Discovery of 4-(phenyl)thio-1H-pyrazole derivatives as ... - Springer Link

Arch. Pharm. Res. (2015) 38:1019–1032 DOI 10.1007/s12272-015-0560-4

RESEARCH ARTICLE

Discovery of 4-(phenyl)thio-1H-pyrazole derivatives as agonists of GPR109A, a high affinity niacin receptor Hyeon Young Kim • Vithal B. Jadhav • Dae Young Jeong • Woo Kyu Park • Jong-Hwan Song • Sunkyung Lee • Heeyeong Cho

Received: 29 September 2014 / Accepted: 8 January 2015 / Published online: 20 January 2015 Ó The Pharmaceutical Society of Korea 2015

Abstract Even though nicotinic acid (niacin) appears to have beneficial effects on human lipid profiles, niacininduced cutaneous vasodilatation called flushing limits its remedy to patient. GPR109A is activated by niacin and mediates the anti-lipolytic effects. Based on the hypothesis that b-arrestin signaling mediates niacin-induced flushing, but not its anti-lipolytic effect, we tried to find GPR109A agonists which selectively elicit Gi-protein-biased signaling devoid of b-arrestin internalization using a b-lactamase assay. We identified a 4-(phenyl)thio-1H-pyrazole as a novel scaffold for GPR109A agonist in a high throughput screen, which has no carboxylic acid moiety known to be important for binding. While 1-nicotinoyl derivatives (5a– g, 6a–e) induced b-arrestin recruitment, 1-(pyrazin-2-oyl) derivatives were found to play as G-protein-biased agonists without GPR109A receptor internalization. The activity of compound 5a (EC50 = 45 nM) was similar to niacin (EC50 = 52 nM) and MK-6892 (EC50 = 74 nM) on calcium mobilization assay, but its activity at 10 lM on

H. Y. Kim D. Y. Jeong W. K. Park H. Cho (&) Research Center for Drug Discovery Technology, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong, Daejeon 305-343, Republic of Korea e-mail: [email protected] V. B. Jadhav J.-H. Song S. Lee (&) Research Center for Medicinal Chemistry, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong, Daejeon 305-343, Republic of Korea e-mail: [email protected] S. Lee H. Cho Korea University of Science and Technology, 141 Gajeong-ro, Yuseong, Daejeon 305-343, Republic of Korea

b-arrestin recruitment were around two and five times weaker than niacin and MK-6892, respectively. The development of G-protein biased GPR109A ligands over b-arrestin pathway is attainable and might be important in differentiation of pharmacological efficacy. Keywords 4-(Phenylthio)-1H-pyrazole Niacin GPR109A G-protein b-Arrestin Biased agonist

Introduction Since Altschul et al. discovered nicotinic acid (niacin) lowered plasma cholesterol (Altschul et al. 1955; Parsons Jr and Flinn 1959), it has been used as a broad-spectrum antiatherosclerosis agent for over 50 years because of its beneficial effects on lipid profiles in human (Carlson 2005), lowering levels of very-low-density lipoprotein (LDL), LDL, and triglycerides, while raising levels of high-density lipoprotein cholesterol (Benyo et al. 2005; Hanson et al. 2010; Eaton et al. 1969; Ganji et al. 2004; Le Goff et al. 2004). However, its clinical use to patient is limited by the severe side effects of cutaneous vasodilation called flushing (Benyo et al. 2005; Hanson et al. 2010). GPR109A (hydroxyl-carboxylic acid receptor 2, human HM74, mouse PUMA-G) was identified in 2003 as a specific and high affinity receptor for niacin and it was known to be expressed primarily on human adipose tissue, Langerhans cells, and macrophage (Soga et al. 2003; Tunaru et al. 2003; Offermanns 2006). It mediates the anti-lipolytic effects of niacin in Gi-coupled manner, leading to reduced hydrolysis of triglycerides to free fatty acids (FFAs) although the precise mechanism remains elusive (Gaidarov et al. 2013). The flushing side effect of niacin has been also induced by a GPR109A dependent vasodilation through the

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generation of prostaglandin D2 from Langerhans cells in the skin (Wise et al. 2003; Li et al. 2010). G proteins can be activated independently from receptor internalization which is regulated by G protein-coupled receptor kinases (GRKs) and b-arrestin by agonist binding to GPR109A (Li et al. 2010). Walters et al. demonstrated that flushing is mediated by b-arrestin, but anti-lipolytic effect is not (Walters et al. 2009). In another study, GPR109A agonists that led to a flushing response induce both ERK1/2 mitogen-activated protein kinase phosphorylation and receptor internalization, whereas non-flushing compounds do not (Richman et al. 2007; Lai et al. 2008; Boatman et al. 2012). The biased GPR109A agonist, MK-0354 activates the antilipolytic pathways in adipose cells but does not signal via ERK1/2 pathways to induce PG production and vasodilation (Lai et al. 2008). These selective agonists may provide therapeutic benefit for anti-atherosclerotic drugs. We tried to identify novel scaffolds of GPR109A agonist, and found a 4-thiopyrazole derivative (5a) in a high throughput screen (HTS) without carboxylic acid moiety known to be important for binding (Jung et al. 2007; Soudijn et al. 2007; Deng et al. 2008), even though there are exceptions (Palani et al. 2012). The synthesis and evaluation including calcium mobilization, b-arrestin recruitment, and induction of receptor internalization assay of these 4-(phenyl)thio-1H-pyrazole derivatives is described here to identify selective GPR109A agonists.

H. Y. Kim et al.

methanol (3 mL) was added and the solution was stirred for 6 h at room temperature. Precipitated piperidine hydrochloride was removed by filtration and washed with ether (3 9 100 mL). The combined filtrates were washed with water (5 9 150 mL) and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (2 % ethyl acetate in hexane) to yield the product. 3-[(4-Chlorophenyl)thio]pentane-2,4-dione (1a) Yield 91 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.31 (s, 6H), 4.32 (s, 1H), 7.34 (d, 2H, J = 8.9 Hz), 7.41 (d, 2H, J = 8.9 Hz). MS 243 (M?). 3-[(4-Bromophenyl)thio]pentane-2,4-dione (1b) Yield 86 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.32 (s, 6H), 4.28 (s, 1H), 7.45 (d, 2H, J = 8.4 Hz), 7.82 (d, 2H, J = 8.4 Hz). MS 286, 288 (M?). 3-[(2-Bromophenyl)thio]pentane-2,4-dione (1c) Yield 92 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.28 (s, 6H), 4.27 (s, 1H), 6.83 (d, 1H, J = 8.7 Hz), 7.01 (m, 1H), 7.24 (m, 1H), 7.52 (d, 1H, J = 8.1 Hz). MS 286, 288 (M?). 3-[(2-Chlorophenyl)thio]pentane-2,4-dione (1d)

Materials and methods Chemistry Anhydrous solvents were dried by conventional methods. Reagents of commercial quality were used from freshly opened containers unless otherwise stated. For purification of products by column chromatography, Merck Silica gel 60 (230–400 mesh) was used. 1H NMR spectra were recorded on a Varian Gemini 300 with TMS as an internal standard. Chemical shifts are reported in d (ppm). Mass spectra (MS) were obtained with a Bruker instrument by using electron impact techniques, and high resolution MS (HRMS) of key compounds were determined.

General procedure for the syntheses of 3-(phenylthio)pentane-2,4-dione 1a–g

Yield 91 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.31 (s, 6H), 4.25 (s, 1H), 6.85 (d, 1H, J = 7.2 Hz), 7.10 (dd, 1H, J = 7.2, 7.4 Hz), 7.18 (dd, 1H, J = 7.4, 7.8 Hz), 7.35 (d, 1H, J = 7.8 Hz). MS 243 (M?). 3-[(4-(Dichloro)phenyl)thio]pentane-2,4-dione (1e) Yield 90 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.34 (s, 6H), 4.25 (s, 1H), 6.93 (d, 2H, J = 3.3 Hz), 7.12 (d, 1H, J = 3.3 Hz). MS 257 (M?). 3-[(4-(Tert-butyl)phenyl)thio]pentane-2,4-dione (1f) Yield 97 %. Off white solid. 1H NMR (CDCl3, d, ppm): 1.29 (s, 9H), 2.34 (s, 6H), 4.25 (s, 1H), 7.02 (d, 2H), 7.30 (d, 1H). MS 264 (M?). 3-[(4-Acetamidophenyl)thio]pentane-2,4-dione (1g)

To a mixture of 3-chloro-2,4-pentanedione (1 g, 7.4 mmol) and benzenethiol (1.1 g, 7.4 mmol) was dropwise added a solution of piperidine (7.4 mmol) in dichloromethane (0.8 mL) at 0 °C over 10 min. After stirring for 5 min,

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Yield 97 %. Off white solid. 1H NMR (CDCl3, d, ppm): 1.29 (s, 9H), 2.34 (s, 6H), 4.25 (s, 1H, J = 8.7 Hz), 7.02 (d, 2H, J = 8.7 Hz), 7.30 (brs, 1H). MS 265 (M?).

Discovery of 4-(phenyl)thio-1H-pyrazole derivatives

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General procedure for the syntheses of 3,5-dimethyl-4(phenylthio)-1H-pyrazoles 3a–g

General procedure for the syntheses of 3-methyl-5phenyl-4-(phenylthio)-1H-pyrazoles 4a–e

To a solution of 3-(phenylthio)-pentane-2,4-dione 1a– g (13.2 mmol) in ethanol (10 mL) was added hydrazine hydrate (0.8 mL, 16.5 mmol) dropwise with vigorous stirring. The mixture became homogeneous on heating and continuously heated at reflux overnight, cooled to room temperature, and then concentrated under reduced pressure. The residue was recrystallized from benzene/hexane (20/ 150 mL) to yield product 3a–g.

To a mixture of 2-chloro-1-phenyl-butane-1,3-dione (5.64 mmol) and benzenethiol (5.64 mmol) was dropwise added a solution of piperidine (0.55 mL, 5.64 mmol) in dichloromethane (0.8 mL) at 0 °C over 10 min. After stirring for 5 min, methanol (3 mL) was added and the solution was stirred for 6 h at rt. Precipitated piperidine hydrochloride was removed by filtration and washed with ether (3 9 100 mL). The combined filtrates were washed with water (5 9 150 mL) and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue (2a–e) was taken in ethanol (10 mL) and hydrazine hydrate (0.32 mL, 6.7 mmol) was added to it. The reaction mixture was heated at reflux for 4 h. All volatiles were removed under reduced pressure, and the residue was purified by silica gel column chromatography (10 % ethyl acetate in hexane) to yield the product 4a–e.

4-[(4-Chlorophenyl)thio]-3,5-dimethyl-1H-pyrazole (3a) Yield 64 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.78 (s, 6H), 7.32 (d, 2H, J = 7.2 Hz), 7.75 (d, 2H, J = 7.2 Hz). MS 238 (M?). 4-[(4-Bromophenyl)thio]-3,5-dimethyl-1H-pyrazole (3b) Yield 79 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.77 (s, 6H), 7.32 (d, 2H, J = 8.2 Hz), 7.75 (d, 2H, J = 8.2 Hz). MS 282, 284 (M?).

4-[(4-Bromophenyl)thio]-5-methyl-3-phenyl-1H-pyrazole (4a)

4-[(2-Bromophenyl)thio]-3,5-dimethyl-1H-pyrazole (3c)

Yield 58 % over two steps. Off white solid. 1H NMR (CDCl3, d, ppm): 2.28 (s, 3H), 6.88 (d, J = 8.7 Hz, 2H), 7.27–7.37 (m, 5H), 7.73 (m, 2H). MS 344, 346 (M?).

Yield 91 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.28 (s, 6H), 6.51 (d, 1H, J = 8.4 Hz), 6.95 (dd, 1H, J = 7.8, 8.1 Hz), 7.10 (dd, 1H, J = 7.8, 8.4 Hz), 7.50 (d, 1H, J = 8.1 Hz). MS 282, 284 (M?).

4-[(2-Bromophenyl)thio]-5-methyl-3-phenyl-1H-pyrazole (4b)

4-[(2-Chlorophenyl)thio]-3,5-dimethyl-1H-pyrazole (3d) Yield 64 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.88 (s, 6H), 6.53 (m, 1H), 7.03 (m, 2H), 7.32 (m, 1H). MS 238 (M?).

Yield 33 % over two steps. Off white solid. 1H NMR (CDCl3, d, ppm): 2.29 (s, 3H), 6.63 (dd, J = 1.5, 6.6 Hz, 1H), 6.96 (m, 1H), 7.35–7.40 (m, 3H), 7.50 (dd, J = 1.2, 6.6 Hz, 1H), 7.72 (m, 2H). MS 344, 346 (M?).

4-[(3,5-Dichlorophenyl)thio]-3,5-dimethyl-1H-pyrazole (3e)

4-[(2-Chlorophenyl)thio]-5-methyl-3-phenyl-1H-pyrazole (4c)

Yield 78 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.27 (s, 6H), 6.81 (d, 2H, J = 1.8 Hz), 7.06 (d, 1H, J = 1.8 Hz). MS 273 (M?).

Yield 39 % over two steps. Off white solid. 1H NMR (CDCl3, d, ppm): 2.30 (s, 3H), 6.65 (m, 1H), 7.03 (m, 2H), 7.31–7.38 (m, 4H), 7.72 (m, 2H). MS 300 (M?).

4-[(4-(Tert-butyl)phenyl)thio]-3,5-dimethyl-1H-pyrazole (3f)

4-[(3,5-Dichlorophenyl)thio]-5-methyl-3-phenyl-1Hpyrazole (4d)

Yield 54 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.27 (s, 6H), 6.92 (d, 2H, J = 3.3 Hz), 7.22 (d, 2H). MS 260 (M?). 4-[(4-Acetamidophenyl)thio]-3,5-dimethyl-1H-pyrazole (3g)

Yield 73 % over two steps. Off white solid. 1H NMR (CDCl3, d, ppm): 2.30 (s, 3H), 6.86 (d, J = 1.8 Hz, 2H), 7.06 (m, 1H), 7.38–7.43 (m, 3H), 7.69–7.72 (m, 2H). MS 335 (M?). 4-(Phenylthio)-5-methyl-3-phenyl-1H-pyrazole (4e)

Yield 75 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.15 (s, 3H), 2.25 (s, 6H, J = 8.1 Hz), 6.95 (d, 2H, J = 8.1 Hz), 7.05 (brs, 1H), 7.34 (d, 2H). MS 261 (M?).

Yield 43 % over two steps. Off white solid. 1H NMR (CDCl3, d, ppm): 2.29 (s, 3H), 6.63 (m, 2H), 7.09 (m, 1H),

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7.35–7.40 (m, 4H), 7.50 (m, 2H), 7.72 (m, 2H). MS 266 (M?).

1-(Pyridin-3-yl)carbonyl-4-[(3,5-dichlorophenyl)thio]-3,5dimethyl-1H-pyrazole (5e)

General procedure for the syntheses of 1-(pyridin-3-yl) carbonyl-4-(phenylthio)-1H-pyrazoles 5a–g, 6a–e, 7a, b

Yield 88 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.21 (s, 3H), 2.74 (s, 3H), 6.68 (s, 2H), 7.13 (s, 1H), 7.45 (m, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.81 (d, J = 3.3 Hz, 1H), 9.26 (s, 1H). HRMS calcd. for C17H13Cl2N3OS (m/z) 377.0156; found 377.0152.

To a solution of 4-(phenylthio)-1H-pyrazole 3a–g, 4a– e (0.21 mmol) in THF (5 mL) were added nicotinyl chloride (0.42 mmol), triethylamine (58 lL, 0.42 mmol), and N,N-dimethylaminopyridine (DMAP; 5 mg, 0.042 mmol). The suspension was stirred at rt for 2 h until the starting material was disappeared on TLC monitoring, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (15 % ethyl acetate in hexane) to give the product 5a–g, 6a–e, 7a, b. 1-(Pyridin-3-yl)carbonyl-4-[(4-chlorophenyl)thio]-3,5dimethyl-1H-pyrazole (5a) Yield 89 %. Off white amorphous solid. 1H NMR (CDCl3, d, ppm): 2.08 (s, 3H), 2.53 (s, 3H), 6.84 (d, 2H, J = 9.0 Hz), 7.25 (d, 2H, J = 9.0 Hz), 7.43 (m, 1H), 8.31(m, 1H), 8.79 (dd, 1H, J = 1.8, 3.0 Hz), 9.23 (d, 1H, J = 1.8 Hz). HRMS calcd. for C17H14ClN3OS (m/z) 343.0546; found 343.0543. 1-(Pyridin-3-yl)carbonyl-4-[(4-bromophenyl)thio]-3,5dimethyl-1H-pyrazole (5b) Yield 90 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.08 (s, 3H), 2.52 (s, 3H), 6.51 (d, 2H, J = 9.4 Hz), 7.42 (d, 2H, J = 8.4 Hz), 7.44 (m, 1H), 8.32 (d, 1H, J = 8.1 Hz), 8.77 (m, 1H), 9.27 (s, 1H). HRMS calcd. for C17H14BrN3OS (m/z) 387.0041; found 387.0039. 1-(Pyridin-3-yl)carbonyl-4-[(2-bromophenyl)thio]-3,5dimethyl-1H-pyrazole (5c) 1

Yield 90 %. Off white solid. H NMR (CDCl3, d, ppm): 2.21 (s, 3H), 2.72 (s, 3H), 6.57 (d, J = 7.8 Hz, 1H), 6.99 (dd, J = 7.5, 7.5 Hz, 1H), 7.15 (dd, J = 7.5, 7.5 Hz, 1H), 7.46 (m, 1H), 7.54 (d, J = 8.1 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.80 (brs, 1H), 9.27 (s, 1H). HRMS calcd. for C17H14BrN3OS (m/z) 387.0041; found 387.0044. 1-(Pyridin-3-yl)carbonyl-4-[(2-chlorophenyl)thio]-3,5dimethyl-1H-pyrazole (5d) Yield 89 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.21 (s, 3H), 2.73 (s, 3H), 6.61 (m, 1H), 7.10 (m, 2H), 7.37 (m, 1H), 7.48 (m, 1H), 8.38 (d, J = 7.8 Hz, 1H), 8.81 (d, J = 3.9 Hz, 1H), 9.27 (s, 1H). HRMS calcd. for C17H14ClN3OS (m/z) 343.0546; found 343.0541.

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1-(Pyridin-3-yl)carbonyl-4-[(4-(tert-butyl)phenyl)thio]3,5-dimethyl-1H-pyrazole (5f) Yield 90 %. Off white solid. 1H NMR (CDCl3, d, ppm): 1.28 (s, 9H), 2.21 (s, 3H), 2.75 (s, 3H), 7.01 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.1 Hz, 1H), 7.44 (m, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.79 (d, J = 4.8 Hz, 1H), 9.26 (s, 1H). HRMS calcd. for C21H23N3OS (m/z) 365.1562; found 365.1572. 1-(Pyridin-3-yl)carbonyl-4-[(4-acetamidophenyl)thio]-3,5dimethyl-1H-pyrazole (5g) Yield 87 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.16 (s, 3H), 2.18 (s, 3H), 2.75 (s, 3H), 7.05 (d, J = 8.7 Hz, 2H), 7.36 (brs, 1H), 7.41 (d, J = 8.7 Hz, 1H), 7.47 (m, 1H), 8.34 (d, J = 8.1 Hz, 1H), 8.79 (d, J = 4.5 Hz, 1H), 9.23 (s, 1H). HRMS calcd. for C19H18N4O2S (m/z) 366.1150; found 366.1151. 1-(Pyridin-3-yl)carbonyl-4-[(4-bromophenyl)thio]-3methyl-5-phenyl-1H-pyrazole (6a) Yield 50 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.80 (s, 3H), 6.94 (d, J = 8.7 Hz, 2H), 7.37 (m, 5H), 7.48 (m, 1H), 7.88 (m, 2H), 8.44 (m, 1H), 8.82 (m, 1H), 9.35 (s, 1H). HRMS calcd. for C22H16BrN3OS (m/z) 449.0197; found 449.0191. 1-(Pyridin-3-yl)carbonyl-4-[(2-bromophenyl)thio]-3methyl-5-phenyl-1H-pyrazole (6b) Yield 60 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.78 (s, 3H), 6.67 (d, J = 8.1 Hz, 1H), 6.98 (dd, J = 7.5, 7.5 Hz, 1H), 7.12 (dd, J = 7.5, 7.5 Hz, 1H), 7.37 (m, 3H), 7.49 (m, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.88 (m, 2H), 8.40 (d, J = 4.5 Hz, 1H), 8.45 (d, J = 8.1 Hz, 1H), 9.37 (s, 1H). HRMS calcd. for C22H16BrN3OS (m/z) 449.0197; found 449.0194. 1-(Pyridin-3-yl)carbonyl-4-[(2-chlorophenyl)thio]-3methyl-5-phenyl-1H-pyrazole (6c) Yield 66 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.79 (s, 3H), 6.69 (m, 1H), 7.04–7.09 (m, 2H), 7.34–7.38


(m, 4H), 7.49 (m, 1H), 7.88 (m, 2H), 8.45 (m, 1H), 8.83 (m, 1H), 9.37 (d, J = 1.5 Hz, 1H). HRMS calcd. for C22H16ClN3OS (m/z) 405.0703; found 405.0710. 1-(Pyridin-3-yl)carbonyl-4-[(3,5-dichlorophenyl)thio]-3methyl-5-phenyl-1H-pyrazole (6d) Yield 60 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.81 (s, 3H), 6.91 (d, J = 1.8 Hz, 2H), 7.10 (m, 1H), 7.35–7.39 (3H), 7.48 (m, 1H), 7.86 (m, 2H), 8.45 (m, 1H), 9.37 (d, J = 2.1 Hz, 1H). HRMS calcd. for C22H15Cl2N3OS (m/z) 439.0313; found 439.0302. 1-(Pyridin-3-yl)carbonyl-4-(phenylthio)-3-methyl-5phenyl-1H-pyrazole (6e) Yield 60 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.77 (s, 3H), 6.67–6.98 (m, 3H), 7.12–7.35 (m, 4H), 7.49 (m, 1H), 7.54 (d, 1H), 7.82 (m, 2H), 8.40 (d, 1H), 8.45 (d, 1H), 9.36 (s, 1H). MS 371 (M?). 1-(6-Chloropyridin-3-yl)carbonyl-4-[(2bromophenyl)thio]-3,5-dimethyl-1H-pyrazole (7a) Yield 80 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.20 (s, 3H), 2.72 (s, 3H), 6.56 (d, J = 8.1 Hz, 1H), 7.01 (dd, J = 7.5, 7.5 Hz, 1H), 7.15 (dd, J = 7.5, 8.1 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.54 (d, J = 6.3 Hz, 1H), 8.34 (dd, J = 1.8, 6.3 Hz, 1H), 9.08 (d, J = 1.8 Hz, 1H). HRMS calcd. for C17H13BrClN3OS (m/z) 420.9651; found 420.9648. 1-(6-Chloropyridin-3-yl)carbonyl-4-[(3,5dichlorophenyl)thio]-3,5-dimethyl-1H-pyrazole (7b) Yield 86 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.21 (s, 3H), 2.74 (s, 3H), 6.88 (m, 2H), 7.13 (s, 1H), 7.48 (d, J = 6.3 Hz, 1H), 8.35 (dd, J = 1.8, 6.3 Hz, 1H), 9.08 (d, J = 1.8 Hz, 1H). MS 412 (M?). 1-(6-Morpholinopyridin-3-yl)carbonyl-4-[(2bromophenyl)thio]-3,5-dimethyl-1H-pyrazole (8a) In 5 mL microwave vial, compound 7a (31 mg, 0.066 mmol), morpholine (28 lL, 0.33 mmol), and DMF (3 mL) were added, and the mixture was subjected to microwave irradiation at 120 °C for 20 min. The reaction mixture was cooled to rt, and partitioned between ethyl acetate (30 mL) and H2O (30 mL). The layers were separated and the organic layer was washed with H2O (30 mL 9 5), dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica

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gel column chromatography (5–15 % ethyl acetate in hexane) to give the off white solid 8a in 38 % yield. 1 H NMR (CDCl3, d, ppm): 2.23 (s, 3H), 2.66 (s, 3H), 3.73 (m, 4H), 3.82 (m, 4H), 6.58 (m, 1H), 6.65 (d, J = 9.0 Hz, 1H), 7.08 (m, 2H), 7.35 (m, 1H), 8.23 (dd, J = 1.8, 6.9 Hz, 1H), 8.99 (d, J = 1.8, Hz, 1H). HRMS calcd. for C21H21BrN4O2S (m/z) 472.0569; found 472.0580. 1-(6-Morpholinopyridin-3-yl)carbonyl-4-[(3,5dichlorophenyl)thio]-3,5-dimethyl-1H-pyrazole (8b) The same reaction as the above ‘‘1-(6-Morpholinopyridin3-yl)carbonyl-4-[(2-bromophenyl)thio]-3,5-dimethyl-1Hpyrazole (8a)’’ section was proceeded except using 7b as a starting material to give an off white solid in 26 % yield. 1 H NMR (CDCl3, d, ppm): 2.29 (s, 3H), 2.68 (s, 3H), 3.72 (m, 4H), 3.82 (m, 4H), 6.55 (d, J = 8.7 Hz, 1H), 6.86 (s, 2H), 7.11 (s, 1H), 8.23 (d, J = 8.7 Hz, 1H), 8.99 (s, 1H). HRMS calcd. for C21H20Cl2N4O2S (m/z) 462.0684; found 462.0669. General procedure for the syntheses of 1-(pyrazin-3yl)carbonyl-4-(phenylthio)-1H-pyrazoles 9a–e, 10a, b To a solution of 4-(phenylthio)-1H-pyrazole 3c–g, 4b, c (0.42 mmol) in CH2Cl2 (5 mL) were added pyrazine-2carboxylic acid (0.84 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (160 mg, 0.84 mmol). The yellow homogeneous reaction mixture was stirred at rt for 3 h until the starting material was disappeared on TLC monitoring, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (15–20 % ethyl acetate in hexane) to give the product 9a– d, 10a, b. 1-(Pyrazin-2-yl)carbonyl-4-[(2-bromophenyl)thio]-3,5dimethyl-1H-pyrazole (9a) Yield 44 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.17 (s, 3H), 2.72 (s, 3H), 6.60 (d, J = 7.8 Hz, 1H), 7.01 (dd, J = 7.2, 7.8 Hz, 1H), 7.16 (dd, J = 7.2, 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 8.78 (s, 2H), 9.14 (s, 1H). HRMS calcd. for C16H13BrN4OS (m/z) 387.9993; found 387.9949. 1-(Pyrazin-2-yl)carbonyl-4-[(2-chlorophenyl)thio]-3,5dimethyl-1H-pyrazole (9b) Yield 59 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.17 (s, 3H), 2.76 (s, 3H), 6.64 (m, 1H), 7.11 (m, 2H), 8.77 (s, 2H), 9.14 (s, 1H). HRMS calcd. for C16H13ClN4OS (m/z) 344.0499; found 344.0467.

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1-(Pyrazin-2-yl)carbonyl-4-[(3,5-dichlorophenyl)thio]-3,5dimethyl-1H-pyrazole (9c) Yield 58 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.18 (s, 3H), 2.78 (s, 3H), 6.80 (s, 2H), 7.14 (s, 1H), 8.78 (s, 2H), 9.15 (s, 1H). HRMS calcd. for C16H12Cl2N4OS (m/z) 378.0109; found 378.0095. 1-(Pyrazin-2-yl)carbonyl-4-[(4-(tert-butyl)phenyl)thio]3,5-dimethyl-1H-pyrazole (9d) Yield 57 %. Off white solid. 1H NMR (CDCl3, d, ppm): 1.28 (s, 9H), 2.17 (s, 3H), 2.78 (s, 3H), 7.03 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 8.75 (s, 2H), 9.12 (s, 1H). HRMS calcd. for C20H22N4OS (m/z) 366.1514; found 366.1506. 1-(Pyrazin-2-yl)carbonyl-4-[(4-acetamidophenyl)thio]-3,5dimethyl-1H-pyrazole (9e) Yield 43 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.15 (s, 3H), 2.16 (s, 3H), 2.78 (s, 3H), 7.07 (d, J = 8.1 Hz, 2H), 7.18 (brs, 1H), 7.41 (d, J = 8.1 Hz, 1H), 8.76 (s, 2H), 9.12 (s, 1H). HRMS calcd. for C18H17N5O2S (m/z) 367.1103; found 367.1070. 1-(Pyrazin-2-yl)carbonyl-4-[(2-bromophenyl)thio]-3methyl-5-phenyl-1H-pyrazole (10a) Yield 11 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.82 (s, 3H), 6.67 (m, 1H), 6.96 (m, 1H), 7.12 (m, 1H), 7.34 (m, 3H), 7.54 (m, 1H), 7.82 (m, 2H), 8.80 (s, 2H), 9.26 (s, 1H). HRMS calcd. for C21H15BrN4OS (m/z) 450.0150; found 450.0142. 1-(Pyrazin-2-yl)carbonyl-4-[(2-chlorophenyl)thio]-3methyl-5-phenyl-1H-pyrazole (10b) Yield 10 %. Off white solid. 1H NMR (CDCl3, d, ppm): 2.82 (s, 3H), 6.70 (m, 1H), 7.05 (m, 1H), 7.35 (m, 5H), 7.73 (m, 1H), 7.82 (m, 1H), 8.80 (s, 2H), 9.26 (s, 1H). HRMS calcd. for C21H15ClN4OS (m/z) 406.0655; found 406.0645.

H. Y. Kim et al.

Applied Science) and the cells expressing GPR109A were selected with 800 lg/mL G418. U2OS b-arrestin2-RrGFP cells were maintained in minimum essential medium supplemented with 10 % heat inactivated FBS, 1 % penicillin/ streptomycin solution, 10 lg/mL gentamicin, 10 mM HEPES, and 800 lg/mL G418. We subcloned GPR109A cDNA into pCMV6-A-Hygro vector (Origene) and transfected the construct to U2OS b-arrestin2-RrGFP cells. And then the cells expressing GPR109A were selected with 200 lg/mL hygromycin B. We cultured TangoTM GPR109A-bla U2OS cells in McCoy’s 5A medium supplemented with 10 % dialyzed FBS, 1 % penicillin/streptomycin solution, 0.1 mM non-essential amino acids, 25 mM HEPES, 1 mM sodium pyruvate, 200 lg/mL ZeocinTM, 50 lg/mL hygromycin B, and 100 lg/mL GeneticinÒ. Calcium fluorescence assay Fluo-4 NW calcium assay kit (Molecular Devices) was employed to measure calcium mobilization by receptor activation in CHO/Ga16 stable cell line expressing GPR109A. The cells were seeded in black with clear bottom 96-well plates and stabled overnight. The growth medium was changed with F-12 0.5 % FBS and incubated for 3 h. And then the medium was removed and the plates were stored with dye loading solution for 1 h. Fluorescence was measured for excitation at 485 nm and emission at 525 nm using FlexStation II (Molecular Devices).

b-Lactamase fluorescence assay To investigate b-arrestin recruitment, LiveBLAzerTM FRET-B/G loading kit (Invitrogen) was used. TangoTM GPR109A-bla U2OS cells were settled overnight and the cells were stimulated with test compounds for 5 h at 37 °C. And then LiveBLAzerTM FRET-B/G substrate mixture (CCF4-AM) was loaded and incubated at room temperature for 2 h protecting from light. Fluorescence was measured for excitation at 409 nm and emission at 460 and 530 nm using FlexStation II.

Cell culture and generation of stable cell lines

Image analysis of b-arrestin internalization

Ga16-coupled CHO cell line (CHO/Ga16) and U2OS barrestin2-RrGFP cells were purchased form Molecular Devices, and TangoTM GPR109A-bla U2OS cells from Invitrogen. We maintained CHO/Ga16 in Ham’s F-12 culture medium supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin/streptomycin solution, and 100 lg/ mL hygromycin B. GPR109A cDNA clone (Origene) was transfected into CHO/Ga16 cells with FuGENEÒ 6 (Roche

U2OS b-arrestin2-RrGFP cells expressing GPR109A were seeded in 96-well glass bottom plates. The cells were stained with 1 lg/mL Hoechst 33342 and treated with compounds. And then the cells were fixed with 4 % paraformaldehyde and washed with PBS three times. b-Arrestin internalization was analyzed by GFP fluorescence in cellomics array scan high content screening (HCS) reader (Thermo Scientific).

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Results and discussion Chemistry From the library screening, several novel scaffolds were discovered as GPR109A agonists (Fig. 1). Compound 5a, 1-nicotinoyl-4-[(4-chlorophenyl)thio]-3,5-dimethyl-1Hpyrazole showed the most potent activity in the HTS campaign (Z0 = 0.656 ± 0.068), and was used as a starting point for SAR studies. Especially, 5a does not have carboxylic acid moiety which is considered as a key pharmacophore of GPR109A agonists. Because carboxylic acid group usually disturbs the absorption and is frequently subjected to Phase II metabolism, 5a may provide a good starting point for optimization. For SAR studies of this series, we sequentially modified the structure of 5a including the substituents at 4-thiophenol, 5-methyl, and 1-nicotinoyl group. As shown in Scheme 1, the key 3,4,5-trisubstituted pyrazole intermediates (3a–g, 4a–e) were prepared by a straightforward route. S-Alkylation of variously substituted thiophenols with 3-chloropentane-2,4-dione and 2-chloro-1phenylbutane-1,3-dione provided 3-(thiophenoxy)pentane2,4-diones (1a–g) and 1-phenyl-2-(thiophenoxy)butane-1,3diones (2a–e) in good yields, respectively (Muhammad et al. 2008). Subsequent cycloaddition reaction of 1,3-diones using hydrazine hydrate in ethanol to form pyrazoles (3a–g, 4a– e) proceeded well at reflux condition. Additionally, the oneposition of pyrazole was substituted with nicotinoyl or 6-chloronicotinoyl group by the coupling with nicotinoyl chloride hydrochloride or 6-chloronicotinoyl chloride hydrochloride in the presence of triethyl amine and catalytic DMAP to give the nicotinamide products (5a–g, 6a–e, 7a, b) in reasonable yields, 50–90 %. 6-Chloronicotinoyl compounds (7a, b) were further diversified to 6-morpholinonicotinoyl derivatives (8a, b) via nucleophilic substitution reaction using microwave irradiation. Amide bond formation of 3,5-dimethylpyrazoles (3c–g) with pyrazine-2-carboxylic

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acid was achieved by using EDClHCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) as the coupling agent in dichloromethane at ambient temperature to provide the corresponding pyrazine-2-carboxamide derivatives (9a–e) in moderate yields (43–59 %). But, pyrazine-2-carboxamides of 3-methyl-5-phenylpyrazoles (10a, b) were obtained in poor yields (10, 11 %, respectively). Evaluation of biological activity Niacin and MK-6892 were used as positive controls. MK6892 discovered by Merck and reported as a full and potent GPR109A agonist, was synthesized in our laboratory (Shen et al. 2010). Cell construction and calcium mobilization assay To investigate GPR109A mediated intracellular Ca2? mobilization, we have established stably transfected CHOK1/Ga16 with GPR109A. A chemical library (the representative library of Korean Chemical Bank, *6,700 compounds) was screened with calcium mobilization assay and the % activation values were calculated comparing with value of niacin at 10 lM set to 100 %. From the library screening several novel scaffolds were discovered as GPR109A agonists (Fig. 1). A 4-thiopyrazole compound 5a showed the most potent activity in the HTS campaign (Z0 = 0.656 ± 0.068), and its derivatives were synthesized and evaluated. As shown in Table 1, a series of 1-nicotinoyl derivatives (5a–e, 6e) and MK-6892 elicited intracellular calcium mobilization to the similar levels to niacin and displayed excellent enhancement for the GPR109A receptor activity by indicating the EC50 values ranged 45–280 nM in a dose dependent manner. Especially, compound 5a showed an EC50 value of 45 nM, comparable to those of niacin (EC50 = 52 nM) and MK-6892 (EC50 = 74 nM). On the contrary, 1-(pyrazin-2-oyl) derivatives (9a–c, e, 10a, b) exhibited 3–30 times lower activities (EC50 values of 2.3–7.8 lM) compared with the corresponding 1-nicotinoyl derivatives. Generally, 3,5-dimethylpyrazoles (5a–g) were more active than 2-methyl-5-phenylpyrazoles (6a–e). The incorporation of Cl or morpholine at the six-position of pyridine (7a, b, 8a, b) was not tolerated, resulting in complete loss of activity (Fig. 2). b-Arrestin recruitment by agonist binding upon GPR109A

Fig. 1 Distribution of compound activities in high-throughput screening campaign for GPR109A agonists

We examined whether agonist binding upon GPR109A promotes b-arrestin recruitment in Tango GPR109A-bla U2OS cells. b-Lactamase fluorescence assay is based on ligand binding to G-protein coupled receptors that triggers desensitization, a process mediated by the recruitment of

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Scheme 1 Conditions and reagents: (a) piperidine, CH2Cl2–MeOH, 0 °C–rt, 6 h, (b) N2H4H2O, EtOH, reflux, 12 h, (c) nicotinoyl chloride hydrochloride, Et3N, THF, rt, 2 h, (d) 6-chloronicotinoyl

chloride, Et3N, THF, rt, 2 h, (e) morpholine, mwave irradiation, 120 °C, 20 min, and (f) piperazine-2-carboxylic acid, EDClHCl, CH2Cl2, rt, 24 h

intracellular arrestin proteins to the activated receptor (Richman et al. 2007). Blue/green emission ratio was calculated by dividing the background-subtracted blue emission values by the background-subtracted green emission values. 1-Nicotinoyl derivatives (5a–g, 6a–e) induced barrestin recruitment although the responses are milder than those induced by niacin and MK-6892 (Fig. 3). Compound 5a showed weaker activation on b-arrestin recruitment as 2.3-fold activation at 10 lM than niacin and MK-6892 representing 4.8- and 13.0-fold activation at 10 lM, while their activities on calcium mobilization were similar. Previously, MK-6892 was reported to have superior therapeutic window over niacin regarding the FFA reduction versus vasodilation in rats and dogs (Richman et al. 2007). However, it highly promoted b-arrestin recruitment in our assay. In contrast, MK-0354 induced G-protein biased signaling without b-arrestin recruitment. Compounds 5f and 6c did not elevate green emission up to 10 lM but

induced around 3-fold activation in b-arrestin signaling at 30 lM. 1-(Pyrazin-2-oyl) derivatives (9a–e, 10a, b) showed no effect on b-arrestin recruitment even at the high concentration of 30 lM, and seem to be G-protein biased GPR109A agonists even considering their relatively weak activities on calcium mobilization.

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Induction of internalization of GPR109A Sequestration of G protein-coupled receptors from the cell surface following agonist-stimulation is regulated by GRKs and b-arrestins. Internalization of GPCR is important for receptor resensitization as well as for signal transduction (Kunapuli et al. 2003). We investigated whether agonist induced stimulation promotes internalization of the GPR109A receptor, and whether b-arrestin plays a role in GPR109A-mediated signal transduction. The GPR109A expressing U2OS b-arrestin2-RrGFP cells


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Table 1 Effects of 4-thiopyrazole derivatives and known agonists on GPR109A

X

S R1

N

R2

O Compounds

N

R1

R2

X

% Activation (at 10 lM) 5a

CH3

5b

CH3

5c

CH3

5d

CH3

5e

CH3

5f

CH3

5g

CH3

6a

Ph

6b

Ph

6c

Ph

6d

Ph

6e

Ph

7a

CH3

N

N

N

N

N

N

N

N

N

N

N

N

N

b-Arrestin assay

Calcium assay EC50 (lM)

a

Efficacy (%)

a

Fold activation (at 10 lM)

4-Cl

94.9

0.045

100.3

2.31

4-Br

101.7

0.13

102.2

2.19

2-Br

99.1

0.23

98.9

3.27

2-Cl

106.7

0.28

106.5

3.46

3,5-diCl

104.7

0.20

99.3

1.49

4-tBu

55.1

1.3

118.2

1.03

4-NHAc

98.2

0.85

98

3.16

4-Br

111.0

0.99

111.1

1.32

2-Br

118.1

1.4

101.2

1.15

2-Cl

114.5

0.85

121.8

1.03

3,5-diCl

108.7

2.5

95.9a

1.54

H

106.9

0.20

101.5

1.75

2-Br

-0.7

–

–

–

Cl

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H. Y. Kim et al.

Table 1 continued R1

Compounds

R2

7b

CH3

8a

CH3

X

N

b-Arrestin assay

Calcium assay % Activation (at 10 lM)

EC50 (lM)a

Efficacy (%)a


3,5-diCl

-1.3

–

–

–

2-Br

-2.9

–

–

–

3,5-diCl

-1.4

–

–

–

2-Br

72.0

7.0

124.2

0.97

2-Cl

80.8

7.8

137.2

1.04

3,5-diCl

61.1

6.7

133.8

1.06

101.0

3.7

103.0

1.06

4-NHAc

69.0

2.3

85.5

1.00

2-Br

59.3

2.8

105.7

1.02

2-Cl

70.5

3.6

112.6

1.07

112.5

0.074

103.5

13.00

64.3

0.77

64.5

1.03

Cl N N O CH3

8b

N N O

CH3

9a

N N

CH3

9b

N N

CH3

9c

N N

CH3

9d

4-tBu

N N

CH3

9e

N N

10a

Ph

10b

Ph

N N N N

MK-6892 HO

N

MK-6892

N N O

MK-0354 N MK-0354

N H

123

N

N

N NH

O N H

COOH


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Table 1 continued R1

Compounds

R2

X

Niacin

b-Arrestin assay

Calcium assay % Activation (at 10 lM)

EC50 (lM)a

Efficacy (%)a


100.0

0.052

110.8

4.82

O Niacin

OH N a

EC50 values and efficacy was calculated using non-linear regression analysis of dose–response activities obtained from a duplicated experiment in range of 1 nM–100 lM (eight different semi-logarithmic concentrations were designed according to the potency of the compounds). Efficacy 100 % was set to the efficacy of 10 lM niacin

Fig. 2 Effects of GPR109A agonists on intracellular Ca2? mobilization and b-arrestin recruitment. Calcium flux was measured using CHO-K1/Ga16/GPR109A cells and the % activation values were calculated from baseline relative to the value of niacin at 10 lM (left

y axis). The b-arrestin recruitment after GPR109A activation was monitored in Tango GPR109A-bla U2OS cells (right y axis). Fold activation values at 10 lM were calculated relative to control cells

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H. Y. Kim et al.

Fig. 3 GPR109A internalization in U2OS/barrestin2-RrGFP cells stably expressing GPR109A. The cells were stimulated with 1 lM of compounds over a time course from 0 to 60 min. Images are obtained from a HCS reader

were activated with 1 lM of compounds and images were analyzed in a HCS reader. As seen in Fig. 3, cells showed a uniform level of GFP-fusion proteins in control (untreated) but internalized GPR109A formed pits

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throughout the cytoplasm upon activation by niacin. MK6892 evoked a potent internalization of GPR109A in U2OS b-arrestin2-RrGFP cells. Compound 5a also displayed pits indicating GPR109A internalization, whereas


compounds 9b and d did not elicit receptor internalization. Those data are well correlated with the results of ‘‘3[(4-Bromophenyl)thio]pentane-2,4-dione (1b)’’ section. The time-dependent experiment demonstrated that the internalization of receptor was achieved within 30 min following compound treatments. Based on both ‘‘3-[(4Bromophenyl)thio]pentane-2,4-dione (1b)’’ and ‘‘3-[(2Bromophenyl)thio]pentane-2,4-dione (1c)’’ sections experiments, 1-(pyrazin-2-oyl) compounds may elicit more G-protein-biased activation over b-arrestin compared with 1-nicotinoyl compounds.

Conclusions We identified 4-(phenyl)thio-1H-pyrazole as a novel scaffold for GPR109A agonist without carboxylic acid moiety known to be important for binding. From SAR studies, we found that 1-nicotinoyl derivatives exhibited 3–30 times potent activities than the corresponding 1-(pyrazin-2-oyl) compounds on calcium mobilization assay. Especially, compound 5a showed an EC50 value of 45 nM, comparable to those of niacin (EC50 = 52 nM) and MK-6892 (EC50 = 74 nM). The incorporation of Cl or morpholine at the six-position of pyridine (7a, b, 8a, b) was not tolerable. Generally, 3,5-dimethylpyrazoles (5a–g) were more active than 3-methyl-5-phenylpyrazoles (6a–e). Additionally, we found that 1-nicotinoyl derivatives are agonists acting on b-arrestin recruitment and GPR109A internalization like niacin and MK-6892. Compound 5a exhibited around two and five times weaker activation on b-arrestin recruitment than niacin and MK-6892, respectively at 10 lM, while their activities on calcium mobilization were similar. On the contrary, 1-(pyrazin-2-oyl) derivatives showed agonistic activity without b-arrestin recruitment and GPR109A internalization effect. Especially, the 1-(pyrazin-2-oyl) compounds 9a–c showed even higher efficacy (124–137 %) at high concentration (100 lM) without barrestin recruitment and GPR109A internalization effect. G-proteins and b-arrestins may contribute differentially to the therapeutic effects of nicotinic acid on lipids and on the undesired effect of cutaneous flushing. Our results demonstrated a clear instance of biased activation of G-protein signaling over b-arrestin recruitment, even though it has not been proven whether the biased activation of GPR109A results in pharmacological dissociation of the anti-lipolytic effect from its vasodilatory effect or not. Acknowledgments The chemical library used in this study was kindly provided by Korea Chemical Bank (http://www.chembank. org) of Korea Research Institute of Chemical Technology. This research was supported by a grant of Korea Research Council for Industrial Science and Technology (KK-1203-D0).

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