Discovery of Simple Diacylhydrazine-Functionalized Cinnamic Acid Derivatives as Potential Microtubule Stabilizers

To develop novel microtubule-binding agents for cancer therapy, an array of N-cinnamoyl-N’-(substituted)acryloyl hydrazide derivatives were facilely synthesized through a two-step process. Initially, the antiproliferative activity of these title compounds was explored against A549, 98 PC-3 and HepG2 cancer cell lines. Notably, compound I23 exhibited the best antiproliferative activity against three cancer lines with IC50 values ranging from 3.36 to 5.99 μM and concurrently afforded a lower cytotoxicity towards the NRK-52E cells. Anticancer mechanism investigations suggested that the highly bioactive compound I23 could potentially promote the protofilament assembly of tubulin, thus eventually leading to the stagnation of the G2/M phase cell cycle of HepG2 cells. Moreover, compound I23 also disrupted cancer cell migration and significantly induced HepG2 cells apoptosis in a dosage-dependent manner. Additionally, the in silico analysis indicated that compound I23 exhibited an acceptable pharmacokinetic profile. Overall, these easily prepared N-cinnamoyl-N’-(substituted)acryloyl hydrazide derivatives could serve as potential microtubule-interacting agents, probably as novel microtubule-stabilizers.


Introduction
Cancer remains the chief and ever-expanding culprit in human mortality that prompts significant concerns in every country [1]. As the International Agency for Research on Cancer (IARC) reported, this intractable disease had been emerging in approximately 18.1 million new cases and thus leading to 9.6 million cancer deaths in 2018 [2,3]. Except for the use of radiotherapy and surgery, chemotherapy is still an effective approach to treat cancer in view of its fast-acting performance against adversaries, the practical applicability and the available effectiveness, but sometimes certain pharmacologically induced side effects coexist [4][5][6][7]. Therefore, exploring and developing a highly effective therapeutic approach to selectively eliminate cancer cells should be actively pursued.
To date, cell cycle modulators or inhibitors, which can arrest uncontrollable tumor growth, are considered as a kind of hopeful antiproliferative agent. Among these modulators, microtubule-interacting agents that target microtubule and subsequently affect multiple cellular processes (e.g., mitosis, cell division, and intracellular transportation) have become a promising group of anticancer agents that have been introduced to the market for cancer therapy over 50 years [8][9][10][11][12][13][14][15]. To date, there have been three key binding sites in tubulin involving paclitaxel (Taxol), colchicine, and vinca alkaloid. These agents have also been divided into two categories: microtubule destabilizing agents and have also been divided into two categories: microtubule destabilizing agents and microtubule-stabilizing agents [16][17][18][19][20][21][22]. Particularly, microtubule-stabilizing agents (MSAs) were considered promising for clinical cancer treatment [23]. For instance, Taxol, the first diterpene isolated from the western yew, had been verified with excellent antiproliferative activity and was used in the clinic. Subsequently, docetaxel, obtained from the semisynthetic derivatives of Taxol, was also approved in 1996 by the Food and Drug Administration (FDA) for clinical treatment [24,25]. Moreover, epothilones and laulimalide, as natural microtubule-stabilizing agents that could promote tubulin self-assembly into microtubules, were also commercialized [26]. However, the wide application of these compounds was restricted due to some urgent problems, including high toxicity, limited sources, complex isolation processes, and the already discovered drug resistance [22]. To solve this issue, some chemically synthesized small molecules were persistently explored and actively developed as microtubule stabilizers, such as GS-164, Synstab A, 4′-methoxy-2-styrylchromone, and compound A ( Figure 1) [22,[27][28][29][30]. It is notable that α, β-unsaturated ketones, as the privileged chemical scaffolds, frequently appeared in the microtubule modulators, exemplified by curcumin and its derivatives that could inhibit tubulin self-assembly by interacting with the unique binding site of tubulin [22,[31][32][33]. Meanwhile, these similar substrates have also been exploited as the correlative olefin polymer materials in the field of advanced materials science [34]. Herein, to discover some potential anti-cancer agents targeting tubulin based on the chemical modifications of natural ingredients of α, β-unsaturated carbonyl compounds, a series of facilely synthetic N-cinnamoyl-N'-(substituted)acryloyl hydrazide derivatives from raw material cinnamic acid were designed, synthesized, and screened for their anticancer activity in vitro ( Figure 1). We expected that these designed frameworks would have the ability to disrupt tubulin assembly, which should be investigated by fluorescence imaging, tubulin polymerization assay and TEM imaging.

Chemistry
To effectively obtain the target compounds, a facile synthetic route was designed as depicted in Scheme 1. Briefly, the cinnamic acid was reacted with 60% hydrazine hydrate, EDCI, and HOBt under basic conditions to give the intermediate cinnamohydrazide [35]. Then, the corresponding cinnamic acid analogue was reacted with cinnamohydrazide, EDCI, and HOBt and subsequently yielded the precipitates. The final target compounds I 1 -I 22 were afforded by being filtered and washed by CH 2 Cl 2 , respectively. Similarly, compounds I 23 -I 26 were synthesized according to the same reaction condition by changing to different starting substrates. Finally, these structures were confirmed by NMR and HRMS analysis.

Chemistry
To effectively obtain the target compounds, a facile synthetic route was designed as depicted in Scheme 1. Briefly, the cinnamic acid was reacted with 60% hydrazine hydrate, EDCI, and HOBt under basic conditions to give the intermediate cinnamohydrazide [35]. Then, the corresponding cinnamic acid analogue was reacted with cinnamohydrazide, EDCI, and HOBt and subsequently yielded the precipitates. The final target compounds I1-I22 were afforded by being filtered and washed by CH2Cl2, respectively. Similarly, compounds I23-I26 were synthesized according to the same reaction condition by changing to different starting substrates. Finally, these structures were confirmed by NMR and HRMS analysis.

Immunofluorescence Staining of Tubulin
The disrupting effect of microtubule triggered by compound I 23 in living cells was disclosed using the immunofluorescence staining assay. Primarily, the HepG2 cells were treated with 0, 3, and 6 µM of compound I 23 for 24 h before cell imaging. Clearly, the agminated microtubule network and wrinkled nucleus were observed in cells after incubating with compound I 23 , thereby leading to the morphological change in HepG2 cells ( Figure 3). By contrast, the cellular microtubule network was well assembled and arranged normally in the control group. This result revealed that compound I 23 might be capable of leading to cell cycle disorder through disturbing microtubule assembly and targeting tubulin.

Effects of Compound I23 on Tubulin Polymerization
Tubulin polymerization assay [36][37][38] triggered by compound I23 was carried out to verify the interaction mode. Meanwhile, paclitaxel, served as a known microtubule stabilizer that could promote the protofilament assembly. As shown in Figure 4, similar to paclitaxel, compound I23 could stabilize tubulin assembly and promote protofilament as-

Effects of Compound I 23 on Tubulin Polymerization
Tubulin polymerization assay [36][37][38] triggered by compound I 23 was carried out to verify the interaction mode. Meanwhile, paclitaxel, served as a known microtubule stabilizer that could promote the protofilament assembly. As shown in Figure 4, similar to paclitaxel, compound I 23 could stabilize tubulin assembly and promote protofilament assembly in a dosage-dependent manner, which was consistent with the outcome from previous reports [38,39], indicating that the potential microtubule-stabilizer might be developed. Figure 3. Effects of compound I23 on the cellular microtubule network were visualized by immunofluorescence assay. HepG2 cells were treated with vehicle control 0.1% DMSO, 3 μM and 6 μM I23 for 24 h. Then, cells were fixed and stained with anti-α-tubulin antibody (red) and counterstained with DAPI (blue). Detection of the fixed and stained cells was performed using fluorescence microscope. Scale bars are 50 μm.

Effects of Compound I23 on Tubulin Polymerization
Tubulin polymerization assay [36][37][38] triggered by compound I23 was carried out to verify the interaction mode. Meanwhile, paclitaxel, served as a known microtubule stabilizer that could promote the protofilament assembly. As shown in Figure 4, similar to paclitaxel, compound I23 could stabilize tubulin assembly and promote protofilament assembly in a dosage-dependent manner, which was consistent with the outcome from previous reports [38,39], indicating that the potential microtubule-stabilizer might be developed.  Taxol (10 µM) was used as positive polymerization control, whereas untreated tubulin was used as negative control.

Tubulin Polymerization Affected by Compound I 23 via TEM
Transmission electron microscopy (TEM) was employed to directly visualize the influence of tubulin polymerization stimulated by compound I 23 . Evidently, without the addition of compound I 23 , the observed microtubules presented uniform fibrous nanostructures, suggesting the spontaneous formation of microtubules occurred in general tubulin buffer solution ( Figure 5A). However, the spontaneous assembly of α/β tubulin heterodimers was disturbed by the existence of 20 µM compound I 23 , subsequently affording more large nonlinear disorganized aggregations ( Figure 5B). These microscopic investigation results manifested that I 23 could promote the microtubular aggregation.
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Tubulin Polymerization Affected by Compound I23 via TEM
Transmission electron microscopy (TEM) was employed to directly visualize the influence of tubulin polymerization stimulated by compound I23. Evidently, without the addition of compound I23, the observed microtubules presented uniform fibrous nanostructures, suggesting the spontaneous formation of microtubules occurred in general tubulin buffer solution ( Figure 5A). However, the spontaneous assembly of α/β tubulin heterodimers was disturbed by the existence of 20 μM compound I23, subsequently affording more large nonlinear disorganized aggregations ( Figure 5B). These microscopic investigation results manifested that I23 could promote the microtubular aggregation.

Cell Cycle Analysis
Microtubules play a critical role during the eukaryotic cell division [40]. Based on the above results, compound I23 could clearly affect the tubulin polymerizations, and this event probably led to the stagnation of cell cycle. To investigate this effect, flow cytometry analysis on HepG2 cell cycle arrest was performed as Figure 6A-C, and the corresponding

Cell Cycle Analysis
Microtubules play a critical role during the eukaryotic cell division [40]. Based on the above results, compound I 23 could clearly affect the tubulin polymerizations, and this event probably led to the stagnation of cell cycle. To investigate this effect, flow cytometry analysis on HepG2 cell cycle arrest was performed as Figure 6A-C, and the corresponding results were displayed as Figure 6D. Interestingly, the G2/M pattern was arrested after treatment with compound I 23 (24 µM), thus providing the relevant percentages from 17.68% (0 µM) to 20.56% (Figure 6), indicating the designed compound could cause cell cycle disorder through targeting tubulin and the subsequent disturbance on microtubule assembly.
Microtubules play a critical role during the eukaryotic cell division [ above results, compound I23 could clearly affect the tubulin polymeriz event probably led to the stagnation of cell cycle. To investigate this effect analysis on HepG2 cell cycle arrest was performed as Figure 6A-C, and th results were displayed as Figure 6D. Interestingly, the G2/M pattern w treatment with compound I23 (24 μM), thus providing the relevant p 17.68% (0 μM) to 20.56% (Figure 6), indicating the designed compound cycle disorder through targeting tubulin and the subsequent disturbance assembly.

Molecular Docking of Compound I 23 with Tubulin
To better understand the possible interaction mode and binding sites between compound I 23 and tubulin, the related software Sybyl X 2.0, PyMOL and Discovery Studio (DS) 2020 were exploited. Meanwhile, the reported 3D crystal structure of tubulin (PDB code: 5syf) was used by the removal of the ligand molecule paclitaxel. Docking study (Figure 7) displayed that compound I 23 could embed in the active pocket around with the amino acid residues, including Leu227, Leu217, His229, Leu230, Ala233, Phe272, Pro360, Arg320, and Ser374. Apart from conventional hydrogen bonds and carbon hydrogen bonds, other non-covalent interactions including π-donor hydrogen bonds, π-alkyl, etc., also were very crucial donations for the interaction [41][42][43]. In detail, the Ser374 residue could form strong hydrogen bond interactions with compound I 23 , thus affording a distance of 2.2 Å located in the S9-S10 stabilizing loop of tubulin, which was the binding site of Taxol. The correlative Ala233, Leu217, Leu230, Phe272, Pro360, and Arg320 residues could form π-alkyl interactions. Simultaneously, π-π stacked interaction was also observed between His229 residue and compound I 23 . These results indicated that a strong interaction occurred between the designed compound and tubulin, thereby disturbing the normal assembly of tubulin, which was consistent with the aforementioned tubulin polymerization assays. could form π-alkyl interactions. Simultaneously, π-π stacked interaction was also o served between His229 residue and compound I23. These results indicated that a stro interaction occurred between the designed compound and tubulin, thereby disturbing t normal assembly of tubulin, which was consistent with the aforementioned tubu polymerization assays.

Effects on Cell Migration of Compound I23
Because the targeting-tubulin agents were validated, having the ability to interfe with cell migration [44,45], the cell migration effect triggered by compound I23 was inv tigated by the calculation of average migration rates of scratched A549 cells monolay which was a widely used model. As depicted in Figure 8, a 12.5% migration rate w

Effects on Cell Migration of Compound I 23
Because the targeting-tubulin agents were validated, having the ability to interfere with cell migration [44,45], the cell migration effect triggered by compound I 23 was investigated by the calculation of average migration rates of scratched A549 cells monolayer, which was a widely used model. As depicted in Figure 8, a 12.5% migration rate was found in the control after 12 h, while the decreased migration rates reached to 8.90%, 6.10%, and 3.98% after treatment of 3 µM I 23 , 6 µM I 23 , and 6 µM gefitinib, respectively. After incubation for another 12 h, the migration rate of A549 cells that coexisted with compound I 23 changed to 13.2% (blank control reached to 24.4%). This outcome indicated that compound I 23 could significantly attenuate the migration of A549 cells in a dosage-dependent manner, which could be a lead structure for the exploration of novel microtubule-stabilizing agents.
6.10%, and 3.98% after treatment of 3 μM I23, 6 μM I23, and 6 μM gefitinib, respectively. After incubation for another 12 h, the migration rate of A549 cells that coexisted with compound I23 changed to 13.2% (blank control reached to 24.4%). This outcome indicated that compound I23 could significantly attenuate the migration of A549 cells in a dosage-dependent manner, which could be a lead structure for the exploration of novel microtubulestabilizing agents. The cancer cells were incubated with 0 μM, 3 μM, 6 μM compound I23, the 6 μM gefitinib was served as the positive control. Scale bars are 100 μm (B) the cell relative migration was calculated using the software.

Apoptosis Effects of HepG2 Cells Caused by Compound I23.
To date, many reports have demonstrated that anti-mitotic cancer agents could also cause cell death through inducing apoptosis [46,47]. Thus, the apoptotic behavior of HepG2 cells triggered by compound I23 should be tested since an appreciable antiproliferative activity and substantial disturbance on microtubule assembly were achieved. As shown in Figure 9, apoptosis effects were observed after incubating cells with 0 μM, 3 μM and 6 μM of compound I23, respectively. Particularly, as the dose was up to 6 μM, a strong

Apoptosis Effects of HepG2 Cells Caused by Compound I 23
To date, many reports have demonstrated that anti-mitotic cancer agents could also cause cell death through inducing apoptosis [46,47]. Thus, the apoptotic behavior of HepG2 cells triggered by compound I 23 should be tested since an appreciable antiproliferative activity and substantial disturbance on microtubule assembly were achieved. As shown in Figure 9, apoptosis effects were observed after incubating cells with 0 µM, 3 µM and 6 µM of compound I 23 , respectively. Particularly, as the dose was up to 6 µM, a strong apoptotic phenomenon happened, indicating that compound I 23 acting as a potential microtubule-stabilizer could slightly induce HepG2 cancer cells apoptosis. apoptotic phenomenon happened, indicating that compound I23 acting as a potential microtubule-stabilizer could slightly induce HepG2 cancer cells apoptosis.

In Silico Drug-Likeness Evaluation
Finally, the early evaluation of the lead compound for its potential is a critical step in drug development. Therefore, to assess whether compound I23 has the potential as the promising lead compound, ADMETlab 2.0 software was used to obtain more information on the pharmacokinetic profile, including ADMET and drug-likeness properties ( Figure  10). The corresponding physicochemical properties, ADMET, and drug-likeness properties of compound I23 are presented in Figure 11 and Table 3, respectively. Notably, the outcomes illustrated that compound I23 exhibited acceptable physicochemical properties, ADMET, and drug-likeness properties. For instance, compound I23 possessed favorable physicochemical properties: molecular weight = 369.13, logS = −4.140, logP = 2.821, and log D = 3.559. Meanwhile, ADMET and drug-likeness properties of compound I23 were provided as follows: (1) compound I23 had appreciable absorption potency: for example, compound I23 was active in both human intestinal absorption (HIA) and had 20% bioavailability (F20%); (2) compound I23 was active in blood-brain barrier (BBB) penetration; (3) compound I23 possessed acceptable safety profiles (e.g., hERG blockers, eye corrosion and respiratory toxicity), and displayed some satisfactory metrics on metabolism (See support information Table S1) and excretion potency; and (4) more interestingly, compound I23 met all drug-likeness properties including Lipinski rule, Pfzer rule, Golden triangle, and GSK rule. Based on the above-mentioned outcomes, compound I23 displayed an acceptable pharmacokinetic profile, making it a promising lead compound to excavate and discover novel microtubule stabilizers.

In Silico Drug-likeness Evaluation
Finally, the early evaluation of the lead compound for its potential is a critical step in drug development. Therefore, to assess whether compound I 23 has the potential as the promising lead compound, ADMETlab 2.0 software was used to obtain more information on the pharmacokinetic profile, including ADMET and drug-likeness properties ( Figure 10). The corresponding physicochemical properties, ADMET, and drug-likeness properties of compound I 23 are presented in Figure 11 and Table 3, respectively. Notably, the outcomes illustrated that compound I 23 exhibited acceptable physicochemical properties, ADMET, and drug-likeness properties. For instance, compound I 23 possessed favorable physicochemical properties: molecular weight = 369.13, logS = −4.140, logP = 2.821, and log D = 3.559. Meanwhile, ADMET and drug-likeness properties of compound I 23 were provided as follows: (1) compound I 23 had appreciable absorption potency: for example, compound I 23 was active in both human intestinal absorption (HIA) and had 20% bioavailability (F20%); (2) compound I 23 was active in blood-brain barrier (BBB) penetration; (3) compound I 23 possessed acceptable safety profiles (e.g., hERG blockers, eye corrosion and respiratory toxicity), and displayed some satisfactory metrics on metabolism (See support information Table S1) and excretion potency; and (4) more interestingly, compound I 23 met all drug-likeness properties including Lipinski rule, Pfzer rule, Golden triangle, and GSK rule. Based on the above-mentioned outcomes, compound I 23 displayed an acceptable pharmacokinetic profile, making it a promising lead compound to excavate and discover novel microtubule stabilizers. Figure 9. The cells were incubated with 0 μM, 3 μM and 6 μM compound I23 for 24 h, subseq stained with Hoechst 33258 (10 μg/mL) and visualized by fluorescence microscopy. Scale ba 100 μm.

In Silico Drug-Likeness Evaluation
Finally, the early evaluation of the lead compound for its potential is a critical s drug development. Therefore, to assess whether compound I23 has the potential promising lead compound, ADMETlab 2.0 software was used to obtain more inform on the pharmacokinetic profile, including ADMET and drug-likeness properties (F 10). The corresponding physicochemical properties, ADMET, and drug-likeness pr ties of compound I23 are presented in Figure 11 and Table 3, respectively. Notabl outcomes illustrated that compound I23 exhibited acceptable physicochemical prop ADMET, and drug-likeness properties. For instance, compound I23 possessed favo physicochemical properties: molecular weight = 369.13, logS = −4.140, logP = 2.821 log D = 3.559. Meanwhile, ADMET and drug-likeness properties of compound I23 provided as follows: (1) compound I23 had appreciable absorption potency: for exa compound I23 was active in both human intestinal absorption (HIA) and had 20% vailability (F20%); (2) compound I23 was active in blood-brain barrier (BBB) penetr (3) compound I23 possessed acceptable safety profiles (e.g., hERG blockers, eye corr and respiratory toxicity), and displayed some satisfactory metrics on metabolism support information Table S1) and excretion potency; and (4) more interestingly, pound I23 met all drug-likeness properties including Lipinski rule, Pfzer rule, Gold angle, and GSK rule. Based on the above-mentioned outcomes, compound I23 disp an acceptable pharmacokinetic profile, making it a promising lead compound to exc and discover novel microtubule stabilizers.

Instruments and Chemicals
NMR spectra were performed using the JEOL-ECX500 instrument (Akishima, Japan) or Bruker Biospin AG-400 instrument (Bruker Optics, Ettlingen, Germany) using DMSOd 6 as the solvent and tetramethylsilane as the internal standard. HRMS spectra were obtained on Waters Xevo G2-S QTOF MS (Waters MS Technologies, Manchester, UK). Immunofluorescence staining assay was performed by the tubulin-tracker red kit (Beyotime Institute of Biotechnology, Shanghai, China) and observed using a Nikon ECLIPSE Ti-S fluorescent microscope (Nikon, Tokyo, Japan). The tubulin polymerization assay was performed by the tubulin polymerization assay kit (cytoskeleton, # BK004P) and recorded by Cytation™ 5 multi-mode readers (BioTek Instruments, Inc., Winooski, VT, USA). The trans-cinnamic acid and analogues were purchased from Aladdin Industrial Inc. (Shanghai, China). The FACSCalibur™ flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) was employed to analyze the cell cycle arrest.

Immunofluorescence Staining Pattern
According to the previous methods [51,52], compound solutions of various concentrations were added to a 6-well plate in which HepG2 cells had been seeded for 12 h. After incubation for 24 h and washed with phosphate-buffered saline (PBS, 10 mM, pH 7.3), each well was treated with 4% formaldehyde solution for 15 min and washed with 0.1% Triton X-100 of phosphate-buffered saline (PBS, 10 mM, pH 7.3). After that, the fixed cells were incubated with diluted tubulin-tracker red for 40 min in a dark environment, followed by washing three times by 0.1% Triton X-100 of phosphate-buffered saline (PBS, 10 mM, pH 7.3). Finally, each well was added DAPI (2 µg/mL). Finally, the results were observed using a Nikon ECLIPSE Ti-S fluorescent microscope (Nikon Co., Tokyo, Japan).

Tubulin Polymerization Assay In Vitro
The tubulin polymerization assay in vitro was determined by HTS-Tubulin Polymerization Assay Kit (#BK004P, Cytoskeleton, Inc., Denver, CO, USA) as described in previous articles [36][37][38]53,54]. The plate was pre-warmed at 37 • C, and the reaction assay contained 100 µL 4 mg/mL tubulin in G-PEM buffer. Then, 10 µL (10×) compounds solution or 100 µM paclitaxel solution as a control. Finally, the polymerization was carried out at 37 • C and recorded at 340 nm each 10 s for 70 min using Cytation™ 5 multi-mode readers.

Tubulin Polymerization Affected by Target Compounds via TEM
Briefly, according to the protocol of tubulin polymerization assay [38,39], the 100 µL centrifuge tubes were pre-warmed at 37 • C, and the reaction assay contained 20 µL 4 mg/mL tubulin in G-PEM buffer. Then, 2 µL (10×) compounds solution was added. Finally, the assay was carried out at 37 • C for 30 min. The formed microtubules were trans-ferred to Formvar-caron-coated copper grids, negatively stained with 1% phosphotungstic acid, and visualized under a transmission electron microscope.

Cell Cycle Analysis
The cells in the 6-well plates were carefully collected 24 h after dosing. After the cells were washed with pre-cooled PBS, the pre-cooled 70% ethanol solution was added for fixation (4 • C overnight). After that, the cells were washed with PBS, and incubated with 1 mg/mL RNase A for 30 min at 37 • C). Then, 20 mg/mL PI staining solution was added and incubated in the dark for 30 min at 4 • C. Finally, the cell arrest was analyzed by the FACSCalibur™ flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) as previously described [40].

Computational Docking Studies
The docking study was performed by Sybyl X 2.0 and the tubulin protein (PDB: 5syf) was downloaded from RCSB Protein Data Bank (www.rcsb.org, accessed on 1 October 2020.). The protein and all ligands were prepared by minimization with the CHARMM force field. Molecular docking was carried out using Sybyl X 2.0 protocol without constraint. All bound water and ligands were eliminated from the protein and the polar hydrogen was added to the proteins. The docking results were performed by PyMol software and Discovery Studio (DS) 2020 [55][56][57].

Scratch Test
According to the previous methods [58], A549 cells of the logarithmic growth stage were cultured in 6-well plates with two lines on the back, with each well containing a density of 1 × 10 6 cells/mL. After the cells adhered to the wall, three uniform thin lines were drawn in each well by a sterile pipette tip. Afterward, the medium containing 1% fetal bovine serum (FBS) and various concentrations of compounds were added in wells. After the different time of incubation, the cells were performed by an inverted fluorescence microscope. Then the scratch healing rate was calculated: Migration distance (n h) = edge distance (0 h) − edge distance (12 h or 24 h).

Hoechst Apoptosis Experiment
The nuclear morphological modifications were exhibited by fluorescence pattern. In this assay [58][59][60][61], HepG2 cells were seeded in 6-well plates, after 24 h of incubation, different compounds were added at various concentrations, and cells were incubated again for 24 h. Afterward, the medium was removed and cells were washed twice with phosphate-buffered saline (PBS, 10 mM, pH 7.3), and fixed with 4% paraformaldehyde for 15 min at room temperature, then stained with 10 mg/mL Hoechst 33258 in PBS for 20 min at 37 • C in the dark. After incubation, the cells were incubated with an anti-fluorescent attenuator and imaged with an inverted fluorescence microscope.

In Silico Pharmacokinetics
The structures of compound I 23 were drawn using ChemDraw (version Ultra 12.0, PerkinElmer Informatics, Waltham, MA, USA) and transformed as SMILES format. In silico drug-likeness predictions were conducted using http://www.swissadme.ch/index.php accessed on 1 May 2022 [62,63].

Conclusions
To develop novel microtubule-binding agents for cancer therapy, an array of diacylhydrazine-functionalized cinnamic acid derivatives were facilely synthesized through a twostep process. Antiproliferative bioassays showed that compound I 23 exhibited the best antiproliferative activity against three cancer lines with IC 50 values ranging from 3.36 to 5.99 µM and yielded a lower cytotoxicity towards the normal cell line. Anticancer mechanism investigations suggested that the highly bioactive compound I 23 could potentially promote the protofilament assembly of tubulin through fluorescence imaging, tubulin polymerization assay and TEM imaging, thus eventually leading to the stagnation of G2/M phase cell cycle of HepG2 cells. Meanwhile, molecular docking studies revealed that compound I 23 could interact with Ser374 residue, Ala233, Leu217, Leu230, Phe272, Pro360, and Arg320 residues, and His229 residue of tubulin directed by the corresponding hydrogen bond interactions, π-alkyl interactions, and π-π stacked interactions, which was similar with that of Taxol located at the S9-S10 stabilizing loop of tubulin. Furthermore, compound I 23 could also reduce the cancer cell migration rate and induce HepG2 cells apoptosis. Additionally, the in silico analysis indicated that compound I 23 exhibited an acceptable pharmacokinetic profile. Based on the present findings, these simple hydrazide derivatives could be considered as potential microtubule-stabilizer lead structures for future anticancer drug discovery.