following 24 h of treatment with 0.5×IC 50 , IC 50 or 2×IC 50 concentrations using Annexin V-FITC/PI dual staining assay.
All data obtained in this work are described in Figure 7, and we can find that compounds C6 and C11 induced apoptotic changes after 24 h treatment, and both compounds initiated apoptosis, in terms of FITC(+)PI(−)   Frontiers in Chemistry | www.frontiersin.org July 2022 | Volume 10 | Article 953523 6 staining, to a larger extent than the control. 12% and 27% apoptotic rates are observed with treatment of compounds C6 and C11 at 2×IC 50 concentrations respectively, whereas 2.14% of apoptosis was observed in control (0.1% DMSO), which indicated that these two potential molecules can induce apoptosis in A875 cells at least partly. However, the clearer mechanism of cell death induction by these compounds still remain to be further investigated.

Determination of Lactate Dehydrogenase
From the preliminary analysis of apoptosis, the results encourage us to further investigate the type of cell death induced by the potential compounds (C6 and C11). As we know, the release of LDH is a typical characteristic feature of necrotic cell death, and so which was detected. From the results indicated in Figure 8, we can find that the release of LDH was significantly increased at 24 h after treatment with compounds C6, C11, and 5-FU (Figure 8), which were consistent with those of flow cytometry.

Kinase Inhibition Assay
Based on the aforementioned results for the potential mechanism of action, the kinase inhibitory profile of compounds C6 and C11 were screened at two dose concentration of 10 and 1 µM over a panel of 15 kinases, and the kinase inhibitory activities are listed in supporting information (Supplementary Table S1). It was found that compound C6 exerted certain inhibitions on the tested MAPK1 and CDK2/A2 protein kinases at 1 μM, and compound C11 also indicated some inhibitions on the tested CDK1/A2, CDK2/A2, MAPK1 protein kinases at 1 μM, however, the inhibitory activity is not very good. These results will provide some reference for further mechanism research.

CONCLUSION
In summary, two series of diarylamine-guided diamide and carboxamide derivatives based on lead molecule have been designed and synthesized, and their potential cytotoxic activities were fully investigated by cell-based assay. The results demonstrated that some of the obtained diarylamineguided carboxamide derivatives (C5, C6, C7, C11) had good cytotoxic effect on cancer cell lines (SGC-7901, A875, HepG2) compared with 5-FU in vitro, especially, compound C11 present significantly selective proliferation inhibition activity between cancer and normal cell lines, and the selectivity index was greater than 8.26, and these interesting results might be helpful to develop diarylamine-guided carboxamides as potential anticancer agents.

Materials and Apparatus
Melting points (m.p.) were measured using a digital model X-5 apparatus (Shanghai Instrument Physical Optics Instrument Co., LTD., Shanghai, China) and were uncorrected. Infrared (IR) Spectra were recorded using Shimadzu FTIR 8400S spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded on a Bruker spectrometer at 600 MHz (Bruker, Bremen, Germany) with CDCl 3 , DMSO-d 6 or CD 3 OD as the solvent. Liquid chromatography-tandem mass spectrometry (LC−MS/MS) analysis were performed on a Waters ACQUITY UPLC ® H-CLASS PDA (Waters ® , Milford, MA, United States) instrument. Analytical thin-layer chromatography was carried out on precoated plates, and spots were visualized with ultraviolet light. All chemicals or reagents used for syntheses were commercially available.