Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents through microtubule stabilization.

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BCP-12238; No. of Pages 14 Biochemical Pharmacology xxx (2015) xxx–xxx

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Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents through microtubule stabilization Hong-Yan Lin a,b, Zi-Kang Li a,b, Li-Fei Bai c, Shahla Karim Baloch a,b, Fang Wang a,b, Han-Yue Qiu a,b, Xue Wang a,b, Jin-Liang Qi a,b, Raong-Wu Yang a,b, Xiao-Ming Wang a,b,*, Yong-Hua Yang a,b,* a

State Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of Plant Molecular Biology, School of Life Sciences, Nanjing University, Nanjing 210023, China b Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China c Jiangsu Key Laboratory of Biofunction Molecule, Jiangsu Second Normal University, Nanjing 210013, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 February 2015 Accepted 28 April 2015 Available online xxx

The high incidence of cancer and the side effects of traditional anticancer drugs motivate the search for new and more effective anticancer drugs. In this study, we synthesized 17 kinds of aryl dihydrothiazol acyl shikonin ester derivatives and evaluated their anticancer activity through MTT assay. Among them, C13 showed better antiproliferation activity with IC50 = 3.14 0.21 mM against HeLa cells than shikonin (IC50 = 5.75 0.47 mM). We then performed PI staining assay, cell cycle distribution, and cell apoptosis analysis for C13 and found that it can cause cell arrest in G2/M phase, which leads to cell apoptosis. This derivative can also reduce the adhesive ability of HeLa cells. Docking simulation and confocal microscopy assay results further indicated that C13 could bind well to the tubulin at paclitaxel binding site, leading to tubulin polymerization and mitotic disruption. ß 2015 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: Shikonin (PubChem CID: 479503) L-Cysteine (PubChem CID: 5862) Colchicine (PubChem CID: 6167) Paclitaxel (PubChem CID: 36314) Benzonitrile (PubChem CID: 7505) 4-(Dimethylamino)benzonitrile (PubChem CID: 70967) 4-Methoxybenzonitrile (PubChem CID: 70129) 4-Methylbenzonitrile (PubChem CID: 7724) 4-Chlorobenzonitrile (PubChem CID: 12163) 2,3-Dichlorobenzonitrile (PubChem CID: 736567) Keywords: Tubulin polymerization Mitotic arrest Shikonin ester derivatives

1. Introduction Mitosis is a complicated process that ensures the faithful inheritance of genetic material by cellular progeny while preventing aneuploidy [1]. Mitosis disruption and chromosomal

Abbreviations: MTT, (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide); NMR, nuclear magnetic resonance; TLC, thin layer chromatography; TMS, tetramethylsilane; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DAPI, 40 ,6-diamidino-2-phenylindole; BSA, bovine serum albumin; SAR, structure–activity relationship. * Corresponding authors. Tel.: +86 25 89681381; fax: +86 25 89681381. E-mail addresses: [email protected] (X.-M. Wang), [email protected] (Y.-H. Yang).

instability are the two established hallmarks of cancer [2]. Microtubules (MTs) are cytoskeletal filaments comprising a- and btubulin proteins and an individual MT is a long and hollow cylinder [3]. However, the collective structure of MTs may assume various configurations. For example, MT aster is a star-shaped structure where MTs radiate from a centrally located centrosome [4]. The mitotic spindle that forms during cell division is composed of two asters located at opposite poles of the cell. MTs emanate from asters toward the spindle midzone and exert forces to separate chromosomes between two daughter cells. The formation of MTs is a dynamic process that involves the polymerization and depolymerization of a- and b-tubulin heterodimers [5]. Disruption of the dynamic equilibrium blocks the cell division machinery at mitosis and leads to cells cycle arrest in metaphase, resulting in cell

http://dx.doi.org/10.1016/j.bcp.2015.04.021 0006-2952/ß 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Lin H-Y, et al. Synthesis of aryl dihydrothiazol acyl shikonin ester derivatives as anticancer agents through microtubule stabilization. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.021

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BCP-12238; No. of Pages 14 H.-Y. Lin et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

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death [5,6]. Given the vital role of MTs in cell growth and function, it has become an important target for the design and development of new anticancer agents. Tubulin-binding agents, also named ‘‘spindle poisons’’ can bind to tubulin and alter MT polymerization, perturb MT dynamics, disrupt mitotic spindle function, and block cell cycle progression [7]. Historically, taxanes and vinca alkaloids represent tubulinbinding agents [7–10]. The former promotes MT polymerization, whereas the latter inhibits MT polymerization. These agents induce sustained mitotic arrest in metaphase/anaphase transition and subsequent apoptotic cell death [11,12]. Shikonin and its derivatives are active naphthoquinone compounds isolated from the root of the Chinese herbal medicine Lithospermum erythrorhizon [13]. Shikonin is attracting considerable attention in the field of natural product chemistry because it has extensive pharmacological activities [14–16], especially good anticancer effects [17–20]. However, the side effects and toxicity of shikonin hinder its development as a new clinical anticancer agent [21]. In our previous study, we found that changing the side chain hydroxyl group into an ester group may reduce the toxicity of shikonin toward non-cancer cells; thus, shikonin ester derivatives may be good anticancer agents [19,20,22–25]. Meanwhile, some researchers have reported aryl thiazole compounds that can disrupt tubulin polymerization, thereby inhibiting the production of functional MTs and cell mitosis [26]. Thiazole also improves the water solubility and pharmacokinetic parameters of the drugs itself [26,27]. Based on the aforementioned results, we proposed in this work that theintroduction of thiazole moiety into shikonin may improve its water solubility and tumor targeting. Based on these studies, we synthesized a series of aryl dihydrothiazol acyl shikonin ester derivatives and evaluated them as tubulin-binding agents. The underlying mechanism was also studied. 2. Results 2.1. Chemistry The routes to synthesizing the novel aryl dihydrothiazol acyl shikonin ester derivatives C1–C17 are outlined in Fig. 1. These compounds were obtained in three steps, as elucidated in Section 3. All synthesized compounds were reported and characterized for the first time by 1H NMR, elemental analysis, melting test, and mass spectroscopy, and results were in accordance with their depicted structures.

2.2. Bioactivity 2.2.1. C13 selectively inhibited cancer-cell proliferation All synthesized derivatives C1–C17 were evaluated for their antiproliferation activities against five cancer cell lines [human hepatoma cell line (HepG2), human lung adenocarcinoma epithelial cell line (A549), carcinoma of cervix cell line (HeLa), human breast cancer cell line (MCF-7) and squamous cell carcinoma (SSC-4)], and two non-cancer cell lines [African green monkey kidney cell (VERO) and human normal liver cell (L02)] by MTT assay. Results shown in Table 3 indicated that all compounds had lower toxicity against the non-cancer cells VERO and L02 than shikonin itself. However, the introduction of dihydrothiazol moiety also generally attenuated the antiproliferation activity of shikonin against cancer cells as well. Fortunately, we obtained some good compounds with strong toxicity toward cancer cells while avoiding VERO and L02 cells. Table 3 shows that most of the target compounds can also effectively inhibit the proliferation of the five tumor cells, but the effects were not better than those of the shikonin. The main reason is dihydrothiazol moiety reduced shikonin cytotoxicity. Interestingly, the introduction of some moieties reduced only the toxicity of shikonin toward the non-cancer cells except for cancer cells, such as C2 (IC50 = 15.6 1.00 mM), C5 (IC50 = 9.91 0.64 mM), and C7 (IC50 = 10.4 0.87 mM) against HepG2 cells (IC50 = 9.36 0.57 mM for shikonin); C1 (IC50 = 7.95 0.32 mM), C12 (IC50 = 9.66 0.37 mM), and C16 (IC50 = 7.93 0.47 mM) against HeLa cells (IC50 = 5.75 0.47 mM for shikonin); C13 (IC50 = 6.52 0.68 mM), C7 (IC50 = 7.24 0.77 mM), and C2 (IC50 = 6.41 0.38 mM) against MCF-7 cells (IC50 = 4.99 0.36 mM for shikonin); and C1 (IC50 = 16.2 1.14 mM), and C12 (IC50 = 11.1 1.00 mM) against SCC-4 cells (IC50 = 10.1 0.49 mM for shikonin). We also obtained C13 (IC50 = 3.14 0.21 mM) and C8 (IC50 = 5.69 0.42 mM), which showed better antiproliferation activity against HeLa cells than shikonin (IC50 = 5.75 0.47 mM). To further determine the effect of the compounds on HeLa cell viability, we performed PI staining assay for C13. HeLa cells (1 105 per well) were seeded in 12-well plates and treated with C13 of various concentrations (0, 1, 3, and 5 mM) at 37 8C and 5% CO2 for 0, 12, 24, and 36 h. Cells were then collected and analyzed using PI staining and flow cytometry analysis. From the results shown in Fig. 2, we observed that C13 induced dose- and timedependent death of cancer cells. Therefore, C13 could efficiently inhibit cancer cell growth.

Fig. 1. Synthesis routes of C1–C17. The detailed process was described in Section 3.


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Fig. 2. Effects of C13 in killing HeLa cells was analyzed using PI staining and flow cytometry analysis. Each image is representative of three experiments. Data shown are the mean S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.

2.2.2. C13 reduced the adhesive ability of HeLa cells Cell adhesion is an important indicator of cancer cells. Herein, we examined the adhesive ability of HeLa cells as influenced by C13 and shikonin, which was used as positive control in this assay.

HeLa cells (2 105 per well) were seeded in 24-well plates and treated with C13 and shikonin of different concentrations for 24 h. After harvesting the cells, their adhesive ability to fibronectin and laminin was detected. The data shown in Fig. 3 indicated that C13


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Fig. 3. Influence of C13 and shikonin on HeLa cell adhesive to fibronectin and laminin (A) Influence of C13 on HeLa cell adhesive to fibronectin; (B) Influence of C13 on HeLa cell adhesive to laminin; (C) Influence of shikonin on HeLa cell adhesive to fibronectin; (D) Influence of shikonin on HeLa cell adhesive to laminin. Data shown are the mean S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.

could significantly reduce the adhesive ability of HeLa cells to fibronectin and laminin but the effects of shikonin were not obvious. 2.2.3. C13 caused cell apoptosis in HeLa cells in a dose- and timedependent manner Subsequently, we investigated the effect of C13 on cell apoptosis using shikonin as positive control. We treated HeLa cells with various concentrations of C13 and shikonin for 24 h and analyzed cells for changes in apoptotic markers with a flow cytometer in vitro. As shown in Fig. 4(A), the percentage of apoptotic cell significantly increased after treatment with high doses of C13. Meanwhile, we treated HeLa cells with 4 mM C13 for 0, 12, 24 and 36 h and found that cells were induced to apoptosis in a time-dependent manner (Fig. 4(B)). Likewise, we treated HeLa cells with different doses of shikonin and found that the 5 mM- and 10 mM-treated groups displayed apoptosis (Fig. 4(C)). We also treated HeLa cells with 4 mM shikonin for 12, 24, and 36 h and found that the cells did not undergo significant apoptosis until the exposure time was extended to 36 h (Fig. 4(D)). By comparison, we drew the conclusion that C13 caused cell apoptosis more effectively than shikonin. 2.2.4. C13 induced cell cycle arrest in HeLa cells in a dose- and timedependent manner We next assessed the cell cycle distribution of HeLa cells by flow cytometry. HeLa cells treated with C13 of different concentrations (0, 1, 3, and 5 mM) for 24 h and cells treated with 5 mM C13 for different times (0, 12, 24, and 48 h) could both arrest cells in G2/M phase. As shown in Fig. 5(A), the accumulation of cells in G2/M

phase increases with increased drug concentration, and 30.16% of cells were arrested in G2/M phase upon exposure to 5 mM C13 for 24 h. Meanwhile, data shown in Fig. 5(B) demonstrated that the maximum accumulation of cells in G2/M phase was observed after treatment with 5 mM C13 for 48 h. 2.2.5. Molecular docking of C13 and tubulin To better understand the potency of C13 and further guide structure–activity relationship (SAR) studies, we examined the interaction of C13 and shikonin with tubulin (PDB code: 1JFF). All docking runs were applied using the Lamarckian genetic algorithm of Auto-Dock 4.0. The interaction of C13 with tubulin amino acid residues is depicted in Fig. 6(A). All amino acid residues of tubulin that interacted with C13 were exhibited. In the binding mode, C13 bound well to the paclitaxel site of tubulin through three p bonds with ARG284 (distance = 4.81 A˚) and HIS 229 (distance = 4.18 and 4.04 A˚). The CDOCK interaction energy value for C13 and tubulin was 40.17 kcal/mol. Fig. 6(B) depicts the 3D models of the interaction between C13 and tubulin. We further investigated the binding mode of shikonin and tubulin at the paclitaxel site. Fig. 6(C) and (D) showed that shikonin bound to the paclitaxel site of tubulin through one p bond with HIS 229 (distance = 4.90 A˚) and two hydrogen bonds with PRO 274 (distance = 1.98 A˚), as well as THR276 (distance = 2.21 A˚). The CDOCK interaction energy value for shikonin and tubulin was 27.36 kcal/mol. We found that C13 could bind to b-tubulin at the paclitaxel site better than shikonin by comparing binding modes and interaction energy. Molecular docking results indicated that C13 could be a potential tubulin polymerization stabilizer.


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Fig. 4. Annexin V/PI dual-immuno-fluorescence staining after treatment with different concentrations of C13 and shikonin for different times revealed significantly increased number of apoptotic and necrotic cells (measured with Annexin V+/PI+ cells). (A) Cells treated with 0, 1, 3, 5, and 10 mM C13 for 24 h were collected and processed for analysis. (B) Cells treated with 4 mM C13 for different times (0, 12, 24, and 36 h) was collected and analyzed. (C) Cells treated with 0, 1.25, 2.5, 5, and 10 mM shikonin for 24 h were collected and processed for analysis. (D) Cells treated with 4 mM shikonin for different times (0, 12, 24, and 36 h) was collected and analyzed. The percentage of early apoptotic cells in the lower right quadrant (Annexin V-FITC positive/PI negative cells), as well as late apoptotic cells located in the upper right quadrant (Annexin V-FITC positive/PI positive cells). Images are representative of three independent experiments. Data are mean S.E.M. of three independent experiments. *P < 0.05, **P < 0.01.


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Fig. 5. Effect of C13 on the cell cycle distribution of HeLa cells. (A) Cells treated with 0, 1, 3, and 5 mM C13 for 24 h were collected and processed for analysis (G1 phase, green; S phase, yellow and G2/M phase, blue). (B) Cells treated with 3 mM C13 for different times (0, 12, 24, and 48 h) were collected and analyzed (G1 phase, green; S phase, yellow and G2/M phase, blue). Images are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 6. Molecular docking analysis of C13, showing proposed binding modes at the paclitaxel binding pocket b-tubulin (PDB code: 1JFF). (A) Interaction of C13 with the amino acid residues of paclitaxel binding site (carbon atom, gray; oxygen atom, red; hydrogen atom, white; sulfur atom, yellow, chlorine atom, green and nitrogen atom; light blue). (B) Binding position of C13 in the protein surface of tubulin (carbon atom, gray; oxygen atom, red; hydrogen atom, white; sulfur atom, yellow, chlorine atom, green and nitrogen atom; light blue). (C) Interaction of shikonin with the amino acid residues of paclitaxel binding site (carbon atom, blue; oxygen atom, red; hydrogen atom, white). (D) Binding position of shikonin in the protein surface of tubulin (carbon atom, blue; oxygen atom, red; hydrogen atom, white). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.2.6. C13 enhanced tubulin polymerization and disrupted cell division To observe the phenotypic effect of C13 on the cellular cytoskeletal network of tubulin, HeLa cells were immunostained and analyzed under a confocal microscope. Fig. 7 clearly shows the substantial stabilization of MTs with bundle-like appearance in paclitaxel (1 mM)-treated cells that were used as positive control in this assay. By contrast, the cell membrane MT of colchicine (1 mM)-treated cells (negative control) showed depolymerization and solubility. With reference to these two groups and the control group, the stabilization of tubulin network in C13-treated cells was not apparent up to the level of the paclitaxel-treated group, but stabilization was more obvious than in the control and colchicine-treated groups. Specifically, substantial stabilization of MTs appeared and formed polymorphonuclear cells when HeLa cells were treated with 3 mM C13. With increased dose to 5 mM, we can observe that a cell (enlarged image on the right in the third line) was arrested in metaphase, and the spindle fiber was unable to form. Meanwhile, the control group also had a cell (enlarged image on the right in the first line) that was going through metaphase. After the comparison, we found that the spindle fiber was unable to form in the 5 mM C13-treated group, whereas the spindle fiber was distinct and strong in the control group. These results suggested that C13 can enhance MT polymerization.

3. Experimental 3.1. Materials and measurements All chemicals (reagent grade) used were purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Shikonin, paclitaxel, colchicine and L-cysteine were purchased from Sigma– Aldrich (St. Louis, MO). Aromatic aldehydes were purchased from Shanghai Shaoyuan Co. Ltd. (Shanghai, China). All the 1H NMR spectra were recorded on a Bruker DPX 300 or DRX 500 spectrometer in CDCl3. Chemical shifts (d) for 1H NMR spectra were reported in ppm (d). Melting points (uncorrected) were measured on a XT4 MP Apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained on a Mariner Biospectrometry Workstation (ESI-TOF) mass spectrometer. Elemental analyses were performed on a CHNO-Rapid instrument and were within 0.4% of the theoretical values. TLC was carried out on the glass-backed silica gel sheets (silica gel 60 A˚ GF254) and visualized in UV light (254 nm). Goat anti-mouse IgG (H + L) was purchased from Invitrogen Trading (Shanghai) Co., Ltd (Shanghai, China). b-Tubulin antibody (#2146) was purchased from Cell Signaling Technology (Beverly, MA). Anti-tubulin (#AT819), Cy3-labeled goat anti-mouse IgG (H + L) (#A0521) were purchased from Cytoskeleton, Inc. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Beyotime Institute of Biotechnology (Haimen, China).


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Fig. 7. Effects of C13 (0, 3, and 5 mM), paclitaxel (1 mM), and colchicine (1 mM) on interphase MTs of HeLa cells. Microtubules tagged with rhodamine (red) and nuclei tagged with DAPI (blue) were observed under a confocal microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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RNase A (#EN0531) was purchased from Thermo Scientific, Fermentas (USA). AnnexcinV-FITC cell apoptosis assay kit (#BA11100) was purchased from BIO-BOX (Nanjing, China). Fibronectin (#F1056) and laminin (#L2020) were purchased from Sigma–Aldrich (St. Louis, MO). 3.2. General procedure for preparation of compounds a1–a17 A mixture of aromatic aldehydes (50 mmol) (1–17), hydroxylamine hydrochloride (62.5 mmol), sodium acetate (125 mmol) were dissolved in the mixture of formic acid and water (60:40) and stirred at 80 8C until TLC analysis indicated the disappearance of aromatic aldehydes. Then, cooling the reaction system to room temperature and put it into water to obtain the target compounds. Some desired products which were dissolved in the mixture of water and formic acid can be obtained by salting out. Then, solid target compounds were obtained by filtration and recrystallized by alcohol, and then dried under vacuum. While, some target compounds are oily. These oily compounds were obtained by extraction with ethyl acetate and the solvent was removed at the vacuum to afford aryl nitriles (a1–a17). 3.3. General procedure for preparation of compounds b1–b17 Methold A: Aryl nitrile (30 mmol), L-cysteine (45 mmol), NaHCO3 (45 mmol) were subsequently added to a degassed mixture of MeOH (2.6 mL/mmol), H2O (1.7 mL/mmol), and a catalytic amount of 1 M NaOH (5 mol%). The reaction mixture, which remained suspended throughout, was stirred at room temperature until TLC analysis indicated disappearance of the nitrile. MeOH was then removed from the mixture by evaporation under reduced pressure (40–50 8C/300–50 mbar).

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Methold B: A mixture of aryl nitrile (30 mmol), L-cysteine (60 mmol), and NaHCO3 (120 mmol) in EtOH (1.5 mL/mmol) was stirred at 100 8C, until TLC analysis indicated disappearance of the nitrile. The solvent was evaporated under reduced pressure and H2O (5 mL/mmol) was added to the residual white solid. The cold solution was then acidified with HCl to pH 2. The precipitated solid was collected by filtration and washed with a small amount of H2O. 3.4. General procedure for the preparation of compounds C1–C17 Aryl dihydrothiazol acids b1–b17, shikonin, 4-dimethyaminopyridine (DMAP) and N,N0 -dicyclohexylcarbodiimide (DCC) were dissolved in dichloromethane and cooled and stirred for 12 h. Adding proper amount of silica gel and condensing solvent by vacuum concentration. Then, collecting target compounds by column chromatography. Chemical structures of the target compounds (C1–C17) were shown in Table 1 and the eluent composition and proportion was shown in Table 2. 3.4.1. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-phenyl-4,5-dihydrothiazole-4carboxylate (C1) Red powder, yield 75%; mp: 97.5–99.7 8C; 1H NMR (500 MHz, CDCl3) d: 12.56 (s, 1H, –OH), 12.40 (s, 1H, –OH), 7.90 (d, J = 7.3 Hz, 2H, Ar–H), 7.46 (dt, J = 14.5, 7.1 Hz, 3H, Ar–H), 7.21 (s, 1H, Ar–H), 7.16 (s, 2H, Ar–H), 6.14 (dd, J = 7.5, 4.4 Hz, 1H, –O–CH), 5.40 (t, J = 8.5 Hz, 1H, N–CH), 5.17 (m, 1H, –C5 5CH), 3.79–3.71 (m, 1H, S– CH2), 3.64 (t, J = 10.2 Hz, 1H, S–CH2), 2.69 (s, 1H, C–CH2–C5 5C), 2.56 (dd, J = 14.6, 7.4 Hz, 1H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3), 1.38–1.21 (m, 3H). ESI-TOF, calcd for C26H23NO6S ([M+Na]+) 500.1246, found 500.1162. Anal. Calcd for C26H23NO6S: C, 65.39; H, 4.85; N, 2.93; O, 20.10; S, 6.71. Found: C, 64.83; H, 4.89; N, 2.96; O, 20.14; S, 6.66.

Table 1 Chemical structures of C1–C17.

Compound

R1

R2

R3

R4

R5

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17

H H H H H H H H F F Cl H Cl Cl Cl H H

H OCH3 H OCH3 CH3 H H H H H H H Cl H H H H

H H OCH3 OCH3 H CH3 OCF3 CH(CH3)2 H H F Cl H Cl H Br I

H H H H H H H H H H H H H H Cl H H

H H H H H H H H OCH3 H H H H H H H H


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10 Table 2 Eluent composition and proportion. Compound

Eluent

C1 C2

Ethyl acetate:petroleum ether (V:V = 1:7) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:12:0.25) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:15:0.025) Ethyl acetate:petroleum ether (V:V = 1:3) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:15:0.025) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:15:3.2) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:12:0.25) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:15:1.6) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:7:3.2) Methyl alcohol:dichloromethane (V:V = 1:50) Ethyl acetate:petroleum ether (V:V = 1:7) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:15:0.4) Ethyl acetate:petroleum ether (V:V = 1:5) Ethyl acetate:petroleum ether (V:V = 1:7) Ethyl acetate:petroleum ether:methyl alcohol (V:V:V = 1:15:0.03) Ethyl acetate:petroleum ether (V:V = 1:7) Ethyl acetate:petroleum ether (V:V = 1:7)

C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17

3.4.2. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(3-methoxyphenyl)-4,5dihydrothiazole-4-carboxylate (C2) Red oil, yield 68%; 1H NMR (300 MHz, CDCl3) d: 12.57 (s, 1H, – OH), 12.40 (s, 1H, –OH), 7.54–7.41 (m, 2H, Ar–H), 7.38–7.29 (m, 1H, Ar–H), 7.22–7.14 (m, 3H, Ar–H), 7.06 (m, 1H, Ar–H), 6.14 (dd, J = 7.4, 4.4 Hz, 1H, –O–CH), 5.45–5.36 (m, 1H, N–CH), 5.16 (dd, J = 16.3, 9.1 Hz, 1H, –C5 5CH), 3.89 (s, 3H, –OCH3), 3.80–3.58 (m, 2H, S–CH2), 2.78–2.47 (m, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C27H25NO7S ([M+Na]+) 530.1352, found 530.1103. Anal. Calcd for C27H25NO7S: C, 63.89; H, 4.96; N, 2.76; O, 22.07; S, 6.32. Found: C, 63.55; H, 5.06; N, 2.81; O, 22.01; S, 6.25. 3.4.3. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(4-methoxyphenyl)-4,5dihydrothiazole-4-carboxylate (C3) Red oil, yield 62%; 1H NMR (500 MHz, CDCl3) d: 12.56 (s, 1H, – OH), 12.40 (s, 1H, –OH), 7.85 (d, J = 8.2 Hz, 2H, Ar–H), 7.23–7.13 (m, 3H, Ar–H), 6.93 (d, J = 8.2 Hz, 2H, Ar–H), 6.13 (m, 1H, –O–CH), 5.42– 5.28 (m, 1H, N–CH), 5.15 (m, 1H, –C5 5CH), 3.86 (s, 3H, –OCH3), 3.66 (ddd, J = 30.7, 19.5, 10.6 Hz, 2H, S–CH2), 2.58 (dd, J = 40.7, 33.5 Hz, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C27H25NO7S ([M+Na]+) 530.1352, found 530.1103. Anal. Calcd for C27H25NO7S: C, 63.89; H, 4.96; N, 2.76; O, 22.07; S, 6.32. Found: C, 63.55; H, 5.06; N, 2.81; O, 22.01; S, 6.25. 3.4.4. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(3,4-dimethoxyphenyl)-4,5dihydrothiazole-4-carboxylate (C4) Red oil, yield 66%; 1H NMR (500 MHz, CDCl3) d: 12.56 (s, 1H, – OH), 12.40 (s, 1H, –OH), 7.21 (s, 2H, Ar–H); 7.17–7.15 (m, 1H, Ar–H, Ar–H); 7.11 (d, J = 1.5 Hz, 1H, Ar–H); 7.08 (s, 1H, Ar–H); 6.91 (d, J = 9.0 Hz, 1H, Ar–H); 6.14 (m, 1H, –O–CH); 5.43–5.35 (m, 1H, N– CH), 5.16 (dd, J = 16.3, 9.1 Hz, 1H, –C5 5CH), 3.89 (s, 3H, –OCH3), 3.86 (s, 3H, –OCH3), 3.80–3.58 (m, 2H, S–CH2), 2.78–2.47 (m, 2H, C– CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C28H27NO8S ([M+Na]+) 560.1457, found 560.1303. Anal. Calcd for C28H27NO8S: C, 62.56; H, 5.06; N, 2.61; O, 23.81; S, 5.96. Found: C, 62.05; H, 5.24; N, 2.73; O, 23.88; S, 6.06.

3.4.5. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(m-tolyl)-4,5-dihydrothiazole-4carboxylate (C5) Red powder, yield 80%; mp: 109.9–111.2 8C; 1H NMR (500 MHz, CDCl3) d: 12.56 (s, 1H, –OH), 12.40 (s, 1H, –OH), 7.77 (s, 1H, Ar–H), 7.69 (s, 1H, Ar–H), 7.33 (d, J = 4.0 Hz, 2H, Ar–H), 7.22 (s, 1H, Ar–H), 7.16 (s, 2H, Ar–H), 6.15 (s, 1H, –O–CH), 5.42 (m, 1H, N–CH), 5.18 (s, 1H, –C5 5CH), 3.70 (dt, J = 20.5, 10.9 Hz, 2H, S–CH2), 2.59 (dd, J = 40.5, 33.1 Hz, 2H, C–CH2–C5 5C), 2.42 (s, 3H, –CH3), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C27H25NO6S ([M+Na]+) 514.1403, found 514.1252. Anal. Calcd for C27H25NO6S: C, 65.97; H, 5.13; N, 2.85; O, 19.53; S, 6.52. Found: C, 65.55; H, 5.36; N, 2.89; O, 20.06; S, 6.15. 3.4.6. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(p-tolyl)-4,5-dihydrothiazole-4carboxylate (C6) Red oil, yield 58%; 1H NMR (300 MHz, CDCl3) d: 12.58 (s, 1H, – OH), 12.40 (s, 1H, –OH), 8.01 (d, J = 8.1 Hz, 2H, Ar–H), 7.71 (d, J = 8.0 Hz, 2H, Ar–H), 7.23–7.15 (m, 3H, Ar–H), 6.15 (dd, J = 7.3, 4.4 Hz, 1H, –O–CH), 5.51–5.39 (m, 1H, N–CH), 5.24–5.07 (m, 1H, – C5 5CH), 3.89–3.62 (m, 2H, S–CH2), 2.78–2.46 (m, 2H, C–CH2–C5 5C), 1.70 (s, 3H, C5 5C–CH3), 1.61 (s, 3H, C5 5C–CH3), 1.26 (s, 3H, –CH3). ESI-TOF, calcd for C27H25NO6S ([M+Na]+) 514.1403, found 514.1252. Anal. Calcd for C27H25NO6S: C, 65.97; H, 5.13; N, 2.85; O, 19.53; S, 6.52. Found: C, 65.55; H, 5.36; N, 2.89; O, 20.06; S, 6.15. 3.4.7. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(4-(trifluoromethoxy)phenyl)-4,5dihydrothiazole-4-carboxylate (C7) Red powder, yield 72%; mp: 96.5–97.7 8C; 1H NMR (500 MHz, CDCl3) d: 12.56 (s, 1H, –OH), 12.39 (s, 1H, –OH), 7.94 (d, J = 8.5 Hz, 2H, Ar–H), 7.28 (d, J = 8.5 Hz, 2H, Ar–H), 7.20 (s, 1H, Ar–H), 7.16 (s, 2H, Ar–H), 6.14 (dd, J = 7.3, 4.3 Hz, 1H, –O–CH), 5.40 (t, J = 8.6 Hz, 1H, N–CH), 5.17 (t, J = 6.9 Hz, 1H, –C5 5CH), 3.87–3.59 (m, 2H, S– CH2), 2.61 (ddd, J = 22.5, 14.9, 8.5 Hz, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.61 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C27H22F3NO7S ([M+Na]+) 584.1069, found 584.0836. Anal. Calcd for C27H22F3NO7S: C, 57.75; H, 3.95; F, 10.15; N, 2.49; O, 19.94; S, 5.71. Found: C, 57.03; H, 4.06; F, 10.07; N, 2.94; O, 20.02; S, 5.23. 3.4.8. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(4-isopropylphenyl)-4,5dihydrothiazole-4-carboxylate (C8) Red oil, yield 65%; 1H NMR (300 MHz, CDCl3) d: 12.58 (s, 1H, – OH), 12.42 (s, 1H, –OH), 7.89–7.80 (m, 2H, Ar–H), 7.30 (d, J = 8.2 Hz, 2H, Ar–H), 7.22–7.11 (m, 3H, Ar–H), 6.14 (dd, J = 7.4, 4.3 Hz, 1H, – O–CH), 5.49–5.34 (m, 1H, N–CH), 5.16 (dd, J = 16.1, 8.5 Hz, 1H, – C5 5CH), 3.69 (dt, J = 20.5, 11.2 Hz, 2H, S–CH2), 2.97 (dd, J = 13.6, 6.7 Hz, 1H, –CH(CH3)2), 2.77–2.48 (m, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3), 1.27 (d, J = 7.0 Hz, 6H– CH(CH3)2). ESI-TOF, calcd for C29H29NO6S ([M+Na]+) 542.1716, found 542.1542. Anal. Calcd for C29H29NO6S: C, 67.03; H, 5.63; N, 2.70; O, 18.47; S, 6.17. Found: C, 66.54; H, 5.82; N, 2.83; O, 18.54; S, 6.01. 3.4.9. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(2-fluoro-6-methoxyphenyl)-4,5dihydrothiazole-4-carboxylate (C9) Red oil, yield 83%; 1H NMR (300 MHz, CDCl3) d: 12.58 (s, 1H, – OH), 12.44 (s, 1H, –OH), 7.36 (td, J = 8.4, 4.3 Hz, 1H, Ar–H), 7.18 (s, 2H, Ar–H), 7.15 (s, 1H, Ar–H), 6.77 (t, J = 9.1 Hz, 2H, Ar–H), 6.23– 6.08 (m, 1H, –O–CH), 5.53–5.36 (m, 1H, N–CH), 5.16 (d, J = 6.1 Hz, 1H, –C5 5CH), 3.88 (s, 3H, –OCH3), 3.72 (m, 2H, S–CH2), 2.74–2.45 (m, 2H, C–CH2–C5 5C), 1.67 (s, 3H, C5 5C–CH3), 1.58 (s, 3H, C5 5C– CH3). ESI-TOF, calcd for C27H24FNO7S ([M+Na]+) 548.1258, found


G Model


548.1053. Anal. Calcd for C27H24FNO7S: C, 61.71; H, 4.60; F, 3.61; N, 2.67; O, 21.31; S, 6.10. Found: C, 61.11; H, 4.73; F, 3.64; N, 2.73; O, 21.36; S, 5.49. 3.4.10. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(2-fluorophenyl)-4,5-dihydrothiazole4-carboxylate (C10) Red oil, yield 61%; 1H NMR (500 MHz, CDCl3) d: 12.57 (s, 1H, – OH), 12.41 (s, 1H, –OH), 8.02 (t, J = 7.5 Hz, 1H, Ar–H), 7.52–7.42 (m, 2H, Ar–H), 7.20 (s, 1H, Ar–H), 7.19 (s, 1H, Ar–H), 7.17 (d, J = 5.5 Hz, 2H, Ar–H), 6.14 (dd, J = 7.3, 3.6 Hz, 1H, –O–CH), 5.34 (dd, J = 17.9, 9.0 Hz, 1H, N–CH), 5.18 (dd, J = 27.6, 20.3 Hz, 1H, –C5 5CH), 3.78– 3.60 (m, 2H, S–CH2), 2.75–2.49 (m, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H22FNO6S ([M+Na]+) 518.1152, found 518.1003. Anal. Calcd for C26H22FNO6S: C, 63.02; H, 4.48; F, 3.83; N, 2.83; O, 19.37; S, 6.47. Found: C, 62.21; H, 4.63; F, 3.76; N, 2.98; O, 19.42; S, 6.05. 3.4.11. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(2-chloro-4-fluorophenyl)-4,5dihydrothiazole-4-carboxylate (C11) Red oil, yield 63%; 1H NMR (500 MHz, CDCl3) d: 12.60 (s, 1H, – OH), 12.43 (s, 1H, –OH), 7.79 (d, J = 8.6 Hz, 1H, Ar–H), 7.26–7.17 (m, 3H, Ar–H), 7.11 (d, J = 22.6 Hz, 2H, Ar–H), 6.18 (s, 1H, –O–CH), 5.40 (dd, J = 18.6, 9.5 Hz, 1H, N–CH), 5.26–5.11 (m, 1H, –C5 5CH), 3.76 (ddd, J = 39.8, 20.1, 10.9 Hz, 2H, S–CH2), 2.79–2.53 (m, 2H, C–CH2– C5 5C), 1.72 (s, 3H, C5 5C–CH3), 1.63 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H21ClFNO6S ([M+Na]+) 552.0762, found 552.0105. Anal. Calcd for C26H21ClFNO6S: C, 58.92; H, 3.99; Cl, 6.69; F, 3.58; N, 2.64; O, 18.11; S, 6.05. Found: C, 58.24; H, 4.15; Cl, 6.65; F, 3.59; N, 2.72; O, 18.24; S, 6.00. 3.4.12. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(4-chlorophenyl)-4,5-dihydrothiazole4-carboxylate (C12) Red powder, yield 65%; mp: 36.5–37.7 8C; 1H NMR (500 MHz, CDCl3) d: 12.59 (s, 1H, –OH), 12.42 (s, 1H, –OH), 7.86 (d, J = 8.0 Hz, 2H, Ar–H), 7.44 (d, J = 8.0 Hz, 2H, Ar–H), 7.21 (d, J = 18.8 Hz, 3H, Ar–H), 6.17 (d, J = 4.3 Hz, 1H, –O–CH), 5.42 (t, J = 8.3 Hz, 1H, N–CH), 5.20 (m, 1H, –C5 5CH), 3.74 (dt, J = 59.9, 10.3 Hz, 2H, S–CH2), 2.80–2.52 (m, 2H, C–CH2–C5 5C), 1.72 (s, 3H, C5 5C–CH3), 1.63 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H22FNO6S ([M+Na]+) 534.0856, found 534.0148. Anal. Calcd for C26H22FNO6S: C, 60.99; H, 4.33; Cl, 6.92; N, 2.74; O, 18.75; S, 6.26. Found: C, 60.01; H, 4.52; Cl, 6.65; N, 2.94; O, 18.94; S, 6.07. 3.4.13. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(2,3-dichlorophenyl)-4,5dihydrothiazole-4-carboxylate (C13) Red oil, yield 64%; 1H NMR (500 MHz, CDCl3) d: 12.60 (s, 1H, – OH), 12.43 (s, 1H, –OH), 7.59 (dt, J = 15.7, 7.8 Hz, 2H, Ar–H), 7.35– 7.30 (m, 1H, Ar–H), 7.25–7.17 (m, 3H, Ar–H), 6.23–6.15 (m, 1H, –O– CH), 5.44 (dd, J = 19.0, 10.1 Hz, 1H, N–CH), 5.19 (dd, J = 13.9, 6.9 Hz, 1H, –C5 5CH), 3.90–3.72 (m, 2H, S–CH2), 2.78–2.53 (m, 2H, C–CH2– C5 5C), 1.72 (s, 3H, C5 5C–CH3), 1.63 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H21Cl2NO6S ([M+Na]+) 568.0467, found 568.0218. Anal. Calcd for C26H21Cl2NO6S: C, 57.15; H, 3.87; Cl, 12.98; N, 2.56; O, 17.57; S, 5.87. Found: C, 56.52; H, 4.15; Cl, 13.05; N, 2.74; O, 17.79; S, 5.56. 3.4.14. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(2,4-dichlorophenyl)-4,5dihydrothiazole-4-carboxylate (C14) Red powder, yield 71%; mp: 131.6–132.7 8C; 1H NMR (300 MHz, CDCl3) d: 12.59 (s, 1H, –OH), 12.42 (s, 1H, –OH), 7.68 (t, J = 10.8 Hz, 1H, Ar–H), 7.45 (dd, J = 15.6, 5.4 Hz, 1H, Ar–H), 7.36–7.28 (m, 1H, Ar–H), 7.18 (s, 3H, Ar–H), 6.15 (dd, J = 11.5, 6.9 Hz, 1H, –O–CH), 5.39 (t, J = 8.7 Hz, 1H, N–CH), 5.17 (t, J = 7.6 Hz, 1H, –C5 5CH), 3.71 (qt,

11

J = 9.1, 4.5 Hz, 2H, S–CH2), 1.63 (dd, J = 28.3, 7.5 Hz, 2H, C–CH2–C5 5C), 1.26 (s, 3H, C5 5C–CH3), 1.22 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H21Cl2NO6S ([M+Na]+) 568.0467, found 568.0218. Anal. Calcd for C26H21Cl2NO6S: C, 57.15; H, 3.87; Cl, 12.98; N, 2.56; O, 17.57; S, 5.87. Found: C, 56.52; H, 4.15; Cl, 13.05; N, 2.74; O, 17.79; S, 5.56. 3.4.15. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(2,5-dichlorophenyl)-4,5dihydrothiazole-4-carboxylate (C15) Red oil, yield 69%; 1H NMR (300 MHz, CDCl3) d: 12.59 (s, 1H, – OH), 12.41 (s, 1H, –OH), 7.72–7.66 (m, 2H, Ar–H), 7.37 (s, 1H, Ar– H), 7.17 (t, J = 3.3 Hz, 3H, Ar–H), 6.20–6.10 (m, 1H, –O–CH), 5.44– 5.35 (m, 1H, N–CH), 5.18 (dd, J = 14.0, 6.7 Hz, 1H, –C5 5CH), 3.82– 3.65 (m, 2H, S–CH2), 2.77–2.48 (m, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H21Cl2NO6S ([M+Na]+) 568.0467, found 568.0218. Anal. Calcd for C26H21Cl2NO6S: C, 57.15; H, 3.87; Cl, 12.98; N, 2.56; O, 17.57; S, 5.87. Found: C, 56.52; H, 4.15; Cl, 13.05; N, 2.74; O, 17.79; S, 5.56. 3.4.16. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(4-bromophenyl)-4,5-dihydrothiazole4-carboxylate (C16) Red powder, yield 87%; mp: 106.4–107.9 8C; 1H NMR (300 MHz, CDCl3) d: 12.57 (s, 1H, –OH), 12.40 (s, 1H, –OH), 7.78 (d, J = 8.4 Hz, 2H, Ar–H), 7.58 (d, J = 8.4 Hz, 2H, Ar–H), 7.22–7.13 (m, 3H, Ar–H), 6.14 (dd, J = 7.5, 4.5 Hz, 1H, –O–CH), 5.39 (t, J = 8.4 Hz, 1H, N–CH), 5.16 (dd, J = 16.3, 8.9 Hz, 1H, –C5 5CH), 3.79–3.64 (m, 2H, S–CH2), 2.78–2.45 (m, 2H, C–CH2–C5 5C), 1.69 (s, 3H, C5 5C–CH3), 1.60 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H22BrNO6S ([M+Na]+) 578.0351, found 578.0203. Anal. Calcd for C26H22BrNO6S: C, 56.12; H, 3.99; Br, 14.36; N, 2.52; O, 17.25; S, 5.76. Found: C, 55.43; H, 4.28; Br, 14.48; N, 2.76; O, 17.39; S, 5.45. 3.4.17. (4S)-1-(5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalen-2yl)-4-methylpent-3-en-1-yl 2-(4-iodophenyl)-4,5-dihydrothiazole-4carboxylate (C17) Red powder, yield 79%; mp: 98.2–99.5 8C; 1H NMR (300 MHz, CDCl3) d: 12.58 (s, 1H, –OH), 12.41 (s, 1H, –OH), 7.79 (d, J = 8.5 Hz, 2H, Ar–H), 7.62 (d, J = 8.5 Hz, 2H, Ar–H), 7.19 (dd, J = 6.3, 2.4 Hz, 3H, Ar–H), 6.14 (dd, J = 7.5, 4.4 Hz, 1H, –O–CH), 5.40 (t, J = 8.5 Hz, 1H, N–CH), 5.16 (dd, J = 16.3, 8.9 Hz, 1H, –C5 5CH), 3.82–3.60 (m, 2H, S– CH2), 2.78–2.48 (m, 2H, C–CH2–C5 5C), 1.70 (s, 3H, C5 5C–CH3), 1.61 (s, 3H, C5 5C–CH3). ESI-TOF, calcd for C26H22INO6S ([M+Na]+) 626.0213, found 626.0048. Anal. Calcd for C26H22INO6S: C, 51.75; H, 3.67; I, 21.03; N, 2.32; O, 15.91; S, 5.31. Found: C, 51.13; H, 4.00; I, 21.06; N, 2.46; O, 15.98; S, 5.17. 3.5. Cell culture Human hepatoma cell line (HepG2), human lung adenocarcinoma epithelial cell line (A549), carcinoma of cervix cell line (HeLa), human breast cancer cell line (MCF-7), squamous cell carcinoma (SSC-4), African green monkey kidney cell (VERO) and human normal liver cell (L02) were purchased from Nanjing Keygen Technology (Nanjing, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) (High Glucose) with L-glutamine supplemented with 10% fetal bovine serum (FBS, BI), 100 U/mL penicillin and 100 mg/mL streptomycin (Hyclone), and incubated 37 8C in a humidified atmosphere containing 5% CO2. 3.6. Anti-proliferation assay Target tumor cell lines were grown to log phase in DMEM medium supplemented with 10% fetal bovine serum. After diluting to 2 104 cells/mL with the complete medium, 100 mL of the


G Model



Table 3 Inhibition of cell proliferation against HepG2, A549, HeLa, MCF-7, SCC-4, VERO and L02 cells by C1–C17 and shikonin. Compt

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 Shikonin

IC50 (mM) HepG2

A549

HeLa

MCF-7

SCC-4

VERO

L02

31.7 1.61 15.6 1.00 29.3 1.01 38.7 1.72 9.91 0.64 19.26 1.26 10.4 0.87 32.8 1.65 42.2 2.86 43.4 1.98 37.6 1.56 47.4 2.76 35.7 1.23 39.7 2.06 28.9 1.86 52.1 2.87 54.6 3.04 9.36 0.57

10.1 0.48 30.3 2.24 32.7 1.64 27.1 1.08 30.0 2.00 17.4 1.54 28.8 1.42 43.2 3.42 53.8 3.65 27.8 1.23 12.3 0.57 34.7 1.69 14.7 0.77 25.5 1.22 38.6 1.68 17.9 1.05 23.6 1.11 3.13 0.25

7.95 0.32 54.6 3.32 29.1 1.17 24.2 0.99 40.1 2.04 27.6 1.04 24.7 1.01 5.69 0.42 53.0 3.25 43.7 1.23 34.9 1.48 9.66 0.37 3.14 0.21 36.5 2.29 38.1 2.54 7.93 0.47 18.8 1.48 5.75 0.47

21.2 1.06 6.41 0.38 34.1 2.07 24.8 1.25 18.4 1.42 12.4 1.43 7.24 0.77 33.7 2.03 51.4 3.08 28.1 1.78 27.9 1.22 16.3 1.27 6.52 0.68 31.2 1.65 25.5 1.75 41.22 2.99 26.8 2.37 4.99 0.36

16.2 1.14 45.3 2.98 34.5 1.68 29.9 1.27 41.2 2.69 35.9 2.27 38.2 1.95 37.3 2.24 21.6 1.09 30.1 1.69 45.0 3.53 11.1 1.00 36.4 1.77 22.7 1.56 31.7 1.22 35.1 2.23 46.2 3.01 10.1 0.49

86.2 5.67 124 6.04 121 5.78 157 7.89 108 5.41 99.6 4.49 101 5.52 103 5.55 89.2 4.01 115 6.47 99.1 4.89 96.1 5.29 92.2 4.48 187 6.88 97.9 3.77 253 7.89 104 6.35 6.76 0.85

105 3.91 167 4.89 106 4.97 99.1 3.36 115 3.36 106 2.98 98.7 2.27 136 4.65 142 4.68 93.4 3.60 102 3.92 118 4.55 107 4.46 147 3.35 108 4.57 189 3.78 113 4.39 83.8 2.26

obtained cell suspension was added to each well of 96-well culture plates and then allowed to adhere for 12 h at 37 8C, 5% CO2 atmosphere. Tested samples at pre-set concentrations (0.1, 1, 10, and 100 mM) were added to 96 wells with shikonin as positive reference. After 24 h exposure period, 20 mL of PBS containing 2.5 mg/mL of MTT was added to each well. Plates were then incubated for further 4 h, and then were centrifuged (1500 rpm at 4 8C for 10 min) to remove supernatant. 150 mL of DMSO was added to each well for coloration. The plates were shaken vigorously to ensure complete solubilization for 10 min at room temperature. The absorbance was measured and recorded on an ELISA reader (ELx800, BioTek, USA) at a test wavelength of 570 nm. In all the experiments three replicate wells were used for each drug concentration. Each assay was carried out at least three times. The results are shown in Table 3. 3.7. PI staining assay Cells were seeded into 12-well plate (1 105 per well) and incubated at 37 8C for 12 h. Then the cells were treated with different concentrations (0, 1, 3, and 5 mM) of C13 and continue to incubate at 37 8C, 5% CO2 for 0, 12, 24, and 36 h. Then, cells were collected and analyzed using PI staining and flow cytometry analysis (BD, USA). 3.8. Cell adhesion assay Cell adhesion assay was performed using method published by Qian et al. [33] with some modifications. Briefly, 96-well flatbottom plates were coated with 50 mL fibronectin and laminin (10 mg/mL) in PBS overnight at 4 8C and then blocked with 0.2% BSA for 2 h at room temperature followed by three times washing. Then, HeLa cells, which had been presented by C13 or shikonin for 24 h, were added to each well (5 103 per well) in triplicate, and incubated at 37 8C, 5% CO2 for 40 min. Next, plates were washed twice with PBS to remove unbound cells. Cells remained adhering to the plated were determined by MTT assay. The binding cells were calculated by dividing the optical density of initial input cells. Each assay was carried out at least three times. 3.9. Cell apoptosis assay For Annexin V/PI assays, HeLa cells were stained with Annexin V-FITC and PI and then monitored for apoptosis by flow cytometry.

Briefly, 5 103 cells were seeded in 6-well plates for 24 h and then were treated with C13 (0, 1, 3, 5, and 10 mM) or shikonin (0, 1.25, 2.5, 5, and 10 mM) for 24 h and treated cells with 4 mM C13 or shikonin for 0, 12, 24, and 36 h. Then cells were collected and washed twice with PBS and stained with 5 mL of Annexin V-FITC and 2.5 mL of PI (5 mg/mL) in 1 binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2) for 30 min at room temperature in the dark. Apoptotic cells were quantified using BD Accuri C6 Flow Cytometer (BD, USA). Statistical analysis was done using Flowjo 7.6.1 software. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (double positive of Annexin V and PI) cells were detected. 3.10. Cell cycle analysis HeLa cells were seeded in 6-well plates (5.0 103 cells/well) and incubated at 37 8C for 24 h. Exponentially growing cells were then incubated with the C13 at 1, 3, and 5 mM. And in the timedependent assays, exponentially growing cells were incubated with 5 mM C13 at 37 8C for 12, 24, and 48 h. Cells treated with the compounds solvent (DMSO) were included. DMSO was used at the highest concentration used in the experiments. After then, cells were centrifuged and fixed in 70% ethanol at 4 8C for at least 12 h and subsequently resuspended in PBS containing 0.1 mg/mL RNase A and 5 mg/mL propidium iodide (PI). Cellular DNA content, for cell cycle distribution analysis, was measured by flow cytometry using BD Accuri C6 Flow Cytometer (BD, USA) plotting 10,000 events per sample. The percentage of cells in the G1, S and G2/M phases of the cell cycle were determined using the Flowjo 7.6.1 software after cell debris exclusion. 3.11. Docking simulation The three-dimensional X-ray structure of tubulin (PDB code: 1JFF) was chosen as the template for the modeling study of C13 and shikonin bound to tubulin. The crystal structure was obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/ home.do). The molecular docking procedure was performed by using Ligand Fit protocol within Auto-Dock 4.0. For ligand preparation, the 3D structure of C13 was generated and minimized using Auto-Dock 4.0. For protein preparation, the hydrogen atoms were added, and the water and impurities were removed. The whole tubulin was defined as a receptor and the site sphere was selected based on the ligand binding location of taxol, then the taxol molecule was removed and C13 or shikonin was placed


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during the molecular docking procedure. Types of interactions of the docked protein with ligand were analyzed after the end of molecular docking. 3.12. Confocal microscopy assay HeLa cells were grown on round cover slips to 70% confluence and incubated with 3 mM or 5 mM C13, 1 mM paclitaxel and 1 mM colchicine for 24 h, respectively. After incubating, cells were washed with PBS three times and fixed with 4% paraformaldehyde for 20 min, permeabilized with 1% Triton X-100 for another 10 min. Then, the cells were blocked with 3% BSA for 1 h. Subsequently, the cells were washed once with PBS, and incubated with anti-tubulin antibody (1:500, Cytoskeleton, Inc.) in 3% BSA overnight at 4 8C. After being washed with 0.5% Triton X-100 (incubate for 5 min), each coverslip was added 200 mL of Cy3labeled goat anti-mouse IgG (H + L) (1:1500, Cytoskeleton, Inc.) in 3% BSA and incubated for 1 h at room temperature followed by DAPI (5 ng/mL). Cells were then observed under an Olympus confocal microscope and data was analyzed using FV-10-ASW 1.7 viewer. 4. Discussion MTs are key cytoskeletal filaments involved in a variety of crucial cellular processes such as cell motility, cell division, shape maintenance, and vesicle transport [28,29]. MTs are an important molecular target for cancer chemotherapeutic agents because of their significant function in cellular functions [30,31]. Herein, we aimed at breaking the dynamic balance of MT polymerization and de-polymerization to disrupt the mitosis process of cancer cells. Meanwhile, it is well known that the proliferation rate of cancer cell is higher than that of non-cancer cell. Generally, the continuous mitotic division of proliferating cancer cells showed better sensitivity to the inhibition of mitosis than the non-cancer cells, especially for the HeLa cell line, which is the fastest proliferated cell line among the five cancer cell lines used in this study. MTT assay results confirmed this phenomenon. Tubulin contains three binding sites: colchicine, paclitaxel and vinblastine sites [32]. In our previous studies, we have synthesized some shikonin derivatives as good anticancer agents and confirmed that they can induce cancer cell apoptosis and inhibit MT polymerization by binding to the colchicine or vinblastine site [19,20,24]. However, the 17 kinds of aryl dihydrothiazol acyl shikonin ester derivatives in this study were synthesized and evaluated as a MT polymerization stabilizer by binding to the paclitaxel site. Confocal microscopy observation results indicated that C13 could enhance MT polymerization, and the effect was similar to that of paclitaxel. By contrast, the podophyllotoxin ester derivative S12 was also synthesized and evaluated as a potent antitubulin agent. S12 could cause cell cycle arrest at G2/M phase by binding to the colchicine site of b-tubulin [33]. The phenotypic effect of S12 on the cellular cytoskeletal network of tubulin was similar to that of colchicine. Their effects on the cellular cytoskeletal network of tubulin were opposite because of the different binding sites. SAR studies were further performed to investigate the different modes of C13 and shikonin bind to tubulin. First, we found that shikonin could bind to the b-tubulin active site and was stabilized by forming one p-bond on the naphthoquinone ring and two hydrogen bonds on the hydroxyl group of the side chain. By contrast, C13 can bind to the active site of b-tubulin better than shikonin and was stabilized by forming three p-bonds on the naphthoquinone ring of shikonin and the benzene ring of aryl dihydrothiazol acid. In addition, the CDOCK interaction energy value of C13 bind to tubulin was lower than that of shikonin, which

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meant that the binding of C13 with tubulin was more stable than that of shikonin. In view of the binding mode and the interaction energy, we can conclude that C13 can bind well to the paclitaxel site of b-tubulin, and this effect was more potent than that of shikonin. Therefore, C13 was the best anticancer agent in this study; it enhanced MT polymerization by binding to the paclitaxel site of tubulin, and the effect was better than that of shikonin. Acknowledgements The authors are grateful to the National Natural Science Foundation of China (NSFC) (Nos. 31470384, 31170275, 31171161), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R27), the Project of New Century Excellent Talents in University (NCET-11-0234) and the Natural Science Foundation of the Jiangsu (BK2011414). References [1] M. Jemaa`, L. Galluzzi, O. Kepp, L. Senovilla, M. Brands, U. Boemer, M. Koppitz, P. Lienau, S. Prechtl, V. Schulze, G. Siemeister, A.M. Wengner, D. Mumberg, K. 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