Supporting Information. Design, Synthesis and Biological Evaluation of Glutathione Peptidomimetics as Components of Antiparkinson Prodrugs

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1 S1 Supporting Information Design, Synthesis and Biological Evaluation of Glutathione Peptidomimetics as Components of Antiparkinson Prodrugs Swati S. More and Robert Vince* Center for Drug Design, Academic ealth Center, and Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, 8-123A WD, 308 arvard St SE, Minneapolis, M 55455, USA Table of Contents General Experimental Procedures Glycidic acid (5) -Adamantylglycidamide (6) 3-(Benzylthio)--adamantyl lactamide (7a) & 3-ydroxy-2-(Benzylthio)-adamantylpropanamide (7b) 3-(Tritylthio)--adamantyl lactamide (8) 3-Mercapto--adamantyl lactamide (9), -Di(tert-butyl-dimethylsilyl)dopamine (10) -[, -di(tert-butyl-dimethylsilyl)dopaminyl]-glycidamide (11) 3-(Tritylthio)--[, -di(tert-butyl-dimethylsilyl)dopaminyl]-lactamide (12) 3-Mercapto--[, -di(tert-butyl-dimethylsilyl)dopaminyl]-lactamide (13) Scheme 1: Synthesis of 14 Boc-Dap- (18) Boc-Dap(Cbz)- (19) Boc-Dap(Cbz)-tBu (20) Boc-Dap-tBu (21) Boc--Gla[-Cys(StBu)-All]-tBu (22) Boc--Gla[-Cys(StBu)-]-tBu (23) Boc--Gla[-Cys(StBu)-Gly-tBu]-tBu (24) Boc-Cys(StBu)-All (26) Cl. 2 -Cys(StBu)-All (27) S2 S2 S2 S3 S4 S4 S5 S5 S5 S6 S7 S7 S7 S8 S8 S9 S10 S10 S11 S11

2 S2 Boc--Gla[-Cys(S)-Gly-tBu]-tBu (14) In vitro BBB permeability prediction of prodrugs 1 and 2 In vitro stability studies: Convertibility of prodrugs into active drugs S12 S13 S14 General Experimental Procedures: All commercial chemicals were used as supplied unless otherwise indicated. Dry solvents (TF, Et 2, C 2 Cl 2 and DMF) were dispensed under argon from an anhydrous solvent system with two packed columns of neutral alumina or molecular sieves. Flash chromatography was performed with Silia-P flash silica gel (silicycle, mesh) with indicated mobile phase. All reactions were performed under inert atmosphere of ultra-pure argon with oven-dried glassware. 1 and 13 C MR spectra were recorded on a Varian 300 Mz spectrometer. igh resolution mass data were acquired on Bruker Biotof II ESI-MS spectrometer capable of positive and negative ion sources, using PPG or PEG as internal standards. Glycidic acid (5). To a stirred solution of (±)-glycidol (2.00 g, mmol) in CCl 4 :C 3 C: 2 (15 ml:15 ml:2 ml), ruthenium dioxide dihydrate (0.11 g, 2.4 mol%) 5 was added. The reaction mixture was stirred for 5 min under argon. Solid ai 4 (23.14 g, 0.11 mol) was added to above solution in small portions over 15 min and then reaction was allowed to stir at room temperature for another 2½ h. The reaction was stopped by adding silica gel and evaporated to dryness. The solid residue was loaded on a short silica gel column and product acid was eluted with EtAc. After evaporation of EtAc, the product was obtained as a light yellow oil (~3.00 g). Without any further purification the product was subjected to next coupling step. 1 MR (300 Mz, CDCl 3 ) (bs, 1, C), (m, 1, C), (m, 2, C 2 ); 13 C MR (75 Mz, CDCl 3 ) (C), 47.2 (C), 46.8 (C 2 ). -Adamantylglycidamide (6). A solution of glycidic acid (5) (2.40 g, mmol) in 6 anhydrous C 2 Cl 2 (25 ml) was cooled to 0 C using ice bath. Then MM (3.00 ml, mmol) was added to the reaction mixture,

3 S3 followed by dropwise addition of isobutyl chloroformate (3.50 ml, mmol). Reaction mixture was allowed to stir at 0 C for 1 h after which adamantamine (4.57 g, mmol) was added to the reaction flask along with MM (3.00 ml, mmol). The solution was allowed to stir overnight before being poured into water (20 ml). The dichloromethane layer was washed with 10% citric acid solution (25 ml), sodium bicarbonate solution (20 ml) and brine, dried over a 2 S 4, the residue obtained after evaporation of C 2 Cl 2 was purified by silica gel column chromatography to isolate product amide as an oil (3.13 g, 52% yield). R f 0.35 (EtAc/hexanes, 2:3); 1 MR (300 Mz, CDCl 3 ) 5.75 (bs, 1, ), 3.16 (dd, J = 4.8 z, 2.7 z, 1, C), 2.79 (dd, J = 4.5 z, 5.4 z, 1, C A B ), 2.58 (dd, J = 2.7 z, 6.0 z, 1, C A B ), (m, 10, C 2, C:Adm), 1.54 (m, 5, C 2 :Adm); 13 C MR (75 Mz, CDCl 3 ) (C(=)), 52.1 (C), 50.1 (C 2 ), 41.8, 36.3, 29.8 (C:Adm); ESI-LRMS m/z (M+) +. SBn SBn, nbuli, BF 3.Et 2,TF + SBn 6 7a 7b 7a:7b 3-(Benzylthio)--adamantyl lactamide (7a) & 3-ydroxy-2-(Benzylthio)-adamantylpropanamide (7b). To a TF (20 ml) solution of benzyl thiol (0.56 g, 4.52 mmol) cooled to -78 C was added nbuli (2.83 ml, 4.52 mmol) and the reaction mixture was allowed to stir for another 30 min at -78 C. A TF solution of epoxamide 6 (1.00 g, 4.52 mmol) was then added to the above solution followed by addition of BF 3.Et 2 (0.57 ml, 4.52 mmol). Reaction mixture was allowed to warm to rt and stirred overnight. The residue obtained after evaporation of TF was partitioned between EtAc (30 ml) and sat. 4 Cl. The organic layer was separated, washed with brine (20 ml), dried over a 2 S 4 and evaporated to get an oil. The crude product was purified by silica gel column chromatography to isolate the alcohol product as an inseparable mixture of regioisomers 7a and 7b (0.87 g, 56% yield). R f 0.60 (EtAc/hexanes, 2:3); 1 MR (300 Mz, CDCl 3 ) (m, 7, Ar), 6.54 (s, 1, ), 5.82 (s, 0.35, ), 4.02 (s, 2, C, C 2 ), 3.70 (s, 2.7, C 2 Ph), 3.29 (m, CS), (m, 3, C 2 S,

4 S4 C, C 2 ), (m, 21, C 2, C:Adm), 1.57 (m, 5, C 2 :Adm); 13 C MR (75 Mz, CDCl 3 ) (C(=)), 138.8, 129.1, 128.8, (C Ar ), 70.3 (C), 51.9, 50.1 (C 2 ), 48.0 (CS), 41.7, 41.6, 36.8, 36.6, 36.5, 29.7, 29.6 (C 2 Ph, C 2 S, C:Adm). 3-(Tritylthio)--adamantyl lactamide (8). To a TF (20 ml) solution of trityl mercaptan (2.50 g, 9.04 mmol) cooled to -78 C was added nbuli (5.66 ml, 9.04 mmol) STrt and the reaction mixture was allowed to stir for another 30 min at -78 C. A TF solution of epoxamide 6 (2.00 g, 9.04 mmol) was 8 then added to the above solution followed by addition of BF 3.Et 2 (1.14 ml, 9.04 mmol). Reaction mixture was allowed to warm to room temperature and stirred overnight. The residue obtained after evaporation of TF was partitioned between EtAc (30 ml) and sat. 4 Cl. The organic layer was separated, washed with brine (20 ml), dried over a 2 S 4 and evaporated to get an oil. The crude product was purified by silica gel column chromatography to isolate the pure alcohol product 8 (3.55 g, 69% yield). R f 0.60 (EtAc/hexanes, 2:3); 1 MR (300 Mz, CDCl 3 ) (m, 15, Ar), 5.86 (s, 1, ), 3.28 (t, J = 6.6 z, 1, C), 2.75 (bs, 1, ), 2.59 (d, J = 6.0 z, 2, C 2 STrt), (m, 10, C 2 :Adm, C:Adm), 1.57 (m, 5, C 2 :Adm); 13 C MR (75 Mz, CDCl 3 ) (C(=)), 144.6, 129.7, 128.2, (C Ar ), 70.1 (C), 52.1 (SC(Ph) 3 ), 41.7, 37.5, 36.6, 29.7 (C 2 S, C:Adm); ESI-LRMS m/z (M+) +. 3-Mercapto--adamantyl lactamide (9). Trityl protected thiol 8 (0.50 g, 1.00 mmol) was dissolved in dry dichloromethane (10 ml) and 0.37 ml (5.00 mmol) of trifluoroacetic acid was added under stirring. The color of S the solution was immediately turned to yellow. Subsequently, 9 triethylsilane (0.24 ml, 1.5 mmol) was added. The color of the solution was slowly recovered to colorless. After the mixture was stirred for 2 h, the solvent was removed under reduced pressure and the residual material was passed through a short plug of silica using EtAc to isolate thiol 9 as an oil (0.22 g, 89% yield). R f 0.30 (EtAc/hexanes, 3:2); 1 MR showed two sets of rotamers. 1 MR (300

5 S5 Mz, CDCl 3 ) 6.38, 5.80 (2s, 1, ), (m, 1, C), 3.40 (d, J = 5.4 z, 1, ), 3.33, 2.70 (2m, 1, S), (m, 2, C 2 S), (m, 15, C 2 :Adm, C:Adm); 13 C MR (75 Mz, CDCl 3 ) (C(=)), 70.3 (C), 41.8, 41.7, 36.5, 29.7 (C 2 S, C:Adm)., -Di(tert-butyl-dimethylsilyl)dopamine (10). To a C 2 Cl 2 solution of dopamine hydrochloride (2.00 g, 6.58 mmol), imidazole (0.67 g, mmol) was added in one 2 portion. A C 2 Cl 2 solution of TBDMSCl (3.85 g, mmol) TBS 10 TBS was then added to above solution slowly over 5 min. The reaction mixture was allowed to stir at rt overnight. The sticky solid obtained after evaporation of C 2 Cl 2 was partitioned between 10% citric acid solution (30 ml) and diethyl ether (20 ml). The aqueous layer was then neutralized to p 10 by solid ac 3 and the precipitated amine was extracted twice with EtAc (2 30 ml), combined EtAc layers was washed with water, brine; dried over a 2 S 4 and evaporated to dryness to obtain product as an oil (4.12 g, 86% yield). 1 MR (300 Mz, CDCl 3 ) (m, 3, Ar), 6.51 (bs, 2, 2 ), 3.08 (m, 2, C 2 ), 2.86 (m, 2, C 2 Ph), 0.98, 0.93 (2s, 18, SiC(C 3 ) 3 ), 0.19, 0.16 (2s, 12, SiC 3 ); 13 C MR (75 Mz, CDCl 3 ) 150.6, 149.6, 134.0, 125.5, 125.4, (C Ar ), 38.6 (C 2 ), 35.6 (C 2 Ph), 29.9, 29.7 (SiC(C 3 ) 3 ), 18.2, 18.1 (SiC(C 3 ) 3 ), 0.0 (SiC 3 ). -[, -di(tert-butyl-dimethylsilyl)dopaminyl]-glycidamide (11). Procedure same as for compound 6. Yield 56%; R f 0.45 (EtAc/hexanes, 3:2); 1 MR (300 Mz, CDCl 3 ) (m, 3, TBS TBS 11 Ar), 6.13 (s, 1, ), 3.41 (q, J = 6.9 z, 13.2 z, 2, C 2 ), 3.34 (dd, J = 2.7 z, 4.8 z, 1, C), 2.87 (t, J = 5.4 z, 1, C A B ), 2.63 (m, 2, C 2 Ph), 2.57 (dd, J = 2.7 z, 6.0 z, 1, C A B ), 0.95, 0.94 (2s, 18, SiC(C 3 ) 3 ), 0.16 (s, 12, SiC 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)), 146.9, 145.6, 131.5, 121.7, 121.6, (C Ar ), 49.8 (C), 47.7 (C 2 ), 40.2 (C 2 ), 35.0 (C 2 Ph), 26.3, 26.2 (SiC(C 3 ) 3 ), 18.8, 18.7 (SiC(C 3 ) 3 ), -3.7 (SiC 3 ); ESI-LRMS m/z (M+) +.

6 S6 3-(Tritylthio)--[, -di(tert-butyl-dimethylsilyl)dopaminyl]-lactamide (12). Procedure same as for compound 8. Yield 65%; R f 0.55 (EtAc/hexanes, 1:1); 1 MR TBS TBS 12 STrt (300 Mz, CDCl 3 ) (m, 15, Ar), (m,3, Ar), 6.20 (d, J = 6.0 z, 1, ), (m, 3, C, C 2 ), (m, 5, C, C 2 S, C 2 Ph), (m, 18, SiC(C 3 ) 3 ), (m, 12, SiC 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)), (C Ar ), 73.7 (C), 71.0 (SC(C 3 ) 3 ), 44.4 (C 2 ), 41.0 (C 2 Ph), 38.9 (C 2 S), 29.9 (SiC(C 3 ) 3 ), 22.4 (SiC(C 3 ) 3 ), 0.0 (SiC 3 ); ESI-LRMS m/z (M+) +. 3-Mercapto--[, -di(tert-butyl-dimethylsilyl)dopaminyl]-lactamide (13). To a C 2 Cl 2 /Et (5 ml: 5 ml) solution of trityl TBS TBS 13 S protected thiol 12 (0.20 g, 0.27 mmol), 26 µl of pyridine (0.32 mmol) was added at room temperature followed by addition of Ag 3 (51 mg, 0.29 mmol) at once. Then reaction mixture was heated to reflux for 1 ½ h. The reaction mixture was evaporated to dryness to get a yellow solid which was washed 4 times with C 3 to get almost pure silver salt of the thiol 13 (0.15 g). The sliver salt was then redissolved in C 2 Cl 2 /C 3 (10 ml: 2 ml) and treated with dithiothreitol (0.17 g, 1.08 mmol) at once at room temperature. Reaction mixture was allowed to stir for 3 h, after which reaction mixture was evaporated to get an oily residue which was washed 5 times with water (6 ml) to remove excess dithiothreitol and then the oil was taken in C 2 Cl 2 (15 ml), dried over a 2 S 4 and evaporated to dryness to obtain the product thiol 13 as an oil (0.14 g, 96% yield). R f 0.35 (EtAc/hexanes, 7:3); 1 MR (300 Mz, CDCl 3 ) 7.17 (m, 1, ), (m, 3, Ar), 4.71 (m, 1, S), 4.36 (m, 1, C), (m, 3, C, C 2 ), (m, 2, C 2 S), 2.71 (m, 2, C 2 Ph), (m, 18, SiC(C 3 ) 3 ), 0.19 (s, 12, SiC 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)), (C Ar ), 72.6 (C), 43.4 (C 2 ), 39.1 (C 2 Ph), 29.9 (C 2 S), 25.4 (SiC(C 3 ) 3 ), 18.6 (SiC(C 3 ) 3 ), -3.4 (SiC 3 ).

7 S7 Scheme 1. Synthesis of 14. C CR 1 S-StBu a e ButC g Boc Boc CR R 2 Boc ; R 1 =,R 2 = b 19; R 1 =,R 2 22; R=Allyl =Cbz f c 23; R= d 20; R 1 = tbu, R 2 =Cbz 21; R 1 = tbu, R 2 = ButC Boc h SR CtBu 24; R=S-StBu 14; R= ButS-S C 25 Boc j i ButS-S R 2 C R 1 26; R 1 =Boc,R 2 = 27; R 1 =,R 2 =Allyl Reagents and conditions: (a) PIDA, EtAc/C 3 C/ 2 (2:2:1), 62%; (b) CbzCl, K, K 2 C 3, TF/ 2 (3:1), 94%; (c) tbu, DCC, DMAP, C 2 Cl 2, 84%; (d) 2,10%Pd/C,C 3,94%;(e)27, CDI, MM, C 2 Cl 2, 67%; (f) (PPh 3 ) 4 Pd, morpholine, C 2 Cl 2, 80%; (g) Cl.GlytBu, EDC, Bt, MM, C 2 Cl 2, 78%; (h) Bu 3 P, TF/ 2, 94%; (i) Allyl bromide, benzene, reflux, 86%; (j) 4 Cl/dioxane, quant. Boc-Dap- (18). A slurry of -Boc-L-asparagine (17) (5.00 g, 21.5 mmol), EtAc (24 ml), acetonitrile (24 ml), water (12 ml), and PIDA (8.32 g, 25.8 mmol) was cooled and C Boc 2 18 stirred at 16 C for 30 min. The temperature was then allowed to reach 20 C, and the reaction was stirred until completion (ca. 4 h). The mixture was cooled to 0 C and filtered. The filter cake was washed with EtAc (10 ml) and dried in vacuo to give 18 (2.73 g, 62% yield). mp 212 C (lit ºC); [] D 14.5 (c 3.0, 2 ) [lit. [] D 16.4 (c 3.0, 2 )]; 1 MR (CD 3 D and a drop of TFA) 4.05 (t, J = 7.8 z, 1, -C), (m, 2, -C 2 ), 1.45 (s, 9, C(C 3 ) 3 ). Boc-Dap(Cbz)- (19). A stirring mixture of the -Boc-Dap- 18 (10.00 g, C Boc Cbz 19 mmol), TF (90 ml), water (30 ml), K 2 C 3 (13.53 g, mmol) and K (2.75 g, mmol) was cooled to 5 C. Cbz-Cl solution (50 wt% in toluene, 25 ml, mmol) was then added dropwise over a period of an hour. The mixture was allowed to stir for 8 h before being concentrated under reduced pressure to remove the TF. The aqueous residue was extracted with diethyl ether (50 ml), acidified to p 4 by solid citric acid and the

8 S8 resulting precipitate was extracted by C 2 Cl 2 (2 50 ml). Combined organic layers were dried (a 2 S 4 ) and evaporated to furnish the free acid 19 (15.57 g, 94% yield) as a gummy white solid which was used without further purification. 1 MR (300 Mz, CDCl 3 ) (bs, 1, C), 7.25 (bs, 5, Ar), 6.24, 5.67 (2s, 1, ), 5.85 (s, 1, ), 5.08, 5.02 (2s, 2, C 2 Ph), (m, 1, -C), (m, 2, -C 2 ), 1.38 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)), 157.5, (C(=)), 136.4, 128.6, (C Ar ), 80.6 (C(C 3 )), 67.3 (C 2 Ph), 54.4 (-C), 42.9 ( -C), 28.6 (C(C 3 ) 3 ). Boc-Dap(Cbz)-tBu (20). To an ice-cold C 2 Cl 2 (60 ml) solution of DCC (9.78 g, mmol), t-bu (17.54 g, 0.24 mol) and DMAP (0.29 g, 2.37 mmol); a C 2 Cl 2 (40 CtBu Boc Cbz 20 ml) solution of 19 (16.00 g, mmol) was added slowly over 20 min. Then reaction mixture was allowed to stir 1 h at 0 C and 10 h at 20 C. Precipitated urea was then filtered off and the filtrate evaporated in vacuo. The residue was taken up in C 2 Cl 2, and if necessary, filtered free of any further precipitated urea. The C 2 Cl 2 solution was washed twice with 10% citric acid solution and with saturated ac 3 solution, and then dried over a 2 S 4. The residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography to obtain the product as a clear viscous oil (15.67 g, 84% yield). R f 0.4 (EtAc/hexanes, 1:2); 1 MR (300 Mz, CDCl 3 ) 7.26 (s, 5, Ar), 5.53 (d, 1, J = 6.6 z, ), 5.45 (s, 1, ), 5.02 (s, 2, C 2 Ph), (m, 1, -C), (m, 2, -C 2 ), 1.38 (s, 9, C(C 3 ) 3 ), 1.37 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)tBu), (C(=)), (C(=)), 136.6, 128.7, (3C, C Ar ), 82.8, 80.1 (C(C 3 ) 3 ), 66.9 (C 2 Ph), 54.7 (-C), 43.3 ( -C), 28.5, 28.1 (C(C 3 ) 3 ); ESI- LRMS m/z (M+) +, (M+a) +. Boc-Dap-tBu (21). A solution of 20 (5.00 g, mmol) in methanol (50 ml) was CtBu Boc 21 2 placed in a Parr hydrogenation bottle and purged with argon. Palladium on Carbon (10 %, 500 mg) was added under a stream of argon. The bottle was evacuated and refilled with hydrogen (3 times), pressurized to 60 psi of hydrogen and shaken for 45 minutes before being evacuated and poured into

9 S9 200 ml of C 2 Cl 2. The suspension was filtered through a fine-porosity filter paper and then through a 45 µm PFTE filter. The resulting clear solution was evaporated under reduced pressure to a colorless oil (21) (3.10 g, 94% yield). [] D 22.3 (c 1.0, Et) [lit. [] D 23.9 (c 1.05, Et)]; R f 0.45 (C 2 Cl 2 /C 3, 9:1); 1 MR (300 Mz, CDCl 3 ) 5.52 (d, 1, J = 7.5 z, ), (m, 1, -C), (m, 2, -C 2 ), 1.95 (s, 2, 2 ), 1.35 (s, 9, C(C 3 ) 3 ), 1.32 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)tBu), (C(=)), 82.3, 79.9 (C(C 3 ) 3 ), 56.8 (-C), 44.4 ( -C), 28.5, 28.2 (C(C 3 ) 3 ). Boc--Gla[-Cys(StBu)-All]-tBu (22). An ice-cold C 2 Cl 2 solution of carbonyl diimidazole (0.63 g, 3.84 mmol) was stirred under argon for 5 min before being added via cannula to a C 2 Cl 2 solution of 27 (1.00 g, 3.49 mmol) ButC Boc 22 S-StBu and 0.77 ml of -methylmorpholine (6.99 mmol) over 20 min. The reaction mixture was stirred further for a 15 min at 0 C. Then a second C 2 Cl 2 solution of 21 (1.37 g, 5.25 mmol) and 0.77 ml of MM was added to reaction mixture in one portion. The reaction mixture was then allowed to stir at rt overnight before being washed with 10% citric acid solution (2 20 ml) and brine. rganic layer was then dried over a 2 S 4, evaporated and the residue obtained was purified by silica gel chromatography using (3:2) EtAc/hexanes, to obtain product 22 as a colorless oil (1.26 g, 67% yield) with 20% yield of homourea of Cys(StBu)-All. R f 0.75 (EtAc/hexanes, 2:3); [] D 28.7 (c 1.0, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) 6.03 (d, J = 7.8 z, 1, ), 5.94 (t, J = 6.3 z, 2, ), (m, 1, C=C 2 ), (m, 2, C=C 2 ), 4.61 (q, J = 5.1 z, 1, -C:Gla), 4.49 (d, J = 5.7 z, 2, C 2 C), 4.14 (m, 1, - C:Cys), (m, 2, -C 2 :Gla), 3.03 (d, J = 5.1 z, 2, -C 2 :Cys), 1.46 (s, 9, SC(C 3 ) 3 ), 1.44, 1.31 (s, 18, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) 171.6, (C(=)), (C(=)), (C(=)), (C=C 2 ), (C=C 2 ), 80.3, 79.9 (C(C 3 ) 3 ), 66.4 (C 2 C), 55.5 (-C:Gla), 53.1 (-C:Cys), 48.2 (SC(C 3 ) 3 ), 43.5 ( -C:Gla), 42.4 ( -C:Cys), 29.9 (SC(C 3 ) 3 ), 28.5, 28.1 (C(C 3 ) 3 ); ESI-RMS m/z (M+a) + ; C S 2 + a + requires

10 S10 Boc--Gla[-Cys(StBu)-]-tBu (23). (Ph 3 P) 4 Pd (0.21 g, 10 mol%) was added under nitrogen to a solution of allyl ester 22 (1.00 g, 1.87 mmol) in C 2 Cl 2 (20 ml) at room S-StBu temperature and the resulting mixture was treated dropwise with (1.63 ml, mmol) morpholine. ButC After completion of the reaction (1 h) as judged by Boc TLC, solvent was removed in vacuo. The residue was 23 redissolved in C 2 Cl 2 (20 ml) and the resulting solution was washed with 1 Cl (15 ml) and water (15 ml). The organic layer was dried over a 2 S 4 and evaporated in vacuo. The crude product was purified by flash column chromatography using C 2 Cl 2 - methanol (0.74 g, 80% yield). Rotamers were seen in proton and carbon MR. 1 MR (300 Mz, CDCl 3 ) 9.36 (bs, 1, C), (m, 1, ), 6.06 (d, J = 7.2 z, 1, ), 4.68 (m, 1, -C:Gla), 4.31, 4.12 (2q, J = 6.9 z, 1, -C:Cys), (m, 2, -C 2 :Gla), (m, 2, -C 2 :Cys), 1.44 (s, 9, SC(C 3 ) 3 ), 1.39, 1.33 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) 175.6, 175.0, (C), 171.9, (C(=)tBu), 159.3, (C(=)), 157.0, (C(=)), 81.8, 80.6 (C(C 3 ) 3 ), 56.5, 54.7 (-C:Gla), 53.7, 53.2 (-C:Cys), 48.5, 48.4 (SC(C 3 ) 3 ), 42.7 ( - C:Gla), 40.9 ( -C:Cys), 31.2, 31.1 (SC(C 3 ) 3 ), 30.2, 28.7 (C(C 3 ) 3 ). Boc--Gla[-Cys(StBu)-Gly-tBu]-tBu (24). EDC (0.23 g, 1.21 mmol), Bt (0.16 g, SStBu 1.21 mmol) and MM (0.33 ml, 3.03 mmol) were added to a ice-cold solution of 23 (0.50 g, ButC 1.01 mmol) and Cl.Gly-tBu (0.25 g, 1.51 Boc CtBu 24 mmol) in C 2 Cl 2 (20 ml). The solution was allowed to stir for 12 h before being poured into water (20 ml). The dichloromethane layer was washed with 10% citric acid solution (25 ml), sodium bicarbonate solution ( 2 20 ml) and brine; dried over a 2 S 4 ; filtered and the residue obtained after evaporation of C 2 Cl 2 was purified by column chromatography using 50% EtAc/hexanes to isolate product as a colorless oil (0.47 g, 78% yield). R f 0.30 (EtAc/hexanes, 2:3); [] D 14.7 (c 1.0, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) 7.13 (t, J = 4.5 z, 1, ), (m, 3, ), 4.61 (m, 1, -C:Gla), 4.22 (m, 1, -

11 S11 C:Cys), 3.91 (t, J = 6.0 z, 2, C 2 :Gly), 3.54 (m, 2, -C 2 :Gla), 3.10 (d, J = 5.1 z, 2, -C 2 :Cys), 1.45, 1.43, 1.32 (s, 36, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)Gly), 170.3, (C(=)), (C(=)), (C(=)), 82.5, 80.1 (C(C 3 ) 3 ), 55.4 (-C:Gla), 53.8 (-C:Cys), 48.2 (SC(C 3 ) 3 ), 43.0 ( -C:Gla), 42.6 (C 2 :Gly), 42.5 ( -C:Cys), 30.2 (SC(C 3 ) 3 ), 28.7, 28.4, 28.3 (C(C 3 ) 3 ); ESI-LRMS m/z (M+) +, (M+a) + ; ESI-RMS m/z (M+) + ; C S requires Boc-Cys(StBu)-All (26). ünig s base (5.6 ml, mmol) was added to a solution of Boc-Cys(StBu)- (25) (5.0 g, mmol) in dry benzene allyl bromide (1:1, 50 ButS-S AllC Boc 26 ml), and the mixture was heated at reflux for 3 h. The residue obtained after evaporation of benzene was dissolved in EtAc (200 ml); washed with 10% aqueous citric acid (3 50 ml), 5% aqueous ac 3 (3 50 ml), brine, dried (a 2 S 4 ) and concentrated in vacuo. The residue was dissolved in EtAc/hexanes (1:2, 50 ml) and passed through a short silica gel plug (10 g). The plug was eluted further with a 100 ml of the same solvent mixture. Combined eluents were evaporated to give 4.40 g (86% yield) of 26 as a colorless oil. R f 0.50 (EtAc/hexanes, 1:2); [] D 46.3 (c 1.0, CCl 3 ); 1 MR (300 Mz, CDCl 3 ) (m, 1, C=C 2 ), 5.37 (d, J = 7.2 z, 1, ), 5.26 (dd, J = 10.2, 14.8 z, 2, C=C 2 ), 4.62 (d, J = 5.4 z, 1, C 2 C), 4.58 (m, 1, -C), 3.14 (m, 2, - C 2 ), 1.42 (s, 9, SC(C 3 ) 3 ), 1.29 (s, 9, C(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)All), 155.1(C(=)), (C=C 2 ), (C=C 2 ), 80.3 (C(C 3 ) 3 ), 66.5 (C 2 C), 53.6 (-C), 48.4 (SC(C 3 ) 3 ), 43.0 ( -C), 30.1 (SC(C 3 ) 3 ), 28.6 (C(C 3 ) 3 ); ESI-LRMS m/z (M+a) +. Cl. 2 -Cys(StBu)-All (27). The Boc-protected amine 26 (2.0 g, 5.73 mmol) was ButS-S AllC 27 2 Cl dissolved in 4 Cl in dioxane (12 ml) and stirred at room temperature for 1 hour. After removal of all volatiles at reduced pressure, the residue was triturated with Et 2 (3 10 ml) providing the hydrochloride salt 27 (1.63 g, 100% yield). 1 MR (300 Mz, CDCl 3 ) 7.78 (bs, 1, 3 ),

12 S (m, 1, C=C 2 ), (m, 2, C=C 2 ), 4.73 (d, J = 6.0 z, 1, C 2 C), 4.54 (m, 1, -C), (m, 2, -C 2 ), 1.34 (s, 9, SC(C 3 ) 3 ); 13 C MR (75 Mz, CDCl 3 ) (C(=)), (C=C 2 ), (C=C 2 ), 68.5 (C 2 C), 53.4 (-C), 49.7 (SC(C 3 ) 3 ), 38.8 ( -C), 29.9 (SC(C 3 ) 3 ); ESI-LRMS m/z (M+) +. Boc--Gla[-Cys(S)-Gly-tBu]-tBu (14). To a TF/ 2 (5 ml, 10:1) solution of urea tripeptide 24 (0.35 mmol), nbu 3 P (0.70 mmol) was added in one portion and the S reaction mixture was allowed to stir at room temperature under argon for 2 h. After ButC completion of the reaction after 2 h (as indicated Boc CtBu 14 by TLC), the reaction mixture was evaporated to dryness to obtain an oil that was purified by flash column chromatography using 70% EtAc/hexanes to isolate the thiol 14 as a colorless oil (94% yield; ESI-LRMS m/z (M+a) + ). The product was incorporated immediately in the next heterodisulfide bond formation step without further characterization.

13 S13 In vitro BBB permeability prediction of prodrugs 1 and 2 A M TEAB in water/ 70% acetonitrile in water = % B linear Assay Buffer B 0.04M TEAB in water/ 70% acetonitrile in water = % B linear 2 Assay Buffer Figure S1. PLC chromatogram of the prodrug transported across MDCK cell monolayer A. adamantamine prodrug (1); B. dopamine prodrug (2).

14 S14 In vitro stability studies: Convertibility of prodrugs into active drugs C D Dopamine 2 Dopamine 2 Figure S2. PLC chromatogram diasplaying prodrug 2 (t R = 12.2 min) to dopamine (t R = 11.2 min) conversion in C. brain homogenate; D. plasma.