1-Alkyl-benzotriazole-5-carboxylic Acids Are Highly Selective Agonists

1-Substituted benzotriazole carboxylic acids have been identified as the first reported examples of selective small-molecule agonists of the human orp...
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Supporting Information 1-Alkyl-benzotriazole-5-carboxylic Acids are Highly Selective Agonists of the Human Orphan G-Protein Coupled Receptor GPR109b Graeme Semple,a* Philip J. Skinner,a Martin C. Cherrier,a Peter J. Webb,a Carleton R. Sage a, Susan Y. Tamura,a Ruoping Chen,b Jeremy G. Richman,b and Daniel T. Connolly.b a) Medicinal Chemistry and, b) Biological Sciences, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA92121, USA

Table of Contents Table of Contents............................................................................................................................... 1 General ............................................................................................................................................ 2 General Procedure for the Synthesis of Aminonitrobenzoic acids (5a-z). ....................................................... 3 General Procedure for the Synthesis of 1-Alkylbenzotriazole-5-carboxylic acids (3a-z) (Method A). .................. 5 General Procedure for the Synthesis of 1-Alkylbenzotriazole-5-carboxylic acids (3a-z) (Method B). .................. 5 General Procedure for the Direct Alkylation Synthesis of 1-Alkylbenzotriazole-5-carboxylic acids (3a-z) (Method C). .................................................................................................................................................. 5 Procedure for the Synthesis of 1-Isopropylbenzotriazole-5-tetrazole (9). ....................................................... 8 General Procedure for the Synthesis of 1-Alkylindazole-5-carboxylic acids (14). ........................................... 9 cAMP Whole Cell Assay. .................................................................................................................. 11 Adipocyte Lipolysis Assay................................................................................................................. 11 CLUSTAL W (1.8) multiple sequence alignment of GPR109a and GPR109b .............................................. 11

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General 1-Methylbenzotriazole-5-carboxylic acid and 1-phenylbenzotriazole-5-carboxylic acid were purchased from Maybridge. 1-Isopropylbenzotriazole-5-carboxylic acid was initially purchased from Maybridge before subsequent resynthesis in house. Polymer supported nitrite resin was purchased from Novabiochem, Amberlite IRA 900 NO2— form, capacity (NO2) ~4 mmol/g, resin. All other reagents were purchased from Aldrich. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Mercury Vx-400 equipped with a 4 nucleus auto switchable probe and z-gradient or a Bruker Avance-400 equipped with a QNP (Quad Nucleus Probe) or a BBI (Broad Band Inverse) and z-gradient. Chemical shifts are given in parts per million (ppm) with the residual solvent signal used as reference. Coupling constants are reported in Hz. NMR abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, br = broad. Microwave irradiations were carried out using the Emyrs Synthesizer (Personal Chemistry).

Thin-layer

chromatography (TLC) was performed on silica gel 60 F254 (Merck) and column chromatography was carried out on prepacked silica gel columns using KP-Sil and supplied by Biotage. Evaporation was done in vacuo on a Buchi rotary evaporator. Celite 545 ® was used during palladium filtrations. Analytical HPLC/MS was conducted on an AB/MDS Sciex API 150EX mass spectrometer with an a) or b) electrospray or c) APC ionisation source , using a Shimadzu Inc. LC-10AD VP HPLC-pump, Shimadzu Inc. SCL10A VP HPLC system controller, Shimadzu Inc. SPD-10A VP UV-Detector, monitoring at 214nm, Leap Scientific CTC HTS, PAL Autosampler, Analyst 1.2 software and a a) Waters® YMC™ ODS-A C18 column (5 µ, 50 × 4.6 mm), using a gradient of 5% v/v CH3CN (containing 1% v/v TFA) in H2O (containing 1% v/v TFA) (t=0.0 min) gradient to 95% v/v CH3CN in H2O (t=4.0 min), 3.5 mL/min; b) Alltech Prevail C18 column (5 µ, 50 × 4.6 mm), using a gradient of 5% v/v CH3CN (containing 1% v/v TFA) in H2O (containing 1% v/v TFA) (t=0.0 min) gradient to 95% v/v CH3CN in H2O (t=4.0 min), 3.5 mL/min; or c) Waters® YMC™ ODS-A C18 column (5 µ, 50 × 4.6 mm), using a gradient of 5% v/v CH3CN (containing 1mmol NH4OAc) in H2O (containing 1mmol NH4OAc) (t=0.0 min) gradient to 95% v/v CH3CN in H2O (t=4.0 min), 3.5 mL/min. Elemental analysis was conducted by West Coast Analytical Service Inc., 9240 Santa Fe Springs Road, Santa Fe Springs, CA90670, USA. Preparative HPLC was conducted on a Varian Prostar reverse phase HPLC using a Phenomenex® Luna C18 column (10 µ, 250 × 50 mm), 5% (v/v) CH3CN (containing 0.1% v/v TFA) in H2O (containing 0.1% v/v TFA) gradient to 100% H2O, 60 ml/min, λ = 220 nm

S3 General Procedure for the Synthesis of Aminonitrobenzoic acids (5a-z). 4-fluoro-3-nitrobenzoic acid (4), amine (1.1 equiv.) and sodium bicarbonate (2.1 equiv) in H2O (3 mL) was heated to 150°C for 20 minutes under microwave irradiation in a sealed heavy walled Pyrex tube. The resulting orange mixture was cooled, poured into 1M HCl (40 mL) and extracted into EtOAc. Solvent was removed under reduced pressure to give a yellow solid (>95% yield) which was used without further purification. 4-Isopropylamino-3-nitro-benzoic acid (5a) 1H NMR (CDCl3): δ 9.05 (d, 1H, J = 2.0, C(2)-H), 8.47 (d, 1H, J = 6.5, NH), 8.16 (dd, 1H, J1 = 9.1, J2 = 2.0, C(6)-H), 7.00 (d, 1H, J = 9.1, C(5)-H), 4.01 (septet, 1H, J = 6.5, NHCH), 1.47 (d, 6H, J = 6.5, CHCH3). 4-(2’-Methoxy-1’-methyl-ethyl)amino-3-nitro-benzoic acid (5b). 1H NMR (CD3OD): δ 8.80 (d, 1H, J=2.1, C(2)-H), 8.04 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 7.13 (d, 1H, J=9.1, C(5)-H), 4.08 (sextet, 1H, J=5.4, NHCH), 3.603.50 (m, 2H, CHCH2), 3.41 (s, 3H, OCH3), 1.33 (d, 3H, J=6.8, CH3). 4-(2’-Ethoxy-ethyl)amino-3-nitro-benzoic acid (5c). 1H NMR (CD3OD): δ 8.80 (d, 1H, J=2.0, C(2)-H), 8.05 (dd, 1H, J1=9.1, J2=2.0, C(6)-H), 7.10 (d, 1H, J=9.1, C(5)-H), 3.74 (t, 2H J=7.0, NHCH2CH2), 3.65-3.55 (m, 4H, NHCH2 & CH2CH3), 1.22 (t, 3H, J=7.0, CH3). 4-tert-Butylamino-3-nitro-benzoic acid (5d) 1H NMR (CD3OD): δ 9.04 (d, 1H, J=2.1, C(2)-H), 8.25 (dd, 1H, J1=9.2, J2=2.1, C(6)-H), 7.54 (d, 1H, J=9.2, C(5)-H), 1.78 (s, 9H, CH3). 4-Pentylamino-3-nitro-benzoic acid (5e). 1H NMR (CD3OD): δ 8.66 (d, 1H, J=2.1, C(2)-H), 7.91 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 6.93 (d, 1H, J=9.1, C(5)-H), 3.29 (t, 2H, J=7.2, NHCH2), 1.62 (quintet, 2H, J=7.0, NCH2CH2), 1.35-1.25 (m, 4H), 0.83 (t, 3H, J=7.1, CH3). 4-(2-Ethylsulfanyl-ethylamino)-3-nitro-benzoic acid (5f). 1H NMR (CDCl3): δ 8.99 (d, 1H, J=1.9, C(2)-H), 8.68 (br s, 1H, NH), 8.12 (dd, 1H, J1=9.0, J2=1.9, C(6)-H), 6.97 (d, 1H, J=9.1, C(5)-H), 3.61 (q, 2H, J=6.3, NHCH2), 2.91 (t, 2H, J=6.8, NCH2CH2), 2.63 (q, 2H, J=7.4, CH2CH3), 1.32 (t, 3H, J=7.4, CH3). 4-(2’-Methoxy-ethyl)amino-3-nitro-benzoic acid (5g). 1H NMR (CD3OD): δ 8.80 (d, 1H, J=2.1, C(2)-H), 8.05 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 7.10 (d, 1H, J=9.1, C(5)-H), 3.69 (t, 2H, J=5.2, NHCH2), 3.60 (t, 2H, J=5.2, OCH2), 3.42 (s, 3H, OCH3). 4-(2’-Butyl)amino-3-nitro-benzoic acid (5h). 1H NMR (CD3OD): δ 8.81 (d, 1H, J=2.1, C(2)-H), 8.04 (dd, 1H, J1=9.2, J2=2.1, C(6)-H), 7.10 (d, 1H, J=9.2, C(5)-H), 3.82 (sextet, 1H, J=6.4, NHCH), 1.75-1.65 (m, 2H, CH2CH3), 1.31 (d, 3H, J=6.4, CHCH3), 1.02 (t, 3H, J=7.5, CH2CH3).

S4 4-Ethylamino-3-nitro-benzoic acid (5j) 1H NMR (CD3OD): δ 8.80 (d, 1H, J=2.1, C(2)-H), 8.05 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 7.07 (d, 1H, J=9.1, C(5)-H), 3.48 (q, 2H, J=7.2, NHCH2), 1.35 (t, 3H, J=7.2, CH3). 4-(3’-Pentyl)amino-3-nitro-benzoic acid (5l). 1H NMR (CDCl3): δ 8.90 (d, 1H, J=2.1, C(2)-H), 8.35 (d, 1H, J=8.2, NH), 7.98 (dd, 1H, J1=9.2, J2=1.8, C(6)-H), 6.83 (d, 1H, J=9.2, C(5)-H), 3.50 (sextet, 1H, J=7.6, NHCH), 1.75-1.50 (m, 4H, CH2CH3), 0.92 (t, 6H, J=7.4, CH2CH3). 4-Propylamino-3-nitro-benzoic acid (5m). 1H NMR (CD3OD): δ 8.80 (d, 1H, J=2.1, C(2)-H), 8.04 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 7.07 (d, 1H, J=9.1, C(5)-H), 3.40 (t, 2H, J=7.1, NHCH2), 1.76 (sextet, 2H, J=7.3, CH2CH3), 1.06 (t, 3H, J=7.4, CH2CH3). 4-Cyclopropylamino-3-nitro-benzoic acid (5r). 1H NMR (CD3OD): δ 8.78 (d, 1H, J=2.0, C(2)-H), 8.09 (dd, 1H, J1=9.0, J2=2.0, C(6)-H), 7.47 (d, 1H, J=9.0, C(5)-H), 2.71 (septet, 1H, J=3.5, NHCH), 1.05-0.95 (m, 2H), 0.750.65 (m, 2H). 4-Cyclobutylamino-3-nitro-benzoic acid (5s). 1H NMR (CD3OD): δ 8.78 (d, 1H, J=2.0, C(2)-H), 8.03 (dd, 1H, J1=9.0, J2=2.0, C(6)-H), 6.93 (d, 1H, J=9.0, C(5)-H), 4.22 (quintet, 1H, J=7.8, NHCH), 2.60-2.50 (m, 2H), 2.152.00 (m, 2H), 2.00-1.85 (m, 2H). 4-Cyclopentylamino-3-nitro-benzoic acid (5t). 1H NMR (CD3OD): δ 8.81 (d, 1H, J=2.1, C(2)-H), 8.06 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 7.12 (d, 1H, J=9.1, C(5)-H), 4.2-4.1 (m, 1H, NHCH), 2.3-2.1 (m, 2H), 1.9-1.6 (m, 6H). 4-Cyclohexylamino-3-nitro-benzoic acid (5u). 1H NMR (CD3OD): δ 8.80 (d, 1H, J=2.1, C(2)-H), 8.03 (dd, 1H, J1=9.2, J2=2.1, C(6)-H), 7.10 (d, 1H, J=9.2, C(5)-H), 3.75-3.65 (m, 1H, NHCH), 2.10-2.05 (m, 2H), 1.85-1.75 (m, 2H), 1.75-1.60 (m, 1H), 1.60-1.30 (m, 5H). 4-Benzylamino-3-nitro-benzoic acid (5v). 1H NMR (CDCl3): δ 9.00 (d, 1H, J=2.0, C(2)-H), 8.79 (t, 1H, J=5.5, NH), 8.06 (dd, 1H, J1=9.1, J2=1.8, C(6)-H), 7.5-7.3 (m, 5H), 6.89 (d, 1H, J=9.1, C(5)-H), 4.63 (d, 2H, NHCH2). 4-Benzhydrylamino-3-nitro-benzoic acid (5w). 1H NMR (CD3OD): δ 8.69 (d, 1H, J=2.0, C(2)-H), 7.79 (dd, 1H, J1=9.1, J2=2.0, C(6)-H), 7.30-7.15 (m, 10H), 6.77 (d, 1H, J=9.1, C(5)-H), 5.88 (br s, 1H, NHCH). 4-(4’-Methoxybenzyl)amino-3-nitro-benzoic acid (5x). 1H NMR (CD3OD): δ 8.81 (d, 1H, J=2.0, C(2)-H), 7.98 (dd, 1H, J1=9.0, J2=2.0, C(6)-H), 7.31 (d, 2H, J=8.8, C(2’)-H), 7.00 (d, 1H, 1=9.0, C(5)-H),6.91 (d, 2H, J=8.8, C(3’)-H) 4.59 (s, 2H, NHCH2), 3.78 (s, 3H, OCH3).

S5 4-(3’-Methoxybenzyl)amino-3-nitro-benzoic acid (5y). 1H NMR (CD3OD): δ 8.82 (d, 1H, J=2.1, C(2)-H), 7.97 (dd, 1H, J1=9.1, J2=2.1, C(6)-H), 7.27 (t, 1H, J=8.1, C(5’)-H), 7.0-6.9 (m, 3H), 6.84 (dd, 1H, J1=7.5, J2=2.5, C(5)H), 4.64 (s, 2H, NHCH2), 3.78 (s, 3H, OCH3). 4-(3’,5’-Difluorobenzyl)amino-3-nitro-benzoic acid (5z). 1H NMR (CD3OD): 8.34 (d, 1H, J=2.0, C(2)-H), 7.99 (dd, 1H, J1=8.6, J2=2.0, C(6)-H), 7.05-6.85 (m, 4H), 4.71 (s, 2H, NHCH2).

General Procedure for the Synthesis of 1-Alkylbenzotriazole-5-carboxylic acids (3a-z) (Method A). The amino-3-nitro-benzoic acid (5) was taken up in ethyl acetate (30mL), palladium (0.1 equiv) added and the suspension shaken at room temperature under a hydrogen atmosphere (1 atm.) for 3 hours. The resulting solution was filtered through celite and solvent removed under reduced pressure to give the diamine (6) as a pale brown glass. The diamine was taken up immediately in glacial acetic acid (5mL), and sodium nitrite (1.1 equiv.) added. The mixture was stirred at room temperature under argon, poured into DCM (50 mL) and washed with saturated aqueous sodium bicarbonate solution (50 mL) and 1M aqueous hydrochloric acid (50 mL). Solvent was removed under reduced pressure to give the desired benzotriazole (3) as a crystalline solid.

General Procedure for the Synthesis of 1-Alkylbenzotriazole-5-carboxylic acids (3a-z) (Method B). The amino-3-nitro-benzoic acid (5) was taken up in ethyl acetate (30mL), palladium (0.1 equiv) added and the suspension shaken at room temperature under a hydrogen atmosphere (1 atm.) for 3 hours. The resulting solution was filtered through celite and solvent removed under reduced pressure to give the diamine (6) as a pale brown glass. The diamine was taken up immediately in glacial acetic acid (5mL), and polymer supported nitrite (2-3 equiv.) added. The mixture was shaken overnight at room temperature under argon, filtered and solvent removed under reduced pressure to give the desired benzotriazole (3) as a brown crystalline solid.

General Procedure for the Direct Alkylation Synthesis of 1-Alkylbenzotriazole-5-carboxylic acids (3a-z) (Method C). Benzotriazole-5-carboxylic acid, the appropriate bromide (1.5 equiv.) and potassium carbonate (2.2 equiv) were stirred for 18 hours at 60°C in DMA (3 mL). The resulting solution was diluted with water and acetonitrile until all solid was dissolved, and purified by preparative HPLC. 1-Isopropyl-1H-benzotriazole-5-carboxylic acid (3a) (B). (0.057g, 81%) HPLC/MS: (column a) 99%, tr=1.73 min, m/z (ES+): 206 [M+H]+. 1H NMR (CD3OD): δ 8.58 (s, 1H, C(4)-H), 8.09 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.79 (dd, 1H, J1=8.8, J2=0.5, C(7)-H), 5.15 (septet, 1H, J=6.7, CH(CH3)2), 1.63 (d, 6H, J=6.7, CH(CH3)2). Found: C: 58.3%, H: 5.24%, N: 19.9%. C10H11N3O2 requires C: 58.5%, H: 5.40%, N: 20.5%.

S6 1-(2’-Methoxy-1’-methyl-ethyl)-1H-benzotriazole-5-carboxylic acid (3b) (B). (0.010g, 11%) HPLC/MS: (column c) 96%, tr=1.61 min, m/z (ES+): 236 [M+H]+. 1H NMR (CD3OD): δ 8.68 (s, 1H, C(4)-H), 8.18 (dd, 1H, J1=8.8, J2=1.3, C(6)-H), 7.88 (d, 1H, J=8.8, C(7)-H), 5.35-5.25 (m, 1H, NCH), 3.93 (dd, 1H, J1=10.0, J2=8.4, CHCHH), 3.85 (dd, 1H, J1=10.0, J2=4.4, CHCHH), 3.25 (s, 3H, OCH3), 1.73 (d, 3H, J=6.8, CH3). 1-(2’-Ethoxy-ethyl)-1H-benzotriazole-5-carboxylic acid (3c) (B). (0.042g, 43%) HPLC/MS: (column c) 97%, tr=1.64 min, m/z (ES+): 236 [M+H]+. 1H NMR (CD3OD): δ 8.68 (d, 1H, J=0.8, C(4)-H), 8.18 (dd, 1H, J1=8.8, J2=1.3, C(6)-H), 7.88 (dd, 1H, J1=8.8, J2=0.6, C(7)-H), 4.92 (t, 2H, J=5.2, NCH2), 3.95 (t, 2H, J=5.0, NCH2CH2), 3.44 (q, 2H, J=7.0, CH2CH3), 1.04 (t, 3H, J=7.0, CH3). 1-t-Butyl-1H-benzotriazole-5-carboxylic acid (3d) (B). (0.036g, 33%) HPLC/MS: (column c) 91%, tr=1.93 min, m/z (ES+): 220 [M+H]+.1H NMR (CD3OD): δ 8.68 (dd, 1H, J1=1.5, J2=0.6, C(4)-H), 8.17 (dd, 1H, J1=8.9, J2=1.5, C(6)-H), 8.07 (dd, 1H, J1=8.8, J2=0.6, C(7)-H), 1.90 (s, 9H, CH3). 1-Pentyl-1H-benzotriazole-5-carboxylic acid (3e) (B). (0.029g, 31%) HPLC/MS: (column c) 95%, tr=2.25 min, m/z (ES+): 234 [M+H]+.1H NMR (CD3OD): δ 8.69 (d, 1H, J=0.8, C(4)-H), 8.21 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.86 (dd, 1H, J1=8.8, J2=0.6, C(7)-H), 4.76 (t, 2H, J=7.2, NCH2), 2.10-1.95 (m, 2H, NCH2CH2), 1.45-1.25 (m, 4H), 0.90 (t, 3H, J=7.2, CH3). 1-(2’-Ethylsulfanyl-ethyl)-1H-benzotriazole-5-carboxylic acid (3f) (B). (0.015g, 16%) HPLC/MS: (column c) 92%, tr=1.94 min, m/z (ES+): 252 [M+H]+. 1H NMR (CD3OD): δ 8.69 (d, 1H, J=0.8, C(4)-H), 8.21 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.89 (d, 1H, J=8.8, C(7)-H), 4.95 (t, 2H, J=6.8, NCH2), 3.18 (t, 2H, J=6.8, NCH2CH2), 2.48 (q, 2H, J=7.6, CH2CH3), 1.18 (t, 3H, J=7.4, CH2CH3). 1-(2-Methoxy-ethyl)-1H-benzotriazole-5-carboxylic acid (3g) (B). (0.054g, 59%) HPLC/MS: (column b) 90%, tr=1.44 min, m/z (ES+): 222 [M+H]+. 1H NMR (CD3OD): δ 8.67 (dd, 1H, J1=1.4, J2=0.7, C(4)-H), 8.18 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.87 (dd, 1H, J1=8.8, J2=0.7, C(7)-H), 4.93 (t, 2H, J=5.1, NCH2), 3.91 (t, 2H, J=5.1, OCH2), 3.29 (s, 3H, OCH3). 1-(2’-Butyl)-1H-benzotriazole-5-carboxylic acid (3h). (B) (0.026g, 29%) HPLC/MS: (column a) 96%, tr=1.87 min, m/z (ES+): 220 [M+H]+. 1H NMR (CD3OD): δ 8.72 (dd, 1H, J1=1.3, J2=0.7, C(4)-H), 8.22 (dd, 1H, J1=8.8, J2=1.3, C(6)-H), 7.91 (dd, 1H, J1=8.8, J2=0.7, C(7)-H), 5.10-5.00 (m, 1H, NCH), 2.30-2.05 (m, 2H, CH2CH3), 1.75 (d, 3H, J=6.8, CHCH3), 0.85 (t, 3H, J=7.4, CH2CH3). 1-Butyl-1H-benzotriazole-5-carboxylic acid (3i) (C). (0.023g, 25%) HPLC/MS: (column c) 97%, tr=2.01 min, m/z (ES+): 220 [M+H]+. 1H NMR (CD3OD): δ 8.68 (dd, 1H, J1=1.4, J2=0.6, C(4)-H), 8.20 (dd, 1H, J1=8.8, J2=1.4,

S7 C(6)-H), 7.86 (dd, 1H, J1=8.4, J2=0.6, C(7)-H), 4.76 (t, 2H, J=7.0, NCH2), 2.05-1.95 (m, 2H, NCH2CH2), 1.341.25 (m, 2H, CH2CH3), 0.97 (t, 3H, J=7.4, CH3). 1-Ethyl-1H-benzotriazole-5-carboxylic acid (3j) (B). (0.043g, 47%) HPLC/MS: (column c) 96%, tr=1.53 min, m/z (ES+): 192 [M+H]+. 1H NMR (CD3OD): δ 8.69 (d, 1H, J=0.7, C(4)-H), 8.21 (dd, 1H, J1=8.8, J2=1.3, C(6)-H), 7.86 (dd, 1H, J1=8.8, J2=0.7, C(7)-H), 4.80 (q, 2H, J=7.4, NCH2), 1.63 (t, 3H, J=7.4, CH3). 1-(3’-Pentyl)-1H-benzotriazole-5-carboxylic acid (3l) (A). (0.036g, 39%) HPLC/MS: (column a) 90%, tr=2.05 min, m/z (ES+): 234 [M+H]+. 1H NMR (CD3OD): δ 8.51 (dd, 1H, J1=1.4, J2=0.6, C(4)-H), 8.00 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.69 (dd, 1H, J1=8.8, J2=0.6, C(7)-H), 4.60 (septet, 1H, J=4.8, NCH), 2.10-1.85 (m, 4H, CH2CH3), 0.58 (t, 6H, J=7.4, CH2CH3). 1-Propyl-1H-benzotriazole-5-carboxylic acid (3m) (A). (0.008g, 9%) HPLC/MS: (column a) 93%, tr=1.70 min, m/z (ES+): 206 [M+H]+. 1H NMR (CD3OD): δ 8.69 (dd, 1H, J1=1.4, J2=0.7, C(4)-H), 8.20 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.86 (dd, 1H, J1=8.8, J2=0.7, C(7)-H), 4.73 (t, 2H, J=7.0, NCH2), 2.06 (sextet, 2H, J=7.2, CH2CH3), 0.96 (t, 3H, J=7.4, CH2CH3). 1-(Allyl)-1H-benzotriazole-5-carboxylic acid (3o) (C). (0.019g, 18%) HPLC/MS: (column c) 99%, tr=1.62 min, m/z (ES+): 204 [M+H]+. 1H NMR (CD3OD): δ 8.73 (s, 1H, C(4)-H), 8.19 (dd, 1H, J1=8.8, J2=1.6, C(6)-H), 7.85 (d, 1H, J=8.4, C(7)-H), 6.1-6.0 (m, 1H, CH=CH2), 5.50-5.40 (m, 1H, CH=CHH trans to H), 5.35-5.30 (m, 1H, CH=CHH cis to H), 4.90-4.85 (m, 2H, NCH2).

1-(Cyclopropylmethyl)-1H-benzotriazole-5-carboxylic acid (3p) (C) (0.085g, 78%) HPLC/MS: (column c) 90%, tr=2.08 min, m/z (ES+): 218 [M+H]+. 1H NMR (CD3OD): δ 8.46 (s, 1H, C(4)-H), 8.03 (dd, 1H, J1=8.9, J2=0.9, C(6)-H), 7.79 (d, 1H, J=8.9, C(7)-H), 4.60 (d, 2H, J=7.6, NCH2), 1.55-1.50 (m, 1H, NCH2CH), 0.70-0.60 (m, 2H), 0.60-0.50 (m, 2H). 1-Cyclopropyl-1H-benzotriazole-5-carboxylic acid (3r) (A). (0.029g, 32%) HPLC/MS: (column b) 92%, tr=1.66 min, m/z (ES+): 204 [M+H]+. 1H NMR (CD3OD): δ 8.67 (dd, 1H, J1=1.4, J2=0.7, C(4)-H), 8.23 (dd, 1H, J1=8.7, J2=1.4, C(6)-H), 7.92 (dd, 1H, J1=8.7, J2=0.7, C(7)-H), 4.05-3.95 (m, 1H, NCH), 1.4-1.3 (m, 4H). 1-Cyclobutyl-1H-benzotriazole-5-carboxylic acid (3s) (A). (0.046g, 20%) HPLC/MS: (column b) 92%, tr=1.91 min, m/z (ES+): 218 [M+H]+. 1H NMR (CD3OD): δ 8.68 (dd, 1H, J1=1.4, J2=0.6, C(4)-H), 8.19 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.86 (dd, 1H, J1=8.8, J2=0.6, C(7)-H), 5.46 (quintet, 1H, J=8.3, NCH), 2.95-2.80 (m, 2H), 2.802.65 (m, 2H), 2.15-2.05 (m, 2H).

S8 1-Cyclopentyl-1H-benzotriazole-5-carboxylic acid (3t) (A). (0.042g, 45%) HPLC/MS: (column a) 93%, tr=2.04 min, m/z (ES+): 232 [M+H]+. 1H NMR (CD3OD): δ 8.67 (dd, 1H, J1=1.3, J2=0.7, C(4)-H), 8.19 (dd, 1H, J1=8.8, J2=1.3, C(6)-H), 7.87 (dd, 1H, J1=8.8, J2=0.7, C(7)-H), 5.45-5.30 (m, 1H, NCH), 2.45-2.20 (m, 4H), 2.10-1.95 (m, 2H), 1.95-1.80 (m, 2H). 1-Cyclohexyl-1H-benzotriazole-5-carboxylic acid (3u) (A). (0.040g, 43%) HPLC/MS: (column a) 96%, tr=2.23 min, m/z (ES+): 246 [M+H]+. 1H NMR (CD3OD): δ 8.67 (s, 1H, C(4)-H), 8.19 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.90 (d, 1H, J=8.8, C(7)-H), 4.95-4.80 (m, 1H, NCH), 2.25-2.05 (m, 4H), 2.05-1.95 (m, 2H), 1.90-1.80 (m, 1H), 1.70-1.55 (m, 2H), 1.50-1.40 (m, 1H). 1-Benzyl-1H-benzotriazole-5-carboxylic acid (3v) (A). (0.005g, 6%) HPLC/MS: (column a) 90%, tr=2.04 min, m/z (ES+): 254 [M+H]+. 1H NMR (CD3OD): δ 8.59 (dd, 1H, J1=1.3, J2=0.7, C(4)-H), 8.04 (dd, 1H, J1=8.8, J2=1.3, C(6)-H), 7.62 (dd, 1H, J1=8.8, J2=0.7, C(7)-H), 7.26-7.20 (m, 5H), 5.87 (d, 2H, NCH2). 1-Benzhydryl-1H-benzotriazole-5-carboxylic acid (3w) (A). (0.0580g, 26%) HPLC/MS: (column c) 95%, tr=1.82 min, m/z (ES+): 167 [M+2H]2+. m/z (ES-): 328 [M-H]-. 1H NMR (CD3OD): δ 8.72 (s, 1H, C(4)-H), 8.07 (d, 1H, J=8.8, C(6)-H), 7.48 (d, 1H, J=8.8, C(7)-H), 7.40-7.20 (m, 10H), 4.96 (s, 1H, NCH). 1-(4’-Methoxybenzyl)-1H-benzotriazole-5-carboxylic acid (3x) (A). (0.070g, 74%) HPLC/MS: (column b) 99%, tr=2.11 min, m/z (ES+): 284 [M+H]+. 1H NMR (CD3OD): δ 8.68 (s, 1H, C(4)-H), 8.14 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.72 (d, 1H, J=8.8, C(7)-H), 7.31 (d, 2H, J=8.7, C(2’)-H), 6.90 (d, 2H, J=8.7, C(3’)-H), 5.90 (s, 2H, NCH2), 3.76 (s, 3H, OCH3). 1-(3’-Methoxybenzyl)-1H-benzotriazole-5-carboxylic acid (3y) (A). (0.043g, 46%) HPLC/MS: (column b) 91%, tr=2.13 min, m/z (ES+): 284 [M+H]+. 1H NMR (CD3OD): δ 8.69 (dd, 1H, J1=1.4, J2=0.7, C(4)-H), 8.15 (dd, 1H, J1=8.7, J2=1.4, C(6)-H), 7.72 (dd, 1H, J1=8.7, J2=0.7, C(7)-H), 7.26 (t, 1H, J=7.9, C(5’)-H), 7.0-6.8 (m, 3H), 5.94 (s, 2H, NCH2), 3.75 (s, 3H, OCH3). 1-(3’,5’-Difluorobenzyl)-1H-benzotriazole-5-carboxylic acid (3z) (A). (0.056g, 61%) HPLC/MS: (column b) 93%, tr=2.19 min, m/z (ES+): 290 [M+H]+. 1H NMR (CD3OD): δ 8.72 (dd, 1H, J1=1.4, J2=0.6, C(4)-H), 8.20 (dd, 1H, J1=8.8, J2=1.4, C(6)-H), 7.78 (dd, 1H, J1=8.8, J2=0.6, C(7)-H), 7.00-6.90 (m, 3H), 6.00 (s, 2H, NCH2).

Procedure for the Synthesis of 1-Isopropylbenzotriazole-5-tetrazole (9). 4-(Isopropylamino)-3-nitro-benzonitrile (11). 4-Fluoro-3-nitrobenzonitrile (10) (0.500g, 3.01 mmol) was taken up in acetonitrile (4 mL) with isopropylamine (0.350g, 5.90 mmol) and heated to 120°C for 20 minutes in a heavy walled tube under microwave irradiation. The resulting solution was poured into dichloromethane (20 mL) and

S9 washed with 1M aqueous hydrochloric acid (20 mL). Solvent was removed under reduced pressure to give 4(isopropylamino)-3-nitro-benzonitrile (11) as a yellow solid (0.615g, 99%). 1H NMR (CDCl3): δ 8.61 (d, 1H, J=2.0, C(2)-H), 8.45 (br s, 1H, NH), 7.68 (dd, 1H, J1=9.1, J2=2.0, C(6)-H), 7.02 (d, 1H, J=9.1, C(5-H), 3.98 (septet, 1H, J=6.5, NHCH), 1.46 (d, 6H, J=6.5, CHCH3). 1-Isopropylbenzotriazole-5-nitrile (13). 4-(Isopropylamino)-3-nitro-benzonitrile (11) (0.540g, 2.63 mmol) was stirred for 2 hours at room temperature in ethyl acetate (50 mL) with 10% palladium on carbon (0.050g) under an atmosphere of hydrogen (balloon pressure). The resulting near colourless solution was filtered through celite and solvent removed under reduced pressure to give 4-(isopropylamino)-3-amino-benzonitrile (12) as a pale yellow solid which was carried on immediately without further purification. 4-(Isopropylamino)-3-amino-benzonitrile (12) was taken up in glacial acetic acid (20 mL) and sodium nitrite (0.200g, 2.90 mmol) added, the resulting solution was stirred under argon at room temperature for 18 hours, poured into dichloromethane (100 mL) and washed with saturated aqueous sodium bicarbonate (100 mL) and 1M aqueous hydrochloric acid (100 mL). Solvent was removed under reduced pressure to give 1-isopropylbenzotriazole-5-nitrile (13) as an off white solid (0.405g, 83%). HPLC/MS: (column a) 95%, tr=2.04 min, m/z (ES+): 187 [M+H]+. 1H NMR (CDCl3): δ 8.39 (s, 1H), 7.61 (s, 2H), 5.04 (septet, 1H, J=6.8, NCH), 1.70 (d, 6H, J=6.8, CHCH3). 1-Isopropylbenzotriazole-5-tetrazole (9). 1-Isopropylbenzotriazole-5-nitrile (13) (0.380g, 2.04 mmol) was heated to 180°C for 20 minutes in a heavy walled tube under microwave irradiation in water (2 mL) with sodium azide (0.150g, 2.40 mmol) and zinc bromide (0.480g, 2.10 mmol). Aqueous sodium bicarbonate (10 mL) was added and the solution extracted into EtOAc (20 mL), solvent removed under reduced pressure and the resulting solid purified by preparative HPLC to give 1-isopropylbenzotriazole-5-tetrazole (9) as an off white solid (0.085g, 0.37 mmol, 18%). HPLC/MS: (column b) 99%, tr=1.42 min, m/z (ES+): 230 [M+H]+, m/z (ES-): 228 [M-H]-. 1H NMR (d6-DMSO): δ 8.50 (s, 1H), 8.05-7.95 (m, 2H), 5.11 (septet, 1H, J=6.8, NCH), 1.47 (d, 6H, J=6.8, CHCH3).

General Procedure for the Synthesis of 1-Alkylindazole-5-carboxylic acids (14). Ethyl-1H-indazole-5-carboxylate, bromide (1.5 equiv) and potassium carbonate (2.2 equiv) were taken up in DMA and heated to 60°C for 18 hours. The resulting mixture of ethyl-1-alkyl-indazole-5-carboxylate and ethyl-2-alkylindazole-5-carboxylate was purified by preparative HPLC, the desired fractions were combined, solvent removed under reduced pressure, and the resulting solid taken up in a 1:5:1 solution of 1M aqueous lithium hydroxide:THF:methanol and heated for 18 hours at 50°C. Volatile solvents were removed under reduced pressure. The resulting aqueous solution was taken to pH1 with the addition of 1M aqueous hydrochloric acid and extracted into ethyl acetate. Solvent was removed under reduced pressure to give the desired 1-alkylindazole-5carboxylic acid as a white solid.

S10 1-Isopropylindazole-5-carboxylic acid (14a). (0.030g, 30%) HPLC/MS: (column c) 93%, tr=1.96 min, m/z (ES+): 205 [M+H]+.1H NMR (CDCl3): δ 8.63 (d, 1H, J=0.6, C(4)-H), 8.20 (s, 1H, C(3)-H), 8.12 (dd, 1H, J1=8.9, J2=1.5, C(6)-H), 7.50 (d, 1H, J=8.9, C(7)-H), 4.90 (septet, 1H, J=6.7, NCH), 1.63 (d, 6H, J=6.7, CHCH3). 1-(2’-Butyl)indazole-5-carboxylic acid (14h). (0.021g, 19%) HPLC/MS: (column c) 91%, tr=2.15 min, m/z (ES+): 219 [M+H]+. 1H NMR (CDCl3): δ 8.63 (d, 1H, J=0.7, C(4)-H), 8.19 (s, 1H, C(3)-H), 8.11 (dd, 1H, J1=8.9, J2=1.5, C(6)-H), 7.48 (d, 1H, J=8.9, C(7)-H), 4.70-4.50 (m, 1H, NCH), 2.20-2.00 (m, 1H, NCHCHH), 2.00-1.80 (m, 1H, NCHCHH), 1.61 (d, 3H, J=6.8, NCHCH3), 0.79 (t, 3H, J=7.4, CH2CH3). 1-(3’-Pentyl)indazole-5-carboxylic acid (14l). (0.028g, 25%) HPLC/MS: (column c) 96%, tr=2.35 min, m/z (ES+): 233 [M+H]+. 1H NMR (CDCl3): δ 8.64 (d, 1H, J=0.6, C(4)-H), 8.22 (s, 1H, C(3)-H), 8.11 (dd, 1H, J1=8.9, J2=1.5, C(6)-H), 7.48 (d, 1H, J=8.9, C(7)-H), 4.40-4.25 (m, 1H, NCH), 2.20-2.00 (m, 2H, NCHCH2), 2.00-1.80 (m, 2H, NCHCH2), 0.74 (t, 6H, J=7.4, CH3). 1-(Methylcyclopropyl)indazole-5-carboxylic acid (14p). (0.035g, 33%) HPLC/MS: (column c) 97%, tr=2.01 min, m/z (ES+): 217 [M+H]+. 1H NMR (CD3OD): δ 8.72 (s, 1H, C(4)-H), 8.61 (s, 1H, C(3)-H), 8.04 (dd, 1H, J1=9.1, J2=1.5, C(6)-H), 7.69 (d, 1H, J=8.9, C(7)-H), 4.41 (d, 2H, J=7.2, NCH2), 1.55-1.45 (m, 1H, NCH2CH), 0.80-0.70 (m, 2H), 0.60-0.50 (m, 2H). 1-Isopropyl-1H-benzoimidazole-5-carboxylic acid (15). 4-(Isopropylamino)-3-nitro-benzoic acid (5a) (0.120g, 0.535 mmol) was taken up in ethyl acetate (10 mL) and shaken for 18 hours with palladium on carbon (0.014g) under hydrogen (60 psi). The resulting colourless solution was filtered through celite, and trimethylorthoformate (0.5g, 4.72 mmol) and TFA (ca 0.01g) added. The resulting solution was stirred at room temperature for 1 hour and solvent removed under reduced pressure. The resulting solid was purified by column chromatography to give an off white solid (0.032g, 0.157 mmol, 30%) HPLC/MS: (column b) 99%, tr=1.0. min, m/z (ES+): 205 [M+H]+. 1

H NMR (CD3CN): δ 10.0 (br s, 1H , CO2H), 8.41 (d, 1H, J=1.1, C(4)-H), 8.25 (s, 1H, C(2)-H), 7.99 (dd, 1H,

J1=8.6, J2=1.6, C(6)-H), 7.65 (d, 1H, J=8.5, C(7)-H), 4.79 (septet, 1H, J=6.7, CH(CH3)2), 1.62 (d, 6H, J=6.7, CH(CH3)2). 1-Isopropyl-2-methyl-1H-benzoimidazole-5-carboxylic acid (16). 4-(Isopropylamino)-3-nitro-benzoic acid (5a) (0.140g, 0.625 mmol) was taken up in ethyl acetate (10 mL) and shaken for 18 hours with palladium on carbon (0.014g) under hydrogen (60 psi). The resulting colourless solution was filtered through celite, and trimethylorthoacetate (0.5g, 4.17 mmol) and TFA (ca 0.01g) added. The resulting solution was stirred at room temperature for 1 hour and solvent removed under reduced pressure. The resulting solid was purified by column chromatography to give an off white solid (0.029g, 0.133 mmol, 21%) HPLC/MS: (column c) 97%, tr=0.88 min, m/z (ES-): 217 [M-H]-. 1H NMR (d6-DMSO): δ 12.5 (br s, 1H , CO2H), 7.97 (s, 1H, C(4)-H), 7.66 (dd, 1H, J1=8.6,

S11 J2=1.5, C(6)-H), 7.60 (d, 1H, J=8.4, C(7)-H), 4.65 (septet, 1H, J=6.9, CH(CH3)2), 2.46 (s, 3H, CH3), 1.45 (d, 6H, J=6.9, CH(CH3)2).

cAMP Whole Cell Assay. GPR109b with an N-terminal HA tag was cloned into pcDNA3.1 (Invitrogen) and GPR109b CHO stable cell lines were generated by G418 selection. Positive clones were selected by anti-HA immunostaining. Compound potencies were determined by the 96 well Adenylyl Cyclase Activation FlashPlate Assay from Perkin Elmer as described by the manufacturer. The cAMP assay was optimized for the GPR109 stable clone 3-1C, 5 µM forskolin was used for stimulation and 50,000 cells were used for each well. Positive controls (200%) were defined as cAMP generated by cells without forskolin stimulation and negative controls (100%) were defined as cAMP generated by cells with 5 µM forskolin stimulation.

Adipocyte Lipolysis Assay. Human subcutaneous adipose was collected from a 54-year old Caucasian woman who had died of Lupus. Adipose was placed in cold Krebs-Ringers-HEPES (KRH) buffer with 2% BSA. Tissue was digested for 40min in KRH with 1mg/ml collagenase, then spun at 100rpm at 37oC for 10min and the adipocytes were isolated. Primary cells were incubated with 1µM Isoproterenol for 1hr in the absence or presence of increasing concentrations of Niacin or 3a.

CLUSTAL W (1.8) multiple sequence alignment of GPR109a and GPR109b GPR109a GPR109b

MNRHHLQDHFLEIDKKNCCVFRDDFIVKVLPPVLGLEFIFGLLGNGLALWIFCFHLKSWK MNRHHLQDHFLEIDKKNCCVFRDDFIAKVLPPVLGLEFIFGLLGNGLALWIFCFHLKSWK ************************** *********************************

GPR109a GPR109b

SSRIFLFNLAVADFLLIICLPFLMDNYVRRWDWKFGDIPCRLMLFMLAMNRQGSIIFLTV SSRIFLFNLAVADFLLIICLPFVMDYYVRRSDWNFGDIPCRLVLFMFAMNRQGSIIFLTV **********************:** **** ** ******** ***:*************

GPR109a GPR109b

VAVDRYFRVVHPHHALNKISNRTAAIISCLLWGITIGLTVHLLKKKMPIQNGGANLCSSF VAVDRYFRVVHPHHALNKISNWTAAIISCLLWGITVGLTVHLLKKKLLIQNGPANVCISF ********************* *************:********** **** ** * **

GPR109a GPR109b

SICHTFQWHEAMFLLEFFLPLGIILFCSARIIWSLRQRQMDRHAKIKRAITFIMVVAIVF SICHTFRWHEAMFLLEFLLPLGIILFCSARIIWSLRQRQMDRHAKIKRAITFIMVVAIVF ******:**********:******************************************

GPR109a GPR109b

VICFLPSVVVRIRIFWLLHTSGTQNCEVYRSVDLAFFITLSFTYMNSMLDPVVYYFSSPS VICFLPSVVVRIRIFWLLHTSGTQNCEVYRSVDLAFFITLSFTYMNSMLDPVVYYFSSPS ************************************************************

GPR109a GPR109b

FPNFFSTLINRCLQRKMTGEPDNNRSTSVELTGDPNKTRGAPEALMANSGEPWSPSYLGP FPNFFSTLINRCLQRKMTGEPDNNRSTSVELTGDPNKTRGAPEALMANSGEPWSPSYLGP

S12 ************************************************************ GPR109a GPR109b Key:

TSP-----------------------TSNNHSKKGHCHQEPASLEKQLGCCIE ** X= Predicted transmembrane domains X=Differences in sequence within the TM domains R=Arg-111 * = identical residues