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Discovery of novel jaspine B analogues as autophagy inducer
Bioorganic & Medicinal Chemistry Letters 28 (2018) 497–502
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel jaspine B analogues as autophagy inducer En Zhang a,b,⇑, Shang Wang a, Li-Li Li a, Yong-Gang Hua a, Jing-Fei Yue a, Jin-Feng Li c, Cheng-Yun Jin a,b,⇑ a School of Pharmaceutical Sciences, Institute of Drug Discovery and Development, Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, PR China b Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China c Kidney Transplantation, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe Road, Zhengzhou 450001, PR China
a r t i c l e
i n f o
Article history: Received 12 September 2017 Revised 22 November 2017 Accepted 5 December 2017 Available online 8 December 2017 Keywords: Jaspine B Pachastrissamine Anti-tumor Anti-proliferative Autophagy
a b s t r a c t A series of 2-alkylaminomethyl jaspine B analogues were synthesized and evaluated for their cytotoxic effects on human lung adenocarcinoma, breast cancer, and prostate cancer cell lines and a mouse melanoma cell line. Most of the compounds exhibited moderate to good activity against the cancer cell lines. Compound 7f showed the best overall cytotoxicity on PC-3 cells (IC50 = 0.85 lM). Further mechanistic studies revealed that compound 7f induced marked changes in PC-3 cell morphology, disrupted the mitochondrial membrane potential, and increased expression of the autophagy proteins beclin-1, LC3, and P62. Ó 2017 Elsevier Ltd. All rights reserved.
Jaspine B (Fig. 1, 1), which was originally isolated from the marine sponge Pachastrissa sp.1and Jaspis sp.2, and is an anhydrophytosphingosine derivative with three chiral centers. Jaspine B inhibits the growth of a range of tumor cell lines in vitro3, and the anti-tumor activity is critically dependent on the stereo configuration4, alkyl chain length5, amide moiety6, and the tetrahydrofuran ring.7 Based on these characteristics, many researchers have synthesized jaspine B8,9 and its derivatives through various synthetic routes. Rives et al.10 developed an approach to generate pyrrolidine-based analogues of jaspine B (Fig. 1, 2) and found that their cytotoxicity towards melanoma cells was comparable to that of jaspine B. Our group synthesized a series of novel jaspine B analogues (Fig. 1, 3) bearing a 1,2,3-triazole functional group11, one of which had more potent anti-proliferative activity against EC-9706, MGC-803 and MCF-7 than either 5-Fu or jaspine B. Kwon et al. prepared a carbocyclic analogue of jaspine B (Fig. 1, 4) that showed comparable cytotoxicity to the parent compound but was a more potent inhibitory of sphingosine kinases.3 Several studies have examined the mechanism of cytotoxicity of jaspine B, Canals et al.4 suggested that jaspine B might be involved ⇑ Corresponding authors at: School of Pharmaceutical Sciences, Institute of Drug Discovery and Development, Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, PR China. E-mail addresses: [email protected] (E. Zhang), [email protected] (C.-Y. Jin). https://doi.org/10.1016/j.bmcl.2017.12.011 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
in dihydroceramide-induced autophagy in A549 cells. Yoo et al.12 reported that jaspine B-mediated inhibition of melanoma cell growth may occur via dual inhibition of extracellular regulatory kinase signaling and cyclin-dependent kinase 2 pathways. Despite these findings, the anti-tumor mechanisms of jaspine B and its analogues remain unclear. Cell survival and death are both strongly influenced by autophagy,13 the process through which aberrant or dysfunctional cellular components are removed by lysosomal degradation and recycling. Three main classes of autophagy have been described; namely, chaperone-mediated autophagy, microautophagy and macroautophagy, which differ in their physiological functions, substrate types, and manner of substrate delivery to lysosomes.14 Many reports indicate that autophagy have a good inhibitory effect for various cancers.15–17 Autophagy is required for normal cell homeostasis and is activated by exposure to a variety of stressors, including hypoxia,18 endoplasmic reticulum stress,19 starvation, toxins, and inflammation,20 cancer and senescence.21 Autophagy is a dynamic process in which intracellular membranes undergo dramatic morphological changes. When autophagy-induced signaling initiates, the cells form a small liposome-like membrane somewhere in the cytoplasm, which then expands, known as phagophore. Phagophore continues to extend the cytoplasm of any component, including the organelles, into which all embrace, then shut up and becomes a closed spherical autophagic bodies. Autophagosomes formed with endocytosis of phagosome
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E. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 497–502
Fig. 1. Jaspine B and its representative derivatives.
Scheme 1. Reagent and conditions: (a) R-NH2, NaBH(OAc)3, 1,2-dichloroethane, room temperature; b) Pd/C, H2, CH3OH, 60 °C, 60 psi.
Scheme 2. Reagent and conditions: (a) R-NH2, NaBH4, CH3OH, room temperature; b) Pd/C, H2, CH3OH, 60 °C, 60 psi.
Table 1 Reaction conditions for reductive amination of aldehydes. Material
Solvent
Reductant
Yield/%
5 5 50 50
CH3OH 1,2-dichloroethane CH3OH 1,2-dichloroethane
NaBH4 NaBH(OAc)3 NaBH4 NaBH(OAc)3
27 84 89 45
pinocytosis, endocytic vesicles and endosomes integration. The autophagosome fuses with the lysosome to form autophagosomes, during which the endomembrane of the autophagosome is degraded by lysosomal enzymes, including integrating the contents of both, and the cargo in the autophagosome is degraded then the product is transported to cytoplasm.22–24 Because excessive autophagy can promote cell death, autophagy-inducing compounds may be useful anti-cancer drugs. Previous studies have shown that such compounds can induce cell death by various mechanisms.25 Here, we have extended our previous efforts to develop jaspine B derivatives with anti-cancer activity.11,26–29 We synthesized a series of jaspine B analogues bearing a 2-alkylaminomethyl group and tested the anti-tumor activity on four tumor cell lines. We identify one of the compounds as a new autophagy inducer that cause loss of mitochondrial permeability, increases the expression of the autophagy proteins LC-3, beclin-1, and P62, and, at high concentrations, results in tumor cell death. Jaspine B analogues were synthesized using as starting material compounds 5 or 50 , which were generated according to our previously developed method 11, 26. The first step reaction was a reductive amination of aldehyde, using sodium triacetoxyborohydride as the reducing agent (Scheme 1). In some cases, the reactions between aldehydes and primary amines generated considerable amounts of dialkylation and other side products. Thus, a stepwise procedure involving imine formation in methanol followed by reduction with sodium borohydride was carried out for some compound, as shown in Scheme 2. The stepwise procedure did not apply to the synthesis of compound 6a-6g. The various reaction conditions were summarized in Table 1.
Table 2 Anti-proliferative activity of jaspine B analogues against four cancer cell lines. Com.
7a 7b 7c 7d 7e 7f 7g 70 a 70 b 70 c 70 d 70 e 70 f 70 g 5-Fu 1
n
6 7 8 9 10 11 13 6 7 8 9 10 11 13
IC50(lM)a A549
B16-F10
MCF-7
PC-3
>128 >128 22.07 ± 2.92 47.84 ± 4.09 11.1 ± 1.761 5.87 ± 0.82 2.85 ± 0.08 >128 71.76 ± 5.38 >128 >128 9.65 ± 1.54 41.10 ± 3.63 19.95 ± 2.48 0.43 ± 0.01 1.87b
>128 >128 55.18 ± 4.46 29.62 ± 3.33 19.31 ± 2.57 10.70 ± 1.68 14.31 ± 2.04 >128 >128 >128 >128 88.82 ± 4.90 58.11 ± 4.13 64.25 ± 4.03 6.29 ± 0.66 0.5c
>128 96.56 ± 5.62 12.46 ± 1.96 34.20 ± 3.55 6.77 ± 1.01 1.80 ± 0.08 0.72 ± 0.18 >128 >128 60.34 ± 4.62 42.71 ± 3.91 13.13 ± 2.01 12.63 ± 1.92 8.83 ± 1.40 6.11 ± 0.01 2.33b
22.36 ± 3.09 13.75 ± 2.15 2.03 ± 0.10 11.86 ± 1.86 3.33 ± 0.07 0.85 ± 0.19 1.20 ± 0.12 >128 12.96 ± 2.06 7.65 ± 1.14 >128 10.01 ± 1.59 2.38 ± 0.04 1.99 ± 0.05 0.53 ± 0.11 0.7d
a Inhibitory activity is expressed as the concentration required to inhibit tumor cell proliferation by 50% after exposure for 72 h (IC50). n, alkyl chain length. Data are presented as the means ± SDs of three independent experiments. b Literature values 9. c Literature values 8. d Literature values 3.
E. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 497–502
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Fig. 2. (A) Cell viability of PC-3 cells after treatment with various concentrations for 48 h; (B) Quantitative analysis of the ratio of the cell viability.
Fig. 3. Morphological changes of PC-3 cells induced by compound 7f.
After reductive amination of the aldehyde, the azide functionality was reduced and the benzyl-protecting group was removed to give the target compound 7a-7g in a one-pot reaction using palladium-catalyzed hydrogenolysis. The method was also applied to synthesize compounds 70 a-70 g. The structures of all newly synthesized compounds were fully characterized by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry. The compounds were then evaluated for their anti-tumor activities against four cancer cell lines: A549 (human lung adenocarcinoma), B16-F10 (mouse melanoma), MCF-7 (human breast cancer) and PC-3 (human prostate cancer) by using the colorimet-
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Fig. 4. Expression changes of autophagy related proteins induced by compound 7f.
ric MTT viability/proliferation assay. 5-Fluorouracil (5-Fu) was included as a positive control compound. The results of the MTT assay, expressed as the concentration resulting in 50% inhibition of maximal proliferation (IC50), are shown in Table 2. The analogues had much better activity against MCF-7 and PC-3 than against A549 and B16-F10. Notably, compounds containing fewer than 10 alkyl carbon showed little or no anti-proliferative activity (7a-7d). while better activity was observed in compounds with 11 or 13 alkyl carbons (7f, 7 g). However, the alkyl carbon number was not directly proportional to
activity; for example, compound 7f had better activity than compound 7 g against B16-F10 and PC-3 cells despite containing fewer alkyl carbons. This suggested that an intermediate alkyl chain length was optimal. Similar results were observed with the 3-epijaspine B derivatives 70 a-70 g. In addition, the stereo configuration of these compounds played an important role in their anti-proliferative activity. For example, compound 7g was 12-fold potent than its stereo isomer compound 70 g against MCF-7. Since compound 7f was the most potent inhibitor of PC-3 proliferation (Table 2), we explored its mechanism of action in more detail. We first examined the viability of cells incubated with various concentrations of 7f for 48 h. As shown in Fig. 2, 7f at 3 lM caused a 56% reduction in viability, as measured by the MTT assay, compared with the untreated control cells. Under high power microscopy, cells treated with 7f also showed a significant change in morphology with abundant small vesicular structures (Fig. 3). To determine whether 7f induced autophagy in PC-3 cells, we examined the expression of beclin-1, LC3B-I, LC3B-II, and P62, which are involved in the production of autophagic vacuoles, as a sign for the initiation of autophagy.30,31 Moreover, the autophagy markers like beclin-1 and LC3-II32,33 were detected by western blot (Fig. 4). The protein of P62 was decreased in the concentration of 1 lM, whereas beclin-1 and LC3-II increased with increasing concentration. We next use the flow cytometry analysis of Annexin V and PI to stain cells, we found that the apoptotic kit assay was only able to detect late stages of cell death, however, just as cell death could not be detected prior to 3 lM, that is to say, the morphological changes could not be detected by apoptotic kit (Fig. 5). Surprisingly, the mitochondrial membrane potential was increased strongly. At the concentration of 4 lM, the mitochondrial membrane potential reduced in the decrease of 42.5% (Fig. 6). Finally,
Fig. 5. Apoptosis effect on PC-3 cell line induced by compound 7f. Apoptotic cells were detected with Annexin V/PI double staining after incubation with compound 7f. (0, 1.0, 2.0, 3.0, 4.0 lM) for 48 h. The lower left quadrants represent live cells, the lower right quadrants are for early/primary apoptotic cells, upper right quadrants are for late/ secondary apoptotic cells, while the upper left quadrants represent cells damaged during the procedure. The experiments were performed three times, and a representative experiment is shown.
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Fig. 6. Compound 7f decreased mitochondrial membrane potential. PC-3 cells were incubated with compound 7f at different concentrations for 24 h. The cells were stained with JC-1 and detection by flow cytometer.
we once again demonstrated the autophagy protein marker of LC3 by immunofluorescence (Fig. 7). We observed changes in the formation of autophagosomes involved in LC-3 by using confocal microscopy in time-dependence, clearly inspecting the increase of LC-3 in green with the extend of time until 24 h. According to the experimental results and analysis, it is indicated that 7f induces cell death by promoting autophagy in a low concentration. The proteins of beclin-1 and LC3-II raise significantly with increased concentrations, which was reported as autophagy markers. Beclin-1 acts as a surprise role in autophagy. When autophagy is blocked at an early stage by depletion of beclin-1 and other autophagy-related proteins, it is formidable to form autophagic vacuoles in the cells, which undergo a typical type I cell death.34 In addition, LC3 also has an essential effect for autophagy.35,36 As expected, both of the two are improved seriously in Fig. 4 and we also use immunofluorescence once again prove elevated protein of LC3. As shown in the results, it is perplexing that we could not detect cell death by flow cytometry at concentrations below 3 lM, while the mitochondrial membrane potential had changed significantly at low concentrations. Many literatures have shown apoptosis and autophagy are closely linked and can even achieve the purpose of interacting with some related proteins including beclin-1,37,38 p6239 and so on. Cho et al. recommend that ATG6/Beclin-1 is a novel caspase substrate in HeLa cells.40 Crighton et al. demonstrate a new p53-induced modulator of autophagy, whereas it is also useful for apoptosis.41 However, in the study, the cell death could not be detected by using apoptotic kit. In a sense, it is possible that 7f purely causes autophagy in PC-3 cells, with no involvement of apoptosis. Mitochondrial membrane potential had a great loss in PC-3 cells, Paglin et al. investigated that rapamycin-sensitive pathway regulates mitochondrial membrane potential and autophagy, but maybe other mitochondrial
changes.42 Others have suggested that changes in mitochondrial membrane potential can trigger autophagy. Just as our study, it is quite possible that mitochondrial transmembrane potential changes do contribute to the autophagic response in PC-3 cells. However, we still need further research on mechanisms. In summary, we have synthesized a series of jaspine B analogues that possess potent cytotoxicity activity against four tumor cell lines. Among these compounds, 7f showed the greatest inhibition of the against human prostate cancer cell line PC-3. Further mechanistic analyses showed that compound 7f inhibited the proliferation of PC-3, induced morphological changes, promoted the expression of autophagy-related proteins and decreased the mitochondrial membrane potential. However, these events were elicited by 7f at concentrations that did not induce apoptosis. Our results suggest that 7f is a novel autophagy-inducing compound. Further studies to understand the mechanism of action of 7f are currently underway.
Acknowledgments We are grateful for the financial support from the Natural Science Foundation of China (Nos. U1204206, 21702190 and No. U1404821), the Education Department of Henan Province (No. 17A350004) and the Scientific Innovation Talent Award from Department of Education of Henan Province (No. 15HASTIT036).
A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2017.12.011.
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Fig. 7. Immunofluorescence for autophagy related proteins. Immunofluorescence was performed for autophagy related protein LC3 (green), DAPI (blue) was utilized as a nuclear counterstain.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Kuroda I, Musman M, Ohtani II, et al. J Nat Prod. 2002;65:1505. Ledroit V, Debitus C, Lavaud C, Massiot G. Tetrahedron Lett. 2003;44:225. Kwon Y, Song J, Bae H, et al. Mar Drugs. 2015;13:824. Canals D, Mormeneo D, Fabrias G, Llebaria A, Casas J, Delgado A. Bioorg Med Chem. 2009;17:235. Genisson Y, Lamande L, Salma Y, Andrieu-Abadie N, Andre C, Baltas M. Tetrahedron-Asymmetry. 2007;18:857. Salma Y, Lafont E, Therville N, et al. Biochem Pharmacol. 2009;78:477. Jeon H, Bae H, Baek DJ, Kwak Y-S, Kim D, Kim S. Org Biomol Chem. 2011;9:7237. Salma Y, Ballereau S, Maaliki C, Ladeira S, Andrieu-Abadie N, Génisson Y. Org Biomol Chem. 2010;8:3227. Ghosal P, Ajay S, Meena S, Sinha S, Shaw AK. Tetrahedron-Asymmetry. 2013;24:903. Rives A, Ladeira S, Levade T, Andrieu-Abadie N, Genisson Y. J Org Chem. 2010;75:7920. Xu J-M, Zhang E, Shi X-J, et al. Eur J Med Chem. 2014;80:593. Yoo H, Lee YS, Lee S, Kim S, Kim T-Y. Phytother Res. 1927;2012:26. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Nature. 2008;1069:451. Levine B, Kroemer G. Cell. 2008;132:27. Amaravadi RK, Yu D, Lum JJ, et al. J Clin Invest. 2007;117:326. Mirzoeva OK, Hann B, Hom YK, et al. J Mol Med. 2011;89:877. Ma X-H, Piao S, Wang D, et al. Clin Cancer Res. 2011;17:3478. Rausch V, Liu L, Apel A, et al. J Pathol. 2012;227:325.
19. Yorimitsu T, Nair U, Yang Z, Klionsky DJ. J Biol Chem. 2006;281:30299. 20. Netea-Maier RT, Plantinga TS, van de Veerdonk FL, Smit JW, Netea MG. Autophagy. 2016;12:245. 21. García-Prat L, Martínez-Vicente M, Perdiguero E, et al. Nature. 2016;529:37. 22. Espina V, Wysolmerski J, Edmiston K, Liotta LA. Womens Health. 2013;9:157. 23. Klionsky DJ, Emr SD. Science. 2000;290:1717. 24. Zhang H, Chang JT, Guo B, et al. Autophagy. 2015;11:9. 25. Tang Z-H, Chen X, Wang Z-Y, et al. Sci Rep. 2016;6:26241. 26. Zhao M-L, Zhang E, Gao J, et al. Carbohydr Res. 2012;351:126. 27. Zhang E, Liu HM, Wang S, et al. Heterocycles. 2018;2016:92. 28. Zhang E, Jiao W-W, Wang S, et al. Med Chem Res. 2016;25:3011. 29. Zhang E, Wang S, Jiao W, et al. Chin J Org Chem. 2017;37:141. 30. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Cell Struct Funct. 1998;23:33. 31. Tanaka Y, Guhde G, Suter A, et al. Nature. 2000;406:902. 32. Høyer-Hansen M, Jäättelä M. Cell Death Differ. 2007;14:1576. 33. Rami A, Kögel D. Autophagy. 2008;4:422. 34. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Nat Rev Mol Cell Biol. 2007;8:741. 35. Huang R, Liu W. Autophagy. 2015;11:852. 36. Huang R, Xu Y, Wan W, et al. Mol Cell. 2015;57:456. 37. Levine B, Sinha SC, Kroemer G. Autophagy. 2008;4:600. 38. Kang R, Zeh H, Lotze M, Tang D. Cell Death Differ. 2011;18:571. 39. Moscat J, Diaz-Meco MT. Cell. 2009;1001:137. 40. Cho D-H, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC. Cancer Lett. 2009;274:95. 41. Crighton D, Wilkinson S, O’Prey J, et al. Cell. 2006;126:121. 42. Paglin S, Lee N-Y, Nakar C, et al. Cancer Res. 2005;65:11061.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel jaspine B analogues as autophagy inducer En Zhang a,b,⇑, Shang Wang a, Li-Li Li a, Yong-Gang Hua a, Jing-Fei Yue a, Jin-Feng Li c, Cheng-Yun Jin a,b,⇑ a School of Pharmaceutical Sciences, Institute of Drug Discovery and Development, Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, PR China b Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China c Kidney Transplantation, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe Road, Zhengzhou 450001, PR China
a r t i c l e
i n f o
Article history: Received 12 September 2017 Revised 22 November 2017 Accepted 5 December 2017 Available online 8 December 2017 Keywords: Jaspine B Pachastrissamine Anti-tumor Anti-proliferative Autophagy
a b s t r a c t A series of 2-alkylaminomethyl jaspine B analogues were synthesized and evaluated for their cytotoxic effects on human lung adenocarcinoma, breast cancer, and prostate cancer cell lines and a mouse melanoma cell line. Most of the compounds exhibited moderate to good activity against the cancer cell lines. Compound 7f showed the best overall cytotoxicity on PC-3 cells (IC50 = 0.85 lM). Further mechanistic studies revealed that compound 7f induced marked changes in PC-3 cell morphology, disrupted the mitochondrial membrane potential, and increased expression of the autophagy proteins beclin-1, LC3, and P62. Ó 2017 Elsevier Ltd. All rights reserved.
Jaspine B (Fig. 1, 1), which was originally isolated from the marine sponge Pachastrissa sp.1and Jaspis sp.2, and is an anhydrophytosphingosine derivative with three chiral centers. Jaspine B inhibits the growth of a range of tumor cell lines in vitro3, and the anti-tumor activity is critically dependent on the stereo configuration4, alkyl chain length5, amide moiety6, and the tetrahydrofuran ring.7 Based on these characteristics, many researchers have synthesized jaspine B8,9 and its derivatives through various synthetic routes. Rives et al.10 developed an approach to generate pyrrolidine-based analogues of jaspine B (Fig. 1, 2) and found that their cytotoxicity towards melanoma cells was comparable to that of jaspine B. Our group synthesized a series of novel jaspine B analogues (Fig. 1, 3) bearing a 1,2,3-triazole functional group11, one of which had more potent anti-proliferative activity against EC-9706, MGC-803 and MCF-7 than either 5-Fu or jaspine B. Kwon et al. prepared a carbocyclic analogue of jaspine B (Fig. 1, 4) that showed comparable cytotoxicity to the parent compound but was a more potent inhibitory of sphingosine kinases.3 Several studies have examined the mechanism of cytotoxicity of jaspine B, Canals et al.4 suggested that jaspine B might be involved ⇑ Corresponding authors at: School of Pharmaceutical Sciences, Institute of Drug Discovery and Development, Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, PR China. E-mail addresses: [email protected] (E. Zhang), [email protected] (C.-Y. Jin). https://doi.org/10.1016/j.bmcl.2017.12.011 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
in dihydroceramide-induced autophagy in A549 cells. Yoo et al.12 reported that jaspine B-mediated inhibition of melanoma cell growth may occur via dual inhibition of extracellular regulatory kinase signaling and cyclin-dependent kinase 2 pathways. Despite these findings, the anti-tumor mechanisms of jaspine B and its analogues remain unclear. Cell survival and death are both strongly influenced by autophagy,13 the process through which aberrant or dysfunctional cellular components are removed by lysosomal degradation and recycling. Three main classes of autophagy have been described; namely, chaperone-mediated autophagy, microautophagy and macroautophagy, which differ in their physiological functions, substrate types, and manner of substrate delivery to lysosomes.14 Many reports indicate that autophagy have a good inhibitory effect for various cancers.15–17 Autophagy is required for normal cell homeostasis and is activated by exposure to a variety of stressors, including hypoxia,18 endoplasmic reticulum stress,19 starvation, toxins, and inflammation,20 cancer and senescence.21 Autophagy is a dynamic process in which intracellular membranes undergo dramatic morphological changes. When autophagy-induced signaling initiates, the cells form a small liposome-like membrane somewhere in the cytoplasm, which then expands, known as phagophore. Phagophore continues to extend the cytoplasm of any component, including the organelles, into which all embrace, then shut up and becomes a closed spherical autophagic bodies. Autophagosomes formed with endocytosis of phagosome
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Fig. 1. Jaspine B and its representative derivatives.
Scheme 1. Reagent and conditions: (a) R-NH2, NaBH(OAc)3, 1,2-dichloroethane, room temperature; b) Pd/C, H2, CH3OH, 60 °C, 60 psi.
Scheme 2. Reagent and conditions: (a) R-NH2, NaBH4, CH3OH, room temperature; b) Pd/C, H2, CH3OH, 60 °C, 60 psi.
Table 1 Reaction conditions for reductive amination of aldehydes. Material
Solvent
Reductant
Yield/%
5 5 50 50
CH3OH 1,2-dichloroethane CH3OH 1,2-dichloroethane
NaBH4 NaBH(OAc)3 NaBH4 NaBH(OAc)3
27 84 89 45
pinocytosis, endocytic vesicles and endosomes integration. The autophagosome fuses with the lysosome to form autophagosomes, during which the endomembrane of the autophagosome is degraded by lysosomal enzymes, including integrating the contents of both, and the cargo in the autophagosome is degraded then the product is transported to cytoplasm.22–24 Because excessive autophagy can promote cell death, autophagy-inducing compounds may be useful anti-cancer drugs. Previous studies have shown that such compounds can induce cell death by various mechanisms.25 Here, we have extended our previous efforts to develop jaspine B derivatives with anti-cancer activity.11,26–29 We synthesized a series of jaspine B analogues bearing a 2-alkylaminomethyl group and tested the anti-tumor activity on four tumor cell lines. We identify one of the compounds as a new autophagy inducer that cause loss of mitochondrial permeability, increases the expression of the autophagy proteins LC-3, beclin-1, and P62, and, at high concentrations, results in tumor cell death. Jaspine B analogues were synthesized using as starting material compounds 5 or 50 , which were generated according to our previously developed method 11, 26. The first step reaction was a reductive amination of aldehyde, using sodium triacetoxyborohydride as the reducing agent (Scheme 1). In some cases, the reactions between aldehydes and primary amines generated considerable amounts of dialkylation and other side products. Thus, a stepwise procedure involving imine formation in methanol followed by reduction with sodium borohydride was carried out for some compound, as shown in Scheme 2. The stepwise procedure did not apply to the synthesis of compound 6a-6g. The various reaction conditions were summarized in Table 1.
Table 2 Anti-proliferative activity of jaspine B analogues against four cancer cell lines. Com.
7a 7b 7c 7d 7e 7f 7g 70 a 70 b 70 c 70 d 70 e 70 f 70 g 5-Fu 1
n
6 7 8 9 10 11 13 6 7 8 9 10 11 13
IC50(lM)a A549
B16-F10
MCF-7
PC-3
>128 >128 22.07 ± 2.92 47.84 ± 4.09 11.1 ± 1.761 5.87 ± 0.82 2.85 ± 0.08 >128 71.76 ± 5.38 >128 >128 9.65 ± 1.54 41.10 ± 3.63 19.95 ± 2.48 0.43 ± 0.01 1.87b
>128 >128 55.18 ± 4.46 29.62 ± 3.33 19.31 ± 2.57 10.70 ± 1.68 14.31 ± 2.04 >128 >128 >128 >128 88.82 ± 4.90 58.11 ± 4.13 64.25 ± 4.03 6.29 ± 0.66 0.5c
>128 96.56 ± 5.62 12.46 ± 1.96 34.20 ± 3.55 6.77 ± 1.01 1.80 ± 0.08 0.72 ± 0.18 >128 >128 60.34 ± 4.62 42.71 ± 3.91 13.13 ± 2.01 12.63 ± 1.92 8.83 ± 1.40 6.11 ± 0.01 2.33b
22.36 ± 3.09 13.75 ± 2.15 2.03 ± 0.10 11.86 ± 1.86 3.33 ± 0.07 0.85 ± 0.19 1.20 ± 0.12 >128 12.96 ± 2.06 7.65 ± 1.14 >128 10.01 ± 1.59 2.38 ± 0.04 1.99 ± 0.05 0.53 ± 0.11 0.7d
a Inhibitory activity is expressed as the concentration required to inhibit tumor cell proliferation by 50% after exposure for 72 h (IC50). n, alkyl chain length. Data are presented as the means ± SDs of three independent experiments. b Literature values 9. c Literature values 8. d Literature values 3.
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Fig. 2. (A) Cell viability of PC-3 cells after treatment with various concentrations for 48 h; (B) Quantitative analysis of the ratio of the cell viability.
Fig. 3. Morphological changes of PC-3 cells induced by compound 7f.
After reductive amination of the aldehyde, the azide functionality was reduced and the benzyl-protecting group was removed to give the target compound 7a-7g in a one-pot reaction using palladium-catalyzed hydrogenolysis. The method was also applied to synthesize compounds 70 a-70 g. The structures of all newly synthesized compounds were fully characterized by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry. The compounds were then evaluated for their anti-tumor activities against four cancer cell lines: A549 (human lung adenocarcinoma), B16-F10 (mouse melanoma), MCF-7 (human breast cancer) and PC-3 (human prostate cancer) by using the colorimet-
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E. Zhang et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 497–502
Fig. 4. Expression changes of autophagy related proteins induced by compound 7f.
ric MTT viability/proliferation assay. 5-Fluorouracil (5-Fu) was included as a positive control compound. The results of the MTT assay, expressed as the concentration resulting in 50% inhibition of maximal proliferation (IC50), are shown in Table 2. The analogues had much better activity against MCF-7 and PC-3 than against A549 and B16-F10. Notably, compounds containing fewer than 10 alkyl carbon showed little or no anti-proliferative activity (7a-7d). while better activity was observed in compounds with 11 or 13 alkyl carbons (7f, 7 g). However, the alkyl carbon number was not directly proportional to
activity; for example, compound 7f had better activity than compound 7 g against B16-F10 and PC-3 cells despite containing fewer alkyl carbons. This suggested that an intermediate alkyl chain length was optimal. Similar results were observed with the 3-epijaspine B derivatives 70 a-70 g. In addition, the stereo configuration of these compounds played an important role in their anti-proliferative activity. For example, compound 7g was 12-fold potent than its stereo isomer compound 70 g against MCF-7. Since compound 7f was the most potent inhibitor of PC-3 proliferation (Table 2), we explored its mechanism of action in more detail. We first examined the viability of cells incubated with various concentrations of 7f for 48 h. As shown in Fig. 2, 7f at 3 lM caused a 56% reduction in viability, as measured by the MTT assay, compared with the untreated control cells. Under high power microscopy, cells treated with 7f also showed a significant change in morphology with abundant small vesicular structures (Fig. 3). To determine whether 7f induced autophagy in PC-3 cells, we examined the expression of beclin-1, LC3B-I, LC3B-II, and P62, which are involved in the production of autophagic vacuoles, as a sign for the initiation of autophagy.30,31 Moreover, the autophagy markers like beclin-1 and LC3-II32,33 were detected by western blot (Fig. 4). The protein of P62 was decreased in the concentration of 1 lM, whereas beclin-1 and LC3-II increased with increasing concentration. We next use the flow cytometry analysis of Annexin V and PI to stain cells, we found that the apoptotic kit assay was only able to detect late stages of cell death, however, just as cell death could not be detected prior to 3 lM, that is to say, the morphological changes could not be detected by apoptotic kit (Fig. 5). Surprisingly, the mitochondrial membrane potential was increased strongly. At the concentration of 4 lM, the mitochondrial membrane potential reduced in the decrease of 42.5% (Fig. 6). Finally,
Fig. 5. Apoptosis effect on PC-3 cell line induced by compound 7f. Apoptotic cells were detected with Annexin V/PI double staining after incubation with compound 7f. (0, 1.0, 2.0, 3.0, 4.0 lM) for 48 h. The lower left quadrants represent live cells, the lower right quadrants are for early/primary apoptotic cells, upper right quadrants are for late/ secondary apoptotic cells, while the upper left quadrants represent cells damaged during the procedure. The experiments were performed three times, and a representative experiment is shown.
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Fig. 6. Compound 7f decreased mitochondrial membrane potential. PC-3 cells were incubated with compound 7f at different concentrations for 24 h. The cells were stained with JC-1 and detection by flow cytometer.
we once again demonstrated the autophagy protein marker of LC3 by immunofluorescence (Fig. 7). We observed changes in the formation of autophagosomes involved in LC-3 by using confocal microscopy in time-dependence, clearly inspecting the increase of LC-3 in green with the extend of time until 24 h. According to the experimental results and analysis, it is indicated that 7f induces cell death by promoting autophagy in a low concentration. The proteins of beclin-1 and LC3-II raise significantly with increased concentrations, which was reported as autophagy markers. Beclin-1 acts as a surprise role in autophagy. When autophagy is blocked at an early stage by depletion of beclin-1 and other autophagy-related proteins, it is formidable to form autophagic vacuoles in the cells, which undergo a typical type I cell death.34 In addition, LC3 also has an essential effect for autophagy.35,36 As expected, both of the two are improved seriously in Fig. 4 and we also use immunofluorescence once again prove elevated protein of LC3. As shown in the results, it is perplexing that we could not detect cell death by flow cytometry at concentrations below 3 lM, while the mitochondrial membrane potential had changed significantly at low concentrations. Many literatures have shown apoptosis and autophagy are closely linked and can even achieve the purpose of interacting with some related proteins including beclin-1,37,38 p6239 and so on. Cho et al. recommend that ATG6/Beclin-1 is a novel caspase substrate in HeLa cells.40 Crighton et al. demonstrate a new p53-induced modulator of autophagy, whereas it is also useful for apoptosis.41 However, in the study, the cell death could not be detected by using apoptotic kit. In a sense, it is possible that 7f purely causes autophagy in PC-3 cells, with no involvement of apoptosis. Mitochondrial membrane potential had a great loss in PC-3 cells, Paglin et al. investigated that rapamycin-sensitive pathway regulates mitochondrial membrane potential and autophagy, but maybe other mitochondrial
changes.42 Others have suggested that changes in mitochondrial membrane potential can trigger autophagy. Just as our study, it is quite possible that mitochondrial transmembrane potential changes do contribute to the autophagic response in PC-3 cells. However, we still need further research on mechanisms. In summary, we have synthesized a series of jaspine B analogues that possess potent cytotoxicity activity against four tumor cell lines. Among these compounds, 7f showed the greatest inhibition of the against human prostate cancer cell line PC-3. Further mechanistic analyses showed that compound 7f inhibited the proliferation of PC-3, induced morphological changes, promoted the expression of autophagy-related proteins and decreased the mitochondrial membrane potential. However, these events were elicited by 7f at concentrations that did not induce apoptosis. Our results suggest that 7f is a novel autophagy-inducing compound. Further studies to understand the mechanism of action of 7f are currently underway.
Acknowledgments We are grateful for the financial support from the Natural Science Foundation of China (Nos. U1204206, 21702190 and No. U1404821), the Education Department of Henan Province (No. 17A350004) and the Scientific Innovation Talent Award from Department of Education of Henan Province (No. 15HASTIT036).
A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmcl.2017.12.011.
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Fig. 7. Immunofluorescence for autophagy related proteins. Immunofluorescence was performed for autophagy related protein LC3 (green), DAPI (blue) was utilized as a nuclear counterstain.
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