Biological reagents and methods for determining the mechanism in the generation of beta-amyloid peptide

Abstract

Disclosed are biological reagents which comprise compounds that inhibit β-amyloid peptide release and/or its synthesis, and, accordingly, have utility in determining the cellular mechanism involved in the generation of β-amyloid peptide.

What is claimed is:  
     
         1 . A biological reagent defined by formula I below:  
       A-B-C  I  
       wherein: 
 A is selected from the group consisting of formulas II, II, IV and V below;  
 B is selected from the group consisting of 
 a) (CH 2 CH(R)Q) n    
 b) alkylene-Q  
 c) substituted alkylene-Q  
 
  wherein R is selected from hydrogen, alkyl, aryl and Q is selected from the group consisting of —O—, —S—, —NH—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, —NHC(O)— and —C(O)NH—; and  
 C is selected from the group consisting of a solid support and a detectable marker wherein C is optionally linked to Q through a linking arm;  
 wherein Formula II is defined as follows:  
 of:  
                     
  wherein R 11  is selected from the group consisting 
 (a) a substituted phenyl group of the formula:  
                     
 wherein  
 
 R c  is selected from the group consisting of acyl, alkyl, alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein R b  and R c  are fused to form a heteroaryl or heterocyclic ring with the phenyl ring wherein the heteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from 1 to 3 are heteroatoms independently selected from the group consisting of oxygen, nitrogen and sulfur;  
 R b  and R b′  are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that R b , R b′  and R c  are not all hydrogen and with the further proviso that when R c  is hydrogen, then neither R b  nor R b′  are hydrogen; 
 (b) 2-naphthyl; and  
 (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy;  
 
 R 12  is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to 4 carbon atoms; and  
 R 13  is selected from the group consisting of —O—, —S—, —O—(CH 2 ) p C(O)—, —O—(CH 2 ) p C(O)O— and —O—(CH 2 ) p C(O)NH—, wherein p is an integer of from 1 to 2;  
 Formula III is defined as follows:  
                     
  wherein R 21  is selected from the group consisting of 
 a) alkyl, alkenyl, alkcycloalkyl, phenyl-(R d ) m —, naphthyl-(R d ) m — wherein R d  is an alkylene group of from 1 to 8 carbon atoms and m is an integer equal to 0 or 1, cycloalkyl, cycloalkenyl, 3-pyridyl, 4-pyridyl and heteroaryl, other than 3- and 4-pyridyl, of 3 to 10 atoms and 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen wherein the heteroaryl group is optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, halo, nitro, thioalkoxy, and thioaryloxy with the proviso that for such heteroaryls when there is at least one nitrogen heteroatom, there is also at least one oxygen and/or sulfur heteroatom;  
 (b) a substituted phenyl group of the formula:  
                     
 wherein  
 
 R is alkylene of from 1 to 8 carbon atoms,  
 m is an integer equal to 0 or 1,  
 R e  and R f  are independently selected from the group consisting of hydrogen, hydroxy, fluoro and methyl;  
 R g  and R h  are independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, cyano, cycloalkyl, halo, heteroaryl, heterocyclic, nitro, trihalomethyl, thioalkoxy, thioaryloxy, thioheteroaryloxy, and —C(O)R j  where R j  is selected from the group consisting of alkyl, aryl, alkoxy and aryloxy; and  
 R i  is selected from the group consisting of hydrogen, alkyl, aryl, cyano, halo, nitro, and where R g  and R i  are fused to form a methylenedioxy ring with the phenyl ring; and  
 when R g  and/or R h  and/or R i  is fluoro, chloro, bromo and/or nitro, then R e  and/or R f  can also be chloro; and 
 (c) 1- or 2-naphthyl-(R k ) m — substituted at the 5, 6, 7 and/or 8 positions with 1 to 4 substituents selected from the group consisting alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy wherein R k  is an alkylene group of from 1 to 8 carbon atoms and m is an integer equal to 0 or 1;  
 
 R 22  is selected from the group consisting of hydrogen, alkyl, phenyl, alkylalkoxy, alkylthioalkoxy;  
 X is oxygen or sulfur;  
 X′ is hydrogen, hydroxy or fluoro; and  
 X″ is hydrogen, hydroxy or fluoro, or X′ and X″ together form an oxo group;  
 Formula IV is defined as follows:  
                     
 wherein:  
 R 31  is selected from the group consisting of 
 (a) phenyl,  
 (b) a substituted phenyl group of the formula:  
                     
 
 wherein R o  is selected from the group consisting of acyl, alkyl, alkoxy, alkylalkoxy, azido, cyano, halo, hydrogen, nitro, trihalomethyl, thioalkoxy, and wherein R o  and R m  are fused to form a heteroaryl or heterocyclic ring with the phenyl ring,  
 R m  and R n  are independently selected from the group consisting of hydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the proviso that when R o  is hydrogen, then R m  and R n  are either both hydrogen or both substituents other than hydrogen, 
 (c) 2-naphthyl,  
 (d) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5 substituents selected from the group consisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, thioalkoxy, aryl, and heteroaryl,  
 (e) heteroaryl, and  
 (f) substituted heteroaryl containing 1 to 3 substituents selected from the group consisting of alkyl, alkoxy, aryl, aryloxy, cyano, halo, nitro, heteroaryl, thioalkoxy and thioaryloxy provided that said substituents are not ortho (adjacent) to the heteroaryl attachment to the —NH group;  
 
 R 32  is selected from the group consisting of hydrogen, alkyl of from 1 to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms, alkylthioalkoxy of from 1 to 4 carbon atoms, aryl, heteroaryl, substituted aryl and substituted heteroaryl provided that the substituents are not ortho (adjacent) to the attachment of the aryl or heteroaryl atom to the carbon atom;  
 R 33  is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, and heterocyclic; and  
 X is selected from the group consisting of oxygen and sulfur; and  
 Formula V is defined as follows:  
                     
 wherein R 41  is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkenyl, substituted alkynyl, aryl, heteroaryl and heterocyclic;  
 R 42  is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclic;  
 each R 43  is independently selected from the group consisting of hydrogen and methyl and R 43  together with R 44  can be fused to form a cyclic structure of from 3 to 8 atoms which is optionally fused with an aryl or heteroaryl group;  
 each R 44  is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl, heterocyclic, substituted alkyl, substituted alkenyl and substituted alkynyl;  
 each R 45  is selected from hydrogen and methyl or together with R 43  forms a cycloalkyl group of from 3 to 6 carbon atoms;  
 X is selected from oxygen, sulfur and NH;  
 X′ is hydrogen, hydroxy or fluoro; and  
 X″ is hydrogen, hydroxy or fluoro, or X′ and X″ together form an oxo group;  
 Z is selected from the group consisting of a bond covalently linking R 41  to —CX′X″—, oxygen and sulfur; and  
 n is an integer equal to 1 to 3;  
 wherein the compounds of formulas II, III, IV and V are effective in inhibiting the cellular release and/or synthesis of β-amyloid peptide.  
 
     
     
         2 . The biological reagent of  claim 1  wherein A comprises Formula II.  
     
     
         3 . The biological reagent of  claim 1  wherein A comprises Formula III.  
     
     
         4 . The biological reagent of  claim 1  wherein A comprises Formula IV.  
     
     
         5 . The biological reagent of  claim 1  wherein A comprises Formula V.  
     
     
         6 . An intermediate useful in the synthesis of compounds of formula I wherein said intermediate is represented by formula VI  
       A-B′  VI  
       wherein: 
 A is selected from the group consisting of formulas II, III, IV and V as defined in  claim 1;  and  
 B′ is selected from the group consisting of 
 a) (CH 2 CH(R)Q) n H  
 b) alkylene-Q-H  
 c) substituted alkylene-Q-H  
 wherein R is selected from hydrogen, alkyl, aryl and Q is selected from the group consisting of —O—, —S—, —NH—.  
 
 
     
     
         7 . A compound selected from the group consisting of: 
 Tert-butyl N-(8-amino-3,6-dioxaoctyl) carbamate    Methyl N-2-aminoethyl-N′-2-t-butylcarbamoylethyl amine    Tert-butyl N-(8-N′-(N″-benzyloxycarbonyl-L-phenylglycine)-3,6-dioxaoctyl) carbamate    Methyl N-(2-N′-(N″-benzyloxycarbamoyl-L-phenylglycine)ethyl)-N′-2-t-butylcarbamoylethyl amine    Tert-butyl N-(2-N′-(N″-benzyloxycarbonyl-L-phenylglycine)-ethyl) carbamate    Tert-butyl N-(8-N′-L-phenylglycine-3,6-dioxaoctyl) carbamate    Methyl N-(2-N-(L-phenylglycine)ethyl)-N′-2-t-butylcarbamoylethyl amine    Tert-butyl N-(2-N′-L-phenylglycine)-ethyl carbamate    Tert-butyl N-[8-N′-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl]carbamate    Methyl N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)ethyl]-N′-2-t-butylcarbamoylethyl amine    Tert-butyl N-[2-N′-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-ethyl]carbamate    Tert-butyl-2-(2′-(N′-benzyloxycarbonyl-L-phenylglycine) aminoethoxy)ethylcarbamate    Tert-butyl-2-(2′-(L-phenylglycine)aminoethoxy)ethylcarbamate.    N-((R/S)-3,5-difluorophenyl-α-hydroxyacetyl)-L-alanine, methyl ester    N-((R/S -3,5-difluorophenyl-α-hydroxyacetyl)-L-alanine    Tert-butyl-2-(2′-(N′-(((R/S)-3,5-difluorophenyl-α-hydroxyacetyl)-L-alanine)-L-phenylglycine)aminoethoxy)ethylcarbamate    8-N′-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)amino-3,6-dioxaoctylamine hydrochloride    Methyl N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)aminoethyl]-N′-2-aminoethyl amine hydrochloride    2-N′-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-ethylamine hydrochloride    N-(8-N′-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycine)-3,6-dioxaoctyl)-biotinamide    2-(2′-(N′-(((R/S)-3,5-difluorophenyl-a-hydroxyacetyl)-L-alanine)-L-phenylglycine)aminoethoxy)ethylamine, hydrochloride salt    1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-6-(N-biotinyl)aminohexane    1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-3-(4-iodophenoxy)propane    1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-6-[N-(4-amino-7-nitrobenzofurazanyl)]hexane    1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-3-(4-trimethylstannylphenoxy) propane    N-{1-N-[N-(3,5-difluorophenylacetyl)-L-alaninyl-(L-phenylglycinyl)amino-6-hexyl]}-4,4-difluoro-5,7-dimethyl-4-bora-3α,4α-diaza-s-indacene-3-propionamide    N-{methyl N-[2-N-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)aminoethyl]-N′-2-aminoethyl}-4,4-difluoro-5,7-dimethyl-4-bora-3α,4α-diaza-s-indacene-3-propionamide    N-[8-N′-((2-(R/S)-hydroxy-2-(3,5-difluorophenyl)-acetyl-L-alaninyl)-L-phenylglycinyl)amino-3,6-dioxaoctyl]-4,4-difluoro-5,7-dimethyl-4-bora-, 3α,4α-diaza-s-indacene-3-propionamide    5-(S)-[N′-((S)-3,5-Difluorophenyl-α-hydroxyacetyl)-L-alaninyl]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one    5-(S)-[N′-((R)-3,5-Difluorophenyl-α-hydroxyacetyl)-L-alaninyl]amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one    5-(S)-(N′-((S)-(+)-2-Hydroxy-3-methylbutyryl)-L-alaninyl)amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one    1-[N-(3,5-difluorophenylacetyl)-L-alaninyl)-(L-phenylglycinyl)]amino-4-p-azidosalicylamidobutane    
     
     
         8 . The biological reagent as defined in  claim 1  wherein C is a detectable marker.  
     
     
         9 . The biological reagent as defined in  claim 8  wherein the detectable marker is selected from the group consisting of radioactive markers and fluorescent markers.  
     
     
         10 . The biological reagent as defined in  claim 9  wherein the radioactive marker is  125 I.  
     
     
         11 . The biological reagent as defined in  claim 1  wherein C is a solid support.  
     
     
         12 . A method for determining the mechanism in the generation of β-amyloid peptide, which method comprises 
 contacting a biological reagent of  claim 1  with a biological solution from a cell capable of generating the β-amyloid peptide under conditions whereby the cellular factor involved in the generation of the β-amyloid peptide attaches to the biological reagent;  
 removing the complex of the biological reagent and the cellular factor from the biological solution; and  
 identifying the cellular factor attached to the biological reagent.