Small-molecule inhibition of TLR8 through stabilization of its resting state

Supplementary Note 1 Small-molecule inhibition of TLR8 through stabilization of its resting state Shuting Zhang 1,2,3, Zhenyi Hu 2, Hiromi Tanji 4, Shuangshuang Jiang 1, Nabanita Das 2, Jing Li 5, Kentaro Sakaniwa 4, Jin Jin 6, Yanyan Bian 6, Umeharu Ohto 4, Toshiyuki Shimizu 4 *, Hang Yin 1,2 * 1 Center of Basic Molecular Science, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100082, China; 2 Department of Chemistry and Biochemistry and Biofrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA; 3 School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China; 4 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan; 5 Department of Rheumatology and Clinical Immunology, Peking Union Medical College Hospital and Chinese Academy of Medical Sciences Key Laboratory of Rheumatology and Clinical Immunology Ministry of Education, Beijing 100032, China; 6 Department of Orthopedics, Peking Union Medical College Hospital, Beijing 100032, China. These authors contributed equally to this work *Email: yin_hang@tsinghua.edu.cn or shimizu@mol.f.u-tokyo.ac.jp

Chemical synthesis and characterizations Supplementary Figure 4: Synthesis of pyrazolo[1,5-a]pyrimidine derivatives. Reagents and conditions: (i) 1,1-dimethoxy-N,N-dimethylmethanamine (DMF-DMA), reflux; (ii) 5-amino-4-ethoxycarbonyl-1H-pyrazole, AcOH, reflux; (iii) NaOH, H 2 O/EtOH, 80 C; then acidify with 1M hydrochloric acid; (iv) SOCl 2, reflux; (v) DCM, NH 3 in THF, or DCM, methylamine in THF, or DCM, diethylamine in THF. Supplementary Figure 8: Synthesis of quinoline derivatives. Reagents and conditions: (i) POCl 3, reflux overnight; (ii) B 2 Pin 2, KOAc, PdCl 2 dppf CH 2 Cl 2, 1,4-dioxane, 90 C; (iii) K 2 CO 3, PdCl 2 dppf CH 2 Cl 2, 1,4-dioxane, H 2 O, 90 C.

Supplementary Figure 9: Synthesis of phthalazinone derivatives. Reagents and conditions: (i) AlCl 3, dichloroethane, reflux overnight; (ii) Hydrazine, EtOH, reflux overnight.

General chemistry methods NMR spectra were acquired on a Bruker 400 spectrometer, running at 400 MHz for 1 H and 101 MHz for 13 C respectively. 1 H NMR spectra were recorded at 400 MHz in CHCl 3 -d and (CH 3 ) 2 SO-d 6 using residual CHCl 3 (7.26 ppm) and DMSO (2.50 ppm) as an internal reference. 13 C NMR spectra were recorded at 101 MHz in CHCl 3 -d and DMSO-d 6 using residual CHCl 3 (77.16 ppm) and DMSO (39.52 ppm) as an internal reference. Thin layer chromatography was performed on Merck Kieselgel 60 Å F254 plates eluting with the solvent indicated, visualized by a 254 nm UV lamp. Compounds were purified using flash chromatography, (Silica gel 60Å, 230-400 mesh, Sorbent Tech.). Mass spectrometry was performed at the mass spectrometry facility of the Department of Chemistry at University of Colorado Boulder on a double focusing high-resolution mass spectrometer. All commercially reagents are purchased from Sigma-Aldrich, Fisher Scientific, VWR, Invivogen, etc. Unless otherwise noted, analytical grade solvents and commercially available reagents were used without further purification. The purity of tested compounds was evaluated by HPLC (Agilent Technologies 1200 serials) using UV detector at 254 nm. Synthesis Synthesis of (E)-3-(Dimethylamino)-(3-(trifluoromethyl)phenyl)prop-2-en-1-one (30) 3 -(Trifluoromethyl)acetophenone (1.6 ml, 10 mmol) and DMF-DMA (2.7 ml, 20 mmol) were refluxed overnight until the starting materials were consumed as determined by thin-layer chromatography (TLC), and then the reaction mixture was cooled to room temperature. The precipitate was filtered off and washed with cold hexane followed by cold ethanol to provide 2.1 g (86%) of 30 as a yellow solid. The product was of sufficient purity to be used directly in the next step. 1 H NMR (400 MHz, CDCl 3 ) δ 8.15 (td, J = 1.8, 0.9 Hz, 1H), 8.13 8.06 (m, 1H), 7.87 (d, J = 12.2 Hz, 1H), 7.72 (ddt, J = 7.8, 1.9, 0.9 Hz, 1H), 7.55 (tt, J = 7.8, 0.8 Hz, 1H), 5.71 (d, J = 12.3 Hz, 1H), 3.20 (s, 3H), 2.97 (s, 3H).

Synthesis of Ethyl 7-(3-(trifluoromethyl)phenyl)pyrazolo [1,5-a]pyrimidine-3-carboxylate (9) 5-Amino-4-ethoxycarbonyl-1H-pyrazole (216 mg, 1.39 mmol) and 30 (260 mg, 1.27 mmol) were added to acetic acid (5 ml). The reaction mixture was refluxed while stirring overnight. The progress of the reaction was monitored by TLC, and after completion of the reaction, the mixture was cooled and acetic acid was removed by co-evaporation with toluene. The crude product was purified with flash column chromatography on silica gel (eluent: dichloromethane/ethyl acetate, 0-20%) to yield 330 mg (78%) of ethyl 7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine -3-carboxylate (9) as white solid. Purity: 95.5%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.88 (d, J = 4.4 Hz, 1H), 8.64 (s, 1H), 8.27 (m, 2H), 7.89 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 7.0 Hz, 1H), 7.15 (d, J = 4.4 Hz, 1H), 4.49 (q, J = 7.1 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H). Ethyl 7-m-tolylpyrazolo[1,5-a]pyrimidine-3-carboxylate (10) 10 was prepared in a similar manner as described for 9. Yield: 73%; ESI MS m/z: 253.1071 [M+H] +. Purity: 99.7%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.83 (d, J = 4.4 Hz, 1H), 8.63 (s, 1H), 7.60 7.47 (m, 3H), 7.19 7.07 (m, 2H), 4.48 (q, J = 7.1 Hz, 2H), 3.90 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 162.57, 159.64, 152.32, 148.94, 147.82, 147.46, 131.38, 129.98, 121.68, 117.13, 115.07, 109.14, 103.16, 60.43, 55.51, 14.61. Synthesis of 7-(3-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine -3-carboxylic acid (6) The 9 (0.2 g, 0.6 mmol) was suspended in ethanol (3 ml) and treated with 7.2 M sodium hydroxide solution (1.1 ml, 7.9 mmol). The mixture was heated to 80 C and then stirred for 3 h. The mixture was cooled and neutralized to ph 6 7 using 1M HCl solution. The slurry was filtered and the solid residue was washed with water and then diethyl ether to obtain 0.12

g (67%) of 6 as a yellow solid. Purity: 97.9%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.65 (s, 1H), 8.48 (s, 1H), 8.24 (s, 2H), 8.07 7.97 (m, 2H), 7.80 (d, J = 3.6 Hz, 1H), 7.33 (s, 1H). 7-(m-Tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (11) 11 was prepared in a similar manner as described for 6. Yield: 67%; ESI MS m/z: 252.0776 [M-H] +. Purity: 94.1%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.42 (s, 1H), 8.84 (d, J = 4.4 Hz, 1H), 8.63 (s, 1H), 7.91 7.85 (m, 2H), 7.55 7.48 (m, 1H), 7.48 7.44 (m, 1H), 7.43 (d, J = 4.5 Hz, 1H), 2.42 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.64, 153.11, 148.75, 147.67, 147.48, 138.32, 132.41, 130.50, 130.49, 128.90, 127.36, 110.09, 103.09, 21.47. Synthesis of 7-(3-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carbox amide (12). A mixture of acid 6 (0.1 g, 0.3 mmol) and thionyl chloride (3 ml) were refluxed in a 10 ml round bottom flask fitted with a calcium chloride guard tube. After the reaction was completed (4 hours), the excess thionyl chloride was removed by rotary evaporation. The flask was put under high vacuum for 30-60 minutes to complete thionyl chloride removal. The residue was dissolved in anhydrous DCM (3 ml), the solution was cooled in an ice bath, and then ammonia (0.5 M in THF, 4 ml, 2 mmol) was added to the chilled solution. The reaction was seated in an ice bath for an additional 30 min, then the ice bath was removed and the reaction was allowed to stir at room temperature for 3 hours. The crude product was purified by recrystallization in DCM and hexane to yield 0.067 g (67.2%) of 12 as a white solid. Purity: 90.8%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.90 (d, J = 4.5 Hz, 1H), 8.64 (s, 1H), 8.51 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.66 7.54 (m, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.02, 152.50, 147.12, 146.28, 145.97, 134.29, 131.39, 130.31, 129.50, 128.40, 126.95, 125.70, 110.16, 106.10. 7-m-Tolylpyrazolo[1,5-a]pyrimidine-3-carboxamide (5 aka. CU-CPT8m)

5 (CU-CPT8m) was prepared in a similar manner that was described for 12. Purity: 99.9%. Yield: 76%. ESI MS m/z: 250.1508 [M+H] + ; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.82 (d, J = 4.6 Hz, 1H), 8.59 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.72 (s, 1H), 7.55 7.45 (m, 3H), 7.41 (d, J = 4.6 Hz, 1H), 2.41 (s, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.12, 152.38, 147.77, 147.24, 146.20, 138.37, 132.60, 130.48, 130.32, 128.95, 127.37, 109.56, 105.79, 21.48. 7-(2-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carbox amide (13) 13 was prepared in a similar manner as described for 11. Yield: 64%; Purity: 93.0%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.86 (d, J = 4.2 Hz, 1H), 8.54 (s, 1H), 8.12 8.05 (m, 1H), 7.94 7.85 (m, 2H), 7.60 7.50 (m, 1H), 7.42 (s, 2H). 7.02 (d, J = 4.2 Hz, 1H). 7-(4-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carbox amide (14) 14 was prepared in a similar manner as described for 11. Yield: 76%; Purity: 99.7%. ESI MS m/z: 307.0835 [M+H] + ; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.91 (d, J = 4.5 Hz, 1H), 8.63 (s, 1H), 8.33 (d, J = 8.1 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 7.61 (s, 2H), 7.56 (d, J = 4.5 Hz, 1H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.00, 152.55, 147.09, 146.26, 134.37, 131.27, 131.18, 125.92, 125.88, 111.68, 110.22, 106.14. 7-Phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide (15) 15 was prepared in a similar manner that described for 11. Yield: 81%; Purity: 98.4%. ESI MS m/z: 238.0835 [M+H] + ; 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.86 (d, J = 4.4 Hz, 1H), 8.63 (s, 1H), 8.15 8.04 (m, 2H), 7.64 (s, 1H), 7.71 7.58 (m, 2H), 7.47 (d, J = 4.4 Hz, 1H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.63, 153.17, 148.72, 147.66, 147.33, 131.80, 130.54, 130.19, 129.00, 110.14, 103.12. 7-(3-Nitrophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (16)

16 was prepared in a similar manner as described for 11. Yield: 72%; ESI MS m/z: 283.0746 [M+H] + ; Purity: 94.7%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.04 9.01 (m, 1H), 8.92 (d, J = 4.5 Hz, 1H), 8.66 (s, 1H), 8.55 8.49 (m, 2H), 7.98 7.92 (m, 1H), 7.66 7.59 (m, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 152.53, 148.06, 147.11, 146.31, 145.27, 136.62, 131.97, 131.81, 130.77, 126.45, 125.19, 110.25, 106.25. 7-(3-Fluorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (17) 17 was prepared in a similar manner that described for 11. Yield: 72%. ESI MS m/z: 257.0865 [M+H] + ; Purity: 99.0%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.88 (d, J = 4.5 Hz, 1H), 8.63 (s, 1H), 8.04 (ddd, J = 10.2, 2.4, 1.6 Hz, 1H), 7.98 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H), 7.69 (td, J = 8.1, 6.1 Hz, 1H), 7.60 (d, J = 7.5 Hz, 2H), 7.57 7.49 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.31, 163.01, 160.89, 152.40, 147.17, 146.25, 132.36, 131.23, 126.45, 118.86, 117.19, 109.86, 106.04. 7-(3-Chlorophenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (18) 18 was prepared in a similar manner that described for 11. Yield: 79%; ESI MS m/z: 273.0547 [M+H] + ; Purity: 94.9%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.87 (d, J = 4.5 Hz, 1H), 8.64 (s, 1H), 8.24 (t, J = 1.9 Hz, 1H), 8.11 8.03 (m, 1H), 7.74 (ddd, J = 8.1, 2.2, 1.1 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.63 7.51 (m, 3H). 13 C NMR (101 MHz, DMSO) δ 163.01, 152.42, 147.14, 146.26, 146.02, 133.59, 132.30, 131.73, 130.99, 129.89, 128.94, 109.92, 106.06. 7-(3,5-Bis(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (19) 19 was prepared in a similar manner that described for 11. Yield: 61%; Purity: 95.4%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.91 (d, J = 4.4 Hz, 1H), 8.67 (s, 1H), 8.61 8.63 (m, 2H), 8.29 8.23 (m, 1H), 7.38 (s, 2H), 7.20 (d, J = 4.3 Hz, 1H).

7-(2-Methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (20) 20 was prepared in a similar manner that described for 11. Yield: 66%; Purity: 99.7%. 1 H NMR (400 MHz, DMSO) δ 8.83 (d, J = 4.3 Hz, 1H), 8.57 (s, 1H), 7.81 7.72 (m, 4H), 7.31 7.23 (m, 2H), 7.06 (m, J = 4.3 Hz, 1H), 3.77 (s, 3H). 7-(3-Methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (21) 21 was prepared in a similar manner as described for 11. Yield: 62%; Purity: 97.2%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.83 (d, J = 4.4 Hz, 1H), 8.63 (s, 1H), 7.71 7.59 (m, 3H), 7.33 7.18 (m, 2H), 7.12 (s, 2H) 3.90 (s, 3H). 7-(Pyridin-3-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (22) 22 as prepared in a similar manner that described for 11. Yield: 73%; ESI MS m/z: 240.0911[M+H] + ; Purity: 96.8%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.25 (dd, J = 2.3, 0.8 Hz, 1H), 8.90 (d, J = 4.5 Hz, 1H), 8.82 (dd, J = 4.8, 1.6 Hz, 1H), 8.63 (s, 1H), 8.56 (ddd, J = 8.0, 2.3, 1.7 Hz, 1H), 7.70 7.65 (m, 2H), 7.40 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.01, 152.48, 152.35, 150.35, 147.05, 146.23, 145.14, 137.91, 126.71, 123.87, 109.90, 106.11. 7-(3-Ethylphenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (23) 23 was prepared in a similar manner as described for 11. Yield: 68%; ESI MS m/z: 267.1307 [M+H] + ; Purity: 99.9%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.84 (d, J = 4.5 Hz, 1H), 8.61 (s, 1H), 7.98 7.93 (m, 2H), 7.63 (s, 1H), 7.57 7.49 (m, 3H), 7.46 (d, J = 4.4 Hz, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 163.09, 152.32, 147.85, 147.25, 146.22, 144.65, 131.43, 130.39, 129.44, 128.99, 127.63, 109.56, 105.86, 28.57, 15.97.

N-Methyl-7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (24) 24 was prepared in a similar manner as described for 11. Yield: 68%; ESI MS m/z: 321.1136 [M+H] + ; Purity: 98.3%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.71 (d, J = 4.4 Hz, 1H), 8.36 8.32 (m, 1H), 8.31 8.27 (m, 1H), 8.01 (d, J = 5.5 Hz, 1H), 7.92 7.88 (m, 1H), 7.80 7.75 (m, 1H), 7.13 (d, J = 4.4 Hz, 1H), 3.11 (d, J = 4.9 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 162.61, 150.37, 146.88, 146.79, 146.48, 132.79, 131.72, 131.39, 130.87, 129.45, 128.29, 126.36, 108.43, 106.57, 25.98. N, N-Diethyl-7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (25) 25 was prepared in a similar manner as described for 11. ESI MS m/z: 363.1605 [M+H] + ; Purity: 98.5%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.71 (d, J = 4.3 Hz, 1H), 8.38 (s, 1H), 8.30 8.25 (m, 2H), 7.91 7.85 (m, 1H), 7.80 7.72 (m, 1H), 7.04 (d, J = 4.3 Hz, 1H), 3.61 (s, 4H), 1.27 (s, 6H). 13 C NMR (101 MHz, CDCl 3 ) δ 163.64, 150.32, 146.48, 145.55, 144.88, 132.64, 131.60, 131.33, 131.27, 129.37, 127.99, 126.25, 108.28, 108.08, 29.72, 14.69. 4-Chloro-7-methoxy-quinoline (31) 7-Methoxy-4-quinolinol (175 mg, 1.0 mmol) was stirred in POCl 3 (5 ml) at 90 C overnight. The reaction was then quenched with saturated sodium carbonate solution and extracted with ethyl acetate (50 ml 3). The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/ethyl acetate, 10-100%) to give the title compound (31) as a white solid (163 mg, 85%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.70 (d, J = 4.8 Hz, 1H), 8.12 (d, J = 9.2 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H), 7.35 (d, J = 4.8 Hz, 1H), 7.29 (dd, J = 9.2, 2.6 Hz, 1H), 3.97 (s, 3H).

2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol (32) Potassium acetate (2.94 g, 30.0 mmol), bis-(pinacolato)-diboron (3.05 g, 12.0 mmol) and bis(diphenylphosphine) ferrocene dichloropalladium (II) complex with dichloromethane (0.36 g, 0.5 mmol) was added to an anhydrous solution of 4-bromo-2-methylphenol (1.87 g, 10.0 mmol) in dioxane (180 ml) under anhydrous conditions in an atmosphere of nitrogen. The mixture was stirred at 90 C overnight. The reaction was then quenched with water and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/ethyl acetate, 10-100%) to give the title compound (32) as a white solid (1.75 g, 75%). ESI MS m/z: 235.1508 [M+H] + ; 1 H NMR (400 MHz, CDCl 3 ) δ 7.60 (s, 1H), 7.55 (d, J = 7.9 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 4.97 (s, 1H), 2.25 (s, 3H), 1.33 (s, 12H); 13 C NMR (101 MHz, CDCl 3 ) δ 156.77, 138.04, 134.46, 123.20, 114.56, 83.71, 24.99, 15.56. 2-Methyl-4-(7-methoxyl-4-quinolinyl)-phenol (7 aka. CU-CPT9a) A mixture of 4-chloro-7-methoxy-quinolone (80 mg, 0.42 mmol), 2-methyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol (97 mg, 0.42 mmol), K 2 CO 3 (158 mg, 1.1 mmol) and [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium (II), complex with dichloromethane (18 mg, 0.02 mmol) in dioxane (5 ml) and H 2 O (1 ml) was heated at 100 C overnight in an atmosphere of nitrogen. The reaction was then quenched with water and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/methanol, 0-10%) to give the title compound as white solid (77 mg, 85%). ESI MS m/z: 266.1181[M+H] + ; Purity: 99.0%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.69 (s, 1H), 8.79 (d, J = 4.5 Hz, 1H), 7.85 (d, J = 9.3 Hz, 1H), 7.44 (d, J = 2.6 Hz, 1H), 7.26 7.20 (m, 3H), 7.20 7.16 (m, 1H), 6.95 (d, J = 8.2 Hz, 1H), 3.93 (s, 3H), 2.21 (s, 3H); 13 C NMR (101 MHz, DMSO) δ 159.84, 155.98, 150.27, 150.14, 147.57, 131.66, 127.96, 127.90, 126.93, 124.32, 121.13, 119.20, 119.12, 114.71, 107.77, 55.46, 16.03.

2-Methyl-4-(7-hydroxyl-4-quinolinyl)-phenol (8 aka. CU-CPT9b) A solution of CU-CPT9a (80 mg, 0.30 mmol) in anhydrous dichloromethane (7 ml) was cooled in ice bath and treated dropwise with a 1 M solution of BBr 3 in dichloromethane (1.9 ml), and the resulting solution was stirred in an ice bath for 30 min and at room temperature for 1 h. The mixture was then diluted with water and adjusted to ph 6 with NaHCO 3 and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/methanol, 0-20%) to give the title compound as a light yellow solid (37 mg, 49%). ESI MS m/z: 252.1024 [M+H] + ; Purity: 95.9%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.15 (s, 1H), 9.66 (s, 1H), 8.71 (d, J = 4.5 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 7.25 7.21 (m, 1H), 7.19 7.09 (m, 3H), 6.94 (d, J = 8.2 Hz, 1H), 2.20 (s, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 158.14, 155.87, 150.16, 150.07, 147.52, 131.60, 128.12, 127.88, 127.05, 124.24, 120.33, 119.12, 118.43, 114.67, 110.46, 16.02. 2-Methyl-4-(2-methyl-4-quinolinyl)-phenol (26) A mixture of 4-Chloroquinaldine (89 mg, 0.50 mmol), 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-phenol (110 mg, 0.50 mmol), K 2 CO 3 (207 mg, 1.5 mmol) and [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium(ii), complex with dichloromethane (19 mg, 0.02 mmol) in dioxane (5 ml) and H 2 O (1 ml) was heated at 100 C overnight in an atmosphere of nitrogen. The reaction was then quenched with water and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/methanol, 0-10%) to give the title compound as white solid (87 mg, 70%). ESI MS m/z: 250.1232 [M+H] + ; Purity: 97.8%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.99 7.93 (m, 1H), 7.92 7.86 (m, 1H), 7.70 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.49 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.27 (s, 1H), 7.25 (dd, J = 2.3, 0.9 Hz, 1H), 7.20 7.15 (m, 1H), 6.95 (d, J = 8.2 Hz, 1H), 2.65 (s, 3H), 2.20 (s, 3H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 158.49, 156.07, 148.06, 148.00, 131.87, 129.35, 128.80, 128.15, 128.00, 125.89, 125.60, 124.75, 124.54,

122.02, 114.86, 24.88, 16.19. 2-Methyl-4-(2-methyl-4-quinolinyl)-phenol (27) A mixture of 1-chloronaphthalene (105 mg, 0.50 mmol), 2-methyl-4-(4,4,5,5-tetramethyl-1,3, 2-dioxaborolan-2-yl)-phenol (110 mg, 0.50 mmol), K 2 CO 3 (209 mg, 1.5 mmol) and [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium(ii), complex with dichloromethane (21 mg, 0.02 mmol) in dioxane (5 ml) and H 2 O (1 ml) was heated at 100 C overnight in an atmosphere of nitrogen. The reaction was then quenched with water and extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/methanol, 0-10%) to give the title compound as white solid (87 mg, 77%). ESI MS m/z: 235.1123 [M+H] + ; Purity: 95.9%. 1 H NMR (400 MHz, CDCl 3 ) δ 7.98 7.86 (m, 2H), 7.87 7.80 (m, 1H), 7.56 7.35 (m, 4H), 7.31 7.23 (m, 1H), 7.22 (dd, J = 8.1, 2.2 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 4.91 (s, 1H), 2.34 (s, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ 153.33, 140.14, 133.95, 133.36, 132.81, 131.96, 128.88, 128.37, 127.38, 126.98, 126.27, 126.00, 125.80, 125.52, 123.69, 114.85, 16.01. 4-Phenyl-1(2H)-phthalazinone (28) To a solution of phthalic anhydride (347 mg, 2.36 mmol) in dichloroethane (10 ml) was added reactant benzene(188 mg, 2.41 mmol). The solution was cooled in an ice bath. Aluminum chloride (640 mg, 4.80 mmol) was added portionwise. The ice bath was removed after 10 min and warmed to room temperature over 1 h. The reaction mixture was refluxed overnight (16 h), cooled to room temperature, and poured carefully into a stirred solution of ice /1 N HCl (500 ml). The organic layer was separated, and the aqueous layer was extracted with EA (250 ml 3). The combined organic layers were dried over magnesium sulfate, filtered, concentrated, and redissolved in EtOH (15 ml). Hydrazine monohydrate (250 μl, 5.22 mmol) was added, the mixture was refluxed overnight, cooled to room temperature and concentrated. The residue was purified by flash column chromatography on silica gel (eluent:

dichloromethane/methanol, 0-10%) to give the title compound as white solid (376 mg, 72%). Purity: 95.4%. 1 H NMR (400 MHz, CDCl 3 ) δ 10.49 (s, 1H), 8.62 8.45 (m, 1H), 7.86 7.69 (m, 3H), 7.62 7.57 (m, 2H), 7.56 7.50 (m, 3H). 4-(4-Hydroxy-3-methylphenyl)-1(2H)-phthalazinone (29) To a solution of phthalic anhydride (103 mg, 0.70 mmol) in dichloroethane (10 ml) was added 2-methylphenol (50 mg, 0.46 mmol). The solution was cooled in an ice bath. Aluminum chloride (185 mg, 1.39 mmol) was added portionwise. The ice bath was removed after 10 min and warmed to room temperature over 1 h. The reaction mixture was refluxed overnight, cooled to room temperature, and poured carefully into a stirred solution of ice/1 N HCl (150 ml). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (100 ml 3). The combined organic layers were dried over magnesium sulfate, filtered, concentrated, and redissolved in EtOH (15 ml). Hydrazine monohydrate (150 μl, 3.13 mmol) was added, the mixture was refluxed overnight, cooled to room temperature and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: dichloromethane/methanol, 0-10%) to give the title compound as white solid (74 mg, 63%). Purity: 97.1%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.73 (s, 1H), 8.35 8.27 (m, 1H), 7.94 7.82 (m, 2H), 7.78 7.71 (m, 1H), 7.29 (dd, J = 2.3, 0.9 Hz, 1H), 7.21 (ddd, J = 8.2, 2.3, 0.6 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 2.19 (s, 3H).