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Title:
HIV/FIV PROTEASE INHIBITORS
Document Type and Number:
WIPO Patent Application WO/2002/068586
Kind Code:
A3
Abstract:
With the help of X-ray structural analyses of drug-resistant HIV proteases and molecular modeling, a new type of inhibitor with a small P3 residue has been developed. These inhibitors are effective against HIV and its drug-resistant mutants, as well as FIV. Modification of existing HIV protease inhibitors by reducing the size of the P3 residue has the same effect. This finding provides a new strategy for the development of HIV protease inhibitors effective against the wild type and drug-resistant mutants and further supports that FIV protease is a useful model for drug-resistant HIV proteases, which often are developed through reduction in size of the binding region for the P3 group or the combined P3 and P1 groups.

Inventors:
WONG, Chi-Huey (P.O. Box 8154, Rancho Santa Fe, CA, 92067, US)
Application Number:
US2002/001695
Publication Date:
November 15, 2007
Filing Date:
January 22, 2002
Export Citation:
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Assignee:
THE SCRIPPS RESEARCH INSTITUTE (10555 North Torrey Pines Road, LaJolla, CA, 92037, US)
WONG, Chi-Huey (P.O. Box 8154, Rancho Santa Fe, CA, 92067, US)
International Classes:
A61P31/18; C07D273/00; C07D273/02; C07D273/08; C07D277/06; C07K5/06; C07K5/065; C07K7/02; A61K31/33; A61K38/00
Foreign References:
US6043357A2000-03-28
Other References:
MAK ET AL.: 'Design, synthesis and biological evaluation of HIV-FIV protease inhibitors incorporating a coformationally contrained macrocyte and a small P3' residue' BIOORGANIC & MEDICINAL CHEMISTRY LETTERS vol. 11, no. 2, 22 January 2001, pages 219 - 222, XP004314852
Attorney, Agent or Firm:
VIKSNINS, Ann, S. (Schwegman, Lundberg Woessner & Kluth,P.O.Box 293, Minneapolis MN, 55402, US)
Download PDF:
Claims:
WHAT IS CLAIMED 1. An HIV protease inhibitor comprising a macrocycline group of formula VII, VIII or VIIIa :

VII

VIII

VIIIa wherein Z is a leaving group or a linkage to a protease inhibitor; and R, 3 is hydrogen or carbobenzyloxy-amino-.

2. A pharmaceutical composition comprising a therapeutically effective amount of the HIV protease inhibitor of claim 1, in combination with a pharmaceutically acceptable diluent or carrier.
3. A compound of formula IX: wherein: R3 is hydrogen, oxygen or hydroxyl ; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and Re are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when Rs and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; and Y is Rg, Rlo or-NH-Rlo R9 is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine-

amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-#-fluoro-phenylalanine, acetyl-#-fluoro-phenylalanine- amino, acetyl-#-fluoro-phenylalanine-valine-, acetyl-#-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino-; and Rio is a macrocycle of formula VII, VIII or VIIIa :

wherein: Z is the linkage to the compound of formula IX; and R13 is hydrogen, carbobenzyloxy-amino-.

4. A compound of Formula X:

wherein: Ph is phenyl ; R3 is hydrogen, oxygen or hydroxyl; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when Rs and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; and Rg is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4-

trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine- amino, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino-.

5. The compound of claim 4 wherein R9 is tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-alanine-valine-, carbobenzyloxy- alanine-valine-amino-, p-methylphenylsulfoxide-alanine-valine, p- methylphenylsulfoxide-alanine-valine-amino, N-acetyl-tryptophan-valine, N- acetyl-tryptophan-valine-amino,acetyl-p-F-phenylalanine-valine-, 4-methyl- phenylsulfoxide-, 4-methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo- phenylsulfoxide-, 4-bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4- nitro-phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 4-trifluoromethoxyphenylamino-carbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenyl-aminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro- phenylalanine-valine-amino-, acetyl-phenylalanine-valine-, acetyl- phenylalanine-valine-amino, acetyl-tyrosine-valine, or acetyl-tyrosine-valine- amino.
6. A compound having Formula XI:

wherein: Ph is phenyl; R3 is hydrogen, oxygen or hydroxyl; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when Rs and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; Rg is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4-

methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine- amino, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, #-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino- ; R, I is hydrogen or lower alkyl ; and R, 2 is hydrogen or lower alkyl.

7. A compound having Formula XIa : wherein: Ph is phenyl ;

R3 is hydrogen, oxygen or hydroxyl; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when Rs and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; R9 is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4-

trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine- amino, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino- ; R"is hydrogen or lower alkyl ; and R, 2 is hydrogen or lower alkyl.

8. A compound having formula XIII: wherein: Ph is phenyl ; and Rg is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine-

amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3- pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine- amino, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino-.

9. A compound having Formula XIIIa :

XIIIa wherein: Ph is phenyl; and R9 is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl-

phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine- amino, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino-.

10. The compound of claim 8 or 9 wherein Rg is carbobenzyloxy-, carbobenzyloxy-serine-, 4-methylphenylsulfoxide-, phenylsulfoxide-, 4- bromophenylsulfoxide-,4-nitrophenylsulfoxide-, 4-methoxyphenyl-sulfoxide, phenylmethylsulfoxide, phenylcarbonyl-, 3-pyridylcarbonyl-, 4- trifluoromethoxyphenylaminocarbonyl-, 4-trifluoromethylphenyl- aminocarbonyl-, 4-methylphenylaminocarbonyl-or 4-methoxyphenyl- aminocarbonyl-.
11. The compound of claim 8 or 9wherein R9 is 4-methylphenyl-sulfoxide-, 3-pyridyl-carbonyl-, carbobenzyloxy-serine-or phenylcarbonyl-.
12. The compound of claim 8 or 9 wherein Rg is 4-methylphenylsulfoxide-, or 3-pyridyl-carbonyl-.
13. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of claims 2-4 or 6-9, in combination with a pharmaceutically acceptable diluent or carrier.
14. A therapeutic method for preventing or treating a pathological condition or symptom in a mammal, such as a human, wherein the activity of HIV protease is implicated and antagonism of its action is desired, comprising administering to a mammal in need of such therapy, an effective amount of a compound of any one of claims 2-4 or 6-9, or a pharmaceutically acceptable salt thereof.
15. A compound of the formula:

and stereoisomers thereof.

16. A compound of the formula:

and stereoisomers thereof.

17. A compound of the formula:

and stereoisomers thereof.

18. A compound of the formula:

and stereoisomers thereof.

19. A compound of the formula :

and stereoisomers thereof.

20. A compound of the formula :

and stereoisomers thereof.

21. A compound of the formula : and stereoisomers thereof.
22. A compound of the formula:

and stereoisomers thereof.

23. A compound of the following formula :

and stereoisomers thereof.

24. A compound of the formula: 10050 and stereoisomers thereof.
25. A compound of the formula:

and stereoisomers thereof.

26. The compound of any one of claims 15-25 wherein said compound can inhibit HIV protease.
27. A composition comprising a therapeutically effective amount of a compound of any one of claims 15-25 and a pharmaceutically acceptable salt.
28. A therapeutic method for preventing or treating a pathological condition or symptom in a mammal, such as a human, wherein the activity of HIV protease is implicated and antagonism of its action is desired, comprising administering to a mammal in need of such therapy, an effective amount of a compound of any one of claims 15-25, or a pharmaceutically acceptable salt thereof.
Description:

HIV/FIV PROTEASE INHIBITORS The invention described herein was made with United States Government support under Grant Number GM48870 awarded by the National Institutes of Health. The United States Government may have certain rights in this invention.

Field Of The Invention This invention relates to a novel series of chemical compounds useful as HIV protease inhibitors, to methods of making those compounds and to the use of such compounds as antiviral agents. The invention provides HIV and FIV protease inhibitors characterized by core structures having a small P3 residue.

The invention is also directed to methods for making such compounds with clinically useful activity and which are potentially resistive against loss of inhibitory activity due to development of resistant strains of HIV.

Background Of The Invention Acquired Immune Deficiency Syndrome (AIDS) causes a gradual breakdown of the body's immune system as well as progressive deterioration of the central and peripheral nervous systems. Since its initial recognition in the early 1980's, AIDS has spread rapidly and has now reached epidemic proportions within a certain segments of the population. Intensive research has led to the discovery of the responsible agent, human T-lymphotropic retrovirus III (HTLV-111), now more commonly referred to as the human immunodeficiency virus or HIV.

HIV is a member of the class of viruses known as retroviruses. The retroviral genome is composed of RNA that is converted to DNA by reverse transcription. This retroviral DNA is then stably integrated into a host cell's chromosome and, employing the replicative processes of the host cells, produces new retroviral particles and advances the infection to other cells. HIV appears to have a particular affinity for the human T-4 lymphocyte cell that plays a vital role in the body's immune system. HIV infection of these white blood cells

depletes this white cell population. Eventually, the immune system is rendered inoperative and ineffective against various opportunistic diseases such as, among others, pneumocystic carini pneumonia, Kaposi's sarcoma, and cancer of the lymph system.

Although the regulation of the HIV viral formation and function is not perfectly understood, identification of the virus has led to some progress in controlling the disease. For example, the drug azidothymidine (AZT) has been found to be effective for inhibiting the reverse transcription of the HIV retroviral genome, thereby providing a measure of control, though not a cure, for patients afflicted with AIDS. The search continues for drugs that can cure or at least provide an improved measure of control of the deadly HIV virus.

Retroviral replication routinely features post-translational processing of polyproteins. Such processing is accomplished by a virally encoded, HIV protease. The mature polypeptides formed by action of the HIV protease will aid in the formation and function of infectious HIV. Inhibition of the HIV protease has been shown to inhibit viral proteolytic processing during the late stage of the HIV-1 life cycle and to prevent proviral integration of infected T- lymphocytes during the early phase of the HIV-1 life cycle.

In the last several years, researchers have searched for therapeutically useful inhibitors of the HIV protease enzyme to stop the progression of AIDS.

Five competitive inhibitors of the enzyme have been approved and several others are in clinical trials (Babine et al. Them. Rev. 1997,97,1359-1472; De Lucca et al. Drug Discovery Today 1997,2,6-18; Vacca et al. Drug Discovery Today 1997,2,261-272; Huff et al. J. Med. Chem. 1991,34,2305; Wlodawer et al.

Ann. Rev. Biochem. 1993,62,543-585).

For example, compound 1 (TL3) with Ala residue in P3 positions has been shown to inhibit wild-type HIV proteases and some drug-resistant HIV mutants at nanomolar concentrations.

Also, compound 1 has been shown to inhibit replication of HIV, Feline Immunodeficiency Virus (FIV) and Simian Immunodeficiency Virus (SIV) with at micromolar concentrations (-I uM) in tissue culture. FIV has been proposed as a model for certain drug-resistant HIV strains and is used as a model for the development of new inhibitors to tackle the problem of drug resistance.

The introduction of macrocyclic structures in HIV protease inhibitors may improve their binding affinity and resistance toward proteolytic enzymes.

Podlogar et al., J. Med. Chem. 1994,37, 3684; Smith et al., Bioorg. Med. Chem.

Lett. 1994,4,2217; Chen et al., Bioorg. Med. Chem. Lett. 1996,6,435; Abbenante et al., J. Am. Chem. Soc. 1995,117,10220; March et al., J. Am.

Chem. Soc. 1996,118,3375; Reid et al., J. Am. Chem. Soc. 1996,118,8511; Fairlie et al., J. Med. Chem. 2000,43,1271. In addition, HIV protease inhibitors containing the norstatine analog (3-amino-2-hydroxy-4-phenylbutyric acid), particularly 2 (JE-2147), have antiviral activities in vitro and exhibit good oral bioavailability and plasma pharmacokinetic profiles.

However, reports indicate that at least forty-five distinct drug-resistant single mutations in the HIV protease exist, and that the number of mutations has increased by 250% over a 3-year period. See Schinazi et al., Int. Antiviral News 1997,5,129. The development of drug-resistance is the consequence of incomplete suppression of HIV replication. The rapid replication rate of HIV and its inherent genetic variation result in the generation of numerous viral variants. Moreover, these mutant enzymes often exhibit cross-resistance to many structurally distinct protease inhibitors. Therefore, development of new broad-based protease inhibitors efficacious against a wide spectrum of HIV variants may be necessary in order to slow down the development of drug resistance. Schinazi et al., Int. Antiviral News 1997,5,129; Wilson et al. J.

Biochim. Biophy. Acta 1997,1339,113-125; Erickson et al. Annu. Rev.

Pharmacol. Toxicol. 1996,36,545-571; Gulnik et al. Biochemistry 1995,34,

9282-9287; Erickson et al. Nature Struct. Biol. 1995,2,523-529; Condra et al.

Nature, 1995,374,569-571; Otto et al. Proc. Natl. Acad. Sci. USA 1993, 90, 7543-7547; Wong et al. Science, 1997,278,1291-1295; Finzi et al. Science, 1997,278,1295-1300).

This mutation rate and the genetic flexibility of the HIV virus compel development of a new generation of HIV inhibitors. Previous attempts to tackle the drug-resistance problems have focused on the modification of inhibitors to overcome the effects of single mutations, or have relied on combination therapy.

However, clinically isolated drug-resistant HIV variants often contain multiple mutations in their protease. The higher number of substitutions in the enzyme also increases the chance of developing cross-resistance to a wide range of structurally diverse inhibitors. Therefore, combination therapy relying on multiple, currently available HIV protease inhibitors may not be a solution to combat resistance selection. Instead, new protease inhibitors are needed that are efficacious against both wild type and drug resistant HIV proteases and that are less prone to the development of drug resistance.

Summary of the Invention The invention provides new types of HIV protease inhibitors that can strongly inhibit HIV and FIV proteases and that are not prone to the development of drug resistance. The present inhibitors are useful for the treatment and prevention of AIDS and of HIV infection. Also provided by the invention are methods of making these HIV protease inhibitors. The protease inhibitors and methods provided by the invention have several advantages. The methods are simpler than currently available methods. The inhibitors are highly specific for the HIV retroviral protease and are not prone to the development of drug resistance. Moreover, the inhibitors of the invention are longer lived in the HIV virus and less toxic than many currently available drugs.

One aspect of the invention is directed to a protease inhibitor represented by Formula I :

In the above structure, R, may be any of the following radicals: hydrogen, carbobenzyloxy-, carbobenzyloxy-valine-, carbobenzyloxy-glycine- valine-, carbobenzyloxy-alanine-valine-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-serine-valine-, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-threonine-valine-and carbobenzyloxy-valine-valine-.

R2 may be any of the following radicals :-CH2-Phenyl, and - CH2-CH (CH3) 2; R3 is hydrogen, oxygen or hydroxyl; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when Rs and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group ; and R7 is a radical represented by the Formula II: wherein R8 is a radical selected from the group consisting of (H) 2, and-H (t- Butyl).

Another aspect of the invention is directed to a protease inhibitor of Formula III:

In Formula III, R, may be any of the following radicals: hydrogen, carbobenzyloxy-, carbobenzyloxy-valine-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-alanine-valine-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-serine-valine-, carbobenzyloxy-threonine-val4ne-, carbobenzyloxy-alanine asparagine-and carbobenzyloxy-valine-valine-. R2 may be any of the following radicals:-CH2- Phenyl, and-CH2-CH (CH3) 2; Rs is either hydrogen or-OH.

Another aspect of the invention is directed to a protease inhibitor of Formula IV: In Formula IV, R, may be any of the following radicals: hydrogen, carbobenzyloxy-, carbobenzyloxy-valine-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-alanine-valine-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-serine-valine-, carbobenzyloxy-threonine-valine-, carbobenzyloxy-alanine-asparagine-and carbobenzyloxy-valine-valine- ; and Rg is either (H) 2 or-H (t-Butyl).

Another aspect of the invention is directed to a protease inhibitor of Formula V:

In Formula V, R, may be any of the following radicals: hydrogen, carbobenzyloxy-, carbobenzyloxy-valine-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-alanine-valine-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-serine-valine-, carbobenzyloxy-threonine-valine-, carbobenzyloxy-valine-valine-and carbobenzyloxy-alanine-asparagine-.

Another aspect of the invention is directed to protease inhibitor of Formula VI: In Formula VI, R, may be any of the following radicals: hydrogen, carbobenzyloxy-, carbobenzyloxy-valine-, carbobenzyloxy-glycine- valine-, carbobenzyloxy-alanine-valine-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-serine-valine-,

carbobenzyloxy-threonine-valine-, carbobenzyloxy-valine-valine-and carbobenzyloxy-alanine-asparagine-.

The invention also provides HIV protease inhibitors having a macrocycline group of formula VII, VIII or VIIIa : VII VIII

wherein Z is a leaving group or a linkage to a protease inhibitor; and R, 3 is hydrogen or carbobenzyloxy-amino-.

The invention further provides HIV protease inhibitors having formula IX:

wherein: R3 is hydrogen, oxygen or hydroxyl ; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when Rs and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; and Y is Rg, Rlo or-NH-Rlo R9 is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino, carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene-

sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4-trifluoromethoxy- phenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl-phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine-amino, acetyl-p- fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine-valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl-phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl-methylene-, p-fluoro- phenyl-methylene-amino, p-fluoro-phenyl-methylene-valine-, p-fluoro-phenyl- methylene-valine-amino-, acetyl-tyrosine-, acetyl-tyrosine-amino, acetyl- tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine- amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino-; and RIO is a macrocycle of formula VII or VIII wherein the Z of the macrocycle is a linkage to the compound of formula IX.

The invention also provides an HIV protease inhibitor having Formula X: x wherein Ph, R3, R4, R5, R6 and Rg are as described herein.

The invention further provides an HIV protease inhibitor of formula XI:

wherein Ph, R3, R4, R5, R6 and Rg are as described herein, and wherein R, I is hydrogen or lower alkyl ; and R, 2 is hydrogen or lower alkyl.

The invention also provides an HIV protease inhibitor having formula XIII:

wherein Ph is phenyl and Rg is as described herein.

In another embodiment, the invention provides an HIV protease inhibitor having Formula XIIIa :

XIIIa wherein Ph is phenyl and Rg is as described herein.

The invention contemplates all stereoisomers of the compounds described herein.

Moreover, the invention is directed to a composition comprising a therapeutically effective amount of a compound of the invention and a

pharmaceutically acceptable salt. Such compositions can include more than one compound of the invention.

The invention is further directed to therapeutic methods for preventing or treating a pathological condition or symptom in a mammal, such as a human, wherein the activity of HIV protease is implicated and antagonism of its action is desired, comprising administering to a mammal in need of such therapy, an effective amount of a compound of any one of claims 14, or a pharmaceutically acceptable salt thereof. For example, the compounds and compositions of the invention can be used to treat a person exposed to HIV, a person having an HIV infection or a person with AIDS.

Description of the Figures Figure 1 illustrates models of HIV protease (top) and FIV protease (middle) complexed with compound lb. The models show the small S3 site in FIV protease and the close proximity of the P3 (CH3) and PI (PhCH2) residues, a structural feature found in many drug-resistant HIV proteases. Bottom: shown is a cut-away view of the molecular surfaces of HIV (pink) and FIV protease (blue), in which the dipping plane is parallel to the plane of the two aspartates and cuts inside the P3 site.

Figure 2 shows the structure of several compounds and the amino acid side chains in the S subsites of HIV protease interacting with an inhibitor.

Figure 3 provides the structures of several dissymmetric inhibitors with small P3 residues. Compounds 2b-6b and 7 contain a methyl group as P3 residue.

Figure 4 shows the proposed mechanism of inhibition by compound 5b.

A water molecule is added, with assistance of the enzyme, to the a-keto group to form a gem-diol.

Figure 5 illustrates models of HIV protease (top) and FIV protease (middle) complexed with R031-8959, and FIV protease complexed with the modified inhibitor 7a (bottom). The P3 group of R031-8959 is too big to fit the S3 subsite of FIV PR, whereas 7a with methyl group as P3 residue shows a good fit.

Figure 6 shows the synthesis of intermediate compound 13 with the following conditions: (a) 2,2-dimethoxypropane, p-TsOH (80%); (b) Pd/C, H2, MeOH (99%); (c) HBTU, Cbz-Val, Et3N, CH3CN (89%); (d) HBTU, Cbz-amino acids, Et3N, CH3CN; (e) p-TsOH, MeOH.

Figure 7 shows the synthesis of compound 2b with the following conditions: (a) NMM, THF, i-BuOCOCI. (b) CH2N2, Et2O. (c) HC 1 (85%, 3 steps) (d) NaBH4, EtOH, (90% d. e, 81%) (e) NaOMe, MeOH (96%). (f) MeOH, Et3N (90W) (g) Pd/C, H2, EtOAc. (h) Cbz-Ala-Val-OH, HBTU, Et3N, CH3CN.

(49k, 2 steps).

Figure 8 shows the synthesis of the a-Ketoamides 5a and 5b with the following conditions: (a) BH3 THF. (b) Swern Oxidation (90%). (c) NaHS03, H20. (d) KCN. (e) HC1 (6N in dioxane), (f) Cbz-Cl, NaOH, H20 (52%, 4 steps).

(g) HBTU, Et3N, CH3CN (73%). (h) Pd/C, H2, EtOAc. (i) Cbz-Ala-Val-OH, HBTU, Et3N, CH3CN (65%, 2 steps). (j) Dess-Martin (63W).

Figure 9 illustrates the synthesis of compound 6b with the following conditions: (a) PhB (OH) 2, PhMe, reflux (b) THF, r. t., (78%, 2 steps); (c) Cbz- Ala-Val-OH, HBTU, Et3N, CH3CN (73%); and drugs 7a, b with the following conditions: (a) MeOH, Et3N, (80%). (b) TFA, CH2Cl2. (c) HBTU, Et3N, CH3CN, Cbz-Ala-Val-OH (76%) or Cbz-Ala-Asn-OH (78%).

Figure 10 tabulates the inhibition of FIV and HIV proteases by small P3 residue containing inhibitors and their parent compounds wherein the superscript are each described as follows: Ki and IC50 values were determined in duplicate using fluorescent substrate (For procedures see Lee et al. Proc. Natl.

Acad. Sci. USA 1998,95,939-944; for HIV protease substrate, see Toth, M. V.; Marshall, G. R. Int. J. Peptide Res. 1990,36,544); b) Data obtained at pH 5.25 at 37°C in 0. IM NaH2PO4, 0. 1 M sodium citrate, 0.2 M NaCl, 0.1 mM DTT, 5% glycerol, and 5% DMSO in volume; c) Data obtained at pH 5.25 at 370 C in 0.1 t M MES, 5% glycerol, and 5% DMSO in volume; d) From Lee et al. Proc. Natl.

Acad. Sci. USA 1998,95,939-944; e) From Slee et al. J. Am. Chem. Soc. 1995, 117,11867-11878; f) From Wilson et al. J. Biochim. Biophy. Acta 1997,1339, 113-125."NI"means"no inhibition"at 800 M of inhibitor.

Figure 11 tabulates the inhibition of FIV and HIV proteases by C2- symmetric diols wherein the superscripts are each described as follows: Ki

values were determined in duplicate. b) Data obtained at pH 5.25 at 37°C in 0. 1M NaH2P04, 0. 1 M sodium citrate, 0.2 M NaCl, 0.1 mM DTT, 5% glycerol, and 5% DMSO in volume; c) data obtained at pH 5.25 at 37°C in 0.1 M MES, 5% glycerol, and 5% DMSO in volume; d) From Slee et al. J. Am. Chem. Soc.

1995,117,11867-11878;"nd"means not determined.

Figure 12 illustrates the synthesis of C2 symmetric inhibitors 1000-1400.

Figure 13 illustrates examples of HIV protease inhibitors tested as inhibitors of FIV protease.

Figure 14 illustrates TL-3-139 Timecourse PPR-FIV Acute Infection.

Figure 15 illustrates SIV p27 ELISA.

Figure 16 shows Days post WEAU-1.6 Infection (25 TCID 50).

Figure 17 provides the structure of VLE776 (compound 10031) within HIV and FIV protease binding pockets.

Detailed Description of the Invention The invention provides compounds active as HIV protease inhibitors.

The compounds of the invention can circumvent the drug-resistance problems of the currently available HIV protease inhibitors.

According to the invention, the feline immunodeficiency virus (FIV) protease can be used to screen for effective inhibitors of the HIV protease. FIV causes an immunodeficiency syndrome in cats comparable to AIDS in humans.

Furthermore, the cat may be used as an animal model to test the effectiveness of potential therapeutic drugs in vivo to accelerate the drug development process.

Both HIV and FIV proteases are C2-symmetric homodimeric enzymes.

The active-site structures of the HIV and FIV proteases are almost superimposable and they facilitate catalysis by an identical mechanism (Slee et al. J. Am. Chem. Soc. 1995,117,11867-11878). Like the HIV protease, the FIV protease processes both structural proteins of gag and the enzymes encoded by pol during FIV replication (Kramer et al. Science 1986,231,1580-1584).

Furthermore, six mutated residues in the HIV protease that cause drug resistance (K20I, V321, 15OV, N88D, L90M, Q92K; Mellors et al. Int. Antiviral News, 1995,3,8-13) are found in the structurally aligned native residues of the FIV protease.

Although the active site structures of HIV and FIV proteases are superimposable and have an identical mechanism of catalysis, all the approved inhibitors with Ki values in the low nanomolar range for HIV protease, only bind to the FIV protease in the micromolar range. Kinetic studies demonstrate that various potent HIV protease inhibitors that interact with the S4 to S4'binding region are less efficient inhibitors of FIV protease by a factor of 100 or more.

However, good inhibitors of FIV protease are often better inhibitors of wild type and drug-resistant HIV proteases. According to the invention, the FIV protease has a more restricted P3 binding subsite than does the wild-type HIV protease.

Moreover, the FIV protease resembles many drug-resistant HIV proteases that were found to have a smaller P3 binding subsite. Good inhibitors of the FIV protease with a small P3 residue are therefore better inhibitors of wild type and drug-resistant HIV proteases.

Testing candidate drugs with respect to their inhibitory activity against both HIV and FIV proteases and determining which inhibitors are simultaneously efficacious against both of these mechanistically identical proteases identifies inhibitors of HIV protease which are potentially less prone to resistance development. Candidate drugs that are successfully screened by in vitro protease assays may then be tested in the cat as a model animal system for HIV progression in vivo.

Accordingly, the invention is directed to potent inhibitors against human immunodeficiency virus protease and feline immunodeficiency virus protease.

More particularly, the invention is directed to a new type of inhibitor with a small P3 residue. According to the invention, existing HIV protease inhibitors can also be modified by reducing the size of the P3 group, or the combined P3 and P I groups, to produce a more effective HIV protease inhibitor. These inhibitors are effective against HIV and its drug-resistant mutants.

The invention also provides a new strategy for the development of HIV protease inhibitors effective against the wild type and drug-resistant mutants that involves using the FIV protease is a model for drug-resistant HIV proteases and screening compounds for potent inhibitory activity against the FIV protease.

Protease Inhibitors The invention provides a variety of HIV and FIN protease inhibitors. In one embodiment, the protease inhibitors of the invention have one, two or three macrocycles. In other embodiments, the protease inhibitors of the invention have a variety of substituents instead of macrocycles. The inhibitors of the invention can inhibit the HIV or FIV protease at low concentration, for example, at nanomolar concentrations.

A macrocycle is a large ring system that mimics the conformationally constrained Ala-Val-Phe motif, where Ala is in the P3 position. Inhibitors containing one or more macrocycles inhibit FIV and drug-resistant HIV proteases at low nanomolar concentrations. In one embodiment, a macrocycle of Formula VII, VIII or VIIIa (below) is used to provide an effective macrocyclic protease inhibitor, wherein Z is a leaving group or a linkage to a protease inhibitor.

VII

Villa wherein Z is a leaving group or a linkage to a protease inhibitor; and Rl3 is hydrogen, or carbobenzyloxy-amino-.

In one embodiment the protease inhibitors of the invention can have formula IX: wherein: R3 is hydrogen, oxygen or hydroxyl; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; Rs is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when R5 and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; and Y is Rs, Rlo or-NH-RIo ; R9 is hydrogen, amino, alkyloxy-valine-, alkyloxy-valine-amino, carbobenzyloxy-, carbobenzyloxy-amino-, alkylamino-valine-carbobenzyloxy-, alkylamino-valine-carbobenzyloxy-amino-, tert-butyloxycarbonyl-, tert- butyloxycarbonyl-amino-, carbobenzyloxy-valine-, carbobenzyloxy-valine- amino-, carbobenzyloxy-serine-, carbobenzyloxy-serine-amino-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-glycine-valine-amino,

carbobenzyloxy-alanine-valine, carbobenzyloxy-alanine-valine-amino-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-leucine-valine-amino-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-phenylalanine-valine- amino, carbobenzyloxy-serine-valine-, carbobenzyloxy-serine-valine-amino, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-alanine-asparagine-amino- , carbobenzyloxy-threonine-valine-, carbobenzyloxy-threonine-valine-amino, carbobenzyloxy-valine-valine-, carbobenzyloxy-valine-valine-amino, p- methylphenylsulfoxide-alanine-valine, p-methylphenylsulfoxide-alanine-valine- amino, N-acetyl-tryptophan-, N-acetyl-tryptophan-valine, N-acetyl-tryptophan- valine-amino, acetyl-p-F-phenylalanine-valine-, 4-methyl-phenylsulfoxide-, 4- methyl-phenylsulfonamide-, phenylsulfonamide-, 4-bromo-phenylsulfoxide-, 4- bromo-phenylsulfonamide-, 4-nitro-phenylsulfoxide-, 4-nitro- phenylsulfonamide-, 4-methoxy-phenylsulfoxide-, 4-methoxy- phenylsulfonamide-, phenylmethylene-sulfoxide-, phenylmethylene- sulfonamide-, phenylcarbonyl-, phenylcarbamide-, 3-pyridylcarbonyl-, 3pyridyl-carbamide-, 3-pyridyl-methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl- ethylene-, 3-pyridyl-ethylamino-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxy-phenylurea-, 4-trifluoromethylphenylaminocarbonyl-, 4- trifluoromethyl-phenylurea-, 4-methylphenylaminocarbonyl-, 4-methyl- phenylurea-, 4-trifluoromethoxyphenylaminocarbonyl-, 4- trifluoromethoxyphenylurea-, 4-trifluoromethyl-phenylurea-, 4-trifluoromethyl- phenylurea-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro-phenylalanine- amino, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro-phenylalanine- valine-amino-, acetyl-phenylalanine-, acetyl-phenylalanine-amino, acetyl- phenylalanine-valine-, acetyl-phenylalanine-valine-amino, p-fluoro-phenyl- methylene-, p-fluoro-phenyl-methylene-amino, p-fluoro-phenyl-methylene- valine-, p-fluoro-phenyl-methylene-valine-amino-, acetyl-tyrosine-, acetyl- tyrosine-amino, acetyl-tyrosine-valine, acetyl-tyrosine-valine-amino, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, or acetylhistidine-valine-amino- ; Rio is a macrocycle of formula VII, VIII or VIIIa ; and Rl3 is hydrogen, carbobenzyloxy-amino-.

Examples of compounds of the invention having formula IX with one and two macrocycles are compounds 10050 and 10055, shown below.

10050 In another embodiment, the invention provides protease inhibitors having Formula X:

x Specific examples of compounds contemplated by the invention that have formula X include those listed in Table 1.

Table 1 Compd. No. R9 R3 R4 R5 + R6 10003 carbobenzyloxy-alanine-valine-NH OH H =O 10004 p-CH3-C6H4SO2-alanine-valine-NH OH H =O 10005 acetyl-tryptophan-valine-NH OH H =O 10006 acetyl-phenylalanine-valine-NH OH H =O 10007 acetyl-tyrosine-valine-NH OH H =O 10009 tert-butyloxycarbonyl OH H =O 10010 tert-butyloxycarbonyl H OH =O 10050 acetyl-p-F-phenylalanine-valine-NH OH H =O

Preferred Rg substituents are acetylphenylalanine-, acetylphenylalanine-valine-, acetylphenylalanine-valine-amino-, acetyltyrosine-, acetyltyrosine-valine-, acetyltyrosine-valine-amino-, acetyl-p-fluoro-phenylalanine, acetyl-p-fluoro- phenylalanine-amino-, acetyl-p-fluoro-phenylalanine-valine-, acetyl-p-fluoro- phenylalanine-valine-amino-, p-fluoro-phenyl-methylene-, p-fluoro-phenyl- methylene-amino, p-fluoro-phenyl-methylene-valine-, p-fluoro-phenyl- methylene-valine-amino-, 3-pyridyl-carbonyl-, 3-pyridyl-carbamide-, 3-pyridyl- methylene-, 3-pyridyl-methyl-amino-, 3-pyridyl-ethylene-, 3-pyridyl- ethylamino-, acetyltrytophan-, acetyltrytophan-amino-, acetyltrytophan-valine-, acetyltrytophan-valine-amino-, acetylhistidine-, acetylhistidine-amino-, acetylhistidine-valine-, and acetylhistidine-valine-amino-.

The invention also provides C2-symmetrical diol inhibitors having Formula XIII provided below. wherein Ph is phenyl, and R9 is as provided herein. Preferably, the compounds of Formula XIII are of the configuration shown in Formula XIIIa below.

Moreover, for compounds of Formula XIII or XIIIa, Rg is preferably CBz, CBz- Ser, 4-MePhSO2, PhS02, 4-BrPhSO2, 4-02NPhS02, 4-MeOPhSO2, PhCH2SO2, PhCO, 3-Pyr-CO, 4-F3COPhNHCO, 4-F3CPhNHCO, 4-MePhNHCO or 4- MeOPhNHCO. More preferably, Rg is 4-methylphenylsulfoxide-, 3-pyridyl- carbonyl-, carbobenzyloxy-serine-or phenylcarbonyl-. Even more preferably, R9 is 4-methylphenylsulfoxide-, or 3-pyridyl-carbonyl-.

The invention also contemplates all the enantiomers and stereoisomers of the compounds of the invention. For example, the configuration of the C-OH stereogenic center formed when R3 or R4 are separate constituents is important for the inhibitory activity. By with of illustration, compound 10009 with R stereochemistry around its essential carbinol function has about 6-fold better inhibitory activity for HIV/FIV proteases than that of the S isomer 10010.

The term"stereoisomer"refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. The different three-dimensional structures are called configurations. The term"diastereomer"refers to those stereoisomers with more than one chiral center that are not mirror images of one another. The term"enantiomer"refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another. The term"racemic mixture" or"racemic modification"refers to a mixture of equal parts of enantiomers. The term"chiral center"refers to a carbon atom to which four different groups are attached. For amino acids, the designations L/D or R/S can be used as described in IUPAC-IUB Joint Commission on Biochemical Nomenclature, Eur. J.

Biochem., 138,9-37 (1984). It is understood that the compounds of the invention may exist in a variety of stereoisomeric configurations. It is further understood that, for example, where the configuration of a compound or formula depicted herein is fixed, the maximum number of enantiomers possible for each

compound is equal to 2°, where n represents the total number of chiral centers located on the compound.

In general, as used herein, the term"alkyl"includes the straight, branched-chain and cyclized manifestations thereof unless otherwise indicated, particularly such moieties as methyl, ethyl, isopropyl, n-butyl, t-butyl,-CH2-t- butyl, cyclopropyl, n-propyl, pentyl, cyclopentyl, n-hexyl, cyclohexyl and cyclohexylmethyl. The term'-'aralkyl", when used, includes those aryl moieties attached to an alkylene bridging moiety, preferably methylene or ethylene.

Alkyls can have up to fifteen carbon atoms (Cl 15) but preferably have up to six carbon atoms (Cl-6) moieties and more preferably have up to three carbon atoms (CI-3)- "Aryl"includes both carbocyclic and heterocyclic moieties of which phenyl, pyridyl, pyrimidinyl, pyrazinyl, indolyl, indazolyl, furyl and thienyl are of primary interest; these moieties being inclusive of their position isomers such as, for example, 2-, 3-, or 4-pyridyl, 2-or 3-furyl and thienyl, 1-, 2-, or 3-indolyl or the 1-and 3-indazolyl, as well as the dihydro and tetrahydro analogs of the furyl and thienyl moieties. Also included within the term"aryl"are such fused carbocyclic moieties as pentalenyl, indenyl, naphthalenyl, azulenyl, heptalenyl, acenaphthylenyl, fluorenyl, phenalenyl, phenanthrenyl, anthracenyl, acephenanthrylenyl, aceanthrylenyl, triphenylenyl, pyrenyl, chrysenyl and naphthacenyl. Also included within the term"aryl"are such other heterocyclic radicals as 2-or 3-benzo [b] thienyl, 2-or 3-naphtho [2,3-b] thienyl, 2-or 3- thianthrenyl, 2H-pyran-3- (or 4-or 5-) yl, 1-isobenzo-furanyl, 2H-chromenyl-3-yl, 2-or 3-phenoxathiinyl, 2-or 3-pyrrolyl, 4-or 3-pyrazolyl, 2-pyrazinyl, 2- pyrimidinyl, 3-pyridazinyl, 2-indolizinyl, 1-isoindolyl, 4H-quinolizin-2-yl, 3- isoquinolyl, 2-quinolyl, 1-phthalazinyl, 1, 8-naphthyridinyl, 2-quinoxalinyl, 2- quinazolinyl, 3-cinnolinyl, 2-pteridinyl, 4H-carbazol-2-yl, 2-carbazolyl, ß- carbolin-3-yl, 3-phenanthridinyl, 2-acridinyl, 2-perimidinyl, 1-phenazinyl, 3- isothiazolyl, 2-phenothiazinyl, 3-isoxazolyl, 2-phenoxazinyl, 3-isochromanyl, 7- chromanyl, 2-pyrrolin-3-yl, 2-imidazolidinyl, 2-imidazolin-4-yl, 2-pyrazolidinyl, 3-pyrazolin-3-yl, 2-piperidyl, 2-piperazinyl, 1-indolinyl, 1-isoindolinyl, 3- morpholinyl, benzo [b] isoquinolinyl and benzo [b] furanyl, including the position isomers thereof except that the heterocyclic moieties cannot be attached directly

through their nitrogen one, two or three substituents independently selected from C,-6 alkyl, haloalkyl, alkoxy, thioalkoxy, aminoalkylamino, dialkylamino, hydroxy, halo, mercapto, nitro, carboxaldehyde, carboxy, carboalkoxy and carboxamide.

A compound of the invention may be in free form, e. g., amphoteric form, or in salt form, for example, in acid addition or anionic salt form. A compound in free form may be converted into a salt form by methods available in the art.

Similarly, a compound in a salt form may also be converted into a free form by methods available in the art.

In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds of the invention as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ketoglutarate, and glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The compounds of the invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i. e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e. g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more

excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compounds may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the

active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pure form, i. e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a

given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions that can be used to deliver the compounds of the invention to the skin are known to the art; for example, see Jacquet et al. (U. S. Pat. No. 4,608,392), Geria (U. S. Pat. No.

4,992,478), Smith et al. (U. S. Pat. No. 4,559,157) and Wortzman (U. S. Pat. No.

4,820,508).

Useful dosages of the compounds of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Pat. No. 4,938,949.

Generally, the concentration of the compounds of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%. Tablets and capsules may have higher concentrations of the present compounds, e. g. concentrations ranging from 1% to 95%.

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e. g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. The dosage may vary depending upon the specific active compound or compounds used in the pharmaceutical composition.

Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.005 to about 75 uM, preferably, about 0.01 to 50 uM, most preferably, about 0.1 to about 30 uM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient.

Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient (s).

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e. g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

The ability of a compound of the invention to act as as a protease inhibitor may be evaluated in vitro and in vivo using pharmacological models that are known to the art, for example, a feline animal model as described herein.

Method of Making The compounds of this invention may be prepared using chemical reactions, reagents and starting materials available in the art. Alternatively, the compounds of the invention can be made using the generalized and specific methods provided by the invention. Specific synthetic schemes are provided in the examples.

In one embodiment, macrocyclization is achieved by joining an alkyl to a phenolic oxygen to form the ether moiety of the macrocycle. The alkyl can have a leaving group to facilitate condensation with the phenolic oxygen. The leaving

group can be any leaving group contemplated for Z. Preferably, the leaving group used for macrocyclization is a halide (chloride, iodide or bromine), mesolate or tosylate. Alternatively, the phenolic oxygen can be coupled to such a leaving group.

The alkyl group can have a reactive group such as an amino, oxy, carbonyl or other group on the end opposite the end to be joined with the phenolic oxygen. Such a reactive group can facilitate linkage of the alkyl to a convenient site in the forming macrocycle to form an amide, ester, ether or carbon-carbon bond.

The final step in the formation of the macrocycle is an intramolecular reaction. However, depending upon the leaving group and reactive group chosen, the joining of the alkyl and the phenolic oxygen can be intramolecular or intermolecular. For example, when the reactive group of the alkyl has been joined to a convenient site in the forming macrocycle, the reaction between the alkyl and the phenolic oxygen is an intramolecular coupling reaction.

Conversely, an intermolecular reaction is used when the reactive group on the alkyl has not yet been joined with a convenient site on the forming macrocycle.

In one embodiment, the macrocyclic protease inhibitors of formula can be made by the following reaction scheme.

wherein: Z is a leaving group; Ph is phenyl; and R9 is as defined hereinabove.

The leaving groups (Z) can be any leaving group available to one of skill in the art. In many embodiments, the leaving group is attached to an amino group the'amino group is attached to an amino protecting group so that, when the amino protecting group leaves an amino group remains. Any amino protecting group available to one of skill in the art can be employed. Among the classes of leaving groups contemplated are: (1) acyl type protecting groups such as formyl, trifluoroacetyl, phthalyl, p-toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfenyl, tritylsulfenyl, and O-nitrophenoxyacetyl ; (2) aromatic urethane type protecting groups such as benzyloxycarbonyl and substituted benzyloxycarbonyls such as p-chlorobenzyloxycarbonyl, p-methoxybenzyloxy- carbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxy-carbonyl, l- (p- biphenylyl)-1-methylethoxycarbonyl, a-, a-dimethyl-3, 5-dimethoxybenzyl- oxycarbonyl, and benzhydryloxycarbonyl; (3) aliphatic urethane protecting groups such as tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (FMOC), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, and allyloxycarbonyl; (4) cycloalkyl urethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; (5) thio urethane type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups such as triphenylmethyl (trityl) and benzyl (Bzl); (7) trialkylsilane protecting groups such as trimethylsilane if compatible. Preferably, the leaving group used for macrocyclization is a halide (chloride, iodide or

bromine), mesolate or tosylate. Preferred a-amino protecting groups are tert- butyloxycarbonyl (Boc) or benzyloxycarbonyl (CBZ). The use of Boc as an a- amino protecting group for amino acids is described by Bodansky et al. in"The Practice of Peptide Synthesis", Springer-Verlag, Berlin (1984), p. 20.

Reagents useful for forming a macrocycle as illustrated above from the 1- amino-, 3-bromo-alkyl include reagents a-d. Reagent mix a is Boc-Val-OH in a mixture of HBTU, DIPEA and tetrahydrofuran. Reagent mix b is a mixture of tetrahydrofuran and CHzClz. Reagent mix c is Boc-Tyr-OH in a mixture of HBTU, DIPEA and tetrahydrofuran. Reagent mix d is a mixture of CsC03, tetrabutylammonium iodide (TBAI) and CH3CN.

Reagents useful for forming compound 20012 from compound 20011 include reagents e-g. Reagent mix e is a mixture of TFA and CH2C12. Reagent mix f is Ac-Trp-ValOH in a mixture of HBTU, DIPEA and tetrahydrofuran.

Reagent mix g is a mixture of LiOH, in a mixture of methanol and water.

Reagents useful for forming a compound of the final formula from a mixture of the macrocyclic compound and 20012 include reagents h and i.

Reagent mix h is a mixture of TFA and CH2C12. Reagent mix i is a mixture of HBTU, DIPEA and dimethylfuran.

The R5 and R6 funtional groups can be formed by available procedures.

Functional groups on the protease inhibitors may be protected as needed with available protecting group. Many of the leaving groups listed herein can be used as protecting groups. The selection of appropriate combinations of protective groups and reagents to selectively remove protective groups is well known in the art. For example, see M. Bodansky,"Peptide Chemistry, A Practical Textbook", Springer-Verlag (1988); J. Stewart, et al.,"Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical Co. (1984).

The following Examples illustrate certain aspects of the invention. These examples are for illustrative purposes and are not intended to limit the scope of the invention.

EXAMPLE 1: Redesign of C2-Symmetric Inhibitors with small P3 Residues Our initial work on the development of protease inhibitors efficacious against both HIV and FIV was focused on the systematic analysis of the S3 and S3'subsite specificities of the enzymes using a series of C2-symmetric inhibitors containing (1S, 2R, 3R, 4S)-1, 4-diamino-1, 4-dibenzyl-2,3-butan-diol as a PI and PI'core and Val as P2 and P2'residues (Lee et al. Proc. Natl. Acad. Sci.

USA 1998,95,939-944). We have demonstrated that FIV protease exhibited a strong preference for small hydrophobic groups at the S3 and S3'subsites in contrast to the high flexibility for the P3 and P3'residues binding to HIV protease (ibid, 939-944). Kinetic studies have also indicated that the binding preference observed in drug-resistant mutant HIV proteases is very similar to that found in FIV protease (id.). In addition, the most potent FIV protease inhibitor lb (Figure 2) strongly inhibits FIV, HIV, and SIV infections in tissue culture with virtually the same degree of effectiveness.

The x-ray structure of FIV protease complexed with inhibitor lb has been determined and has shown that the PI and P3 side chains are positioned very closely, and that the S 1 and S3 subsites are neighboring hydrophobic pockets that accommodate the corresponding PI and P3 side chains (The x-ray structures of lb complexed with HIV, FIV (3X), FIV (V59I), and FIV (Q99V) proteases have been determined and will be published separately: Li et al.

Biochemistry, unpublished as of filing). Models of lb bound to HIV and FIV protease (Figure 1) indicate that the S 1 and S3 subsites in HIV protease constitute a much larger hydrophobic pocket than the corresponding subsites found in the FIV protease. Because of this smaller hydrophobic size, the FIV protease can only accommodate inhibitors with a smaller size for P 1 and P3 residues together, and several drug-resistant HIV proteases are indeed found to have a smaller domain composed of S3 and S 1 subsites.

Based on the x-ray structures of HIV and FIV proteases complexed with inhibitors, the residues that define the S subsites are shown in Figure 2. In HIV protease, Pro 81 and Val 82 are components for both S 1 and S3 subsites since they can affect binding of both PI and P3 moieties, whereas Ile 98 and Gln 99 are the structurally aligned residues for FIV protease (Hosur et al. J. Am. Chem.

Soc. 1994,116,847-855). The x-ray structures also revealed that three residues at S3 and S3'subsites, Gly 48, Pro 81, and Val 82 of HIV protease, were replaced with Ile 57, Ile 98, and Gln 99 in FIV protease. As a result, the S3 and S3'subsites of FIV protease are sterically more congested than those in HIV protease, and these three different residues may define the S3 and S3'subsite specificities of the enzymes. The results of these studies point to a new direction for development of inhibitors effective against both HIV protease and its drug- resistant variants.

EXAMPLE 2: Dissymmetric Inhibitors with Small P3 Groups The S3 and S3'subsite specificities of FIV protease and drug-resistant HIV proteases with mutations affecting the S3 subsite were investigated further to determine if there is a correlation between them. The new inhibitors of HIV protease were synthesized with Ala at P3, Val or Asn at P2, and various Phe-Pro isosteric cores for the P 1-P 1'residues (Figure 3). The inhibitory activities of each compound against FIV, HIV, and drug-resistant mutant HIV proteases were determined as described previously (Lee et al. Proc. Natl. Acad.

Sci. USA 1998,95,939-944) and the results are summarized in Figure 10.

Each compound tested in this study is a competitive inhibitor and is significantly more potent against HIV protease than FIV protease, as expected.

However, the new inhibitors lb-6b, and 7a showed a remarkably different pattern of inhibitory activities compared to their parent compounds la-5a, ABT- 538 and R031-8959. Compounds 2a-4a, containing no P3 moieties, exhibited marginal activities with IC50 values in the range of 2-300 uM against HIV protease and did not show any significant inhibition of FIV protease at 800 pM.

Only the a-keto amide 5a showed reasonable potency against HIV protease with a Ki value of 214 nM (Slee et al. J. Am. Chem. Soc. 1995,117, 11867-11878).

On the other hand, the modified inhibitors 2b-5b, which contain a methyl group as P3 residue, displayed 120-to 1000-fold improved inhibitory activities against HIV protease and at least three orders of magnitude higher potency for FIV protease compared to their parent compounds. In particular, 5b was found to be a slow binding inhibitor with Ki of 2.5 and 46 nM against HIV and FIV proteases,

respectively. This level of potency against FIV protease by the inhibitor with a molecular weight of only 649 was truly remarkable, considering the smallest efficient substrate for the enzyme is an eight-residue peptide, Ac-pro-Gln-Ala- TyrPro-Ile-Gln-Thr (Schnlzer et al. Virology 1996,224,268-275).

Since the ketone moiety of 5b is not hydrated in aqueous solution according to 13C NMR studies, the increased inhibitory activity of 5b may occur via enzyme-assisted hydration of the ketone moiety within the active site to form a gem-diol as transition-state mimic, similar to the case of a related a-keto amide inhibitor observed previously by x-ray structure and 13C NMR analyses (Figure 4; Slee et al., ibid).

The relative inhibitory activities of inhibitors 2b-5b against FIV protease are similar to the relative activities against HIV protease. For example, 5b is a superior inhibitor of both enzymes, and the hydroxyketone 4b is 57-and 44-fold more effective than its diastereomer 3b against HIV and FIV protease, respectively. In addition, the relative effectiveness of the P1-P1' core structures in 2b-5b against HIV protease is also consistent with the binding pattern of their parent compounds 2a-5a. These results indicate that introduction of Val and Ala as P2 and P3 residues can improve the inhibition against both enzymes despite some changes for the P 1-P 1'core unit. In addition, extension of the backbone of an inhibitor to contain an appropriate P3 moiety is essential to exhibit high potency against FIV protease, which is also consistent with our previous results with C2-symmetric inhibitor lb (Lee et al., ibid).

The kinetic results of inhibitors 6b and 7a, which were modified from existing drugs ABT-538 and R031-8959 (Saquinavir), respectively were even more promising. Compound 7a was found to have Ki values of 1.5 nM and 2.6 uM against HIV and FIV protease, respectively. The inhibitory activities of the original drug R031-8959 were also evaluated under the same assay conditions, and found to have Ki values of 1.6 nM and 76 uM for HIV and FIV proteases, respectively. These results clearly indicated that compound 7a retained the original inhibitory activity of R031-8959 against HIV protease, which was already optimized for the enzyme, but showed 29-fold enhanced potency against FIV protease. Inhibitor 6b also exhibited comparable activities to compound 7a, with Ki values of 3.0 nM and 3.7 uM for HIV and FIV proteases, respectively.

However, compound 7b was 7.5-and 51-fold less potent than 7a against HIV and FIV protease, respectively. Changing the P2 Val residue of 7a to Asn increased hydrophilicity of the inhibitor, which could be a major cause of lower activity found in 7b for both enzymes. In fact, compound 7b was more soluble in water than 7a and thus would require higher desolvation energy to bind the hydrophobic active sites of enzymes.

The above results suggest that inhibitors with a small P3 residue are effective against both the FIV protease and the HIV protease. These results are consistent with the results of molecular modeling that shows HIV and FIV protease binding to Saquinavir and its modified derivative with a small P3 residue (Figure 5).

EXAMPLE 3: Inhibition of Drug-Resistant Mutant HIV proteases The modified inhibitors with dual efficacy against FIV and HIV proteases (compounds lb, 4b, 5b, 6b, and 7a) were also tested against drug- resistant mutant HIV proteases G48V and V82F. These mutant enzymes were selected because Gly 48 and Val 82 are within the S3 and S3'subsites and have been identified as some of the most frequently mutated residues associated with development of drug resistance.

Against these mutant G48V and V82F enzymes, all modified inhibitors retained most of their original potency, and their relative inhibitory activity was directly proportional to the efficacy against wild-type HIV protease. In particular, modification of the FDA approved drugs ABT-538 and R031-8959 containing a bulky P3 group to the ones with methyl group at P3 (i. e. 6b and 7a) provided significantly improved inhibitory activity against the mutant proteases.

Only 4.8- and 6.5-fold higher IC50 values were observed for the V82F and G48V mutant variants, respectively, compared to 90-and 27-fold higher IC50 values for the parent compounds. It is noteworthy that V82F and G48V mutants are known to be less efficient enzymes compared to the wild-type HIV protease.

Therefore, inhibitory activities of compounds against these mutant enzymes are expected to be lower than against the wild-type HIV protease. Compound 1 b was also active in cell culture (Bacheler et al. J. Antiviral Chem. Chemother. 1994,5,

111). The IC90 values of lb against the wild type HIV protease, the I84V and 82F/84V mutants are 0.1,0.4 and 0.9 uM respectively.

EXAMPLE 4: C2-Symmetric Inhibitors with an iso-Butyl-2. 3-diol Core as Pl and PI' The x-ray structure of lb complexed with HIV, FIV (3X), FIV (V59I), and FIV (Q99V) proteases have been determined and will be published in Biochemistry. Since the side chains of PI and P3 residues of lb are positioned very close to each other in the S 1 and S3 subsites of the enzyme, we have investigated the activities of new C2-symmetric inhibitors (8-11) with iso-butyl groups at PI and P 1'. In addition, since water molecules were identified in the S3 and S3'subsites of both HIV and FIV protease complexed with compound lb, compounds 12 and 13, with P3 and P3'hydroxy groups, were synthesized to improve in by providing favorable electrostatic interactions with water.

The inhibitory effects of each C2-symmetric inhibitor on HIV and FIV proteases were determined, and the results are provided in Figure 11. For comparison, the Ki values for inhibition of FIV and HIV protease by the C2- symmetric inhibitors with a benzyl group at P I and P 1'are also included (lb, 9b- lOb).

All the C2-symmetric diols tested in this study showed competitive inhibition of both the feline and human lentivirus proteases in every case, but with at least an order of magnitude higher potency against HIV protease. Among the inhibitors with iso-butyl groups at the PI-PI'core (8-11), compound 8 with Ala at P3 and P3'is the best inhibitor of FIV. This maximum inhibitory activity observed in compound 8 was reduced by increasing the size of the side chain of the P3 and P3'residues. In fact, the measured Ki values of inhibitors 9,10, and 11 were 3.7-, 7.9-, and 4-fold higher than the Ki value observed for inhibitor 8.

This specificity for small hydrophobic groups at P3 and P3'sites found among the C2-symmetric inhibitors 8-10 with iso-butyl groups as the PI-PI' core against FIV protease was consistent with the observation for the analogous inhibitors lb, 9b-lOb. In addition, compounds 8 and 9 exhibited almost the same degree of potency against FIV protease compared to the benzyl analogs lb and 9b, respectively. This result indicates that Leu can bind the S 1 and S 1'

subsites of the enzyme as effectively as Phe. Previously, the observed preference for small P3 and P3'moieties in FIV protease was explained by hypothesizing that bulky groups at the P3 and P3'positions of the inhibitor could provoke unfavorable interactions at the sterically congested S3 and S3'subsites of FIV protease. See, Lee et al. Proc. Natl. Acad. Sci. USA 1998,95,939-944.

In particular, compound 10b (Ki = 7.0 uM) exhibited 170-fold lower potency than lb. This proposal is still valid for the lower inhibitory activities of 9,10, and 11 compared to 8 against FIV protease. However, it is noteworthy that compound 10 showed 14-fold higher inhibitory activity than its analog lOb. This improved potency by compound 10 against FIV protease can not be explained by the above proposal since both inhibitors contain Phe as P3 and P3'residues.

Therefore, current observations also suggest that the steric interaction between neighboring PI and P3 side chains is another crucial factor to be considered for the design of new inhibitors. Indeed, compound 10, containing smaller P 1 and P 1'side chains than inhibitor lOb, can provide more room at S3 and S3'binding sites to accommodate bulky benzyl groups. This proposal was further confirmed by the modeling of FIV protease complexed with compound 8.

The structure reveals that the side chain of PI and P3 residues are positioned closely, and the combining S 1 and S3 pocket is not wide enough to accommodate two benzyl groups. It is also expected that binding of PI and P3 moieties can be affected by each other, and an appropriate combination of PI and P3 residues is essential for good binding.

It has been determined that HIV protease exhibits a high degree of flexibility in binding at the S3 and S3'subsites (Lee et al., ibid). Inhibition studies of the diols 8-10 against HIV protease provided very different patterns compared to their analogs lb, 9b-lOb. The Ki values of compounds 8 and 10 were 4.3- and 2.0-fold higher than lb and lOb, respectively. This indicates that Phe at PI and P 1'is better than Leu for binding to HIV protease. However, compound 9 is a more effective inhibitor than compound 9b and also showed 7.5- and 6.3-fold higher potency against HIV protease compared to 8 and 10, respectively. This significant improvement was not observed with compounds lb, 9b and lOb. These kinetic results indicate that introduction of iso-butyl groups at S3 and S3'subsites can improve the potency of an inhibitor containing

a medium size Pl-Pl'core. These results also suggest that the overall size of the PI and P3 residues will significantly affect the binding specificity of the enzymes.

Finally, compounds containing a hydroxy group at P3 and P3'side chains (compounds 12 and 13) were also effective inhibitors of both HIV and FIV proteases. In particular, compound 12 displayed the highest potency with Ki values of 0.58 nM and 32 nM against HIV and FIV proteases, respectively. It is noteworthy that compound 12 is more hydrophilic than lb by two additional hydroxy groups and would require more desolvation energy in order to bind to the hydrophobic active sites of enzymes. However, compared to compound lb, compound 12 exhibited a 3-fold higher potency against HIV protease but a similar potency against FIV protease. These results indicate that binding of 12 is significantly enhanced by the two hydroxyl groups of the P3 and P3'side chains, presumably by promoting favorable electrostatic interactions with the water molecules observed by crystallography studies at the S3 and S3'subsites.

The ability of compound 12 to prevent infection of FIV in tissue culture was also examined. Compound 12 was, however, not as effective as lb. It has been known that introduction of hydrophilic functional groups, such as a hydroxyl or carboxyl group, to the P2 and P2'positions of C2-symmetric inhibitors would cause a dramatic loss of potency against HIV protease in tissue culture, though the inhibitory activity in vitro has little change (Budt et al., Bioorg. Med. Chem. 1995,3,559-571). Our ex vivo assay results have confirmed a similar activity loss in tissue culture when a hydrophilic group at P3 and P3'is present. These results suggest that increasing the overall polarity and hydrophilicity of compounds may result in the reduction of efficacy in vivo.

The above results indicate that the interaction of PI and P3 residues in the active sites of HIV and FIV proteases is a crucial factor to be considered for maximizing the binding affinity of inhibitors. Although it has been considered that Phe can provide binding at the S 1 subsite of HIV protease, our observations suggest that with appropriate P3 and P3'moieties, one can also develop potent inhibitors with P 1 and P 1'side chains smaller than the benzyl group.

EXAMPLE 5: Synthesis of Key Intermediates and Inhibitors The key intermediates (IS, 2R, 3R, 4S)-1, 4-bis [(N-Cbz) amino]-1, 4- dibenzyl-2,3-diol and (IS, 2R, 3R, 4S)-1, 4-bis [(N-Cbz) amino]-1, 4-diisobutyl-2,3- diol 14 (Scheme 1) were prepared by the stereoselective Pinacol coupling of L- Cbz-phenylalanal and L-Cbz-leucinal, respectively, using Pedersenis procedure (Konradi et al. J. Org. Chem. 1992,57,28-32). The minor diastereomeric impurities of the coupling reaction were removed by flash column chromatography after protection of the diol as an isopropylidene derivative to form compound 15. The Cbz groups of compound 15 were removed to yield the diamine 16 by hydrogenation. Amine 16 was directly coupled with Cbz-Val using HBTU to give adduct 17 (Dourtoglou et al. Synthesis 1984,572-574).

Four different P3 and P3'residues were then introduced to adduct 17 by applying the same deprotection and coupling procedures described above to give compounds 1§-22. Finally, the target inhibitors 8-11 were prepared by deprotection of the isopropylidene group from the corresponding precursors (Figure 6). Compounds 12 and 13 were obtained by applying the same procedure described previously using (lS, 2R, 3R, 4S)-1, 4-bis [ (N-Cbz) amino]- 1, 4-dibenzyl- 2,3-diol as a starting material.

The hydroxyethylamine inhibitor 2b was prepared by coupling the proline derivative 24 to the epoxide 25 (Slee et al. J. Am. Chem. Soc. 1995,117, 11867-11878; Hung et al. J. Org. Chem. 1991,56,3849-3855) via reflux in methanol, using triethylamine as shown in Figure 7. The synthesis of the core isostere 5a has been modified from the method previously employed by our group. The a-hydroxy acid 26, prepared by known procedures (Munoz et al.

Bioorg. Med. Chem. 1994,2,1085-1090) was coupled to the proline derivative 24 to give the a-hydroxy amides 3a and 3b. Hydrogenation of the diasteromers 3a and 3b followed by HBTU mediated coupling with Cbz-Ala-Val-OH gave 4a and 4b in 65% yield. Dess-Martin oxidation of the a-hydroxy amides 3a-4b gave the corresponding a-keto amides 5a and 5b in moderate yield (Figure 8; Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991,113,7277-7287).

The synthesis of 6b began with the cyclic boronate 28 prepared by refluxing the diamine 27 with phenylboric acid (Kempf et al. J. Med. Chem.

1998,41,602-617). Condensation of the phenyl boronate 28 with carbonate 29 followed by HBTU mediated coupling with Cbz-Ala-Val-OH gave the desired product 6b (Figure 9). The synthesis of the modified Saquinavifrm derivatives 7a and 7b began with condensation of the isoquinoline derivative 31 with epoxide 30 to give the adduct 7 in 80% yield (Thompson et al. J. Am. Chem.

Soc. 1993,115,801-803). Removal of the BOC group in 7 with TFA followed by HBTU coupling with Cbz-Ala-Val-OH or Cbz-Ala-Asn-OH gave the modified inhibitors 7a and 7b respectively (Figure 9).

In summary, manipulation of the backbone of HIV protease inhibitors to create appropriate P3 residues can improve potency against FIV, HIV, and mutant HIV proteases, regardless of the structure of the P 1-P 1'core units. The inhibitors with small P3 and bulky PI described in this study are effective against FIV and HIV proteases, and exhibit high potency against drug-resistant mutant HIV proteases. In addition, modification of some existing HIV protease inhibitors to have a small P3 group also enhances their inhibition of FIV protease and mutant HIV proteases without affecting the activity against wild- type HIV protease. The development of broad-based inhibitors described in this study contributes significantly to our understanding of the specificity and resistance development of the aspartyl proteases and provides a new strategy for the development of new antiviral pharmaceuticals.

The current results indicate that using FIV protease as a general model for the drug-resistant mutant KIV proteases is clearly an effective strategy. One may thus use cats as a drug-resistant animal model to accelerate the new drug development process.

EXAMPLE 6: Analysis of S3 and S3'Subsite Specificities of FIV Protease: Development of a Broad-based Protease Inhibitor Efficacious Against FIV, SIV, and HIV in vitro and ex vivo The S3 and S3'subsite binding specificities of HIV and FIV proteases have been explored using C2-symmetric competitive inhibitors. The inhibitors evaluated contained (1S, 2R, 3R, 4S)-1, 4-diamino-1, 4-dibenzyl-2,3-diol as PI and P 1'units, Val as P2 and P2'residues, and a variety of amino acids at the P3

and P3'positions. All inhibitors showed very high potency against HIV protease in vitro, and their Ki's ranged between 1.1 and 2.6 nM. In contrast to the low restriction of P3 and P3'residues observed in HIV protease, FIV protease exhibited strong preference for small hydrophobic groups at the S3 and S3' subsites. Within this series, the most effective inhibitor against FIV protease contained Ala at P3 and P3'. This inhibitor had a Ki of 41 nM that was 415-and 170-fold lower than those of the inhibitors without P3 and P3'moieties or with Phe at these positions, respectively.

In addition, these compounds were tested against mutant FIV proteases, which contain amino acid substitutions corresponding to those in native HIV protease at homologous sites, and their efficacy of inhibition progressively increased up to 5-fold. The most potent FIV protease inhibitor was selected for examination of its effectiveness in tissue culture, and it was able to block nearly 100% of virus production in an acute infection at 1 pg/ml (1.1 against HIV, FIV and SIV. Furthermore, it was not toxic to cells, and even after 2 months of culture there was no sign of resistance development by virus. The findings suggest that the more stringent requirements for FIV protease may lead to the development of broad-based inhibitors that will be efficacious against a broad range of HIV quasispecies as well as interspecies proteases.

At least six mutated residues in HIV protease that cause drug resistance are also found in the structurally aligned native residues of FIV protease. Kinetic studies also showed that various potent HIV protease inhibitors containing the P3 to P3'residues, including the FDA approved drug Ro 31-8959 (Slee et al., JACS 117, 11867-11878), are less efficient inhibitors of FIV protease by a factor of 100 or more. Although the significance of these observations is yet to be appreciated, it suggests that FIV protease may serve as a model for drug resistant mutant HIV proteases and may contribute to the understanding of HIV resistance to protease inhibitors. Therefore, we are developing potent inhibitors of FIV protease containing residues that bind to the S3 to S3'region with the aim of developing broad-based therapeutic agents against AIDS that may be less prone to resistance development. An additional advantage is that this strategy facilitates the in vivo testing of candidate inhibitors in an animal system (Figure 13).

Since the active sites of both HIV and FIV proteases are C2-symmetric, it has been predicted that the axis of symmetry of an inhibitor with a C2-symmetric unit would co-align with the C2-axis of the enzymes resulting in specific inhibition. In fact, C2-symmetric inhibitors containing diol cores have been identified as extremely potent inhibitors of HIV protease in vitro. The x-ray crystal structure of HIV protease complexed with the inhibitor A-76889, containing (IS, 2R, 3R, 4S)-1, 4-diamino-1, 4-dibenzyl-2, 3-diol as the PI and P1' unit and N-protected Val as P2-P3 and P2'-P3', also displayed the high degree of structural correspondence at P 1-P3, P 1'-P3, although the R, R diol core bound in an asymmetric mode. Condra et al., 1995 Nature, 374,569-571. Therefore, evaluating the binding affinities of C2-symmetric inhibitors represents a convenient strategy for systematic probing of enzyme specificities at certain sites.

The kinetic parameters of the FIV protease for peptide substrates also indicated that replacing Val at P2 position by Ile led to a significant decrease in binding. Therefore, compound 500 (Figure 12) was chosen as a core unit. A variety of amino acids were then introduced to the core as the P3 and P3' residues by a combinatorial approach to rapidly create a number of potential inhibitors for the analysis of amino acid restriction and tolerance at the S3 and S3'subsites of HIV and FIV proteases. Promising compounds revealed by in vitro analyses were then tested ex vivo for efficacy against FIV, SIV, and HIV infection.

Chemical Syntheses.

The (1S, 2R, 3R, 4S-1, 4-bis [(N-Cbz) amino]-1, 4-dibenzyl-2,3-diol 100 was prepared by Pedersens procedure (Konradi et al J. Org. Chem 57,28-32), along with its diastereomers. After protection of the diol as the isopropylidene 200, these minor diastereomeric impurities were removed by flash column chromatography. The Cbz groups of compound 200 were deprotected by hydrogenation, then the diamine 300 was coupled with Cbz-Val using HBTU, providing adduct 400. Four different P3 and P3'residues were then introduced to adduct 400 by applying the same deprotection and coupling procedures described above to give compounds 600-900. Finally, the target inhibitors 1100-

1400 were obtained by removal of the isopropylidene from the corresponding precursor under acidic conditions. The same procedure was applied in the synthesis of other compounds that were not shown here (Wlodawer et al, 1995, Nat. Struc. Biol. 2,480-488). The reference inhibitor 1000 was also synthesized from compound 400 by the same deprotection procedure (Figure 12).

In Vitro Inhibitory Activities Against Proteases.

The inhibitory effects of each inhibitor were evaluated against HIV and FIV proteases along with two mutant FIV proteases that contain amino acid substitutions corresponding to those in HIV protease at homologous sites. The results of some selective inhibitors, each of which is a competitive inhibitor of all four enzymes, are summarized in the Figures.

All the C2-symmetric diols tested in this study showed very high potency against HIV protease, and their Ki's ranged between 1.1 and 2.6 nM.

Considering experimental error, there was no significant difference in the overall efficacy of these diols for inhibition of the HIV protease. In part, this reflects the low restriction of amino acid residues at the S3 and S3'subsites of the HIV protease. In addition, the Cbz groups of the reference inhibitor 1000, which does not contain P3 and P3'residues, could be positioned tightly at the S3 and S3' subsites of HIV protease to make compound 1000 an effective inhibitor.

However, inhibition of FIV protease by inhibitors 1000-1400 showed a remarkably different pattern. First, the inhibitory activity of the reference compound 1000 was decreased by almost 1.7 x 104 fold compared to its Ki for HIV protease. This striking activity loss observed for 1000 was recovered by extending the backbone of the inhibitor using Gly as P3 and P3'residues, with the Ki of 1100 being 110-fold lower than 1000. This preference of the extended inhibitor backbone found in FIV protease is also supported by the observation that the HIV protease will cleave a six residue peptide substrate, Ac-Gln-Ala- Tyr-Pro-Ile-Gln (SEQ ID NO : 1), whereas the smallest FIV protease substrate is an eight residue peptide, Ac-Pro-Gln-Ala-Tyr-Pro-Ile-Gln-Thr (SEQ ID NO : 2).

The best residue for S3 and S3 binding was Ala. In fact, inhibitor 1200 (Ki = 41 nM) is the most potent inhibitor of FIV protease known to date. The inhibitory activity of 1200 against FIV protease was reduced by increasing the size of the

side chain of the P3 and P3'residues to produce compound 1300, and the Ki of 1300 is 4-fold higher than the Ki for 1200. Furthermore, the diol 1400 showed 45-and 170-fold lower potency compared to 1100 and 1200, respectively, and this result suggests that the benzyl side chain of P3 and P3 residues may cause unfavorable interaction with FIV protease or the neighboring P I and P I'side chains. This severe restriction of P3 and P3'moieties in the FIV protease partly explains the total loss of potency against FIV protease by HIV protease inhibitor Ro 31-8959, since it contains bulky aromatic group at the P3 position.

The result was further confirmed by comparison of the x-ray structures of FIV and HIV proteases complexed with inhibitors. The structures revealed that only two residues (Arg 13, Asp 34) in the S3 and S3'subsites of FIV protease were conserved at the structurally aligned KIV protease positions. Three other residues at the subsites Gly 48, Pro 81, and Val 82 of HIV protease were replaced with Ile 57, Ile 98, and Gln 99 in FIV protease. As a result, the S3 and S3'subsites of FIV protease is sterically more congested than those in HIV protease, and these three different residues may define the S3 and S3'subsite specificities of the enzymes.

In addition, the Gly 48 and Val 82 of HIV protease have been identified as frequently mutated residues in the development of drug resistance. For example, the potency of the FDA approved drugs Ro 31-8959 and ABT-538, which contain bulky P3 moieties, against the G48V HIV mutant was decreased by 27 and 17 fold, respectively. Among the HIV protease variants containing mutations at Val 82, the V82F mutant becomes 15,7, and 90 fold less sensitive toward the licensed drugs AG-1343, MK-639, and ABT-538, respectively. It is noteworthy that both AG-1343 and MK-639 contain no P3 residues.

Restrictions observed in mutant HIV proteases with inhibitors containing inappropriate P3 and P3 moieties are very similar to binding preferences found in FIV protease. These observations further support utility of FIV protease as a model for drug resistant variants.

The results from the inhibition studies of the diols 1000-1200 and 1400 against the mutant FIV proteases are also intriguing. The less effective inhibitors 1000 and 1400 showed very similar activities against mutants compared to wild type FIV protease. However, the efficacy of inhibition by the more potent

inhibitors 1100 and 1200 was progressively increased up to 5 fold in mutant enzymes. Overall, these results provide new insights into the specificity and resistance development of the aspartyl proteases and may help development of new inhibitors better than those that are currently available as described above.

Ex Vivo Inhibitory Activities of Compound 1200.

The ability of the most potent FIV protease inhibitor 1200 to prevent infection of FIV, HIV and SIV in tissue culture was examined. The results are summarized in Figure 14.

For FIV, the assays were performed in FIV-infected feline T-cells that were cultured in the presence of compound 1200 at different concentrations over the course of 1 month. Each data point in Figure 14 represents the amount of pelletable FIV reverse transcriptase in the culture supernatant. Compound 1200 was able to markedly inhibit FIV replication at 0.5 µg/ml (0.55 mM) and found to be most effective at 1.0 µg/ml (1.1 µM). Furthermore, this inhibitor was not toxic to feline T-cells.

After one month, the drug-treated cultures were split and re-plated with and without compound 1200. No virus was detected in the absence or presence of drug after two weeks in culture (data not shown). No sign of resistance development against the drug was observed after eight weeks of continuous culture.

The results from tissue culture assays against SIV (Figure 14) and HIV (Figure 14) were equally encouraging. Compound 1200, at 1 pg/ml, reduced virus expression levels to near background, as judged by reduction in release of p27 antigen into the culture supernatant in the presence of drug.

The effectiveness of compound 1200 against HIV was measured by determining the percentage of viable cells remaining in culture over time in the presence and absence of drug (Figure 14). In the control, HIV caused formation of multinucleated syncytia and 100% cell death by 9 days post infection.

However, the cells cultured with 1 pg/ml (l. 1 pM) or 5 g/ml (5.5 uM) of inhibitor 1200 remained 100% viable after 1 month, identical to results obtained in the absence of virus infection. To test for virus in these cultures, supernatants were removed and added to 1 x 105 uninfected MT-2 cells after a 1 : 5 dilution

with fresh CM. After 3 weeks the MT-2 cells remained uninfected, demonstrating the absence of free virus in cultures of infected MT-2 cells treated with compound 1200. In contrast, when 2 x 105 infected MT-2 cells that had been treated with compound 1200 for two weeks were removed and re-plated in fresh medium with or without compound 1200, only MT-2 cells cultured in the absence of compound 1200 were dead within 4 days (data not shown).

These results clearly demonstrated strong potency of compound 1200 against HIV as well as its minimal toxicity to host cells.

Cultures are being carried continuously in the presence of compound 1200 to look for resistance development. Tests are also underway to determine the level of efficacy of the compound against a defined panel of drug-resistant HIVs.

It is clear that FIV protease exhibits a specific preference for amino acids containing small side chains at the P3 and P3'positions, especially for Ala. In addition, extension of inhibitor backbone can increase the potency of inhibitors in FIV protease. Our in vitro inhibition studies of mutant FIV protease also showed a direct relationship between the inhibition of FIV protease and HIV protease. This observation suggests that potent inhibitors of FIV protease, containing P3 to P3 residues, become even more efficient against HIV protease.

The most potent inhibitor 1200 has also shown strong ability to control lentiviral infections in tissue culture. In fact, this is the first compound that inhibits replication of FIV, HIV and SIV with virtually the same degree of effectiveness. This remarkable versatility of compound 1200 also suggests the strong possibility of sustaining its potency against mutant HIV proteases.

Finally, the FIV system is clearly an effective and relevant strategy for advancing HIV therapies. As a natural animal system, FIV offers the ability to perform in vivo tests of efficacy and assessment of drug resistance that is not readily feasible in primate systems. In addition, it is hoped that the broad-based nature of inhibitors arising from these studies will afford a reduced level of resistance development.

While a preferred form of the invention has been shown in the drawings and described, since variations in the preferred form will be apparent to those skilled in the art, the invention should not be construed as limited to the specific

form shown and described, but instead is as set forth in the following claims.

Synthetic Protocols.

General Procedures: Analytical TLC was performed on pre-coated plates (Merck, silica gel 60F-254). Silica gel used for flash column chromatography was Mallinckrodt Type 60 (230-400 mesh).

NMR ('H,'C) spectra were recorded on either a Bruker AMX-400 or Am-500 MMz Fourier transform spectrometer. Coupling constants (J) are reported in hertz (Hz), and chemical shifts are reported in parts per million (8) relative to tetramethylsilane (TMS, 0.0 ppm) with CDC13, DMSO or CD30D as solvent. All other reagents were commercial compounds of the highest purity available and purchased from the Aldrich, Sigma, Nova Biochem, or Bachem. It should be noted that"x"as shown in the figure 3 is selected from the group consisting of hydrogen, carbobenzyloxy-, carbobenzyloxy-valine-, carbobenzyloxy-glycine-valine-, carbobenzyloxy-alanine-valine-, carbobenzyloxy-leucine-valine-, carbobenzyloxy-phenylalanine-valine-, carbobenzyloxy-serine-valine-, carbobenzyloxy-alanine-asparagine-, carbobenzyloxy-threonine-valine-and carbobenzyloxy-valine-valine-all linked to the inhibitors using the procedures as described below and wherein all groups obtained from sources disclosed above or synthesized using procedures well known in the art or stated herein.

Molecular modeling: Models of the protease inhibitor, lb, complexed with HIV-1 protease and FIV protease were built using the Brookhaven Protein Data Bank entries 1HVI and 1FIV6 as starting points. The entry 1HVI has a resolution of 1.8, and contains the C2-symmetric Abbott inhibitor A77003 (R, S) co-crystallized with the HIV-1 protease of strain HIVLAI expressed in E. coli. The structure of A77003 is quite similar to that of lb. The stereochemistry of one of the core hydroxyls had to be inverted, and the pyrimidine groups were replaced by the Cbz groups. The FIV structure has a resolution of 2.0 and contained the inhibitor LP-149 co-crystallized with the Feline immunodeficiency virus protease from Felis catus and expressed in E. coli.

The model of lb bound to HIV-1 protease was built first, using InsightII 97.0is Biopolymerl8 and Discover modules, and the AMBER force field.

Molecular mechanics minimization was performed using the conjugate gradients minimizer until the minimization converged, i. e. the derivative of the energy was less than 0.001. Distance constraints were used in the early stages of the modeling to ensure that the hydrogen bonds were preserved between the inhibitor, water and protease.

Initially, only the inhibitor model, five active-site water molecules, and the residues of the active site that were in contact with the inhibitor were allowed to move. These five waters were the water bound between the tips of the flaps, and four waters, two in each sub-unit of the dimer, that bind inside a pocket adjacent to the Arg-8 residue in HIV (Arg-13 in FIV). The resulting model was minimized while allowing all atoms to move. A similar procedure was applied to the modeling shown in Figure 5, using HIV protease complexed with R031- 8959.

Biological Assays For determination of IC50 values for HIV protease, a backbone engineered HIV-1 protease was prepared by total chemical synthesis (Kent et. al.

Science 1992,256,221) and used in the protease assay at final concentration of 450 nM. The HIV-1 protease was added to a solution (152 L final volume) containing 28 pM of the fluorogenic peptide inhibitor having sequence Abz-Thr- Ile-Nle-Phe- (p-NO2)-Gln-Arg-NH2 (Toth et. al., Int. J. Peptide Res. 1990,36, 544)) (SEQ ID NO : 3) and 1.8 % dimethylsulfoxide in assay buffer. Assay buffer contained 100mM MES buffer, pH 5.5, with 0.5 mg/mL BSA. The solution was mixed and incubated over 5 minutes, during which time the rate of substrate cleavage was monitored by continuously recording the change in fluorescence of the assay solution. An excitation filter of 325 nm, and an emission filter of 420 nm were used. The fluorescence data were converted into uM substrate cleaved per minute, using a predetermined standard calibration curve of change in fluorescence against concentration of substrate cleaved.

Determination of Ki for HIV protease was performed similarly with the following modifications. The substrate concentrations used were 57,43,28 and

14 pM. All other concentrations were as above. The curve fit for the data was determined and the subsequent Ki derived using a computer program based on the equation of Morrison et. al. BioChim. Biophys. Acta 1969,185,269, for tight binding inhibitors.

For determination of Ki and ICso for FIV protease, 0.125 pg of the enzyme was added to a solution (10011L final volume) containing inhibitor, 560 uM peptide substrate (sequence Gly-Lys-Glu-Glu-Gly-Pro-Pro-Gln-Ala-Tyr- Pro-Ile-Gln-Thr-Val-Asn-Gly) (SEQ ID NO : 4) and 2% dimethyl sulfoxide in a 1 : 3 mixture of assay buffer (as above) and 4M NaCI aqueous solution. The solution was mixed and incubated for 10 minutes at 37 °C and the reaction quenched by addition of 8M guanidine HC 1 solution containing 0.2 M sodium acetate at pH 4.2 (100 L). The cleavage products and substrate were separated by reverse phase HPLC. Absorbance was measured at 215 nm, peak areas were determined and percent conversion to product was calculated using relative peak areas.

The data were plotted as IN (V = rate substrate is cleaved in nmol/min) against inhibitor concentration and the-Ki determined as the point at which the resulting line intersects with 1/Vmax (Vmax = 6.85 nmol/min). IC50 was determined as the inhibitor concentration at 50% inhibition. Vmax (6.85 0.7 nmol min-1) and Km (707 70um) for the FIV protease were determined from a plot of IN (V = rate in nmol/min). The data used was generated similarly to that for Ki with the following modifications. The substrate concentrations used were 560,448,336,224,111 and 56 uM, in the absence of inhibitor.

The FIV protease was obtained from the H 1 fragment containing the coding sequence of FIV protease. The H 1 fragment was cloned from FIV- 34TF10 (Talbott et. al. Proc. Natl. Acad. Sci. USA 86 1989,5743) into the pT7- 7 vector (Tabor et. al. Proc. Natl. Acad. Sci USA 82 1985,1074). The 5'end of the insert was modified by the addition of an Ndel adaptor, which provided the proper reading frame with initiation of translation from the methionine encoded in the latter site. Translation resulted in production of an 18.6 kDa precursor, which autoprocessed to a 13.2 kDa FIV protease plus N-and C-terminal fragments of 3.6 kDa and 1.8 kDa, respectively. The construct was transformed into E. coli strain BL21. DE3, lys S (Studier et. al. Meth. Enzymol. 1990,185,

60). Overnight cultures were used to inoculate 15 liter fermentations.

Circlegrow medium (Bio 101) plus 100 pL ampicillin, 20 pM chloramphenicol, at 37° C was used for the fermentation cultures. The cells were allowed to reach mid-log phase, then the temperature was reduced to 24° C and IPTG (isopropyl- p-thiogalactopyranoside) was added to a final concentration of 1 mM. The fermentation was allowed to proceed for 16 hours, at which time the cells were harvested by centrifugation and frozen at-70 °C in 100 g aliquots for future use.

Pelleted cells (100 g) were frozen and lysed by addition of 600 mL, 50 mM Tris-HCl, pH 8,5 mM EDTA and 2 mM 2-mercaptoethanol. The cells lysed upon thawing and the viscous mixture was homogenized at 4 °C for 2 min in a Waring blender. The sample was centrifuged at 8,000 x g for 20 min and the pellet discarded. The sample was diluted to 1 liter, and subjected to tangential flow against a 300 K cut-off membrane (Filtron). The protease was washed through the membrane using five liters of the same buffer. The retentate was discarded and the flow-through supernatant concentrated by tangential flow against a 10 K cut-off membrane. The retentate was passed over a DE52 anion exchange column (5 x 20 cm) equilibrated in the same buffer.

The flow-through from this column was passed over an S-Sepharose Fast Flow matrix (2.5 x 20 cm column, Pharmacia), again equilibrated at pH 8 in the same buffer. The flow-through from S-Sepharose was made 1M with respect to ammonium sulfate and applied to a phenyl sepharose column (Pharmacia, 1.5 x 10 cm), washed with lysis buffer containing 1M ammonium sulfate, then eluted with a 100-0% linear ammonium sulfate gradient.

Peak fractions containing protease were pooled, concentrated using Centripreps (Amicon), and dialyzed against 10 mM Tris-HCI, pH 8,5 mM EDTA, 2 mM 2-mercaptoethanol. MOPS was added to the sample to a concentration of 10 mM, and the pH was further adjusted to 5.5 with HCI. The sample was then applied to a Resource S column (Pharmacia) equilibrated in 10 mM Tris-MOPS, pH 5.5,5 mM EDTA and 2 mM 2mercaptoethanol. Protease was eluted using a linear 0-300 mM NaCl gradient in the same buffer. Peak fractions were pooled, concentrated, and stored as aliquots at-20 °C for further studies. The integrity of the isolated FIV protease was confirmed by ion spray mass spectrometry.

Chemical Synthesis All manipulations were conducted under an inert atmosphere (argon or nitrogen). All solvents were reagent grade. Anhydrous ether, tetrahydrofuran (THF), and toluene were distilled from sodium and/or benzophenone ketyl.

Dichloromethane (CH2C12) was distilled from calcium hydride (CaH2). N, N, Dimethylformamide (DMF) and acetonitrile were distilled from phosphorous pentoxide and calcium hydride. Methanol was distilled from magnesium and iodine. Organic acids and bases were reagent grade. All other reagents were commercial compounds of the highest purity available. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel (60 F-254) plates (0.25 mm). Visualization was effected using standard procedures unless otherwise stated. Flash column chromatography was carried out on Merck silica gel 60 particle size (0.040-0.063 mm, 230-400 Mesh). Melting points were determined with a Thomas-Hoover capillary melting point apparatus and are uncorrected. Proton and carbon magnetic resonance spectra (lH-NMR, 3C- NMR) were recorded either on a Bruker AM-500, AMX-400 or on a AC250MHz Fourier transform spectrometer. Coupling constants (J) are reported in hertz and chemical shifts are reported in parts per million (8) relative to tetramethylsilane (TMS, 0 ppm), MeOH (3.30 ppm for'H and 49.0 ppm for 13C) or CHC13 (7.24 ppm for'H and 77.0 ppm for 13C) as internal reference. Infrared spectra (IR) were recorded on a Perkin-Elmer 1600 series FT-IR spectrophotometer. Absorptions are reported in wave numbers (cm~').

Peptide fragments described herein were synthesized using traditional peptide coupling methodologies [EDC (1- (3 dimethylaminopropyl)-3- ethylcarbodiimide HC1), HOBt (1-hydroxybenzotriazole) and DIEA (diisopropylethylamine)]. Esters were hydrolyzed either by base (LiOH for methyl esters) or acid (TFA for t-butyl esters).

General Procedure for Coupling Reaction (steps c and d, Figure 6; step h, Figure 7; step i, Figure 8; and step c, Figure 9).

To a solution of free amine (1.0 mol equiv. as shown in the Figure and either purchased from Aldrich, Sigma, Nova Biochem, or Bachem or synthesized

herein) and carboxylic acid (1. 0 mol equivalent as shown in Figure and either purchased from Aldrich, Sigma, Nova Siochem, or Bachem or synthesized herein) in dry CH3CN (0.1-0.15 M) was added HBTU (1.0 mol equiv. ; Aldrich) followed by Et3N (1.0 mol equiv. Aldrich) at 20 °C under Argon atmosphere.

The reaction mixture was stirred for 15 min then quenched by addition of brine and extracted with EtOAc. The organic layer was then washed sequentially with IN HCI, sat. aq. NaHC03, and brine, dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by flash chromatography to give the desired product.

Standard workup procedure.

The reaction mixture was stirred for 15 min, then quenched by addition of brine and extracted with EtOAc. Next, the organic layer was washed sequentially with IN HCl or saturated ammonium chloride solution (or combination thereof-normality only approximate), sat. aq. NaHC03, and brine, dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by flash chromatography to give a desired product.

Standard Dess Martin Oxidation.

In Figure 8, step j, the substrate 3a, 3b, 4a, or 4b (21 mg, 0.044 mmol) was dissolved in dry CH2C12 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 mmol) added. The reaction mixture was stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and quenched by addition of saturated sodium bicarbonate (aq.) (5 mL) and sodium thiosulfate. The aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts washed with water (10 mL), brine (10 mL), dried (MgS04) and concentrated in vacuo to give the crude product. Flash chromatography eluting with 30% ethyl acetate in hexane gave the desired product as a 3: 1 mixture of diastereomers (colorless oil).

Preferred synthesis of compound 15 as illustrated in Figure 6.

To a solution of 1, 4-bis {(N-Cbz) amino-1, 4-diisobutyl-2,3-diol 14 (450 mg, 0.90 mmol) in 2,2-dimethoxypropane (24 ml), was added catalytic amounts

of p-TsOH. The reaction mixture was heated at 600°C for 5 hr and cooled to 200°C. The reaction mixture was diluted with EtOAc (200 ml), and the resulting solution was washed with sat. aq. NaHC03 and sat. aq. NaC 1, dried over MgSO4, filtered and concentrated in vacuo. The residue was then purified by flash chromatography to give 2,3-protected (1S, 2R, 3R, 4S)-diastereomer 15 (467 g, 96%) as a white solid: 1H NMR (400 MHz, DMSO-d6,80 °C) d 0.80-0.87 (6H, m), 1.11-1.20 (1H, m), 1.27 (3H, s), 1.42-1.49 (1H, m), 1.53-1.61 (1H, m), 3.58 (1H, s), 3.72-3.83 (1H, m), 5.00 (1H, d, J = 12.8 Hz), 5.08 (1H, d, J = 12.8 Hz), 6.42 (1H, br s), 7.26-7.32 (5H, m) ; 13C NMR (100 MHz, DMSO-d6,80 OC) 8 21.1,22.3,23.8,26.4,41.0,48.1,64.8,78.9,126.8,127.0,127.6,13 6.7, 153.4; HRMS (FAB+), calcd for MCs+ C3lH44N206Cs m/e 673.2254, found m/e 673.2228.

Synthesis of compound 17 as illustrated in Figure 6.

Compound 15 (480 mg, 0.89 mmol) in EtOAc (30 ml) containing 10% Pd/C (170 mg) was stirred under H2 (1 atm) at 20 OC for 20 hr. The reaction mixture was filtered through Celite and then concentrated in vacuo to give diamine 16 (226 mg, 93%) as a colorless oil, which was used for the coupling reaction without purification. To a solution of diamine 16 (194 mg, 0.71 mmol) and N-Cbz-L-Valine (377 mg, 1.50 mmol) in CH3CN (8 ml) was added HBTU (569 mg, 1.50 mmol) followed by Et3N (166 mg, 1.64 mmol). The reaction mixture was stirred for 15 min at 20 °C under Ar then quenched by addition of brine (20 ml) and extracted with EtOAc (4 x 20 ml). The organic layer was washed sequentially with 1M HCl (5 ml), sat. aq. NaHC03 (5 ml), and sat. aq.

NaCI (5 ml), dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by flash chromatography to give compound 17 (430 mg, 82%) as a white solid: 1H NMR (400 MHz, DMSO-d6,25 OC) 6 0.78-0.88 (12H, m), 1.05-1.15 (1H, m), 1.22 (3H, s), 1.47-1.63 (2H, m), 2.01 (1H, q, J = 6.4 Hz), 3.42 (1H, s), 3.97 (1H, dd, J = 9.0,6.2), 4.02-4.10 (1H, m), 5.01 (2H, dd, J = 17.3,12.6 Hz), 7.27-7.41 (7H, m); 13C NMR (100 MHz, DMSO-d6,20 °C) d 17.6,19.3,21.4,23.9,27.0,30.1,41.7,44.5,60.2,65.4,79.2,107. 3,127.6, 127.7,128. 3,136.8,156.0,171.1; HRMS (FAB+), calcd for MCs+ C41H62N408Cs m/e 871. 3622, found m/e 871.3648.

Synthesis of compound 19 as illustrated in Figure 6.

Compound 17 (170 mg, 0.23 mmol) was hydrogenated with 10% Pd/C (50 mg) in EtOAc (8 ml) to give compound 18 (106 mg, 99%) as a colorless viscous oil. It was used for coupling reaction without purification. Next, compound 18 (20 mg, 0.043 mmol) was coupled with N-Cbz-Ala (20 mg, 0.089mmol) to give compound 19 (28 mg, 75%) as a white solid.

Preferred Synthesis of compound 20 as shown in Figure 6.

To a solution of compound 19 (25 mg, 0.030 mmol) in MeOH (1.5 ml) was added catalytic amounts of p-TsOH. The reaction mixture was heated at 60°C for 24 hr then diluted with EtOAc (10 ml). The organic solution was washed with sat. aq. NaHC03 (2 ml) and sat. aq. NaCI (2 ml), dried over MgS04, filtered and concentrated in vacuo to give free diol 18 (14 mg, 57%) as a white solid. The preparations of compound 9-13 were carried out using the general procedures for coupling and deprotection. In a same manner, compound 18 (19 mg, 0.041 mmol) was coupled to N-Cbz-Leu (23 mg, 0.086 mmol) to give compound 20 (23 mg, 58%) as a white solid.

Preferred synthesis of compound 21 as illustrated in Figure 6.

Compound 18 (22 mg, 0.047 mmol) was coupled to N-Cbz-Phe (30 mg, 0.098 mmol) to give compound 21 (30 mg, 63%) as a white solid: 1H NMR (500 MHz, DMSO-d6,80 OC) 8 0.76-0.92 (12H, m), 1.15-1.20 (1H, m), 1.29 (3K, s), 1.45-1.60 (2H, m), 1.98-2.10 (1H, m), 2.78-2.85 (1H, m) 3.51 (1H, s), 4.08-4.15 (1H, br), 4.25-4.35 (2H, m), 4.95 (2H s), 7.12-7.35 (12H, m), 7.56 (1H, d, J = 8.5 Hz); HRMS (FAB+), calcd for MCs+ C59H80N6010Cs m/e 1165.4990, found m/e 1165.4936.

Preferred synthesis of compound 10 as illustrated in Figure 6.

Compound 21 (20 mg, 0.019) was deprotected to give compound 10 (11 mg, 58%) as a white solid: 1H NMR (400 MHz, DMSO-d6,80 °C) 6 0.83-0.89 (12H, m), 1.18-1.25 (1H, m), 1.43-1.58 (2H, m), 2.02 (1H, q, J = 6.7 Hz), 2.82 (1H, dd, J = 14.1,9.8 Hz), 3.04 (1H, dd, J = 14. 2,4.4 Hz), 3.23 (1H, s), 4.10-

4.20 (2H, m), 4.32-4.38 (1H, m), 4.95 (2H, s), 6.95-7.00 (2H, m), 7.15-7.32 (1OH, m), 7.54 (1H, d, J = 8. 5 Hz); 13C NMR (100 MHz, DMSO-d6,25'C) d- 17.3,18.7,21.5,22.6,23.6,29.7,37.0,41.5,47.3,55.7,57.7,64.9, 72.5,125.5, 126.7,127.0,127.3,127.6,128.5,137.4,139.1,156.0,169.6,170.6; HRMS (FAB+), calcd for MCs+ C56H76N6010Cs m/e 1125.4677, found m/e 1125.4709.

Preferred synthesis of compound 22 as illustrated in Figure 6.

Compound 18 (22 mg, 0.047 mmol) was coupled to N-Cbz-Val (25 mg, 0.098 mmol) to give compound 22 (31 mg, 70%) as a white solid: 1 H NMR (500 MHz, DMSO-d6, 80 °C) # 0.75-0.88 (18H, m), 1.12-1.19 (1H, m), 1.28 (3H, s), 1.45-1.60 (2H, m), 2.00 (2H, br s), 3.50 (1H, s), 3.92-3.99 (1 H, m), 4.03-4.12 (1H, br s), 4.25-4.32 (1H, m), 4.98-5.08 (2H m), 6.89-6.95 (1H, br), 7.25-7.38 (6H, m), 7.48 (1H, d, J = 8.9 Hz); HRMS (FAB+), calcd for MCs+ C51 H80N6010Cs m/e 1069.4990, found m/e 1069.4943.

Compound 22 (24 mg, 0.025) was deprotected to give compound 11 (11 mg, 50%) as a white solid: 1H NMR (400 MHz, DMSO-d6,80 °C) d 0.80-0.88 (18H, m), 1.13-1.22 (1H, m), 1.41-1.56 (2H, m), 1.95-2.04 (2H, m), 3.20 (1H, s), 3.94 (1H, dd, J = 8. 9,6.6 Hz), 4.11 (1 H, se, J= 4. 4 Hz), 4.17 (1H, dd, J = 8. 8,6.6 Hz), 5.04 (2H, s), 6.74 (1H, d, J = 8.2 Hz), 6.94 (1H, d, J = 9.3 Hz), 7.25-7.35 (5H, m), 7.43 (1H, d, J = 8.8 Hz); 13C NMR (100 MHz, DMSO-d6,80 OC) 8 17.3,18.5,18.7,21.5,22.6,23.6,29.6,29.7,41.5,47.3,57.6,60.0, 65.0,72.5, 126.9,127.0,127.6,137.4,156.0,169.7,170.3; HRMS (FAB+), calcd for MCs+ C48H76N6010Cs m/e 1029.4677, found m/e 1029.4701.

Preferred synthesis of compound 12 as illustrated in Figure 6.

(IS, 2R, 3R, 4S)-1,4-bis [(N-Cbz) amino]-1, 4-dibenzyl-2,3-diol derivative 23 was prepared by applying the procedure described (Lee et al. Proc. Natl.

Acad. Sci. USA 1998,95,939-944).

Compound 23 (40 mg, 0.074 mmol) was coupled to N-Cbz-Ser (38 mg, 0.16 mmol) to give the adduct (60 mg, 83%) as a white solid: (500 MHz, DMSO-d6,80 °C) 8 0.66 (3H, d, J = 6.5), 0.72 (3H, d, J = 6.0), 1.31 (3H, s), 1.90 (1H, d, J = 6.2 Hz), 2.65-2.80 (2H, m), 3.13-3.17 (1H, m), 3.58 (2H, s),

4.07-4.16 (2H, m), 4.23-4.31 (1H, m), 4.60-4.70 (1H, m), 5.05 (2H, s), 6.90-6.99 (1H, br), 7.07-7.19 (6H, m), 7.25-7.35 (5K, m) 7.45-7.50 (1H, br); HRMS (FAB+), calcd for MCs+ C53H68N6012Cs m/e 1113.3990, found m/e 1113.3897. The adduct (30 mg, 0.031 mmol) was then deprotected as above to give compound 12 (15 mg, 51 %) as a white solid: 1 H NMR (400 MHz, DMSO- d6,80 OC) 6 0.70 (3H, d, J = 6.5 Hz), 0.72 (3H, d, J = 7.0 Hz), 1.91 (1H, se, J = 6.5), 2.68-2.82 (2H, m), 3.33 (1H, s), 3.60-3.65 (2H, m), 4.07 (1H, dd, J = 9.0,6.5 Hz), 4.16 (1H, q, J = 8.0), 4.29 (1H, s), 4.37-4.43 (1H, m), 4.63-4.70 (1H, m), 5.07 (2H, s), 6.82-6.92 (1H, br), 7.05-7.40 (12H, m); 13C NMR (100 MHz, DMSO-d6,80 °C) 6 17.6,19.3,30.3,38.5,50.5,57.0,57.8,61.8,65.5,73.2, 125.7,127.8,128.4,129.1,136.9,139.0,155.9,169.9,170.2; HRMS (FAB+), calcd for MCs+ C50H64N6012Cs m/e 1073.3637, found m/e 1073.3685.

Preferred synthesis of compound 13 as described in Figure 6.

Compound 23 (33 mg, 0.061 mmol) was coupled to N-Cbz-Thr (33 mg, 0.13 mmol) to give the adduct (52 mg, 85%) as a white solid: (500 MHz, DMSO-d6, 80 °C) # 0.69 (3H, d, J = 7.0 Hz), 0.74 (3H, d, J = 6.5 Hz), 1.03 (3H, d, J = 6.5 Hz), 1.32 (3H, s), 1.93 (1H, se, J = 6. 0 Hz), 2.68-2.80 (2H, m), 3.60 (1H, s), 3.90-3.98 (1H, m), 4.02 (1H, dd, J= 8. 5,5.0), 4.20 (1H, dd, J = 8. 5,6.0 Hz), 4.27-4.32 (1H, m), 4.56 (1H, d, J = 5. 5 Hz), 5.06 (2H, s), 6.65-6.75 (1H, br), 7.10-7.19 (6H, m), 7.28-7.35 (5H, m), 7.44 (1H, d, J = 9. 0 Hz); HRMS (FAB+), calcd for MCs+C55H72N6012Cs m/e 1141.4263, found m/e 1141.4316. The adduct (35 mg, 0.037 mmol) was then deprotected to give compound 13 (20 mg, 56%) as a white solid: 1H NMR (500 MHz, DMSO-d6,80 OC) 6 0.69 (3H, d, J = 6.5 Hz), 0.72 (3H, d, J = 7.0 Hz), 1.04 (3H, d, J = 6.5 Hz), 1.87-1.93 (1H, m), 2.68-2.80 (2H, m), 3.33 (1H, s), 3.90-4.15 (3H, m), 4.22-4.38 (2H, m), 5.06 (2H, s), 6.60-6.70 (IH, br), 7.05-7.40 (11 H, m), 7.50 (1H, d, J = 8. 0 Hz); 13C NMR (100 MHz, DMSO-d6, 20 °C) # 17.6,19.3,19.7,30.6,38.5,50.6,57.5, 60.3,65.5,66.8,73.1,125.5,127.7,127.8,128.0,128.4,129.0,136. 9,138.9, 156.0,169.7,170.2; HRMS (FAB+), calcd for MCs+ C52H68N6012Cs m/e 1101. 3950, found m/e 1101.3900.

Synthesis of compound 14 and compound 23 as shown in Figure 6 The key intermediates (lS, 2R, 3R, 4S)-1, 4-bis [(N-Cbz) amino]-1, 4- dibenzyl-2,3-diol (used for preparation of compound 23) and (IS, 2R, 3R, 4S)-1,4- bis [(N-Cbz) amino]-1, 4-diisobutyl-2,3-diol 14 were prepared by the stereoselective Pinacol coupling of L-Cbz-phenylalanal (for 23) or L-Cbz- leucinal (for 14) (Each from Aldrich or Sigma), respectively. Using conditions exactly as described in Konradi, A. W.; Pedersen, S. F. J. Org. Chem. 1992,57, 28-32. Compound 14 was carried onto the next step.

Compound 23 was formed by reacting the formed diol intermediate with . 10 equiv. p-TsOH in 1.0 Molar 2,2-dimethoxypropane at 25 °C for 1 hour until complete, followed by standard workup and purification conditions.

Synthesis of compounds 8,9,10 and 11 as shown in Figure 6.

Compound 14 was reacted with 1.0 Molar 2,2-dimethoxypropane with 0.10 equiv. p-TsOH (80%) at 25 °C for 1 hour until complete, followed by standard workup and purification conditions as described herein to form 15; (b) Intermediate 15 from step a was suspended in 0.10 M MeOH at 25 °C and catalytic amount of 10% Pd/C was added under H2 balloon and stirred for 1 hour or until complete, followed by standard workup and purification conditions.

(99%) to form 16; (c) General Procedure for Coupling Reaction using HBTU, Cbz-Val, Et3N, CH3CN (898), followed by standard workup and purification conditions to form 17. Intermediate 17 was suspended in 0.10 M MeOH at 25 °C and catalytic amount of 10% Pd/C was added under H2 balloon and stirred for 1 hour or until complete, followed by standard workup and purification conditions. (99%) to form 18 (d) General Procedure for Coupling Reaction as described above using Intermediate 18, HBTU, Cbz-amino acids (Aldrich, Sigma, Nova Biochem, or Bachem), Et3N, CH3CN, followed by standard workup and purification conditions to form intermediate (depending on amino acid used to form 19-22); (e) Intermediate (either 19-22) was finally suspended in a catalytic amount of p-TsOH (0.10 equiv.) in 0.10 M MeOH at 25 °C for 1 hour until complete, followed by standard workup and purification conditions to separately form 8,9,10, or 11.

Synthesis of compounds 12 or 13 as shown in Figure 6 General Procedure for Coupling Reaction as described above using Intermediate 23, HBTU, Cbz-amino acids (Aldrich, Sigma, Nova Biochem, or Bachem), Et3N, CH3CN, followed by standard workup and purification conditions to form intermediate (depending on amino acid used to form intermediate); (e) Intermediate was finally suspended in a catalytic amount of p- TsOH (0.10 equiv.) in 0.10 M MeOH at 25 °C for 1 hour until complete, followed by standard workup and purification conditions to separately form 12 or 13.

Synthesis of compound 25 as shown in Figure 7: Compound 25 was formed from the multi-step process as shown in Figure 7 (with all intermediates purified by silica gel chromatography). Step (a) Free acid 1.0 equiv. (Aldrich), NMM 1.1 equiv. (Aldrich), 0.10 M THF, 1.1 equiv. i-BuOCOCI were mixed together for 1 hour at 0 °C until complete by TLC monitoring. The reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography.

Step (b) 1.0 equiv. of intermediate compound from step a was suspended in solution with 1.1 equiv. CH2N2 (diazomethane formed by standard methods-see March, Carey and Sundburg, Caruthers or any advanced organic text for standard procedure of generating diazomethane), 0.10 M Et20, mixed together for 1 hour at 0 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography. Step (c) 1.0 equiv. of intermediate compound from step b was suspended in solution with 0.01 M HCI/0. I OM THF, mixed together for 1 hour at 8 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography (85%, for the 3 above steps from step a-c).

Step (d) 1.0 equiv. of intermediate compound from step c was suspended in solution with 1.1 equiv. NOSH4 (1.0 M/THF added dropwise at 0 °C),. 10 M EtOH, (90% d. e, 81 %) mixed together for 1 hour at 8 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described

above and carried on to the next step after column chromatography. Step (e) 1.0 equiv. of intermediate compound from step d was suspended in solution with 10 equiv. NaOMe, 1.0 M MeOH, 25 °C for 1 hour until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography (96%) to form compound 25.

Synthesis of compound 2b as shown in Figure 7: Compound 25 (18mg, 0.039 mmol in 0.10 M MESH, 1. 1 equiv. Et3N was added 1.1 equiv. 24 (Slee et al. ibid; Aldrich/Sigma) to 25; stir 0 °C until complete by TLC, standard workup/chromatography (90W)) and prepared exactly as conditions described in Slee et al. J. Am. Chem. Soc. 1995,117, 11867-11878). The intermediate was then hydrogenated with 10% Pd/C (15mg) in EtOAc (2ml) to give an amine 2a as a colorless oil (standard reduction conditions as described above) filtration of Pd/C from solution then condensation of EtOAc and carry onto next step. The General Procedure for Coupling Reaction as described above, was next used with the crude amine 2a, Cbz-Ala-Val-OH (12mg, 0.039 mmol; Aldrich/Sigma, Nova Biochem, or Bachem), HBTU (14mg, 0.038 mmol) and Et3N (4.7mg, 0.046 mmol) in CH3CN (I ml) and gave compound 2b (12mg, 49%) as a white solid: 1H NMR (500 MHz, DMSOd6,60 °C) 6 0.76 (6H, d, J = 6. 7 Hz), 1.20 (3H, d, J = 7.1 Hz), 1.26 (9H, s), 1.64-1.70 (3H, br), 1.86-1.90 (1H, m), 1.98-2.00 (1H, m), 2.67 (1H, m), 2.69 (1H, m), 2.93 (1H, b), 2.96 (1H, b), 3.03 (3H, m), 3.50 (1H, b), 4.00-4.13 (3H, m), 5.04 (2H, s), 7.10-7.50 (14H, m); 13C NMR (100 MHz, DMSO-d6,60 OC) d-18. 0,19.2,28.3,30.0,30.8,34.7,38.2,49.5,50.1,52.8, 55.5,57.7,59.4,65.3,67.8,71.0,125.7,127.6,127.7,127.8,128.3, 129.2,137.0, 138. 8,155.6,170.2,170.3,172.0; HRMS (FAB+), calcd for MCs+ C35H51N506Cs m/e 770.2894, found m/e 770.2922.

Synthesis of aldehyde as shown in Figure 8: To 1.0 equivalents of acid (Aldrich/Sigma) is added 1.1 equiv. of a 1.0 Molar solution of BH3 (THF solution) in 0.10 Molar THF total reaction solution mixed together for 1 hour at 0 °C until complete by TLC monitoring, the

reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography. (b) Intermediate from step a is exposed to standard Swern Oxidation conditions well known in the art using standard amounts of oxalyl chloride/triethyl amine in methylene chloride and mixed together for 1 hour at 0 C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography. (90%).

Synthesis of a-hydroxy-acid compound 26 as shown in Figure 8 The above synthesized intermediate aldehyde (1.0 equivalents) was suspended with 1.1 equiv. NaMS03 in 1.0 THF/H2O combination mixed together for 1 hour at 0 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography. (d) Above synthesized intermediate (1.0 equivalents) was suspended with 1.1 equiv KCN in 1.0 Molar acetonitrile mixed together for 1 hour at 0 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography. (e) Above synthesized intermediate (1.0 equivalents) was next suspended with 1.1 equiv HCl in 6N in dioxane mixed together for 1 hour at 0 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography. (f) Finally synthesized intermediate (1.0 equivalents) was suspended with Cbz-Cl (Aldrich), 1.0 Molar solution of 1.1 equiv. NaOH in 0.01 Molar THF/H20 mixed together for 1 hour at 0 °C until complete by TLC monitoring, the reaction was next quenched via standard workup as described above and carried on to the next step after column chromatography (52%, 4 steps).

Synthesis of compound 3a as shown in Figure 8: (g) HBTU, Et3N, CH3CN (73%); pyrolidine amine from Aldrich/Sigma and coupling was achieved using exactly the protocol described herein for general peptide coupling conditions.

Synthesis of compound 3b as shown in Figure 8: A mixture of diastereomer 3a (85mg, 0.170 mmol) was hydrogenated with 10% Pd/C (20mg) in EtOAc (3ml) to give an amine as a colorless oil. The crude amine, Cbz-Ala-Val-OH (55mg, 0.170 mmol), HBTU (65mg, 0.170 mmol) and Et3N (20mg, 0.20 mmol) in CH3CN (4ml) gave separable mixture of compounds 3b and 4b (72mg, 65%) as a white solid; Compound 3b: 1H NMR (400 MHz, CD30D, 20 C) d 0.82 (separable mixture of compounds 3b and 4b (72mg, 65%) as a white solid; Compound 4b: I H NMR (400 MHz, DMSO-d6, 60 C) 6-0. 74-0.77 (6H, m), 1.21-1.23 (3H, overlapping), 1.23 (9H, s), 1.81-1.91 (so, b), 2.70 (1H, m), 2.71 (1H, m), 2.78 (1H, b), 2.89 (1H, b), 3.00 (1H, b), 3.36-3.41 (4H, m), 5.04 (2H, s), 7.16-7.44 (14H, m); 13C NMR (100 MHz, DMSO-d6,60 °C) d-17.1,17.6,18.7,23.3,28.1,30.1,36.8,37.8,45.7,49.6, 50.0,57.3,60.1,65.1,69.4,125.6,127.1,127.2,127.6,127.8,128.6 ,136.6, 138.2,155.1,169.9,171.6; HRMS (FAB+), calcd for MCs+ C35H51N506Cs m/e 770.2894, found m/e 770. 2947.

Synthesis of compound 5a as illustrated in Figure 8: Dess-Martin reagent (22mg, 0.074 mmol) was added to a solution of diastereomer 3b (16mg, 0.025 mmol; as described above and in Slee et al., ibid) in methylene chloride (2ml). The reaction mixture was stirred for 12 hrs then quenched by addition of brine and extracted with EtOAc. The organic layer was then washed with sat. aq. NaHC03, and brine, dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by flash chromatography to give compound 5a (lOmg, 63%) as a mixture of isomers (1: 1).

Synthesis of compound 5b as illustrated in Figure 8: Dess-Martin reagent (22mg, 0.074 mmol; procedure described above) was added to a solution of diasteromers 3b and 4b (16mg, 0.025 mmol) in CH2CI2 (2ml). The reaction mixture was stirred for 12 hrs then quenched by addition of brine and extracted with EtOAc. The organic layer was then washed with sat. aq. NaHC03, and brine, dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by flash chromatography to give compound 5b (lOmg, 63%) as a mixture of isomers (1: 1): 1H NMR (500 MHz,

DMSO-d4,60 °C) d 0.76-0.85 (6H, m), 1.16,1.18 (3H, d, J= 7.0 Hz), 1.24,1.26 (9H, s), 1.70-1.80 (3H, m), 1.82-2.08 (3H, m), 2.15-2.22 (1H, m), 2.60 (1H, dd, J= 10. 5,14.6 Hz), 2.96 (IH, dd, J= 9. 1,14.2 Hz), 3.17 (1 H, dd, J= 4. 9,14.2), 3.24-3.26 (1H, m), 3.38-3.65 (5H, m), 4.07-4.12 (3H, m), 4.19 (1H, t, J= 6.6 Hz), 4.24 (2H, t, J= 5.7 Hz), 4.62 (1H, q, J= 4.3 Hz), 4.95-4.99 (1H, m), 5.01 (2H, s), 5.26 (1H, m), 7.15-7.45 (12H, m); 13C NMR (100 MHz, DMSO-d4, 60 oC) d 17.6,18.0,19.1,21.7,24.4,28.4,29.1,31.0,32.4,33.7,34.2,35.6, 47.4,50.0,50.2,55.9,56.7,56.8,57.1,57.5,59.8,60.4,65.3,126.2 ,126.5, 127.7,127.7,128.1,128.2,128.3,128.4,128.6,128.9,137.1,137.2, 138.0, 140.5,155.6,162.0,162.2,169.8,170.7,171.0,171.4,172.1,172.2, 196.0, 198.1; HRMS (FAB+), calcd for MCs+ C35H47N507Cs m/e 782.2530, found m/e 782.2558.

Synthesis of compound 6b as described in Figure 9: The amine 6a (20mg, 0.047 mmol ; prepared according to the figure and exactly as described in Kempf et al. J. Med. Chem. 1998,41,602-617), Cbz-Ala-Val-OH (15mg, 0.047 mmol), HBTU (18mg, 0.047 mmol) and Et3N (5.7mg, 0.056 mmol) in CH3CN (I ml) gave the title compound 6b (25mg, 73%) as a white solid (see general coupling protocol for exact procedure as described herein): 1H NMR (400 MHz, DMSO-d6,80 OC) 6 0.75-0.78 (6H, m), 1.21 (3H, d, J= 7.1 Hz), 1.48-1.56 (2H, m), 1.85-1.94 (1H, m), 2.62-2.79 (3H, m), 3.58 (1H, m), 3.79 (1H, m), 4.05-4.08 (1H, m), 4.10-4.13 (2H, m), 5.04 (2H, d, J= 2.5 Hz), 5.14 (2H, s), 7.08-7.34 (15H, m), 7.80 (lem, s), 8.98 (1H, s); 13C NMR (100 MHz, DMSO-d6,80"C) 5 17.4,18.7,30.2,37.0,38.0,46.9,50.0, 52.9,55.5,57.0,57.5,65.1,68.6,125.3,127.1,127.2,127.4,127.4, 127.8,128.5, 128.6,128.7,133.5,136.9,138.2,138.8,142.3,154.4,155.0,169.2, 170.0,171.5 ; HRMS (FAB+), calcd for MCs+ C39H47N507SCs m/e 862.2277, found m/e 862.2251.

Synthesis of compound 7a as illustrated in Figure 9: Compound 7a: The amine of 7 (30mg, 0.075 mmol; Thompson et al. J.

Am. Chem. Soc. 1993,115,801-803) prepared from the BOC derivative 7 (Thompson et al, ibid) was coupled with Cbz-Ala-Val-OH (24mg, 0.075 mmol;

Aldrich), using standard coupling conditions (e. g. HBTU (28mg, 0.075 mmol) and Et3N (9. lmg, 0.09 mmol) in CH3CN (Iml)) gave the title compound 7a (40mg, 76%) as a white solid: 1H NMR (500 MHz, DMSO-d6,80 °C) 60.73 (3H, d, J= 6.8 Hz), 0.75 (3H, d, J= 6.8 Hz), 1.19 (3H, d, J= 7.08 Hz), 1.26 (9H, s), 1.31-1.43 (4H, b), 1.48 (2H, b), 1. 59 (1H, b), 1.63 (1H, b), 1.73 (1H, b), 1.82- 1.98 (3H, m), 2.05-2.13 (2H, m), 2.57-2.66 (3H, m), 2.93-2.98 (2H, m), 4.06- 4.20 (3H, m), 5.03 (2H, s), 7.09-7.39 (14H, m); 13C NMR (100 MHz, DMSO- d6,80 OC) 516.9,17.8,18.2,18.6,19.3,20.4,24.8,25.9,28.4,29.8,30.6,31.2 , 32.4,33.3,35.7,49.9,52.1,57.3,58.0,65. 3,69.8,125.4,127.6,127.7,127.8, 128.3,129.3,137.0,140.2,155.6,170.1,172.0,172.7; HRMS (FAB+), calcd for MCs+ C40H59N506Cs m/e 838. 3520, found m/e 838.3546.

Preferred synthesis of compound 7b as illustrated in Figure 9 The amine of 7 ( (49mg, 0.122 mmol); Thompson et al. J. Am. Chem.

Soc. 1993,115,801-803) was prepared from the BOC derivative 7, was coupled with Cbz-Ala-Asn-OH (41mg, 0.122 mmol; Aldrich or as described above), using the standard coupling conditions e. g. (HBTU (46mg, 0.122 mmol) and Et3N (12. 4mg, 0.09 mmol) in DMF (Iml)) gave the title compound 7b (42mg, 78%) as a white solid: 81.22 (3H, d, J= 7.1 Hz), 1.27 (9H, s), 1.31-1.85 (1 OH, m), 1.90 (2H, m), 2.22 (1H, b), 2.33 (1H, b), 2.49-2.58 (3H, m), 2.71 (1H, dd, J= 9.4,14.3 Hz), 2.81 (1H, m), 2.91-2.94 (1H, m), 3.18-3.25 (2H, m), 3.74 (1H, m), 4.08 (1H, q, J= 7.3 Hz), 4.52 (1H, q, J= 6.3 Hz), 5.03 (2H, s), 7.13-7.35 (1OH, m); 13C NMR (100 MHz, DMSO-d6,20"C) 818.3,20.2,21.3,25.2,26.0,28.3, 29.5,30.5,32.8,34.7,35.6,37.5,49.5,50.1,50.3,58.3,58.5,61.2, 65.5,68.0, 126.7,127.8,127.9,128.4,128.7,129.6,137.1,137.4,155.7,158.0, 158.3, 172.1,173.2; HRMS (FAB+), calcd for MH+ C39H57N607 m/e 721.4262, found m/e 721.4289.

Synthesis of compound 100 as illustrated in Figure 12.

1, 4Bis [ (N-Cbz) amino]-1, 4-dibenzyl-2,3-diol 100 was prepared using literature procedure, Konradi et al. J. Org. Chem., 57,28-32. All new compounds were homogeneous by TLC and were characterized by satisfactory lH, 13C NMR, and mass spectra.

Synthesis of Compound 200 as illustrated in Figure 12. T To a suspension of diastereomeric mixture of 1, 4-bis [ (N-Cbz) amino] 1, 4- dibenzyl-2, 3-diol 100 (1.5 g, 2.63 mmol) in 2,2dimethoxypropane (50 ml) was added catalytic amounts of p-TsOH. The reaction mixture was heated at 60"C for 5 hr and cooled to 20"C. The reaction mixture was diluted with EtOAc (200 ml), and the resulting solution was washed with sat. aq. NaHC03 and sat. aq.

NaCI, dried over MgS04, filtered and concentrated in vacuo. The residue was then purified by flash chromatography (hexanes/EtOAc 80/20) to give 2,3- protected (IS, 2R, 3R, 4S) diastereomer 200 (1.28 g, 80%) as a white solid.

Synthesis of compound 400 as illustrated in Figure 12 (General Procedure for the Coupling Reaction).

Compound 2 (1.20 g, 1.97 mmol) in EtOAc (75 ml) containing 10% Pd/C (400 mg) was stirred under H2 (I atm) at 20oC for 20 hr. The reaction mixture was filtered through Celite and then concentrated in vacuo to give diamine 300 (664 mg, 99%) as a colorless viscous oil, which was used for coupling reaction without purification.

To a solution of diamine 300 (650 mg, 1.91 mmol) and NCbz-L-Valine (961 mg, 3.82 mmol) in CH3CN (16 ml) was added HBTU (1.45 g, 3.82 mmol) followed by Et3N (425 mg, 4.2 mmol). The reaction mixture was stirred for 15 min at 20"C under Ar then quenched by addition of brine (60 ml) and extracted with EtOAc (4 x 50 ml). The organic layer was washed sequentially with I M HCl (10 ml), sat. aq. NaHC03 (10 ml), and sat. aq. NaCI (10 ml), dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by flash chromatography to give compound 400 (1.16 g, 75%) as a white solid.

Synthesis of compound 1000 as illustrated in Figure 12 (General Procedure for Deprotection).

To a solution of compound 400 (55 mg, 0.068 mmol) in MeOH (3 ml) was added catalytic amounts of p-TsOH. The reaction mixture was heated at 60 °C for 24 hr then diluted with EtOAc (20 ml). The organic solution was washed with sat. aq. NaHC03 (5 ml) and sat. aq. NaCI (5 ml), dried over

MgS04, filtered and concentrated in vacuo to give free diol 1000 (36 mg, 69%) as a white solid.

The preparations of compound 1100-1400 were carried out using the general procedures for coupling and deprotection.

Synthesis of compound 1100 as illustrated in Figure 12.

In a same manner, compound 400 (1.10 g, 1.36 mmol) was hydrogenated to give compound 500 (665 mg, 99%) as a colorless viscous oil. Compound 500 (20 mg, 0.037 mmol) was coupled to give Compound 600 (27 mg, 79%) as a white solid. Compound 600 (22 mg, 0.024) was deprotected to yield compound 1100 (13 mg, 62%) as a white solid: I H NMR (400 MHz, DMSO-d6,80"C) d 0.74 (3H, d, J = 4.9), 0.76 (3H, d, J = 4.9), 1.90 (1H, se, J = 6.4), 2.72-2.80 (2H, m), 3.37 (1 H, s), 3.683.73 (3H, m), 4.10 (1H, dd, J = 8.3,6.7), 4.33 (1H, s), 4.364.42 (1H, m), 5.09 (2H, s), 7.02-7.09 (1H, br), 7.10-7.41 (12H, m); 13C NMR. (100 MHz, DMSO-d6,80to) d 17.1,19.0,30.1,39.0,42.5,50.7,58.0, 65.7,72.5,125.5,125.7,128.2,128.5,128.8,129.4,136.6,138.8,15 5.7,168.5, 170.2; HRMS (FAB+), calcd for MCs+ C4sH60N601oCs m/e 1013. 3425, found m/e 1013.3447.

Synthesis of compound 1200 as illustrated in Figure 12.

Compound 500 (68 mg, 0.13 mmol) was converted to compound 700 (80 mg, 67%) as a white solid. Compound 700 (55 mg, 0.058) was deprotected to give compound 1200 (42 mg, 80%) as a white solid: 1H NMR (400 MHz, DMSO-d6,80"C) d 0.70 (3H, d, J = 2.4), 0.72 (3H, d, J = 2.4), 1.21 (3H, d, J = 7.0), 1.87 (1H, se, J = 6. 7), 2.69-2.79 (2H, m), 3.32 (1H, s), 4.03 (1H, dd, J = 8.8,6.4), 4.10 (1H, qu, J = 7.0), 4.27 (1H, s), 4.34-4.40 (1H, m), 5.04 (2H, s), 6.926.96 (1H, br), 7.05-7.34 (12H, m); 13C NMR (100 MHz, DMSO-d6,80 C) d 17.3,17.6,18.7,29.7,38.0,49.9,50.4,57.8,65.1,72.5,125.1,127. 0,127.2, 127.3,127.8,128.6,136.6,138.4,155.1,169.8,171.6; HRMS (FAB+), calcd for MCs+ C50M64N6010C5 m/e 1041.3738, found m/e 1041.3780.

Synthesis of compound 1300 as illustrated in Figure 12.

Compound 500 (50 mg, 0.093 mmol) was converted to compound 800 (52 mg, 54%) as a white solid. Compound 1300 (22 mg, 65%) was prepared from compound 800 (35 mg, 0.034) as a white solid: 1H NMR (400 MHz, DMSO-d6,80to) d 0.73 (6H, d, J = 6.8), 0.87 (3H, d, J = 6.5), 0.89 (3H, d, J = 6.5), 1.48 (2H, t, J = 6. 8), 1.59-1.67 (1H, m), 1.88 (1H, se, J = 6.7), 2.70-2.80 (2H, m), 3.34 (1H, s), 4.04-4.93 (2H, m), 4.23 (lem, s), 4.32-4.38 (1H, m), 5.05 (2H, s), 7.02-7. 36 (13H, m); 13C NMR (100 MHz, DMSO-d6,80 C) d 17.2, 18.7,21.1,22.3,23.8,29.8,37.9,40.3,50.4,53.2,57.4,65.1,72.2, 125.1,126.9, 127.1,127.2,127.7,128.5,136.5,138.3,155.3,169.7,171.3; HRMS (FAB+), calcd for MCs+ C56H76N6010CS m/e 1125.4677, found m/e 1125.4720.

Synthesis of compound 1400 as illustrated in Figure 12.

Compound 500 (49 mg, 0.091 mmol) was converted to compound 900 (68 mg, 68%) as a white solid. Compound 900 (43 mg, 0.039) was then deprotected to give compound 1400 (30 mg, 72%) as a white solid: 1K NMR (400 MHz, DMSO-d6,80 C) d 0.73 (6H, d, J = 6.8), 1.88 (1H, se, J = 6.6), 2.70- 2.81 (4H, m), 3.36 (1H, s), 4.09 (1H, dd, J = 8.6,6.4), 4.28-4.42 (3H, m), 4.95 (2H, s), 7.04-7.08 (2H, m), 7.12-7.32 (15H, m), 7.40-7.43 (1H, m); 13C NMR (100 MHz, DMSO-d6,80'C) d 17.3,18.7,29.8,37.0,37.9,50.5,55.7,57.7, 65.0,72.3,125.1,125.6,126.8,127.1,127.3,127.5,127.7,128.5,12 8.6,137.6, 137.9,138.3,155.4,169.8,170.6; HRMS (FAB+), calcd for MCs+

C62H72N6010Cs m/e 1193.4364, found m/e 1193.4323.

Biological Assays.

Kinetic determinations for both HIV protease and FIV proteases were performed at 37°C at pH 5.25 in duplicate using an F-2000 fluorescence spectrophotometer (Hitachi). For HIV protease, the m and Vmax values for the fluorogenic peptide substrate 2-aminobenzoyl (Abz)-Thr-Ile-Nle-Phe- (p- N02)- Gln-Arg-NH2 (Toth et al Int. J. Peptide Res. 36,544-550) (SEQ ID NO : 3) were determined by measuring the initial rate of hydrolysis at different substrate concentrations (5.0,7.5,10,20,35,50,100, and 200 RM) by monitoring the change in fluorescence at an excitation wavelength of 325 nm and an emission wavelength of 420 nm, and fitting the obtained data to the Michaelis-Menten equation using the Grafit program (version 3.0, Erithacus Software Ltd., UK).

Assays were run in 0.1 M MES buffer, containing 5% (v/v) glycerol, and 5% (v/v) DMSO (200 Al final volume). The enzyme concentration (30 Rg/ml) that gave ideal progress curve was used for assays, but the dimeric active HIV protease concentration was not accurately determined. The Ki for each inhibitor of HIV protease was determined by obtaining the progress curve with the inhibitor (2.0-9.0 nM) at different substrate concentrations (7.5,10,20,35, and 50 mM), under the same reaction conditions as above. The curve fit the data was determined, and the subsequent Ki was derived using the Grafit program.

For FIV proteases, the kinetic data were determined under the similar reaction conditions as for HIV PR. The KM and Vmax for the fluorogenic substrate Arg-Ala-Leu-Thr-Lys (Abz)-Val-Gln-nPhe-Val-Gln-Ser-Lys-Gly-Arg (SEQ ID NO : 5) were determined by monitoring the change in fluorescence at an excitation filter of 325 nm and an emission filter of 410 nm with the Grafit program under the following reaction conditions: substrate concentration (6.0, 10,20,35,50,100, and 200 Rum), 0. 1 M NaH2PO4 buffer containing 0.1 M Na citrate, 0.2 M NaCI, 1.0 mM DTT, 5% (v/v) glycerol, 5% (v/v) DMSO and 7.5 , ug/ml (FIV (3X) and FIV (V59I)) or 2.5, ug/ml (FIV (Q99V)) of the enzyme. The Ki for each inhibitor of FIV proteases was also determined by obtaining the progress curve with the inhibitor (50 nM-20 RM) at different substrate concentrations (10,20,35, and 50 zM).

Protease Constructs.

Autoproteolysis-resistant FIV Protease. FIV (3X) was constructed as described Laco et al, J. Virol. 71,5505-5511 and contains the G5I, N55T, and C84K codon mutations which block three primary autoproteolysis sites in the FIV protease. All clones were sequenced to confirm the modifications made to the FIV protease ORF. Kinetic analyses revealed no significant change in KM or kcat values between the autoproteolysis-resistant 3X protease and wild type FIV protease.

Mutant FIV Proteases Mutant FIV Proteases were prepared that contained substitutions of HIV residues noted to be associated with drug resistance in HIV at equivalent sites in the three dimensional structure.

FIV (Q99V). The feline immunodeficiency virus 34TF 10 infectious molecular clone (FIV-34TF10) was used as the template in a polymerase chain reaction (PCR) using a negative strand primer (5'-ATCTCTCCCCAATAA TGGTACTATTAATGAGTTATCTTCTAAGAC-3' ; complementary to nucleotides 2252-2297) (SEQ ID NO : 6) which mutated the FIV protease Gln 99 codon to Val. The positive strand primer (5'-ACTAT TGGACATATGGCATATAATAAAGTAGGTACTACTAC-3' ; nucleotides 19642005) (SEQ ID NO : 7) which, when incorporated into the PCR product, added an initiation Met and an Ala codon to the determined 5'Tyr codon of the FIV protease open reading frame (ORF) as well as a 5'Nde I restriction site. The 300 bp PCR product was purified and used in a second PCR with the same template and with a negative strand primer (5'-ATCAGAAAGCTTTT ACATTACTAACCTGATATTAAATTT-3' ; complementary to nucleotides 2306-2345) (SEQ ID NO : 8). This reaction added a stop codon after the determined C-terminal Met codon of the protease ORF in addition to a 3'Hind III restriction site, to facilitate cloning. The resulting PCR product was digested with Nde I and Hind III and ligated into pT7-7, which had been digested with Nde I and Hind III, to give FIV (Q99V).

FIV (V59I). FIV-34TF 10 was the template in a PCR reaction with the positive strand primer (5'-GGAAGGCAAAATATGATTGGAATTGGAGG AGGAAAGAGAGGAACA-3' ; nucleotides 2135-2178) (SEQ ID NO : 9) which mutated the FIV protease Val codon 59 to Ile, and the second negative strand primer used for FIV (Q99V). The-200 bp PCR product was purified and used in a second PCR with the same template and the positive strand primer used for FIV (Q99V). The resulting PCR product was digested with Nde I and Hind III and ligated into pT7-7, which had been digested with Nde I and Hind III, to give FIV (V59I).

FIV Protease Expression and Purification.

All protease expression constructs were transformed into the E. coli cell line BL21 (DE3) that contains the T7 polymerase gene under control of the Lac promoter. Cultures were induced at OD600 = 0. 5 with 1 mM IPTG for 5 hrs with the protease inclusion bodies isolated and then solubilized in 8 M Urea, 10 mM Tris, pH 8.0,5 mM EDTA. The proteases were subsequently purified and renatured as described Laco et al. J. Virol. 71,5505-5511, and either stored at- 70 °C, or brought to 50% glycerol and stored at-20 °C.

HIV protease Expression and Purification.

A recombinant plasmid bearing a portion of the Pol gene of the BH 10 clone of HIV was used for amplification of sequence encoding protease. The 5' primer was constructed so as to insert an initiator methionine as part of the coding sequence for an Ndel site, eight amino acids prior to the beginning of protease. This primer also encoded a nucleotide change to mutate Gln 7 to Lys, in order to block a major site of autoproteolysis Rose et al. J. Biol Chem 268, 11939-11945 and thus increase stability of the enzyme. The 3'primer was designed to insert a stop codon immediately following residue 99 of the protease, with a Hind III site engineered 30 of the stop codon, to facilitate directional cloning. The PCR product was then cut with Ndel and Hind III and inserted into the pET 21+ vector (Novagen) for protein expression.

The recombinant plasmid was transformed into the BL21. DE3, p lys S strain of E. coli. Inclusion bodies were prepared and solubilized essentially as

described for preparation of FIV protease. The washed inclusion body pellet was then solubilized in 200 ml 20 mM Tris-HCI, pH 8,1 mM DTT, 5 mM EDTA, 8 M urea with stirring at 4 C for 1 hr. Insoluble material was removed by centrifugation at 8,000 x g for 30 min. The supernatant from this centrifugation was treated batch-wise by the addition of 20 gm DE 52 anion exchange resin and the mixture was stirred at 4°C for 1 hr. After centrifugation, protease was found in the supernatant. The resin was washed once with 50 ml resuspension buffer (above) and the wash and supernatant fractions were combined.

The supernatant/wash fraction was then passed over Resource Q anion exchange resin equilibrated in resuspension buffer, using a Pharmacia FPLC apparatus. The fraction that failed to bind to the column was concentrated using 5K cutoff UltraFree centrifugal concentrators (Millipore). The retentate was then dialyzed overnight against deionized water, which caused precipitation of protease. The pellet was recovered by centrifugation at 3,000 x g for 20 min, then resuspended in 20 mM sodium acetate, pH 5.3,1 mM DTT, 5 M GuHCI, to a concentration of 1 mg/ml (determined by Lowry assay of the pellet suspended in a known volume of water prior to final pelleting and solubilization in sodium acetate, DTT, GuHCI buffer). MALDI analysis indicated a mass of 10,792, which is within 1 mass unit of the predicted mass for the properly processed PR.

Activity was monitored using a flourogenic substrate, as detailed above.

Aliquots were stored at-70 C for subsequent use.

Ex Vivo Inhibitor analyses against FIV.

The lymphocytic cell line 104-C 1 (provided by C. Grant) was used as the target for infection. Cells were cultured in RPMI-1640 media supplemented with 10% FBS, 200 nM L-Glutamine, 1X MEM-Vitamins, 100 pM Sodium Pyruvate, 1X Non-Essential Amino Acids, 5. 5X10-sM b-ME, 50 pg/ml Gentamicin, 50 U/ml human recombinant Interleukin-2 (provided by Hoffmann-LaRoche) and 7.5 pg/ml Concanavalin-A. For inhibitor assessment, 5 x 106 cells were infected with FIV-PPR (4 x 105 RT units) in 1 mL culture for 2 hr at 37°C. No virus and virus only controls were incubated in a similar manner. The cells were then washed with Hanks-buffered saline solution (HBSS) and resuspended in 10 ml of complete medium. The compound 1200 (10 mg/mL in DMSO) was then

added to final concentrations of 0.1,0.5,1, and 5 llg/ml in duplicate. The cells were then incubated at 37 C. The culture supernatants were monitored for the presence of pelletable reverse transcriptase activity at weekly intervals, as follows. Cell-free culture supernatants (4 ml) were centrifuged at 60K for 30 min and the pellets were resuspended in 100 pl of lysis buffer containing 40 mM TrisHCI (pH 8.1), 360 mM NaCl, 20 mM DTT and 0.2 % NP40. Twenty five p 1 of lysate was added to 25 1 of a mixture containing polyrA-pdT (Pharmacia, Piscataway, NJ), MgC12, and 3H-labeled deoxythymidine 5'-triphosphate (DuPont NEN, Boston, MA), and incubated for 1 hr at 37°C. The mixture was spotted on DE81 paper, air-dried, fixed in 0.1 M sodium pyrophosphate, washed three times in 0.3 M ammonium formate, and an additional time in 95% ethanol.

The paper was then dried and counted on a scintillation counter.

Cells were split 1: 5 after the second, third, and fourth time points and fresh inhibitor was added at the appropriate concentration at each of these intervals. The data were expressed as values +/-standard deviation of the mean.

(Fig. 2A).

Ex Vivo inhibitor analyses against HIV.

WEAU-1.6, a kind gift of George Shaw, University of Alabama, a CXCR4 utilizing isolate, was used for all studies. 2x105 MT-2 cells in 1 ml were infected for 4 hr, at 37°C, using WEAU-1.6 at 25 TCID50. Before establishing cultures for inhibitor assessment infected cells were washed 3 times with complete medium (CM), which was RPMI-1640 supplemented with 10% FBS, 100 mM sodium pyruvate, 200 mM L-glutamine, and 50 mg/ml of gentamicin sulfate, to remove any unbound virus. For inhibitor assessment, cultures were established in duplicate or triplicate using 5x104 infected cells and 1 x 105 uninfected cells in a total volume of 1.5 mls in Costar 6 well-plates. Once cultures were established they were split 1: 4 every 3 days, and given fresh CM.

Compound 12 (from a 10 mg/ml stock in DMSO) was added at 1 or 5 mg/ml at initiation of the culture and after each split.

For testing of the antiviral efficacy of compound 1200 in MT-2, cells were assessed every day using a Olympus (model) inverted microscope equipped with phase contrast objectives. When MT-2 cells are infected with WEAU-1.6

they form syncytia and die with in 24 hours, events easily discernible visually. A total of 200 cells were counted in each well, and when syncytia were noted, cells were removed and tested for viability by using trypan blue. Results are expressed as the average of viable cells.

Ex Vivo inhibitor analyses against SIV.

Stocks of SIVmac251 (provided by R. Desrosiers) were prepared in 174xCEM cells (provided by the NIH AIDS Research and Reference Program) grown in 88% RPMI medium containing 20 mM HEPES and up to 12% heat- inactivated fetal calf serum. A 24 hr supernatant was collected at day 14 post infection and aliquoted and stored at-80 °C for subsequent experiments.

Cells were acutely infected with approximately 400 TCIDso units of SIVmac251 for 90 min at 37 °C. The cells were collected by centrifugation, washed twice with medium to remove free virus, then plated in 0.45 ml medium in 48-well tissue culture plates, at 105 cells per well. Compound 1200, prepared as a 10 mg/ml stock in DMSO, was then added to final concentrations of 10,1.0, 0.1, and 0.001 llg/ml final concentrations, in triplicate cultures. Triplicate control cultures received medium only. Uninfected cells were also cultured with the above concentrations of Compound 1200, with no effects noted. Wells were observed for the presence of syncytia at 72 and 96 hr post infection, and supernatants collected at 96 hr were assayed for relative amounts of p27 antigen, using a quantitative ELISA assay (Coulter).

EXAMPLE 7: Protease Inhibitors with Macrocycles A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle that structurally mimics the tri-peptide (Ala-Val-Phe) were synthesized and tested for inhibitory activity. These compounds were effective HIV/FIV protease inhibitors in the low nanomolar range of concentration. The inhibitors were also effective against some drug-resistant as well as TL3-resistant HIV proteases.

Protease inhibitors having Formula X were synthesized:

The structures of several compounds that have formula X are provided in Table 1 (reproduced below).

Table 1 Compd. No. R9 R3 R4 R5 + R6 10003 carbobenzyloxy-alanine-valine-NH OH H =0 10004 p-CH3-C6H4S02-alanine-valine-NH OH H =O 10005 acetyl-tryptophan-valine-NH OH H =O 10006 acetyl-phenylalanine-valine-NH OH H =O 10007 acetyl-tyrosine-valine-NH OH H =O 10009 Tert-butyloxycarbonyl OH H =O 10010 Tert-butyloxycarbonyl H OH =O 10050 acetyl-p-F-phenylalanine-valine-NH OH H =O Compound 10055 is provided below.

The inhibitory activities against FIV, HIV, drug-resistant mutant HIV, and TL3-resistant mutant HIV proteases were determined as described previously in Lee et al. Proc. Natl. Acad. Sci. U. S. A. 1998,95,939 and Lee et al.

J. Am. Chem. Soc. 1999,121,1145. The concentration of inhibitor needed to provide 50% inhibition of a protease (ICso) was recorded as a measure the effectiveness of each compound. The results for each type of protease (PR) are summarized in Table 2 below, where the HIV Hexa PR is a mutant HIV protease having six mutations: L24I, M46I, F53L, L63P, V77I and V82A.

Table 2 Inhibitor FIV PR HIV PR HIV HIV HIV HIV G48V V82F V82A Hexa PR PR PR PR TL3 72 4 21 (Sx) 15 (4x) 16 (4x) 144 (36x) 10003 95 10 Nd Nd nd Nd 10004 147 13 Nd Nd nd Nd 10005 49 10 71 (7x) 3Q' (3x) 10 (lx) 49 (5x) 10006 35 7 94 (13x) 32 (4x) 12 (2x) 79 (l lx) 10007 27 6 57 (10x) 26 (4x) 11 (2x) 70 (12x) 10050 20 3 27 (9x) 20 (7x) 7 (2x) 33 (llx) 10055 88 6 34 (6x) 23 (4x) 3 (0. 5x) 23 (4x) The term"nd"means"not determined."Numbers in parentheses denote fold higher IC50 values in inhibition compared to that of the wild-type HIV protease.

Although TL3 displayed significant inhibitory activity against the HIV protease, its activity against the FIV protease and the G48V and V82F drug- resistant HIV proteases is significantly lower. As expected, TL3 has significantly lower activity against the V82A and Hexa TL3-resistant proteases.

Compounds 10005,10006,10007,10050 and 10055 were potent inhibitors of FIV protease with ICso values about 1-to 3-fold lower than TL3. In particular, compound 10007 and compound 10050 with ICso values of only 27 nM and 20 nM, are the most potent cyclic inhibitor of FIV proteases--more potent than any cyclic inhibitors of FIV protease reported to date.

Compounds 10005,10006,10007,10050 and 10055 also have excellent inhibitory activity when evaluated against several drug-resistant and TL3- resistant HIV proteases. All these mutant enzymes have their mutated residues affect the S3 and S3'subsites, which are the important areas associated with the development of drug resistance.

The cyclic inhibitors 10005,10006,10007,10050 and 10055 have higher potency than TL3 when tested on the TL3-resistant mutant proteases. In particular, 10005,10006,10007,10050 and 10055 showed an improved inhibitory activity against the hexa-mutant induced by TL3. While, showing somewhat less inhibitory activity toward the G48V and V82F drug resistant mutants, the results on the TL3-resistant mutant protease indicate that inhibitors with cyclic structures like those of inhibitors 10005,10006,10007,10050 and 10055 are good candidates as theraeutic agents effective against variant HIV proteases.

Compound 10009 has an ICso of 100 nM against wild type HIV and an ICso of 48 M against wild type FIV.

9 10 Compound 10010 has an ICso of 600 nM against wild type HIV and provides only 43% inhibition at 200 uM against wild type FIV protease.

The effect of the configuration of a C-OH stereogenic center present in the macrocycline protease inhibitors when Y is hydrogen is illustrated by the activities of compounds 9 and 10. Compound 10009 with R stereochemistry around this carbinol provides approximately 6-fold better inhibitory activity for HIV/FIV proteases than does the S isomer 10010.

The macrocycle was found to be important for the overall activity of the inhibitors, as the hydroxyl methyl esters 10013-10015 (abbreviated as 13-15 below) were found to be less potent than that of the cyclic inhibitor 10009 against HIV/FIV proteases.

13 X = Ac-Trp IC50 14 µM (HIV-WT) IC5X 66 µM (FIV-WT) 14 X = Ac-Phe IC50 113 tuM (HIV-WT) 24% inhibition at 200 pM (FIV-WT) IS X = Ac-Tyr) IC50 71 µM (HIV-WT) 32°h inhibition at 200 pM (FIV-WT)

16 R=NH2 IC50 212 nM (HIV-WT) IC50 560 nM (FIV-W'T) Also hexapeptide 10016 (abbreviated as 16 above), which is the acetyl analog of 10007, displayed about 20-or 35-fold higher ICso values for wild type FIV and HIV proteases, respectively as compared to 10007. Such observations may be attributed to the macrocycle that is pre-organized in a favorable conformation for binding, resulting in an entropy advantage over the acyclic compound.

It is also of note that the keto amide 10017 has an ICSO of 13 nM against wild type HIV and an IC50 of 4 uM against wild type FIV protease. These data indicate that the keto amide is about 10-fold more active than the corresponding hydroxyl amide 9 against HIV/FIV proteases.

10017 The best monocyclic protease inhibitor is compound 10060, shown below.

Compound 10060 has an ICso for wild type HIV protease of 3 nM and an ICso for wild type FIV protease of 20 nM.

Other compounds of the invention include compounds 10061-10065.

Compound 10061 is provided below.

Compound 10061 has an IC50 for wild type HIV protease of 10 nM and an ICso for wild type FIV protease of 49 nM.

Compound 10062 is provided below. Compound 10062 has an IC50 for wild type HIV protease of 7 nM and an IC50 for wild type FIV protease of 35 nM.

Compound 10063 is provided below.

Compound 10063 has an ICso for wild type HIV protease of 18 nM and an IC50 for wild type FIV protease of 51 nM.

Compound 10064 is provided below.

Compound 10064 has an ICso for wild type HIV protease of 17 nM and an ICso for wild type FIV protease of 138 nM.

Compound 10065 is provided below.

Compound 10065 has an IC5o for wild type HIV protease of 6 nM and an IC5o for wild type FIV protease of 27 nM.

Other compounds of the invention had a double ether macrocycle. One example of a compound with such an ether macrocycle is compound 10516.

Another example of an HIV protease inhibitor with a double ether macrocycle is compound 10515.

-Compound 10515 has an ICso for wild type HIV protease of 5 nM and an IC5o for wild type FIV protease of 45 nM.

An efficient synthetic route toward inhibitors 10003-10007, which can readily be modified to provide compounds 10050 and 10055 is shown in Scheme 1 (below), providing cyclic inhibitor 10005 (abbreviated as 5 in Scheme 1) by way of illustration. Ph BocHN OMe 1 11 45% overall ! ! a-d 719 ovra3 "", O t . 0 "W ow . y'". C ? H 8 12

The synthesis involves the preparation of the hydroxyl acid 10012 (abbreviated as 12 in Scheme 1) derived from 10011 (abbreviated as 11 in Scheme 1). See Yuan et al., J. Med. Chem. 1993,36,211. The macrocyle 10008 (abbreviated as 8 in Scheme 1) is incorporated after Boc removal.

Macrocyclization of 10008 was achieved efficiently by the treatment of Cs2CO3 in CH3CN (10 mM) in the presence of 1 equiv of tetrabutylammonium iodide at room temperature. Despite introduction of the flexible trimethylene ether linker, the NMR spectrum indicates four unequivalent aromatic protons in the cycle, suggesting that the 15-membered ring system is highly constrained and the aromatic ring is not able to rotate freely.

Compound 10055 can be made by the following procedure, where P (P1') and P3 (P3') residues are connected via ether linkages.

Sn 1.LiBI-14, TFIF, 2. Dess-Martin Peziodinaane tu 0 NHBac H JS H I Zn, VC130 3 2,p-TsOH kdeOs OMe » ° °>O ° BotHN NHBoc 3.separation of diastereoinen 60% overall Z BCf OBn 1. Pd/C, H2 2. eHCbz cbz ClC02EtoEt3N, NABEI4 HO °sy 47°. 4 overall Ho., o ''"' sf \of 3, OBli4 Ho 47 ?, 4 overall HO 0 o HM NH '. O p-' 'NHCtu GbzHPJ,, O-.,.. r \v0 0 1 _ TFAJCH2Ct2 2. BOP, DIPEA 42% overall 3.TFA/iII0 0 0 H OU N H ))) t U J 0 OH \

10055 One example of a synthetic scheme for macrocycle compounds such as compound 10065 is provided below. H-Val-OIvle Ph c 1. Ac-Tfp-OH OH JOME 83% overall HBTU, DIPEA 2. LiOH !-R-Boc R m Soc TFAfCHaCIa Ac-TEal-OH W R W H 1 1 13'0 4vexall 1. HBTU, DIPEA 7! % overaJl' 2. I. iOH P3-Plhydroxyiacid HM-*", y Ph v/'h Data OH

The P3-P 1 hydroxy acid is then joined with a de-protected macrocycle.

The deprotected macrocycle can be formed by reaction with tetrahydrofuran and methylene chloride as illustrated below.

Reaction between the P3-P I hydroxy acid and the de-protected macrocycle in the presence of HBTU and DIPEA yields a compound 10061 (61% yield).

10061 Compounds with such a double ether macrocycle can be synthesized by available methods or by the methods provided herein. One scheme for synthesizing such ether macrocycles is provided below.

10529 10530 (X = OH) 10531 (X = Br)

10532 The macrocycle is then joined to the core structure of the protease inhibitor.

In summary, the invention provides compounds that structurally and functionally mimic the tripeptide (Ala-Val-Phe) as protease inhibitors by utilizing a ring system with approximatelyl 5-members. Several of the inhibitors of the invention were able to inhibit HIV/FIV proteases when used in only low nanomolar concentrations.

Experimental Protocols: In general, reagents and solvents were used as purchased without further purification. Methylene chloride (CH2Cl2) and acetonitrile (CH3CN) were distilled over calcium hydride and tetrahydrofuran (THF) was freshly distilled from sodium/benzophenone ketyl under argon. Analytical TLC was performed using silica gel 60 F2s4 glass plates (Merck). Flash column chromatography was performed on silica gel 60 Geduran (35-75 urn, EM Science). NMR (tH, 3C) spectra were recorded either on a Bruker AMX-400, AMX-500 or AMX-600 MHz spectrometer. Coupling constants (J) are reported in hertz, and chemical shifts are reported in parts per million (8) relative to tetramethylsilane (TMS, 0.0 ppm) or DMSO (2.49 ppm for'H and 39.5 ppm for 13C) or CD30D (3.30 ppm for 'H and 49.0 ppm for 3C) as internal reference.

Method A: General Procedure for Peptide Bond Formation. To a solution of a carboxylic acid (1.0 mol equiv) and a free amine (1.0 mol equiv) in dry THF, CH3CN or DMF (0.2-0.5 M) was added HBTU (1. 1 mol equiv) followed by DIPEA (1. 1 mol equiv or 2.2 mol equiv if the free amine is in the form of any acid salt). The reaction mixture was stirred at room temperature for 2-4 h and then diluted with EtOAc. The organic layer was washed with 1M HCI, saturated NaHC03, brine, dried over MgS04 and filtered. The filtrate was concentrated in vacuo and the crude product purified as described in the text below.

Method B: General Procedure for Boc Removal or Hydrolysis of t-Butyl Ester. The protected peptide dissolved in 50% TFA in dry CH2C12 (-0. 3 M) or in 4 M HCI in 1,4-dioxane (-0. 5 M) was stirred at room temperature for 1-3 h.

The solvent was removed in vacuo and the remaining TFA/HC1 was removed by

repeated evaporation from toluene in vacuo to give the TFA/HC1 salt of the amine or the free acid.

Method C: General Procedure for the Hydrolysis of Methyl Ester. To the methyl ester of any peptide dissolved in a mixture of 40% MeOH in THF (-0. 5 M) was added LiOH (4.0 mol equiv) and water (0.5-2 mL). The reaction was monitored by TLC and usually took about 2-4 h to complete. Most solvents were removed in vacuo and the residue was diluted in EtOAc. The organic layer was washed with 10% citric acid, brine, dried over MgS04 and filtered. The free acid was obtained after removal of the solvent in vacuo.

Compound 10529. This was prepared from Boc-Tyr-OH (0.50 g, 1.8 mmol) and H-Val-OMe HCl (1.33 g, 2.0 mmol) in THF according to the method A. The crude product was triturated in EtOAc-Et20 (1: 3 v/v) and the solid filtered to afford 10529 (0.67 g, 95%) as a white solid : 1H NMR (600 MHz, MeOH-d4) 0.91 (3H, d, J= 6.6 Hz), 0.92 (3H, d, J= 7. 0 Hz), 1.37 (9H, s), 2.05-2.15 (1H, m), 2.73 (1H, dd, J= 13.6,8.8 Hz), 2.80 (3H, s), 2.96 (1H, dd, J= 14. 0,5.9 Hz), 3.67 (3H, s), 4.27 (1H, dd, J= 8.5,5.9 Hz), 4.30 (1H, d, J= 6.3 Hz), 6.68 (2H, d, J= 8.1 Hz), 7.03 (2H, d, J= 8.5 Hz) ;'3C NMR (150 MHz, MeOH-d4) 18.4, 19.4,28.7,32.1,38.3,52.5,57.5,59.1,80.6,116.1,129.1,131.4,15 7.2,157.6, 173.2,174.6; HRMS (MALDI), calcd for MNa+ C2oH3oN206Na mle 417.1996, found mle 417. 2004.

Compound 10530. Compound 10529 (0.73 g, 1.9 mmol) was deprotected (method C) to give the corresponding free acid followed by coupling with 2- (2- aminoethoxy) ethanol (0.20 g, 1.9 mmol) in CH3CN-DMF (25 mL, 4: 1 v/v) according to the method A. After stirring at room temperature for 2.5 h and normal aqueous work up, the crude product was purified by flash chromatography on silica gel (EtOAc) to afford 10530 (0.34 g, 39%) as a white foam :'H NMR (400 MHz, MeOH-d4) 0.92 (6H, d, J= 6. 8 Hz), 1.37 (9H, s), 1.96-2.09 (1H, m), 2.73 (1H, dd, J= 13.5,8.8 Hz), 2.97 (1H, dd, J= 12.6,5.9 Hz), 3.32-3.39 (1H, m), 3.48-3.56 (4H, m), 3.62-3.69 (2H, m), 4.13 (1H, d, J= 7.3 Hz), 4.24 (1H, dd, J= 8. 8,5.9 Hz), 6.68 (2H, d, J= 8.5 Hz), 7.03 (2H, d, J= 8.2 Hz) ;'3C NMR (150 MHz, MeOH-d4) 18.5,19.6,28.6,32.2,38.1,40.3,57.7, 60.0,62.2,70.4,73.4,80.7,116.2,129.2,131.3,157.2,173.3,174.4 ; HRMS (MALDI), calcd for MNa+ C23H37N307Na m/e 490. 2524, found mle 490.2508.

Cyclic precursor 10531. To a solution of 10530 (1.0 g, 2.2 mmol) and triphenylphosphine (0.64 g, 2.4 mmol) in CH2Cl2 (20 mL) was added carbon tetrabromide (0.80 g, 2.4 mmol). The reaction mixture was stirred at room temperature for 2 h and the solvent removed in vacuo. Purification by flash chromatography on silica gel (CHC13/EtOAc = 1 : 1) afforded 10531 (0.70 g, 66%) as a white solid :'H NMR (500 MHz, MeOH-d4) 0.92 (3H, d, J= 6.3 Hz), 0.93 (3H, d, J= 6. 6 Hz), 1.37 (9H, s), 2.00-2.10 (1H, m), 2.73 (1H, dd, J= 14. 0, 8.8 Hz), 2.98 (1H, dd, J= 13.6,5.5 Hz), 3.29-3.32 (1H, m), 3.35-3.42 (1H, m), 3.48 (2H, t, J=5. 9 Hz), 3.54 (2H, t, J=5. 5 Hz), 3.75 (2H, t, J= 6.2 Hz), 4.13 (1H, d, J=7. 3 Hz), 4.25 (1H, dd, J=8. 8,5.9 Hz), 6.68 (2H, d, J= 8.5 Hz), 7.03 (2H, d, J= 8. 1 Hz) ; 13C NMR (150 MHz, MeOH-d4) 18.6,19.7,28.7,31.3,32.2, 38.1,40.3,57.7,60.0,70.2,72.1,80.7,116.2,129.2,131.3,157.2,1 57.7,173.3, 174.4; HRMS (MALDI), calcd for MNa+ C23H36BrN306Na mle 552.1680, found mle 552.1669.

Macrocycle 10532. Preparation was as described for 10528 and the crude product was purified by flash chromatography on silica gel (hexane/EtOAc = 1: 3) to afford 10532 (75 mg, 36%) as a white solid : 1H NMR (500 MHz, MeOH- d4) 0.80 (3H, d, J= 6. 6 Hz), 0.82 (3H, d, J= 6.6 Hz), 1.44 (9H, s), 2.00-2.13 (1H, m), 2.77 (1H, t, J = 12. 5 Hz), 2.89 (1H, dd, J= 12.9,4.8 Hz), 2.59-3.06 (1H, m), 3.65-3.81 (2H, m), 4.00-4.08 (1H, m), 418-4. 29 (2H, m), 4.30-4.39 (1H, m), 6.86 (1H, d, J= 7.4 Hz), 6.90 (2H, d, J= 8.8 Hz), 7.00 (1H, br s), 7.42 (1H, d, J=8. 1 Hz) ; 13C NMR (150 MHz, MeOH-d4) 17.9,19.4,28.7,32.9,38.4, 41.1,58.2,59.5,68.4,71.2,73.3,80.5,117.0,129.7,131.0,157.4,1 59.6,172.7, 174.2; HRMS (MALDI), calcd for MNa+ C23H35N3O6Na m/e 472.2418, found mle 472. 2418.

EXAMPLE 8: FIV and HIV Protease Inhibitors Containing Allophenylnorstatine The interaction of PI and P3 side chains with the S 1 and S3 hydrophobic subsites of HIV and FIV proteases was explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S, 3S)-3-amino-2-hydroxy-4- phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5, 5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2'

residues, and a variety of amino acids at the P3 and P3'positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro.

Within this series, compound 10031 (VLE776) was the most effective inhibitor against FIV protease.

This compound contains phenylalanine at P3, but no P3'residue. Compound VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and against several TL3-resistant HIV mutants.

Structure-Activity Relationship To improve the activity of TL3, the P4 and P4'residues associated with the inhibitors of the invention were structurally modified. A C2-symmetrical diol inhibitors having Formula XIII and a variety of R groups were tested in this study. Formula XIII is provided below.

wherein Ph is phenyl, and Rg is as provided herein. Exemplary compounds were tested that had Formula XIIIa below.

The IC50 values of several compounds of formula XIIIa, having a variety of P4 and P4'residues (R groups), are shown in Table 3.

Table 3a Structure and Activity of TL3 Derivatives Against FIV, HIV and Drug-Resistant HIV Proteases Inhibito Rg FIV HIV HIV HIV r PRb PRc (G48V)c (V82F)c ICso IC50 ICso (nM) ICso (nM) (nM) (nM) TL3 CBz 100 4 21 (x5) 15 (x4) 10101 CBz-Ser 128 17 202 (x6) 58 (x3) 10102 4-MePhSO2 17 5 28 (x6) 6 (xl) 10103 PhS02 138 46 10104 4-BrPhSO2 141 70 10105 4-02NphS02 228 79 10106 4-MeOPhSO2 310 20 10107 PhCH2S02 118 23 10108 PhCO 156 17 10109 3-Pyr-CO 62 11 53 (x5) 43 (x4) 10110 4-119,000 1800 F3COPhNHCO 10111 4-F3CPhNHCO 55% 2000 (200 PM) 10112 4-MePhNHCO 37% 160 (200 PM) 10113 4-61% 14 MeOPhNHCO (200 µM)

a IC50 values were determined in duplicate. bData obtained at pH 5.25 at 37°C in 0.1 M NaH2PO4, 0. 1 M sodium citrate, 0.2 M NaCl, 0.1 mM dithiothreitol, 5% glycerol and 5% dimethylsulfoxide in volume.

'Data obtained at pH 5.25 at 37°C in 0.1 M MES, 5% glycerol and 5% dimethylsulfoxide in volume.

All the C2-symmetric diols tested in this study showed competitive inhibition of both the feline and human viral proteases, but with higher potency against HIV proteases by at least an order of magnitude. The TL3 inhibitor used as a standard for comparison, with R as CBz, had an ICso value of 100 nM against the FIV protease and an ICSO value of 4 nM against the HIV protease.

Compounds with R9 as 4-MePhSO2 (10102) and as 3-Pyr-CO (10109) displayed the highest potency against FIV protease. Against the FIV protease the compound, with 4-MePhSO2 had an ICso value of 17 nM and the 3-Pyr-CO compound had an ICSO value of 62 nM, indicating that these compounds are significantly better inhibitors than TL3. These data indicate that compounds with R9 as 4-MePhSO2 and as 3-Pyr-CO will be less prone to drug-resistance than the TL3 compound. Compounds with Rg as 4-MePhSO2 and as 3-Pyr-CO were tested against drug-resistant mutant HIV proteases, G48V and V82F, and were found to be quite effective. In particular, the compound containing R9 as 4- MePhS02 had a similar ICso value against wild type HIV protease (5 nM) as against the drug resistant HIV mutant V82F protease (6 nM).

In order to ascertain the optimal length of the C2-symmetrical inhibitors derived from TL3, the inhibitory activities of asymmetrically substituted derivatives 10114 and 10115 (abbreviated as 114 and 115 below) were compared to TL3.

115

The chain length significantly affected the inhibitory activity. Removal of the P3'residue from TL3 reduces the potency against HIV and FIV proteases by 4-fold and 22-fold respectively. Compound 10115, with no P3'and P2' residues, exhibited marginal activity with IC50 value of 200 M against FIV protease and 100 nM against HIV protease. Such reduction in potency in 10114 and 10115 may be attributed to Cbz group not being properly bound within S2 and S3 pockets.

In an effort to increase the antiviral activity and pharmacokinetic profiles of TL3 derivatives, the allophenylnorstatine [(2S, 3S)-3-amino-2hydroxy-4- phenylbutyric acid] was used as a transition-state isostere. See Mimoto et al., J.

Med. Chem. 1999,42,1789; Kiso, Y. J. Synth. Org. Chem., Jpn. 1998,56,896; Tam et al., J. Med. Chem. 1992,35,1318. Using hydroxymethyl-carboxamide as a transition-state mimic has produced the potent HIV protease inhibitor JE- 2147 (2) now in clinical trials. See Yoshimura et al., Proc. Natl. Acad. Sci. USA 1999,96,8675.

Compounds 10021-24 fall with Formula XI and have a thiazolidine ring as a bio-isostere of proline at the P 1'position.

wherein: Ph is phenyl; R3 is hydrogen, oxygen or hydroxyl ; R4 is hydrogen, oxygen or hydroxyl, wherein R3 and R4 are not both hydroxyl and wherein, when R3 and R4 are both oxygen, they form a single combined oxygen that is a carbonyl group; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when R5 and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; R9 is as defined herein; R, t is hydrogen or lower alkyl ; and R, 2 is hydrogen or lower alkyl.

Compound 10021 has the following structure.

Compound 10022 has the following structure.

Compound 10023 has the following structure.

Compound 10024 has the following structure.

Compounds 10021-10024 were tested against both the wild type FIV and HIV proteases and the IC50 values in nM are provided below in Table 5.

Table 5 Protease TL3 10021 10022 10023 10024 FIV 100 95 200 5200 19, 200 HIV 4 20 18 15 475

Compound 10021 with the same residues as TL3, except for the PI' residue, exhibited similar FIV protease inhibitory activity (IC50 = 95 nM) to TL3 (IC50 = 100 nM). However, the IC50 value for compound 21 when tested with the HIV protease (20 nM) is significantly lower than with the FIV protease (95 nM).

Removal of the P3'residue from compound 10021 to form compound 10022, resulted in a 2-fold decrease in FIV protease inhibitory activity.

However, the inhibitory activities of compounds 10021 and 10022 against HIV protease were almost the same (IC50 =20 and 18 nM, respectively).

Deletion of the 1-oxo group in 10022 led to compound 10023, which showed a significant less inhibitory activity against FIV protease (IC50 = 5. 2 ? M), but still retained its binding affinity for HIV protease. The configuration of the hydroxyl, which forms hydrogen bonds with the catalytic aspartic acids, plays an important role in binding. Compound 10024 with the (2R)-alcohol displays a 96-and 26-fold reduction in potency against FIV and HIV PRs respectively as compared to compound 10022 with the (2S)-alcohol. These results are consistent with the results reported by Mimoto et al., Chem. Pharm.

Bull. 1991,39,3088.

Compounds 10025 and 10026 (abbreviated as 25 and 26, respectively, below) have phenylalanine and tryptophan, respectively, in the P3 positions.

The results for compound 10025 are consistent with the theory that large hydrophobic P3 groups reduce the potency of inhibitors against FIV protease considerably. However, compound 10026, which contains a tryptophan as P3 residue and N-acetyl as the N-terminal group, has a reasonable binding affinity for the FIV protease (212 nM). This result is in agreement with independent results reported by the Dunn laboratory indicating that in this case the P3 residue has moved away from the P3 binding site and is oriented toward outside the

cleft. See Dunn et al., Biopolymers (Peptide Science 1999, 51, 69; Kervinen et al., Protein Science 1998,7,2314.

Compounds 27-33 fall with Formula XII and have a thiazolidine ring as a bio-isostere of proline at the P1' position.

XII wherein: Ph is phenyl; R5 is hydrogen, hydroxyl or oxygen; R6 is hydrogen, hydroxyl or oxygen, wherein, when R5 and R6 are both oxygen, they are a single combined oxygen forming a carbonyl group; and R, 1 is hydrogen or lower alkyl ; R12 is hydrogen or lower alkyl ; R14 is Cbz-Ala, Cbz-Phe, acetyl-N-Trp, CbzTrp, acetyl-Trp, acetyl-Phe, or acetyl-Ala; and R, 5 is-0-alkyl,-NH-alkyl or-Ala-Cbz.

The substituents present on compounds 10027-10033 are listed in Table 6.

Table 6 Inhibitor R14 R R12 R15 R5 and R6 10027 Cbz-Trp--CH3-CH3-O-CH3 Together they form =O 10028 Ac-Trp- -CH3 -CH3 -O-CH3 Together they form =O 10029 Ac-Trp- -H -H -O-CH3 Together they form =O 10030 Ac-Trp--CH3-CH3-NH-CH3 Together they form =O 10031 Ac-Phe--CH3-CH3-O-CH3 Together they form-0 10032 Ac-Phe--CH3-CH3-NH-CH3 Together they forum-0 10033 Ac-Ala--CH3-CH3-0-CH3 Together they form =O

Compounds 10027-10033 were tested against both the wild type FIV and HIV proteases and the ICso values in nM are provided below in Table 7.

Table 7 Protease 10027 10028 10029 10030 10031 10032 10033 FIV 1000 256 1300 80 48 117 182 HIV 25 18 12 15 8 10 8

In the allophenylnorstatine series, compound 10027, with tryptophan as a P3 residue and N Cbz as the terminal group, had an IC50 value of 1 M against FIV protease (Table 7). Interestingly, compound 10028, with an N-acetyl group, displays a 4-fold higher activity.

Removal of two methyl groups from the thiazolidine ring to form compound 10029 reduces the inhibitory potency against FIV protease by about 5-fold, as compared to compound 10028. However, the presence or absence of such methyl groups appears to have no effect on inhibition of the HIV protease (compare IC50 values of compounds 10028 and 10029).

Conversion of the methyl ester on compound 10028 into a methyl amide on compound 10030 was found to increase the inhibitory activity against FIV protease by a factor of 3.

Replacing the tryptophan group in compound 10028 with a phenylalanine group in compound 10031 (VLE776) at the P3 site resulted in a 2-and 5-fold increase in the binding affinity to HIV and FIV proteases respectively.

However, compound 10033, with an alanine group at the P3 site has only a 1.4-fold increase in IC50 against FIV protease compared to that of compound 10028. This observation suggests that the steric interaction between neighboring PI and P3 side chains is still a crucial factor for proper binding in the active site of FIV protease. A phenylalanine in the P3 binding site is not too big or too small compared to tryptophan and alanine, respectively. Therefore with phenylalanine in both PI and P3 is the appropriate combination for good binding of 31.

A chemical structure of VLE776 (compound 10031) within HIV and FIV protease binding pockets was constructed and is shown in Figure 17. This structure is partially based on the x-ray crystallography studies of LP130 complex with FIV protease and JE2147 complex with HIV protease. See Dunn et al., Biopolymers (Peptide Science 1999,51,69; Kervinen et al., Protein Science 1998,7,2314; Balden et al., Structure 1995,3,581.

The orientation of the phenylalanine side chain in HIV protease is completely different from that observed in FIV protease, due to the presence of the Phe53 side chain in the flap region of HIV protease. The phenyl ring at P3 is tightly packed between Pro81 and Phe53. In FIV protease, the phenyl ring at P3 rotates away from the flap and is in closed contact with the three residues, Q99, 198 and I57.

Compound 10031/VLE776 is a Resistance Surmountable Inhibitor Over time, the HIV protease develops a high level of resistance to TL3 by acquiring mutations in the protease-encoding gene. Mutations conferring TL3-resistance that have been observed include L24I, M46I, F53L, L63P, V77I and V82A. The ICso values of TL3 against wild type HIV protease and TL3- resistant HIV protease were 4 nM and 144 nM respectively.

In this study, compound 10031 (VLE776) was found to be active against both wild-type HIV protease (ICso = 8 nM) and the TL3-resistant protease (IC50 = 40 nM). Other FDA approved drugs were also tested against the TL3-resistant HIV protease and they showed about 12-22-fold decrease in potency as compared to the wild-type HIV protease. The results for such drugs with each type of protease are summarized in Table 8 below, where"Hexa"is a mutant HIV protease having six mutations: L24I, M46I, F53L, L63P, V77I and V82A.

"Triple"is a mutant HIV protease with mutationsV82A, F53L and M46I.

Table 8

Inhibitor WT G48V V82F V82A Triple Hexa HIV TL3 4 21 (5x) 15 (4x) 13 (3x) 51 (3x) 144 (36x) SQV 2 Nd nd 7 (4x) 20 (10x) 37 (19x) NFV 3 Nd nd 6 (2x) 19 (6x) 35 (12x) RTV 2 Nd nd 7 (4x) 25 (13x) 43 (22x) 10031 8 68 (8x) 28 (4x) 16 (2x) Nd 40 (5x) The term"nd"means"not determined."Numbers in parentheses denote fold higher ICso values compared to that of the wild-type HIV protease.

In summary, the invention provides a series of novel HIV inhibitors containing allophenylnorstatine with a hydroxymethylcarboxamide as transition- state analogs capable of secure binding to HIV and FIV proteases. While several compounds show promise, compound 10031 (VLE776) appears to be the best FIV protease inhibitor to date. Compound 10031 also showed a strong inhibition against the drug-resistant mutant proteases (G48V and V82F) and against TL3-resistant HIV proteases.

Materials and Methods Analytical TLC was performed on pre-coated plates (Merck, silica gel 60F-254). Silica gel used for flash column chromatography was Mallinckrodt Type 60 (230-400 mesh). NMR (1 H, 13C) spectra were recorded either on a Bruker AMX-400, AMX-500 or AMX-600 MHz fourier transform spectrometer.

Coupling constants (J) are reported in hertz, and chemical shifts are reported in parts per million (relative to tetramethylsilane (TMS, 0.0 ppm) or DMSO (2.49 ppm for 1H and 39.5 ppm for 13C) or CD30D (3.30 ppm for'H and 49.0 ppm for 3C) as internal reference. All new compounds were homogeneous by TLC and were characterized by satisfactory H, C NMR, and mass spectra.

Scheme 2 illustrates the procedures to synthesize the tripeptide derivatives 10016a, b, 10017a, b, 10018a, b and 10019 (abbreviated as 16a, b, 17a, b, 18a, b and 19 in Scheme 2). BOC-protected 1, 3-thiazolidine-4-carboxylic acid derivatives 10016a, b were prepared according to known procedures.

Samanen et al., J. Med. Chem. 1989,32,466; Mimoto et al., J. Med. Chem.

1999,42,1789; Kiso et al., Synth. Org. Chem., Jpn. 1998,56,896; Krantz, A. J.

Med. Chem. 1992,35,1318.

Compounds 10017a, b were prepared in good yields from the acids 10016a, b by using valine methyl ester and HBTU as a condensation reagent.

Removal of the BOC group in 10017a, b followed by coupling of the acid (Yuan et al., J. Med. Chem. 1993,36,211) gave the tripeptides 10018a, b. Hydrolysis of the ester 10018a with LiOH followed by activation of the acid with N hydroxysuccinimide gave the activated ester. This activated ester was condensed with aqueous methylamine to give the tripeptide 10019 in good yields.

Scheme 2 Reagents used in Scheme 2 include reagents a-h. Reagent mixture (a) is a combination of HCHO, py and water. (Reaction yield 85%.) Reagent mixture (b) is a mixture of BOC20, tetrahydrofuran and water, containing NaHC03 (yield 77%). Reagent mixture (c) is a mixture of Val-OMe, HBTU, dimethylfuran (DMF), and triethylamine (Et3N) (yield 90%). Reagent mixture (d) is a mixture of 4N HCI in dioxane. Reagent mixture (e) is a dilute buffer that

neutralizes the pH to pH7. Reagent (f) is a mixture of HBTU, dimethylfuran and triethylamine (yield 89%). Reagent (g) is a mixture of LiOH in 20% MeOH/THF and water (yield 100%). Reagent (h) is a mixture of NHS, HOBt, DCC, DCE then MeNH2 (yield 78%).

Scheme 3 illustrates the synthesis of compounds 10020 and 10023 (abbreviated as 20 and 23 in Scheme 3) which began with the removal of the BOC group in 10017a (abbreviated as 17 in Scheme 3) followed by condensation with an epoxide to give compound 10020. Deprotection of the BOC group in 10020 followed by peptide coupling with Cbz-AlaVal-OH gave compound 10023 in 80% yield.

20 Scheme 3 The reagents used in Scheme 3 for synthesis of compound 10020 include reagents a-e. Reagent (a) is 4N HC1 in dioxane. Reagent (b) is any convenient buffer or solution for neutralization to pH7. Reagent (c) is a mixture of methanol, triethylamine at 80 °C (yield 90%). Reagent (d) is 4NHC1 in dioxane. Reagent (e) is Cbz-Ala-Val-OH in a mixture of HBTU, dimethylfuran and triethylamine (yield 68%).

The inhibitors 10022,10027-10033 were prepared from the corresponding tripeptides 10018a, b and 10019. The reaction scheme for synthesis of compounds 10022,10027,10028,10030,10031,10032 and 10033 is provided in Scheme 4, below (where compounds 10022,10027,10028,10030, 10031,10032 and 10033 are indicated by the numbers 22,27,28,30,31,32 and 33, respectively).

18aR = OMe 19 R = NHMe Scheme 4 The reagents used in Scheme 4 for synthesis of compounds 10022,10027-10033 include reagents a-b. Reagent (a) is 4N HCl in dioxane. Reagent (b) is the appropriate dipeptide in HBTU, dimethylfuran and triethylamine (yield 70- 90%).

The reaction scheme for synthesis of compounds 10029 is provided in Scheme 5, below (where compounds 10018b and 10029 are indicated by the numbers 18b and 29, respectively). lSb Scheme 5 The reagents used in Scheme 5 for synthesis of compound 10029 include reagents a-b. Reagent (a) is 4N HCl in dioxane. Reagent (b) is acetyl-Trp-Val- OH, in HBTU, dimethylfuran and triethylamine (yield 82%).

Compound 10001. To a solution of compound TL3 (1 g, l. lOmmol) in 2,2- dimethoxypropane (20 ml) was added catalytic amounts ofp-TsOH. The reaction mixture was heated at room temperature for 24 hr then diluted with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq.

NaCl, dried over MgS04, filtered and concentrated in vacuo to give acetonide as a white solid. The above acetonide in MeOH (30 ml) containing 10% Pd/C (170 mg) was stirred under H2 (latm) at 20°C for 6 hr. The reaction mixture was filtered through Celite and then concentrated in vacuo to give diamine in nearly quantitative yields. To the above diamine (20mg, 0.029mmol) in DMF (2ml) were added Cbz-Ser-OH (14mg, 0.058mmol) and HBTU (22.4mg, 0.058mmol).

The reaction mixture was stirred at room temperature for overnight then diluted

with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq.

NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was dissolved in MeOH (2ml) andpTsOH (4mg) was added. The reaction mixture was stirred for 24 h and then water (2ml) was added. Compound 10001 which precipitated out was collected and dried (78%): 1 H NMR (600 MHz, DMSO-d6,) 8.08 (d, 1H, J= 7.4), 7.62 (d, 1H, J= 9.0), 7.29-7.37 (m, 7H), 7.14-7.16 (m, 3H), 7.07-7.10 (m, 1H), 5.02 (d, 1H, J = 12.7), 5.00 (d, 1H, J = 12.7), 4.41-4.45 (m, 1H), 4.28 (dt, 1H, J = 14.1,7.0), 4.09 (dd, 1H, J = 13. 8,6.4), 4.03 (dd, 1H, J = 8.6,6.9), 3.58 (dd, 1H, J= 10.9,5.8), 3.52 (dd, 1H, J = 10.8,6.7), 3.22 (s, 1H), 2.74 (dd, 1H, J=13. 9,10.3), 2.59 (dd, 1H, J= 13.7,3.8), 1.75-1.81 (m, 1H), 1.15 (d, 3H, J= 7. 0), 0.69 (d, 3H, J= 6.7), 0.62 (d, 3H, J= 6.7); 13C NMR (150 MHz, DMSO-d6,) 171.8,170.3,169.9,155.9,138.9,136.9,129.1,128.3,127.8, 127.7,125.7,73.2,65.5,61.8,58.0,57.0,50.4,48.3,38.5,30.4,19. 2,18.1,18.0; HRMS (FAB+), calcd for C56H74N8O14Na m/e 1105.5222, found m/e 1105.5181 Compound 10002. To the above diamine (31mg, 0.046mmol) in dry pyridine (2ml) at 0 °C was added a solution ofp-toluenesulfonyl chloride (17. 4mg, 0.091mmol) in CH2C12 (0. 5ml). The reaction mixture was stirred at room temperature for 24 h then diluted with EtOAc. The organic solution was washed with 1 N HCI, sat. aq. NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was dissolved in MeOH (2ml) and pTsOH (4mg) was added. The reaction mixture was stirred for 24 h and then water (2ml) was added. Compound 10002 which precipitated out was collected and dried (88%): 1H NMR (500 MHz, DMSO-d6,) 7.85 (d, 1H, J= 8.5), 7.64 (d, 2H, J= 8.0), 7.58 (d, 1H, J= 8.5), 7.32 (m, 4H), 7.14 (m, 7H), 4.64 (s, 2H), 4.45 (m, 1H), 3.91 (dd, 1H, J = 9.0,6.5), 3.82 (m, 1H), 3.18 (m, 1H), 2.75 (m, 1H), 2.57 (m, 1H), 2.34 (s, 3H), 1.70 (m, 1H), 0.95 (d, 3H, J= 7.0), 0.58 (d, 3H, J= 7.0), 0.54 (d, 3H, J= 7.0); HRMS (FAB+), calcd for C4gH64N601 OS2Cs mle 1081.3180, found m/e 1081.3219 The preparation of compounds 10003-13 was carried out using the general procedures for coupling and deprotection.

Compound 10003. In a same manner, diamine was reacted with phenylsulfonyl chloride followed by deprotection to give compound 10003 (58%) as a white solid: 1H NMR (500 MHz, DMSO-d6,) 8.00 (d, 1H, J = 8. 5), 7.78 (m, 2H), 7.46-7.64 (m, 4H), 7.33 (d, 1H, J= 9.5), 7.15-7.16 (m, SH), 7.07-7.10 (m, 1H), 4.65 (s, 1H), 4.46 (m, 1H), 3.93 (dd, 1H, J= 9.0,6.5), 3.87 (m, 1H), 3.20 (s, 1H), 2.74 (dd, 1H, J = 13.5,10.0), 2.60 (dd, 1H, J = 14.0,10.0), 1.73 (m, 1H), 0.97 (d, 3H, J= 7.0); HRMS (FAB+), calcd for C46H60N60lOS2Cs mle 1053.2867, found m/e 1053.2904 Compound 10004. In a same manner, diamine was reacted with 4- bromophenylsulfonyl chloride followed by deprotection to give compound 10004 (70%) as a white solid; 1H NMR (500 MHz, DMSO-d6,) 8.09 (bs, 1H), 7.65-7.75 (m, SH), 7.32 (d, 1H, J= 9.5), 7.14-7.15 (m, 1H), 7.07-7.10 (m, 1H), 4.65 (s, 2H), 4.44 (m, 1H), 3.84-3.92 (m, 2H), 3.19 (s, 1H), 2.73 (dd, 1H, J = 14. 0,10.0), 2.56 (m, 1H), 1.65-1.70 (m, 1H), 1.00 (d, 3H, J = 7. 0), 0.58 (d, 3H, J = 7.0), 0.54 (d, 3H, J= 7. 0); HRMS (FAB+), calcd for C46H58N60ioS2Br2Cs mle 1211.1056, found mle 1211.1021 Compound 10005. In a same manner, diamine was reacted with 4- nitrophenylsulfonyl chloride followed by deprotection to give compound 10005 (74%) as a white solid: 1H NMR (500 MHz, DMSO-d6,) 8.35 (d, 2H, J= 8.5), 8.02 (d, 2H, J= 8.5), 7.70 (d, 1H, J= 8.5), 7.34 (d, 1H, J= 9.0), 7.13-7.14 (m, SH), 7.08 (m, 1H), 4.64 (s, 2H), 4.42 (m, lH), 4.00 (m, 1H), 3.89 (dd, 1H, J = 8.5,6.5), 3.18 (s, 1H), 2.72 (m, 1H), 2.54 (m, 1H), 1.65 (m, 1H), 1. 01 (d, 1H, J = . 0), 0.56 (d, 1H, J= 6.5), 0.5 (d, 1H, J= 6.5) Compound 10008. In a same manner, diamine was reacted with benzoyl chloride followed by deprotection to give compound 10008 (88%) as a white solid: 1H NMR (500 MHz, DMSO-d6,) 8.52 (d, 1H, J= 7.4), 7.88 (d, 2H, J= 7.0), 7.40-7.61 (m, 6H), 7.06-7.18 (m, 6H), 4.50-4.55 (m, 1H), 4.43-4.49 (m, 1H), 4.07 (dd, 1H, J= 8.4,6.6), 3.27 (s, 1H), 2.77 (dd, 1H, J = 13.6,10.3), 1.81- 1.88 (m, 1H), 1.30 (d, 3H, J = 7.4), 0.70 (d, 3H, J= 7. 0), 0.66 (d, 3H, J= 7.0); 13C NMR (125 MHz, DMSO-d6,) 171.9,170.2,166.2,138.9,134.1,131.3, 129.0,128.2,127.7,127.4,125.6,73.2,57.8,50.4,48.9,38.5,30.4, 19.3,17.8,

17.6; HRMS (FAB+), calcd for C48H6pN608Na mle 871.4370, found m/e 871.4373 Compound 10009. In a same manner, diamine was coupled with 6- methylnicotinic acid followed by deprotection to give compound 10009 (55%) as a white solid: 1H NMR (500 MHz, DMSO-d6,) 8.91 (s, 1H), 8.67 (d, 1H, J= 7.4), 8.11 (d, 1H, J = 8.0), 7.64 (d, 1H, J = 8.8), 7.42 (d, 1H, J = 9.6), 7.35 (d, 1H, J= 8.1), 7.08-7.28 (m, 7H), 4.68 (s, 1H), 4.47-4.53 (m, 2H), 4.06 (m, 1H), 3.26 (s, 1H), 2.76 (m, 1H), 2.61 (m, 1H), 2.55 (s, 3H), 1.82-1.86 (m, 1H), 1.29 (d, 3H, J= 7.0), 0.70 (d, 3H, J= 6.6), 0.66 (d, 3H, J= 6.6); 13C NMR (125 MHz, DMSO-d6,) 179.8,174.2,170.4,157.6,148.5,144.9,138.5,137.6,137.3,136.4, 135.1,132.1,82.9,67.3,59.9,58.4,51.2,40.0,33.6,28.8,27.4,27. 1; HRMS (FAB+), calcd for C48H62N8O8Na m/e 901.4583, found m/e 901.4608 Compound 10010. In a same manner, diamine was reacted with 4- trifuoromethylphenyl isocyanate followed by deprotection to give compound 10010 (67%) as a white solid: 1H NMR (400 MHz, DMSO-d6,) 8.87 (s, 1H), 7.84 (d, 1H, J= 8. 9), 7.44 (d, 2H, I= 8.9), 7.13-7.21 (m, 8H), 7.08-7.10 (m, 1H), 6.43 (d, 1H, J = 7.6), 4.67 (s, 1H), 4.46-4.51 (m, 1H), 4.26-4.33 (m, 1H), 4.06 (dd, 1H, J = 8.6,7.0), 3.24 (s, 1H), 2.76 (dd, 1H, J = 13.8,10.5), 2.58-2.63 (m, 1H), 1.77-1.86 (m, 1H), 1.14 (d, 3H, J= 6.8), 0.70 (d, 3H, J= 6.8), 0.65 (d, 3H, J= 6.5); HRMS (FAB+), calcd for C50H60N8O10F6Cs m/e 1179.3391, found m/e 1179.3350 Compound 10012. In a same manner, diamine was reacted with 4- methylylphenyl isocyanate followed by deprotection to give compound 10012 (70%) as a white solid: 1H NMR (400 MHz, DMSO-d6,) 8.46 (d, 1H, J= 7.3), 7.79 (d, 2H, J= 7.9), 7.59 (d, 1H, J= 9.1), 7.42 (d, 1H, J= 9.4), 7.26 (d, 2H, J= 7.9), 7.08-7.17 (m, SH), 4.38-4.51 (m, 4H), 4.06 (dd, 1H, J = 8.8,6.4), 3.25 (s, 1H), 2.76 (dd, 1H, J= 13.8,10.3), 2.61 (dd, 1H, J= 13.8,3.8), 2.35 (s, 3H), 1.80-1.88 (m, 1H), 1.28 (d, 3H, J= 7. 04), 0.69 (d, 3H, J= 7.0), 0.65 (d, 3H, J = 6.8); 13C NMR (100 MHz, DMSO-d6,) 172.1,170.3,166.1,141.2,139.0,131.3, 129.1,128.8,128.1,127.8,127.5,125.7,73.3,57.8,50.4,48.9,38.6 ,30.5,21.0,

20.8,19.3,17.8,17.6; HRMS (FAB+), calcd for C5oH64N608Na mle 899.4683, found mle 899.4690 Compound 10013. In a same manner, diamine was reacted with 4- methoxyphenyl isocyanate followed by deprotection to give compound 10013 (70%) as a white solid: 1H NMR (500 MHz, DMSO-d6,) 8.45 (s, 1H), 7.80 (d, 1H, J= 9.0), 7.33 (d, 1H, J= 8.9) 7.25 (d, 1H, J= 8.6), 7.14-7.17 (m, 4H), 7.08- 7.11 (m, 1H), 6.74 (d, 1H, J= 0.3), 6.25 (d, 1H, J= 7.7), 4.65 (s, 1H), 4.48 (m, 1H), 4.27 (dt, 1H, J= 13.8,6.7), 4.05 (t, 1H, J= 7.5), 3.70 (s, 3H), 3.25 (s, 1H), 2.76 (dd, 1H, J= 13.8,10.7), 2.60 (dd, 1H, J= 13.7,3.5), 1.79-1.85 (m, 1H), 1.14 (d, 3H, J= 6.74), 0.70 (d, 3H, J= 6.7), 0.65 (d, 3H, J= 6.6); HRMS (FAB+), calcd for C5oH66N80loCs mle 1071.3956, found m/e 1071.3929 Compound 10014. To a solution of amine 10014a (Kempf et al., Bioorg. Med.

Chem., 1994,2,847) (50mg, 0.13mmol) in DMF (2ml) was added Cbz-AlaVal- OH (40.3mg, 0J13mmol) and HBTU (47.4mg, 0.13mmol). The reaction mixture was stirred for 2 h then diluted with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was dissolved in 4 N HC1 in dioxane (2ml) and stirred for 2 hr. After removal of the solvents to dryness, the residue was dissolved in DMF and Cbz-Val-OH (31mg, 0. 13mmol) and HBTU (47mg) and Et3N ( ? ?? L, 0.38mmol) was added. The reaction mixture was stirred for 6 hr then diluted with EtOAc. The organic solution was washed with sat. aq.

NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo to give compound 10014 (65%): 1H NMR (500 MHz, DMSO-d6,) 7.09- 7.35 (m, 20H), 5.04 (d, 2H, J= 12.5), 5.02 (s, 2H), 4.71-4.77 (m, 2H), 4.47-4.50 (m, 2H), 4.14 (m, 1H), 4.08 (t, 2H, J= 8.1), 3.76 (t, 1H, J = 8.8), 3.25 (m, 3H), 2.76 (m, 2H), 2.60 (m, 2H), 1.80 (m, 2H), 1.18 (d, 3H, J= 7. 0), 1. 15 (d, 3H, J= 7.0), 0.61-0.72 (m, 12H); HRMS (FAB+), calcd for C47H59N5O9Na mle 860.4313, found mle 860.4390 Compound 10015. In a same manner, amine was coupled with Cbz-Cl to give compound 10015 (70%) as a white solid: IH NMR (400 MHz, CD30D) 7.13- 7.35 (m, 15H), 6.39-6.47 (m, 1H), 5.09 (s, 2H), 5.04 (s, 2H), 4.05-4.30 (m, 4H), 3.76 (m, 2H), 2.75-2.87 (m, 4H), 1.82-1.90 (m, 1H), 1.20 (d, 3H, J= 7.2), 0.86

(m, 3H), 0.75 (m, 3H); HRMS (FAB+), calcd for C42H5oN408Na mle 761.3629, found mle 761.3640 Compound 10017a. To a solution of acid 10016a (Samanen et al., J. Med.

Chem. 1989,32,466) (261mg, lmmol) in DMF (4ml) was added Val-OMe. HCl (168mg, 1 mmol), HBTU (379mg, Immol) and Et3N (0.28ml). The reaction mixture was stirred overnight and then diluted with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash chromatography in 10% EtOAc/Hexane to give compound 10017a (85%): 1H NMR (500 MHz, CD30D) 4.65 (d, 1H, J= 9.1), 4.60 (d, J= 9.1), 4.21-4.37 (m, 2H), 3.70 (s, 3H), 2.12-2.19 (m, 1H), 1.56 (s, 3H), 1.43 (s, 9H), 1.38 (s, 3H), 0.98 (m, 6H); HRMS (FAB+), calcd for C17H3pN205SNa mle 397.1768, found m/e 397.1787.

Compound 10017b. In a similar manner, acid 10016b (Samanen et al., J. Med.

Chem. 1989,32,466) was coupled with ValOMe. » Cl to give compound 10017b (80%) as a white solid: 1H NMR (500 MHz, CDC13) 4.73 (bs, 1H), 4.66 (m, 1H), 4.53 (m, 1H), 4.38 (bs, 1H), 3.73 (s, 3H), 2.14-2.20 (m, 1H), 1.50 (s, 9H), 0.93 (d, 3H, J= 6. 6), 0.89 (d, 3H, J= 6.6) ; 13C NMR (125 MHz, CDC13) 171.6, 169.8,153.7,81.7,56.9,51.8,49.2,31.1,27.9,18.7,17.3; HRMS (FAB+), calcd for C15H26N2O5SNa m/e 369.1455, found mle 369.1490.

Compound 10018a. To a solution of compound 10017a (374mg, lmmol) in CH2C12 (4ml) was added 4N HC1 in dioxane (4ml). The reaction mixture was stirred for 4 hr and the solvent was removed to dryness. To a solution of this residue in DMF (8ml) was added the acid" (295mg, lmmol) HBTU (379mg, 1mmol) and Et3N (0.14ml). The reaction mixture was stirred overnight and then diluted with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash chromatography in 20% EtOAc/Hexane to give compound 10018a (72%): 1H NMR (600 MHz, CD30D) 7.14-7.26 (m, SH), 5.05 (d, 1H, J= 9.2), 4.93 (d, 1H, J= 9.2), 4.62 (s, 1H), 4.50 (d, 1H, J= 3,9), 4.44 (d, 1H, J = 5.7), 4.01 (m, lH), 3.70 (s, 3H), 2.81 (dd, 1H, J = 14.0,3.5), 2.62 (dd, 1H, J= 13.6,11.0), 2.17 (m, 1H), 1.60 (s, 3H), 1.44 (s, 3H), 1.31 (s, 9H),

0.96 (t, 6H, J= 7.0); 13C NMR (150 MHz, CD30D) 173.4,172.3,170.9,157.8, 140.2,130.6,129.2,127.1,80.1,73.3,73.1,59.0,56.0,52.4,35.7,3 2.0,30.1, 28.7,25.3,19.5,18.4; HRMS (FAB+), calcd for C27H41N307SNa m/e 574.2665, found mle 574.2693.

Compound 10018b. To a solution of compound 10017b (346mg, Immol) in CH2C12 (4ml) was added 4N HC1 in dioxane (4ml). The reaction mixture was stirred for 4 hr and the solvent was removed to dryness. To a solution of this residue in DMF (8ml) was added the acid (295mg, 1 mmol), HBTU (379mg, lmmol) and Et3N (0.14ml). The reaction mixture was stirred overnight and then diluted with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash chromatography in 20% EtOAc/Hexane to give compound 10018b (68%); 1H NMR (500 MHz, CD30D) 7.13-7.29 (m, 5H), 5.00 (d, 1H, J= 9.5), 4.96 (t, IN, J= 7.0), 4.77 (d, 1H, J= 9.9), 4.59 (d, 1H, J= 3.3), 4.41 (d, 1H, J= 5.5), 4.02-4.05 (m, 2H), 3.70 (s, 3H), 3.40 (dd, 1H, J= 11.7,7.7), 3.14 (dd, 1H, J= 11. 7,6.6), 2.89 (dd, 1H, J= 14.0,3.0), 2.66 (m, 1H), 2.16 (m, 1H), 1.32 (s, 9H), 0.95 (d, 6H, J= 6. 6); 13C NMR (125 MHz, CD30D) 173.4,172.2,171.8,157.7,140.0,130.5,129.1,127.1,80.0,73.3,65 .3, 64.0,61.4,59.1,56.0,52.5,51.0,34.0,32.0,28.7,19.5,18.4; HRMS (FAB+), calcd for C25H37N307SNa mle 546.2244, found mle 546.2239 Compound 10019. To a solution of compound 10018a (SSlmg, lmmol) in 20% MeOH in THF (8ml) was added a solution of LiOH (72mg, 3mmol) in water (0. 5ml). The reaction mixture was stirred for 4 hr and then diluted with EtOAc.

The organic solution was washed with 10% citric acid and water, dried over MgS04, filtered and concentrated in vacuo. To a solution of the residue in 1,2- dichloroethane (1 Oml) was added N-hydroxysuccinimide (11 Smg, lmmol), HOBt (153mg, 1 mmol) and DCC (206mg, 1 mmol). The reaction mixture was stirred for 24 hr and excess of solvents was removed to dryness. The residue was dissolved in THF (10mol) and methylamine (0.7ml, 2 M in THF, 1.4mmol) was added. The reaction mixture was stirred for 1 hr and the precipitate which form was removed by filtration. Excess of solvents were removed and the crude product was purified by flash chromatography to give compound 10019 (90%)

as a white solid: 1H NMR (500 MHz, CD30D) 7.13-7.18 (m, 5H), 5.09 (d, 1H, J = 9.6), 4.95 (d, 1H, J= 8. 8), 4.57 (s, 1H), 4.54 (d, 1H, J= 4. 4), 4.18 (m, 1H), 3.94 (m, 1H), 2.90 (m, 1H), 2.70 (s, 3H), 2.03 (m, 1H), 1.57 (s, 3H), 1.40 (s, 3H), 1.31 (s, 9H), 0.96 (d, 3H, J= 6.6), 0.91 (d, 3H, J= 7.0); 13C NMR (125 MHz, CD30D) 173.9,172.5,170.5,157.7,140.0,130.5,129.1,127.1,80.0, 73.5,61.4,56.1,52.3,35.9,32.0,30.4,28.7,26.3,26.1,25.1,20.9, 19.7,19.1 14.5; HRMS (FAB+), calcd for C27H42N406SNa mle 573.2717, found m/e 573.2726 Compound 10020. To a solution of compound 10017a (374mg, lmmol) in CH2C12 (4ml) was added 4N HCl in dioxane (4ml). The reaction mixture was stirred for 4 hr and the solvent was removed to dryness. The residue was dissolved in water and neutralized with 2MNaOH. The aqueous solution was extracted with EtOAc and the organic layer was dried over MgS04, filtered and concentrated, in vacuo. To a solution of this residue in dry MeOH (10ml) was added epoxide (263mg, 1mmol) and Et3N (0.28ml). The reaction mixture was stirred at 80 0C for 24 hr. After allowing to cool to room temperature, excess of solvent was removed and the crude product was purified by flash chromatography to give compound 10020 (62%): 1H NMR (500 MHz, CDC13) 7.97 (d, 1H, J= 9.0), 7.17-7.30 (m, SH), 4.71 (d, 1H, J= 8. 7), 4.52 (d, 1H, J = 10.0), 4.48 (dd, 1H, J= 9.4,4.9), 4.42 (d, 1H, J= 4.7), 3.95 (d, 1H, J= 10. 0), 3.80 (m, 1H), 3.75 (s, 3H), 3.59 (m, 1H), 3.16 (s, 1H), 2.98 (d, 2H, J= 12.3), 2.86 (dd, 1H, J = 12.7,9.6), 2.76 (m, 1H), 2.21 (m, 1H), 1.63 (s, 3H), 1.41 (s, 3H), 1.31 (s, 9H), 0.98 (d, 3H, J= 6.9), 0.93 (d, 3H, J= 6.8); 13C NMR (125 MHz, CDC13) 174.4,171.4,155.5,138.0,129.4,128.2,126.2,80.7,79.2,72.0, 62.0,60.3,58.6,56.8,55.6,54.6,52.5,35.9,30.4,29.6,28.2,25.3, 19.4,17.7, 14.1; HRMS (FAB+), calcd for C27H43N306SNa m/e 560.2765, found mle 560.2742 Compound 10023. To a solution of compound 10020 (54mg, 0. 1mmol) in CH2Cl2 (2ml) was added 4N HCl in dioxane (2ml). The reaction mixture was stirred for 4 hr and the solvent was removed to dryness. To a solution of crude amine in DMF (2ml) was added Cbz-AlaVal-OH (32mg, 0. 1mmol), HBTU (38mg, 0. Immol) and Et3N (28? L, 0.2mmol). The reaction mixture was stirred

for 12 hr and then diluted with EtOAc. The organic solution was washed with sat. aq. NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash chromatography in 15% EtOAc/Hexane to give compound 10023 (80%) as a white solid: 1H NMR (600 MHz, CD30D) 7.12-7.35 (m, 10H), 5.09 (s, 2H), 5.05 (s, 1H), 4.53 (d, 1H, J= 9.5), 4.34 (dd, 1H, J= 10.6,5.4), 3.95-4.18 (m, 4H), 3.73 (s, 3H), 2.94-2.98 (m, 1H), 2.72-2.76 (m, 2H), 2.16-2.21 (m, 1H), 1.87-1.92 (m, 1H), 1.57 (s, 3H), 1.37 (s, 3H), 1.32 (d, 3H, J= 7.1), 0.94-0.96 (m, 6H), 0.77 (t, 3H, J= 7.0), 0.60 (t, 3H, J = 7. 0); 13C NMR (150 MHz, CD30D,) 175.5,174.3,173.6,173.1,158.4, 140.0,138.1,130.6,129.5,129.3,129.0,127.2,82.6,74.1,67.7,61. 5,60.4,58.7, 58.5,55.5,53.1,54.9,52.8,52.1,37.0,32.0,31.4,30.3,26.7,19.9, 19.7,18.4, 18.0; HRMS (FAB+), calcd for C3gH55N50gSNa mle 764.3664, found m/e 764.3689 Compound 10024.1H NMR (600 MHz, CD30D) 8.27 (d, 1H, J= 8.76), 7.86 (d, 1H, J = 9.24), 7.26-7.34 (m, 10H), 7.18-7.20 (m, 1H), 5.10 (d, 1 H, J = 12.7), 5.06 (d, 1H, J = 12.2), 4.65 (d, 1H, J= 8.3), 4.53 (m, 1H), 4.47 (d, 1H, J = 7.86), 4.35 (s, 1H), 4.30-4.32 (m, 2H), 4.18 (dd, 1H, J= 14.0,7.0), 4.13 (t, 1H, J= 6.6), 3.67 (s, 3H), 2.99 (dd, 1H, J= 12.6,8.8), 2.87 (dd, 1H, J= 12.3,6.6), 2.11 (dt, 1H, J= 21.1,14.5,7.5), 1.91-1.93 (m, 1H), 1.53 (s, 3H), 1.35 (s, 3H), 1.32 (d, 3H, J= 7.0), 0.94 (t, 3H, J= 5.7), 0.80 (dd, 3H, J= 9.7,7.0); 13C NMR (150 MHz, CD30D,) 173.3,173.1,172.1,171.7,171.6,158.5,139.3,138.1,130.5, 129.6,129.5,129.0,128.7,127.7,73.4,70.6,67.7,60.1,59.3,52.9, 52.6,52.4, 51.9,38.8,32.0,31.9,31.3,24.3,20.0,19.5,18.7,18.5,18.0; HRMS (FAB+), calcd for C38H53N509SNa m/e 778.3564, found mle 778.3590 Compound 10025. See Lee, T.; Le, V.-D.; Lim, D.; Lin, Y. C.; Wong, A. L.; Morris, G. M.; Olson, A. J.; Elder, J. H.; Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 1145.

Compound 10026.1H NMR (600 MHz, DMSO-d6,) 8.10 (d, 1H, J= 8. 3), 7.68 (d, 1H, J= 9.2), 7.60 (d, 1H, J= 7. 9), 7.46 (d, 1H, J = 7. 9), 7.31 (d, 1H, J = 7.9), 6.95-7.21 (m, 15H), 4.52-4.55 (m, 1H), 4.44-4.48 (m, 1H), 4.08 (dd, 1H, J= 8. 8, 6.5), 3.29 (s, 1H), 3.01 (dd, 1H, J= 12.6,1.6), 2.85 (dd, 1H, J= 14.9,10.5),

2.76-2.81 (m, 1H), 2.64 (dd, 1H, J= 14.0,4.0), 1.83-1.86 (m, 1H), 1.74 (s, 3H), 0.68 (d, 3H, J= 7.0), 0.65 (d, 3H, J= 7.0); 13C NMR (150 MHz, DMSO-d6,) 171.6,170.3,169.2,139.0,136.0,129.1,128.8,128.0,127.8,127.3, 125.7, 125.5,123.3,120.8,118.5,118.1,111.2,110.5,73.3,57.9,53.2,50. 6,38.6,30.5, 27.4,22.5,19.3,17.9; HRMS (FAB+), calcd for C54H66N80gNa mle 977.4896, found mle 977.4882 General Procedure for the synthesis of compounds 10022,10027,10028, 10029 and 10031. To a solution of compound 10018a (551mg, 1mmol) or 10018b (523mg, Immol) or 10019 (550mg, Immol) in CH2Cl2 (10ml) was added 4NHC1 in dioxane (lOml). The reaction mixture was stirred for 4 hr and the solvent was removed to dryness. To a solution of the amine (49mg, 0.1 mmol) in DMF (2ml) was added dipeptide (0. 1mmol), HBTU (38mg, 0. lmmol) and Et3N (28? L, 0.2mmol). The reaction mixture was stirred for 12 hr and then diluted with EtOAc. The organic solution was washed with sat. aq.

NaHC03 and sat. aq. NaCl, dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash chromatography in 15% EtOAc/Hexane to give the title compound.

Compound 10022. The above amine (49mg, 0. 1mmol) was coupled with Cbz- AlaVal-OH (32mg, 0. 1mmol) to give compound 10022 (75%) as a white solid: 1H NMR (400 MHz, CD30D) 7.11-7.34 (m, 10H), 5.07 (s, 2H), 5.04 (m, 1H), 4.62 (s, 1H), 4.35-4.48 (m, 3H), 4.05-4.13 (m, 2H), 3.70 (s, 3H), 2.79-2.86 (m, 2H), 2.13-2.16 (m, 1H), 1.60 (s, 3H), 1.45 (s, 3H), 0.93-0.97 (m, 9H), 0.77-0.79 (m, 3H); HRMS (FAB+), calcd for C3gH53N509SNa mle 778.3564, found m/e 778.3580 Compound 10029. The above amine (46mg, 0.1 mmol) was coupled with Ac- TrpVal-OH (35mg, 0. lmmol) to give compound 10029 (66%) as a white solid: 1H NMR (500 MHz, CD30D) 7.60 (m, 1H), 6.94-7.32 (m, 9H), 5.14 (d, 1H, J= 9.2), 4.87 (d, 1H, J = 9.2), 4.64 (s, 1H), 4.53 (m, 1H), 4.34-4.43 (m, 3H), 4.05 (d, 1H, J= 6. 3), 3.70 (s, 3H), 3.15-3.20 (m, 1H), 3.06 (m, 1H), 2.69 (m, 1H), 3.15- 3.20 (m, 1H), 3.06 (m, 1H), 2.88 (m, 1H), 2.69 (m, 1H), 2.13 (m, 1H), 1.90 (s, 3H), 1.60 (s, 3H), 1.45 (s, 3H), 0.94 (t, 3H, J= 7.0), 0.40 (dd, 3H, J= 24.6,7.0); HRMS (FAB+), calcd for C38H50N6O8SNa m/e 773.3411, found m/e 773.3450

Compound 10030. The above amine (49mg, 0. lmmol) was coupled with Ac- PheVal-OH (35mg, O. lmmol) to give compound 10030 (55%) as a white solid: 1H NMR (500 MHz, CD30D) 7.58 (d, 1H, J= 7.7), 7.32 (d, 1H, J = 8.0), 7.17- 7.24 (m, 6H), 7.03-7.14 (m, 2H), 6.97-7.00 (m, 1H), 5.04 (d, 1H, J = 9. 2), 4.86 (d, 1H, J = 9.2), 4.65 (m, 1H), 4.52 (dd, 1H, J= 9.2,3.7), 4.32 (m, 1H), 4.07- 4.14 (m, 2H), 3.18 (dd, 1H, J= 14.7,5.9), 3.04 (dd, 1H, J = 14.7,8.1), 2.90 (m, 1H), 2.75-2.80 (m, 1H), 2.70 (s, 3H), 1.94-1.98 (m, 2H), 1.54 (s, 3H), 1.40 (s, 3H), 0.89-0.95 (m, 6H), 0.72 (d, 3H, J= 8.0), 0.70 (d, 3H, J= 7.0); 13C NMR (125 MHz, CD30D,) 174.1,173.9,173.4,173.2,172. 3,170.7,139.9,138.1, 130.6,129.3,127.5,124.6,122.4,119.8,119.4,112.3,110.9,73.5,7 2.6,60.5, 60.4,55.5,55.0,52.5,34.8,31.9,31.6,30.3,28.7,26.1,25.2,22.5, 19.8,19.7, 19.6,19.2,18.5; HRMS (FAB+), calcd for C40H55N707SNa mle 800.3776, found mle 800.3793 Compound 10031. The above amine (49mg, 0. 1 mmol) was coupled with Ac- PheVal-OH (31mg, 0. 1mmol) to give compound 10031 (72%) as a white solid: HNMR (400MHz, CD30D) 7.15-7.31 (m, 10H), 5.08 (d, lH, J=9. 4), 4.91 (d, 1H, J= 9.4), 4.63 (s, 1H), 4.57 (d, 1H, J= 10.0,5.3), 4.50 (d, 1H, J= 3.52), 4.43 (d, 1H, J= 5.88), 4.34-4.37 (m, 1H), 4.07 (d, 1H, J= 7.3), 3.70 (s, 3H), 2.98 (dd, 1H, J = 14.1,5.0), 2.73-2.87 (m, 3H), 2.11-2.19 (m, 1H), 1.90-1.97 (m, 1H), 1.86 (s, 3H), 1.60 (s, 3H), 1.45 (s, 3H), 0.95 (dd, 3H, J= 6.8,3.2), 0.81 (d, 3H, J = 6. 8); 13C NMR (100 MHz, CD30D,) 173.7,173.5,173.2,173.1,172.0,170.9, 139.9,138.5,130.6,130.2,129.4,129.3,127.7,127.3,73.0,72.7,60 .3,59.1, 56.0,54.8,52.5,52.4,38.5,34.7,32.0,30.2,25.3,22.3,19.7,19.5, 18.6,18.5; HRMS (FAB+), calcd for C3gH53N50gSNa mle 762.3507, found m/e 762.3539 Compound 10032. The above amine (49mg, 0. lmmol) was coupled with Ac- PheVal-OH (3 lmg, 0. lmmol) to give compound 10032 (79%) as a white solid: 1H NMR (400 MHz, CD30D) 8.03 (d, 1H, J = 8.8), 7.30 (d, 1H, J = 7.4), 7.18- 7.22 (m, 10H), 5.08 (d, 1H, J= 9. 4), 4.91 (m, 1H), 4.58 (dd, 1H, J = 11.4,5.0), 4.55 (s, 1H), 4.52 (d, 1H, J= 4.1), 4.33-4.38 (m, 1H), 4.16 (d, 1H, J= 8.2), 4.09 (d, 1H, J= 7.3), 3.00 (dd, 1H, J= 9.4,5.0), 2.90 (dd, 1H, J= 14.1,3.2), 2.79 (dd, 1H, J = 14.1,10), 2.70 (s, 3H), 1.96-2.00 (m, 1H), 1.86 (s, 3H), 1.56 (s, 3H),

1.40 (s, 3H), 0.96 (m, 3H), 0.79 (t, 3H, J= 6.8); 13C NMR (100 MHz, CD30D,) 173.9,173.6,173.4,172.8,172.2,170.6,139.9,138.5,130.5,130.2, 129.4, 129.3,127.7,127.3,73.4,72.8,60.4,60.3,55.9,54.9,52.5,38.5,34 .9,32.0, 31.9,30.3,26.1,25.2,22.3,19.8,19.6,18.6,17.7; HRMS (FAB+), calcd for C3gH54N607SNa m/e 761. 3667, found m/e 761.3640 Compound 10033. The above amine (49mg, 0. lmmol) was coupled with Ac-AlaVal-OH (23mg, 0. lmmol) to give compound 10033 (85%) as a white solid: 1H NMR (600 MHz, CD30D) 7.16-7.25 (m, 5H), 5.07 (d, 1H, J= 9.2), 4.92 (d, 1H, J = 9.2), 4.62 (s, 1H), 4.48 (d, 1H, J= 3. 5), 4.43 (d, 1H, J = 5.7), 4.39 (m, 1H), 4.32 (dd, 1H, J = 14.0,7.0), 4.06 (d, 1H, J = 7.4), 3.71 (s, 3H), 2.83 (dd, 1H, J= 14.0,3.5), 2.73 (dd, 1H, J = 14.0,11.0), 2.16 (m, 1H), 1.95 (s, 3H), 1.60 (s, 3H), 1.45 (s, 3H), 0.96 (t, 3H, J= 7.0), 0.80 (t, 3H, J= 6.5); 13C NMR (150 MHz, CD30D) 175.0,173.5,173.3,173.2,172.0,170.9,139.8, 130.5,129.3,127.2,73.0,72.8,60.4,59.1, 54.6, 52.5,52.4,50.4,34.9,32.0, 31.9,30.1,25.3,22.3,19.7,19.5,18.5,17.7; HRMS (FAB+), calcd for C32H49N508SNa mle 686.3302, found mle 686.3320.

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