M. F. Parker et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5790–5795
5795
pared by dilution into 40 mM Tris–HCl (pH 7.4) with
0.2% BSA and added to assay plates. For the Ab40
measurements, antibodies specific for the Ab40 neoepitope
(TSD, developed at BMS; conjugated to the Wallac
reagent (Perkin-Elmer)) and 26D6 as described above
were mixed and 20 ll of the mixture was added to the 10 ll
aliquots which had been removed previously from the cell
plate yielding a final concentration of 1.6 ng/well TSD and
17.5 ng/well 26D6. Assay plates containing antibodies
were sealed with aluminum foil and incubated overnight at
4 ꢁC. Signal was determined using a Viewlux counter
(Perkin-Elmer) and IC50 values determined using curve
fitting in CurveMaster (Excel-Fit based data analysis
package). Also, see Smith, D. W.; Munoz, B.; Srinirvasan,
K.; Bergstrom, C. P.; Chaturvedula, P. V.; Deshpande, M.
S.; Keavy, D. J.; Lau, W. Y.; Parker, M. F.; Sloan, C. P.;
Wallace, O. B.; Wang, H. H. WO 0050391.
fold more potent than the aniline 25, 25 has higher rel-
ative brain and plasma exposures. This may explain
why the in vivo potencies of these compounds are
comparable.
In summary, potent c-secretase inhibitors emerged from
SAR studies and optimization of an initial amino capro-
lactam screening hit. The most potent compounds to
emerge from this study were the 4-methoxy benzyl com-
pound 23 and the N,N-dimethyl analog 25. These com-
pounds were approximately 50-fold more potent than
the original screening hit and markedly reduced the con-
centration of Ab in the brain and plasma in transgenic
Tg2576 mice. However, 23 and 25 were also found to
be nanomolar inhibitors of the human CYP450 isoforms
3A4 and 2C19. Further investigations are focused on the
removal of these liabilities from this and similar sulfon-
amide series.
7. Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.;
Smith, D. W.; Pak, R. Isolation of functionally active c-
secretase presenilin 1 complex and fluorescence assay for
c-secretase activity and inhibitors. US 2004, 0121411 A1.
8. Purchased from ComGenex Corporation as part of a
collection of sulfonamides.
References and notes
9. Recently disclosed in a patent, Martin, N.; preparation of
N-substituted heterocyclic sulfonamides for treating cog-
nitive disorders. US 2006/0270657A1.
1. Scheff, S. W.; Price, D. A.; Schmitt, F. A.; Mufson, E. J.
Neurobiol. Aging 2006, 27, 1372.
10. Pellegata, R.; Pinza, G.; Pifferi, G. Synthesis 1978, 614.
11. The 3 amino-2-piperidone was synthesized from D-orni-
thine following the method of Pellegata (9). The 3-amino-
2-pyrrolidinone was purchased from Ramidus AB.
12. Possible clarifications for the 10-fold increase in activity
include but are not limited to: antagonism or partial
agonist activity by the inactive enantiomer, chiral ligand
regulated enzyme activity, allosteric binding by one of the
enantiomers or even relative error in the binding assay.
These hypothetical explanations are being explored
further.
2. (a) Clark, C. M.; Karlawish, J. H. Ann. Intern. Med. 2003,
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13. Determination of Ab, full length APP, and APP carboxy-
terminal fragments, brain tissues was performed following
the procedure as described previously. Barten, D. M.;
Guss, V. L.; Corsa, J. A.; Loo, A.; Hansel, S. B.; Zheng,
B.; Munoz, K.; Srinivasan, K.; Wang, B.; Roberson, B. J.;
Polson, C. T.; Wang, J.; Roberts, S. B.; Hendrick, J. P.;
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Smith, D. W.; Felsenstein, K. M. J. Pharmacol. Exp. Ther.
2005, 312, 635.
6. H4 cells stably transfected with APP751 containing the
Swedish mutation (H4 APP751 SWE clone 8.20, devel-
oped at BMS) were maintained in log phase through twice
weekly passage at a 1:20 split. For IC50 determinations,
1.5 · 104 cells/well in DMEM containing 0.0125% BSA
(Sigma A8412) were plated directly into 384-well com-
pound plates (Costar 3709) containing 0.1 ll serially
diluted compound in DMSO. Following incubation for
19 h in 5% CO2 at 37 ꢁC, plates were briefly centrifuged
(1000 rpm, 5 min). A 10 ll aliquot from each well was
transferred to a second assay plate (Costar 3709) for Ab1–
40 measurements. Antibody cocktails were freshly pre-
14. Hsiao, K.; Chapman, P.; Nilsen, S.; Eckman, C.; Hari-
gaya, Y.; Younkin, S.; Yang, F.; Cole, G. Science 1996,
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