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Article

Design, Synthesis and Cytotoxicity Screening of New Thiazole Derivatives as Potential Anticancer Agents through VEGFR-2 Inhibition

by
Tarfah Al-Warhi
1,
Matokah Abualnaja
2,
Ola A. Abu Ali
3,
Najiah M. Alyamani
4,
Fahmy G. Elsaid
5,6,
Ali A. Shati
5,
Sarah Albogami
7,
Eman Fayad
7,
Ali H. Abu Almaaty
8,
Khaled O. Mohamed
9,
Wael M. Alamoudi
10 and
Islam Zaki
11,*
1
Department of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia
2
Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah Al Mukarrama 24381, Saudi Arabia
3
Department of Chemistry, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
4
Biology Department, College of Science, University of Jeddah, Jeddah 23218, Saudi Arabia
5
Biology Department, Science, College, King Khalid University, Abha 61421, Saudi Arabia
6
Zoology Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
7
Department of Biotechnology, Faculty of Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
8
Zoology Department, Faculty of Science, Port Said University, Port Said 42526, Egypt
9
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
10
Department of Biology, Faculty of Applied Science, Umm Al-Qura University, Makkah Al Mukarrama 24382, Saudi Arabia
11
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt
 
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Author to whom correspondence should be addressed.
Symmetry 2022, 14(9), 1814; https://doi.org/10.3390/sym14091814
Submission received: 5 August 2022 / Revised: 25 August 2022 / Accepted: 26 August 2022 / Published: 1 September 2022
(This article belongs to the Section Chemistry: Symmetry/Asymmetry)
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Abstract

:
Z-configurated isomers are kinetically preferred molecules. Compounds with Z-configuration are contained in many natural products, biologically active compounds and as synthons for organic synthesis. Two series of new thiazole-based analogs were synthesized from appropriate starting materials hydrazinecarbothioamide derivatives (Z)-2a,b to be evaluated for their inhibitory activity towards VEGFR-2. The prepared thiazole compounds 3a-5b were screened for their cytotoxic potency against the MDA-MB-231 breast cancer cell line and their percentage inhibition against VEGFR-2. Compound 4d exhibited good VEGFR-2 inhibitory activity. A DNA flow cytometry analysis was conducted, and compound 4d demonstrated cell cycle arrest at the G1 and G2/M phases of the cell cycle profile and an apoptosis-inducing effect by increasing the percentage of pre-G1 phase. Compound 4d was further evaluated for its apoptosis-inducing effect by studying the effect on mitochondrial membrane potential (MMP) and p53 activation. It was found to boost the level of p53 and reduce the level of MMP compared with the untreated control cells.

1. Introduction

Cancer is a major health problem with a complex pathogenesis, which threatens human life greatly [1,2]. Breast cancer is the second leading cause of cancer death for women [3]. A number of chemotherapeutic therapies have been developed to treat breast cancer, which include antiangiogenic agents and antimitotic agents [4,5]. Many natural compounds are known for their effective cancer treatment [6]. However, the complex chemical structure, difficult synthesis and formulation, in addition to the loss of oral availability, make these agents not suitable for the clinical treatment of breast cancer [7]. Therefore, there is considerable interest in the design and development of novel molecules that inhibit angiogenesis [8].
Tyrosine kinases are responsible for the transfer of phosphate group from ATP to the tyrosine residues in specific proteins inside a cell [9]. This phosphorylation leads to a change in many cellular functions [10]. They are considered as important mediators involved in signaling pathways [11]. Mutation can cause some tyrosine kinases to become continuously active, leading to the development of malignancy [12]. Vascular endothelial growth factor receptor-2 (VEGFR-2) is a receptor of tyrosine kinase and is one of the major regulators of physiological and pathological vascular endothelial cells growth [13]. VEGFR-2 is overexpressed in tumor vascular cells which promote vascular proliferation [14]. Some VEGFR-2-targeted inhibitor agents have been developed and used in experimental studies to monitor tumor angiogenesis and treatment efficacy [15]. The main structural characteristics of VEGFR-2 inhibitors consist of two main head and tail parts [16]. The head part is formed from a heterocyclic ring of five-, six- or even seven-membered heterorings such as furan, pyrazole, pyridine or benzodiazepine rings that bind the hydrophobic ATP-binding domain, and the tail part is bound to the allosteric site of the enzyme [17,18]. The tail part is sectioned into two main parts: a hydrophilic linker and a hydrophobic tail [19]. The hydrophilic linker group has an H-bond acceptor–donor pair (HBA/HBD) domain which can be urea, thiourea or carbohydrazide [20]. The hydrophobic tail consists of aryl groups capable of forming hydrophobic interactions [21]. Sorafenib I is a multitarget receptor tyrosine kinase inhibitor of several receptors mainly VEGFR-2 [22]. It represents a typical pharmacophoric feature of VEGFR-2 inhibitors where a central aryl ring attached to a pyridine ring via an ether linker as the head part and is also attached to the terminal aryl ring via a urea moiety [23].
Thiazoles are considered to be important chemical synthons found in a variety of pharmacologically active compounds [24]. They possess a wide range of biological activities as anticancer, antimicrobial and anti-inflammatory agents [25,26,27]. Hydrazinyl thaizole molecule II displayed potent antitumor activity against the C6 cell line with an IC50 value of 3.83 μM compared with that of cisplatin as a reference drug (IC50 = 12.67 μM) [28]. In addition, compound III possessing a 4-chlorophenylthiazole ring was found to inhibit the kinase activity of VEGFR-2 with an IC50 value of 51.09 nM compared with sorafenib’s IC50 of 51.41 nM [29]. Moreover, hydrazinyl thiazole derivative IV exhibited a promising cytotoxic activity via good EGFR-TK inhibition [30] (Figure 1).
In an attempt to discover potential VEGFR-2 inhibitors, a new series of thiazole-based derivatives 3a-5b was designed and synthesized as VEGFR-2 tyrosine kinase inhibitors (Figure 2). The prepared thiazole compounds were evaluated for their cytotoxic activity in vitro against the MDA-MB-231 breast cancer cell line. In order to investigate the mechanistic pathways of antiproliferative activity of the constructed thiazole compounds, the most potent cytotoxic compounds were chosen to perform extra investigations such as the VEGFR-2 inhibitory activity, cell cycle analysis and apoptosis related assays.

2. Results and Discussion

2.1. Chemistry

In Scheme 1, we report the synthetic route utilized to obtain the target thiazolyl derivatives 3a-5b. The treatment of oxazolones (Z)-1a,b with thiosemicarbazide in absolute ethanol under reflux gave the Z-configurated isomer hydrazine carbothioamides (Z)-2a,b. The hydrazine carbothioamides (Z)-2a,b were utilized as synthons for the synthesis of thiazole derivatives 3a-5b. The 1H-NMR spectrum of hydrazinecarbothioamide derivative (Z)-2a, for example, revealed the presence of five signals at δ 8.04, 8.64, 9.43, 10.03 and 10.32 ppm assigned to NH and NH2 protons. In addition, the 13C-NMR spectrum of compound (Z)-2a exhibited three signals at δ 164.17, 16.78 and 181.65 ppm attributed to two carbonyl (C=O) and thioamide (C=S) groups, respectively.
The thioamide group in the aforementioned hydrazinecarbothioamide derivatives (Z)-2a,b was subjected to a cyclization reaction through the reaction of the appropriate hydrazinecarbothioamide derivative (Z)-2a,b with a respective (un)substituted phenacyl bromide in pure ethanol and sodium acetate anhydrous (NaOAc) to yield the corresponding arylthiazolyl derivatives 3a-4e. The chemical structures of the prepared arylthiazolyl derivatives 3a-4e were elucidated on the basis of the 1H-NMR and 13C-NMR spectral data. The 1H-NMR spectra of 3-nitrophenylthiazolyl derivative 4d, as a representative example, displayed the appearance of a characteristic signal at δ 7.59 ppm corresponding t the H-4 thiazole ring as well as the presence of characteristic signals at δ 9.73, 9.90 and 10.62 ppm corresponding to three NH protons. In addition, the 1H-NMR spectrum of 3-nitrophenylthiazolyl derivative 4d showed the appearance of new proton peaks in the aromatic region corresponding to the phenyl protons of the 3-nitrophenylthiazolyl moiety. Moreover, the 13C-NMR spectrum of 3-nitrophenylthiazolyl derivative 4d showed the appearance of signal at δ 173.51 ppm due to the C2 thiazole carbon in addition to the presence of the characteristic signals of aromatic carbons related to phenyl carbons of the 3-nitrophenylthiazolyl moiety.
In addition, the thiazolyl ester derivatives 5a,b were synthesized by the reaction of appropriate hydrazinecarbothioamide (Z)-2a,b with ethyl 4-chloroacetoacetate in dry DMF and potassium carbonate anhydrous (K2CO3). The structure of the novel thiazolyl ester derivatives 5a,b was established using 1H-NMR and 13C-NMR spectral data. The 1H-NMR spectrum of 5b, for example, displayed a triplet signal at δ 1.19 ppm attributed to methyl (CH3) ester and a quartet signal at δ 3.75 ppm for the methylene (CH2) ester, in addition to a singlet signal at δ 3.54 ppm representing the methylene (CH2) group of the thiazole ring. Moreover, the 1H-NMR spectrum of compound 5b showed three characteristic NH signals at δ 9.41, 9.86 and 10.48 ppm. The 13C-NMR spectrum of compound 5b highlighted new upfield signals at δ 14.57 and 60.69 ppm for the OCH2CH3 ester carbons as well as a signal at δ 37.52 ppm attributed to the methylene (CH2) carbon of the thiazole ring. Furthermore, the 13C-NMR spectrum of compound 5b showed a signal at δ 173.03 ppm due to the carbonyl (C=O) ester carbon.

2.2. Biology

2.2.1. Cytotoxic Activity against MDA-MB-231 Breast Cancer Cell Line

All the newly synthesized aryl thiazole molecules 3a-4e and ester thiazole derivatives 5a,b were evaluated for their in vitro cytotoxic activity against the MDA-MB-231 breast cell line utilizing an MTT antiproliferative assay using sorafenib as the reference drug. The results were recorded as half-maximal inhibitory concentration (IC50) values. The results presented in Table 1 reveal that in general all the tested compounds possessed a moderate to good cytotoxic activity against the MDA-MB-231 cell line. On the basis of the obtained IC50 values against MDA-MB-231 cells, compounds 4-chlorophenylthiazolyl 4b, 4-bromophenylthiazolyl 4c and 3-nitrophenylthiazolyl 4d were found to be the most effective cytotoxic compounds against MDA-MB-231 cells with IC50 values of 3.52, 4.89 and 1.21 μM, respectively, in comparison to sorafenib (IC50 = 1.18 μM) (Table 1). Besides 4b, 4c and 4d, compounds 3a-3c and 4e showed a moderate cytotoxic activity against MDA-MB-231 cells. According to the current study, a comparative analysis between the synthesized compounds revealed that in general compounds containing a halogen or nitro substituent showed better cytotoxic activity than the unsubstituted phenyl 4a or 3-methoxy-substituted 4e derivatives. In addition, regarding the corresponding 3-nitrophenyl group, the results revealed that the replacement of the nitro group with another group resulted in a decrease of the cytotoxic activity.

2.2.2. Vascular Endothelium Growth Factor-2 (VEGFR-2) Inhibitory Activity

To cast light onto the mechanism of action of the prepared thiazole derivatives, the VEGFR-2 inhibitory activity of our designed compounds 4-chlorophenylthiazolyl 4b, 3-nitrophenylthiazolyl 4d and 3-methoxyphenylthiazolyl 4e was studied using an ELISA analysis. Sorafenib was utilized as a reference standard in the current study. The results in Figure 3 demonstrate that 4-chlorophenylthiazole 4b and 3-nitrophenylthiazole 4d showed a good inhibitory activity against VEGFR-2 which caused an 81.36 and 85.72% inhibition percentage compared with sorafenib (86.93%). These data pointed out that the presence of an electron-withdrawing group at the phenyl group of the arylthiazolyl moiety might be the reason for a better VEGFR-2 enzyme inhibitory activity. On the other hand, the VEGFR-2 percentage inhibition showed by 3-methoxyphenylthiazolyl derivative 4e was recorded as 38.22% in comparison with sorafenib. In conclusion, the results correlated the good cytotoxic activity of compounds 4b and 4d to their abilities to inhibit VEGFR-2 enzyme.

2.2.3. Cell Cycle Analysis of Compound 4d

To confirm the mechanism of action of these thiazole derivatives, a cell cycle analysis was performed. For this experiment, MDA-MB-231 cells were treated with compound 4d for 48 h at a concentration equal to its IC50 concentration (IC50 = 1.21 μM). The results showed that 3-nitrophenylthiazole molecule 4d displayed both G1 and G2/M phases accumulation, which strongly suggested that its cytotoxic activity was due to the effect on VEGFR-2 in MDA-MB-231 cells. Furthermore, the results demonstrated that 3-nitrophenylthiazolyl molecule 4d increased the accumulation of cells at both G1 and G2/M phases by 1.2- and 2.2-fold, respectively, compared to untreated controls, which indicated a cell cycle blockade at the G1 and G2 phases. Finally, the results suggested that the test 3-nitrophenylthiazole molecule 4d resulted in a cell cycle blockade at the G1 and G2/M phases in the treated MDA-MB-231 cells (Figure 4).

2.2.4. Annexin V/FITC Apoptosis Staining Assay

In order to study whether cell death induced by 3-nitrophenylthiazolyl derivative 4d treatment was related to apoptosis or necrosis, the Annexin V-FITC/PI staining assay was carried out using a FACS analysis. MDA-MB-231 cells were treated with compound 4d at a concentration equal to its IC50 concentration (IC50 = 1.21 μM) for 48 h. Thiazole derivative 4d caused a marked accumulation of annexin V positive cells and induced both early and late apoptosis compared to untreated control cells. Results presented in Figure 5 revealed that subjecting MDA-MB-231 cells to compound 4d led to 57.1-fold increase in early apoptosis and a 51.4-fold increase in the percentage of late apoptotic cells compared with the negative control cells. On the other hand, the percentage of necrotic cells was increased by fivefold compared to the negative control. Based on the findings of the cell cycle arrest and apoptosis assays, it appears that 3-nitrophenylthiazolyl molecule 4d could induce cellular apoptosis in MDA-MB-231 cells.

2.2.5. Mitochondrial Membrane Potential (MMP)

Apoptosis is an important potential target for therapeutic intervention [31]. Most cells are programmed to stop proliferation or kill themselves via the mitochondrial apoptotic pathway if survival signals are not regularly received from their environment due to the altering of the mitochondrial membrane potential (MMP) [32]. Studies have demonstrated that VEGFR-2 and tubulin inhibitors are capable of decreasing the level MMP resulting in a release of cytochrome c and other proapoptotic markers, which in turn leads to the activation of the intrinsic cellular apoptosis pathway [33]. To evaluate the effect of thiazole molecule 4d on the level of MMP, MDA-MB-231 cells were treated with 3-nitrophenylthiazolyl derivative 4d at its IC50 concentration (IC50 = 1.21 μM) for 48 h (Figure 6). The data showed that compound 4d reduced the level of MMP by almost 3.70-fold compared with negative control cells, indicating that 4d possessed the ability to induce intrinsic cellular apoptosis in MDA-MB-231 breast cancer cells.

2.2.6. In Vitro ELISA Measurement of the Level of p53

p53 is a transcription factor which regulates cell cycle growth and is very important in cancer because of its large number of downstream targets [34]. p53 can either inhibit cell cycle entry or induce apoptosis, thus it is a multifaceted tumor suppressor and plays a key role in the defense against cancer [35]. Moreover, p53 can decrease the synthesis of VEGF and inhibit tumor-cell-induced angiogenesis [36]. In order to study the mediated mechanistic pathway of apoptosis in treated cells with 3-nitrophenylthiazolyl derivative 4d, the concentration of p53 was measured using an ELISA. The data demonstrated that there was an increased level of p53 induction after treatment with 3-nitrophenylthiazolyl molecule 4d by 9.14-fold more than in the negative control group (Figure 7). According to the obtained data, the proapoptotic-inducing abilities of 3-nitrophenylthiazolyl molecule 4d were caused by p53 activation, which boosted the induction of cellular apoptosis through the mitochondrial apoptotic pathway.

2.2.7. Molecular Docking Study

Molecular docking simulation was performed with the VEGFR-2 crystal structure (PDB ID: 4ASD) by molecular operating environment MOE 2015.10. The molecular docking study was performed for 3-nitrophenylthiazolyl molecule 4d into the active site of VEGFR-2 with the aim to gain more insight on the binding mode of this compound. Docking results revealed that the docking score achieved by 3-nitrophenylthiazolyl molecule 4d was −12.18 kcal/mol. As can be seen in (Figure 8), the 3-nitrophenylthiazolyl 4d interacted with the carbonyl group (C=O) of the hydrazine group as H-bond acceptor with the key amino acid Arg 1027. As a hydrogen bond donor, it interacted with the nitro group (NO2) of the 3-nitrophenyl thiazolyl moiety with the key amino acid Cys 817. The phenyl group of the 3-nitrophenylthiazolyl moiety interacted as a hydrogen bond donor with Glu 818. Additionally, there was a hydrophobic interaction between the phenyl group of the 3-nitrophenylthiazolyl moiety with the hydrophobic side chains of the amino acid Glu 815. From the obtained results, molecular modeling studies of 3-nitrophenylthiazolyl derivative 4d revealed that the interaction result is in line with the obtained data against VEGFR-2 inhibition and justifies the observed chemicals findings.

3. Conclusions

In conclusion, two new series of synthetic thiazole-based molecules 3a-5b were designed and synthesized as anti-VEGFR-2 agents. The chemical structure of the prepared thiazole derivatives 3a-5b was elucidated on the basis of 1H-NMR and 13C-NMR spectra along with an elemental microanalysis. The prepared thiazole derivatives 3a-5b were screened for their cytotoxic potency against the MDA-MB-231 breast cancer cell line and their percentage inhibition against VEGFR-2. The cytotoxicity screening demonstrated that 4-chlorophenylthiazolyl 4b and 3-nitrophenylthiazolyl 4d derivatives displayed the highest cytotoxic activity with IC50 values of 3.52 and 1.21 μM, respectively, compared with sorafenib (IC50 = 1.18 μM) as a reference compound. 4-Chlorophenylthiazolyl 4b and 3-nitrophenylthiazolyl 4d derivatives showed good VEGFR-2 enzyme inhibition which was 81.36 and 85.72% inhibition compared to sorafenib (86.93% enzyme inhibition). A DNA flow cytometry analysis for the 3-nitrophenylthiazolyl 4d derivative demonstrated a cell cycle arrest at the G1 and G2/M phases by 1.23- and 2.20-fold, respectively, compared to the negative control group. Moreover, 3-nitrophenylthiazolyl 4d molecule showed proapoptotic activity by inducing a marked increase in the percentage of the pre-G1 phase by almost 23.80-fold in an apoptosis-staining study compared with the negative control group. The apoptosis mechanistic pathway of 3-nitrophenylthiazolyl derivative 4d indicated that it boosted the level of p53 by 9.14-fold as well as reduced the level of MMP by 3.70-fold compared with the control MDA-MB-231 cells.

4. Experimental Methods

4.1. Chemistry

4.1.1. General

Melting points were determined in open capillaries tube using Electrothermal Digital melting point apparatus and were uncorrected. 1H-NMR and 13C-NMR spectra were obtained with a Bruker 400 MHz DRX-Avance NMR spectrometer, peaks positions are given in ppm downfield from tetramethylsilane (TMS) as the internal standard. Elemental analyses were performed on Elementar, Vario El, Microanalytical unit, Cairo, Egypt and were found within ±0.4% of the theoretical values.

4.1.2. General Procedure for the Synthesis of (Z)-N-(3-(2-Carbamothioylhydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)arylamides (Z)-2a,b

A mixture of 2-aryl-4-(furan-2-ylmethylene)oxazol-5(4H)-one 1a,b (10 mmol) and thiosemicarbazide (12 mmol) in 20 mL of pure ethanol was heated to reflux with stirring for 6–8 h. The reaction mixture was concentrated under reduced pressure to remove the excess ethanol. The obtained solid residue was filtered, washed with ethanol, dried and purified by crystallization using ethanol/H2O (3:1) to obtain the title compound 2a,b.

(Z)-N-(3-(2-Carbamothioylhydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4-dimethoxybenzamide ((Z)-2a)

Pale yellow powder (231 mg, 59%), m.p. 184–186 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.85 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.62 (s, 1H, furan CH), 6.78 (s, 1H, furan CH), 7.01 (s, 1H, arom.CH), 7.11 (d, J = 8.5 Hz, 1H, arom.CH), 7.20 (s, 1H, olefinic CH), 7.62 (s, 1H, arom.CH), 7.81 (s, 1H, furan CH), 8.04 (s, 1H, NH), 8.64 (s, 1H, NH), 9.43 (s, 1H, NH), 10.03 (s, 1H, NH), 10.32 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.15 (OCH3), 56.19 (OCH3), 111.41 (C5 dimethoxybenzamide), 111.81 (C2 dimethoxybenzamide), 115.26 (C3 furan), 116.79 (C4 furan), 120.41 (C olefinic), 122.14 (C6 dimethoxybenzamide), 126.10 (C olefinic), 126.18 (C1 dimethoxybenzamide), 145.36 (C5 furan), 148.65 (C2 furan), 149.96 (C3 dimethoxybenzamide), 152.41 (C4 dimethoxybenzamide), 164.17 (C=O thiosemicarbazone), 166.78 (C=O dimethoxybenzamide), 181.65 (C=S). Anal. Calcd. for C17H18N4O5S (390.41): C, 52.30; H, 4.65; N, 14.35. Found: C, 52.44; H, 4.73; N, 14.18.

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

Orange powder (259 mg, 62%), m.p. 194–196 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.87 (s, 6H, 2OCH3), 6.62 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.81 (d, J = 3.4 Hz, 1H, furan CH), 7.05 (s, 1H, arom.CH), 7.13 (s, 1H olefinic CH), 7.37 (s, 2H, arom.CH), 7.83 (d, J = 1.5 Hz, 1H, furan CH), 8.04 (s, 1H, NH), 8.64 (s, 1H, NH), 9.45 (s, 1H, NH), 10.12 (s, 1H, NH), 10.32 (s, 1H, NH). Anal. Calcd. for C18H20N4O6S (420.44): C, 51.42; H, 4.79; N, 13.33. Found: C, 51.53; H, 4.71; N, 13.21.

4.1.3. General Procedure for the Synthesis of (Z)-N-(3-(2-(4-Arylthiazol-2-yl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)arylamides 3a-4e

A mixture of appropriate hydrazinecarbothioamide 2a,b (10 mmol), appropriate (un)substituted phenacyl bromide (10 mmol) and sodium acetate anhydrous (984 mg, 12 mmol) in 20 mL pure ethanol was heated to reflux for 2–3 h. The reaction progress was followed up by a TLC analysis. After completion of the reaction, the obtained solid residue was filtered, washed with 10 mL ethanol, dried and crystallized from ethanol/H2O (3:1) to give the title products 3a-4e.

(Z)-N-(3-(2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4-dimethoxybenzamide (3a)

White powder (382 mg, 67%), m.p. 218–220 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.85 (s, 6H, 2OCH3), 6.58–6.66 (m, 1H, furan CH), 6.78 (d, J = 3.4 Hz, 1H, furan CH), 7.10 (d, J = 8.4 Hz, 1H, arom.CH), 7.14 (s, 1H, olefinic CH), 7.32 (s, 1H, thiazole CH), 7.45 (d, J = 8.5 Hz, 2H, arom.CH), 7.65 (s, 1H, arom.CH), 7.70 (d, J = 8.5 Hz, 1H, furan CH), 7.81 (s, 1H, arom.CH), 7.86 (d, J = 8.4 Hz, 2H, arom.CH), 9.60 (s, 1H, NH), 9.79 (s, 1H, NH), 10.56 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.10 (OCH3), 56.14 (OCH3), 104.39 (C5 thiazole), 111.37 (C5 dimethoxybenzamide), 111.80 (C2 dimethoxybenzamide), 112.86 (C3 furan), 114.80 (C4 furan), 118.22 (C olefinic), 121.93 (C6 dimethoxybenzamide), 126.43 (C olefinic), 126.95 (C1 dimethoxybenzamide), 127.72 (C2,6 chlorophenyl), 129.02 (C3,5 chlorophenyl), 132.27 (C1 chlorophenyl), 134.06 (C4 chlorophenyl), 145.21 (C5 furan), 148.66 (C2 furan), 149.70 (C4 thiazole), 149.97 (C3 dimethoxybenzamide), 152.16 (C4 dimethoxybenzamide), 165.37 (C=O hydrazide), 165.76 (C=O dimethoxybenzamide), 173.20 (C2 thiazole). Anal. Calcd. for C25H21BrN4O5S (569.43): C, 52.73; H, 3.72; N, 9.84. Found: C, 52.66; H, 3.78; N, 9.98.

(Z)-N-(3-(2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4-dimethoxybenzamide (3b)

White powder (374 mg, 71%), m.p. 215–217 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.85 (s, 6H, 2OCH3), 6.61 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.78 (d, J = 3.4 Hz, 1H, furan CH), 7.10 (d, J = 8.5 Hz, 1H, arom.CH), 7.14 (s, 1H, olefinic CH), 7.33 (s, 1H, thiazole CH), 7.58 (d, J = 8.6 Hz, 2H, arom.CH), 7.65 (d, J = 1.9 Hz, 1H, arom.CH), 7.70 (dd, J = 8.4, 1.9 Hz, 1H, furan CH), 7.78 (s, 1H, arom.CH), 7.81 (d, J = 8.4 Hz, 2H, arom.CH), 9.60 (s, 1H, NH), 9.79 (s, 1H, NH), 10.56 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.10 (OCH3), 56.14 (OCH3), 104.49 (C5 thiazole), 111.37 (C5 dimethoxybenzamide), 111.80 (C2 dimethoxybenzamide), 112.86 (C3 furan), 114.80 (C4 furan), 118.22 (C olefinic), 120.86 (C4 bromophenyl), 121.93 (C6 dimethoxybenzamide), 126.43 (C olefinic), 126.95 (C1 dimethoxybenzamide), 128.04 (C2,6 bromophenyl), 131.93 (C3,5 bromophenyl), 134.40 (C1 bromophenyl), 145.22 (C5 furan), 148.66 (C2 furan), 149.74 (C4 thiazole), 149.97 (C3 dimethoxybenzamide), 152.16 (C4 dimethoxybenzamide), 165.37 (C=O hydrazide), 165.76 (C=O dimethoxybenzamide), 173.20 (C2 thiazole). Anal. Calcd. for C25H21ClN4O5S (524.98): C, 57.20; H, 4.03; N, 10.67. Found: C, 57.33; H, 3.95; N, 10.76.

(Z)-N-(1-(Furan-2-yl)-3-(2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazinyl)-3-oxoprop-1-en-2-yl)-3,4-dimethoxybenzamide (3c)

Pale yellow powder (318 mg, 59%), m.p. 222–224 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.85 (s, 6H, 2OCH3), 6.62 (dd, J = 3.3, 1.8 Hz, 1H, furan CH), 6.79 (d, J = 3.4 Hz, 1H, furan CH), 7.10 (d, J = 8.5 Hz, 1H, arom.CH), 7.15 (s, 1H, olefinic CH), 7.59 (s, 1H, thiazole CH), 7.66 (s, 1H, arom.CH), 7.70 (t, J = 8.0 Hz, 2H, arom.CH), 7.80–7.83 (m, 1H, arom.CH), 8.14 (dd, J = 8.1, 1.7 Hz, 1H, furan CH), 8.29 (d, J = 7.8 Hz, 1H, arom.CH), 8.65 (s, 1H, arom.CH), 9.72 (s, 1H, NH), 9.81 (s, 1H, NH), 10.61 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.10 (OCH3), 56.14 (OCH3), 106.36 (C5 thiazole), 111.37 (C5 dimethoxybenzamide), 111.80 (C2 dimethoxybenzamide), 112.86 (C3 furan), 114.85 (C4 furan), 118.21 (C olefinic), 120.39 (C5 nitrophenyl), 121.94 (C6 dimethoxybenzamide), 122.42 (C4 nitrophenyl), 126.42 (C olefinic), 126.91 (C1 dimethoxybenzamide), 130.66 (C2 nitrophenyl), 132.08 (C6 nitrophenyl), 136.71 (C1 nitrophenyl), 145.25 (C5 furan), 148.53 (C3 nitrophenyl), 148.66 (C2 furan), 148.76 (C4 thiazole), 149.97 (C3 dimethoxybenzamide), 152.17 (C4 dimethoxybenzamide), 165.41 (C=O hydrazide), 165.78 (C=O dimethoxybenzamide), 173.56 (C2 thiazole). Anal. Calcd. for C25H21N5O7S (535.53): C, 56.07; H, 3.95; N, 13.08. Found: C, 55.88; H, 4.03; N, 13.19.

(Z)-N-(1-(Furan-2-yl)-3-oxo-3-(2-(4-phenylthiazol-2-yl)hydrazinyl)prop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4a)

Buff powder (283 mg, 65%), m.p. 197–199 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.63 (s, 1H, furan CH), 6.80 (d, J = 2.8 Hz, 1H, furan CH), 7.18 (s, 1H, olefinic CH), 7.25 (s, 1H, thiazole CH), 7.29 (t, J = 7.3 Hz, 1H, arom.CH), 7.38 (d, J = 7.8 Hz, 2H, arom.CH), 7.41 (s, 2H, arom.CH), 7.83 (s, 2H, arom.CH), 7.85 (s, 1H, furan CH), 9.56 (s, 1H, NH), 9.88 (s, 1H, NH), 10.57 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.55 (2OCH3), 60.59 (OCH3), 103.63 (C5 thiazole), 106.08 (C2,6 trimethoxybenzamide), 112.90 (C3 furan), 115.12 (C4 furan), 118.46 (C olefinic), 126.01 (C2,6 phenyl), 126.62 (C olefinic), 127.90 (C4 phenyl), 129.01 (C3,5 phenyl), 129.31 (C1 trimethoxybenzamide), 135.19, (C1 phenyl) 140.83 (C4 trimethoxybenzamide), 145.39 (C5 furan), 149.88 (C2 furan), 150.96 (C4 thiazole), 153.02 (C3,5 trimethoxybenzamide), 165.27 (C=O hydrazide), 165.75 (C=O trimethoxybenzamide), 173.03 (C2 thiazole). 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.

(Z)-N-(3-(2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4b)

Yellow powder (383 mg, 69%), m.p. 218–220 °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), 6.81 (d, J = 3.4 Hz, 1H, furan CH), 7.18 (s, 1H, olefinic CH), 7.32 (s, 1H, thiazole CH), 7.41 (s, 2H, arom.CH), 7.45 (d, J = 8.6 Hz, 2H, arom.CH), 7.83 (d, J = 1.4 Hz, 1H, furan CH), 7.86 (d, J = 8.6 Hz, 2H, arom.CH), 9.61 (s, 1H, NH), 9.88 (s, 1H, NH), 10.56 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.55 (2OCH3), 60.58 (OCH3), 104.42 (C5 thiazole), 106.10 (C2,6 trimethoxybenzamide), 112.90 (C3 furan), 115.13 (C4 furan), 118.45 (C olefinic), 126.63 (C olefinic), 127.72 (C2,6 chlorophenyl), 129.02 (C3,5 chlorophenyl), 129.31 (C1 trimethoxybenzamide), 132.27 (C1 chlorophenyl), 134.05 (C4 chlorophenyl), 140.84 (C4 trimethoxybenzamide), 145.39 (C5 furan), 149.70 (C2 furan), 149.88 (C4 thiazole), 153.02 (C3,5 trimethoxybenzamide), 165.25 (C=O hydrazide), 165.71 (C=O trimethoxybenzamide), 173.17 (C2 thiazole). Anal. Calcd. for C26H23ClN4O6S (555.00): C, 56.27; H, 4.18; N, 10.09. Found: C, 56.39; H, 4.23; N, 9.93.

(Z)-N-(3-(2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazinyl)-1-(furan-2-yl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4c)

Pale yellow powder (413 mg, 69%), m.p. 216–218 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.87 (s, 6H, 2OCH3), 6.62 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.80 (d, J = 3.0 Hz, 1H, furan CH), 7.17 (s, 1H, olefinic CH), 7.33 (s, 1H, thiazole CH), 7.41 (s, 2H, arom.CH), 7.58 (d, J = 8.6 Hz, 2H, arom.CH), 7.77–7.81 (m, 2H, arom.CH), 7.83 (d, J = 1.3 Hz, 1H, furan CH), 9.61 (s, 1H, NH), 9.88 (s, 1H, NH), 10.57 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.55 (2OCH3), 60.58 (OCH3), 104.50 (C5 thiazole), 106.09 (C2,6 trimethoxybenzamide), 112.89 (C3 furan), 115.12 (C4 furan), 118.54 (C olefinic), 120.86 (C4 bromophenyl), 126.64 (C olefinic), 128.03 (C2,6 bromophenyl), 129.31 (C1 trimethoxybenzamide), 131.93 (C3,5 bromophenyl), 134.40 (C1 bromophenyl), 140.84 (C4 trimethoxybenzamide), 145.38 (C5 furan), 149.74 (C2 furan), 149.89 (C4 thiazole), 153.02 (C3,5 trimethoxybenzamide), 165.28 (C=O hydrazide), 165.70 (C=O trimethoxybenzamide), 173.16 (C2 thiazole). Anal. Calcd. for C26H23BrN4O6S (599.45): C, 52.09; H, 3.87; N, 9.35. Found: C, 51.97; H, 3.96; N, 9.43.

(Z)-N-(1-(Furan-2-yl)-3-(2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazinyl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4d)

Buff powder (374 mg, 66%), m.p. 214–216 °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, 1.8 Hz, 1H, furan CH), 6.81 (d, J = 3.3 Hz, 1H, furan CH), 7.18 (s, 1H, olefinic CH), 7.41 (s, 2H, arom.CH), 7.59 (s, 1H, thiazole CH), 7.70 (t, J = 8.0 Hz, 1H, arom.CH), 7.80–7.88 (m, 1H, arom.CH), 8.10–8.20 (m, 1H, arom.CH), 8.29 (d, J = 7.9 Hz, 1H, furan CH), 8.66 (s, 1H, arom.CH), 9.73 (s, 1H, NH), 9.90 (s, 1H, NH), 10.62 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 56.55 (2OCH3), 60.59 (OCH3), 106.10 (C2,6 trimethoxybenzamide), 106.37 (C5 thiazole), 112.90 (C3 furan), 115.17 (C4 furan), 118.43 (C olefinic), 120.43 (C5 nitrophenyl), 122.43 (C4 nitrophenyl), 126.59 (C olefinic), 129.30 (C1 trimethoxybenzamide), 130.66 (C2 nitrophenyl), 132.07 (C6 nitrophenyl), 136.70 (C1 nitrophenyl), 140.85 (C4 trimethoxybenzamide), 145.42 (C5 furan), 148.52 (C3 nitrophenyl), 148.75 (C2 furan), 149.88 (C4 thiazole), 153.02 (C3,5 trimethoxybenzamide), 165.27 (C=O hydrazide), 165.79 (C=O trimethoxybenzamide), 173.51 (C2 thiazole). Anal. Calcd. for C26H23N5O8S (565.55): C, 55.22; H, 4.10; N, 12.38. Found: C, 55.11; H, 4.19; N, 12.31.

(Z)-N-(1-(Furan-2-yl)-3-(2-(4-(3-methoxyphenyl)thiazol-2-yl)hydrazinyl)-3-oxoprop-1-en-2-yl)-3,4,5-trimethoxybenzamide (4e)

Yellow powder (333 mg, 61%), m.p. 231–233 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 3.75 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.60–6.66 (m, 1H, furan CH), 6.80 (d, J = 3.1 Hz, 1H, furan CH), 6.86 (dd, J = 8.2, 2.2 Hz, 1H, arom.CH), 7.18 (s, 1H, olefinic CH), 7.28 (s, 1H, thiazole CH), 7.31 (d, J = 7.9 Hz, 1H, arom.CH), 7.42 (d, J = 9.7 Hz, 4H, arom.CH), 7.83 (s, 1H, furan CH), 9.57 (s, 1H, NH), 9.87 (s, 1H, NH), 10.57 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 55.52 (OCH3), 56.55 (2OCH3), 60.59 (OCH3), 104.03 (C5 thiazole), 106.04 (C2,6 trimethoxybenzamide), 111.37 (C2 methoxyphenyl), 112.90 (C3 furan), 113.63 (C4 methoxyphenyl), 115.10 (C4 furan), 118.42 (C olefinic), 118.46 (C6 methoxyphenyl), 126.64 (C olefinic), 129.32 (C1 trimethoxybenzamide), 130.05 (C5 methoxyphenyl), 136.59 (C1 methoxyphenyl), 140.83 (C4 trimethoxybenzamide), 145.37 (C5 furan), 149.89 (C2 furan), 150.79 (C4 thiazole), 152.99 (C3,5 trimethoxybenzamide), 159.94 (C3 methoxyphenyl), 165.28 (C=O hydrazide), 165.71 (C=O trimethoxybenzamide), 172.90 (C2 thiazole). Anal. Calcd. for C27H26N4O7S (550.58): C, 58.90; H, 4.76; N, 10.18. Found: C, 59.04; H, 4.88; N, 10.07.

4.1.4. General Procedure for the Synthesis of (E)-Ethyl 2-(2-(2-((Z)-2-(arylamido)-3-(furan-2-yl)acryloyl)hydrazinyl)thiazol-4(5H)-ylidene)acetates 5a,b

A mixture of appropriate hydrazinecarbothioamide 2a,b (10 mmol), ethyl 4-chloroacetoacetate (1.35 mL, 10 mmol) and potassium carbonate anhydrous (164 mg, 12 mmol) was stirred in dry DMF (20 mL) for 12 h. After completion of the reaction as monitored by a TLC analysis, the solution was poured onto crushed ice, filtered off, dried and crystallized from DMF/ethanol (1:1) to furnish the title compound 5a,b.

(E)-Ethyl 2-(2-(2-((Z)-2-(3,4-dimethoxybenzamido)-3-(furan-2-yl)acryloyl)hydrazinyl)thiazol-4(5H)-ylidene)acetate (5a)

Yellow powder (267 mg, 53%), m.p. 211–213 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.20 (t, J = 7.1 Hz, 3H, CH3), 3.54 (s, 2H, CH2 thiazole), 3.85 (s, 6H, 2OCH3), 4.08 (q, J = 7.1 Hz, 2H, CH2), 6.57 (s, 1H, olefinic CH), 6.59–6.62 (m, 1H, furan CH), 6.76 (d, J = 3.3 Hz, 1H, furan CH), 7.09 (d, J = 8.5 Hz, 1H, arom.CH), 7.11–7.15 (m, 1H, olefinic CH), 7.64 (s, 1H, arom.CH), 7.69 (d, J = 8.4 Hz, 1H, arom.CH), 7.79 (s, 1H, furan CH), 9.40 (s, 1H, NH), 9.79 (d, J = 5.9 Hz, 1H, NH), 10.50 (d, J = 5.9 Hz, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.56 (OCH2CH3), 37.51 (C5 thiazole), 55.38 (OCH3), 56.14 (OCH3), 60.70 (OCH2CH3), 105.54 (C olefinic), 111.35 (C5 dimethoxybenzamide), 111.80 (C2 dimethoxybenzamide), 112.84 (C3 furan), 114.72 (C4 furan), 118.22 (C olefinic), 121.93 (C6 dimethoxybenzamide), 126.43 (C1 dimethoxybenzamide), 126.93 (C olefinic), 145.15 (C5 furan), 145.54 (C2 furan), 148.64 (C2 thiazole), 149.96 (C3 dimethoxybenzamide), 152.14 (C4 dimethoxybenzamide), 165.30 (C4 thiazole), 165.73 (C=O hydrazide), 170.51 (C=O dimethoxybenzamide), 173.06 (C=O ester). Anal. Calcd. for C23H24N4O7S (500.52): C, 55.19; H, 4.83; N, 11.19. Found: C, 55.10; H, 4.91; N, 11.34.

(E)-Ethyl 2-(2-(2-((Z)-3-(furan-2-yl)-2-(3,4,5-trimethoxybenzamido)acryloyl)hydrazinyl)thiazol-4(5H)-ylidene)acetate (5b)

White powder (371 mg, 70%), m.p. 216–218 °C. 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.19 (t, J = 7.1 Hz, 3H, CH3), 3.54 (s, 2H, CH2 thiazole), 3.75 (s, 3H, OCH3), 3.87 (s, 6H, 2OCH3), 4.08 (q, J = 7.1 Hz, 2H, CH2), 6.57 (s, 1H, olefinic CH), 6.62 (dd, J = 3.4, 1.8 Hz, 1H, furan CH), 6.79 (d, J = 3.4 Hz, 1H, furan CH), 7.14 (s, 1H, olefinic CH), 7.39 (s, 2H, arom.CH), 7.82 (d, J = 1.4 Hz, 1H, furan CH), 9.41 (s, 1H, NH), 9.86 (s, 1H, NH), 10.48 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6, δ ppm): 14.57 (OCH2CH3), 37.52 (C5 thiazole), 56.54 (2OCH3), 60.58 (OCH3), 60.69 (OCH2CH3), 105.56 (C olefinic), 106.07 (C2,6 trimethoxybenzamide), 112.88 (C3 furan), 115.03 (C4 furan), 118.38 (C olefinic), 126.66 (C olefinic), 129.31 (C1 trimethoxybenzamide), 140.82 (C4 trimethoxybenzamide), 145.34 (C5 furan), 145.59 (C2 furan), 149.88 (C2 thiazole), 153.01 (C3,5 trimethoxybenzamide), 165.15 (C4 thiazole), 165.66 (C=O hydrazide), 170.50 (C=O trimethoxybenzamide), 173.03 (C=O ester). Anal. Calcd. for C24H26N4O8S (530.55): C, 54.33; H, 4.94; N, 10.56. Found: C, 54.42; H, 5.02; N, 10.44.

4.2. Biological Study

4.2.1. MTT Cytotoxicity Assay

An MTT colorimetric assay was carried out to investigate the impact of the newly synthesized thiazolyl derivatives 3a-5b on a breast carcinoma (MDA-MB-231) cell line. See Section S4.2.1 in Supplementary Materials.

4.2.2. VEGFR-2 Inhibition Assay

A VEGFR-2 inhibition assay was performed for thiazolyl derivatives 4b, 4d and 4d compared with sorafenib according to a previously reported method [37]. See Section S4.2.2 in Supplementary Materials.

4.2.3. Cell Cycle Analysis

A cell cycle analysis in MDA-MB-231 cells was carried out by a FACS analysis according to the manufacturer’s directions. See Section S4.2.3 in Supplementary Materials.

4.2.4. Annexin V/FITC Staining Assay

An annexin V/FITC double staining analysis in MDA-MB-231 cells was carried out by a FACS analysis according to the manufacturer’s directions. See Section S4.2.4 in Supplementary Materials.

4.2.5. Mitochondrial Membrane Potential

The MMP was measured by a flow cytometry analysis in MDA-MB-231 cells according to the manufacturer’s directions. See Section S4.2.5 in Supplementary Materials.

4.2.6. Effect on p53, Bax and Bcl-2

Apoptotic marker p53 was measured in MDA-MB-231 cells using an ELISA according to the manufacturer’s directions. See Section S4.2.6 in Supplementary Materials.

4.2.7. Molecular Docking Study

The docking study was carried out using molecular operating environment (MOE 2015.10). See Section S4.2.7 in Supplementary Materials.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/sym14091814/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: 1H-NMR spectrum of compound 3a, Figure S5: 13C-NMR spectrum of compound 3a, Figure S6: 1H-NMR spectrum of compound 3b, Figure S7: 13C-NMR spectrum of compound 3b, Figure S8: 1H-NMR spectrum of compound 3c, Figure S9: 13C-NMR spectrum of compound 3c, Figure S10: 1H-NMR spectrum of compound 4a, Figure S11: 13C-NMR spectrum of compound 4a, Figure S12: 1H-NMR spectrum of compound 4b, Figure S13: 13C-NMR spectrum of compound 4b, Figure S14: 1H-NMR spectrum of compound 4c, Figure S15: 13C-NMR spectrum of compound 4c, Figure S16: 1H-NMR spectrum of compound 4d, Figure S17: 13C-NMR spectrum of compound 4d, Figure S18: 1H-NMR spectrum of compound 4e, Figure S19: 13C-NMR spectrum of compound 4e, Figure S20: 1H-NMR spectrum of compound 5a, Figure S21: 13C-NMR spectrum of compound 5a, Figure S22: 1H-NMR spectrum of compound 5b, Figure S23: 13C-NMR spectrum of compound 5b.

Author Contributions

Conceptualization, I.Z., E.F., M.A., N.M.A. and T.A.-W.; methodology, O.A.A.A., F.G.E., A.A.S., S.A. and K.O.M.; data curation, T.A.-W., E.F., S.A., A.H.A.A., W.M.A., O.A.A.A. and I.Z.; software, F.G.E., K.O.M., W.M.A. and I.Z.; resources, T.A.-W., N.M.A., F.G.E., A.A.S., S.A. and I.Z.; supervision, K.O.M. and I.Z.; funding acquisition, T.A.-W., M.A., E.F., F.G.E., 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, King Khalid University, KSA (research group project number (RGP. 2/113/43).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are available within the manuscript.

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, King Khalid University, KSA (research group project number (RGP. 2/113/43). The authors are grateful to Taif University for carrying out the cytotoxicity testing. The authors thank all members of the department of biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia for carrying out VEGFR-2 inhibition assay.

Conflicts of Interest

The authors declare no conflict of interest.

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Symmetry 14 01814 g001 550
Figure 1. Chemical structure of sorafenib (I) and some bioactive thiazole derivatives (II–IV).
Figure 1. Chemical structure of sorafenib (I) and some bioactive thiazole derivatives (II–IV).
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Figure 2. Design strategy of the prepared thiazole-based compounds 3a-5b.
Figure 2. Design strategy of the prepared thiazole-based compounds 3a-5b.
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Symmetry 14 01814 sch001 550
Scheme 1. Synthesis of the target compounds 3a-5b. Reagents and reaction condition: (i) thiosemicarbazide, EtOH; (ii) respective (un)substituted phenacyl bromide, NaOAc and EtOH; (iii) ethyl 4-chloroacetoacetate, K2CO3 and DMF.
Scheme 1. Synthesis of the target compounds 3a-5b. Reagents and reaction condition: (i) thiosemicarbazide, EtOH; (ii) respective (un)substituted phenacyl bromide, NaOAc and EtOH; (iii) ethyl 4-chloroacetoacetate, K2CO3 and DMF.
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Figure 3. Compounds 4b, 4d and 4e inhibited VEGFR-2 in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 3.52, 1.21, 13.33 and 1.18 μM of compounds 4b, 4d and 4e and sorafenib, respectively, and VEGFR-2 concentration was determined by ELISA analysis. (B) VEGFR-2 inhibition percentage induced by compounds 4b, 4d and 4e compared to sorafenib at their IC50 concentration (μM).
Figure 3. Compounds 4b, 4d and 4e inhibited VEGFR-2 in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 3.52, 1.21, 13.33 and 1.18 μM of compounds 4b, 4d and 4e and sorafenib, respectively, and VEGFR-2 concentration was determined by ELISA analysis. (B) VEGFR-2 inhibition percentage induced by compounds 4b, 4d and 4e compared to sorafenib at their IC50 concentration (μM).
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Figure 4. Compound 4d induced both G1 and G2/M phase arrest in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 1.21 μM of compound 4d for 48 h. Cell cycle analysis was quantized by PI and FACS analysis using image-based cytometry. (B) The percentage of cells in different phases was quantified.
Figure 4. Compound 4d induced both G1 and G2/M phase arrest in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 1.21 μM of compound 4d for 48 h. Cell cycle analysis was quantized by PI and FACS analysis using image-based cytometry. (B) The percentage of cells in different phases was quantified.
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Figure 5. Compound 4d induced apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 1.21 μM of compound 4d for 48 h. Cell apoptosis was quantized by an Annexin V-FITC/PI dual staining assay using image-based cytometry. (B) The quantification of MDA-MB-231 cellular apoptosis.
Figure 5. Compound 4d induced apoptosis in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 1.21 μM of compound 4d for 48 h. Cell apoptosis was quantized by an Annexin V-FITC/PI dual staining assay using image-based cytometry. (B) The quantification of MDA-MB-231 cellular apoptosis.
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Figure 6. Compound 4d promoted MMP depolarization in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 1.21 μM of compound 4d for 48 h. MMP was quantified using image-based flow cytometry. (B) The quantification of MMP in MDA-MB-231 cells after treatment with compound 4d at its IC50 concentration (μM).
Figure 6. Compound 4d promoted MMP depolarization in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 1.21 μM of compound 4d for 48 h. MMP was quantified using image-based flow cytometry. (B) The quantification of MMP in MDA-MB-231 cells after treatment with compound 4d at its IC50 concentration (μM).
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Figure 7. Graphical representation of the effect of 3-nitrophenylthiazolyl molecule 4d on the level of p53 in MDA-MB-231 cells for 48 h.
Figure 7. Graphical representation of the effect of 3-nitrophenylthiazolyl molecule 4d on the level of p53 in MDA-MB-231 cells for 48 h.
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Figure 8. The binding poses (2D and 3D) of 3-nitrophenylthiazolyl molecule 4d in VEGFR-2 active site (PDB ID: 4ASD). (A) 2D representation; (B) 3D representation.
Figure 8. The binding poses (2D and 3D) of 3-nitrophenylthiazolyl molecule 4d in VEGFR-2 active site (PDB ID: 4ASD). (A) 2D representation; (B) 3D representation.
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Table
Table 1. Cytotoxic screening of the tested thiazole derivatives 3a-5b. Data expressed as the mean ± SD.
Table 1. Cytotoxic screening of the tested thiazole derivatives 3a-5b. Data expressed as the mean ± SD.
Comp No.IC50 Value (μM)
MDA-MB-231
3a8.12 ± 0.42
3b9.55 ± 0.46
3c7.39 ± 0.41
4a29.77 ± 3.27
4b3.52 ± 0.18
4c4.89 ± 0.24
4d1.21 ± 0.09
4e13.33 ± 0.93
5a24.89 ± 1.19
5b19.25 ± 0.68
Sorafenib1.18 ± 0.06
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Al-Warhi, T.; Abualnaja, M.; Abu Ali, O.A.; Alyamani, N.M.; Elsaid, F.G.; Shati, A.A.; Albogami, S.; Fayad, E.; Abu Almaaty, A.H.; Mohamed, K.O.; et al. Design, Synthesis and Cytotoxicity Screening of New Thiazole Derivatives as Potential Anticancer Agents through VEGFR-2 Inhibition. Symmetry 2022, 14, 1814. https://doi.org/10.3390/sym14091814

AMA Style

Al-Warhi T, Abualnaja M, Abu Ali OA, Alyamani NM, Elsaid FG, Shati AA, Albogami S, Fayad E, Abu Almaaty AH, Mohamed KO, et al. Design, Synthesis and Cytotoxicity Screening of New Thiazole Derivatives as Potential Anticancer Agents through VEGFR-2 Inhibition. Symmetry. 2022; 14(9):1814. https://doi.org/10.3390/sym14091814

Chicago/Turabian Style

Al-Warhi, Tarfah, Matokah Abualnaja, Ola A. Abu Ali, Najiah M. Alyamani, Fahmy G. Elsaid, Ali A. Shati, Sarah Albogami, Eman Fayad, Ali H. Abu Almaaty, Khaled O. Mohamed, and et al. 2022. "Design, Synthesis and Cytotoxicity Screening of New Thiazole Derivatives as Potential Anticancer Agents through VEGFR-2 Inhibition" Symmetry 14, no. 9: 1814. https://doi.org/10.3390/sym14091814

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