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Title:
INHIBITORS OF β-SECRETASE, AND THEIR USE FOR THE PREVENTION OR TREATMENT OF ALZHEIMER’S DISEASE OR MILD COGNITIVE IMPAIRMENT
Document Type and Number:
WIPO Patent Application WO/2004/041211
Kind Code:
A2
Abstract:
One aspect of the present invention relates to urea-containing compounds. In certain embodiments, the urea-containing compounds of the present invention comprise a heteroaromatic moiety. Another aspect of the present invention relates to a method of inhibiting a beta-secretase using a urea-containing compound of the present invention. A third aspect of the present invention relates to a method of treating Alzheimer’s Disease or mild cognitive impairment using a urea-containing compound of the present invention.

Inventors:
KOZIKOWSKI ALAN P (US)
AISEN PAUL S (US)
PETUKHOV PAVEL (US)
Application Number:
PCT/US2003/035219
Publication Date:
May 21, 2004
Filing Date:
November 03, 2003
Export Citation:
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Assignee:
UNIV GEORGETOWN (US)
KOZIKOWSKI ALAN P (US)
AISEN PAUL S (US)
PETUKHOV PAVEL (US)
International Classes:
A61K31/34; A61K31/38; A61K31/40; C07C233/00; C07C275/14; C07C275/16; C07C275/20; C07C275/24; C07D207/10; C07D307/02; C07D307/66; C07D333/36; C07D405/00; A61K; (IPC1-7): A61K/
Foreign References:
US6531610B12003-03-11
Attorney, Agent or Firm:
Gordon, Dana M. (Foley Hoag LLP 155 Seaport Boulevar, Boston MA, US)
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Claims:
WHAT IS CLAIMED IS:
1. A compound represented by formula I : wherein: R is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, alkenyl, and alkynyl; Ri, R2, R3, and R4 are, independently of each other, selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl ; wherein the aromatic ring in aryl, heteroaryl, aralkyl, and heteroaralkyl may be substituted 1 to 4 times with Xl ; and Xi is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, hydroxy, alkoxy, nitro, amido, amino, halide, methylalcohol, trifluoromethyl, and formamide.
2. The compound of claim 1, wherein R is H.
3. The compound of claim 1, wherein Ri is heteroaralkyl.
4. The compound of claim 1, wherein R2 is aralkyl.
5. The compound of claim 1, wherein R3 is aralkyl.
6. The compound of claim 1, wherein R4 is aralkyl.
7. The compound of claim 1, wherein R is H, Rl is heteroaralkyl, Ra is aralkyl, R3 is aralkyl, and R4 is aralkyl.
8. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is pethoxybenzyl, and R4 is mnitrobenzyl.
9. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oethoxybenzyl, and R4 is mnitrobenzyl.
10. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 ispCONH2benzyl, and R4 is onitrobenzyl.
11. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oCONH2benzyl, and R4 is maminobenzyl.
12. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 is oiodobenzyl, and R4 is mNHCHObenzyl.
13. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is ofluorobenzyl, and R4 is pnitrobenzyl.
14. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 is oiodobenzyl, and R4 is oethoxybenzyl.
15. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is pNHCHObenzyl, and R4 is mCONH2benzyl.
16. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 isphydroxymethylbenzyl, and R4 is onitrobenzyl.
17. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 isphydroxymethylbenzyl, and R4 is methoxybenzyl.
18. The compound of claim 7, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is otrifluoromethylbenzyl, and R4 is methoxybenzyl.
19. The compound of claim 7, wherein Rl isCH2imidazolyl, Ra is phydroxybenzyl, R3 is otrifluoromethylbenzyl, and R4 is mfluorobenzyl.
20. A compound represented by formula II: wherein: R is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, alkenyl, and alkynyl; Rl and R2 are, independently of each other, selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl ; wherein the aromatic ring in aryl, heteroaryl, aralkyl, and heteroaralkyl may be substituted 1 to 4 times with Xi ; Xi is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, hydroxy, alkoxy, nitro, amido, amino, halide, methylalcohol, trifluoromethyl, and formamide ; and X2 is selected from the group consisting of O, S, NR, and C (R) 2.
21. The compound of claim 20, wherein R is H.
22. The compound of claim 20, wherein Rl is aralkyl.
23. The compound of claim 20, wherein R2 is aryl or aralkyl.
24. The compound of claim 20, wherein X2 is S.
25. The compound of claim 20, wherein X2 is O.
26. The compound of claim 20, wherein R is H, Ri is aralkyl, Ra is aryl or aralkyl, and X2 is O or S.
27. The compound of claim 26, wherein Rl is (CH2) 3PhpI, R2 is pbiphenyl, and X2 is S.
28. The compound of claim 26, wherein R1 is (CH2)3PhpF, R2 is pbiphenyl, and X2 is S.
29. The compound of claim 26, wherein R1 is (CH2)4PhpCH2OH, R2 is pbiphenyl, and X2 is S.
30. The compound of claim 26, wherein R1 is(CH2) 3PhpI, R2 is benzyl, and X2 is O.
31. The compound of claim 26, wherein Rlis(CH2) 4PhpNO2, R2 is pbiphenyl, and X2 is S.
32. The compound of claim 26, wherein R1 is (CH2)3PhpCH2OH, R2 is benzyl, and X2 is 0.
33. The compound of claim 26, wherein Rlis(CH2) 3PhpCF3, R2 ispbiphenyl, and X2 is S.
34. The compound of claim 26, wherein R1 is (CH2)5PhpCH2OH, R2 is benzyl, and X2 is 0.
35. The compound of claim 26, wherein R1 is (CH2)3PhpF, R2 is benzyl, and X2 is O.
36. The compound of claim 26, wherein Rlis(CH2) 3PhpF, R2is benzyl, and X2 is S.
37. The compound of claim 26, wherein R1 is (CH2)5PhpCF3, R2 is benzyl, and X2 is 0.
38. The compound of claim 26, wherein Ri is(CH2) 3Ph, R2 is benzyl, and X2 is S.
39. The compound of claim 26, wherein Ri is(CH2) 3PhpI, R2 is benzyl, and X2 is S.
40. The compound of claim 26, wherein R1 is(CH2) 3PhpCF3, R2 is benzyl, and X2 is S.
41. A method of inhibiting Psecretase comprising the step of contacting (3secretase with an effective amount of a compound of claim 1.
42. The method of claim 41, wherein R is H.
43. The method of claim 41, wherein R1 is heteroaralkyl.
44. The method of claim 41, wherein R2 is aralkyl.
45. The method of claim 41, wherein R3 is aralkyl.
46. The method of claim 41, wherein R4 is aralkyl.
47. The method of claim 41, wherein R is H, Rl is heteroaralkyl, R2 is aralkyl, R3 is aralkyl, and R4 is aralkyl.
48. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is pethoxybenzyl, and R4 is mnitrobenzyl.
49. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oethoxybenzyl, and R4 is mnitrobenzyl.
50. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is pCONH2benzyl, and R4 is onitrobenzyl.
51. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oCONH2benzyl, and R4 is maminobenzyl.
52. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oiodobenzyl, and R4 is mNHCHObenzyl.
53. The method of claim 47, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 is ofluorobenzyl, and R4 ispnitrobenzyl.
54. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oiodobenzyl, and R4 is oethoxybenzyl.
55. The method of claim 47, wherein Ri isCH2imidazolyl, R2 is phydroxybenzyl, R3 is pNHCHObenzyl, and R4 is mCONH2benzyl.
56. The method of claim 47, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is phydroxymethylbenzyl, and R4 is onitrobenzyl.
57. The method of claim 47, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 isphydroxymethylbenzyl, and R4 is methoxybenzyl.
58. The method of claim 47, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 is otrifluoromethylbenzyl, and R4 is methoxybenzyl.
59. The method of claim 47, wherein Rl isCH2imidazolyl, R2 is phydroxybenzyl, R3 is otrifluoromethylbenzyl, and R4 is mfluorobenzyl.
60. A method of inhibiting Psecretase comprising the step of contacting 3secretase with an effective amount of a compound of claim 20.
61. The method of claim 60, wherein R is H.
62. The method of claim 60, wherein Ri is aralkyl.
63. The method of claim 60, wherein R2 is aryl or aralkyl.
64. The method of claim 60, wherein Xa is S.
65. The method of claim 60, wherein X2 is O.
66. The method of claim 60, wherein R is H, Rl is aralkyl, R2 is aryl or aralkyl, and X2 is O or S.
67. The method of claim 66, wherein R1 is(CH2) 3PhpI, R2 is pbiphenyl, and X2 is S.
68. The method of claim 66, wherein R1 is (CH2) 3PhpF, R2 is pbiphenyl, and X2 is S.
69. The method of claim 66, wherein R1 is (CH2) 4PhpCH20H, R2 is pbiphenyl, and X2 is S.
70. The method of claim 66, wherein Ri is(CH2) 3PhpI, R2 is benzyl, and X2 is O.
71. The method of claim 66, wherein R1 is (CH2)4PhpNO2, R2 is pbiphenyl, and X2 is S.
72. The method of claim 66, wherein R1 is (CH2) 3PhpCH20H, R2 is benzyl, and X2 is 0.
73. The method of claim 66, wherein Ri is (CH2)3PhpCF3, R2 is pbiphenyl, and X2 is S.
74. The method of claim 66, wherein R1 is (CH2)5PhpCH2OH, R2 is benzyl, and X2 is 0.
75. The method of claim 66, wherein R1 is (CH2) 3PhpF, R2 is benzyl, and X2 is O.
76. The method of claim 66, wherein R1 is(CH2) 3PhpF, R2 is benzyl, and X2 is S.
77. The method of claim 66, wherein R1 is(CH2) sPhpCF3, R2 is benzyl, and X2 is O.
78. The method of claim 66, wherein R1 is (CH2) 3Ph, R2 is benzyl, and X2 is S.
79. The method of claim 66, wherein R1 is(CH2) 3PhpI, R2 is benzyl, and X2 is S.
80. The method of claim 66, wherein R1 is (CH2) 3PhpCF3, R2 is benzyl, and X2 is S.
81. A method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of claim 1.
82. The method of claim 81, wherein R is H.
83. The method of claim 81, wherein R1 is heteroaralkyl.
84. The method of claim 81, wherein R2 is aralkyl.
85. The method of claim 81, wherein R3 is aralkyl.
86. The method of claim 81, wherein R4 is aralkyl.
87. The method of claim 81, wherein R is H, Rl is heteroaralkyl, R2 is aralkyl, R3 is aralkyl, and R4 is aralkyl.
88. The method of claim 87, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 ispethoxybenzyl, and R4 is mnitrobenzyl.
89. The method of claim 87, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oethoxybenzyl, and R4 is mnitrobenzyl.
90. The method of claim 87, wherein Ri isCH2imidazolyl, R2 isphydroxybenzyl, R3 is pCONH2benzyl, and R4 is onitrobenzyl.
91. The method of claim 87, wherein Rl isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oCONH2benzyl, and R4 is maminobenzyl.
92. The method of claim 87, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oiodobenzyl, and R4 is mNHCHObenzyl.
93. The method of claim 87, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 is ofluorobenzyl, and R4 is pnitrobenzyl.
94. The method of claim 87, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 is oiodobenzyl, and R4 is oethoxybenzyl.
95. The method of claim 87, wherein R1 isCH2imidazolyl, R2 is phydroxybenzyl, R3 ispNHCHObenzyl, and R4 is mCONH2benzyl.
96. The method of claim 87, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 isphydroxymethylbenzyl, and R4 is onitrobenzyl.
97. The method of claim 87, wherein R1 is CH2imidazolyl, R2 is phydroxybenzyl, R3 isphydroxymethylbenzyl, and R4 is methoxybenzyl.
98. The method of claim 87, wherein R1 isCH2imidazolyl, R2 isphydroxybenzyl, R3 is otrifluoromethylbenzyl, and R4 is methoxybenzyl.
99. The method of claim 87, wherein R1 is CH2imidazolyl, R2 is phydroxybenzyl, R3 is otrifluoromethylbenzyl, and R4 is mfluorobenzyl.
100. A method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of claim 20.
101. The method of claim 100, wherein R is H.
102. The method of claim 100, wherein R1 is aralkyl.
103. The method of claim 100, wherein R2 is aryl or aralkyl.
104. The method of claim 100, wherein X2 is S.
105. The method of claim 100, wherein X2 is O.
106. The method of claim 100, wherein R is H, Rl is aralkyl, R2 is aryl or aralkyl, and X2 isOorS.
107. The method of claim 106, wherein R1 is (CH2) 3PhpI, R2 is pbiphenyl, and X2 is S.
108. The method of claim 106, wherein R1 is (CH2) 3PhpF, R2 is pbiphenyl, and X2 is S.
109. The method of claim 106, wherein R1 is(CH2) 4PhpCH2OH, R2 ispbiphenyl, and X2 is S.
110. The method of claim 106, wherein Ri is (CH2)3PhpI, R2 is benzyl, and X2 is O.
111. The method of claim 106, wherein Ri is (CH2)4PhpNO2, R2 is pbiphenyl, and X2 is S.
112. The method of claim 106, wherein R1 is(CH2) 3PhpCH2OH, R2 is benzyl, and X2 is O.
113. The method of claim 106, wherein R1 is (CH2)3PhpCF3, R2 is pbiphenyl, and X2 is S.
114. The method of claim 106, wherein R1 is (CH2)5PhpCH2OH, R2 is benzyl, and X2 is O.
115. The method of claim 106, wherein R1 is (CH2) 3PhpF, R2 is benzyl, and X2 is O.
116. The method of claim 106, wherein R1 is (CH2) 3PhpF, R2 is benzyl, and X2 is S.
117. The method of claim 106, wherein R1 is (CH2) 5PhpCF3, R2 is benzyl, and X2 is O.
118. The method of claim 106, wherein R1 is (CH2)3Ph, R2 is benzyl, and X2 is S.
119. The method of claim 106, wherein R1 is (CH2)3PhpI, R2 is benzyl, and X2 is S.
120. The method of claim 106, wherein R1 is (CH2)3PhpCF3, R2 is benzyl, and X2 is S.
121. The method of any of claims 81120, wherein said compound is administered intranasally.
Description:
Inhibitors of ß-Secretase, and Their Use for the Prevention or Treatment of Alzheimer* Disease orMild Cogniíive Impairsnerzt Background of Invention Alzheimer's disease (AD) is among the most important health care problems in the world. The past decade has seen the adoption of the first class of medications, the cholinesterase inhibitors, effective in improving cognitive symptoms in AD. These drugs provide symptomatic relief; effective disease-modifying therapy remains a major, elusive goal. S ubstantial efforts have been made to apply findings from 1 aboratory research, as well as genetic and epidemiologic studies, to the identification of potential strategies for influencing AD pathology.

The two pathologic hallmarks of AD are the extracellular neuritic amyloid plaques and the intracellular neurofibrillary tangles. While there has been great debate over the past few decades on the relative importance of plaques versus tangles, in recent years many investigators have focused on the former. Perhaps the most convincing evidence that generation of the amyloid B peptide (AB), which comprises the core of the amyloid plaque, and is the inciting event in AD pathophysiology, is the demonstration that each of the genetic causes of familial autosomal dominant AD (FAD) influences cleavage of the amyloid precursor protein (APP) to release AB. Mutations of three distinct genes can cause FAD, the genes coding for APP, and presenilin (PS) 1 and 2. Mutations of the APP gene increase the susceptibility of the protein to cleavage (by B-and y-secretases) at the two ends of the AB sequence to release the amyloidogenic fragment. Mutations of PS-1 and PS- 2 are associated with increased activity of γ-secretase. The inescapable implication is that overproduction of AB is the cause of FAD. Since FAD and sporadic AD have the same neuropathology (though FAD h as a m uch e arlier a ge o f onset), it also se ems 1 ikely t hat accumulation of AB is causative in sporadic AD.

If this formulation is correct, then the most direct method of interrupting the AD process is to inhibit (3-or y-secretase. Inhibition of y-secretase may be problematic, as this enzyme apparently serves essential functions such as cleavage of the transmembrane protein Notch. By contrast, 0-secretase activity does not appear to be essential for the health of the organism. Studies in genetically altered mice suggest that eliminating ß- secretase activity prevents the generation of the amyloid peptide without adverse effects.

Thus, an effective inhibitor of (3-secretase activity in brain may represent a safe disease- halting treatment for AD.

The sequence and conformation of P-secretase is now known, and the amino acid sequence and cleavage site of its natural substrate (APP) is also known. The amino acid sequence of the cleavage site of a mutated form of APP with increased affinity for ß- secretase is likewise known. Utilizing this information, computer technology was used to design optimal, specific inhibitors of P-secretase, with small molecular weight to allow penetration into brain. Initial efforts have yielded compounds that compare favorably with any in the published literature. The next step in the process is screening the best compounds for selective (3-secretase inhibition using cell culture systems. The last step is testing the few most promising (3-secretase inhibitors for activity and toxicity in transgenic mice with AD-type amyloid deposition.

Demonstration that each of the genetic causes of AD (APP mutations, PS-1 and PS- 2 mutations, Down syndrome) is associated with increased cleavage of APP into Ap is strong evidence that this cleavage is the cause of AD in these instances, and is presumably the pivotal step in the pathophysiology of sporadic AD. Selkoe DJ, Ann N Y Acad Sci, 2000, 924 : 17-25. Evidence suggests that Ap generation results in neurodegeneration by several mechanisms, including direct neurotoxicity, oxidative stress, and induction of destructive inflammatory processes. If generation of AP is indeed the inciting event, then inhibition of (3-or y-secretase will halt the disease process.

Since mutations of PS-1 and PS-2 that cause familial AD increase Ap generation be increasing the activity of y-secretase, development of y-secretase inhibitors has been pursued. Selkoe DJ, Nature, 1999,399 : A23-31. However, this strategy has not yet been fruitful. Evidence that-secretase is essential in Notch processing suggests that its inhibition may not be feasible. Hartmann D, Tournoy J, Saftig P, Annaert W, De Strooper B, J Mol Neurosci, 2001,17 : 171-181 ; Geling A, Steiner H, Willem M, Bally-Cuif L, Haass C, EMBO Rep, 2002,3 : 688-694. Since (3-secretase cleavage appears to be the rate-limiting step in generation of Ap, and since knocking out (3-secretase blocks Ap production without untoward effects in mice, P-secretase inhibition emerges as the most promising strategy for the development of disease-modifying therapy for AD. Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman S, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME,

Sinha S, Svensson KA, Tatsuno G, Tintrup H, Wijsman J, Wright S, McConlogue L, Hum Mol Genet, 2001,10 : 1317-1324; Vassar R., J Mol Neurosci, 2001,17 : 157-170.

Sunzmary of the Iiiventioii In one embodiment, the present invention relates to a compound of formula 1 : wherein: R is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, heteroaryl, alkenyl, alkynyl, aralkyl, and heteroaralkyl ; Ri, R2, R3, and R4 are, independently of each other, selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl ; wherein the aromatic ring in aryl, heteroaryl, aralkyl, and heteroaralkyl may be substituted 1 to 4 times with Xl ; and Xi is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, hydroxy, alkoxy, nitro, amido, amino, halide, methylalcohol, trifluoromethyl, and formamide.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein R is H.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is heteroaralkyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Ra is aralkyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein R3 is aralkyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein R4 is aralkyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein R is H, Rl is heteroaralkyl, R2 is aralkyl, R3 is aralkyl, and R4 is aralkyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-ethoxybenzyl, and R4 is m-nitrobenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-ethoxybenzyl, and R4 is m-nitrobenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-CONH2-benzyl, and R4 is o-nitrobenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-CONH2-benzyl, and R4 is m-aminobenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Ri is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-iodobenzyl, and R4 is m-NHCHO-benzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-fluorobenzyl, and R4 is p-nitrobenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-iodobenzyl, and R4 is o-ethoxybenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-NHCHO-benzyl, and R4 is m-CONH2-benzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-hydroxymethylbenzyl, and R4 is o-nitrobenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-hydroxymethylbenzyl, and R4 is in-ethoxybenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-trifluoromethylbenzyl, and R4 is m-ethoxybenzyl.

In a further embodiment, the present invention relates to a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-trifluoromethylbenzyl, and R4 is m-fluorobenzyl.

In another embodiment, the present invention relates to a compound represented by formula II : wherein: R is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, heteroaryl, alkenyl, alkynyl, aralkyl, heteroaralkyl ; Rl and R2 are, independently of each other, selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl ; wherein the aromatic ring in aryl, heteroaryl, aralkyl, and heteroaralkyl may be substituted 1 to 4 times with Xl ; Xi is, independently for each occurrence, selected from the group consisting of H, alkyl, aryl, hydroxy, alkoxy, nitro, amido, amino, halide, methylalcohol, trifluoromethyl, and formamide ; and X2 is selected from the group consisting of O, S, NR, and C (R) 2.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R is H.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein Rl is aralkyl.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R2 is aryl or aralkyl.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein X2 is O.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R is H, Rl is aralkyl, R2 is aryl or aralkyl, and X2 is O or S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is-(CH2) 3Ph-p-I, R2 isp-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is-(CH2) 3Ph-p-F, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is- (CH2) 4Ph-p-CH2OH, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is-(CH2)3Ph-p-I, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is- (CH2) 4Ph-p-N02, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph-p-CH2OH, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is- (CH2) 3Ph-p-CF3, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is-(CH2) sPh-p-CH2OH, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph-p-F, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is- (CHa) 3Ph p-F, R is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein Ri is-(CH2) sPh-p-CF3, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein Ri is-(CH2) 3Ph-p-I, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a compound of formula II and the attendant definitions, wherein Ri is -(CH2)3Ph-p-CF3, R2 is benzyl, and X2 is S.

In another embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R is H.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is heteroaralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R2 is aralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R3 is aralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R4 is aralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R is H, Rl is heteroaralkyl, R2 is aralkyl, R3 is aralkyl, and R4 is aralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-ethoxybenzyl, and R4 is m-nitrobenzyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a

compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-ethoxybenzyl, and R4 is m-nitrobenzyl.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-CONH2-benzyl, and R4 is o-nitrobenzyl.

In a further embodiment, the present invention relates to a method of inhibiting p- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-CONH2-benzyl, and R4 is m-aminobenzyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-iodobenzyl, and R4 is m-NHCHO-benzyl.

In a further embodiment, the present invention relates to a method of inhibiting p- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-fluorobenzyl, and R4 is p-nitrobenzyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-iodobenzyl, and R4 is o-ethoxybenzyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-NHCHO-benzyl, and R4 is m-CONH2-benzyl.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-hydroxymethylbenzyl, and R4 is o-nitrobenzyl.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a

compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 isp-hydroxymethylbenzyl, and R4 is m-ethoxybenzyl.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-trifluoromethylbenzyl, and R4 is m-ethoxybenzyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting 3-secretase with an effective amount of a compound of formula I and the attendant definitions, wherein R1 is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-trifluoromethylbenzyl, and R4 is m-fluorobenzyl.

In another embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R is H.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is aralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R2 is aryl or aralkyl.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein X2 is O.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting ß-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R is H, Rl is aralkyl, R2 is aryl or aralkyl, and X2 is O or S.

In a further embodiment, the present invention relates to a method of inhibiting p- secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph-p-I, R2 is p- biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein Ri is -(CH2)3Ph-p-F, R2 is p- biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is- (CH2) 4Ph-p-CH20H, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph-p-I, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is -(CH2)4Ph-p-NO2, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph-p-CH2OH, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is-(CH2) 3Ph-p-CF3, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein Ri is -(CH2)5Ph-p-CH2OH, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of inhibiting ß- secretase comprising the step of contacting P-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein Ri is- (CH2) 3Ph-p-F, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is- (CH2) 3Ph-p-F, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is -(CH2)5Ph-p-CF3, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is-(CH2) 3Ph, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is- (CH2) 3Ph-p-I, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a method of inhibiting secretase comprising the step of contacting (3-secretase with an effective amount of a compound of formula II and the attendant definitions, wherein R1 is -(CH2)3Ph-p-CF3, R2 is benzyl, and X2 is S.

In another embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein R is H.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Ri is heteroaralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein R2 is aralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein R3 is aralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein R4 is aralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein R is H, Rl is heteroaralkyl, R2 is aralkyl, R3 is aralkyl, and R4 is aralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein R, is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-ethoxybenzyl, and R4is m-nitrobenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Rlis-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-ethoxybenzyl, and R4 is iti-nitrobenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of

administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Ri is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-CONH2-benzyl, and R4 is o-nitrobenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Ri is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-CONH2-benzyl, and R4 is m-aminobenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-iodobenzyl, and R4 is m-NHCHO-benzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-fluorobenzyl, and R4 isp-nitrobenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-iodobenzyl, and R4 is o-ethoxybenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein RI is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-NHCHO-benzyl, and R4 is m-CONH2-benzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Ri is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-hydroxymethylbenzyl, and R4 is o-nitrobenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein RI is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is p-hydroxymethylbenzyl, and R4 is m-ethoxybenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Ri is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-trifluoromethylbenzyl, and R4 is m-ethoxybenzyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula I and the attendant definitions, wherein Rl is-CH2-imidazolyl, R2 is p-hydroxybenzyl, R3 is o-trifluoromethylbenzyl, and R4 is m-fluorobenzyl.

In another embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein R is H.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is aralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein R2 is aryl or aralkyl.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of

administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Xa is O.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein R is H, Rl is aralkyl, R2 is aryl or aralkyl, and X2 is OorS.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is-(CH2) 3Ph-p-I, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph-p-F, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 4Ph-p-CH20H, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is-(CH2) 3Ph-p-I, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 4Ph-p-N02, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is-(CH2) 3Ph-p-CH2OH, R2 is benzyl, and X2 is 0.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph-p-CF3, R2 is p-biphenyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is-(CH2) 5Ph-p-CH2OH, R2 is benzyl, and X2 is 0.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph-p-F, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph-p-F, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is-(CH2) sPh-p-CF3, R2 is benzyl, and X2 is O.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of

administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph-p-I, R2 is benzyl, and X2 is S.

In a further embodiment, the present invention relates to a method of treating Alzheimer's Disease or mild cognitive impairment in a subject comprising the step of administering to said subject a therapeutically effective amount of a compound of formula II and the attendant definitions, wherein Rl is- (CH2) 3Ph-p-CF3, R2 is benzyl, and X2 is S.

Brief Description of the Drawings Figure 1 depicts known OM99-2-based BACE 1 inhibitors.

Figure 2 depicts a 1, 3-diaminopropan-2-ol scaffold for BACE 1 inhibitors and two new classes of ligands based on this scaffold.

Figure 3 depicts a general view of BACE 1, binding site, and ligands OM99-2 and the best ligand from DAPOL1 library (upper). Overlay of the best ligand from DAPOL1 library (middle) and DAPOL2 library (bottom) with OM99 original ligand.

Detailed Description of the Invention Definitions For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

The articles"a"and"an"are used herein to refer to one or to more than one (i. e. , to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The terms"comprise"and"comprising"are used in the inclusive, open sense, meaning that additional elements may be included.

The term"including"is used to mean"including but not limited to"."Including" and"including but not limited to"are used interchangeably.

The term"alkyl"refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred

embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e. g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5,6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified,"lower alkyl"as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise,"lower alkenyl"and"lower alkynyl"have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.

The term"aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e. g. , an aromatic or heteroaromatic group).

The terms"alkenyl"and"alkynyl"refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term"aryl"as used herein includes 5-, 6-and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as"aryl heterocycles"or "heteroaromatics. "The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,-CF3,-CN, or the like. The term"aryl"also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are"fused rings") wherein at least one of the rings is aromatic, e. g. , the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1, 3- and 1,4-disubstituted benzenes, respectively. For example, the names 1, 2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms"heterocyclyl"or"heterocyclic group"refer to 3-to 10-membered ring structures, more preferably 3-to 7-membered rings, whose ring structures include one to

four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, azetidine, azepine, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety,-CF3,-CN, or the like.

The terms"polycyclyl"or"polycyclic group"refer to two or more rings (e. g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e. g. , the rings are"fused rings". Rings that are joined through non-adjacent atoms are termed"bridged"rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety,- CF3,-CN, or the like.

The term"carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

As used herein, the term"nitro"means-NO2 ; the term"halogen"designates-F,-Cl, -Br or-I ; the term"sulfhydryl"means-SH ; the term"hydroxyl"means-OH ; and the term "sulfonyl"means-SO2-.

The terms"amine"and"amino"are art-recognized and refer to both unsubstituted and substituted amines, e. g. , a moiety that can be represented by the general formula : wherein Rg, Rl 0 and R'10 each independently represent a group permitted by the rules of valence.

The term"acylamino"is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R9 is as defined above, and R'l 1 represents a hydrogen, an alkyl, an alkenyl or -(CH2) m-Rg, where m and R8 are as defined above.

The term"amido"is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: wherein Rg, Rlo are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.

The term"alkylthio"refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the"alkylthio"moiety is represented by one of-S-alkyl,-S-alkenyl,-S-alkynyl, and-S-(CH2) m-Rg, wherein m and R8 are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.

The term"carbonyl"is art recognized and includes such moieties as can be represented by the general formula: wherein X is a bond or represents an oxygen or a sulfur, and Rl l represents a hydrogen, an alkyl, an alkenyl,-(CH2) m-Rg or a pharmaceutically acceptable salt, R'11 represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-R8, where m and R8 are as defined above.

Where X is an oxygen and Rl l or R'l l is not hydrogen, the formula represents an"ester".

Where X is an oxygen, and Rl 1 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Rl 1 is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R ! 11 is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a"thiolcarbonyl"group. Where X is a sulfur and Rl 1 or R ! 11 is not hydrogen, the formula represents a"thiolester. "Where X is a sulfur and Rl 1 is hydrogen, the formula represents a"thiolcarboxylic acid. "Where X is a sulfur and Rl l'is hydrogen, the formula represents a"thiolformate."On the other hand, where X is a bond, and Rl 1 is not hydrogen, the above formula represents a"ketone"group. Where X is a bond, and Rl 1 is hydrogen, the above formula represents an"aldehyde"group.

The terms"alkoxyl"or"alkoxy"as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An"ether"is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of-O-alkyl,-O- alkenyl,-0-alkynyl,-O- (CH2) m-Rg, where m and R8 are described above.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry ; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.

Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e. g. alkyl, m, n, etc. , when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

It will be understood that"substitution"or"substituted with"includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e. g., which does not spontaneously undergo undesired transformation, such as by rearrangement, cyclization, elimination, etc.

As used herein, the term"substituted"is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

The phrase"protecting group"as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.

Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2"d ed.; Wiley: New York, 1991).

Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis-and trans-isomers, R-and S-enantiomers, diastereomers, (D) -isomers, (L) -isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, it may be isolated using chiral chromatography methods, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group,

such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed. , 1986-87, inside cover.

Experimental Design and Methods Although considerable effort is being expended to identify BACE inhibitors in both industry and academia, it is likely that just as in the case of HIV protease inhibitors, it will be highly valuable to develop several such compounds.

Design and synthesis of candidate BACE1 inhibitors.

Using the available x-ray structure of B ACE 1, we are using structure-based drug design methods to identify potent and selective inhibitors of this key processing enzyme.

The first stage of our work will focus on the screening of chemical databases, de novo/rational drug design using programs like Ludi, and the creation of virtual combinatorial libraries for in silico screening. The best ligands obtained from the 3D database search or de novo/rational drug design methods will be subjected to retrosynthetic analysis in order to identify major components of the ligand molecule, such as a"core"and "sidechains". The resulting cores and sidechains as well as"aspartic protease-compatible" cores and sidechains of known aspartic protease inhibitors (e. g. , published candidate BACE1 inhibitors and HIV protease inhibitors) will be used to generate virtual combinatorial libraries. The newly modeled compounds will then be screened in silico, and those that give excellent consensus scores indicative of their good fit to BACE1 will be synthesized i n o ur 1 aboratories, and then assayed in a c ellular system for their ability t o inhibit the formation of Ap. Compounds that show an ICso of at least 1 u. M in the cell assay will then be refit to the enzyme active site, and further modeling studies performed in order to optimize their interaction with the enzyme. Additionally, the interaction of the compounds with BACE2 will be modeled (structure to be built from that of BACE 1 using homology modeling methods), in order to further refine our inhibitors so as to enhance their selectivity for BACE1. A further round of synthesis and screening will take place in which

inhibitory activity at both BACE1 and BACE2 will be measured. During the computational part o f o ur i nhibitor d esign, Lipinski's rule o f 5 w ill b e u sed a s a filter, t o i ncrease t he likelihood of identifying structures that will cross the blood-brain-barrier. Candidate molecules showing a BACE1 inhibitory activity of at least 50 nM and a selectivity of at least 3-fold for BACE1 over BACE2 will then be ready for testing in the transgenic animal models.

The x-ray crystal structure of BACE1 in complex with the potent peptide-based inhibitor OM99-2 has been published and is available from the Protein Data Bank (1FKN).

OM99-2 shows low nM potency for inhibition of BACE 1 and it contains a non- hydrolyzable Leu-Ala hydroxyethylene dipeptide bioisostere (Figure 1, left). Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC, Tang J, Science, 2000, 290: 150-153. Additional SAR studies conducted by Ghosh et al. have allowed further reduction in size of the inhibitor while maintaining potency (Figure 1, right); however, these modified inhibitors are still of relatively high molecular weight, and still contain five amide bonds. Ghosh AK, Bilcer G, Harwood C, Kawahama R, Shin D, Hussain KA, Hong L, Loy JA, Nguyen C, Koelsch G, Ermolieff J, Tang J, J Med Chem, 2001,44 : 2865-2868; Ghosh AK, Shin DW, Downs D, Koelsch G, Lin XL, Ermolieff J, Tang J, Journal of the American Chemical Society, 2000,122 : 3522-3523. Thus, considerably more research will be required to identify more drug-like molecules that possess appropriate metabolic stability and PK parameters necessary to entering the brain.

As a r esult o f our preliminary structure-based de n ovo d esign, two series o f 1,3- diaminopropan-2-ol (DAPOL) -based ligands emerged as potential BACE1 inhibitors (Figure 2). Two virtual combinatorial libraries consisting of several thousands of compounds were constructed and screened in silico to optimize sidechains in the core molecule. In our studies we used a combination of well known scoring functions (DOCK, FlexX, PMF, Chemscore, GOLD) implemented in the FlexX and CScore modules in Sybyl.

Sybyl (2002) SYBYL In :, 6.8 Edition. 1699 South Hanley Rd. , St. Louis, Missouri, 63144, USA : Tripos Inc. To increase robustness and reliability of the results, a consensus of the above scoring functions was used to choose the best pose and calculate the score for each ligand. It has been reported that a consensus of several independent functions outperforms a single scoring function. Bissantz C, Folkers G, Rognan D, J Med Chem, 2000,43 : 4759-4767; Clark RD, Strizhev A, Leonard JM, Blake JF, Matthew JB, J Mol Graph Model, 2002,20 : 281-295. The best ligands from both virtual combinatorial libraries

are shown in Table 1 and Table 2. Interestingly, compounds from both series overlay reasonably well with OM-99 (Figure 3) suggesting that they will also block the binding of APP to BACE1 (Figure 2).

Table 1. Top 25 structures in virtual combinatorial library DAPOL1. Cmpd Structure PMF MW LogF Cmpd Structure PMF MW JLogP score score j 1. X1 = p-OEt, X2 = m-NO2 -168 746 1.35 13. X1 = nothing, X2 = o-OEt -145 701 1.53 2. X1=p-OEt, X2=m-NO2 -156 746 1.35 14. X1 =p-OEt, X2 = m-I - 145 827 2.70 3. X1 = p-CONH2, X2 = o- -152 745 -0.48 15.X1 = o-OEt, X2 = m-CONH2 -145 744 0. 28 2 4. X1 =o=CONH2, X2 = m- -150 715 -1.21 16. X1 = p=NHCHO, X2 = m-I -144 826 1.27 INH2 5. Xl=o-I, X2=m-NHCHO-149 826 1.27 17. 1 = o-CONH2, X2 = p-NO2 -144 745-1.21 6. Xl=o-F, X2=j ?-N02-149 719 0.98 18. Xl=p-F, X2=p-CF3-144 743 2.12 7. Xl=o-I, X2=o-OEt-147 827 2.70 19. 1 = p-OEt, X2 = m-CONH2 -144 744 0.28 8. X1 = p-NHCHO, X2 = m- -147 743 -1.16 20.X1 = o-I, X2 = p-NO2 -143 828 1.94 CON2 9. X1 = p-CH2OH, X2 = o- -147 732 0.27 21.X1 = o-NH2, X2 = p-NHCHO -143 715 -0.82 NO2 | 10. X1 = p-CH2OH, X2 = m- -147 731 1.02 22. 768 1. 06 OEt .X1 = p-NHCHO, X2 = m-CF3 -142 ll. X1 = o-CF3, X2 = m-OEt -146 769 2.49 23. X1 = o-F, X2 = p-CF3 -142 743 2.12 12. Xl=o-CF3, X2=m-F-145 743 2. 12 24. X1 = p-CF3, X2 = m-CONH2 -142 768 0.67 25. X1 = p-CF3, X2 = p-NO2 -141 770 1.74

Table 2. Top 14 structures in virtual combinatorial library DAPOL2.

Cmpd Structure PMF MW LogP score 1. R1 ~ (CH2) 3Ph-p-I, X2=S, R2 =p-biphenyl-112 613 6. 75 (. __. _. z) 3 2. Rl = CH Ph--F X2 = S R2 =-bi hen 1-111 505 5. 78 3. Rl-- (CHz) 4Ph p-CH20H, X2=S, R2 =p-biphenyl-111-531 5. 56 4 Rl-(CH2) 3Ph-p-Ï ; X2-Ö, R2-CH2Ph iös 534 4 56 5. Rl = CH Ph--NO X2 = S R2 =-bi hen 1-107 545 5. 89 ... ............... . .. .-. -...-.. ..... "3. y-pj........ ... _.. (CHz) sPhP-CHzOH, X2=.. '__= 3. 87. R. ..., _,..,, ~ ~,,,,, _ _ _,,,., __ 8 R1= (CH2) sPh-p-CH2OH, X2= O, R2=CH2Ph-104 467 3 87 9. Rl-\CH Ph-F, X2-O, R2-CH Ph-102 427 3. 60 10. Rl = CH Ph--F X2 = S R2 = CH Ph-101 443 4. 51 ... .......... _.... _. _... . (__... _ i) s p'_ __..... 3 _. __. . _... _.... __= 11. Rl-CH Ph-CF X2-O, R2-CHZPh-99 505 5. 34 12. Rl = \CH Ph X2 = S, R2 = CH Ph-99 425 4. 31 . _... ____... (_) 3___. p ; 13. Rl = CH Ph--I X2 = S R2 = CHZPh-99 551 5. 47 14. Rl= (CH2) 3Ph-p-CF3, X2=S, R2=CH2Ph 93 493 5. 27 "12RTCHJsPh99 ; ''''-' "13i (CHPhIpIlTX2'199y"'g" "llRl' (CH2) 3PhIp3,'X2'=s7M93'"Y7"

Although, DAPOL1-based ligands (listed in Table 1) are about as large as the OM99-based ligands, and their molecular weights are in the range of 700-830, unlike the OM99-based compounds, they possess only two amide bonds that are susceptible to hydrolysis by amidases. Moreover, DAPOL1 ligands exhibit PMF score of up to-168, which is better than the PMF score of-120 for OM99-2 and-92 for ligand 22 from Ghosh et al. Ghosh AK, Bilcer G, Harwood C, Kawahama R, Shin D, Hussain KA, Hong L, Loy JA, Nguyen C, Koelsch G, Ennolieff J, Tang J, J Med Chem, 2001,44 : 2865-2868. The urea function has been employed in the design of clinically relevant HIV protease inhibitors, thus supporting the use of this group in the preparation of BACE1 aspartase inhibitors. Once candidate molecules from this series are identified as effective BACE inhibitors, we will conduct further rounds of synthesis and modeling to replace the amide bonds with bioisosteric groups and hence arrive at structures that will be more stable when used in vivo and likely to penetrate the blood-brain-barrier (having a logP of approximately 3).

The second series of ligands that emerged from our modeling work is the DAPOL2- based compounds (see Table 2 and Figure 3). Unlike the OM-99-based ligands, these ligands have molecular weights around 500 and possess an appropriate lipophilicity (logP) that is within the limits o f"drug-like"molecules (Lipinski's rule of 5). Moreover, these ligands possess no amide bonds, and appear to be only slightly less active than their

DAPOL1 counterparts and OM-99-based ligands based upon their PMF scores (compare data in Figure 1, Table 1, and Table 2). Accordingly, we anticipate that these ligand candidates may prove active under in vivo conditions. These compounds represent our first series of non-peptide ligands, and after initial synthesis and screening, will be further modified to increase their in silico activity, followed by further rounds of synthesis and testing. Additional modifications in the substituents in Rl and R2 will be explored in order to bring their logP into the range of 3, which is ideal for access to the brain.

Further studies into the de novo/rational design of BACE inhibitors will target the identification of compounds exhibiting"drug-like"properties possessing improved PMF scores, as well as showing selectivity for BACE1 over BACE2. For this stage of the process, the 3D model of BACE2 will be generated and optimized using the Homology and CHARMm modules in InsightII. Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, K arplus M, J. C omput. C hem., 1983,4 : 187-217; Homology (2002) hl :. 9685 Scranton Rd. , San Diego, CA 92121-3752: Accelrys Inc. The resulting 3D model of BACE2 will be used to test the in silico ligands for BACE1 ; the comparison modeling will be used to guide structural changes likely to further enhance BACE1 versus BACE2 selectivity. The most selective ligands for BACE1 will be prioritized for synthesis.

Combinatorial Libraries The subject compounds may be synthesized using the methods of combinatorial synthesis described in this section. Combinatorial libraries of the compounds may be used for the screening of pharmaceutical, agrochemical or other biological or medically-related activity or material-related qualities. A combinatorial library for the purposes of the present invention is a mixture of chemically related compounds which may be screened together for a desired property; said libraries may be in solution or covalently linked to a solid support.

The preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for the appropriate biological, pharmaceutical, agrochemical or physical property may be done by conventional methods.

Diversity in a library can be created at a variety of different levels. For instance, the substrate aryl groups used in a combinatorial approach can be diverse in terms of the core aryl moiety, e. g. , a variegation in terms of the ring structure, and/or can be varied with respect to the other substituents.

A variety of techniques are available in the art for generating combinatorial libraries of small organic molecules. See, for example, Blondelle et al. (1995) Trends Anal. Chem.

14: 83; the Affymax U. S. Patents 5,359, 115 and 5,362, 899: the Elhnali U. S. Patent 5,288, 514: the Still et al. PCT publication WO 94/08051; Chen et al. (1994) JACS 116: 2661: Kerr et al. (1993) JACS 115: 252; PCT publications W092/10092, W093/09668 and W091/07087 ; and the L emer et al. PCT publication W093/20242). Accordingly, a variety of libraries on the order of about 16 to 1,000, 000 or more diversomers can be synthesized and screened for a particular activity or property.

In an exemplary embodiment, a library of substituted diversomers can be synthesized using the subject reactions adapted to the techniques described in the Still et al.

PCT publication WO 94/08051, e. g. , being linked to a polymer bead by a hydrolyzable or photolyzable group, e. g. , located at one of the positions of substrate. According to the Still et al. technique, the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead. In one embodiment, which is particularly suitable for discovering enzyme inhibitors, the beads can be dispersed on the surface of a permeable membrane, and the diversomers released from the beads by lysis of the bead linker. The diversomer from each bead will diffuse across the membrane to an assay zone, where it will interact with an enzyme assay. Detailed descriptions of a number of combinatorial methodologies are provided below.

A. Direct Characterization A growing trend in the field of combinatorial chemistry is to exploit the sensitivity of techniques such as mass spectrometry (MS), e. g. , which can be used to characterize sub- femtomolar amounts of a compound, and to directly determine the chemical constitution of a compound selected from a combinatorial library. For instance, where the library is provided on an insoluble support matrix, discrete populations of compounds can be first released from the support and characterized by MS. In other embodiments, as part of the MS sample preparation technique, such MS techniques as MALDI can be used to release a compound from the matrix, particularly where a labile bond is used originally to tether the compound to the matrix. For instance, a bead selected from a library can be irradiated in a MALDI step in order to release the diversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis The libraries of the subject method can take the multipin library format. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS 81: 3998-4002) introduced a method for generating compound libraries by a parallel synthesis on polyacrylic acid-grated polyethylene pins arrayed in the microtitre plate format. The Geysen technique can be used to synthesize and screen thousands of compounds per week using the multipin method, and the tethered compounds may be reused in many assays. Appropriate linker moieties can also been appended to the pins so that the compounds may be cleaved from the supports after synthesis for assessment of purity and further evaluation (c. f. , Bray et al. (1990) Tetrahedron Lett 31: 5811-5814; Valerio et al. (1991) Anal Biochem 197: 168-177; Bray et al. (1991) Tetrahedron Lett 32: 6163-6166).

C) Divide-Couple-Recombine In yet another embodiment, a variegated library of compounds can be provided on a set of beads utilizing the strategy of divide-couple-recombine (see, e. g. , Houghten (1985) PNAS 82: 5131-5135; and U. S. Patents 4,631, 211; 5,440, 016; 5,480, 971). Briefly, as the name implies, at each synthesis step where degeneracy is introduced into the library, the beads are divided into separate groups equal to the number of different substituents to be added at a particular position in the library, the different substituents coupled in separate reactions, and the beads recombined into one pool for the next iteration.

In one embodiment, the divide-couple-recombine strategy can be carried out using an analogous approach to the so-called"tea bag"method first developed by Houghten, where compound synthesis occurs on resin sealed inside porous polypropylene bags (Houghten et al. (1986) PNAS 82: 5131-5135). Substituents are coupled to the compound- bearing resins by placing the bags in appropriate reaction solutions, while all common steps such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end of the synthesis, each bag contains a single compound.

D) Combinatorial Libraries by Light-Directed. Spatially Addressable Parallel Chemical Synthesis A scheme of combinatorial synthesis in which the identity of a compound is given by its locations on a synthesis substrate is termed a spatially-addressable synthesis. In one embodiment, the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations on a solid support (Dower et al. (1991) Annu Rep

Med Chem 26: 271-280; Fodor, S. P. A. (1991) Science 251: 767; Pirrung et al. (1992) U. S.

Patent No. 5,143, 854; Jacobs et al. (1994) Trends Biotechnol 12: 19-26). The spatial resolution of photolithography affords miniaturization. This technique can be carried out through the use protection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al. (1994) J Med Chem 37: 1233-1251. A synthesis substrate is prepared for coupling through the covalent attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers. Light is used to selectively activate a specified region of the synthesis support for coupling. Removal of the photolabile protecting groups by light (deprotection) results in activation of selected areas. After activation, the first of a set of amino acid analogs, each bearing a photolabile protecting group on the amino terminus, is exposed to the entire surface. Coupling only occurs in regions that were addressed by light in the preceding step. The reaction is stopped, the plates washed, and the substrate is again illuminated through a second mask, activating a different region for reaction with a second protected building block. The pattern of masks and the sequence of reactants define the products and their locations. Since this process utilizes photolithography techniques, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be addressed with appropriate resolution. The position of each compound is precisely known; hence, its interactions with other molecules can be directly assessed.

In a light-directed chemical synthesis, the products depend on the pattern of illumination and on the order of addition of reactants. By varying the lithographic patterns, many different sets of test compounds can be synthesized simultaneously; this characteristic leads to the generation of many different masking strategies.

E) Encoded Combinatorial Libraries In yet another embodiment, the subject method utilizes a compound library provided with an encoded tagging system. A recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using tags that uniquely encode the reaction steps a given bead has undergone and, by inference, the structure it carries. Conceptually, this approach mimics phage display libraries, where activity derives from expressed peptides, but the structures of the active peptides are deduced from the corresponding genomic DNA sequence. The first encoding of synthetic combinatorial libraries employed DNA as the code. A variety of other forms of encoding

have been reported, including encoding with sequenceable bio-oligomers (e. g., oligonucleotides and peptides), and binary encoding with additional non-sequenceable tags.

1) Tagging with sequenceable bio-oligomers The principle of using oligonucleotides to encode combinatorial synthetic libraries was described in 1992 (Brenner et al. (1992) PNAS 89: 5381-5383), and an example of such a library appeared the following year (Needles et al. (1993) PNAS 90: 10700-10704). A combinatorial library of nominally 77 (= 823,543) peptides composed of all combinations of Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acid code), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), was prepared by a series of alternating rounds of peptide and oligonucleotide synthesis on solid support. In this work, the amine linking functionality on the bead was specifically differentiated toward peptide or oligonucleotide synthesis by simultaneously preincubating the beads with reagents that generate protected OH groups for oligonucleotide synthesis and protected NEt2 groups for peptide synthesis (here, in a ratio of 1: 20). When complete, the tags each consisted of 69-mers, 14 units of which carried the code. T he b ead-bound library w as incubated with a f luorescently 1 abeled antibody, and beads containing bound antibody that fluoresced strongly were harvested by fluorescence- activated cell sorting (FACS). The DNA tags were amplified by PCR and sequence, and the predicted peptides were synthesized. Following such techniques, compound libraries can be derived for use in the subject method, where the oligonucleotide sequence of the tag identifies the sequential combinatorial reactions that a particular bead underwent, and therefore provides the identity of the compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive tag analysis.

Even so, the method requires careful choice of orthogonal sets of protecting groups required for alternating co-synthesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the choice of reagents and conditions that can be employed for the synthesis of non-oligomeric libraries. In preferred embodiments, the libraries employ linkers permitting selective detachment of the test compound library member for assay.

Peptides h ave a Iso b een e mployed a s t agging m olecules for c ombinatorial libraries. Two exemplary approaches are described in the art, both of which employ branched linkers to solid phase upon which coding and ligand strands are alternately

elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115: 2529-2531), orthogonality in synthesis is achieved by employing acid-labile protection for the coding strand and base-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6: 161-170), branched linkers are employed so that the coding unit and the test compound can both be attached to the same functional group on the resin. In one embodiment, a cleavable linker can be placed between the branch point and the bead so that cleavage releases a molecule containing both code and the compound (Ptek et al. (1991) Tetrahedron Lett 32: 3891- 3894). In another embodiment, the cleavable linker can be placed so that the test compound can be selectively separated from the bead, leaving the code behind. This last construct is particularly valuable because it permits screening of the test compound without potential interference of the coding groups. Examples in the art of independent cleavage and sequencing of peptide library members and their corresponding tags has confirmed that the tags can accurately predict the peptide structure.

2) Non-sequenceable Tagging : Binary Encoding An alternative form of encoding the test compound library employs a set of non-sequencable electrophoric tagging molecules that are used as a binary code (Ohlmeyer et al. (1993) PNAS 90: 10922-10926). Exemplary tags are haloaromatic alkyl ethers that are detectable as their trimethylsilyl ethers at less than femtomolar levels by electron capture gas chromatography (ECGC). Variations in the length of the alkyl chain, as well as the nature and position of the aromatic halide substituents, permit the synthesis of at least 40 such tags, which in principle can encode 240 (e. g. , upwards of 1012) different molecules. In the original report (Ohlmeyer et al. , supra) the tags were bound to about 1% of the available amine groups of a peptide library via a photocleavable o-nitrobenzyl linker.

This approach is convenient when preparing combinatorial libraries of peptide-like or other amine-containing molecules. A more versatile system has, however, been developed that permits encoding of essentially any combinatorial library. Here, the compound would be attached to the solid support via the photocleavable linker and the tag is attached through a catechol ether linker via carbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem 59 : 4723-4724). This orthogonal attachment strategy permits the selective detachment of library members for assay in solution and subsequent decoding by ECGC after oxidative detachment of the tag sets.

Although several amide-linked libraries in the art employ binary encoding with the electrophoric tags attached to amine groups, attaching these tags directly to the bead matrix provides far greater versatility in the structures that can be prepared in encoded combinatorial libraries. Attached in this way, the tags and their linker are nearly as unreactive as the bead matrix itself. Two binary-encoded combinatorial libraries have been reported where the electrophoric tags are attached directly to the solid phase (Ohlmeyer et al. (1995) PNAS 92: 6027-603 1) and provide guidance for generating the subject compound library. Both libraries were constructed using an orthogonal attachment strategy in which the library member was linked to the solid support by a photolabile linker and the tags were attached through a linker cleavable only by vigorous oxidation. Because the library members can be repetitively partially photoeluted from the solid support, library members can be utilized in multiple assays. Successive photoelution also permits a very high throughput iterative screening strategy: first, multiple beads are placed in 96-well microtiter plates; second, compounds are partially detached and transferred to assay plates; third, a metal binding assay identifies the active wells ; fourth, the corresponding beads are rearrayed singly into new microtiter plates; fifth, single active compounds are identified; and sixth, the structures are decoded.

Intranasal Administration The intranasal administration of a pharmacologically active compound generally results in more rapid bioavailability of the compound, or of its desired active metabolite, than if the compound is administered orally. Moreover, the time required to achieve a given concentration of the active compound in the bloodstream, e. g. , within a period of about thirty minutes after administration, is generally less via the intranasal route compared to oral administration.

Accordingly, in a preferred embodiment of the instant methods of treating Alzheimer's disease, a therapeutically effective amount of a compound of the present invention is administered intranasally to a mammal in need thereof. In a preferred embodiment, said mammal is a human. This aspect of the present invention relates to compositions and methods having utility for the administration, via the respiratory tract, of compounds of the present invention. Reference to a compound of the present invention herein, unless indicated otherwise, is also understood to encompass allpharmaceutically acceptable forms of the compound, either as the free base and/or as its pharmaceutically

acceptable derivatives, salts and active metabolite and/or combinations thereof, all of which are contemplated to be used in the present compositions and methods. Reference to the term "salt"herein is also intended to encompass the haloacids, e. g., HC1 salts and the like, unless explicitly indicated.

There are many advantages to intranasal administration of medications and other compositions, including a direct route to the blood stream, avoidance of hepatic first-pass metabolism, bioavailability, ease and convenience, and proximity to the central nervous system. S ee Y. W. C hien et al., Anatomy and Physiology o f the Nose, Nasal S ystemic Drug Delivery, Chapter 1, 1-26,1989. Various types of compositions, therapeutics, prophylactics or otherwise, may be delivered intranasally including, but not limited to, topical anesthetics, sedatives, hypnotics, analgesics, ketamines, opiates, glucagons, vaccines, anti-nausea and motion sickness medications, antihistamines, antihypertensive drugs, psychoactive medications, antibiotics, and hormones. See, e. g. , M. R. Nott et al., Topical Anaesthesia for the Insertion of Nasogastric Tubes, European Journal of Anaesthesiology, 12 (3), May 1995; R. J. Henry et al, A pharmacokinetic Study of Midazolam in Dogs: Nasal Drop Versus Atomizer Administration, Journal of the American Academy of Pediatric Dentistry, 20 (5), 321-326,1998 ; J. Lithander et al., Sedation with nasal Ketamine and Midazolam for Cryotherapy in Retinopathy of Prematurity, British Journal of Ophthalmology, 77 (8), 529-530,1993 ; F. E. Ralley, Intranasal Opiates: Old Route For New Drugs, Canadian Journal of Anesthesiology, 36 (5) 491-493,1989 ; B.

Haneberg et al, Intranasal Administration of Mengiococcal outer membrane vesicle vaccine induces persistent local Mucosal Antibodies and Serum Antibodies with Strong Bactericidal Activity in Humans, Infection and Immunity, 66 (4), 1334-1341,1998 ; B. K. Wager et al, A Double Blind Placebo-Controlled Evaluation of Intranasal Metoclopramide in the Prevention of Postoperative nausea and Vomiting, Pharmacotherapy, 16 (6), 1063-1069 1996; and J. Q. Wang, et al., An Experimental Study on Nasal Absorption of Gentamycin in Dogs, Chinese Medical Journal, 107 (3), 219-221,1994.

The intranasal route of administration provides numerous advantages over intravenous (IV) and intramuscular (IM) injections. One principal advantage of intranasal administration is convenience. An injectable system requires sterilization of the hypodermic syringe and in the institutional setting, leads to concerns among medical personnel about the risk of contracting disease if the they are accidentally stuck by a contaminated needle.

Strict requirements for the safe disposal of the used needle and syringe must also be

imposed in the institutional setting. In contrast, intranasal administration requires little time on the part of the patient and the attending medical personnel, and is far less burdensome on the institution than injectables. There is no significant risk of infection of medical personnel or others in the institutional setting that is associated with nasal spray devices.

A second important advantage of intranasal administration over IM and IV is patient acceptance of the drug delivery system. Many, if not most, patients experience anxiety and exhibit symptoms of stress when faced with hypodermic injections via the IM or IV routes.

In some cases, the after-effects of the injection include burning, edema, swelling, turgidity, hardness and soreness. In contrast, intranasal administration is perceived as non-invasive, is not accompanied by pain, has no significant after-effects and produces the gratification of prompt relief in the patient exhibiting the symptom. Most people have some familiarity with nasal sprays in the form of over-the-counter decongestants for alleviating the symptoms of colds and allergies that they or a family member have used routinely. Another important consideration is that the patient can self-administer the prescribed dosage (s) of nasal spray. An empty nasal spray device, or one containing a non-medicated solution can be given to the patient to practice the technique for proper insertion, inhalation and activation for self-administration.

Other ingredients, such as preservatives, colorants, lubricating or viscous mineral or vegetable oils, perfumes, natural or synthetic plant extracts, such as aromatic oils, and humectants and viscosity enhancers, e. g. , glycerol, can also be included to provide additional viscosity, moisture retention and a pleasant texture and odor for the formulation.

For nasal administration of solutions or suspensions according to the invention, various devices are available in the art for the generation of drops, droplets and sprays. For example, solutions can be administered into the nasal passages by means of a simple dropper (or pipet) that includes a g lass, plastic o r metal dispensing tube from which the contents are expelled drop by drop by means of air pressure provided by a manually powered pump, e. g. , a flexible rubber bulb, attached to one end. Fine droplets and sprays can be provided by a manual or electrically powered intranasal pump dispenser or squeeze bottle as well known to the art, e. g. , that is designed to blow a mixture of air and fine droplets into the nasal passages. The tear secretions of the eye drain from the orbit into the nasal passages, thus, if desirable, a suitable pharmaceutically acceptable ophthalmic solution can be readily provided by the ordinary artisan as a carrier for the analgesic

compounds to be delivered and the analgesic can be administered to the orbit of the eye in the form of eye drops to provide for both ophthalmic and intranasal administration.

Each intranasal carrier must be"acceptable"in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient. See generally US Patent 6,610, 271; U. S. Patent 6,017, 963; U. S. Patent 4,973, 596; U. S. Patent 4,880, 813; U. S. Patent 4,778, 810; and U. S. Patent 4,464, 378. The carrier must be biologically acceptable and inert. To prepare formulations suitable for intranasal administration, solutions and suspensions are sterilized and are preferably isotonic to blood. The formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the art. Such methods include the step of bringing the active ingredient into association with the carrier which itself may encompass one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided semi-solid carriers or both. Various unit dose and multidose containers, e. g., sealed ampules and vials, may be used, as is well known in the art. In addition to the ingredients particularly mentioned above, the formulations of this invention may also include other agents conventional in the art for this type of intranasal pharmaceutical formulation.

Solutions for intranasal administration of a compound of the present invention may be prepared by using 0. 05 M phosphate buffer at pH 6.0. Aqueous solutions of a compound of the present invention may be administered through the nostril using a microsyringe. The deposition of a composition or dose after intranasal delivery depends upon particle inertia, sedimentation due to gravity, and diffusion due to Brownian motion. M. Dolovich, Principles Underlying Aerosol Therapy, Journal of Aerosol Medicine, Vol. 2, No. 2,1989 ; see also A. Brown and J. Slusser, Propellent-driven Aerosols of Function Proteins as Potential Therapeutic Agents in the Respiratory Tract, Immmunopharmacology 28,241- 257,1994. Each of these mechanisms can be dependent upon the particle size of the dose or composition delivered. As disclosed by M. Dolovich, particles having a diameter of less than about 1 micrometer can remain suspended as the time required for the particle to diffuse to an airway wall tends to be greater than the time to complete the inspiratory phase of a normal breath. Optimum deposition in the lung may be achieved with particles having a diameter of about 3 micrometers. Larger particles having a diameter of greater than about 5 micrometers are often deposited in the upper airways. M. Dolovich, at pages 173-174. As

such the proper particle size should be selected depending on where in the airway or lung compartment deposition is to occur.

Dosages The dosage of any composition of the present invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the subject composition.

Any of the subject formulations may be administered in a single dose or in divided doses. Dosages for the compositions of the present invention may be readily determined by techniques known to those of skill in the art or as taught herein.

In certain embodiments, the dosage of the subject compounds will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.

An effective dose or amount, and any possible affects on the timing of administration of the formulation, may need to be identified for any particular composition of the present invention. This may be accomplished by routine experiment as described herein, using one or more groups of animals (preferably at least 5 animals per group), or in human trials if appropriate. The effectiveness of any subject composition and method of treatment or prevention may be assessed by administering the composition and assessing the effect of the administration by measuring one or more applicable indices, and comparing the post-treatment values of these indices to the values of the same indices prior to treatment.

The precise time of administration and amount of any particular subject composition that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a subject composition, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e. g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.

While the subject is being treated, the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during the treatment period. Treatment, including composition, amounts, times of administration and formulation, may be optimized according to the results of such monitoring. The patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters. Adjustments to the amount (s) of subject composition administered and possibly to the time of administration may be made based on these reevaluations.

Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained.

The use of the subject compositions may reduce the required dosage for any individual agent contained in the compositions because the onset and duration of effect of the different agents may be complimentary.

Toxicity and therapeutic efficacy of subject compositions may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e. g. , for determining the LDso and the EDso.

The data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of any subject composition lies preferably within a range of circulating concentrations that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.

Formulation The compositions of the present invention may be administered by various means, depending on their intended use, as is well known in the art. For example, if compositions of the present invention are to be administered orally, they may be formulated as tablets, capsules, granules, powders or syrups. Alternatively, formulations of the present invention may be administered parenterally as injections (intravenous, intramuscular or subcutaneous), drop infusion preparations or suppositories. For application by the ophthalmic mucous membrane route, compositions of the present invention may be formulated as eyedrops or eye ointments. These formulations may be prepared by

conventional means, and, if desired, the compositions may be mixed with any conventional additive, such as an excipient, a binder, a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a suspension aid, an emulsifying agent or a coating agent.

In formulations of the subject invention, wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may be present in the formulated agents.

Subject compositions may be suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of composition that may be combined with a carrier material to produce a single dose vary depending upon the subject being treated, and the particular mode of administration.

Methods of preparing these formulations include the step of bringing into association compositions of the present invention with the carrier and, optionally, one or more accessory ingredients. I n general, the formulations are prepared by uniformly and intimately bringing into association agents with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), each containing a predetermined amount of a subject composition thereof as an active ingredient.

Compositions of the present invention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca

starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. S olid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-

irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for transdermal administration of a subject composition includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.

The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Compositions of the present invention may alternatively be administered by aerosol.

This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e. g. , fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchasglycine, buffers, salts, sugars or sugar alcohols.

Aerosols generally are prepared from isotonic solutions.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise a subject composition in combination with one or more pharmaceutically- acceptable s terile i sotonic aqueous o r n on-aqueous s olutions, dispersions, s uspensions o r emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Kits This invention also provides kits for conveniently and effectively implementing the methods of this invention. Such kits comprise any subject composition, and a means for facilitating compliance with methods of this invention. Such kits provide a convenient and effective means for assuring that the subject to be treated takes the appropriate active in the correct dosage in the correct manner. The compliance means of such kits includes any means which facilitates administering the actives according to a method of this invention.

Such compliance means include instructions, packaging, and dispensing means, and combinations thereof. Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. In other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.

Exernplification The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example 1 Synthesis of DAPOLI-based compounds The synthesis o f the D APOLl-based i s o utlined in S cheme 1. The epoxy ring in tosyl-protected epoxyalcohol 1 is opened regio-and stereoselectively with trimethylsilylazide and Ti (O-i-Pr) 4 as a catalyst to give corresponding acyclic optically active tosyl-protected alcohol 2. Sutowardoyo KI, Sinou D, Tetrahedron-Asymmetry, 1991,2 : 437-444. Replacement of OTs group with amino group in compound 2 and reaction of amine 4 with isocyanate 5 affords urea 6. Further reduction of azide 6, reaction of the resulting amine 7 with isocyanate 8, and final removal ofp-methoxybenzenesulfonyl, p-methoxybenzyl, and 3,4-dimethoxybenzyl groups furnishes DAPOLl-based compounds 10. Maiti SN, Singh MP, Micetich RG, Tetrahedron Letters, 1986,27 : 1423-1424; Kitagawa K, Kitade K, Kiso Y, Akita T, Funakoshi S, Fujii N, Yajima H, J. Chem. Soc., Chem. Commun. , 1979,: 955; White JD, Amedio JC, Journal of Organic Chemistry, 1989, 54: 736-738; Wood JL, Stoltz BM, Dietrich HJ, Journal of the American Chemical Society, 1995,117 : 10413-10414. Isocyanate 5 and 8 are readily available from the protected amino acid amides by the reaction with di-or triphosgene. Kurita K, Iwakura Y, Org Synth, 1988, 50: 715-718. See Scheme 1.

Scheme 1. 0 0 O=G=N + R j X p Me3SiN3/cat. OSiMe3 e OHO \ f OH 5 I i > OTs | N<OTs ~ N<NHz Ti (OjPr) 4 HCI N3 N N3""NHz (from the free amine with 1 (Aldrich) 2 3 4 di-or triphosgene ; R = 3, 4-dimethoxybenzyl ; PMB = p-methoxybenzyl) NiN=C=O N N=C=O XI OH H H O OH H H O N N3, xl R R /X2 SnClz IOI \R /X S02CsH40Me-p _ Mesh 6 OPMB OPMB X1 X2 OHHOHHHO N N N, , N CF3COOH, Me2S y 0 H H OH H H O i N F N HtXN° N N I H N O N 9 OPMB SO2C6H40Me-p N H H

Example 2 Synthesis of DAPOL2-based compounds DAPOL2-based compounds will be prepared from the same intermediate that is used for preparation of DIPOLl-based ligands, tosyl-protected alcohol 2, and five- membered aminoheterocycles 11 as described in Scheme 2. White AD, Creswell MW, Chucholowski AW, Blankley CJ, Wilson MW, Bousley RF, Essenburg AD, Hamelehle KL, Krause BR, Stanfield RL, Dominick MA, Neub M, Journal of Medicinal Chemistry, 1996, 39: 4382-4395. Nucleophilic substitution of OTs group in 2 and further reduction of azide goup in 12 leads to amine 13. Next, amine 13 is reacted with readily available isocyanates 14 to give DIPOL2-based ligands 14. A large set of different isocyanates 14 is available at major suppliers or can be synthesized from corresponding commercially available amines.

See Scheme 2.

Scheme 2.

Example 3 Testing of compounds for BACE inhibition.

The design and synthesis activities described above will yield a series of compounds expected, on the basis of in silico testing results and drug development principles, to be effective inhibitors of BACE1 in brain. These compounds will be supplied to Dr.

Wroblewski'a lab for in vitro assessment of BACE inhibition. The compounds are first screened in a high-throughput fluorescent assay using BACE-transfected cells and synthetic fluorogenic substrates. Two types of cells are used as a source of the BACE enzymes: the naturally BACE-expressing human neuroblastoma cells (SH-SY5Y), and Chinese hamster ovary (CHO) cell lines transfected with cDNAs for human BACE1 and BACE2. The activity of the lead compounds is then confirmed using cells co-transfected with BACE and full-length APP.

Example 4 Preparation of SH-SYSY cells.

Human neuroblastoma cells SH-SY5Y (ATTC CRL-2266) are cultured in a culture medium containing 90% EMEM and Ham's F12 (1: 1), and 10% fetal bovine serum at 37°C in 5% C02. Cells are grown to confluency with the medium being exchanged every 4 days and then harvested in PBS pH 7.4, and sedimented by centrifugation. Cell pellets are lysed in ice-cold medium containing 100 mM Na-acetate buffer, pH 4.5, 100 mM NaCI, 0.06% Triton X-100, sonicated (3 x 20 s) and centrifuged (50,000 g for 10 min). Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J, Proc Natl Acad Sci U S A, 2000,97 : 1456-1460.

Protein concentration is measured in the resulting supernatant, and aliquots are frozen and stored at-80°C.

Example 5 Expression of BACEI, BACE2 and APP in CHO cells Full-length cDNAs for human BACE1 and BACE2 are obtained from ResGen, Invitrogen Corp. APP cDNA is purchased from ATCC. BACE1 and BACE2 cDNAs are subcloned into pcDNA3.1 vector (Invitrogen) containing neomycin resistance gene for selection of stable clones. Farzan M, Schnitzler CE, Vasilieva N, Leung D, Choe H, Proc Natl Acad Sci U S A, 2000,97 : 9712-9717. APP cDNA is subcloned into pcDNA3. 1/Zeo vector (Invitrogen) c ontaining Zeocin s electable marker. CHO c ells, t ransfected w ith t he above constructs by Effectene (Qiagen) are selected for resistance to the corresponding antibiotic. Different selectable markers allow for selection of cell lines expressing both APP and BACE genes. Positive clones with stable expression of BACE1 or BACE2 are identified based on their functional activity in the fluorescent assay. Ermolieff J, Loy JA, Koelsch G, Tang J, Biochemistry, 2000,39 : 16263. APP producing cells are verified using immunoblotting with the anti-APP antibodies. For fluorescent assays, CHO cell lines with stable expression of either BACE1 or BACE2 are harvested and the enzyme is prepared as described above.

Example 6 Fluorescent BACE assay BACE activity is measured using a fluorescence assay based on the fluorescent resonance energy transfer properties of the synthetic fluorogenic substrates. Ermolieff J, Loy JA, Koelsch G, Tang J, Biochemistry, 2000,39 : 16263. The substrates used include two synthetic peptides: MCA-EVKMDAEFK (DNP) (wild type) and MCA- SEVNLDAEFK (DPN) (Swedish mutation) (Calbiochem). The concentration of active BACE in cell extracts is determined by active site titration using the tight-binding inhibitor OM99-2 (Calbiochem). Ghosh AK, Shin DW, Downs D, Koelsch G, Lin XL, Ermolieff J, Tang J, Journal of the American Chemical Society, 2000,122 : 3522-3523. Enzyme aliquots are incubated for 1 hr at 37°C in 96-well assay plates in a reaction buffer containing 100 mM Na-acetate, pH 4.5, 100 mM NaCI, 0.06% Triton X-100 and the appropriate concentration of the fluorogenic BACE substrate. Ermolieff J, Loy JA, Koelsch G, Tang J, Biochemistry, 2000,39 : 16263. The change in fluorescence is monitored in time with an automated fluorescence plate reader (Dynex), using excitation at 328 nm and emission at 393 nm. Calibration curves are prepared using the control fluorogenic substrate DAEFK (DPN) (Calbiochem). For the determination of the enzyme kinetic properties curves are constructed using varying concentrations of the fluorogenic substrates and the apparent Km values are calculated using non-linear regression.

Example 7 Evaluation of BACE inhibitors.

Newly synthesized compounds are assayed for their ability to inhibit BACE activity in the presence of the fluorogenic substrates, as described above. In initial screening experiments, various concentrations of the putative inhibitor (1-1000 nM) are included during the assay and the inhibitory potency (IC50) is calculated from the dose-response curves by non-linear regression. In all experiments a synthetic BACE inhibitor (KTEEISEVN (St) VAEF, Calbiochem) is used as a reference compound to determine the maximal enzyme inhibition. Active inhibitors are evaluated in further experiments where BACE activity is measured in the presence of a dose-response of the fluorogenic substrate in the absence and in the presence of several (at least 4) concentrations of the inhibitor. The

obtained results are used to ascertain the competitive character of the inhibition and to calculate the inhibitor Ki values.

Example 8 Validation of inhibitor action.

The activity of new potent inhibitors is validated in an assay using the full-length ß- amyloid precursor protein (APP) co-expressed in cells expressing BACE1 or BACE 2 enzymes. Farzan M, Schnitzler CE, Vasilieva N, Leung D, Choe H, Proc Natl Acad Sci U S A, 2000,97 : 9712-9717. After incubation in the absence and presence of several inhibitor concentrations, cells are harvested and lysed. Samples of cell extracts are run on SDS- PAGE, transferred to PVDF membranes and probed with terminal-specific antibodies (Biosource) to determine the amount of total amount of APP and its cleavage products.

Mallender WD, Yager D, Onstead L, Nichols MR, Eckman C, Sambamurti K, Kopcho LM, Marcinkeviciene J, Copeland RA, Rosenberry TL, Mol Pharmacol, 2001,59 : 619-626.

Bound antibodies are visualized with HRP-labeled secondary antibodies and an enhanced chemiluminescence detection kit. The intensity of the bands is determined using a chemiluminescence imaging system (Alpha-Innotech) and compared with immunoblots of standards of APP and its cleavage products.

Example 9 Transgenic mouse studies To confirm the effect of the lead (3-secretase inhibitors on A (3 generation in vivo, we will conduct treatment studies using mutant APP-PS1 double transgenic mice kindly supplied by Dr. David Borchelt; these mice develop progressive Ap deposition beginning at 9 months of age. Borchelt DR, Ratovitski T, Van Lare J, Lee MK, Gonzales V, Jenkins NA, Copeland NG, Price DL, Sisodia SS, Neuron, 1997,19 : 939-945. The primary measures in these studies will be quantification of Ap by ELISA and plaque density b y image analysis; secondary measures will include inflammatory markers. Six month old transgenic mice will be randomly assigned to one of seven treatment groups: placebo treatment, or treatment with low, medium or high dose of one of the two leading candidate secretase inhibitors. We will study 12 mice in each of the seven groups; this number will be adequate to demonstrate the anticipated large effect size in analysis of Ap levels and plaque deposition. Cohen J (1988) Statistical power analysis for the behavioral sciences.

Hillsdale, New Jersey: Lawrence Erlbaum Assoc. The doses will be selected based on the effective concentrations in the cell culture studies and estimates of CNS penetrability. Four mice per group will be sacrificed at 8 months of age to assess soluble Ap prior to significant plaque accumulation. The remaining 8 mice per group will be sacrificed at 11 months of age to assess plaque density and Ap levels.

Animals will be perfused before brain dissection with 0.9% normal saline followed by Hepes buffer pH 7.2 containing protease inhibitor. One hemisphere of the brain will be fixed in 4% paraformaldehyde and processed in 10-20% sucrose. The tissue will be frozen and processed for cryostat sectioning for immunohistochemistry. The other hemisphere of the brain will be trimmed quickly to obtain the hippocampal and cortical regions and would be snapped frozen in liquid nitrogen. Tissue samples will be homogenized in TBS containing protease inhibitor homogenized briefly and centrifuged 20 mins in 10,000 g. The soluble fraction will be used for Ap, IL-1 (3 and IL-6 ELISAs. To analyze insoluble AP the pellet will be solubilized and homogenized with 70% formic acid. The extract will be neutralized with 0.25 M tris, pH 8.0 containing 30% acetonitrile and 5 M NaOH, before loading onto ELISA plate.

Prophetic Embodiment A n ew 1 ine o f t ransgenic m ice w as r ecently c reated t hrough c o-microinjection o f mutant arnyloid precursor protein (APP) and tau into presenilin-l (PS-1) knock-in mice, and a homozygous line was established. The mice breed well and the mortality rate is low, i. e. , around 10% up to 1 year of age. This transgenic mouse line has a less complicated genetic background (129/C57BL6) than transgenic mice obtained by crossing independent lines. Therefore, this mouse can breed and be maintained with minimal genotyping cost and labor, reduced risk of error and high efficiency of breeding. This mouse model includes plaque and tangle pathology and synaptic dysfunction. However, further detailed characterization is necessary prior to utilization of this model to evaluate therapeutic approaches and drug candidates.

First, w e will o btain d etailed c haracterization o f A ß and t au p athologies a nd c ell damage (biochemistry and histology), brain volumetric analysis (MRI imaging), synaptic dysfunction (electron physiology), and cognitive function (behavior assay). Massive neuronal cell loss is a critical feature of Alzheimer's disease (AD), but is not seen in standard mouse models. However, homocysteine elevation by dietary maniputlation causes

hippocampal neuron loss in transgenic mice; since homocysteine levels are elevated in AD patients, homocysteine-induced neuronal damage may be an appropriate model for AD neurodegeneration. Dietary manipulation of the triple transgenic mouse line will produce an o ptimal, r eliable m odel o f A D n eurodegeneration, i deal for t esting d isease-modifying therapeutic interventions.

Example 10 Sandwich ELISA for Aß Monoclonal 4G8 against AP17-24 (Senetek) will be used as capture antibody. The antibody will be dissolved in the alkaline (pH 9.6) carbonate buffer and attached to the 96 well plate from Dynex. The wells will be blocked with 2% BSA in TBS. Processed and neutralized samples will be diluted with EC buffer (containing EDTA, BSA) containing protease inhibitors. Equal volumes of samples and detector antibody (biotynalated 10G4) will be loaded onto wells overnight at 4°C. The antibody binding will be detected using sreptavidin alkaline phosphate conjugated with luminescence. The product will be monitored using a Luminometer from Dynatech using excitation wavelength of 450 and emission wavelength of 580 nm.

Example 11 Sandwich ELISA for IL-l ß and IL-6 Monoclonal antibodies for the cytokines will be purchased from R&D System and attached to the 96 well plates from Dynex overnight at 4°C. The wells will then be blocked and the brain homogenates will be added to the wells and incubated overnight at 4 °C. The binding will then be detected using the paired detection antibody from R&D System and reported by measuring the luminiscence tagged to the substrate as described for the Ap ELISA.

Example 12 Immunostaining and image analysis The brain tissue will be sectioned by cryostat to have 10 micron thick sections.

Antiphosphotyrosine staining will be performed to demonstrate evidence of activated microglia clustered within and around the plaque by quantitative morphometric image analysis. Frautschy SA, Yang FS, Irrizarry M, Hyman B, Saido TC, Hsiao K, Cole GM

(1998) Microglial response to amyloid plaques in A PPsw t ransgenic m ice. 152 : 307-317.

To identify neuritic plaques the section will be stained immunohistologically for either antibody against the synthetic A, Bl l3 (DAE). The sections will be incubated overnight at 4 °C with the antibody and will be developed using ABC kit from Vector Lab. Dystrophic neurites will be labeled using polyclonal antibody against ubiquitin. Image analysis will be per formed using the immunolabelled coronal section. All images will be obtained from an Olympus microscope and with an Optronix video system. The video signals will be analyzed by NIH Image public domain software. Positively stained areas for Ap and ubiquitin antibodies will be compared to the total plaque area; analysis of anti phosphotyrosine will be performed using a quantitative ring analysis as previously described. Frautschy SA, Yang FS, Irrizarry M, Hyman B, Saido TC, Hsiao K, Cole GM (1998) Microglial response to amyloid plaques in APPsw transgenic mice. 152: 307-317.

Incorporation by Reference All of the patents and publications cited herein are hereby incorporated by reference.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.