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Synthesis of New 4-(tert-Octyl)phenol Derivatives and Their Anticancer Activity against Human Prostate and Lung Cancer Cell Lines
Synthesis of New 4-(tert-Octyl)phenol Derivatives and Their Anticancer Activity against Human Prostate and Lung Cancer Cell Lines
Bulletin of the Korean Chemical Society. 2014. Jul, 35(7): 2038-2042
Copyright © 2014, Korea Chemical Society
  • Received : September 23, 2013
  • Accepted : March 17, 2014
  • Published : July 20, 2014
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About the Authors
Haiyan Che
Yuanying Fang
Santosh K. Gurung
Jun Luo
Deok Hyo Yoon
College of Natural Science (Biochemistry), Kangwon National University, Chunchon 200-701, Korea
Gi-Ho Sung
Rural Development Administration, Suwon 441-707, Korea
Tae Woong Kim
College of Natural Science (Biochemistry), Kangwon National University, Chunchon 200-701, Korea
Haeil Park

Abstract
4-( tert -Octyl)phenol derivatives bearing the D-mannitol substructure ( 6a, 6b, 7 ) were prepared from D-mannitol and evaluated their anticancer activity against human lung (A549) and prostate (Lncap, Du145, PC3) cancer cell lines. Among derivatives tested, the bis( tert -octyl)phenoxy compound 7 exhibited strongest proliferation inhibitory activities against human cancer cell lines tested, especially high sensitivity to human Du145 prostate cancer cells (IC 50 = 7.3 μM).
Keywords
Introduction
Cordyceps militaris ( C. militaris ), a caterpillar-grown traditional insect-flower (medicinal mushroom), is a fungus that parasitizes Lepidoptera Larvae and is known to possess diverse biological activities in the human body including immune, respiratory, circulatory and glandular systems. C. militaris has been used in traditional chinese medicine to treat numerous illnesses, promote longevity, relieve exhaustion and increase athletic prowess. Precedent literatures showed various biological activities such as antitumor and antidiabetic, 1 antiangiogenesis, 2 antimutagenic 3 and hypoglycemic effect. 4 It was also reported that butanol fraction of C. militaris produces a number of bioactive constituents and one of the major components was identified as cordycepin which markedly inhibited the phosporylation of Akt and MAP kinase in LPS-activated macrophage, suppressed tumor necrosis factor (TNF-α) expression. 5 Cordycepin was reported to possess various biological properties in precedent literatures. 6-12 Bioactivity-guided further fractionation of butanol fraction of C. militaris yielded a novel anticancer substance with polyethylene glycol moiety in its structure named by us as militarin ( Figure 1 ). An efficient synthetic method of militarin was developed by us to prepare multigram scale sample for bioassay in diverse therapeutic areas. 13 Also a number of militarin derivatives bearing different numbers of ethylene glycol unit ( Figure 1 ) were synthesized. It was proved from bioassay that 4-( tert -octyl)phenol derivatives with 1 and 4 ethylene glycol unit exhibited strong anticancer and antiinflammatory activities. 14
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Structures of militarin and new target compounds (6a, 6b, 7).
Structure-activity relationship study (SARs) of bioactive natural products is a general and powerful procedure, and one of very promising approach to discover new lead compounds as proved in many precedent drug discovery. Recently, several polysaccharide components have been isolated from C. militaris and suggested as anticancer therapeutic agents in preliminary in vitro and animal studies. 15-17 Militarin derivatives functionalized with a carbohydrate group (D-mannitol) as the congener of the ethylene glycol substructure of militarin were designed ( Figure 1 ) since the introduction of a carbohydrate moiety allowed the generation of glycosides linked through an ethylene ether bridge; this is a very promising approach for the development of novel drugs with improved cellular membrane permeability and stability as therapeutic reagents.
Experimental
General. All chemicals were obtained from commercial suppliers, and used without further purification. All solvents used for reaction were freshly distilled from proper dehydrating agent under nitrogen gas. All solvents used for chromatography were purchased and directly applied without further purification. 1 H-NMR spectra were recorded on a Varian Gemini 3000 instrument (300 MHz) and a Bruker DPX 400 (400 MHz) spectrometers. Chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal standard. Peak splitting patterns are abbreviated as m (multiplet), s (singlet), bs (broad singlet), d (doublet), bd (broad doublet), t (triplet), dd (doublet of doublets) and ddd (doublet of double doublet). 13 C-NMR spectra were recorded on a Bruker DPX 400 (100 MHz) spectrometer, fully decoupled and chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal standard. Melting points were recorded on a Fisher-Johns microscopic scale melting point apparatus. Mass spectra were recorded on a Autospec M363 and MALDI-TOM MS (Voyager DE STR). Analytical thin-layer chromatography (TLC) was performed using commercial glass plate with silica gel 60F 254 purchased from Merck. Chromatographic purification was carried out by flash chromatography using Kieselgel 60 (230-400 mesh, Merck).
(2R,3R,4R,5R)-1,2,3,4,5,6-Hexakis(benzyloxy)hexane (1). D-Mannnitol (5.0 g, 26.90 mmol) was added to a solution of powdered KOH (22.64 g, 403.47 mmol) and tetrabutylammonium bromide (TBAB, 0.87 g, 2.69 mmol) in 25 mL DMSO. The mixture was stirred for 1 h at room temperature. The suspension was cooled in an ice bath, and benzyl chloride (BnCl, 25 mL, 215.18 mmol) was immediately added dropwise. The reaction was allowed to come slowly to room temperature by allowing the ice to melt. After stirring for 24 h, the reaction was diluted with 60 mL of cold water and then extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with water (100 mL) and sat brine (100 mL), dried (MgSO 4 ) and concentrated to give a cream-colored solid. Purification by column chromatography (hexane) afforded the title product 1 (18.26 g, 94%) as colorless oil. 1 H-NMR (300 MHz, CDCl 3 ) δ 7.27-7.31 (m, 30H, 6xPh), 4.41-4.70 (m, 12H, 6x-C H 2 -Ph), 4.02-4.03 (dd, 2H, H3, H4), 3.85-3.88 (m, 4H, H1a, H6a, H2, H5), 3.68-3.74 (dd, 2H, J = 11.1 Hz, 5.8 Hz, H1b, H6b).
(2R,3R,4R,5R)-1,6-Dimesyl-2,3,4,5-tetrakis(benzyloxy)- hexane (2). To ZnCl 2 (11.36 g, 83.34 mmol) was added 1:5 HOAc–Ac 2 O (65 mL). The mixture was cooled down to 0 ℃, and then a solution of compound 1 (12.04 g, 16.67 mmol) in 1:5 HOAc–Ac 2 O (65 mL) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours, and then 300 mL of ice water was added. The resulting organic layer was washed with water (3 × 200 mL) and brine (100 mL), dried (MgSO 4 ) and filtered to gave 1,6-diacetate compound as a light-yellow oil. K 2 CO 3 (11.5 g, 83.35 mmol) was added to a solution of crude l,6-diacetate compound in 80 mL MeOH. The reaction mixture was stirred at room temperature for 3 h, and filtered, concentrated to provided the corresponding alcohol as colorless oil. The reaction mixture of the crude alcohol (1.62 g, 2.99 mmol), methanesulfonyl chloride (0.50 mL, 6.27 mmol) and DMAP (0.0 g, 0.24 mmol) in pyridine (15 mL) was stirred at 0 ℃ and the mixture was allowed to warm to room temperature. The reaction mixture was quenched with saturated NH 4 Cl aqueous solution, extracted with CH 2 Cl 2 and 3% HCl. The organic layer was dried over anhydrous MgSO 4 and the solvent was removed under reduced pressure to obtain the title product 2 (1.80 g, 86%) as colorless oil. 1 H-NMR (300 MHz, CDCl 3 ) δ 7.28-7.35 (m, 20H, 4xPh), 4.56-4.74 (m, 8H, J = 11.4 Hz, 4x-C H 2 -Ph), 4.35-4.43 (m, 4H, H1a, H1b, H6a, H6b), 3.85 (m, 4H, H2, H3, H4, H5), 2.84 (s, 6H, 2xOMs).
(2R,3R,4R,5R)-6-Mesyl-1-(4-(tert-octyl)phenoxy)-2,3,4,5- tetrakis(benzyloxy)hexane (3). Compound 2 (4.63 g, 6.63 mmol) and potassium iodide (0.11 g, 0.66 mmol) were dissolved in CH 3 CN (33 mL), and stirred for 5 min. A mixture of 4-( tert -octyl)phenol (1.69 g, 7.96 mmol) and K 2 CO 3 (2.75 g, 15.9 mmol) dissolved in acetonitrile (33 mL) was added. The reaction mixture was refluxed for 24 h. The white solid in the mixture was removed by filtration and the solvent was removed in vacuo to give yellow oil. Purification of crude product by chromatography (silica, 10:1 to 5:1 hexane-acetone) provided the title product 3 (2.3 g, 39%) and 4 (0.99 g, 15%) as colorless oil. 1 H-NMR (300 MHz, CDCl 3 ) δ 7.23-7.36 (m, 22H, Ph), 6.81-6.83 (d, 2H, J = 7.7 Hz, Ph), 4.57-4.76 (m, 8H, 4x-C H 2 -Ph), 4.35-4.51 (m, 4H, H1a, H1b, H6a, H6 b ), 4.12-4.15 (br, 1H, H5), 4.00 (br, 2H, H3, H4), 3.89 (br, 1H, H2), 2.80 (s, 3H, OMs), 1.71 (s, 2H, -CH 2 -), 1.35 (s, 6H, 2xMe), 0.72 (s, 9H, 3xMe).
(2R,3R,4R,5R)-1,6-Bis(4-(tert-octyl)phenoxy)-2,3,4,5- tetrakis(benzyloxy) hexane (4). 1 H-NMR (300 MHz, CDCl 3 ) δ 7.22-7.31 (m, 24H, Ph), 6.81-6.84 (d, 4H, J = 8.8 Hz, Ph), 4.60-4.76 (m, 6H, J = 11.7 Hz, 11.2 Hz, 3x-C H 2- Ph) 4.89-4.53 (d, 2H, J = 11.7 Hz, -C H 2 -Ph), 4.37-4.42 (m, 2H, H1a, H6a), 4.12-4.18 (m, 4H, J = 10.6 Hz, 5.2 Hz, H1b H6b, H2, H5), 4.05 (br, 2H, H3, H4), 1.71 (s, 4H, 2x-CH 2 -), 1.35 (s, 12H, 4xMe), 0.72 (s, 18H, 6xMe).
(2R,3R,4R,5R)-2,3,4,5-Tetrakis(benzyloxy)-6-(2-hydroxyethoxy)- 1-(4-(tert-octyl)phenoxy)hexane (5a). A solution of the ethylene glycol (0.19 mL, 3.41 mmol) in anhydrous THF (9 mL) was treated with NaH (0.50 g; 11.37 mmol, 55% in mineral oil) and stirred for 1 h at room temperature. The reaction mixture was cooled to 0 ℃, and the compound 3 (2.30 g; 2.84 mmol) in dry THF (9 mL) was added dropwise over 30 min at 0-5 ℃. After completed addition, the mixture was stirred at room temperature for 10 h and filtered. The filtrate was washed with THF and concentrated by using rotary evaporation, redissolved in chloroform (100 mL), washed with brine, and dried over anhydrous MgSO 4 . Evaporation of the solvent gave a pale yellow oil, which was subjected to flash chromatography on silica with up to 5% gradient elution of methanol in dichloromethane to give 5a (0.70 g; 32%) as colorless oil. 1 H-NMR (300 MHz, CDCl 3 ) δ 7.13-7.33 (m, 22H, Ph), 6.70-6.73 (d, 2H, J = 8.8 Hz, Ph), 4.62-4.73 (m, 6H, 3x-C H 2 -Ph), 4.44-4.50 (m, 2H, J = 11.7 Hz, -C H 2 -Ph), 4.25-4.37 (m, 2H, H1a, H1b), 3.77-4.01 (m, 5H, H6a, H2, H3, H4, H5), 3.66-3.76 (m, 3H, H6b, C H 2 ), 3.54-3.61 (m, 2H, C H 2 ), 1.71 (s, 2H, -CH 2 -octyl), 1.34 (s, 6H, 2xMe), 0.70 (s, 9H, 3xMe).
(3R,4R,5R,6R)-4,5,6-Tris(benzyloxy)-1-phenyl-3-((4- (2,4,4-trimethylpentan-2-yl)phenoxy)methyl)-2,8,11,14,17- pentaoxanonadecan-19-ol (5b). A solution of the tetraethylene glycol (0.13 mL, 0.65 mmol) in anhydrous THF (3 mL) was treated with NaH (0.09 g; 1.95 mmol, 55% in mineral oil) at room temperature, stirred for 1 h, and cooled to 0 ℃, and the compound 3 (0.53 g; 0.65 mmol) in dry THF (4 mL) was added dropwise over 30 min at 0-5 ℃. After complete addition, the mixture was stirred at room temperature for 1 day and filtered. The filtrate was washed with THF and concentrated by using rotary evaporation, redissolved in chloroform (100 mL), washed with brine, and dried over anhydrous MgSO 4 . Evaporation of the solvent gave a pale yellow oil, which was subjected to flash chromatography on silica with up to 5% gradient elution of methanol in dichloromethane to give 5b (0.14 g; 23%) as colorless oil. 1 H-NMR (300 MHz, CDCl 3 ) δ 7.23-7.33 (m, 22H, Ph), 6.78- 6.81 (d, 2H, J = 8.8 Hz, Ph), 4.59-4.75 (m, 6H, 3x-C H 2-Ph), 4.46-4.53 (m, 2H, J = 11.7 Hz, -C H 2 -Ph), 4.33-4.37 (m, 1H, J = 10.4 Hz, 2.6 Hz, H18a), 4.08-4.16 (m, 2H, J = 10.4 Hz, 5.5 Hz, 4.9 Hz, 4.0Hz, H18b, H17), 3.87-4.03 (m, 4H, H13a, H14, H15, H16, H17), 3.66-3.76 (m, 3H, H13b, -OC H 2 - CH 2 OH), 3.54-3.61 (m, 14H, 3x-OC H 2 -C H 2 O-, C H 2 OH), 1.71 (s, 2H, -CH 2 -octyl), 1.34 (s, 6H, 2xMe), 0.72 (s, 9H, 3xMe).
(2R,3R,4R,5R)-1-(2-Hydroxyethoxy)-6-(4-(2,4,4-trimethylpentan- 2-yl)phenoxy)hexane-2,3,4,5-tetraol (6a). To a solution containing compound 5a (0.21 g, 0.27 mmol) and HCOOH (0.3 mL) in dry MeOH (3 mL) under an argon atmosphere, Pd 10 wt % on activated carbon (0.5 g) was added and the mixture was stirred for overnight at reflux. After removal of the catalyst by filtration, the organic layer was evaporated. Purification by flash chromatography (100:1-10:1 CHCl 3 :MeOH) to get product 6a (0.05 g, 47%) as colorless oil. 1 H-NMR (400 MHz, CDCl 3 ) δ 7.23-7.25 (d, 2H, J = 8.8 Hz, Ph), 6.80-6.82 (d, 2H, J = 8.8 Hz, Ph), 4.08- 4.16 (m, 2H, J = 10.1 Hz, 5.7 Hz, 4.3 Hz, H6a, H6b), 3.89-3.93 (m, 1H, H5), 3.61-3.85 (m, 9H, H1a H1b, H2, H3, H4, -OC H 2 -C H 2 OH), 1.69 (s, 2H, -CH 2 -octyl), 1.31 (s, 6H, 2xMe), 0.70 (s, 9H, 3xMe). 13 C-NMR (100 MHz, CDCl 3 ) δ 156.0 (C-1-Ph), 142.8 (C-4-Ph), 127.1 (C-3-Ph, C-5-Ph), 113.7 (C-2-Ph, C-6-Ph), 72.6 (C-3, C-4), 72.2 (- C H 2 -CH 2 OH), 71.3 (C-2, C-5), 67.6 (C-1), 64.2 (C-6), 61.4 (CH 2 OH), 56.9 (- C H 2 - octyl), 38.0 ( C -Me 2 ), 32.3 ( C -Me 3 ), 31.8 (Me 3 ), 31.7 (Me 2 ). Calc’d exact mass for C 22 H 35 Na 3 O 7 : 480.2076. HRMS (MALDI-TOF): m/z [M + 3Na] + 480.0655.
(14R,15R,16R,17R)-18-(4-(2,4,4-Trimethylpentan-2-yl)- phenoxy)-3,6,9,12-tetraoxaoctadecane-1,14,15,16,17-pentaol (6b). Following the procedure applied for synthesis of 6a , but replacing compound 5b (0.14 g, 0.15 mmol) for compound 5a , the title product 6b was obtained as colorless oil. Yield 55%; 1 H-NMR (400 MHz, CDCl 3 ) δ 7.25-7.27 (d, 2H, J = 8.8 Hz, Ph), 6.85-6.87 (d, 2H, J = 8.8 Hz, Ph), 4.25-4.28 (m, 1H, H18a), 4.07-4.14 (m, 2H, H18b, H17), 3.89-3.98 (m, 3H, H14, H13a H13b), 3.57~3.79 (m, 18H, H15, H16, 4x- OC H 2 -C H 2 O-), 1.90 (s, 1H, OH), 1.70 (s, 2H, -CH 2 -), 1.33 (s, 6H, 2xMe), 0.71 (s, 9H, 3xMe). 13 C-NMR (100 MHz, CDCl 3 ) δ 156.3 (C-1-Ph), 142.6 (C-4-Ph), 127.1 (C-3-Ph, C-5-Ph), 113.8 (C-2-Ph, C-6-Ph), 72.9 (C-15), 72.7 (C-16), 71.0 (C-11), 70.6 (C-14), 70.4 (C-17), 70.3 (C-4, C-5, C-7, C-8, C-10, C-11), 70.2 (C-2) 69.9 (C-13), 69.8 (C-18), 61.3 (CH 2 OH), 57.0 (-C H 2 -octyl), 38.0 ( C -Me 2 ), 32.3 ( C -Me 3 ), 31.8 (Me 3 ), 31.7 (Me 2 ). Calc’d exact mass for C 28 H 49 NaO 10 : 568.3223. HRMS (MALDI-TOF): m/z [M + Na] + 568.3385.
(2R,3R,4R,5R)-1,6-Bis(4-(2,4,4-trimethylpentan-2-yl)- phenoxy)hexane-2,3,4,5-tetraol (7). Following the procedure applied for synthesis of 6a , the title product 7 was obtained as colorless oil. Yield 50%; 1 H-NMR (400 MHz, CDCl 3 ) δ 7.27-7.29 (d, 4H, J = 8.8 Hz, Ph), 6.84-6.86 (d, 4H, J = 8.8 Hz, Ph), 4.23-4.26 (dd, 2H, J = 8.8 Hz, 3.1 Hz, H1a, H6a), 4.11-4.19 (m, 4H, J = 8.8 Hz, 6.2 Hz, H1b, H6b, H2, H5), 4.04-4.07 (t, 2H, J = 5.8 Hz, H3, H4), 3.01-3.02 (d, 2H, 2xOH), 2.83 (s, 2H, 2xOH), 1.71 (s, 4H, 2x-CH 2 -), 1.34 (s, 12H, 4xMe), 0.71 (s, 18H, 6xMe). 13 C-NMR (100 MHz, CDCl 3 ) δ 155.9 (C-1-Ph), 143.1 (C-4-Ph), 127.2 (C3-Ph, C-5-Ph), 113.8 (C-2-Ph, C-6-Ph), 71.3 (C-3, C-4), 70.6 (C-2, C-5), 69.2 (C-1, C-6), 56.9 (- C H 2 -octyl), 38.00 ( C -Me 2 ), 32.3 ( C -Me 3 ), 31.8 (Me 3 ), 31.7 (Me 2 ). Calc’d exact mass for C 33 H 51 NaO 6 : 566.3583. HRMS (MALDI-TOF): m/z [M + Na] + 566.3637.
Evaluation of Anticancer Activity against Human Lung (A549) and Prostate (Lncap, Du145, PC3) Cancer Cell Lines: Militarin derivatives ( 6a, 6b, 7 ) were dissolved in DMSO and diluted with media. The solutions were added to cancer cell lines at the concentration of 0-200 ug/mL each. For the growth inhibition assay, non adherent (10,000 cells) or adherent (10,000 cells) cancer cell were cultured in the 96-well culture plate in the presence or absence of the extracts or fractions of C. militaris . Cell growth inhibition was observed by MTT colorimetric method. Briefly, 20 uL of 5 ug/mL MTT was added to each 96 well. After 30 minutes incubation, 100 uL of the culture medium was replaced with 100 uL of isopropanol for the extraction of dye. All crystals were dissolved by repeated pipeting of the medium and measured by ELISA reader (570 nm). The anticancer activities of compounds 6a, 6b and 7 were determined using the MTT assay after the incubation of A549 cells in the presence of the militarin derivatives (0, 10, 30, 50 and 100 μM) for 24 h. Also anticancer activities of compound 7 against three human prostate cancer cell lines (DU145, LNCap and PC3) and a human lung adenocarcinoma (A549) were evaluated. The cells were treated with compound 7 (0, 2.5, 5, 7.5 and 10 μM) for 24 h and determined using the MTT assay.
Results and Discussion
Chemistry. Militarin derivatives bearing the D-mannitol substructure ( 6a, 6b, 7 ) were prepared following the synthetic pathway as shown in Scheme 1 . The target compound 6a and 6b were generated from commercially available Dmannitol, 4-( tert -octyl)phenol, ethylene glycol and tetraethylene glycol. The compound 7 was prepared from the compound 4 , which was obtained as a by-product from coupling reaction between the dimesylate intermediate ( 2 ) and 4-( tert -octyl)phenol. D-Mannitol was converted to the hexabenzylated compound 1 by reaction with BnCl, KOH and tetra-butylammonium bromide (TBAB) in DMSO at room temperature. 18 Selective debenzylation–acetylation of compound 1 using ZnCl 2 –Ac 2 O–HOAc, then deacetylation with K 2 CO 3 in MeOH at room temperature gave alcohol intermediate, 18 which was converted to dimesylate compound 2 by reaction with MsCl and DMAP in pyridine at room temperature. 12 Compound 2 was converted to mono- and disubstituted phenyl ether compound 3 and 4 by reaction with 4-( tert -octyl)phenol, K 2 CO 3 and KI in CH 3 CN at reflux. 13 Compound 3 was converted to the compound 5a (or 5b ) by reaction with ethylene glycol (or tetraethylene glycol) and NaH in dry THF at room temperature. 19 Hydrogenolysis of compounds 5a, 5b and 4 gave carbohydrate bearing militarin derivatives 6a, 6b and 7 , respectively.
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Synthesis of 4-(tert-octyl)phenol derivatives with D-mannitol substructure.
Biological Evaluation. The anticancer activities of compounds 6a , 6b and 7 were determined using the MTT assay 14 after the incubation of A549 cells in the presence of the militarin derivatives (0, 10, 30, 50 and 100 μM) for 24 h. The treatment of compound 6a or 6b resulted in a slight decrease in cell viability at 100 μM, whereas treatment of compound 7 significantly suppressed A549 cell viability (36.9%) at 10 μM ( Figure 2(a) ) without cytotoxicity (data not shown). Thus, the compound 7 , which has additional 4-( tert -octyl)phenol in its structure, showed better anticancer activity than that of compound 6a or 6b ( Figure 2(a) ). We next examined whether compound 7 affects proliferation of three human prostate cancer cell lines (DU145, LNCap and PC3) and a human lung adenocarcinoma (A549). The cells were treated with compound 7 (0, 2.5, 5, 7.5 and 10 μM) for 24 h and determined using the MTT assay. As shown in Figure 2(b) , the compound 7 treated in A549, DU145, LNCap and PC3 decreased cell viability in a concentrationdependent manner after 24 h incubation. From the more precise comparison from MTT analysis from Figure 2(b) , compound 7 showed an anticancer effect towards A549, Du145, LNCap and PC3 cells with an IC 50 value of 8.5, 7.3, 12.1 and 9.3 μM, respectively. Human Du145 prostate cancer cells showed the highest sensitivity to the anti-cancer effect on compound 7 among the 4 cancer cell lines tested as shown in Figure 2(b) . Our previous results also indicated similar trends that the 4-( tert -octyl)phenol derivatives bearing different numbers of ethylene glycol substructure exhibited generally reduced anticancer activities against HeLa, B16F19 and HT-29 cancer cell lines than those of the parent compound (militarin). 14 Based on the previous and present results, we found that increased number (length) of ethylene glycol unit generally resulted in decreased anticancer activity.
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Cell viability as determined by the MTT assay. (a) A549 cells were treated for 24 h with three militarin derivatives (Compound 6a, 6b and 7). (b) A549, Du145, LNCap and PC3 cells were treated for 24 h with compound 7 at the indicated doses. Data are presented as the mean ± SEM of three independent experiments. *; p < 0.05 vs. control, **; p < 0.01 vs. control.
Conclusion
In summary, three militarin derivatives bearing a carbohydrate (D-mannitol) as the congener of the polyethylene glycol substructure of militarin were successfully synthesized to decipher the effects of structural alterations on the anticancer activity. Anticancer activities of the synthesized derivatives ( 6a, 6b and 7 ) were evaluated against human lung (A549) and prostate (Lncap, Du145, PC3) cancer cell lines. Among tested derivatives, the compound with bis-4- tert -octylphenoxy group ( 7 ) exhibited strong proliferation inhibitory activities against tested human cancer cell lines, especially high sensitivity to human Du145 prostate cancer cells (IC 50 = 7.3 μM). The proliferation inhibitory activity of compound 7 against Du145 prostate cancer cells was slightly potent than that of militarin (IC 50 = 9.4 μM). On the other hand, derivatives bearing a carbohydrate (D-mannitol) and ethylene glycol substructures ( 6 a and 6b ) exhibited less potent anticancer activities than those of compound 7 . Our present results may imply that the polyethylene glycol substructure is not essential for the anticancer activity and could be replaced by other congeners. Further SARs results related with militarin derivatives bearing diverse carbohydrates will be reported in due course.
Acknowledgements
This work was supported by a grant from the Regional Innovation which was conducted by the Ministry of Commerce Industry and Energy, and Biogreen 21 Program, Rural Development Administration, Republic of Korea and also was supported by University-Industry Foundation of Kangwon National University (Foreign Post- Doc Invitation Program for Dr. Santosh K. Gurung). The authors also thank to New Drug Development Research Institute and Central Laboratory of Kangwon National University for the use of analytical instruments and bioassay facilities.
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