Iap binding compounds

ES 2 318 167 T3

DESCRIPTION

IAP binding compounds.

This application claims the benefit of US Provisional Application No. 60 / 395,918, filed July 15, 2002.

Field of the invention

The present invention relates to the field of drug design and development for the diagnosis, prevention and treatment of cell proliferative disease. Specifically, the invention offers peptidomimetics of the N-terminal tetrapeptide of the mitochondrial protein Smac / DIABLO (hereinafter Smac), which promote apoptosis in cells through a pathway involving the inhibitor of apoptosis proteins (IAP ), shown as an example using XLAP. These peptidomimetics bind to IAPs and offer improved pharmacological characteristics compared to tetrapeptide.

Background of the invention

Apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multicellular organisms. Alterations in apoptotic pathways have been implicated in many types of human pathologies, including developmental disorders, cancer, autoimmune diseases, as well as neurodegenerative disorders.

Thus, programmed cell death pathways have become attractive targets for the development of therapeutic agents. In particular, since it is conceptually easier to kill than to maintain cells, attention has focused on anticancer treatments using pro-apoptotic agents such as conventional chemotherapy and radiation. In general, these treatments are believed to trigger the activation of mitochondrial-mediated apoptotic pathways. However, these treatments lack molecular specificity and more specific molecular targets are needed.

Apoptosis is carried out mainly by activated caspases, a family of cysteine ​​proteases with specificity for aspartate on its substrates. Caspases are produced in cells as catalytically inactive zymogens and must be proteolytically processed to become active proteases during apoptosis. In normal surviving cells that have not received an apoptotic stimulus, most of the caspases remain inactive. Even if some caspases are activated abnormally, their proteolytic activity can be completely inhibited by a family of proteins conserved in evolution called IAPs (inhibitors of apoptosis proteins) (Deveraux & Reed, Genes Dev. 13: 239-252, 1999) . Each of the IAPs contains 1-3 copies of the so-called BIR domain (baculovirus IAP repeat) and interacts directly with and inhibits the enzymatic activity of mature caspases. Several different mammalian IAPs have been identified including XIAP, survivin, and livin / ML-LAP (Kasof & Gomes, J. Biol. Chem. 276: 3238-3246, 2001; Vucic et al. Curr. Biol. 10: 1359-1366 , 2000; Ashhab et al. FEBS Lett. 495: 56-60, 2001), and all show antiapoptotic activity in cell culture (Deveraux & Reed, 1999, cited above). Because IAPs are expressed in most cancer cells, they can directly contribute to tumor progression and subsequent resistance to drug treatment.

However, in normal cells destined to undergo apoptosis, the IAP-mediated inhibitory effect must be eliminated, a process performed, at least in part, by a mitochondrial protein called Smac (a second mitochondrial-derived caspase activator; Du et al. Cell 102: 33-42, 2000) or DIABLO (low pI direct IAP-binding protein; Verhagen et al. Cell 102: 43-53, 2000). The Smac, synthesized in the cytoplasm, targets the intermembrane space of the mitochondria. Following apoptotic stimuli, Smac is released from the mitochondria back into the cytosol, along with cytochrome c. While cytochrome c induces Apaf-1 multimerization to activate procaspase-9 and -3, Smac eliminates the inhibitory effect of multiple IAPs. Smac interacts with all IAPs that have been examined to date, including XIAP, c-IAP1, c-IAP2, and survivin (Du et al., 2000, cited above; Verhagen et al., 2000, cited above). Thus, Smac appears to be a master regulator of apoptosis in mammals.

Smac is synthesized as a 239 amino acid precursor molecule; the 55 N-terminal residues serve as the mitochondrial targeting sequence that is removed upon transfer (Du et al., 2000, cited above). The mature form of Smac contains 184 amino acids and behaves like an oligomer in solution (Du et al., 2000, cited above). Smac and various fragments thereof have been proposed for use as targets for the identification of therapeutic agents. US Patent No. 6,110,691 issued to Wang et al. describes the Smac polypeptide and fragments ranging from at least 8 amino acid residues in length. However, the patent neither discloses nor teaches a structural basis for choosing a particular peptide fragment of Smac for use as a target or therapeutic agent.

Similar to mammals, flies contain two IAPs, DIAP1 and DIAP2, which bind and inactivate various Drosophila caspases (Hay, Cell Death Differ. 7: 1045-1056, 2000). DIAP1 contains two BIR domains; the second BIR domain (BIR2) is necessary and sufficient to block cell death in many contexts. In Drosophila cells, the anti-death function of DIAP1 is eliminated by three pro-apoptotic proteins, Hid, Grim and Reaper,

ES 2 318 167 T3 that physically interact with the BIR2 domain of DIAP1 and eliminate its inhibitory effect on caspases. Thus Hid, Grim and Reaper represent the functional homologues of the mammalian protein Smac. However, except for their 10 N-terminal residues, Hid, Grim, and Reaper do not share sequence homology with each other, and there is no apparent homology between the three Drosophila and Smac proteins.

In co-pending US application number 09 / 965,967, it is disclosed that the previously described biological activity of Smac is related to the binding of its four N-terminal residues to a characteristic surface groove in a part of XIAP called the BIR3 domain. This binding prevents XIAP from exerting its apoptosis-suppressing function in the cell. Furthermore it was disclosed that the N-terminal tetrapeptides of the IAP-binding proteins of the Drosophila Hid, Grim and Veto proapoptotic proteins function in the same way.

Co-pending international application co-owned and co-owned number PCT / US02 / 17342 describes assays for use in high-throughput screening or rational drug design of agents that can, such as the Smac tetrapeptide or its homologues in other species bind to a BIR domain of an IAP, thereby alleviating the IAP-mediated suppression of apoptosis. Those assays are based on competitive displacement of a labeled IAP-binding tetrapeptide by a test compound. That application also describes a library of peptides and N-methyl-peptide analogs which, by the assay method, were shown to bind to the BIR3 domain of XIAP.

The use of peptides for in vivo administration as therapeutic or diagnostic agents is associated with certain disadvantages. These include a short half-life due to proteolytic degradation in the body, low absorption through the intestinal walls, and possible immunogenic reactions, as well as the expense involved in peptide synthesis. For these reasons, many current drug development efforts focus on non-peptide mimetics that mimic the structure and biological activity of bioactive peptides, but have improved pharmacological properties and are easier and cheaper to synthesize.

W002 / 26775 discloses peptides and peptidomimetics that can modulate apoptosis through their interaction with cellular IAPs. In this connection the peptide sequence of AVPF is disclosed.

In relation to the Smac tetrapeptides and homologues described above, then, it would be a significant advance in the art to develop non-peptide or partial peptide mimetics of those molecules. Such mimetics should exhibit the apoptosis-promoting and IAP-binding bioactivity of Smac peptides, while also having improved properties associated with non-peptide mimetics, for use as therapeutic and diagnostic agents in the treatment of cancer.

Summary of the invention

The present invention relates generally to oligopeptides and peptidomimetics, to pharmaceutical compositions containing these compounds, and to methods for their use as therapeutic and diagnostic agents. The compounds offered herein are mimetics of the N-terminal tetrapeptide of IAP (inhibitor of apoptosis protein) binding proteins, such as the mammalian Smac / DIABLO and its homologues, hid, grim and reaper.

In one embodiment, the invention relates to compounds of formula I:

in which:

R¹ is methyl, ethyl, n-propyl, isopropyl, or ethenyl;

R^1st is H or methyl;

X is -O-, -S-, -CH₂- or -NH-, and J is -CH- or -N-, provided that when J is -N-, X is -CH₂- or -NH-;

Y is H, methyl, ethyl, n-propyl, or isopropyl;

ES 2 318 167 T3

R² it is:

<img file="ES2318167T3_D0001.tif" />

R^2nd is cycloalkyl or cycloalkylalkyl, alpha-naphthylmethyl, beta-naphthylmethyl or a benzyl substituted with one or more halogen, aryl, carboxyl, alkoxycarbonyl or aroyl, or combinations thereof;

R^2b is H or alkyl;

Month:

<img file="ES2318167T3_D0002.tif" />

Ar is:

<img file="ES2318167T3_D0003.tif" />

R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen, cyano, - (CH2) pC (-O) OH, - (CH₂) pC (= O) O-alkyl, - (CH2) pC (= O) NH2;

n and p are each independently the integer 0, 1, 2, or 3, and the sum of (n + p) is the integer 2 or 3; provided that at least one of R³, R⁴ and R⁵, or at least two of R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen or cyano;

provided that when one or more of R³ and R⁵ is isopropyl, R⁴ it is different from isopropyl;

provided that when R⁴ is isopropyl, R³ and R⁵ are each independently different from isopropyl;

provided that when R⁸ is isopropyl, R⁹ it is different from isopropyl; and as long as R^1st is H, X is -NH-, J is -CH-, Y is H, methyl or isopropyl, and R² it is:

<img file="ES2318167T3_D0004.tif" />

L-amino acid then

R¹ is ethenyl;

or a pharmaceutically acceptable salt thereof.

ES 2 318 167 T3

<img file="ES2318167T3_D0005.tif" />

R¹ is methyl, ethyl, n-propyl, isopropyl, or ethenyl;

R^1st is H or methyl;

Q¹ and Q³ are each independently -O-, S-, or -NH-;

Q² is -CH- or -N-;

Q⁴ is -N-;

R² it is:

<img file="ES2318167T3_D0006.tif" />

R^2nd is cycloalkyl, cycloalkylalkyl, alpha-naphthyl-methyl, beta-naphthylmethyl or a benzyl substituted with one or more halogen, aryl, carboxyl, alkoxycarbonyl or aroyl or combinations thereof;

R^2b is H or alkyl;

Month:

<img file="ES2318167T3_D0007.tif" />

Ar is:

<img file="ES2318167T3_D0008.tif" />

R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen, cyano, - (CH₂) pC (-O) OH, - (CH₂) pC (= O) O-alkyl, - (CH₂) pC (= O) NH2;

ES 2 318 167 T3

R¹⁰ is H or methyl;

n and p are each independently the integer 0, 1, 2, or 3, and the sum of (n + p) is the integer 2 or 3;

provided that at least one of R³, R⁴ and R⁵ or at least two of R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen or cyano;

provided that when one or more of R³ and R⁵ is isopropyl, R⁴ it is different from isopropyl;

provided that when R⁴ is isopropyl, R³ and R⁵ are each independently different from isopropyl;

provided that when R⁸ is isopropyl, R⁹ it is different from isopropyl;

or a pharmaceutically acceptable salt thereof.

The above compounds can be formulated as pharmaceutical compositions or as diagnostic agents or both. As described in greater detail herein, these pharmaceutical compositions and diagnostic agents are useful for the treatment or detection of cell proliferative disorders, as well as in screening assays for the discovery and development of additional therapeutic and diagnostic agents. .

Other features and advantages of the invention will be understood with reference to the detailed description and examples that follow.

Detailed Description of Illustrative Embodiments

As used above and throughout the description, unless otherwise indicated, the following terms are to be understood to have the following meanings.

The terms "pharmaceutically active" and "biologically active" refer to the ability of the compounds of the invention to bind to IAP (inhibitor of apoptosis protein), specifically to the BIR binding groove of IAP, more specifically to the groove of BIR3 binding of IAP. This biological activity can be measured with respect to any IAP, with XIAP being particularly suitable, and an exemplary embodiment being a peptide fragment comprising the BIR3 binding domain of XIAP.

The pharmaceutically active compounds of the invention are sometimes referred to herein as drugs, to emphasize their therapeutic utility in promoting apoptosis by binding IAP. However, another embodiment of the invention uses the compounds as diagnostic agents, for the detection of IAP in vitro, in situ or in vivo, or for IAP binding assays. In this embodiment, the compounds of the invention are detectably labeled, for example, with a fluorophore as described in co-pending international application number PCT / US02 / 17342. Another embodiment of the invention uses the compounds as targeting agents, that is, incorporating tumor cell killing agents or other therapeutic or antitumor agents, such as radionuclides, into their structure. Accordingly, the term "drug" as used herein is intended to refer to all pharmaceutically / biologically active (ie, IAP-binding) compounds of the invention, for use as therapeutic or diagnostic agents.

As used herein, "alkyl" refers to a saturated, linear, branched, or cyclic hydrocarbon having from about 1 to about 10 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms). carbon therein), with about 1 to about 7 carbon atoms being preferred. Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl, cyclooctyl , adamantyl, 3-methylpentyl, 2,2 -dimethylbutyl and 2,3-dimethylbutyl.

As used herein, "halogen" refers to the Cl or F moiety.

As used herein, "cyano" refers to the remainder:

<img file="ES2318167T3_D0009.tif" />

As used herein, "aryl" refers to an optionally substituted mono- or bicyclic aromatic ring system, having from about 5 to about 14 carbon atoms (and all combinations and subcombinations of specific ranges and numbers carbon atoms therein), with about 6 to about 10 carbons being preferred. Non-limiting examples include, for example, phenyl and naphthyl.

ES 2 318 167 T3

As used herein, "aralkyl" refers to alkyl radicals bearing aryl substituents having from about 6 to about 20 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms in the itself), with about 6 to about 12 carbon atoms being preferred. Aralkyl groups can be optionally substituted. Non-limiting examples include, for example, benzyl, naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl.

As used herein, "cycloalkyl" refers to an optionally substituted alkyl group, having one or more rings in its structures having from about 3 to about 14 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with about 3 to about 10 carbon atoms being preferred. Multiple ring structures can be groups of bridged or fused ring structures include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and adamantyl.

As used herein, "cycloalkylalkyl" refers to alkyl radicals bearing a cycloalkyl substituent and having from about 4 to about 20 carbon atoms (and all combinations and subcombinations of specific ranges and numbers of carbon atoms therein), with about 6 to about 12 carbon atoms being preferred.

As used herein, the terms "alkoxy" and "alkoxy" refer to an optionally substituted alkyl-O group in which alkyl is as defined above. Exemplary alkoxy and alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, α-butoxy, and heptoxy.

As used herein, "carboxyl" refers to a group -C (= O) OH.

As used herein, "alkoxycarbonyl" refers to a group -C (-O) O-alkyl, where alkyl is as defined above.

As used herein, "aroyl" refers to a group -C (= O) -aryl, where aryl is as defined above. Exemplary aroyl groups include benzoyl and naphthoyl.

Typically, substituted chemical moieties include one or more substituents that replace hydrogen. Exemplary substituents include, for example, halogen (eg, F, Cl), alkyl, cycloalkyl, aralkyl, aryl, hydroxyl (-OH), alkoxy, cyano (-CN), carboxyl (-COOH), - C (= O) O-alkyl, aminocarbonyl (-C (= O) Nh₂), -N-substituted aminocarbonyl (-C (= O) NHR "), CF₃, CF₂CF₃, and the like. In relation to the substituents mentioned above, each R "moiety can independently be any of H, alkyl, cycloalkyl, aryl or aralkyl, for example.

As used herein, "L-amino acid" refers to any of the naturally occurring levorotatory alpha amino acids normally present in proteins or the alkyl esters of those alpha amino acids. Alpha-amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, asparagine, glutamine, serine, threonine, aspartic acid, glutamic acid, tyrosine, cysteine, lysine, arginine, and histidine.

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by preparing acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; organic or alkaline salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymax, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic , fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like. These physiologically acceptable salts are prepared by methods known in the art, for example, by dissolving the free amine bases with an excess of the acid in aqueous alcohol or by neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with a amine.

When any variable appears more than once in any constituent or in any formula, its definition in each case is independent of its definition in any other case. Combinations of substituents and / or variables are acceptable only if such combinations result in stable compounds.

The chemical names and formulas used herein are believed to accurately and correctly reflect the underlying chemical compounds. However, the nature and value of the present invention do not depend on the theoretical accuracy of these formulas, in whole or in part. It is therefore understood that the formulas used herein, as well as the chemical names attributed to the correspondingly indicated compounds, are not intended to limit the invention in any way, including restricting it to any specific tautomeric form.

ES 2 318 167 T3 ca or to any specific optical shape; or geometric isomer, except where such stereochemistry is clearly defined.

The compounds described throughout this document can be used or prepared in alternate ways. For example, many amino-containing compounds can be used or prepared as an acid addition salt. Such salts often improve the isolation and handling properties of the compound. For example, depending on reagents, reaction conditions and the like, compounds as described herein can be used or prepared, for example, as their hydrochloride or tosylate salts.

Accordingly, in one embodiment, the invention provides novel pharmaceutically active compounds of formula I:

<img file="ES2318167T3_D0010.tif" />

Ϊ in which:

R¹ is methyl, ethyl, n-propyl, isopropyl, or ethenyl;

R^1st is H or methyl;

X is -O-, -S-, -CH2- or -NH-, and J is -CH- or -N-, provided that when J is -N-, X is -CH2- or -NH-;

Y is H, methyl, ethyl, n-propyl, or isopropyl;

R² it is:

<img file="ES2318167T3_D0011.tif" />

R^2nd is cycloalkyl, cycloalkylalkyl, alpha-naphthylmethyl, beta-naphthylmethyl or a benzyl substituted with one or more halogen, aryl, carboxyl, alkoxycarbonyl or aroyl, or combinations thereof;

R^2b is H or alkyl;

Month:

<img file="ES2318167T3_D0012.tif" />

Ar is:

<img file="ES2318167T3_D0013.tif" />

ES 2 318 167 T3

R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen, cyano, - (CH₂) pC (= O) OH, - (CH₂)_P-C (= O) O-alkyl, - (CH₂)_P-C (= O) NH₂;

n and p are each independently the integer 0, 1, 2, or 3, and the sum of (n + p) is the integer 2 or 3;

provided that at least one of R³, R⁴ and R⁵ or at least two of R⁶, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen or cyano;

provided that when one or more of R³ and R⁵ is isopropyl, R⁴ it is different from isopropyl;

provided that when R⁴ is isopropyl, R³ and R⁵ are each independently different from isopropyl;

provided that when R⁸ is isopropyl, R⁹ it is different from isopropyl; and as long as R^1st is H, X is -NH-, J is -CH-, Y is H, methyl or isopropyl and, R² it is:

<img file="ES2318167T3_D0014.tif" />

then

R¹ is ethenyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments of the compounds of formula I, R¹ it is methyl, ethyl or ethenyl. More preferably, R¹ is methyl.

In other embodiments of the compounds of formula I, R^1st it's H.

In some embodiments of the compounds of formula IY is H, methyl, ethyl, n-propyl, or isopropyl. Most preferably Y is H, methyl or isopropyl. Even more preferably Y is methyl or isopropyl. Still more preferably Y is isopropyl.

In other embodiments of the compounds of formula I, Ar is:

<img file="ES2318167T3_D0015.tif" />

In certain more preferred embodiments of the compounds of formula I, when Ar is:

<img file="ES2318167T3_D0016.tif" />

one of R³, R⁴ and R⁵ is - (CH2) pC (= O) OH, - (CH2) pC (= O) O-alkyl, - (CH2) pC (= O) NH2. More preferably, when one of R³, R⁴ and R⁵ is - (Ch2)_p-C (= O) OH, - (Ch₂)_p-C (= O) O-alkyl, - (Ch₂)_p-C (-O) NH₂, p is the integer 0.

ES 2 318 167 T3

Even more preferably one of one of R³, R⁴ and R⁵ is - (CH2) pC (= O) OH or - (CH2) pC (= O) O-alkyl. More preferably still, when one of one of R³, R⁴ and R⁵ is - (CH₂)_p-C (= O) OH or - (CH₂)_p-C (= O) O-alkyl, p is the integer 0. Even more preferably, one of R³, R⁴ and R⁵ is - (CH2) pC (= O) OH. Most preferably, when one of R³, R⁴ and R⁵ is - (CH2) pC (= O) OH, p is the integer 0.

In some embodiments of the compounds of formula I, the sum of (n + p) is the integer 2 or 3. More preferably it is the integer 2.

In some embodiments of the compounds of formula I in which R² it is

<img file="ES2318167T3_D0017.tif" />

M is preferably

2 + X _t / Wx

<img file="ES2318167T3_D0018.tif" />

In other embodiments of the compounds of formula I, Ar is:

<img file="ES2318167T3_D0019.tif" />

In still other embodiments of the compounds of formula I, R² it is:

<img file="ES2318167T3_D0020.tif" />

Preferably R^2nd it is cyclohexyl, alpha-naphthylmethyl, beta-naphthylmethyl, or cyclohexylmethyl. Alternatively, R^2nd is benzyl substituted with one or more halogen, aryl, carboxyl, alkoxycarbonyl, or aroyl, or combinations thereof.

In another embodiment, the invention provides novel pharmaceutically active compounds of formula IIa or IIb:

<img file="ES2318167T3_D0021.tif" />

ES 2 318 167 T3 in which:

R¹ is methyl, ethyl, n-propyl, isopropyl, or ethenyl;

R^1st is H or methyl;

Q¹ and Q³ are each independently -O-, -S- or -NH-;

Q² is -CH- or -N-;

Q⁴ is -N-;

R² it is:

<img file="ES2318167T3_D0022.tif" />

R^2nd is cycloalkyl, cycloalkylalkyl, alpha-naphthylmethyl, beta-naphthylmethyl or a benzyl substituted with one or more halogen, aryl, carboxyl, alkoxycarbonyl or aroyl, or combinations thereof;

R^2b is H or alkyl;

Month:

<img file="ES2318167T3_D0023.tif" />

Ar is:

<img file="ES2318167T3_D0024.tif" />

R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen, cyano, - (CH₂) pC (= O) OH, - (CH₂) pC (= O) O-alkyl, - (CH2) pC (= O) NH2;

R¹⁰ is H or methyl, n and p are each independently the integer 0, 1, 2 or 3, and the sum of (n + p) is the integer 2 or 3;

provided that at least one of R³, R⁴ and R⁵ or at least two of R⁶, R⁷, R⁸ and R⁹ are each independently H, methyl, ethyl, n-propyl, isopropyl, halogen or cyano;

provided that when one or more of R³ and R⁵ is isopropyl, R⁴ it is different from isopropyl;

provided that when R⁴ is isopropyl, R³ and R⁵ are each independently different from isopropyl;

provided that when R⁸ is isopropyl, R⁹ it is different from isopropyl;

ES 2 318 167 T3 or a pharmaceutically acceptable salt thereof.

In some embodiments of the compounds of formula IIa or IIb, R¹ it is methyl, ethyl or ethenyl. More preferably, R¹ is methyl.

In other embodiments of the compounds of formula IIa or IIb, R^1st it's H.

In certain embodiments of the compounds of formula IIa or IIb, Y is H, methyl, or isopropyl. More preferably, it is isopropyl.

In other embodiments of the compounds of formula IIa or IIb, Ar is:

<img file="ES2318167T3_D0025.tif" />

In certain more preferred embodiments of the compounds of formula IIa or IIb, when Ar is:

R³ one of R³, R⁴ and R⁵ is - (CH2) pC (= O) OH, - (CH2) pC (= O) O-alkyl, - (CH2) pC (= O) NH2. More preferably, when one of R³, R⁴ and R⁵ is - (CH2)_p-C (= O) OH, - (CH₂)_p-C (= O) O-alkyl, - (CH₂)_p-C (= O) NH₂, p is the integer 0. Even more preferably one of one of R³, R⁴ and R⁵ is - (CH2) pC (= O) OH or - (CH2) pC (= O) O-alkyl. More preferably still, when one of one of R³, R⁴ and R⁵ is - (CH₂)_p-C (= O) OH or - (CH₂)_p-C (= O) O-alkyl, p is the integer 0. Even more preferably, one of R³, R⁴ and R⁵ is - (CH2) -C (= O) OH. Most preferably, when one of R³, R⁴ and R⁵ is - (CH2) -C (= O) OH, p is the integer 0.

In some embodiments of the compounds of formula IIa or IIb, the sum of (n + p) is the integer 2 or 3. More preferably it is the integer 2.

In some embodiments of the compounds of formula IIa or IIb in which R² it is

M is preferably

ES 2 318 167 T3

In other embodiments of the compounds of formula IIa or IIb, Ar is:

<img file="ES2318167T3_D0026.tif" />

In still other embodiments of the compounds of formula I, R² it is:

<img file="ES2318167T3_D0027.tif" />

Preferably R^2nd it is cyclohexyl, alpha-naphthylmethyl, beta-naphthylmethyl, or cyclohexylmethyl. Alternatively, R^2nd is benzyl substituted with one or more halogen, aryl, carboxyl, alkoxycarbonyl or aroyl or combinations thereof.

In some embodiments of the compounds of formula IIa or IIb, R¹⁰ is H or methyl.

Preferably R¹⁰ is methyl.

Certain acidic or basic compounds of the present invention can exist as zwitterions. All forms of the compounds, including free acid, free base, and zwitterions, are contemplated to be within the scope of the present invention. It is well known in the art that compounds containing both amino and carboxyl groups often exist in equilibrium with their zwitterionic forms. Thus, any of the compounds described throughout this document that contain, for example, both amino and carboxyl groups, also include reference to their corresponding zwitterions.

In any of the above teachings, a compound of the invention can be either a compound of one of the formulas described herein or a stereoisomer, prodrug, salt form, hydrate, solvate, hydrated acid salt, N- oxide or pharmaceutically acceptable isomorphic crystalline thereof.

The compounds employed in the methods of the present invention can be prepared in various ways well known to those of skill in the art. The compounds can be synthesized, for example, by the methods described below or variations thereof as appreciated by one of ordinary skill in the art. It is contemplated that all methods disclosed in association with the present invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram, or commercial industrial scale.

As discussed in detail above, the compounds employed in the present methods can contain one or more asymmetrically substituted carbon atoms and can be isolated in optically active or racemic forms. Thus, all racemic, diastereomeric, chiral, and all geometric isomeric forms of a structure are intended, unless the specific isomeric or stereochemical form is specifically indicated. How to prepare and isolate such optically active forms is well known in the art. For example, mixtures of stereoisomers can be separated by conventional techniques including, but not limited to, resolution of racemic forms, normal, reverse phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis or from chiral starting materials or by deliberate synthesis of target chiral centers.

As will be readily understood, the functional groups present may contain protecting groups during the course of synthesis. Protecting groups per se are known as chemical functional groups that can be selectively added to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to provide such functionality inert to the chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups can be employed with the present invention. Preferred protecting groups include benzyloxycarbonyl group and tert-butyloxycarbonyl group. Other Preferred Protecting Groups That May Be Employed In Accordance With The Present Invention

ES 2 318 167 T3 can be described in Greene, TW and Wuts, PGM, Protective Groups in Organic Synthesis 2^to Ed., Wiley &amp; Sons, 1991.

The ability of compounds of the invention to bind IAP is typically measured by an in vitro displacement assay using the BIR3 binding domain and a labeled Smac tetrapeptide analog, such as AVPC-badan dye. Compositions and methods for conducting such an assay are described in co-pending international application number PCT / US02 / 17342. Briefly, a protein comprising the BIR3 domain of an IAP is placed in an assay medium comprising a suitable buffer. Preferably this is a recombinant protein comprising the BIR3 domain, but a full length IAP protein can also be used. An aliquot of the AVP dye is added to the reaction mixture, in the presence of the test compound. Controls comprise BIR3 and the dye in the absence of the test compound and optionally BIR3 and the dye in the presence of the naturally occurring tetrapeptide, AVPI. The fluorescence of the reaction mixture is measured at a selected excitation and emission wavelength, eg, 387 nm excitation, 545 nm emission. Alternatively, an emission spectrum is measured at the selected excitation wavelength. In one type of measurement, the test compound is added and an emission spectrum is measured by scanning from, for example, 460-480 nm. In another type of measurement, the emission intensity is measured at a particular wavelength, for example 470 nm. The emission spectrum of the dye bound to BIR3 is clearly different from the spectrum of the dye in solution. Thus, the binding affinity of the test compound can be calculated as a function of its ability to displace the dye from the BIR3 domain.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate the invention.

Example 1

Synthesis of certain compounds of the invention and components thereof

<img file="ES2318167T3_D0028.tif" />

50a (4-Bromo-butyl) -benzene (50a) (PAA 2-68). Phosphorous tribromide (1.10 ml, 11.2 mmol) was added dropwise to (4-hydroxy-butyl) -benzene (5.04 g, 33.5 mmol) and stirred for 1 h under argon at 23 ° C. The flask was then heated to 100 ° C by means of a silicone oil bath and allowed to stir for 4 h. The reaction mixture was cooled, quenched with several ml of cold H2O, diluted with ether, washed with brine (2x20 ml), dried over sodium sulfate, and concentrated in vacuo to give 6.32 g (88%) colorless oil. ¹H NMR (CdC13, 300 MHz) δ 7.18-7.33 (m, 5H), 3.44 (t, J = 6.9 Hz, 2H), 2.66 (t, J = 6.9 Hz , 2H), 1.76-1.96 (m, 4H); ¹³C NMR (CDCl3, 75 MHz) δ 147.8, 128.4, 125.8, 35.0, 33.7, 32.2, 29.8.

<img file="ES2318167T3_D0029.tif" />

(5-Bromo-pentyl) -benzene (50 b) (PAA 67). Phosphorous tribromide (0.47 ml, 5.0 mmol) was added dropwise to (5-hydroxy-pentyl) -benzene (1.98 g, 12.0 mmol) and stirred for 1 h under argon at 23 ° C . The flask was then heated to 100 ° C by means of a silicone oil bath and allowed to stir for 4 h. The reaction mixture was cooled, quenched with several ml of cold H2O, diluted with ether, washed with brine (2x20 ml), dried over sodium sulfate, and concentrated in vacuo to give 2.48 g (91%) colorless oil. ¹H NMR (CdC13, 300 MHz) δ 7.18-7.33 (m, 5H), 3.45 (dt, J = 1.8, 6.9 Hz, 2H), 2.68 (t, J = 7.2 Hz, 2H), 1.95 (d quintets, J = 1.84, 7.2 Hz, 2H), 1.71 (d quintets, J = 1.84, 7.2 Hz, 2H), 1.49-1.59 (m, 2H); ¹³C NMR (CDCl3, 75 MHz) δ 142.2, 128.3, 128.2, 125.7, 35.7, 33.7, 32.6, 30.6, 27.8.

<img file="ES2318167T3_D0030.tif" />

ES 2 318 167 T3

Phenylmethyl-triphenylphosphonium bromide (51a) (PAA 59). Triphenylphosphine (2.364 g, 9.00 mmol) was added to benzyl bromide (1.348 g, 8.00 mmol) in 35 ml of toluene in a 100 ml round bottom flask. The mixture was heated to reflux under argon and stirred for 24 h, then concentrated in vacuo. The resulting white amorphous solid was triturated with ether (3x20 ml) to provide 3.268 g (94%) of white powder.¹H NMR (CDCl₃, 300 MHz) δ 7.58-7.78 (m, 15H), 7.10-7.26 (m, 5H), 5.34-5.42 (m, 2H).

<img file="ES2318167T3_D0031.tif" />

(2-Phenyl-ethyl) -triphenylphosphonium (51b) bromide (PAA 62). Triphenylphosphine (2.364 g, 9.00 mmol) was added to (2-bromo-ethyl) -benzene (1.496 g, 8.00 mmol) in 35 ml of toluene in a 100 ml round bottom flask. The mixture was heated to reflux under argon and stirred for 48 h, then concentrated in vacuo to give a thick yellow oil. The oil was dissolved in boiling EtOH under argon. Benzene was added until an oil separated from the solution. The solution was heated in vacuo removing all solvent and providing 3.6 g (100%) of white foam. ¹H NMR (CDCl3, 300 MHz) δ 7.67-7.91 (m, 15H), 7.17-7.32 (m, 5H), 4.25 (dt, J = 12.6, 7, 8Hz, 2H), 3.08 (dt, J = 12.9, 7.8Hz, 2H).

<img file="ES2318167T3_D0032.tif" />

(3-Phenyl-propyl) -triphenylphosphonium bromide (51 c) (PAA 61). Triphenylphosphine (2.364 g, 9.00 mmol) was added to (3-bromo-propyl) -benzene (1.552 g, 8.00 mmol) in 35 ml of toluene in a 100 ml round bottom flask. The mixture was heated to reflux under argon and stirred for 48 h, then concentrated in vacuo. The resulting white amorphous solid was triturated with ether (3x20 ml) to provide 1.984 g (55%) of white solid.¹H NMR (CDCl₃, 300 MHz) δ 7.667.83 (m, 15H), 7.19-7.27 (m, 5H), 3.92-4.02 (m, 2H), 3.06 (t, J = 7, 2Hz, 2H), 1.92-2.00 (m, 2H).

® ®Br

PPhs

<img file="ES2318167T3_D0033.tif" />

d

(4-Phenyl-butyl) -triphenylphosphonium bromide (51 d) (PAA 71). Triphenylphosphine (3.932 g, 15.0 mmol) was added to 50 to (3.070 g, 14.4 mmol) in 35 ml of toluene in a 100 ml round bottom flask. The mixture was heated to reflux under argon and stirred for 65 h, then concentrated in vacuo to give a thick yellow oil. The oil was dissolved in boiling EtOH under argon. Benzene was added until an oil separated from the solution. The solution was heated in vacuo removing all solvent and yielding 6.59 g (96%) of white foam. ¹H NMR (CDCl₃, 300 MHz) δ 7.64-7.84 (m, 5H), 7.09-7.20 (m, 5H), 3.81-3.90 (m, 2H), 2.67 (t, J = 7.2 Hz, 2H), 2.01 (quint, 7.2 Hz, 2H), 1.58-1.67 (m, 2H).

<img file="ES2318167T3_D0034.tif" />

ES 2 318 167 T3

(5-Phenyl-phenyl) -triphenylphosphonium bromide (51 e) (PAA 70). Triphenylphosphine (2.620 g, 10.0 mmol) was added to 50 μg (1.971 g, 8.70 mmol) in 35 ml of toluene in a 100 ml round bottom flask. The mixture was heated to reflux under argon and stirred 65 h, then concentrated in vacuo to give a thick yellow oil. The oil was dissolved in boiling EtOH under argon. Benzene was added until an oil separated from the solution. The solution was heated in vacuo removing all solvent and providing 3.99 g (94%) of white foam. ¹H NMR (CDCl₃, 300 MHz) δ 7.61-7.79 (m, 5H), 7.04-7.29 (m, 5H), 3.63-3.71 (m, 2H), 2.49 (m, 7.2 Hz, 2H, 1.52-1.63 (m, 5H).

<img file="ES2318167T3_D0035.tif" />

(S) -2-Styryl-N-Boc-pyrrolidine (45 a) (PAA 2-18). N-BuLi (0.730 ml, 1.05 mmol) was added dropwise via syringe over 10 minutes at 51 a (0.442 g, 1.021 mmol) in 20 ml of THF under argon at -78 ° C. The mixture was stirred cold for 1 hr, then heated to 23 ° C over a period of 30 minutes. The mixture was immediately cooled back to -78 ° C and Boc-L-prolinal (0.200 ml, 1.035 mmol) in 20 ml THF was added dropwise via cannula over 5 minutes. The reaction was allowed to stir cold for 1 hr, then an additional 24 hr at 23 ° C. The reaction was quenched with several ml of water, diluted with ethyl acetate, washed with brine (2x25 ml), dried over sodium sulfate, and concentrated in vacuo. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 242 mg (73%) of a light yellow solid. R_F: 0.51 (5: 1 hexanes / EtOAc). ¹H NMR (CDCl₃, 300 MHz) δ 7.22-7.37 (m, 5H), 6.38-6.43 (m, 1H), 5.59-6.12 (m, 1H), 4.40-4, 74 (m, 1H), 3.47 (br, 1H), 1.80-2.20 (m, 4H), 1.43 (s, 9H).

<img file="ES2318167T3_D0036.tif" />

(S) -2- (3-Phenyl-propenyl) -N-Boc-pyrrolidine (45 b) (PAA 2-19). n-BuLi (1.2 ml, 1.9 mmol) was added dropwise via syringe over 10 min at 51 b (0.769 g, 1.72 mmol) in 20 ml of THF under argon at -78 ° C. The mixture was stirred cold for 1 hr, then heated to 23 ° C over a period of 30 minutes. The mixture was immediately cooled back to -78 ° C and Boc-L-prolinal (0.325 ml, 1.72 mmol) in 20 ml THF was added dropwise via cannula over 5 minutes. The reaction was allowed to stir cold for 1 hr, then an additional 24 hr at 23 ° C. The reaction was quenched with several ml of water, diluted with ethyl acetate, washed with brine (2x25 ml), dried over sodium sulfate, and concentrated in vacuo. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 330 mg (67%) of light yellow oil. R_F: 0.78 (3: 1 hexanes / EtOAc). ¹H NMR (CDCl₃, 300 MHz) δ 7.17-7.32 (m, 5H), 5.43-5.59 (m, 2H), 4.65 (bs, 1H), 3.38-3.57 (m, 4H), 2.08-2.14 (m, 1H), 1.79-1.96 (m, 2H), 1.651.73 (m, 1H), 1.47 (s, 9H).

<img file="ES2318167T3_D0037.tif" />

c (S) -2- (4-Phenyl-but-1-enyl) -N-Boc-pyrrolidine (45 c) (PAA 82). N-BuLi (0.730 ml, 1.05 mmol) was added dropwise via syringe over 10 minutes at 51 ° C (0.478 g, 1.04 mmol) in 20 ml of THF under argon at -78 ° C. The mixture was stirred cold for 1 h, then heated to 23 ° C over a 30 minute period. The mixture was immediately cooled back to -78 ° C and Boc-L-prolinal (0.200 ml, 1.04 mmol) in 20 ml THF was added dropwise via cannula over 5 minutes. The reaction was allowed to stir cold for 1 hr, then an additional 24 hr at 23 ° C. The reaction was quenched with several ml of water, diluted with ethyl acetate, washed with brine (2x25 ml), dried over sodium sulfate, and concentrated in vacuo. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 230 mg (73%) light yellow oil. R_F: 0.82 (4: 1 hexanes / EtOAc). ¹H NMR (CdC1₃, 300 MHz) δ 7.17-7.31 (m, 5H), 5.30-5.39 (m, 2H), 4.43 (bs, 1H), 3.36-3.40 (m, 4H), 2.31-2.82 (m, 4H), 1.72-1.95 (m, 2H), 1.47 (s, 9H).

ES 2 318 167 T3

Boc &lt;

<img file="ES2318167T3_D0038.tif" />

&lt;1 (S) -2- (5-Phenyl-pent-1-enyl) -N-Boc-pyrrolidine (45 d) (PAA 82). N-BuLi (0.900 ml, 1.29 mmol) was added dropwise via syringe over 10 minutes at 51 d (0.620 g, 1.29 mmol) in 20 ml of THF under argon at -78 ° C. The mixture was stirred cold for 1 hr, then heated to 23 ° C over a period of 30 minutes. The mixture was immediately cooled back to -78 ° C and Boc-L-prolinal (0.250 ml, 1.29 mmol) in 20 ml THF was added dropwise via cannula over 5 minutes. The reaction was allowed to stir cold for 1 hr, then an additional 24 hr at 23 ° C. The reaction was quenched with several ml of water, diluted with ethyl acetate, washed with brine (2x25 ml), dried over sodium sulfate, and concentrated in vacuo. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 232 mg (60%) light yellow oil. R_F: 0.55 (5: 1 hexanes / EtOAc). ¹H NMR (CdC1₃, 300 MHz) δ 7.18-7.31 (m, 5H), 5.36-5.38 (m, 2H), 4.47 (bs, 1H), 3.40 (bs, 1H), 2 , 64 (t, J = 7.2Hz, 2H), 1.61-2.20 (m, 8H), 1.47 (s, 9H).

45e (S) -2- (6-Phenyl-hex-1-enyl) -N-Boc-pyrrolidine (45 e) (PAA 81). N-BuLi (0.900 ml, 1.29 mmol) was added dropwise via syringe over 10 minutes at 51 d (0.620 g, 1.29 mmol) in 20 ml of THF under argon at -78 ° C. The mixture was stirred cold for 1 h, then heated to 23 ° C over a 30 minute period. The mixture was immediately cooled back to -78 ° C and Boc-L-prolinal (0.250 ml, 1.29 mmol) in 20 ml THF was added dropwise via cannula over 5 minutes. The reaction was allowed to stir cold for 1 hr, then an additional 24 hr at 23 ° C. The reaction was quenched with several ml of water, diluted with ethyl acetate, washed with brine (2x25 ml), dried over sodium sulfate, and concentrated in vacuo. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 280 mg (69%) light yellow oil. R_F: 0.64 (5: 1 hexanes / EtOAc). ¹H NMR (CdC1₃, 300 MHz) δ 7.17-7.32 (m, 5H), 5.29-5.34 (m, 2H), 4.49 (bs, 1H), 3.38-3.42 (m, 2H), 2.63 (t, J = 7.2 Hz, 2H), 1.38-2.11 (m, 10H), 1.44 (s, 9H).

Boc

44 «(S) -2-Phenethyl-N-Boc-pyrrolidine (44 a) (PAA 2-28). A 10 ml round bottom flask equipped with a three-way stopcock and a balloon was loaded with 160 mg of 10% Pd -C. The flask was emptied and filled with hydrogen gas 10 times. EtOH (5 ml) was added via syringe and stirred for several minutes. Alkene 45 to (260 mg) in 2 ml EtOH was added via syringe and stirred for 24 h. Hydrogen gas was added as the balloon deflated. After completion of the reaction, the catalyst was removed by filtration. The product was concentrated in vacuo to provide 251 mg (97%) of colorless oil.¹H NMR (CDCl₃, 300 MHz) δ 7.19-7.30 (m, 5H), 3.76-3.88 (ma, 1H), 3.34-3.43 (ma, 2H), 2.59-2, 62 (m, 2H), 1.62-2.16 (m, 6H), 1.46 (s, 9H).

Boc «

N,

44b

ES 2 318 167 T3 (S) -3-Phenpropyl-N-Boc-pyrrolidine (44 b) (PAA 2-29). A 10 ml round bottom flask equipped with a three-way stopcock and a balloon was loaded with 157 mg of 10% Pd -C. The flask was emptied and filled with hydrogen gas 10 times. EtOH (5 ml) was added via syringe and stirred for several minutes. Alkene 45 b (145 mg) in 2 ml EtOH was added via syringe and stirred for 24 h. Hydrogen gas was added as the balloon deflated. After completion of the reaction, the catalyst was removed by filtration. The product was concentrated in vacuo to provide 149 mg (100%) of colorless oil. '11 NMR (CDCl₃, 300 MHz) δ 7.16-7.30 (m, 5H), 3.713.83 (ma, 1H), 3.30-3.39 (ma, 2H), 2.60 (t, J = 7, 2Hz, 2H), 1.62-1.90 (m, 5H), 1.46 (s, 9H), 1.29-1.42 (m, 3H).

<img file="ES2318167T3_D0039.tif" />

(S) -4-Fenbutyl-N-Boc-pyrrolidine (44 c) (PAA 2-160). A 50 ml round bottom flask equipped with a three-way stopcock and a balloon was loaded with 400 mg of 10% Pd -C. The flask was emptied and filled with hydrogen gas 10 times. MeOH (30 ml) was added via syringe and stirred for several minutes. 45c alkene (405 mg,) in 5 ml EtOH was added via syringe and stirred for 2 h. Hydrogen gas was added as the balloon deflated. After completion of the reaction, the catalyst was removed by filtration. The product was concentrated in vacuo to provide 376 mg (93%) of colorless oil. '11 NMR (CDCl₃, 300 MHz) δ 7.15-7.31 (m, 5H), 3.70-3.76 (ma, 1H), 3.26-3.41 (m, 2H), 2.58-2, 64 (m, 2H), 1.59-1.93 (m, 7H), 1.49 (s, 9H), 1.30-1.35 (m, 3H).

<img file="ES2318167T3_D0040.tif" />

(S) -5-Fenpentyl-N-Boc-pyrrolidine (44 d) (PAA 2-27). A 10 ml round bottom flask equipped with a three-way stopcock and a balloon was loaded with 100 mg of 10% Pd -C. The flask was emptied and filled with hydrogen gas 10 times. EtOH (3 ml) was added via syringe and stirred for several minutes. Alkene 45 d (100 mg) in 2 ml EtOH was added via syringe and stirred for 24 h. Hydrogen gas was added as the balloon deflated. After completion of the reaction, the catalyst was removed by filtration. The product was concentrated in vacuo to provide 93.7 mg (94%) of colorless oil. 'II NMR (CDCl₃, 300 MHz) δ 7.17-7.31 (m, 5H), 3.70-3.76 (ma, 1H), 3.30-3.40 (m, 2H), 2.61 (7, J = 8.0 Hz, 2H), 1.73-1.90 (m, 3H), 1.59-1.65 (m, 3H), 1.47 (s, 9H), 1.29-1 , 40 (m, 6H).

<img file="ES2318167T3_D0041.tif" />

(S) -6-Fenhexyl-N-Boc-pyrrolidine (44 e) (PAA 2-27). A 10 ml round bottom flask equipped with a three-way stopcock and a balloon was loaded with 151 mg of 10% Pd -C. The flask was emptied and filled with hydrogen gas 10 times. EtOH (3 ml) was added via syringe and stirred for several minutes. Alkene 45e (301 mg) in 2 ml EtOH was added via syringe and stirred for 24 h. Hydrogen gas was added as the balloon deflated. After completion of the reaction, the catalyst was removed by filtration. The product was concentrated in vacuo to provide 285 mg (95%) of colorless oil. '11 NMR (CDCl₃, 300 MHz) δ 7.17-7.30 (m, 5H), 3.69-3.76 (ma, 1H), 3.30-3.39 (m, 2H), 2.61 (7, J = 7.6 Hz, 2H), 1.73-1.90 (m, 3H), 1.62-1.64 (m, 3H), 1.47 (s, 9H), 1.28-1 , 43 (m, 8H).

<img file="ES2318167T3_D0042.tif" />

ES 2 318 167 T3

Boc-Ala-Val-OMe (53) (PAA 2-7). EDC (1.521 g, 7.93 mmol) was added to Boc-Ala (1,500 g, 7.93 mmol) in 25 ml of chloroform at 0 ° C and the mixture was allowed to stir for 30 minutes. Valine methyl ester (1.329 g, 7.93 mmol), triethylamine (1.11 ml, 7.93 mmol) and 25 ml of chloroform were combined in a separate 50 ml flask and stirred. This mixture was transferred to the activated Boc-alanine solution and stirred at 0 ° C for 3 h. The ice bath was removed and the reaction mixture was stirred for an additional 27 h. Three drops of acetic acid were added and stirring was continued for 15 minutes. The mixture was concentrated in vacuo and then redissolved in EtOAc (50 ml). The organic phase was washed with NaHCO₃ 10% (2x25 ml), 1N HCl (2x25 ml), water (25 ml), dried over sodium sulfate and concentrated in vacuo to give 1.848 g (81%) of product. ¹H NMR (CDCl₃, 300 MHz) δ 6.68 (da, J = 5.4 Hz, 1H), 4.99 (sa, 1H), 4.53 (dd, J = 4.8, 8.7 Hz, 1H), 4.19 (rt, J = 7.5 Hz, 1H), 3.74 (s, 3H), 2.18 (septet, J = 6.9 Hz, 1H), 1.45 (s, 9H), 0.93 (d, J = 6.9Hz, 3H), 0.90 (d, J = 6.9Hz, 3H).

<img file="ES2318167T3_D0043.tif" />

Boc-Ala-Val-OH (54) (PAA 2-32). Boc-Ala-Val-OMe 53 (1,750 g, 5.79 mmol) was stirred in 40 ml of acetone. NaOH solution (8.7 ml of 2.0 M aqueous solution) was added and stirred for 2 h at 23 ° C. Acetone was removed under reduced pressure and the resulting solution was diluted with EtOAc (20 ml). The organic fraction was washed with water (2x20 ml), dried over sodium sulfate and concentrated in vacuo to provide 1.561 g (99%) of product.¹H NMR (CDCI3, 300 MHz) δ 12.28 (sa, 1H), 7.69 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 4.15 (dd, J = 5.4, 8.4 Hz, 1H), 4.04 (quint, J = 7.2 Hz, 1H), 3.35 (sa, 2H), 2.04 (octet , J = 6.6 Hz, 1H), 1.37 (s, 9H), 1.16 (d, J = 7.2 Hz, 3H), 0.87 (d, J = 7.2 Hz, 3H ); ¹³C NMR (CDCI3, 75 MHz) δ 172.9, 155.1, 78.1, 56.8, 30.2, 28.2, 19.0, 18.0, 17.8.

<img file="ES2318167T3_D0044.tif" />

Boc-L-Prolinal (38) (PAA 2-226). Boc-L-proline (20.0 g, 92.9 mmol) was combined with 200 ml of THF in a 500 ml flask equipped with a reflux condenser. Borane-methyl sulfide complex (70 mL, 140 mmol) was added over a 30 minute period at 0 ° C under argon. When the evolution of gas ceased, the flask was heated to reflux by means of a silicone oil bath and stirred for 1 hr. The reaction was cooled to 23 ° C and slowly quenched with MeOH. An additional 100 ml of MeOH was added and then concentrated in vacuo. The reaction mixture was redissolved and concentrated with 200 ml of MeOH and then 2x100 ml of toluene. The sample was concentrated on the vacuum pump overnight to provide 18.78 g of white solid alcohol product. This material was dissolved in 200 ml of dichloromethane and stirred under argon. PCC (28 g, 141 mmol) followed by 4 A molecular sieves (35 g) and 1 ml of AcOH were added to the mixture at 0 ° C. The reaction mixture was allowed to stir for 2.5 h at 23 ° C. The flask was filled with ether then filtered over a small pad of silica gel. Silica gel chromatography provided 13.1 g (70% in two steps) of colorless oil. R_F: 0.39 (5: 1 hexanes / EtOAc). ¹H NMR (CDCl₃, 300 MHz) δ 9.46-9.56 (m, 1H), 4.03-4.21 (m, 1H), 3.43-3.58 (m, 2H), 1.87-2, 14 (m, 4H), 1.43-1.48 (m, 9H).

<img file="ES2318167T3_D0045.tif" />

(S) -2- (1-Hydroxy-4-phenyl-butyl) -N-Boc-pyrrolidine (57 and 58) (PAA 2-138). Mg chips (1.6 g, 67 mmol) were stirred under argon in 34 ml of ether. (3-Bromopropyl) -benzene (8 ml, 52 mmol) in 10 ml of ether was added slowly to minimize boiling of the solvent. After 1 hr, the resulting Grignard solution was transferred from excess magnesium by cannula and stored under argon. A flask was charged with 16 ml of 1.25 M Grignard solution and cooled to 0 ° C under argon. Prolinal (1.529 g, 7.7 mmol) in 10 ml of ether was transferred dropwise to the flask via syringe within 10 min. The reaction mixture was stirred for 1 hr, then removed

ES 2 318 167 T3 the ice bath and the mixture was stirred for an additional 3 h. The reaction was slowly quenched with several ml of NH₄Saturated Cl, extracted with 50 ml of ether, dried over sodium sulfate and concentrated in vacuo. Chromatography on silica gel (hexanes / EtOAc 5: 1) allowed the separation of the diastereomers, 0.98 g of oil and 0.63 g of solid (overall yield 67%). Rf: 0.32 (5: 1 hexanes / EtOAc). 1H NMR (CDCl₃, 300 MHz) δ; ¹³C NMR (CDCI3, 75 MHz) δ 142.4, 128.4, 128.2, 125.6, 80.4, 75.2, 62.6, 47.1, 35.8, 34.2, 28 , 5, 28.4, 26.7, 24.0. Rf: 0.28 (5: 1 hexanes / EtOAc).¹H NMR (CDCl3, 300 MHz) δ; ¹³C NMR (CDCl₃, 75 MHz) δ 124.4, 128.4, 128.2, 125.6, 79.8, 73.0, 62.8, 47.9, 35.8, 34.2, 32.0, 28 , 5, 28.4, 27.8, 24.2.

V (S) -2- (4-Phenyl-butyryl) -N-Boc-pyrrolidine (59) (PAA 2-245). A mixture of alcohols 57 and 58 (0.77 g, 2.4 mmol) in 25 ml of dichloromethane was stirred and stirred under argon. PCC (0.8 g, 3.6 mmol) followed by 4 A molecular sieves (0.8 g) and several drops of AcOH were added to the mixture at 0 ° C. The reaction mixture was allowed to stir for 48 h at 23 ° C. The flask was filled with ether, then filtered over a small pad of silica gel and concentrated. The resulting oil was stirred in saturated sodium bisulfite for 1 hr. The product was extracted with dichloromethane and concentrated in vacuo to provide 588 mg (77%) of an unpleasant, white solid. R_F: 0.32 (5: 1 hexanes / EtOAc). ¹H NMR (CDCI3, 300 MHz) δ 7.17-7.30 (m, 5H), 4.33 (dd, J = 4.4, 8.8 Hz) + 4.21 (dd, J = 5, 2, 8.8 Hz) 1H, 3.40-3.55 (m, 2H), 2.60-2.66 (m, 2H), 2.39-2.53 (m, 2H), 2, 06-2.18 (m, 1H), 1.93 (quint, J = 7.2 Hz, 1H), 1.74-1.86 (m, 3H), 1.532 + 1.37 (s, 9H) ; ¹³C NMR (CDCl₃, 75 MHz) δ 209.9, 154.5, 153.8, 141.6, 141.3, 128.4, 128.3, 128.2, 125.9, 125.8, 80.0, 79 , 7, 65.1, 64.6, 46.8, 46.6, 38.4, 37.5, 35.0, 29.9, 28.8, 28.4, 28.2, 24.6 , 24.6, 24.3, 23.6.

BooVal ,,

<img file="ES2318167T3_D0046.tif" />

¢ 0 (S) -2- (4-Phenyl-butyryl) -Boc-Val-pyrrolidine (60) (PAA 2-211). Ketone 59 was stirred in 50:50 TFA / dichloromethane for 2 hours. After removal of the solvent, the resulting TFA amine salt (0.784 mmol) was combined with Boc-Val-OH (170 mg, 0.784 mmol), NEt₃ (0.330 ml, 2.35 mmol), BOP-Cl (300 mg, 1.18 mmol) in 15 ml of dichloromethane at 0 ° C under argon. The reaction mixture was allowed to stir for 24 h, then washed with 1N HCl (10 mL), NaHCO₃ saturated (10 ml) and brine (20 ml). The organic fraction was dried over sodium sulfate and concentrated in vacuo. Silica gel chromatography (50:50 hexanes / ether) provided 226 mg (71%) of colorless oil. R_F: 0.32 (50:50 hexanes / ether). ¹H NMR (CDCI3, 300 MHz) δ 7.13-7.27 (m, 5H), 5.20 (d, J = 9.3 Hz, 1H), 4.55 (dd, J = 6.0, 8.4 Hz) 1H, 4.25 (dd, J = 6.0, 9.3 Hz, 1H), 3.71-3.77 (m, 1H), 2.39-2.62 (m, 4H), 1.89-2.12 (m, 6H), 1.71 (septet, J = 6.6 Hz, 1H), 1.40 (s, 9H), 1.00 (d, J = 6 , 6Hz, 3H), 0.90 (d, J = 6.6Hz, 3H); ¹³C NMR (CDCI3, 75 MHz) δ 208.0, 170.7, 156.7, 141.4, 128.3, 128.2, 125.7, 64.2, 56.5, 47.2, 39 , 3, 34.8, 31.1, 28.1, 27.9, 24.8, 24.6, 19.2, 17.2.

"to

3-p-Tolyl-propionaldehyde (63 a) (PAA 2-259). A flask was charged with p-iodotoluene (2.18 g, 10. mmol), allyl alcohol (1.0 ml, 15 mmol), sodium bicarbonate (1.68 g, 20 mmol), tetrabutylammonium chloride (2, 78 g, 10.0 mmol), palladium acetate (45 mg, 2 mol%) and 10 ml of DMF. The mixture was stirred under argon at 40 ° C for 18 h. The reaction mixture was cooled, diluted with ether, and filtered over Celite. Silica gel chromatography (5: 1 hexanes / EtOAc) provided g (%) of yellow oil. R_F: 0.47 (3: 1 hexanes / ether). ¹H NMR (CDCl3, 300 MHz) δ; ¹³C NMR (CDCl3, 75 MHz) δ

ES 2 318 167 T3

<img file="ES2318167T3_D0047.tif" />

3-Naphthalen-2-yl-propionaldehyde (63 b) (PAA 2-260). A flask was charged with 2-iodo-naphthalene (2.095 g, 8.25 mmol), allyl alcohol (0.83 ml, 12.38 mmol), sodium bicarbonate (1.39 g, 16.5 mmol), chloride of tetrabutylammonium (8.25 mmol), palladium acetate (37 mg, 2 mol%) and 10 ml of DMF. The mixture was stirred under argon at 40 ° C for 18 h. The reaction mixture was cooled, diluted with ether, and filtered over Celite. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 1.23 g (67%) of yellow oil. R_F: 0.40 (3: 1 hexanes / ether). ¹H NMR (CDCl3, 300 MHz) δ 9.87 (t, J = 1.2 Hz, 1H). 7.80-7.85 (m, 3H), 7.65 (s, 1H), 7.46-7.50 (m, 2H), 7.35 (dd, 1.8, 8.7 Hz, 1H), 3.14 (t, J = 7.5 Hz, 2H), 2.87 (ddt, 0.6, 1.2, 7.5 Hz, 2H);¹³C NMR (CDCl3. 75 MHz) δ 201.4, 137.8, 133.5, 132.1, 128.2, 127.6, 127.4, 126.9, 126.4, 126.1, 125 , 4, 45.1, 28.2.

<img file="ES2318167T3_D0048.tif" />

4- (3-Oxo-propyl) -benzoic acid (63 c) ethyl ester (PAA 2-264). A flask was charged with p-iodobenzoic acid ethyl ester (2.78 g, 10.0 mmol), allyl alcohol (1.0 ml, 15. mmol), sodium bicarbonate (1.68 g, 20 mmol), Tetrabutylammonium chloride (2.78 g, 10.0 mmol), palladium acetate (37 mg, 2 mol%) and 10 ml of DMF. The mixture was stirred under argon at 40 ° C for 18 h. The reaction mixture was cooled, diluted with ether, and filtered over Celite. Silica gel chromatography (5: 1 hexanes / EtOAc) provided 1.27 g (61%) of yellow oil. R_F: 0.33 (5: 1 hexanes / ether). ¹H NMR (CDCl₃, 300 MHz)) δ 9.83 (t, J = 0.9 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.27 (d, 8.4 Hz, 2H ), 4.37 (quartet, J = 7.2 Hz, 2H), 3.02 (t, J = 7.5 Hz, 2H), 2.82 (d, J = 7.5 Hz, 2H), 1.39 (t, 7.2 Hz, 3H).

<img file="ES2318167T3_D0049.tif" />

3-Naphthalen-2-yl-propanol (64 b) (PAA 2-266). Sodium borohydride (53 mg, 1.36 mmol) was added to 63 b in 10 ml of EtOH under argon. The mixture was allowed to stir for 10 h at 23 ° C. The reaction was quenched with dilute acetic acid. The reaction mixture was diluted with EtOAc, washed with water (2x20 ml), dried over sodium sulfate, and concentrated in vacuo to provide 470 mg (94%) of light brown solid.¹H RmN (CDCl₃, 300 MHz) δ 7.78-7.83 (m, 3H), 7.65 (d, 8.4 Hz, 2H), 7.43-7.47 (m, 2H), 7.35-7 , 37 (m, 1H), 3.74-3.90 (m, 2H), 2.89 (t, J = 6.9 Hz, 2H), 2.00 (quintet, J = 6.9 Hz, 2H).

<img file="ES2318167T3_D0050.tif" />

4- (3-Hydroxy-propyl) -benzoic acid (64 c) ethyl ester (PAA 2-267). Sodium borohydride (47 mg, 1.21 mmol) was added at 63 c (500 mg, 2.42 mmol) in 10 ml of EtOH under argon. The mixture was allowed to stir for 10 h at 23 ° C. The reaction was quenched with dilute acetic acid. The reaction mixture was diluted with EtOAc, washed with water (2x20 ml), dried over sodium sulfate, and concentrated in vacuo to provide 422 mg (85%) of colorless oil.¹H NMR (CDCl3, 300 MHz) δ 7.96 (d, J = 8.4 Hz, 2H), 7.25-7.27 (m, 2H), 4.36 (quartet, J = 6.9 Hz , 2H), 3.68 (t, J = 6.0 Hz, 2H), 2.75 (quintet, J = 6.9 Hz, 2H), 2.09 (s, 1H), 1.93 (quintet , J = 6.9 Hz, 2H), 1.39 (t, 6.0 Hz, 3H).

ES 2 318 167 T3

<img file="ES2318167T3_D0051.tif" />

2- (3-bromo-propyl) naphthalene (65 b) (PAA 2-271). PBr was added₃ (60 µl, 0.53 mmol) by syringe at 64 b (246 mg, 1.32 mmol) in 1 ml of toluene under argon at 0 ° C. The mixture was stirred overnight, then refluxed for 16 h. The mixture was cooled, washed with water (5 ml), brine (5 ml), dried over sodium sulfate, and concentrated in vacuo.¹H NMR (CDCI3, 300 MHz) δ 7.81-7.86 (m, 3H), 7.68 (d, 8.4 Hz, 2H), 7.44-7.53 (m, 2H), 7 , 37 (dd, J = 1.5, 8.4 Hz, 1H), 3.45 (t, J = 6.6 Hz, 2H), 2.98 (t, J = 7.2 Hz, 2h) , 2.29 (quint, J = 6.6 Hz, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 137.9, 133.5, 132.1, 128.1, 127.1, 126.7, 126.0, 125.3, 34.1, 34.0, 33.1.

<img file="ES2318167T3_D0052.tif" />

4- (3-Bromo-propyl) -benzoic acid ethyl ester (65 c) (PAA 2-272). PBr was added₃ (40 µl, 0.34 mmol) by syringe at 64 b (175 mg, 0.84 mmol) in 1 ml of toluene under argon at 0 ° C. The mixture was stirred overnight, then refluxed for 16 h. The mixture was cooled, washed with water (5 ml), brine (5 ml), dried over sodium sulfate, and concentrated in vacuo.¹H NMR (CDCl3, 300 MHz) δ 8.01 (d, J = 7.8 Hz, 2H), 7.29-7.31 (m, 2H), 4.40 (quartet, J = 6.9 Hz , 2H), 3.42 (t, J = 6.6 Hz, 2H), 2.87 (quintet, J = 7.2 Hz, 2H), 2.21 (quintet, J = 6.6 Hz, 2H ), 1.42 (t, J = 6.6 Hz, 3H); ¹³C NMR (CDCl3, 75 MHz) δ 166.5, 145.8, 129.8, 128.5, 128.4, 60.8, 33.9, 33.7, 32.7, 14.3.

Example 2

Synthesis of certain oxazole compounds

Synthetic Scheme for Oxazole Compounds

<img file="ES2318167T3_D0053.tif" />

ES 2 318 167 T3

I. Formation of Boc-Ala-Ser-OMe (3)

1 eq. of Boc-Ala-succinimide ester (2) to an ice-cold solution of DIPEA (2 eq.) and H-Ser-methyl ester hydrochloride (R = H) or H-Thr-methyl ester hydrochloride (R = CH3) (1) in dry THF under argon. The solution was allowed to warm to RT and stirred overnight (Rocchi, R. et al. (1987) Int. J. Peptide Protein Res. 30, 240-256). THF was removed in vacuo, the resin was taken up in ethyl acetate and washed three times, each with saturated sodium bicarbonate, 5% citric acid, and saturated sodium chloride. The solution was dried over magnesium sulfate and the solvent was removed in vacuo leaving a white solid. Single point in TLC (MeOH / CHCl₃ 1:10). No purification was necessary. 75% yield.

II. Formation of Boc-Ala-Ser-OMe-oxazoline (4)

1.05 eq. of Burgess reagent in one portion to a stirring solution of 3 in dry THF and then the resulting solution was heated at 70 ° C for 12 h under an argon atmosphere (Mink et al., (1998) Tetrahedron Lett. 39, 5709-5712). The reaction was run as described in I above. The product was purified on a silica gel column in 70/30 ethyl acetate / hexanes. 25% yield.

III. Formation of Boc-Ala-Ser-OMe-oxazole (5)

A solution of 4 in CH was cooled₂Cl₂ to 0 ° C and 1.1 eq. by DBU. 1.1 eq. of bromotrichloromethane dropwise via syringe over 10 min. The reaction was stirred under argon until complete (8 hr). The product was purified by a silica gel column in 30-50% ethyl acetate in hexane. 55% yield.

IV. Deprotection of carboxylic acid from Boc-Ala-Ser-OMe-oxazole (6)

2.3 eq. of LiOH monohydrate to a 5 solution with stirring in McOH: H₂O (3: 1) at 0 ° C. The solution was stirred with gradual warming to room temperature, until the reaction was complete (monitored by TLC in MeOH / CHCl₃ 1:10) (Aquilar, E. and Meyers, A. (1994) Tetrahedron Lett. 35, 2472-2480).

V. Formation of Boc-Ala-Ser-Pro-AA-OMe-oxazole (7)

Pro-AA (where AA is phenylalanine, isoleucine, tyrosine, or tryptophan) was synthesized as in I and AA-NH2 or AA-OMe was used to provide the amide, methyl ester, or free acid, respectively, dissolved 1.1 eq. of Pro-Phe and 3 eq. of DIPEA in dry THF and added to a solution of 6 and 1.4 eq. of BOP-Cl at 0 ° C (Palomo-Coll, AL and Diago-Meseguer, J. (1980) Synthesis. 7, 547-551). The reaction was stirred for 12-14 hr under Ar. The Boc group was removed by TFA. Then, the Phe-OMe was deprotected as in IV. The methyl ester, the amide and the free acid were purified by HPLC and characterized by mass spectrometry and¹H NMR.

Synthetic Scheme for Non-Peptide Oxazole Analogs

<img file="ES2318167T3_D0054.tif" />

*" Y

OR

The coupling reaction between the oxazole analog and the Pro-Phe analogs was carried out as in V above. Compounds were purified by HPLC and characterized by mass spectrometry and¹ H NMR.

Example 3

Synthesis of compounds containing proline or phenylalanine derivatives

Materials. Unless otherwise noted, materials were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Fisher Scientific (Pittsburgh, PA) and used without further purification. All phenylalanine derivatives

Non-natural ES 2 318 167 T3 were purchased from Advanced ChemTech (Louisville, KY) or Novabiochem (Calbiochem, San Diego, CA) as the Fmoc-protected amino acid. These and the proline derivatives were used in peptide synthesis in the same way that a naturally occurring amino acid would be used. Methylbenzhydrylamine (MBHA) solid phase peptide synthesis resin, Rink-amide resin, and 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids were obtained from Advanced ChemTech (Louisville, KY) and NovaBiochem (San Diego, CA). 6-bromoacetyl-2-dimethylaminonaphthalene (badan) dye was obtained from Molecular Probes (Eugene, O).

Peptide synthesis. Peptide molecules were synthesized on an Advanced ChemTech 396 MPS automated peptide synthesizer using the Fmoc protocol on a Rink-amide resin. The side chains of amino acids that are susceptible to side reactions were protected as follows: the hydroxyl groups on 4-hydroxyproline and p-carboxy-substituted phenylalanine were protected with t-butyl and a pentamethyldihydrobenzofuran group was used to protect the arginine. After the final amino acid was added, deprotection and cleavage of the peptides from the resin was effected by adding 1 ml of a 95% solution of TFA, 2.5% of water and 2.5% of triisopropylsilane (TlS) to each well and shaking for 1 hour. The cleavage solution was collected and an additional 0.5 ml of the cleavage solution was added to each well and mixed for another hour. The combined cleavage solutions were added to 20 ml of water, lyophilized to dryness, then made up to 5 ml of water before being filtered through syringe filters (0.2 pm) and lyophilized again. Peptides containing proline derivatives were purified by HPLC on a C18 Vydac preparative scale column. The presence of the desired molecules was confirmed by mass spectroscopy.

Example 4

essays

Test compounds were reconstituted in water and test solutions that were approximately 200 pM in test compounds were prepared. The exact concentrations were determined for 10 representative test solutions for the phenylalanine library, and for all members of the proline and oxazole libraries. The concentrations were determined by^' H-NMR using a dioxane solution of known concentration as external reference. The concentrations of the other peptide solutions of the phenylalanine library were considered to be the average value of the known solutions of the same library synthesis.

The concentrations of the methylene bridged compounds were estimated using the molar absorptivity of a phenyl ring in water at 250 nm (E = 200 M 'cm'). These compounds were not soluble in water, so they were brought into a 10% aqueous dimethylsulfoxide (DMSO) solution. The test solution then was about 1% DMSO, which had no effect on the emission observed at that concentration.

Fluorescence protocol. Luminescence spectra were recorded using a Photon Technologies, Inc. fluorometer with an Xe arc lamp and a PMT detector. The absorbance of all solutions was less than 0.2 at the excitation wavelength (387 nm). The buffer used in all fluorescence experiments was 50 mM potassium phosphate, 100 mM NaCl, 2 mM 1,4-dithio-DL-threitol (DTT), pH 7.

Assay of test compounds. The samples were prepared in a 96-well plate covered with glass tubes, to avoid adsorption of the dye to the plastic. The plate was stored on ice in the dark between measurements. A small volume cuvette, with a path length of 2 mm, was used to collect the emission spectrum. A 44 pM aqueous solution of AVPC-badan, a 63 pM BIR3 solution and buffer were mixed to give a stock solution that was 5.6 pM in both AVPC-badan and BIR3. 390 µl of this stock solution was added to each well in the 96-well plate. 50 µl of the test compound solutions were added and mixed immediately before taking emission spectra. The final solutions were 5 pM in both badan and BIR3 and approximately 20-30 pM in the test compound solutions. 50 µl of water was added to three of the wells as controls, to determine the intensity observed when AVPC-badan bound BIR3. 190 µl AVPC-badan and 1020 µl buffer were mixed and added to three wells in 390 µl aliquots. 50 µl of water was added to these wells, again as controls, to determine the intensity of the unbound dye. The equilibrium constants were determined by relating the observed intensity of the test solution (as determined by the area under the emission curve) with the average values ​​obtained from the control experiments.

Test results

Test results are reported relative to K_D found for AVPI, the tetrapeptide based on the sequence of the natural binding component, in each assay. In this way, the results were normalized for variation from day to day. The results are reported as follows, as ratios of the KD of the test compound to the K_D for AVPI: "-" = no binding was observed at the concentration used in the assay; "NA" = not tested; ± = ratio greater than 10^-4 (i.e. test compound bound with K_D greater than 10 ⁴ than AVPI); + = ratio greater than 10^-3; ++ = ratio greater than 10²; +++ = ratio greater than 10^-'.

ES 2 318 167 T3

Methylene bridged compounds

<img file="ES2318167T3_D0055.tif" />

<td rowspan="2">Number of carbons</td><td colspan="2">K_D (AVPI) / K_D (Counterpart)</td>

<td>no double bond</td><td>with double bond</td>

<td> 2</td><td> +</td><td> +</td>

<td> 3</td><td> —</td><td> +</td>

<td> 4</td><td> +++</td><td> +++</td>

<td> 5</td><td> ++</td><td> +++</td>

<td> 6</td><td> +</td><td>NA</td>

Oxazole-based compounds

<td>Counterpart</td><td>Name</td><td>K_D (AVPI) / K_D (Counterpart)</td>

<td></td><td>AoxSPF-OCH3</td><td> + +</td>

<td></td><td>AoxSPF-OH</td><td> + +</td>

<td></td><td>AOXTPF-0CH3</td><td> + +</td>

<td></td><td>AoxTPF-OH</td><td> + + +</td>

<td></td><td>AoxSPF-insat.</td><td> +</td>

ES 2 318 167 T3

<td></td><td>AoxSPF-ketone</td><td></td>

<td></td><td>AoxSPW</td><td> +</td>

<td></td><td>AoxSPY</td><td> + +</td>

<td></td><td>AoxSPI</td><td></td>

Phenylalanine derivatives

<td></td><td></td><td colspan="2">K_D (AVPI) / K_D (Counterpart)</td>

<td>Derived from phenylalanine</td><td>Name</td><td>Derived from Wing-Val- Pro-Phe</td><td>Derived from Wing-Arg- Pro-Phe</td>

<td>CO Mr</td><td>1-Naphthalene</td><td> +++</td><td>NA</td>

<td>ά _ / - \ HBrff * - /</td><td>Phenylglycine</td><td> +++</td><td> + + +</td>

<td></td><td>3-Chlorophenylalanine</td><td> +++</td><td> +++</td>

<td>Xoo</td><td>Biphenylalanine</td><td> +++</td><td> ++ +</td>

<td></td><td>2-Naphthalene</td><td> +++</td><td>NA</td>

<td>~ x</td><td>Cyclohexylglycine</td><td> +++</td><td>NA</td>

ES 2 318 167 T3

<td>lo&gt; c</td><td>4-Chlorophenylalanine</td><td> +++</td><td> + + +</td>

<td></td><td>2-Chlorophenylalanine</td><td> +++</td><td> +++</td>

<td></td><td>3,4- Dichlorophenylalanine</td><td> + + +</td><td> +++</td>

<td>»O&gt; C</td><td>4-Methylphenylalanine</td><td> +++</td><td> +++</td>

<td></td><td>Homophenylalanine</td><td> +++</td><td>NA</td>

<td>"Vw \</td><td>Cyclohexylalanine</td><td> +++</td><td> +++</td>

<td></td><td>Benzoylphenylalanine</td><td> +++</td><td> ++ +</td>

<td>MCM *</td><td>4- Carboxyphenylalanine</td><td> +++</td><td>NA</td>

Proline derivatives

<td>Proline derivative</td><td>Name</td><td>K_D (AVPI) / K_D (Counterpart)</td>

<td> \</td><td>Homoproline</td><td> +++</td>

<td>¿L</td><td>4- Hydroxyproline</td><td> +++</td>

ES 2 318 167 T3

<td> 5%,</td><td>1-amino- acid 1-cyclohexane- carboxylic</td><td> +</td>

<td></td><td>Cycloleucine</td><td> +</td>