Synthesis and biological evaluation of novel 1-aryl, 5-(phenoxy-substituted)aryl-1,4-pentadien-3-one derivatives

Kai Yuan; Baoan Song; Linhong Jin; Shuai Xu; Deyu Hu; Xiaoqing Xu; Song Yang

doi:10.1039/C1MD00038A

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DOI: 10.1039/C1MD00038A (Concise Article) Med. Chem. Commun., 2011, 2, 585-589

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Synthesis and biological evaluation of novel 1-aryl, 5-(phenoxy-substituted)aryl-1,4-pentadien-3-one derivatives†

Kai Yuan , Baoan Song *, Linhong Jin , Shuai Xu , Deyu Hu , Xiaoqing Xu and Song Yang *
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang, 550025, China. E-mail: songbaoan22@yahoo.com

Received 7th February 2011 , Accepted 25th March 2011

First published on 14th April 2011

Abstract

A series of 1-aryl, 5-(phenoxy-substituted)aryl-1,4-pentadien-3-one derivatives were synthesized and evaluated for anticancer activity. Amongst the synthesized ethers, 4A and 4Y exhibited substantial antiproliferative activity with IC₅₀ values ranging from 6.6–8.6 μmol L⁻¹ against a variety of human cancer cell lines. Preliminarily mechanism of antitumor action of 4A by DNA Ladder, Annexin V/PI double staining and related studies indicated growth inhibition of PC3, BGC-823 and Bcap-37 cells by induction of tumor cells apoptosis.

Introduction

Curcumin is the principal curcuminoid of the popular Indian spice turmeric, which is a member of the ginger family. Curcumin and its derivatives have extensive bioactivities such as bactericidal,¹ antioxidative,² anti-inflammatary³ and anti-HIV⁴ (Fig. 1). They are particularly known for their ability to resist mutation and raise anticancer activity.⁵ The research in this area indicates that curcumin derivatives can induce apoptosis, e.g. carcinoma of colorectal, renal and hepatocarcinoma cancer cells.⁶ Great interest has been devoted to the synthesis of new curcumin analogues exhibiting enhanced biological properties, e.g. the novel ferrocenyl curcuminoids were synthesized by covalent anchorage of three different ferrocenyl ligands. They were more effective in inhibiting B16 melanoma cells proliferation in vitro (Fig. 1).⁷ Structurally, the new analogues are symmetrical 1,5-diarylpentadienones whose aromatic rings possess alkoxy substituents at 3 and 5 positions. Analysis of the effects of the analogues on the expression of cancer-related genes indicated that some of them could induce down-regulation of β-catenin, Ki-ras, cyclin D1, c-Myc, and ErbB-2 at as low as one eighth of the concentration at which curcumin normally exerts an effect.⁸ Shibata's group reported some newly synthesized curcumin analogs containing methoxymethyl group (compound A) having improved potential to prevent colorectal carcinogenesis in vivo,⁹ whereas Hiroyuki's group obtained similar class of compounds by studying structure–activity relationship of C5-curcuminoids Ai-ii.¹⁰


	Fig. 1 Organic curcumin derivatives and the target compounds 4A and 4Y.

In an attempt to obtain more potent compounds without disturbing the core structure of curcumin to keep the medicinal properties and safety profile intact, minute structural variations were made by omitting the active methylene group and one carbonyl group leading to 1,4-pentadiene-3-ones to yield new structures which still displayed antioxidative properties. A series of diphenyl-1,4-pentadiene-3-ones, together with cyclopentanone and cyclohexanone analogues were prepared and tested for inhibition of lipid peroxidation by Sardjiman's group.¹¹ Similarly, Adams's group have reported 3,5-bis(2-flurobenzylidene)piperidin-4-one C with better antimour activity but lower toxicity than curcumin.¹² Furthermore, W. M. Weber, et al. obtained curcumin and related enones which inhibited TNFα-induced activation of NFκB thereby contributing to the development of pro-survival and anti-apoptotic state. Among these compounds, 1,5-bis(3-pyridyl) -1, 4-pentadien-3-one has been proved to be the most effective one with an IC₅₀ value of 3.4 ± 0.2 μM.¹³ Nevertheless, to the best of our knowledge, there has been no report on the inhibition of PC3, BGC-823 and Bcap-37 cells with 1,5-diarylpentadien-3-one derivatives bearing a phenolic ether pharmacophore. This prompted us to study the new class of compounds 4 and investigate their preliminarily mechanism of action as potent anticancer agents.

Results and discussion

Chemistry

The key intermediate 3 was synthesized starting from the 4-(2-hydroxyphenyl)-3-butene-2-ones or 4-(4-hydroxyphenyl)-3-butene-2-one 2 with substituted benzaldehyde, water and ethanol in water at 5–10 °C, followed by dropwise addition of NaOH solution. The mixture was stirred at 5–10 °C for 3 h, then stirred at room temperature for 7 h, with carbon dioxide in solution about 45 min. The resulting precipitate was filtered and washed with water, dried. The solid was purified by thin layer chromatography with silica gel (ethyl acetate/dichloromethane/petroleum ether, V [thin space (1/6-em)]

V = 2

1) to afford intermediates 3. The target compounds 4A–4AF were synthesized by reaction of halogenated hydrocarbons with intermediates 3, in the presence of K₂CO₃ and KI in refluxing acetone for 2 h, as can be seen in Scheme 1. The solvent was then removed under reduced pressure and the crude yellow products were purified by recrystallization using acetone/petroleum ether (V [thin space (1/6-em)]

V = 1

2) to give the target compounds 4A–4AF.


	Scheme 1 Synthetic route to the title compounds. Reagents and conditions: (a) NaOH, EtOH–H₂O, r.t. 11 h, 76%; (b) gently bubbling CO₂, r.t., 45 min, 74%; (c) substituted benzaldehyde, NaOH, EtOH–H₂O, 5–10 °C for 3h, r.t. for 7 h; (d) gently bubbling CO₂, r.t., 45 min, 51–58% for two steps; (e) RX, KI/K₂CO₃/acetone, 58 °C, 2 h, 61–81%.

Biological evaluation

(A) The results of in vitro antitumor activity by MTT (thiazolyl blue tetrazolium bromide) method. The antitumor activity in vitro of phenolic ether analogs of curcumin derivatives 4A–4AF were investigated against PC3 cells, BGC-823 cells and Bcap-37 cells. The results of antitumor activity in vitro of these compounds are shown in Table S1 by support information.† It can be seen from Table S1 that antitumor activity in vitro of 4A and 4Y were obviously higher than other curcumin analogues. Then, IC₅₀ values of this novel compound 4A and 4Y were also tested and the results are provided in Table 1. It can be seen from Table 1 that 4A inhibited growth of three kinds of tumor cells. Meanwhile, the inhibition of 4A and 4Y varied in a dose-dependent manner.

Table 1 Inhibition activity (IC₅₀) of compounds 4A and 4Y to PC3, Bcap37, BGC823 cancer cells

Compd^a	IC₅₀ (μM)^b
Compd^a	PC3^c	Bcap37^d	BGC823^e
a These compounds were tested as the free base. b IC₅₀ concentrations needed to inhibit cell growth by 50% as determined from the dose response curve. Determination was done in three separate experiments and each was performed in triplicate. c Prostate cancer. d Breast cancer. e Stomach cancer. f ADM (adriamycin) was used as positive control. g The value was determined through MTT assay.
4A	7.1 ± 0.5	7.3 ± 0.1	7.2 ± 0.1
4Y	8.0 ± 0.3	8.6 ± 0.3	6.6 ± 0.2
ADM^f	1.4^g ± 0.1	2.0 ± 0.3	2.6 ± 0.4

(B) The results of AO/EB (acridine orange/ethidium bromide) staining. The PC3 cells treated with 4A were treated with AO/EB staining. The stained cells revealed four different types under fluorescence microscope: the chromatin of living cells was green and the structure was normal; the chromatin of non-apoptotic dead cells was orange and the structure was normal; the chromatin of early apoptotic cells was green and the morphous was in the form of pycnosis or bead; the chromatin of late apoptotic cells was orange and the morphous was in the form of pycnosis or bead. Cytotoxicity of 4A at the concentration of 10 μmol L⁻¹ against PC3 cells for 72 h was detected by AO/EB staining. The results are given in Fig. 2.


	Fig. 2 The AO/EB staining of compound 4A on PC3 cells for 72 h.

It can be seen from Fig. 2 that the cells treated with 4A changed little, the nucleus was green and the morphous was in the form of pycnosis. The cells presented apoptotic morphous. Little red cells indicated that 4a was associated with low cytotoxicity. Therefore, we concluded that 4A could induce apoptosis without any significant cytotoxicity.

(C) The results of Hoechst 33258 staining. The PC3 cells treated with 4A were treated with Hoechst 33258 dying. The cells were observed under fluorescence microscope, Nucleus of normal cells were normal blue; nucleus of apoptotic cells appeared compact and condensed, the cells exhibited strong blue fluorescence; but nucleus of dead cells did not show any staining under fluorescence microscope. 4A induced apoptosis at different concentrations against PC3 cells for 72 has detected by Hoechst 33258 dying. The results are shown in Fig. 3.


	Fig. 3 The Hoechst 33258 staining of compound 4A on PC3 cells for 72 h.

It can be seen from Fig. 3 that cells of the negative group were normal blue. Nucleus of cells at high concentration group and HCPT group appeared compact and condensed. The cells exhibited strong blue fluorescence and revealed typical apoptotic morphology. Therefore, it appears that 4A induced apoptosis against PC3 cells. The results were identical with the previous AO/EB double staining.

(D) The results of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) coloration. The PC3 cells treated with 4A were treated with TUNEL coloration. The cells were observed under fluorescence microscope, brown precipitate was the result of positive apoptosis. 4A induced apoptosis at different concentrations against PC3 cells for 48 h as detected by TUNEL coloration. The results are provided in Fig. 4.


	Fig. 4 The TUNEL staining of compound 4A on PC3 cells for 48 h.

It can be seen from Fig. 4 that cells of the negative group do not appear as brown precipitate. The cells at high concentration group and HCPT group appeared as brown precipitate. Therefore, we further concluded that 4A induced apoptosis against PC3 cells. The results were identical with the previous experiment.

(E) The results of agarose gel electrophoresis. Endonucleases were activated when cells exhibited apoptosis, then DNA was degraded selectively, forming 50–300 kb big fragments, then cleaved in the vicinity of nucleosome and formed DNA fragments of or multiples of 180–200 bp. These DAN fragments could be extracted from cells. The DNA appeared by agarose gel electrophoresis and EB staining. We presumed that the cells showed apoptosis according to DNA Ladder. The compound 4A induced apoptosis at the concentration of 10 μmol L⁻¹ against PC3 cells for 72 h as detected by agarose gel electrophoresis. The results are shown in Fig. 5.


	Fig. 5 The DNA Ladder of compound 4A on PC3 cells for 72 h.

It can be seen from Fig. 5 that cells of the negative group did not appear as DNA Ladder. The cells in compound 4A and HCPT group appeared as typical DNA Ladder. Therefore, it again leads to the fact that 4A induced apoptosis against PC3 cells. The results are similar to the previous experiments.

(F) The results of FCM (flow cytometry). Phosphatidylserine distributes inside the lipid bilayer in the normal cells. But in early apoptotic cells, PS is being transferred from inside to outside of lipid bilayer. Annexin V with a molecular weight of 35–36 KD is Ca²⁺ dependent phospholipid binding protein with high affinity to phosphatidylserine, Annexin V can be affiliated with cytomembrane of early apoptotic cells by phosphatidylserine exposed outside of cells. Therefore, early apoptosis of cells was detected by Annexin V as a sensitive indicator. The FITC labeled Annexin V detects apoptosis by FCM. PI (Propidium Iodide) is not able to pass intact through cytomembrane, a nucleic acid dye, but can do so at a slightly later stage through cells and dead cells. PI can however pass through cytomembrane and dye nucleus red. The different periods of apoptotic cells could be distinguished if Annexin V matched with PI. The first quadrant of apoptosis detected by flow cytometry PI/Annexin V FITC dying scatter diagrams demonstrated that mechanically injured cells presented Annexin−/PI+; the second quadrant demonstrated that the late apoptotic cells presented Annexin+/PI+; the third quadrant demonstrated that the normal cells presented Annexin−/PI−; the forth quadrant demonstrated that the early apoptotic cells presented Annexin+/PI−. The compound 4A induced apoptosis at the concentration of 10 μmol L⁻¹ against PC3 cells for 72 h was detected by FCM. The results are shown in Fig. 6.


	Fig. 6 The scatter diagram of FCM tested apoptosis.

It is evident from Fig. 6 that the second and forth quadrant of 4A and HCPT group presented positive results. These observations demonstrated that 4A and HCPT group exhibited simultaneously early and late apoptotic cells against PC3 cells for 72 h after being treated with the compound. This is in line with our assumption that 4A induced apoptosis against PC3 cells. The results were identical with the previous finding. The effects of action time and concentration of 4A against PC3 cells are provided in Fig. 7.


	Fig. 7 Effects of concentration of 4A on apoptosis rate.

It can be seen from Fig. 7 that the apoptosis rate increased with the action time and concentration of 4A. Moreover, the apoptosis rate changed in a dose-dependent manner. The highest rate of apoptosis was 21.9% when the cells were treated with 4A at the concentration of 10 μmol L⁻¹ for 72 h. Thus, 4A induced apoptosis in a dose-dependent manner.

Conclusion

In summary, we prepared a series of novel 1, 5-diaryl-1, 4-pentadien-3-one derivatives containing phenolic ether moieties as potential anticancer agents. The results show that in vitro inhibitory activities IC₅₀ of 4A and 4Y were 7.1 μmol L⁻¹ and 8.0μmol L⁻¹, 7.3 μmol L⁻¹ and 8.6μmol L⁻¹, 7.2μmol L⁻¹ and 6.6 μmol L⁻¹ against PC3, BGC-823 and Bcap-37 cells, respectively. It shows that 4A and 4Y has high inhibitory activity and obvious dose dependent relationship on the three cancer cells.

The results also show that 4A induces apoptosis against PC3 cells as detected by AO/BO staining, Agarose Gel Electrophoresis and FCM. To conclude, we found that 4A possesses high antitumor activity and can inhibit the growth of tumor cells by inducing tumor cells apoptosis, the effect of which largely depends on the concentration of 4A. Further studies on the mechanism of interaction need to be conducted in the future.

Acknowledgements

The authors wish to thank the National Key Program for Basic Research (Nos. 2010CB126105, 2010CB134504), and the National Natural Science Foundation of China (Nos. 20872021, 20662004) for the financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1md00038a