BRPI9917881B1 - caspases inhibiting compounds, pharmaceutical composition and use of said compounds - Google Patents

Abstract

caspase inhibiting compounds, pharmaceutical composition and use of said compounds. The present invention relates to novel classes of compounds which are caspase inhibitors, in particular interleukin-1 beta ("ice") converting enzyme inhibitors. This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are particularly well suited for inhibiting caspase activity and therefore can be advantageously used as agents against interleukin-1 ("il-1"), apoptosis, interferon-inducing factor mediated diseases. - <sym> (i-gif), or interferon- <sym> ("ifn- <sym>"), including inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, and degenerative diseases. This invention also relates to methods for inhibiting caspase activity and decreasing igif production and ifn- <sym> production and methods for treating interferon- <sym>, apoptosis, and interleukin-mediated diseases. 1 using the compounds and compositions of this invention. This invention also relates to methods of preparing the compounds of this invention.

Description

(54) Title: CASPASE INHIBITING COMPOUNDS, PHARMACEUTICAL COMPOSITION AND USE OF COMPOUNDS (73) Holder: VERTEX PHARMACEUTICALS INCORPORATED, North American Society. Address: 130 Waverly Street- Cambridge, Massachusetts 02139-4242, UNITED STATES OF AMERICA (US) (72) Inventor: MARION W. WANNAMAKER; GUY W. BEMIS; PAUL S. CHARIFSON; DAVID J. LAUFFER; MICHAEL

D. MULLICAN; MARK A. MURCKO; KEITH P. WILSON; JAMES W. JANETKA; ROBERT J. DAVIES; ANNE-LAURE GRILLOT; ZHAN SHI; CORNELIA J. FORSTER.

Validity Term: 10 (ten) years from 11/21/2018, subject to legal conditions

Issued on: 11/21/2018

Digitally signed by:

Alexandre Gomes Ciancio

Substitute Director of Patents, Computer Programs and Topographies of Integrated Circuits

Invention Patent Descriptive Report for CASPASE INHIBITING COMPOUNDS, PHARMACEUTICAL COMPOSITION AND USE OF THOSE COMPOUNDS.

PI9917842-7 Division Application, filed on March 19, 1999.

Technical Field of the Invention

The present invention relates to new classes of compounds that are caspase inhibitors, in particular interleukin-1beta converting enzyme (ICE) inhibitors. This invention also relates to

pharmaceutical compositions comprising these compounds. The pharmaceutical compositions and compositions of this invention are particularly well suited for inhibiting caspase activity and therefore can be advantageously used as agents against interleukin-1 (IL-1) -mediated diseases, apoptosis, interferon-inducing factor γ (IGIF), or interferon-γ (IFN-γ), including inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, and degenerative diseases. This invention also relates to methods for inhibiting caspase activity and decreasing IGIF production and IFN-γ production and methods for treating γ-interferon-mediated diseases, apoptosis, and interleukin-1 using the compositions and compositions of this invention. This invention also relates to

methods of preparing the compounds of this invention.

Fundamentals of the Invention

Interleukin 1 (IL-1) is a major immunoregulatory and pro-inflammatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. Oppenheim, J. H. et al., Immunology Today, 7, p. 45 - 46 (1986). As such, it is involved in the pathogenesis of acute autoimmune and inflammatory diseases. For example, in rheumatoid arthritis, IL-1 is both a mediator 30 of inflammatory symptoms and the destruction of cartilage proteoglycan in afflicted joints. Wood, D. D. and others. Arthitis Rheum. 26 975, (1983); Pettipher, E. J. et al., Proc. Natl. Acad. Know. USA 71, 295 (1986);

Arend, W. P. and Dayer, J. M .. Arthrite Rheum. 38, 151 (1995). IL-1 is also a highly potent bone resorption agent. Jandiski, J. J., J. Oral Path 17, 145 (1988); Dewhirst, F. E. et al., J. Immunol. 8, 2562 1985). It is alternatively called an osteoclast activating factor in two destructive bone segments such as osteoarthritis and multiple myeloma. Bataille, R. et al., Int. J. Clin. Lab. Res. 21 (4), 283 (1992). In certain proliferative disorders, such as acute myelogenous leukemia and multiple myeloma, IL-1 can promote tumor cell adhesion and growth. Bani, M. R., J. Natl. Cancer Inst. 83, 123 (1991); Vidal-Vanaclocha, F., Cancer Res. 54, 2667 (1994). In these disorders, IL-1 also stimulates the production of other cytokines such as IL-6, which can modulate tumor development (Tartour et al., Cancer Res. 54, pp. 6243 (1994). IL-1 is predominantly produced by monocytes peripheral blood samples as part of the inflammatory response and exists in two different agonist forms, IL-1 alpha and IL-1 beta. Mo15 seley, BS and others, Proc. Nat. Acad. Sci., 84, pp. 4572 - 4576 (1987);

Lonnemann, G. et al., Eur. J. Immunol., 19, pgs. 1531-1536 (1989).

IL-1 beta is synthesized as a biologically inactive precursor,

plL-1beta. plL-1beta lacks a conventional leader sequence and is not processed by a signal peptidase. March, C. J., Nature, 315, pgs. 641 20 - 647 (1985). Instead, plL-1beta is cleaved by interleukin-1 beta (ICE) converting enzyme between Asp-116 and Ala-117 to produce the biologically active C-terminal fragment in synovial fluid and human serum.

Sleath, P.R., et al., J. Biol. Chem., 265, p. 14526 - 14528 (1992); A. D.

Howard et al., J. Immunol., 147, pgs. 2964 - 2969 (1991). ICE is a cysteine protease located mainly in monocytes. It converts precursor IL-1 beta to mature form. Black. R. A. et al., FEBS Lett., 247, pgs. 386 - 390 (1989); Kostura, M. J. et al., Proc. Natl. Acad. I know USA, 86, pgs. 5227 - 5231 (1989). ICE processing is also necessary for the transport of mature IL-1 beta across the cell membrane.

ICE (or caspase-1) is a member of a family of homologous enzymes called caspases. These homologues have sequence similarities in the active site regions of the enzymes. Such counterparts (caspases)

include TX (or ICE _re | .n or ICH-2) (caspase-4) (Faucheu et al., EMBO J., 14, p. 1914 (1995); Kamens J., et al., J. Biol. Chem. , 270, page 15250 (1995); Nicholson et al., J. Biol. Chem., 270 15870 (1995)), TY (or ICE _re ) (caspase-5) (Nicholson et al., J. Bio. Chem ., 270, page 15870 (1995);

ICH-1 (or Nedd-2) (caspase-2) (Wang, L. et al., Cell. 78, p. 739 (1994)), MCH-2 (caspase-6), (Fernandes-Alnemri, T. and others, Câncer Res., 55 p. 2737 (1995), CPP32 (or YAMA or apopaína) (caspase-3) (Fernandes-Alnemri, T. et al., J. Biol. Chem. 269, p. 30761 (1994 ); Nicholson, DW et al., Nature, 376, page 37 (1995)), CMH-1 (or MCH-3) (caspase10 7) (Lippke et al., J. Biol. Chem., 271 (4), p1825-1828 (1996)); (FernandesAlnemri, T. et al., Cancer Res., (1995)), Mch5 (caspase-8) (Muzio, M. et al., Cell 85 (6), 817 - 827, ( 1996)), MCH-6 (caspase-9) (Duan, H. et al., J. Biol. Chem., 271 (34), pp. 16720-16724 (1996)), Mch4 (caspase-10) (Vincenz , C. et al., J. Biol. Chem. 272, pp. 6578 - 6583 (1997); Fernan15 des-Alnemri, T. et al., Proc. Natl. Acad. Sci. 93, pp. 7464 - 7469 (1996 )), lch-3 (caspase-11) (Wang, S. et al., J. Biol. Chem., 271, pp. 20580 - 20587 (1996)), mCASP-12 (caspase-12), (Van de Craen, M. and others

others, FEBS Lett. 403, p. 61-69 (1997); Yuan, Y. and Miura, M. PCT Publication WO 95/00160 (1995), ERICE (caspase-13), (Humke, E. W „and others, 20 J. Biol. Chem., 273 (25) pages 15702-15707 (1998)), and MICE (caspase-14) (Hu, S. et al., J. Biol. Chem., 273 (45) pp. 29648 - 29653 (1998)).

Each of these ICE homologues, as well as the ICE itself, is capable of inducing apoptosis when overexpressed in transfected cell lines. Inhibition of one more of these homologs with a peptidyl ICE inhibitor Tyr-Val-Ala-Asp-chloromethylketone results in inhibition of apoptosis in primary cells or cell lines. Lazebnik et al., Nature, 371, p. 346 (1994).

Caspases also appear to be involved in the regulation of apoptosis or programmed cell death. Yuan, J. et al., Cell, 75, pgs. 641 - 652 (1993); Miura, M. et al., Cell, 75, pgs. 653 - 660 (1993);

Nett-Fiordalisi, Μ. A. et al., J. Cell Biochem, 17B, p. 117 (1993). In particular, ICE or ICE counterparts are thought to be associated with

regulation of apoptosis in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Marx, J. and M. Baringa, Science, 259, p. 760 - 762 (1993); Gagliardini, V. et al., Science, 263, pgs. 826 828 (1994). Therapeutic applications for inhibiting apoptosis may include the treatment of Alzheimer's disease, Parkinson's disease, septic shock, myocardial infarction, spinal atrophy and aging.

ICE has been shown to mediate apoptosis (programmed cell death)

on certain types of fabric. Steller, H., Science, 267, p. 1445 (1995); Whyte, M. and Evan, G., Nature, 376, p. 17 (1995); Martin, S. J. and Green, D. R., Cell.

82, p. 349 (1995); Alnemri, E. S. et al., J. Biol. Chem., 270, p. 4312 (1995); Yuan, J. Curr. Opin. Cell Biol., 7, p. 211 (1995). A transgenic mouse with a disruption of the ICE gene is deficient in Fas-mediated apoptosis (Kuida, K. et al., Science 267, 2000 (1995). This ICE activity is distinct from its role as the processing enzyme for pro-IL-1beta. It is conceivable that in certain types of tissue, inhibition of ICE may not affect secretion of mature IL-1beta, but may inhibit apoptosis.

Enzyme-active ICE was previously described as a heterodimer consisting of two subunits, p20 and p10 (molecular weight

20 kDa and 10 kDa, respectively). These subunits are derived from a 45 kDa (p45) proenzyme as a p30 form, through an activation mechanism that is autocatalytic. Thornberry, N.A. et al., Nature, 356, pgs. 768 - 744 (1992), The ICE proenzyme has been divided into several functional domains; a pro-domain: a pro-domain (p14), a subunit of p22 / 20, a polypeptide linker and a subunit of p10. Thornberry et al., Supra; Casano et al., Genomics, 20, pgs. 474 - 481 (1994).

Complete tablet p45 was characterized by its cDNA and amino acid sequences. PCT patent applications WO 91/15577 and

WO 94/00154. The p20 and p10 cDNA and amino acid sequences are also known. Thornberry et al., Supra. Rat and mouse ICE were also sequenced and cloned. They have high sequence homology of nucleic acids and amino acids to human ICE.

Miller, D. K. and others. Ann. N. Y. Acad. Sci., 696, p. 133-148 (1993); Molineaux, S. M. et al., Proc. Nat. Acad. Sci. 90, p. 1809 - 1813 (1993). The three-dimensional structure of ICE was determined by atomic resolution by X-ray crystallography. Wilson, K. P. et al., Nature, 370, pp. 270 - 275 5 (1994). The active enzyme exists as a tetramer of two p20 subunits and two p10 subunits.

Recently, ICE and other members of the ICE / CED-3 family have been linked to converting pro-IGIF to IGIF or producing IFN-γ in vivo (PCT application US 96/20843, publication No. WO 97/22619 , which is 10 incorporated here by reference). IGIF is synthesized in vivo as the precursor protein pro-IGIF.

Gamma-interferon induction factor (IGIF) is a polypeptide of about 18 kDa that stimulates production of interferon-gamma T cells (IFNγ). IGIF is produced by activated Kupffer cells and macrophages in vivo and is exported out of such cells after endotoxin stimulation. Thus, a compound that decreases IGIF production would be useful as an inhibitor of such T-cell stimulation that would in turn reduce the levels of IFN-γ production by those cells.

IFN-γ is a cytokine with immunodulatory effects on a variety of immune cells. In particular, IFN-γ is involved in macrophage activation and Th1 cell selection (F. Belardelli. APMIS, 103, page 161 (1995)). IFN-γ exerts its effects in part by modulating gene expression via the STAT and IRF pathways (C. Schindler and JE Darnell. Ann. See. Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer Res. Clin. Oncol. 25 121, page 516 (1995)).

Mice that lack IFN-γ or its receptor have multiple defects due to immune cells and are resistant to endotoxic shock (S. Huang et al., Science, 259, p. 1742 (1993); D. Dalton et al., Science, 259, page 1739 (1993); BD Car et al., J. Exp. Med., 179, page 30 1437 (1994)). Along with IL-12, IGIF appears to be a potent inducer of IFN-γ production by células cells (H. Okamura et al., Infection and Immunity,

63, p. 3966 (1995); H. Okamura et al., Nature, 378, p. 88 (1995); S.

Ushio et al., J. Immunol., 156, p. 4274 (1996)).

IFN-γ has been shown to contribute to pathology associated with a variety of inflammatory, infectious and autoimmune diseases and disorders. Thus, compounds capable of decreasing IFN-γ production would be useful to improve the effects of pathologies related to IFN-γ.

Correspondingly, compositions and methods capable of regulating the pro-IGIF to IGIF conversion would be useful for decreasing IGIF and IFN-γ production in vivo, and thus for improving harmful effects.

of these proteins that contribute to human disorders and diseases.

Caspase inhibitors represent a class of compounds useful for the control of inflammation or apoptosis or both. ICE peptide and peptidyl inhibitors have been described (PCT patent applications WO 91/15577, WO 93/05071, WO 93/09135, WO 93/12076, WO 93/1477, WO

93/16710, WO 95/35308, WO 96/30395, WO 96/33209 and WO 98/01133, WO

96/30395, WO 96/33209 and WO 98/01133; European Patent Applications 503

561, 547 699, 618 223, 623 592, and 623 606; and U.S. patents nos. 5/434/248, 5,710,153, 5,716,929 and 5,744,451). Such ICE peptidyl inhibitors have been observed to block production of mature IL-1beta in a mouse model of inflammation (see below) and to suppress growth

of leukemia cells in vitro (Estrov et al., Blood, 84 380a (1994)). However, due to their peptide nature, such inhibitors are typically characterized by undesirable pharmacological properties, such as poor cellular activity and cell penetration, poor oral absorption, fast installability and metabolism, Plattner, JJ and DW Norbeck, in Drug Discovery Technology, CR Clark and WH Moos, Eds. (Ellis Horwood, Chichester,

England, 1990), p. 92 - 126. These properties prevented their development into effective drugs.

Non-peptidyl compounds have been reported to inhibit ICE in vitro. PCT patent application WO 95/26958; U.S. Patent No. 5,552,400; Dolle et al., J. Med. Chem., 39, pgs. 2438 - 2440 (1996).

It is not clear, however, whether these compounds have the appropriate pharmacological profiles to be therapeutically useful.

Correspondingly, there is a need for compounds that can effectively inhibit caspase, and that have favorable in vivo activity, for use as agents for the prevention and treatment of chronic and acute forms of IL-1 mediated diseases, apoptosis, IGIF or IFN- γ, as well as inflammatory, autoimmune, bone destructive, proliferative, infectious or degenerative diseases.

Summary of the Invention

The present invention provides new classes of compounds, and their pharmaceutically acceptable derivatives, which are useful as inhibitors of caspase, in particular, as inhibitors of ICE. These compounds can be used alone or in combination with other therapeutic or prophylactic agents, such as antibiotics, immunomodulators or other anti-inflammatory agents, for the treatment or prophylaxis of diseases mediated by

IL-1, apoptosis, IGIF or IFN-γ. According to a preferred embodiment, the compounds of this invention are able to bind to the active site of a caspase and inhibit the activity of that enzyme.

It is a main objective of this invention to provide new classes of compounds represented by formula I, which have favorable in vivo profiles:

where various substituents are described here.

It is another object of this invention to provide pharmaceutical compositions, including multi-component compositions. This invention also provides methods for the use and preparation of the compounds of this invention and related compounds.

Detailed Description of the Invention

In order that the invention described here can be more fully understood, the following detailed description is given.

Abbreviations

Ac ₂ O Acetic anhydride MeCN Acetonitrile AMC aminomethyl coumarin n-Bu normal butyl DMF Dimethylformamide DIEA N, N-diisopropylethylamine DMA N, N-dimethylacetamide EDC 1- (3-dimethylaminopropyl) - 3-ethylcarbodiimide hydrochloride Et ₂ O diethyl ether EtOAc ethyl acetate Fmoc 9-fluorenylmetoxycarbonyl HBTU O-benzotriazole-1-yl-N, N, N, N'-tetramethyluronium hexafluorophosphate HOBT 1-hydroxybenzotriazole hydrate MeOH Methanol NMP N-methylpyrrolidone TFA trifluoroacetic acid pNA p-nitrophenyl

The term caspase refers to an enzyme that is a member of the enzyme family that includes ICE (see H. Hara, Natl. Acad. Sci., 94, pages 2007-2012 (1997)).

The terms HBV, HCV and HGV refer to hepatitis-B virus, hepatitis-C virus and hepatitis-G virus, respectively.

The term Kj refers to a numerical measure of the effectiveness of a compound in inhibiting the activity of a target enzyme such as ICE. Lower Ki values reflect superior effectiveness. The K value, is derived by adjusting data from experimentally determined rate for standard enzyme kinetic equations (see I. H. Segei, Wiley-Interscience, 1975).

The term gamma interferon induction factor or IGIF refers to a factor that is able to stimulate the endogenous production of IFN-γ.

The term caspase inhibitor refers to a compound that is capable of demonstrating detectable inhibition of one or more caspases. The term ICE inhibitor refers to a compound that is capable of demonstrating detectable inhibition of ICE and optionally one or more additional caspases.

Inhibition of these enzymes can be determined using the methods described and incorporated by reference here.

The skilled person realizes that an enzyme inhibitor in vivo is not necessarily an enzyme inhibitor in vitro. For example, a prodrug form of a compound typically shows little or no activity in in vitro assays. Such pro-drug forms can be altered by metabolic or other biochemical processes in the patient to provide an ICE inhibitor in vivo.

The term cytokine refers to a molecule that mediates interactions between cells.

The term condition refers to any disease, disorder or effect that produces harmful biological consequences for an individual.

The term individual refers to an animal, or to one or more cells derived from an animal. Preferably the animal is a mammal, more preferably a human being. Cells can be in any form, including but not limited to cells retained in tissue, cell groups, immortalized cells, transfected or transformed cells and cells derived from an animal that has been physically or phenotypically altered.

The term patient as used in this application refers to any mammal, preferably humans.

The term alkyl refers to a saturated, straight or branched aliphatic hydrocarbon containing 1 to 6 carbon atoms.

The term alkenyl refers to an unsaturated, straight or branched chain hydrocarbon containing from 2 to 6 carbon atoms and at least one double bond.

The term alkynyl refers to an unsaturated straight or branched hydrocarbon containing 2 to 6 carbon atoms and at least one triple bond.

The term cycloalkyl refers to a non-aromatic mono- or polycyclic hydrocarbon ring system that can optionally contain unsaturated bonds in the ring system. Examples include cyclohexyl, adamantyl, norbornyl, and spirocyclopentyl.

The term aryl refers to a mono- or polycyclic ring system that contains 6, 10, 12 or 14 carbons in which at least one ring of the ring systems is aromatic. The aryl groups of this invention are optionally individually or multiply substituted with R ¹¹ . Examples of 5 aryl ring systems include phenyl, naphthyl and tetrahydronaphthyl.

The term heteroaryl refers to a mono- or

polycyclic containing 1 to 15 carbon atoms and 1 to 4 hetero atoms, and in which at least one ring of the ring system is aromatic. Heteroatoms are sulfur, nitrogen or oxygen. The heteroaryl groups of this invention are optionally individually or multiply substituted with R ¹¹ .

The term heterocyclic refers to a mono- or

polycyclic containing 1 to 15 carbon atoms and 1 to 4 hetero atoms, and where the mono- or polycyclic ring system can optionally contain unsaturated bonds but is not aromatic. Heteroatoms independently of sulfur, nitrogen or oxygen.

The term alkylaryl refers to an alkyl group, in which a hydrogen atom of the alkyl group is replaced by an aryl radical.

The term alkyletheroaryl refers to an alkyl group, wherein a hydrogen atom of the alkyl group is replaced by a heteroaryl radical.

The term amino acid side chain refers to any group attached to the alpha carbon of a naturally occurring or unnatural amino acid.

The term substitute refers to the replacement of a hydrogen atom in a compound with a substituent group.

The term straight chain refers to a contiguous non-branching strand of covertly linked atoms, but these substituents are not a part of the straight chain.

In chemical formulas, parentheses are used here to mean connectedness in molecules or groups. In particular, parentheses are used to indicate: 1) that more than one atom or group is attached to a

particular atom; or 2) branching point (ie the atom immediately before the open parenthesis is linked to both the atom or group in the parentheses and the atom or group immediately after the closed parenthesis). An example of the first use is -N- (alkyl) 2, indicating attachment of two alkyl groups to an N atom. An example of the second use is -C (O) NH ₂ indicating a carbonyl group and an amino group (NH ₂ ) both attached to the indicated carbon atom. A -C (O) NH _{2 group} can be represented by other means, including the following structure: o

Substituents can be represented in several forms. These various forms are known to the skilled practitioner and can be used interchangeably. For example, a methyl substituent or a phenyl ring can be represented in any of the following ways:

0- “. θ '.

Various forms of substituents such as methyl are used interchangeably.

Further definitions are given in the report where necessary. Compounds of this Invention

The compounds of an embodiment A of this invention are those of formula I:

on what:

Yé:

(The)

with the proviso that when R ⁷ is -OH then Y can also be:

(B)

m is 0 or 1;

R ¹ is H, -C (O) R ⁸ , -C (O) C (O) R ⁸ , -S (O) 2R ⁸ , -S (O) R ⁸ , -C (O) OR ⁸ , - C (O) N (H) R ⁸ , -S (O) 2N (H) -R ⁸ , -S (O) N (H) -R ⁸ , -C (O) C (O) N (H) R ⁸ , -C (O) CH = CHR ⁸ , (C (O) CH2OR ⁸ , -C (O) CH2N (H) R ⁸ , -C (O) N (R ⁸ ) 2i -S (O) 2N (R ⁸ ) 2i -S (O (R ⁸ ) 2, -C (O) C (O) N (R ⁸ ) 2- -C (O) CH ₂ N (R ⁸ ) 2, -CH2R ⁸ , - CH2-alkenyl-R ⁸ , or -CH ₂ alkynyl-R ^B ;

R ₂ is -H and each R ³ is independently -H, an amino acid side chain, -R ⁸ , alkenyl-R ⁹ , or alkynyl-R ⁹ , or R ² and an R ³ together with the atoms to which they are connected, form a ring system

3- to 7-membered heterocyclic or cyclic, where a hydrogen atom attached to any -alkyl or -cycloalkyl carbon atom is optionally substituted by -R ¹⁰ , a hydrogen atom attached to any -aryl carbon atom or - heteroaryl is optionally substituted by -R ¹¹ , a hydrogen atom attached to any nitrogen atom in the ring system is optionally substituted by -R ¹ ;

R ⁴ is -H and each R ⁵ is independently -H, an amino acid side chain, -R ⁸ , -alkenyl-R ⁹ , or -alkynyl-R ⁹ , or R ⁴ and an R ⁵ together with the atoms to which they are linked form a 3- to 7-membered heterocyclic or cyclic ring system, in which a hydrogen atom 25 attached to any carbon atom of -alkyl or -cycloalkyl is optionally substituted by R ¹⁰ , a hydrogen atom attached to any carbon atom of -aryl or -heteroaryl is optionally substituted by R ¹¹ , and

a hydrogen atom attached to any nitrogen atom in the ring system is optionally substituted with R ¹ ;

R ⁶ is -H;

R ⁷ is -OH, -OR ⁸ , or -N (H) OH;

each R ⁸ is independently -alkyl, -cycloalkyl, -aryl, heteroaryl, -heterocyclyl, -alkylcycloalkyl, -alkylaryl, -alkyletheroaryl, or alkyletherocyclyl,

wherein a hydrogen atom attached to any -alkyl or -cycloalkyl carbon atom is optionally substituted by R ¹⁰ , a hydrogen atom attached to any -aryl or heteroaryl carbon atom is optionally substituted by R ¹¹ , and one hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ;

each R ⁹ is independently -aryl, -heteroaryl, cycloalkyl, or

-heterocyclyl, where a hydrogen atom attached to any -alkyl or -cycloalkyl carbon atom is optionally substituted by R ¹⁰ , a hydrogen atom attached to any -aryl or heteroaryl carbon atom is optionally substituted by R ¹¹ , and a hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ;

each R ¹⁰ is independently -OH, -SH, -F, -Cl,

-Br, -I, -N0 ₂ , -CN, -NH ₂ , -CO ₂ H, -C (O) NH ₂ , -N (H) C (O) H, -N (H)

C (O) NH ₂ , -perfluoroalkyl, -O-alkyl, -O-aryl, -o-alkylaryl, -N (H) alkyl, -N (H) aryl, -N (H) -alkylaryl, -N ( alkyl) ₂ , -C (O) N (H) alkyl, -C (O) N (alkyl) ₂ , -N (H) C (O) alkyl, -N (H) C (O) N (H) alkyl, -N (H) C (O) N (alkyl1) ₂ , -S-alkyl, -Sari, -S-alkylaryl, -S (0) ₂ alkyl, -S (O) alkyl, -C (O) alkyl, -CH ₂ NH ₂ , -CH ₂ N 25 (H) alkyl, or -CH ₂ N (alkyl) ₂ , -alkyl, -cycloalkyl, -aryl, -heteroaryl, heterocyclyl, -alkylcycloalkyl, -alkylaryl, -alkyleteroaryl , or alkyletherocyclyl, in which a hydrogen atom attached to any -aryl or -heteroaryl carbon atom is optionally substituted by R ¹¹ and a hydrogen atom attached to any nitrogen atom is optionally replaced by R ¹ ; and each R ¹¹ is independently -OH, -SH, -F, -Cl, -Br, -I, -NO ₂ , -CN, -NH ₂ , -C0 ₂ H, -C (O) NH ₂ , -N (H) C (O) H, -N (H) C (O) NH _2i -alkyl, -

cycloalkyl, -perfluoroalkyl, -O-alkyl, -O-aryl, -O-alkylaryl, -N (H) alkyl, -N (H) aryl, -N (H) -alkylaryl, -N (alkyl) ₂ , - C (O) N (H) alkyl, -C (O) N (alkyl) ₂ , -N (H) C (O) aylyl, -N (H) C (O) N (H) alkyl, -N ( H) C (O) N (alkyl) ₂ , -S-alkyl, S-aryl, -S-alkylaryl, -S (O) ₂ alkyl, -S (O) alkyl, -C (O) alkyl, -CH ₂ NH ₂ , 5 CH ₂ N (H) alkyl, or -CH ₂ N (alkyl) ₂ .

In an alternative form of embodiment A:

R ¹ is H, -R ⁸ , -C (O) R ⁸ , -C (O) C (O) R ⁸ , -S (0) 2R ⁸ , -S (O) R ⁸ , -C (O) OR ⁸ , -C (O) N (H) R ⁸ , -S (O) 2N (H) -R ⁸ , -S (O) N (H) -R ⁸ , -C (O) C (O) N (H) R ⁸ , -C (O) CH = CHR ⁸ , -C (O) CH2OR ⁸ , -C (O) CH2N (H) R ⁸ , -C (O) N (R ⁸ ) 2, - S (0) 2N (R ⁸ ) 2, -S (O) N 10 (R ⁸ ) 2, -C (O) C (O) N (R ⁸ ) 2í -C (O) CH ₂ N (R ⁸ ) 2i -CH2R ⁸ , -CH2-alkenyl-R ⁸ , or -CH ₂ alkynyl-R ⁸ ;

R ² is -H and each R ³ is independently -H, an amino acid side chain, -R ⁸ , alkenyl-R ⁹ , or alkynyl-R ⁹ , or each R ³ , together with the atom to which they are attached, form a 3- to 7-membered heterocyclic or cyclic ring system, or R ² and R ³ together with the atoms to which they are attached, form a 3- to 7-membered heterocyclic or cyclic ring system, in which one atom of hydrogen attached to any carbon atom of -alkyl or -cycloalkyl is optionally substituted by -R ¹⁰ , a hydrogen atom attached to any carbon atom of -aryl or 20 heteroaryl is optionally substituted by -R ¹¹ , a hydrogen atom

attached to any nitrogen atom in the ring system is optionally replaced by -R ¹ ;

each R ¹⁰ is independently -OH, -SH, -F, -Cl, -Br, -I, -NO ₂ ,

-CN, -NH ₂ , -CO ₂ H, -C (O) NH ₂ , -N (H) C (O) H, -N (H) C (O) NH _2i -perfluoroalkyl,

-O-alkyl, -O-aryl, -O-alkylaryl, -N (H) alkyl, -N (H) aryl, -N (H) -alkylaryl,

-N (alkyl) ₂ , -C (O) N (H) alkyl, -C (O) N (alkyl) ₂ , -N (H) C (O) alkyl, -N (H) C (O) Oalkyl , -N (H) C (O) Oaryl, -N (H) C (O) Oalkylaryl, -N (H) C (O) Oheteroaryl, -N (H) C (O) Oalkyletheroaryl, -N (H) C (O) Ocycloalkyl, -N (H) C (O) N (H) alkyl, -N (H) C (O) N (alkyl) ₂ , -N (H) C (O) N (H) aryl , -N (H) C (O) N (H) alkylaryl, -N (H) C (O)

N (H) heteroaryl, -N (H) C (C) N (H) alkyletheroaryl, -N (H) C (O) N (H) cycloalkyl, -S-alkyl, -S-aryl, -S-alkylaryl , -S (0) ₂ alkyl, -S (O) alkyl, -C (O) alkyl,

-CH ₂ NH ₂ , -CH ₂ N (H) alkyl, or -CH ₂ N (alkyl) ₂ , -alkyl, -cycloalkyl, -aryl,

-heteroaryl, -heterocyclyl, -alkylcycloalkyl, -alkylaryl, -alkyletheroaryl, or -alkyletherocyclyl, in which a hydrogen atom attached to any carbon-aryl or -heteroaryl atom is optionally substituted by R ¹¹ and a hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ; and the other substituents are as defined above.

Preferably, in any of the above embodiments:

m is 0;

R ² is -H;

one R ³ is -H and the other R ³ is -R ⁸ ,

-alkenyl-R ⁹ , or -alkynyl-R ⁹ ; or

R ⁴ and R ⁵ together with the atoms to which they are attached form a 3- to 7-membered heterocyclic or cyclic ring system, in which a hydrogen atom attached to any 15-alkyl or -cycloalkyl carbon atom is optionally substituted with R ¹⁰ , a hydrogen atom attached to any heteroaryl carbon atom is optionally replaced by R ¹¹ , and a hydrogen atom attached to any nitrogen atom in the ring system is optionally replaced with R ¹ , where the ring is:

In an alternative preferred embodiment, X is C (R ³ ) ₂ or an R ³ is an amino acid side chain, -R ⁸ , alkenyl-R ⁹ , or alkynyl-R ⁹ , more preferably, an R ³ is -H and the other R ³ is -alkyl; or

R ⁴ and R ⁵ together with the atom to which they are attached form a 3- to 7-membered heterocyclic or cyclic ring system, in which any hydrogen atom attached to a carbon atom of the ring system is optionally substituted by R ¹⁰ and any hydrogen atom 5 attached to a nitrogen atom in the ring system is optionally substituted by R ¹ , selected from:

more preferably, one R ³ is -H and the other is

R ³ is - C (H) (CH ₃ ) ₂ or - C (CH ₃ ) ₃ ; and

R ⁴ and R ⁵ together with the atoms to which they are attached form a 3- to 7-membered heterocyclic or cyclic ring system, in which any hydrogen atom attached to a carbon atom of the ring system is optionally substituted by R ¹⁰ and any hydrogen hi15 atom attached to a nitrogen atom in the ring system is optionally substituted by R ¹ , selected from:

N.

In an alternative most preferred embodiment, one R ³ is -H and the other R ³ is -CH ₃ , -C (H) (CH ₃ ) ₂ or -C (OH ₃ ) ₃ and R ⁴ and

R ⁵ are as defined directly above.

According to another embodiment B, the present invention

provides a compound of formula I, where Y is:

with the proviso that when R ⁶ is not hydrogen, R ⁶ and Y, together with the nitrogen to which they are attached, form a ring (g):

R ¹² is -C (O) alkyl, -C (O) cycloalkyl, -C (O) alkenyl, C (O) alkylaryl, -C (O) alkyletheroaryl, -C (O) heterocyclyl, or C (O) alkyleterocyclyl ;

R ¹³ is -H, -alkyl, -aryl, -alkylaryl or

-alkyletheroaryl; and the other substituents are as described above.

Preferably, in (c), (d), (e) or (f), R ⁸ is methyl, ethyl, n-pzopyl, isopropyl, cyclopentyl, phenethyl, or benzyl.

Preferred definitions for the other individual components of embodiment B are the same as those indicated above for concretization A.

A preferred embodiment C of this invention provides compounds of formula I:

on what

Yé:

(a) (b)

m is 0 or 1;

X is -C (R ³ ) 2R ¹ is H, -R ⁸ , -C (O) R ⁸ , -C (O) C (O) R ⁸ , -S (0) 2R ⁸ , -S (O) R ⁸ , -C (O) OR ⁸ , -C (O) N (H) R ⁸ , -S (0) 2N (N) -R ⁸ , -S (O) N (H) -R ⁸ , - C (O) C (O) N (H) R ⁸ , -C (O) CH = 15 CHR ⁸ , -C (O) CH20R ⁸ , -C (O) CH2N (H) R ⁸ , -C (O ) N (R ⁸ ) 2i -S (O) ₂ N (R ⁸ ) 2, -S (O) N (R ⁸ ) 2i -C (O) C (O) N (R ⁸ ) _2i -C (O ) CH ₂ N (R ⁸ ) 2, -CH2R ⁸ , -CH2-alkenyl-R ⁸ . or -CH ₂ alkynyl-R ⁸ ;

R ² is -H and each R ³ is independently -H, an amino acid side chain, -R ⁸ alkenyl-R9, or alkynyl-R ⁹ , or each R ³ together with the atom to which they are attached, form a system of a 3- to 7-membered heterocyclic or cyclic ring, where a hydrogen atom attached to either a -alkyl or -cycloalkyl carbon atom is optionally substituted by R ¹⁰ , a hydrogen atom attached to any carbon atom of

-aryl or -heteroaryl carbon atom is optionally substituted by R11, a hydrogen atom attached to any nitrogen atom in the ring system is optionally substituted by R ¹ ;

R ⁴ is -H and each R ⁵ is independently -H, an amino acid side chain, -R ⁸ , -alkenyl-R ⁹ , or -alkynyl-R ⁹ , or R ⁴ and an R ⁵ together with the atoms to which they are linked form a ring system

3- to 7-membered heterocyclic or cyclic, where a hydrogen atom attached to any -alkyl or -cycloalkyl carbon atom is optionally substituted by R ¹⁰ , a hydrogen atom attached to any -aryl carbon atom or - heteroaryl is optionally substituted by R ¹¹ , and a hydrogen atom attached to any nitrogen atom in the ring system is optionally substituted with R ¹ ;

R ⁶ is -H;

R ⁷ is -OH, -OR ⁸ , -N (H) OH, or -N (H) S (O) ₂ R ⁸ ;

each R ⁸ is independently -alkyl, -cycloalkyl, -aryl, heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkyletheroaryl, or alkyletherocyclyl, in which a hydrogen atom attached to any -alkyl or -cycloalkyl carbon atom is optionally substituted by R ¹⁰ , a hydrogen atom attached to any carbon atom of -aryl or 20 heteroaryl is optionally replaced by R ¹¹ , and a hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ;

each R ⁹ is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, in which a hydrogen atom attached to any carbon atom of -alkyl or -cycloalkyl is optionally substituted by R ¹⁰ , a hydrogen atom attached to any atom carbon-aryl or heteroaryl is optionally substituted by R ¹¹ , and a hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ;

each R ¹⁰ is independently -OH, -SH, -F, -Cl, -Br, -I, -N0 ₂ ,

-CN, -NH ₂ , -CO ₂ H, -C (O) NH ₂ , -N (H) C (O) H, -N (H) C (O) NH ₂ , -perfluoroalkyl,

-O-alkyl, -O-aryl, -O-alkylaryl, -N (H) alkyl, -N (H) aryl, -H (H) -alkylaryl, -N (alkyl) ₂ , -C (O) N (H) alkyl, -C (O) N (alkyl) ₂ , -N (H) C (O) alkyl, -N (H) C (O) Oalkyl, -N (H) C (O) Oaryl, - N (H) C (O) Oalkylaryl, -H (H) C (O) 0 heteroaryl, -N (H) C (O) Oalkyleteroaryl, -N (H) C (O) Ocycloalkyl, -N (H) C ( O) N (H) alkyl, -N (H) C (O) N (alkyl) ₂ , -N (H) C (O) N (H) aryl, -N (H) C (O) N (H ) alkylaryl, -N (H) C (O) N (H) heteroaryl, -N (H) C (O) N (H) alkyletheroaryl,

N (H) C (O) N (R) cycloalkyl, -S-alkyl, -S-aryl, -S-alkylaryl, -S (O) ₂ alkyl, S (O) alkyl, -C (O) alkyl, -CH ₂ NR ₂ , -CH ₂ N (H) alkyl, or -CH ₂ N (alkyl) ₂ , alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl, alkylaryl, -alkyletheroaryl, or -alkyleterocyclyl , wherein a hydrogen atom attached to any -aryl or -heteroaryl carbon atom is optionally substituted by R ¹¹ and a hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ; and each R ¹¹ is independently -OH, -SH, -F, -Cl, -Br, -I, -NO ₂ , -CN, -NH ₂ , -CO ₂ H, -C (O) NH ₂ , -N (H) C (O) H, -N (H) C (O) NH ₂ , -alkyl, cycloalkyl, -perfluoroalkyl, -O-alkyl, -O-aryl, -O-alkylaryl, -N (H) alkyl , -N (H) aryl, -N (H) -alkylaryl, -N (alkyl) ₂ , -C (O) N (H) alkyl, -C (O) N (alkyl) ₂ , -N (H) C (O) alkyl, -N (H) C (O) N (H) alkyl, -N (H) C (O) N (alkyl) ₂ , -S-alkyl, S-aryl, -S-alkylaryl, -S (0) ₂ alkyl, -S (0) alkyl, -C (O) alkyl, -CH ₂ NH ₂ , CH ₂ N (H) alkyl, or -CH ₂ N (alkyl) ₂ ;

with the proviso that if one R ³ is -H, then the other R ³ is not -H.

Another preferred embodiment D of the present invention provides a compound of formula I, wherein Y is:

R ¹² is -C (O) alkyl, -C (O) cycloalkyl, -C (O) alkenyl,

C (O) alkylaryl,

-C (O) alkyletheroaryl, -C (O) heterocyclyl, or

C (O) alkyl etherocyclyl; and the other substituents are as described above except that both R ³ groups can be -H.

In any of the A-D embodiments, preferred compounds are those in which:

R ¹ is -C (O) R ⁸ or -C (O) C (O) R ⁸ ;

R ² and one R ³ are both -H and the other R ³ is a side chain

amino acid, -R ⁸ , alkenyl-R ⁹ , or alkynyl-R ⁹ ; or

R ⁴ and R ⁵ together with the atoms to which they are attached form a ring system selected from:

R10 THE t THE r G t River - f f '^ N- ^ Y-Rio River 4 t 0 » , ep f P RtO ϊ 6 1 THE f Γ ^ζ ι ° f ^ v ^Rie r ^z t f Rio, .φ r River 9 f çx * ° 1 f

kò

Rto , ço ', or t

with the proviso that each of the ring systems is optionally substituted with one or more R ¹⁰ groups.

Alternatively, preferred compounds of the AD embodiments are those in which R ³ is -H and the other R ³ is methyl, isopropyl, tert-butyl, -CH2SR ⁸ , -CH2SO2R ⁸ , -CH2CH2SR ⁸ , -CH2CH ₂ S (O) ₂ R ⁸ .

Most preferred compounds of the AD embodiments are those in which R ⁴ and an R ⁵ together with the atoms to which they are attached form the ring system:

and the other R ⁵ is H; or one R ³ is -H and the other R ³ is methyl.

Alternatively, more preferred compounds in AD embodiments are those in which R ⁴ and an R ⁵ together with the atoms to which they are attached form the ring system:

and the other R ⁵ is H.

In the above alternative embodiment, R ¹⁰ is preferably 4-fluorine or 4,4-difluorine.

Most preferred compounds of this invention are those in which R ³ is methyl; and

R ⁴ and R ⁵ together with the atoms to which they are attached form the ring system;

and the other R ⁵ is H.

Alternatively, more preferred compounds of the embodiments

AD are those in which R ³ is methyl; and R ⁴ and an R ⁵ together with the atoms to which they are attached form the ring system:

other ^{5 is} H; and

R ¹⁰ is 4-fluorine or 4,4-difluorine.

Preferred compounds of embodiments (B) or (D) are those

0% cc

Specific compounds of this invention include, but are not limited to, examples 5a-5bd, 7a-7at, 9a-9g, 15a-15f, 16a-16b, 17a-17e, 18a-18f, 20a-20t, 23a-23i, 24a-24e, 25a-25e, 26a-26h, 27a-27n, 28a-28c, 29a-29s, 32a-32e, 34, G1, G2, 41, 42, 45, 46, 51, 52, 56, 57, 60, 61, 64, 65, 68, 69, 72, 73, 76-93, 98a-z, aa-az, and ba-bb, 101, 102a, 102b, 108a-d, 110, 111, 116a-h , 120a and b, 121, 122 av, and 123 ac.

The compounds of this invention can contain one or more asymmetric carbon atoms and thus can occur as racemic mixtures and racemates, simple enantiomers, diastereomeric mixtures and individual diastereomers. Each stereogenic carbon can be of the R or S configuration. Although specific frames and compounds exemplified in this application can be shown in a stereochemical configuration, compounds and frames having either the opposite stereochemistry at any chiral center or mixtures thereof are also provided for.

All such isomeric forms of these compounds are expressly included in the present invention, as well as their pharmaceutically acceptable derivative.

The term pharmaceutically acceptable derivative means any pharmaceutically acceptable salt, ester or salt of such an ester, of a compound of this invention or any other compound which after administration to a receptor, is capable of providing (directly or indirectly) a compound of this invention or a its active metabolite or residue.

Pharmaceutically acceptable salts of the compounds of this invention include, for example, those derived from pharmaceutically acceptable organic and inorganic 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, malonic, naphthalene-2-sulfonic and benzenesulfonic. Other acids, such as oxalic,

although not in themselves pharmaceutically acceptable, they can be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (eg sodium), alkaline earth metal (eg magnesium), ammonium and N- (C-m alkyl) / salts.

This invention also provides for the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Basic nitrogen can be quaternized with any agents known to those skilled in the art including, for example, lower alkyl halides, such as methyl, ethyl and propyl butyl bromides, iodides and butyl chloride; dialkyl sulfates including dimethyl, diethyl, dibutyl, and diamyl sulfates;

long chain halides such as stearyl iodides, bromides and chlorides,

myristyl, lauryl and decila; and aralkyl halides including benzyl and phenethyl bromides. Dispersible or oil-soluble or water-soluble products can be obtained by such quaternization.

When multiple substituted, each substituent can be collected independently of any other substituent as long as the combination of substituents results in the formation of a stable compound.

Combinations of substituents and variables provided by this invention are only those that result in the formation of stable compounds. The term stable, as used herein, refers to compounds that have sufficient stability to permit manufacture and administration to a mammal by methods known in the art. Typically, such compounds are stable at a temperature of 40 ° C or less, in the absence of moisture from other chemically reactive conditions, for at least one week.

Preferred compounds of this invention can be readily absorbed into the bloodstream of patients after oral administration. This oral availability makes such compounds excellent agents for orally administered treatment and prevention regimes against diseases mediated by IL-1-apoptosis, IGIF or IFN-γ.

It should be understood that the compounds of this invention can exist in various forms of equilibrium depending on conditions including choice of solvent, pH and others known to the person skilled in the art. All such forms of these compounds are expressly included in the present invention. In particular, many compounds of this invention, especially those containing aldehyde or ketone groups and Y-carboxylic acid groups, can take hydrate or semi-acetal forms. For example, compounds of embodiment A are in a semi-acetal form when Y is:

hcAi

Depending on the choice of solvent and other known conditions27

For those skilled in the art, compounds of this invention may also take hydrated, acyloxy acetal, acetal or enol forms. For example, compounds of this invention are in hydrated forms when Y is:

and R ⁸ is H;

acyloxy acetal forms when Y is:

acetal forms when Y is and R ⁸ is other than H:

and forms of enol when Y is:

In addition, it should be understood that the equilibrium forms of the compounds of this invention can include tautomeric forms. All such forms of these compounds are expressly included in the present invention.

The compounds of formula I can be synthesized using conventional techniques. Advantageously, these compounds are conveniently synthesized from readily available starting materials.

Compounds of this invention can be prepared using the processes described here. As can be appreciated by the converse, these pro28

processes are not the only means by which the compounds described and claimed in this application can be synthesized. In addition, the methods will be evident by those normally skilled in the art. In addition, the various synthetic steps described here can be performed in an order or substitute sequence to give the desired compounds.

It would be understood that the compounds of this invention can be modified by appropriate functionalities to increase selective biological properties. Such modifications are known in the art and include those that increase biological penetration into a given biological system (eg, blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter the rate of excretion. In addition, the compounds can be changed to the prodrug form so that the desired compound is created in the patient's body 15 as a result of the action of metabolic or other biochemical processes on the prodrug. Such pro-drug forms typically show little or no activity in in vitro assays. Some examples of pro-drug forms include ketal, acetal, oxime, imine and hydrazone forms of compounds that contain ketone or aldehyde groups, especially

where they occur in the group Y of the compounds of this invention. Other examples of prodrug forms include the semi-acetal, semiacetal, acyloxy ketal, acyloxy acetal, ketal, acetal and enol forms that are described here.

Compositions and Methods

The compounds of this invention are caspase inhibitors, and in particular ICE inhibitors. Correspondingly, these compounds are able to target and inhibit events in diseases mediated by IL-1, apoptosis, IGIF, and IFN-γ, and, thus, the final activity of that protein in inflammatory diseases, autoimmune diseases, proliferative disorders, destructive bones, infectious diseases and degenerative diseases. For example, the compounds of this invention inhibit the conversion of precursor IL-1beta to mature IL-beta by inhibiting ICE. Since ICE is essential for the production of mature IL-1, inhibition of that enzyme effectively blocks initiation of

SO symptoms and physiological effects mediated by IL-1, such as inflammation, by inhibiting the production of mature IL-1. Thus, by inhibiting precursor activity of IL-1 beta, the compounds of this invention effectively function as inhibitors of IL-1.

Compounds of this invention also inhibit the conversion of proIGIF to mature, active IGIF by inhibiting ICE. Since ICE is essential for the production of mature IGIF, inhibition of ICE effectively blocks initiation of symptoms and physiological effects mediated by IGIF, by inhibiting the production of mature IGIF. IGIF is in turn essential for the production of IFN-γ 10. ICE therefore effectively blocks initiation of symptoms and physiological effects mediated by IFN-γ, by inhibiting production of mature IGIF and thus production of IFN-γ.

The compounds of this invention are surprisingly available when compared to peptidyl inhibitors, such as those described in, for example, EP 618 223, EP 623 592, WO 93/09135, WO

93/16710, U.S. Patent No. 5,434,248, WO 95/35308 or WO 96/33209.

Thus, the pharmaceutical methods and compositions of this invention will be useful for the control of caspase activity in vivo. The compositions and methods of this invention will therefore be useful for controlling levels of IL-1, IGIF or IFN-γ in vivo and for treating or reducing the progress, severity or effects of IL-1 mediated conditions, apoptosis, IGIF or IFNγ, including diseases, disorders or effects.

Pharmaceutical compositions of this invention comprise a compound of the formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such compositions can optionally comprise an additional therapeutic agent. Such agents include, but are not limited to, an anti-inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an antiviral agent, a cytokine, 30 a growth factor , an immunomodulator, a prostaglandin or an antivascular hyperproliferation compound.

The term pharmaceutically acceptable carrier refers to a

Λ

non-toxic vehicle that can be administered to a patient, together with a compound of this invention, and that does not destroy its pharmacological activity.

Pharmaceutically acceptable vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts zinc, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, wool fat, and auto drug delivery systems -emulsification (SEDDS) such as alpha-tocopherol, polyethylene glycol 1000 succinate or other similar polymeric delivery matrices s.

In pharmaceutical composition comprising only one compound of embodiments A-D as the active component, methods for administering these compositions may additionally comprise the step of administering an additional agent to the individual. Such agents include, but are not limited to, an anti-inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an antiviral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an antivascular hyperproliferation compound.

The term pharmaceutically acceptable amount refers to an amount effective in treating or ameliorating an IL-1 mediated disease, apoptosis, IGIF, or IFN-γ in a patient. The term prophylactically active amount refers to an amount effective in preventing or substantially reducing diseases mediated by IL-1, apoptosis, IGIF, or IFN-γ in a patient.

The compounds of this invention can be employed in a

conventional way for the control of IGIF and IFN-γ levels in vivo and for the treatment of diseases or reduction of the advance or severity of the effects that are mediated by IL-1, apoptosis, IGIF, or IFN-γ. Such treatment methods, their dosage levels and requirements can be selected by those normally skilled in the art from the available methods and techniques.

For example, a compound of this invention can be combined with a pharmaceutically acceptable auxiliary for administration to a patient suffering from an IL-1 mediated disease, apoptosis, IGIF, or IFN-γ in a pharmaceutically acceptable medium and in an effective amount. to reduce the severity of that disease.

Alternatively, the compounds of this invention can be used in compositions and methods for the treatment or protection of individuals against diseases mediated by IL-1, apoptosis, IGIF, or IFN-γ for extended periods of time. The compounds can be used in such compositions either alone or together with other compounds of this invention in a manner consistent with the conventional use of enzyme inhibitors in pharmaceutical compositions. For example, a compound of this invention can be combined with pharmaceutical auxiliaries.

conventionally used in vaccines and administered in prophylactically effective amounts to protect individuals over an extended period of time against the measured diseases IL-1, apoptosis, IGIF, or IFN-γ.

The compounds of formula I can also be co-administered with other caspase or ICE inhibitors to increase the effect of therapy or prophylaxis against various diseases mediated by IL-1, apoptosis, IGIF, or

IFN-γ.

In addition, the compounds of this invention can be used in combination either with conventional anti-inflammatory agents or with matrix metalloprotease inhibitors, lipoxygenase inhibitors and cytokine antagonists other than IL-1 beta.

The compounds of this invention can also be administered in combination with immunomodulators (for example, bropyrimine, anti-human alpha-interferon antibody, IL-2, GM-CSF, methionine encephalin, alpha-interferon, diethyldithiocarbamate, tumor necrosis factor , naltrexone and

EPO), with prostaglandins or with antiviral agents (for example, 3TC, polysulfated polysaccharides, geniclovir, ribavirin, acyclovir, alpha interferon, trimetotrexate and fanciclovir) or prodrugs of these or correlated compounds

to prevent or combat symptoms of IL-1 mediated disease such as inflammation.

When the compounds of this invention are administered in combination therapy with other agents, they can be administered sequentially or concurrently to the patient. Alternatively, prophylactic or pharmaceutical compositions according to this invention comprise a combination of a compound of formula I and another therapeutic or prophylactic agent.

The pharmaceutical compositions of this invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer to prefer oral administration. The pharmaceutical compositions 20 may contain any conventional non-toxic pharmaceutically acceptable carriers, auxiliaries or carriers. In some cases, the pH of the formulation can be adjusted with pharmaceutically acceptable buffers, bases or acids to increase the stability of the formulated compound or its form of delivery. The term parenteral as used here 25 includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial infusion or injection techniques.

The pharmaceutical compositions can be in the form of a sterile injectable preparation, for example, a sterile injectable oleaginous or aqueous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and <4

suspension. The sterile injectable preparation can also be a suspension or solution in a non-toxic parenterally acceptable solvent or diluent, for example, as a solution in 1,3-butanediol. Among the acceptable solvents and vehicles that can be employed are mannitol, water, Ringer's solution 5 and isotonic sodium chloride solution. In addition, fixed, sterile oils are conventionally employed as a suspending medium or solvent. For this purpose, any soft fixed oil can be used including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil suspensions or solutions may also contain a long-chain alcohol dispersant or diluent, such as those described in Pharmacopeia Helvetica, or a similar alcohol.

Pharmaceutical compositions of this invention can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets and aqueous solutions or suspensions. In the case of tablets for oral use, vehicles that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dry corn starch. When solutions or suspensions and propylene glycol are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents can be added.

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

&

Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For topical application to the skin, the pharmaceutical composition should be formulated 5 with a suitable ointment containing the active components suspended or dissolved in a vehicle. Vehicles for topical administration of drugs

posts of this invention include, but are not limited to, mineral oil, liquid oil, white oil, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsification wax and water. Alternatively, the pharmaceutical composition 10 can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a vehicle. Suitable vehicles include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention can also be applied topically to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically administered transdermal patches are also

included in this invention.

The pharmaceutical compositions of this invention can be administered by inhalation or nasal spray. Such compositions are prepared according to techniques well known in the pharmaceutical formulation technique and can be prepared as brine solutions, which use benzyl alcohol or other preservatives, absorption enhancers to increase bioavailability, fluorocarbons, and / or other dispersing agents25. or solubilizing agents known in the art.

Dosage levels of between about 0.01 and about 100 mg / kg in body weight per day, preferably between 0.5 and about 75 mg / kg in body weight per day and more preferably between about 1 and 50 mg / kg in body weight per day of the active ingredient compound are useful in a monotherapy for the prevention and treatment of diseases mediated by IL-1, apoptosis, IGIF, or IFN-γ, including inflammatory diseases, autoimmune diseases , destructive bone disorders, proliferative disorders, internal diseases

feces, degenerative diseases, necrotic diseases, inflammatory peritonitis, osteoarthritis, acute pancreatitis, chronic pacreatitis, asthma, respiratory distress syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, gastritis au5-to5 insulin dependent diabetes mellitus (type I), autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, psoriasis, atopic dermatitis, graft disease vs. host, osteoporosis, multiple myeloma-related bone disorder, leukemias and related disorders, 10 myelodysplastic syndrome, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kapose sarcoma, multiple myeloma, sepsis, septic shock, Shigellosis, Disease, Alzheimer's disease Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, neurological damage due to septic shock, ulcerative colitis, infectious hepatitis, juvenile diabetes, lichenplanus, acute dermatitis, eczema, primary cirrhosis, uveitis, Behcet's disease, atopic skin disease, pure red cell aplasia, aplastic anemia, amyotrophic lateral sclerosis, nephrotic syndrome and systemic diseases or

diseases with localized effects on the liver or other organs having an apoptic or inflammatory component caused by ingestion of excess diet alcohol or viruses, such as HBV, HCV, HGV, yellow fever virus, dengue fever virus, and encephalitis virus Japanese.

Typically, the pharmaceutical compositions of this invention will be administered about 1 to 5 times a day or alternatively, as a continuous infusion. Such administration can be used as an acute or chronic therapy. The amount of active ingredient that can be combined with the vehicle materials to produce a single dosage form will vary depending on the treated host and the particular mode of administration. A typical preparation will contain about 5% to about 95% active compound (w / w). Preferably, such preparations contain from about 20% to about 80% of the active compound.

When the compositions of this invention comprise a combination of a compound of formula I and one or more additional prophylactic or therapeutic agents, both the compound and the additional agent should be present at dosage levels of between about 10% to 80% of the dosage normally administered on a monotherapy regimen.

After the improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention can be administered, if necessary. Subsequently, the dosage and frequency of administration, or both, may be reduced, as a function of the symptoms, to a level where the improved condition is retained when the symptoms have been relieved to the desired level, the treatment would cease. Patients may, however, need intermittent treatment on a long-term basis after any recurrence or symptoms of illness.

As the skilled person will appreciate, higher and lower doses than those reported above may be necessary. Treatment regimens and specific dosage for any patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, excretion rate, drug combination, severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician.

Diseases mediated by apoptosis or IL-1 that can be treated or prevented by the compounds of this invention include, but are not limited to, inflammatory diseases, autoimmune diseases, proliferative disorders, infectious diseases and degenerative diseases. Apoptosis-mediated diseases that can be treated or prevented by the compounds of this invention include degenerative diseases.

Inflammatory diseases mediated by apoptosis or IL-1 can be treated or prevented, but are not limited to osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma and adult respiratory distress syndrome. The inflammatory disease is preferably osteoarthritis or a37 pacreatitis

guda.

Automatic IL-1 mediated diseases or apoptosis that can be treated or prevented include, but are not limited to, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis 5, Graves' disease, autoimmune gastritis, diabetes milluts dependent insulin (type I), autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, severe myasteinia, multiple sclerosis, inflammatory bowel disease, Crohn's disease, psoriasis, atopic dermatitis and graft vs. disease. host. Preferably auto10 immune disease is rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, psoriasis or atrophic dermatitis.

Destructive bone disorders mediated by apoptosis or IL-1 that can be treated or prevented include, but are not limited to, osteoporosis and bone disorder enhanced with multiple myeloma.

Proliferative disorders mediated by apoptosis or IL-1 that can be treated or prevented include, but are not limited to, leukemias and related disorders, such as myeloplastic syndrome, myelogenous leukemia

acute, chronic myelogenous leukemia, metatatic melanoma, Kaposi's sarcoma, and multiple myeloma.

Infectious diseases mediated by apoptosis or IL-1 that can be treated or prevented include, but are not limited to, sepsis, septic shock, and Shigellosis.

Necrotic or degenerative diseases mediated by apoptosis or

IL-1 that can be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia and myocardial ischemia.

Preferably, the degenerative disease is Alzheimer's disease.

Degenerative diseases mediated by apoptosis or IL-1 that can be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, sclerosis

multiple, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia and neurological damage due to septic shock.

Other diseases having an apoptotic or inflammatory component can be treated or prevented by the compounds of this invention. Such 5 diseases can be systemic diseases or diseases with localized effects on the liver and other organs and can be caused by, for example, ingestion of excess diet alcohol or viruses, such as HBV, HCV, HGV, yellow fever virus , dengue fever virus, and Japanese encephalitis virus.

Diseases mediated by IGIF or IFN-γ that can be treated or prevented by the compounds of this invention include, but are not limited to, inflammatory, infectious, autoium, proliferative, neurodegenerative and necrotic conditions.

Inflammatory diseases mediated by IGIF or IFN-γ that can be treated or prevented by the compounds of this invention include, but are not limited to, osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, cerebral ischemia, myocardial ischemia and adult respiratory distress syndrome. Preferably, the inflammatory disease is rheumatoid arthritis, ulcerative colitis, Crohn's disease, hepatitis or adult respiratory distress syndrome.

Infectious diseases mediated by IGIF or IFN-γ that can be treated or prevented include, but are not limited to, hepatitis, sepsis, septic shock and Shigellosis.

Autoimmune diseases mediated by IGIF or IFN-γ that can be treated or prevented include, but are not limited to, glomerulonephritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin-dependent diabetes milluts (type I), juvenile diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, severe myasteinia, multiple sclerosis, psoriasis, lichenplanus, graft vs. disease. host, acute dermatomyositis, eczema, primary cirrhosis, hepatitis, uveitis, Behcet's disease, atopic skin disease,

pure red cell aplasia, aplastic anemia, amyotrophic lateral sclerosis and nephrotic syndrome. Preferably, the autoimmune disease is glomerulonephritis, insulin-dependent diabetes mellitus (type I), juvenile dibetes, psoriasis

ase, graft disease vs. host or hepatitis.

Most preferred diseases that can be treated or prevented include rheumatoid arthritis, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, inflammatory peritonitis, septic shock, pancreatitis, traumatic brain injury, organ transplant rejection, osteoarthritis, asthma, psoriasis, Alzheimer's disease, atopic dermatitis, or leukemia and 10 related disorders, such as myelodysplastic syndrome or multiple myeloma.

Correspondingly, an embodiment of this invention provides a method for treating or preventing an apoptosis-mediated disease or IL-1 in an individual comprising the step of administering to the individual any compound, pharmaceutical composition, or combination described herein and a pharmaceutically carrier. acceptable.

Another embodiment of this invention provides a method of decreasing IGIF production in an individual comprising the step of administering to the individual any compound, pharmaceutical composition or combination described and a pharmaceutically acceptable carrier.

Yet another embodiment of this invention provides a method for decreasing the production of IFN-γ in an individual comprising the step of administering to the individual any compound, pharmaceutical composition or combination described herein and a pharmaceutically acceptable carrier.

Although this invention focuses on the use of the compounds disclosed herein for the prevention and treatment of diseases mediated by IL-1, apoptosis, IGIF and IFN-γ, the compounds of this invention can also be used as inhibitory agents for other cysteine proteases.

The compounds of this invention are also useful as commercial reagents that effectively bind caspases or other cysteine proteases including, but not limited to, ICE. As commercial reagents, the compounds of this invention, and their derivatives, can be used to block

protein proteolysis of a target peptide in cell or biochemical assays for ICE and ICE counterparts or can be derivatized to bind to a stable resin as a bonded substrate for affinity chromotography applications. These and other uses that characterize commercial cysteine protease inhibitors will be evident to those normally skilled in the art.

In order for this invention to be more fully understood, the following examples are given. These examples are for the purpose of illustration only and should not be construed as limiting the scope of the invention in any way.

General Methods

Analytical HPLC conditions:

Column: C-18, particular size: 5 μ, pore size:

100Â,

Column Size: 4.6 x 150 mm

Solvent A: 0.1% TFA / 1% MeCN / 98.9% water

Solvent B: 0.1% TFA / 99.9% MeCN

Gradient: A to B lasts 20 minutes at a flow rate of 1 ml / min.

Column: Cyan, particle size: 5 μ, pore size:

Column Size: 4.6 x 150 m

Solvent A: 0.1% TFA / 1% MeCN / 98.9% water

Solvent B: 0.1% TFA / 99.9% MeCN

Gradient: A / B = 99% / 1 to 50% / 50% for 20 minutes at a flow rate of 1 m / min.

HPLC Mass Spectral Analysis

Mass Spectral Analysis: All mass spectrum data was collected using a qua30 drupole Micromass Quattro II triple mass spectrometer (Beverley, MA) equipped with a cross-current electrospray ionization source. The mass spectrometer was coupled to an HPLC system manufactured by Hewlett-Packard

(ΗΡ1100). The auto-sampler for the system was a Gilson 215 liquid handler (Middleton, Wl). All equipment was controlled by the MassLynx program package purchased from Micromass.

Mass spectrum analysis was performed by chromatography

liquid-EM to determine purity and confirm molecular weight simultaneously. In cases, where sample purity has been determined by other means, a flow injection analysis (FIA) has been used instead of the total chromatography analysis. In all cases, both positive and negative ion spectra were collected.

Mass Spectrum Acquisition Conditions: For all experiments, the mass spectrometer was configured in electrospray mode with the transverse current counter electrode. A flow splitter was used to reduce the HPLC flow to 40% of the original flow. The inlet temperature was adjusted to 140 ° C and the drying gas flow was adjusted to maximize the signal. The resolution of the mass spectrometer was adjusted to 0.65 amu FWHM and data were collected in centroid mode. In positive ion mode, the cone voltage was set to 25V, the capillary voltage was 3.8 kV. In negative ion mode, the cone voltage was adjusted to 25V and the capillary voltage was adjusted to 3.5 kV. 20 Both in positive and negative ion mode, the time to acquire a

total spectrum was 1 s with a switching time of 0.25 seconds between scans. The swept mass range for molecules with an expected molecular weight of less than 350 amu was 70-500 m / z while for molecules with an expected mass of more than 350 amu the mass 25 for loading swept ratio was 200 -1000 m / z.

Chromatography Conditions: Liquid chromatography was performed using a YMC AQ C18 column (150 mm x 3 mm with a 5 pm particle and a pore size of 120A). For all analyzes, MeCN with 0.2% formic acid was combined with water with 0.2% formic acid 30 to form the elution gradient. The gradient profile consisted of starting with 15% MeCN: water and increasing the amount of MeCN linearly for 10 minutes at 90%. That concentration was kept constant

for two minutes before returning to the initial conditions. During the total analysis, the flow rate was 0.9 ml / min.

Flow Injection Conditions: A 1: 1 mixture of water for MeCN (also with 2% formic acid added) was used to acquire the FIA data. The flow rate was adjusted to 0.3 ml / min.

¹ H NMR

All ¹ H NMR spectra were acquired using a Bruker Instruments AMX-500 NMR spectrometer in the given solvent. Synthetic Methods

General procedure for the preparation of compounds of the formula

I, embodiment C (schemes l-VI).

Procedure for the preparation of 5a-5bd analogs.

Scheme I

In schemes l-VIII, the variable LR refers to the bonding resin and

is defined as shown above in Scheme I.

Step 1: A 6.7 g portion (0.8 mmol / gram loading,

5.36 mmoles) of 4-methyl benzhydrylamine hydrochloride salt resin (es5 diagram I) were washed with DMF (3 x 50 ml). DIEA / DMF 10% (3 x 50 ml and

N-methylpyrrolidone (NMP) (3 x 50 ml). To a suspension of the washed resin in 25 ml of NMP, successively compound 1 (1.1 eq, 3.5 g, 5.90 mmoles) DIEA (3.33 eq, 3.1 ml, 17.70 mmoles) was successively added , 1hydroxybenzotriazole hydrate (HOBt) (1.1 eq, 797 mg, 5.90 mmol), and 0-benzotriazole 10 Ν, Ν, Ν, Ν'-tetramethyluronium hexafluorophosphate (HBTU) (1.1 eq, 2.24 g , 5.90 mmoles). Compound 1 was prepared according to the literature procedure of A. M, Murphi et al., J. AM, Chem, Sog., 114, pages, 3156-3157 (1992). The mixture was spun at room temperature overnight using a wrist arm shaker.

The resulting mixture was filtered, and the resin was rinsed with

DMF was then treated with 12 mL of a 20% acetic anhydride solution in DMF for 30 minutes at room temperature. The mixture was filtered, and the resin was washed successively with DMF (2 x 50 ml), CH ₃ OH (50 ml), 1: 1 DMF / CH ₂ CI ₂ (2 x 50 ml), CH 3 OH (50 ml) and CH ₂ CI ₂ (3 x 50 ml.). After vacuum drying, 9.0 grams of resin 2 were obtained (0.48 mmol / gram load).

Step 2: To 4.5 g of resin 2 (0.48 mmol / gram, 2.16 mmol) was added 25 ml of a 20% solution of piperidine in DMF. The suspension was spun at room temperature for 5 minutes and drained. The procedure was repeated for 20 minutes. The resin was then washed successively with DMF (2 x 40 ml), CH ₃ OH (40 ml), CH 2 Cl 2 (2 x 40 ml), CH ₃ OH (40 ml; and NMP (40 ml). To a resin suspension in 40 mL of NMP, 2.92 g of N-Fmoc-proline (4 eq, 8.64 mmoles), 3.0 ml of DIEA (8 eq, 17.28 mmoles), 1.17 g of HOBt (4 eq, 30 8.64 mmoles) and 3.27 g of HBTU (14 eq, 5.64 mmoles) .The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 3 The resin was then washed

successively with DMF (2 x 40 ml), CH ₃ OH (40 ml), 1: 1 DMF / CH ₂ CI ₂ (2 x ml), CH ₃ 0H (40 ml) and CH ₂ CI ₂ (3 x 40 ml ), and briefly vacuum dried to provide resin 3,

Step 3: A suspension of resin 3 in 25 mL of a 20% solution of piperidine in DMF was spun at room temperature for 5 minutes. The suspension was drained. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (2 x 40 ml), CH ₃ OH (40 ml), CH 2 Cl 2 (2 x 40 ml), CH ₃ OH (40 ml) and NMP (2 x 40 ml). To a suspension of the resin in 40 ml of NMP were added successively 2.93 g of N10 FMOc-valine (4 eq, 8.64 mmoles), 3.0 ml of DIEA (8 eq, 17.28 mmoles), 1, 17 g of HOBt (4 eq, 8.64 mmoles) and 3.27 g of HBTU (4 eq, 8.64 mmoles). The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 3 hours. The resin was then washed successively with DMF (2 x 40 ml), C ₃ OH (40 ml), 15 1: 1 DMF / CH2 Cl2 (2 x 40 ml), C ₃ OH (40 ml) and CH ₂ CI ₂ ( 3 x 40 ml), and dried in vacuo to provide resin 4 (0.45 mmol / gram).

Step 4: To a 0.05 mmol portion of resin 4 was added mL of a 20% solution of piperidine in DMF. The suspension was spun at room temperature for 5 minutes, and drained. The procedure was repeated for 20 minutes. The resulting resin was washed successively

with DMP (3x5 ml), CH ₃ OH (5 ml), and NMP (3x5 ml). The desired carboxylic acid was then added (4 eq, 0.2 mmol), followed by 0.8 ml of a 0.25 M HoBt solution in NMP, 0.14 ml DIEA (8 aq, 0.4 mmol ) and 0.8 ml of a 0.25 M solution of HBTU in NMP. The mixture was spun at room temperature overnight and drained. The resin was washed successively with DMF (2x5 ml), CH3OH (5 ml), 1: 1 DMF / CH ₂ CI ₂ (2x5 ml), CH3OH (5 ml) and CH ₂ CI ₂ (3x5 ml), and was dried in a vacuum. A 2 ml portion of a 95% solution of TFA in water was then added to the resin. The mixture was stirred at room temperature for one hour, and was filtered. The filtrate was evaporated, and the residue was taken up in acetonitrile-water and purified by preparative HPLC to provide compounds 5a-5bd.

Product yield, analytical HPLC condition, run time

HPLC retention, product purity, and mass spectrum data obtained, for example, 5a-5bd, 7a-7at, 9a-9 g, I5a-15f, 16a-16b, 17a-17e, 18a18f, 20a-20t, 23a-23i, 24a-24e, 25a-25e, 26a-26h, 27a-27n, 28a-28c, 29a29s, 32a-32e are provided in table 1 unless otherwise noted,

Table 1. Physical data for selected examples

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 5th 1.0 5-45% / 10 ' 4.66 92 475.2 5b 1.0 5-45% / 10 * 6.89 85 457.2 5c 4.0 5-45% / 10 ' 5.98 85 469.2 5d 1.5 5-45% / 10 ' 6.82 95 467.5 5e 1.0 5-45% / 10 ' 5.52 95 418.2 5f 0.6 5-45% / 10 ' 4.28 93 434.2 5g 6.4 10-60% / 10 ’ 8.57 97 504.7 (+ Na) 5 am 15.6 10-60% / 10 ' 4.51 99 459.2 5i 6.6 5% -90% / 10 ' 9.34 90 506.1 5j 5.1 5% -90% / 10 ' 10.04 95 506.1 5k 10.74 5% -90% / 10 ' 8.64 85 520.1 5I 6.6 5% -90% / 10 ' 8.72 85 540.1 5 m 6.8 5% -90% / 10 ' 7.89 85 502.0 5n 1.9 5% -0% / 10 ’ 6.46 85 494.1 5th 3.8 5% -90% / 10 ' 7.04 85 506.1 5p 6.8 5% -90% / 10 ' 11.62 95 576.0 5q 2.2 5% -90% / 10 ’ 9.72 90 508.1 5r 3.8 5% -90% / 10 ’ 7.27 85 462.1 5s 4.3 5% -90% / 10 ' 6.45 90 470.1 5t 5.9 5% -60% / 9 ' 60% -90% / 2 ' 10.27 90 486.2 5u 3.1 5% -60% / 9 ' 60% -90% / 2 ’ 9.09 80 522.1 5v 1.0 5% -60% / 9 ' 60% -90% / 2 ' 11.63 85 50.2.2

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 5w 10.3 5% -60% / 9 ' 60% -90% / 2 ' 8.75 95 470.2 5x 8.8 5% -60% / 9 ' 60% -90% / 2 ' 8.88 95 565.1 5y 6.6 5% -60% / 9 ' 60% -90% / 2 ' 12.32 95 518.2 5z 10.2 5% -60% / 9 ' 60% -90% / 2 ' 12.63 95 502.2 5aa 2.5 5% -60% / 9 ' 60% -90% / 2 ' 9.57 95 554.1 5ab 7.8 5% -60% / 9 ' 60% -90% / 2 ' 10.54 85 538.2 5ac 1.4 5% -60% / 9 ' 60% -90% / 2 ' 9.29 95 476.2 5ad 5.3 5% -60% / 9 ' 60% -90% / 2 ' 6.51 85 469.2 5ae 4.3 5% -60% / 9 ' 60% -90% / 2 ' 9.81 95 551.1 5af 0.9 5% -60% / 9 ' 60% -90% / 2 ' 9.98 90 547.4 5ag 5.7 5% -60% / 9 ' 60% -90% / 2 ' 10.31 90 526.2 5ah 1.4 5% -60% / 9 ' 60% -90% / 2 ' 8.13 85 542.1 5ai 10.9 10% -90% / 10 * 5.88 85 584.2 5aj 4.2 10% -90% / 10 ’ 5.89 90 556.2 5ak 7.8 10% -90% / 10 ' 5.54 85 568.3 5al 8.4 10% -90% / 10 ’ 6.25 95 516.2 5am 7.6 10% -90% / 10 ' 6.49 95 474.3 5an 6.2 10% -90% / 10 ' 6.00 95 500.2 5th 9.4 10% -90% / 10 ' 6.68 95 581.3 5ap 6.4 10% -90% / 10 ' 4.30 90 500.3 5aq 5.2 10% -90% / 10 ' 6.45 95 559.2 5th 1.4 10% -90% / 10 ' 5.38 90 561.3

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 5th 16.2 10% -90% / 10 ' 4.25 95 475.2 5at 15.4 10% -90% / 10 ' 6.68 95 560.3 5au 5.9 10% -90% / 10 ’ 6.70 90 498.3 5av 4.1 10% -90% / 10 * 5.13 85 531.2 5aw 5.5 10% -90% / 10 ' 6.53 85 570.3 5ax 14.0 10% -90% / 10 ’ 6.26 90 557.3 5ay 10.4 10% -90% / 10 ’ 6.52 90 510.3 5az 9.2 10% -90% / 10 ' 5.96 95 522.3 5ba 8.5 10% -90% / 10 ’ 6.69 95 562.3 5bb 4.6 10% -90% / 10 ’ 6.00 85 520.2 5bc 8.8 10% -90% / 10 ' 4.96 90 546.2 5bd 8.2 10% -90% / 10 ’ 8.01 95 536.3 7th 2.1 5-45% / 10 ’ 5.28 86 440.2 7b 1.7 5-45% / 10 ' 4.12 94 390.2 7c 0.5 5-45% / W ' 4.04 94 434.2 7d 1.1 5-45% / W , 429 95 441.2 7e 1.1 5-45% / W 3.28 98 447.2 7f 1.0 5-45% / W 3.96 97 420.2 7g 11.0 10% -90% / W 4.00 95 483.4 7am 6.0 10% -90% / 10 ' 4.90 95 439.3 7i 12.0 10% -90% / W 6.40 95 474.3 7j 6.6 10% -90% / W 4.50 95 461.4 7k 4.0 10% -90% / 10 ’ 5.40 95 480.4 71 8.6 10% -90% / W 2.61 95 435.3 7m 6.0 10% -90% / 10 ' 4.29 95 438.4 7n 15.4 10% -90% / W 2.00 85 433.4 7th 8.4 10% -90% / 10 ’ 4.01 90 470.0 7p 3.0 10% -90% / W 4.61 95 434.4 7q 5.7 10% -90% / W 3.89 95 434.3 7r 8.8 10% -90% / 10 ’ 2.94 95 425.3 7s 10.5 10% -90% / 10 ’ 3.89 90 433.4 7t 6.9 10% -90% / 10 ' 2.45 85 419.3

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 7u 11.6 10% -90% / 10 ' 1.98 85 433.3 7v 4.6 10% -90% / 10 ' 4.60 95 489.4 7w 13.8 10% -90% / 10 ' 4.20 95 453.3 7x 6.0 10% -90% / 10 ' 3.90 95 483.2 7y 12.0 10% -90% / 10 ' 5.40 95 489.2 7z 10.0 10% -90% / 10 ' 5.40 95 517.2 7th 11.0 10% -90% / 10 ' 5.30 95 453.8 7ab 10.0 10% -90% / 10 ' 5.60 95 595.1 7ac 3.9 10-60% / 10 ' 4.59 88 469.2 7th 13.0 5-45% / 10 ' 4.48 88 415.2 7ae 4.9 5-45% / 10 ' 4.37 88 426.2 7af 20.6 5-45% / 10 ' 4.68 86 405.3 7ag 14.9 5-45% / 10 ' 4.78 82 406.3 7ah 9.5 5-45% / 10 ' 7.40 81 447.3 (+ Na) 7ai 10.8 5-45% / 10 ' 9.21 85 480.8 (+ Na) 7aj 8.3 5-45% / 10 ' 9.14 92 481.1 (+ Na) 7ak 13.6 5-45% / 10 ' 8.05 96 464.8 (+ Na) 7al 8.1 5-45% / W 7.04 91 448.8 (+ Na) 7am 7.8 5-45% / 10 ' 8.54 90 480.4 (+ Na) 7an 4.3 5-45% / 10 ' 7.48 88 460.9 (+ Na) 7th 13.5 5-45% / 10 ' 7.45 92 460.7 (+ Na) 7ap 2.5 5-45% / 10 ' 6.52 93 440.3 (+ Na) 7aq 3.4 5-45% / 10 ' 3.30 84 405.2 7th 15.10 5-45% / 10 ' 3.79 97 413.30 7th 15.70 5-45% / 10 ' 5.50 88 441.30 7at 22.20 5-45% / 10 ' 4.49 94 415.30 9a 0.5 10-60% / 10 ' 7.08 98 527.4 (+ Na) 9b 1.2 10-60% / 10 ' 8.31 93 570.5 (+ Na) 9c 2.5 10-60% / 10 ' 8.46 97 569.6 9d 1.2 10-60% / 10 ' 7.22 85 562.1 9 and 0.4 10-60% / 10 ' 7.86 95 543.2 (+ Na) 9f 4.3 10-60% / 10 ' 8.11 96 542.5

7th

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 9g 2.1 10-60% / 10 ' 7.18 93 526.3 15th 1.0 10% -90% / 10 ’ 5.76 95 477.6 15b 3.9 10% -90% / 10 ' 6.32 91 483.8 15c 2.9 10% -90% / 10 ’ 3.30 85 463.3 15d 1.3 10% -90% / 10 ' 4.26 95 531 15e 1.0 10% -90% / 10 ' 3.10 85 482.3 15f 1.2 10% -90% / 10 ' 3.60 95 484.6 16th 2.5 10% -90% / 10 · 2.80 95 456.3 16b 6.0 10% -90% / 10 ' 1.90 95 454.2 17th 1.0 10% -90% / 10 ’ 3.30 85 496.8 17b 1.1 10% -90% / 10 ’ 3.62 85 498.3 17c 2.3 10% -90% / 10 ’ 5.80 95 573.2 17d 1.6 10% -90% / 10 ’ 1.60 95 525.1 17e 1.5 10% -90% / 10 ’ 2.62 95 526.3 18th 1.0 10% -90% / 10 ' 3.90 95 528.3 18b 1.2 10% -90% / 10 ' 5.27 95 562.3 18c 2.2 10% -90% / 10 ’ 4.09 95 499.2 18d 1.5 10% -90% / 10 ' 3.78 95 498.3 18th 1.2 10% -90% / 10 ' 4.78 95 541.3 18f 7.1 10% -90% / 10 ’ 3.84 98 526.1 20th 2.0 5-45% / 10 ' 6.95 91 483.2 20b 1.3 5-45% / 10 ' 7.58 99 482.2 20c 2.5 10% -90% / 10 ’ 5.89 85 483.3 20d 4.3 10% -90% / 10 ’ 4.09 90 471.3 20e 3.6 10% -90% / 10 ’ 4.65 95 455.3 20f 12.2 10% -90% / 10 ' 3.25 95 518.3 20g 12.1 10% -90% / 10 · 5.01 90 487.2 8 pm 3.3 10% -90% / 10 ’ 4.30 90 533.2 20i 5.0 10% -90% / 10 * 4.16 90 485.2 20j 1.3 10% -90% / 10 ' 3.45 85 531.2 20k 9.7 10% -90% / 10 ’ 5.41 90 516.2 20I 3.8 10% -90% / 10 · 3.73 85 504.2

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 20m 6.6 10% -90% / 10 ' 4.52 90 488.2 20n 1.8 10% -90% / 10 ' 2.85 90 551.2 20th 7.0 10% -90% / 10 ' 4.70 95 520.2 20p 1.2 10% -90% / 10 ' 4.00 95 566.2 20q 2.3 10% -90% / 10 ‘ 4.93 95 481.3 20r 3.6 10% -90% / 10 ' 4.45 95 519.3 20s 3.0 10% -90% / 10 ' 4.18 95 552.2 20t 6.0 10% -90% / 10 ' 3.80 90 517.4 23a 21.0 10-60% / 10 ' 8.11 99 495.4 23b 25.0 10-60% / 10 ' 8.94 99 539.8 (+ Na) 23c 26.0 10-60% / 10 ' 8.55 99 539.9 (+ Na) 23d 12.6 10% -90% / 10 ' 3.90 85 453.3 23e 8.3 10% -90% / 10 ' 5.16 85 501.3 23f 12.5 10% -90% / 10 ' 3.41 80 425.3 23g 1.5 10% -90% / 10 ' 3.34 85 452.3 11 pm 8.4 10% -90% / 10 ' 3.84 90 451.3 23i 9.0 10% -90% / 10 ' 3.78 85 469.4 24a 1.1 10-60% / 12 ' 8.76 90 480.5 24b 3.1 10-60% / 10 ’ 5.14 90 375.4 24c 7.2 10-60% / 10 ' 10.33 96 531.0 24d 3.4 10-60% / 10 ’ 6.51 95 426.5 (+ Na) 24e 6.9 10-60% / 10 ' 7.22 99 455.5 25a 1.9 5-45% / 10 ' 5.38 85 455.5 25b 1.5 5-45% / 10 ’ 6.90 97 483.2 25c 1.0 5-45% / 10 ' 8.09 94 497.2 25d 12.9 5-45% / 10 ‘ 5.75 88% 453.3 25e 9.5 5-45% / 10 ' 7.76 90% 495.2 26a 10.2 5-45% / 10 ' 7.36 95 455.1 (+ Na) 26b 1.1 5-45% / 10 ' 7.38 89 476.3 26c 13.8 5-45% / 10 ' 8.13 98 483.2 26d 2.3 5-45% / 10 ' 10.35 99 503.0 26e 12.8 5-45% / 10 ' 11.11 99 523.2

Τ

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 26f 13.2 10-60% / 10 ’ 12.12 99 545.0 26g 0.7 10-60% / 10 ' 10.89 87 523.2 26h 4.4 10-60% / 10 ' 11.62 99 545.8 27a 5.0 10% -90% / 10 ' 4.42 95 475.3 27b 16.4 10-60% / 10 ' 5.20 92 505.1 27c 2.7 5-45% / 10 ' 7.50 82 476.6 (+ Na) 27d 1.6 5-45% / 12 ' 8.70 90 503.2 27e 4.4 5-45% / 12 ' 7.80 82 489.2 27f 1.2 5-45% / 12 ' 6.95 85 476.3 27g 2.5 5-45% / 12 ' 6.67 82 510.1 27h 1.1 5-45% / 12 ' 8.49 95 524.1 27i 0.9 5-45% / 12 ' 7.34 90 484.3 27j 4.3 5-45% / 12 ' 5.77 82 470.3 27k 1.3 5-45% / 12 ' 5.33 95 551.1 271 16.6 5-45% / 10 ' 5.90 91 477.2 27m 7.0 5-45% / 10 ' 7.70 93 494.2 27n 15.1 5-45% / 10 ' 3.99 86 466.2 28th 1.2 5-45% / 10 ' 5.91 86 487.1 28b 0.5 5-45% / 10 ' 6.86 98 486.1 28c 1.5 5-45% / 10 ' 7.47 93 515.1 29th 4.5 5-45% / 12 ' 8.21 98 392 29b 28.0 5-45% / 12 ' 11.80 96 443.3 29c 1.7 5-45% / 10 ' 3.73 97 415.2 29d 1.7 5-45% / 10 ' 4.62 89 414.2 29e 0.6 5-45% / 10 ' 4.94 85 436.2 29f 1.1 5-45% / 10 ' 6.23 97 442.2 29g 1.7 5-45% / 10 ' 6.39 90 457.2 29h 0.7 5-45% / 10 ' 3.56 92 408.2 29i 0.7 5-45% / 10 ' 6.50 96 431.2 29j 0.4 5-45% / 10 ' 7.24 89 445.2 29k 1.6 5-45% / 10 ' 7.07 90 456.2 291 0.9 5-45% / 10 ' 3.08 99 408.2

Ex. Yield (mg) HPLC gradient / time (min) RT (min) Purity (%) Mass spectrum (M + H or M + Na) 29m 1.5 10-60% / 10 ' 6.68 90 406.3 29n 6.4 10-60% / 10 ' 4.27 87% 422.3 29th 8.4 10-60% / 10 ' 4.42 86% 422.3 29p 13.2 10-60% / 10 ' 5.62 83% 450.3 29q 15.1 5-45% / W 3.79 97% 413.3 29r 15.7 5-45% / 10 ‘ 5.50 88% 441.3 29s 19.9 5-45% / 10 ‘ 4.30 95% 394.3 32a 7.7 5-60% / 20 ' 14.2 90 469.3 32b 4.0 5-60% / 20 ' 13.4 85 485.1 (+ Na) 32c 2.5 5-60% / 20 ' 11.1 90 459.3 32d 3.0 5-60% / 20 ' 8.19 95 472.3 32e 4.9 5-60% / 20 ' 14.2 90 486.2

in

S * d

x °

• m

Μ ·

Ci

<. ►

Procedure for the preparation of 7a-7at analogs.

Scheme II

Analogs of 7a-7at were prepared as described above in scheme I only using Fmoc-alanine as a substitute for Fmoc-valine in step 3 (scheme II).

Step 3: A suspension of resin 3 (3.5 g, 1.75 mmoles) in 20 mL of a 20% solution of piperidine in DMF was spun at room temperature for 5 minutes. The suspension was drained. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (2 x 30 ml), CH ₃ OH (30 ml) CH ₂ CI ₂ (2 x 30 ml), CH ₃ OH (30 ml) and NMP ( 2 x 30 ml) To a resin suspension in 30 ml of NMP, successively 1.44 g of N-Fmoc-alanine (4 eq, 7.0 mmoles), 2.4 ml of DIEA (8 eq, 14 , 0 mmoles), 0.95 g of HOBt (4 eq, 7.0 mmoles) and 2.66 of HBTU (4 eq,

7.0 mmoles). The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated during

3 hours. The resin was then washed successively with DMF (2 x 30 ml),

CH ₃ OH (30 ml), 1: 1 DMF / CH ₂ CI ₂ (2 x 30 ml), CH ₃ OH (30 ml) and CH ₂ CI ₂ (3 x 30 ml), and was vacuum dried to provide resin 6 (0.50 mmol / gram).

Step 4: To a 0.125 mmol portion of resin 6, 5 ml of a 20% solution of piperidine in IOKF was added. The suspension was spun at room temperature for 5 minutes, and drained. The procedure was repeated for 20 minutes. The resulting resin was washed successively with

DMF (3x5 ml), CH ₃ OH (5 ml), and NMP (3x5 ml). The desired carboxylic acid was then added (4 eq, 0.6 mmol), followed by 2.0 ml of a 0.25 ml solution of HOBt in NMP, 0.35 ml of DIEA (8 eq, 1.0 mmol) and 2.0 mL of a 0.25 M solution of HBTU in NMP. The mixture was spun at room temperature overnight and drained. The resin was washed successively with DMF (3x5 ml), CH ₃ OH (5 ml), 1: 1 DMF / CH ₂ CI ₂ (2x5 ml), CH3OH (5 ml) and CH ₂ CI ₂ (3x5 ml), and was vacuum dried. A 5 ml portion of a 95% solution of TFA in water was then added to the resin. The mixture was stirred at room temperature for one hour, and was filtered. The filtrate was evaporated and the residue was dissolved in acetonitrile-water and purified by preparative HPLC to provide compounds 7a-7at.

Ο

7π

7q x

ο

F3

Procedure for the preparation of analogs 9a-9g.

Scheme III

Step 1: A 10.0 g portion (0.75 mmol / gram load, 7.5 mmoles) Agropore-aminomethyl resin (catalog number 800047) was

washed with DMF (3 x 40 ml). DIEA / DMF 10% (3 x 40 ml). DMF and NMP (3 x 40 ml). The above resin was added successively compound 1 (0.87 eq, 3.88 g, 6.55 mmol), HBTU (1.14 eq, 3.13 g, 8.25 mmol), HOBt (1.14 eq , 1.26 g, 8.25 mmoles) and NMP (40 ml). The reagents were then mixed by bubbling nitrogen through the bottom of the flask for two minutes at room temperature. N, N-diisopropylethylamine (3.33 eq, 4.35 ml, 25 mmol) was added and the resulting suspension was mixed at room temperature overnight, filtered, then washed successively.

with NMP (3 x 40 ml) and DMF (3 x 40 ml). The resin was then treated with 50 ml of a 20% acetic anhydride solution in DMF for 38 minutes at room temperature. The mixture was filtered, and the resin was washed successively with NMP (3 x 40 ml) CH ₂ CI ₂ (3 x 40 ml), 1: 1 CH ₃ OH / CH ₂ CI ₂ (3x 40 ml), and CH ₃ OH (3 x 40 ml). After vacuum drying, 13.76 grams of resin 2 were obtained (0.35 mmol / gram of load).

Step 2: Seven reaction vessels were each loaded with 181 mg of resin 2 (0.48 mmol / gram, 0.063 mmol) then were washed

with CH ₂ CI ₂ (3x1 ml) and NMP (3x1 ml). Then each container was treated with 1 ml of a 25% solution of piperidine in DMF and mixed (vortex) at room temperature for 15 minutes. This procedure was repeated in triplicate. Each container was then washed three times with (3x1 ml).

The containers were then treated with 500 μΙ of an acid solution of

0.4 M (2S, 4H) -Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic at 0.4 M / HOBt /

0.4 M NMP, 500 μΙ of a 0.4 M HBTU / NMP solution, and 250 μΙ of a 1.6 M DIEA / MMP solution and are mixed for 3 hours at room temperature. After mixing, the containers were drained and the procedure was repeated.

Step 3: The resulting resin was washed with NMP (3 x ml) and then treated with 1 ml of a 25% solution of piperidine in DMF and was mixed (vortex) at room temperature for 15 minutes. The procedure was repeated in triplicate. The resulting resin was washed with NMR (3x1 ml) then treated with either acetic anhydride, or propyl isocyanate, or methane, sulfonyl chloride, or methyl chloroformate. For acetic anhydride:

use 300 μΙ of a 1.6 M DIEA / NMP solution and 1 ml of a 0.5 M acetic anhydride / 0.125 M DIEA / 0.015 M HOBt solution in NMP. For

isopropyl isocyanate: add 300 μΙ of a DIEA / NMP solution to

1.6 M and 1 mL of a 1 M propyl isocyanate solution in NMP. For 5 sulfonyl methane chloride: add 600 μΙ of a 1% Pyridine solution

M in CH ₂ CI ₂ and 600 μΙ of a solution of methane sulfonyl chloride in CH ₂ CI ₂ , Per methyl chloroformate: add 500 μΙ of a solution of DIEA /

1.6 M NMP and 1 ml of a 0.7 M methyl chloroformate solution in CH ₂ CI ₂ , The resulting suspensions were mixed for 6 hours at room temperature, the solvent was drained and the coupling procedure was repeated .

Step 4: The resulting resin was washed with MNP (3 x 1ml) and then treated with a 1: 1 mixture of TFA / CH ₂ CI ₂ at room temperature for 30 minutes. The resulting resin was then washed with CH ₂ CI ₂ (3x1 ml) 15 and NMP (3x1 ml). The resin was then treated with 500 μΙ of a 0.4 M Fmoc-valine-carboxylic acid solution / 0.4 M / HOBt / NMP, 500 μΙ of a 0.4 M HBTU / NMP solution, and 250 μΙ of a 1.6 M DIEA / NMP solution and were mixed for 3 hours at room temperature. After mixing, the containers were drained and the coupling procedure was repeated.

Step 5: The resulting resin was washed with NMP (3x1 ml), then treated with 1 ml of a 25% solution of piperidine in DMF and mixed (vortex) at room temperature for 15 minutes. This procedure was repeated in triplicate. The resulting resin was washed with NMP (3 x 25 1 ml) then treated or with 500 μΙ of a 0.4 M / HOBt / NMP 0.4 M or 500 μΙ solution of 1-isoquinoline carboxylic acid or 500 μΙ of a solution of 0.4 M p-anisic acid / HOBt / 0.4 M NMP, The resulting mixtures were treated with 500 μΙ of a 0.4 M HBTU / NMP solution and 250 μΙ of a DIEA / NMP solution at 1.6 M then they were mixed for 3 hours at room temperature the solvent was drained and the procedure was repeated. The resulting resin was treated with 1.5 ml of a 95% solution of TFA in water and was stirred at room temperature for one hour then filtered. The filtrate

it was evaporated, and the residue was placed in a 2: 1 mixture of DMF / acetonitrile / water and was purified by preparative HPLC to provide compounds 9a-9g.

Sd

Procedure for the synthesis of analogs 15-18

Scheme IV

15, R ^ CHiCH ^ j

15. Rj-CHj

17, F ^ -CH (Ch%) ₂

15. Rs ^ CH,

Preparation of analogs 15 and 16 (scheme IV):

Synthesis of 2- (S) -piperamino-1,2,4-tricarboxylic acid 1- (9H-fluoren-9-ylmethyl) ester

To a solution of 2- (S) -piperamino carboxylic acid (Lonza) (3 g, 15 mmol) in 1: 1 H ₂ 0: dioxane (30 ml) was added a solution of (BOC) ₂ 0 in dioxone (3 , 3 g, 15 mmoles, in 5 ml of dioxone) while maintaining the pH at 11 with 1 N NaOH. The pH was maintained for 3 hours at room temperature. The solution was adjusted to pH 9.5 with HCL A 1 N, 10 was cooled to 0 ° C and treated with Fmoc-CI (3.87 g, 15 mmoles, pH was maintained at 9.5 for 1 hour and the mixture was stirred at room temperature overnight The resulting suspension was filtered and the filtrate was treated

with ₄ to 1 N KHS (O) at pH 2 then it was extracted with ethyl acetate (2 x 75 ml). The organic layer was dried with brine and MgSO ₄ , filtered, and concentrated to give colorless oil. The oil was dissolved in ethyl acetate and added to hexane to give 3.5 g (51% yield) of white solid after isolation. ¹ H NMR (500 MHz, DMSO-dg) δ 1.55 (s, 9H) 2.80-3.5 (m, 3H), 3.8-4.9 (m, 5H), 5.7 ( bs, 1H), 7.3 (m, 2 H), 7.3-7.9 ppm (m, 8H), LC / MS (ES) m / e 451.3 (mH)

Step 1: To 5 g of resin 2 (0.375 mmol / gram, 1.82 mmoles) was added 25 ml of a 20% solution of piperidine in DMF. The suspension was spun at room temperature for 5 minutes and drained. The procedure was repeated for 20 minutes. The resin was then washed successively with DMF (2 x 50 ml), CH ₃ OH (50 ml), CH 2 Cl 2 (2 x 50 ml), CH 3 OH (50 ml) and NMP (50 ml). To a suspension of resin in 25 mL of

NMP was added successively 3.5 g of N-Fmoc-Boc piperamine carboxylic acid (4 eq, 7.48 mmoles), 1.0 ml of DIEA (8 eq, 14.96 mmoles), 1.01 g of HOBt ( 4 eq., 7.48 mmoles) and 2.83 g HBTU (4 eq, 7.48 moles). The mixture was spun at room temperature overnight and drained.

This coupling procedure was repeated for 3 hours. The resin was then washed successively with DMF (2 x 50 ml), CH ₃ OH (50 ml), 1: 1

DMF / CH2CI2 (2 x 50 ml), CH ₃ OH (3 x 50 ml) and CH ₂ CI ₂ (3 x 50 ml), and soon

it was vacuum dried to provide resin 10.

Step 2: To 5 g (0.335 mmol / gram of load, 1.675 mmol) 25 ml of a 20% solution of piperidine in DMF was added.

The suspension was spun at room temperature for 5 minutes and drained.

The procedure was repeated for 20 minutes. The resin was then washed successively with DMF (2 x 50 ml), CH ₃ OH (50 ml) CH 2 Cl 2 (2 x 50 ml), CH ₃ OH (50 ml) and NMP (2 x 50 ml). To a resin suspension in 25 ml of

NMP was added successively 2.08 g of N-Fmoc-valine or N-Fmocalanine (4 eq, 6.7 mmoles), 1.17 DIEA (4 eq, 6.7 mmoles), 0.905 g of

HOBt (4 eq, 6.7 mmoles) and 1.38 g of HBTU (4 eq, 3.66 mmoles). The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 3 hours. The resin was

so washed successively with DMF (2 x 50 ml), CH ₃ OH (50 ml), 1: 1 DMF / CH ₂ CI ₂ (2 x 50 ml), CH ₃ OH (50 ml) and CH ₂ CI ₂ (3 x 50 ml), and was vacuum dried to provide resin 11 or 12 respectively (0.35 mmol / gram, 5 g)

Step 3: To a 1.5 g (0.165 mmol) portion of resin 11 or 12 was added 2 mL of a 20% solution of piperidine in DMF. The suspension was spun at room temperature for 5 minutes, and drained. The procedure was repeated for 20 minutes. The resulting resin was washed

successively with DMF (3x15 ml), CH ₃ OH (15 ml), and NMP (3x15 ml). The desired carboxylic acid was then added (4 eq, 0.66 mmol), followed by 0.25 g HOBt (0.66 mmol), 0.12 ml DIEA (4 eq, 0.66 mmol) and 0, 89 g (0.66 mmol) HBTU in NMP. The mixture was spun at room temperature overnight and drained. The resin was successively filled with DMF (2x15 ml), CH ₃ OH (15 ml), 1: 1 DMF / CH ₂ CI ₂ (2 x

15 ml), CH ₃ OH (15 ml) and CH ₂ CI ₂ (3x15 ml), and dried in vacuo to provide 13 or 14,

Step 4: A 2 ml portion of a 95% solution of TFA in water was then added to the resin. The mixture was stirred at room temperature for one hour, and was filtered. The filtrate was evaporated, and residue 20 was placed in acetonitrile-water and purified by preparative HPLC to

isb o

Cl o

Procedure for the synthesis of analogs 17 and 18 scheme IV):

Step 5: Resin 13 or 14 was treated with 2 mL of TFA / CH2Cl2 at

25% for 30 min and was washed with DMF (2x5 ml). DIEA / CH ₂ CI ₂ to 10 (2 x 5 ml) DMF / CH ₂ CI ₂ (2x5 ml), CH 3 OH (5 ml) and CH ₂ CI ₂ (3x5 ml) and was dried for 5 minutes. The resulting resin was washed with NMP (3x1 ml) then treated with acetic anhydride, or methoxyacetic acid, or 2propanesulfonyl chloride, or propyl isocyanate, or methane sulfonyl chloride, or methyl chloroformate according to the procedure used for prepa10 rar analogues 9 (Scheme III). Compounds 17 and 16 were obtained as described in step 4 for compounds 15 and 16.

Compounds 17a and 17b were prepared by reductive amination using Na (OAc) ₃ BH and HCHO (38% in H ₂ 0, (0.2 ml) and CH3COOH (0.02 ml) before step 4 and the compound was prepared by treatment with phospho15 genius followed by ammonia before step 4.

Procedure for the preparation of analogs 20, compounds 20a-20t were prepared according to the procedure described for compounds 5 (Scheme I) only using the appropriate Fmoc-amino acid 5 as a substitute for FmocValine acid in step 3 (Scheme V ).

Scheme V

Step 4

Ri-COjH

Preparation of 3 - ({1- [2-4-amino-3-chloro-benzoylamino) -3-methylsulfonylpropionill-pyrrolidine-2-carbonyl} -amino) -4-oxo-butyric acid (20i).

A suspension of 0.132 mmol of resin 3 in 4 ml of 20% piperidine in DMF was spun at room temperature for 5 minutes, and the mixture was drained. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (twice), CH0 ₃ H (once), CH ₂ CI ₂ (twice), CH3OH (once) and NMP (twice). To a suspension of the resin in 4 ml of NMP, 189 mg of N-Fmoc-methyl cysteine (4 eq, 0.528 mmol), 0.185 ml of DIEA (8 eq, 1.056 mmol), 71 mg HOBt (4 eq) were successively added , 0.58 mmol) and 200 mg of HBTU (4 eq, 0.528 mmol). The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 15 3 hours. The resin was then washed successively with DNF (twice),

CH3OH (once), and 1: 1 DMF / CH2Cl2 (twice), CH ₃ OH (once) and CH ₂ CI ₂ (three times), and was vacuum dried.

A 100 mg suspension of this resin in 2 ml of piperidine a

20% DMF was spun at room temperature for 5 minutes, and drained20. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (twice), CH ₃ OH (once), CH2Cl2 (twice), CH3OH (once) and NMP (twice). To a resin suspension in 2 ml mL of NMP, 38 mg of 4 amino-3- chlorobenzoic acid (4 eq, 0.2 mmol), 0.140 ml of DIEA (8 eq, 0.4 mmol) were successively added,

27 mg of HOBt (4 eq, 0.2 mmol) and 76 mg of HBTU (4 eq, 0.4 mmol). The mixture was spun at room temperature overnight and drained.

The resin was then washed successively with DMF (twice), CH ₃ OH (once), and 1: 1 DMF / CH ₂ CI ₂ (twice), CH ₃ OH (once) and CH ₂ CI ₂ (three) times), and was vacuum dried. The resin was then treated with 2 ml of 95% TFA in water for 1 h. The suspension was filtered, the filtrate was concentrated in 5 vacuum and was purified by preparative HPLC to provide the title compound (20i).

Preparation of 3 - ((142- (3,5-dichloro-4-hydroxy-benzoylamino) -4methanesulfonyl-butyryl] -pyrrolidine-2-carbonyl) -amino) -4-oxo-butyric (20p).

compound 20p was prepared according to procedure 10 used for the preparation of 20i using N-Fmoc-methionine as the first component coupled to resin 3, and 3,5-dichloro-4-hydroxybenzoic acid as the second component.

Preparation of 3-1 (1- {2-i (isoquinoline-1-carbonyl) -amino] -3methanesulfonyl-propionyl} -pyrrolidine-2-carbonyl) -aminol-4-oxo-butyric acid (20r).

N-Fmoc methyl cysteine was oxidized to give the corresponding sulfone using the method of B. M. Trost and 3). P. Curran, Tetrahedron

Lett. 22, pp. 2297-190 (1981). To a solution of 0.714 g (2 mmoles) of FFmoc methyl cysteine in 24 mL of a 1: 1 solution of CH ₃ OH-water was stirred at 0 ° C, 3.68 g (3 eq, 6 mmoles) were added oxone®. The mis-

The mixture was stirred at room temperature for 48 h, diluted with water, acidified to pH 2 using 6 N HCI, and extracted with three 100 ml portions of ethyl acetate. The combined organic extracts were dried (MgSO ₄ ) and concentrated in vacuo to provide 0.700 g (89% yield) of sulfone; ¹ H NMR (DMSO-d ₆ , 500 MHz) δ 2.97 (s, 3H), 3.4925 3.59 (m, 2H), 4.25 (m, 1H), 4.30-4.18 (m, 2H), 4.46 (m, 1H), 7.33 (t, 2H), 7.42 (t, 2H), 7.70-8.00 (m, 4H); exact mass calculated for Ci9H ₁₉ NO ₆ S m / e

389.09, found m / e 390.2,

A suspension of 0.250 mmol of resin 3 in 10 mL of 20% piperidine in DMF was spun at room temperature for 5 minutes, and the mixture was drained. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (twice), CH ₃ OH (once), CH ₂ CI ₂ (twice), CH ₃ OH (once) and NMP (twice). A sus-

resin resin in 6 ml of NMP 200 mg of N-Fmoc-methyl cysteine sulfone (4 eq, 0.50 mmol), 0.175 ml of DIEA (8 eq, 1.00 mmol), 70 mg HOBt ( 4 eq, 0.50 mmol) and 188 mg HBTU (4 eq, 0.50 mmol).

The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 3 hours. The resin was washed successively with DMF (twice), CH ₃ OH (once), 1: 1 DMF / CH ₂ CI ₂ (twice), CH ₃ OH (once), and CH ₂ CI ₂ (three times) ), and was vacuum dried.

A suspension of 150 mg of this resin in 4 mL of 20% piperidine in DMF was spun at room temperature for 5 minutes, and drained. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (twice) CH ₃ OH (once), CH ₂ CI ₂ (twice), CH ₃ OH (once) and NMP (twice). To a resin suspension

in 3 ml of NMP, 52 mg of isoquinolinecarboxylic acid (4 eq, 0.3 mmol) were added successively 0.104 ml of DIEA (8 eq, 0.6 mmol), 37 mg of HOBt (4 eq, 0.3 mmol) and 104 mg of HBTU (4 eq, 0.3 mmol). The mixture was spun at room temperature overnight and drained. The resin was washed successively with DMF (twice), CH ₃ OH (once), and 1: 1 DMF / CH ₂ CI ₂ (twice) CH ₃ OH (once) and CH ₂ CI ₂ (three times) , and was vacuum dried. The resin was then treated with 2 ml of 95% TFA in water for 1 hour. The suspension was filtered, the filtrate was concentrated in vacuo and was purified by preparative HPLC to provide the title compound (20r).

Preparation of 3 - ({1-1,2- (3,5-dichloro-4-hydroxybenzoylamino) -3methanesulfonyl-propionin-pyrrolidine-2-carbonyl} -amino) -4-oxo-butyric (20s).

Compound 20s was prepared according to the procedure used for the preparation of 20i, using 3,5-dichloro-4hydroxybenzoic acid in place of isoquinolinecarboxylic acid.

ΊΑ

201

Oh, OK

201

20m

ό | 6

20ο

20ρ

20q

Procedure for preparing analogs 23

Compounds 23a-23i were prepared according to the procedure described for compounds 7 (scheme II) only using the

Fmoc-amino acid suitable as a substitute for Fmoc-proline in step 2 (scheme VI)

Scheme VI

0T

Preparation of 3 - ({2- [2- (4-amino-3-chloro-benzoylamino) -propionill-4methyl-3,4-dihydro-2H-pyrazol-3-carbonyl} -amino) -4-oxo- butyric (23 g)

Compound 23g was prepared according to the procedure described for compounds 7 only using 4-methyl-4,5-dihydro-pyrazole-1,5-dicarboxylic acid 1 - (9H) -fluorophen-9ylmethyl) as a substitute for Fmoc-proline (Scheme II) in step 2.

Preparation of 4-methyl-4,5-dihydro10-pyrazole-1,3-dicarboxylic acid 1- (9H-fluoren-9-ylmethyl) ester:

To a solution of 650 mg (2 mmol) of (10,10-dimethyl-3,3dioxo-76-thia-4-aza-tricycle [5,2,1.0 ⁰⁰ ] dec-4-yl) (4-methyl- 3,4-dihydro-2H-pyrazazol3-yl) -methanone (J. Am, Chem, Soc., 119, pp. 8379-8380 (1997)) in 6 ml of water and 14 ml of THF stirred at 0 ° C 420 mg (10 mmo15 les, 5 eq) of lithium hydroxide were added. The mixture was stirred at 0 ° C for 2 hours and at room temperature for 30 minutes, diluted with 20 ml of water and was washed with ether (20 ml). The pH of the solution was then adjusted to 9, and a solution of 519 mg (2 mmoles, 1 eq) of Fmoc-CI in 3 ml of dioxone was added. The mixture was stirred at room temperature overnight, washed with ether, acidified to pH 2-3 and extracted with 3 40 ml portions of ethyl acetate. The combined organic extracts were washed with brine, dried (MgSO ₄ ) and concentrated in vacuo to provide 690 mg (98% yield) of a colorless foam that was identified as the title compound. ¹ H NMR (DMSO-d ₆ , 500 MHz) δ 1.2 25 (d, 3H), 3.2 (m, 1H), 4.2-4.6 (m, 3H), 7.1 (s , 1H), 7.2-7.5 (m, 5H), 7.7-8.0 (m,

4Η). Exact mass calculated for C2OH18N2O4 m / e 350.13, found m / e

351.3

Preparation of 3 - ({1- [2- (4-amino-3-chlorobenzoylamino) -propionyl] -4methoxy-pyrrolidine-2-carbamoyl} -amino) -4-oxo-butyric acid (23i).

Compound 23i was prepared according to the procedure described for compound 7 using only N-Fmoc-4-methoxyproline as a substitute for Fmoc-proline (Scheme II) in step 2.

Preparation of N-Fmoc-4-methoxyproline:

To a solution of 735 mg (3 mmoles) of N10 BOC-4-hydroxyproline methyl ester in 20 ml of THF stirred at 0 ° C was added 79 mg (1.1 eq, 3.3 mmoles) of sodium hydride 60% in mineral oil. The mixture was stirred at 0 ° C for 1 hour, and methyl iodide (0.56 ml, 3 eq, 9 mmol) was added. The mixture was stirred at room temperature overnight, quenched by the addition of saturated aqueous ammonium chloride, diluted with water, and was extracted with 3 80 ml portions of ethyl acetate. The combined organic extracts were washed with brine, dried (MgSO ₄ ), and concentrated in vacuo to provide a pale yellow oil. The oil was placed in 9 ml of CH ₃ OH and 3 ml of water, and 378 mg (3 eq, 9 mmol) of lithium hydroxide was added. The mixture was stirred at

overnight at room temperature, it was diluted with water, acidified to pH 3 and extracted with 3 80 ml portions of ethyl acetate. The combined organic extracts were washed with brine, dried (MgSO ₄ ) and concentrated in vacuo. The residual oil was placed in 10 ml of TFA and the solution was stirred at room temperature for 2 hours, and was concentrated in vacuo. The residual oil was diluted with 6 ml of 10% aqueous sodium carbonate and 3 ml of dioxone, and the solution of 9 fluorenylmethyl chloroformate (779 mg, 1 eq, 3 mmol) in 5 ml of dioxone was added. The mixture was stirred at room temperature overnight, diluted with water, acidified to pH 3 and extracted with 3 x 30 ml portions of ethyl acetate. The organic extracts were washed with brine, dried (MgSO ₄ ) and concentrated in vacuo to provide an oil, which was purified by column chromatography on silica

gel was eluted with CH2Cl2 / CH3OH 20: 1 to provide 600 mg (55%) of NFmoc-4-methoxyproline: exact mass calculated for C21H21NO5 m / e 367.14 found m / e 368.4.

To a 0.125 mol portion of resin 2 was added 4 ml 5 of 20% piperidine in DMF. The mixture was spun at room temperature for 5 minutes and drained. The procedure was repeated for 20 minutes.

The resin was washed successively with DMF (twice), CH ₃ OH (once), CH ₂ CI ₂ (twice), CH ₃ OH (once) and NMP (twice). To a suspension of the resin in 4 ml of NMP was successively added 10 184 mg of N-Fmoc-4-methoxyproline (4 eq, 0.50 mmol), 0.175 ml of DIEA (8 eq, 1.00 mmol), 70 mg HOBt (4 eq, 0.50 mmol) and 188 mg HBTU (4 eq,

0.50 mmol). The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 3 hours. The resin was washed successively with DMF (twice), CH ₃ OH 15 (once), 1: 1 DMF / CH ₂ CI ₂ (twice), CH3OH (once) and CH ₂ CI ₂ (three times), and was vacuum dried.

To the resin, 4 ml of 20% piperidine in DMF was added. The mixture was spun at room temperature for 5 minutes and drained. The procedure was repeated for 20 minutes. The resin was washed successively

with DMF (twice), CH ₃ OH (once), CH ₂ CI ₂ (twice),

CH3OH (once) and NMP (twice). To a suspension of the resin in 4 ml of NMP, 156 mg of N-Fmoc-alanine (4 eq, 0.50 mmol), 0.275 ml of DIEA (a eq, 1.00 mmol), 70 mg HOBt ( 4 eq, 0.50 mmol) and 188 mg HBTU (4 eq, 0.50 mmol). The mixture was spun at room temperature overnight and drained. This coupling procedure was repeated for 3 hours. The resin was washed successively with DMF (twice), CH ₃ OH (once) 1: 1 DMF / CH ₂ CI ₂ (twice), CH ₃ OH (once) and CH ₂ CI ₂ (three times) and was vacuum dried.

To the resin, 4 ml of 20% piperidine in DMF was added. The mixture was spun at room temperature for 5 minutes and drained. The procedure was repeated for 20 minutes. The resin was washed successively (ΰθ again with DMF (twice), CH ₃ OH (once), CH2Cl2 (twice), CH3OH (once) and NMP (twice). To a suspension of the resin in 4 ml of NMP 50 mg of 4-amino-acid were successively added

chlorobenzoic acid (4 eq, 0.50 mmol), 0.175 mL DIEA (8 eq, 1.00 mmol), 70 mg HOBt (4 eq, 0.50 mmol) and 188 mg HBTU (4 eq, 0.50 mmol). The mixture was spun at room temperature overnight and drained. The resin was washed successively with DMF (twice), CH ₃ OH (twice, 1: 1 DMF / CH ₂ CI ₂ (twice), CH ₃ OH (once), and CH ₂ CI ₂ (three times) , and was vacuum dried.

The resin was treated with 4 ml of 95% TFA in water for one hour. The mixture was filtered. The filtrate was concentrated in vacuo to provide an oil, which was purified by HPLC to provide the title compound

| Ο

Procedure for the preparation of analogues 24a-e

Compounds 24a-24e were prepared according to the procedure described for compounds 5 (scheme I) using either Fmoc-azatidine carboxylic acid or trans-2-phenyl-Fmoc-azetidine acid only

Procedure for the preparation of analogs 25,

Compounds 25a-25a were prepared according to the procedures described for compounds 5 and 7 (Scheme T and Scheme II) only using Fmoc-2 (S) -pipecolic acid as a substitute for Fmocproline in step 2 and coupling or Fmoc -valine or Fmoc-alanine or Fmoc15 tert-leucine in step 3.

Procedure for the preparation of analogs 26a-h

Compounds 26a-26h were prepared according to the procedure described for compounds 23 (Scheme VI) using only

Fmoc-valine as a substitute for Fmoc-alinine in step 3.

Procedure for the preparation of analogs 27,

Compounds 27a-27n were prepared according to the procedure described for compounds 7 (Scheme II) using only 5 Fmoc-4,4-difluoroproline as a substitute for Fmoc-proline in step 2.

Preparation of N-Boc 4,4-difluoroproline methyl ester

To a solution of 9.63 ml (7.2 mmoles) of oxolyl chloride in 10.6 ml of CH2 Cl2 stirred at -78 ° C was added a solution of 0.94 ml (13.2 mmoles) of methyl sulfoxide in 15 ml of CH2 Cl2. the solution was stirred at -78 ° C for 30 min. A solution of 1.47 g (6 mmol) of N-Boc-4-hydroxyproline methyl ester in 19 ml of CH2 Cl2 was added dropwise. The mixture was stirred at -78 ° C for 1.5 h, and 3.34 ml (24 mmol) of triethylamine were added. The solution was allowed to warm to room temperature and was stirred overnight. It was then diluted with 100 ml of CH ₂ CI ₂ , washed successively with 100 ml of water, 100 ml of HCI a

N, and 100 ml of brine, was dried (MgSO ₄ ) and concentrated in vacuo. O

The residue was purified by column chromatography on silica gel (eluted with ethyl acetate / hexanes, 1: 3), to provide 1.294 g (89% redness) of N-Boc-4-oxo-proline methyl ester. ¹ H NMR (500 MHz, CDCI ₃ ) δ 20 1.45 (m, 9H), 2.60 (m, 1H), 2.95 (m, 1H), 3.75 (m, 3H), 3, 90 (m, 2H), 4.80 (m,

1H).

To a solution of 808 mg (3.33 mmoles) of N-Boc-4-oxoproline methyl ester in 13 ml of CH2 Cl2 stirred at 0 ° C was added 0.88 ml (7.19 mmoles, 2.2 eq ) of DAST. The mixture was stirred at 0 ° C for 2 hours, 25 at room temperature for 16 hours, and was poured into ice water, the mixture was stirred at room temperature for 2 hours. The organic phase was separated, washed with water, dried (MgSO ₄ ) and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate-hexanes, 1: 1), to provide 754 mg (79% yield) of difluoro derivative as a pale yellow oil. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.50 (m, 9H), 2.45 (m, 1H), 2.70 (m, 1H),

3.75 (m, 3H), 3.80 (m, 2H), 4.50 (m, 1H).

Preparation of N-Fmoc-4,4-difluoroproline:

To a solution of 754 mg (2.85 mmoles) of N-BOC 4,4-difluoroproline methyl ester in 5 ml of THF stirred at 0 ° C was added a solution of 179 mg (4.27 mmoles) of hydroxide of lithium in 5 ml of water. The solution was stirred at 0 ° C for 3 hrs, at room temperature for 1 hour, diluted with water, extracted with ether, acidified to pH 2-3, and extracted with two 30 ml portions of ethyl acetate. The combined organic extracts were washed with brine, dried (MgSO ₄ ) and concentrated in vacuo to provide 652 mg (91%) of acid as a pale yellow solid.

A solution of 652 mg (2 mmol) of N-BOC-4,4 difluoroproline in 10 mL of 1: 1 TFA / CH ₂ CI ₂ was stirred at 0 ° C for 45 min, and was concentrated in vacuo. The residue was placed in 3 ml of dioxone, and 5 ml of 10% aqueous sodium carbonate were added, followed by a solution of 675 mg (1 eq) of Fmoc-CI in 5 ml of dioxane. The mixture was stirred at room temperature for 16 h, it was eluted with 20 ml of water, it was extracted with 2 portions of 20 ml of ethyl ether, it was acidified to pH 2, and it was extracted with three 30 ml portions of acetate. ethyl. The combined organic extracts were washed with 50 ml of brine, dried (MgSO ₄ ) and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluted with CH ₂ CI ₂ / CH ₃ OH 10: 1), to provide 850 mg (88%) of N-Fmoc-4,4-difluoroproline as a brownish solid. . ¹ H NMR (500 MHz, CDCI ₃ ) δ 2.55 (m, 1H), 2.95 (m, 1H), 3.80 (m, 2H), 4.20 (m, 1H), 4.30 (m, 2H), 4.55 (m, 1H), 7.32 (m, 2H), 7.45 (m, 2H), 7.70 (m, 2H), 7.90 (m, 2H) . Exact mass calculated for C _2O Hi ₇ F ₂ NO ₄ m / e 373.11, found m / e 174.4.

7ΙΛ

Ζ7 ·

27]

Compounds 28a-28C were prepared according to the procedure described for compounds 5 and 7 (Scheme I and Scheme II) only using Fmoc-dimethylthioproline as a substitute for Fmoc-proline in (C &

2U _t X «N

Mb, X = C

28c

General Procedures for the Preparation of the Formula I Embodiment Compounds (Schemes VII-VIII)

Compounds of the embodiment Formula I where R ₄ = H and an R ₅ = H

Procedure for the preparation of analogs 29,

Compounds 29a-29s were prepared according to the procedure described for compounds 5 (Scheme I) only using Fmoc-alanine as a substitute for Fmoc-proline in step 2 and using or

Fmoc-valine or Fmoc-alanine or Fmoc-tert-leucine in step 3

2··

Md

Compounds 32a-32e were prepared according to the procedure described for compounds 5 (Scheme I) using only 2- (3-tert-butoxycarbonylamino-2-oxo-pyrrolidin-1-yl) -4-methyl- pentanoic (30) (catalog number Neosystem BB02101) as a substitute for proline Fmoc, in step 2 followed by Step 4 (Scheme VII).

Compounds of the embodiment Formula I in which R ₂ and R ₃ join (0% with the atoms to which they are attached form a 5-membered ring.

Scheme VII

Compound of embodiment Formula I where X = N-CH ₃

Scheme VIII

Μ

Preparation of 3 - ({1- [N- (isoquinoline-1-carbonyl) -N-methylhydrazinocarbonill-pyrrolidine-2-carbonyl} amino) -4-oxo-butyric acid (34).

A 0.250 mmol suspension of resin 3 (scheme VIII) in

10 mL of 20% piperidine in DMF was spun at room temperature for 5 minutes and drained. The procedure was repeated for 20 minutes. The resin was washed successively with DMF (twice), CH ₃ OH (once), 1: 1 DMF / CH2Cl2 (twice), CH ₃ OH (once) and CH ₂ CI ₂ (three times), and was soon dry. To the resin were added 5 ml of dry CH ₂ CI ₂ , 0.128 ml of DIEA (3 eq, 0.75 mmol) and 0.400 ml of a 20% solution of phosphogen in toluene (3 eq, 0.75 mmol). The suspension was spun at room temperature for 1.5 hours. The mixture was drained, and the resin was washed with CH ₂ CI ₂ several times. To a suspension of resin in 5 ml of CH ₂ CI ₂ was added 0.133 ml of methyl hydramine (10 eq, 2.5 mmoles). The mixture was spun at room temperature overnight and drained. The resin was washed successively with DMF (twice), CH ₃ OH (once), 1: 1 DMF / CH2Cl2 (twice), CH ₃ OH (once) and CH ₂ CI ₂ (three times), and was vacuum dried.

To a 0.075 mmol portion of the resin in 3 ml of NMP, 52 mg of 1-isoquinolinocarboxylic acid (4 eq,

0.3 mmol), 0.19 ml DIEA (8 eq, 0.6 mmol), 37 mg HOBt (4 eq, 0.3 (ΙΟ mmol) and 104 mg HBTU (4 eq, 0.3 mmol The mixture was spun at room temperature overnight and drained The resin was washed successively with DMF (twice), CH ₃ OH (once), 1: 1 DMF / CH ₂ CI ₂ (twice) CH ₃ OH (once) and CH ₂ CI ₂ (three times), and was vacuum dried.

The resin was treated with 4 ml of 95% TFA in water for 1 hour.

The mixture was filtered, the filtrate was concentrated in vacuo to provide an oil, which was purified by HPLC to provide the title compound (34). Embodiment Compounds Formula I where R ₃ = R ₃ = H:

3 - ({1 - [(4-amino-3-chloro-benzoylamino) -aceti1-3-pyrrolidine-2-carbonyl} amino) -4-oxo-butyric acid (G1).

Prepared as described for compounds 7 only using Fmoc glycine as a substitute for Fmoc-alanine in step 3 (scheme II) to provide 4.3 mg of the title compound, LC-EM (ES ⁺ ) m / e = 425.2 ( M + H)

3 - ({1 - (4-amino-3-chloro-benzoylamino) -acetyl] -4,4, -difluoro-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric (G2)

Prepared as described for compounds 7 and 27 only using Fmoc-glycine with Fmoc-alanine substitute in step 3 (Scheme II) 20 to provide 10.0 mg of the title compound. LCMS (ES ⁺ ) m / e = 461.2 (M + H)

General procedures for the preparation of the compounds of Embodiment C Formula I and Embodiment D Formula I where Y = C (Schemes IX-XXII) Scheme IX

Hl

1) H ₂

2) EDC. R1CQ2H

FtiCOzH

EDC

EDC

((l

Sources for Selected Ring Systems

Structure Reference 1 Blvthin, D.J., J. Orq. Chem., 59, oo. 6098-6100 (1994). - £ ~ \ θ2Κ 0 Decicco, C.P. etal., Svn. Lett., Oo. YX 615-616, (1995). • y-f Bennion, C. et al., J. Med. Chem., 34, pp. 439-447 (1991), COjR Jensen, K.A. et al., Acta Chemica Scand., 13, pp. 1097-1103 (1961). Commercially available TC Mish, M. R., J. Am. Chem. Soc., 119, - Αγ CO ^ R pp. 8379-8380 (1997). Xi, N. et al., J. Am. Chem. Soc., 120 • /O CO2R pp. 80-86 (1998). ry V, CO; jH Merour, J.Y. et al., Tetrahedron Lett., 32, pp. 2469-2470 (1991); Rossen, K., Tetrahedron Lett., 36, pp. 6419-6422 (1995).

31 ((3

38, R-CHj

44, RM-MeOPh-

45. R = 4-MeOPh46. R «4-W | eOPh-

5-tert-butyl-3- [2- (9H-fluoren-9-ylmethoxycarbonylamino) ethyl ester 5 3-methyl butyrill-2,3-dihydro [1,3,4ltiadiazole-2-carboxylic acid (37).

A stirred suspension of polyvinylpyridine (2.63 g, 25 mmol) in a solution of 5-tert-butyl-2,3-dihydyl [1,3,4] thiadiazole-2-carboxylic ethyl ester (36), J. Med Chem ,, 34, p. 439 (1991)), (2.16 g, 10 mmol) in dry toluene was treated with the dropwise addition of 10 9H-fluoren-9-yl methyl ester (1-chlorocarbonyl-2-methyl- propyl) -carbamic acid (4.76 g, 12.1 mmol) in 20 mL of anhydrous toluene. After stirring for 16 hours, the suspension was filtered and the filtrate was washed with saturated sodium bicarbonate solution. The organic layer was separated, washed with water, dried over anhydrous sodium sulfate, and evaporated to give a yellow oil. Purification by flash chromatography eluting with 9/2 hexane / ethyl acetate gave 2.66 g (49% yield) of the title compound (37) as a viscous oil, chlorine. ¹ H NMR (500 MHz, CD ₃ 0D) δ 0.9 (d, 1.5H), 0.93 (d, 1.5H), 1.00 (m, 1.5H), 1.06 (d , 1.5H), 1.22 (t, 3H), 1.28 (s, 9H), 2.12-2.22 (m, 0.5H), 2.32-2.42 (m, 0 , 5H), 4.18-4.28 (m, 2H), 4.31 - 4.45 (m, 2H), 4.96-5.01 20 (m, 0.5H), 5.02- 5.3.0 (m, 0.5H), 5.52 (d, 0.5H), 5.61 (d, 0.5H), 6.10 (s, 0.5H),

6.13 (s, 0.5H), 7.27-7.34 (m, 2H), 7.35-7.42 (m, 2H), 7.56-7.64 (m, 2H), 7.73UH

7.78 (m, 2H).

3- (2-Acetylamino-3-methylbutyryl) -5-tert-butyl-2,3-dihydroΓ1,2,41thiadiazole-2-carboxylic acid ethyl ester (38).

To a solution of (37) (Scheme IX) (0.508 g, 0.94 mmol) in

CH ₃ CN (10 ml) was added diethylamine (1 ml). The solution was stirred at room temperature for 2 hours, the solvent was removed in vacuo and the resulting oil was azeotroped with CH2 Cl2 (4x). The crude oil was dissolved in

CH ₂ CI ₂ (5 ml) and triethylamine (0.26 ml, 1.96 mmoles) and acetyl chloride (80 μΙ,

1.1 mmoles) were added. The solution was stirred at room temperature under an atmosphere of N ₂ for 2 hours. The solvent was evaporated, and the crude material was dissolved in EtOAc and was washed with 0.5 N (2x) NaHS (O) ₄ ,

Saturated NaHCO ₃ (2x) and brine and dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give a yellow oil. Purification by flash column chromatography on silica gel using hexanes / EtOAc (95/5 to 90/10%) gave the product as a yellow oil (0.301 g, 89% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 0.88 (dd, 3H), 0.99 (dd, 3H), 1.16-1.45 (m, 12H),

2.02 (s, 3H), 2.09-2.19 (m, 0.5H), 2.30-2.40 (m, 0.5H), 4.12-4.29 (m, 2H ), 5.20-5.27 (m, 0.5H), 5.30-5.36 (m, 0.5H), 6.60 (s, 0.5H), 6.90 (s, 0 , 5H), 6.206,31 (m, 1H). Analytical HPLC (column C18), (mixture of diastereomers) 20 7.77.7.98 min. LC-MS (ES ⁺ ) m / e = 358.3 (M + H).

3- (2-acetylamino-3-methylbutyryl) -5-tert-butyl-2,3-dihydro- [1,2,4] thiadiazole-2-carboxylic acid (39).

To a solution of 38 (0.301 g, 0.84 mmol) in MeOH (10 ml) was added 1N NaOH solution (1.7 ml, 1.7 mmol). Reaction 25 was stirred at room temperature for 2 hours and the solvent was evaporated. The residue was dissolved in EtOAc and washed with 0.5 N (2x) NaHS (O) ₄ and brine and dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give the title compound as a yellow solid. (0.277 g, quantitative) Preparation of carbamic 2- (benzyloxy-5-oxo-tetrahydro-furan-3-yl) 30 allyl ester (40).

Compound 40 was prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester by modifying the product

procedure described in Bioorg. Med, Chem, Lett, Vol. 2, No. 6, pp. 613-615 (1992).

To a solution of DMSO (27.52 g, 352 mmoles) in CH ₂ CI ₂ (240 ml) at -78 ° C was added oxolyl chloride (24.4 g, 192 mmoles). After 5 15 min, a solution of 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester (41.44 g, 160 mmol) in CH ₂ CI ₂ (100 ml) was slowly added and the mixture was stirred at -78 ° C for an additional 1.5 hours, DIEA (62.0 g, 480 mmoles) was added and the mixture was allowed to warm to room temperature for 15 min. The resulting solution was diluted 10 with CH ₂ CI ₂ (300 ml), washed with 0.5 N NaHSO ₄ (500 ml x 2), water (300 ml x 2), and brine (400 ml x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to a volume of 200

ml. To this solution, benzyl alcohol (48 g, 444 mmoles) was added, followed by 3Â ° molecular sieves (30 g) and p-toluenesulfonic acid (0.8 15 g). The reaction mixture was allowed to stir for 4 days and TFA (96 ml) was added. The resulting suspension was stirred for one hour and then evaporated in vacuo. Ethyl acetate (500 ml) was added and the mixture was filtered through Celite. The filtrate was washed with saturated NaHCO ₃ (500 ml x

2), water (400 ml x 2), and brine (300 ml x 2). The organic solution was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated in vacuo to give 90 g of pale yellow oil, which was stirred with hexane (400 mL x 2) to give 31 g of the crude product from the residue bottom layer. Chromatography using ethyl acetate / hexane (4/96 to 22/78) provided 6.97 g of anti-2- (benzyloxy5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester (Rf higher)

4.53 of diastereomer syn and 12.97 g of the mixture of diastereomers (total yield 53%. ¹ H NMR (500 MHz, CDCI ₃ ) for antidiastereomer: δ 2,412.45 (m, H) 3.02-3.01 (m, H) 1.28 (br, H), 4.50-4.80 (m, 3H), 4.80-5.15 (m, 2H), 5.24,5.32 (m, 2H), 5.48 (s, H) 5.88-6.00 (m, H), 7.31-7.56 (m, 5H); for syn diastereomer: ô 2.49-2.53 ( m, H), 2.83-2.89 (m, H) 4.57-4.65 (m, 4H), 30 4.87-4.90 (m, H), 5.12-5, 30 (m, 3H) 5.52-5.53 (d, H), 5.88-6.00 (m, H), 7.317.39 (m, 5H); retention time on analytical HPLC: 10, 49 min for antidiastereomer and 10.37 min for syn diastereomer; LC-MS: m / z = 292 (m95

Η ⁺ ).

3- (2-Acetylamino-3methylbutyryl) -5-tert-butyl-2 ₁ 3-dihydro- [1,2,41 thiadiazole-2- (2-benzyloxy-5-oxo-tetrahydrofuran-3-yl) carboxylic (41).

To a solution of allyl ester of (2-benzyloxy-5-oxo5 tetrahydrofuran-3-yl) -carbamic acid (40) (0.365 g, 1.32 mmol) in DMF (2 ml) and CH2 Cl2 (2 ml) were DMBA (0.456 g, 2.92 mmol) and Pd (PPh ₃ ) ₄ (0.136 g, 0.12 mmol) were added and the solution was stirred at room temperature for 15 min. A solution of (39) in CH ₂ CI ₂ (4.5 ml) and DMF (0.5 ml) was added, followed by HOBT (0.168 g, 1.24 mmol) and EDC (0.256 g, 1.33 mmol) ). The reaction was stirred at room temperature for 18 hours under N ₂ , The solvent was evaporated. The crude material was dissolved in EtOAc and washed with

0.5 N NaHSO ₄ (2x), saturated Na ₂ CO ₃ (2x) and brine and was dried over

Anhydrous NaHSO ₄ , was filtered and evaporated to give a yellow solid. Purification by flash column chromatography gave the title compound (41) as a mixture of diastereomers (374 mg, 88% yield) ¹ H NMR (500 MHz, CDCI ₃ ) δ 0.75-1.05 (m 6H ), 1.19-1.34 (m, 9H), 1.93-2.08 (m, 3H), 2.19-2.50 (m, 2H), 2.80-3.30 (m , 1H), 4.56-4.93 (m, 3H), 5.02-5.20 (m, 1H), 5.46-5.56 (m, 1H), 5.95-6.16 (m, 2H), 6.86-6.95 (m, 1H), 7.2-7.43 (m, 5H).

Analytical HPLC (column C18), (mixtures of diastereomers) 8.58 min. LC-MS 20 (ES ⁺ ) M / e = 519.2 (M + H).

Preparation of 3- {f3- (2-acetylamino-3-methyl-butyryl) -5-tert-butyl-2,3-dihydro-Π, 3,41thiadiazol-2-carbonill-amino} -4-oxo-butyric acid (42 ).

A 45 mg (0.087 mmol) sample of 41 was hydrolyzed according to method A (see Scheme XXIII) to give 17 mg (45% yield) of the title compound. Analytical HPLC (column C18): 5.15 mins, LCEM (ES ⁺ ) m / e = 429.3 (M + H).

5-tert-Butyl-3-f2- (4-methoxy-benzoylamino) -3-methyl-butyrill-2,3-dihydro-1,3,31-thiadiazole-2-carboxylic acid ethyl ester (43).

It was prepared by the method reported above for compound 38 using anisoyl chloride to give 216 mg (50%) of the title compound as an amorphous solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 0.92 (d, 1.5H), 0.98 (d, 1.5H), 1.03 (d, 1.5H), 1.07 (d, 1.5H), 1.21 (t, 3H), 1.28 (s, (H), 2.21-2.18 (m, ((?

0.5Η), 2.41-2.48 (m, 0.5Η), 3.83 (s, 3Η), 4.15-4.28 (m, 2H), 5.411-5.46 ( m, 0.5H), 5.48-5.53 (m, 0.5H), 6.08 (s, 0.5H), 6.13 (s, 0.5H), 6.75 (d, 0.5H), 6.85 (d, 0.5H), 6.91 (d, 2H), 7.59 (d, 2H).

5-tert-Butyl-3- [2- (4-methoxy-benzoylamino) -3-methylbutyrin-2,3-dihydro- [1,3,4l 5 thiadiazole-2-carboxylic acid (44).

Prepared by the procedure described for 39 to give 180 mg (quantitative) of the title compound as a white solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 0.92 (d, 1.5H), 0.96 (d, 1.5H), 1.03 (d, 1.5H), 1.07 (d, 1.5H), 2.22-2.30 (m, 0.5H), 2.37-2.45 (m, 0.5H), 3.83 (5.1.5H), 3.84 ( s, 1.5H), 5.4110 5.48 (m, 1H), 6.14 (s, 0.5H), 6.15 (s, 0.5H), 6.87-5.95 (m , 2H), 7.75-7.83 (m,

3H).

5-tert-Butyl-3- [2- (4-methoxy-benzylamino) -3-methyl-butyryl-2,3-dihydro- (2-bentyloxy-5-oxo-tetrahydro-furan-3-yl) -3-methyl-butyryl-2,3-dihydro- [1,3,41 thiadiazole-2-carboxylic (45a and 45b).

It was prepared by the procedure reported for compound 41 to give the crude title compound as 4 diastereomers. The crude matter was purified by flash chromatography, eluting with a gradient from CH ₂ CI ₂ to CH ₂ CI ₂ / ethyl acetate (6/4) to give 31 mg of the upper Rf component as a single diastereomer (45a) . Analytical HPLC (column 20 of Microsb C18) 19.87 min. ¹ H NMR (500 MHz, CDCI ₃ ) (single diastereomer) δ 1.04 (d, 3H), 1.14 (d, 3H), 1.28 (s, 9H), 2.77 (d, 0, 5H), 2.81 (d, 0.5H), 2.90 (d, 0.5H), 2.95 (d, 0.5H), 3.84 (s, 3H), 4.44-4 , 49 (m, 1H), 4.53 (d, 1H), 4.85 (d, 1H), 5.02.508 (m, 1H), 6.37 (s, 1H), 6.41 (d, 1H), 6.93 (d, 2H), 7.267-40 (m, 5H), 1.75 (d, 2H), 7.92-7.96 (m, 1H).

The Rf fraction less than 185 mg of a solid as a mixture of 3: 1: 2 diastereomers (45b). Analytical HPLC: Microsorb C18 column. 19.00, 19.26, 20.02 mins, ¹ H NMR (500 MHz, CDCI ₃ ) (3 diastereomer mixture 3: 3: 2) δ 0.89 (d, 2.25 H), 0.98 (d, 0.75H), 1.02 (d, 0.5H), 1.03 (d, 1.5H), 1.08 (d, 0.25H), 1.10 (d, 0.15H), 1.16 (s, 0.75H), 1.17 (s, 2.25H), 1.23 (s, 0.375H), 1.24 (5.1.125H), 1.28 (s, 1,125H) , 1.29 (s, 3,315H), 2.12-2.18 (m, 0.33H), 2.32-2.42 (m, 0.67H), (m, 0.5H), 2.61 -2-67 (m, 0.5H) 2.84-2.92 (m, 0.5H), 2.96-3.07 (m, 0.0.5H), 3.65 (s, 3H ) 4.58-4.71 (m, 2H), 4.81 (d,

II?

0.16Η), 4.86 (d, 0.32 Η), 4.91 (d, 0.52H), 5.09-5.13 (m, 0.33H), 5.14-5.18 (m , 0.67H), 5.35 (dd, 1H), 5.46 (s, 0.16H), 5.53 (d, 0.32H), 5.58-5.62 (d, 0.52H ), 6.17 (s, 0.52H), 6.20 (s, 0.16H), 5.34 (s, 0.32H), 6.50 (d, 0.32H), 6.62 (d, 0 , 16H), 6.67 (d, 0.52H), 6.86 (d, 0.33H), 16.51 (d, 0.67H), 6.94 (d, 1, OH), 7, 24-7.43 (m, 5H), 7.61 (d, 1H), 7.70 (d, 0.33H), 7.71 (d, 0.67H), 7.76 (d, 1H).

Preparation of 3 - ({5-tert-butyl-3- [2- (4-methoxy-benzoylamino) -3-methylbutyryl] -2,3-diidrori, 3,41thiadiazol-2-carbonyl} -amino acid -4 -oxo-butyric (46a),

A 30 mg sample of 45a was hydrolyzed according to method B (see scheme XXIII) to give 8 mg (30% yield) of the desired product. Analytical HPLC (microsorb C-18 column, acetonitrile / water, with TFA buffer) 12.85 min, 1H NMR (500 MHz, CD ₃ OD) δ 0.98-1.1 (m, 6H), 1.28 (s, 9H), 2.20-2.32 (m, 1H), 2.40-2.48 (m, 1H), 2.6-2.72 (m, 1H), 3.84 (s , 3H), 4.18-4.26 (m, 1H), 4.56-4.62 (m, 1H), 5.25-5.32 (m, 1H), 6.24-6.28 (m, 1H), 6.98 (d, 2H), 7.85 (d, 2H).

Preparation of 3 - ({5-tert-butyl-3-12- (4-methoxy-benzoylamino) -3-methylbutyrill-2,3-dihydro [1 ₁ 3,4ltiadiazol-2-carbonyl} -amino) -4 -oxo-butyric (46b),

A 30 mg sample of 45b (mixture of 3 diastereomers) was hydrolyzed according to method B (see Scheme XXIII) to give 22 mg (84% yield) of the desired product as a 3: 2 mixture of diastereomers. Analytical HPLC (Microsor cyan column) 7.08, 7.78 min. ¹ H NMR (500 MHz, CD ₃ OD) δ 0.98-1.08 (m, 4H), 1.09-1.12 (m, 2H), 1.29 - 1.31 (2 uniplets, 9H ), 2.23-2.30 (m, 0.5H), 2.36-2.55 (m, 1.5H), 2.62-2.72 (m, 1H), 3.85 (s , 3H), 4.18-4.27 (m, 1H), 4.58-4.65 (m, 1H), 5.27-5.33 (m, 2, H), 6.23-6 , 27 (m, 1H), 7.00 (d, 2H), 7.70-7.86 (m, 2H).

íd

1- (2-Benzyloxycarbonylamino-2-methyl-propionyl) pyrrolidine-2-carboxylic acid tert-butyl ester (49).

To a solution of proline-tert-butyl ester (47) (2.00 g, 12 mmoles, in CH2 Cl2 (15 ml) was added N-carbobenzyloxy-2-methylalanine (3.05 g, 13 mmoles), HOBT (2 , 36 g, 17 mmoles) and EDC (3.43 g, 18 mmoles) and the solution was stirred at room temperature under N ₂ for 48 hours.The solvent was evaporated, the crude material was dissolved in EtOAc and was washed 10 with 0.5N _NaHSO4 (2x), saturated _NaHCO3 (2x) and brine and dried over anhydrous Na ₂ SO _4, filtered and evaporated to give a white solid (4.68 g, 100%). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.20-2.15 (m, 4H), 1.43 (s, 9H),

1.59 (d, 6H), 2.21-3.79 (m, 2H), 4.35 (br, 5.1H), 4.82-5.19 (m, 3H), 5.74 ( br s,

1H), 7.17-7.49 (m, 5H). Analytical HPLC (column C18) 10.66 min. LC-MS 15 (ES ⁺ ) m / e = 391.3 (M + H).

1-r2- (4-methoxybenzoylamino) -2-methyl-propionylpyrrolidine-2-carboxylic acid tert-butyl ester (50).

A solution of 49 (1.00 g, 2.56 mmoles) in MeOH (20 ml) was added 10% Pd / C (200 mg) and the mixture was stirred under H ₂ for 2 hours. The mixture was filtered through a 0.45 pm PTFE filter and the solvent was removed in vacuo to provide colorless oil. This oil was dissolved in CH ₂ CI ₂ (25 ml) and DIEA (660 μΙ, 3.79 mmoles) and p-anisoyl chloride (480 mg, 2.8 mmoles) were added. The solution was stirred at room temperature under N ₂ for 18 hours. The solvent was removed in vacuo and the oil was dissolved

in EtOAc. The organic phase was washed with 0.5 N NaHSO ₄ (2x), water, saturated NaCO ₃ (2x) and brine. The organic phase was dried over Na ₂ HSO _4, filtered and evaporated to give a white solid which was purified by flash column chromatography, eluting with CH ₂ CI ₂ / MeOH (99/1 to 98/2%) to give the title compound as a white solid (655 mg, 65% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.47 (s, 9H), 1.68-2.24 (m, 5H), 1.80 (d, 6H), 3.55-3.68 ( m, 1H), 3.72-3.93 (m, 1H), 3.84 (s, 3H), 4.43-4.55 (m, 1H), 6.90 (d, 2H), 7 , 60 (br s, 3H), 7.77 (d, 2H) analytical HPLC (Column C18) 8.98 min.

1- [2- (4-Methoxybenzoylamino) -2-methyl-propioninpyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (51).

To a solution of 50 (325 mg, 0.83 mmol) in dioxane (5 ml) was added triethylamine (463 μΙ, 3.32 mmoles) and TMS-triflate (642 μΙ, 3.32 mmoles) and the solution was stirred at 100 ° C for 5 hours, then at room temperature for 18 hours. The reaction was diluted with water, adjusted to pH 8 with saturated NaHCO ₃ and extracted with Et ₂ O, dried over Na ₂ SO ₄ , filtered and evaporated to give a white solid (230 mg, 83% yield ) that was used directly in the next step.

To a solution of allyl ester of acid (2-benzyloxy-5-oxo-

tetrahydrofuran-3-yl) -carbamic (40) (1.027 g, 3.5 mmol) in CH ₂ CI ₂ (20 ml) DMBA (543 mg, 3.48 mmol) and Pd (PPh ₃ ) ₄ (280 mg, 0.24 mmol) and the solution was stirred at room temperature under N ₂ for 20 minutes. A solution of 1- [2- (4-methoxy-benzoylamino) -2-methyl-propionyl) pyrrolidine-2-carboxylic acid (818 mg, 2.45 mmol) in CH ₂ CI ₂ (5 ml) was added, followed by HOBT (0.534 g, 3.95 mmoles) and EDC (738 mg, 3.84 mmoles). The reaction was stirred at room temperature for 18 hours under N ₂ .

The solvent was evaporated, the crude material was dissolved in EtOAC and was washed with 0.5 N NaHSO ₄ (2x), saturated NaHC0 ₃ (2x) and brine and dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. to give a yellow solid.

Purification by flash column chromatography, eluting with ethyl acetate / hexanes (20/80 to 50/50%), gave the product as a pale yellow solid (760 mg, 61% yield): ¹ H NMR (500 MHz , CD ₃ 0D) δ 1.53 (d, 6H),

100

1.65-1.93 (m, 3H), 1.96-2.14 (m, 1H), 2.60 (dd, 0.1H), 2.77 (dd, 0.85H), 2, 94 (dd, 0.85H), 3.04-3.11 (m, 0.2H), 3.42-3.52 (m, 1H), 3.57-3-67 (m, 1H), 3.84 (s, 3H), 4.38-4.76 (m, 3H), 4.84 (d, 1H), 5.64-5.70 (m, 1H), 6.96-7, 00 (m, 2H), 7.23-7.43 (m, 5H), 7.76-7.97 (m, 2H). Analytical HPLC (column C18) 5 13.32, 14.37 min, LC-MS (ES ⁺ ) m / e = 524.3 (M + H).

Preparation of 3 - ({1- [2- (4-methoxy-benzoylamino) -2-methyl-propionylpyrrolidine-2-carbonyl} -amino) -4-oxobutyric acid (52)

A 61 mg (0.14 mmol) sample of 51 was hydrolyzed according to method C (see Scheme XXIII to provide 30 mg (60%

yield) of the title compound: Analytical HPLC (Column C18) 6.79 min

LC-MS (ES ⁺ ) m / e = 434.3 (M + H).

Scheme XII

HC | H ^ü C02 + BU

Μ, X «H. Υ · Μ · Ο ίβ, Χ-Q. Υ · ΝΗζ

I

8β, Χ-Η, Υ-ΜβΟ

60, X «CI, Y« NHj

M.X »H, Y = MeO

Y-Nbfe

1- [2- (4-Methoxy-benzoylamino) -3-methylbutyryl] pyrrolidine-2-carboxylic acid tert-butyl ester (54)

To a suspension of H-val-pro-OtBu.HCI (53) (2.011 g, 7.44 mmoles) in CH2CI2 (20 ml) was added DIEA (3.2 ml, 18.4 mmoles) 20 followed by a solution of 4-methoxy-benzoyl chloride (1.26 g, 7.4 mmo-

101 les) in CH2 Cl2 (5 ml). The solution was stirred at room temperature under nitrogen for 1 hour, it was concentrated. The resulting oil was dissolved in EtOAc, and was washed with KHS (O) ₄ at 0.5 N KHSO ₄ (2x), saturated NaHCO ₃ (2x)

and brine, then it was concentrated in vacuo to give the title compound as a white solid (2.814 g, 94% yield). ¹ H NMR (500 MHz, CDCl3) δ 1.05 (dd, 6H) 1.46 (s, 9H), 1.88-2.29 (m, 5H), 1.65-3.74 (m, 1H), 3.81 - 3.92 (m, 1H), 3.85 (s, 3H), 4.32 - 4.42 (m, 1H), 4.81 - 4.91 (m, 1H) , 6,796.66 (m, 1H), 6.91 (d, 2H), 7.78 (d, 2H). Analytical HPLC (cyan column) 10.18 mins.

1-f2- (4-methoxybenzoylamino) -3-methylbutyryl] -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydrofuran-3-yl) (56).

A sample of 1.079 g (2.67 mmoles) of 54 was dissolved in CH2 Cl2 (40 ml) and was stirred at room temperature for 4 hours. The solvent was concentrated in vacuo to give 55 as a white solid (0.93 g, 15 100%) which was used in the next step.

To a solution of 40 (1,796 g, 6.17 mmoles) in CH2 Cl2 (20 ml) were added DMBA (1,119 g, 7.17 mmoles) and Pd (PPh ₃ ) ₄ (0.683 g, 0.59 mmol) and a The solution was stirred at room temperature for 20 minutes. A solution of 55 (0.928 g, 2.67 mmoles) in CH ₂ CI ₂ (17 ml) and DMF (2 ml) was

added, followed by HOBT (0.81 g, 6.01 mmoles) and EDC (1.16 g, 6.04 mmoles). The reaction was stirred at room temperature for 18 hours under N ₂ . The solvent was evaporated, the raw material was dissolved in EtOAc and washed with 0.5N _NaHSO4 (2x), saturated NaHCO ₃ (2x) and brine and dried over Na ₂ SO ₄ andiro, filtered and evaporated to give a yellow solid.

Purification by flash chromatography eluting with ethyl acetate / CH ₂ CI ₂ (10/90 to 40/60%) gave the title compound as pale yellow solid (910 mg, 63% yield), ¹ H NMR (500 MHz, CDCI ₃ ) δ 0.96 (dd, 6H) 1.84-2.19 (m, 4H), 2.25-2.38 (m, 1H), 2.45 (dd, 1H), 2 , 80-2.98 (m, 1H), 3.60-3.72 (m, 1H), 3.82-3.95 (m, 1H), 3.86 (s, 3H), 4.26 -4.35 (m, 6H), 5.41 (m, 0.2H), 5.53 (d, 0.8H), 6.67-6.77 (m, 1H), 6.88-6 , 99 (d, 2H), 7.22-7.57 (m, 5H), 7.71-7.82 (d, 2H). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 9.21 min. LC-MS (ES ⁺ ) m / e = 538.3 (M + H).

102

3 - ({l- [2- (4-methoxy-benzoylamino) -3-methyl-butyrill pyrrolidine-2-carbonyl} amino) -4-oxo-butyric acid (57).

A 125 mg (0.23 mmoles) sample of 56 was hydrolyzed according to method A (see Scheme xx11) to provide 60 mg (58% yield) of the title compound: Analytical HPLC 5.71 min. LC-MS (ES ⁺ ) m / e = 448.2 (M + H).

Preparation of 4-amino-3-chloro-benzoic acid

A suspension of 4-amino-3-chlorobenzonitrile (4.82 g, 31.58 mmol) was heated to reflux in 6 N HCI (140 ml). The precipitate was dissolved after heating to give a colorless solution. After further heating the solution became cloudy. After 9 hours the reaction was cooled to room temperature. The resulting precipitate was filtered, then it was dissolved in THF and the solvent was evaporated. The residue was repeatedly concentrated from toluene to give a white solid (3.18 g, 59% yield). ¹ H NMR (500 MHz, CD ₃ 0D: CDCI ₃ 1: 4) δ 6.80 (d, 1H),

7.75 (dd, 1H), 7.94 (d, 1H). Analytical HPLC (cyan column) 8.73 min.

1-1,2- (4-Amino-3-chlorobenzoylamino) -3methylbutyrill pyrrolidine-2-carboxylic acid tert-butyl ester (58).

To a suspension of 53 (1.707 g, 6.31 mmoles) in CH ₂ CI ₂ (25 ml) at 0 ° C was added DIEA (3.2 ml, 18.4 mml) followed by a solution of 4-amino acid -3-chlorobenzoic (1.298 g, 7.56 mmoles), HOBT (1.005 g, 7.44 mmoles) and EDC (1.456 g, 7.58 mmoles). The resulting mixture was stirred at 0 ° C for 15 minutes then it was allowed to warm to room temperature and stir for 18 hours. The solvent was evaporated and the resulting oil 25 was dissolved in EtOAc, washed with 0.5 N NaSO ₄ (2x), saturated NaHCO ₃ (2x) and brine to give a white solid (2.68 g). Flash chromatography using MeOH / CH ₂ CI ₂ (1/99 to 2/98%) gave 2.049 (76% yield) as a white solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.05 (dd, 6H) 1.47 (s, 9H), 1.86-2.29 (m, 5H), 3.62-3.78 (m , 1H), 3.76-3.94 (m, 1H), 4.39 (dd,

1H), 4.79-4.89 (dd, 1H) 6.73 (d, 1H), 6.78 (d, 1H), 7.52 (dd, 1H), 7.75 (d, 1H)

Analytical HPLC (cyan column), 16.18 min. LC-ME (ES ⁺ ) m / e = 424.3 (M + R).

103 1- [2- (4-Amino-3-chlorobenzoylamino) -3-methylbutirill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -3 (60).

A 0.632 g (1.49 mmoles) sample of 58 was dissolved in 50% TFA in CH ₂ CI ₂ (20 ml) and the solution was stirred at room temperature for 2 hours. Residual TFA was removed by repeated concentration from CH ₂ CI ₂ (3x) to give the product as a white solid.

A sample of 385 mg (1.04 mmoles) was allowed to react with the method used for compound 56, the title compound (60) was isolated as a yellow solid (265 mg, 45% yield). ¹ H NMR (500

MHz, CD ₃ OD) δ 0.89-1.12 (m, 6H), 1.72-2.26 (m, 5H), 2.49 (dd, 0.25H), 2.60 (dd, 0 , 7H), 2.80 (dd, 0.75H), 2.96-3.09 (m, 0.3H), 3.64-3.17 (m, 1H), 3,944.10 (m, 1H ), 4.20-4-74 (m, 4H), 4.76-4.95 (m, 1H), 5.51 (s, 0.5H), 5.61 - 5.70 (m, 1 , 5H), 6.79 (dd, 1H), 7.23-7.43 (m, 5H), 7.48-7.61 (m, 1.4H), 7.68-7.81 (m , 1H), 7.99-8.12 (m, 0.6H).

Analytical HPLC (cyan column) (mixture of 2 diastereomers)

14.90, 15.20 mins, LC-MS (ES ⁺ ) m / e = 557.2 (M + H).

3 - ({1- [2- (4-amino-3-Chloro-benzoylamino) -3-methylbutyrin-pyrimidine-2carbonyl} -amino) -4-oxo-butyric acid (61).

A 45 mg (0.08 mmol) sample of 60 was hydrolyzed to a20 according to method A (see Scheme XXIII) to provide 30 mg (80% yield) of the title compound: ¹ H NMR (500 MHz, CD ₃ OD) δ 1.06 (dd, 6H), 1.78-2.38 (m, 5H), 2.38-2.86 (m, 2H), 3.62-3.83 (m, 1H ), 4.12-4.76 (m, 4H), 7.04-7.21 (m, 1H), 7.58-8.01 (m, 2H); Analytical HPLC 8.16 min. (ES ⁺ ) m / e = 467.3 (M + H).

Scheme XIII

1- [2- (4-hydroxy-3,5-dimethyl-benzoylamino) -3-methylbutyrill-pyrrolidine-2-carboxylic acid tert-butyl ester (63),

To a solution of 62 (prepared from 53 and Fmoc-CI) (600 mg, 1.22 mmmoles) in anhydrous DMF (10 ml) was added diethylamine (3 ml).

The solution was stirred at room temperature under N ₂ for 3 hours and the solvent was evaporated. The resulting oil was dissolved in CH ₂ CI ₂ (8 ml) and 3,5-dimethyl-4-hydroxybenzoic acid (0.302 g, 1.82 mmoles), HOBT (338 mg, 2.5 mmoles) and EDC (0.456 g , 2.43 mmoles) were added to the solution was agi. 10 at room temperature under N ₂ for 18 hours. The solvent was concentrated in vacuo and the resulting oil was dissolved in EtOAc, washed with

0.5N _NaHSO4 (2x) , saturated _NaHCO3 (2x) and brine to give the crude product as a white solid (0.80 g). Flash chromatography eluting with

MeOH / CH ₂ CI ₂ (1/99 to 2/99%) gave 380 mg (75% yield) of a white solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.06 (dd, 6H), 1.47 (s, 9H), 1.90-2.32 (m, 5H), 2.24 (s, 6H) , 3.65-3.75 (m, 1H), 3.84-3.92 (m, 1H), 4.36-4.42 (m,

1H), 4.82-4.82 (m, 1H), 5.53-5.61 (m, 1H) 6.77-6.85 (m, 1H), 7.42 (s, 2H) HPLC analytical (cyan column) 17.53 min. LC-MS (ES ⁺ ) m / e = 419.3 (M + H).

1-1,2- (4-hydroxy-3,5dimethyl-benzoylamino) -3-methylbutyryl-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -3-carboxylic acid (64).

Prepared from 63 and 40 by the method used to prepare to give the title compound (64) as a pale yellow solid (352 mg, 72% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 0.83-1.28 (m, 6H),

105

1.66-2.37 (m, 3H), 2.23 (s, 6H), 2.48-2.54 (m, 0.2H), 2.61 (ddd, 0.8H), 2, 72 (ddd, 0.9H), 3.01-3.09 (m, 1H), 3.66-3.76 (m, 1H), 3.95-4.07 (m, 1H), 4.444, 73 (m, 3H), 4.75-4.92 (m, 2H), 5.45-S, 48 (m, 0.1H), 5.62-5.64 (m, 0.1 H) , 5.64-5-70 (m, 0.8H), 7.21-7.62 (m, 5H), 7.88-6.04 (m, 1H). Analytical HPLC 5 (cyan column) 2 diastereomer mixture) 17.73 min. LC-MS (ES ⁺ ) m / e = 552.3 (M + H).

3 - ({1 - [2- (4-Hydroxy-3,5-dimethyl-benzoylamino) -3-methyl-butyrill-pyrrolidine2-carbonyl} -amino) -4-oxobutyric acid (65),

A 160 mg (0.29 mmol) sample of 64 was hydrolyzed to

according to method A (see Scheme XXIII) to provide 13.1 mg (10% yield) of the title compound: Analytical HPLC (cyan column) 10.28 min. LC-MS (ES ⁺ ) m / e = 462.2 (M + H),

Scheme XIV

es

and?

1- [2- {2-9H-Fluoren-9-yl-acetylamino) -3,3dimethylbutyrill-pyrrolidine-2-carboxylic acid tert-butyl ester (66),

To a solution of H-pro-OtBu (53) (1.033 g, 6.0 mmol, II, Scheme 5) in CH ₂ CI ₂ (20 ml) and DMF (5 ml) were added Fmoc-tLeu-OH 20 ( 2.337 g, 6.60 mmoles, Scheme 5), HOBT (1.63 g, 12.1 mmoles) and EDC (2.30 g, 12.0 mmoles) and the solution was stirred at room temperature under N ₂ for 18 hours. The solvent was removed in vacuo, and the residue was dissolved in EtOAc then washed with 0.5 N NaHSO ₄ (2x), saturated NaHCO ₃ (2x) and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and evaporated to give a pale yellow solid (3.65 g). Flash chromatography using

EtOAc / hexanes (10/90 to 20/80%) gave the title compound (66) (2.25 g, 74% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.09 (s, 9H), 1.47 (s, 9H), 1,792.28, (m, 3H), 3.62-3.72 (m, 1H ), 3.76-3.83 (m, 2H), 4.18-4.43 (m, 4H), 5.485 5.67 (m, 1H), 7.28-7.44 (m, 4H) , 7.55-7.64 (m, 2H), 7.72-7-82 (m, 2H). HPLC

Analytical (cyan column) 11.95 min. LC-MS (ES ⁺ ) m / e = 507.3 (M + H).

1-F2- (4-Methoxy-benzoylamino) -3,3-dimethyl-butyryl) pyrrolidine-2-carboxylic acid tert-butyl ester (67).

To a solution of 66 (0.503 g, 0.99 mmoles) in DMF (8 ml) was added diethylamine (2.5 ml) and the solution was stirred at room temperature for 1 hour and the solvent was evaporated. The residue was repeatedly concentrated from CH2 Cl2 (3x). The resulting oil was dissolved in CH ₂ CI ₂ (9 ml) and DIEA (260 μΙ, 1.49 mmoles) and 4-methoxy-benzoyl chloride (190 mg, 1.05 mmoles) was added. The solution was stirred under N ₂ for 18 hours and the solvent was concentrated in vacuo. The residue was dissolved in EtOAc and was

washed with 0.5 N NaHS (O) ₄ (2x), saturated NaHCO ₃ (2x) and brine then dried over Na ₂ SO ₄ and evaporated to give a white solid (0.529 g). Flash chromatography on silica gel using MeOH / CH ₂ Cl2 (1/99 to 2/98%) gave the title compound (2.23 g, 74% yield). ¹ H NMR (500 MHz, 20 CD ₃ 0D) δ 1.01 (s, 1.4H), 1.11 (s, 7.6H), 1.73-2.25 (m, 4H), 2, 47-2.77 (m, 1H),

2.81 (dd, 0.7H), 2.92-3.11 (m, 0.3H), 3.61 - 4.03 (m, 3H), 3.84 (s, 3H), 4.294, 49 (m, 1H), 4.49-5.00 (m, 5H), 5.46 (s, 0.15H), 5.58-5.73 (m, 0.85H), 6.947-04 (m , 2H), 7.27-7.41 (m, 4H), 7.61-7.73 (m, 1H), 7.74-7.84 (m, 2H). Analytical HPLC (cyan column) 13.10 min.

13-1,2- (4-methoxybenzoylamino) -3,3-dimethyl-butyrin-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (68).

To a solution of 67 (0.90 g, 1.74 mmoles) in CH2 Cl2 (25 ml) were added 2,6-lutidine (2.1 ml, 18.0 mmol) and TMS-triflate (2.3 ml,

11.9 mmoles) and the reaction was stirred at room temperature under N ₂ for 1.5 30 hours. The resulting mixture was diluted with CH2 Cl2, washed with NaHCO ₃ at

10% (2x) and brine was then dried over Na ₂ SO ₄ , filtered and evaporated. The residue was dissolved in CH ₂ CI _{2 and} then treated with DIBA (0.6 ml,

107

3.5 mmoles) and 4-methoxybenzoyl chloride (0.355 g, 2.09 mmoles) and was allowed to stir under N ₂ at room temperature for 18 hours. The crude product was purified by flash chromatography, eluting with CH ₂ CI ₂ / MeOH (99%) to provide the title compound (274 mg, 25% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.01 (s, 1.4H), 1.11 (s, 7.6H), 1.73-2.25 (m, 4H), 2,472-77 (m, 1H), 2.81 (dd, 0.7H), 2.91 - 3.11 (m, 0.3H), 3.61 - 4.03 (m, 3H), 3.84 (s , 3H), 4.29-4.49 (m, 1H), 4.49-5.00 (m, 5H), 5.46 (s, 0.15H), 5.58-5.73 (m , 0.8H), 6.94-7.04 (m, 2H), 7.27-7.41 (m, 4H), 7.61-7.73 (m, 1H), 7.74-7 , 84 (m, 2H). Analytical HPLC (cyan column) (mixture of 2 diastereomers), 17.03,

17.39 min, LC-MS (ES ⁺ ) m / e = 552.3 (M + H).

________ 3 - ({1- [2- (4-Methoxy-benzoylamino) -3,3-dimethyl-butyryl-1-pyrrolidine-2carboinyl} -amino) -4-oxo-butyric acid (69).

A sample (0.21 mmoles) of 68 was hydrolyzed according to method C (see Scheme XXIII) to provide 40 mg (41% yield) of the title compound: Analytical HPLC 7.16 min. LC-MS (ES ⁺ ) M / e = 462.3 (M + H).

Scheme XV

Acid benzyl ester 1- (2-tert-butoxycarbonylamino-3 _liter 3-dimethyl-butyryl) -pyrrolidine-2-carboxylic acid (70).

To a suspension of H-pro-OBzl.HCI (2.00 g, 8.66 mmoles) in

CH ₂ CI ₂ (20 ml) was added DIEA (2.25 ml, 12.92 mmoles) to give a colorless solution. Boc-tLeu-OH (1.95 g, 9.52 mmoles), HOBT (2.76 g, 13.03 ίΠ

108 mmoles) and EDC (2.49 g, 12.95 mmoles) were added and the solution was stirred under N ₂ at room temperature for 18 hours. Solvent removed in vacuo, dissolved in EtOAc and washed with H ₂ O, 0.5 N NaHSO ₄ (2x), saturated NaHCO ₃ (2x) and brine. Dry over anhydrous Na ₂ SO ₄ and evaporate 5 to give the title compound (3.57 g, 99% yield). ¹ H NMR (500

MHz, CDCI3)

0.99 (s, 9H), 1.40 (s, 9H), 1.88-2.33 (m, 4H), 3.58-3.90 (m, 2H), 4.21 - 4, 35 (d, 1H), 4.53-4.66 (m, 1H), 5.04-5.35 (m, 3H), 7.14-7.42 (m, 5H). LC-MS (ES ⁺ ) m / e = 419.4 (M + H).

{1-1,2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3ylcarbamoyl) -pyrrolidine-1-carbonin-2,2-dimethylpropyl} -carbamic acid tert-butyl ester (71).

A sample of 871 mg (2.08 mmoles) of 70 was dissolved in

MeOH (15 ml) and 10% Pd / C (200 mg) was added. The suspension was stirred under H ₂ for 1 hour then it was filtered through Celite and the solvent was evaporated. This resulting residue was reacted with 40 according to the procedure used to prepare 56 to give 889 mg (71% yield) of the title compound (71), ¹ H NMR (500 MHz, CDCI ₃ ) δ 0.93 (s , 9H), 1.44 (s, 9H), 1.78-2.18 (m, 4H), 2.29-2-49 (m, 2H), 2.76-3.04 (m, 1H ), 3.50-3.70 (m,

1H), 3.70-3.85 (m, 1H), 4.20-4.37 (m, 1H), 4.49-4.78 (m, 3H), 4.78-4.98 ( m, 20 1H), 5.12-5.26 (m, 1H), 5.40-5.59 (m, 1H), 7.10-7.76 (m, 5H). Analytical HPLC (cyan column) 11.17 min. LC-MS (W) m / e = 518.3 (M + H).

1- (2- (4-Amino-3-chlorobenzoylamino) -3,3-dimethylbutirill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -72 (72) .

A solution of 456 mg (0.088 mmoles) of 71 in CH ₂ CI ₂ (20 ml) was treated with anhydrous TFA (5 ml) then stirred at room temperature under N ₂ for 1 hour and evaporated to dryness. The residue it was repeatedly concentrated from CH ₂ CI ₂ (3x) then dried under vacuum. The resulting residue was dissolved in CH ₂ CI ₂ (20 ml), cooled to 0 ° C, then treated with DIEA (1.3 ml, 8 eq, 2.46 mmoles) followed by 4-amino-330 chlorine -benzoic acid (202 mg, 1.17 mmol), HOBT (183 mg, 1.35 mmol), and

EDC (279 mg, 1.45 mmol). The resulting mixture was allowed to warm to room temperature and stir for 18 hours. The solvent was removed in vacuo and the residue was dissolved in EtOAc then washed with distilled water (3x), 0.5 N NaHSO ₄ (2x), saturated NaHCÜ3 (2x) and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated to give a residue which was purified by flash chromatography, eluting with 5 CH ₂ CI ₂ / MeOH (99/1 at 97/3%, providing 285 mg (57% yield) of the title compound (72) as a yellow solid.

¹ H NMR (500 MHz, CD ₃ OD) δ 0.91 - 2.24 (m, 9H), 1.70 - 2.27 (m, 4H), 2,472.6.5 (m, 1.5H) , 2.99-3.13 (m, 0.5H), 3.39-3.53 (m, 0.5H), 3.60-3.78 (m,

1.5H), 3.05-4.04 (m, 1H), 4.24-4.47 (m, 2H), 4.53-4.97 (m, 4H), 5.46 (s,

0.3H), 3.88-4.02 (m, 0.1 H), 5.60-5.69 (m, 0.6H), 6.80 (d, 1H), 7.22-7 , 77 (m,

7H). Analytical HPLC (cyan column) (mixture of 2 diastersomers) 15.90, 16.23 min. LC-MS (ES ⁺ ) m / e = 571.2 (M + H).

3 - ({1-r2- (4-amino-3-chlorobenzoylamino) -3,3-dimethylbutyryl-1-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric (73).

A 40 mg (0.07 mmol) sample of 72 was hydrolyzed to method A (see Scheme XXIII) to provide 25 mg (74% yield) of the title compound: Analytical HPLC (cyan column) 10.66 min. LC-MS (ES ⁺ ) m / e = 481.3 (M + R)

Scheme XVI

{2- [2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3ylcarbamoyl) -pyrrolidin-1-yl1-1-methyl-2-oxo-ethyl} carbonic acid tert-butyl ester (75).

To a solution of 40 (6.69 g, 23.0 mmol) in anhydrous CH ₂ CI ₂ fo / 3 /

110 rams added 1,3-dimethylbarbituric acid (DMBA) (3.97 g, 25.4 mmoles) and

Pd (PPh ₃ ) 4 (1.12 g, 0.97 mmol). The solution was stirred under N ₂ at room temperature for 15 min, cooled to 0 ° C, followed by the addition of Boc-alapro-OH (BaChem) (5.087 g, 17.8 mmoles), HOBT (3.60 g, 26.7 mmoles) and 5 EDC (5.12 g, 26.7 mmoles). The resulting solution was allowed to warm to room temperature and stir under N ₂ for 18 hours. The solvent was concentrated in vacuo and the residue was dissolved in EtOAc, then it was washed with 0.5 N NaHSO ₄ (2x), saturated NaHCO ₃ (2x) and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and evaporated to give an orange oil (12.23 g). Column chromatography on silica gel using CH ₂ CI ₂ / EtOAc (80/20 to 60/40) gave the title compound 75 as a yellow solid (7.29 g, 86% remedy). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.19-1.31 (m, 3H), 1.42 (5,9H), 1.69 to 2.29 (m, 4H), 2.45 -2.67 (m, 0.9H), 2.71-2.86 (m, 0.5H), 2.99-3.10 (m, 0.6H), 3.49-3.64 ( m, 2H), 4.24-4.45 (m, 2.5H), 4.57-4.73 (m, 1.5H), 4.76-4.92 (m, 1H), 5- 45 (s, 0.45H), 5.63-5.68 (m, 0.55H), 7.257.40 (m, 5H). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 15.99, 16.33 min. LC-MS (ES ⁺ ) m / e = 476.3 (M + H).

F1- [2- (4-amino-3-chloro-benzoylamino) -propionill pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (76),

A sample of 1.899 g (3.99 mmoles) of 75 in CH ₂ CI ₂ (20 ml) was treated with anhydrous TFA (5 ml) then stirred at room temperature under N ₂ for 1 hour and evaporated to dryness. The residue was repeatedly concentrated from CH ₂ CI ₂ (3x) then dried under vacuum. The resulting residue was dissolved in CH ₂ CI ₂ (20 ml), cooled to 0 ° C, then treated with DIEA (5 , 6 ml, 8 eq, 32.1 mmoles), 4-amino-3-chloro-benzoic acid (0.910 g, 5.3 mmoles), HOBT (0.824 g, 6.1 mmoles), and EDC (1.197 g, 6.23 mmol). The resulting mixture was warmed to room temperature and was stirred for 18 hours. The solvent was removed in vacuo and the residue was dissolved

111 solvent in EtoAc was then washed with distilled water (3x), 0.5 N NaHSO ₄ (2x), saturated NaHCO ₃ (2x) and brine. The organic layer was dried over Na ₂ S (O) 4, filtered and evaporated to give a residue which was purified by flash chromatography using C ^Cb / MeOH (99/1 at 97/3%). The title composition was obtained as a white solid (1.221 g, 58% yield).

¹ H NMR (500 MHz, CD ₃ OD) δ 1.15 (d, 0.25H), 1.29-1.660 (m, 2.75H), 2.412.54 (m, 0.5H), 2.55- 2.70 (m, 0.5H), 2.77 (dd, 0.5H), 3.03 (ddd, 0.5H), 3.59-

3.75 (m, 1H), 3.75-3.98 (m, 1H), 4.26-5.01 (m, 5H), 5.41-5.57 (m, 1H), 5.60-

5.76 (m, 0.5H), 6.70-6.92 (m, 0.5H), 7.15-7.48 (m, 5H), 7.48-7.68 (m, 1H),

7.68-7.88 (m, 1H), 8.15-8.34 (m, 1H). Analytical HPLC (cyan column) (2 diastereomer mixture) 14.44, 14.89 min. LC-MS (ES ⁺ ) m / e = 529.3 (M + H).

(2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) amide] [1- (2- (415 Methoxy-3,5-dimethyl-benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (77) was synthesized from 75 and 3,5-dimethyl-4-methoxy-benzoic acid according to the procedure used to prepare 76 to provide the title compound (1.18 g, 44% yield). ¹ H NMR ( 500 MHz, CD ₃ OD) δ 1.40 (m,

3H), 1.67-2.41 (m, 4H), 2.20 (s, 5H), 2.48 (ddd, 0.5H), 2.62 (dd, 0.5H), 2.78 20 (ddd, 0.5H), 3.04 (ddd, 0.5H), 3.62-3.94 (m, 3H), 3.72 (9.3H), 4.21-4.51 ( m,

2H), 4.59-4.85 (m, 4H), 5.46 (s, 0.25H), 5.52 (s, 0.25H), 5.63 (d, 0.4H), 5, 67 (d, 0.1 H), 7.17-7.45 (m, 5H), 7.45-7.65 (m, 2H), Analytical HPLC (column of

Cyan) (mixture of 2 diastereomers) 15.06, 15.39 min. LC-MS (ES ⁺ ) m / e = 538 (M + H)

Preparation of 4-Acetylamino-3-chlorobenzoic acid

To a solution of 4-amino-3-chloro-benzoic (10.0 g, 58.3 mmoles) in anhydrous THF (100 ml) was added acetyl chloride (20.7 ml, 291.1 mmoles) and the solution it was stirred at room temperature for 48 hours. The solvent was evaporated and the product was precipitated from hexanes then it was

133

112 filtered and dried to give a white solid (11.73 g, 94% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 2.28 (s, 3H), 7.92 (dd, 1H), 7.99-8.16 (m, 2H).

Analytical HPLC (cyan column) 7.84 min.

1-1,2- (4-acetylamino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (78).

Prepared from 75 and 4-acetylamino-3-chloro-benzoic acid according to the procedure used to prepare 76 to provide the title compound (146 mg, 19% yield). ¹ H NMR (500 MHz, 10 CD ₃ OD) δ 1.25-1.52 (m, 3H), 1.68-2.38 (m, 4H), 2.20 (s, 3H), 2, 41-2.88 (m, 1-5H), 2.96-3.10 (m, 0.5H), 2.96-3.10 (m, 0.5H), 3.43-3.79 (m, 1H), 3.80-3.96 (m, 1H), 4.25-5.00 (m,% H), 5.42-5.54 (m, 0.5H), 5, 63-5.76 (m, 0.5H), 7,137.48 (m, 0.5H), 7.79-8.14 (m, 2.5H), 8.56-8.70 (m, 0 , 52). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 8.64 min. LC-MS (ES ⁺ ) m / e = 15 571.2 (M + H).

1-f2- (3-isopropoxybenzoylamino) -pripionyl1-pyrrolidine-2-carboxylic acid (2-benzyloxy-3-oxo-tetrahydro-furan-3-yl)-79 (79).

Prepared from 75 and 3-isopropoxybenzoic acid according to the procedure used to prepare 76 to provide the title compound (120 mg, 58% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.27 (d, 5H), 1.33 to 1.52 (m, 3H), 1.69 to 2.31 (m, 4H), 2.49 (dd, 0.3H), 2.63 (dd, 0.7H), 2.78 (dd, 0.7H), 3.03 (dd, O, 3H), 3.43-3-73 (m , 1H), 3.78-3.94 (m, 1H), 4,274.47 (m, 2H), 4.47-4.87 (m, 4H), 5.47 (s, 0.7H), 5.53 (d, 0.3H), 5.64 (d, 0.8H),

5.72 (d, 0.2H), 6.98-7.12 (m, 1H), 7.19-7.47 (m, 9H). Analytical HPLC (Cyan column) (2 diastereomer mixture) 14.54, 14.85 min. LC-EM (ES ⁺ ) m / e ι3Η

113 = 538 (M + R).

Quinoxaline-2-carboxylic acid {2- [2- (2-benzyloxy-5-oxo-tetrahydrofuran-3-ylcarbamoyl) -pyrrolidin-1-yl] -1-methyl-2oxo-ethyl} -amide (80).

Prepared from 75 and 2-quinoxoline carboxylic acid according to the procedure used to prepare 76 to provide the title compound (122 mg, 60% yield). ¹ H NMR (500 MHz, CD ₃ CD) δ 1.12-1.67 (m, 3H), 1.68-2.34 (m, 4H), 2.35-2.70 (m, 0.85H ), 2.70-2.95 (m, 0.75H), 3.06 (dd, 0.4H), 3.41-3.49 (m, 2H), 4.18-5.03 (m , 6H), 5.47 (d, 0.5H), 10 5.55, (d, 2H), 5.67 (dd, 1H), 5.71 (dd, 0.3H), 7.03- 7.53 (m, 5H), 7.80-8.06 (m,

2H), 8.06-8.34 (m, 2H), 9.43-9.48 (m, 1H). Analytical HPLC (cyan column) (mixture of 2 diasteromers) 9.06 min. LC-MS (ES ⁺ ) M / e = 532.3 (M + H).

1- [2- (3-Benzyloxy-415 methoxy-benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -81-81.

Prepared from 75 and 3-benzyloxy-4-methoxy-benzoic according to the procedure used to prepare 76 to provide the title compound (142 Mg, 58% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.14 (d, 0.3H), 1.27-1.52 (m, 2.7H), 1.66-2.30 (m, 4H) , 2.47 (dd, 0.4H), 2.59 (dd, 20 0.6H), 2.77 (dd, 0.6H), 3.02 (dd, 0.4H), 3.41 3.72 (m, 1H), 3.72-3.99 (m, 2H),

3.86 (6.3H), 4.29-4.86 (m, 5H), 4.99-5.15 (m, 2H), 5.45 (m, 0.5H), 5.65 ( m,

1.2H), 6.98 (dd, 2H) 7.11-7.63 (m, 12H). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 12.28, 12.44 min LC-MS (ES ⁺ ) m / e = 616.3 (M + H).

4-Allyloxy-3,5-dimethyl-benzoic acid.

114

A Mixture of 4-hydroxy-3,5-dimethyl-benzoic acid (3.32 g, 20 mmol), allyl bromide (7.26 g, 60 mmol), benzyltriethylammonium chloride (455 mg, 2 mmol) and K ₂ CO ₃ (6.9 g, 50 mmoles) in DMF (50 ml) was stirred at room temperature for 16 hours. The mixture was diluted with 5 ethyl acetate (200 ml), washed with water, brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated in vacuo to give 5.3 g of the ester as an oil. The ester was refluxed with NaOH (5 g, 125 mmoles) in water / methanol (50 ml / 50 ml) for 6 hours. The mixture was evaporated in vacuo to remove methanol and the resulting solution was diluted with water (200 x 10 ml), washed with ethyl acetate / hexane (30 ml / 70 ml). The aqueous layer was acidified at 0 ° C with HCI solution concentrated to pH 2. The resulting precipitate was collected by filtration and washed with water, dried under high vacuum to provide 3.86 g (94% yield) of the compound of the title. ¹ H NMR (500 MHz, CDCI ₃ ): δ 2.23 (s, 6H), 4.35-4.37 (m, 2H), 5.28-5.30 (m, 15 H), 5, 42-5.46 (m, H), 6.07-6.15 (m, H), 7.79 (s, 2H); retention time on analytical HPLC: 12.28 mins; LC-MS: m / z = 205 (MH ⁺ ) -

1-1,2- (4-allyoxy-3,5-dichloro-benzoylamino) -propionill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-2-yl) (82),

Prepared from 75 and 4-allyloxy-3,5-dicloxo-benzoic acid according to the procedure used to prepare 76 to provide the title compound (208 mg, 47% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.05-1.58 (m, 3H), 1.68-3.21 (m, 7H), 3.39-3-90 (m, 3H), 4.05-5.01 (m, 6H), 5.22-5.62 (m, 3H), 6.04-6.25 (m, 1H), 6.94-7.63 (m, 8H ). Analytical HPLC (cyan lumine) (2 diastereomer mixture) 9.69, 9.89 min. LC-MS (ES ⁺ ) m / e = 604.2 (M + H).

í

1- [2- (3,5-dichloro-4hydroxy-benzoylamino) -propionin pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (83).

A 140 mg sample of 82 (0.23 mmol) was dissolved in

CH2 Cl2, (4 ml) and was treated with DMBA (35.4 mg, 0.26 mmol) and Pd (PPh ₃ ) 4 (32 mg, 0.028 mmol). The solution was stirred at 0 ° C for 15 minutes, was warmed to room temperature for 2 hours, then it was diluted with CH ₂ CI ₂ and was washed with water (2x) and brine. The solvent was concentrated in vacuo and the residue was purified by flash chromatography on silica gel using

MeOH / CH ₂ Cl2 (1/99 to 3/97) to give the title compound (93.2 mg, 71% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.16 (d, 0.25H), 1.28-1.49 (m, 2.75H), 1.63-2.33 (m, 4H), 2 , 48 (dd, 0.4H), 3.39-3.59 (m, 0.2H) 3.60-3.73 (m, 0.8H), 3.73-3.96 (m, 1H ), 4.24-4.48 (m, 2H), 4.57-4.92 (m, 7H), 5.44 (s,

0.4H), 5.50 (d, 0.4H), 5.64 (d, 0.8H), 5.75 (d, 0.5H), 7.16-7.43 (m, 5H) , 7.7815 7.89 (m, 1.6H), 8.40-8.63 (m, 0.4H). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 11.57, 11.82 min. LC-MS (ES ⁺ ) m / em = 564.1 (M + H).

3- (2-Benzoylamino20 propionyl) -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -pyrrolidine (84),

Prepared from 75 and benzoyl chloride according to the procedure used to prepare 76 to provide the title compound as a colorless oil (8 mg, 38% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.35-2.54 (m, 3H), 1.72-2.30 (m, 4H), 2.42-2.70 (m, 1, 3H), 2.7425 2.84 (m, 0.5H), 3.03 (dd, 0.2H), 3.41-3.75 (m, 2H), 3.81-3.96 (m , 1H), 4,224.86 (m, 4H), 5.46 (s, 0.3H), 5.51-5.54 (m, 0.1H), 5.66 (d, 0.5H), 5.72 (d, 0.1H), 7.20-7.57 (m, 7H), 7.77-7.89 (m, 2H), 8.42-8.67 (m, 1H). Analytical HPLC (3?

116 typical (cyan column) (mixture of 2 diastereomers) 15.23, 15.67 min .: LCEM (ES ⁺ ) (ES ⁺ ) m / e = 481.2 (M + H).

{2- (2-benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamoyl) -pyrrolidin-1-ill-2-imethyl-25 oxo-ethyl} -isoquinoline-1-carboxylic acid amide (85).

Prepared from 75 and 1-isoquinolinecarboxylic acid according to the procedure used to prepare 76 to provide the title compound (732 mg, 53% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ

1.22-1.56 (m, 3H), 1.70-2.34 (m, 4H), 2.43-2.71 (m, 0.9H), 2.73-2.89 (m ,

0.5H), 3.06 (ddd, O, 6H), 3.42-3.81 (m, 2H), 3.04-4.01 (m, 1H), 4.29-5.00 ( m,

5H), 5.47 (d, 0.65H), 5.55 (s, 0.3H), 5.67 (d, 0.8H), 5.72 (d, 0.25H), 7.21- 7.43 (m, 5H), 7.49-7.83 (m, 2.8H), 7.88-8.04 (m, 2.8H), 8.45-8.54 (m, 0 , 5H), 8,979.06 (m, 0.6H). Analytical HPLC (mixture of 2 diastereomers) 15.71, 16.04 min. LC-MS (ES ⁺ ) m / e = 531.2 (M + H).

1-F2- (4-Amino-5-chloro2-methoxy) -benzoylamino) propionill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (86).

Prepared from 75 and 4-amino-5-chloro-2-methoxybenzoic acid according to the procedure used for 76 to provide the title compound (330 mg, 61% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.22 (d, 0.25H), 1.29-1.50 (m, 0.75H), 1.68-2.36 (m, 4H), 2.382.99 (m, 1.5H), 2.94-3.34 (m, 0.5H), 3.37-3.96 (m, 6H), 4.27-4.98 (m, 6H ), 5.44-5.50 (m, 0.4H), 5.53-5.56 (9, 0.1 H), 5.60-5.75 (m, 0.5H), 6, 50 (s, 1H), 7.17-7.45 (m, 4H), 7.73-7.90 (m, 1H), 8.49-8.70 (m, 1H). Analytical HPLC (cyan lumine) (mixture of 2 diastereomers) 26-39, 16.82 min. LC-NS (ES ⁺ ) m / e = 559.2 (M + H).

I3Í

117

1-1,2- (4-acetylamino-3-chloro-2-methoxy-benzoylamino) propionill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (87).

Prepared from 73 and 4-acetylamino-5-chloro-2-methoxy5 benzoic acid according to the procedure used by 76 to provide the title compound (364 mg, 64% yield) ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.20-1.27 (m, 0.25), 1.35-1.49 (m, 0.75H), 1.72-2.30 (m, 4H), 2.23 ( s, 3H), 2.42-2.58 (m, 0.6H), 2.59-2.68 (m, 0.5H), 2.73-2-86 (m, 0.7H), 2 , 99-3.1: (m, 0.7H), 3.41-4.07 (m, 5H), 4.29-4.97 (m, 5H), 4.79-5.56 (m , 0.5H), 5.65-5.73 10 (m, 0.5H), 7.18-7.44 (m, 4.3H), 7.90-8.03 (m, 2H), 8.71-8.05 (m, 0.7H). HPLC

Analytical (cyan column) (mixture of 2 diastereomers) 15.61, 16.01 min. LC-MS (ES ⁺ ) m / e = 601.1, (M + H).

2- {2- (2-benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamoyl) -pyrrolidin-1-yl1-1-methyl-215 oxo-ethyl} -pyridine-2-carboxylic acid amide (88) .

Prepared from 75 and pyridine-2-carboxylic acid according to the procedure used by 76 to provide the title compound (233 mg, 42% yield), ¹ H NMR (500 MHz, CD ₃ 0D) δ 1, 30-1.59 (m, 3H), 1.68-2.36 (m, 4H), 2.39-2.57 (m, 0.6H), 2.57-2.69 (m, 0 , 35H), 2.71-2.87 (m, 20 0.4H), 3.05 (dd, 0.65H), 3.39-3.93 (m, 3H), 4.24-4.99 (m, 5H), 5.49-5.55 (m,

0.8H), 5.63-5.77 (m, 1.2H), 7.17-7.46 (m, 5H), 7.49-7.60 (m, 1H), 7.89-7 , 99 (m, 1H), 8.03-8.12 (m, 1H), 8.58-8.67 (m, 1H). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 8.63 min. LC-MS (ES ⁺ ) m / e = 481.3 (M + H).

(35

118

[1- [2- (4-Amino-3,5dichloro-benzoylamino) -propionyl1-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-89-propionyl.

Prepared from 75 and 3,5-dichloro-4-aminobenzoic acid according to the procedure used by 76 to provide the title composition (162 mg, 70% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.21 to 1.58 (m, 3H), 1.56 to 2.37 (m, 4H), 2.37 to 3.13 (m, 2H) , 3.43-3.74 (m, 1.5H), 3.77-3.94 (m, 1H), 4.28-4.51 (m, 1.5H), 4.50-5, 01 (m, 3H), 5.41 - 5.77 (m, 1H), 7.15-7.49 (m, 5H), 7.66-7.88 (m, 2H). Analytical HPLC (cyan column) (mixture of 2 10 diastereomers) 8.361 min. LC-MS (ES ⁺ ) m / e = 563.2 (M + H).

1-1,2- (4-Methoxybenzoylamino) -propionyl) -pyridine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (90).

Prepared from 75 and 4-methoxy-benzoyl chloride according to the procedure used by 76 to provide the title compound (404 mg, 50%). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.19 (d, O, 3H), 1.29 to 1.58 (m, 2.7H), 1.58 to 2.38 (m, 4H) , 2.43-2.69 (m, 1H), 2.74-2.66 (m, 0.6H), 2.99-3.11 (m, 0.4H), 3.39-3, 75 (m, 1.5H), 3.77-3-94 (m, 2H), 3.84 (s, 3, H), 4.29-4.94 (m, 4.5H), 5, 45-5.55 (m, 4.5H), 5.63-5.71 (m, 0.5H), 5.73 (d, 0.1 H), 6.85-7.09 20 (m , 2H), 7.19-7.44 (m, 4H), 7.73-7.92 (m, 2H), 8.26-8.44 (m, 1H). Analytical HPLC (cyan column) (mixture of 2 diastereomers). LC-MS (ES ⁺ ) m / e = 510.2

1- {2 - [(9-oxo-9H | ({0-acid-2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -amide ({0

119 fluorene-4-carbonyl) -aminol-propionyl) pyrrolidine-2-carboxylic (91).

Prepared from 75 and 9-oxo-9H-fluorene-carboxylic acid according to the procedure used for 76 to provide the title compound (403 mg, 44% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.38-1.59 (m, 3H), 2.75-2.37 (71, 4H), 2.43-2.59 (m, 0.659), 2.59-2.72 (m, 0.35H), 2.792.89 (m, 0.35H), 3.01-3.11 (m, 0.65H), 3.68-3.86 (m, 1H), 3.92-4.09 (m, 2H), 4.35-5.03 (m, 7H), 5.42-5.90 (m, 1H), 7.06-8.00 ( m, 12H). Analytical HPLC (cyan column) (mixture of 2 diastereomers) 12.30 min. LC-MS (ES ⁺ ) m / e = 582.1 (M + H).

1- [2- (3,5 ^ ίοΙθΓθ-4methoxy-benzoylamino) -propionill pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-92-carboxylic acid (92).

Prepared from 75 and 3,5-dichloro-4-methoxy-benzoic acid according to the procedure used by 76 to provide the title compound (394 mg, 46% yield), ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.17 (d, 0.25H), 1.28-1.53 (m, 2.75H), 1.64-2.33 (m, 4H), 2.39-2.94 (m, 1.5H), 2,943.12 (m, O, 5H), 3.41-3.74 (m, 2H), 3.74-4.00 (m, 1H), 3.91 (s , 3H), 4.26-5.02 (m, 5H), 5.42-5.82 (m, 1H), 7.08 (d, 0.4H), (m, 0.5H), 7 , 21-7.43 (m, 4.6H), 7.53-7.69 (m, 0.8H), 7.85-7.97 (m, 1.2H). Analytical HPLC (coia column 20 no) (mixture of 2 diastereomers) 10.79 min. LC-MS (ES ⁺ ) m / e = 578.2 (M + H).

> Quinoline-6-carboxylic acid {2- (2-benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbomoyl) -pyrrolidin-1-ill-1-methyl-2oxo-ethyl} -amide (93).

Prepared from 75 and 6-quinolinecarboxylic acid (4ί

120 with the procedure used for 76 to provide the title compound (344 mg, 71% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.11-1.58 (m,

3H), 1.69-2.40 (m, 4H), 2.42-3.15 (m, 2H), 3.80-4.01 (m, 1H), 4.29-4.99 ( m,

5H), 5.44-5.54 (m, 0.1H), 5.63-5.73 (d, 0.4H), 5.73-5.79 (d, 0.1H), 8, 13-8.25 (m, 1H), 8.40-8.56 (m, 2H), 8.88-8.99 (m, 1H).

Analytical HPLC (cyan column) (mixture of 2 diastereomers) 10.27, 10.50 min. LC-MS (ES ⁺ ) m / e = 531.2 (M + H).

Scheme XVII “vM? — CO2H ⁰ COsBu-t

M «6

I

1- (2-Benzyloxycarbonylamino-propionyl) pyrrolidine-2-carboxylic acid tert-butyl ester (95),

Prepared according to the method described in Pierre Chevallet, Patrick Garrouste, Barbara Malawaska & Jean Martinez in Tetrahedron

Letters, Vol. 34, p. 7409-7412 (1993). The mixture of Cbz-ala-pro-OH (10.0 g, 31.2 mmoles), tert-butyl bromide (180 g, 1.31 moles), benzyltriethylammonium chloride (7.11 g, 31.2 mmoles ) and K ₂ CO ₃ (180 g, 1.30 moles) in N, Ndimethylacetamide (DMA) (225 ml) was stirred at 55 ° C for 24 hours. The reaction mixture was cooled to room temperature and was diluted with one liter

121 of ice water, was extracted with ethyl acetate (200 ml x 3). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated in vacuo to give 14 g of oil, which was purified by flash chromatography using hexane / ethyl acetate (95/5 to 50/50) to provide 11.73 g (yield of

99.7%) of the title compound as a chlorine oil. ¹ H NMR (500 MHz, CDCl ₃ ); δ 1.25-1.50 (m, 12H), 1.85-2.25 (m, 4H), 3.42-3-70 (m, 2H), 4.25-4.57 (m, 2H), 5.07-5.11 (m, 2H), 5.69 (d, H), 7.28-7.38 (m, 5H); retention time on analytical HPLC; 11.07 min; LC-MS: m / z = 377 (M + H ⁺ ).

eea, X = CI, Z-H Mt>, X = Cf, Y “AcNH. Ζ · Η

Mc, X-CI. Y-AcNH, ZKXaO

W ·. X «CI, Y-NHj, Z« = H Wb, X-CI, Y «AcNH, Z« H • Tc, X-CI, Y * AcNH, Z <H, 0

1-r2- (4-amino-3-chloro-benzoylamino) propionin pyrrolidine-2-carboxylic acid tert-butyl ester

To a solution of 95 (10.50 g, 21.9 mmol) in MeOH (100 ml) was added a suspension of 10% Pd / C (5.00 g) in EtOAc (50 ml). The mixture was stirred under H ₂ for 48 hours, filtered through celite and the solvent was evaporated to provide a waxy solid. This was dissolved in CH ₂ CI ₂ (100 ml) and DMF (50 ml) and the solution was cooled to 0 ° C. 4 amino-3-chlorobenzoic acid (5.82 g, 27.2 mmoles). DIEA (14.58 ml, 93.7 mmoles), HOBT (0.77 g, 27.9 mmoles) and EDC (6.68 g, 34.8 mmoles) were added and the solution was stirred at 0 ° C for Then 15 mins at room temperature for 24 hours. The reaction mixture was diluted with MAO, washed with NaHS (O) ₄ (2x), 10% NaHCO ₃ (2x) and brine then dried over MgSO ₄ , filtered and evaporated. The crude product was purified by flash column chromatography, using CH ₂ CI ₂ / MeOH (99/1 to 97/3%) to provide the title compound as a white solid (7.75 g, 70% yield 25) ment). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.27-1.67 (m, 12H), 1.82-2.3.4 (m, 4H), 3.48-3.85 (m, 2H), 4.26-4.53 (m, 3H), 4.81 - 4.98 (m, 3H), 6.71 (d, 1H), 7.15 (m, 1H), 7.50 (dd, 1H), 7.75 (d, 1H). Analytical HPLC 10.83 min. LC-EM

Ι * 0

122 (ES ⁺ ) m / e = 396.3 (ΜΗ ⁺ ).

___________ 242- (4-amino-3-chloro-benzoylamino) propionyl1-1-pyrrolidine-2-carboxylic acid (97a).

Prepared from 96a by treatment with TFA / CH ₂ CI ₂ . After the complete reaction, the solvent is removed in vacuo and the residue is repeatedly concentrated from toluene. The resulting residue was dried in vacuo to a constant weight.

1-F2- (4-acetylamino-3-chloro-benzoylamino) propioninpyrrolidine-2-carboxylic acid tert-butyl ester (96b).

Prepared from 95 and 4-acetylamino-3-chlorobenzoic acid according to the method used for 96a to provide the title compound as a white solid (9.18 g, 77% yield). ¹ H NMR (100 MHz,

CD ₃ 0D) δ 1.30-1.62 (m, 12H), 1.85-2.26 (m, 3H), 2.16-2.44 (m, 1H), 2.27 (s,

3H), 3.47-3.83 (m, 2H), 4.34-4.64 (m, 1H), 4.89 (m, 1H), 7.27-7.3.9 (m, 1H),

7.59-7.71 (m, 2H), 7.83-7-97 (m, 1H), 8.47 (d, 1H), Analytical HPLC 9.43 min.

_______ 1-r2- (4-Acetylamino-3-chloro-benzoylamino) -Propioninpyrrolidine-2-carboxylic acid (97b).

Prepared from 96b by treatment with TFA / CH ₂ CI ₂ . After the complete reaction, the solvent is removed in vacuo and the residue is repeatedly concentrated from toluene. The resulting residue was dried

under vacuum to a constant weight.

4-Acetylamino-5-chloro-2-methoxy-benzoic acid.

4-Acetylamino-5-chloro-2-methoxy-benzoic acid methyl ester (2.09 g, 8.11 mmoles) was dissolved in MeOH (110 ml) and LiOH solution (25.48 mmoles in 30 ml, 1: 1 MeOH: H ₂ O) was added and the solution was stirred at room temperature for 6 hours. The solvent was concentrated in vacuo, EtOAc was added and the organic phase was washed with 0.5 N HCI then it was extracted with saturated NaHCO ₃ (2x). The aqueous phase was acidified with 12 N HCI to pH 1 and the resulting precipitate was extracted into CH ₂ CI ₂ . The combined extracts were dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to give the title compound as a white solid (0.933 g, 50% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ

123

2.31 (s, 3Η), 4.10 (s, 3H), 7.78-7.92 (br s, 1H), 8.17 (s, 1H), 8.45 (s, 1H). Analytical HPLC 5.62 min.

1- [2- (4-Acetylamino-5-chloro-2-methoxybenzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester (96c).

To a solution of 95 (1.534 g, 4.07 mmoles) in MeOH (40 ml) was added 10% Pd / C (650 mg) and the mixture was stirred under H ₂ for 2 hours. The suspension was filtered through celite and was evaporated to give a yellow oil. This was allowed to react with 4-acetyl-5-chloro-2-methoxy acid

benzoic following the procedure used for the preparation of 96a to give the title compound (497 mg, 52% yield), ¹ H NMR (500 MHz, CD ₃ OD) δ 1.46 (d, 39), 1.49 (s, 9H), 1.80-2.01 (m, 3H), 2.19-2.40 (m, 1H), 2.22 (s, 3H), 3.58-3.72 (m , 1H), 3.79-3.89 (m, 1H), 3.99-4.09 (s, 3H), 4.314.45 (s, 1H), 4.78-4.95 (m, 1H ), 7.89-8.10 (m, 2H). HPLC 11.31 min.

Acid 1 - [2- (4-acetylamino-5-chloro-2-methoxy-benzoylamino) propionill-pyrolidine

2-carboxylic (97c).

Prepared from 96c by treatment with TFA / CH ₂ CI ₂ . After the complete reaction, the solvent is removed in vacuo and the residue was suddenly concentrated from toluene. The resulting residue was dried under vacuum to a constant weight

1-1,2- (4-Amino-3-chlorobenzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (5-oxo-2-phenethyloxy-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98a).

To a solution of allyl ester of (5-oxo-2-phenethyloxytetrahydrofuran-3-yl) -carbamic acid (194 mg, 0.54 mmol) (prepared as described for (40) using phenethyl alcohol) in CH ₂ anhydrous CI ₂ (5 ml) at 0 ° C DMBA (196 mg, 1.26 mmol) and Pd (PPh ₃ ) ₄ (32 mg, 0.03 mmol) were added. The solution was stirred for 15 min and a solution of 97a (prepared from 96a by treatment with TFA in CH ₂ CI ₂ ) (166 mg, 0.49 mmol) and DIEA (680 μΙ, 3.90 mmol) in CH ₂ CI ₂ (2 ml) was added followed by HOBT (98

124 mg, 0.73 mmol) and EDC (122 mg, 0.63 mmol). The solution was stirred at 0 ° C for 15 min then at room temperature for 18 hours. The solvent was removed in vacuo and the residue was dissolved in EtOAc then washed with 0.5 N NaHSO ₄ (2x), saturated NaHCO ₃ (2x) and brine. Dried over Na ₂ SO ₄ andiro and evaporated to give an orange solid which was purified by flash column chromatography, using CH ₂ CI ₂ / MeOH (99/1 to 97/3%) to provide the title compound as a white solid (190 mg, 73% yield). ¹ H NMR (500 MHz, CD ₃ OD), δ 1.29 (d, 0.6H), 1.41 (d, 2.4H), 1.78 (m, 1H), 2.08 (m, 3H), 2.56 (m, 1H), 2.77 (dd, 1H), 2.94 (t, 2H), 3.53 (m, 0.3H), 3.67 (m,

0.8H), 3.85 (m, 2H), 3.96-4.08 (m, 1H), 4.40 (m, 2H), 4.62 (m, 1H), 4.67-4 , 79 (m, 1H), 5.57 (d, 0.7H), 5.60 (d, 0.3H), 6.78 (dd, 1H), 7.21 (m, 5H), 7, 58 (m, 1H), 7.79 (m, 1H), 8.26 (d, 1H). Analytical HPLC 14.52 min. LC-MS (ES ⁺ ) m / e = 543.2 (MH ⁺ ).

1- [2- (4-Amino-3-chlorobenzoylamino-propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-98-carboxylic acid (98b).

It was prepared from the syn diastereomer of allyl ester of (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid (40) and 97a after the method used for 98a. The title compound was isolated as a pale yellow solid 20 (720 mg, 51% yield) ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.16 (d, 0.5H), 1.40 (d , 2.59), 1.64-2.25 (m, 4H), 2.61 (dd, 1H), 2.79 (dd, 1H), 3.37-3.59 (m, 1H), 3.59-3.74 (m, 1H), 3.77-3.92 (m, 1H), 4.29-4.47 (m, 1H), 4.47-5.02 (m, 4H ), 5.48 (s, 0.5H), 5.66 (d, 1H), 5.68 (d, 0.5H), 6.79 (d, 1H), 7.11-7.52 ( m, 5H), 7.48-7.62 (m, 1H), 7.68-7.83 (m, 1H). Analytical HPLC 25 15.98 min. LC-MS (BS +) m / e = 529.2 (MH ⁺ ) ·

125

1- [2- (4-Amino-3-chlorobenzoylamino) -propionill pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98c).

Prepared from the anti- (2-benzyloxy-5oxo-tetrahydrofuran-3-yl) -carbamic acid allyl ester (40) and 97a after the method used for 98a. The title compound was isolated as a white solid (186.6 mg, 46% yield).

¹ H NMR (500 MHz, CD ₃ 0D) δ 1.30-1.52 (m, 3H), 1.76-2.33 (m, 4H), 2,412.59 (m, 1H), 2.90 (dd, 0.15H), 3.04 (dd, 0.85H), 3.44-3.75 (m, 1.5H), 3.823.95 (m, 1H), 4.27-4.42 (m , 2H), 4.42-4.56 (m, 0.5H), 4.56-4.86 (m, 4H), 5.42-5.56 (m, 1H), 6.79 (d , 1H), 7.21-7.42 (m, 4.6H), 7.54-7.63 (m, 1.4H), 7.76-7-83 (m, 0.65H), 8 , 60-8.68 (m, 0.35H). Analytical HPLC 15.19 min. LCEM (ES ⁺ ) m / e = 529.3 (MH ⁺ ).

2- (ethoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester.

Prepared from 2-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for (40) using ethanol. Chromatography using hexane / ethyl acetate (95/5 to 80/20) gave 0.94 g of anti-2- (ethoxy-5-oxo-tetrahydro-furan-3-yl) carbonyl allyl ester ( Upper Rf), 1.96 g of syn diastereomer (lower Rf) and 8.08 g of the mixture of diatereomers (total global yield 60%). ¹ H NMR (500 MHz, CDCI ₃ ) per antidiastereomer. δ 1.13-1.31 (m, 3H), 2.31-2.45 (m, 1H), 2.92-1.08 (m, 1H), 3.52-3.72 (m, 1H), 3.78-3.92 (m, 1H), 4.10-4.25 (m, 1H), 4.45-4.70 (m, 2H), 5.00 (bs, 1H) , 5.12-5.45 (m, 3H), 5.80-5.95 (m, 1H); for syn diastereomer 1.23-1.35 (m, 3H), 2.38-2.50 (m, 1H), 2.75-2.92 (m,

126

1Η), 3.60-3.73 (m, 1H), 3.82-3.95 (m, 2H), 4.40-4.70 (m, 3H), 5.20-5.52 ( m, 4H), 5.80-3.94 (m, 1H); LC-MS: m / z = 230 (M + H ⁺ ) for both diastereomers.

1- [2- (4-Amino-3-chloro-5-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -98-carboxylic acid (98d).

Prepared from allyl ester of (2-ethoxy-5-oxotetrahydro-furan-3-yl) carbamic acid and 97a after the method used for 98a. The title compound was isolated as a white solid (175 mg, 77% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.13 (t, 0.5H) 1.23 (7, 2.5H), 1.36 (d, 10 0.5H), 1.44 (d , 2.5H), 1.75-2.38 (m, 4H), 2.56 (dd, 1H), 2.76 (dd, 1H), 3,453.97 (m, 5H), 4.47 ( dd, 1H), 4.59-4.67 (m, 1H), 4.74 (q, 1H) 5.55 (d, 0.2H),

5.56 (d, 0.8H), 6.75-6.82 (m, 1H), 7.56 (dd, 1H), 7.77 (d, 1J), 8.39 (d, 1H) .

Analytical HPLC 8.17 min. LC-MS (ES ⁺ ) m / e = 467.4 (MH ⁺ ) ·

Allyl ester of (2-cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using cyclopentanol to provide the title compound as a mixture of diastereomers. Flash column chromatography using hexanes / EtOAc 90/10 a

80/20) provided the syn diastereomer of the title compound: syn diastereomer ¹ H NMR (500 MHz, CDCl3) δ 1.5-2.0 (m, 8H), 2.45 (dd, 1H), 2.81 (dd, O, 9H), 3.0 (dd, 0.1 H), 4.32 (m, 1H), 4.59 (m, 4H), 5.23 (m, 1H), 5.32 (m, 1H), 5.45 (s, 0.1H), 5.51 (s, 0.9H), 5.92, (m, 1H) ppm; antidiastereomer ¹ H NMR (500 MHz, CDCl 3) δ 1.50 (m, 2H), 1.67 (m, 6H), 2.36 (d, 1H), 2.8 (dd, 0.08H),

2.96 (dd, 0.92H), 4.13 (m, 1H), 4.25 (m, 1H), 4.55 (br, 2H), 5.20 (d, 1H), 5.30 (m, 2H), 5.43 (s, 0.92H), 5.5 (d, 0.08H), 5.89 (s, 1H) ppm.

1- [2- (4-amino-3-chloro-benzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (2-cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98e).

Prepared from allyl ester of (2-cyclopentyloxy-5127 oxo-tetrahydro-furan-3-yl) -carbamic acid and 97a after the method used for 98a to give the title compound (280 mg, 51% yield) , ¹ H NMR (500 MH,

CD ₃ 0D) δ 1.38 (d, 0.5H), 1.44 (d, 2.5H), 1.49-2.35 (m, 12H), 2.47 (dd, 0.7H) ,

2.66 (dd, 0.3H), 2.75 (dd, 0.3H), 2.81-2.98 (m, 0.1 H), 2.97 (dd, 0.6H), 3.475 3.76 (m, 0.2H), 3.82-3.96 (m, 1H), 4.10-4.40 (m, 2H), 4.40-4.46 (m, 1H), 5.44 (d, 0.5H), 5.50 (d, 0.2H), 5.65 (d, 0.3H), 6.79 (d, 1H), 7.54-7.64 ( m, 1H), 7.78 (d, 1H), 8.21-8.31 (m, 1H). Analytical HPLC 15.02, 15.34 min. LC-EM (ES ⁺ )

2-Cyclohexyl-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester.

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using cyclohexanol to provide the title compound as a mixture of diastereomers (pale yellow oil) (4.62 g, 85% income). Flash column chromatography using hexanes / EtOAC (90/10 to 80/20) gave 394 mg (74% yield) of the diastereomer syn of the title compound. ¹ H NMR (500 MHz, CDCh) δ 1.11-2.09 (m, 10H), 2.35-2.91 (dd, 1H), 2.72-2.98 (dd, 1H), 3 , 60-3.63 (m, 1H), 4.32-4.72 (m, 3H), 5.06-5.43 (m, 2H), 5.60 (d, 1H), 5.826.03 (m, 1H).

1-F2- (4-Acetylamino3-chloro-benzoylamino) -propionylpyrrolidine-2-carboxylic acid (2-cyclohexyloxy-5-oxo-tetrahydro-furan-3-yl) -98-propionylpyrrolidine-98-carboxylic acid.

Prepared from syn- (2-cyclohexyloxy-5oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester and 97b after the method used for 98a to give the title compound (121 mg, 33% yield ). ¹ H NMR (500 25 MHz, CD3OD) δ 1.06-1.61 (m, 9H), 1.61-2.37 (m, 7H), 2.22 (s, 3H), 2.52- 2.81 (m, 2H), 3.49-3.78 (m, 2H), 3.84-3.97 (m, 1H), 4.42-4.57 (m, 1H), 4, 57-4.69 (m, 1H), 5.67-5.81 (m, 1H), 7.72-7.89 (m, 1H), 7.89-8.12 (m, 2H). Analytical HPLC 9.84 min. LC-MS (ES ⁺ ) m / e = 563.3 (MH ⁺ ).

t F 4

128

1- [2- (4-amino-3-) acid (2-cyclohexyloxy-5-oxo-tetrahydro-furan-3-yl) -amide

chloro-benzoylamino) -propionylalpyrrolidine-2-carboxylic (98q).

Prepared from syn- (2-cyclohexyloxy-55 oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester and 97a after the method used for 98a to give the title compound (153 mg, 47% Yield). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.06-2.38 (m, 14H), 1.42 (d, 3H), 2.50-2.66 (m, 1H), 2,692.82 (dd, 1H), 3.06-3.75 (m, 2H, 3.80-3.94 (m, 1H, 4.40-4.52 (m, 1H), 4,574.65 (m, 1H ), 4.70-4.80 (m, 1H), 4.72 (d, 1H), 6.71 (m, 1H), 7.50-7.63 (m,

1H), 7.78 (d, 0.6H), 8.42 (d, 0.4H). Analytical HPLC 10.30 min. LC-MS (ES ⁺ ) m / e = 521.2 (MH ⁺ ).

1-1,2- (4-amino-3-chlorobenzoylamino) -propionyl} -pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) (98h)

Prepared from carbamic acid (2-ethoxy-5-oxotatrahydro-furan-1-yl) allyl ester and 97a after the method used for 98a. The title compound was isolated as a white solid (195 mg, 82% yield). ¹ H NMR (50 ° MHz, CD ₃ 0D) δ 1.32-1.55 (m, 3H), 1.58-1.77 (m, 3H), 1.96-2.54 (m, 4H ), 2.68-2.76 (d, 0.3H), 2.79-2.89 (m, 0.7H), 2.96-3.10 (m,

0.7H), 3.18-3.27 (dd, 0.3H), 3.72-4.18 (m, 4H), 4.46-5.12 (m, 3H), 5.60 ( s,

0.4H), 5.74-5.84 (m, 0.6H), 7.03 (d, 0.8H), 7.75-7.86 (m, 1H), 8.01 (d, 0.7H),

8.35 (d, 0.3H), 8.74 (d, 0.2H). Analytical HPLC 8.31 min. LC-MS (ES ⁺ ) m / e = 467.3 (MH ⁺ ).

129

Allyl ester of [5-oxo-2- (tricycle [3.3.1.1 ^0.0 ldec-2-yloxy) -tetrahydrofuran-3ill-carbamic acid.

Prepared from 3- tert-butyl ester

allyloxycarbonylamino-4-hydroxy-butyric as described for 40 using 2adamantanol (6.21 g, 5 equivalents) to provide the title compound as a pale yellow oil (1.52 g, 61% yield). ¹ H NMR (500 MHz,

CDCIs) δ 1.38-2.22 (m, 14H), 2.40 (d, 0.2H), 2.53 (dd, 0.7H), 2.87 (dd, 0.7H), 2 , 87 (dd, 0.8H), 3.00-3.12 (m, 0.3H), 8.84-3.97 (m, 1H), 4.40- 4.71 (m, 3H) , 10 5.18-5.44 (m, 2H), 5.53-5.69 (m, 1H), 5.82-6.02 (m, 1H).

[5-oxo-2- (tricycle [3.3.1.1 ⁰ ' ⁰ ] dec-2-yloxy) -tetrahydro-furan-3-yl] amide 1 [2- (4-amino-3-chloro-benzoylamino) amide) -propionill-pyrrolidine-2-carboxylic (98i),

Prepared from [5-oxo-2- (tricycle [3.3.1.1 ^0.0 ] dec-2-yloxy) -tetrahydro-furan-3-yl] -carbamic acid allyl ester and 97a after the method

used for 98a. The title compound was isolated as a white solid (76 mg, 13% yield): ¹ H NMR (500 MHz, CD ₃ CD) δ 1.38-2.22 (m, 14H), 2.40 (d , 0.2H), 2.53 (dd, 0.7H), 2.87 (dd, 0.8H), 3.00-3.12 (m, 0.3H), 3,843.97 (m, 1H ), 4.40-4.71 (m, 3H), 5.18-5.44 (m, 2H), 5.53-5.69 (m, 1H), 5.826.02 (m, 1H). Analytical HPLC 11.89 min. LC-MS (ES ⁺ ) m / e = 573.2 (MH ⁺ ).

1- [2- (4-acetylamino-5-chloro-2-methoxy-benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-98-carboxylic acid (98j) ,

Prepared from syn- {2- [2- (2-benzyloxy-5-oxo-tetrahydrofuran-3-ylcarbamoyl) -pyrrolidin-1-yl] -1-methyl-2-oxo-ethyl} tert-butyl ester} 130 carbamic and 97c after the procedure used for 98a to provide the title compound (222 mg, 82% yield). ¹ H NMR (500 MHz, CD ₃ OD) δ

1.23 (d, 0.6H), 1.42 (d, 2.4H), 1.72-2.27 (m, 4H), 2.23 (s, 3H), 2.63 (dd, 1H ),

2.77-2.88 (m, 1H), 3.43-3.52 (m, 0.5H), 3.56-3.71 (m, 1.5H), 3.74-3.85 (m,

1H), 3.98 (s, 3H), 4.38-4.50 (m, 1.5H), 4.51-4.92 (m, 4.5H), 5.63-5.76 ( m,

1H), 7.23-7.40 (m, 5H), 7.97 (s, 1H), 8.45 (d, 1H), 8.69-8.80 (m, 1H) analytical HPLC 11, 63 min LC-MS (ES ⁺ ) m / e = 601.2 (MH ⁺ ).

Synthesis of 1- [2- (4-amino10 3-chloro-benzoylamino) -propionill-pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-98k.

Prepared from anti- (2-ethoxy-5-oxotetrahydro-furan-3-yl) -carbamic acid allyl ester and 97a after the method used for 98a to provide 175 mg of the title compound (59%). ¹ H 1: 1 NMR (500 MHz, CDCI ₃ : CD ₃ OD) δ 1.10-1.28 (m, 3H), 1.42 (d, 0.6H), 1.46 (d, 2, 4H), 1.75-2.45 (m, 15 4H), 2.45-2.70 (m, 1H), 2.80-3.05 (m, 1H), 3.50-3.95 (m, 4H), 4.20-4.75 (m,

3H), 4.75-4.90 (m, 1H), 5.32 (s, 0.8H), 5.38 (s, 0.2H), 6.80 (d, 1H), 7.55 -7.84 (m, 2H). Analytical HPLC I 0.47 min. LC-MS (ES ⁺ ): m / e = 467.3 (M + H ⁺ ).

Synthesis of 1- [2- (4-amino20 3,5-dichloro-benzoylamino) propionill-Pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-bore-3-yl) -98-carboxylic acid (98I)

Prepared from (2-ethoxy-5-oxotetrahydro-furan-3-yl) -carbamic acid allyl and 1- [2- (4-amino3,5-dichlorobenzoylamino) -propionyl acid tert-butyl ester ] -pyrrolidine-2-carboxylic according to the method used for 98a to provide 158 mg of the title compound (54% yield 25) ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1, 08-1.30 (m, 3H), 1.32-1.52 (m, 3H), 1.72-2.44 (m, 4H), 2.40-3.05 (m, 2, H ), 3.50-3.97 (m, 4H),

131

4.25-4.70 (m, 3H), 4.70-4.86 (m, 1H), 5.33 (s, 0.4H), 5.47 (s, 0.1H) 5.56 (d,

0.4H), 5.62 (d, 0.1H), 7.50 (s, 1H), 7.80 (s, 1H). Analytical HPLC: 10.84 min.

LC-EM (ES ⁺ ); m / e 501.2 (M + M +).

1-1,2- (4-amino-3-chlorobenzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionill-2-carboxylic acid (98m).

Prepared according to the procedure used to prepare 98a using Cbz-Ala-D-pro-0H to provide 230 mg of the title compound (69% yield). 1H-NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.30 (d, 10 1.2H), 1.45 (d, 1.85M), 1.62-2.40 (m , 4H), 2.40-3.10 (m, 2H), 3.30-3.97 (m,

2H), 4.33-4.95 (m, 5H), 5.30 (s, 0.5H), 5.68 (d, 0.5H), 6.80 (d, 1H), 7.25 -7.95 (m, 7H). Analytical HPLC: 11.56, 11.91 min. LC-MS (ES ⁺ ): m / e = 529.2 (M + H ⁺ ).

1- | ~ 2- (4-acetylamino-3-chloro-benzoylamino) -propionin pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionin-2-carboxylic acid (98n).

Prepared from 97b and syn- (2-benzyloxy-5-oxotetrahydro-furan-3-yl) -carbamic acid ally according to the procedure used to prepare 98a to provide 210 mg of the title compound (64% yield 20 to). ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.33 (d, 0.6H), 1.44 (d, 2.4H), 1.68-2.40 (m, 4H), 2.26 (s, 3H), 2.55-3.05 (m, 2H), 3.40-3.90 (m, 2H), 4.204.95 (m, 5H), 5.68 (d, 0.8H), 5.84 (d, 0.2H), 7.15-8.30 (m, 8H), analytical HPLC; 15.67 min, LC-MS (ES ⁺ ): m / e = 571.1 (M + H ⁺ ).

132

Allyl ester of (2-isopropoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid Prepared as described for compound 40 using isopropanol to provide 3.80 grams (81% yield) of the compound title 5 as a colorless oil. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.10-1.35 (m, 6H), 2.322.60 (m, 1H), 2.82 (dd, 0.5H), 3.02 (dd, 0.5H), 3.82-4.11 (m, 1H), 4.48-4.66 (m, 3H), 5.20-5.36 (m, 2H), 5.54 (dd, 1H), 5.82-6.05 (m, 1H). LC-MS (ES ⁺ ) m / e = 244.2 (M + H).

1- [2- (4-Amino-310-chlorobenzoylamino) -propionill pyrrolidine-2-carboxylic acid (2-isopropoxy-5-oxo-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98o).

Prepared from 97a and 2-isopropoxy5-oxo-tetrahydro-furan-3-yl-carbamic acid allyl ester according to the procedure used to prepare 98a to provide 200 mg of the title compound (66% yield) , ^1H NMR (500 MHz 1: 1 CDCl _3: CD ₃ 0D) δ 1.05-1.35 (m, 6H), 1.3515 1.50 (m, 3H), 1.70 to 2.45 (m, 4H), 2.45-3.05 (m, 2H), 3.55-4.10 (m, 1H), 4.154.88 (m, 4H), 5.48 (s, 0.4H ), 5.58 (s, 0.1 H), 5.64 (d, 0.4H), 5.70 (d, 0.1 H), 6.78 (d, 1H), 7.58 ( d, 1H), 7.80 (s, 1H) Analytical HPLC: 12.19; 12.40 min. LC-MS (ES ⁺ ): m / e = 581.2 (M + H ⁺ ).

1-1,2- (4-acetylamino3,5-dichloro-benzoylamino) propionill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tatrahydro-furan-3-yl) -propionyl-pyrrolidine-2-carboxylic acid (98p).

Prepared from 1- [2- (4acetylamino-3,5-dichloro-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and syn- (2-benzyloxy-5) allyl ester -oxo-tetrahydrofuran-3-yl) -carbamic 25 according to the procedure used to prepare 98a to provide 230 mg of the title compound (72% yield). ¹ H NMR (500 MHz, 1: 1 CD-

133

Cl ₃ : CD ₃ 0D) δ 1.36 (d, 0.6H), 1.47 (d, 2.4H), 1.68-2.47 (m, 4H), 2.23 (s, 3H ),

2.60-3.15 (m, 2H), 3.40-3.90 (m, 2H), 4.15-4.95 (m, 5H), 5.68 (d, 0.8H), 5.84 (d, 0.2H), 7.20-7.98 (m, 7H). Analytical HPLC: 13.07 min. LC-MS (ES ⁺ ): m / e =

605.1 (M + H ⁺ ).

1- (2- (4-Acetylamino-3-chloro-benzoylamino) -propionill pyrrolidine-2-carboxylic acid (2-cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl) -propionill pyrrolidine-2-carboxylic (98q),

Prepared from 97b and allyl ester of (2cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl] carbamic acid according to the procedure used to prepare 98a to provide 215 mg of the title compound (69% yield ) ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ OD) δ 1.35-1.90 (m, 11H), 1.90-2.35 (m, 4H), 2.24 (s , 3H), 2.40-3.10 (m, 2H), 3.50-3.95 (m, 3H), 4.15-4.90 (m, 3H), 5.44 (s, 0 , 5H), 5.56 (s, 0.15H), 5.64 (d, 0.22H), 5.71 (d, 0.08H), 7.70-8.25 (m, 3H). : 12.13 min LC-MS (ES ⁺ ): m / e =

549.2 (M + H ⁺ ).

1- (2- (4-Acetylamino-3-chloro-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -amide (98r),

Prepared from 97b and syn- (2-ethoxy20 5-oxo-tetrahydrofuran-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 68 mg of the title compound (24%) . ¹ H

NMR (500 MHz, 1: 1 CDCI ₃ : CD3OD) δ 1.13 (t, 0.6H), 1.28 (t, 2.4H), 1.38 (d,

0.6H) 1.48 (d, 2.4H), 1.75-2.40 (m, 4H), 2.22 (s, 3H), 2.55-2.88 (m, 2H), 3,503.92 (m, 4H), 4.40-4.90 (m, 3H), 5.57 (d, 0.8H), 5.61 (d, 0.2H), 7.60-8, 20 (m,

3H). Analytical HPLC: 8.64 min. LC-MS (ES ⁺ ): m / e = 509.2 (M + H ⁺ ).

134

Preparation of (2-cyclopentylmethoxy-5-oxo-tetrahydrofuran-3-yl) -carbamic acid allyl ester.

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric tert-butyl ester as described for compound 40 using cyclopentylmethanol (6.5 mL, 60 mmol) to provide 2.98 grams (52% total yield) of the title compound as a mixture of epimers. Purification provided 0.97 grams (17% yield) of 4 (S), 5 (R) as a colorless oil. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.19 (m, 2H), 1.54 (m, 4H), 1.71 (m, 2H), 2.16 (m, 1H), 2.44 (dd, J = 17.2, 10.4 Hz, 1H), 2.82 (dd, J = 17.2, 8.4 Hz, 1H), 3.44 (dd, J = 9.3, 7 , 2 Hz, 1H), 3.71 (dd, J = 9.3, 7.2 Hz, 1H), 4.57 (m, 3H), 5.32 (m, 3H), 5.41 (d , J = 5.2 Hz, 1H), 5.91 (ddt, J = 17.1 10.4, 5 Hz, 1H) ppm, LC-MS, (ES ⁺ ): m / e = 284.

Also isolated was the epimer mixture (0.66 grams, 11% yield) and epimer 4 (S), 5 (S) (1.35 grams, 24% yield) as a waxy solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.20 (m, 2H), 1.54 (m, 4H), 1.69 (m, 2H), 2.10 (m, 1H), 2.37 (d, J = 8.1 Hz, 1H), 2.97 (dd, J = 18.0, 7.6 Hz, 1H), 3.42 (dd, J = 7.3, 1.7 Hz, 1H ), 3.49 (m, 2H), 3.64 (dd, J = 9.0, 7.33 Hz, 1H), 4.19 (br, 1H), 4.55 (m, 2H), 5 , 25 (m, 2H), 5.36 (s, 1H), 5.87 (m, 1H) ppm, LC-MS (ES ⁺ ): m / e = 284 (M + H).

1- [2- (4-Amino-3-chloro-benzoylamino) -propionyl1-3-pyrrolidine-2-carboxylic acid (2-cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl) (98s).

Prepared from 97a and syn- (2cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 195 mg of the title compound (51 % yield) ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1,151.90 (m, 11H), 1.90-2.40 (m, 5H), 2.55-2, 78 (m, 2H), 3.50-3.90 (m, 4H), 4,384.92 (m, 4H), 5.53 (d, 0.8H), 5.57 (d, 0.2H) , 6.78 (d, 1H), 7.50-8.15 (m, 2H). Analytical HPLC; 10.48 min. LC-MS (ES ⁺ ): m / e = 521.2 (M + H ⁺ )

135

Allyl ester of (5-oxo-2- (3-phenyl-propoxy) -tetrahydro-furan-3-yl) carbamic acid.

Prepared from 35 allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for compound 40 using 3-phenylpropanol to provide 1.15 grams (32% yield) of the title compound as an oil colorless. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.622-05 (m, 2H), 2.38 (dd, 1H), 2.68 (m, 2H), 2.82 (dd, 1H), 3.55 -3.65 (m, 1H), 3.82-3.92 (m, 1H), 4.48-4.72 (m, 3H), 5.12-5.59 (m, 3H), 5 , 82-6.03 (m, 1H), 10 7.11-7.45 (m, 5H). Analytical HPLC: 9.08 min. LC-MS (ES ⁺ ): m / e = 320.2

M + H ⁺ ).

1- [2- (4-amino3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (5-oxo-2- (3-phenyl-propoxyl) -tetrahydro-furan-3-yl) -amide 98t).

Prepared from 97b and syn- (5-oxo-215 (3-phenyl-propoxyl) -tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 200 mg of the title compound (57% yield). ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.34 (d, 0.6H), 1.44 (d, 2.4H), 1.75-2.40 (m, 6H), 2.50-2.95 (m, 4H), 3.47-3-95 (m, 4H), 4.38-4.82 (m, 3H), 5.52 (d, 0, 8H), 5.56 (d, 0.2H), 6.75-8.25. Analytical HPLC: 10.79 min. LC-MS (ES ⁺ ): m / e = 557.2 (M + H ⁺ ).

Synthesis of 1 [2- (4-acetylamino-3-chloro-benzoylamino) -propionyl1-pyrrolidine-2-carboxylic acid (2-cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl) (98u).

Prepared from 97b and syn- (2cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl] -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 215 mg of the (sT compound)

136 title (67% yield). ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.38 (d,

0.6H), 1.47 (d, 2.4H), 1.11-1.88 (m, 8H), 1.92-2.40 (m, 5H), 2.24 (s, 3H) ,

2.53-2.86 (m, 2H), 3.30-3.90 (m, 4H), 4.38-4.89 (m, 3H), 5.53 (d, 0.8H), 5.60 (d, 0.2H), 7.68-8.22 (m, 3H). Analytical HPLC: 9.90 min. LC-MS (ES ⁺ ): m / e =

563.3 (M + H ⁺ ).

Λ

142- (4-acetylamino-3-chloro-benzoylamino) -propionill-pyrrolidine-2-carboxylic acid (5-oxo-2- (3-phenyl-propoxill-tetrahydro-furan-3-yl) -propionill-pyrrolidine-2-carboxylic (98v),

Prepared from 1- (2- (410 acetylamino-3-chloro-benzoylamino) -piopionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and syn- (5-oxo-2- acid allyl ester (3-phenyl-propoxyl) tetrahydro-furan-1-yl) -carbamic according to the procedure used to prepare 98a to provide 238 mg of the title compound (75% yield), ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.33 (d, 0.6H), 1.56 (d, 2.4H), 1.78-2.45 (m, 6H), 2.27 (s, 3H), 15 2.53-2.97 (m, 4H), 3.53-3.94 (m, 4H), 4.47 ^, 86 (m, 3H), 5.53 (d, 0, 8H), 5.62 (d, 0.2H), 7.11-8.26 (m, 8H) Analytical HPLC: 10.27 min LC-MS (ES ⁺ ): m / e = 599.2 M + H ⁺ ).

1-f2- (4-amino-320 trifluoromethyl-benzoylamino) propionin-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-fruan-3-yl) -propionin-pyrrolidine-2-carboxylic acid (98w).

Prepared from {2- (2- (2- (2-benzyloxy-5-oxo-tetrahydrofuran-3-ylcarbamoyl) -pyrrolidin-1-yl] -methyl-2-oxo-ethyl} carbamic acid tert-butyl ester 4 -amino-3-trifluoromethyl-benzoic according to the procedure used to prepare 98a to provide 56 mg of the title compound 25 (48% yield). ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.20-1.55 (m,

3H) 1.75-2.50 (m, 4H), 2.50-3.10 (m, 2H) 3.50-4.00 (m, 2H), 4.30-5.00 (m, 5H), 5.42 (s, 0.4H), 5.62 (d, 0.3H), 5.78 (d, 0.1H), 6.84 (d, 1H), 7.20-8 , 15 (m,

137

7Η). Analytical HPLC: 14.90, 15.20 min. LC-MS (ES ⁺ ): m / e = 563.2 (M + H ⁺ ).

1-1,2- (3-chloro-4-dimethylamino-benzoyl amido) propionyl] -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) (98x).

Prepared from {2- (2- (2-benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbmoyl) -pyrrolidin-1-yl] -l-methyl-2-oxo-ethyl} tert-butyl ester} carbamic and 3-chloro-4-dimethylamino-benzoic acid according to the procedure used to prepare 98a to provide 82 mg of the title compound (44% yield). ¹ H NMR (500 MHZ, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.18-1.53 (m,

3H), 1.70-2.40 (m, 4H), 2.55-3.10 (m, 2H), 2.84 (s, 6H), 3.45-3.94 (m, 2H) ,

4.25-4.95 (m, 5H), 5.46 (s, 0.3H), 5.51 (s, 0.2H), 5.63 (d, 0.4H), 5.73 ( d, 0.1H), 7.05 (d, 1H), 7.15-7.95 (m, 7H). Analytical HPLC: 11.85, 12.19 min. LCEM (ES ⁺ ): m / e = 557.3 (M + H ⁺ ).

1-f2- (4-dimethylamino-) acid (2-benzyloxy-5-oxo-tetraido-furan-3-yl) -amide

3,5-difluoro-benzoylamino) propionin-pyrrolidine-2-carboxylic (98y).

Prepared from carbonic {2- (2- (2- (2-benzyloxy-5-oxo-tetrahydrofuran-3-ylcarbamoyl) -pyrrolidin-1-yl] -1-methyl-2-oxo-ethyl} carbamic acid tert-butyl ester and 4-dimethylamino-3,5-dichloro-benzoic acid according to the procedure used to prepare 98a to provide 106 mg of the title compound (65% yield), ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.10-155 (m, 3H), 1.75-2.30 (m, 4H), 2.45-3.15 (m, 2H), 2.84 (s, 6H) , 3-40-3.95 (m, 2H), 4.15-4.95 (m, 5H), 5.47 (s, 0.35H), 5.54 (s, 0.15H), 5 , 67 (d, 0.4H), 5.77 (d, 0.1 H), 7.20-7.70 (m, 7H) Analytical HPLC: 12.21, 12.51 min. (ES ⁺ ): 25 m / e = 559.2 (M + H ⁺ ).

138

1- [2- (4-Amino-2,3,5,6tetrafluoro-benzoylamino) propionyl-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-98-carboxylic acid (98z ).

Prepared from {2- [2- (2-benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbmoyl) -pyrrolidin-1-yl] -1-methyl-2-oxo-ethyl} tert-butyl ester} carbamic and 4-amino-2,3,5,6-tetrafluoro-benzoic acid according to the procedure used to prepare 98a to provide 58 mg of the title compound (73% yield), ¹ H NMR (500 MHz, 1 : 1 CDCI ₃ : CD ₃ 0D) δ 1.301.50 (m, 3H), 1.62-2.35 (m, 4H), 2.45-3.12 (m, 2H), 3.50-3 , 90 (m, 2H), 4,204.95 (m, 5H), 5.42 (s, 0.4H), 5.52 (s, 0.1 H), 5.64 (d, 0.4H) , 5.82 (d, 0.1 H), 7.25-7.65 (m, 5H). Analytical HPLC: 16.56, 16.90 min. LC-MS (ES ⁺ ): m / e = 567.2 (M + H ⁺ ).

1- {2- (3-Chloro-4- (2,215 dimethylpropionylamino) -benzoylamino-propionyl} -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -amide )

To a suspension of 98b (100 mg, 0.19 mmol) and poly (4vinylpyridine) (200 mg) was added pivaloyl chloride (70 μΙ, 0.57 mmol). The resulting suspension was stirred overnight at room temperature, filtered and diluted with EtOAc (25 ml). The organic layer was washed with 10% NaHC0 ₃ (2 x 25 ml), saturated NaCl (I x 25 ml), dried (MgSO ₄ ), and evaporated to dryness to provide 98 mg of the title compound ( 85% yield) after chromatography.

¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.10-1.55 (m, 3H), 1.38 (s, 9H), 1.65-2.40 (m, 4H), 2.60-3.10 (m, 2H), 3.46-3.88 (m, 2H), 4.20-4.95 25 (m, 5H), 5.62 (d, 0 , 8H), 5.78 (d, 0.2H), 7.15-8.30 (m, 8H). Analytical HPLC:

11.82 min. LC-MS (ES ⁺ ): m / e = 613.2 (M + H ⁺ ).

139

1- [2- (3-chloro-4-propionylamino) -benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-98-carboxylic acid,

Prepared from 98b and propionyl chloride according to the procedure used to prepare 98aa to provide 104 mg of the title compound (95% yield). ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ OD) δ 1.16 (t, 0.6H), 1.18 (d, 0.6H), 1.27 (t, 2.4H) , 1.38 (d, 2.4H), 1.72-2.35 (m, 4H), 2.45-2.58 (m, 2H), 2.58-3.05 (m, 2H) 3.45-3.65 (m, 2H), 4.20-4.88 (m, 5H), 5.64 (d, 0.8H), 5.76 (d, 0.2H), 7, 20-8.35 (m, 8H). Analytical HPLC: 9.89 min.

LC-MS (ES ⁺ ): m / e = 585.2 (M + H ⁺ ).

1- [2- (3-chloro-4-phenylacetylamino) -benzoylamino) propionill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propyl-2-carboxylic acid (98ac),

Prepared from 98b and phenylacetyl chloride according to the procedure used to prepare 98aa to provide 85 mg of the title compound (77% yield). ¹ H NMR (CDCI ₃ : CD ₃ OD) δ 1.18 (d, 0.6H), 1.40 (d, 2.4H), 1.72-2.38 (m, 4H), 2.58 -3.05 (m, 2H), 3.46-3.78 (m, 2H), 3.85 (s, 2H), 4.18-4.92 (m, 5H), 5.63 (d , 0.8H), 5.75 (d, 0.2H), 7.15-8.34 (m, 13H). Analytical HPLC: 11.63 min. LC-MS (ES ⁺ ): m / e = 647.2 (M + H ⁺ ).

1- (2- (3-chloro-4-methylbutyrylamino) -benzoylamino) propionill-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-98-carboxylic acid,

Prepared from 98b and isovaleryl chloride according to the procedure used to prepare 98aa to provide 60 mg of the compound (6 (

140 of the security (58% yield). ¹ H NMR (500 MHz, 1: 1 CDCI ₃ : CD ₃ 0D) δ 1.07 (d, 5H), 1.15 (d, 0.8H), 1.27 (d, 1H), 1.45 (d, 2.2H), 1.67-2.30 (m, 5H), 2.34 (d, 2H), 2.58-3.05 (m, 2H), 3.48-3.88 (m, 2H), 4.10-4.98 (m, 5H), 5.68 (d,

0.7H), 5.78 (m, 0.3 H), 7.18-8.33 (m, 8H). Analytical HPLC: 10.74 min. LC-MS (ES ⁺ ): m / e = 613.2 (M + H ⁺ ).

142- (4-Methoxy-3,5-dimethylbenzoylamino) -propionylal-pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) (98ae).

Prepared from 1 - [2- (4-methoxy10 3,5-dimethyl-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and syn- (2-ethoxy-) allyl ester 5-oxo-tetrahydrofuran-3-yl) -carbamic according to the procedure used to prepare 94a to provide 174 mg (81% yield) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.04 (t, 0.45H), 1.27 (t, 2.55H), 1.34-1.45 (m, 3H), 1.95- 2.45 (m, 10H), 2.78-2.84 (m, H), 15 3.60-3.90 (m, 8H), 4.50-4.70 (m, 2H), 4 , 90-4.94 (m, H), 5.45 (d, 0.85H), 5.61 (d, 0.15H), 6.99 (d, H), 7.15 (d, H ), 7.45 (s, 2H), retention time on analytical HPLC: 10.09 min; LC-MS: m / z = 476 (M + H ⁺ ).

1- [2- (4-Methoxy-3,5-dimethyl20 benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) (98af)

Prepared from 1- [2- (4-methoxy-) acid tert-butyl ester

3,5-dimethyl-benzoylamino) propionyl] -pyrrolidine-2-carboxylic and anti- (2-ethoxy-5-oxo-tetrahydrofuran-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 168 mg (77% yield) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ, 1.10 (m, 3H), 1.35-1.60 (m, 3H), 1.90-2.45 (m, 10H), 2, 60-3.00 (m, H), 3.55-3.95 (m, 8H), 4.15-4.60 (m, 2H), 4.83-5.00 (m, H), 5.29 (s, H), 6.95-7.06 (m, H), 7.50 (s,

141

2H), 7.92 (d, H); retention time on analytical HPLC: 10.14 min; LCEM: m / z = 476 (M + H ⁺ ).

1- [2- (4-Methoxy-3,55 dimethyl-benzoylamino) -propionillpyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionillpyrrolidine-2-carboxylic acid (98aq).

Prepared from 1- [2- (4-methoxy-) acid tert-butyl ester

3,5-dimethyl-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid and syn- (2-benzyloxy-5-oxo-tetrahydrofuran-3-yl) -carbamic acid allyl ester (40) according to the procedure used to prepare 98a to provide 406 mg (yield 71%) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.09 (d, 0.6H), 1.35 (m, 2.4H), 1.90-2.20 (m, 3H), 2.22 -2.50 (m, 10H), 2.84-2.90 (m, H), 3.52-3.62 (m, 1.6H), 3.65-3.80 (m, 3, 4H), 4.10-4.40 (m, H), 4.50-4.75 (m, 3H), 4.82-4.95 (m, 2H), 5.54 (d, 0, 8H), 5.80 (d, 0.2H), 6.87 (d, H), 7.10-7.40 (m, 6H), 7.45 (m, 2H): retention time on HPLC analytical: 16.71 min ¹ ;

1- [2- (4-Allyoxy-3,5dimethyl-benzoylamino) -propionylal-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionylal-pyrrolidine-2-carboxylic acid (98ah).

Prepared from 1, - [2- (4-allyoxy20 3,5-dimethyl-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and 40 according to the procedure used to prepare 98a for provide 264 mg (46% yield) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 1,091.43 (m, 3H), 1.90-2.20 (m, 3H), 2.20-2.38 (m, 7H), 2.38 -2.52 (m, H), 2.802.95 (m, H), 3.52-3.67 (m, H), 3.70-3.80 (m, H), 4.10-4 , 40 (m, 2H), 4.40-4.95 25 (m, 5H), 5.26-5.55 (m, 3H), 9.00-6.14 (m, H), 6, 87 (d, H), 7.10-7.70 (m, 8H);

retention time on analytical HPLC: 18.56, 18.92 min 1; LC-MS: m / z =

142

{2- [1R- (2S-lsopropyl-5R-methyl-cyclohexyloxy) 1-5-oxotetrahydro-furan-3-yl} -carbamic acid allyl ester.

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using (1R, 2S, 5R) - (-) - menthol to provide 0.32 g of syn diastereomer (lower Rf ) of the title compound and 4.25 g of the anti / syn diastereomer mixture; (67% total yield). ¹ H NMR (500 MHz, CDCI ₃ ) mixture: δ 0.70-1.05 (m, 13H), 10 1.20-1.47 (m, 2H), 1.60-1.80 (m, 2H), 1.94-2.20 (m, 2H), 2.35-2.50 (m, H),

2.82- 3.04 (m, H), 3.40-3.61 (m, H), 4.43-4.70 (m, 3H), 5.15-5.35 (m, 2H), 5,485.61 (m, H)) 5.90-5.94 (m, H); for diastereomer syn 0.70-1.05 (m, 13H),

1.20-1.47 (m, 2H), 1.60-1.80 (m, 2H), 1.94-2.18 (m, 2H), 2.40-2.50 (m, H ),

2.82- 2.92 (m, H), 3.54-3.61 (m, H), 4.45-4.70 (m, 3H), 5.18-5.35 (m, 2H),

5.58-5.61 (m, H), 5.90-5.93 (m, H); LC-MS: m / z = 340 (M + H ⁺ ) for the mixture

of anti / syn diastereomers.

4-Benzyloxy-3,5-dimethyl-benzoic acid.

Prepared by the method used to synthesize 4-allyloxy-3,5-dimethyl-benzoic acid to provide 2.43 g (56% yield) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ) δ 4.87 (s, 2H), 7.36-7.48 (m, 5H), 7.92 (s, 2H); LC-MS: m / zM 255 (MH ⁺ ).

1- [2- (4-Benzyloxy-3,5-dimethyl-) acid [2-f 1 R- (2S-isopropyl-5R-methyl-cyclohexyloxy) 1- [2- (4-benzyloxy-3,5-dimethyl-) -amide benzoylamino) propionill-pyrrolidine-2-carboxylic (98ai).

Prepared from l- [2- (4benzyloxy-3,5-dimethyl-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester {2- [IR- (2S- lsopropyl-5R-methyl-cyclohexyloxy)] - 5-oxo-tetrahydrofuran-3-yl) -carbamic according to the procedure used to prepare

143

98a to provide 130 mg (39% yield) of the title compound. ¹ H

NMR (500 MHz, CDCI ₃ ): δ 0.45-1.10 (m, 12H), 1.15-1.90 (m, 8H), 1.90-2.45 (m, 12H), 2 , 80-2.84 (m, H), 3.50-3.85 (m, 3H), 4.45-4.70 (m, 2H), 4.80-4.95 (m, 3H) , 5.62 (d, H), 7.05 (d, H), 7.17 (d, H), 7.30-7.60 (m, 7H), 7.62-7.75 (m ,

H); retention time on analytical HPLC: 15.90 and 16.08 min; LC-MS: m / z = 662 (M + H ⁺ ).

1-r2- (4-hydroxy-3,5-dimethyl-benzoylamino) acid {2-11R- (2S-isopropyl-5R-methyl-cyclohexyloxy) 1-5-oxo-tetrahydro-furan-3-yl} amide -propionyl) pyrrolidine-210 carboxylic (98aj)

A solution of {2- [1R- (2S-isopropyl-5R-methyl-cyclohexyloxy)] - 5oxo-tetrahydro-furan-3-yl) -amide of l- [2- (4-benzyloxy-3,5-) acid dimethylbemzoylamino) -propionyl] -pyrrolidine-2-carboxylic (110 mg, 0.17 mmol) in ethyl acetate (2 ml) was stirred with 10% palladium on carbon (20 mg) 15 under a hydrogen atmosphere for 24 hours then it was filtered through Celite and evaporated in vacuo. The resulting residue was purified by chromatography using CH ₂ CI ₂ / methanol (99/1 to 96/4) to provide 58 mg of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 0.70-1.00 (m, 10H), 1.20-1.80 (m, 10H), 1.90-2.40 (m, 11H) , 2.82-2.86 (m, H), 3.57-3.78 (m, 3H), 4.55-4.67 20 (m, 2H), 4.90-4.94 (m , H), 5.29 (s, H), 5.62 (d, H), 6.90 (d, H), 7.14 (d, H),

7.42 (s, 2H), retention time on analytical HPLC: 12.84 and 13.05 min; LC-MS: m / z = 572 (M + H ⁺ )

1-1,2- (4-hydroxy-3,525-dimethyl-benzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98ak).

144

A solution of 98ah (230 mg, 0.41 mmol) in CH2 Cl2 (10 mL) was treated with DMBA (65 mg, 0.42 mmol) and Pd (PPh ₃ ) 4 (50 Mg) at room temperature for 20 hours. The mixture was concentrated to dryness in vacuo and was purified by flash chromatography using CH ₂ CI ₂ / methanol (99.5 / 0.5 to 97/3) to provide 1.81, mg of the title compound. ¹ H NMR (500 MHz,

CDCI ₃ : δ 1.08 (d, 0.75H), 1.20-1.35 (m, 2.25H), 1.70-2.50 (m, 12H), 2.802.90 (m, H), 3.50-3.65 (m, H), 3.70-3.80 (m, H), 4.10-4.25 (m, H), 4.35-4.98 (m, 3H ), 5.53 (d, 0.75H), 5.85 (d, 0.25H), 6.81 (d, H), 7.13-7.60 (m, 8H); retention time on analytical HPLC: 10.38 and 10.56 min; LC-MS: m / z = 524 10 (M + H ⁺ ).

1- [2- (4-dimethylaminobenzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionyl (98al).

Prepared from 1- [2- (415 dimethylamino) -benzoylamino) propionyl) -pyrrolidine-2-carboxylic acid tert-butyl ester and syn- (2-benzyloxy-5-oxo-tetrahydro-) allyl ester furan-3-yl) -carbamic according to the procedure used to prepare 98a to provide 60 mg (45% yield). ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.04 (d, 0.75H), 1.35 (d, 2.25H), 1.80-2.50 (m, 5H), 2.75 -3.20 (m, 8H), 3.45-3.75 (m, 2H), 4.05-4.20 (m, 0.5H), 20 4.30-4.60 (m, 3 , 5H), 4.80-4.95 (m, 1.5H), 5.52 (d, H), 5.75-6.00 (m, 0.5H),

6.60-6.90 (m, 3H), 7.10-7.50 (m, 4H), 7.50-7.80 (m, 2H); retention time on analytical HPLC: 10.46 min; LC-MS: m / z = 523 (M + H ⁺ ).

Acid {2R- [1R- (2S-isopropyl-5R-methyl-cyclohexyloxy) 1-5-oxo-tetrahydro-furan-3-yl} -amide 1 - [2- (4-amino-3-chlorobenzoylamino) - propionill-pyrrolidine-2-

145 carboxylic (98am).

Prepared from 1- [2- (4-amino3-chloro-benzoylamino) propionyl) -pyrrolidine-2-carboxylic acid tert-butyl ester (97a) and syn- (2- [1 R acid allyl ester) - (2S-isopropyl-5R-methylcyclohexyloxy) -5-oxo-tetrahydro-furan5 3-yl) -carbamic according to the procedure used to prepare 98a to provide 1.03 mg (67% yield) of the title compound. ¹ H NMR (500 KHZ, CDCh): δ 0.70-1.10 (m, 1.2H), 1.20-1.50 (m, 5H), 1.50-1.85 (m, 2H ), 1.90-2.30 (m, 5H), 2.75-2.85 (m, H), 3.50-3.70 (m, 2H), 3.70-3.82 (m , H),

4.20-4.65 (m, 4H), 4.80-4.95 (m, H), 5.61, (d, H), 6.70-6.73 (m, H), 6 , 95 (d, 10 H), 7.15 (d, H), 7.49-7.51 (m, H), 7.73 (s, H): retention time on analytical HPLC: 12.88 min; LC-MS: m / z = 577 (M + H ⁺ ).

{2- [1S- (2R-isopropyl-5S-methyl-cyclohexyloxy) 1-5-oxotetrahydro-furan-3-yl} -carbamic acid allyl ester.

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using (1S, 2R, 5S) - (+) - menthol to provide 55 mg of antidiastereomer (upper Rf) of title compound, 503 mg of syn diastereomer (lower Rf) and 459 mg of the mixture of anti / syn diastereomer (total yield 66%) ¹ H NMR 20 (500 MHz, CDCI3) antidiastereomer: δ 0.74-1.00 (m, 12H), 1.20-1.45 (m,

2H), 1.58-1.72, (m, 2H), 1.98-2.12 (m, 2H), 2.18-2.40 (m, H), 2.98-3 , 03 (m,

H), 3.49-3.54 (m, H), 4.17 (br, H), 4.59 (br, 2H), 4.97 (br, H), 5.22-5.33 (m, 2H), 5.58 (s, H), 5.87-5.93 (m, H); for syn diastereomer 0.75-1.02 (m, 12H) 1.25-1.45 (m, 2H), 1.57-1.70 (m, 2H), 2.00-2.16 (m , 2H), 2.40-2.52 (m, 25 H), 2.78-2.90 (m, H), 3.40-3.50 (m, H), 4.58 (br, 2H), 5.24-5.35 (m, 2H), 5.515.52 (d, H), 5.85-5.98 (m, H); L-MS: m / z = 340 (M + H ⁺ ) for both diastereomers.

{2R- [1S- (2R-isopropyl-5S-methyl-cyclohexyloxy)] - 5-oxo-tetrahydro-furan-3-yl) amide

146 of 1- [2- (4-amino-3-chloro-benzoylamino) -propionill pyrrolidine-2- acid

carboxylic acid (98an).

Prepared from 97- and syn- {2- [1S (2R-isopropyl-5S-methyl-cyclohexyloxy) acid - 5-oxo-tetrahydro-furan-2-yl} allyl ester according to the procedure used to prepare 98a to provide 80 mg (50% yield) of the title compound. ¹ H NMR (500 MHz, CDCb): δ 0.70-1.10 (m, 12H), 2.20-1.50 (m, 14H), 1.50-1.70 (br, 2H), 1.90-2.25 (m, 4H), 2.27-2.37 (m, H), 2.40-2.50 (m, H), 2.75-2.79 (m, H ), 3.35-3.80 (m, 3H),

4.20-4.57 (m, 3H), 4.60-4.70 (m, H), 4.88 ^, 92 (m, H), 5.53 (d, H), 6.71 -6.75 (m, H), 6.90 (d, H), 7.20 (d, H), 7.50-7.53 (m, H), 7.75 (d, H); retention time on analytical HPLC:

13.20 min; LC-EM; m / z = 577 (M + H ⁺ ).

Allyl ester of carbamic acid (2-cyclohexylmethoxy-5-oxo-tetrahydro-furan-3-yl).

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using cyclic

clohexylmethanol to provide 1.04 g (higher Rf) (35% yield) of antidiastereomer of the title compound and 1.295 g (lower Rf) (44% yield) of syn diastereomer. ¹ H NMR (500 MHz, CDCI ₃ ) for antidiastereomer: δ 0.90-0.96 (m, 2H), 1.10-1.30 (m, 3H), 1.55-1.85 (m, 6H), 2,372.41 (d, H), 2.97-3.03 (m, H), 3.34-3.38 (m, H), 3.58-3.62 (m, H) , 4.55-4.70 (m, 2H), 4.70-4.73 (m, H), 5.03 (bs, H), 5.22-5.37 (m, 3H), 5 , 87-5.93 (m, H);

for syn diastereomer 0.91-0.97 (m, 2H), 1.10-1.31, (m, 3H), 1.56-1.90 (m,

7H), 2.44-2.48 (m, H), 2.81-2.87 (m, H), 3.35-3.39 (m, H), 3.63-3.67 ( m, H),

4.53-4.70 (m, 3H), 5.20-5.50 (m, 3H), 5.89-5-95 (m, H); LC-MS: m / z = 298 (M + H ⁺ ) for both diastereomers.

1- [2- (4acetylamino-3-chloro-benzoylamino) -propionill-pyrrolidine-2-carboxylic acid (2-cyclohexylmethoxy-5-oxo-tetrahydro-furan-3-yl) -propionill-pyrrolidine-2-carboxylic (98ao).

147

Prepared from 97b and syn- (2cyclohexylmethoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 212 mg (64% yield) of title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 0.70-1.30 (m, 5H),

1.30-1.85 (m, 9H), 1.85-2.60 (m, 8H), 2.75-3.00 (m, H), 3.10-3.80 (m, 4H ),

4.30-4.95 (m, 3H), 5.42 (d, 0.85H), 5.62 (d, 0.15H), 6.87 (d, 0.15H) 7.08 (d, 0.85H), 7.25 (d, H), 7.60-7.90 (m, 3H), 8.08 (d, 0.15H), 8.50 (d, 0.85H); time on analytical HPLC: 11.81 min; LC-MS: m / z = 577 (M + H ⁺ )

1- [2- (4-Acetylamino-3- (chloro-) -5- oxo-tetrahydro-furan-3-yl} -amido {2- [1S- (2R-isopropyl-5S-methyl-cyclohexyloxy) benzoylamino) -propionin pyrrolidine-2-carboxylic (98ap).

Prepared from 97b and syn- {2- [1S (2R-isopropyl-5S-methyl-cyclohexyloxy) acid - 5-oxo-tetrahydro-furan-3-yl) allyl ester according to the procedure used to prepare 98a to provide 223 mg (63% yield) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 0.70-1.15 (m, 12H), 1.20-1.85 (m, 8H), 1.85-2.60 (m, 9H) , 2.74-2.88 (m, H), 3.35-3.85 (m, 3H), 4.40-4.55 (m, H), 4.65-4.78 (m, H), 4.88-4.91, (m, H), 5.53 (d, H), 7.00-7.25 (m, 2H), 7.60-7.90 (m, 3H ), 8.50 (d, H); retention time on analytical HPLC: 13.31, min; LC-EM; m / z = 919 (M + H ⁺ ).

148 (2-cyclohexylmethoxy-5-oxo-tetrahydro-furan-3-yl) -amide 1- [2- (4-amino-3-chlorobenzoylamino) -propionyl-pyrrolidine-2-carboxylic (98aq).

Prepared from 97a and syn- (2cyclohexylmethoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 113 mg (56% yield) (from the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 0.70-1.35 (m, 5H), 1.35-1.90 (m, 8H), 1.90-2, 20 (m, 3H), 2.30-2.60 (m, H), 2.80-3.00 (m, H), 3.15-3.80 (m, 4H), 4.28- 4.75 (m, 4H), 4.89-4.93 (m, H), 5.42 (d, H), 6.74 (d, H), 6.87 (d, H), 7 , 30 (d, H), 7.51-7.53 (m, H), 7.74 (d, H): retention time on analytical HPLC: 12.02 min .; LC-MS: m / z = 535 (M + H ⁺ )

Allyl ester of (2-Butoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using n15 butanol to provide 878 mg (29% yield) of the title compound (313 mg antidiastereomer, 260 mg of syn diastereomer and 305 mg of the mixture). ¹ H NMR (500 MHz, CDCI ₃ ) for anti diastereomer: δ (upper Rf) 0.89-0.96 (t, 3H), 1.32-1.40 (m, 2H), 1.54-1 , 63 (m, 2H), 2.37-2.41 (d, H), 2.98-3.04 (q, H), 3.55-3.60 (m, H), 3.77 -3.82 (m, H), 4.19-4.22 (m, H), 4.58 20 (br, 2H), 5.03 (br, H), 5.23-5.40 ( m, 3H), 5.87-5.93 (m, H), for syn diastereomer (lower Rf) 0.91-0.95 (m, 3H), 1.34-1.39 (m, 2H) , 1.56-1.63 (m, 2H), 2.42-2.50 (m, H), 2.83-2.87 (m, H), 4.07-4.11, (t , H), 4.45-4.50 (m, 0.5H), 4.51-4.70 (m, 2.5H), 5.23-5.35 (m, 2H), 5.42 -5.43 (d, H), 5.80-5.95 (m, H); LC-MS: m / z = 258 (M + H ⁺ ) for both diastereomers.

1- [2- (4-Amino-3-chlorobenzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (2-butoxy-5-oxo-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98ar).

Prepared from 97a and syn- (2-butoxy5-oxo-tetrahydrofuran-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 118 mg (48% yield) of the compound

149 of the title as a diastereomer syn. ¹ H NMR (500 MHz, CDCI ₃ ): δ 0.80-102 (m, 2H), 1.35-1.51, (m, 5H), 1.51-1.70 (m, 2H), 1.90-2.27 (m, 3H), 2.302.46 (m, H), 2.80-2.90 (m, H), 3.55-3-94 (m, 4H), 4, 30-4.75 (m, 4H), 4.905.00 (m, H), 5.44-5.46 (m, H), 6.73-6.80 (m, H), 6.80- 6.93 (m, H), 7.16-7.25 (m, H), 7.49-7.60 (m, H), 7.70-7.84 (m, H); retention time on analytical HPLC; 9.71, min; LC-MS: m / z = 495; (M + H ⁺ ).

Allyl ester of (3-isobutoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid

Prepared from 310 allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using iso-

butanol to provide 190 mg (7.3% yield) of the title compound as an antidiastereomer and 290 mg (11% yield) of the diastereomer syn, ¹ H NMR (500 MHz, CDCI ₃ ) for antidiastereomer: δ (Rf higher ) 0.85-1.05 (m, 6H), 1-82-1.98 (m, H), 2.37-2.42 (m, H), 2.98-3.04 (m, H), 3.3115 3.35 (m, H), 3.55-3.58 (m, H), 4.20-4.30 (t, H), 4.58 (br, 2H), 5.07 (br, H),

5.22-5.43 (m, 3H), 5.84-5.96 (m, H), for syn diastereomer (lower Rf) 0.85-1.05 (m, 6H), 1.88- 1.95 (m, H), 2.40-2.51, (m, H), 2.83-2.90 (m, H),

3.33-3.36 (m, H), 3.61-3.65 (t, H), 3.87-3.88 (d, H), 4.40-4.68 (m, 3H ), 5.205.40 (m, 2H), 5.42-5.43 (d, H), 5.80-5.97 (m, H); LC-MS: m / z = 258 (M + H ⁺ ) 20 for both diastereomers.

1-F2- (4-Amino-3-chlorobenzoylamino) -propionyl-1-pyrrolidine-2-carboxylic acid (2-isobutoxy-5-oxo-tetrahydro-furan-3-yl) (98as).

Prepared from 97a and syn- (2isobutoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 93 mg (38% yield) of the title compound. ¹ H NMR (500 MHz, CDCI ₃ ): δ 0.74-0.76 (t, 0.6H), 0.801.00 (m, 5.4H), 1.40-1.50 (m, 3H) 1.90-2.22 (m, 3H), 2.33-2.45 (m, H), 2.802.90 (m, H) 3.32-3.38 (m, H), 3.55 -3.80 (m, 3H), 4.38 (br, H), 4.50-4.60 (m, H), 5.42-5.45 (m, H), 6.74-6 , 76 (d, H), 6.86-6.88 (d, H) 7.31-7.33 (d, H), 7.5101

150

7.53 (m, Η), 7.74-7.75 (d, Η): retention time on analytical HPLC: 9.63 & 9.80 min; LC-MS: m / z = 495 (M + H ⁺ ).

2- (Indan-2-yl) oxy-5-oxo-tetrahydro-furan-3-yl] carbamic acid allyl ester.

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester (5.2 grams, 20 mmol) as described for 40 using 2-indanol (8.05 grams, 60 mmol) to provide 4, 10 grams (65% yield) of the title compound as a mixture of epimers. Purification provided 1.76 grams (28% yield) of the 4 (S), 10 5 (R) epimer as a yellow oil. ¹ H NMR (500 MHz, CDCI ₃ ) δ 2.42 (dd,

J = 17.2, 10.5 Hz, 1H), 2.79 (dd, J = 17.2, 8.4 Hz, 1H), 2.99 (dd, J = 16.7, 4.1 HZ , 1H), 3.04 (dd, J = 16.7, 4.1 Hz, 1H), 3.22 (dd, J = 17.2, 6.6 Hz, 1H), 3.26 (dd, J = 17.2, 6.6 Hz, 1H), 4.53 (m, 3H), 4.70 (m, 1H), 5.20 (m, 2H), 5.60 (d,

J = 5.3 Hz, 1H), 5.87 (m, 1H), 7.17 (m, 4H) ppm. LC-MS (ES ⁺ ): m / e = 318 15 (M + H) analytical HPLC (column C18): 17.094 Min.

Also isolated was the epimer mixture (0.75 gram, 12% yield), and the 4 (S) epimer (1.59 gram, 25%) as a white solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 2.38 (d, J = 17.9 Hz, 1H), 3.0 (m, 3H), 3.22 (m, 2H), 4.13 (m , 1H), 4.58 (m, 2H), 4.68 (m, 2H), 4.98 (m, 1H), 5.26 (m, 1H), 20 5.57 (s, 1H), 5.88 (ddt, J = 18.0, 11.1, 5.4 Hz, 1H), 7.20 (m, 4H) ppm. LC-MS (ES ⁺ ): m / e = 318 (M + H). Analytical HPLC (column C18): 17.025 (5.5%), 17.325 (94.5%) min.

1- [2- (4-amino-3-chloro-benzoylamino) -propionyl-1-pyrrolidine-2-carboxylic acid 2- (indan-2-yloxy) -5-oxo-tetrahydro-furan-3-ill-amide (98at) .

Prepared from 97a and a [2- (indanol-2yl] oxy-5-oxo-tetrahydro-furan-3-yl) -carbamic allyl ester according to the procedure used to prepare 98a to provide the title compound as a 71:29 mixture of epimer as an off-white solid (0.20 g, 58%

151 yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.0-1.5 (m, 3H), 1.6-2.3 (m, 4H),

2.42 (m, 1H), 2.6-3.4 (m, 5H), 3.5-4.1, (m, 3H), 4.2-4.9 (m, 4H), 5 , 65 (d, J =

5.0 Hz, 0.80H), 5.8 (m, 0.07H), 5.85 (d, J = 5.0 HZ, 0.13H), 6.8-7.3 (m, 6H),

7.4-7.9 (m, 3H) ppm. Analytical HFLC (column C18) 16.035 (71.4%) 16.476 (28.6%) min. LC-MS (ES ⁺ ): m / e = 555 (M + H).

142- (4-acetylamino-3-chloro-benzoylamino) -propionin-pyrrolidine-2-carboxylic acid 2- (indan-2-yloxy) -3-oxo-tetrahydro-furan-3-yl-amide (98au),

Prepared from 97b and [2- (indanol-210 yl] oxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to provide the compound of title as a mixture of 76:24 epimers as an off-white solid (0.22 g, 57% yield). ¹ H NMR (500 MHz, CDCL ₂ ) δ 1.08 (d, J = 6.9 Hz, 0.4H), 1.26 (d, J = 6.9 Hz, 0.6H), 1.35 (d, J = 6.9 Hz, 2H), 1.8-2.3 (m, 3H), 2.28 (s, 2H), 15 2.29 (s, 1H), 2.4 (m , 1H), 2.8 (m, 1H), 3.10 (m, 2H), 3.27 (m, 2H), 3.58 (m,

2H), 3.69 (m, 1H), 4.5-4.9 (m, 4H), 5.65 (d, J = 5.3 Hz, 0.68H), 5.84 (d, J = 5 , 3 Hz, O, 18H), 6.38 (br, 0.14H), 6.9-7.7 (m, 6H), 7.6-7.9 (m, 3H), 8.33 (br d, J = 6.8 Hz, 0.18H), 8.51, (br d, J = 8.0 Hz, 0.82H) ppm. Analytical HPLC (column C18) 15.596 (76.2%), 15.932 (23.8%) min. LC-MS (ES ⁺ ): m / e = 597 20 (M + H).

1- [2- (4-Amino-3-chloro-benzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (2-cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl) -propionill pyrrolidine-2-carboxylic acid (98av).

Prepared from 97a and syn- (225 cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure

152 ment used to prepare 98a to provide the title compound as an off-white solid (0.19 g, 59% yield). ¹ H NMR (500 MHz, CDCl ₃ ) δ 1.2-2.4 (m, 15H), 2.4-3.1 (m, 2H), 3.6-3.9 (m, 2H), 4.2-4.4 (m, 2H), 4.55.0 (m, 4H), 5.40 (m, J = 5.0 Hz, 0.35H), 5.55 (d, J = 5.0 Hz, 0.65H), 6.8-8.2 5 (m, 5H) ppm. Analytical HPLC (column C18) 14.065 min. LC-MS (ES ⁺ ): m / e =

507 (M + H).

1-1,2- (3,5-dichloro-4-hydroxy-benzoylamino) -propionyl-1-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionyl-2-carboxylic acid (98aw).

Prepared from 1- [2- (4-allyoxy3,5-dimethyl-benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester according to the procedure used to prepare 98a for provide the title compound as a pale yellow solid (1.087 g, 64% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.09 (d, J = 6.9 Hz, 0.6H), 1.33 (d, J = 6.9 Hz,

2.4H), 1.96 (m, 1H), 2.03 (m, 1H), 2.10 (m, 1H), 2.28 (m, 0.8H), 2.40 (dd, J =

17.3, 1.02 Hz, 0.8H), 2.56 (m, 0, 2H), 2.85 (dd, J = 17.3, 8.5 HZ, 0.8H), 3.09 ( dd, J = 17.7 1.02 Hz, 0.2H), 3.57 (m, 1H), 3.73 (dt, J = 9.2, 7.9 Hz, 0.8H), 4, 09 (m, 0.2H), 4.21, (d, J = 7.9 Hz, 0.2H), 4.44 (d, J = 9.8 Hz, 0.2H), 4.55 ( dd, J = 8.0, 3.0 Hz, 0.8H), 4.62 (d, J = 11.6 Hz, 1H), 4.70 (m, 1H), 4.80 (m,

1H), 4.89 (d, J = 11.6 Hz, 0.8H), 5.52 (d, J = 5.2 Hz, 0.8H), 5.82 (m, J = 5.2

Hz. 0.2H), 6.51, (br, 0.2H), 6.62 (br, 0.8H), 7.0-7.4 (m, 7H), 7.43 (s, 0 , 4H), 7.66 (d, J = 1.0, Hz, 1.6H) ppm. Analytical HPLC (column C18) 10.135 min. LC-MS (ES ⁺ ): m / e = 564.566 (6: 4) (M + H).

1-1,2- (4-amino-3-chloro-benzoylamino) -propionillpyrrolidine-2-carboxylic acid (2-cyclopentyloxy-5-oxo-tetrahydro-furan-3-yl) -propionillpyrrolidine-2-carboxylic acid (98ax).

/ Μ

153

Prepared according to the procedure used to prepare (98av) using anti- (2-cyclopentyloxy-5-oxo-tetrahydro-furan-3 yl) amide to provide the title compound as an off-white solid (0.24 g, 74% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.41 (d, J = 6.5 Hz, 3H), 1.7 (m, 7H), 1.98 (br, 2H), 2.13 (br , 2H), 2.27 (m, 1H), 2.69 (m, 1H), 2.86 (dd, J =

18.0, 6.8 Hz, 0.7H), 2.98 (dd, J = 18.3, 8.2 Hz, 0.3H), 3.60 (br, 1.4H), 3.77 (br, 0.6H), 4.1-4.6 (m, 5H), 4.82 (m, 3H) 5.27 (m, 0.65H), 5.51 (d, J = 5.3 Hz, 0.05H), 5.59 (br s, 0.3H), 6.76 (br, 1H), 7.00 (br, 1H), 7.49 (br, 1H), 7.74 ( br, 1H), 7.89 (br, 1H) ppm. Analytical HPLC (column C18) 9.756 min. LC-MS 10 (ES ⁺ ): m / e = 507 (M + H).

1- [2- (4-Acetylamino-3-chloro-benzoylamino) -propioninpyrrolidine-2-carbaxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -propioninpyrrolidine-2-carbaxyl (98ay).

Prepared from carbamic acid (2-ethoxy-5-oxo15 tetrahydro-furan-3-yl) allyl ester and 97b after the method used for 98a. The title compound was isolated as a white solid (51 mg, 18% yield). ¹ H NMR (500MHz, 1: 1 CDCI ₃ : CD3OD) δ 1.08-1.35 (m, 3H), 1.35-1.55 (m, 3H) 1.75-2.44 (m, 4H), 2.26 (s, 3H), 2.44-3.07 (m, 2H), 3.48-3.97 (m, 2H), 4.18-4.92 (m, 5H) , 5.32 (d, 0.4H), 5.47 (d, 0.1H), 5.58 (d, 0.4H), 5.64 (d,

0.1 H), 7.70-8.35 (m, 3H). Analytical HPLC 10.37, 10.54 min. LC-MS (ES ⁺ ) m / e = 509.2 (M + H ⁺ ).

2- (2-Chloro-ethoxy) -3-oxo-tatrahydrO-furan-3-illcarbamic acid allyl ester.

Prepared from 3-allyloxide acid tert-butyl ester

154 carbonylamino-4-hydroxy-butyric (5.2 g, 20 mmol) as described for 40 using chloroethanol (4.05 ml, 60 mmol) to provide 1.84 g (35% yield) of the title compound as a mixture of epimers. For the antidiastereomer: ¹ H NMR (500 MHz, CDCI ₃ ) δ 2.42 (dd, J = 18.1 Hz, 1, H), 5 3.00 (dd, J = 18.1, 7.8, 1H), 3.63 (m, 2H), 3.85 (m, 1H), 4.02 (m, 1, H), 4.23 (m, 1H), 4.57 (br s, 2H) , 5.17 (br s, 1H), 5.22 (d, J = 11.5 Hz, 1H), 5.29 (d, J = 16.8 Hz, 1H), 5.44 (s, 1H ), 5.89 (m, 1H) ppm; LC-MS (ES ⁺ ) m / e = 264 (M + H). For syn-diastereomers: ¹ H NMR (500 MHz, CDCI ₃ ) δ 2.47 (dd, J = 17.3, 10.1 Hz, 1H), 2.83 (dd, J = 17.3, 8 , 4 Hz, 1H), 3.65 (m, 2H), 3.83 (m,

1, H), 4.11, (m, 1H), 4.57 (m, 3H), 5.22 (d, H = 10.4 Hz, 1H), 5.30 (d, J = 17, 2

Hz, 1, H), 5.33, (m, 1, H), 5.47 (d, J = 5.2 Hz, 1H), 5.89 (ddt, J = 17.1, 11.0 , 5.4 Hz, 1H) ppm; LC-MS (ES ⁺ ) m / e = 264 (M + H).

[2- (2-Morpholin-4-yl-ethoxy) -5-oxo-tetrahydro-furan-3-in- acid allyl ester

carbamic

It is prepared from allyl ester of acid (2- (2-chloro-ethoxy) 5-oxo-tetrahydrofuran-3-yl] -carbamic by reaction with morpholine (2 eq.) And Kl (1 eq.) In DMF .

1- (2- (4 amino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2- (2-morpholin-4-yl-ethoxy) -5-oxo-tetrahydro-furan-3-yl-amide carboxylic (98az),

It is prepared from 97- and syn- [2- (2morpholin-4-yl-ethoxy) -5-oxo-tetrahydro-furan-5-yl] -carbamic acid allyl ester after the method used for 98a.

98be

[2- (4-Chloro-benzyloxy) -5-oxo-tetrahydro-furan-3-yl125 carbamic acid allyl ester

Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using 4-chlorobenzyl alcohol to provide the title compound as a white solid. Antidiastereomer: HPLC (column C18) 10.924 min; ¹ H NMR (500

155

Hz, CDCI3) δ 2.41, (d, J = 8.0 Hz, 1H), 3.02 (dd, J = 18.1, 7.8 Hz, 1H), 4.25 (br, 1H) , 4.56 (m, 2H), 4.58 (d, J = 11.7 Hz, 1H), 4.79 (d, J = 11, 7 Hz, 1H), 4.99 (br, 1H) , 5.22 (dd, J = 10.4, 1.1 Hz, 1H), 5.28 (dd, J = 17.2, 1.3 Hz, 1H), 5.44 (s, 1H), 5.86 (m, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H): 5 ppm; LC-MS (ES ⁺ ) m / e = 326 (M + H); Syn-diastereomer. HPLC (column C18) 10.780 min; ¹ H NMR (500 MHz, CDCI ₃ ) δ 2.47 (dd, J = 17.3, 10.5 Hz, 1, H), 2.85 (dd, J = 17.3, 8.4 Hz, 1H ), 4.55 (m, 3H), 4.58 (d, J = 11.7 Hz, 1H), 4.84 (d, J = 11.7 Hz, 1H), 5.23 (dd, H = 10.4, 1.1 Hz, 1H), 5.30 (d, J = 16.6 Hz, 1H), 5.49 (d, J = 5.0 Hz, 1H), 5.89 (ddt , J = 171.1, 11.0, 5.4 Hz, 1H), 7.23 10 (d, J = 8.3 Hz, 2H), 7.31, (d, J = 8.3 Hz, 2H) ppm; LC-MS (ES ⁺ ) m / e = 326 (M + H).

1- [2- (4-amino3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid 2- (4-chloro-benzyloxy) 5-oxo-tetrahydro-furan-3-yl-amide (98ba) .

Prepared from 97a and syn- [2- (415 chloro-benzyloxy) -5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester after the method used for 98a to provide 154 mg (65% yield) of the title compound as a pale pink solid. HPLC (C18 column) 10.597 min; ¹ H NMR (500 MHz, CDCI3) δ 1.14 (d, J = 6.8 Hz, 0.75H), 1.34, (d, J = 6.8 Hz, 2.25H), 1.6 (br , 0.25H), 1.91, (m, 1, H), 2.03 (m, 1H), 2.10 (m, 1H), 2.29 (m, 0.75H), 2.40 20 (dd, J = 17.3, 10.3 Hz, 0.75H), 2.51, (m, 0.25H), 2.82 (dd, J = 17.3, 8.5 HZ, 0.75H ), 3.08 (dd, J = 17.9, 10.9 Hz, 0.25H), 3.58 (m, 1H), 3.72 (dd, J = 1.65 Hz, 0.75H) , 4.10 (m, 0.25H), 4.22 (d, J = 8.0 Hz, 0.23H), 4.39 (d, J = 10.85 Hz, 0.25H), 4.54 (dd , J = 9.1, 2.9 Hz, 0.75H), 4.60 (d, J = 11.9 Hz, 0.75H), 4.68 (m, 1H), 4.85 (d, J = 11.7 Hz, 0.75H), 4.86 (m, 1H), 5.49 (d, J = 5.2 Hz, 25 0.75H), 5.81, (d, J = 5.2 Hz, 0.25H), 6.2 (br, 0.25H), 6.74 (m, 2H), 7.05 (m, J = 8.5 Hz, 0.5H), 7.17 (d, J = 8.4 Hz, 0.5H), 7.30 (m, 3.25H), 7.48 (dd, J = 8.4, 2.0 HZ, 0.75H), 7.56 (d, J = 1.9 Hz, 0.25H), 7.73 (d, J = Ι.9 Hz, 0.75H), 8.42 (d, J = 5.7 Hz, 0.25H) ppm; LC-MS (ES ⁺ ) m / e = 563, 565 (M + H).

156

Nbb

1- [2- (4acetylamino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid 2- (4-chloro-benzyloxy) -5-oxo-tetrahydro-furan-3-ill-amide (98bb) ,

Prepared from 97b and syn- [2- (45 chloro-benzyloxy) -5-oxo-tetrahydro-furan-3-yl] -carbamic acid allyl ester according to the procedure used to prepare 98a to provide 165 mg (64% yield) of the title compound with pale yellow solid. HPLC (column C18)

10.491, min: ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.16 (d, J = 6.8 HZ, 0.6H), 1.35, (d, J = 6.8 Hz, 2.4H ), 1.94 (η, 1H), 2.04 (, 1H), 2.10 (m, 1H), 2.25 (s, 3H), 10 2.28 (m, 1H), 2.40 (dd, J = 17.3, 10.4 Hz, 0.8H), 2.53 (m, 0.2H), 2.84 (dd, J =

17.3, 8.5 Hz, 0.8H), 3.02 (dd, J = 17.5, 1.05 Hz, 0.2H), 3.58 (m, 1, H), 3.72 (ddd, J = 17.2, 8.3, 8.3 Hz, 0.8H), 4.13 (m, 0.2H), 4.22 (d, J = 8.2 Hz, 0.2H ),

4.40 (d, J = J = 10.9 Hz, 0.2H), 4.54 (dd, J = 8.1, 3.0 Hz, 0.8H), 4.60 (d, J =

11.8 Hz, 0.8H), 4.69 (m, 1H), 4.85 (d, J = 11.8 Hz, 0.8H), 4.87 (m, 1H), 5.49 15 (d, J = 5.2 Hz, 0.8H), 5.80 (d, J = 5.2 HZ, 0.2H), 6.47 (br, 0.2H), 6.95 (d, J =

8.3 HZ, 0.8H), 7.05 (d, J = 8.3 Hz, 0.4H) 7.18 (d, J = 8.3Hz, 0.4H), 7.29 (m,

3.2H), 7.49 (dd, J = 8.6, 1.9 Hz, 0.2H), 7.63 (dd, J = 8.6, 1.9 Hz, 0.8H), 8 , 25 7.74 (d, J = 1.9 Hz), 7.85 (d, J = 1.9 Hz, 0.84), (d, J = 6.4 Hz, 0.2H), 8.51 (m, 0.8H) ppm; LC-MS (ES ⁺ ) m / e = 605, 607 (M + H).

Scheme XVIII

100

157

102a, Y = AcNH

102b, Y = NHi

2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamyl) piperidine-1-carboxylic acid tert-butyl ester (100),

Prepared from 99 and 40 piperidine-1.25 dicarboxylic acid tert-butyl ester after the method used in the preparation of 75 to give the title compound as a yellow solid (2.63 g, 57% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.15-1.79 (m, 15H), 2.12-2.50 (m, 2H), 2.56-2.83 (m, 1H), 2.89 (dd, 0.5H), 3.05 (dd, 0.5H), 3.81 - 4.15 (brs, 1, H), 4.36-4.97 (m, 3H), 5.37-5.61, (m, 1H), 6.42-6.89 (br s, 1, H), 7.17-7.51, (m, 5H). LC-MS 10 (ES ⁺ ) m / e = 419.4 (MH ⁺ ).

{2- [2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamoyl) -piperidin-1-ill-1-methyl-2-oxo-ethyl} carbamic acid tert-butyl ester (101) ,

2- (2-Benzyloxy-5-oxo-tetrahydro-furan3-ylcarbamoyl) piperidine-1-carboxylic acid tert-butyl ester (100) was dissolved in 20% TFA (25-15 ml) and was stirred at room temperature for 50 mins. The solvent was evaporated and the residual acid was azeotroped with CH ₂ CI ₂ (4x). The resulting oil was dissolved in CH ₂ CI ₂ (20 ml) and DMF (5 ml), cooled to 0 ° C and treated with DIEA (4.7 ml, 27 mmoles), Boc-alanine (970 mg, 5 , 1 mmoles), HOBT (924 mg, 6.8 mmoles) and EDC (1.31 g, 6.8 mmoles) and the solution was stirred under N ₂ for 1.8 hours. The solvent was concentrated in vacuo then it was dissolved in EtOAc and was washed with NaHS (O) ₄ (2x), saturated NaHC0 ₃ (2x) and brine. The organic layer was dried over anhydrous NaHSO ₄ and evaporated to give an orange solid which was dissolved in CH ₂ CI ₂ and diethyl ether was added dropwise to provide a white precipitate. The title compound 25 with a white solid (1.21 g, 73% yield). ¹ H NMR (500

MHz, CDCI ₃ ) δ 1.10-1.79 (m, 18H), 1.98-2.19 (m, 0.5H), 2.28-2.88 (m, 3H), 2.89 -3.13 (m, 0.5H), 3.78-3.95 (m, 0.5H), 4.21-5.16 (m, 5.5H), 5.38-5.59 ( m, 0.3H), 5.66 (d, 0.4H), 5.80 (d, 0.3H), 7.24-7.40 (m, 5H). LC-MS (ES ⁺ ) m / e =

158

490.3 (ΜΗ ⁺ ).

1-1,2- (4-acetylamino-3-) acid (1,2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) amide

chloro-benzoylamino) -propionyl1-piperidine-2-carboxylic (102a).

Prepared from [2- (2- (25 benzyloxy-5-oxo-tetrahydro-furan-ylcarbamoyl) -piperidin-1-yl] -l-methyl-2-oxo-ethyl} carbamic acid tert-butyl ester and 4-acetylamino-3-chlorobenzoic acid by the procedure used in the preparation of 98a to give the title compound (71 mg, 47% yield) ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.10-1.97 ( m, 10H), 2.10-2.68 (m, 5H), 2.73-3.24 (m, 2H), 3.62-3.92 (m, 1H), 4.24-5, 27 (m, 5H), 5.48-5.59 (m, 0.5H),

5.75-5.85 (m, 0.5H), 6.51-6.61, (d, 1, H), 7.05-7.45 (m, 4, H), 7.52- 8.12 (m,

4H). Analytical HPLC 8.30 min. LC-MS (ES ⁺ ) m / e = 585.3 (MH ⁺ ). 1-f2- (4-amino-3-chlorobenzoylamino) -propioninpiperidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propioninpiperidine-102-carboxylic acid (102b).

Prepared as above for 102a to give the title compound (0.06%, 27% yield) as a yellow solid. ¹ H NMR (500MHz,

CDCI ₃ ) δ 1.2-1.8 (m, 7H), 2.1-2.6 (m, 2H), 2.7-3.2 (m, 4H), 3.6-4.0 (m, 1H),

4.3-4.9 (m, 7H), 5.0-5.8 (m, 2H), 6.5-7.0 (m, 2H), 7.2-7.8 (m, 8H ) ppm. Analytical HPLC (cyan column) 14.559 (39.6%), 15.198 (60.4%). LC-EM (ES ⁺ ):

103

COjBwt

IM lao

159

4-hydroxy-pyrrolidine-1,2-dicarboxylic ester 1-benzyl ester (104).

Compound 104 was prepared according to the procedure used to prepare compound 95.

A suspension of Cbz-Hyp-OH (4.854 g, 18 mmoles) in DMA (135 ml, benzyltriamonium chloride (4.105 g, 18 mmoles), K ₂ CO ₃ (64 g, 46 mmoles) and 2-bromo-2- methylpropane (99 ml, 859 mmoles) was stirred at 55 ° C for 18 hours The mixture was diluted with ice water and extracted with EtO10 Ac (3x). The organic phase was washed with water, NaHS (O) solution ₄ a 0.5 N and brine was dried and the solvent in vacuo to provide the title compound as a yellow oil (5.368 g, 98% yield) ¹ H NMR (500 MHz,

CDCIs) δ 1.33 (s, 5H), 1.47 (s, 4H), 2.01-2.4 (m, 1H), 2.22-2.38 (m, 1H), 3,503.72 (m, 2H), 4.34-4.45 (m, 1, H), 4.45-4.53 (m, 1H), 5.04-5.20 (m, 2H), 7.2215 7.42 (m, 5H). Analytical HPLC 10.14 min, LC-MS (ES ⁺ ) m / e = 322.2 (MH ⁺ ).

4-Fluoro-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester tert-butyl ester (105).

A solution of 104 (4.262 g, 13.96 mmoles) in CH ₂ CI ₂ (100 ml) at -78 ° C was treated with DAST (1.80 ml, 13.6 mmoles), was stirred for 1.0 min . then it was warmed to room temperature for 60 hours under N ₂ . The mixture was poured into ice-cold NaHCO ₃ (10% solution, 350 ml) and was extracted with CH ₂ CI ₂ (2X). The organic phase was washed with water, brine, dried over anhydrous Na ₂ SO ₄ and concentrated to give an oil

160 brown (4.299 g) which was purified by flash column chromatography on silica gene using hexanes / EtOAc (90/10 to 80/20%). The title compound was obtained as a yellow oil (2.805 g, 64% yield) ¹ H

NMR (500 MHz, CDCI ₃ ): δ 1.37 (s, 4.5H), 1.45 (s, 4.5H), 2.20-2.55 (m, 2H),

3.61 - 3.93 (m, 2H), 4.41, (d, 0.5H), 4.49 (d, 0.5H), 5.03-5.21, (m, 3H), 7,237.44 (m, 5H). Analytical HPLC 12.15 min. LC-MS (ES ⁺ ) m / e = 324.2 (MH ⁺ ) 1- (2-) acid 1-benzyl ester 2-tert-butyl ester

benzyloxycarbonylamino-propionyl) -4-fluoropyrrolidine-1,2-dicarboxylic (106).

A solution of 1.03 (2.72 g, 8.42 mmoles) in MeOH (50 ml) and 10 Pd / C 10% (1.27 g) was stirred under H ₂ for two hours then it was filtered through Celite and the solvent was evaporated to give a yellow oil (1.526 g).

This oil was dissolved in CH ₂ CI ₂ (30 ml) and treated with DIEA (1.5 ml, 8.6 mmoles), Cbz-ala-OH, (2.34 g, 10.5 mmoles) and EDC ( 2.32 g, 12 mmol) at 0 ° C. The mixture was stirred for an additional 10 mins. at 0 ° C it was then allowed to warm to room temperature and stir for 18 hours. The solvent was concentrated in vacuo and the residue was dissolved in EtOAc between

so it was washed with 0.5 N NaOH (O) ₄ (2X), saturated NaHC0 ₃ (2X) and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and evaporated to give a white solid which was purified by flash column chromatography, eluting using hexanes / EtoAc (80/20 at 60/40%). The title compound was isolated as a white solid (286 g, 86% 4-fluoropyrrolidine-1,2-dicarboxylic acid 2-tert-butyl ester) ¹ H NMR (500

MHz, CD ₃ 0D) δ 1.26 to 1.59 (m, 12H), 2.20 to 2.67 (m, 2H), 3.45 to 4.13 (m, 2H), 4,25- 4.47 (m, 1H), 4.58-4.72 (m, 1H), 4, 96-5.17 (m, 2H), 5.19-5.45 (m, H), 25 7 , 23-7.40 (m, 5H). Analytical HPLC 16.36 min. LC-RS (ES ⁺ ) m / e = 395.3 (MH ⁺ ).

1- (2- (4-Amino-3-chloro-benzoylamino) -propionyl4-fluoro-pyrrolidine-2-carboxylic acid tert-butyl ester (107).

A suspension of 106 (2.65 g, 6.72 mmoles) in MeOH (40 ml) and 10% Pd / C (1.32 g) was stirred under an atmosphere of H ₂ for 1.5 hours, filtered through celite and was concentrated to give a waxy solid (1,694

g). The solid was dissolved in CH ₂ CI ₂ (25 ml) and treated with DIEA (3.4 ml,

161

19.5 mmoles), 4-amino-3-chlorobenzoic acid (1.362 g, 7.9 mmoles), HOBT (1.164 g, 8.62 mmoles) and EDC (1.645 g, 8.5 mmoles) at 0 ° C under N2. The mixture was allowed to warm to room temperature and was stirred for 18 hours. The solvent was concentrated in vacuo. The residue was dissolved in Ethanol

Ac, was washed with water (4x), 0.5 N NaHS (O) 4 (2x), saturated NaHCO ₃ (2X) and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and evaporated to give a white solid which was purified by flash column chromatography, using CH ₂ CI ₂ / MeOH (99/1, 98/2%). The product obtained as a white solid (2.705 g, 97%). ¹ H NMR (500 MHz, CD ₃ OD) δ 1.33 (s, 9H), 10 1.48 (d, 3H), 2.31-2.55 (m, 2H), 3.93 (dd, 1, H), 4.02-4.21, (m, 1H), 4.59-4.76 (m, 1H), 5.31, (brs, 0.5H), 5.41, (brs , 0.5H), 6.78 (d, 1H), 7.57 (dd, 1H), 7.78 (s, 1, H), 8.31, (d, 1H). Analytical HPLC 14.14 min. LC-MS (ES ⁺ ) m / e = 414.2 (MH ⁺ ).

1- [2- (4-Amino-3-chlorobenzoylamino) -propionill-4-fluoro-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -amide (108a) .

Prepared from syn- (2-benzyloxy-5oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester and 107a after the method used for the synthesis of 98a. The title compound was isolated as a white solid (41 mg, 20 15% yield): ¹ H NMR (500 MHz, CD ₃ 0D) δ 0.94 (d, 0.3H), 1.07 (d,

1H), 1.40 (m, 1.7H), 2.21-2.65 (m, 2.2H), 2.70-2.85 (m, 1.4H), 2.96-3, 08 (m, 1.4H), 2.96-3.08 (dd, 0.4H), 3.57-4.24 (m, 3H), 4.41-4.93 (m, 4H), 5.14-5.45 (m, 1H), 5.60-5.67 (m, 0.6H), 5.77 (d, 0.4H), 6.77 (dd, 1H), 7, 15-7.41, (m, 5H), 7.51-7.62 (m, 1H), 7.77 (dd, 1H). Analytical HPLC 12.83min. LC-EM 25 (ES ⁺ ) m / e = 547.1, (MH ⁺ ) ·

162

1-F2- (4-Amino-3-chlorobenzoxyamino) -propionyl] -4-fluoro-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -propionyl (108b) .

Prepared from (2-benzyloxy-5-oxo5 tetrahydro-furan-3-yl) -carbamic acid allyl ester 107a after the method used for the synthesis of

98a. The title compound was isolated as a white solid (654 mg, 54% yield); ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.07 (d, 0.5H), 1.25 to 1.56 (m, 2.5H), 2.21 to 2.65 (m, 2, 3H), 2.68-2.89 (m, 1H), 2.91-3.10 (m, 0.7H), 3.57-4.23 (m, 2H), 4.32-4, 95 (m, 5H), 5.16-5.52 (m, 1, H), 5.45-5.50 (m, 0.3H), 5.5410 5.58 (m, 0.2H) 5.61 - 5.67 (m, 0.3H), 5.77, (d, 0.2H), 6.72-6.84 (m, 1H), 7,167.41, (m, 5H), 7.50-7-65 (m, 1H), 7.71-7.87 (m, 1H). Analytical HPLC 12.83 min: LC-MS (ES ⁺ ) m / e = 547.1.

Acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -amide

- [2- (4-Amino-3-chloro15 benzoylamino) -propionill-4-fluoro-pyrrolidine-2-carboxylic (108c),

Prepared from syn- (2-ethoxy-5-oxotetrahydro-furan-3-yl) -carbamic acid allyl ester and 107a after the method used for the synthesis of 98a to give the title compound (100.3 mg , 38% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.09 (t, 1.2H), 1.25 (t, 1.8H), 1.40 (d, 1H), 1.49 (d, 20 2H), 2.33-2.61, (m, 2H), 2.65-2.95 (m, 2H), 3.44-4.30 (m, 4H), 4.47-4.79 (m,

3H), 5.18-5.25 (m, 0.2H), 5.27-5.36 (m, 0.5H), 5.39-5.46 (m, 0.3H), 5, 56 (m, 1H), 6.72-6.94 (m, 0.8H), 7.54-7.69 (m, 0.8H), 7.79 (d, 0.55H), 8, 06 (d, 0.55H), 9.00 (d, 0.3H). Analytical HPLC 8.46 min. LC-MS (ES ⁺ ) m / e = 485.2 (MH ⁺ ).

163

[2- (2-Isopropyl-5-methyl-cyclohexyl) -5-oxo-tetrahydro-furan-3-ill-amide 1 [2- (4-amino-3-chloro-benzoylamino) -propionill-4- fluorine-pyrrolidine-2-carboxylic, (108d).

Prepared from {2- [1 R- (2S-isopropyl5R-methyl-cyclohexyloxy)] - 5-oxo-tetrahydro-furan-3-yl} -carbamic acid allyl ester and 107a after the method used for the synthesis 98a to give the title compound (95 mg, 31% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 0.42 (d, 2H), 0.57 (d, 2H), 0.60-1.10 (m, 10H), 1.22-1.76 (m, 6H), 1.96-2.17 (m, 1H), 2.29-2.60 (m, 2H), 10 2.61-2.88 (m, 1.5H), 3, 02-3.23 (dd, 0.5H), 3.37-3.47 (m, 0.5H), 3.50-3.61, (m,

0.5H), 3.63-4.24 (m, 2H), 4.48-4.62 (m, 3H), 5.18-5.48 (m, 1H), 5.72 (d,

0.4H), 5.82 (d, 0.6H), 6.77-6.84 (m, 1H), 7.53-7.67 (m, 1H), 7.78 (d, 0, 4H), 7.84 (d, 1H) analytical HPLC 8.34 min. LC-MS (ES ⁺ ) m / e = 595 (MH ⁺ ).

Scheme XX

{2- [2- (2-Ethoxy-5-oxo-tetrahydro-furan-3-ylylcarbamoyl) -pyrrolidin-1 -ill-1-methyl-2-oxo-ethyl} carbamic acid tert-butyl ester (109 ).

Prepared from acid allyl ester (2-ethoxy-5-oxo-

164 tetrahydro-furan-yl) carbamic 74 after the method used in the synthesis of 75 to give the title compound as a pale yellow solid (660 Mg, 73% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.14-1.36 (m, 6H), 1.42 (m, 9H), 1.75-2.29 (m, 4H), 2.48 (dd, 0.5H), 2.58 (dd, 0.5H), 2.72-2.85 (m, 0.5H), 2.99 5 (dd, 0.5H), 3.43- 3.91, (m, 4H), 4.07-4.52 (m, 2.5H), 4.53-4.72 (m, 0.5H), 5.37 (s, 0.5H) , 5.57 (d, 0.5H). Analytical HPLC (mixture of 2 diastereomers) 7.92, 8.14 min. LC-MS (ES ⁺ ) m / e = 414.3 (MH ⁺ ) (1- [2- (4-allyoxy-3-acid-2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -amide] , 5-dichlorobenzoylamino) -propionyl] pyrrolidine-2-carboxylic (110).

Prepared from 109 and 4-allyloxy-3,5-dichloro-benzoic acid after the method used in the synthesis of 82 to give the title compound as a white solid (228 mg, 65%). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.10-1.30 (m, 4H), 1.32-1.52 (m, 3H), 1.63-2.31, (m, 4H ), 2.41-2.50 (d, 0.5H), 2.52-2.61, (dd, 0.5H), 2.67-2.81, (m, 0.5H), 2 , 94-3.05 (dd, 0.5H), 3.47-3.96 (m, 4H),

4.21 - 4.81, (m, 5H), 5.22-5.32 (m, 1H), 5.35-5.49 (1.0, 1.5H), 5.55-5, 63 (m,

0.5H), 6.06-6.21, (m, 1H), 7.90 (s, 2H). Analytical HPLC (mixture of 2 diastereomers) 12.56 min. LC-MS (ES ⁺ ) m / e = 542.3 (MH ⁺ ).

1- [2- (3,5-dichloro-4-hydroxybenzoylamino) -propionyl-pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-111 (111).

(2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -amide 1-I2- (3 _l 5-dichloro-4-hydroxybenzoylamino) -propionillpirrolidina-2-carboxylic acid (111).

To a solution of 110 (194 mg, 0.36 mmol) in CH ₂ CI ₂ (5 ml) was added DMBA (70.7 mg, 0.45 mmol) and Pd (PPh ₃ ) ₄ (50.3 mg, 0.044 mmol) at 0 ° C. The solution was warmed to room temperature after 15 minutes, was stirred for two hours, was diluted with CH ₂ CI ₂ then washed with water (2x) and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and evaporated to give the crude product. Flash chromatography using

CH ₂ CI ₂ / MeOH (99/1 at 95/5%) provided the title compound (138.6 mg, 77% yield). ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.13-1.31, (m, 3H), 1.35-1.49 30 (m, 3H), 1.84-2.35 (m, 4H), 2.43-3.05 (m, 2H), 3.48-3.93 (m, 4H), 4.22-4.80 (m, 3H), 5.38 (d, 0, 4H), 5.46 (s, 0.1H), 5.55-5.61, (m, 0.5H), 7.76-7.94 (m, 2H). Analytical HPLC 8.70 min. LC-MS (ES ⁺ ) m / e = 502.2 (MH ⁺ ).

165

Scheme XXI

116b, X * CJ, Y * = AcNH, Z »K 116q X« C !, YMcNH. Z = CHjO

Compounds of 116a-116L were prepared as described a5 above for compounds 98 only using 1- (2-benzyloxycarbonylamino-propionyl) -4,4-difluoro-pyrrolidine-2-carboxylic acid tert-butyl ester (114) as a substitute for 1- (2benzyloxycarbonylamino-propionyl) -pyrrolidine-2-carboxylic acid tert-butyl ester (95).

Preparation of 1- (2-benzyloxycarbonylamino propionyl) -4,4-difluoro-pyrrolidine-2-carboxylic acid tert-butyl ester (114).

A solution of 2-tert-butyl ester of 4,4-difluoro-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester (113) (Karanowaky, et.al., J.

Med. Chem. 33, p. 1459-1469 (1990) (0.42 g, 1.23 mmol) and 10% palladium on carbon (0.22 g) in methanol (6 mL) was stirred at 1 atm hydrogen pressure for 3 hours. The mixture was filtered through Celite and was evaporated. The residue was dissolved in CH ₂ CI ₂ (4 ml) and DMF (2 ml) and was cooled to 0 ° C. 2-Benzyloxycarbonylamino-propionic acid (0.30 g, 1.35 mmol),

EDC (0.30, 1.54 mmoles). DIEA (0.65 mL) and HOBt (0.17 g, 1.23 mmol) were added and the reaction was stirred for 0.5 h at 0 ° C, then 16 h at room temperature

Zp

166 ambient temperature under nitrogen. The solvent was removed in vacuo and the residue was dissolved with ethyl acetate, then it was washed with sodium bisulfate a

10%, saturated sodium bicarbonate, water and brine, dried over sodium sulfate and evaporated. Purification by flash chromatography on silica,

eluted with 25:75 ethyl acetate: hexanes provided 1- [2-benzyloxycarbonylaminopropionyl) -4,4-difluoro-pyrrolidine-2-carboxylic acid tert-butyl ester (0.39 g, 77% yield) as an oil colorless. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.3-1.6 (m, 12H), 2.5 (m, 8H), 2.7 (m, 1.2H), 3.9 (m, 1H), 4.1, (m, 1H), 4.4 (m, 1H), 4.7 (m, 1H), 5.1, (m, 2H), 5.59 (br d, J = 7.7 Hz, 0.8H), 10 5.7 (br d, J = 7.7 Hz, 0.2H), 7.35 (m, 5H) ppm. Analytical HPLC (cyano column8) 17.069 Min. LC-MS (ES ⁺ ): m / e = 413 (M + H), 357 (M + H-tert-butyl), 313 [(M + H-CO ₂ ter- butyl) J.

1-1,2- (4-Amino-3-chloro-15-benzoylamino) -proprionill-4,4-difluoro-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) 116a).

Prepared from 115a and syn-40 to provide the title compound as an off-white solid (0.14 g, 73% yield). ¹ H NMR (500 MHz, CD30D) δ 1.0-1.5 (m, 3H), 2.0-3.5 (m, 4R + CH30H), 3.5-5.5 (m, 6H ⁺ H2O), 5.6-5.8 (m, 1H), 6.7-6.8 (m, 1H), 7.1-7.8 (m, 8H), 8.2-8.6 ( m, 20 1H) ppm. Analytical HPLC (cyan column) 13.744 min. LC-EM (ES ⁺ ); m / e =

565 (M ⁺ H).

1- (2- (4-acetylamino-3chloro-benzoylamino) -propionill-4,4-difluoro-pyrrolidine-2-carrboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) amide (116b ),

Prepared from 115b and syn-40 to provide the compound

167 of the title as an off-white solid (0.08 g, 38% yield). ¹ H

NMR (500 MHz, CDCI ₃ ) δ 1.03 (d, J = 6.9 Hz, 0.4H), 1.30 (d, J = 6.9 Hz,

0.6H), 2.25 (d, J = 2.9 Hz, 3H), 2.4-3.2 (m, 4H), 3.6-4.4 (m, 4H), 4.6 -4.9 (m,

3H), 5.52 (d, J = 5.2 Hz, 0.6H), 5.78 (d, J = 5.2 Hz, 0.4H), 6.6 (br s, 1H), 6 , 95 7.9 (m, 8H), 8.39 (d, J = 8.1 Hz, 0.4H), 8.44 (d, J = 8.3 Hz, 0.6H), 8.74 (d, J =

6.8 Hz, 1H) ppm. Analytical HPLC (cyan column) 11,830 min, LC-MS (ES ⁺ ): m / e = 607 (M + H).

1- [2- (4-acetylamino-510 chloro-2-methoxy-benzylamino) propionill-4,4-difluoro-pyrrolidine- (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -amide 2-carboxylic (116c).

Prepared from 115c and syn-40 to provide the title compound as an off-white (0.07 g, 29% yield). ¹ H NMR (500 MHz, CDCIs) δ 0.99 (d, J = 6.9 Hz, 1.35H), 1.32 (d, J = 6.9 Hz, 1.65H), 2.25 15 (m, 1.5H), 2.26 (s, 1.5H), 2.3-3.2 (m, 4H), 3.95 (s, 0.55H), 3.98 (s, 0.45H),

3.7-4.1, (m, 2.5H), 4.2-4.5 (m, 1.5H), 4.6-4.9 (m, 3H), 5.52 (d, J = 5.3 Hz, 0.55H), 5.80 (d, J = 5.3 Hz, 0.45H), 7.0-7.4 (m, 4H), 7.7-7.9 (m, 2H), 8.0-8.4 (m, 2H), 8.49 (d, J = 6.5 Hz, 1H), 8.93, (d, J = 6.7 Hz, 1H ) ppm. Analytical HPLC (cyan column) 12.959 min. LC-MS (ES ⁺ ): m / e = 637 (M + H).

1-1,2- (4-acetylamino-3-chlorobenzoylamino) -propionyl1-4,4-difluoro-pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) -propionyl (116d ).

Prepared from 115b and syn- (2-ethoxy5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester to provide the title compound as a 92: 8 mixture of epimers. Off-white solid (0.27 g, 66% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.0-1.5 (m, 6H), 2.25 (s, 1.8H),

168

-3.2 (m, 4H), 3.3-4.3 (m, 4H), 4.5-4.9 (m, 3H), 5.45 (d, J =

2.26 (s, 1.2H), 2.3

5.3 Hz, 0.75H), 5.59 (d, J = 5.2 Hz, 0.25H), 6.7-7.1, (m, 2H), 7.62 (dd, J = 8.7 Hz, 2.0 Hz, 1H), 7.76 (m, 1H), 7.85 (d, J = 2.0 Hz, 1H), 8.48 (m, 1H) ppm. Analytical HPLC (column C18) 13,300 (91.8%), 14.046 (8.2%) min. LC-EM 5 (ES ⁺ ): m / e 545 (M + H).

1- [2- (4-acetylamino3-chloro-benzoylamino) -propionill 4,4-difluoro-pyrrolidine-2-carboxylic acid (2-cyclohexyloxy-5-oxo-tetrahydro-furan-3-i [) 116e).

Prepared from 115b and syn- (210 cyclohexyloxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester to provide the title compound as a 93: 7 mixture of epimers. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.02.0 (m, 13H), 2.25 (s, 2H), 2.26 (s, 1H), 2.40 (dd, J = 17, 3, 10.1 Hz, 1H), 2.84 (dd, J = 17.3, 8.5 Hz, 1H), 2.5-3.0 (m, 2H), 3.5-4.3 (m, 5.5H), 4.5-4.9 (m, 2.5H), 5.59 (d, J = 5.3 H, 0.75H), 5.76 (d, J = 5 , 2 Hz, 0.25H), 6.74 (br d, J = 15 5.7 Hz, 0.25H), 6.93 (br d, J = 7.1 Hz, 1H), 7.06 (br d , J = 7.8 Hz, 0.75H),

7.62 (dd, J = 8.6, 2.0 Hz, 1H), 7.78, (m, 1H), 7.85 (d, J = 2.0 Hz, 1H), 8.35 ( br d, J = 6.6 Hz, 0.25H), 8.50 (br d, J = 8.2 Hz, 0.75H) ppm. Analytical HPLC (column C18) 17.112 (93%), 17.433 (7%) Min. LC-MS (ES ⁺ ): m / e = 599 (M + H).

1- (2- (4-Acetylamino-3-chloro-benzoylamino) -propionill4,4-difluoro-pyrrolidine- acid 2- (indanol-2-yl) oxy-5-oxo-tetrahydro-furan-3-ill-amide) 2 carboxylic (116f).

Prepared from 115b and [2- (indanol2-yl] oxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester to provide the compound of

169 title as a 62:38 mixture of epimers. Off-white solid (0.34 g, 71% yield). ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.09 (d, J = 6.9 Hz, 0.6H), 1.21, (d, J = 6.9 Hz, 0.9H), 1, 33 (d, J = 6.9 Hz, 0.9H), 1.42 (d, J = 6.9 Hz, 0.6H), 2.28 (s, 2H), 2.29 (s, 1H ), 2.40 (dd, J = 17.4, 10.3 Hz, 1H), 2.4-3.3 5 (m. 7H), 3.6-4.2 (m, 2H), 4 , 5-4.8 (m, 4H), 5.66 (m, 0.6H), 5.84 (d, J = 4.3 Hz,

0.2H), 6.22 (m, 0.2H), 6.7-7.0 (m, 2H), 7.2-7.3 (m, 4H), 7.5-7.7 ( m, 1H), 7.88.0 (m, 2H), 8.52 (m, 0.6H), 8.62 (br d, J = 6.5 Hz, 0.4H) ppm. Analytical HPLC (column C18) 16.556 (62.0%), 16.824 (38%) Min. LC-MS (ES ⁺ ): m / e = 633 (M + H).

1- [2- (4acetylamino-3-chloro-benzoylamino) -propionill-4,4-difluoro-pyrrolidine-2-carboxylic acid (2-cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl) (116q).

Prepared from 115b and syn- (215 cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester to provide the title compound as an off-white solid (0.20 g, 44% yield), ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.0-1.8 (m, 11H), 1.9-3.0 (m, 5H), 2.26 (s, 3H),

3.29 (m, 0.25H), 3.47 (m, 0.75H), 3.58 (m, 0.25H), 3.74 (m, 0.75H), 3.8 (m,

0.75H), 4.1 (m, 0.25H), 4.25 (m, 1H), 4.4-4.8 (m, 3H), 5.44 (d, J = 5.2 Hz,

0.75H), 5.62 (d, J = 5.2 Hz, 0.25H), 6.7 (br, 0.25H), 6.91 (d, J = 7.1 Hz, 1H),

7.1, (m, 0.75H), 7.59 (d, J = 8.5 Hz, 0.25H), 7.63 (dd, J = 8.5, 2.5 Hz, 0.75H),

7.75 (m, 1H), 7.86 (d, J = 1.8 Hz, 1H), 86.33 (br d, J = 6.5 Hz, 0.25H), 8.49 (br d , J = 8.4 Hz, 0.75H) ppm. Analytical HPLC (column C18) 17.705 min. LC-MS (ES ⁺ ): m / e = 599 (M + H).

1H

170 [2-phenylethoxy-5-oxo-tetrahydro-furan-3-yl) -amide 1- [2- (4-acetylamino-3-

chloro-benzoylamino) -propionin 4,4-difluoro-pyrrolidine-2-carboxylic (116h),

It was prepared from 115b and syn- (5oxo-2-phenethyloxytetrahydro-furan-3-yl) -carbamic acid allyl ester to provide the title compound as an off-white solid (0.15 g, 24% yield ). ¹ H NMR (500 MHz, CDCI3) δ 1.29 (d, J = 6.9 Hz, 0.75H), 1.40 (d, J = 6.9 Hz, 2.25H), 2.25 (s , 2.25H), 2.26 (s, 0.75H), 2.3-3.0 (m, 6H), 3.7-4.8 (m, 7H), 5.38 (d, J = 5.3 Hz, 0.75H), 5.67 (d, J = 5.1 Hz, 0.25H), 6.65 (m, 1H), 6.90 (d, J = 7.0 Hz, 0.75H), 7.06 (d, J = 7.6 Hz, 0.25H), 7.1-7.3 (m, 5H), 7.57 (d, J = 8.6 Hz, 10 0 , 25H), 7.63 (d, J = 8.6 Hz, 0.75H), 7.75 (m, 1H), 7.86 (d, J = 1.8 Hz, 1H),

8.35 (d, J = 6.2 Hz, 0.25H), 8.49 (d, J = 8.3 Hz, 0.75H) ppm. Analytical HPLC (column C18) 17.265 Min. LC-MS (ES ⁺ ): m / e 621, (M + H).

Scheme XXII

tert-butyl ylcarbamoyl) pyrrolidine-1-carboxylic (118).

ISOtoFsyn

2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3- acid ester

Prepared from 40 (1.16 g, 4.0 mmoles) and Boc-Pro-OH according to the procedure used to prepare 100 (scheme XVIII) to provide 1.53 g (94% yield) of the compound of the title as a white solid. ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.61 (b, 9H), 1.88 (br, 2H), 2.00-2.50 (m, 3H), 2.80-3.10 ( m, H), 3.20-3.60 (m, 2H), 4.05-4.45 (m, 1.5H), 4.58-4.80 (m, 1.5H), 4, 83-4.98 (m, H), 5.43-5.53 (d, H), 7.26-7.45 (m, 5H), 7.60-7.80 (d,

171

H); Analytical HPLC: 11.32 min; LC-MS: m / e = 405 (M + H ⁺ ). 2-Phenylaminopropionic acid (219).

The mixture of alanine (356 mg, 4.0 mmoles), iodobenzene (81.6 mg, 4.0 mmoles), tans-dichlorobis (tri-o-tolylphosphine) palladium (II) {Pd [P (o-tol) ₃ ] ₂

Cl ₂ ) (160 mg, 0.2 mmol), copper (I) iodine (40 mg, 0.2 mmol) K ₂ CC ₃ (552 mg,

4.0 mmoles), benzyltriethylammonium chloride (1.60 mg, 0.8 mmol), triethylamine (1.6 ml) and water (0.8 ml) in DMF (8 ml) was stirred under an atmosphere of nitrogen at 100 ° C for 20 hours. The mixture was cooled to room temperature, diluted with ethyl acetate (50 ml) and water (50 ml) was acidified 10 with 6 N HCI to pH = 2 to 3. The aqueous layer was extracted with ethyl acetate (50 ml x 4). The combined organic layers were washed with water, brine, dried over anhydrous Na ₂ SO ₄ , filtered and

evaporated in vacuo to give a red oil. Flash chromatography using hexane / ethyl acetate / acetic acid (95/5 / 0.5 to 80/20 / 0.5) to provide 300 mg (45% yield) of the title compound as a pink solid. ¹ H NMR (500 MHz, CDCI ₃ / C0 ₃ OD = 0.5 ml / 3 drops) δ 1.45 (d, 3H), 4.02-4.15 (m, H), 6.57-6 , 10 (m, 3H), 7.11-7.25 (m, 2H); Analytical HPLC: 61.0 min. LC-MS: m / e = 166 (M + H ⁺ ).

1- (2-phenylamino20 propionyl) -pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -pyrrolidine (120a and 120b).

A solution of 118 (405 mg, 1.0 mmol) was treated with TFA (2 ml) in CH ₂ CI ₂ (2 ml) for one hour. The reaction solution was evaporated in vacuo and azeotroped with CH ₂ CI ₂ four times to give pyrrolidine-2-carboxylic acid (2-benzyloxy-5oxo-tatrahydro-furan-3-yl) -amide as a pale yellow solid . ¹ H NMR (500 MHz, CDCI ₃ ) δ 1.87-2.17 (m, 4H), 2.302-70 (m, 2H), 2.80-3.08 (m, H), 3.45 ( br, 2H), 4.35-4.98 (m, 5H), 5.30-5.56 (m, H), 7.10-7.60 (m, 5H); Analytical HPLC 7.78 / 8.20 Min .; LC-MS: m / e = 305 (M + B +).

2-Phenylaminopropionic acid (119) (300 mg, 1.8 mmoles) in CH? CI ₂ (10 ml) 30 was treated with HOBT (270 mg, 2.0 mmoles) and EDC (2.1 q, 11 mmoles) at 0 ° C for 10 mins.

Diisopropylethylamine (2 ml) was added followed by a solution

172 pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl) -amide in CH2 Cl2 (1.0 mL). The mixture was stirred at room temperature for 4 hours, diluted with CH2 Cl2 (40 ml), washed with water then brine. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated in vacuo to give a pale yellow solid. Flash chromatography using CH ₂ CI ₂ / Methanol (99/1 to 98/2) provided 151 mg (33% yield) of antidiastereomer of the title compound (120a) and 129 mg (29% yield) in syn diastereomer (120b) as a white solid. ¹ H NMR (500 MHz, CDCI3) for the antidiastereomer: δ 1.37-1.41, (m, 3H), 1.50-2.45 (m, 4H), 3.60-2.70 (m , 0.3H), 2.89-2.94 (m, 0.7H), 3.40-3.80 (m, 2H), 4.104.50 (m, 3H), 4.50-4.90 (m, 3H), 5.26 (s, 0.3H), 5.38 (s, 0.7H), 6.45-6.60 (m, 2.3H), 6.65-6.80 (m, H), 7.10-7.20 (m, 2.5H), 7.25-7.50 (m, 4.5H), 7.53-7.70 (m, 0.7H, ), 7.82 (d, H). For the diastereomer syn: δ 0.86-0.89 (m, H), 1.201.40 (m, 4H), 1.80-2.45 (m, 4H), 2.80-2.86 (m , H), 3.56-3.65 (m, 2H), 4.2015 4.40 (m, H), 4.50-4.75 (m, 2H), 4.90 (d, H) , 5.52 (d, H), 6.45-6.70 (m, 3H),

6.75-6.85 (m, H), 7.30-7.20 (m, 2.3H) 7.10-7.50 (m, 5.7H); Analytical HPLC: 10.55 min for antidiastereomer and 10.62 min for syn diastereomer; LC / MS: m / e = 452 (M + H ⁺ ) for both diastereomers.

4-ΟΧΟ-34Τ1- (2-phenylamino-propionyl) -pyrrolidine-2-carbonyl1-amino} butyric acid (121),

Prepared from 120 (151 mg, 0.33 mmol) using hydrolysis method A to provide 1.01 mg (83% yield) of the title compound as a white solid. ¹ H NMR (500 MHz, CDCI ₃ / CD ₃ OD = 1/1): δ 1.20-1.65 (m, 2H), 1.65-2.35 (m, 3H), 2.40- 3.00 (m, H), 3.20-3.80 (m, 2H), 25 3.90-4.90 (m, 7H), 7.25-7.80 (m, 5H); Analytical HPLC: 6.38 min. LC-MS: m / e = 362 (M + H ⁺ ).

General Procedures for the Preparation of Compounds of Embodiment C

Formula I (Schemes XXIII-XXV)

Scheme XXIII

173

Hydrolysis Method A:

A 0.005-50 mole sample of the alkylemiacetal was dissolved in HCl at 2.5 N / CH ₃ CN (10/1) and stirred at room temperature until the reaction is complete. The resulting aqueous layer was washed with diethyl ether (2 x 20 ml) and was lyophilized to provide the product.

Hydrolysis Method B:

A sample of 0.005-50 moles of alkylemia ketal was placed in pure formic acid and was stirred overnight at room temperature. The mixture was ground with a 3: 1 mixture of hexane / ether

diethyl to give a precipitate. The solvent was decanted and the precipitate was washed with diethyl ether to provide the product.

Hydrolysis Method C,

A sample of 0.005-50 moles of alkylemiacetal was dissolved 15 in CH ₃ OH and 20% Pd (OH) ₂ / C and stirred under H ₂ until the reaction is complete. The resulting suspension was filtered and the solution was concentrated in vacuo, then it was triturated with a 3: 1 mixture of hexane / diethyl ether to give a precipitate. The solvent was decanted and the precipitate was washed with diethyl ether to provide the product.

Hydrolysis Method D

A sample of 0.005-50 moles of alkylemiacetal in CH ₃ CN / water (½) was stirred with acidic resin (Dowex 50w x 2, type H ⁺ ) until the reaction is complete. The solution was filtered and the resin was washed with CH ₃ CN / water (¼). The resulting aqueous layer was washed with diethyl ether, 25 was concentrated to give a smaller volume in a vacuum then it was lyophilized (1½

174 to supply the product.

Scheme XXIV

________ 4-oxo-3 - [(1- {2- [9-oxo-9H-fluorene-4-carbonyl) -amino1-propionyl} pyrrolidine-2-carbonyl) -amino1-butyric acid (122a).

A 109.0 mg (0.19 mmol) sample of 91, was hydrolyzed according to method A to provide 88 mg (96% yield) of the title compound; Analytical HPLC 7.15min. LC-MS (ES ⁺ ) m / e = 492.2 (M + E).

12 »

_________ 3 - ({142- (4-amino-3-chloro-benzoylamino) -propionyl1-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric acid (122b),

A 51.0 mg (0.096 mmol) sample of 76 was hydrolyzed according to method A to provide 43.0 mg (100% yield) of the title compound: ¹ H NMR (500 MHz, CD3OD / D2O; 0.5 ml_ / 10 drops): δ 1,371.52 (m, 3H), 1.80-2.20 (m, 3H), 2.20-2.37 (m, H), 2.49-2 , 60 (m, H), 2.602.75 (m, H), 3.70-3.80 (m, H), 3.80-3.95 (m, H), 4.20-4-35 (m, H), 4.40-4.50 (m, H), 4.50-4.70 (m, H), 4.70-4.85 (m, H), 6.85-6 , 87 (d, H), 7.58-7.60 (m, H), 7.77 (s, H); retention time on analytical HPLC: 6.54 min; LC-MS: 20 m / z = 439 (M + H ⁺ ).

175

3 - ({1- [2- (3,5-dichloro-4-motoxy-benzoylamino) propionyl) -pyrrolidine-2carbonyl} -amino) -4-oxo-butyric acid (122c).

A 51.0 mg (0.088 ml) sample of 92 was hydrolyzed from a5 according to method A to provide 24.0 mg (56% yield) of the title compound: analytical HPLC 6.41 min. LC-MS (ES ⁺ ) m / e = 488.3 (M + H).

3 - ({1-1,2- (4-methoxy-3,5-dimethyl-benzoylamino) propionyl1-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric acid (122d),

A 55.0 mg (0.102 mmol) sample of 77 was hydrolyzed from

according to method A to provide 44.0 mg (96% yield) of the title compound: analytical HPLC (C18) 8.70 min, ¹ H NMR (CDCh, 500 MHz): δ 1.23-1.70 (m, 3H), 1.80-2.70 (m, 10H), 2.70-3.15 (m, 2H), 3.58-4.20 (m, 5H), 4.32-5 , 50 (m, 3H), 5.60-6.00 (m, H), 6.80-7.90 (m, 4H), LC-EM 15 (ES ⁺ ) m / e = 448.2 ( M + H).

________ 4-oxo-3- (í1- {2-Pyridine-2-carbonyl) -amino1-propionyl} pyrrolidine-2carbonyl) -aminol-butyric acid (122e),

A 55.0 mg (0.114 mmol) sample of 88 was hydrolyzed according to method A to provide 30.0 g (67% yield) of the title compound: analytical HPLC 4.60 min. LC-MS (ES ⁺ ) m / e = 391.3 (M + H).

176

3 - ({1- [2- (4-acetylamino-3-chloro-benzoylamino) propionyl1-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric (122f),

A 52 mg (0.091 mmol) sample of 78 was hydrolyzed according to method A to provide 40 mg (91,% yield) of the title compound; ¹ H NMR (500 MHz, CD ₃ OD) δ 1.08-1.61, (m, 3H), 1.77-2.41, (m, 3H), 2.21, (s, 3H), 2.41 - 2.77 (m, 2H), 3.43 - 3.63 (m, 0.3H), 3.65 - 3.76 (m, 1, H), 3.81 - 3.94 (m, 1H), 4.18A, 34 (m, 1H), 4.42-4.64 (m, 1.7H), 4.77 (q, 1H), 7.79 (dd, 1H); Analytical HPLC 4.97 min. LC-MS (ES ⁺ ) m / e = 481.3 (M + H).

3 - ({1-f2- (4-amino-3,5-dichloro-benzoylamino) propionill-pyrrolidine-215 carbonyl} -amino) -4-oxo-butyric (122q),

A 44.3 mg (0.079 mmol) sample of 89 was hydrolyzed according to method A to provide 30 mg (81% yield) of the title compound: analytical HPLC 5.40 min. LC-MS (ES ⁺ ) m / e = 473.2 (M + H).

3 - ({142- (3-isopropoxy-benzoylamino) -propionill-pyrrolidine-2-carbonyl} amino acid) -4-oxo-butyric (122h),

A 52.0 mg (0.097 mmol) sample of 79 was hydrolyzed according to method A to provide 30.0 mg (69% yield) of the title compound. Analytical HPLC 8.92 min, LC-MS (ES ⁺ ) m / e = 448.3 (M + H).

177

3 - ({1- [2- (3-benzyloxy-4-methoxy-benzylamino) -propionin-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric acid (122i),

A 50.8 mg (0.082 mmol) sample of 81, was hydrolyzed 5 according to method A to provide 22.4 mg (52% yield) of the title compound: analytical HPLC 6.72 min. LC-MS (ES ⁺ ) m / e = 526.3 (M + H).

4-Oxo-3 - [(1 - [(2-quinoxaline-2-carbonyl) -aminolpropionyl} -pyrrolidine-2carbonyl} -amino] -butyric acid (122j).

A sample of 38.0 mg (0.072 mmol) of 80 was hydrolyzed according to method A to provide 32.0 mg (100% yield) of the title compound: analytical HPLC 5.95 min. LC-MS (ES ⁺ ) m / e = 442.3 (M + H).

3 - ({142- (3,5-dichloro-4-hydroxy-benzoylamino) propionill-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric acid (122k),

A 35 mg (0.060 mol) 83 sample was hydrolyzed according to method A to provide 29.4 mg (75% yield) of the title compound; Analytical HPLC 7.91 min. ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.47 (m,

178

3Η), 1.8-2.3 (m, 4H), 2.49 (m, 1H), 2.61, (m, 1H), 3.5 (br m, 0.2H), 3.69 (br m, 0.9H), 3.84 (br m, 0.9H), 4.27 (m, 1H), 4.46 (m, 1H), 4.57 (m, 1H), 4, 73 (m, 1H), 7.83 (m, 2H) ppm, LC-MS (ES ⁺ ) m / e = 474.1 (M + H).

3 - ({1-r2- (4-amino-3-trifluoromethyl-benzoylamino) propionill-pyrrolidine-2carbonyl} -amino) -4-oxo-butyric (1221),

A 10 mg (0.021 mmol) 98w sample was hydrolyzed according to method A to provide 7.9 mg (94% yield) of the title compound: analytical HPLC 6.64 Min. LC-EM (ES ⁺ ) m / e = 473.3 (M + H).

3 - ({1-r2- (3-chloro-4-dimethylamino-benzoylamino) propionill-pyrrolidine-2carbonyl) -amino) -4-oxo-butyric (122m),

A sample of 10.0 mg (0.021 mmol) of 98x was hydrolyzed according to method A to provide 7.0 mg (84% yield) of the title compound: analytical HPLC 5.15 min. LC-MS (ES ⁺ ) m / e = 467.3 (M + H).

3 - ({1- [2- (4-dimethylamino-3,5-difluoro-benzoylamino) -propionyl-2-carbonyl} amino) -4-oxa-butyric acid (122n).

A sample of 20.0 mg (0.043 mmol) of 98y was hydrolyzed according to method A to provide 16.8 mg (100% yield) of the title compound. Analytical HPLC 5.86 Min. LC-MS (ES ⁺ ) m / e = 469.3 (M + H).

179

122q

_________ 3 - ({1- [2- (4-amino-3-chloro-benzoylamino) -propionylpyrrolidine-2carbonyl} -amino) -4-oxo-butyric acid (122o),

A sample of 20.0 mg (0.046 mmol) of 98m was hydrolyzed according to method A to provide 16.7 mg (100% yield) of the title compound: analytical HPLC 8.47 min. LC-MS (ES ⁺ ) m / e = 439.2 (M + H).

Acid 3 - ({142- (4-amino-2,3,5 ₁ 6-tetrafluoro-benzoylamino) propionin-pyrrolidine

2-carbonyl} -amino) -4-oxo-butyric (122p),

A sample of 20.0 mg (0.042 mmol) 98z was hydrolyzed to

according to method A to provide 15.3 mg (91% yield) of the title compound: analytical HPLC 7.90 min. LC-MS (ES ⁺ ) m / e = 477.2 (M + H).

4-Oxo-3 - [(1- {2 - [(quinoline-6-carbonyl) -aminol-propionyl} -pyrrolidine-2carbonyl) -aminol-butyric acid (122q),

A sample of 44 mg (0.080 mmol) of 93 was hydrolyzed according to method A to provide 41 mg (100% yield) of the title station COM20: ¹ H NMR (500 MHz, CD ₃ 0D) δ 1.24 -1.69 (m, 3H), 1.75-2.37, (m, 4H), 2.39-2.87 (m, 2H), 3.46-4.04 (m, 2H), 4.11 - 4.77 (m, 3H), 8.19 (dd, 1H), 8.33 (d, 1H), 8.56-8.58 (m, 1H), 8.85 (s, 1H), 9.27-9.39 (m, 2H); HPLC

180 analytical ole 4.91, min. LC-MS (ES ⁺ ) m / e = 441.2 (M + H).

_______ 3 - ({1- [2- (4-acetylamino-5-chloro-2-methoxy-benzoylamino) -propioninpyrrolidine-2-carbonyl} -amino) -4-oxo-butyric acid (122r).

A sample of 44.5 mg (0.074 mmol) 87 was hydrolyzed according to method A to provide 34.5 mg (91% yield) of the title compound: analytical HPLC 6.88 min. LC-MS (ES ⁺ ) m / e = 511.2 (M + H).

3 - [(1- {2- [3-chloro-4- (2,2-dimethyl-propionylamino) benzoylaminol-propionyl} pyrrolidine-2-carbonyl) amino] -4-oxo-butyric acid (222s).

A sample of 19.0 mg (0.036 mmol) of 98aa was hydrolyzed according to method A to provide 1.45 mg (90% yield) of the analytical HPLC title compound 7.28 min. LC-MS (ES ⁺ ) m / e = 523.3 (M + H).

3 - ({1-1,2- (3-chloro-4-propionylamino-benzoylamino) propionill-pyrrolidine2-carbonyl} -amino) -4-oxo-butyric (122t),

A 21.0 mg (0.042 mmol) sample of 98ab was hydrolyzed according to method A to provide 17.5 mg (97% yield) of the title compound: analytical HPLC 5.72 min. LC-MS (ES ⁺ ) m / e = 495.2 (M + H).

181

1Z2U

3 - ({1 - [2- (3-Chloro-4-phenylacetylamino-benzoylamino) -propionill-pyrrolidine2-carbonyl} amino) -4-oxo-butyric acid (122u),

A sample of 10.0 mg (0.017 mol) of 98ac was hydrolyzed according to method A to provide 7.9 mg (85% yield) of the title compound: analytical HPLC 7.52 min. LC-MS (ES ⁺ ) m / e = 557.2 (M + H).

________ 3-i (1- {2-r3-chloro-4- (3-methyl-butyrylamino) benzoylamino1-propionyl} pyrrolidine-2-carbonyl} -amino1-4-oxo-butyric acid (122v).

A sample of 8.0 mg (0.015 mmol) of 98ad was hydrolyzed according to method A to provide 6.5 mg (96% yield) of the title compound: analytical HPLC 6.92 min. LC-MS (ES ⁺ ) m / e = 523.2 (M + H). Scheme XXV

108 ·. X »ci, Y-NH1 Z« H, Qt “F, Qj-H 118b, X« CI, Y-AcNH, Z = H, Qt »Qi * 1180, X« C (, Y = ACNH, Z ^ CHsO .0, = 01 = ^

123 ^ X * CI, Υ-ΝΗχ Z-H, Qi = F. QsPH 123b, X «CI, Y-AcNH, Ζ · Η. Qi «CbF 123C, X» CI, Y = ACNH, Z »CHjO, Qi = Qí“ F

3 - ({1-1,2- (4-amino-3-chloro-benzoylamino) -propionyl] -4-fluoro-pyrrolidine-2-carbonyl} -amino) -4-oxo-butyric (123a),

A 12.4 mg (0.022 mol) sample of 108b was hydrolyzed according to method A to provide 9.6 mg (93% yield) of the title compound: analytical HPLC 6.99 min. LC-MS (ES ⁺ ) m / e = 473.2 (M + H).

Acid ______ 3 - ({1-r2- (4-acetylamino-3-chloro-benzoylamino) propionill-4,4-diflúor3ό3

182 pyrrolidine-2-carbonyl} -amino) 4-oxo-butyric (123b).

A 26.2 mg (0.043 mmol) sample of 116b was hydrolyzed according to method A to provide 10.8 mg (49% yield) of the title compound: analytical HPLC 9.89 min. LC-MS (ES ⁺ ) m / e = 517.2

3 - ({1-r2- (4-acetylamino-3-chloro-2-methoxy-benzoylamino) propionill-4,4difluoro-pyrrolidine-2-carbonyl} -amino) 4-oxo-butyric (123c),

A 23.1 mg (0.036 mmol) sample of 116c was hydrolyzed according to method A to provide 1.8 mg (9% yield) of the title compound: analytical HPLC 11.87 min. LC-MS (ES ⁺ ) m / e = 547.1 10 (M + H).

Biological Methods

In vitro, ex vivo and in vivo data for selected compounds of this invention are obtained using the methods described below. The results are shown in tables 2-8. The designation ND indicates that the compound was not tested in the described test.

In ICE Caspase assays, category A indicates inhibition of <10-1000 nM. Category B indicates inhibition of 10-1000 mM. Category C indicates inhibition of> 1000 nM. See tables 2 and 3.

In the PBMC assay, category A indicates inhibition of <500 nM.

Category B indicates inhibition of 500-1000 nM. Category C indicates 1001-2000 nM inhibition. Category D indicates inhibition of> 2000 nM. See table 4.

In the whole blood assay, category A indicates inhibition of <2500 nM. Category B indicates inhibition of 2500-7500 nM. Category C indicates> 7500 nM. See table 5.

In the in situ metabolism assay, values of [f (g) x f (h)] are revealed as follows: category A indicates <0.25. Category B indicates 0.25-0.49. Category C indicates 0.5-0.75. Category D indicates> 0.75. In the measurement of bile excretion category A indicates <5%. Category B indicates 5-10%. Category C indicates> 10%. See table 6.

In the i.v. clearance test, values are reported as follows: category A indicates <50. Category B indicates 50-80. Category C indicates> 80. See table 7.

183

In the bioavailability test, Cmax values (pg / ml) are revealed as follows: category A indicates <2.5. Category> 5.0. AUC values frg x hr / ml) are revealed as follows: category A indicates <2.5. Category B indicates 2.5 - 5.0. Category C indicates> 5.0. Half-life (h) ranges are revealed as follows: category A indicates <1.5. Category B indicates 1.5 - 2.0. Category C indicates> 2.0. F (%) values are revealed as follows: category A indicates <33. Category B indicates 33-67. Category C indicates> 67. See table 8.

In Vitro Tests

Enzyme Inhibition

Ki values for test compounds with the various caspases were obtained by the method of Margolin et al., (J. Biol. Chem., 272, pp. 7223-7228 (1997)). Assays were performed on 10 mM Tris (Sigma Corpo. St. Louis MO) pH 7.5, 1 mM Dithiothreitol (DTT, Research Organic INC.

Cleveland, OH) and 0.1% CHAPS (Pierce, Rockford IL) at 37 ° C. For caspase-3, an 8% glycerol solution was added to the assay buffer to improve enzyme stability. A 65 μΙ aliquot of the assay buffer and 5 μΙ aliquot of the appropriate dilutions of inhibitor in DMSO were pipetted into a 96 well plate, treated

with 10 μΙ of caspase, then they were diluted in assay buffer (active protein at 0.5- 40 nM by active site titration). A control containing DMSO but no compound was included for each determination. The plates were then incubated for 15 minutes at 37 ° C, before adding the appropriate substrate (20 μΙ, final concentration 1-4 XK _M , final test volume 100 μΙ) to initiate the reaction. Reaction rates were measured at 37 ° C or following the time dependent increase in absorbance at 405 nM (for pNA substrates) or fluorescence (Ex 390, Em 460) (for substrates of

AMC). The obtained rates were plotted against the inhibitor concentration and the data were fitted to the tight binding equation for competitive inhibitors (Morrison, J. F. Biochem. Biophys. Acta, 185 pages. 269-286 (1969)). The substrates used for the individual tests were as follows:

184

Caspase-1 Suc-YVAD-pNA (Bachem, King of Prussia, PA) (final concentration in the 80 μΜ assay).

Caspase-3 AC-DEVD-Pna (Bachem, King of Prussia, PA) (final concentration in the assay, 60 μΜ).

Caspase-4 Ac-WEHD-AMC (Synpep, Dublin, CA) (final concentration in the 20 μΜ assay).

Caspase-7 Ac-DEVD-AMC (Bachem, King of Prussia, (final concentration in the 50 μΜ assay).

Caspase-8 Ac-DEVD-pNA (Bachem, King of Prussia, (

final tensile strength in the 80 μΜ test).

Table 2. Caspase-1 inhibition data

Example Caspase-1-ki (nM) 5th THE 5b THE 5c THE 5d THE 5e B 5f B 5g B 5 am THE 5i THE 5j THE 5k THE 5I B 5 m THE 5n THE ⁵⁰ B 5p B 5q B 5r B 5s B 5t Ç 5u B 5v B

185

Example Caspase-1-ki (nM) 5w B 5x THE 5y THE 5z THE 5aa THE 5ab B 5ac THE 5ad THE 5ae B 5af B 5ag THE 5ah B 5ai THE 5aj B 5ak B 5al THE 5am THE 5na B 5th B 5ap B 5aq B 5th THE 5th THE 5at B 5au B 5av B 5aw THE 5ax THE 5ay THE 5az THE 5ba THE 5bb THE 5bc B 5bd THE

186

Example Caspase-1-ki (nM) 7th THE 7b B 7c THE 7d THE 7e B 7f B 7g THE 7am B 7i B 7j Ç 7k B 7I B 7m B 7n B 7th THE 7p THE 7q B 7r B 7s B 7t B 7u B 7V B 7W THE 7x B 7y B 7z B 7th THE 7ab B 7ac B 7th B 7ae B 7af B 7ag B 7ah THE

187

Example Caspase-1-ki (nM) 7ai THE 7aj THE 7ak THE 7al B 7am THE 7na THE 7th B 7ap B 7aq B 7th THE 7th THE 7at THE 9a B 9b THE 9c THE 9d THE 9 and THE 9f THE 9g THE 15th THE 15b THE 15c THE 15d B 15e B 15f B 16th B 16b THE 17th B 17b B 17c THE 17d B 17e B 18th B 18b THE

188

Example Caspase-1-ki (nM) 18c B 18d B 18th THE 18f B 20th THE 20b THE 20c THE 20d B 20e THE 20f THE 20g THE 8 pm THE 20i B 20j B 20K THE 20I THE 20m THE 20n THE 20th THE 20p THE 20q B 20r B 20s THE 20t B 23a THE 23b B 23c THE 23d THE 23e THE 23f B 23g THE 11 pm THE 23i B 24a THE

> 40

189

Example Caspase-1-ki (nM) 24b Ç 24c THE 24d B 24e B 25a THE 25b THE 25c THE 25d THE 25e THE 26a THE 26b THE 26c THE 26d THE 26e THE 26f THE 26g THE 26h THE 27a B 27b B 27c B 27d THE 27e B 27f B 27g THE 27h B 27i B 27j B 27k B 27I B 27m B 27n B 28th THE 28b THE 28c THE

190

Example Caspase-1-ki (nM) 29th THE 29b THE 29c THE 29d THE 29e THE 29f THE 29g THE 29h THE 29i THE 29k THE 29I B 29m THE 29n B 29th B 29p THE 29q THE 29r THE 29s B 32a Ç 32b Ç 32c Ç 32d Ç 32e B 34 Ç G1 Ç G2 B 42 B 46b THE 46a Ç 57 THE 65 THE 61 THE 69 THE 73 THE

191

Example Caspase-1-ki (nM) 121 Ç 122a THE 122b THE 122c THE 122d THE 122e B 122f THE 122g THE 122h B 122i THE 122j B 122k THE 1221 B 122m B 122n B 122o Ç 122p THE 122q B 122r B 122s B 122t B 122u THE 122v B 123a B 123b B 123c B

Table 3. Caspase-3, Caspase-4 and Caspase-8 Inhibition Data

Example Caspase-3 ki (nM) Caspase-4 ki (nM) Caspase-8 ki (nM) 7c Ç ND Ç 7d Ç ND B 7f ç ND Ç 24a ç ND ND 29th ç ND ND

513

192

Example Caspase-3 ki (nM) Caspase-4 ki (nM) Caspase-8 ki (nM) 29b Ç ND ND 32d B ND ND 46b B ND ND 69 Ç ND B 122b Ç THE B 122d Ç THE Ç 122f Ç ND B 122k Ç ND B

PBMC Cell Assay

IL-1beta assay with a mixed population of human peripheral blood mononuclear cells (PBMC) or enriched adherent mononuclear cells.

Pre-IL-1beta processing by ICE can be measured in cell culture using a variety of cell sources. Human PBMC obtained from healthy donors provides a mixed population of lymphocyte and mononuclear cell subtypes that produce a spectrum of interleukin and cytokines in response to many classes of physiological stimulators. Adherent mononuclear cells from PBMC provide an enriched source of normal monocytes for selective studies of cytokine production by activated cells.

Experimental Procedures:

An initial dilution series of test compound in DMSO or ethane is prepared, with a subsequent dilution in 10% RPMI-FBS media (containing 2 mM L-glutamine, 10 mM HEPES, 50 U and 50 ug / ml pen / strep) respectively to deliver drugs at 4x final test concentration containing 0.4% DMSO or 0.4% ethanol. The final concentration of DMSO is 0.1% for all drug dilutions. A concentration titration that groups K, apparent for a test compound determined in an ICE inhibition assay is generally used for primary compound screening.

In general 5-6 dilutions of compound are tested and the component

193

Cell assay is performed in duplicate, with ELISA determinations in duplicate on each cell culture supernatant.

PBMC isolation and IL-1 assay:

Buffer coating cells isolated from a pint of human blood (providing 40 - 45 ml of final volume plasma plus cells) are diluted with 80 ml media and LeukiPREP (Becton Dickinson) separation tubes are each coated with 10 ml of cell suspension. After centrifugation for 15 minutes at 1500-1800 xg, the plasma / media layer is aspirated and then the mononuclear cell layer is collected with a Pasteur pipette and transferred to a 15 ml conical centrifuge tube (Corning). Means are added to bring the volume to ml, lightly mix the cells by inversion and centrifuge at 300 xg for minutes. The PBMC pellet is resuspended in a small volume of media, cells are counted and adjusted to 6 x 10 ⁶ cells / ml.

For the cell assay, 1.0 ml of the cell suspension is added to each well of a 24-well flat-bottom tissue culture plate (Corning), 0.5 ml of test compound dilution and 0.5 ml of LPS solution (Sigma No. L-3012; 20 ng / ml of solution prepared in complete RPMI media; final LPS concentration of 5 ng / ml). Additions of

0.5 ml of test compound and LPS are usually sufficient to mix the contents of the wells. Three control mixtures are performed by experiment, or with LPS alone, solvent vehicle control and / or additional means to adjust the final culture volume to 2.0 ml. Cell cultures are incubated for 16-18 h at 37 ° C in the presence of 5% CO ₂ .

At the end of the incubation period, cells are collected and transferred to 15 ml conical centrifuge tubes. After centrifugation for 10 mins at 200 xg, supernatants are collected and transferred to 1.5 ml Eppendorf tubes. It can be seen that the cell pellet can be used for biochemical assessment of pre-IL-1beta and / or mature IL-1beta content in cytosol extracts by Western blotting or ELISA with specific pre-IL-1beta antisera .

Isolation of Adherent Mononuclear Cells:

194

PBMCs are isolated and are prepared as described above.

Media (1.0 ml) are first added to wells followed by 0.5 ml of the PBMC suspension. After a 1 hour incubation, plates are gently shaken and non-adherent cells are aspirated from each well. Wells are then gently washed three times with 1.0 ml of media and resuspended in 1.0 ml of media. Enrichment for adherent cells generally provides 2.5-3.0 x 10 ⁵ cells per well. The addition of test compounds, LPS, cell incubation conditions and processing of supernatants proceeds as described above.

ELISA:

Quanticine kits (R&D Systems) can be used for the measurement of mature IL-1 beta. Tests are carried out according to the manufacturers' directions. Mature IL-1 beta levels of about 1-3 ng / ml in both PBMC and positive adherent mononuclear cell controls are observed. ELISA assays are performed on 1: 5, 1:10, and 1:20 dilutions of supernatants from positive LPS control to select the optimal dilution for the supernatants on the test panel.

The inhibitory potency of the compounds can be represented by an IC ₅₀ value, which is the concentration of inhibitor at which 50% of mature IL1beta is detected in the supernatant when compared to the positive controls.

The skilled person realizes that values obtained in cell tests can depend on multiple factors. Values cannot necessarily represent higher quantitative results.

Table 4 PBMC Cell Assay Data

Example PBMC IC50 (nM) 5th D 5b B 5c B 5d B 5e B 5f Ç 5g Ç

195

Example PBMC IC50 (nM) 5 am THE 5i Ç 5j D 5k D 5I THE 5 m THE 5n B 5r D 5s B 5u Ç 5v D 5w B 5x B 5y B 5z B 5aa B 5ab B 5ac THE 5ag B 5ai THE 5aj B 5ak B 5al D 5am D 5th D 5aq D 5th D 5th D 5at D 5au D 5av D 5aw Ç 5ax B 5ay B

196

Example PBMC IC50 (nM) 5az B 5ba Ç 5bb B 5bd Ç 7th D 7b B 7c THE 7d THE 7e D 7f D 7g THE 7am B 7k B 7I B 7m B 7n B 7th THE 7p D 7q D 7s B 7t D 7u D 7V D 7W Ç 7x D 7y D 7z Ç 9a B 9b B 9c THE 9d B 9 and Ç 9f B 9g Ç

197

Example PBMC IC50 (nM) 15th D 15b Ç 15c B 15d Ç 15e D 15f D 16th THE 16b Ç 17b Ç 17c D 17d D 17e B 18th B 18b B 18c B 18d B 18th Ç 18f D 20th B 20b B 20c Ç 20d D 20e Ç 20f B 20g THE 8 pm THE 20i B 20j D 20K THE 20I B 20m B 20n B 20th THE 20p THE

198

Example PBMC IC50 (nM) 20q B 20r B 20s Ç 20t B 23a Ç 23b B 23c Ç 23d B 23e B 23f Ç 23g Ç 11 pm Ç 23i THE 24a B 24b D 24c THE 24d B 24e Ç 25a THE 25b B 25c B 26a Ç 26b B 26c B 26d B 26e THE 26f THE 26g THE 26h THE 27a THE 28th B 28b B 28c B 29th THE

199

Example PBMC IC50 (nM) 29b Ç 29c B 29d B 29e THE 29g B 29h THE 29i THE 29j B 29k THE 29I B 29m B 42 D 46b D 57 B 65 B 61 B 69 THE 73 B 122a D 122b B 122c D 122d B 122e Ç 122f B 122g B 122h Ç 122i Ç 122j D 122k THE 1221 B

Whole Blood Assay for IL-1beta Production

Whole blood assay IC 50 values for the compounds of this invention are obtained using the method described below:

Goal:

200

The whole blood assay is a simple method for measuring the production of IL-1beta (or other cytones) and the activity of potential inhibitors. The complexity of this assay system, with its total complement of inflammatory and lymphoid cell types, plasma protein spectrum and red blood cells, is an ideal in vitro representation of human in vivo physiological conditions.

Materials:

Pyrogen-free syringes (~ 30 cc)

Sterile pyrogen-free vacuum tubes containing Na ₂ EDTA

lyophilisate (4.5 mg / 10 ml per tube)

Whole human blood sample (~ 30-50 cc)

Eppendorf tubes of 1.5 ml

Test compound stock solutions (~ 25 mM in DMSO or other solvent)

Endotoxin - free sodium chloride solution (0.9%) and HBSS

Lipopolysaccharide stock solution (Sigma); cat. No. L3012) to 1 mg / ml in HBSS

IL-1beta ELISA kit (R & D Systems; cat. No. DLB50)

TNFalpha ELISA Kit (R & D Systems; Cat. No. DTA50)

Water bath or incubator

Experimental Whole Blood Assay Procedure

Adjust incubator or water bath to 30 ° C. Divide into 0.25 ml aliquots of blood in 1.5 ml eppendorf tubes. Observe: be sure to invert the whole blood sample tubes after every two quarters. Differences in replicates can result if the cells settle and are not uniformly suspended. Use of a positive replacement pipette will also minimize differences between replicate rates.

Prepare drug dilutions in sterile pyrogen-free brine by serial dilution. A dilution series that matches K, apparent 30 for a test compound determined in the ICE inhibition assay is generally used for primary compound screening. For extremely hydrophobic compounds, prepare compound dilutions in fresh plasma

201 obtained from the same blood as the donor or in 5% DMSO containing

PBS to increase solubility.

Add 25 μΙ of test compound or control dilution

vehicle and gently mix the sample. Then add 5.0 μΙ of LPS solution (250 ng / ml of fresh prepared stored: 5.0 ng / ml of final concentration LPS), and mix again. Incubate the tubes at 30 ° C in a water bath for 16-18 h with occasional mixing. Alternatively, the tubes can be placed in a rotary set at 4 rpm for the same incubation period. This assay should be adjusted in duplicate or in 10 triplicates with the following controls: negative control - no LPS; positive control - no test inhibitor; vehicle control - the highest concentration of DMSO or compound solvent used in the experiment. Additional brine is added to all control tubes to normalize volumes for both control and whole blood test samples.

After the incubation period, whole blood samples are centrifuged for 10 minutes at ~ 2000 rpm in the microfuge, plasma is transferred to a fresh microfuge tube and is centrifuged at 1000 x g to transform residual platelet pellets if necessary. Plasma samples 20 can be stored frozen at -70 ° C prior to testing for cytokine levels by ELISA.

ELISA:

Quanticine R & D Systems kits (614 McKinley Place N. E. Minneapolis, MN 55413) can be used for measurement of IL-1 beta and 25 TNF-alpha. The tests are carried out according to the manufacturers' directions. IL-1 beta levels of ~ 1-5 ng / ml in positive controls among a range of individuals can be observed. A 1: 200 dilution of plasma for all samples is usually sufficient for the experiments for ELISA results to fall within the linear range of the standard ELISA curves. It may be necessary to optimize standard dilutions if you see differences in the whole blood assay. Nerad, J. L. et al., J. Leukcyte Biol. 52, p. 687692 (1992).

202

Table 5. Whole Blood Test Data

Example Whole blood IC50 (nM) 5th THE 5b THE 5c THE 5d B 5e THE 5f B 5 am THE 5i Ç 5j Ç 5k B 51 Ç 5 m THE 5n B 5r Ç 5s Ç 5u THE 5w THE 5x THE 5y B 5z Ç 5aa THE 5ab B 5ac THE 5ad THE 5ag B 5ai Ç 5aj Ç 5ak Ç 5ax B 5ay B 5az THE 5bb B 7th B

203

Example Whole blood IC50 (nM) 7b THE 7c THE 7d B 7e Ç 7f B 7g B 7am THE 7k THE 71 THE 7m B 7n THE 7th THE 7p B 7q THE 7s B 7t B 7u B 7v THE 7w THE 7x Ç 7y Ç 7z THE 7th B 7ab THE 7ac B 7th B 7ah B 7ai B 7aj Ç 7ak B 7am B 7an B 7th B 7ap Ç

204

Example Whole blood IC50 (nM) 9a B 9b B 9c THE 9d THE 9 and B 9f THE 9g B 15th B 15b B 15c THE 16b THE 18th THE 18b THE 18c THE 18d THE 18th THE 18f B 20th THE 20b Ç 20c B 20d B 20e B 20f THE 20g THE 8 pm THE 20i THE 20k THE 20I THE 20m THE 20n THE 20 ° THE 20p THE 20q Ç 20r B

205

Example Whole blood IC50 (nM) 20s THE 20t THE 23a B 23b B 23c B 23d THE 23e B 23f THE 23g THE 11 pm THE 23i B 24a B 24c B 24d B 24e B 25a B 25b B 25c B 25d THE 25e Ç 26c B 26d THE 26e THE 26f B 26g B 26h THE 27a THE 27b THE 27d THE 27e THE 27f B 27g B 27h B 27i B

206

Example Whole blood IC50 (nM) 271 B 27m B 27n THE 28th B 28b B 28c Ç 29 ^to THE 29c THE 29d THE 29e THE 29g THE 29h THE 29i THE 29j THE 29k THE 291 THE 29n B 29th THE 29p THE 29q THE 29r THE 29s THE G2 B 42 B 46b B 57 Ç 65 THE 61 THE 69 THE 73 THE 122a THE 122b THE 122c B 122d THE

207

Example Whole blood IC50 (nM) 122e B 122f THE 122g THE 122h THE 122i THE 122j B 122k THE 1221 THE 122m B 122p B 122q B 122r B 122s B 123a THE 123b B

Ex Vivo Essays

Metabolism and Excretion

Single-pass perfusion studies in rats were performed to ensure gastrointestinal (GI) (f (g)) metabolism, liver (f (h)) metabolism, and biliary excretion. The method used was described in Pang, C. S., J. Pharmacol. Exp. Therapeutics, 333, p. 788-798 (1984). Table 6. Excretion and Metabolism Data

Example F (g) Xf (h) Biliary execution 5c THE Ç 5k B THE 5 m B Ç 7d D THE 7f Ç THE 7ac Ç Ç 18f D THE 20f B Ç 20g B THE 23b B B

208

Example F (g) Xf (h) Biliary execution 24a Ç THE 24c THE Ç 24e THE Ç 25d Ç Ç 25e Ç B 26c THE Ç 26e B B 26f THE Ç 27a Ç THE 27b B THE 29b THE B 29g B B 29n Ç THE 29th Ç THE 29p B Ç 29q Ç THE 29r Ç B 46b B Ç 57 B B 65 B Ç 69 Ç THE 122a B THE 122b Ç THE 122c Ç B 122d Ç THE 122r B THE

In Vivo Tests

In vivo Mouse Clearance Assay - Clearance Rates

The rat clearance rate (ml / min / kg) for the compounds of this invention can be obtained using the method described below:

Representative Procedure

Cannulations of the carotid and jugular vessels of rats under anesthesia were performed one day before the pharmacokinetic study. M. J. Free,

209

R.A. Jaffee; Cannulation techniques for the collection blood and other bodily fluids; in: Animal Models; p. 480 - 495; N. J. Alexander, Ed; Academic

Press; (1978). Drug (10 mg / ml is administered via the jugular vein in a vehicle usually consisting of: propylene glycol / brine, containing 100 mM sodium bicarbonate in a 1: 1 ratio. Animals are administered

dosed with 10 - 20 mg of drug / kg and blood samples are taken at 0, 2, 5, 7, 10, 20, 30, 60 and 90 minutes from a permanently present carotid catheter. The blood is centrifuged for plasma and is stored at -20 ° C until analysis. Pharmacokinetic data analysis is performed 10 by non-linear regression using a standard program such as Rstrip (program MicroMath, UT) and / or Pcnonlin (program SCI, NC) to obtain clearance values.

Representative Analysis:

Rat plasma is extracted with an equal volume of acetonitrile 15 (containing 0.1% TFA). Samples are then centrifuged at about 1,000 x g and the supernatant is analyzed by gradient HPLC. A typical test procedure is described below.

200 μΙ of plasma are precipitated with 200 μΙ of 0.1% trifluoroacetic acid (TFA) in acetonitrile and 10 μΙ of 50% aqueous zinc chloride solution 20, are swirled then are centrifuged at -1000 xgeo supernatant is collected and is analyzed by HPLC.

HPLC procedure:

Column:

Column temperature

Zorbax SB-CN (4.6 x 150 mm) (particle size 5 μ) 50 ° C

Flow rate

Injection volume

1.0 ml / min μΙ

Mobile phase

Gradient employed

A = 0.1% TFA in water in

B = 100% acetonitrile

100% A to 30% A in 15.5 min

0% from A to 16 min

210

100% A at 19.2 min

Wavelength 214 nm

A standard curve is performed at concentrations of 20, 10, 5, 2 and 1 gg / ml.

Table 7. Debug Data _______________________________________

Example I.V. clearance in rat (ml / min / kg) 7d THE 7f B 8 pm B 20m THE 65 Ç 122b B 122c Ç 122d B 122f THE

Availability

Oral Pharmacokinetic Studies

Male Sprague-Dawley rats (Harlan, Indianopolis, IN, 300350 g) were anesthetized by an intramuscular injection of ce10 tamina / ropum mixture. A PE-50 cannula was inserted into the right carotid artery for sampling arterial blood. The rats were allowed to recover from surgery overnight (·> 16 hours) before being used in the study. Test compounds were administered orally in 25% Cremophor El / water (w / w) or 100% propylene glycol (PG) in a dose volume of

10 ml / kg. Blood samples (-0.30 ml) were removed at 0.25, 0.50, 1.0,

1.5, 2, 3, 4, 6, and 8 hours post-dose, plasma was separated by centrifugation and was stored at -20 ° C pending analysis. Quantification of plasma samples was conducted using an HPLC / EM / EM gradient or enzymatic method detailed below:

HPLC / EM / EM method for the quantification of ICE inhibitors in rat plasma

Sample preparation

- 50 μΙ of plasma are divided into aliquots in vials of

211 Ependorf trifuge.

- An equal volume of acetonitrile is added to the plasma to precipitate plasma proteins.

- Samples are swirled for 5 minutes, and centrifuged at 5 to 14,000 rpm for 5 minutes.

- 75 μΙ of the supernatant is loaded into 12 mm HPLC liquid sampler vials.

- 50 μΙ of sample is injected for analysis via mass spectrometer

Instrumental HPLC Parameters

HPLC: Hewlett Packard Binary Solvent Delivery System

HP1100

HPLC Gradient Conditions

A = H ₂ 0.2% formic acid

B = 0.2% formic acid acetonitrile

Mobile Phase

Time% A

0100

2100

50

110

11.5100

17100% B

100

100

HPLC Analytical Column: Keystone Phenyl -2 Hypersil 2.0 x 100 mm, pore size 120 Â 5 μ, P / N No. 105-39-2

Injection volume: 50 μΐ

Flow rate: 0.20 ml / min.

Instrumental Parameters of Mass Spectrometry

Instrument: Mass Spectrometer Ρ E Sciex API-365 Tan30 dem

Ionization technique: Turbo-lonspray (ESI)

Polarity: positive

253

212

Downtime: 300 ms Pause time: 5 ms Scan time: 0.9 s Scan mode: MRM (multiple reaction monitoring)

ICE Enzyme Assay for the Quantification of ICE Inhibitors in

Mouse Plasma

μΙ of plasma were extracted as 150 μΙ of acetonitrile, sonicated, swirled, centrifuged at 10,000 xg and 180 μΙ of the supernatant were dried in a Sor10 vall vortex evaporator at room temperature. Samples were reconstituted in 100 μΙ of buffer (10 mM Tris-HCI, pH 7.5 with 0.1% CHAPS, 1 mM DTT) with sonication. 10 μΙ of each sample was mixed with 10 μΙ of ICE (1.1 mg / ml) in a microtiter plate with 60 μΙ of buffer. Samples were incubated for 15 m. at room temperature then 20 μΙ of Succ YVAD15 pNA (400 μΜ, preheated to 37 ° C) was added, and the plate was monitored at 405 nm for 20 mins. at 37 ° C, a SpectraMax reader was used. The data were adjusted using a parameter setting 4 with the SpectraMax program using an extracted standard curve. The assay was linear from 0.15 to 2.0 - 3.0 pg / ml of aldehyde.

Pharmacokinetic Parameters

Pharmacokinetic analysis of these plasma concentration data was conducted using non-behavioral methods. The area under the curve (AUC (ot) was estimated from time zero until the last time point measured using the linear trapezoidal rule. The elimination rate (Ke) was estimated by log-linear regression from the terminal phase of the plasma-time concentration curves. The area under the end of the curve was estimated as the ratio of the last measured concentration to ke. The area under the curve from time zero to infinity (AUC (O-oo) ) was obtained by adding the area under the end to AUC (ot). Elimination half-life was estimated to be 0.693 / ke. The observed values for peak plasma concentration (Cmax) were recorded. : availability of aldehyde (bioavailability) was calculated as: (AUC _to id / prodrug

4

213

po) / (Auc _a ici / ald iv) x (ald dose, ald iv / prodrug dose, prodrug po) x (PM drug / PM aldehyde).

Table 8. Bioavailability data

Example Cmax (pg / mL) AUC (pgXh / mL) ^T1 / ₂ (hrs) F (%) 56 THE B THE 45 THE B 90 Ç Ç THE Ç 85 THE B B THE 68 THE Ç B 76 Ç Ç Ç 77 B B THE THE 78 THE THE B THE 89 B Ç Ç 83 THE Ç Ç 98d THE THE B 98h THE Ç B 98e Ç Ç B 98c B Ç Ç 98k B Ç B 98e THE THE B 98f THE THE B 98b Ç Ç B 111 THE THE Ç 98th THE THE B 108a THE THE B 98ag Ç Ç B Ç 98a B B Ç 98am THE THE B 116a Ç Ç B 98an THE THE B 116g THE THE Ç 116h THE THE B 116e THE THE B 108b THE THE B

Animal Testing

214

The effectiveness of the compounds of this invention in the treatment or prevention of conditions, disorders, related antiviral diseases can be evaluated in various in vitro and in vivo assays. For example, assays can be performed to determine the ability of these compounds to inhibit inflammatory responses associated with viral infections. In vitro assays can employ isolated cell components or whole cells. In vivo trials include animal models for viral diseases.

Examples of such animal models include, but are not limited to, rodent models for HBV or HCV infection, the 10 groundhog model for HBV infection, and chimpanzee model for HCV infection.

The compounds of this invention can also be evaluated in animal models for diet alcohol-induced disease.

Other assays that can be used to evaluate the compounds of this invention are disclosed in PCT Application PCT / US96 / 20843, published on June 26, 1997, under publication No. WO 97/22619. Such assays include in vivo pharmacokinetic studies in the mouse, inhibition of ICE homologs, inhibition of apoptosis, acute in vivo assay for anti-inflammatory efficacy, measurement of drug blood levels, IGIF 20 assays, carrageenan peritoneal inflammation assay , and type II collagen-induced arthritis.

As the compounds of this invention are able to inhibit caspases, particularly ICE, in vitro and moreover, they can be delivered orally to mammals, they are of evident clinical utility for the treatment of diseases mediated by IL-1, apoptosis, IGIF and IFN-γ.

Although numerous embodiments of this invention have been described, it is evident that our basic constructions can be altered to provide other embodiments using the products and processes of this invention.

Claims (10)

1. Compound, characterized by the fact that it presents the formula I: O R ¹ Y where: THE) Y is: (The) H R ⁷ or R ¹ -C (O) N (H) R ⁸ ; R ² R ³ O X is -C (R3) 2-; is -C (O) R ⁸ , -C (O) C (O) R ⁸ , -S (O) ₂ R ⁸ , -C (O) OR ⁸ , is -H; is independently -H or an amino acid side chain selected from -H, -CH3, -CH (CH3) CH2CH3, -CH2CH (CH3) 2, - (CH2) 2SCH3, -CH (OH) CH3, and -CH (CH3) 2, pyridine or CH2CH2S (O) 2CH3; R ⁴ and R ⁵ together with the atoms to which they are attached form an unsubstituted pyrrole ring; ₅ and the other R ⁵ is H; R ⁶ is -H; R ⁷ is -OH; each R ⁸ is independently -cycloalkyl, -aryl, -heteroaryl, where a hydrogen atom attached to any carbon atom of -alkyl or -cycloalkyl is optionally substituted by R ¹⁰ and a hydrogen atom attached to any carbon atom of -aryl or -heteroaryl is optionally substituted by R ¹¹ ; each R ⁹ is independently -aryl, -heteroaryl, cycloalkyl, or Petition 870160044562, of 08/17/2016, p. 4/20 -heterocyclyl, where a hydrogen atom attached to any -alkyl or -cycloalkyl carbon atom is optionally substituted by R ¹⁰ , a hydrogen atom attached to any -aryl or heteroaryl carbon atom is optionally substituted by R ¹¹ and a hydrogen atom attached to any nitrogen atom is optionally substituted by R ¹ ; each R ¹⁰ is -N (H) C (O) H, where m is 0; each R ¹¹ is independently selected from the group consisting of: -OH, -F, -Cl, -Br, -I, -CN, -NH2, -C (O) NH2, -N (H) C (O) H, -alkyl, -O-alkyl, -O-aryl, -O-alkylaryl, -N (H) -alkyl, -C (O) N (H) -alkyl, -N (H) C (O) -alkyl, -S-alkyl and -C (O) aryl; and R ¹² is -C (O) -alkyl, -C (O) -cycloalkyl, -C (O) -alkylaryl, -C (O) alkyletheroaryl, -C (O) -heterocyclyl, or -C (O) -alkyleterocyclyl ; and wherein each alkyl is a straight or branched, saturated aliphatic hydrocarbon containing 1 to 6 carbon atoms; each alkenyl is a straight or branched, unsaturated aliphatic hydrocarbon containing 2 to 6 carbon atoms and at least one double bond; each alkynyl is a straight or branched, unsaturated aliphatic hydrocarbon containing 2 to 6 carbon atoms and at least one triple bond; each cycloalkyl is a mono or polycyclic hydrocarbon ring system, non-aromatic, containing 5 to 10 carbon atoms, which can optionally contain unsaturated bonds in the ring system; each aryl is a mono- or polycyclic ring system containing 6, 10, 12 or 14 carbon atoms, in which at least one ring of the ring system is aromatic; each heteroaryl is a mono- or polycyclic ring system containing 1 to 15 carbon atoms and 1 to 4 hetero atoms, in which at least one ring of the ring system is aromatic; Petition 870160044562, of 08/17/2016, p. 5/20 each heterocyclyl is a mono- or polycyclic ring system containing 1 to 15 carbon atoms and 1 to 4 heteroatoms, in which the mono- or polycyclic ring system can optionally contain unsaturated bonds, but is not aromatic; and each heteroatom is sulfur, nitrogen or oxygen. 2. Compound according to claim 1, characterized by the fact that an R ³ is -H. Compound according to claim 2, characterized by the fact that R ¹ is -C (O) R ⁸ or -C (O) C (O) R ⁸ . 4. Compound according to claim 1, characterized by the fact that it is selected from the group consisting of: 5a-5bd, 7 a- 7at, 20c-20f, 20h-20n, 20p-20t, 52, 57, 61.65, 69, 73, and 122a-v. 5b O 5c CO2H H 5d 5e .CO2H V O O 5f 5g 5l Petition 870160044562, of 08/17/2016, p. 6/20 5q Petition 870160044562, of 08/17/2016, p. 7/20 5ac O 5al O O Petition 870160044562, of 08/17/2016, p. 8/20 O O Petition 870160044562, of 08/17/2016, p. 9/20 O 1 --- l h ₂ n U ^N OJ co ₂ h cr L ^ H O 7am H O ^ co ₂ h O 7k 7m O /- A N N Y X H η 1 , co ₂ h N ^ I . The J A-H | j O ^ H 1 7g O 7n Petition 870160044562, of 08/17/2016, p. 10/20 r \ 7th 7q 7x 7th Petition 870160044562, of 08/17/2016, p. 11/20 7ac 7aj Γ \ 7ak 7al Γ \ 7ah O ^Cl \ CO ₂ H H The JL J L ^ h O 'N Cl H O 7ai 7ap Petition 870160044562, of 08/17/2016, p. 12/20 7aq 7th Petition 870160044562, of 08/17/2016, p. 13/20 O CO ₂ H 20r O 20s H O 122a Petition 870160044562, of 08/17/2016, p. 14/20 Pr ⁰ 1 r- λ 0 0H ^ N ^ H h ₂ n ^/ 0 OJ ^Z ^ N ^ H Cl H 0 122b Ck 0 | ___ 0 0 1 1— \ 0 Γϊ '' ^ N ' H k ^ N. Oh | H k ^ N. 0H ^X / ^Z 44 0 J 4 ^ N ^ .H -0> 44 0 J 0 ^ ^ N ^ H Cl H 0 H 0 122c 122d 122e 122f 122k 122l 0 1 0 0 1 0 Cr ^ N H \ ^ N. 0H> Pr ^ N ^ H L „n. 0H 44 Cl 0 J 0 ' ^k N ^ H 0 , H x) N 44 F 0 J 0 ^{Z k} N ^ H 0 ,H 122m 122n Petition 870160044562, of 08/17/2016, p. 15/20 O 122p 122o 5. Pharmaceutical composition, characterized by the fact that it comprises (a) a compound, as defined in any one of claims 1 to 4; and (b) a pharmaceutically acceptable vehicle, auxiliary or carrier. 6. Use of a compound, as defined in any of claims 1 to 4, or a pharmaceutical composition, as defined in claim 5, characterized by the fact that it is for the preparation of a medicament for the treatment or prevention of a disease selected from an IL-1-mediated disease, an apoptosis-mediated disease, an inflammatory disease, an autoimmune disease, a destructive bone disorder, a proliferative disorder, an infectious disease, a degenerative disease, a necrotic disease, an ingestion disease of excess diet alcohol, a viral-mediated disease, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis Petition 870160044562, of 08/17/2016, p. 16/20 te, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin-dependent diabetes mellitus (type I), autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis , myasthenia gravis, inflammatory bowel disease, Crohn's disease, psoriasis, atopic dermatitis, graft vs. disease. host, osteoporosis, leukemias and related disorders, myelodysplastic syndrome, bone disorder related to multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kapose sarcoma, multiple myeloma, sepsis, septic shock, 10 Alzheimer's disease Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, neurological damage due to septic shock, ulcerative colitis, traumatic brain injury, organ transplant rejection , hepatitis-B, hepatitis-C, 15 hepatitis-G, yellow fever, dengue fever, or Japanese encephalitis, in a patient. 7. Use according to claim 6, characterized by the fact that the disease is rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory peritonitis, septic shock, pan- 20 creatitis, traumatic brain injury, organ transplant rejection, osteoarthritis, asthma, psoriasis, Alzheimer's disease, atopic dermatitis, leukemia and related disorders, myelodysplastic syndrome or multiple myeloma. 8. Use of a compound, as defined in any of claims 1 to 4, or a pharmaceutical composition, as defined in Claim 5, characterized by the fact that it is for the preparation of a medication for inhibiting an ECI-mediated function in a patient. 9. Use of a compound, as defined in any one of claims 1 to 4, or a pharmaceutical composition, as defined in claim 5, characterized by the fact that it is for the preparation of a 30 dication to decrease the production of IGIF or IFN-γ in a patient. 10. Compound according to claim 1, characterized by the fact that R ¹ is -C (O) R ⁸ or -C (O) C (O) R ⁸ . Petition 870160044562, of 08/17/2016, p. 17/20