Concise synthesis and biological activity evaluation of novel pyrazinyl–aryl urea derivatives against several cancer cell lines, which can especially induce T24 apoptotic and necroptotic cell death

Abstract

Based on the structural modification of regorafenib, 28 pyrazinyl–aryl urea derivatives were synthesized and their in vitro antiproliferative activities were evaluated. Six compounds (5-16, 5-17, 5-18, 5-19, 5-22, and 5-23) exhibited favorable inhibitory activity against the human bladder cancer T24 cell line, and 5-23 demonstrated the strongest inhibitory activity (IC₅₀ = 4.58 ± 0.24 μM) with high selectivity. Compound 5-23 induced apoptosis in the low concentration range (≤7.5 μM) combined with shorter incubation time (≤10 h) via the activation of caspases, while high concentrations and prolonged incubation times led to necroptotic cell death by activating the RIPK1/RIPK3/MLKL signaling pathway. Induced apoptosis and necroptosis were closely associated with intracellular reactive oxygen species generation and decreased mitochondrial membrane potential. Compared with regorafenib, 5-23 displayed improved pharmacokinetic profiles in an in vivo rat model. Molecular docking and structure–activity relationship analyses were in agreement with the biological data. Compound 5-23 may be a potent anti-bladder cancer agent and this small molecule can be considered as a promising structure for further optimization.

Based on structural modification of regorafenib, 28 pyrazinyl–aryl urea derivatives were synthesized and the in vitro anti-cancer effects were evaluated. Compound 5-23 possessed the strongest inhibitory activity against bladder cancer T24 cell line.

Introduction

Bladder cancer (BC), characterized by a high recurrence rate, is the most common malignancy involving the urinary system.^1,2 According to global cancer statistics, approximately 500 000 cases are newly diagnosed annually and there were 200 000 BC-associated deaths worldwide.³ Despite the strenuous efforts of researchers, the development of therapeutic drugs for BC is in slow progress in the past 10 years. BC can be categorized as non-muscle-invasive BC (NMIBC) and muscle-invasive BC (MIBC) subtypes, according to the distinct genetic background and clinical prognosis.⁴ For NMIBC, complete resection of the tumor followed by intravesical instillation of chemotherapeutic agents remains the mainstay of treatment. For MIBC, a multimodal treatment approach is necessary, which at least involves radical cystectomy with neoadjuvant chemotherapy.⁵ Cisplatin-based combination chemotherapies such as MVAC (methotrexate, vinblastine, adriamycin, and cisplatin) and GC (gemcitabine, cisplatin) represent the standard regimens.¹ However, the utilization of these agents is severely limited by substantial toxicity, including neutropenia, mucositis, cardiac and neurologic toxicity.^1,6 Hence, it is imperative to develop novel strategies, especially the discovery of new chemotherapeutic agents with high potency and minimal side effects. Unlike conventional chemotherapy, targeted chemotherapy specifically attacks signaling pathways controlling cancer cell proliferation and hindering tumor mass growth.^7,8 Over the last decade, important advances have been made in cancer targeted therapy; in particular, the successful development of molecular-targeted agents (MTAs) with various chemical structures has led to a significant change in treatment strategies.^8–11

Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis, and its receptor vascular endothelial growth factor receptors (VEGFRs) are key regulators of this process.¹² The VEGFR family proteins consist of VEGFR-1 (FMS-like tyrosine kinase [FLT]-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (FLT-4).¹³ Several VEGFR inhibitors have been marketed as antitumor drugs and a range of inhibitors are undergoing clinical or preclinical studies, such as regorafenib (BAY 73-4506, Stivarga®, Reg),^14–16 sunitinib,¹⁷ pazopanib,¹⁸ apatinib (YN968D1, Apat),^19,20 and so on. Small MTA Reg is an oral multiple kinase inhibitor that targets several protein kinases at least including VEGFR 1–3, platelet-derived growth factor receptor-β (PDGFR-β), fibroblast growth factor receptor (FGFR), and Raf.^14–16 Reg was proved to be effective in a variety of solid tumor models and approved by the U.S. Food and Drug Administration (FDA) for treating colorectal carcinoma, gastrointestinal stromal tumors, and hepatocellular carcinoma (HCC). In BC models, its therapeutic effects are being evaluated by several groups.²¹ Apat is a small-molecule anti-angiogenic agent that selectively inhibits VEGFR-2.^19,20

Programmed cell death (PCD) is a genetically regulated process of cellular suicide at a special time and position with endogenous or environmental induction signals for multicellular organisms.²² It plays an important role in multiple biological processes of mammals such as organismal development, physiological homeostasis, epithelial cell renewal, and lymphocyte selection.²³ Apoptosis, also called type I PCD, is the most common mode of cell death, which is initiated and executed by a unique family of cysteine-dependent aspartate-directed proteases known as caspases.²⁴ Apoptosis has been considered the only type of PCD for a long time, however, current studies have revealed that necrosis can also be executed as a programmed process and is regulated by specific intracellular signaling pathways, and multiple modes of cell death have been discovered, such as necroptosis.^23,25–27 Necroptosis was coined by Prof. Yuan and her colleagues in 2005.²⁷ Accumulating evidence suggests that necroptosis offers the potential for treating a wide variety of pathologies, including inflammation and cancer.^26,28–30 Necroptosis is mainly mediated by the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like pseudokinase (MLKL).^31,32 The classic necroptosis pathway involves the activation of RIPK1, which binds and phosphorylates downstream RIPK3 to generate a complex called necrosome, also called RIP1/RIP3 signaling complexes. The complexes recruit and phosphorylate downstream MLKL, which then oligomerizes and translocates to the plasma membrane.^25,28,31,32 Necroptosis can be pharmacologically inhibited by several chemical agents, such as necrostatin-1 (Nec-1, RIPK1 inhibitor) and necrosulfonamide (NSA, MLKL inhibitor).^25,26,32,33

In the past few years, our group has been devoted to exploring small MTAs with high potency and selectivity.^34–36 Not long ago, we covered a series of novel quinazolinyl–diaryl urea derivatives and investigated the anti-tumor effects on several cancer cell lines.^34,35 To find compounds with superior bioactivity, more aryl urea derivatives with various structural types were prepared by combinatorial synthesis. In this study, 28 pyrazinyl–aryl urea derivatives are reported. Among these target compounds, 5-23 exhibited favorable proliferation inhibitory activity against T24 cells with the lowest IC₅₀ value, showing high selectivity towards normal human cell line HCV29; therefore, we have focused on T24 cell death induced by 5-23 and try to give reasonable explanations at both the molecular and genetic levels. Our results suggest that different concentrations and incubation times of 5-23 could induce two different death modes: apoptosis and necroptosis; however, the positive reference drug, Reg, inhibited T24 cell necroptosis.

Results and discussion

Synthesis

The design concept of these pyrazinyl–aryl urea derivatives is as follows: the urea group in the chemical structure of Reg was kept intact, which is a key pharmacophore; the modifications included the left-most pyridyl moiety, intermediate linker, and right-most aryl group. The pyridyl moiety was replaced with the pyrazinyl group, while, the linker moiety of phenoxyl was replaced with the benzylamino group. Various substituents were introduced to the distal phenyl ring, or the phenyl ring was replaced by naphthenic groups for structure–activity relationship (SAR) studies. Based on the above, a total of twenty-eight novel pyrazinyl–aryl urea derivatives 5-1–5-28 were designed (Fig. 1). A general synthetic route to pyrazinyl–aryl urea derivatives (5-1–5-28) is outlined in Scheme 1. The commercially available starting material 2,3-dichloropyrazine (1) was reacted with 3-aminobenzylamine at 100 °C. It should be mentioned that under this condition, only one chlorine atom in the 2,3-dichloropyrazine molecule could be replaced; it was very difficult to replace another chlorine atom. A series of isocyanates (4) were prepared by treating the corresponding aromatic or naphthenic amines (3) with triphosgene (BTC) in ClCH₂CH₂Cl, which has been covered by our group.³⁴ Target compounds (5-1–5-28) were successfully obtained via the reaction of the intermediate (2) with the above-prepared isocyanates (4) in the presence of Et₃N. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography. The chemical structures of these novel compounds were confirmed by melting point (m.p.), hydrogen nuclear magnetic resonance (¹H NMR), ¹³C NMR, and high-resolution mass spectra (HRMS). Original spectra of typical compounds can be found in the ESI† data (see additional file 1).

Fig. 1. Rationale for the design of pyrazinyl–aryl urea derivatives (5-1–5-28) based on the reference drug regorafenib (Reg).

Scheme 1. The synthesis of pyrazinyl–aryl urea derivatives (5-1–5-28). Reagents and conditions: (a) 3-aminobenzylamine, 1,4-dioxane, 100 °C, N₂ atmosphere; (b) ClCH₂CH₂Cl, Et₃N, triphosgene, 0 °C for 15 min, then r.t. for 0.5 h, and finally 85 °C for 8 h; (c) ClCH₂CH₂Cl, Et₃N, r.t.

Compound 5-23 exhibited favorable proliferation inhibitory activity against the T24 cell line

The in vitro anti-proliferation effect of synthesized pyrazinyl–aryl urea derivatives against five human tumor cell lines including Hep G2 (liver), MGC-803 (stomach), T24 (bladder), NCI-H460 (lung), and PC-3 (prostate) were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. Reg and gemcitabine (Gem) served as dual positive reference drugs. IC₅₀ values (μM) of the target compounds are presented in Tables 1 and S1.† Compared with other cell lines, T24 was sensitive to most of the compounds. IC₅₀ values of six compounds (5-16, 5-17, 5-18, 5-19, 5-22, and 5-23) against human BC T24 cells were <6 μM. Among them, 5-23 gave the most favorable results, which was also superior to Reg and Gem. It was found that 5-23 inhibited the proliferation of T24 cells in a time- and dose-dependent manner with the IC₅₀ values of 9.18 ± 0.34, 4.58 ± 0.24, and 3.55 ± 0.13 μM after 24, 48, and 72 h of incubation, respectively (Fig. S1†). Moreover, 5-23 displayed very low cytotoxicity against normal human bladder epithelial cell line HCV29 (IC₅₀ > 30 μM) with a favorable selectivity index (SI > 6). Promising anti-proliferative activity and high selectivity drove us to further explore the possible molecular mechanisms and elucidate the modes of T24 cell death induced by this compound, as presented in the following sections.

Proliferation inhibitory effects of prepared pyrazinyl–aryl urea derivatives (5-1–5-28) on several human tumor cell lines, and their selectivity index (SI) values.

Compd.	R	IC50a,b (μM)				SId
		Hep G2	MGC-803	T24	HCV29
5-1	Phenyl	15.55 ± 1.03	16.31 ± 0.94	19.82 ± 1.19	36.19 ± 0.94	1.83
5-2	2-Methylphenyl	15.31 ± 0.76	14.02 ± 0.89	13.62 ± 0.95	33.61 ± 1.02	2.47
5-3	3-Methylphenyl	11.24 ± 0.61	14.56 ± 0.72	9.57 ± 0.73	28.33 ± 0.98	2.96
5-4	4-Methylphenyl	11.49 ± 0.88	11.91 ± 0.66	9.82 ± 0.79	20.09 ± 0.62	2.05
5-5	2,4-Dimethylphenyl	9.29 ± 0.49	10.07 ± 0.77	9.23 ± 0.59	23.77 ± 1.09	2.58
5-6	3,4-Dimethylphenyl	10.74 ± 0.61	9.87 ± 0.55	8.93 ± 0.43	12.81 ± 0.58	1.43
5-7	2-Ethylphenyl	7.86 ± 0.73	9.37 ± 0.49	8.72 ± 0.58	16.35 ± 0.49	1.88
5-8	2,6-Diethylphenyl	10.81 ± 0.75	6.13 ± 0.57	6.39 ± 0.59	18.74 ± 0.76	2.93
5-9	4-(tert-Butyl)phenyl	7.55 ± 0.34	4.82 ± 0.41	6.01 ± 0.47	15.29 ± 0.66	2.54
5-10	2-Methoxyphenyl	13.69 ± 0.63	12.19 ± 0.53	10.41 ± 0.51	18.16 ± 0.58	1.74
5-11	4-Methoxyphenyl	12.03 ± 0.95	9.24 ± 0.84	9.82 ± 0.73	20.92 ± 0.61	2.13
5-12	4-Ethoxyphenyl	7.05 ± 0.52	7.24 ± 0.45	9.08 ± 0.44	30.52 ± 0.95	3.36
5-13	2-Fluorophenyl	10.51 ± 0.73	6.44 ± 0.58	7.45 ± 0.36	25.12 ± 0.83	3.37
5-14	2-Chlorophenyl	8.56 ± 0.59	7.13 ± 0.61	6.68 ± 0.44	28.09 ± 1.18	4.21
5-15	3-Chlorophenyl	9.14 ± 0.51	5.33 ± 0.47	6.81 ± 0.47	21.37 ± 0.84	3.14
5-16	4-Chlorophenyl	8.35 ± 0.43	4.98 ± 0.49	5.47 ± 0.33	19.38 ± 0.97	3.54
5-17	3-Chloro-4-fluorophenyl	6.75 ± 0.33	5.02 ± 0.46	4.99 ± 0.31	24.66 ± 0.89	4.94
5-18	2,4-Dichlorophenyl	7.23 ± 0.36	6.43 ± 0.29	5.74 ± 0.51	25.38 ± 1.37	4.42
5-19	3,4-Dichlorophenyl	6.17 ± 0.51	4.97 ± 0.37	5.21 ± 0.28	27.21 ± 1.29	5.22
5-20	3-Chloro-4-methylphenyl	11.29 ± 0.97	17.53 ± 1.19	7.39 ± 0.42	21.49 ± 1.02	2.91
5-21	2-Bromo-4-chlorophenyl	6.72 ± 0.48	7.48 ± 0.47	6.65 ± 0.41	24.03 ± 1.57	3.61
5-22	3-Trifluoromethylphenyl	6.21 ± 0.44	7.29 ± 0.53	5.08 ± 0.26	28.65 ± 1.31	5.64
5-23	4-Trifluoromethylphenyl	5.05 ± 0.46	6.91 ± 0.67	4.58 ± 0.24	30.03 ± 1.69	6.56
5-24	4-(N,N-Dimethyl)phenyl	>40	11.53 ± 0.44	20.19 ± 0.76	>40	N.D.
5-25	4-Methylsulfonylphenyl	15.08 ± 0.92	12.32 ± 0.78	29.34 ± 1.39	>40	N.D.
5-26	α-Naphthyl	>40	32.71 ± 1.09	>40	>40	N.D.
5-27	Cyclopentyl	18.41 ± 1.21	23.41 ± 0.91	>40	>40	N.D.
5-28	Cyclohexyl	>40	>40	>40	>40	N.D.
Reg	—	10.43 ± 0.89	9.46 ± 0.62	5.98 ± 0.31	12.89 ± 0.57	2.16
Gem	—	N.D.c	N.D.	10.15 ± 0.62	21.48 ± 1.07	2.12

^aAnti-proliferative activity was evaluated by MTT assay in cancer or normal cell lines (Hep G2, MGC-803, T24, HCV29) treated with different concentrations of pyrazinyl–aryl urea derivatives and positive reference drugs (Reg, Gem) for 48 h, respectively. Data shown are means ± S.D. of four independent experiments.

^bCompounds with IC₅₀ values > 40 μM are considered to be inactive.

^cN.D. = not determined.

^dSI = The mean IC₅₀ value of the compound against HCV29 cells/the mean IC₅₀ value against T24 cells.

Lower concentrations combined with shorter co-incubation times of 5-23 led to T24 apoptotic cell death

Induced T24 cell apoptosis by 5-23 was investigated using Annexin V-FITC/propidium iodide (Annexin/PI) dual staining assay.³⁷ As demonstrated in Fig. 2A and Table S2,† after T24 cells were co-incubated with 5-23 for 10 h, the apoptotic ratios (early plus late apoptotic cells, Q2 + Q3) of the cells were 3.24% ± 0.31%, 9.01% ± 0.74%, 14.83% ± 1.26%, and 21.41% ± 1.39% at concentrations of 0, 2.5, 5, and 7.5 μM, respectively. We also investigated whether 5-23-induced apoptosis was due to caspase activation. T24 cells were co-incubated with 5-23 and a cell-permeable, irreversible pan-caspase inhibitor z-VAD-fmk (zVAD, 30 μM) for 10 hours. As expected, the addition of zVAD significantly decreased the cell apoptotic ratios, in comparison with the group treated with 5-23 alone (Fig. 2A and Table S2†). Moreover, compared with Reg and Gem, 5-23 showed a stronger ability to induce apoptosis at the same concentration based on the higher apoptotic ratios (Q2 + Q3) in the groups of 5-23-treated T24 cells. The Hoechst 33342/propidium iodide (Hoe/PI) double-labeling technique was used and the results are shown in Fig. S2.† For the negative control (Con), very weak blue fluorescence and almost no red fluorescence were observed. However, with increased concentrations of 5-23, a strong red fluorescence signal was observed in T24 cells, which suggested that induced apoptosis by 5-23 occurred in a concentration-dependent manner.

Fig. 2. Lower concentrations and shorter co-incubation times of 5-23 induced T24 apoptotic cell death. (A) T24 cell apoptosis analysis by flow cytometry. (B–F) Apoptosis-related protein expressions by Western blotting analysis. T24 cells were treated with different concentrations of 5-23 in the presence of an irreversible pan-caspase inhibitor Z-VAD-FMK (zVAD, 30 μM) for 10 h, subsequently stained with Annexin and PI. The population of apoptotic cells was determined using flow cytometry. In figure (A), only representative flow cytometry graphs are shown. The expressions of cytochrome c (cyto c), Bax, Bcl-2, caspase-3 (casp-3), casp-8, cleaved casp-3, and cleaved casp-8 were analyzed by Western blotting. β-Actin was used as a loading control. * p < 0.05, ** p < 0.01, and *** p < 0.01 versus control. In figure (E and F), # p < 0.05, ## p < 0.01, and ### p < 0.01 versus the group treated with the same concentration of 5-23 (absence of zVAD).

To more deeply understand 5-23-induced apoptotic cell death, apoptosis-associated proteins including cytochrome c (cyto c), Bax, Bcl-2, caspase-3 (casp-3), caspase-8 (casp-8), cleaved caspase-3 (cleaved casp-3), and cleaved caspase-8 (cleaved casp-8) were analyzed by Western blotting. As shown in Fig. 2B–F, the expressions of Bax and cyto c were up-regulated, while the level of Bcl-2 was decreased. For casp-3, casp-8, and their active forms (cleaved casp-3 and -8), different results were obtained. Within the range of 0–5 μM, 5-23 treatment caused a concentration-dependent increase; at the concentration of 5 μM, their expression levels reached the peak, and then gradually decreased. When the concentration of 5-23 was set at 10 μM, their respective expression levels in the experimental group were lower than that in the control group (Fig. 2D–F). Caspase-dependent apoptosis occurs principally via two distinct but interrelated mechanisms: the extrinsic pathway initiated by the activation of death receptors, and the intrinsic mitochondria-mediated pathway triggered by the release of cyto c from the mitochondrial matrix.³⁸ Abnormally elevated caspase activity after exposure to chemotherapeutic agents is usually considered as a sign of apoptosis.³⁹ Our results demonstrated that T24 cells treated with lower concentrations of 5-23 within a short period of time (≤10 h) resulted in caspase-dependent apoptotic cell death as evidenced by the activation of casp-3 and -8, and the release of cyto c.

High concentrations combined with prolonged incubation time of 5-23 could induce T24 necroptotic cell death

When the concentrations of 5-23 were ≥7.5 μM and the incubation time was prolonged (>10 h), a significant decrease (but no increase) in apoptotic ratios of T24 cells was detected; meanwhile, the expressions of casp-3 and -8 were down-regulated (Fig. 2). The following two conclusions were drawn: (1) induced apoptosis of T24 cells was closely associated with the concentration and co-incubation time; (2) there should be another death mode that is distinct from apoptosis.

Necroptosis is now being considered as a backup for apoptosis when the catalytic activity of caspases (particularly casp-8) is lost.^40,41 To explore whether 5-23 could trigger T24 cell necroptosis, T24 cells were co-incubated with 5-23 and Nec-1 (60 μM). For the groups without the addition of Nec-1, the ratio of necrotic cells (Q4 quadrant) was dose-dependently increased after 16 hour incubation. After the addition of Nec-1, the ratio decreased significantly, while the ratio of viable cells was increased as compared with the group treated with 5-23 alone (Fig. 3A and S3†). The ratio of necrotic cells was much higher than that of late apoptotic cells (Q3 quadrant) at the same concentration of 5-23 (Fig. S3†). The results in the trypan blue exclusion assay showed that the percent of living cells was considerably reduced in the groups treated with 5-23 from 51.03% ± 2.83%, to 38.01% ± 2.91%, then to 24.79% ± 3.39% at concentrations of 7.5, 10, and 12.5 μM, respectively; the addition of Nec-1 resulted in a significant increase in the percent of living cells (Fig. 3B). In previous studies, the cytotoxicity of Nec-1 to cells was underestimated; however, an obvious cytotoxic effect could be detected once its concentration was over 80 μM (Fig. 3C). In this study, the concentration of Nec-1 was set at 60 μM. The addition of Nec-1 was unable to attenuate Reg- or Gem-induced cell death but led to lower cell survival ratios (Fig. 3D and E), and this result suggested that Reg and Gem-triggered T24 cell death was not mediated through necroptosis, which was first reported.

Fig. 3. High concentration combined with prolonged incubation of 5-23 led to T24 cell necroptosis. (A) Flow cytometric analysis. (B, D and E) Trypan blue staining assay. (C) Cytotoxicity effect of Nec-1 on T24 cells. In figure (A, B, D and E, T24 cells were treated with 5-23 (or positive reference drugs) in the presence or absence of Nec-1 (60 μM); incubation time: 16 h. * p < 0.05 and ** p < 0.01 versus the groups treated with the same concentration of 5-23 (absence of Nec-1).

To determine the molecular mechanisms involved in necroptosis after treatment with 5-23, necroptosis-related proteins were analyzed. Reg dose-dependently down-regulated the expressions of RIPK1, RIPK2, RIPK3, and MLKL (Fig. 4A). The expressions of phosphorylated proteins (p-RIPK1, p-RIPK3, and p-MLKL) were also down-regulated (Fig. 4B), which suggested that Reg probably inhibited necroptosis of T24 cells. Quite distinct from these results, there was a significant increase in the expression levels of RIPK1, RIPK2, and MLKL in the groups treated with high concentrations (≥7.5 μM) of 5-23. Meanwhile, the expressions of p-RIPK1, p-RIPK3, and p-MLKL were significantly up-regulated; the addition of Nec-1 led to decreases in the expression levels (Fig. 4B). In the literature,^25,26,33 Nec-1 was typically regarded as an inhibitor of RIPK1. In our study, the addition of Nec-1 could also attenuate the phosphorylation of RIPK1 besides inhibiting the activation of RIPK1. Moreover, in our study Nec-1 may have suppressed the phosphorylation of RIPK3 and MLKL in a cascaded way. In another experiment, the addition of NSA reduced the levels of phosphorylated MLKL as compared with the groups treated with 5-23 alone (Fig. S4A†). After this, the concentrations of the above three kinases (RIPK1, RIPK3, and MLKL) in T24 cells were monitored using commercially available ELISA kits. It was found that the concentrations of RIPK1, RIPK3, and MLKL in those groups treated with 5-23 were significantly increased as compared with the negative control (Fig. S4B–D†). However, in a lower concentration range (≤7.5 μM) combined with shorter incubation time, 5-23 treatment had little effect on the levels of RIPK1 and MLKL (Fig. S4E†), which suggested that intracellular RIPK1 and MLKL could be activated only when T24 cells were exposed to higher concentrations of 5-23. In contrast, Reg incubation led to a steady concentration-dependent decrease in the expression levels of the above proteases, which once again proved that Reg inhibited the activation of the RIPK1/RIPK3/MLKL signaling pathway (Fig. S4B–D and F†). The RT-PCR technique was then used to quantitatively analyze apoptosis- and necroptosis-related gene expressions. There was a significant increase in the expression levels of caspase-8 mRNA within the first 10 hours, while prolonged incubation times led to down-regulation (Fig. 5A). Moreover, it was found that high concentrations of 5-23 incubation led to dose-dependent increases in the expression levels of the three genes RIPK1 mRNA, RIPK3 mRNA, and MLKL mRNA (Fig. 5B–D). From these, the changing trend of protein expressions was consistent with that of the corresponding gene expressions. Our results suggest that exposure to high concentrations (≥7.5 μM) combined with prolonged incubation time (>10 h) of 5-23 led to T24 necroptotic cell death, while Reg inhibited necroptosis.

Fig. 4. The effects of 5-23 on necroptosis-related proteins in T24 cells in the presence or absence of Nec-1 (60 μM). After the T24 cell line was co-incubated with 5-23 for 16 h, the expressions of intracellular RIPK1–3 and MLKL were measured via western blotting (A). Meanwhile, the expressions of the phosphorylated forms including p-RIPK1, p-RIPK3, and p-MLKL were analyzed (B). In figure (A) and (B), * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control. # p < 0.05, ## p < 0.01, and #### p < 0.001 versus the group treated only with the same concentration of 5-23 (absence of Nec-1).

Fig. 5. Intracellular mRNA expression levels of casp-8 (A), RIPK1 (B), RIPK3 (C), and MLKL (D) after treatment with 5-23. All samples were compared with corresponding controls. Samples were normalized to GAPDH for RT-PCR. In figure (A), # p < 0.05 and ## p < 0.01 versus control. In figure (B and D), * p < 0.05; incubation time: 16 h.

It is well known that Reg is an inhibitor of VEGFRs and has shown excellent proliferation inhibitory activity against colorectal cancer cell lines.^14,15 Given this fact, the changes in the gene and protein expressions of VEGFR-1 and -2 in T24 cells before and after exposure to 5-23 were examined. Unexpectedly, the mRNA content of VEGFR-1 and -2 in T24 cells was very low, and their respective expression levels were about 1% of that of caspase-8. In our opinion, it is quite possible that the levels of VEGFRs may be strikingly different among different types of cell lines. Even so, their content in T24 cells was measurable. Compound 5-23 down-regulated the expressions of VEGFR-1 and -2 mRNA in a concentration-dependent manner. After the addition of Apat, their expression levels were further decreased (Fig. S5A and B†).

For similar reasons, the proteases of VEGFR-1 and -2 were not detectable. We analyzed phosphorylated VEGFR-2 including p-VEGFR-2 (Tyr951) and p-VEGFR-2 (Tyr1175), and the results are shown in Fig. S5C.† There were only two very shallow bands and their molecular weights were about 150 kDa. According to the results published on CST's official website, their molecular weight should be between 200 and 250 kDa. However, we failed to obtain any bands within this range. Even if we added appropriate inhibitors to prevent the degradation of VEGFR-1, VEGFR-2, and their phosphorylated forms when extracting the total proteins, the bands in their correct positions were still not detected. In other words, although VEGFRs (especially VEGFR-2) are excellent targets for the development of colorectal cancer therapeutic drugs, whether they are suitable targets for anti-BC agents has yet to be studied. Of course, multiple BC cell lines should be analyzed and compared to help to understand the differences in intracellular VEGFRs' expressions. This is in progress.

Reactive oxygen species (ROS) production and loss of mitochondrial membrane potential induced by 5-23

The abnormal generation of intracellular ROS induced by various chemotherapeutic agents can damage the function of mitochondria, resulting in cell apoptosis/necroptosis.⁴² Intracellular RIPK3 physically interacts with specific metabolic enzymes and leads to ROS production during cell necroptosis; in the apoptosis process, ROS have been shown to regulate the Bcl-2 family protein activity via multiple mechanisms.^43,44 As shown in Fig. 6A, after treatment with 5-23, intracellular DCF fluorescence intensity was increased in a concentration-dependent manner. Intracellular ROS levels were elevated to 153.2% ± 4.6%, 189.4% ± 7.7%, and 243.5% ± 7.3% of the negative control at concentrations of 5, 7.5, and 10 μM, respectively (Fig. 6B). The addition of Nec-1 (60 μM) or z-VAD (30 μM) led to a significant decrease in ROS levels, compared with the group treated with 5-23 alone (Fig. 6C). Then, we evaluated whether 5-23-induced necroptosis and apoptosis were associated with the dissipation of the mitochondrial membrane potential (ΔΨ_m). As shown in Fig. S6,† JC-1 emitted very strong red fluorescence and almost no green fluorescence was observed in the control group, which indicated high ΔΨ_m. For the experimental groups, the green fluorescence intensity increased rapidly with the increased concentration of 5-23, indicating a decrease in the ΔΨ_m. In our opinion, the 5-23-induced ROS production and loss of ΔΨ_m are closely related; both directly resulted in mitochondrial dysfunction and are probably important factors responsible for T24 apoptotic/necroptotic cell death.

Fig. 6. ROS generation induced by 5-23. (A and B) ROS level analyses of T24 cells using flow cytometry (qualitatively) and luminescence spectroscopy (quantitatively), respectively. Incubation time: 16 h. (C) Relative fluorescence intensity (% of control) in the presence (or absence) of zVAD (30 μM) or Nec-1 (60 μM). After T24 cells were treated with different concentrations of 5-23, intracellular ROS levels were monitored using the fluorescent indicator DCFH-DA. Data represent the mean ± S.D. of three independent experiments. In figure (B), * p < 0.05 and ** p < 0.01 versus control. In figure (C), # p < 0.05 versus the group treated with the same concentration of 5-23 (absence of Nec-1).

Pharmacokinetic evaluation

After many attempts, we found that the concentration–time data were fitted to a one-compartment model with a weight of 1. The relevant pharmacokinetic parameters of 5-23 and Reg are summarized in Table 2, and plasma concentration-time profiles are shown in Fig. 7. It should be noted that although compound 5-23 was an analogue of Reg, the chromatographic conditions for the determination of Reg were not suitable for analyzing 5-23. HPLC methods are described in detail and typical chromatograms are listed in ESI† (see additional file 1). The K_a of the Reg suspension, low and high doses of 5-23 suspension were 0.138 ± 0.036 h, 0.098 ± 0.015, and 0.107 ± 0.028 h⁻¹, respectively. At the same dose (12 mg kg⁻¹), Reg was absorbed a little faster than 5-23, but without significant difference (p > 0.05); the C_max of Reg suspension was slightly lower than that of 5-23 suspension. More importantly, the AUC_{0–48 h} and AUMC of the 5-23 suspension were significantly higher than those of the Reg suspension, and the p values were 0.046 and 0.0089, respectively. The Cl of the 5-23 suspension was lower than that of the Reg suspension, and the T_max value of the former was significantly greater than that of the latter. These results suggest that compared with the Reg suspension, the 5-23 suspension had slower absorption and elimination, and longer action time in vivo.

Pharmacokinetic parameters of 5-23 and regorafenib after intragastric administration (mean ± S.D., n = 6).

Parameters	Unit	Reg (12 mg kg−1)	Compd. 5-23 (6 mg kg−1)	Compd. 5-23 (12 mg kg−1)
C max	ng mL−1	3105 ± 183	1193 ± 121	3361 ± 198
T max	h	8.07 ± 0.43	12.03 ± 0.22	12.39 ± 0.37*
AUC0–48h	h ng mL−1	50 696 ± 5876	30 821 ± 3997	75 828 ± 6313*
AUMC	h2 ng mL−1	737 185 ± 86 972	606 261 ± 53 957	1 494 860 ± 103 279**
K a	h−1	0.138 ± 0.036	0.098 ± 0.015	0.107 ± 0.028
Cl	mL h−1	59.18 ± 8.13	48.67 ± 4.76	39.56 ± 6.36
t 1/2	h	5.06 ± 0.41	6.56 ± 0.24	7.17 ± 0.37
MRT0–48h	h	7.29 ± 0.62	9.17 ± 0.49	10.34 ± 0.68

Fig. 7. The pharmacokinetic profiles of 5-23 and Reg in rats after intragastric administration.

Molecular docking and structure–activity study

As key signal transduction molecules, MLKL and its phosphorylated form (p-MLKL) play an important role in regulating necroptosis.⁴⁵ In our study, higher concentrations and prolonged incubation times of 5-23 up-regulated the expressions of MLKL and p-MLKL protease. Given these facts, we tried to find the probable binding mode of 5-23 to the receptor p-MLKL. Unfortunately, to date, the three-dimensional structure of p-MLKL has not been unveiled and the ligand bound to MLKL has to be used. MLKL consists of two chains (a and b) and 5-23 mainly interacted with chain b (Fig. 8A). Four H-bonds were formed between 5-23 and MLKL: two hydrogen bonds (NH₂⋯O) with distances of 1.8 and 1.9 Å formed, respectively, between two imino groups (NH) in the urea moiety and the carboxyl oxygen atoms of the residue LEU209; a H-bond with a distance of 2.3 Å formed between the oxygen atom and the amino of the residue LYS367 (O⋯HN); the last bond, with a distance of 1.9 Å, formed between the amino and one carboxyl oxygen atom of the residue GLU293 (NH⋯O) (Fig. 8B). There was a binding pocket “M” that was neither big nor small (Fig. 8C–E), which was constituted by a series of amino acid residues and could just accommodate those molecules like 5-23. Additionally, a strongly hydrophobic interaction between the distal trifluoromethyl of the ligand and multiple amino acid residues of the receptor strengthened the affinity. In comparison, a smaller molecular volume of 5-1 resulted in a weaker affinity. As shown in Fig. S7,† there were only three H-bonds formed between 5-1 and the receptor, which led to a decrease in the binding energy. The calculated binding energies of 5-23 and 5-1 were −7.34 and −5.61 kcal mol⁻¹, respectively. For the positive reference drug Reg, very different results were obtained. Besides the three H-bonds mentioned above, two additional H-bonds were formed between the carbonyl oxygen atom in the Reg molecule and two NH of the residue ARG297 (O⋯HN) of chain b (Fig. 8F). For this reason, there was a strong interaction between Reg and the receptor MLKL with the binding energy of −9.82 kcal mol⁻¹. More importantly, opposite pharmacological behaviors were shown: from an antagonistic effect (for Reg) to activation (for 5-23 and 5-1) effect on MLKL. It should be noted that the above-mentioned binding model can explain why intracellular MLKL is activated after the exposure of T24 cells to 5-23, but it cannot interpret why the activation only occurs at high concentrations of 5-23 with prolonged co-incubation time. We also realized that it is more meaningful to study the binding model of these ligands (e.g., 5-23) bound to p-MLKL (rather than MLKL), which can better elucidate the role of 5-23 in inducing the necroptosis of T24 cells.

Fig. 8. Binding model of 5-23 (or Reg) with the receptor MLKL (PDB ID: 6O5Z). (A and B) Docked conformation of 5-23 when it interacted with MLKL. Ligand and key residues are represented as stick models and colored by atom type, whereas the proteins are represented as cartoons. (C) Receptor MLKL binding pocket “M”. (D) Compound 5-23 and Reg (E) were embedded in the binding pocket of MLKL. (F) Reg interacted with the receptor MLKL and five hydrogen bonds were formed. In figure (B and F), hydrogen bonds are represented by red dotted lines. For the ligands, white: hydrogen atom; red: oxygen atom; dark blue: nitrogen atom; light blue: fluorine atom; green: chlorine atom; orange: the backbone and the carbon atoms of 5-23 (or Reg).

We also performed docking experiments to obtain the binding model of 5-23 (or Reg) in complex with another receptor VEGFR-2, and the results are presented in Fig. S8.† Four H-bonds were formed between 5-23 and VEGFR-2: two hydrogen bonds (NH₂⋯O) with distances of 2.2 and 2.1 Å formed respectively between two imino groups (NH) in the urea moiety and the carboxyl oxygen atoms of the residue GLU885; one H-bond with a distance of 2.1 Å formed between the oxygen atom and the amino of the residue ASP1046 (O⋯HN); the last bond with a distance of 2.6 Å was formed between a nitrogen atom in the pyrazinyl group and one carboxyl oxygen atom of the residue CYS919 (NH⋯O) (Fig. S8A and B†). Five H-bonds between Reg and VEGFR-2 were formed (Fig. S8C and D†). Compound 5-23 and Reg could be well embedded into the cavity “P”, which was comprised of a series of amino acid residues of VEGFR-2 (Fig. S8E and F†). The calculated binding energies of 5-23 and Reg were −5.04 and −6.61 kcal mol⁻¹, respectively. Although the intracellular VEGFR-2 content was very low and the molecular docking analysis was of little significance in our study, the above binding models could reasonably explain why those molecules, such as 5-23 and other structural analogues of Reg could effectively suppress the activity of VEGFR-2, and are more likely to inhibit the proliferation of a variety of human cancer cell lines with overexpressed VEGFR-2.

Next, we studied the effects of different substituents on the proliferation inhibitory activity of T24 cells. Compared with compound 5-1 (without any substitution), the introduction of electron-donating groups at the ortho or para positions, such as methyl, methoxy, ethoxy, and tert-butyl (5-2–5-12) groups, could moderately improve the proliferative inhibitory activity. The introduction of halogen atoms, especially the substituents such as trifluoromethyl, fluoro-, and chloro- remarkably enhanced activity (e.g., 5-13–5-23). We speculate that the hydrophobic interactions between the substituent(s) on the distal phenyl ring and the receptor are very important. A typical case was that 5-23, bearing the trifluoromethyl group at the para position, demonstrated superior activity. The position of the trifluoromethyl substituent had a significant impact: the para-position was more favorable than the meta-position (Fig. S1†), however, the introduction of N,N-dimethyl or 4-methylsulfonyl (5-24, 5-25) was unfavorable. Additionally, when the distal phenyl ring was replaced by naphthyl, cyclopentyl, or cyclohexyl (5-26–5-28), the inhibitory activity decreased significantly. Therefore, besides the pharmacophore urea moiety, the distal phenyl ring and suitable substituents are important factors that synergistically affect the activity against T24 cells.

Finally, we speculate on the possible molecular mechanisms underlying the inhibitory activity of 5-23 and associated death modes. As shown in Fig. 9, lower concentrations (≤7.5 μM) of 5-23 induced T24 apoptotic cell death via the activation of caspases and the release of cyto c within the initial 10 h (mode I). With the extension of incubation time and elevated concentrations, the activity of intracellular casp-8 was inhibited and 5-23 induced T24 cell necroptosis by regulating the RIPK1/RIPK3/MLKL signaling pathway (mode II).

Fig. 9. Proposed molecular mechanisms of 5-23-induced T24 cell death. Compound 5-23 triggered caspase-dependent cell apoptosis in the case of lower concentrations (≤7.5 μM) and shorter incubation times (≤10 h) (mode I). High concentration (≥7.5 μM) and prolonged incubation time (>10 h) led to necroptotic cell death (mode II). The above two modes were closely related to 5-23-induced ROS production.

Conclusion

In summary, a series of novel pyrazinyl–aryl urea derivatives were succinctly synthesized and tested for their antiproliferative activities. Compound 5-23 displayed the best profile against T24 cells with a favorable IC₅₀ value and high selectivity. Highlights of this study are as follows: (1) compound 5-23 could induce two different death modes (apoptosis and necroptosis) of T24 cells; the mode that would occur was dependent on its concentration and incubation time. (2) In comparison with 5-23, the positive reference drug Reg inhibited necroptosis. Although cell necroptosis induced by chemotherapeutic agents has been observed in many experiments within the last decade, aryl urea derivatives-induced necroptosis in BC cell lines has hardly been observed. Our results prove once again that inhibiting the activity of casp-8 could lead to necroptotic cell death. Both cell apoptosis and necroptosis would occur by inducing a high level of intracellular ROS. The question of why 5-23 could induce T24 cell necroptosis but Reg exhibited inhibitory effects, remains to be answered?

Immune checkpoint inhibitors approved by the FDA during 2016 and 2017 transformed the treatment landscape of various cancers, including BC.⁴⁶ However, moderate or severe side effects, such as pneumonitis and hepatotoxicity impact patients benefiting from these agents.⁴⁷ Luckily, in 2019 the FDA granted accelerated approval of erdafitinib (JNJ-42756493, Balversa™) for patients with locally advanced or metastatic urothelial carcinoma.⁴⁸ Due to the limitations of the number of cases, the safety of erdafitinib needs further evaluation. Taken together, pyrazinyl–aryl urea derivative 5-23 may serve as a promising candidate for the further development of potent molecular-targeted anti-BC agents. Further structural optimization of 5-23 is in progress.

Experimental

Chemistry

The ¹H and ¹³C NMR spectra were recorded in DMSO-d₆ (or CD₃OD) solution on a Bruker Avance III 500 MHz NMR spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) at 500 and 125 MHz, respectively; chemical shifts (δ) were reported in parts per million (ppm) with tetramethylsilane as an internal standard. Melting points (m.p.) were measured on an X-4 electro-thermal digital melting point apparatus and were uncorrected. HRMS data were recorded on an UltrafleXtreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). Column chromatography was conducted on silica gel (300–400 mesh). Unless otherwise noted, all solvents and reagents were obtained from commercial suppliers and used without further purification.

Synthesis of the intermediate N-(3-aminobenzyl)-3-chloro-pyrazin-2-amine (2)

3-Aminobenzylamine (3.66 g, 30.0 mmol) was slowly added to a solution of 2,3-dichloropyrazine (3.70 g, 25.0 mmol) and triethylamine (4 mL) dissolved in 1,4-dioxane (60 mL). The mixture was stirred at 100 °C for 8 h under an N₂ atmosphere. After the reaction mixture was cooled to room temperature and concentrated in vacuo, 100 mL of distilled water was added and the products were extracted with CH₂Cl₂ (3 × 150 mL). The combined organic layers were dried over sodium sulfate. The crude residue was purified through silica gel column chromatography using petroleum/EtOAc (2/1) as the eluent to afford 2 as a white solid (5.13 g, yield: 88%). m.p. 124.2–125.7 °C. ¹H NMR (500 MHz, CD₃OD) δ 7.83 (d, J = 2.7 Hz, 1H), 7.52–7.45 (m, 3H), 7.42 (d, J = 2.7 Hz, 1H), 7.30 (s, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 4.55 (s, 2H).¹³C NMR (125 MHz, DMSO-d₆) δ 151.44, 149.04, 141.41, 140.59, 133.98, 130.26, 129.15, 114.91, 112.79, 112.69, 44.31. ESI-HRMS (m/z): calcd. for C₁₁H₁₁ClN₄ [M + H]⁺: 235.07505; found: 235.07429.

A general procedure for the synthesis of pyrazinyl–aryl urea derivatives (5-1–5-28)

Various isocyanates could be synthesized by the reaction of the corresponding amines with triphosgene (BTC).³⁴ Aryl isocyanates 4 (1.1 equiv.) were added slowly to a mixture of N-(3-aminobenzyl)-3-chloropyrazin-2-amine (2, 1 equiv.) and triethylamine (1.5 equiv.) dissolved in dry ClCH₂CH₂Cl under an N₂ atmosphere. The mixture was stirred overnight at room temperature, then the solvent was removed under reduced pressure. After deionized water was added to quench the reaction, the mixture was extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na₂SO₄. After filtration and concentration, the crude product was purified through silica gel column chromatography using MeOH/CHCl₃ as an eluent to afford the target compounds 5-1–5-28.

N-Phenyl-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-1)

Yield: 78%. m.p. 241.4–243.1 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.05 (s, 1H), 8.47 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 5.0 Hz, 1H), 7.64 (t, J = 5.0, 10.0 Hz, 1H), 7.53 (d, J = 2.7 Hz, 1H), 7.34 (d, J = 3.5 Hz, 1H), 7.29 (s, 1H), 7.20 (t, J = 5.0, 15.0 Hz, 1H), 7.10 (d, J = 10 Hz, 2H), 6.84 (d, J = 5.0 Hz, 2H), 6.80 (d, J = 3.4 Hz, 2H), 4.54 (d, J = 5.0 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.96, 151.33, 141.40, 140.72, 140.22, 137.55, 133.98, 131.05, 130.45, 129.61, 127.10, 126.53, 124.87, 121.34, 120.81, 118.68, 116.84, 44.20. ESI-HRMS (m/z): calcd. for C₁₈H₁₆ClN₅O [M]⁺: 353.10434; found: 353.19748.

N-(2-Methylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-2)

Yield: 60%. m.p. 212.4–214.3 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 9.09 (s, 1H), 8.44 (d, J = 3.5 Hz, 1H), 7.97 (d, J = 5.0 Hz, 1H), 7.83 (t, J = 13.5 Hz, 2H), 7.70 (dd, J = 12.4, 7.3 Hz, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.30 (s, 1H), 7.20 (t, J = 5.0, 9.5 Hz, 1H), 7.14 (m, 2H), 6.92 (dd, J = 12.7, 5.7 Hz, 2H), 4.55 (d, J = 5.3 Hz, 2H), 2.22 (s, 3H). 13C NMR (125 MHz, DMSO-d₆) δ (ppm): 153.03, 151.34, 141.42, 140.79, 140.31, 137.84, 133.99, 130.63, 130.48, 129.18, 127.84, 126.60, 123.07, 121.36, 120.85, 116.76, 116.66, 44.23, 18.32. ESI-HRMS (m/z): calcd. for C₁₉H₁₈ClN₅O [M + H]⁺: 368.12781; found: 368.11023.

N-(3-Methylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-3)

Yield: 65%. m.p. 202.4–203.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.10 (s, 1H), 8.33 (d, J = 3.0 Hz, 1H), 7.97 (d, J = 5.0 Hz, 1H), 7.69 (t, J = 5.0, 10.0 Hz, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.30 (m, 3H), 7.18 (t, J = 5.0, 15.0 Hz, 1H), 7.01 (d, J = 10 Hz, 2H), 6.94 (d, J = 5.0 Hz, 2H), 4.54 (d, J = 5.0 Hz, 2H), 2.46 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 153.48, 151.71, 142.06, 141.35, 140.68, 138.29, 135.17, 132.44, 130.68, 129.64, 127.43, 127.01, 121.62, 120.71, 118.41, 117.21, 116.24, 44.21, 19.36. ESI-HRMS (m/z): calcd. for C₁₉H₁₈ClN₅O [M + H]⁺: 368.12781; found: 368.13938.

N-(4-Methylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-4)

Yield: 38%. m.p. 200.2–201.3 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.47 (s, 1H), 7.98 (d, J = 2.7 Hz, 1H), 7.70 (t, J = 6.2 Hz, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.32 (s, 1H), 7.29 (d, J = 3.8 Hz, 2H), 7.20 (dd, J = 15.8, 8.1 Hz, 1H), 7.07 (d, J = 8.3 Hz, 2H), 6.90 (d, J = 7.6 Hz, 1H), 4.55 (d, J = 6.1 Hz, 2H), 2.23 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.96, 151.33, 141.40, 140.72, 140.22, 137.55, 133.98, 131.05, 130.45, 129.61, 127.10, 126.53, 121.34, 120.81, 118.68, 116.96, 116.84, 44.20, 20.79. ESI-HRMS (m/z): calcd. for C₁₉H₁₈ClN₅O [M + H]⁺: 368.12781; found: 368.10843.

N-(2,4-Dimethylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-5)

Yield: 67%. m.p. 201.7–203.2 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.61 (s, 1H), 8.44 (s, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.79 (d, J = 12.7 Hz, 1H), 7.69 (dd, J = 15.4, 9.3 Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.55 (dd, J = 24.3, 3.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.30 (s, 1H), 7.20 (t, J = 7.8 Hz, 1H), 6.92 (dd, J = 19.4, 7.9 Hz, 2H), 4.59 (t, J = 12.2 Hz, 2H), 2.30–2.13 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 153.14, 151.33, 141.41, 140.75, 140.41, 135.20, 133.98, 132.03, 131.15, 130.46, 129.14, 128.16, 127.02, 121.79, 120.73, 116.70, 116.61, 44.24, 20.79, 18.25. ESI-HRMS (m/z): calcd. for C₂₀H₂₀ClN₅O [M + H]⁺: 382.14346; found: 382.12439.

N-(3,4-Dimethylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-6)

Yield: 67%, m.p. 164.2–166.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.42 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.72 (t, J = 6.2 Hz, 1H), 7.57 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.30 (s, 1H), 7.23 (d, J = 1.9 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 7.18 (d, J = 7.9 Hz, 1H), 7.01 (s, 1H), 6.90 (d, J = 7.8 Hz, 1H), 4.55 (d, J = 6.1 Hz, 2H), 2.29 (d, J = 17.3 Hz, 3H), 2.17 (d, J = 17.3 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.94, 151.33, 141.42, 137.94, 136.78, 136.75, 130.46, 130.07, 129.73, 129.10, 120.76, 119.93, 119.87, 116.80, 116.15, 116.08, 44.21, 20.12, 19.14. ESI-HRMS (m/z): calcd. for C₂₀H₂₀ClN₅O [M + H]⁺: 382.14346; found: 382.12784.

N-(2-Ethylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-7)

Yield: 69%. m.p. 206.0–207.4 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.57 (s, 1H), 8.42 (s, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.57 (d, J = 2.6 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.25–7.10 (m, 3H), 7.00 (t, J = 7.3 Hz, 1H), 6.91 (d, J = 7.4 Hz, 1H), 4.56 (d, J = 6.0 Hz, 2H), 2.60 (q, J = 12.5, 7.5 Hz, 2H), 1.17 (t, J = 7.5 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 153.25, 151.33, 141.41, 140.78, 140.35, 136.96, 134.13, 133.99, 130.47, 129.17, 128.86, 126.49, 123.58, 122.42, 120.82, 116.74, 116.63, 44.24, 24.24, 14.73. ESI-HRMS (m/z): calcd. for C₂₀H₂₀ClN₅O [M + H]⁺: 382.14346; found: 382.12576.

N-(2,6-Diethylphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-8)

Yield: 69%. m.p. 215.8–217.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.98 (d, J = 2.7 Hz, 1H), 7.68 (t, J = 6.1 Hz, 1H), 7.60 (s, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.16 (dd, J = 9.4, 7.2 Hz, 2H), 7.11–7.07 (m, 2H), 6.87 (d, J = 7.7 Hz, 1H), 4.54 (d, J = 6.1 Hz, 2H), 2.55 (q, J = 7.6 Hz, 4H), 1.11 (t, J = 7.6 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 154.25, 151.35, 142.40, 141.42, 140.78, 140.69, 134.41, 134.00, 130.45, 129.08, 127.20, 126.44, 124.78, 124.02, 120.42, 118.39, 116.53, 44.24, 24.91, 19.04. ESI-HRMS (m/z): calcd. for C₂₂H₂₄ClN₅O [M + H]⁺: 410.17476; found: 410.15840.

N-((4-tert-Butyl)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-9)

Yield: 79%. m.p. 209.4–211.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.53 (s, 1H), 7.97 (d, J = 5.0 Hz, 1H), 7.69 (t, J = 5.0, 10.0 Hz, 1H), 7.56 (d, J = 2.7 Hz, 2H), 7.37–7.27 (m, 4H), 7.01 (d, J = 10 Hz, 2H), 6.89 (d, J = 5.0 Hz, 2H), 4.54 (d, J = 5.0 Hz, 2H), 1.46 (s, 9H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.96, 151.33, 141.40, 140.72, 140.22, 137.55, 133.98, 131.05, 130.45, 129.61, 127.10, 126.53, 121.34, 120.81, 118.68, 116.96, 116.84, 42.86, 36.27, 31.40. ESI-HRMS (m/z): calcd. for C₂₂H₂₄ClN₅O [M + H]⁺: 410.17476; found: 410.16477.

N-(2-Methoxyphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-10)

Yield: 89%. m.p. 198.2–199.8 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 9.36 (s, 1H), 8.18 (s, 1H), 8.12 (dd, J = 7.9, 1.4 Hz, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.71 (s, 1H), 7.57 (d, J = 2.6 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.29 (s, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.99–6.81 (m, 3H), 4.56 (d, J = 6.1 Hz, 2H), 3.87 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 156.97, 152.92, 149.69, 143.30, 141.82, 135.71, 134.24, 129.75, 128.73, 128.14, 124.21, 122.56, 121.22, 120.31, 119.65, 119.24, 112.84, 56.27, 44.25. ESI-HRMS (m/z): calcd. for C₁₉H₁₈ClN₅O₂ [M + H]⁺: 384.12273; found: 384.10331.

N-(4-Methoxyphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-11)

Yield: 89%. m.p. 184.2–186.2 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.37 (s, 1H), 7.98 (d, J = 2.7 Hz, 1H), 7.70 (s, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.39–7.33 (m, 3H), 7.29 (s, 1H), 7.19 (s, 1H), 6.95–6.82 (m, 3H), 4.55 (d, J = 6.1 Hz, 2H), 3.90 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 154.78, 153.41, 151.35, 141.43, 140.73, 140.34, 134.00, 133.40, 133.16, 130.46, 129.10, 120.37, 116.79, 114.42, 55.63, 45.58. ESI-HRMS (m/z): calcd. for C₁₉H₁₈ClN₅O₂ [M + H]⁺: 384.12273; found: 384.14418.

N-(4-Ethoxyphenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-12)

Yield: 86%. m.p. 181.3–183.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (s, 1H), 8.36 (s, 1H), 7.98 (d, J = 2.7 Hz, 1H), 7.70 (s, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.38–7.28 (m, 4H), 7.18 (t, J = 7.8 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.87–6.80 (m, 2H), 4.55 (d, J = 6.1 Hz, 2H), 3.96 (d, J = 7.0 Hz, 2H), 1.30 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 154.02, 153.41, 151.35, 141.44, 140.72, 140.23, 134.74, 133.32, 133.01, 130.47, 129.10, 120.41, 120.34, 116.81, 115.17, 114.99, 63.55, 45.59, 15.21. ESI-HRMS (m/z): calcd. for C₂₀H₂₀ClN₅O₂ [M + H]⁺: 398.13838; found: 398.11832.

N-(2-Fluorophenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-13)

Yield: 63%. m.p. 182.4–183.1 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.47 (s, 1H), 8.31 (s, 1H), 8.14 (t, J = 7.6 Hz, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.71 (t, J = 4.8 Hz, 1H), 7.57 (d, J = 5.0 Hz, 1H), 7.38 (d, J = 5.3 Hz, 1H), 7.31 (s, 1H), 7.22 (m, 2H), 7.13 (t, J = 6.4 Hz, 1H), 7.00 (dd, J = 9.5, 5.4 Hz, 1H), 6.93 (d, J = 10.0 Hz, 1H), 4.56 (d, J = 9.8 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 152.57, 151.33, 141.42, 140.88, 139.88, 134.00, 130.50, 129.23, 124.96, 122.88, 122.82, 121.20, 120.90, 116.84, 116.80, 115.48, 115.32, 44.19. ESI-HRMS (m/z): calcd. for C₁₈H₁₅ClFN₅O [M + H]⁺: 372.10274; found: 372.11981.

N-(2-Chlorophenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-14)

Yield: 60%. m.p. 224.6–225.3 °C.¹H NMR (500 MHz, DMSO-d₆) δ 9.05 (s, 1H), 8.50 (s, 1H), 8.15 (t, J = 4.2 Hz, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.74 (t, J = 6.1 Hz, 1H), 7.57 (d, J = 2.7 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.30 (s, 1H), 7.26–7.18 (m, 2H), 7.14 (t, J = 7.8 Hz, 1H), 7.05–6.96 (m, 1H), 6.94 (d, J = 7.6 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.56, 151.31, 141.42, 140.88, 139.87, 133.98, 130.49, 129.23, 127.98, 126.35, 124.96, 122.81, 121.17, 120.87, 116.75, 115.48, 115.33, 44.18. ESI-HRMS (m/z): calcd. for C₁₈H₁₅Cl₂N₅O [M + H]⁺: 388.07319; found: 388.06128.

N-(3-Chlorophenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-15)

Yield: 62%. m.p. 181.1–182.9 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.47 (s, 1H), 8.32 (s, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.69 (m, 1H), 7.55 (d, J = 2.3 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.30 (s, 1H), 7.28 (m, 2H), 7.14 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 7.6 Hz, 2H), 4.55 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 152.76, 151.82, 141.70, 141.39, 140.80, 139.82, 134.00, 133.64, 130.83, 130.47, 129.14, 121.86, 121.22, 117.95, 117.14, 117.04, 44.19. ESI-HRMS (m/z): calcd. for C₁₈H₁₅Cl₂N₅O [M + H]⁺: 388.07319; found: 388.06778.

N-(4-Chlorophenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-16)

Yield: 62%. m.p. 227.8–229.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.74 (s, 1H), 8.69 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.73 (t, J = 6.1 Hz, 1H), 7.57 (d, J = 2.7 Hz, 1H), 7.51–7.48 (t, J = 8.7 Hz, 1H), 7.38 (s, 1H), 7.35–7.32 (m, 2H), 7.31 (d, J = 5.4 Hz, 2H), 7.21 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 152.81, 151.32, 141.41, 140.78, 139.00, 133.99, 130.47, 129.14, 129.09, 129.07, 125.94, 125.73, 121.07, 120.28, 120.10, 117.01, 116.98, 44.18. ESI-HRMS (m/z): calcd. for C₁₈H₁₅Cl₂N₅O [M + H]⁺: 388.07319; found: 388.05382.

N-((3-Chloro-4-fluoro)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl) urea (5-17)

Yield: 49%. m.p. 190.9–192.3 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.83 (s, 1H), 8.79 (s, 1H), 7.98 (d, J = 2.7 Hz, 1H), 7.79 (dd, J = 6.7, 2.3 Hz, 1H), 7.63–7.52 (m, 2H), 7.36 (s, 1H), 7.35–7.25 (m, 3H), 7.21 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.9 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 156.57, 152.33, 143.42, 141.88, 136.88, 135.00, 130.50, 129.23, 124.96, 123.78, 122.83, 121.26, 120.90, 116.84, 116.80, 115.98, 115.62, 46.17. ESI-HRMS (m/z): calcd. for C₁₈H₁₄Cl₂FN₅O [M + H]⁺: 406.06377; found: 406.05128.

N-(2,4-Dichlorophenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-18)

Yield: 57%. m.p. 163.4–164.1 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.62 (s, 1H), 8.56 (s, 1H), 7.99 (d, J = 2.7 Hz, 1H), 7.71 (t, J = 6.1 Hz, 1H), 7.57 (d, J = 2.7 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.28 (s, 1H), 7.24–7.19 (m, 2H), 7.18–7.11 (m, 1H), 6.91 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 7.3 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.95, 151.35, 141.43, 140.77, 140.17, 140.11, 140.06, 138.41, 130.48, 129.09, 122.99, 120.90, 119.12, 116.88, 115.78, 44.22. ESI-HRMS (m/z): calcd. for C₁₈H₁₄Cl₃N₅O [M + H]⁺: 422.03422; found: 422.01908.

N-(3,4-Dichlorophenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-19)

Yield: 62%. m.p. 181.2–183.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm): 8.50 (s, 1H), 8.38 (s, 1H), 7.97 (d, J = 2.6 Hz, 1H), 7.69 (m, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.30 (m, 2H), 7.28 (m, 1H), 6.99 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 7.6 Hz, 1H), 4.55 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm): 152.70, 151.32, 141.39, 140.81, 140.41, 139.69, 134.00, 131.47, 131.00, 130.47, 129.14, 123.49, 122.18, 121.34, 119.67, 118.74, 117.24, 44.19. ESI-HRMS (m/z): calcd. for C₁₈H₁₄Cl₃N₅O [M + H]⁺: 422.03422; found: 422.02508.

N-((3-Chloro-4-methyl)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl) urea (5-20)

Yield: 78%. m.p. 186.8–188.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.69 (s, 2H), 7.98 (d, J = 2.5 Hz, 1H), 7.73 (t, J = 6.2 Hz, 1H), 7.68 (s, 1H), 7.57 (d, J = 2.6 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 7.19 (s, 2H), 7.17–7.14 (m, 1H), 6.92 (d, J = 7.5 Hz, 1H), 4.56 (d, J = 6.1 Hz, 2H), 2.26 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.85, 151.36, 141.41, 140.78, 139.95, 139.35, 134.02, 133.57, 131.62, 130.49, 129.13, 128.67, 121.14, 118.57, 117.41, 117.12, 44.23, 19.25. ESI-HRMS (m/z): calcd. for C₁₉H₁₇Cl₂N₅O [M + H]⁺: 402.08884; found: 402.09854.

N-((2-Bromo-4-chloro)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl) urea (5-21)

Yield: 49%. m.p. 209.3–211.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.44 (s, 1H), 8.34 (s, 1H), 8.13 (d, J = 2.6 Hz, 1H), 7.79 (m, 1H), 7.75 (d, J = 2.6 Hz, 1H), 7.71 (d, J = 2.6 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.47 (m, 2H), 7.30 (s, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 4.55 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 156.57, 152.33, 143.42, 141.88, 136.88, 135.00, 130.50, 129.23, 124.96, 123.78, 122.83, 121.26, 120.90, 116.84, 116.80, 115.98, 115.62, 46.17. ESI-HRMS (m/z): calcd. for C₁₈H₁₄BrCl₂N₅O [M + H]⁺: 465.98370; found: 465.96558.

N-((3-Trifluoromethyl)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl) urea (5-22)

Yield: 60%. m.p. 200.1–201.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.91 (s, 1H), 8.50 (s, 1H), 7.97 (d, J = 5.0 Hz, 1H), 7.69 (t, J = 5.0, 10.0 Hz, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.44–7.34 (m, 3H), 7.30 (s, 1H), 7.28 (d, J = 10 Hz, 1H), 7.21 (d, J = 10 Hz, 1H), 7.10 (d, J = 5.0 Hz, 1H), 7.02 (d, J = 5.0 Hz, 1H), 4.54 (d, J = 5.0 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.96, 151.33, 141.40, 140.72, 140.22, 137.55, 133.98, 131.05, 130.45, 129.61, 127.10, 126.53, 124.55, 121.34, 120.81, 118.68, 116.96, 116.84, 42.47. ESI-HRMS (m/z): calcd. for C₁₉H₁₅ClF₃N₅O [M + H]⁺: 422.09955; found: 422.10341.

N-((4-Trifluoromethyl)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl) urea (5-23)

Yield: 59%. m.p. 201.4–203.1 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.02 (s, 1H), 8.78 (s, 1H), 7.98 (d, J = 2.6 Hz, 1H), 7.71 (t, J = 6.1 Hz, 1H), 7.68–7.59 (m, 4H), 7.57 (d, J = 2.6 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.34 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 4.57 (d, J = 6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 152.69, 151.35, 143.92, 141.41, 140.86, 139.75, 134.03, 130.49, 129.17, 126.55, 126.51, 126.38, 123.68, 122.36, 122.05, 121.37, 118.27, 117.22, 44.21. ESI-HRMS (m/z): calcd. for C₁₉H₁₅ClF₃N₅O [M + H]⁺: 422.09955; found: 422.10764.

N-((N,N-Dimethyl)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-24)

Yield: 49%, m.p. 203.7–205.1 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.79 (s, 1H), 8.54 (s, 1H), 7.87 (dd, J = 1.4, 7.8 Hz, 1H), 7.54 (d, J = 4.8 Hz, 1H), 7.46 (t, J = 6.1, 12.2 Hz, 2H), 7.45 (d, J = 2.7 Hz, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.32 (s, 1H), 7.12 (t, J = 7.8, 15.6 Hz, 2H), 6.57 (d, J = 6.3 Hz, 2H), 4.55 (d, J = 6.0 Hz, 2H), 2.85 (s, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 156.97, 152.92, 149.69, 143.30, 141.82, 135.71, 134.24, 129.75, 128.73, 128.14, 124.21, 122.57, 121.14, 120.81, 120.41, 116.46, 112.76, 42.51, 40.27. ESI-HRMS (m/z): calcd. for C₂₀H₂₁ClN₆O [M + Na]⁺: 419.13631; found: 419.12335.

N-((4-Methanesulfyl)phenyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl) urea (5-25)

Yield: 44%. m.p. 280.6–281.3 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.09 (s, 1H), 8.74 (s, 1H), 7.87 (d, J = 2.6 Hz, 1H), 7.54 (m, 1H), 7.47 (d, J = 2.6 Hz, 2H), 7.46 (d, J = 2.6 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.32 (s, 1H), 7.12 (d, J = 3.2 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 4.55 (d, J = 6.1 Hz, 2H), 3.39 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 156.57, 152.03, 142.01, 141.64, 135.70, 134.00, 129.00, 128.50, 128.49, 126.48, 122.67, 122.44, 119.00, 118.87, 116.20, 112.19, 56.20, 41.70. ESI-HRMS (m/z): calcd. for C₁₉H₁₈ClN₅O₃S [M + H]⁺: 432.08971; found: 432.07238.

N-(α-Naphthyl)-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-26)

Yield: 73%. m.p. 220.3–221.8 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.19 (s, 1H), 9.04 (s, 1H), 8.25 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 8.6 Hz, 1H), 8.00 (dd, J = 12.6, 4.8 Hz, 2H), 7.92 (d, J = 8.1 Hz, 1H), 7.72 (t, J = 6.0 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.57 (m, 3H), 7.46 (m, 1H), 7.34 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 153.30, 151.36, 141.45, 140.87, 140.24, 134.84, 134.23, 134.01, 130.51, 129.24, 128.96, 126.38, 123.39, 121.86, 121.73, 121.01, 117.92, 116.86, 116.77, 44.24. ESI-HRMS (m/z): calcd. for C₂₂H₁₈ClN₅O [M + H]⁺: 404.12781; found: 404.12862.

N-Cyclopentyl-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-27)

Yield: 46%. m.p. 220.4–222.1 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.44 (s, 1H), 8.18 (s, 1H), 7.87 (d, J = 2.6 Hz, 1H), 7.54 (m, 1H), 7.45 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 2.6 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.80 (m, 2H), 4.55 (d, J = 6.1 Hz, 2H), 3.61 (d, J = 8.5 Hz, 1H), 1.86 (d, J = 6.1 Hz, 2H), 1.64 (m, 2H), 1.57–1.50 (m, 4H). ¹³C NMR (125 MHz, DMSO-d₆) δ 155.49, 153.28, 141.44, 139.49, 135.23, 134.51, 129.03, 128.64, 121.38, 119.42, 119.03, 56.18, 42.36, 34.45, 24.91. ESI-HRMS (m/z): calcd. for C₁₇H₂₀ClN₅O [M + H]⁺: 346.14346; found: 346.16475.

N-Cyclohexyl-N′-(3-(((3-chloropyrazin-2-yl)amino)methyl)phenyl)urea (5-28)

Yield: 49%. m.p. 212.6–214.2 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.31 (s, 1H), 8.04 (s, 1H), 7.87 (d, J = 2.6 Hz, 1H), 7.54 (m, 1H), 7.45 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 2.6 Hz, 1H), 7.30 (s, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.80 (m, 2H), 4.55 (d, J = 6.1 Hz, 2H), 3.54 (d, J = 8.4 Hz, 1H), 1.78 (d, J = 5.8 Hz, 4H), 1.49 (d, J =7.6 Hz, 4H), 1.46 (d, J = 10.1 Hz, 1H). 13C NMR (125 MHz, DMSO-d₆) δ 156.57, 154.69, 142.01, 141.94, 134.99, 134.00, 128.77, 128.43, 122.60, 119.61, 119.20, 56.20, 42.48, 38.16, 28.00, 22.50. ESI-HRMS (m/z): calcd. for C₁₈H₂₂ClN₅O [M + H]⁺: 360.15911; found: 360.20188.

Biological evaluation

In vitro proliferation inhibitory activity evaluation

The proliferation inhibitory activity of the synthesized pyrazinyl–aryl urea derivatives was assessed using the MTT assay, according to the previous report.⁴⁹ Human cancer cell lines including Hep G2, MGC-803, T24, NCI-H460, and PC-3 were purchased from the Typical Culture Preservation Committee Cell Bank of the Chinese Academy of Science (Shanghai, China). Normal bladder epithelial cell line HCV29 was provided by the Department of Cell Biology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine. Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and cultivated overnight at 37 °C. After the cells were co-incubated with compounds 5-1–5-28 or positive reference drugs (Reg and Gem; their final concentrations: 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 30, and 40 μM) for the indicated periods, 20 μL of MTT solution in PBS was added to the medium. After the cells were incubated for another 4 h at 37 °C, the culture medium was removed, then 100 μL of DMSO was added to dissolve the formed formazan crystals. The percentage of cell viability was determined using a microplate reader. The selectivity index (SI) was used to evaluate the harmfulness of pyrazinyl–aryl urea derivatives to normal cell line HCV29.⁵⁰ Corresponding equation: SI = (IC₅₀ of HCV29 cells)/(IC₅₀ of T24 cells). The IC₅₀ values were defined as the drug concentrations that killed 50% of cells relative to the negative controls (Con). The experiment was replicated for four parallel samples.

Detection of cell death by flow cytometry

Flow cytometric measurements were performed to differentiate the two cell death modes necroptosis and apoptosis: For the former, the necroptosis blocker Nec-1 was added during incubation; for the latter, the pan-caspase blocker zVAD was added.^51–54 T24 cell death could be evaluated using an Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific (China) Co., Ltd, Shanghai). In brief, cells in 60 mm dishes were treated with 5-23 in the presence or absence of the zVAD (or Nec-1) at 37 °C for the indicated periods of time. Cells were collected, washed with PBS thrice, and resuspended in 500 μL of binding buffer. Then Annexin (5 μL) along with PI (5 μL) were added and incubated for 15 min at room temperature in the dark. The samples were analyzed using a FACSCalibur flow cytometry system (Becton Dickinson, San Jose, CA, USA). The percentages of viable (Annexin−/PI−), early apoptotic (Annexin+/PI−), late apoptotic (Annexin+/PI+), and necrotic (Annexin−/PI+) cells were determined using FlowJo software. Both the early apoptotic cells and the late apoptotic cells were designated as apoptotic cells. The percentages of necrotic and late apoptotic cells in the presence (or absence) of Nec-1 were compared to determine the cell death mode.^53,54 For each sample, at least 1 × 10⁴ events were collected and analyzed. All experiments were performed in triplicate.

Hoe/PI double-labeling assays

After T24 cells were treated with the indicated concentrations of 5-23, floating and adherent subpopulations were merged. The Hoe/PI dual staining Kit (Solarbio Science & Technology Co., Ltd., Beijing, China) was used and the following procedures were performed according to the manufacturer's protocol.⁵⁵ The samples were analyzed under an Olympus BX61 fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Trypan blue exclusion assay

The Trypan blue dye exclusion assay was performed to investigate induced cell death by 5-23.⁵⁶ T24 cells seeded in 6-well plates (2 × 10⁵/well) were incubated with different concentrations of 5-23 in the absence or presence of necroptosis inhibitor Nec-1. Then, the cells were stained with trypan blue solution, and the numbers of viable and dead cells were counted. The results were expressed as the percentage of viable cells relative to untreated control cells.

Western blot analysis

T24 cells treated with 5-23 were collected in lysis buffer. The protein concentration of the supernatant was determined by the bicinchoninic acid assay. Protein samples (40 μg) were separated by SDS-PAGE and then transferred to a poly(vinylidene difluoride) membrane. After blocking, the membrane was incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The bands were visualized and analyzed using an enhanced chemiluminescence (ECL) western blotting detection system. β-Actin was used as a loading control. The gray value of the protein bands was analyzed using ImageJ software (Bethesda, MD, USA). All of the antibodies were purchased from CST Biological Reagents Co. Ltd. (Shanghai, China) and diluted according to the manufacturer's instructions.

Kinase activity analyses by ELISA

To quantitatively determine the levels of RIPK1, RIPK3, and MLKL in cell lysates, enzyme-linked immunosorbent assay (ELISA) kits were used. Human RIPK1 (product no. SEE640Hu), RIPK3 (no. SEE639Ra), and MLKL (no. SER645Hu) test kits were purchased from Cloud-Clone Corp. (Wuhan, Hubei Province, China). The detection method has been reported previously.⁵⁷ In brief, T24 cells pretreated with 5-23 (or Reg) were collected, diluted with PBS, counted, and stored overnight at −20 °C. After the cell membranes were broken, lysates were centrifuged at 5000 × g for 5 min (4 °C) and analyzed by ELISA. The following procedures were carried out according to the manufacturer's instructions.

RNA extraction and RT-PCR

Total RNA from T24 cells was extracted using TRIzol reagent (Thermo Fisher Scientific (China) Co. Ltd., Shanghai) and reverse transcribed using the PrimeScript™ RT Reagent Kit (Takara Biotechnology Co. Ltd., Dalian, China). Quantitative real-time PCR (RT-PCR) was performed on the ABI 7300 real-time PCR thermal cycle instrument (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Rockford IL). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene in the normalization process of the corresponding sample. Primers were acquired from Invitrogen as follows: caspase-8, forward: 5′-TACTACCGAAACTTGGACC-3′, reverse: 5′-GTGAAAGTAGGTTGTGGC-3′; RIP1: forward: 5′-AATTTCCCAGGCAGTTGTTG-3′, reverse: 5′-CATTTCCTGTTCCCTC-TCCA-3′, RIP3, forward: 5′-TGGAGACAACAACTACTTGACTATG-3′, reverse: 5′-GGTGCTTTATTTCCCGCTATGATTA-3′; MLKL, forward: 5′-AGGAGGCTAATG-GGGAGATAGA-3′, reverse: 5′-TGGCTTGCTGTTAGAAACCTG-3′; GAPDH, forward: 5′-ACCACAGTCCATGCCATCAC-3′, reverse: 5′-CCACCACCCTGTTGC-TGTA-3′; VEGFR-1, forward: 5′-CTTCCCTCAGGCGACTGC-3′, reverse: 5′-CGACG-TGTGGTCTTACGGAGTA-3′; VEGFR-2, forward: 5′-ACCTCATATCTGTCCTGAT-GTGATAT-3′, reverse: 5′-TTGAACCTCCCGCATTCAGT-3′. Relative expressions of selected genes were calculated and compared to the reference transcript using the 2^−ΔΔCt method.⁵⁸ At least three independent experiments were performed in triplicate for each gene.

Intracellular ROS level measurement

The production of ROS in T24 cells induced by 5-23 was measured using 2′,7′-dichlorofluorescein diacetate (DCF-DA).⁵⁹ In brief, after treatment with 5-23, floating and adherent cells were merged. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was added to each well (final concentration: 10 μM) and incubated for 30 min at 37 °C in the dark. Cells were washed, resuspended in PBS, and analyzed using flow cytometry. To further characterize the ROS generated upon 5-23 stimulation, the intracellular ROS levels were measured quantitatively with a Perkin-Elmer LS55 luminescence spectrometer (PerkinElmer, Inc., Waltham, MA, USA). Results were expressed as ratios relative to the control group. All experiments were conducted with three parallel samples.

Mitochondrial membrane potential (ΔΨ_m) assay

The lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China) was used to detect the ΔΨ_m.⁶⁰ Briefly, floating and adherent T24 cells pretreated with 5-23 were merged, washed with cold PBS, and dyed with JC-1 (10 μg mL⁻¹) in the dark. Fluorescence microscopic images were acquired under a laser scanning confocal microscope (Carl Zeiss LSM710, Oberkochen, Germany). Five fields of vision were randomly selected for each sample.

In vivo pharmacokinetic study

Healthy Sprague-Dawley rats (250 ± 20 g) were obtained from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, Hunan Province, China). All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Guangxi Normal University and approved by the Animal Ethics Committee of Guangxi Normal University. Rats were fasted for 12 h but allowed free access to water before the experiments. The suspension of 5-23 (or Reg) was prepared at a final concentration of 2 mg mL⁻¹ in a mixture of Tween 80 and 0.9% sodium chloride (1.5 : 5, v/v). Eighteen rats were randomly divided into 3 groups (6 in each group). One group was given Reg suspension at a single dose of 12 mg kg⁻¹. The other two groups were administered with 5-23 at low (6 mg kg⁻¹) and high (12 mg kg⁻¹) doses, respectively. Blood samples (0.25 mL) were collected from the orbital vein into the heparinized tubes at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. Blood samples were centrifuged at 3000 rpm for 10 min and the resultant plasma samples were transferred into capped tubes and stored at −20 °C until analysis. The concentrations of 5-23 and Reg in the plasma were determined by HPLC. The Shimadzu LC-10A HPLC analysis system equipped with an ultraviolet detector (SPD-10A) was used. Chromatographic separation was achieved on a reversed-phase C18 column (250 mm × 4.6 mm, 5 μm, Promosil®, Bonna-Agela Technologies Ltd., Tianjin, China). The post-processing steps were described in our previous report.⁶¹ HPLC method and its validation for the determination of 5-23 and Reg were described in detail in ESI† (see additional file 1). The pharmacokinetic parameters were calculated using Kinetica (version: 4.4.1) software. All experiments were repeated three times.

Molecular docking

A docking study was performed using the AutoDock software package (Scripps Research Institute, La Jolla, CA, USA; version: 4.2.6) and MGLTools (version: 1.5) to explore the interactions between the target compounds and MLKL kinase (receptor) and to visualize the probable binding mode. Before docking, the three-dimensional structures of the ligands were achieved via ChemBio3D Ultra 12.0 software (PerkinElmer Inc., USA). Compounds 5-23 and 5-1 were selected as representative examples of the above pyrazinyl–aryl urea derivatives. The three-dimensional structure of MLKL can be obtained from the Protein Data Bank (http://www.rcsb.org, PDB ID: 6O5Z).⁶² Before implementing molecular simulation and docking procedures, the original ligands LN4 (CA name: 1-[2-fluoranyl-5-(trifluoromethyl)phenyl]-3-[4-[methyl-[2-[(3-sulfamoyl-phenyl)amino]pyrimidin-4-yl]amino]phenyl]urea), EDO (CA name: 1,2-ethanediol), and water molecules were removed, then polar hydrogen atoms were added. In the docking model, a grid (100 × 100 × 100 Å) was set and the binding energies were obtained by the AutoDock scoring function. After the complex of the ligand bound to the receptor was obtained, the following analyses were carried out using PyMOL open-source software (Schrodinger LLC, New York, NY, USA; version: 2.5). All calculations were performed on a Linux workstation (4 × 2 cores) running Centos 7.9. The binding model of 5-23 (or Reg) in complex with another receptor VEGFR-2 was also constructed using the same procedures mentioned above (PDB ID of VEGFR-2: 3WZE).⁶³

Statistical analysis

Data are presented as means ± standard deviation (S.D.). The differences between groups were analyzed by the one-way analysis of variance (one-way-ANOVA) using GraphPad Prism 7.0 software, and p < 0.05 was considered to indicate a statistically significant difference.

Abbreviations used

Annexin

Annexin V-fluorescein isothiocyanate (Annexin V-FITC)

Apat

Apatinib

BC

Bladder cancer

Casp-3

Caspase-3

Casp-8

Caspase-8

cyto c

Cytochrome c

DCFH-DA

2′,7′-Dichlorodihydrofluorescein diacetate

ECL

Enhanced chemiluminescence

FDA

U.S. Food and Drug Administration

FGFR

Fibroblast growth factor receptor

Gem

Gemcitabine

HCC

Hepatocellular carcinoma

Hoe

Hoechst 33342

JC-1

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide

MIBC

Muscle-invasive bladder cancer

MLKL

Mixed lineage kinase domain-like pseudokinase

MTA

Molecular-targeted agent

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Nec-1

Necrostatin-1

NMIBC

Non-muscle invasive bladder cancer

NSA

Necrosulfonamide

PCD

Programmed cell death

PDGFR-β

Platelet-derived growth factor receptor-β

PI

Propidium iodide

Reg

Regorafenib

RIPK1

Receptor-interacting protein kinase 1

ROS

Reactive oxygen species

SAR

Structure–activity relationship

SDS-PAGE

SDS-polyacrylamide gel electrophoresis

SI

Selectivity index

VEGF

Vascular endothelial growth factor

VEGFR

Vascular endothelial growth factor receptor

zVAD

Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone

Ψ _m

Mitochondrial membrane potential

Author contributions

J. C. designed the project and performed molecular docking. C. C. and Y. H. synthesized and characterized compounds 5-1–5-28, performed western blotting analysis and in vivo pharmacokinetic study. T. Q. and L. C. performed the following experiments: proliferation inhibitory activity analysis, FACS analysis, Hoe/PI double-labeling, and trypan blue exclusion assay. Y. S., K. Y., and Q. C. performed ROS level measurement, membrane potential assay, and enzyme activity analysis. C. Y. and Y. W. performed RT-PCR analysis. All authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts of interest to declare.