Growth inhibition and anticancer activity of phenoxyartemisinin derivatives in various cancer cell lines
Article information
Abstract
Cancer remains a significant global health challenge, underscoring the need for novel antineoplastic agents with improved efficacy and reduced toxicity. Artemisinin, a naturally occurring sesquiterpene lactone, and its derivatives have demonstrated promising anticancer potential. In this study, we synthesized and evaluated the anticancer activity of C-10-substituted artemisinin derivatives using the MTT assay across six cancer cell lines: DLD-1, SNU-16, MCF7, HepG2, A549, and U87. Growth inhibition 50% (GI50) values were determined via nonlinear regression analysis. Among the tested compounds, 4b, 5d, 4e, and 4f exhibited potent anticancer activity, with GI50 values below 0.1 μM in DLD-1 cells and below 0.18 μM in SNU-16 cells. Notably, 5d and 4e demonstrated potent inhibitory effects in U87 glioblastoma cells at concentrations as low as 0.2 μM. These compounds demonstrated broad-spectrum anticancer effects across all tested cell lines, highlighting their potential as promising candidates for further development. Furthermore, the structure-activity relationship analysis suggests that specific substitutions at the 10-position of artemisinin may significantly enhance its anticancer efficacy.
INTRODUCTION
Cancer remains a major global health concern, with incidence rates varying across different types of neoplasms. In 2020 alone, approximately 600,000 people died from cancer in the United States, making it the second leading cause of death after cardiovascular diseases [1]. The most frequently diagnosed cancers in high-income countries include lung cancer, colorectal cancer, breast cancer, melanoma, and prostate cancer. Although cancer incidence and mortality in children and adolescents are relatively low, they increase exponentially with age. Early detection plays a particularly important role in reducing the mortality rates of cancers such as female breast cancer, cervical cancer, colon and rectal cancer, lung cancer, and prostate cancer. With the increasing burden of cancer, significant advancements have been made in treatment modalities, including chemotherapy, targeted therapy, and immunotherapy [2]. However, the adverse effects associated with these treatments remain a critical challenge, highlighting the need for novel anticancer agents with improved efficacy and reduced toxicity.
Artemisinin (1), a naturally occurring endoperoxide sesquiterpene lactone derived from Artemisia annua L., has been widely used as an antimalarial drug.3 Over the years, various semisynthetic 10-substituted derivatives of artemisinin, including dihydroartemisinin (2), artemether, arteether, artesunate, and artemisone, have been developed to enhance its therapeutic efficacy [4-7]. Chen et al. [8] reported that dihydroartemisinin (2) and acetal-type artemisinin derivatives (3) exhibit both antiangiogenic and antitumor activity in in vitro models of angiogenesis.
In our laboratory, we have also demonstrated that artemisinin, dihydroartemisinin, and acetal-type artemisinin derivatives (3) possess antiangiogenic activity.
In this study, we investigate the anticancer properties of phenoxyatemisinin (4, 5), a subclass of acetal-type artemisinin compounds (3), by evaluating their growth-inhibitory effects on multiple cancer cell lines. Given their established role as potent antimalarial agents and their emerging potential in oncology, artemisinin-based compounds offer a promising avenue for cancer therapy. By systematically analyzing their effects on cancer cell proliferation, we aim to contribute to the development of novel therapeutic strategies with improved safety and efficacy.
METHODS
1. Reagents and material
Trimethylsilyl triflate (TMSOTf, 1.0 mmol) and silver perchlorate (AgCIO4, 0.2 mmol) were added to a stirred solution of dihydroartemisinin (284 mg, 1.0 mmol) and phenol (186 mg, 2.0 mmol) in CH2Cl2 (50 mL) at room temperature. The solution was stirred for 12 hours, then diluted with CH2Cl2 (100 mL), and washed with saturated NaHCO3 and brine. The organic layer was separated, dried over MgSO4, filtered, and evaporated to dryness. The crude product was purified by flash chromatography (hexane:EtOAc=5:1) yielding 210 mg (52%) of compound 4a.
Compound 4a means 1Η-NMR (300 MHz, CDCl3) δ 7.25 (t, J=4.4 Hz, 2H), 7.11 (d, J=5.8 Hz, 2H), 7.00 (t, J=7.1 Hz, 1H), 5.49 (s, 1H), 5.05 (d, J=9.5 Hz, 1H), 2.73 (m, 1H), 2.42 (dt, 1H), 2.10 (m, 10H), 1.43 (s, 3H), 0.99 (d, J=3.2 Hz, 3H), 0.97 (d, J=1.8 Hz, 3H) ppm; 13C-NMR (75 MHz, CDCl3) δ 145, 129, 122, 117, 104.5, 100, 99, 91, 52, 45, 37, 36, 33, 26, 25, 22, 20, 12.5 ppm.
2. Cell culture
Human cancer cells lines used in this study included DLD-1 (colorectal adenocarcinoma), SNU-16 (gastric carcinoma), MCF7 (breast adenocarcinoma), HepG2 (hepatocellular carcinoma), A549 (lung carcinoma), and U87 (glioblastoma), all of which were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). As the cell lines were obtained from ATCC, Institutional Review Board (IRB) approval was not required. Cells were cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cultures were incubated at 37℃ in a humidified atmosphere containing 5% CO2.
3. MTT assay
Cancer cells were plated in 96-well culture plates at a density of 5,000 cells/well in a final volume of 100 μL of DMEM containing 10% FBS. After a 4-hour preincubation, cells were treated with serial concentrations of artemisinin derivatives for 72 hours. Following treatment, cells were incubated for an additional 4 hours at 37°C with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/mL). The culture supernatant was aspirated, and 100 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate spectrophotometer (SpectraMax 250; Molecular Devices, San Jose, CA, USA). Each assay was performed in triplicate. Growth inhibition 50% (GI50) values were calculated using nonliner regression analysis of the sigmoidal dose-response curve, with R2>0.95, using GraphPad Prism software (GraphPad Software, Boston, MA, USA).
RESULTS
To evaluate anticancer activity, phenoxyartemisin derivatives (4, 5), semi-synthetic acetal-type artemisinin compounds, were synthesized from dihydroartemisinin (2), which was itself obtained from artemisinin (1) (Fig. 1). As shown in Fig. 2, C-10 substituted artemisinin derivatives were produced via the reaction of dihydroartemisinin (2) with various phenols (2 eq) under catalytic conditions using TMSOTf (1 eq) and AgCIO4 (0.2 eq) at room temperature for 12 hours [9]. Given the similarity between the C-10 position of compound 2 and the anomeric center of a carbohydrate, acetalization with various phenol compounds (a-j) yielded a major α-anomer (5) and a minor β-anomer (4). To assess the anticancer activity of the resulting C-10 substituted artemisinin derivatives, along with reference compounds (artemisinin and dihydroartemisinin), a cell proliferation inhibition assay was conducted. GI50 values (μM) were determined using the MTT assay across multiple cancer cell lines, including DLD-1, SNU-16, MCF7, HepG2, A549, and U87 [10,11]. The results are summarized in Table 1.
Chemical structures of artemisinin (1), dihydroartemisinin (2), and phenoxyartemisinin (3). Natural artemisinin (1), isolated from Artemisia annua L., has served as a lead compound in the development of agents with high activity against drug-resistant malaria and anticancer effects. In the course of drug discovery research, phenoxyartemisin (3) was developed as a semi-synthetic, acetal-type artemisinin derivative of dihydroartemisinin (2), which itself is derived from artemsinin (1).
Reagents and conditions. (a) ROH (2 eq), TMSOTf (1 eq), AgClO4 (0.2 eq), CH2Cl2, room temperature, 12 hours. A separable diastereomeric mixture of 10α- (5) and 10β-phenoxyartemisinins (4) was prepared by reacting dihydroartemisinin (2) with phenol derivatives (a-j) under the catalysis of TMSOTf and AgCIO4. TMSOTf: trimethylsilyl triflate.
Proliferation inhibition assay results for various cancer cell lines
Artemisinin exhibited relatively high GI50 values (13.4-40.6 μM) across most cancer cell lines, indicating weak anticancer activity. In contrast, dihydroartemisinin (DHA) demonstrated significantly lower GI50 values (0.5-9.0 μM), suggesting a stronger inhibitory effect on cancer cell proliferation. Most of the synthesized derivatives showed lower GI50 values than artemisinin and exhibited inhibitory activity comparable to or exceeding that of DHA. Among the tested compounds, 4b, 5d, 4e, and 4f demonstrated the strongest anticancer activity, with GI50 values below 1 μM in most cancer cell lines. Compounds 4b, 5d, and 4e were particularly effective against DLD-1, SNU-16, and U87 cells, while 4f exhibited potent activity against DLD-1 and SNU-16. In the DLD-1 cell line, 4b, 5d, 4e, and 4f demonstrated GI50 values of 0.1 μM, indicating strong anticancer effects even at very low concentrations. Similarly, in the SNU-16 cell line, compounds 4b, 5d, 4e, and 4i also exhibited GI50 values of 0.1 μM. In U87 cells, a brain tumor cell line, compounds 5d and 4e displayed particularly potent effects, with GI50 values of 0.2 μM, outperforming other compounds in this model. In MCF7 and HepG2 cells, compound 4e was the most effective, with GI50 values of 1.3 μM and 1.1 μM, respectively. However, in A549 cells, the anticancer activity of the 10-substituted artemisinin derivatives appeared to be lower, as indicated by the higher GI50 values (3.6-39.57 μM). Notably, compounds 4b, 5d, 4e, and 4f exhibited strong anticancer activity at low concentrations across multiple cancer cell lines, indicating broad-spectrum efficacy and potential for further development.
Compounds 4a, 4d, 5b, and 5f displayed moderate growth inhibition, with GI50 values ranging from 0.5 to 12.4 μM, depending on the cancer cell type. In contrast, compounds 4g and 5g exhibited significantly higher GI50 values (>17.5 μM), suggesting lower potency or selective activity. Similarly, compound 4j demonstrated weaker inhibition, with GI50 values ranging from 13 to 39 μM. Some derivatives exhibited cell-line-specific cytotoxicity. For instance, DHA and the most potent derivatives (4b, 5d, 4e, and 4f) demonstrated stronger inhibitory effects against SNU-16 and U87, as indicated by consistently low GI50 values. Conversely, compound 4g exhibited weak inhibition across most cancer cell lines, with GI50 values exceeding 43 μM, suggesting that certain structural modifications may reduce anticancer potency.
The strong cytotoxicity observed for 4b, 5d, 4e, and 4f indicates that specific substitutions at the 10-position of artemisinin can significantly enhance anticancer activity. These compounds exhibit cytotoxic effects comparable to or greater than DHA and represent promising candidates for future anticancer drug development. The structure-activity relationship findings support the conclusion that targeted modifications at the 10-position of artemisinin may significantly enhance its anticancer potential.
DISCUSSION
Cancer is characterized by genetic mutations and uncontrolled cell proliferation. Thus, potential therapeutic agents are commonly evaluated for their ability to affect the cell cycle, induce cytotoxicity, or inhibit cell growth. The initial studies on the anticancer effects of artemisinin and its derivatives were conducted in vitro using various cancer cell lines, including oral, pancreatic, prostate, breast, liver, and ovarian cancer cells [12-16].
Other artemisinin derivatives have also been investigated for their effects on cancer cell lines. One major mechanism by which biologically active compounds exert anticancer effects is through the induction of programmed cell death [17]. Artemisinin derivatives have been shown to promote cytotoxicity via a mitochondria-mediated pathway, characterized by a reduction in mitochondrial membrane potential, increased intracellular reactive oxygen species (ROS) and Ca2+ levels, resulting in G1 phase arrest and acceleration of apoptosis [18].
Recent studies have demonstrated that artemisinin and its derivatives exhibit promising anticancer properties by inducing apoptosis, inhibiting angiogenesis, and modulating oxidative stress in cancer cells [19,20]. Notably, the introduction of functional groups at specific positions, such as triazole substitutions via Huisgen 1,3-dipolar cycloaddition, has produced compounds with enhanced cytotoxicity against various cancer cell lines, including DLD-1, U-87, HeLa, SiHa, A172, and B16 [21,22].
Based on our preliminary results, we hypothesized that many acetal-type artemisinin derivatives, specifically phenoxyartemisinins (4, 5), would exhibit inhibitory effects on cancer cell proliferation and demonstrate greater activity than non-acetal artemisinin (1). Among the synthesized compounds, 10α-substituted (5) and 10β-substitued (4) derivatives, the 10β-epimers generally showed higher inhibitory potency than their 10α counterparts. It is particularly noteworthy that these compounds, differing only in C-10 stereochemistry, exhibited substantial differences in anticancer activity. As reported by Chen et al. [8], phenoxyartemisinins may serve as lead compounds in anticancer drug development.
Compared with findings from our previous work, compounds 4e and 4f demonstrated anticancer activity comparable to the conventional chemotherapeutic agent paclitaxel [21,22]. This suggests that substituted artemisinin derivatives may suppress cancer cell growth similar to paclitaxel. Compounds 4e and 4f displayed GI50 values below 0.2 μM in multiple cancer cell lines, similar to reported values for paclitaxel in comparable models. This potent cytotoxicity may be attributed to structural modifications at the C-10 position of artemisinin, particularly the inclusion of electron-donating or hydrophobic phenoxy substituents that could enhance cellular uptake or target-binding affinity. While paclitaxel primarily exerts its effects by stabilizing microtubules and inhibiting mitosis, artemisinin derivatives (including 4e and 4f) are known to induce ROS, disrupt mitochondrial membrane potential, and trigger apoptosis through iron-mediated activation [23-25]. Despite differing mechanisms of action, the comparable efficacy may arise from convergent disruption of cancer cell survival pathways. Further mechanistic studies, such as ROS quantification, apoptosis marker analysis, or tubulin polymerization assays, will be essential to elucidate the underlying pathways of these compounds.
Regarding the structure-activity relationship (SAR), derivatives with methoxy or alkyl-substituted phenoxy groups at the C-10 position (e.g., 4e and 4f) showed markedly improved activity. This observation suggests that such substituents may enhance interactions with molecular targets or improve compound stability and cell permeability. Future SAR studies focusing on electronic and steric influences at the C-10 position may lead to further optimization of anticancer activity.
In conclusion, this study assessed the anticancer activity of various 10-substituted artemisinin derivatives. Among them, 4b, 5d, 4e, and 4f exhibited exceptionally low GI50 values and potent anticancer activity. In particular, 4e and 4f demonstrated growth inhibition comparable to paclitaxel, indicating their potential as anticancer candidates. These findings highlight the therapeutic potential of artemisinin-based compounds in cancer treatment. Further studies are warranted to elucidate their precise mechanisms of action and explore their clinical applications.
Supplementary Material
Supplementary Materials 1.
Reagents and material
Notes
CONFLICT OF INTEREST
The authors report no conflicts of interest.
FUNDING
This research was supported by Regional Innovation Strategy (RIS) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2022RIS-005) and this work was supported by research fund of Catholic Kwandong University.