Supporting Information

Design, synthesis, and biological evaluation of deuterated

Apalutamide with improved pharmacokinetic profiles

Xuehai Panga, b, Yingwei Wanga, b, Yuanwei Chen a, b, c, *

aUniversity of Chinese Academy of Sciences, Chengdu institute of organic chemistry, Chengdu, 610041, China.bUniversity of Chinese Academy of Sciences, Beijing, 100049, China. cHinova Pharmaceuticals Inc., Suite 301, Building B, #5 South KeYuan Road, Chengdu 610041, China.

Chemistry 2-5

Pharmacology 5-6

Analytical data 7-16

Chemistry. Unless specified otherwise, starting materials and reagents were obtained from commercial

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sources and were used without further purification. Analytical TLC (thin-layer chromatography) was performed using 0.2mm silica gel plates (Yantai Jiangyou). And flash column chromatography was performed using silica gel (40-60 μm, Agela) with the indicated solvents. 1H and 13C NMR spectra were recorded on a Bruker 400 MHz instrument using deuteriochloroform (CDCl3), methanol-d4 (CD3OD), or DMSO-d6 as the solvent and TMS as the internal reference. Chemical shifts (δ) were reported with parts per million (ppm) relative to the residual solvent peak (for chloroform-d, δ (1H) = 7.26 ppm and δ (13C) = 77.16 ppm; for methanol-d4, δ (1H) = 3.31 ppm and δ  (13C) =49.00 ppm; and for DMSO-d6, δ  (1H) = 2.50 ppm and δ  (13C) = 39.52 ppm). Abbreviations used for signal patterns are as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; t, triplet; q, quadruplet; m, multiplet; and br, broad. High-resolution mass spectrometry (HRMS) was performed with a Bruker micrOTOF-Q (ESI)Ⅱ instrument. Liquid chromatography-Mass (LC/MS) was recorded on an Agilent LCMS 1200-6110 (ESI). All of the test compounds had purity ≥95% as determined by LCMS.Preparation of compound 5.5-nitro-3-(trifluoromethyl)pyridin-2-ol (2) To a  solution   of   compound  1  (25g,   0.15mol)   in cocnH2SO4  (100ml) was added the mixture HNO3  and cocnH2SO4  (v/v=1/1)(100ml) drop-wise  at room temperature. The reaction mixture was stirred for 4h. This reaction mixture was added into ice (1kg) portion-wise, stirred for 2h,  filtrated, washed with water (50ml), then, dried to afford 5-nitro-3-(trifluoromethyl)pyridin-2-ol (2) (23.3g, 74%). 1H NMR (400 MHz, DMSO-d6) δ 13.49 (br, 1H), 8.97 (s, 1H), 8.47 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 158.0, 142.4, 134.1 (q, J =5.1Hz), 128.7, 121.9 (q, J = 272.7 Hz), 116.8 (q, J = 30.3 Hz). 2-bromo-5-nitro-3-(trifluoromethyl)pyridine (3) Compound 9 (2.5g, 12mmoles) and POBr3 (10g, 34.8mmoles) were added in  round-bottom flask with a magnetic stir bar, followed by DMF (0.1 ml). The round-bottom flask was heated to 110°C and stirred for 4 h. This reaction mixture was added into ice (100g) portion-wise, then, adjusted pH to 7, and extracted with EA. The organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silics gel (PE) to afford 2-bromo-5-nitro-3-(trifluoromethyl)pyridine (3) (3.2g, 98%).5-nitro-3-(trifluoromethyl)picolinonitrile (4) To a solution of compound 3 (2.55g, 9.4mmoles)  in DMF (10ml) was added CuCN (1.01 g, 11.2 mmoles) under N2 at room temperature. The reaction mixture was heated to 110°C and stirred for 4 h. After completion of the reaction water (100ml) was added, and extracted with EA (25ml) 3 times. The organic  layer was washed with water (50ml) 2 times,   then, dried over anhydrous sodium sulfate,  and concentrated  under reduced pressure. The residue was purified by flash column chromatography on silics gel (PE) to afford 5-nitro-3-(trifluoromethyl)picolinonitrile (4) (1.15g, 52%). 5-amino-3-(trifluoromethyl)picolinonitrile (5) To a solution of compound 4 (1.15g, 4.9mmoles) in EA (20ml) and AcOH (4ml) was added Fe (890mg) portion-wise under N2 at room temperature. The  reaction mixture was   refluxed  for  16h.  After  cooling  to  room temperature,   the  reaction mixture was filter, then, the filtrate was concentrated under reduced pressure. The residue was purified by  flash column chromatography on silics gel (DCM/MeOH=50/1)  to  afford  5-amino-3-(trifluoromethyl)picolinonitrile   (5)  (540mg,  54%).  1H NMR (400 MHz, DMSO-d6) δ 8.21  (d, J=2.8Hz, 1H)，7.30 (d, J=2.8Hz, 1H)，7.02 (br, 2H).  13C NMR (101 MHz, DMSO-d6) δ 147.97 , 139.37, 129.9 (q, J =30.3 Hz), 122.2 (q, J = 274.7 Hz), 116.0 (d, J = 1.8 Hz), 115.0 (q, J = 4.0 Hz), 112.9.

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General procedure for preparation of compounds 7 and 8.2-fluoro-N-methyl-4-nitrobenzamide (7) To a solution of compound 6 (5.0g, 27.0mmol) and NEt3 

(8.2g, 81.1mmol) in DCM (50ml) was added CDI (5.3g, 32.5mmol) portion-wise under N2 at room temperature.   The  reaction  mixture  was  stirred  for  1h.   To   this  reaction  mixture   were  added MeNH2HCl  (2.9g, 40.5mmol), then the  reaction  mixture  was  stirred  for 4h. After completion of the reaction water (50ml) was added. The organic layer was washed with 1M HCl (50ml) and water   (50ml)   orderly.   The   organic   layer   was   dried   over   anhydrous   sodium   sulfate,   and concentrated under reduced pressure. The residue was purified by flash column chromatography on silics gel (ethyl acetate/ petroleum ether=1/2) to afford 2-fluoro-N-methyl-4-nitrobenzamide (7) (10.3g, 95%). MS (ESI): m/z 198.8 (M+H+, positive mode).2-fluoro-N-trideuteriomethyl-4-nitrobenzamide (8) 1H NMR (400 MHz, CDCl3) δ 7.95 (t, J = 8.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 13.0 Hz, 1H), 6.75 (br, 1H). 13C NMR (101 MHz, CDCl3) δ 163.1, 160.1 (d, J =252.5 Hz), 133.1, 128.5, 126.1 (d, J = 10.1 Hz), 120.2 (d, J =12.1 Hz), 119.5 (d, J = 28.3 Hz), 26.2 (m). MS (ESI): m/z 201.7 (M+H+, positive mode).General procedure for preparation of compounds 9 and 10.4-amino-2-fluoro-N-methylbenzamide (9) To a solution of compound 7 (14.6g, 73.7mmol) in EA (100ml)   and   AcOH   (9.2ml,   0.50mol)   was   added   Fe  (39g)  portion-wise  under   N2  at   room temperature. The reaction mixture was refluxed for 16h. After cooling to room temperature, the reaction  mixture  was  filter,   then,   the  filtrate  was  concentrated  under   reduced pressure.  The residue was purified by  flash column chromatography on silics  gel   (ethyl  acetate/  petroleum ether=1/1)  to afford 4-amino-2-fluoro-N-methylbenzamide (9)  (7.6g,  62%).  1H NMR (400 MHz, CDCl3) δ 7.92 (t, J=8.8 Hz, 1H)，6.60 (s, 1H)，6.49 (d, J=8.4 Hz, 1H), 6.32 (d, J=14Hz, 1H), 4.10 (s, 2H), 2.99 (d, J=4.4Hz, 3H). MS (ESI): m/z 169.1 (M+H+, positive mode).4-amino-2-fluoro-N-trideuteriomethylbenzamide (10) 1H NMR (400 MHz, CDCl3) δ 7.89 (t, J = 8.6 Hz, 1H), 6.61 (d, J = 12.2 Hz, 1H), 6.48 (dd, J = 8.6, 2.2 Hz, 1H), 6.33 (dd, J = 14.4, 2.2 Hz, 1H), 4.15 (br, 2H). 13C NMR (101 MHz, CDCl3) δ 164.4, 162.2 (d, J =245.7Hz), 151.5 (d, J = 12.8 Hz), 133.3 (d, J = 4.3 Hz), 110.9 (d, J = 1.8 Hz), 110.2 (d, J = 11.9 Hz), 100.8 (d, J =28.8Hz), 25.8 (m). MS (ESI): m/z 172.2 (M+H+, positive mode).General procedure for preparation of compounds 14 and 15.3,5-dideuterio-4-amino-2-fluoro-N-methylbenzamide (14) In a microwave reaction vial with a magnetic stir bar, compound 9 (336mg, 2.0 mmol) was added, followed by 0.2 M HCl in D2O (10ml, 2.0mmol). The vial was capped and sealed and heated in the microwave synthesis apparatus for 30 minutes at a temperature of 180°C. After cooling to room temperature 2M NaOH (1.2ml) and EA (10ml) was added. The   organic   layer   was   washed  with brine,  dried   over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silics gel (ethyl acetate/ petroleum ether=1/1) to afford 3,5-dideuterio-4-amino-2-fluoro-N-methylbenzamide   (14) (290.1mg, 84%).1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 9.2 Hz, 1H), 6.73 (br, 1H) , 4.74 (br, 2H), 2.96 (dd, J = 4.7, 1.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.5, 162.1 (d, J = 245.4 Hz), 152.2 (d, J = 12.8 Hz), 132.7 (d, J = 4.4 Hz), 110.3 (t, J = 25.2 Hz), 109.4 (d, J = 12.0 Hz), 100.3 (t, J = 27.8 Hz), 26.5. MS (ESI): m/z 170.7 (M+H+, positive mode). D% (ph) =97%.3,5-dideuterio-4-amino-2-fluoro-N- trideuteriomethylbenzamide (15) 1H NMR (400 MHz, CDCl3) δ 7.81  (d, J  = 9.2 Hz,  1H),  6.72  (br,  1H),  6.60  (br, 1H).  13C NMR (101 MHz, CDCl3) δ 164.5, 162.1(d, J = 245.4 Hz), 152.1(d, J = 12.7 Hz), 132.6, 110.3 (t, J = 24.9Hz), 109.4 (d, J = 12.1 Hz),

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100.3 (t, J = 25.4Hz), 25.8 (t, J = 20.5 Hz). MS (ESI): m/z 238.0 (M+H+, positive mode). D% (ph) =95%.General procedure for preparation of compounds 11-13, 16 and 17.4-((1-cyanocyclobutyl)amino)-2-fluoro-N-methylbenzamide (11) In a sealed tube with a magnetic stir bar, compound 9 (1g, 6.0mmol) was added, followed by TMSCN (1.77g, 17.8mmol), cyclobutanone (0.89ml, 11.9mmol) and AcOH (10ml). The sealed tube was heated to 80°C and stirred for 16h. After cooling to room temperature, EA (20ml) was added and then washed with water   (20ml). The organic   layer  was  dried  over  anhydrous  sodium sulfate,  and concentrated under reduced pressure. The residue was purified by flash column chromatography on silics gel (EA/PE=1/2)  to  afford  4-((1-cyanocyclobutyl)amino)-2-fluoro-N-methylbenzamide   (11) (1.32g, 90%). 1H NMR (400 MHz, CDCl3) δ 7.99 (t, J=8.4Hz, 1H), 6.70 (s, 1H), 6.49 (d, J=8.8Hz, 1H), 6.30 (d, J=14.4Hz, 1H), 4.62(s, 1H), 3.01 (d, J=4.8Hz, 3H), 2.84(m, 2H), 2.40(m, 2H), 2.27 (m, 1H), 2.20 (m, 1H). MS (ESI): m/z 247.7 (M+H+, positive mode).4-((1-cyanocyclobutyl)amino)-2-fluoro-N-trideuteriomethylbenzamide (12) 1H NMR (400 MHz, CDCl3) δ 7.98 (t, J = 8.8 Hz, 1H), 6.62 (d, J = 13.3 Hz, 1H), 6.49 (dd, J = 8.8, 2.2 Hz, 1H), 6.30 (dd, J = 14.3, 2.2 Hz, 1H), 4.72 (br, 1H), 2.93-2.75 (m, 2H), 2.44-2.36 (m, 2H), 2.33 – 2.10 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 164.1, 162.0 (d, J = 246.6 Hz), 148.0 (d, J = 12.3 Hz), 133.3 (d, J = 4.3 Hz), 120.7, 111.6 (d, J = 12.1 Hz), 110.7, 100.2 (d, J = 30.1 Hz), 49.5, 34.0, 15.7. MS (ESI): m/z 251.0 (M+H+, positive mode).4-((3,3,4,4-tetradeuterio-1-cyanocyclobutyl)amino)-2-fluoro-N-methylbenzamide (13) 1H NMR (400 MHz, DMSO-d6) δ 7.82 (t, J = 4.7 Hz, 1H), 7.57 (t, J = 8.6 Hz, 1H), 7.37 (s, 1H), 6.47 (dd, J = 8.6, 2.1 Hz, 1H), 6.32 (dd, J = 13.7, 2.2 Hz, 1H), 2.75 (d, J = 4.5 Hz, 3H), 2.04 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 160.7 (d, J = 248.0 Hz), 148.5 (d, J = 11.5 Hz), 131.6, 131.6, 121.5, 112.0 (d, J = 13.8 Hz), 109.4, 99.5 (d, J = 27.4 Hz), 48.6, 32.4 (m), 26.3, 14.9. MS (ESI): m/z 251.7 (M+H+, positive mode). D% (methene) =88%.3,5-dideuterio-4-((1-cyanocyclobutyl)amino)-2-fluoro-N-methylbenzamide (16) 1H NMR (400 MHz, DMSO-d6) δ 7.90 – 7.71 (m, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.36 (s, 1H), 2.84 – 2.68 (m, 5H), 2.39-2.32 (m, 2H), 2.10-2.04 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 160.7 (d, J = 248.0 Hz), 148.4 (d, J = 11.5 Hz), 131.5, 121.5, 112.0 (d, J = 13.8 Hz), 48.9, 33.0, 26.3, 15.3. MS (ESI): m/z 250.1 (M+H+, positive mode). D% (methene) =94%.3,5-dideuterio-4-((1-cyanocyclobutyl)amino)-2-fluoro-N-trideuteriomethylbenzamide (17) 1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 4.5 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.38 (s, 1H), 2.79-2.72 (m, 2H), 2.40-2.32 (m, 2H), 2.17-2.00 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 160.7 (d, J = 248.0 Hz), 148.4 (d, J = 11.5 Hz), 131.5, 121.5, 112.0 (d, J = 13.8 Hz), 48.9, 33.0, 15.3. MS (ESI): m/z 252.9 (M+H+, positive mode). D% (methene) =97%.General procedure for preparation of compounds 18-22.4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide (18) To a solution of compound 5 (50mg, 0.26mmol) and compound 11 (66mg, 0.26mmol) in DMA (0.5ml) was added thiophosgene (32mg, 0.26mmol) under N2. The reaction mixture was stirred for 16h at 60°C. To this mixture were added MeOH (2ml) and 2M HCl (2ml),   then  the  reaction  mixture  was   reflux   for  2h.  After   cooling   to  room  temperature,   the reaction mixture was poured into ice water (10ml) and extracted with EtOAc (10ml). The organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silics gel (EA/PE=1/1) to afford 4-(7-(6-

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cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide (18)  (51.3mg, 41%). 1H NMR (400 MHz, Methanol-d4) δ 9.17 (d, J = 2.1 Hz, 1H), 8.65 (d, J = 2.3 Hz, 1H), 7.95 (t, J = 8.2 Hz, 1H), 7.52 – 7.32 (m, 2H), 2.98 (s, 3H), 2.75-2.70 (m, 2H), 2.65-2.48 (m, 2H), 2.29-2.09 (m, 1H), 1.72-1.62 (m, 1H). MS (ESI):  m/z  478.1  (M+H+,  positive mode).4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-trideuteriomethylbenzamide (19) 1H NMR (400 MHz, Methanol-d4) δ 9.17 (d, J = 2.2 Hz, 1H), 8.65 (d, J = 2.2 Hz, 1H), 7.95 (t, J = 8.2 Hz, 1H), 7.59 – 7.32 (m, 2H), 2.81 – 2.67 (m, 2H), 2.67 – 2.52 (m, 2H), 2.29 – 2.05 (m, 1H), 1.72-1.62 (m, 1H). 13C NMR (101 MHz, Chloroform-d) δ 179.0, 174.2, 162.8, 160.5 (d, J=252.0 Hz), 159.3, 137 (d, J=10.5 Hz), 134.1 (q, J=4.3 Hz), 133.5 (d, J=3.4 Hz), 132.3, 130.4 (q, J=34.6 Hz), 129.9 (d, J=9.3 Hz), 126.5 (d, J=3.0 Hz), 123.1 (q, J=12.0 Hz), 121.3 (q, J=275.4 Hz), 118.3 (d, J=26.3 Hz), 67.7, 31.8, 26.4 (m), 13.7. MS (ESI):  m/z  481.2  (M+H+, positive mode). D% (methyl) >99%.3,5-dideuterio-4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide (20) 1H NMR (400 MHz, Methanol-d4) δ 9.17 (d, J = 1.6 Hz, 1H), 8.65 (d, J = 1.6 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 2.98 (s, 3H), 2.75-2.69 (m, 2H), 2.64 -2.56 (m, 2H), 2.19-2.12 (m, 1H), 1.69 -1.64 (m, 1H). MS (ESI): m/z 480.2 (M+H+, positive mode). D% (methene)=94%.3,5-dideuterio-4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-trideuteriomethylbenzamide (21) 1H NMR (400 MHz, Methanol-d4) δ 9.17 (d, J = 2.2 Hz, 1H), 8.65 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 2.76-2.70 (m, 2H), 2.67 -2.54 (m, 2H), 2.09-2.22 (m, 1H), 1.78 -1.57 (m, 1H).   MS (ESI):  m/z 483.3  (M+H+, positive mode). D% (methene) =97%, (methyl) >99%.4-(1,1,3,3-tetradeuterio-7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide (22) 1H NMR (400 MHz, Methanol-d4) δ 9.17 (d, J = 1.6 Hz, 1H), 8.65 (d, J = 1.2 Hz, 1H), 7.95 (t, J = 8.4 Hz, 1H), 7.44-7.40 (m, 2H), 2.98 (s, 1H), 2.17-2.09 (m, 1H), 1.66 -1.63 (m, 1H).  MS (ESI):  m/z  482.2  (M+H+,  positive mode).  D% (methyl) =88%.PharmacologyAnimals and cellsICR mouse (males, 23-33g) and Sprague-Dawley (SD) rats (males, 180-220g) were purchased from Shanghai Lingchang biological technology co., LTD, (Shanghai, China). LNCaP/AR cell lines were obtained from Sichuan University and Collaborative Innovation Center for Biotherapy (Chengdu, China).All animals were kept in an animal room with controlled illumination (12/12h light/dark cycle), temperature (20-25°C), and humidity (over 40 %). The animals were fasted for 12 h before dose administration. All PK studies performed at Shanghai Institute of Materia Medica (Chinese Academy of Sciences), and all PD studies performed at Sichuan Kangcheng biological co., LTD. The animal studies were carried out under protocols approved by the animal protection law of the People's Republic of China Care and Use Committees and institutional guidelines for the proper, humane use of animals. All rodent studies were also carried out in accordance with the Guide lines for Care and Use of Laboratory Animals approved by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Shanghai, China).Pharmacokinetic Parameters in Balb/c mice.

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The compounds 18, 19, 20, 21 and 22 were dissolved in 0.5% HPMC at a concentration of 1 mg/ml, respectively. Three male ICR mice were orally administered with 10 mg/kg of each compound. The blood sampling time points were pre-dose (0) and 0.5, 1, 2, 4, 6 and 24h post-doses. Three-hundred microliters of blood was collected from the orbital plexus into tubes after the animal was temporarily anesthetized by ether inhalation. The tubes were filled full for about 12h with a saline solution containing heparin (1000 units/ml), the saline was removed. Immediately after collection, blood collection tubes were gently inverted at least five times, ensuring complete mixing, and were then immediately placed on ice. The blood was centrifuged for 10 min at 3500 rpm and 4°C to separate the plasma from the red blood cells. After separation, 200μl disposable pipettes were used to take 100μl aliquots from the top plasma layer into tubes, with each being labeled by the name of analysis unit. Plasma was stored at −20 °C until removal for LC-MS/MS analysis for the test compound concentration.Pharmacokinetic Parameters in SD Rats.The compounds 18 and 19 were dissolved in 0.5% HPMC at a concentration of 1mg/ml, respectively. Four male SD rats were orally administered with 10 mg/kg of each compound. The blood sampling time points were pre-dose (0) and 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 24h post-doses. Three-hundred microliters of blood was collected from the orbital plexus into tubes after the animal was temporarily anesthetized by ether inhalation. The tubes were filled full for about 12h with a saline solution containing heparin (1000 units/ml), the saline was removed. Immediately after collection, blood collection tubes were gently inverted at least five times, ensuring complete mixing, and were then immediately placed on ice. The blood was centrifuged for 10 min at 3500 rpm and 4°C to separate the plasma from the red blood cells. After separation, 200μl disposable pipettes were used to take 100μl aliquots from the top plasma layer into tubes, with each being labeled by the name of analysis unit. Plasma was stored at −20 °C until removal for LC-MS/MS analysis for the test compound concentration.AR binding assaysThe LNCap/AR cell was cultured with RPMI1640 +10% FBS in the incubator at 37°C, 5% CO2. 6×104 cells were added to the 96-well plate (Poly-D precoated), and incubated for overnight. The medium was removed, then, 25μl compound dose (top concentration: 200μM, three-fold dilution) was added. Firstly, the cells were incubated for 30 min, then 25μM [3H]-R1881 (final top concentration is 100μM, three-fold dilution, final [3H]-R1881 concentration is 7nM) was added, the cells were incubated for 30 min again. The medium was removed, then the cells were washed with uptake buffer (10mM HEPEs, pH 7.5, 125mM NaCl, 4.8mM KCl, 1.2mM CaCl 2, 1.2mM MgSO4, 1.2mM KH2PO4, 5.6mM D-Glucose), and 100μl lysis buffer was added. Shaking the plate for 1 hour, the 50μl lysis buffer was transfered to an isotope plate, 200μl scintillation cocktail was added, and the dates were counted on the MicroBeta.

Analytical dataCompound 2

6

Compound 5

7

Compound 8

8

Compound 12

9

Compound 13

10

Compound 16

11

Compound 17

12

Compound 18

13

Compound 19

14

Compound 20

Compound 21

15

Compound 22

16