Discovery of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation with potent liver cancer inhibitory activity

Abstract

In this work, a series of novel arylamide derivatives containing piperazine moiety were designed and synthesised as tubulin polymerisation inhibitors. Among 25 target compounds, compound 16f (MY-1121) exhibited low nanomolar IC₅₀ values ranging from 0.089 to 0.238 μM against nine human cancer cells. Its inhibitory effects on liver cancer cells were particularly evident with IC₅₀ values of 89.42 and 91.62 nM for SMMC-7721 and HuH-7 cells, respectively. Further mechanism studies demonstrated that compound 16f (MY-1121) could bind to the colchicine binding site of β-tubulin and directly act on β-tubulin, thus inhibiting tubulin polymerisation. Additionally, compound 16f (MY-1121) could inhibit colony forming ability, cause morphological changes, block cell cycle arrest at the G2 phase, induce cell apoptosis, and regulate the expression of cell cycle and cell apoptosis related proteins in liver cancer cells. Overall, the promising bioactivities of compound 16f (MY-1121) make the novel arylamide derivatives have the value for further development as tubulin polymerisation inhibitors with potent anticancer activities.

Keywords:

• Tubulin
• arylamide
• piperazine
• antiproliferative activities

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Introduction

Microtubules, which are mainly composed mainly of α, β-tubulin heterodimers, serve as key component of the cytoskeleton¹. Given the important roles in the multiple biological functions, microtubule has been one important and successful target for the human cancer treatment. At present, a total of seven binding sites of tubulin have been identified^2,3, and there are already microtubule inhibitors targeting taxane and vinca alkaloid sites approved by the FDA for clinical tumour treatment. Colchicine binding site, as one of the tubulin binding sites, is also receiving increasing attention due to their ability to overcome the shortcomings and limitations associated with taxane or vinca alkaloid binding site inhibitors^4,5. Although there are no FDA-approved colchicine binding site inhibitors (CBSIs) for clinical tumour treatment, several CBSIs (e.g. CA-4P⁶, AVE8062⁷, BNC-105p⁸, and VERU-111⁹; Figure 1) have been or are being studied in clinical trials due to their significant antitumor activities. Additionally, many groups also have reported potent anticancer effective CBSIs with different chemical structure types^10–17, and the arylamide tubulin inhibitors have also attracted much attention because of their strong antitumor abilities^18–21. For example, compound 1¹⁸, benzamide derivative 2¹⁹, and N-benzylbenzamide derivative 3²⁰ could potently inhibit the polymerisation of tubulin and exhibit significant antiproliferative activities on several cancer cell lines. Recently, we also reported N-benzylarylamide 4 as a tubulin polymerisation inhibitor with potent antiproliferative activities in two-digit nanomolar IC₅₀ values²¹.

Figure 1. Structures of typical CBSIs under clinical trials and reported CBSIs.

The structures of FDA approved marketed drugs generally contain nitrogen heterocycles, especially piperazines, which might help to adjust the lipid–water partition coefficient and acid–base equilibrium constant of drugs^22–24. In fact, piperazine groups are also frequently used to design antitumor drugs²⁵. Recently, many groups have also reported high anticancer potency tubulin polymerisation inhibitors with piperazine moieties^26–35. Müller and coworkers reported a N‑heterocyclic (4-phenylpiperazin-1-yl)methanone 5 as a tubulin polymerisation inhibitor (Figure 2), which was derived from the phenoxazine and phenothiazine²⁶. Compound 5 exhibited potent antiproliferative potency against a panel of cancer cell lines with a mean GI₅₀ value of 3.3 nM and potent inhibitory effect on tubulin polymerisation by acting on the colchicine binding site. Compound 6 posing a N-acyl-piperazine moiety, as the tubulin polymerisation inhibitor, showed significant antiproliferative activities against SGC-7901, A549, and Hela cells (IC₅₀ values of 0.084–0.221 μM)³¹. In addition, arylpiperazinyl oxazole 7 exhibited potent anticancer effects in vitro and in vivo by the inhibition of tubulin polymerisation and excellent vascular-disrupting activity³². Piperazine-based compound 8 was identified as a tubulin polymerisation inhibitor with the activity of sensitising cancer cells to apoptotic ligands³⁵.

Figure 2. Structures of piperazine-based tubulin inhibitors.

In a continuation of our interest in developing novel tubulin polymerisation inhibitors^21,36, we designed and synthesised a series of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation. In this work, we assumed that linking N-benzylarylamide fragment to the piperazine moieties through the principle of molecular hybridisation strategy could afford novel arylamide derivatives containing piperazine moiety, which might bind to the colchicine binding site of β-tubulin with potent anticancer activities (Figure 3). Therefore, a total of 25 target compounds were designed, synthesised and explored for antiproliferative activities against three selected tumour cell lines. Among them, the representative compound 16f (MY-1121) was identified as a tubulin polymerisation inhibitor by binding to the colchicine binding site with potent anticancer activities.

Figure 3. Design strategy of arylamide derivatives as inhibitors of tubulin polymerisation.

Synthesis of novel arylamide derivatives 16a–16y

The synthetic route of the designed arylamide derivatives 16a–16y is outlined in Scheme 1. A condensation reaction between commercially available 3-hydroxy-4-methoxybenzaldehyde (10) and 3,4,5-trimethoxyaniline (9) afforded the imine intermediate 11, which was further reduced with sodium borohydride (NaBH₄) to get 2-methoxy-5-(((3,4,5-trimethoxyphenyl)amino)methyl)phenol (12). Then, 12 was subjected to a substitution reaction in the presence of triethylamine (TEA) to obtain 2-chloro-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (13), which were then treated with commercially available morpholine, thiomorpholine, or different substituted piperazines and piperidines to give the corresponding designed arylamide derivatives 16a–16y.

Scheme 1. Synthesis of target compounds 16a–16y.

Results and discussion

Antiproliferative activities of compounds 16a–16y against MGC-803, HCT-116, and SMMC-7721 cells

To explore the antiproliferative activities of designed arylamide derivatives 16a–16y, we determined the MTT assay to test the antiproliferative potency on three selected human cancer cells, including human liver cancer cells SMMC-7721, human gastric cancer cells MGC-803, and human colon cancer cells HCT-116. Additionally, the well-known tubulin polymerisation inhibitor colchicine was selected as the positive control. The results of the antiproliferative activities of designed arylamide derivatives 16a–16y are shown in Tables 1–3.

Table 1. Antiproliferative activities of compounds 16a–16k and colchicine.

Comp.		IC50 (μM)^a
		MGC-803	HCT-116	SMMC-7721
16a		0.25 ± 0.010	0.32 ± 0.032	0.18 ± 0.022
16b		0.26 ± 0.023	0.15 ± 0.014	0.21 ± 0.018
16c		0.78 ± 0.033	0.88 ± 0.078	0.65 ± 0.018
16d		0.88 ± 0.046	0.91 ± 0.065	0.8 ± 0.021
16e		0.094 ± 0.006	0.12 ± 0.027	0.092 ± 0.008
16f (MY-1121)		0.092 ± 0.005	0.098 ± 0.034	0.088 ± 0.006
16g		0.089 ± 0.004	0.104 ± 0.013	0.095 ± 0.004
16h		0.095 ± 0.002	0.098 ± 0.004	0.097 ± 0.005
16i		0.17 ± 0.022	0.16 ± 0.026	0.10 ± 0.012
16g		0.11 ± 0.007	0.098 ± 0.008	0.091 ± 0.004
16k		0.15 ± 0.023	0.16 ± 0.012	0.13 ± 0.014
Colchicine		0.066 ± 0.003	0.077 ± 0.002	0.087 ± 0.002

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values. IC₅₀ values were indicated as the mean ± SD of three independent experiments.

Table 2. Antiproliferative activities of compounds 16l–16s and colchicine.

Comp.		IC50 (μM)^a
		MGC-803	HCT-116	SMMC-7721
16l		1.21 ± 0.14	0.88 ± 0.21	1.58 ± 0.28
16m		1.44 ± 0.38	1.57 ± 0.41	1.79 ± 0.32
16n		1.48 ± 0.011	1.66 ± 0.18	1.77 ± 0.15
16o		2.17 ± 0.25	2.41 ± 0. 18	2.21 ± 0.19
16p		5.36 ± 0.42	3.53 ± 0.27	3.77 ± 0.31
16q		6.21 ± 0.38	5.91 ± 0.52	5.68 ± 0.48
16r		6.29 ± 0.38	6.31 ± 0.17	6.69 ± 0.11
16s		2.86 ± 0.014	2.28 ± 0.12	2.87 ± 0.066
Colchicine		0.066 ± 0.003	0.077 ± 0.002	0.087 ± 0.002

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values.

^b IC₅₀ values are indicated as the mean ± SD of three independent experiments.

Table 3. Antiproliferative activities of compounds 16t–16y and colchicine.

Comp.		IC50 (μM)^a
		MGC-803	HCT-116	SMMC-7721
16t		5.87 ± 0.39b	5.75 ± 0.42	6.82 ± 0.44
16u		7.55 ± 0.36	5.45 ± 0.42	7.12 ± 0.67
16v		6.37 ± 0.54	5.29 ± 0.38	8.16 ± 0.48
16w		5.48 ± 0.28	5.18 ± 0.41	6.11 ± 0.46
16x		5.13 ± 0.27	6.07 ± 0.16	5.12 ± 0.28
16y		5.18 ± 0.14	6.22 ± 0.28	5.01 ± 0.25
Colchicine		0.066 ± 0.003	0.077 ± 0.002	0.087 ± 0.002

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values. IC₅₀ values are indicated as the mean ± SD of three independent experiments.

As described in Table 1, we first explored the antiproliferative activities of compounds 16a–16k with aryl piperazine groups. As expected, most of compounds exhibited potent antiproliferative activities against these three human cancer cells with IC₅₀ values less than 0.1 μM. Among them, the compound 16f (also named MY-1121) bearing the 1-(4-chlorophenyl)piperazine group exhibited the most potent inhibitory activities on MGC-803 (IC₅₀ = 0.092 μM), HCT-116 (IC₅₀ = 0.098 μM), and SMMC-7721 (IC₅₀ = 0.088 μM) cells, which was similar to the positive control colchicine. In addition, the SMMC-7721 cells were more sensitive to compounds 16a–16k with lower IC₅₀ values. Therefore, we discussed the structure–activity relationships based on the antiproliferative activities of compounds 16a–16k on SMMC-7721 cells. Compared with compound 16a, it was found that the substituents and their positions of the aryl piperazines showed a certain influence on the inhibitory activities. Among compounds 16b–16d, the antiproliferative activity of compound 16b with substituent in the para position of aryl piperazine was superior to that in the meta (compound 16c) and ortho (compound 16d) positions. Compared with compound 16a, the introduction of electron withdrawing groups (–CN, –OH, –F, –Cl, –Br, and –COOCH₃) on the para position of benzene ring helped to improve the antiproliferative activities, while the introduction of electron donating group –OCH₃ in the para, meta, or ortho position could impair the inhibitory potency.

We further explored the effects of replacement of aryl piperazine groups with alkyl piperazines, carbonyl piperazines, morpholine, or thiomorpholine on the antiproliferative activities. As shown in Table 2, the target compounds 16l–16s exhibited reduced antiproliferative activities with IC₅₀ values ranging from 0.88 to 6.69 μM than that of compounds 16a–16k. Among them, compound 16l bearing the 1-methylpiperazine group exhibited the most potent inhibitory activities with IC₅₀ values of 1.21 (for MGC-803), 0.88 (for HCT-116), and 1.58 μM (for SMMC-7721). Comparing the antiproliferative activities of compounds 16l–16s, it was obvious to figure out that the compounds 16l–16o bearing the alkyl piperazines were more effective in inhibiting the proliferation of cancer cells than that of compounds 16p–16s bearing the carbonyl piperazines (compounds 16p–16q), morpholine (compound 16r), or thiomorpholine (compound 16s). In addition, the extension of alkyl chains (compounds 16l–16o) reduced inhibitory activities.

In addition, we also explored the effects of replacement of piperazine groups with piperidine groups on the antiproliferative activities (Table 3). Unfortunately, when the substituent piperazine groups were replaced with the piperidine groups, the antiproliferative activities of the compounds were obviously reduced with IC₅₀ values ranging from 5.01 to 8.16 μM, indicating that piperazine groups were more help to maintain inhibitory effects than that of the piperidine groups.

Antiproliferative activities of compound MY-1121 against other 7 cells

Given the most potent antiproliferative activities of compound MY-1121 against MGC-803, HCT-116, and SMMC-7721 cells, six additional cancer cell lines (human liver cancer cells MHCC-97H, HuH-7 and HepG2, human gastric cancer cells BGC-823 and NCI-N87, and human oesophageal cancer cells KYSE450) and one normal liver cells HL-7702 were chosen to further explore the broad-spectrum antiproliferative activities. As shown in Table 4, compound MY-1121 also exhibited potent antiproliferative activities against these selected cancer cells with IC₅₀ values from 91.6 to 238.2 nM. Among them, human liver cancer cells HuH-7 and HepG2 were more sensitive to compound MY-1121 with IC₅₀ values of 91.6 and 100.8 nM, respectively. Importantly, compound MY-1121 displayed more reduced antiproliferative activity against normal liver cells HL-7702.

Table 4. Antiproliferative activities of compound MY-1121 against six additional cancer cell lines.

Cell types	Cell lines	IC50 (nM)^a
Liver cancer	MHCC-97H	131.9 ± 10.31
	HuH-7	91.6 ± 5.92
	HepG2	100.8 ± 6.09
Gastric cancer	BGC-823	146.1 ± 23.38
	NCI-N87	110.1 ± 4.30
Oesophageal cancer	KYSE450	238.4 ± 14.75
Normal liver cells	HL-7702	3276.2 ± 531.23

^a Cells were treated with different concentrations of compounds for 48 h to obtain the IC₅₀ values. IC₅₀ values are indicated as the mean ± SD of three independent experiments.

Compound MY-1121 inhibited tubulin polymerisation by acting on the colchicine binding site of β-tubulin

To explore whether these compounds we designed interacted with tubulin system, the most potent compound MY-1121 was selected to determine the inhibitory effect on tubulin polymerisation by the cell-free tubulin polymerisation assay. Additionally, the well-known tubulin polymerisation inhibitor colchicine and microtubule-stabilising agent paclitaxel were chosen as the reference drugs. As shown in Figure 4, compound MY-1121 significantly decreased the absorbance, indicating that it could inhibit the polymerisation of tubulin, which was consistent with the effect of colchicine, and the effect was stronger than that of colchicine. While the paclitaxel obviously increased the absorbance, suggesting that it could enhance the polymerisation of tubulin. These results indicated that compound MY-1121 exhibited slightly less potent tubulin polymerisation inhibitory activity than that of colchicine.

Figure 4. Effects of compound MY-1121 on tubulin polymerisation under cell free condition. The vertical coordinate indicates the degree of tubulin polymerisation. The experiments were repeated thrice.

To confirm whether the compound MY-1121 could act on the colchicine binding site of β-tubulin, the EBI competitive assay and alkaline protease assay were carried out in SMMC-7721 and HuH-7 cells. In the EBI competitive assay, if the tested compound exhibit good affinity with the colchicine binding site of β-tubulin, the β-tubulin adduct band in the western blot results will be reduced. As shown in Figure 5(A,B), compound MY-1121 significantly reduced the signal level of the β-tubulin adduct band in a concentration-dependent manner, which was consistent with the effect of the colchicine. These results indicated that compound MY-1121 showed good affinity with the colchicine binding site of β-tubulin. In the alkaline protease assay (Figure 5(C,D)), compound MY-1121 could directly bind to β-tubulin, leading to a decrease in the degradation ability of alkaline protease on β-tubulin. These results indicated that compound MY-1121 could act on the colchicine binding site of β-tubulin.

Figure 5. Compound MY-1121 bind to β-tubulin directly on colchicine binding site. (A&B). EBI competition assay, the affinity of the compound with colchicine binding site was negatively correlated with the level of the tubulin adduct band; (C&D). Alkaline protease assay. Cells were treated with Alkaline Proteinase and different concentration of compound MY-1121. The band signal of β-tubulin was negatively correlated with the ability of Alkaline Protease to hydrolyze β-tubulin. The binding of the compound to β-tubulin was able to inhibit the hydrolysis of β-tubulin by Alkaline Protease.

Inhibition of intracellular microtubules

To profile the intracellular inhibitory abilities of the representative compound MY-1121 against microtubules, the immunofluorescence staining assay was performed in SMMC-7721 and HuH-7 cells. As shown in Figure 6, with the increase concentrations of compound MY-1121, the proportion of tubulin (green) polymerised into filamentous structures (microtubules) gradually decreased, indicating that compound MY-1121 inhibited the ordered polymerisation of tubulin. However, the cell nucleus did not stopped replicating, resulting in a phenomenon of multiple nuclei in the high-dose group in SMMC-7721 and HuH-7 cells. Additionally, these intracellular inhibitory effects were consistent with the positive control colchicine. Taken together, these above results indicated that MY-1121 could inhibit microtubules at the cellular level.

Figure 6. Effects of compound MY-1121 on microtubule network in liver cancer cells SMMC-7721 and HuH-7. Cells were treated for 48 h. β-tubulin was stained green and cell nuclei were stained blue.

Binding model of compound MY-1121 with tubulin

Given the compound MY-1121 was identified as a novel tubulin polymerisation inhibitor, a molecular docking was performed to study the possible binding mode of compound MY-1121 with tubulin (PDB: 1SA0). We analysed the interactions between protein and ligand (Figure 7), all functional residues were identified and classified according to their interactions. There were multiple groups of residues used to form interactions between receptor protein and ligand, such as the hydrogens bond formed by GLN11 and TYR224 of TUBA1D and ligand. With these interaction forces, the binding energy of protein–ligand complex was −6.6 kcal/mol, which was a good performance and might help to exhibit potent activities.

Figure 7. Molecular docking results of compound MY-1121 with tubulin (PDB: 1AS0). The hydrogen bond, ionic interactions, and hydrophobic interactions are depicted as yellow, magentas, and green dashed lines, respectively.

Compound MY-1121 inhibited cell proliferation

As the executor of cell proliferation, when the polymerisation of tubulin is inhibited, cell proliferation is also inhibited. Therefore, after verifying the inhibitory effects of compound MY-1121 on tubulin polymerisation, we further explored the effects of compound MY-1121 on the proliferation of liver cancer cells SMMC-7721 and HuH-7.

The results in Figure 8 suggested that compound MY-1121 caused strong arresting effects on the cell cycle in SMMC-7721 and HuH-7 cells, and the percentage of G2/M phase cells in two liver cancer cells increased by 40% (SMMC-7721 cells) and 57% (HuH-7 cells), respectively, indicating that compound MY-1121 arrested SMMC-7721 and HuH-7 cell cycle at the G2 phase.

Figure 8. Effects of compound MY-1121 on cell cycle distribution. Liver cancer cells SMMC-7721 (A, B) and HuH-7 (C, D) were treated for 48 h. The cells were stained with propidium iodide and analysed via flow cytometry to measure the cell cycle profile.

Considering that the colony forming ability of cells is a manifestation of cell proliferation activity, we further investigated the effects of compound MY-1121 on the colony forming ability of HuH-7 cells by the colony formation assay. As shown in Figure 9, when cells were treated with compound MY-1121 for seven days, the ability of cells to format colonies was concentration-dependently inhibited, and the number of colonies was also significantly reduced in a concentration-dependent manner.

Figure 9. Effects of compound MY-1121 on colony formatting ability. Liver cancer cells SMMC-7721 (A) and HuH-7 (B) were treated for seven days.

The effects of compound MY-1121 on the expression of cell cycle regulatory proteins were further tested by Western blotting assay. At the protein levels, compound MY-1121 induced down-regulation on the expression of G2 phase-related protein p-cdc2, and up-regulation on the expression of M phase marker protein p-histone H3 (Figure 10).

Figure 10. The effects of compound MY-1121 on cell cycle related proteins in SMMC-7721 cell were conducted via Western blotting assay. Cells were treated with indicated concentrations of compound MY-1121 for 48 h.

In summary, compound MY-1121 could arrest liver cancer cells SMMC-7721 and HuH-7 cells in the G2/M phase and regulate the expression of related proteins.

Effects of compound MY-1121 on morphology changes

Effects of compound MY-1121 on morphology changes in SMMC-7721 and HuH-7 cells were further explored. As shown in Figure 11, under the compound MY-1121 effects, the SMMC-7721 and HuH-7 cells became smaller and rounder (upper panel in Figure 11), the cell density decreased and the dead cells (red) increased (middle panel in Figure 11), and the nuclei became brighter and concentrated (lower panel in Figure 11).

Figure 11. Effects of compound MY-1121 on morphology changes of liver cancer cells. Liver cancer cells were treated for 48 h. (A) Cell morphology in the bright field; (B) number of live cells (green) to dead cells (red); and (C) cell nuclei changes.

Compound MY-1121 induced apoptosis in liver cancer cells SMMC-7721 and HuH-7

Previous studies have shown that tumour cell cycle arrest and inhibition of proliferation have the potential to trigger the development of apoptosis. Therefore, the effects of compound MY-1121 on apoptosis of liver cancer cells SMMC-7721 and HuH-7 were examined. The changes in cell morphology after the treatment of compound MY-1121 suggested that it could induce apoptosis in liver cancer cells SMMC-7721 and HuH-7 (Figure 12). The annexin-V/PI assay further validated the concentration-dependent induction of apoptosis by compound MY-1121 in SMMC-7721 and HuH-7 cells (Figure 12). As shown in Figure 12, after 48 h of treatment with compound MY-1121, the apoptosis rates of SMMC-7721 and HuH-7 cells were both close to 70%, indicating that compound MY-1121 exhibited a clear apoptosis-inducing effect.

Figure 12. Cell apoptosis induced by compound MY-1121. Liver cancer cell SMMC-7721 (A, B) and HuH-7 (C, D) were treated indicated concentrations of compound MY-1121 for 48 h.

Considering that compound MY-1121 could induce apoptosis in SMMC-7721 and HuH-7 cells, further detection of the effects on apoptosis-related proteins were conducted by the Western blotting assay. As shown in Figure 13, it was found that compound MY-1121 caused a decrease in the expression of anti-apoptotic protein Bcl-xL, and an increase in the expression of apoptosis executor cleaved caspase-7, as well as its cleavage substrate cleaved-PARP. These above results indicated that compound MY-1121 effectively induced apoptosis and regulated the expression of apoptosis related proteins in liver cancer cells SMMC-7721 and HuH-7.

Figure 13. The effects of compound MY-1121 on apoptosis-related proteins in SMMC-7721 cell were conducted via Western blotting assay. Cells were treated indicated concentrations of compound MY-1121 for 48 h.

Conclusions

In summary, a series of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation have been designed, synthesised, and evaluated. The representative compound MY-1121 exhibited low nanomolar IC₅₀ values ranging from 0.089 to 0.238 μM against nine cancer cells. Its inhibitory effects on liver cancer cells were particularly evident, with IC₅₀ values of 89.42 and 91.62 nM for SMMC-7721 and HuH-7 cells, respectively. Further research found that compound MY-1121 could bind to the colchicine binding site of β-tubulin and directly act on β-tubulin, thus inhibiting tubulin polymerisation both in liver cancer cells and cell-free conditions. The mechanism studies demonstrated compound MY-1121 could cause cell cycle arrest at G2 phase, induce cell apoptosis, and regulate the expression of cell cycle and cell apoptosis related proteins in liver cancer cells. In addition, compound MY-1121 could also inhibit the colony forming ability of SMMC-7721 cells and cause morphological changes in MMC-7721 and HuH-7 cells. Therefore, novel arylamide derivatives containing piperazine moiety could serve as effective tubulin polymerisation inhibitors, which caused inhibition of cell proliferation and induction of apoptosis in liver cancer cells.

Experimental

General

All the commercially available materials (Aladdin Co., Ltd., Shanghai, China, Macklin Co., Ltd., Shanghai, China, or Innochem Co., Ltd., Beijing, China), solvents (Aladdin Co., Ltd., Shanghai, China, Macklin Co., Ltd., Shanghai, China, or Innochem Co., Ltd., Beijing, China), antibodies (Cell Signaling Technology, Beverly, MA), and cancer cells (Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were purchased from commercial companies. NMR and HRMS spectra were recorded using the Bruker av400 and Bruker Micromass (Billerica, MA), respectively. The purity of compounds was determined by reversed phase C18 high performance liquid chromatography with UV detection at 254 nm and 25 °C.

Synthesis of target compounds 16a–16y

The synthesis methods of compounds 11, 12, and 14 refer to our previous collaboration and were provided in the supporting information²¹.

To a solution of 14 (1.0 mmol, 1.0 equiv.) in anhydrous acetonitrile (20 mL), commercially available morpholine, thiomorpholine, or different substituted piperazines and piperidines (1.0 mmol, 1.0 equiv.), and K₂CO₃ (1.0 mmol, 0.5 equiv.) were added, and then the mixture was stirred for 6–8 h at 80 °C. Upon completion, the resultant mixture was concentrated to get the white crude product, which was further purified by chromatography on silica gel (PE:EA = 1:1) to afford corresponding designed arylamide derivatives 16a–16y.

N-(3-Hydroxy-4-methoxybenzyl)-2-(4-phenylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl) acetamide (16a)

White powder; yield: 58.2%; m.p. 118–119 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (s, 1H), 7.20 (t, J = 7.8 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H), 6.84–6.69 (m, 3H), 6.57 (d, J = 8.0 Hz, 1H), 6.43 (s, 2H), 4.68 (s, 2H), 3.72 (s, 3H), 3.68 (s, 6H), 3.66 (s, 3H), 3.06 (s, 6H), 2.55 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.05, 153.25, 151.47, 147.30, 146.77, 137.78, 137.18, 130.82, 129.37, 119.75, 119.24, 116.19, 115.80, 112.41, 106.38, 60.55, 59.42, 56.42, 56.10, 52.86, 52.03, 48.69. HRMS: calcd. C₂₉H₃₆N₃O₆ [M + H]⁺, 522.2599, found: 522.2607.

N-(3-Hydroxy-4-methoxybenzyl)-2-(4-(4-methoxyphenyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16b)

White powder; yield: 62.1%; m.p. 108–109 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 6.88–6.76 (m, 5H), 6.70 (d, J = 1.8 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.43 (s, 2H), 4.68 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.04 (s, 2H), 2.99–2.89 (m, 4H), 2.60–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.98, 153.33, 153.24, 147.30, 146.79, 145.87, 137.79, 137.17, 130.84, 119.73, 117.76, 116.22, 114.71, 112.42, 106.39, 60.54, 59.45, 56.42, 56.10, 55.64, 52.97, 52.04, 50.06. HRMS: calcd. C₃₀H₃₈N₃O₇ [M + H]⁺, 522.2704, found: 522.2710.

N-(3-Hydroxy-4-methoxybenzyl)-2-(4-(3-methoxyphenyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16c)

White powder; yield: 49.7%.; m.p. 112–113 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.08 (t, J = 8.2 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 1.7 Hz, 1H), 6.58–6.32 (m, 6H), 4.67 (s, 2H), 3.72 (s, 3H), 3.70 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.11–2.99 (m, 6H), 2.55–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.52, 160.17, 152.75, 152.35, 146.81, 146.31, 137.32, 136.73, 130.37, 129.51, 119.23, 115.74, 111.99, 107.97, 105.94, 104.06, 101.41, 60.00, 58.94, 55.99, 55.59, 54.78, 52.34, 51.56, 48.18. HRMS: calcd. C₃₀H₃₈N₃O₇ [M + H]⁺, 522.2704, found: 522.2714.

N-(3-Hydroxy-4-methoxybenzyl)-2-(4-(2-methoxyphenyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16d)

White powder; yield: 59.2%; m.p. 121–122 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 6.95–6.79 (m, 5H), 6.70 (d, J = 1.6 Hz, 1H), 6.57 (d, J = 7.8 Hz, 1H), 6.43 (s, 2H), 4.68 (s, 2H), 3.75 (s, 3H), 3.72 (s, 3H), 3.68 (s, 6H), 3.65 (s, 3H), 3.05 (s, 2H), 2.94–2.82 (m, 4H), 2.61–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.07, 153.24, 152.44, 147.29, 146.77, 141.70, 137.81, 137.16, 137.13, 130.83, 122.86, 121.32, 119.73, 118.37, 116.18, 112.42, 106.38, 60.56, 59.52, 56.43, 56.10, 55.78, 53.10, 52.02, 50.51. HRMS: calcd. C₃₀H₃₈N₃O₇ [M + H]⁺, 522.2704, found: 522.2709.

2-(4-(4-Fluorophenyl)piperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16e)

White powder; yield: 49.7%; m.p. 124–125 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.05–6.98 (m, 2H), 6.91 (dd, J = 9.1, 4.6 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 1.8 Hz, 1H), 6.57 (d, J = 5.8 Hz, 1H), 6.41 (d, J = 12.4 Hz, 2H), 4.67 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.05 (s, 2H), 3.02–2.96 (m, 4H), 2.58–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.52, 157.09, 154.76, 152.75, 147.89 (d, J = 1.9 Hz), 146.81, 146.31, 137.01 (d, J = 58.8 Hz), 130.37, 119.23, 116.97, 115.28, 115.06, 111.96, 105.94, 60.00, 58.89, 55.98, 55.90, 55.67, 52.34, 51.55, 49.00. HRMS: calcd. C₂₉H₃₅FN₃O₆ [M + H]⁺, 540.2504, found: 540.2511.

2-(4-(4-Chlorophenyl)piperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16f, MY-1121)

White powder; yield: 62.1%; m.p. 132–133 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H), 7.21 (d, J = 9.0 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 1.7 Hz, 1H), 6.57 (d, J = 7.5 Hz, 1H), 6.43 (s, 2H), 4.68 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.12–3.02 (m, 6H), 2.57–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.99, 153.25, 150.26, 147.30, 146.76, 137.75, 137.16, 130.80, 129.05, 122.72, 119.74, 117.25, 116.20, 112.41, 106.38, 60.55, 59.33, 56.42, 56.10, 52.66, 52.05, 48.50. HRMS: calcd. C₂₉H₃₅ClN₃O₆ [M + H]⁺, 556.2209, found: 556.2211.

2-(4-(4-Bromophenyl)piperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16g)

White powder; yield: 58.7%; m.p. 119–120 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.32 (d, J = 8.9 Hz, 2H), 6.83 (dd, J = 19.1, 8.6 Hz, 3H), 6.70 (d, J = 1.5 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.42 (s, 2H), 4.67 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.33 (s, 2H), 3.07–3.04 (m, 4H), 2.57–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.50, 152.76, 150.11, 146.81, 146.31, 137.29, 136.73, 131.43, 130.36, 119.23, 117.20, 115.75, 111.95, 109.84, 105.93, 60.00, 58.86, 55.99, 55.90, 55.59, 52.14, 47.91. HRMS: calcd. C₂₉H₃₅BrN₃O₆ [M + H]⁺, 600.1704, found: 600.1696.

2-(4-(4-Cyanophenyl)piperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16h)

White powder; yield: 67.1%; m.p. 142–142 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.57 (t, J = 9.2 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.69 (d, J = 1.6 Hz, 1H), 6.56 (d, J = 7.9 Hz, 1H), 6.42 (s, 2H), 4.67 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.31–3.23 (m, 4H), 3.06 (s, 2H), 2.57–2.52 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.46, 153.12, 152.78, 146.82, 146.31, 137.22, 133.23, 130.33, 119.98, 119.25, 115.73, 114.01, 113.96, 111.96, 105.93, 98.16, 60.08, 58.69, 55.99, 55.91, 55.68, 51.88, 46.44. HRMS: calcd. C₃₀H₃₅N₄O₆ [M + H]⁺, 547.2551, found: 547.2554.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-(4-nitrophenyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16i)

White powder; yield: 67.1%; m.p. 118–119 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H), 8.04 (d, J = 9.3 Hz, 2H), 7.00 (d, J = 9.3 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.70 (s, 1H), 6.57 (d, J = 7.6 Hz, 1H), 6.42 (s, 2H), 4.67 (s, 2H), 3.72 (s, 3H), 3.68 (s, 6H), 3.65 (s, 3H), 3.43–3.38 (m, 4H), 3.08 (s, 2H), 2.63–2.53 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.94, 155.16, 153.29, 147.30, 146.76, 137.68, 137.65, 137.27, 130.77, 126.18, 119.76, 116.20, 113.07, 112.41, 106.38, 60.55, 59.02, 56.44, 56.10, 52.29, 52.05, 46.89. HRMS: calcd. C₂₉H₃₅N₄O₈ [M + H]⁺, 567.2449, found: 567.2453.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-(4-hydroxyphenyl)piperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16j)

White powder; yield: 59.7%; m.p. 122–123 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.90 (s, 1H), 8.78 (s, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.72 (dd, J = 17.6, 5.4 Hz, 3H), 6.63 (d, J = 8.9 Hz, 2H), 6.56 (d, J = 6.5 Hz, 1H), 6.42 (s, 2H), 4.67 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.03 (s, 2H), 2.95–2.82 (m, 4H), 2.54–2.51 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.57, 152.73, 150.81, 146.81, 146.31, 144.18, 137.35, 136.70, 130.38, 124.50, 119.20, 117.64, 115.40, 111.99, 105.94, 60.09, 60.00, 55.98, 55.90, 52.58, 51.54, 49.98. HRMS: calcd. C₂₉H₃₆N₃O₇ [M + H]⁺, 538.2548, found: 538.2559.

Methyl 4-(4-(2-((3-hydroxy-4-methoxybenzyl)(3,4,5-trimethoxyphenyl)amino)-2-oxoethyl)piperazin-1-yl)benzoate (16k)

White powder; yield: 59.7%; m.p. 131–132 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H), 7.77 (d, J = 8.9 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 1.7 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.43 (s, 2H), 4.67 (s, 2H), 3.77 (s, 3H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.29–3.19 (m, 4H), 3.06 (s, 2H), 2.61–2.52 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.95, 166.61, 154.33, 153.27, 147.29, 146.77, 137.73, 137.11, 131.16, 130.80, 119.74, 118.51, 116.19, 113.77, 112.36, 106.33, 60.53, 59.22, 56.41, 56.07, 52.51, 52.02, 51.92, 47.15. HRMS: calcd. C₃₁H₃₈N₃O₈ [M + H]⁺, 580.2653, found: 580.2653.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-methylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16l)

White powder; yield: 48.2%; m.p. 127–128 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 1.7 Hz, 1H), 6.55 (d, J = 7.7 Hz, 1H), 6.40 (s, 2H), 4.65 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 2.96 (s, 2H), 2.42–2.32 (m, 4H), 2.30–2.16 (m, 4H), 2.11 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.12, 153.19, 147.27, 146.75, 137.85, 137.10, 130.83, 119.68, 116.16, 112.39, 106.35, 60.54, 59.58, 56.40, 56.10, 55.15, 52.76, 51.99, 46.20. HRMS: calcd. C₂₄H₃₄N₃O₆ [M + H]⁺, 460.2442, found: 460.2452.

2-(4-Ethylpiperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16m)

White powder; yield: 47.8%; m.p. 122–123 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 1.9 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 6.40 (s, 2H), 4.66 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 2.96 (s, 2H), 2.41–2.31 (m, 5H), 2.30–2.21 (m, 5H), 0.95 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.13, 153.19, 147.27, 146.77, 137.88, 137.10, 130.85, 119.68, 116.17, 112.40, 106.34, 60.54, 59.63, 56.40, 56.10, 52.92, 52.83, 52.10, 52.00, 12.40. HRMS: calcd. C₂₅H₃₆N₃O₆ [M + H]⁺, 474.2559, found: 474.2607.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-isopropylpiperazin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16n)

White powder; yield: 67.1%; m.p. 126–127 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 1.8 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.40 (s, 2H), 4.66 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 2.97 (s, 2H), 2.64–2.56 (m, 1H), 2.42–2.33 (m, 8H), 0.95 (s, 3H), 0.93 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.62, 152.69, 146.79, 146.30, 137.42, 136.65, 130.39, 119.18, 115.74, 111.97, 105.86, 60.06, 59.20, 55.95, 55.58, 53.73, 52.61, 51.54, 47.88, 18.04. HRMS: calcd. C₂₆H₃₈N₃O₆ [M + H]⁺, 488.2755, found: 488.2758.

2-(4-Cyclopropylpiperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16o)

White powder; yield: 59.5%; m.p. 134–135 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.65 (s, 1H), 6.57 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 1.7 Hz, 1H), 6.32 (d, J = 8.0 Hz, 1H), 6.16 (s, 2H), 4.42 (s, 2H), 3.48 (s, 3H), 3.44 (s, 6H), 3.41 (s, 3H), 2.72 (s, 2H), 2.26–2.17 (m, 4H), 2.16–2.02 (m, 4H), 1.35–1.27 (m, 1H), 0.12 (q, J = 6.0, 4.1 Hz, 2H), 0.00 (q, J = 2.5 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.64, 152.70, 146.79, 146.29, 137.38, 136.67, 130.38, 119.19, 115.68, 111.94, 105.92, 60.08, 59.12, 55.96, 55.58, 52.73, 52.38, 51.51, 37.97, 5.50. HRMS: calcd. C₂₆H₃₆N₃O₆ [M + H]⁺, 486.2599, found: 486.2608.

2-(4-(Cyclopropanecarbonyl)piperazin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16p)

White powder; yield: 61.3%; m.p. 124–125 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.68 (s, 1H), 6.56 (d, J = 4.0 Hz, 1H), 6.44 (d, J = 30.2 Hz, 2H), 4.66 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.62–3.36 (m, 4H), 3.04 (s, 2H), 2.49–2.27 (m, 4H), 1.99–1.86 (m, 1H), 0.74–0.61 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.85, 169.44, 168.47, 152.78, 146.82, 146.31, 137.20, 130.32, 119.28, 115.75, 111.99, 105.91, 60.08, 59.97, 56.00, 55.91, 55.67, 55.59, 51.55, 10.17, 6.86. HRMS: calcd. C₂₇H₃₆N₃O₇ [M + H]⁺, 514.2548, found: 514.2553.

Tert-butyl 4-(2-((3-hydroxy-4-methoxybenzyl)(3,4,5-trimethoxyphenyl)amino)-2-oxoethyl)piperazine-1-carboxylate (16q)

White powder; yield: 56.3%; m.p. 129–130 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J = 8.5 Hz, 1H), 6.81 (t, J = 8.3 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.56 (s, 1H), 6.40 (d, J = 8.5 Hz, 2H), 4.65 (s, 2H), 3.71 (s, 3H), 3.66 (s, 6H), 3.64 (s, 3H), 3.30–3.20 (m, 4H), 3.02 (s, 2H), 2.43–2.30 (m, 4H), 1.40 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.54, 153.54, 153.28, 147.79, 147.30, 146.79, 137.70, 130.81, 119.76, 116.23, 112.43, 106.37, 60.52, 59.13, 56.44, 56.11, 52.91, 52.45, 46.20, 41.37, 21.61. HRMS: calcd. C₂₈H₄₀N₃O₈ [M + H]⁺, 546.2810, found: 546.2819.

N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-N-(3,4,5-trimethoxyphenyl) acetamide (16r)

White powder; yield: 58.8%; m.p. 132–133 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 6.87–6.51 (m, 3H), 6.41 (s, 2H), 4.66 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.57–3.43 (m, 4H), 2.99 (s, 2H), 2.47–2.30 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.88, 153.26, 147.29, 146.79, 137.75, 137.15, 130.83, 119.73, 116.20, 112.38, 106.34, 66.69, 60.52, 59.70, 56.40, 56.07, 53.36, 52.01. HRMS: calcd. C₂₃H₃₁N₂O₇ [M + H]⁺, 447.2126, found: 447.2132.

N-(3-hydroxy-4-methoxybenzyl)-2-morpholino-N-(3,4,5-trimethoxyphenyl) acetamide (16s)

White powder; yield: 65.1%; m.p. 127–128 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.67 (d, J = 1.8 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.39 (s, 2H), 4.65 (s, 2H), 4.03 (q, J = 7.1 Hz, 1H), 3.85 (t, J = 13.4 Hz, 1H), 3.71 (s, 3H), 3.67 (s, 6H), 3.64 (s, 3H), 3.05 (s, 2H), 2.72–2.60 (m, 4H), 2.51–2.49 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.26, 153.24, 147.27, 146.75, 137.80, 137.10, 130.81, 119.72, 116.18, 112.37, 106.32, 60.53, 60.29, 56.42, 56.08, 54.37, 52.00, 27.76. HRMS: calcd. C₂₃H₃₁N₂O₆S [M + H]⁺, 463.1897, found: 463.1897.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-methylpiperidin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16t)

White powder; yield: 68.2%; m.p. 141–131 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (s, 1H), 6.86–6.36 (m, 5H), 4.65 (s, 2H), 3.72 (s, 3H), 3.66 (s, 6H), 3.64 (s, 3H), 2.94 (s, 2H), 2.67 (d, J = 10.1 Hz, 2H), 1.96 (t, J = 10.8 Hz, 2H), 1.48 (d, J = 11.7 Hz, 2H), 1.28–1.16 (m, 1H), 1.02 (dd, J = 21.6, 10.8 Hz, 2H), 0.84 (d, J = 6.4 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.35, 153.16, 147.25, 146.75, 137.98, 137.04, 130.89, 119.67, 116.16, 112.36, 106.31, 60.52, 60.11, 56.37, 56.07, 53.49, 51.99, 34.43, 30.36, 22.33. HRMS: calcd. C₂₅H₃₅N₂O₆ [M + H]⁺, 459.2490, found: 459.2496.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-methoxypiperidin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16u)

White powder; yield: 48.3%; m.p. 131–132 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 1.3 Hz, 1H), 6.55 (d, J = 7.9 Hz, 1H), 6.40 (s, 2H), 4.65 (s, 2H), 3.72 (s, 3H), 3.66 (s, 6H), 3.65 (s, 3H), 3.19 (s, 3H), 3.02 (d, J = 46.4 Hz, 3H), 2.66–2.53 (m, 2H), 2.13 (t, J = 9.7 Hz, 2H), 1.74 (d, J = 9.8 Hz, 2H), 1.31 (d, J = 9.4 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.30, 153.20, 147.27, 146.78, 137.93, 137.09, 130.89, 119.68, 116.18, 112.38, 106.33, 75.96, 60.52, 59.62, 56.38, 56.08, 55.20, 51.99, 50.87, 31.20. HRMS: calcd. C₂₅H₃₅N₂O₇ [M + H]⁺, 475.2439, found: 475.2446.

N-(3-hydroxy-4-methoxybenzyl)-2-(4-hydroxypiperidin-1-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (16v)

White powder; yield: 55.7%; m.p. 146–147 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.69 (s, 1H), 6.55 (d, J = 7.8 Hz, 1H), 6.39 (s, 2H), 4.66 (s, 2H), 4.48 (s, 1H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 2.95 (s, 2H), 2.61 (d, J = 9.5 Hz, 2H), 2.09 (t, J = 9.8 Hz, 2H), 1.63 (d, J = 9.9 Hz, 2H), 1.30 (d, J = 9.5 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.86, 152.69, 146.78, 146.30, 137.44, 136.66, 130.43, 119.18, 115.69, 111.94, 105.91, 66.06, 60.07, 59.22, 55.95, 55.57, 51.52, 50.74, 34.43. HRMS: calcd. C₂₄H₃₃N₂O₇ [M + H]⁺, 461.2282, found: 461.2282.

2-(4-Chloropiperidin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16w)

White powder; yield: 58.2%; m.p. 135–136 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 1.4 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 6.40 (s, 2H), 4.66 (s, 2H), 4.12 (s, 1H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.01 (s, 2H), 2.64 (s, 2H), 2.29 (t, J = 9.1 Hz, 2H), 1.96 (d, J = 10.2 Hz, 2H), 1.66 (d, J = 9.0 Hz, 2H). ¹³C NMR δ 168.69, 152.74, 146.80, 146.31, 137.35, 136.71, 130.37, 119.21, 115.73, 111.97, 105.89, 60.07, 58.81, 57.89, 55.89, 55.58, 51.55, 50.47, 35.41. HRMS: calcd. C₂₄H₃₂ClN₂O₆ [M + H]⁺, 479.1943, found: 479.1944.

2-(4-Chloropiperidin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16x)

White powder; yield: 61.7%; m.p. 126–126 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.23 (dt, J = 13.2, 6.9 Hz, 5H), 6.81 (d, J = 8.2 Hz, 1H), 6.71 (s, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.43 (s, 2H), 4.68 (s, 2H), 3.72 (s, 3H), 3.67 (s, 6H), 3.65 (s, 3H), 3.04 (s, 2H), 2.83 (d, J = 9.7 Hz, 2H), 2.40 (t, J = 11.9 Hz, 1H), 2.14 (t, J = 10.7 Hz, 2H), 1.69–1.50 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.92, 152.73, 146.79, 146.31, 146.25, 137.59, 136.64, 130.45, 128.30, 126.57, 125.93, 119.22, 115.72, 111.96, 105.85, 60.00, 59.60, 55.98, 55.67, 53.34, 51.58, 41.42, 33.09. HRMS: calcd. C₃₀H₃₇N₂O₆ [M + H]⁺, 521.2646, found: 521.2654.

2-(4-Chloropiperidin-1-yl)-N-(3-hydroxy-4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (16y)

White powder; yield: 67.7%; m.p. 151–152 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.2 Hz, 1H), 6.71 (s, 1H), 6.57 (d, J = 8.1 Hz, 1H), 6.43 (s, 2H), 4.69 (s, 2H), 3.72 (s, 3H), 3.68 (s, 6H), 3.66 (s, 3H), 3.04 (s, 2H), 2.83 (d, J = 9.7 Hz, 2H), 2.42 (t, J = 11.9 Hz, 1H), 2.15 (t, J = 10.8 Hz, 2H), 1.56 (dt, J = 21.9, 11.6 Hz, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.87, 152.74, 146.80, 146.32, 145.21, 137.55, 136.66, 130.45, 128.48, 128.46, 128.20, 119.21, 115.74, 111.97, 105.87, 59.99, 59.48, 55.89, 55.58, 53.19, 51.57, 40.73, 32.94. HRMS: calcd. C₃₀H₃₆ClN₂O₆ [M + H]⁺, 555.2256, found: 555.2260.

Cell culture

All the cells were obtained from the ATCC (Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China). The cells were cultured in the RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin, and 10 mg/mL penicillin streptomycin. All the cells were incubated in a humidified atmosphere containing 5% CO₂ at 37 °C.

In vitro tubulin polymerisation assay

Blank solvent was used as the blank control group, the test compound was used as the experimental group, colchicine was used as the positive drug group, and paclitaxel was used as the negative drug group. The temperature of the fluorescence microplate reader was set to 37 °C, and the above drug mixture was added to each well in a 96-well plate. Fluorescence microplate reader parameters were set with an excitation wavelength of 360 nm and emission wavelength of 450 nm, and the fluorescence intensity was detected every 1 min for a total of 60 times. The reaction system for tubulin polymerisation was prepared. After preheating, the culture plate was removed and the prepared tubulin polymerisation reaction system was quickly added to each well. The plate was then placed in the microplate reader, and the program was started to begin measuring. Data were collected and a curve was drawn.

EBI competitive assay

Different liver cancer cells in logarithmic growth phase were treated with blank solvent, different concentrations of compound, and positive control drugs for 2 h. EBI was added to each group to make the final concentration 100 μM, and incubated for 2 h. A blank control group without EBI treatment was also set up. Cells from each group were collected, and total protein was extracted. Western blot was used to detect the tubulin adduct band. The signal of the tubulin adduct band is negatively correlated with the affinity of the compound and the colchicine binding site of β-tubulin.

Alkaline protease assay

Tumour cells were collected and lysate. Different concentrations of compounds were used to treat the lysate, with a blank solvent group set up, and incubated at 4 °C for 1 h. Proteins in the lysates were hydrolysed by adding subtilisin at a ratio of 1:500, incubated at room temperature for 15 min, and 1 mM PMSF was added to terminate the protein hydrolysis. A control group without hydrolysis was also set up. Western blot was used to detect changes in the level of the target protein.

Molecular docking study

The X-ray crystal structures of TUBA1D (PDB: 1SA0) were retrieved from the Protein Data Bank. The protonation state of all the compounds was set at pH = 7.4, and the compounds were expanded to 3D structures using Open Babel. AutoDock Tools (ADT3) were applied to prepare and parametrise the receptor protein and ligands. The docking grid documents were generated by AutoGrid of sitemap, and AutoDock Vina (1.2.0) was used for docking simulation. The optimal pose was selected to analysis interaction. Finally, the protein–ligand interaction figure was generated by PyMOL. The TUBA1D protein is represented as a slate cartoon model, ligand is shown as a cyan stick, and their binding sites are shown as magentas stick structures. Nonpolar hydrogen atoms are omitted. The hydrogen bond, ionic interactions, and hydrophobic interactions are depicted as yellow, magentas, and green dashed lines, respectively.

Calcein-AM/PI staining

Different liver cancer cells in the logarithmic growth phase were treated with blank solvent, different concentrations of compounds for 48 h. Calcein-AM and PI dyes were added to 1× assay buffer to prepare a double staining solution. After washing the liver cancer cells, they were incubated with the double staining solution for 0.5 h. The results were observed and recorded under a fluorescence microscope.

DAPI staining

Liver cancer cells SMMC-7721 and HuH-7 were treated with drugs at different concentrations of compound for 48 h. After incubation, the cells were washed and fixed in a 4% paraformaldehyde solution at room temperature for 10 min. The paraformaldehyde solution was removed, and the cells were washed again. DAPI staining solution was added to the cells and incubated in the dark at room temperature for 30 min. After incubation, the DAPI staining solution was removed and the cells were washed again. The cells were then observed using a fluorescence microscope to visualise the nuclei and images were recorded.

Immunofluorescence staining

Cells were seeded in 24-well cell culture plates and then treated with vehicle control 0.1% DMSO, different concentrations of compound MY-1121 or colchicine for 6 h. After the treatment, cells were fixed with 4% paraformaldehyde, and then infiltrated for three times with PBS containing 0.1% Triton X-100. Five percent BSA was added into each well before blocking for 30 min at 25 °C. After that, cells were incubated with anti-β-tubulin at 4 °C overnight. Then, we discarded the anti-β-tubulin, added PBS to each well for washing three times, added a fluorescent secondary antibody, and incubated it at room temperature under the dark condition for 1 h. Next, we discarded the fluorescent secondary antibody, washed wells with PBS for three times, and then each well was added DAPI (2 μg/mL) and further incubated at room temperature under the dark condition for 25 min. Finally, the fluorescence microscope was used to visualise the cells.

Western blotting assay

Cells treated with compound MY-1121 were collected, and then lysed to obtain prepared protein samples. The prepared protein samples were denatured and separated by SDS-PAGE, and then transferred to the PVDF membrane. The membrane was sealed with skim milk (5%), and then incubated at 4 °C overnight with the first antibody conjugate. After the incubation, the membrane was washed with TBST/TBS for three times (10 min each time), and then was incubated with the second antibody for another 1.5 h. The target proteins were analysed after washing ECL Ultrasensitive Luminol and Ultrasensitive Imaging Analyzer.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. U2004123 and 82273782 for Sai-Yang Zhang).

Disclosure statement

The authors declared that there was no conflict of interest about this paper.

Correction Statement

This article was originally published with errors, which have now been corrected in the online version. Please see Correction (http://dx.doi.org/10.1080/14756366.2023.2255447)

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Nos. U2004123 and 82273782 for Sai-Yang Zhang). This work was also supported by the Henan Provincial Medical Science and Technology Research Project (No. LHGJ20220394).