US20070129353A1

US20070129353A1 - Alpha-helix mimetics and method relating to the treatment of cancer stem cells

Info

Publication number: US20070129353A1
Application number: US11/594,576
Authority: US
Inventors: Michael Kahn
Original assignee: Institute for Chemical Genomics
Current assignee: Choongwae Pharmaceutical Co Ltd; Institute for Chemical Genomics
Priority date: 2005-11-08
Filing date: 2006-11-08
Publication date: 2007-06-07
Also published as: WO2007056593A3; US20100069333A1; KR101486490B1; JP5536336B2; WO2007056593A2; CA2629136C; KR20080070060A; EP1957516A2; JP2009515890A; US8293743B2; CA2629136A1; AU2006311433B2; ES2570994T3; AU2006311433A1; EP1957516B1

Abstract

The invention provides α-mimetic structures and a chemical library relating thereto. Additionally, the invention provides methods wherein a-mimetic compounds are used to treat cancer stem cells.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional Application Ser. No. 60/734,655, filed on Nov. 8, 2005, which application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to α-helix mimetic structures and to a chemical library relating thereto. The invention specifically relates to applications in the treatment of cancer and particularly cancer stem cells and pharmaceutical compositions comprising the α-helix mimetics.

BACKGROUND OF THE INVENTION

Despite the clonal origin of many cancers, most primary tumors display a notable degree of cellular heterogeneity. Although modern chemotherapies kill a majority of the cells in a tumor, evidence clearly indicates that cancer stems cells often remain. The cancer stem cell hypothesis posits that a very rare population of cells within tumors are the only tumor cells with the capacity for limitless self-renewal. This concept has important therapeutic implications, and may explain why it is possible to treat many cancers until the tumor can no longer be detected and yet the cancer returns. There is a need in the art for compositions and methods that will inhibit, reduce, and/or eliminate cancer stem cells from a patient.
The present invention also fulfills these needs, and provides further related advantages by providing conformationally constrained compounds which mimic the secondary structure of α-helix regions of biologically active peptides and proteins and particularly selectively disrupt the β-catenin/CBP interaction.

SUMMARY OF THE INVENTION

Provided is a compound having the following general formula (I):

wherein A is —(C═O)—CHR₃—, or —(C═O), B is N—R₅— or —CHR₆—, D is —(C═O)—(CHR₇)— or —(C═O)—, E is -(ZR₈)— or (C═O), G is —(XR₉)_n—, —(CHR₁₀)—(NR₆)—,—(C═O)—(XR₁₂)—, —(C═N—W—R₁)—, —(C═O)—, X—(C═O)—R₁₃, X—(C═O)—NR₁₃R₁₄, X—(SO₂)—R₁₃, or X—(C═O)—OR₁₃, W is —Y(C═O)—, —(C═O)NH—, —(SO₂)—, —CHR₁₄, (C═O)—(NR₁₅)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=0 or 1; and R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers, salts, and prodrugs thereof, provided that where B is CHR₆and W is —Y(C═O)—, —(C═O)NH—, —(SO₂)—, —CHR₁₄, or (C═O)—(NR₁₅)—, G cannot be CHR₉, NR₉, (C═O)—CHR₁₂, (C═O)—NR₁₂, or no atom at all.
Also provided is a compound, salts, and prodrugs thereof of formula (I), wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, are R₁₅are independently selected from the group consisting of aminoC_2-5alkyl, guanidinoC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidinoC_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, subsitituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC_1-4alkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylC_0-2alkyl.
Further provided is the compound, salts, and prodrugs thereof of compound (I) wherein A is —(CHR₃)—(C═O)—, B is —(NR₄)—, D is (C═O)—, E is -(ZR₆)—, G is —(C═O)—(XR₉)—, and the compound has the following general formula (III):

wherein R₁, R₂, R₄, R₆, R₉, W and X are as defined in claim 1, Z is nitrogen or CH (when Z is CH, the X is nitrogen).
Also provided is a compound, salts, and prodrugs thereof of formula (I) wherein A is —O—CHR₃—, B is —NR₄—, D is —(C═O)—, E is -(ZR₆)—, Gi is (XR₇)_n—, the α-helix mimetic compounds of this invention have the following formula (IV):

wherein R₁, R₂, R₄, R₆, R₇, R₈W, X and n are as defined above, Y is —C═O, —(C═O)—O—, —(C═O)—NR₈, —SO₂—, or nothing, and Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero). In a preferred embodiment, R₁, R₂, R₆, R₇, and R₈represent the remainder of the compound, and R₄is selected from an amino acid side chain moiety. In this case, R₆or R₇may be selected from an amino acid side chain moiety when Z and X are CH, respectively.
Further provided is a compound, salts, and prodrugs thereof of formula (I) wherein A is —(C═O), B is —(CHR₆)—, D is —(C═O)—, E is -(ZR₈)—, and G is —(NH)— or —(CH₂)—, and W is a substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, the α-helix mimetic compounds of this invention have the following formula (V):

wherein K is nitrogen, oxygen, or sulfur, L is nitrogen, oxygen, —(CH)—, or —(CH₂)—, J is nitrogen, oxygen, or sulfur, Z is nitrogen or CH, and R₁, R₂, R₆, R₈, and R₁₃are selected from an amino acid side chain moiety.
Also provided is a compound having the general formula (VI):

wherein B is —(CHR₂)—, —(NR₂)—, E is —(CHR₃)—, V is —(XR₄)— or nothing, W is —(C═O)—(XR₅R₆), —(SO₂)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, X is indepentently nitrogen, oxygen, or CH, and R₁, R₂, R₃, R₄, R₅and R₆are selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and solid support, and stereoisomers, salts, and prodrugs thereof.
Further provided is a compound, salts, and prodrugs thereof of formula (I), wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, are R₁₅are independently selected from the group consisting of aminoC_2-5alkyl, guanidinoC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidinoC_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, subsitituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC_1-4alkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylC_0-2alkyl. Further provided is a compound, salts, and prodrugs thereof wherein B is —(CH)—(CH₃), E is —(CH)—(CH₃), V is —(XR₄)— or nothing, and W is substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, and X is independently introgen or CH, the compounds have the following general formula (VII):

wherein K is nitrogen, oxygen, or sulfur, L is nitrogen, oxygen, —(CH)—, or —(CH₂)—, J is nitrogen, oxygen, or sulfur, and R₅is independently selected from the group consisting of aminoC_2-5alkyl, guanidinoC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidinoC_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, Phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, subsitituted pyridyl, (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC_1-4alkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylC_0-2alkyl.
Provided is a pharmaceutical composition comprising a compound of the following general formula (I):

wherein A is —(C═O)—CHR₃—, or —(C═O), B is N—R₅— or —CHR₆—0, D is —(C═O)(CHR₇)— or 13 (C═O)—, E is —(ZR₈)— or (C═O), G is —(XR₉)_n—, —(CHR₁₀)—(NR ₆ 13 ,—(C═O)—(XR₁₂)—, -(or nothing)-, —(C═O)—, X—(C═O)—R₁₃, X—(C═O)—NR₁₃R₁₄, X—(SO₂)—R₁₃, or X—(C═O)—OR₁₃, W is —Y(C═O)—, —(C═O)NH—, —(SO₂)—, —CHR₁₄, (C═O)—(NR₁₅)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=0 or 1; and R_1,R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers, salts, and prodrugs thereof, and a pharmaceutically acceptable carrier.
Also provided is a pharmaceutical composition comprising the compound of formula (I), wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, are R₁₅are independently selected from the group consisting of aminoC_2-5alkyl, guanidinoC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidinoC_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, substituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC_1-4alkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylC_0-2alkyl. Further provided is a pharmaceutical composition of formula (I) wherein A is —(CHR₃)—(C═O)—, B is —(NR₄)—, D is (C═O)—, E is -(ZR₆)—, G is —(C═O)—(XR₉)—, and the compound has the following general formula (III):

wherein Z is nitrogen or CH (when Z is CH, the X is nitrogen).
Also provided is a pharmaceutical composition of formula (I) wherein A is —O—CHR₃—, B is —NR₄—, D is —(C═O)—, E is -(ZR₆)—, Gi is (XR₇)_n—, the α-helix mimetic compounds have the following formula (IV):

wherein R₁, R₂, R₄, R₆, R₇, R₈W, X and n are as defined above, Y is —C═O, —(C═O)—O—, —(C═O)—NR₈, —SO₂—, or nothing, and Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero). In a preferred embodiment, R₁, R₂, R₆, R₇, and R₈represent the remainder of the compound, and R₄is selected from an amino acid side chain moiety. In this case, R₆or R₇may be selected from an amino acid side chain moiety when Z and X are CH, respectively. Also provided is a pharmaceutical composition wherein A is —(C═O), B is —CHR₆)—, D is —(C═O)—, E is -(ZR₈)—, and G is —(NH)— or —(CH₂)—, and W is a substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, the α-helix mimetic compounds of this invention have the following formula (V):

wherein K is nitrogen, oxygen, or sulfur, L is nitrogen, oxygen, —(CH)—, or —(CH₂)—, J is nitrogen, oxygen, or sulfur, Z is nitrogen or CH, and R₁, R₂, R₆, R₈, and R₁₃are selected from an amino acid side chain moiety.
Further provided is a pharmaceutical composition comprising a compound having the general formula (VI):

wherein B is —(CHR₂)—, —(NR₂)—, E is —(CHR₃)—, V is —(XR₄)— or nothing, W is —(C═O)—(XR₅R₆), —(SO₂)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, X is indepentently nitrogen, oxygen, or CH, and R₁, R₂, R₃, R₄, R₅and R₆are selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and solid support, and stereoisomers, salts and prodrugs thereof. In this pharmaceutical composition, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, are R₁₅are independently selected from the group consisting of aminoC_2-5alkyl, guanidinoC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidinoC_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, substituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC_1-4alkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylC_0-2alkyl. In certain embodiments, wherein B is —(CH)—(CH₃), E is —(CH)—(CH₃), V is —(XR₄)— or nothing, and W is substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, and X is independently introgen or CH, the compounds have the following general formula (VII):

wherein K is nitrogen, oxygen, or sulfur, L is nitrogen, oxygen, —(CH)—, or —(CH₂)—, J is nitrogen, oxygen, or sulfur, and R₅is independently selected from the group consisting of aminoC_2-5alkyl, guanidinoC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidinoC_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, Phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bis-phenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, subsitituted pyridyl, (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC_1-4alkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylCO-₂alkyl.
Provided is a compound selected from the group consisting of Compounds 1-2217, and pharmaceutical compositiona comprising at least one compound of Compounds 1-2217. The pharmaceutical composition may comprise an effective amount of the compound and a pharmaceutically acceptable carrier.
Compounds of the invention may be used in the preparation of a medicament for eradicating pathologic stem cells in cancer therapy. The stem cells are leukaemic stem cells, the stem cells may be derived from solid tumors, and the solid tumor may be derived from breast, brain, lung, colon, liver, and intestine.
Therapeutically effective amount of the compounds are provided, wherein the amount is sufficient to cause cell death or inhibit proliferation and cause differentiation of stem cells in solid tumors or leukemias. The compound according to the invention may be used in the preparation of a medicament for achieving the differentiation of pathologic stem cells by causing a switch from CBP/catenin to p300/catenin transcription in cancer therapy. The catenin may be β-catenin or γ/p120-catenin.
The compounds of the invention may inhibit CBP/catenin signaling in cancer stem cells, such as by inhibiting CBP/catenin signaling in cancer stem cells thereby inducing differentiation of cancer stem cells and making them more susceptible to apoptosis induced by at least one specific pathway inhibitor. The specific pathway may be selected from the group consisting of EGFR pathway; Herceptin, Abl or Kit tyrosine kinase pathway (Imantinib).
Also provided are compounds of the invention delivered to the subject orally, transdermally, intravenously, topically, by inhalation or rectally; delivery may be by sustained release. The pharmaceutical composition may be administered by a method selected from the group consisting of capsules, tablets, powders, granules, syrups, injectable fluids, creams, ointments, hydrophilic ointments, inhalable fluids, and suppositories.
Further provided are methods of treating a cancerous condition by administering at least one compound or pharmaceutical composition of the invention, wherein the cancerous condition is at least one selected from the group consisting of acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervix cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer, penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginal cancer, cancer of the vulva, and Wilm's tumor.
Further provided is a method for eliminating teratoma-forming stem cells prior to transplant into a mammalian subject, comprising incubating a stem cell culture with at least one compound of the invention, wherein the compound inhibits CBP-β-catenin interaction and thereby causes stem cell differentiation.
Also provided is a pharmaceutical composition used in the preparation of a medicament for eradicating pathologic stem cells in cancer therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-Z shows the chemical structures of compounds 1-200.
FIG. 2A-2AD shows the chemical structures of compounds 201-400.
FIG. 3A-3AC shows the chemical structures of compounds 401-600.
FIG. 4A-4Y shows the chemical structures of compounds 601-800.
FIG. 5A-5Y shows the chemical structures of compounds 801-1000.
FIG. 6A-6Y shows the chemical structures of compounds 1001-1200.
FIG. 7A-7Z shows the chemical structures of compounds 1201-1400.
FIG. 8A-8AC shows the chemical structures of compounds 1401-1600.
FIG. 9A-9AE shows the chemical structures of compounds 1601-1800.
FIG. 10A-10AA shows the chemical structures of compounds 1801-2000.
FIG. 11A-11AA shows the chemical structures of compounds 2001-2200.
FIG. 12A-12C shows the chemical structures of diasteric and enantiomeric stereo isomers of Compounds 2203-2217.
FIG. 13A-C. FIG. 13A shows the structure of the compound ASN 06387747. FIG. 13B shows the structure of the compound ICG001. FIG. 13C shows the structures of ASN 06387747 (green) and ICG001 (red) superimposed. In accordance with an certain embodiments of the present invention, each compound has three pharmacophore rings. Distances measured from the center of each pharmacophore ring may be based on a conformation generated by flexible alignment calculations. As shown in this figure, the distance between F1 and F4 is approximately 9.6 Å, the distance between F1 and F6 is approximately 9.2 Å, and the distance between F4 and F6 is approximately 10.3 Å.
FIG. 14A-C shows the levels of cytosolic and nuclear β-catenin as measured by immunoblotting (FIG. 14A), and immunofluorescence microscopy (FIG. 14B) as compared to drug sensitive counterparts. The increased nuclear β-catenin was blocked using a dominant negative TCF4 construct (FIG. 14C).
FIG. 15A-E shows that in MES-SA cells, Wnt3a but not Wnt5a increased luciferase activity, which was blocked by cotransfection with a dominant negative TCF4 construct (FIG. 15A). Wnt5a conditioned media showed no enhancement of expression of the MDR-1/luciferase reporter construct (FIG. 15B). MDR-1 wild-type HCT-116 cells and Hβ18 (KO/*) cells is shown in FIG. 15C (MDR-1/luciferase activity) and FIG. 15D (RT-PCR). Recruitment of TCF4 and β-catenin to the MDR-1 promoter is shown in FIG. 15E.
FIG. 16A-E shows the effect of ICG-001 on transcriptional regulation of the MDR-1 gene in MES-SA cells: MDR-1/luciferase activity (16A); MDR-1 protein expression by immunofluorescence (16B) and immunoblotting (16C); message level by RT-PCR in MES-SA/Dx5 cells (16D) and K562 cells (16E).
FIG. 17A-C shows MDR-1 transcriptional regulation in HCT116 cell lines: MDR-1/luciferase expression (17A); effect of ICG-001 (17B); and blocking occupancy of the MDR-1 promoter by CBP (17C).
FIG. 18A-E shows the mRNA level of endogenous CBPP coactivator compared to p300 (FIG. 18A); the level of CBP (FIG. 18B); the association of β-catenin with p300 (FIG. 18C); the level of p300 (FIG. 18D); and the effect of p300 siRNA (FIG. 18E).
FIG. 19A-F compares MES-SA/Dx5 cells with K562 cells: growth rate (19A, 19B); message levels for survivin and cyclin D1 (19C, 19D); and protein levels for survivin and cyclin D1 (19E, 19F).
FIG. 20. RT-PCR shows an increased expression of Oct 4, hTert, Bmi-1 and ABCG-2 in MES-SA/Dx5 and K562 cells. Protein levels for Oct 4 and CD133 were increased in these cell lines.
FIG. 21A-D. FIG. 21A shows that ICG-001 in combination with the respective chemotherapeutic agent was more effective that the chemotherapeutic agent alone or ICG-001 alone in decreasing cell proliferation/viability. FIG. 21B: ICG does not effect CD34+ normal hematopoeitic cells. FIG. 21C: ICG-001* aka PRI-004 completely blocks colony formation at 500 nM concentration. FIG. 21D shows that combination treatment with ICG-001 and imatinib reduced colony forming units more than did either drug treatment alone.
FIG. 22A-E. The effect of ICG-001 at different doses, with and without imatinib, is shown in FIG. 22A and 22B. FIG. 22C and D: RT-PCR analysis for Beta-Catenin, BMI-1, MDR-1, ABCG1, survivin and survivin splice variant delta Ex3in CD34+ cells isolated form bone marrow from an imatinib naïve CML blast crisis patient. Reference is CD34− cells from the same patient. FIG. 22D: colony formation assay with CD34+ cells from an imatinib naïve blast crisis CML patient. FIG. 22E: hematoxylin and eosin staining for CD34+ blasts treated with 0.5 μM imatinib alone (top) or in combination with ICG-001 5 μM.
FIG. 23. FIG. 23 shows the sensitivity of IGROV-1 (FIG. 23A), A2780 (FIG. 23B) and CP70 (FIG. 23C) to ICG-001, as tested in repeat experiments with different concentrations.
FIG. 24. FIG. 24 shows the sensitivity of ovarian cell lines A2780 and CP70 to ICG-001.
FIG. 25. FIG. 25 shows that increasing concentrations of compounds PRI-001, PRI-002, PRI-003, PRI-004, PRI-005, and PRI-006 were effective, as compared with ICG-001, on SW480 cells.
FIG. 26. FIG. 26 shows pluc-6270 expression (luciferase) in SW480 cells treated with varying concentrations of ICG-001, PRI-003, and PRI-004.
FIG. 27 shows the chemical structures of Compounds 2203-2217.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to conformationally constrained compounds which mimic the secondary structure of α-helix regions of biological peptide and proteins (also referred to herein as “α-helix mimetics” and chemical libraries relating thereto, for the inhibition and/or eradication of cancer cells, particularly cancer cells having significant self-renewal potential, such as cancer stem cells.
Although there have been remarkable advances in the development of molecularly targeted drugs against cancer, for example imantinib (Gleevec) for the treatment of chronic phase CML, these agents in the end often fail. It is clear that new agents are needed to eradicate the cancer stem cells—literally the root of the problem.
Some parallels can be drawn between somatic stem cells and cancer stem cells (Pardal et al. Nat. Rev. Cancer. 3, 895, 2003). Both somatic stem cells and cancer stem cells are endowed with the ability to self renew and to differentiate. However, crucial differences exist. Whereas somatic stem cells differentiate to normal tissues, cancer stem cells differentiate aberrantly (Reya et al, Nature 2001, 414, 105-111). Despite the clonal origin of many cancers, most primary tumors display a notable degree of cellular heterogeneity. Thus, although modern chemotherapies kill a majority of the cells in a tumor, it is believed that the cancer stems cells often remain. ATP-binding cassette (ABC) multidrug resistance (MDR) transporters are believed to play important roles in protecting cancer stem cells from chemotherapy (Dean et al, Nat. Rev. Cancer 5, 275, 2005). The overexpression of P-glycoprotein (Pgp), energy-dependent efflux pumps of a variety of chemotherapeutic agents, resulting in multidrug resistant tumor cells was first demonstrated over two decades ago (Ling V. Cancer Chemother. Pharm. 40, S3-8, 1997; Sharom, F. J. J. Membr. Biol. 160, 161-175, 1997). MDR1 is a “TATA-less” gene, which belongs to a group of proteins whose genes lack a consensus TATA box within the proximal promoter region (Cornwell, M. M. Cell Growth Differ. 1, 607-615, 1990). Cells selected for their resistance to drugs often exhibit constitutive overexpression of MDR1. Additionally, efflux of Hoechst 33342 from normal murine hematopoietic cells identifies a “side population” (SP(+)) of negatively staining cells that are enriched for primitive progenitors (Feuring-Buske M., et al., Blood, 15:3882-9, 2001).
Mutations in the gene APC (adenomatous polyposis coli), which is a common early event in the majority of both hereditary and sporadic colorectal cancer, leads to the nuclear accumulation of β-catenin where it forms a complex with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF-1) family of transcription factors (8). To generate a transcriptionally active complex, β-catenin recruits the transcriptional coactivators Creb-Binding Protein (CBP) or its closely related homolog, p300 (9, 10) as well as other components of the basal transcription machinery. The MDR1 promoter contains several TCF/LEF binding sites between positions −275 and −1813. A link between APC mutations and enhanced MDR-1 expression via TCF/β-catenin driven transcription has been described (Yamada T., et al. Cancer Res. 60, 4761-4766, 2000).
It is becoming apparent that despite their high degree of homology and similar patterns of expression, CBP and p300 play unique and distinct roles in gene regulation. Data disclosed herein were generated using siRNA, ChIP assay and the chemogenomic tool ICG-001, which selectively disrupts the β-catenin/CBP interaction but not the corresponding β-catenin/p300 interaction (Emami et al PNAS, 2004) thereby interfering with a subset of Wnt/1-catenin regulated gene expression including survivin (Ma et al Oncogene 2005). The present disclosure demonstrates that TCF/β-catenin/CBP driven gene expression is essential for MDR-1 transcription. Furthermore, in the broader context, the disclosure shows that a CBP/β-catenin driven transcriptional cassette is critical for the expression of a “cancer stem cell-like” profile.
Embryonic stem cells can proliferate readily, in vitro and in vivo. In vivo, they can form teratocarcinoma-like tumors in adult mice if injected subcutaneously, intramuscularly, or into the testis. Thomson, J. A., et al., Science 282:1145-7:1998; Odorico, J. S., Stem Cells 19:193-204, 2001; Chung, Y., et al., Nature 439:216-9, 2006. Thus, hES cell-based therapy may lead to unwanted tumor formation.
To eliminate contamination of transplant material with residual undifferentiated ES cells, two different approaches have been reported. In one case, ES cell-specific expression in an engineered cell line of a compound that is toxic to undifferentiated ES cells is used and the culture conditions are modified to allow expression. This approach was used to eliminate mouse ES cells from a mixed cell population prior to transplant, Billon, N., et al., J Cell Sci, 115: 3657-65, 2002, and to express a suicide gene in the differentiated stem cells following transplantation, Schuldiner, M., J., Stem Cells 21:257-65, 2003. In another approach, the mixed cell population is treated with the ceramide analogue N-oleoyl serinol (S18) to selectively induce apoptosis of ES cells, Bieberich, E., et al., J Cell Biol. 167:723-34, 2004. In this case, subsequent teratocarcinoma formation following transplantation of mixed populations containing both ES stem and ES-derived neural stem cells was prevented, Bieberich, E., et al., J Cell Biol 167:723-34, 2004.
The compounds and methods disclosed herein provide another option for eliminating teratoma-forming stem cells prior to transplant. An advantage is that the treatment used a small molecule that has no toxicity in humans at the doses that would be used.
The synthesis and identification of conformationally constrained α-helix mimetics and their application to diseases are discussed in Walensky, L. D. et al Science 305, 1466, 2004; and Klein, C. Br. J Cancer. 91, 1415, 2004. This disclosure further demonstrates that in conjunction with other chemotherapeutic agents, targeting cancer stem cells by antagonizing the CBP/β-catenin interaction not only eliminates the cancer stem cells which are resistant to normal chemotherapy, but also has an additive effect on the killing of other cancer cells that are normally sensitive to chemotherapy, by decreasing the transcription of anti-apoptotic genes such as survivin.
As shown in detail in the examples, compounds disclosed herein ICG-001 reduced MDR-1/luciferase activity in a doxorubin-resistant ovarian sarcoma line MES-SA/Dx5 and in the CML derived cell line K562. In these cell lines, there is an increased level of cytosolic and nuclear β-catenin. This activated Wnt/β-catenin pathway leads in twin to activation of the multiding resistance gene (MDR-1) in the cell lines.
By reducing MDR-A/luciferase activity, ICG-001 was a candidate for tsting against patient CML cells. The examples further show that ICG-001 in combination with imatinib reduced total colony forming units in comparison with either drug alone. Morphological examination showed that the treated colonies had an increased state of differentiation.
In addition to being effective against ovarian sarcoma and CML cells, ICG-001 reduced stem cell markers in cells for other ovarian cell lines and melanoma B 16 cells. ICG-100 and several other compounds, including PRI-001, PRI-002, PRI-003, PRI-004, PRI-005, and PRI-006 inhibited β-catenin interaction with CBP in SW480 cells, a cell line derived from intestinal carcinoma.
The wide range of cancers amenable to treatment with the compounds disclosed herein is consistent with β-catenin's role in several cancer-related events. These include expression of survivin, expression of MDR-1, and maintenance of a cancer stem cell population.
The compounds and methods herein are therefore suitable for treating cancers including but not limited to acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervix cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer, penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginal cancer, cancer of the vulva, and Wilm's tumor.
The α-helix mimetic structures of the present invention are useful as bioactive agents, including (but not limited to) use as diagnostic, prophylactic and/or therapeutic agents. The α-helix mimetic structure libraries of this invention are useful in the identification of such bioactive agents. In the practice of the present invention, the libraries may contain from tens to hundreds to thousands (or greater) of individual α-helix structures (also referred to herein as “members”).
In one aspect of the present invention, a α-helix mimetic structure is disclosed having the following formula (I):

wherein A is —(C═O)—CHR₃—, or —(C═O), B is N—R₅— or —CHR₆—, D is —(C═O)—(CHR₇)— or —(C═O)—, E is -(ZR₈)— or (C═O), G is —(XR₉)_n—, —(CHR₁₀)—(NR₆)—,—(C═O)—(XR₁₂)—, —(C═N—W—R₁)—, —(C═O)—, X—(C═O)—R₁₃, X—(C═O)—NR₁₃R₁₄, X—(SO₂)—R₁₃, or X—(C═O)—OR₁₃, W is —Y(C═O)—, —(C═O)NH—, —(SO₂)—, —CHR₁₄, (C═O)—(NR₁₅)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=0 or 1; and R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers thereof.
More specifically, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅are independently selected from the group consisting of aminoC_2-5alkyl, guanidineC_2-5alkyl, C_1-4alkylguanidinoC_2-5alkyl, diC_1-4alkylguanidino-C_2-5alkyl, amidinoC_2-5alkyl, C_1-4alkylamidino C_2-5alkyl, diC_1-4alkylamidinoC_2-5alkyl, C_1-3alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl (where the substituents on the benzyl are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-3alkyl, nitro, carboxy, cyano, sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), bisphenyl methyl, substituted bis-phenyl methyl (where the subsitituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridyl, substituted pyridyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC_1-4alkyl, substituted pyridylC_1-4alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC_1-4alkyl, substituted pyrimidylC_1-4alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C_1-4alkyl, substituted triazin-2-yl-C_1-4alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC_1-4alkyl, substituted imidazol C_1-4alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C_1-4alkylamino, C_1-4dialkylamino, halogen, perfluoro C_1-4alkyl, C_1-4alkyl, C_1-3alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl or methyl), imidazolinylCalkyl, N-amidinopiperazinyl-N—C_0-4alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, hydroxyC_2-5alkyl, C_1-5alkylaminoC_2-5alkyl, C_1-5dialkylaminoC_2-5alkyl, N-amidinopiperidinylC_1-4alkyl and 4-aminocyclohexylC_0-2alkyl.
In one embodiment, R₁, R₂, R₆of E, and R₇, R₈and R₉of G are the same or different and represent the remainder of the compound, and R₃or A, R₄of B or R₅of D is selected from an amino acid side chain moiety or derivative thereof. As used herein, the term “remainder of the compound” means any moiety, agent, compound, support, molecule, linker, amino acid, peptide or protein covalently attached to the α-helix mimetic structure at R₁, R₂, R₅, R₆, R₇, R₈and/or R₉positions. This term also includes amino acid side chain moieties and derivatives thereof.

As used herein, the term “amino acid side chain moiety” represents any amino acid side chain moiety present in naturally occurring proteins including (but not limited to) the naturally occurring amino acid side chain moieties identified in Table 1. Other naturally occurring amino acid side chain moieties of this invention include (but are not limited to) the side chain moieties of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine, γ-carboxyglutamate, phosphotyrosine and phosphoserine. In addition, glycosylated amino acid side chains may also be used in the practice of this invention, including (but not limited to) glycosylated threonine, serine and asparagine.

TABLE 1


Amino Acid Side Chain Moieties

	Amino Acid Side Chain Moiety	Amino Acid

	—H	Glycine
	—CH₃	Alanine
	—CH(CH₃)₂	Valine
	—CH₂CH(CH₃)₂	Leucine
	—CH(CH₃)CH₂CH₃	Isoleucine
	—(CH₂)₄NH₃ ⁺	Lysine
	—(CH₂)₃NHC(NH₂)NH₂ ⁺	Arginine
		Histidine
	—CH₂COO⁻	Aspartic acid
	—CH₂CH₂COO⁻	Glutamic acid
	—CH₂CONH₂	Asparagine
	—CH₂CH₂CONH₂	Glutamine
		Phenylalanine
		Tyrosine
		Tryptophan
	—CH₂SH	Cysteine
	—CH₂CH₂SCH₃	Methionine
	—CH₂OH	Serine
	—CH(OH)CH₃	Threonine
		Proline
		Hydroxyproline

In addition to naturally occurring amino acid side chain moieties, the amino acid side chain moieties of the present invention also include various derivatives thereof. As used herein, a “derivative” of an amino acid side chain moiety includes modifications and/or variations to naturally occurring amino acid side chain moieties. For example, the amino acid side chain moieties of alanine, valine, leucine, isoleucine and pheylalanine may generally be classified as lower chain alkyl, aryl, or arylalkyl moieties. Derivatives of amino acid side chain moieties include other straight chain or brached, cyclic or noncyclic, substitutes or unsubstituted, saturated or unsaturated lower chain alkyl, aryl or arylalkyl moieties.
As used herein, “lower chain alkyl moieties” contain from 1-12 carbon atoms, “lower chain aryl moieties” contain from 6-12 carbon atoms and “lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, in one embodiment, the amino acid side chain derivative is selected from a C_1-2alkyl, a C_6-12aryl and a C_7-12arylalkyl, and in a more preferred embodiment, from a C_1-7alkyl, a C_6-10aryl and a C_7-11arylalkyl.
Amino side chain derivatives of this invention further include substituted derivatives of lower chain alkyl, aryl, and arylalkyl moieties, wherein the substituents is selected from (but are not limited to) one or more of the following chemical moieties: —OH, —OR, —COOH, —COOR, —CONH₂, —NH₂, —NHR, —NRR, —SH, —SR, —SO₂R, —SO₂H, —SOR and halogen (including F, Cl, Br and I), wherein each occurrence of R is independently selected from straight chain or branched, cyclic or noncyclic, substituted or unsubstituted, saturated or unsaturated lower chain alkyl, aryl, and aralkyl moieties. Moreover, cyclic lower chain alkyl, aryl and arylalkyl moieties-of this invention include naphthalene, as well as heterocyclic compounds such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole. Amino acid side chain derivatives further include heteroalkyl derivatives of the alkyl portion of the lower chain alkyl and aralkyl moieties, including (but not limited to) alkyl and aralkyl phosphonates and silanes.
Representative R₁, R₂, R₅, R₆, R₇, R₈and R₉moieties specifically include (but are not limited to) —OH, —OR, —COR, —COOR, —CONH₂, —CONR, —CONRR, —NH₂, —NHR, —NRR, —SO₂R and —COSR, wherein each occurrence of R is as defined above.
In a further embodiment, and in addition to being an amino acid side chain moiety or derivative thereof (or the remainder of the compound in the case of R₁, R₂, R₅, R₆, R₇, R₈and R₉), R₁, R₂, R₅, R₆, R₇, R₈or R₉may be a linker facilitating the linkage of the compound to another moiety or compound. For example, the compounds of this invention may be linked to one or more known compounds, such as biotin, for use in diagnostic or screening assay. Furthermore, R₁, R₂, R₅, R₆, R₇, R₈or R₉may be a linker joining the compound to a solid support (such as a support used in solid phase peptide synthesis) or alternatively, may be the support itself. In this embodiment, linkage to another moiety or compound, or to a solid support, is preferable at the R₁, R₂, R₇or R₈position, and more preferably at the R₁or R₂position.
In the embodiment wherein A is —(C═O)—CHR₃—, B is —N—R₄, D is —(C═O)—, E is -(ZR₆)—, G is —(C═O)—(XR₉)—, the α-helix mimetic compounds of this invention have the following general formula (III):

wherein R₁, R₂, R₄, R₆, R₇, R₈, W and X are as defined above, Y is —C═O, —(C═O)—O—, —(C═O)—NR₈, —SO₂—, or nothing, and Z is nitrogen or CH (when Z is CH, then X is nitrogen). In a preferred embodiment, R₁, R₂, R₆, R₇and R₈represent the remainder of the compound, and R₄is selected from an amino acid side chain moiety. In a more specific embodiment wherein A is —O—CHR₃—, B is —NR₄—, D is —(C═O)—, E is -(ZR₆)—, Gi is (XR₇)_n—, the α-helix mimetic compounds of this invention have the following formula (IV):

wherein R₁, R₂, R₄, R₆, R₇, W, X and n are as defined above, and Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero). In a preferred embodiment, R₁, R₂, R₆, and R₇represent the remainder of the compound, and R₄is selected from an amino acid side chain moiety. In this case, R₆or R₇may be selected from an amino acid side chain moiety when Z and X are CH, respectively.
In the embodiment of structure (I) wherein A is —(C═O), B is —(CHR₆)—, D is —(C═O)—, E is -(ZR₈)—, and G is —(NH)— or —(CH₂)—, and W is a substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, the α-helix mimetic compounds of this invention have the following general formula (V):

wherein K is nitrogen, oxygen, or sulfur, L is nitrogen, oxygen, —(CH)—, or —(CH₂)—, J is nitrogen, oxygen, or sulfur, Z is nitrogen or CH, and R₁, R₂, R₆, R₈, and R₁₃are selected from an amino acid side chain moiety.
Alternative embodiments of the invention relate to compounds having the general formula (VI):

wherein B is —(CHR₃)—, —(NR₃)—, E is —(CHR₄)—, V is —(XR₅)— or nothing, W is —(C═O)—(XR₆R₇), —(SO₂)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, X is indepentently nitrogen, oxygen, or CH, and R₁, R₂, R₃, R₄, R₅, R₆, and R₇are selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and solid support, and stereoisomers thereof.
In the embodiments of formula (VI) wherein V is —(XR₅)— or nothing, and W is substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, and X is independently introgen or CH, the compounds have the following general formula (Vll):

wherein K is nitrogen, oxygen, or sulfur, L is nitrogen, oxygen, —(CH)—, or —(CH₂)—, J is nitrogen, oxygen, or sulfur, and R₂and R₅are defined as described above.
In preferred embodiments of the invention, R₂in structures I through VII comprises an aromatic ring substituent such as a phenyl or naphthyl group that is substituted with a basic moiety such a primary or secondary amine. The aromatic ring substituent may also be a heterocycle, such as a purine or indole. Some embodiments of the invention also provide for aromatic ring substituents that may be substituted with one or two halogen moieties.
A feature of many α-helix mimetic compounds is that they provide a scaffolding that places three hydrophobic functional groups, which may also be referred to as pharmacophore rings, in a specific, spatially-defined orientation referred to as an “optimized chemical space”. The optimized chemical space may be triangular, with the centers of three functional groups forming the three points of the triangle. An example of an optimized chemical space is one in which the lengths of the three sides of the triangle are around 9.6±0.5 Angstroms (symbolized hereafter by “A”), 9.2±0.5 Å, and 10.3±0.5 Å. FIG. 13C depicts two superimposed structures having three such pharmacophore rings forming a triangle in space. A number of different compounds exhibit such an optimized chemical space, and may be considered to be within the scope of the invention.
The compounds of general formula (I) of the present invention have one or more asymmetric carbons depending on it's substituents. For example, where the compounds of general formula (I) contains one or more asymmetric carbons, two kinds of optical isomers exist when the number of asymmetric carbon is 1, and when the number of asymmetric carbon is 2, four kinds of optical isomers and two kinds of diastereomers exist. Pure stereoisomers including opticalisomers and diastereoisomers, any mixture, racemates and the like of stereoisomers all fall within the scope of the present invention. Mixtures such as racemates may sometimes be preferred from viewpoint of easiness for manufacture.
When the compounds of general formula (I) of the present invention contains a basic functional group such as amino group, or when the compounds of general formula (I) of the present invention contains an aromatic ring which itself has properties of base (e.g., pyridine ring), the compound can be converted into a pharmaceutically acceptable salt (e.g., salt with inorganic acids such as hydrochloric acid and sulfuric acid, or salts with organic acids such as acetic acid and citric acid) by a known means. When the compounds of general formula (I) of the present invention contains an acidic functional group such as carboxyl group or phenolic hydroxyl group, the compound can be converted into pharmaceutically acceptable salt (e.g., inorganic salts with sodium, ammonia and the like, or organic salts with triethylamine and the like) by a known means. When the compounds of general formula (I) of the present invention contains a prodrugable functional group such as phenolic hydroxyl group, the compound can be converted into prodrug (e.g., acetylate or phosphonate) by a known means. Any pharmaceutically acceptable salt and prodrug all fall within the scope of the present invention.
The various compounds disclosed by the present invention can be purified by known methods such as recrystallization, and variety of chromatography techniques (column chromatography, flash column chromatography, thin layer chromatography, high performance liquid chromatography).
The α-helix mimetic structures of the present invention may be prepared by utilizing appropriate starting component molecules (hereinafter referred to as “component pieces”). Briefly, in the synthesis of α-helix mimetic structures having formula (II), first and second component pieces are coupled to form a combined first-second intermediate, if necessary, third and/or fourth component pieces are coupled to form a combined third-fourth intermediate (or, if 1.5 commercially available, a single third intermediate may be used), the combined first-second intermediate and third-fourth intermediate (or third intermediate) are then coupled to provide a first-second-third-fourth intermediate (or first-second-third intermediate) which is cyclized to yield the α-helix mimetic structures of this invention. Alternatively, the α-helix mimetic structures of formula (II) may be prepared by sequential coupling of the individual component pieces either stepwise in solution or by solid phase synthesis as commonly practiced in solid phase peptide synthesis.
Within the context of the present invention, a “first component piece” has the following formula S1

Wherein R₂as defined above, and R is a protective group suitable for use in peptide synthesis. Suitable R groups include alkyl groups and, in a preferred embodiment, R is a methyl group. Such first component pieces may be readily synthesized by reductive amination or substitution reaction by displacement of H₂N—R₂from CH(OR)₂—CHO or CH(OR)₂—CH₂-Hal (wherein Hal means a halogen atom).
A “second component piece” of this invention has the following formula S2:

Where L₁is carboxyl-activation group such as halogen atom, R₃, R₄is as defined above, and P is an amino protective group suitable for use in peptide synthesis. Preferred protective groups include t-butyl dimethylsilyl (TBDMS), t-Butyloxycarbonyl (BOC), Methylosycarbonyl (MOC), 9H-Fluorenylmethyloxycarbonyl (FMOC), and allyloxycarbonyl (Alloc). When L is —C(O)NHR, —NHR may be an carboxyl protective group. N-Protected amino acids are commercially available. For example, FMOC amino acids are available for a variety of sources. The conversion of these compounds to the second component pieces of this invention may be readily achieved by activation of the carboxylic acid group of the N-proctected amino acid. Suitable activated carboxylic acid groups include acid halides where X is a halide such as chloride or bromide, acid anhydrides where X is an acyl group such as acetyl, reactive esters such as an N-hydroxysuccinimide esters and pentafluorophenyl esters, and other activated intermediates such as the active intermediate formed in a coupling reaction using a carbodiimide such as dicyclohexylcarbodiimide (DCC).
In the case of the azido derivative of an amino acid serving as the second component piece, such compounds may be prepared from the corresponding amino acid by the reaction disclosed by Zaloom et al. (J. Org. Chem. 46:5173-76, 1981).
A “third component piece” of this invention has the following formula S3:

where G, E, and L₁are as defined above. Suitable third component pieces are commercially available from a variety of sources or can be prepared by known methods in organic chemistry.
More specifically, the α-helix mimetic structures of this invention of formula (II) are synthesized by reacting a first component piece with a second component piece to yield a combined first-second intermediate, followed by either reacting the combined first-second intermediate with third component pieces sequentially to provide a combined first-second-third-fourth intermediate, and the cyclizing this intermediate to yield the α-helix mimetic structure.
The general synthesis of an α-helix having structure I′ may be carried out by the following technique. A first component piece I is coupled with a second component piece 2 by using coupling reagent such as phosgene to yield, after N-deprotection, a combined first-second intermediate 1-2 as illustrated below:

wherein R₁, R₂, R₄, R₇.Fmoc, Moc and X are as defined above, and Pol represents a polymeric support.
The synthesis of representative component pieces of this invention are described in the Examples.
The α-helix mimetic structures of formula (III) and (IV) may be made by techniques analogous to the modular component synthesis disclosed above, but with appropriate modifications to the component pieces.
As mentioned above, the reverse-turn mimetics of U.S. Pat. No. 6,013,458 to Kahn, et al. are useful as bioactive agents, such as diagnostic, prophylactic, and therapeutic agents. The opiate receptor binding activity of representative reverse-turn mimetics is presented in Example 9 of said U.S. Pat. No. 6,013,458, wherein the reverse-turn mimetics of this invention were found to effectively inhibit the binding of a radiolabeled enkephalin derivative to the δ and μ opiate receptors, of which data demonstrates the utility of these reverse-turn mimetics as receptor agonists and as potential analgesic agents.
The α-helix mimetic structures of the present invention will be useful as bioactive agents, such as diagnostic, prophylactic, and therapeutic agents.
Therefore, since the compounds according to the present invention are of α-helix mimetic structures, it may be useful for modulating a cell signaling transcription factor related peptides in a warm-blooded animal, comprising administering to the animal an effective amount of the compound of formula (I). Besides being useful for human treatment, the compounds of the present invention are also useful for veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to dogs, cats, horses, cows, sheep, and pigs.
Further, the α-helix mimetic structures of the present invention may also be effective for inhibiting transcription factor/coactivator and transcription factor corepressor interactions.
In another aspect of this invention, libraries containing α-helix mimetic structures of the present invention are disclosed. Once assembled, the libraries of the present invention may be screened to identify individual members having bioactivity. Such screening of the libraries for bioactive members may involve, for example, evaluating the binding activity of the members of the library or evaluating the effect the library members have on a functional assay. Screening is normally accomplished by contacting the library members (or a subset of library members) with a target of interest, such as, for example, an antibody, enzyme, receptor or cell line. Library members, which are capable of interacting with the target of interest, are referred to herein as “bioactive library members” or “bioactive mimetics”. For example, a bioactive mimetic may be a library member which is capable of binding to an antibody or receptor, which is capable of inhibiting an enzyme, or which is capable of eliciting or antagonizing a functional response associated, for example, with a cell line. In other words, the screening of the libraries of the present invention determines which library members are capable of interacting with one or more biological targets of interest. Furthermore, when interaction does occur, the bioactive mimetic (or mimetics) may then be identified from the library members. The identification of a single (or limited number) of bioactive mimetic(s) from the library yields α-helix mimetic structures which are themselves biologically active, and thus useful as diagnostic, prophylactic or therapeutic agents, and may further be used to significantly advance identification of lead compounds in these fields.
In another aspect of this invention, methods for constructing the libraries are disclosed. Traditional combinatorial chemistry techniques (see, e.g., Gallop et al., J. Med. Chem. 37:1233-1251, 1994) permit a vast number of compounds to be rapidly prepared by the sequential combination of reagents to a basic molecular scaffold. Combinatorial techniques have been used to construct peptide libraries derived from the naturally occurring amino acids. For example, by taking 20 mixtures of 20 suitably protected and different amino acids and coupling each with one of the 20 amino acids, a library of 400 (i.e., 20²) dipeptides is created. Repeating the procedure seven times results in the preparation of a peptide library comprised of about 26 billion (i.e., 20⁸) octapeptides.
Specifically, synthesis of the peptide mimetics of the library of the present invention may be accomplished using known peptide synthesis techniques, for example, the General Scheme of [4,4,0] α-helix Mimetic Library as follows:
thesis of the peptide mimetics of the libraries of the present invention was accomplished using a FlexChem Reactor Block which has 96 well plates by known techniques. In the above scheme ‘Pol’ represents a bromoacetal resin (Advanced ChemTech) and detailed procedure is illustrated below.
Step 1
A bromoacetal resin (37 mg, 0.98 mmol/g) and a solution of R₂-amine in DMSO (1.4 mL) were placed in a Robbins block (FlexChem) having 96 well plates. The reaction mixture was shaken at 60° C. using a rotating oven [Robbins Scientific] for 12 hours. The resin was washed with DMF, MeOH, and then DCM
Step 2
A solution of available Fmoc hydrazine Amino Acids (4 equiv.), PyBop (4 equiv.), HOAt (4 equiv.), and DIEA (12 equiv.) in DMF was added to the resin. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM.
Step 3
To the resin swollen by DMF before reaction was added 25% piperidine in DMF and the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and the resin was washed with DMF, Methanol, and then DCM. A solution of hydrazine acid (4 equiv.), HOBt (4 equiv.), and DIC (4 equiv.) in DMF was added to the resin and the reaction mixture was shaken for 12 hours at room temperature. The resin was washed with DMF, MeOH, and then DCM.
Step 4a (Where Hydrazine Acid is MOC Carbamate)
The resin obtained in Step 3 was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure using SpeedVac [SAVANT] to give the product as oil. The product was diluted with 50% water/acetonitrile and then lyophilized after freezing.
Step 4b (Where Fmoc Hydrazine Acid is Used to Make Urea Through Isocynate)
To the resin swollen by DMF before reaction was added 25% piperidine in DMF and the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and the resin was washed with DMF, Methanol, then DCM. To the resin swollen by DCM before reaction was added isocynate (5 equiv.) in DCM. After the reaction mixture was shaken for 12 hours at room temperature the resin was washed with DMF, MeOH, then DCM. The resin was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure using SpeedVac [SAVANT] to give the product as oil. The product was diluted with 50% water/acetonitrile and then lyophilized after freezing.
Step 4c (Where Fmoc-Hydrazine Acid is Used to Make Urea Through Active Carbamate)
To the resin swollen by DMF before reaction was added 25% piperidine in DMF and the reaction mixture was shaken for 30 min at room temperature. This deprotection step was repeated again and the resin was washed with DMF, MeOH, and then DCM. To the resin swollen by DCM before reaction was added p-nitrophenyl chloroformate (5 equiv.) and diisopropyl ethylamine (5 equiv.) in DCM. After the reaction mixture was shaken for 12 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM. To the resin was added primary amines in DCM for 12 hours at room temperature and the resin was washed with DMF, MeOH, and then DCM. After reaction the resin was treated with formic acid (1.2 mL each well) for 18 hours at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure using SpeedVac [SAVANT] to give the product as oil. The product was diluted with 50% water/acetonitrile and then lyophilized after freezing.
To generate these block libraries the key intermediate hydrazine acids were synthesized according to the procedure illustrated in the examples.
Administration and Dosage
The inventive compounds may be administered by any means known to one of ordinary skill in the art. For example, the inventive compounds may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial, and intraosseous injection and infusion techniques. The exact administration protocol will vary depending upon various factors including the age, body weight, general health, gender and diet of the patient; the determination of specific administration procedures would be routine to an one of ordinary skill in the art.
The inventive compounds may be administered by a single dose, multiple discrete doses or continuous infusion. Pump means, particularly subcutaneous pump means, are useful for continuous infusion.
Dose levels on the order of about 0.001 mg/kg/d to about 100 mg/kg/d of an inventive compound are useful for the inventive methods. In one embodiment, the dose level is about 0.1 mg/kg/d to about 100 mg/kg/d. In another embodiment, the dose level is about 1 mg/kg/d to about 10 mg/kg/d. The specific dose level for any particular patient will vary depending upon various factors, including the activity and the possible toxicity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; the drug combination; the severity of the disease; and the form of administration. Typically, in vitro dosage-effect results provide useful guidance on the proper doses for patient administration. Studies in animal models are also helpful. The considerations for determining the proper dose levels are well known in the art and within the skills of an ordinary physician.
Any known administration regimen for regulating the timing and sequence of drug delivery may be used and repeated as necessary to effect treatment in the inventive methods. The regimen may include pretreatment and/or co-administration with additional therapeutic agent(s).
The inventive compounds can be administered alone or in combination with one or more additional therapeutic agent(s) for simultaneous, separate, or sequential use. Examples of an additional therapeutic agent include, without limitation, compounds of this invention; steroids (e.g., hydrocortisones such as methylprednisolone); anti-inflammatory or anti-immune drug, such as methotrexate, azathioprine, cyclophosphamide or cyclosporin A; interferon-β; antibodies, such as anti-CD4 antibodies; chemotherapeutic agents; immunotherapeutic compositions; electromagnetic radiosensitizers; and morphine. The inventive compounds may be co-administered with one or more additional therapeutic agent(s) either (i) together in a single formulation, or (ii) separately in individual formulations designed for optimal release rates of their respective active agent.
The pharmaceutical composition may comprise at least one compound disclosed herein, in combination with at least one cancer chemotherapeutic wherein said cancer chemotherapeutic works by a mechanism other than blocking CPB/catenin interaction. The cancer therapeutic can be selected from the group consisting of, but not limited to, cis-platinum, retinoic acid, histone deacetylase (HDAC) inhibitors such as Vorinostat (SAHA), and imatinib.
The pharmaceutical composition may comprise at least one pathway-specific inhibitor such as Her1/Her2 inhibitors; Notch inhibitors; Hedgehog inhibitors; EGF inhibitors; and PI3K pathway inhibitors. The Notch inhibitor can be a gamma secretase inhibitor, the Hedgehog inhibitor can be cyclopamine, the EGF inhibitor can be Iressa, and the PI3K pathway inhibitor can be rapamycin.
Pharmaceutical Compositions
This invention further provides a pharmaceutical composition comprising: (i) an effective amount of a compound of formula I, II or III; and (ii) a pharmaceutically acceptable carrier.
The inventive pharmaceutical composition may comprise one or more additional pharmaceutically acceptable ingredient(s), including without limitation one or more wetting agent(s), buffering agent(s), suspending agent(s), lubricating agent(s), emulsifier(s), disintegrant(s), absorbent(s), preservative(s), surfactant(s), colorant(s), flavorant(s), sweetener(s) and additional therapeutic agent(s).
The inventive pharmaceutical composition may be formulated into solid or liquid form for the following: (1) oral administration as, for example, a drench (aqueous or non-aqueous solution or suspension), tablet (for example, targeted for buccal, sublingual or systemic absorption), bolus, powder, granule, paste for application to the tongue, hard gelatin capsule, soft gelatin capsule, mouth spray, emulsion and microemulsion; (2) parenteral administration by, for example, subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution, suspension or sustained-release formulation; (3) topical application as, for example, a cream, ointment, or controlled-release patch or spray applied to the skin; (4) intravaginal or intrarectal administration as, for example, a pessary, cream or foam; (5) sublingual administration; (6) ocular administration; (7) transdermal administration; or (8) nasal administration.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

EXAMPLE 1

Intermediate Synthesis

Synthesis of 2-Boc-amino-benzothiazoleyl-4-methylamine

Step-1 (2-Boc-amino-4-methyl benzothiazole)

A solution of 2-Amino-4-methyl benzothiazole (25.0 g, 152 mmol) in 456 mL of dry THF was treated with Et₃N (42 mL, 300 mmol), (Boc)₂O (40.0 g, 183 mmol) and DMAP (3.7 g, 30 mmol) at 20° C. and stirred at 30° C. for 12 h. The resulting solution was concentrated in vacuo, diluted with EtOAc (200 mL) and filtered through a glass filter (Celite) washing with EtOAc (200 mL). The filtrate was washed with NaHCO₃(saturated aqueous solution, 100 mL) and NaCl (saturated aqueous solution, 100 mL), dried over MgSO₄and concentrated in vacuo. The residue was filtered through a silica gel plug (flash column chromathography) eluting with toluene:Et₂O=15:1 to 8:1 to afford 2-Boc-amino-4-methyl benzothiazole as a colorless oil (41.4 g, quant.) R_f=0.48 (toluene:Et₂O=10:1); ¹H NMR (400 MHz, CDCl₃) δ 9.75 (1H, br s), 7.61 (1H, d, J=7.8 Hz), 7.19 (3H, m), 2.64 (3H, s), 1.47 (9H, s).

Step-2 (2-Boc-amino-4-bromomethyl benzothiazole)

A solution of 2-Boc-amino-4-methyl benzothiazole (152 mmol) in 456 mL of dry CCl4 was treated with NBS (27.1 g, 152 mmol) and AIBN (3.2 g, 20 mmol) at 20° C. and stirred at 80° C. for 3.5 h. The mixture was retreated with NBS (7.2 g, 41 mmol) and AIBN (0.84 g, 5.1 mmol) at 20° C. and stirred at 80° C. for 11 hr. The resulting mixture was cooled to 20° C. and filtered through a glass filter (Celite) washing with Et₂O (200 mL). The filtrate was concentrated in vacuo. The residue was filtered through a silica gel column (flash column chromathography) eluting with toluene:Et₂O=20:1 to 10:1 to afford 2-BocNH-4-bromomethyl benzothiazole (46.7 g, 136 mmol, 90%) as a yellowish oil. R_f=0.51 (toluene:Et₂O=15:1); ¹H NMR (400 MHz, CDCl₃) δ 8.27 (1H, br s), 7.72 (1H, d, J=8.2 Hz), 7.43 (1H, d, J=7.2 Hz), 7.24 (1H, dd, J=8.2, 7.2 Hz), 4.91 (2H, s), 1.56 (9H, s).

Step-3 (2-Boc-amino-4-azidemethyl benzothiazole)

A solution of 2-Boc-amino-4-bromomethyl benzothiazole (46.7 g, 136 mmol) in 205 mL of dry DMF was treated with NaN₃(8.80 g, 136 mmol) at 15° C. and stirred at 20° C. for 45 min. The resulting mixture was diluted with Et₂O (400 mL), quenched by addition of NaCl (1 g in 150 mL of H₂O) at 0° C. The solution was extracted with Et₂O (100 mL). The organic phase was washed with NaCl (2 g in 100 mL of H₂O) twice, dried over MgSO₄and concentrated in vacuo. The residue was filtered through a silica gel plug (flash column chromathography) eluting with toluene:Et₂O=100:0 to 10:1 to afford 2-Boc-amino-4-azidemethyl benzothiazole (33.2 g, 109 mmol, 80%) as a colorless oil. R_f=0.48 (toluene:Et₂O=10:1); ¹H NMR (400 MHz, CDCl₃) δ 7.75 (1H, d, J=8.2 Hz), 7.37 (1H, d, J=7.2 Hz), 7.27 (1H, m), 4.74 (2H, s), 1.52 (9H, s); ¹³C NMR (99.5 MHz, CDCl₃) δ 159.8, 151.9, 147.6, 132.5, 127.6, 125.8, 123.5, 121.3, 83.4, 51.4, 28.1.

Step-4 (2-Boc-amino-benzothiazoleyl-4-methylamine)

A solution of 2-Boc-amino-4-azidemethyl benzothiazole (11.6 g, 38.0 mmol) in 183 mL of MeOH was treated with Pd(OH)₂(20% on carbon, 2.9 g), placed under an atmosphere of hydrogen and stirred at 20° C. for 1.5 hr. The resulting mixture was filtered through Celite washing with MeOH:NH₄OH (100:3, 100 mL) and concentrated in vacuo. The obtained yellowish solid was triturated with toluene (35 mL) and filtered to afford 2-Boc-amino-benzothiazoleyl-4-methylamine (6.90 g, 24.7 mmol, 65%) as a colorless powder. R_f=0.32 (CHCl₃:MeOH:NH₄OH=100:25:1); ¹H NMR (400 MHz, CDCl₃) δ 7.67 (1H, d, J=7.7 Hz), 7.25-7.15 (2H, m), 4.85 (2H, br s), 1.58 (9H, s); ¹³C NMR (99.5 MHz, CDCl₃) δ 160.0, 152.8, 148.0, 134.5, 132.7, 124.4, 123.1, 120.0, 82.4, 44.3, 28.3; LC/MS [ESI+] (m/z) 280.2 (M+1)⁺.

Synthesis of Benzothiazoleyl-4-methylamine

Step-1(4-Methyl benzothiazole)

A solution of 2-amino-4-methylbenzothiazolee (24.5 g, 149 mmol) in 745 mL of 1,4-dioxane was treated with isoamylnitrile (40.0 mL, 300 mmol) at 20° C. and stirred at 70° C. for 0.5 hr. After the nitrogen evolution had subsided, the mixture was stirred at the same temperature for 1.5 h and concentrated in vacuo. The residue was submitted to silica gel column chromathography with hexane:Et₂O=3:1 to 2:1 as eluate to afford 4-methyl benzothiazole as a yellowish oil. (16.0 g, 107 mmol, 72%) R_f=0.45 (toluene:Et₂O=10:1); ¹H NMR (400 MHz, CDCl₃) δ 8.98 (1H, s), 7.79 (1H, d, J=6.8 Hz), 7.33 (2H, m), 2.80 (3H, s).

Step-2 (4-Bromomethyl benzothiazole)

A solution of 4-Methyl benzothiazole (16.0 g, 107 mmol) in 535 mL of CCl₄was treated with NBS (19.0 g, 107 mmol) and AIBN (2.28 g, 13.9 mmol) at 20° C. and stirred at 70° C. for 2.5 h. The resulting mixture was filtered through Celite washing with Et₂O (150 mL) and concentrated in vacuo. The residue was submitted to a silica gel column chromatography with toluene:Et₂O=50:3 to 50:5 as eluate to afford 4-bromomethyl benzothiazole as a yellowish solid. (20.4 g, 89.9 mmol, 84%) R_f=0.61 (toluene-Et₂O 10:1); ¹H NMR (400 MHz, CDCl₃) δ 9.07 (1H, s), 7.90 (1H, d, J=7.5 Hz), 7.55 (1H, d, J=7.5 Hz), 7.41 (1H, t, J=7.5 Hz), 5.08 (2H, s); ¹³C NMR (99.5 MHz, CDCl₃) δ 154.1, 151.4, 134.3, 132.6, 127.0, 125.6, 122.3, 29.5.

Step-3 (4-Azidemethyl benzothiazole)

A solution of 4-Bromomethyl benzothiazole (20.4 g, 89.9 mmol) in 272 mL of dry DMF was treated with NaN₃(7.00 g, 108 mmol) at 20° C. and stirred at the same temperature for 5 min. The resulting mixture was quenched by addition of NaCl (5 g in 150 mL of H₂O) at 0° C., diluted with Et₂O (200 mL) and extracted with Et₂O (200 mL×6). The organic phase was washed with NaCl (2 g in 100 mL of H₂O) twice and brine (100 mL). The resulting solution was dried over MgSO₄and concentrated in vacuo. The residue was submitted to silica gel column chromathography with toluene:Et₂O=50:3 to 50:5 as eluate to afford 4-azidemethyl benzothiazole as a colorless oil (15.5 g, 81.5 mmol, 91%). R_f=0.48 (toluene:Et₂O=10: 1); ¹H NMR (400 MHz, CDCl₃) δ 9.03 (1H, s), 7.95 (1H, d, J=7.7 Hz), 7.49 (2H, m), 5.01 (2H, s); ¹³C NMR (99.5 MHz, CDCl₃) δ 154.2, 151.7, 134.3, 130.6, 126.0, 125.7, 122.1, 51.6.

Step-4 (Benzothiazole-4-methylamine)

To a solution of 4-Azidemethyl benzothiazole (15.4 g, 81.0 mmol) in 243 mL of MeOH was added Pd(OH)₂(20% on carbon, 3.1 g) and then hydrogenolysis at 20° C. After 1.5 hr, additional Pd(OH)₂(20% on carbon, 0.87 g) was added and then hydrogenolysis. After further 1.5 hr, additional Pd(OH)₂(20% on carbon, 1.27 g).was added and then hydrogenolysis for 1 hr. The resulting mixture was replaced with N₂and then filtered through Celite washing with MeOH:NH₄OH (25:1, 260 mL) and concentrated in vacuo. The residue was submitted to silica gel column chromathography eluting with CHCl₃:MeOH:NH₄OH (100:0:0 to 20:5:1) followed by trituration with toluene to afford 4-aminomethyl benzothiazole as a white solid (10.5 g, 63.9 mmol, 79%). R_f=0.49 (CHCl₃:MeOH:NH₄OH=100:25:1); ¹H NMR (400 MHz, CD₃OD) δ 9.23 (1H, s), 7.97 (1H, d, J=7.7 Hz), 7.46 (2H, m), 4.30 (2H, s); ¹³C NMR (99.5 MHz, CD₃OD) δ 184.2, 180.1, 165.3, 163.5, 154.9, 154.1, 150.1, 72.0; LC/MS [ESI+] (m/z) 165.4 (M+1)⁺.

Synthesis of 4-Benzyl-3-Boc-2-methylsemicarbazidylacetatic acid

Step-1 (4-Benzyl-2-methylsemicarbazide)

A solution of Benzyl isocyanate (1.85 mL, 15.0 mmol) in 7.5 mL of CHCl₃was treated with methyl hydrazine (795 μL, 15.0 mmol) at 0° C. and stirred at the same temperature for 2 h. The resulting mixture was dissolved in 1N HCl (200 mL) and the solution was washed with CHCl₃(50 mL×3). The aqueous phase was adjusted to pH 12 with 2 M NaOHaq and then extracted with CHCl₃(100 mL×3). The organic phase was dried over Na₂SO₄and concentrated in vacuo. The residue was recrystalized from hexane-CHCl₃to afford (1.7 g, 9.5 mmol, 63%) as a colorless crystal. R_f=0.44 (CHCl₃:MeOH=9:1); ¹H NMR (400 MHz, DMSO-d6) δ 7.28-7.19 (5H, m), 4.47 (2H, s), 4.20 (2H, d, J=6.3 Hz), 2.96 (3H, s); ¹³C NMR (99.5 MHz, DMSO-d6) δ 159.3, 141.1, 128.1, 127.1, 126.5, 43.1, 37.8; LC/MS [ESI+] (m/z) 180.3 (M+1)⁺.

Step-2 (Ethyl 4-benzyl-2-methylsemicarbazidylacetate)

To the solution of 4-Benzyl-2-methylsemicarbazide (5.24 g, 29.2 mmol) in Toluene (58 mL) were added DIPEA (7.63 mL, 43.8 mmol) and Ethyl bromoacetate (4.86 mL, 43.8 mmol) and then stirred at 856 for 24 hr. The reaction mixture was allowed to cool to room temperature followed by dilution with EtOAc (100 mL). The mixture was washed with H₂O (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered and concentrated. The crude was submitted to silica gel (250 g) column chromatography with Hex:EtOAc=1:1 to 1:9 as elute to afford a pale yellow oil (5.75 g, 21.7 mmol, 74%). Rf=0.36 (Hex:EtOAc=1:3); ¹H NMR (400 MHz, CDCl₃) δ 7.34-7.21 (5H, m), 6.88 (1H, br s), 4.40 (2H, d, J=5.8 Hz), 4.18 (2H, q, J=7.2 Hz), 3.69 (1H, br t, J=4.8 Hz), 3.58 (2H, d, J=4.8 Hz), 3.08 (3H, s), 1.26 (3H, t, J=7.2 Hz); ¹³C NMR (99.5 MHz, CDCl₃) δ 170.8, 159.3, 139.9, 128.6, 127.6, 127.1, 61.4, 50.1, 44.4, 33.1, 14.2; LC/MS [ESI+] (m/z) 266.3 (M+1)⁺.

Step-3 (Ethyl 4-benzyl-3-Boc-2-methylsemicarbazidylacetate)

To the solution of Ethyl 4-benzyl-2-methylsemicarbazidylacetate (5.70 g, 21.5 mmol) in CH₂Cl₂(43 mL) were added DIPEA (7.5 mL, 43 mmol), DMAP (1.1 g, 8.6 mmol) and (Boc)₂O (9.4 g, 43 mmol) and then stirred for 1 hr at room temperature. The reaction miture was concentrated and then submitted to SiO₂(250 g) column chromatography with Hex:EtOAc=7:1 to 1:2 as eluate to afford product (2.58 g, 7.06 mmol, 33%) as a pale yellow oil, and starting material (2.80 g, 10.6 mmol, 49%) was recovered. Rf=0.76 (Hex:EtOAc=1:3); ¹H NMR (400 MHz, CDCl₃) δ 7.54 (1H, br s), 7.33-7.20 (5H, m), 4.59-4.46 (2H, m), 4.27-4.19 (4H, m), 3.72 (1H, br d, J=17 Hz), 3.03 (3H, br s), 1.39 (9H, s), 1.26 (3H, t, J=7.2 Hz); ¹³C NMR (99.5 MHz, CDCl₃) δ 170.7, 158.3, 139.8, 128.3, 127.6, 126.9, 82.7, 62.0, 51.6, 44.3, 34.4, 28.0, 14.1; LC/MS [ESI+] (m/z) 366.3 (M+1)⁺.

Step-4 (4-Benzyl-3-Boc-2-methylsemicarbazidylacetatic acid)

To the solution of Ethyl 4-benzyl-3-Boc-2-methylsemicarbazidylacetate (2.30 g, 6.29 mmol) in THF/MeOH/H₂O (2/3/1, 24 mL) was added LiOH H₂O (528 mg, 12.6 mmol) at 0δ. After stirred for 1 hr at room temperature, the reaction mixture was diluted with EtOAc (40 mL) at 0δ. The mixture was acidified with 1N HCl and then extracted with EtOAc. The combined extracts were washed with H₂O (30 mL) and brine (30 mL), dried over Na₂SO₄, added Et₃N (2 mL), filtered and concentrated. The crude was submitted to SiO₂column chromatography with CHCl₃:MeOH=100:0 to 85:15 as eluante to afford a pale yellow sticky oil 4-Benzyl-3-Boc-2-methylsemicarbazidylacetatic acid8Et₃N salt (1.99 g, 4.56 mmol, 72%); ¹H NMR (400 MHz, CDCl₃) δ 8.45 (1H, br s), 7.32-7.18 (5H, m), 4.58-4.22 (3H, m), 3.71-3.57 (1H, m), 3.08 and 3.01 (3H, br s), 2.82 (2.4H, q, J=7.3 Hz, Et₃N), 1.40 (9H, br s), 1.08 (3.6H, t, J=7.3 Hz, Et₃N); ¹³C NMR (99.5 MHz, CDCl₃) δ 174.2, 159.2, 154.1, 140.1, 128.2, 127.4, 12.7, 81.8, 52.2, 45.1 (Et₃N), 44.1, 34.5, 28.1, 8.3 (Et₃N); LC/MS [ESI+] (m/z) 338.3 (M+1)⁺.

Synthesis of 4-Benzyl-3-Boc-2-allylsemicarbazidylacetatic acid

Step-1 (4-Benzyl-2-allylsemicarbazide)

To the solution of Allyl hydrazine (1.55 mL, 15.0 mmol) in 7.5 mL of CHCl₃was added benzyl isocyanate (1.85 mL, 15.0 mmol) slowly at 0° C. and stirred at the same temperature for 2 h. The resulting mixture was dissolved in 1N HCl (200 mL) and the solution was washed with CHCl₃(50 mL×3). The aqueous phase was adjusted to pH 12 with 2 M NaOH aq and then extracted with CHCl₃(100 mL×3). The organic phase was dried over Na₂SO₄and concentrated in vacuo. The residue was recrystalized from hexane-CHCl₃to afford a colorless crystal (2.20 g, 10.7 mmol, 70%). R_f=0.50 (CHCl₃:MeOH=9:1); ¹H NMR (400 MHz, CDCl₃) δ7.34-7.23 (5H, m), 6.77 (1H, br s), 5.77 (1H, ddt, J=16.9, 10.1, 6.3 Hz), 5.28 (1H, d, J=10.1 Hz), 5.22 (1H, dd, J=16.9, 1.5 Hz), 4.42 (2H, d, J=6.3 Hz), 4.14 (2H, d, J=6.3 Hz), 3.47 (2H, s); ¹³C NMR (99.5 MHz, CDCl₃) δ159.0, 139.9, 132.7, 128.6, 127.6, 127.2, 119.2, 52.8, 44.3; LC/MS [ESI+] (m/z) 206.3 (M+1)⁺.

Step-2 (Ethyl 4-benzyl-2-allylsemicarbazidylacetate)

To the solution of 4-Benzyl-2-allylsemicarbazide (8.60 g, 41.9 mmol) in toluene (50 mL) were added DIPEA (14.6 mL, 83.8 mmol) and Ethyl bromoacetate (8.1 mL, 73 mmol) and then stirred at 958 for 39 hr. The reaction mixture was allowed to cool to room temperature followed by dilution with EtOAc (150 mL). The mixture was washed with H₂O (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered and concentrated. The crude was submitted to silica gel (250 g) column chromatography with Hex:EtOAc=2:1 to 1:1 as eluate to afford a pale yellow oil (7.60 g, 26.1 mmol, 62%). Rf=0.30 (Hex:EtOAc=2:3); ¹H NMR (400 MHz, CDCl₃) δ 7.32-7.23 (5H, m), 7.02 (1H, br,s), 5.78 (1H, ddt, J=17.4, 10.1, 6.3 Hz), 5.25 (2H, m), 4.42 (2H, d, J=5.8 Hz), 4.16 (3H, q and br m, J=7.2 Hz), 3.98 (1H, t, J=4.8Hz), 3.55 (2H, d, J=4.8Hz), 1.25 (3H, t, J=7.2 Hz); ¹³C NMR (99.5 MHz, CDCl₃) δ 170.5, 158.9, 139.8, 132.5, 128.5, 127.6, 127.1, 119.2, 61.3, 50.0, 46.7, 44.3, 14.1; LC/MS [ESI+] (m/z) 292.3 (M+1)⁺.

Step-3 (Ethyl 4-benzyl-3-Boc-2-allylsemicarbazidylacetate)

To the solution of Ethyl 4-benzyl-2-allylsemicarbazidylacetate (7.10 g, 24.4 mmol) in CH₂Cl₂(50 mL) were added DIPEA (8.5 mL, 49 mmol), DMAP (1.19 g, 9.76 mmol) and (Boc)₂O (10.6 g, 48.8 mmol). After the mixture was stirred for 3.5 hr at room temperature, additional DIPEA (2.12 mL, 12.2 mmol) and (Boc)₂O (2.66 g, 12.2 mmol) were added. After the reaction mixture was stirred for additional 6 hr, the mixture was diluted with CH₂Cl₂(100 mL) and then sat.NaHCO₃(50 mL) was added at 0δ. The separated aqueous phase was extracted with CH₂Cl₂(100 mL×2). The combined organic phases were washed with H₂O (100 mL) and brine (100 mL), dried over Na₂SO₄, filtered and concentrated. The crude was submitted to SiO₂(300 g) column chromatography with Hex:EtOAc=7:1 to 1:1 as eluate to afford product as a pale yellow oil (6.61 g, 16.9 mmol, 69%). Rf=0.57 (Hex:EtOAc=1:1); ¹H NMR (400 MHz, CDCl₃) δ 7.77 (1H, br s), 7.34-7.21 (5H, br m), 5.88 (1H, br m), 5.20 (2H, br m), 4.62-4.46 (3H, m), 4.37-4.13 (3H, m), 3.92-3.65 (2H, m), 1.48 and 1.38 (9H, s), 1.26 (3H, t, J=7.2 Hz); ¹³C NMR (99.5 MHz, CDCl₃) δ 170.8, 157.8, 154.1, 139.8, 128.4, 127.6, 127.0, 119.6, 82.7, 62.0, 51.2, 44.3, 30.9, 28.0, 14.1; LC/MS [ESI+] (m/z) 392.4 (M+1)⁺.

Step-4 (4-Benzyl-3-Boc-2-allylsemicarbazidylacetatic acid)

To the solution of Ethyl 4-benzyl-3-Boc-2-allylsemicarbazidylacetate (3.20 g, 8.17 mmol) in THF/MeOH/H₂O (2/3/1, 25 mL) was added LiOH H₂O (685 mg, 16.3 mmol) at 0δ. After stirred for 40 min at room temperature, the reaction mixture was diluted with CH₂Cl₂(50 mL) at 0δ. The mixture was acidified with 1N HCl and then extracted with CH₂Cl₂. The combined extraction were washed with H₂O (30 mL) and Brine (30 mL), dried over Na₂SO₄, added Et₃N (3 mL), filtered and concentrated. The crude was submitted to SiO₂column chromatography with CHCl₃:MeOH=100:0 to 85:15 as eluate to afford orange sticky oil 4-Benzyl-3-Boc-2-allylsemicarbazidylacetatic acidδEt₃N salt (3.66 g, 7.87 mmol, 96%); ¹H NMR (400 MHz, CDCl₃, rotamer) δ 9.44 and 9.34 (1H, br s), 7.35-7.18 (5H, m), 5.91 (1H, m), 5.17 (2H, m), 4.58 and 4.87 (2H, dd, J=15.5, 6.3 and 14.5, 5.8 Hz), 4.39-4.23 (2H, m), 3.89 and 3.80 (1H, dd, J=14.0, 8.2 and 14.5, 8.2 Hz), 3.58 and 3.52 (1H, d, J=17.4 and 16.9 Hz), 2.81 (5H, q, J=7.2 Hz, Et₃N), 1.44 and 1.42 (9H, s), 1.11 (7.5H, t, J=7.2 Hz, Et₃N); ¹³C NMR (99.5 MHz, CDCl₃) δ 158.9, 154.3, 153.6, 140.6, 134.2, 128.1, 127.4, 126.5, 118.8, 81.1, 55.6, 51.4, 44.9 (Et₃N), 44.2, 28.2, 8.3 (Et₃N); LC/MS [ESI+] (m/z) 364.3 (M+1)⁺.

Synthesis of Compound No. 61

Step-1

The hydroxy-functionalized resin (5.0 g, 0.68 mmol/g, Novabiochem) was placed in 200 mL round-bottom flask. To the mixture of the resin and PPTS (1.7 g, 6.8 mmol) in 1,2-dichloromethane (51 mL) was added bromoacetaldehyde diethylacetal (4.2 mL, 27 mmol) at room temperature. After being stirred under reflux for 4.0 hr, the mixture was filtered and the resin was washed with DMF 50 mL×3, DMSO 50 mL×3, 1,4-dioxane 50 mL×3, CH₂Cl₂50 mL×3, MeOH 50 mL×3, Et₂O 50 mL×3. The resin was dried under reduced pressure for over night to afford the desired bromoacetal resin (5.5 g).

Step-2

Bromoacetal resin (1.0 g, 0.9 mmol/g) was placed in 30 mL round-bottom flask. The resin was swollen with DMF (9.0 mL×5 min×1) and then treated with 1.0 M solution of 1-naphtylmethylamine (1.4 g, 9.0 mmol) in DMSO (9.0 mL) at 70° C. After being stirred for 12 hr, the resin was filtered and rinsed with DMSO (9.0 mL×5 min×3). The resin was washed with DMF (5.0 mL×5 min×3) and CH₂Cl₂(5.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin (1.1 8g).

Step-3

Naphthylmethylamino resin (1.18 g, 0.84 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen with DMF (9.0 mL×5 min×1) and then DMF (9.0 mL), Fmoc-Tyr(t-Bu)-OH (620 mg, 1.35 mmol), DIPEA (470 μL, 2.70 mmol) and HATU (513 mg, 1.35 mmol) were added at room temperature. After being shaken for 12 hr, in case of Kaiser test was positive, the same procedure was repeated. The mixture was filtered and the resin was washed with DMF (10.0 mL×5 min×3) and CH₂Cl₂(10.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin (1.50 g).

Step-4

The 1-Naphthylmethylamino-Fmoc-Tyr(tBu) resin (1.50 g, 0.61 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen in DMF (10.0 mL) and DMF was sucked out. The resin was treated with 20 v/v % piperidine/DMF (10.0 mL) at room temperature. After being shaken for 1.0 hr, the mixture was filtered and the resin was washed with DMF (10 mL×5 min×3) and CH₂Cl₂(10 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin (1.48 g).

Step-5

The Amino resin (300 mg, 0.71 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen in DMF (3.0 mL) and DMF was sucked out. To the resin was added 0.3 M stocked CH₂Cl₂soltuion of 4-Benzyl-3-Boc-2-methylsemicarbazidylacetatic acid (2.5 mL, 0.75 mmol), DIPEA (260 μL, 1.49 mmol) and HATU (284 mg, 0.75 mmol) at room temperature. After being shaken for 12 hr, the mixture was filtered and the resin was washed with DMF (5.0 mL×5 min×3) and CH₂Cl₂(5.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin.

Step-6

The resin (115 mg, 0.58 mmol/g) was placed in 5.0 mL plastic disposable syringe. After addition of 99% HCO₂H (1.0 mL), the mixture was shaken for 12 hr at room temperature, the solution was collected by filteration. The resin was washed with 99% HCO₂H (1.5 mL×5 min×2). The combined HCO₂H solutions were concentrated and then submitted to silica gel column chromatography to afford Compound No. 61 (7.1 mg, 19% from bromoacetal resin). Rf=0.63 (CHCl₃:MeOH=9:1); ¹H NMR (400 MHz, CDCl₃) δ 8.06 (1H, d, J=8.2 Hz), 7.89 (1H, m), 7.84 (1H, d, J=8.2 Hz), 7.56 (2H, m), 7.38 (1H, dd, J=8.2, 7.2 Hz), 7.20 (3H, m), 7.12 (1H, d, J=6.8 Hz), 7.05 (2H, dd, J=7.7, 2.9 Hz), 7.02 (2H, d, J=8.2 Hz), 6.88 (0.5H, br s), 6.71 (2H, d, J=8.2 Hz), 6.05 (1H, t, J=5.8 Hz), 5.06 (2H, ABq, J=14.5 Hz), 4.80 (1H, dd, J=5.8, 2.5 Hz), 4.23 (2H, ABX, J=14.5, 5.8 Hz), 3.67-3.44 (4H, m), 3.21 (1H, dd, J=14.0, 5.8 Hz), 3.12 (1H, dd, J=11.0, 3.9 Hz), 2.86 (1H. dd. J=11.0, 9.1 Hz), 2.59 (3H, s); LC/MS [ESI+] (m/z) 564.4 (M+1)⁺.

Synthesis of Compound No. 71

Step-1

The Amino resin (100 mg, 0.71 mmol/g) was placed in 5 mL plastic disposable syringe. The resin was swollen in DMF (1.0 mL) and DMF was sucked out. To the resin was added 0.3 M stocked CH₂Cl₂soltuion of 4-Benzyl-3-Boc-2-allylsemicarbazidylacetatic acid (830 μL, 0.25 mmol), DIPEA (87 μL, 0.50 mmol) and HATU (95 mg, 0.25 mmol) at room temperature. After being shaken for 12 hr, the mixture was filtered and the resin was washed with DMF (1.0 mL×5 min×3) and CH₂Cl₂(1.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin.

Step-2

The resin (100 mg, 0.57 mmol/g) was placed in 5.0 mL plastic disposable syringe. After addition of 99% HCO₂H (1.0 mL), the mixture was shaken for 12 hr at room temperature, the solution was collected by filteration. The resin was washed with 99% HCO₂H (1.5 mL×5 min×2). The combined HCO₂H solutions were concentrated and then submitted to silica gel column chromatography to afford Compound No. 71 (11 mg, 26% from bromoacetal resin). Rf=0.63 (CHCl₃:MeOH=9:1).
Similar synthesis was carried out to obtain the compounds as shown as Compounds 1-1200 in FIGS. 1-6.

Synthesis of Compound No. 1273

Step-1

Bromoacetal resin (1.0 g, 0.9 mmol/g) was placed in 30 mL round-bottom flask. The resin was swollen with DMF (9.0 mL×5 min×1) and then treated with 1.0 M suspension of 2-tert-Butoxycarbonylaminobenzothiazole-4-methylamine (2.5 g, 9.0 mmol) in DMSO (9.0 mL) at 70° C. After being stirred for 12 hr, the resin was filtered and rinsed with DMSO (9.0 mL×5 min×3). The resin was washed with DMF (5.0 mL×5 min×3) and CH₂Cl₂(5.0 mL×5 min×3). 10 The resin was dried under reduced pressure to afford desired resin (1.16 g).

Step-2

2-tert-Butoxycarbonylaminoebenzothiazole-4-methylamino resin (1.16 g, 0.76 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen with DMF (9.0 mL×5 min×1) and then DMF (9.0 mL), Fmoc-Tyr(t-Bu)-OH (620 mg, 1.35 mmol), DIPEA (470 μL, 2.70 mmol) and HATU (513 mg, 1.35 mmol) were added at room temperature. After being shaken for 12 hr, in case of Kaiser test was positive, the same procedure was repeated. The mixture was filtered and the resin was washed with DMF (10.0 mL×5 min×3) and CH₂Cl₂(10.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin (1.76 g).

Step-3

The 2-tert-Butoxycarbonylbenzothiazole-4-methylamino-Fmoc-Tyr(tBu) resin (1.76 g, 0.57 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen in DMF (10.0 mL) and DMF was sucked out. The resin was treated with 20 v/v % piperidine/DMF (10.0 mL) at room temperature. After being shaken for 1.0 hr, the mixture was filtered and the resin was
washed with DMF (10 mL×5 min×3) and CH₂Cl₂(10 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin (1.42 g).

Step-4

The Amino resin (350 mg, 0.65 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen in DMF (3.0 mL) and DMF was sucked out. To the resin was added 0.3 M stocked CH₂Cl₂soltuion of 4-Benzyl-3-Boc-2-methylsemicarbazidylacetatic acid (2.7 mL, 0.80 mmol), DIPEA (277 μL, 1.59 mmol) and HATU (302 mg, 0.80 mmol) at room temperature. After being shaken for 12 hr, the mixture was filtered and the resin was washed with DMF (5.0 mL×5 min×3) and CH₂Cl₂(5.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin.

Step-5

The resin (350 mg, 0.54 mmol/g) was placed in 20 mL plastic disposable syringe. After addition of 99% HCO₂H (4.0 mL), the mixture was shaken for 12 hr at room temperature, the solution was collected by filteration. The resin was washed with 99% HCO₂H (4.0 mL×5 min×2). The combined HCO₂H solutions were concentrated and then submitted to silica gel column chromatography to afford Compound No. 1273 (9.1 mg, 6.8% from bromoacetal resin). Rf=0.47 (CHCl₃:MeOH=9:1).

Synthesis of Compound No. 1285

Step-1

The Amino resin (350 mg, 0.65 mmol/g) was placed in 20 mL plastic disposable syringe. The resin was swollen in DMF (3.0 mL) and DMF was sucked out. To the resin was added 0.3 M stocked CH₂Cl₂soltuion of 4-Benzyl-3-Boc-2-allylsemicarbazidylacetatic acid (2.7 mL, 0.80 mmol), DIPEA (277 μL, 1.59 mmol) and HATU (302 mg, 0.80 mmol) at room temperature. After being shaken for 12 hr, the mixture was filtered and the resin was washed with DMF (5.0 mL×5 min×3) and CH₂Cl₂(5.0 mL×5 min×3). The resin was dried under reduced pressure to afford desired resin.

Step-2

The resin (350 mg, 0.53 mmol/g) was placed in 20 mL plastic disposable syringe. After addition of 99% HCO₂H (4.0 mL), the mixture was shaken for 12 hr at room temperature, the solution was collected by filteration. The resin was washed with 99% HCO₂H (4.0 mL×5 min×2). The combined HCO₂H solutions were concentrated and then submitted to silica gel column chromatography to afford Compound No. 1285 (18 mg, 13% from bromoacetal resin). Rf=0.52 (CHCl₃:MeOH=9:1).
Similar synthesis was carried out to obtain Compounds 1201-2200 as shown in FIGS. 7-11.

Synthesis of Compound No. 2201

To the cooled (0δ) solution of Compound No. 61 (18 mg, 0.032 mmol) in THF (500 δL) were added Et₃N (13.4 μL, 0.096 mmol) and POCl₃(14.9 μL, 0.160 mmol) and then the mixture was stirred till SM was disappeared on TLC (4 hr). The mixture was diluted with H₂O (1 mL) and then NaHCO₃was added at 0δ to pH 8. After stirred overnight, the mixture was acidified to pH 3 with 1N HCl followed by extraction with CHCl₃(5 mL×3). The combined extracts were dried over Na₂SO₄, filtered and concentrated to afford pale yellow powder Compound No. 2201 (17.1 mg, 83%). TLC: Rf=0.458Silica gel F254, CHCl₃:MeOH:EtOH:H₂O:AcOH:nBuOH=100:40:10:10:8:5δ; ¹H NMR (400 MHz, CDCl₃) δ 7.98 (1H, d, J=7.7 Hz), 7.83 (1H, m), 7.77 (1H, d, J=8.2 Hz), 7.51 (2H, m), 7.35 (1H, t, J=7.3 Hz), 7.24-6.93 (10H, m), 6.07 (1H, br s), 5.86 (3H, br s), 5.34 (1H, br d, J=15.0 Hz), 4.76 (2H, m), 4.11 (2H, br ABX, J=15.5, 5.3 Hz), 3.62 (2H, m), 3.47 and 3.31 (2H, br ABq, J=15.0 Hz), 3.22 (2H, br m), 3.02 (1H, br m), 2.77 (1H, br t, J=10.6 Hz), 2.56 (3H, s); ³¹P NMR (160.26 MHz, CDCl₃) δ −3.57.

Synthesis of Compound No. 2202

To the cooled (0δ) solution of Compound No. 71 (21 mg, 0.036 mmol) in THF (1.0 mL) were added Et₃N (14.9 μL, 0.107 mmol) and POCl₃(16.6 μL, 0.178 mmol) and then the mixture was stirred till SM was disappeared on TLC (4 hr). The mixture was diluted with H₂O (1 mL) and then NaHCO₃was added at 0δ to pH 8. After stirred overnight, the mixture was acidified to pH 3 with 1N HCl followed by extraction with CHCl₃(5 mL×3). The combined extracts were dried over Na₂SO₄, filtered and concentrated to afford pale yellow powder Compound No. 2202 (21.0 mg, 88%). TLC: Rf=0.536Silica gel F254, CHCl₃:MeOH:EtOH:H₂O:AcOH:nBuOH=100:40:10:10:8:56.
Similar synthesis was carried out to obtain Compounds 2203-2217 as shown in FIG. 27. Diastereomeric and Enantiomeric stereo isomers of Compounds 2203-2217 were obtained and are shown FIG. 12.

Table 2 below shows the molecular weight (M.W.) and mass for compounds 1-2217.

TABLE 2


Compound
No.	M.W.	Mass

1	533	534
2	551	552
3	563	564
4	602	603
5	457	458
6	561	562
7	579	580
8	591	592
9	630	631
10	485	486
11	559	560
12	577	578
13	589	590
14	628	629
15	483	484
16	557	558
17	575	576
18	587	588
19	626	627
20	481	482
21	561	562
22	579	580
23	591	592
24	630	631
25	485	486
26	558	559
27	576	577
28	588	589
29	627	628
30	482	483
31	547	548
32	565	566
33	577	578
34	616	617
35	471	472
36	575	576
37	593	594
38	605	606
39	644	645
40	499	500
41	573	574
42	591	592
43	603	604
44	642	643
45	497	498
46	571	572
47	589	590
48	601	602
49	640	641
50	495	496
51	575	576
52	593	594
53	605	606
54	644	645
55	499	500
56	572	573
57	590	591
58	602	603
59	641	642
60	496	497
61	563	564
62	581	582
63	593	594
64	632	633
65	487	488
66	591	592
67	609	610
68	621	622
69	660	661
70	515	516
71	589	590
72	607	608
73	619	620
74	658	659
75	513	514
76	587	588
77	605	606
78	617	618
79	656	657
80	511	512
81	591	592
82	609	610
83	621	622
84	660	661
85	515	516
86	588	589
87	606	607
88	618	619
89	657	658
90	512	513
91	563	564
92	581	582
93	609	610
94	648	649
95	503	504
96	607	608
97	625	626
98	637	638
99	676	677
100	531	532
101	605	606
102	623	624
103	635	636
104	674	675
105	529	530
106	603	604
107	621	622
108	633	634
109	672	673
110	527	528
111	607	608
112	625	626
113	637	638
114	676	677
115	531	532
116	604	605
117	622	623
118	634	635
119	673	674
120	528	529
121	562	563
122	580	581
123	592	593
124	631	632
125	486	487
126	590	591
127	608	609
128	620	621
129	659	660
130	514	515
131	588	589
132	606	607
133	618	619
134	657	658
135	512	513
136	586	587
137	604	605
138	616	617
139	655	656
140	510	511
141	590	591
142	608	609
143	620	621
144	659	660
145	514	515
146	587	588
147	605	606
148	617	618
149	656	657
150	511	512
151	590	591
152	608	609
153	620	621
154	659	660
155	514	515
156	618	619
157	636	637
158	648	649
159	687	688
160	542	543
161	616	617
162	634	635
163	646	647
164	685	686
165	540	541
166	614	615
167	632	633
168	644	645
169	683	684
170	538	539
171	618	619
172	636	637
173	648	649
174	687	688
175	542	543
176	615	616
177	633	634
178	645	646
179	684	685
180	539	540
181	666	667
182	684	685
183	696	697
184	735	736
185	590	591
186	694	695
187	712	713
188	724	725
189	763	764
190	618	619
191	692	693
192	710	711
193	722	723
194	761	762
195	616	617
196	690	691
197	708	709
198	720	721
199	759	760
200	614	615
201	694	695
202	712	713
203	724	725
204	763	764
205	618	619
206	691	692
207	709	710
208	721	722
209	760	761
210	615	616
211	696	697
212	714	715
213	726	727
214	765	766
215	620	621
216	724	725
217	742	743
218	754	755
219	793	794
220	648	649
221	722	723
222	740	741
223	752	753
224	791	792
225	646	647
226	720	721
227	738	739
228	750	751
229	789	790
230	644	645
231	724	725
232	742	743
233	754	755
234	793	794
235	648	649
236	721	722
237	739	740
238	751	752
239	790	791
240	645	646
241	590	591
242	608	609
243	620	621
244	659	660
245	514	515
246	618	619
247	636	637
248	648	649
249	687	688
250	542	543
251	616	617
252	634	635
253	646	647
254	685	686
255	540	541
256	614	615
257	632	633
258	644	645
259	683	684
260	538	539
261	618	619
262	636	637
263	648	649
264	687	688
265	542	543
266	615	616
267	633	634
268	645	646
269	684	685
270	539	540
271	592	593
272	610	611
273	622	623
274	661	662
275	516	517
276	620	621
277	638	639
278	650	651
279	689	690
280	544	545
281	618	619
282	636	637
283	648	649
284	687	688
285	542	543
286	616	617
287	634	635
288	646	647
289	685	686
290	540	541
291	620	621
292	638	639
293	650	651
294	689	690
295	544	545
296	617	618
297	635	636
298	647	648
299	686	687
300	541	542
301	577	578
302	595	596
303	607	608
304	646	647
305	501	502
306	605	606
307	623	624
308	635	636
309	674	675
310	529	530
311	603	604
312	621	622
313	633	634
314	672	673
315	527	528
316	601	602
317	619	620
318	631	632
319	670	671
320	525	526
321	605	606
322	623	624
323	635	636
324	674	675
325	529	530
326	602	603
327	620	621
328	632	633
329	671	672
330	526	527
331	635	636
332	653	654
333	665	666
334	704	705
335	559	560
336	663	664
337	681	682
338	693	694
339	732	733
340	587	588
341	661	662
342	679	680
343	691	692
344	730	731
345	585	586
346	659	660
347	677	678
348	689	690
349	728	729
350	583	584
351	663	664
352	681	682
353	693	694
354	732	733
355	587	588
356	660	661
357	678	679
358	690	691
359	729	730
360	584	585
361	716	717
362	734	735
363	746	747
364	785	786
365	640	641
366	744	745
367	762	763
368	774	775
369	813	814
370	668	669
371	742	743
372	760	761
373	772	773
374	811	812
375	666	667
376	740	741
377	758	759
378	770	771
379	809	810
380	664	665
381	744	745
382	762	763
383	774	775
384	813	814
385	668	669
386	741	742
387	759	760
388	771	772
389	810	811
390	665	666
391	565	566
392	583	584
393	595	596
394	634	635
395	489	490
396	593	594
397	611	612
398	623	624
399	662	663
400	517	518
401	591	592
402	609	610
403	621	622
404	660	661
405	515	516
406	589	590
407	607	608
408	619	620
409	658	659
410	513	514
411	593	594
412	611	612
413	623	624
414	662	663
415	517	518
416	590	591
417	608	609
418	620	621
419	659	660
420	514	515
421	578	579
422	596	597
423	608	609
424	647	648
425	502	503
426	606	607
427	624	625
428	636	637
429	675	676
430	530	531
431	604	605
432	622	623
433	634	635
434	673	674
435	528	529
436	602	603
437	620	621
438	632	633
439	671	672
440	526	527
441	606	607
442	624	625
443	636	637
444	675	676
445	530	531
446	603	604
447	621	622
448	633	634
449	672	673
450	527	528
451	634	635
452	652	653
453	664	665
454	703	704
455	558	559
456	662	663
457	680	681
458	692	693
459	731	732
460	586	587
461	660	661
462	678	679
463	690	691
464	729	730
465	584	585
466	658	659
467	676	677
468	688	689
469	727	728
470	582	583
471	662	663
472	680	681
473	692	693
474	731	732
475	586	587
476	659	660
477	677	678
478	689	690
479	728	729
480	583	584
481	677	678
482	695	696
483	707	708
484	746	747
485	601	602
486	705	706
487	723	724
488	735	736
489	774	775
490	629	630
491	703	704
492	721	722
493	733	734
494	772	773
495	627	628
496	701	702
497	719	720
498	731	732
499	770	771
500	625	626
501	705	706
502	723	724
503	735	736
504	774	775
505	629	630
506	702	703
507	720	721
508	732	733
509	771	772
510	626	627
511	607	608
512	625	626
513	637	638
514	676	677
515	531	532
516	635	636
517	653	654
518	665	666
519	704	705
520	559	560
521	633	634
522	651	652
523	663	664
524	702	703
525	557	558
526	631	632
527	649	650
528	661	662
529	700	701
530	555	556
531	635	636
532	653	654
533	665	666
534	704	705
535	559	560
536	632	633
537	650	651
538	662	663
539	701	702
540	556	557
541	640	641
542	658	659
543	670	671
544	709	710
545	564	565
546	668	669
547	686	687
548	698	699
549	737	738
550	592	593
551	666	667
552	684	685
553	696	697
554	735	736
555	590	591
556	664	665
557	682	683
558	694	695
559	733	734
560	588	589
561	668	669
562	686	687
563	698	699
564	737	738
565	592	593
566	665	666
567	683	684
568	695	696
569	734	735
570	589	590
571	587	588
572	605	606
573	617	618
574	656	657
575	511	512
576	615	616
577	633	634
578	645	646
579	684	685
580	539	540
581	613	614
582	631	632
583	643	644
584	682	683
585	537	538
591	615	616
592	633	634
593	645	646
594	684	685
595	539	540
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1619	605	606
1620	647	648
1621	685	686
1622	703	704
1623	715	716
1624	754	755
1625	609	610
1626	651	652
1627	682	683
1628	700	701
1629	712	713
1630	751	752
1631	606	607
1632	648	649
1633	738	739
1634	756	757
1635	768	769
1636	807	808
1637	662	663
1638	704	705
1639	766	767
1640	784	785
1641	796	797
1642	835	836
1643	690	691
1644	732	733
1645	764	765
1646	782	783
1647	794	795
1648	833	834
1649	688	689
1650	730	731
1651	762	763
1652	780	781
1653	792	793
1654	831	832
1655	686	687
1656	728	729
1657	766	767
1658	784	785
1659	796	797
1660	835	836
1661	690	691
1662	732	733
1663	763	764
1664	781	782
1665	793	794
1666	832	833
1667	687	688
1668	729	730
1669	587	588
1670	605	606
1671	617	618
1672	656	657
1673	511	512
1674	553	554
1675	615	616
1676	633	634
1677	645	646
1678	684	685
1679	539	540
1680	581	582
1681	613	614
1682	631	632
1683	643	644
1684	682	683
1685	537	538
1686	579	580
1687	611	612
1688	629	630
1689	641	642
1690	680	681
1691	535	536
1692	577	578
1693	615	616
1694	633	634
1695	645	646
1696	684	685
1697	539	540
1698	581	582
1699	612	613
1700	630	631
1701	642	643
1702	681	682
1703	536	537
1704	578	579
1705	600	601
1706	618	619
1707	630	631
1708	669	670
1709	524	525
1710	566	567
1711	628	629
1712	646	647
1713	658	659
1714	697	698
1715	552	553
1716	594	595
1717	626	627
1718	644	645
1719	656	657
1720	695	696
1721	550	551
1722	592	593
1723	624	625
1724	642	643
1725	654	655
1726	693	694
1727	548	549
1728	590	591
1729	628	629
1730	646	647
1731	658	659
1732	697	698
1733	552	553
1734	594	595
1735	625	626
1736	643	644
1737	655	656
1738	694	695
1739	549	550
1740	591	592
1741	656	657
1742	674	675
1743	686	687
1744	725	726
1745	580	581
1746	622	623
1747	684	685
1748	702	703
1749	714	715
1750	753	754
1751	608	609
1752	650	651
1753	682	683
1754	700	701
1755	712	713
1756	751	752
1757	606	607
1758	648	649
1759	680	681
1760	698	699
1761	710	711
1762	749	750
1763	604	605
1764	646	647
1765	684	685
1766	702	703
1767	714	715
1768	753	754
1769	608	609
1770	650	651
1771	681	682
1772	699	700
1773	711	712
1774	750	751
1775	605	606
1776	647	648
1777	699	700
1778	717	718
1779	729	730
1780	768	769
1781	623	624
1782	665	666
1783	727	728
1784	745	746
1785	757	758
1786	796	797
1787	651	652
1788	693	694
1789	725	726
1790	743	744
1791	755	756
1792	794	795
1793	649	650
1794	691	692
1795	723	724
1796	741	742
1797	753	754
1798	792	793
1799	647	648
1800	689	690
1801	727	728
1802	745	746
1803	757	758
1804	796	797
1805	651	652
1806	693	694
1807	724	725
1808	742	743
1809	754	755
1810	793	794
1811	648	649
1812	690	691
1813	629	630
1814	647	648
1815	659	660
1816	698	699
1817	553	554
1818	595	596
1819	657	658
1820	675	676
1821	687	688
1822	726	727
1823	581	582
1824	623	624
1825	655	656
1826	673	674
1827	685	686
1828	724	725
1829	579	580
1830	621	622
1831	653	654
1832	671	672
1833	683	684
1834	722	723
1835	577	578
1836	619	620
1837	657	658
1838	675	676
1839	687	688
1840	726	727
1841	581	582
1842	623	624
1843	654	655
1844	672	673
1845	684	685
1846	723	724
1847	578	579
1848	620	621
1849	662	663
1850	680	681
1851	692	693
1852	731	732
1853	586	587
1854	628	629
1855	690	691
1856	708	709
1857	720	721
1858	759	760
1859	614	615
1860	656	657
1861	688	689
1862	706	707
1863	718	719
1864	757	758
1865	612	613
1866	654	655
1867	686	687
1868	704	705
1869	716	717
1870	755	756
1871	610	611
1872	652	653
1873	690	691
1874	708	709
1875	720	721
1876	759	760
1877	614	615
1878	656	657
1879	687	688
1880	705	706
1881	717	718
1882	756	757
1883	611	612
1884	653	654
1885	609	610
1886	627	628
1887	639	640
1888	678	679
1889	533	534
1890	575	576
1891	637	638
1892	655	656
1893	667	668
1894	706	707
1895	561	562
1896	603	604
1897	635	636
1898	653	654
1899	665	666
1900	704	705
1901	559	560
1902	601	602
1903	633	634
1904	651	652
1905	663	664
1906	702	703
1907	557	558
1908	599	600
1909	637	638
1910	655	656
1911	667	668
1912	706	707
1913	561	562
1914	603	604
1915	634	635
1916	652	653
1917	664	665
1918	703	704
1919	558	559
1920	600	601
1921	609	610
1922	627	628
1923	639	640
1924	678	679
1925	533	534
1926	575	576
1927	637	638
1928	655	656
1929	667	668
1930	706	707
1931	561	562
1932	603	604
1933	635	636
1934	653	654
1935	665	666
1936	704	705
1937	559	560
1938	601	602
1939	633	634
1940	651	652
1941	663	664
1942	702	703
1943	557	558
1944	599	600
1945	637	638
1946	655	656
1947	667	668
1948	706	707
1949	561	562
1950	603	604
1951	634	635
1952	652	653
1953	664	665
1954	703	704
1955	558	559
1956	600	601
1957	626	627
1958	644	645
1959	656	657
1960	695	696
1961	550	551
1962	592	593
1963	654	655
1964	672	673
1965	684	685
1966	723	724
1967	578	579
1968	620	621
1969	652	653
1970	670	671
1971	682	683
1972	721	722
1973	576	577
1974	618	619
1975	650	651
1976	668	669
1977	680	681
1978	719	720
1979	574	575
1980	616	617
1981	654	655
1982	672	673
1983	684	685
1984	723	724
1985	578	579
1986	620	621
1987	651	652
1988	669	670
1989	681	682
1990	720	721
1991	575	576
1992	617	618
1993	626	627
1994	644	645
1995	656	657
1996	695	696
1997	550	551
1998	592	593
1999	654	655
2000	672	673
2001	684	685
2002	723	724
2003	578	579
2004	620	621
2005	652	653
2006	670	671
2007	682	683
2008	721	722
2009	576	577
2010	618	619
2011	650	651
2012	668	669
2013	680	681
2014	719	720
2015	574	575
2016	616	617
2017	654	655
2018	672	673
2019	684	685
2020	723	724
2021	578	579
2022	620	621
2023	651	652
2024	669	670
2025	681	682
2026	720	721
2027	575	576
2028	617	618
2029	586	587
2030	604	605
2031	616	617
2032	655	656
2033	510	511
2034	552	553
2035	614	615
2036	632	633
2037	644	645
2038	683	684
2039	538	539
2040	580	581
2041	612	613
2042	630	631
2043	642	643
2044	681	682
2045	536	537
2046	578	579
2047	610	611
2048	628	629
2049	640	641
2050	679	680
2051	534	535
2052	576	577
2053	614	615
2054	632	633
2055	644	645
2056	683	684
2057	538	539
2058	580	581
2059	611	612
2060	629	630
2061	641	642
2062	680	681
2063	535	536
2064	577	578
2065	640	641
2066	658	659
2067	670	671
2068	709	710
2069	564	565
2070	606	607
2071	668	669
2072	686	687
2073	698	699
2074	737	738
2075	592	593
2076	634	635
2077	666	667
2078	684	685
2079	696	697
2080	735	736
2081	590	591
2082	632	633
2083	664	665
2084	682	683
2085	694	695
2086	733	734
2087	588	589
2088	630	631
2089	668	669
2090	686	687
2091	698	699
2092	737	738
2093	592	593
2094	634	635
2095	665	666
2096	683	684
2097	695	696
2098	734	735
2099	589	590
2100	631	632
2101	637	638
2102	655	656
2103	667	668
2104	706	707
2105	561	562
2106	603	604
2107	665	666
2108	683	684
2109	695	696
2110	734	735
2111	589	590
2112	631	632
2113	663	664
2114	681	682
2115	693	694
2116	732	733
2117	587	588
2118	629	630
2119	661	662
2120	679	680
2121	691	692
2122	730	731
2123	585	586
2124	627	628
2125	665	666
2126	683	684
2127	695	696
2128	734	735
2129	589	590
2130	631	632
2131	662	663
2132	680	681
2133	692	693
2134	731	732
2135	586	587
2136	628	629
2137	659	660
2138	677	678
2139	689	690
2140	728	729
2141	583	584
2142	625	626
2143	687	688
2144	705	706
2145	717	718
2146	756	757
2147	611	612
2148	653	654
2149	685	686
2150	703	704
2151	715	716
2152	754	755
2153	609	610
2154	651	652
2155	683	684
2156	701	702
2157	713	714
2158	752	753
2159	607	608
2160	649	650
2161	687	688
2162	705	706
2163	717	718
2164	756	757
2165	611	612
2166	653	654
2167	684	685
2168	702	703
2169	714	715
2170	753	754
2171	608	609
2172	650	651
2173	559	560
2174	577	578
2175	589	590
2176	628	629
2177	483	484
2178	525	526
2179	587	588
2180	605	606
2181	617	618
2182	656	657
2183	511	512
2184	553	554
2185	585	586
2186	603	604
2187	615	616
2188	654	655
2189	509	510
2190	551	552
2191	583	584
2192	601	602
2193	613	614
2194	652	653
2195	507	508
2196	549	550
2197	587	588
2198	605	606
2199	617	618
2200	656	657
2203	661	662
2204	673	674
2205	671	672
2206	669	670
2207	687	688
2208	683	684
2209	695	696
2210	693	592
2211	691	692
2212	709	710
2213	559	560
2214	701	702
2215	713	714
2216	711	712
2217	709	710

EXAMPLE 3

Effect of ICG-001 and Imatinib on Cancer Cell Lines

The human ovarian sarcoma cells MES-SA and the corresponding doxorubicin-resistant line MES-SA/Dx5 (Hua J et al Gynecologic Oncol. 2005) and the CML derived cell line K562 and the corresponding imatinib mesylate resistant K562 cells (Dai Y et al JBC 279, 34227, 2004) were used for this example. Both resistant (R) cell lines showed dramatically increased levels of both cytosolic and nuclear β-catenin as judged by both immunoblotting (FIG. 14A) and immunofluoresence microscopy (FIG. 14B) compared to their drug sensitive (S) counterparts. The increased nuclear β-catenin was reflected in dramatically increased TCF/β-catenin transcriptional activity as judged by the TOPFLASH reporter, which could be completely blocked using a dominant negative TCF4 construct (FIG. 14C).
To confirm that activation of the Wnt/β-catenin pathway was critical for the activation of MDR-1 expression in MES-SA cells, the following set of experiments were performed. MES-SA cells were transfected with either the TOPFLASH or FOPFLASH reporters and treated with media alone, or with added Wnt3a or Wnt5a. Addition of “canonical” Wnt3a but not “non-canonical” Wnt5a increased luciferase activity ˜4 fold and the increased activation was completely blocked by cotransfection of a dnTCF4 construct (FIG. 15A). Similarly, an ˜2 fold increase in MDR-1/lucifearse activity was observed upon treatment with Wnt3a. This activation was also completely inhibited by cotransfection of the dnTCF4 construct. Wnt5a conditioned media showed no enhancement of expression of the MDR-1/luciferase reporter construct (FIG. 15B).
To further confirm the importance of the role of nuclear β-catenin in driving MDR-1 expression, isogenic HCT-116 cell lines were utilized (Waldmann 2002). Wild-type HCT-116 cells demonstrated the highest MDR-1 expression as judged by both MDR-1/luciferase activity and real time RT-PCR (FIG. 15C, D). Hβ18(ko/*) cells, in which the wild type allele of β-catenin is deleted but the oncogenic allele is maintained, and have somewhat lower levels of nuclear β-catenin, showed slightly reduced MDR-1/luciferase activity and a reduction in MDR-1 message (FIG. 15C, D). Hβ92 (wt/ko) cells, in which the wild type allele is retained and the oncogenic allele is deleted, showed even more dramatic reduction of MDR-1/luciferase activity and message (FIG. 15C, D).
TCF/β-catenin recruitment at the MDR-1 promoter in MES-SA and MES-SAJDx5 cells was investigated. In the MES-SA/Dx5 cells, in which MDR-1 is actively transcribed as judged by the level of acetylated Histone H3 at the promoter, and expressed, there was obvious recruitment of both TCF4 and β-catenin to the promoter, which was absent in the parental MES-SA cell line (FIG. 15E).
To investigate differential coactivator usage for the transcriptional regulation of the MDR-1 gene in MES-SA cells, the chemogenomic tool ICG-001 was used (Emami et al. 2004). ICG-001 reduced MDR-1/luciferase activity in MES-SA/Dx5 cells with an IC₅₀˜16 uM (FIG. 16A). The level of MDR-1 protein expression in the MES-SA/Dx5 cells was also significantly reduced by ICG-001 as judged by immunofluoresence (FIG. 16B) and immunoblotting (FIG. 16C) in a dose dependent manner. This effect was reflected at the message level as judged by real time RT-PCR in both MES-SA/Dx5 cells (FIG. 16D) and the imatinib mesylate resistant K562 cells (FIG. 16E).
MDR-1 transcriptional regulation in the isogenic HCT116 cell lines was also investigated. In all of the isogenic HCT116 cell lines, cotransfection of point mutant constitutively translocating β-catenin and CBP increased MDR-1/luciferase expression (FIG. 17A), whereas transfection of point mutant β-catenin alone only increased luciferase activity compared to non-transfected control in the Hβ92(wt/ko) cells (FIG. 17A), which have severely limiting amounts of nuclear β-catenin. Transfection of p300 decreased MDR-1/luciferase activity below control levels in all 3 cell lines (FIG. 17A). ICG-001 dose dependently decreased MDR-1/luciferase activity in the HCT-116 wild type and Hβ18(ko/*) cell lines, whereas essentially no further reduction below basal levels was observed in the Hβ92(wt/ko) cells (FIG. 17B), consistent with a lack of β-catenin/CBP driven transcription in these cells (H Ma et al Oncogene 2005).
ChIP assay in the MES-SA/Dx5 cells demonstrated that in untreated cells, there was significant occupancy of the MDR-1 promoter by CBP, which was blocked in a dose dependent fashion by ICG-001 (FIG. 17C). On the contrary, in the absence of ICG-001, there was minimal occupancy of the MDR-1 promoter by p300, however occupancy increased with 25uM ICG-001 treatment (FIG. 17C). Similar ICG-001 induced p300 recruitment at the survivin promoter has been previously observed, which was associated with recruitment of proteins associated with transcriptional repression (i.e., HDAC6 and PML) (H Ma et al. Oncogene 2005). A proposed non-binding mechanism is repressive transcriptional apparatus recruitment to the MDR-1 promoter by p300.
The mRNA level of endogenous CBP coactivator was also significantly increased in the MES-SA/Dx5 cells compared to the MES-SA cells, whereas p300 levels message remained essentially equal (FIG. 18A). Immunofluoresence also demonstrated a substantial increase in CBP (FIG. 18B) as did immunoblotting in the MES-SA/Dx5 compared to the MES-SA parental line; although p300 protein levels remained essentially equal (FIG. 18C).
Coimmunoprecipitation of CBP or p300 showed a strong association of β-catenin with CBP in the MES-SA/Dx5 cells that was not present in the MES-SA cells while virtually no association of β-catenin with p300 could be detected in either cell line (FIG. 18D). Finally, coactivator specific siRNA was utilized (H Ma Oncogene 2005) to knockdown either CBP or p300 in the MES-SA/Dx5 cells. MDR-1 message was specifically decreased by treatment with siRNA to CBP compared to the siRNA control treated cells, whereas p300 siRNA increased MDR-1 message levels compared to control (FIG. 18E). In culture, the MES-SA/Dx5 and K562 imatinib resistant cells grew at a somewhat faster rate than the corresponding sensitive cell lines (FIG. 19A, B). Consistent with previous data (Emami et al PNAS 2004, H. Ma et al Oncogene 2005, and J Teo et al 2005), enhanced β-catenin/CBP driven transcription was reflected at both the message (FIG. 19C, D) and protein levels (FIG. 19E, F) for both survivin and cyclin D1, in both resistant cell lines compared to their sensitive counterparts.
To further investigate the “cancer stem cell” nature of these resistant cell lines, the expression of a number of markers associated with stem cell pluripotency and survival was evaluated. Real time RT-PCR demonstrated an increased expression of Oct4, hTert, Bmi-1 and ABCG-2 in the MES-SA/DX5 and imatinib resistant K562 cells compared to their sensitive counterparts (FIG. 20A). Protein levels for both Oct4 and the stem cell surface marker CD133 were also increased in both resistant cell lines (FIG. 20B).
Although modern chemotherapies kill a majority of the cells in a tumor, it is believed that the resistant “cancer stems cells” are significantly associated with disease relapse. MDR transporters are believed to play important roles in protecting cancer stem cells from chemotherapy (Dean et al, Nat. Rev. Cancer 5, 275, 2005). To further study this phenomenon, a series of experiments was performed. Drug resistant MES-SA/Dx5 and K562 imatinib resistant cells were treated with Doxorubicin±ICG-001 or Imatinib mesylate±001. As can be seen in FIG. 21 A, ICG-001 in combination with the respective chemotherapeutic agent was significantly more effective than the chemotherapeutic agent alone or ICG-001 alone in decreasing cell proliferation/viability. The addition of ICG-001 to MES-SA/Dx5 cells treated with either 1 mg/ml or 5 mg/ml of Doxorubicin increased caspase3/7 activation significantly.

EXAMPLE 4

Effect of ICG-001 on Chronic Myelocytic Leukemia (CML)

Despite the significant clinical success achieved in CML patients with imatinib to date, in advanced phase disease, the responses are often short-lived and patients invariably undergo disease progression (Melo J Hematology, 2003). This is the result of the emergence of leukemic drug resistant clones associated with increased nuclear β-catenin levels, a hallmark of increased TCF/β-catenin transcription (Weissman NEJM 2003). The efficacy of ICG-001 either alone or in combination with imatnib mesylate was investigated in both normal CD34+blast cells (mostly early stem/progenitors) and from bone barrow of CML patients at various stages of progression. CD34+ CML blasts showed significantly higher expression of β-catenin, ABCB1, htert, survivin/variant AEx3 and BMI-1 relative to CD34− cells, indicating constitutive activation of Wnt/catenin signaling and confirming the increased “stem/progenitor-like” features of this CD34+ CML blast cell population (FIG. 21C) (Jamieson et al., 2004).
Combination ICG-001 and imatinib treatment resulted in the most significant reduction in total colony forming units (CFU) as compared to the control of either drug treatment alone in all samples (FIG. 21D). Moreover, the morphological features of the colonies after drug treatment are also altered; the colonies became small and dispersed, and the dispersed colony phenotypes were more profound in the combination treatments, indicating that the treated colonies have an increased state of differentiation. In sharp contrast, the control colonies were large and compact. The H&E staining displayed reduced nuclear/cytoplasmic ratio in the treated cells (FIG. 21 E). Importantly, treatment of normal CD34+ cells with ICG-001 had minimal effects on total cellularity, CFU-Es and BFU-Es. ICG-001 did not affect colony formation of normal CD34+ hematopoietic cells.
In summary, whereas imatinib itself had limited effect, imatinib plus IGC-001 had a significant additive effect. ICG-001 up to 20CM did not have significant adverse effects on normal CD34+ cells and induced differentiation but not capase activation in K562 cells.

EXAMPLE 5

The Effect of ICG-001 and of Cisplatin on Cultured Ovarian Carcinoma and Melanoma Cells Expressing the Stem Cell Markers CD133 or Prominin-1, Respectively

This example describes measurements of the sensitivity of ovarian carcinoma cells and to ICG-001.

Colony inhibition assays were performed, in which plated cells from A2780, CP70, IGROV-1 and B 16 cells were exposed to doses of ICG-001 within the range of 0.625 to 10 μM. An exemplary experiment is illustrated in Table 3.

TABLE 3


Colony numbers formed by plated cells from the
cisplatin-sensitive A2780 exposed in vitro to ICG-001.

CONCENTRATION	COLONIES
OF ICG-001	(n = 4)
(μM)	M +/− SD	P-VALUE ≦ *

Control	160 +/− 21.5	—
0.625	74 +/− 4.7	0.003
1.25	28 +/− 13.2	0.004
2.5	0.25 +/− 0.5	0.001
5	0	0.000
*10	0	0.000

Statistical difference, according to t-test, when compared to control.

As shown Table 3, there were statistically significant differences between the control group (medium containing DMSO) and all the experimental groups (medium containing ICG-001 dissolved in DMSO) even at an ICG-001 concentration of 0.625 μM.
Table 4 presents data on the plating efficiencies of cultured cells from A2780, CP70, IGROV-1 and B16 in control wells as well as in wells exposed to ICG-001. The data indicate that the plating efficiency of the various cell lines was high, varying between 21 and 83%, which is commensurate with the fact that most of the plated cells expressed the CD133 marker of CSC.

TABLE 4

Average plating efficiency of 80 cells/well of the ovarian carcinoma

lines and the mouse melanoma line treated with ICG-001.

CONCENTRATION A2780 CP70 IGROV-1 IGROV-

OF ICG-001 (μM) % % % 1/CP % B16 %

Control

83 23 36 54 21

0.625 25 35 31 59 24

1.25 35 35 24 25 18

2.5 6 13 8 13 3

5 0 1 1 6 0

10 0 0 0 0 0
The cells were tested at range of concentrations of ICG-001 between 0.625 and 10 μM and at cisplatin concentrations between 1.25 to 20 μM. All three ovarian cancer lines tested (A2780, CP70 and IGROV-1) were more sensitive to ICG-001 than to cisplatin. For the cisplatin-resistant line CP70, >90% inhibition was achieved at 5 μM of ICG-001, as compared to 20 μM of cisplatin (FIG. 23C). The cisplatin-sensitive lines, IGROV-1 and A2780, had similar sensitivity to ICG-001 as to cisplatin (FIG. 23A and B). FIG. 24 shows experiments in which the sensitivity of ovarian carcinoma lines to ICG-001 and cisplatin were compared.
The cells were tested at range of concentrations of ICG-001 between 0.625 and 10 μM and at cisplatin concentrations between 1.25 to 20 μM. All three ovarian cancer lines tested (A2780, CP70 and IGROV-1) were more sensitive to IC G-001 than to cisplatin. For the cisplatin-resistant line CP70, >90% inhibition was achieved at 5 μM of ICG-001.

EXAMPLE 6

Inhibition of CBP-β-Catenin Interaction in SW480 Cells

The effect of several compounds on CBP-β-catenin binding was tested using the TOPFlash reporter system in SW480 cells.
As shown in FIG. 25, increasing concentrations of compounds PRI-001, PRI-002, PRI-003, PRI-004, PRI-005 and PRI-006 were effective, as compared with ICG-001. FIG. 26 shows pluc-6270 expression (luciferase) in SW480 cells treated with varying concentrations of ICG-001, PRI-003, and PRI-004.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1-8. (canceled)

9. A pharmaceutical composition comprising a compound of the following general formula (I):

wherein A is —(C═O)—CHR₃—, or —(C═O), B is N—R₅— or —CHR₆—, D is —(C═O)—(CHR₇)— or —(C═O)—, E is -(ZR₈)— or (C═O), G is —(XR₉)_n—, —(CHR₁₀)—(NR₆)—,—(C═O)—(XR₁₂)—, -(or nothing)-, —(C═O)—, X—(C═O)—R₁₃, X—(C═O)—NR₁₃R₁₄, X—(SO₂)—R₁₃, or X—(C═O)—OR₁₃, W is —Y(C═O)—, —(C═O)NH—, —(SO₂)—, —CHR₁₄, (C═O)—(NR₁₅)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=0 or 1; and R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers, salts, and prodrugs thereof, and a pharmaceutically acceptable carrier.

10-16. (canceled)

17. A compound selected from the group consisting of Compounds 1-2217.

18. A pharmaceutical composition comprising at least one compound of claim 17.

19-20. (canceled)

21. The pharmaceutical composition according to claim 18 where the composition comprises an effective amount of the compound and a pharmaceutically acceptable carrier.

22. A compound according to claim 17 wherein said compound is used in the preparation of a medicament for eradicating pathologic stem cells in cancer therapy.

23. (canceled)

24. The compound of claim 22, wherein said stem cells are derived from solid tumors.

25-26. (canceled)

27. The compound according to claim 17 wherein said compound is used in the preparation of a medicament for achieving the differentiation of pathologic stem cells by causing a switch from CBP/catenin to p300/catenin transcription in cancer therapy.

28. The compound of claim 17 wherein said catenin is β-catenin.

29. The compound of claim 17 wherein said catenin is γ/p120-catenin.

30. The compound of claim 17 wherein said compound inhibits CBP/catenin signaling in cancer stem cells.

31-35. (canceled)

36. The compound selected according to claim 17 wherein said compound inhibits CBP/catenin signaling in cancer stem cells thereby inducing differentiation of cancer stem cells and making them more susceptible to apoptosis induced by at least one specific pathway inhibitor.

37-39. (canceled)

40. The compound according to claim 17 wherein said compound is used in the preparation of a medicament for achieving the differentiation of pathologic stem cells by causing a switch from CBP/catenin to p300/catenin transcription in cancer therapy, thereby rendering the cancer cell more susceptible to treatment with other pathway-specific inhibitors.

41. The compound of claim 40 wherein said pathway-specific inhibitor is selected from the group consisting of imatinib; Her1/Her2 inhibitors; Notch inhibitors; Hedgehog inhibitors; EGF inhibitors; and PI3K pathway inhibitors.

42-45. (canceled)

46. The compound according to claim 17 wherein said compound blocks the CBP/β-catenin interaction.

47-51. (canceled)

52. A method of treating a cancerous condition by administering at least one compound of claim 17, wherein the cancerous condition is at least one selected from the group consisting of acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervix cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer, penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginal cancer, cancer of the vulva, and Wilm's tumor.

53-55. (canceled)

56. A pharmaceutical composition comprising at least one compound of claim 17, wherein said pharmaceutical composition is administered by a method selected from the group consisting of capsules, tablets, powders, granules, syrups, injectable fluids, creams, ointments, hydrophilic ointments, inhalable fluids, eye drops, and suppositories.

57. A pharmaceutical composition comprising at least one compound of claim 17, in combination with at least one cancer chemotherapeutic wherein said cancer chemotherapeutic works by a mechanism other than blocking CPB/catenin interaction.

58. (canceled)

59. A method for eliminating teratoma-forming stem cells prior to transplant into a mammalian subject, comprising incubating a stem cell culture with at least one compound of claim 17, wherein said compound inhibits CBP-β-catenin interaction and thereby causes stem cell differentiation.

60-96. (canceled)

97. A method for eliminating teratoma-forming stem cells prior to transplant into a mammalian subject, comprising incubating a stem cell culture with at least one compound of claim 17, wherein said compound inhibits CBP-β-catenin interaction and thereby causes stem cell differentiation.

98. A method for eliminating teratoma-forming stem cells prior to transplant into a mammalian subject, comprising incubating a stem cell culture with a compound of the following general formula (I):

wherein A is —(C═O)—CHR₃—, or —(C═O), B is N—R₅— or —CHR₆—, D is —(C═O)—(CHR₇)— or —(C═O)—, E is -(ZR₈)— or (C═O), G is —(XR₉)_n—, —(CHR₁₀)—(NR₆)—,—(C═O)—(XR₁₂)—, -(or nothing)-, —(C═O)—, X—(C═O)—R₁₃, X—(C═O)—NR₁₃R₁₄, X—(SO₂)—R₁₃, or X—(C═O)—OR₁₃, W is —Y(C═O)—, —(C═O)NH—, —(SO₂)—, —CHR₁₄, (C═O)—(NR₁₅)—, substituted or unsubstituted oxadiazole, substituted or unsubstituted triazole, substituted or unsubstituted thiadiazole, substituted or unsubstituted 4,5 dihydrooxazole, substituted or unsubstituted 4,5 dihydrothiazole, substituted or unsubstituted 4,5 dihydroimidazole, or nothing, Y is oxygen or sulfur, X and Z is independently nitrogen or CH, n=0 or 1; and R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and stereoisomers salts, and prodrugs thereof.