Arch Pharm Res Vol 30, No 9, 1055-1061, 2007
http://apr.psk.or.kr
Synthesis and Biological Activity of 5-{4-[2-(Methyl-p-substituted phenylamino)ethoxy]benzyl}thiazolidine-2,4-diones Hyo Jin Gim, BoMi Kang, and Raok Jeon
College of Pharmacy, Sookmyung Women's University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, Korea (Received January 25, 2007)
Thiazolidinedione derivatives are potential antidiabetic drugs that bind and activate peroxisome proliferator activated receptor γ (PPARγ), which is a member of the nuclear hormone receptor superfamily and enhances insulin sensitivity. In an effort to develop a novel and effective thiazolidindione derivative, 5-{4-[2-(methyl-p-substituted phenylamino)ethoxy]benzyl}thiazolidine-2,4-diones 7 have been prepared by Mitsunobu reaction of the hydrophobic segment, methyl-p-substituted phenylaminoethanol 4 with hydroxybenzylthiazolidinedione 5 and their ability to activate PPARγ and inhibit LPS-induced NO production were evaluated. Key words: Thiazolidinedione, PPARγ, Nitric oxide, Diabetes, Inflammation
INTRODUCTION Peroxisome proliferator activated receptors (PPARs) belong to the nuclear hormone receptor superfamily that consists of three members, PPAR-α, -γ and -δ. They act as ligand-activated transcription factors and play a major role in the regulation of lipid metabolism and storage (Desvergne and Wahli, 1999; Kersten ., 2000; Berger and Moller, 2002). Therefore, PPARs are legitimate molecular targets for the development of antidiabetic agents. In particular, PPARγ activators can act as insulin sensitivity enhancers that improve glucose utilization without stimulating insulin secretion, representing an attractive approach to the treatment of type 2 diabetes. The synthetic ligands thiazolidinediones (TZD) are known to be high-affinity ligands of PPARγ (Kletzien ., 1992; Ibrahimi ., 1994; Lehmann ., 1995). To date, a large number of compounds containing the TZD moiety have been synthesized to produce new antidiabetic agents. Among them, rosiglitazone (Cantello ., 1994) and pioglitazone (Sohda ., 1990) are in clinical use. These drugs, however, have been associated with liver, cardiovascular, and hematological toxicity and body weight gain (Aronoff ., 2000). This situation emphasizes the need to develop new antidiabetic agents that could et al
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Correspondence to: Raok Jeon, College of Pharmacy, Sookmyung Women's University, 52 Hyochangwon-Gil, Yongsan-Ku, Seoul 140-742, Korea Tel: 82-2-710-9571, Fax: 82-2-715-9571 E-mail:
[email protected]
retain the insulin sensitizing properties of TZDs, but be safer and have better efficacy. For this reason many research groups are endeavoring to find new PPARγ activators (Lohray ., 1998; Oguchi ., 2000; Henke ., 1998). In addition, PPARγ appears to play a pivotal role in the regulation of cellular proliferation and inflammation including down-regulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. The mechanisms of these functions are not fully understood. The role of PPARγ in suppressing inflammation in mouse macrophage cell lines has been described (Alleva ., 2002; Jiang ., 1998; Ricote ., 1998). PPARγ agonists such as TZDs inhibit iNOS expression and NO production (Colville-Nash ., 1998; Reilly ., 2000; Dobashi ., 2000; Heneka ., 1999; Crosby ., 2005). These previous reports prompted us to investigate novel selective PPARγ agonists as candidates for the development of antidiabetic and anti-inflammatory agents. The structural feature of our compounds were based on rosiglitazone as the lead compound. We modified the lipophilic segment of rosiglitazone into a series of substituted phenyl-linked TZD derivatives in order to investigate the effect of substitution at the lipophilic tail. Herein, we report the synthesis of 5-{4-[2-(methyl- -substituted phenylamino)ethoxy]benzyl}thiazolidine-2,4-diones, their activities in a PPAR transactivation assay and their inhibitory activities on NO production in a LPS-activated macrophage cell culture system. et al
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Fig. 1. Structures of two members of the thiazolidinedione class of antidiabetic drugs
MATERIALS AND METHODS
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Materials
Most of the reagents and solvents were purchased from Aldrich Chemicals and were used without purification, with the following exceptions. Ethyl ether and tetrahydrofuran were distilled from sodium benzophenone ketyl. Acetonitrile, methylene chloride, benzene, toluene, triethylamine, pyridine, dimethyl formamide, and diisopropylamine were distilled from calcium hydride under a nitrogen atmosphere. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco Laboratories (Detroit, MI). Lipopolysaccharide (LPS, , 0127:B8), bovine serum albumin, sodium nitrite, naphthylethylene diamine, sulfanilamide, aminoguanidine, L-arginine, N-(1-naphthyl) ethylenediamine and N -monomethyl-L-arginine (L-NMMA) were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-mouse iNOS polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY) and antibactin monoclonal antibody from Sigma Chemical Co. Flash column chromatography (FCC) was performed using silica gel 60 (230-400 mesh, Merck) with the indicated solvents. Thin-layer chromatography (TLC) was performed using Kieselgel 60 F plates (Merck). IR spectra were recorded on a JASCO FT/IR 430 spectrophotometer. Hand C-NMR spectra were recorded on a Varian YH 400 spectrometer as solutions in CDCl , CH OH- or DMSO. Chemical shifts are expressed in parts per million (ppm, δ) downfield from the internal standard, tetramethylsilane. Escherichia
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A solution of -toluidine (1.01 g, 9.38 mmol) and formic acid (30 mL) was stirred at 120 C overnight. The reaction mixture was dispersed between ethyl acetate and saturated aqueous sodium bicarbonate and the organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluent, -hexane/EtOAc = 2:1) to yield the title compound (1.09 g, 86%) as a yellow oil: R = 0.21 ( -hexane/EtOAc = 2:1); IR (neat) 3269, 3123, 3037, 2922, 2869, 2774, 1894, 1694, 1607, 1519, 1406, 1313, 1230, 1254, 815 cm ; H-NMR (400 MHz, CDCl ) δ 8.61 (d, 1H, = 5.6 Hz), 8.31 (s, 0.5H), 7.77 (br s, 0.5H), 7.40 p
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N-(4-Methoxyphenyl)formamide (2b)
This compound was prepared using the procedure described for the preparation of : Yield 61%; R = 0.11 ( -hexane/EtOAc = 2:1); IR (neat) 3271, 3131, 3063, 3005, 2956, 2874, 2837, 2773, 1683, 1604, 1512, 1465, 1442, 1412, 1300, 1247, 1180, 1148, 1111, 1033, 828, 808 cm ; H-NMR (400 MHz, CDCl ) δ 8.49 (d, 0.5H, = 11.2 Hz), 8.29 (d, 1H, = 1.6 Hz), 7.63 (br s, 0.5H), 7.42 (m, 1H), 7.01 (m, 1H), 6.84 (m, 2H), 3.77 (s, 1.5H), 3.76 (s, 1.5H); C-NMR (100 MHz, CDCl ) δ 163.2, 159.1, 157.6, 156.7, 129.8, 129.5, 121.8, 129.5, 121.8, 121.6, 114.8, 114.5, 55.5, 55.4. 2a
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N-(4-Isopropylphenyl)formamide (2c)
This compound was prepared using the procedure described for the preparation of : Yield 93%; R = 0.27 ( -hexane/EtOAc = 2:1); IR (neat) 3270, 3194, 3122, 3052, 2960, 2871, 2776, 2596, 1898, 1682, 1610, 1519, 1462, 1412, 1363, 1312, 1255, 1055, 832 cm ; H-NMR (400 MHz, CDCl ) δ 8.90 (d, 0.5H, = 11.2 Hz), 8.63 (d, 0.5H, = 11.6 Hz), 8.30 (d, 0.5H, = 2.0 Hz), 8.07 (br s, 0.5H), 7.45 (m, 1H). 7.16 (m, 2H), 7.01 (m, 1H), 2.87 (sp, 0.5H, = 6.8 Hz), 2.84 (sp, 0.5H, = 6.8 Hz) 1.21 (d, 3H, = 7.2 Hz), 1.20 (d, 3H, = 6.8 Hz); C-NMR (100 MHz, CDCl ) δ 163.2, 159.5, 146.0, 145.3, 134.6, 134.4, 127.5, 126.8, 120.2, 119.0, 33.5, 33.4, 23.9. 2a
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N-p-Tolylformamide (2a)
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(m, 1H), 7.11 (m, 2H), 6.97 (m, 1H), 2.30 (s, 1.5H), 2.28 (s, 1.5H); C-NMR (100 MHz, CDCl ) δ 163.0, 159.2, 135.1, 134.4, 134.3, 134.1, 130.2, 129.5, 120.0, 119.0, 20.8, 20.7.
N-(4-tert-Butylphenyl)formamide (2d)
This compound was prepared using the procedure described for the preparation of : Yield 94%; R = 0.48 ( -hexane/EtOAc = 2:1); IR (neat) 3267, 3192, 3114, 3056, 2962, 2904, 2870, 2778, 1898, 1682, 1610, 1521, 1405, 1319, 1301, 1267, 833 cm ; H-NMR (400 MHz, CDCl ) δ 8.90 (d, 0.5H, = 10.8 Hz), 8.68 (br s, 0.5H), 8.33 (s, 0.5H), 8.11 (br s, 0.5H), 7.46 (m, 1H), 7.30 (m, 2H), 7.03 (m, 1H), 1.29 (s, 4.5H), 1.27 (s, 4.5H); C-NMR (100 MHz, CDCl ) δ 163.1, 159.4, 148.3, 147.7, 134.3, 134.1, 126.5, 125.8, 119.9, 118.7, 34.32, 34.3, 31.2. 2a
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Synthesis and Biological Activity of Thiazolidinedione Derivatives
Methyl-p-tolylamine (3a)
LAH (554 mg, 14.59 mmol) was added dropwise at 0 C with stirring to a solution of (0.99 g, 7.29 mmol) in ether (25 mL). The reaction mixture was gradually warmed to room temperature, and was stirred at room temperature for 6 h. The resulting mixture was quenched by the addition of saturated ammonium chloride solution. The mixture was then extracted with ether. The combined organic extracts were washed with water then brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography to yield the title compound (0.82 g, 93%) as a yellow oil: R = 0.57 ( -hexane/EtOAc = 2:1); IR (neat) 3409, 2923, 1617, 1522, 1316, 1260, 807 cm ; H-NMR (400 MHz, CDCl ) δ 7.02 (m, 2H), 6.56 (m, 2H), 3.55 (br s, 1H), 2.82 (s, 3H), 2.26 (s, 3H); C-NMR (100 MHz, CDCl ) δ 147.0, 129.7, 126.4, 112.6, 31.1, 20.3. o
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This compound was prepared using the procedure described for the preparation of : Yield 88%; R = 0.21 ( -hexane/EtOAc = 2:1); IR (neat) 3405, 2934, 2831, 1515, 1237, 1034, 820 cm ; H-NMR (400 MHz, CDCl ) δ 6.80 (m, 2H), 6.59 (m, 2H), 3.74 (s, 3H), 3.44 (br s, 1H), 2.79 (s, 3H); C-NMR (100 MHz, CDCl ) δ 152.0, 143.6, 114.8, 113.6, 55.8, 31.6. 3a
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2-[(4-Methoxyphenyl)methylamino]ethanol (4b)
This compound was prepared using the procedure described for the preparation of : Yield 58%; R = 0.17 ( -hexane/EtOAc = 3:1); IR (neat) 3385, 2946, 2832, 1514, 1244, 1038, 816 cm ; H-NMR (400 MHz, CDCl ) δ 6.82-6.76 (m, 4H), 3.73 (s, 3H), 3.71 (t, 2H, = 5.6 Hz), 3.29 (t, 2H, = 5.6 Hz), 2.82 (s, 3H); C-NMR (100 MHz, CDCl ) δ 152.4, 145.0, 115.8, 114.5, 59.6, 56.8, 55.6, 39.4. 4a
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2-[(4-Isopropylphenyl)methylamino]ethanol (4c)
This compound was prepared using the procedure described for the preparation of : Yield 98%; R = 0.18 ( -hexane/EtOAc = 4:1); IR (neat) 3376, 2957, 2870, 1615, 1521, 1362, 1194, 1048, 815 cm ; H-NMR (400 MHz, CDCl ) δ 7.11 (m, 2H), 6.78 (m, 2H), 3.78 (t, 2H, = 5.6 Hz), 3.41 (t, 2H, = 5.6 Hz), 2.91 (s, 3H), 2.82 (sp, 2H, = 6.8 Hz), 1.21 (d, 6H, = 6.8 Hz); C-NMR (100 MHz, CDCl ) δ 148.3, 138.3, 127.1, 113.6, 59.9, 56.0, 38.9, 33.0, 24.2. 4a
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This compound was prepared using the procedure described for the preparation of : Yield 90%; R = 0.63 ( -hexane/EtOAc = 3:1); IR (neat) 3410, 3349, 2958, 2869, 2809, 1616, 1522, 820 cm ; H-NMR (400 MHz, CDCl ) δ 7.06 (m, 2H), 6.59 (m, 2H), 3.72 (br s, 1H), 2.82 (s, 3H), 2.81 (sp, 1H, = 6.8 Hz), 1.20 (d, 6H, J = 6.8 Hz); C NMR (100 MHz, CDCl ) δ 147.4, 138.1, 127.1, 112.7, 33.2, 31.1, 24.3. 3a
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2-[(4-tert-Butylphenyl)methylamino]ethanol (4d)
This compound was prepared using the procedure described for the preparation of : Yield 84%; R = 0.38 ( -hexane/EtOAc/MeOH = 9:3:1); IR (neat) 3366, 2960, 2901, 2868, 1614, 1521, 1363, 1203, 1046, 815 cm ; HNMR (400 MHz, CDCl ) δ 7.28 (m, 2H), 6.80 (m, 2H), 3.80 (t, 2H, = 5.6 Hz), 3.48 (t, 2H, = 5.6 Hz), 2.94 (s, 3H), 1.29 (s, 9H); C-NMR (100 MHz, CDCl ) δ 126.0, 113.3, 59.9, 56.0, 39.0, 33.8, 31.5. 4a
(4-tert-Butylphenyl)methylamine (3d)
This compound was prepared using the procedure described for the preparation of : Yield 63%; R = 0.68 ( -hexane/EtOAc = 3:1); IR (neat) 3410, 3055, 2961, 2900, 2868, 1617, 1523, 1263, 1194, 821 cm ; H-NMR (400 MHz, CDCl ) δ 7.22 (m, 2H), 6.57 (m, 2H), 2.81 (s, 3H), 1.27 (s, 9H); C NMR (100 MHz, CDCl ) δ 126.0, 112.6, 33.9, 31.5, 31.2. 3a
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2-(Methyl-p-tolylamino)ethanol (4a)
60% NaH (dispersed in mineral oil, 279.5 mg, 6.99 mml) was added to a solution of methyl- -tolylamine (846.8 mg, 6.99 mmol) in DMF (30 mL) at room temperature with stirring under nitrogen. The mixture was stirred for 1 h. 2-Iodoethanol (0.654 mL, 8.39 mmol) was then added dropwise and was stirred at 60 C for 48 h. The p
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reaction mixture was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluent, -hexane/EtOAc = 3:1) to yield the title compound (772.0 mg, 67%) as a yellow oil: R = 0.24 ( -hexane/EtOAc = 3:1); IR (neat) 3358, 2919, 2870, 1618, 1522, 1360, 1045, 803 cm ; HNMR (400 MHz, CDCl ) δ 7.04 (m, 2H), 6.74 (m, 2H), 3.76 (t, 2H, = 5.2 Hz), 3.39 (t, 2H, = 5.2 Hz), 2.89 (s, 3H), 2.25 (s, 3H); C-NMR (100 MHz, CDCl ) δ 148.1, 129.7, 127.0, 113.8, 59.9, 56.1, 39.0, 20.2.
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5-{4-[2-(Methyl-p-tolylamino)ethoxy]benzyl}-3-tritylthiazolidine-2,4-dione (6a)
A solution of 1.64 mL (0.82 mmol) tributyl phosphine (0.5 M) in anhydrous toluene was added dropwise to a solution of 2-(methyl- -tolylamino)ethanol ( ) (67.5 mg, 0.41 mmol), 5-(4-hydroxy-benzyl)-3-tritylthiazolidine-2,4dione ( ) (125.5 mg, 0.27 mmol) and 1,1'-(azodicarbonyl) dipiperidine (206.1 mg, 0.82 mmol) in THF. The mixture was stirred at room temperature for 8 h. The reaction p
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mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (eluent, -hexane/EtOAc = 3:1) to yield the title compound (86.8 mg, 35%) as a white powder: R = 0.42 ( -hexane/EtOAc = 3:1); IR (neat) 3060, 3029, 2925, 2867, 1756, 1688, 1516, 1299, 1248, 1133, 734 cm ; H-NMR (400 MHz, CDCl ) δ 7.32-7.15 (m, 15H), 7.10 (d, 2H, = 8.8 Hz), 7.05 (d, 2H, = 8.0 Hz), 6.80 (d, 2H, = 8.8 Hz), 6.69 (d, 2H, = 8.8 Hz), 4.35 (dd, 1H, = 8.8, 4.0 Hz), 4.09 (t, 2H, = 6.4 Hz), 3.72 (t, 2H, = 6.4 Hz), 3.40 (dd, 1H, = 14.0, 4.0 Hz), 3.05 (dd, 1H, = 14.0, 9.2 Hz), 3.01 (s, 3H), 2.25 (s, 3H); C-NMR (100 MHz, CDCl ) δ 173.2, 169.6, 158.2, 146.9, 141.5, 130.5, 129.8, 128.5, 127.8, 127.6, 126.7, 125.9, 114.7, 112.5, 76.4, 65.3, 52.2, 50.6, 39.3, 37.7, 20.2. n
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5-(4-{2-[(4-Methoxyphenyl)methylamino]ethoxy}benzyl)-3-tritylthiazolidine-2,4-dione (6b)
This compound was prepared using the procedure described for the preparation of : Yield 22%; R = 0.31 ( -hexane/EtOAc = 3:1); IR (neat) 3059, 3023, 2933, 2833, 1693, 1683, 1514, 1300, 1246, 753 cm ; H-NMR (400 MHz, CDCl ) δ 7.32-7.15 (m, 15H), 7.10 (d, 2H, = 8.4 Hz), 6.84 (d, 2H, = 9.2 Hz), 6.81 (d, 2H, = 8.8 Hz), 6.75 (d, 2H, = 8.8 Hz), 4.35 (dd, 1H, = 8.8, 4.0 Hz), 4.09 (t, 2H, = 6.0 Hz), 3.75 (s, 3H), 3.67 (t, 2H, = 6.0 Hz), 3.40 (dd, 1H, = 14.0, 4.0 Hz), 3.05 (dd, 1H, = 14.0, 8.8 Hz), 2.98 (s, 3H); C-NMR (100 MHz, CDCl ) δ 173.2, 169.6, 158.2, 151.8, 143.9, 141.5, 130.5, 128.5, 127.8, 127.5, 126.7, 114.8, 114.7, 114.3, 76.4, 65.4, 55.8, 53.0, 50.6, 39.7, 37.7. 6a
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5-(4-{2-[(4-Isopropylphenyl)methylamino]ethoxy}benzyl)-3-tritylthiazolidine-2,4-dione (6c)
This compound was prepared using the procedure described for the preparation of : Yield 27%; R = 0.57 ( -hexane/EtOAc = 3:1); IR (neat) 3058, 3026, 2957, 2925, 2869, 1692, 1512, 1299, 1248, 1133, 754 cm ; HNMR (400 MHz, CDCl ) δ 7.31-7.13 (m, 15H), 7.10 (d, 2H, = 8.4 Hz), 7.09 (d, 2H, = 8.4 Hz), 6.80 (d, 2H, = 8.4 Hz), 6.70 (d, 2H, = 8.8 Hz), 4.35 (dd, 1H, = 8.8, 4.0 Hz), 4.10 (t, 2H, = 6.0 Hz), 3.72 (t, 2H, = 6.0 Hz), 3.40 (dd, 1H, = 14.0, 4.0 Hz), 3.05 (dd, 1H, = 14.0, 8.8 Hz), 3.02 (s, 3H), 2.82 (sp, 1H, = 6.8 Hz), 1.22 (d, 6H, = 6.8 Hz); C NMR (100 MHz, CDCl ) δ 173.2, 169.6, 158.2, 147.1, 141.5, 137.0, 130.5, 128.5, 127.8, 127.5, 127.1, 126.7, 114.7, 112.2, 76.4, 65.3, 52.1, 50.6, 39.2, 37.7, 33.0, 24.2. 6a
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( -hexane/EtOAc = 3:1); IR (neat) 3059, 3030, 2958, 2925, 2869, 1688, 1515, 1299, 1248, 733, 704 cm ; HNMR (400 MHz, CDCl ) δ 7.32-7.14 (m, 17H), 7.10 (d, 2H, = 8.8 Hz), 6.81 (d, 2H, = 8.8 Hz), 6.71 (d, 2H, = 8.8 Hz), 4.36 (dd, 1H, = 8.8, 4.0 Hz), 4.10 (t, 2H, = 6.0 Hz), 3.73 (t, 2H, = 6.0 Hz), 3.40 (dd, 1H, = 14.0, 4.0 Hz), 3.05 (dd, 1H, = 14.0, 8.8 Hz), 3.03 (s, 3H), 1.29 (s, 9H); C-NMR (100 MHz, CDCl ) δ 173.2, 169.6, 158.2, 146.7, 141.5, 139.2, 130.5, 128.5, 127.8, 127.5, 126.7, 126.1, 114.7, 111.9, 76.4, 65.3, 52.0, 50.6, 39.2, 37.7, 33.7, 31.5. n
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5-{4-[2-(Methyl-p-tolylamino)ethoxy]benzyl}thiazolidine-2,4-dione (7a)
A mixture of (86.8 mg, 0.14 mmol) and trifluoroacetic acid (0.77 mL, 9.90 mmol) was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and neutralized by the addition of saturated potassium carbonated solution, then extracted with ethyl acetate. The combined organic extracts were washed with water and brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (eluent, -hexane/EtOAc = 3:1) to yield the title compound (35.5 mg, 68%): R = 0.20 ( -hexane/EtOAc=3:1); IR (neat) 3209, 3035, 2922, 2870, 1748, 1695, 1683, 1613, 1516, 1246, 732 cm ; H-NMR (400 M Hz, CDCl ) δ 7.11 (d, 2H, = 8.4 Hz), 7.05 (d, 2H, = 8.0 Hz), 6.82 (d, 2H, = 8.8 Hz), 6.69 (d, 2H, = 8.4 Hz), 4.47 (dd, 1H, = 9.2, 4.0 Hz), 4.10 (t, 2H, = 6.0 Hz), 3.71 (t, 2H, =6.0 Hz), 3.43 (dd, 1H, = 14.0, 4.0 Hz), 3.07 (dd, 1H, = 14.0, 9.2 Hz), 3.00 (s, 3H), 2.25 (s, 3H); C-NMR (100 MHz, CDCl ) δ 174.2, 170.5, 158.2, 146.8, 130.3, 129.8, 127.8, 114.7, 112.6, 65.2, 53.7, 52.2, 39.3, 37.7, 20.2. 6a
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5-(4-{2-[(4-Methoxyphenyl)methylamino]ethoxy}benzyl)thiazolidine-2,4-dione (7b)
This compound was prepared using the procedure described for the preparation of : Yield 37%; R = 0.10 ( -hexane/EtOAc = 3:1); IR (neat) 3193, 3064, 2933, 2834, 1752, 1696, 1513, 1246, cm ; H-NMR (400 MHz, CDCl ) δ (d, 2H, = 8.4 Hz), 6.84 (d, 2H, = 9.2 Hz), 6.82 (d, 2H, = 9.2 Hz), 6.76 (d, 2H, = 9.2 Hz), 4.48 (dd, 1H, = 9.6, 4.0 Hz), 4.09 (t, 2H, = 6.0 Hz), 3.76 (s, 3H), 3.66 (t, 2H, = 6.0 Hz), 3.43 (dd, 1H, J = 14.0, 4.0 Hz), 3.09 (dd, 1H, = 14.0, 9.6 Hz), 2.97 (s, 3H); C-NMR (100 MHz, CDCl ) δ 173.9, 170.2, 158.2, 151.9, 143.9, 130.3, 127.7, 114.8, 114.8, 114.5, 65.4, 55.8, 53.7, 53.1, 39.8, 37.7. 7a
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5-(4-{2-[(4-tert-Butylphenyl)methylamino]ethoxy}benzyl)-3-tritylthiazolidine-2,4-dione (6d) 5-(4-{2-[(4-Isopropylphenyl)methylamino]ethoxy}benThis compound was prepared using the procedure zyl)thiazolidine-2,4-dione (7c) described for the preparation of
: Yield 12%; R = 0.35
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This compound was prepared using the procedure
Synthesis and Biological Activity of Thiazolidinedione Derivatives
described for the preparation of : Yield 74%; R = 0.21 ( -hexane/EtOAc = 3:1); IR (neat) 3216, 3034, 2958, 2871, 2814, 1753, 1696, 1612, 1515, 1247, 756 cm ; HNMR (400 MHz, CDCl ) δ 7.12-7.11 (m, 4H), 6.82 (d, 2H, = 8.8 Hz), 6.71 (d, 2H, = 8.4 Hz), 4.48 (dd, 1H, = 9.6, 3.6 Hz), 4.10 (t, 2H, = 6.0 Hz), 3.71 (t, 2H, = 6.0 Hz), 3.43 (dd, 1H, = 14.4, 3.6 Hz), 3.08 (dd, 1H, = 14.4, 9.6 Hz), 3.02 (s, 3H), 2.82 (sp, 1H, = 6.8 Hz), 1.22 (d, 6H, = 6.4 Hz); C-NMR (100 MHz, CDCl ) δ 174.2, 170.4, 158.2, 147.1, 137.1, 130.1, 130.3, 127.8, 127.1, 114.7, 112.3, 65.3, 53.7, 52.1, 39.2, 37.7, 33.0, 24.2. 7a
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5-(4-{2-[(4-tert-Butylphenyl)methylamino]ethoxy}benzyl)thiazolidine-2,4-dione (7d)
This compound was prepared using the procedure described for the preparation of : Yield 72%; R = 0.23 ( -hexane/EtOAc = 3:1); IR (neat) 3217, 3039, 2961, 2868, 1754, 1695, 1682, 1613, 1515, 1248, 733 cm ; HNMR (400 MHz, CDCl ) δ 7.27 (d, 2H, = 9.2 Hz), 7.12 (d, 2H, = 8.4 Hz), 6.83 (d, 2H, = 8.8 Hz), 6.72 (d, 2H, = 8.4 Hz), 4.49 (dd, 1H, = 9.6, 4.0 Hz), 4.11 (t, 2H, = 6.0 Hz), 3.72 (t, 2H, = 6.0 Hz), 3.44 (dd, 1H, = 14.0, 4.0 Hz), 3.14 (dd, 1H, = 14.0, 9.6 Hz), 3.03 (s, 3H), 1.29 (s, 9H); C-NMR (100 MHz, CDCl ) δ 173.9, 170.1, 158.2, 146.6, 139.5, 130.3, 114.8, 111.9, 65.3, 53.7, 52.0, 39.2, 37.7, 33.7, 31.5. 7a
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PPAR Transactivation assay
GAL4 fusions were made by fusing murine PPAR ligand binding domain to the C-terminal end of yeast GAL4 DNA binding domain. CV-1 cells were seeded at 2 × 10 cells/ well and cultured for 24 h at 37°C. Cells were cotransfected for 3 h at 37 C with pUAS, pRL-TK, and pCMX-GalPx. Transfected cells were treated with 2 µM of the test compounds for 24 h. DMSO (0.1%) was used as a blank. 4
o
Scheme 1. Preparation of 2-(methyl-p-substituted phenylamino)ethanols
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GW409544, which is a potent full agonist of both PPARα and PPARγ was used as positive control. Luciferase activity was determined as ‘fold activation’ relative to the positive control.
Nitrite assay
: The murine macrophage cell line (RAW 264.7) was obtained from American Type Culture Collection (Rockville, MD, U.S.A.). Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 U/mL) and streptomycin (10 mg/mL). Cells were grown at 37 C, in an atmosphere of 5% CO in fully humidified air, and were split twice a week. RAW 264.7 cells were seeded at 5×10 cells/mL in 24-well plates and activated for 20 h by incubation in 1% FBS medium containing LPS (1 µg/mL) and various concentrations of test compounds dissolved in DMSO (final concentration 0.1 % in media). The supernatant was collected as a source of secreted NO. NO released from macrophages was assessed by the determination of NO − concentration in the culture supernatant. Samples (100 µL) of culture media were incubated with 150 µL Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine in 2.5% phosphoric acid solution) at room temperature for 10 min in 96-well microplates (Green, ., 1982). Absorbance at 540 nm was read using an ELISA plate reader. Standard calibration curves were prepared using sodium nitrite. Cell culture
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RESULTS AND DISCUSSION Preparation of the lipophilic tail, 2-(methyl- -substituted phenylamino)ethanol is outlined in Scheme 1. Formylation of the -substituted anilines - was followed by reduction with lithium aluminum hydride to give p
para
1a d
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Hyo Jin Gim et al.
Scheme 2. Preparation of 5-{4-[2-(methyl-p-substituted phenylamino)ethoxy]benzyl}thiazolidine-2,4-diones
substituted phenylmethylamines - , which were converted to compounds - by -alkylation of the compounds - with iodoethanol in the presence of sodium hydride. The target compounds were prepared by reaction of compound with 5-(4-hydroxybenzyl)thiazolidine-2,4-dione ( ), which was prepared by modification of the reported method (Morita , 1998), under Mitsunobu conditions using tributylphosphine and 1,1’-(azodicarbonyl)dipiperidine (ADDP). Removal of the trityl group of the resulting with trifluoroactic acid gave the final compounds (Scheme 2). The prepared compounds were evaluated for their ability to activate murine PPARα and PPARγ in a transactivation assay in CV-1 cells. The results are shown in Table I. The tested compounds showed very marginal PPARγ activities with no PPARα activities. The most active compound , which was substituted with isopropyl at the position of phenyl ring, caused 21% transactivation of PPARγ compared to the positive control GW409544. In addition, the amount of NO accumulation in LPSactivated RAW 264.7 cells was measured. Most of the compounds tested caused less than 20% inhibition of NO production except compound , which caused 52% inhibition. Although the activities of the compounds were low to moderate, the tendency of the compounds to inhibit LPS-induced NO production was consistent with their ability to transactivate PPARγ. Compound was the most active activator of PPARγ and inhibitor of NO production. In summary, we synthesized 5-{4-[2-(methyl- -substituted phenylamino)ethoxy]benzyl}thiazolidine-2,4-diones and evaluated their biological activities on PPARγ activation and inhibition of NO production. Although the activities of the compounds were very low according to our criteria, 3a d
4a d
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In vitro activities of the compounds on transactivation of PPARa and inhibition of NO productionb Table I.
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7c
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Compound
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7a 7b 7c 7d
Me OCH3 i-Pr t-Bu
Transactivation (RR %)a PPARα
PPARγ
Inhibition (%)b
NAc 2.4 13.0 NA 10.7 10.9 NA 21.2 51.5 NA 9.8 18.0 GW409544 100 100.0 KRP-297 NA 63.3 Compounds were assayed for agonist activity on PPAR-GAL4 chimeric receptors in transiently transfected CV-1 cells as described in the Materials and Methods section. Data are represented as relative response (RR%), which is [(test drug-negative control)/(positive control-negative control)] × 100. GW409544 and DMSO were used as positive and negative controls, respectively. Cells were incubated with LPS (1 mg/mL) in the presence or absence of each of the compounds. After treatment, NO accumulation was measured. Unstimulated cells served as controls. Values represent the inhibition (%) of NO production relative to the LPS control at 20 mM concentration of the compounds. Each value is the mean ± S.D. of three determinations. NA: no activity. c
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this initial data showed us that it probably would be possible to design more promising compounds. In particular, our findings that the agonistic action of the compounds on PPARγ correlated with inhibitory action against NO production suggest that our PPARγ activator might be a useful therapeutic candidate in the treatment of inflammatory diseases as well as for the treatment of type 2 diabetes. Further SAR study on the lipophilic tail of the TZD derivatives is currently being performed.
Synthesis and Biological Activity of Thiazolidinedione Derivatives
ACKNOWLEDGEMENTS This research was supported by the Sookmyung Women's University Research Grants (2005). The authors would like to thank Dong-A Pharm. Co., Ltd. and Prof. Jae-Ha Ryu for providing biological data form the PPAR transactivation assay and nitrite assay, respectively.
REFERENCES Alleva, D. G., Johnson, E. B., Lio, F. M., Boehme, S. A., Conlon, P. J., and Crowe, P. D., Regulation of murine macrophage proinflammatory and anti-inflammatory cytokines by ligands for peroxisome proliferator-activated receptor-gamma: counterregulatory activity by IFN-gamma. J. Leukoc. Biol., 71, 677685 (2002). Aronoff, S., Rosenblatt, S., Braithwaite, S., Egan, J. W., Mathisen, A. L., and Schneider, R. L., Pioglitazone hydrochloride monotherapy improves glycemic control in the treatment of patients with type 2 diabetes: a 6-month randomized placebo-controlled dose-response study. The Pioglitazone 001 Study Group. Diabetes Care, 23, 1605-1611 (2000). Berger, J. and Moller, D. E., The mechanisms of action of PPARs. Annu. Rev. Med., 53, 409-435 (2002). Cantello, B. C., Cawthorne, M. A., Cottam, G. P., Duff, P. T., Haigh, D., Hindley, R. M., Lister, C. A., Smith, S. A., and Thurlby, P. L., [[omega-(Heterocyclylamino)alkoxy]benzyl]2,4-thiazolidinediones as potent antihyperglycemic agents. J. Med. Chem., 37, 3977-3985 (1994). Colville-Nash, P. R., Qureshi, S. S., Willis, D., and Willoughby, D. A., Inhibition of inducible nitric oxide synthase by peroxisome proliferator-activated receptor agonists: correlation with induction of heme oxygenase 1. J. Immunol., 161, 978-984 (1998). Crosby, M. B., Svenson, J., Gilkeson, G. S., and Nowling, T. K., A novel PPAR response element in the murine iNOS promoter. Mol. Immunol., 42, 1303-1310 (2005). Desvergne, B. and Wahli, W., Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev., 20, 649-688 (1999). Dobashi, K., Asayama, K., Nakane, T., Kodera, K., Hayashibe, H., and Nakazawa, S., Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo study in 3T3-L1 cells and Otsuka Long-Evans Tokushima Fatty rats. Life Sci., 67, 2093-2101 (2000). Heneka, M. T., Feinstein, D. L., Galea, E., Gleichmann, M., Wullner, U., and Klockgether, T., Peroxisome proliferatoractivated receptor gamma agonists protect cerebellar granule cells from cytokine-induced apoptotic cell death by inhibition of inducible nitric oxide synthase. J. Neuroimmunol., 100, 156-168 (1999). Henke, B. R., Blanchard, S. G., Brackeen, M. F., Brown, K. K., Cobb, J. E., Collins, J. L., Harrington, W. W., Jr., Hashim, M. A., Hull-Ryde, E. A., Kaldor, I., Kliewer, S. A., Lake, D. H.,
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Leesnitzer, L. M., Lehmann, J. M., Lenhard, J. M., OrbandMiller, L. A., Miller, J. F., Mook, R. A., Jr., Noble, S. A., Oliver, W., Jr., Parks, D. J., Plunket, K. D., Szewczyk, J. R., and Willson, T. M., N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents. J. Med. Chem., 41, 5020-5036 (1998). Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R., Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem., 126, 131-138 (1982). Ibrahimi, A., Teboul, L., Gaillard, D., Amri, E. Z., Ailhaud, G., Young, P., Cawthorne, M. A., and Grimaldi, P. A., Evidence for a common mechanism of action for fatty acids and thiazolidinedione antidiabetic agents on gene expression in preadipose cells. Mol. Pharmacol., 46, 1070-1076 (1994). Jiang, C., Ting, A. T., and Seed, B., PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature, 391, 82-86 (1998). Kersten, S., Desvergne, B., and Wahli, W., Roles of PPARs in health and disease. Nature, 405, 421-424 (2000). Kletzien, R. F., Clarke, S. D., and Ulrich, R. G. Enhancement of adipocyte differentiation by an insulin-sensitizing agent. Mol. Pharmacol., 41, 393-398 (1992). Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and Kliewer, S. A., An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J. Biol. Chem., 270, 12953-12956 (1995). Lohray, B. B., Bhushan, V., Rao, B. P., Madhavan, G. R., Murali, N., Rao, K. N., Reddy, A. K., Rajesh, B. M., Reddy, P. G., Chakrabarti, R., Vikramadithyan, R. K., Rajagopalan, R., Mamidi, R. N., Jajoo, H. K., and Subramaniam, S., Novel euglycemic and hypolipidemic agents. 1. J. Med. Chem., 41, 1619-1630 (1998). Oguchi, M., Wada, K., Honma, H., Tanaka, A., Kaneko, T., Sakakibara, S., Ohsumi, J., Serizawa, N., Fujiwara, T., Horikoshi, H., and Fujita, T., Molecular design, synthesis, and hypoglycemic activity of a series of thiazolidine-2,4-diones. J. Med. Chem., 43, 3052-3066 (2000). Reilly, C. M., Oates, J. C., Cook, J. A., Morrow, J. D., Halushka, P. V.,and Gilkeson, G. S., Inhibition of mesangial cell nitric oxide in MRL/lpr mice by prostaglandin J2 and proliferator activation receptor-gamma agonists. J. Immunol., 164, 14981504 (2000). Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K., The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature, 391, 79-82 (1998). Sohda, T., Momose, Y., Meguro, K., Kawamatsu, Y., Sugiyama, Y., and Ikeda, H., Studies on antidiabetic agents. Synthesis and hypoglycemic activity of 5-[4-(pyridylalkoxy)benzyl]-2,4thiazolidinediones. Arzneimittelforschung, 40, 37-42 (1990).
