Supplemental Data Alexander et al. Experimental Procedures General Methods for Inhibitor Synthesis All chemicals were obtained from Aldrich, Acros, Fisher, or Fluka and were used without further purification, except where noted. Dry solvents (tetrahydrofuran, dichloromethane, and toluene) and triethylamine were obtained by passing these through activated alumina columns. All reactions were carried out under an inert nitrogen atmosphere using oven-baked glassware unless otherwise noted. Flash chromatography was performed using 230-400 mesh silica gel 60. 1 H spectra were recorded on a Bruker AMX-400 MHz spectrometer. Chemical shifts are reported in δ values relative to tetramethylsilane, and coupling constants (J) are reported in Hz. Synthesis of N-oleyl-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (JP23) 1-Isocyanato-octadec-9-ene Using a modification of the procedure from Nowick and colleagues [1], oleylamine (70% technical grade, 1.23 ml, 3.7 mmol, 1.0 equiv) was dissolved in dichloromethane (22 ml, 0.10 M), cooled to 0 C, and a saturated aqueous solution of sodium bicarbonate added. The biphasic mixture was stirred for 25 min at 0 C, the layers were allowed to separate, and a solution of phosgene (20% in toluene, 4.0 ml, 7.6 mmol, 2.1 equiv) was added directly to the organic layer via syringe. After 10 min, the aqueous layer was extracted with dichloromethane (3 x 40 ml). The extracts were combined with the organic layer of the reaction, dried over Na 2 SO 4, and the solvent removed by rotary
evaporation under reduced pressure to provide a yellow oil (1.19 g, 100%). A clean shift of the protons on C-11 from δ 2.66 ppm to δ 3.28 ppm indicated the isocyanate had formed so this material was carried on without further purification. 1 H NMR (CDCl 3 ) δ 5.36 (m, 2H), 3.28 (t, J=6.7, 2H), 2.01(m, 4H), 1.61 (m, 3H), 1.27 (m, 25H), 0.88 (m, 3H). N-oleyl-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (JP23) Using a modification of the procedure of Mor and colleagues [2], 3'-hydroxy-biphenyl-3- carboxylic acid amide (0.05 g, 0.23 mmol, 1.0 equiv), which was synthesized as described [2], was suspended in a mixture of toluene/tetrahydrofuran (2:1, 3 ml) and triethylamine (33 µl, 0.23 mmol, 1.0 equiv) was added. To this was added a solution of 1-isocyanato-octadec-9-ene (0.076 g, 0.26 mmol, 1.1 equiv) in toluene (1 ml, 0.26 M). The reactants were heated at reflux for 13 hrs. After cooling the reaction to room temperature, dichloromethane was added (40 ml) and the organic phase washed with HCl (1 M, 1 x 40 ml), saturated sodium bicarbonate solution (1 x 40 ml), and brine (1 x 40 ml). The organic layer was dried over Na 2 SO 4 and solvent was removed via rotary evaporation under reduced pressure. Column chromatography (2:1 hexanes/ethyl acetate followed by 10:1 ethyl acetate/methanol) afforded JP23 as a white powder (0.045 g, 38%). 1 H NMR (CDCl 3 ) δ 7.99 (s, 1H), 7.76 (d, J=7.7, 1H), 7.70 (d, J=7.7, 1H), 7.48 (t, J=7.8, 1H), 7.41 (d, J=5.0, 2H), 7.35 (bs, 1H), 7.13 (m, 1H), 6.34 (bs, 1H), 5.85 (bs, 1H), 5.35 (m, 2H), 5.10 (m, 1H), 3.29 (m, 2H), 1.99 (m, 3H), 1.58 (m, 2H), 1.28 (m, 22H), 0.88 (t, J=6.8, 3H). ESI-TOF m/z 507.3575 (C 25 H 30 N 2 O 3 + H + requires 507.3581).
Synthesis of (6-Phenyl-hexyl)-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (JP83) 2-(6-Phenyl-hexyl)-isoindole-1,3-dione Using a modification of the procedure of Govoni and colleagues [3], 6-phenyl-1-hexanol (0.32 ml, 1.68 mmol, 1.0 equiv), triphenylphosphine (0.441 g, 1.68 mmol, 1.0 equiv) and isoindole-1,3-dione (0.247 g, 1.68 mmol, 1.0 equiv) were stirred vigorously in dry THF (2.4 ml) in a flask cooled to 0 C. Diisopropyl azodicarboxylate (0.32 ml, 1.67 mmol, 0.99 equiv) in dry THF (2.4 ml) was added dropwise to the cooled flask. After stirring at room temperature overnight, the solvent was removed via rotary evaporation under reduced pressure. Column chromatography (4:1 hexanes/ethyl acetate) afforded the desired product (0.365 g, 71%). 1 H NMR (CDCl 3 ) δ 7.82 (m, 2H), 7.68 (m, 2H), 7.23 (m, 2H), 7.15 (m, 3H), 3.66 (t, J=7.2, 2H), 2.58 (t, J=2.6, 2H), 1.64 (m, 4H), 1.37 (m, 4H). ESI-TOF m/z 308.1648 (C 20 H 21 NO 2 + H + requires 308.1645). 6-Phenyl-1-hexylamine To a flask containing 2-(6-Phenyl-hexyl)-isoindole-1,3-dione (0.364 g, 1.19 mmol, 1 equiv) dissolved in EtOH (18 ml) was added hydrazine monohydrate (0.18 ml, 3.71 mmol, 3.1 equiv). The solution was heated at reflux. After 5 hrs, the solution was cooled to room temperature and the white precipitate removed by filtration. The solvent was removed from the filtrate by rotary evaporation under reduced pressure. The residue was dissolved in sodium hydroxide (1M, 50 ml) and extracted with chloroform (3 x 50 ml). The combined organic extracts were washed with brine (1 x 100 ml) and dried over Na 2 SO 4. Solvent was removed via rotary evaporation under reduced pressure to provide a
yellow oil. Column chromatography (10:1 chloroform/methanol with 2% triethylamine) afforded 6-phenyl-1hexylamine as a yellow oil (0.168 g, 80%). 1 H NMR (CDCl 3 ) δ 7.26 (m, 2H), 7.16 (m, 3H), 7.23 (m, 2H), 2.68 (t, J=7.0, 2H), 2.60 (t, J=2.6, 2H), 2.35 (s, 2H), 1.62 (m, 2H), 1.45 (m, 2H), 1.34 (m, 4H). ESI-TOF m/z 178.1593 (C 11 H 21 N + H + requires 178.1590). (6-Isocyanato-hexyl)-benzene Using a modification of the procedure from Nowick and colleagues [1], 6-phenyl-1- hexylamine (0.165 g, 0.93 mmol, 1.0 equiv) was dissolved in dichloromethane (9.3 ml), cooled to 0 C, and a saturated aqueous solution of sodium bicarbonate (9.3 ml) was added. The biphasic mixture was stirred for 10 min at 0 C, the layers were allowed to separate, and a solution of phosgene (20% in toluene, 0.98 ml, 1.86 mmol, 2 equiv) was added directly to the organic layer via syringe. After 15 min, the aqueous layer was extracted with dichloromethane (3 x 25 ml). The organic extracts were combined with the organic layer and dried over Na 2 SO 4. The solvent was removed by rotary evaporation under reduced pressure to provide a pale yellow oil (0.178 g, 94%). A clean shift of the protons on C-1 from δ 2.66 ppm to δ 3.14 ppm indicated the isocyanate had formed so this material was carried on without further purification. 1 H NMR (CDCl 3 ) δ 7.17 (m, 2H), 7.07 (m, 3H), 3.14 (t, J=6.6, 2H), 2.51 (t, J=7.7, 2H), 1.51 (m, 4H), 1.27 (m, 4H). (6-Phenyl-hexyl)-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (JP83) Using a modification of the procedure of Mor and colleagues [2], 3'-hydroxy-biphenyl-3- carboxylic acid amide (0.093 g, 0.44 mmol, 1.0 equiv) which was synthesized as
described [2] was suspended in a mixture of toluene/tetrahydrofuran (4:1, 5 ml) and triethylamine (6.1 µl, 0.435 mmol, 1.0 equiv) was added. To this was added a solution of (6-isocyanato-hexyl)-benzene (0.177 g, 0.87 mmol, 2.0 equiv) in toluene/thf (4:1, 2.5 ml, 0.35 M). The reactants were heated at reflux for 9 hrs. Solvent was removed via rotary evaporation under reduced pressure. Column chromatography (10:1 chloroform/methanol) afforded JP83 as a white solid (0.114 g, 63%). 1 H NMR (CDCl 3 ) δ 7.95 (s, 1H), 7.73 (d, J=7.1, 1H), 7.63 (d, J=7.3, 1H), 7.36 (m, 3H), 7.27 (m, 3H), 7.16 (m, 3H), 7.08 (m, 1H), 6.71 (bs, 1H), 6.20 (bs, 1H), 5.39 (m, 1H), 3.23 (m, 2H), 2.58 (t, J=7.5, 2H), 1.57 (m, 4H), 1.35 (m, 4H). ESI-TOF m/z 417.2186 (C 26 H 28 N 2 O 3 + H + requires 417.2173). Synthesis of N-oleyl-carbamic acid phenyl ester (JP87) To a flask containing phenol (0.259 g, 2.75 mmol, 1.0 equiv) in toluene (16 ml) was added triethylamine (0.38 ml, 2.8 mmol, 1.0 equiv). To this was added a solution of 1- isocyanato-octadec-9-ene (1.40 g, 4.8 mmol, 1.7 equiv).. The reactants were heated at reflux for 8 hrs. The solvent was removed via rotary evaporation under reduced pressure. Column chromatography (4:1 hexanes/ethyl acetate) afforded JP87 as a white powder (0.045g, 38%). 1 H NMR (CDCl 3 ) δ 7.34 (t, J=7.8, 2H), 7.18 (t, J=7.3, 1H), 7.12 (d, J=7.9, 2H), 5.36 (m, 2H), 5.01 (bs, 1H), 3.27 (m, 2H), 2.00 (m, 4H), 1.57 (m, 2H), 1.29 (m, 22H), 0.88 (t, J=6.9, 3H). ESI-TOF m/z 388.3216 (C 25 H 41 NO 2 + H + requires 388.3210).
Synthesis of Undec-10-ynyl-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (JP104) 2-Undec-10-ynyl-isoindole-1,3-dione Using a modification of the procedure of Govoni and colleagues [3], 10-undecyn-1-ol (1.0 ml, 5.21 mmol, 1.0 equiv), triphenylphosphine (1.37 g, 5.21 mmol, 1.0 equiv) and isoindole-1,3-dione (0.767 g, 5.21 mmol, 1.0 equiv) were stirred vigorously in dry THF (7.45 ml) under argon in a flask cooled to 0 C. Diisopropyl azodicarboxylate (1.0 ml, 5.16 mmol, 0.99 equiv) in dry THF (7.45 ml) was added dropwise to the cooled flask. After stirring at room temperature overnight, the solvent was removed via rotary evaporation under reduced pressure. Upon dilution of the yellow oil in hexanes/ethyl acetate (2:1) a white precipitate formed and was removed by filtration. Column chromatography (2:1 hexanes/ethyl acetate) of the filtrate afforded the desired product as a pale yellow solid (1.27 g, 82%). 1 H NMR (CDCl 3 ) δ 7.84 (m, 2H), 7.71 (m, 2H), 3.68 (t, J=7.2, 2H), 2.17 (td, J=2.6, J=7.1, 2H), 1.94 (t, J=2.6, 1H), 1.67 (m, 2H), 1.51 (m, 2H), 1.33 (m, 6H), 1.28 (m, 4H). ESI-TOF m/z 298.1805 (C 19 H 23 NO 2 + H + requires 298.1801). Undec-10-ynylamine To a flask containing 2-undec-10-ynyl-isoindole-1,3-dione (1.27g, 4.27 mmol, 1.0 equiv) dissolved in EtOH (64 ml) was added hydrazine monohydrate (0.62 ml, 12.8 mmol, 3.0 equiv). The flask was fitted with a condenser and flushed with argon. The solution was heated at reflux for 5 hrs, cooled to room temperature, and the white precipitate was
removed by filtration. The solvent was removed from the filtrate by rotary evaporation under reduced pressure. The residue was dissolved in saturated sodium bicarbonate solution (75 ml) and extracted with chloroform (3 x 75 ml). The combined organic extracts were washed with brine (1 x 75 ml) and dried over Na 2 SO 4. Solvent was removed via rotary evaporation under reduced pressure to provide a pale yellow solid. Column chromatography (10:1 chloroform/methanol with 2% triethylamine) afforded undec-10-ylylamine as a white solid (0.604 g, 85%). 1 H NMR (CDCl 3 ) δ 2.67 (t, J=7.0, 2H), 2.15 (m, 2H), 1.93 (dt, J=0.8, J=2.6, 1H), 1.86 (s, 2H), 1.50 (m, 2H), 1.37 (m, 4H), 1.28 (m, 8H). ESI-TOF m/z 168.1749 (C 11 H 21 N + H + requires 168.1747). 11-Isocyanato-undec-1-yne Using a modification of the procedure from Nowick and colleagues [1], undec-10- ylylamine (0.368 g, 2.2 mmol, 1.0 equiv) was dissolved in dichloromethane (22 ml), cooled to 0 C, and a saturated aqueous solution of sodium bicarbonate (22 ml) added. The biphasic mixture was stirred for 10 min at 0 C, the layers were allowed to separate, and a solution of phosgene (20% in toluene, 2.2 ml, 4 mmol, 2.0 equiv) was added directly to the organic layer via syringe. After 25 min, the reaction was diluted in 50 ml dichloromethane, the layers separated, and the aqueous layer was extracted with dichloromethane (3 x 50 ml). The organic layer and organic extracts were combined and washed with water (1 x 50 ml), dried over Na 2 SO 4, and the solvent removed by rotary evaporation under reduced pressure to provide a pale yellow oil (0.396 g, 93%). A clean shift of the protons on C-11 from δ 2.67 ppm to δ 3.28 ppm indicated the isocyanate had formed so this material was carried on without further purification. 1 H NMR (CDCl 3 ) δ
3.28 (t, J=6.7, 2H), 2.17 (dt, J=2.6, J=7.1, 2H), 1.93 (t, J=2.6, 1H), 1.6 (m, 2H), 1.50 (m, 2H), 1.38 (m, 4H), 1.30 (m, 6H). Undec-10-ynyl-carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (JP104) Using a modification of the procedure of Mor and colleagues [2], 3'-hydroxy-biphenyl-3- carboxylic acid amide (0.218 g, 1.02 mmol, 1.0 equiv) which was synthesized as described [2] was suspended in a mixture of toluene/tetrahydrofuran (7:1, 8 ml) and triethylamine (0.14 ml, 1.02 mmol, 1.0 equiv) was added. To this was added a solution of 11-isocyanato-undec-1-yne (0.396 g, 2.05 mmol, 2.0 equiv) in toluene (4 ml). The reactants were heated at reflux for 8 hrs. The reaction was cooled to room temperature and filtered to remove a small amount of precipitate. Solvent was removed from the filtrate via rotary evaporation under reduced pressure. Column chromatography (10:1 chloroform/methanol) afforded JP104 as an off-white powder (0.246 g, 59%). 1 H NMR (CDCl 3 ) δ 7.97 (s, 1H), 7.75 (d, J=7.6, 1H), 7.67 (d, J=7.6, 1H), 7.44 (t, J=7.7, 1H), 7.39 (d, J=4.9, 2H), 7.32 (bs, 1H), 7.10 (m, 1H), 6.61 (bs, 1H), 6.10 (bs, 1H), 5.36 (m, 1H), 3.28 (m, 2H), 2.17 (dt, J=2.6, J=7.0, 2H), 1.95 (t, J=2.6, 1H), 1.53 (m, 4H), 1.31 (m, 10H). ESI-TOF m/z 407.2331 (C 25 H 30 N 2 O 3 + H + requires 407.2329). References 1. Nowick, J.S., Holmes, D.L., Noronha, G., Smith, E.M., Nguyen, T.M., and Huang, S.-L. (1996). Synthesis of Peptide Isocyanates and Isothiocyanates. Journal of Organic Chemistry 61, 3929-3934. 2. Mor, M., Rivara, S., Lodola, A., Plazzi, P.V., Tarzia, G., Duranti, A., Tontini, A., Piersanti, G., Kathuria, S., and Piomelli, D. (2004). Cyclohexylcarbamic Acid 3'- or 4'-Substituted Biphenyl-3-yl Esters as Fatty Acid Amide Hydrolase Inhibitors: Synthesis, Quantitative Structure-Activity Relationships and Molecular Modeling Studies. J. Med. Chem. 47, 4998-5008.
3. Govoni, M., Bakker, R.A., van de Wetering, I., Smit, M.J., Menge, W.M.B.P., Timmerman, H., Elz, S., Schunack, W., and Leurs, R. (2003). Synthesis and Pharmacological Identification of Neutral Histamine H1-Receptor Antagonists. Journal of Medicinal Chemistry 46, 5812-5824. Supplemental Figures and Table Supplemental Figure 1. Selective labeling of FAAH in brain membrane extracts by JP104 in vitro. A, Identification of targets of JP104 in brain membrane extracts by CC with an RhN 3 tag. The 65 kda JP104-labeled protein was confirmed as FAAH by its absence in brain proteomes either pre-treated with the FAAH inhibitor URB597 (data not shown) or from FAAH(-/-) mice (Figure 3C). B, Competitive ABPP with the serine hydrolase directed probe FP-Rh. JP104 did not block FP-Rh labeling of brain membrane serine hydrolases other than FAAH up to a concentration of 10 µm (right lanes). See Experimental Procedures for more details. Fluorescence images shown in grayscale.
Supplemental Figure 2. In vivo JP104 reactivity profiles of soluble proteomes from various mouse tissues. JP104 did not modify any soluble brain proteins except for a weakly labeled 70 kda target at the highest administered dose (10 mg/kg, left panel). This target was also observed at greater abundance in soluble kidney and liver proteomes, along with several additional proteins (middle and right panels). These JP104-labeled proteins were also observed in membrane proteomes from kidney and liver (Figure 3B). Fluorescence images shown in grayscale.
Supplemental Figure 3. JP104 and URB597 show similar dose-response profiles for the inactivation of brain FAAH. The extent of FAAH labeling by JP104 and URB597 in vivo was estimated by competitive ABPP experiments using membrane proteomes from brain tissue of inhibitor-treated mice and the FP-Rh activity-based probe. From these data sets, IC 50 values of 0.19 mg/kg (0.12-0.28 mg/kg, 95% confidence limits) and 0.28 mg/kg (0.15-0.54 mg/kg, 95% confidence limits) were estimated for the in vivo inactivation of FAAH by URB597 and JP104, respectively. Data represent an average of two trials per dose. Supplemental Table 1. IC 50 values determined by competitive ABPP for CE-6, Est31, and FAAH with JP104 and URB-597. IC 50 (µm) Compound CE-6 Est31 FAAH JP104 0.052 (0.035 0.076) 1.95 (1.31 2.90) 0.0016 (0.0012 0.0021) URB597 0.196 (0.119 0.322) > 100 0.045 (0.031 0.065) Data are presented as averages (n=3) with 95% confidence limits.