(en)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.

1.ApplicationNumber: US-59457606-A
1.PublishNumber: US-2007129353-A1
2.Date Publish: 20070607
3.Inventor: KAHN MICHAEL

4.Inventor Harmonized: KAHN MICHAEL(US)

5.Country: US
6.Claims:
(en)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.

7.Description:
(en)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)—CHR 3 —, or —(C═O), B is N—R 5 — or —CHR 6 —, D is —(C═O)—(CHR 7 )— or —(C═O)—, E is -(ZR 8 )— or (C═O), G is —(XR 9 ) n —, —(CHR 10 )—(NR 6 )—,—(C═O)—(XR 12 )—, —(C═N—W—R 1 )—, —(C═O)—, X—(C═O)—R 13 , X—(C═O)—NR 13 R 14 , X—(SO 2 )—R 13 , or X—(C═O)—OR 13 , W is —Y(C═O)—, —(C═O)NH—, —(SO 2 )—, —CHR 14 , (C═O)—(NR 15 )—, 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 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are 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 CHR 6 and W is —Y(C═O)—, —(C═O)NH—, —(SO 2 )—, —CHR 14 , or (C═O)—(NR 15 )—, G cannot be CHR 9 , NR 9 , (C═O)—CHR 12 , (C═O)—NR 12 , or no atom at all.

Also provided is a compound, salts, and prodrugs thereof of formula (I), wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , are R 15 are independently selected from the group consisting of aminoC 2-5 alkyl, guanidinoC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidinoC 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC 1-4 alkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylC 0-2 alkyl.
Further provided is the compound, salts, and prodrugs thereof of compound (I) wherein A is —(CHR 3 )—(C═O)—, B is —(NR 4 )—, D is (C═O)—, E is -(ZR 6 )—, G is —(C═O)—(XR 9 )—, and the compound has the following general formula (III):


wherein R 1 , R 2 , R 4 , R 6 , R 9 , 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—CHR 3 —, B is —NR 4 —, D is —(C═O)—, E is -(ZR 6 )—, Gi is (XR 7 ) n —, the α-helix mimetic compounds of this invention have the following formula (IV):


wherein R 1 , R 2 , R 4 , R 6 , R 7 , R 8 W, X and n are as defined above, Y is —C═O, —(C═O)—O—, —(C═O)—NR 8 , —SO 2 —, 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, R 1 , R 2 , R 6 , R 7 , and R 8 represent the remainder of the compound, and R 4 is selected from an amino acid side chain moiety. In this case, R 6 or R 7 may 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 —(CHR 6 )—, D is —(C═O)—, E is -(ZR 8 )—, and G is —(NH)— or —(CH 2 )—, 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 —(CH 2 )—, J is nitrogen, oxygen, or sulfur, Z is nitrogen or CH, and R 1 , R 2 , R 6 , R 8 , and R 13 are selected from an amino acid side chain moiety.

Also provided is a compound having the general formula (VI):


wherein B is —(CHR 2 )—, —(NR 2 )—, E is —(CHR 3 )—, V is —(XR 4 )— or nothing, W is —(C═O)—(XR 5 R 6 ), —(SO 2 )—, 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 R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , are R 15 are independently selected from the group consisting of aminoC 2-5 alkyl, guanidinoC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidinoC 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC 1-4 alkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylC 0-2 alkyl. Further provided is a compound, salts, and prodrugs thereof wherein B is —(CH)—(CH 3 ), E is —(CH)—(CH 3 ), V is —(XR 4 )— 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 —(CH 2 )—, J is nitrogen, oxygen, or sulfur, and R 5 is independently selected from the group consisting of aminoC 2-5 alkyl, guanidinoC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidinoC 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, Phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC 1-4 alkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylC 0-2 alkyl.

Provided is a pharmaceutical composition comprising a compound of the following general formula (I):


wherein A is —(C═O)—CHR 3 —, or —(C═O), B is N—R 5 — or —CHR 6 — 0 , D is —(C═O)(CHR 7 )— or 13 (C═O)—, E is —(ZR 8 )— or (C═O), G is —(XR 9 ) n —, —(CHR 10 )—(NR 6 13 ,—(C═O)—(XR 12 )—, -(or nothing)-, —(C═O)—, X—(C═O)—R 13 , X—(C═O)—NR 13 R 14 , X—(SO 2 )—R 13 , or X—(C═O)—OR 13 , W is —Y(C═O)—, —(C═O)NH—, —(SO 2 )—, —CHR 14 , (C═O)—(NR 15 )—, 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, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , are R 15 are independently selected from the group consisting of aminoC 2-5 alkyl, guanidinoC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidinoC 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC 1-4 alkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylC 0-2 alkyl. Further provided is a pharmaceutical composition of formula (I) wherein A is —(CHR 3 )—(C═O)—, B is —(NR 4 )—, D is (C═O)—, E is -(ZR 6 )—, G is —(C═O)—(XR 9 )—, 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—CHR 3 —, B is —NR 4 —, D is —(C═O)—, E is -(ZR 6 )—, Gi is (XR 7 ) n —, the α-helix mimetic compounds have the following formula (IV):


wherein R 1 , R 2 , R 4 , R 6 , R 7 , R 8 W, X and n are as defined above, Y is —C═O, —(C═O)—O—, —(C═O)—NR 8 , —SO 2 —, 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, R 1 , R 2 , R 6 , R 7 , and R 8 represent the remainder of the compound, and R 4 is selected from an amino acid side chain moiety. In this case, R 6 or R 7 may 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 —CHR 6 )—, D is —(C═O)—, E is -(ZR 8 )—, and G is —(NH)— or —(CH 2 )—, 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 —(CH 2 )—, J is nitrogen, oxygen, or sulfur, Z is nitrogen or CH, and R 1 , R 2 , R 6 , R 8 , and R 13 are 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 —(CHR 2 )—, —(NR 2 )—, E is —(CHR 3 )—, V is —(XR 4 )— or nothing, W is —(C═O)—(XR 5 R 6 ), —(SO 2 )—, 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 R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , are R 15 are independently selected from the group consisting of aminoC 2-5 alkyl, guanidinoC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidinoC 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC 1-4 alkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylC 0-2 alkyl. In certain embodiments, wherein B is —(CH)—(CH 3 ), E is —(CH)—(CH 3 ), V is —(XR 4 )— 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 —(CH 2 )—, J is nitrogen, oxygen, or sulfur, and R 5 is independently selected from the group consisting of aminoC 2-5 alkyl, guanidinoC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidinoC 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, Phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy or nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl, or methyl), imidazolinylC 1-4 alkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylCO- 2 alkyl.

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 - 2 AD shows the chemical structures of compounds 201-400.
FIG. 3A - 3 AC 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 - 8 AC shows the chemical structures of compounds 1401-1600.
FIG. 9A - 9 AE shows the chemical structures of compounds 1601-1800.
FIG. 10A - 10 AA shows the chemical structures of compounds 1801-2000.
FIG. 11A - 11 AA 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)—CHR 3 —, or —(C═O), B is N—R 5 — or —CHR 6 —, D is —(C═O)—(CHR 7 )— or —(C═O)—, E is -(ZR 8 )— or (C═O), G is —(XR 9 ) n —, —(CHR 10 )—(NR 6 )—,—(C═O)—(XR 12 )—, —(C═N—W—R 1 )—, —(C═O)—, X—(C═O)—R 13 , X—(C═O)—NR 13 R 14 , X—(SO 2 )—R 13 , or X—(C═O)—OR 13 , W is —Y(C═O)—, —(C═O)NH—, —(SO 2 )—, —CHR 14 , (C═O)—(NR 15 )—, 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 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are 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, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 R 10 , R 11 , R 12 , R 13 , R 14 , and R 15 are independently selected from the group consisting of aminoC 2-5 alkyl, guanidineC 2-5 alkyl, C 1-4 alkylguanidinoC 2-5 alkyl, diC 1-4 alkylguanidino-C 2-5 alkyl, amidinoC 2-5 alkyl, C 1-4 alkylamidino C 2-5 alkyl, diC 1-4 alkylamidinoC 2-5 alkyl, C 1-3 alkoxy, phenyl, substituted phenyl (where the substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-3 alkyl, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, 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 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyridylC 1-4 alkyl, substituted pyridylC 1-4 alkyl (where the pyridine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), pyrimidylC 1-4 alkyl, substituted pyrimidylC 1-4 alkyl (where the pyrimidine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), triazin-2-yl-C 1-4 alkyl, substituted triazin-2-yl-C 1-4 alkyl (where the triazine substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl or hydroxyl), imidazoC 1-4 alkyl, substituted imidazol C 1-4 alkyl (where the imidazole substituents are independently selected from one or more of amino, amidino, guanidino, hydrazino, amidrazonyl, C 1-4 alkylamino, C 1-4 dialkylamino, halogen, perfluoro C 1-4 alkyl, C 1-4 alkyl, C 1-3 alkoxy, nitro, carboxy, cyano, sulfuryl, hydroxyl or methyl), imidazolinylCalkyl, N-amidinopiperazinyl-N—C 0-4 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, hydroxyC 2-5 alkyl, C 1-5 alkylaminoC 2-5 alkyl, C 1-5 dialkylaminoC 2-5 alkyl, N-amidinopiperidinylC 1-4 alkyl and 4-aminocyclohexylC 0-2 alkyl.
In one embodiment, R 1 , R 2 , R 6 of E, and R 7 , R 8 and R 9 of G are the same or different and represent the remainder of the compound, and R 3 or A, R 4 of B or R 5 of 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 R 1 , R 2 , R 5 , R 6 , R 7 , R 8 and/or R 9 positions. 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 —CH 3 Alanine —CH(CH 3 ) 2 Valine —CH 2 CH(CH 3 ) 2 Leucine —CH(CH 3 )CH 2 CH 3 Isoleucine —(CH 2 ) 4 NH 3 + Lysine —(CH 2 ) 3 NHC(NH 2 )NH 2 + Arginine Histidine —CH 2 COO − Aspartic acid —CH 2 CH 2 COO − Glutamic acid —CH 2 CONH 2 Asparagine —CH 2 CH 2 CONH 2 Glutamine Phenylalanine Tyrosine Tryptophan —CH 2 SH Cysteine —CH 2 CH 2 SCH 3 Methionine —CH 2 OH Serine —CH(OH)CH 3 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 1-2 alkyl, a C 6-12 aryl and a C 7-12 arylalkyl, and in a more preferred embodiment, from a C 1-7 alkyl, a C 6-10 aryl and a C 7-11 arylalkyl.
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, —CONH 2 , —NH 2 , —NHR, —NRR, —SH, —SR, —SO 2 R, —SO 2 H, —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 R 1 , R 2 , R 5 , R 6 , R 7 , R 8 and R 9 moieties specifically include (but are not limited to) —OH, —OR, —COR, —COOR, —CONH 2 , —CONR, —CONRR, —NH 2 , —NHR, —NRR, —SO 2 R 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 R 1 , R 2 , R 5 , R 6 , R 7 , R 8 and R 9 ), R 1 , R 2 , R 5 , R 6 , R 7 , R 8 or R 9 may 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, R 1 , R 2 , R 5 , R 6 , R 7 , R 8 or R 9 may 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 R 1 , R 2 , R 7 or R 8 position, and more preferably at the R 1 or R 2 position.
In the embodiment wherein A is —(C═O)—CHR 3 —, B is —N—R 4 , D is —(C═O)—, E is -(ZR 6 )—, G is —(C═O)—(XR 9 )—, the α-helix mimetic compounds of this invention have the following general formula (III):


wherein R 1 , R 2 , R 4 , R 6 , R 7 , R 8 , W and X are as defined above, Y is —C═O, —(C═O)—O—, —(C═O)—NR 8 , —SO 2 —, or nothing, and Z is nitrogen or CH (when Z is CH, then X is nitrogen). In a preferred embodiment, R 1 , R 2 , R 6 , R 7 and R 8 represent the remainder of the compound, and R 4 is selected from an amino acid side chain moiety. In a more specific embodiment wherein A is —O—CHR 3 —, B is —NR 4 —, D is —(C═O)—, E is -(ZR 6 )—, Gi is (XR 7 ) n —, the α-helix mimetic compounds of this invention have the following formula (IV):


wherein R 1 , R 2 , R 4 , R 6 , R 7 , 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, R 1 , R 2 , R 6 , and R 7 represent the remainder of the compound, and R 4 is selected from an amino acid side chain moiety. In this case, R 6 or R 7 may 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 —(CHR 6 )—, D is —(C═O)—, E is -(ZR 8 )—, and G is —(NH)— or —(CH 2 )—, 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 —(CH 2 )—, J is nitrogen, oxygen, or sulfur, Z is nitrogen or CH, and R 1 , R 2 , R 6 , R 8 , and R 13 are selected from an amino acid side chain moiety.

Alternative embodiments of the invention relate to compounds having the general formula (VI):


wherein B is —(CHR 3 )—, —(NR 3 )—, E is —(CHR 4 )—, V is —(XR 5 )— or nothing, W is —(C═O)—(XR 6 R 7 ), —(SO 2 )—, 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are 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 —(XR 5 )— 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 —(CH 2 )—, J is nitrogen, oxygen, or sulfur, and R 2 and R 5 are defined as described above.

In preferred embodiments of the invention, R 2 in 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 R 2 as 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 H 2 N—R 2 from CH(OR) 2 —CHO or CH(OR) 2 —CH 2 -Hal (wherein Hal means a halogen atom).

A “second component piece” of this invention has the following formula S2:


Where L 1 is carboxyl-activation group such as halogen atom, R 3 , R 4 is 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 L 1 are 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 R 1 , R 2 , R 4 , R 7 .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., 20 2 ) dipeptides is created. Repeating the procedure seven times results in the preparation of a peptide library comprised of about 26 billion (i.e., 20 8 ) 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 R 2 -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 Et 3 N (42 mL, 300 mmol), (Boc) 2 O (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 NaHCO 3 (saturated aqueous solution, 100 mL) and NaCl (saturated aqueous solution, 100 mL), dried over MgSO 4 and concentrated in vacuo. The residue was filtered through a silica gel plug (flash column chromathography) eluting with toluene:Et 2 O=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:Et 2 O=10:1); 1 H NMR (400 MHz, CDCl 3 ) 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 Et 2 O (200 mL). The filtrate was concentrated in vacuo. The residue was filtered through a silica gel column (flash column chromathography) eluting with toluene:Et 2 O=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:Et 2 O=15:1); 1 H NMR (400 MHz, CDCl 3 ) 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 NaN 3 (8.80 g, 136 mmol) at 15° C. and stirred at 20° C. for 45 min. The resulting mixture was diluted with Et 2 O (400 mL), quenched by addition of NaCl (1 g in 150 mL of H 2 O) at 0° C. The solution was extracted with Et 2 O (100 mL). The organic phase was washed with NaCl (2 g in 100 mL of H 2 O) twice, dried over MgSO 4 and concentrated in vacuo. The residue was filtered through a silica gel plug (flash column chromathography) eluting with toluene:Et 2 O=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:Et 2 O=10:1); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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:NH 4 OH (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 (CHCl 3 :MeOH:NH 4 OH=100:25:1); 1 H NMR (400 MHz, CDCl 3 ) δ 7.67 (1H, d, J=7.7 Hz), 7.25-7.15 (2H, m), 4.85 (2H, br s), 1.58 (9H, s); 13 C NMR (99.5 MHz, CDCl 3 ) 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:Et 2 O=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:Et 2 O=10:1); 1 H NMR (400 MHz, CDCl 3 ) 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 CCl 4 was 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 Et 2 O (150 mL) and concentrated in vacuo. The residue was submitted to a silica gel column chromatography with toluene:Et 2 O=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-Et 2 O 10:1); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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 NaN 3 (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 H 2 O) at 0° C., diluted with Et 2 O (200 mL) and extracted with Et 2 O (200 mL×6). The organic phase was washed with NaCl (2 g in 100 mL of H 2 O) twice and brine (100 mL). The resulting solution was dried over MgSO 4 and concentrated in vacuo. The residue was submitted to silica gel column chromathography with toluene:Et 2 O=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:Et 2 O=10: 1); 1 H NMR (400 MHz, CDCl 3 ) δ 9.03 (1H, s), 7.95 (1H, d, J=7.7 Hz), 7.49 (2H, m), 5.01 (2H, s); 13 C NMR (99.5 MHz, CDCl 3 ) 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 N 2 and then filtered through Celite washing with MeOH:NH 4 OH (25:1, 260 mL) and concentrated in vacuo. The residue was submitted to silica gel column chromathography eluting with CHCl 3 :MeOH:NH 4 OH (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 (CHCl 3 :MeOH:NH 4 OH=100:25:1); 1 H NMR (400 MHz, CD 3 OD) δ 9.23 (1H, s), 7.97 (1H, d, J=7.7 Hz), 7.46 (2H, m), 4.30 (2H, s); 13 C NMR (99.5 MHz, CD 3 OD) 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 CHCl 3 was 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 CHCl 3 (50 mL×3). The aqueous phase was adjusted to pH 12 with 2 M NaOHaq and then extracted with CHCl 3 (100 mL×3). The organic phase was dried over Na 2 SO 4 and concentrated in vacuo. The residue was recrystalized from hexane-CHCl 3 to afford (1.7 g, 9.5 mmol, 63%) as a colorless crystal. R f =0.44 (CHCl 3 :MeOH=9:1); 1 H 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); 13 C 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 H 2 O (50 mL) and brine (50 mL), dried over Na 2 SO 4 , 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); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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 CH 2 Cl 2 (43 mL) were added DIPEA (7.5 mL, 43 mmol), DMAP (1.1 g, 8.6 mmol) and (Boc) 2 O (9.4 g, 43 mmol) and then stirred for 1 hr at room temperature. The reaction miture was concentrated and then submitted to SiO 2 (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); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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/H 2 O (2/3/1, 24 mL) was added LiOH H 2 O (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 H 2 O (30 mL) and brine (30 mL), dried over Na 2 SO 4 , added Et 3 N (2 mL), filtered and concentrated. The crude was submitted to SiO 2 column chromatography with CHCl 3 :MeOH=100:0 to 85:15 as eluante to afford a pale yellow sticky oil 4-Benzyl-3-Boc-2-methylsemicarbazidylacetatic acid8Et 3 N salt (1.99 g, 4.56 mmol, 72%); 1 H NMR (400 MHz, CDCl 3 ) 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, Et 3 N), 1.40 (9H, br s), 1.08 (3.6H, t, J=7.3 Hz, Et 3 N); 13 C NMR (99.5 MHz, CDCl 3 ) δ 174.2, 159.2, 154.1, 140.1, 128.2, 127.4, 12.7, 81.8, 52.2, 45.1 (Et 3 N), 44.1, 34.5, 28.1, 8.3 (Et 3 N); 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 CHCl 3 was 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 CHCl 3 (50 mL×3). The aqueous phase was adjusted to pH 12 with 2 M NaOH aq and then extracted with CHCl 3 (100 mL×3). The organic phase was dried over Na 2 SO 4 and concentrated in vacuo. The residue was recrystalized from hexane-CHCl 3 to afford a colorless crystal (2.20 g, 10.7 mmol, 70%). R f =0.50 (CHCl 3 :MeOH=9:1); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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 H 2 O (50 mL) and brine (50 mL), dried over Na 2 SO 4 , 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); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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 CH 2 Cl 2 (50 mL) were added DIPEA (8.5 mL, 49 mmol), DMAP (1.19 g, 9.76 mmol) and (Boc) 2 O (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) 2 O (2.66 g, 12.2 mmol) were added. After the reaction mixture was stirred for additional 6 hr, the mixture was diluted with CH 2 Cl 2 (100 mL) and then sat.NaHCO 3 (50 mL) was added at 0δ. The separated aqueous phase was extracted with CH 2 Cl 2 (100 mL×2). The combined organic phases were washed with H 2 O (100 mL) and brine (100 mL), dried over Na 2 SO 4 , filtered and concentrated. The crude was submitted to SiO 2 (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); 1 H NMR (400 MHz, CDCl 3 ) 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); 13 C NMR (99.5 MHz, CDCl 3 ) 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/H 2 O (2/3/1, 25 mL) was added LiOH H 2 O (685 mg, 16.3 mmol) at 0δ. After stirred for 40 min at room temperature, the reaction mixture was diluted with CH 2 Cl 2 (50 mL) at 0δ. The mixture was acidified with 1N HCl and then extracted with CH 2 Cl 2 . The combined extraction were washed with H 2 O (30 mL) and Brine (30 mL), dried over Na 2 SO 4 , added Et 3 N (3 mL), filtered and concentrated. The crude was submitted to SiO 2 column chromatography with CHCl 3 :MeOH=100:0 to 85:15 as eluate to afford orange sticky oil 4-Benzyl-3-Boc-2-allylsemicarbazidylacetatic acidδEt 3 N salt (3.66 g, 7.87 mmol, 96%); 1 H NMR (400 MHz, CDCl 3 , 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, Et 3 N), 1.44 and 1.42 (9H, s), 1.11 (7.5H, t, J=7.2 Hz, Et 3 N); 13 C NMR (99.5 MHz, CDCl 3 ) 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 (Et 3 N), 44.2, 28.2, 8.3 (Et 3 N); 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, CH 2 Cl 2 50 mL×3, MeOH 50 mL×3, Et 2 O 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 CH 2 Cl 2 (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 CH 2 Cl 2 (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 CH 2 Cl 2 (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 CH 2 Cl 2 soltuion 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 CH 2 Cl 2 (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% HCO 2 H (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% HCO 2 H (1.5 mL×5 min×2). The combined HCO 2 H 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 (CHCl 3 :MeOH=9:1); 1 H NMR (400 MHz, CDCl 3 ) 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 CH 2 Cl 2 soltuion 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 CH 2 Cl 2 (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% HCO 2 H (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% HCO 2 H (1.5 mL×5 min×2). The combined HCO 2 H 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 (CHCl 3 :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 CH 2 Cl 2 (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 CH 2 Cl 2 (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 CH 2 Cl 2 (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 CH 2 Cl 2 soltuion 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 CH 2 Cl 2 (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% HCO 2 H (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% HCO 2 H (4.0 mL×5 min×2). The combined HCO 2 H 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 (CHCl 3 :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 CH 2 Cl 2 soltuion 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 CH 2 Cl 2 (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% HCO 2 H (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% HCO 2 H (4.0 mL×5 min×2). The combined HCO 2 H 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 (CHCl 3 :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 Et 3 N (13.4 μL, 0.096 mmol) and POCl 3 (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 H 2 O (1 mL) and then NaHCO 3 was added at 0δ to pH 8. After stirred overnight, the mixture was acidified to pH 3 with 1N HCl followed by extraction with CHCl 3 (5 mL×3). The combined extracts were dried over Na 2 SO 4 , filtered and concentrated to afford pale yellow powder Compound No. 2201 (17.1 mg, 83%). TLC: Rf=0.458Silica gel F254, CHCl 3 :MeOH:EtOH:H 2 O:AcOH:nBuOH=100:40:10:10:8:5δ; 1 H NMR (400 MHz, CDCl 3 ) 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); 31 P NMR (160.26 MHz, CDCl 3 ) 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 Et 3 N (14.9 μL, 0.107 mmol) and POCl 3 (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 H 2 O (1 mL) and then NaHCO 3 was added at 0δ to pH 8. After stirred overnight, the mixture was acidified to pH 3 with 1N HCl followed by extraction with CHCl 3 (5 mL×3). The combined extracts were dried over Na 2 SO 4 , filtered and concentrated to afford pale yellow powder Compound No. 2202 (21.0 mg, 88%). TLC: Rf=0.536Silica gel F254, CHCl 3 :MeOH:EtOH:H 2 O: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 99 676 677 100 531 532 101 605 606 102 623 624 103 635 636 104 674 675 105 529 530 106 603 604 107 621 622 108 633 634 109 672 673 110 527 528 111 607 608 112 625 626 113 637 638 114 676 677 115 531 532 116 604 605 117 622 623 118 634 635 119 673 674 120 528 529 121 562 563 122 580 581 123 592 593 124 631 632 125 486 487 126 590 591 127 608 609 128 620 621 129 659 660 130 514 515 131 588 589 132 606 607 133 618 619 134 657 658 135 512 513 136 586 587 137 604 605 138 616 617 139 655 656 140 510 511 141 590 591 142 608 609 143 620 621 144 659 660 145 514 515 146 587 588 147 605 606 148 617 618 149 656 657 150 511 512 151 590 591 152 608 609 153 620 621 154 659 660 155 514 515 156 618 619 157 636 637 158 648 649 159 687 688 160 542 543 161 616 617 162 634 635 163 646 647 164 685 686 165 540 541 166 614 615 167 632 633 168 644 645 169 683 684 170 538 539 171 618 619 172 636 637 173 648 649 174 687 688 175 542 543 176 615 616 177 633 634 178 645 646 179 684 685 180 539 540 181 666 667 182 684 685 183 696 697 184 735 736 185 590 591 186 694 695 187 712 713 188 724 725 189 763 764 190 618 619 191 692 693 192 710 711 193 722 723 194 761 762 195 616 617 196 690 691 197 708 709 198 720 721 199 759 760 200 614 615 201 694 695 202 712 713 203 724 725 204 763 764 205 618 619 206 691 692 207 709 710 208 721 722 209 760 761 210 615 616 211 696 697 212 714 715 213 726 727 214 765 766 215 620 621 216 724 725 217 742 743 218 754 755 219 793 794 220 648 649 221 722 723 222 740 741 223 752 753 224 791 792 225 646 647 226 720 721 227 738 739 228 750 751 229 789 790 230 644 645 231 724 725 232 742 743 233 754 755 234 793 794 235 648 649 236 721 722 237 739 740 238 751 752 239 790 791 240 645 646 241 590 591 242 608 609 243 620 621 244 659 660 245 514 515 246 618 619 247 636 637 248 648 649 249 687 688 250 542 543 251 616 617 252 634 635 253 646 647 254 685 686 255 540 541 256 614 615 257 632 633 258 644 645 259 683 684 260 538 539 261 618 619 262 636 637 263 648 649 264 687 688 265 542 543 266 615 616 267 633 634 268 645 646 269 684 685 270 539 540 271 592 593 272 610 611 273 622 623 274 661 662 275 516 517 276 620 621 277 638 639 278 650 651 279 689 690 280 544 545 281 618 619 282 636 637 283 648 649 284 687 688 285 542 543 286 616 617 287 634 635 288 646 647 289 685 686 290 540 541 291 620 621 292 638 639 293 650 651 294 689 690 295 544 545 296 617 618 297 635 636 298 647 648 299 686 687 300 541 542 301 577 578 302 595 596 303 607 608 304 646 647 305 501 502 306 605 606 307 623 624 308 635 636 309 674 675 310 529 530 311 603 604 312 621 622 313 633 634 314 672 673 315 527 528 316 601 602 317 619 620 318 631 632 319 670 671 320 525 526 321 605 606 322 623 624 323 635 636 324 674 675 325 529 530 326 602 603 327 620 621 328 632 633 329 671 672 330 526 527 331 635 636 332 653 654 333 665 666 334 704 705 335 559 560 336 663 664 337 681 682 338 693 694 339 732 733 340 587 588 341 661 662 342 679 680 343 691 692 344 730 731 345 585 586 346 659 660 347 677 678 348 689 690 349 728 729 350 583 584 351 663 664 352 681 682 353 693 694 354 732 733 355 587 588 356 660 661 357 678 679 358 690 691 359 729 730 360 584 585 361 716 717 362 734 735 363 746 747 364 785 786 365 640 641 366 744 745 367 762 763 368 774 775 369 813 814 370 668 669 371 742 743 372 760 761 373 772 773 374 811 812 375 666 667 376 740 741 377 758 759 378 770 771 379 809 810 380 664 665 381 744 745 382 762 763 383 774 775 384 813 814 385 668 669 386 741 742 387 759 760 388 771 772 389 810 811 390 665 666 391 565 566 392 583 584 393 595 596 394 634 635 395 489 490 396 593 594 397 611 612 398 623 624 399 662 663 400 517 518 401 591 592 402 609 610 403 621 622 404 660 661 405 515 516 406 589 590 407 607 608 408 619 620 409 658 659 410 513 514 411 593 594 412 611 612 413 623 624 414 662 663 415 517 518 416 590 591 417 608 609 418 620 621 419 659 660 420 514 515 421 578 579 422 596 597 423 608 609 424 647 648 425 502 503 426 606 607 427 624 625 428 636 637 429 675 676 430 530 531 431 604 605 432 622 623 433 634 635 434 673 674 435 528 529 436 602 603 437 620 621 438 632 633 439 671 672 440 526 527 441 606 607 442 624 625 443 636 637 444 675 676 445 530 531 446 603 604 447 621 622 448 633 634 449 672 673 450 527 528 451 634 635 452 652 653 453 664 665 454 703 704 455 558 559 456 662 663 457 680 681 458 692 693 459 731 732 460 586 587 461 660 661 462 678 679 463 690 691 464 729 730 465 584 585 466 658 659 467 676 677 468 688 689 469 727 728 470 582 583 471 662 663 472 680 681 473 692 693 474 731 732 475 586 587 476 659 660 477 677 678 478 689 690 479 728 729 480 583 584 481 677 678 482 695 696 483 707 708 484 746 747 485 601 602 486 705 706 487 723 724 488 735 736 489 774 775 490 629 630 491 703 704 492 721 722 493 733 734 494 772 773 495 627 628 496 701 702 497 719 720 498 731 732 499 770 771 500 625 626 501 705 706 502 723 724 503 735 736 504 774 775 505 629 630 506 702 703 507 720 721 508 732 733 509 771 772 510 626 627 511 607 608 512 625 626 513 637 638 514 676 677 515 531 532 516 635 636 517 653 654 518 665 666 519 704 705 520 559 560 521 633 634 522 651 652 523 663 664 524 702 703 525 557 558 526 631 632 527 649 650 528 661 662 529 700 701 530 555 556 531 635 636 532 653 654 533 665 666 534 704 705 535 559 560 536 632 633 537 650 651 538 662 663 539 701 702 540 556 557 541 640 641 542 658 659 543 670 671 544 709 710 545 564 565 546 668 669 547 686 687 548 698 699 549 737 738 550 592 593 551 666 667 552 684 685 553 696 697 554 735 736 555 590 591 556 664 665 557 682 683 558 694 695 559 733 734 560 588 589 561 668 669 562 686 687 563 698 699 564 737 738 565 592 593 566 665 666 567 683 684 568 695 696 569 734 735 570 589 590 571 587 588 572 605 606 573 617 618 574 656 657 575 511 512 576 615 616 577 633 634 578 645 646 579 684 685 580 539 540 581 613 614 582 631 632 583 643 644 584 682 683 585 537 538 591 615 616 592 633 634 593 645 646 594 684 685 595 539 540 586 611 612 587 629 630 588 641 642 589 680 681 590 535 536 596 612 613 597 630 631 598 642 643 599 681 682 600 536 537 601 551 552 602 579 580 603 577 578 604 565 566 605 593 594 606 591 592 607 581 582 608 609 610 609 607 608 610 497 498 611 525 526 612 523 524 613 511 512 614 539 540 615 537 538 616 527 528 617 555 556 618 553 554 619 513 514 620 541 542 621 539 540 622 527 528 623 555 556 624 553 554 625 543 544 626 571 572 627 569 570 628 483 484 629 511 512 630 509 510 631 497 498 632 525 526 633 523 524 634 513 514 635 541 542 636 539 540 637 518 519 638 546 547 639 544 545 640 532 533 641 560 561 642 558 559 643 548 549 644 576 577 645 574 575 646 553 554 647 581 582 648 579 580 649 567 568 650 595 596 651 593 594 652 583 584 653 611 612 654 609 610 655 553 554 656 581 582 657 579 580 658 567 568 659 595 596 660 593 594 661 583 584 662 611 612 663 609 610 664 563 564 665 591 592 666 589 590 667 577 578 668 605 606 669 603 604 670 593 594 671 621 622 672 619 620 673 545 546 674 573 574 675 571 572 676 559 560 677 587 588 678 585 586 679 575 576 680 603 604 681 601 602 682 518 519 683 546 547 684 544 545 685 532 533 686 560 561 687 558 559 688 548 549 689 576 577 690 574 575 691 497 498 692 525 526 693 523 524 694 511 512 695 539 540 696 537 538 697 527 528 698 555 556 699 553 554 700 497 498 701 525 526 702 523 524 703 511 512 704 539 540 705 537 538 706 527 528 707 555 556 708 553 554 709 497 498 710 525 526 711 523 524 712 511 512 713 539 540 714 537 538 715 527 528 716 555 556 717 553 554 718 541 542 719 569 570 720 567 568 721 555 556 722 583 584 723 581 582 724 571 572 725 599 600 726 597 598 727 554 555 728 582 583 729 580 581 730 568 569 731 596 597 732 594 595 733 584 585 734 612 613 735 610 611 736 554 555 737 582 583 738 580 581 739 568 569 740 596 597 741 594 595 742 584 585 743 612 613 744 610 611 745 554 555 746 582 583 747 580 581 748 568 569 749 596 597 750 594 595 751 584 585 752 612 613 753 610 611 754 561 562 755 589 590 756 587 588 757 575 576 758 603 604 759 601 602 760 591 592 761 619 620 762 617 618 763 562 563 764 590 591 765 588 589 766 576 577 767 604 605 768 602 603 769 592 593 770 620 621 771 618 619 772 568 569 773 596 597 774 594 595 775 582 583 776 610 611 777 608 609 778 598 599 779 626 627 780 624 625 781 603 604 782 631 632 783 629 630 784 617 618 785 645 646 791 555 556 792 553 554 793 541 542 794 569 570 795 567 568 786 643 644 787 633 634 788 661 662 789 659 660 790 527 528 796 557 558 797 585 586 798 583 584 799 544 545 800 572 573 801 570 571 802 558 559 803 586 587 804 584 585 805 574 575 806 602 603 807 600 601 808 526 527 809 554 555 810 552 553 811 540 541 812 568 569 813 566 567 814 556 557 815 584 585 816 582 583 817 526 527 818 554 555 819 552 553 820 540 541 821 568 569 822 566 567 823 556 557 824 584 585 825 582 583 826 519 520 827 547 548 828 545 546 829 533 534 830 561 562 831 559 560 832 549 550 833 577 578 834 575 576 835 534 535 836 562 563 837 560 561 838 548 549 839 576 577 840 574 575 841 564 565 842 592 593 843 590 591 844 569 570 845 597 598 846 595 596 847 583 584 848 611 612 849 609 610 850 599 600 851 627 628 852 625 626 853 603 604 854 631 632 855 629 630 856 617 618 857 645 646 858 643 644 859 633 634 860 661 662 861 659 660 862 534 535 863 562 563 864 560 561 865 548 549 866 576 577 867 574 575 868 564 565 869 592 593 870 590 591 871 534 535 872 562 563 873 560 561 874 548 549 875 576 577 876 574 575 877 564 565 878 592 593 879 590 591 880 484 485 881 512 513 882 510 511 883 498 499 884 526 527 885 524 525 886 514 515 887 542 543 888 540 541 889 484 485 890 512 513 891 510 511 892 498 499 893 526 527 894 524 525 895 514 515 896 542 543 897 540 541 898 534 535 899 562 563 900 560 561 901 548 549 902 576 577 903 574 575 904 564 565 905 592 593 906 590 591 907 534 535 908 562 563 909 560 561 910 548 549 911 576 577 912 574 575 913 564 565 914 592 593 915 590 591 916 519 520 917 547 548 918 545 546 919 533 534 920 561 562 921 559 560 922 549 550 923 577 578 924 575 576 925 519 520 926 547 548 927 545 546 928 533 534 929 561 562 930 559 560 931 549 550 932 577 578 933 575 576 934 537 538 935 565 566 936 563 564 937 551 552 938 579 580 939 577 578 940 567 568 941 595 596 942 593 594 943 573 574 944 601 602 945 599 600 946 587 588 947 615 616 948 613 614 949 603 604 950 631 632 951 629 630 952 501 502 953 529 530 954 527 528 955 515 516 956 543 544 957 541 542 958 531 532 959 559 560 960 557 558 961 501 502 962 529 530 963 527 528 964 515 516 965 543 544 966 541 542 967 531 532 968 559 560 969 557 558 970 501 502 971 529 530 972 527 528 973 515 516 974 543 544 975 541 542 976 531 532 977 559 560 978 557 558 979 552 553 980 580 581 981 578 579 982 566 567 983 594 595 984 592 593 985 582 583 986 610 611 987 608 609 988 566 567 989 594 595 990 592 593 991 580 581 992 608 609 993 606 607 994 596 597 995 624 625 996 622 623 997 523 524 998 551 552 999 549 550 1000 537 538 1001 565 566 1002 563 564 1003 553 554 1004 581 582 1005 579 580 1006 537 538 1007 565 566 1008 563 564 1009 551 552 1010 579 580 1011 577 578 1012 567 568 1013 595 596 1014 593 594 1015 523 524 1016 551 552 1017 549 550 1018 537 538 1019 565 566 1020 563 564 1021 553 554 1022 581 582 1023 579 580 1024 537 538 1025 565 566 1026 563 564 1027 551 552 1028 579 580 1029 577 578 1030 567 568 1031 595 596 1032 593 594 1033 561 562 1034 589 590 1035 587 588 1036 575 576 1037 603 604 1038 601 602 1039 591 592 1040 619 620 1041 617 618 1042 523 524 1043 551 552 1044 549 550 1045 537 538 1046 565 566 1047 563 564 1048 553 554 1049 581 582 1050 579 580 1051 537 538 1052 565 566 1053 563 564 1054 551 552 1055 579 580 1056 577 578 1057 567 568 1058 595 596 1059 593 594 1060 671 672 1061 699 700 1062 697 698 1063 685 686 1064 713 714 1065 711 712 1066 701 702 1067 729 730 1068 727 728 1069 561 562 1070 589 590 1071 587 588 1072 575 576 1073 603 604 1074 601 602 1075 591 592 1076 619 620 1077 617 618 1078 561 562 1079 589 590 1080 587 588 1081 575 576 1082 603 604 1083 601 602 1084 591 592 1085 619 620 1086 617 618 1087 524 525 1088 552 553 1089 550 551 1090 538 539 1091 566 567 1092 564 565 1093 554 555 1094 582 583 1095 580 581 1096 538 539 1097 566 567 1098 564 565 1099 552 553 1100 580 581 1101 578 579 1102 568 569 1103 596 597 1104 594 595 1105 538 539 1106 566 567 1107 564 565 1108 552 553 1109 580 581 1110 578 579 1111 568 569 1112 596 597 1113 594 595 1114 540 541 1115 568 569 1116 566 567 1117 554 555 1118 582 583 1119 580 581 1120 570 571 1121 598 599 1122 596 597 1123 523 524 1124 551 552 1125 549 550 1126 537 538 1127 565 566 1128 563 564 1129 553 554 1130 581 582 1131 579 580 1132 539 540 1133 567 568 1134 565 566 1135 553 554 1136 581 582 1137 579 580 1138 569 570 1139 597 598 1140 595 596 1141 539 540 1142 567 568 1143 565 566 1144 553 554 1145 581 582 1146 579 580 1147 569 570 1148 597 598 1149 595 596 1150 540 541 1151 568 569 1152 566 567 1153 554 555 1154 582 583 1155 580 581 1156 570 571 1157 598 599 1158 596 597 1159 524 525 1160 552 553 1161 550 551 1162 538 539 1163 566 567 1164 564 565 1165 554 555 1166 582 583 1167 580 581 1168 540 541 1169 568 569 1170 566 567 1171 554 555 1172 582 583 1173 580 581 1174 570 571 1175 598 599 1176 596 597 1177 554 555 1178 582 583 1179 580 581 1180 568 569 1181 596 597 1182 594 595 1183 584 585 1184 612 613 1185 610 611 1186 583 584 1187 611 612 1188 609 610 1189 597 598 1190 625 626 1191 623 624 1192 613 614 1193 641 642 1194 639 640 1195 558 559 1196 586 587 1197 584 585 1198 572 573 1199 600 601 1200 598 599 1201 555 556 1202 573 574 1203 585 586 1204 624 625 1205 479 480 1206 521 522 1207 583 584 1208 601 602 1209 613 614 1210 652 653 1211 507 508 1212 549 550 1213 581 582 1214 599 600 1215 611 612 1216 650 651 1217 505 506 1218 547 548 1219 579 580 1220 597 598 1221 609 610 1222 648 649 1223 503 504 1224 545 546 1225 583 584 1226 601 602 1227 613 614 1228 652 653 1229 507 508 1230 549 550 1231 580 581 1232 598 599 1233 610 611 1234 649 650 1235 504 505 1236 546 547 1237 569 570 1238 587 588 1239 599 600 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1317 659 660 1318 698 699 1319 553 554 1320 595 596 1321 627 628 1322 645 646 1323 657 658 1324 696 697 1325 551 552 1326 593 594 1327 625 626 1328 643 644 1329 655 656 1330 694 695 1331 549 550 1332 591 592 1333 629 630 1334 647 648 1335 659 660 1336 698 699 1337 553 554 1338 595 596 1339 626 627 1340 644 645 1341 656 657 1342 695 696 1343 550 551 1344 592 593 1345 584 585 1346 602 603 1347 614 615 1348 653 654 1349 508 509 1350 550 551 1351 612 613 1352 630 631 1353 642 643 1354 681 682 1355 536 537 1356 578 579 1357 610 611 1358 628 629 1359 640 641 1360 679 680 1361 534 535 1362 576 577 1363 608 609 1364 626 627 1365 638 639 1366 677 678 1367 532 533 1368 574 575 1369 612 613 1370 630 631 1371 642 643 1372 681 682 1373 536 537 1374 578 579 1375 609 610 1376 627 628 1377 639 640 1378 678 679 1379 533 534 1380 575 576 1381 612 613 1382 630 631 1383 642 643 1384 681 682 1385 536 537 1386 578 579 1387 640 641 1388 658 659 1389 670 671 1390 709 710 1391 564 565 1392 606 607 1393 638 639 1394 656 657 1395 668 669 1396 707 708 1397 562 563 1398 604 605 1399 636 637 1400 654 655 1401 666 667 1402 705 706 1403 560 561 1404 602 603 1405 640 641 1406 658 659 1407 670 671 1408 709 710 1409 564 565 1410 606 607 1411 637 638 1412 655 656 1413 667 668 1414 706 707 1415 561 562 1416 603 604 1417 688 689 1418 706 707 1419 718 719 1420 757 758 1421 612 613 1422 654 655 1423 716 717 1424 734 735 1425 746 747 1426 785 786 1427 640 641 1428 682 683 1429 714 715 1430 732 733 1431 744 745 1432 783 784 1433 638 639 1434 680 681 1435 712 713 1436 730 731 1437 742 743 1438 781 782 1439 636 637 1440 678 679 1441 716 717 1442 734 735 1443 746 747 1444 785 786 1445 640 641 1446 682 683 1447 713 714 1448 731 732 1449 743 744 1450 782 783 1451 637 638 1452 679 680 1453 718 719 1454 736 737 1455 748 749 1456 787 788 1457 642 643 1458 684 685 1459 746 747 1460 764 765 1461 776 777 1462 815 816 1463 670 671 1464 712 713 1465 744 745 1466 762 763 1467 774 775 1468 813 814 1469 668 669 1470 710 711 1471 742 743 1472 760 761 1473 772 773 1474 811 812 1475 666 667 1476 708 709 1477 746 747 1478 764 765 1479 776 777 1480 815 816 1481 670 671 1482 712 713 1483 743 744 1484 761 762 1485 773 774 1486 812 813 1487 667 668 1488 709 710 1489 612 613 1490 630 631 1491 642 643 1492 681 682 1493 536 537 1494 578 579 1495 640 641 1496 658 659 1497 670 671 1498 709 710 1499 564 565 1500 606 607 1501 638 639 1502 656 657 1503 668 669 1504 707 708 1505 562 563 1506 604 605 1507 636 637 1508 654 655 1509 666 667 1510 705 706 1511 560 561 1512 602 603 1513 640 641 1514 658 659 1515 670 671 1516 709 710 1517 564 565 1518 606 607 1519 637 638 1520 655 656 1521 667 668 1522 706 707 1523 561 562 1524 603 604 1525 614 615 1526 632 633 1527 644 645 1528 683 684 1529 538 539 1530 580 581 1531 642 643 1532 660 661 1533 672 673 1534 711 712 1535 566 567 1536 608 609 1537 640 641 1538 658 659 1539 670 671 1540 709 710 1541 564 565 1542 606 607 1543 638 639 1544 656 657 1545 668 669 1546 707 708 1547 562 563 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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 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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 IC 50 ˜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.