Hydroxytriamides as potent γ-secretase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.01.086

Using a cell-based assay, we have identified optimal residues and key recognition elements necessary for inhibition of γ-secretase. An (S)-hydroxy group or 3,5-difluorophenylacetyl group at the amino terminus and N-methyltertiary amide moiety at the carbo

Full text of DOI:10.1016/j.bmcl.2004.01.086

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1917–1921
Hydroxytriamides as potent ꢀ-secretase inhibitors
C. V. C. Prasad,a,* Jeffrey W. Noonan,a Charles P. Sloan,a Wai Lau,a Shikha Vig,a
Michael F. Parker,a David W. Smith,a Steven B. Hansel,b Craig T. Polson,c
Donna M. Barten,c Kevin M. Felsensteinc and Susan B. Robertsc
aDepartment of Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
bDepartment of Drug Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
cDepartment of Neuroscience Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492, USA
Received 20 November 2003; revised 26 January 2004; accepted 28 January 2004
Abstract—Using a cell-based assay, we have identified optimal residues and key recognition elements necessary for inhibition of
g-secretase. An (S)-hydroxy group or 3,5-difluorophenylacetyl group at the amino terminus and N-methyltertiary amide moiety at
the carboxy terminus provided potent g-secretase inhibitors with an IC50 <10 nM.
# 2004 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a chronic neurodegenerative
disorder affecting the elderly. The disease is associated
with dementia, loss of neurons, reduced levels of several
major neurotransmitters, senile plaques and neurofi-
brillary tangles.1 To date all therapeutic approaches
have relied on neurotransmitter replacement therapies.
Although these treatments have provided modest pal-
liative improvement, the underlying disease progression
remains unchecked. Recent genetic evidence strongly
implicates abnormal proteolytic processing of b-amy-
loid precursor protein (b-APP) to Ab40 and elevated
amounts of Ab42 in amyloid plaque formation.2 Amy-
loid deposition is one of the major pathological hall-
marks of Alzheimer’s disease and therapeutic
approaches that modulate abnormal processing of APP
may delay the onset and progression of the disease.3
Ab40 and Ab42 are generated through the combined
action of b- and g-secretases. Recently, b-secretase has
been cloned and recombinant protein isolated in pure
form. g-Secretase activity has been isolated in associ-
ation with the presenilin complex.4 Cellular assays to
measure Ab40 and Ab42 levels have also been descri-
bed.5 These assays have permitted the discovery of sev-
eral selective small-molecule inhibitors (A, B, C, D and
E) of Ab production that inhibit g-secretase activity
(Fig. 1).6,7 A substrate-based difluoro ketone F was also
developed as a selective inhibitor of g-secretase.8
Keywords: Hydroxytriamides; g-Secretase.
* Corresponding author. Tel.: +1-203-677-6699; fax: +1-203-677-
7702; e-mail: prasad.chaturvedula@bms.com
Figure 1. Known inhibitors of g-secretase.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.086

1918
C. V. C. Prasad et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1917–1921
Herein we report the SAR within a novel series of g-
secretase inhibitors evaluated in a cell-based assay mea-
suring Ab production.9 Screening of our corporate
compound collection provided the hydroxyethylene iso-
stere 1 (IC50=4.7 mM) as a lead compound that inhib-
ited Ab production in tissue culture cells. The lead
compound and subsequent analogues exhibited a profile
consistent with inhibition of g-secretase. Cells treated
with the compounds produced dose dependent decrea-
ses in Ab levels and concomitant increases in 9 and 11
kDa membrane bound substrates of g-secretase. A cell
free assay that specifically detects g-secretase cleavage of
membrane bound precursors was used to profile select
compounds in the series and show that g-secretase is
specifically inhibited.10À13
Scheme 1. Reagents and conditions: (a) EtOC(O)Cl, EtN(i-Pr)2,
CH2Cl2, then NH(Me)n-Bu (90%); (b) 20% CF3CO2H in CH2Cl2
(95%); (c) Boc leucine, EtOC(O)Cl, EtN(i-Pr)2 (85%); (d) 20%
CF3CO2H in CH2Cl2 (95%); (e) PhCH2CO2H, PyBop, EtN(i-Pr)2
(80%).
Truncation of both the amino- and carboxy-termini of 1
afforded ketone 2 (IC50=300 nM) with improved
potency. Treatment of Weinreb amide 3 with iso-pen-
tylmagnesium bromide afforded ketone 2 in 50% yield.
Surprisingly, amide 3 (IC50=130 nM) was found to be
more potent than either ketone 2 or alcohol 4
(IC50=1200 nM) respectively. Incorporation of struc-
tural features fromthe inhibitors 2 and 3 led to the
design and synthesis of N-methylbutyl amide 5 (Fig. 2).
Synthesis of the triamide is readily achieved through
peptide coupling of N-(t-butoxycarbonyl)-amino acids
with N-methylbutyl amine followed by trifluoroacetic
acid (TFA) mediated deprotection.14 For example,
treatment of N-(t-butoxycarbonyl)-cyclohexylalanine
(Boc-Cha) 6 with ethyl chloroformate and diisopropyl-
ethylamine afforded the mixed anhydride which was
Table 1. Structure–activity relationships at the carboxy terminus
Compd
R
R1
R2
IC50 (nM)a
5
9
NR1R2
NR1R2
NR1R2
NR1R2
NR1R2
NR1R2
NR1R2
piperidyl
OR1
Me
H
Me
H
Me
Me
Et
na
Me
Me
(CH2)3Me
(CH2)3Me
CH2CH(Me)2
CH2CH(Me)2
CH2Ph
Me
(CH2)3Me
na
na
17
240
13
150
39
102
55
50
10
11
12
13
14
15
16
17
subsequently
reacted
with
N-methylbutylamine
(Scheme 1). The resulting amide was treated with TFA
to afford the amine salt 7. Reaction of N-(t-butoxy-
carbonyl)leucine under mixed anhydride coupling con-
ditions (diisopropylethylamine and ethyl chloroformate)
with amine salt 7 afforded the N-Boc dipeptide which
was subsequently deprotected via treatment with TFA
affording amine salt 8. Triamide 5 was obtained in 55%
overall yield by PyBop-mediated coupling of aminodi-
peptide 8 with phenylacetic acid.
800
900
OR1
na
na, not applicable.
a Values are means of two experiments with 16 data points in each
experiment; intra-assay variance <10%.
potent than the secondary amides (compound 5 and 9).
These tertiary amides likely possess enhanced cellular
penetration as a result of fewer solvated amide bonds.15
The conformational constraint imposed by the N-
methyl group also may play a role in influencing the
potency as the proton NMR spectrumof 5 reveals cis–
trans isomerism about the Cha-R bond. Among the
tertiary amides assayed, n-butyl and iso-butyl moieties
were optimal (compound 5 and 10). In contrast, the
methyl and ethyl esters displayed only modest potencies
(16 and 17).
Analogue 5 proved to be a potent g-secretase inhibitor
(IC50=17 nM). Encouraged by the potency of 5, we
elected to assess the effect of various secondary and
tertiary C-terminal amides on g-secretase inhibition
(Table 1). Within the N-phenylacetylLeu-Cha-R series
of compounds, tertiary amides were consistently more
Several strategies were employed to identify the optimal
structural requirements at the amino terminus. A vari-
ety of substituents were introduced on the phenylacetyl
ring to probe electrostatic effects (Table 2). In general,
meta-halogens afforded greater potency than either
ortho- or para-substitution. Introduction of an amino
group or electron donating methoxy group provided
less active compounds. The most potent compound in
this series was obtained through the introduction of two
Figure 2. Lead compound and analogues modified.

C. V. C. Prasad et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1917–1921
Table 2. Structure–activity relationships at the amino terminus Table 3. Substitution at the benzylic position
1919
Compd
X
Y
IC50 (nM)a
Compd
G
IC50 (nM)a
32
33
34
35
36
37
38
39
H
OH
O
OH
H
O
OMe
H
OMe
Me
OH
1
240
1
2000
>2500
>2500
>2500
>2500
5
H
2-F
3-F
4-F
17
280
20
36
52
18
19
20
21
22
23
24
25
26
27
28
H
OMe
Me
OMe
Me
3-Cl
3-CH3
3-OMe
3-NH2
4-NH2
3,5-di-F
3,5-di-CF3
3,5-di-OMe
454
480
>2500
1100
6
a Values are means of two experiments with 16 data points in each
experiment; intra-assay variance <10%.
408
800
a Values are means of two experiments with 16 data points in each
experiment; intra-assay variance <10%.
As revealed in Table 3, methylation of the hydroxy
group or substitution on the carbon carrying the
hydroxy group was not tolerated.
meta-fluorine atoms on the aromatic ring. This SAR
finding is consistent with other recently disclosed func-
tional g-secretase inhibitors.7
It is conceivable that compounds of the triamide class
represented by 5 are undergoing oxidation to give
hydroxylated compounds such as 32 under the condi-
tions of the cell-based assay. To address the possibility
of cellular oxidation of these compounds, cells treated
with compound for 18 h were lyopholized, extracted
with acetonitrile, and analyzed by HPLC as well as
mass spectrometry. These analytical methods were
unable to detect the presence of hydroxy analogues 31,
32 or keto compound 33 in the cellular extracts.
Removal of the methylene group tethering the phenyl
ring to the amide carbonyl (Fig. 3, compound 29) dra-
matically attenuated activity. Insertion of an additional
methylene group (compound 30) resulted in a 10-fold
loss in potency. Furthermore, introduction of an unsa-
turated bond in 30 led to a dramatic loss of potency
(compound 31). Clearly the methylene group was play-
ing a pivotal role in the inhibitory profile of these com-
pounds. Hydroxylated compounds 32 and 33 were
synthesized to investigate the impact of hydrogen
bonding and stereospecificity (Table 3). Introduction of
a (S)-hydroxyl moiety (compound 32) resulted in a sig-
nificant enhancement in potency (IC50=1 nM). The
ketoamide 34, which is expected to be in the hydrate
formalso was found to be very potent (IC 50=1 nM). To
investigate the role of the hydroxyl group further, the
methoxy (35 and 36), substituted methoxy (37 and 38)
and substituted hydroxy (39) analogues were prepared.
To identify optimal substituents appended to the inter-
nal amino acid residues, a variety of amino acid sub-
stitutions were explored (Table 4). Cyclohexylalanine in
compound 5 was replaced with basic, acidic, and neutral
amino acids. The hydrophobic nature of the recognition
subsite was evident fromthe reduced potency of
Table 4. Structure–activity relationships at the sub-sites
Compd
G
R
R1
IC50 (nM)a
40
41
5
42
26
43
44
45
46
47
3,5-di-F (S)-Leu
3,5-di-F (S)-Leu
(S)-Asp
(S)-Lys
(S)-Cyclohexylalanine
na
na
17
1000
6
32
300
2400
na
H
H
(S)-Leu
(S)-Leu (R)-Cyclohexylalanine
3,5-di-F (S)-Leu
(S)-Cyclohexylalanine
(S)-Cyclohexylalanine
(S)-Cyclohexylalanine
(S)-Cyclohexylalanine
(S)-Cyclohexylalanine
3,5-di-F
3,5-di-F
3,5-di-F
3,5-di-F
(S)-Ala
(S)-Gly
(S)-Phe
(S)-Pro
3,5-di-F (R)-Leu (S)-Cyclohexylalanine
na
na=not active; IC50 >2500 nM.
a Values are means of two experiments, with 16 data points in each
experiment; intra-assay variance <10%.
Figure 3. Modifications at the amino terminus.

1920
C. V. C. Prasad et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1917–1921
compounds 40 and 41 compared with the more potent
compounds 26 and 43. A similar SAR emerged when
small (44), medium (43) and large (45) hydrophobic
residues were introduced in place of leucine. It was clear
fromthe inhibition data that leucine and alanine were
the preferred residues among those assayed. The ste-
reochemical requirements at both subsites were investi-
gated by incorporating R-amino acids. The strong
preference for S-stereochemistry was evident from poor
activity displayed by compounds 42 and 47.
1833Æ196 nM in the plasma, but only 65Æ7 nM in the
brain three h after dosing. The lack of significant activ-
ity in the brain is likely due to its inadequate CNS
penetration.
In summary we have explored the SAR of a series of
g-secretase inhibitors exemplified by compound 5 and
its analogues. We have identified two moieties for opti-
mal g-secretase inhibition. An hydroxy group with (S)-
stereochemistry or a keto group is optimal at the amino
terminus. A N-alkyl tertiary amide functionality at the
carboxy terminus is optimal for potency in this series.18
Mediumand large hydrophobic groups are preferred at
the central hydrophobic residues. Compounds 6, 32 and
34 are the most potent g-secretase inhibitors identified
so far in this series. Investigations are underway in our
laboratories to improve the pharmacokinetic properties of
these hydroxy triamides. Compounds such as hydroxy
amides with good pharmacokinetic profiles will be
required to determine the role of amyloid in Alzheimer’s
disease and to evaluate g-secretase as a target for thera-
peutic intervention.
Incorporation of constrained amino acids was con-
ducted to define key interactions with the enzyme. The
inhibition data for the inhibitors 48–52 against g-secre-
tase is presented in Figure 4. Pipecolic acid at the amino
terminus resulted in a dramatic loss of affinity (48: IC50
>2500 nM). On the other hand, introduction of (S)-
tetrahydroisoquinoline-3-carboxylic acid (Tic) at the
carboxy terminus in compound 49 afforded moderate
inhibition (IC50=66 nM). Replacement of either the C-
terminal carbonyl or the central carbonyl with a meth-
ylene moiety lowered activity (compound 50 and 51).
However, the activity could be restored by the incor-
poration of a b-sheet mimetic such as cis-aminoindanol
(compound 52).16
References and notes
Although there are potential limitations in the use of a
cell-based assay to determine subtle enzyme–inhibitor
interactions, the relative potency of compounds within
this series provided direction for synthesis of com-
pounds that consistently demonstrated g-secretase clea-
vage inhibition. The situation is more complex for the
demonstration of in vivo efficacy, where many other
parameters can influence the outcome. Oral administra-
tion of a single 200 mmol/kg dose to Tg2576 bAPP-
Swedish transgenic mice17 of compound 32 resulted in
little (15%) or no reduction of Ab levels in the CNS.
This single dose of 200 mmol/kg gave a concentration of
1. (a) Hardy, J.; Higgins, G. A. Science 1992, 256, 184. (b)
Selkoe, D. J. Trends Neurosci. 1993, 16, 403. (c) Selkoe,
D. J. Science 1997, 275, 630.
2. (a) Hardy, J.; Higgins, G. A. Science 1992, 256, 184. (b)
Hardy, J. Ann. Med. 1996, 28, 255. (c) Citron, M.;
Oltersdorf, T.; Haass, C.; Teplow, D. B.; Selkoe, D. J.
Nature 1992, 360, 672. (d) Suzuki, N.; Cheung, T. T.; Cai,
X. D.; Odaka, A.; Otvos, L., Jr.; Eckman, C.; Golde,
T. E.; Younkin, S. G. Science 1994, 264, 1336. (e) Borch-
elt, D. R.; Thinakaran, G.; Eckman, C. B.; Lee, M. K.;
Davenport, F.; Ratovisky, T.; Prada, C. M.; Kim, G.;
Seekins, S.; Yager, D.; Slunt, H. H.; Wang, R.; Seeger,
M.; Levey, A. I.; Gandy, S. E.; Copeland, N. G.; Jenkins,
N. A.; Price, D. L.; Younkin, S. G.; Sisodia, S. S. Neuron
1996, 17, 1005.
3. (a) Glenner, G. G.; Wong, C. W. Biochem. Biophys. Res.
Commun. 1994, 120, 885. Selkoe, D. J. J. Neuropathol.
Exp. Neurol. 1984, 53, 438. (b) Schenk, D. B.; Rydel,
R. E.; May, P.; Little, S.; Panetta, J.; Lieburburg, I.;
Sinha, S. J. Med. Chem. 1995, 38, 4141. (c) Wolfe, M. S.
J. Med. Chem. 2001, 44, 2039.
4. (a) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.;
Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.;
Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.;
Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.;
Curran, E.; Burgess, T.; Louis, J.-C.; Collins, F.; Treanor,
J.; Rogers, G.; Citron, M. Science 1999, 286, 735. (b)
Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Cac-
cavello, R.; Davis, D.; Doan, M.; Dovey, H. F.; Frigon,
N.; Hong, J.; Jacobson-Croak, K.; Keim, P.; Knops, J.;
Lieberberg, I.; Power, M.; Tan, H.; Tatsuno, G.; Tung, J.;
Schenk, D.; Zhao, J.; McConlogue, L.; Gurney, M. E.
Nature 1999, 402, 537. (c) Yan, R.; Bienkowski, M. J.;
Shuck, M. E.; Miao, H.; Tory, M. C.; Pauley, A. M.;
Brashier, J. R.; Stratman, N. C.; Mathews, W. R.; Buhl,
A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.;
Heinrikson, R. L.; Gurney, M. E. Nature 1999, 402, 533.
(d) Wolfe, M. S.; Xia, W.; Ostaszewski, B. L.; Diehl, T. S.;
Kimberly, W. T.; Selkoe, D. J. Nature 1998, 398, 513. (e)
De Strooper, B.; Saftig, P.; Craessaerts, K.; Vander-
Figure 4. Constrained inhibitors of g-secretase.

C. V. C. Prasad et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1917–1921
1921
stichele, H.; Guhde, G.; Annaert, W.; Von Figura, K.;
Van Leuven, F. Nature 1999, 391, 387. (f) Tsai, J.-Y.;
Wolfe, M. S.; Xia, W. Curr. Med. Chem. 2002, 9, 1087.
5. Citron, M.; Diehl, T.; Gordon, G.; Biere, A. L.; Seubert,
P.; Selkoe, D. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
13170.
6. (a) Felsenstein, K.; Smith, D. W.; Poss, M. A.; Chaturve-
dula, C.; Sloan, C. P. U.S. Patent 5,703,129, 1997; (b)
Rishton, G. M.; Retz, D. M.; Tempest, P. A.; Novotny,
J.; Kahn, S.; Treanor, J. J. S.; Lile, J. D.; Citron, M. J.
Med. Chem. 2000, 43, 2297. (c) Nadin, A.; Owens, A. P.;
Castro, J. L.; Harrison, T.; Shearman, M. S. Bioorg. Med.
Chem. Lett. 2003, 13, 37. (d) Owens, A. P.; Nadin, A.;
Talbot, A. C.; Clarke, E. E.; Harrison, T.; Lewis, H. D.;
Reilly, M.; Wrigley, J. D. J.; Castro, J. L. Bioorg. Med.
Chem. Lett. 2003, 13, 4143.
7. Dovey, H. F. V.; John, V.; Anderson, J. P.; Chen, L. Z.;
de Saint, A.; Fang, L. Y.; Freedman, S. B.; Folmer, B.;
Goldbach, E.; Holsztynska, E. J.; Hu, K. L.; Johnson-
Wood, K. L.; Kennedy, S. L.; Kholodenko, D.; Knops,
J. E.; Latimer, L. H.; Lee, M.; Liao, Z.; Lieberburg, I. M.;
Motter, R. N.; Mutter, L. C.; Nietz, J.; Quinn, K. P.;
Sacchi, K. L.; Seubert, P. A.; Shopp, G. M.; Thorsett,
E. D.; Tung, J. S.; Wu, J.; Yang, S.; Yin, C. T.; Schenk,
D. B.; May, P. C.; Altstiel, L. D.; Bender, M. H.; Boggs,
L. N.; Britton, T. C.; Clemens, J. C.; Czilli, D. L.; Dieck-
man-McGinty, D. K.; Droste, J. J.; Fuson, K. S.; Gitter,
B. D.; Hyslop, P. A.; Johnstone, E. M.; Li, W.-Y; Little,
S. P.; Mabry, T. E.; Miller, F. D.; Ni, B.; Nissen, J. S.;
Porter, W. J.; Potts, B. D.; Reel, J. K.; Stephenson, D.;
Su, Y.; Shipley, L. A.; Whitesitt, C. A.; Yin, T.; Audia,
J. E. J. Neurochem. 2001, 76, 173.
ium, Joklik Modified, 5% heat inactivated calf serum, 1%
MEM Non-Essential Amino Acids, 1% L-Glutamine,
1% Penicillin/Streptomycin) in suspension. Cells were
harvested by centrifugation and washed in ice cold PBS.
Hypotonic buffer HB (10 mM HEPES pH 7.5–7.9, 10
mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF
or Pefabloc) was added and cells carefully resuspended
and washed. Cells were collected by centrifugation, resus-
pended carefully in HB and incubated on ice for 10 min to
swell. Cells were dounced and the homogenate was cen-
trifuged at low speed (1000gÂ10 min). Supernatant was
added to ice cold 10x TRIS buffered saline. The post-
nuclear supernatant was recentrifuged 12,000gÂ30 min.
Â4 ꢀC and the pellet was resuspended in glycerol (20%)/
HEPES (0.02M), leupeptin (10 mg/mL), 1,10 phenanthro-
line. Membrane suspensions were flash frozen and stored
at À80ꢀ C. For an assay the crude membrane fraction (2–
6 mg/mL) was diluted 10-fold in ice-cold wash buffer (50
mM HEPES, pH 7.4, NaCl (1M), 10% glycerol) contain-
ing protease inhibitors leupeptin (10 mg/mL), 1,10 phe-
nanthroline (1 mM) and collected by centrifugation.
Washed membranes were reconstituted in reaction buffer
(50 mM HEPES, pH 7.4, 10% glycerol; 30 mL/assay) and
used at 45 mg/well. Test compounds were diluted to 40x
the highest final concentration desired in 100% DMSO
and diluted serially 1 to 3. Diluted drug was added to each
reaction (final DMSO concentration of 1%). The reaction
(40 mL) was incubated at 37 ꢀC for 90 min. Ice cold PBS/
1%BSA (120 mL) was added to the reaction and the mix-
ture was vortexed briefly. The plate was centrifuged at
6000;g at 4 ꢀC for 45 min. Two 50 mL aliquots of the
supernatant were removed to a microtiter plate for assay
by ELISA as described above.
8. Wolfe, M. S.; Citron, M.; Diehl, T. S.; Xia, W.; Donkor,
I. O.; Selkoe, D. J. J. Med. Chem. 1998, 41, 6.
14. Bodansky, M.; Bodansky, A. The Practise of Peptide
Synthesis; Springer-Verlag, 1984.
15. Conradi, R. A.; Hilgers, A. R.; Ho, N. F. H.; Burton, P. S.
Pharmaceutical Research 1992, 9, 435.
16. Lyle, T. A.; Wiscount, C. M.; Guare, J. P.; Thompson,
W. J.; Anderson, P. S.; Darke, P. L.; Zugay, J. A.; Emini,
E. A.; Schleif, W. A.; Quintero, J. C.; Dixion, R. A. F.;
Sigal, I. S.; Huff, J. R. J. Med. Chem. 1991, 34, 1228.
17. Hsiao, K.; Chapman, R.; Nilsen, S.; Eckman, C.;
Harigaya, Y.; Younkin, S.; Yang, R.; Cole, G. Science
1996, 274, 99.
9. H4 human neuroglioma cells expressing HPLAPÂ
bAPP164SFAD were grown in high glucose (4.5 g/l)
DMEM (Invitrogen) media supplemented with 10% FBS,
100 mg/mL pen-strep, 2 mM glutamine and 100 mg/mL
geneticin. Cells were aliquoted into a 96-well plate, and
after attachment the medium was replaced with Ultra-
culture (Whittaker Bioproducts) containing individual
compounds of interest (final DMSO concentration of 1%.
After an overnight incubation the conditioned medium
was removed and evaluated for the presence of Ab in a
sandwich ELISA using a monoclonal C-terminal Ab40
specific capture antibody and an HRP labeled monoclonal
antibody to the N-terminus of Ab for detection. The
endpoint measurement of Ab1-40 level was developed
using TMB reagent followed by the addition of 1M
phosphoric acid. The plates were read at 450 nm.
18. All compounds gave satisfactory spectroscopic data con-
sistent with the proposed structures. Data for 5: IR (KBr):
3294, 3065, 2925, 2870, 1631, 1553, 1495, 1449, 1367,
1254, 696 cmÀ1 1H NMR (CDCl3) d 7.4–7.2 (m, 5H),
;
6.6–6.4 (m, 2H), 5.85–5.75 (m, 1H), 5.0–4.9 (m, 1H), 4.5–
4.4 (m, 1H), 3.6 (s, 2H), 3.45–3.15 (m, 2H), 2.9 (m, 3H),
1.9–1.8 (m, 1H), 1.8–0.8 (m, 27H); MS (ES), 470 (M-H)À;
Data for 26: IR (KBr): 3282, 3055, 2946, 2830, 1636,
10. Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.;
Smith, D. W.; Pak, R. Isolation of a functionally active
g-secretase presenilin 1 complex and fluorescence assay
for g-secretase activity and inhibitors. PCT Int. Appl.
2001, WO 01/75435 A2.
1558, 1490, 1459, 1373, 1264, 694 cmÀ1 1H NMR
;
(300 MHz, CDCl3) d 6.84–6.78 (m, 3H), 6.73–6.66 (m,
1H), 6.44–6.37 (m, 1H), 4.97–4.92 (m, 1H), 4.53–4.46 (m,
1H), 3.51–3.39 (s, 2H), 3.33–3.20 (m, 2H), 3.02–2.90 (s,
3H), 1.89–0.86 (m, 29H); MS (ESI) 508 (M+H); Data for
32: IR (film): 3285, 2924, 2852, 1679, 1630, 1519, 1449,
11. Zhang, L.; Song, L.; Terracina, G.; Liu, Y.; Pramanik, B.;
Parker, E. Biochemistry 2001, 40, 5049.
12. 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.
13. Human HeLa cells stably expressing HPLAPÂ
b-APPSwedish164 were grown (Minimum Essential Med-
1
1277, 1061, 729, 696 cmÀ1; H NMR (CDCl3) d 7.4–7.2
(m, 5H), 6.85–6.6 (m, 2H), 5.1 (s, 1H), 4.95–4.80 (m, 1H),
4.4–4.3 (m, 1H), 3.4–2.8 (m, 6H), 1.9–1.75 (m, 1H), 1.70–
0.70 (m, 28H); MS (ES), 488 (M+H)+.