Helvetica Chimica Acta Vol. 87 (2004)
2339
General Procedure (GP) for EDAC/HOBt Couplings2). A soln. of the N-terminally protected amino acid or
peptide6) (2.9 mmol, 1 equiv.), EDAC (1 equiv.), and HOBt (1 equiv.) in anh. CH2Cl2 (20 ml) was stirred for
10 min at r.t. Then, Et3N (0.693 ml, 5 mmol, 1.7 equiv.) and either a C-terminally protected amino acid or (for
ester formation) the corresponding alcohol (1 equiv.) were added. The mixture was stirred for ca. 22 h at r.t.,
extracted with 0.1n aq. NaOH soln. (3Â), dried (Na2SO4), and evaporated in vacuo. The resulting colorless oil
was purified by HPLC (SiO2; hexane/AcOEt 1:1).
1-Cyclopropylethyl (2S)-({N-[(3,5-Difluorophenyl)acetyl]-l-alanyl}amino)(phenyl)acetate (11). Prepared
in 23% yield (1:1 diastereoisomer mixture) according to the GP. HPLC: tR 5.293 min. 1H-NMR (300 MHz,
CDCl3): 7.47 7.19 (m, 5 arom H); 6.76 6.63 (m, 3 arom. H); 6.15 (d, NH); 5.41 (d, CH); 4.52 4.48 (m, CH);
4.14 4.05 (m, CH); 3.48 (d, CH2); 1.32 (d, Me); 1.25 (d, Me); 1.16 1.09 (m, 2 CH2); 0.85 0.79 (m, CH). EI-
MS: 444.5 (22, M ), 331 (26), 127 (26), 106 (100), 44 (63).
Cyclopropylmethyl (2S)-({N-[(3,5-Difluorophenyl)acetyl]-l-alanyl}amino)(phenyl)acetate (12). Prepared
in 28% yield according to the GP. HPLC: tR 5.292 min. 1H-NMR (300 MHz, CDCl3): 7.36 7.26 (m, 5 arom. H);
7.19 (d, NH); 6.84 6.69 (m, 3 arom. H); 6.34 (d, NH); 5.49 (d, CH); 4.58 4.54 (m, CH2); 4.21 4.12 (m, CH);
3.46 (d, CH2); 1.39 (d, Me); 1.30 1.18 (m, 2 CH2); 0.88 0.85 (m, CH). EI-MS: 430.5 (31, M ), 331 (42), 127
(32), 106 (100), 44 (87).
Dicyclopropylmethyl (2S)-({N-[(3,5-Difluorophenyl)acetyl]-l-alanyl}amino)(phenyl)acetate (13). Pre-
pared in 38% yield according to the GP. HPLC: tR 5.206 min. 1H-NMR (300 MHz, CDCl3): 7.41 7.23 (5 arom.
H); 6.81 6.70 (3 arom. H); 5.52 (d, CH); 5.13 (d, CH), 4.19 4.12 (m, CH); 3.46 (d, CH2); 1.71 (d, Me); 1.38
1.26 (m, CH2); 1.01 (d, CH2); 0.88 0.85 (m, CH); 0.54 0.48 (m, CH2); 0.31 0.27 (m, CH2). EI-MS: 471.0 (2,
M ), 453 (22), 311 (20), 44 (84), 91 (100).
N-{(1S)-2-[(Cyclopropylmethyl)amino]-2-oxo-1-phenylethyl}-l-alanine-N-[(3,5-difluorophenyl)acetyl]-
amide (14). Obtained in trace amounts only via the GP. However, via the alternative route, starting from Boc-
Phg-Gly-OH, described in the text, and later on following the EDAC/HOBt protocol, 77% of 14 were obtained.
EI-MS: 430 (100, M ), 412 (15), 365 (45), 359 (100).
Cyclopropyl(phenyl)methyl (2S)-({N-[(3,5-Difluorophenyl)acetyl]-l-alanyl}amino)(phenyl)acetate (15).
Prepared in 73% yield (1:1 diastereoisomer mixture) according to the GP. HPLC: tR 5.30 min. EI-MS: 507.6 (9,
M ); 496 (100), 410 (47), 290 (25), 107 (48), 91 (98).
N-(Benzyloxycarbonyl)-l-alanine-N-({(1S)-2-[(cyclopropylmethyl)amino]-2-oxo-1-phenyl}ethyl)amide
1
(16). Prepared in 45% yield according to the GP. HPLC: tR 5.24 min. H-NMR (300 MHz, CDCl3): 7.21 7.10
(10 arom. H); 6.09 (d, NH); 5.39 (d, CH); 5.02 (s, CH2); 4.19 4.08 (m, CH); 3.04 2.90 (m, CH2); 1.29 (d, Me);
0.74 0.72 (m, CH); 0.32 0.29 (m, CH2); 0.05 0.01 (m, CH2). EI-MS: 409.0 (63, M ), 384 (84), 338 (100).
Bioassay. g-Secretase activity was monitored by de novo production of AICD (APP intracellular domain)
in vitro. Membrane fractions of HEK-293 cells, stably transfected with Swedish mutant APP, were prepared as
described in [5], except that membranes were directly resuspended in assay buffer (150 mm sodium citrate, pH
6.4; 1 Â protease inhibitors (Complete, Roche)) without any further washing step. To allow generation of AICD,
samples were incubated at 378 for 2 h. Control samples were kept on ice. Where indicated, g-secretase inhibitors
were added to the samples. After termination of the assay reactions on ice, samples were subjected to
ultracentrifugation (1 h at 48, 105g). The soluble fractions were subjected to SDS-PAGE on 10 20% Tris-Tricine
gels (Invitrogen), and AICD was analyzed by immuno-blotting with antibody 6687, using enhanced
chemiluminescence (ECL; Amersham Pharmacia Biotech).
REFERENCES
[1] D. J. Selkoe, J. Clin. Invest. 2002, 110, 1375; M. Citron, Neurobiol. Aging 2002, 23, 1017; S. Roggo, Curr.
Top. Med. Chem. 2002, 2, 359.
[2] B. Schmidt, ChemBioChem 2003, 4, 366.
[3] H. F. Dovey, V. John, J. P. Anderson, L. Z. Chen, P. De Saint Andrieu, L. Y. Fang, S. B. Freedman, B.
Folmer, E. Goldbach, E. J. Holsztynska, K. L. Hu, K. L. Johnson-Wood, S. L. Kennedy, D. Kholodenko,
J. E. Knops, L. H. Latimer, M. Lee, Z. Liao, I. M. Lieberburg, R. N. Motter, L. C. Mutter, J. Nietz, K. P.
Quinn, K. L. Sacchi, P. A. Seubert, G. M. Shopp, E. D. Thorsett, J. S. Tung, J. Wu, S. Yang, C. T. Yin, D. B.
Schenk, P. C. May, L. D. Altstiel, M. H. Bender, L. N. Boggs, T. C. Britton, J. C. Clemens, D. L. Czilli, D. K.
6
)
For the synthesis of 11 15, peptide acid 1a ((2S)-({N-[(3,5-difluorophenyl)acetyl]-l-alanyl}amino)-
(phenyl)acetic acid) was used.