Inhibitors of interleukin-1 converting enzyme

The invention relates to novel peptide compounds that are inhibitors of interleukin-ΐβ ("ICE") converting enzyme, and pharmaceutical compositions containing such compounds. The compounds according to the invention are characterized by particular structural and physicochemical characteristics.

The compounds and pharmaceutical compositions of the invention are particularly suitable for inhibiting the activity of ICE and, consequently, may be advantageously used as agents against interleukin-1 ("IL-1") dependent diseases, including inflammatory, autoimmune and neurodegenerative diseases.

Interleukin-1 ("IL-1") is a major proinflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, degranulation of basophils and eosinophils, and activation of neutrophilic cells. JH Oppenheim et al., Immunology Today, 7, pp. 45-56 (1986). As such, it is therefore associated with the pathogenesis of chronic and acute inflammations and autoimmune diseases. IL-1 is produced mainly by peripheral blood monocytes as part of the inflammatory response and exists in two distinct agonist forms, IL-Ια and IL-Ιβ. BS Mosely et al., Proc. Nat. Acad. Sci. 84, pp 4572-4576 (1987); G. Lonnemann et al., Enr. J. Immunol., 19, pp. 1531-1536 (1989).

IL-Ιβ is synthesized as a biologically inactive precursor, pIL-Ιβ. pIL-Ιβ does not have a conventional leader sequence and is not processed by the signal peptidase CJ March, Nature, 315, pp. 641-647 (1985). In contrast, pIL-Ιβ is cleaved by interleukin-ΐβ converting enzyme ("ICE") between Asp-116 and Ala-117 to form a biologically active C-terminal fragment present in human serum and synovial fluid. PR Sleat et al., J. Biol. Chem., 265, pp. 14526-14528 (1992); AD Howard et al., J. Immunol., 147 pp. 2964-2969 (1991). Treatment with the ICE enzyme is also necessary for the transport of parent 1L-1 β across the cell membrane.

ICE is a cysteine ​​protease mainly located in monocytes. It transforms IL-Ιβ into a mature form. RA Black et al., FEES Lett., 247, pp. 386-390 (1989); MJ Kostura et al., Proc. Natl. Acad, Sci. USA, 86, pp. 6227-5231 (1989). ICE or its homologues are also involved in regulating cell death or apotosis. J. Yuan et al., Cell, 75, pp. 641-652 (1993); M. Miura et al., Cell, 75, pp. 653-660 (1993); MA Nett-Fiordalisi et al., J. Cell Blochem., 17B, p. 117 (1993). In particular, ICE or ICE homologues are believed to be associated with the regulation of apoptosis in neurodegenerative diseases such as Alzheimer's disease

185 693 and Parkinson's disease. J. Marx and M. Baringa, Science, 259, pp. 760-762 (1993); V. Gagliardini et al., Science, 263, pp. 826-828 (1994).

ICE has previously been described as a heterodimer composed of two subunits, p20 andplO (20 kDa and 10 kDa molecular weight, respectively). These subunits are derived from the 45 kDa proenzyme (p45) in the form of p30, through an activation mechanism that is autocatalytic. NA Thomberry et al., Nature, 356, pp. 768-774 (1992). The ICE proenzyme has been divided into several functional domains: the prodomain (p14), the p22 / 20 subunit, the polypeptide linker and the p10 subunit. Thomberry et al., Supra; Casano et al., Genomics, 20, pp. 474-481 (1994).

The entire length of p45 was characterized by cDNA and amino acid sequences. PCT patent applications WO 91/15577 and WO 94/00154. cDNA and the amino acid sequences of p20 and p10 are also known. Thomberry et al., Supra. Mouse and rat ICE have also been sequenced and cloned. They show a high degree of amino acid and nucleic acid sequence homology with human ICE. DK Miller et al., Ann, NY Acad. Sci., 696, pp. 133-148 (1993); SM Molineaux, Proc. Nat. Acad. Sci., 90, pp. 1809-18813 (1993). However, the knowledge of the primary structure of ICE makes it impossible to predict its tertiary structure. It does not provide knowledge of the structural, conformational and chemical interactions of ICE and its substrate pIL-1β or other substrates or inhibitors.

ICE inhibitors are a class of compounds useful in combating inflammation or apoptosis, or both. Peptide and peptidyl ICE inhibitors have been described in PCT patent applications WO 91/15577; WO 93/05071; WO 93/09135; WO 93/14777 and WO 93/16710 and European Patent Application 0 547 699.

In the publication of NA Thomberry et al, "Inactivation of Interleukin-β Converting Enzyme by Peptide (Acyloxy) methyl Ketones" in Biochemistry, 33 pp. 3934-3940; ICE inhibitors of the type Ac-Tyr-Val-Ala-Asp-CH are shown₂-O-CO-aryl.

In RE Dolle et al., "P, Aspartate-Based Peptide a- (2,6-Dichlorobenzoyl) oxy) methyl Ketones as Potent Time-Dependent Inhibitors of Interleukin-ΐβ Converting Enzyme", J. Med. Chim. 37, pp. 563-564 (1994), describes non-cyclic amino acid and peptide derivatives in which the N-terminal amino group is protected as a carbobenzyloxy derivative. These are ICE inhibitors similar to those described in the Thornberry publications, and like them also contain an acyloxymethylketone group.

. European patent EP 0519748 describes non-cyclic ICE inhibitors of the type XTry-Val-Asp-CH₂-O-CO-aryl.

WO 93/09135 also relates to non-cyclic derivatives of amino acids and peptides.

In turn, WO 93/16710 relates to aspartic acid derivatives which are linked by peptide bonds to at least two other non-cyclic amino acids. These are ICE inhibitors of the type (AA) |.₃-Asp-CH₂-O-CO-aryl and (AA / .j-Asp-CH1-O-aryl.

In MD Mullican et al., "The Synthesis and Evaluation of Peptidyl Aspartyl Aldehydes as Inhibitor of ICE", Bioorg. Med. Chem. Lett., 4, pp. 2359-2364 (1994) describe asparaginate-based peptidyl derivatives which are also useful as ICE inhibitors.

However, due to their peptide nature, such inhibitors usually have undesirable pharmacological properties, such as poor absorption when administered orally, poor stability and rapid metabolism. JJ Plattnęr and DW Norbeck, in Drug Discoyery Technologies, CR Clark and WH Moos, ed. (Ellis Horwood, Chichester, England, 1990), pp. 92-126. This is an obstacle to their use as effective drugs.

Accordingly, there is a need for compounds that can effectively inhibit the action of ICE and are useful as agents for the prevention and treatment of chronic and acute forms of IL-1 related diseases, including a variety of cancers, as well as inflammation, autoimmune and neurodegenerative diseases. .

The present invention provides a new class of compounds and pharmaceutically acceptable derivatives thereof that are useful as ICE inhibitors. These compounds can be used alone or in combination with other therapeutic and prophylactic agents, such as antibiotics, immunomodulators, or other anti-inflammatory agents, to treat or prevent IL-1 dependent diseases. According to a preferred embodiment of the invention, the compounds of

185 693 of the invention are capable of binding to the active site of ICE and inhibiting the activity of this enzyme.

The main object of the invention is to provide a new class of ICE inhibitors. This new class of ICE inhibitors is characterized by the following structural and physicochemical features:

a) first and second hydrogen bonding moieties, each capable of hydrogen bonding to a different atom of the ICE chain, which atom is selected from the group consisting of carbonyl oxygen Arg-341, amide-NH-wArg-341, carbonyl oxygen Ser-339 and amide-NH- in Ser-339;

b) first and second moderately hydrophobic residues each of which are capable of combining a separate ICE binding cavity (pocket) when the inhibitor binds to the enzyme; such binding cavity is selected from the group consisting of P2, P3, P4 and P 'binding cavities and

c) an electronegative moiety consisting of one or more electronegative atoms that are attached to the same atom in the rest or to adjacent atoms, the rest being capable of forming one or more hydrogen bonds or salt bridges with the moieties in PI binding cavity in ICE.

ABBREVIATIONS AND DEFINITIONS

Shortcuts

Reagent designation or fragment

Ala alanine

Arg arginine

Asn asparagine

Asp aspartic acid

Cys cysteine

Gin glutamine

Glu glutamic acid

Gly glycine

His Histidine

How much isoleucine

Leu leucine

Lys Lysine

Methionine

Phe phenylalanine

Pro proline

Serine cheese

Thr threonine

Trp tryptophan

Tyr tyrosine

Val valina

Definitions

The following terms are used:

The term "active site" means any or all of the following sites in ICE: substrate binding site, inhibitor binding site, and site where cleavage of the substrate occurs. The active center is characterized by at least the following amino acid residues: 173, 176, 177, 178, 179, 180, 236, 237, 238, 239, 244, 248, 283, 284, 285, 290, 338, 339, 340, 341, 342 , 343, 344, 345, 348, 352, 381, 383 (sequence and numbering according to Thomberry et al., Supra).

The terms "binding cavity", "S sub-site", "S cavity", and the like mean binding sites or portions of a substrate binding site in an ICE molecule. The amino acid residues of the substrate are determined according to their position relative to the broken bond, ie the bond that is broken by the protease. Residues towards the N-terminus of the substrate are designated as P1, P2, etc. "Arrests towards the C-terminus of the substrate are designated as PT, P2 'etc. Parts of the inhibitor that correspond to the P or P 'residues of the substrate are also designated PI and PI'.

185 693 etc., by analogy to the substrate. Binding sub sites in an ICE molecule that adopt residues labeled P1, ΡΓ, etc. are designated SI, SI 'etc. or, alternatively, may be labeled "P1 binding cavity", "ΡΓ binding cavity", etc. [I. Schechter and A. Berger, "On the Size of the Active Site in Proteases", Biochem. Biophys. Res. Commun. vol. 25, pp. 157-162 (1967)].

The terms "P2 binding cavity" or "S2 sub-site" in the active site of ICE are synonymous and are referred to as the space comprised of the amino acid residues Pro-290, Val-338 and Trp-340.

The terms "P3 binding cavity" or "S3 sub-site" in the active site of ICE are synonymous and are defined as the space encompassed by the amino acid residues Pro-177, Arg-178, Thr-180, Arg-341, or Pro-343.

The terms "P4 binding cavity" or "S4 sub-site" in the active center of ICE are equivalent and are defined as the space covered by the amino acid residues His-342, Met-345, Val-348, Arg-352, Asp-381, Arg-383 or Trp-340.

The terms "P1 binding cavity" or "SI subsite" in the ICE active site are synonymous and are referred to as the space encompassed by amino acid residues Arg-179, His237, Gln-383, or Arg-341.

The terms "P1-binding cavity" or "S-sub-site" in the active center of ICE are synonymous and are defined as the space encompassed by the amino acid residues Phe-173, Ile-176, His-237, Gly-238, Ile-239, Cys-244 or His-248.

The term "hydrophobic" refers to a residue that is non-water-soluble and fat-soluble. Hydrophobic residues include, but are not limited to, hydrocarbons such as alkanes, alkenes, alkynes, cycloalkanes, cycloalkenes, cycloalkines, and aromatics such as aryls, certain saturated and unsaturated heterocycles, and residues that are substantially similar to natural and unnatural hydrophobic side chains α-amino acids, including valine, leucine, isoleucine, methionine, phenylalanine, α-aminoisobutyric acid, alloisoleucine, tyrosine, and tryptophan.

The term "moderately hydrophobic" refers to a hydrophobic moiety in which one or two carbon atoms have been replaced with more polar atoms such as oxygen or nitrogen atoms.

The term "heterocycle" or "heterocyclic" denotes a stable mono- or polycyclic compound which may optionally contain one or two bonds or may optionally contain one or more aromatic rings. Each heterocycle is composed of carbon atoms and from one to four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The terms "nitrogen heteroatoms" and "sulfur heteroatoms" as used herein include any oxidized form of nitrogen or sulfur and a quaternized form of any basic nitrogen. The heterocycles as defined above include, for example, pyrimidinyl, tetrahydroquinolyl, tetrahydroisoquinolinyl, purinyl, pyrimidyl, indolinyl, benzimidazolyl, imidazolyl, imidazolinoyl, imidazolidinyl, quinolyl, isoquinolyl, indolyl, pyridinyl, pyrrolinyl, pyrrolinyl, pyrrolinyl, pyrrolinyl, pyrrolinyl, pyrrolinyl, pyrrolinyl, , furyl, thienyl, triazolyl, thiazolyl, βcarbolinyl, tetrazolyl, thiazolidinyl, benzofuranoyl, thiamorpholinylsulfone, benzoxazolyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, isoxazolyl, tetrahydropyranyl, tetrahydrofuranyl, thiadiazolyl, benzodioxolyl, benzothienyl, tetrahydrothiophenyl and sulfolanyl. Other heterocycles are described in AR Katritzky and CW Rees, Ed., Comprehensive Heterocyclic Chemistry, The Structure, Reactions, Synthesis and Use of Heterocyclic Compounds, Vol. 1-8, Pergamon Press, NY (1984).

The term "cycloalkyl" refers to mono- or polycyclic groups which contain 3 to 15 carbon atoms and optionally one or two double bonds. Examples include cyclohexyl, adamantyl, and norbomyl.

The term "aryl" denotes a mono- or polycyclic group which contains 6, 10, 12 or 14 carbon atoms and in which at least one ring is aromatic. Examples include phenyl, naphthyl, and biphenyl.

The term "heteroaromatic" denotes a mono- or polycyclic group that contains 1 to 15 carbon atoms and 1 to 4 heteroatoms, each independently being

185 693 is selected from the group consisting of sulfur, nitrogen and oxygen, and which further comprises 1 to 3 5 or 6 membered rings, at least one of which is aromatic.

The term "alpha-amino acid" (α-amino acid) refers to both naturally occurring amino acids and to other "non-protein" α-amino acids typically used by those skilled in the art of peptide chemistry to prepare synthetic analogs of naturally occurring peptides, including the D forms and L. The naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutoxyglutamic acid, arginamine, γ-carbonyl acid and lysine. Examples of "non-protein" alpha amino acids include hydroxy lysine, homoserine, homotyrosine, homophenylalanine, citrulline, kynurenine, 4-aminophenylalanine, 3- (2-naphthyl) alanine, 3- (1-naphthyl) alanine, methionine sulfone, t-butylalanine , t-butylglycine, 4-hydroxyphenylglycine, aminoalanine, phenylglycine, vinylalanine, propargylglycine, 1,2,4-triazole-3-alanine, 4,4,4-trifluorotreonine, tyronine, 6-hydroxytryptophan, 5-hydroxytryptoxycin, 3-hydroxytryptophan , 3-aminotyrosine, trifluoromethylalanine, 2-thienylalanine, (2- (4-pyridyl) ethyl) -cysteine, 3, 4-dimethoxy-phenylalanine, 3- (2-thiazolyl) -alanine, ibotenic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, quisqualic acid, 3-trifluromethylphenylalanine, 4-trifluoromethylphenylalanine, cyclohexylalanine, cyclohexylglycine, thiohistidine, 3-methoxytyrosine, elastatinal, norleucine, thioproline, thioproline, norleucine dehydrine, isonipectonic acid, homoproline, cyclohexylglycine, α-amino-nbutyric acid, cyclohexylalanine, aminophenylbutyric acid, ortho, meta or para-substituted phenylalanines with one or two of the following groups: (C, C₄) alkyl, (C, -C₄) alkoxy, halogen or nitro groups or substituted with methylenedioxy; β-2- and 3-thienylalanine, β-2- and 3-furanylalanine, β-2- 3- and 4-pyridylalanine, β (benzothienyl-2- and 3-yl) alanine, β- (1- and 2- naphthyl) alanine, O-alkylated serine, threonine or tyrosine derivatives, S-alkylated cysteine, S-alkylated homocysteine, O-sulphate, tyrosine O-phosphate and O-carboxylate, 3-sulfo-tyrosine, 3-carboxy-tyrosine, 3-phospho - tyrosine, tyrosine 4-methanesulfonic acid ester, tyrosine 4-methanesulfonic acid ester, 3,5-diiodotyrosine, 3-nitrotyrosine, ε-alkyl lysine and delta-alkylmithine. All of these α-amino acids can be substituted with a methyl group at the alpha position, a halogen at any aromatic residue on the α-amino side chain, or a suitable protecting group on the O, N or S atoms in the side chain. Suitable protecting groups are disclosed in "Protective Groups In Organic Synthesis", TW Greene and PGM Wuts, J. Wiley & Sons, NY, NY, 1991.

The term "α-amino acid side chain residue" means a chemical residue that is attached to the a-carbon atom in the alpha-amino acid.

The term "bioisosteric replacement group -CO."₂H "refers to a group that can replace an acid carboxyl group in bioactive molecules. Examples of such groups are disclosed in Christopher A. Lipiński, "Bioizosteres in Drug Design" Annual Report in Medical Chemistry, 21, pp. 286-288 (1986) and CW Thomber, "Izoesterism and Molecular Modification in Drug Design," Chemical Society Ręyiews, pp 563-580 (1979).

The term "association" is used to refer to a state of proximity between an inhibitor or portions thereof and an ICE molecule or portions thereof, this juxtaposition being energetically favored by electrostatic or van der Waals interactions.

The term "hydrogen bond" refers to the beneficial interaction that occurs when the respective donor atom, X, containing a proton, H, and the corresponding acceptor atom, Y, are between 0.25 nm - 0.35 nm and the angle Χ-Η ------- Y is greater than 90 degrees. Suitable donor and acceptor atoms are well known in medical chemistry (GC Pimentel and AL McClellan, The Hydrogen Bond, Freeman, San Francisco, 1960; R. Taylor and O. Kennard, "Hydrogen Bond Geometry and Organic Crystals", Account of Chemical Research, 17, pp. 320236 (1984)).

The term "electrolytic bridge" refers to a non-covalent attraction interaction between a positively charged residue (P) and a negatively charged residue

185 693 (N) when it is in the range of 0.2 nm and 0.6 nm between the P and N mass centers. When calculating the center of mass, the atoms that carry a formal charge and the atoms immediately adjacent to them are taken into account. For example, an electrolytic bridge may be formed between a positively charged guanidinium side chain in the arginine residue and a negatively charged carboxylate side chain in the glutamate residue. Electrolytic bridges are well known in medical chemistry (L. Stryer, Biochemistry, Freeman, San Francisco, (1975); KA Diii, "Dominant Forces in Protein Folding", Biochemistry. 29, No. 31, pp. 7133-7155 (1990)).

The term "center of mass" refers to a point in three-dimensional space that represents the weighted average position of the component masses.

The terms "backbone" or "backbone" refer to that portion of a polypeptide that comprises -CO-CH-NH- repeat units.

The term "scaffold" refers to a block of the structural model that forms the basis of an ICE inhibitor according to the invention. A variety of residues and functional groups can be included in the scaffold. In the present invention, the scaffolds are shown to contain open valences. The various ICE inhibitor scaffolds of the invention include parts:

X

NC

And 0

HO or

X \ / \ /

NSO

IU

HO

In such scaffolds, the residuals NH and CO or SO₂ represent first and second hydrogen bonding residues, each of which is capable of hydrogen bonding to an atom in the backbone of ICE which is selected from the group consisting of carbonyl oxygen Arg-341, amide -NH- in Arg-341, carbonyl oxygen Ser-339 and amide-NH- in Ser-339.

The term "substituted" refers to the replacement of a hydrogen atom in a compound with a substituent group. In the present invention, no substitution is made for those hydrogen atoms which form part of a hydrogen bond-forming moiety capable of hydrogen bonding with the carbonyl oxygen in Arg-341 ICE or the carbonyl oxygen in Ser-339 ICE. Such excluded hydrogens include those atoms which are included in the NH- group, which is alpha to Z or the -CO- group and is shown as -NH and not as the X group or other designation in the formulas: (a ) to (t), (v) to (y) and (I) to (VIID).

The term "straight chain" denotes a continuous, unbranched string of covalently linked members, ie, atoms that form part of a ring. A straight chain and the ring of which it forms part may be substituted, but such substituents are not part of a straight chain.

The symbol "Kj" refers to the numerical measurement of a compound's effectiveness in inhibiting the activity of a target enzyme, such as ICE. The lower the K values, the higher the efficiency. The K i value is derived by fitting the experimentally determined rate values ​​to a standard enzyme kinetic equation (see IH Segel, Enzyme Kinetics, Wiley-Interscience, 1975).

185 693

The term "minimize" refers to a systematic alteration of the spatial arrangement of atoms ("atomic geometries") in a molecule or molecular complex such that any further minor perturbations in the atomic geometry would increase the total energy of the system as measured by molecular force-field mechanisms. Minimization and molecular force-field mechanisms are well known in computer chemistry [U. Burkert and NL Allinger, Molecular Mechanics, ACS Monograph 177, America Chemical Society, Washington, 1982, pp. 59-78].

The term "strain energy" is used in this application to refer to the difference between the "free conformation energy" and the "bound conformation energy" of an ICE. The stress energy can be determined by the following steps. Assessment of the energy of a molecule that has the conformation needed to bind to ICE. Then minimize and re-evaluate the energy which is the conformational energy of the free compound. The stress energy for binding a potential inhibitor to ICE is the difference between the conformational energy of the free compound and the conformational energy of the bound compound. In a preferred embodiment, the stress energy of an inhibitor of the invention is less than about 0.0418 KJ / mol.

The term "patient" as used herein refers to any mammal, especially humans.

The term "pharmaceutically effective amount" means an amount effective to treat or ameliorate IL-1 related diseases in a patient.

The term "prophylactically effective amount" means an amount effective to prevent or substantially reduce the symptoms of an IL-1 related disease in a subject.

The term "pharmaceutically acceptable carrier or excipient" refers to a non-toxic carrier or excipient that can be administered to a patient with the compound of the invention without abolishing its pharmacological activity.

The term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, or salt of such ester of a compound of the invention, or any other compound which, when administered to a patient, is capable of providing (directly or indirectly) a compound of the invention or an anti-metabolite. ICE or the rest of it.

Pharmaceutically acceptable salts of the compounds of the invention include, for example, salts formed with pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene -2-sulfonic and benzenesulfonic. Other acids, such as oxalic, while not themselves pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts formed with suitable bases include alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium salts and N- (C, -C₄alkyl)₄⁺.

The invention also contemplates the "quaternization" of any basic nitrogen containing groups in the compounds of the invention. Basic nitrogen can be quaternized by means known to those skilled in the art including, for example, lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides, including benzyl and phenethyl bromides. By such quaternization, water-soluble or oil-soluble or dispersible products can be obtained.

The ICE inhibitors of the invention may contain one or more "asymmetric" carbon atoms and thus may exist as racemates or racemic mixtures, single enantiomers, diastereomeric mixtures and single diastereomers. All isomeric forms of the compounds according to the invention are within the scope of the invention

185 693 of the invention. Each stereogenic carbon atom may be in the R or S configuration. While specific compounds and scaffolds exemplified in this application may be represented in a particular stereochemical configuration, compounds and scaffolds and mixtures thereof of opposite stereochemistry at a given chiral center are also contemplated.

The ICE inhibitors of the invention can include ring structures that may be optionally substituted on carbon, nitrogen or other atoms with a variety of substituents. Such ring structures can be single or multiple substituted. Preferably, the ring structures contain from 0 to 3 substituents. For multiple substitution, each substituent may be selected independently of the other substituents, as long as the combination thereof results in the formation of a stable compound.

Only combinations of substituents and variables that result in the formation of a stable compound are included in the invention. The term "stable" as used herein refers to compounds which are stable enough to be produced and administered to mammals by methods known in the art. Typically, such compounds are stable for at least one week at temperatures of 40 ° C or less in the absence of moisture or other chemically reactive conditions.

The following detailed description is provided below for a more complete understanding of the present invention.

We have found compounds with the following new combination of features to be surprisingly effective inhibitors of ICE:

a) first and second hydrogen bonding moieties each capable of hydrogen bonding to a different atom of the ICE chain, which atom is selected from the group consisting of carbonyl oxygen Arg-341, amide-NH- in Arg-341, carbonyl Ser-339 oxygen and the amide-NH- group in Ser-339;

b) first and second moderately hydrophobic residues each of which are capable of combining a separate ICE binding cavity (pocket) when the inhibitor binds to the enzyme; such binding cavity is selected from the group consisting of P2, P3, P4 and P 'binding cavities and

c) an electronegative moiety comprising one or more electronegative atoms that are linked to the same atom in the rest or to adjacent atoms, the moiety being capable of forming one or more hydrogen bonds or electrolytic bridges with the moieties in the ICE binding cavity of the Beer.

Preferably, each of the moderately hydrophobic residues connecting to the P2 binding cavity of the ICE connects thereto such that:

a) the distance from the mass center of the moderately hydrophobic residue in the P2 binding cavity to the carbonyl oxygen of Arg-341 ICE is in the range of about 0.71 nm and about 1.25 nm;

b) the distance from the mass center of the moderately hydrophobic residue in the P2 binding cavity to the amide nitrogen in Arg-341 ICE is in the range of about 0.6 nm and about 1.2 nm;

c) the distance from the mass center of the moderately hydrophobic residue in the P2 binding cavity to the carbonyl oxygen in Ser-339 ICE is in the range of about 0.37 nm and about 0.95 nm.

Preferably, a moderately hydrophobic residue communicating with the P3 binding cavity of the ICE connects thereto such that:

a) the distance from the mass center of the moderately hydrophobic residue in the P3 binding cavity for Arg-341 ICE carbonyl oxygen is in the range of about 0.39 nm to about 0.95 nm;

b) the distance from the mass center of the moderately hydrophobic residue in the P 'binding cavity to the nitrogen of the Arg-341 ICE amide group is between about 0.54 nm and about 1.1 nm; and

c) the distance from the mass center of the moderately hydrophobic residue in the P 'binding cavity to Ser-339 ICE carbonyl oxygen is in the range of about 0.7 nm and about 1.3 nm.

Preferably, a moderately hydrophobic residue communicating with the P4 binding cavity of the ICE connects thereto such that:

a) the distance from the mass center of the moderately hydrophobic residue in the P4 binding cavity for Arg-341 ICE carbonyl oxygen is in the range of about 0.45 nm to about 0.75 nm;

185 693

b) the distance from the mass center of the moderately hydrophobic residue in the cavity which binds P4 to the nitrogen of the Arg-341 ICE amide group is between about 0.55 nm and about 0.85 nm; and

c) the distance from the mass center of the moderately hydrophobic residue in the P4 binding cavity for Ser-339 ICE carbonyl oxygen is in the range of about 0.8 nm and about 1.1 nm.

Preferably, a moderately hydrophobic residue communicating with the P binding cavity of the ICE connects thereto such that:

a) the distance from the mass center of the moderately hydrophobic residue in the binding cavity of PP 'to Arg-341 ICE carbonyl oxygen is in the range of about 1.1 nm to about 1.6 nm;

b) the distance from the mass center of the moderately hydrophobic residue in the P-binding cavity to the nitrogen of the Arg-341 ICE amide group is between about 1 nm and about 1.5 nm; and

c) distance from the center of mass of the moderately hydrophobic residue in the P binding cavity¹ to the carbonyl oxygen Ser-339 ICE is in the range of about 0.8 nm and about 1.2 nm.

Preferably, all of the above combination conditions are met in the compounds of the invention.

One skilled in the art will appreciate that there are many ways to design the inhibitors of the invention. The same methods can be used to select a candidate compound as an ICE inhibitor in a screening assay. Such design and selection can start with selecting the various residues that fill the binding cavities.

There are many ways to select the residues that fill individual binding cavities. These methods include visually inspecting the physical model or computer model of the active site, and manually placing the selected residue models into different binding cavities. You can also use the known and available computer modeling. Such modeling is included in the QUANTA program [Molecular Simulations, Inc., Burlington, MA, 1992], SYBYL [Molecular modeling Software, Tripos Associates, Inc., St. Louis, MO, 1992], AMBER [SJ Weiner, PA Kollman, DA Case, UC Singh, C. Ghio, G. Alagon, and P. Weiner, J, Am, Chem. Soc., Vol. 106, pp. 765-784 (1984)] or CHARMM [BR Brooks, RE Bruccoleri, BD Olafson, DJ Slates, S. Swaminathan and M. Karpłus, J, Comp. Chem. Vol. 4, pp. 187-217 (1983)]. After the modeling step, an energy minimization step can be performed using standard molecular force-field mechanisms such as CHARMM and AMBER. In addition, there are several more specialized computer programs that can assist in the selection of binding moieties according to the invention. These programs include:

1. GRID (PJ Goodford, A Computational Procedure for Determing Energetically Favorable Binding Sites on Biologically Important Macromolecules. J, Med. Chem., 28 pp. 849857 (1985). GRID is available from Oxford University, Oxford, UK.

2. MCSS (A. Miranker, M. Karplus, Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method. Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is available from Molecular Simulations, Burlington, MA.

3. AUTODOCK (DS Goodsell, AJ Olsen, Automated Docking of Substrates to Proteins by Simmulated Annealing. Proteins: Structure, Function and Genetics, 8, pp. 195-202 (1990). AUTODOCK is available from the Scripps Research Institute, La Jolla, CA.

4. DOCK (ID Kuntz, JM Blaney, SJ Oatley, R. Langridge, TE Ferrin, A Geometrie Approach is Macromolecule-Ligand Interactions. J. Mol. Biol., 161, pp. 269-288 (1982)). DOCK is available from the University of California, San Francisco, CA.

As soon as suitable binding moieties are selected, they can be assembled into a single inhibitor. This assembly can be performed by attaching the various residues to the central scaffold. This compilation process can be performed, for example, by visually checking and building the model by hand, and again using software such as Quanta or Sybyl. There are many other programs to help choose how to attach the various residues. These programs include:

1. CAVEAT (PA Barlett, GT Shea, SJ Telfer, S. Waterman. CAVEAT: A Program to Facilitate the Structure-Derived Desing of Biological Active Molecules. In "Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc. ., 78, pp. 182-196 (1989)). CAYEAT is available from the University of California, Berkeley, CA.

185 693

2. 3D database system such as MACCS-3D (MDL Information System, San Leandro, CA). This program was recently reviewed by Martin (YC Martin, 3D Database Searching in Drug Design. J. Med. Chem. 35, pp. 2145-2154 (1992)).

3. HOOK (available from Molecular Simulations, Burlington, MA).

In addition to the above computer programs to assist in the modeling of inhibitor compounds, the inhibitors of the invention may be constructed "de novo" using either the empty active site or optionally including some portions of known inhibitors. Such methods are well known in the art. These include, for example:

1. LUDI (HJ Bohm, The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors. J. Comp. Aid. Molec. Design, 6, 61-78 (1992)). LUDI is available from Biosym Technologies, San Diego, CA.

2. LEGEND (Y. Nishibata, A. Itai, Tetrahedron, 47, 8985 (1991)). LEGEN is available from Molecular Simulations, Burlington, MA.

3. LeapFrog (available from Tripos associates, St. Louis, MO).

Several techniques commonly used in drug modeling can be used (see, e.g., NC Cohen; JM Blaney; C. Humblet; P. Gund; DC Barry, "Molecular Modeling Software and Methods for Medicinal Chemistry", J, Med. Chem., 33 , pp. 883-894 (1990)). Likewise, there are many examples of techniques in the chemical literature that can be used to design a particular drug. See, e.g., MA Navia and MA Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202210 (1992). Examples of specific publications include: JJ Baldwin et al., "Tienothipyran-2sulfonamides: Novel Topically Active Carbonic Anhydrase Inhibitors for the Treatment of Glaucoma", J. Med. Chem, 32, pp. 2510-2513 (1989); K. Appelt et al. "Design of Enzyme Inhibitors Using Iterative Protein Crystallographic Analysis" J. Med. Chem., 34, pp. 1925-1934 (1991); and SE Ealick et al., "Application of Crystallographic and Modeling Methods in the Design of Purine Nucleotide Phosphorylase Inhibitors", Proc. Nat. Acad. Sci. USA, 88, pp. 11540-11544 (1991).

Many of the conventional techniques can be used to evaluate the enzyme inhibitory activity of individual compounds, as well as for evaluation necessary in screening a candidate compound for inhibiting ICE activity. Generally, such techniques include determining the distribution and bonding proximity of a given residue, the space occupied by the bound inhibitor, the bond deformation energy of a given compound, and electrostatic interaction energy. Examples of conventional techniques useful for the above research include: quantum mechanics, molecular mechanics, molecular dynamics, Monte Carlo method, systems research method and spatial geometry. (GR Marshall, Ann. Ref. Pharmacol. Toxicol., 27, p. 193 (1987)). Specific computer software has also been developed for use in such methods. Examples of computer programs for such applications include: Gaussian 92, revision E.2 (MJ Frisch, Gaussian, Inc. "Pittsburgh, PA © 1993); AMBER, version 4.0 (PA Kollman, University of California, San Francisco, © 1993); QUANTA / CHARMM [Molecular Simulation, Inc. Burlington, MA © 1992); and Insight II / Discover (Biosysm Technologies Inc., San Diego, CA © 1992). These programs can be implemented, for example, on a Silicon Graphics Indigo 2 or IBM RISC / 6000 model 550 workstation. Other computer systems and software packages will be known and will be readily employed by those skilled in the art.

The different classes of active ICE inhibitors of the invention may similarly interact with different binding cavities at the active center of ICE. The spatial distribution of these important groups is often referred to as a pharmacophore. The pharmacophore concept has been thoroughly described in the literature (D. Mayer, CB Naylor, I. Motoc and GR Marshall, J. Comp. Aided Molec. Design, vol. 1, pp. 3-16 (1987); A. Hopfmger and BJ Burke , in Concepts and Applications of Molecular Similarity, MA Johnson and GM Maggiora, Eds., Wiley (1990)).

Different scaffolds or core structures may be used in the different classes of ICE inhibitors of the invention, but in all such cores the necessary residues will be placed in the active site so that the specific interactions necessary for

185 693 bindings. These compounds are best defined in terms of their ability to conform to the pharmacophore, i.e. their structural similarity to the shape and nature of the ICE active site.

The compounds of the invention (C) are represented by the formula σ:

<img file="PL185693B1_D0001.tif" />

wherein the ring is optionally substituted with one or more R's, preferably 0, 1 or 2; and in which:

R] is R₅-(AND)_p-;

R₅ is selected from the group consisting of:

-H,

-Ar_b

-CO-Ar ,,

-SO₂-Ar "

-CO-R ,,

-CO-O-Ry,

-SO₂-R9, / Ar!

-CO-N \ Ro, / Αη

-SO, -N '\ R₁₀,

-CO-N and \ R, o

-SO₂-N \ R₁₀each A is independently selected from the group consisting of any one of a-amino acids:

p is 2 or 3;

Y is -O -, - S-, or -NH;

R is -H, -OC 4 alkyl, -NH (C 1-4 alkyl), -N (C 6.₆alkyl), -S-Cj.₆alkyl, - C 1-4 alkyl, or -Q₂;

each Ry is a straight or branched C1-4 alkyl group optionally singly or multiply substituted with -OH, -F or = O and optionally substituted with one Αη;

each of the R.₁₀ is independently selected from the group consisting of -H or C 1-4 straight or branched alkyl;

each of Ij is independently selected from the group consisting of -CH = CH-, -O-, -S-, -SO-, -SO₂-, -NR₁₀-, -NR_I0-CO-, -CO-, -O-CO-, -CO-O-, -CO-NR₁₀-, -O-CO-NO_l0-, -NR₁₀-CO-O-, -NR '_1O-CO-NO₁₀-, -SO₂-NR₁₀-, -NR₁₀-SO₂ or -NR₁₀-SO₂-NR₁₀-,

185 Each Ar, is a cyclic group independently selected from the group consisting of an aryl group that contains 6, 10, 12, or 14 carbon atoms and 1 to 3 rings, a cycloalkyl group that contains 3 to 15 carbon atoms and 1 to 3 rings, and optionally fused to a benzene ring, and a heterocyclic group having 5 to 15 ring atoms and 1 to 3 rings which contains at least one heteroatom selected from -O-, -S-, -SO-, -SO₂-, = N- and -NH- and optionally contains one or more double bonds, and optionally includes one or more aromatic rings, this cyclic group being optionally singly or multiply substituted with: -NH₂. -WHAT₂H, -Cl, -F, -Br, -J, -NO₂, -CN, = 0, -OH, -perfluoro-C | ₃alkyl, O / \ ch₂, \ / or Qg

Each Q] is independently selected from the group consisting of -Αη, -Rg, -T_rRg and - (CH₂)| _{2 3}-T] -Rg;

every Q₂ is independently selected from the group consisting of -OH, -NH₂, -C0₂H, -Cl, -F, -Br, -J, -N0₂, -CN, -CF₃ and O / \

CH₂;

\ /

O provided that when -Αη is substituted with a group Q "that includes one or more additional groups -Αη, these additional groups -Αη are not substituted with Q ,.

Preferred compounds of embodiment C of the invention include, but are not limited to, compounds of the formulas:

<img file="PL185693B1_D0002.tif" />

185 693

<img file="PL185693B1_D0003.tif" />

Preferred compounds of embodiment C according to the invention are also those in which each A is independently selected from the group consisting of α-amino acids: alaninine, histidine, lysine, phenylalanine, proline, tyrosine, valine, leucine, isoleucine, glutamine, methionine, homoproline, 3- (2-thienyl) alanine and 3- (3-thienyl) alanine.

The compounds according to a further embodiment of the invention (D) are represented by the formula π:

<img file="PL185693B1_D0004.tif" />

wherein:

R is R.₅- (A)_p-;

each T, is independently selected from the group consisting of -CH = CH-, -0-, -S-, -S0-, -S0₂-, -NR₁₀-, -NR₁₀-CO-, -C0-, -0-C0-, -C0-0-, -CO-NR₁₀-, -O-CO-NO₎₀-, -NR₁₀-CO-O-, -NR₁₀-CO-NO₁₀-, -SO₂-NR_I0-, -NR₁₀-SO₂ and -NR₁₀-SO₂-NR₁₀-,

R₅ is selected from the group consisting of -Η, -Ar, -CO-Ar_1; -S0₂- Αη, -R, -CO-K, -CO-O-Re, -SO₂-R5, / Ar,

-CO-N \ Ro / Ar, so₂-n '' Rio?

-CO-N \ R₁₀,

-so₂-n \ R10?

185 693 each A is independently selected from the group consisting of any a-amino acid;

p is 2 or 3;

each R, is a straight or branched C 1-4 alkyl group optionally singly or multiply substituted with -OH or -F and optionally substituted with Ar ,;

each of the R.₁₀ is independently selected from the group consisting of -H or straight or branched C,.₆alkyl

Ar, is a cyclic group independently selected from the group consisting of an aryl group that has 6, 10, 12, or 14 carbon atoms and 1 to 3 rings, a cycloalkyl group that has 3 to 15 carbon atoms and 1 to 3 rings, and optionally is fused to a benzene ring, and a heterocyclic group having 5 to 15 ring atoms and 1 to 3 rings which contains at least one heteroatom selected from -O-, -S-, -SO-, -SO₂-, = N- and -NH- and optionally contains one or more double bonds, and optionally includes one or more aromatic rings, this cyclic group being optionally singly or multiply substituted with: -NH₂, -WHAT₂H, -Cl, -F, -Br, -J, -NO₂, -CN, = 0, -OH, -perfluoro-C, ₃alkyl, OR, or -IjRg.

/ \

CH₂\ / about

Preferred compounds according to embodiment D of the invention are those in which R_g represents a straight or branched group C._M.alkyl substituted with Ar 'when Ar is phenyl.

Preferred compounds of embodiment D of the invention include, but are not limited to, compounds of the formulas:

<img file="PL185693B1_D0005.tif" />

18S 693

<img file="PL185693B1_D0006.tif" />

Preferred compounds of embodiment D of the invention are also those in which A is independently selected from the group consisting of the α-amino acids: alaninine, histidine, lysine, phenylalanine, proline, tyrosine, valine, leucine, isoleucine, glutamine, methionine, homoproline, 3- ( 2-thienyl) alanine; and 3- (3-thienyl) alanine.

The compounds according to a further embodiment of the invention (E) are represented by the formula v:

<img file="PL185693B1_D0007.tif" />

wherein:

m is 0, 1 or 2;

T is -CO₂H or any bioisosteric -CO replacement group₂H, selected from -CO-CH₂OH, -CO-NHOH, -SO₂-NHR, -SO₃H, -PO (OH) NH₂, -CHNHCN, -oso₃h, -CO-NHSO₂R₁₆, PO (OH)₂, -PO (OH) (OR₁₆), -PO (OH) (R_!6), -OPO (OH)₂, -OPO (OH) (OR₁₆), -OPO (OH) (R_l6), -NHPO (OH)₂ -NHPO (OH) (OR₁₆), -NHPO (OH) (R₁₆),

<img file="PL185693B1_D0008.tif" />

where R,₅ is -H, a -C 1-6 alkyl group, or a T-bond of (CH₂)_m;

185 693

R₁₆ represents the -C group] ₆ alkyl;

^5

R₃ means -CN, -COR₁₃ or -CO-CO-N \ Rio&gt;

R₅ is selected from the group consisting of -H, -AT], -CO-Ar "-SO₂-Ar, -R, -CO-R, -CO-OR, -SO₂-R), / Ar,

-CO-N \ R₁₀, / Ar, so₂-n

-CO-N \ R₁₀,

-SOj-N ^ \ R_I0;

each A is independently selected from the group consisting of any amino acid;

p is 2;

each R, is a straight or branched C 1-4 alkyl group optionally singly or multiply substituted with -OH, -F or = 0 and optionally substituted with one Ar i, the alkyl group being optionally unsaturated;

each T, is independently selected from the group consisting of -CH = CH-, -0-, -S-, -S0-, S0₂-, -NRio-, -NR₁₀-CO-, -C0-, -0-C0-, -C0-0-, -CO-NR₁₀-, -O-CO-NO₁₀-, -NR₁₀-CO-O-, NO₁₀-CO-NO₁₀-, -SO₂-NR_l0-, -NR,₀-SO₂ or -NR₁₀-SO₂-NR_l0-, each of R,_about is independently selected from the group consisting of -H or straight or branched C,.₆alkyl;

each of R,₃ is independently selected from the group consisting of H, R 2, Ar₂ their₂-T, -Rj, each Ar, is a cyclic group independently selected from the group consisting of ary Iowa which contains 6, 10, 12 or 14 carbon atoms and 1 to 3 rings, cycloalkyl group which contains 3 to 15 atoms carbon and from 1 to 3 rings, and is optionally fused to a benzene ring, and a heterocycle with up to 15 ring atoms and 1 to 3 rings which contains at least one heteroatom selected from -O-, -S-, -SO- , -S0₂-, = N- and -NH- and optionally contains one or more double bonds, and optionally includes one or more aromatic rings, this cyclic group being optionally singly or multiply substituted with: -NH₂, -C0₂H, -Cl, -F, -Br, -J, -N0₂, -CN, = 0, -OH, -perfluoro-C, ₃alkyl, O or -Q; a / \

CH₂\ / o every Ar₂ is independently selected from the following groups in which any ring may be optionally singly or multiply substituted with -Q, and -Q₂:

<img file="PL185693B1_D0009.tif" />

185 693

<img file="PL185693B1_D0010.tif" />

each X is independently = N- or -CH- and each Y is independently -O- or -S-;

each Qj is independently selected from the group consisting of -Ar, -O-Ar_1; -R, -T ^ R, and (CH₂) ". ^ - T.-R ,; .

each of Q₂ is independently selected from the group consisting of -OH, -NH₂, -WHAT₂H, -Cl, -F, Br, -J, -NO₂, -CN, -CF₃ and O / \ ch₂\ / o provided that when -Αη is substituted by the group Q, which contains one or more additional groups -Ar, these additional groups -Αη are not substituted by Q_P.

Preferred compounds of embodiment E of the invention include, but are not limited to, compounds of the formulas:

<img file="PL185693B1_D0011.tif" />

185 693

<img file="PL185693B1_D0012.tif" />

185 693

Preferred compounds of embodiment E according to the invention are those in which A is independently selected from the group consisting of α-amino acids: alanine, histidine, lysine, phenylalanine, proline, tyrosine, valine, leucine, isoleucine, glutamine, methionine, homoproline, 3- ( 2-thienyl) alanine; and 3- (3-thienyl) alanine.

The compounds according to a further embodiment of the invention (F) are represented by the formula δ:

<img file="PL185693B1_D0013.tif" />

wherein:

R is R.₅- (A)_p-;

R₅ is selected from the group consisting of:

-H,

-Ar "

-CO- Ai !,

-SO₂- Ar ,,

-CO-Rg,

-CO-OR ,,

-SO₂-Rg, / Ar,

-CO-N \ Ro, / Ar, so₂-n \ R, o,

-CO-N \ Rio&gt; i

-so₂-n \ R1 (b each A is independently selected from the group consisting of any a-amino acid;

p is 2 or 3;

each Rg is a straight or branched C 1-4 alkyl group optionally singly or multiply substituted with -OH, -F, or = O and optionally substituted with one Ar ,;

each R_l0 is independently selected from the group consisting of -H or straight or branched C,.₆alkyl;

each T, is independently selected from the group consisting of -CH = CH-, -O-, -S-, -S0-;

each Ar, is a cyclic group independently selected from the group consisting of ary I that contains 6, 10, 12, or 14 carbon atoms and from 1 to 3 rings, a cycloalkyl group that has from 3 to 15 carbon atoms and from 1 to 3 rings, and optionally fused to a benzene ring, and a heterocyclic group having 5 to 15 ring atoms and 1 to 3 rings which contains at least one heteroatom selected from -O-, -S-, -SO-, -SO₂-, = N- and -NH- and optionally includes one or more double bonds, and optionally includes one or more aromatic rings, wherein

185 This cyclic group is optionally singly or multiply substituted with: -NH₂, -WHAT₂H, -Cl, -F, -Br, -J, -NO₂, -CN, = 0, -OH, -perfluoro-C] ₃alkyl, O or Qi;

/ \ ch₂ \ / about

each Ar₂ is independently selected from the following groups, wherein any ring may be optionally singly or multiply substituted with - (/ and -Q₂:

<img file="PL185693B1_D0014.tif" />

<img file="PL185693B1_D0015.tif" />

(kk)

<img file="PL185693B1_D0016.tif" />

each Q, is independently selected from the group consisting of:

- Αη,

- O-Atj,

- R ,,.

- Tj-Rg i

- (CH2 -T.-R6;

each of Q₂ is independently selected from the group consisting of -OH, -NH, and -CO₂H, -Cl, -F, Br, -J, -N0₂, -CN, -CF₃ i, O '/ \ ch₂.

\ / ' about

provided that when -Ar! is substituted with the group Q_]; that contains one or more additional groups Αη, these additional groups - Αη are not substituted with (/;

each X is independently selected from the group consisting of = N- and = CH-, and each Y is independently selected from the group consisting of -O-, -S-, and -NH.

Preferred embodiments F of the invention include, but are not limited to, compounds of the formulas:

<img file="PL185693B1_D0017.tif" />

185 693

<img file="PL185693B1_D0018.tif" />

185 693

<img file="PL185693B1_D0019.tif" />

Preferred compounds of embodiment F of the invention are those in which A is independently selected from the group consisting of the α-amino acids: alaninine, histidine, lysine, phenylalanine, proline, tyrosine, valine, leucine, isoleucine, glutamine, methionine, homoproline, 3- ( 2-thienyl) alanine and 3- (3-thienyl) alanine.

The compounds of the invention have a molecular weight less than or equal to about 700 Daltons, and more preferably between about 400 and 600 Daltons. Such beneficial compounds can be readily absorbed into the patient's bloodstream after oral administration. Such oral availability makes the compounds excellent agents for oral administration in the treatment or prevention of IL-1 related diseases.

The ICE inhibitors of the invention can be synthesized using conventional methods. Preferably, these compounds are conveniently synthesized from readily available starting materials.

Among the known ICE inhibitors, the compounds according to the invention are among the most easily produced. ICE inhibitors described so far often contain four or more chiral centers and multiple peptide bonds. The relative ease with which the compounds of the invention can be synthesized offers great advantages for their commercial production.

It should be understood that the compounds of the invention may exist in a variety of equivalent forms depending on the conditions including the choice of solvent, pH, and others known to those skilled in the art. All such forms are expressly within the scope of the present invention. In particular, many of the compounds of the invention, especially those which contain aldehyde or ketone groups in R.₃ and the carboxylic acid groups on T, can take the hemi-ketal (or hemi-acetal) form or the hydrated form as shown below:

(EQ1)

O / On

Rr ~ N-Xioh

H \ (O-Dg-e-Rn

OH hydrated form

O (CJ ^ mC ^ He

Ri-N-Xi

H \ (at) gCR₁₃

ABOUT

<img file="PL185693B1_D0020.tif" />

Ri-N-Xi H '(CH) g-C-Ri3 04 hemi-ketal or hemi-acetal

185 693

Depending on the choice of the solvent and other conditions known to the person skilled in the art, the compounds of the invention may also exist in the acyloxyketal, acyloxyacetal, ketal or acetal form:

(EQ2) o

(CJ2) nr-Ck 'Uri

Ri-Xi

H. ^x '0 (OOg — C — Rn OR

Rf — N-Xi

H '

Ri-H-Xi

H '

OR I (Ofe) g — ę — Rn

Acyloxyketal or

Ketal

OR or acyloxyacetal acetal

Furthermore, it should be understood that the equivalent forms of the compounds of the invention may include tautomeric forms. All such forms are expressly included within the scope of the present invention.

It should be understood that the compounds of the invention may be modified with appropriate functional groups to increase selective biological properties. Such modifications are known in the art and include methods of increasing biological penetration into a given biological system (e.g. blood, lymphatic system, central nervous system), increasing oral availability, increasing solubility to enable administration by injection, altering metabolism, or changing secretion rate. Furthermore, the compounds of the invention can be converted into prodrugs such that the desired compound is formed in the body of the patient as a result of a metabolic or other biochemical process acting on the prodrug. Examples of such prodrug forms are the ketal, acetal, oxime and hydrazone of compounds that contain ketone or aldehyde groups, especially when they are in the R group.₃ compounds of the invention.

The compounds of the invention are excellent ligands for ICE. Thus, these compounds are capable of target inhibiting the processes in IL-1 mediated diseases such as the conversion of the IL-1β precursor to mature IL-β, and ultimately inhibiting the activity of this protein in inflammatory diseases, autoimmune and neurodegenerative diseases. For example, compounds of the invention inhibit the conversion of the IL-Ιβ precursor to mature IL-β by inhibiting ICE. Since ICE is essential in the production of mature IL-1, inhibition of this enzyme is effective in blocking the onset, effects, and physiological symptoms associated with IL-1 by preventing the production of mature IL-1. Thus, by inhibiting IL-β precursor activity, the compounds of the invention are effective as IL-1 inhibitors.

The compounds of the invention can be used in conventional manner to treat IL-1 mediated diseases. Treatment methods, dosage levels, and requirements can be selected by one skilled in the art from methods and techniques available to them. For example, a compound of the invention can be combined with a pharmaceutically acceptable excipient and administered to a patient suffering from an IL-1 related disease in a pharmaceutically acceptable manner and in an amount effective to ameliorate the condition of the disease.

Alternatively, the compounds of the invention can be used in compositions and methods for treating or preventing IL-1 related diseases in humans over an extended period of time.

The compounds in such compositions may be used alone or together with other compounds of the invention in a manner compatible with the conventional use of ICE inhibitors in pharmaceutical compositions. For example, a compound of the invention can be combined with pharmaceutically acceptable adjuvants used for vaccines and administration

185 693 should be administered in a prophylactically effective amount to protect the patient from IL-1 related diseases over an extended period of time.

Compounds of the invention may also be co-administered with other ICE inhibitors to increase the effectiveness of treatment or prophylaxis against various IL-1 related diseases.

In addition, the compounds of the invention may be used in combination with other conventional anti-inflammatory agents or with matrix metalloprotease inhibitors, lipoxygenase inhibitors, and antagonists of cytokines other than IL-β.

To prevent or combat the symptoms of IL-1 related diseases such as inflammation, the compounds of the invention can also be administered in combination with immunomodulators (e.g. bropyrimine anti-human interferon alpha, IL-2, GM-CSF, methionine enkephalin, interferon alpha, diethyl dithiocarbamate, tumor necrosis factor, naltrexone and rEPO), or with prostaglandins.

When the compounds of the invention are administered in combination therapy with other agents, they may be administered sequentially or simultaneously to the patient. Alternatively, the pharmaceutical or prophylactic compositions of the invention may contain a combination of an ICE inhibitor of the invention and another therapeutic or prophylactic agent.

The pharmaceutical compositions of the invention include any of the compounds of the invention, pharmaceutically acceptable salts thereof, and any pharmaceutically acceptable carrier, excipient or diluent. Pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated fatty acids of vegetable origin, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polypropylene-blocking polymers , polyethylene glycol and lanolin.

The pharmaceutical compositions of the invention may be administered orally, parenterally, by inhalation inhalation, topically, rectally, nasally, buccally, vaginally and via an implanted reservoir. Oral administration is preferred. The pharmaceutical compositions of the invention may contain any conventional non-toxic pharmaceutically acceptable carriers, excipients and diluents. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection and infusion.

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous or oleaginous suspension. The suspension is prepared according to methods known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) or suspending agents. The sterile injectable preparation may also be in the form of injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable diluents and solvents, mannitol, water, Ringer's solution, and isotonic sodium chloride solution can be mentioned. In addition, it is preferred to use sterile, liquid oils as a solvent or suspending medium. For this purpose any bland liquid oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are suitable for the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated oils.

185 693 characters. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as Ph. Helv. or the like.

The pharmaceutical compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, suspensions, and aqueous solutions. In the case of tablets for oral administration, common carriers can be used, including lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. In capsules for oral administration, suitable diluents include lactose and dry corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. Some sweetening and / or flavoring agents and / or coloring agents can also be added if desired.

The pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration. Such compositions are prepared by mixing a compound of the invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and thus will melt in the rectum to release the active ingredient. Such excipients include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

When the desired treatment involves sites or organs readily accessible by topical application, the compositions of the present invention for topical administration are particularly useful. For topical application to the skin, the pharmaceutical compositions of the invention should be formulated as a suitable ointment containing the active compounds suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene-polyoxypropylene compound, emulsifying wax, and water. Alternatively, the pharmaceutical compositions may be formulated as suitable lotions or creams containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan mono stearate, polysorbate 60, cetyl ester waxes, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the invention may also be applied topically to the lower intestinal tract in the form of rectal suppositories or enema preparations. Topical transdermal patches are also within the scope of the invention.

The pharmaceutical compositions of the present invention may be administered by nasal spray or inhalation. Such compositions are prepared by methods well known in the art and may be formulated in saline solutions using benzyl alcohol or other suitable preservatives, bioavailability enhancing absorption promoters, fluorocarbons and / or other art-known solubilizing or dispersing agents.

IL-1 related diseases that can be treated or prevented using the compounds of the invention include, but are not limited to, inflammatory, autoimmune, and neurodegenerative diseases.

Inflammatory diseases that may be treated or prevented include, for example, septic shock, sepsis, and adult respiratory distress syndrome. Autoimmune diseases include, for example, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin-dependent diabetes autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, and sclerosis. scattered. The target neurodegenerative diseases include, for example, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and primary lateral sclerosis. The ICE inhibitors of the invention may also be used as wound healing aids. Finally, the ICE inhibitors of the invention can also be used to treat infectious diseases.

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While the present invention focuses on the use of the compounds disclosed herein for the treatment and prevention of IL-1 related diseases, the compounds of the invention may also be used as inhibitors of other cysteine ​​proteases.

The compounds of the invention are also useful as commercial reagents that bind effectively to ICE or other cistern proteases. As commercial reagents, the compounds of the invention, and their derivatives, may be used to block the proteolysis of a given peptide or may be derivatized to bind to a stable resin as binding substrates for use in affinity chromatography. These and other uses, characteristic of commercially available cysteine ​​protease inhibitors, will be apparent to one skilled in the art.

For a more complete understanding of the present invention, the following examples are provided below. These examples are intended to be illustrative only and are not intended to limit the scope of the invention in any way.

Example 1

The following example shows how to design a compound that is encompassed by the present invention:

Step 1) The 2 hydrogen bonding residues in ICE, here, the C = O backbone and NH in Arg-341 are selected.

Step 2) The scaffold is selected, here a pyridone derivative, ensuring that the hydrogen bonding moieties in the scaffold are capable of forming satisfactory hydrogen bonds with the hydrogen bonding moieties selected in step 1. Such confirmation is achieved using molecular mechanics techniques to minimize the fragment scaffolding in the context of the ICE active center:

(Z1)

<img file="PL185693B1_D0021.tif" />

Step 3) Select the hydrophobic cavity here S2 as the next target and the hydrophobic moiety here benzene. To obtain a hydrophobic overlap, the benzene group within the cavity of S2 is minimized.

<img file="PL185693B1_D0022.tif" />

Step 4) The next hydrophobic cavity here S4 is selected as the next target and the hydrophobic moiety here benzene. The benzene group within the S4 cavity is minimized to obtain a hydrophobic overlap.

(Z3)

<img file="PL185693B1_D0023.tif" />

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Step 5) The S1 polar cavity is filled with an electronegative residue, here with a carboxylate side chain provided by aspartic acid, in which the C-terminus has been reduced to the aldehyde. Minimized to ensure that the carboxylate side chain is in favorable electrostatic interaction with the polar S1 cavity

<img file="PL185693B1_D0024.tif" />

Step 6) Binds the scaffold to the residues of steps 3, 4 and 5, preferably using the minimum number of bonds consistent with a chemically justified structure. The entire complex molecule in the active center of ICE is reduced.

and²⁵)

<img file="PL185693B1_D0025.tif" />

Step 7) The energy of the molecule is calculated when it has the conformation necessary to bind to ICE. Then the energy is minimized and recalculated - this is the conformational energy of a free compound. The strain energy of binding of a potential inhibitor to ICE is the difference between the conformational energy of the free compound and the conformational energy of the bound compound. The stress energy should be less than about 0.0418 kJ / mol. When the conformational energy of the bond is -0.0067 kJ / mol and the conformational energy of the free compound is -0.0498 kJ / mol, the stress energy is 0.0422 kJ / mol.

Step 8) An inhibitor designed according to the above steps was prepared having a K i value of 150 nM.

Example 2

The inhibition constants (KJ and IC₅₀ for several compounds of the invention.

1. Enzyme test with UV visible substrate

The study was conducted with the succinyl-Tyr-Val-Ala-Asp-p-nitroanilide substrate. The synthesis of analogous substrates was described by LA Reiter (Int. J. Peptide Protein Res. 43, 87-96 (1994). The test mixture contained:

μΐ buffer (10 mM Tris, 1 mM DTT, 0.1% CHAPS @ pH 8.1) μΐ ICE (50 nM final concentration, rate 1 mOD / min) μΐ DMSO / inhibitor mixture μΐ 400 μΜ substrate (final concentration 80 μΜ )

100 μΐ total volume of the reaction mixture.

The visible ICE assay was performed in a 96-well microtiter plate. Buffer, ICE, and DMSO (when inhibitor is present) were added to the wells in that order. Compounds were incubated at room temperature for 15 minutes, starting when all compounds were placed in all wells. The microtiter plate reader was set to the incubation temperature of 37 ° C. After 15 minutes of incubation, substrate was added directly to the wells and the reaction monitored by chromophore (pNA) release study at 405-603 nm at 37 ° C for 20 minutes. A linear data plot was drawn up and the rate was calculated in mOD / min. DMSO was used only in the inhibitor studies and buffer was used to bring the volume up to 100 µL in the remaining experiments.

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2. Enzymatic studies with a fluorescent substrate

The study was conducted essentially according to Thomberry et al. (Nature 356. 768-774 (1992)) using substrate 17 presented in this article. This substrate is Acetyl-TyrVal-Ala-Asp-amino-4-methyl coumarin (AMC).

The following ingredients were mixed:

μΐ buffer (10 mM Tris, mM DTT,

0.1% CHAPS @pH 8.1) 1 μΐ ICE (2-10 nM final concentration) μΐ DMSO solution / inhibitor μΐ 150 μΜ substrate (30 μΜ final concentration)

100 μΐ total volume of the reaction mixture.

The assay was carried out in a 96-well microtiter plate. Buffer and ICE were added to the wells. Compounds were allowed to incubate at 37 ° C for 15 minutes while controlling the temperature in the wells. After 15 minutes of incubation, the reaction was started by direct addition of substrate to the well and monitored at 37 ° C for 30 minutes by AMC fluorophore release using an excitation wavelength of 380 nm and an emission wavelength of 460 nm. A linear plot of the data was made for each well and the rate was determined in units of fluorescence per second.

To determine the enzyme inhibition constant (KJ or inhibition mode (competitive, non-competitive and non-competitive), data obtained from enzyme studies with varying inhibitor concentrations were computer fitted to standard enzyme kinetics equations (see H. Segel, Enzyme Kinetics, Wiley-Interscience, 1975) .

3. Cell research

IL-1 β studies using mixed populations of human peripheral blood mononuclear cells (PBMCs) or enriched adherent mononuclear cells

The processing of pre-IL-Ιβ by ICE can be measured in cell culture using a variety of cell sources. Human PBMCs from healthy donors have produced a mixed population of lymphocyte subtypes and mononuclear cells that produce multiple interleukins and cytokines in response to many types of physiological stimulators. An enriched source of normal monocytes for selective studies of cytokine production by activated cells was obtained from adherent mononuclear cells from PBMC.

Experimental procedure:

A series of initial dilutions of test compounds in DMSO or ethanol were prepared, with serial dilutions in RPMI-10% FBS medium (containing 2 mM Lglutamine, 10 mM HEPES, 50 U, and 50 µg / ml pen / strep), respectively, to obtain drugs at 4x final test concentration containing 0.4% DMSO or 0.4% ethanol. Final DMSO concentration is 0.1% for all diluted media. A concentration titer, which includes a putative K value for a test compound as determined by the ICE inhibition assay, is generally used for primary compound screening.

Typically, compounds were tested at 5-6 dilutions and the cellular component of the assay was run in duplicate, with duplicate ELISA determinations for each cell culture supernatant.

PBMC isolation and IL-1 assay

The buffy coat cells were isolated from 0.568 liters of human blood (yielding 40-45 ml final plasma volume with cells), diluted to 80 ml with medium and 10 ml of the cell suspension was applied to each Leuko-PREP separation tube (Becton Dickinson). After 15 minutes of centrifugation at 1500-1888 xg, the plasma / medium layer was aspirated, and then the mononuclear cell layer was collected with a pasteur pipette and transferred to a 15 ml conical centrifuge tube (Corning). Medium was added to bring the volume to 15 ml, cells were gently mixed by inversion and centrifuged at 300 xg for 15 minutes. The PBMC pellet was resuspended in a small volume of medium, cells were counted and diluted to 6 x 10 cells / ml.

For the cell assay, 1.0 mL of cell suspension, 0.5 mL of test compound dilution was added to each well of a 24-well flat bottom culture plate (Corning).

185 693 ku and 0.5 ml LPC solution (Sigma # L-3012; 20 ng / ml solution made in complete RPMI medium; final LPS concentration 5 ng / ml). Usually, the addition of 0.5 ml of the test compound and LPS is sufficient to mix the contents of the wells. Controls were performed with three mixes per test, with LPS alone, a solvent-vehicle control, and / or additional medium to bring the final culture volume to 2.0 mL. Cell cultures were incubated for 16-18 hours at 37 ° C in the presence of 5% CO₂.

At the end of the incubation period, cells were harvested and transferred to 15 ml conical centrifuge tubes. After centrifugation for 10 minutes at 200 xg, the supernatants were collected and transferred to 1.5 Eppendorf tubes. It should be noted that cell pellets can be used for biochemical studies of the pre-IL--β and / or mature IL-β content in cytosolic extracts by western blotting or ELISA with antisera specific for pre-IL-β.

Isolation of Adherent Mononuclear Cells PBMCs were isolated and prepared as described above. Medium (1.0 ml) was added to the wells first, followed by 0.5 ml of the PBMC suspension. After 1 hour of incubation, the plates were gently shaken and non-adherent cells were aspirated from each well. The wells were then washed gently three times with 1.0 ml of medium and finally resuspended in 1.0 ml of medium. The enrichment for adherent cells was 2.5-3.0 x 10⁵ cells per well. The addition of test compounds, LPS, cell incubation conditions and processing of the supernatants were performed as described above.

ELISA

The Qantikine kit (R&amp;D Systems) was used to measure mature IL-1 β. The test was performed according to the manufacturer's recommendations. Mature IL-1β levels of about 1-3 ng / ml were observed in both PBMC and positive adherent mononuclear cells. ELISA studies were performed with 1: 5, 1:10 and 1:20 dilutions of the positive control supernatants to select the optimal dilution for the supernatant in the test panel.

The inhibitory capacity of the compounds is shown in IC values₅₀which is the inhibitor concentration at which 50% of the mature IL-1β is detected in the supernatant compared to the positive control.

Using the tests described, the following K, and IC values ​​were determined₅₀ for compounds A to N. The formulas of compounds A to N are shown in the table below

Compound Κ, (μΜ, in the indicated studies)

<td></td><td>UV-visible K, μΜ</td><td>Fluorescence Kj μΜ</td><td>IC cells₅₀(gM)</td>

<td>AND</td><td> 5.5</td><td></td><td> 25.0</td>

<td>B</td><td> 8.6</td><td></td><td> 20.0</td>

<td>C.</td><td> 10</td><td></td><td> &gt;30</td>

<td>D</td><td> 4.7</td><td></td><td></td>

<td>E.</td><td> 3.2</td><td></td><td></td>

<td>F.</td><td> 0.15</td><td></td><td> 2-4</td>

<td>G.</td><td> 4.8</td><td></td><td></td>

<td>H.</td><td> 0.023</td><td> 0.0047</td><td> 6-11</td>

<td>AND</td><td> 0.0072</td><td> 0.0052</td><td> 2.6</td>

<td>J.</td><td> 0.012</td><td> 0.0039</td><td> 5-7</td>

<td>K.</td><td> 0.010</td><td> 0.002</td><td> 2- 11</td>

<td>L.</td><td> 0.014</td><td></td><td></td>

<td>M.</td><td> 0.15</td><td></td><td></td>

<td>N</td><td> 0.95</td><td></td><td></td>

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Compound formulas A to N;

<img file="PL185693B1_D0026.tif" />

<img file="PL185693B1_D0027.tif" />

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<img file="PL185693B1_D0028.tif" />

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<img file="PL185693B1_D0029.tif" />

Example 3

The compounds of Example 2 were synthesized as follows:

H. N- (N-Acetyl-tyrosinyl-valinyl-pipecolyl) -3-amino-4-oxobutanoic acid

Step A. N- (tert-butoxycarbonylpipecolyl) -4-amino-5-benzyloxy-2-oxotetrahvdrofuran

The reaction of N-tert-butoxycarbonylpipecolinic acid (460 mg, 2.0 mmol) and N-allyloxycarbonyl-4-amino-5-benzyloxy-2-oxotetrahydrofuran (530 mg, 1.82 mmol) was carried out analogously to that described by Chapman (Bioorg . &amp; Med. Chem. Lett. 1992, 2, 613-618) to obtain 654 mg of the title compound.

1 H NMR (500 MHz, CDCl₃) (in the form of rotamers)) δ 7.35 (m, 5H), 6.88 (br s, 1H), 4.9-4.45 (m, 4H), 3.95 (br m, 2H) , 3.06 (m, 1H), 2.9 (m, 1H), 2.7 (br m, 1H), 2.45 (m, 1H), 2.2 (m, 1H), 1.7 -1.5 (m, 3H), 1.45 (two s, 9H).

Step B. N-pipecolyl-4-amino-5-benzyloxy-2-oxotetrahydrofuran

N- (N-tert-butoxycarbonylpipecolyl) -4-amino-5-benzyloxy-2-oxo-tetrahydrofuran (654 mg) was dissolved in 15 mL of 25% trifluoroacetic acid in dichloromethane and stirred at room temperature. The mixture was concentrated to give a gummy residue. This residue was dissolved in dichloromethane and washed with 10% sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give 422 mg of the title compound as a beige solid.

Ή NMR (500 MHz, CDC1₃) δ 7.38 (m, 5H), 7.15 (d, 1H), 5.55 (d, 1H), 4.95-4.8 (m, 1H), 4.78 (m, 1H) , 4.65 (d, 1H), 4.45 (m, 1H), 3.2 (m, 0.5H), 3.05 (m, 0.5H), 2.95 (m, 0.5H ), 2.85 (m, 0.5H), 2.65 (m, 1H), 2.55-2.38 (m, 1H), 1.95 (m, 1H), 1.8 (m, 1H), 1.6 (m, 2H), 1.38 (m, 2H).

Step C. N-iN-acetyl-tyrosinyl-valinyl-pipecolyl) -4-amino-5-benzyloxy-2-oxotetrahydrofuran

N-acetyl-tyrosinyl-valine (464 mg, 1.44 mmol) and N-pipecolyl-4-amino-5-benzyloxy-2-oxotetrahydrofuran (412 mg, 1.3 mmol) were dissolved in 5 ml of dimethylformamide and 5 ml of dichloromethane and cooled to 0 ° C. 1-Hydroxybenzotriazole (HOBT; 210mg, 1.56mmol) was added to the cooled solution followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC; 326mg, 1.7mmol). After stirring for 18 hours, the mixture was diluted with ethyl acetate and washed with water, 10% sodium hydrogen sulfate solution, 10% sodium bicarbonate solution and water. The organic layer was concentrated to give a crude solid which was purified by flash chromatography (SiO₂), eluting with a mixture of dichloromethane: isopropanol: pyridine, 94: 6: 1, to give 370 mg of the title compound.

1 H NMR (500 MHz, CD₃OD) (both diastereomers and rotamers)) δ 7.35 (m, 5H), 7.05 (m, 2H), 6.68 (m, 2H), 5.65 &amp; 5.25 ( m, 1H), 4.9-3.95 (m, 8H).

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3.4- 2.6 (m, 4H), 2.5-2.1 (m, 1H), 1.98 (s, 1H), 1.9 (s, 1H), 1.85 (s, 1H), 1.8-1.6 (m, 2H), 1.551.3 (m, 4H), 0.95-0.85 (m, 6H).

Stage D. N- (N-Acetyl-tvrosinyl-valinyl-nipekolyl) -3-amino-4-oxobutanoic acid

To a solution of 100 mg of N- (N-acetyl-tyrosinyl-valinyl-pipecolyl) -4-amino-5-benzyloxy-2-oxotetrahydrofuran in 10 ml of methanol was added 60 mg of Pd (OH)₂ on carbon and the mixture under a balloon under an atmosphere of hydrogen. The mixture was filtered through Celite and concentrated to give a white solid. This crude material was dissolved in 2 mL of methanol and triturated with Iowa diethyl ether to give 26 mg of the title compound.

1 H NMR (500 MHz, CD₃OD) (both diastereomers and rotamers)) δ 7.1 (m, 2H), 6.7 (m, 2H), 5.2 (br m, 1H), 4.8-3.6 (m, 6H), 3.2-3.5 (m, 4H), 2.52.1 (m, 1H), 1.95 (three s, 3H), 1.9-1.3 (m, 6H), 1.1-0.7 (m, 6H).

The following compounds were prepared in a manner analogous to that described for compound H:

J. N- [N-Acetyl-tvrosinyl-valinyl) - (4-hydroxyprolinyl) 1-3-amino-4-oxobutanoic acid

N-tert-butoxycarbonyl-4-benzyloxyproline was used instead of N-tert-butoxycarbonylpipecolinic acid.

L. N- [2- (N-Acetyl-tetrazyl-valinyl) - (S) -1,2,3,4-tetrahyldroisoquinoline-3-carbonyl-3-amino-oxobutanoic acid

N-tert-butoxycarbonylpipecolinic acid was replaced with (S) -N-tert-butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

I. N- (N-acetyl-tyrosinyl-valinyl) - (4-phenoxyprolinyl)) - 3-amino-4-oxobutanoic acid

Step A. N-tert-butoxycarbonyl-4-phenoxyproline methyl ester

To a cooled (0 ° C) solution of N-tert-butoxy-cis-4-hydroxyproline (2.0 g, 8.15 mmol), phenol (0.77 g, 8.15 mmol) and triphenylphosphine (2.14 g , 8.15 mmol) in 20 mL of tetrahydrofuran was added dropwise over 30 minutes to diethyl azodicarboxylate (1.4 mL, 9 mmol). After stirring at room temperature for 16 hours, the reaction mixture was concentrated to a sticky residue. The crude residue was purified by flash chromatography (SiO₂), eluting with a 3: 7 mixture of ethyl acetate and hexane to give 1.89 g of the title compound.

1 H NMR (500 MHz, CDCl₃) δ 7.3 (m, 2H), 6.95 (m, 1H), 6.85 (d, 2H), 4.9 (br m, 1H), 4.55-4.15 (m, 2H ), 3.88-3.65 (m, 1H), 3.70 (s, 3H), 2.58 (m, 1H), 2.22 (m, 1H), 1.4 (3xs, 9H ).

Step B. 4-phenoxyproline methyl ester hydrochloride

A cooled solution (ice bath) of N-tert-butoxycarbonyl-4-phenoxyproline methyl ester (0.6 g) in 20 mL of ethyl acetate was bubbled with anhydrous hydrogen chloride until saturation. The mixture was warmed to room temperature and stirred for 3 hours then concentrated to give 480 mg of the title compound.

1 H NMR (500 MHz, CDCl₃) δ 7.22 (m, 2H), 6.95 (m, 1H), 6.83 (m, 2H), 5.1 (br., 1H), 4.6 (br m, 1H), 4 .06 (br m, 1H), 3.75 (s, 3H), 3.55 (brm, 1H), 2.58 (m, 2H).

Step C. N-acetyl-tyrosinyl-valinyl-f 4-phenoxy) proline methyl ester

N-acetyl-tyrosinyl-valine (0.524 g, 1.63 mmol) and 4-phenoxyproline methyl ester (0.381 g, 1.48 mmol) were dissolved in 4 mL of dimethylformamide and 4 mL of dichloromethane and cooled to 0 ° C. To the cooled solution was added diisopropylethylamine (258 µΐ, 1.86 mmol), HOBT (0.24 g, 1.78 mmol), and EDC (0.37 g, 1.92 mmol) and the reaction mixture was stirred for 18 hours. The mixture was diluted with 400 mL of ethyl acetate and washed with water, 10% sodium hydrogen sulfate solution, 10% sodium bicarbonate, and water. The organic layer was concentrated to a residue which was purified by flash chromatography (SiO₂), eluting with a mixture of CH₂Cl₂: I-PrOH: pyridine, 94: 6: 1 to give 360 ​​mg of the title compound.

1 H NMR (500 MHz, CDCl₃ (occurs in the form of rotamers)) δ 7.3 (m, 2H), 7.05 (m, 1H), 6.95 (d, 2H), 6.9-6.2 (4 xd, 4H), 5 .05 (br s, 1H), 4.7-3.94 (m, 5H), 2.93 (m, 1H), 2.82 (m, 1H), 2.65 (m, 1H), 2 . 2 (m, 1H), 2.05 (m, 1H), 1.95 (s, 3H), 1.86 (m, 1H), 0.98 (d, 3H), 0.88 (d, 3H).

Step D. N-acetyl-tyrosinyl-valinyl- (4-phenoxy) proline

To a solution of N-acetyl-tyrosinyl-valinyl- (4-phenoxy) proline methyl ester (360 mg, 0.685 mmol) in 8 ml of a mixture of tetrahydrofuran and water (1: 1) was added lithium hydroxide (57 mg,

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1.37 mmol) and the mixture was stirred at room temperature for 1 hour. The mixture was acidified with 10% hydrochloric acid to give a white solid which was collected to give 175 mg of the title compound.

Ή NMR (500 MHz, DMSO-d₆) δ 9.2 (br s, 1H), 8.05-7.95 (m, 2H), 7.3 (m, 1H), 7.06.9 (m, 4H), 6.65 (d , 2H), 4.42 (m, 1H), 4.35 (m, 1H), 4.05-3.95 (m, 2H), 3.3 (br s, 2H), 2.75 (m , 1H), 2.55-2.38 (m, 2H), 2.2 (m, 1H), 2.0 (m, 1H), 1.7 (s, 3H), 0.95 (d, 3H). 0.85 (d, 3H).

Step E. N- [N-acetyl-tyrosinyl-valinyl- (4-phenoxy) prolinyl-4-amino-5-benzyloxy -2-oxotetrahydrofuran

The title compound was prepared as described for H, step A, by reacting N-acetyl-tyrosinyl-valinyl- (4-phenoxy) proline and N-allyloxycarbonyl-4-amino-5-benzyloxytetrahydrofuran.

Ή NMR (500 MHz, CDC1₃ (present in the form of a diastereomeric mixture of 1: 1 hemiacetal)) δ 7.8-6.3 (m, 17H), 5.6 (d, 1H), 5.1-4.15 (m, 5H), 4 15-3.75 (m, 2H), 2.952.15 (m, 5H), 2.15-1.95 (m, 1H), 1.9-1.85 (2xs, 3H), 1.1 -0.75 (m, 6H).

Step F. N - [(N-acetyl-tYrosinyl-valinyl) - (4-phenoxy) prolinyl-1-3-amino-4-oxobutanoic acid

The title compound was prepared by the hydrogenolysis method described for compound E in step D.

Ή NMR (500 MHz, CD₃OD (present as a diastereomeric mixture of 1: 1 hemiacetal)) δ 7.25 (m, 2H), 7.10-6.85 (m, 5H), 6.65 (d, 2H), 5.1 (br m, 1H), 4.65-4.05 (m, 5H), 4.0-3.40 (m, 2H), 2.95-2.35 (m, 5H), 2.25 (m, 1H), 2.05 (m, 1H), 1.85 (s, 3H), 1.0 (d, 3H), 0.95 (d, 3H).

K. Acid No. (N-acetyl-tetrozvnyl-valinyl) - (4-benzyloxy) prolinyl-3-amino-4-oxobutanoic acid

Step A. Semicarbazone of N- (N-allyloxycarbonyl-4-benzyloxvprolinyl) -3-amino-4-oxobutanoic acid tert-butyl ester

The title compound was prepared by reaction of N-allyloxycarbonyl-4-benzyloxyproline and 3-amino-4-oxobutanoic acid tert-butyl ester semicarbazone (TL Graybill et al., Abstract of papers, 206th National Meeting of the American Chemical Society, Abstract MEDI-235 Chicago, IL (1993)) under similar peptide coupling conditions as described above (compound H, step C).

Ή NMR (500 MHz, CDC1₃) δ 9.05 (br s, 1H), 7.85 (br m, 1H), 7.4-7.2 (m, 5H), 7.15 (br s, 1H), 6.55 (br s, 1H), 5.9 (m, 1H), 5.1-4.9 (br m, 2H), 4.65-4.4 (m, 4H), 4.2 (br m, 1H) , 3.75-3.5 (m, 2H), 2.75-2.55 (m, 2H), 2.5 (br m, 1H), 2.25 (brm, 1H), 1.4 ( s, 9H).

Step B. Semicarbazone of N- (acetyl-tyrosinyl-valinyl- (4-benzYloxylprolinyl)) -3-amino-4-oxobutanoic acid tert-butyl ester

The title compound was prepared by reacting N-acetyl-tyrosinyl-valine and N- (N-allyloxycarbonyl-4-benzyloxyprolinyl) -3-amino-4-oxobutanoic acid tert-butyl ester semicarbazone under the reaction conditions described for compound H, step A.

Ή NMR (500 MHz, CD₃OD) δ 7.35-7.2 (m, 6H), 7.0 (d, 2H), 6.65 (d, 2H), 4.85 (m, 1H), 4.6-4.45 (m, 4H), 4.3 (br m, 1H), 4.15 (m, 1H), 3.7 (m, 1H), 2.95 (m, 1H), 2.75-2.6 (m, 3H), 2.35 (m, 1H), 2.1 (m, 1H), 1.9 (s, 3H), 1.4 (s, 9H), 0.95 (d, 3H) . 0.90 (s, 3H).

Step C. N- (N-Acetyl-Thyrosyl-valinyl- (4-benzyloxYprolinyl)) - 3-amino-4-oxobutanoic acid

N- (N-acetyl-tyrosinyl-valinyl- (4-benzyloxyprolinyl)) - 3-amino-4-oxobutanoic acid tert-butyl semicarbazone (270 mg) was dissolved in 10 ml of 25% trifluoroacetic acid in dichloromethane and stirred at room temperature for Three hours. The mixture was concentrated to a solid residue. This residue was dissolved in 10 ml of a mixture of methanol, acetic acid and 37% formaldehyde (3: 1: 1) and stirred at room temperature for 1 hour. The mixture was concentrated and the resulting residue was purified by flash chromatography (SiO₂), eluting with a mixture of dichloromethane / methanol / formic acid (100: 5: 0.5). 37 mg of the title compound were obtained.

185 693 H NMR (500 °, CD₃OD (exists as a 1: 1 diastereomeric hemiacetal mixture)) δ 7.4-7.25 (m, 5H), 7.0 (d, 2H), 6.65 (d, 2H), 4.65-4 .05 (m, 7H), 3.75-3.4 (m, 2H), 3.0-2.3 (m, 5H), 2.2-1.95 (m, 2H), 1.90 (d, 3H), 1.0 (d, 3H), 0.95 (d, 3H).

Example 4

Inhibition constants (K i) and IC values₅₀ Several compounds of the invention were prepared by enzyme studies using a UV visible substrate and by cell studies as described in Example 2. The following K, and IC values ​​were obtained in the indicated tests₅₀ for compounds 7a, 7b, 20a-d, 21c-f, 22e, 25, 28, 33a-c, 36a, 39, 43, 47a, 47b, 54a1, 63, 69a, 69b, 84a and 84 b. the corresponding letter designations of the compounds. Compound formulas are shown in Examples 2 and 5.

<td rowspan="2">Relationship</td><td colspan="2">Research</td><td rowspan="2">Relationship</td><td colspan="2">Research</td>

<td>UV-visible K, (μΜ)</td><td>Cells 1₅₀(μΜ)</td><td>UV-visible K, (μΜ)</td><td>Ι cells₅ο (μΜ)</td>

<td>7a</td><td> 35</td><td></td><td>47a</td><td> 0.019</td><td> 2.1</td>

<td>7b</td><td> 1.2</td><td></td><td>47b</td><td> 0.027</td><td> 1.8</td>

<td>20a (= Ę)</td><td> 3.2</td><td></td><td>54a (= F)</td><td> 0.15</td><td> 2.7</td>

<td>20b</td><td> 0.85</td><td> 16.4</td><td>54b (= M)</td><td> 0.15</td><td> 9.1</td>

<td>20c (= N)</td><td> 0,95</td><td></td><td>54c</td><td> 1.2</td><td> &gt;19.0</td>

<td>20d</td><td> 0,1</td><td> 6,2</td><td>54d</td><td> 1.0</td><td></td>

<td>21c</td><td> 0.64</td><td></td><td>54e</td><td> 3.5</td><td></td>

<td>21d</td><td> 0.24</td><td> 4.8</td><td>54f</td><td> 0.9</td><td></td>

<td>21e</td><td> 0,22</td><td> 2,9</td><td>54g (= G)</td><td> 4.8</td><td> &gt;20.0</td>

<td>21f</td><td> 0,17</td><td> 2,9</td><td>54h</td><td> 0.97</td><td></td>

<td>22e</td><td> 0.19</td><td></td><td>54i</td><td> 0.054</td><td> 2.4</td>

<td> 25</td><td> 6.2</td><td></td><td>54j</td><td> 0.28</td><td></td>

<td> 28</td><td> 12.0</td><td></td><td>54k</td><td> 0.085</td><td></td>

<td>33a (= A)</td><td> 5.5</td><td> 25.0</td><td> 541</td><td> 0.215</td><td> 7.0</td>

<td>33b (= C)</td><td> 10.0</td><td> &gt;30.0</td><td> 63 (=£»)</td><td> 0.85</td><td> 4.1</td>

<td>33c (= B)</td><td> 8.6</td><td> 20.0</td><td>69a (= R)</td><td> 0.011</td><td> 0.735</td>

<td>36a (= D)</td><td> 4.7</td><td></td><td>69b (= S)</td><td> 0.050</td><td> 0.745</td>

<td>36b</td><td> 0.8</td><td> 17.0</td><td>84a (= V)</td><td> 0.100</td><td> 3.3</td>

<td> 39</td><td> 2.5</td><td></td><td>84b (= W)</td><td> 0.019</td><td> 0.50</td>

<td> 43</td><td> 20.0</td><td></td><td></td><td></td><td></td>

Example 5

Compounds of Example 4 were synthesized as follows:

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<img file="PL185693B1_D0031.tif" />

<img file="PL185693B1_D0032.tif" />

<img file="PL185693B1_D0033.tif" />

3-Benzylamino-4-oxo-4,6,7,8-tetrahydro-pyrrolo [1,2-a] pyrimidine-6-carboxylic acid methyl ester (3).

A mixture of (4S) -2-amino-1-pyrroline-5-carboxylic acid ethyl ester hydrochloride (1.44 g, 2.38 mmol); prepared by analogy to the preparation of the methyl ester as described by Lee and Lown, J. Org. Chem. 52, 5717-21 (1987)); 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one (2.50 g, 2.31 mmol) and sodium methanol (0.12 g, 2.22 mmol) in ethanol (10 mL) was heated to reflux for 2 hours. The mixture was allowed to cool to room temperature and concentrated in vacuo. The residue was suspended in water and 1N sulfuric acid was added until the pH was 1. The aqueous mixture was extracted with dichloromethane, the organic layer was separated and concentrated in vacuo to give 0.6 g of an orange solid. After chromatography (fast, SiO₂, 60% ethyl acetate / hexane, gradually increasing the solvent gradient to 100% ethyl acetate then 10% methanol / dichloromethane) afforded 0.5 g of an orange solid. A mixture of this solid and potassium cyanide (0.03 g, 0.5 mmol) in methanol (10 ml) was heated to reflux overnight. After cooling, the reaction mixture was concentrated in vacuo to give a yellow solid. As a result of chromatography (fast, SiO₂40% ethyl acetate / hexane, gradual increase of the solvent gradient to 100% ethyl acetate) gave 0.22 g (31.6%) of the title compound:

Ή NMR (d₆-DMSO) δ 2.25 (m, 1H), 2.65 (m, 1H), 3.15 (m, 2H), 3.75 (s, 3H), 5.15 (dd, 1H), 7 . 5 (t, 2H), 7.6 (t, 1H), 7.95 (d, 2H), 8.6 (s, 1H), 9.5 (s, 1H).

(3S) - [(3-Benzoylamino-4-oxo-4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine-6-carbonyl) -amino] -4-oxobutanoic acid semicarbazone (5a and 5b)

A mixture of 3-benzoylamino-4-oxo-4,6,7,8-tetrahydro-pyrrolo [1,2-a] pyrimidine-6-carboxylic acid ethyl ester (3.22 g, 0.70 mmol) and hydrate lithium hydroxide (0.032 g, 0.76 mmol) in methanol (5 ml) and tetrahydrofuran (5 ml) were stirred for 18 hours at room temperature. The reaction mixture was concentrated to give 3-benzoylamino-4-oxo-4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine-6-carboxylic acid lithium salt (4) as a white solid. This material was used in the next reaction without further purification.

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A cooled to 0 ° C mixture of (3S) -amino-4-oxo-butanoic acid tert-butyl ester semicarbazone (0.163 g, 0.71 mmol; Graybill et al., Int. J, Protein Res., 44, pp. 173-83 (1994)) and 3-benzoylamino-4-oxo-4,6,7,8-tetrahydro-pyrrolo [1,2-a] pyrimidine-6-carboxylic acid lithium salt (4) in dimethylformamide (5 ml) and dichloromethane (5 mL) was treated with hydroxybenzotriazole (0.104 g, 0.77 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.148 g, 0.37 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 18 hours, then poured into water (50 ml) and extracted with ethyl acetate (2 x 50 ml). The combined organic layers were washed with 1M aqueous sodium hydrogen sulfate solution, diluted with aqueous sodium bicarbonate (50 ml) and saturated aqueous sodium chloride. The layer was concentrated in vacuo to give 0.43 g of a yellow solid. As a result of chromatography (fast, SiO₂, ammonium hydroxide / methanol / dichloromethane (1: 1: 99, step gradient, to 1:10:90) gave 0.11 g (30.9%) of the diastereomer with the higher R f (5a): 1 H NMR (500 MHz, CD₃OD) δ 1.45 (s, 9H), 2.29-2.35 (m, 1H), 2.6-2.7 (m, 2H), 2.8 (dd, 1H), 3.1 -3.15 (m, 1H), 3.2-3.3 (m, 1H), 4.9-4.95 (m, 1H), 5.2 (dd, 1H), 7.25 (d , 1H), 7.5-7.55 (m, 2H), 7.557.6 (m, 1H), 7.95 (d, 2H), 8.9 (s, 1H) and 0.11 g (30 , 9%) of the lower Rf diastereomer (5b): Ή NMR (500 MHz, CD₃OD) δ 1.45 (s, 9H), 2.3-2.4 (m, 1H), 2.6-2.7 (m, 1H), 2.7-2.8 (m, 2H) , 3.1-3.15 (m, 1H), 3.2-3.3 (m, 1H), 4.85-4.95 (m, 1H), 5.15 (dd, 1H), 7 , 25 (d, 1H), 7.55 (t, 2H), 7.95 (d, 2H), 8.9 (s, 1H). Diastereomers 5a and 5b were obtained separately.

(3S) - [(3-Benzoylamino-4-oxo-4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine-6-carbonyl) -amino] -4-oxobutanoic acid (7a). Semicarbazone suspension of (3S) - [(3-Benzoylamino-4-oxo-4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine-6-carbonyl) -amino] acid tert-butyl ester] 4- oxo-butanoic acid (5a, 0.11 g, 0.22 mmol) in dichloromethane (7.5 mL) and trifluoroacetic acid (2.5 mL) was stirred for 5 hours. The reaction mixture was concentrated in vacuo, the residue was taken up in dichloromethane, concentrated in vacuo, suspended in toluene and concentrated in vacuo to give 0.07 g of the acid semicarbazone (33) - [(3-benzoylamino-4-oxo-4,6,7,8-tetrahydro-pyrrole [1,2-a] pyrimidine-6-carbonyl) -amino] -4-oxo-butane (6a) as a white solid. This material was suspended in a mixture of 37% aqueous formaldehyde, acetic acid and methanol (1: 1: 5) and stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and the residue was suspended in acetonitrile and concentrated in vacuo to yield 0.1 g of a white solid. Chromatography (HPLC, reverse phase Cl8, gradient elution from 1% to 75% acetonitrile / water (buffered with 0.1% trifluoroacetic acid)) gave 0.05 g (60%) of 7a as a white solid: RT = 7.9 min. (HPLC, C18, reverse phase, 1 to 100% acetonitrile / water (0.1% trifluoroacetic acid buffer); gradient elution 20 min); 1 H NMR (CD₃OD (exists as a mixture of hemiacyloxyacetal anomers, 1: 1) δ 2.25-2.4 (m, 1H), 2.45-2.8 (m, 4H), 3.05-3.15 ( m, 1H), 4.25-4.35 (m, 1H), 4.55-4; 6 (m, 1H), 5.1-5.2 (m, 1H), 7.45-7, 65 (m, 3H), 7.9-8.0 (m, 2H), 8.9 (s, 1H).

(3S) - [(3-Benzoylamino-4-oxo-4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine-6-carbonyl) -amino] -4-oxobutanoic acid (7b) was prepared as described for the diastereomer 7a to give 0.03 g (35%) of 7b as a white solid: RT = 8.1 min. (HPLC, C18 reverse phase, 1 to 100% acetonitrile / water (0.1% trifluoroacetic acid buffer); 20 min gradient elution);

Ή NMR (d ₆-DMSO (exists as a mixture of anomers in the form of hemiacyloksya cetal, 1: 1) δ 2.1-2.2 (m, 1H), 2.4 (d, 1H), 2.7-2.8 (m 1H), 3.0-3.2 (m, 3H), 5.0 (dd, 1H), 5.1-5.2 (m, 1H), 5.5 (s, 1H), 5.7 -5.8 (m, 1H), 7.55 (t, 2H), 7.67 (t, 1H), 7.95 (d, 2H), 8.55 (s, 1H), 9.0- 9.15 (m, 1H), 9.4-9.5 (m, 1H).

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<img file="PL185693B1_D0035.tif" />

<img file="PL185693B1_D0036.tif" />

<img file="PL185693B1_D0037.tif" />

<img file="PL185693B1_D0038.tif" />

<img file="PL185693B1_D0039.tif" />

<img file="PL185693B1_D0040.tif" />

Ph

About and

<img file="PL185693B1_D0041.tif" />

Pb

<img file="PL185693B1_D0042.tif" />

a R = H b R - PhCH₂ c R = Ph (CH₂)₂ d R = Ph (CH₂)₃ e R = 4MeO-Ph (CH₂)₃ f R = 4H0-Ph (CH₂)₃

The imidazole-2-carboxylic acids were prepared by modifications of the methods described (Yamaka et al., Chem. Pharm, Bull, 31, pp. 4549-52 (1983)); Suzuki et al., J. Org. Chem ... 38, pp. 3571-75 (1973)); and Oliver et al. J. Org. Chem., 38, pp. 1437-38 (1973)).

Imidazole-2-carboxylic acid (13a) was prepared according to Curtis and Brown, J. Org. Chem. 45, pp. 4038-40 (1980).

4-benzylimidazole-2-carboxylic acid (13b) was isolated as an off-white solid; mp temp. 153-155 ° C; IR (KBr) 3026-2624, 1630, 1515, 1498, 1438, 1405; NMR (d-DMSO) δ 7.31 (5H, m), 7. 4 (1H, s), 3.95 (2H, s).

4- (2-Phenylethyl) imidazole-2-carboxylic acid (13c) was isolated as a pale yellow solid; mp temp. 151-153 ° C; IR (KBr) 3054-2617, 1637, 1497, 1376; Ή NMR (d₆-DMSO) δ 7.27 (5H, m), 7.11 (1H, s), 2.92 (4H, s).

4- (3-Phenylpropyl) imidazole-2-carboxylic acid (13d) was isolated as a pale yellow solid; mp temp. 148-150 ° C; IR (KBr) 3020-2615, 1636, 1509, 1498, 1383; Ή NMR (d₆-DMSO) δ 7.35-7.22 (5H, m), 7.01 (1H, s), 2.62 (4H, m), 1.94 (2H, m).

4- [3- (4-Methoxyphenyl) propyl] imidazole-2-carboxylic acid (13e), isolated as a white crystalline solid; mp temp. 155-156 ° C (decomposition); IR (KBr) 33002300, 1633, 1513, 1376, 1244; 'RNMR (d₆-DMSO) δ 9.50-7.50 (2H, bs), 7.15 (1H, s), 7.11 (2H, d, J = 8.5), 6.84 (2H, d, J = 8.5), 3.71 (3H, s), 2.60-2.50 (4H, m), 1.86 (2H, m) ..

Analysis for C |₄H.₁₆N₂ABOUT₃.

Calculated C, 64.60; H, 6.20; N, 10.76.

Found C, 64.45; H, 6.21; N, 10.70.

185 693

4- [3- (4-Hydroxyphenyl) propyl] imidazole-2-carboxylic acid (13f). A solution of compound 13e ethyl ester (1.15 g, 4.0 mmol) in dry dichloromethane (50 mL) at 0 ° C was treated with boron tribromide (16 mL, 1.0 M solution in CH₂C1₂, 16.0 mmol). After 15 minutes at 0 ° C, the mixture was warmed to 25 ° C and stirred for 16 hours. The reaction mixture was cooled in an ice bath and quenched by dropwise addition of water (20 mL). The resulting mixture was stirred briefly at 25 ° C and then filtered. The filtrate was thoroughly neutralized by the addition of solid NaHCO₃to give compound 13f (700 mg, 71%) as a white solid: mp. 186-187 ° C (decomposition) (recrystallized from MeOH); IR (KBr) 3500-2400, 2935, 16.40, 1516, 1396, 1232;

Ή NMR (d₆-DMSO) δ 9.83 (3H, bs), 7.16 (1H, s), 6.98 (2H, d, J = 8.2), 6.66 (2H, d, J = 8.2 ), 2.60-2.40 (4H, m), 1.84 (2H, m).

Analysis for C.₁₃H.₁₄N₂ABOUT₃.

Calculated C, 63.40; H, 5.73; N, 11.38.

Found C, 62.96; H, 5.70; N, 11.27.

(2R, S, 3S) N²-tert-butoxycarbonyl-N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -Lalaninamide (14).

To a solution of (2R, S, 3S) 3- (N-allyloxycarbonyl) amino-2-benzyloxy-5-oxotetrahydrofuran (Chapman, Biorg, Med, Chem. Lett. 2, pp. 613-618 (1992); (2 , 91 g, 10 mmol)), N-tert-butoxycarbonyl-L-alanine (2.08 g, 11 mmol) and bis (triphenylphosphine) palladium (II) chloride in dichloromethane were added dropwise tri-n-butyltin hydride (4.0 ml, 14.9 mmol) until the color of the solution turned dark orange. Hydroxybenzotriazole (2.70 g, 20 mmol) was added and the mixture was cooled to 0 ° C. 1- (3-Dimethylamino-propyl) -3-ethylcarbodiimide hydrochloride (2.30 g, 12 mmol) was added and the mixture was allowed to slowly warm to room temperature over 4 hours. The mixture was diluted with ethyl acetate (250 ml) and washed with IN hydrochloric acid (3 x 150 ml), saturated aqueous sodium bicarbonate (3 x 150 ml) and brine (2 x 150 ml) then dried (MgSO₄), filtered and concentrated. The crude product was purified by column chromatography (50-70% ethyl acetate / hexane) to afford 3.17 g (84%) of a mixture of diastereomers. After recrystallization (ethyl acetate-hexane), colorless crystals were obtained: mp. 132-145 ° C; IR (KBr) 3357, 3345, 1781, 1688, 1661, 1535, 1517, 1165;

Ή NMR (d_ex-DMSO) δ 8.49 (d, J = 6.8), 8.23 ​​(d, J = 7.4), 7.40 (5H, m), 7.01 (1H, m), 5, 68 (d, J = 5.0), 4.75 (m), 4.31 (m), 3.97 (1H, m), 2.82 (m), 3.11 (m), 2, 82 (m), 2.59 (m), 2.45 (m), 1.40 (9H, s), 1.20 (d, J = 7.2), 1.16 (d, J = 7 , 2).

Analysis for C.₁₉H.₂₆N₂ABOUT₆.

Calculated C, 60.31; H, 6.92; N, 7.40.

Found C, 60.30; H, 6.91; N, 7.38.

(2R, S, 3S) tert-butoxycarbonyl-N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyI) -L -prolinamide (15) was prepared by the method described for compound 14 to give 1.64 g (81%) of a colorless glassy substance. IR (KBr) 3317, 2978, 1797, 1697, 1670, 1546, 1400, 1366, 1164, 1121; Ή NMR (CDC1₃) δ 7.68 (1H, brm), 7.35 (5H, m); 5.53 (d, J = 5.2), 5.43 (s), 4.93-4.61 (m), 4.44 (m), 4.25 (brm), 3.39 (2H , brm), 3.10-2.81 (1H, m), 2.44 (1H, m), 2.32 (brm), 1.88 (brm), 1.67 (brm), 1.42 (9H, s).

(2R, S, 3S) N- (N-tert-butoxycarbonyl- (4 (R) -phenoxy-L-prolinyl) -3-amino-2-benzyloxy-5-oxotetrahydrofuran (16) was prepared by the method described for compound 14 and 530 mg (84%) of a colorless amorphous solid were obtained: 1 H NMR (CDCl₃) δ 7.65 (1H, m), 7.4-7.2 (7H, m), 6.95 (1H, m), 6.85 (1H, m), 5.55 (1H, d) , 4.95 (1H, d), 4.8-4.7 (1H, brm), 4.65 (1H, d), 4.55-4.45 (1H, brm), 4.4-4 , 3 (0.5H, brm), 3.95-3.85 (0.5H, brm), 3.75-3.58 (2H, m), 2.95-2.8 (1H, m \) 2.7-2.55 (1H, m), 2.54-2.4 (1H, m), 2.35-2.2 (1H, m), 1.4 (9H, s).

(2R, S, 3S) N - [4- (3-phenylpropyl) imidazole-2-carbonyl] -N- (tetrahydro-2-benzyloxy-4-oxo-3-furanyl) -L-alaninamide (17d). For a solution of (2R, S, 3S) N²-tert-butoxycarbonyl-N (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (14) (1.00 g, 2.64 mmol) in dichloromethane (7 mL) at 0 [Deg.] C trifluoroacetic acid (7 ml) was added. The mixture was stirred at 0 ° C for 75 minutes. The mixture was concentrated and the residue was treated with diethyl ether and the ether was removed in vacuo. This procedure was repeated two times

185 693 times to obtain a pale yellow glassy substance. This material was dissolved in DMF (20 mL). Then to this solution was added diisopropylethylamine (1.38 mL, 7.92 mmol) followed by 4- (3-phenylpropyl) imidazole-2-carboxylic acid (13d) (0.67 g, 290 mmol), 1- ( 3-dimethylaminopropyl) -3-ethylcarbodiimide (0.56 g, 2.90 mmol) and hydroxybenzotriazole (0.71 g, 5.28 mmol).

The mixture was stirred at room temperature for 20 hours then poured into brine. The mixture was extracted with ethyl acetate (3 x 50 ml). The combined organic extracts were washed with saturated aqueous sodium bicarbonate (2 x 100 ml) and brine (2 x 100 ml), dried (MgSO₄), filtered and concentrated. The residue was purified by column chromatography (ethyl acetate) to yield 0.99 g (76%) of 17d as a mixture of diastereomers: IR (KBr) 3293, 3064, 2937, 1793, 1650, 1530, 1451, 1446, 1119; Ή NMR (CDC1₃) δ 7.96 (brm), 7.62 (brd), 7.36-7.10 (10H, m), 6.88 (s), 6.86 (s), 5.53 (d, J = 5.2), 5.48 (s), 4.87-4.52 (4H, m), 3.11-2.38 (2H, m), 2.65 (4H, m), 1. 99 (2H, m), 1.47 (d, J = 6.9), 1.46 (d, J = 7.0).

The following compounds were prepared in a similar manner: (2R, S, 3S) N²- (imidazole-2-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (17a) was isolated as a pale yellow solid: IR (KBr) 3289, 3056, 2937, 1793, 1664, 1642, 1528, 1453, 1440, 1124; Ή NMR (d-DMSO) δ 13.13 (1H, brs), 8.67 (d, J = 7.0), 8.48 (d, J = 7.8), 8.29 (d, J = 6.8), 8.25 (d, J = 7.6), 7.40-7.34 (6H, m), 7.11 (1H, s), 5.69 (d, J = 5 0.0), 5.49 (d, J = 0.8), 4.85-4.31 (4H, m), 3.19-2.42 (2H, m), 1.38 (d, J = 7.4), 1.34 (d, J = 7.4).

(2R, S, 3S) N²- (4-benzylimidazole-2-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (17b) was isolated (75%) as a pale yellow glassy substance: IR (KBr) 3294, 3031, 2937, 1792, 1650, 1530, 1453, 1444, 1119; Ή NMR (CDCl 3) δ 7.99 (brm), 7.75 (brd), 7.36-7.11 (10H, m), 6.81 (1H, s), 5.51, 5.45 ( d, s, J = 5.3), 4.85-4.47 (4H, m), 3.95 (2H, s), 3.04-2.72 (1H, m), 2.48- 2.35 (1H, m), 1.44 (d, J = 6.9), 1.43 (d, J = 7.1). (2R, S, 3S) N²- [4- (2-phenylethyl) imidazole-2-carbonyl] -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (17c) was isolated (79%) as a pale yellow glassy substance: IR (KBr) 3292, 3029, 2936, 1793, 1650, 1530, 1453, 1444, 1119; Ή NMR (CDCl 3) δ 8.06 (brm), 7.70 (brs), 7.39-7.15 (10H, m), 6.82 (s), 6.81 (s), 5.52 (d, J = 5.2), 5.48 (s), 4.87-4.53 (4H, m), 2.95 (4H, m), 3.14-2.37 (2H, m ), 1.48 (d, J = 6.5), 1.45 (d, J = 6.7). (2R, S, 3S) 1- [4 (2-phenylethyl) imidazole-2-carbonyl] -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-prolinamide (18c) was isolated (79 %) as a pale yellow glassy substance: IR (KBr) 3422, 2959, 1795, 1685, 1611, 1497, 1116; Ή NMR (d-DMSO) δ 12.78-12.59 (1H, m), 8.61-8.34 (1H, m), 7.39-7.22 (10H, m), 6.99 -6.61 (1H, m), 5.71-5.26 (1H, m), 4.85-4.02 (4H, m), 3.63 (1H, m), 3.18-1 . 74 (11H, m).

(2R, S, 3S) 1 - [4- (3-phenylpropyl) imidazole-2-carbonyl] -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-prolinamide (18d) was isolated (87% ) as a colorless glassy substance: IR (CH₂C1₂) 3422, 3214, 2945, 1794, 1685, 1604, 1496, 1117; Ή NMR (d₆-DMSO) δ 12.71 (1H, brm), 8.61-8.34 (1H, m), 7.45-7.18 (10H, m), 7.05-6.64 (1H, m ), 5.70-5.28 (1H, m), 4.85-4.02 (4H, m), 3.62 (1H, m), 3.18-1.71 (13H, m).

(2R, S, 3S) l- {4- [3- (4-methoxyphenyl) propylimidazole-2-carbonyl} -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-prolinamide (18e) was isolated (72%) as a white glassy solid: mp. 62-65 ° C; (IR) (KBr) 3213, 2937, 1793, 1680, 1606, 1512, 1245; Ή NMR (d₆-DMSO) δ 12.71, 12.67, 12.58 (1H, 3 x bs), 8.60-8.30 (1H, m), 7.40-7.20 (5H, m), 7 , 15-6.55 (5H, m), 5.66-5.20 (1H, m), 4.81-4.59 (2H, m), 4.55-4.05 (2H, m) , 3.71 (3H, s), 3.65-3.45 (1H, m), 3.15-1.50 (13H, m). FABSMS m / e 547 (M⁺, 100%), 439, 412, 340, 312,243, 177, 154.

(2R, S, 3S) 1- {4- [3- (4-hydroxyphenyl) propyl] imidazol-2-carbonyl} -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-prolinamide (18f) was isolated (70%) as a light yellow glassy solid: mp. 86-90 ° C; IR (KBr) 3298, 1790, 1669, 1606, 1515, 1242; Ή NMR (d₆-DMSO) δ 12.66, 12.56 (1H, 2 x bs), 9.14 (1H, s), 8.57-8.30 (1H, m), 7.367.30 (5H, m), 7.02-6.83 (3H, m), 6.70-6.57 (2H, m), 5.65-5.28 (1H, m), 4.80-4.49 (2H, m ), 4.50-4.05 (2H, m), 3.65-3.45 (1H, m), 3.15-1.55 (13H, m). FABMS m / e 533 (M⁺, 100%) 425, 298,229, 176, 154.

185 693

- {5 - [3 - (4-methoxyphenyl) propyl] -1H-imidazole-2-carbonyl} -4 (R) -phenoxypyrrolidine-2 (S) -carbonyl- ( tetrahydro-2 (R, S) - benzyloxy-5-oxofuran-3 (S) -yl) amide (19e) was isolated (77%) as a clear colorless amorphous solid. 1 H NMR (CDCl₃) δ 9.95-9.75 (IH, m), 7.95 (IH, brs), 7.40-7.2 (7H, m), 7.2-6.78 (7H, m), 5.65-5.6 (IH, m), 5.55-5.45 (IH, m), 5.3-5.2 (IH, m), 5.15-5.0 (IH, m ), 4.95-4.75 (IH, m), 4.7-4.6 (IH, m), 4.54.4 (IH, m), 4.35-4.25 (IH, m ), 3.8 (3H, s), 3.05-1.75 (10H, m).

(3S) 3- {N- [4- (3-Phenylpropyl) imidazole-2-carbonyl] -L-alaninyl} amino-4-oxo-butanoic acid (20d). A mixture of (2R, S, 3S) N²- [4- (3-phenylpropyl) imidazole-2-carbonyl] -N (tetrahydro-2-benzyloxy-4-oxo-3-furanyl) -L-alaninamide (0.93 g, 1.90 mmol) and 10% palladium on activated carbon (0.93 g) in methanol (100 ml) was stirred under an atmosphere of hydrogen for 5 hours. The resulting mixture was filtered and concentrated to give a colorless glass. After recrystallization from a mixture of methanol and diethyl ether, 401 mg (53%) of 20d were obtained as a colorless solid; mp temp. 94-96 ° C; [a] D²⁷+ 16.4 ° (c 0.5, MeOH); IR (KBr) 3300, 3287, 1786, 1732, 1659, 1651, 1532, 1451; Ή NMR (CD3OD) δ 7.19 (5H, m), 6.91 (IH, s), 4.60-4.46 (2H, m), 4.27 (IH, m), 2.63 ( 4H, m), 2.752.40 (2H, m), 1.96 (2H, m), 1.44 (3H, d, J = 7.0).

The following compounds were prepared in a similar manner: (3S) 3- [N- (imidazole-2-carbonyl) -L-alaninyl] amino-4-oxobutanoic acid (20a, Ę) was isolated (83%) as a colorless solid: m.p. . 115 ° C; [and]_D²⁵+ 4.4 ° (c 0.5, MeOH); IR (KBr) 3303, 1782, 1658, 1650, 1563, 1521, 1454; Ή NMR (CD₃OD) δ 7.18 (2H, s), 4.55 (2H, m), 4.27 (IH, m), 2.56 (2H, m), 1.45 (d, J = 7.1 ), 1.44 (d, J = 7.0).

(3S) 3- [N- (4-benzylimidazole-2-carbonyl) -L-alaninyl] amino-4-oxobutanoic acid (20b) was isolated (56%) as a colorless solid: m.p. 113-115 ° C; [and]/⁹ + 18.2 ° (c 0.5, MeOH). IR (KBr) 3301, 3288, 1783, 1727, 1650, 1521, 1452; Ή NMR (CD₃OD) δ 7.25 (5H, m), 6.90 (IH, s), 4.59-4.45 (2H, m), 4.26 (IH, m), 3.95 (2H, s) ), 2.742.39 (2H, m), 1.42 (3H, d, J = 7.0).

Analysis for C._]8H.₂₀N₄ABOUT₅.

Calculated C, 56.69; H, 5.55; N, 14.69.

Found C, 57.06; H, 5.54; N, 14.41.

(3S) 3- {N- [4- (2-Phenylethyl) imidazole-2-carbonyl] -L-alaninyl} amino-4-oxobutanoic acid (20c; N) was isolated (53%) as a colorless solid: temp maturity 102-104 ° C; [and]_D²⁷ + 13.7 ° (c 0.5, MeOH); IR (KBr) 3299, 3289, 1785, 1732, 1531, 1452; Ή NMR (CD₃OD) δ 7.20 (5H, m), 6.82 (IH, s), 4.60-4.46 (2H, m), 4.29 (IH, m), 2.92 (4H, s ). 2.76-2.41 (2H, m), 1.44 (3H, 2xd, J = 7.1).

Analysis for C.₁₉H.₂, N₄ABOUT₅.

Calculated: C, 56.43; H, 5.98; N, 13.85.

Found C, 56.65; H, 5.84; N, 13.91.

(3S) 3- {N- [4- (2-Phenylethyl) imidazole-2-carbonyl] -L-prolinyl} amino-4-oxobutanoic acid (21c) was isolated (85%) as a glassy colorless substance; mp temp. 101-103 ° (methanol-diethyl ether); [α] i - 63.8 ° (c 0.25, MeOH); IR (KBr) 3275, 1784, 1728, 1664, 1606, 1498, 1429; Ή NMR (CD₃OD) δ 7.24 (5H, m), 6.83 (s), 6.79 (s), 4.58-4.14 (3H, m), 3.69 (IH, m), 2, 93 (4H, br s), 2.75-1.99 (6H, m).

Analysis for C.₂| H24N₄ABOUT₅ H.₂ABOUT.

Calculated C, 58.60; H, 6.90; N, 13.02.

Found C, 58.34; H, 5.96; N, 12.67.

(3S) 3- {N- [4- (3-phenylpropyl) imidazole-2-carbonyl] -L-prolinyl} amino-4-oxobutanoic acid (21d) was isolated (81%) as a glassy colorless substance; mp temp. 91-94 ° C; (methanol-diethyl ether); [and]_D² -68 ° (c 0.25, MeOH); IR (KBr) 3277, 2939, 1784, 1727, 1662, 1606, 1498, 1429; Ή NMR (CD₃OD) δ 7.29-7.16 (5H, m), 6.92 (s), 6.86 (s), 4.58-4.16 (3H, m), 3.71 (IH, m ), 2.75-1.92 (13H, m).

Analysis for C.₂₂H.₂₆N₄ABOUT₅ H.₂ABOUT.

Calculated C, 59.45; H, 6.35; N, 12.60.

Found C, 59.75; H, 6.21; N, 12.41.

185 693

(3S) 3- {N- [4- [3- (4-methoxyphenyl) propyl] imidazole-2-carbonyl] -L-prolinyl} amino-4-oxobutanoic acid (21e) was isolated (65%) as a glassy white solid substance; mp temp. 101-105 ° C; [and]_D²³ -60.5 (c 0.05, MeOH); IR (KBr) 3231, 1784, 1726, 1611, 1512, 1245; Ή NMR (CD₃OD) δ 7.09 (2H, d, J = 8.6), 6.85 (1H, 2 xs), 6.81 (2H, d, J = 8.6), 5.45-5.30 (1H, m), 4.64-4.46 (1H, m), 4.28-4.10 (2H, m), 3.75 (3H, s), 3.74-3.66 (1H , m), 2.67-1.84 (13H, m).

Analysis for C.₂₃H.₂₈N₄ ABOUT₆ H.₂ABOUT.

Calculated C, 58.22; H, 6.37; N, 11.81.

Found C, 58.39; H, 6.34; N, 11.45;

FABMS m / e 457 (M⁺), 405, 312, 243, 215, 176, 154 (100%).

(3S) 3 - {N- [4- [3 - (4-Hydroxyphenyl) propyl] imidazole-2-carbonyl] -L-prolinyl} amino-4-oxobutanoic acid (21f) was isolated (43%) as a glassy white solid; mp temp. 114-118 ° C; [and]_D²⁵ -55.7 (c 0.05, MeOH); IR (KBr) 3288, 2935, 1780, 1715, 1662, 1610, 1515, 1441; Ή NMR (CD₃OD) δ 6.99 (2H, d, J = 8.5), 6.91, 6.85 (1H, 2 xs), 6.68 (2H, d, J = 8.5), 5.45 -5.30 (1H, m), 4.60-4.47 (1H, m), 4.30-4.10 (2H, m), 3.80-3.55 (1H, m), 2 , 70-1.80 (13H, m).

Analysis for C.₂₂H.₂₆N₄ABOUT_Æ H.₂ABOUT.

Calculated C, 57.38; H, 6.13; N, 12.17.

Found C, 57.68; H, 6.25; N, 11.66.

FABMS m / e 443 (M⁺), 298, 229, 154 (100%).

Acid 3 (S) - [(1 - {5- [3- (4-methoxyphenyl) propyl] -1H-imidazole-2-carbonyl} -4 (R) -phenoxypyrrolidine-2 (S) -carbonyl) amino] -4-oxobutane (22e) was isolated (43%) as a beige solid: 1 H NMR (CD₃OD) 6 7.35-7.2 (3H, m), 7.15-7.0 (2H, m), 6.986.85 (3H, m), 6.83-6.77 (2H, d) , 5.4-5.1 (1H, m), 4.65-4.5 (1H, m), 4.35-4.2 (2H, m), 4.153.90 (1H, m), 3 , 78 (3H, s), 3.62-3.48 (1H, m), 2.78-2.25 (8H, m), 2.02-1.9 (2H, m).

<img file="PL185693B1_D0043.tif" />

185 693

<img file="PL185693B1_D0044.tif" />

{Phenethyl- {5- (3-propyl) -1H-imidazole-2-carbonyl] amino} -acetic acid tert-butyl ester (23).

A solution of 4- (3-phenylpropyl) -imidazole-2-carboxylic acid (13d) (150 mg, 0.65 mmol) and N- (2-phenethyl) glycine tert-butyl ester (140 mg, 0 , 59 mmol) in 5 ml of anhydrous dimethylformamide was treated with diisopropylethylamine (154 μΐ, 0.89 mmol), hydroxybenzotriazole (160 mg, 1.18 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (136 mg, 0.18 mmol). 71 mmol). After stirring for 36 hours, the reaction mixture was poured into a saturated aqueous sodium chloride solution and extracted with ethyl acetate (3 x 50 ml). The combined organic extracts were washed twice with saturated aqueous sodium bicarbonate (2x) and saturated aqueous sodium chloride (1x), dried (Na₂SO₄), filtered and concentrated in vacuo to yield a brown oil. After chromatography (fast, SiO₂30% EtOAc / Hexane) afforded 160 mg (61%) of compound 23 as a white solid. 1 H NMR (CDCl₃) δ 7.38-7.14 (10H, m), 6.85-6.8 (1H, m), 4.84-4.76 (1H, d), 4.5-4.42 (1H , m), 4.07-4.0 (1H, d), 3.78-3.72 (1H, m), 3.12-2.94 (2H, 2xm), 2.75-2. 55 (4H, m), 2.1-1.95 (2H, m), 1.5-1.45 (9H, 3xs).

(3S) - (2-Phenethyl- [5- (3-phenyl-propyl) -1H-imidazole-2-carbonyl] amino} acetylamino) -4-oxobutanoic acid tert-butyl semicarbazone (24). Ester 23 (160 mg, 0.375 mmol) was treated with a 25% solution of trifluoroacetic acid and dichloromethane (7 ml) for 4 hours. The mixture was concentrated in vacuo to give 180 mg of the acid. This acid (180 mg, 0.357 mmol) was coupled with (3S) -3amino-4-oxobutanoic acid tertbutanoic acid semicarbazone (161 mg, 0.357 mmol) as described for the preparation of compounds 5a and 5b to give 86 mg (33% ) compound 24 (one diastereomer) as a white solid. Ή NMR (CDC1₃) δ 10.08-9.78 (1H, 2d), 9.25-9.15 (1H, m), 8.35-8.10 (1H, 2m), 7.9-7.85 (1H .2s), 7.40-7.05 (10H, m), 6.9-6.75 (1H, m), 6.3-5.8 (1H, br s), 5.2-4 , 65 (2H, m), 4.35-3.5 (3H, m), 3.25-3.0 (2H, m), 2.9-2.45 (6H, m), 2.05 -1.8 (2H, m), 1.4 (9H, s).

(3S) - (2- {Phenethyl- [5- (3-phenylpropyl) -1H-imidazole-2-carbonyl] amino} acetylamino) -4-oxobutanoic acid trifluoroacetate (25) was prepared as described for 7a and obtained 32 mg (82%) compound as a white solid: 1 H NMR (CD₃OD) δ 7.05-7.35 (m, 11H), 4.65 m, 1H), 4.4 (m, 1H), 4.3 (s, 2H), 3.6-4.0 ( m, 2H), 2.52.95 (m, 8H), 2.05 (m, 2H).

7- [5- (3-Phenyl-propyl) -1H-imidazole-2-carbonyl] -1,4-dithia-7-azaspiro [4.4] nonane-8 (S) -carboxylic acid methyl ester (26). 4- (3-Phenylpropyl) imidazole-2-carboxylic acid (13d) was coupled with 1,4-dithia-7-azaspiro [4.4] nonane-8 (S) -carboxylic acid methyl ester hydrobromide (Smith et al. J, Med. Chem. .. 31, pp. 875-85 (1988)) as described for compound 23 to give 140 mg (65%) of the title compound as a yellow gum: Ή NMR (CDCL₃) δ 7.34-7.15 (5H, m), 6.98-6.8 (1H, 3s), 5.7-5.65 (0.5H, m), 5.2-5.1 (1H, m), 4.82-4.75 (0.5H, m), 4.4-4.35 (1H, m), 4.05 (1H, d), 3.75-3.7 (3H, 2s), 3.4-3.3 (4H, m), 2.95-2.45 (8H, m), 2.05-1.95 (2H, m).

(3S) - ({7- [5- (3-phenyl-propyl) -1H-imidazole-2-carbonyl] -1,4-dithia-7-azaspiro [4.4] nonane-8 (S) acid tert-butyl semicarbazone -carbonyl} -amino) -4-oxobutanoic (27). According to the procedure described for compound 4, ester 26 was converted to him

185 693 acid which was then coupled with (3S) -3-Amino-4-oxobutanoic acid tert-butyl ester semicarbazone as described for compound 24 to give 70 mg (33%) brown solid: Ή NMR (CD₃OD) δ 7.28-7.10 (5H, m), 6.90 (1H, br s), 4.94 (1H, m), 3.963.86 (2H, q), 3.35-3, 25 (4H, d), 3.0 (2H, s), 2.73-2.59 (6H, m), 2.0-1.92 (2H, m), 1.44 (9H, s) .

(3 S) - {{7- [5- (3-Phenylpropyl) -1H-imidazole-2-carbonyl] -1,4-dithia-7-azaspiro [4.4] nonane-8 (S) -carbonyl} acid -amino) -4-oxobutane (28) was prepared as described for 7a to give 17 mg (26%) of a light brown solid. 1 H NMR (CD₃OD) δ 7.4 (s, 1H), 7.1-7.25 (m, 5H), 4.9 (m, -1H), 4.6 (m, 1H), 4.3 (m , 1H), 3.95 (s, 2H), 3.25-3.4 (m, 4H), 3.0 (d, 2H), 2.6-2.8 (m, 5H), 2. 45 (m, 1H). 2.05 (m, 2H).

<img file="PL185693B1_D0045.tif" />

<img file="PL185693B1_D0046.tif" />

<img file="PL185693B1_D0047.tif" />

36 31

<img file="PL185693B1_D0048.tif" />

4,5-dihydroimidazole-4-carboxylic acid esters (29) were prepared by modifying the method described by Jones et al., Tetrahedron Lett., 29, pp. 3853-56 (1988).

Methyl (4R, S) 2- (2-phenylethyl) -4,5-dihydroimidazole-4-carboxylate (29a). Dry hydrogen chloride was bubbled through a solution of hydrocinnamonitrile (3.28 mL, 25 mmol) in methanol (125 mL) at 0 ° C for 45 minutes. The solvents were removed to give the imidate which was dissolved in methanol (125 mL) along with methyl 2,3-diaminopropionate (25 mmol) (Jones et al., Supra). The mixture was kept at room temperature for 2.5 hours and then concentrated to a yellow oil. The crude product was purified by column chromatography (10-20% methanol / dichloromethane) to give 3.52 g (61%) of a colorless glassy substance: Ή NMR (CDCl₃): δ 7.30-7.15 (5H, m), 4.63 (1H, t, J = 9.7), 3.96 (2H, d, J = 9.7), 3.72 ( 3H, s), 3.10 (4H, m), ^,3C NMR (CDC1₃): δ 171.3, 138.3, 128.4, 128.2, 126.6, 57.3, 53.0, 47.7, 31.7, 27.9.

Methyl (4R, S) 2- [2- (4-trifluoromethylphenyl) ethyl] -4,5-dihydroimidazole-4-carboxylate (29b) was prepared as described for 29a to give 6.80 g (78%) of a colorless solid : mp. 136-141 ° C; Ή NMR (CDC1₃): δ 7.45 (4H, s), 4.71 (1H, dd, J = 8.6, 10.8), 4.02 (2H, m), 3.73 (3H, s), 3 . 19 (4H, m).

The imidazole-4-carboxylic acid esters were prepared by modifying the method described by Martin et al., J. Org. Chem., 33, pp. 3758-61 (1968).

Methyl 2- (2-phenylethyl) imidazole-4-carboxylate (30a). A mixture of methyl (4R, S) 2- (2-phenylethyl) -4,5-dihydroimidazole-4-carboxylate (29a) (3.40 g, 14.64 mmol), chloroform (75 ml) and manganese (IV) oxide (13.0 g, 150 mmol) was heated to reflux for 21 hours then filtered hot. The solids were washed with chloroform and methanol. The combined filtrates were concentrated to give a yellow / brown solid which was purified by column chromatography (2-5% methanol / dichloromethane)

185 693 to afford 1.46 g (43%) of a pale yellow solid: mp. 151-155 ° C; IR (KBr) 3028, 2946, 1720, 1533, 1433, 1348, 1195, 1166; 'HNMR (CDC1₃): δ 7.62 (1H, s), 7.26-7.02 (5H, m), 3.82 (3H, s), 3.03 (4H, brs), ¹³C NMR (CDC1₃): δ 162.9, 150.2, 140.3, 128.5, 128.2, 126.3, 51.5, 34.5, 30.4.

Analysis for C.₁₃H.₁₄N₂ABOUT₂.

Calculated C, 67.81; H, 6.13; N, 12.16.

Found C, 67.70; H, 6.15; N, 12.16.

Methyl 2- [2- (4-trifluoromethylphenyl) ethyl] imidazole-4-carboxylate (30b) was prepared by the method described for 30a. After recrystallization from ethyl acetate, 1.89 g (33%) of cream-colored crystals were obtained: mp. 225-26 ° C; IR (KBr) 3239, 2951, 1715, 1532, 1331, 1158, 1105, 1068; Ή NMR (CDC1₃): δ 7.61 (1H, s), 7.54 (2H, d, J = 8.1), 7.26 (2H, d, J = 8.1), 3.89 (3H, s) . 3.10 (4H, m).

Analysis for C.₁₄H.₁₃F.₃N₂ABOUT₂.

Calculated C, 56.38, H, 4.39; N, 9.39; F, 19.11.

Found C, 56.23; H, 4.44; N, 9.33; F, 19.08.

2- (2-Phenylethyl) imidazole-4-carboxylic acid (3a).

A mixture of methyl 2- (2-phenylethyl) imidazole-4-carboxylate (31 a) (1.38 g, 6 mmol), methanol (30 ml) and 1M aqueous sodium hydroxide solution (30 ml) was heated to reflux for 16 hours. The methanol was removed under reduced pressure and the resulting aqueous solution was neutralized with 4M hydrochloric acid, resulting in a yellow solid precipitating. The material was collected, washed with water, and dried. 1.18 g (91%) of a pale yellow solid were obtained: mp. 117-120 ° C; IR (KBr) 3375, 3131, 2616, 2472, 1638, 1592, 1551, 1421, 1388, 1360; 'HNMR (d₆-DMSO): δ 7.59 (1H, s), 7.26 (5H, m), 2.99 (4H, m).

Analysis for C.₁₂H.₁₂N₂ABOUT₂ 0.25H₂ABOUT.

Calculated C, 65.29; H, 5.71; N, 12.69.

Found C, 65.00; H, 5.64; N, 12.58.

2- [2- (4-Trifluoromethylphenyl) ethyl] imidazole-4-carboxylic acid (3Ib) was prepared as described for 3a to give 1.09 g (76%) of a pale yellow solid: mp. 126-130 ° C; IR (KBr) 3339, 2640-2467, 1638, 1589, 1545, 1383, 1323; Ή NMR (d₆-DMSO): δ 7.69 (2H, d, J = 8.0), 7.59 (1H, s), 7.47 (2H, d, J = 8.0), 3.06 (4H, m).

(2R, S, 3S) N²- [2- (2-phenylethyl) imidazole-4-carbonyl] -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (32a). To a 0 ° C solution of (2R, S, 3S) N²-tert-butoxycarbonyl-N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-aIaninamide (14) (1.59 g, 4.20 mmol; Chapman, Biorg. Med.Chem. Lett., 2 , pp. 613-18 (1992)) in dichloromethane (15 ml) was added trifluoroacetic acid (15 ml). The mixture was stirred at 0 ° C for 1 hour and then concentrated. The residue was treated with ether and then the ether was removed in vacuo. This procedure was repeated twice to give a pale yellow glassy substance. This material was dissolved in DMF (20 mL), then diisopropylethylamine (2.19 mL, 12.6 mmol), 2- (2-phenylethyl) imidazole-4-carboxylic acid (3la) (1.0 g, 4.62 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.89 g, 4.62 mmol), and hydroxybenzotriazole (1.14 g, 8.40 mmol). The reaction mixture was stirred at room temperature for 20 hours then poured into brine. The mixture was extracted with ethyl acetate (3 x 50 ml). The combined organic extracts were washed with saturated aqueous sodium bicarbonate then brine, dried (MgSO₄) and concentrated. The residue was purified by column chromatography (2-10% isopropanol in dichloromethane then 0-6% isopropanol in ethyl acetate) to give 1.10 g (55%) of compound 32a as a mixture of diastereomers: IR (KBr) 3278, 3065, 1790 , 1641, 1577, 1545, 1499, 1454, 1120; Ή NMR (CDC1₃): δ 10.26 (1H, s), 8.14 (1H, s), 7.66 (d, J = 7.0), 7.56 (d, J = 7.0), 7.43 (1H, s), 7.31-7.11 (10H, m), 5.49 (d, J = 5.6), 5.48 (s), 4.83-4.41 (4H, m ), 3.04-2.41 (2H, m), 2.99 (4H, s), 1.45 (d, J - 7.0), 1.44 (d, J = 7.0).

(2R, S, 3S) N²- {2- [2- (4-trifluoromethylphenyl) ethyl] imidazole-4-carbonyl} -4- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (32b) was prepared as described for 32a to give 1.08 g (62%) of a pale yellow glassy substance: IR (KBr) 3376,

185 693

3284, 3070, 2938, 1791, 1642, 1578, 1546, 1327, 1165, 1122, 1068; Ή NMR (CDC1₃): δ 7.95 (0.5H, m), 7.55-7.25 (11.5H, m), 5.53 (s), 5.49 (d, J = 5.3), 4 , 88-4.48 (4H, m), 3.11-2.96 (4H, m), 2.91 (1H, m), 2.51 (1H, m), 1.47 (3H, d , J = 7.1).

(2R, S, 3S) N²- (2-benzylimidazole-4-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (32c) was prepared as described for compound 32a from 2-benzylimidazole-4-carboxylic acid (Ger. Offen DE 3427136) to give 1.13 g (83%) of a yellow glass: IR (CH₂C1₂) 3433, 3062, 2990, 1803, 1693, 1584, 1504, 1429, 1285, 1258; Ή NMR (CDC1₃) δ 9.50 (s), 9.37 (s), 7.86 (0.5H, d, J = 6.1), 7.56-7.21 (10.5H, m), 7, 48 (1H, s), 5.51 (d, J = 5.2), 5.48 (s), 4.87-4.41 (4H, m), 4.08 (s), 4.07 (s), 3.03-2.39 (2H, m), 1.46 (3H, d, J = 7.0).

(3S) 3 - {N- [2- (2-Phenylethyl) imidazole-4-carbonyl] -L-alaninyl} amino-4-oxobutanoic acid (33aA). A mixture of (2R, S, 3S) N²- [2- (2-phenylethyl) imidazole-4-carbonyl] -N (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (32a) (1.0 g, 2.10 mmol) and 10% palladium on activated carbon (1.0 g) in methanol (50 ml) was stirred under a hydrogen atmosphere for 4.5 hours. The resulting mixture was filtered and concentrated to give a colorless glass. After recrystallization from a mixture of methanol and diethyl ether, 510 mg (63%) of a colorless solid are obtained: mp. 127 ° C; IR (KBr) 3360, 3279, 2981, 1781, 1732, 1646, 1577, 1547; Ή NMR (CD3OD): δ 7.54 (1H, s), 7.29-7.12 (5H, m), 4.60-4.47 (2H, m), 4.28 (1H, m) , 3.01 (4H, s), 2.76-2.39 (2H, m), 1.43 (3H, 2 xd, J = 7.0, J = 7.0),¹³C NMR (CD3OD): δ 176.2, 176.0, 174.7, 174.6, 164.4, 164.3, 150.5, 141.9, 134.8, 129.3, 127.3 , 122.3, 98.8, 52.3, 52.0, 50.3, 35.6, 31.2, 18.8, 18.7.

Analysis for C.₁₉H.₂₂N₄ABOUT₅ H.₂ABOUT.

Calculated C, 56.43; H 5.98; N, 13.85;

Found C, 56.78; H, 5.70; N, 13.77.

(3S) 3- {N- [2- (2- (4-Trifluoromethylphenyl] ethyl) imidazole-4-carbonyl] -Lalaninyl} -amino-4-oxobutanoic acid (33b; C) was prepared as described for compound 33a and was prepared 612 mg (73%) of a colorless solid: mp 120-124 ° C; [a]_D²³+ 14.3 ° (c 0.5, MeOH); IR (KBr) 3287, 2985, 2937, 1782, 1732, 1646, 1579, 1547, 1327; Ή NMR (CD₃OD): δ 7.56 (2H, d, J = 8.0), 7.54 (1H, s), 7.36 (2H, d, J = 8.0), 4.60-4.48 (2H, m), 4.28 (1H, m), 3.08 (4H, m), 2.75-2.41 (2H, m), 1.43 (3H, d, J = 7.0 ).

Analysis for C.₂₀H.₂| F₃Ń₄ABOUT₅ 0.5H₂ABOUT.

Calculated C, 51.84; H, 4.78, N 12.09; F, 12.30.

Found C, 51.83; H, 4.72; N, 12.14; F, 12.36.

(3S) 3- [N- (2-benzylimidazole-4-carbonyl) -L-alaninyl] amino-4-oxobutanoic acid (33c, B) was prepared as described for compound 33a to give 426 mg (64%) of a colorless solid : [a]_D²³ + 13.4 ° (c 0.407, MeOH);

IR (KBr) 3260, 3150, 2980, 1779, 1727, 1649, 1573, 1547; Ή NMR (CD₃OD): δ 7.58 (1H, s), 7.34-7.22 (5H, m), 4.59-4.47 (2H, m), 4.28 (1H, m), 4, 07 (2H, s), 2.74-2.41 (2H, m), 1.42 (3H, d, J = 6.7); ^l3C NMR (CD₃OD): δ 175.6, 175.5, 175.0. 164.6, 164.5, 150.1, 138.7, 135.3 130.0, 129.9, 128.2, 122.9, 98.9, 98.5, 52.5, 52.2 , 35.5, 35.1, 35.0, 19.0, 18.9.

Analysis for Ć |₈H.₂₀N₄ABOUT₅ H.₂ABOUT.

Calculated C, 55.37; H, 5.68; N, 14.35.

Found C, 55.83; H, 5.75; N, 13.96.

MS (FAB, m / z): 373 (M⁺), 228, 185, 91.

<img file="PL185693B1_D0049.tif" />

(a) R = H (b) R = CH₂Ph

185 693

5-Benzylpyrrole-2-carboxylic acid (34b). A mixture of ethyl 5-benzylpyrrole-2-carboxylate (0.7 g, 3.05 mmol; Elder et al., Synthetic Communications, 19, 763-767 (1989)), ethanol (20 ml) and 1M sodium hydroxide (9, 2 ml, 9.2 mmol) was stirred and heated to reflux for 3 hours. Most of the methanol was removed and the remaining liquid was diluted with water, washed with ether, cooled in ice, and acidified with concentrated hydrochloric acid. The mixture was extracted with ether. The combined extracts were washed with brine, dried (a₂SO₄) and concentrated to afford 0.567 g (92%) of an off-white solid; mp temp. 130-134 ° C; Ή NMR (CDC1₃): δ (1H, brs), 7.37-6.95 (5H, m), 6.97 (1H, m), 6.07 (1H, m), 4.00 (2H, s).

(2R, S, 3S) N²- (pyrrole-2-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -Lalaninamide (35a).

A solution of (2R, S, 3S) N²-tert-butoxycarbonyl-N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (14) (756 mg, 2.0 mmol) in dry dichloromethane (8 ml) at 0 ° C treated for 1 hour with trifluoroacetic acid (8 ml) then evaporated to dryness. Dry ether was added to the residue, and the mixture was concentrated to a viscous oil. This oil was dissolved in dry DMF (10 ml). Pyrrole-2-carboxylic acid (34a) (244mg, 2.2mmol) was added and the solution was cooled in an ice bath, then N, N-diisopropylamine (0.78g, 6.0mmol), hydroxybenzotriazole (0.54g 4.0 mmol) and dimethylaminopropylethylcarbodiimide hydrochloride (0.42 g, 2.2 mmol). The resulting mixture was stirred at 25 ° C for 17 hours, then a saturated aqueous sodium chloride solution (30 mL) was added. The mixture was extracted with ethyl acetate (3 x 20 mL) and the combined organic extracts were washed with 5% aqueous sodium bicarbonate (3 x 10 mL) and brine (10 mL), dried (MgSO₄) and concentrated. Flash chromatography (25% hexane-ethyl acetate) provided 557 mg (75%) of a white glassy solid as a 1: 1: mp mixture of diastereomers. 85-90 ° C; IR (KBr) 3288, 1789, 1665, 1629, 1557, and 1122; Ή NMR (d₆-DMSO): δ 11.46 (1H, bs), 8.55 (0.5H, d, J = 7.0), 8.30 (0.5H, d, J = 7.6), 8, 06 (0.5H, d, J = 7.0), 8.04 (0.5H, d, J = 7.6), 7.36-7.30 (5H, m), 6.88-6 , 85 (2H, m), 6.10-6.07 (1H, m), 5.63 (0.5H, d, J = 5.0), 5.42 (0.5H, s), 4 , 72 (2H, q, J = 12.2), 4.74-4.25 (2H, m), 3.14-2.35 (2H, m), 1.29, 1.25 (3H, 2 xd, J = 7.2).

(2R, S, 3S) N.²- (5-benzylpyrrole-2-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-alaninamide (35b) was prepared from 5-benzylpyrrole-2-carboxylic acid (34b) by the method described in for compound 35a (65%). Data are given for a single diastereomer. Ή NMR (d₆-DMSO): δ 11.37 (1H, brs), 8.27 (1H, d, J = 7.4), 7.93 (1H, d, J = 7.6), 7.33-7, 16 (10H, m), 6.76 (1H, m), 5.82 (1H, m), 5.62 (1H, d, J = 5.2), 4.76 (1H, d, J = 12.0), 4.65 (1H, m), 4.62 (1H, d, J = 12.2), 4.47 (1H, m), 3.88 (2H, s), 2.77 (1H, dd, J = 9.0, 18.0), 2.5 (dd), 1.23 (3H, d, J = 7.0).

(3S) 3- [N- (pyrrole-2-carbonyl) -L-alaninyl] amino-4-oxobutanoic acid (36a; D). A mixture of compound (35a) (612 mg, 1.65 mmol), methanol (40 ml) and 10% palladium on carbon (500 mg) was stirred vigorously under a hydrogen atmosphere for 4 hours. The mixture was filtered through 0.2 µΜ nylon membrane and then concentrated. The residue was purified by flash chromatography (5-10% methanol in methylene chloride) and precipitation from a mixture of ethyl acetate and ether gave the hemihydrate of compound (36a) (223 mg, 48%) as a white solid. There were traces of a solvent in the product: mp. 96-100 ° C; IR (KBr) 3381, 1774, 1729 (EtOAc), 1632, 1558, 1523, 1123; Ή NMR (CD₃OD): δ 6.94-6.85 (2H, m), 6.17 (1H, dd, J = 3.8 and 2.6), 4.58 (0.5H, d, J = 3, 94), 4.56 (0.5H, d, J = 4.24), 4.51 (1H, q, J - 7.16), 4.35-4.20 (1H, m), 2, 74-2.40 (2H, m), 1.42 (3H, 2xd, J = 7.13).

(3 S) 3 - [N - (5-benzylpyrrole-2-carbonyl) -L-alaninyl] amino-4-oxobutanoic acid (36b) was prepared (41%) from 35b as described for 36a to give off white solid: mp. 109-112 ° C; [and]_D²⁵ + 6.3 ° (c 0.3, methanol); IR (KBr) 3368, 1724, 1630, 1530, 1453, 1414, 1233, 1049; Ή NMR (d₄ methanol): δ 7.25-7.11 (5H, m), 6.76 (1H, d, J = 3.5), 5.84 (1H, d, J = 3.5), 4.51 (1H, m), 4.43 (1H, q, J = 7.1), 4.23 (1H, m), 2.5 (2H, m), 1.35 (3H, d, J = 7 , 0).

Analysis for C._I9H.₂₁N₃ABOUT₅. 1.75H₂ABOUT.

Calculated C, 56.64; H, 6.13; N, 10.43.

Found C, 56.34; H, 5.72; N, 10.00.

185 693

<img file="PL185693B1_D0050.tif" />

<img file="PL185693B1_D0051.tif" />

(2R, S, 3S) l- (indole-2-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L -prolinamide (38).

To a solution of (2R, S, 3S) 1-tert-butoxycarbonyl-N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-prolinamide (15) (0.607 g, 1.5 mmol) in dichloromethane (4 ml) at 0 ° C, trifluoroacetic acid (4 ml) was added. The mixture was stirred at 0 ° C for 75 minutes. The mixture was concentrated and the residue was treated with diethyl ether and then the ether was removed in vacuo. This procedure was repeated twice to give a yellow oil which was dissolved in DMF (12 ml). Diisopropylethylamine (0.78 mL, 4.5 mmol) was added to the solution followed by indole-2-carboxylic acid (266 mg, 1.65 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (316 mg, 1 65 mmol) and hydroxybenzotriazole (405 mg, 3 mmol). The mixture was stirred at room temperature for 20 hours then poured into brine. The mixture was extracted with ethyl acetate (3 x 30 ml). The combined organic extracts were washed with saturated aqueous sodium bicarbonate (2 x 60 mL) followed by brine (2 x 60 mL), dried (MgSO₄) and concentrated. The residue was purified by column chromatography (ethyl acetate) to obtain 518 mg (77%) of a mixture of diastereomers: IR (KBr) 3314, 1780, 1677, 1609, 1524, 1435, 1406, 1344; Ή NMR (d₆-DMSO): δ 11.58 (1H, m), 8.81-8.41 (1H, m), 7.71-6.67 (10H, m), 5.70 (d, J = 5, 2), 5.48 (s), 4.89-4.29 (4H, m), 3.99-3.74 (2H, m), 3.20-2.44 (2H, m), 2 , 39-1.77 (4H, m).

(3S) 3- [1- (indole-2-carbonyl) -L-prolinyl] amino-4-oxobutanoic acid (39). A mixture of (2R, S, 3S) 1- (indole-2-carbonyl) -N- (tetrahydro-2-benzyloxy-5-oxo-3-furanyl) -L-prolinamide (38) (478 mg, 1.07 mmol ), 10% palladium on carbon (475 mg) and methanol (150 ml) were stirred under a hydrogen atmosphere for 6 hours. The resulting mixture was filtered and concentrated to give a colorless glass. After recrystallization from a mixture of methanol and diethyl ether, 202 mg (53%) of a colorless glassy solid were obtained: mp. 135-138 ° C; [and]_D²⁴ - 44 ° (0.25, CH₃OH); IR (KBr) 3287, 2977, 2879, 1781, 1725, 1716, 1667, 1662, 1600, 1529, 1441, 1346; Ή NMR (CD₃OD): δ 7.65 (1H, d, J = 8.0), 7.44 (1H, d, J = 8.4), 7.22 (1H, m), 7.09-6.64 (2H, m), 4.62 (2H, m), 4.29 (1H, m), 4.15-3.73 (2H, m), 2.74-1.72 (6H, M).

<img file="PL185693B1_D0052.tif" />

<img file="PL185693B1_D0053.tif" />

185 693

Methyl 2- (3,5-dihydro-7-methyl-4-oxo-4H-pyrrolo [3,2-d] pyrimidin-3-yl) acetate (40). To a solution of ethyl 3- [N- (dimethylamino) methylene) arnino-4-methylpyrrole-2-carboxylate (1.56 g, 7.0 mmol; Lim et al., J. Org. Chem., 44, p. 3826 -29 (1979)) in dry methanol (60 ml) was added with stirring freshly prepared methyl glycinate (1.25 g, 14 mmol). The resulting mixture was kept at 70 ° C. After 18 and 42 hours of heating, two portions of methyl glycinate (1.25, 14.0 mmol) were added. The mixture was cooled and filtered 24 hours after the last addition. The filtrate was concentrated and the residue was purified by flash chromatography (2-5% methanol / chloroform) to afford 0.54 g (35%) of a white crystalline solid: mp. 233-235 ° C (recrystallized from ethyl acetate); IR (KBr) 3135, 2958, 1745, 1675, 1254; Ή NMR (d₆-DMSO): δ 11.90 (1H, s), 8.07 (1H, s), 7.23 (1H, s), 4.83 (2H, s), 3.69 (3H, s), 2.16 (3H, s).

Analysis for C.₁₀H._nN₃ABOUT₃ 0.1 H.₂ABOUT.

Calculated C, 53.85; H, 5.07; N, 18.84.

Found C, 53.85; H, 4.96; N, 18.81;

MS (70 eVe.I) m / e 222,221 (M⁺, 100%), 189, 162, 133, 105.

2- (3,5-Dihydro-7-methyl-4-oxo-4H-pyrrolo [3,2-d] pyrimidin-3-yl) -acetic acid sodium salt (41). A suspension of compound 40 (354 mg, 1.6 mmol) in methanol (15 mL) was treated with 0.5N sodium hydroxide (4.8 mL), and the resulting mixture was stirred at 25 ° C for 1 hour. The reaction mixture was filtered to give compound 41 hemihydrate (354 mg, 97%) as a white crystalline solid: mp. &gt; 340 ° C (recrystallized from methanol); IR (KBr) 3461, 3143, 1676, 1666, 1605, 1415; Ή NMR (d₆-DMSO): δ 11.63 (1H, s), 7.83 (1H, s), 7.11 (1H, d, J = 2.0), 4.24 (2H, s), 2.14 (3H, s).

Analysis for C.₉H.₈N₃ABOUT₃On. 0.5H₂ABOUT.

Calculated C, 45.39; H, 3.81; N, 17.64.

Found C, 45.57; H, 4.05; N, 17.39.

(2R, S, 3S) 2- (3,5-dihydro-7-methyl-4-oxo-4H-pyrrolo [3,2-d] pyrimidin-3-yl) -N- (tetrahydro-2-benzyloxy- 5-oxo-3-furanyl) acetamide (42). A suspension of compound 41 sodium (344 mg, 1.5 mmol) in dry DMF (15 mL) was treated with ethyldimethylaminopropylcarbodiimide hydrochloride (373 mg, 1.95 mmol) and 1-hydroxybenzotriazole (405 mg, 3.0 mmol). The mixture was held at 25 ° C for 1 hour then (2R, S, 3S) N-allyloxycarbonyl-3-amino-2-benzyloxy-5-oxotetrahydrofuran (437 mg, 1.5 mmol; Chapnam, Biorg) was added. Med. Chem. Lett., 2. pp. 613-618 (1992)) and (Ph₃P)₂PdCl₂(25 mg) then n-tributyltin hydride (0.6 mL, 2.25 mmol) was added dropwise. The resulting mixture was stirred at 25 ° C for 1 hour, then water (20 mL) was added. The mixture was extracted with ethyl acetate (3 x 15 ml) and the combined organic extracts washed with water (5 ml), dried (MgSO₄) and concentrated to give a mixture of diastereomers. Evaporation of the aqueous phase and purification of the residue by flash chromatography (5% methanol / chloroform) gave additional compound for a total of 182 mg of compound 42 (31%); mp temp. 240-242 ° C; IR (KBr) 3274, 1772, 1691, 1664, 1562; Ή NMR (d₆-DMSO): δ 11.81 (1H, s), 8.85 (0.6H, d, J = 6.6), 8.72 (0.4H, d, J = 7.4), 7, 98 (O, 6H, s), 7.95 (, 0.4H, s), 7.40-7.30 (5H, m), 7.20 (1H, d, J = 2.2), 5 . 61 (0.4H, d, J = 7.5), 5.46 (s), 4.85-4.60 (m), 4.28 (m), 3.20-2.35 (2H , m), 2.16 (3H, s).

(3S) -3- [2- (3,5-Dihydro-7-methyl-4-oxo-4H-pyrrolo [3,2-d] pyrimidin-3-yl) -1-oxo-ethylamino] acid -4-oxobutane (43). A mixture of compound 42 (131 mg, 0.33 mmol) in methanol (50 ml) and 10% palladium on carbon (100 mg) was vigorously stirred under a hydrogen atmosphere for 2 hours. More catalyst (100 mg) was added and the mixture was hydrogenated for an additional 2 hours. The mixture was filtered through 0.2 µΜ nylon membrane and concentrated. The residue was recrystallized from methanol and diethyl ether to give 79 mg (78%) of compound 43 as a hygroscopic white solid; mp temp. 222-226 ° C (decomp.); [and]_D³²+ 0.5 ° (c 0.02, MeOH); IR (KBr) 3282, 1680, 1558, 1425, 1275; Ή NMR (CD₃OD): δ 8.03 (1H, s), 7.18 (1H, d, J = 0.7), 4.79-4.74 (2H, m), 4.63-4.59 (1H , 2 xd, J = 3.6), 4.36-4.25 (1H, m), 2.78-2.39 (2H, m), 2.24 (3H, d, J = 0.7 ).

185 693

Analysis for C.₁₃H.₁₄N₄ABOUT₅Na · 1.4 H.₂ABOUT.

Calculated C, 47.10; H, 5.12; N, 16.90.

Found C, 47.00; H, 4.79; N, 16.59.

FABMS m / e 307, 306 (M⁺), 244, 207, 190, 152, 115 (100%).

<img file="PL185693B1_D0054.tif" />

<img file="PL185693B1_D0055.tif" />

<img file="PL185693B1_D0056.tif" />

<img file="PL185693B1_D0057.tif" />

(a) Σ eo (b) X = H.₂ T-Butyl (1S, 9S) 6,10-dioxo-octahydro-9- (3-phenylpropionylamino) -6H-pyridazine [1,2-a] [1,2] diazepine-1-carboxylate (44a). To a solution of t-butyl (1S, 9S) 9-amino-6,10-dioxo-octahydro-6H-pyridazine [1,2-a] [1,2] diazepine-1-carboxylate (690 mg, 2.32 mmol ; GB 2128984) in dioxane (16 ml) and water (4 ml) at 0 ° C was added solid sodium bicarbonate (292 mg, 3.48 mmol) followed by dropwise addition of 3-phenylpropyl chloride (470 mg, 2 78 mmol). The mixture was stirred at room temperature for 2 hours then additional sodium bicarbonate (200 mg; 2.38 mmol) and 3-phenylpropionyl chloride (100 mg, 0.6 mmol) were added. The mixture was stirred at room temperature for an additional 2 hours, diluted with ethyl acetate (50 ml), washed with saturated sodium bicarbonate solution (2 x 25) then dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (0-50% ethyl acetate / chloroform) and finally crystallized by trituration with ether to afford 860 mg (86%) of a white solid: mp. 137-138 ° C; [and]_D²³ -95.1 ° (c 0.549, CH₂Customs₂); IR (KBr) 3327, 1736, 1677, 1664, 1536, 1422, 1156; Ή NMR (CDC1₃): δ 7.24 (5H, m), 6.50 (1H, d, J = 7.5), 5.24 (1H, m), 4.90 (1H, m), 4.60 (1H , m), 3.44 (1H, m), 2.93 (2H, m), 2.84 (1H, m), 2.64 (1H, m), 2.54 (2H, m), 2 , 26 (2H, m), 1.70 (4H, m), 1.70 (9H, s). MS (FAB, m / z): 430 (M.⁺ + 1), 374, 242, 105, 91.

T-Butyl (1S, 9S) octahydrodro-10-oxo-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxylate (44b) was prepared from (1S , 9S) 9-amino-octahydro-10-oxo-6H-pyridazine [1,2-a] [1,2] t-butyl diazepine-1-carboxylates (Attwood et al., J, Chem. Soc. Perkin 1, p. 1011-19 (1986)) as compound 44a to give 810 mg (81%) as a colorless oil; [and]_D²³ -33.5 ° (c 0.545, CH₂C1₂); IR (KBr) 3334, 2935, 1737, 1728, 1659, 1642; Ή NMR (CDCl₃): δ 7.24 (5H, m), 6.75 (1H, d, J = 6.7), 5.27 (1H, m), 4.92 (1H, m), 3.39 (1H, m ), 3.03 (4H, m), 2.55 (3H, m), 2.33 (1H, m), 2.17 (1H, m), 1.80 (5H, m), 1.47 (9H, s), 1.39 (1H, m). MS (FAB, m / z): 416 (M.⁺ + 1), 360, 211, 143, 97.

185 693

(1S, 9S) 6,10-Dioxo-octahydro-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxylic acid (45a). To a solution of (1S, 9S) t-butyl (44a) 6,10-dioxo-octahydro-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxylate (800 mg, 1.863 mmol) in dry dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) at 0 ° C. The solution was stirred at room temperature for 3 hours and then concentrated. Dry ether (10 mL) was added to the residue then removed in vacuo. This operation was repeated three times to obtain a crystalline solid. This material was triturated with ether and filtered to give 590 mg (85%) of a white crystalline solid: mp. 196-197.5 ° C; [and]_D²³ -129.5 ° (c 0.2, CH₃OH); IR (KBr) 3237, 1688, 1660, 1633, 1574, 1432, 1285, 1205; Ή NMR (CD₃OD): δ 8.28 (1H, d, J = 7.4), 7.22 (5H, m), 5.32 (1H, dd, J = 5.9, 2.9), 4.75 (1H, m), 4.51 (1H, m), 3.50 (1H, m), 3.01 (1H, m), 2.91 (2H, m), 2.55 (2H, m) , 2.29 (3H, m), 1.95 (2H, m), 1.71 (2H, m).

Analysis for C.₁₉H.₂₃N₃ABOUT₅.

Calculated C, 61.12; H, 6.21; N, 11.25.

Found C, 60.80; H, 6.28; N, 10.97.

MS (FAB, m / z) 374 (M.⁺ + 1), 242, 105, 91.

(1S, 9S) octahydro-10-oxo-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxylic acid (45b) was prepared from octahydro-10- t-butyl oxo-9- (3-phenylpropionyl-amino) -6H-pyridazine [1,2-a] - [1,2] diazepine-1-carboxylate (44b) as described for compound 45a to give 657 mg (96 %) of 45b as a crystalline solid: mp. 198-202 ° C; [and]_D²³ -86.2 ° (c 0.5, CH₃OH); IR (KBr) 3294, 2939, 1729, 1645, 1620, 1574, 1453, 1214; Ή NMR (CD₃OD): δ 7.92 (1H, d, J = 7.9), 7.20 (5H, m), 5.29 (1H, m), 4.90 (1H, m), 3.47 ( 1H, m), 3.08 (2H, m), 2.90 (2H, m), 2.55 (3H, m), 2.36 (1H, m), 1.81 (5H, m), 1.43 (2H, m). MS (FAB, m / z) 360 (M.⁺ + 1), 211, 143, 91.

[3S, 2R, S, (IS, 9S)] N- (2-benzyloxy-S-oxotetrahydrofuran-3-yl) -6.10-dioxo-octahydro-9- (3-phenylpropionylamino) -6H- pyridazine- [1,2-a] [1,2] diazepine-1-carboxamide (46a). For a solution of (1S, 9S) 6,10-dioxooctahydro-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] -diazepine-1-carboxylic acid (45a) (662 mg , 1.773 mmol) in dry dichloromethane (9 ml) and dry dimethylformamide (3 ml) at room temperature were added bis (triphenylphosphine) palladium chloride (30 mg) and (3S, 2R, S) -3-allyloxycarbonylamino-2benzyloxy-5-oxotetrahydrofuran (Chapnam, Biorg. Med, Chem. Lett., 2 p. 613-618 (1992)) and then tri-n-butyltin hydride (1.19 g, 4.09 mmol) was added dropwise. 1-Hydroxy-benzotriazole (479 mg, 3.546 mmol) was added to the mixture and the mixture was cooled to 0 ° C, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (408 mg, 2.128 mmol) was added. The mixture was stirred at room temperature for 3.25 hours, then diluted with ethyl acetate (50 ml), washed twice with dilute hydrochloric acid (20 ml), twice with saturated sodium bicarbonate solution (20 ml), once with brine, then dried (MgSO 4).₄) and concentrated. The resulting oil was purified by flash chromatography (0-100% ethyl acetate / chloroform) to afford 810 mg (81%) of 46a as a mixture of anomers: mp. 92-94 ° C; IR (KBr) 3311, 1791, 1659, 1651, 1536; Ή NMR (CDC1₃): δ 7.49, 6.56 (1H, 2d, J = 6.7, 7.8), 7.29 (10H, m), 6.37, 6.18 (1H, 2d, J = 7 , 7, 7.6), 5.56, 5.34 (1H, d, s, J = 5.2), 5.08-4.47 (6H), 3.18-2.80 (5H) , 2.62-2.28 (5H), 2.04-1.53 ​​(5H). MS (FAB, m / z) 563 (M.^r + 1), 328, 149, 91.

[3S, 2R, S, (1S, 9S)] N- (2-benzyloxy-5-oxotetrahydrofuran-3-yl) -octahydro-10-oxo-9- (3-phenylpropionylamino) -6H-pyridazine- [1 , 2-a] [1,2] diazepine-1-carboxamide (46b) was prepared from 45b as described for 46a to give 790 mg (96%) of a glass: mp. 58-60 ° C; IR (KBr) 3316, 2940, 1793, 1678, 1641, 1523, 1453, 1120; Ή NMR (CDC1₃): δ 7.28 (10H, m), 6.52, 6.42 (1H, 2d, J = 7.2, 7.1), 5.53, 5.44 (1H, d, s, J = 5.2), 5.35 (1H, m), 4.34 (4H, m), 3.1-2.8 (6H, m). 2.6-2.1 (7H), 1.95-1.05 (5H). MS (FAB, m / z) 549 (M.⁺ + 1), 400, 310, 279, 91.

Acid [3S, (1S, 9S)] 3- (6,10-dioxooctahydro-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxamido) - 4-oxobutane (47a). A mixture of [3S, 2R, S, (1S, 9S)] N- (2-benzyloxy-5-oxotetrahydrofuran-3-yl) -6.10-dioxooctahydro-9- (3-phenyl)

185 693 propionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxamide (46a) (205 mg; 0.364 mmol), 10% palladium on carbon (200 mg) and methanol (20 ml ) was stirred under an atmospheric pressure of hydrogen for 5 hours. The mixture was filtered and then concentrated to give 154 mg (90%) of a glass: mp. 116-118 ° C; [and]_D²³ -140 ° (c 0.1, CH₃OH); IR (KBr) 3323 (br), 1783, 1731, 1658, 1539, 1455, 1425; Ή NMK (CD₃OD): δ 7.21 (5H, m), 5.17 (1H, m), 4.73 (1H, m), 4.50 (2H, m), 4.23 (1H, m), 3 , 38 (1H, m), 3.06 (1H, m), 2.91 (2H, m), 2.73-2.18 (6H, m) and 2.01-1.59 (5H, m ).

Analysis for C.₂₃H.₂₇N₄O + H.₂ABOUT.

Calculated C, 56.32; H, 6.16; N, 11.42.

Found C, 56.29; H, 6.11; N, 11.25.

MS (FAB, m / z) 473 (M.⁺ + 1), 176, 149, 105, 91.

Acid [3S, (1S, 9S)] 3- (octahydro-10-oxo-9- (3-phenylpropionylamino) -6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxamido) - 4-Oxobutane (47b) was prepared from 46a by the method described for 47a. The residue was purified by flash chromatography (0-10% methanol / chloroform) to give 65 mg (52%) of a glass: mp. 8790 ° C; [and]_D²³ -167.0 ° (c 0.1, methanol); IR (KBr) 3329, 2936, 1786, 1727, 1637; Ή NMR (CD₃OD): δ 7.23 (5H, m), 5.29 (1H, m), 4.83 (1H, m), 4.59 (1H, d, J = 3.6), 4.29 ( 1H, m), 3.3-3.0 (3H, m), 2.91 (2H, m), 2.70-2.34 (5H, m), 2.19 (2H, m), 1 , 75 (4H, m), 1.36 (2H, m).

Analysis for C.₂₃H.₃₀N₄ABOUT₆ + 0.5H₂ABOUT.

Calculated C, 59.09; H, 6.68; N, 11.98.

Found C, 58.97; H, 6.68; N, 11.73.

MS (FAB, m / z) 459 (M.⁺+ 1),310, 149, 105,91.

<img file="PL185693B1_D0058.tif" />

<img file="PL185693B1_D0059.tif" />

49

<td></td><td>R,</td><td>r₂</td><td>Rj</td>

<td>(and)</td><td>PhCH₂</td><td>H.</td><td>(S) Me</td>

<td>(b)</td><td>PhCH₂</td><td>CH₂Ph</td><td>H.</td>

<td>(c)</td><td>PhCH₂</td><td>(CH₂)₂Ph</td><td>H.</td>

<td>(d)</td><td>PhCH₂</td><td>nBu</td><td>H.</td>

<td>(e)</td><td>PhCH₂</td><td>Me</td><td>H.</td>

<td>(f)</td><td>PhCH₂</td><td>Ph</td><td>H.</td>

<td>(g)</td><td>PhCH₂</td><td>H.</td><td>H.</td>

<td>(h)</td><td>PhCH₂</td><td>CH₂Ph</td><td>(S) -Me</td>

<td>(and)</td><td>Ph (CH₂)₂</td><td>CH₂Ph</td><td>H.</td>

Pyridones 48 were prepared as described by Damewood et al., J. Med. Boor,. 37, pp. 3303-12 (1994)). Compound 48d is new.

3-benzyloxycarbonylamino-6-butyI-pyrid-2-one (48d) was isolated as a cream solid: mp. 158-160 ° C; IR (KBr) 3382, 2953, 2930, 2866, 1729, 1643, 1524, 1468, 1202, 1044. NMR (d₆-DMSO): δ 8.26 (1H, s), 7.72 (1H, d), 7.39 (5H, m), 6.00 (1H, d), 5.14 (2H, s), 2.41 (2H, t), 1.52 (2H, m), 1.24 (2H, m), 0.87 (3H, t).

185 693

Analysis for C.₁₇H.₂₀N₂ABOUT₃.

Calculated C, 67.98; H, 6.71; N, 9.33.

Found C, 67.69; H, 6.68; N, 9.20.

MS CI M⁺ = 300 (m)) 28%.

Methyl (2S) 2- [3-benzyloxycarbonylamino-1,2-dihydro-2-oxo-1-pyridyl] propionate (49a). To a mixture of 3- (benzyloxycarbonylamino) pyrid-2-one (48a) (2.58 g, 10.58 mmol) and tetrahydrofuran (100 ml) at room temperature was added sodium hydride (80% oil dispersion) (0.35 g, 11.64 mmol). The mixture was stirred for 10 minutes. To a solution of methyl 2 (R) 2 ((trifluoromethane) sulfonyloxy) propionate (2.5 g, 10.58 mmol; Feenstra et al. Tetrahedron Lett., 28, p. 1215-18 (1987)) in tetrahydrofuran (5 ml) at room temperature was added the solution prepared above in 10 minutes. The mixture was stirred at room temperature for 80 minutes then poured into ethyl acetate. The mixture was washed twice with 1M HCl, twice with aq. Sodium bicarbonate, then with brine then dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (30% ethyl acetate / hexane) to afford 2.945 g (84%) of a colorless solid: mp. 96-7 ° C; [and]_D²⁰ -71.36 (c 2.5, CHCl₂); IR (KBr) 3370, 1764, 1729, 1648, 1602, 1564, 1523, 1515, 1503, 1449, 1359, 1203, 1064; Ή NMR (CDC1₃): δ 8.04 (1H, d, J = 7.2), 7.86 (1H, s), 7.36 (5H, m), 6.98 (1H, dd, J = 7.1, J = 7.1), 6.30 (1H, t, J = 7.2), 5.46 (1H, q, J = 7.4), 5.20 (2H, s), 3.74 ( 3H, s), 1.66 (3H, d, J = 7.4).

Analysis for C._l7H._lgN₂ABOUT₅.

Calculated C, 61.81; H, 5.49; N, 8.48.

Found C, 61.49; H, 5.51; N, 8.41.

MS (FAB, m / z) 331 (M⁺ + 1), 299, 223, 196, 163, 91.

Methyl [6-benzyl-3-benzyloxycarbonylamino-1,2-dihydro-2-oxo-1-pyridyl] acetate (49b). Sodium hydride (80% oil dispersion) was added to a mixture of 6-benzyl-3- (benzyloxycarbonylamino) pyrid-2-one (48b) (7.3 g, 2.18 mmol) and tetrahydrofuran (150 ml) at room temperature ( 0.65 g, 26.2 mmol). The mixture was stirred for 10 minutes, treated with methyl bromoacetate (2.5 mL, 26.2 mmol) and left for 3 hours. The resulting mixture was poured into a mixture of ice and 1M HCl. The resulting solid was filtered off and then dissolved in dichloromethane. The resulting solution was dried (MgSO₄), decolorized with charcoal and concentrated. The residue was purified by chromatography (2-5% ethyl acetate / dichloromethane) to give 7.2 g (81%) of colorless crystals: mp. 117-9 °; IR (KBr) 3375, 1753, 1730, 1651, 1605, 1513, 1384, 1223, 1185, 1071; Ή NMR (CDC1₃): δ 8.02 (1H, d, J = 7.5), 7.78 (1H, s), 7.31 (8H, m), 7.10 (2H, m), 6.15 (1H , d, J = 7.45), 5.20 (2H, s), 4.70 (2H, s), 3.88 (2H, s), 3.66 (3H, s).

The following compounds were prepared in a similar manner: Methyl [3-benzyloxycarbonylaminol, 2-dihydro-2-oxo-6-phenethyl-1-pyridyl] -acetate (49c). 97% yield; mp temp. 102-4 ° C. IR (KBr) 3245, 2323, 1741, 1725, 1648, 1600, 1526, 1216; Ή NMR (d₆-DMSO): δ 8.45 (1H, s), 7.76 (1H, d, J = 7.6), 7.35 (10H, m), 6.15 (1H, d, J = 7, 6), 5.15 (2H, s), 4.85 (2H, s), 3.68 (3H, s), 2.86 (4H, s).

Methyl [3-benzyloxycarbonylamino-6-butyl-1,2-dihydro-2-oxo-1-pyridyl] -acetate (49d). 90% efficiency; mp temp. 112 ° C. IR (KBr) 3393, 1738, 1731, 1645, 1598, 1517, 1225, 1208; Ή NMR (d₆-DMSO): δ 8.39 (1H, s), 7.78 (1H, d, J = 7.7), 7.35 (5H, m), 6.17 (1H, d, J = 7, 7), 5.15 (2H, s), 4.80 (2H, s), 3.67 (3H, s), 1.38 (6H, m), 0.89 (3H, t).

Methyl [3-benzyloxycarbonylamino-1,2-dihydro-6-methyl-2-oxo-1-pyridyl] -acetate (49e). Yield 84% as a colorless solid: mp. 115-6 °; IR (KBr) 3246, 1740, 1725, 1649, 1598, 1535, 1417, 1365, 1259, 1219, 1193, Ή NMR (d₆-DMSO): δ 8.40 (1H, s), 7.75 (1H, d, J = 7.6), 7.38 (5H, m), 6.20 (1H, d, J = 7, 6), 5.15 (2H, s), 4.85 (2H, s), 3.68 (3H, s), 2.26 (3H, s).

Methyl [3-benzyloxycarbonylamino-1,2-dihydro-6-phenyl-1-pyridyl] -acetate (49f). Yield 67% as colorless oil: IR (KBr) 3266, 1739, 1727, 1646, 1606, 1566, 1517, 1490, 1365, 1213, 1163, 1075; Ή NMR (CDC1₃) δ 8.16 (1H, d), 7.85 (1H, s), 7.39 (10H, m), 6.22 (1H, d), 5.22 (2H, s), 4.57 (2H, s), 3.74 (3H, s).

185 693

Methyl [3-benzyloxycarbonylamino-1,2-dihydro-2-oxo-1-pyridyl] -acetate (49g). Yield 80% in the form of a colorless crystalline substance: mp. 110-111 ° C. IR (KBr) 3385, 1745, 1650, 1601, 1512, 1502, 1378, 1369, 1358, 1215, 1195, 1162, 1067; Ή NMR (CDC1₃): δ 8.06 (1H, d), 7.84 (1H, s), 7.36 (5H, m), 6.88 (1H, dd), 6.27 (1H, t), 5, 20 (2H, s), 4.68 (2H, s), 3.78 (3H, s).

Analysis for C.₁₆H.₁₆N₂ABOUT₅.

Calculated C, 60.75; H, 5, 10; N, 8.85.

Found C, 60.55; H, 5.15; N, 8.85.

MS FAB (+) M + = 317 (M + 1).

Methyl 2-methyl- [6-benzyl- (3-benzyloxycarbonylamino) -1,2-dihydro-2-oxo-1-pyridyl] -acetate (49h) was prepared as used for 49a to afford an oil (58%); [and]/⁵ -25.0 ° (c 1, CH₂C1₂); IR (KBr) 3381, 1736, 1650, 1604, 1513, 1218, 1190, 1068; Ή NMR (CDC1₃): δ 7.97 (1H, d), 7.78 (1H, s), 7.4-7.14 (10H, m), 6.17 (1H, d), 5.19 (2H, s ), 4.64 (1H, q), 3.98 (2H, s), 3.62 (3H, s), 1.31 (3H, d).

Methyl [6-benzyl-1,2-dihydro-2-oxo-3 - (2-phenylethoxy) carbonylamino-1-pyridyl] acetate (49i) was prepared (88%) as a colorless solid: mp. 130-133 ° C; IR (KBr) 3363, 1746, 1732, 1651, 1604, 1515, 1368, 1231, 1212, 1185; Ή NMR (CDC1₃): δ 8.00 (1H, d, J = 7.0), 7.68 (1H, s), 7.36-7.10 (10H, m), 6.15 (1H, d, J = 7.6), 4.7 (2H, s), 4.38 (2H, t, J = 7.0), 3.88 (2H, s), 3.67 (3H, s), 2.98 (2H, t, J = 7).

<img file="PL185693B1_D0060.tif" />

<img file="PL185693B1_D0061.tif" />

Methyl (S) 2 [3-amino-1,2-dihydro-2-oxo-1-pyridyl] propionate (50a). A mixture of methyl 2 (S) -2 [3-benzyloxycarbonylamino-1,2-dihydro-2-oxo-1-pyridyl] propionate (49a) (2.75 g, 8.33 mmol), methanol (100 ml) and 10 % palladium on carbon (300 mg) was stirred under a hydrogen atmosphere for 30 minutes. The mixture was filtered and concentrated to give 1.63 g (100%) of a colorless solid; Ή NMR (d₆-DMSO): δ 8.35 (1H, brs), 7.46 (1H, d), 7.22 (1H, d), 6.29 (1H, t), 5.22 (1H, q), 3.63 (3H, s), 1.55 (3H, d).

The following compounds were prepared in a similar manner:

Methyl [3-amino-6-benzyl-1,2-dihydro-2-oxo-1-pyridyl] acetate (50b). Yield: 100% as a gray solid: mp. 134-6 ° C; IR (KBr) 3418, 3312, 1723, 1658, 1596, 1548, 1435, 1290, 1245, 1011; Ή NMR (d₆-DMSO): δ 7.25 (5H, m), 6.45 (1H, d, J = 7.4), 5.92 (1H, d, J = 7.4), 5.00 (2H, s), 4.63 (2H, s), 3.88 (2H, s), 3.51 (3H, s).

Methyl [3-amino-1,2-dihydro-2-oxo-6-phenethyl-1-pyridyl] acetate (50c). Yield 99%. as a viscous oil: IR (KBr) 3456, 341, 2953, 1745, 1649, 1600, 1548, 1219; Ή NMR (CDC1₃): δ 7.25 (5H, m), 6.51 (1H, d, J = 7.4), 5.92 (1H, d, J = 7.4), 4.79 (2H, s) , 3.77 (3H, s), 2.80 (4H, m). Methyl [3-amino-6-butyl-1,2-dihydro-2-oxo-1-pyridyl] acetate (50d). Yield: 97% as a brown solid: mp. 75-7 ° C; IR (KBr) 3437, 3342, 2955, 1745, 1655, 1609, 1550, 1432, 1301, 1222, 1200; Ή NMR (CDC1₃) δ 6.53 (1H, d, J = 6.8), 5.93 (1H, d, J = 6.8), 4.81 (2H, s), 3.77 (3H, s), 2.44 (2H, t), 1.45 (4H, m), 0.93 (3H, t).

Methyl [3-amino-1,2-dihydro-6-methyl-2-oxo-1-pyridyl] acetate (50e) was isolated (100%) as a colorless crystalline solid: mp. 87-9 ° C; IR (KBr) 3442, 3326, 1735, 1647, 160.0, 1549, 1434, 1407, 1383, 1366, 1225, 1209; Ή NMR (d₆-DMSO): δ 6.40 (1H, d, J = 7.3), 5.93 (1H, d, J = 7.3), 4.86 (2H, s), 4.79 (2H, s), 3.67 (3H, s), 2.15 (3H, s).

Methyl [3-amino-1,2-dihydro-2-oxo-6-phenyl-1-pyridyl] acetate (50e) was isolated (86%) as a gray solid: mp. 207-9 ° C; IR (KBr) 3473, 3345, 1750, 1644, 1600, 1536, 1443, 1336, 1309, 1212, 1184, 1156; Ή NMR (d₆-DMSO): δ 7.30 (5H, m), 6.54 (1H, d), 6.03 (1H, d), 5.25 (2H, s), 4.49 (2H, s), 3.61 (3H, s).

185 693

Methyl [3-amino-1,2-dihydro-2-oxo-1-pyridyl] acetate (50g) was obtained as a colorless oil and used directly in the next step.

Methyl (2S) 2-methyl- [3-amino-6-benzyl-1,2-dihydro-2-oxo-1-pyridyl] acetate (50h) isolated (58%) as a colorless oil: IR (shell) 3354 , 1743, 1646, 1600, 1548, 1494, 1309, 1268, 1227, 113; Ή NMR (C.₆D₆): δ 7.29-6.76 (5H, m), 5.86 (1H, d, J = 7.2), 5.51 (1H, d, J 7.2), 4.43 (1H , q, J = 6.7), 3.69 (2H, s), 3.21 (2H, s), 3.36 (3H, s), 1.43 (3H, d, J = 6.7 ).

<img file="PL185693B1_D0062.tif" />

51

<td>R,</td><td>R&gt;</td><td>r₃</td><td></td>

<td>(and)</td><td>Ph (CH₂)₂WHAT</td><td>H.</td><td>(S) Me</td>

<td>(b)</td><td>Ph (CH₂)₂WHAT</td><td>CH₂Ph</td><td>H.</td>

<td>(c)</td><td>Ph (CH₂)₂WHAT</td><td>(CH₂)₂Ph</td><td>H.</td>

<td>(d)</td><td>Ph (CH₂)₂WHAT</td><td>nBu</td><td>H.</td>

<td>(e)</td><td>Ph (CH₂)₂WHAT</td><td>Me</td><td>H.</td>

<td>(f)</td><td>Ph (CH₂)₂WHAT</td><td>Ph</td><td>H.</td>

<td>(g)</td><td>Ph (CH₂)₂WHAT</td><td>H.</td><td>H.</td>

<td>(h)</td><td>Ph (CH₂)₂WHAT</td><td>CH₂Ph</td><td>(S) -Me or (R, S) -Me</td>

<td>(and)</td><td>AcTyr</td><td>CH₂Ph</td><td>H.</td>

<td>(and)</td><td>Ph (CH₂)₂SO₂</td><td>CH₂Ph</td><td>H.</td>

<td>(k)</td><td>Ph (CH₂)₂ABOUT WHAT</td><td>CH, Ph</td><td>H.</td>

<td>(ABOUT</td><td>Ph (CH₂)₃WHAT</td><td>CH₂Ph</td><td>H.</td>

Methyl 2 (S) 2- [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] propionate (5a). To a mixture of methyl 2S 2- [3-amino-1,2-dihydro-2-oxo-1-pyridyl] propionate (50a) (1.63 g, 8.33 mmol), dioxane (60 ml), water (15 ml) and sodium bicarbonate (1.54 g, 16.7 mmol) were added dropwise with stirring 3-phenylpropionyl chloride (1.5 g, 9 mmol). The mixture was left for 1 hour and then extracted with ethyl acetate. The extracts were washed with aqueous sodium bicarbonate, dried (MgSO₄) and concentrated. The resulting red oil was purified by flash chromatography to give 2.54 g (93%) of an oil: [a]_D²⁰ -68 ° (1, CH₂Customs₂); IR (CH₂C1₂) 3369, 1747, 1690, 1650, 1602, 1512, 1267, 1260, 1217; Ή NMR (CDC1₃): δ 8.41 (1H, dd), 8.36 (1H, s), 7.24 (5H, m), 7.02 (1H, dd), 6.32 (1H, t), 5, 44 (1H, q), 3.75 (3H, s), 3.03 (2H, t), 2.70 (2H, t), 1.66 (3H, d). FAB M + = 329 (M + 1), 197, 165, 131, 110.91.

The following compounds were prepared in a similar manner: Methyl [6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -acetate (51b) was isolated (93%) as crystals: temp. maturity 95-7 °; IR (KBr) 3265, 1747, 1686, 1642, 1590, 1563, 1511, 1454, 1401, 1220, 1183, 1133; Ή NMR (CDC1₃): δ 8.39 (1H, d, J = 7.7), 8.27 (1H, s), 7.21 (10H, m), 6.17 (1H, d, J = 7.7) , 4.70 (2H, s), 3.89 (2H, s), 3.67 (3H, s), 3.02 (2H, m), 2.70 (2H, m).

Methyl [1,2-dihydro-2-oxo-6-phenethyl-3- (3-phenylpropionyl) amino-1-pyridyl] -acetate (51c) was isolated (81%) as colorless crystals: m.p. 105-8 ° C; IR (KBr)

185 693

3378 1746, 1680, 1646, 1597, 1517, 1221; Ή NMR (CDC1₃): δ 8.34 (1H, d, J = 7.7), 8.25 (1H, s), 7.23 (10H, m), 6.11 (1H, d, J = 7.7) , 4.77 (2H, s), 3.78 (3H, s), 2.88 (8H, m).

Methyl [6-butyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -acetate (5Id) was isolated (88%) as colorless crystals: m.p. 84-5 ° C; IR (KBr) 3345 2958, 2930, 1756, 1693, 1650, 1602, 1510, 1227, 1180, 1137; Ή NMR (CDC1₃): δ 8.34 (1H, d, J = 7.7), 8.22 (1H, s), 7.26 (5H, m), 6.12 (1H, d, J = 7.7) , 4.80 (2H, s), 3.79 (3H, s), 3.03 (2H, t), 2.68 (2H, t), 2.50 (2H, t), 1.46 ( 4H, m), 0.95 (3H, t).

Methyl [1,2-dihydro-6-methyl-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -acetate (51e) was isolated (100%) as a pale yellow oil: IR (shell) 3264, 1745, 1691, 1644, 1587, 1566, 1518, 1495, 1400, 1215, 1183, 1136; Ή NMR (CDC1₃): δ 8.33 (1H, d, J 7.6), 7.26 (5H, m), 6.13 (1H, d, J = 7.6), 4.83 (2H, s), 3.79 (3H, s), 3.03 (2H, m), 2.69 (2H, m), 2.28 (3H, s).

Methyl [1,2-dihydro-2-oxo-6-phenyl-3- (3-phenylpropionyl) amino-1-pyridyl] acetate (51f) was isolated (99%) as a pale yellow oil: IR (shell) 3365. 3299, 1751, 1689, 1643, 1600, 1563, 1519, 1493, 1419, 1370, 1224; Ή NMR (CDC1₃): δ 8.46 (1H, d, J - 7.7), 8.32 (1H, s), 7.32 (10H, m), 6.24 (2H, d, J = 7.7) , 4.57 (2H, s), 3.73 (3H, s), 3.06 (2H, s), 2.72 (2H, m).

Methyl [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -acetate (51f) was isolated (81%) as an oil: IR (shell) 3330, 1753, 1689, 1650 , 1600, 1560, 1517, 1374, 1225, 1208; Ή NMR (CDCI₃): δ 8.43 (1H, dd, J - 7.4, 1.7), 8.33 (1H, s), 7.28 (5H, m), 6.92 (1H, dd, J = 6.9, 1.7), 6.29 (1H, t), 4.67 (2H, s), 3.79 (3H, s), 3.04 (2H, m), 2.70 (2H , m). MS FAB (+) M + = 315 (M + 1).

Methyl 2 (S) 2-methyl- [6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] acetate (5Ig) was isolated (93%) as colorless oil; [and]_D³⁰ -19 ° (c 1, CH₂C1,); IR (coating) 3354, 3313, 3028, 2950, ​​1745, 1687, 1645, 1600, 1567, 1514, 1454, 1225; Ή NMR (CDC1₃): δ 8.35 (1H, d, J = 7.5), 8.26 (1H, s), 7.27 (10H, m), 6.20 (1H, d, J = 7.5) , 4.65 (1H, q, J = 6.8), 3.99 (2H, s), 3.71 (3H, s), 3.03 (2H, m), 2.68 (2H, m ). 1.31 (3H, d, J = 6.8).

Methyl [3- (N-acetyl-O-benzyl-L-tyrosine) amino-6-benzyl-1,2-dihydro-2-oxopyridyl] acetate (5l). A mixture of methyl [3-amino-6-benzyl-1,2-dihydro-2-oxo-1-pyridyl] acetate (100 mg, 0.367 mmol), Boc-Tyr (Bn) -OH (136 mg, 0.367 mmol), dimethylformamide (1 mL), diisopropylethylamine (0.25 mL, 1.468 mmol), and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (118 mg, 0.367 mmol) was kept at room temperature while stirring. night. The mixture was diluted with ethyl acetate, washed twice with 1M hydrochloric acid, twice with aq. Sodium bicarbonate, once with brine, then dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (10% ethyl acetate / dichloromethane) to afford 162 mg (70%) of a colorless oil. The oil (160 mg, 0.255 mmol) was dissolved in dichloromethane (1 mL) and treated at 0 ° C with trifluoroacetic acid (1 mL). The resulting solution was allowed to warm to room temperature for 40 minutes, then evaporated to dryness at 30 ° C. The residue was dissolved in dichloromethane then re-evaporated to dryness. This procedure was repeated three times. The residue was dissolved in pyridine (0.5 mL) and treated with acetic anhydride (0.03 mL, 0.3 mmol) at 0 ° C. The resulting mixture was allowed to warm to room temperature and held at that temperature for 3.5 hours. It was then diluted with ethyl acetate, washed twice with 1M hydrochloric acid, twice with aq. Sodium bicarbonate solution, dried (MgSO₄) and concentrated to give 128 mg (86%) of a colorless oil; IR (coating) 3290, 1751, 1649, 1602, 1568, 1513, 1455, 1438, 1375, 1224, 1179; Ή NMR (CDC1₃): δ 8.78 (1H, s), 8.33 (1H, d, J = 7.6), 7.33 (8H, m), 7.11 (4H, m), 6.86 (2H , d, J = 8.5), 6.47 (1H, d, J = 7.6), 6.12 (1H, d, J = 7.6), 4.99 (2H, s), 4 , 85 (1H, m), 4.69 (2H, s), 3.87 (2H, s), 3.62 (3H, s), 3.08 (2H, m), 1.96 (3H, s).

Methyl [6-benzyl-1,2-dihydro-2-oxo-3- (2-phenylethanesulfonyl) amino-1-pyridyl] acetate (51j). To a mixture of methyl [3-amino-6-benzyl-2-oxo-1,2-dihydro-1-pyridyl] -acetate (49b) (1.0 g, 3.67 mmol), dichloromethane (15 ml) and triethylamine (1.0 mL, 7.34 mmol) was added with stirring 3-phenylethanesulfonyl chloride (Zhong et al. J. Atu. Chem. Soc. 113, pp. 2259-63 (1991)). The mixture was allowed to stand overnight then poured into ethyl acetate.

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The resulting mixture was washed twice with aq. Sodium bicarbonate, three times with 1M hydrochloric acid, then with brine, then dried (MgSO₄) and concentrated. The resulting pale brown solid was purified by flash chromatography (10% ethyl acetate / dichloromethane) to afford 1.25 g (77%) of a pale yellow solid: mp. 92-4 ° C; IR (KBr) 3181, 1737, 1646, 1595, 1565, 1454, 1241, 1220, 1150; Ή NMR (CDC1₃): δ 7.53 (1H, d, J = 7.5), 7.29 (10H, m), 6.10 (1H, d, J = 7.5), 4.75 (2H, s) , 3.89 (2H, s), 3.67 (3H, s), 3.34 (2H, m), 3.14 (2H, m).

Methyl (6-benzyl-1,2-dihydro-2-oxo-3- (4-phenylbutyryl) amino-1-pyridyl] -acetate (511) was isolated (74%) as colorless crystals: mp 93- 95 ° C; IR (KBr) 3285, 1747, 1683, 1642, 1591, 1563, 1512, 1455, 1220, 1181; Ή NMR (CDC1₃): δ 8.39 (1H, d, J = 7.6), 8.24 (1H, s), 7.2 (10H, m), 6.18 (1H, d, J = 7.6) , 4.7 (2H, s), 3.90 (2H, s), 3.67 (3H, s), 2.69 (2H, t), 2.40 (2H, t), 2.04 ( 2H, m).

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2 (S) 2- [1,2-Dihydro-2-oxo-3- (3-phenylpropionyl) amino) -1-pyridyl] propionic acid (52a). To a solution of methyl 2 (S) 2- [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) propionate (5la) (2.39 g, 7.3 mmol) in methanol (30 mL) at 0 ° C was added 1M sodium hydroxide solution (15 mL, 15 mmol). The mixture was kept at this temperature for 2 hours, acidified with 1M hydrochloric acid (15.1 ml) and extracted with ethyl acetate. The extracts were washed with brine, dried (MgSO₄) and concentrated to give 1.98 g (87%) of a colorless solid: [α] _D^{2 (f}-75 ° (1, CH₂C1₂); IR (KBr) 3301, 1724, 1693, 1637, 1563, 1523, 1453, 1233, 1216, 765; Ή NMR (CDC1₃): δ 8.47 (2H, m), 7.20 (5H, m), 7.03 (1H, d), 6.36 (1H, t), 5.35 (1H, q), 3, 01 (2H, m), 2.70 (2H, m), 1.69 (3H, m).

The following compounds were prepared in a similar manner: [6-Benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetic acid (52b) was isolated (100%) as a pale amber oil: IR ( coating) 3291, 1738, 1686, 1644, 1591, 1554, 1519, 1496, 1454, 1403, 1215, 1182; Ή NMR (CDC1₃): δ 8.44 (1H, d, J = 7.8), 8.4 (1H, s), 7.21 (10H, m), 6.19 (1H, d, J = 7.8) , 4.71 (2H, s), 3.90 (2H, s), 2.99 (2H, m), 2.71 (2H, m).

[1,2-Dihydro-2-oxo-6-phenethyl-3- (3-phenylpropionyl) amino-1-pyridyl] acetic acid (52c) was isolated (92%) as a beige solid: mp. 214-6 °; IR (KBr) 3289, 1740, 1680, 1640; Ή NMR (d₆-DMSO): δ 9.24 (1H, s), 8.14 (1H, d, J = 7.7), 7.22 (10H, m), 6.11 (1H, d, J = 7, 8), 4.78 (2H, s), 2.81 (8H, m).

[6-Butyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] acetic acid (52d) was isolated (99%) as a pale brown solid: mp. 132-4 ° C; IR (KBr) 3286, 1739, 1676, 1641, 1584, 1555, 1535, 1455, 1414, 1249, 1227, 1204; Ή NMR (CDC1₃): δ 8.42 (1H, d, J = 7.8), 8.37 (1H, s), 7.24 (5H, m), 6.19 (1H, d, J = 7.8) , 4.82 (2H, s), 3.55 (1H, s), 3.00 (2H, t), 2.67 (2H, t), 2.53 (2H, t), 1.41 ( 4H, m), 0.94 (3H, t).

[1,2-Dihydro-6-methyl-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] acetic acid (52e) was isolated as a solid (100%): mp. 159-61 ° C; IR (KBr) 3335, 1731, 1686, 1642, 1536, 1516, 1430, 1420, 1401, 1222, 1195; Ή NMR (d₆-DMSO): δ 9.21 (1H, s), 8.13 (1H, d. J = 7.6), 7.20 (5H, m), 6.15 (1H, d, J = 7.6) , 4.77 (2H, s), 2.87 (2H, m), 2.70 (2H, m), 2.25 (3H, s).

[1,2-Dihydro-2-oxo-6-phenyl-3- (3-phenylpropionyl) amino-1-pyridyl] acetic acid (52f) was isolated (100%) as a pale yellow foam: IR (KBr) 3271, 1747 , 1683, 1634, 1580, 1536, 1490, 1406, 1392, 1365, 1235, 1219, H NMR (CDC1₃): δ 8.62 (1H, d, J = 7.7), 7.31 (10H, m), 6.48 (2H, s), 6.30 (1H, d, J = 7.7) , 4.60 (2H, s), 3.03 (2H, m), 2.71 (2H, m).

[1,2-Dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] acetic acid (52g) was isolated (94%) as a colorless solid: mp. 195-7 ° C; IR (KBr) 3324, 1724, 1693, 1644, 1569, 1555, 1512, 1427, 1370, 1240; Ή NMR (d₆-DMSO): δ 9.31

185 693 (1Η, s), 8.23 ​​(1H, d, J = 6.8), 7.36 (1H, dd, J = 6.8, 1.71), 7.25 (5H, m), 6.25 (1H, t), 4.66 (2H, s), 2.84 (4H, m).

2 (R, S) 2- [6-Benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -propionic acid (52h) was prepared by hydrolysis of compound 5 Ih in aqueous tetrahydrofuran for 5 hours at 40 ° C. Yellow oil (95%) was obtained: IR (coating) 3330, 1734, 1686, 1643, 1600, 1587, 1553, 1524, 1498, 1208; Ή NMR (d₆-DMSO): δ 9.29 (1H, s), 8.18 (1H, d, J = 7.6), 7.21 (10H, m), 6.22 (1H, d, J = 7, 6), 4.82 (1H, q, J = 6.6), 4.08 (2H, m), 2.76 (4H, m), 1.05 (3H, d, J = 6.6) .

[3- (Acetyl-Tyr (Bn)) amino-6-benzyl-1,2-dihydro-2-oxo-1-pyridyl] acetic acid (52i) was isolated (93%) as a foam: IR (KBr) 3302 , 1731, 1646, 1603, 1562, 1512, 1454, 1428, 1379, 1231, 1178; Ή NMR (CDC1₃): δ 9.48 (1H, s) , 8.36 (1H, d, J = 7.6), 7.30 (8H, m), 7.10 (2H, m), 6.85 (2H , d, J = 8.3), 6.91 (2H, d, J = 8.3), 6.71 (1H, d, J = 7.6), 4.95 (1H, m), 4 , 90 (2H, s), 4.68 (2H, s), 3.92 (2H, s), 3.17-2.83 (2H, m), 1.92 (3H, s).

[6-Benzyl-1,2-dihydro-2-oxo-3- (2-phenylethanesulfonyl) amino-1-pyridyl] acetic acid (52j) was isolated (100%) as a colorless solid: mp. 165-7 ° C;

IR (KBr) 3174, 1760, 1646, 1593, 1567, 1497, 1453, 1424, 1326, 1225, 1140, 1127; Ή NMR (d₆-DMSO): δ 13.09 (1H, s), 9.08 (1H, s), 7.30 (11H, m), 6.02 (1H, d), 4.68 (2H, s), 4.99 (2H, s), 3.29 (2H, m), 3.03 (2H, m).

[6-Benzyl-1,2-dihydro-2-oxo-3 - (2-phenylethoxy) carbonylamino-1-pyridyl] acetic acid (52k) was prepared (70%) by hydrolysis of 49i for 1 hour at 60 ° C ; IR (CH₂C1₂) 1797, 1689, 1649, 1601, 1512, 734; Ή NMR (CDC1₃): δ 8.39 (1H, s), 8.03 (1H, d), 7.81 (1H, s), 7.33-7.07 (10H, m), 6.13 (1H, d , J = 7.8), 4.72 (2H, s), 4.33 (2H, t, J = 7.0), 3.86 (2H, s), 2.93 (2H, t, J = 7.0).

[6-Benzyl-1,2-dihydro-2-oxo-3- (4-phenylbutyryl) amino-1-pyridyl] acetic acid (521) was isolated (100%) as a white foam: m.p. 159-161 ° C; IR (KBr) 3373-3310, 1787, 1726, 1691, 1649, 1599, 1567, 1517, 1367, 1215; Ή NMR (CDC1₃): δ 8.43 (1H, d, J = 7.7), 8.25 (1H, s), 7.37-7.09 (10H, m), 6.21 (1H, d, J = 7.7), 4.73 (2H, s), 4.15 (3H, s), 3.91 (2H, s), 2.67 (2H, t), 2.39 (2H, t), 2.02 (2H, m).

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⁵² 53

2 (S), N-3 (S) 2- [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran-3 -yl) propionamide (53a). To a mixture of 2 (S) -2- [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] propionic acid (52a) (1.1 g, 3.49 mmol), 3 ( 3), 2 (R, S) 3-allyloxycarbonylamino-2-benzyloxy-5-oxo-tetrahydrofuran (1.02, 3.49 mmol; Chapman, Biorg. Med. Chem. Łett., 2, p. 613-18 (1992)), bis (triphenylphosphine) palladium (II) chloride (55 mg), dichloromethane (35 ml) and dimethylformamide (1 ml) were added dropwise with stirring tri-n-butyltin hydride (1.7 ml, 6 3 mmol). The resulting mixture was stirred for 5 minutes and then 1-hydroxybenzotriazole (946 mg, 7 mmol) was added. The mixture was cooled to 0 ° C and 1- (3-dimethylaminopropyl) -2-ethylcarbodiimide hydrochloride (740 mg, 3.84 mmol) was added. The mixture was allowed to stand overnight at room temperature then poured into ethyl acetate. The mixture was washed twice with 1M hydrochloric acid, twice with aq. Sodium bicarbonate, then with brine, dried (MgSO₄) and concentrated.

The residue was triturated with pentane. The resulting solid was purified by flash chromatography (40-60% ethyl acetate / hexane) to afford 1.28 g (73%) of a colorless solid: IR (KBr) 1796, 1692, 1647, 1595, 1557, 1512, 1119; Ή NMR (d H 2 -DMSO): δ 9.28, 9.26 (1H, 2xs), 8.77, 8.69 (1H, 2xd), 8.24, 8.20 (1H, 2x dd), 7.20 (11H, m), 6.31.

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6.26 (IH, 2 xt), 5.65 (0.5H, d), 5.46 (0.5H, d), 5.41, 5.28 (IH, 2 xq), 4.7 ( 2.5H, m), 4.24 (0.5H, t), 3.24 (2H, m), 2.80 (4H, m), 1.51, 1.46 (3H, 2xd).

The following compounds were prepared in a similar manner:

N (3 (S)) 2 [6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl] amino-1-pyridyl] -N (2-benzyloxy-5-oxotetrahydrofuran-3-yl) acetamide (53b) prepared as foam (86%): IR (KBr) 3345, 3297, 1807, 1791, 1688, 1679, 1650, 1602, 1525, 1497, 1453, 1372, 1257, 1119; Ή NMR (d₆-DMSO): δ 9.25 (0.5H, s), 9.23 (0.5H, s), 8.75 (0.5H, d, J = 6.5), 8.67 (0, 5H, d, J = 7.4), 8.18 (IH, 2d), 7.21 (15H, m), 6.07 (IH, 2d), 5.65 (0.5H, d, J = 5.0), 5.38 (0.5H, s), 4.83-4.45 (4.5H, m), 4.19 (0.5H, m), 3.94, 3.83 ( 2H, m), 3.10-2.31 (6H, m).

N (3 (S)) 2 (1,2-dihydro-2-oxo-2-phenethyl-3- (3-phenylpropionyl) amino-1-pyridyl] -N (2-benzyloxy-5-oxotetrahydrofuran-4-yl ) acetamide (53c) was prepared (74%) as a mixture of anomers: Ή NMR (d₆-DMSO): δ 9.71 (IH, d), 9.41 (0.5H, d), 9.25 (0.5H, d), 8.64 (IH, d, J = 7.7) , 7.75 (15H, m), 6.61 (IH, 2d), 6.11 (0.5H, d), 5.93 (0.5H, s), 5.17 (5H, m), 4.77 (0.5H, m), 3.68-2.94 (2H, m), 3.32 (8H, m).

N (3 (S)) 2 [6-butyl-1,2-dihydro-2-oxo-3 - (3-phenylpropionyl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran-3-yl) acetamide (53d) was prepared (74%) as a mixture of anomers: IR (KBr) 3300, 1791, 1689, 1645, 1597, 1566, 1546, 1514, 1454, 1417, 1378; Ή NMR (CDC1₃): δ 8.38 (IH, d, J = 7.7), 8.13 (IH, s), 7.30 (10H, m), 6.18 (IH, t), 5.47 (0 , 5H, d, J = 5.2), 5.43 (0.5H, s), 4.75 (4.5H, m), 4.38 (0.5H, m), 3.08-2 , 35 (8H, m), 1.43 (4H, m), 0.95 (3H, t).

N (3 (S)) 2 [1,2-dihydro-6-methyl-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -N- (2benzyloxy-5-oxotetrahydrofuran-3-yl) acetamide (53e) was prepared (67%) as a mixture of anomers: IR (KBr) 3282, 1774, 1667, 1651, 1596, 1556, 1498, 1265, 1254, 1236, 1199, 1143; Ή NMR (d₆-DMSO): δ 9.17 and 9.15 (IH, 2 xs), 8.89 (0.5H, d, J = 6.5), 8.73 (0.5H, d, J = 7, 4), 7.25 (10H, m), 6.13 (IH, t), 5.64 (0.5H, d, J = 5.0), 5.45 (0.5H, s), 4 , 89-4.61 (4.5H, m), 4.26 (0.5H, m), 3.17-2.36 (6H, m), 2.23 and 2.15 (3H, 2s) . N (3 (S)) 2- [1,2-dihydro-2-oxo-6-phenyl-3- (3-phenylpropionyl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran-3-yl) acetamide (53f) was prepared (73%) as a mixture of anomers: IR (KBr) 3296, 1792, 1691, 1643, 1595, 1514, 1489, 1453, 1420, 1373, 1230, 1118; Ή NMR (d₆DMSO): δ 9.40, 9.36 (IH, 2s), 8.70 (0.5H, d, J = 7.6), 8.52 (0.5H, d, J = 7.5) , 8.29 (IH, dd), 7.25 (15H, m), 6.20 (IH, d, J = 7.6), 5.61 (0.5H, d, J = 5.0) , 5.28 (0.5H, s), 4.78-4.20 (5H, m), 3.12-2.24 (6H, m).

N (3 (S)) 2- [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran-3-yl) acetamide (53g) was prepared (70%) as a mixture of anomers: IR (KBr) 3336, 3290, 1791, 1691, 1646, 1595, 1582, 1556, 1518, 1454, 1376, 1351, 1150, 1122; Ή NMR (d₆-DMSO): δ 9.26 (IH, 2s), 8.86 (0.5H, d, J = 6.4), 8.67 (0.5H, d, J = 7.5), 8, 23 (IH, m), 7.40-7.13 (11H, m), 6.24 (IH, 2t, J = 7.2), 5.61 (0.5H, d, J = 5.0 ), 5.44 (0.5H, s), 4.83-4.59 (2.5H, m), 4.25 (0.5H, m), 3.15-2.34 (2H, m ), 2.91-2.70 (4H, m).

Analysis for C.₂₇H.₂₇N₃OH₂ABOUT.

Calculated C, 63.90; H, 5.76; N, 8.28.

Found C, 63.70; H, 5.68; N, 8.22.

MS FAB M⁺ = 490 (M + 1).

2 (R, S), N (3 (S)) 2- [6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -N- (2- benzyloxy-5-oxotetrahydrofuran-3-yl) propionamide (53h) was prepared (89%) as a mixture of diastereomers. Data are given for a single diastereomer: IR (coating) 3356, 1788, 1677, 1645, 1602, 1517, 1455, 1377, 1203, 1167, 1120; Ή NMR (CDC1₃): δ 8.34 (IH, d, J = 7.6), 8.19 (IH, s), 7.38-7.13 (10H, m), 6.26 (IH, d, J = 7.6), 5.58 (IH, t), 5.31, 5.24 (IH, 2 xs), 4.62 (2H, 2q), 4.60 (IH, m), 4.27 (IH, m), 2.98, 2.68 (4H, 2m), 3.0-2.0 (2H, m), 1.42 (3H, d).

N (3 (S)) 2- [6-benzyl-1,2-dihydro-2-oxo-3- (N-acetyl-O-benzyltyrosinyl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran -3-yl) acetamide (53i) was prepared (76%) as a mixture of anomers: IR (KBr) 1794, 1698, 1651, 1612, 1514, 1454, 1374, 1247, 1177, 1126; Ή NMR (d₆-DMSO): δ 9.34, 9.31 (2 x 0.5H, 2s), 8.71 (IH, 2d), 8.38 (IH, m),

185 693

8.17 (1Η, d), 7.48-6.88 (19H, m), 6.08 (1H, 2d), 5.65 (0.5H, d, J = 5.0), 5, 40 (0.5H, s), 5.04 (2H, s), 4.68 (5.5H, m), 4.15 (0.5H, m), 3.95, 3.84 (2H, s + abq), 3.20-2.40 (4H, m), 1.78 (3H, s).

N (3 (S)) 2- [6-benzyl-1,2-dihydro-2-oxo-3- (2-phenylethanesulfonyl) amino-1-pyridyl] N- (2-benzyloxy-5-oxotetrahydropyran-3- yl) acetamide (53j) was prepared (78%) as a mixture of anomers: IR (KBr) 3344, 1792, 1691, 1647, 1599, 1454, 1365, 1150, 1121, 973; Ή NMR (d₆-DMSO): δ 9.02, 8.99 (1H, 2s), 8.80 (0.5H, d, J = 6.4), 8.70 (0.5H, d, J = 7.4 ), 7.26 (15H, m), 6.00 (1H, dd), 5.63 (0.5H, d, J = 5.0), 5.39 (0.5H, s), 4. 68 (4.5H, m), 4.18 (0.5H, m), 3.90 (2H, m), 3.30-2.30 (6H, m).

N (3 (S)) 2- [6-benzyl-1,2-dihydro-2-oxo-3- (2-phenylethoxy) carbonylamino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran-3 -yl) acetamide (53k) was prepared (78%) as a mixture of anomers: IR (KBr) 3386, 1794, 1726, 1650, 1603, 1518, 1366, 1214, 699; Ή NMR (CDC1₃): δ 8.03 (1H, bd), 7.63, 7.61 (1H, 2 xs), 7.34-7.04 (15H, m), 6.21, 6.18 (1H, 2d ), 5.44 (0.5H, d, J = 5.4), 5.37 (0.5H, s), 4.85, 4.83 (1H, 2d, J = 11.6, 11, 5), 4.61-4.48, 4.32 (4H, 2m), 4.4 (2H, t), 4.08, 4.03 (2H, 2bs), 3.07-2.78 ( 3H, m), 2.47-2.30 (1H, m).

N (3 (S)) 2- [6-benzyl-1,2-dihydro-2-oxo-3- (4-phenylbutyryl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran-3) -yl) acetamide (531) was prepared (86%) as a colorless oil: IR (CH, C1₂) 1797, 1689, 1649, 1601, 1512, 734; Ή NMR (CDC1₃): δ 8.42, 8.40 (1H, 2d, J = 7.6), 7.35-7.07 (15H, m), 6.21, 6.19 (1H, 2d, J = 7 , 6), 5.44 (0.5H, d), 5.37 (0.5H, s), 4.84, 4.81 (1H, 2d, J = 11.7, 11.4), 4 , 73-4.48, 4.34 (4H, 2m), 4.05 (2H, m), 3.052.63, 2.46-2.30 (6H, 2m), 2.01 (2H, m) .

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3 (S), N (2 (S)) 3- (2- (1,2-dihydro-2-oxo-3- (3-phenylpropionylamino-1-pyridyl) propionylamino) -4-oxobutanoic acid (54a; F A mixture of 2 (S), N (3 (S)) 2- [1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl] -N- (2-benzyloxy-5-oxotetrahydrofuran) -3-yl) propionamide 53a (1.28 g, 2.5 mmol), methanol (140 ml), ethyl acetate (60 ml) and 10% palladium on carbon (1.4 g) were stirred under a hydrogen atmosphere. , 5 hours more catalyst was added

185 693 (300 mg) and the hydrogenation was continued for 1 hour. The mixture was filtered through 0.2 µΜ nylon membrane and concentrated. The residual oil was triturated with a mixture of methanol and ether to give 916 mg (87%) of colorless crystals: mp. 198-200 ° C; [and]²⁸_D -120 ° (0.1, CH₃OH); IR (KBr) 3330, 1794, 1688, 1644, 1583, 1556, 1515, 1427; Ή NMR (CD₃OD): δ 8.28 (1H, d), 7.35 (1H, d), 7.20 (5H, m), 6.36 (1H, t), 5.49 (1H, q), 4 . 59 (1H, t), 4.25 (1H, m), 2.98, 2.74 (2 × 2H, 2 × m), 2.59 (2H, m), 1.57 (3H, d) .

Analysis for C.₂₁H.₇₃N₃ABOUT₆ 0.75H₂ABOUT.

Calculated: C, 59.08; H, 5.78; N, 9.84.

Found: C. 59.24; H, 5.96; N, 9.84.

FAB M⁺ = 414 (M + 1), 297, 165, 91.

The following compounds were prepared in a similar manner:

3 (S) 3- (6-Benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54b; M) was isolated (59%) in the form of colorless crystals: mp. 115 ° C (decomposition); IR (KBr) 3440, 3297, 1718, 1646, 1598, 1565, 1526, 1496, 1260; 1 H NMR (CD₃OD): δ 8.25 (1H, d, J = 7.7), 7.25 (10H, m), 6.15 (1H, 2d, each J = 7.7), 4.73 (2H, 2q), 4.59 (1H, m), 4.30 (1H, m), 3.95 (2H, s), 2.98 (2H, m), 2.75 (2H, m), 2, 8-2. 42 (2H, m).

Analysis for C.₂₇H.₂₇N₃ABOUT₆ 0.7H₂ABOUT

Calculated C, 64.58; H, 5.70; N, 8.37.

Found C, 64.51; H, 5.63; N, 8.38.

MS FAB + M + = 490 (M + 1).

3 (S) 3- (1,2-Dihydro-2-oxo-6-phenethyl-3- (3-phenylpropionyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54c) was isolated (46%) as white solid: IR (KBr) 3375, 1694, 1643, 1586, 1561, 1515, 1377, 1254, 1188, 1070; Ή NMR (CD₃OD): δ 8.18 (1H, d, J = 7.8), 7.22 (10H, m), 6.15 (1H, d, J = 7.8), 4.75 (2H, s ), 4.58 (1H, m), 4.30 (1H, ml), 3.01-2.28 (10H, m); MS FAB + M + = 504 (M + 1).

3 (S) 3- (6-Butyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54d) was isolated (90%) as colorless crystals: mp. 120-5 ° C; IR (KBr) 3315, 1784, 1679, 1644, 1589, 1561, 1520, 1415, 1379, 1186; 1 H NMR (CD₃OD): δ 8.22 (1H, d, J = 7.8), 7.24 (5H, m), 6.22 (1H, d, J = 7.8), 4.80 (2H.m ), 4.60 (1H, s), 4.28 (1H, m), 2.98 (2H, m), 2.72 (2H, m), 2.58 (4H, m), 1.48 (4H, m). 0.97 (3H, t, J = 7.1).

Analysis for C.₂₄H.₂₉N₃ABOUT₆ 0.5H₂ABOUT.

Calculated C, 62.06; H, 6.51; N, 9.05.

Found C, 62.08; H, 6.43; N, 9.01.

MS FAB + M + = 456 (M + 1).

3 (S) 3- (1,2-Dihydro-6-methyl-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54e) was isolated (85%) of a colorless substance constant: temp, mp. 129-138 ° C; IR (KBr) 327, 3294, 1710, 1695, 1682, 1554, 1525, 1379, 1272, 1240; Ή NMR (CD₃OD): δ 8.19 (1H, d, J = 7.6), 7.19 (5H, m), 6.21 (1H, d, J = 7.6), 4.80 (2H, m ), 4.59 (1H, m), 4.30 (1H, m), 2.98 (2H, m), 2.72 (2H, m), 2.80-2.40 (2H, m) . 2.30 (3H, s).

Analysis for C.₂₁H.₂₂N₃ABOUT₆ H.₂ABOUT

Calculated C, 58.46; H, 5.84; N, 9.74.

Found C, 58.82; H, 60.5, N, 9.42.

3 (S) 3- (1,2-Dihydro-2-oxo-6-phenyl-3- (3-phenylpropionyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54f), 73% as an off-white substance constant: mp. 140 ° C (decomposition). [and]_D²⁴ - 8.5 ° (c 0.1, MeOH). IR (KBr) 3302, 1796, 1726, 1679, 1643, 1590, 1560, 1516, 1490, 1449, 1420, 1398, 1376, 1231; Ή NMR (CD₃OD): δ 8.36 (1H, d), 7.49-7.14 (10H, m), 6.27 (1H, dd), 4.54 (3H, m), 4.30 (1H, m), 3.0.2.73 (2 · 2H, 2xm), 2.7-2.9 (2H, m).

3 (S) 3- (1,2-Dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54g; G) was isolated (73%) as a foam: mp temp. 140-5 ° C (decomposition); IR (KBr) 3352, 3314, 1719, 1668, 1649, 1600, 1559, 1514, 1379, 1261; Ή NMR (CD₃OD): δ 8.32 (1H, d, J = 7.5), 7.19 (6H, m), 6.34 (1H, t), 5.1-4.6 (3H, m), 4.32 (1H, m), 2.7 (6H, m).

185 693

Analysis for C.₂₀H.₂₎N₃ABOUT₆ 0.6H₂ABOUT.

Calculated C, 58.50; H, 5.45; N, 10.24.

Found C, 58.43; H, 5.35; N, 9.85.

MS FAB + M + = 400 (M + 1).

Acid 3 (S) N (2 (R, S)) 3- (2- (6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylepropionyl) amino-1-pyridine! O) propionylamino ) -4-oxobutane (54h) was prepared (69%) as a colorless foam: mp. 120 ° C; [and]_D²⁰ -16.0 ° (c, 0.11, CH₂C1₂). IR (KBr) 3315, 1783, 1727, 1666, 1644, 1599, 1564, 1517, 1454, 1379; Ή NMR (CD₃OD): δ 8.23 ​​(1H, m), 7.27 (10H, m), 6.28 (1H, m), 4.84 (1H, m), 4.53 (1H, m), 4 , 22 (1H, m), 4.10 (2H, m), 2.96 (2H, m), 2.72 (2H, m), 2.39 (2H, m), 1.21 (3H, m).

Analysis for C.₂₈H.₂₉N₃ABOUT₆ 1.25H₂ABOUT.

Calculated C, 63.93; H, 6.03; N, 7.99.

Found C, 63.98; H, 5.85; N, 7.86.

MS FAB (+) M + = 504 (M + 1).

3 (S) 3 - (3 - (2-acetyl-L-tyrosinyl) amino-6-benzyl-1,2-dihydro-2-oxo-1-pyridyl) acetylamino-4-oxobutanoic acid (54i) was isolated (79 %) in the form of colorless crystals: mp. 193-6 ° C (decomp.); IR (KBr) 3284, 1644, 1565, 1519, 1455, 1429, 1407, 1375, 1267, 1251; Ή NMR (d₆-DMSO / CDCl₃): δ 8.16 (1H, d, J = 7.7), 7.26 (5H, m), 7.03 (2H, d, J = 8.4), 6.61 (2H, d, J = 8.4), 6.03 (1H, d, J = 7.7), 4.58 (3H, m), 4.44 (1H, m), 4.13 (1H, m), 3 M. 84 (2H, s), 3.07-2.30 (4H, m).

Analysis for C.₂₉H.₃₀N₄ABOUT₈ 2H₂ABOUT.

Calculated: 0.58.19; H, 5.72; N, 9.36.

Found: 0.58.11; H, 5.63; N, 9.29.

MS FAB + M + = 563 (M + 1).

3 (S) 3- (6-Benzyl-1,2-dihydro-2-oxo-3- (2-phenylethanesulfonyl) amino-1-pyridyl) acetylamino-4-oxobutanoic acid (54j) was isolated (85%) as colorless solid: mp. 102-5 ° C; [and]_D²³ -9.9 ° (c 0.1, MeOH); IR (KBr) 3452, 3328, 3155, 1719, 1679, 1645, 1594, 1567, 1453, 1425, 1357, 1307, 1225, 1148, 1132; Ή NMR (CD₃OD): δ 7.52 (1H, d, J = 7.6), 7.33 (10H, m), 6.12 (1H, d, J = 7.6), 4.73 (2H, m ), 4.58 (1H, d, J = 3.7), 4.34 (1H, m), 3.97 (2H, s), 3.29 (2H, m), 3.08 (2H, m), 2.75-2.37 (2H, m).

Analysis for C.₂₆H.₂₇N₃ABOUT₇S 1.7H₂ABOUT.

Calculated: 0.56.14; H, 5.51; N, 7.55.

Found C, 55.20; H, 5.49; N, 7.29.

MS FAB + M + = 526 (M + 1).

3 (S) 3 - (6-Benzyl-1,2-dihydro-2-oxo-3- (2-phenylethoxy) carbonylamino-1-pyridyl) acetylamino-4-oxobutanoic acid (54k) was isolated (54%) as off-white solid: mp. 84-86 ° C; IR (KBr) 3373-3310, 1787, 1726, 1691, 1649, 1599, 1567, 1517, 1367, 1215; Ή NMR (CD₃OD): δ 7.93 (1H, bd, J = 7.4), 7.37-7.18 (10H, m), 6.15 (1H, d, J = 7.4), 4.77 (1H, d, J = 3.7), 4.67 and 4.58 (2H, 2m), 4.35 (2H, t, J = 6.9), 4.35 (1H, m), 3 , 94 (2H, s), 2.98 (2H, t, J = 6.9), 2.76-2.39 (2H, m).

3 (S) 3- (6-Benzyl-1,2-dihydro-2-oxo-3- (4-phenylbutyryl) carbonylamino-l-pyridyl) acetylamino-4-oxobutanoic acid (541) was isolated (50%) as a white substance constant: mp. 89-93 ° C; IR (KBr) 3369-3302, 1678, 1645, 1594, 1565, 1517, 1379, 1258; Ή NMR (d₄-methanol): δ 8.25 (1H, d, J = 7.6), 7.37-7.18 (10H, m), 6.15 (1H, d, J = 7.4), 4, 74 (2H, m), 4.60 (1H, m), 4.30 (1H, m), 3.97 (2H, s), 2.76-2.37 (2H, m), 2.67 (2H, t), 2.45 (2H, t), 1.98 (2H, m).

Analysis for C.₂₈H.₂₉N₃ABOUT₆ 1.5H₂ABOUT.

Calculated C, 63.39; H, 6.08; N, 7.92.

Found C, 63.69; H, 5.74; N, 7.83.

185 693

<img file="PL185693B1_D0068.tif" />

T-Butyl N-2- (3-benzyloxycarbonylamino-1,2-dihydro-2-oxo-1-pyridyl) acetyl-3-amino-5 (2,6-dichlorobenzoyloxy) -4-oxo-pentanoate (56a). Acetic acid (55a) (WO 93 21213) in THF (2 ml) was stirred at room temperature and 1-hydroxybenzotriazole (60 mg, 0.448 mmol) and dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (47 mg, 0.246 mmol) were added. After 5 minutes, (2 drops) of water was added dropwise and stirring was continued for 20 minutes. Bis (triphenylphosphine) palladium (II) chloride (6 mg) was added followed by a solution of t-butyl 3- (allyloxycarbonylamino) -4-oxo-5- (2,6-dichlorobenzoyloxy) pentanoate (WO 93 16710) (103 mg, 0.224 mmol) in THF (1 ml). Tributyltin hydride (0.09 mL, 0.336 mmol) was added dropwise over 1 hour at room temperature. The mixture was stirred for an additional 3 hours and poured into ethyl acetate, washed with 1M HCl, aq. NaHCO₃, brine, dried over MgSO₄ and concentrated in vacuo. The residue was triturated with pentane and the supernatant was discarded. The residual solid was purified by flash chromatography (50% ethyl acetate / hexane) to yield 92 mg (63%) of the title compound as a colorless oil: [a]_D²⁶-29.6 ° (c 1.1, CH₂C1,); IR (coating) 3377, 3365, 3332, 3312, 1733, 1691, 1650, 1599, 1515, 1366, 1261, 1153, 1068, 747; Ή NMR (CDC1₃): δ 8.09 (1H, d, J = 6.8), 7.84 (1H, s), 7.58 (1H, d, J = 8.3), 7.33 (8H, m) , 7.02 (1H, dd, J = 6.9, 1.7), 6.33 (1H, t, J = 7.2), 5.20 (2H, s), 5.12 (2H, m), 4.89 (1H, dt), 4.65 (2H, m), 2.80 (2H, m), 1.38 (9H, s).

N-2- (6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetyl-3amino-5- (2,6-dichlorobenzyloxy) -4-oxo- t-Butyl pentanoate (56b) was prepared as described for 56a to give the title compound (66%) as a colorless oil: IR (coating) 3364, 3313, 1738, 1688, 1648, 1600, 1566, 1514, 1433, 1369, 1254 , 1152; Ή NMR (CDC1₃): δ 8.40 (1H, d, J = 7.6), 8.30 (1H, s), 7.28 (13H, m), 6.20 (1H, d, J = 7.6) , 5.12 (2H, q), 4.86 (1H, m), 4.65 (2H, q), 4.06 (2H, s), 3.07-2.61 (6H, m), 1.39 (9H, s).

<img file="PL185693B1_D0069.tif" />

<img file="PL185693B1_D0070.tif" />

185 693

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N-2 (3-Benzyloxycarbonylamino-1,2-dihydro-2-oxo-1-pyridyl) acetyl-3-amino-5- (2,6-dichlorobenzoyloxy) -4-oxo-pentanoic acid (57a; Q). The ester of compound 56a (210 mg, 0.356 mmol) in dichloromethane (0.5 ml) was cooled to 0 ° C and treated with trifluoroacetic acid (0.5 ml), stirred and heated to 20 ° C for 30 minutes. The solution was evaporated to dryness under reduced pressure, redissolved in dichloromethane and concentrated (x 3). The residue was triturated with ethyl acetate and diluted with ether to give 162 mg (85%) of the title compound as a colorless solid: mp. 165-8 ° C (decomp.); [and]_D²³ -38.8 ° (c 0.1 CH₃OH); IR (KBr) 3332, 3275, 1723, 1658, 1649, 1581, 1562, 1526, 1432, 1385, 1258, 1218, 1206; Ή NMR (d₆-DMSO): δ 8.96 (1H, d, J = 7.3), 8.34 (1H, s), 7.85 (1H, dd, J = 7.3), 7.58 (3H, m), 7.35 (5H, m), 6.29 (1H, t, J = 7.3), 5.26 (2H, m), 5.15 (2H, s), 4.69 (3H , m), 2.75 (2H, m).

Analysis for C.₂₇H.₂₃N₃ABOUT₉C1₂.

Calculated C, 53.66; H, 3.84; N, 6.95.

Found C, 53.36; H, 3.90; N, 6.81.

MS (+ FAB); 604 (M + 1), 285, 241, 195, 173, 149, 91.

N-2- (6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetyl-3-amino-5- (2,6-dichlorobenzoyloxy) -4 acid -oxo-pentane (57b; P) was prepared as described for 57a to give the title compound (78%) as colorless crystals: mp. 116-120 ° C (decomp.); [and]_D²⁶ -41.1 ° (c 0.1, CH₃OH); IR (KBr) 3299, 1739, 1715, 1689, 1666, 1645, 1598, 1563, 1518, 1432, 1209, 1151; Ή NMR (d₆-DMSO): δ 9.24 (1H, s), 8.88 (1H, d, J 6 = 7.6), 8.18 (1H, d, J = 7.7), 7.60 (3H , m), 7.26 (10H, m), 6.06 (1H, d, J = 7.7), 5.23 (2H, ABq), 4.69 (3H, m), 3.93 ( 2H, s). 2.78 (6H, m).

Analysis for C.₃₅H.₃₁N₃ABOUT₈C1₂ H.₂ABOUT.

Calculated C, 59.16; H, 4.68; N, 5.91.

Found C, 59.38; H, 4.53; N, 5.84.

MS (+ FAB); 694, (Cl = 35.37), (M + 1), 692 (Cl = 35.35), (M + 1).

<img file="PL185693B1_D0071.tif" />

<img file="PL185693B1_D0072.tif" />

<img file="PL185693B1_D0073.tif" />

185 693

<img file="PL185693B1_D0074.tif" />

<img file="PL185693B1_D0075.tif" />

T-Butyl CO2H (3S, 4R, S) N- (benzyloxycarbonyl) -3-amino-4- (2-benzoxazolyl) -4-hydroxybutanoate (59). To a solution of benzoxazole (250.2 mg, 2.1 mmol) in anhydrous THF (10.5 ml) at -78 ° C under N₂ 2.3M solution of n-butyllithium in hexanes (0.96 mL, 2.2 mmol) was added dropwise. After stirring at -78 ° C for 20 minutes, dry solid MgBr was added₂OEt₂ (594.0 mg, 2.3 mmol). The resulting heterogeneous mixture was warmed to -45 ° C and stirred for 15 minutes. The reaction mixture was then re-cooled to -78 ° C and aldehyde 58 solution (Graybill et al., Int. J, Peptide Protein Res., 44, pp. 173-82 (1993)) (644.6 mg, 2.1) was added dropwise. mmol) in THF (10.5 mL). The mixture was stirred at -78 ° C for 30 minutes, warmed to 0 ° C for 1 hour, and then stirred at room temperature for 16 hours. The reaction was quenched with 5% sodium bicarbonate (2.0 mL) and the THF was removed in vacuo. The resulting aqueous residue was extracted four times with methylene chloride. The combined extracts were washed with brine, dried (MgSO₄), filtered and concentrated in vacuo to give 880.0 mg of crude product. Flash chromatography (45:55 ethyl acetate / hexane) provided 567.2 mg (63%) of the title compound as an oil and a mixture of diastereomers at C-4. IR (coating) 3324, 2976, 1726, 1517, 1455, 1368, 1243, 1159, 1048, 747; Ή NMR (CDC1₃): δ 7.71-7.64 (1H, m), 7.52-7.48 (1H, m), 7.37-7.20 (7H, m), 5.91 (1H, brd, J = 9.0), 5.79 (1H, d, J = 9.0), 5.41-4.78 (4H, m), 4.75-4.54 (1H, m), 2, 91-2.51 (2H, m), 1.42 (9H, s), 1.37 (9H, s).

T-Butyl (3S, 4R, S) 3-amino-4- (2-benzoxazolyl) -4-hydroxybutanoate (60). A solution of ester 59 (189.0 mg, 0.44 mmol) in ethanol (5.0 mL) was treated with 10% palladium on carbon (20.5 mg) and stirred under an atmosphere of H₂About for 21 hours. The mixture was filtered through Celite® and the solvent was evaporated to afford 125.0 mg (98%) of the crude amine (60) as an oil. The product was used without further purification. Ή NMR (CDClJ: δ 7.73-7.64 (1H, m), 7.51-7.42 (1H, m), 7.35-7.22 (2H, m), 6.48 (3H , brs), 5.58 1H, d, J = 3.0), 5.27 (1H, d, J = 6.5), 4.23-4.05 (1H, m), 2.92- 2.63 (2H, m), 1.36 (9H, s), 1.33 (9H, s).

T-Butyl (3S, 4R, S) N- (N-benzyloxycarbonyl- (S) -valinyl- (S) -alaninyl) -3-amino-4- (2-benzoxazolyl) -4-hydroxybutanoate (61) . A solution of amine 60 (261.4 mg, 0.89 mmol), ZVal-Ala-OH (286.9 mg, 0.89 mmol) (prepared by standard peptide synthesis methods) and hydroxybenzotriazole (120.3 mg, 0.89 mmol) ) in DMF (3.0 ml) at 0 ° C was treated with 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride (179.2 mg, 0.93 mmol). The mixture was warmed to room temperature and stirred for 16 hours. Diluted with ethyl acetate and washed twice with 1M sodium hydrogen sulfate solution, twice with saturated sodium bicarbonate solution, then with water and brine. The organic layer was dried (MgSO₄), filtered and concentrated in vacuo to yield 494.8 mg of crude product. Flash chromatography (methylene chloride / methanol 95: 5) provided 480.9 mg (91%) of the title compound as a yellow solid: mp. 81-83 ° C; IR (KBr) 3312, 2974, 1723, 1709, 1529, 1455, 1368, 1243, 1156, 747; 1 H NMR (CDCl₃): δ 7.79 (0.5H, d, J = 8.0), 7.73-7.20 (9.5H, m), 6.15 (1H, t, J = 8.5), 5.74 (0.5H, brd, J = 5.5), 5.45 (1H, brd, J = 7.5), 5.28-5.20 (0.5H, m), 4.82 -4.11 (3.5H, m), 4.78-4.55 (1H, m),

185 693

40-4.22 (1Η, m), 2.95-2.51 (2H, m), 2.12-1.95 (1H, m), 1.45-1.32 (12H, m), 1.11-0.81 (6H, m), ¹³C NMR (CDC1₃) δ 173.14, 172.94, 171.82, 171.03, 170.78, 165.98, 165.45, 157.29, 157 17 151.23, 151.10, 140.92, 140, 82. 136.83, 136.79, 128.91, 128.52, 125.75, 124.97, 120 ^ 60, 120.40, 111.38, 81.82, 81.68, 70.27, 68, 97, 67.44, 60.43, 50.74, 50.55, 49.18, 49.07, 36.87, 36.57, 32.37, 28.51, 19.88, 19.80, 18.53.

Analysis for C._3!H.₄₀N₄ABOUT₈ H.₂ABOUT.

Calculated C, 60.57; H, 6.89; N, 9.11.

Found C, 60.84; H, 6.64; N, 9.09.

MS (+ FAB); 597 (M + 1); 541, 91.

T-Butyl (3S) N- (N-benzyloxycarbonyl- (S) -valinyl- (S) -alaninyl) -3-amino-4- (2-benzoxazolyl) -4-oxobutanoate (62). Alcohol 61 (100.3 mg, 0.17 mmol) was dissolved in methylene chloride (2.0 mL) and the Dess-Martin reagent (142.6 mg, 0.34 mmol) was added (Ireland et al. J. Org, Chem. , 58, p. 2899 (1993); Dess et al. J. Org. Chem., 48, pp. 4155-4156 (1983)). The resulting mixture was stirred for 22 minutes, then partitioned between saturated sodium thiosulfate solution, saturated sodium bicarbonate solution (1: 1, 10 ml) and ethyl acetate (10 ml). The resulting organic phase was washed with saturated sodium thiosulfate solution, saturated sodium bicarbonate solution (1: 1), saturated sodium bicarbonate solution and brine. The organic phase was dried (MgSO₄), filtered and concentrated in vacuo to yield 111.3 mg of crude product. Flash chromatography (methylene chloride / methanol 95: 5) provided 97.3 mg (96%) of the title compound as an oil: [a]_D²³ 11.74 ° (c 0.95, CH 2 Cl 2); IR (CH2 Cl2) 3419, 2974, 1721, 1677, 1501, 1369, 1221, 1156; Ή NMR (CDC13): δ 7.89-7.74 (1H, m), 7.73-7.22 (10H, m), 5.89 (1H, d, J = 9.0) 5.72 (1H, m), 5.10 (2H, q, J = 12.5), 4.73 (2H, m), 4.20 (1H, dd, J = 7.0, 8.5), 3 , 30; 1H, dd, J = 5.0, 16.5), 3.03 (1H, dd, J = 5.5, 16.5), 2.18-1.97 (1H, m), 1.39 (3H, d, J = 7.0), 1.34 (9H, s), 0.94 (3H, d, J = 6.0), 0.90 (3H, d, J = 6 0),^,3C NMR (CDC13) δ 186.46, 172.73, 171.90, 170.13, 157.17, 156.28, 151.16, 140.99, 136.99, 129.39, 129.08, 128.66, 128.59, 126.49, 123.06, 112.55, 82.73, 67.60, 60.84, 53.75, 49.41, 38.58, 32.05, 28, 52, 19.85, 19.32, 18.51. MS (+ FAB); 595 (M + 1); 539, 91.

(3S) N- (N-benzyloxycarbonyl- (S) -valinyl- (S) -alaninyl) -3-amino-4- (2-benzoxazolyl) -4-oxobutanoate (63Q). A solution of ester 62 (95.0 mg, 0.16 mmol) in a 1: 1 mixture of methylene chloride and trifluoroacetic acid (10.0 ml) was stirred for 1 hour under a dry atmosphere of N₂. The solution was concentrated in vacuo, taken up in ether and concentrated again. This was repeated six times to give the crude product as an off-white solid. Flash chromatography (methylene chloride / methanol 95: 5) gave 60.0 mg (69%) of the title compound as a white solid. The product was in the form of a mixture of three isomers in CD₃OD, consisting of a ketone (one isomer, c 44%), its acyloxyketal (two isomers at C-4, c 56%): m.p. 156-159 ° C; [and]_D²⁶ -45.6 ° (c 0.13, methanol); IR (KBr) 3440, 2967, 1713, 1703, 1638, 1531, 1427; Ή NMR (CD₃OD): δ 7.93-7.24 (9H, m), 5.59 (1H, brt), 5.16-5.00 (2H, m), 5.0-4.78 (1H, m), 4.50-4.22 (1H, m), 3.95-3.81 (1H, m), 3.11 (2H, d, J = 6.5), 3.05-2.92 (1H , m), 2.70-2.39 (1H, m), 2.08-1.89 (1H, m), 1.19-0.78 (9H, m).

Analysis for C.₂₇H.₃₀N₄ABOUT₈ 0.5H₂ABOUT,

Calculated C, 59.22; H, 5.71; N, 10.23.

Found C, 59.48; H, 5.36; N, 10.17.

MS (+ FAB); 539 (M + 1), 91.

185 693

<img file="PL185693B1_D0076.tif" />

(b) R¹ e H, R.² «= OH₃

7-methoxybenzoxazole (65a). A mixture of 2-nitro-6-methoxyphenol (2.62 g, 15.5 mmol) (EP 333176) and 10% palladium on carbon (130 mg) in ethanol (50.0 ml) was stirred under an atmosphere of H₂for 75 minutes. The mixture was filtered through Celite® and treated immediately with p-toluenesulfonic acid (32.0 mg) and triethyl orthoformate (6.45 mL, 38.8 mmol) then heated to reflux under N atmosphere.₂. After 20 hours, p-toluenesulfonic acid (30.0 mg) and triethyl orthoformate (6.45 ml, 38.8 mmol) were added. After heating a total of 44 hours, the mixture was allowed to cool and concentrated in vacuo. The resulting residue was purified by flash chromatography (ethyl acetate / hexane 25:75) to yield 1.97 g (85%) of the title compound as a yellow solid: mp. 28-31 ° C; IR (coating) 1629, 1497, 1434, 1285, 1097; 1 H NMR (CDCl₃): δ 8.09 (1H, s), 7.40 (1H, d, J = 8.0), 7.28 (1H, t, J = 8.0), 6.89 (1H, d, J = 8.0), 4.02 (3H, s); ¹³C NMR (CDC1₃): δ 152.84, 145.82, 142.50, 139.99, 125.75, 113.42, 108.80, 56.97.

185 693

Analysis for C._gN₇WELL₂ 0.1H₂0.

Calculated C, 63.65; H, 4.81; N, 9.29.

Found C, 63.43; H, 4.88, N, 9.05.

MS (+ FAB); 150 (M + 1).

4-methoxybenzoxazole (65b). To a suspension of 4-hydroxybenzoxazole (2.00 g, 14.8 mmol) (Musser et al., J. Med. Chem., 30, pp. 62-67 (1987)) in acetone (80.0 ml) was added dried K.₂WHAT₃ (2.25 g, 16.3 mmol) followed by iodomethane (1.38 mL, 22.2 mmol). The mixture was heated to reflux under N atmosphere₂ for 4.5 hours, then filtered and concentrated in vacuo to give the crude product. The resulting residue was purified by flash chromatography (ethyl acetate / hexane 25:75) to provide 2.0 g (91%) of the title compound as a crystalline solid: mp. 72-84 ° C; IR (KBr) 3089, 1619, 1610, 1503, 1496, 1322, 1275, 1090, 1071, 780, 741, H NMR (CDC1₃): δ 8.02 (1H, s), 7.32 (1H, t, J = 8.0), 7.18 (1H, d, J = 8.0), 6.81 (1H, d, J = 8.0), 4.04 (3H, s).

Analysis for C.₈H.₇WELL₂.

Calculated C, 64.42; H, 4.73; N, 9.39.

Found C, 64.40; H, 4.84; N, 9.31;

m / z (EI) 149 (M⁺+ 1, 100%).

T-Butyl (3S, 4R, S) N- (allyloxycarbonyl) -3-amino-4-hydroxy-4- (2- (7-methoxybenzoxazolyl)) butanoate (66a).

To a solution of 7-methoxybenzoxazole 65a (548.6 mg, 3.68 mmol) in anhydrous THF (18.5 ml) with stirring at -78 ° C under N₂ 1.56M n-butyllithium solution in hexanes (2.47 mL, 3.86 mmol) was added dropwise and a yellow colored solution was formed. After stirring at -78 ° C for 20 minutes, dry MgBr was added₂OEt₂(1.045 g, 4.05 mmol). The resulting heterogeneous mixture was warmed to -45 ° C and stirred for 15 minutes. The reaction mixture was then cooled back to -78 ° C and a solution of (S) -Alloc-Asp (t-Bu) H was added dropwise.^lb (946.4 mg, 3.68 mmol) in THF (18.5 mL). The mixture was stirred at -78 ° C for 30 minutes, warmed to 0 ° C, and stirred for 1 hour. The resulting homogeneous mixture was warmed to room temperature and stirred for 16 hours. The reaction was quenched with 5% sodium bicarbonate (3.5 mL) and the THF was removed in vacuo. The resulting aqueous residue was extracted with methylene chloride (6 x). The combined extracts were washed with brine, dried (MgSO4), filtered and concentrated in vacuo to give 1.8 g of the crude product. Flash chromatography (ethyl acetate / hexane 40:60) provided 1.21 g (81%) of the title compound, an oil, as a mixture of diastereomers at C-4; IR (CH2 Cl2) 3425, 2983, 1725, 1504, 1290, 1157, 1101; Ή NMR (CDC13): δ 7.35-7.19 (2H, m), 6.89-6.81 (1H, m), 6.00-5.57 (2H, m), 5.32- 5.05 (3H, m), 4.68-4.35 (3H, m), 4.01 (3H, s), 2.86-2.59 (2H, m), 1.45 (9H, s), 1.41 (9H, s);¹³C NMR (CDC13): δ 171.18, 171.09, 165.80, 165.30, 156.71, 156.60, 145.65, 142.76, 142.71, 140.82, 140.72 , 133.23, 125.72, 118.41, 118.21, 113.07, 112.87, 108.95, 82.16, 70.28, 69.98, 66.52, 66.39, 57 .03, 52.57, 52.29, 37.83, 36.86, 28.65.

Analysis for C.₂₀H.₂₆N₂ABOUT₇ 0.6H, O.

Calculated C, 57.57; H, 6.57; N, 6.72.

Found C, 57.49; H; 6.34, N, 6.60.

MS (+ FAB); 407 (M + 1); 351, 307, 154.

T-butyl (3S, 4R, S) N- (allyloxycarbonyl) -3-amino-4-hydroxy-4- (2- (4-methoxybenzoxazolyl) butanoate (66b) was prepared according to the method described for compound 66a to obtain 1. 29 g (26%, 68% based on recovered starting material) of the title compound as an oil and mixture of diastereomers at C-4: IR (CH₂C1₂) 3400, 1725, 1625, 1505, 1369, 1354, 1281, 1263, 1226, 1158, 1092, 1048; 'HNMR (CDC1₃): δ 7.34-7.24 (1H, m), 7.16 (1H, d, J = 8.2), 6.79 (1H, d, J = 7.9), 6.00- 5.50 (2H, m), 5.30-5.05 (3H, m), 4.70-4.35 (4H, m), 4.02 (3H, s), 2.90-2, 45 (2H, m), 1.45-1.41 (9H, 2xs).

Analysis for C.₂₀H.₂₆N₂ABOUT₇ 0.4H₂ABOUT.

Calculated C, 58.07; H, 6.53; N, 6.77.

Found C, 58.09; H, 6.41; N, 6.63.

MS (+ FAB); 407 (M + 1.88%); 351 (100).

185 693 (3S, 4R, S) N- (N-acetyl- (S) - (O-tert-butyl-tyrosinyl) - (S) -valinyl- (S) -alaninyl) -3_andmino-4-hydroxy-4- (t-butyl 2- (7-methoxybenzoxazolyl) butanoate (67a).

To a solution of benzoxazole 66a (481.9 mg, 1.19 mmol) and Ac-Tyr ('Bu) -Val-Ala-OH (585.3 mg, 1.30 mmol) in methylene chloride (3.5 ml) and DMF (3.5 ml) was added bis (triphenylphosphine) palladium (II) chloride (18.0 mg) with stirring followed by dropwise addition of tributyltin hydride (0.80 ml, 2.96 mmol). Hydroxybenzotriazole (320.4 mg, 2.37 mmol) was added and the mixture was cooled to 0 ° C. Ethyl-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride (278.2 mg, 1.42 mmol) was added and the mixture was allowed to warm to room temperature and stirred for 16.5 hours. The reaction mixture was diluted with ethyl acetate and washed twice with 1M sodium hydrogen sulfate solution, twice with saturated sodium bicarbonate solution, water and brine. The organic layer was dried (MgSO₄), filtered and concentrated to give 2.0 g of crude product. Flash chromatography (methylene chloride / methanol 95: 5) gave 844.0 mg (94%) of the title compound as a white solid: mp. 205 ° C; IR (KBr) 3399, 3304, 2977, 1729, 1643, 1506, 1367, 1290, 1161; 'HNMR (d₆-DMSO): δ 8.24-7.78 (4H, m), 7.43-7.32 (2H, m), 7.23 (2H, d, J = 8.5), 7.16- 7.07 (1H, m), 6.93 (2H, d, J = 8.5), 6.52, 6.40 (1H, 2 xd, J = 5.5, J - 5.0), 5.03, 4.78-4.49, 4.45-4.16 (5H, brt, 2xm), 4.05, 4.04 (3H, 2xs), 3.08-2.35 ( 14H, m), 2.11-1.89 (1H, m), 1.83 (3H, s), 1.49-1.32, 1.15, 1.0-0.81 (27H, x 2.xm, J = 7.0); ^l3C NMR (d₆-DMSO): δ 175.55, 175.18, 173.88, 173.75, 173.05, 169.23, 157.28, 148.55, 146.16, 143.21, 136.63, 133 , 55, 128.87, 127.17, 115.78, 111.92, 84.02, 81.50, 71.40, 61.15, 60.05, 57.79, 53.39, 51.62 , 43.76, 40.52, 34.58, 32.52, 31.60, 26.35, 23.11, 22.71, 21.76.

Analysis for C.₃₉H.₅₅N₅ABOUT₁₀ 0.5 H.₂ABOUT.

Calculated C, 61.40; H, 7.40; N, 9.18

Found C, 61.43; H, 7.31; N, 9.07.

MS (+ FAB); 754 (Nf + 1); 698, 338, 267.

(3S, 4R, S) N- (N-acetyl- (S) - (O-tert-butyl-tyrosinyl) - (S) -valinyl- (S) -alaninyl) -3amino-4-hydroxy-4 - t-Butyl - (2- (4-methoxybenzoxazolyl)) butanoate (67b) was prepared according to the method described for 67a to give 1.05 g (94%) of the title compound as a fine white powder: mp. 210-213 ° C (decomposition); IR (KBr) 3284, 2977, 1736, 1691, 1632, 1536, 1505, 1452, 1392, 1367, 1258, 1236, 1161, 1091; Ή NMR (d₆DMSO) δ 8.20-7.75 (4H, m), 7.40-7.10 (4H, m), 7.00-6.80 (3H, m), 6.45, 6.34 ( 1H, 2xd, J = 5.3, J = 5.0), 5.00-4.10 (5H, m), 4.00, 3.99 (3H, 2xs), 3.00-2 , 25 (4H, m), 1.95 (1H, m), 1.79 (3H, s), 1.39-0.80 (27H, m).

Analysis for C.₃₉H.₅₅N₅ABOUT₁₀ 0.5H₂ABOUT.

Calculated C, 61.40; H, 7.40; N, 9.18.

Found C, 61.58; H, 7.38; N, 8.91.

MS (+ FAB); 754 (M⁺ + 1, 30%); 72 (100).

(3S) N- (N-acetyl- (S) -O-tert-butyl-tyroshyl) - (S) -valinyl- (S) -alaninyl) -3-amino-4 (2- (7-methoxybenzoxazolyl)) T-Butyl-4-oxobutanoate (68a). To a suspension of alcohol 67a (641.0 mg, 0.85 mmol) in methylene chloride (46.0 mL) with stirring was added Dess-Martin reagent (1.082 g, 2.55 mmol) (Ireland et al., J. Org. Chem. 58, p. 2399 (1993); Dess et al. J. Org. Chem., 48, pp. 4155-4156 (1983). The resulting mixture was stirred for 1 hour and then partitioned between saturated sodium thiosulfate solution, saturated sodium bicarbonate solution (1: 1, 86.0 mL) and ethyl acetate (86.0 mL). The resulting organic phase was washed successively with saturated sodium thiosulfate solution, saturated sodium bicarbonate solution (1: 1), saturated sodium bicarbonate solution and brine. The organic phase was dried (MgSO₄), filtered and concentrated in vacuo to yield 660.0 mg of crude product. Flash chromatography (methylene chloride / methanol, 94: 6) gave 636.0 mg (100%) of the title compound as a white solid: mp. 209C; [and]_D²⁴ - 21.8 ° (c 0.16, methanol); IR (KBr) 3395, 3294, 2977, 1722, 1641, 1535, 1505, 1161; 'HNMR (CDC13) δ 8.43-8.16 (1H, m), 7.97-7.62 (2H, m), 7.49-7.14 (3H, m), 7.08-6 , 95 (3H, m), 6.89-6.73 (2H, m), 5.81-5.68 (1H, m), 5.16-4.86 (2H, m), 4.53 (1H, brt), 4.03 (3H, s), 3.16-2.84 (4H, m), 2.11-1.84 (4H, m), 1.46-1.14 (21H , m), 0.78 (6H, m);^,3C NMR (CDC13) δ 186.28, 173.39, 171.90, 171.19, 171.03, 169.89, 156.43, 154.75, 146.32, 142.88, 140.98, 132.31, 130.54,

185 693

126.98, 124.73, 114.95, 111.42, 82.44, 78.71, 58.92, 57.20, 54.91, 53.47, 48.77, 39.43, 38, 15, 32.79, 29.44, 28.60, 23.55, 20.27, 19.70.19.34. MS (+ FAB); 752 (M⁺ + 1); 696, 336, 265.

(3S) N- (N-acetyl- (S) - (O) -tert-butyl-tyrosinyl) - (S) -valinyl- (S) -alaninyl) -3-amino4- (2- (4-methoxybenzoxazolyl) T-Butyl-4-oxobutanoate (68b) was prepared according to the method described for ketone 68a to give 420 mg (55%) of the title compound as a white solid: mp. 211-213 ° C (decomp.); [and]_D²⁴ - 23.9 ° (c 0.82, methanol); IR (KBr) 3277, 3075, 1723, 1690, 1632, 1530, 1506, 1392, 1269, 1234, 1160, 1094; Ή NMR (CDC1₃) δ 8.15 (1H, (brs), 7.7 (2H, brs), 7.46 (1H, t, J = 8.3), 7.24 (2H, d, J = 8.3) , 7.10 (1H, brs), 7.03 (2H, d, J = 8.3), 6.83 (3H, m), 5.74 (1H, q, J = 6.9), 5 .00 (2H, m), 4.51 (1H, t, J = 7.0), 4.07 (3H, s), 3.20-2.95 (4H, m), 2.00 (4H , m), 1.42 (3H, d, J = 6.8), 1.35 (9H, s), 1.23 (9H, s), 0.86 (6H, d, J = 6.7 ). MS (+ FAB): 752 (M.⁺ + 1,7%); 72 (100).

(3S) N- (N-acetyl- (S) -tyrosinyl- (S) -valinyl- (S) -alaninyl) -3-amino-4- (2- (7-methoxybenzoxazolyl)) - 4-oxobutanoate (69a ; R). A solution of ester 68a (600.0 mg, 0.80 mmol) in a 1: 1 mixture of methylene chloride and trifluoroacetic acid (65.0 ml) was stirred for 1 hour under dry N atmosphere. The solution was concentrated in vacuo, taken up in ether and concentrated again. This was repeated six times to give the crude product as an off-white solid. Flash chromatography (95: 5 to 80:20 gradient of methylene chloride and methanol) provided 420.8 mg (83%) of the title compound as a hygroscopic white solid. The product was in the form of a mixture of three wCD isomers₃OD, consisting of a ketone (c 50%), its acyl ketone (two isomers at C-4, c 50%): temp, mp. decomposes above 150 ° C; [and]_D²⁴ - 33.2 ° (c 0.17, methanol); IR (KBr) 3300, 1715, 1658, 1650, 1531, 1517, 1204; Ή NMR (CD₃OD) δ 7.46-7.19 (2H, m), 7.16-6.91 (3H, m), 6.70-6.59 (2H, m), 5.62-5.49 ( 1H, m), 5.00-4.72 (1H, indistinct m), 4.69-4.51 (1H, m), 4.49-4.08 (2H, m), 4.05-3 , 89 (3H, m), 3.16-2.47 (4H, m), 2.05-1.78 (4H, m), 1.41-1.11, 1.050.70 (9H, 2 xm ).

Analysis for C.₃₁H.₃₇N₅ABOUT₁₀ 3H₂ABOUT.

Calculated C, 53.67; H, 6.25; N, 10.10.

Found: 53.76; H, 5.56; N, 10.28.

MS (+ FAB); 640 (M⁺ + 1); 435, 147.

(3S) N- (N-acetyl- (S) -tyrosinyl- (S) -valinyl- (S) -alaninyl) -3-amino-4- (2- (4-methoxybenzoxazolyl)) -4-oxobutanoate t- butyl (69b; S), prepared according to the method described for the acid 69a to give the hygroscopic title compound, 252 mg (96%). The product was in the form of a mixture of the three isomers in CD₃OD, consisting of a ketone and its acyloxyketal (two isomers at C-4). The product existed as a single DW isomer₆DMSO: mp. 200-203 ° C (decomposition); [and]_D²⁴ - 38.0 ° (c 0.23, methanol); IR (KBr) 3289, 2968, 1718, 1713, 1658, 1634, 1548, 1517, 1506, 1461, 1453, 1393, 1713, 1658, 1634, 1548, 1517, 1506, 1461, 1453, 1393, 1369, 1268, 1228, 1174, 1092; Ή NMR (d₆-DMSO) δ 9.20 (1H, brs), 8.71 (1H, d, J = 6.2), 8.10 (2H, m), 7.83 (1H, d, J = 8.7 ), 7.61 (1H, t, J = 8.2), 7.46 (1H, d, J = 8.2), 7.08 (3H, m), 6.65 (2H, d, J = 8.3), 5.50 (1H, q, J = 6.5), 4.50 (1H, m), 4.37 (1H, m), 4.20 (1H, m), 4. 05 (3H, s), 3.09-2.77 (4H, m), 1.94 (1H, m), 1.79 (3H, s), 1.23 (3H, d, J = 7, 0), 0.82 (6H, m).

Analysis for C.₃₁H.₃₇N₅ABOUT₁₀ 1.5H₂ABOUT.

Calculated C, 55.85; H, 6.05; N, 10.51.

Found C, 55.21; H, 5.69; N, 10.13.

MS (+ FAB); 640 (M⁺ + 1, 22%); 107 (100).

<img file="PL185693B1_D0077.tif" />

<img file="PL185693B1_D0078.tif" />

185 693

<img file="PL185693B1_D0079.tif" />

T-Butyl (3S) N- (allyloxycarbonyl) -3-amino-4-oxo-5- (1,2-dioxo-phenylethyloxy) -pentanoate (80).

To a solution of t-butyl (3S) N- (allyloxycarbonyl) -3-amino-5-bromo-4-oxoentanoate (WO 93 16710) (2.17 g, 6.20 mmol) in dimethylformamide (30 ml) was added during a mixture of potassium fluoride (792 mg, 13.6 mmol) followed by benzoylformic acid (1.02 g, 6.82 mmol). The mixture was stirred for 140 minutes, then quenched with water (50 ml) and extracted with ethyl acetate (2 x 50 ml). The combined organic extracts were washed with water (4 x 50 ml) followed by brine (50 ml), dried (MgSO₄) and concentrated.

An oil was obtained which was purified by flash chromatography (20-45% ethyl acetate in hexane) to give 2.44 g (94%) of a colorless oil: [a]_D²⁰ -35.0 ° (c 1.41, CH₂C1₂); IR (coating) 3359, 2981, 2938, 1752, 1740, 1726, 1712, 1512, 1369, 1285, 1177, 1053.991, 939, 688; Ή NMR (CDC1₃) δ 8.15 (2H, m), 7.66 (1H, m), 7.53 (2H, m), 5.90 (2H, m), 5.33 (2H, m), 5.31 (1H, d, J = 16.9), 5.18 (1H, d, J = 16.9), 4.63 (3H, m), 3.03 (1H, dd, J = 17.3, 4.6), 2.74 (1H, dd, J = 17.3, 4.9), 1.44 (9H, s). MS (CI) 420 (M.⁺ + 1, 20%); 364 (100).

T-Butyl (3S) N- (allyloxycarbonyl) -3-amino-5-hydroxy-4-oxopentanoate (81). A mixture of ester 80 (2.40 g, 5.71 mmol), tetrahydrofuran (200 ml) and 1M aqueous potassium bicarbonate (200 ml) was stirred vigorously at room temperature for 18 hours. The layers were separated and the aqueous portion was extracted with ethyl acetate (100 ml). The combined organic extracts were washed with brine (100 ml), dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (ethyl acetate in hexane) to give 1.48 g of a pale yellow oil: [a]_D²⁰ 5.9 ° (c 1.06, CH 2 Cl 2); IR (coating) 3345, 2981, 2936, 1739, 1725, 1712, 1692, 1515, 1368, 1259, 1158, 1051;¹H NMR (CDCl3) δ 5.92 (2H, m), 5.30 (2H, m), 4.36-4.69 (5H, m), 3.05 (1H, dd, J = 17.4 , 4.3), 2.93 (1H, t), 2.70 (IH, dd, J = 17.4.4.9), 1.43 (9H, s).

185 693

Analysis for C.₁₀H._2]WELL₆ 0.25H₂ABOUT

Calculated C, 53.51; H, 7.43; N, 4.80.

Found C, 53.61; H, 7.18; N, 4.71.

MS (CI) 280 (M⁺ + 1, 87%); 232 (100).

T-Butyl (3S) N- (allyloxycarbonyl) -3-amino-5- (2,6-dichlorophenyl-methoxy) -4-oxo-pentanoate (82). A mixture of alcohol 81 (1.44 g, 5.01 mmol), 2,6-dichlorobenzyl iodide (Abraham et al., J. Chem, Soc., Pp. 1605-1607 (1936)) (4.31 g, 15 0 mmol), silver oxide (2.32 g, 10.0 mmol) and dichloromethane (25 ml) was heated to reflux for 45 hours with stirring. The mixture was allowed to cool to room temperature, then diluted with water (50 ml) and then extracted with ethyl acetate (50 ml, 25 ml). The organic layer was washed with water (50 ml) then brine (50 ml), dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (10-100% ethyl acetate in hexane) to give 1.65 g (74%) of a colorless oil: [a]_D²"+ 8.8 ° (c 1.13, CH₂C1₂); IR (coating) 3339, 2980, 2935, 1724, 1712, 1503, 1438, 1368, 1246, 1156, 1106, 770; Ή NMR (CDC1₃) δ 7.33 (2H, m), 7.22 (1H, dd), 5.92 (2H, m), 5.28 (2H, m), 4.87 (2H, m), 4.67 (1H, m), 4.58 (2H, br d), 4.56 (1H, d, J = 16.9), 4.31 (1H, d, J = 16.9), 3.04 ( 1H, dd, J = 16.9.4.5), 2.77 "(1H, dd, J = 16.7, 4.9), 1.40 (9H, s).

Analysis for C.₂₀H.₂₅Cl₂WELL₆ 0.25H₂ABOUT

Calculated C, 53.28; H, 5.70; N. 3.11.

Found C, 53.15; H, 5.52; N, 2.98.

MS (CI); 446 (M⁴, 27%); 390 (100).

T-Butyl (3R, S) N- [N-phenyto-loxycarbonylvalaninylalaninyl] -3-amino-5 - (2,6-dichlorophenylmethyloxy) -4-oxo-pentanoate (83a). To a stirred solution of N-phenyl-methyloxycarbonylvalinylalanine (637 mg, 1.98 mmol) in tetrahydrofuran (40 ml) and water (1 ml) was added 1- (3-dimethylamino-propyl) -3-ethylcarbodiimide hydrochloride (379 g, 1 ml). , 98 mmol) and 1-hydroxybenzotriazole (486 mg, 3.60 mmol). The mixture was stirred for 15 minutes and then ether 82 (802 mg, 1.80 mmol) and bis (triphenylphosphine) palladium (II) chloride (ca. 5 mg) were added. Tributyltin hydride (785 mg, 725 L, 2.70 mmol) was then added dropwise over 20 minutes and the resulting solution was stirred for 3.75 h before quenching with 1M hydrochloric acid (50 mL). The mixture was extracted twice with ethyl acetate. The combined organic extracts were washed with 1M hydrochloric acid, twice with saturated aqueous sodium bicarbonate, water then brine, dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (10-30% ethyl acetate-dichloromethane) to afford 941 mg (79%) of a pale yellow solid: mp. 148-52 °; IR (KBr) 3287, 3070, 1730, 1691, 1641, 1536, 1369, 1289, 1247, 1156; Ή NMR (CDC1₃) δ 7.33 (8H, m), 7.23 (1H, dd), 6.61 (1H, br, d). 5.42 (1H, br, d), 5. -1 (2H, s), 4.85 (3H, m), 4.50 (1H, m), 4.40 (1H, d, J = 16, 9), 4.26 (1H, d, J = 16.9), 4.02 (1H, m), 2.99 (1H, dd, J = 16.8, 4.7), 2.73 ( 1H, dd, J = 16.8, 5.0), 2.09 (1H, m), 1.37 (12H, m), 0.96 (3H, d, J = 6.9), 0, 91 (3H, d, J = 6.8).

Analysis for C.₃₂H.₄₁C1₂N₃ABOUT₈ 0.25H₂ABOUT.

Calculated C, 57.25; H, 6.23; Cl, 10.57; N, 6.26.

Found C, 57.18; H, 6.23; Cl 10.58; N, 5.95.

MS (+ FAB); 667 (M⁺- 1,1%); 666 (3), 159 (25), 91 (100).

T-Butyl (3R, S) N - [(N-acetyl-Ot-butyltyrosinyl) -valaninyl-alaninyl] -3-amino-5- (2,6-dichlorophenylmethyloxy) -4-oxo-pentanoate (83b) was prepared by the method described for compound 83a to give 554 mg (64%) of a colorless solid: mp. 184-6 °; IR (KBr) 3282, 3075, 1736, 1690, 1633, 1536, 1508, 1366, 1236, 1161; Ή NMR (d₆-DMSO) 58.49 (1H, d), 8.14 (1H, d), 8.08 (1H, d), 7.84 (1H, d), 7.43 (3H, m), 7, 14 (2H, d), 6.83 (2H, d), 4. -71 (2H, s), 4.51 (2H, m), 4.36 (2H, dd), 4.17 (2H, m), 2.93 (1H, m), 1.94 (1H, m), 1.74 (3H, s), 1.37 (9H, s), 1.23 (12H, m), 0, 83 (6H, m). MS (+ FAB); 793 (M⁺ + 1.4%); 737 (5), 681 (1), 178 (40), 159 (45), 136 (100), 107 (40). MS (- FAB); 792 (20), 791 (40) 447 (100).

(R, S) N- [N- (Phenylmethyloxy) carbonylvalinyl-αIaninyl] -3-amino-5- (2,6-dichlorophenylmethyloxy) -4-oxo-pentanoic acid (84a, V). To a stirred solution of ester 83a (918 mg, 1.38 mmol) in dichloromethane (20 mL) was added trifluoroacetic acid (5 mL). Mixed

185 The mixture was stirred for 2.5 hours then evaporated to dryness. The residue was treated with ether (25 ml) and evaporated to dryness. This operation was repeated three times. The resulting product was triturated with ether (10 mL) then dried to give 730 mg (87%) of a light brown powder: mp. 156-60 ° C; IR (KBr) 3282, 2965, 1702, 1694, 1642, 1536, 1438, 1246, 1230; Ή NMR (d₆-DMSO) δ 8.48 (1H, d), 8.09 (1H, d), 7.47 (9H, m), 5.02 (2H, s), 4.70 (2H, s), 4 , 49 (1H, m), 4.37 (2H, dd), 4.27 (1H, m), 3.88 (1H, m), 2.75 (1H, dd), 2.54 (1H, dd) 1.96 (1H, m), 1.19 (3H, s), 0.84 (6H, m).

Analysis for C.₂₈H.₃₃C1₂N₃ABOUT₈ 0.5H₂ABOUT.

Calculated C, 54.27; H, 5.53; Cl, 11.45; N, 6.78.

Found C, 54.49; H, 5.39; Cl, 11.33; N, 6.73.

MS (+ FAB); 610 (M⁺ + 1, 10%); 91 (100).

(R, S) N- [N- (acetyl) tyrosinyl-valinyl-alaninyl] -3-amino-5- (2,6-dichlorophenylmethyloxy) -4-oxo-pentanoic acid (84b; W) was obtained as a colorless powder (95%) as used for compound 84a. Temp. top 165-8 °; IR (KBr) 3295, 2968, 1733, 1642, 1517, 1438, 1231, 1105; Ή NMR (d₆-DMSO) δ 9.1 (1H, br, d), 8.48 (1H, br, d), 8.14 (1H, br, d), 8.02 (1H, br, d), 7.81 (1H, br, d), 7.45 (3H, m), 7.02 (2H, d), 6.62 (2H, d), 4.70 (2H, s), 4.12-4. 53 (3H, m), 3.60 (3H, m), 2.51-2.92 (4H, m), 1.96 (1H, m), 1.75 (3H, s), 1.21 (3H, d). 0.83 (6H, m).

Analysis for C.₃, H.₃₈C1₂N₄ABOUT₉ H.₂ABOUT.

Calculated C, 53.22; H, 5.76; Cl, 10.14; N, 8.09.

Found C, 53.33; H, 5.54; Cl, 10.02; N, 7.85.

MS (+ FAB); 682 (M⁺ 2, 30%); 681 (67), 158 (100).

Example 6

Inhibition constants (Ki) and IC values ​​were obtained₅₀ for some compounds of the invention, using enzyme assays with UV visible substrate, with a fluorescent substrate and assays with cells as described in example 2. For compounds 22e, 54b, 54j, 54k, 57b, 85, 86, 87, 88, 89, 90, 91, 92, 88, 102a-c, 106a-c, 108a-c, 114a, 114b, 115, 121, 125a, 125b, 126, 127, 128, 129, 130, 131, 132a, 132b, 133, 135a, 136, 137, 138, 139, 140, 141, 142, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162 and 163, using the tests indicated above, determined the following Kj and IC values₅₀. Compound formulas 22e, 54b, 54j, 54k, and 57b are provided in Example 5. Other compound formulas are provided in Example 7.

185 693

<td rowspan="2">Relationship</td><td colspan="2">Test</td><td rowspan="2">Relationship</td><td colspan="2">Test</td>

<td>UV-visible K, (μΜ)</td><td>Cells I.₅₀ (μΜ)</td><td>UV-visible K, (μΜ)</td><td>Cells I.₅₀ (μΜ)</td>

<td>22e</td><td> 0,19</td><td> &gt;20</td><td> 131</td><td> 12,0</td><td> 30,0</td>

<td>54b</td><td></td><td> 20</td><td>132a</td><td> 5,0</td><td> &gt;30,0</td>

<td>54j</td><td></td><td> 10</td><td>132b</td><td> 12,5</td><td></td>

<td>54k</td><td></td><td> 6,6</td><td> 133</td><td> 50,0</td><td> &gt;30,0</td>

<td>57b</td><td></td><td> 2,2</td><td>135a</td><td> 0,090</td><td> 0,90</td>

<td> 85</td><td> 0,0035</td><td> 9,8</td><td>135b</td><td> 0,32</td><td> 0,95</td>

<td> 86</td><td> 0,175</td><td> 4,0</td><td> 136</td><td> 1,0</td><td></td>

<td> 87</td><td> 7,2</td><td> 35,0</td><td> 137</td><td> 0,04</td><td> 0,25</td>

<td> 88</td><td> 0,9</td><td></td><td> 138</td><td></td><td> 0,375</td>

<td> 89</td><td> 0,018</td><td></td><td> 139</td><td> 0,350</td><td> 2,0</td>

<td> 90</td><td> 0,42</td><td> 6,2</td><td> 140</td><td> 0,87</td><td> &gt;30,0</td>

<td> 91</td><td> 0,26</td><td> &gt;25</td><td> 141</td><td> 0,670</td><td></td>

<td> 92</td><td> 3,8</td><td></td><td> 142</td><td></td><td> 1,75</td>

<td> 98</td><td> 0,535</td><td> 4,0</td><td> 144</td><td> 0,32</td><td> &gt;20,0</td>

<td>102a</td><td></td><td> 4,0</td><td> 145</td><td> 0,34</td><td> 8,5</td>

<td>102b</td><td> 0,29</td><td> 1,75</td><td> 146</td><td> 0,16</td><td> 3,8</td>

<td>102c</td><td> 0,68</td><td></td><td> 147</td><td> 0,26</td><td> 8,5</td>

<td>106a</td><td> 2,3</td><td> 30,0</td><td> 148</td><td> 6,3</td><td> 30,0</td>

<td>l, 06b</td><td> 0,2</td><td> 2,9</td><td> 149</td><td> 14,0</td><td> &gt;30,0</td>

<td>106c</td><td> 3,8</td><td> &gt;30,0</td><td> 150</td><td> 10,0</td><td> 30,0</td>

<td>108a</td><td></td><td> 17,5</td><td> 151</td><td> 13,0</td><td> 30,0</td>

<td>108b</td><td> 0,4</td><td> 25,0</td><td> 152</td><td> 8,8</td><td></td>

<td>108c</td><td> 0,43</td><td></td><td> 153</td><td> 0,24</td><td></td>

<td>114a</td><td> 0,12</td><td> 3,8</td><td> 154</td><td> 0,042</td><td> 2,4</td>

<td>114b</td><td> 3,7</td><td></td><td> 155</td><td> 0,023</td><td></td>

<td> 115</td><td> 0,345</td><td> 6,0 1</td><td> 156</td><td> 0,001</td><td> 2,7</td>

<td> 121</td><td> 4,3</td><td></td><td> 157</td><td> 0,26</td><td></td>

<td>125a</td><td> 0,39</td><td> &gt;30,0</td><td> 158</td><td> 1,1</td><td></td>

<td>125b</td><td> 0,060</td><td> 0,30 |</td><td> 159</td><td> 0,0017</td><td> 8,0</td>

<td> 126</td><td> 0,45</td><td> 1,5 |</td><td> 160</td><td> 0,145</td><td> 2,25</td>

<td> 127</td><td> 0,39</td><td> 8,0 [</td><td> 161</td><td> 0,011</td><td></td>

<td> 128</td><td> 0,04</td><td> 7,5 |</td><td> 162</td><td> 0,0025</td><td></td>

<td> 129</td><td> 0,59</td><td> 25,0 |</td><td> 163</td><td> 0,0028</td><td> 1,2</td>

<td> 130</td><td></td><td> 1,20 |</td><td></td><td></td><td></td>

185 693

Example 7

The following compounds were prepared in a manner similar to that used in the synthesis of 69a: 126, 127, 128, 129, 135a, 135b, 137, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155 , 156, 157, 159, 160, 162 and 163.

<img file="PL185693B1_D0080.tif" />

185 693

<img file="PL185693B1_D0081.tif" />

185 693

<img file="PL185693B1_D0082.tif" />

185 693

<img file="PL185693B1_D0083.tif" />

Ο

<img file="PL185693B1_D0084.tif" />

Compound 158 was synthesized in a similar manner to that used in the synthesis of compound (K).

<img file="PL185693B1_D0085.tif" />

185 693

Compound 130 was synthesized in a similar manner to that used in the synthesis of compound 56b.

<img file="PL185693B1_D0086.tif" />

Compounds 131, 136, 138 in the synthesis of 57b.

and 142 was synthesized by a method similar to the one used

<img file="PL185693B1_D0087.tif" />

142

<img file="PL185693B1_D0088.tif" />

Compounds 132, 132b, 139, 140 and 141 were synthesized in a similar manner to that used in the synthesis of 47a. The starting material for compound 140 was prepared as described in Robi, et al. J. Am. Chem. Soc., 116, pp. 2348-2355 (1994). Starting compound for

100

185 693 of compound 141 was prepared as described in Wyvratt, et al. Pept. Struct. Funct. Proc. (8th Am. Pept. Symp.) (1983) or in US Patent 4,415,496.

<img file="PL185693B1_D0089.tif" />

Compound 133 was synthesized in a similar manner to that used in the synthesis of compound 47b.

<img file="PL185693B1_D0090.tif" />

185 693

101

Compound 161 of compound 125 a.

to that used in the synthesis were synthesized in a similar manner

<img file="PL185693B1_D0091.tif" />

Compounds 22e, 54b, 54j, 54k and 57b were synthesized as described in Example 5.

Compounds 85, 86, 87, 88, 89, 90, 91, 92, 98, 102a, 102b, 102c, 106a, 106b, 106c, 108a, 108b, 108c, 114a, 114b, 115, 121, 125a and 125b were synthesized. as follows.

<img file="PL185693B1_D0092.tif" />

N- (N-acetyl-tyrosinyl-valinyl- (4 (R) -allyloxyprolinyl)) - 3 (S) -amino-4-oxobutanoic acid (85).

Stage. A. N-tert-butoxycarbonyl-4 (R) -allyloxyproline. N-tert-butoxycarbonyl (4R) -hydroxyproline (9.25 g, 40 mmol) was added to a solution of 60% sodium hydride (3.36 g, 84 mmol) in 100 ml of anhydrous tetrahydrofuran and stirred for 2 hours at room temperature. Allyl bromide (6.9 ml, 80 mmol) was added to the mixture and the mixture was heated to reflux for 6 hours. The reaction was quenched with ice flakes, more water was added, and the mixture was washed with hexane. The aqueous layer was acidified with 10% sodium bisulfate solution and extracted with ethyl acetate (2 x 150 ml). The combined extracts were dried over anhydrous sodium sulfate, filtered and evaporated to yield 5 g of the title compound without further purification. 1 H NMR (CDCl₃; occurs in the form of rotamers) δ 5.92-5.82 (1H, m), 5.3-5.14 (2H, m), 4.5-4.31 (1H, m), 4.16-4 . 05 (1H, m), 4.04-3.9 (1H, m), 3.79-3.5 (3H, m), 2.43-2.2 (1.5H, m), 2 , 15-2.10 (0.5H, m), 1.45 (4.5H, s), 1.35 (4.5H, s).

Step B. 4 (R) -allyloxyproline methyl ester hydrochloride. N-tert-butoxycarbonyl-4 (R) -allyloxyproline (5 g, 18.4 mmol) was heated to reflux for 6 hours in 50 ml of saturated methanolic hydrogen chloride solution. The mixture was evaporated in vacuo to give 3.78 g of the title compound as a yellow gum; Ή NMR (CDC1₃) δ 5.83-5.72 (1H, m), 5.24-5.14 (1H, d), 5.13-5.08 (1H, d), 4.55-4.3 (3H , m), 4.25-4.15 (1H, m), 3.9 (1.5H, s), 3.78 (1.5H, s), 3.7-3.28 (3H, m ), 2.452.32 (1H, m), 2.2-2.05 (1H, m).

Step C. N-Acetyl-tyrosinyl-valinyl- (4 (R) -allyloxyproline) methyl ester. 4 (R) -allyloxyproline methyl ester hydrochloride (1.05 g, 4.75 mmol) and N-acetyl-Tyr-ValOH (1.68 g, 5.21 mmol) was dissolved in 10 ml of a 1: 1 mixture of dichloromethane and dimethylformamide and cooled to 0 ° C. To the cooled mixture was added diisopropylethylamine (1 mL, 5.93 mmol) followed by N-hydroxybenzotriazole (0.769 g, 5.69 mmol)

102

185 693 and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.18 g, 6.2 mmol). After stirring for 2 hours, the mixture was warmed to room temperature and stirred for 16 hours. The mixture was poured into 150 ml of ethyl acetate and washed each time with 50 ml of water, 10% sodium hydrogen sulfate solution and 10% sodium hydrogen carbonate solution. The organic layer was dried over sodium sulfate, filtered and evaporated to give a light yellow solid. This material was purified by flash chromatography eluting with dichloromethane / methanol / pyridine (100: 3: 0.5) to give 780 mg of the title compound. Ή NMR (CD₃OD) δ 7.02-6.96 (2H, d), 6.67-6.63 (2H, d), 5.95-5.85 (1H, m), 5.34-5.27 ( 1H, d), 5.16-5.13 (1H, d), 4.53-4.38 (3H, m), 4.28-4.22 (1H, m), 3.82-3. 73 (1H, m), 3.72 (3H, s), 3.04-2.88 (2H, m), 2.85-2.72 (2H, m), 2.45-2.34 ( 1H, m), 2.08-1.95 (2H, m), 1.92 (3H, s), 1.00-0.92 (6H, 2xd).

Step D. Semicarbazone of N- (N-acetyl-tyrosinyl-valinyl (4 (R) -allyloxyprolinyl)) -3 (S) -amino-4-oxobutanoic acid tert-butyl ester. N-acetyl-tyrosinyl-valinyl- (4-allyloxyproline) methyl ester (770 mg, 1.57 mmol) was dissolved in 20 mL of tetrahydrofuran and 4 mL of methanol. Lithium hydroxide (145 mg, 3.46 mmol) was added to the mixture and stirred at room temperature. After two hours, 10% hydrogen chloride solution was added to the mixture and the mixture was evaporated in vacuo to give a solid residue and then partitioned between 5 mL of water and 50 mL of ethyl acetate. The organic layer was separated and evaporated in vacuo to give 430 mg of the acid which was used directly in the next step.

From N-acetyl-tyrosinyl-valinyl-4-allyloxyproline (420 mg, 0.88 mmol) and 3-amino-4-oxobutyric acid tert-butyl ester semicarbazone (184 mg, 0.8 mol, Graybill et al., Int J. Protein Res .. 44, pp. 173-82 (1994)) obtained 100 mg (20%) of the title compound as a white amorphous solid: 1 H NMR (CD, OD) δ 7.24-7.2 (1H, m), 7.046.97 (2H , d), 6.73-6.65 (2H, d), 5.98-5.86 (1H, m), 5.35-5.24 (1H, d), 5.17-5.12 (1H, m), 4.12-3.98 (2H, m), 3.72-3.72 (1H, m), 2.98-2.92 (3H, m), 2.38-2 , 32 (1H, m), 2.1-2.02 (2H, m), 1.92 (3H, s), 0.98-0.89 (6H, 2xd).

Step E. N- (N-Acetyl-tyrosinyl-valinyl- (4 (R) -allyloxyprolinyl)) -3 (S) -amino-4-oxobutanoic acid (85). The protecting group was removed from the semicarbazone of N- (N-acetyl-tyrosinyl-valinyl- (4 (R) -allyloxyprolinyl)) -3 (S) -amino-4-oxobutanoic acid tert-butyl ester (100 mg) as described in example 3, compound K, step C to afford 44.2 mg (53%) of the title compound: Ή NMR (CD₃OD) δ 7.04-6.97 (2H, d), 6.72-6.65 (2H, d), 5.97-5.86 (1H, m), 5.32-525 (1H, d), 5.17-5.12 (1H, d), 4.62-4.40 (3H, m), 4.30-4.13 (2H, m), 4.12-3.96 ( 3H, m), 3.75-3.68 (1H, m), 2.99-2.92 (1H, m), 2.78-2.70 (1H, m), 2.70-2. 48 (2H, m), 2.352.30 (1H, m), 2.17-1.95 (2H, m), 1.92 (3H, s), 0.98-0.88 (6H, 2 xd ).

Compounds 86 and 87 were prepared by a method similar to that described for the synthesis of 69a in Example 5:

<img file="PL185693B1_D0093.tif" />

<img file="PL185693B1_D0094.tif" />

N-Acetyl- (S) -valinyl- (4- (S) -phenoxy) prolinyl-3 (S) -amino-4- (7-methoxybenzoxazol-2-yl) -4-oxo-butanoic acid (86). N-acetyl- (S) -valinyl- (S) - (4- (S) -phenoxy) proline was converted to compound 86 to give a white powder: 1 H NMR (DMSO-d₆) δ 8.75 (d, 1H), 7.6-7.2 (m, 4H), 7.0-6.8 (m, 4H), 5.5 (m, 1H), 5.05 ( s, 1H), 4.5 (t, 1H), 4.29 (t, 1H), 4.0 (s,

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3Η), 4.03.8 (m, 2H), 3.0-2.8 (dd, 2H), 2.3 (m, 1H), 2.09 (m, 1H), 1.95-1 . 8 (m, 2H), 1.78 (s, 3H), 1-0.7 (dd, 6H).

<img file="PL185693B1_D0095.tif" />

N-Acetyl (4- (R) -phenoxy) prolinyl-3 (S) -amino-4- (7-methoxybenzoxazol-2-yl) 4-oxo-butanoic acid (87). N-acetyl- (S) - (4- (S) -phenoxy) proline was converted to compound 87 to give a white powder: Ή NMR (DMSO-d₆) δ 9, 1 (d, 1H), 8.76 (d, 1H), 7.6-7.2 (m, 4H), 7.0-6.9 (m, 4H), 5.55 ( m, 1H), 5.45 (m, 1H), 5.0 (m, 2H), 4.56 (t, 1H), 4.40 (t, 1H), 4.0 (s, 3H), 3.9 (dd, 1H), 3.76 (d, 1H), 3.64 (d, 1H), 3.1-2.9 (m, 1H), 2.8 (m, 1H), 2 , 50 (m, 1H), 2.32.2 (m, 1H), 2.09 (m, 1H), 1.95 and 1.75 (2xs, 3H, rotamers).

<img file="PL185693B1_D0096.tif" />

N-2- (6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetyl-3 (S) -amino-5-hydroxy-4-oxo- acid pentane (88). N-2- (6-Benzyl-1,2-dihydro-2-oxo-3- (3-phenyl-propionyl) amino-1-pyridyl) acetyl-3 (S) -amino-5-hydroxy- acid tert-butyl ester 4-Oxo-pentane was prepared from compounds 52b and 81 according to the method described for the synthesis of compound 83a to give a white solid (45%): Ή NMR (CDC1₃) 6 8.40 (d, 1H), 8.20 (s, 1H), 7.4-7.1 (m, 1H), 6.18 (s, 1H), 4.72 (m, 1H) , 4.65-4.5 (q, 2H), 4.4-4.2 (dd, 2H), 4.0 (s, 2H), 3.04 (t, 2H), 2.9 (dd , 1H), 2.76 (t, 2H), 2.55 (dd, 1H), 1.39 (s, 9H).

The resulting product was converted to compound 89 as described in Example 5 for compound 84a to afford the title compound (42%) as a white solid: Ή NMR (CDCl₃) δ 8.5 (d, 1H), 8.1 (d, 1H), 8.0 (m, 1H), 7.4-7.1 (m, 11H), 6.3 (d, 1H) , 4.9-4.8 (m, 2H), 4.6-4.4 (m, 2H), 4.3 (dd, 1H), 4.1 (s, 2H), 3.3 (t , 1H), 3.05 (t, 2H), 2.8-2.6 (m, 3H).

Compounds 89 and 90 were prepared by a method similar to that described in Example 5 for the preparation of 84a.

104

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<img file="PL185693B1_D0097.tif" />

N-Acetyl- (S) -tyrosinyl- (S) -valinyl- (S) -alaninyl-3 (S) -amino-5- (2-chlorobenzyloxy) -4-oxo-pentanoic acid (89) was prepared from Ac- Tyr-Val-Ala-OH and t-butyl (3S) N- (allyloxycarbonyl) -3-amino-5- (chlorophenylmethoxyl) -4-oxo-pentanoate (prepared in a manner similar to compound 82) to give a white solid: Ή NMR (DMSO-d₆) δ 9.15 (s, 1H), 8.5 (d, 1H), 7.98 (d, 1H), 7.75 (d, 1H), 7.55-7.3 (m, 4H) , 7.0 (d, 1H), 6.6 (d, 2H), 4.6-4.3 (m, 6H), 4.3-4.1 (m, 2H), 2.9 (d , 1H), 2.76 (dd, 1H), 2.7-2.5 (m, 2H), 1.95 (m, 1H), 1.75 (s, 3H), 1.2 (d, 3H), 0.9-0.7 (dd, 6H).

<img file="PL185693B1_D0098.tif" />

N-2- (6-benzyl-1,2-dihydro-2-oxo-3 - (3-phenylpropionyl) amino-1-pyridyl) acetyl-3-amino-5- (2-chlorobenzyloxy) -4-oxo acid -pentane (90) was prepared from 52b and t-butyl N- (allyloxycarbonyl) -3-amino-5- (2-chloro- phenylmethoxyl) -4-oxo-pentanoate (prepared in a similar manner to compound 82) to give a white solid : Ή NMR (DMSO-d₆) δ 9.2 (s, 1H), 8.75 (d, 1H), 7.7-7.1 (m, 14H), 6.4 (d, 1H), 4.65 (d, 6H) , 4.56 (s, 1H), 4.6-4.35 (dd, 1H), 3.9 (s, 2H), 2.9-2.6 (m, 6H).

<img file="PL185693B1_D0099.tif" />

N-2- (6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionyl) amino-1-pyridyl) acetyl-3 (S) -amino-5- (5- (2, 6-Dichlorophenyl) thiazol-2-yl) -4-oxo-pentanoic acid (91) was prepared from compound 52b and 3- (allyloxy) -amino-4 - [(2,6-dichloro-phenyl) thiazol2 acid tert-butyl ester -yl] -4-hydroxybutyric (99) as described for the preparation of compound 69a to give an off-white powder: Ή NMR (DMSO-d₆) δ 9.32 (s, 1H), 9.05 (d, 1H), 8.27 (d, 1H), 8.18 (d, 1H), 7.7 (d, 1H), 7.6 (t, 1H), 7.4-7.1 (m, 11H), 6.1 (d, 1H), 5.64 (m, 1H), 4.8-4.6 (dd, 2H), 3.85 (s, 2H), 3.02 (m, 1H), 2.9-2.7 (m, 4H).

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<img file="PL185693B1_D0100.tif" />

3- (S) - (2- (3 [3- (S) - (4-Hydroxy-phenyl) -propionylamino] -2-oxo-azepan-1-yl) -acetylamino) -4-oxo-butyric acid ( 92) was prepared from 2- (3 [3- (S) - (4-hydroxy-phenyl) -propionylamino] -2-oxoazepan-1-yl) -acetic acid and N-allyloxycarbonyl-4-amino-5 -benzyl-xy-2-oxotetrahydrofuran (Chapman, Biorg, Med. Chem. Lett. 2, pp. 613-18 (1992)) in a manner similar to that described for the synthesis of compound 54a to afford the title compound as a white solid: Ή NMR (DMSO-d₆) δ 9.10-9.20 (s, 1H), 8.40 (s, 1H), 7.88 (d, 1H), 7.0 (d, 2H), 6.64 (d, 2H) , 4.60 (t, 1H), 4.10 (q, 2H), 3.9-4.2 (m, 2H), 3.6 (m, 1H), 3.18 (d, 2H), 2.70 (t, 2H), 2.40 (m, 2H), 1.85-1.40 (m, 8H).

<img file="PL185693B1_D0101.tif" />

4-ethoxymethylene-2-styryl-4H-oxazol-5-one (94) was prepared according to Comforth, The Chemistry of Penicillin, Ciarka, Johnson, Robinson (ed.), Princeton University Press, p. 804 (1949).

4-Oxo-3- (3-phenyl-acryloylamino) -4,6,7,8-tetrahydropyrrolo [1,2a] pyrimidine- (6S) -carboxylic acid ethyl ester (95) was prepared from compound 94 as described in Example 5 for compound 3, yielding 4.5 g (30%) of the title compound: Ή NMR (CD₃OD) δ 1.3 (t, 3H), 2.35 (m, 1H), 2.65 (m, 1H), 3.1 (m, 1H), 3.15 (m, 1H), 4.25 ( q, 2H), 5.15 (dd, 1H), 6.95 (d, 1H), 7.4 (m, 3H), 7.6 (m, 2H), 7.65 (d, 1H), 8.95 (s, 1H).

4-Oxo-3- (2-phenyl-acryloylamino) -4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine (6S) -carboxylic acid (96). A mixture of 4-Oxo-3- (3-phenyl-acryloylamino) -4,6,7,8-tetrahydro-pyrrolo [1,2-a] pyrimidine- (6S) -carboxylic acid ethyl ester (95.3.1 g , 8.8mmo

106

185 693 [mu] L) and a 1N solution of aqueous lithium hydroxide (8.8 mL, 8.8 mmol) in methanol (10 mL) was stirred for 18 hours at room temperature. The mixture was diluted with water and washed with diethyl ether (1 x 20 mL). The aqueous layer was acidified with concentrated hydrochloric acid. The solid was collected by filtration and washed with water, dried in a vacuum oven at 50 ° C for 18 hours to give 2.2 g (75%) of the title compound as a tan solid: 1 H NMR (CD₃OD) δ 2.4 (m, 1H), 2.7 (m, 1H), 3.1 (m, 1H), 3.2 (m, 1H), 5.15 (dd, 1H), 7, O (d, 1H), 7.4 (m, 3H), 7.6 (m, 2H), 7.65 (d, 1H), 8.95 (s, 1H).

4-Oxo-3- (2-phenyl-acryloylamino) -4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine 2-benzyloxy-5-oxo-tetrahydrofuran- (3S) -yl) -amide - (6S) -carboxylic acid (97) was prepared from compound 96 as described in Example 3 for compound H, step ai gave 0.52 g (75%) of the title compound as a mixture of diastereomers: 1 H NMR (CDCl₃) 2.3-2.7 (m, 3H), 2.9 (dd, 1H), 3.05 (m, 1H), 3.3 (m, 1H), 4.4-4.8 (m , 2H), 4.2 (2xd, 1H), 5.05 (m, 1H), 5.55 (2xs, 1H), 6.6 (2xd, 1H), 7.4 (m, 6H ), 7.55 (m, 4H), 7.65 (2xd, 1H), 8.0 (m, 2H), 9.2 (sx2.1H).

Acid - (3S) - {[4-oxo-3- (3-phenyl-propionylamino) -4,6,7,8-tetrahydro-pyrrolo [1,2-a] pyrimidine- (6S) -carbonyl] -amino } -butyric (98) was prepared as described in Example 3 for Compound H, step D to give 0.13 g (45%) of the title compound: Ή NMR (CD₃OD) δ 2.35 (m, 1H), 7.15 (m, 1H), 7.3 (m, 4H 2.45-2.75 (m, 3h), 2.8 (t, 2H), 3.0 (t, 2H), 3.1 (m, 1H), 3.25 (m, 1H), 4.3 (m, 1H), 6.65 (dd, 1H), 5.15 (m , 1H), 8.8 (a, 1H).

<img file="PL185693B1_D0102.tif" />

<img file="PL185693B1_D0103.tif" />

<img file="PL185693B1_D0104.tif" />

3 (S) - (allyloxycarbonyl) -amino-4 - [(2,6-dichlorophenyl) oxazol-2-yl] -4 (R, S) -hydroxybutyric acid tert-butyl ester (99). A solution of 5- (2,6-dichlorophenyl) oxazole (2.71 g, 12.7 mmol; prepared by a similar procedure as described in Tet. Lett. 23, p. 2369 (1972)) in tetrahydrofuran (65 mL) was cooled to -78 ° C under nitrogen atmosphere. To this solution was added n-butyllithium (1.5 M hexanes solution, 8.5 mL, 13.3 mmol) and stirred at 78 ° C for 30 minutes. Magnesium bromide etherate was added and the solution was allowed to warm to -45 ° C for 15 minutes. The solution was cooled to -78 ° C and aldehyde 58 (3.26 g, 12.7 mmol; Graybill et al., Int. J. Protein Res. 44, pp. 173-82 (1993)) in tetrahydrofuran (65) was added dropwise. ml). The reaction mixture was stirred for 25 minutes, then allowed to warm to -40 ° C for 3 hours and then at room temperature for 1 hour. The reaction was quenched with 5% NaHCO₃ (12 ml) and stirred for 3 hours. The tetrahydrofuran was removed in vacuo and the tetrahydrofuran residue was extracted with dichloromethane. The organic layer was washed with saturated sodium chloride solution, dried over magnesium sulfate, filtered and concentrated to provide 6.14 g of the title compound. After purification, 4.79 g (80%) of compound 99 was obtained: Ή NMR (CDCl₃) Ó 2.7-2.5 (m, 2H), 2.8 (dd, 1H), 4.2, 4.4 (2 xd, 1H), 4.7-4.5 (m, 3H) , 5.35-5.1 (m, 2H), 5.6, 5.7 (2xd, 1H), 6.0-5.8 (m, 1H), 7.2 (d, 1H), 7.3 (m, 1H), 7.4 (m, 2H).

4-Oxo-3- (3-phenyl-propionylamino) -4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine- (6S) -carboxylic acid (100). A mixture of 4-oxo-3- (3-phenyl-acryloylamino) -4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine- (6S) -carboxylic acid (96, 2.1 g, 6.5 mmol) and 20% palladium hydroxide on carbon (0.5 g) in methanol (50 ml) was stirred under a hydrogen atmosphere for 4 hours. The resulting mixture was filtered and concentrated to afford 2.1 g (100%) of the title compound as a white solid: 1 H NMR (CD₃OD) δ 2.35 (m, 1H), 2.65 (m, 1H), 2.75 (t, 2H), 3.0 (t, 2H), 3.1 (m, 1H), 3, 15 (m, 1H), 5.1 (dd, 1H), 7.15 (m, 1H), 7.25 (m, 4H), 8.75 (s, 1H).

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<img file="PL185693B1_D0105.tif" />

<img file="PL185693B1_D0106.tif" />

100

<img file="PL185693B1_D0107.tif" />

4-tert-butoxycarbonyl-2-oxo- (3S) - {[4-oxo-3- (3-phenyl-propionylamino) -4,6 7,8-tetrahydropyrrolo [1,2-a] pyrimidine- ester 2,6-Dichlorobenzoic acid (6S) -carbonyl] amino} -butyl (10aa) was prepared as described in Example 5 for compound 56a to give 0.16 g (20%) of the title compound: Ή NMR (CD₃OD) δ 1.45 (s, 9H), 2.3 (m, 1H), 2.6 (m, 1H), 2.7 (m, 3H), 2.95 (m, 3H), 4. 8 (m, 1H), 5.1 (m, 1H), 5.2 (q, 2H), 7.1 (m, 1H), 7.2 (m, 4H), 7.4 (m, 3H ). 8.75 (s, 1H).

4- (7-Methoxy-benzoxazol-2-yl) -4-oxo- (3S) - {[4-oxo-3- (3-phenyl-1-propionylamino) -4,6,7,8 acid tert-butyl ester -tetrahydro-pyrrolo [1,2-a] pyrimidine- (6S) -carbonyl] amino} -butyric (101b). 4-Hydroxy-4- (7-methoxy-benzoxazol-2-yl) - (33) - {[4-oxo-3- (3-phenyl-1-propionylamino) -4,6,7,8 acid tert-butyl ester -tetrahydropyrrolo [1,2-a] pyrimidine - (6S) -carbonyl] amino} -butyric was prepared from 100 and 66a as described in Example 5 for 67a to give 0.95 g (quantitative) of the product in as a mixture of diastereomers: 1 H NMR (CD₃OD) δ 1.45 (2xs, 9H), 2.2 (2xm, 1H), 2.35-3.0 (m, 9H), 4.0 (m, 3H), 4.75 (m , 1H), 4.85 (m, 1H), 5.05 (2 x dd, 1H), 7.1 (2 x dd, 1H), 7.15-7.3 (m, 4H), 7. Δ (2xt, 1H), 7.8 (2xd, 1H), 8.55 (2xdd, 1H), 8.7 (2xs, 1H).

The resulting product was converted to 10Ib as described in Example 5 for 68a to obtain 0.36 g (50%) of the title compound: Ή NMR (CD₃OD) δ 1.4 (s, 9H), 2.35 (m, 1H), 2.55 (m, 1H), 2.75 (t, 2H), 2.95 (t, 2H), 3, 00 (m, 1H), 3.1 (dd, 2H), 3.15 (m, 1H), 5.15 (dd, 1H), 5.65 (t, 1H), 7.1 (m, 2H ), 7.2 (m, 4H), 7.4 (m, 2H), 8.7 (s, 1H).

4- [5- (2,6-Dichloro-phenyl) -oxazol-2-yl] -4-oxo- (3S) - {[4-oxo-3- (3-phenyl-1-propionylamino) -4 acid tert-butyl ester , 6,7,8-tetrahydro-pyrrolo [1,2-a] pyrimidine- (6S) -carbonyl] amino} -butyric (10c). 4- [5- (2,6-Dichloro-phenyl) oxazol-2-yl] -4-hydroxy- (3S) - {[4-oxo-3- (3-phenyl-1-propionyl) acid tert-butyl ester -amino) -4,6,7,8-tetrahydro

108

185 693 pyrrolo [1,2-a] pyrimidine- (6S) -carbonyl] amino} -butyric was prepared from compounds 100 and 99 using the method described in Example 5 for compound 67a.

0.09 g (60%) of the product was obtained in the form of a mixture of diastereomers: Ή NMR (CD₃OD) δ 1.45 (2 xs, 9H), 2.2 (m, 1H), 2.5 (m, 2H), 2.7 (2 x dd, 1H), 2.75 (t, 2H) , 2.9-3.1 (m, 4H), 4.7 (m, 1H), 5.1 (m, 1H), 7.1 (m, 1H), 7.1-7.25 (m , 4H), 7.4 (t, 1H), 7.5 (t, 1H), 8.55 (d, 1H), 8.75 (s, 1H). The resulting product was converted to 10Ic as described in Example 5 for 68a to give 0.04 g (45%) of the title compound: 1 H NMR (CD₃OD) δ 1.4 (s, 9H), 2.3 (m, 1H), 2.6 (m, 1H), 2.75 (t, 2H), 2.95 (t, 2H), 2, 9-3.2 (m, 4H), 5.2 (dd, 1H), 5.55 (t, 1H), 7.1 (m, 1H), 7.25 (m, 4H), 7.55 (m, 3H, 8.75 (s, 1H).

4-carboxy-2-oxo- (3S) - {[4-oxo-3- (3-phenyl-propionylamino) -4,6,7,8-tetrahydropyrrolo [1,2-a] pyrimidine- ( 2,6-Dichloro-benzoic acid 6S) -carbonyl] amino} -butyl (102a) was prepared from 10la as described in Example 5 for 57a to give 0.12 g (80%) of the title compound: Ή NMR (CD₃OD) δ 2.35 (m, 1H), 2.65 (m, 1H), 2.75 (m, 2H), 2.85 (dd, 1H), 2.95 (m, 2H), 3, 0 (dd, 1H), 3.15 (m, 1H), 3.25 (m, 1H), 4.55 (dd, 1H), 5.15 (m, 1H), 5.25 (q, 2H ), 7.15 (m, 1H), 7.25 (m, 4H), 7.45 (m, 1H), 8.8 (s, 1H).

4- (7-Methoxy-benzoxazol-2-yl) -4-oxo- (3S) - {[4-oxo-3- (3-phenyl-propionylamino) -4,6,7,8-tetrahydro-pyrrolo acid [1,2-a] pyrimidine (6S) -carbonyl] amino} -butyric (102b) was prepared from 10Ib as described in Example 5 for 69a to give 0.12 g (35%) of the title compound: 1 H NMR (DMSO-d₆) Δ 2.1 (m, 1H), 2.55 (m, 1H), 2.7-3.1 (m, 8H), 4.05 (s, 3H), 5.1 (dd, 1H) , 5.55 (t, 1H), 7.2 (m, 1H), 7.25 (m, 5H), 7.5 (t, 1H), 7.55 (d, 1H), 8.7 ( s, 1H), 9.2 (d, 1H), 9.4 (s, 1H), 12.7 (br, 1H).

4- (5- (2,6-Dichloro-phenyl) -oxazol-2-yl] -4-oxo- (3S) - {[4-oxo-3- (3-phenyl-propionylamino) -4.6 acid , 7,8-tetrahydro-pyrrolo [1,2-a] pymidine- (6S) -carbonyl] amino} -butyric (102c) was prepared from 10Ic as described in Example 5 for 69a to afford 0.01 g of ( 40%) of the title compound: Ή NMR (CD₃OD) δ 2.35 (m, 1H), 2.6 (m, 1H), 2.75 (t, 2H), 2.95 (t, 2H), 3.05 (m, 1H), 3, 15 (m, 3H), 5.15 (dd, 1H), 5.55 (t, 1H), 7.15 (m, 1H), 7.2 (m, 4H). 7.55 (m, 3H), 8.8 (s, 1H).

<img file="PL185693B1_D0108.tif" />

Λ

Step A

<img file="PL185693B1_D0109.tif" />

<img file="PL185693B1_D0110.tif" />

<img file="PL185693B1_D0111.tif" />

(3-tert-Butoxycarbonylamino-2-oxo-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl) -acetic acid methyl ester (103).

Step A. 2 (S) -tert-Butoxycarbonylamino-3- (2-nitrophenyl-amino) -propionic acid. 2-tert-butoxycarbonylamino-3-aminopropionic acid (10 g, 49 mmol), 2-fluoronitrobenzene (5.7 ml, 54 mmol) and sodium bicarbonate (8.25 g, 98 mmol) were taken up in 130 ml of dimethylformamide and heated in at 80 ° C for 18 hours. The mixture was evaporated in vacuo to give a sticky orange colored residue which was dissolved in 300 ml of water and extracted with diethyl ether (3 x 150 ml). The aqueous solution was acidified to pH 5 with 10% sodium bisulfate solution and extracted with ethyl acetate (3 x 250 ml). The combined extracts were dried over anhydrous sodium sulfate, filtered and evaporated to yield 12.64 g (83%) of the title compound as an orange amorphous solid. Ή NMR (CD₃OD) δ 8.15-8.10 (1H, d), 7.54-7.48 (1H, t), 7.13-7.08 (1H, d), 6.73-6.65 ( 1H, t), 4.45-4.35 (1H, m), 3.9-3.8 (1H, dd), 3.65-3.55 (1H, dd), 1.45 (9H, s).

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109

Step B. 2 (S) -tert-butoxycarbonylamino-3- (2-aminophenylamino) -propionic acid. A mixture of 2-tert-butoxycarbonylamino-3- (2-nitrophenylamino) propionic acid (12.65 g, 40.5 mmol) and 0.5 g of 10% Pd / C in 100 ml of methanol was stirred at 1 atm. hydrogen for 4 hours. The solution was filtered through Celite 545 and the filtrate was evaporated in vacuo to yield 11.95 g of the title compound in quantitative yield as a dark brown solid that was used without purification. 1 H NMR (CD₃OD) δ 6.75-6.70 (3H, m), 6.65-6.58 (1H, m), 4.35-4.3 (1H, m), 3.6-3.38 ( 2H, m), 1.45 (9H, s).

Step C. 3 (S) -tert-Butoxycarbonylamino-1,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-2-one. To a cooled (0 ° C) solution of 2-tert-butoxycarbonylamino-3- (2-aminophenylamino) propionic acid (11.95 g, 40.5 mmol) in 100 ml dimethylformamide was added 1- (3-dimethylaminopropyl) -3- hydrochloride. ethylcarbodiimide (8.54 g, 44.5 mmol) and stirred for 18 hours. The mixture was poured into 700 ml of ethyl acetate and washed four times with 100 ml of water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to give a brown solid which was purified by flash chromatography eluting with a 3: 7 mixture of ethyl acetate and hexane to give 8 g (71%) of the title compound: Ή NMR (CDCl₃) δ 7.78 (1H, s), 7.02-6.95 (1H, m), 7.02-6.95 (1H, m), 6.886.82 (1H, m), 6.82- 6.78 (1H, m), 6.75-6.70 (1H, m), 5.8-5.7 (1H, d), 4.55-4.45 (1H, m), 3. 95 (IH, s), 3.9-3.82 (IH, m), 3.48-3.40 (IH, m), 1.45 (9H, s).

Step D. (3 (S) -tert-butoxycarbonylamino-2-oxo-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl) -acetic acid methyl ester (103). To a cooled to -78 ° C solution of 3-t-butoxycarbonylamino-1,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-2-one (0.94 g, 3.38 mmol) in 20 mL anhydrous tetrahydrofuran, a solution of lithium bis (trimethylsilyl) amide (3.4 mL, 3.4 mmol) in THF was added dropwise and stirred for 30 minutes. Methyl bromoacetate (0.44 mL, 4 mmol) was added dropwise to the reaction mixture, and then the mixture was warmed to room temperature. The mixture was diluted with 100 ml of ethyl acetate and washed with 0.3N potassium bisulfate solution (50 ml), water (2 x 50 ml) and brine. The combined organics were dried over anhydrous sodium sulfate, filtered and evaporated to give a gum which was purified by flash chromatography eluting with a 3: 7 mixture of ethyl acetate and hexane. 0.98 g (83%) of the title compound was obtained as a white solid. Ή NMR (CDC1₃) δ 7.15-7.07 (2H, m), 6.98-6.94 (1H, m), 6.88-6.84 (1H, m), 5.62-5.55 (1H , d), 4.71-4.65 (1H, d), 4.65-4.6 (1H, m), 4.33-4.27 (1H, d), 3.96-3.90 (1H, m), 3.78 (3H, s), 3.44-3.37 (1H, m), 1.4 (9H, s).

<img file="PL185693B1_D0112.tif" />

103

<img file="PL185693B1_D0113.tif" />

<img file="PL185693B1_D0114.tif" />

105 ¹⁰⁶ a R = H b R = COCH₂CH₂Ph c R = CH₂Ph

110

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[2-Oxo-3 (S) - (3-phenyl-propionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] -acetic acid methyl ester (104a). Solution of (3 (S) -tert-butoxycarbonylamino-2-oxo-2,3,4,5-tetrahydro-benzo [b] -1,4] diazep-1-yl) -acetic acid methyl ester 103.1 g, 2.86 mmol) in 25 mL of ethyl acetate was bubbled with anhydrous hydrogen chloride for 2 minutes, followed by stirring for 1 hour at room temperature. The solution was evaporated to give 2-oxo-3 (S) -amino-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-ylacetic acid methyl ester hydrochloride as a white solid. The hydrochloride salt and hydrocinnamic acid (0.47 g, 3.15 mmol) were dissolved in 20 mL of dimethylformamide and cooled to 0 ° C. Diisopropylethylamine (1 mL, 5.72 mmol) was added to the solution followed by N-hydroxybenzotriazole and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. After stirring for 18 hours at room temperature, the mixture was diluted with 150 ml of ethyl acetate and washed with 10% sodium bisulfate, 10% sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to a crude solid which was purified by flash chromatography eluting with a 7: 3 mixture of ethyl acetate and dichloromethane to afford 600 mg (55%) of the title compound as a white solid: H NMR (CDCl₃) δ 7.36.85 (9H, m), 6.55-6.0 (IH, d), 4.88-4.82 (IH, m), 4.72-4.65 (IH, d ), 4.28-4.22 (IH, m), 3.953.9 (IH, m), 3.78 (3H, s), 3.65 (IH, br.s), 3.28-3, 2 (1H, m), 2.95-2.84 (2H, m), 2.55-2.4 (2H, m).

(3 (S) - (3-Phenylpropionylamino) -2-oxo-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl) acetic acid (105a). (3 (S) - (3-Phenyl-propionylamino) -2-oxo-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepinyl) -acetic acid methyl ester (104a) was dissolved in 90% methanol. Lithium hydroxide hydrate was added to the reaction mixture and stirred at room temperature for 4 hours. The mixture was evaporated in vacuo to give a white solid. This material was dissolved in 20 mL of water, acidified to pH 5, and extracted with ethyl acetate. 304 mg (88%) of the title compound was obtained as a white solid. Ή NMR (CDC1₃) δ 7.5-6.9 (1HI, m), 4.92-4.8 (IH, m), 4.7-4.58 (IH, m), 4.38-4.25 ( IH, d), 3.88-3.78 (IH, m), 3.45-3.25 (IH, m), 3.05-2.85 (2H, m), 2.55-2, 45 (2H, m).

4-oxo-3 (S) - {2- [2-oxo-3 (S) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepine- acid l-ylacetylamino} butter (106a). N- [1- (2-benzyloxy-5-oxotetrahydrofuran-3-ylcarbamoyl-methyl) -2-oxo-2,3,4,5-tetrahydro-1H-benzo [b] 1,4] diazepin-3-yl ] -3-phenylpropionamide was prepared from 105a as described in Example 3 for H (step A). 390 mg (93%) of the product were obtained in the form of diastereomers. 1 H NMR (CD₃OD) δ 7.58-7.22 (14H, m), 5.78-5.73 (0.5H, d), 5.64 (0.5H, s), 5.0-4 / 72 ( 4H, m), 4.54-4.42 (2H, m), 3.82-3.76 (0.5H, m), 3.68-3.62 (0.5H, m), 3. 28-3.21 (0.5H, m), 3.19-3.12 (0.5H, m), 3.07-2.98 (2H, m), 2.78-2.48 (4H , m).

The resulting product was converted to compound 106a as described in Example 3, compound H (Step D) to afford the title compound as a white solid (17%): Ή NMR (CD₃OD) δ 7.54-6.98 (9H, m), 5.58-5.54 (IH, m), 4.8-4.2 (4H, m), 3.96-3.3 ( 2H, m), 3.30-3.05 (1H, m), 2.98-2.25 (5H, m).

[2-Oxo-5- (3-phenyl-propionyl) -3 (S) - (3-phenyl-propionyl-amino) 2,3,4,5-tetrahydrobenzo [b] 1,4] diazepin-l- acid methyl ester yl] acetic acid (104b). Solution of (3 (S) -tert-butoxycarbonyl-amino-2-oxo-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-yl) -acetic acid methyl ester (103.1 g, 2.86 mmol ) in 25 ml of ethyl acetate was bubbled with anhydrous hydrogen chloride for 2 minutes, then stirred for 1 hour at room temperature. The mixture was evaporated to give 2-oxo-3 (S) -amino-2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-ylacetic methyl ester hydrochloride as a white solid. This hydrochloride was suspended in 20 ml of dichloromethane and cooled to 0 ° C. Triethylamine (1.6 mL, 11.5 mmol) was added to the suspension followed by the dropwise addition of dihydrocinnamoyl chloride (0.9 mL, 6 mmol) dropwise. The mixture was warmed to room temperature for 18 hours, diluted with 25 ml of dichloromethane and washed twice with 50 ml of water and once with 50 ml of brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. A viscous yellow oil was obtained which was purified by flash chromatography eluting with a 1: 1 mixture of ethyl acetate and dichloromethane to give 1.35 g (92%) of the title compound as a white solid. Ή NMR (CDC1₃) δ 7.45-7.02 (14H, m), 6.37-6.32 (IH, d), 4.78-4.72

185 693

111 (1Η, m), 4.52-4.3 (3H, m), 3.82-3.77 (1H, m), 3.74 (3H, s), 3.03-2.87 (4H , m), 2.58-2.45 (2H, m), 2.45-2.35 (1H, m), 2.25-2.16 (1H, m).

[2-Oxo-5- (3-phenylpropionyl) -3- (3 (S) -phenyl-propionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acid acetic (105b). [2-Oxo-5- (3-phenyl-propionyl) -3- (3-phenyl-propionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] -acetic acid methyl ester (104b, 680 mg, 1.32 mmol) was hydrolyzed as in Example 105a to yield 645 mg (98%) of the title compound as a white solid. Ή NMR (CDC1₃) δ 7.58 (1H, br.s), 7.5-7.42 (1H, m), 7.35-6.95 (14H, m), 4.95-4.88 (1H, m ), 4.64-4.55 (1H, d), 4.54-4.45 (1H, t), 4.15-4.05 (1H, d), 3.75 (1H, m), 3.05-2.75 (4H, m), 2.58-2.45 (2H, m), 2.45-2.28 (1H, m), 2.25-2.14 (1H, m ).

2-Oxo-3 (S) - {2- [2-oxo-5- (3-phenylpropionyl) -3 (S) -O-phenylpropionyl-amino) 2,3,4,5-tetrahydro-benzo [b] [ 1,4] diazepin-1-yl] acetylamino} butyric (106b). [2-oxo5- (3-phenylpropionyl) -3- (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetic acid and tert ester semicarbazone 3-amino-4-oxobutyric acid -butyl was coupled as in Example 3 for compound K (step A) to give 350 mg (85%) of a white solid. Ή NMR (CDC1₃) δ 9.05 (1H, br.s), 7.58-7.55 (1H, d), 7.5-7.35 (1H, m), 7.35-6.95 (14H, m ), 6.75-6.72 (1H, d), 6.25 (1H, br. S), 5.25 (1H, br. S), 4.95-4.88 (1H, m), 4.8-4.72 (1H, m), 4.55-4.4 (2H, m), 3.92-3.88 (1H, d), 3.73-3.68 (1H, m ), 2.95-2.8 (4H, m), 2.8-2.72 (1H, m), 2.62-2.55 (1H, m), 2.55-2.45 (2H , m), 2.4-2.32 (IH, m), 2.2-2.12 (IH, m), 1.45 (9H, s).

By the method described in Example 3, compound K (step C) from 4-oxo-3- {2- [2-oxo-5- (3-phenylpropionyl) -3- (3-phenylpropionylamino) acid tert-butyl ester semicarbazone, The 4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] -acetylamino} butyric group was removed to give 118 mg (47%) of the title compound as a white solid. Ή NMR (CD₃OD) δ 7.48-6.95 (14H, m), 4.65-4.15 (6H, m), 3.5-3.4 (1H, m), 2.85-2.72 ( 4H, m), 2.652.5 (1H, m), 2.5-2.34 (3H, m), 2.34-2.15 (2H, m).

[5-Benzyl-2-oxo-3 (S) - (3-phenyl-propionylamino) -2,3,4-tetrahydro-benzo [b] [1,4] diazepin-1-yl] -acetic acid methyl ester (104c). [2-Oxo-3- (3-phenyl-propionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] -acetic acid methyl ester (104a, 500 mg, 1.31 mmol ), calcium carbonate (155 mg, 1.58 mmol) and benzyl bromide (170 µΐ, 1.44 mmol) were taken up in 10 ml of dimethylformamide and heated to 80 ° C for 8 hours. The mixture was diluted with 150 ml of ethyl acetate and washed 4 times with 50 ml of water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. A viscous yellow oil was obtained which was purified by flash chromatography eluting with a mixture of dichloromethane and ethyl acetate (8: 2) to give 460 mg (75%) of the title compound as a white solid. Ή NMR (CDC1₃) δ 7.34-7.05 (14H, m), 6.32-6.28 (1H, d), 4.84-4.76 (1H, d), 4.76-4.70 (1H , m), 4.43-4.37 (1H, d), 4.26-4.18 (1H, d), 4.06-4.00 (1H, d), 3.79 (3H, s ), 3.45-3.37 (1H, m), 3.02-2.95 (1H, m), 2.90-2.82 (2H, m), 2.5-2.34 (2H , m).

[5-Benzyl-2-oxo-3 (S) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetic acid (105c) was prepared on hydrolysis of the ester (102c) as described for 105a gave 450 mg (98%) of the title compound as a white solid. Ή NMR (CD₃OD) δ 7.5-7.05 (14H, m), 6.4 (1H, br.s), 4.85-4.55 (2H, m), 4.54.21 (2H, m) , 4.12-3.92 (1H, d), 3.45-3.3 (1H, m), 3.1-2.8 (3H, m), 2.55-2.28 (3H, m).

3 (S) - {2- [5-benzyl-2-oxo-3 - (3 (S) -phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1 acid -yl] -acetylamino} -4-oxobutyric (106c). [5-benzyl-2-oxo-3 (S) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetic acid and tert-butyl acid semicarbazone The 3 (S) -amino-4-oxobutyric compound was coupled as described in Example 3 to compound K (step A) to afford 260 mg (85%) of a white solid. Ή NMR (CD₃OD) δ 7.35-7.0 (15H, m), 4.94-4.88 (1H, m), 4.68-4.58 (1H, d), 4.57-4.52 ( 1H, m). 4.41-4.34 (1H, d), 4.3-4.23 (1H, d), 4.1-4.04 (1H, d), 3.18-3.11 (1H, m ), 3.09-2.98 (1H, m), 2.78-2.72 (2H, t), 2.65-2.57 (1H, m), 2.42-2.33 (3H , m).

By the method described in Example 3, compound K (step C) from 3 (S) - {2- [5-benzyl-2-oxo-3 (S) - (3-phenylpropionylamino) acid tert-butyl ester semicarbazone -2.3.4 , 5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetylamino} -4-oxobutyro removed

112

185 693 to give 168 mg (81%) of the title compound as a white solid. 1 H NMR (CD₃OD) δ 7.37-7.0 (14H, m), 4.75-4.62 (1H, m), 4.6-4.45 (2H, m), 4.4-4.21 ( 2H, m), 4.153.95 (2H, m), 3.15-3.0 (2H, m), 2.82-2.67 (2H, m), 2.65-2.52 (1H, m), 2.5-2.32 (3H, m).

<img file="PL185693B1_D0115.tif" />

<img file="PL185693B1_D0116.tif" />

<img file="PL185693B1_D0117.tif" />

4-tert-butoxycarbonyl-2-oxo-3 (S) - {2- [2-oxo-5- (3-phenylpropionyl) -3 (S) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo ester 2,6-Dichlorobenzoic acid [b] [1,4] diazepin-1-yl] acetyl-amino} butyl (107a).

The obtained semicarbazone was prepared by coupling 105b and t-butyl 3- (allyloxycarbonylamino) -4-oxo-5- (2,6-dichloro-benzoyloxy) pentanoate (WO 93 16710) as described for 56a to afford 256 mg (58 %) of the title compound as a white solid. Ή NMR (CDC1₃) δ 7.45-7.04 (17H, m), 6.45-6.34 (2H, m), 5.28-5.21 (1H, m), 5.1-5.0 (1H , m), 4.95-4.90 (1H, m), 4.75-4.70 (1H, m), 4.55-4.44 (1H, m), 4.32-4.22 (1H, dd), 3.99-3.85 (1H, dd), 3.85-3.76 (1H, m), 3.06-2.83 (5H, m), 2.83-2 , 74 (1H, m), 2.6-2.44 (2H, m), 2.43-2.33 (1H, m), 2.24-2.15 (1H, m), 1.45 (9H, s).

4-carboxy-2-oxo-3 (S) - {2- [2-oxo-5- (3-phenylpropionyl) -3 (S) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo ester 2,6-dichlorobenzoic acid [b] [1,4] diazepin-1-yl] acetylamino} butyl (108a) was prepared from 107a as described for 57a to afford 156 mg (68%) of the title compound as a white solid. 1 H NMR (CD₃OD) δ 7.5-7.9 (17H, m), 5.16-5.02 (1H, dd), 4.88-4.71 (2H, m), 4.62-4.44 ( 2H, m), 4.444.28 (2H, m), 4.27-4.18 (1H, m), 3.47-3.41 (1H, m), 2.90-2.60 (5H, m), 2.46-2.4 (2H, m), 2.39-2.18 (2H, m).

4- (7-Methoxy-benzoxazol-2-yl) -4-oxo-3 (S) - {2- [2-oxo-5 (3-phenyl-propionyl) -3 (S) - (3) tert-butyl ester -phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetylamino} butyric (107b). 4 (R, S) -hydroxy-4- (7-methoxy-benzoxazol-2-yl-3 (S) - {2- [2-oxo-5- (3-phenyl-propionyl) -3 (S) acid tert-butyl ester - (3-phenylpropionylamino) -2,3,4,5-tetrahydro [b] [1,4] diazepin-1-yl-acetylamino} butyric was prepared from 105b and 66a as described in Example 5 for compound 67 yielding 56% white solid. NMR (CDCl₃) δ 7.72-6.78 (19H, m), 6.37-6.28 (1H, m), 5.17-5.08 (0.5H, m), 4.92-4.82 (0.5H, m), 4.81-4.6 (1H, m), 4.6-4.35 (3H, m), 4.05-3.9 (1H, m), 3.95 (3H, s), 3.82-3.7 (1H, m), 2.96-2.05 (10H, m), 1.45 (4.5H, s), 1.38 (4.5H , s).

The resulting product was converted to compound 107b as described in Example 5 for compound 69a to afford the title compound (56%) as a white solid. Ή NMR (CD₃OD) δ 7.62-6.8 (17H, m), 5.64-5.58 (0.5H, t), 5.52-5.46 (0.5H, t), 4.624.47 ( 2H, m), 4.40-4.32 (1H, m), 3.9 (1.5H, s), 3.88 (1.5H, s), 3.43-3.37 (1H, m), 3.0-2.92 (1H, m), 2.90-2.62 (6H, m), 2.5-2.4 (2H, m), 2.28-2.15 ( 2H, m), 1.32 (4.5H, s), 1.25 (4.5H, s).

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113

4- (7-Methoxy-benzoxazol-2-yl) -4-oxo-3 (S) - {2- [2-oxo-5- (S-phenylpropionyl) 3 (S) - (3-phenylpropionylamino) -2 acid, 3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetylamino} butyric (108b) was prepared as in Example 5 for 69a to afford the title compound (50%) as white solid. 1 H NMR (CD₃OD) δ 7.4Ιό, 88 (17H, m), 5.6-5.55 (0.5H, t), 5.48-5.43 (0.5H, t), 4.64-4, 45 (2H, m), 4.45-4.30 (1H, m), 3.93 (1.5H, s), 3.90 (1.5H, s), 3.47-3.34 ( 1H, m), 3.10-2.85 (2H, m), 2.84-2.63 (5H, m), 2.62.4 (2H, m), 2.3-2.1 ( 2H, m).

4- [5- (2,6-Dichlorophenyl) oxazol-2-yl) -4-oxo-3 (S) - {2- [2-oxo-5- (3-phenyl-propionyl) -3 (S) tert-butyl ester ) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] acetylamino} butyl (107c). 4- [5- (2,6-Dichlorophenyl) oxazol-2-yl] -4 (R, S) -hydroxy-3 (S) - {2- [2-oxo-5- (3) tert-butyl ester -phenylpropinyl) -3 (S) - (3-phenylpropionylamino) -2,3,4,5-tetrahydro-benzo [b] [1,4] diazepin-1-yl] -acetylamino} butyric was prepared from compounds 106c and 99 , in a similar manner to that described for compound 67a in Example 5, giving 72% of a white solid. Ή NMR (CDC1₃) δ 7.71-7.64 (1H, m), 7.58-7.42 (2H, m), 7.42-6.92 (15H, m), 6.5-6.37 (2H , m), 5.15-5.04 (1H, m), 4.88-4.68 (2H, m), 4.57-4.37 (2H, m), 4.28-4.13 (1H, m), 3.87-3.64 (2H, m), 3.04-2.80 (4H, m), 2.76-2.68 (1H, m), 2.672.42 (3H , m), 2.41-2.31 (1H, m), 2.22-2.12 (1H, m), 1.45 (9H, s).

The resulting product was converted to compound 107c in a similar manner to that described for compound 68a in Example 5 to give the title compound in quantitative yield as a white solid. Ή NMR (CDC1₃) δ 7.47-6.98 (18H, m), 6.52-6.42 (1H, d), 5.6-5.52 (1H, m), 4.78-4.71 (1H , m), 4.52-4.40 (2H, m), 4.03-3.94 (0.67H, m), 3.94-3.85 (0.33H, m), 3.85 -3.75 (1H, m), 3.45-3.33 (1H, m), 3.08-2.98 (1H, m), 2.97-2.84 (4H, m), 2 , 55-2.43 (2H, m), 2.43-2.32 (1H, m), 2.23-2.13 (1H, m), 1.35 (9H, s).

4- [5- (2,6-Dichlorophenyl) oxazol-2-yl] -4-oxo-3 (S) - {2- [2-oxo-5- (3-phenylpropionyl) -3 (S) - acid (3-phenylpropionylamino) -2,3,4,5-tetrahydrobenzo [b] [1,4] diazepin-1-yl] -acetylamino} butyric acid (108c) was prepared from 107c, similar to 69a in Example 5 to give 72% of the title compound as a white solid. Ή NMR (CD₃OD) δ 7.58-7.0 (18H, m), 5.62-5.53 (0.67H, m), 5.52-5.47 (0.33H, m), 4.68 ( 3H, m), 3.54-3.42 (1H, m), 3.1-2.92 (2H, m), 2.88-2.68 (5H, m), 2.63-2. 45 (2H, m), 2.40-2.22 (2H, m).

<img file="PL185693B1_D0118.tif" />

<img file="PL185693B1_D0119.tif" />

114

185 693

<img file="PL185693B1_D0120.tif" />

and Ri = CH₃bRi = H.

3 (S) - {2 (R, S) - [4-benzyl-7-oxo-6 (S) - (N-benzyloxycarbonylamino) - [1,4-diazepan-1-yl] propionylamino} -4-oxo salt -butyric acid with trifluoroacetic acid (114a).

Stage. A. To a solution of tert-butyl 2-N-benzyloxycarbonyl-3-N-benzyl- (S) -2,3-diaminopropionate (110; 0.85 g, 2.2 mmol), 3- (N- tert-butoxycarbonyl) amino-2-methyl-5-oxo-pentanoic acid (109a; 0.65 g, 2.7 mmol), acetic acid (0.1 ml, 1.8 mmol), sodium acetate (0.36 g , 2 mmol) and 0.4 nm molecular sieves (1 g) in methanol (45 ml) were added sodium cyanoborohydride (0.33 g, 5.3 mmol). The mixture was stirred overnight at 25 ° C, then filtered through Celite and concentrated in vacuo. The residue was dissolved in IN NaOH and extracted with ethyl acetate (3 x 40 ml). The organic layer was dried (MgSO₄), filtered and evaporated to yield an oil. Chromatography (silica gel, 4: 1 hexane: ethyl acetate as eluent) gave 0.92 g (68% yield) of Compound 111 as an oil.

Step B. The product obtained above was dissolved in chilled to 0 ° C dichloromethane (3 ml) and treated with a 25% solution of trifluoroacetic acid in dichloromethane (20 ml) then allowed to warm to 25 ° C and stirred until TLC indicated the reaction was complete (hexane: ethyl acetate, 4: 1). The solvent was removed under reduced pressure and the residue was dried in vacuo then dissolved in dichloromethane (40 mL) and treated with 4-methylmorpholine (1 mL, 9 mmol), HOBT (0.2 g, 1.5 mmol) and EDC (0.1 61 g, 3.2 mmol). The resulting mixture was stirred overnight at 25 ° C then diluted with dichloromethane and washed with water. The organic layer was dried (MgSO₄), filtered and evaporated to yield an oil. Chromatography (silica gel, 3: 2 hexane: ethyl acetate as eluant) provided 0.49 g (74% yield) of 112a as a viscous oil.

Step C. A solution of 2 (R, S) - [4-benzyl-7-oxo-6 (S) - (N-benzyloxycarbonylamino) - [1,4] diazepan-1-yl} -propionic acid methyl ester (112a; 0.15 g, 0.32 mmol) was dissolved in methanol, treated with 1M LiOH (0.32 ml) and stirred for 5.5 hours at 25 ° C, then evaporated to dryness. The residue was azeotroped with ethanol (2 x 10 ml), acetonitrile (2 x 10 ml) and benzene (2 x 10 ml) and dried to dryness. The residue obtained was converted to compound 114a in a similar manner to Example 3 for compound K (steps A, B, and C), and then purified by reverse phase HPLC (C18 column) using 0.1% TFA: water / 0.1% TFA: acetonitrile. 17 mg (10% yield) of a viscous oil were obtained: 1 H NMR (500 MHz, CD₃OD) δ 1.15 (m, 3h), 2.30-2.70 (m, 6H), 2.72-2.95 (bm, 6H), 3.30-3.80 (m, 4H) , 4.10 (m, 1H), 4.40 (m, 4H), 4.95 (m, 1H), 6.95-7.10 (bs, 5H), and 7.12-7.20 ppm ( bs, 5H).

3 (S) - {2- [4-benzyl-7-oxo-6 (S) - (N-benzyloxycarbonylamino) - [1,4] diazepan-1-yl] acetylamino} -4-oxo-butyric acid salt with trifluoroacetic acid (114b) was prepared from 109b by a method similar to that described for the synthesis of 114a to give 85 mg of a viscous oil: Ή NMR (500 MHz, CD₃OD) δ (d, J = 7Hz, 3H), 2.28 (m, 2H), 2.60 (m, 2H), 3.18 (bs, 6H), 3.35-3.45 (m , 2H), 3.60-3.95 (m, 2H), 4.15 (m, 1H), 4.32 (m, 1H), 4.42 (m, 1H), 5.00 (bm, 2H), 7.20 (bs, 5H) and 7.40 ppm (bs, 5H); Ή NMR (470 MHz, CD₃OD) δ 10.72 ppm (s, 3 F).

4-oxo-3 (S) - {2 (R, S) - [7-oxo-4- (3-phenyl-propionyl) -6 (S) - (3-phenyl-propionylamino) - [1,4 ] diazepan-1-yl] -propionylamino) -butyric (115).

185 693

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Step D. Suspension of 2 (R, S) - [4-benzyl-7-oxo-6 (S) - (Nbenzyloxycarbonylamino) - [1,4] diazepan-1-yl] propionic acid methyl ester (112b; 0.22 g, 0.49 mmol) and 20% Pd (OH)₂ on carbon (50 ml) in ethanol was stirred under an atmosphere of hydrogen for 7 hours. The solvent was evaporated under reduced pressure and the residue was dissolved in dichloromethane (20 ml) and then treated with triethylamine (1 ml) and dihydrocinnamoyl chloride (170 mg, 1 mmol). The resulting mixture was stirred overnight then diluted with ethyl acetate and washed with IN NaOH. The organic layer was dried (MgSO₄), filtered and evaporated to yield an oil. Chromatography (silica gel, 4: 1 hexane: ethyl acetate) gave 0.175 g (75% yield) of compound 113 as an oil.

Step C. 0.15 g of compound 113 (0.32 mmol) was dissolved in methanol, treated with 1M LiOH (0.32 ml), stirred at 40 ° C overnight then evaporated to dryness. The residue was azeotroped with ethanol (2 x 10 ml), acetonitrile (2 x 10 ml), benzene (2 x 10 ml) then dried in vacuo. The resulting residue was converted to compound 115 in a similar manner to that described in Example 3 for compound K (steps A, B, and C).

<img file="PL185693B1_D0121.tif" />

<img file="PL185693B1_D0122.tif" />

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<img file="PL185693B1_D0124.tif" />

11«

<img file="PL185693B1_D0125.tif" />

120

<img file="PL185693B1_D0126.tif" />

121

3- {2- [2,4-Dibenzyl-3,7-dioxo-6- (N-benzyloxycarbonylamino) - [1,4] diazepan-1-yl] acetylaraine} -4-oxo-butyric acid (121).

Step E. A solution of tert-butyl 2-N-carbobenzoxy-3-N-benzyl- (S) -2,3-diaminopropionate (100; 1.77 g, 4.6 mmol), N-allyl-N-tert-butoxycarbonyl- (S) -phenylalanine (116; 1.04 g, 4.8 mmol), HOBT (0.74 g, 5.5 mmol) and EDC (1.33 g, 6.9 mmol) in dichloromethane (50 ml) stirred at 25 ° C for 16 hours then diluted with dichloromethane (100 ml) and washed with water. The organic layer was dried (MgSO₄), filtered and evaporated to yield an oil. After chromatography (silica gel, hex

116

185 693 san: ethyl acetate, 85:15) gave 1.34 g (43% yield) of compound 117 as a colorless viscous oil.

Step F. 1.34 g of compound 117 was dissolved in dichloromethane (3 mL) and treated with a 50% solution of trifluoroacetic acid in dichloromethane (20 mL). After 1.5 hours, the dissolution was removed under reduced pressure and the residue was dried in vacuo, then dissolved in dichloromethane (50 ml) and combined with 4-methylmorpholine (0.2 ml, 2 mmol,), HOBT (0.27 g, 2 mmol,). mmol) and EDC (0.8 g, 4 mmol). The mixture was stirred overnight at 25 ° C, then diluted with dichloromethane and washed with water. The organic layer was dried (MgSO₄), filtered and evaporated to yield an oil. Chromatography (silica gel, hexane: ethyl acetate 7: 3) provided 0.8 g (80% yield) of compound 118 as a viscous oil.

Step G. A 0.8 g portion of compound 188 was dissolved in methanol (400 mL), cooled to -78 ° C, and saturated with ozone until the solution turned blue. Excess ozone was removed by purging with argon, then dimethyl sulfide (5 mL) was added and the mixture was allowed to warm to 25 ° C and stirred for 3 hours. Removal of the solvent and chromatography (silica gel, hexane-ethyl acetate, 1: 1) provided 0.74 g (74% yield) of compound 119 as a white solid.

Step H. A 0.2 g (0.4 mmol) of compound 119 was dissolved in acetone (25 mL), cooled to 0 ° C, and Jones's reagent solution was added dropwise until the solution turned orange. 2-Propanol (5 ml) was then added to the mixture and the resulting solution was filtered through Celite and washed with acetone. Removal of the solvent gave a green-white solid which was dried in vacuo to yield compound 120. The resulting residue was converted to compound 121 in a similar manner to Example 3 for compound K (steps A, B, and C). After chromatography (SiO₂, dichloromethane: methanol: acetic acid, 95: 4.5: 0.5 as eluant) gave 85 mg (53% yield) of a cream-colored solid, which was identified as the acid 3- {2- [2,4- dibenzyl-3,7-dioxo6- (N-benzyloxycarbonylamino) - [1,4] diazepan-1-yl-acetylamino} -4-oxobutyric (121) based on the following spectral data: Ή NMR (500 MHz, CD₃OD) δ 2.38 (m, 1H), 2.45 (m, 1H), 3.21 (bs, 2H), 3.32-3.39 (bm, 6H), 3.85 (m, 1H ), 4.05 (m, 1H), 4.21 (bm, 1H), 4.31 (bs, 1H), 4.45 (dm, J = 11Hz, 1H), 4.95 (bs, 4H ), 7.20 (bs, 5H) and 7.33-7.45 ppm (m, 5H); ^,9F NMR (470 MHz, CD, OD) δ 10.62 (s, 3F).

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T-Butyl 3 (S) -N- (allyloxycarbonyl) -3-amino-5- (2-chlorophenylmethylthio) -4-oxo-pentanoate (123). To a solution of t-butyl 3 (3) N- (allyloxycarbonyl) -3-amino-5-bromo-4-oxo-pentanoate (122; 749 mg, 2.14 mmol; WO 93 16710) in dimethylformamide (20 ml) was added with stirring potassium fluoride (273 mg, 4.70 mmol) followed by 2-chlorophenylmethylthiol (373 mg, 2.35 mmol). The mixture was stirred for 3.5 hours, quenched with water (50 ml) and extracted with ethyl acetate (2 x 50 ml). The combined organic extracts were washed with water (4 x 50 ml) followed by brine (50 ml) then dried (MgSO₄) and concentrated to give an oil which was purified by flash chromatography (1035% ethyl acetate / hexane). 832 mg (91%) of a colorless solid were obtained: mp. 45-6 ° C; [and]_D²⁰-19.0 ° (c 1.0, CH₂C1,); IR (coating) 3340, 2980, 2935, 1725, 1712, 1511, 1503, 1474, 1446, 1421, 1393, 1281, 1244, 1157, 1052, 1040, 995, 764, 739; Ή NMR (CDC1₃) δ 7.36 (2H, m), 7.21 (2H, m), 5.91 (2H, m), 5.27 (2H, m), 4.76- (1H, m), 4, 59 (2H, d), 3.78 (2H, s), 3.36 (2H, m), 2.91 (1H, dd), 2.74 (1H, dd), 1.43 (9H, s ).

Analysis for C.₂₀H.₂₆ClNO₅S.

Calculated C, 56.13; H, 6.12; N, 3.27; S, 7.49.

Found C, 56.08; H, 6.11; N, 3.26; S, 7.54.

MS (CI); 430/28 (M⁺ + 1,3%); 374/2 (100).

(3 S) t-butyl 3 (2 (6-benzyl-1,2-dihydro-2-oxo-3 (3-phenyl) propionylamino) -1-pyridyl) acetylamino-5- (2-chlorophenylmethylthio) -4-oxopentanoate (124a). 6-Benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionylamino) -pyridylacetic acid (52b; 300mg, 0.76mmol) in THF (7ml) was mixed with 1-hydroxybenzotriazole (205 mg, 1.52 mmol) and 1- (3-dimethylaminopropoxy-3-ethylcarbodiimide) hydrochloride. After 3 hours, water (12 drops) was added and the mixture was stirred for 10 minutes, then t-butyl (3S) N- (allyloxycarbonyl) -3-amino-5- (2-chlorophenylmethylthio) -4-oxopentanoate (123) ( 325 mg, 0.76 mmol), bis (triphenylphosphine) palladium II chloride (20 mg), and tributyltin hydride (0.6 ml, 2.28 mmol). The mixture was stirred for 5 hours at room temperature, poured into ethyl acetate and washed with aq. 1M HCl (x 2), aq. Sodium bicarbonate, brine, dried (MgSO₄) and concentrated. The residue was triturated with pentane and the supernatant was discarded. Chromatography (silica gel, 50% ethyl acetate / hexane) provided a colorless foam (439 mg, 81%): [a]_D²¹ -18.3 ° (c 0.5, CH₂C1₂); IR (KBr) 3356, 3311, 1722, 1689, 1646, 1599, 1567, 1513, 1367, 1154; Ή NMR (CDC1₃) δ 8.39 (1H, d), 8.23 ​​(1H, s), 7.24 (14H, m), 6.16 (1H, d), 4.95 (1H, m), 4.63 (2H, m), 4.02 (2H, s), 3.74 (2H, s), 3.27 (2H, s), 2.85 (6H, m), 1.40 (9H, s) .

Analysis for C.₃₉H.₄₂C1N₃ABOUT₆S:

Calculated; C, 65.39 H, 5.91; N, 5.87.

Found C, 65.51; H, 5.99; N, 5.77.

[3S (1S, 9S)] - 3- (6,10-dioxo-1,2,3,4,7,8,9,10-octahydro) -9- (3-phenylpropionylamino) -6H-pyridazine- [ 1,2-a] [1,2] diazepine-1-carboxamido-5- (2-chlorophenylmethylthio) -4-t-butyl oxopentanoate (124b) was prepared by a method similar to compound 124a from thioether 123 and 3S acid (1S, 9S ) -3- (6,10-dioxo-1,2,3,4,7,8,9,10-octahydro) -9- (3-phenylpropionylamino) -6H-pyridazine [1,2-a] [l , 2] diazepine-1-carboxylic acid (45a) to give 452 mg (50%) of a colorless foam: mp. 55-7 ° C; [and]_D²¹ -94.0 ° (c 0.12, CH₂C1₂); IR (KBr) 3288, 2934, 1741, 1722, 1686, 1666, 1523, 1433, 1260, 1225, 1146, 757; H NMR (CDCl₃) δ 7.35 (3H, m), 7.20 (7H, m), 6.46 (1H, d), 5.21 (1H, m), 4.97 (2H, m), 4.56 (1H, m), 3.75 (2H, s), 3.25 (3H, m), 2.93 (5H, m), 2.71 (1H, dd), 2.55 (2H, m) , 2.30 (1H, m), 1.92 (3H, m), 1.66 (2H, m), 1.42 (9H, s).

Analysis for C.₃₅H.₄₃C1N₄ABOUT₇S · Ó, 25H₂ABOUT.

Calculated C, 59.73; H, 6.23; Cl, 5.04; N, 7.96; S, 4.56.

Found C, 59.73; H, 6.19; Cl, 5.10; N, 7.79; S, 4.58.

MS (-FAB) 697 (M1, 100).

(3S) 3 (2 (6-Benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionylamino) -1-pyridyl) acetylamino-5- (2-chlorophenylmethylthio) -4-oxopentanoic acid (125a) T-Butyl 3- (2 (6-benzyl-1,2-dihydro-2-oxo-3- (3-phenylpropionylamino) -1-pyridyl) acetylamino-5- (2-chlorophenylmethylthio) -4-oxopentanoate (124a) (400 mg, 0.56 mmol) in dichloromethane (3 ml) at 0 ° C, treated with trifluoroacetic acid (3 ml) and stirred at

118

185 693 at 0 ° C for 1 hour and at room temperature for 0.5 hours. The solution was concentrated then redissolved in dichloromethane and concentrated again. This operation was repeated three times. The residue was stirred in ether for 1 hour and filtered to give a colorless solid (364 mg, 99%); mp temp. 165-7 ° C; [and]_D²² -27.7 ° (c 0.2, CH₂C1₂); IR (KBr) 3289, 1712, 1682, 1657, 1645, 1593, 1562, 1527, 1497, 1416, 1203, 1182; Ή NMR (CDC1₃) δ 8.47 (1H, d), 8.21 (1H, s), 7.70 (1H, d), 7.22 (14H, m), 6.24 (1H, d), 5.03 (1H, m), 4.65 (2H, m), 4.06 (2H, s), 3.69 (2H, m), 3.23 (2H, m), 2.88 (6H, m) .

Acid [3 (lS, 9S)] - 3- (6,10-dioxo-1,2,3,4,7,8,9,10-octa-hydro) -9- (3-phenylpropionyl-amino) - 6H-pyridazine- [1,2-a] [1,2] diazepine-1-carboxamido-5- (2-chlorophenyl-methylthio) -4-oxopentane (125b) was prepared in a similar manner as described for compound 125a from the t-butyl ester 124b to obtain 362 mg (93%) of a colorless powder: mp. 76-80 ° C; [and]_D²¹ -134 ° (c 0.10, MeOH); IR (KBr) 3309, 2935, 1725, 1658, 1528, 1445, 1417, 1277, 1219, 1175; Ή NMR (D₆-DMSO) δ 8.80 (1H, d), 8.19 (1H, d), 7.31 (9H, m), 5.09 (1H, m), 4.74 (1H, m), 4 , 63 (1H, m), 4.35 (1H, m), 3.76 (2H, m), 3.28 (3H, m), 2.80 (5H, m), 2.52 (4H, m), 2.16 (2H, m), 1.90 (3H, m).

Analysis for C.₃₁H.₃₅C1₂N₄O, S 0.25H₂ABOUT

Calculated C, 57.49; H, 5.53; N, 8.65; S, 4.95.

Found C, 57.35; H, 5.43; N, 8.45; S, 4.88.

The data of the above examples demonstrate that the compounds of the invention exhibit inhibitory activity on the IL-β converting enzyme.

Since the compounds of the invention are capable of inhibiting ICE in vitro and, moreover, can be administered orally to mammals, they have obvious clinical utility in the treatment of IL-1 dependent diseases. From these assays, the ability of compounds to inhibit ICE in vivo can be predicted.

Notwithstanding the many embodiments of the present invention described, it is evident that the basic structure may be varied to provide other embodiments with the products and methods of the invention. Thus, it should be considered that the scope of the present invention is defined by the appended claims and not by the specific embodiments presented by way of illustration only.

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