Novel Benzo[a]quinolizidine Analogs Induce Cancer Cell Death

Article pubs.acs.org/jmc

Novel Benzo[a]quinolizidine Analogs Induce Cancer Cell Death through Paraptosis and Apoptosis Hongbo Zheng,†,# Yiwen Dong,‡,# Lin Li,† Bin Sun,†,§ Lei Liu,† Huiqing Yuan,*,‡ and Hongxiang Lou*,† †

Department of Natural Products Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, ‡Department of Biochemistry and Molecular Biology, School of Medicine, and §National Glycoengineering Research Center, Shandong University, No.44 Western Wenhua Road, Jinan 17923, China S Supporting Information *

ABSTRACT: Paraptosis is nonapoptotic cell death characterized by massive endoplasmic reticulum (ER)- or mitochondriaderived vacuoles. Induction of paraptosis offers significant advantages for the treatment of chemotherapy-resistant tumors compared with anticancer drugs that rely on apoptosis. Because some natural alkaloids induce paraptotic cell death, a novel series of benzo[a]quinolizidine derivatives were synthesized, and their antiproliferative activity and ability to induce cytoplasmic vacuolation were analyzed. Structural optimization led to the identification of the potent compound 22b, which inhibited cancer cell proliferation in vitro and in vivo and profoundly facilitated paraptosis-like cell death and induced caspase-dependent apoptosis. Further investigation revealed that 22b-mediated vacuolation originated from persistent ER stress and upregulation of LC3B. Paraptosis induced by benzo[a]quinolizidine derivatives thus represents an alternative strategy for cancer chemotherapy.

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both α and β isoforms). Extensive efforts have focused on the identification of ER stress/UPR activators that specifically trigger these alternate cell death mechanisms as novel cancer treatment options. For example, the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN55, 212-2) selectively inhibits cell proliferation via paraptosis-like cell death after activating pro-death UPR signaling.6 The immunological inhibitor celastrol also induces paraptosis-like cytoplasmic vacuolation in cancer cells, possibly by affecting a variety of pathways, including the proteasome, ER stress, and Hsp90.8,9 Many UPR activators, such as radicicol and tanespimycin, are also used in the treatment of various cancers.10 Activation of nonapoptotic cell death in parallel with apoptosis is thought to facilitate the tumor response to the drug in anticancer treatments. Many natural alkaloids are employed in clinical chemotherapy. Taxol, a diterpene alkaloid, causes ER stress and paraptosis in a large range of human cancer cells in addition to inducing apoptotic cell death as a major mechanism.11 Bromocriptine (Figure 1) is another typical chemotherapeutic drug for the treatment of pituitary tumors primarily via

INTRODUCTION Most chemotherapeutic agents exert antitumor activity via caspase-dependent apoptosis.1 However, chemotherapy resistance in recurrent tumors is directly linked to the limited success of cytotoxic drugs against cancers. Chemotherapy resistance has also been attributed to the acquisition of antiapoptotic mechanisms because cancer cells often develop defects in apoptosis that provide a survival advantage and contribute to chemotherapy resistance.2−4 Therefore, therapies based on the induction of nonapoptotic cell death, such as paraptosis, autophagic cell death, and necroptosis, may provide an effective strategy for the treatment of malignant cancers that are multidrug resistant and frequently resistant to apoptosis.5,6 Paraptosis, a form of nonapoptotic cell death characterized by extensive cytoplasmic vacuolation resulting from swelling of the ER and/or mitochondria, is caspase-independent and lacks the morphological hallmarks of apoptosis.1,2,7 Although the mechanisms involved in triggering dilatation of the ER and/or mitochondria are poorly defined, de novo protein synthesis and ER stress are likely essential because inhibition of protein synthesis attenuates vacuolation.1,7 The unfolded protein response (UPR), which can be initiated by ER stress, also contributes to cell death through three ER stress sensors: protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), or activating transcription factor 6 (ATF6; © 2016 American Chemical Society

Received: April 1, 2016 Published: April 14, 2016 5063

DOI: 10.1021/acs.jmedchem.6b00484 J. Med. Chem. 2016, 59, 5063−5076

Journal of Medicinal Chemistry

Article

benzo[a]quinolizidine derivatives were synthesized, and their antiproliferative activity was evaluated in cell-based assays. Chemistry. We developed a new method for constructing the benzo[a]quinolizidine ring according to Gawley’s strategy.22 The general procedure for benzo[a]quinolizidine synthesis is shown in Scheme 1. Tetrahydroisoquinolines II Scheme 1. General Procedure for Benzo[a]quinolizidine Synthesisa

Figure 1. Examples of bioactive compounds with specific quinolizidine or benzo[a]quinolizidine skeletons.11−15

paraptosis.12,13 Benzo[a]quinolizidine derivatives, such as berberine, emetine, and deoxytubulosine (Figure 1), are also important naturally occurring antiproliferative agents, and their “privileged” structure always provides unique activity.14−16 Thus, the development of a rapid chemical synthesis for benzo[a]quinolizidines and further chemical modifications of these potential products may facilitate the identification of more potent and selective derivatives. Our ongoing research on antitumor compounds prompted us to synthesize a series of benzo[a]quinolizidine derivatives for the discovery of novel agents with unique activity.17−19 On the basis of a structure−activity relationship (SAR) investigation, an optimized compound, 22b, was identified that exhibited antitumor activity at low concentration accompanied by massive cytoplasmic vacuoles in cell-based assays. We then examined the dependence of the cytoplasmic vacuolation induced by 22b on ER stress, autophagy, and protein translation. The results demonstrated that 22b triggers ER vacuolation with specific characteristics of paraptosis-like cell death in addition to inducing apoptosis. The antitumor efficiency of 22b in PC3 tumor-bearing mice indicated that 22b potently suppresses tumor growth without significant toxicity.

Reagents and conditions: (a) paraformaldehyde, formic acid, 50 °C, 12 h. (b) ditert-butyl dicarbonate, Et3N, DMF or THF, r.t., 2−10 h. (c) HBr, reflux, 4 h. (d) benzyl bromide, K2CO3, DMF, r.t., 3 h. (e) TEMPO, EtOH, CH2Cl2, r.t., 24 h. (f) Yb(OTf)3, CH2Cl2, r.t., 2−3 h. (g) TFA, CH2Cl2, 0 °C, 4 h. (h) NH3·H2O/MeOH, 0 °C, 2 h. a

were first prepared, followed by the N-Boc protection III and introduction of different O-modifications IV. N-Boc ethoxy derivatives V, the key intermediates, were synthesized by adding the oxidant TEMPO and alcohol under conditions similar to those described previously.23 N-Boc ethoxy V was then coupled with various trimethylsilyl enol ethers VI prepared from methyl vinyl ketones. We performed reactions in the presence of different Lewis acids, such as TMSOTf, BF3· EtO2, and M(OTf)x, and Yb(OTf)3 was selected as the most useful additive to facilitate the generation of 1-substituted tetrahydronisoquinolines VII under mild conditions. The protective group was then removed by TFA, followed by intramolecular Michael addition in NH3·H2O/methanol to afford the desired benzo[a]quinolizidines VIII as trans isomers with high diastereoselectivity. The total yield of V from VIII (40−60%) was acceptable for further modification. Reaction details can be obtained in Schemes S1−S2 and Table S1. Ketone VIII was further modified via the Grignard reaction to afford IX. Reduction of VIII using the Wolff−Kishner− Huang reaction or other reductants (NaBH4 or L-selectride) led to the generation of products X and XI, respectively (Scheme 2). All benzo[a]quinolizidine derivatives were prepared as racemates for biological evaluation. The relative configuration was determined by analysis of NOESY spectral data (Figure S1). In Vitro Antiproliferative Activity Screening and SAR Analysis. The effect of these benzo[a]quinolizidine derivatives on cell proliferation was investigated in a panel of human cancer cell lines by MTT assay. Non-neoplastic prostate epithelial RWPE1 cells were included to test the cytotoxicity of these compounds. The bioactive structures are shown in Chart 1, and their IC50 values are listed in Table 1. The remaining derivatives and related antiproliferative activity are provided in Tables S2−S5. The preliminary screening of 9,10-substituted benzo[a]quinolizidines revealed that only compounds 1−3 exhibited an inhibitory effect on most of the tested tumor cell lines. The basic ring-fused skeleton, particularly the type b

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RESULTS AND DISCUSSION Design. The novel benzo[a]quinolizidines were designed on the basis of an analysis of naturally occurring alkaloids that target subcellular organelles and induce paraptosis.20,21 Biologyoriented modifications of the structures were performed to target three potential active positions (Figure 2): (a) the aromatic substituent effect on tetrahydroisoquinoline ring system; (b) functionalization and optimization of position 4; and (c) modification and exploration of the important hydroxyl group by introducing various chemical moieties while retaining the benzo[a]quinolizidine skeleton. Thus, a novel series of

Figure 2. Design and analysis of the potential bioactive positions of benzo[a]quinolizidine. 5064



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Scheme 2. Synthesis of Benzo[a]quinolizidine Derivativesa

Reagents and conditions: (a) Grignard reagent, THF, 0 °C, 1 h. (b) N2H4·H2O, KOH, 80−180 °C, 5 h. (c) NaBH4, MeOH, 0 °C, 1 h. (d) Lselectride, THF, −78 °C, 2 h.

a

Chart 1. Bioactive Benzo[a]quinolizidine Derivativesa

a

methyl group at C-2 increased cytotoxicity, whereas the activity of analogues with a longer alkyl chain or phenyl ring decreased or was absent. Third, changes in substituents at the phenyl ring of the benzyloxy group at C-9 and C-10 affected activity. The IC50 values of 20−27 in Table 1 revealed that derivatives with the π-rich aryl ring were significantly active, and these substituents enhanced activity in the order 3,5-CF3 > 3,5-Me > 4-tBu > 4-Br > 3,5-F > 3-F > H. Similarly, derivatives 28−34 featuring a methyl group (type IX analogues) displayed a similar pattern, with the exception of the 3-fluorobenzyl derivative 31, which exhibited superior antitumor activity. Cell-based activity assays indicated that most type VIII derivatives were inactive, whereas several 4-methyl analogues were moderately cytotoxic. Notably, compound 35 (3-F benzyl) displayed strong inhibition of MDA-MB-231 cells (IC50 = 1.3 μM, Table S4). Octahydroazocino[2,1-a]isoquinolines with specific 8-membered rings (37 and 38), which were obtained unexpectedly, also exhibited moderate cytotoxicity (IC50 = 8.3−13.0 μM). The SAR analysis (Figure 3) indicated that 9,10-bisbenzoxyl groups, 4-methyl, and 2-hydroxyl, were very important for activity. Prostate cancer (PCa) and breast cancer cells appeared to be more sensitive to the novel benzo[a]quinolizidine derivatives. PC3, an androgen-independent tumor cell line, exhibits elevated expression of antiapoptotic proteins, enabling apoptosis resistance;26 therefore, we investigated the potent compounds 20b, 22a,b, 24a,b, 27b, 28, 30, and 31 in PC3 cells. These compounds profoundly inhibited cell proliferation in association with the induction of massive cytoplasmic vacuoles (Table 1 and Figure 4), and the electron-rich benzoxyl groups were essential for vacuolation-associated cell death. 3,5Dimethylbenzyl derivatives (e.g., 20b and 22b; Figure 4) induced vacuole production more potently than 3,5-bis(trifluoromethyl)benzyl (e.g., 30) and other electron-rich benzoxyl derivatives (or electron-deficient benzoxyl derivatives such as 26b). However, the positive control, docetaxel, did not induce vacuolation. Notably, among these compounds, 22b exhibited smaller inhibitory effects against RWPE1 cells with a higher IC50 (Table 1). Compounds 22b-1 and 22b-2, optical isomers (Figure S2), displayed similar potency in vacuoleassociated cell death, and 22b was selected for subsequent analysis in PC3 cells. 22b Induces Cytoplasmic Vacuolation and Apoptosis. Given the inhibitory effect of compound 22b on PCa cells, we analyzed changes in cell cycle distribution and apoptosis in response to 22b. Flow cytometry analysis of PC3 cells treated with 22b (0.5, 1, and 2 μM) for 24 h revealed that compared with the control (62.5% of cells in G0/G1 phase), 22b induced cell cycle arrest at G0/G1 phase (Figure 5A). The percentage

Only the type b configuration was obtained for these compounds.

configuration shown in Chart 1 (cis relationship between C-2 and C-4), and the two benzyloxy groups at position 9 and 10 were essential for high cytotoxicity.24,25 By contrast, all derivatives with alkoxyl moieties instead of benzyloxy substituents at positions 9 and 10 were completely inactive. Next, we focused on modification of lead compound 1 in three positions. The results of the MTT assay indicated that the following features contributed to optimal activity. First, among the benzo[a]quinolizidines with various substituted phenyl rings introduced at C-4, the order of antiproliferation potency was −NO2 (no cytotoxicity) < 3,4,5-OMe < 4-OMe < H < 3,4OMe < 4-Ph, indicating that electron-donating groups (phenyl or methoxyl moiety) were critical for cytotoxicity. In another series of alkyl chain compounds 11−14, the paired diastereoisomers exhibited superior bioactivity. The length of the alkyl chain modulated the potency, and the methyl group was the optimal pharmacophore vector for the antiproliferative effect. Second, reductive product 15 was devoid of activity, implying that the 2-hydroxyl is essential. Additionally, introduction of a 5065



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Table 1. In Vitro Antiproliferative Activity of Benzo[a]quinolizidine Derivatives against Various Human Cancer Cell Lines IC50 (μM)a compound

b

MDA-MB-231

Hela

1

11.8 ± 0.63

11.2 ± 0.29

5 8b 10b 11a 11b 12a 12b 13a 13b

5.01 1.15 3.28 7.22 1.15 10.2 2.88 6.72 2.57

16b 18 19

5.48 ± 0.52 6.47 ± 0.59 2.37 ± 0.19

20b 21b 22a 22b 23a 23b 24a 24b 25a 25b 26a 26b 27a 27b 28 29 30 31 32 33 34

0.97 1.30 1.33 0.82 3.16 2.11 0.56 0.51 1.46 3.25 2.25 0.88 1.23 0.58 2.35 2.81 0.53 0.59 1.20 0.98 2.52

38b docetaxele

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.13 0.22 0.36 0.40 0.02 0.01 0.54 0.14 0.31

0.11 0.20 0.35 0.20 0.57 0.24 0.14 0.16 0.15 0.63 0.24 0.13 0.21 0.03 0.34 0.42 0.12 0.13 0.25 0.14 0.24

2.72 ± 0.17 0.032 ± 0.003

7.22 3.46 3.42 1.06 2.66 5.27 2.43 5.49 2.38

± ± ± ± ± ± ± ± ±

6.57 ± 5.21 ± 6.34 ± 3.05 4.67 3.24 1.65 3.04 1.96 2.84 2.01 2.56 2.06 2.88 1.16 1.31 1.73 1.25 1.29 1.21 0.96 1.88 0.91 2.99

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.43 ± 0.061 ±

K562

PC3

DU145

6.96 ± 0.25 8.37 ± 0.06 12.0 ± 0.73 Various 4-Substituted Derivatives 0.52 5.44 ± 0.21 NTc NT 0.15 5.42 ± 0.53 1.51 ± 0.12 1.59 ± 0.10 0.025 3.24 ± 0.22 1.16 ± 0.07 1.36 ± 0.27 0.18 3.26 ± 0.37 7.67 ± 0.45 1.95 ± 0.13 0.08 1.52 ± 0.34 2.81 ± 0.54 2.18 ± 0.35 0.28 3.81 ± 0.47 3.52 ± 0.24 3.37 ± 0.21 0.43 1.41 ± 0.07 2.15 ± 0.36 1.68 ± 0.06 0.28 3.88 ± 0.36 NT NT 0.26 2.32 ± 0.02 NT NT Modification of C-2 Position, Addition, and Reduction 0.81 1.98 ± 0.16 NT 11.6 ± 0.46 0.36 NT 2.60 ± 0.28 2.11 ± 0.11 0.58 6.81 ± 0.79 NT NT Various 9,10-Benzyloxy Groups 0.09 3.95 ± 0.17 1.15 ± 0.04 4.53 ± 0.57 0.38 3.92 ± 0.41 1.71 ± 0.26 3.96 ± 0.22 0.58 2.35 ± 0.23 0.72 ± 0.06 1.51 ± 0.14 0.27 1.44 ± 0.18 0.66 ± 0.07 1.96 ± 0.23 0.53 2.29 ± 0.27 NT NT 0.46 1.89 ± 0.33 1.35 ± 0.27 1.88 ± 0.25 0.09 1.15 ± 0.07 0.68 ± 0.11 1.87 ± 0.36 0.05 1.35 ± 0.06 0.45 ± 0.07 1.74 ± 0.04 0.07 2.88 ± 0.21 NT NT 0.28 2.04 ± 0.32 3.79 ± 0.63 2.22 ± 0.17 0.53 3.15 ± 0.61 NT NT 0.22 1.22 ± 0.10 1.24 ± 0.16 2.58 ± 0.12 0.16 1.08 ± 0.14 1.36 ± 0.09 3.05 ± 0.42 0.25 1.42 ± 0.17 0.82 ± 0.10 3.85 ± 0.27 0.10 1.46 ± 0.15 0.89 ± 0.04 1.95 ± 0.33 0.19 1.13 ± 0.14 1.29 ± 0.17 1.33 ± 0.23 0.11 1.84 ± 0.19 0.61 ± 0.04 2.24 ± 0.20 0.09 1.01 ± 0.18 0.41 ± 0.06 2.14 ± 0.22 0.04 1.79 ± 0.06 0.79 ± 0.05 1.97 ± 0.02 0.10 1.35 ± 0.16 1.11 ± 0.19 1.46 ± 0.28 0.41 d 2.27 ± 0.37 4.25 ± 0.11 Octahydroazocino[2,1-a]isoquinoline Derivatives 0.63 5.61 ± 0.26 NT NT 0.015 0.039 ± 0.007 0.013 ± 0.001 0.026 ± 0.005

MCF-7

RWPE-1

20.1 ± 0.39

>40 (86.8)

NT 2.00 0.28 2.50 2.20 3.02 2.79 NT NT

NT 2.08 4.42 NT 1.84 NT 2.20 NT NT

± ± ± ± ± ±

0.18 0.01 0.25 0.09 0.28 0.44

9.2 ± 0.33 1.39 ± 0.16 NT 1.32 3.52 0.65 0.51 NT 1.71 1.87 0.52 NT 0.86 NT 1.29 0.62 0.49 0.50 1.98 0.81 0.47 0.84 2.61 2.23

± ± ± ±

0.12 0.37 0.08 0.06

± 0.18 ± 0.28 ± 0.06 ± 0.05 ± ± ± ± ± ± ± ± ± ±

0.19 0.08 0.02 0. 03 0.06 0.07 0.03 0.15 0.44 0.39

NT 0.022 ± 0.003

± 0.02 ± 0.28 ± 0.03 ± 0.15

2.02 ± 0.14 NT NT 3.04 2.24 1.35 5.06 NT 4.42 2.32 1.15 NT 1.91 2.89 NT 4.45 1.49 4.77 4.45 2.57 1.86 2.95 4.03 NT

± ± ± ±

0.18 0.04 0.04 0.29

± 0.19 ± 0.31 ± 0.10 7 ± 0.16 ± 0.30 ± ± ± ± ± ± ± ±

0.21 0.14 0.22 0.38 0.47 0.18 0.61 0.16

NT 1.73 ± 0.08

a

Results are expressed as the mean IC50 of three experiments. The standard deviations were 20 μM) or are shown in the Supporting Information. cNot tested. dNot active (IC50 > 20 μM). eDocetaxel was used as the positive control.

with the pan-caspase inhibitor Z-VAD-fmk to block caspasedependent apoptosis. As shown in Figure 5D, Z-VAD-fmk partially rescued 22b-induced cell death, indicating that caspase-independent mechanisms also partially contributed to 22b-induced cell death. The cells underwent cytoplasmic vacuolation in the presence of 22b; thus, we determined if the characteristics of the vacuoles were correlated with cell death. Cellular vacuoles were readily observed by phasecontrast microscopy as shown in Figure 5C. Small vacuoles appeared in 22b-treated cells after approximately 4 h and increased in size and number in a time-dependent manner. Robust vacuolation persisted up to 24 h of treatment: At this time point, nonviable cells were evident, and most of the cells had detached from the dish. Time-course studies were also performed to examine the change in cleavage of poly(ADPribose) polymerase (PARP), an apoptosis marker, by activated

Figure 3. SAR summary of requirements for cytotoxicity.

of cells in G0/G1 phase was 68.1, 72.4, and 81.4% at 0.5, 1, and 2 μM, respectively. The results in Figure 5B indicate that 22b induced apoptosis in PC3 cells in a dose-dependent manner, with apoptosis levels of 3.7, 8.5, and 56.9% at concentrations of 0.5, 1.0, and 2.0 μM, respectively. To determine the role of apoptosis in 22b-mediated cell death, PC3 cells were pretreated 5066



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Figure 4. Morphological changes of PC3 cells treated with the corresponding compounds (1 μM) for 12 h. All IC50 values were determined using PC3 cells.

Meanwhile, 22b-mediated activation of XBP1 mRNA splicing was also attenuated when combined with 4-PBA. More importantly, 4-PBA alone did not cause any detectable change in cellular morphology and cytotoxicity, whereas 4-PBA markedly alleviated vacuole production and cell death induced by 22b, highlighting the importance of ER stress in the regulation of vacuole formation, which is closely correlated with 22b-mediated cell death. Given the importance of ER stress in 22b-induced vacuole formation and cell death, we determined if proteasome inhibitors that trigger ER stress facilitate 22binduced vacuolation and cell death. CompuSyn software was used to analyze the effect of 22b combined with 39 (MG132) or bortezomib, proteasome inhibitors that trigger ER stress.30−32 The results in Figure 6D and Table S6 demonstrate that the combination of 22b at IC30 and 39 at various concentrations yielded combination index (CI) values of 0.1 to 0.3, indicating a strong synergistic effect. For example, 22b at IC30 resulted in 54% inhibition of PC3 cell growth when combined with 39 at its IC10 concentration. Bortezomib also displayed synergistic effects (Table S7). Protein overload in the ER lumen may induce the stress response; therefore, we hypothesized that abrogating protein synthesis with cycloheximide (CHX) would promote cell survival in the presence of 22b. As shown in Figure 6E, halting protein synthesis via the addition of CHX significantly reduced 22b-induced cytoplasmic vacuolation and cell death. We also demonstrated that the transcriptional inhibitor actinomycin-D (Act-D) dramatically reduced cellular vacuolation as well as cell death, revealing an important role of de novo gene expression in cytoplasmic vacuolation and the cell death response. Thus, the characteristics of cell death induced by 22b included cytoplasmic vacuolation, induction of ER stress, the requirement for active gene expression, and the absence of inhibition by caspase inhibitors, fulfilling the criteria for paraptosis. 22b-Induced Cytoplasmic Vacuolation Requires Elevated LC3. Vacuolation is also a feature of autophagy, and CHX was unable to completely abolish the formation of vacuoles induced by 22b (Figure 6E). We therefore examined whether 22b activates autophagy in PC3 cells. As shown in Figure 7A, the expression of LC3, an autophagosome marker, was slightly upregulated in treated cells compared with the vehicle control, whereas conversion of LC3B-I to lipidated LC3B-II (a PE-conjugated form) was observed in cells after 4 h of treatment and markedly robust at up to 24 h of treatment with 22b, suggesting activation of autophagic flux. However, the

caspase 3. As shown in Figure 5D, only modest cleavage of PARP was observed after 24 h of treatment, implying a critical role of cytoplasmic vacuolation in 22b-induced cell death because caspase-dependent apoptosis occurred after vacuole production. Thus, alternative mechanisms are involved in 22binduced cell death because apoptosis occurred as a late event during treatment. More importantly, Z-VAD-fmk partially restored 22b-induced cell death but did not significantly affect cytoplasmic vacuolation. Cells were partially rescued from 22binduced cell death by another pan-caspase inhibitor, Ac-DEVDCHO. (Figure 5D). Thus, the formation of vacuoles plays an important role in 22b-induced caspase-independent cell death. 22b-Mediated Paraptosis Depends on ER Stress and Protein Synthesis. Vacuoles were observed at the perinuclear region and in the cytosol; therefore, we utilized ER-Tracker Red staining to determine if the vacuoles were derived from the ER. As shown in Figure 6A, untreated cells displayed a smear pattern upon ER staining, whereas fluorescence intensity notably increased in cells treated with 22b for 4 h. Furthermore, vacuoles were readily observed after 8 h of exposure and were surrounded by a positive ER-specific marker, suggesting that the vacuoles were of ER origin. The generation of ER vacuoles is usually associated with persistent ER stress; therefore, we reasoned that 22b-induced vacuolation may be preceded by the induction of ER stress. The results in Figure 6B demonstrate that 22b upregulated glucose-regulated protein GRP78, a hallmark of the ER stress response. Expression of GRP78 significantly increased after 4 h of exposure to 22b, and vacuoles started to appear, reaching a peak at approximately 8− 12 h and being sustained at high levels for up to 24 h. Activation of p-PERK and PERK-mediated phosphorylation of eukaryotic initiation factor (p-eIF2α) were also observed (Figure 6B), indicating activation of the PERK branch of UPR signaling. 27,28 Persistent UPR ultimately initiates apoptosis if these adaptive responses are insufficient to protect cells from ER stress.29 We also observed a change in the expression of CHOP, a protein involved in the ER-mediated apoptosis. Upregulation of CHOP was observed at 8 h of treatment and was sustained up to 24 h, confirming the ability of 22b to trigger ER stress, which may also contribute to caspase-dependent apoptosis. To investigate the requirement for ER stress in the formation of vacuoles, we used 4phenylbutyric acid (4-PBA), an ER stress inhibitor, to suppress 22b-mediated ER stress. As shown in Figure 6C, enhancement of GRP78 by 22b was abolished in the presence of 4-PBA. 5067



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Figure 5. Compound 22b induces cell cycle arrest, apoptosis, and cytoplasmic vacuolization in PC3 cells. (A) Flow cytometry analysis of cell cycle populations after treatment with 0.5−2 μM 22b for 24 h and the frequency distribution graph. (B) Flow cytometry analysis of apoptosis. Cells were exposed to 0.5−2 μM 22b for 24 h. Cells were collected and stained with Annexin V-fluorescein isothiocyanate (FITC) and PI. Necrotic, late apoptotic, normal, and early apoptotic cells were labeled as B1−B4. (C) Phase-contrast images of PC3 cells treated with 22b at indicated time points after 1 μM 22b treatment for 0−24 h. All images were acquired at the same magnification. (D) The effect of Z-VAD-fmk (10 μM) or Ac-DEVDCHO (50 μM) on 22b-treated cell viability was analyzed by MTT assay, **, p < 0.01, and ***, p < 0.001, vs Control. The relative enzyme activity of Caspase 3, ***, p < 0.01, vs Control. The control cells received an equivalent volume of DMSO vehicle. Phase-contrast images of PC3 cells in the presence or absence of Z-VAD-fmk (10 μM) for 24 h. Western blotting analysis of PARP after 22b or Z-VAD-fmk treatment. (E) RealTime Glo assays were performed after treatment with 1 μM 22b for 24−72 h.

22b for prolonged treatment despite the increased LC3-II.33 The lack of decrease in LC3B-II and p62 levels over time during the treatments indicates that autophagosomes may not

expression level of p62/SQSTM1, a protein preferentially degraded by the autophagic pathway, remained unaffected following 4 h of treatment and was elevated in cells exposed to 5068



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Figure 6. Effect of 22b on ER stress and protein synthesis in PC3 cells. (A) Cells were incubated with vehicle or 22b for 0, 4, or 8 h and analyzed using the ER-Tracker staining assay. Images were obtained by fluorescence microscopy. (B) Western blotting was performed to examine the changes in protein levels of GRP78, CHOP, PERK, p-PERK, eIF2α, p-eIF2α, and XBP1 mRNA splicing analysis in cells treated with 22b at the indicated time points. GAPDH and β-actin were used as loading controls. (C) Effect of 4-PBA on cell proliferation and morphology. Cells were pretreated with 4-PBA (100 μM) for 2 h prior to exposure to 22b for an additional 22 h. Cell viability was monitored by MTT assays, and morphological images were obtained by phase-contrast microscopy. ***, p < 0.001, compared to the 22b treatment alone. Western blotting analysis of GRP78 and XBP1 mRNA splicing analysis in cells treated with 22b and 4-PBA. (D) Combined effect of 22b and 39 (up) or bortezomib (down) in PC3 cells. After incubating cells with increasing concentrations of 22b and 39 or bortezomib, cell viability was determined, and the combination index (CI) was calculated by CompuSyn software.30−32 (E) Cell viability and vacuolization in response to ActD and CHX. Cells were pretreated with 10 μM ActD or 5 μM CHX and then treated with 22b for 24 h. MTT assays and image collection were performed as described above. **, p < 0.01, and ***, p < 0.001, compared to 22b-treated cells.

be efficiently fused with lysosomes. To determine if 22b interrupts the fusion of autophagosomes with lysosomes and

causes defective autophagy, a pH-sensitive LC3-reporter plasmid containing both GFP and RFP fusion tags (mRFP5069



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Figure 7. Nonautophagic effect of LC3 on vacuolization induced by 22b. (A) Western blotting analysis of the protein levels of LC3B and p62 in cells treated with 22b. (B) Cells were transfected with a pcDNA3.1-mRFP-GFP-LC3 plasmid, then treated with 1 μM 22b or 0.5 μM rapamycin for 8 h. GFP/mRFP-LC3 signals were visualized by fluorescence microscopy. (C) Cells were pretreated with the autophagy inhibitor 3-MA or E64d and then exposed to 1 μM 22b for 24 h. Morphologic changes were observed by phase-contrast microscopy, and cell viability was measured by MTT assays, *, p < 0.05, **, p < 0.01, and ***, p < 0.001, compared to 22b-treated cells. (D) After transfection with an LC3-targeting siRNA for 48 h, cells were exposed to 1 μM 22b for an additional 24 h. Western blotting analysis and morphological images were obtained as described above.

GFP-LC3) was transfected into cells prior to exposure to 22b.34 The green GFP signal in the LC3-reporter is quenched in acidic lysosomes. Therefore, autophagosomes appear yellow because of overlapped red and green fluorescence at less acidic pH, and autolysosomes appear red. As shown in Figure 7B,

rapamycin, a well-known inducer of autophagy, activated an obvious autophagy flux from autophagosomes to autolysosomes, as evidenced by the strong red punctate dots. However, green/yellow fluorescence of LC3 puncta was clearly observed in 22b-treated cells, indicating that 22b caused impaired 5070



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Figure 8. Compound 22b exerts antitumor efficiency in vivo. Three groups of mice (n = 5, each group) were injected with placebo, 22b, or docetaxel every 2 days. (A) Differences in tumor weight in mice treated with vehicle, 22b, and docetaxel. *, p < 0.05, and ***, p < 0.001, compared to the placebo group. (B) Differences in tumor size in mice exposed to vehicle, 22b, and docetaxel. (C) Changes in body weight of mice treated with vehicle, 22b, and docetaxel. (D) Tumor growth in each mouse was monitored on day 0, 9, and 18 by IVIS-100, and representative images of control and treated mice showing tumor size are shown. (E) Summary of changes in bioluminescence intensity (Photon Flux; photons/s/cm2/square root) in each group. ***, p < 0.001. (F) H&E (40×) and Ki-67 (20×) staining of tumor tissues treated with vehicle and 22b. (G) Western blotting and immunohistochemical staining for analysis of LC3, p62, and GRP78 protein levels in tumor tissues from vehicle- and 22b-treated groups. 5071



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observations in cell culture, the increased LC3, p62, and GRP78 levels in tumors (Figure 8G) revealed that 22b activated an immature autophagic response and induced remarkable ER stress in vivo. Together, our data clearly demonstrate that 22b exhibits antitumor effects by eliciting paraptosis-like cell death with low toxicity.

autophagosome−lysosome fusion, in agreement with the observations that 22b treatment resulted in the accumulation of lipid-conjugated LC3 and p62 (Figure 7A). Notably, increased granulation of LC3 was observed during rapamycininduced autophagy, whereas the LC3 in 22b-treated cells displayed a perivacuolar distribution, although several LC3positive vacuoles were clearly observed. These results prompted us to explore the role of autophagy in 22b-induced vacuole formation and cell death. As shown in Figure 7C, in the presence of 3-methyladenine (3-MA), which is a PI3K inhibitor to inactivate autophagy, 22b-induced cell viability was partially reversed but had no detectable impact on cytoplasmic vacuolation. The cell response to E-64d, an inhibitor of lysosomal enzymes, was similar to the response to 3-MA. The persistent upregulation of LC3-II and p62 and the lack of protective effect of 3-MA and E-64d demonstrated that 22binduced vacuolation was independent of classic autophagy and lysosomal degradation. However, elevated LC3-II was critical in vacuole formation induced by 22b, and the depletion of LC3 by LC3 siRNA nearly completely abolished the 22b-induced vacuole level (Figure 7D). This finding is consistent with reports that increased LC3-II is not specific for autophagy but plays a nonautophagic role during cytoplasmic vacuolation.35,36 Thus, 22b-induced vacuolation associated with the formation of LC3-II-positive nonautophagic vacuoles contributes to the cytotoxicity of 22b. In Vivo Anticancer Activity of 22b. The inhibitory efficiency of 22b against the growth of PCa cells was evaluated in a PC3M-luc-C6 (modified PC3M cells expressing luciferase) xenograft model. The mice were administered 22b (15 mg/kg) intraperitoneally every other day for 18 consecutive days and were compared with placebo-treated mice. A group of mice injected with docetaxel (15 mg/kg) served as a positive control. As shown in Figure 8A,B, 22b significantly reduced the growth of tumors by 40.5% compared with the placebo control, as indicated by the reduction in volume (410.2 ± 180.3 for treatment versus 845.6 ± 84.3 for vehicle) and weight (0.42 ± 0.08 for treatment versus 0.71 ± 0.06 for vehicle). Although docetaxel exerted higher antitumor efficacy than 22b (percent growth inhibition was 92% on day 18), docetaxel exhibited higher toxicity as indicated by the significant body weight loss compared with 22b treatment, which did not affect mouse body weight or other morbidity (Figure 8C, 13.3 vs 16.3 g). Additionally, one mouse died in the docetaxel-treated group, whereas no mice were lost during 22b treatment, supporting the conclusion that 22b was less toxic in animal trials at the same dosage. Bioluminescence measurements of tumors in each mouse on days 0, 9, and 18 of the treatments revealed that all tumorbearing mice exhibited ∼108 photons/s/cm2 in photon intensity prior to treatment. Mice treated with 22b exhibited 1.8 × 109 photons/s/cm2 on day 18, and the placebo group exhibited 2.5 × 1010 photons/s/cm2 on day 18 (Figure 8E), indicating that 22b caused a significant decrease in tumor burden with diminished cell activity compared with the placebo group. Immunohistochemical staining supported the notion that 22b inhibited tumor cell proliferation because a significant decrease in Ki-67-positive cells was observed in xenograft tumors treated with 22b compared with the placebo group (Figure 8F), consistent with the bioluminescence data. Importantly, H&E staining clearly showed extensive vacuoles in mice treated with 22b, providing evidence that 22b induces paraptosis in vivo (Figure 8F). In addition, consistent with the

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CONCLUSIONS A new series of novel benzo[a]quinolizidines were synthesized based on SAR analysis. Screening assays of these derivatives identified lead compound 22b as a potential antitumor agent. Novel compound 22b, which features 3,5-dimethylbenzyloxy groups at positions 9 and 10, is particularly attractive for future studies because of the following features: (i) Inhibition of cell proliferation with a low IC50 via the induction of both apoptosis and paraptosis by a combination of mechanisms, which will be beneficial for chemotherapy-resistant cancers. (ii) The impairment of autophagy maturation by 22b contributes to its antitumor efficacy because the activation of autophagy is generally cytoprotective. (iii) The low toxicity in in vivo experiments supports potential human applications. The activation of more than one type of cell death by 22b highlights its potential therapeutic applications in cancer treatment.

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EXPERIMENTAL SECTION

General Materials and Methods. Melting points were determined with an X-6 melting point apparatus (Beijing TECHInstrument Co., Ltd.) and are uncorrected. IR spectra were recorded on a Nicolet iN 10 Micro FTIR spectrometer. NMR spectra were obtained using a Bruker Avance AVIII-400 spectrometer operating at 400 (1H) or 101 (13C) MHz. HR-ESI-MS was conducted with an LTQ Orbitrap XL instrument. HPLC was performed with an Agilent 1200 system equipped with a G1311A isopump, a G1322A degasser, and a G1315D DAD detector using an Eclipse XDB-C18 (150 mm × 4.6 mm × 5 μm) and Chiralpak AS-H (250 mm × 4.6 mm × 5 μm). Column chromatography was conducted on silica gel or alumina (200−300 mesh). All solvents used were of analytical grade. Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.) were used for thin-layer chromatography. All reactions were conducted under a nitrogen atmosphere with dry solvents under anhydrous conditions. The purities of all biologically evaluated compounds were established as ≥95% by HPLC analysis with UV detection. Preparation of Benzo(α)quinolizidine Derivatives. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline (II-22b). In formic acid (13 mL) at 0 °C was dissolved 2-(3,4-dimethoxyphenyl)ethanamine I-22b (5 g, 27.6 mmol), followed by strring for 5 min. Then, paraformaldehyde (830 mg, 27.6 mmol) was added, and the mixture was stired at 50 °C overnight. The solvent was removed by evaporation and the residue poured into 6 M aqueous sodium hydroxide to yield white precipitate. The suspension was filtered; the crude material can be purified by recrystallization from dichloromethane to give II-22b as pale-yellow needles (4.96 g, 93%). 1H NMR (400 MHz, D2O, δ) 6.88 (s, 1H), 6.82 (s, 1H), 4.27 (s, 2H), 3.81 (d, J = 4.9 Hz, 6H), 3.48 (t, J = 6.4 Hz, 2H), 3.03 (t, J = 6.2 Hz, 2H). 13C NMR (101 MHz, D2O, δ) 147.84, 147.15, 124.11, 119.97, 111.85, 109.71, 55.77, 55.72, 44.08, 41.65, 24.01. ESI-MS m/z 194 [M + H]+. tert-Butyl-6,7-dihydroxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (III-22b). The solid of II-22b (2.5 g, 12.9 mmol) was dissolved in 48% aqueous hydrobromic acid (50 mL), and the resulting mixture was heated at reflux for 5 h. Evaporation of the hydrobromic acid afforded 6,7-digydroxy-1,2,3,4-tetrahydroisoquinoline hydrobromide as white powder (3.17 g, quantitative); the product was used without further purification. 1H NMR (400 MHz, D2O, δ) 6.71 (s, 1H), 6.66 (s, 1H), 4.19 (s, 2H), 3.43 (t, J = 6.4 Hz, 2H), 2.94 (t, J = 6.2 Hz, 2H). 13 C NMR (101 MHz, D2O, δ) 143.8, 143.0, 123.7, 119.6, 115.8, 113.7, 44.0, 41.7, 23.8. ESI-MS m/z 166 [M + H]+. Tetrahydroisoquinoline 5072



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oil (570 mg, 86%). 1H NMR (400 MHz, CDCl3, rotamer seen, δ) 7.08 (s, 4H), 6.95 (s, 2H), 6.90−6.64 (m, 3H), 6.31−5.97 (m, 1H), 5.68− 5.37 (m, 1H), 5.18−4.86 (m, 4H), 4.20 (d, J = 11.7 Hz, 0.67H), 3.90 (d, J = 12.0 Hz, 0.37H), 3.34 (s, 0.38H), 3.18 (t, J = 10.2 Hz, 0.64H), 3.07−2.70 (m, 3H), 2.64 (d, J = 16.0 Hz, 1H), 2.32 (s, 12H), 1.91 (d, J = 6.5 Hz, 3H), 1.45 (s, 9H). 13C NMR (101 MHz, CDCl3, rotamer seen, δ) 197.7, 154.3, 148.2, 147.6, 143.2, 142.9, 138.0, 138.0, 137.2, 132.7, 132.2, 129.8, 129.4, 127.3, 125.3, 125.1, 115.1, 114.2, 113.9, 80.3, 71.7, 71.5, 51.6, 51.1, 47.5, 39.1, 37.4, 28.3, 28.1, 21.3, 18.4. ESIMS m/z 584 [M + H]+. trans-9,10-Bis((3,5-dimethylbenzyl)oxy)-4-methyl-3,4,6,7-tetrahydro-1H-pyrido[2,1-α]isoquinolin-2(11bH)-one (VIII-22b). To a solution of VII-22b (570 mg, 0.98 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (1.5 mL, 20 mmol) dropwise at 0 °C. The reaction stirred at 0 °C for 4 h; then, the reaction was quenched by adding saturated NaHCO3 solution, adjusting the pH to 6.5, and extracting with CH2Cl2 (3 × 10 mL). The combined organic layers were concentrated in vacuo giving the amine intermediate as a yellowish oil. ESI-MS m/z 484. The resulting yellowish oil was diluted with MeOH (8 mL) and treated with NH4OH (28%, 8 mL) for 2 h at room temperature. The reaction mixture was extracted with dichloromethane (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4. Filtration and evaporation in vacuo gave a crude mixture. The residue was purified via flash-column chromatography on silica gel to yield compound VIII-22b as a colorless crystal (336 mg, 71%). 1H NMR (400 MHz, CDCl3, δ) 7.07 (d, J = 6.1 Hz, 4H), 6.94 (s, 2H), 6.72 (s, 1H), 6.64 (s, 1H), 5.04 (d, J = 11.4 Hz, 4H), 4.18−4.08 (m, 1H), 3.64−3.53 (m, 1H), 3.16−3.05 (m, 1H), 2.99−2.76 (m, 4H), 2.67−2.56 (m, 1H), 2.48 (dd, J = 13.9, 11.3 Hz, 1H), 2.32 (s, 12H), 2.26−2.22 (m, 1H), 1.13 (d, J = 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3, δ) 209.4, 148.3, 147.7, 138.2, 137.4, 137.4, 130.3, 129.6, 129.6, 127.3, 125.4, 125.3, 115.5, 113.3, 72.0, 71.7, 57.8, 54.1, 47.6, 47.2, 46.5, 29.7, 21.5, 15.1. ESI-MS m/z 484 [M + H]+. HR-EI-MS m/z 484.2846 [M + H]+. Calcd for C32H38NO3+, 484.2835. Synthesis of (±)-(9,10-Bis((3,5-dimethylbenzyl)oxy)-4α-methyl2,3,4,6,7,11bα-hexahydro-1H-pyrido[2,1-α]isoquinolin-2α-ol (22a) and (±)-(9,10-Bis((3,5-dimethylbenzyl)oxy)-4α-methyl2,3,4,6,7,11bα-hexahydro-1H-pyrido[2,1-α]isoquinolin-2β-ol (22b). To a solution of VIII-22b (100 mg, 0.21 mmol) in MeOH (5 mL) at 0 °C was added sodium borohydride (76 mg, 2 mmol), and the resulting mixture was stirred for 1 h. The reaction was quenched with water (3 mL) and extracted with dichloromethane (3 × 10 mL). The combined organic layers were washed with brine, dried, filtered, and evaporated in vacuo. The residue was purified via flash-column chromatography on silica gel to yield compounds 22a (40 mg, 39%) and 22b (42 mg, 41%). Compound 22a: yellowish solid, mp 85−87 °C. IR νmax 3396, 2916, 1609, 1509, 1383, 1249, 1224, 850 cm−1. 1H NMR (400 MHz, CDCl3, δ) 7.08 (s, 4H), 6.95 (s, 2H), 6.78 (s, 1H), 6.69 (s, 1H), 5.04 (d, J = 1.6 Hz, 4H), 4.13−3.98 (m, 1H), 3.81 (d, J = 10.4 Hz, 1H), 3.35 (s, 1H), 3.02−2.84 (m, 2H), 2.79−2.60 (m, 2H), 2.40−2.24 (m, 13H), 1.89 (d, J = 11.6 Hz, 1H), 1.78 (td, J = 11.7, 5.1 Hz, 1H), 1.57 (s, 1H), 1.45 (dd, J = 22.0, 11.1 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3, δ) 148.00, 147.54, 138.13, 137.61, 137.56, 131.46, 129.56, 129.53, 128.03, 125.50, 125.34, 115.50, 113.66, 72.31, 71.71, 66.13, 55.80, 52.36, 48.30, 41.59, 39.81, 29.86, 21.49, 12.77. ESI-MS m/z 486 [M + H]+. HR-EI-MS m/z 486.2997 [M + H]+. Calcd for C32H40NO3+, 486.3003. Compound 22b: yellowish solid, mp 82−84 °C. IR νmax 3369, 2916, 1609, ,1514, 1370, 1253, 1201, 849 cm−1. 1H NMR (400 MHz, CDCl3, δ) 7.09 (d, J = 6.6 Hz, 4H), 6.96 (d, J = 5.1 Hz, 2H), 6.86 (s, 1H), 6.69 (s, 1H), 5.29−4.72 (m, 4H), 4.31 (s, 1H), 3.65 (s, 1H), 3.37 (dd, J = 12.8, 5.0 Hz, 1H), 3.05 (td, J = 12.7, 4.2 Hz, 1H), 3.00−2.88 (m, 1H), 2.87−2.71 (m, 1H), 2.52−2.39 (m, 1H), 2.38−2.14 (m, 13H), 2.02− 1.73 (m, 3H), 1.42 (dt, J = 12.5, 9.3 Hz, 1H). 13C NMR (101 MHz, CDCl3, δ) 147.9, 147.4, 138.0, 138.0, 137.5, 137.3, 129.5, 129.4, 129.3, 128.3, 125.3, 125.2, 115.4, 113.6, 72.2, 71.5, 65.3, 55.6, 48.0, 47.0, 42.2, 37.9, 24.0, 21.3, 21.3, 18.9. ESI-MS m/z 486 [M + H]+. HR-EI-MS m/ z 486.3002 [M + H]+. Calcd for C32H40NO3+, 486.3003.

(6.3 g, 26 mmol) was suspended in H2O (30 mL), di-tertbutyldicarbonate (6.6 mL, 28.6 mmol ), and TEA (7.6 mL, 55 mmol) in THF (40 mL) was added dropwise. The resulting mixture was stirred overnight. The mixture was concentrated, dissolved in EtOAc and washed with brine. The organic phase was dried, concentrated and chromatographed on silica to give III-22b as a yellow powder (4.96 g, 72%). 1H NMR 1H NMR (400 MHz, CDCl3) δ 6.66 (s, 2H), 5.78 (s, 2H), 4.44 (s, 2H), 3.61 (t, J = 5.9 Hz, 2H), 2.71 (t, J = 5.8 Hz, 2H), 1.50 (s, 5H). 13C NMR (101 MHz, CDCl3) δ 155.6, 143.4, 142.8, 126.6, 125.3, 115.1, 112.9, 80.8, 45.5, 42.5, 41.3, 28.7, 28.3, 27.9. ESI-MS m/z 266 [M + H]+. tert-Butyl-6,7-bis((3,5-dimethylbenzyl)oxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate (IV-22b). Potassium carbonate (2.1 g, 15 mmol) was added to a solution of 3,5-dimethylbenzyl bromide (1.49 g, 7.5 mmol) and III-22b (800 mg, 3 mmol) in DMF (5 mL). The resulting reaction mixture was stirred at room temperature for 1 h and water (5 mL) was added. Then, the organic layer was washed with brine (3 × 10 mL), dried with sodium sulfate, filtered, and evaporated under reduced pressure. The resulting light-orange residue was purified via flash-column chromatography on silica gel to yield IV22b as colorless oil (1.45 g, 96%). 1H NMR (400 MHz, CDCl3, δ) 7.09 (s, 4H), 6.96 (s, 2H), 6.74 (s, 1H), 6.71 (s, 1H), 5.06 (s, 4H), 4.48 (s, 2H), 3.63 (s, 2H), 2.74 (s, 2H), 2.33 (s, 12H), 1.51 (s, 9H). 13 C NMR (101 MHz, CDCl3, δ) 154.9, 147.9, 147.8, 138.0, 137.3, 137.2, 129.4, 129.4, 127.6, 126.4, 125.1, 115.4, 113.1, 79.7, 71.8, 71.7, 45.5 41.9, 28.5, 21.3. ESI-MS m/z 502 [M + H]+. (±)-6,7-Bis((3,5-dimethylbenzyl)oxy)-1-ethoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (V-22b). The 2,2,6,6-tetramethylpiperidine N-oxide salt (T+BF4−) (340 mg, 1.4 mmol) was added to a solution of compound IV-22b (700 mg, 1.4 mmol) and ethanol (1.5 mL) in anhydrous dichloromethane (14 mL), and the reaction was allowed to stir for 24 h until the complete consumption of the starting material. Then, 1 M aqueous hydrochloric acid (10 mL) was added dropwise at 0 °C. The organic layer was separated and washed with aqueous sodium bicarbonate and brine, dried, and evaporated. The resulting residue was purified via flash-column chromatography on silica gel to yield the compound V-22b as a pale-yellow oil (618 mg, 81%). 1H NMR (400 MHz, CDCl3, rotamer seen, δ) 7.11 (d, J = 18.3 Hz, 4H), 7.03−6.87 (m, 3H), 6.81−6.61 (m, 1H), 6.35−5.67 (m, 1H), 5.27− 4.95 (m, 4H), 4.23 (d, J = 8.4 Hz, 0.52H), 4.00 (d, J = 11.0 Hz, 0.40H), 3.80−3.55 (m, 2H), 3.51−3.22 (m, 1H), 2.83 (d, J = 14.1 Hz, 1H), 2.62 (d, J = 15.8 Hz, 1H), 2.34 (d, J = 4.4 Hz, 12H), 1.53 (s, 9H), 1.29 (t, J = 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3, rotamer seen, δ) 155.0, 154.4, 149.2, 148.0, 138.2, 138.0, 138.0, 137.7, 137.3, 137.2, 136.5, 129.6, 129.4, 129.4, 128.6, 128.1, 127.5, 127.2, 125.3, 125.0, 115.3, 114.9, 114.5, 81.1, 80.6, 80.1, 77.5, 77.2, 76.9, 71.9, 71.5, 63.4, 62.9, 39.0, 37.8, 36.2, 34.2, 28.5, 28.5, 28.0, 27.6, 21.3, 15.4. ESI-MS m/z 546 [M + H]+. Preparation of (E)-Trimethyl(penta-1,3-dien-2-yloxy)silane (VI-22b) and Derivatives. Methyl vinyl ketone (0.2 mL, 2 mmol) was dissolved in THF (1 mL) and added dropwise to the a solution of LDA (1.1 mL, 2.2 mmol) at −78 °C over 1 min. After stirring for 1 h, TMSCl was added, and the reaction mixture was allowed to warm to room temperature for 1 h. The reaction was quenched by adding saturated aqueous NH4Cl and extracting with ether (3 × 10 mL). The organic layer was combined, dried (Na2SO4), filtered, and concentrated to yield compound VI-22b as a yellow oil. ESI-MS m/z 157 [M + H]+. The crude product was used directly without further purification. (E)-tert-Butyl-6,7-bis((3,5-dimethylbenzyl)oxy)-1-(2-oxopent-3en-1-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (VII-22b). To a fresh solution of crude VI-22b (312 mg, 2 mmol) in dichloromethane (5 mL) was added V-22b (618 mg, 1.13 mmol), and to the mixture was added Yb(OTf)3 (400 mg, 0.6 mmol) in portions at 0 °C. The resulting mixture was allowed to stir at room temperature for 3 h until the complete consumption of V-22b, as monitored by TLC. Aqueous NaHCO3 was added, and the aqueous layer was extracted with dichloromethane (3 × 10 mL). The organic phase was combined, dried with NaSO4, and evaporated. The residue was purified via flashcolumn chromatography on silica gel to yield the VII-22b as a yellow 5073



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(±)-9,10-Bis((3,5-dimethylbenzyl)oxy)-2,4α-dimethyl2,3,4,6,7,11bα-hexahydro-1H-pyrido[2,1-α]isoquinoline-2α-ol (28). To a solution of VIII-22b (100 mg, 0.21 mmol) in THF (1 mL) at 0 °C was added methylmagnesium bromide (1.0 M solution in THF, 1 mL). The reaction mixture was allowed to stir for 1 h, and saturated aqueous NH4Cl (3 mL) was added. The product was extracted with dichloromethane (3 × 10 mL), dried (MgSO4), and the solvent removed in vacuo. The residue was purified via flash-column chromatography on silica gel to yield compound 28 as a yellow oil (74 mg, 72%). IR νmax 3374, 2919, 1508, 1381, 1249, 1223, 699 cm−1. 1 H NMR (400 MHz, CDCl3, δ) 7.09 (s, 4H), 6.95 (s, 2H), 6.80 (s, 1H), 6.69 (s, 1H), 5.05 (s, 4H), 4.09 (d, J = 11.0 Hz, 1H), 3.28−3.16 (m, 1H), 3.06−2.86 (m, 2H), 2.86−2.75 (m, 1H), 2.63 (d, J = 15.5 Hz, 1H), 2.32 (s, 12H), 2.06−1.91 (m, 2H), 1.63−1.52 (m, 2H), 1.29 (d, J = 6.8 Hz, 3H), 1.24 (s, 3H). 13C NMR (101 MHz, CDCl3, δ) 147.8, 147.5, 138.1, 138.1, 137.6, 137.6, 132.0, 129.5, 129.5, 128.4, 125.6, 125.3, 115.6, 113.9, 72.3, 71.7, 70.5, 54.5, 50.1, 48.4, 45.3, 43.1, 32.6, 29.4, 21.5, 14.1. ESI-MS m/z 500 [M + H]+. HR-EI-MS m/z 500.3156 [M + H]+. Calcd for C33H42NO3+, 500.3159. trans-9,10-Bis(benzyloxy)-4-phenyl-2,3,4,6,7,11b-hexahydro-1Hpyrido[2,1-α]isoquinoline (15). A mixture of VIII-15 (200 mg, 0.41 mmol) and 80% aqueous hydrazine hydrate (72 μL, 1.2 mmol) in diethylene glycol (2 mL) was heated to 80 °C for 2 h. Potassium hydroxide (114 mg, 2 mmol) was added, and the reaction was heated to 180 °C for 2 h. The mixture was poured into ice water and extracted with ethyl acetate (3 × 10 mL). The organic layer was combined, dried, and evaporated. The residue was purified via flash-column chromatography on silica gel to yield compound 15 as a yellowish oil (54 mg, 28%). 1H NMR (400 MHz, CDCl3, δ) 7.68−7.23 (m, 15H), 6.85 (s, 1H), 6.73 (s, 1H), 5.26−5.02 (m, 4H), 4.35 (s, 1H), 3.64 (t, J = 6.7 Hz, 1H), 3.11−2.69 (m, 3H), 2.51−2.20 (m, 2H), 2.20−1.94 (m, 1H), 1.70 (d, J = 2.7 Hz, 2H), 1.57 (dt, J = 13.4, 3.7 Hz, 1H), 1.37− 1.28 (m, 1H). 13C NMR (101 MHz, CDCl3, δ) 147.8, 147.3, 137.6, 137.5, 128.5, 128.5, 128.4, 127.8, 127.8, 127.6, 127.5, 127.4, 127.1, 115.4, 114.0, 72.1, 71.3, 59.2, 57.1, 47.3, 34.6, 28.0, 23.4, 19.9. ESI-MS m/z 476 [M + H]+. HR-EI-MS m/z 476.2582 [M + H]+. Calcd for C33H34NO2+, 476.2584. Cell Culture and Treatments. The human prostate carcinoma cell lines PC3 and DU145, the breast carcinoma cell line MDAMB231, MCF-7 cells, HeLa cells, and the myelogenous leukemia cell line K562 (The American Type Culture Collection (ATCC), Rockville, MD) were cultured in RPMI 1640 medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone). The human prostate epithelial cell line RWPE-1 was maintained in Keratinocyte1 medium (K-SFM) supplemented with 50 mg/L bovine pituitary extract and 5 μg/L epidermal growth factor (Gibco, Grand Island, NY). Luciferase-expressing PC3M cells (PC3MLuc-C6) (Shanghai Genomics, China) were cultured in MEM medium (HyClone) supplemented with 1% nonessential amino acids (SigmaAldrich), 1% L-glutamine (ICN), and 1 mM sodium pyruvate (Sigma− Aldrich). The cells were incubated with 100 units/mL penicillin and 100 μg/mL streptomycin and were maintained in 5% CO2 at 37 °C until they reached approximately 50−70% confluence and were then treated with various concentrations of compounds. Dimethyl sulfoxide (DMSO) was used as the control vehicle. Antiproliferation Assay. The effects of the compounds on the proliferation of the human cell lines were evaluated by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma, St. Louis, MO) assay. Briefly, cells were seeded into 96-well plates for 24 h and then treated with vehicle, the desired compounds alone, or docetaxel as a positive control for 24 h. After incubation with 10 μL of MTT (5 mg/mL) for 4 h, absorbance of the soluble MTT product was measured at 570 nm. The antiproliferation assay was performed in triplicate. The RealTime Glo assay was also performed for individual experiments. Cell Cycle and Apoptosis Assay. For cell cycle analysis, after treatment for 24 h, the PC3 cells were fixed in 70% cold ethanol overnight at 4 °C, incubated for 30 min in PBS containing 100 μg/mL RNase (Invitrogen), and then incubated with 50 μg/mL PI (Propidium iodide, Sigma-Aldrich) at 4 °C for 30 min in the dark.

Data were processed using ModFit LT Software. Apoptotic cells were detected using an Annexin V−fluorescein isothiocyanate (FITC)/ propidium iodide (PI) apoptosis detection kit (BD, San Jose, CA, USA). Flow cytometry was performed with a FACScan cytometry instrument (FACSCalibur, Becton Dickinson, USA). Data were analyzed using MODFIT or CELLQUEST software (Verity Software House, Topsham, Maine, USA). In the apoptotic analysis, necrotic (Annexin V−/PI+), late apoptotic (double-stained), normal (Annexin V−/PI−), and early apoptotic (Annexin V+/PI−) cells were labeled as B1−B4. Microscopy. PC3 cells in various stages after treatment were incubated at 37 °C for 15 min with ER-Tracker (1:3000, Beyotime, China), Lyso-Tracker (1:20000, Beyotime, China), or MitoTracker (200 nM, Beyotime, China) and 10 μg/mL of Hoechst 33342 (Sigma−Aldrich). The cells were then washed with PBS for confocal microscopy (Carl Zeiss). We used ER-Tracker, Lyso-Tracker, MitoTracker, or Hoechst 33342 for endoplasmic reticulum, lysosomal, mitochondrial, and nuclear integrity assays, respectively. Western Blotting. The cells were lysed in RIPA lysis buffer. A total of 40 μg of protein was quantified and loaded onto an SDSPAGE gel. After electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane and incubated with primary antibodies at 4 °C overnight. The membrane was washed and incubated with HRP-conjugated secondary antibodies for 45 min. The immunoblot bands were detected by an ECL system, and the membranes were exposed to X-ray films. Drug Combination Analysis. Three replicates were averaged, and repeats of these data sets were entered into CalcuSyn (Biosoft, Ferguson, MO) for analysis. This program uses the median effect analysis algorithm, which produces the combination index (CI) value as a quantitative indicator of the degree of synergy or antagonism. Using this analysis method, CI < 1, CI = 1, and CI > 1 indicate synergism, an additive effect, and antagonism, respectively. Autophagy Assays. To evaluate the formation of autophagosomes and autolysosomes, the mRFP-GFP-LC3 reporter plasmid was transfected into PC3 cells and incubated with 22b for 8 h. Images were acquired using confocal microscopy. siRNA Transfection. PC3 cells were plated into six-well plates at 20−30% confluency, and 24 h later, the cells were transfected with 50 nM siRNA using Lipofectamine 2000 (Invitrogen). The siRNAs targeting LC3 and the scramble control siRNA were designed, modified, and synthesized by Invitrogen. Inhibition of Tumor Growth in Vivo. Male athymic (BALB/cnu) mice (6 weeks old) were obtained from the Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). Our experimental protocol was reviewed and approved by the Institutional Animal Care Committee of Shandong University. PC3M-luc-C6 cells (5 × 106 cells used for each injection) were injected subcutaneously into the right anterior flanks of the mice. When the tumor volume reached approximately 0.1 cm3, the mice were sorted into three groups (n = 5), and administration was started. Compound 22b suspension (15 mg/kg) was injected via intraperitoneal injection, and docetaxel (15 mg/kg) was injected as a positive control. Administration was performed every 2 days for three consecutive weeks. The percentages of growth inhibition were defined as the ratio of tumor weight to that in the vehicle control. Tumor dimensions were determined using calipers, and the tumor volume (mm3) was calculated using the following the formula: volume = length × (width)2 × 0.5. Tumor size was assessed in each mouse via in vivo bioluminescence measurement using the IVIS Imaging System (Caliper Life Sciences, USA). The photometry of the tumor was calculated using Living Image 3.1.0 (Caliper Life Sciences) software, and tumor growth curve was calculated. Bioluminescent PC3M-luc-C6 cells constitutively expressing luciferase were implanted under the skin of mice for primary tumor assessment. For the luciferase detection imaging, luciferin (Shanghai Genomics, China) was dissolved in PBS and injected (i.p., 150 mg/kg, 200 μL) before imaging. During in vivo imaging, the mice were immobilized using the anesthetic isoflurane (1.5−2.5%). Immunocytochemistry. H&E staining was performed using routine methods. The frozen tissue sections were rewarmed at 60 5074



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°C for 30 min, washed with PBS and incubated overnight at 4 °C with primary antibody (anti-Ki67 (1:500, mouse; Santa Cruz Biotechnologies), anti-LC3 (1:800, rabbit; Nonvus), anti-p62 (1:500, mouse; Santa Cruz Biotechnologies), and anti-GRP78 (1:500, rabbit; Stressgen)) diluted in PBS. After staining with IgG-conjugated HRP and DAB (Vector Laboratories), the samples were counterstained with hematoxylin, and images were captured by microscopy (Nikon). Statistical Analysis. Data are presented as the mean ± SD for triplicate experiments. Statistically significant differences between treated groups and controls were analyzed using Student’s t test, and p < 0.05 was considered statistically significant.

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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.6b00484. HR-ESI-MS and 1H and 13C NMR data for all benzo[a]quinolizidines; key 1H−1H COSY, HSQC, HMBC, and NOESY correlations of compounds 1, 4, 11b, 16a, and 18; IR spectra of compounds 22a,b and 28; whole MTT results of all the derivatives; optimization for the Z-VAD-fmk assays; western blot analysis of the decrease of IκB-α and p-P65 reponse to CHX, ActD, and CI values. (PDF) Structual and IC50 data for compounds 1−38, SD1−10, SE1−10, SF1−9, SG1−6, SH1,2, IV-22b, V-22b, and VII-22b. (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-531-8838-2346. Fax: +86-531-8838-2019. *E-mail: [email protected]. Tel.: +86-531-8838-2012. Fax: +86-531-8838-2019. Author Contributions #

Z. H. and Y. D. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81172956 and 81473107), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13028).

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ABBREVIATIONS USED ER, endoplasmic reticulum; LC3, microtubule-associated protein 1 light chain 3; Hsp90, Heat shock protein 90; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxy; SAR, structure− activity relationship; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; Z-VAD-fmk, Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone; GRP78, gastric inhibition polypeptide 78; UPR, unfolded protein response; CHOP, C/EBP-homologous protein; PERK, PKR-like endoplasmic reticulum kinase; eIF2α, PERK-mediated phosphorylation of eukaryotic initiation factor 2α; 4-PBA, 4-phenylbutyricacid; 3-MA, 3-methyladenine; CHX, cycloheximide; Act-D, actinomycin-D; mRFP, monomeric red fluorescence protein; GFP, green fluorescence protein; PC3M-luc-C6, modified PC3M cells that express luciferase 5075



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Novel Benzo[a]quinolizidine Analogs Induce Cancer Cell Death

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