Development and Pharmacological Characterization of Selective. Blockers of 2-Arachidonoyl Glycerol Degradation with Efficacy in

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1 SUPPORTING INFORMATION Development and Pharmacological Characterization of Selective Blockers of 2-Arachidonoyl Glycerol Degradation with Efficacy in Rodent Models of Multiple Sclerosis and Pain Margherita Brindisi, Samuele Maramai, Sandra Gemma, Simone Brogi, Alessandro Grillo, Lorenzo Di Cesare Mannelli, Emanuele Gabellieri, Stefania Lamponi, Simona Saponara, Beatrice Gorelli, Daniele Tedesco, Tommaso Bonfiglio, & Christophe Landry, Kwang-Mook Jung, Andrea Armirotti, Livio Luongo, # Alessia Ligresti, Fabiana Piscitelli, Carlo Bertucci, Marie-Pierre Dehouck, Giuseppe Campiani,, * Sabatino Maione, # Carla Ghelardini, Anna Pittaluga, &, Daniele Piomelli, Vincenzo Di Marzo, and Stefania Butini *To whom correspondence may be addressed: , campiani@unisi.it; Tel Figures S1-S10 Tables S1-S6 1 H and 13 C NMR spectra of compounds (±)-4a, (±)-5a and (±)-6a Elemental analysis 1

2 Revised pharmacophore model for 3,4-disubstituted β-lactam-based MGL Inhibitors. Our revised pharmacophore model for 3,4-disubstituted β-lactam-based MGL Inhibitors consists in the combination of appropriate aromatic systems geometry with a suitable linker and a polar functionality, to establish interactions with Ser122. This model allowed us to attain potency and selectivity. In fact, the key feature of the trans 3,4-disubstituted β-lactam system of compounds 4a-h is the longer distance between the centroids of the aromatic systems (~7 Å) compared to the diphenylmethane scaffold of JZL184, SAR629 and JJKK048 (5-6 Å) (Figure S1), which may provide improvement in MGL inhibition potency. The spatial arrangement of the aryl moieties, constrained by a dihedral angle of 119.4, is critical to maximize the interaction with the hydrophobic pockets of the enzyme and provides the correct accommodation of the molecule within the catalytic site. Figure S1. Pharmacophoric model proposed for diphenylmethane-based MGL inhibitors, revised pharmacophore distances, spatial arrangement of the key structural motifs of 4a in its lower energy 2

3 conformation (relevant interactions with catalytic site residues of the enzyme are highlighted in black). HPLC separation of racemic mixture (±)-6a Figure S2. HPLC separation of racemic mixture (±)-6a. and analytical control of optical purity for compounds (+)-6a and (-)-6a. 3

4 Top-down and bottom-up proteomics studies Purified rmgl was incubated with 4a (1 h at 37 C, 1:10 molar ratio) and the intact MGL mass was determined by high-resolution mass spectrometry. From the multiply charged mass spectrum of naïve MGL (Figure 2A), incubated with DMSO as vehicle, we calculated a deconvoluted mass of 35,528 Da. A corresponding deconvoluted mass of 35,922 Da was observed for MGL following incubation with 4a (Figure 2B), indicating a mass increase of 394 Da, which is consistent with a covalent interaction with 4a. This result suggests that the hydroxyl group of the catalytic serine may carry out a nucleophilic attack on the ureidic portion of 4a, thereby producing a net increase of +C 22 H 20 FN 2 O 4 in the brute formula of MGL. Accordingly, this corresponds to a mass increase of Da on the intact protein mass (Figure 2C). Our results also indicate that a full conversion of naïve MGL to the covalent MGL-4a adduct was achieved with 1:1 stoichiometry under the assay conditions. We also performed bottom-up proteomic analyses to map the binding site of 4a in MGL. After digesting MGL with trypsin, analysis of the tryptic fragments identified the peptide as an adduct with compound 4a, because its mass shifted from 5,330 Da to 5,724 Da, with a net increase of +394 Da. This peptide contains six serine residues, including the catalytic Ser122. Tandem mass analysis carried out on charge state 5 precursor ions of the naïve and modified peptide allowed us to unequivocally assign Ser122 as the site of modification induced by the nucleophilic attack on the urea moiety of 4a. Results of tandem mass analysis are reported in Figures S2-S5. 4

5 Figure S3. Bottom-up proteomics analysis for the 4a-MGL interaction. Figure S4. Bottom-up proteomics analysis for 4a-MGL interaction. 5

6 Figure S5. Tandem mass analysis for the 4a-MGL binding site mapping. Figure S6. Tandem mass analysis for the 4a-MGL binding site mapping 6

7 Pharmacokinetic profiling of 4a. A B Peak area Solubility Peak area Solubility Chem. Stability (µm) (µm) (% remaining) ph = 7.4 ph = 3.0 ph = C Metabolic Stability Permeability on CYP3A assay (PAMPA) (% remaining) P e (x10-6 cm/sec) Figure S7. Pharmacokinetic profiling of 4a. (A) solubility and chemical stability, (B) metabolic stability on human CYP3A4; (C) permeability assay (PAMPA). 7

8 Analgesic effect of 4a in the mouse Hot plate test Table S1. Analgesic effect of acute administration of 4a in the mouse Hot plate test Hot plate (s) Treatment Before treatment After treatment 15 min 30 min 45 min 60 min 4a 3 mg kg -1 p.o ± ± ± ± ± 2.2 4a 10 mg kg -1 p.o ± ± ± ± ± 0.8 Compounds were suspended in 1% carboxymethylcellulose sodium salt (CMC) and administered (p.o.). Pain-related behavior (i.e. lifting and licking of the hind paw) were observed and the time (seconds) of the first sign was recorded. **P<0.01 and *P<0.05 in respect to the value before treatment. Each value represents the mean of 12 mice. 8

9 Determination of the absolute configuration of (3R,4S)-6a. Figure S8. Arbitrary atom numbering scheme for compound (3R,4S)-6a. Table S2. List of dihedral angles used for the conformational analysis on compound (3R,4S)-6a. Label ψ α φ 1 φ 2 φ 3 β 1 β 2 Dihedral angle O5 C2 N1 C6 C2 C3 C10 C11 C2 N1 C6 C7 N1 C6 C7 C8 C6 C7 C8 N9 N1 C4 C12 C13 C13 C14 O15 C16 9

ID ψ (deg) α (deg) φ 1 (deg) φ 2 (deg) φ 3 (deg) β 1 (deg) β 2 (deg) Smallest RMSD (Å) (R,S)-6a1 5.36 46.52 118.21 179.30 57.98 125.00 172.03 (R,S)-6a4, 0.00014 (R,S)-6a2 6.70 42.82 112.41 179.36 58.

10 Figure S9. Graphical representation of the dihedral angles used for the conformational analysis on compound (3R,4S)-6a. Table S3. Geometric parameters for the conformers of compound (3R,4S)-6a, as obtained by conformational analysis after B97D/TZ2P optimization. RMSD values below 0.1 Å are also reported. Conf. ID ψ (deg) α (deg) φ 1 (deg) φ 2 (deg) φ 3 (deg) β 1 (deg) β 2 (deg) Smallest RMSD (Å) (R,S)-6a (R,S)-6a4, (R,S)-6a (R,S)-6a5, (R,S)-6a (R,S)-6a1, (R,S)-6a (R,S)-6a1, (R,S)-6a (R,S)-6a2, (R,S)-6a (R,S)-6a8, (R,S)-6a (R,S)-6a (R,S)-6a6, (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a

Table S4. Free energies and Boltzmann populations at 298.15 K and 1 bar for the conformers of compound (3R,4S)-6a, as obtained after B97D/TZ2P optimization. MM energies are also reported. Conf.

128487 1.679 (R,S)-6a12 10.89 0.345 1247.128401 2.663 (R,S)-6a2 10.34 0.376 1247.128352 0.080 (R,S)-6a6 6.80 0.624 1247.127956 2.214 (R,S)-6a3 4.20 0.910 1247.127501 1.395 (R,S)-6a11 3.40 1.034 1247.

11 Table S4. Free energies and Boltzmann populations at K and 1 bar for the conformers of compound (3R,4S)-6a, as obtained after B97D/TZ2P optimization. MM energies are also reported. Conf. ID χ (%) G (kcal mol 1 ) G (Ha) E MM (kcal mol 1 ) (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a (R,S)-6a Figure S10. Optimized structure of the lowest-energy conformer of compound (3R,4S)-6a, (R,S)- 6a13. 11

12 Table S5. Rotational strengths in dipole velocity formalism (R j ) and excitation wavelengths (λ j ) for the first 50 excited states of the conformers of compound (3R,4S)-6a, as obtained by PBE0/TZ2P//B97D/TZ2P calculations. J R j, erg cm 3 (λ j, nm) (R,S)-6a (R,S)-6a2 (R,S)-6a3 (R,S)-6a5 (R,S)-6a (262.71) (265.58) (262.91) (265.60) (265.25) (251.80) (249.24) (252.14) (249.35) (256.41) (248.84) (239.11) (248.87) (239.12) (250.63) (238.98) (238.75) (238.98) (238.75) (238.02) (234.69) (232.59) (235.03) (232.62) (233.62) (223.68) (229.11) (223.81) (229.21) (228.78) (222.96) (223.49) (222.98) (223.62) (223.14) (221.46) (222.83) (221.49) (222.74) (221.54) (216.83) (216.44) (216.79) (216.32) (219.59) (214.22) (215.89) (214.18) (215.92) (219.08) (213.13) (213.39) (213.12) (213.42) (217.83) (212.02) (212.25) (212.07) (212.24) (212.38) (210.06) (209.83) (210.03) (209.86) (210.36) (209.85) (208.75) (209.97) (208.69) (207.91) (206.77) (205.68) (206.96) (205.89) (207.78) (206.25) (204.73) (206.20) (204.53) (206.59) (205.12) (203.80) (205.12) (203.73) (204.90) (202.29) (203.27) (202.36) (203.40) (204.18) (200.56) (201.11) (200.68) (201.15) (201.22) (200.18) (199.25) (200.13) (199.30) (201.18) (198.78) (197.31) (198.80) (197.24) (200.43) (195.97) (195.16) (196.19) (195.23) (196.02) (194.09) (192.71) (194.20) (192.87) (194.13) (191.25) (192.31) (191.33) (192.42) (191.66) (188.17) (191.68) (188.28) (191.70) (189.09) (187.83) (188.98) (187.84) (188.99) (188.29) (186.52) (187.84) (186.55) (187.81) (186.37) (186.50) (184.29) (186.48) (184.23) (185.47) (184.01) (182.42) (184.00) (182.39) (185.04) (182.11) (180.85) (182.14) (180.82) (180.60) (180.79) (180.56) (180.63) (180.40) (180.35) (180.49) (179.76) (180.49) (179.67) (179.55) (179.91) (178.62) (179.52) (178.57) (178.84) (178.78) (178.10) (178.81) (177.90) (177.96) (177.49) (176.75) (177.73) (176.67) (175.58) (174.96) (175.05) (174.90) (175.33) (175.17) (171.41) (171.93) (171.46) (172.04) (172.85) (169.85) (169.45) (169.88) (169.48) (170.77) (169.42) (168.30) (169.46) (168.29) (169.97) (167.94) (168.07) (167.91) (168.03) (169.01) (164.93) (166.70) (165.10) (166.71) (166.82) (164.36) (164.47) (164.53) (164.47) (166.33) (163.62) (162.65) (163.71) (162.53) (164.69) (163.11) (162.08) (163.09) (162.11) (164.34) (162.81) (161.43) (162.80) (161.47) (162.70) (161.71) (160.77) (161.70) (160.80) (162.64) (161.06) (160.38) (161.06) (160.42) (162.51) (159.48) (159.54) (159.48) (159.67) (161.98) (159.04) (159.08) (159.18) (159.03) (160.45) (158.48) (158.76) (158.51) (158.66) (159.24) 12

13 Table S5. (continued) j R j, erg cm 3 (λ j, nm) (R,S)-6a7 (R,S)-6a8 (R,S)-6a9 (R,S)-6a10 (R,S)-6a (266.12) (265.31) (264.41) (265.85) (266.09) (258.40) (256.55) (252.45) (248.22) (249.42) (252.00) (250.74) (250.10) (243.50) (239.32) (239.37) (238.59) (239.67) (240.03) (239.02) (236.95) (233.74) (237.27) (239.11) (232.94) (235.09) (233.18) (234.91) (234.05) (231.72) (228.59) (227.49) (233.05) (230.27) (228.25) (224.22) (222.98) (229.22) (225.44) (222.67) (221.54) (220.83) (222.97) (223.63) (218.84) (218.99) (219.46) (218.61) (221.57) (216.11) (217.83) (218.71) (216.73) (219.32) (214.78) (215.07) (212.35) (213.49) (214.06) (213.93) (211.35) (211.96) (210.39) (211.97) (212.53) (210.35) (210.03) (208.44) (210.76) (208.20) (207.05) (207.61) (206.43) (207.18) (207.31) (206.48) (206.57) (205.84) (205.77) (204.66) (206.19) (204.79) (205.02) (202.39) (204.09) (203.99) (203.98) (202.04) (202.28) (203.61) (201.33) (201.42) (201.41) (200.70) (201.84) (200.09) (201.03) (198.96) (200.01) (199.43) (199.84) (200.16) (198.79) (198.12) (198.02) (195.00) (196.36) (196.92) (196.42) (196.69) (194.28) (194.07) (195.90) (195.14) (194.42) (193.77) (193.38) (194.88) (192.60) (193.17) (190.10) (189.07) (192.30) (191.71) (192.61) (188.12) (188.13) (189.33) (190.91) (192.32) (185.96) (186.07) (188.55) (188.50) (188.03) (185.28) (185.22) (187.01) (187.30) (185.40) (184.26) (184.61) (186.03) (183.22) (183.04) (181.90) (180.30) (184.79) (182.36) (182.34) (180.86) (180.17) (182.55) (181.97) (180.30) (180.61) (179.28) (181.42) (180.69) (179.97) (179.72) (178.73) (180.92) (180.31) (179.22) (178.98) (177.87) (180.06) (178.73) (177.81) (177.94) (175.41) (178.99) (178.04) (175.80) (176.89) (174.82) (176.47) (175.87) (174.59) (174.36) (172.85) (174.83) (174.80) (172.38) (173.01) (170.90) (173.83) (173.79) (168.45) (172.68) (169.01) (171.63) (171.34) (167.39) (170.15) (168.47) (171.42) (168.62) (166.71) (169.02) (166.50) (165.75) (168.26) (166.35) (166.72) (165.90) (164.65) (164.55) (163.66) (166.06) (164.43) (164.28) (164.31) (162.37) (164.65) (164.19) (164.15) (164.20) (161.58) (164.07) (162.24) (163.65) (163.22) (161.11) (163.82) (162.08) (163.25) (162.67) (160.44) (163.34) (161.73) (162.78) (162.35) (159.98) (163.13) (161.50) (161.92) (162.21) (159.85) (162.50) (160.04) (160.84) (161.95) (159.18) (160.93) (159.83) (160.58) (161.18) (158.13) 13

14 Table S5. (continued) j R j, erg cm 3 (λ j, nm) (R,S)-6a12 (R,S)-6a13 (R,S)-6a14 (R,S)-6a (267.65) (266.66) (262.97) (265.04) (257.14) (249.90) (251.64) (257.13) (247.12) (239.24) (246.50) (249.86) (239.84) (239.06) (239.13) (240.08) (234.38) (235.94) (233.92) (236.79) (233.07) (230.96) (232.68) (235.20) (230.00) (227.82) (226.66) (229.16) (223.28) (223.31) (224.53) (222.61) (221.12) (218.42) (218.33) (221.15) (220.41) (216.38) (214.53) (220.37) (217.69) (215.15) (213.67) (215.63) (215.93) (213.00) (212.15) (214.71) (213.25) (212.34) (209.85) (211.16) (211.30) (208.09) (209.37) (208.82) (208.69) (206.97) (209.06) (207.30) (205.24) (204.03) (206.63) (207.07) (204.33) (203.62) (204.19) (203.70) (202.09) (203.05) (203.74) (203.57) (200.88) (201.87) (202.09) (200.84) (199.75) (198.69) (199.15) (199.74) (199.63) (197.34) (198.50) (199.35) (195.40) (195.63) (198.09) (197.28) (194.26) (193.32) (197.27) (195.49) (192.80) (192.36) (195.14) (193.46) (190.55) (192.19) (191.11) (189.23) (188.70) (190.83) (188.83) (188.26) (188.06) (186.93) (187.80) (186.26) (184.86) (184.63) (186.41) (184.86) (182.80) (182.32) (185.61) (184.73) (181.18) (181.69) (183.15) (182.55) (181.11) (180.49) (180.31) (181.01) (180.09) (179.94) (180.08) (180.38) (178.42) (178.99) (179.71) (180.01) (178.03) (178.23) (179.53) (179.36) (177.60) (177.27) (178.05) (177.81) (175.59) (175.11) (177.14) (177.13) (174.86) (171.81) (173.42) (175.91) (171.37) (168.52) (172.15) (173.13) (170.71) (168.08) (170.03) (172.55) (169.77) (166.93) (169.31) (170.02) (168.66) (166.00) (165.48) (169.38) (167.77) (163.88) (164.46) (166.46) (166.09) (163.49) (163.74) (165.91) (165.01) (163.03) (163.31) (164.36) (164.36) (161.60) (163.00) (163.76) (163.52) (161.17) (162.54) (163.38) (163.33) (160.41) (162.27) (162.25) (161.97) (160.37) (161.73) (161.92) (161.85) (160.07) (160.59) (161.56) (160.96) (158.50) (160.36) (160.69) 14

15 Table S6. Oscillator strengths (f j ) and excitation wavelengths (λ j ) for the first 50 excited states of the conformers of compound (3R,4S)-6a, as obtained by PBE0/TZ2P//B97D/TZ2P calculations. j f j (λ j, nm) (R,S)-6a1 (R,S)-6a2 (R,S)-6a3 (R,S)-6a5 (R,S)-6a (262.71) (265.58) (262.91) (265.60) (265.25) (251.80) (249.24) (252.14) (249.35) (256.41) (248.84) (239.11) (248.87) (239.12) (250.63) (238.98) (238.75) (238.98) (238.75) (238.02) (234.69) (232.59) (235.03) (232.62) (233.62) (223.68) (229.11) (223.81) (229.21) (228.78) (222.96) (223.49) (222.98) (223.62) (223.14) (221.46) (222.83) (221.49) (222.74) (221.54) (216.83) (216.44) (216.79) (216.32) (219.59) (214.22) (215.89) (214.18) (215.92) (219.08) (213.13) (213.39) (213.12) (213.42) (217.83) (212.02) (212.25) (212.07) (212.24) (212.38) (210.06) (209.83) (210.03) (209.86) (210.36) (209.85) (208.75) (209.97) (208.69) (207.91) (206.77) (205.68) (206.96) (205.89) (207.78) (206.25) (204.73) (206.20) (204.53) (206.59) (205.12) (203.80) (205.12) (203.73) (204.90) (202.29) (203.27) (202.36) (203.40) (204.18) (200.56) (201.11) (200.68) (201.15) (201.22) (200.18) (199.25) (200.13) (199.30) (201.18) (198.78) (197.31) (198.80) (197.24) (200.43) (195.97) (195.16) (196.19) (195.23) (196.02) (194.09) (192.71) (194.20) (192.87) (194.13) (191.25) (192.31) (191.33) (192.42) (191.66) (188.17) (191.68) (188.28) (191.70) (189.09) (187.83) (188.98) (187.84) (188.99) (188.29) (186.52) (187.84) (186.55) (187.81) (186.37) (186.50) (184.29) (186.48) (184.23) (185.47) (184.01) (182.42) (184.00) (182.39) (185.04) (182.11) (180.85) (182.14) (180.82) (180.60) (180.79) (180.56) (180.63) (180.40) (180.35) (180.49) (179.76) (180.49) (179.67) (179.55) (179.91) (178.62) (179.52) (178.57) (178.84) (178.78) (178.10) (178.81) (177.90) (177.96) (177.49) (176.75) (177.73) (176.67) (175.58) (174.96) (175.05) (174.90) (175.33) (175.17) (171.41) (171.93) (171.46) (172.04) (172.85) (169.85) (169.45) (169.88) (169.48) (170.77) (169.42) (168.30) (169.46) (168.29) (169.97) (167.94) (168.07) (167.91) (168.03) (169.01) (164.93) (166.70) (165.10) (166.71) (166.82) (164.36) (164.47) (164.53) (164.47) (166.33) (163.62) (162.65) (163.71) (162.53) (164.69) (163.11) (162.08) (163.09) (162.11) (164.34) (162.81) (161.43) (162.80) (161.47) (162.70) (161.71) (160.77) (161.70) (160.80) (162.64) (161.06) (160.38) (161.06) (160.42) (162.51) (159.48) (159.54) (159.48) (159.67) (161.98) (159.04) (159.08) (159.18) (159.03) (160.45) (158.48) (158.76) (158.51) (158.66) (159.24) 15

16 Table S6. (continued) j f j (λ j, nm) (R,S)-6a7 (R,S)-6a8 (R,S)-6a9 (R,S)-6a10 (R,S)-6a (266.12) (265.31) (264.41) (265.85) (266.09) (258.40) (256.55) (252.45) (248.22) (249.42) (252.00) (250.74) (250.10) (243.50) (239.32) (239.37) (238.59) (239.67) (240.03) (239.02) (236.95) (233.74) (237.27) (239.11) (232.94) (235.09) (233.18) (234.91) (234.05) (231.72) (228.59) (227.49) (233.05) (230.27) (228.25) (224.22) (222.98) (229.22) (225.44) (222.67) (221.54) (220.83) (222.97) (223.63) (218.84) (218.99) (219.46) (218.61) (221.57) (216.11) (217.83) (218.71) (216.73) (219.32) (214.78) (215.07) (212.35) (213.49) (214.06) (213.93) (211.35) (211.96) (210.39) (211.97) (212.53) (210.35) (210.03) (208.44) (210.76) (208.20) (207.05) (207.61) (206.43) (207.18) (207.31) (206.48) (206.57) (205.84) (205.77) (204.66) (206.19) (204.79) (205.02) (202.39) (204.09) (203.99) (203.98) (202.04) (202.28) (203.61) (201.33) (201.42) (201.41) (200.70) (201.84) (200.09) (201.03) (198.96) (200.01) (199.43) (199.84) (200.16) (198.79) (198.12) (198.02) (195.00) (196.36) (196.92) (196.42) (196.69) (194.28) (194.07) (195.90) (195.14) (194.42) (193.77) (193.38) (194.88) (192.60) (193.17) (190.10) (189.07) (192.30) (191.71) (192.61) (188.12) (188.13) (189.33) (190.91) (192.32) (185.96) (186.07) (188.55) (188.50) (188.03) (185.28) (185.22) (187.01) (187.30) (185.40) (184.26) (184.61) (186.03) (183.22) (183.04) (181.90) (180.30) (184.79) (182.36) (182.34) (180.86) (180.17) (182.55) (181.97) (180.30) (180.61) (179.28) (181.42) (180.69) (179.97) (179.72) (178.73) (180.92) (180.31) (179.22) (178.98) (177.87) (180.06) (178.73) (177.81) (177.94) (175.41) (178.99) (178.04) (175.80) (176.89) (174.82) (176.47) (175.87) (174.59) (174.36) (172.85) (174.83) (174.80) (172.38) (173.01) (170.90) (173.83) (173.79) (168.45) (172.68) (169.01) (171.63) (171.34) (167.39) (170.15) (168.47) (171.42) (168.62) (166.71) (169.02) (166.50) (165.75) (168.26) (166.35) (166.72) (165.90) (164.65) (164.55) (163.66) (166.06) (164.43) (164.28) (164.31) (162.37) (164.65) (164.19) (164.15) (164.20) (161.58) (164.07) (162.24) (163.65) (163.22) (161.11) (163.82) (162.08) (163.25) (162.67) (160.44) (163.34) (161.73) (162.78) (162.35) (159.98) (163.13) (161.50) (161.92) (162.21) (159.85) (162.50) (160.04) (160.84) (161.95) (159.18) (160.93) (159.83) (160.58) (161.18) (158.13) 16

17 Table S6. (continued) j f j (λ j, nm) (R,S)-6a12 (R,S)-6a13 (R,S)-6a14 (R,S)-6a (267.65) (266.66) (262.97) (265.04) (257.14) (249.90) (251.64) (257.13) (247.12) (239.24) (246.50) (249.86) (239.84) (239.06) (239.13) (240.08) (234.38) (235.94) (233.92) (236.79) (233.07) (230.96) (232.68) (235.20) (230.00) (227.82) (226.66) (229.16) (223.28) (223.31) (224.53) (222.61) (221.12) (218.42) (218.33) (221.15) (220.41) (216.38) (214.53) (220.37) (217.69) (215.15) (213.67) (215.63) (215.93) (213.00) (212.15) (214.71) (213.25) (212.34) (209.85) (211.16) (211.30) (208.09) (209.37) (208.82) (208.69) (206.97) (209.06) (207.30) (205.24) (204.03) (206.63) (207.07) (204.33) (203.62) (204.19) (203.70) (202.09) (203.05) (203.74) (203.57) (200.88) (201.87) (202.09) (200.84) (199.75) (198.69) (199.15) (199.74) (199.63) (197.34) (198.50) (199.35) (195.40) (195.63) (198.09) (197.28) (194.26) (193.32) (197.27) (195.49) (192.80) (192.36) (195.14) (193.46) (190.55) (192.19) (191.11) (189.23) (188.70) (190.83) (188.83) (188.26) (188.06) (186.93) (187.80) (186.26) (184.86) (184.63) (186.41) (184.86) (182.80) (182.32) (185.61) (184.73) (181.18) (181.69) (183.15) (182.55) (181.11) (180.49) (180.31) (181.01) (180.09) (179.94) (180.08) (180.38) (178.42) (178.99) (179.71) (180.01) (178.03) (178.23) (179.53) (179.36) (177.60) (177.27) (178.05) (177.81) (175.59) (175.11) (177.14) (177.13) (174.86) (171.81) (173.42) (175.91) (171.37) (168.52) (172.15) (173.13) (170.71) (168.08) (170.03) (172.55) (169.77) (166.93) (169.31) (170.02) (168.66) (166.00) (165.48) (169.38) (167.77) (163.88) (164.46) (166.46) (166.09) (163.49) (163.74) (165.91) (165.01) (163.03) (163.31) (164.36) (164.36) (161.60) (163.00) (163.76) (163.52) (161.17) (162.54) (163.38) (163.33) (160.41) (162.27) (162.25) (161.97) (160.37) (161.73) (161.92) (161.85) (160.07) (160.59) (161.56) (160.96) (158.50) (160.36) (160.69) 17

18 1 H and 13 C NMR spectra of compounds (±)-5a, (±)-6a and (±)-4a 18

19 19

20 20

21 Elemental Analysis Compound Formula Calcd Found C H N C H N 4a C 24 H 22 FN 5 O b C 28 H 24 FN 3 O c C 25 H 23 FN 4 O d C 25 H 21 F 7 N 2 O e C 25 H 25 N 5 O f C 26 H 29 N 5 O g C 27 H 31 N 5 O h C 25 H 27 N 5 O a C 28 H 27 FN 2 O a C 21 H 21 FN 2 O C 24 H 24 FN 5 O C 16 H 12 FNO C 8 H 12 N 4 O REFERENCES (1) Di Marzo, V.; Stella, N.; Zimmer, A.; Endocannabinoid signalling and the deteriorating brain. Nat Rev Neurosci 2015, 16,

MS-based proteomics to investigate proteins and their modifications

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