2.13 (dd, 1H, J = 5.0, 15.0), 2.32 (dd, 1H, J = 8.5, 14.5), 2.92 (dd, 1H, J = 6.3, 12.8), 3.02–3.08 (m, 2H), 7.35–7.40 (m, 1H), 7.45– 7.51 (m, 2H), 7.68 (s, 1H), 7.83–7.89 (m, 3H), 10.32 (s, 1H), 12.30 (br s, 1H). Procedure for the Preparation of Amides 8a–10b Acid 7a (140 mg, 0.5 mmole) was dissolved in anhydrous THF (5 ml) and anhydrous DMF (1 ml) and cooled to 220uC. A solution of triethylamine (0.075 ml, 0.54 mmole) in THF (1 ml) was added dropwise to acid 7a, followed by a solution of ethyl chloroformate (0.048 ml, 0.5 mmole) in THF (1 ml). The reaction mixture was stirred at 220uC for 1h and the treated with NH2-Arg(Pbf)- Allyl?HCl ( 380 mg, 0.55 mmole) and triethylamine (0.093 ml, 0.67 mmole) and stirred 1h at 220uC before slowly warmed to room temperature overnight. The reaction mixture was diluted with EtOAc (150 ml), washed sequentially with 0.5N HCl (100 ml), saturated sodium bicarbonate (100 ml), saturated sodium chloride (100 ml) and then dried over anhydrous sodium sulfate, filtered, evaporated to give a white foam. The resultant foam was purified by silica gel column chromatography with 5% MeOH in DCM to give 8a (foam, 270 mg, 74%). The diastereomers 8a was separated by silica gel column chromatography with 3% MeOH in DCM to give 9a (foam, (R)-form, 125 mg, 34%) as first eluting peak: 1H NMR (500 MHz, CDCl3): d 1.18 (s, 9H), 1.40–1.80 (m, 4H), 1.46 (s, 6H), 1.66–1.72 (m, 1H), 1.85–1.91 (m, 1H), 2.09 (s, 3H), 2.21–2.24 (m, 1H), 2.52 (s, 3H), 2.53–2.60 (m, 1H, overlap with s), 2.58 (s, 3H), 2.67 (m, 1H), 2.95 (s, 2H), 2.95–2.97 (m, 1H), 3.16 (broad, 2H), 4.45–4.61 (m, 3H), 5.25–5.33 (m, 2H), 5.82– 5.92 (m, 1H), 6.35 (broad d, 1H, J = 7.5), 6.44 (broad s, 1H), 7.15– 7.16 (m, 2H), 7.20–7.23 (m, 1H), 7.26–7.30 (m, 2H), 8.12 (broad s, 1H), while 10a as second eluting peak (foam, (S)-form, 145 mg, 40%) peak: 1H NMR (500 MHz, CDCl3): d 1.21 (s, 9H), 1.30– 1.38 (m, 2H), 1.45 (s, 6H), 1.54–1.62 (m, 2H), 1.70–1.78 (m, 1H), 2.09 (s, 3H), 2.35–2.38 (m, 1H), 2.44–2.50 (m, 1H), 2.52–2.58 (m, 1H), 2.53 (s, 3H), 2.58 (s, 3H), 2.78–2.83 (m, 1H), 2.95 (s, 2H), 2.96–2.98 (m, 1H, overlap with s), 3.08 (broad, 1H), 3.15–3.21 (m, 1H), 4.31 (broad, 1H), 4.56–4.62 (m, 2H), 5.23–5.31 (m, 2H), 5.83–5.91 (m, 1H), 6.10 (broad, 1H), 6.34 (broad, 1H), 6.75 (broad, 1H), 7.17–7.20 (m, 3H), 7.24–7.25 (m, 2H), 8.40 (broad s, 1H), The diastereomers 8b was also separated by silica gel column chromatography with 2.5% MeOH in DCM to give 9b (foam, (R)-form, 120 mg, 40%) as first eluting peak, peak: 1H NMR (500 MHz, CDCl3): d 1.16 (s, 9H), 1.36–1.66 (m, 4H), 1.45 (s, 6H), 1.85 (broad, 1H), 2.08 (s, 3H), 2.23–2.26 (m, 1H), 2.51 (s, 3H), 2.58 (s, 3H), 2.60–2.66 (m, 1H), 2.84–2.88 (m, 1H), 2.93 (s, 2H), 3.10–3.62 (broad m, 4H), 4.34–4.42 (m, 2H), 4.51–4.55 (m, 1H), 5.17–5.24 (m, 2H), 5.69–5.77 (m, 1H), 5.98 (broad, 1H), 6.43 (broad s, 1H), 6.49 (broad d, 1H, J = 8.0), 7.29–7.31 (m, 1H), 7.42–7.47 (m, 2H), 7.58 (s, 1H), 7.73–7.80 (m, 2H), 8.36 (broad s, 1H), while 10b as second eluting peak (foam, (S)-form, 160 mg, 52%) peak: 1H NMR (500 MHz, CDCl3): d 1.07 (broad, 2H), 1.20 (s, 9H), 1.43–1.44 (m, 6H), 1.47–1.70 (broad m, 3H), 2.09 (s, 2H), 2.42–2.47 (m, 1H), 2.52–2.58 (m, 1H), 2.52 (s, 3H), 2.58 (s, 3H), 2.88–2.98 (m, 3H, overlap with s), 2.93 (s, 2H), 3.07–3.12 (m, 1H), 3.19–3.23 (m, 1H), 4.26 (broad, 1H), 4.53 (broad d, 2H, J = 6.0), 5.20–5.28 (m, 2H), 5.79–5.85 (m, 1H), 6.26 (broad, 1H), 6.90 (broad, 1H), 7.32–7.34 (m, 1H), 7.37–7.43 (m, 2H), 7.61 (s, 1H), 7.70–7.76 (m, 3H), 8.60 (broad, 1H). Procedure for the Deprotection of Allyl Esters 9a–10b to acids 11a–12b A solution of the allyl ester 9a (125 mg, 0.17 mmole) in anhydrous THF (20 ml) was treated with tetrakis(triphenylphosphine)palladium(0) (20 mg, 0.017 mmole) and morpholine ( 0.2 ml, 2.29 mmole) at room temperature for 4 h. The reaction mixture was diluted with EtOAc (100 ml), washed with 0.5N HCl (100 ml), saturated sodium chloride (100 ml), dried over anhydrous sodium sulfate, filtered, evaporated to give a waxy solid. The resultant solid was Inhibitors of IDE purified by silica gel column chromatography with 5% MeOH in DCM to give 11a (waxy solid, 60 mg, 51%) and used without further purification. Other Acids 11b–12b were prepared in the same manner with similar yields and used in the next step without further purification. Procedure for the Preparation of Protected Amide 14 The protected dipeptide 14 was obtained as a white foam (1.16 g, 93%) starting from acid 13 and the HCl salt of NH2- Glu(tBu)-CONH2 following the general procedure described for 6a and 6b: 1H NMR (500 MHz, DMSO-d6): d 1.39 (s, 9H), 1.61 (s, 9H), 1.70–1.78 (m, 1H), 1.87–1.93 (m, 1H), 2.12–2.22 (m, 2H), 2.90 (dd, 1H, J = 10.5, 15.0), 3.09 (dd, 1H, J = 3.5, 15.0), 4.18– 4.23 (m, 1H), 4.36–4.40 (m, 1H), 4.92–4.98 (m, 2H), 7.10 (s, 1H), 7.22–7.33 (m, 8H), 7.56 (s, 1H), 7.60 (d, 1H, J = 8.0), 7.74 (d, 1H, J = 8.0), 8.03 (d, 1H, J = 8.0), 8.09 (d, 1H, J = 8.0). Procedure for the Deprotection of Amide 14 to 15 The deprotected dipeptide 15 was obtained as a white foam (230 mg, 90%) starting from protected amide 14 following the general procedure described for 7a and 7b: 1H NMR (500 MHz, CDCl3): d 1.45 (s, 9H), 1.68 (s, 9H), 1.88 (sextet, 1H, J = 7.5), 2.06–2.12 (m, 1H), 2.22 (dt, 1H, J = 6.8, 17.0), 2.38 (dt, 1H, J = 16.5, 7.5), 2.86–2.90 (m, 1H), 3.33 (dd, 1H, J = 3.5, 15.0), 3.75 (dd, 1H, J = 4.3, 9.3), 4.39–4.43 (m, 1H), 5.41 (br s, 1H), 6.47 (br s, 1H), 7.23–7.26 (m, 1H), 7.30–7.34 (m, 1H), 7.48 (s, 1H), 7.61 (d, 1H, J = 8.0), 7.95 (d, 1H, J = 8.0), 8.12 (br s, 1H). Procedure for the Preparation of Protected Tetrapeptide 16a, 16b, 17a and 17b The protected tetrapeptide 16a was obtained as a white foam (138 mg, 70%) starting from (R)-acid 11a and amine 15 following the general procedure described for 6a and 6b. See 1H NMRs below. Procedure for the Preparation of Deprotected Tetrapeptide 18a, 18b, 19a and 19b A solution of the protected tetrapeptide 16a (100 mg, 0.086 mmole) in TFA (6 ml) was treated with anisole (0.3 mg, 2.76 mmole) and stirred at 40uC for 4.5 h. The reaction mixture was cooled and the volatiles were removed in vacuo. The resultant oil was washed with ether (10 ml), dissolved in water (5 ml) and filtered. The filtrates were lyophilized to give white powders that were further purified by reverse-phase liquid chromatography using a linear acetonitrile:water gradient to give 18a as a white powder: 1H NMR (500 MHz, DMSO-d6): d 1.42–1.53 (m, 3H), 1.65–1.77 (m, 2H), 1.86–1.93 (m, 2H), 2.16–2.20 (m, 2H), 2.26 (dd, 1H, J = 9.8, 15.3), 2.45–2.47 (m, 1H, overlap with DMSO), 2.80 (dd, 1H, J = 6.0, 13.5), 2.96–3.06 (m, 4H), 3.14 (dd, 1H, J = 4.8, 14.3), 4.16–4.24 (m, 2H), 4.50–4.54 (m, 1H), 6.76 (broad, 1H), 6.96 (t, 1H, J = 7.5), 7.04 (t, 1H, J = 7.8), 7.09–7.24 (m, 8H), 7.29 (d, 1H, J = 7.5), 7.32–7.34 (m, 2H), 7.56 (d, 1H, J = 8.5), 7.97 (d, 2H, J = 7.5), 8.09 (d, 1H, J = 7.5), 8.76 (s, 1H), 10.45 (s, 1H), 10.81 (d, 1H, J = 2.0), 12.11 (broad s, 1H); MALDI-MS obsd 694.3308, calcd 694.3235 [(M + H)+ , M = C33H43N9O8]. 18b: 1H NMR (400 MHz, DMSO-d6): d 1.44–1.56 (m, 3H), 1.68–1.79 (m, 2H), 1.87–1.95 (m, 2H), 2.16–2.23 (m, 2H), 2.28–2.31 (m, 1H, overlap with DMSO), 2.45–2.55 (m, 1H, overlap with DMSO), 2.61–2.64 (m, 1H), 2.94–3.16 (m, 5H), 4.15–4.30 (m, 2H), 4.51– 4.59 (m, 1H), 6.80 (broad, 1H), 6.96 (t, 1H, J = 7.2), 7.03 (t, 1H, J = 6.8), 7.09 (s, 1H), 7.14–7.18 (m, 2H), 7.27–7.36 (m, 3H), 7.41– 7.47 (m, 2H), 7.56 (d, 1H, J = 8.0), 7.65 (s, 1H), 7.74 (d, 1H, J = 8.8), 7.80–7.82 (m, 2H), 7.95–8.00 (m, 2H), 8.19 (d, 1H, J = 7.6), 8.75 (s, 1H), 10.45 (s, 1H), 10.82 (s, 1H); MALDI-MS obsd 744.3103, calcd 744.3391 [(M + H)+ , M = C39H45N9O8]. 19a: 1H NMR (500 MHz, DMSO-d6): d 1.06–1.09 (m, 2H), 1.14– 1.23 (m,1H), 1.46–1.54 (m, 1H), 1.80–1.88 (m, 1H), 1.98–2.05 (m, 1H), 2.14 (dd, 1H, J = 4.0, 14.5), 2.22–2.31 (m, 2H), 2.41 (dd, 1H, J = 10.5, 15.5), 2.61 (dd, 1H, J = 6.5, 13.0), 2.71 (dd, 1H, J = 9.0, 13.5), 2.84 (q, 2H, J = 7.5), 2.93–2.99 (m, 1H), 3.16–3.24 (m, 2H), 3.76–3.80 (m, 1H), 4.09–4.13 (m, 1H), 4.37–4.41 (m, 1H), 6.78 (broad, 1H), 6.86 (s, 1H), 6.97 (t, 1H, J = 7.3), 7.05 (t, 1H, J = 7.8), PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10504