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1 Supporting Information Evaluation of the analgesic effect of 4-anilidopiperidine scaffold containing ureas and carbamates Ludovica Monti [1], Azzurra Stefanucci [2], Stefano Pieretti [3], Francesca Marzoli [3], Lorenzo Fidanza [3], Adriano Mollica [4] *, Sako Mirzaie [5], Simone Carradori [4], Luciano De Petrocellis [6], Aniello Schiano Moriello [6], Sándor Benyhe [7], Ferenc Zádor [7], Edina Szűcs [7], Ferenc Ötvös [7], Anna I. Erdei [7], Reza Samavati [7], Szabolcs Dvorácskó [7], Csaba Tömböly [7], Ettore Novellino [8] Contents MOR binding affinity of compounds DOR, KOR and CBR binding affinity of 585 and 586. G-protein activity of and Maximal G-protein efficacy and potency of Relative orientation of the ligands lowest docking energies. Selected atom pairs for interaction models. Docked poses of fentanyl in the binding pocket of -receptor. Fig. S1 Fig. S2 Tab.S1 Fig. S3 Tab. S2 Tab. S3 Tab. S4 Fig. S4 1

2 Figure S1. MOR binding affinity of compounds (A) and (B). Supplementary Figure 1. MOR binding affinity of compounds (A) and (B) compared to fentanyl in [ 3 H]DAMGO competition binding assays in rat brain membrane homogenates. Figure represents the specific binding of [ 3 H]DAMGO in percentage in the presence of increasing concentrations ( M) of the indicated unlabeled ligands. The vertical grey stripes represent the minimum and maximum range of the calculated logic50 of compounds based on Table 1. Total on the x-axis indicates the total specific binding of [ 3 H]DAMGO, which is measured in the absence of the unlabeled compounds. The level of total specific binding was defined as 100% and is presented with a dotted line. Points represent means S.E.M. for at least three experiments performed in duplicate. 2

3 Figure S2. DOR (A), KOR (B) CBR (C) binding affinity of 585 and 586 in [ 3 H]IleDelt II, [ 3 H]U and [ 3 H]WIN55,212-2 competition binding assays in rat (A, C) and guinea pig (B) brain membrane homogenates. Supplementary Figure 2. DOR (A), KOR (B), and CBR (C) binding affinity of 585 and 586 in [ 3 H]IleDelt II, [ 3 H]U-69593, and [ 3 H]WIN55,212-2 competition binding assays in rat (A and C) and guinea pig (B) brain membrane homogenates. Figures represent the specific binding of the indicated radioligands in percentage in the presence of increasing concentrations ( M) of 585 and 586 and corresponding homologous ligand. Total on the x-axis indicates the total specific binding of radioligands, which is measured in the absence of the unlabeled compounds. The level of total specific binding was defined as 100% and is presented with a dotted line. Points represent means S.E.M. for at least three experiments performed in duplicates. 3

4 Table S1. The affinity values (logic50 S.E.M.) of 585 and 586 compounds in [ 3 H]IleDelt II, [ 3 H]U and [ 3 H]WIN55,212-2 competition binding assays in rat and guinea pig brain membrane homogenates. For control the affinity values of unlabeled IleDelt II, U and WIN55,212-2 are also indicated. The logic50 values were calculated according to the competition binding curves (see Supplementary Figure 2). Compounds Homologous ligand [ 3 H]IleDelt II a [ 3 H]U b [ 3 H]WIN55,212-2 a (5.68 nm) (15.97 nm) (20.07 nm) (62480 nm) (15040 nm) (1116 nm) (3555 nm) (8334 nm) (31990 nm) a : performed in rat brain membrane homogenate b : performed in guinea pig membrane homogenate 4

5 Figure S3. The effect of compounds (A and B) and (C and D) on G-protein activity compared to fentanyl in [ 35 S]GTP S binding assays in the absence (A and C) or presence (B and D) of the opioid antagonist naloxone in rat brain membrane homogenates. Supplementary Figure 3. The effect of compounds (A and B) and (C and D) on G-protein activity compared to fentanyl in [ 35 S]GTP S binding assays in the absence (A and C) or presence (B and D) of the opioid antagonist naloxone in rat brain membrane homogenates. Figures represents the specific binding of [ 35 S]GTP S in percentage in the presence of increasing concentrations ( M) of the indicated ligands and in the presence or absence of 10 M naloxone. The grey vertical and horizontal stripes represent the maximum and minimum range of logec50 and Emax values, respectively of compounds based on Table 2. Basal on the x-axis indicates the basal activity of the monitored G-protein, which is measured in the absence of the compounds and also represents the total specific binding of [ 35 S]GTP S. The level of basal activity was defined as 100% and is presented with a dotted line. Points represent means S.E.M. for at least three experiments performed in triplicate. 5

6 Table S2. The maximal G-protein efficacy (Emax) and ligand potency (logec50) of compounds and fentanyl in the absence or presence of the opioid antagonist naloxone in [ 35 S]GTP S binding assays in rat brain membrane homogenates. The values were calculated according to dose-response binding curves in Supplementary Figure 3. For better comparison the two different basic structures are presented side by side. Compounds Emax S.E.M. (%) logec50 S.E.M. (M) (EC50) Compounds Alone + naloxone Alone + naloxone Fentanyl Fentanyl (110.4 nm) ambiguous Y O N X (5691 nm) (3079 nm) (4710 nm) (1938 nm) (3265 nm) (2986 nm) (4939 nm) (3353 nm) (3289 nm) (3100 nm) (5333 nm) (3266 nm) (3064 nm) (2909 nm) (3848 nm) (4367 nm) (3629 nm) (3043 nm) (4273 nm) (3037 nm) (3295 nm) (2658 nm) (4628 nm) (4554 nm) 1 : since the compound did not alter G-protein basal activity significantly, logec50 cannot be interpreted Note: X and Y indicates the chemical modifications performed in the basic structure of fentanyl (see Scheme 1 in the manuscript). 6

7 Table S3. Relative orientation of the ligands lowest docking energies of fentanyl analogues. (+): the analogues orientation calculated for phenylalanine; [16] (-): opposite orientation of the analogues. Ligands Orientation Table S4. Selected atom pairs for interaction models. Column A: ligand atoms in fentanyl-like binding; Column B: ligand atoms for opposite orientation. A 1: proton on the piperidine N of the ligands and side chain carboxylate of Asp147; 2: phenethyl aromatic ring of the ligands and Tyr326 side chain; 3: anilide aromatic ring of the ligands and Ile144 side chain; 4: phenethyl aromatic ring of the ligands and Ile322. B 1: piperidine nitrogen and Asp147 (OD2); 2: para aromatic carbon of the anilide ring and the aromatic carbon of Tyr326 holding the phenolic OH (CZ); 3: para aromatic carbon of the phenethyl group and Ile144 (CD1). Atom pair (i) Receptor (ri) Ligand (li) 1 carboxylate of Asp147 proton on piperidine N proton on piperidine N 2 Tyr326 side chain phenethyl aromatic ring anilide aromatic ring 3 Ile144 side chain anilide aromatic ring phenethyl aromatic ring 4 Ile322 side chain phenethyl aromatic ring A B 7

8 Figure S4. Docked poses of fentanyl in the binding pocket of -receptor; A: orientation similar to that reported by Micovic et al. [15] ; B: opposite orientation. A B 8