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. 2019 Jun 12;10(8):1370–1378. doi: 10.1039/c9md00246d

Synthesis and antitumor activity of fluorouracil – oleanolic acid/ursolic acid/glycyrrhetinic acid conjugates†

Chun-Mei Liu a, Jia-Yan Huang a, Li-Xin Sheng a, Xiao-An Wen b, Ke-Guang Cheng a,✉
PMCID: PMC6786008  PMID: 31673307

graphic file with name c9md00246d-ga.jpgDue to the obvious adverse effects of 5-fluorouracil and considering the diverse biological activities of pentacyclic triterpenes, twelve pentacyclic triterpene-5-fluorouracil conjugates were synthesized and their antitumor activities were evaluated.

Abstract

Due to the obvious adverse effects of 5-fluorouracil that limit its clinical usefulness and considering the diverse biological activities of pentacyclic triterpenes, twelve pentacyclic triterpene-5-fluorouracil conjugates were synthesized and their antitumor activities were evaluated. The results indicated that all the single substitution targeted hybrids (7a–12a) possessed much better antiproliferative activities than the double substitution targeted hybrids (7b–12b). Hybrid 12a exhibited good antiproliferative activities against all the tested MDR cell lines. Furthermore, it was revealed that 12a could induce intracellular calcium influx, the generation of ROS, arrest the cell proliferation at the G1 phase, and activate the apoptotic signaling caspase-8, which eventually activates the apoptotic effector caspase-3 and causes the later nuclear apoptosis.

Introduction

5-Fluorouracil (5-FU, Fig. 1), as well as the oral pro-drug capecitabine, is a highly effective cell cycle-specific antitumor antimetabolite that interferes with DNA synthesis and inhibits RNA formation.1–4 It has been an essential part of the treatment of a wide range of solid tumors, such as esophageal adenocarcinoma, breast cancer, metastatic colorectal cancer and so on, since being introduced into clinical use in the 1950s.5–8 However, besides its broad antitumor spectrum, 5-FU also displays obvious adverse effects such as headache, depression, anxiety, myelosuppression, stomatitis, nausea and diarrhea,2,7,9 which limit its clinical usefulness. As such, structural modification of 5-FU is required to enhance its selectivity and minimize the toxic side effects.10–12

Fig. 1. Chemical structures of OA, UA, GA and 5-FU.

Fig. 1

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Natural products, which have a long history in disease therapy, play a key role in anti-tumor drug investigations for their important pharmacological properties.13,14 Extensive research has revealed that pentacyclic triterpenes are the main active ingredients of many traditional Chinese medicines.15,16 Pentacyclic triterpenes, including oleanolic acid (OA, 1, Fig. 1), ursolic acid (UA, 2, Fig. 1) and glycyrrhetinic acid (GA, 3, Fig. 1), which are the effective elements of many natural plants, have been widely researched for their diverse biological activities, such as anti-hepatodamage, anti-HIV, anti-inflammatory, anti-cancer, anti-diabetic, anti-bacterial and anti-proliferation.13,15,17–20 Particularly, the mechanism of the anti-tumour activity of natural pentacyclic triterpenes and their semisynthetic derivatives were summarized and analyzed.21 Regrettably, although there are many publications covering this research area, it is still not clear which proteins are the primary targets and which master regulators are switched on in response to these compounds.21

Nowadays, the pharmacophore hybridization principle is one of the important methods for designing and developing new drug lead compounds. The principle mainly refers to combining two known potential pharmacophores by covalent bonds to generate a novel hybrid molecule that could optimize certain pharmacological activities to get over the adverse effects related to a single drug.22–24

Alkyl chains are usually used as the linkers of two pharmacophores to increase the liposolubility. The different lengths of the alkyl chains could be used to obtain the targeted molecules with distinct physical and chemical properties for finding the optimal molecule.25,26 Therefore, modification through introducing active pharmacophores into natural products has become a research hot topic in drug development.27 Based on pharmacophore hybridization, our group has already reported several series of pentacyclic triterpene conjugates and some of them possess much better inhibition of proliferation as well as the cytotoxicity selectivity as compared to monomer pentacyclic triterpenes and the commercial anticancer drug 5-FU.28,29 For example, the dimeric oleanolic acid linked at C-28 by 1,6-hexanediamine displayed moderate cytotoxicity towards Hep-G2, A549, BGC-823, MCF-7 and PC-3 tumor cell lines with the IC50 value under 10.0 μM, while low cytotoxicity against the normal human liver cell HL-7702.30 Inspired by the evidence, in this study, 5-FU was respectively attached to the natural products OA/UA/GA to afford conjugates of pentacyclic triterpene-5-fluorouracil (Schemes 1 and 2, 7a/7b–12a/12b) and their antitumor activities were evaluated (Table 1). Finally, hybrid 12a was chosen for further preliminary investigation of its mechanism on A549 cell lines (Fig. 2–6).

Scheme 1. Synthesis of OA/UA/-5-FU derivatives, where n is the number of methylene groups.

Scheme 1

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Scheme 2. Synthesis of GA–5-FU derivatives, where n is the number of methylene groups.

Scheme 2

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Table 1. In vitro cytotoxicity of the target compounds after 48 h of treatment.

Compd IC50 a (μM)
A549 MCF-7 Bel-7402 K562 L-O2 A549/T MCF-7/ADR Bel-7402/FU K562/ADR
5-FU 50.43 52.07 53.33 41.66 >200 84.28 54.72 38.24 31.90
OA 41.65 >200 59.94 >200 >200 >200 >200 >200 >200
UA 77.65 70.12 64.64 41.01 58.37 — b — — 38.06
GA >200 >200 >200 >200 >200 99.85 74.49 69.77 86.46
7a 23.44 24.33 25.22 14.92 46.64 — — — —
7b >200 >200 >200 >200 >200 — — — —
8a 50.54 53.63 43.82 22.99 >200 43.07 166.2 31.42 >200
8b >200 >200 >200 >200 >200 — — — —
9a 17.35 18.86 32.50 14.89 22.25 — — — —
9b >200 >200 >200 >200 >200 — — — —
10a >200 >200 >200 >200 >200 — — — —
10b >200 >200 >200 >200 >200 — — — —
11a 49.35 50.31 40.92 35.05 73.24 — — — —
11b >200 >200 >200 >200 >200 — — — —
12a 15.68 12.89 20.98 9.80 20.90 20.73 23.42 19.77 26.98
12b >200 >200 >200 >200 >200 — — — —
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aThe IC50 values are the concentrations of the compounds that inhibit tumor cell growth by 50%, and are presented as the mean from three separate experiments.

bInhibition effects have not been detected.

Fig. 2. Analysis of Ca2+ production by flow cytometry after A549 cells were treated with hybrid 12a for 48 h.

Fig. 2

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Fig. 3. Analysis of ROS production by flow cytometry after A549 cells were treated with hybrid 12a for 48 h.

Fig. 3

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Fig. 4. Cell cycle progress of A549 cells treated with hybrid 12a at the specified concentration for 48 h, which were analyzed by flow cytometry assay after PI staining.

Fig. 4

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Fig. 5. Apoptosis effects on A549 cells treated with hybrid 12a at the specified concentration for 48 h, which were analyzed by flow cytometry after Annexin V-FITC/PI staining.

Fig. 5

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Fig. 6. Activation of caspase-3/8 on A549 cells treated with hybrid 12a was measured by flow cytometry in active caspase-3/8 staining kits.

Fig. 6

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Results and discussion

Synthesis

Pentacyclic triterpene–5-FU conjugates were synthesized as described in Schemes 1 and 2. Firstly, OA/UA/GA (1 equiv.) was esterified with the corresponding α,ω-dibromoalkane (3 equiv.) in the presence of potassium carbonate (1 equiv.) to prepare compounds 4a/4b–6a/6b28,31 in moderate to good yield (62–89%). Compounds 4a/4b–6a/6b (1 equiv.) were treated with 5-FU (3 equiv.) under potassium carbonate (3 equiv.) at 50 °C by means of a nucleophilic substitution reaction to generate targeted products 7a/7bb–12a/12b in the yields of 21–45%. Targeted compounds 7a/7b–12a/12b were fully characterized by 1H-NMR, 13C-NMR spectroscopy and mass spectrometry.

Antitumor activities evaluation

The cytotoxicities of the synthesized targeted pentacyclic triterpene–5-FU hybrids (7a/7b–12a/12b) were evaluated by MTT assay,32,33 firstly against the A549, MCF-7, Bel-7402 and K562 tumor cell lines as well as human normal liver cells L-O2 with 5-FU as positive controls (Table 1). However, the results showed that with the exception of 8a, all the targeted hybrids did not display selectivity against the tumor cell lines and liver cell L-O2 cell line. The selectivity of 8a was consistent with the proliferation activity of 5-FU on the tested cell lines. It was found that all the double substitution targeted hybrids (7b–12b) did not possess any anti-tumor activity, while the single substitution targeted hybrids (7a–12a), with the exception of 10a, exhibited moderate to good anti-tumor activity against tested cell lines (A549, MCF-7, Bel-7402 and K562).

Among the single substitution targeted hybrids, though hybrids 7a/9a/12a showed good antiproliferative activities, only hybrid 8a showed potential selectivity against tumor cells. 8a and 12a were tested against multidrug resistant (MDR) cell lines (A549/T, MCF-7/ADR, Bel-7402/FU and K562/ADR). The results showed that 8a exhibited moderate antiproliferative activity against A549/T and Bel-7402/FU cell lines, which was the same level as against A549 and Bel-7402 cell lines; 12a exhibited good antiproliferative activities against all the tested MDR cell lines.

Flow cytometry analysis

The detection of intracellular Ca2+ production

As the important second messenger in the cell, calcium ions regulate most of the signaling pathways in cells and play an important role in the physiological activities of cells.34 The change in the free calcium ion concentration is closely related to the cell function, signal transmission, damage and apoptosis.35 In this study, the Fluo-3 AM (Calcium Indicator, CAS 121714-22-5) was used to detect the variety of calcium ion to determine whether the apoptosis was related to the intracellular calcium influx. As shown in Fig. 2, the A549 cells treated with hybrid 12a showed a right-shift fluorescence peak as compared with the control group, which indicated that the hybrid 12a could induce intracellular calcium influx.

Evaluation of ROS levels

It was reported that ROS (reactive oxygen species) could induce apoptosis through cellular oxidative stress levels and affect cell signal transduction pathways to regulate tumor cell apoptosis and proliferation.36 Thus, in order to investigate whether the hybrid 12a could influence the ROS production in A549 cells, the fluorescent probe DCFH-DA (Reactive Oxygen Species Assay Kit) combined with flow cytometry was used to detect the level of ROS. As shown in Fig. 3, the A549 cells treated with hybrid 12a demonstrated a right-shift fluorescence peak as compared with the control group, which indicated that hybrid 12a could induce the generation of ROS.

Cell cycle analysis

The cell cycle regulates cell differentiation and proliferation, and also regulates cell function decline and cell senescence. The regulation mechanism of the cell cycle could reveal the nature of tumor development and explain the mechanism of cancer. The cell cycle can be divided into four phases: G1 phase (synthesis of RNA and ribosomes), G2 phase (mass synthesis of RNA and protein), S phase (synthesis of histones, DNA and corresponding enzymes) and M phase (average distribution of genetic material). The influence of 12a on the cell cycle of A549 cells was detected by propidium iodide (PI) through flow cytometry (Fig. 4). The results revealed that the A549 cell ratio in the G1 phase rose from 70.63% to 92.18%, indicating that the hybrid 12a could block the cell cycle in the G1 phase.

Annexin V/propidium iodide assay

Apoptosis is a mode of programmed cell death, it occurs through the regulation of intracellular genes and their products during development or under certain factors. It involves the activation, expression, and regulation of a range of genes, and it is a gradual process that can be divided into four periods. At different stages of apoptosis, the cells undergo the corresponding apoptotic molecular signals, produce different cellular stress responses, and exhibit different morphological features such as cell morphology. Therefore, to investigate cell apoptosis in the presence of 12a, FITC-Annexin V/PI dual staining and flow cytometric analysis were performed on A549 cells (Fig. 5). The results showed that as the concentration of 12a increased, the proportion of the A549 cells in the Q2 area increased from 4.19% to 5.43%, 31.90% and 46.40% in a dose-dependent manner. Hence, flow cytometric analysis indicated that 12a could induce later apoptosis in A549 cells in a concentration-dependent way.

Caspase-3/8 activation assay

Caspase-8 plays a key role in triggering apoptosis upon endoplasmic reticulum stress.37 Caspase-3, which can promote cell apoptosis through intracellular or extracellular pathways, is the main effector in the process of apoptosis.38 The activation of caspase-3 is a sign that apoptosis has entered an irreversible phase. In this study, the activation of caspase-3/8 was measured by flow cytometry in active caspase-3/8 staining kits. As shown in Fig. 6, activated caspase-8 levels increased to 12.70% and 32.40% after treatment with hybrid 12a (15 μM, 20 μM, respectively) as compared to the control. Similarly, activated caspase-3 levels increased significantly from 5.99% to 24.50%, 50.90% and 64.70% after treatment 12a (0 μM, 10 μM, 15 μM, 20 μM, respectively). These results indicate that 12a could induce apoptosis via activating caspase-3/8 in A549 cells.

Experimental

General

All the chemical reagents and solvents were of analytical grade and were used without further purification unless specified. All commercial reagents were purchased from Aladdin (Shanghai) Industrial Corporation. Melting points were measured on a RY-1 melting point apparatus. 1H and 13C NMR-spectra were recorded on a Bruker AV-500 or AV-300 spectrometer. Chemical shifts are reported as values from an internal tetramethylsilane standard. Mass spectra were recorded on an Agilent 6500 Series Q-TOF LC/MS System, or on a Thermo Scientific Accela – Exactive High-Resolution Accurate Mass spectrometer. The item numbers of the cell lines from Nanjing Kaiji Biotechnology Development Co. Ltd. (Nanjing, China) are as follows: Hep-G2 (KG020), A549 (KG007), Bel-7402 (KG022), BGC-823 (KG025), MCF-7 (KG031), MDA-MB-231 (KG033), PC-3 (KG044), L-O2 (HL-7702, KG063), A549/T (KG124), MCF-7/ADR (KG0311), Bel-7402/FU (KG125) and K562/ADR (KG301).

Synthesis

General procedure for N-alkylation reaction of 5-FU to afford 7a/7b to 12a/12b

To a solution of ω-bromoalkyl oleanate/ursolate/glycyrrhetinate28,31 (0.404 mmol) in DMF (5 mL), K2CO3 (167.2 mg, 1.21 mmol) and 5-fluorouracil (167.2 mg, 1.29 mmol) were added. After stirring at 50 °C for 4 hours, the mixture was diluted with EtOAc (50 mL). Then, the organic layer was washed successively with 1 N HCl, saturated NaHCO3 and saturated brine, dried over anhydrous Na2SO4, filtered and concentrated to obtain the residue, which was purified by flash column chromatography.

Compound 7a

Prepared from compound 4a28,31 (250 mg, 0.404 mmol) and 5-fluorouracil (167.2 mg, 1.29 mmol) according to the general procedure, compound 7a was obtained by flash column chromatography. Yield 63 mg, 24%, white solid, mp 93–95 °C; Rf = 0.28 (petroleum ether : EtOAc = 1 : 1). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.88 (s, 6 H, 2 × CH3), 0.70 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.90 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 1.11 (s, 3 H, CH3), 0.67–2.05 (m, 30 H), 2.84 (dd, J = 4.0, 13.9 Hz, 1 H, 18-H), 3.20 (dd, J = 4.6, 11.2 Hz, 1 H, 3-H), 3.69 (t, 1 H, J = 7.4 Hz, NCHa), 3.86–4.00 (m, 3 H, COOCH2 and NCHb), 5.25 (t, 1 H, J = 3.5 Hz, 12-H), 7.24 (d, J = 5.4 Hz, 1 H, 6-CHflu), 9.72 (s, 1 H, 3-NHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.4, 15.7, 17.2, 18.4, 23.1, 23.5, 23.7, 25.8, 26.0, 26.1, 27.2, 27.8, 28.2, 28.5, 28.9, 30.8, 32.6, 32.9, 33.2, 34.0, 37.1, 38.5, 38.8, 39.4, 41.4, 41.8, 46.0, 46.8, 47.7, 49.1, 55.3, 64.0, 79.1, 122.5, 128.6, 139.6, 143.9, 149.6, 157.5, 177.9. HRMS (ESI) m/z: [M + Cl]+ calcd for C40H61ClFN2O5 703.4253; found 703.4279.

Compound 7b

Prepared from compound 4a28,31 (500 mg, 0.807 mmol) and 5-fluorouracil (314.8 mg, 2.42 mmol) according to the general procedure, compound 7b was obtained by flash column chromatography. Yield 242 mg, 25%, white solid, mp 135–137 °C; Rf = 0.288 (petroleum ether : EtOAc = 2 : 1). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.89 (s, 12 H, 4 × CH3), 0.72 (s, 6 H, 2 × CH3), 0.77 (s, 6 H, 2 × CH3), 0.91 (s, 6 H, 2 × CH3), 0.98 (s, 6 H, 2 × CH3), 1.12 (s, 6 H, 2 × CH3), 0.67–2.19 (m, 60 H), 2.85 (dd, J = 4.6, 14.9 Hz, 2 H, 18-H and 18′-H), 3.22 (dd, J = 4.8, 10.4 Hz, 2 H, 3-H and 3′-H), 3.71 (t, J = 7.4 Hz, 2 H, NCHa and NCHa′), 3.81–4.31 (m, 6 H, 2 × OCH2, NCHb and NCHb′), 5.27 (brs, 2 H, 12-H, 12′-H), 7.20 (d, J = 5.1 Hz, 1 H, 6-CHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.4, 15.7, 17.1, 17.2, 18.5, 23.2, 23.6, 23.8, 25.8, 25.9, 26.0, 26.2, 26.7, 27.3, 27.5, 27.8, 28.2, 28.57, 28.63, 29.0, 30.8, 32.60, 32.63, 32.9, 33.20, 33.23, 34.0, 37.2, 38.6, 38.9, 39.5, 41.5, 41.8, 42.1, 46.00, 46.01, 46.79, 46.81, 47.72, 47.76, 50.0, 55.34, 55.37, 64.0, 64.2, 79.08, 79.13, 122.5, 126.3, 126.6, 139.2, 141.1, 143.9, 149.9, 157.4, 177.8. HRMS (ESI) m/z: [M + Cl]+ calcd for C76H119ClFN2O8, 1241.8639; found 1241.8646.

Compound 8a

Prepared from compound 4b28,31 (250 mg, 0.386 mmol) and 5-fluorouracil (150.9 mg, 1.16 mmol) according to the general procedure, compound 8a was obtained by flash column chromatography. Yield 61 mg, 23%, white solid, mp 92–94 °C; Rf = 0.281 (petroleum ether : EtOAc = 1 : 1). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.88 (s, 6 H, 2 × CH3), 0.71 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.90 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 1.11 (s, 3 H, CH3), 0.67–2.08 (m, 34 H), 2.84 (dd, J = 4.0, 13.8 Hz, 1 H, 18-H), 3.21 (dd, J = 4.6, 11.1 Hz, 1 H, 3-H), 3.68 (t, J = 7.4 Hz, 1 H, NCHa), 3.85–4.00 (m, 3 H, COCH2 and NCHb), 5.25 (t, J = 3.3 Hz, 1 H, 12-H), 7.24 (d, J = 5.4 Hz, 1H, 6-CHflu), 9.81 (s, 1 H, 3-NHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.4, 15.7, 17.1, 18.4, 23.1, 23.5, 23.7, 25.97, 26.03, 26.4, 27.2, 27.7, 28.2, 28.6, 29.0, 29.1, 29.2, 30.8, 32.6, 32.9, 33.2, 34.0, 37.1, 38.5, 38.8, 39.4, 41.4, 41.8, 46.0, 46.8, 47.7, 49.2, 55.3, 64.2, 79.1, 122.4, 128.7, 139.6, 143.9, 149.6, 157.5, 178.0. HRMS (ESI) m/z: [M – H]+ calcd for C42H64FN2O5, 695.4799; found 695.4811.

Compound 8b

Prepared from compound 4b28,31 (500 mg, 0.772 mmol) and 5-fluorouracil (301.8 mg, 2.32 mmol) according to the general procedure, compound 8b was obtained by flash column chromatography. Yield 202 mg, 21%, white solid, mp 118–120 °C; Rf = 0.356 (petroleum ether : EtOAc = 2 : 1). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.72 (s, 6 H, 2 × CH3), 0.77 (s, 6 H, 2 × CH3), 0.89 (s, 12 H, 4 × CH3), 0.92 (s, 6 H, 2 × CH3), 0.98 (s, 6 H, 2 × CH3), 1.12 (s, 6 H, 2 × CH3), 0.65–2.12 (m, 68 H), 2.86 (dd, J = 3.75, 13.9 Hz, 2 H, 18-H and 18′-H), 3.22 (dd, J = 4.8, 10.5 Hz, 2 H, 3-H and 3′-H), 3.68–3.73 (t, J = 7.4 Hz, 2 H, NCHa and NCHa′), 3.91–3.99 (m, 6 H, 2 × OCH2, NCHb and NCHb′), 5.27 (t, J = 3.0 Hz, 2 H, 12-H and 12′-H), 7.19 (d, J = 5.1 Hz, 1 H, 6-CHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.4, 15.7, 17.2, 18.5, 23.2, 23.6, 23.7, 25.99, 26.04, 26.1, 26.5, 26.9, 27.3, 27.6, 27.8, 28.2, 28.66, 28.71, 29.0, 29.1, 29.20, 29.24, 29.3, 30.8, 32.6, 32.9, 33.2, 34.0, 37.2, 38.6, 38.9, 39.5, 41.5, 41.8, 42.2, 46.0, 46.8, 47.8, 50.0, 55.4, 64.3, 79.1, 122.5, 126.5, 139.2, 144.0, 149.9, 157.3, 177.9. HRMS (ESI) m/z: [M + Cl]+ calcd for C80H127ClFN2O8, 1297.9265; found 1297.9274.

Compound 9a

Prepared from compound 5a31 (250 mg, 0.403 mmol) and 5-fluorouracil (157.42 mg, 1.21 mmol) according to the general procedure, compound 9a was obtained by flash column chromatography. Yield 70 mg, 26%, white solid, mp 85–87 °C; Rf = 0.433 (petroleum ether : EtOAc = 1 : 1). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.72 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.89 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 1.06 (s, 3 H, CH3), 0.67–2.08 (m, 30 H), 2.19 (d, J = 11.2 Hz, 1 H, 18-H), 3.20 (dd, J = 4.6, 11.1 Hz, 1 H, 3-H), 3.69 (t, J = 7.4 Hz, 2 H, OCH2), 3.71–4.22 (m, 3 H, OH and NCH2), 5.21 (t, J = 3.5 Hz, 1 H, 12-H), 7.24 (d, J = 5.4 Hz, 1 H), 9.89 (s, 1 H, 3-Hflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.5, 15.7, 17.0, 17.1, 18.3, 21.2, 23.3, 23.5, 24.2, 25.7, 26.0, 27.2, 28.0, 28.1, 28.4, 28.8, 30.7, 33.1, 36.8, 37.0, 38.6, 38.7, 38.9, 39.1, 39.6, 42.1, 47.5, 48.1, 49.0, 52.9, 55.2, 63.9, 79.0, 125.5, 128.5, 138.2, 139.5, 149.5, 157.3, 177.6. HRMS (ESI) m/z: [M – H]+ calcd for C40H60FN2O5, 667.4486; found 667.4494.

Compound 9b

Prepared from compound 5a31 (500 mg, 0.807 mmol) and 5-fluorouracil (314.83 mg, 2.42 mmol) according to the general procedure, compound 9b was obtained by flash column chromatography. Yield 316 mg, 32%, white solid, mp 71–73 °C; Rf = 0.156 (petroleum ether : EtOAc = 2 : 1). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.74 (s, 6 H, 2 × CH3), 0.77 (s, 6 H, 2 × CH3), 0.85 (d, J = 6.2 Hz, 6 H, 2 × CH3), 0.90 (s, 6 H, 2 × CH3), 0.94 (d, J = 6.2 Hz, 6 H, 2 × CH3), 0.98 (s, 6 H, 2 × CH3), 1.07 (s, 6 H, 2 × CH3), 0.53–2.19 (m, 60 H), 2.21 (d, J = 11.2 Hz, 2 H, 18-H and 18′-H), 3.21 (dd, J = 4.6, 10.8 Hz, 2 H, 3-H and 3′-H), 3.71 (t, J = 7.3 Hz, 2 H, NCHa and NCHa′), 3.93–3.99 (m, 6 H, 2 × COOCH2, NCHb and NCHb′), 5.23 (brs, 2 H, 12-H and 12′-H), 7.19 (d, J = 5.1 Hz,1 H, 6-CHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 14.2, 15.6, 15.8, 17.1, 17.3, 18.4, 21.5, 22.8, 23.4, 23.7, 24.3, 25.8, 25.9, 26.2, 26.7, 27.3, 27.5, 28.1, 28.3, 28.56, 28.62, 29.0, 29.5, 29.77, 29.82, 30.8, 32.0, 33.2, 36.9, 37.1, 38.8, 38.9, 39.0, 39.2, 39.7, 42.2, 47.7, 48.2, 50.0, 53.0, 55.3, 64.0, 64.2, 79.1, 125.6, 126.5, 138.3, 141.1, 149.9, 157.3, 177.7. HRMS (ESI) m/z: [M + Cl]+ calcd for C76H119ClFN2O8, 1241.8639; found 1241.8586.

Compound 10a

Prepared from compound 5b31 (250 mg, 0.386 mmol) and 5-fluorouracil (150.6 mg, 1.16 mmol) according to the general procedure, compound 10a was obtained by flash column chromatography. Yield 71 mg, 26%, white solid, mp 83–85 °C; Rf = 0.393 (petroleum ether : EtOAc = 1 : 1). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.73 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.84 (d, J = 6.3 Hz, 3 H, CH3), 0.89 (s, 3 H, CH3), 0.93 (d, J = 6.3 Hz, 3 H, CH3), 0.97 (s, 3 H, CH3), 1.06 (s, 3 H, CH3), 0.67–2.11 (m, 34 H), 2.20 (d, J = 11.4 Hz, 1 H, 18-H), 3.21 (dd, J = 4.7, 11.1 Hz, 1 H, 3-H), 3.67 (t, J = 7.4 Hz, 1 H, NCHa), 3.96 (t, J = 6.6 Hz, 1 H, NCHb), 3.91–4.01 (m, 2 H, COOCH2), 5.21 (t, J = 3.6 Hz, 1 H, 12-H), 7.24 (d, J = 5.4 Hz, 1H, 6-CHflu), 9.72 (s, 1 H, 3-NHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.6, 15.8, 17.1, 17.2, 18.4, 21.3, 23.4, 23.7, 24.5, 26.0, 26.4, 27.3, 28.4, 28.6, 29.0, 29.1, 29.2, 30.8, 33.2, 36.9, 37.1, 38.7, 38.8, 39.0, 39.2, 39.7, 42.2, 47.6, 48.2, 49.2, 53.0, 55.3, 64.2, 79.1, 125.6, 128.6, 138.3, 139.6, 149.6, 157.4, 177.8. HRMS (ESI) m/z: [M – H]+ calcd for C42H64FN2O5, 695.4799; found 695.4810.

Compound 10b

Prepared from compound 5b31 (500 mg, 0.772 mmol) and 5-fluorouracil (301.2 mg, 2.32 mmol) according to the general procedure, compound 10b was obtained by flash column chromatography. Yield 294 mg, 30%, white solid, mp 59–61 °C; Rf = 0.213 (petroleum ether : EtOAc = 2 : 1). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.74 (s, 6 H, 2 × CH3), 0.77 (s, 6 H, 2 × CH3), 0.85 (d, J = 6.2 Hz, 6 H, 2 × CH3), 0.90 (s, 6 H, 2 × CH3), 0.94 (d, J = 6.2 Hz, 6 H, 2 × CH3),0.98 (s, 6 H, 2 × CH3), 1.07 (s, 6 H, 2 × CH3), 0.65–2.02 (m, 68 H), 2.22 (d, J = 11.3 Hz, 2 H, 18-H and 18′-H), 3.21 (dd, J = 4.5, 10.5 Hz, 2 H, 3-H and 3′-H), 3.67 (t, J = 7.4 Hz, 2 H, NCHa and NCHa′), 3.82–4.03 (m, 6 H, 2 × OCH2, NCHb and NCHb′), 5.23 (t, J = 3.5 Hz, 2 H, 12-H and 12′-H), 7.19 (d, J = 5.1 Hz, 1 H, 6-CHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 14.1, 15.5, 15.6, 17.0, 17.1, 18.3, 21.2, 22.7, 23.3, 23.6, 24.2, 25.9, 26.0, 26.4, 26.8, 27.2, 27.4, 28.0, 28.3, 28.5, 28.6, 28.9, 29.1, 29.2, 29.4, 29.7, 30.7, 31.9, 33.1, 36.7, 37.0, 38.7, 38.8, 38.9, 39.1, 39.6, 42.1, 47.6, 48.1, 49.9, 52.9, 55.2, 64.2, 79.0, 125.5, 126.3, 138.2, 141.0, 149.8, 157.2, 177.6. HRMS (ESI) m/z: [M + Cl]+ calcd for C80H127ClFN2O8, 1297.9265; found 1297.9225.

Compound 11a

Prepared from compound 6a31 (250 mg, 0.395 mmol) and 5-fluorouracil (153.95 mg, 1.18 mmol) according to the general procedure, compound 11a was obtained by flash column chromatography. Yield 66 mg, 25%, white solid, mp 111–113 °C; Rf = 0.283 (petroleum ether : EtOAc = 1 : 1). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.77 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.98 (s, 3 H, CH3), 1.12 (s, 3 H, CH3), 1.10 (s, 9 H, 3 × CH3), 0.62–2.33 (m, 27 H), 2.32 (s, 1 H, 9-H), 2.73 (m, 1 H, 18-H), 3.20 (dd, J = 5.1, 11.1 Hz, 1 H, 3-H), 3.72 (m, 2 H, NCH2), 3.89–4.19 (m, 3 H, COOCH2 and 3-OH), 5.57 (s, 1 H, 12-H), 7.54 (d, J = 5.6 Hz, 1H, 6-CHflu), 9.87 (s, 1 H, 3-NHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.7, 16.4, 17.6, 18.8, 23.5, 26.2, 26.3, 26.5, 26.6, 27.3, 28.2, 28.6, 28.7, 28.8, 29.0, 31.2, 32.0, 32.8, 37.2, 37.7, 39.2, 41.2, 43.4, 44.1, 45.6, 48.8, 48.9, 55.0, 62.0, 64.3, 78.8, 128.3, 129.2, 139.6, 149.7, 169.9, 176.6, 200.6. HRMS (ESI) m/z: [M – H]+ calcd for C40H58FN2O6, 681.4279; found 681.4282.

Compound 11b

Prepared from compound 6a31 (500 mg, 0.789 mmol) and 5-fluorouracil (307.89 mg, 2.36 mmol) according to the general procedure, compound 11b was obtained by flash column chromatography. Yield 204 mg, 21%, white solid, mp 108–109 °C; Rf = 0.203 (petroleum ether : EtOAc = 2 : 1). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.80 (s, 12 H, 4 × CH3), 1.00 (s, 12 H, 4 × CH3),1.13 (s, 18 H, 6 × CH3), 0.49–2.19 (m, 54 H), 2.34 (s, 2 H, 9-H and 9′-H), 2.75 (d, J = 4.3 Hz, 2 H, 18-H and 18′-H), 3.23 (brs, 2 H, 3-H and 3′-H), 3.76 (brs, 2 H, NCH2), 3.96–4.15 (m, 6 H, 2 × OCH2, 2 × OH), 5.62 (s, 2 H, 12-H and 12′-H), 7.48 (d, 1 H, J = 5.1 Hz, 6-CHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 14.2, 15.7, 16.5, 17.6, 18.8, 22.8, 23.5, 25.8, 26.2, 26.4, 26.6, 27.5, 28.2, 28.6, 28.8, 28.9, 29.1, 29.5, 29.8, 31.2, 32.0, 32.9, 37.2, 37.8, 37.9, 39.3, 41.2, 41.3, 42.0, 43.3, 43.4, 44.10, 44.13, 45.5, 45.6, 48.5, 48.8, 49.8, 55.1, 62.0, 64.4, 78.8, 126.9, 128.4, 139.2, 150.0, 157.4, 169.5, 176.5, 200.3. HRMS (ESI) m/z: [M + Cl]+ calcd for C76H115ClFN2O10, 1269.8224; found 1269.8228.

Compound 12a

Prepared from compound 6b31 (300 mg, 0.453 mmol) and 5-fluorouracil (176.9 mg, 1.36 mmol) according to the general procedure, compound 12a was obtained by flash column chromatography. Yield 75 mg, 23%, white solid, mp 65–67 °C; Rf = 0.295 (petroleum ether : EtOAc = 1 : 1). 1H NMR (500 MHz, CDCl3) δ (ppm): 0.76 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.97 (s, 6 H, 2 × CH3), 1.08 (s, 3 H, CH3), 1.09 (s, 3 H, CH3), 1.11 (s, 3 H, CH3), 0.61–2.35 (m, 31 H), 2.31 (s, 1 H, 9-H), 2.71 (m, 1H, 18-H), 3.20 (dd, J = 4.9, 11.3 Hz, 1 H, 3-H), 3.69 (t, J = 7.5 Hz, 1 H, NCHa), 4.00–4.15 (m, 3 H, OCH2 and NCHb), 5.58 (s, 1 H, 12-H), 7.37 (d, J = 5.5 Hz, 1 H, 6-CHflu), 10.02 (s, 1 H, 3-NHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 14.2, 15.7, 16.4, 17.5, 18.6, 23.4, 25.9, 26.0, 26.3, 26.45, 26.53, 26.7, 27.2, 28.2, 28.5, 28.6, 28.7, 28.8, 29.0, 29.1, 31.2, 31.9, 32.8, 37.1, 37.8, 39.2, 41.1, 43.3, 44.1, 45.5, 48.6, 49.1, 55.0, 61.9, 64.5, 78.8, 128.4, 129.0, 139.5, 149.2, 169.7, 176.6, 200.4. HRMS (ESI) m/z: [M + Na]+ calcd for C42H63FN2O6Na, 733.4568; found 733.4575.

Compound 12b

Prepared from compound 6b31 (900 mg, 1.36 mmol) and 5-fluorouracil (530.71 mg, 4.08 mmol) according to the general procedure, compound 12b was obtained by flash column chromatography. Yield 793 mg, 45%, white solid, mp 126–128 °C; Rf = 0.164 (petroleum ether : EtOAc = 2 : 1). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.80 (s, 18 H, 6 × CH3), 1.00 (s, 6 H, 2 × CH3), 1.12 (s, 12 H, 4 × CH3), 1.13 (2 s, 6 H, 2 × CH3), 0.60–2.43 (m, 62 H), 2.34 (s, 2 H, 9-H and 9′-H), 2.77 (m, 2 H, 18-H and 18′-H), 3.23 (m, 2 H, 3-H and 3′-H), 3.73 (t, J = 7.5 Hz, 2 H, NCHa and NCHa′), 3.94 (t, J = 7.5 Hz, 2 H, NCHb and NCHb′), 4.00–4.09 (m, 4 H, 2 × OCH2), 5.62 and 5.63 (2 s, each 1 H, 12-H and 12′-H), 7.30 (d, 1 H, J = 5.1 Hz, 6-CHflu). 13C NMR (125 MHz, CDCl3) δ (ppm): 15.7, 16.5, 17.6, 18.8, 23.5, 26.0, 26.1, 26.4, 26.57, 26.62, 26.9, 27.40, 27.44, 27.5, 28.2, 28.5, 28.6, 28.7, 28.8, 28.9, 29.2, 31.3, 32.0, 32.9, 37.2, 37.9, 39.3, 41.2, 42.1, 43.4, 44.1, 45.5, 48.4, 48.6, 50.0, 55.1, 62.0, 64.6, 78.9, 126.7, 128.6, 139.2, 149.9, 157.4, 169.4, 176.6, 200.3. HRMS (ESI) m/z: [M + H]+ calcd for C80H124FN2O10, 1291.9240; found 1291.9290.

In vitro cytotoxicity

The MTT assay32,33 was carried out as follows. Cells were seeded in 96-well plates and incubated in the CO2 (5%) incubator at 37 °C. When the cells adhered to the plate, compounds at different concentrations (20, 10, 5, 2.5, 1.25 μM) were added to each well. After incubation for another 48 h, 20 μL MTT (5%) was added to each well and incubated for an additional 4 h. The viable cells were stained with MTT and scanned with a spectrophotometer at 570 nm. Each treatment was done in triplicate.

Intracellular Ca2+ generation

The intracellular Ca2+ production was measured by a Fluo-3 AM probe. The cells were seeded in six-well plates for 24 h, and then with the selected compound (0 μM, 10 μM, 15 μM) for 48 h. After that, the cells were harvested and washed three times with serum-free medium (RPMI medium 1640 basic (1×)). The Fluo-3 AM (0.5 μM, 500 μL) was added to each sample and then incubated in an incubator for 60 min. The mixture was washed twice with a serum-free medium (RPMI medium 1640 basic (1×)) and suspended with 500 μL of corresponding serum-free medium. The generation of Ca2+ was immediately measured by flow cytometry.

Detection of intracellular reactive oxygen species

Intracellular reactive oxygen species (ROS) production was detected with a DCFH-DA probe. Appropriate A549 cells were incubated with the selected compound (0 μM, 20 μM, 25 μM) in six-well plates for 48 h. The cells were then harvested and washed twice with PBS, followed by suspension in 500 μL of the serum-free medium (RPMI medium 1640 basic (1×)) containing 10 μM L–1 DCFH-DA probe. After 30 min of incubation at 37 °C in the dark, the cells were centrifuged and washed three times with serum-free medium. The generation of ROS in the A549 cells was detected by flow cytometry.

Annexin V/propidium iodide assay

In the apoptosis experiments, the Annexin V-FITC kit was used. A549 cells were seeded in six-well plates and incubated with the selected compound at different concentrations (0 μM, 10 μM, 15 μM, 20 μM) for 48 h. The A549 cells were then collected and suspended in annexin binding buffer (1×, 200 μL). In the next step, 5 μL Annexin V and 5 μL propidium iodide were added to the corresponding sample, and the mixture was incubated on the ice in the dark for 30 min. Subsequently, the extra binding buffer (300 μL) was added to the mixture, and the cell apoptosis was analyzed by flow cytometry.

Cell cycle assay

To investigate the influence of the selected compound on the cell cycle of tumor cells, flow cytometry was used to measure the changes in the cell cycle distribution in A549 cells at different concentrations. The A549 cells were seeded in cell and tissue culture dishes (70 mm) for 24 h, then treated with the selected compound (0 μM, 10 μM, 15 μM, 20 μM) for 48 h. The cells were then collected and washed with cool phosphate buffer saline (PBS) solution. The cells were fixed with cold 75% ethanol and stored at –20 °C for 36 h. Later, the cells were centrifuged and washed with cold PBS. RNase A (100 μg mL–1, 500 μL) was added to each sample and incubated in an incubator for 30 min. Propidium iodide (1 mg mL–1, 25 μL) was added to each sample for 10 min in the dark, and then detected by flow cytometry.

Detection of caspase-3/8

The activation of caspase-3/8 was detected by flow cytometry via the Caspase-3/8 Staining Kit. Appropriate A549 cells in six-well plates were exposed to the selected compounds (0 μM, 10 μM, 15 μM, 20 μM) for 48 h. Cells were collected and washed with PBS solution, suspended with PBS 300 μL containing 0.5 μL FITC-DEVD-FMK, and incubated for 50 minutes. The mixture was centrifuged and washed with wash buffer twice, and suspended in 500 μL wash buffer. A flow cytometer was immediately employed to detect caspase-3/8 activity.

Statistics

Data processing included the Student's t-test with P ≤ 0.05 taken as the significance level, using SPSS 17.0.

Conclusions

Twelve pentacyclic triterpene–5-FU conjugates were synthesized and their antitumor activities were evaluated. The results outlined that all the single substitution targeted hybrids (7a–12a) possessed much better antiproliferative activities than the double substitution targeted hybrids (7b–12b). Hybrid 8a showed potential selectivity against tumor cells, and also possessed moderate antiproliferative activities against MDR cell lines A549/T and Bel-7402/FU, which were at the same levels as against A549 and Bel-7402 cell lines. However, only 12a exhibited good antiproliferative activities against all the tested MDR cell lines and possessed much better antiproliferative activities than 8a. Hybrid 12a was chosen for further preliminary investigation of its mechanism on A549 cell lines. It was revealed that after the treatment of A549 cells with 12a, the changes in cellular morphology could induce intracellular calcium influx and the generation of ROS as a result of significant apoptosis induction. Subsequently, cell proliferation in the G1 phase was arrested and apoptotic signaling of caspase-8 was activated. Furthermore, as a protease, caspase-8 could activate the apoptotic effector caspase-3, which eventually caused the later nuclear apoptosis. The other specific mechanisms of 12a are currently underway.

Conflicts of interest

There are no conflicts of interest to declare.

Supplementary Material

Click here for additional data file. (3MB, pdf)

Acknowledgments

This study was financially supported by BAGUI Scholar Program of Guangxi Province of China (2016A13) and grants from the National Natural Science Foundation of PRC (21562006), Guangxi Natural Science Foundation of China (2015GXNSFAA139186), Guangxi's Medicine Talented Persons Small Highland Foundation (1506), Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), and Ministry of Education of China (CMEMR2013-A01, CMEMR2013-C02 and CMEMR2014-B12) and IRT_16R15. Miss Liu specially thanks to the Innovation Project of Guangxi Graduate Education (XYCSZ2018070).

Footnotes

†Electronic supplementary information (ESI) available: Supplementary data contains NMR and HRMS spectra for all of the synthetic compounds. See DOI: 10.1039/c9md00246d

References

  1. Zhao D., Zhang H., Tao W., Wei W., Sun J., He Z. Biomater. Sci. 2017;5:502. doi: 10.1039/c6bm00884d. [DOI] [PubMed] [Google Scholar]
  2. Rejhová A., Opattová A., Čumová A., Slíva D., Vodička P. Eur. J. Med. Chem. 2018;144:582. doi: 10.1016/j.ejmech.2017.12.039. [DOI] [PubMed] [Google Scholar]
  3. Fang X. J., Jeyakkumar P., Avula S. R., Zhou Q., Zhou C. H. Bioorg. Med. Chem. Lett. 2016;26:2584. doi: 10.1016/j.bmcl.2016.04.036. [DOI] [PubMed] [Google Scholar]
  4. Zhang B. H., Leng C., Wu C., Zhang Z. G., Dou L., Luo X., Zhang B. X., Chen X. P. Oncol. Rep. 2016;35:1807. doi: 10.3892/or.2015.4479. [DOI] [PubMed] [Google Scholar]
  5. Hishinuma E., Akai F., Narita Y., Maekawa M., Yamaguchi H., Mano N., Oda A., Hirasawa N., Hiratsuka M. Biochem. Pharmacol. 2017;143:118. doi: 10.1016/j.bcp.2017.06.121. [DOI] [PubMed] [Google Scholar]
  6. Eads J. R., Beumer J. H., Negrea L., Holleran J. L., Strychor S., Meropol N. J. Cancer Chemother. Pharmacol. 2016;77:333. doi: 10.1007/s00280-015-2939-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Wang J., Fan X., Yang L. Y., He H., Huang R., Jiang F. L., Liu Y. MedChemComm. 2016;7:2016. [Google Scholar]
  8. Lu S., Obianom O. N., Ai Y. MedChemComm. 2018;9:1722. doi: 10.1039/c8md00284c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Li Y., Fan L., Niu Y., Mian W., Zhang F., Xie M., Sun Y., Mei Q. Eur. J. Pharmacol. 2017;804:13. doi: 10.1016/j.ejphar.2017.04.001. [DOI] [PubMed] [Google Scholar]
  10. Jiang Y., Li X., Hou J., Huang Y., Wang X., Jia Y., Wang Q., Xu W., Zhang J., Zhang Y. Eur. J. Med. Chem. 2018;143:334. doi: 10.1016/j.ejmech.2017.11.074. [DOI] [PubMed] [Google Scholar]
  11. Ai Y., Obianom O. N., Kuser M., Li Y., Shu Y., Xue F. ACS Med. Chem. Lett. 2019;10:127. doi: 10.1021/acsmedchemlett.8b00539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Thysiadis S., Katsamakas S., Dalezis P., Chatzisideri T., Trafalis D., Sarli V. Future Med. Chem. 2017;9:2181. doi: 10.4155/fmc-2017-0139. [DOI] [PubMed] [Google Scholar]
  13. Chen Y., Li C., Zheng Y., Gao Y., Hu J., Chen H. Bioorg. Med. Chem. Lett. 2017;27:1007. doi: 10.1016/j.bmcl.2016.12.071. [DOI] [PubMed] [Google Scholar]
  14. Zígolo M. A., Salinas M., Alché L., Baldessari A., Liñares G. G. Bioorg. Chem. 2018;78:210. doi: 10.1016/j.bioorg.2018.03.018. [DOI] [PubMed] [Google Scholar]
  15. Zhang B. W., Xing Y., Wen C., Yu X. X., Sun W. L., Xiu Z. L., Dong Y. S. Bioorg. Med. Chem. Lett. 2017;27:5065. doi: 10.1016/j.bmcl.2017.09.027. [DOI] [PubMed] [Google Scholar]
  16. Li H., Li M., Xu R., Wang S., Zhang Y., Zhang L., Zhou D., Xiao S. Eur. J. Med. Chem. 2019;163:560. doi: 10.1016/j.ejmech.2018.12.006. [DOI] [PubMed] [Google Scholar]
  17. Wang G. W., Deng L. Q., Luo Y. P., Liao Z. H., Chen M. Nat. Prod. Res. 2017;31:1855. doi: 10.1080/14786419.2016.1261348. [DOI] [PubMed] [Google Scholar]
  18. Salvador J. A. R., Leal A. S., Valdeira A. S., Gonçalves B. M. F., Alho D. P. S., Figueiredo S. A. C., Silvestre S. M., Mendes V. I. S. Eur. J. Med. Chem. 2017;142:95. doi: 10.1016/j.ejmech.2017.07.013. [DOI] [PubMed] [Google Scholar]
  19. Xiao S., Tian Z., Wang Y., Si L., Zhang L., Zhou D. Med. Res. Rev. 2018;38:951. doi: 10.1002/med.21484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ren Y., Anaya-Eugenio G. D., Czarnecki A. A., Ninh T. N., Yuan C., Chai H. B., Soejarto D. D., Burdette J. E., de Blanco E. J. C., Kinghorn A. D. Bioorg. Med. Chem. 2018;26:4452. doi: 10.1016/j.bmc.2018.07.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Markov A. V., Zenkova M. A., Logashenko E. B. Curr. Med. Chem. 2017;24:1277. doi: 10.2174/0929867324666170112115313. [DOI] [PubMed] [Google Scholar]
  22. Ortega J. A., Riccardi L., Minniti E., Borgogno M., Arencibia J. M., Greco M. L., Minarini A., Sissi C., De Vivo M. J. Med. Chem. 2018;61:1375. doi: 10.1021/acs.jmedchem.7b01388. [DOI] [PubMed] [Google Scholar]
  23. Abbot V., Sharma P., Dhiman S., Noolvi M. N., Patel H. M., Bhardwaj V. RSC Adv. 2017;7:28313. [Google Scholar]
  24. Thomas K., Moody T. W., Jensen R. T., Tong J., Rayner C. L., Barnett N. L., Fairfull-Smith K. E., Ridnour L. A., Wink D. A., Bottle S. E. Eur. J. Med. Chem. 2018;147:34. doi: 10.1016/j.ejmech.2018.01.077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Guo L., Chen W., Cao R. H., Fan W. X., Ma Q., Zhang J., Dai B. Eur. J. Med. Chem. 2018;147:253. doi: 10.1016/j.ejmech.2018.02.003. [DOI] [PubMed] [Google Scholar]
  26. Jackson T. W., Kojima M., Lambrecht R. M. Aust. J. Chem. 2000;53:983. [Google Scholar]
  27. Huang Z., Mou Y., Xu X., Zhao D., Lai Y., Xu Y., Chen C., Li P., Peng S., Tian J., Zhang Y. J. Med. Chem. 2017;60:8847. doi: 10.1021/acs.jmedchem.7b00971. [DOI] [PubMed] [Google Scholar]
  28. Cheng K. G., Su C. H., Huang J. Y., Liu J., Zheng Y. T., Chen Z. F. Chem. Biol. Drug Des. 2016;88:329. doi: 10.1111/cbdd.12758. [DOI] [PubMed] [Google Scholar]
  29. Mo W. B., Su C. H., Huang J. Y., Liu J., Chen Z. F., Cheng K. G. Chem. Cent. J. 2016;10:69. doi: 10.1186/s13065-016-0217-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Cheng K. G., Su C. H., Yang L. D., Liu J., Chen Z. F. Eur. J. Med. Chem. 2015;89:480. doi: 10.1016/j.ejmech.2014.10.066. [DOI] [PubMed] [Google Scholar]
  31. Huang J. Y., Yang L. D., Su C. H., Chu X. W., Zhang J. Y., Deng S. P., Cheng K. G. Chem. Nat. Compd. 2018;54:106. [Google Scholar]
  32. Ciapetti G., Cenni E., Pratelli L., Pizzoferrato A. Biomaterials. 1993;14:359. doi: 10.1016/0142-9612(93)90055-7. [DOI] [PubMed] [Google Scholar]
  33. Liu Y., Peterson D. A., Kimura H., Schubert D. J. Neurochem. 1997;69:581. doi: 10.1046/j.1471-4159.1997.69020581.x. [DOI] [PubMed] [Google Scholar]
  34. Britzolaki A., Saurine J., Flaherty E., Thelen C., Pitychoutis P. M. Cell. Mol. Neurobiol. 2018;38:981. doi: 10.1007/s10571-018-0583-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Chen Y., Xie X., Hu T., Dong X., Zhang X., Huang F. RSC Adv. 2018;8:12449. doi: 10.1039/c8ra00260f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shree G. S., Prasad K. Y., Arpitha H. S., Deepika U. R., Kumar K. N., Mondal P., Ganesan P. Mol. Cell. Biochem. 2017;436:1. doi: 10.1007/s11010-017-3071-4. [DOI] [PubMed] [Google Scholar]
  37. Lam M., Lawrence D. A., Ashkenazi A., Walter P. Cell Death Differ. 2018;25:1530. doi: 10.1038/s41418-018-0155-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kuo L. M., Chen P. J., Sung P. J., Chang Y. C., Ho C. T., Wu Y. H., Hwang T. L. Mar. Drugs. 2018;16:19. doi: 10.3390/md16010019. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Click here for additional data file. (3MB, pdf)

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