Design, Synthesis, and Antitumor Activity Evaluation of Artemisinin Bivalent Ligands

Five artemisinin bivalent ligands molecules 4a–4e were designed, synthesized, and confirmed by 1H NMR, 13C NMR, and low-resolution mass spectrometry, and the bioactivities of the target compounds were investigated against four human tumor cell lines in vitro, including BGC-823, HepG-2, MCF-7, and HCT-116. The results showed 4a, 4d, and 4e exhibited significantly tumor cell inhibitory activity compared with the artemisinin and dihydroartemisinin; compound 4e has good biological activity inhibiting BGC-823 with an IC50 value of 8.30 μmol/L. Then, the good correlations with biological results were validated by molecular docking through the established bivalent ligands multi-target model, which showed that 4e could bind well with the antitumor protein MMP-9.


Introduction
Artemisinin (ART) is a novel sesquiterpene containing an endoperoxide structure, with significant antimalarial activity of a fast-acting, high-efficiency, and low-toxicity nature [1].Some artemisinin-based derivatives have also become excellent clinical drugs such as dihydroartemisinin (DHM), artemether, artesunate, and so on [2][3][4].During the research of structural modification, artemisinin derivatives have shown superior antitumor activity [5], such as against ovarian cancer [6,7], leukemia [8], liver cancer, colon cancer [9], bladder cancer, and breast cancer [10].Dihydroartemisinin and dihydroartemisitene acetal dimers were synthesized and tested for their antitumor activity against human tumor 60 cell lines, and the GI 50 concentration ranged from 0.019 to 8.7 µmol/L against non-small-cell lung cancer, central nervous system tumors, leukemia cells, and other cancer cells [11].Chadwick et al. [12] synthesized a series of C-10 carba artemisinin dimers with good inhibitory effects on human promyelocytic leukaemia HL-60 cells with an IC 50 value in the micromolar to nanomolar range.
Cancer is a serious threat to human health [13].Despite remarkable progress in cancer prevention and treatment over the past decades, traditional tumor chemotherapeutic drugs often fail to distinguish between tumor cells and normal cells, and the development of new safe and effective drugs for cancer treatment remains extremely challenging [14].Natural products provide a rich source of leads for the discovery of new anticancer drugs [15].
"Bivalent ligands" molecules are antitumor star molecules, consisting of a variablesized linker group connecting two pharmacodynamic groups [16].Bivalent ligands show the advantages of enhanced binding affinity and significantly improved targeting to a tumor cell [17][18][19], which provides new ideas for drug design.Previous studies have shown that artemisinin bivalent ligands have higher biological activity than the corresponding monomers [20][21][22][23][24].And appropriate substitution in the linker of two artemisinin units can improve the antitumor activity of the analogs [4].Porphyrins and porphyrin derivatives are important functional molecules for cancer therapy [25][26][27].Huang et al. bridged the natural product with porphyrin and the synthesized derivatives showed a promising cleavage effect on pBR322 plasmid DNA [28,29].Heterocycles have always been key elements in medicinal chemistry as well as being found in the structure of numerous drugs, drug candidates, and biologically active molecules [30].
Guided and impelled by these results, five artemisinin bivalent ligands linked by different molecules were designed and synthesized with the purpose of enhancing the targeting and antitumor activity of artemisinin (Figure 1); their anti-proliferative activities were evaluated and the potential mechanisms were investigated preliminarily on representative tumor cells by Western blot assay.Furthermore, molecular docking models were performed to validate the artemisinin-multitarget molecules via LeDock.win32software.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 15 "Bivalent ligands" molecules are antitumor star molecules, consisting of a variablesized linker group connecting two pharmacodynamic groups [16].Bivalent ligands show the advantages of enhanced binding affinity and significantly improved targeting to a tumor cell [17][18][19], which provides new ideas for drug design.Previous studies have shown that artemisinin bivalent ligands have higher biological activity than the corresponding monomers [20][21][22][23][24].And appropriate substitution in the linker of two artemisinin units can improve the antitumor activity of the analogs [4].Porphyrins and porphyrin derivatives are important functional molecules for cancer therapy [25][26][27].Huang et al. bridged the natural product with porphyrin and the synthesized derivatives showed a promising cleavage effect on pBR322 plasmid DNA [28,29].Heterocycles have always been key elements in medicinal chemistry as well as being found in the structure of numerous drugs, drug candidates, and biologically active molecules [30].
Guided and impelled by these results, five artemisinin bivalent ligands linked by different molecules were designed and synthesized with the purpose of enhancing the targeting and antitumor activity of artemisinin (Figure 1); their anti-proliferative activities were evaluated and the potential mechanisms were investigated preliminarily on representative tumor cells by Western blot assay.Furthermore, molecular docking models were performed to validate the artemisinin-multitarget molecules via LeDock.win32software.

Chemistry
Artemisinin bivalent ligands 4a-4e were catalyzed by 4-dimethylaminopyridine (DMAP) and imine according to the synthetic routes outlined in Scheme 1.The yields of compounds 4a-4c were improved as the amounts of DMAP used increased.Compound 4d was generated by the reaction in the presence of DMAP and 1,3dicyclohexylcarbodiimide (DCC), and it was difficult to purify for the by-product 1, 3dicyclohexylurea (DCU) as well as being produced in low yield, as was 4e.Yet, these disadvantages can be effectively overcome by changing the condensing agents to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI .HCl) instead of DCC.

Chemistry
Artemisinin bivalent ligands 4a-4e were catalyzed by 4-dimethylaminopyridine (DMAP) and imine according to the synthetic routes outlined in Scheme 1.The yields of compounds 4a-4c were improved as the amounts of DMAP used increased.Compound 4d was generated by the reaction in the presence of DMAP and 1,3-dicyclohexylcarbodiimide (DCC), and it was difficult to purify for the by-product 1, 3-dicyclohexylurea (DCU) as well as being produced in low yield, as was 4e.Yet, these disadvantages can be effectively overcome by changing the condensing agents to 1-ethyl-3-

Screening of Anti-Cancer Activity
Anti-proliferation activity of 4a-4e in vitro was evaluated by four tumor cell lines, including MCF-7, HepG-2, HCT-116, and BGC-823, using the MTT assay, and the results were shown in Table 1.

Biological Evaluation 2.2.1. Screening of Anti-Cancer Activity
Anti-proliferation activity of 4a-4e in vitro was evaluated by four tumor cell lines, including MCF-7, HepG-2, HCT-116, and BGC-823, using the MTT assay, and the results were shown in Table 1.Compared to the parent compound ART, compounds 4a, 4d, and 4e displayed promising antiproliferative activity against the abovementioned tumor cells lines.Among them, compound 4a showed better inhibitory activity against each tumor cell line, exhibiting 2-4-fold increased activity compared to ART, indicating it possesses a broad spectrum of antitumor activity.Compounds 4d and 4e, which possessed a heterocyclic ring as the linker, improved the inhibitory activity against some tumor cell lines.In particular, compound 4e showed the best inhibitory activity against BGC-823 with an IC 50 value of 8.30 µmol/L.Unfortunately, the activity of compounds 4b and 4c were not as good as expected, proving that the introduction of bulky substituents on the linker had a detrimental effect on the activity.It may be related to the permeability of the molecule, which faces difficulty in penetrating through the cell membrane due to the large structural skeleton.
Preliminary structure-activity relationship (SAR) studies demonstrated that the linker between two artemisinin molecules had an effect on their antitumor activity.The use of linkers with small backbones or high polarity is more helpful in improving antitumor activity.The SAR studies suggest that other heterocycles, such as thiazole and pyrimidine, can be used as linkers to further investigate the effect of the type of linker on antitumor activity in future studies.

Western Blot Analysis
The latest research has revealed that artemisinin can work on multiple targets and affect multiple signaling pathways against cancer cells, such as inducing apoptosis, triggering ferroptotic cell death, inducing autophagy, and causing cell cycle arrest [31,32].In this study, we further evaluated the effects of artemisinin derivatives on apoptosis-related proteins in the four tumor cell lines.As shown by Western blotting in Figure 2, the effect of derivatives on the expression of different apoptotic proteins is variable in diverse tumor cells.
The Western blot assay results showed that compounds 4a, 4d, and 4e effectively decreased the protein levels of apoptosis-related proteins BCL-2, CDK-4, MMP-9, and VEGFR-2 in BGC-823 and HepG-2 cells (Figure 2A,B).In particular, compound 4e reduced the expression of the four apoptosis-associated proteins in BGC-823 cells more completely.Compounds 4a and 4d significantly inhibited the expression of protein VEGFR-2 in HepG-2 in a dose-dependent manner.However, all three of the tested compounds exhibited no major differences in the levels of expression of the four proteins over a 48-hour period in HCT-116 and MCF-7 cells (Figure 2C,D).Positive control Paclitaxel downregulated the protein level of CDK-4, MMP-9, and VEGFR-2 in the BGC-823 cells, decreased the expression of CDK-4 and MMP-9 in HepG-2, and reduced the expression of BCL-2, CDK-4, MMP-9, and VEGFR-2 in MCF-7 (Figure 2A-D).The Western blot assay results showed that compounds 4a, 4d, and 4e effectively decreased the protein levels of apoptosis-related proteins BCL-2, CDK-4, MMP-9, and VEGFR-2 in BGC-823 and HepG-2 cells (Figure 2A,B).In particular, compound 4e reduced the expression of the four apoptosis-associated proteins in BGC-823 cells more completely.Compounds 4a and 4d significantly inhibited the expression of protein VEGFR-2 in HepG-2 in a dose-dependent manner.However, all three of the tested compounds exhibited no major differences in the levels of expression of the four proteins over a 48-hour period in HCT-116 and MCF-7 cells (Figure 2C,D).Positive control Paclitaxel downregulated the protein level of CDK-4, MMP-9, and VEGFR-2 in the BGC-823 cells, decreased the expression of CDK-4 and MMP-9 in HepG-2, and reduced the expression of BCL-2, CDK-4, MMP-9, and VEGFR-2 in MCF-7 (Figure 2A-D).

Molecular Docking
Docking studies were performed to investigate the molecular binding patterns of artemisinin divalent ligands 4a-4e in the active pockets of the crystal structures of anticancer targets.The targets of the divalent ligand 4a-4e used for docking analysis are BCL-2, CDK-4, VEGFR-2, and MMP-9, which are widely noted to contribute to cellular proliferation and apoptosis regulation, cell cycle progression, transcriptional regulation,

Molecular Docking
Docking studies were performed to investigate the molecular binding patterns of artemisinin divalent ligands 4a-4e in the active pockets of the crystal structures of anticancer targets.The targets of the divalent ligand 4a-4e used for docking analysis are BCL-2, CDK-4, VEGFR-2, and MMP-9, which are widely noted to contribute to cellular proliferation and apoptosis regulation, cell cycle progression, transcriptional regulation, DNA damage repair, stem cell self-renewal, and antiangiogenic effects [33][34][35].The binding of ligands at the active site of the target protein suggests the possibility that ligands may have the ability to direct functional changes in target molecules.Ligand-target protein interaction is also decoded in terms of interacting amino acid residues, hydrogen bonding, docking energy analysis, and comparison of active site amino acid residues and possible binding sites.
MMP-9 (PDB ID: 2OVZ) is identified as the target protein for anticancer reagents.Upon evaluation of the docking results, it is clear that bivalent ligand 4a can fit perfectly into the binding pocket of MMP-9, demonstrating good uniformity between the in vitro MMP-9 screening and the in silico prediction.The docking results of bivalent ligand 4a exhibited that it engaged in the formation of hydrogen bonds with GLU111 and LEU187 amino acid residues (Figure 3).Additionally, bivalent ligand 4a is involved in numerous hydrophobic interactions, such as six Pi alkyl interactions with PHE110, HIS401, HIS405, HIS411, LEU188 and MET422.But, it forms an unfavorable acceptor-acceptor with ALA189.
Upon evaluation of the docking results, it is clear that bivalent ligand 4a can fit perfectly into the binding pocket of MMP-9, demonstrating good uniformity between the in vitro MMP-9 screening and the in silico prediction.The docking results of bivalent ligand 4a exhibited that it engaged in the formation of hydrogen bonds with GLU111 and LEU187 amino acid residues (Figure 3).Additionally, bivalent ligand 4a is involved in numerous hydrophobic interactions, such as six Pi alkyl interactions with PHE110, HIS401, HIS405, HIS411, LEU188 and MET422.But, it forms an unfavorable acceptor-acceptor with ALA189.The results of the present in silico docking simulation revealed the significant bivalent ligand 4b, which showed good hydrogen bonding interactions with the most important residues in the active site pockets of the proteins.The best confirmation of bivalent ligand 4b with MMP-9 showed hydrogen bonds with ARG424 amino acid residues, as well as several hydrophobic interactions including Pi-sigma interaction with HIS405 and PHE110, Pi-Pi stacked interaction with MET422, Pi-sigma interaction with Leu607, carbon hydrogen bonding with VAL398, and Pi alkyl interaction (8) with HIS411, LEU187, LEU188, LEU397, LEU418, TYR423, PRO421, and MET422, as depicted in Figure 4.The results of the present in silico docking simulation revealed the significant bivalent ligand 4b, which showed good hydrogen bonding interactions with the most important residues in the active site pockets of the proteins.The best confirmation of bivalent ligand 4b with MMP-9 showed hydrogen bonds with ARG424 amino acid residues, as well as several hydrophobic interactions including Pi-sigma interaction with HIS405 and PHE110, Pi-Pi stacked interaction with MET422, Pi-sigma interaction with Leu607, carbon hydrogen bonding with VAL398, and Pi alkyl interaction (8) with HIS411, LEU187, LEU188, LEU397, LEU418, TYR423, PRO421, and MET422, as depicted in Figure 4.
exhibited that it engaged in the formation of hydrogen bonds with GLU111 and LEU187 amino acid residues (Figure 3).Additionally, bivalent ligand 4a is involved in numerous hydrophobic interactions, such as six Pi alkyl interactions with PHE110, HIS401, HIS405, HIS411, LEU188 and MET422.But, it forms an unfavorable acceptor-acceptor with ALA189.The results of the present in silico docking simulation revealed the significant bivalent ligand 4b, which showed good hydrogen bonding interactions with the most important residues in the active site pockets of the proteins.The best confirmation of bivalent ligand 4b with MMP-9 showed hydrogen bonds with ARG424 amino acid residues, as well as several hydrophobic interactions including Pi-sigma interaction with HIS405 and PHE110, Pi-Pi stacked interaction with MET422, Pi-sigma interaction with Leu607, carbon hydrogen bonding with VAL398, and Pi alkyl interaction (8) with HIS411, LEU187, LEU188, LEU397, LEU418, TYR423, PRO421, and MET422, as depicted in Figure 4.The results of the present in silico docking simulations show the strongest docking binding energy of the divalent ligand 4c to MMP-9, which exhibits good multiple interactions with the most important residues in the active site pocket of the protein.The best confirmation of bivalent ligand 4c with MMP-9 showed several hydrophobic interactions, including Pi-Pi T-shaped interaction with HIS401, Pi-Pi stacked interaction (5) with MET422, HIS401, HIS405, HIS411, and PHE110, carbon hydrogen bond (2) with ARG424 and GLN402, amide-Pi stacked interaction with (5) MET422, HIS401, HIS405, HIS411, and PHE110, and Pi alkyl interaction (8) with HIS401, LEU187, LEU188, LEU397, LEU418, ARG424, PRO430, and VAL398, as depicted in Figure 5.
Docking results show strong docking binding of 4d to MMP-9 and the presence of multiple interaction forces (Figure 6).The docking results of bivalent ligand 4d exhibited that it engaged in the formation of H bonds with GLN402 amino acid residues.Also, it was engaged in the formation of many hydrophobic interactions such as two carbon hydrogen bonds with LEU418 and TYR423, Pi-Pi stacked interaction (2) with HIS401 and MET422, amide-Pi stacked interaction with (2) HIS401 and MET422, and Pi alkyl interaction (8) with HIS401, HIS405, HIS411, LEU187, LEU397, LEU418, ARG424, and PRO430.
(5) with MET422, HIS401, HIS405, HIS411, and PHE110, carbon hydrogen bond (2) with ARG424 and GLN402, amide-Pi stacked interaction with (5) MET422, HIS401, HIS405, HIS411, and PHE110, and Pi alkyl interaction (8) with HIS401, LEU187, LEU188, LEU397, LEU418, ARG424, PRO430, and VAL398, as depicted in Figure 5. Docking results show strong docking binding of 4d to MMP-9 and the presence of multiple interaction forces (Figure 6).The docking results of bivalent ligand 4d exhibited that it engaged in the formation of H bonds with GLN402 amino acid residues.Also, it was engaged in the formation of many hydrophobic interactions such as two carbon hydrogen bonds with LEU418 and TYR423, Pi-Pi stacked interaction (2) with HIS401 and MET422, amide-Pi stacked interaction with (2) HIS401 and MET422, and Pi alkyl interaction (8) with HIS401, HIS405, HIS411, LEU187, LEU397, LEU418, ARG424, and PRO430.Analysis of the docking results revealed that the docking studies were consistent with the antitumor cell experiment.From the inspection of the docking results, bivalent ligand 4e can fit perfectly into the catalytic binding pocket of MMP-9.As presented in Figure 7, bivalent ligand 4e was incorporated in the formation of one hydrogen bond with  Docking results show strong docking binding of 4d to MMP-9 and the presence of multiple interaction forces (Figure 6).The docking results of bivalent ligand 4d exhibited that it engaged in the formation of H bonds with GLN402 amino acid residues.Also, it was engaged in the formation of many hydrophobic interactions such as two carbon hydrogen bonds with LEU418 and TYR423, Pi-Pi stacked interaction (2) with HIS401 and MET422, amide-Pi stacked interaction with (2) HIS401 and MET422, and Pi alkyl interaction (8) with HIS401, HIS405, HIS411, LEU187, LEU397, LEU418, ARG424, and PRO430.Analysis of the docking results revealed that the docking studies were consistent with the antitumor cell experiment.From the inspection of the docking results, bivalent ligand 4e can fit perfectly into the catalytic binding pocket of MMP-9.As presented in Figure 7, bivalent ligand 4e was incorporated in the formation of one hydrogen bond with Analysis of the docking results revealed that the docking studies were consistent with the antitumor cell experiment.From the inspection of the docking results, bivalent ligand 4e can fit perfectly into the catalytic binding pocket of MMP-9.As presented in Figure 7, bivalent ligand 4e was incorporated in the formation of one hydrogen bond with GLN402 and Pi-Pi T-shaped interaction with PHE110, as well as Pi alkyl interaction (5) with HIS401, HIS405, HIS411, LEU188, and MET422.Hydrogen bonding was also evaluated for the interaction of bivalent ligands 4a-4e with these four targets.The amino acid residues involved in hydrogen bonding at the binding sites of the bivalent ligands 4a-4e in BCL-2, CDK-4, VEGFR-2, and MMP-9 are summarized in Table 2.The total binding strength is the result of many types of bonding, including ionic, hydrophobic interactions and van der Waals forces, although hydrogen bonding is the major contributor.Hydrogen bonding also depends on the composition and three-dimensional arrangement of the contacting amino acid residues at the prominent and active binding sites.
Table 2. Binding energies and amino acid residues involved in hydrogen bonding of bivalent ligands 4a-4e within the binding sites of BCL-2, CDK-4, VEGFR-2, and MMP-9.Hydrogen bonding was also evaluated for the interaction of bivalent ligands 4a-4e with these four targets.The amino acid residues involved in hydrogen bonding at the binding sites of the bivalent ligands 4a-4e in BCL-2, CDK-4, VEGFR-2, and MMP-9 are summarized in Table 2.The total binding strength is the result of many types of bonding, including ionic, hydrophobic interactions and van der Waals forces, although hydrogen bonding is the major contributor.Hydrogen bonding also depends on the composition and three-dimensional arrangement of the contacting amino acid residues at the prominent and active binding sites.Binding energies and theoretical inhibition constants (Ki) of artemisinin, dihydroartemisinin, and compounds 4a-4e within the binding sites of BCL-2, CDK-4, VEGFR-2, and MMP-9 are shown in Table 3.In Table 3, the binding energies of the target compounds with each target protein were significantly stronger than that of ART and DHA, except for compound 4c, which showed lower binding activity to the VEGFR-2 protein than ART and DHA.The results were similar to those of the antitumor cell proliferation assay, indicating that compounds 4a-4e have good interaction power with the four target proteins.However, the cellular activity of the target compounds 4b and 4c was opposite to the docking activity, probably because the structural backbone of the compounds was too large to penetrate the cell membrane and could not eventually intervene to inhibit the tumor cells.

Chemistry
The commonly used reagents were purchased from Sinopharm Group Chemical Reagent Co., Ltd. and used without further purification.Thin-layer chromatography (TLC, silica gel HSGF254, Yantai Jiang you Silicone Development Co., Ltd.(Yantai, Shandong, China)) was used to monitor for completeness of the reaction visualized by UV light (λ = 254 nm or λ = 365 nm).The target compounds were purified by silica gel column chromatography.NMR spectra were recorded on the Bruker Avance III spectrometer at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR, with tetramethylsilane (TMS) as the internal standard, and DMSO-d6, CDCl 3 , or CD 3 OD as the solvent.Coupling constant (J) values were estimated in hertz (Hz).Splitting patterns are designated as follows: s, singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet.Mass spectra were measured on an LCMS 6400 Series Triple Quadrupole Mass Spectrometer (Agilent).Intermediates 1a-1d, 2a, 2b, 3a, and 3c were synthesized according to the references [36][37][38][39], and the detailed synthesis process and details can be seen in Supplementary Materials.

Cytotoxicity In Vitro
Cell cytotoxicity was determined by methyl thiazolyl tetrazolium (MTT) assay.Four tumor cell lines, MCF-7, HepG-2, HCT-116, and BGC-823, were inoculated at 5 × 10 3 cells per well in a 96-well plate.The cells were incubated with compounds for 48 h at five different concentrations.Then, the cells were incubated with 5 mg/mL MTT reagent and incubated for 4 h at 37 • C.After 4 h, MTT supernatant was removed and DMSO (100 µL, Sigma, St. Louis, MO, USA) was added.The absorbance of formazan was measured at 490 nm (OD 490 ).Cell viability was calculated from three independent experiments.The growth inhibitory rates of the complexes were calculated as (OD control − OD test )/OD control × 100%.IC 50 values were calculated using the percentage of growth versus untreated control.
The closer the candidate conformation is to the conformation of the natural complex under ideal conditions, the lower the evaluation score derived from the scoring function, and the output of LeDock is the binding energy ∆G.Therefore, using the LeDock.win32software molecular docking, the binding free energy of artemisinin, dihydroartemisinin, and compounds 4a-4e was predicted to interact with the four target proteins, and combined with the scoring function of the original ligand of each target site as the threshold value.The target with the lowest binding energy to the target compound was selected.The Ki of the target compound for the potential target was further calculated according to the theoretical inhibition constant (Ki) formula.

Conclusions
In summary, five artemisinin bivalent ligands were designed, synthesized, and tested for their antitumor activity against four human cancer cell lines, and verified by the "single ligand-multiple target" docking model.The experimental results showed that the antitumor

Table 3 .
Docking data of compounds and target proteins.