Novel Endoperoxide Steroid Derivatives Containing Thiazole Moiety as Potential Antitumor Agents: Design,Synthesis,and Cytotoxic Evaluation

Abstract

As part of the search for new steroid substances with potential application in oncology, a series of endoperoxide steroid derivatives (7a-n) containing a thiazole system were synthesized and evaluated for their cytotoxicities in four tumor cell lines. Among them, compound 7h exhibited the most significant cytotoxic activity against HepG2 cells (IC₅₀ = 4.54 μM), being more potent than ergosterol peroxide. Furthermore, 7h was able to promote apoptosis in HepG2 cells and decrease the mitochondrial membrane potential, accompanied by activating the expression of cytochrome c, caspase-9, caspase-3, and Bax, while Bcl-2 expression was suppressed. The molecular mechanism may refer to the mitochondrial apoptotic pathway. On the whole, the above results incentivize the further study of 7h derivatives as potent anticancer agents.

Graphical Abstract

This is a visual representation of the abstract.

Keywords

natural endoperoxide ergosterol peroxide thiazole dehydroepiandrosterone anticancer activity

Introduction

Cancer is caused by malignant cell proliferation and is recognized worldwide as a disease with a high mortality rate.^1,2 Chemotherapy remains one of the main effective cancer treatments available today, but serious side effects greatly limit the clinical use of many chemotherapy drugs.^3,4 Therefore, the development of new chemotherapy drugs with low toxicity and high efficiency is a meaningful and important task in the field of pharmaceutical chemistry. Natural active products have a very long history of medicinal use, and structural modification based on a natural active product skeleton is one of the main sources and methods of production of novel anticancer drugs.⁵

Natural endoperoxides are a class of compounds with peroxy-bridged structures (-O-O-) that are abundant and biologically diverse in nature, and many endoperoxide natural products have been shown to have significant antitumor activities and potential medicinal values.⁶ Artemisinin,⁷ gracilioether A,⁸ schinalactone A,⁹ and other natural peroxides have been verified to have significant anticancer activities. It is desirable that the novel structure of endoperoxide is utilized to provide a research direction for the research and development of new pharmacophores or new drugs.

Ergosterol peroxide (EP) is a natural endoperoxide steroid that was extracted from the traditional Chinese medicine Ganoderma lucidum,^10,11 which has broad-spectrum antitumor activity (Figure 1).^12‐18 As a lead compound with potential research value, appropriate structural modification of EP is the key to increase its antitumor activity.

Figure 1.

Structures of ergosterol peroxide (EP) and its derivatives.

Previously, we have prepared a series of C-17 modified derivatives of EP, and many of them exhibited strong inhibitory efficacy against different kinds of tested tumor cell lines, such as compound A in Figure 1.^19‐23 In addition, we have reported that the introduction of acrylate and propionate groups into the C-3 hydroxy group of EP has strong efficacy against three kinds of human tumor cell lines. Among them, compound B showed significant cytotoxicity against the HepG2 cell line with an IC₅₀ value of 2.70 μM (Figure 1).²⁴ We also successfully introduced a carbamate side chain into the C-3 hydroxy of EP, and most of these synthetic ester derivatives exhibited nanomole cytotoxic activity, such as compound C in Figure 1.²⁵

Thiazole is a five-membered heterocyclic molecule containing nitrogen and sulfur atoms and is the central reactive group in many natural products.²⁶ The biological activities of thiazoles and compounds containing thiazole structures cover a wide range of aspects, including antitumor, antioxidant, anti-inflammatory, antiviral, antibacterial, and other activities, with antitumor activity being particularly prominent.^27‐31 Compounds containing thiazole can exhibit anti-cancer activities through different pathways of action. Clinically applied antitumor agents containing thiazole nuclei are partially exhibited in Figure 2. Therefore, the synthesis and pharmacological studies of compounds containing thiazole and other multitarget groups are of great significance.

Figure 2.

Representative antitumor drugs with thiazole structure.

Based on the above-mentioned encouraging results, our research group investigated the further modification of EP by introducing richer types of heterocyclic substituents. In the present work, fourteen endoperoxide steroid derivatives (7a-n) containing thiazole moieties at the C-17 position were designed and synthesized. The cytotoxicity of all obtained compounds was evaluated using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay in four different human cancer cell lines (HCT-116, HepG2, MCF-7, and A549). In addition, the action mechanism of the most active compound was explored.

Results and Discussion

Chemistry

The general synthesis route of new endoperoxide steroid derivatives 7a-n is shown in Scheme 1. Dehydroepiandrosterone (DHEA, 1) was acetylated with acetic anhydride to give intermediate 2. This was treated with N-bromosuccinimide (NBS) and 2,4,6-collidine in sequential order to give intermediate 3. Then, this was hydrolyzed in the presence of potassium hydroxide to obtain intermediate 4. Subsequently, intermediate 4 was subjected to photoirradiation with phloxine B under highly pure oxygen to give endoperoxide intermediate 5, which was reacted with 4-methyl-3-thiosemicarbazide to obtain semicarbazone intermediate 6. Finally, intermediate 6 was reacted with a variety of 2-bromoacetophenones to obtain compounds 7a-n. The chemical structures of all new compounds 7a-n were characterized by spectroscopic methods, including ¹H and ¹³C NMR spectroscopy and HRMS.

Scheme 1.

Synthesis of endoperoxide steroid derivatives 7a-n. Reagents and conditions: (i) collidine, Ac₂O, DCM, rt, 93%; (ii) N-iodosuccinimide, cyclohexane, reflux, 1 h; xylene, 2,4,6-trimethylpyridine, reflux, 3h, 32%; (iii) light petroleum, MeOH, KOH, 80 °C, 1 h, 85%; (iv) oxygen, phloxine B, MeOH, light, 0 °C, 0.5 h, 74%; (v) 4-methyl-3-thiosemicarbazide, AcOH, EtOH, acetic acid, 60 °C, 8 h, 55%; (vi) 2-bromoacetophenones, EtOH, 60 °C, 2-3 h, 77%-86%.

Biology

In vitro cytotoxic effects of all new compounds 7a-n against four different human cancer cell lines were carried out using MTT assays. Mitomycin C and EP were used as positive drugs. The IC₅₀ values of 7a-n are listed in Table 1. The MTT results showed that some of the new derivatives (7g and 7h) have significant cytotoxic effects against all four tested tumor cell lines, and have more potential than lead compound EP.

Table 1.

In Vitro Cytotoxic Activities of Compounds 7a-n.

Compound	R	IC₅₀ (μM)
Compound	R	HepG2	MCF-7	HCT-116	A549
7a	H	27.60 ± 1.33	29.22 ± 1.04	46.34 ± 2.36	50.78 ± 3.25
7b	4-CF₃	18.72 ± 0.94	21.52 ± 0.83	26.30 ± 1.09	33.85 ± 1.42
7c	4-CN	11.15 ± 0.58	14.35 ± 0.79	19.88 ± 0.81	25.27 ± 0.99
7d	4-OCH₃	25.64 ± 1.26	28.47 ± 1.22	33.38 ± 1.87	42.30 ± 2.26
7e	2-NO₂	9.10 ± 0.35	15.22 ± 0.84	18.57 ± 1.13	22.12 ± 1.34
7f	4-OCF₃	26.40 ± 1.57	32.45 ± 1.74	47.43 ± 2.58	53.11 ± 2.83
7g	4-NO₂	6.22 ± 0.82	7.46 ± 0.55	8.13 ± 0.73	9.84 ± 1.04
7h	3-NO₂	4.54 ± 0.60	5.73 ± 0.87	7.71 ± 0.62	8.65 ± 0.71
7i	4-F	12.66 ± 1.08	13.46 ± 0.79	19.79 ± 1.50	25.86 ± 1.68
7j	4-Br	14.33 ± 0.95	16.43 ± 1.20	25.42 ± 1.25	33.17 ± 1.48
7k	4-CH₃	22.62 ± 0.82	20.45 ± 0.96	27.18 ± 0.80	42.06 ± 2.32
7l	3-OCH₃	28.04 ± 1.24	32.12 ± 1.75	43.58 ± 2.44	56.64 ± 2.78
7m	4-Cl	8.25 ± 0.42	12.60 ± 0.72	14.58 ± 0.90	24.36 ± 0.79
7n	4-OH	35.04 ± 1.39	40.60 ± 1.62	55.16 ± 2.30	54.10 ± 2.70
EP	—	21.20 ± 0.76	16.24 ± 0.57	28.15 ± 1.42	21.30 ± 1.35
Mitomycin C	—	20.34 ± 0.85	15.74 ± 0.58	14.02 ± 0.46	17.50 ± 0.84

For the HepG2 cell line, most of the derivatives showed conspicuous cytotoxic activities to EP (IC₅₀ = 21.20 μM) against HepG2 cells. Among them, compounds 7e (IC₅₀ = 9.10 μM), 7g (IC₅₀ = 6.22 μM), 7h (IC₅₀ = 4.54 μM), and 7m (IC₅₀ = 8.25 μM) were the most promising endoperoxide derivatives. Moreover, compound 7h containing a 3-methyl-4-(3′-nitrophenyl)thiazol side chain was about 4.6-fold stronger than EP. For the MCF-7 cell line, compounds 7c (IC₅₀ = 14.35 μM), 7g (IC₅₀ = 7.46 μM), 7h (IC₅₀ = 5.73 μM), 7i (IC₅₀ = 13.46 μM) and 7m (IC₅₀ = 12.60 μM) showed stronger cytotoxicity than EP (IC₅₀ = 16.24 μM). Among them, compound 7h had the most potent inhibitory effect, which was 2.8-fold more potent than EP. For most of the compounds, the inhibitory activity against MCF-7 cells was not as remarkable as that against HepG2 cells. For the HCT-116 cell line, compounds 7e, 7g, 7h, 7i, and 7m showed moderate cytotoxicity against HCT-116 cells, with IC₅₀ values of 7.71–19.79 μM. Among them, compounds 7g (IC₅₀ = 8.13 μM) and 7 h (IC₅₀ = 7.71 μM) had the most remarkable inhibitory effect, which was almost 3.5- and 3.7-fold stronger than EP (IC₅₀ = 28.15 μM). For the A549 cell line, most of the compounds were not very active, with only compounds 7g (IC₅₀ = 9.84 μM) and 7h (IC₅₀ = 8.65 μM) exhibiting moderate to potent cytotoxicity compared to EP (IC₅₀ = 21.30 μM).

Taken together, the above results suggested that both the thiazole moiety and aromatic ring conjugated substituents are significant for the cytotoxicity of the novel endoperoxide steroid derivatives. In addition, compounds 7c, 7e, 7g, 7h, 7i, 7j, and 7m, with electron-withdrawing groups (CN, NO₂, Br, Cl and F) on the benzene ring, showed more ideal cytotoxicity. Notably, both compounds 7g and 7h had the most potent inhibitory effect in all four tested cancer cell lines. Given their potent inhibitory effects, compound 7h was selected to explore further its possible action mechanism in HepG2 cells.

To assess whether 7h was able to promote apoptosis in HepG2 cells, apoptosis-associated cellular morphological changes were detected by the acridine orange (AO) and ethidium bromide (EB) double-staining method. After being treated with different concentrations of 7h (0, 2, 4, and 8 μM) for 48 h, the AO/EB double staining method was used to detect the cellular morphological changes. As shown in Figure 3, HepG2 cells generated obvious bright red nuclear vesicles, nuclear roundness, and nuclear shrinkage in a dose-dependent manner. The morphology of untreated cells was almost intact and stained bright green. In addition, in order to quantify the percentage of apoptosis in HepG2 cells induced by 7h, Annexin V (AV) and propidium iodide (PI) double staining was carried out. As shown in Figure 4, after treatment with 7h at concentrations of 2, 4, and 8 μM for 48 h, the percentage of apoptotic cells markedly increased from 12.90% to 24.55% in a dose-dependent manner.

Figure 3.

The effect of 7h on the cellular morphological changes of HepG2 cells was determined by the AO/EB method. Abbreviations: AO, acridine orange; EB, ethidium bromide.

Figure 4.

Compound 7h induced HepG2 cell apoptosis. (A) HepG2 cells were treated with 7h and then apoptosis was detected by annexin V-FITC/PI staining. (B) Quantitative analysis of apoptotic cells after treatment with 7h. Data are represented as average ± SD (n = 3). *P < .05, **P < .01.

The loss of mitochondrial membrane potential (MMP) is an important sign of early events in the tumor cells apoptosis cascade. Hence, we decided to evaluate the effect of 7h on the MMP of HepG2 cells by MitoProbe JC-1 staining. As shown in Figure 5, HepG2 cells were treated with different concentrations of 7h (0, 2, 4, and 8 μM) for 48h, and the percentage of low MMP cells increased correspondingly from 1.69% to 36.02%, which proved that 7h led to the collapse of MMP and then induced tumor cell apoptosis.

Figure 5.

Compound 7h induced HepG2 cells mitochondrial depolarization. (A) HepG2 cells were treated with 7h and then detected by JC-1 staining. (B) Quantitative analysis of the ratio of JC-1 monomers. Data are represented as average ± SD (n = 3). *P<.05, **P<.01, ***P<.001.

It is well known that changes in MMP trigger cytochrome c release, and subsequently apoptosis via caspase-9 activation. Here, we measured the expression levels of mitochondrial apoptosis related proteins using Western blotting. As shown in Figure 6, the content of cytochrome c in the cytoplasm increased, while cytochrome c triggered the activation of cleaved-caspase-9. Accordingly, as the end point of the signaling pathway, after treatment with 7h, the expression of apoptosis-related protein Bax began to increase, while the Bcl-2 expression was significantly suppressed. These effects were all achieved in a concentration-dependent manner. These results illustrate that 7h was able to induce HepG2 cell death through the mitochondrial apoptosis pathway.

Figure 6.

Effects of 7h on the expression of apoptosis-related proteins. (A) The expressions of related proteins in HepG2 cells were analyzed by Western blotting. (B) Quantitative analysis of the related protein expression. Data are represented as average ± SD (n = 3). *P < .05; **P < .01.

Experimental

Chemistry

All chemical reagents and materials were commercially available from Energy Chemical (Zesheng Technology Co., LTD). The reaction process was monitored by TLC (Rongman Biotechnology Co., Ltd). The crude product was purified by flash column chromatography (200-300 mesh silica gel, Yantai Chemical Industry Research Institute). Melting points (mp) of new derivatives were obtained with an MP120 melting point apparatus (Haineng Electrical Co., Ltd). NMR spectra were recorded using an AVANCE NEO 600 Bruker spectrometer (Bruker Optics). HRMS data were measured on a Waters G2-XS UPLC QTOF (Waters Corporation).

Syntheses

Synthesis of Intermediate 2

DHEA (1, 3.00 g, 10.4 mmol) in pyridine (50 mL) was added to DMAP (0.1 g, 1 mmol) and acetic anhydride (1.00 mL, 12.0 mmol). The reaction mixture was stirred at room temperature for 4–6 h. The organic layer was washed with distilled water and saturated brine, and dried with Na₂SO₄. The crude product was purified by silica gel column chromatography to afford intermediate 2 as a white solid (3.2 g, yield 93%).¹⁹

Synthesis of Intermediate 3

Intermediate 2 (1.60 g, 4.80 mmol), NBS (0.50 g, 2.80 mmol) and AIBN (0.10 g, 0.60 mmol) were placed in a 200 mL flask and cyclohexane (100 mL) was added as solvent. The reaction system was heated to 100 °C and refluxed for 1 h until no new precipitate was generated. The precipitate in the reaction system was removed by filtration. The organic solvent was evaporated to obtain a brown solid. Subsequently, this was dissolved in xylene (100 mL) and 24,6-collidine (20 mL). The reaction system was reacted at 135 °C for 4 h, then filtered and the solvent collected. The solvent was evaporated, and the residue recrystallized in methanol to give compound 3 as a pale brown solid (0.5 g, yield 32%).¹⁹

Synthesis of Intermediate 4

A solution of intermediate 3 (0.50 g, 1.50 mmol) and potassium hydroxide (0.50 g, 8.90 mmol) in 80 mL methanol was heated at 80 °C for 1 h. After being cooled overnight to precipitate crystals, the solvent was removed by filtration. The crude crystals were washed with methanol to give compound 4 as a brown solid (0.36 g, yield 85%).¹⁹

Synthesis of Intermediate 5

A mixture of intermediate 4 (1.00 g, 3.50 mmol) and phloxine B (0.1 g, 0.1 mmol) in methanol (100 ml) under high purity oxygen was irradiated with visible light (300 W) at 0 °C for 2 h. The reaction mixture was concentrated in vacuo and the residue purified by silica gel chromatography to afford intermediate 5 as white needles (0.80 g, yield 74%).¹⁹

Synthesis of 2-(3β-Hydroxy-17H-5α,8α-epidioxyandrost-6-en-17-ylidene)-N-phenylhydrazine-1-carbothioamide (6)

Intermediate 5 (100 mg, 0.30 mmol) and 4-methyl-3-thiosemicarbazide (0.45 mmol) in 20 mL anhydrous ethanol were dissolved at 60 °C, and acetic acid (0.1 mL) was added. Then the mixture was stirred at rt for 8 h. The reaction was monitored by TLC. The mixture was concentrated in vacuo and the residue was purified by column chromatography on silica gel (dichloromethane and methanol as eluent) to afford intermediate 6. Yellow solid; yield 55%). mp 134.3–135.5 °C; ¹H NMR (600 MHz, CDCl₃) δ 9.16 (s, 1H, NH), 8.39 (s, 1H, -NH), 7.63 (d, J = 8.8 Hz, 2H, Ar-H), 7.41–7.36 (m, 2H, Ar-H), 7.23 (s, 1H, Ar-H), 6.50 (d, J = 8.6 Hz, 1H, C6-H), 6.33 (d, J = 8.5 Hz, 1H, C7-H), 3.97 (s, 1H, C3-H), 2.55 (dd, J = 19.2, 8.5 Hz, 1H), 2.41 (dt, J = 18.0, 8.6 Hz, 1H), 2.13 (d, J = 13.9 Hz, 1H), 2.05 (s, 1H), 2.00–1.89 (m, 4H), 1.84 (d, J = 13.0 Hz, 2H), 1.77 (d, J = 5.6 Hz, 1H), 1.74 (s, 1H), 1.68 (d, J = 10.1 Hz, 1H), 1.56 (dd, J = 13.4, 4.7 Hz, 2H), 1.48 (s, 1H), 1.31 (d, J = 9.8 Hz, 1H), 1.06 (s, 3H), 0.91 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 176.2, 164.5, 137.8, 136.5, 129.6, 128.8, 126.1, 124.4, 82.4, 78.7, 66.2, 53.4, 51.5, 49.2, 46.2, 37.1, 36.6, 34.7, 34.0, 30.1, 26.2, 22.8, 20.5, 18.4, 18.2; MS (ESI) m/z: [M + H]⁺ 468.2.

General Procedure for Synthesis of 7a-n

Intermediate 6 and different 2-bromoacetophenone substituents (1.5 e.q.) were added to a 50 mL round bottom flask, to which was added 20 mL anhydrous ethyl alcohol. Stirring of the mixture was continued for 2–3 h at 60 °C. TLC followed the reaction process to the end. The mixture was diluted with ethyl acetate. The ethyl acetate layer was washed with brine, and dried with Na₂SO₄. The crude mixture was concentrated and purified by column chromatography (dichloromethane/methanol) to afford the target compounds 7a-n.

17-((3-Methyl-4-phenylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7a)

Yellow solid, yield 82%, mp 147.8–149.5 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.53 (s, 1H, Ar-H), 7.35 (s, 2H, Ar-H), 7.27 (s, 2H, Ar-H), 6.54 (d, J = 8.6 Hz, 1H, C6-H), 6.30 (d, J = 8.5 Hz, 1H, C7-H), 5.89 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, OH), 3.29 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.64 (s, 1H), 2.13 (s, 1H), 2.06 (s, 1H), 1.96 (d, J = 12.7 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.66 (s, 1H), 1.58 (s, 1H), 1.54 (s, 1H), 1.45 (s, 1H), 1.32 (s, 1H), 1.25 (s, 2H), 1.08 (s, 3H), 0.92 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 172.7, 168.5, 140.8, 135.8, 130.4, 129.1, 128.8, 99.0, 82.4, 79.3, 66.4, 65.6, 51.6, 49.5, 45.8, 37.2, 36.9, 34.7, 34.4, 33.5, 30.6, 30.1, 29.7, 27.6, 23.1, 20.5, 18.5, 18.3, 13.8; HRMS (ESI) Calcd for C₂₉H₃₅N₃O₃S [M + H]⁺ 506.2477, found: 506.2442.

17-((4-(4-(Trifluoromethyl)phenyl)-3-methylthiazol-2(3H)-ylidene)hydraziney-lidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7b)

Yellow solid, yield 79%, mp 173.4–175.6 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.70 (d, J = 8.1 Hz, 2H, Ar-H), 7.48 (d, J = 8.0 Hz, 2H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C₆-H), 6.30 (d, J = 8.5 Hz, 1H, C₇-H), 5.96 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, OH), 3.29 (s, 3H, N-CH₃), 2.71 (s, 1H), 2.63 (s, 1H), 2.13 (d, J = 13.8 Hz, 1H), 2.06 (d, J = 13.2 Hz, 1H), 1.96 (d, J = 11.8 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.67 (s, 1H), 1.58 (s, 1H), 1.50 (s, 1H), 1.44 (d, J = 7.5 Hz, 1H), 1.33 (s, 1H), 1.26 (s, 2H), 1.07 (s, 3H), 0.94 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.3, 168.1, 139.3, 135.9, 131.0, 130.4, 128.9, 125.8, 100.6, 82.4, 79.3, 66.4, 65.6, 51.6, 49.5, 45.8, 37.1, 36.9, 34.6, 34.4, 33.5, 30.6, 30.1, 27.6, 23.1, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₃₀H₃₄F₃N₃O₃S [M + H]⁺ 574.2351, found: 574.2302.

17-((4-(4-(Cyano)phenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7c)

Yellow solid, yield 86%, mp 156.1–168.2 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.73 (d, J = 8.2 Hz, 2H, Ar-H), 7.48 (d, J = 8.2 Hz, 2H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C₆-H), 6.30 (d, J = 8.5 Hz, 1H, C₇-H), 6.01 (s, 1H, C = CH), 4.31 (s, 1H, C₃-H), 3.97 (s, 1H, OH), 3.31 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.63 (s, 1H), 2.12 (s, 1H), 2.06 (d, J = 13.1 Hz, 1H), 1.96 (d, J = 13.6 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.64 (s, 1H), 1.57 (s, 1H), 1.50 (s, 1H), 1.44 (d, J = 7.6 Hz, 1H), 1.31 (d, J = 13.5 Hz, 1H), 1.26 (s, 2H), 1.07 (s, 3H), 0.93 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.6, 167.9, 135.9, 132.6, 131.0, 130.3, 128.9, 112.6, 101.7, 82.4, 79.2, 66.3, 65.6, 51.6, 49.4, 45.8, 37.1, 36.9, 34.6, 34.3, 33.8, 30.6, 30.1, 27.6, 23.0, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₃₀H₃₄N₄O₃S [M + H]⁺ 531.2430, found: 531.2397.

17-((4-(4-Methoxyphenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7d)

Yellow solid, yield 84%, mp 165.5–167.4 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.62 (d, J = 102.5 Hz, 1H, Ar-H), 7.25 (s, 1H, Ar-H), 6.95 (d, J = 6.9 Hz, 2H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C6-H), 6.30 (d, J = 8.5 Hz, 1H, C7-H), 5.83 (s, 1H, C-CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, -OH), 3.84 (s, 3H, OCH₃), 3.28 (s, 3H, N-CH₃), 2.73 (s, 1H), 2.64 (s, 1H), 2.12 (s, 1H), 2.05 (s, 1H), 1.96 (d, J = 11.6 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.64 (s, 1H), 1.58 (s, 1H), 1.51 (s, 1H), 1.44 (d, J = 9.7 Hz, 1H), 1.32 (s, 1H), 1.25 (s, 2H), 1.07 (s, 3H), 0.92 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 167.8, 160.2, 135.8, 132.3, 131.0, 130.4, 130.1, 128.8, 114.1, 82.3, 79.3, 66.4, 65.6, 55.4, 51.6, 49.4, 45.8, 37.1, 36.9, 34.6, 34.4, 30.6, 30.1, 27.6, 23.1, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₃₀H₃₇N₃O₄S [M + H]⁺ 536.2583, found: 536.2539.

17-((4-(2-Nitrophenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α, 8α-epidioxyandrost-6-en-3β-ol (7e)

Yellow solid, yield 77%, mp 172.8–174.1 °C; ¹H NMR (600 MHz, CDCl₃) δ 8.11 (s, 1H, Ar-H), 7.71 (d, J = 3.2 Hz, 2H, Ar-H), 7.65 (s, 1H, Ar-H), 7.53 (s, 1H, C6-H), 7.43 (s, 1H, C7-H), 5.86 (s, 1H, C = CH), 3.94 (s, 1H, C3-H), 3.35 (s, 1H, OH), 3.14 (s, 3H, N-CH₃), 2.98 (s, 1H), 2.71 (s, 1H), 2.44 (s, 1H), 2.38 (s, 1H), 2.20 (s, 2H), 2.06 (s, 1H), 1.83 (s, 1H), 1.71 (d, J = 7.8 Hz, 2H), 1.64–1.62 (m, 1H), 1.44 (d, J = 7.7 Hz, 2H), 1.25 (s, 2H), 1.20 (s, 1H), 0.96 (s, 3H, C18), 0.84 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 167.8, 149.0, 135.8, 135.3, 133.0, 132.3, 131.0, 130.8, 128.9, 126.2, 124.8, 68.5, 67.1, 65.8, 65.6, 62.6, 46.9, 44.2, 39.1, 38.2, 30.8, 30.6, 29.7, 23.6, 23.0, 22.7, 19.2, 16.3, 13.7; HRMS (ESI) Calcd for C₂₉H₃₄N₄O₅S [M + H]⁺ 551.2328, found: 551.2294.

17-((4-(4-(Trifluoromethoxy)phenyl)-3-methylthiazol-2(3H)-ylidene)hydrazine-ylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7f)

Yellow solid, yield 81%, mp 174.4–176.6 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.62 (d, J = 105.6 Hz, 1H, Ar-H), 7.39 (d, J = 8.7 Hz, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 6.54 (d, J = 8.4 Hz, 1H, C6-H), 6.30 (d, J = 8.4 Hz, 1H, C7-H), 5.91 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, OH), 3.29 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.63 (s, 1H), 2.12 (s, 1H), 2.05 (s, 1H), 1.96 (d, J = 13.8 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.65 (s, 1H), 1.57 (s, 1H), 1.50 (s, 1H), 1.44 (d, J = 7.7 Hz, 1H), 1.32 (d, J = 9.7 Hz, 1H), 1.25 (s, 2H), 1.07 (s, 3H), 0.93 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.1, 168.2, 135.9, 131.0, 130.4, 130.2, 128.9, 121.3, 99.8, 82.4, 79.3, 66.3, 65.6, 51.6, 49.5, 45.8, 37.1, 36.9, 34.6, 34.4, 33.4, 30.6, 30.1, 27.6, 23.1, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₃₀H₃₄F₃N₃O₄S [M + H]⁺ 590.2300, found: 590.2245.

17-((4-(4-Nitrophenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α, 8α-epidioxyandrost-6-en-3β-ol (7g)

Yellow solid, yield 86%, mp 175.0–176.5 °C; ¹H NMR (600 MHz, CDCl₃) δ 8.30 (s, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.54 (d, J = 6.7 Hz, 2H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H,C6-H), 6.30 (d, J = 8.4 Hz, 1H, C7-H), 6.09 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.98 (s, 1H, OH) 3.35 (s, 3H, N-CH₃), 2.74 (s, 1H), 1.96 (d, J = 11.4 Hz, 2H), 1.73 (s, 2H), 1.45 (s, 1H), 1.44 (s, 1H), 1.34–1.32 (m, 1H), 1.25 (s, 2H), 1.08 (s, 3H), 0.93 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 167.8, 147.9, 135.9, 132.3, 130.9, 130.3, 129.1, 128.9, 124.2, 82.4, 79.2, 66.4, 65.6, 51.6, 49.4, 45.9, 37.2, 36.9, 34.7, 34.3, 30.6, 30.1, 29.7, 23.1, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₂₉H₃₄N₄O₅S [M + H]⁺ 551.2328, found: 551.2286.

17-((4-(3-Nitrophenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α, 8α-epidioxyandrost-6-en-3β-ol (7h)

Yellow solid, yield 85%, mp 174.6–166.1 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.70 (d, J = 13.7 Hz, 2H, Ar-H), 7.64 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C6-H), 6.30 (d, J = 8.5 Hz, 1H, C7-H), 6.03 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.98 (s, 1H, OH), 3.32 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.63 (s, 1H), 2.14 (d, J = 8.8 Hz, 1H), 2.06 (d, J = 13.2 Hz, 1H), 1.96 (d, J = 13.8 Hz, 2H), 1.90 (s, 1H), 1.73 (d, J = 7.3 Hz, 2H), 1.65 (s, 1H), 1.58 (s, 1H), 1.44 (d, J = 7.5 Hz, 2H), 1.33 (s, 1H), 1.25 (s, 2H), 1.08 (s, 3H), 0.93 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.6, 167.8, 148.5, 135.9, 134.2, 131.0, 130.3, 129.9, 128.9, 123.4, 101.4, 82.4, 79.2, 66.4, 65.6, 51.6, 49.4, 45.8, 37.1, 36.9, 34.3, 33.5, 30.6, 29.7, 27.6, 23.1, 20.4, 19.2, 18.2, 13.7; HRMS (ESI) Calcd for C₂₉H₃₄N₄O₅S [M + H]⁺ 551.2328, found: 551.2294.

17-((4-(4-Fluorophenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7i)

Yellow solid, yield 80%, mp 167.3–168.7 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.25 (s, 2H, Ar-H), 7.06 (s, 2H, Ar-H), 6.47 (d, J = 8.5 Hz, 1H, C6-H), 6.23 (d, J = 8.5 Hz, 1H, C7-H), 5.81 (s, 1H, C = CH), 4.23 (s, 1H, C3-H), 3.90 (s, 1H, OH), 3.21 (s, 3H, N-CH₃), 2.66 (s, 1H), 2.56 (s, 1H), 2.06 (s, 1H), 1.98 (s, 1H), 1.88 (d, J = 12.6 Hz, 2H), 1.83 (s, 1H), 1.65 (s, 2H), 1.57 (s, 1H), 1.51 (s, 1H), 1.38 (s, 1H), 1.36 (s, 1H), 1.25 (s, 1H), 1.18 (s, 2H), 1.00 (s, 3H), 0.85 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 167.8, 135.9, 132.3, 131.0, 130.6, 130.4, 128.9, 116.0, 115.9, 82.4, 79.3, 66.4, 65.6, 51.6, 49.5, 45.8, 37.2, 36.9, 34.7, 34.4, 30.6, 30.1, 29.8, 23.1, 20.5, 19.2, 18.5, 18.3, 13.7; HRMS (ESI) Calcd for C₂₉H₃₄FN₃O₃S [M + H]⁺ 524.2383, found: 524.2344.

17-((4-(4-Bromophenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7j)

Yellow solid, yield 75%, mp 171.2–172.5 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.57 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.22 (d, J = 8.1 Hz, 2H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C6-H), 6.30 (d, J = 8.5 Hz, 1H, C7-H), 5.90 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, OH), 3.28 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.63 (s, 1H), 2.12 (s, 1H), 2.05 (s, 1H), 1.95 (d, J = 13.9 Hz, 2H), 1.90 (s, 1H), 1.72 (s, 2H), 1.64 (s, 1H), 1.57 (s, 1H), 1.44 (d, J = 7.6 Hz, 2H), 1.32 (s, 1H), 1.25 (s, 2H), 1.07 (s, 3H), 0.92 (s, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 173.1, 167.8, 135.8, 132.0, 130.9, 130.2, 128.8, 123.3, 82.3, 79.2, 66.4, 65.6, 51.6, 49.4, 45.8, 37.1, 36.9, 34.6, 34.4, 30.6, 30.1, 29.7, 27.6, 23.1, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₂₉H₃₄BrN₃O₃S [M + H]⁺ 584.5744, found: 584.5762.

17-((4-(p-Tolyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7k)

Yellow solid, yield 79%, mp 165.7–166.4 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.72 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.27 (s, 2H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C6-H), 6.30 (d, J = 8.4 Hz, 1H, C7-H), 5.84 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, OH), 3.28 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.63 (s, 1H), 2.40 (s, 3H), 2.13 (s, 1H), 2.06 (s, 1H), 1.96 (d, J = 11.4 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.63 (s, 1H), 1.58 (s, 1H), 1.45 (d, J = 7.5 Hz, 1H), 1.44 (s, 1H), 1.32 (s, 1H), 1.25 (s, 2H), 1.07 (s, 3H), 0.92 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 172.6, 167.8, 135.8, 132.3, 131.0, 130.4, 129.4, 128.9, 128.6, 82.4, 79.3, 66.4, 65.6, 51.6, 49.5, 45.7, 37.2, 36.9, 34.7, 34.4, 30.6, 30.1, 27.6, 23.1, 21.3, 20.5, 19.2, 18.5, 18.3, 13.8; HRMS (ESI) Calcd for C₃₀H₃₇N₃O₃S [M + H]⁺ 520.2634, found: 520.2594.

17-((4-(4-Methoxyphenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7l)

Yellow solid, yield 82%, mp 167.6–168.2 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.34 (s, 1H, Ar-H), 6.94 (d, J = 10.8 Hz, 2H, Ar-H), 6.86 (s, 1H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C6-H), 6.30 (d, J = 8.4 Hz, 1H, C7-H), 5.90 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.98 (s, 1H, OH), 3.83 (s, 3H, N-CH₃), 3.30 (s, 3H), 2.73 (s, 1H), 2.64 (s, 1H), 2.13 (s, 1H), 2.05 (s, 1H), 1.96 (d, J = 11.4 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.64 (s, 1H), 1.57 (s, 1H), 1.44 (d, J = 7.7 Hz, 2H), 1.32 (s, 1H), 1.26 (s, 2H), 1.07 (s, 3H), 0.92 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 167.8, 159.7, 135.8, 132.3, 131.0, 130.4, 129.8, 128.9, 121.0, 114.4, 82.3, 79.3, 66.4, 65.6, 55.4, 51.6, 45.8, 37.1, 36.9, 34.6, 34.4, 30.6, 30.1, 29.7, 23.1, 20.4, 19.2, 18.5, 18.2, 13.7; HRMS (ESI) Calcd for C₃₀H₃₇N₃O₄S [M + H]⁺ 536.2583, found: 536.2554.

17-((4-(4-Chlorophenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7m)

Yellow solid, yield 86%, mp 174.1–175.5 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.62 (d, J = 105.5 Hz, 1H, Ar-H), 7.41 (d, J = 8.5 Hz, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 6.54 (d, J = 8.5 Hz, 1H, C6-H), 6.30 (d, J = 8.5 Hz, 1H, C7-H), 5.90 (s, 1H, C = CH), 4.31 (s, 1H, C3-H), 3.97 (s, 1H, OH), 3.28 (s, 3H, N-CH₃), 2.72 (s, 1H), 2.63 (s, 1H), 2.15 (s, 1H), 2.05 (s, 1H), 1.95 (d, J = 13.8 Hz, 2H), 1.90 (s, 1H), 1.73 (s, 2H), 1.64 (s, 1H), 1.57 (s, 1H), 1.50 (s, 1H), 1.44 (d, J = 7.6 Hz, 1H), 1.32 (s, 1H), 1.25 (s, 2H), 1.07 (s, 3H), 0.92 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 173.0, 167.8, 139.6, 135.8, 132.3, 130.9, 130.4, 129.9, 129.1, 128.8, 82.3, 79.2, 66.3, 65.6, 51.6, 49.4, 45.8, 37.1, 36.8, 34.6, 34.4, 30.5, 30.1, 27.6, 23.1, 20.4, 19.2, 18.2, 13.7; HRMS (ESI) Calcd for C₂₉H₃₄ClN₃O₃S [M + H]⁺ 540.2088, found: 540.2062.

17-((4-(4-Hydroxyphenyl)-3-methylthiazol-2(3H)-ylidene)hydrazineylidene)-2H-5α,8α-epidioxyandrost-6-en-3β-ol (7n)

Yellow solid, yield 85%, mp 165.5–167.3 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.11 (d, J = 8.5 Hz, 2H, Ar-H), 6.79 (d, J = 8.4 Hz, 2H, Ar-H), 6.46 (d, J = 8.4 Hz, 1H, C6-H), 6.22 (d, J = 8.4 Hz, 1H, C7-H), 5.74 (s, 1H, C = CH), 4.24 (s, 1H, C3-H), 4.05 (s, 1H, OH), 3.92 (s, 1H, OH), 3.19 (s, 3H, N-CH₃), 2.64 (s, 1H), 2.55 (s, 1H), 2.06 (s, 1H), 2.01 (s, 1H), 1.89 (d, J = 11.6 Hz, 2H), 1.80 (s, 1H), 1.65 (s, 2H), 1.57 (s, 1H), 1.50 (s, 1H), 1.38 (s, 1H), 1.36 (s, 1H), 1.23 (s, 1H), 1.18 (s, 2H), 0.99 (s, 3H), 0.85 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 166.8, 134.8, 131.3, 130.0, 129.4, 129.2, 127.8, 114.7, 81.3, 78.3, 65.5, 64.6, 59.5, 50.5, 48.4, 44.8, 36.1, 33.6, 29.5, 29.1, 28.7, 26.5, 22.1, 19.4, 18.2, 17.5, 17.2, 13.1, 12.7; HRMS (ESI) Calcd for C₃₄H₃₇N₃O₄S [M + H]⁺ 522.2427, found: 522.2403.

in Vitro Cytotoxic Activity

Human MCF-7, HepG2, HCT-116, and A549 cancer cells in exponential growth period were cultured in moderate RPMI 1640 medium (Corning, USA) containing 100 kU/L of penicillin, 10% fetal bovine serum (FBS) and 100 mg/L streptomycin. The culture vessels were placed in a humidified atmosphere of 5% CO₂. The cytotoxic activity was evaluated by MTT assay (Beyotime). Cells were cultured into 96-well plates (100 μL per well) at a concentration of 1 × 10⁴/mL for 12 h. Then the cancer cells were treated with different concentrations of synthesized compounds for 48 h before MTT solution (10 µL) was added to each well for another 2 h. The MTT solution was poured off and then dimethyl sulfoxide (DMSO) 100 μL was added. The optical density (OD) value of each well was recorded using a microplate reader. The IC₅₀ value of the compound was calculated by SPSS analysis.³²

Determination of Morphological Changes of Cells

HepG2 cells were seeded into 6-well plates at a concentration of 1 × 10⁶ cells/well and cultured for 12 h. Following 7h treatment for 48 h, the tumor cells were double stained with AO and EB dye solutions for 20 min. The AO/EB staining kit was obtained from Beyotime Biotechnology. The tumor cells were collected carefully, resuspended in PBS, and then the samples were recorded under an Observe.A1 fluorescence microscope (Zeiss, Germany).³³

Apoptosis Analysis by Flow Cytometry

HepG2 cells were seeded on six-well plates, and then incubated with 7h (0, 2, 4 and 8 μM) for 48 h. Cells were washed with PBS and then resuspended in binding buffer (500 μL). Annexin V-FITC (10 μL) and PI (5 μL) were added to stain the cells at room temperature for 15 min. The kit was purchased from BD Biosciences. The stained cells were analyzed by flow cytometry (BD FACSCalibur).³⁴

Analysis of Mitochondrial Membrane Potential

HepG2 cells were treated with 7h for 48 h, then washed with cold PBS and stained with MitoProbe JC-1 at 37 °C for 20 min (BD Biosciences). The stained cells were collected and washed with cold PBS three times. The percentage of cells with normal or collapsed MMP was monitored using a flow cytometer.³⁵

Western Blot Analysis

HepG2 cells were treated with 7h for 48 h. Then the cells were lysed in RIPA lysis buffer, and boiled at 100 °C for 10 min. Equal amounts of proteins (30 μg) were separated on an polyacrylamide gel electrophoresis (SDS-PAGE) gel (10%) and transferred to nitrocellulose membranes. Then, these membranes were blocked with BSA (5%) and probed primary antibody against cleaved-caspase-9, cleaved-caspase-3, Bcl-2, Bax and cytochrome c. Then, the membranes were incubated with a secondary antibody for another 2 h. The positive bands were made visible on an x-ray film using an enhanced chemiluminescence system.³⁶

Conclusions

In summary, a series of endoperoxide steroid derivatives (7a-n) containing a thiazole system were prepared and characterized successfully. The cytotoxic activities of 7a-n were tested in four human carcinoma cell lines. Among them, compound 7h displayed marked cytotoxic activity against HepG2 cells, with an IC₅₀ value of 4.54 μM, which was 4.6-fold more potent than EP. SAR analysis suggested that the introduction of a thiazole system at the C-17 position of the endoperoxide steroid led to a remarkable increase in cytotoxic activity. Furthermore, the preliminary cellular mechanism of compound 7h was investigated. The results showed that 7h caused morphological changes, reduced MMP, and induced apoptosis by triggering the mitochondrial apoptotic pathway in HepG2 cells. The above findings incentivize the further study of 7h derivatives as potent anticancer agents.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X231175588 - Supplemental material for Novel Endoperoxide Steroid Derivatives Containing Thiazole Moiety as Potential Antitumor Agents: Design, Synthesis, and Cytotoxic Evaluation

Supplemental material, sj-doc-1-npx-10.1177_1934578X231175588 for Novel Endoperoxide Steroid Derivatives Containing Thiazole Moiety as Potential Antitumor Agents: Design, Synthesis, and Cytotoxic Evaluation by Gang Li, Xiaoshan Guo, Wenkang Ren, Jiafeng Wang, Zhen Lv, Hongling Li, Mingrui Sun, Xiaoming Li, Gang Chen, Zhiguo Zhang, Wenting Zhang and Ming Bu in Natural Product Communications

Footnotes

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 research was funded by Fundamental Research Funds for Education Department of Heilongjiang Province (No. 2019-KYYWF-1217).

ORCID iDs

Zhen Lv

Ming Bu

Hongling Li

Supplemental Material

Supplemental material for this article is available online.

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