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Article

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

by
Tarfah Al-Warhi
1,
Adil Aldhahrani
2,
Fayez Althobaiti
3,
Eman Fayad
3,
Ola A. Abu Ali
4,
Sarah Albogami
3,
Ali H. Abu Almaaty
5,
Amgad I. M. Khedr
6,
Syed Nasir Abbas Bukhari
7 and
Islam Zaki
8,*
1
Department of Chemistry, College of Science, Princess Nourah Bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia
2
Clinical Laboratory Sciences Department, Turabah University Faculty, Taif University, Taif 21995, Saudi Arabia
3
Department of Biotechnology, Faculty of Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
4
Department of Chemistry, College of Science, Taif University, P.O. Box 11099 Taif 21944, Saudi Arabia
5
Zoology Department, Faculty of Science, Port Said University, Port Said 42526, Egypt
6
Department of Pharmacognosy, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
7
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka 72388, Saudi Arabia
8
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(12), 3960; https://doi.org/10.3390/molecules27123960
Submission received: 1 May 2022 / Revised: 21 May 2022 / Accepted: 7 June 2022 / Published: 20 June 2022
(This article belongs to the Section Bioorganic Chemistry)
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Abstract

:
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.

1. 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 has been shown in a broad range of diseases including several types of cancer, cardiovascular and neurological diseases [12,13,14]. Therefore, HDACs inhibition is considered as highly attractive therapeutic targets for numerous disorders especially, malignancies and generating the interest toward vast number of HDACs inhibitors in various clinical trials [15,16].
HDAC inhibitors employing zinc chelating functionalities such as hydroxamates, benzamides, short chain fatty acids and ketones have shown promising results in cancer treatment [17]. Chidamide (I) is a benzamide HDAC inhibitor approved by the China food and drug administration for the treatment of refractory PTCL [18]. The 2-amino benzamide molecule MS-275 (II) represents another HDAC inhibitor in phase I/II clinical trials for the treatment of solid tumors [19]. In addition, compound III containing bulky group exhibited 10-fold and 20-fold potencies for HDAC1 compared with HDAC2 and HDAC3, respectively [20]. Moreover, compound IV displayed comparable pan HDAC inhibitory activity compared with IC50 4.648 μM, as compared with BG45 (IC50 = 5.506 μM) [21] (Figure 1).
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.

2. Results and Discussion

2.1. 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 1H-NMR and 13C-NMR spectral studies. The 1H-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, 13C-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. 1H-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 13C-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, 1H-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 13C-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 1H-NMR and 13C-NMR spectra. 1H-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. 13C-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.

2.2. Biology

2.2.1. 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 IC50 values (μM) as shown in Table 1. Compounds 6a, 6b and 6c were the most potent in this investigation with IC50 values 0.65, 0.92 and 1.12 μM, respectively. Compound 6a (IC50 = 0.65 μM) was four folds more active than the SAHA (IC50 = 2.91 μM) and nearly equipotent to reference compound CA-4 (IC50 = 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 (IC50 = 16.24 μM) and compounds 2a–c (IC50 = 22.03–68.90 μM). In the triamide series 4a,b compound 4b bearing pyridyl function favors the anticancer activity (IC50 = 8.36 μM) than 3-hydroxyphenyl moiety (IC50 = 13.37 μM). Regarding the tetraamide series 5 and 6a–e, compound 6a was the most effective in cell proliferation in HepG2 cells with IC50 value 0.65 μM. Moreover, compound 6a proved to be selective toward normal liver cell line HL-7022 with selectivity ratio of 14.8.

2.2.2. HDAC Inhibitory Activity

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 IC50 values 0.047 and 0.086 μM, respectively compared with values of 0.028 and 0.072 μM for SAHA, respectively.

2.2.3. 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.

2.2.4. 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 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.

2.2.5. 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 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.

2.2.6. 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.

2.2.7. Mitochondrial Membrane Potential (MMP)

To investigate the mitochondrial events, ΔΨ dissipation was monitored after treatment with compound 6a with the concentration induced cytotoxicity for 48 h. Results presented in Figure 7 revealed that the ΔΨ 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 ΔΨ after 6a treatment with the concentration induced cytotoxicity concluded that the activation of caspase 3/7 may occur via mitochondrial apoptotic pathway.

3. Conclusions

In the present study, a novel series of amide derivatives containing TMP moiety have been conveniently synthesized and characterized by 1H-NMR and 13C-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 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. In conclusion, the tetraamide analogs could be considered as lead templates for further development to obtain more potent anticancer agents.

4. Experimental

4.1. 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.

4.2. Chemistry

4.2.1. General Procedure for the Preparation of N-(3-(arylamino)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamides 2a–c

A mixture of oxazolone 1 (0.01 mol, 3.29 g) with respective aryl amine (0.01 mol) in glacial acetic acid (20 mL) was refluxed for 1–2 h. After completion of the reaction, the reaction mixture was cooled down and poured into ice/cold water and then filtered. The obtained residue was crystallized from DMF/H2O to get pure diamide compound 2a–c.

N-(3-(3-chlorophenylamino)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (2a)

White powder (2.48 g, 54.33%), m.p. 248–250 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.76 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.64 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.81 (d, J = 3.4 Hz, 1H, furan CH), 7.11 (s, 1H, olefinic CH), 7.14 (ddd, J = 8.0, 2.1, 0.8 Hz, 1H, arom.CH), 7.36 (t, J = 8.1 Hz, 1H, arom.CH), 7.42 (s, 2H, arom.CH), 7.66–7.71 (m, 1H, 1H, arom.CH), 7.84 (d, J = 1.4 Hz, 1H, furan CH), 7.91 (t, J = 2.0 Hz, 1H, 1H, arom.CH), 9.97 (s, 1H, NH), 10.24 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.56 (OCH3), 60.60 (2OCH3), 106.02 (C2,6 trimethoxybenzamide), 112.87 (C olefinic), 114.88 (C3 furan), 117.21 (C4 furan), 118.91 (C6 chlorophenyl), 119.95 (C2 chlorophenyl), 123.56 (C4 chlorophenyl), 128.26 (C1 trimethoxybenzamide), 129.04 (C olefinic), 130.68 (C5 chlorophenyl), 133.31 (C3 chlorophenyl), 140.96 (C4 trimethoxybenzamide), 141.16 (C1 chlorophenyl), 145.27 (C5 furan), 150.04 (C2 furan), 153.11 (C3,5 trimethoxybenzamide), 164.31 (C=O trimethoxybenzamide), 165.57 (C=O amide). Anal. Calcd. for C23H21ClN2O6 (456.88): C, 60.46; H, 4.63; N, 6.13. Found: C, 60.64; H, 4.74; N, 6.02.

N-(3-(4-chlorophenylamino)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (2b)

White powder (2.32 g, 50.89%), m.p. 241–243 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.87 (s, 6H, 2OCH3), 6.63 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.80 (d, J = 3.4 Hz, 1H, furan CH), 7.10 (s, 1H, olefinic CH), 7.41–7.35 (m, 2H, arom.CH), 7.42 (s, 2H, arom.CH), 7.77 (d, J = 8.9 Hz, 2H, arom.CH), 7.83 (d, J = 1.5 Hz, 1H, furan CH), 9.95 (s, 1H, NH), 10.21 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.55 (OCH3), 60.59 (2OCH3), 106.02 (C2,6 trimethoxybenzamide), 112.84 (C olefinic), 114.76 (C3 furan), 117.12 (C4 furan), 122.14 (C2,6 chlorophenyl), 127.49 (C4 chlorophenyl), 128.38 (C1 trimethoxybenzamide), 128.87 (C3,5 chlorophenyl), 129.08 (C olefinic), 138.64 (C1 chlorophenyl), 140.95 (C4 trimethoxybenzamide), 145.19 (C5 furan), 150.08 (C2 furan), 153.10 (C3,5 trimethoxybenzamide), 164.14 (C=O trimethoxybenzamide), 165.54 (C=O amide). Anal. Calcd. for C23H21ClN2O6 (456.88): C, 60.46; H, 4.63; N, 6.13. Found: C, 60.28; H, 4.76; N, 6.22.

N-(1-(furan-2-yl)-3-oxo-3-(p-tolylamino)prop-1-en-2-yl)-3,4,5-trimethoxybenzamide (2c)

White powder (2.51 g, 57.51%), m.p. 261–263 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.28 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.62 (dd, J = 3.3, 1.7 Hz, 1H, furan CH), 6.77 (d, J = 3.4 Hz, 1H, furan CH), 7.12 (d, J = 5.3 Hz, 2H, arom.CH), 7.14 (s, 1H, olefinic CH), 7.42 (s, 2H, arom.CH), 7.61 (d, J = 8.4 Hz, 2H, arom.CH), 7.77–7.86 (m, 1H, furan CH), 9.91 (s, 1H, NH), 9.97 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 20.95 (CH3), 56.54 (OCH3), 60.59 (2OCH3), 106.01 (C2,6 trimethoxybenzamide), 112.80 (C olefinic), 114.50 (C3 furan), 117.03 (C4 furan), 120.68 (C2,6 methylphenyl), 128.61 (C1 trimethoxybenzamide), 129.22 (C olefinic), 129.33 (C3,5 methylphenyl), 132.85 (C1 methylphenyl), 137.09 (C4 methylphenyl), 140.89 (C4 trimethoxybenzamide), 145.04 (C5 furan), 150.19 (C2 furan), 153.08 (C3,5 trimethoxybenzamide), 163.79 (C=O trimethoxybenzamide), 165.54 (C=O amide). Anal. Calcd. for C24H24N2O6 (436.46): C, 66.04; H, 5.54; N, 6.42. Found: C, 65.88; H, 5.68; N, 6.33.

4.2.2. N-(1-(furan-2-yl)-3-(naphthalen-2-ylamino)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (3)

A mixture of 1 (0.01 mol, 3.29 g) with 2-naphthyl amine (0.01 mol, 1.43 g) in glacial acetic acid (20 mL) was refluxed for 3 h. After completion of the reaction, the reaction mixture was cooled down, poured into ice/cold water and then filtered. The obtained residue was crystallized from DMF/H2O to get pure diamide compound 3.
Buff powder (2.35 g, 49.79%), m.p. 270–272 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.89 (s, 6H, 2OCH3), 6.65 (dd, J = 3.1, 1.8 Hz, 1H, furan CH), 6.84 (d, J = 3.4 Hz, 1H, furan CH), 7.30 (s, 1H, olefinic CH), 7.47 (s, 2H, arom.CH), 7.51–7.60 (m, 4H, arom.CH), 7.80–7.89 (m, 2H, furan CH), 7.93–8.01 (m, 1H, furan CH), 8.05–8.14 (m, 1H, arom.CH), 10.03 (s, 1H, NH), 10.15 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.58 (OCH3), 60.60 (2OCH), 106.13 (C2,6 trimethoxybenzamide), 112.85 (C olefinic), 114.83 (C3 furan), 117.85 (C4 furan), 123.97 (C1 naphthyl), 124.19 (C3 naphthyl), 125.93 (C6 naphthyl), 126.22 (C8 naphthyl), 126.47 (C7 naphthyl), 126.54 (C5 naphthyl), 128.20 (C4″ naphthyl), 128.38 (C1 trimethoxybenzamide), 129.43 (C olefinic), 129.68 (C4 naphthyl), 134.17 (C8″ naphthyl), 134.43 (C2 naphthyl), 140.88 (C4 trimethoxybenzamide), 145.24 (C5 furan), 150.23 (C2 furan), 153.06 (C3,5 trimethoxybenzamide), 164.67 (C=O trimethoxybenzamide), 166.01 (C=O amide). Anal. Calcd. for C27H24N2O6 (472.49): C, 68.63; H, 5.12; N, 5.93. Found: C, 68.69; H, 5.22; N, 6.01.

4.2.3. General Procedure for the Preparation of N-(3-(2-(4-Aroyl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamides (4a,b)

A mixture of oxazolone 1 (0.01 mol, 3.29 g) with respective aryl carbohydrazide (0.01 mol) in pure ethanol was refluxed for 4–5 h. After completion of the reaction, the reaction mixture was cooled down and then filtered. The buff residue that formed was crystallized from ethanol (70%) as buff crystals.

N-(1-(furan-2-yl)-3-(2-(3-hydroxybenzoyl)hydrazinyl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4a)

Buff powder (2.67 g, 55.81%), m.p. 235–237 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.62 (dd, J = 3.3, 1.8 Hz, 1H, furan CH), 6.78 (d, J = 3.4 Hz, 1H, furan CH), 6.93–7.00 (m, 1H, arom.CH), 7.25 (s, 1H, olefinic CH), 7.26–7.31 (m, 2H, arom.CH), 7.34 (d, J = 7.8 Hz, 1H, arom.CH), 7.41 (s, 2H, arom.CH), 7.81 (d, J = 1.4 Hz, 1H, furan CH), 9.71 (s, 1H, OH), 9.85 (s, 1H, NH), 10.15 (s, 1H, NH), 10.35 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.52 (OCH3), 60.61 (2OCH3), 106.04 (C2,6 trimethoxybenzamide), 112.90 (C olefinic), 114.87 (C3 furan), 115.09 (C2 hydroxyphenyl), 118.50 (C4 furan), 119.03 (C4 hydroxyphenyl), 119.19 (C6 hydroxyphenyl), 126.25 (C1 trimethoxybenzamide), 129.41 (C olefinic), 130.01 (C5 hydroxyphenyl), 134.43 (C1 hydroxyphenyl), 140.77 (C4 trimethoxybenzamide), 145.31 (C5 furan), 149.92 (C2 furan), 152.99 (C3,5 trimethoxybenzamide), 157.71 (C3 hydroxyphenyl), 164.63 (C=O amide), 165.93 (C=O amide), 166.30 (C=O trimethoxybenzamide). Anal. Calcd. for C24H23N3O8 (481.45): C, 59.87; H, 4.82; N, 8.73. Found: C, 60.03; H, 4.69; N, 8.56.

N-(1-(furan-2-yl)-3-(2-isonicotinoylhydrazinyl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4b)

Pale buff powder (2.44 g, 52.24%), m.p. 228–230 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.63 (dd, J = 3.3, 1.7 Hz, 1H, furan CH), 6.80 (d, J = 3.4 Hz, 1H, furan CH), 7.25 (s, 1H, olefinic CH), 7.41 (s, 2H, arom.CH), 7.82 (d, J = 1.4 Hz, 1H, furan CH), 7.83 (s, 2H, arom.CH), 8.78 (d, J = 5.3 Hz, 2H, arom.CH), 9.90 (s, 1H, NH), 10.32 (s, 1H, NH), 10.80 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.54 (OCH3), 60.59 (2OCH3), 106.10 (C2,6 trimethoxybenzamide), 112.89 (C olefinic), 115.09 (C3 furan), 118.94 (C4 furan), 121.83 (C3,5 pyridyl), 126.40 (C1 trimethoxybenzamide), 129.40 (C olefinic), 140.07 (C4 pyridyl), 140.82 (C4 trimethoxybenzamide), 145.38 (C5 furan), 149.95 (C2 furan), 150.87 (C2,6 pyridyl), 153.01 (C3,5 trimethoxybenzamide), 164.48 (C=O amide), 164.52 (C=O amide), 165.81 (C=O trimethoxybenzamide). Anal. Calcd. for C23H22N4O7 (466.44): C, 59.22; H, 4.75; N, 12.01. Found: C, 58.97; H, 4.87; N, 11.93.

4.2.4. General Procedure for the Synthesis of N-(4-(2-(3-(furan-2-yl)-2-(3,4,5 trimethoxybenzamido)acryloyl)hydrazinecarbonyl)phenyl)nicotinamide (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.
Pale yellow powder (3.49 g, 59.66%), m.p. 217–219 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.62 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.79 (d, J = 3.4 Hz, 1H, furan CH), 7.25 (s, 1H, olefinic CH), 7.41 (s, 2H, arom.CH), 7.60 (dd, J = 7.5, 4.8 Hz, 1H, arom.CH), 7.82 (d, J = 1.4 Hz, 1H, furan CH), 7.90 (d, J = 8.9 Hz, 2H, arom.CH), 7.96 (d, J = 8.8 Hz, 2H, arom.CH), 8.32 (dt, J = 8.0, 1.9 Hz, 1H, arom.CH), 8.75–8.83 (m, 1H, arom.CH), 9.13 (s, 1H, arom.CH), 9.87 (s, 1H, NH), 10.18 (s, 1H, NH), 10.40 (s, 1H, NH), 10.68 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.54 (OCH3), 60.58 (2OCH3), 106.10 (C2,6 trimethoxybenzamide), 112.86 (C olefinic), 114.88 (C3 furan), 118.78 (C4 furan), 120.00 (C3,5 phenyl), 124.01 (C5 pyridyl), 126.66 (C1 trimethoxybenzamide), 128.24 (C1 phenyl), 128.82 (C2,6 phenyl), 129.48 (C olefinic), 130.87 (C3 pyridyl), 136.04 (C4 pyridyl), 140.78 (C4 trimethoxybenzamide), 142.38 (C4 phenyl), 145.27 (C5 furan), 149.21 (C3 furan), 150.02 (C6 pyridyl), 152.77 (C2 pyridyl), 152.99 (C3,5 trimethoxybenzamide), 164.61 (C=O amide), 164.90 (C=O amide), 165.45 (C=O amide), 165.75 (C=O trimethoxybenzamide). Anal. Calcd. for C30H27N5O8 (585.56): C, 61.53; H, 4.65; N, 11.96. Found: C, 61.66; H, 4.73; N, 11.88.

4.2.5. General Procedure for the Synthesis of N-((1Z)-3-(2-(3-(aryl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamides 6a–e

A mixture of oxazolone 1 (0.01 mol 3.29 g) with respective acrylic acid hydrazide (0.01 mol) in DMF (20 mL) and catalytic amount glacial acetic acid (10 drops) was refluxed for 6–8 h. After completion of the reaction the reaction mixture was then cooled and poured into ice/cold water. The formed precipitate was crystallized from DMF/H2O to afford pure compound 6a–e.

N-((1Z)-3-(2-(3-(4-chlorophenyl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (6a)

White powder (3.72 g, 50.54%), m.p. 209–211 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.74 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.85 (s, 6H, 2OCH3), 3.87 (s, 6H, 2OCH3), 6.61 (dd, J = 3.2, 1.7 Hz, 1H, furan CH), 6.76 (d, J = 3.4 Hz, 1H, furan CH), 7.22 (s, 1H, olefinic CH), 7.25 (s, 1H, olefinic CH), 7.35 (s, 2H, arom.CH), 7.39 (s, 2H, arom.CH), 7.47 (d, J = 8.6 Hz, 2H, arom.CH), 7.62 (d, J = 8.6 Hz, 2H, arom.CH), 7.81 (d, J = 1.3 Hz, 1H, furan CH), 9.81 (s, 1H, NH), 9.93 (s, 1H, NH), 10.20 (s, 1H, NH), 10.26 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.53 (4OCH3), 60.57 (2OCH3), 106.06 (C2,6 trimethoxybenzamide), 106.08 (C2,6 trimethoxybenzamide), 112.85 (C olefinic), 114.74 (C3 furan), 118.74 (C4 furan), 119.68 (C olefinic), 126.64 (C1 trimethoxybenzamide), 128.66 (C1 trimethoxybenzamide), 129.11 (C3,5 chlorophenyl), 129.47 (C olefinic), 130.18 (C olefinic), 131.57 (C2,6 chlorophenyl), 133.48 (C1 chlorophenyl), 133.69 (C4 chlorophenyl), 140.76 (C4 trimethoxybenzamide), 140.88 (C4 trimethoxybenzamide), 145.19 (C5 furan), 150.03 (C2 furan), 152.99 (C3,5 trimethoxybenzamide), 153.00 (C3,5 trimethoxybenzamide), 164.24 (C=O amide), 164.75 (C=O amide), 165.62 (C=O trimethoxybenzamide), 165.77 (C=O trimethoxybenzamide). Anal. Calcd. for C36H35ClN4O11 (735.14): C, 58.82; H, 4.80; N, 7.62. Found: C, 59.02; H, 4.88; N, 7.43.

N-((1Z)-3-(2-(3-(4-cyanophenyl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (6b)

Yellow powder (4.43 g, 61.03%), m.p. 223–225 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.73 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.84 (s, 6H, 2OCH3), 3.86 (s, 6H, 2OCH3), 6.60 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.76 (d, J = 3.4 Hz, 1H, furan CH), 7.22 (s, olefinic CH), 7.25 (s, olefinic CH), 7.33 (s, 2H, arom.CH), 7.39 (s, 2H, arom.CH), 7.75 (d, J = 8.4 Hz, 2H, arom.CH), 7.80 (d, J = 1.4 Hz, 1H, furan CH), 7.86 (d, J = 8.4 Hz, 2H, arom.CH), 9.81 (s, 1H, NH), 10.04 (s, 1H, NH), 10.23 (s, 1H, NH), 10.36 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): Anal. Calcd. for C37H35N5O11 (725.70): C, 61.24; H, 4.86; N, 9.65. Found: C, 61.06; H, 4.98; N, 9.58.

N-((1Z)-3-(2-(3-(4-(dimethylamino)phenyl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (6c)

Orange powder (3.77 g, 50.73%), m.p. 215–217 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.93 (s, 6H, 2CH3), 3.75 (s, 6H, 2OCH3), 3.87 (s, 12H, 4OCH3), 6.60 (s, 1H, furan CH), 6.69 (d, J = 8.7 Hz, 2H, arom.CH), 6.74 (d, J = 3.0 Hz, 1H, furan CH), 7.21 (s, 1H, olefinic CH), 7.28 (s, 1H, olefinic CH), 7.39 (s, 2H, arom.CH), 7.40 (s, 2H, arom.CH), 7.47 (d, J = 8.7 Hz, 2H, arom.CH), 7.80 (s, 1H, furan CH), 9.75 (s, 1H, NH), 9.80 (s, 1H, NH), 9.97 (s, 2H, 2NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 40.40 (2CH3), 56.52 (4OCH3), 60.57 (2OCH3), 106.03 (2C2,6 trimethoxybenzamide), 112.14 (C3,5 dimethylaminophenyl), 112.83 (C olefinic), 114.60 (C3 furan), 118.55 (C4 furan), 121.74 (C olefinic), 124.28 (C1 dimethylaminophenyl), 126.78 (C olefinic), 129.50 (C1 trimethoxybenzamide), 129.60 (C1 trimethoxybenzamide), 131.70 (C2,6 dimethylaminophenyl), 131.94 (C olefinic), 140.65 (C4 trimethoxybenzamide), 140.74 (C4 trimethoxybenzamide), 145.11 (C5 furan), 150.09 (C2 furan), 151.08 (C4 dimethylaminophenyl), 152.97 (2C3,5 trimethoxybenzamide), 164.18 (C=O amide), 165.19 (C=O amide), 165.59 (C=O trimethoxybenzamide), 165.66 (C=O trimethoxybenzamide). Anal. Calcd. for C38H41N5O11 (743.76): C, 61.36; H, 5.56; N, 9.42. Found: C, 61.47; H, 5.63; N, 9.31.

N-(3-(2-((Z)-3-(furan-2-yl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)-1-(4-methoxyphenyl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (6d)

Red powder (3.61 g, 49.41%), m.p. 206–208 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.74 (s, 6H, 2OCH3), 3.77 (s, 3H, OCH3), 3.86 (s, 6H, 2OCH3), 3.87 (s, 6H, 2OCH3), 6.59–6.64 (m, 1H, furan CH), 6.75 (d, J = 3.4 Hz, 1H, furan CH), 6.97 (d, J = 8.8 Hz, 2H, arom.CH), 7.22 (s, 1H, olefinic CH), 7.29 (s, 1H, olefinic CH), 7.38 (s, 2H, arom.CH), 7.39 (s, 2H, arom.CH), 7.58 (d, J = 8.8 Hz, 2H, arom.CH), 7.81 (s, 1H, furan CH), 9.80 (s, 1H, NH), 9.83 (s, 1H, NH), 10.12 (s, 1H, NH), 10.15 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 55.70 (OCH3), 56.52 (4OCH3), 60.57 (2OCH3), 106.04 (2C2,6 trimethoxybenzamide), 112.82 (C olefinic), 114.57 (C3,5 methoxyphenyl), 114.71 (C3 furan), 118.71 (C4 furan), 126.66 (C olefinic), 127.13 (C1 methoxyphenyl), 127.15 (C1 trimethoxybenzamide), 129.35 (C olefinic), 129.47 (C1 trimethoxybenzamide), 130.53 (C olefinic), 131.51 (), 131.74 (C2,6 methoxyphenyl), 140.75 (2C4 trimethoxybenzamide), 145.17 (C5 furan), 150.03 (C2 furan), 152.98 (2C3,5 trimethoxybenzamide), 160.23 (C4 methoxyphenyl), 163.98 (C=O amide), 164.24 (C=O amide), 165.05 (C=O trimethoxybenzamide), 165.76 (C=O trimethoxybenzamide). Anal. Calcd. for C37H38N4O12 (730.72): C, 60.82; H, 5.24; N, 7.67. Found: C, 61.00; H, 5.37; N, 7.51.

N-(3-(2-((Z)-3-(furan-2-yl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4,5-trimethoxybenzamide (6e)

Orange powder (3.68 g, 46.51%), m.p. 212–214 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.65 (s, 6H, 2OCH3). 3.67 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.84 (s, 6H, 2OCH3), 3.87 (s, 6H, 2OCH3), 6.59–6.65 (m, 1H, furan CH), 6.76 (d, J = 3.4 Hz, 1H, furan CH), 7.00 (s, 2H, arom.CH), 7.24 (s, 1H, olefinic CH), 7.36 (s, 1H, olefinic CH), 7.40 (s, 2H, arom.CH), 7.42 (s, 2H, arom.CH), 7.81 (s, 1H, furan CH), 9.81 (s, 1H, NH), 9.89 (s, 1H, NH), 10.21 (s, 2H, 2NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.07 (2OCH3), 56.53 (4OCH3), 60.52 (OCH3), 60.57 (OCH3), 60.63 (OCH3), 106.02 (C2,6 trimethoxybenzamide), 106.06 (C2,6 trimethoxybenzamide), 107.68 (C2,6 trimethoxyphenyl), 112.85 (C olefinic), 114.73 (C3 furan), 118.78 (C4 furan), 126.67 (C olefinic), 128.51 (C1 trimethoxyphenyl), 129.26 (C1 trimethoxybenzamide), 129.51 (C1 trimethoxybenzamide), 129.76 (C olefinic), 131.18 (C olefinic), 138.62 (C4 trimethoxyphenyl), 140.76 (C4 trimethoxybenzamide), 140.84 (C4 trimethoxybenzamide), 145.18 (C5 furan), 150.05 (C2 furan), 152.94 (C3,5 trimethoxyphenyl), 152.99 (C3,5 trimethoxybenzamide), 153.05 (C3,5 trimethoxybenzamide), 164.20 (C=O amide), 164.75 (C=O amide), 165.64 (C=O trimethoxybenzamide), 165.74 (C=O trimethoxybenzamide). Anal. Calcd. for C39H42N4O14 (790.77): C, 59.24; H, 5.35; N, 7.09. Found: C, 59.41; H, 5.52; N, 6.90.

4.3. Biological Studies

4.3.1. Cytotoxic Activity Evaluation

Cytotoxic activity was carried out using MTT colorimetric antiproliferative assay to investigate the effect of the prepared molecules on HepG2 as well as HL-7702 cell lines. See Section 4.3.1 in Supplementary Data.

4.3.2. In Vitro HDAC Inhibition Assay

The in vitro HDAC inhibitory activities of compound 6a and SAHA against two HDAC isoforms (HDAC1, 2) were measured using ELISA assay kits {Mybiosource, Inc. [#MBS2020012 and #MBS2510971]} according to manufacturer’s directions. See Section 4.3.2 in Supplementary Data.

4.3.3. In Vitro Tubulin Inhibition Assay

Compound 6a and CA-4 were evaluated for their tubulin inhibitory activity according to manufacturer’s instructions using # abcam Human Beta-tubulin sim-plestep ELISA Kit ab245722. See Section 4.3.3 in Supplementary Data.

4.3.4. Cell Cycle Analysis

Cell cycle analysis in HepG2 cells was investigated using fluorescent Annexin V-FITC/ PI detection kit (BioVision EZCellTM Cell Cycle Analysis Kit Catalog #K920) by flow cytometry assay. See Section 4.3.4 in Supplementary Data.

4.3.5. Apoptosis Assay

Apoptosis in HepG2 cells was investigated using fluorescent Annexin V-FITC/ PI detection kit (BioVision Annexin V-FITC Apoptosis Detection Kit, Catalog #: K101) by flow cytometry assay. See Section 4.3.5 in Supplementary Data.

4.3.6. Caspase 3/7 Assay

Caspase 3/7 in HepG2 cells was investigated using CellEvent® Caspase 3/7 Green Detection Flow Cytometry Assay Kit according to manufacturer’s directions. See Section 4.3.6 in Supplementary Data.

4.3.7. Mitochondrial Membrane Potential (MMP) Assay

MMP was measured by FACS analysis using abcam ab113852TMRE Mitochondri-al Membrane Potential Assay Kit according to manufacturer’s directions. See Section 4.3.7 in Supplementary Data.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27123960/s1, Figure S1: 1H-NMR spectrum of compound 2a, Figure S2: 13C-NMR spectrum of compound 2a, Figure S3: 1H-NMR spectrum of compound 2b, Figure S4: 13C-NMR spectrum of compound 2b, Figure S5: 1H-NMR spectrum of compound 2c, Figure S6: 13C-NMR spectrum of compound 2c, Figure S7: 1H-NMR spectrum of compound 3, Figure S8: 13C-NMR spectrum of compound 3, Figure S9: 1H-NMR spectrum of compound 4a, Figure S10: 13C-NMR spectrum of compound 4a, Figure S11: 1H-NMR spectrum of compound 4b, Figure S12: 13C-NMR spectrum of compound 4b, Figure S13: 1H-NMR spectrum of compound 5, Figure S14: 13C-NMR spectrum of compound 5, Figure S15: 1H-NMR spectrum of compound 6a, Figure S16: 13C-NMR spectrum of compound 6a, Figure S17: 1H-NMR spectrum of compound 6b, Figure S18: 1H-NMR spectrum of compound 6c, Figure S19: 13C-NMR spectrum of compound 6c, Figure S20: 1H-NMR spectrum of compound 6d, Figure S21: 13C-NMR spectrum of compound 6d, Figure S22: 1H-NMR spectrum of compound 6e, Figure S23: 13C-NMR spectrum of compound 6e. Section 4.1. Chemistry: General. Section 4.3. Biological studies.

Author Contributions

Conceptualization, I.Z. and T.A.-W.; methodology, T.A.-W., F.A., O.A.A.A., A.H.A.A. and I.Z.; data curation, A.A., E.F., S.A., S.N.A.B., A.I.M.K. and I.Z.; software, A.I.M.K., S.N.A.B. and I.Z.; resources, T.A.-W., A.A., A.H.A.A., F.A., S.A. and I.Z.; supervision, I.Z.; funding acquisition, T.A.-W., A.A., F.A., O.A.A.A. and S.A.; original draft preparation, I.Z. and T.A.-W.; Writing, review, and editing, all authors. All authors have read and agreed to the published version of the manuscript.

Funding

The authors extend their appreciation to the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R25), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia & Deanship of Scientific Research at Taif University for funding this work through Taif University Researchers Supporting Project number (TURSP-2020/197), Taif University, Taif, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors extend their appreciation to the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R25), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia & Deanship of Scientific Research at Taif University for funding this work through Taif University Researchers Supporting Project number (TURSP-2020/197), Taif University, Taif, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

The authors confirm that the data supporting the findings of this study are available within the article.

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Figure 1. The structures of HDAC inhibitors in clinical trials (I–IV) and dual HDAC-tubulin inhibitors (V and VI). Cap group showed in blue color and zinc binding group showed in red color.
Figure 1. The structures of HDAC inhibitors in clinical trials (I–IV) and dual HDAC-tubulin inhibitors (V and VI). Cap group showed in blue color and zinc binding group showed in red color.
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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.
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.
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Figure 2. (A) Dose response curve for in vitro HDAC inhibitory activity of tetraamide 6a and SAHA against HDAC1 and HDAC2 isoforms. (B) Graphical representation for in vitro HDAC inhibitory activity of tetraamide 6a and SAHA against HDAC1 and HDAC2 isoforms.
Figure 2. (A) Dose response curve for in vitro HDAC inhibitory activity of tetraamide 6a and SAHA against HDAC1 and HDAC2 isoforms. (B) Graphical representation for in vitro HDAC inhibitory activity of tetraamide 6a and SAHA against HDAC1 and HDAC2 isoforms.
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Figure 3. (A) Tubulin polymerization inhibition activity induced by compound 6a compared with no treatment control cells using ELISA assay for β-tubulin. (B) Dose response curve for in vitro β-tubulin inhibitory activity of tetraamide 6a and CA-4 against tubulin.
Figure 3. (A) Tubulin polymerization inhibition activity induced by compound 6a compared with no treatment control cells using ELISA assay for β-tubulin. (B) Dose response curve for in vitro β-tubulin inhibitory activity of tetraamide 6a and CA-4 against tubulin.
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Figure 4. (A) Graphical representation of cell cycle analysis in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptograms shows cell cycle analysis after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control.
Figure 4. (A) Graphical representation of cell cycle analysis in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptograms shows cell cycle analysis after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control.
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Figure 5. (A) Graphical representation shows the apoptosis effect by Annexin V-FITC versus PI uptake method in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptograms show the apoptosis effect by the Annexin V-FITC versus PI uptake method in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control.
Figure 5. (A) Graphical representation shows the apoptosis effect by Annexin V-FITC versus PI uptake method in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptograms show the apoptosis effect by the Annexin V-FITC versus PI uptake method in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control.
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Figure 6. (A) Graphical representation of caspase 3/7 in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptograms show caspase 3/7 in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control.
Figure 6. (A) Graphical representation of caspase 3/7 in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptograms show caspase 3/7 in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control.
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Figure 7. (A) Graphical representation of MMP in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptogram shows the loss of MMP during compound 6a treatment in in HepG2 cells after 48 h compared with the untreated control.
Figure 7. (A) Graphical representation of MMP in HepG2 cells after 48 h of compound 6a exposure at its IC50 concentration (μM) compared with the untreated control. (B) Cryptogram shows the loss of MMP during compound 6a treatment in in HepG2 cells after 48 h compared with the untreated control.
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Table
Table 1. Cytotoxic screening of the tested amides 2a-6e. Values are the mean of three independent replicates ± SE.
Table 1. Cytotoxic screening of the tested amides 2a-6e. Values are the mean of three independent replicates ± SE.
Comp NoIC50 Value (μM)
HepG2HL-7702
2a68.90 ± 3.32NT
2b23.28 ± 1.03NT
2c22.03 ± 0.96NT
316.24 ± 0.79NT
4a13.37 ± 0.56NT
4b8.36 ± 0.51NT
53.25 ± 0.25NT
6a0.65 ± 0.039.62 ± 0.23
6b0.92 ± 0.1011.09 ± 0.18
6c1.12 ± 0.129.88 ± 0.14
6d3.81 ± 0.18NT
6e3.98 ± 0.09NT
SAHA2.91 ± 0.15NT
CA-40.54 ± 0.048.86 ± 0.67
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Al-Warhi, T.; Aldhahrani, A.; Althobaiti, F.; Fayad, E.; Abu Ali, O.A.; Albogami, S.; Abu Almaaty, A.H.; Khedr, A.I.M.; Bukhari, S.N.A.; Zaki, I. Design, Synthesis and Cytotoxic Activity Evaluation of Newly Synthesized Amides-Based TMP Moiety as Potential Anticancer Agents over HepG2 Cells. Molecules 2022, 27, 3960. https://doi.org/10.3390/molecules27123960

AMA Style

Al-Warhi T, Aldhahrani A, Althobaiti F, Fayad E, Abu Ali OA, Albogami S, Abu Almaaty AH, Khedr AIM, Bukhari SNA, Zaki I. Design, Synthesis and Cytotoxic Activity Evaluation of Newly Synthesized Amides-Based TMP Moiety as Potential Anticancer Agents over HepG2 Cells. Molecules. 2022; 27(12):3960. https://doi.org/10.3390/molecules27123960

Chicago/Turabian Style

Al-Warhi, Tarfah, Adil Aldhahrani, Fayez Althobaiti, Eman Fayad, Ola A. Abu Ali, Sarah Albogami, Ali H. Abu Almaaty, Amgad I. M. Khedr, Syed Nasir Abbas Bukhari, and Islam Zaki. 2022. "Design, Synthesis and Cytotoxic Activity Evaluation of Newly Synthesized Amides-Based TMP Moiety as Potential Anticancer Agents over HepG2 Cells" Molecules 27, no. 12: 3960. https://doi.org/10.3390/molecules27123960

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