Bioorganic & Medicinal Chemistry 23 (2015) 4884–4890
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioevaluation of heterocyclic derivatives of Cleistanthin-A Yu Zhao, Rui Zhang, Yapeng Lu, Jinlong Ma, Li Zhu ⇑ Institute of Nautical Medicine, Nantong University, Nantong 226001, China
a r t i c l e
i n f o
Article history: Received 31 March 2015 Revised 13 May 2015 Accepted 15 May 2015 Available online 27 May 2015 Keywords: Cleistanthin-A Synthesis Vacuolar H+-ATPase Inhibitor
a b s t r a c t The vacuolar H+-ATPase (V-ATPase) was proposed as a key target for new strategies in cancer treatment recently. We have synthesized a novel class of derivatives of Cleistanthin-A bearing heterocyclic moieties. Most of these compounds displayed potent antiproliferative effects on four cancer cells at submicromolar concentration, and they have no cytotoxicity on normal WRL-68 cells at 200 nM. The most potent compound 3a has been shown to inhibit the activity of vacuolar H+-ATPase at submicromolar concentration, and it could also significantly decrease the cytosolic pH values in HepG2 cells. The current findings provide valuable insights for future development of novel V-ATPase inhibitors as anticancer agents. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Vacuolar H+-ATPases (V-ATPases) are a class of ATP-driven multisubunit proton pumps found in all eukaryotic cells.1 They play a major role in the regulation of cellular pH conditions and microenvironment acidification, which is critical to managing cancer development, progression and metastasis.2,3 V-ATPases were proposed as key targets for new strategies in cancer treatment.4–8 Therefore, inhibitors of V-ATPases could be promising novel therapeutics for cancer treatment. Most of the known V-ATPases inhibitors are natural compounds of microbial origin such as salicylihalamides and bafilomycin, and they showed potent cell death inducing activity.9,10 Recently, a natural arylnaphthalene lignan lactone, diphyllin, has been noted to potently inhibit V-ATPase and thereby lysosomal acidification in osteoclasts.11 Moreover, it was found that some diphyllin glycosides possessed strong inhibitory effects on cancer cell proliferation.12,13 We have successfully synthesized a natural cytotoxic diphyllin glycoside CleistanthinA, which is indentified as a potent V-ATPase inhibitor (Fig. 1).14 It is hoped that by modifying the structure of Cleistanthin-A more effective V-ATPase inhibitor could be developed. Therefore, we design and synthesize a series of derivatives of Cleistanthin-A by introducing glycosyl and long alkyl chains to its C00 -2 hydroxyl group.15 It is proved that substitution with long alkyl chains substituents on C00 -2 hydroxyl group of Cleistanthin-A does not cause significant loss of its antiproliferative activity. The long alkyl chains ⇑ Corresponding author. Tel./fax: +86 513 55003376. E-mail address: [email protected] (L. Zhu). http://dx.doi.org/10.1016/j.bmc.2015.05.033 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
could play a key role in regulating the octanol/water partition coefficient, improving chemical behavior and enhancing activities of the molecules. These new derivatives displayed potent antiproliferative activity and V-ATPase inhibitory activity, which inspired us to explore more potent V-ATPase inhibitors. Further preclinical development of these derivatives has encountered certain limitations such as poor water solubility and high lipophilicity, which could cause poor pharmacokinetic properties. Therefore, we intend to synthesize a new class of Cleistanthin-A derivatives by adding various heterocycles to the C00 -2 hydroxyl group of Cleistanthin-A through long alkyl chains with the aim to increase their water solubility and improve their release rates to acidic tumor position (Scheme 1). In this paper, we report the synthesis of these derivatives, their antiproliferative activity and the V-ATPase inhibitory activity. These compounds prepared in order to understand the effect of side basic chains substitution on the C00 -2 hydroxyl group of xylosyl moiety. Most of these compounds displayed potent antiproliferative effects, and the most potent compound 3a has been shown to inhibit the activity of vacuolar H+-ATPase at submicromolar concentration. 2. Results and discussion 2.1. Chemistry Previously, we have reported the synthesis of Cleistanthin-A from diphyllin and 2-acetyl-3,4-dimethoxy xylopyranosyl bromide in good yield.10 Therefore, we used the synthesized Cleistanthin-A as staring material in this study. Etherification reactions of
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Y. Zhao et al. / Bioorg. Med. Chem. 23 (2015) 4884–4890 Table 1 In vitro anti-proliferative activity of compounds 3a–3n Compd
Figure 1. Cleistanthin-A and 3a.
Cleistanthin-A with respective dibromoalkanes in the presence of NaOH in DMSO were performed to afford 2a, 2b and 2c in 68– 76% yields. The basic side chains in the C-2 position of the xylosyl moiety were generally added by substitution with nitrogen heterocyclic rings to product target compounds 3a–3n in 70–84% yield. (Scheme 1) 2.2. Pharmacological studies With fourteen Cleistanthin-A derivatives in hand, four cancer cell lines from various solid tumors, HCT-116, HepG2, A549, Hela and a normal cell WRL68, were used to preliminarily screen their antiproliferation activity employing a MTT assay (Table 1). Most of these compounds displayed antiproliferative effects on four cancer cells at submicromolar concentration, but they were less potent than Cleistanthin-A. In these targeted compounds, 3a possessed the best antiproliferative activity on HepG2, HCT-116, A549 and Hela cancer cell lines with IC50 value 20–30 nM. These Compounds showed selectivity for HCT-116 cell line, the inhibitory activity was obviously higher than other three cell lines. Neither the alkyl chains nor heterocyclics has apparent effects on their antiproliferative activity against HCT-116 cell line. But in other three cell lines, variation in the alkyl chain length had practically significant effects on their antiproliferative activity. The shorter chain compounds possess better antiproliferative activity than that
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n Cleistanthin-A
IC50 (nM) HepG2
HCT-116
A549
Hela
WRL-68
30 33 39 30 34 43 >200 41 >200 26 25 39 >200 52 8
21 23 32 22 23 28 29 27 73 25 27 11 12 15 1
26 19 36 23 26 23 >200 23 >200 26 29 25 26 >200 4
20 25 32 32 36 28 >200 51 >200 45 55 91 100 >200 19
>200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200
of longer chain compounds. The shortest series of derivatives 3a– 3e possessed the best antiproliferation activity. We deduced that the longer basic chains could hinder the binding of glycosyl lignan to the target enzyme. Moreover, these compounds showed no cytotoxic effects on normal WRL-68 cell line. Further MTT assay indicated that 3a displayed potent inhibitory effects on cell viability of HepG2 cells (Fig. 2). After treatment with different concentration of 3a (15 nM, 30 nM and 60 nM) for 24, 48, 72 h, HepG2 cell viability was decreased significantly. Based on the above MTT results, 3e was used for V-ATPase activity assay to determine its effect on V-ATPase activity by using diphyllin as positive control (Fig. 3). Firstly, the lysosome acidity was examined in HepG2 cells after incubation with 1 lM diphyllin and various concentrations of 3a (0, 15 nM, 30 nM, 60 nM) (Fig. 3A). HepG2 cells were stained with a pH-sensitive fluorescence dye LysoTracker Red and sequentially loaded with diphyllin and 3a. It was found that the amount of red fluorescence decreased in cells treated with diphyllin and 3a compared with untreated cells. The V-ATPase activity was completely suppressed after
Scheme 1. Synthesis of Cleistanthin-A derivatives.
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Y. Zhao et al. / Bioorg. Med. Chem. 23 (2015) 4884–4890
Figure 2. Cell viability was determined using the MTT assay. HepG2 cells were treated for 24, 48, 72 h with different concentrations of 3a (0, 15, 30, 60 nM).
treatment of 60 nM 3a or 1 lM diphyllin, which indicated 3a was more potent than diphyllin. Introducing side basic chains did not cause significant loss of the inhibitory of V-ATPase activity of Cleistanthin-A.
Nextly, the human liver hepatocellular carcinoma (HepG2) was treated with diphyllin and 3a in a range of concentrations (0, 15 nM, 30 nM, 60 nM) and then, the activity of V-ATPase was examined by V-ATPase activity assay kit. As shown in Fig. 3B, treatment of HepG2 with 3a at 30 nM and 60 nM significantly decreased the percentage of activity of V-ATPase to 62% and 56%, respectively, which were better than that of the control drug diphyllin. These results implied that the modulation of lysosomes acidity by 3a was consistent with its inhibitory effect on VATPase activity. Finally, we measured the changes in the cytosolic pH following treatment with 3a in HepG2 cells. The cytosolic pH values were decreased gradually with the increase of concentration of 3a (Fig. 3C). The inhibition of V-ATPase activity in HepG2 cells was responsible for the acidification of cytoplasm. 3. Conclusion To explore novel structural V-ATPase inhibitors and understand their anticancer mechanisms, we have designed and synthesized a
Figure 3. Effects of compound 3a on V-ATPase activity and pH of HepG2 cells. (A) Cells were treated with increasing doses of 3a for 48 h, and the pH of lysosomal was analyzed by confocal microscopy. (B) The activity of V-ATPase in HepG2 cells was decreased after treated with 3a (0, 15, 30, 60 nM) for 48 h (⁄P
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioevaluation of heterocyclic derivatives of Cleistanthin-A Yu Zhao, Rui Zhang, Yapeng Lu, Jinlong Ma, Li Zhu ⇑ Institute of Nautical Medicine, Nantong University, Nantong 226001, China
a r t i c l e
i n f o
Article history: Received 31 March 2015 Revised 13 May 2015 Accepted 15 May 2015 Available online 27 May 2015 Keywords: Cleistanthin-A Synthesis Vacuolar H+-ATPase Inhibitor
a b s t r a c t The vacuolar H+-ATPase (V-ATPase) was proposed as a key target for new strategies in cancer treatment recently. We have synthesized a novel class of derivatives of Cleistanthin-A bearing heterocyclic moieties. Most of these compounds displayed potent antiproliferative effects on four cancer cells at submicromolar concentration, and they have no cytotoxicity on normal WRL-68 cells at 200 nM. The most potent compound 3a has been shown to inhibit the activity of vacuolar H+-ATPase at submicromolar concentration, and it could also significantly decrease the cytosolic pH values in HepG2 cells. The current findings provide valuable insights for future development of novel V-ATPase inhibitors as anticancer agents. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Vacuolar H+-ATPases (V-ATPases) are a class of ATP-driven multisubunit proton pumps found in all eukaryotic cells.1 They play a major role in the regulation of cellular pH conditions and microenvironment acidification, which is critical to managing cancer development, progression and metastasis.2,3 V-ATPases were proposed as key targets for new strategies in cancer treatment.4–8 Therefore, inhibitors of V-ATPases could be promising novel therapeutics for cancer treatment. Most of the known V-ATPases inhibitors are natural compounds of microbial origin such as salicylihalamides and bafilomycin, and they showed potent cell death inducing activity.9,10 Recently, a natural arylnaphthalene lignan lactone, diphyllin, has been noted to potently inhibit V-ATPase and thereby lysosomal acidification in osteoclasts.11 Moreover, it was found that some diphyllin glycosides possessed strong inhibitory effects on cancer cell proliferation.12,13 We have successfully synthesized a natural cytotoxic diphyllin glycoside CleistanthinA, which is indentified as a potent V-ATPase inhibitor (Fig. 1).14 It is hoped that by modifying the structure of Cleistanthin-A more effective V-ATPase inhibitor could be developed. Therefore, we design and synthesize a series of derivatives of Cleistanthin-A by introducing glycosyl and long alkyl chains to its C00 -2 hydroxyl group.15 It is proved that substitution with long alkyl chains substituents on C00 -2 hydroxyl group of Cleistanthin-A does not cause significant loss of its antiproliferative activity. The long alkyl chains ⇑ Corresponding author. Tel./fax: +86 513 55003376. E-mail address: [email protected] (L. Zhu). http://dx.doi.org/10.1016/j.bmc.2015.05.033 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
could play a key role in regulating the octanol/water partition coefficient, improving chemical behavior and enhancing activities of the molecules. These new derivatives displayed potent antiproliferative activity and V-ATPase inhibitory activity, which inspired us to explore more potent V-ATPase inhibitors. Further preclinical development of these derivatives has encountered certain limitations such as poor water solubility and high lipophilicity, which could cause poor pharmacokinetic properties. Therefore, we intend to synthesize a new class of Cleistanthin-A derivatives by adding various heterocycles to the C00 -2 hydroxyl group of Cleistanthin-A through long alkyl chains with the aim to increase their water solubility and improve their release rates to acidic tumor position (Scheme 1). In this paper, we report the synthesis of these derivatives, their antiproliferative activity and the V-ATPase inhibitory activity. These compounds prepared in order to understand the effect of side basic chains substitution on the C00 -2 hydroxyl group of xylosyl moiety. Most of these compounds displayed potent antiproliferative effects, and the most potent compound 3a has been shown to inhibit the activity of vacuolar H+-ATPase at submicromolar concentration. 2. Results and discussion 2.1. Chemistry Previously, we have reported the synthesis of Cleistanthin-A from diphyllin and 2-acetyl-3,4-dimethoxy xylopyranosyl bromide in good yield.10 Therefore, we used the synthesized Cleistanthin-A as staring material in this study. Etherification reactions of
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Y. Zhao et al. / Bioorg. Med. Chem. 23 (2015) 4884–4890 Table 1 In vitro anti-proliferative activity of compounds 3a–3n Compd
Figure 1. Cleistanthin-A and 3a.
Cleistanthin-A with respective dibromoalkanes in the presence of NaOH in DMSO were performed to afford 2a, 2b and 2c in 68– 76% yields. The basic side chains in the C-2 position of the xylosyl moiety were generally added by substitution with nitrogen heterocyclic rings to product target compounds 3a–3n in 70–84% yield. (Scheme 1) 2.2. Pharmacological studies With fourteen Cleistanthin-A derivatives in hand, four cancer cell lines from various solid tumors, HCT-116, HepG2, A549, Hela and a normal cell WRL68, were used to preliminarily screen their antiproliferation activity employing a MTT assay (Table 1). Most of these compounds displayed antiproliferative effects on four cancer cells at submicromolar concentration, but they were less potent than Cleistanthin-A. In these targeted compounds, 3a possessed the best antiproliferative activity on HepG2, HCT-116, A549 and Hela cancer cell lines with IC50 value 20–30 nM. These Compounds showed selectivity for HCT-116 cell line, the inhibitory activity was obviously higher than other three cell lines. Neither the alkyl chains nor heterocyclics has apparent effects on their antiproliferative activity against HCT-116 cell line. But in other three cell lines, variation in the alkyl chain length had practically significant effects on their antiproliferative activity. The shorter chain compounds possess better antiproliferative activity than that
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n Cleistanthin-A
IC50 (nM) HepG2
HCT-116
A549
Hela
WRL-68
30 33 39 30 34 43 >200 41 >200 26 25 39 >200 52 8
21 23 32 22 23 28 29 27 73 25 27 11 12 15 1
26 19 36 23 26 23 >200 23 >200 26 29 25 26 >200 4
20 25 32 32 36 28 >200 51 >200 45 55 91 100 >200 19
>200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200
of longer chain compounds. The shortest series of derivatives 3a– 3e possessed the best antiproliferation activity. We deduced that the longer basic chains could hinder the binding of glycosyl lignan to the target enzyme. Moreover, these compounds showed no cytotoxic effects on normal WRL-68 cell line. Further MTT assay indicated that 3a displayed potent inhibitory effects on cell viability of HepG2 cells (Fig. 2). After treatment with different concentration of 3a (15 nM, 30 nM and 60 nM) for 24, 48, 72 h, HepG2 cell viability was decreased significantly. Based on the above MTT results, 3e was used for V-ATPase activity assay to determine its effect on V-ATPase activity by using diphyllin as positive control (Fig. 3). Firstly, the lysosome acidity was examined in HepG2 cells after incubation with 1 lM diphyllin and various concentrations of 3a (0, 15 nM, 30 nM, 60 nM) (Fig. 3A). HepG2 cells were stained with a pH-sensitive fluorescence dye LysoTracker Red and sequentially loaded with diphyllin and 3a. It was found that the amount of red fluorescence decreased in cells treated with diphyllin and 3a compared with untreated cells. The V-ATPase activity was completely suppressed after
Scheme 1. Synthesis of Cleistanthin-A derivatives.
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Y. Zhao et al. / Bioorg. Med. Chem. 23 (2015) 4884–4890
Figure 2. Cell viability was determined using the MTT assay. HepG2 cells were treated for 24, 48, 72 h with different concentrations of 3a (0, 15, 30, 60 nM).
treatment of 60 nM 3a or 1 lM diphyllin, which indicated 3a was more potent than diphyllin. Introducing side basic chains did not cause significant loss of the inhibitory of V-ATPase activity of Cleistanthin-A.
Nextly, the human liver hepatocellular carcinoma (HepG2) was treated with diphyllin and 3a in a range of concentrations (0, 15 nM, 30 nM, 60 nM) and then, the activity of V-ATPase was examined by V-ATPase activity assay kit. As shown in Fig. 3B, treatment of HepG2 with 3a at 30 nM and 60 nM significantly decreased the percentage of activity of V-ATPase to 62% and 56%, respectively, which were better than that of the control drug diphyllin. These results implied that the modulation of lysosomes acidity by 3a was consistent with its inhibitory effect on VATPase activity. Finally, we measured the changes in the cytosolic pH following treatment with 3a in HepG2 cells. The cytosolic pH values were decreased gradually with the increase of concentration of 3a (Fig. 3C). The inhibition of V-ATPase activity in HepG2 cells was responsible for the acidification of cytoplasm. 3. Conclusion To explore novel structural V-ATPase inhibitors and understand their anticancer mechanisms, we have designed and synthesized a
Figure 3. Effects of compound 3a on V-ATPase activity and pH of HepG2 cells. (A) Cells were treated with increasing doses of 3a for 48 h, and the pH of lysosomal was analyzed by confocal microscopy. (B) The activity of V-ATPase in HepG2 cells was decreased after treated with 3a (0, 15, 30, 60 nM) for 48 h (⁄P
