Design, Synthesis and Cytotoxic Activity Evaluation of Newly Synthesized Amides-Based TMP Moiety as Potential Anticancer Agents over HepG2 Cells

A novel series of amides based TMP moiety was designed, synthesized and evaluated for their antiproliferative as well as enzyme inhibition activity. Compounds 6a and 6b showed remarkable cytotoxic activity against HepG2 cells with IC50 values 0.65 and 0.92 μM, respectively compared with SAHA and CA-4 as reference compounds. In addition, compound 6a demonstrated good HDAC-tubulin dual inhibition activity as it showed better HDAC activity as well as anti-tubulin activity. Moreover, compound 6a exhibited G2/M phase arrest and pre-G1 apoptosis as demonstrated by cell cycle analysis and Annexin V assays. Further apoptosis studies demonstrated that compound 6a boosted the level of caspase 3/7. Caspase 3/7 activation and apoptosis induction were evidenced by decrease in mitochondrial permeability suggesting that activation of caspase 3/7 may occur via mitochondrial apoptotic pathway.


Introduction
Targeted-based anticancer therapy is one of the most important tactics for optimizing antitumor agents to avoid several drawbacks associated with traditional chemotherapeutic agents such as systemic toxicity, adverse side effects, absence of selective target and emergence of drug resistance [1][2][3][4]. Recently, researchers have focused on designing dual or multi-target anticancer agents which hold great advantages such as reverse drug resistance, improve therapeutic efficacy and seems to be an ideal solution to control cancer [5][6][7].
Histone deacetylases (HDACs) are epigenetic enzymes that have been regarded validated targets in inhibition of cancer cell proliferation and apoptosis induction [8,9]. HDACs enzymes catalyze the deacetylation from lysine residue in histone tails [10]. In addition, HDACs regulate signaling pathways via deacetyling large number of other nonhistones involved in gene expression [11]. High expression of aberrant recruitment of these enzymes

Chemistry
The general approach leading to the synthesis of the target amide derivatives is outlined in Scheme 1. Ring opening of oxazolone 1 with respective aryl amine; namely 3chloro aniline, 4-chloro aniline, 4-methyl aniline or 2-naphthyl amine in glacial acetic acid for 1-3 h provided the corresponding diamide compounds 2a-c or 3, respectively [30]. Structures of compounds 2a-c and 3 were elucidated from their 1 H-NMR and 13 C-NMR spectral studies. The 1 H-NMR spectra, the presence of two NH groups of the two amide functions was supported by two signals at δ 9.91-9.97 and 9.97-10. 24 ppm, in addition to the presence of new signals at aromatic region at δ 6.62-8.14 ppm ascribed to new phenyl Several reported experimental studies proved that 3,4,5-trimethoxyphenyl (TMP) moiety is a privileged ring in several anticancer molecules such Colchicine (Col), combretastatin A-4 (CA-4) and their analogues [22][23][24]. The TMP ring exhibited promising anticancer activity mainly via inhibition of tubulin assembly into microtubules [25]. Interestingly, synergistic effect of HDACs inhibition and tubulin inhibition has been observed in many studies. For example, compound V and VI possessing TMP moiety and were demonstrated as potential HDAC-tubulin inhibitor [26,27]. Inspired by the above mentioned aspects and in continue efforts to discover new anticancer agents with better apoptotic properties [28,29], the present study concerned with the design and synthesis of novel series of amide-based compounds in the hopes of obtaining novel dual HDAC and tubulin inhibitors with promising anticancer potency. All the prepared amide compounds were screened for cytotoxicity against hepatocellular carcinoma HepG2 and normal liver cell line HL-7702 cell lines utilizing MTT antiproliferative assay. Moreover, apoptosis assays and cell cycle analysis of the most active molecule was carried out to detect if the cytotoxic potency is accompanied by change in cell cycle analysis and apoptosis induction. Furthermore, its ability to boost caspase 3/7 and decrease the MMP was investigated to show the apoptotic pathway mechanism.

Chemistry
The general approach leading to the synthesis of the target amide derivatives is outlined in Scheme 1. Ring opening of oxazolone 1 with respective aryl amine; namely 3-chloro aniline, 4-chloro aniline, 4-methyl aniline or 2-naphthyl amine in glacial acetic acid for 1-3 h provided the corresponding diamide compounds 2a-c or 3, respectively [30]. Structures of compounds 2a-c and 3 were elucidated from their 1 H-NMR and 13 C-NMR spectral studies. The 1 H-NMR spectra, the presence of two NH groups of the two amide functions was supported by two signals at δ 9.91-9.97 and 9.97-10.24 ppm, in addition to the presence of new signals at aromatic region at δ 6.62-8.14 ppm ascribed to new phenyl or naphthyl ring protons. In addition, 13 C-NMR spectra of compounds 2a-c and 3 revealed the presence of two peaks at δ 163.79-166.01 ppm corresponds to carbonyl (C=O) groups of the two amide functions. The desired triamide derivatives 4a,b were obtained through oxazolone 1 reaction with respective aryl carbohydrazide in boiling pure ethanol. 1 H-NMR spectra of the product 4b as representative example exhibited signals of the three NH protons of the triamide function at δ 9.90, 10.32 and 10.80 ppm as well as the presence of extra proton signals in the region at δ 6.63-8.78 ppm related pyridyl function. In addition, signals of the carbonyl (C=O) groups of the triamide function were recorded in 13 C-NMR spectra of compound 4b at δ 164.48, 164.52 and 165.81 ppm. The target tetraamide derivative 5 was achieved by refluxing oxazolone 1 with N-[4-(hydrazine carbonyl]phenyl]nicotinamide in DMF containing catalytic amount of glacial acetic acid. In confirmation, 1 H-NMR spectrum of compound 5 exhibited four signals at δ 9.87, 10.18, 10.40 and 10.68 ppm attributed to NH protons of four amide functions, in addition to new signals between δ 6.62-9.13 ppm integrating thirteen aromatic protons, and olefinic (=CH) proton. In the 13 C-NMR spectrum, compound 5 exhibited four carbon signals at δ 164.61, 164.90, 165.45 and 165.75 ppm ascribed to carbonyl (C=O) groups of tetraamide function as well as the presence of extra signals related to phenyl and pyridyl carbons. In order to obtain the target tetraamide derivatives 6a-e, various acrylic acid hydrazide molecules were used to synthesize the target compounds. The structure confirmations of tetraamide molecules 6a-e were based on spectral studies such as 1 H-NMR and 13 C-NMR spectra. 1 H-NMR spectra of 6a-e exhibited four new signals in the region between δ 9.75-10.36 ppm ascribed to the four amide protons in addition to extra proton signals in the aromatic region corresponds to phenyl groups. 13 C-NMR spectra of tetraamide 6a-e confirmed the carbon skeleton due to the presence of four carbon signals at δ 163.98-165.77 ppm attributed to the carbonyl (C=O) functions of the four amide groups.
the target tetraamide derivatives 6a-e, various acrylic acid hydrazide molecules were used to synthesize the target compounds. The structure confirmations of tetraamide molecules 6a-e were based on spectral studies such as 1 H-NMR and 13 C-NMR spectra. 1 H-NMR spectra of 6a-e exhibited four new signals in the region between δ 9.75-10.36 ppm ascribed to the four amide protons in addition to extra proton signals in the aromatic region corresponds to phenyl groups. 13 C-NMR spectra of tetraamide 6a-e confirmed the carbon skeleton due to the presence of four carbon signals at δ 163.98-165.77 ppm attributed to the carbonyl (C=O) functions of the four amide groups. Scheme 1. Synthesis of the target compounds 2-6e. Reagent and reaction condition: (i) respective aryl amine, AcOH, reflux 1-2 h; (ii) 2-naphthyl amine, AcOH, reflux 3 h; (iii) respective aryl carbohydrazide, ethanol, reflux 4-5 h; (iv) N- [4-(hydrazinecarbonyl]phenyl]nicotinamide, DMF, AcOH, reflux 6 h; (v) respective acrylic acid hydrazide, DMF, AcOH, reflux 6-8 h.

In Vitro Cytotoxic Activity against HepG2 Cell Line
The synthesized amide based compounds were subjected to MTT cell proliferation assay using suberoylanilide hydroxamic acid (SAHA) and CA-4 as positive reference compounds in this investigation. Results were reported as IC 50 values (µM) as shown in Table 1. Compounds 6a, 6b and 6c were the most potent in this investigation with IC 50 values 0.65, 0.92 and 1.12 µM, respectively. Compound 6a (IC 50 = 0.65 µM) was four folds more active than the SAHA (IC 50 = 2.91 µM) and nearly equipotent to reference compound CA-4 (IC 50 = 0.54 µM). Structurally, in the diamide series 2a-c and 3, naphthalene favors the anticancer activity rather substituted phenyl ring. This is obvious upon compound 3 (IC 50 = 16.24 µM) and compounds 2a-c (IC 50 = 22.03-68.90 µM). In the triamide series 4a,b compound 4b bearing pyridyl function favors the anticancer activity (IC 50 = 8.36 µM) than 3-hydroxyphenyl moiety (IC 50 = 13.37 µM). Regarding the tetraamide series 5 and 6a-e, compound 6a was the most effective in cell proliferation in HepG2 cells with IC 50 value 0.65 µM. Moreover, compound 6a proved to be selective toward normal liver cell line HL-7022 with selectivity ratio of 14.8. In order to cast light onto the mechanism of action of the prepared tetraamide based molecules, the most potent compound in the present study was investigated for its in vitro HDAC1 and HDAC2 inhibitory activity using human colorimetric simple ELISA kits and SAHA was taken as reference compound. Results in Figure 2 revealed that the tested tetraamide molecule 6a showed significant inhibitory activity against HDAC1 and HDAC2 isoforms. It could be noticed that compound 6a strongly inhibited HDAC1 and HDAC2 isoforms with IC 50 values 0.047 and 0.086 µM, respectively compared with values of 0.028 and 0.072 µM for SAHA, respectively.

Tubulin Polymerization Inhibition Assay
To evaluate the effect of the prepared amide derivatives on tubulin assembly in vitro, compound 6a was evaluated for its tubulin polymerization inhibition activity using ELISA analysis. The results in Figure 3A revealed that the tetraamide derivative 6a inhibited assembly of tubulins into microtubules with a percentage inhibition value of 66.39% compared with the untreated control cells. Additionally, the IC50 value for compound 6a was recorded as 0.27 μM. CA-4 was used as a reference compound with IC50 value 0.083 μM and 88.72% tubulin inhibition. These results indicate that cytotoxicity of compound 6a related mainly to good HDAC than β-tubulin polymerization inhibition activity.

Tubulin Polymerization Inhibition Assay
To evaluate the effect of the prepared amide derivatives on tubulin assembly in vitro, compound 6a was evaluated for its tubulin polymerization inhibition activity using ELISA analysis. The results in Figure 3A revealed that the tetraamide derivative 6a inhibited assembly of tubulins into microtubules with a percentage inhibition value of 66.39% compared with the untreated control cells. Additionally, the IC 50 value for compound 6a was recorded as 0.27 µM. CA-4 was used as a reference compound with IC 50 value 0.083 µM and 88.72% tubulin inhibition. These results indicate that cytotoxicity of compound 6a related mainly to good HDAC than β-tubulin polymerization inhibition activity.
activity of tetraamide 6a and SAHA against HDAC1 and HDAC2 isoforms.

Tubulin Polymerization Inhibition Assay
To evaluate the effect of the prepared amide derivatives on tubulin assembly in vitro, compound 6a was evaluated for its tubulin polymerization inhibition activity using ELISA analysis. The results in Figure 3A revealed that the tetraamide derivative 6a inhibited assembly of tubulins into microtubules with a percentage inhibition value of 66.39% compared with the untreated control cells. Additionally, the IC50 value for compound 6a was recorded as 0.27 μM. CA-4 was used as a reference compound with IC50 value 0.083 μM and 88.72% tubulin inhibition. These results indicate that cytotoxicity of compound 6a related mainly to good HDAC than β-tubulin polymerization inhibition activity.

Cell Cycle Analysis
Inhibition of tubulin assembly into microtubule and the antiproliferative effects are characterized by cell cycle arrest in the G2/M phase [31]. Cell cycle analysis on the most active compound was performed using FACS analysis following treatment of HepG2 cells with tetraamide derivative 6a at its IC50 dose level for 48 h. As shown in Figure 4, the tested tetraamide molecule 6a showed good ability to block cells in G2/M phase of the cells cycle (39.09%) compared with untreated control (7.28%). In addition, tetraamide

Cell Cycle Analysis
Inhibition of tubulin assembly into microtubule and the antiproliferative effects are characterized by cell cycle arrest in the G2/M phase [31]. Cell cycle analysis on the most active compound was performed using FACS analysis following treatment of HepG2 cells with tetraamide derivative 6a at its IC 50 dose level for 48 h. As shown in Figure 4, the tested tetraamide molecule 6a showed good ability to block cells in G2/M phase of the cells cycle (39.09%) compared with untreated control (7.28%). In addition, tetraamide derivative 6a increase the percentage of cells at pre-G1 phase (37.51%) compared with the untreated control (1.59%). The results in this study indicate that the newly prepared tetraamide derivative 6a cause cell cycle perturbation in the G2/M phase which is the main gauge of HDAC and tubulin inhibitors confirming the mode of action under study.

Apoptosis Assay
G2/M blockade is often followed by cellular apoptosis [32]. To quantify the percentage of cellular apoptosis induced by compound 6a in HepG2 cells, Annexin V fluorescein isothiocyanate versus propidium iodide (PI) dual staining analysis was performed after treatment with compound 6a at its IC50 concentration for 48 h. The results were presented graphically in Figure 5. From the results in Figure 5, it can be observed that the total apoptosis percentage increased in HepG2 cells (37.51%) after treatment with compound 6a

Apoptosis Assay
G2/M blockade is often followed by cellular apoptosis [32]. To quantify the percentage of cellular apoptosis induced by compound 6a in HepG2 cells, Annexin V fluorescein isothiocyanate versus propidium iodide (PI) dual staining analysis was performed after treatment with compound 6a at its IC 50 concentration for 48 h. The results were presented graphically in Figure 5. From the results in Figure 5, it can be observed that the total apoptosis percentage increased in HepG2 cells (37.51%) after treatment with compound 6a compared with the untreated control cells (1.59%). In addition the early and late apoptotic cell percentages were increased in HepG2 cells; 23.41 and 12.08%, respectively after treatment with compound 6a compared with the untreated control cells (0.52 and 0.13%, respectively). Therefore, it can be concluded that compound 6a can be considered as apoptotic inducer.

Caspase 3/7 Assay
Further, the activation of caspase 3/7 in HepG2 cells treated with compound 6a at its IC50 concentration for 48 h was carried out to investigate the apoptotic pathway mechanism. The results were presented graphically in Figure 6. From the results in Figure 6, it can be showed that the treatment of HepG2 cells with compound 6a for 48 h the level of caspase 3/7 was increased by −9.73 fold in comparison with the no treatment control. It can be concluded that compound 6a induced apoptosis through the activation of caspase 3/7.

Caspase 3/7 Assay
Further, the activation of caspase 3/7 in HepG2 cells treated with compound 6a at its IC 50 concentration for 48 h was carried out to investigate the apoptotic pathway mechanism. The results were presented graphically in Figure 6. From the results in Figure 6, it can be showed that the treatment of HepG2 cells with compound 6a for 48 h the level of caspase 3/7 was increased by −9.73 fold in comparison with the no treatment control. It can be concluded that compound 6a induced apoptosis through the activation of caspase 3/7.

Caspase 3/7 Assay
Further, the activation of caspase 3/7 in HepG2 cells treated with compound 6a at its IC50 concentration for 48 h was carried out to investigate the apoptotic pathway mechanism. The results were presented graphically in Figure 6. From the results in Figure 6, it can be showed that the treatment of HepG2 cells with compound 6a for 48 h the level of caspase 3/7 was increased by −9.73 fold in comparison with the no treatment control. It can be concluded that compound 6a induced apoptosis through the activation of caspase 3/7.  To investigate the mitochondrial events, ∆Ψ dissipation was monitored after treatment with compound 6a with the concentration induced cytotoxicity for 48 h. Results presented in Figure 7 revealed that the ∆Ψ was decreased from 31,559 for control untreated HepG2 cells to 13,914 when the cells were treated with the test compound. Therefore, the loss of ∆Ψ after 6a treatment with the concentration induced cytotoxicity concluded that the activation of caspase 3/7 may occur via mitochondrial apoptotic pathway.

Conclusions
In the present study, a novel series of amide derivatives containing TMP moiety have been conveniently synthesized and characterized by 1 H-NMR and 13 C-NMR spectral analyses. The prepared amide derivatives were tested for their antiproliferative as well enzyme inhibition activity. Compounds 6a and 6b showed remarkable cytotoxic activity against HepG2 cells with IC50 values 0.65 and 0.92 μM, respectively compared with SAHA and CA-4 as reference compounds. In addition, compound 6a demonstrated good HDACtubulin dual inhibition activity as it showed better HDAC activity as well as anti-tubulin activity. Moreover, compound 6a exhibited G2/M phase arrest and pre-G1 apoptosis as demonstrated by cell cycle analysis and Annexin V assays. Further apoptosis studies demonstrated that compound 6a boosted the level of caspase 3/7. Caspase 3/7 activation

Conclusions
In the present study, a novel series of amide derivatives containing TMP moiety have been conveniently synthesized and characterized by 1 H-NMR and 13 C-NMR spectral analyses. The prepared amide derivatives were tested for their antiproliferative as well enzyme inhibition activity. Compounds 6a and 6b showed remarkable cytotoxic activity against HepG2 cells with IC 50 values 0.65 and 0.92 µM, respectively compared with SAHA and CA-4 as reference compounds. In addition, compound 6a demonstrated good HDACtubulin dual inhibition activity as it showed better HDAC activity as well as anti-tubulin activity. Moreover, compound 6a exhibited G2/M phase arrest and pre-G1 apoptosis as demonstrated by cell cycle analysis and Annexin V assays. Further apoptosis studies demonstrated that compound 6a boosted the level of caspase 3/7. Caspase 3/7 activation and apoptosis induction were evidenced by decrease in mitochondrial permeability suggesting that activation of caspase 3/7 may occur via mitochondrial apoptotic pathway. In conclusion, the tetraamide analogs could be considered as lead templates for further development to obtain more potent anticancer agents.

General
Melting points, NMR spectra and elemental analyses were carried out to elucidate the chemical structure of target amide derivatives 2a-6e. For experimental details see Section 4.1 in Supplementary Data.
Buff powder ( (5) N- [4-(hydrazinecarbonyl]phenyl]nicotinamide (0.01 mol, 2.56 g) was added to a suspension of compound 1 (0.01 mol, 3.29 g) in dry in DMF (20 mL) containing catalytic amount glacial acetic acid (10 drops) and the mixture was refluxed for 6 h. After completion of the reaction, the reaction mixture was then cooled and poured into ice/cold water. The residue was purified by crystallization from pure ethanol to furnish pure compound 5.