New derivatives based on zerumbone scaffold as anticancer inhibitors: Synthesis,in vitro anticancer evaluation,docking,and MD simulation studies

Abstract

A three-step procedure was applied to synthesize seven novel zerumbone ethers 7a–g with N-(4-hydroxybenzyl) benzamide, N-(4-hydroxybenzyl) substituted benzamide, and N-(4-hydroxybenzyl) cinnamamide moieties, including simultaneous O- and N-acylations, ester hydrolysis, and O-alkylation. The structure of intermediates and target compounds was elucidated using one-dimensional and two-dimensional nuclear magnetic resonance, and high-resolution mass spectrometry data. Their cytotoxic activities were screened on three human cancer cell lines HepG2, LU-1, and HeLa. The results showed that ethers 7a–g exhibited activity against all tested cell lines with IC₅₀ values ranging from 1.71 to 6.86 µM and stronger than zerumbone, approximately 10–35 folds. Theoretically, the mode of their anticancer action was studied based on predicting the ability to inhibit Fibroblast Growth Factor Receptor 1 and Vascular Endothelial Growth Factor Receptor 2 proteins using docking and molecular dynamic simulation studies. The interaction of 7a–g with important amino acids in the active binding sites of protein Fibroblast Growth Factor Receptor 1 and Vascular Endothelial Growth Factor Receptor 2, and Umbrella Effect formation explained the significant improvement in their cytotoxic activities compared to the parent compound zerumbone.

Keywords

4-hydroxy benzylamine cytotoxic Gromacs MD simulation molecular docking zerumbone

Introduction

Ethers are organic molecules with a C–O–C bond connecting alkyl or aryl groups. Many known natural and synthetic organic compounds have one or more ether groups presented in their molecules.^1,2 The ether bond can be located in different positions in the molecule such as intracyclic, exocyclic, or acting as a bridge between two molecules in their conjugates.^3
–5 In pharmaceutical chemistry, the presence of an ether group can improve the solubility in polar solvents such as water due to acting as a hydrogen bond acceptor. Moreover, ether group-containing molecules can enhance lipophilicity⁶ and promote the balance between hydrophilic and lipophilic properties.⁷ The ether group also serves as a proton acceptor forming hydrogen bonds when small molecule-target protein complexes are formed. Notably, ether linkages are often used as a common structure for the hydroxyl-protecting group in drug synthesis⁸ and a useful moiety in studying the inhibition of target proteins in virtual screening.⁹

Zerumbone is a natural sesquiterpene abundantly found in the Vietnamese traditional medicinal plant Zingiber zerumbet.¹⁰ This sesquiterpene possesses strong anticancer activity, which has been explored in the last two decades.^11,12 The pentadienone structural moiety and the original zerumbone scaffold are the required structures for its bioactivity, which must be kept in converting zerumbone into derivatives.^13,14 This approach has resulted in impressive anticancer activity results and diverse structures reported in recent studies.^15
–18 Accordingly, the bromination of the allyl structure moiety of zerumbone has yielded bromine derivatives suitable for the Williamson etherification as described in several studies.^15
–18 To date, this approach has received intensive attraction from scientists to explore potential candidates of zerumbone for research and development of anticancer drugs.

In oncology, both proteins FGFR1 (Fibroblast Growth Factor Receptor 1, PDB ID: 1agw) and VEGFR2 (Vascular Endothelial Growth Factor Receptor 2, PDB ID: 4asd) play important roles in the development of cancer.¹⁹ FGFR1 is a member of the tyrosine kinase family of molecules that plays a role in many biological processes, such as regulating tissue growth and repair. Alterations in FGFR1, such as gain, splicing, mutations, aberrant epigenetic or metabolic modifications, and altered tumor-tissue interactions in the tumor environment, can lead to the development and/or tumor progression for cancer cells. FGFR1 is an important component of FGF-FGFR signaling, playing a key role in the oncogenic process. VEGFR2 is a key component of VEGF-VEGFR signaling, the most important pathway for stimulating angiogenesis.^20,21 The inhibition of this signaling chain has been a successful method in treating cancer. FGF/FGFR inhibitors are considered a promising approach for treating tumors with FGFR alterations. Several FGFR1 inhibitors have demonstrated benefit in clinical trials with groups of patients whose tumors contain FGFR1 alterations.²² Several FGFR1 inhibitors are approved by the Food and Drug Administration (FDA), such as erdafitinib to treat metastatic bladder cancer with FGFR3 alterations and pemigatinib to treat inoperable biliary cancer with FGFR2 alterations.²³ Studies show that the effectiveness and anticancer range of the above drugs are limited and have side effects. Therefore, combining FGF/FGFR inhibitors with VEGF/VEGFR inhibitors is a good way to optimize treatment efficacy and expand the anticancer spectrum. This combination can target cancer cells and the tumor’s microbial environment.²⁴ Therefore, the new derivatives inhibiting FGFR1 and VEGFR2 proteins may lead to new candidates for further research on anticancer drug development.

In organic chemistry, 4-hydroxy benzylamine has been used to synthesize spirooxazoline.²⁵ In addition, its structural moiety has been found in synthesizing modified chitosan derivatives.^26,27 However, its structure is also suitable for the role of a bonding bridge to connect bioactive compounds such as zerumbone with other compounds due to the ether and amide linkage formation from its hydroxy and amine groups. In molecular modeling, 4-hydroxybenzylamine moiety has been predicted to enhance the inhibition of target proteins, as the hydroxy and amine groups act as proton acceptors and donors, facilitating π-cation interaction. Interestingly, no studies on the synthesis, cytotoxic valuation, and in silico FGFR1/VEGFR2 inhibitory activities of zerumbone ethers with aromatic acids through the 4-hydroxy benzyl bridge are available. In the present work, we introduce herein the synthesis, cytotoxic activity evaluation, and docking studies on in silico FGFR1 and VEGFR2 inhibitory activity of novel zerumbone ethers with seven various aromatic acids.

Results and discussion

The synthesis of 7a–g was divided into two parts, including the synthesis of intermediates 4a–g and the synthesis of target derivatives 10a–g, as figured out in Scheme 1. The first part is the synthesis of intermediates 4a–g. First, a step protocol for the direct N-acylation of 4-hydroxybenzyl amine with acyl chlorides 2a, 2b, and 2c in CH₂Cl₂ catalyzed by triethylamine (TEA) was introduced to obtain 4a, 4b, and 4c in 55%–61% yields as described by Tariq and Wesley.^25,28 Next, the synthesis of 4f was also mentioned in several references.^29,30 In the current report, a two-step procedure was applied to synthesize 4a–g. First, the starting compound 4-hydroxy benzylamine was simultaneously esterified and amidated with acyl chlorides 2a–g in dry acetone in the presence of a base potassium tert-butoxide to give crudes 3a–g in quantitative yields. A small amount of representative 3d was purified by column chromatography on silica gel using n-hexane:acetone 3:1 as eluent. The structure of the representative 3d was elucidated by ¹H NMR and ¹³C NMR spectra. Subsequently, the ester group of crudes 3a–g was cleaved by their hydrolysis with 2 equiv. of KOH powder in methanol at 50 °C for 5 h to give 4a–g in 85% to 91% yields. The spectral data of new intermediates 4d, 4e, and 4g were in excellent agreement with their structures.

Scheme 1.

The synthesis of zerumbone derivatives 7a–g.

In the second part, bromide 6 was prepared from zerumbone in quantitative yield according to the protocols of Kitayama et al.¹³ Finally, series 7a–g were obtained by the O-alkylation of the intermediates 4a–g with the bromide 6 catalyzed by a weak base K₂CO₃ in the DMF to give series 7a–g in 55% to 73% yields. The structures of 7a–g were elucidated by ¹H NMR and ¹³C NMR spectra. The NMR spectra of 7a–g had the similarity of chemical shifts and multiplicity of structural parts derived from zerumbone and 4-hydroxy benzylamine. The signal difference was only observed in the amide parts due to their different substituents attached to the benzamide parts. Ether 7e was an example of its structural interpretation using ¹H NMR, ¹³C NMR, HSQC, and HMBC spectra (Supplementary Material 1). The ¹H NMR of 7e was divided into two signal regions including the signal of aromatic protons that appeared at intervals 6.91–7.47 ppm and those of zerumbone that had chemical shifts ranging from 1.04 to 6.02 ppm. First, the assignment of proton signals was for zerumbone scaffold. The triplet of H-3 overlapped with two doublets of H-10 and H-11, as seen in its ¹H NMR spectrum like other ethers, to give a multiplet at 6.01 ppm. Another triplet at 5.52 ppm was attributed to H-7. The overlapped multiplets from six protons, including H-4a, H4b, H5a, H-5b, H8a, and H-8c, were found at 2.50, 2.46, 2.23, and 1.96 ppm that were assigned to H-5a, H-8a and H-4a, H-5b and H-4b, and H-8b, respectively. The singlet at 1.69 ppm arose from three equivalent protons H-12. Two other singlet signals at 1.04 and 1.13 ppm were identified as H-13 and H-14, but they could not be distinguished exactly. Two of the remaining doubles at 4.66 ppm (J = 8.4 Hz), 4.40 ppm (J = 6.0 Hz), and the broad singlet at 4.27 ppm were assigned to H-15a, 1′-CH₂-, and H-15b, respectively. The ¹³C NMR spectrum and directly linked carbons with the above protons were assigned as: 148.8 ppm (C-3), 159.6 ppm (C-10), 127.0 ppm (C-11), 129.8 ppm (C-7), 23.4 ppm (C-4), 34.8 ppm (C-5), 41.5 ppm (C-8), 11.8 ppm (C-12), 29.8 ppm (C-13 or C-14), 23.6 ppm (C-14 or C-13), 64.6 ppm (C-15), and 42.1 ppm (1′-CH₂-) as shown by the related correlation in HSQC spectrum. The cross-peaks in HMBC spectra (see SI) provided assignments of quaternary carbons C-1, C-2, C-6, and C-9 at 202.7, 137.7, 135.5, and 37.0 ppm, respectively. Last but not least, the spectral data of zerumbone scaffold were in excellent agreement with those reported previously.^16
–18 Next, except a triplet was observed at 8.94 ppm (J = 6.0 Hz) of proton NH due to the interaction with two equivalent neighbor protons 1′-CH₂-, the resonance of eight aromatic protons gave rise to the signals in intervals 6.91–7.47 ppm. Specifically, the first doublet (J = 7.46 Hz) at 7.46 ppm was attributed to H-6″. Proton H-2″ interacted with two neighbor protons H-6″ and H-4″ giving rise to a triplet (J = 1.8 Hz) at 7.43 ppm. The interaction of protons H-4″ and H-6″ with H-5″ resulted in a triplet of the H-5″ identified at 7.37 ppm. Two couples of equivalent protons (H-2′, H-6′) and (H-3′, H-5′) resonated giving a doublet (J = 7.8 Hz) at 7.25 ppm and another multiplet at 6.92 ppm, respectively. Next, proton H-4″ appeared at 7.03 ppm under doublet of doublet signal with J₁ = 1.8 Hz and J₂ = 7.8 Hz. The correlation in the HSQC spectrum of known protons with the directly linked carbons indicated the position of these carbons as follows: 119.4 ppm (C-6″), 112.3 ppm (C-2″), 114.4 (C-5″), 128.5 (C-2′, C-6′), 114.4 (C-3′, C-5′), and 117.0 (C-4″), respectively. Subsequently, the signals at 159.1, 157.1, 135.8, and 131.8 ppm showed no correlation in the HSQC spectrum, attributed to quaternary carbons. The cross-peaks in the HMBC spectrum of quaternary carbon at 159.1, 157.1, 135.8, and 131.8 ppm with the related protons resulted in the assignment of their positions: C-1′, C4′, C-1″, and C-3″, respectively. In summary, the structure of 7e was exactly determined by the 1D and 2D NMR data. The structure of the remaining ethers was also elucidated based on the structural data of 7e, as shown in the experimental section.

The synthesized derivatives in series 7a–g were evaluated for their cytotoxic activity. Cell lines used for screening cytotoxic profiles were human hepatocellular carcinoma HepG2, human lung adenocarcinoma A549, and HeLa cervical cancer cells HeLa. IC₅₀ values were calculated using the table curve program and listed in Table 1.

Table 1.

The cytotoxic activities of derivatives 7a–g.

No	Compounds	IC₅₀ (µM)
No	Compounds	HepG2	A549	HeLa
1	7a	2.08 ± 0.14	2.69 ± 0.44	2.55 ± 0.39
2	7b	1.95 ± 0.15	2.56 ± 0.15	2.47 ± 0.21
3	7c	2.18 ± 0.10	2.58 ± 0.20	2.60 ± 0.34
4	7d	2.08 ± 0.09	2.56 ± 0.18	2.47 ± 0.38
5	7e	1.71 ± 0.08	2.17 ± 0.07	2.41 ± 0.17
6	7f	1.84 ± 0.10	2.21 ± 0.13	1.71 ± 0.04
7	7g	2.60 ± 0.19	2.66 ± 0.34	6.86 ± 1.15
8	4e	>100	>100	>100
9	Zerumbone	61.19 ± 2.96	27.75 ± 1.88	69.90 ± 2.05
10	Ellipticine	1.38 ± 0.07	1.50 ± 0.06	1.42 ± 0.06

Data represent the mean ± standard error of the mean of three independent wells.

Ellipticine (Merck, Germany) was employed as a positive control.

According to the IC₅₀ data in Table 1, the derivatives 7a–g demonstrated strong cytotoxic effects toward three tested cell lines, HepG2, A549, and HeLa, with IC₅₀ values ranging from 1.71 to 2.69 µM, except 7g containing cinnamamide moiety, which had weaker cytotoxicity against the HeLa cell line with IC₅₀ value of 6.86 µM. The results showed that the presence of zerumbone scaffolds in 7a–g was the required structural moieties to improve their cytotoxicity. This conclusion agreed with comparing cytotoxic activity results of representative 7e containing 3-methoxy benzamide moiety and its starting compound 4e. While 7e exhibited strong activity against the tested cell lines HepG2, A549, and HeLa cell lines with IC₅₀ values in intervals 1.71–2.41 µM, 4e showed no activity toward these cell lines. Structurally, ethers 7a, 7b, 7c, 7d, 7e, 7f, and 7g were distinguished by the methyl and methoxy groups on the benzene ring of benzamide, and the hydrocarbon skeleton of cinnamic acid. Except for 7g cytotoxicity against HeLa cells, the data in Table 1 indicated that the variation in cytotoxic activities caused by the methyl, methoxy groups, and hydrocarbon skeleton of cinnamic acid in the amide part of series 7a–g with three tested cell lines was not significant. Specifically, the methyl and methoxy groups had the same electron-donating properties which increased electron density in the benzene ring. Moreover, the difference in spatial blocking between the methyl and methoxy groups did not create a clear effect. Therefore, their contribution to the cytotoxic activities of ethers 7a–f was the same. In terms of IC₅₀ value, the cytotoxic activities toward human cancer cell lines: HepG2, A549, and HeLa of ethers 7a–g, except for 7g cytotoxicity toward HeLa, were stronger than those of zerumbone, approximately: 23–40, 10–12, and 26–40 folds, respectively. On the contrary, the structural part of acrylamide in cinnamamide moiety decreased the electron density on the benzene ring resulting in the decrease of sensibility toward HeLa. As a result, 7g exhibited cytotoxic activity against HeLa cells with an IC₅₀ value of 6.86 µM, weaker than the remaining ethers. In summary, increasing electron density in the benzene ring of amide parts could improve hydrophobic and π-cations bond formation, and the stability of ligand–protein complexes in virtual screening on related target proteins. This is a crucial finding that can be applied in designing new zerumbone candidates for their anticancer activity enhancement.

Docking study

The structural difference in the active binding pocket area between the two receptors FGFR1 (ID: 1agw) and VEGFR2 (ID: 4asd) can be recognized due to the three-dimensional model, as illustrated in Figure 1(a) and (b). The active binding domain of PDB ID 1agw is characterized by a bilobed structure typical of kinase domains. The N-terminal lobe (N-lobe) consists mainly of β-sheets, while the C-terminal lobe (C-lobe) is predominantly α-helical. The ATP-binding site is located in the cleft between these two lobes, where the inhibitor binds, stabilizing the inactive conformation of the kinase.³¹

Figure 1.

3D surface of active binding pocket: (a) receptor PDB ID 1agw and (b) receptor PDB ID 4asd.

For PDB ID 4asd, the structure of the active binding domain includes two regions: the Juxtamembrane domain, located adjacent to the cell membrane, plays a crucial role in regulating receptor activity. This domain often participates in dimerization (combining two receptors) and receptor activation through interactions with other molecules. The kinase domain is responsible for the receptor’s enzymatic activity, known as the process of phosphorylation (adding a phosphate group to target molecules). The kinase domain typically has a bilobed structure, with an N-terminal lobe and a C-terminal lobe, and the ATP-binding site is situated between these two lobes.³² This structural arrangement significantly impacts the ability of the ligand or substrate to make contact, create interactions, and lead to biological reactions. Receptor 1agw (a) has higher flexibility than receptor 4asd (b).

Analysis docking result of receptor 1agw

Autodock Vina software is selected to dock the receptors (PDB ID 1agw and 4asd) with ligands 7a–g along with zerumbone, SU2, BAX, and infigratinib and calculates the energy-binding affinities. The results are shown below (Figure 2, Table 2, and Appendix 1, Supplementary Material 2).

Figure 2.

Binding affinity of the complexes between receptor (PDB ID 1aw) and ligands 7a-g, SU2, and zerumbone by Autodock Vina.

Table 2.

Summarize the interactions of the ligands with the receptor FGFR1 (PDB ID 1agw).

Ligand	Binding affinity (kcal mol⁻¹)	Hydrogen bonding	Hydrophobic	Others
Zerumbone	-7.9	ALA 564	LEU 484 (), VAL 492, ALA 512, LYS 514, ILE 545, LEU 630(*)	-
7a	-9.2	ASN 568, ASP 641, THR 658	LEU 484(), VAL 492(), ALA 512, LYS 514, ILE 545, VAL 561, TYR 563, LEU 630(), ALA 640, THR 658	-
7b	-9.2	ALA 564	LEU 484, VAL 492, ILE 545, VAL 561, ARG 627, LEU 630(*)	-
7c	-8.9	GLN 491, ALA 564	LEU 484(), VAL 492(), ALA 512, LYS 514, LEU 528, LEU 630, LEU 644	LYS 514 (π-Cation)
7d	-9.4	LYS 514, ALA 564	LEU 484(), VAL 492(), ALA 512, LYS 514, ASP 527, LEU 528, VAL 561, LEU 630(), LEU 644(),
7e	-9.2	GLN 491, LYS 514	VAL 492(*), LYS 514, LEU 516, LEU 528, ILE 545, LEU 630
7f	-8.5	LYS 514, ASN 568, ARG 570, GLU 571	LEU 484, VAL 492, ALA 512, LYS 514, ILE 545(), VAL 561, LEU 630()	-
7g	-9.1	GLN 491, LYS 514	LEU 484(), VAL 492(), LYS 514, ASP 527, VAL 561, LEU 630(), LEU 644(), ALA 645	-
SU2	-8.2	GLU 562, ASN 568, ARG 570(*)	LEU 484, VAL 492(*), ALA 512, TYR 563, ALA 564, LEU 630	-

(*) There are two interactions between the key residues of the receptor and the ligand molecules.

(**) There are three interactions between the key residues of the receptor and the ligand molecules.

The docking data in Table 2 show a significant improvement in the energy-binding affinity of series 7a–g (from −8.5 to −9.4 kcal mol⁻¹) compared to SU2 (−8.2 kcal mol⁻¹) and zerumbone (−7.9 kcal mol⁻¹). The interaction analysis between the protein FGFR1 and the inhibitor SU2 shows that the reference SU2 forms four hydrogen bonds with the receptor PDB ID 1agw (at GLU 562, ASN 568, and ARG 570), where the carbonyl group of SU2 interacts simultaneously with the residues ASN 568 and ARG 570. In addition, hydrophobic interactions with other amino acids, as listed in Table 2 and described in Figure 3, have also reinforced the inhibitory ability of SU2 against the receptor 1agw at the active binding domain, as defined in Table 6, Docking Study.

Figure 3.

3D interaction of SU2, zerumbone, and ligand 7d, 7e with the receptor (PDB ID 1agw).

The interaction analysis of complexes formed by the receptor FGFR1, PDB ID 1agw with derivatives 7a–g reveals that the hydrogen bonds are mainly formed with residues GLN 491, LYS 514, and ALA 564, which is quite different from the case of complex FGFR1-SU2 (GLU 562, ASN 568, and ARG 570), except for the case of ligand 7f. This ligand has hydrogen bonds with residues ASN 568 and ARG 570, like SU2, and additional hydrogen interactions with residues LYS 514 and GLU 571 (Appendix 2, Supplementary Material 2). Subsequently, the evaluation of the hydrophobic interaction between ligands 7a–g and the residues of receptor PDB ID 1agw leads to an exciting result that ligands 7a–g have hydrophobic interactions with similar residues as in the case of SU2, which are LEU 484, VAL 492, ALA 512, and LEU 630. Especially in the case of ligands 7c–g, hydrophobic interactions are additionally formed with neighboring residues GLN 491, LYS 514, VAL 561, and THR 568, as presented above. This is the basis to assert that the derivatives 7a–g give better docking results with receptor PDB ID 1agw than the reference SU2 and zerumbone.

Analysis docking result of receptor 4asd

The binding affinity data of complexes between the references BAX (sorafenib), infigratinib, 7a–g, and zerumbone with the receptor VEGFR2 (PDB ID 4asd) are represented in Figure 4 and Table 3.

Figure 4.

Binding affinity diagram of the docking results between the receptor (PDB ID 4asd) and the ligands 7a-g, BAX, infigratinib, and zerumbone by Autodock Vina.

Table 3.

Summarize the interactions of the ligands with the receptor (PDB ID 4asd).

Ligand	Binding affinity (kcal mol⁻¹)	Hydrogen bonding	Hydrophobic	Others
Zerumbone	-6.7	-	GLU 885, LEU 889, LEU 889, ILE 892, LEU 1019, LEU 1019, ILE 1044, ASP 1046	-
BAX	-9.3	LYS 868, GLU 885(), CYS 919(), GLY 922, ASP 1046	VAL 848, ALA 866, GLU 885, LEU 889, VAL 916, LEU 1035, PHE 1047(*)	ILE 1044 (Halogen Bonds)
Infigratinib	-9.3	LYS 868, GLU 885, ASP 1046(*)	LEU 840, GLU 885, VAL 899(*), VAL 916, ASP 1046	PHE 1047 (π-Stacking)
7a	-9.8	ASP 1046	VAL 848, ALA 866, GLU 885, LEU 889(*), ILE 892, VAL 899, VAL 916, LEU 1019, LEU 1035, LEU 1035, PHE 1047	LYS 868 (π-Cation Interactions)
7b	-10.7	ASP 1046	ASP 814, LEU 840, VAL 848, ALA 866, GLU 885, ILE 888, LEU 889(), ILE 892, VAL 916(), VAL 916, PHE 918, LEU 1019, LEU 1035, ASP 1046, PHE 1047	LYS 868 (π-Cation Interactions)
7c	-11.2	CYS 919, ASP 1046	LEU 840, VAL 848, ALA 866, GLU 885, ILE 888, LEU 889(), ILE 892, VAL 899, VAL 916(), LEU 1019, LEU 1035, ASP 1046, PHE 1047	LYS 868 (π-Cation Interactions)
7d	-11.4	ASP 1046	LEU 840, VAL 848, ALA 866, GLU 885, ILE 888(), LEU 889, VAL 899, VAL 916(), LEU 1019, LEU 1035(*), ASP 1046, PHE 1047	LYS 868 (π-Cation Interactions)
7e	-10.4	ASP 1046	VAL 848, ALA 866, LYS 868, GLU 885, LEU 889, ILE 892(), VAL 899, VAL 916, LEU 1019(), LEU 1035(*), ILE 1044, PHE 1047	LYS 868 (π-Cation Interactions)
7f	-8.6	GLU 885, ASP 1046	LEU 889, VAL 899, ASP 1046, LEU 1049, ILE 1053	LYS 868 (π-Cation Interactions)
7g	-11.4	-	LEU 840(), VAL 848, GLU 885, ILE 888, LEU 889(), ILE 892, VAL 899, VAL 916(), PHE 918, LEU 1019, ASP 1046, PHE 1047()	LYS 868 (π-Cation Interactions)

(*)

There are two interactions between the key residues of the receptor and the ligand molecules.

The residue ASP 1046 is crucial for coordinating the binding of ligand. According to the binding affinity data in Figure 4 and Table 3, the derivatives 7a–g (except 7g) interact strongly with residue ASP 1046 and give better docking results with the receptor PDB ID 4asd than the cases of BAX and infigratinib. The interaction efficiency of derivatives 7a–g has been improved due to the appearance of zerumbone moiety. In addition, the interaction between the receptor (PDB ID 4asd) and the ligands BAX and infigratinib shows that both inhibitors have binding affinity energy of −9.3 kcal mol⁻¹ and forming hydrogen bonds with residues LYS 868, GLU 885, and ASP 1046. However, the BAX molecule forms two hydrogen bonds at the position of secondary nitrogen and the oxygen atoms in the carboxylic group of GLU 885, in which the secondary nitrogen behaves as an acceptor (the bonds number 5 and 6, Figure 5(a)). For the case of infigratinib, three hydrogen bonds are also formed between the secondary nitrogen atom on infigratinib and the residues LYS 868, GLU 665, and ASP 1046 (the bonds number 2, 3, and 4, Figure 5(a)). Interestingly, in this case, the secondary nitrogen atom plays a dual role as donor and acceptor.

Figure 5.

3D interaction of: (a) sorafenib (BAX) and infigratinib; (b) zerumbone and ligand 7c; with receptor PDB ID 4asd.

The summarized data in Table 3 confirm that the derivatives 7a–g (except 7f) form hydrophobic interactions with specific amino acids of the receptor (PDB ID 4asd), and like BAX and infigratib. The derivatives 7a–e and 7g show higher similarity than the remaining one. The amino acid LYS 868 plays important role for the interaction with the phosphate groups of ATP and inhibitors, and it forms a hydrogen bond with BAX and infigratinib. However, in the cases of 7a–g, it forms a π-cation interaction with the electrons π in the structure of the substituent group attached to zerumbone. The mentioned π-cation interaction of LYS 868 contributes to the stability of complexes 7a–g and receptor (PDB ID 4asd). Thus, their absolute binding energy is higher than that of BAX and infigratinib.

Furthermore, in the 3D surface simulation images from the docked poses (as described in Figure 6), an umbrella effect outside the active binding domain of the receptor has been formed due to the hydrophobic interactions of zerumbone. This effect prevents the substrate ATP (adenosine triphosphate) from approaching the active binding site, inhibiting ATP to ADP biotransformation reaction. This unique feature differentiates the derivatives 7a–g from BAX and infigratinib in the docking study with the receptor PBD ID 4asd.

Figure 6.

3D surface simulation of the docked poses of the derivatives 7e interacting with the receptor (PDB ID 4asd)—Umbrella Effect.

Through docking studies of receptors PDB ID 1agw and 4asd with derivatives 7a–g and reference compounds, the results show that the addition of N-(4-hydroxybenzyl) benzamide moieties to the zerumbone molecule significantly enhances its interaction with key amino acids in the active binding domains of the receptors.

Moreover, it can be predicted that the derivatives 7a–e and 7g have the same ability to inhibit VEGFR2 as BAX and infigratinib. However, the interaction analyses between the receptors and the derivatives 7a–g were conducted under ideal conditions. In the subsequent part, kinetic simulations have been used to elucidate hypotheses about the improvement in inhibiting VEGFR2 and FGFR1 proteins of the derivatives 7a–g from zerumbone, compared to the references SU2, BAX, and infigratinib.

Molecular dynamics simulation

RMSD analysis

The RMSD analysis results of complexes between the receptors FGFR1 and VEGFR2 and derivatives 7a–g, zerumbone, SU2, BAX, and infigratinib are illustrated in Figures 7 and 8, and Appendices 8 and 9, Supplementary Material 2.

Figure 7.

Comparison RMSD of the complexes formed by the receptor (PDB ID 1agw) with zerumbone, SU2, and derivatives 7c, g.

Figure 8.

Comparison RMSD of the complexes formed by the receptor (PDB ID 4asd) with zerumbone, SU2, and derivatives 7c, f.

The RMSD values of complexes between 7a–g with receptor FGFR1 (PDB ID 1agw)

The complexes with reference compounds (including zerumbone and SU2) show relatively stable RMSD values. Meanwhile, the complexes between the receptor and derivatives 7a–g show significant changes during the simulation, leading to higher RMSD values (Appendix 8, Supplementary Material 2). Analysis of simulation results using the MD movie function of Chimera 1.17.3 shows diversity in the interaction of derivatives 7a–g at various positions in the active binding domain of the receptor. This leads to more changes in the size and shape of the receptor than the complexes formed from the reference compounds. In addition, the RMSD stability of reference compounds is also explained by their smaller size and fewer atoms than that of the derivatives 7a–g. Finally, the number of interactions in Figure 9 and Appendix 6, Supplementary Material 2 shows that the number of contact values of the complexes 1agw-7a–g are always larger than that of the complexes formed with the reference compounds, which is entirely appropriate and clarifies the above assertion, affirming that the compounds in the series 7a–g have many outstanding differences compared to SU2 in interaction with the receptor (PDB ID 1agw).

Figure 9.

Comparison of the number of contacts of the complex formed by the receptor (PDB ID 1agw) with the specific compound of derivatives 7b,c, zerumbone, and SU2.

The RMSD values of complexes between 7a–g with receptor VEGFR2 (ID: 4asd)

Due to the difference in the structure of the active binding domain of receptor VEGFR2 (PDB ID 4asd) compared to FGFR1 (PBD ID 1agw) as described in Figure 1(a) and (b), the interaction ability of zerumbone and its derivatives with key residues in the active binding pocket of receptor PDB ID 4asd is better than receptor PDB ID 1agw (see Appendix 9, Supplementary Material 2). The contact numbers in the complexes formed by zerumbone with receptors (PDB ID 1agw and 4asd) are shown in Figures9 –11.

Figure 10.

Comparison of the number of contacts (<0.6 nm) in the complexes formed by the receptor (PDB ID 4asd) with zerumbone, BAX, and infitinib.

Figure 11.

Comparison of the number of contacts of the complex formed by the receptor (PDB ID 4asd) with the specific compound of derivatives 7a,g, zerumbone, BAX, and infigratinib.

RMSF analysis

This metric measures a particle’s root mean square fluctuation (e.g. a protein residue) over time compared to a reference position (usually the time-averaged position of the particle). Therefore, RMSF analyzes the parts of the structure that fluctuate the most (or least) compared to their average structure. For the receptor, RMSF describes local changes along the protein chain. A high RMSF value for a protein residue indicates that this part has large fluctuations during the simulation, while a low RMSF value suggests that this part fluctuates less.

The RMSF data of the receptors (PDB IDs 1agw and 4asd) are presented in Figures 12 and 13, and Appendices 10 and 11, Supplementary Material 2. The results show the differences in the shape of spectral and RMSF values of the receptors (1agw and 4asd) in their complexes with zerumbone. This confirms that interactions between zerumbone with the key residues in the active binding domain of proteins (PDB ID 4asd and 1agw) are different. However, in their interactions with derivatives 7a–g, the RMSF results of the receptors in their complexes are similar to those of their complexes with SU2 (receptor PDB ID 1agw) and BAX, infigratinib (receptor PDB ID 4asd). A unique point is that the RMSF receptor values (PDB ID 4asd) in its complexes with series 7a–g are higher than that of its complexes with BAX and infigratinib, as shown in Figure 13 and Appendix 11, Supplementary Material 2. This affirms that the derivatives 7a–g interact more positively with the residues of the receptor (PDB ID 4asd) than BAX and infigratinib.

Figure 12.

Comparison RMSF of the receptor (PDB ID 1agw) in the complexes formed with reference compounds (zerumbone, SU2) and derivatives 7e, g.

Figure 13.

Comparison RMSF of the receptor (PDB ID 4asd) in the complexes formed with reference compounds (zerumbone, SU2) and derivatives 7c, d.

Hydrogen bond analysis (Hbond)

Hydrogen bond formation during the interaction between the residues of protein and the atoms in the functional group of ligands contributes to the stability of the complex. Furthermore, hydrogen bonds are a supportive factor in evaluating improvements in new drug design. Thus, this study analyzes the number of hydrogen bonds formed between ligands 7a–g and reference compounds with receptors 1agw and 4asd.

As the simulation results in Figures 14 and 15, and Appendices 4 and 5, Supplementary Material 2, the derivatives 7a–g and the reference compounds (SU2, BAX, infigratinib, and zerumbone) form hydrogen bonds with the residues of the receptors (PDB ID 1agw and 4asd). However, the number and stability of these hydrogen bonds over the simulation time vary significantly depending on the ligand structure and the simulation environment (solvent, electrolytes), leading to continuous variations in the receptor structure, ligand position, and conformation at the active binding pocket.

Figure 14.

Hydrogen bond formation in the complexes formed by the receptor PDB ID 1agw with derivatives 7d, e and reference compounds (SU2 and zerumbone).

Figure 15.

Hydrogen bond formation in the complexes of receptor PDB ID 4asd with derivatives 7a,b,d and reference compounds (BAX, infigratinib, zerumbone).

The observation of hydrogen bond formation during the MD simulation of ligands 7a–g and SU2 with receptor PDB ID 1agw (Appendix 4, Supplementary Material 2) leads to important judgments, the SU2 can form up to four hydrogen bonds with the receptor, but only three bonds are relatively stable. In the cases of 7a–g, the ethers 7e, 7d, and 7f, the number of hydrogen bonds can reach a maximum of four to five hydrogen bonds in which the hydrogen bonds of ether 7e are more stable than those of the SU2. For the receptor PDB ID 4asd (Appendix 5, Supplementary Material 2), the number of hydrogen bonds formed between the receptor and the BAX, and infigratinib are evaluated to be stable at 2 and 3 bonds, respectively. Meanwhile, the hydrogen bonds between the receptor and derivatives 7a–g have fewer numbers and stability than those of BAX and infigratinib. This significant difference is attributed to the number of multiple heteroatoms (N, F, Cl, O) presented in SU2, BAX, and infigratinib, which generate and sustain strong electrostatic energy and enhance ligand activity and interaction potential. However, it also results in unpredictable chemical behavior.

Number of contact analysis

The number of contact metric measures the number of contacts between two related groups of protein and ligand. Each contact is determined based on the shortest distance between the two groups. The number of contacts between the protein and ligand can help evaluate their interaction level. The ligand–protein complex has many contacts in the active binding domain referring to their strong interaction and resulting in its stability. As a result, this ligand is potent in inhibiting protein overexpression.

Comparing the number of contacts during the complex formation between the receptor (PDB ID 4asd) and the compounds: zerumbone, BAX, infigratinib, and 7a–g is shown in Figures 10 and 11, and Appendix 7, Supplementary Material 2. The data indicate that infigratinib has the same number of contacts with the residues of the receptor (PDB ID 4asd) as BAX and is higher than zerumbone. Next, as shown in Figures 10 and 11, the ethers 7a, 7b, 7c, and 7g show fewer contact levels than BAX and infigratinib. An Umbrella Effect has formed by hydrophobic interactions and shielded the protein pocket domain, which may be a part of the explanation for this phenomenon, as presented in Figure 6. It prevents zerumbone atoms from contacting with the amino acids in the protein pocket domain to form interactions.

Binding free energy analysis

In this research, the MMPBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) method is applied to calculate the binding free energy of protein–ligand complexes. The binding free energy is calculated by the difference in energy between the bound and unbound states of two molecules in a solvent. This value reflects the ability of the ligand to interact with the key residues in the active binding domain of the receptor. Furthermore, free binding energy values can reveal the inhibitory ability of ligands toward the receptor. The mentioned energies of the complexes between 7a–g and the reference compounds with the receptors (PDB ID 1agw and 4asd) are presented in Tables 4 and 5, and Figures 16 and 17.

Table 4.

Interaction energy of receptor (PDB ID 4asd) with specific compounds.

Ligand	ΔVDWAALS	ΔEEL	ΔEGB	ΔESURF	ΔGGAS	ΔGSOLV	ΔTOTAL
7a	-56.66	0.11	2.34	-7.38	-56.55	-5.04	-61.59
7b	-61.58	-0.32	2.83	-8.44	-61.89	-5.60	-67.50
7c	-59.58	-0.02	2.19	-7.79	-59.59	-5.61	-65.2
7d	-58.49	-1.30	3.81	-8.21	-59.79	-4.40	-64.19
7e	-53.52	-0.96	2.81	-6.99	-54.47	-4.17	-58.64
7f	-49.64	-0.49	2.61	-6.71	-50.12	-4.10	-54.22
7g	-49.53	-1.83	3.70	-6.72	-51.36	-3.02	-54.38
Zerumbone	-30.38	-0.58	1.72	-4.14	-30.96	-2.42	-33.39
Infigratinib	-59.54	-3.31	6.40	-7.49	-62.85	-1.09	-63.94
Sorafenib (BAX)	-58.50	-0.97	3.90	-8.15	-59.46	-4.26	-63.72

Table 5.

Interaction energy of receptor (PDB ID 1agw) with specific compounds.

Ligand	ΔVDWAALS	ΔEEL	ΔEGB	ΔESURF	ΔGGAS	ΔGSOLV	ΔTOTAL
7a	-32.03	-0.63	1.73	-4.43	-32.66	-2.69	-35.35
7b	-30.45	-1.04	2.14	-4.15	-31.49	-2.00	-33.50
7c	-42.15	-1.43	2.81	-6.01	-43.58	-3.20	-46.78
7d	-33.08	-2.11	3.57	-4.60	-35.18	-1.04	-36.22
7e	-37.48	-2.05	3.19	-4.95	-39.52	-1.76	-41.28
7f	-44.72	-2.30	3.83	-6.32	-47.01	-2.49	-49.51
7g	-24.27	-0.77	1.56	-3.38	-25.04	-1.82	-26.86
Zerumbone	-21.79	-0.36	0.99	-3.05	-22.15	-2.06	-24.21
SU2	-30.28	-2.50	2.90	-4.04	-32.78	-1.14	-33.92

All energies are in kcal mol⁻¹.

ΔTOTAL, total binding free energy; ΔVDWAALS, van der Waals energy; ΔEEL, electrostatic energy; ΔEGB, polar solvation energy in Generalized-Born method; ΔESURF, nonpolar solvation energy in Generalized-Born method; ΔGGAS, gas-phase molecular mechanics free energy; ΔGSOLV, solvation free energy; MM/GB(PB)SA, Molecular Mechanics/Poisson-Boltzmann (Generalized-Born) Surface Area.

Figure 16.

Binding free energy of the complexes formed by the receptor (PDB ID 4asd) with the ligands.

Figure 17.

Binding free energy of the complexes formed by the receptor (PDB ID1agw) with the ligands.

Analysis of the free binding energy of complexes between receptors (PDB ID 1agw and 4asd) with derivatives 7a–g and zerumbone reveals that the complexes with zerumbone have significantly higher absolute free binding energy values compared to that of complexes with derivatives 7a–g. Therefore, the presence of N-(4-hydroxybenzyl) benzamide moieties connected with zerumbone has significantly enhanced the inhibitory ability of the receptors (PDB ID 1agw and 4asd).

For the receptor FGFR1 (PDB ID 1agw), its complexes with derivatives 7b–d yield higher absolute free binding energy results than those of BAX and infigratinib. Notably, all complexes of the receptor VEGFR2 (PDB ID 4asd) with derivatives 7a–g (except 7b) have higher absolute free binding energy results than those of reference compound SU2.

In conclusion, derivatives 7a–g are evaluated as potential candidates to replace SU2, BAX, and infigratinib in inhibiting FGFR1 and VEGFR2. These derivatives can be further studied in managing the activity and development of blood vessels in chemotherapy to prevent tumor growth.

Conclusion

Zerumbone ethers 7a–g were prepared and evaluated in vitro cytotoxic activity using three human cancer cell lines: HepG2, A549, and HeLa. The derivatives 7a–g improved their cytotoxic activities toward the tested cell line compared to zerumbone. Their IC₅₀ values were found in intervals 1.71–6.86 µM, which were more potent than zerumbone, approximately 10–40 folds. Docking studies of 7a–g on FGFR1 and VEGFR2 give improved results of the binding affinity energy compared to references SU2, BAX, and infigratinib due to the hydrophobic interactions and the Umbrella Effect formation. Analysis of parameters in MD simulation of 7a–g with FGFR1 and VEGFR2 showed that the number of contact values of complexes between 7a–g-FGFR1 and VEGFR2 is often larger than that of complexes between reference SU2-above proteins. The derivatives 7a–g interact more positively with the residues of receptors FGFR1 and VEGFR2. Moreover, the number of hydrogen bonds formed during MD simulation between 7a–g with FGFR1 was higher and more stable than those of reference SU2. Finally, the fewer hydrogen bonds observed in the complexes of 7a–g with VEGFR2, compared to those of references BAX and infigratinib, can be attributed to the lower number of heteroatoms in their molecules. However, molecules containing too many heteroatoms often result in unpredictable chemical behavior.

Experimental

The preparation of zerumbone was mentioned in our previous studies.^16
–18 The intermediate 6 were then prepared from zerumbone using the protocol described by Kitayama.¹³ Other chemicals were purchased from Meck and used for the reactions without further purification. 1D and 2D NMR spectra for DMSO-d₆ solutions were measured on a Bruker Avance 600 MHz spectrometer. Chemical shifts δ are quoted relative to internal TMS referenced to the residual solvent peak (DMSO at 2.50, 3.32, and 39.5 ppm). HRMS spectra were acquired on an Agilent LC/MSD Trap SL operating in the positive mode. Thin-layer chromatography (TLC) was performed on a sheet of aluminum foil coated with a Silica Gel 60 F₂₅₄. Spots were visualized under a UV lamp at 254 nm. Products were purified by column chromatography on Silica gel (40-230 mesh) using n-hexane:acetone as eluent. Three human cancer cell lines, HepG2, LU-1, and HeLa, were supplied by Prof. J. M. Pezzuto from Long-Island University, United States, and Prof. Jeanette Maier from the University of Milan, Italia.

General procedure for the synthesis of intermediates 3a–g

A solution of 4-(aminomethyl) phenol 1 (617.0 mg, 5.0 mmol) in dry acetone (20 mL) was stirred with potassium tert-butoxide (855.0 mg, 7.5 mmol) for 30 min at room temperature. To this was added each acyl chloride, including benzoyl chloride 2a, 4-methylbenzoyl chloride 2b, 4-methoxybenzoyl chloride 2c, 3-methylbenzoyl chloride 2d, 3-methoxybenzoyl chloride 2e, 3,4,5-trimethoxybenzoyl chloride 2f, and cinnamoyl chloride 2g (12.5 mmol). The mixture was stirred overnight at room temperature until TLC (n-hexane:acetone 3:1 v/v) showed the absence of 4-(aminomethyl) phenol 1. The solvent was removed under reduced pressure; then, the reaction mass was poured into cold water and acidified with dilute HCl to pH = 5. The precipitate was filtered on a Buchner funnel, washed thoroughly with water, and dried in a vacuum desiccator to give corresponding crude 3a–g (quantitative yield) which was used for the next step without further purification. A part of crude 3e was purified by column chromatography using n-hexane:acetone 3:1 v/v as eluent.

4-((3-methoxybenzamido)methyl)phenyl 3-methoxybenzoate (3e): Quantitative yield, white solid, m.p.: 123 °C; R_f = 0.43 (n-hexane: acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 7.72 (d, J = 7.8 Hz, 1H, H-6), 7.59 (d, J = 1.2 Hz, 1H, H-2), 7.51 (m, 2H, H-6″, H-5), 7.47 (s, 1H, H-2″), 7.40 (m, 3H, H-3′, H-5′, H-5″), 7.31 (dd, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4″), 7.24 (d, J = 7.8 Hz, 2H, H-2′, H-6′), 7.10 (dd, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4), 4.52 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 3.85 (s, 3H, 3-OCH₃), 3.81 (s, 3H, 3″-OCH₃). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 165.9 (C=O amide), 164.5 (C=O ester), 159.4 (C-3), 159.2 (C-3″), 149.3 (C-4′), 137.4 (C-1″), 135.7 (C-1), 130.3 (C-1′), 130.1 (C-5″), 129.4 (C-5), 128.3 (C-2′, C-6′), 122.0 (C-6), 121.7 (C-3′, C-5′), 120.1 (C-6″), 119.4 (C-4), 117.1 (C-4″), 114.2 (C-2), 112.4 (C-2″), 55.4 (3-OCH₃), 55.2 (3″-OCH₃), 42.1 (1′-CH₂-).

General procedure for the synthesis of intermediates 4a–g

A solution of each crude 3a–g (1 mmol) and powder KOH (2 mmol) in methanol (3 mL) was stirred at 50 °C for 5 h until TLC (n-hexane:acetone 3:1 v/v) showed the absence of starting material 3a–g. The solvent was removed under reduced pressure and dissolved in water (5 mL). The resulting solution was extracted with EtOAc (3 × 10 mL) and then acidified with HCl 2M to pH = 4. The precipitate was filtered on a Buchner funnel, washed thoroughly with water, and dried in a vacuum desiccator to give corresponding crudes 4a–g that were purified by column chromatography eluting with n-hexane:acetone.

N-(4-hydroxybenzyl)-3-methylbenzamide (4d): Yield: 88%, white solid, m.p.: 183–184 °C; R_f = 0.39 (n-hexane:acetone-2:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 9.24 (s, 1H, 4′-OH), 8.84 (t, J = 6.0 Hz, 1H, -NH-), 7.70 (s, 1H, H-2″), 7.67 (m, 1H, H-5″), 7.33 (m, 2H, H-4″, H-6″), 7.13 (d, J = 8.4 Hz, 2H, H-2′, H-6′), 6.71 (m, 2H, H-3′, H-5′), 4.36 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 2.35 (s, 3H, 3″-CH₃). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 166.1 (C=O, amide), 156.2 (C-4′), 137.5 (C-3″), 134.5 (C-1″), 131.6 (C-1′), 129.9 (C-2″), 128.6 (C-2′, C-6′), 128.1 (C-4″), 127.8 (C-5″), 124.3 (C-6″), 115.0 (C-3′, C-5′), 42.1 (1′-CH₂-), 20.9 (3″-CH₃). ESI-HRMS calculated for C₁₅H₁₆NO₂: [M+H]⁺ (m/z): 242.1181, found: 242.1170.

N-(4-hydroxybenzyl)-3-methoxybenzamide (4e): Yield: 91%, white solid, m.p.: 135–137 °C; R_f = 0.35 (n-hexane:acetone-2:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 9.24 (s, 1H, 4′-OH), 8.89 (s, 1H, -NH-), 7.45 (d, J = 7.8 Hz, H-6″), 7.43 (s, 1H, H-2″), 7.36 (t, J = 7.8 Hz, 1H, H-5″), 7.12 (d, J = 7.8 Hz, 2H, H-2′, H-6′), 7.08 (d, J = 7.8 Hz, 1H, H-4″), 6.70 (d, J = 7.8 Hz, 2H, H-3′, H-5′), 4.36 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 3.80 (s, 3H, 3″-OCH₃). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 165.7 (C=O amide), 159.1 (C-3″), 156.2 (C-4′), 135.9 (C-1″), 129.8 (C-1′), 129.4 (C-5″), 128.6 (C-2′, C-6′), 119.4 (C-6″), 117.0 (C-4″), 115.0 (C-3′, C-5′), 112.3 (C-2″), 55.2 (3″-OCH₃), 42.2 (1′-CH₂-). ESI-HRMS calculated for C₁₅H₁₆NO₃: [M+H]⁺ (m/z): 258.1130, found: 258.1115.

N-(4-hydroxybenzyl)cinnamamide (4g): Yield: 85%, white solid, m.p.: 157–158 °C; R_f = 0.37 (n-hexane:acetone-2:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 9.31 (s, 1H, 4′-OH), 8.46 (t, J = 5.7 Hz, 1H, -NH-), 7.57 (d, J = 8.4 Hz, 2H, H-2″, H-6″), 7.47 (d, J = 15.6 Hz, 1H, H-7″), 7.41 (m, 2H, H-3″, H-5″), 7.37 (m, 1H, H-4″), 7.11 (dd, J₁ = 1.8 Hz, J₂ = 8.7 Hz, 2H, H-2′, H-6′), 6.73 (ddd, J₁ = 1.8 Hz, J₂ = 4.8 Hz, J₃ = 8.4 Hz, 2H, H-3′, H-5′), 6.69 (d, J = 15.6 Hz, 2H, H-8″), 4.29 (d, J = 6.0 Hz, 2H, 1′-CH₂-). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 164.7 (C=O, amide), 156.3 (C-4′), 138.8 (C-7″), 134.9 (C-1″), 129.5 (C-1′), 129.4 (C-4″), 128.9 (C-3″, C-5″), 128.8 (C-2′, C-6′), 127.5 (C-2″, C-6″), 122.2 (C-8″), 115.1 (C-3′, C-5′), 41.9 (1′-CH₂-). ESI-HRMS calculated for C₁₆H₁₆NO₂: [M+H]⁺ (m/z): 254.1181, found: 242.1165.

General procedure for the synthesis of derivatives 7a–g

A mixture of each 4a–g (1.0 mmol), potassium carbonate (1.0 mmol, 1 equiv.), and the intermediate 6 (1.1 mmol, 1,1 equiv.) in dry dimethylformamide (DMF, 5 mL) was stirred overnight at room temperature until TLC (n-hexane:EtOAc 2:1 v/v) indicated the absence of 4a–g. The mixture was poured into cold water and then extracted with ethyl acetate (EtOAc, 3 × 15 mL). The combined EtOAc was dried on anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain corresponding crudes 7a–g that were purified by column chromatography/silica gel using n-hexane:acetone as the eluent.

N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)benzamide (7a): Yield: 57%, white solid, m.p.: 95–96 °C; R_f = 0.38 (n-hexane:acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.96 (t, J = 6.0 Hz, 1H, -NH-), 7.88 (m, 2H, H-2″, H-6″), 7.52 (m, 1H, H-4″), 7.46 (m, 2H, H-3″, H-5″), 7.24 (m, 2H, H-2′, H-6′), 6.92 (m, 2H, H-3′, H-5′), 6.00 (t, J = 5.7 Hz, 1H, H-3), 5.97 (m, 2H, H-10, H-11), 5.53 (t, J = 8.1 Hz, 1H, H-7), 4.66 (s, br, 1H, H-15a), 4.40 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 4.27 (s, brd, 1H, H-15b), 2.58 (m, 1H, H-5a), 2.46 (m, 2H, H-4a, H-8a), 2.21 (m, 2H, H-4b, H-5b), 1.97 (m, 1H, H-8b), 1.69 (s, 3H, H-12), 1.23 (s, 3H, H-13 or H-14), 1.04 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.7 (C-1), 166.0 (C=O, amide), 159.7 (C-10), 157.4 (C-4′), 148.8 (C-3), 137.7 (C-2), 135.5 (C-6), 134.4 (C-1″), 131.8 (C-1′), 131.1 (C-4″), 129.8 (C-7), 128.5 (C-2′, C-6′), 128.2 (C-3″, C-5″), 127.2 (C-2″, C-6″), 127.0 (C-11), 114.4 (C-3′, C-5′), 64.6 (C-15), 42.0 (1′-CH₂-), 41.5 (C-8), 37.0 (C-9), 34.8 (C-5), 28.9 (C-13 or C-14), 24.3 (C-4), 23.7 (C-14 or C-13), 11.8 (C-12). ESI-HRMS calculated for C₂₉H₃₄NO₃: [M+H]⁺ (m/z): 444.2538, found: 444.2515.

4-methyl-N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)benzamide (7b): Yield: 61%, white solid, m.p.: 76–77 °C; R_f = 0.38 (n-hexane:acetone-3:1).¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.86 (t, J = 6.0 Hz, 1H, -NH-), (t, J = 8.4 Hz, 2H, H-2″, H-6″), 7.24 (m, 4H, H-3″, 5H″, H-2′, H-6′), 6.92 (m, 2H, H-3′, H-5′), 6.01 (t, J = 6.0 Hz, 1H, H-3), 5.97 (m, 2H, H-10, H-11), 5.52 (t, J = 8.4 Hz, 1H, H-7), 4.66 (d, J = 9.0 Hz, 1H, H-15a), 4.39 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 4.27(d, J = 9.0 Hz, 1H, H-15b), 2.58 (m, 1H, H-5a), 2.46 (m, 2H, H-4a, H-8a), 2.35 (s, 3H, 4″-CH₃) 2.20 (m, 2H, H-4b, H-5b), 1.96 (m, 1H, H-8b), 1.69 (s, 3H, H-12), 1.21 (s, 3H, H-13 or H-14), 1.04 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.7 (C-1), 165.9 (C=O, amide), 159.6 (C-10), 157.4 (C-4′), 148.8 (C-3), 140.9 (C-4″), 137.7 (C-2), 135.5 (C-6), 131.9 (C-1″), 131.6 (C-1′), 129.8 (C-7), 128.7 (C-3″, C-5″), 128.5 (C-2″, C-6″), 127.2 (C-2′, C-6′), 114.4 (C-3′, C-5′), 64.6(C-15), 42.0 (1′-CH₂-), 41.5 (C-8), 37.0 (C-9), 34.8 (C-5), 28.9 (C-13 or C-14), 24.3 (C-4), 23.6 (C-14 or C-13), 20.9 (4″-CH₃), 11.8 (C-12). ESI-HRMS calculated for C₃₀H₃₆NO₃: [M+H]⁺ (m/z): 458.2695, found: 458.2667.

4-methoxy-N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)benzamide (7c): Yield: 73%, white solid, m.p.: 72–73 °C; R_f = 0.37 (n-hexane:acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.80 (t, J = 6.0 Hz, 1H, -NH-), 7.86 (d, J = 8.7 Hz, 2H, H-2″, H-6″), 7.23 (d, J = 8.7 Hz, 2H, H-2′, H-6′), 6.99 (d, J = 8.7 Hz, 2H, H-3″, H-5″), 6.91 (dd, J₁ = 2.4 Hz, J₂ = 8.7 Hz, 2H, H-3′, H-5′), 6.02 (t, J = 6.0 Hz, 1H, H-3), 5.97 (m, 2H, H-10, H-11), 5.52 (t, J = 8.4 Hz, 1H, H-7), 4.66 (d, J = 9,0 Hz, 1H, H-15a), 4.38 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 4.27 (d, J = 9,0 Hz, 1H, H-15b), 3.80 (s, 3H, 4″-OCH₃), 2.58 (m, 1H, H-5a), 2.46 (m, 2H, H-4a, H-8a), 2.20 (m, 2H, H-4b, H-5b), 1.96 (m, 1H, H-8b), 1.69 (s, 3H, H-12), 1.21 (s, 3H, H-13 or H-14), 1.05 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.7 (C-1), 165.5 (C=O, amide), 161.5 (C-4″), 159.7 (C-10′), 157.4 (C-4′), 148.8 (C-3), 137.7 (C-2), 135.5 (C-6), 132.1 (C-1′), 129.8 (C-7), 129.0 (C-2″, C-6″), 128.5 (C-2′, C-6′), 127.0 (C-11), 126.6 (C-1″), 114.4 (C-3′, C-5′), 113.4 (C-3″, C-5″), 64.7 (C-15), 55.3 (4″-OCH₃), 42.0 (1′-CH₂-), 41.5 (C-8), 37.0 (C-9), 34.8 (C-5), 28.9 (C-13 or C-14), 24.3 (C-4), 23.7 (C-14 or C-13), 11.8 (C-12). ESI-HRMS calculated for C₃₀H₃₆NO₄: [M+H]⁺ (m/z): 474.2644, found: 474.2619.

3-methyl-N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)benzamide (7d): Yield: 63%, white solid, m.p.: 80–81 °C; R_f = 0.39 (n-hexane:acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.89 (t, J = 6.0 Hz, 1H, -NH-), 7.70 (s, 1H, H-2″), 7.67 (m, 1H, H-5″), 7.33 (m, 2H, H-4,″ H-6″), 7.21 (dd, J₁ = 2.7 Hz, J₂ = 8.4 Hz, 2H, H-2′, H-6′), 6.92 (ddd, J₁ = 2.7 Hz, J₂ = 4.8 Hz, J3 = 8.4 Hz), 6.01 (t, J = 5.7 Hz, 1H, H-3), 5.98 (d, J = 16.8 Hz, 1H, H-10), 5.96 (d, J = 16.8 Hz, 1H, H-11), 5.52 (t, J = 8.4 Hz, 1H, H-7), 4.66 (d, J = 8.4 Hz, 1H, H-15a), 4.40 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 4.27 (d, J = 8.4 Hz, 1H, H-15b), 2.58 (m, 1H, H-5a), 2.46 (m, 2H, H-4a, H-8a), 2.35 (s, 3H, 3″-CH₃), 2.22 (m, 2H, H-4a, H-5a), 1.96 (m, 1H, H-8b), 1.69 (s, 3H, H-12), 1.21 (s, 3H, H-13 or H-14), 1.04 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.7 (C-1), 166.1 (C=O, amide), 159.6 (C-10), 157.4 (C-4′), 148.8 (C-3), 137.7 (C-2), 137.5 (C-3″), 135.5 (C-6), 134.4 (C-1″), 131.9 (C-1′), 131.6 (C-2″), 129.8 (C-7), 128.5 (C-2′, C-6′), 128.1 (C-4″), 127.7 (C-5″), 127.0 (C-11), 124.3 (C-6″), 114.4 (C-3′, C-5′), 64.7 (C-15), 42.0 (1′-CH₂-), 41.5 (C-8), 37.0 (C-9), 34.8 (C-5), 28.9 (C-13 or C-14), 24.3 (C-4), 23.6 (C-14 or C-13), 20.9 (3″-CH₃), 11.8 (C-12). ESI-HRMS calculated for C₃₀H₃₆NO₃: [M+H]⁺ (m/z): 458.2695, found: 458.2670.

3-methoxy-N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)benzamide (7e): Yield: 68%, white solid, m.p.: 90–91 °C; R_f = 0.37 (n-hexane:acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.94 (t, J = 6.0 Hz, 1H, -NH-), 7.46 (d, J = 7.8 Hz, 1H, H-6″), 7.43 (t, J = 1.8 Hz, 1H, H-2″), 7.37 (t, J = 7.8 Hz, 1H, H-5″), 7.25 (d, J = 7.8 Hz, 2H, H-2′, H-6′), 7.,08 (d, J₁ = 1.8 Hz, J₂ = 7.8 Hz, 1H, H-4″), 6.92 (m, 2H, H-3′, H-5′), 6.00 (m, 3H, H-3, H-10, H-11), 5.52 (t, J = 8.4 Hz, 1H, H-7), 4.66 (d, J = 8.4 Hz, 1H, H-15a), 4.40 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 4.27 (s, brd, 1H, H-15b), 3.80 (s, 3H, 3″-OCH₃), 2.50 (m, 1H, H-5a), 2.46 (m, 2H, H-8a, H-4a), 2.33 (m, 2H, H-5b, H-4b), 1.96 (m, 1H, H-8b), 1.69 (s, 3H, H-12), 1.21 (s, 3H, H-13 or H-14), 1.04 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.7 (C-1), 165.7 (C-amide), 159.6 (C-10), 159.1 (C-3″), 157.4 (C-4′), 148.8 (C-3), 137.7 (C-2), 135.8 (C-1″), 135.5 (C-6), 131.8 (C-1′), 129.8 (C-7), 129.3 (C-5″), 128.5 (C-2′, C-6′), 127.0 (C-11), 119.4 (C-6″), 117,0 (C-4″); 114,4 (C-3′, C-5′); 112,3 (C-2″); 64,6 (C-15); 55,2 (2″-OCH₃); 42,1 (1′-CH₂-), 41.5 (C-8), 37.0 (C-9), 34.8 (C-5), 28.9 (C-13 or C-14), 24.3 (C-4), 23.6 (C-14 or C-13), 11.8 (C-12). ESI-HRMS calculated for C₃₀H₃₆NO₄: [M+H]⁺ (m/z): 474.2644, found: 474.2617.

3,4,5-trimethoxy-N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)benzamide (7f): Yield: 67%, white solid, m.p.: 91–92 °C; R_f = 0.35 (n-hexane:acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.90 (t, J = 5.7 Hz, 1H, -NH-), 7.24 (m, 4H, H-2′, H-6′, H-2″, H-6″), 6.93 (d, J = 8.4 Hz, 2H, H-3′, H-5′), 6.01 (t, J = 5.7 Hz, 1H, H-3), 5.97 (m, 2H, H-10, H-11), 5.52 (t, J = 8.1 Hz, 1H, H-3), 4.66 (s, brd, 1H, H-15a), 4.41 (d, J = 6.0 Hz, 2H, 1′-CH₂-), 4.28 (s, brd, 1H, H-15b), 3.82 (s, 6H, 3″-OCH₃-, 5″-OCH₃), 3.71 (s, 3H, 4″-OCH₃), 2.58 (m, 1H, H-5a), 2.47 (m, 2H, H-4a, H-8a), 2.20 (m, 2H, H-4b, H-5b), 1.97 (m, 1H, H-8b), 1.69 (s, 3H, 2 -CH₃- or H-12), 1.21 (s, 3H, H-13 or H-14), 1.05 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.7 (C-1), 165.4 (C=O, amide), 159.6 (C-10), 157.4 (C-4′), 152.5 (C-3″, C-5″), 148.8 (C-3), 139.9 (C-4″), 137.7 (C-2), 135.5 (C-6), 131.8 (C-1″), 129.8 (C-7), 129.5 (C-1′), 128.6 (C-2′, C-6′), 127.0 (C-11), 114.4 (C-3′, C-5′), 104.8 (C-2″, C-6″), 64.7 (C-15), 60.0 (4″-OCH₃), 55.9 (3″-OCH₃, 5″-OCH₃), 42.1 (1′-CH₂-), 41.5 (C-8), 37.0 (C-9), 34.8 (C-5), 28.9 (C-13 or C-14), 24.3 (C-4), 23.6 (C-14 or C-13), 11.8 (C-12). ESI-HRMS calculated for C₃₂H₄₀NO₆: [M+H]⁺ (m/z): 534.2825, found: 534.2855.

N-(4-(((1Z,5E,8E)-4,4,8-trimethyl-7-oxocycloundeca-1,5,8-trien-1-yl)methoxy)benzyl)cinnamamide (7g): Yield: 55%, white solid, m.p.: 73–74 °C; R_f = 0.39 (n-hexane:acetone-3:1). ¹H NMR (600 MHz, DMSO-d₆, δ (ppm)): 8.52 (t, J = 5.7 Hz, 1H, -NH-), 7.56 (d, J = 8.4 Hz, 2H, H-2″, H-6″), 7.46 (d, J = 15.9 Hz, 1H, H-7″), 7.39 (m, 3H, H-3″, H-4″, H-5″), 7.22 (d, J = 8.4 Hz, 2H, H-3′, H-5′), 6.93 (dd, J₁ = 1.8 Hz, J₂ = 8.4 Hz, 2H, H-3′, H-5′), 6.68 (d, J = 15.9 Hz, 1H, H-8″), 6.01 (t, J = 6.3 Hz, 1H, H-3), 5.99 (m, 2H, H-10, H-11), 5.53 (t, J = 8.4 Hz, 1H, H-7), 4.67 (d, J = 9.6 Hz, 1H, H-15a), 4.30 (s, brd, 1H, H-15b), 2.58 (m, 1H, H-5a), 2.47 (m, 2H, H-4a, H-8a), 2.22 (m, 2H, H-4b, H-5b), 1.96 (m, 1H, H-8b), 1.69 (s, 3H, H-12), 1.21 (s, 3H, H-13 or H-14), 1.05 (s, 3H, H-14 or H-13). ¹³C NMR (150 MHz, DMSO-d₆, δ (ppm)): 202.8 (C-1), 164.8 (C=O, amide), 159.7 (C-10), 157.5 (C-4′), 148.9 (C-3), 138.8 (C-7″), 137.7 (C-2), 135.6 (C-6), 134.9 (C-1″), 131.5 (C-1′), 129.9 (C-7), 129.4 (C-1′), 128.9 (C-3″, C-5″), 128.8 (C-2′, C-6′), 127.5 (C-2″, C-6″), 127.1 (C-11), 122.1 (C-8″), 114.5 (C-3′, C-5′), 64.7 (C-15), 41.8 (1′-CH₂-), 41.6 (C-8), 37.1 (C-9), 34.8 (C-5), 29.0 (C-13 or C-14), 24.4 (C-4), 23.7 (C14 or C-13), 11.9 (C-12). ESI-HRMS calculated for C₃₁H₃₆NO₃: [M+H]⁺ (m/z): 470.2695, found: 470.2644.

Biology

The in vitro cytotoxic evaluation against HepG2, A549, and HeLa cancer cell lines was carried out according to the described protocols.^33,34 Accordingly, stock solutions of the derivatives 7a–g were prepared in dimethyl sulfoxide (DMSO) at a concentration of 4 mM and then were diluted 200-fold to obtain solutions with the highest concentration (20 μM) tested on 96-well plates. These solutions were serially diluted for bioassay. Determination of IC₅₀ values was carried out using three human cancer cell lines: HepG2, A549, and HeLa. Ellipticine was used as a positive control. The IC₅₀ values were determined from dose-dependent curves plotted from five different concentration regimens (0–20 μM). At each regimen, the meaning of triplicate experiments was used for a point in the curve. Calculations were performed using the GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA).

Docking study

The Autodock Vina integrated with Chimera version 1.17.3 software³⁵ was applied to dock ligands and receptors (PDB IDs 1agw and 4asd). In addition, MolAICal software³⁶ and SwissDock web tool³⁷ were also referenced to improved Autodock tools for evaluation.

The molecules infigratinib, BAX, SU2, DHA, zerumbone and derivatives 7a–g were sketched using MarvinSketch version 32.11 and formatted as Mol2.

The docking process with Chimera version 1.17.3 was carried out following a similar our previous procedure¹⁸ in which a grid box was established to identify the active binding site with coordinate parameters (x,y,z) and size (x,y,z) of the receptors as shown in Table 6.

Table 6.

Parameters identifying the active binding site of the receptor.

Receptor (PDB ID)	Grid box dimension	Size
1agw	(8.8; 7; 21)	(21; 20; 24)
4asd	(-23,5; -1; -9)	(19; 26; 21)

Protein-Ligand Interaction Profiler (PLIP)³⁸ at the address https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index was used to analyze the interaction results between ligand and protein. The docking results are presented in Appendix 1, Supplementary Material 2.

Ligand–protein molecular dynamics simulation studies

In the current study, molecular dynamics (MD) simulation was carried out using the Gromacs software version 2024.1 on the SuperMicro GPU SuperWorkstation 7048GR-TR system, equipped with 2 x Intel Xeon E5-2696 V4 (2.2Ghz Turbo 3.6Ghz) 44 Core/88 Threads; 4 x Nvidia Tesla P100 16Gb HBM2, 128 DDR4 Ecc Buss 2400, 2 x SSD Nvme PCie 2TB. The simulation process was performed according to the method described by Truong et al.,¹⁸ but with the simulation time extended up to 100 ns.

To ensure the accuracy and reliability of the MD simulation results, preliminary simulation steps were performed to achieve temperature and pressure equilibrium in the system. Initially, an NVT (Number of particles, Volume, and Temperature) simulation was performed with fixed temperature (T) and volume (V) for the system to achieve temperature equilibrium and appropriate energy distribution. Subsequently, an NPT (Number of particles, Pressure, and Temperature) simulation was performed with fixed temperature (T) and constant pressure (P) for the system to achieve pressure equilibrium and adapt to environmental pressure, preparing for the next step of running MD.

To minimize the ligand moving far from the active binding domain during the simulation, at the energy minimization stage, the energy step size (emstep) is set to 0.01 nm, the maximum number of steps to perform (nsteps) was 500,000; the maximum force was lower than 100 kJ mol⁻¹ nm⁻¹ (emtol = 100) using the position restraint technique with tension for constraints in all three directions x,y,z is 1000 kJ mol⁻¹ nm⁻². During the simulation process from NVT to NPT and MD, the RMSD and number of contacts of the protein–ligand complex were evaluated to ensure the simulations achieve stability.

During the MD simulation process, kinetic values and interactions between particles in the system were recorded according to the change in position and speed of the particles over time. The recorded results for interaction analysis include RMSD, RMSF, Hbond, and number of contacts. The binding free energy was calculated using the “g_mmpbsa” tool on GROMACS according to the method described by Valdés-Tresanco et al.³⁹ and applied by Truong et al.¹⁸

Supplemental Material

sj-docx-1-chl-10.1177_17475198241312456 – Supplemental material for New derivatives based on zerumbone scaffold as anticancer inhibitors: Synthesis, in vitro anticancer evaluation, docking, and MD simulation studies

Supplemental material, sj-docx-1-chl-10.1177_17475198241312456 for New derivatives based on zerumbone scaffold as anticancer inhibitors: Synthesis, in vitro anticancer evaluation, docking, and MD simulation studies by Ngoc Hung Truong, Minh Ha Le, Hanh Trang Luu, Tien Chinh Vu, Van Chung Pham and Van Chinh Luu in Journal of Chemical Research

Supplemental Material

sj-docx-2-chl-10.1177_17475198241312456 – Supplemental material for New derivatives based on zerumbone scaffold as anticancer inhibitors: Synthesis, in vitro anticancer evaluation, docking, and MD simulation studies

Supplemental material, sj-docx-2-chl-10.1177_17475198241312456 for New derivatives based on zerumbone scaffold as anticancer inhibitors: Synthesis, in vitro anticancer evaluation, docking, and MD simulation studies by Ngoc Hung Truong, Minh Ha Le, Hanh Trang Luu, Tien Chinh Vu, Van Chung Pham and Van Chinh Luu in Journal of Chemical Research

Footnotes

Author contributions

N.H.T.: Investigation, Formal analysis; M.H.L.: Formal analysis, Data curation; H.T.L.: Investigation; T.C.V.: Resource, Writing-review & Editing; V.C.P.: Software, Validation, Writing original draft; V.C.L.: Conceptualization, Supervision, Writing original draft.

Data availability statement

The data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported under the project with the code: VAST04.01/23-24 by the Vietnam Academy of Science and Technology.

ORCID iDs

Ngoc Hung Truong

Van Chung Pham

Van Chinh Luu

Supplemental material

Supplemental material for this article is available online.

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