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Article

Design, Synthesis, and Investigation of Cytotoxic Activity of cis-Vinylamide-Linked Combretastatin Analogues as Potential Anticancer Agents

by
Tarfah Al-Warhi
1,
Leena S. Alqahtani
2,
Ghadi Alsharif
3,
Matokah Abualnaja
4,
Ola A. Abu Ali
5,
Safa H. Qahl
6,
Hussam Awwadh E. Althagafi
7,
Fahad Alharthi
8,
Ibrahim Jafri
9,
Fahmy G. Elsaid
10,11,
Ali A. Shati
10,
Salman Aloufi
9,
Eman Fayad
9,
Islam Zaki
12,* and
Martha M. Morcoss
13
1
Department of Chemistry, College of Science, Princess Nourah Bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia
2
Department of Biochemistry, College of Science, University of Jeddah, Jeddah 23445, Saudi Arabia
3
College of Clinical Laboratory Sciences, King Saud Bin Abdulaziz University for Health Sciences, Jeddah 21423, Saudi Arabia
4
Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah Al Mukarrama 24381, Saudi Arabia
5
Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
6
Department of Biology, College of Science, University of Jeddah, P.O. Box 80327, Jeddah 21589, Saudi Arabia
7
Biology Department, Faculty of Science and Arts, Al-Mikhwah, Al-Baha University, Al-Baha 1988, Saudi Arabia
8
Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
9
Department of Biotechnology, College of Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
10
Biology Department, Science, College, King Khalid University, Abha 61421, Saudi Arabia
11
Zoology Department, Faculty of Science, Mansoura University, P.O. Box 70, Mansoura 35516, Egypt
12
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
13
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni-Suef 62513, Egypt
 
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*
Author to whom correspondence should be addressed.
Symmetry 2022, 14(10), 2088; https://doi.org/10.3390/sym14102088
Submission received: 19 September 2022 / Revised: 29 September 2022 / Accepted: 4 October 2022 / Published: 7 October 2022
(This article belongs to the Section Chemistry: Symmetry/Asymmetry)
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Abstract

:
The combretastatins (cis-stilbenoid molecules) have received significant interest because of their simple chemical structures, excellent antiproliferative activity, and novel anti-tubulin molecular mechanism of action. Significant efforts have been carried out aiming at stabilizing the active cis-isomers. A new series of cis-vinylamide derivatives containing trimethoxyphenyl moiety were synthesized and characterized. Their anticancer activities were evaluated in vitro against MCF-7 breast cancer cell line. Compounds 2f, 3, and 5 displayed potent cytotoxic activity against the breast cancer cell line compared with CA-4 as the reference compound. The microtubule polymerization assay and flow cytometry analysis confirmed that the cytotoxic activity of compound 3 was related to inhibitory activity against tubulin polymerization. Compound 3 showed pro-apoptotic activity by inducting a significant increase in the percentage of pre-G1 phase in DNA flow cytometry compared to untreated control. The pro-apoptotic activity of compound 3 was inferred by a significant increase in the percentage of fluorescent annexin V/PI positive apoptotic cells. It also increased the level of caspase 3 compared to the untreated control.

1. Introduction

In recent years, target-based chemotherapeutic agents have become an effective choice for cancer treatment [1,2]. Targeted-based chemotherapeutic agents have been developed in order to avoid the dangerous side effects associated with traditional chemotherapeutic agents [3]. Targeted-based chemotherapeutic agents are focused on certain cellular enzymes or genes such as microtubules, angiogenesis, DNA, etc. [4,5]. Microtubules are a major component of the cytoskeletons, play a crucial role in cell division, and are also involved in protein trafficking [6,7]. During cell division, microtubules pull replicated chromosomes apart and into the two daughter cells in the M stage of the mitotic process [8]. As the microtubules grow, each chromosome is guided to the splitting cells by the growing microtubules [9]. There are three different classes of compounds that bind to tubulin [10]. One of the most tubulin polymerization inhibitors is a simple compound isolated from the bark of the South African tree Combretum caffrum, known as combretastatin A-4 (CA-4) [11]. CA-4 displayed potent cytotoxic activity against numerous cancer cell lines [12]. The binding of CA-4 to the colchicine binding site on tubulin causes a cell signaling cascade which causes cell morphology to change and leads to cellular apoptosis [13,14]. CA-4 can be described as a cis-stilbenoid (1,2-diaryl-cis-ethene) with a conserved 3,4,5-trimethoxyphenyl (TMP) moiety [15]. The opposing side of the stilbenoid consists of a 3-hydroxy-4-methoxyphenyl moiety which is more tolerant to modifications and substitutions [16]. Unfortunately, CA-4 showed a large decrease in its cytotoxic and anti-tubulin potency because of the spontaneous conversion of the olefinic bond from the less stable cis-isomer to the more stable trans-isomer [17]. Therefore, the development of synthetic analogs of CA-4 either in a cis-configuration or in conjugation with other compounds has been a rapidly accelerating area of research for well over a decade [18,19,20]. The conjugation of CA-4 with piperlongumine II displayed potent broad-spectrum antitumor activity with IC50 below 1.5 μM against all tested cell lines [21]. In addition, chalcone III possessed cytotoxic and antimitotic activity due to its ability to inhibit tubulin polymerization [22]. Additionally, chalcone-guanidine conjugate IV showed strong inhibitory activity against the growth of MCF-7 cells with IC50 of 0.09 μM and the polymerization of tubulin with IC50 = 8.40 μM [23]. Moreover, quinoline-hydrazone hybrid V emerged as the most potent cytotoxic compound with IC50 values of 0.04, 0.026, 0.022, and 0.038 μM, respectively, in HL-60, MCF-7, HCT-116, and Hela cancer cell lines compared with CA-4 as the reference compound (IC50 values range between 0.028–0.37 μM) [24]. Furthermore, N,N-dimethyl thiosemicarbazone molecule VI showed high antiproliferative activity in vitro against a variety of cancer cell lines including SK-N-MC neuroepithelioma cells (Figure 1) [25].
In this work, a new series of cis-vinylamide derivatives related to the cis-configurated isomer of CA-4 were designed and synthesized as possible antitumor agents. The designed compounds consisted of two aryl rings connected through a vinylamide group. The two aryl rings were decorated with 3,4-dimethoxyphenyl (DMP) and TMP moieties. In addition, the target compounds were subjected to structural extension with different side chains having an H-bond acceptor–donor (A–D) pair forming group. These side chains may be 2-arylethylidene hydrazinyl, N-ethyl thiosemicarbazide, N-phenyl thiosemicarbazide, or 2-(2,5-dioxopyrrolidinyl)amino groups with the aim to study the impact of these structural modification on the cytotoxic activity (Figure 2). The prepared cis-vinylamide compounds were evaluated for their cytotoxic activity in vitro against the MCF-7 breast cancer cell line. Furthermore, a tubulin polymerization inhibition assay and cell cycle analysis of the most active compounds were carried out to investigate if the antiproliferative activity is accompanied by a change in microtubule assembly. Moreover, the ability to activate caspase 3 was conducted to detect the apoptosis-inducing effect of the tested cis-vinylamide derivatives.

2. Results and Discussion

2.1. Chemistry

The title compounds were synthesized as shown in Scheme 1. As depicted in Scheme 1, the key starting material 1 was synthesized according to the synthetic method in the literature reported [26].
Condensation of the key starting material (Z)-1 with different aromatic ketones in absolute ethanol provided the corresponding 2-arylethylidene hydrazinyl derivatives 2a–g. The chemical structure of 2-arylethylidene hydrazinyl molecules 2a–g was characterized by 1H-NMR, 13C-NMR, and elemental analysis data. The 1H-NMR spectrum of compound 2a, as a representative example, revealed the disappearance of the signal corresponding to NH2 of the parent compound. In addition, 1H-NMR of 2a displayed two NH proton signals at δ 10.54 and 10.02 ppm and additional aromatic signals at δ 7.84–7.04 ppm. Furthermore, the 1H-NMR spectrum of compound 2a showed the presence of two singlet signals at δ 7.21 and 2.29 ppm attributed to olefinic and methyl protons, respectively. The 13C-NMR spectrum of 2a showed the presence of three signals at δ 166.15, 162.77, and 148.73 ppm corresponding to two carbonyls (C=O) and azomethine (C=N) groups, respectively.
The interaction of the hydrazide (Z)-1 with ethyl isothiocyanate in boiling absolute ethanol yielded the title compound N-ethyl thiosemicarbazide molecule 3. Its structure was confirmed by spectral and elemental data. The 1H-NMR spectrum of compound 3 revealed the presence of four proton signals at δ 10.32, 10.27, 9.41, and 7.67 ppm in addition to the presence of triplet and quartet signals at δ 3.47 and 1.20 ppm attributed to N-ethyl function. The 13C-NMR of compound 3 confirmed the carbon skeleton due to the presence of a new signal at δ 181.04 ppm corresponding to the thiocarbonyl (C=S) group in addition to additional carbon signals at the aliphatic region at δ 38.84 and 14.07 ppm attributed to N-ethyl (CH3CH2N-) group. In addition, the N-phenyl thiosemicarbazide molecule 4 was accomplished by treatment of the hydrazide derivative (Z)-1 with phenyl isothiocyanate in boiling n-butanol. The 1H-NMR spectrum of compound 4 revealed the presence of four singlet signals at δ 10.56, 10.45, 9.88, and 9.37 ppm related to four NH protons as well as the presence of extra proton signals at the aromatic region at δ 7.80–7.02 ppm corresponding to extra phenyl group of the introduced phenyl isothiocyanate group. The 13C-NMR spectrum of compound 4 revealed a new signal at δ 180.35 ppm assigned for the thiocarbonyl (C=S) group in addition to the presence of additional signals due to extra phenyl carbons.
Furthermore, the hydrazide (Z)-1 was subjected to a cyclization reaction by refluxing with succinic anhydride in acetic acid glacial to provide the 2-(2,5-dioxopyrrolidinyl)-amino derivative 5. The 1H-NMR spectrum of compound 5 lacked the hydrazinyl NH2 proton at δ 5.41 ppm and revealed the presence of two NH proton signals δ 10.01 and 9.79 ppm as well as the presence of two multiplet signals at the aliphatic region at δ 2.49–2.44 and 2.44–2.38 ppm corresponding to two methylene (CH2) protons of 2,5-dioxopyrrolidinyl moiety. 13C-NMR spectrum of compound 5 revealed signals of four carbonyl (C=O) groups at δ 174.04, 170.48, 165.86, and 164.66 ppm as well as the presence of two new signals at aliphatic region at δ 29.36 and 28.63 ppm attributed to two methylene (CH2) carbons of the 2,5-dioxopyrrolidinyl group.

2.2. Biology

2.2.1. Cytotoxic Activity against MCF-7 Breast Cancer Cell Line

The prepared cis-vinylamide derivatives 2a–g and 3–5 were screened for their in vitro cytotoxic activity against the MCF-7 breast cancer cell line through an MTT colorimetric assay. CA-4 was utilized as a reference drug in the current study. Results presented in Table 1 reveals that in general all the tested cis-vinylamide derivatives 2a–g and 3–5 influenced the survival response which showed moderate to good cytotoxic potency against MCF-7 cells. On the basis of the obtained IC50 values against the MCF-7 cell line, three compounds 3,4-dimethoxyphenylethylidine hydrazinyl 2f, N-ethyl thiosemicarbazide 3 and 2-(2,5-dioxopyrrolidinyl)amino 5 exhibited higher cytotoxic activity with IC50 values of 3.22, 1.04, and 1.81 μM, respectively, compared with CA-4 (IC50 = 0.67 μM). In addition to 2f, 3, and 5, compounds 2d, 2e, 2g, and 4 showed moderate cytotoxic activity (IC50 4.11–8.45 μM). According to the present study, a comparative analysis of the synthesized compounds revealed that in general the potency of 2-arylethylidene hydrazinyl derivatives 2a–g seems to be influenced by the type and nature of substituents on the arylidene moiety as electron donating groups exhibited higher cytotoxic activity than unsubstituted or electron withdrawing groups. In addition, regarding the corresponding N-ethyl thiosemicarbazide molecule 3, the results revealed that the replacement of N-ethyl group (compound 3) with the N-phenyl group (compound 4) resulted in a decrease in the cytotoxic activity.

2.2.2. Tubulin Assay

Targeting the tubulin-microtubule protein system in the M stage of the mitotic process during cell division is a viable methodology for the treatment of cancer [27]. To further explore the molecular target of the prepared cis-vinylamide derivatives, a microtubule polymerization assay was carried out. MCF-7 cells were treated with compounds 2f, 3, and 5 at their IC50 concentration in comparison with CA-4 as a reference compound, and the percentage of β-tubulin was determined using ELISA assay. The data presented in Figure 3 indicated that N-ethyl thiosemicarbazide derivative 3 and 2-(2,5-dioxopyrrolidin-1-yl)amino derivative 5 elicited potent tubulin polymerization inhibition with percentage inhibitions of 83.26% and 81.91%, respectively, compared with CA-4 (87.08% polymerization inhibition percentage). On the other hand, 3,4-dimethoxyphenylethylidine hydrazinyl derivative 2f showed moderate inhibition activity which caused 71.69% polymerization inhibition at its IC50 concentration. The obtained results mirror those obtained on MCF-7 cells, suggesting that the mechanism of action of the tested cis-vinylamide compounds may be tubulin polymerization inhibition.

2.2.3. Cell Cycle Analysis

The cell cycle of living tissue follows a highly regulated path of growth, division, and death [28]. Genetic mutations to cellular cycle are the root cause of cancerous growth [29]. Compound 3 was the most active compound against the MCF-7 cancer cell line (IC50 = 1.04 ± 0.13 μM). Therefore, it was selected to test its effect on the cell cycle stages in MCF-7 cells using DNA flow cytometry analysis. The results revealed an increase in the pre-G1 phase for compound 3 (33.26%) relative to no treatment control MCF-7 cell line (0.97%). In addition, the percentage of cells in G2/M phase increased from 8.69% to 46.75% after the treatment of MCF-7 cells with compound 3. Furthermore, there was a decrease in the percentage of G1 and S phases (23.52% and 46.75%, respectively) compared with no treatment control (49.07 and 42.24%, respectively). The data suggested that N-ethyl thiosemicarbazide molecule 3 induced a pro-apoptotic effect in the pre-G1 phase and cell cycle arrest in the G2/M phase through an increase in the percentage of population during the pre-G1 and G2/M phases (Figure 4).

2.2.4. Apoptosis Assay

Classically, there are two types of cell death in biological systems: necrosis (accidental cell death) and apoptosis (programmed cell death) [30]. Apoptosis involved many genes which safely dispose of cells once they have fulfilled their intended biological role [31]. The reduced apoptotic response is an important factor contributing to the tendency of the cancerous cells to resist treatment with traditional chemotherapeutic agents [32]. To confirm and quantify the mode of cellular death induced by compound 3 in MCF-7 cells, annexin V-FITC/PI dual staining assay was carried out using DNA flow cytometry analysis. As depicted in Figure 5, compound 3 at 48 h had already induced an accumulation of annexin V positive cells in comparison with no treatment control. It could be noticed that the total apoptotic cells (early and late apoptosis) increase in MCF-7 cells (27.94%) after treatment with compound 3 compared with no treatment control (0.65%). From the obtained results, it could be concluded that N-ethyl thiosemicarbazide derivative 3 can be considered an apoptosis inducer.

2.2.5. Caspase Assay

Activation of caspases plays a vital role in the initiation and termination of the apoptotic pathway [33]. Caspase 3 is initiated by the death cascade. It is activated by the upregulation of caspase 8 and 9. Thus, it acts as a convergence point in both intrinsic and extrinsic pathways [34]. The effect of N-ethyl thiosemicarbazide molecule 3 on caspase 3 was investigated using an ELISA assay. The results showed an increase in the level of active caspase 3 by 11.8-fold compared to the control MCF-7 cells(Figure 6).

3. Conclusions

In this study, a new series of cis-vinylamide derivatives 2a–g and 3–5 containing trimethoxyphenyl moiety were designed and synthesized as potential anticancer agents. 3,4-dimethoxyphenylethylidine hydrazinyl 2f, N-ethyl thiosemicarbazide 3, and 2-(2,5-dioxopyrrolidinyl)amino 5 exhibited potent cytotoxic activity against MCF-7 breast cancer cell line with IC50 value of 3.22, 1.04, and 1.84 μM, respectively, compared with CA-4 (IC50 = 0.67 μM) as the reference compound. N-ethyl thiosemicarbazide derivatives 3 and 2-(2,5-dioxopyrrolidinyl)amino 5 exhibited good β-tubulin polymerization inhibition percentage (83.26% and 81.91% polymerization inhibition, respectively) in comparison with CA-4 (87.08% polymerization inhibition). DNA flow cytometric analysis of N-ethyl thiosemicarbazide derivatives 3 caused MCF-7 cells to exhibit cell cycle arrest at G2/M phase (46.75%) of the cell cycle profile compared to the untreated control (8.69%). In addition, N-ethyl thiosemicarbazide derivatives 3 showed pro-apoptotic activity by inducting a significant increase in the percentage of pre-G1 phase (33.26%) in DNA flow cytometry compared to the control (0.97%). The pro-apoptotic activity of N-ethyl thiosemicarbazide derivatives 3 was inferred by a significant increase in the percentage of annexin V-FITC/PI-positive apoptotic cells. The ELISA measurement of the effect of N-ethyl thiosemicarbazide derivatives 3 on MCF-7 cells demonstrated that it boosted the apoptotic cascade through increased the level of caspase 3 by 11.8-fold compared to the untreated control. Accordingly, the novel cis-vinylamide derivatives represent potential lead candidates that might be useful as anticancer agents on MCF-7 cells for further in vivo study.

4. Experimental

4.1. Chemistry

4.1.1. General

See Section S4.1.1 in Supplementary Data.

4.1.2. General Procedure for the Synthesis of N-((Z)-3-((E)-2-(arylethylidene)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamides 2a–g

A mixture of the hydrazide (Z)-1 (431 mg, 1 mmol) and respective alkyl aryl ketone (1 mmol) in absolute ethanol (20 mL) containing 0.5 mL of glacial acetic acid was refluxed for 6–8 h. After cooling, the crude product was obtained by filtration and crystallized from ethanol/H2O (3:1) to afford pure compound 2a–g.

3,4-Dimethoxy-N-((Z)-3-oxo-3-((E)-2-(1-phenylethylidene)hydrazinyl)-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)benzamide (2a)

Pale yellow powder (415 mg, 78%), m.p. 235–237 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.29 (s, 3H, CH3), 3.67 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.04 (s, 2H, arom.CH), 7.09 (d, J = 8.1 Hz, 1H, arom.CH), 7.21 (s, 1H, olefinic CH), 7.43 (s, 3H, arom.CH), 7.68 (s, 1H, arom.CH), 7.72 (d, J = 8.4 Hz, 1H, arom.CH), 7.84 (s, 2H, arom.CH), 10.02 (s, 1H, NH), 10.54 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.89 (CH3), 56.10 (OCH3), 56.12 (OCH3), 56.14 (2OCH3), 60.56 (OCH3), 107.77 (C-aromatic), 111.41 (C-aromatic), 121.83 (C-aromatic), 125.93 (C-aromatic), 126.86 (C-aromatic), 128.78 (C-olefinic), 129.40 (C-aromatic), 129.44 (C-aromatic), 129.64 (C-aromatic), 129.87 (C-olefinic), 130.02 (C-aromatic), 138.52 (C-O), 142.70 (C-aromatic), 148.73 (C=N), 152.29 (C-O), 153.05 (2C-O), 155.70 (C-O), 162.77 (C=O), 166.15 (C=O). Anal. Calcd. for C29H31N3O7 (533.57): C, 65.28; H, 5.86; N, 7.88. Found: C, 65.13; H, 6.04; N, 7.97.

N-((Z)-3-((E)-2-(1-(4-Chlorophenyl)ethylidene)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamide (2b)

Pale yellow powder (387 mg, 66%), m.p. 212–214 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.28 (s, 3H, CH3), 3.67 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.04 (s, 2H, arom.CH), 7.09 (d, J = 8.7 Hz, 1H, arom.CH), 7.19 (s, 1H, olefinic CH), 7.50 (s, 2H, arom.CH), 7.66 (s, 1H, arom.CH), 7.71 (d, J = 8.3 Hz, 1H, arom.CH), 7.85 (d, J = 7.4 Hz, 2H, arom.CH), 10.01 (s, 1H, NH), 10.58 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.79 (CH3), 56.09 (OCH3), 56.12 (OCH3), 56.14 (2OCH3), 60.56 (OCH3), 107.78 (C-aromatic), 111.41 (C-aromatic), 121.82 (C-aromatic), 125.89 (C-aromatic), 128.60 (C-olefinic), 128.76 (C-aromatic), 128.82 (C-aromatic), 129.42 (C-aromatic), 129.57 (C-olefinic), 130.01 (C-aromatic), 137.35 (C-aromatic), 138.52 (C-O), 142.52 (C-aromatic), 148.72 (C=N), 152.29 (C-O), 153.05 (2C-O), 155.64 (C-O), 162.68 (C=O), 166.07 (C=O). Anal. Calcd. for C29H30ClN3O7 (568.02): C, 61.32; H, 5.32; N, 7.40. Found: C, 61.48; H, 5.41; N, 7.26.

N-((Z)-3-((E)-2-(1-(4-Bromophenyl)ethylidene)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamide (2c)

Pale yellow powder (422 mg, 69%), m.p. 210–212 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.28 (s, 3H, CH3), 3.67 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.04 (s, 2H, arom.CH), 7.09 (d, J = 8.5 Hz, 1H, arom.CH), 7.19 (s, 1H, olefinic CH), 7.65 (s, 2H, arom.CH), 7.70 (d, J = 8.5 Hz, 2H, arom.CH), 7.75–7.86 (m, 2H, arom.CH), 10.01 (s, 1H, NH), 10.58 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.88 (CH3), 56.09 (OCH3), 56.13 (OCH3), 56.15 (2OCH3), 60.56 (OCH3), 107.78 (C-aromatic), 111.42 (C-aromatic), 121.82 (C-aromatic), 122.99 (C-aromatic), 124.15 (C-aromatic), 125.89 (C-aromatic), 128.87 (C-olefinic), 129.77 (C-olefinic), 130.00 (C-aromatic), 131.75 (C-aromatic), 134.62 (C-aromatic), 137.72 (C-O), 142.40 (C-aromatic), 148.72 (C=N), 152.31 (C-O), 153.05 (2C-O), 155.45 (C-O), 161.16 (C=O), 166.11 (C=O). Anal. Calcd. for C29H30BrN3O7 (612.47): C, 56.87; H, 4.94; N, 6.86. Found: C, 57.07; H, 5.11; N, 6.70.

N-((Z)-3-((E)-2-(1-(4-Hydroxyphenyl)ethylidene)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamide (2d)

Pale yellow powder (368 mg, 67%), m.p. 229–231 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.21 (s, 3H, CH3), 3.66 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.81 (d, J = 8.3 Hz, 2H, arom.CH), 7.02 (s, 2H, arom.CH), 7.09 (d, J = 8.4 Hz, 1H, arom.CH), 7.22 (s, 1H, olefinic CH), 7.67 (d, J = 2.0 Hz, 2H, arom.CH), 7.69–7.73 (m, 2H, arom.CH), 9.79 (s, 1H, OH), 9.96 (s, 1H, NH), 10.38 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.70 (CH3), 56.10 (OCH3), 56.11 (OCH3), 56.12 (2OCH3), 60.55 (OCH3), 107.72 (C-aromatic), 111.39 (C-aromatic), 111.50 (C-aromatic), 115.51 (C-aromatic), 121.82 (C-aromatic), 126.00 (C-aromatic), 128.49 (C-olefinic), 129.28 (C-aromatic), 129.71 (C-olefinic), 130.05 (C-aromatic), 130.33 (C-aromatic), 138.47 (C-O), 148.71 (C=N), 152.26 (C-O), 153.04 (2C-O), 156.54 (C-O), 158.60 (C-O), 159.38 (C=O), 166.14 (C=O). Anal. Calcd. for C29H31N3O8 (549.57): C, 63.38; H, 5.69; N, 7.65. Found: C, 63.52; H, 5.59; N, 7.54.

3,4-Dimethoxy-N-((Z)-3-((E)-2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)benzamide (2e)

Pale yellow powder (411 mg, 73%), m.p. 225–227 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.25 (s, 3H, CH3), 3.66 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.99 (d, J = 7.5 Hz, 2H, arom.CH), 7.03 (s, 2H, arom.CH), 7.09 (d, J = 8.7 Hz, 1H, arom.CH), 7.20 (s, 1H, olefinic CH), 7.66 (d, J = 2.1 Hz, 1H, arom.CH), 7.69–7.74 (m, 1H, arom.CH), 7.80 (d, J = 8.3 Hz, 2H, arom.CH), 9.98 (s, 1H, NH), 10.44 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.76 (CH3), 55.71 (OCH3), 56.09 (OCH3), 56.13 (3OCH3), 60.56 (OCH3), 107.74 (C-aromatic), 111.46 (C-aromatic), 114.15 (C-aromatic), 114.95 (C-aromatic), 121.82 (C-aromatic), 125.98 (C-aromatic), 128.39 (C-olefinic), 129.31 (C-aromatic), 129.70 (C-olefinic), 130.42 (C-aromatic), 130.87 (C-aromatic), 138.47 (C-O), 148.71 (C=N), 152.27 (C-O), 153.04 (2C-O), 155.98 (C-O), 156.29 (C-O), 160.41 (C=O), 166.05 (C=O). Anal. Calcd. for C30H33N3O8 (563.60): C, 63.93; H, 5.90; N, 7.46. Found: C, 64.12; H, 5.78; N, 7.35.

N-((Z)-3-((E)-2-(1-(3,4-Dimethoxyphenyl)ethylidene)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamide (2f)

Pale yellow powder (380 mg, 64%), m.p. 202–204 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.26 (s, 3H, CH3), 3.67 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.81 (s, 6H, 2OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 7.00 (s, 1H, arom.CH), 7.03 (s, 2H, arom.CH), 7.09 (d, J = 8.4 Hz, 1H, arom.CH), 7.20 (s, 1H, olefinic CH), 7.37 (d, J = 8.4 Hz, 1H, arom.CH), 7.47 (s, 1H, arom.CH), 7.66 (s, 1H, arom.CH), 7.71 (d, J = 8.6 Hz, 1H, arom.CH), 9.98 (s, 1H, NH), 10.48 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.97 (CH3), 56.00 (OCH3), 56.09 (OCH3), 56.12 (OCH3), 56.14 (3OCH3), 60.56 (OCH3), 107.72 (C-aromatic), 109.74 (C-aromatic), 111.39 (C-aromatic), 111.47 (C-aromatic), 111.54 (C-aromatic), 120.37 (C-aromatic), 121.81 (C-aromatic), 126.00 (C-olefinic), 129.51 (C-olefinic), 130.07 (C-aromatic), 131.02 (C-aromatic), 132.00 (C-aromatic), 137.55 (C-aromatic), 138.47 (C-O), 148.71 (C=N), 148.90 (C-O), 152.27 (C-O), 153.05 (2C-O), 156.44 (C-O), 160.72 (C=O), 162.39 (C=O). Anal. Calcd. for C31H35N3O9 (593.62): C, 62.72; H, 5.94; N, 7.08. Found: C, 62.64; H, 6.04; N, 7.16.

3,4-Dimethoxy-N-((Z)-3-oxo-1-(3,4,5-trimethoxyphenyl)-3-((E)-2-(1-(3,4,5-trimethoxyphenyl)ethylidene)hydrazinyl)prop-1-en-2-yl)benzamide (2g)

Pale yellow powder (305 mg, 49%), m.p. 196–198 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.30 (s, 3H, CH3), 3.67 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.82 (s, 6H, 2OCH3), 3.84 (s, 6H, 2OCH3), 7.03 (s, 2H, arom.CH), 7.06–7.16 (m, 3H, arom.CH), 7.18 (s, 1H, olefinic CH), 7.66 (s, 1H, arom.CH), 7.71 (d, J = 8.4 Hz, 1H, arom.CH), 10.00 (s, 1H, NH), 10.55 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 15.25 (CH3), 56.08 (OCH3), 56.12 (OCH3), 56.15 (3OCH3), 56.43 (OCH3), 60.56 (2OCH3), 104.53 (C-aromatic), 107.73 (C-aromatic), 111.39 (C-aromatic), 111.46 (C-aromatic), 121.80 (C-aromatic), 125.97 (C-olefinic), 129.43 (C-olefinic), 130.05 (C-aromatic), 130.22 (C-aromatic), 134.05 (C-aromatic), 138.48 (C-O), 144.00 (C-O), 148.71 (C=N), 152.27 (C-O), 153.05 (2C-O), 153.10 (2C-O), 155.58 (C-O), 162.53 (C=O), 166.06 (C=O). Anal. Calcd. for C32H37N3O10 (623.65): C, 61.63; H, 5.98; N, 6.74. Found: C, 61.74; H, 6.06; N, 6.61.

4.1.3. General Procedure for the Synthesis of (Z)-N-(3-(2-(Ethylcarbamothioyl)hydrazinyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamide (3)

A mixture of the hydrazide (Z)-1 (431 mg, 1 mmol) and ethyl isothiocyanate (87 mg, 1 mmol) in absolute ethanol (20 mL) containing 0.5 mL of glacial acetic acid was refluxed for 6 h. The solution was allowed to reach ambient temperature and the resulting solid was collected by filtration and crystallized from 70% ethanol to provide the title compound 3.
Pale yellow powder (270 mg, 56%), m.p. 243–245 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.20 (t, J = 7.2 Hz, 3H, CH3), 3.47–3.57 (m, 2H, CH2), 3.67 (s, 6H, 2OCH3), 3.68 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 7.00 (s, 2H, arom.CH), 7.06 (s, 1H, olefinic CH), 7.12 (d, J = 8.5 Hz, 1H, arom.CH), 7.67 (t, J = 5.0 Hz, 1H, NH), 7.71 (d, J = 1.8 Hz, 1H, arom.CH), 7.75 (dd, J = 8.4, 1.9 Hz, 1H, arom.CH), 9.41 (s, 1H, NH), 10.27 (s, 1H, NH), 10.32 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.07 (CH3), 38.84 (CH2), 55.63 (OCH3), 55.67 (2OCH3), 55.70 (OCH3), 60.07 (), 107.40, 110.93, 111.19, 121.72, 124.96, 127.96, 128.22 (C-olefinic), 129.04 (C-olefinic), 138.17 (C-O), 148.17 (C-O), 152.11 (C-O), 152.61 (2C-O), 164.52 (C=O), 166.67 (C=O), 181.04 (C=S). Anal. Calcd. for C24H30N4O7S (518.58): C, 55.59; H, 5.83; N, 10.80. Found: C, 55.48; H, 6.02; N, 10.91.

4.1.4. General Procedure for the Synthesis of (Z)-3,4-Dimethoxy-N-(3-oxo-3-(2-(phenylcarbamothioyl)hydrazinyl)-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)benzamide (4)

A mixture of the hydrazide (Z)-1 (431 mg, 1 mmol) and phenyl isothiocyanate (135 mg, 1 mmol) in n-butanol (20 mL) was refluxed for 6 h. After the completion of the reaction, the solution was evaporated to dryness and the obtained solid residue was crystallized from DMF/H2O (1:1) to afford the title compound 4.
Pale yellow powder (357 mg, 63%), m.p. 226–228 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.68 (s, 6H, 2OCH3), 3.69 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 7.02 (s, 2H, arom CH), 7.11 (d, J = 2.2 Hz, 1H, arom CH), 7.13 (d, J = 2.1 Hz, 1H, arom CH), 7.15–7.20 (m, 1H, olefinic CH), 7.38 (t, J = 7.9 Hz, 2H, arom CH), 7.67 (d, J = 2.1 Hz, 1H, arom CH), 7.76 (dd, J = 8.5, 2.2 Hz, 1H, arom CH), 7.91–7.80 (m, 2H, arom CH), 9.37 (s, 1H, NH), 9.88 (s, 1H, NH), 10.45 (s, 1H, NH), 10.56 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.01 (OCH3), 56.18 (2OCH3), 56.20 (OCH3), 60.57 (OCH3), 107.98 (C-aromatic), 111.44 (C-aromatic), 111.67 (C-aromatic), 122.15 (C-aromatic), 123.94 (C-aromatic), 125.11 (C-aromatic), 125.36 (C-aromatic), 128.24 (C-aromatic), 128.60 (C-olefinic), 128.93 (C-aromatic), 129.44 (C-olefinic), 138.78 (C-O), 139.60 (C-aromatic), 148.71 (C-O), 152.66 (C-O), 153.13 (2C-O), 165.09 (C=O), 167.48 (C=O), 180.35 (C=S). Anal. Calcd. for C28H30N4O7S (566.63): C, 59.35; H, 5.34; N, 9.89. Found; 59.43; H, 5.42; N, 9.77.

4.1.5. General Procedure for the Synthesis of (Z)-N-(3-((2,5-Dioxopyrrolidin-1-yl)amino)-3-oxo-1-(3,4,5-trimethoxyphenyl)prop-1-en-2-yl)-3,4-dimethoxybenzamide (5)

A mixture of the hydrazide (Z)-1 (431 mg, 1 mmol) and succinic anhydride (100 mg, 1 mmol) in glacial acetic acid (20 mL) was heated to reflux for 5 h. Upon cooling, the reaction mixture was filtered off and crystallized from 70% ethanol to provide pure compound 5.
Pale yellow powder (354 mg, 69%), m.p. 190–192 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.38–2.44 (m, 2H, CH2), 2.44–2.49 (m, 2H, CH2), 3.62 (s, 6H, 2OCH3), 3.66 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.97 (s, 2H, arom CH), 7.06 (d, J = 8.5 Hz, 1H, arom CH), 7.25 (s, 1H, olefinic CH), 7.65 (d, J = 2.1 Hz, 1H, arom CH), 7.68 (dd, J = 8.5, 2.0 Hz, 1H, arom CH), 9.79 (s, 1H, NH), 10.01 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 28.63 (CH2), 29.36 (CH2), 56.08 (2OCH3), 56.11 (OCH3), 56.13 (OCH3), 60.51 (OCH3), 107.63 (C-aromatic), 111.25 (C-aromatic), 111.71 (C-aromatic), 121.90 (C-aromatic), 126.33 (C-aromatic), 128.63 (C-olefinic), 129.84 (C-olefinic), 130.63 (C-aromatic), 138.55 (C-O), 148.57 (C-O), 152.13 (C-O), 153.03 (C-O), 164.66 (C=O), 165.86 (C=O), 170.48 (C=O), 174.04 (C=O). Anal. Calcd. for C25H27N3O9 (513.50): C, 58.48; H, 5.30; N, 8.18. Found; 58.56; H, 5.22; N, 8.09.

4.2. Biological Study

4.2.1. MTT Cytotoxicity Assay

The MTT colorimetric assay was carried out to investigate the cytotoxic activity of the newly prepared cis-vinylamide derivatives 2a–g and 3–5 on the breast carcinoma (MCF-7) cell line. See Section S4.2.1 in the Supplementary Materials.

4.2.2. Tubulin Assay

The tubulin polymerization inhibition assay was performed for derivatives 2f, 3, and 5 compared with CA-4. See Section S4.2.2 in the Supplementary Materials.

4.2.3. Cell Cycle Analysis

Cell cycle analysis in MCF-7 cells was carried out by flow cytometry assay according to the manufacturer’s directions. See Section S4.2.3 in the Supplementary Materials.

4.2.4. Annexin V/FITC Staining Assay

The annexin V-FITC/PI dual staining assay in MCF-7 cells was carried out by flow cytometric analysis according to the manufacturer’s directions. See Section S4.2.4 in the Supplementary Materials.

4.2.5. Caspase 3 Assay

Caspase 3 was measured by ELISA analysis in MCF-7 cells according to the manufacturer’s directions. See Section S4.2.5 in the Supplementary Materials.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/sym14102088/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 2d; Figure S8: 13C-NMR spectrum of compound 2d; Figure S9: 1H-NMR spectrum of compound 2e; Figure S10: 13C-NMR spectrum of compound 2e; Figure S11: 1H-NMR spectrum of compound 2f; Figure S12: 13C-NMR spectrum of compound 2f; Figure S13: 1H-NMR spectrum of compound 2g; Figure S14: 13C-NMR spectrum of compound 2g; Figure S15: 1H-NMR spectrum of compound 3; Figure S16: 13C-NMR spectrum of compound 3; Figure S17: 1H-NMR spectrum of compound 4; Figure S18: 13C-NMR spectrum of compound 4; Figure S19: 1H-NMR spectrum of compound 5; Figure S20: 13C-NMR spectrum of compound 5; And detailed descriptions for Section 4.1.1 and Section 4.2.1, Section 4.2.2, Section 4.2.3, Section 4.2.4 and Section 4.2.5.

Author Contributions

Conceptualization, T.A.-W., O.A.A.A., G.A. and M.M.M.; methodology, L.S.A., M.A. and F.A.; data curation, S.H.Q., I.J., I.Z. and M.M.M.; software, A.A.S., F.G.E., E.F., T.A.-W. and H.A.E.A.; resources, O.A.A.A., S.A., F.A., I.J. and A.A.S.; supervision, F.G.E., L.S.A., G.A., M.A. and M.M.M.; funding acquisition, T.A.-W., E.F., S.H.Q. and I.J.; original draft preparation, M.M.M., T.A.-W. and H.A.E.A.; 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), and Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia & Deanship of Scientific Research, King Khalid University, KSA (Research group project number (RGP. 2/113/43).

Acknowledgments

The authors extend their appreciation to the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R25), and Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia & Deanship of Scientific Research, King Khalid University, KSA (Research group project number (RGP. 2/113/43). The authors are grateful to Saba Beigh, Department of Public Health, Faculty of Medical Science, Albaha University, Albaha, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

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Symmetry 14 02088 g001 550
Figure 1. Chemical structure of CA-4 I, some reported tubulin polymerization inhibitor II–V, and N,N-dimethyl thiosemicarbazone VI as anticancer agents.
Figure 1. Chemical structure of CA-4 I, some reported tubulin polymerization inhibitor II–V, and N,N-dimethyl thiosemicarbazone VI as anticancer agents.
Symmetry 14 02088 g001
Symmetry 14 02088 g002 550
Figure 2. Design strategy of the prepared cis-vinylamide derivatives 2a–g and 3–5.
Figure 2. Design strategy of the prepared cis-vinylamide derivatives 2a–g and 3–5.
Symmetry 14 02088 g002
Symmetry 14 02088 sch001 550
Scheme 1. Synthetic route for the synthesis of the target cis-vinylamide derivatives 2a–g and 3–5. Reagents and reaction conditions: (i) respective aromatic ketone, EtOH, AcOH; (ii) N-ethyl isothiocyanate, EtOH; (iii) N-phenyl isothiocyanate, n-butanol; (iv) succinic anhydride, AcOH.
Scheme 1. Synthetic route for the synthesis of the target cis-vinylamide derivatives 2a–g and 3–5. Reagents and reaction conditions: (i) respective aromatic ketone, EtOH, AcOH; (ii) N-ethyl isothiocyanate, EtOH; (iii) N-phenyl isothiocyanate, n-butanol; (iv) succinic anhydride, AcOH.
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Figure 3. Tubulin polymerization inhibition percentage induced by cis-vinylamide compounds 2f, 3, and 5 compared to CA-4 at their IC50 concentration (μM).
Figure 3. Tubulin polymerization inhibition percentage induced by cis-vinylamide compounds 2f, 3, and 5 compared to CA-4 at their IC50 concentration (μM).
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Figure 4. N-ethyl thiosemicarbazide 3 induced G2/M phase arrest in MCF-7 cells. (A) MCF-7 cells were treated with the 1.04 μM of N-ethyl thiosemicarbazide 3 for 48 h. Cellular cycle analysis was quantized by flow cytometry analysis using image-based cytometry. (B) The percentage of cells in different phases was quantized.
Figure 4. N-ethyl thiosemicarbazide 3 induced G2/M phase arrest in MCF-7 cells. (A) MCF-7 cells were treated with the 1.04 μM of N-ethyl thiosemicarbazide 3 for 48 h. Cellular cycle analysis was quantized by flow cytometry analysis using image-based cytometry. (B) The percentage of cells in different phases was quantized.
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Figure 5. N-ethyl thiosemicarbazide 3 induced apoptosis in MCF-7 cells. (A) MCF-7 cells were treated with 1.04 μM of N-ethyl thiosemicarbazide 3 for 48 h. Cellular apoptosis was quantized by annexin V-FITC/PI double staining assay using image-based cytometry. (B) The quantification of MCF-7 cell apoptosis.
Figure 5. N-ethyl thiosemicarbazide 3 induced apoptosis in MCF-7 cells. (A) MCF-7 cells were treated with 1.04 μM of N-ethyl thiosemicarbazide 3 for 48 h. Cellular apoptosis was quantized by annexin V-FITC/PI double staining assay using image-based cytometry. (B) The quantification of MCF-7 cell apoptosis.
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Figure 6. Graphical representation for the effect of N-ethyl thiosemicarbazide molecule 3 on the level of caspase 3 in MCF-7 cells for 48 h.
Figure 6. Graphical representation for the effect of N-ethyl thiosemicarbazide molecule 3 on the level of caspase 3 in MCF-7 cells for 48 h.
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Table
Table 1. Cytotoxic screening of the tested cis-vinylamide derivatives 2a–g and 3–5. Data expressed as mean ± SD.
Table 1. Cytotoxic screening of the tested cis-vinylamide derivatives 2a–g and 3–5. Data expressed as mean ± SD.
Comp NoIC50 Value (μM)
MCF-7
2a19.43 ± 1.02
2b27.78 ± 1.43
2c25.41 ± 1.07
2d6.93 ± 0.33
2e8.45 ± 0.53
2f3.22 ± 0.40
2g4.82 ± 0.69
31.04 ± 0.13
44.11 ± 0.49
51.81 ± 0.15
CA-40.67 ± 0.08
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Al-Warhi, T.; Alqahtani, L.S.; Alsharif, G.; Abualnaja, M.; Abu Ali, O.A.; Qahl, S.H.; Althagafi, H.A.E.; Alharthi, F.; Jafri, I.; Elsaid, F.G.; et al. Design, Synthesis, and Investigation of Cytotoxic Activity of cis-Vinylamide-Linked Combretastatin Analogues as Potential Anticancer Agents. Symmetry 2022, 14, 2088. https://doi.org/10.3390/sym14102088

AMA Style

Al-Warhi T, Alqahtani LS, Alsharif G, Abualnaja M, Abu Ali OA, Qahl SH, Althagafi HAE, Alharthi F, Jafri I, Elsaid FG, et al. Design, Synthesis, and Investigation of Cytotoxic Activity of cis-Vinylamide-Linked Combretastatin Analogues as Potential Anticancer Agents. Symmetry. 2022; 14(10):2088. https://doi.org/10.3390/sym14102088

Chicago/Turabian Style

Al-Warhi, Tarfah, Leena S. Alqahtani, Ghadi Alsharif, Matokah Abualnaja, Ola A. Abu Ali, Safa H. Qahl, Hussam Awwadh E. Althagafi, Fahad Alharthi, Ibrahim Jafri, Fahmy G. Elsaid, and et al. 2022. "Design, Synthesis, and Investigation of Cytotoxic Activity of cis-Vinylamide-Linked Combretastatin Analogues as Potential Anticancer Agents" Symmetry 14, no. 10: 2088. https://doi.org/10.3390/sym14102088

APA Style

Al-Warhi, T., Alqahtani, L. S., Alsharif, G., Abualnaja, M., Abu Ali, O. A., Qahl, S. H., Althagafi, H. A. E., Alharthi, F., Jafri, I., Elsaid, F. G., Shati, A. A., Aloufi, S., Fayad, E., Zaki, I., & Morcoss, M. M. (2022). Design, Synthesis, and Investigation of Cytotoxic Activity of cis-Vinylamide-Linked Combretastatin Analogues as Potential Anticancer Agents. Symmetry, 14(10), 2088. https://doi.org/10.3390/sym14102088

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