Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Amino acid or sequencing procedure
Reexamination Certificate
1997-11-12
2001-02-20
Celsa, Bennett (Department: 1627)
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Amino acid or sequencing procedure
C436S086000, C436S501000, C435S004000, C530S317000
Reexamination Certificate
active
06190920
ABSTRACT:
INTRODUCTION
1. Technical Field
The invention relates to a method for detecting cyclization of acyclic compounds. In particular, the invention relates to a method of screening for macrocyclic peptidase inhibitors, and is especially useful for screening a combinatorial library of compounds.
2. Background
Recently there has been much interest in using combinatorial techniques for preparing libraries of organic compounds, including peptides, oligonucleotides, and the like. See, for example, U.S. Pat. Nos. 5,480,971, 5,506,337, 5,504,190, 5,539,083, 5,541,061, 5,550,215, 5,556,762, 5,565,324, 5,565,325, and 5,618,825. One advantage of such an approach is that initially a large number of compounds can be prepared in small quantities, and the compounds thus produced screened in order to discover ligands having useful activity. Those compounds found to exhibit useful activity may then be prepared on a larger scale by conventional techniques for further testing. Additionally, once a preferred compound has been identified from the initial screening, other combinatorial libraries of compounds closely related to the initially selected compound may be assembled using the same combinatorial techniques. In this fashion, a rapid and efficient method is employed for identifying active compounds.
An area of increasing interest relates to macrocyclic peptidase inhibitors. Such inhibitors are useful as antihypertensives, HIV protease inhibitors, thrombin inhibitors, etc. Because it requires enormous effort to make complex macrocyclic analogs by conventional synthetic techniques, the ability to identify those ring systems useful as macrocyclic peptidase inhibitors before undertaking such a synthetic effort would be desirable. Thus, the preparation of a combinatorial library of compounds coupled with a rapid and convenient method for identifying the components of that library that are useful as macrocyclic peptidase inhibitors would be especially valuable.
A method of determining the potential of compounds for utility as macrocyclic peptidase inhibitors has been discovered. This invention is based upon the observation that a ring system that is favorably bound as an inhibitor can be generated from a linear molecule by action of the target enzyme. Linear molecules are easier to synthesize than macrocycles, and therefore a method for identifying acyclic compounds that are cyclized readily by a target enzyme has utility for discovering macrocyclic peptidase inhibitors. The method is particularly useful for the screening of a large number of acyclic compounds generated in a combinatorial library for utility as macrocyclic peptidase inhibitors.
Relevant Literature
A review of combinatorial techniques is given in “Combinatorial Chemistry” by Gordon Lowe,
Chemical Society Reviews,
1995, pp 309-317.
SUMMARY OF THE INVENTION
The present invention concerns a method for detecting enzyme-catalyzed cyclization of acyclic amino compounds, comprising;
a) contacting a peptidase with an acyclic amino compound of Formula I:
NH
2
—R
1
—X—R
2
—C(O)—Y Formula I
wherein:
C(O)Y represents a carboxylic acid, ester, or amide derivative capable of acylating or being hydrolyzed by a peptidase;
R
1
and R
2
each represent a series of subunits connected by covalent bonds, in which
one of said subunits in R
1
is linked to a dye molecule or a resin;
one of said subunits in R
2
is linked to a dye molecule or to a resin; and
X is a group cleavable under reaction conditions that do not cleave an amide;
with the proviso that the acyclic amino compound contains only one dye molecule and one resin-linked subunit;
b) contacting the product of step a) with an agent capable of cleaving subunit X under reaction conditions that do not cleave an amide; and
c) isolating the resin;
wherein cyclization of said acyclic amino compound is detected by retention of said dye on the resin.
Preferred is where Y is —OR
3
, —SR
3
, or —NHR
3
, in which;
R
3
is hydrogen, alkyl, cycloalkyl, aryl, or heteroaryl; and
R
1
and R
2
each represent a series of 1-20 subunits, wherein the number of subunits represented by R
1
and R
2
combined represent between 2 and 30 subunits.
Another aspect of the invention relates to a method of discovering effective peptidase inhibitors, comprising:
a) contacting an acyclic amino compound of Formula I with a peptidase;
b) contacting the product of step a) with an agent capable of cleaving group X under reaction conditions that do not cleave an amide; and
c) isolating the resin;
wherein the presence of an effective peptidase inhibitor is indicated by retention of said dye on the resin, and the effective peptidase inhibitor is a compound of Formula II:
where R
1
, R
2
, and X are as defined above.
DESCRIPTION OF SPECIFIC EMBODIMENTS
As used herein:
“Alkyl” means a branched or unbranched saturated monovalent hydrocarbon radical containing 1 to 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-hexyl, 2-methylhexyl, n-octyl, 4-ethyloctyl, n-decyl, n-dodecyl, and the like.
“Lower alkyl” means a branched or unbranched saturated monovalent hydrocarbon radical containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, tert-butyl, butyl, n-hexyl and the like.
“Cycloalkyl” means a saturated monovalent monocyclic hydrocarbon radical containing 3-8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
“Lower alkoxy” means the group —O—(lower alkyl) wherein lower alkyl is as herein defined.
“Halo” or “halogen” means fluoro, chloro, bromo, or iodo.
“Alkylene” means a branched or unbranched saturated divalent hydrocarbon radical containing 1 to 12 carbon atoms, such as methylene, ethylene, 1,2-propylene, 1,4-butylene, 1,3-butylene, 1,5-pentylene, 1,3-pentylene, 1,6-hexylene, 1,12-docecylene, and the like. “Optionally substituted alkylene” means alkylene as defined optionally mono-, di- or tri-substituted, independently, with lower alkyl, lower alkoxy, halo, nitro, trifluoromethyl and/or cyano, such as difluoromethylene. The alkylene chain is optionally interrupted by a heteroatom chosen from oxygen, sulfur, and nitrogen,
“Alkenylene” means a branched or unbranched unsaturated divalent hydrocarbon radical containing 2 to 12 carbon atoms, such as ethene, 1-propene, 1-butene, 3-methylbut-1-ene, 1-pentene, 2-methylpent-1-ene, 1-hexene, 1-docecene, and the like.
The term “aryl” refers to a monovalent unsaturated aromatic carbocyclic radical having a single ring (e.g., phenyl) or two rings (e.g., naphthyl, biphenyl, indanyl, 1,2,3,4-tetrahydronaphthyl, benzocycloheptane), which can optionally be mono-, di- or tri-substituted, independently, with OH, COOH, lower alkyl, lower alkoxy, halo, nitro, amino, alkylamino, dialkylamino, trifluoromethyl and/or cyano.
The term “heteroaryl” refers to a monovalent aromatic carbocyclic radical having 1-3 heteroatoms within one or two rings, (e.g., thiophenyl, furanyl, pyridyl, thiazolyl, pyrimidine, oxazolyl, benzoxazole, benzofuran, benzothiophene, indolinyl, quinoline), which can optionally be mono-, di- or tri-substituted, independently, with OH, COOH, lower alkyl, lower alkoxy, halo, nitro, amino, alkylamino, dialkylamino, trifluoromethyl and/or cyano.
The term “peptidase” refers to the class of enzymes that are capable of hydrolyzing the amide linkage such as that between amino acids in an oligopeptide or polypeptide.
The term “dye” or “dye molecule” refers to an entity that might be visible under UV light or via fluorescence, but preferably can be seen in the visible spectrum. Examples of such dyes are analogs of Disperse Red 1, xylenol orange, malachite green, Coomassie blue, dabsyl or dabcyl derivatives, fluoroscein, and the like. They may be attached to one of the subunits comprising R
1
and R
2
by any means; the attachment must be stable to conditions used for the cleavage of X. Examples include, but are not limited to, attachment of an alkyl, aryl, sulfonyl, or acyl group to a nitrogen atom of a subunit; attachment of an alkyl or aryl group to an oxygen or sulfur atom of
Bartlett Paul A.
Burger Matthew T.
Celsa Bennett
Cooley & Godward LLP
The Regents of the University of California
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