Description of the Related Art Mitochondria are the subcellular organelles that are the main energy source in cells of higher organisms, and provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes.

The electron transport chain (ETC) machinery resides in the mitochondrion, and drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP). Mitochondria also play a critical role in maintaining intracellular calcium homeostasis. In addition to their role in energy production in growing cells, mitochondria (or, at least, mitochondrial components) are required for at least some forms of programmed cell death (PCD), also known as apoptosis (Newmeyer et al., Cell 79 : 353-364,1994; Liu et al., Cell 86 : 147-157,1996). Apoptosis is required for normal development of the nervous system and functioning of the immune system. However, some disease states are thought to be associated with either insufficient or excessive levels of apoptosis (e. g., cancer and autoimmune diseases in the first instance, and stroke damage and neurodegeneration in Alzheimer's disease in the latter case). For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed (Science 281 : 1309-1312,1998), Green (Cell : 695-698, 1998) and Kroemer (Nature Medicine 3: 614-620,1997).

Defective mitochondrial activity, including but not limited to failure at any step of the elaborate multi-complex mitochondrial assembly, known as the electron transport chain (ETC), may result in (i) decreases in ATP production, (ii) increases in the generation of highly reactive free radicals (e. g., superoxide, peroxynitrite and hydroxyl radicals, and hydrogen peroxide), (iii) disturbances in intracellular calcium homeostasis and (iv) the release of factors that initiate the apoptosis cascade. Because of these biochemical changes, mitochondrial dysfunction has the potential to cause widespread damage to cells and tissues. For example, oxygen free radical induced lipid

peroxidation is a well established pathogenic mechanism in central nervous system (CNS) injury such as that found in a number of degenerative diseases, and in ischemia (i. e., stroke).

Cells from long-lived tissue that have high energy demands such as neurons, pancreatic islet cells, cardiac and muscle cells are particularly vulnerable to mitochondrial dysfunction. A number of degenerative diseases may thus be caused by or associated with either direct or indirect alterations in mitochondrial function. These include Alzheimer's Disease, diabetes mellitus, Parkinson's Disease, neuronal and cardiac ischemia, Huntington's disease and other related polyglutamine diseases (spinalbulbar muscular atrophy, Machado-Joseph disease (SCA-3), dentatorubro- pallidoluysian atrophy (DRPLA) and spinocerebellar ataxias, dystonia, Leber's hereditary optic neuropathy, schizophrenia, and myodegenerative disorders such as mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS), and myoclonic epilepsy ragged red fiber syndrome (MERRF).

Increasing evidence points to the fundamental role of mitochondrial dysfunction in neurodegenerative diseases (Beal, Biochim. Biophys. Acta 1366 : 211- 223,1998), and recent studies implicate mitochondria for regulating the events that lead to necrotic and apoptotic cell death (Susin et al., Biochim. Biophys. Acta 1366 : 151-168, 1998). Stressed (stressors include free radicals, high intracellular calcium, loss of ATP, among others) mitochondria may release pre-formed soluble factors that can initiate apoptosis through an interaction with novel apoptosomes (Marchetti et al., Cancer Res.

56 : 2033-38,1996; Li et al., Cell 91 : 479-89,1997). Release of preformed soluble factors by stressed mitochondria, like cytochrome c, may occur as a consequence of a number events. In some cases, release of apoptotic molecules (apoptogens) occurs when mitochondria undergo a sudden change in permeability to cytosolic solutes. This process has been termed"permeability transition". There is strong evidence that suggests that the loss of mitochondrial function may be due to the activation of the mitochondrial permeability transition pore, a Ca2+ regulated inner membrane megachannel. Opening of the mitochondrial permeability transition pore results in the exchange of solutes that are less than 1500 daltons in size, collapse of the mitochondrial membrane potential, and uncoupling of the electron transport chain. In other cases, the permeability may be more subtle and perhaps more localized to restricted regions of a mitochondrion. In still other cases, overt permeability transition may not occur but apoptogens can still be released as a consequence of mitochondrial abnormalities. The magnitude of stress (ROS, intracellular calcium levels) influences the changes in mitochondrial physiology that ultimately determine whether cell death occurs via a

necrotic or apoptotic pathway. To the extent that apoptotic cell death is a prominent feature of degenerative diseases, mitochondrial dysfunction may be a critical factor in diseaseprogression.

Whereas mitochondria-mediated apoptosis may be critical in degenerative diseases, it is thought that disorders such as cancer involve the unregulated and undesirable growth (hyperproliferation) of cells that have somehow. escaped a mechanism that normally triggers apoptosis in such undesirable cells. Enhanced expression of the anti-apoptotic protein, Bcl-2 and its homologues is involved in the pathogenesis of numerous human cancers. Bcl-2 acts by inhibiting programmed cell death and overexpression of Bel-2 and the related Bcl-xL block mitochondrial release of cytochrome c from mitochondria and the activation of caspase 3 (Yang et al, Science 275 : 1129-1132,1997; Kluck et al., Science 275 : 1132-1136,1997; Kharbanda et al., Proc. Natl. Acad. Sci. USA 94 : 6939-6942,1997). Bcl-2 also binds to several proteins that are involved in death regulation (Reed, Nature 387 : 773-779,1997). Over expression of Bcl-2 and Bcl-xL protect against the mitochondrial dysfunction preceding nuclear apoptosis that is induced by chemotherapeutic agents. In addition, acquired multi-drug resistance to cytotoxic drugs is associated with inhibition cytochrome c release that is dependent on overexpression of Bcl-xL (Kojima et al., J Biol. Chem.

273: 16647-16650,1998). Because mitochondria have been implicated in apoptosis, it is expected that agents that interact with mitochondrial components will effect a cell's capacity to undergo apoptosis. Thus, agents that induce or promote apoptosis in hyperproliferative cells are expected to be useful in treating such hyperproliferative disorders and diseases.

Thus, alteration'of mitochondrial function has great potential for a broad- based therapeutic strategy for designing drugs to treat degenerative diseases as well as hyperproliferative diseases. The mitochondrial permeability transition pore is a key target for the prevention of mitochondrial function collapse that leads to cell death, or the induction of apoptosis in cancer cells via dysregulation of mitochondria. This megachannel is a multi-protein complex in which the adenine nucleotide translocator (ANT) has been implicated as the critical molecular component.

ANT is located in the inner mitochondrial inner membrane and it facilitates transport of ADP and ATP across the mitochondrial inner membrane. In humans there are three genetic isoforms (ANT1, ANT2 and ANT3), each with different tissue expression patterns. ANT1 is highly expressed in heart and skeletal muscle and is induced during myoblast differentiation. ANT2 is overexpressed in a variety of hyperproliferative cells, tumors and neoplastically transformed cells with high

glycolytic rates (Battini et al., R Biol. Chem. 262 : 4355-4359,1987 ; Torroni et al., J.

Biol. Chem. 265 : 20589-20593,1990; Faure-Vigny et al., Mol. Carcinogen. 16 : 165-172, 1996 ; Heddi et al., Biochim. Biophys. Acta 1316 : 203-209, 1996; and Giraud et al., J Mol. Biol. 281 : 409-418,1998). ANT3 is ubiquitously expressed in all tissues. ANT3 transcript level is proportional to the level of oxidative metabolism in a given tissue.

Two different conformations of ANT have been demonstrated on the basis of interactions with specific ligands, namely the inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BKA). Ligands can bind to ANT in an asymmetric fashion, either from the matrix (m) or from the cytosolic (c) side of the inner mitochondrial membrane. For example, CATR binds to ANT in the c-conformation and induces permeability transition, while BKA interacts with ANT in the m- conformation and inhibits permeability transition in response to a variety of apoptotic stimuli (see, Budd et al., PNAS US 97 : 6161-6166,2000) Different small molecule ligands of ANT isoforms therefore can possess a spectrum of activities-that is, they can act as cell protective agents targeted for degenerative diseases and as cytotoxic agents for hyperproliferative diseases.

Bongkrekic acid (BKA) is a polyenoic triacid that is produced by the microorganism Pseudomonas cocovenenans.

In the protonated form, BKA diffuses through the lipid phase of the inner mitochondrial membrane and binds to the matrix side of ANT with high affinity (2 x 10-8 M). Upon binding, BKA is believed to stabilize ANT in the m-conformation. Accordingly, BKA is believed to have significant potential as a cell-protective agent. Unfortunately, there are a number of problems associated with BKA. For example, large quantities of BKA are difficult to obtain by fermentation. While a convergent total synthesis of BKA has been reported (R Am. Chem. Soc. 106 : 462-463,1984), this technique involves 33 steps which makes it tedious to produce.

Accordingly, there is a need for agents that are cytotoxic with regard to undesirable cells and tissues, as well as agents that limit or prevent damage to desirable organelles, cells and tissues resulting from various consequences of mitochondrial dysfunction. In the former instance, such agents are desired to treat hyperproliferative

diseases and disorders, or as species-specific antibiotics, herbicides or insecticides. In the latter instance, because mitochondria are essential organelles for producing metabolic energy, agents that protect mitochondria against injury are desired for the prevention, treatment and management of degenerative diseases, including mitochondria associated diseases. The present invention fulfills these needs and provides other related advantages.

BRIEF SUMMARY OF THE INVENTION In brief, the present invention generally relates to compounds which inhibit mitochondrial permeability transition by binding to the adenine nucleotide translocator (ANT) in the m-conformation, and thereby have activity as cell-protective agents. In addition, this invention is directed to compositions containing a compound of this invention in combination with a pharmaceutically acceptable carrier or diluent, as well as to methods related to the administration of such compounds and/or composition to an animal in need thereof (including humans).

The compounds of this invention have the following general structure (I): including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein RI, R2, R3 and A are as defined below.

The compounds of the invention are, in some aspects, mimics of bongkrekic acid (BKA) and function as agonists or antagonists of protein targets of BKA that elicit cell-protective or cytotoxic responses. In one embodiment, the target (s) of BKA that is (are) affected by a compound of the invention is (are) one or more isoforms of ANT. In a related embodiment, a compound of the invention binds preferentially to a specific isoform of ANT, but not to other ANT isoforms, from a single species of organism.

In embodiments wherein a compound of the invention is cytotoxic, the compound has, for example, remedial, therapeutic, palliative, rehabilitative,

preventative, disease-impeditive or prophylactic activity with regard to hyperproliferative diseases and disorders such as cancer, psoriasis and the like. In such embodiments, a cytotoxic compound of the invention (a) binds preferentially to a specific isoform of ANT, but not to other ANT isoforms, from a single species of organism, wherein the preferentially-bound isoform of ANT is overexpressed in undesirable hyperproliferative cells; or (b) preferentially enters undesirable hyperproliferative cells.

In further embodiments wherein a compound of the invention is cytotoxic, the compound acts as a species-specific antibiotic, herbicide or insecticide.

Such compounds binds preferentially to, respectively, (i) one or more ANT proteins from an undesirable parasitic or infective species (e. g., a eukaryotic parasite such as members of the Trypanosoma or Leishmania genera), but not to the corresponding ANT protein (s) from the host species (i. e., a mammal such as a human); (ii) one or more ANT proteins from an undesirable plant species (e. g., a weed), but not to the corresponding ANT protein (s) from desirable plants having economic value (e. g., crops or decorative plants), desirable insects (e. g., bees) or mammals including humans ; or (iii) one or more ANT proteins from an undesirable insect (e. g., members of the genus Lepidoptera), but not to the corresponding ANT protein (s) from desirable insects, desirable plants or mammals including humans.

In embodiments wherein a compound of the invention is cyto-protective, the compound is used, for example, to prevent, treat or manage neurodegenerative diseases and disorders, (e. g., Alzheimer's disease and Parkinson's disease, or to ameliorate the undesirable effects of acute events such as ischemia or cardiac arrest.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. To that end, various references are set forth herein which describe in more detail certain aspects of this invention, and are each incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention is generally directed to compounds and to pharmaceutical compositions containing the same, as well as to methods for treating diseases by altering mitochondrial function that affects cellular processes. The compounds of this invention have the following general structure (I) :

including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein: A is a direct bond, alkyldiyl, substituted alkyldiyl,-O- (alkyldiyl)-, - O- (substituted alkyldiyl)-,- (alkyldiyl)-O-,- (substituted alkyldiyl)-O- ,-N(R')-(alkyldiyl)-,-N (R')-(substituted alkyldiyl)-,-(alkyldiyl)- N (R')-,- (substituted alkyldiyl)-N (R')-, heterocyclediyl, substituted heterocyclediyl, heterocyclealkyldiyl or substituted heterocyclealkyldiyl, wherein R'is hydrogen or alkyl ; Rl is hydroxy, alkoxy, aryloxy, arylalkyloxy, amino, or mono-or di- alkylamino; R2 is hydrogen, alkyl, substituted alky, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocylealkyl or substituted heterocyclealkyl ; and R3 is alkyl, substituted alky, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocylealkyl or substituted heterocyclealkyl.

As used herein, the terms used above have the following meaning: "Alkyl"means a straight chain or branched, saturated or unsaturated, cyclic or non-cyclic hydrocarbon having from 1 to 10 carbon atoms, while"lower alkyl" has the same meaning but only has from 1 to 6 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (also referred to as an"alkenyl"or"alkynyl", respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1- butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like ; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like. Representative saturated

cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, (cycloalkyl) CH2-, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like. Cycloalkyls are also referred to herein as"carbocyclic"rings systems, and include bi-and tri-cyclic ring systems having from 8 to 14 carbon atoms such as a cycloalkyl (such as cyclo pentane or cyclohexane) fused to one or more aromatic (such as phenyl) or non-aromatic (such as cyclohexane) carbocyclic rings.

"Alkyldiyl"means a divalent alkyl from which two hydrogen atoms are taken from the same carbon atom or from different carbon atoms, including divalent alkyl, alkenyl and alkynyl, as well as saturated and unsaturated carbocyclic rings as defined above.

"Halogen"means fluorine, chlorine, bromine or iodine.

"Oxo"means a carbonyl group (ive., =0).

"Mono-or di-alkylamino"means an amino substituted with one alkyl or with two alkyls, respectively. <BR> <BR> <BR> <P> "Alkoxy"means-O- (alkyl).<BR> <BR> <BR> <BR> <BR> <BR> <P> "Aryloxy"means-0- (aryl).<BR> <BR> <BR> <BR> <BR> <P> "Arylalkyloxy"means-O- (arylalkyl).

"Aryl"means an aromatic carbocyclic moiety such as phenyl or naphthyl.

"Arylalkyl"means an alkyl having at least one alkyl hydrogen atom replaced with an aryl moiety, such as benzyl,-(CH2) 2phenyl,-(CH2) 3phenyl, - CH (phenyl) 2, and the like.

"Heteroaryl"means an aromatic heterocycle ring of 5-to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono-and bicyclic ring systems.

Representative heteroaryls are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl.

"Heteroarylalkyl"means an alkyl having at least one alkyl. hydrogen atom replaced with a heteroaryl moiety, such as-CH2pyridinyl,-CH2pyrimidinyl, and the like.

"Heterocycle"means a 5-to 7-membered monocyclic, or 7-to 10- membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally

oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined above. Thus, in addition to the heteroaryls listed above, heterocycles also include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

"Heterocyclediyl"means a divalent heterocycle from which two hydrogen atoms are taken from the same atom or from different atoms.

"Heterocyclealkyl"means an alkyl having at least one alkyl hydrogen atom replaced with a heterocycle, such as-CH2morpholinyl, and the like.

"Heterocylcealkyldiyl"means a divalent heterocyclealkyl from which two hydrogen atoms are taken from the same atom or from different atoms.

The term"substituted"as used herein means any of the above groups (i. e., alkyl, aryl, arylalkyl, heterocycle, heterocyclealkyl, alkyldiyl, heterocyclediyl and heterocylcealkyldiyl) wherein at least one hydrogen atom is replaced with a substituent.

In the case of an oxo substituent ("=O") two hydrogen atoms are replaced. Substituents include halogen, hydroxy, oxoalkyl, substituted alkyl (such as haloalkyl, mono-or di- substituted aminoalkyl, alkyloxyalkyl, and the like), aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,-NRaRb,-NRaC (=O) Rb,-NRcC (=O) NRaRb,-NRaC (=O) ORb <BR> <BR> <BR> - NRaS02Rb,-ORa,-C (=O) Ra-C (=O) ORa-C (=O) NRaRb,-OC (=O) Ra,-OC (=O) ORa, -OC (=O) NRaRb,-NRaSO2Rb"or a radical of the formula-Y-Z-Ra where Y is alkanediyl, substituted alkanediyl, or a direct bond, Z is-O-,-S-,-S (-O)-,-S (=O) 2-, -N (Rb)-,-C (=O)-,-C (=O) O-,-OC (=O)-,-N (Rb) C (=O)-,-C (=O) N (Rb)- or a direct bond, wherein Ra, Rb and Re are the same or different and independently hydrogen, amino, alkyl, substituted alkyl (including halogenated alkyl), aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle, heterocylealkyl or substituted heterocyclealkyl, or wherein Ra and Rb taken together with the nitrogen atom to which they are attached form a heterocycle or substituted heterocycle.

In one embodiment, A is a direct bond and the compounds have the following structure (II) :

In another embodiments, and depending upon the choice of A, the compounds have one of the following structures (MI) through (IX), wherein each of alkyldiyl, heterocyclediyl and/or heterocylcealkyldiyl moieties may be unsubstituted or substituted with one or more substituents as defined above. 0 0 RZ R2 Rt O R1 O H0"0" HO--O" R3 o H Rs HO O HO O (HI) (IV) 0 0 Rz Rz RI--Iy 0 N/ (alkyldiyl) O/ N'N (R') (alkyldiyl)/ j"T Tm f T Tis Ho, 00 H HOo 0 H HO O HO O (V) (VI) o o RyRz R I. RZ N (alkyldiyl) N (R'/N/ (lleterocyclediyl)/ y N) , R3 1-1 N R3 HO 0 HO O (Vil) (VIII) o R2 Ri O N/ (heterocyclealkyldiyl) (1X) (in) OX) HO p \ H

The compounds of the present invention may generally be utilized as the free acid or base. Alternatively, the compounds of this invention may be used in the form of acid or based addition salts. Acid addition salts of the free base amino compounds of the present invention may be prepared by methods well known in the art, and may be formed from organic and inorganic acids. Suitable organic acids include maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Based addition salts include the ammonium ion, other suitable cations. Thus, the term"pharmaceutically acceptable salt"of structure (I) is intended to encompass any and all acceptable salt forms. Suitable salts in this context may be found in Remington's Pharmaceuitcal Sciences, 17 th ed., Mack Publishing Co., Easton, PA, 1985, which is hereby incorporated by reference.

In addition, prodrugs are also included within the context of this invention. Prodrugs are any covalently bonded carriers that release a compound of structure (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound.

With regard to stereoisomers, the compounds of structure (I) may have chiral centers and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers. All such isomeric forms are included within the present invention, including mixtures thereof. Furthermore, some of the crystalline forms of the compounds of structure (I) may exist as polymorphs, which are included in the present invention. In addition, some of the compounds of structure (I) may also form solvates with water or other organic solvents. Such solvates are similarly included within the scope of this invention.

The compounds of this invention are typically formulated in conjunction with a suitable pharmaceutical carrier or diluent, and such compositions may be in any

form that allows for the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral (e. g., sublingually or buccally), sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrameatal, intraurethral injection or infusion techniques. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.

Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.

For oral administration, which is the route of administration in preferred embodiments, an excipient and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose.

Coloring and/or flavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e. g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to one or more agents that impair MCA activity, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oral administration should contain an amount of an agent that impairs MCA activity as provided herein such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 wt% of the agent in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition.

Preferred oral compositions contain between about 4% and about 50% of the agent (s) that alter mitochondrial function. Preferred compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of active compound.

The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the agent that impairs MCA activity of from about 0.1 to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form, e. g., of a suppository that will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol. In the methods of the invention, the agent (s) that alter mitochondrial function identified as described herein may be administered through use of insert (s), bead (s), timed-release formulation (s), patch (es) or fast-release formulation (s).

It will be evident to those of ordinary skill in the art that the optimal dosage of the compound may depend on the weight and physical condition of the patient; on the severity and longevity of the physical condition being treated; on the particular form of the active ingredient, the manner of administration and the composition employed. The use of the minimum dosage that is sufficient to provide effective therapy is usually preferred. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those having ordinary skill in the art and which, as noted above, will typically involve determination of whether circulating

insulin and/or glucose concentrations fall within acceptable parameters according to well-known techniques. Suitable dose sizes will vary with the size, condition and metabolism of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg individual. It is to be understood that according to certain embodiments the agent may be membrane permeable, preferably permeable through the plasma membrane and/or through mitochondrial outer and/or inner membranes. According to certain other embodiments, the use of a compound as disclosed herein in a chemotherapeutic composition can involve such an agent being bound to another compound, for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said agent.

Another embodiment of the invention involves the creation and identification of compounds that increase the degree or enhance the rate of apoptosis in hyperproliferative cells present in diseases and disorders such as cancer and psoriasis (note that, for the purposes of the disclosure, the term"hyperproliferative disease or disorder"specifically excludes pregnancy). Because oncogenic changes render certain tumors more susceptible to apoptosis (Evan and Littlewood, Science 281 : 1317,1998), such agents are expected to be useful for treating such hyperproliferative diseases or disorders. In a related embodiment, a biological sample from a patient having or suspected of having a hyperproliferative disease or disorder are evaluated for their susceptibility to such agents using the methods of the invention. Cybrid cells are a preferred biological sample in this embodiment.

A further embodiment of the invention involves the creation and identification of agents that alter mitochondrial function and/or selectively affect MPT in mitochondria and/or cell death in a species-specific manner. By"species-specific manner"it is meant that such agents affect MPT or cell death in a first organism belonging to one species but not in a second organism belonging to another species.

This embodiment of the invention is used in a variety of methods.

For example, this embodiment of the invention to identify agents that selectively induce MPT and/or apoptosis in biological samples comprising cells or mitochondria derived from different species, e. g., in trypanasomes (Ashkenazi and Dixit, Science 281 : 1305,1998), and other eukaryotic pathogens and parasites, including but not limited to insects, but which do not induce MPT and/or apoptosis in their mammalian hosts. Such agents are expected to be useful for the prophylactic or therapeutic management of such pathogens and parasites.

As another example, this embodiment of the invention is used to create and identify agents that selectively induce MPT and/or apoptosis in biological samples

comprising cells or mitochondria derived from undesirable plants (e. g., weeds) but not in desirable plants (e. g., crops), or in undesirable insects (in particular, members of the family Lepidoptera and other crop-damaging insects) but not in desirable insects (e. g., bees) or desirable plants. Such agents are expected to be useful for the management and control of such undesirable plants and insects. Cultured insect cells, including for example, the Sf9 and Sf21 cell lines derived from Spodoptera frugiperda, and the HIGH FIVE (cell line from Trichopolusia ni (these three cell lines are available from InVitrogen, Carlsbad, California) may be biological sample in certain such embodiments of the invention.

The suitability of a compound for treatment of a subject having a disease associated with altered mitochondrial function may be determined by various assay methods. Such compounds are active in one or more of the following assays for measuring mitochondrial permeability transition, or in any other assay known in the art that directly or indirectly measures induction of MPT, MPT itself or any downstream sequelae of MPT, or that may be useful for identifying mitochondrial permeability pore components (i. e., molecules that regulate MPT). Accordingly, it is also an aspect of the invention to provide compositions and methods for treating a disease associated with altered mitochondrial function by administering a composition that regulates MPT. In embodiments of the invention, agents to be formulated into such compositions may be identified by the following assay methods.

A. Assay for Mitochondrial Permeability Transition (MPT) Using 2-, 4- Dimethylaminostvtyl-N-Methylpvridinium (DASPMI) According to this assay, one may determine the ability of a compound of the invention to inhibit the loss of mitochondrial membrane potential that accompanies mitochondrial dysfunction. As noted above, maintenance of a mitochondrial membrane potential (atom) may be compromised as a consequence of mitochondrial dysfunction.

This loss of membrane potential, or mitochondrial permeability transition (MPT), can be quantitatively measured using the mitochondria-selective fluorescent probe 2-, 4- dimethylaminostyryl-N-methylpyridinium (DASPMI).

Upon introduction into cell cultures, DASPMI accumulates in mitochondria in a manner that is dependent on, and proportional to, mitochondrial membrane potential. If mitochondrial function is disrupted in such a manner as to compromise membrane potential, the fluorescent indicator compound leaks out of the membrane bounded organelle with a concomitant loss of detectable fluorescence.

Fluorimetric measurement of the rate of decay of mitochondria associated DASPMI

fluorescence provides a quantitative measure of loss of membrane potential, or MPT.

Because mitochondrial dysfunction may be the result of multiple factors that directly or indirectly induce MPT as described above (e. g., ROS, calcium flux), agents that retard the rate of loss of DASPMI fluorescence may be effective agents for treating diseases associated with altered mitochondrial function, according to the methods of this invention.

B. Assay of Apoptosis in Cells Treated with Mitochondria Protecting Agents As noted above, mitochondrial dysfunction may be an induction signal for cellular apoptosis. According to this assay, one may determine the ability of a compound agent to inhibit or delay the onset of apoptosis. Mitochondrial dysfunction may be present in cells known or suspected of being derived from a subject having a disease associated with altered mitochondrial function, or mitochondrial dysfunction may be induced in normal cells by one or more of a variety of physiological and biochemical stimuli, with which those having skill in the art will be familiar.

In one aspect of the apoptosis assay, cells that are suspected of undergoing apoptosis may be examined for morphological, permeability or other changes that are indicative of an apoptotic state. For example, apoptosis in many cell types may cause altered morphological appearance such as plasma membrane blebbing, cell shape change, loss of substrate adhesion properties or other morphological changes that can be readily detected by those skilled in the art using light microscopy. As another example, cells undergoing apoptosis may exhibit fragmentation and disintegration of chromosomes, which may be apparent by microscopy and/or through the use of DNA specific or chromatin specific dyes that are known in the art, including fluorescent dyes. Such cells may also exhibit altered plasma membrane permeability properties as may be readily detected through the use of vital dyes (e. g., propidium iodide, trypan blue) or by the detection of lactate dehydrogenase leakage into the extracellular milieu. These and other means for detecting apoptotic cells by morphologic criteria, altered plasma membrane permeability and related changes will be apparent to those familiar with the art.

In another aspect of an apoptosis assay, translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is detected by measuring outer leaflet binding by the PS-specific protein annexin (Martin et al., J. Exp. Med. 182 : 1545,1995; Fadok et al., J Immunol. 148 : 2207,1992.) In another aspect of the apoptosis assay, induction of specific protease activity in a family

of apoptosis-activated proteases known as the caspases is measured, for example, by determination of caspase-mediated cleavage of specifically recognized protein substrates. These substrates may include, for example, poly- (ADP-ribose) polymerase (PARP) or other naturally occurring or synthetic peptides and proteins cleaved by caspases that are known in the art (see, e. g., Ellerby et al., J Neurosci. 17 : 6165,1997).

The synthetic peptide Z-Tyr-Val-Ala-Asp-AFC, wherein"Z"indicates a benzoyl carbonyl moiety and AFC indicates 7-amino-4-trifluoromethylcoumarin (Kluck et al., Science 275 : 1132,1997 ; Nicholson et al., Nature 376 : 37,1995), is one such substrate.

Other substrates include nuclear proteins such as U1-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, J. Cell. Biochem. 64 : 50,1997; Cohen, Biochem. J 326 : 1,1997).

As described above, the mitochondrial inner membrane may exhibit highly selective and regulated permeability for many small molecules, including certain cations, but is impermeable to large (>-10 kDa) molecules (see, e. g., Quinn, 1976 The Molecular Biology of Cell Membranes, University Park Press, Baltimore, Maryland).

Thus, in another aspect of the apoptosis assay, detection of the mitochondrial protein cytochrome c that has leaked out of mitochondria in apoptotic cells may provide an apoptosis indicator that can be readily determined (Liu et al., Cell 86 : 147,1996). Such detection of cytochrome c may be performed spectrophotometrically, immunochemically or by other well established methods for determining the presence of a specific protein.

Release of cytochrome c from cells challenged with apoptotic stimuli (e. g., ionomycin, a well-known calcium ionophore) can be followed by a variety of immunological methods. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry coupled with affinity capture is particularly suitable for such analysis since apo-cytochrome c and holo-cytochrome c can be distinguished on the basis of their unique molecular weights. For example, the Surface-Enhanced Laser Desorption/Ionization (SELDITM) system (Ciphergen, Palo Alto, California) may be utilized to follow the inhibition by mitochondria protecting agents of cytochrome c release from mitochondria in ionomycin treated cells. In this approach, a cytochrome c specific antibody immobilized on a solid support is used to capture released cytochrome c present in a soluble cell extract. The captured protein is then encased in a matrix of an energy absorption molecule (EAM) and is desorbed from the solid support surface using pulsed laser excitation. The molecular mass of the protein is determined by its time of flight to the detector of the SELDITM mass spectrometer.

The person of ordinary skill in the art will readily appreciate that there may be other suitable techniques for quantifying apoptosis, and such techniques for

purposes of determining the effects of mitochondria protecting agents on the induction and kinetics of apoptosis are within the scope of the assays disclosed here.

C. Assay of Electron Transport Chain (ETC) Activity in Isolated Mitochondria As described above, mitochondria associated diseases may be characterized by impaired mitochondrial respiratory activity that may be the direct or indirect consequence of elevated levels of reactive free radicals such as ROS, of elevated cytosolic free calcium concentrations or other stimuli. Accordingly, a compound for use in the treatment of a disease associated with altered mitochondrial function may restore or prevent further deterioration of ETC activity in mitochondria of individuals having mitochondria associated diseases. Assay methods for monitoring the enzymatic activities of mitochondrial ETC Complexes I, II, III, IV and ATP synthetase, and for monitoring oxygen consumption by mitochondria, are well known in the art (see, e. g., Parker et al., Neurology 44 : 1090-96,1994; Miller et al., J Neurochem..

67 : 1897,1996). It is within the scope of the methods provided by this invention to identify a suitable compound using such assays of mitochondrial function, given the relationship between mitochondrial membrane potential and ETC activity as described above. Further, mitochondrial function may be monitored by measuring the oxidation state of mitochondrial cytochrome c at 540 nm. Also as described above, oxidative damage that may arise in mitochondria associated diseases may include damage to mitochondrial components such that the oxidation state of cytochrome c, by itself or in concert with other parameters of mitochondrial function including, but not limited to, mitochondrial oxygen consumption, may be an indicator of reactive free radical damage to mitochondrial components. Accordingly, the invention provides various assays designed to test the inhibition of such oxidative damage by compounds that may influence mitochondrial membrane permeability. The various forms such assays may take will be appreciated by those familiar with the art, and are not intended to be limited by the disclosures herein, including in the Examples.

For example, Complex IV activity may be determined using commercially available cytochrome c that is fully reduced via exposure to excess ascorbate. Cytochrome c oxidation may then be monitored spectrophotometrically at 540 nm using a stirred cuvette in which the ambient oxygen above the buffer is replaced with argon. Oxygen reduction in the cuvette may be concurrently monitored using a micro oxygen electrode with which those skilled in the art will be familiar, where such an electrode may be inserted into the cuvette in a manner that preserves the argon

atmosphere of the sample, for example through a sealed rubber stopper. The reaction may be initiated by addition of a cell homogenate or, preferably a preparation of isolated mitochondria, via injection through the rubber stopper. In the assay described here, for example, a defect in complex IV activity may be correlated with an enzyme recognition site. This assay, or others based on similar principles, may permit correlation of mitochondrial respiratory activity with mitochondria membrane permeability, which may be determined according to other assays described herein.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES Example 1 SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF STRUCTURE (I) "First Component" L I R2 0 BrCH CO H Br tB2CNH2 Solid OH Solid > Support DIC/DMAP Support DIEA/DMSO, rt DMF, rt DMF, rt "SecondComponent') tBU02 R2 N02 0 o Y H02C, y O H02C Solid A Solid PyBrop/D1EA/DMAP Support 'NO "ThirdComponent" tau02 Ra o V SnCl2.2H20 A R3COX/DIEA Solid,> DMF, rt Fport 4 ° \/ o 4 0 tBu02 R, AYR2 0 y 0 TFA 0 Solid o' 1--- ' A Solid N A/I I-- N A/ Support N R3 5 HO 0

STEP 1 : Coupling Bromoacetic Acid on to Wang Resin Polystyrene Wang resin (10.0 g, 1.25 mmol/g) was shaken at room temperature with bromoacetic acid (8. 68 g, 62.5 mmol), diisopropylcarbodiimide (DIC) (9.79 ml, 62.5 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) in DMF (60 ml) in a polypropylene bottle for 4 hours. The resin was collected via vacuum filtration using a 50 ml polypropylene syringe fitted with a polyethylene frit, and washed with DMF (3 x 40 ml), methanol (3 x 40 ml), DMF (3 x 40 ml), methanol (3 x 40 ml), DCM (3 x 40 ml), methanol (3 x 40 ml), and air dried. The resulted bromoacetate resin 1 (12.0 g) was used in the next step without further analysis.

STEP 2: Displacement Reaction with Amino Esters a. Bromoacetate resin 1 (4.0 g) was shaken with glycine t-butyl ester HOAc salt (3.82 g, 20.0 mmol) and diisopropylethylamine (DIEA) (7.2 ml, 75 mmol) in DMSO (13 ml) in a 20 ml polypropylene syringe fitted with a polyethylene frit at room temperature for 24 hours. The resin was washed with DMF (3 x 20 ml), methanol (3 x 20 ml), DMF (3 x 20 ml), methanol (3 x 20 ml), DCM (3 x 20 ml), methanol (3 x 20 ml), and air dried. The resulted resin 2A was used in the next step without further analysis. b. Bromoacetate resin 1 (4.0 g) was treated with aspartic acid di-t- butyl ester HC1 salt in the same manner as described in 2a. The resulted resin 2B was used in the next step without further analysis. c. Bromoacetate resin 1 (4.0 g) was treated with glutaric acid di-t- butyl ester HC1 salt in the same manner as described in 2a. The resulted resin 2C was used in the next step without further analysis.

STEP 3: Coupling Reaction with Nitrobenzoic Acids (A = direct bond) a. One-third of resin 2A was shaken with 2-nitrobenzoic acid (1. 16 g, 6.9 mmol), DIEA (2.0 ml, 11.5 mmol), and bromo- (tris-pyrrolidino) phosphonium hexafluorophasphate (PyBrop) (3.26 g, 7.0 mmol) in DMF (10 ml) at room temperature overnight. The resin was washed with DMF (3 x 10 ml). The reaction was repeated to ensure complete coupling. The resin was washed with DMF (3 x 10 ml), methanol (3 x 10 ml), DMF (3 x 10 ml), methanol (3 x 10 ml), DCM (3 x 10 ml), mathanol (3 x 10 ml), and air dried. A small sample (-50 mg) of the resulted resin 3AA was treated with TFA/water (95/5,1.0 ml) for 1 hour at room temperature. The solution was collected via filtration. The resin was washed with acetic acid (3 x 1 ml). The combined solution was lyophilized. The residue was analyzed by 1H NMR and mass spectrometry. 1H

NMR (CD30D) _ 8. 25 (m, 1H), 7.82 (m, 1H), 7.72 (m, 1H), 7.54 (m, 1H), 4.38 (s, 2H), 4.07 (s, 2H). MS calcd. for CllHioN207 : 282. 05, found : 281 (M-H). b. One-third of resin 2A was reacted with 3-nitrobenzoic acid in the same manner as described in 3a to yield resin 3AB. 1H NMR (CD30D) _ 8. 37 (m, 1H), 8.32 (m, 1H), 7.84 (m, 1H), 7.74 (m, 1H), 4.31 (s, 2H), 4.12 (s, 2H). MS calcd. for CuHloN207 : 282.05, found: 281 (M-H). c. One third of resin 2A was reacted with 4-nitrobenzoic acid in the same manner as described in 3a to yield resin 3AC. lH NMR (CD30D) 8. 33 (d, 2H), 7.67 (d, 2H), 4.31 (s, 2H), 4.09 (s, 2H). MS calcd. for CnHioN207 : 282.05, found: 281 (M-H). d. One-third of resin 2B was reacted with 2-nitrobenzoic acid in the same manner as described in 3a to yield resin 3BA. MS calcd. for C13Hl2N209 : 340. 05, found: 339 (M-H). e. One-third of resin 2B was reacted with 3-nitrobenzoic acid in the same manner as described in 3a to yield resin 3BB. MS calcd. for C13Hl2N209 : 340.05, found: 339 (M-H). f. One-third of resin 2B was reacted with 4-nitrobenzoic acid in the same manner as described in 3a to yield resin 3BC. MS calcd. for C13Hl2N209 : 340.05, found: 339 (M-H). g. One-third for resin 2C was reacted with 2-nitrobenzoic acid in the same manner as described in 3a to yield resin 3CA. MS calcd. for C14H14N2O9: 354.07, found: 353 (M-H). h. One-third of resin 2C was shaken with 3-nitrobenzoic acid in the same manner as described in 3a to yield resin 3CB. MS calcd. for C14H14N2O9 : 354.07, found: 353 (M-H). i. One-third of resin 2C was shaken with 4-nitrobenzoic acid in the same manner as described in 3a to yield resin 3CC. MS calcd. for C14Hl4N209 : 354.07, found: 353 (M-H).

STEP 4. Reduction of nitro groups to amines a. Resin 3AA was shaken with tin dichloride dihydrate (2.0 M, 20ml) in DMF at room temperature overnight. The resin was washed with DMF (5 x 10 ml), methanol (3 x 10 ml), DMF (3 x 10 ml), methanol (3 x 10 ml), DCM (3 x 10 ml), methanol (3 x 10 ml), and air dried. A small sample (-50 mg) of the resulting resin 4AA was treated with TFA/water (95/5, 1.0 ml) for 1 hour at room temperature. The solution was collected via filtration. The resin was washed with acetic acid (3 x 1 ml).

The combined solution was lyophilized. The residue was analyzed by mass spectrometry. MS calcd. for CIlHl2N205 : 252.07, found: 251 (M-H). b. Resin 3AB was treated in the same manner as described in 4a to yield 4AB. MS calcd. for C11H12N2O5 : 252.07, found: 251 (M-H). c. Resin 3AC was treated in the same manner as described in 4a to yield 4AC. MS calcd. for CllHl2N205 : 252.07, found: 251 (M-H). d. Resin 3BA was treated in the same manner as described in 4a to yield 4BA : MS calcd. for Cl2Hl4N205 : 310.08, found: 309 (M-H). e. Resin 3BB was treated in the same manner as described in 4a to yield 4BB. MS calcd. for C12Hl4N205 : 310.08, found: 309 (M-H). f. Resin 3BC was treated in the same manner as described in 4a to yield 4BC. MS calcd. for C12Hl4N205 : 310.08, found 309 (M-H). g. Resin 3CA was treated in the same manner as described in 4a to yield 4CA. MS calcd. for Cl2Hl4N205 : 324.10, found 323 (M-H). h. Resin 3CB was treated in the same manner as described in 4a to yield WCB. MS calcd. for C12H14N2O5 : 324.10, found 323 (M-H). i. Resin 3CC was treated in the same manner as described in 4a to yield 4CC. MS calcd. for C12H14N2O5 : 324.10, found 323 (M-H).

STEP 5. Coupling of Carboxylic Acids on to Resin and TFA cleavage a. Resin 4AA was divided into 7 equal portions.

Portion A was shaken with acetic anhydride (A) (0.24 ml, 2.5 mmol), DIEA (0.87 ml, 5.0 mmol) and DMAP (10 mg) in DMF (5.0 ml) at room temperature overnight. The resin was washed with DMF (3 x 5 ml), methanol (3 x 5 ml), DMF (3 x 5 ml), methanol (3 x 5 ml), DCM (3 x 5 ml), methanol (3 x 5 ml), and air dried. The resulted resin 5AAA was treated with TFA/water (95/5,3.0 ml) for 1 hour at room temperature. The solution was collected via filtration. The resin was washed with acetic acid (3 x 5 ml). The combined was lyophilized to give the desired product 6AAA. Its purity and identity were assessed using HPLC-MS spectrometry.

Portion B was shaken with benzoic acid (B) (0.31 g, 2.5 mmol), DIC (0.47 ml, 3.0 mmol), DIEA (0.87 ml, 5.0 mmol) and DMAP (10 mg) in DMF (5.0 ml) at room temperature overnight. The resin was washed with DMF (3 x 5 ml), methanol (3 x 5 ml), DMF (3 x 5 ml), methanol (3 x 5 ml), DCM (3 x 5 ml), methanol (3 x 5 ml), and air dried. The resulting resin 5AAB was treated with TFA/water (95/5,3.0 ml) for 1 hour at room temperature. The solution was collected via filtration. The resin was washed with acetic acid (3 x 5 ml). The combined solution was lyophilized to give the

desired product 6AAB Its purity and identity were assessed using HPLC-MS spectrometry.

Portion C was treated with decanoic acid (C) in the same manner as described for Portion B.

Portion D was treated with glutaric anhydride (D) in the same manner as described for Portion A.

Portion E was treated with heptanoyl chloride (E) in the same manner as described for Portion A.

Portion F was treated with heptanoyl chloride (F) in the same manner as described for Portion A.

Portion G was treated with heptanoyl chloride (G) in the same manner as described for Portion A. b. Resin 4AB, 4AC, 4BA, 4BB, 4BC, 4CA, 4CB, and 4CC were treated using the same procedure as described in Step Sa..

The compounds made according to the above procedures are summarized in the following Table 1. In Table 1, it should be noted that compounds are identified with three-letter codes. Within these three-letter codes, the first letter codes for the first component piece, the second letter codes for the second component piece, and the third letter codes for third component piece. Such first, second and third component pieces are identified in the reaction scheme presented above in this example.

TABLE 1 REPRESENTATIVECOMPOUNDS (R1 OF STRUCTURE (I) = HYDROXY)

MW Cpd. First Second Third MW found No. Component Component Component Formula Calcd (M-H) 6AAA glycine t-butyl 2-nitrobenzoic acetic C13H14N2O6 294.3 293 ester (A) acid (A) anhydride 6AAB glycine t-butyl 2-nitrobenzoic benzoic acid C1sHl6N206 356. 3 355 ester (A) acid (A) (B) 6AAC glycine t-butyl 2-nitrobenzoic decanoic acid C2IH3oN206 406.5 405 ester (A) acid (A) (C) 6AAD glycine t-butyl 2-nitrobenzoic glutaric C16H18N2O8 366. 5 365 ester (A) acid (A) anhydride (D) 6AAE glycine t-butyl 2-nitrobenzoic heptanoyl C18H24N2O6 364.4 363 ester (A) acid (A) chloride 6AAF glycine t-butyl 2-nitrobenzoic methyl C22H30N208 450.5 449 ester (A) acid (A) sebacoyl chloride 6AAG glycine t-butyl 2-nitrobenzoic methyl C20H26N2O8 422.4 421 ester (A) acid (A) suberyl chloride (G) 6ABA glycine t-butyl 3-nitrobenzoic acetic C13H14N2O6 294.3 293 ester (A) acid (B) anhydride (A) 6ABB glycine t-butyl 3-nitrobenzoic benzoic acid C18Hl6N206 356.3 355 ester A acid 6ABC glycine t-butyl 3-nitrobenzoic decanoic acid C21H30N2O6 406.5 405 ester (A) acid (B) (C) 6ABD glycine t-butyl 3-nitrobenzoic glutaric C16H18N2O8 366.3 365 ester (A) acid (B) anhydride (D) 6ABE glycine t-butyl 3-nitrobenzoic heptanoyl C18H24N206 364.4 363 ester (A) acid (B) chloride (E) 6ABF glycine t-butyl 3-nitrobenzoic methyl C22H30N2O8 450.5 449 ester (A) acid (B) sebacoyl chloride 6ABG glycine t-butyl 3-nitrobenzoic methyl C2oH26N20s 422.4 421 ester (A) acid (B) suberyl chloride (G) 6ACA glycine t-butyl 4-nitrobenzoic acetic C13H14N2O6 294.3 293 ester (A) acid (C) anhydride (A) MW Cpd. First Second Third MW found No. Component Component Component Formula Calcd (M-EI) 6ACB glycinet-butyl 4-nitrobenzoic benzoic acid C18Hl6N206 356.3 355 ester (A) acid (C) (B) 6ACC glycine t-butyl 4-nitrobenzoic decanoic acid C2lH3oN206 406.5 405 ester (A) acid (C) (C) 6ACD glycine t-butyl 4-nitrobenzoic glutaric C16H18N2O8 366. 3 365 ester (A) acid (C) anhydride (D) 6ACE glycine t-butyl 4-nitrobenzoic heptanoyl Ci8H24N206 364.4 363 ester (A) acid (C) chloride (E) 6ACF glycine t-butyl 4-nitrobenzoic methyl C22H3oN20s 450.5 449 ester (A) acid (C) sebacoyl chloride (F) 6ACG glycine t-butyl 4-nitrobenzoic methyl C2oH26N208 422.4 421 ester (A) acid (C) suberyl chloride (G) 6BAA aspartic acid 2-nitrobenzoic acetic C15H16N2O8 352.3 351 di-t-butyl ester acid (A) anhydride (A) (B) 6BAB aspartic acid 2-nitrobenzoic benzoic acid C20H18N2O8 414.4 413 di-t-butyl ester acid (A) (B) (B) 6BAC aspartic acid 2-nitrobenzoic decanoic acid C23H32N2O8 464.5 464 di-t-butyl ester acid (A) (C) (B) 6BAD aspartic acid 2-nitrobenzoic glutaric C18H20N2O1 424.4 423 di-t-butyl ester acid (A) anhydride (D) 0 (B) 6BAE aspartic acid 2-nitrobenzoic heptanoyl C20H25N2O8 422.4 421 di-t-butyl ester acid (A) chloride (E) (B) 6BAF aspartic acid 2-nitrobenzoic methyl C24H32N2O1 508.5 508 di-t-butyl ester acid (A) sebacoyl o (B) chloride (F) 6BAG aspartic acid 2-nitrobenzoic methyl C22H28N201 480.5 479 di-t-butyl ester acid (A) suberyl o (B) chloride (G) 6BBA aspartic acid 3-nitrobenzoic acetic C15Hl6N208 352. 3 351 di-t-butyl ester acid (B) anhydride (A) (B) MW Cpd. First Second Third MW found No. Component Component Component Formula Calcd (M-EI) 6BBB aspartic acid 3-nitrobenzoic benzoic acid C20H18N2O8 414.4 413 di-t-butyl ester acid (B) (B) (B) 6BBC aspartic acid 3-nitrobenzoic decanoic acid C23H32N2O8 464.5 423 di-t-butyl ester acid (B) (C) (B) 6BBd aspartic acid 3-nitrobenzoic glutaric C18H20N2O1 424.4 423 di-t-butyl ester acid (B) anhydride (D) 0 (B) 6BBE aspartic acid 3-nitrobenzoic heptanoyl C20H26N2O8 422.4 421 di-t-butyl ester acid (B) chloride (E) (B) 6BBF aspartic acid 3-nitrobenzoic methyl C24H32N201 508.5 508 di-t-butyl ester acid (B) sebacoyl o (B) chloride (F) 6BBG aspartic acid 3-nitrobenzoic methyl C22H2sN20i 480.5 479 di-t-butyl ester acid (B) suberyl o (B) chloride (G) 6BCA aspartic acid 4-nitrobenzoic acetic C15Hl6N20s 352.3 351 di-t-butyl ester acid (C) anhydride (A) (B) 6BCB aspartic acid 4-nitrobenzoic benzoic acid C20H18N2O8 414.4 413 di-t-butyl ester acid (C) (B) (B) 6BCC aspartic acid 4-nitrobenzoic decanoic acid C23H32N2O8 464.5 464 di-t-butyl ester acid (C) (C) (B) 6BCD aspartic acid 4-nitrobenzoic glutaric C18H20N2O1 424.4 423 di-t-butyl ester acid (C) anhydride (D) 0 (B) 6BCE aspartic acid 4-nitrobenzoic heptanoyl C20H26N2O8 422.4 421 di-t-buyl ester acid (C) chloride (E) (B) 6BCF aspartic acid 4-nitrobenzoic methyl C24H32N2O1 508.5 508 di-t-butyl ester acid (C) sebacoyl o (B) chloride (F) 6BCG aspartic acid 4-nitrobenzoic methyl C22H28N201 480.5 479 di-t-butyl ester acid (C) suberyl o (B) chloride (G) MW Cpd. First Second Third MW found No. Component Component Component Formula Calcd (M-H) 6CAA glutaric acid 2-nitrobenzoic acetic C16H18N2O8 366.3 365 di-t-butyl ester acid (A) anhydride (A) (C) 6CAB glutaric acid 2-nitrobenzoic benzoic acid C21H20N2O8 428.4 427 di-t-butyl ester acid (A) (B) (C) 6CAC glutaric acid 2-nitrobenzoic decanoic acid C24H34N208 478.5 478 di-t-butyl ester acid (A) (C) (C) 6CAD glutaric acid 2-nitrobenzoic glutaric C19H22N2O1 438.4 437 di-t-butyl ester acid (A) anhydride (D) o (C) 6CAE glutaric acid 2-nitrobenzoic heptanoyl C21H28N2O8 436.6 435 di-t-butyl ester acid (A) chloride (E) (C) 6CAF glutaric acid 2-nitrobenzoic methyl C25H34N20, 522.6 522 di-t-butyl ester acid (A) sebacoyl o (C) chloride (F) 6CAG glutaric acid 2-nitrobenzoic methyl C23H3oN201 494.5 493 di-t-butyl ester acid (A) suberyl o (C) chloride (G) 6CBA glutaric acid 3-nitrobenzoic acetic C16Hl8N208 366.3 365 di-t-butyl ester acid (B) anhydride (A) (C) 6CBB glutaric acid 3-nitrobenzoic benzoic acid C2lH2oN20s 428.4 427 di-t-butyl ester acid (B) (B) (C) 6CBC glutaric acid 3-nitrobenzoic decanoic acid C24H34N2O8 478.5 478 di-t-butyl ester acid (B) (C) (C) 6CBD glutaric acid 3-nitrobenzoic glutaric C19H22N2O1 438.4 437 di-t-butyl ester acid (B) anhydride (D) o (C) 6CBE glutaric acid 3-nitrobenzoic heptanoyl C2lH28N208 436.5 435 di-t-butyl ester acid (B) chloride (E) (C) 6CBF glutaric acid 3-nitrobenzoic methyl C2sH34N201 522.6 522 di-t-butyl ester acid (B) sebacoyl o (C) chloride MW Cpd. First Second Third MW found No. Component Component Component Formula Calcd (M-H) 6CBG glutaric acid 3-nitrobenzoic methyl C23H30N2O1 494.5 493 di-t-butyl ester acid (B) suberyl o (C) chloride (G) 6CCA glutaric acid 4-nitrobenzoic acetic C16Hl8N208 366.3 365 di-t-butyl ester acid (C) anhydride (A) (C) 6CCB glutaric acid 4-nitrobenzoic benzoic acid C2lH2oN20s 428.4 427 di-t-butyl ester acid (C) (B) (C) 6CCC glutaric acid 4-nitrobenzoic decanoic acid C24H34N20g 478.5 478 di-t-butyl ester acid (C) (C) (C) 6CCD glutaric acid 4-nitrobenzoic glutaric Cl9H22N201 438.4 437 di-t-butyl ester acid (C) anhydride (D) o (C) 6CCE glutaric acid 4-nitrobenzoic heptanoyl C2lH28N20s 436.5 435 di-t-butyl ester acid (C) chloride (E) (C) 6CCF glutaric acid 4-nitrobenzoic methyl C25H34N2O1 522.6 522 di-t-butyl ester acid (C) sebacoyl o (C) chloride (F) 6CCG glutaric acid 4-nitrobenzoic methyl C23H30N2O1 494.5 493 di-t-butyl ester acid (C) suberyl o (C) chloride (G)

Example 2 SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF STRUCTURE (I) Using the same procedures as illustrated in Example 1, additional representative compounds were prepared using glycine methyl ester (A) or glycinamide (B) as the first component. The corresponding compounds are listed below in Table 2.

TABLE 2 REPRESENTATIVE COMPOUNDS (Rl OF STRUCTURE (I) = METHOXY OR AMINO)

Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 7AAA glycine methyl 3-nitrobenzoic glutaric C17H20N2O8 380.4 381 ester (A) acid (A) anhydride (A) 7AAB glycine methyl 3-nitrobenzoic heptanoyl Cl9H26N206 378.4 379 ester (A) acid (A) chloride 7AAC glycine methyl 3-nitrobenzoic decanoic acid C22H32N2O6 420.5 421 ester (A) acid (A) (C) 7AAD glycine methyl 3-nitrobenzoic methyl C21H28N2O8 436.5 437 ester (A) acid (A) suberyl chloride (D) 7AAE glycine methyl 3-nitrobenzoic methyl C23H32N20g 464.5 465 ester (A) acid (A) sebacoyl chloride 7ABA glycine methyl 4-nitrobenzoic glutaric C17H20N2O8 380.4 381 ester (A) acid (B) anhydride 7ABB glycine methyl 4-nitrobenzoic heptanoyl Cl9H26N206 378.4 379 ester (A) acid chloride 7ABC glycine methyl 4-nitrobenzoic decanoic acid C22H32N2O6 420.5 421 ester (A) acid (B) (C) 7ABD glycine methyl 4-nitrobenzoic methyl C21H28N2O8 436.5 437 ester (A) acid (B) suberyl chloride (D) 7ABE glycine methyl 4-nitrobenzoic methyl C23H32N208 464.5 465 ester (A) acid (B) sebacoyl chloride 7BAA glycinamide 3-nitrobenzoic glutaric C61H19N3O7 365.3 366 acid (A) anhydride (A 7BAB glycinamide 3-nitrobenzoic heptanoyl C18H25N305 363.4 364 acid A chloride 7BAC glycinamide 3-nitrobenzoic decanoic acid C21H31N3O5 405.5 406 acid (A) (C 7BAD glycinamide 3-nitrobenzoic methyl C20H27N307 421.4 422 (B) acid (A) suberyl chloride (D) Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 7BAE glycinamide 3-nitrobenzoic methyl C22H31N3O7 449.5 450 (B) acid (A) sebacoyl chloride (E) 7BBA glycinamide 4-nitrobenzoic glutaric C16Hl9N307 365.3 366 (B) acid (B) anhydride (A) 7BBB glycinamide 4-nitrobenzoic heptanoyl C18H25N305 363.4 364 (B) acid (B) chloride (B) 7BBC glycinamide 4-nitrobenzoic decanoic acid C21H31N3O5 405.5 406 (B) acid (B) (C) 7BBD glycinamide 4-nitrobenzoic methyl C2oH27N307 421.4 422 (B) acid (B) suberyl chloride (D) 7BBE glycinamide 4-nitrobenzoic methyl C22H31N3O7 449.5 450 (B) acid (B) sebacoyl chloride (E)

Example 3 SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF STRUCTURE (I) Using the same procedures as illustrated in Example 1, additional representative compounds were prepared using alanine benzyl ester (A), valine benzyl ester (B), leucine benzyl ester (C) or phenylalanine benzyl ester (D) as the first component. The corresponding products are listed below in Table 3.

TABLE 3 REPRESENTATIVECOMPOUNDS (Rl OF STRUCTURE (I) = BNO) Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M) 8AAA alanine benzyl 3-nitrobenzoic glutaric C26H3oN20s 498. 5 499 ester (A) acid (A) anhydride 8AAB alanine benzyl 3-nitrobenzoic heptanoyl C28H36N206 496.6 497 ester (A) acid (A) chloride (B) Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 8AAC alanine benzyl 3-nitrobenzoic decanoic acid C3iH4aN206 538. 7 539 ester (A) acid (A) (C) 8AAD alanine benzyl 3-nitrobenzoic methyl C3oH38N20s 554.6 555 ester (A) acid (A) suberyl chloride (D) 8AAE alanine benzyl 3-nitrobenzoic methyl C32H42N2O8 582.7 583 ester (A) acid (A) sebacoyl chloride (E) 8ABA alanine benzyl 4-nitrobenzoic glutaric C2oH26N20g 422. 4 423 ester (A) acid (B) anhydride 8ABB alanine benzyl 4-nitrobenzoic heptanoyl C22H32N206 420.5 421 ester (A) acid (B) chloride (B) 8ABC alanine benzyl 4-nitrobenzoic decanoic acid C25H38N206 462.6 463 ester (A) acid (B) (C) 8ABD alanine benzyl 4-nitrobenzoic methyl C24H34N20s 478.5 479 ester (A) acid (B) suberyl chloride (D) 8ABE alanine benzyl 4-nitrobenzoic methyl C26H38N208 506.6 507 ester (A) acid (B) sebacoyl- chloride 8BAA valine benzyl 3-nitrobenzoic glutaric C27H32N2O8 512.6 513 ester (B) acid (A) anhydride 8BAB valine benzyl 3-nitrobenzoic heptanoyl C29H3sN206 510. 6 511 ester (B) acid (A) chloride (B) 8BAC valine benzyl 3-nitrobenzoic decanoic acid C32H44N206 552.7 553 ester (B) acid (A) (C) 8BAD valine benzyl 3-nitrobenzoic methyl C31H40N2O8 568.7 569 ester (B) acid (A) suberyl chloride (D) 8BAE valine benzyl 4-nitrobenzoic methyl C33H44N208 596.7 597 ester (B) acid (B) sebacoyl chloride 8BBA valine benzyl 4-nitrobenzoic glutaric C2oH26N208 422.4 423 ester (B) acid (B) anhydride (A) 8BBB valine benzyl 4-nitrobenzoic heptanoyl C22H32N206 420.5 421 ester acid chloride 8BBC valine benzyl 4-nitrobenzoic decanoic acid C25H38N2O6 462.6 463 ester (B) acid (B) (C) Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 8BBD valine benzyl 4-nitrobenzoic methyl C24H34N2O8 478.5 479 ester (B) acid (B) suberyl chloride (D) 8BBE valine benzyl 4-nitrobenzoic methyl C26H38N2Os 506.6 507 ester (B) acid (B) sebacoyl chloride (E) 8CAA leucine benzyl 3-nitrobenzoic glutaric C27H32N20$ 512.6 513 ester (C) acid (A) anhydride (A) 8CAB leucine benzyl 3-nitrobenzoic heptanoyl C29H38N2O6 510.6 511 ester (C) acid (A) chloride 8CAC leucine benzyl 3-nitrobenzoic decanoic acid C32H44N206 552.7 553 ester (C) acid (A) C 8CAD leucine benzyl 3-nitrobenzoic methyl C3iH4oN20s 568.7 569 ester (C) acid (A) suberyl chloride (D) 8CAE leucine benzyl 3-nitrobenzoic methyl C33H44N20g 596.7 597 ester (C) acid (A) sebacoyl chloride (E) 8CBA leucine benzyl 4-nitrobenzoic glutaric C23H24N20g 456.6 457 ester (C) acid anh anhydride 8CBB leucine benzyl 4-nitrobenzoic heptanoyl C25H30N2O6 454.5 455 ester (C) acid chloride 8CBC leucine benzyl 4-nitrobenzoic decanoic acid C28H36N206 496.6 497 ester (C) acid (B) (C) 8CBD leucine benzyl 4-nitrobenzoic methyl C27H32N2O8 512.6 513 ester (C) acid (B) suberyl chloride (D) 8CBE leucine benzyl 4-nitrobenzoic methyl C29H36N2Os 540.6 541 ester (C) acid (B) sebacoyl chloride (E) 8DBA phenylalanine 4-nitrobenzoic glutaric C3oH3oN208 546. 6 547 benzyl ester acid (B) anhydride (A) (D) 8DBB phenylalanine4-nitrobenzoic heptanoyl C32H36N2O6 544.6 545 benzyl ester acid (B) chloride (B) (D) 8DBC phenylalanine 4-nitrobenzoic decanoic acid C35H42N2O6 586.7 587 benzyl ester acid (B) (C) (D) Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 8DBD phenylalanine 4-nitrobenzoic methyl C34H38N2O8 602.7 603 benzyl ester acid (B) suberyl chloride 8DBE phenylalanine 4-nitrobenzoic methyl C36H42N2O8 630.7 631 benzyl ester acid (B) sebacoyl (D) chloride (E)

Example 4 SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF STRUCTURE (I) Each of the compounds of Example 3 was divided into two portions.

One portion was treated with hydrogen (10 psi) in the presence of 10% Palladium on activated carbon (15 mg) in acetic acid/methanol (1/4,5 ml) at room temperature overnight. The Pd/C was removed by filtration and washed with acetic acid (3 x 5 ml).

This resulted in the conversion of the benzyl ester (R1 = BnO) to the corresponding acid (Ri = OH). The resulting compounds are summarized in Table 4.

TABLE 4 REPRESENTATIVE COMPOUNDS (R1 OF STRUCTURE (I) = OH) Cpd. No. First Second Third Formula Calcd found Component Component Component (M+H) 9AAA alanine benzyl 3-nitrobenzoic glutaric Cl7H2oN208 380.4 381 ester (A) acid (A) anhydride 9AAB alanine benzyl 3-nitrobenzoic heptanoyl Cl9H26N206 378.4 379 ester (A) acid (A) chloride (B) 9AAC alanine benzyl 3-nitrobenzoic decanoic acid C22H32N206 420. 5 421 ester (A) acid (A) (C) 9AAD alanine benzyl 3-nitrobenzoic methyl C2IH28N208 436.5 437 ester (A) acid (A) suberyl chloride Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 9AAE alanine benzyl 3-nitrobenzoic methyl C23H32N208 464.5 465 ester (A) acid (A) sebacoyl chloride (E) 9ABA alanine benzyl 4-nitrobenzoic glutaric C24H26N2°8 470.5 471 ester (A) acid (B anhydride (A) 9ABB alanine benzyl 4-nitrobenzoic heptanoyl C26H32N206 468.5 469 ester (A) acid chloride 9ABC alanine benzyl 4-nitrobenzoic decanoic acid C29H38N206 510.6 511 ester (A) acid (B (C) 9ABD alanine benzyl 4-nitrobenzoic methyl C28H34N208 526.6 527 ester (A) acid (B suberyl chloride (D) 9ABE alanine benzyl 4-nitrobenzoic methyl C3oH38N208 554.6 555 ester (A) acid (B sebacoyl chloride 9BAA valine benzyl 3-nitrobenzoic glutaric C17H20N2O8 380.4 381 ester acid (A) anhydride (A 9BAB valine benzyl 3-nitrobenzoic heptanoyl Cl9H26N206 378.4 379 ester (B) acid (A) chloride (B) 9BAC valine benzyl 3-nitrobenzoic decanoic acid C22H32N206 420.5 421 ester (B) acid (A) (C) 9BAD valine benzyl 3-nitrobenzoic methyl C2iH28N20g 436.5 437 ester (B) acid (A) suberyl chloride (D) 9BAE valine benzyl 3-nitrobenzoic methyl C23H32N208 464. 5 465 ester (B) acid (A) sebacoyl chloride (E) 9BBA valine benzyl 4-nitrobenzoic glutaric C24H26N2Og 470.5 471 ester (B) acid (B) anhydride (A) 9BBB valine benzyl 4-nitrobenzoic heptanoyl C26H32N206 468.5 469 ester acid chloride 9BBC valine benzyl 4-nitrobenzoic decanoic acid C29H38N2o6 510. 6 511 ester (B) acid (B) (C) 9BBD valine benzyl 4-nitrobenzoic methyl C28H34N2O8 526.6 527 ester (B) acid (B) suberyl chloride (D) 9BBE valine benzyl 4-nitrobenzoic methyl C3oH38N208 554.6 555 ester (B) acid (B) sebacoyl chloride Cpd. MW MW No. First Second Third Formula Calcd found Component Component Component (M+H) 9CAA leucine benzyl 3-nitrobenzoic glutaric C19H24N2O8 408.4 409 ester (C) acid (A) anhydride (A) 9CAB leucine benzyl 3-nitrobenzoic heptanoyl C2lH3oN206 406.5 407 ester (C) acid (A) chloride (B) 9CAC leucine benzyl 3-nitrobenzoic decanoic acid C24H36N2O6 448.6 449 ester (C) acid (A) (C) 9CAD leucine benzyl 3-nitrobenzoic methyl C23H32N2O8 464.5 465 ester (C) acid (A) suberyl chloride (D) 9CAE leucine benzyl 3-nitrobenzoic methyl C25H36N2O8 492.6 493 ester (C) acid (A) sebacoyl chloride 9CBA leucine benzyl 4-nitrobenzoic glutaric C26H3oN208 498. 5 499 ester (C) acid (B) anhydride (A) 9CBB leucine benzyl 4-nitrobenzoic heptanoyl C28H36N206 496.6 497 ester (C) acid chloride 9CBC leucine benzyl 4-nitrobenzoic decanoic acid C3iH42N206 538.7 539 ester (C) acid (B) (C) 9CBD leucine benzyl 4-nitrobenzoic methyl C3oH38N208 554.6 555 ester (C) acid (B) suberyl chloride (D) 9CBE leucine benzyl 4-nitrobenzoic methyl C32H42N2O8 582.7 583 ester (C) acid (B) sebacoyl chloride 9DBA phenylalanine 4-nitrobenzoic glutaric C19H24N2O8 408.4 409 benzyl ester acid (B) anhydride (A) (D) 9DBB phenylalanine 4-nitrobenzoic heptanoyl C21H30N2O6 406.5 407 benzyl ester acid (B) chloride (B) (D) 9DBC phenylalanine 4-nitrobenzoic decanoic acid C24H36N2O6 448.6 449 benzyl ester acid (B) (C) (D) 9DBD phenylalanine 4-nitrobenzoic methyl C23H32N2O8 464.5 465 benzyl ester acid (B) suberyl chloride 9DBE phenylalanine 4-nitrobenzoic methyl C25H36N2O8 492.6 493 benzyl ester acid (B) sebacoyl chloride

Example 5 SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF STRUCTURE (I) Using the same procedures as illustrated in Example 1, additional representative compounds were prepared using leucine benzyl ester (A) or phenylalanine benzyl ester (B) as the first component, and using various second components to illustrate embodiments wherein the"A"moiety of structure (I) is other than a direct bond. The corresponding compounds are listed below in Table 5.

TABLE 5 REPRESENTATIVE COMPOUNDS (R1 OF STRUCTURE (1) = BNO) Cpd. Third Third No. First Second Componen Formula Calc found Component Component t d (M+H) Leucine 2-nitrophenyl-glutaric benzyl ester acetic acid (A) anhydride 10AAA (A) (A) C28H34N2O8 526. 6 527 Leucine 2-nitrophenyl- benzyl ester acetic acid (A) heptanoyl 10AAB (A) chloride (B) C30H40N2O6 524.7 525 Leucine 2-nitrophenyl- benzyl ester acetic acid (A) decanoic 10AAC (A) acid (C) C33H46N206 566.7 567 Leucine 2-nitrophenyl-methyl benzyl ester acetic acid (A) suberyl 10AAD (A) chloride (D) C32H42N20s 582.7 583 Leucine 2-nitrophenyl-methyl benzyl ester acetic acid (A) sebacoyl 10AAE (A) chloride (E) C34H46N208 610.7 611 Phenylalanin 2-nitrophenyl-glutaric e benzyl ester acetic acid (A) anhydride 10BAA (B) (A) C3lH32N208 560.6 561 Phenylalanin 2-nitrophenyl- e benzyl ester acetic acid (A) heptanoyl 10BAB (B) chloride (B) C33H38N2O6 558.7 559 Phenylalanin 2-nitrophenyl- e benzyl ester acetic acid (A) decanoic 10BAC (B) acid (C) C36H44N206 600.8 601 Phenylalanin 2-nitrophenyl-methyl e benzyl ester acetic acid (A) suberyl 10BAD (B) chloride (D) C35H4pN208 616.7 617 Phenylalanin 2-nitrophenyl-methyl e benzyl ester acetic acid (A) sebacoyl 10BAE (B) chloride (E) C37H44N2O8 644. 8 645 Leucine 3-nitrophenyl-glutaric benzyl ester acetic acid (B) anhydride 10ABA (Å) (A) C28H34N208 526.6 527 Leucine 3-nitrophenyl- benzyl ester acetic acid (B) heptanoyl 10ABB (A) chloride (B) C30H40N2O6 524.7 525 Leucine 3-nitrophenyl- benzyl ester acetic acid (B) decanoic 10ABC (A) acid (C) C33H46N206 566.7 567 Leucine 3-nitrophenyl-methyl benzyl ester acetic acid (B) suberyl 10ABD (A) chloride (D) C32H42N2O8 582. 7 583 Leucine 3-nitrophenyl-methyl benzyl ester acetic acid (B) sebacoyl lOABE (A) chloride (E) C34H46N20s 610.7 611 Phenylalanin 3-nitrophenyl-glutaric e benzyl ester acetic acid (B) anhydride lOBBA (B) A) C31H32N2O8 560. 6 561 Phenylalanin 3-nitrophenyl- e benzyl ester acetic acid (B) heptanoyl lOBBB (B) chloride (B) C33H38N206 558.7 559 Phenylalanin 3-nitrophenyl- e benzyl ester acetic acid (B) decanoic lOBBC (B) acid (C) C36H44N206 600.8 601 Phenylalanin 3-nitrophenyl-methyl e benzyl ester acetic acid (B) suberyl lOBBD (B) chloride (D) C35H40N2O8 616.7 617 Phenylalanin 3-nitrophenyl-methyl e benzyl ester acetic acid (B) sebacoyl lOBBE (B) chloride (E) C37H44N208 644.8 645 Leucine 4-nitrophenyl-glutaric benzyl ester acetic acid (C) anhydride 10ACA (A) (A) C28H34N208 526.6 527 Leucine 4-nitrophenyl- benzyl ester acetic acid (C) heptanoyl lOACB (A) chloride (B) C3oH4oN206 524.7 525 Leucine 4-nitrophenyl- benzyl ester acetic acid (C) decanoic 10ACC (A) acid (C) C33H46N2O6 566.7 567 Leucine 4-nitrophenyl-methyl benzyl ester acetic acid (C) suberyl IOACD. (A) chloride (D) C32H42N208 582.7 583 Leucine 4-nitrophenyl-methyl benzyl ester acetic acid (C) sebacoyl 10ACE (A) chloride (E) C34H46N20s 610.7 611 Phenylalanin 4-nitrophenyl-glutaric e benzyl ester acetic acid (C) anhydride lOBCA (B) (A) C31H32N208 560.6 561 Phenylalanin 4-nitrophenyl- e benzyl ester acetic acid (C) heptanoyl lOBCB (B) chloride (B) C33H38N206 558.7 559 Phenylalanin 4-nitrophenyl- e benzyl ester acetic acid (C) decanoic lOBCC (B) acid (C) C36H44N2O6 600.8 601 Phenylalanin 4-nitrophenyl-methyl e benzyl ester acetic acid (C) suberyl 10BCD (B) chloride (D) C35H40N2O8 616.7 617 Phenylalanin 4-nitrophenyl-methyl e benzyl ester acetic acid (C) sebacoyl 1OBCE- (B) chloride (E) C37H44N208 644.8 645 Leucine 2-glutaric benzyl ester nitrophenoxy-anhydride 10ADA (A) acetic acid (D) (A) C28H34N209 542.6 543 Leucine 2-methyl benzyl ester nitrophenoxy-suberyl 10ADD (A) acetic acid (D) chloride (D) C32H42N2O9 598.7 599 Leucine 2-methyl benzyl ester nitrophenoxy-sebacoyl lOADE (A) acetic acid (D) chloride E) C34H46N2O9 626.7 627 Phenylalanin 2-glutaric e benzyl ester nitrophenoxy-anhydride 10BDA (B) acetic acid (D) (A) C31H32N2O9 576.6 577 Phenylalanin 2- e benzyl ester nitrophenoxy-heptanoyl IOBDB (B) acetic acid (D) chloride (B) C33H38NiO7 574. 7 575 Phenylalanin 2- e benzyl ester nitrophenoxy-decanoic 10BDC (B) acetic acid (D) acid (C) C36H44N2O7 616. 8 617 Leucine 3-glutaric benzyl ester nitrophenoxy-anhydride 10AEA (A) acetic acid (E) (A) C28H34N2Og 542.6 543 Leucine 3- benzyl ester nitrophenoxy-heptanoyl 10AEB (A) acetic acid (E) chloride (B) C30H40N2O7 540. 7 541 Leucine 3- benzyl ester nitrophenoxy-decanoic 10AEC (A) acetic acid (E) acid (C) C33H46N2O7 582.7 583 Leucine 3-methyl benzyl ester nitrophenoxy-suberyl 10AED (A) acetic acid (E) chloride (D) C32H42N209 598.7 599 Leucine 3-methyl benzyl ester nitrophenoxy-sebacoyl lOAEE (A) acetic acid (E) chloride (E) C34H46N2O9 626.7 627 Phenylalanin 3-glutaric e benzyl ester nitrophenoxy-anhydride lOBEA (B) acetic acid (E) (A) C3lH32N209 576.6 577 Phenylalanin 3- e benzyl ester nitrophenoxy-heptanoyl lOBEB (B) acetic acid (E) chloride (B) C33H38N207 574.7 575 Phenylalanin 3- e benzyl ester nitrophenoxy-decanoic 10BEC (B) acetic acid (E) acid (C) C36H44N2O7 616.8 617 Phenylalanin 3-methyl e benzyl ester nitrophenoxy-suberyl ROBED (B) acetic acid (E) chloride (D) C35H40N2O9 632.7 633 Phenylalanin 3-methyl e benzyl ester nitrophenoxy-sebacoyl 10BEE (B) acetic acid (E) chloride (E) C37H44N2O9 660.8 661 Leucine 4-glutaric benzyl ester nitrophenoxy-anhydride 10AFA A) acetic acid (F) (A) C28H34N2O9 542.6 543 Leucine 4- benzyl ester nitrophenoxy-heptanoyl 10AFB (A) acetic acid (F) chloride (B) C30H40N2O7 540.7 541 Leucine 4- benzyl ester nitrophenoxy-decanoic lOAFC (A) acetic acid (F) acid (C) C33H46N207 582.7 583 Leucine 4-methyl benzyl ester nitrophenoxy-suberyl lOAFD (A) acetic acid (F) chloride (D) C32H42N209 598.7 599 Leucine 4-methyl benzyl ester nitrophenoxy-sebacoyl lOAFE (A) acetic acid (F) chloride (E) C34H46N2O9 626.7 627 Phenylalanin 4-glutaric e benzyl ester nitrophenoxy-anhydride lOBFA (B) acetic acid (F) (A) C31H32N209 576.6 577 Phenylalanin 4- e benzyl ester nitrophenoxy-heptanoyl lOBFB (B) acetic acid (F) chloride (B) C33H38N207 574.7 575 Phenylalanin 4- e benzyl ester nitrophenoxy-decanoic lOBFC (B) acetic acid (F) acid (C) C36H44N2O7 616.8 617 Phenylalanin 4-methyl e benzyl ester nitrophenoxy-suberyl 10BFD (B) acetic acid (F) chloride (D) C35H40N2O9 632.7 633 Phenylalanin 4-methyl e benzyl ester nitrophenoxy-sebacoyl 10BFE (B) acetic acid (F) chloride (E) C37H44N209 660.8 661 Leucine 2-glutaric benzyl ester nitrocinnamic anhydride 10AGA (A) acid (G) (A) C29H34N2Os 538.6 539 Leucine 2- benzyl ester nitrocinnamic heptanoyl 10AGB (A) acid (G) chloride (B) C31H4N2O6 536.7 537 Leucine 2- benzyl ester nitrocinnamic decanoic 10AGC (A) acid (G) acid (C) C34H46N206 578.7 579 Leucine 2-methyl benzyl ester nitrocinnamic suberyl 10AGD (A) acid (G) chloride (D) C33H42N2O8 594.7 595 Leucine 2-methyl benzyl ester nitrocinnamic sebacoyl 10AGE (A) acid (G) chloride (E) C35H46N2O8 622.8 623 Phenylalanin 2-glutaric e benzyl ester nitrocinnamic anhydride 10BGA (B) acid (G) (A) C25H26N208 572.6 573 Phenylalanin 2- e benzyl ester nitrocinnamic heptanoyl 10BGB (B) acid (G) chloride (B) C27H32N206 570. 3 571 Phenylalanin 2- e benzyl ester nitrocinnamic decanoic 10BGC (B) acid (G) acid (C) C30H38N206 612.8 613 Phenylalanin 2-methyl e benzyl ester nitrocinnamic suberyl 10BGD (B) acid (G) chloride (D) C29H34N208 628.7 629 Phenylalanin 2-methyl e benzyl ester nitrocinnamic sebacoyl 10BGE (B) acid (G) chloride (E) C31H38N2O8 656.8 657 Leucine 3-glutaric benzyl ester nitrocinnamic anhydride 10AHA (A) acid (H) (A) C29H34N2O8 538.6 539 Leucine 3- benzyl ester nitrocinnamic heptanoyl 1OAHB (A) acid (H) chloride (B) C3tH4pN206 536.7 537 Leucine 3- benzyl ester nitrocinnamic decanoic 10AHC (A) acid (H acid (C) C34H46N206 578.7 579 Leucine 3-methyl benzyl ester nitrocinnamic suberyl 10ABD (A) acid (H) chloride (D) C33H42N20s 594.7 595 Leucine 3-methyl benzyl ester nitrocinnamic sebacoyl 10ABC (A) acid (H) chloride (E) C3sH46N20s 622.8 623 Phenylalanin 3-glutaric e benzyl ester nitrocinnamic anhydride 10BHA (B) acid (H) (A) C32H32N208 572.6 573 Phenylalanin 3- e benzyl ester nitrocinnamic heptanoyl 10BIB (B) acid (H) chloride (B) C34H38N206 570.7 571 Phenylalanin 3- e benzyl ester nitrocinnamic decanoic 10BHC (B) acid (H) acid (C) C37H44N206 612.8 613 Phenylalanin 3-methyl e benzyl ester nitrocinnamic suberyl 10BHD (B acid (H) chloride (D) C36H40N2O8 628.7 629 Phenylalanin 3-methyl e benzyl ester nitrocinnamic sebacoyl 10BHE (B) acid (H) chloride (E) C38H44N2O8 656.8 657 Leucine 4-glutaric benzyl ester nitrocinnamic anhydride 10AIA (A) acid (1) (A) C29H34N208 538.6 539 Leucine 4- benzyl ester nitrocinnamic heptanoyl 10AIB (A) acid (I) chloride (B) C31H40N2O6 536.7 537 Leucine 4- benzyl ester nitrocinnamic decanoic 10AIC (A) acid (1) acid (C) C34H46N206 578.7 579 Leucine 4-methyl benzyl ester nitrocinnamic suberyl 10AID (A) acid (I) chloride (D) C33H42N2O8 594.7 595 Leucine 4-methyl benzyl ester nitrocinnamic sebacoyl 10AIE (A) acid (I) chloride (E) C35H46N208 622.8 623 Phenylalanin 4-glutaric e benzyl ester nitrocinnamic anhydride 10BIA (B) acid (1) (A) C32H32N208 572.6 573 Phenylalanin 4- e benzyl ester nitrocinnamic heptanoyl 1OBIT (B) acid (I) chloride (B) C34H38N206 570.7 571 Phenylalanin 4- e benzyl ester nitrocinnamic decanoic 10BIC (B) acid (I) acid (C) C37H44N2O6 612.8 613 Phenylalanin 4-methyl e benzyl ester nitrocinnamic suberyl 10BID (B) acid (I) chloride (D) C36H4oN20s 628.7 629 Phenylalanin 4-methyl e benzyl ester nitrocinnamic sebacoyl lOBIE (B) acid (I) chloride (E) C38H44N2O8 656.8 657 Leucine 5- (2- benzyl ester nitrophenyl)-glutaric (A) 2-furoic acid anhydride 10AJA (J) (A) C31H34N2O9 578. 6 579 Leucine 5- (2- benzyl ester nitrophenyl)- (A) 2-furoic acid heptanoyl 10AJB (J) chloride (B) C33H40N2O7 576.7 577 Leucine 5- (2- benzyl ester nitrophenyl)- (A) 2-furoic acid decanoic 10AJC J) acid (C) C36H46N207 618. 8 619 Leucine 5- (2- benzyl ester nitrophenyl)-methyl (A) 2-furoic acid suberyl 10AJD (J) chloride (D) C3sH42N209 634.7 635 Leucine 5- (2- benzyl ester nitrophenyl)-methyl (A) 2-furoic acid sebacoyl 10AJE (J) chloride (E) C37H46N209 662.8 663 Phenylalanin 5- (2- e benzyl ester nitrophenyl)-glutaric (B) 2-furoic acid anhydride 10BJA (J) (A) C34H32N209 612.6 613 Phenylalanin 5- (2- e benzyl ester nitrophenyl)- (B) 2-furoic acid heptanoyl 1OBJB (J) chloride (B) C36H38N207 610.7 611 Phenylalanin 5- (2- e benzyl ester nitrophenyl)- (B) 2-furoic acid decanoic 10BJC (J) acid (C) C391144N207 652.8 653 Phenylalanin 5- (2- e benzyl ester nitrophenyl)-methyl (B) 2-furoic acid suberyl 10BJD (J) chloride (D) C3sH4oN209 668.7 669 Phenylalanin 5- (2- e benzyl ester nitrophenyl)-methyl (B) 2-furoic acid sebacoyl 10BJE (J) chloride (E) C40H44N2O9 696.8 697 Leucine 5- (3- benzyl ester nitrophenyl)-glutaric (A) 2-furoic acid anhydride 10AKA (K) (A) C3lH34N209 578.6 579 Leucine 5- (3- benzyl ester nitrophenyl)- (A) 2-furoic acid heptanoyl 10AKB (K) chloride (B) C33H40N207 576.7 577 Leucine 5- (3- benzyl ester nitrophenyl)- (A) 2-furoic acid decanoic 10AKC (K) acid (C) C36H46N2O7 618.8 619 Leucine 5- (3- benzyl ester nitrophenyl)-methyl (A) 2-furoic acid suberyl 10AKD (K) chloride (D) C35H42N209 634.7 635 Leucine 5- (3- benzyl ester nitrophenyl)-methyl (A) 2-furoic acid sebacoyl 10AKE (K) chloride (E) C37H46N209 662.8 663 Phenylalanin 5- (3- e benzyl ester nitrophenyl)-glutaric (B) 2-furoic acid anhydride 10BKA (K) (A) C34H32N209 612.6 613 Phenylalanin 5- (3- e benzyl ester nitrophenyl)- (B) 2-furoic acid heptanoyl 10BKB (K) chloride (B) C36H38N2O7 610.7 611 Phenylalanin 5- (3- e benzyl ester nitrophenyl)- (B) 2-furoic acid decanoic 10BKC (K) acid (C) C39H44N2O7 652.8 653 Phenylalanin 5- (3- e benzyl ester nitrophenyl)-methyl (B) 2-furoic acid suberyl 10BKD (K) chloride (D) C381-LoN209 668. 7 669 Phenylalanin 5- (3- e benzyl ester nitrophenyl)-methyl (B) 2-furoic acid sebacoyl 10BKE (K) chloride (E) C40H44N2O9 696.8 697 Leucine 5- (4- benzyl ester nitrophenyl)-glutaric (A) 2-furoic acid anhydride 10ALA (L) (A) C31H34N209 578.6 579 Leucine 5- (4- benzyl ester nitrophenyl)- (A) 2-furoic acid heptanoyl 10ALB (L) chloride (B) C33H40N207 576.7 577 Leucine 5- (4- benzyl ester nitrophenyl)- (A) 2-furoic acid decanoic 10ALC (L) acid (C) C36H46N2O7 618.8 619 Leucine 5- (4- benzyl ester nitrophenyl)-methyl (A) 2-furoic acid suberyl 10ALD (L) chloride (D) C35H42N2O9 634.7 635 Leucine 5- (4- benzyl ester nitrophenyl)-methyl (A) 2-furoic acid sebacoyl 10ALE (L) chloride (E) C37H46N2O9 662.8 663 Phenylalanin 5- (4- e benzyl ester nitrophenyl)-glutaric (B) 2-furoic acid anhydride 1 OBLA (L) (A) C34H32N209 612.6 613 Phenylalanin 5- (4- e benzyl ester nitrophenyl)- (B) 2-furoic acid heptanoyl lOBLB (L) chloride (B) C36H38N207 610. 7 611 Phenylalanin 5- (4- e benzyl ester nitrophenyl)- (B) 2-furoic acid decanoic lOBLC (L) acid (C) C39H44N207 652.8 653 Phenylalanin 5- (4- e benzyl ester nitrophenyl)-methyl (B) 2-furoic acid suberyl 10BLD (L) chloride (D) C38H4oN209 668.7 669 Phenylalanin 5- (4- e benzyl ester nitrophenyl)-methyl (B) 2-furoic acid sebacoyl NOBLE (L) chloride (E) C40H44N2O9 696.8 697

Example 6 SYNTHESIS OF REPRESENTATIVE COMPOUNDS OF STRUCTURE (I) Using the same procedures as illustrated in Example 1, additional representative compounds were prepared using leucine benzyl ester (A) or phenylalanine benzyl ester (B) as the first component, and using various second components to illustrate embodiments wherein the"A"moiety of structure (I) is other than a direct bond. The corresponding compounds are listed below in Table 6.

TABLE 6 REPRESENTATIVECOMPOUNDS (R1 OF STRUCTURE (1) = OH) MU MU Cpd. No. First Second Third Formula Calcd found Component Component Component (M+H) Leucine 2-nitrophenyl-glutaric benzyl ester acetic acid (A) anhydride 11AAA (A) (A) C2tH28N20s 436.5 437 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (n Leucine 2-nitrophenyl- ben2yl ester acetic acid (A) heptanoyl 11AAB (A) chloride (B) C23H34N206 434.5 435 Leucine 2-nitrophenyl- benzyl ester acetic acid (A) decanoic 11AAC (A) acid (C) C26H4pN206 476.6 477 Leucine 2-nitrophenyl-methyl benzyl ester acetic acid (A) suberyl 11AAD (A) chloride (D) C25H36N 492.6 493 Leucine 2-nitrophenyl-methyl benzyl ester acetic acid (A) sebacoyl 11AAE (A) chloride (E) C27H40N2O8 520.6 521 Phenylalanin 2-nitrophenyl-glutaric e benzyl ester acetic acid (A) anhydride 11BAA (B) (A) C24H26N208 470.5 471 Phenylalanin 2-nitrophenyl- e benzyl ester acetic acid (A) heptanoyl 11BAB (B) chloride (B) C26H32N206 468.5 469 Phenylalanin 2-nitrophenyl- e benzyl ester acetic acid (A) decanoic 11BAC (B) acid (C) C2gH38N206 510.6 511 Phenylalanin 2-nitrophenyl-methyl e benzyl ester acetic acid (A) suberyl 11BAD (B) chloride (D) C28H34N2O8 526.6 527 Phenylalanin 2-nitrophenyl-methyl e benzyl ester acetic acid (A) sebacoyl 11BAE (B) chloride (E) C3oH38N208 554.6 555 Leucine 3-nitrophenyl-glutaric benzyl ester acetic acid (B) anhydride 11ABA (A) (A) C2iH2sN208 436.5 437 Leucine 3-nitrophenyl- benzyl ester acetic acid (B) heptanoyl 11ABB (A) chloride (B) C23H34N206 434.5 435 Leucine 3-nitrophenyl- benzyl ester acetic acid (B) decanoic 11ABC (A) acid (C) C26H40N206 476.6 477 Leucine 3-nitrophenyl-methyl benzyl ester acetic acid (B) suberyl 11ABD (A) chloride (D) C25H36N208 492.6 493 Leucine 3-nitrophenyl-methyl benzyl ester acetic acid (B) sebacoyl 11ABE (A) chloride (E) C27H4ON2Os 520.6 521 Phenylalanin 3-nitrophenyl-glutaric e benzyl ester acetic acid (B) anhydride 11BBA (B) (A) C24H26N208 470.5 471 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (M+H) Phenylalanin 3-nitrophenyl- e benzyl ester acetic acid (B) heptanoyl 11BBB (B) chloride (B) C26H32N206 468.5 469 Phenylalanin 3-nitrophenyl- e benzyl ester acetic acid (B) decanoic 11BBC (B) acid (C) C29H38N206 510.6 511 Phenylalanin 3-nitrophenyl-methyl e benzyl ester acetic acid (B) suberyl 11BBD (B) chloride (D) C2sH34N208 526.6 527 Phenylalanin 3-nitrophenyl-methyl e benzyl ester acetic acid (B) sebacoyl 11BBE (B) chloride (E) C30H38N2O8 554.6 555 Leucine 4-nitrophenyl-glutaric benzyl ester acetic acid (C) anhydride 11ACA (A) (A) C2lH28N2Os 436.5 437 Leucine 4-nitrophenyl- benzyl ester acetic acid (C) heptanoyl 11ACB (A) chloride (B) C23H34N206 434.5 435 Leucine 4-nitrophenyl- benzyl ester acetic acid (C) decanoic 11ACC (A) acid (C) C26H40N2O6 476.6 477 Leucine 4-nitrophenyl-methyl benzyl ester acetic acid (C) suberyl 11ACD (A) chloride (D) C2sH36N208 492.6 493 Leucine 4-nitrophenyl-methyl benzyl ester acetic acid (C) sebacoyl 11ACE (A) chloride (E) C27H40N2O8 520.6 521 Phenylalanin 4-nitrophenyl-glutaric e benzyl ester acetic acid (C) anhydride 11BCA (B) (A) C24H26N2Os 470.5 471 Phenylalanin 4-nitrophenyl- e benzyl ester acetic acid (C) heptanoyl 11BCB (B) chloride (B) C26H32N206 468.5 469 Phenylalanin 4-nitrophenyl- e benzyl ester acetic acid (C) decanoic 11BCC (B) acid (C) C29H38N206 510.6 511 Phenylalanin 4-nitrophenyl-methyl e benzyl ester acetic acid (C) suberyl 11BCD (B) chloride (D) C28H34N208 526.6 527 Phenylalanin 4-nitrophenyl-methyl e benzyl ester acetic acid (C) sebacoyl 11BCE (B) chloride (E) C30H38N208 554.6 555 Leucine 2-glutaric benzyl ester nitrophenoxy-anhydride 11ADA (A) acetic acid (D) (A) C2lH28N209 452.5 453 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (M+H) Leucine 2- benzyl ester nitrophenoxy-heptanoyl 11ADB (A) acetic acid (D) chloride (B) C23H34N207 450.5 451 Leucine 2- benzyl ester nitrophenoxy-decanoic 11ADC (A) acetic acid (D) acid (C) C26H40N207 492.6 493 Leucine 2-methyl benzyl ester nitrophenoxy-suberyl 11ADD (A) acetic acid (D) chloride (D) C25H36N209 508.6 509 Leucine 2-methyl benzyl ester nitrophenoxy-sebacoyl CLADE (A) acetic acid (D) chloride (E) C27H40N2O9 536. 6 537 Phenylalanin 2-glutaric e benzyl ester nitrophenoxy-anhydride 11BDA (B) acetic acid (D) (A) C24H26N209 486.5 487 Phenylalanin 2- e benzyl ester nitrophenoxy-heptanoyl 11BDB (B) acetic acid (D) chloride (B) C26H32N2O7 484.5 485 Phenylalanin 2- e benzyl ester nitrophenoxy-decanoic 11BDC (B) acetic acid (D) acid (C) C29H38N207 526.6 527 Phenylalanin 2-methyl e benzyl ester nitrophenoxy-suberyl 11BDD (B) acetic acid (D) chloride (D) C28H34N209 542.6 543 Phenylalanin 2-methyl e benzyl ester nitrophenoxy-sebacoyl 11BDE (B) acetic acid (D) chloride (E) C3oH38N209 570.6 571 Leucine 3-glutaric benzyl ester nitrophenoxy-anhydride 11AEA (A) acetic acid (E) (A) C21H28N2O9 452. 5 453 Leucine 3- benzyl ester nitrophenoxy-heptanoyl 11AEB (A) acetic acid (E) chloride (B) C23H34N2O7 450.5 451 Leucine 3- benzyl ester nitrophenoxy-decanoic 11AEC (A) acetic acid (E) acid (C) C26H4oN207 492.6 493 Leucine 3-methyl benzyl ester nitrophenoxy-suberyl 11AED (A) acetic acid (E) chloride (D) C25H36N2O9 508.6 509 Leucine 3- methyl benzyl ester nitrophenoxy-sebacoyl 11AEE (A) acetic acid (E) chloride (E) C27H40N2O9 536.6 537 Phenylalanin 3-glutaric e benzyl ester nitrophenoxy-anhydride 11BEA (B) acetic acid (E) (A) C24H26N2O9 486.5 487 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (M+H) Phenylalanin 3- e benzyl ester nitrophenoxy-heptanoyl 11BEB (B) acetic acid (E) chloride (B) C26H32N207 484.5 485 Phenylalanin 3- e benzyl ester nitrophenoxy-decanoic.. 11BEC (B) acetic acid (E) acid (C) C29H38N207 526.6 527 Phenylalanin 3-methyl e benzyl ester nitrophenoxy-suberyl 11BED (B) acetic acid (E) chloride (D) C28H34N2O9 542.6 543 Phenylalanin 3-methyl e benzyl ester nitrophenoxy-sebacoyl 11BEE (B) acetic acid (E) chloride (E) C3oH38N2o9 570.6 571 Leucine 4-glutaric benzyl ester nitrophenoxy-anhydride 11AFA (A) acetic acid (F) (A) C2lH28N209 452.5 453 Leucine 4- benzyl ester nitrophenoxy-heptanoyl 11AFB (A) acetic acid (F) chloride (B) C23H34N207 450. 5 451 Leucine 4- benzyl ester nitrophenoxy-decanoic 11AFC (A) acetic acid (F) acid (C) C26H40N2O7 492.6 493 Leucine 4-methyl benzyl ester nitrophenoxy-suberyl 11AFD (A) acetic acid (F) chloride C25H36N2O9 508.6 509 Leucine 4-methyl benzyl ester nitrophenoxy-sebacoyl 11AFE (A) acetic acid (F) chloride (E) C27H40N2O9 536.6 537 Phenylalanin 4-glutaric e benzyl ester nitrophenoxy-anhydride 11BFA (B) acetic acid (F) (A) C24H26N209 486. 5 487 Phenylalanin 4- e benzyl ester nitrophenoxy-heptanoyl 11BFB (B) acetic acid (F) chloride (B) C26H32N207 484.5 485 Phenylalanin 4- e benzyl ester nitrophenoxyac decanoic 11BFC (B) etic acid (F) acid (C) C29H38N207 526.6 527 Phenylalanin 4-methyl e benzyl ester nitrophenoxy-suberyl 11BFD (B) acetic acid (F) chloride (D) C28H34N2Og 542.6 543 Phenylalanin 4-methyl e benzyl ester nitrophenoxy-sebacoyl 11BFE (B) acetic acid (F) chloride (E) C3oH38N209 570.6 571 Leucine 2-. glutaric benzyl ester nitrocinnamic anhydride 11AGA (A) acid (G) (A) C22H28N208 448.5 449 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (M+ Leucine 2- benzyl ester nitrocinnamic heptanoyl 11AGB (A) acid (G) chloride (B) C24H34N206 446.5 447 Leucine 2- benzyl ester nitrocinnamic decanoic 11AGC (A) acid (G) acid (C) C27HtoN206 488.6 489 Leucine 2-methyl . benzyl ester nitrocinnamic suberyl 11AGD (A) acid (G) chloride (D) C26H36N2°8 504.6 505 Leucine 2-methyl benzyl ester nitrocinnamic sebacoyl 11AGE (A) acid (G) chloride (E) C28H40N2O8 532.6 533 Phenylalanin 2-glutaric e benzyl ester nitrocinnamic anhydride 11BGA (B) acid (G) (A) C25H26N2O8 482.5 483 Phenylalanin 2- e benzyl ester nitrocinnamic heptanoyl 11BGB (B) acid (G) chloride (B) C27H32N206 480. 6 481 Phenylalanin 2- e benzyl ester nitrocinnamic decanoic 11BGC (B) acid (G) acid (C) C30H38N2O6 522.6 523 Phenylalanin 2-methyl e benzyl ester nitrocinnamic suberyl 11BGD (B) acid (G) chloride (D) C29H34N208 538.6 539 Phenylalanin 2-methyl e benzyl ester nitrocinnamic sebacoyl 11BGE (B) acid (G) chloride (E) C3lH38N208 566.6 567 Leucine 3-glutaric benzyl ester nitrocinnamic anhydride 11AHA (A) acid (H) (A) C22H28N208 448.5 449 Leucine 3- benzyl ester nitrocinnamic heptanoyl 11AHB (A) acid (H) chloride (B) C24H34N2O6 446.5 447 Leucine 3- benzyl ester nitrocinnamic decanoic 11AHC (A) acid (H) acid (C) C27H40N2O6 488.6 489 Leucine 3-methyl benzyl ester nitrocinnamic suberyl 11AHD (A) acid (H) chloride (D) C26H36N208 504.6 505 Leucine 3-methyl benzyl ester nitrocinnamic sebacoyl 11AHE (A) acid (H) chloride (E) C28H40N2O8 532.6 533 Phenylalanin 3-glutaric e benzyl ester nitrocinnamic anhydride 11BHA B) acid (H) (A) C25H26N2O8 482.5 483 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (M+I) Phenylalanin 3- e benzyl ester nitrocinnamic heptanoyl 11BHB (B) acid (H) chloride (B) C27H32N206 480.6 481 Phenylalanin 3- e benzyl ester nitrocinnamic decanoic 11BHC (B) acid (H) acid (C) C30H38N2O6 522.6 523 Phenylalanin.3-methyl e.benzyl ester nitrocinnamic suberyl 11BHD (B) acid (H) chloride (D) C29H34N208 538.6 539 Phenylalanin 3-methyl e benzyl ester nitrocinnamic sebacoyl 11BHE (B) acid (H) chloride (E) C31H38N208 566.6 567 Leucine 4-glutaric benzyl ester nitrocinnamic anhydride 11AIA (A) acid (I) (A) C22H28N2O8 448. 5 449 Leucine 4- benzyl ester nitrocinnamic heptanoyl 11AIB (A acid (I) chloride (B) C24H34N206 446.5 447 Leucine 4- benzyl ester nitrocinnamic decanoic 11AIC (A) acid (1) acid (C) C27H4oN206 488.6 489 Leucine 4-methyl benzyl ester nitrocinnamic suberyl 11AID (A) acid (1) chloride (D) C26H36N208 504.6 505 Leucine 4-methyl benzyl ester nitrocinnamic sebacoyl 11AIE (A) acid (I) chloride (E) C28H40N2O8 532.6 533 Phenylalanin 4-glutaric e benzyl ester nitrocinnamic anhydride 11BIA (B) acid (I) (A) C25H26N2O8 482.5 483 Phenylalanin 4- e benzyl ester nitrocinnamic heptanoyl 11BIB (B) acid (I) chloride (B) C27H32N206 480.6 481 Phenylalanin 4- e benzyl ester nitrocinnamic decanoic 11BIC (B) acid (1) acid (C) C30H38N206 522.6 523 Phenylalanin 4-methyl e benzyl ester nitrocinnamic suberyl 11BID (B) acid (I) chloride (d) c29h34n2o8 538.6 539 Phenylalanin 4-methyl e benzyl ester nitrocinnamic sebacoyl 11bie (B) acid (1) chloride (E) C31H38N208 566.6 567 Leucine 5- (2- glutaric benzyl ester nitrophenyl)-2-anhydride 11AJA (A) furoic acid (J) (A) C24H28N209 488.5 489 Cpd. No. First Second Third Formula Calcd found Component Component Component + Leucine 5- (2- benzyl ester nitrophenyl)-2-heptanoyl 11AJB (A) furoic acid (chloride (B) C26H34N2O7 486.6 487 Leucine 5- (2- benzyl ester nitrophenyl)-2-decanoic. 11AJC (A) furoic acid acid (C) C29H40N2O7 528.6 529 Leucine 5- (2- methyl benzyl ester nitrophenyl)-2-suberyl 11AJD (A) furoic acid (J) chloride (D) C28H36N2O9 544. 6 545 Leucine 5- (2- methyl benzyl ester nitrophenyl)-2-sebacoyl 11AJE (A) furoic acid (J) chloride (E) C3oH4oN209 572.7 573 Phenylalanin 5- (2- glutaric e benzyl ester nitrophenyl)-2-anhydride 11BJA (B) furoic acid ( (J) A) C27H26N2O9 522.5 523 Phenylalanin 5- (2- e benzyl ester nitrophenyl)-2-heptanoyl 11BJB (B) furoic acid (J) chloride (B) C29H32N2O7 520.6 521 Phenylalanin 5- (2- e benzyl ester nitrophenyl)-2-decanoic 11BJC (B) furoic acid (J) acid (C) C32H38N207 562.7 563 Phenylalanin 5- (2- methyl e benzyl ester nitrophenyl)-2-suberyl 11BJD (B) furoic acid (J) chloride (D) C31H34N209 578.6 579 Phenylalanin 5- (2- methyl e benzyl ester nitrophenyl)-2-sebacoyl 11BJE (B) furoic acid (4 chloride (E) C33H38N209 606.7 607 Leucine 5- (3- glutaric benzyl ester nitrophenyl)-2-anhydride 11AKA (A) furoic acid (K) (A) C24H28N209 488.5 489 Leucine 5- (3- benzyl ester nitrophenyl)-2-heptanoyl 11AKB (A) furoic acid (K) chloride (B) C26H34N207 486.6 487 Leucine 5- (3- benzyl ester nitrophenyl)-2-decanoic 11AKC (A) furoic acid (K) acid (C) C29H4oN207 528.6 529 Leucine 5- (3- methyl benzyl ester nitrophenyl)-2-suberyl 11AKD (A) furoic acid (K) chloride (D) C28H36N209 544.6 545 Leucine 5- (3- methyl benzyl ester nitrophenyl)-2-sebacoyl 11AKE (A) furoic acid (K) chloride (E) C30H40N2O9 572.7 573 Phenylalanin 5- (3- glutaric e benzyl ester nitrophenyl)-2-anhydride 11BKA (B) furoic acid (K) (A) C27H26N209 522.5 523 MW MW Cpd. No. First Second Third Formula Calcd found Component Component Component (M+H) Phenylalanin5- (3- e benzyl ester nitrophenyl)-2-heptanoyl 11BKB (B) furoic acid (K) chloride (B) C29H32N207 520.6 521 Phenylalanin5- (3- e benzyl ester nitrophenyl)-2-decanoic. 11BKC (B) furoic acid (K) acid (C) C32H38N207 562.7 563 Phenylalanin 5- (3- methyl. e benzyl ester. nitrophenyl)-2-suberyl. 11BKD (B) furoic acid (K) chloride (D) C31H34N2O9 578.6 579 Phenylalanin5- (3- methyl e benzyl ester nitrophenyl)-2-sebacoyl 11BKE (B) furoic acid (K) chloride (E) C33H3gN209 606.7 607 Leucine 5- (4- glutaric benzyl ester nitrophenyl)-2-anhydride 11ALA (A) furoic acid (L) (A) C24H28N209 488.5 489 Leucine 5- (4- benzyl ester nitrophenyl)-2-heptanoyl 11ALB (A) furoic acid (L) chloride (B) C26H34N207 486.6 487 Leucine 5- (4- benzyl ester nitrophenyl)-2-decanoic 11ALC (A) furoic acid (L) acid (C) C29H40N2O7 528.6 529 Leucine 5- (4- methyl benzyl ester nitrophenyl)-2-suberyl 11ALD (A) furoic acid (L) chloride 9D) C28H36N2O9 544.6 545 Leucine 5- (4- methyl benzyl ester nitrophenyl)-2-sebacoyl HALE (A) furoic acid (L) chloride (E) C30H40N2O9 572.7 573 Phenylalanin 5-(4- glutaric e benzyl ester nitrophenyl)-2-anhydride 11BLA (B) furoic acid (L) (A) C27H26N209 522.5 523 Phenylalanin5- (4- e benzyl ester nitrophenyl)-2-heptanoyl 11BLB (B) furoic acid (L) chloride (B) C29H32N207 520.6 521 Phenylalanin5- (4- e benzyl ester nitrophenyl)-2-decanoic 11BLC (B) furoic acid (L) acid (C) C32H38N2O7 562.7 563 Phenylalanin5- (4- methyl e benzyl ester nitrophenyl)-2-suberyl 11BLD (B) furoic acid (L) chloride (D) C31H34N20g 578.6 579 Phenylalanin5- (4- methyl e benzyl ester nitrophenyl)-2-sebacoyl 11BLE (B) furoic acid (L) chloride (E) C26H32N209 606.7 607

Example 7 BIOLOGICAL ACTIVITY Neuronal Viability Assay This assay is used to assess the ability of compounds of this invention to protect neurons from glutamate-induced excitotoxic cell death. Primary cell culture is performed with embryonic day 18 rat hippocampal and cortical neurons that are plated into Biocoat Poly-D-Lysine precoated 96-well plates (Becton-Dickinson, Bedford, MA; catalog no. 356461) at a density of 21,000 cells/well. The. cells are grown in Neurobasal media (Gibco/Life Technologies, Rockville, MD, catalog no. 21103049) supplemented with B27, penicillin (100 IU/ml), streptomycin (100 zg/ml) and 500 uM L-glutamine.

This media supports growth of pure neuronal cultures as contaminating glial cells that may be initially present do not survive in the media conditions. At 17 days after culture, media is aspirated from wells and 100 ul solution of test compound in assay buffer (HBSS supplemented with 25 mM HEPES) is added. After 10 min of incubation, 100 u, L of test compound solution in assay buffer supplemented with 200 uM glutamate/20 uM glycine is added. Ten min later, cells are treated with 100 ul of a 20 LM solution of MK-801 (an NMDA receptor antagonist that blocks Ca2+ influx ; Sigma-Aldrich, St.

Louis, MO, catalog no. M-107) in Neurobasal media. After 24 hrs, the extent of cell death is quantitated by measuring lactate dehydrogenase activity released by lysed cells using a colorimetric Cytotoxicity Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany, catalog no. 1 644 793) and following the manufacturer's instructions.

In this assay, preferred compounds have a mean neuronal viability of 0.6 or less. To this end, preferred compounds of this invention include the following: 6AAD, 6AAE, 6ABE, 6ABF, 6ACE, 6BAA, 6BAE, 6BAG, 6BCA, 6BCB, 6CCA, 7AAC, 7AAE, 7ABA, 7BAA, 7BBD, 9AAA, 9AAB, 9AAE, 9ABA, 9ABB, 9ABC, 9BAD, 9BAE, 9BBA, 9BBD, 9CAA, 9CAB, 9CAC, 9CAD, 9CBA, 9CBC, 9CBD, 9CBE, 9DBA, 9DBB, 9DBC, 8AAB, 8AAE, 8ABB, 8ABE, 8BAB, 8BBA, 8BBB, 8CAB, 8CAD and 8CBA.

Displacement Assays of an Adenine Nucleotide Translocase (ANT) Ligand from Isolated Mitochondria using Test Compounds Compound 1 below is a 12sI-labeled atractyloside derivative that binds to the mitochondrial adenine nucleotide translocase with high affinity (IC5o =300 nom in a displacement assay using [3H]-ADP as ligand). Thus, Compound 1 may be used as the radioligand to measure efficacy of binding of the compounds of this invention.

Compound 1

Competition binding assays are performed using bovine cardiac mitochondria. One microgram aliquots of mitochondrial protein are incubated with 100 ul binding buffer (10 mM Tris, 120 mM KC1, 6 mM MgCl2, 1 mM EDTA, pH 7.4) containing 0. 5 nM Compound 1 and a test compound at various concentrations (10 or 100 uM). Mixtures are incubated for one hour on ice, at the end of which unbound ligand is separated by centrifugation. Supernatants containing unbound Compound 1 are aspirated and discarded. Mitochondrial pellets are washed with four volumes of cold binding buffer and counted in a Micromedic 4/200 automatic gamma counter. For higher throughput, assays are performed in a 96 well microtitre well format. Unbound ligand is removed by filtration and washes through glass fiber filter mats (Whatman GF/B paper, catalog no. FPXLR-196, Brandel, Inc., Gaithersburg, MD). The radioactivity associated with the mitochondrial pellets retained in the filter mat is determined in a 1450 Wallac MicroBeta TriLux liquid scintillation and luminescence counter (EG&G Wallac, Gaithersburg, MD). In this assay, preferred compounds of this invention (at 10 uM) displace the radioligand (i. e., Compound 1) such that 80% or less of the radioactivity of the mitochondrial pellets is detected. To this end, preferred compounds are 6AAB, 6AAD, 6ABA, 6ABB, 6ABD, 6ACB, 6ACC, 6BAB, 6BAC, 6BAD, 6BBA, 6BCB, 6BCF, 6CAB, 6CAC, 6CAD, 6CAG, 6CBB, 6CBD, 6CBE, 6CCC, 6CCD, 7AAE, 8AAE, 8BBC and 8CBC.

Example 8 CHONDROCYTE CYTOPROTECTION This example illustrates the ability of a test compound, 9DBC, to mediate chondrocyte cytoprotection. More specifically, the test compound was found to protect against (1) trigger-induced cell death (i. e., viability), (2) trigger-induced inhibition of collagen synthesis, (3) trigger-induced GAG release, and (4) IL-1-mediated GAG release and NO generation. The procedures employed are set forth below, while the results of these assays are summarized in Table 7.

TABLE 7 SUMMARY OF RESULTS Viability1(cells Collagen2(cells) GAG3(cells) GAG(Slices)NO4 (Slices Trigger NOc-12 SIN-1 NOC-12 SIN-1 IL-1 NOC-12 SIN-1 IL-1 IL-1 IL-1 - o # 1 nm 1 nm # 1 µm >1 µm >1 um 100 nm 100 nm 100 nm 10 µm 10 µm 1. Protection against trigger-induced cef) death. TC 28 cefis as monoiayer culture. Trigger NOC-12, 250 pM ; SIN-1, 100 pM.

2. Protection against trigger-induced inhibition of collagen synthesis. TC 28 cells cultured in polyHEME plates. trigger. NOC-12, 25 uM 3. Protection against trigger-induced GAG release. TC 28 cells cultured in polyHEME plates. trigger : NOC-12, 25 pM ; SIN-1, 10 uM ; IL 4. Protection against IL-1-mediated GAG release and NO generation in bovine cartilage slices. IL-1 trigger, 10 ng/ml.

Chondrocyte Function Screening Assays Cell culture: Chondrocytic TC28 cells were maintained in monolayer culture in DMEM/Ham's F12 (1: 1) and supplemented with 10% FCS, 1% L- glutamine, 100 units/ml Penicillin and 50 mg/ml Streptomycin (Omega Scientific, Tarzana, CA) and cultured at 37~C with 5% C02. Additionally, to further study chondrocytic cells in a more physiologic nonadherent state, in some experiments, TC28 cells were transferred to 6 well plates that had been previously coated for 18 hours at 22°C with 10% (v/v) in 95% ethanol solution of the cell adhesion inhibitor poly 2- Hydroxyethyl methacrylate (polyHEME), followed by two washes in PBS. Complete DMEM/Ham's F12 medium was then added to the wells and the cells studied for up to 72 hours in culture. Type II collagen and aggrecan expression were confirmed using RT-PCR, which verified maintenance of chondrocyte phenotype.

Screening Assays : Compound 9DBC was screened for chohdrocyte protective effects in vitro. The agonists employed have included a donor of nitric oxide (NOC-12, 250 uM), a donor of peroxynitrite (SIN-1,100 uM), and human recombinant IL-lbeta (10 ng/ml). Cytotoxicity was measured via standard lactate dehydrogenase (LDH) release assay as described below. Collagen synthesis was monitored by 3H proline incorporation into TC28 cells, TCA precipitation of proteins, followed by assay of radioactivity in collagenae sensitive proteibn as outlined in Johnson et al (Arthritis Rhum. 43 : 1560-70,2000). NO was detected by using the Greiss reaction.

Cytotoxicityassay 105 TC28 cells (DMEM/F12 media with 10% FCS, 1% glutamine, 1% P/S) were plated each well in a 96 well plate and allowed to adhere overnight. The cells were washed once with PBS and media changed to contain only 1% FCS. Compound 9DBC at various concentrations was added to the cells for a pretreatment of 1 hr. The media was removed and fresh compound +/-the toxic stimuli are added. The cells were

then incubated for 24 hrs at 37°C. Following the incubation the media was collected and used for analysis in the CytTox 96 Nonradioactive Cytotoxicity Assay. Briefly, the LDH release from the dead cells was quantified in a 30 min enzymatic reaction that resulted in the conversion of a tetrazolium saletin to a red formazan product. The results were expressed as the percent of cells dead as to the release of LDH by the control cells.

Gylcosaminoglycans (GAG) Release Assay The enhanced release from chondrocytes of glycosaminoglycans (GAG) is a central feature of osteoarthritic chondrocytes, and is known to be stimulated potently by IL-1, which, like NO and peroxynitrite is held to be a major pathogenic factor in osteoarthritis. Thus, GAG release assays were carried out on Compound 9DBC, in which, to optimize the screening assay, a one hour digestion of the cartilage "nodules"formed in the polyheme system was carried out using 300ug/ml of papain in 20 mM sodium phosphate, 1mM EDTA, and 2mM DTT (pH 6.8). The digestion of the interfering proteins accomplished in this manner allowed the GAG release to be more readily detectable, and the GAG release was quantified by the standard dimethylene blue (DMB) dye binding colorimetric assay. In brief, the cell extract digested from above was combined with 46 uM DMB, 40 mM glycine and 40 mM NaCI (pH 3.0) and immediately read at 525, nm and compared again a standard curve of 1-50 ug/ml chondroitin sulfate.

Bovine Cartilage Organ Culture Methods Mature bovine knees were obtained and cartilage from the femoral condyles and patellar groove was removed in full thickness slices (1-3mm). Circular cores (6-7 mm in diameter) were punched out of the tissue. The cores were washed twice with media (1 % FCS, 1 % P/S, 1% glutamine containing DMEM high glucose) and then placed in 96 wells plates. The slices were incubated in media (as above) at 37°C for 48 hrs to allow for recovery from the isolation process. After the recovery period, the media was removed and fresh media with Compound 9DBC was added to the slices for a pretreatment period of 6 hrs. Then the media was removed and fresh compound plus/minus IL-1 was added and incubated at 37°C for 24 hrs. The conditioned media was collected and the GAG and NO release were analyzed. Finally the slices were weighed to correct for slight variations in size or thickness.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.