Mitochondria-targeted lupane triterpenoid derivatives and their

Jul 3, 2017 - ... selective apoptosis-inducing anticancer mechanisms. Yaqing Ye, Tao Zhang, Huiqing Yuan, Defeng Li, Hong-Xiang Lou, and Peihong Fan...
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Mitochondria-Targeted Lupane Triterpenoid Derivatives and Their Selective Apoptosis-Inducing Anticancer Mechanisms Yaqing Ye,† Tao Zhang,‡,∥ Huiqing Yuan,§ Defeng Li,∥ Hongxiang Lou,† and Peihong Fan*,† †

Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China ‡ Department of Medicinal Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P. R. China § Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, P. R. China ∥ Shandong Qidu Pharmaceutical Co. Ltd., Shandong Provincial Key Laboratory of Neuroprotective Drugs, Zibo 255400, P. R. China S Supporting Information *

ABSTRACT: Betulin and betulinic acid have been widely studied for their anticancer activities. However, their further development is limited due to low bioavailability, poor aqueous solubility, and limited intracellular accumulation. In the present study, a triphenylphosphonium cation moiety was linked to betulin and betulinic acid to specifically target them to cancer cell mitochondria. Biological characterization established that uptake of mitochondria-targeted compound 1a in the mitochondria of cancer cells was increased compared to betulin. The mitochondria-targeted derivatives of betulin and betulinic acid showed stronger cytotoxicity than their parent drugs and exhibited more cytotoxic effects in cancer cells than normal cells. The mechanisms may involve the mitochondrial apoptotic pathway, probably caused by the induction of reactive oxygen species production and reducing mitochondrial membrane potential. More importantly, 1a significantly inhibited cancer cell proliferation and migration in an in vivo zebrafish xenograft model. Collectively, these results encourage further study of 1a analogs as anticancer agents.

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and selectivity, e.g., chlorambucil,10 gallic acid,8 and ascorbate,11 among others. Recent studies have revealed a wide range of pharmacological activities, including the antitumor activity of pentacyclic triterpene compounds, such as boswellic acid, oleanolic acid, betulinic acid, and betulin.12,13 Betulinic acid and betulin are naturally occurring triterpenes found in birch bark, and studies have shown that betulinic acid and betulin exert cytotoxic effects on a variety of malignant tumor cells.14,15 However, the high hydrophobicity of these natural products hampers their further development as cytotoxic drugs. A number of studies were attempted by structural modifications to improve bioavailability, cytotoxicity, and selectivity.16−18 Mechanistic studies suggested that the antitumor activity of betulinic acid

INTRODUCTION One of the major challenges in developing cancer chemotherapeutics is to improve selectivity and to reduce severe toxic side effects for normal cells and tissues.1 Since tumor cells easily gain resistance to apoptosis and mitochondria are tightly associated with both intrinsic and extrinsic apoptotic pathways,2,3 a promising strategy is to target therapeutic agents to the mitochondria of cancer cells to improve selectivity and the toxicity profile.4,5 Furthermore, recent evidence of the mitochondrial membrane potential (Δψm) difference between normal and cancer cells has provided further confidence for the strategy of mitochondria targeting in drug design.6,7 This strategy was exemplified by employment of delocalized lipophilic cations (DLCs) to target parent compounds to the mitochondrial matrix.8,9 The most studied DLC is the triphenylphosphonium cation (TPP+), and many successful examples have been reported with improved antitumor effect © 2017 American Chemical Society

Received: May 7, 2017 Published: July 3, 2017 6353

DOI: 10.1021/acs.jmedchem.7b00679 J. Med. Chem. 2017, 60, 6353−6363

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Figure 1. Chemical structures of the mito-derivatives of betulin (1) and betulinic acid (2).

and betulin is associated with the mitochondrial apoptotic pathway by Δψm depolarization and the release of cytochrome c and Smac protein from the mitochondrial membrane. This consequently results in activation of caspase-9, caspase-3, and caspase-7 as well as cleavage of poly (ADP-ribose) polymerase (PARP), ultimately leading to apoptosis.14,19,20 Thus, we could hypothesize that specifically targeting betulinic acid and betulin to cancer cell mitochondria would improve their cytotoxicity and selectivity. In the present study, we attempted to transport betulinic acid and betulin to the mitochondria by conjugating them to TPP+ to improve their antitumor effect. Spivak et al. have prepared some TPP+ derivatives of betulinic acid and evaluated their antitumor properties.18,21 Their studies demonstrated that those analogs markedly enhanced the cytotoxic action as compared to betulinic acid, thus serving as a proof of concept for such a strategy, and indicated the potential of new cationic derivatives of betulinic acid. The structural modifications of their compounds are mainly focused on position 2 or 30 of the betulinic acid skeleton structure via an alkyl chain. Such modifications may potentially influence the overall activity of analogs since the structural skeleton was modified. Unlike these biochemically stable linkages using carbon−carbon bonds, conjugates with ester bonds can be easily hydrolyzed in the cell under the action of endogenous esterases and have other biokinetic characteristics. Spivak et al. also reported the potential of linking a TPP cation to a 28COOH group of betulinic acid by an ester bond.18 However, continuing to design analogs with an ester linkage at the OH groups of positions of C-28 or C-3 of betulin or betulinic acid can explore the optimal positions to incorporate the TPP+ moiety. Such analogs may also provide benefits to gain insight into the roles of only mitochondria targeting by the TPP+

moiety, since the scaffolds of betulin and betulinic acid remain untouched. Herein we report the design, synthesis, and biological characterization of a series of novel mitochondriatargeted derivatives of betulin and betulinic acid with an ester linkage at the OH groups of the structures to evaluate their mitochondrial uptake and selectivity by cancer cells and to test the anticancer activities of these analogs. We also report the preliminary mechanistic studies of these analogs for observed cytotoxic effects.

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RESULTS Molecular Design and Chemical Synthesis. As shown in Figure 1, a TPP+ moiety was introduced to betulin (1) and betulinic acid (2) by an ester linkage. For betulin analogs, the OH group at the C-28 position was utilized to incorporate the ester linkage, since the reactivity of the hydroxyl group at C-28 is much higher than that of the C-3 one. For betulinic acid analogs, the TPP+ moiety was linked at the C-3 OH group. The chemical synthesis of these analogs is fairly straightforward by esterification of betulin or betulinic acid with ω-bromoalkanoic acid following standard conditions. 22 As these agents’ anticancer activities can be influenced by the introduction of substituents,17 we further designed and synthesized analogs by modification of the remaining hydroxyls. The purity of target compounds was confirmed by high-performance liquid chromatography (HPLC) to be ≥95%. Cytotoxic Effects of the Mito-Derivatives of Betulin and Betulinic Acid. After chemical synthesis, the designed analogs were evaluated for their cytotoxic effects against five human cancer cell lines (K562, HL-60, ECA-109, A549, and HepG2). A normal human hepatocyte cell line (HL-7702) was included to compare the selectivity of these analogs. As shown 6354

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Table 1. Cytotoxic Effects of the Mito-Derivatives on K562, HL-60, ECA-109, A549, and HepG2 Cancer Cell Linesa IC50 (μM) compd

K562

HL-60

ECA-109

A549

HepG2

HL-7702

1 1a 1b 1c 9e 2 2a 2b 2c

12.89 ± 1.44 0.57 ± 0.03b 0.81 ± 0.07b 0.62 ± 0.06b >200 21.96 ± 1.15 1.24 ± 0.12c 3.58 ± 0.41c,d 4.03 ± 0.91c,d

30.94 ± 0.52 0.60 ± 0.03b 0.71 ± 0.06b 0.58 ± 0.05b 118.13 ± 3.0 18.47 ± 1.07 0.32 ± 0.09c 3.11 ± 0.49c,d 2.75 ± 0.21c,d

26.64 ± 2.63 0.78 ± 0.08b 0.95 ± 0.12b 1.41 ± 0.07b 111.73 ± 6.30 28.07 ± 3.28 0.92 ± 0.11c 4.75 ± 0.13c,d 6.04 ± 0.26c,d

18.15 ± 0.46 0.61 ± 0.07b 1.10 ± 0.03b 0.83 ± 0.20b 172.21 ± 28.80 17.13 ± 0.25 1.26 ± 0.12c 4.99 ± 0.57c,d 4.79 ± 0.05c,d

10.70 ± 0.06 1.09 ± 0.05b 1.11 ± 0.05b 2.42 ± 0.46b 158.47 ± 25.10 25.30 ± 3.81 1.07 ± 0.02c 4.35 ± 0.39c,d 4.54 ± 0.38c,d

26.82 ± 2.25 6.62 ± 0.52b 8.32 ± 0.36b 7.18 ± 2.42b >200 35.07 ± 3.12 4.92 ± 0.92c 6.30 ± 1.11c 7.85 ± 1.65c

a The results are expressed as the mean ± standard deviation (SD). bP < 0.0001 compared to 1. cP < 0.0001 compared to 2. dP < 0.001 compared to 2a. e4-Carboxybutyltriphenylphosphonium bromide (9) was used as a control.

Figure 2. Cellular uptake of 1a and 1 to the mitochondria of K562 cells. K562 cells were treated with 10 μM 1a or 1 for 4 h, then mitochondria were isolated and 1a and 1 were extracted with organic solvent for analysis. (A) RP-HPLC chromatograms of 1a standards and of extracts from the mitochondria of K562 cells. (B) RP-HPLC chromatograms of 1 and of extracts from the mitochondria of K562 cells. (C) Quantitative data of 1a and 1 in mitochondria after normalization to the protein content. The values are presented as the mean ± SD (n = 3): (∗) P < 0.01.

increased 3.4-fold when compared to that of betulin after treatment of K562 cells for 4 h. The results demonstrated that the TPP + moiety effectively delivered betulin to the mitochondria. Induction of Apoptotic Effects by Mito-Derivatives. To examine whether the observed cytotoxicity of 1a and 2a is due to induction of apoptosis, an annexin V-FITC/PI dual staining assay was performed after treatment of K562 cells. As shown in Figure 3, after 48 h of treatment, significant apoptotic effects were observed for both 1a and 2a in K562 cells. The analysis of the apoptotic effects in Figure 3B clearly indicated a dose-dependent manner, especially for late apoptosis. At the maximal tested concentration, 1a (1.2 μM) caused early and late apoptosis in 12.86% and 38.18% of K562 cells, respectively, and 2a (3.0 μM) caused early and late apoptosis in 12.62% and 29.77% of K562 cells, respectively. The results suggested that the observed cytotoxicity of 1a and 2a in cancer cells is mainly through the apoptotic pathway. Effects on the Mitochondrial Membrane Potential (Δψm) by Mito-Derivatives. Changes in Δψm have been shown to play a significant role in both extrinsic and intrinsic apoptosis.23 Since the new analogs were designed to target the mitochondria, we therefore decided to examine the influence of 1a and 2a on the Δψm of K562 cells with the JC-1 staining method. As shown in Figure 4A, treatment of K562 cells with 1a caused significant loss of Δψm as reflected by the increase of J-monomers (showing green fluorescence and indicating depolarized mitochondria) and concurrent decrease of Jaggregates (showing red fluorescence and indicating hyperpolarized mitochondria). The loss of Δψm was also confirmed by flow cytometry analysis (Figure 4B) as the intensity of the

in Table 1, the mito-derivatives exhibited improved cytotoxic potency (low or submicromolar potency, and approximately 5to 58-fold increase) against all five human cancer cell lines compared to their parent compounds betulin and betulinic acid, respectively. To rule out the possibility that the observed cytotoxicity could be solely due to the TPP+ moiety, 4carboxybutyltriphenylphosphonium bromide (9) was included as control. As can be seen from Table 1, no cytotoxicity was observed for 9 in any of the tested cancer cells, thus confirming the essential role of the whole structure of the mito-derivatives. Although the mito-derivatives showed increased cytotoxic effects toward the normal HL-7702 cells compared to their parent compounds, comparisons of the IC50 values between tumor line HepG2 cells and HL-7702 cells indicated that these analogs are more cytotoxic to tumor cells than normal cells, exhibiting some selectivity to cancer cells. No significant change on the potency of cytotoxicity was observed for analogs with modification of the C-3 OH group of 1a, as evidenced by 1b and 1c. Nevertheless, a slight increase in the IC50 values (up to ∼6-fold) was observed when blocking the hydroxyl of the carboxyl group of 2a. Among the tested cancer cell lines, the leukemia cells K562 and HL-60 were relatively more sensitive to the lethal effects of the new analogs. Moreover, 1a and 2a exhibited the most potent cytotoxic effects compared to other analogs. Therefore, 1a and 2a were selected for the following pharmacological studies in K562 cells. Cellular Uptake of 1a in the Mitochondria of K562 Cells. To confirm that the improved cytotoxicity of the mitoderivatives was a result of mitochondria targeting, the uptake of 1a and 1 to the mitochondria was evaluated in K562 cells. As shown in Figure 2, the mitochondrial uptake of 1a was 6355

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Figure 3. Apoptotic effects of 1a and 2a on K562 cells. (A) K562 cells were treated with the indicated compounds at indicated concentrations for 48 h. Apoptotic effects were measured by flow cytometry using annexin V-FITC/PI staining protocol (upper figure for 1a and lower figure for 2a). (B) Representative histograms for the numbers of cells (% of total) in the early and late stages of apoptosis for the control and treatment groups. The values are presented as the mean ± SD (n = 3): (∗) P < 0.01 compared to control.

red fluorescence decreased in a dose-dependent manner after treatment with both compounds. Induction of the Production of Intracellular Reactive Oxygen Species (ROS). Mitochondria are the main organelle that produce ROS, and ROS has been indicated to have a double sword role in the cytotoxicity in cancer cells.24 Therefore, we next evaluated how treatment with mitoderivatives in K562 cells impacts the production of ROS by flow cytometry analysis. The reagent ROSup from the ROS assay kit was used as a positive control. Both 1a and 2a induced ROS production at the tested concentrations (0.5 and/or 1.0 μM) (Figure 5). 1a at 0.5 μM or 2a at 1.0 μM increased the ROS level by approximately 50% compared to the control group. Mechanistic Studies of the Apoptotic Effects Induced by Mito-Derivatives. To confirm that the observed apoptotic effects in K562 cells by the mito-derivatives were exerted via the mitochondrial pathway, we measured the expression levels of cytochrome c, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, and cleaved PARP using Western blotting. As shown in Figure 6, the content of cytochrome c in the mitochondria decreased whereas its amount in the cytoplasm increased, indicating that treatment with the mito-derivatives resulted in loss of Δψm and an increase in the release of cytochrome c from the mitochondria into the cytosol.

Consequently, cytochrome c initiated the activation of caspase-9, which further caused an increase in the quantity of cleaved fragments of the downstream effector caspase-3. As the end point of this signaling pathway, cleaved PARP was significantly up-regulated and executed the apoptosis program. Treatment with 1a at 4.0 μM for 48 h dramatically increased the activities of caspase-9 and caspase-3, with 1.5- and 2.1-fold increases, respectively, compared with the control. In Vivo Activity of Mito-Derivatives on the Growth and Metastasis of K562 Cells in Zebrafish. To confirm in vivo activity, 1a was assayed for its effects on the proliferation and metastasis of K562 cells in zebrafish, a well-studied and adopted in vivo model that is widely used for drug screening purposes.25,26 Specifically, CM-Dil-labeled K562 cells (red) were microinjected into zebrafish embryos, and different concentrations of 1a or 1 were added. Taxol was used as a positive control. As shown in Figure 7A, 48 h after xenotransplantation, the K562 cells in the control group widely migrated away from the primary site. Notably, the K562-cell xenografts treated with Taxol (10.0 μg/mL) or 1a (1.0 μg/mL, 2.0 μg/mL) (Figure 7A, parts b, d, and e) exhibited significantly reduced cell migration. Moreover, the K562-cell xenografts treated with Taxol or 1a showed reduced fluorescence intensities than the control group (Figure 7C), indicating that 1a could inhibit the growth of K562 cells in zebrafish 6356

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Figure 4. Effects on Δψm by 1a and 2a in K562 cells. K562 cells were treated with 1a or 2a at the indicated concentrations for 48 h and then analyzed by fluorescence microscopy and flow cytometry after JC-1 staining. (A) Representative fluorescence microcopy images of K562 cells treated with 1a. The change of Δψm was reflected by the disappearance of red-stained mitochondria (negative Δψm) and an increase in green-stained mitochondria (loss of Δψm). (B) Flow cytometry analysis of K562 cells treated with 1a (upper figure) or 2a (lower figure), along with the quantification of % of cells with red aggregates. The values are presented as mean ± SD (n = 3): (∗) P < 0.01 compared to control.

designed and developed to improve antitumor activity and pharmacokinetic properties.17,27 Although promising new analogs were identified with improved therapeutic potential, more effort is required to improve activity and selectivity and to identify their precise mechanisms of action. Cancer cells commonly exhibit elevated Δψm of at least 60 mV compared to normal cells.28 The difference of Δψm between cancer cells and normal cells could be exploited therapeutically to selectively target compounds to the mitochondria of cancer cells. DLCs can easily penetrate the lipid bilayer due to the even distribution of charge over a large hydrophobic surface area, thereby lowering the activation

xenografts. It is worth noting that 1a effectively inhibited the proliferation and metastasis of K562 cells in zebrafish xenografts at a low concentration (1.0 μg/mL), which was 1 /10 the concentration of the positive control Taxol, and that the in vivo activity of 1a was obviously increased compared with that of betulin.

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DISCUSSION AND CONCLUSIONS The triterpenoids betulin and betulinic acid have attracted extensive attention due to their broad-spectrum biological activities and as potential anticancer treatments. Analogs based on the structures of betulin and betulinic acid have been 6357

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Figure 5. Effects of 1a and 2a on ROS production. K562 cells were treated with 1a (A) or 2a (B) for 48 h, then DCFH-DA (10 μM) was loaded and cells were analyzed by flow cytometry quantification of mito-derivative-induced oxidative stress in K562 cells. ROSup was tested as a positive control. The values are presented as the mean ± SD (n = 3): (∗) P < 0.01 compared to control.

energy required for cation movement across the membrane.4 According to the Nernst equation, an approximate 10-fold increase of mitochondrial uptake should be expected for a cation for every 60 mV increase in Δψm.29 Generally, Δψm is approximately 180−200 mV, the maximum value that a bilayer can sustain while maintaining its integrity.29−31 The wellcharacterized and most widely used DLC for mitochondria targeting is the TPP+ cation.29 Proof-of-concept studies for utilizing TPP+ as a mitochondria-targeting moiety have been achieved by in vivo studies of novel mito-derivatives in animal models of human cancer.8,10,32 In the present study, based on the hypothesis that the cytotoxicity of a given compound to cancer cells is closely associated with its accumulation in the mitochondria, we designed and synthesized six TPP-linked derivatives of betulin and betulinic acid and compared their biological activity with betulin and betulinic acid. Our results revealed that the antiproliferative effects of the mito-derivatives are significantly improved compared to the parent compounds against all tested tumor cells (Table 1). In contrast, a simple TPP+ derivative alone exerted no cytotoxicity to either cancer cells or normal cells, confirming the essential role of the whole structure of the designed compounds. Moreover, the lower toxicities of these mito-derivatives to the normal hepatocyte cell line HL-7702 compared with hepatoma carcinoma cells were observed. A similar effect was observed in a recent study of delocalized lipophilic cations of gallic acid derivatives.8 The tumor cellselective cytotoxicity of these derivatives could be further explored by testing more cell lines in the future. The results also demonstrated that the OH groups of the positions of C-28 of betulin and C-3 of betulinic acid were optional positions to incorporate the TPP+ moiety. This design left the scaffold of

betulin and betulinic acid untouched but improved cytotoxicity significantly, supporting the important role of mitochondria targeting by the TPP+ moiety. Importantly, another advantage of such a design is the simple synthetic procedure with an ester linkage. Our studies also suggested that masking the 3-OH group of betulin with an ester moiety did not significantly change the cytotoxic potency or selectivity to cancer cells. This may serve as indirect evidence to support the essential role of the TPP+ moiety in mitochondria uptake. A similar effect was observed in a recent study of resveratrol analogs showing that acetylated or free hydroxyl analogs exhibited comparable cytotoxicity.33 Interestingly, it was reported that alkylation of the 28-COOH group of betulinic acid led to the loss of its antitumor activity.18 Here, when the carboxyl group of the C-28 position of betulinic acid was masked with an ester, the potency was decreased compared to the free carboxylic group 2a, but they are much more potent than betulinic acid. However, the decrease of potency when masking the C-28 position might suggest that the C-3 OH could be a better position for incorporation of the TPP+ moiety than C-28 COOH, but more studies are warranted to further support this. It has been reported that ROS induction and change of Δψm trigger cytochrome c release and subsequently apoptosis through caspase-3 activation.34 Our mechanistic study results are consistent with this by showing that treatment of K562 cells with mito-derivatives induced significant production of ROS and reduced Δψm. The activation of caspase-9 and caspase-3 and the cleavage of PARP were observed under the same experimental conditions. In addition, the level of cytochrome c in the mitochondria was decreased, an event accompanied by an increase of cytosolic level. The results are also consistent 6358

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Figure 6. Effects of 1a and 2a on cytochrome c, caspase-9, caspase-3, and PARP. K562 cells were treated with indicated compounds at indicated concentrations for 48 h. Cells were lysed, and the cell lysates were analyzed by Western blotting using the corresponding antibodies. (A) Representative image of Western blotting analysis of cytochrome c in the cytoplasm or mitochondria. (B) Representative image of Western blotting analysis of caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, and cleaved PARP. (C, D) Quantitative analysis of Western blotting from (A) and (B) by Image Lab program with β-actin as the internal control. The values are presented as the mean ± SD (n = 3). Statistical analysis of protein expression, significantly different from the control: (∗) P < 0.01. China). TLC for monitoring was performed with precoated silica gel GF-254 glass plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). Betulin and betulinic acid were purchased from the Baoji Herbest Bio-Tech Co. Ltd., Shaanxi, China. All other organic compounds, inorganic salts, acids, and solvents were purchased from the Shanghai Xianding Biological Science & Technology Co. Ltd., China. All final compounds were analyzed for purity by highperformance liquid chromatography (HPLC) and were found to have ≥95% purity. HPLC analysis was performed on an Agilent 1100 series with a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). The solvent for compounds was 0.1% formic acid in acetonitrile, and eluent was 90% acetonitrile/water. The absorbance was detected at 220 nm. 3β-Hydroxylup-20(29)-en-28-yl 5-(Triphenylphosphonio)pentanoate Bromide (1a). Triphenylphosphine (325 mg, 1.24 mmol) was added to a solution of 3β-hydroxylup-20(29)-en-28-yl 5bromopentanoate (intermediate 3, 200 mg, 0.33 mmol; for structure, see Supporting Information) in CH3CN (10 mL). The mixture was then stirred at 80 °C until the reaction was complete according to TLC detection. The solvent was subsequently removed under

with previous studies on the anticancer mechanisms of betulin and betulinic acid. Notably, treatment of zebrafish with 1a exhibited significant inhibition on the xenograft proliferation and migration of K562 cells, thus confirming the in vivo activity of 1a. In summary, our studies provide further evidence that natural pentacyclic triterpene compounds can be optimized by the mitochondria-targeting strategy to improve potency and selectivity.

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EXPERIMENTAL SECTION

Chemistry. We recorded the 1H and 13C NMR spectra with a Bruker AV spectrometer operating at 400 (1H) and 100 (13C) MHz or a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz, with tetramethylsilane (TMS) as an internal standard. The chemical shifts are reported as δ (ppm) downfield from TMS for 1 H NMR. Column chromatography (CC) was performed with silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, 6359

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Figure 7. Inhibitory effect of 1a on the proliferation and metastasis of K562 cells in zebrafish xenografts. CM-Dil-labeled K562 cells (red) were microinjected into zebrafish embryos, and different concentrations of 1a or 1 were added. Taxol was used as a positive control. After 48 h, the proliferation and metastasis of the xenografts of K562 cells were imaged under a confocal microscope. (A) Metastasis of K562-cell xenografts. (B) Quantification of the fluorescent area of the tumor xenografts, representing K562 cell metastasis. (C) Fluorescence intensity of the tumor xenografts, representing the number of K562 cells. The results are presented as the mean ± SD (n = 15): (∗) P < 0.01 compared to control. diminished pressure, and the residue was purified by CC using (30:1 → 20:1) DCM/MeOH to obtain 30 mg (15%) of 1a as a white solid. Mp: 124−129 °C. [α]D20 +3.6 (c 0.1, CH3OH). 1H NMR (CDCl3, 600 MHz): δ 7.68−7.89 (m, 15H, Ph-H), 4.67 (s, 1H, H-29a), 4.58 (s, 1H, H-29b), 4.18 (d, J = 11.0 Hz, 1H, H-28a), 3.96−3.95 (m, 2H, CH2-P-), 3.77 (d, J = 11.0 Hz, 1H, H-28b), 3.18 (dd, J = 11.5, 4.7 Hz, 1H, H-3), 2.42 (t, J = 6.8 Hz, 2H, -COCH2), 2.41−2.36 (m, 1H, H19), 2.04−0.90 (m, 28H, CH, CH2 in pentacyclic skeleton or carbon chain), 1.66, 1.00, 0.97, 0.95, 0.82, 0.76 (each, s, 3H, CH3-23, 24, 25, 26, 27, 30). 13C NMR (CDCl3, 150 MHz): δ 173.6 (O-CO-), 150.1 (C-20), 134.9 (d, JC,P = 3.0 Hz, Ph-C), 133.8 (d, JC,P = 10.0 Hz, Ph-C), 130.5 (d, JC,P = 12.5 Hz, Ph-C), 118.4 (d, JC,P = 85.9 Hz, Ph-C), 109.9 (C-29), 78.9 (C-3), 77.3, 77.0, 76.8, 62.5, 55.3, 53.5, 50.3, 48.7, 47.7, 46.3, 42.7, 40.8, 38.8, 38.7, 37.6, 37.1, 34.5, 34.2, 33.5, 29.7, 29.5, 28.0, 27.4, 27.0, 25.5, 25.2, 22.8, 22.5, 22.0, 20.8, 19.1, 18.3, 16.0, 15.4, 14.7. HPLC purity: 99%. HR-ESI-MS m/z calculated for C53H72O3P+ [M − Br]+ 787.5214, found 787.5231. 3β-Formyloxylup-20(29)-en-28-yl 5-(Triphenylphosphonio)pentanoate Bromide (1b). Triphenylphosphine (310 mg, 1.18 mmol) was added to a solution of 3β-formyloxylup-20(29)-en-28-yl 5bromopentanoate (intermediate 4, 150 mg, 0.235 mmol; for structure,

see Supporting Information) in CH3CN (3 mL). The mixture was then stirred at 80 °C until the reaction was complete according to TLC detection. The solvent was subsequently removed under diminished pressure, and the residue was purified by preparative TLC by elution with DCM/MeOH (10:1) to obtain 55 mg (36.7%) of 1b as a yellow solid. Mp: 118−123 °C. [α]D20 +13.5 (c 0.1, CH3OH). 1 H NMR (CD3OD, 600 MHz): δ 8.13 (s, 1H, -CHO), 7.89−7.75 (m, 15H, Ph-H), 4.73 (s, 1H, H-29a), 4.62 (s, 1H, H-29b), 4.58 (dd, J = 12.0, 6.0 Hz, 1H, H-3), 4.37 (d, J = 11.1 Hz, 1H, H-28a), 3.80 (d, J = 11.1 Hz, 1H, H-28b), 3.45 (t, J = 14.7 Hz, 2H, CH2-P-), 2.49−2.48 (m, 1H, H-19), 2.46 (t, J = 6.9 Hz, 2H, -COCH2-), 1.68, 1.05, 1.01, 0.90 (each, s, 3H, CH3-25, 26, 27, 30), 0.88 (s, 6H, CH3-23, 24), 1.91−1.09 (m, 28H, CH, CH2 in pentacyclic skeleton or carbon chain). 13C NMR (CD3OD, 150 MHz): δ 173.6 (O-CO-), 161.7 (-CHO), 149.9 (C-20), 134.9 (d, JC,P = 2.9 Hz, Ph-C), 133.4 (d, JC,P = 10.0 Hz, Ph-C), 130.2 (d, JC,P = 12.6 Hz, Ph-C), 118.5 (d, JC,P = 86.4 Hz, Ph-C), 109.2 (C-29), 80.9 (C-3), 62.2 (C-28), 55.3, 50.2, 48.6, 46.4, 42.4, 40.7, 38.1, 37.6, 37.4, 36.8, 34.2, 33.9, 32.7, 29.4, 29.2, 26.9, 26.8, 25.5, 25.4, 25.1, 23.5, 21.7, 21.6, 21.2, 20.9, 20.5, 17.9, 17.9, 15.6, 6360

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15.3, 15.2, 13.8. HPLC purity: 96%. HR-ESI-MS m/z calculated for C54H72O4P+ [M − Br]+ 815.5163, found 815.5170. 3β-Acetyloxylup-20(29)-en-28-yl 5-(Triphenylphosphonio)pentanoate Bromide (1c). Triphenylphosphine (430 mg, 1.66 mmol) was added to a solution of 3β-acetyloxylup-20(29)-en-28-yl 5bromopentanoate (intermediate 5, 210 mg, 0.33 mmol; for structure, see Supporting Information) in CH3CN (3 mL). The mixture was then stirred at 80 °C until the reaction was complete according to TLC detection. The solvent was subsequently removed under diminished pressure, and the residue was purified by preparative TLC by elution with DCM/MeOH (10:1) to obtain 70 mg (23%) of 1c as a white solid. Mp: 147−156 °C. [α]D20 +8.6 (c 0.1, CH3OH). 1H NMR (CD3OD, 600 MHz): δ 7.93−7.77 (m, 15H, Ph-H), 4.73 (s, 1H, H-29a), 4.62 (s, 1H, H-29b), 4.46 (dd, J = 11.4, 4.9 Hz, 1H, H-3), 4.40 (d, J = 10.7 Hz, 1H, H-28a), 3.83 (d, J = 11.1 Hz, 1H, H-28b), 3.47 (ddd, J = 13.6, 9.4, 7.0 Hz, 2H, CH2-P-), 2.49−2.47 (m, 1H, H-19), 2.46 (t, J = 7.1 Hz, 2H, -COCH2-), 2.04 (s, 3H, CH3CO-), 1.71, 1.07, 1.03, 0.92, 0.89, 0.88 (each, s, 3H, CH3-23, 24, 25, 26, 27, 30), 1.91− 1.25 (m, 28H, CH, CH2 in pentacyclic skeleton or carbon chain). 13C NMR (CD3OD, 150 MHz): δ 173.5 (O-CO-), 171.5 (-COCH3), 149.9 (C-20), 135.0 (d, JC,P = 3.0 Hz, Ph-C), 133.4 (d, JC,P = 10.0 Hz, Ph-C), 130.2 (d, JC,P = 12.6 Hz, Ph-C), 118.5 (d, JC,P = 86.4 Hz, PhC), 109.2 (C-29), 81.0 (C-3), 62.2 (C-28), 55.4, 50.2, 48.6, 46.4, 42.4, 40.7, 38.1, 37.6, 37.5, 36.8, 34.1, 33.9, 32.7, 29.4, 29.2, 27.1, 26.7, 25.5, 25.4, 25.1, 23.3, 21.7, 21.2, 20.9, 20.5, 19.8, 17.9, 15.6, 15.3, 15.2, 13.8. HPLC purity: 96%. HR-ESI-MS m/z calculated for C55H74O4P+ [M − Br]+ 829.5319, found 829.5333. 3β-(5-(Triphenylphosphonio)pentanoyl)oxylup-20(29)-en28-oic Acid Bromide (2a). Triphenylphosphine (292 mg, 1.11 mmol) was added to a solution of 3β-(5-bromopentanoyl)oxylup20(29)-en-28-oic acid (intermediate 6, 230 mg, 0.37 mmol; for structure, see Supporting Information) in CH3CN (10 mL). The mixture was then stirred at 80 °C until the reaction was complete according to TLC detection. The solvent was subsequently removed under diminished pressure, and the residue was purified by preparative TLC by elution with DCM/MeOH (10:1) to obtain 83 mg (28%) of 2a as a white solid. Mp: 135−143 °C. [α]D20 +15.2 (c 0.1, CH3OH). 1 H NMR (CDCl3, 400 MHz): δ 7.88−7.67 (m, 15H, Ph-H), 4.73 (s, 1H, H-29a), 4.61 (s, 1H, H-29b), 4.37 (dd, J = 10.6, 5.8 Hz, 1H, H-3), 3.91 (t, J = 14.1 Hz, 2H, CH2-P-), 3.01 (td, J = 10.5, 4.5 Hz, 1H, H19), 2.38 (t, J = 6.8 Hz, 2H, -OCOCH2-), 1.69, 0.96, 0.88, 0.81 0.75, 0.72 (each, s, 3H, CH3-23, 24, 25, 26, 27, 30), 2.29−1.31 (m, 29H, CH, CH2 in pentacyclic skeleton or carbon chain). 13C NMR (CDCl3, 100 MHz): δ 181.0 (C-28), 172.9 (CH2CO-), 150.5 (C-20), 135.0 (d, JC,P = 2.9 Hz, Ph-C), 133.7 (d, JC,P = 10.0 Hz, Ph-C), 130.5 (d, JC,P = 12.5 Hz, Ph-C), 118.8 (d, JC,P = 85.9 Hz, Ph-C), 109.7 (C-29), 80.9 (C-3), 77.4, 77.0, 76.7, 65.3, 56.3, 55.4, 50.4, 49.3, 46.9, 42.4, 41.9, 40.7, 38.4, 37.8, 37.1, 34.2, 33.9, 32.2, 30.6, 30.1, 29.7, 29.1, 28.0, 25.4, 23.7, 23.3, 23.1, 22.9, 21.9, 20.9, 19.4, 18.1, 16.6, 16.1, 14.6, 14.1. HPLC purity: 97%. HR-ESI-MS m/z calculated for C53H70O4P+ [M − Br]+ 801.5006, found 801.5020. Methyl-3β-(5-(triphenylphosphonio)pentanoyl)oxylup20(29)-en-28-oate Bromide (2b). Triphenylphosphine (376 mg, 1.24 mmol) was added to a solution of methyl-3β-(5bromopentanoyl)oxylup-20(29)-en-28-oate (intermediate 7, 180 mg, 0.28 mmol; for structure, see Supporting Information) in CH3CN (10 mL). The mixture was then stirred at 80 °C until the reaction was complete according to TLC detection. The solvent was subsequently removed under diminished pressure, and the residue was purified by preparative TLC by elution with DCM/MeOH (10:1) to obtain 66 mg (37%) of 2b as a yellow solid. Mp: 168−177 °C. [α]D20 +11.3 (c 0.1, CH3OH). 1H NMR (CD3OD, 600 MHz): δ 7.90−7.75 (m, 15H, Ph-H), 4.71 (s, 1H, H-29a), 4.60 (s, 1H, H-29b), 4.40 (dd, J = 11.6, 4.9 Hz, 1H, H-3), 3.65 (s, 3H, CH3O-), 3.49−3.44 (m, 2H, CH2P-), 3.00 (td, J = 10.9, 5.1 Hz, 1H, H-19), 2.40 (t, J = 7.1 Hz, 2H, -CH2CO), 1.69, 1.01, 0.94, 0.86, 0.80, 0.77 (each, s, 3H, CH3-23, 24, 25, 26, 27, 30), 2.27−1.08 (m, 28H, CH, CH2 in pentacyclic skeleton or carbon chain). 13C NMR (CD3OD, 150 MHz): δ 176.2 (C-28), 173.1 (CH2CO-), 150.4 (C-20), 134.9 (d, JC,P = 3.0 Hz, Ph-C), 133.5 (d, JC,P= 10.0 Hz, Ph-C), 130.1 (d, JC,P = 11.3 Hz, Ph-C), 118.5 (d, JC,P =

86.4 Hz, Ph-C), 108.9 (C-29), 81.1 (C-3), 59.6, 56.3, 55.4, 50.4, 49.2, 42.2, 40.6, 38.2, 38.1, 37.5, 36.9, 36.5, 34.0, 33.1, 31.7, 30.3, 29.4, 27.2, 25.6, 25.5, 25.4, 23.3, 21.7, 21.7, 21.3, 20.9, 20.7, 18.2, 17.8, 15.7, 15.4, 15.2, 13.7, 13.3. HPLC purity: 98%. HR-ESI-MS m/z calculated for C54H72O4P+ [M − Br]+ 815.5163, found 815.5176. Ethyl-3β-(5-(triphenylphosphonio)pentanoyl)oxylup-20(29)en-28-oate Bromide (2c). Triphenylphosphine (244 mg, 0.89 mmol) was added to a solution of ethyl-3β-(5-bromopentanoyl)oxylup-20(29)-en-28-oate (intermediate 8, 115 mg, 0.18 mmol; for structure, see Supporting Information) in CH3CN (10 mL). The mixture was then stirred at 80 °C until the reaction was complete according to TLC detection. The solvent was subsequently removed under diminished pressure, and the residue was purified by preparative TLC by elution with DCM/MeOH (10:1) to obtain 60 mg (52%) of 2c as a white solid. Mp: 150−154 °C. [α]D20 +16.2 (c 0.1, CH3OH). 1 H NMR (CD3OD, 600 MHz): δ 7.91−7.75 (m, 15H, Ph-H), 4.72 (s, 1H, H-29a), 4.60 (s, 1H, H-29b), 4.40 (dd, J = 11.6, 4.9 Hz, 1H, H-3), 4.18−4.10 (m, 2H, CH3CH2O-), 3.48−3.42 (m, 2H, CH2P-), 3.03− 2.99 (td, J = 10.8, 4.8 Hz, 1H, H-19) 2.40 (t, J = 7.1 Hz, 2H, CH2CO), 1.26 (t, J = 7.1 Hz, 3H, CH3CH2O-) 1.70, 1.02, 0.95, 0.87, 0.80, 0.77 (each, s, 3H, CH3-23, 24, 25, 26, 27, 30), 2.29−1.29 (m, 28H, CH, CH2 in pentacyclic skeleton or carbon chain). 13C NMR (CD3OD, 150 MHz): δ 176.2 (C-28), 173.1 (CH2CO-), 150.4 (C-20), 135.0 (d, JC,P = 3.0 Hz, Ph-C), 133.4 (d, JC,P = 10.0 Hz, Ph-C), 130.2 (d, JC,P = 12.6 Hz, Ph-C), 118.5 (d, JC,P = 86.4 Hz, Ph-C), 108.9 (C-29), 81.1(C3), 59.6, 56.3, 55.4, 50.4, 49.2, 42.2, 40.6, 38.3, 38.1, 37.5, 36.9, 36.6, 34.0, 33.1, 31.7, 30.3, 29.4, 27.2, 25.6, 25.5, 25.4, 23.3, 21.7, 21.7, 21.3, 20.9, 20.7, 18.2, 17.9, 15.7, 15.4, 15.2, 13.8, 13.3. HPLC purity: 97%. HR-ESI-MS m/z calculated for C55H74O4P+ [M − Br]+ 829.5316, found 829.5332. Biological Study. Cells and Materials. Human chronic myeloid leukemia cells (K562) and human promyelocytic leukemia cells (HL60) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Human alveolar adenocarcinoma cells (A549), human esophageal squamous carcinoma cells (ECA-109), human hepatoblastoma cells (HepG2), and human normal liver cells (HL-7702) were obtained from the School of Pharmaceutical Science of Shandong University. K562 cells, A549 cells, ECA-109 cells, HepG2 cells, and HL-7702 cells were cultured in RPMI-1604 medium (Macgene, Beijing, China), and HL-60 cells were grown in IMDM (Macgene, Beijing, China). The media were supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Solarbio, Beijing, China), and the cells were incubated at 37 °C with 5% CO2. The annexin V-FITC apoptosis detection kit was obtained from Keygen Biotech. JC-1 and the ROS assay kit was from Beyotime (Nanjing, China). Finally, the primary antibodies against caspase-3, cleaved caspase-3, PARP, cytochrome c, caspase-9, and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). MTT Assay. Cells were seeded in 96-well plates at a density of 5000 cells per well. For the solid tumor lines (A549, ECA-109, HepG2, and HL-7702), after 16−24 h, the cells were treated with a solution of one of the novel synthesized compounds or with control for 48 h. After the medium was refreshed, MTT solution (5 mg/mL, 10 μL; Solarbio, Beijing, China) was added to each well, and the cells were incubated with the MTT for 4 h at 37 °C. After the medium was removed, 100 μL of DMSO (Sigma, St. Louis, MO, USA) was added to each well, and the OD570 was measured using a microplate reader (Bio-Rad, CA, USA). For hematological cells (K562 and HL-60), at the end of the drug treatment, 10 μL of MTT solution was added to each well, and the samples were incubated at 37 °C for 4 h. After the plates were centrifuged at 3000 rpm for 30 min, the medium was removed, 100 μL of DMSO (Sigma, St. Louis, MO, USA) was added to each well, and the OD570 was measured as described above. The test results are expressed as the concentration of the test compound that inhibited cell growth by 50% (IC50). All assays were performed in triplicate. Flow Cytometry Assay. Cell Culture. K562 cells at 4 × 105 cells/ well in a final volume of 2 mL were seeded in six-well plates. After 12 h 6361

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of incubation, various concentrations of 1a or 2a were added to the wells for various periods of time. Cell Apoptosis Analysis. Cells were harvested at 2000 rpm for 5 min and then washed with ice-cold PBS twice, followed by resuspension in binding buffer. Next, the samples were incubated with 5 μL of annexin V and 5 μL of PI for 15 min at room temperature in the dark. Finally, the cells were analyzed by flow cytometry (Becton Dickinson, NJ, USA) within 1 h. Mitochondrial Membrane Potential. Cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in JC-1 (5 mg/mL) and incubation at 37 °C for 15 min. After the cells were rinsed three times and suspended in PBS, the JC-1 fluorescence was observed by flow cytometry. Detection of Intracellular ROS. Intracellular ROS generation was examined using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). For this purpose, cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in 0.5 mL of FBS-free medium containing 10 mM DCFH-DA and incubation at 37 °C for 30 min. After the cells were rinsed three times and suspended in PBS, ROS production in the cells was immediately monitored by flow cytometry. Western Blotting. Protein Extraction. Cells were washed with icecold PBS and centrifuged for 5 min at 2000 rpm. The pellets were then resuspended in 100 μL of lysis buffer (Solarbio, Beijing, China) and incubated on ice for 30 min. The cells were subsequently centrifuged for 10 min at 14 000 rpm, after which the supernatants were collected as whole-cell lysates. The cytosolic and mitochondrial fractions were isolated from the treated cells using the cell mitochondrial isolation kit (Beyotime, Nanjing, China) following the manufacturer’s recommended protocol. The protein levels were quantified with a BCA assay kit (Beyotime). Western Blotting. Protein samples (60 μg/lane) were subjected to SDS−PAGE (Bio-Rad) and electrotransferred to PVDF membranes (Millipore, MA, USA). The membranes were then blocked with 5% skim milk in TBST at room temperature for 4 h, followed by three washes with TBST. Target proteins, including caspase-3, cleaved caspase-3, cytochrome c, PARP, caspase-9, and cleaved caspase-9, were subsequently incubated with the corresponding primary antibodies, and detection was performed using secondary antibodies conjugated to alkaline phosphatase. Measurement of Mitochondrial Uptake of Compounds 1a and 1. HPLC was used to detect and quantify 1a and 1. For this purpose, K562 cells were grown in 100 mm dishes and then treated with 10 μM 1a or 1 for 4 h. After the treatment, the cells were washed twice with ice-cold PBS. Mitochondrial isolation was then performed using the cell mitochondrial isolation kit (Beyotime) according to the manufacturer’s recommended protocol. The mitochondria pellet was diluted in PBS and extracted twice with a dichloromethane/methanol (2:1) mixture. After the organic layers were combined and dried, the dry residue was dissolved in acetonitrile alone or acetonitrile containing 0.1% of formic acid and was then used for HPLC analysis. The extract of betulin-treated cells was dissolved in acetonitrile, and 20 μL of the solution was injected into the HPLC system (Agilent Technologies, CA, USA). Analyses were performed on a ZORBAX SBC18 column (250 mm × 4.6 mm, 5 μm) using 100% acetonitrile as the mobile phase, and peaks were detected at 220 nm. Meanwhile, the extract of 1a-treated cells was dissolved in acetonitrile containing 0.1% formic acid, and the mobile phase was 80% acetonitrile. The wavelength for the detection of 1a was 267 nm. Growth Inhibition and Metastasis in Vivo. Wild-type zebrafish were obtained from the Biology Institute of the Shandong Academy of Sciences. Embryos were produced by pairwise mating in a fish hatch box and maintained in embryonic medium (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4) at 28 °C. Normally developed embryos were selected with a stereoscopic microscope. K562 cells labeled with CM-Dil and suspended in FBS-free medium at a density of (1 × 107)/mL were then microinjected into the zebrafish. After 4 h of incubation, zebrafish embryos with the same injection spot were selected and incubated in a six-well plate with 15−20 embryos per well. Different concentrations of 1 or 1a were added, and Taxol

(Sigma) was used as a positive control. After 48 hpt, confocal microscopy (Olympus, Japan) was used to examine K562 cell death and migration in the zebrafish in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00679. Schemes illustrating synthesis of compounds; experimental description and analytical data for intermediates compounds 3−8 and a control compound 4carboxybutyltriphenylphosphonium bromide (9); NMR and HR-ESI-MS spectra for the final compounds and their HPLC chromatograms for purity analysis; the standard curves for 1 and 1a quantitative analysis (PDF) SMILES molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0086 531 88382012. E-mail: [email protected]. ORCID

Hongxiang Lou: 0000-0003-3300-1811 Peihong Fan: 0000-0001-5529-8922 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported partly by the National Natural Science Foundation of China (Grant 81473323). We thank the Shandong Academy of Sciences for conducting the zebrafish xenograft experiment. We also thank Professor of Medicinal Chemistry Shijun Zhang in School of Pharmacy, Virginia Commonwealth University, and Dr. Claudia Simões-Pires at University of Geneva for revising the manuscript and Professor of Pharmacology Xiuli Guo at Shandong University for useful discussions.

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ABBREVIATIONS USED TPP, triphenylphosphine; Δψm, mitochondrial membrane potential; DLC, delocalized lipophilic cation; caspase, cysteinyl aspartate specific proteinase; PARP, poly (ADP-ribose) polymerase; RP-HPLC, reverse-phase high performance liquid chromatography; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; ROS, reactive oxygen species; NMR, nuclear magnetic resonance; TMS, tetramethylsilane; CC, column chromatography; TLC, thin-layer chromatography; DCM, dichloromethane; MeOH, methanol; Mp, melting point; [α], observed optical rotation in degrees; J, coupling constant; ppm, parts per million; HR-ESI-MS, high resolution electrospray ionization mass spectrometry; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; FBS, fetal bovine serum; rpm, revolutions per minute; hpt, hours post-treatment

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REFERENCES

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