924
G. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 922–925
Acknowledgments
This work was supported by NIH Grant AG026660 (Y.M.L.), the
100
80
60
40
20
0
kDa
37
3
2
Alzheimer’s Association (Zenith Fellows Award to Y.M.L.), Mr. Wil-
liam H. Goodwin and Mrs. Alice Goodwin and the Commonwealth
Foundation for Cancer Research, the Experimental Therapeutics
Center of MSKCC, and the William Randolph Hearst Fund in Exper-
imental Therapeutics. C.C.S. is supported by an NIH NRSA predoc-
toral fellowship 5F31NS053218-02. We thank Dr. George
Sukenick, Ms. Sylvi Rusli (NMR Core Facility, Sloan-Kettering Insti-
tute) for mass spectral analyses and Dr. Louis Todaro (Hunter Col-
lege, New York) for X-ray structure analyses.
PS-1
25
20
3
+
+
L-685,458 -
+
0.01
0.1
1
10
100
Concentration (nM)
References and notes
1. Hardy, J.; Allsop, D. Trends Pharmacol. Sci. 1991, 12, 383.
2. De Strooper, B. Neuron 2003, 38, 9.
3. Wolfe, M. S.; Xia, W.; Ostaszewski, B. L.; Diehl, T. S.; Kimberly, W. T.; Selkoe, D.
J. Nature 1999, 398, 513.
Binding
+
4. Li, Y. M.; Xu, M.; Lai, M. T.; Huang, Q.; Castro, J. L.; DiMuzio-Mower, J.; Harrison,
T.; Lellis, C.; Nadin, A.; Neduvelil, J. G.; Register, R. B.; Sardana, M. K.; Shearman,
M. S.; Smith, A. L.; Shi, X. P.; Yin, K. C.; Shafer, J. A.; Gardell, S. J. Nature 2000,
405, 689.
5. Esler, W. P.; Kimberly, W. T.; Ostaszewski, B. L.; Diehl, T. S.; Moore, C. L.; Tsai, J.
Y.; Rahmati, T.; Xia, W.; Selkoe, D. J.; Wolfe, M. S. Nat. Cell Biol. 2000, 2, 428.
6. Sherrington, R.; Rogaev, E. I.; Liang, Y.; Rogaeva, E. A.; Levesque, G.; Ikeda, M.;
Chi, H.; Lin, C.; Li, G.; Holman, K., et al Nature 1995, 375, 754.
7. Wolfe, M. S.; Kopan, R. Science 2004, 305, 1119.
3
Target Protein
Photolysis
8. Brown, M. S.; Ye, J.; Rawson, R. B.; Goldstein, J. L. Cell 2000, 100, 391.
9. Shearman, M. S.; Beher, D.; Clarke, E. E.; Lewis, H. D.; Harrison, T.; Hunt,
P.; Nadin, A.; Smith, A. L.; Stevenson, G.; Castro, J. L. Biochemistry 2000,
39, 8698.
10. Chun, J.; Yin, Y. I.; Yang, G.; Tarassishin, L.; Li, Y. M. J. Org. Chem. 2004, 69, 7344.
11. Chen, P.; Cheng, P. T. W.; Spergel, S. H.; Zahler, R.; Wang, X. B.; Thottathil, J.;
Barrish, J. C.; Polniaszek, R. P. Tetrahedron Lett. 1997, 38, 3175.
12. Chai, W.; Murray, W. V. Tetrahedron Lett. 1999, 40, 7185.
13. Orito, K.; Sato, S.; Suginome, H. J. Chem. Soc., Perkin Trans. 1 1995, 1, 63.
Western
Blotting
Streptavidin
Column
Figure 3. Both 2 and 3 are potent
presenilin-1. (A) Inhibitory potencies of compounds 2 and 3 against
Scheme of photoaffinity labeling procedure. After photo-crosslinking, the biotinyl-
ated proteins were captured, eluted and analyzed by Western analysis. (C) Analysis
of photolabeled proteins. The photo-crosslinked proteins were resolved by SDS–
PAGE and probed with PS-1-NTF (N-terminal fragment) antibody.
c
-secretase inhibitors that directly bind to
-secretase. (B)
14. Preparion
of
8:
tert-Butyl
(S)-2-(4-(S)-tert-butyldimethylsilyloxy
phenylmethylphenyl)-1-((S)-oxiran-2-yl)ethylcarbamate (8). To an ice-cold
solution of 7 (1.25 g, 2.41 mmol) in EtOH (30 mL) was added KOH (163 mg,
2.9 mmol), and the reaction mixture was stirred at rt for 2 h. The reaction
mixture was concentrated under reduced pressure, and the residue was
partitioned between EtOAc (200 mL) and H2O (50 mL). The organic layer was
washed with saturated NH4Cl solution, H2O, and brine, dried with Na2SO4, and
concentrated under reduced pressure. Purification of the residue by column
chromatography (20% EtOAc in hexane) gave 8 (1.11 g, 95%) as a yellow syrup:
c
pound 2 in reasonable yield (Scheme 3).21 In order to facilitate the
purification of the labeled proteins or fragments thereof, biotinyl-
ated compound 3 was prepared (Scheme 3). Mild saponification
of the methyl ester in 2 led to the corresponding carboxylic acid,
which was coupled with 5-(biotinamido)pentylamine in the pres-
ence of EDC and HOBt and resulted in compound 3.
½ ꢂ
a 2D5 9.1 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): 7.38 (d, J = 7.4 Hz, 2H), 7.33
(m, 4H), 7.22 (t, J = 7.1 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 5.75 (s, 1H), 4.47 (br s,
1H), 3.70 (br s, 1H), 3.93 (m, 2H), 2.80 (m, 3H), 1.38 (s, 9H), 0.94 (s, 9H), 0.01 (s,
3H), 0.00 (s, 3H); 13C NMR: 155.2, 145.1, 143.7, 135.3, 129.3, 128.1, 126.9,
126.5, 126.2, 79.5, 76.4, 53.2, 46.8, 37.2, 28.2, 25.8, 18.3, ꢀ4.8; EIMS: 506.3
[M+Na+], HRMS (ESI) Calcd for C28H41NSiO4Na: 506.2703, found 506.2698.
15. Evans, B. E.; Rittle, K. E.; Homnick, C. F.; Springer, J. P.; Hirshfield, J.; Veber, D. F.
J. Org. Chem. 1985, 50, 4615.
We next examined the biological activities of 2 and 3. First, we
16. Nadin, A.; Lopez, J. M. S.; Neduvelil, J. G.; Thomas, S. R. Tetrahedron 2001, 57, 1861.
17. Preparation of 13: tert-Butyl (S)-1-((2R,4R)-4-benzyl-5-oxotetrahydrofuran-2-
yl)-2-(4-(S)-hydroxyphenyl methylphenyl) ethyl carbamate (13). A solution of
determined their inhibitory potency against c-secretase using an
in vitro assay.22 The IC50 values of 2 and 3 are 0.7 and 0.6 nM,
respectively (Fig. 3A), which is similar to the parent compound,
L-685,458 (1). These findings have demonstrated that incorporat-
ing BPA into the P1 position and attaching a biotin tag at the C-ter-
silyl ether 12 (390 mg, 0.63 mmol) in THF (10 mL) was transferred to
a
polyethylene vial and PyꢁHF (70% HF, 1.5 mL) was dropwise added at 0 °C. The
reaction mixture was stirred at rt overnight. The reaction mixture was
quenched with saturated NaHCO3 solution, and extracted with EtOAc (3ꢃ
50 mL). The combined organic layer was washed with brine, dried with
Na2SO4. The residue was purified by column chromatography (50% EtOAc in
minus do not affect their potency for inhibition of
Second, we tested whether 3 was capable of photo-crosslinking
to -secretase. HeLa cell membranes were incubated with 3 at a fi-
nal concentration of 10 nM in the absence and the presence of
M of L-685,458 for 2.5 h. Then samples were irradiated with
c-secretase.
hexane) to give 13 (264 mg, 83%) as a white solid: mp 59–60 °C; ½a D25
ꢂ
ꢀ58.8 (c
0.7, CHCl3); 1H NMR (400 MHz, CDCl3): 7.35–7.21 (m, 10 H), 7.15 (d, J = 7.2 Hz,
2 H), 7.10 (d, J = 7.9 Hz, 2H), 5.77 (d, J = 2.7 Hz, 1H), 4.43 (br, 1H), 4.09 (br, 1H),
3.85 (br, 1H), 3.24 (dd, J = 4.0, 13.8 Hz, 1H), 2.87–2.68 (m, 4H), 2.63 (d,
J = 3.3 Hz, 1H, OH), 2.18 (m, 1H), 1.75 (m, 1H), 1.31 (s, 9H); 13C NMR: 177.6,
155.3, 143.9, 142.5, 138.4, 135.9, 129.6, 128.8, 128.7, 128.5, 128.4, 127.5, 127.3,
127.2, 126.8, 126.7, 126.5, 79.8, 78.8, 75.9, 54.3, 43.0, 36.1, 31.6, 28.2; EIMS:
524.1 [M+Na+], HRMS (ESI) Calcd for C31H35NO5Na: 524.2413, found 524.2402.
18. Fauq, A. H.; Cherif-Ziani, C.; Richelson, E. Tetrahedron: Asymmetry 1998, 9, 2333.
19. Preparation of 14: tert-Butyl (S)-2-(4-benzoylphenyl)-1-((2R,4R)-4-benzyl-5-
oxotetrahydrofuran-2-yl) ethyl carbamate (14). To an ice-cold solution of 13
(240 mg, 0.478 mmol) in CH2Cl2 (10 mL) was added MnO2 (415 mg,
4.78 mmol). The suspension was stirred at rt overnight. The reaction mixture
was filtered through Celite, washed with EtOAc. The combined organic layer
was concentrated, the residue was purified by column chromatography (40%
c
2
l
UV light (>350 nm) and the labeled proteins were solubilized and
isolated with streptavidin beads.4
The biotinylated proteins were eluted and analyzed by Western
blotting with antibodies against presenilin-1 (PS-1). Inhibitor 3 di-
rectly photolabels PS-1 (Fig. 3C). Moreover, an excess of L-685,458
is able to block photoinsertion of this probe into presenilin-1.
Taken together, these results have demonstrated that compounds
2 and 3 are potent
c-secretase inhibitors that can specifically label
EtOAc in hexane) to give 14 (185 mg, 76%) as a white solid: mp 59–60 °C; ½a D25
ꢂ
the catalytic core of
c-secretase. Therefore, compounds 2 and 3
ꢀ68.7 (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3): 7.74 (dd, J = 7.2, 11.9 Hz, 4H),
7.57 (t, J = 7.5 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.28 (m, 4 H), 7.17 (m, 3H), 4.68
(d, J = 9.4 Hz, 1H), 4.32 (br, 1H), 3.93 (br, 1H), 3.26 (dd, J = 4.1, 13.9 Hz, 1H),
should be valuable probes for mapping the active site of
secretase.
c-