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Publication numberCA2203123 A1
Publication typeApplication
Application numberCA 2203123
PCT numberPCT/US1995/012861
Publication date2 May 1996
Filing date18 Oct 1995
Priority date20 Oct 1994
Also published asEP0789566A1, EP0789566A4, US5734082, US5892113, WO1996012482A1
Publication numberCA 2203123, CA 2203123 A1, CA 2203123A1, CA-A1-2203123, CA2203123 A1, CA2203123A1, PCT/1995/12861, PCT/US/1995/012861, PCT/US/1995/12861, PCT/US/95/012861, PCT/US/95/12861, PCT/US1995/012861, PCT/US1995/12861, PCT/US1995012861, PCT/US199512861, PCT/US95/012861, PCT/US95/12861, PCT/US95012861, PCT/US9512861
InventorsSteve Gallion, Paul S. Furth, David Casebier, Joseph C. Hogan, Jr., Alan Kaplan
ApplicantSteve Gallion, Paul S. Furth, David Casebier, Joseph C. Hogan, Jr., Alan Kaplan, Arqule, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: CIPO, Espacenet
Hydroxyethyl aminimides
CA 2203123 A1
This invention relates to a class of aminimides structurally characterized as an acyl nitrogen-nitrogen ylide such that the acyl moiety possesses the structural diversity element G, the quaternary nitrogen possesses structural diversity elements E and F, and the quaternary nitrogen is bonded to a hydroxyethyl substituent, which in turn is bonded to an aminomethylene moiety that possesses structural diversity elements A and B from the amino group and diversity element C from the methylene substituent, as shown in chemical formula (I), wherein structural diversity elements A, B, C, D, E, F and G are chosen from the set of elements consisting of substituted and unsubstituted as well as branched and straight chain alkyl, aryl, alkaryl, aralkyl, carbocyclic, heterocyclic, hydrogen, amino acid, peptide, polypeptide, protein, depsipeptide, carbohydrate derivatives, nucleotide derivatives, oligonucleotide derivatives, naturally occurring or synthetic organic structural motifs, reporter elements, organic moieties containing at least one polymerizable group, macromolecular component, resin, silicate, other surface and particle support, the diversity elements can be the same, can be different, and can also be connected to form a ringed to several ringed species.
1. A chemical backbone having formula I as follows:


a. structural diversity elements A, B, C, E, F and G
are the same or different, and each is selected from the group consisting of a chemical bond; hydrogen; electrophilic group;
a nucleophilic group; an amino acid derivative; a nucleotide derivative, a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety, optionally containing a polymerizable group; a macromolecular component, wherein the structural diversity elements A, B, C, E, F and G
are optionally connected to each other or to other structures;
D is a selected from the group consisting of hydrogen, branched or straight chain lower alkyl having from 1-8 carbon atoms, ether and ester groups, and wherein n is an integer greater than O.
2. A chemical backbone according to claim 1 wherein D is hydrogen.
3. A chemical backbone according to claim 1 wherein D is straight or branched chain lower alkyl having from 1-8 carbon atoms.
4. A chemical backbone according to claim 1 wherein n is an integer greater than 1.
5. A chemical backbone according to claim 1 wherein B is hydrogen.
6. A chemical backbone according to claim 1 wherein the backbone carbon atom attached to structural diversity element C is chiral.
7. A method of making a first chemical compound having a complementary structure to a second chemical compound comprising the steps of preparing a chemical backbone according to claim 1 and testing its complementary properties with a second chemical compound.
8. A method according to claim 7 wherein structural diversity element D is hydrogen.
9. A method according to claim 8 wherein structural diversity element B is hydrogen.
10. A peptide isostere as defined in claim 1 capable of binding to an active site of a receptor or to an enzyme.
Description  (OCR text may contain errors)

2 P~ llu~35/12861 HYDRO~Y~nY~ AMINIMIDES

Background of the Invention Recent research in the fields of separation technology 5 and pharmaceutical compounds reveals that many reactions between chemical compounds result from the three dimensional structure, and the molecular interactions between different compounds. A complementary structural relationship has been tied to a particular chemical compounds ability to react with 10 a second chemical compound (see, for example, "The Concept of Molecular Structure in Structure-Activity Relationship Studies and Drug Design", Testa et al., Medicinal Research Reviews, 1991, Vol. 11, No. 1). The present invention relates to hydroxyethyl aminimide chemical structures which can be used 15 as molecular scaffolding on which to hang different substituent groups. By varying the different substituent groups on an aminimide chemical backbone as disclosed herein it is possible to develop chemical compounds having a complementary structural and molecular relationship to a 20 target compound, enzyme, molecular recognition site or receptor. Use of the present invention represents an improvement in both the cost and time efficiency of identifying lead compounds.
Hydroxyethyl aminimides (herein after referred to as 25 aminimides), can be prepared from the one-step reaction of an ester or acid chloride, a hydrazine, and an epoxide according to a method such as that disclosed by Middleton, United States Patent 3,963,776 and Culbertson, United States Patent 3,963,703. Alternatively, hydroxyethyl aminimides can be 30 formed from the alkylation of a disubstituted hydrazide through the opening of an epoxide such as by the reactions disclosed in Grimm, United states Patent 3,850,969. An extensive discussion of aminimides including their preparation and uses is set forth in PCT application PCT/US93/12612 filed 35 December 28, 1993 in the name of ArQule Partners, L.P. which is herein incorporated by reference in its entirety.
Aminimides have a diversity of properties and are known W O 96/12482 PC~rrUS95/12861 to be useful as surfactants, (see, for example, Falk, United States Patent 4,102,916, and Middleton, United States Patent 3,963,776), resin hardeners, precursors to isocyanates (see, for example, Brutchen, United States Patent 3,898,087), in the 5 formation of polyurethanes (see, for example, Kresta, United States Patent 4,067,830) and polyisocyanurates ( see Kresta, United States Patent 3,925,284).
Much less is known about the biological activity of aminimides. Kabara has shown that specific aliphatic derived 10 dimethyl-hydroxyethyl aminimides possess antimicrobial (see United States Patents 4,189,481 and 4,217,364) and antifungal properties (see United States Patents 3,934,029 and 3,934,031). L. Boutis, et al., have observed antineoplastic activity (Current Chemotherapy, 1978, 2, 1213-1216), while 15 M. Tichniouin, et al. demonstrated that certain dimethyl-hydroxyethyl aminimides possess a noticeable vasodilating activity (Eur. J. Med. Chem., 1982, 17, 265-270). Yet, to date, no one has used an aminimide moiety, and, in particular, a hydroxyethyl aminimide moiety as a peptide isostere in the 20 design of pharmacologically active compounds.
The present invention relates to the use of hydroxyethyl aminimides as isosteres in the design and synthesis of chemical compounds capable of binding to an active site of a receptor or enzyme or to a molecular recognition site in, for 25 example, separation chemistry.

Summary of the Invention It is an object of this invention to provide a novel class of hydroxyethyl aminimide compounds useful for their 30 complementary properties to molecular recognition sites and/or enzymes.
Another object of this invention is to provide a method of making chemical compounds which are complementary to other chemical compounds.
Other objects of this invention will be apparent to those skilled in the art to which this invention applies.
The objects of this can be accomplished using a chemical W096/1~2 PCT~S95/1~61 backbone having the following chemical formula I:

(~) N~l~lt~ ~

a. structural diversity elements A, B, C, E, F and G
are the same or different, and each is selected from the group lO consisting of a chemical bond; hydrogen; electrophilic group;
a nucleophilic group; an amino acid derivative; a nucleotide derivative, a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety, optionally containing a polymerizable group; a macromolecular component, 15 wherein the structural diversity elements A, B, C, E, F and G
are optionally connected to each other or to other structures;
D is a selected from the group consisting of hydrogen, branched or straight chain lower alkyl having from 1-8 carbon atoms, ether and ester groups, and wherein n is an integer 20 greater than 0.

Detailed Description Of The Invention The present invention relates to the use of hydroxyethyl aminimides and to their use as molecular recognition agents in 25 the design and synthesis of compounds for use as biologically active compounds and in separations technology.
A peptide isostere is a moiety that, when substituted for the peptide bond, will confer upon the analog certain stearic and/or electronic configurations similar to the parent 30 compound, thereby allowing the analog to possess biological properties similar to the parent compound. However, the isostere is designed to be resistant to degradation by the same pathways as a nominal peptide.
For the purposes of the present invention, the term 35 complementary is given that definition currently used in the chemical and biochemical arts that refers to a close three WO96/1~2 PCr~S95/1~61 dimensional and/or molecular interaction relationship between two different compounds.
The term "backbone" is defined, for the purpose of the present invention, as an organic chain of elements that can be 5 substituted with one or more structural diversity elements to yield a chemical compound or a class of chemical compounds which are complementary to a second chemical compound or class of chemical compounds.
The hydroxyethyl aminimides of the present invention have lO a chemical backbone with the following chemical formula I:

(~) N ~ N f ~ ~
~ o ~l...n) wherein: `~' a. structural diversity elements A, B, C, E, F and G
are the same or different, and each is selected from the group 20 consisting of a chemical bond; hydrogen; electrophilic group;
a nucleophilic group; an amino acid derivative; a nucleotide derivative, a carbohydrate derivative; an organic structural motif; a reporter element; an organic moiety, optionally containing a polymerizable group; a macromolecular component, 25 wherein the structural diversity elements A, B, C, E, F and G
are optionally connected to each other or to other structures, D is a selected from the group consisting of hydrogen, branched or straight chain lower alkyl having from 1-8 carbon atoms, ether and ester groups, and n is an integer greater 30 than O. For this invention, it is understood that each integer that n can be, represents a structural embodiment of the backbones of this invention. Thus each integer from l to at least about lO0,000 to 500,000 is expressly disclosed as preferred embodiments of the present invention. Higher values 35 of n can be used, if desired, for specialty polymers.
The particular structural diversity elements can vary greatly, depending upon the specific compound to be prepared.

WO96/1~2 PCT~S95/12861 One of ordinary skill in the chemical arts is well aware of the bonding properties, and capabilities, of these elements, and can easily select the appropriate element for attachment to the particular location on the backbone. Thus, the backbone 5 represents a very valuable tool for constructing any one of a wide variety of compounds, and it represents an essential feature of this invention.
The synthesis and design of aminimide compounds is well known in the art and is detailed for example in PCT/US93/12612 l0 referenced above. The aminimide compound of the present invention can be synthesized in a similar manner by many routes. It is well known in the art of organic synthesis that many different synthetic protocols can be used to prepare a given compound. Different routes can involve more or less 15 expensive reagents, easier or more difficult separation or purification procedures, straightforward or cumbersome scale-up, and higher or lower yield. The skilled synthetic organic chemist knows well how to balance the competing characteristics of synthetic strategies. Thus the compounds 20 of the present invention are not limited by the choice of synthetic strategy, and any synthetic strategy that yields the compounds described above can be used.
Accordingly, any known method of producing the subject hydroxyethyl aminimide compounds can be to produce a wide 25 variety of chiral aminimide conjugates of the following general structures:

IN~y~ ~ and N~l~ N ~

~N~Nt~ ~ and ~N~Nt~

od N~Nt~N~

W O 96/12482 PC~rrUS95/12861 The structural diversity elements A, B, C, E, F and G may be the same or different, may be of a variety of structures and may differ markedly in their physical or functional properties, or may be the same; they may also be chiral or 5 symmetric. The structural diversity elements A, B, C, E, F
and G are preferably selected from:
1) amino acid derivatives of the form (AA) n~ which would include, for example, natural and synthetic amino acid residues (n = 1) including all of the naturally occurring 10 alpha amino acids, especially alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine; the naturally occurring disubstituted amino acids, 15 such as amino isobutyric acid, and isovaline, etc.; a variety of synthetic amino acid residues, including alpha-disubstituted variants, species with olefinic substitution at the alpha position, species having derivatives, variants or mimetics of the naturally occurring side chains; N-substituted 20 glycine residues; natural and synthetic species known to functionally mimic amino acid residues, such as statine, bestatin, etc. Peptides (n = 2 - 30) constructed from the amino acids listed above, such as angiotensinogen and its family of physiologically important angiotensin hydrolysis 25 products, as well as derivatives, variants and mimetics made from various combinations and permutations of all the natural and synthetic residues listed above. Polypeptides (n = 31 -70), such as big endothelin, pancreastatin, human growth hormone releasing factor and human pancreatic polypeptide.
30 Proteins (n > 70) including structural proteins such as collagen, functional proteins such as hemoglobin, regulatory proteins such as the dopamine and thrombin receptors.
Depsipeptides which include a derivatives of amino acids, peptides, polypeptides and proteins that contain a hydroxy and 35 amino acid residual linked by amide or ester bonds, and include peptide-related compounds such as azinothricin, actinomycin, and echinomycin.

WO96/12~2 PCT~S9S/12861 2) a nucleotide derivative of the form (NUCL)n, which includes natural and synthetic nucleotides (n = 1), such as adenosine, thymine, guanidine, undine, cytosine, derivatives of these and a variety of variants and mimetics of the purine 5 ring, the sugar ring, the phosphate linkage and combinations of some or all of these. Nucleotide probes (n = 2 - 25) and oligonucleotides (n > 25) including all of the various possible; homo and hetero-synthetic combinations and permutations of the naturally occurring nucleotides;
10 derivatives and variants containing synthetic purine or pyrimidine species, or mimics of these; various sugar ring mimetics; and a wide variety of alternate backbone analogs, including but not limited to phosphodiester, phosphorothionate, phosphorodithionate, phosphoramidate, alkyl 15 phosphotriester, sulfamate, 3'-thioforimacetal, methylene (methylimino), 3-N-carbamate, morpholino carbamate and peptide nucleic acid analogs.
3) a carbohydrate derivative of the form (CH)ol which would include natural physiologically active carbohydrates;
20 related compounds, such as glucose, galactose, sialic acids, ~-D-glucosylamine and nojorimycin, which are both inhibitors of glucosidase; pseudo sugars, such as 5~-carba-2-D-galactopyranose, which is known to inhibit the growth of Klebsiella pneumonia (n = 1); synthetic carbohydrate residues 25 and derivatives of these (n = 1) and all of the complex oligomeric permutations of these as found in nature, including high mannose oligosaccharides, the known antibiotic streptomycin (n > 1).

4) a naturally occurring or synthetic organic 30 structural motif. The term 'motif' is defined as an organic molecule having or containing a specific structure that has molecular recognition characteristics, such as a molecule having a complementary structure to an enzyme active site, for example. This term includes any of the well known basic 35 structures of pharmaceutical compounds including pharmacophores, or metabolites thereof. These basic structures include beta-lactams, such as penicillin, known to - - ~

inhibit bacterial cell wall biosynthesis; dibenzazepines, known to bind to CNS receptors and used as antidepressants;
polypeptide macrolides, known to bind to bacterial ribosymes, etc. These structural motifs are generally known to have 5 specific desirable binding properties to ligand acceptors.
5) a reporter element, such as a natural or synthetic dye or a residue capable of photographic amplification which possesses reactive groups that may be synthetically incorporated into the aminimide structure or reaction scheme, ~0 and may be attached through the groups without adversely interfering or affecting with the reporting functionality of the group. Preferred reactive groups are amino, thio, hydroxy, carboxylic acid, carboxylic acid ester, particularly methyl ester, acid chloride, isocyanate alkyl halides, aryl 15 halides and oxirane groups.

6) an organic moiety containing a polymerizable group such as a double bond, or other functionalities capable of undergoing condensation polymerization or copolymerization.
Suitable groups include vinyl groups, oxirane group, 20 carboxylic acids, acid chlorides, esters, amides, azlactones, lactones and lactams. Other organic moieties may also be used.

7) a macromolecular component, such as a macromolecular surface or structures which may be attached to 25 the aminimide modules via the various reactive groups outlined above, in a manner where the binding of the attached species to a ligand-receptor molecule is not adversely affected and the interactive activity of the attached functionality is determined or limited by the macromolecule. Examples of 30 macromolecular components include porous and non-porous inorganic components, such as, for example, silica, alumina, zirconia, titania and the like~ as commonly used for various applications, such as normal and reverse phase chromatographic separations, water purification, pigments for paints, etc.;
35 porous and non-porous organic macromolecular components, including synthetic components such as styrene-divinyl benzene beads, various methacrylate beads, PVA beads, and, the like, WO96/12482 PCT~S95112861 commonly used for protein purification, water softening; and a variety of other applications, natural components such as native and functionalized celluloses, such as, for example, - agarose and chitin, sheet and hollow fiber membranes made from 5 nylon, polyether sulfone or any of the materials mentioned above. The molecular weight of these macromolecules may range from about lO00 Daltons to as high as possible. They may take the form of nano-particles (dp = lO0 - lO00 Angstroms), latex particles (dp = lO00 - 5000 Angstroms), porous or non-porous lO beads (dp = 0.5 - lO00 microns), membranes, gels, macroscopic surfaces or functionalized or coated versions or composites.
The structural diversity elements A, B, C, E, F and G may also be a chemical bond to a suitable organic moiety, a hydrogen atom, an organic moiety which contains a suitable 15 electrophilic group, such as an aldehyde, ester, alkyl halide, ketone, nitrile, epoxide or the like; a suitable nucleophilic group, such as a hydroxyl, amino, carboxylate, amide, carbanion, urea or the like; or one of the structural diversity elements C and/or D groups defined below. In 20 addition, structural diversity elements A, B, C, D, E, F
and/or G may join to form a ring, bi-cyclic or tri-cyclic ring system; or structure which connects to the ends of the repeating unit of the compound defined by the preceding formula; or may be separately connected to other moieties.
A more generalized structure of the composition of this invention can be represented by the following structural formulas:


~ n ~ ~

W096/1~W2 ~ ~ PCT~S95/12861 \ ~7 o ~ ~ O

~ n wherein:
a. at ~east one of the structural diversity elements A, B, C, E, F and G are as defined above and are optionally ~0 connected to each other or to other bonds and/or may each represent a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen, phosphorus, silicon or combinations thereof;
b. structural diversity elements A, B, C, E, F and G
15 are the same or different and each represents A, B, E, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein structural diversity elements E and F may be different in adjacent n units and have 20 a selected stereochemical arrangement about the carbon atom to which they are attached.
As used herein, the phrase linear chain or branched chained alkyl groups means any substituted or unsubstituted acyclic carbon-containing compounds, including alkanes, 25 alkenes and alkynes. Alkyl groups having up to 30 carbon atoms are preferred. Examples of alkyl groups include lower alkyl, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl; upper alkyl, for example, octyl, nonyl, decyl, and the like; lower alkylene, for 30 example, ethylene, propylene, propyldiene, butylene, butyldiene; upper alkenyl such as l-decene, 1-nonene, 2,6-dimethyl-5-octenyl, 6-ethyl-5-octenyl or heptenyl, and the like; alkynyl such as 1-ethynyl, 2-butynyl, 1-pentynyl and the like. The ordinary skilled artisan is familiar with numerous 35 linear and branched alkyl groups, which are within the scope of the present invention.

WO96/1~2 PCT~S9Sl12861 In addition, such alkyl group may also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group. Functional groups include but are not limited to hydroxyl, amino, carboxyl, amide, ester, 5 ether, and halogen (fluorine, chlorine, bromine and iodine), to mention but a few. Specific substituted alkyl groups can be, for example, alkoxy such as methoxy, ethoxy, butoxy, pentoxy and the like, polyhydroxy such as l,2-dihydroxypropyl, l,4-dihydroxy-l-butyl, and the like; methylamino, ethylamino, lO dimethylamino, diethylamino, triethylamino, cyclopentylamino, benzylamino, dibenzylamino, and the like; propionic, butanoic or pentanoic acid groups, and the like; formamido, acetamido, butanamido, and the like, methoxycarbonyl, ethoxycarbonyl or the like, chloroformyl, bromoformyl, l,l-chloroethyl, l5 bromoethyl, and the like, or dimethyl or diethyl ether groups or the like.
As used herein, substituted and unsubstituted carbocyclic groups of up to about 20 carbon atoms means cyclic carbon-containing compounds, including but not limited to 20 cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and the like.
Such cyclic groups may also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group. Such functional groups include those described above, and lower alkyl groups as described above.
25 The cyclic groups of the invention may further comprise a hetero-atom. For example, in a specific embodiment, structural diversity element A is cyclohexanol.
As used herein, substituted and unsubstituted aryl groups means a hydrocarbon ring bearing a system of conjugated double 30 bonds, usually comprising (4p - 2) pi bond electrons, where p is an integer equal to or greater than l. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anisyl, toluyl, xylenyl and the like. According to the present invention, aryl also includes aryloxy, aralkyl, 35 aralkyloxy and heteroaryl groups, e.g., pyrimidine, morpholine, piperazinc, piperidine, benzoic acid, toluene or thiophene and the like. These aryl groups may also be W096/1~2 PCT~S95/1~61 substituted with any number of a variety of functional groups.
In addition to the functional groups described above in connection with substituted alkyl groups and carbocyclic groups, functional groups on the aryl groups can be nitro 5 groups.
As mentioned above, structural diversity elements can also represent any combination of alkyl, carbocyclic or aryl groups; for example, l-cyclohexylpropyl, benzylcyclohexylmethyl, 2-cyclohexyl-propyl 2,2-lO methylcyclohexylpropyl, 2,2-methylphenylpropyl, 2,2 methylphenybutyl, and the like.
C. A and G may be a chemical bond or a connecting group that includes a terminal carbon atom for attachment to the quaternary nitrogen and G may be different in adjacent n 15 units; and d. n is an integer greater than 0.

In one embodiment of the invention, at least one of A, B, C, D, E, F and G represents, an organic or inorganic 20 macromolecular surface. Examples of preferred macromolecular surfaces include ceramics such as silica and alumina, porous and non-porous beads, polymers such as a latex in the form of beads, membranes, gels, macroscopic surfaces or coated versions or composites or hybrids thereof. This 25 functionalized surface may be represented as follows.


In a further embodiment of the invention, the above roles of diversity elements A and E are reversed, so that E is the substituent selected from the foregoing list and A represents 35 a functionalized surface, as shown below.

WO96J1~82 PCT~S95/12861 .. ~

SURFACE~ ~ ~N ~) In a further embodiment of the invention, the above roles of diversity elements A and B are reversed, so that A is the substituent selected from the foregoing list and B represents lO a functionalized surface, as shown below.

~ C

In a third preferred embodiment of the invention, either diversity elements A, B, E, two, three or all four contain one or more double bonds capable of undergoing free-radical 20 polymerization or co-polymerization to produce achiral or chiral oligomers, polymers, copolymers, etc.
From the preceding, it is seen that the skilled artisan can design a particular compound in an attempt to achieve a desired goal. In the area of pharmaceuticals, for example, the 25 backbone can be used as a starting point for attachment of a wide variety of diversity elements. The pharmacological activity of the resulting compounds can be easily and accurately screened, since the final structure of the compound is predictable and highly controlled.
Methods of screening or testing hydroxyethyl aminimide compounds for their reactivity and/or complementary nature with respect to a second chemical compound or class of compounds or enzyme, molecular recognition structure receptor is detailed in U.S. Patent application 08/248,263 by Joseph C.
35 Hogan, filed May 23, 1994, the entire content of which is specifically incorporated by reference herein.

WO96/1~2 PCT~S95/1~61 The human immunodeficiency virus (HIV) has been implicated as the causative agent of acquired immune deficiency syndrome (AIDS) (see Popovic; et at., Science, 1984, 198, 497). The RNA genome of the HIV retrovirus encodes 5 an aspartic protease known as the HIV-1 protease (see Kramer, H. A.; et al., Science, 1986, 231, 1580). This protease is required for malitration and proliferation of the infectious virion. The role of the HIV-1 protease is to cleave, viral precursor proteins, in particular the Gag and Pol precursor 10 proteins, into, their active forms (see Darke, P. L.; et al., Biochem. Biophys. Res. Comm., 1988, 156, 297). The HIV-1 protease is formed by the homodimerization of a 99 amino acid polypeptide; the active site of the protease is at the interface of the two subunits, with each subunit contributing 15 one of the essential aspartic acid residues required for catalysis. The X-ray crystal structure of the HIV-1 protease has been solved and shows that the dimer structure is of C2 symmetry in its unbound form (see Navia, M. A.; et al., Nature, 1989, 337, 615). The structure clearly illustrates 20 the extended active site of the protein, which incorporates processing site of S4-S3' (using the subsite nomenclature of Schechter and Berger, Biochem. Biophys. Res. Comm., 1967, 27, 157). Independent substrate cleavage assays have indicated that substrate specificity of HIV-1 protease is significantly 25 determined by subsites S2-S2' (see Pettit, S. C.; et al., Persp. in Drug Disc. Des. 1993, l, 69).
To date, numerous inhibitors of HIV-1 protease have been reported in the literature (see, for instance, Wlodawer, A.;
Erickson, J. W., Annu. Rev. Biochem., 1993, 62, 543). In 30 certain examples, co-crystal structures of the protein/inhibitor complex have been solved, for example see C.
L. Waller et al., ~. Med. Chem., 1993, 36, 4152-4160, allowing researchers to better understand the structure/activity relationships of the active inhibitors. Approaches towards 35 structure-based inhibitor design have taken three routes: 1.
transition-state analog inhibitors (see Grobelny, D.; et al., Biochem. Biophys. Res. Comm., 1990, 169, 1111); 2. substrate WO96/1~2 PCT~S95/12861 analog inhibitors (see Moore, M. L.; Dreyer, G . B , Persp . in Drug Disc. Des . 1993, 1, 85); and, 3. de novo designed inhibitors (see Lam, P. Y. S.; et al., Science, 1994, 263, 380). In the design of transition-state analogues, 5 researchers have attempted to replace the scissile amide bond with a non-hydrolyzable peptide isostere. A selection of successful inhibitor designs is illustrated in Figure l. Some of the more potent inhibitors of HIV-l protease reported are shown in Figure 2.

NonnalPeptide H , N ~ ~ ~ N
Hy~ lami,~e ~u~Amide R O R

_ ~ H
~iy~o~ lene S~tine ~, OH O

H~ I Y H ~/,~Y

Dlhy~ xy-~tl.yle~e Pl.~, 1. .sile Figure l: Examples of transition-state isosteres employed as inhibitors of proteases.

WO 96/12482 PCI~/US95/12861 Va U 8~61~ Kl~ 1 nU

10 >¶~ ~ .~N-- J~3 Ro~1~88 Kl ~ 0.3 n~

OH ~ ~

O H ~ ~OH

L~9~02 ~=o~n~ ~

25 Figure 2: Previous Inhibitors of HIV-l protease.

This invention discloses a novel transition-state analog inhibitor structure, which is an effective antagonist of the HIV-l protease. In this peptide isostere, the scissile amide 30 bond has been replaced with an aminimide linkage as illustrated in Figure 3. Among the advantages of using an aminimide isostere are their ease of syntheses, and the ability to modify the target structures in a modular fashion by varying either the epoxide, hydrazine, or ester components 35 used in the syntheses of new compounds. Furthermore, the aminimide moiety confers an increase water solubility of the synthesized compounds.

W096/12482 PCT~S9511~61 Q

5 ~ ~o,~

Aminimide Figure 3: Structure of an aminimide isostere.

Our prospective inhibitor I was designed using a model based on data from previous inhibition studies with HIV-l protease. Compound I was modeled in the active-site of HIV-l protease using the crystallographic data of the protein complexed with the inhibitor U-855488e (Brookhaven PDB
15 identification code 8HVP). Energy refinements of the inhibitor in the presence of the fixed protein and in vacuo showed that no large conformational changes in the inhibitor were required in order to adopt the bound conformation.

Examples In order to exemplify the results achieved using the chemical backbone of the present invention, the following examples are provided without any intent to limit the scope of the instant invention to the discussion therein, all parts are 25 by weight unless otherwise indicated.

Example l The following is one example of the utility of a hydroxyethyl aminimide moiety being used as a motif for the 30 design of complementary molecular structures.

Experimental: Synthesis Synthesis of compound I required the three building blocks for the aminimide: l. The enantiomerically pure 35 t-butoxycarbonyl (t-BOC) protected epoxide (l), derived from phenylalanine; 2. the benzyl methyl hydrazine (2) and 3.
methyl benzoate (3).

WO 96~12482 P~ 9SI1286 phenylalanine; 2. the benzyl methyl hydrazine (2) and 3.
methyl benzoate (3).
Ph S ~ol N~ N~ h Compound I

Pn Synthetic route 1 Ph ~olN~ ~ ~ \N- N~ ~

The synthesis of compound I is depicted in Scheme l. To an isopropanol solution of 1.43 mmol of a chiral epoxide 1 (prepared using the method of Luly, et al . , J . Org . Chem ., 1987, 52, 1487) and 1.43 mmol of hydrazine 2 (synthesized from 20 the procedure of Ohme, R.; Preuschof, H ., ~ . Pract . Chem ., 1970, 312, 349) is added 1.43 mmol of the commercially available methyl ester 3. The reaction is stirred at 60 C
for 5 h. HPLC analysis of the crude reaction indicated the presence of two diastereomers, which is expected due to the 25 fixed chirality of the hydroxyethyl component and the resulting quaternary nitrogen of compound I. The solvent is removed under reduced pressure, and the crude oil is partially purified via silica gel column chromatography. A sample for enzymological testing is acquired by further purifying the 30 isolated material by recrystallization to afford a white, crystalline solid product. The product is shown to be the desired compound I by Hl NMR and mass spectroscopy. HPLC
analysis of the product indicates that isomer (a) of compound I is obtained free of isomer (b) after column chromatography 35 and repeated crystallization.

CA 02203l23 l997-04-l8 WO96/1~2 PCT~S95/12861 Experimental: Enzymology The inhibition constant (Kj) of compound Ia is determined using HIV-l protease kit supplied by NovaBiochem (cat. #10-39-OOOl, which supplies a solid phase synthesized analog of the 5 protease, incorporating the unnatural residue aminobutyric acid in place of both cysteine residues 67 and 95, along with a thioester linkage in place of the normal peptide bond between residues 51-52). A fluorometric assay is performed using a fluorogenic substrate supplied by NovaBiochem (cat.
lO #05-23-5216, Abz-Thr-Ile-Nle-Phe(N02)-Gln-Arg-NH2). Initial rates of substrate hydrolysis are determined by following the increase in the fluorescence emission at 420 nm (excitation is at 325 nm) as the substrate is digested by the protease. The initial rates of substrate cleavage is determined in the 15 absence and presence of compound Ia (up to a concentration of Ia = 500 nM). A Lineweaver-Burk analysis of Ia indicates that inhibition follows classical competitive inhibition (Figure 4). Replotting the slopes from the Lineweaver-Burk plot (Figure 5) gives a calculated Kj value of 137 nM.
2 o Llneweaver-Burk Plot tor Compound 0.3 C
o no inhibltor = 500 nM
[1] = 50 nM
~ r [1~=5 nM
30 -'~

0.0 . . . . , . ~ , 0.00 0.05 0.10 0.15 l/t~ubstrate] (ll~lA) Figure 4: A Lineweaver-Burk analysis of compound Ia.

wo 96/12482 Pcrlus9sll286 Kl Determlnation for Compound I

y - 0.58147 1 4.230~e-3x R^2 0.992 E
dope Y


O ..............
-200 -1ŻO 0 100 200 300 400 500 600 [11 nl'~
Figure 5: A Replot of the slopes obtained Lineweaver-Burk plot for compound Ia.

Given the potency of this inhibitor for HIV-1 protease, the model of the bound complex was used to rationalize structure-activity relationships for a variety of substituents and to propose additional compounds for synthesis and evaluation. In particular, fragments from all three 25 components of the inhibitor (the epoxide, the ester and the hydrazine) were evaluated as to the most feasible ways to maximize both hydrophobic and hydrogen bonding interactions complementary with the protein.
The scope of the following claims is intended to 30 encompass all obvious changes in the elements, details, materials and arrangement of parts that will occur to one of ordinary skill in the art:

International ClassificationA61K31/16, C07D213/82, A61K31/66, A61K38/00, C07D213/81, C07D307/20, C07C243/24, C07C243/40, C07D309/12, A61P43/00, C07C271/20
Cooperative ClassificationC07D307/20, C07D309/12, C07D213/81, C07D213/82, C07C243/40, C07C271/20, C07C2601/14, C07C2601/02
European ClassificationC07D307/20, C07D309/12, C07C271/20, C07C243/40, C07D213/82, C07D213/81
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