AU2010224367A1 - Inhibitors of Caspases - Google Patents

Inhibitors of Caspases Download PDF

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AU2010224367A1
AU2010224367A1 AU2010224367A AU2010224367A AU2010224367A1 AU 2010224367 A1 AU2010224367 A1 AU 2010224367A1 AU 2010224367 A AU2010224367 A AU 2010224367A AU 2010224367 A AU2010224367 A AU 2010224367A AU 2010224367 A1 AU2010224367 A1 AU 2010224367A1
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alkyl
aryl
optionally replaced
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Guy W. Bemis
Paul S. Charifson
Robert J. Davies
Cornelia J. Forster
Anne-Laure Grillot
James W. Janetka
David J. Lauffer
Michael D. Mullican
Mark A. Murcko
Zhan Shi
Marion W. Wannamaker
Keith P. Wilson
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Vertex Pharmaceuticals Inc
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Vertex Pharmaceuticals Inc
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Description

- 1 Australian Patents Act 1990 - Regulation 2.3 Original Complete Specification, Standard Patent Invention Title: Inhibitors of Caspases The following statement is a full description of this invention, including the best method of performing it known to the applicant: TECHNICAL FIELD OF THE INVENTION The present invention relates to novel classes of compounds which are caspase inhibitors, in 10 particular interleukin-10 converting enzyme ("ICE") 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 is caspase activity and consequently, may be advantageously used as agents against interleukin-l ("IL-1"), apoptosis-, interferon-y inducing factor (IGIF) , or interferon-y- ("IFN-y") mediated diseases, including inflammatory diseases, autoimmune diseases, 20 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-y production and methods for treating interleukin 25 1, apoptosis-, and interferon-y- mediated diseases using the compounds and compositions of this invention. This invention also relates to methods of preparing the compounds of this invention. 30 BACKGROUND OF THE INVENTION Interleukin 1 ("IL-1") is a major pro inflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, - 2 collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. Oppenheim, J.H. et al, Immunology Today, 7, pp. 45-56 (1986). As such, it is 5 involved in the pathogenesis of chronic and acute inflammatory and autoimmune diseases. For example, in rheumatoid arthritis, IL-1 is both a mediator of inflammatory symptoms and of the destruction of the cartilage proteoglycan in afflicted joints. Wood, D.D. 10 et al., Arthritis Rheum. 26, 975, (1983); Pettipher, E.J. et al., Proc. Natl. Acad. Sci. USA 71, 295 (1986); Arend, W.P. and Dayer, J.M., Arthritis Rheum. 38, 151 (1995). IL-1 is also a highly potent bone resorption agent. Jandiski, J.J., J. Oral Path 17, 145 (1988); 15 Dewhirst, F.E. et al., J. Immunol. 8, 2562 1985). It is alternately referred to as "osteoclast activating factor" in destructive bone diseases such as osteoarthritis and multiple myeloma. Bataille, R. et al., Int. J. Clin. Lab. Res. 21(4), 283 (1992). In 20 certain proliferative disorders, such as acute myelogenous leukemia and multiple myeloma, IL-1 can promote tumor cell growth and adhesion. Bani, M.R., J. Natl. Cancer Inst. 83, 123 (1991) ; Vidal-Vanaclocha, F., Cancer Res. 54, 2667 (1994). In these disorders, 25 IL-1 also stimulates production of other cytokines such as IL-6, which can modulate tumor development (Tartour et al., Cancer Res. 54, p. 6243 (1994). IL-1 is predominantly produced by peripheral blood monocytes as part of the inflammatory response and exists in two 30 distinct agonist forms, IL-la and IL-1p. Mosely, B.S. et al., Proc. Nat. Acad. Sci., 84, pp. 4572-4576 (1987); Lonnemann, G. et al., Eur. J. Immunol., 19, pp., 1531-1536 (1989). IL-1p is synthesized as a biologically 35 inactive precursor, pIL-1p. pIL-10 lacks a - 3 conventional leader sequence and is not processed by a signal peptidase. March, C.J., Nature, 315, pp.641-647 (1985) . Instead, pIL-10 is cleaved by interleukin-1p converting enzyme ("ICE") between Asp-116 and Ala-117 5 to produce the biologically active C-terminal fragment found in human serum and synovial fluid. Sleath, P.R., et al., J. Biol. Chem., 265, pp.14526-14528 (1992); A.D. Howard et al., J. Immunol., 147, pp.2964-2969 (1991) . ICE is a cysteine protease localized primarily 10 in monocytes. It converts precursor IL-lp to the mature form. Black, R.A. et al., FEBS Lett., 247, pp.
386-390 (1989); Kostura, M.J. et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp.5227-5231 (1989) . Processing by ICE is also necessary for the transport of mature IL-1p 15 through the cell membrane. ICE (or caspase-1) is a member of a family of homologous enzymes called caspases. These homologs have sequence similarities in the active site regions of the enzymes. Such homologs (caspases) include TX 20 (or ICErel-Ii or ICH-2) (caspase-4) (Faucheu, et al., EMBO J., 14, p. 1914 (1995); Kamens J., et al., J. Biol. Chem., 270, p. 15250 (1995); Nicholson et al., J. Biol. Chem., 270 15870 (1995) ), TY (or ICErel-III) (caspase-5) (Nicholson et al., J. Biol. Chem., 270, p. 25 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. et al., Cancer Res., 55, p. 2737 (1995), CPP32 (or YAMA or apopain) (caspase-3) (Fernandes-Alnemri, T. et al., J. Biol. Chem., 269, p. 30 30761 (1994); Nicholson, D.W. et al., Nature, 376, p. 37 (1995)), CMH-1 (or MCH-3) (caspase-7) (Lippke, et al., J. Biol. Chem., 271(4), p 1 825-1828 (1996)); Fernandes -Alnemri, T. et al., Cancer Res., (1995)), Mch5 (caspase-8) (Muzio, M. et.al., Cell 85(6), 817 35 827, (1996)), MCH-6 (caspase-9) (Duan, H. et.al., 3.
-4 Biol. Chem., 271(34), p. 16720-16724 (1996)), Mch4 (caspase-10) (Vincenz, C. et.al., J. Biol. Chem., 272, p. 6578-6583 (1997); Fernandes-Alnemri, T. et.al., Proc. Natl. Acad. Sci. 93, p. 7464-7469 (1996)), Ich-3 5 (caspase-11) (Wang, S. et.al., J. Biol. Chem., 271, p. 20580-20587 (1996)), mCASP-12 (caspase-12), (Van de Craen, M. et.al., FEBS Lett. 403, p. 61-69 (1997); Yuan, Y.and Miura, M. PCT Publication W095/00160 (1995)), ERICE (caspase-13), (Humke, E.W., et.al., J. 10 Biol. Chem., 273(25) p. 15702-15707 (1998)), and MICE (caspase-14) (Hu, S. et.al., J. Biol. Chem., 273(45) p. 29648-29653 (1998)). Each of these ICE homologs, as well as ICE itself, is capable of inducing apoptosis when 15 overexpressed in transfected cell lines. Inhibition of one or more of these homologs with the 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). 20 Caspases also appear to be involved in the regulation of programmed cell death or apoptosis. Yuan, J. et al., Cell, 75, pp.641-652 (1993); Miura, M. et al., Cell, 75, pp. 653-660 (1993.); Nett-Fiordalisi, M.A. et al., J. Cell Biochem., 17B, p. 117 (1993). In 25 particular, ICE or ICE homologs are thought to be associated with the regulation of apoptosis in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Marx, J. and M. Baringa, Science, 259, pp. 760-762 (1993); Gagliardini, V. et al., 30 Science, 263, pp. 826-828 (1994). Therapeutic applications for inhibition of apoptosis may include treatment of Alzheimer's disease, Parkinson's disease, stroke, myocardial infarction, spinal atrophy, and aging.
- 5 ICE has been demonstrated to mediate apoptosis (programmed cell death) in certain tissue types. Steller, H., Science, 267, p. 1445 (1995); Whyte, M. and Evan, G., Nature, 376, p. 17 (1995); 5 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, 10 K. et al., Science 267, 2000 (1995)). This activity of ICE is distinct from its role as the processing enzyme for pro-IL-1p. It is conceivable that in certain tissue types, inhibition of ICE may not affect secretion of mature IL-10, but may inhibit apoptosis. 15 Enzymatically active ICE has been previously described as a heterodimer composed of two subunits, p20 and p1O (20kDa and 10kDa molecular weight, respectively). These subunits are derived from a 45kDa proenzyme (p45) by way of a p30 form, through an 20 activation mechanism that is autocatalytic. Thornberry, N.A. et al., Nature, 356, pp.768-774 (1992). The ICE proenzyme has been divided into several functional domains: a prodomain (p14), a p22/20 subunit, a polypeptide linker and a plo subunit. 25 Thornberry et al., supra; Casano et al., Genomics, 20, pp. 474-481 (1994). Full length p45 has been characterized by its cDNA and amino acid sequences. PCT patent applications WO 91/15577 and WO 94/00154. The p20 and p1o cDNA and 30 amino acid sequences are also known. Thornberry et al., supra. Murine and rat ICE have also been sequenced and cloned. They have high amino acid and nucleic acid sequence homology to human ICE. Miller, D.K. et al., Ann. N.Y. Acad. Sci., 696, pp. 133-148 (1993); 35 Molineaux, S.M. et al., Proc. Nat. Acad. Sci., 90, pp.
1809-1813 (1993). The three-dimensional structure of ICE has been determined at atomic resolution by X-ray crystallography. Wilson, K.P., et al., Nature, 370, pp. 270-275 (1994). The active enzyme exists as a 5 tetramer of two p20 and two p1o subunits. Recently, ICE and other members of the ICE/CED-3 family have been linked to the conversion of pro-IGIF to IGIF or to the production of IFN-y in vivo (PCT application PCT/US96/20843, publication no. WO 10 97/22619, which is incorporated herein by reference). IGIF is synthesized in vivo as the precursor protein "pro-IGIF". Interferon-gamma inducing factor (IGIF) is an approximately 18-kDa polypeptide that stimulates T-cell 15 production of interferon-gamma (IFN-y). IGIF is produced by activated Kupffer cells and macrophages in vivo and is exported out of such cells upon endotoxin stimulation. Thus, a compound that decreases IGIF production would be useful as an inhibitor of such T 20 cell stimulation which in turn would reduce the levels of IFN-y production by those cells. IFN-y is a cytokine with immunomodulatory effects on a variety of immune cells. In particular, IFN-y is involved in macrophage activation and Thi cell 25 selection (F. Belardelli, APMIS, 103, p. 161 (1995)). IFN-y exerts its effects in part by modulating the expression of genes through the STAT and IRF pathways (C. Schindler and J.E. Darnell, Ann. Rev. Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer Res. Clin. 30 Oncol., 121, p. 516 (1995)). Mice lacking IFN-y or its receptor have multiple defects in immune cell function and are resistant to endotoxic shock (S. Huang et al., Science, 259, p.1742 (1993); D. Dalton et al., Science, 259, p.
35 1739 (1993); B.D. Car et al., J. Exp. Med., 179, p.1437 (1994)). Along with IL-12, IGIF appears to be a potent inducer of IFN-y production by T cells (H. Okamura et al. , Infection *and Immunity, 63, p.3966 (1995); H. Okamura et al., Nature, 378, p..88 (1995); S. Ushio et 5 al., J.Immunol., 156, p.4274 (1996)). IFN-y has been shown to contribute to the pathology associated with a variety of inflammatory, infectious and autoimmune disorders and diseases. Thus, compounds capable of decreasing IFN-y production 10 would be useful to ameliorate the effects of IFN-y related pathologies. Accordingly, compositions and methods capable of regulating the conversion of pro-IGIF to IGIF would be useful for decreasing IGIF and IFN-y production in 15 vivo, and thus for ameliorating the detrimental effects of these proteins which contribute to human disorders and diseases. Caspase inhibitors represent a class of compounds useful for the control of inflammation or 20 apoptosis or both. Peptide and peptidyl inhibitors of ICE have been described (PCT patent applications WO 91/15577, WO 93/05071, WO 93/09135, WO 93/12076, WO 93/14777, WO 93/16710, WO 95/35308, WO 96/30395, WO 96/33209 and WO 98/01133; European patent 25 applications 503 561, 547 699, 618 223, 623 592, and 623 606; and US patent nos. 5,434,248, 5,710,153, 5,716,929, and 5,744,451). Such peptidyl inhibitors of ICE have been observed to block the production of mature IL-1P in a mouse model of inflammation (vide 30 infra) and to suppress growth of leukemia cells in vitro (Estrov et al., Blood, 84, 380a (1994)). However, due to their peptidic nature, such inhibitors are typically characterized by undesirable pharmacologic properties, such as poor cellular 35 penetration and cellular activity, poor oral - 8 absorption, instability and rapid metabolism. Plattner, J.J. and D.W. Norbeck, in Drug Discovery Technologies, C.R. Clark and W.H. Moos, Eds. (Ellis Horwood, Chichester, England, 1990) , pp.92-126. These 5 properties has hampered their development into effective drugs. Non-peptidyl compounds have also been reported to inhibit ICE in vitro. PCT patent application WO 95/26958; US Patent 5,552,400; Dolle et 10 al., J. Med. Chem., 39, pp. 2438-2440 (1996). It is not clear however whether these compounds have the appropriate pharmacological profiles to be therapeutically useful. Accordingly, the need exists for compounds 15 that can effectively inhibit caspases, and that have favorable in vivo activity, for use as agents for preventing and treating chronic and acute forms of IL 1-, apoptosis-, IGIF-, or IFN-y-mediated diseases, as well as inflammatory, autoimmune, destructive bone, 20 proliferative, infectious, or degenerative diseases. SUMMARY OF THE INVENTION The present invention provides novel classes of compounds, and pharmaceutically acceptable 25 derivatives thereof, that are useful as caspase inhibitors, in particular, as ICE inhibitors. These compounds can be used alone or in combination with other therapeutic or prophylactic agents, such as antibiotics, immunomodulators or other anti 30 inflammatory agents, for the treatment or prophylaxis of diseases mediated by IL-1, apoptosis, IGIF, or IFN According to a preferred embodiment, the compounds of this invention are capable of binding to the active site of a caspase and inhibiting the activity of that 35 enzyme.
It is a principal object of this invention to provide novel classes of compounds represented by formula I, which have favorable in vivo profiles: 0 s R5 R2 RR
R
6 N R" X N y
R'
4 0 5 wherein the various substituents are described herein. It is a further objective of this invention to provide pharmaceutical compositions, including 10 multi-component compositions. This invention also provides methods for using and preparing the compounds of this invention and related compounds. DETAILED DESCRIPTION OF THE INVENTION 15 In order that the invention described herein may be more fully understood, the following detailed description is set forth. The following abbreviations and definitions are used throughout the application. 20 Abbreviations Ac20 acetic anhydride MeCN acetonitrile AMC aminomethyl coumarin Fn-Bu Inormal-butyl DMF dimethylformamide DIEA N, N- diisopropylethylamine DMA N,N-dimethylacetamide EDC 1- (3-Dimethylaminopropyl) -3 ethylcarbodiimide hydrochloride Et 2 0 diethyl ether - 10 EtOAc ethy acetate Fmoc 9- fluorenylmethyoxycarbonyl HBTU O-benzotriazol- 1 -yl -NNlN,N' tetramethyluronium hexafluorophosphate HOBT 1-hydroxybenzodriazole hydrate MeOH methanol NMP N-methylpyrrolidinone TFA trafluoroacetic acid pNAJ. p-nitroanilimne The term "caspase" refers to an enzyme that is a member of the family of enzymes that includes ICE (see H. Hara, Natl. Acad. Sci., 94, pp. 2007-2012 5 (1997)). The terms "HBV", "HCV" and "HGV" refer to hepatitis-B virus, hepatitis-c virus and hepatitis-G virus, respectively. The term "Ki" refers to a numerical measure 10 of the effectiveness of a compound in inhibiting the activity of a target enzyme such as ICE. Lower values of Ki reflect higher effectiveness. The Ki value is a derived by fitting experimentally determined rate data to standard enzyme kinetic equations (see I.H. Segel, 15 Enzyme Kinetics, Wiley-Interscience, 1975). The term "interferon gamma inducing factor" or "IGIF" refers to a factor which is capable of stimulating the endogenous production of IFN-y. The term "caspase inhibitor" refer to a 20 compound which is capable of demonstrating detectable inhibition of one or more caspases. The term "ICE inhibitor" refers to a compound which is capable of demonstrating detectable inhibition of ICE and optionally one or more additional caspases. Inhibition 25 of these enzymes may be determined using the methods described and incorporated by reference herein. The skilled practitioner realizes that an in vivo enzyme inhibitor is not necessarily an in vitro - 11 enzyme inhibitor. For example, a prodrug form of a compound typically demonstrates little or no activity in in vitro assays. Such prodrug forms may be altered by metabolic or other biochemical processes in the 5 patient to provide an in vivo ICE inhibitor. The term "cytokine" refers to a molecule which mediates interactions between cells. The term "condition" refers to any disease, disorder or effect that produces deleterious biological 10 consequences in a subject. The term "subject" refers to an animal, or to one or more cells derived from an animal. Preferably, the animal is a mammal, most preferably a human. Cells may be in any form, including but not limited to cells 15 retained in tissue, cell clusters, immortalized cells, transfected or transformed cells, and cells derived from an animal that have been physically or phenotypically altered. The term "patient" as used in this 20 application refers to any mammal, preferably humans. The term "alkyl" refers to a straight-chained or branched, saturated aliphatic hydrocarbon containing 1 to 6 carbon atoms. The term "alkenyl" refers to a straight 25 chained or branched unsaturated hydrocarbon containing 2. to 6 carbon atoms and at least one double bond. The term "alkynyl" refers to a straight chained or branched unsaturated hydrocarbon containing 2 to 6 carbon atoms and at least one triple bond. 30 The term "cycloalkyl" refers to a mono- or polycyclic, non-aromatic, hydrocarbon ring system which may optionally contain unsaturated bonds in the ring system. Examples include cyclohexyl, adamantyl. norbornyl, and spirocyclopentyl.
- 12 The term "aryl" refers to a mono- or polycyclic ring system which contains 6, 10, 12 or 14 carbons in which at least one ring of the ring system is aromatic. The aryl groups of this invention are 5 optionally singly or multiply substituted with R 11 . Examples of aryl ring systems include, phenyl, naphthyl, and tetrahydronaphthyl. The term "heteroaryl" refers to a mono- or polycyclic ring system which contains 1 to 15 carbon 10 atoms and 1 to 4 heteroatoms, 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 singly or multiply substituted with R 11 . 15 The term "heterocyclic" refers to a mono- or polycyclic ring system which contains 1 to 15 carbon atoms and 1 to 4 heteroatoms, in which the mono- or polycyclic ring system may optionally contain unsaturated bonds but is not aromatic. Heteroatoms are 20 independently sulfur, nitrogen, or oxygen. The term "alkylaryl" refers to an alkyl group, wherein a hydrogen atom of the alkyl group is replaced by an aryl radical. The term "alkylheteroaryl" refers to an alkyl 25 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 of carbon of a naturally or non-naturally occuring amino acid. 30 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 unbranching string of covalently bound 35 atoms. The straight chain may be substituted, but - 13 these substituents are not a part of the straight chain. In chemical formulas, parenthesis are used herein to denote connectivity in molecules or groups. 5 In particular, parentheses are used to indicate: 1) that more than one atom or group is bonded to a particular atom; or 2) a branching point (i.e., the atom immediately before the open parenthesis is bonded both to the atom or group in the parentheses and the 10 atom or group immediately after the closed parenthesis). An example of the first use is "-N(alkyl) 2 ", indicating two alkyl groups bond to an N atom. An example of the second use is "-C(0)NH2", indicating a carbonyl group and an amino ("NH 2 ") group 15 both bonded to the indicated carbon atom. A "-C(O)NH 2 " group may be represented in other ways, including the following structure:
NH
2 20 Substituents may be represented in various forms. These various forms are known to the skilled practitioner and may be used interchangeably. For example, a methyl substituent on a phenyl ring may be 25 represented in any of the following forms: H I H H CH Me SCH cYCH 3 ~Ye c Various forms of substituents such as methyl are used herein interchangeably. 30 Other definitions are set forth in the specification where necessary.
-14 Compounds of this Invention The compounds of one embodiment A of this invention are those of formula I: 0 R 5
R
5
R
2 R6 10 X N Ra o wherein: Y is: (a) R 7 H 20 0 provided that when R 7 is -OH then Y can also be: (b) 4 0 HO H 25 X is -C(R 3
)
2 - or -N(R3) m is 0 or 1; 30
R
1 is H, -C(O)R 8 , -C(o)C(O)R 8 , -S(0) 2
R
8 , -S(0)R 8 , -C(0)OR 8 , -C(O)N(H)R 8 , -S(0)2N(H)-R 8 , -S(0)N(H)-R 8 , -C(0)C(O)N(H)RS, -C(0)CH=CHR 8 -C(0)CH2OR 8 , -C(O)CH 2 N(H)R8, -C(O)N(R8) 2 , -S(0)2N(RB) 2
,
- is -S(O)N(R 8
)
2 , -C(O)C(O)N(R8) 2 , -C(O)CH2N(RS) 2 , -CH 2
R
8 ,
-CH
2 -alkenyl-R 8 , or -CH 2 -alkynyl-R 8 ;
R
2 is -H and each R 3 is independently -H, an 5 amino acid side chain, -R 8 , alkenyl-R 9 , or alkynyl-R 9 , or R 2 and one R 3 together with the atoms to which they are bound, form a 3 to 7 membered cyclic or heterocyclic ring system, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally 10 replaced by -R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by -R 11 , a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -RI;
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 one R 5 together with the atoms to which they are bound form a 3 to 7 membered cyclic or heterocyclic ring system, wherein a hydrogen atom bound 20 to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced with R 1 ; 25
R
6 is -H;
R
7 is -OH, -OR 8 , or -N(H)OH; 30 each R 8 is independently -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, 16 wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a 5 hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 9 is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, wherein a hydrogen atom 10 bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 1 0 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; 15 each R 10 is independently -OH, -SH, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -CO 2 H, -C(O)NH 2 , -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-alkylaryl, -N(H)alkyl, -N(H)aryl, -N(H)-alkylaryl, 20 -N(alkyl) 2 , -C(O)N(H)alkyl, -C(O)N(alkyl) 2 , -N(H)C(O)alkyl, -N(H)C(0)N(H)alkyl, -N(H)C(0)N(alkyl) 2 , -S-alkyl, -S-aryl, -S-alkylaryl, -S(O)2alkyl, . -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl) 2 , -alkyl, -cycloalkyl, -aryl, -heteroaryl, 25 -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 and a hydrogen atom bound to any nitrogen atom is optionally replaced by 30 RI; and each R 11 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -C0 2 H, -C(O)NH 2 , -N(H)C(O)H,
-N(H)C(O)NH
2 , -alkyl, -cycloalkyl, -perfluoroalkyl, -0 35 alkyl, -0-aryl, -0-alkylaryl, -N(H)alkyl, -N(H)aryl, - 17 -N(H)-alkylaryl, -N(alkyl) 2 , -C(O)N(H)alkyl, -C(O)N(alkyl)2, -N(H)C(0)alkyl, -N(H)C(0)N(H)alkyl, -N(H)C(O)N(alkyl) 2 , -S-alkyl, -S-aryl, -S-alkylaryl, -S(O)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH 2 , 5 -CH2N(H)alkyl, or -CH2N(alkyl) 2 . In an alternative form of embodiment A:
R
1 is H, -R 8 , -C(O)R 8 , -C(O)C(0)R8,
-S(O)
2 R8, 10
-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)CH
2 0R 8 , -C(0)CH 2 N(H)R8, -C(O)N(R8) 2 , -S (O)2N(R 8
)
2 , -S(O)N(R8) 2 , -C(O)C(O)N(R 8
)
2 , -C(O)CH 2
N(R
8
)
2 , -CH 2
R
8 ,
-CH
2 -alkenyl-R 8 , or -CH 2 -alkynyl-R8; 15
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 bound, form a 3 to 7 membered cyclic or heterocyclic 20 cyclic ring system, or R 2 and one R 3 together with the atoms to which they are bound, form a 3 to 7 membered cyclic or heterocyclic ring system, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -R1 0 , a hydrogen atom bound to 25 any -aryl or -heteroaryl carbon atom is optionally replaced by -R 11 , a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -R 1 ; each R 10 is independently -OH, -SH, -F, -Cl, 30 -Br, -I, -NO 2 , -CN, -NH 2 , -CO2H,
-C(O)NH
2 , -N(H)C(O)H,
-N(H)C(O)NH
2 , -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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, - 18 -N(H)C(O)Oalkylaryl, -N(H)C(O)Oheteroaryl, -N(H)C(O)Oalkylheteroaryl, -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, 5 -N(H)C(O)N(H)heteroaryl, -N(H)C(O)N(H)alkylheteroaryl, -N(H)C(O)N(H)cycloalkyl, -S-alkyl, -S-aryl, -S-alkylaryl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl) 2 , -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, 10 -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by RIl and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; and 15 the other substituents are as defined above. Preferably, in any of the above embodiments: 20 m is 0;
R
2 is -H; one R 3 is -H and the other R 3 is -R 8 , 25 -alkenyl-R 9 , or -alkynyl-R 9 ; or
R
4 and one R 5 together with the atoms to which they are bound form a 3 to 7 membered cyclic or heterocyclic ring system, wherein a hydrogen atom bound 30 to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 1 0 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by Ril, and a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced with R 1 , wherein the 35 ring system is: - 19 1i R1i o:\R1O
R
1 N s R 10 N N R1o 10R10
R
1 10N ,-N ',N ' N -IN N N , N or N In an alternative preferred embodiment, X is 5 C(R3)2 or one R 3 is an amino acid side chain,
-R
8 , alkenyl-R 9 , or alkynyl-R 9 . More preferably, one R 3 is -H and the other
R
3 is -alkyl; or 10
R
4 and one RS together with the atoms to which they are bound form a 3 to 7 membered cyclic or heterocyclic ring system, wherein any hydrogen atom bound to a carbon atom of the ring system is optionally replaced by R 1 0 and any hydrogen atom bound to a 15 nitrogen atom of the ring system is optionally replaced by R1, selected from: -20 N N - N
H
3 C
CH
3 R N
CH
3 S N NCH3 N , or NC3 N 5 Most preferably, one R 3 is -H and the other
R
3 is -C(H) (CH 3
)
2 or -C(CH3) 3 ; and
R
4 and one R 5 together with the atoms to which they are bound form a 3 to 7 membered cyclic or 10 heterocyclic ring system, wherein any hydrogen atom bound to a carbon atom of the ring system is optionally replaced by R 10 and any hydrogen atom bound to a nitrogen atom of the ring system is optionally replaced by RI, selected from: - 21 /N N ' N N N
H
3 H3 R N 8 CH 3 S CH3 I or N CH3 In an alternative most preferred embodiment one R 3 is -H and the other
R
3 is -CH 3 , -C(H) (CH3) 2 or 5
-C(CH
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, wherein Y is: 0 Re O Re Mm m
R
8 0
R
8 , ,or OR" OR1 2 R 10 (C) (d) (e) provided that when
R
6 is not hydrogen,
R
6 and Y, together with the nitrogen to which they are bound, form a ring (g): - 22 NNY N A R13
R
1 3 (g)
R
12 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)alkyenyl, -C(O)alkylaryl, -C(O)alkylheteroaryl, -C(0)heterocyclyl, or -C(0)alkylheterocyclyl; 5
R
13 is -H, -alkyl, -aryl, -alkylaryl or -alkylheteroaryl; and the other substituents are as described 10 above. Preferably, in (c), (d), (e)., or (f), R 8 is methyl, ethyl, n-propyl, isopropyl, cyclopentyl, phenethyl, or benzyl. 15 Preferred definitions for the other individual components of embodiment B are the same as those set forth above for embodiment A. A preferred embodiment C of this invention provides compounds of formula I: 20 5 RS
R
6 R4 0 wherein: Y is: -23 (a) R7 H 0 or (b) 50 O 5 Ho H. m is 0 or 1; X is -C(R 3
)
2 10
R
1 is H, -R 8 , -C(O)R 8 , -C(O)C(O)R8, -S(0)2R 8 ,
-S(O)R
8 , -C(o)OR 8 , -C(0)N(H)R 8 , -S(O)2N(H)-R 8 ,
-S(O)N(H)-R
8 , -C(O)C(O)N(H)R8, -C(0)CH=CHR 8 ,
-C(O)CH
2 OR8,
-C(O)CH
2
N(H)R
8 , -C(O)N(R8) 2 , -S(0)2N(R 8
)
2 , 15 -S(O)N(R8) 2 , -C(O)C(O)N(R 8
)
2 , -C(0)CH2N(R 8
)
2 , -CH 2
R
8 ,
-CH
2 -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 , 20 or each R 3 . together with the atom to which they are bound, form a 3 to 7 menbered cyclic or heterocyclic ring system, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -R 1 0 , a hydrogen atom bound to any -aryl or 25 -heteroaryl carbon atom is optionally replaced by -R 11 , a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -R 1
;
- 24 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 one R 5 together with the atoms to which they are bound form a 3 to 7 membered cyclic 5 or heterocyclic ring system, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 1 0 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by RIl, and a hydrogen atom bound to any 10 nitrogen atom of the ring system is optionally replaced with
R
1 ;
R
6 is -H; 15
R
7 is -OH, -OR 8 , -N(H)OH, or -N(H)S(0) 2 R8; each R 8 is independently -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, 20 wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally 25 replaced by R 1 ; each
R
9 is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is 30 optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by Ril, and a hydrogen atom bound to any nitrogen atom is optionally replaced by RI- - 25 each R 10 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2,
-CO
2 H, -C(O)NH 2 , -N(H)C(O)H,
-N(H)C(O)NH
2 , -perfluoroalkyl, -0-alkyl, -0-aryl, 5 -0-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)Oalkylheteroaryl, -N(H)C(O)Ocycloalkyl, 10 -N(H)C(0)N(H)alkyl, -N(H)C(0)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) alkylheteroaryl, -N(H)C(O)N(H)cycloalkyl, -S-alkyl, -S-aryl, -S-alkylaryl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, 15 -CH2NH 2 , -CH2N(H)alkyl, or -CH2N(alkyl) 2 , -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally 20 replaced by R 11 and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; and each R 11 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH 2 , -CO2H,
-C(O)NH
2 , -N(H)C(O)H, 25
-N(H)C(O)NH
2 , -alkyl, -cycloalkyl, -perfluoroalkyl, -0 alkyl, -0-aryl, -0-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, 30 -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH 2 , -CH2N(H)alkyl, or -CH2N(alkyl) 2 ; provided that if one R 3 is -H, then the other
R
3 is not -H.
- 26 Another preferred embodiment D of the present invention provides a compound of formula I, wherein Y is: 0 OR 8 0 OR' m m
R
8 0 H
R
8 , ,or H H OR 8
OR
12 N H 5 (c) (d) (e) (f)
R
12 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)alkyenyl, -C(O)alkylaryl, -C(O)alkylheteroaryl, -C(O)heterocyclyl, or -C(O)alkylheterocyclyl; and 10 the other substituents are as described above except that both of the R 3 groups may be -H. In any of embodiments A-D, preferred compounds are those wherein: Rl is -C(0)R 8 or -C(0)C(0)R 8 ; 15
R
2 and one R 3 are both -H and the other R 3 is an amino acid side chain, -R 8 , alkenyl-R 9 , or alkynyl-R 9 ; or
R
4 and one R 5 together with the atoms to which they are bound form a ring system selected from: 1 Rio S R10 Ni ,NN R10 N R10 R1, ~ N~5~R10 f N N N 20 - 27 R1o Rio (CH2 )n N S Rio N N N Rio 10 R 10 NN NN N N J R Rio
R
1 0 Rio N 1 R 0 N N N
NR
H ,or| provided that each of the ring systems are optionally substituted with one or more R 10 groups.
-28 Alternatively, preferred compounds of embodiments A-D are those wherein R 3 is -H and the other R 3 is methyl, isopropyl, tert-butyl,
-CH
2
SR
8 ,
-CH
2
SO
2 R, -CH 2
CH
2 SRS, -CH 2
CH
2
SO
2
R
8 . 5 More preferred compounds of embodiments A-D are those wherein R 4 and one R 5 together with the atoms to which they are bound form the ring system: 10 and the other RS is H; or one R 3 is -H and the other R 3 is methyl. Alternatively, more preferred compounds of 15 embodiments A-D are those wherein R 4 and one R 5 together with the atoms to which they are bound form the ring system: -N 20 and the other R 5 is H. In the above alternative embodiment,
R
10 is preferably, 4-fluoro or 4,4-difluoro. Most preferred compounds of this invention 25 are those wherein R 3 is methyl; and
R
4 and one R 5 together with the atoms to which they are bound form the ring system: gN -29 and the other R 5 is H. Alternatively, most preferred compounds of embodiments A-D are those wherein
R
3 is methyl; and 5 R 4 and one R 5 together with the atoms to which they are bound form the ring system: R1o and the other R 5 is H; and
R
1 0 is 4-fluoro or 4,4-difluoro. Preferred compounds of embodiments (B) or (D) are those wherein Y is: 0 m 25 0 H z wherein Z represents
-OR
8 and Z is: CH 3 0, O >-0 0&0 0 2 I o 0 00 -30 0 04,0 0" 0r 0 N .0 Co H 0N R8 H 00 I IN*.-Nr R8 0R 8 0, I0, - 31 H 2 N N ON O S 0 ,NNO, N 0 0 N O or N Specific compounds of this invention include, but are not limited to, Examples 5a-Sbd, 7a-7at, 9a-9g, 15a-15f, 16a-16b, 17a-17e, 18a-18f, 20a-20t, 23a-23i, 5 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 a-v, and 123 a-c. 10 The compounds of this invention may contain one or more "asymmetric" carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Each stereogenic carbon may be of the R 15 or S configuration. Although specific compounds and scaffolds exemplified in this application may be depicted in a particular stereochemical configuration, compounds and scaffolds having either the opposite stereochemistry at any given chiral center or mixtures 20 thereof are also envisioned. All such isomeric forms of these compounds are expressly included in the present invention, as well as pharmaceutically acceptable derivative thereof.
- 32 The term pharmaceuticallyy acceptable derivative" means any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of this invention or any other compound which, upon 5 administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an active metabolite or residue thereof. Pharmaceutically acceptable salts of the compounds of this invention include, for example, those 10 derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, 15 tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as 20 intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-((Cl- 4 alkyl) 4 + 25 salts. This invention also envisions the "quaternization" of any basic nitrogen-containing -groups of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to 30 those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, 35 lauryl, myristyl and stearyl chlorides, bromides and - 33 iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization. 5 When multiply substituted, each substituent may be picked 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 10 envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and administration to a mammal by methods known in the art. 15 Typically, such compounds are stable at a temperature of 40 *C or less, in the absence of moisture or other chemically reactive conditions, for at least a week. Preferred compounds of this invention may be readily absorbed by the bloodstream of patients upon 20 oral administration. This oral availability makes such compounds excellent agents for orally-administered treatment and prevention regimens against IL-1-, apoptosis-, IGIF-, or IFN-y-mediated diseases. It should be understood that the compounds of 25 this invention may exist in various equilibrium forms, depending on conditions including choice of solvent, pH, and others known to the practitioner skilled in the art. All such forms of these compounds are expressly included in the present invention. In particular, many 30 of the compounds of this invention, especially those which contain aldehyde or ketone groups and carboxylic acid groups in Y, may take hemi-acetal or hydrated forms. For example, compounds of embodiment A are in a hemiacetal form when Y is: 35 - 34 m HO Depending on the choice of solvent and other conditions known to the practitioner skilled in the 5 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: ORO m R8 10 H OR 8 and R 8 is H; acyloxy acetal forms when Y is: 40. 0 - Rao H 15 acetal forms when Y is and R 8 is other than H: 0 R8 R8 H OR 8 20 and enol forms when Y is: - 35 R8 R12 In addition, it should be understood that the 5 equilibrium forms of the compounds of this invention may include tautomeric forms. All such forms of these compounds are expressly included in the present invention. The compounds of formula I may be synthesized 10 using conventional techniques. Advantageously, these compounds are conveniently synthesized from readily available starting materials. Compounds of this invention may be prepared using the processes described herein. As can be 15 appreciated by the skilled practitioner, these processes are not the only means by which the compounds described and claimed in this application may be synthesized. Further methods will be evident to those of ordinary skill in the art. Additionally, the 20 various synthetic steps described herein may be performed in an alternate sequence or order to give the desired compounds. It should be understood that the compounds of this invention may be modified by appropriate 25 functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral 30 availability, increase solubility to allow - 36 administration by injection, alter metabolism and alter rate of excretion. In addition, the compounds may be altered to pro-drug form such that the desired compound is created in the body of the patient as the result of 5 the action of metabolic or other biochemical processes on the pro-drug. Such pro-drug forms typically demonstrate little or no activity in in vitro assays. Some examples of pro-drug forms include ketal, acetal, oxime, imine and hydrazone forms of compounds which 10 contain ketone or aldehyde groups, especially where they occur in the Y group of the compounds of this invention. other examples of pro-drug forms include the hemi-ketal, hemi-acetal, acyloxy ketal, acyloxy acetal, ketal, acetal and enol forms that are described 15 herein.
- 37 Compositions and Methods The compounds of this invention are caspase inhibitors, and in particular ICE inhibitors. Accordingly, these compounds are capable of targeting 5 and inhibiting events in IL--, apoptosis-, IGIF-, and IFN-y-mediated diseases, and, thus, the ultimate activity of that protein in inflammatory diseases, autoimmune diseases, destructive bone, proliferative disorders, infectious diseases, and degenerative 10 diseases. For example, the compounds of this invention inhibit the conversion of precursor IL-10 to mature IL 10 by inhibiting ICE. Because ICE is essential for the production of mature IL-1, inhibition of that enzyme effectively blocks initiation of IL-1-mediated 15 physiological effects and symptoms, such as inflammation, by inhibiting the production of mature IL-1. Thus, by inhibiting IL-10 precursor activity, the compounds of this invention effectively function as IL-1 inhibitors. 20 Compounds of this invention also inhibit conversion of pro-IGIF into active, mature IGIF by inhibiting ICE. Because ICE is essential for the production of mature IGIF, inhibition of ICE effectively blocks initiation of IGIF-mediated 25 physiological effects and symptoms, by inhibiting production of mature IGIF. IGIF is in turn essential for the production of IFN-y. ICE therefore effectively blocks initiation of IFN-y- mediated physiological effects and symptoms, by inhibiting production of 30 mature IGIF and thus production of IFN-y. The compounds of this invention are surprisingly bioavailable when compared with peptidyl inhibitors, such as those described in, for example, EP 618 223, EP 623 592, WO 93/09135, WO 93/16710, US 35 patent no. 5,434,248, WO 95/35308, or WO 96/33209.
- 38 Thus, the pharmaceutical compositions and methods of this invention will be useful for controlling caspase activity in vivo. The compositions and methods of this invention will therefore be useful for controlling
IL
5 1, IGIF, or IFN-y levels in vivo and for treating or reducing the advancement, severity or effects of IL-l-, apoptosis-, IGIF-, or IFN-y-mediated conditions, including diseases, disorders or effects. Pharmaceutical compositions of this invention 10 comprise a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such compositions may optionally comprise an additional therapeutic agent. Such agents include, but are not limited to, an anti-inflammatory 15 agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation 20 compound. The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that may be administered to a patient, together with a compound of this invention, and which does not destroy the 25 pharmacological activity thereof. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum 30 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 35 hydrogen phosphate, potassium hydrogen phosphate, - 39 sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, 5 polyethylene-polyoxypropylene-block polymers, wool fat and self-emulsifying drug delivery systems (SEDDS) such as a-tocopherol, polyethyleneglycol 1000 succinate, or other similar polymeric delivery matrices. In pharmaceutical composition comprising only 10 a compound of embodiments A-D as the active component, methods for administering these compositions may additionally comprise the step of administering to the subject an additional agent. Such agents include, but are not limited to, an anti-inflammatory agent, a 15 matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound. 20 The term "pharmaceutically effective amount" refers to an amount effective in treating or ameliorating an IL-1-, apoptosis-, IGIF-, or IFN y-mediated disease in a patient. The term "prophylactically effective amount" refers to an amount 25 effective in preventing or substantially lessening IL 1-, apoptosis-, IGIF-, or IFN-y-mediated diseases in a patient. The compounds of.this invention may be employed in a conventional manner for controlling IGIF 30 and IFN-y levels in vivo and for treating diseases or reducing the advancement or severity of effects which are mediated by IL-1, apoptosis, IGIF, or IFN-y. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill 35 in the art from available methods and techniques.
- 40 For example, a compound of this invention may be combined with a pharmaceutically acceptable adjuvant for administration to a patient suffering from an IL-1-, apoptosis-, IGIF-, or IFN-y-mediated disease in 5 a pharmaceutically acceptable manner and in an amount effective to lessen the severity of that disease. Alternatively, the compounds of this invention may be used in compositions and methods for treating or protecting individuals against IL-1, 10 apoptosis-, IGIF, or IFN-y mediated diseases over extended periods of time. The compounds may be employed in such compositions either alone or together with other compounds of this invention in a manner consistent with the conventional utilization of enzyme 15 inhibitors in pharmaceutical compositions. For example, a compound of this invention may be combined with pharmaceutically acceptable adjuvants conventionally employed in vaccines and administered in prophylactically effective amounts to protect 20 individuals over an extended period of time against IL 1-, apoptosis-, IGIF, or IFN-y mediated diseases. The compounds of formula I may also be co administered with other caspase or ICE inhibitors to increase the effect of therapy or prophylaxis against 25 various IL-1-, apoptosis-, IGIF-, or IFN-1 mediated diseases. In addition, the compounds of this invention may be used in combination either conventional anti inflammatory agents or with matrix metalloprotease 30 inhibitors, lipoxygenase inhibitors and antagonists of cytokines other than IL-1. The compounds of this invention can also be administered in combination with immunomodulators (e.g., bropirimine, anti-human alpha-interferon 35 antibody, IL-2, GM-CSF, methionine enkephalin, - 41 interferon-alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexone and EPO), with prostaglandins, or with antiviral agents (e.g., 3TC, polysulfated polysaccharides, ganiclovir, ribavirin, 5 acyclovir, alpha interferon, trimethotrexate and fancyclovir) or prodrugs of these or related compounds to prevent or combat IL-1-mediated disease symptoms such as inflammation. When the compounds of this invention are 10 administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this invention comprise a combination of a compound 15 of formula I and another therapeutic or prophylactic agent. The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, 20 buccally, vaginally or via an implanted reservoir. We prefer oral administration. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH 25 of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, 30 intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, 35 as a sterile injectable aqueous or oleaginous - 42 suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile 5 injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are 10 mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. 15 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 solutions 20 or suspensions may also contain a long-chain alcohol diluent or dispersant, such as those described in Pharmacopeia Helvetica, or a similar alcohol. The pharmaceutical compositions of this invention may be orally administered in any orally 25 acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium. 30 stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and solutions and propylene glycol are administered orally, the active ingredient is combined 35 with emulsifying and suspending agents. If desired, - 43 certain sweetening and/or flavoring and/or coloring agents may be added. The pharmaceutical compositions of this invention may also be administered in the form of 5 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 therefore will melt in the 10 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 15 when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a'suitable ointment containing the active components suspended or 20 dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying 25 wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, 30 polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this- invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema - 44 formulation. Topically-administered transdermal patches are also included in this invention. The pharmaceutical compositions of this invention may be administered by nasal aerosol or 5 inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance 10 bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between 0.5 and about 75 mg/kg body weight per day and most 15 preferably between about 1 and 50 mg/kg body weight per day of the active ingredient compound are useful in a monotherapy for the prevention and treatment of IL-I-, apoptosis-, IGIF-, and IFN-y mediated diseases, including inflammatory diseases, autoimmune diseases, 20 destructive bone disorders, proliferative disorders, infectious diseases, degenerative diseases, necrotic diseases, inflammatory peritonitis, osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis, 25 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 30 active hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, psoriasis atopic dermatitis, graft vs. host disease, osteoporosis, multiple myeloma related bone disorder, leukemias and related disorders, myelodysplastic syndrome, acute myelogenous leukemia, 35 chronic myelogenous leukemia, metastatic melanoma, - 45 Kaposi's sarcoma, multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer's disease, Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related 5 encephalitis, HIV-related encephalitis, aging, alopecia, neurological damage due to stroke, ulcerative collitis, infectious hepatitis, juvenile diabetes, lichenplanus, acute dermatomyositis, eczema, primary cirrhosis, uveitis, Behcet's disease, atopic skin 10 disease, pure red cell aplasia, aplastic anemia, amyotrophic lateral sclerosis, nephrotic syndrome and systemic diseases or diseases with effects localized in the liver or other organs having an inflammatory or apoptotic component caused by excess dietary alcohol 15 intake or viruses, such as HBV, HCV, HGV, yellow fever virus, dengue fever virus, and Japanese encephalitis virus. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to 5 20 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host 25 treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound. 30 When the compositions of this invention comprise a combination of a compound of formula I and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 10% - 46 to 80% of the dosage normally administered in a monotherapy regime. Upon improvement of a patient's condition, a maintenance dose of a compound, composition or 5 combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been 10 alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence or disease symptoms. As the skilled artisan will appreciate, lower 15 or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health 20 status, s'x, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician. IL-1 or apoptosis mediated diseases which may 25 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. The apoptosis-mediated diseases which may be treated or 30 prevented by the compounds of this invention include degenerative diseases. IL-1 or apoptosis mediated inflammatory diseases which may be treated or prevented include, but are not limited to osteoarthritis, acute pancreatitis, 35 chronic pancreatitis, asthma, and adult respiratory - 47 distress syndrome. Preferably the inflammatory disease is osteoarthritis or acute pancreatitis. IL-I or apoptosis mediated autoimmune diseases which may be treated or prevented include, but 5 are not limited to, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin-dependent diabetes mellitus (Type I), autoimmune hemolytic anemia, autoimmune 10 neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, psoriasis, atopic dermatitis and graft vs. host disease. Preferably the autoimmune disease is rheumatoid 15 arthritis, inflammatory bowel disease, Crohn's disease, psoriasis, or atopic dermatitis. IL-1 or apoptosis mediated destructive bone disorders which may be treated or prevented include, but are not limited to, osteoporosis and multiple 20 myeloma-related bone disorder. IL-1 or apoptosis mediated proliferative diseases which may be treated or prevented include, but are not limited to, leukemias and related disorders, such as myelodysplastic syndrome, acute myelogenous 25 leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma. IL-1 or apoptosis mediated infectious, diseases which may be treated or prevented include, but are not limited to, sepsis, septic shock, and 30 Shigellosis. IL-1 or apoptosis mediated degenerative or necrotic diseases which may be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, 35 cerebral ischemia, and myocardial ischemia.
- 48 Preferably, the degenerative disease is Alzheimer's disease. IL-1 or apoptosis-mediated degenerative diseases which may be treated or prevented by the 5 compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, and 10 neurological damage due to stroke. Other diseases having an inflammatory or apoptotic component may be treated or prevented by the compounds of this invention. Such diseases may be systemic diseases or diseases with effects localized in 15 the liver or other organs and may be caused by, for example, excess dietary alcohol intake or viruses, such as HBV, HCV, HGV, yellow fever virus, dengue fever virus, and Japanese encephalitis virus. IGIF- or IFN-y-mediated diseases which may be 20 treated or prevented by the compounds of this invention include, but are not limited to, inflammatory, infectious, autoimmune, proliferative, neurodegenerative and necrotic conditions. IGiF- or IFN-y-mediated inflammatory diseases 25 which may be treated or prevented include, but are not limited to osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative collitis, cerebral ischemia, myocardial ischemia and 30 adult respiratory distress syndrome. Preferably, the inflammatory disease is rheumatoid arthritis, ulcerative collitis, Crohn's disease, hepatitis or adult respiratory distress syndrome. IGIF- or IFN-y-mediated infectious diseases 35 which may be treated or prevented include, but are not -49 limited to infectious hepatitis, sepsis, septic shock and Shigellosis. IGIF- or IFN-y-mediated autoimmune diseases which may be treated or prevented include, but are not 5 limited to glomerulonephritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin dependent diabetes mellitus (Type I), juvenile diabetes, autoimmune hemolytic anemia, autoimmune 10 neutropenia, thrombocytopenia, myasthenia gravis, multiple sclerosis, psoriasis, lichenplanus, graft vs. host disease, acute dermatomyositis, eczema, primary cirrhosis, hepatitis, uveitis, Behcet's disease, atopic skin disease, pure red cell aplasia, aplastic anemia, 15 amyotrophic lateral sclerosis and nephrotic syndrome. Preferably, the autoimmune disease is glomerulonephritis, insulin-dependent diabetes mellitus (Type I), juvenile diabetes, psoriasis, graft vs. host disease or hepatitis. 20 More preferred diseases which may 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 25 rejection, osteoarthritis, asthma, psoriasis, Alzeheimer's disease, atopic dermatitis, or leukemias and related disorders, such as myelodysplastic syndrome or multiple myeloma. Accordingly, one embodiment of this invention 30 provides a method for treating or preventing an IL-1 or apoptosis mediated disease in a subject comprising the step of administering to the subject any compound, pharmaceutical composition, or combination described herein and a pharmaceutically acceptable carrier.
-50 Another embodiment of this invention provides a method for decreasing IGIF production in a subject comprising the step of administering to the subject any compound, pharmaceutical composition, or combination 5 described herein and a pharmaceutically acceptable carrier. Yet another embodiment of this invention provides a method for decreasing IFN-y production in a subject comprising the step of administering to the 10 subject 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 preventing and 15 treating IL-1, apoptosis-, IGIF, and IFN-y-mediated diseases, 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 which effectively bind to 20 caspases or other cysteine proteases including, but not limited to ICE. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block proteolysis of a target peptide in biochemical or cellular assays for ICE and ICE homologs or may be 25 derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial cysteine protease inhibitors will be evident to those of ordinary skill in the art. 30 In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way. 35 - 51 GENERAL METHODS Analytical HPLC conditions: Column: C-18, Particle size: 5 p, Pore size: 100A, Column size: 4.6 x 150 mm 5 Solvent A: 0.1% TFA / 1% MeCN / 98.9% water Solvent B: 0.1% TFA / 99.9% MeCN Gradient: A to B over 20 min at a flow rate of 1 mL/min 10 Column: Cyano, Particle size: 5 y, Pore size: 1oo, 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 / B = 99% / 1% to 50% / 50% over 20 min at 15 a flow rate of 1 mL/min HPLC Mass Spectral Analysis Mass Spectral Analysis: All mass spectral data were collected using a Micromass Quattro II triple 20 quadrupole mass spectrometer (Beverly, MA) equipped with a cross-flow electrospray ionization source. The mass spectrometer was coupled to a HPLC system manufactured by Hewlett-Packard (HP1100). The autosampler for the system was a Gilson 215 (Middleton, 25 WI) liquid handler. All of the equipment was controlled by the MassLynx software package purchased from Micromass. Mass spectral analysis was performed by liquid chromatography-MS to determine purity and 30 confirm molecular weight simultaneously. In instances - 52 where the sample purity had been determined by other means, a flow injection analysis (FIA) was used instead of the full chromatography analysis. In all cases, both positive and negative ion spectra were collected. 5 Mass Spectrum Acquisition Conditions: For all experiments, the mass spectrometer was configured in electrospray mode with the cross-flow counter electrode. A flow splitter was used to reduce the flow from the HPLC to 40% of the original flow. The inlet 10 temperature was set to 140 *C and the drying gas flow was set to maximize signal. The resolution of the mass spectrometer was adjusted to 0.65 amu FWHM and data was collected in centroid mode. In positive ion mode, the cone voltage was set to 25V, the capillary voltage was 15 3.8 kV. In negative ion mode, the cone voltage was set to 25 V and the capillary voltage was set to 3.5 kv. In both.positive and negative ion mode, the time to acquire a full spectrum was is with a switching time of 0.25 seconds between scans. The mass range scanned for 20 molecules with an expected molecular weight of less than 350 amu was 70-500 m/z while for molecules with a expected mass of more than 350 amu the mass to charge ratio scanned was 200-1000 m/z. Chromatography Conditions:. Liquid 25 chromatography was performed using a YMC AQ C18 column (150 mm X 3mm with 5pm particle and a 120A pore size) . For all analysis, MeCN with 0.2 % formic acid was combined with water with 0.2% formic acid to form the elution gradient. The gradient profile consisted of 30 starting with 15 % MeCN: water and increasing the amount of MeCN linearly over ten minutes to 90%. That concentration was held constant for 2 minutes before returning to initial conditions. During the entire analysis the flow rate was 0.9mL/min.
- 53 Flow Injection Conditions: A 1:1 mixture of the water to MeCN (both with 0.2% formic acid added) was used to acquire the FIA data. The flow rate was set to 0.3 ml/min. 5 -H NMR All 1 H NMR spectra were acquired using a Bruker Instruments AMX-500 NMR spectrometer in the solvent given. 10 SYTMETIC METHODS General Procedure for the Preparation of Compounds of Formula I, Embodiment C (Schemes
I-VI)
- 54 Procedure for the preparation of analogs Sa-Sbd. Scheme I 02Bu-t Step 1 2B Fmoc' H H Fmo c'N N'NH H 2 NRei H N N LNR 1 H OH F O Step 2 Fmoc O2Bu-t Step 3 Fmod- O2Bu-t R N N 0 Oy H NI Se I-VIII t v N LR H 0 3 SteS 4 Ri-CO2H LR= -NH 00N "' RiH. y 0* Q~ 5 0 In Schemes I-VIII, the variable LR refers to the 5 linker-resin and is def ined as shown above in Scheme 1. Step 1: A 6.7 g portion (0.8 mmol/gram loading, 5.36 mmol) of 4-methyl benzhydrylamine hydrochloride salt resin (Scheme I) was washed with DMF (3 x 50 mL) , 10% 10 DIEA/DMF (3 x 50 mL) and N-methylpyrrolidinone (NMP) (3 - 55 x 50 mL). To a suspension of the washed resin in 25 mL of NMP was added successively compound 1 (1.1 eq, 3.5 g, 5.90 mmol) DIEA (3.33 eq, 3.1 mL, 17.70 mmol), 1 hydroxybenzotriazole hydrate (HOBt) (1.1 eq, 797 mg, 5 5.90 mmol), and 0-benzotriazole-N,N,,NN tetramethyluronium hexafluorophosphate (HBTU) (1.1 eq, 2.24 g, 5.90 mmol). Compound 1 was prepared according to the literature procedure of A. M. Murphy et al, J. Am. Chem. Soc., 114, pp. 3156-3157 (1992). The mixture 10 was rotated at room temperature overnight using a wrist arm shaker. The resulting mixture was filtered, and the resin was rinsed with DMF then treated with 12 mL of a 20% 15 solution of acetic anhydride in DMF for 30 minutes at room temperature. The mixture was filtered, and the resin was washed successively with DMF (2 x 50mL),
CH
3 0H (50mL), 1:1 DMF/ CH 2 Cl 2 (2 x 50mL), CH 3 0H (SOmL) and CH 2 Cl 2 (3 x 5OmL). After drying in vacuo, 9.0 20 grams of resin 2 were obtained (0.48 mmol/gram loading). 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. 25 The suspension was rotated at room temperature for 5 minutes and drained. The procedure was repeated over 20 minutes. The resin was then washed successively with DMF (2 x 40 mL) , CH 3 0H (40 mL), CH 2 Cl 2 (2 x 40 mL) ,
CH
3 0H (40 mL) and NMP (40 mL). To a supension of resin 30 in 40 mL of NMP was added successively 2.92 g of N Fmoc-proline (4 eq, 8.64 mmol), 3.0 mL of DIEA (8 eq, 17.28 mmol), 1.17 g of HOBt (4 eq, 8.64. mmol) and 3.27 g of HBTU (4 eq, 8.64 mmol). The mixture was rotated at room temperature overnight and drained. This - 56 coupling procedure was repeated over 3 hours. The resin was then washed successively with DMF (2 x 40 mL) , CH 3 0H (40 mL) , 1:1 DMF/ CH 2 Cl 2 (2 x 40 ML) , CH 3 0H (40 mL) and CH 2 Cl 2 (3 x 40 mL), and briefly dried in 5 vacuo to afford resin 3. Step 3: A suspension of resin 3 in 25 mL of a 20% solution of piperidine in DMF was rotated at room temperature for 5 minutes. The suspension was drained. 10 The procedure was repeated over 20 minutes. The resin was washed successively with DMF (2 x 40 ML) , CH 3 0H (40 ML), CH 2 Cl 2 (2 x 40 mL) , CH 3 0H (40 mL) and NMP (2 x 40 mL). To a suspension of resin in 40 mL of NMP was added successively 2.93 g of N-Fmoc-valine (4 eq, 8.64 15 mmol), 3.0 mL of DIEA (8 eq, 17.28 mmol), 1.17 g of HOBt (4 eq, 8.64 mmol) and 3.27 g of HBTU (4 eq, 8.64 mmol) . The mixture was rotated at room temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was then washed 20 successively with DMF (2 x 40 mL) , CH 3 0H (40 mL) , 1:1 DMF/ CH 2 Cl 2 (2 x 40 mL) , CH 3 0H (40 mL) and CH 2 Cl 2 (3 x 40 mL), and dried in vacuo to afford resin 4 (0.45 mmol/gram). 25 Step 4: To a 0.05 mmol portion of resin 4 was added 2 mL of a 20% solution of piperidine in DMF. The suspension was rotated at room temperature for 5 minutes, and drained. The procedure was repeated over 20 minutes. The resulting resin was washed 30 successively with DMF (3 x 5 mL) , CH 3 OH (5 mL), and NMP (3 x 5 mL) . The desired carboxylic acid was then added (4 eq, 0.2 mmol), followed by 0.8 mL of a 0.25M solution of HOBt in NMP, 0.14 mL of DIEA (8 eq, 0.4 - 57 mmol) and 0.8 mL of a 0.25M solution of HBTU in NMP. The mixture was rotated at room temperature overnight and drained. The resin was washed successively with DMF (2 x 5 ML) , CH 3 0H (5 mL) , 1:1 DMF/ CH 2 Cl 2 (2 x 5 5 mL) , CH 3 0H (5 mL) and CH 2 Cl 2 (3 x 5 mL) , and dried in vacuo. 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 filtered. The filtrate was evaporated, and the residue was taken 10 up in acetonitrile-water and purified by preparative HPLC to afford compounds 5a-Sbd. Product yield, analytical HPLC conditions, HPLC retention time, product purity, and mass spectral data obtained for examples 5a-Sbd, 7a-7at, 9a-9g, 15a 15 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 are provided in Table 1 unless noted otherwise. Table 1. Physical Data for Selected Examples Ex. Yield HPLC RT (min) Purity Mass Spec (mg) Gradient (%) (M+H or /time (min) M+Na) 5a 1.0 5-45%/100 4.66 92 475.2 5b 1.0 5-45t/10 6.86 85 457.2 5c 4.0 5-45%11 5.98 as 469.2 5d 1.5 5-45%11 6.82 95 467.5 Se 1.0 5-4st110' 5.52 95 418.2 5f 0.6 5-45%11 4.28 93 434.2 5g 6.4 10-60%/101 8.57 97 504.7(+Na) 5h 15.6 10-60%-11 4.51 99 459.2 Si 6.6 st%-90%/10' 9.34 90 506.1 5j 5.1 S%-90%/b0- 10.04 95 506.1 5k 10.7 St-90%/b0' 8.64 85 520.1 51 6.6 s%-90%/10' 8.72 85 540.1 Sm 6.8 SC-90t/10' 7.89 85 502.0 Sn 1.9 5*-0%/101 6.46___ 85 494.1 So 3.8 S*-90%/10' 7.04 85506.1 sp 6.8 S%-90%/10' 11.62 95576.0 !1 2.2 st-g0*/16' 9.72_ 90 508.1 Sr 3.8 s%-90%/10' 7.27 85 462.1 Ss 4.3 S*- 90%/b; ' 6.46 .90 470.31
-________
- 58 Ex. Yield HPLC RT (min) Purity Mass Spec (nug) Gradient M% (M+H or /time (min) ______M+Na) St 5.9 5%-60%/9' 10.27 90 486.2 _____ 60%-90%/21 _____ Su 3.1 Sk-60*/9- 9.09 80 522.1 60%-904;/2- _____ 5v 1.0 5t-60%/9, 11.63 5S 02.2 60%-90%/2 ____ 5w 10.3 t-60%/9 8.75 9- 5 470.2 60%-90%/21 _____ 5x 8.8 5-60%/9 8.88 95 565.1 _____ 60%-90%/2' _____ Sy 6.6 5%-60*/91 12.32 95 518.2 60t-90%/2' I__________ Sz 10.2 5%-60%/q, 12.63 95 502.2 60%-90%/21 ___ Saa 2.5 5%-60%/9' 9.57 95 554.1 60%-90%/2' ____ Sab 7.8 5%-60%/9, 10.54 53 60*-90%/2' 538.2___ Sac 1.4 5%-60%/g, 9.28 476-2 60k-90t/2' Sad 5.3 5%-60%/g, 6.51 469.2 _____60t-90%/21 Sae 4.3 5%-60k%/9' 9.81 95551.1 60'k-90%/2' _____ -a f -0 .9 5*k-60*/91 9.98544 60k-90%/21 90__547.4 Sag 5.7 Sk-60V/9 f- 10.3.1 90 526.2 60%-90*/21 _________ 5al 1.4 i -60l/9i 8.13 85 542.1 60%-90%/21 9a 10.9 l0%-90.%/1o' 5.88 85__ 584.2 T -4 .2 1Ot-90't/1b 5.89 90 556.2 5ak . 10*-90*/10' 5.54 65 568.3 Sal 8.4 10%-90%/ao' 6.25 95 516.2 Sanm 7. 6 10%4;- 9 0;%/0-O 6.49 95 474.3 San 6.2 10%-90%/1. 6.00 9 .....
2 .Sao 9.4 10%-90t/iol 6.68 95 581.3 S-ap 6.4 104;- 9 0 %/[1 4.30 90 500.3 Sa -.- 0-9*1' 64 95 559.2 Sar 1.4 10%-90%/1o'- 5.38 90 561.3 Sas .16.,2 10%-90%/10 4.25 95 475.2 Sat 1S. 4 10%-90t/lo' 6.68 95560.3 5au 5.9 10 %-9 0%-/l0 6.70 90 498.3 Say 4. 10%-.90%/1o't 5.13 5i 31.2 Saw T. 5 10%-90%t/ of 6.53 85570.3 Sax 14.0:: %90/O 6.26 90__ 557.3 - 59 Ex. Yield HPLC RT (min) Purity Mass Spec (mg) Gradient (%) (M+H or /time (min) M+Na) Say 10.4 10%-90%/10' 6.52 90 510.3 5az 9.2 10%-90%/10' 5.96 95 522.3 Sba 8.5 10%-90%/10' 6.69 95 562.3 Sbb 4.6 10%-90%/10' 6.00 85 520.2 Sbc 8.8 10%-90%/10' 4.96 90 546.2 sbd 8.2 10%-90%/10' 8.01 95 536.3 7a 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%/10' 4.04 94 434.2 7d 1.1 5-45%/10' 4.29 95 441.2 7e 1.1 5-45%/10' 3.28 98 447.2 7f 1.0 5-45%/10' 3.96 97 420.2 7g 1 10%-90%/10' 4.00 95 483.4 7h 6.0 10%-90%/10' 4.90 95 439.3 7i 12.0 10-90%/10' 6.40 95 474.3 7j 6.6 10-90%/10- 4.50 95 461.4 7k 4.0 10%-90%/10' 5.40 95 480.4 71 8.6 10%-90%/10' 2.61 95 435.3 7m 6.0 10%-90%/10' 4.29 95 438.4 7n 15.4 l01-90i/i1i 2.00 85 433.4 70 8.4 10-90/10- 4.01 90 470.0 7p 3.0 10%-90%/10' 4.61 95 434.4 7q 5.7 10-9it0%/i0 3.89 95 434.3 7r 8.8 10-90*/l1 2.94 95 425.3 7s 10.5 EI!-90E/tO' 3.89 90 433.4 7t 6.9 101-90%/10' 2.45 85 419.3 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 10-904i/.0i 3.90 95 483.2 7y 12.0 tii90ii 0 5.40 95 489.2 7z 10.0 10%-90%/10' 5.40 95 517.2 7aa 11.0 10%-90%/.bO' 5.30 95 453.8 7ab 10.0 10%-90%/10' 5.60 95 595.1 7ac 3.9 10-6011 4.59 88 469.2 7ad 13.0 5-45%/10' 4.48 88 41S.2 7ae 4.9 5-45%/10E 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 91 447.3(+Na) 7ai 10.8 5-45/10i 9.21 95 480.8 7aj 8.3 5-45t/10 9.14 92 J481.1(+Na) 7ak 13.6 5-45!/jO' 8.05 96 464.8( Na) 7al 8.1 5-45t/10 7.04 91 448.8(+Na) 7am 7.8 5-45%/10' 8.54 90 480.4 (+Na) - 60 Ex. Yield HPLC RT (min) Purity Mass Spec (mg) Gradient (%) (M+H or /time (min) M+Na) 7an 4.3 5-45%/10' 7.48 88 460.9(+Na) 7ao 13.5 5-45/jo' 7.45 92 460.7(+Na) 7ap 2.5 5-45%/10 6.52 93 440.3(+Na) 7aq 1.4 5-45%/10' 3.30 84 405.2 7ar 15.10 5-45t*lf ' 3.79 97 413.30 7as 15.70 5-45k11 5.50 88 441.30 7at 22.20 5-45t/10' 4.49 94 415.30 9a 0.5 10-60%/101 7.08 98 527.4(+Na) 9b 1.2 10- 60%o'T- 8.31 93 570.5(+Na) 9c 2.5 10-60*110' 8.46 97 569.6 9d 1.2 10-60%/1o' 7.22 85 562.1 9e 0.4 10-60%/10 7.86 95 543.2(+Na) 9f 4.3 10-60*110' 8.11 96 542.5 9g 2.1 10-60%/1o' 7.18 93 526.3 15a 1.0 10 -60%/10' 5.76 95 477.6 15b 3.9 -- -- 0---- 6.32 91 483.8 15c 2.9 b0%-90%/b0' 3.30 85 463.3 15d 1.3 0%-90%/bo' 4.26 95 531 15e 1.0 10%-90%ljo' 3.10 a5 482.3 1Sf 1.2 10%-90%/jo' 3.60 95. 484.6 16a 2.5 ---- 90-/-0- 2.80 95 456.3 16b 6.0 10%-90%/b0' 1.90 95 454.2 17a 1.0 10---90%-10- 3.30 85 496.8 17b 1.1 10%-90%/10- 3.62 85 498.3 17c 2.3 1o*-90%/10' 5.80 95 573.2 17d 1.6 10%-901/10' 1.60 95 525.1 17e 1.5 10%-- -/- - - 2.62 95 526.3 18a 1.0 10%-90*/10 3.90 95 528.3 38b 1.2 0%-90%/10' 5.27 95 562.3 18c 2.2 : %-90:/10' 4.09 95 499.2 18d 1.5 lI-90I/1i' 3.78 95 498.3 18e 1.2 10%90%i10i 4.78 95 541.3 18f 7.1 10%-90*/j01 3.84 98 526.1 20a 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%/1O' 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%/101 3.25 95 518.3 20g 12.1 1o%-90%/jO' 5.01 90 487.2 20h 3.3 10%-90%/O' 4.30 90 533.2 I-20i 5.0 "ot-90*/10 4.16 90 48.2 _20__ 1.3 10%-90*/I0' 3.45 85 531.2 20k 9.7 10E-90%Ei0l 5.41 90 516.2 201 3.8 10%-90%/ O: 3.73 85 504.2 - 61 Ex. Yield HPLC RT (min) Purity Mass Spec (mg) Gradient (%) (M+H or /time (min) M+Na) 20m 6.6 1Eli790/0 4.52 90 488.2 20n 1.8 mna-mm/10 2.85 90 551.2 20o 7.0 10%-90%/10' 4.70 95 520.2 20p 1.2 101-90%/b', 4.00 95 566.2 20q 2.3 101--90I;10- 4.93 95 481.3 20r 3.6 11-g90i/10i 4.45 95 519.3 20s 3.0 10i-90%/10 4.18 95 552.2 20t 6.0 10-90%/10 3.80 90 517.4 23a 21.0 10-60110, 8.11 99 495.4 23b 25.0 10-60W/10' 8.94 99 539.8(-Na) 23c 26.0 10-60%/10 8.55 99 39.9(+Na) 23d 12.6 1090/10 3.90 85 453.3 23e 8.3 10%-90%110' 5.16 85 501.3 23f 12.5 10*- 90%/150'1 3.41 80 425.3 23g 1.5 10%-90%/101 3.34 85 452.3 23h 8.4 10t-90i/10 3.84 90 451.3 23i 9.0 10%-904/30' 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 37S.4 24c 7.2 10-60%/101 10.33 96 531.0 24d 3.4 10-60%/i0' 6.51 95 4-i26. 5(+Na) 24e 6.9 10-60110' 7.22 99 455.5 -25a 1.9 5-45%-/10, 5.38 85 455.2 25b, 1.5 54%/10- 6.90 97 483.2 25c 1.0 5-451/10' - 8.09 94 497.2 25d 12.8 5-45%/10'I 5.75 8 8% 453.3 25e 9.5 5-45k/10' 7.76 go0- 495.2 26a 10.2 5-45%/la' ?.36 95 455.1(.Na) 26b 1.1 5-45%/101 7.38 89 476.3 26c 13.8 5-454;/10' 8.13 98 483.2 26d 2.3 5-451/10' 10.35 99 503.0 26e 12.'8 5-45*/10' 11.11 99 523.2 16 f 13.2 10-60%/10- 12.11 99 545.0 26g 0.7 16-601/10'- 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%/100' 5.20 92 505.1 27c 2.7 5-45%/101 7.50 82 476.6(+Nai) 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%/121 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-l - 62 Ex. Yield HPLC RT (tm) P-furity Mass Spec (mng) Gradient ()(M+H or Etime (m in) _+Na) 27k 1.3 5-45%153 95 551.1 271 16.6 5-45t/10- 5.90 91 477.2 27M 7.0 5-45%/10' 7.70 93 494.2 27n 15.1 5-45*/i0' 3.99" 86 7 466.2 28a 1.2 5 -45%.1-6-, 5.91 86 487.1 28b 0.5 5-45/ 6.86 98 486.1 28c 1.5 5-45%/36-m'' 7.47 93 515.13 29a 4 5-45*112- 8.21 98 392 29b 28.0 5-45%11 11.80 96 443.3 29c 1.7" 5-'45%-/3.0-' 3.73 97 415.2 29d 1.7 5-45*/la' 4.62 89 414.2 29e 0.6 5-4510 4.94 85 436.2 29f 1.1 5-45t/l0' 6.23 97 442.2 299 1.7 5-45t 10, 6.39 90 457.2 29h1 06.7 5-45k/10' 3.56 92 408.2 2i 0.7 5-45t/10' 6.50 96 431.2 29j 0.4 5-45%/10' 7.24 - 89 445.2 29k 1.6 -5-45t/10' 7.07 90 456.2 291 -0.9 5 -4 5 t1 3.08 99 408.2 -29m 1.5 10-60;-1101 6.68 90 406.3 29n 6.4 10O-60*/l' 4.27 87* 422.3 29o 8. 4 10-60*%/10 i' 4.42 861 422.3 -29p 13.2 110-604*/10' 5.62 83* 450.3 29q 15.1 5-45t/10' 3.9 97* 413.3 29r 15.7 15-45%/10' 5.50 88% 441.3 29s 19.9 15-45!k/10 4.30 95% 394.3 32a 7.7 5-60%/201 14.2 90 469.3 32b 4.0 5-60%/201 13.4 85 485.1(+Na) 32c 2.5 5 60*/20' 11.90 459.3 32 d 3.0 S-604/207 -8.19 95471.3 -32e 4.9 5-604;/20E': 14.2 9___ 486.2 - 63 NN&'4H NA ~H 0?J~ 2 H 0 0N 5a 09H Sh H0 H N W H N c 0 0 0 NN0 2 H N. N H cZ9H44TA 0 0 N N Sc 0' 0 0 N 02H ~NN 02 HO W 0 F 0 0 -64 0 NXH 0 H 5o 5v CI 0 N N .q N?402H 5NH O 050 0 5q 2 N~CX(NCg N 2 H I N HH 01 4H 06 00 N N N H N502 If H44y - 65 o9H Oy<2 0 NH 5ab 5ah - 0 Sac Sa0 0 N b0(H N N N H- 0 Sad 05aj 0 H 0 -N f oC02 4F24l 5ae oSk H 0 0 5at l 2 0 oe 0 0~ N Saal 0 Sa *am 0 - 66 N _No2Hf N'CNg OH 00 cl an H 05au H Cl N ~C02HN02 Q5 g 5awY iH 0 I 0 NN 530 0a 020 aw NI~I4H Na 020Ha H Sa - 67 F 5bc 00 N OH ON 02H H 0 ( . 5bb 0 bd Procedure for the preparation of analogs 7a-7at. 5 Scheme II Fmo-N f 2Bu-t Fm Ste 3 FmcOOB 0 0 1 NLR H ON'L 3 62 Step 4 R1-CO2H 7 N22 0 0 O Analogs of 7a-7at were prepared as described above in Scheme I only substituting Fmoc-alanine for Fmoc-valine 10 in Step 3 (Scheme II) - 68 Step 3: A suspension of resin 3 (3.5 g, 1.75 mmol) in 20 mL of a 20% solution of piperidine in DMF was rotated at room temperature for 5 minutes. The suspension was drained. The procedure was repeated over 5 20 minutes. The resin was washed successively with DMF (2 x 30 mL) , CH 3 0H (30 mL), CH2CI 2 (2 x 30 mL) , CH 3 0H (30 mL) and NMP (2 x 30 mL). To a suspension of resin in 30 mL of NMP was added successively 1.44 g of N Fmoc-alanine (4 eq, 7.0 mmol), 2.4 mL of DIEA (8 eq, 10 14.0.mmol), 0.95 g of HOBt (4 eq, 7.0 mmol) and 2.66 g of HBTU (4 eq, 7.0 mmol). The mixture was rotated at room temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was then washed successively with DMF (2 x 30 mL), CH 3 0H 15 (30 mL), 1:1 DMF/ CH 2 Cl 2 (2 x 30 ML), CH 3 0H (30 mL) and
CH
2 C1 2 (3 x 30 mL), and dried in vacuo to afford resin 6 (0.50 mmol/gram). Step 4: To a 0.125 mmol portion of resin 6 was added 5 20 mL of a 20% solution of piperidine in DMF. The suspension was rotated at room temperature for 5 minutes, and drained. The procedure was repeated over 20 minutes. The resulting resin was washed successively with DMF (3 x 5 mL), CH 3 0H (5 ML), and NMP 25 (3 x 5 mL). The desired carboxylic acid was then added (4 eg, 0.6 mmol), followed by 2.0 mL of a 0.25M solution of HOBt in NMP, 0.35 mL of DIEA (8 eq, 1.0 mmol) and 2.0 mL of a 0.25M solution of HBTU in NMP. The mixture was rotated at room temperature overnight 30 and drained*. The resin was washed successively with DMF (3 x 5 mL), CH30H (5 mL), 1:1 DMF/ CH 2 Cl 2 (2 x 5 mL), CH 3 0H (5 mL) and CH 2 Cl 2 (3 x: 5 mL), and dried in vacuo. A 5 mL portion of a 95% solution of TFA in water was then added to the resin. The mixture was - 69 stirred at room temperature for one hour, and filtered. The filtrate was evaporated, and the residue was dissolved in acetonitrile-water and purified by preparative HPLC to afford compounds 7a-7at.
- 70 0 N N02H H 2 N N HN K NNg H I C0 2 H 7a7h 0 0 b N c C02 7 02 0) 0 N F;o 0 0H HO HON 0 0 0 Nf 0 hm NJ Hc 0 H7j ?7 0 H 0 0 N 0 0 0 -71 HO C0 2 HN 7o 7v0 00 H 2 N l 7p 7w S N N0 2 0- OHH 7q 0 x0 oyH2N 0KN H y 0 F0 7r 0<r F Hy N IyN2 7N &I 7H 00 1HN 0t 0 7s7aa ~ N ? 4 2 H 02H%% 0 0 7ab - 72 .Y? H HN HII1II H C IN 0 0 H0 J Y . 4H 0 J? NJ7 I',--I, HH 0l 0 N 7ad a 0 00 0 0 7ae a 0 00 2 H N F 0 7af H 0 F F 7am 0 0 7ag 07an0 q2H jg HN A2H 7ah 7a CI0 H. 0 OH~JN 02H cl00 ~II 7i7ap 0
-
73 H2N N 02H O N N 2H 7aq 0 7as 0 H 0X #N(~I
H
2 N HiI HO H 7ar O 7at - 74 Procedure for the preparation of analogs 9a-9g. Scheme III
CO
2 Bu-t Step I CO 2 Bu Fmoc, Hb Fmo c *NH 2NRe N, LR O H ,NH F m oc C02H
.NHR
1 ,NHFmoc BOo--
O
2 Bu-t Step 3 Boo- 02Bu-t 7 LR 'LR 6 Fmoc. OH Step 4
INHR
1 Fmoc 0 Step 5 R 1 , O 2 H LR 9 Step 1: A 10.0 g portion (0.75 mmol/gram loading, 7.5 5 mmol) AgroPore-aminomethyl resin (catalog number 800047) was washed with DMF (3 x 40 mL) , 10% DIEA/DMF (3 x 40 mL) , DMF and NMP (3 x 40 ML) . To above resin was added successively compound 1. (0.87 eg, 3.88 g, 6.55 mmol), HBTU (1.14 eq, 3.13 g, 8.25 mmol), HOBt - 75 (1.14 eq, 1.26 g, 8.25 mmol), 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 5 mL, 25 mmol) was added and the resulting suspension mixed at room temperature overnight, filtered, then washed successively with NMP (3x 40 mL) and DMF (3x 40 mL) . The resin was then treated with 50 mL of a 20% solution of acetic anhydride in DMF for 38 minutes at 10 room temperature. The mixture was filtered, and the resin was washed successively with NMP (3x 40 mL)
CH
2 Cl 2 (3x 40 mL), 1:1 CH 3 0H / CH 2 C1 2 (3x 40 mL), and
CH
3 0H (3x 40 mL). After drying in vacuo, 13.76 grams of resin 2 were obtained (0.35 mmol/gram loading). 15 Step 2: Seven reaction vessels were each charged with 181 mg of resin 2 (0.48 mmol/gram, 0.063 mmol) then washed with CH 2 Cl 2 (3 x 1 mL) and NMP (3 x 1 mL). Then each vessel was treated with 1 mL of a 25% solution of 20 piperidine in DMF and mixed (vortex) at room temperature for 15 minutes. This procedure was repeated in triplicate. Each vessel was then washed three times with NMP (3 x 1 mL). The vessels were then treated with 500 p1 of a solution of 0.4 M (2S,4R)-Fmoc-4 25 amino-1-Boc-pyrrolidine-2-carboxylic acid /0.4 M HOBt/ NMP, 500 pl of a solution of 0.4 M HBTU/ NMP, and 250 pl of a solution of 1.6 M DIEA/ NMP and mixed for 3 hours at room temperature. After mixing, the vessels were drained and the procedure was repeated. Step 3: The resulting resin was washed with NMP (3 x imL) and then treated with 1 mL of a 25% solution of piperidine in DMF and mixed (vortex) at room temperature for 15 minutes. The procedure was repeated - 76 in triplicate. The resulting resin was washed with NMP (3 x imL) then treated with either acetic anhydride, or isopropyl isocyanate, or methane sulphonyl chloride, or methyl chloroformate. For acetic anhydride: add 300 pl 5 of a 1.6 M DIEA/ NMP solution and 1 mL of a solution of 0.5 M acetic anhydride/0.125 M DIEA/ 0.015 M HOBt in NMP. For isopropyl isocyanate: add 300 pl of a 1.6 M DIEA/ NMP solution and 1 mL of a solution of 1 M isopropyl isocyanate in NMP. For methane sulphonyl 10 chloride: add 600 pl of a solution of 1 M pyridine in
CH
2 C1 2 and 600 pl of a solution of IM methane sulphonyl chloride in CH 2 C1 2 . For methyl chloroformate: add 500 pl df a 1.6 M DIEA/ NMP solution and 1 mL of a solution of 0.7 M methyl chloroformate in CH 2 Cl 2 The resulting 15 suspensions were mixed for 6 hours at room temperature, the solvent drained and the coupling procedure repeated. Step 4: The resulting resin was washed with NMP (3 x 20 imL) then treated with a 1:1 mixture of TFA/ CH 2 Cl 2 at room temperature for 30 minutes. The resulting resin was then washed with CH 2 C1 2 (3 x 1mL) and NMP (3 x 1mL). The resin was then treated with 500 pl of a solution of 0.4 M Fmoc-valine-carboxylic acid/0.4 M 25 HOBt/ NMP, 500 pl of a solution of 0.4 M HBTU/ NMP, and 250 pl of a solution of 1.6 M DIEA/ NMP and mixed for 3 hours at room temperature. After mixing, the vessels were drained and the coupling procedure was repeated. 30 Step 5: The resulting resin was washed with NMP (3 x imL) 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 - 77 (3 x 1mL) then treated with either 500 p1 of a solution of 0.4 M 1-isoquinoline carboxylic acid/0.4 M HOBt/ NMP or 500 pLl of a solution of 0.4 M p-anisic acid acid/0.4 M HOBt/ NMP. The resulting mixtures were treated with 5 500 pl of a solution of 0.4 M HBTU/ NMP and 250 pl of a solution of 1.6 M DIEA/ NMP then mixed for 3 hours at room temperature, the solvent drained and the procedure repeated. The resulting resin was treated with 1.5 mL of a 95% solution of TFA in water and stirred at room 10 temperature for one hour then filtered. The filtrate was evaporated, and the residue was taken up in a 2:1:2 mixture of DMF /acetonitrile/water and purified by preparative HPLC to afford compounds 9a-9g.
- 78 0 ,,NH :INH O N HN H 0 0 N'0 -0 N 9a Se O N O O O2H NH NH ., NO 2 H N2H 0 H H4 N OH 9b 0 9df Procedure for the synthesis of analogs 15-18:
N
- 79 Scheme IV Fm u( N'Bc~ 1BOC Fmo2B Fmoj ciNy Fmc N) HCO 2 H
CO
2 Bu-t NLR Step 1 0. N 2 10 NL Fmoc , H StepH
R
3 = CH 3 or CH (CH Sep Rj- N 2B-t RIC02H Fmnoc,. N VN B o ~LH _____ N 13. R 3 =HC~L 11, R 3
=CH(CH
3
)
2 14 3 = C H 3 12, R 3 =CH3 Step 4
R
1
R
1 N *R N 0 0 15, R 3 =CH(CH3) 2 17, R 3
=CH(CH
3
)
2 16, R 3 =C-1 18, R 3
=CH
3 Preparation of analogs 15 and 16 (Scheme IV) : 5 Synthesis of 2-(S) -Piperazine-1,,2,4-tricarbo~cylic acid 4-tert-butyl ester 1- (9H-fluoren-9-ylmethyl) ester.
- 80 To a solution of 2-(S)-piperazine carboxylic acid (Lonza) (3g, 15 mmol) in 1:1 H 2 0:dioxane (30 mL) was added a solution of (Boc) 2 0 in dioxane (3.3g, 15mmol, in 5mL dioxane) while maintaining the pH at 11 with iN 5 NaOH . The pH was maintained over 3 hours at room temperature. The solution was adjusted to pH9.5 with iN HCl, cooled to OC and treated with Fmoc-Cl (3.87g,15 mmol). The pH was maintained at 9.5 for 1 hour and the mixture stirred at room temperature overnight. The 10 resulting suspension was filtered and the filtrate treated with IN KHSO 4 to pH 2 then 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 15 and added to hexane to give 3.5g (51k yield) of white solid after isolation. 1H NMR (500 MHz, DMSO-d 6 ) 5 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 (M-H) 20 Step 1: To 5 g of resin 2 (0.375 mmol/gram 1.82 mmol) was added 25 mL of a 20% solution of piperidine in DMF. The suspension was rotated at room temperature for 5 minutes and drained. The procedure was repeated over 25 20 minutes. The resin was then washed successively with DMF (2 x 50 mL), CH 3 0H (50 n), CH 2 C1 2 (2 x 50 mL),
CH
3 0H (50 mL) and NMP (50 mL). To a supension of resin in 25 mL of NMP was added successively 3.5g of N Fmoc-Boc piperazine carboxlyic acid (4 eq, 7.48 mmol), 30 1.0 mL of DIEA (8 eq, 14.96 mmol), 1.01 g of HOBt (4 eg, 7.48 mmol) and 2.83g of HBTU (4 eq, 7.48 mmol). The mixture was rotated at room temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was then washed successively with - 81 DMF (2 x 50 mL), CH 3 OH (50 mL), 1:1 DMF/ CH 2 Cl 2 (2 x 50 mL), CH 3 0H (1 x 50 mL) and CH 2 Cl 2 (3 x 50 mL), and briefly dried in vacuo to afford resin 10. 5 Step 2: To 5 g (0.335 mmol/gram loading, 1.675 mmol) of 10 was added 25 mL of a 20% solution of piperidine in DMF. The suspension was rotated at room temperature for 5 minutes and drained. The procedure was repeated over 20 minutes. The resin was then washed 10 successively with DMF (2 x 50 mL) , CH30H (50 mL), CH 2 Cl 2 (2 x.50 mL), CH 3 0H (50 mL) and NMP (2 x 50 mL). To a suspension of resin in 25 mL of NMP was added successively 2.08g of N-Fmoc-valine or N-Fmoc-alanine (4 eq, 6.7 mmol), 1.17mL of DIEA (4 eq, 6.7 mmol), 15 0.905 g of HOBt (4 eq, 6.7mmol) and 1.38 g of HBTU (4 eq, 3.66 mmol). The mixture was rotated at room temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was then washed successively with DMF (2 x 50 mL), CH 3 0H 20 (50 ML), 1:1 DMF/ CH 2 Cl 2 (2 x 50 mL), CH 3 0H (50 mL) and
CH
2 Cl 2 (3 x 50 ML)), and dried in vacuo to afford resin 11 or 12 respectively (0.35 mmol/gram, 5g). Step 3: To a 1.5g ( 0.165 mmol) portion of resin 11 or 25 12 was added 2 mL of a 20% solution of piperidine in DMF. The suspension was rotated at room temperature for 5 minutes, and drained. The procedure was repeated over 20 minutes. The resulting resin was washed successively with DMF (3 x 15 mL), CH 3 0H (15 mL), and 30 NMP (3 x 15 mL) . The desired carboxylic acid was then added (4 eq, 0.66 mmol), followed by 0.25g HOBt (0.66mmol), 0.12 mL of DIEA (4 eq, 0.66 mmol) and 0.89g (0.66mmol) HBTU in NMP. The mixture was rotated at room temperature overnight and drained. The resin was - 82 washed successively with DMF (2 x 15 mL), CH 3 0H (15 mL), 1:1 DMF/ CH 2 Cl 2 (2 x 15 mL), CH 3 0H (15 mL) and
CH
2 Cl 2 (3 x 15 mL), and dried in vacuo to afford 13 or 14. 5 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 filtered. The filtrate was evaporated, and the residue was taken 10 up in acetonitrile-water and purified by preparative HPLC to afford compounds 15 and 16.
- 83 NH rl NH HNH O2H C NN O2H HO H 00 O CIO 15a 15d N NH 2H2N H H H 0 C 0 16b 1Se NH N2HH I Sc N & O N H H2N N 2H 16Ba 1 I I6b Procedure for the synthesis of analogs 17 and 18 (see Scheme IV) : 5 Step 5: Resin 13 or 14 was treated with 2 mL 25%
TFA/CH
2 Cl 2 for 30 min and washed with DMF (2 x 5 mL) , 10% DIEA/CH 2 Cl 2 (2 x 5 mLs) DMF/CH 2 Cl 2 (2 x 5 miL) , CH 3 OH (5 mL) and CH 2 Cl 2 (3 x 5 miL) and dried for five. 10 minutes . The resulting resin was washed with NMP .(3 x 1 miL) then treated with acetic anhydride, or methoxacetic - 84 acid, or 2-propanesulfonyl chloride, or isopropyl isocyanate, or methane sulphonyl chloride, or methyl chloroformate according to the procedure used to prepare analogs 9 (Scheme III) . Compounds 17 and 18 5 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 H20, 0.2 10 mL) and CH 3 COOH (0.02 mL) prior to Step 4 and compound 18c was prepared by treatment with phosgene followed by ammonia prior to Step 4.
- 85 0N N 0I. H r 0 X H2N 0 N H 2 N(0H 1 7a 17d 0 -qHC02H HH 0 C, , 0 2 H 1 7c 0' H 0 le8a le8d N ~02 lb0 8e NH2 j 02H 2 N)' I I H H Sc ,H H2 7 - 86 Procedure for the preparation of analogs 20. Compounds 20a-20t were prepared according to the procedure described for compounds 5 (Scheme I) only substituting the appropriate Fmoc-amino acid for Fmoc 5 Valine in Step 3 (Scheme V). Scheme V
R
3 Frmc N BN Step 3 F moctJN
CC
2 E3U-t
R
3 N H HO H N, LR Fmoc, N Y.H NLR 3 H 19 Step 4 1RI-CO2H 20 20 O Preparation of 3- ((1-[2-(4-amino-3-chloro 10 benzoylamino) -3 -methylsulfonyl-propionyl] -pyrrolidine 2 -carbonyl)-amino) -4-oxo-butyric acid (201). A suspension of 0.132 mmol of resin 3 in 4 mL of 20% piperidine in DMF was rotated at room temperature for 5 minutes, and the mixture was drained. The procedure 15 was repeated over 20 minutes. The resin was washed successively with DMF (twice),
CH
3 0H (once),
CH
2 C1 2 (twice), CH 3 0H (once) and NMP (twice). To a suspension of the resin in 4 mL of NMP was added successively 189 mg of N-Fmoc-methyl cysteine (4 eg, 0.528 mmol), 0.185 20 mL of DIEA (8 eq, 1. 056 mmol) , 71 mg of HOBt (4 eq, - 87 0.528 mmol) and 200 mg of HBTU (4 eq, 0.528 mmol). The mixture was rotated at room temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was then washed successively with DMF 5 (twice), CH 3 0H (once), and 1:1 DMF/ CH 2 C1 2 (twice),
CH
3 0H (once) and CH 2 C1 2 (three times), and dried in vacuo. A suspension of 100 mg of this resin in 2 mL of 20% 10 piperidine in DMF was rotated at room temperature for 5 minutes, and drained. The procedure was repeated over 20 minutes. The resin was washed successively with DMF (twice), CH 3 0H (once), CH 2 C1 2 (twice), CH 3 0H (once) and NMP (twice). To a suspension of resin in 2 mL of NMP is was added successively 38 mg of 4-amino-3-chlorobenzoic acid (4 eq, 0.2 mmol), 0.140 mL of DIEA (8 eq, 0.4 mmol), 27 mg of HOBt (4 eq, 0.2 mmol) and 76 mg of HBTU (4 eq, 0.4 mmol) . The mixture was rotated at room temperature overnight and drained. The resin was then 20 washed successively with DMF (twice), CH 3 0H (once), and 1:1 DMF/ CH 2 C1 2 (twice), CH30H (once) and CH 2 Cl 2 (three times), and dried in vacuo. The resin was then treated with 2 mL of 95% TFA in water for lh. The suspension was filtered, the filtrate was concentrated In vacuo 25 and purified by preparative HPLC to afford the title compound (201). Preparation of 3-({1-[2-(3,5-dichloro-4-hydroxy benzoylamino) -4 -methanesulfonyl-butyryl] -pyrrolidine- 2 30 carbonyl)-amino)-4-oxo-butyric acid (20p). Compound 20p was prepared according to the procedure used for the preparation of 20i using N-Fmoc-methionine - 88 as the first component coupled to resin 3, and 3,5 dichloro-4-hydroxybenzoic acid as the second component. Preparation of 3-[(1-{2-[(isoquinoline-1-carbonyl) 5 amino] -3-methanesulfonyl-propionyl)-pyrrolidine-2 carbonyl) -amino] -4-oxo-butyric acid (20r). N-Fmoc methyl cysteine was oxidized to the corresponding sulfone using the method of B. M. Trost and D. P. Curran, Tetrahedron Lett. 22, pp. 1287-190 10 (1981). To a solution of 0.714 g (2 mmol) of N-Fmoc methyl cysteine in 24 mL of a 1:1 solution of CH 3 0H water stirred at 0 0 C was added 3.68 g (3 eq, 6 mmol) of Oxonelm. The mixture was stirred at room temperature for 48 h, diluted with water, acidified to pH 2 using 15 6N HCl, and extracted with three 100 mL portions of ethyl acetate. The combined organic extracts were dried (MgSO 4 ) and concentrated in vacuo to afford 0.700 g (89% yield) of sulfone: IH NMR (DMSO-ds, 500 MHz) 8 2.97 (s, 3H), 3.49-3.59 (m, 2H), 4.25 (m, 1H), 4.30 20 4.38 (m, 2H), 4.46 (m, 1H), 7.33 (t, 2H), 7.42 (t, 2H,), 7.70-8.00 (m, 4H); exact mass calculated for Ci 9 HigNO 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% 25 piperidine in DMF was rotated at room temperature for 5 minutes, and the mixture was drained. The procedure was repeated over 20 minutes. The resin was washed successively with DMF (twice), CH 3 0H (once), CH 2 Cl 2 (twice), CH 3 0H (once) and NMP (twice). To a suspension 30 of the resin in 6 mL of NMP was added successively 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 of HOBt (4 eq, 0.50 mmol) and 188 mg of HBTU (4 eq, 0.50 mmol). The mixture was rotated at room temperature overnight - 89 and drained. This coupling procedure was repeated over 3 hours. The resin was washed successively with DMF (twice), CH 3 0H (once), 1:1 DMF/ CH 2 Cl 2 (twice), CH 3 0H (once) and CH 2 C1 2 (three times), and dried in vacuo 5 A suspension of 150 mg of this resin in 4 mL of 20% piperidine in DMF was rotated at room temperature for 5 minutes, and drained. The procedure was repeated over 20 minutes. The resin was washed successively with DMF 10 (twice), CH 3 0H (once), CH 2 C1 2 (twice), CH 3 0H (once) and NMP (twice). To a suspension of resin in 3 mL of NMP was added successively 52 mg of 1 isoquinolinecarboxylic acid (4 eq, 0.3 mmol), 0.104 mL of DIEA (8 eq, 0.6 mmol), 37 mg of HOBt (4 eq, 0.3 15 mmol) and 104 mg of HBTU (4 eq, 0.3 mmol). The mixture was rotated at room temperature overnight and drained. The resin was washed successively with DMF (twice),
CH
3 0H (once), and 1:1 DMF/ CH 2 C1 2 (twice), CH 3 0H (once) and CH 2 C1 2 (three times), and dried in vacuo. The 20 resin was then treated with 2 mL of 95% TFA in water for hour. The suspension was filtered, the filtrate was concentrated in vacuo and purified by preparative HPLC to afford the title compound (20r). 25 Preparation of 3-({1-[2-(3,5-dichloro-4-hydroxy benzoylamino) -3-me thanesulfonyl -prop ionyl I -pyrrolidine 2-carbonyl}-amino) -4-oxo-butyric acid (20s). Compound 20s was prepared according to the procedure used for the preparation of 201, using 3,5-dichloro-4 30 hydroxybenzoic acid in place of 1 isoquinolinecarboxylic acid.
- 90 02CH3
NCN
2 H0H Qg4R 4 HO2 HO ~OZ04H HOz 0 Nl 0Q 20a 20h -s
CO
2 H N 0 2 H '0 Hi 0 o 2 20b 201 HOCH 0e 20c20 HO,.,H0 2H N H0 20H 00 0 0OZI H HO h024H CN 0 20d 0c 20k - 91 C0 2
CH
3 O H H2N ON H2 CI N4N AO2 N N 4N OH H H H HO 200o N 0 20r 02CH32CH3 HO N 2 H ciq 0 HO 0 20p H CI 0 0 20s H2N 0 N 2H 2040 H2N O 20q 0 N 0 N CI H 0 20t Procedure for the preparation of analogs 23. 5 Compounds 23a-23i were prepared according to the procedure described for compounds 7 (Scheme II) only substituting the appropriate Fmoc-amino acid for Fmoc proline in Step 2 (Scheme VI).
- 92 Scheme VI O2Bu-t Step 1 02Bu-t Fmoc 'N HF C O BH N IFmoc..f; H NH H2N$4 I H N,LR 1 0 2 Fmo Step 2 FOH Fmoc 5 2Bu-t Step 3 Fmo'O O27Bu-t 0 N, FmocN H N' R 22 LR HO 21 Step 4 Ri-CO2H Ri, NRs 2 H R1s 04H 23 0 Preparation of 3-({2- [2- (4-Amino-3-chloro benzoylamino) -propionyll -4-methyl-3, 4-dihydro-2H 5 pyrazole-3-carbonyl) -amino) -4-oxo-butyric acid (23g). Compound 23g was prepared according to the procedure described for compounds 7 only substituting 4-methyl 4, 5-dihydro-pyrazole-1,S-dicarboxylic acid 1- (9H- - 93 fluoren-9-ylmethyl) ester for Fmoc-proline (Scheme II) in Step 2. Preparation of 4-methyl-4, 5-dihydro-pyrazole-1, 5 5 dicarboxylic acid 1-(9H-fluoren-9-ylmethyl) ester: To a solution of 650 mg (2 mmol) of (10,10-dimethyl 3,3-dioxo-X 6 -thia-4-aza-tricyclo[5.2.1.0 0
'
0 ldec-4-yl) (4-methyl-3,4-dihydro-2H-pyrazol-3-yl) -methanone (. 10 Am. Chem. Soc., 119, pp. 8379-8380 (1997)) in 6 mL of water and 14 mL of THF stirred at OC was added 420 mg (10 mmol, 5 eg) of lithium hydroxide. The mixture was stirred at 04C for 2 hours and at room temperature for 30 minutes, diluted with 20 mL of water and washed with 15 ether (20 mL). The pH of the solution was then adjusted to 9, and a solution of 519 mg (2 mmol, 1 eq) of Fmoc-Cl in 3 mL of dioxane was added.' The mixture was stirred at room temperature overnight, washed with ether, acidified to pH 2-3 and extracted with 3 40-mL 20 portions of ethyl acetate. The combined organic extracts were washed- with brine, dried (MgSO 4 ) and concentrated in vacuo to afford 690 mg (98% yield) of a colorless foam which was identified as the title compound. 1 H NMR (DMSO-d 6 , 500 MHz) 8 1.2 (d, 3H) , 3.2 25 (m, -11), 4.2-4.6 (m, 3H), 7.1 (s, 1H), 7.2-7.5 (m, 5H), 7. 7 -8 . 0 (m, 4H) . Exact mass calculated for C 20
H
18
N
2 0 4 m/e 350.13, found m/e 3.51.3 Preparation of 3-A{l-[2-(4-amino-3-chloro 30 benzoylamino) -propionyll -4 -methoxy-pyrrolidine -2 carbonyl}-amino) -4-oxo-butyric acid (231) .
- 94 Compound 231 was prepared according to the procedure described for compounds 7 only substituting N-Fmoc-4 methoxyproline for Fmoc-proline (Scheme II) in Step 2. 5 Preparation of N-Fmoc-4-methoxyproline: To a solution of 735 mg (3 mmol) of N-Boc-4 hydroxyproline methyl ester in 20 mL of THF stirred at 0 0 C was added 79 mg (1.1 eq, 3.3 mmol) of 60% sodium hydride in mineral oil. The mixture was stirred at 04C 10 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 addition of saturated aqueous ammonium chloride, diluted with water, and extracted with three 8OmL portions of ethyl acetate. The 15 combined organic extracts were washed with brine, dried (MgSO 4 ), and concentrated in vacuo to afford a pale yellow oil. The oil was taken up in 9 mL of CH 3 0H and 3 mL of water, and 378 mg (3 eq, 9 mmol) of lithium hydroxide was added. The mixture was stirred at room 20 temperature overnight, diluted with water, acidified to pH 3 and extracted with three 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 taken up in 10 mL of TFA and the 25 solution was stirred at room temperature.for 2 hours, and concentrated In vacuo. The residual oil was diluted with 6 mL of 10% aqueous sodium carbonate and 3 mL of dioxane; and a solution of 9-fluorenylmethyl chloroformate (779 mg, leg, 3 mmol) in 5 mL of dioxane 30 was added. The mixture was stirred at room temperature overnight, diluted with water, acidified to pH 3 and extracted with three 80 mL portions of ethyl acetate. The combined organic extracts were washed with brine, dried (MgSO 4 ) and concentrated in vacuo to afford an - 95 oil, which was purified by column chromatography over silica gel eluted with CH 2 Cl2/ CH 3 0H 20:1, to afford 600 mg (55%) of N-Fmoc-4-methoxyproline: exact mass calculated for C 21
H
2 1
NO
5 m/e 367.14 found m/e 368.4. 5 To a 0.125 mmol portion of resin 2 was added 4 mL of 20% piperidine in DMF. The mixture was rotated at room temperature for 5 minutes and drained. The procedure was repeated over 20 minutes. The resin was washed 10 successively with DMF (twice), CH30H (once), CH 2 Cl 2 (twice), CH 3 0H (once) and NMP (twice). To a suspension of the resin in 4 mL of NMP was added successively 184 mg of N-Fmoc-4-methoxyproline (4 eq, 0.50 mmol), 0.175 mL of DIEA (8 eq, 1.00 mmol), 70 mg 15 of HOBt (4 eq, 0.50 mmol) and 188 mg of HBTU (4 eq, 0.50 mmol). The mixture was rotated at room temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was washed successively with DMF (twice), CH 3 0H (once), 1:1 20 DMF/ CH 2 Cl 2 (twice), CH 3 0H (once) and CH 2 Cl 2 (three times), and dried in vacuo. To the resin was added 4 mL of 20% piperidine in DMF. The mixture was rotated at room temperature for 5 25 minutes and drained. The procedure was repeated over 20 minutes. The resin was washed successively with DMF (twice), CH 3 0H (once), CH 2 Cl 2 (twice)., CH 3 0H (once) and NMP (twice). To a suspension of the resin in 4 mL of NMP was added successively 156 mg of N-Fmoc-alanine (4 30 eq, 0.50 mmol), 0.175 mL of DIEA (8 eq, 1.00 mmol), 70 mg of HOBt (4 eq, 0.50 mmol) and 188 mg of HBTU (4 eq, 0.50 mmol). The mixture was rotated at room. temperature overnight and drained. This coupling procedure was repeated over 3 hours. The resin was - 96 washed successively with DMF (twice), CH 3 0H (once), 1:1 DMF/ CH 2 Cl 2 (twice), CH 3 0H (once) and CH 2 Cl1 (three times), and dried in vacuo. 5 To the resin was added 4 mL of 20% piperidine in DMF. The mixture was rotated at room temperature for 5 minutes and drained. The procedure was repeated over 20 minutes. The resin was washed successively with DMF (twice), CH 3 0H' (once), CH 2 Cl 2 (twice), CH 3 0H (once) and 10 NMP (twice) . To a suspension of the resin in 4 mL of NMP was added successively 80 mg of 4-amino-3 chlorobenzoic acid (4 eq, 0.50 mmol), 0.175 mL of DIEA (8 eq, 1.00 mmol), 70 mg of HOBt (4 eq, 0.50 mmol) and 188 mg of HBTU (4 eq, 0.50 mmol). The mixture was 15 rotated at room temperature overnight and drained. The resin was washed successively with DMF (twice), CH 3 0H (once), 1:1 DMF/ CH 2 C1 2 (twice),
CH
3 0H (once) and CH 2 C1 2 (three times), and dried in vacuo. 20 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 afford an oil, which was purified by HPLC to afford the title compound (231).
- 97 N N
CO
2 H N 0H IH N N C O 23f 23 \ O2H OO N H2 H H 2H 2 N O O N§H H0 23b ON N N O2H H2N 0f4H N N JIr N 2 H CI 23h a C O 23c NN N 2 H2N 0 H 02 H2N 0 N A6 0 H 2N 0 23i CI 23d
H
2 N 03 Procedure for the preparation of analogs 24a-e. Compounds 24a-24e were prepared according to the 5 procedure described for compounds 5 (Scheme I) only substituting either Fmoc-azetidine carboxylic acid or - 98 trans-2-phenyl- Fmoc-azetidine carboxylic acid for Fmoc-proline in Step 2. NN C 0 2 H N N 2 H H2 HH H 2H 0 24a 24d 24b 0 24e H 5 24c Procedure for the preparation of-analogs 25. Compounds 25a-25e were prepared according to the 10 procedures described for compounds 5 and 7 (Scheme I and Scheme II) only substituting Fmoc-2 (S) -pipecolic acid for Fmoc-proline in Step 2 and coupling either Fmoc-valine or Fmoc-alanine or Fmoc-tert-leucine in Step 3. 15 - 99 N 022XH H20 2 N NH N H HH H N c0 00 2 25b 25d H H o CI 0 25b26 H 0 25c 5 Procedure for the preparation of.analogs 26a-h. Compounds 26a- 26h were prepared according to the procedure described for compounds 23 (Scheme VI) only substituting Fmoc-valine for Fmoc-alanine in Step 3.
-100 00 HO4 0 HC0 2 H 0 N 226f H~ 0 NO N 00H 26bc N1 o 22h 4 N 00 26cd I0N 0 6 06 NS2 rcdrefrThepeaaino nlg 7 - 101 Compounds 27a- 27n were prepared according to the procedure described for compounds 7 (Scheme II) only substituting Fmoc-4,4-difluoroproline for Fmoc-proline in Step 2. 5 Preparation of N-Boc 4,4-difluoroproline methyl ester: To a solution of 9.63 mL (7.2 mmol) of oxalyl chloride in 10.6 m.L of CH 2 C1 2 stirred at -78 0 C was added a solution of 0.94 mL (13.2 mmol) of methyl sulfoxide in 10 15 mL of CH 2 Cl 2 . 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 CH 2 C1 2 was then added dropwise. The mixture was stirred at -78 0 C for 1.5 h, and 3.34 mL (24 mmol) of triethylamine was 15 added. The solution was allowed to warm up to room temperature and stirred overnight. It was then diluted with 100 mL of CH 2 C1 2 , washed successively with 100 mL of water, 100 mL of 1N HC1, and 100 mL of brine, dried (MgSO 4 ) and concentrated in vacuo. The residue was 20 purified by column chromatography over silica gel (eluted with ethyl acetate/hexanes, 1:3), to afford 1.294 g (89% yield) of N-Boc-4-oxo-proline methyl ester. 1H NMR (500 MHz, CDCl 3 ) 5 1.45 (m, 9H), 2.60 (m, 1H), 2.95 (m, 1H), 3.75 (m, 3H), 3.90 (m, 2H), 4.80 (m, 25 1H). To a solution of 808 mg (3.33 mmol) of N-Boc-4-oxo proline methyl ester in 13 ML of CH 2 Cl 2 stirred at 0 0 C was added 0.88 mL (7.19 mmol, 2.2 eq) of DAST. The 30 mixture was stirred at 0 0 C for 2 hours, at room temperature for 16 hours, and 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 - 102 residue was purified by column chromatography over silica gel (eluted with ethyl acetate-hexanes, 1:8) , to afford 754 mg (79% yield) of difluorinated derivative as a pale yellow oil. 1H NMR (500 MHz, CDCl 3 ) 8 1.50 (M, 5 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 mmol) of N-Boc 4,4 10 difluoroproline methyl ester in 5 mL of THF stirred at 04C was added a solution of 179 mg (4.27 mmol) of lithium hydroxide in 5 mL of water. The solution was stirred at 04C for 3 h, at room temperature for 1 hour, diluted with water, extracted with ether, acidified to 15 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 afford 652 mg (91%) of acid as a pale yellow solid. 20 A solution of 652 mg (2 mmol) of N-Boc-4,4 difluoroproline in 10 mL of 1;1 TFA/ CH 2 Cl 2 was stirred at 0 0 C for 45 min, and concentrated in vacuo. The residue was taken up in 3 mL of dioxane, and 5 mL of 10% aqueous sodium carbonate was added, followed by a 25 solution of 675 mg (1 eq) of Fmoc-Cl in 5 mL of dioxane. The mixture was stirred at room temperature for 16 h, diluted with 20 mL of water, extracted with 2 20-mL portions of diethyl ether, acidified to pH 2, and extracted with three 30-mL portions of ethyl acetate. 30 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 over silica gel (eluted with CH 2 Cl 2 / CH 3 0H 10:1), to afford 850 mg (88%) of N-Fmoc-4,4-difluoroproline as a - 103 brownish solid. 1 H NMR (500 MHz, CDCl 3 ) S 2.55 (m, IH) , 2.95 (m, 1H), 3.80 (m, 2H), 4.20 (m, 1H), 4.30 (m, 2H), 4.55 (m, 1H), 7.32 (m, 21H), 7.45 (m, 2H), 7.70 (in, 2H), 7.90 (m, 2H) . Exact mass calculated for C 2 0 H2-7F 2
NO
4 m/e 5 373.11, found m/e 374.4.
-104 F CIN rJY N F4H C N F 042H
H
2 N 0 c 27a H 0Ct 7 0 F N F H ~ N j H2N 0~J~ ~ 0 N(N0 0 2 0 cl H CI0 0 27b 27i O )A ojy N F N y N F 2 HOH 0 0 27c 27i N FiC 2H CON F 2 H
H
2 N : , jH ,C A00 ? wH a 27d 27k R F ? N Fg2H N F 2 H 2 N N C4 27e N 0271 H 0 E F aj F NJ Y 4 a ,1 0 CF7 N - 0 27g7 - 105 Compounds 28a- 28c were prepared according to the procedure described for compounds 5 and 7 (Scheme I and Scheme II) only substituting Fmoc-dimethylthioproline for Fmoc-proline in Step 2. 5 /- 0 X N 02H N NO O H H 28a, X=N 28c 28b, X=C GENERAL PROCEDURES FOR THE PREPARATION OF COMPOUNDS OF 10 EMBODIMENT A FORMULA I (SCHEMES VII-VIII) Compounds of Embodiment A Formula I where R 4 =H and one
R
5 =H: 15 Procedure for the preparation of analogs 29. Compounds 29a- 29s were prepared according to the procedure described for compounds 5 (Scheme I) only substituting Fmoc-alanine for Fmoc-proline in Step 2 and using either Fmoc-valine or Fmoc-alanine or Fmoc 20 tert-leucine in Step 3.
-106 0 0HIk 0 C2H 29a 29g 0 C0HCOH02H HO 0
CO
2 H H 0y- 0O N 0 29b 29h 0 C0 2 H 0
C
2 H N ~ H N0 c NH 0 29c 29i JJH 0 C2H 0CH 2 H HO 0 0 29d 29j 0 C 2 H CO 00 2 H N N )N %N HOv 0 0 29e 29k - 107 02H00 02 H0
H
2 N 29m 29q 0 0 O.C2H O2H O H 0 0Nj~~ 0 j 2
H
2 N 20 CI 29r 4H2 0H KH 29o 29r X2H H TOJN 0 NI y H 0 0 .CO O O2H 29p29s Procedure for the preparation of analogs 32. Compounds 32a-32e were prepared according to the 5 procedure described for compounds 5 (Scheme I) only substituting 2- (3- tert-butoxycarbonylamino-2-oxo pyrrolidin-1-yl) -4-methyl-pentanoic acid (30.) (Neosystem catalog number BB02101)for Fmoc-proline in Step 2 followed by Step 4 (Scheme VII). 10 15 - 108 Compounds of Embodiment A Formula I wherein R 2 and R 3 together with the atoms to which they are bound form a 5 membered ring. Scheme VII
CO
2 Bu-t r ' - Step 1 O 2 Bu-t FmoH H
-
Fmocf OH N I Boc 7N Step 2 O OH 30 R N 02H Ri-CO2H BO ON N 2Bu-t 32 H N, LR 5 31 - 109 N 02H - ON y02H HCOH 32a O 32d H N0 2 H N 02H H H% HO ZNfci 32b 32e 0 N N02H NI HI0 40 H 32c 5 Compound of Embodiment A Formula I wherein X=N-CH 3 - 110 Scheme VIII Step 3 H3 N CBu-1 H NN N C02 N PmocNB 1) 20% COCl 2 ITol 2 0 H N 2) CHN-NH2 3 NLR 3s' N' LR CO2H Step 4 N 0 CH 5 I ' ,N Y N C t H C0 34 Preparation of 3- ({- [N- (isoquinoline-1-carbonyl)
-N
uethyl-hydrazinocarbonyl] -pyrrolidine-2 -carbonyl} amino)-4-oxo-butyric acid (34). A suspension of 0.250 mmol of resin 3 (Scheme VIII) in S 10 mL of 20% piperidine in DMF was rotated at room temperature for S minutes and drained. The procedure was repeated over 20 minutes. The resin was washed successively with DMF (twice) , CH,OH (once) , 1:1 DMF/
CH
2 Cl, (twice) , CH30H (once) and CHCl2 (three times), 10 and briefly dried. To the resin was added 5 mL of dry CH2,Cla, 0.128 mL of DISA (3eq, 0.7S mmol) and 0.400 mL of a 20% solution of phosgene in toluene (3 eq, 0.75 mmol) . The suspension was rotated at room temperature for 1.5 hours. The mixture was drained, and the resin 15 was washed with CH 2 C1 2 several times. To a suspension of resin in 5 mL of CH2Cla was added 0.133 mL of methyl hydrazine (10 eq, 2.5 mmol) . The mixture was rotated at room temperature overnight and drained. The resin - 111 was washed successively with.DMF (twice), CH 3 0H (once), 1:1 DMF/ CH 2 Cl 2 (twice)., CH 3 0H (once) and CH 2 C1 2 (three times), and dried in vacuo. 5 To a 0.075 mmol portion of the resin in 3 mL of NMP was added successively 52 mg of 1-isoquinolinecarboxylic acid (4 eq, 0.3 mmol) , 0.19 mL of DIEA (8 eq, 0. 6 mmol) , 37mg of HOBt (4 eq, 0. 3 mmol) and 104 mg of HBTU (4 eq, 0.3 mmol). The mixture was rotated at room 10 temperature overnight and drained. The resin was washed successively with DMF (twice), CH 3 0H (once), 1:1 DMF/ CH 2 C1 2 (twice), CH 3 0H (once) and CH 2 C1 2 (three times), and dried in vacuo. 15 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 afford. an oil, which was purified by.HPLC to afford the title compound (34). 20 Compounds of Embodiment A Formula I wherein R 3 = R 3 = H: 0 N ,N C2H H 2 N O H 00 G1 25 3- ({l- [ (4-Amino-3-chloro-benzoylamnino) -acetyl] pyrrolidine-2-carbonyl) - amino) -4-oxo-butyric acid (GI). Prepared as described for compounds 7 only substituting Fmoc-glydine for Fmoc-alanine in Step 3 (Scheme II) to afford 4.3mg of the title compound. LC-MS (ES*) 30 m/e=425.2 (M+H) - 112 F 0F N -"' N ~ CH N 00 2H
H
2 00 N04 C1 H 0 G2 3 -({1- [(4 -Amino- 3 - chloro -benzoylamino) -acetyl] -4, 4 s difluoro-pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric acid (G2) . Prepared as described for compounds 7 and 27 only substituting Fmoc-glycine for Fmoc-alanine in Step 3 (Scheme II) to afford 10.0mg of the title compound. LC 10 MS (ES*) m/e=461.2 (M+H) - 113 GENERAL PROCEDURES FOR THE PREPARATION OF COMPOUNDS OF EMBODIMENT C FORMULA I AND EMBODIMENT D FORMULA I WHEREIN Y= C (SCHEMES IX-XXII) 5 Scheme IX Route A H2N H2Nl
EDR
3 CO Boc N RN R5 H><4 D 0 N TFA N N s R RCO2H R3 R3 R 5 0 NEDC 0 2 Route B R4 ~1) H2 %R CbH 2) EDC. RICO2H R1 -t TFA R 1 R34N R4 R ss RN N H H40 - 114 Sources for Selected Ring Systems Structure Reference Blythin, D.J., J. Org. Chem., 59, pp. 6098-6100 (1994). N
CO
2 R 0 Decicco, C.P. et al., Syn. Lett., N pp . 615-616, (1995) . o Co 2 R Bennion, C. et al., J. Med. Chem., N34, pp. 439-447 (1991); N
CO
2 R Jensen, K.A. et al., Acta Chemica Scand., 13, pp. 1097-1103 (1961). S commercially available o C02R N R Mish, M. R., J. Am. Chem. Soc., 119, pp. 8379-8380 (1997) .
CO
2 R N N. et al., J. Am. Chem. Soc., N .120 pp. 80-86 (1998). CO2
R
- 115 Structure Reference r N1 IMerour, J.Y. et al., Tetrahedron N Lett., 32, pp. 2469-2470 (1991)
CO
2 H Rossen, K., Tetrahedron Lett., 36, pp. 6419-6422 (1995). Scheme X polyvinyl Bu-t N- pyridine N N H toluene 44,W R4-MeO N4 0) Et O B 37 35 36 Bu-t 0 N= Bu-t R O O MeOH R N 39, R=CH3 O H0Et 44, R=4-MeOPh- 38, R=CH3 43. R=4-MeOPh 0 0 Pd(Ph 3
P)
4 H2N DMBA Alloc
CH
2 C/ DMF C6H 5 40 O Bu-t Bu-t R
CHO
2 H 41. R=CH 3 42, R=CH 3 45. R=4-MeOPh- 46, R=4-MeOPh- - 116 5-tert-Butyl-3- [2- (9H-fluoren-9 ylmethoxycarbonylamino)-3-methyl butyryl]-2,3-dihydro [1,3,4]thiadiazole-2-carboxylic acid ethyl ester (37). 5 A stirred suspension of polyvinylpyridine (2.63g, 25mmol) in a solution of 5-tert-butyl-2,3-dihydro [1,3,4]thiadiazole-2-carboxylic acid ethyl ester (36), (J. Med. Chem., 34, p. 439 (1991)), (2.16g, 10mmol) in dry toluene was treated with the dropwise addition of 10 (1-chlorocarbonyl-2-methyl-propyl)-carbamic acid 9H fluoren-9-ylmethyl ester (4.76g, 12.1mmol) in 20mL of anhydrous toluene. After stirring for 16 hours, the suspension was filtered and the filtrate was washed with saturated sodium bicarbonate solution. The 15 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 9/1 hexane/ ethyl acetate gave 2.66 g (49% yield) of the title compound (37) as a clear, viscous 20 oil. IH NMR (50OMHz, CD 3 0D) 5 0.89(d, 1.SH), 0.93 (d, 1.5H), 1.00 (d, 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 (m, 0.5H), 5.02-5.10 (m, 0.5H), 5.52 (d, 0.5H), 5.61 (d, 25 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), 7.73-7.78 (m, 2H). 3- (2-Acetylamino-3-methylbutyryl) -5- tert-butyl-2,3 30 dihydro-[1,2,4] thiadiazole-2-carboxylic acid ethyl ester (38). To a solution of (37) (Scheme IX) (0.508g, 0.94 mmol)in
CH
3 CN (10 mL) was added diethylamine (1 mL). The solution was stirred at room temperature for 2 hours, - 117 the solvent removed in vacuo and the resultant oil azeotroped with CH 2 C1 2 (4x). The crude oil was dissolved in CH2Cl 2 (5 mL) and triethylamine (0.26mL, 1.86mmol) and acetyl chloride (80pil, 1.1mmol) were 5 added. The solution was stirred at room temperature under an N 2 atmosphere for 2 hours. The solvent was evaporated, and the crude material dissolved in EtOAc and washed with 0.5N NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine and was dried over anhydrous Na 2
SO
4 , filtered 10 and evaporated to give a yellow oil. Purification by flash column chromatography on silica gel using hexanes/EtOAc (95/5 to 90/10%) yielded the product as a yellow oil (0.301g, 89% yield). 1 H-NMR (500MHz, CDCl 3 ) 5 0.88 (dd, 3H), 0.99 (dd, 3H), 1.16-1.45 (m, 12H), 15 2.02 (s, 3H), 2.09-2.19 (m, 0.5 H), 2.30-2.40 (m, 0.51H), 4.12-4.29 (m, 2H), 5.20-5.27 (m, 0.5H), 5.30-5.36 (m, 0.5H), 6.60(s, O.5H), 6.90 (s, 0.5H), 6.20-6.31 (m, 1H). Analytical HPLC (C18 column), (mixture of diastereomers) 7.77, 7.98min. LC-MS (ES+) m/e=358.3 20 (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 (0.301g, 0.84mmol) in MeOH (10mL) 25 was added 1N NaOH solution (1.7mL, 1.7mmol). The reaction was stirred at room temperature for 2 hours and solvent was evaporated. The residue was dissolved in EtOAc and washed with 0.5N NaHSO 4 (2x) and brine and was dried over anhydrous Na 2
SO
4 , filtered and 30 evaporated to give the title compound as a yellow solid (0.277g, quantitative). Preparation of 2-(Benzyloxy-5-oxo-tetrahydro-furan-3 yl)-carbamic acid allyl ester (40).
- 118 Compound 40 was prepared from 3-allyloxycarbonylamino 4-hydroxy-butyric acid tert-butyl ester by a modification of the procedure described in Bioorg. Med. Chem. Lett. Vol. 2, No. 6, pp. 613-618, (1992). 5 To a solution of DMSO (27.52 g, 352 mmol) in CH 2 Cl 2 (240 ML) at -78 0 C was added oxalyl chloride (24.4 g, 192 mmol) .. After 15 min, a solution of 3 allyloxycarbonylamino- 4 -hydroxy-butyric acid tert-butyl 10 ester (41.44 g, 160 mmol) in CH 2 C1 2 (100 mL) was slowly added and the mixture was stirred at -78 0 C for an additional 1.5 hours. DIEA (62.0 g, 480 mmol) was added and the mixture allowed to warm to room temperature for 15 min. The resulting solution was 15 diluted with
CH
2 Cl 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 200mL volume. To this solution was added, benzyl alcohol (48 g, 444 20 mmol) , followed by 3A molecular sieves (30 g) and p toluenesufonic acid (0.8 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 then evaporated in vacuo. Ethyl acetate (500 mL) was added 25 and the mixture was filtered through Celite. The filtrate was washed with saturated NaHCO 3 (500 niL x 2), water (400 mL x 2), and brine (300 mL x 2). The organic solution was dried over anhydrous Na2SO 4 , filtered and evaporated in vacuo to give 90 g of pale 30 yellow oil, which was stirred with hexane (400 mL x 2) to give 31 g of crude product from the lower layer residue. Chromatography using ethyl acetate/hexane (4/96 to 22/78) afforded 6.97 g of anti-2-(benzyloxy-S oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester - 119 (higher Rf), 4.53 g of syn diastereomer and 12.97 g of the mixture of the diastereomers (overall yield 53%). 1H-NMR (500 MHz, CDCl 3 ) for anti diastereomer: 6 2.41 2.45 (m, H), 3.02-3.07 (m, H), 4.28 (br, H), 4.50-4.80 5 (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, SH); for syn diastereomer: 52.49-2.53 (m, H), 2.83-2.89 (m, H), 4.57-4.65 (m, 4H), 4.87-4.90 (m, H), 5.12-5.30 (m, 3H), 5.52-5.53 (d, H), 5.88-6.00 (m, H), 7.31-7.39 (m, SH); 10 retention time on analytical HPLC: 10.49 min for anti diastereomer and 10.37 min for syn diastereomer; LC-MS: m/z = 292 (M+H*) 15 3- ( 2 -Acetylamino-3 -ethylbutyryl) -5-tert-butyl-2,3 dihydro- 11,2,4] thiadiazole-2-carboxylic acid (2 benzyloxy-5-oxo-tetrahydrofuran-3-yl)-amide (41). To a solution of ( 2 -benzyloxy-5-oxo-tetrahydrofuran-3 yl)-carbamic acid allyl ester (40) (0.385g, 1.32mmol) 20 in DMF (2ml) and CH 2 Cl 2 (2ml) was added DMBA (0.456g, 2.92mmol) and Pd(PPh 3
)
4 (0.136g, 0.12mmol) and the solution was stirred at room temperature for 15min. A solution of (39) in CH 2 Cl 2 (4.5ml) and DMF (0.5ml) was added, followed by HOBT (0.168g, 1.24mmol) and EDC 25 (0.256g, 1.33mmol). The reaction was stirred at room temperature for 18hours under N 2 . The. solvent was evaporated. The crude material was dissolved in EtOAc and washed with 0.5N NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine and was dried over anhydrous Na 2
SO
4 , filtered 30 and evaporated to give a yellow solid. Purification by flash column chromatography gave the title compound (41) as a mixture of diastereomers (374mg, 88% yield). IH-NMR (500MHz, CDCl 3 ) 8 0.75-1.05 (in, 6H), 1.19-1.34 ,m, 9H), 1.93-2.08 (m, 3M), 2.19-2.50 (m, 2H), 2.80- - 120 3.03 (m, 1H), 4.56-4.93 (m, 3H), 5.02-5.20 (m, 1H), 5.46-5.56 (m, 1H), 5.95-6.16 (to, 2H), 6.86-6.95 (m, 1H), 7.20-7.43 (m, 5H). Analytical HPLC (C18 column), (mixture of diastereomers) 8.58min. LC-MS 5 (ES*) m/e=519.2 (M+H). Preparation of 3-{[3-(2-acetylamino-3-methyl-butyryl) 5-tert-butyl-2,3-dihydro- [1,3,4] thiadiazole-2 carbonyl] -amino)-4-oxo-butyric acid (42). 10 A 45 mg (0.087mmol) sample of 41 was hydrolyzed according to method A (see Scheme XXIII) to give 17 mg (45% yield) of the title compound. Analytical HPLC (C18 column): 5.15min. LC-MS (ES*) m/e=429.3 (M+H). 15 5-tert-Butyl-3- [2- (4-methoxy-benzoylamino) -3-methyl butyryl) -2,3-dihydro- [1,3,41 thiadiazole-2-carboxylic acid ethyl ester (43).. Was prepared by the method reported above for compound 38 using anisoyl chloride to give 216mg (50%) of the 20 title compound as an amorphous solid. 1H NMR (500 MHz, CDCl 3 ) 8 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), 2.21-2.28 (m, 0.5H), 2.41-2.48 (m, 6.5H), 3.83 (s, 3H), 4.15-4.28 (m, 2H), 5.41-5.46 (m, 0.5H), 5.48-5.53 (m, 0.5H), 6.08 25 (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-methyl butyryl]-2,3-dihydro-[1,3,4]thiadiazole-2-carboxylic 30 acid (44). Prepared by the procedure described for 39 to give 180mg (quantitative) of the title compound as a white solid. 1 H NMR (500 MHz, CDCl 3 ) 6 0.92 (d, 1.51H), 0.96 (d, 1.5H), 1.03 (d, 1.5H), 1.07 (d, 1.5H), 2.22-2.30 - 121 (M, 0.5H), 2.37-2.45 (m, 0.51H), 3.83 (s, 1.51H) 3.84 (s, 1.5H), 5.41-5.48 (M, 1H), 6.14 (S, 0.5H), 6.15 (s, 0.SH), 6.87-6.95 (M, 2H), 7.75-7.83 (m, 3H). 5 5- tert-Butyl-3-[2- (4-methoxy-benzoylamino) -3-methyl butyryl] -2,3-dihydro- (1,3,41thiadiazole-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-amide (45a and 45b). 10 Was prepared by the procedure reported for compound 41 to give the crude title compound as 4 diastereomers. The crude material was purified by flash chromatography, eluting with a gradient of CH 2 C1 2 to
CH
2 Cl 2 /ethyl acetate (6/4) to give 31mg of the higher Rf 15 component as a single diastereomer (45a). Analytical HPLC (Microsorb C18 column) 19.87 min. 1H NMR (500 MHz, CDCl 3 ) (single diastereomer) 8 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, 20 1H), 4.53 (d, 1H), 4.85 (d, 1H), 5.02-5.08 (m, 1H), 6.37 (s, 1H), 6.41 (d, 1H), 6.93 (d, 2H), 7.26-7.40 (m, 5H), 7.75 (d, 2H), 7.92-7.96 (m, 1H). The lower Rf fraction contained 185mg of a solid as a 25 3:1:2 mixture of diastereomers (45b). Analytical HPLC: Microsorb C18 column. 19.00, 19.26, 20.02 mins, 'H NMR (500 MHz, CDCl 3 ) (3:1:2 mixture of 3 diastereomers) 5 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, 0.75H), 1.16 30 (s, 0.75H), 1.17 (s, 2.25H), 1.23 (s, 0.375H), 1.24 (s, 1.125H), 1.28 (s, 1.125 H), 1.29 (s, 3.375H), 2.12-2.18 (m, 0.33H), 2.32-2.42 (m, 0.67H), 2.43-2.51 (m, 0.5H), 2.61-2.67 (m, 0.5H), 2.84-2.92 (m, 0.5H), 2.96-3.07 (m, 0.5H), 3.85 (s, 3H), 4.58-4.71 (m, 2H), 4.81 (d, - 122 0.16H), 4.86 (d, 0.32H), 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), 6.34 (s, 0.32H), 6.50 (d, 5 0.32H), 6.62 (d, 0.16H), 6.67 (d, 0.52H), 6.86 (d, 0.33H), 6.91 (d, 0.67H), 6.94 (d, 1.01H), 7.24-7.43 (m, 5H), 7.61 (d, 1H), 7.70 (d; 0.33H), 7.71 (d, 0.67H), 7.76 (d, 1H). 10 Preparation of 3-({S-tert-butyl-3-[2-(4-methoxy benzoylamino) -3 -methyl-butyryl] -2, 3 -dihydro [1, 3, 41 thiadiazole-2 -carbonyl) -amino) -4 -oxo-butyric acid (46a). A 30mg sample of 45a was hydrolyzed according to method 15 B (see Scheme XXIII) to give 8mg (30% yield) of the desired product. Analytical HPLC (Microsorb C-18 column, acetonitrile/ water, with TFA buffer) 12.85min, 1H NMR (500 MHz, CD 3 0D) 6 0.98-1.1 (m, 6H), 1.28 (s, 9H), 2.20-2.31 (m, 1H), 2.40-2.48 (m, 1H), 2.6-2.72 (m, 20 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, 21H), 7.85 (d, 2H). Preparation of 3- ({5-tert-butyl-3- [2- (4-methoxy 25 benzoylamino) -3 -methyl-butyryll -2, 3-dihydro [1, 3,41 thiadiazole-2-carbonyl) -amino) -4-oxo-butyric acid (46b). A 30mg sample of 45b (mixture of 3 diastereomers) was hydrolyzed according to method B (see Scheme XXIII) to 30 give 22mg (84% yield) of the desired product as a 3:2 mixture of diastereomers. Analytical HPLC (Microsorb cyano column) 7.08, 7.78min. 1H NMR (500MHz, CD 3 0D) 6 0.98-1.08 (m, 4H), 1.09-1.12 (m, 2H), 1.29 & 1.31 (2 singlets, 9H), 2.23-2.30 (m, 0.-5H), 2.36-2.55 (m, - 123 1.5H), 2.62-2.72 (m, 1H), 3.85 (s, 3H), 4.18-4.27 (m, 1R), 4.58-4.65 (m, 1H), 5.27-5.33 (m, 1H), 6.23-6.27 (m, 1H), 7.00 (d, 2H), 7.70-7.88 (m, 2H). 5 Scheme XI HN> + H---0N Q~~~o 0~H03 NN
CO
2 Bu-t + OO 2 Bu-t 47 48 49 HH
C
2 Bu-t 51 50 0 N02H 52 1- (2 -Benzyloxycarbonylamino-2 -nethyl-propionyl) pyrrolidine-2-carboxylic acid tert-butyl ester (49). 10 To a solution of proline-tert-butyl ester (47) (2.00g, 12mmol, in CH 2 C1 2 (1.5ml) was added N-carbobenzyloxy-2 methylalanine (3.05g, 13mmol), HOBT (2.36g, 17mmol) and EDC (3.43g, 18mmol) and the solution was stirred at room temperature under N 2 for 48hours. The solvent was 15 evaporated, the crude material dissolved in EtOAc and - 124 washed with 0.5N NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine and was dried over anhydrous Na 2
SO
4 , filtered and evaporated to give a white solid (4.68g, 100%). 1 H-NMR (500MHz, CDCl 3 ) 6 1.20-2.15 (m, 4H), 1.43 (s, 9H), 1.59 5 (d, 6H), 3.21-3.79 (m, 2H), 4.35 (br s, 1H), 4.82-5.19 (m, 3H), 5.74 (br s, 1H), 7.17-7.49 (m, 5H). Analytical HPLC (CI8 column) 10.66min. LC-MS (ES*) m/e= 391.3 (M+H). 10 1- [2- (4-Methoxy-benzoylamino) -2-methyl-propionyl] pyrrolidine-2-carboxylic acid tert-butyl ester (50). To solution of 49 (1.00g, 2.56mmol) in MeOH (20ml) was added 10% Pd/C (200mg) and the mixture was stirred under H 2 for 2 hours. The mixture was filtered through 15 a 0.45pm PTFE filter and the solvent removed in vacuo to yield a colorless oil. This oil was dissolved in
CH
2 C1 2 (25mL) and DIEA (660pl, 3.79mmol) and p-anisoyl chloride (480mg, 2.8mmol) were added. The solution was stirred at room temperature under N 2 for 18hours. The 20 solvent was removed in vacuo and the oil dissolved in EtOAc. The organic phase was washed with 0.5N NaHSO 4 (2x), water, saturated NaHCO 3 (2x) and brine. The organic phase was dried over Na 2
SO
4 , filtered and evaporated to give a white solid which was purified by 25 flash column chromatography, eluting with CH 2 Cl 2 /MeOH (99/1 to 98/2%) to give the title compound as a white solid (655mg, 65% yield) .
1 H-NMR (500MHz, CDCl 3 ) 5 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, 30 1H), 6.90 (d, 2H), 7.60 (br s, 1H), 7.77 (d, 2H). Analytical HPLC (C18 column)8.98min.
- 125 1- [2- (4-Methoxy-benzoylamino) -2-methyl-propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide (51). To a solution of 50 (325mg, 0.83mmol) in dioxane (5mL) 5 was added triethylamine (463pl, 3.32mmol) and TMS triflate (642pl, 3.32mmol) 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 0, 10 dried over Na 2 SO4, filtered and evaporated to give a white.solid (230mg, 83% yield) which was used directly in the next step. To a solution of ( 2 -benzyloxy-5-oxo-tetrahydrofuran-3 15 yl)-carbamic acid allyl ester (40) (1.027g, 3.5mmol) in
CH
2 Cl 2 (20ml) was added DMBA (543mg, 3.48mmol) and Pd(PPh 3
)
4 (280mg, 0.24mmol) and the solution was stirred at room temperature under N2 for 20 minutes. A solution of 1-[2-(4-methoxy-benzoylamino)-2-methyl-propionyl) 20 pyrrolidine-2-carboxylic acid (818mg, 2.45mmol) in
CH
2 Cl 2 (Sml) was added, followed by HOBT (0.534g, 3.95mmol) and EDC (738mg, 3.84mmol). The reaction was stirred at room temperature for 18 hours under N 2 . The solvent was evaporated, the crude material dissolved in 25 EtOAc and washed with 0.5N NaBSO 4 (2x), saturated NaHCO 3 (2x) and brine and was dried over anhydrous Na 2 SO, 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 30 product as pale yellow solid (760mg, 61% yield). 1
H
NMR (500MHz, CD 3 0D) 8 1.53 (d, 6H), 1.65-1.93 (n, 3H), 1.96-2.14 (m, 1H), 2.60 (dd, 0.lH), 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, 3-H), 4.38-4.76 (m, - 126 3H), 4.84 (d, 1H), 5.64-5.70 (m, 1H), 6.96-7.03 (m, 2H), 7.23-7.43 (M, 5H), 7.78-7.97 (M, 2H). Analytical HPLC (C18 column) 13.32, 14.37min. LC-MS (ES*) m/e=524.3 (M+H). 5 Preparation of 3- ({1- [2- (4-methoxy-benzoylamino) -2 methyl-propionyl] -pyrrolidine-2-carbonyl) -amino) -4-oxo butyric acid (52). A 61mg (0.14mmole) sample of 51 was hydrolyzed 10 according to method C (see Scheme XXIII) to afford 30mg (60% yield) of the title compound: Analytical HPLC (C18 column) 6.79min. LC-MS (ES*) m/e=434.3 (M+H) - 127 Scheme XII HCI H2N N? N 0 C02-t-Bu
.
O C02-t-Bu 53 x 54, X=H. Y=MeO 58, X=C, Y= NH2 0 O' N N 02 X x 0 Y. C02H x 56, X=H, Y=MeO 5. X=H, Y=MeO 60, X=CI, Y=NH 2 59. X=CI, Y= NH2 2H 67, X=H, Y=MeO 0 61, X=CI, Y= NH2 1- [2- (4-Methoxy-benzoylamino) -3-methylbutyryl] pyrrolidine-2-carboxylic acid tert-butyl ester (54). 5 To a suspension of H-val-pro-OtBu.Hcl (53) ( 2 .011g, 7.44mmol) in CH 2 C1 2 (20ml) was added DIEA (3.2ml, 18.4mmol) followed by a solution of 4-methoxy-benzoyl chloride (1.26g, 7.4mmol) in CH 2 Cl 2 (5ml) . The solution was stirred at room temperature under nitrogen for 10 hour then 'concentrated. The resulting oil was dissolved in EtOAc and washed with 0.5N KHS0 4 (2x), - 128 saturated NaHCO 3 (2x) and brine, then concentrated in vacuo to give the title compound as a white solid (2.814g, 94% yield). 'H-NMR (500MHz, CDCl 3 ) 8 1.05 (dd, 6H), 1.46 (s, 9H), 1.88-2.29 (m, SH), 3.65-3.74 (m, 5 1H), 3.81-3.92 (m, 1H), 3.85 (s, 3H), 4.32-4.42 (m, 1H), 4.81-4.91 (m, 1H), 6.79-6.86 (m, 1H), 6.91 (d, 2H), 7.78 (d, 2H). Analytical HPLC (cyano column) 10.18min. 10 1- [2- (4-Methoxy-benzoylamino) -3-methylbutyryl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydrofuran-3-yl) amide (56). A 1.079g (2.67mmol) sample of 54 was dissolved In 15% TFA in CH 2 Cl 2 (40mL) and stirred at room temperature 15 for 4hours. The solvent was concentrated in vacuo to give 55 as a white solid (0.93g, 100%) which was used in the next step. To a solution of 40 (1.796g, 6.17mmol) in CH 2 Cl 2 (20ml) 20 was added DMBA (1.119g, 7.17mmol) and Pd(PPh 3
)
4 (0.683g, 0.59mmol) and the solution stirred at room temperature for 20 minutes. A solution of 55 (0.928g, 2.67mmol) in
CH
2 Cl 2 (17ml) and DMF (2ml) was added, followed by HOBT (0.811g, 6.01mmol) and EDC (1.16g, 6.04mmol). The 25 reaction was stirred at room temperature for 18hours under N 2 . The solvent was evaporated, the crude material dissolved in EtOAc and washed with o.SN NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine and was dried over anhydrous Na 2
SO
4 , filtered and evaporated to give 30 a yellow solid. Purification by flash chromatography eluting with ethyl acetate/ CH 2 C1 2 (10/90 to 40/60%) gave the title compound as pale yellow solid (910mg, 63% yield). 1H-NMR (500MHz, CDCl 3 ) 6 0.96 (dd, 6H), 1.84-2.19 (m, 4H), 2.25-2.38 (m, 1H), 2.45 (dd, 1H), - 129 2.80-2.98 (m, 1H), 3.60-3.72 (m, 1H), 3.82-3.95 (m, 1H), 3.86 (s, 3H), 4.26-4.95 (m, 6H), 5.41(s, 0.2H), 5.53 (d, 0.8H), 6.67-6.77 (m, 1H), 6.88-6.99 (d, 2H), 7.22-7.57 (m, SH), 7.71-7.82 (d, 2H). 5 Analytical HPLC (cyano column) (mixture of 2 diastereomers) 9.21min. LC-MS (ES4) m/e= 538.3 (M+H). 3- ({1- [2- (4-Methoxy-benzoylamino) -3-methyl-butyryl] pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric acid (57). 10 A 125mg (0.23mmol) sample of 56 was hydrolyzed according to method A (see Scheme XXIII) to afford 60mg (58% yield) of the title compound: Analytical HPLC 5.71min. LC-MS (ES*) m/e=448.2 (M+H). 15 Preparation of 4-Amino-3-chloro-benzoic acid: A suspension of 4-amino-3-chlorobenzonitrile (4.82g, 31.5 Bmmol) was heated to reflux in 6N HCI (140ml). The precipitate dissolved upon heating to give a colorless solution. Upon further heating the solution became 20 cloudy. After hours the reaction was cooled to room temperature. The resulting precipitate was filtered, then dissolved in THF and the solvent evaporated. The residue was repeatedly concentrated from toluene to give a white solid (3.18g, 59% yield). 1 H-NMR (500MHz, 25 CD 3 0D:CDCl 3 1:4) 6 6.80 (d, 1H), 7.75 (dd, 1H), 7.94 (d, 1H). Analytical HPLC (cyano column) 8.73min. 1- [2- (4-Amino-3-chloro-benzQylamino) -3-methylbutyryl] pyrrolidine-2-carboxylic acid tert-butyl ester (58). .30 To a suspension of 53 (1.707g, 6.31mmol) in CH 2 Cl 2 (25ml) at O'C was added DIEA (3.2ml, 18.4mmol) followed by a solution of 4-amino-3-chlorobenzoic acid (1.298g, 7.56mmol), HOBT (1.005g, 7.44mmol) and EDC (1.456g, 7.58mmol). The resulting mixture was stirred at O'C 120 for 15 minutes then allowed to warm to room temperature and stir for 18 hours. The solvent was evaporated and the resulting oil dissolved in EtOAc, washed with 0.sN NaHSO 4 (2x) , saturated NaIKC03 (2x) and brine to give a 5 white solid (2.68g). Flash chromatography using MeOH/CH2Cl 2 (1/51 to 2/961) gave 2.04c (16* yield) of 58asa whte slid. 1 )FJM (500MH2, CDCI 3 )&1.5 dd 6w),1.47(S.9H)- 1.86-2.29 (m,. SH), 3.62-3.78 (m, 10 1i). 6.73 (a, ilN1), 6.78 (d. IN), -7.S2 (dd, 11%), 7.75 (d, 1H)l. Analytical )IPLC (cyano colun) 16.18mii. LC.
MS (ES') infe=424.3 (M+H). I- t2 - ( 4 -Amito-3 -chlorobeuxoylaxno)-3 methylbutyr1] is pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo. tetrahydro-fu.ran-3-yl) -amide (GO-).. A 0.6329 (1.49iMol1) sample of 58.was dissolved in So% T'A in CH 2 C1 2 (2OinL) and the solution stirred at room temperature for2 hours. Residual TFA was removed by 20 repeated concentration from CH 2 ChZ (3x) to give the product as a white solid. A 385mg (1.O4mnol) sample was allowed to react with 40 by the method used for compound 56. The title compound 25 (60) was isolated as a yellow solid (2G6ing, 4S% yield) 1 H-ZIMR (500MHZ,
CD
3 0D) 6 0.89-1.12 (m, GH), 1.72-2.26 (m, SM), 2.49 (fid, 0.25H), 2.60 (dd. 0.7H), 2.80 (dd, 4.10 (m, IN), 4.20-4.74 Wm 4H), 4.76-4.95 (m, IH), So .51. Is, D.SH). 5.61-5.70 (in, 1.H .*79 (dd. IN), 7.23-7.43 (M. SH), -7.48-7.61 0m, 1.4R1), 7.68-7.81 cm, aMF). 7.99-8.12 (mn, 0.GH).
- 131 Analytical HPLC (cyano column) (mixture- of 2 diastereomers) 14.90, 15.20min. LC-MS (ES*) m/e=557.2 (M+H) . 5 3 - ({l- [2- (4 -Amino-3 - chloro -benzoylamino) -3 -methyl butyryl] -pyrrolidine-2 -carbonyl)- amino) -4-oxo-butyric acid (61). A 45mg (0.08mmol) sample of 60 was hydrolyzed according to method A (see Scheme XXIII) to afford 30mg (80% 10 yield) of the title compound: IH NMR (500MHz, CD 3 0D) 6 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 8.16min. LC-MS (ES*) m/e=467.3 (M+H). 15 Scheme XIII Fmo N N CO-t-Bu HO C0 2 t-Bu 62 63 0 H O 0H HO 65 64 1- [2- (4 -Hydroxy-3 , 5-dimethyl-benzoylamino) -3 -methyl butyryll -pyrrolidine-2-carboxylic acid tert-butyl ester 20 (63) To a solution of 62 (prepared from 53 and Fmoc-Cl) (600mg, 1.22mmol) in anhydrous DMF (10ml) was added - 132 diethylamine (3ml). The solution was stirred at room temperature under N 2 for 3hours and the solvent was evaporated. The resulting oil was dissolved in CH 2 C1 2 (8ml) and 3,5-dimethyl-4-hydroxybenzoic acid (0.302g, 5 1. 82mmol), HOBT (338mg, 2.5mmol) and EDC (0.456g, 2.43mmol) were added and the solution stirred at room temperature under N 2 for 18hours. The solvent was concentrated in vacuo and the resulting oil dissolved in EtOAc, washed with 0.5N NaHSO 4 (2x), saturated 10 NaHCO 3 (2x) and brine to give the crude product as a white solid (0.80g). Flash chromatography eluting with MeOH/CH 2 Cl 2 (1/99 to 2/98%) gave 380mg (75% yield) of a white solid. 1 H-NMR (500MHz, CDCl 3 ) 6 1.06 (dd, 6H), 1.47 (s, 9H), 1.90-2.32 (m, 5H), 2.24 (s, 6H), 3.65 15 3.75 (m, 1H), 3.84-3.92 (m, 1H), 4.36-4.42 (m, 1H), 4.82-4.88 (m, 1H), 5.53-5.61 (m, 1H), 6.77-6.85 (m, 1H), 7.42 (s, 2H). Analytical HPLC (cyano column) 17.53min. LC-MS (ES*) m/e=419.3 (M+H). 20 1- [2- (4-Hydroxy-3,5-dimethyl-benzoylamino) -3-methyl butyryl]-pyrrolidine-2-carboxylic acid (2-benzyloxy-5 oxo-tetrahydro-furan-3-yl)-amide (64). Prepared from 63 and 40 by the method used to prepare 56 to give title compound (64) as a pale yellow solid 25 (352mg, 72% yield) .- 1 H-NMR (500MHz, CD 3 0D) 5 0.83-1.28 (m, 6H), 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), 4.48 4.73 (m, 3H), 4.75-4.92 (m, 1H), 5.45-5.48 (m, 0.1H), 30 5.61-5.64 (m, 0.1H), 5.64-5.70 (m, 0.8H), 7.21-7.62 (m, 6H), 7.88-8.04 (m, 1H). Analytical HPLC (cyano column)-(mixture of 2 diastereomers) 17.73min. LC-MS (ES*) m/e= 552.3(M+H).
- 133 3- ({1- [2- (4-Hydroxy-3, 5-dimethyl-benzoylamino) -3 methyl-butyryll -pyrrolidine-2-carbonyl)-amino) -4-oxo butyric acid (65). A 160mg (0.29mmol) sample of 64 was hydrolyzed 5 according to method A (see Scheme XXIII) to afford 13.1mg (10% yield) of the title compound: Analytical HPLC (cyano column) 10.28min. LC-MS (ES*) m/e=462.2 (M+H). Scheme XIV Fmoc t N? N 0 C02-t-Bu o co2-t-Bu 66 67 2H N 1 H 0 H H H0 0 69 68 1- [2- ( 2
-
9 H-Fluoren-9-yl-acetylamino) -3,3-dimethyl butyryl] -pyrrolidine-2-carboxylic acid tert-butyl ester (66). 15 'To a solution of H-pro-OtBu (53) (1. 0339, 6. Ommol, II, Scheme 5) in CH 2 Cl 2 (20ml) and DMF (5ml) was added Fmoc-tLeu-OH (2.337g, 6.60mmol, I, Scheme 5), HOBT (1.63a, 12.1mmol) and EDC (2.30g, 12.Ommol) and the solution stirred at room temperature under N 2 for 0 18hours. The solvent was removed in vacuo, and the res.due dissolved in EtOAc then washed with 0.SN NaHSO 4 saturated NaHCO 3 (2x) and brine. The organic - 134 layer was dried over anhydrous Na 2
SO
4 and evaporated to give a pale yellow solid (3.65g). Flash chromatography using EtOAc/hexanes (10/90 to 20/80%) give the title compound (66) (2.25g, 74% yield). 1 H-NMR (500MHz, 5 CDCl3) 5 1.09 (s, 9H), 1.47 (s, 9H), 1.79-2.28 -(m, 3H), 3.62-3.72 (m, 1H), 3.76-3.83 (m, 1H), 4.18-4.43 (m, 4H), 5.48-5.67 (m, 1H), 7.28-7.44 (n, 4H), 7.55-7.64 (n, 2H), 7.72-7.82 (m, 2H). Analytical HPLC (cyano column) 11.95min. LC-MS (ES*) m/e=507.3 (M+H). 10 1- [2- (4-Methoxy-benzoylamino) -3,3-dimethyl-butyryl] pyrrolidine-2-carboxylic acid tert-butyl ester (67). To a solution of 66 (0.503g, 0.99mmol) in DMF (8ml) was added diethylamine (2.5ml) and the solution stirred at 15 room temperature for ihour and the solvent evaporated. The resulting residue was repeatedly concentrated from
CH
2 C1 2 (3x) . The resulting oil was dissolved in CH 2 Cl 2 (9ml) and DIEA (260gl, 1.49mmol) and 4-methoxy-benzoyl chloride (190mg, 1.05mmol) was added. The solution was 20 stirred under N 2 for 18hours and the solvent concentrated in vacuo. The residue was dissolved in EtOAc and washed with O.5N NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine then dried over anhydrous Na 2
SO
4 and evaporated to give a white solid (0.529g). Flash 25 chromatography on silica gel using MeOH/CH 2 Cl 2 (1/99 to 2/98%) gave the title compound (2.25g, 74% yield). 'H NMR (500MHz, CD 3 0D) 8 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), 2.91-3.11 (m, 0.3H), 3.61-4.03 (M, 3H), 3.84 (s, 3H), 30 4.29-4.49 (m, 1H), 4.49-5.00 (m, 5H), 5.46 (s, 0.15H), 5.58-5.73 (m, 0.85H), 6.94-7.04 (in, 2H), 7.27-7.41 (m, 4H), 7.61-7.73 (m, 1H), 7.74-7.84 (W, 2H). Analytical HPLC (cyano column) 13.10min.
- 135 1- [2- (4-Methoxy-benzoylamino) -3, 3-dimethyl-butyryl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl) -amide (68).. To a solution of 67 (0.90g, 1.74mmol) in CH 2 C1 2 (25ml) 5 was added 2,6-lutidine (2.1ml, 18.Ommol) and TMS triflate (2.3ml, 11.9mmol) and the reaction stirred at room temperature under N 2 for 1.Shours. The resulting mixture was diluted with CH 2 C1 2 , washed with 10% NaHCO 3 (2x) and brine then dried over Na 2
SO
4 , filtered and 10 evaporated. The residue was dissolved in CH 2 Cl 2 then treated with DIEA (0.6ml, 3.5mmol) and 4-methoxy benzoyl chloride (0.355g, 2.09mmol) and allowed to stir under N 2 at room temperature for 18hours. The crude product was purified by flash chromatography, eluting 15 with CH 2 Cl 2 /MeOH (99/1) to yield the title compound (274mg, 28% yield) .
1 H-NMR (500MHz, CD 3 0D) 5 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), 2.91-3.11 (m, 0.3H), 3.61-4.03 (m, 3H), 3.84 (s, 3H), 4.29-4.49 (m, 1H), 4.49-5.00 (m, 20 5H), 5.46 (s, 0.15H), 5.58-5.73 (m, 0.85H), 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 (cyano column) (mixture of 2 diastereomers) 17.03, 17.39min. LC-MS (ES*) m/e=552.3 (M+H). 25 3 -({l- (2 - (4 -Methoxy-benzoylamino) -3, 3 -dimethyl butyryll -pyrrolidine-2 -carbonyl) - amino) -4 -oxo-butyric acid (69). A 117mg (0.21mmol) sample of 68 was hydrolyzed 30 according to method C (see Scheme XXIII) to afford 40mg (41% yield) of the title compound: Analytical HPLC 7.16min. LC-MS (ES*) m/e=462.3 (M+H).
- 136 Scheme XV Bo, N N 1N 0 CO 2
CH
2 Ph O 70 70 71 0 N0 ZL H2N C H H2N C C1 0 C1 73 72 1- (2- tert-Butoxycarbonylamino-3 , 3 -dimethyl-butyryl) pyrrolidine-2-carboxylic acid benzyl ester (70) . 5 To a suspension of H-pro-OBzl.HC1 (2.00g, 8.66mmol) in
CH
2 C1 2 (20ml) was added DIEA (2.25ml, 12.92mmol) to give a colorless solution. Boc-tLeu-OH (1.95g, 9.52mmol), HOBT (1.76g, 13.03mmol) and EDC (2.49g, 12.95mmol) were added and the solution stirred under N 2 10 at room temperature for 18hours. Removed solvent in vacuo, dissolved in EtOAc and washed with H 2 0, 0.5N NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine. Dried over anhydrous Na 2
SO
4 and evaporated to give the title compound. (3.57g, 99% yield) .
1 H-NMR (500MHz, CDCl 3 ) 5 15 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), 5.04-5.38 (m, 3H), 7.14-7.42 (m, SH). LC-MS (ES*) m/e=419.4 (M+H). 20 (1- [2-(2-Benzyloxy-5-oxo-tetrahydro-furan-3 ylcarbamoyl) -pyrrolidine-l-carbonyl] -2, 2-dimtethyl propyl}-carbamic acid tert-butyl ester (71).
- 137 An 871mg (2.08mmol) sample of 70 was dissolved in MeOH (15mL) and 10% Pd/C (200mg) added. The suspension was stirred under H 2 for hour then filtered through Celite and the solvent evaporated. This resulting residue was 5 reacted with 40 according to the procedure used to prepare 56 to give 889mg (71% yield) of the title compound (71). 'H-NMR (500MHz, CDCl 3 ) 6 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 (in, 1H), 3.70-3.85 (m, 10 1H), 4.20-4.37 (m, 1H), 4.49-4.78 (m, 3H), 4.78-4.98 (m, 1H), 5.12-5.26 (m, 1H), 5.40-5.59 (m, 1H), 7.10 7.78 (m, SH). Analytical HPLC (cyano column) 11.17min. LC-MS (ES*) m/e=518.3 (M+H). 15 1- [2- (4-Amino-3-chloro-benzoylamino) -3,3-dimethyl butyryll-pyrrolidine-2-carboxylic acid (2-benzyloxy-5 oxo-tetrahydro-furan-3-yl)-amide (72). A solution of 456mg (0.088mmol) of 71 in CH 2 Cl 2 (20ml) was treated with anhydrous TFA (5mL) then stirred at 20 room temperature under N 2 for 1 hour and evaporated to dryness. The residue was repeatedly concentrated from
CH
2 Cl 2 (3x) then dried under vacuum. The resulting residue was dissolved in CH 2 Cl 2 (20ml), cooled to 0 C, then treated with DIEA (1.3ml, 8eq, 2.46mmol) followed 25 by 4-amino-3-chloro-benzoic acid (202mg, 1.17mmol), HOBT (183mg, 1.35mmol), and EDC (279mg, 1.45mmol). The resulting mixture was allowed to warm to room temperature and stir for 18hours. The solvent was removed in vacuo and the residue dissolved in EtOAc 30 then washed with distilled water (3x), 0.5N NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine. The organic layer was dried over Na 2
SO
4 , filtered and evaporated to give a residue that was purified by flash chromatography, eluting with CH 2 Cl 2 /MeOH (99/1 to - 138 97/3%), affording 265mg (57% yield) of the title compound (72) as a yellow solid. 1H-NMR (500MHz, CD 3 0D) 8 0.91-1.24 (m, 9H), 1.70-2.27 (m, 4H), 2.47-2.85 (m, 1.5H), 2.99-3.13 (m, 0.5H), 5 3.39-3.53 (m, 0.5H), 3.60-3.78 (m, 1.5H), 3.85-4.04 (m, 1H), 4.24-4.47 (m, 2H), 4.53-4.97 (m, 4H), 5.46 (s, 0.3H), 3.88-4.02 (m, 0.1H), 5.60-5.69 (m, 0.6H), 6.80 (d, 1H) , 7.22-7.77 (m, 7H) . Analytical HPLC (cyano column) (mixture of 2 diastereomers) 15.90, 16.23min. 10 LC-MS (ES*) m/e=571.2 (M+H). 3 -({1- [ 2 - (4 -Amino-3 -chloro-benzoylamino) -3, 3-dinethyl butyryll -pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric acid (73). 15 A 40mg (0.07mmol) sample of 72 was hydrolyzed according to method A (see Scheme XXIII) to afford 25mg (74% yield) of the title compound: Analytical HPLC (cyano column) 10.66min. LC-MS (ES+) m/e=481.3 (M+H) . Scheme XVI Boc. NNr, + Alc w ~ Alloo 0 CO 2 H 4- 6HS 0 74 40 75 )~ -4I 20- - 139 (2- [2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3 ylcarbamoyl) -pyrrolidin-1-yl] -1-methyl-2-oxo-ethyl) carbamic acid tert-butyl ester (75). To a solution of 40 (6.69g, 23.Ommol) in anhydrous 5 CH 2 C1 2 was added 1,3-dimethylbarbituric acid (DMBA) (3.97g, 25.4mmol) and Pd(PPh 3
)
4 (1.12g, 0.97mmol). The solution was stirred under N 2 at room temperature for 15min, cooled to O'C, followed by the addition of Boc ala-pro-OH (BaChem) (5.087g, 17.8mmol), HOBT (3.60g, 10 26.7mmol) and EDC (5.12g, 26.7mmol). The resulting solution was allowed to warm to room temperature and stir under N 2 for 18hours. The solvent was concentrated in vacuo and the residue dissolved in EtOAc, then washed with.O.5N NaHSO 4 (2x), saturated 15 NaHCO 3 (2x) and brine. The organic layer was dried over anhydrous Na 2
SO
4 and evaporated to give an orange oil (12.23g). Flash column chromatography on silica gel using CH 2 Cl 2 /EtOAc (80/20 to 60/40) gave the title compound 75 as a yellow solid (7.28g, 86% yield). 'H 20 NMR (500MHz, CD 3 0D) 5 1.19-1.31 (m, 3H), 1.42 (s, 9H), 1.69-2.29 (m, 4H), 2.45-2.67 (m, 0.9H), 2.71-2.86 (m, 0.5H), 2.99-3.10 (m, 0.6H), 3.49-3.84 (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), 7.25-7.40 (m, 25 5H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 15.99, 16.33min. LC-MS (ES*) m/e=476.3 (M+H). 0 N 0
H
2 N 7 76 30 - 140 [1- [2- (4-Amino-3-chloro-benzoylanino) -propionyl] pyrrolidine-2-carboxylic acid (2 -benzyloxy-5-oxo tetrahydro-furan-3-yl) -amide] (76). A 1.899g (3.99mmol) sample of 75 in CH 2 C1 2 (20ml) was 5 treated with anhydrous TFA (5ml) then stirred at room temperature under N 2 for 1 hour and evaporated to dryness. The residue was repeatedly concentrated from
CH
2 Cl 2 (3x) then dried under vacuum. The resulting residue was dissolved in CH 2 Cl 2 (20ml), cooled to O'C, 10 then treated with DIEA (5.6ml, 8eq, 32.1mmol), 4-amino 3-chloro-benzoic acid (0.9109, 5.3mmol), HOBT (0.824g, 6.Immol) , and EDC (1.197g, 6.23mmol) . The resulting mixture was warmed to room temperature and stirred for 18hours. The solvent was removed in vacuo and the 15 residue dissolved in EtOAc then washed with distilled water (3x), 0.SN NaHSO 4 (2x), saturated NaHCO 3 (2x) and brine. The organic layer was dried over Na 2
SO
4 , filtered and evaporated to give a residue that was purified by flash chromatography using CH 2 Cl 2 /MeOH 20 (99/1 to 97/3%) . The title compound was obtained as a white solid (1.221g, 58% yield) .
1 H-NMR (500MHz, CD 3 0D) 6 1.15 (d, 0.25H), 1.29-1.60 (m, 2.75H), 2.41-2.54 (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 25 (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, SH), 7.48-7.68 (m, 1H), 7.68-7.88 (m, 1R), 8.15-8.34 (m, 1H) . Analytical HPLC (cyano column) (mixture of 2 diastereomers) 14.44, 14.89min. LC-MS (ES*) m/e=S29.3 (M+H). 30 0 N7 .77
H
- 141 [1- [2- ( 4 -Methoxy-3,5-dimethyl-benzoylamino) propionyl) -pyrrolidine-2 -carboxylic .acid (2 -benzyloxy 5-oxo-tetrahydro-furan-3-yl) -amide] (77) was 5 synthesized from 75 and 3 ,5-dimethyl-4-methoxy benzoic acid according to the procedure used to prepare 76 to afford the title compound (1.i1g, 44% yield). 1 H-NMR (500MHz, CD 3 0D) 5 1.40 (m, 3H), 1.67-2.41 (m, 4H), 2.28 (s, 6H), 2.48 (ddd, 0.5H), 2.62 (dd, 0.5H), 2.78 (ddd, 10 0.5H), 3.04 (ddd, 0.5H), 3.62-3.94 (m, 3H), 3.71 (s, 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, 0.1H), 7.17-7.45 (m, 5H), 7.45-7.65 (m, 2H). Analytical HPLC (cyano column) (mixture of 2 15 diastereomers) 15.06, 15.39min. LC-MS (ES*) m/e=538 (M+H) Preparation of 4-Acetylamino-3-chlorobenzoic acid To a solution of 4 -amino-3-chloro-benzoic acid (10.0g, 20 58.3nmol) in anhydrous THF (100mL) was added acetyl chloride (20.7ml, 291.1mmol) and the solution stirred at room temperature for 48hours. The solvent was evaporated and the product precipitated from hexanes then filtered and dried to give a white solid (11.73g, 25 94% yield). 1 H-NMR (500MHz,
CD
3 0D) 8 2.28 (s, 3H) , 7.92 (dd, 1H), 7.99-8.16 (m, 2H). Analytical HPLC (cyano column) 7.84min. 0 0 N N AN H rr1 H IN 78 30 - 142 1- [2- (4-Acetylanino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2 -benzyloxy-5-oxo tetrahydro-furan-3-yl) -amide (78). Prepared from 75 and 4 -acetylamino-3-chloro-benzoic 5 acid according to the procedure used to prepare 76 to afford the title compound (146mg, 19% yield) . IH-NMR (500MHz, CD 3 0D) 6 1.28-1.52 (m, 3H), 1.68-2.38 (m, 4H), 2.20 (s, 3H), 2.41-2.88 (m, 1.5H), 2.96-3.10 (m, O.5H), 2.96-3.10 (m, 0.5H), 3.43-3.75 (m, 1H), 3.80-3.96 (m, 10 1H), 4.25-5.00 (m, %H), 5.42-5.54 (m, 0.5H), 5.63-5.78 (m, 0.5H), 7.13-7.48 (m, OSH), 7.79-8.14 (m, 2.5H), 8.56-8.70 (m, 0.5H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 8.64min. LC-MS (ES*) m/e=571.2 (M+H). 15 0 N N O O N 79 1- [2 - ( 3 -Isopropoxy-benzoylamino) -propionyl] pyrrolidine-2 -carboxylic acid (2-benzyloxy-5-oxo 20 tetrahydro-furan-3-yl)-amide (79). Prepared from 75 and 3-isopropoxybenzoic acid according to the procedure used to prepare 76 to afford the title compound (120mg, 58% yield).. 1 H-NMR (500MHz, CD 3 0D) S 1.27 (d, 6H), 1.33-1.52 (m, 3H), 1.69-2.31 (m, 4H), 25 2.49 (dd, 0.3H), 2.63 (dd, 0.7H), 2.78 (dd, 0.7H), 3.03 (dd, 0.3H), 3.43-3.73 (m, 1H), 3.78-3.94 (m,1H), 4.27 4.47 (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 (cyano - 143 column) (mixture of 2 diastereomers) 14.54, 14.85min. LC-MS (ES*) m/e=538 (M+H). N N N 80 0 H Quinoxaline-2-carboxylic acid (2-[2- (2-benzyloxy-5-oxo tetrahydrofuran-3 -ylcarbamoyl) -pyrrolidin-1-yl] -1 methyl-2-oxo-ethyl)-amide (80). 10 Prepared from 75 and 2-quinoxaline carboxylic acid according to the procedure used to prepare 76 to afford the title compound (122mg, 60% yield) , 'H-NMR (500MHz,
CD
3 0D) 6 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), 15 3.41-3.49 (m, 2H), 4.18-5.03 (m, 6H), 5.47 (d, 0.5H), 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 (cyano column) (mixture of 2 diastereomers) 9.06min. LC-MS (ES+) 20 m/e=532.3 (M+H). 8 N 81O - 144 1- [2- ( 3 -Benzyloxy-4-methoxy-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide (81). Prepared from 75 and 3 -benzyloxy-4-methoxy benzoic acid 5 according to the procedure used to prepare 76 to afford the title compound (142mg, 58% yield) 1 H-NMR (500MHz,
CD
3 0D) 6 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, 0.6H), 2.77 (dd, 0.6H), 3.02 (dd, 0.4H), 3.41-3.72 (m, 1H), 3.72-3.99 10 (m, 2H), 3.86 (s, 3H), 4.19-4.86 (m, 5H), 4.99-5.15 (M, 2H), 5.45 (m, 0.8H), 5.65 (m, 1.2H), 6.98 (dd, 1H), 7.11-7.63 (m, 12H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 12.28, 12.44min. LC-MS (ES*) m/e=616.3 (M+H). 15 4-Allyloxy-3,5-dimethyl-benzoic acid. A mixture of 4 -hydroxy-3,5-dimethyl-benzoic acid (3.32 20 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 mmol) in DMF (50 mL) was stirred at -room temperature for 16 hours. The mixture was diluted with ethyl acetate (200 mL), washed with water, brine. 25 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 mmol) in water/methanol (50 mL/50 mL) for 6 hours. The mixture was evaporated in vacuo to remove methanol and 30 the resulted solution was diluted with water (200 mL), washed with ethyl acetate/hexane (30 mL/70 mL). The aqueous layer was acidified at 0*C with concentrated HCl solution to pH 2. The resulted precipitate was collected by filtration and washed with water, dried - 145 over high vacuum to afford 3.86 g (yield 94%) of the title compound. 1H-NMR (500 MHz, CDCl 3 ): 8 2.33 (s, 6H), 4.35-4.37 (m, 2H), 5.28-5.30 (m, H), 5.42-5.46 (m, H), 6.07-6.15 (m, H), 7.79 (s, 2H); retention time on 5 analytical HPLC: 11.28 min; LC-MS: m/z = 205 (M-H*). C N 1- [2 - (4-Allyloxy-3, 5 -dichloro-benzoylamino) -propionyll 10 pyrrolidine-2-carboxylic acid (2-benzyloxy-S-oxo tetrahydro-furan-3-yl) -amide (82). Prepared from 75 and 4-allyloxy-3,5-dichloro-benzoic acid according to the procedure used to prepare 76 to afford the title compound (208mg. 47% yield). 1 H-NMR 15 (500MHz, CDCl 3 ) 0 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 (cyano column) (mixture of 2 diastereomers) 9.69, 9.89min. LC-MS (ES*) m/e=604.2 (M+H). 20 C HO 0 N C1 1- [2 - (3, 5-Dichloro-4-hydroxy-benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid (2-benzyloxy-S-oxo 25 tetrahydro-furan-3-yl) -amide (83). A 140 mg sample of 82 (0.23mmol) was dissolved in
CH
2 C1 2 - (4mL) and treated with DMBA (35.4mg, 0.26mmol) and Pd(PPh 3
)
4 (32mg, 0.028mmol). The solution was - 146 stirred at 0*C for 15mins, warmed to room temperature for 2hours, then diluted with CH 2 Cl 2 and washed with water (2x) and brine. The solvent was concentrated in vacuo and the residue purified by flash chromatography 5 on silica gel using MeOH/CH 2 Cl 2 (1/99 to 3/97) to give the title compound (93.2mg, 71% yield). 'H-NMR (500MHz, CD 3 0D) 5 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 10 4.48 (m, 2H), 4.57-4.92 (m, 7H), 5.44 (s, 0.4 H), 5.50 (d, 0.4H)., 5.64 (d, 0.8H), 5.75 (d, 0.5H), 7.16-7.43 (m, 5H), 7.78-7.89 (m, 1.6H), 8.40-8.63 (m, 0.4H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 11.57, 11.82min. LC-MS (ES*) m/e=564.1 15 (M+H) oN 84 1- (2-Benzoylamino-propionyl) -pyrrolidine-2-carboxylic 20 acid ( 2 -benzyloxy-5-oxo-tetrahydro-furan-3-yl) -amide (84). Prepared from 75 and benzoyl chloride according to the procedure used to prepare 76 to afford the title compound as a colorless oil (8mg, 38% yield). 1 H-NMR 25 (500MHz, CD 3 0D) 8 1.35-1.54 (m, 3H), 1.72-2.30 (m, 4H) , 2.42-2.70 (m, 1.3H), 2.74-2.84 (m, 0.51H), 3.03 (dd, 0.2H), 3.41-3.75 (m, 2H), 3.81-3.96 (m, 1H), 4.22-4.86 (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, 30 2H), 8.42-8.67 (m, 1H). Analytical HPLC (cyano - 147 column) (mixture of 2 diastereomers) 15.23, 15.67min. LC-MS (ES*) m/e=481.2 (M+H) 0N 85OON 5 Isoquinoline-1-carboxylic acid {2- (2 -benzyloxy-5-oxo tetrahydro-furan-3-ylcarbamoyl) -pyrrolidin-1-yl] -1 methyl-2-oxo-ethyl}-amide (85). Prepared from 75 and 1-isoquinolinecarboxylic acid 10 according to the procedure used to prepare 76 to afford the title compound (732mg, 53% yield) .
1 H-NMR (500MHz,
CD
3 0D) 6 1.22-1.56 (m, 3H), 1.70-2.34 (m, 4H), 2.43-2.71 (m, 0.9H), 2.73-2.89 (m, O.5H), 3.06 (ddd, 0.6H), 3.42 3.81 (m, 2H), 3.84-4.01 (m, IH), 4.29-5.00 (m, 5H), 15 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, SH), 7.49-7.83 (m, 2.8H), 7.88-8.04 (m, 1.8H), 8.45-8.54 (m, 0.8H), 8.97-9.06 (m, 0.6H) . Analytical HPLC (mixture of 2 diastereomers) 15.71, 16.04min. LC-MS (ES*) m/e=531.2 (M+H).
20
H
2 N NC 86 1- 12 - (4 -Amino-5 -chloro-2,-methoxy-benzoylamino) propionyll -pyrrolidine-2-carboxylic acid (2-benzyloxy 25 5-oxo-tetrahydro-furan-3-yl) -amide (86). Prepared from 75 and 4-amino-5-chloro-2-methoxy benzoic acid according to the procedure used for 76- to afford - 148 the title compound (330mg, 61% yield) .
1 H-NMR (500MHz,
CD
3 0D) 6 1. 22 (d, 0.25H), 1.29-1.50 (m, 0.75H), 1.68 2.36 (m, 4H), 2.38-2.89 (m, 1.5H), 2.94-3.14 (m, 0.5H), 3.37-3.98 (m, 6H), 4.27-4.98 (M, 6H), 5.44-5.50 (m, 5 0.4H), 5.53-5.56 (s, 0.1H), 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 (cyano column) (mixture of 2 diastereomers) 16.39, 16.82min. LC-MS (ES*) m/e= 559.2 (M+H). 10 0 ojrN H 87 N 1- [2- ( 4 -Acetylamino-5-chloro-2-methoxy-benzoylamino) propionyl] -pyrrolidine- 2- carboxylic acid (2-benzyloxy 15 5-oxo-tetrahydro-furan-3-yl)-amide (87). Prepared from 75 and 4 -acetylamino-5-chloro-2-methoxy benzoic acid according to the procedure used for 76 to afford the title compound (364mg, 64k yield). 1 H-NMR (500MHz,
CD
3 0D) 5 1.20-1.27 (m, 0.25), 1.35-1.49- (m, 20 0.75H), 1.72-2.30 (m, 4W), 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 3.11 (m, 0.7H), 3.41-4.07 (m, SH), 4.29-4.97 (m, SH), 4.79-5.56 (m, 0.5H), 5.65-5.73 (m, 0.5H), 7.18-7.44 (m, 4.3H), 7.90-8.09 (mn, 2H), 8.71-8.85 (m, 0.7H). 25 Analytical HPLC (cyano column) (mixture of 2 diastereomers) 15.61, 16.01min. LC-MS (ES*) m/e= 601.1
(M+H).
- 149 N N 88 Pyridine-2-carboxylic acid(2- 12- (2-benzyloxy-5-oxo tetrahydro-furan-3-ylcarbamoyl) -pyrrolidin-1-yl]
-I
5 methyl-2-oxo-ethyl)-amide (88). Prepared from 75 and pyridine-2-carboxylic acid according to the procedure used for 76 to afford the title compound (233mg, 42% yield) . 'H-NMR (500MHz,
CD
3 0D) 5 1.30-1.59 (m, 3H), 1.68-2.36 (m, 4H), 2.39-2.57 10 (m, 0.6H), 2.57-2.69 (m, 0.35H), 2.71-2.87 (m, 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, SH), 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 (cyano 15 column) (mixture of 2 diastereomers) 8.63min. LC-MS (ES*) m/e=481.3 (M+H). C N
H
2 N C C1 89 20 11- [2- (4-Amino-3, 5-dichloro-benzoylazino) -propionyll pyrrolidine-2 -carboxylic acid (2 -benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide] (89). Prepared from 75 and 3,5-dichloro-4-aminobenzoic acid according to the procedure used for 76 to afford the 25 title compound (162mg, 70% yield) .
1 H-NMR (500MHz,
CD
3 0D) 5 1.21-1.58 (m, 3H), 1.58-2.37 (m, 4H), 2.37-3.13 (m, 2H), 3.43-3.74 (m, 1.5H), 3.77-3.94 (m, 1H), 4.28- - 150 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 (cyano column) (mixture of 2 diastereomers) 8.36min.
LC
MS (ES*) m/e=563.2 (M+H) . S 9 NO CH 90 1- [2- (4 -Methoxy-benzoylamino) -propionyl) -pyrrolidine- 2 10 carboxylic acid ( 2 -benzyloxy-5-oxo-tetrahydro-furan-3 yl)-amide (90). Prepared from 75 and 4 -methoxy-benzoylchloride according to the procedure used for 76 to afford the title compound (404mg, 50%). 1 H-NMR (500MHz, CD 3 0D) 5 15 1.19 (d, 0.3H), 1.29-1.58 (m, 2.7H), 1.58-2.38 (m, 4H), 2.43-2.69 (m, 1H), 2.74-2.86 (m, 0.6H), 2.99-3.11 (m, 0.4H), 3.39-3.75 (in, 1.5H), 3.77-3.94 (m, 1H), 3.84 (s, 3H), 4.29-4.94 (m, 4.5H), 5.45-5.55 (m, 4.5H), 5.63 5.71 (n, 0.5H), 5.73 (d, 0.1H), 6.85-7.09 (m, 2H), 20 7.19-7.44 (m, 4H), 7.73-7.92 (m, 2H), 8.26-8.44 (m, 1H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 15.18, 15.65min. LC-MS (ES*) m/e=510.2 (M+H). 25 N 25 91 - 151 1.-{2- [( 9 -Oxo-9H-fluorene-4-carbonyl) -amino] -propionyl) pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide (91). Prepared from 75 and 9 -oxo-9H-fluorene-carboxylic acid 5 according to the procedure used for 76 to afford the title compound (403mg, 44% yield). 1 H-NMR (500MHz, CDC1 3 ) 5 1.38-1.59 (m, 3H), 1.75-2.37 (m, 4H), 2.43-2.59 (m, 0.65H), 2.59-2.72 (m, 0.35H), 2.79-2.89 (m, 0.35H), 3.01-3.11 (Im, 0.65H), 3.68-3.86 (m, 1H), 3.92-4.09 (m, 10 1H), 4.35-5.03 (m, 7H), 5.42-5.90 (m, 1H), 7.06-8.00 (m, 12H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 12.30min. LC-MS (ES+) m/e=582.1 (M+H) IH C N 92 15 1- [2- (3, 5-Dichloro-4-methoxy-benzoylamino) -propionyll pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide (92). Prepared from 75 and 3,5-dichloro-4-methoxy-benzoic 20 acid according to the procedure used for 76 to afford the title compound (364mg. 46% yield). 1 H-NMR (500MHz,
CD
3 0D) 6 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), 2.94-3.12 (m, 0.5H), 3..41-3.74 (m, 2H), 3.74-4.00 (m, 1H), 3.91 (s, 3H), 25 4.26-5.02 (m, SH), 5.42-5.81 (m, 1H), 7.08 (d, 0.4H), 7.21-7.43 (m, 4.6H), 7.53~-7.69 (m, 0.8H), 7.85-7.97 (m, 1.2H). Analytical HPLC (cyano column) (mixture of 2 diastereomers) 10.79min. LC-MS (ES*) m/e=578.2
(M+H)
- 152 N N 0 N 93 Quinoline-6 -carboxylic acid {2- ( 2 -benzyloxy-5-oxo tetrahydro-furan-3-ylcarbamoyl)-pyrrolidin-1-ylj-1 5 methyl-2-oxo-ethyl}-aide (93). Prepared from 75 and 6 -quinolinecarboxylic acid according to the procedure used for 76 to afford the title compound (344mg, 71* yield) . H-NM (500MHz,
CD
3 oD) S 1.11-1.58 (m, 3H), 1.69-2.40 (m, 4H), 2.42-3.15 10 (m, 2H), 3.80-4.01 (m, 1H), 4.29-4.99 (m, 5H), 5.44 5.54 (m, 0.5H), 5.63-5.73 (d, 0.4H), 5.73-5.79 (d, 0.1H), 7.18-7.43 (m, 5H), 7.56-7.67 (m, 1H), 8.08 (d, 1H), 8.13-8.25 (m, 1H), 8.40-8.56 (m, 2H), 8.88-8.99 (m, 1H) . Analytical HPLC (cyano column) (mixture of 2 15 diastereomers) 10.27, 10.50min. LC-MS (ES*) m/e=531.2
(M+H).
- 153 Scheme XVII Cbz..- N7 CbzJ>" N o CO2H O CO2BU-t 94 95 R1. N R1 N O CO 2 H O CO2Bu-t 97 96 H2N4 OR8Y R1 N0 N 98 1- (2 -Benzyloxycarbonylamino-propionyl) -pyrrolidine-2 carboxylic acid tert-butyl ester (95) . 5 Prepared according to the method described in Pierre Chevallet, Patrick Garrouste, Barbara Malawaska & Jean Martinez in Tetrahedron Letters, Vol. 34, pp. 7409 7412, (1993). A mixture of Cbz-ala-pro-OH (10.0 g, 31.2 mmol), tert-butyl bromide (180 g, 1.31 mol), 10 benzyltriethylammonium chloride (7.11 g, 31.2 mmol) and
K
2
CO
3 (180 9, 1.30 mol) in N,N-dimethylacetamide (DMA) (225 mL) was stirred at 55 0 C for 24 hours. The reaction mixture was cooled to room temperature and diluted with one liter of ice-water, extracted with 15 ethyl acetate (200 mL x 3) . The organic layer was dried over anhydrous Na 2
SO
4 , filtered and evaporated in - 154 vacuo to give 14 g of oil, which was purified by flash chromatography using hexane/ethyl acetate (95/5 to 50/50) to afford 11.73 g (yield 99.7%) of the title compound as a clear oil. IH-NMR (500 MHz, CDCl 3 ) : 5 1.25-1.50 (m, 12 H), 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, SH); retention time on analytical HPLC: 11.07 min; LC-MS: m/z = 377 (M+H*). 0 N 0N N 0 N
CO
2 Bu-t O CO 2 H X X 96a, X=CI, Y=NH 2 , Z=H 97a, X=CI, Y=NH 2 , Z=H 96b, X=CI, Y=AcNH, Z=H 97b, X=CI, Y=AcNH, Z=H 10 96c, X=CI, Y=AcNH, Z<,H 3 0 97c, X=CI, Y=AcNH, Z=CH 3 0 1- [2- ( 4 -Amino-3 -chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid tert-butyl ester (96a). To a solution of 95 (10.50g, 27.9mmol) in MeOH (100ml) 15 was added a suspension of 10% Pd/C (5.00g) in EtOAc (Soml) . The mixture was stirred under H 2 for 48hours, filtered through celite and the solvent evaporated to yield a waxy solid. This was dissolved in CH 2 C1 2 (100ml) and DMF (50ml) and the solution cooled to 0 0 C. 20 4 -Amino-3-chlorobenzoic acid (5.82g, 27.2mmol), DIEA (14.58ml, 83.7mmol), HOBT (3.77g, 27.9mmol) and EDC (6.68g, 34.8mmol) were added and the solution stirred at 0*C for 15mins then at room temperature for 24hours. The reaction mixture was diluted with EtOAc, washed 25 with NaHSO 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 Cl 2 /MeOH (99/1 to 97/3%) to yield the title compound - 155 as a white solid .(7.75g, 70% yield). 1 H-NMR (500MHz,
CD
3 0D) 5 1.27-1.67 (m, 12H), 1.82-2.14 (M, 4H), 3.48 3.85 (m, 2H), 4.26-4.53- (m, 3H), 4.81-4.98 (m, 1H), 6.71 (d, 1H), 7.15 (m, 1H), 7.50 (dd, 1H), 7.75 (d, 5 1H). Analytical HPLC 10.83min. LC-MS (ES*) m/e=396.3 (MH*). 1- [2- ( 4 -Amino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (97a). 10 Prepared from 96a by treatment with TFA/ CH 2 Cl 2 . After complete reaction, the solvent is removed in vacuo and the residue repeatedly concentrated from toluene. The resulting residue-was dried under vacuum to a constant weight. 15 1- [2- ( 4 -Acetylamino-3-chloro-benzoylamaino) -propionyl) pyrrolidine-2-carboxylic acid tert-butyl ester (96b). Prepared from 95 and 4 -acetyl amino-3-chlorobenzoic acid according to the method used for 96a to afford the 20 title compound as a white solid (9.18g, 77% yield). H-NMR (500MHz,
CD
3 0D) 6 1.30-1.62 (m, 12H), 1.85-2.16 (m, 3H), 2.16-2.44 (m, 1H), 2.27 (s, 3H), 3.47-3.83 (m, 2H), 4.34-4.54 (m, 1H), 4.8.9 (m, 1H), 7.27-7.39 (m, 1H), 7.59-7..71 (m, 2H), 7.83-7.97 (m, 1H), 8.47. (d, 25 1H). Analytical HPLC 9.43min. 1- [2- (4 -Acetylmino-3 - chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (97b). Prepared from 96b by treatment with TFA/ CH 2 C1 2 . After 30 complete reaction, the solvent is removed in vacuo and the residue repeatedly concentrated from toluene. The resulting residue was dried under vacuum to a constant weight.
- 156 4-Acetylamino-5-chloro-2-methoxy-benzoic acid. 4 -Acetylamino-5-chloro-2-methoxy-benzoic acid methyl ester (2.09g, 8.11mmol) was dissolved in MeOR (110ml) 5 and LiOH solution (25.48mmol in 30ml, 1:1 MeOH:H 2 0) added and the solution stirred at room temperature for 6hours. The solvent was concentrated in vacuo, EtOAc added and the organic phase was washed with O.5N HCl then extracted with saturated NaHCO 3 (2x) . The aqueous 10 phase was acidified with 12N HCl to pH 1 and the resulting precipitate extracted into CH 2 Cl 2 . The combined extracts were dried over anhydrous Na 2
SO
4 , filtered and evaporated to give the title compound as a white solid (0.933g, 50% yield). IH-NMR (500MHz, CDCl3) 15 8 2.31 (s, 3H), 4.10 (s, 3H), 7.78-7.92 (br s, 1H), 8.17 (s, 1H), 8.45 (s, 1H). Analytical HPLC 5.62min. 1- [2- ( 4 -Ace tylamino -5-chloro -2-methoxy-benzoylamino) propionyl] -pyrrolidine-2 -carboxylic acid tert-butyl 20 ester (96c). To a solution of 95 (1.534g, 4.07mmol) in MeOH (40ml) was added 10%Pd/C (650mg) and the mixture stirred under
H
2 for 2hours. The suspension was filtered through celite and evaporated to give a yellow oil. This was 25 allowed to react with 4 -acetyl-5-chloro-2-methoxy benzoic acid following the procedure used for the preparation of 96a to give the title compound (497mg, 52% yield). 1 H-NMR (500MHz,
CD
3 OD) 8 1.46 (d, 3H) , 1.49 (s, 9H), 1.80-2.01 (m, 3H), 2.19-2.40 (m, 1H), 2.22 (s, 30 3H), 3.58-3.72 (m, 1H), 3.78-3.89 (m, 1H), 3.98-4.09 (s, 3H), 4.31-4.45 (s, 1H), 4.78-4.95 (m, 1H), 7.89 8.10 (m, 2H) . Analytical HPLC 11.31min.
- 157 1- [2- (4-Acetylamino-5-chloro-2 -methoxy-benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (97c). Prepared from 96c by treatment with TFA/ CH 2 C1 2 . After complete reaction, the solvent is removed in vacuo and 5 the residue repeatedly concentrated from toluene. The resulting residue was dried under vacuum to a constant weight N N
H
2 N / O N CI 98a H 10 1- [2- (4 -Amino- 3 -chloro-benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid (5-oxo-2-phenethyloxy tetrahydro-furan-3-yl) -amide (98a). To a solution of (5-oxo-2-phenethyloxy-tetrahydro 15 furan-3-yl)-carbamic acid allyl ester (194mg, 0.54mmol) (prepared as described for (40) using phenethyl alcohol) in anhydrous
CH
2 Cl 2 (5mL) at OC was added DMBA (196mg, 1.26mmol) and Pd(PPh 3
)
4 (32mg, 0.03mmol). The solution was stirred for 15min and a solution of 97a 20 (prepared from 96a by treatment with TFA in
CH
2 C1 2 ) (166mg, 0.49mmol) and DIEA (680gl, 3.90mmol) in
CH
2 C1 2 (2mL) was added followed by HOBT (98mg, 0.73mmol) and EDC (122mg, 0.63mmol) . The solution was stirred at OC for 15min then at room temperature for 25 18hours. The solvent was removed in vacuo and the residue dissolved in EtOAc then washed with 0.5N NaHSO 4 (2xl , saturated NaHCO 3 (2x) and brine. Dried over an'hydrous NIa2SO 4 and evaporated to give an orange solid which was purified by flash column chromatography, - 158 using CH 2 Cl 2 /MeOH (99/1 to 97/3%) to yield the title compound as a white solid (190mg, 73% yield). 1 H-NMR (500MHz,
CD
3 0D) 6 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), 5 2.94 (t, 2H), 3.53 (M, 0.3H), 3.67 (m, 0.8 H), 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 14.52min. LC-MS (ES*) 10 m/e=543.2 (MH*) 0 N H2N C 98b 1- [2- (4 -Amino-3 -chloro-benzoylamino) -propionyl] 15 pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan.-3-yl)-amide (98b). Was prepared from the syn diastereomer of (2-benzyloxy 5-oxo-tetrahydro-furan-3-y1) -carbamic acid allyl ester (40) and 97a following the method used for 98a. The 20 title compound was isolated as a pale yellow solid (720mg, 51% yield) . 1H-NMR (500MHz,
CD
3 0D) 8 1.16 (d, O.5H), 1.40 (d, 2.5H), 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 (in, 1H), 4.29-4.47 (m, 1H), 4.47-5.02 25 (m, 4H), 5.48 (s, 0.5H), 5.66 (d, 1H), 5.68 .(d, 0.5H), 6.79 (d, 1H), 7.17-7.52 (m, 5H), 7.48-7.62 (m, 1H), 7 .68-7.83 (m, 1H) . Analytical HPLC 15.98min. LC-MS (ES*) m/e=529.2
(MH*)
- 159 N rN H2N C: 98c 1- [2- ( 4 -Amino- 3 -chloro-benzoylamino) -propionyl] 5 pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-aimide (98c). Prepared from the anti- ( 2 -benzyloxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester (40) and 97a following the method used for 98a. The title compound 10 was isolated as a white solid (186.6mg, 46% yield) 1 H-NMR (500MHz,
CD
3 0D) 6 1.30-1.52 (m, 3H) , 1.76-2.33 (m,4H), 2.41-2.59 (m, 1H), 2.90 (dd, 0.15H), 3.04 (dd, 0.85H), 3.44-3.75 (m, 1.5H), 3.82-3.95 (m, 1H), 4.27 4.42 (m, 2H), 4.42-4.56 (m, 0.5H), 4.56-4.86 (m, 4H), 15 5.42-5.55 (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 15.19min. LC-MS (ES+) m/e=529.3 (MH+) . N H2 O004 CI 20 98d 2- (Ethoxy-5-oxo- tetrahydro-furan- 3 -yl) -carbamic acid allyl ester. Prepared from 3 - allyloxycarbonylamino -4 -hydroxy- butyric 25 acid tert-butyl ester as described for (40) using - 160 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)-carbamic acid allyl ester (higher Rf), 1.96 9 of syn diastereomer (lower Rf) and 5 8.08 g of the mixture of the diastereomers (total overall yield 60%). IH-NMR (500 MHz, CDCl-) for the anti diastereomer: 5 1.13-1.31 (m, 3H), 2.31-2.45 (m, 1H), 2.92-3.08 (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 10 (bs, 1H), 5.12-5.45 (m, 3H), 5.80-5.95 (m, 1H); for syn diastereomer 1.13-1.35 (m, 3H), 2.38-2.50 (m, 1H), 2.75-2.92 (m, 1H), 3.60-3.73 (m, 1H), 3.82-3.95 (m, 1H), 4.40-4.70 (m, 3H), 5.10-5.52 (m, 4H), 5.80-5.94 (m, 1H); LC-MS: m/z = 230 (M+H*) for both 15 diastereomers. 1-[2-( 4 -Amino-3-chloro-benzoylamino)-propionyl] pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo tetrahydro-furan-3-yl)-amide (98d). 20 Prepared from (2-ethoxy-5-oxo-tetrahydro-furan-3-yl) carbamic acid allyl ester and 97a following the method used for 98a. The title compound was isolated as a white solid (175mg, 77% yield). 1 H-NMR (500MHz,
CD
3 0D) . 1.13 (t, 0.5H), 1.23 (t, 2.5H), 1.36 (d, 0.SH), 1.44 25 (d, 2.5H), 1.75-2.38 (m, 4H), 2.56 (dd, 1H), 2.76 (dd, 1H), 3.45-3.97 (m, 5H), 4.47 (dd, 11), 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, 1H), 8.39 (d, 1H) . Analytical HPLC 8.17min. LC-MS (ES*) 3C m/e=467.4
(MH).
- 161 N- N H2N 0 0 N C1 H 98e (2-Cyclopentyloxy-5-oxo-tetrahydro-furan- 3 -yl) -carbamic acid allyl ester. 5 Prepared from 3-allyloxycarbonylamino-4 -hydroxy-butyric acid tert-butyl ester as described for 40 using cyclopentanol to afford the title compound as a mixture of diastereomers. Flash column chromatography using hexanes/EtOAc (90/10 to 80/20) afforded the syn 10 diastereomer of the title compound: syn diastereomer 1H NMR (500MHz, CDCl 3 ) 6 1.5-2.0 (m, 8H), 2.45 (dd, 1H), 2.81 (dd, 0.9H), 3.0 (dd, 0.1H), 4.31 (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; anti diastereomer IH-NMR 15 (500 MHz, CDC1 3 ) 6 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. 20 1- [2- (4-Amino-3-chloro-benzoylamino) -propionyll pyrrolidine-2-carboxylic acid (2-cyclopentyloxy-5-oxo tetrahydro-furan-3-yl)-amide (98e). Prepared from ( 2 -cyclopentyloxy-5-oxo-tetrahydro-furan 3-yl)-carbamic acid allyl ester and 97a following the 25 method used for 98a to give the title compound (280mg, 51% yield). 1 H-NMR (500MHz, CD 3 0D) 6 1.38 (d, 0.55H), 1.44 (d, 2.51H), 1.49-2.35 (m, 12H), 2.47 (dd, 0.7H), 2.56 (dd, 0.3H), 2.75 (dd, 0.3H), 2.81-2.88 (m, 0.1H), 2.97 (dd, 0.6H), 3.47-3.76 (m, 0.2H), 3.82-3.96 (m, - 162 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, 15.34min. LC-MS (ES*) m/e=507.3 5 (MH*) 0 NN N O C1 H4 98f
(
2 -Cyclohexyloxy-5-oxo-tetrahydro-furan-3-yl) -carbamic 10 acid allyl ester. Prepared from 3 -allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using cyclohexanol to afford the title compound as a mixture of diastereomers (pale yellow oil) (4 .62g, 85% yield) . 15 Flash column chromatography using hexanes/EtOAc (90/10 to 80/20) gave 394 mg (7% yield) of the syn diastereomer of the title compound. 1H NMR (500MHz, CDC1 3 ) 6.1.11-2.09 (m, 10H), 2.35-2.61 (dd, 1H), 2.72 2.98 (dd, iH), 3.60-3.83 (m, 1H), 4.32-4.72 (m, 3H), 20 5.06-5.43 (m, 2H), 5.60 (d, lH), 5.82-6.03 (m, .1H). 1- [2- ( 4 -Acetylamino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-cyclohexyloxy-5-oxo tetrahydro-furan-3-yl)-amide (98f). 25 Prepared from syn- (2-cyclohexyloxy--oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester and 97b following the method used for 98a to give the title compound (121mg, 33% yield). 1 H-NMR (500MHz,
CD
3 0D) 6 1.06-1.61 r.. 9H), 1.61-2.37 (m, 7H), 2.22 (s, 3H), 2.52-2.81 (m, 3C 2HR , 1.45-3.78 (mr, 2H), 3.84-3.97 (m, 1H), 4.42-4.57 - 163 (m, IH), 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 9.84min. LC-MS (ES+) m/e=563.3 (MH*). 0 CI NO N 5 98g O 1- [2 - ( 4 -Amino-3 -chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid ( 2 -cyclohexyloxy-5-oxo tetrahydro-furan-3-yl) -amide (98g) 10 Prepared from syn- ( 2 -cyclohexyloxy-5-'xo-tetrahydro furan-3-yl)-carbamic acid allyl ester and 97a following the method used for 98a to give the title compound (153mg, 47% yield). 1 H-NMR (500MHz,
CD
3 0D) 5 1.06-2.38 (m, -14H), 1.42 (d, 3H), 2.50-2.66 (m, 1H), 2.69-2.82 15 (dd, 1H), 3.06-3.75 (m, 2H), 3.80-3.94 (m, 1H), 4.40 4.52 (m, 1H), 4.57-4.65 (m, iH), 4.70-4.80 (m, 1H), 5.72 (d, IH), 6.71 (m, 1H), 7.50-7.63 (m, 1H), 7.78 (d, 0.6H), 8.42 (d, 0.4H). Analytical HPLC 10.30min. LC-MS (ES*) m/e=521.2 (MH*). 20 N CI 98h I - 2 - (4 -Amino- 3 - chloro-benzoylamino) -propionyl] -vrrclidine-2-carboxy.lic acid (2-ethoxy-5-oxo tetrahydro- furan-3-yli -amide (98h).
- 164 Prepared from ( 2 -ethoxy-5-oxo-tetrahydro-furan-3-yl) carbamic acid allyl ester and 97a following the method used for 98a. The title compound was isolated as a white solid (195mg, 82% yield) . IH-NMR (500MHz,
CD
3 0D) 5 6 1.32-1.55 (m, 3H), 1.58-1.77 (m, 3H), 1.98-2.54 (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, 10 0.7H), 8.35 (d, 0.3H), 8.74 (d, 0.2H). Analytical HPLC 8.31min. LC-MS (ES*) m/e=467.3 (MH*). H N
H
2 N / 0 N C1 H 0 98i 15 [S-Oxo-2-(tricyclo[3.3.1.1*odec-2-ylory)-tetrahydro furan-3-yll-carbamic acid allyl ester. Prepared from 3 -allyloxycarbonylamino-4 -hydroxy-butyric acid tert-butyl ester as described for 40 using 2 adamantanol (6.21g, 5 equivalents) to afford the title 20 compound as a pale yellow oil (1.52g, 61% yield). 1H NMR (500MHz, CDCl 3 ).6.1.38-2.
2 2 (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), 3.84-3.97 (m, 1H), 4.40 4.71 (m, 3H), 5.18-5.44 (m, 2H), 5.53-5.69 (m, 1H), 25 5.82-6.02 (m, 1H). 1- [2- ( 4 -Amino-3-chloro-benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid [5-oxo-2- - 165 (tricYlo[3.3..l*'*)dec-2-yloxy) -tetrahydro-furan-3-yl] amide (981). Prepared from [S-oxo-2- (tricyclo [3 .3.1.10' 01dec-2 yloxy) -tetrahydro-furan-3-ylj-carbamic acid allyl ester 5 and 97a following the method used for 98a. The title compound was isolated as a white solid (76mg, 13% yield). 1 H-NMR (500MHz,
CD
3 0D) 6 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), 3.84-3.97 (in, 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). Analytical HPLC. 11.89min. LC-MS (ES*) m/e= 573.2 (MH*). O NJ H C 98j 15 1- [2 - ( 4 -Ace tylarmino -5S - chloro -2-me thoxy-benzoylamino) propionyll -pyrrolidine-2-carboxylic acid (2 -benzyloxy S-Oxo-tetrahydro-furan-3-yl) -amide (98j) . Prepared from syn-{2- [2- ( 2 -benzyloxy-5-oxo-tetrahydro 20 furan-3-ylcarbamoyl) -pyrrolidin-1-yl) -1-methyl-2-oxo ethyll-carbamic acid tert-butyl ester and 97c following the procedure used for 98a to afford. the title compound (222mg, 82% yield). 1 H-NMR (SOOMH2,
CD
3 OD) 6 1.23 (d, 0.6H), 1.42 (d, 2.4H), 1.72-2.27 (m, 4H), 2.23 (s, 3H), 25 2.63 (dd, IH), 2.7'1-2.88 (m, 1H), 3.43-3.52 (m, 0.5H), .56-3.71 (m, 1.5H), 3.74-3.85 (m, 1H), 3.98 (s, 3H), .. 3E-4.50 (W, 1.5H.), 4.51-4.92 (M, 4.5H), 5.63-5.76 (m, 7.23-7.40 ( m, 5H), 7.97 (s, 1H), 8.45 (d, 1H), - 166 8.69-8.80 .(m, 1H) Analytical HPLC 11.63min LC-MS (ES+) m/e= 601.2 (MH*) H2N O N C1 H 98k 5 Synthesis of 1-12- ( 4 -amino-3 -chloro -benzoylamino) propionyll -pyrrolidine-2-carboxylic acid (2-ethoxy-5 oxo-tetrahydro-furan-3 -yl) -amide (98k). Prepared from anti- (2-ethoxy-5-oxo-tetrahydro- furan-3 10 yl)-carbamic acid allyl ester and 97a following the method used for 98a to afford 175 mg of title compound (59%). 1 H-NMR (500 MHz, 1:1 CDCl 3
:CD
3 0D) 5 1.10-1.28 (m, 3H), 1.42 (d, 0.6H), 1.46 (d, 2.4H), 1.75-2.45 (m, 4H), 2.45-2.70 (m, 1H), 2.80-3.05 (m, 1H), 3.50-3.95 (n, 15 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, IH), 7.55-7.84 (m, 2H). Analytical HPLC: 10.47 min. LC-MS
(ES
4 ): m/e = 467.3 (M+H*). C N
H
2 N N 20 981 Synthesis of 1- 12 - ( 4 -amino - 3, 5-dichloro-benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (2-ethoxy-S oxo- tetrahydro- furan-3-yl) -amide (981). -~Prepared from (2-ethoxy--oxo-tetrahydro-furan-3-yl) carbamic acid ally'L ester and l-( 2 -(4-amino-3, 5 dichloro-benzoylamino) -propionyl] -pyrrolidine-2
-
- 167 carboxylic acid tert-butyl ester according to the method used for 98a to afford 158 mg of title compound (54% yield) . 'H-NMR (500 MHz, 1:1 CDC1 3
:CD
3 0D) 5 1.08 1.30 (m, 3H), 1.32-1.52 (m, 3H), 1.72-2.44 (m, 4H), 5 2.40-3.05 (m, 2H), 3.50-3.97 (m, 4H), 4.25-4.70 (m, 3H), 4.70-4.86 (m, IH), 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-MS (ES*): m/e = 501.2 (M+H*). 10 0 N ND H2N C08O CI 98m 1- (2- ( 4 -amino-3 -chloro-benzoylamino) -propionyl) pyrrolidine-2-carborylic acid (2-benzyloxy--oxo 15 tetrahydro- furan-3 -yl) -amide (98m). Prepared according to the procedure used to prepare 98a using Cbz-Ala-D-pro-OH to afford 230 mg of title compound (69% yield). 1 H-NMR (-500 MHz, 1:1 CDC1 3
:CD
3 0D) 5 1.30 (d, 1.2H), 1.45 (d, 1.8H), 1.62-2.40 (m, 4H), 20 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, I ), 7.25-7.95 (m, 7H). Analytical HPLC: 11.56, 11.91 min. LC-MS (ES') Tn/e = 529.2 (M+H*). N N OiN H ' 'H ~~ Cl 98nH - 168 1- [2- ( 4 -acetylamino-3-chloro-benzoylonino) -propionyl]. pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide (98n). Prepared from 97b and syn- (2-benzyloxy-5-oxo 5 tetrahydro-furan-3-yl)-carbamic acid allyl according to the procedure used to prepare 98a to afford 220 mg of title compound (64% yield). IH-NMR (500 MHz, 1:1 CDCl 3
:CD
3 0D) 6 1.33 (d, 0.GH), 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, 10 2H), 4.20-4.95 (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
4 ): m/e = 571.1 (M+H*). 0 O N N O HH H 2 N 98o 15 (2-Isopropoxy-5-oxo-tetrahydro-furan- 3 -yl)-carbamic acid allyl ester Prepared as described for compound 40 using isopropanol to afford 3.80 grams (81% yield) of the title compound 20 as a colorless oil. IH-NMR (500 MHz, CDCl 3 ) 5 1.10-1.35 (m, 6H), 2.32-2.60 (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, IH). LC-MS
(ES
4 ): m/e = 244.2 (M+H+). 25 1- 12- ( 4 -amino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid ( 2 -isopropoxy-S-oxo tetrahydro-furan-3-yl)-amide (98o). Prepared from 97a and (2-isopropoxy-5-oxo-tetrahydro ;- rar-2-,.lI-carbamic acid allyl ester according to the - 169 procedure used to prepare 98a to afford 200 mg of title compound (66% yield) . 'H-NMR (500 MHz, 1:1 CDC1 3
:CD
3 0D) 5 1.05-1.35 (m, 6H), 1.35-1.50 (m, 3H), 1.70-2.45 (m, 4H), 2.45-3.05 (m, 2H), 3.55-4.10 (m, 3H), 4.15-4.88 5 (m, 4H), 5.48 (s, 0.4H), 5.58 (s, 0.1 H), 5.64 (d, 0.4H), 5.70 (d, 0.1H), 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*). C N H CI 9 8P H 10 1- [2 - ( 4 -acetylamino-3, S-dichloro-benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (2-benzyloxy 5-oxo-tetrahydro-.furan-3-yl)-amide
(
9 8 p). 15 Prepared from 1- [2- ( 4 -acetylamino-3, 5-dichloro benzoylamino) -- propionyl] -pyrrolidine-2- carboxylic acid tert-butyl ester and syn- ( 2 -benzyloxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 230 mg of title 20 compound (72% yield). 1 H-NMR (500 MHz, 1:1 CDCl 3
:CD
3 0D) 8 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, 7R). Analytical HPLC: 13.07 min. LC-MS 25 (ES*) : m/e = 605.1 (M+H*) N N H C 1 98qH 0 -1 - 170 1-12- (4-acetylamino-3-chloro-benzoylamino) -propionyll pyrrolidine-2-carboxylic acid (2-cyclopentyloxy-5-oxo tetrahydro-furan-3-yl)-amide (98q). 5 Prepared from 97b and (2-cyclopentyloxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 215 mg of title compound (69% yield) .
1 H-NMR (500 MHz, 1:1 CDC1 3
:CD
3 0D) 6 1.35-1.90 (m, 11H), 1.90 10 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, 0.55H), 5.56 (s, 0.15H), 5.64 (d, 0.22H), 5.71 (d, 0.08H), 7.70-8.25 (m, 3H). Analytical HPLC: 12.13 min. LC-MS (ES*) m/e = 549.2 (M+H*). 15 N H C 98r H Synthesis of 1-[2-(4-acetylamino-3-chloro benzoylamino)-propionyl]-pyrrolidine-2-carboxylic acid 20 ( 2 -ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (98r). Prepared from 97b and syn- ( 2 -ethoxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 68 mg of title compound (24%). 1 H-NMR (500 MHz, 1:1 CDC1 3 :CD30D) 8 1.13 25 (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), 3.50-3.92 (m, 4H), 4.40-4.90 (m, 3H), 5.57 (d, 0.8R), 5.61 (d, 0.2H), 7.60-8.20 (m, 3H). Analytical HPLC: 8.64 mir. LC-MS (ES*): m/e = 509.2 (M+H*).
- 171 N N
H
2 N C CI 98S H4 Preparation of of ( 2 -cyclopentylmethoxy-5-oxo 5 tetrahydro-furan-3-yl)-carbamic acid allyl ester. Prepared from 3 -allyloxycarbonylamino-4 -hydroxy-butyric acid tert-butyl ester as described for compound 40 using cyclopentylmethanol (6.5 mL, 60 mmol) to afford 2.98 grams (52% total yield) of the title compound as a 10 mixture of epimers. Purification provided 0.97 grams (17% yield) of the 4(S), 5(R) as a colorless oil. 1H NMR (500 MHz, CDCl 3 ) 6 1.19 (m, 2H) , 1.54 (m, 4H) , 1.71 (m,. 2H), 2.16 (m, 1H), 2.44 (dd, J=17.
2 , 10.4Hz, 1H), 2.82 (dd, J=17.2, 8.4Hz, 1H), 3.44 (dd, J=9.3, 7.2Hz, 15 1H), 3.71 (dd, J=9.3, 7.2Hz, 1H), 4.57 (m, 3H), 5.32 (m, 3H), 5.41 (d, J=5.2Hz, 1H), 5.91 (ddt, J=17.1, 10.4, 5Hz, 1H) ppm. LC-MS. (ES+): m/e=284. Also isolated was epimer mixture (0.66 grain, 11% yield) 20 and the 4 (S) , 5 (S) epimer (1.35 gram, 24% yield) as a waxy solid. 1 H-NMR (500MHz, CDCl 3 ) 8 1.20 (m, 2H), 1.54 (n, 4H), 1.69 (m, 2H), 2.10 (m, 1H), 2.37 (d, J=8.lHz, 1H), 2.97 (dd, J=18.0, 7.6Hz, 1H), 3.42 (dd, J=7.3, 1.7Hz, 1H), 3.49 (m, 2H), 3.64 (dd, J=9.0, 7.3Hz, 1H), 25 4.19 (br, 1H), 4.55 (n, 2H), 5.25 (n, 2H), 5.36 (s, 1H) , 5. 87 *(m, 1H) ppm. LC-MS (ES+) : m/e=284 (M+H) . 1- [2- ( 4 -amino- 3 -chloro-benzoylaino) -propionyll pyrrolidine-2-carboxylic acid
(
2 -cyclopentylmethoxy-5 oxo-tetrahydro-furan-3-yl)-amide (98s).
- 172 Prepared from 97a and syn- ( 2 -cyclopentylmethoxy-5-oxo tetrahydro-furan-3-yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to afford 195 mg of title compound (51% yield) . 'H-NMR 5 (500 MHz, 1:1 CDCl 3
:CD
3 0D) 6 1.15-1.90 (m, 11H), 1.90 2.40 (m, 5H), 2.55-2.78 (m, 2H), 3.50-3.90 (m, 4H), 4.38-4.92 (m, 3H), 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*). 10 0 N
H
2 N 98t (5-oxo-2- (3-phenyl-propoxy) -tetrahydro- furan-3 -yl) carbamic acid allyl ester. 15 Prepared from 3 -allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for compound 40 using 3 -phenylpropanol to afford 1.15 grams (32% yield) of the title compound as a colorless oil. 1 H-NMR (500 MHz, CDCl 3 ) 6 1.82-2.05 (m, 2E), 2.38 (dd, 1H), 2.68 (m, 20 2H), 2.82 (dd, 1H), 3.55-3.65 ( I, 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), 7.11-7.45 (m, 5H). Analytical HPLC: 9.08 min. LC-MS (ES*) : m/e = 320.2 (M+H*) . 25 1- [2- ( 4 -amino-3-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-2-
(
3 -phenyl-propoxyl) t e trahdro- uran -3.-1) -carbamic acid allyl ester according to the procedure used to prepare 98a to afford 200 ma of title compound (57% yield) .
1 Ii-NMR - 173 (500 MHz, 1:1 CDCl 3
:CD
3 0D) 8 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 (n, 3H), 5.52 (d, 0.8H), 5.56 (d, 0.2H), 6.75-8.25 (m, 8H). Analytical HPLC: 10.79 min. 5 LC-MS (ES*) : m/e = 557.2 (M+H*) 0 -11N
-
O N H H 0 Synthesis of 1- [2-( 4 -acetylamaino-3-chloro benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid 10 (2-cyclopentylmethoxy-5-oxo-tetrahydro-furan-3-yl) amide (98u). Prepared from 97b and syn-(2-cyclopentylmethoxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to 15 afford 215 mg of title compound (67% yield) .
1 H-NMR (500 MHz, 1:1 CDC1 3 :CD3OD) 6 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, 20 3H). Analytical HPLC: 9.90 min. LC-MS (ES+): m/e = 563.3 (M+H*). N C1 98v 25 1- [2- ( 4 -acetylamino -3-chloro-benzoylamino) -propionyll pyrrolidine-2-carboxylic acid (5-oxo-2- (3-phenyl propoxyl) -tetrahydro- furan-3 -yl) - amide (98v).
- 174 Prepared from 1- (2- (4-acetylamino-3-chloro benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and syn-(5-oxo-2-(3-phenyl-propoxyl) tetrahydro-furan-3-yl)-carbamic acid allyl ester 5 according to the procedure used to prepare 98a to afford 238 mg of title compound (75% yield) .
1 H-NMR (500 MHz, 1:1 CDC1 3
:CD
3 0D) 8 1.33 (d, 0.6H), 1.56 (d, 2.4H), 1.78-2.45 (m, 6H), 2.27 (s, 3H), 2.53-2.97 (m, 4H), 3.53-3.94 (m, 4H), 4.47-4.86 (m, 3H), 5.53 (d, 10 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*). 0 O N
CF
3 98w H 15 1- [2- ( 4 -aino-3-trifluoromethyl-benzoylam-ino) propionyll -pyrrolidine-2- carboxylic acid (2-benzyloxy 5-oxo-tetrahydro-furan-3-yl) -amide (98w). Prepared from {2- [2- ( 2 -benzyloxy-5-oxo-tetrahydro furan-3-ylcarbmoyl) -pyrrolidin-1-yl) -l-methyl-2-oxo 20 ethyl}-carbamic acid tert-butyl ester and 4-amino-3 trifluoromethyl-benzoic acid according to the procedure used to prepare 98a to afford 56 mg of title compound (48% yield). 1 H-NMR (500 MHz, 1:1 CDCl 3 :CD30D) 8 1.20 1.55 (m, 3H), 1.75-2.50 (m, 4H), 2.50-3.10 (m, 2H), 25 3.50-4.00 (m, 2H), 4.30-5.00 (m, SH), 5.42 (s, 0.4H), 5.51 (s, 0.2H), 5.62 (d, 0.3H),-5.78 (d, 0.1H), 6.84 (d, 1H), 7.20-8.15 (m, 7H). Analytical HPLC: 14.90, 15.20 min. LC-MS (ES*): m/e = 563.2 (M+H*).
- 175 N N \0 O N4 CI ~98x 1- [2- (3-chloro-4-dimethylamino-benzoylamino) propionyl] -pyrrolidine -2 -carboxylic acid (2 -benzyloxy 5 5-oxo-tetrahydro-furan-3-yl) -amide (98x) . Prepared from {2-[2-(2-benzyloxy-5-oxo-tetrahydro furan-3-ylcarbmoyl) -pyrrolidin-1-yl -1-methyl-2-oxo ethyl)-carbamic acid tert-butyl ester and 3-chloro-4 dimethylamino-benzoic acid according to the procedure 10 used to prepare 98a to afford 82 mg of title compound (44% yield) . 3H-NMR (500 MHz, 1:1 CDCl 3
:CD
3 0D) 5 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, SH), 5.46 (s, 0.3H), 5.51 (s, 0.2H), 5.63 (d, 0.4H), 5.73 15 (d, 0.1H), 7.05 (d, 1H), 7.15-7.95 (m, 7H). Analytical HPLC: 11.85, 12.19 min. LC-MS (ES*): m/e = 557.3 (M+H*). 0 20 1- [2- (4-dimaethylamino-3, 5-difluoro-benzoylamino) propionyl] -pyrrolidine -2 - carboxylic acid (2 -benzyloxy 5-oxo-tetrahydro-furan-3-yl) -amide ( 9 8y). Prepared from {2- [2- ( 2 -benzyloxy-5-oxo-tetrahydro 25 furan-3-ylcarbmoyl) -pyrrolidin-1-yl -1-methyl-2-oxo ethyl}-carbamic acid tert-butyl ester and 4 dimethylamino-3,5-dichloro-benzoic acid according to - 176 the procedure used to prepare 98a to afford 106 mg of title compound (65% yield) . 'H-NMR (500 MHz, 1:1 CDCl 3 :CD30D) 5 1.10-1.55 (m, 3H), 1.75-2.30 (m, 4H) , 2.45-3.15 (m, 2H), 2.84 (s, 6H), 3.40-3.95 (m, 2H), 5 4.15-4.95 (m, 5H), 5.47 (s, 0.35H), 5.54 (s, 0.15H), 5.67 (d, 0.4H), 5.77 (d, 0.1H), 7.20-7.70 (m, 7H). Analytical HPLC: 12.21, 12.51 min. LC-MS (ES 4 ): m/e = 559.2 (M+H*). F F N 10 2 N F98Z 1- [2- ( 4 -amino-2,3,5, 6-tetrafluoro-benzoylamino) propionyll -pyrrolidine-2-carboxylic acid (2-benzyloxy 5-oxo-tetrahydro-furan-3-yl)-amide (98z). 15 Prepared from {2- [2- (2-benzyloxy-5-oxo-tetrahydro-. furan-3-ylcarbmoyl) -pyrrolidin-1-yl) -1-methyl-2-oxo ethyl)-carbamic acid tert-butyl ester and 4-amino 2,3,5,6-tetrafluoro-benzoic acid according to the procedure used to prepare 98a to afford 58 mg of title 20 compound (73% yield). 1 H-NMR (500 MHZ, 1:1 CDCl 3
:CD
3 0D) 5 1.30-1.SO (m, 3H), 1.62-2.35 (m, 4H), 2.45-3.12 (m, 2H), 3.50-3.90 (m, 2H), 4.20-4.95. (m, 5H), 5.42 (s, 0.4H), 5.52 (s, 0.1H),-5.64 (d, 0.4H), 5.82 (d, 0.1H), 7.25-7.65 (m, 5H). Analytical HPLC: 16.56, 16.90 min. 25 LC-MS (ES*): m/e 567.2 (M+H*). O N - N C1 98aa O OJ9: - 177 1-{2- [3-chloro-4- (2,2-dimethylpropionylamino) benzoylamino] -propionyl}-pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-amide (98aa). To a suspension of 98b (100 mg, 0.19 mmol) and poly(4 5 vinylpyridine) (200 mg) was added pivaloyl chloride (70pL, 0.57 mmol). The resulting suspension was stirred overnight at room temperature the filtered and diluted with EtOAc (25 mL). The organic layer was washed with 10% NaHCO3 (2 x 25 mL) , saturated NaCl (1 x 25 mL), 10 dried (MgSO4), and evaporated to dryness to afford 98 mg of title compound (85% yield) after chromatography. 1 H-NMR (500 MHz, 1:1 CDC1 3
:CD
3 0D) 6 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 (m, 5H), 5.62 (d, 0.8H), 15 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*). 0 N O0 N H C1 98ab H N 20 1- [2- (3-chloro-4-propionylamino) -benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid (2-benzyloxy 5-oxo-tetrahydro-furan-3-yl) -amide (98ab). Prepared from 98b and propionyl chloride according to the procedure used to prepare 98aa to afford 104 mg of 25 title compound (95% yield). 1 H-NMR (500 MHz, 1:1 CDCli: CD 3 0D) 6 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, 2W), 2.58-3.05 (m, 2H), 3.45-3.85 (m, 2H), 4.20-4.88 n, 5H), 5.64 (d, 0.8H), 5.76 (d, 0.2H), 7.20-8.35 (m, S: S. Analytical HPLC: 9.89 min. LC-MS (ES*): m/e = - 178 C N H C1 98ac HZ 1- (2- (3-chloro-4-phenylacetylamino) -benzoylamino) 5 propionyl) -pyrrolidine-2-carboxylic acid (2-benzyloxy 5-oxo-tetrahydro-furan-3-yl) -amide (98ac). Prepared from 98b and phenylacetyl chloride according to the procedure used to prepare 98aa to afford 85 mg of title compound (771 yield) .
1 H-NMR (500 MHz, 1:1 10 CDC1 3 : CD 3 0D) 5 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*). 15 0 4N CI 98ad 1- [2- (3-chloro-4-methyl-butyrylamino) -benzoylanino) propionyl] -pyrrolidine-2 -carboxylic acid (2-benzyloxy 20 5-oxo-tetrahydro-furan-3-yl) -amide (98ad) . Prepared from 98b and isovaleryl chloride according to the procedure used to prepare 98aa to afford 60 mg of title compound (58% yield). 1 H-NMR (500 MHz, 1:1 CDCl 3
:CD
3 0D) 8 1.07 (d, 5H), 1.15 (d, 0.8H), 1.27 (d, 25 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, 0.3H), 7.18-8.33 (m, 8H).
- 179 Analytical HPLC: 10.74 min. LC-MS (ES*) m/e = 613.2 (M+H*) . H00 O N 98ae H 5 1- (2- (4-Methoxy-3, 5-dimethyl-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo tetrahydro-furan-3-yl)-amide (98ae). Prepared from 1- [2- (4-methoxy-3,5-dimethyl 10 benzoylamino) -propionyl) -pyrrolidine-2-carboxylic acid tert-butyl ester and syn- (2-ethoxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 174 mg (81% yield) of the title compound. 1 H-NMR (500 MHz, CDCl 3 ): 15 5 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), 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; 20 LC-MS: m/z = 476 (M+H*). O H -U N N HwH 0 98af H 1- [2- (4-Methoxy-3,5-dimethyl-benzoylamino) -propionyl] 25 pyrrolidine-2-carboxylic acid (2-ethoxy-S-oxo tetrahydro-furan-3-yl) -amide (98af). Prepared from 1- [2- (4-methoxy-3, 5-dimethyl benzovlaminc) -propionyl] -pyrrolidine-2-carboxylic acid - 180 tert-butyl ester and anti- ( 2 -ethoxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 168 mg (77% yield) of the title compound. 1 H-NMR (500 MHz, CDC1 3 ) 5 5 1.10-1.35 (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, 2H), 7.92 (d, H); retention time on analytical HPLC: 10.14 min; LC-MS: m/z = 476 (M+H*) . 10 0 N N 00 98ag 1- [2- (4 -Methoxy-3, 5-dimethyl-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid ( 2 -benzyloxy-5-oxo 15 tetrahydro-furan-3-yl) -amide (98ag) . Prepared from 1- [2- ( 4 -methoxy-3,S-dimethyl benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and syn- ( 2 -benzyloxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester (40) according to 20 the procedure used to prepare 98a to afford 406 mg (yield 71%) of the title compound. 1 H-NMR (500 MHz, CDC1 3 ): 5 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 25 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 (s, 2H? ; retention time on analytical HPLC: 16.71 min'; LC MS: m/z = 538 (M+H*) - 181 O N 9 NO 0 98ah 1- [2- (4-Allyloxy-3, 5-dimethyl-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo 5 tetrahydro-furan-3-yl) -amide (98ah). Prepared from 1- [2- (.
4 -allyloxy-3,5-dimethyl benzoylamino) -propionyl] -pyrrolidine-2-carboxylic acid tert-butyl ester and 40 according to the procedure used to prepare 98a to afford 264 mg (yield 46%) of the 10 title compound. 1 H-NMR (500 MHz, CDCl 3 ): 5 1.09-1.43 (m, 3H), 1.90-2.20 (m, 3H), 2.20-2.38 (m, 7H), 2.38 2.52 (m, H), 2.80-2.95 (m, H), 3.52-3.67 (m, H), 3.70 3.80 (m, H), 4.10-4.40 (m, 2H), 4.40-4.95 (m, 5H), 5.26-5.55 (m, 3H), 6.00-6.14 (m, H), 6.87 (d, H), 7.10 15 7.70 (m, 8H); retention time on analytical HPLC: 18.56 and 18.92 minI; LC-MS: m/z = 564 (M+H*) 98ai 20 (2- [1R- ( 2 S-Isopropyl-5R-methyl-cyclohexyloxy) -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 (1R, 2S, 5R)-(-)-menthol to afford 0.32 g of syn 25 diastereomer (lower Rf) of the title compound and 4.25 - 182 g of the mixture of anti/syn diastereomers (overall yield 67%). 1 H-NMR (500 MHz, CDCl 3 ) mixture: 8 0.70 1.05 (mn, 13H), 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), 5 3.40-3.61 (m, H), 4.43-4.70 (m, 3H), 5.15-5.35 (m, 2H), 5.48-5.61 (m, H), 5.90-5.94 (m, H); for syn diastereomer 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), 10 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. 15 Prepared by the method used to synthesize 4-allyloxy 3,5-dimethyl-benzoic acid to afford 2.43 g (yield 56%) of the title compound. 1 H-NMR (500 MHz, CDCl 3 ): 6 4.87 (s, 2H), 7.36-7.48 (m, 5H), 7.92 (s, 2H); LC-MS: M/z = 255 (M-H*). 20 1- [2- (4 -Benzyloxy- 3, 5-dimethyl-benzoylamino) propionyll-pyrrolidine-2-carboxylic acid (2-[1R-(2S isopropyl-SR-methyl-cyclohexyloxy) I -S-oxo-tetrahydro furan-3-yl)-amide (98ai). 25 Prepared from 1-[2-(4-benzyloxy-3,5-dimethyl benzoylamino) -propionyll -pyrrolidine-2-carboxylic acid tert-butyl ester {2-[1R-(2S-Isopropyl-5R-methy1 cyclohexyloxy)]-S-oxo-tetrahydro-furan-3-yl}-carbamic acid allyl ester according to the procedure used to 30 prepare 98a to afford 130 mg (yield 39%) of the title compound. 'H-NMR (500 MHz, CDCl 3 ): 8 0.45-1.10 (m, 12H), 1.15-1.90 (m, 8H), 1.90-2.45 (m, 12H), 2.80-2.84 Cm, 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- - 183 7.60 (m, 7H) , 7.62-7.75 (n, H); retention time on analytical HPLC: 15.90 and 16.08 min; LC-MS: m/z = 662 (M+H*) . N- N HO / O0 o N 9 8aj 5 O 1- [2- ( 4 -Hydroxy-3,5-dimethyl -benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2- [1R- (2S-isopropyl-5R methyl-cyclohexyloxy) I -5-oxo-tetrahydro-furan-3-yl) 10 aide (98aj). A solution of 1- [2- (4-benzyloxy-3,5-dimethyl benzoylamino) -propionyl] -pyrrolidine-2 -carboxylic acid (2- [1R- (2S-isopropyl-5R-methyl-cyclohexyloxy) I -5-oxo tetrahydro-furan-3-yl}-amide (110 mg, 0.17 mmol) in 15 ethyl acetate (2 mL) was stirred with 10% palladium on carbon (20 mg) under hydrogen atmosphere for 24 hours then filtered through Celite and evaporated in vacuo. The resulting residue was purified by chromatography using CH 2 C1 2 /methanol (99/1 to 96/4) to afford 58 mg of 20 the title compound. IH-NMR (500 MHz, CDC1 3 ): 5 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 (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 25 analytical HPLC: 12.84 and 13.05 min; LC-MS: M/z = 572
(M+H*).
- 184 0 N N HOO 98ak 1- [2- ( 4 -Hydroxy-3,5-dimethyl - benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo 5 tetrahydro-furan-3-yl)-amide (98ak). A solution of 98ah (230 mg, 0.41 mmol) in CH 2 C1 2 (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 10 purified by flash chromatography using CH 2 Cl 2 /methanol (99.5/0.5 to 97/3) to afford 181 mg of the title compound. 1 H-NMR (500 MHz, CDCl 3 ): 8 1.08 (d, 0. 75H) , 1.20-1.35 (m, 2.25H), 1.70-2.50 (m, 12H), 2.80-2.90 (m, H), 3.50-3.65 (m, H), 3.70-3.80 (m, H), 4.10-4.25 (m, 15 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 (n, 8H); retention time on analytical HPLC: 10.38 and 10.56 min; LC-MS: m/z = 524 (M+H*). NN 98al 20 1- [2- (4 -Dimethylamino-benzoylamino) -propionyl] pyrrolidine 2- carboxylic acid (2 -benzyloxy-5 -oxo 25 tetrahydro-furan -3 -yl) -aide (98al) . Prepared from 1- [2- (4 -dimethylamino-benzoylamino) propionyl] -pyrrolidine-2-carboxylic acid tert-butyl - 185 ester and syn- (2-benzyloxy-5-oxo-tetrahydro-furan-3 yl) -carbamic acid allyl ester according to the procedure used to prepare 98a to afford 60 mg (45% yield)
.
1 H-NMR (500 MHz, CDC1 3 ) : 6 1. 04 (d, 0. 75H) , 5 1.35 (d, 2.25H), 1.80-2.50 (m, SH), 2.75-3.20 (i, 8H), 3.45-3.75 (m, 2H), 4.05-4.20 (m, 0.5H), 4.30-4.80 (M, 3.5H), 4.80-4.95 (m, 1.5H), 5.52 (d, H), 5.75-6.00 (m, 0.SH), 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; 10 LC-MS: m/z = 523 (M+H*). 0 - N H2N / O Cl 98am 1- [2- (4-Amino-3 -chloro-benzoylamino) -propionyl] 15 pyrrolidine-2-carboxylic acid {2R-[lR-(2S-isopropy-5R methyl-cyclohexyloxy)] -5-oxo-tetrahydro-furan-3-yl} amide (98am). Prepared from 1- [2- (4 -amino-3 -chloro-benzoyl amino) propionylJ -pyrrolidine-2 -carboxylic acid tert -butyl 20 ester (97a) and syn-{2-[1R-(2S-isopropyl-5R-methyl cyclohexyloxy)]-S-oxo-tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 103 mg (yield 67%) of the title compound. H-NMR (500 MHz, CDC1 3 ): 5 0.70-1.10 (m, 25 12H), 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, H), 7.15 (d, H), 7.49- - 186 7.51 (m, H), 7.73 (s, H); retention time on analytical HPLC: 12.88 min; LC-MS: m/z = 577 (M+H*). N N H2N /0O 0N CI 98an H 5 {2- [ IS- ( 2 R- Isopropyl-5S-me thyl-cyclohexyloxy) 5 -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 (IS, 10 2R, SS)-(+)-menthol to afford 855 mg of anti diastereomer (higher Rf) of the title compound, 503 mg of syn diastereomer (lower Rf) and 459 mg of the mixture of anti/syn diastereomers (overall yield 66%). 1H-NMR (500 MHz, CDC1 3 ) anti diastereomer: 6 0.74-1.00 15 (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), 20 1.57-1.70 (m, 2H), 2.00-2.16 (m, 2H), 2.40-2.52 (m, H), 2.78-2.90 (m, H), 3.40-3.50 (m, H), 4.58 (br, 2H), 5.24-5.35 (m, 2H), 5.51-5.52 (d, H), 5.85-5.98 (m, H); AC-MS: m/z = 340 (M+H*) for both of diastereomers. 1- [2- ( 4 -Amino-3-chloro.-benzoylamino) -propionyl] pyrrolidine-2.-carboxylic acid {2R - [IS- ( 2 R-isopropy1- -SS methyl-cyclohexyloxy) I -S-oxo-tetrahydro.-furan-3-yl) amide (98an).
- 187 Prepared from 97a and syn-{2-[1S-(2R-isopropyl-5S methyl-cyclohexyloxy)]-5-oxo-tetrahydro-furan-3-yl carbamic acid allyl ester according to the procedure used to prepare 98a to afford 88 mg (50% yield) of the 5 title compound. 1 H-NMR (500 MHz, CDCl 3 ) : 0 0.70-1.10 (m, 12H), 1.20-1.50 (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), 4.88-4.92 (m, H), 5.53 (d, H), 6.71 10 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-MS: m/z = 577 (M+H*). 0 O N N AN H O C1 98ao 13
(
2 -Cyclohexylzmethoxy-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 20 cyclohexylmethanol to afford 1.04 g (higher RF) (35% yield) of anti diastereomer of the title compound and 1.295 g (lower Rf) (44% yield) of syn diastereomer. 1
H
NMR (500 MHz, CDCl 3 ) for anti diastereomer: 6 0.90-0.96 (m, 2H), 1.10-1.30 (m, 3H), 1.55-1.85 (m, 6H), 2.37 25 2.41 (d, H), 2.97-3.03 (m, H), 3.34-3.38 (m, R), 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), 30 3.35-3.39 (m, H), 3.63-3.67 (m, H), 4.53-4.70 (m, 3H), - 188 5.20-5.50 (m, 3H), 5.89-5.95 (m, H); LC-MS: M/z = 298 (M+H*) for both of diastereomers. 1- (2- ( 4 -Acetylamino-3-chloro-benzoylamino) -propionyl) 5 pyrrolidine-2-carboxylic acid (2-cyclohexylmethoxy-5 oxo-tetrahydro-furan-3-yl)-amide (98ao). Prepared from 97b and syn-( 2 -cyclohexylmethoxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester -according to the procedure used to prepare 98a to 10 afford 212 mg (64% yield) of the title compound. 1
H
NMR (500 MHz, CDCl 3 ): 5 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), 15 7.60-7.90 (m, 3H), 8.08 (d, 0.15H), 8.SO (d, 0.85H); retention time on analytical HPLC: 11.81 min; LC-MS: m/z = 577 (M+H*) . 0 N N H C1 98ap H 20 1- [2- ( 4 -Acetylamino-3-chloro-benzoylAiflo) -propionyl) pyrrolidine-2-carboxylic acid {2R- [IS- (2R-isopropyl-5sS methyl-cyclohexyloxy)] -S-oxo-tetrahydro-furan -3 -y} amide (98ap). 25 Prepared from 97b and syn-{2-[1S- (2R-isopropy1-5S methyl-cyclohexyloxy)) -5-oxo-tetrahydro-furan-3 -y1} carbamic acid allyl ester according to the procedure used to prepare 98a to afford 223 mg (63% yield) of the title compound. 1 H-NMR (500 MHz, CDC1 3 ): 6 0.70-1.15 - 189 (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 5 on analytical HPLC: 13.31 min; LC-MS: m/z = 619 (M+H*). N
H
2 N 0r H { Cl 98aq H O 1- [2- (4 -Amino-3-chloro-benzoylamino) -propionyl] 10 pyrrolidine 2- carboxylic acid (2- cyclohexylmethoxy-5.
oxo-tetrahydro-furan-3-yl) -amide (98ag) . Prepared from 97a and syn- (2-cyclohexylmethoxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to 15 afford 113 mg (56% yield) of the title compound. 1
H
NMR (500 MHz, CDCl 3 ): 5 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), 20 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*) .
H
2 N K N 98ar
(
2 -Butoxy-5-oxo-tetrahydro-furan-3-yl)-carbamic acid allyl ester.
- 190 Prepared from 3-allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using n butanol to afford 878 mg (29% yield) of the title compound (313 mg of anti diastereomer, 260 mg of syn 5 diastereomer and 305 mg of the mixture)
.
1 H-NMR (500 MHz, CDCl 3 ) for anti diastereomer: 6 (higher 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 (br, 2H), 5.03 10 (br, H), 5.23-5.40 (m, 3H), 5.87-5.93 (M, H), for syn diastereomer (lower Rf) 0.91-0.95 (t, 3H), 1.34-1.39 (m, 2H), 1.56-1.63 (m, 2H ), 2.42-2.50 (m, HK), 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), 15 5.80-5.95 (m, H); LC-MS: m/z = 258 (M+H+) for both of diastereomers. 1- [2- ( 4 -Amino 3 - chloro-benzoylamino) -propionyl pyrrolidine-2-carboxylic acid (2 -butoxy-5S-oxo 20 tetrahydro- furan-3-yl)-amide (98ar). Prepared from 97a and syn-( 2 -butoxy-5-oxo-tetrahydro furan-3-yl).-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 118 mg (48% yield) of the title compound as a syn diastereomer. 25 H-NMR (500 MHZ, CDC1 3 ): 5 0.80-1.02 (m, 2H), 1.35-1-.51 (m, SH), 1.51-1.70 (m, 2H), 1.90-2.27 (m, 3H), 2.30 2.46 (m, H), 2.80-2.90 (m, R), 3.55-3.94 (m, 4H), 4.30 4.75 (m, 4H), 4.90-5.~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 30 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).
- 191 N N
H
2 N / O Cl 98as O O
(
2 -Isobutoxy-5-oxo-tetrahydro-furan-3-yl) -carbamic acid allyl ester. 5 Prepared from 3 -allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester as described for 40 using isobutanol to afford 190 mg (yield 7.3%) of the title compound as an anti diastereomer and 290 mg (yield 11%) of the syn diastereomer. 1 H-NMR (500 MHz, CDC1 3 ) for 10 anti diastereomer: 5 (higher Rf) 0.85-1.05 (m, 6H), 1.82-1.98 (m, H), 2.37-2.42 (d, H), 2.98-3.04 (m,-H), 3.31-3.35 (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 15 (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 (m, H), 3.87-3.88 (d, H), 4.40-4.68 (m, 3H), 5.20-5.40 (m, 2H), 5.42-5.43 (d, H), 5.80-5.97 (m, H); LC-MS: m/z = 258 (M+H*) for both of diastereomers. 20 1- 12- ( 4 -Aamino-3-chloro-benzoylamino) -propionyl pyrrolidine-2-carboxylic acid (2-isobutoxy-5-oxo tetrahydro-furan-3 -yl) -amide (98as). Prepared from 97a and syn- ( 2 -isobutoxy-5-oxo 25 tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 93 mg (38% yield) of the title compound. 1 H-NMR (500 MHz, CDCl 3 ): 8 0.74-0.76 (t, 0.6H), 0.80-1.00 (m, 5.4H), 1.40-1.50 (m, 3H), 1.90-2.22 (m, 3H), 2.33-2.45 30 (m, H), 2.80-2.90 (m, H), 3.32-3.38 (in, H), 3.55-3.80 - 192 (m, 3H), 4.38 (br, H), 4.50-4.60 (m, H), 4.70-4.80 (m, H), 4.90-5.00 (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), 7.51-7.53 (m, H), 7.74-7.75 (d, H); retention time on analytical 5 HPLC: 9.63 & 9.80 min; LC-MS: m/z = 495 (M+H*). N N H2N / O 98at (2-(indan-2-y1)oxy-5-oxo-tetrahydro-furan-3-yl] 10 carbamic acid allyl ester. Prepared from 3 -allyloxycarbonylamino-4 -hydroxy-butyric acid tert-butyl ester (5.2 gram, 20 mmol) as described for 40 using 2-indanol (8.05 gram, 60 mmol) to afford 4.10 grams (65% yield) of the title compound as a is mixture of epimers. Purification provided 1.76 gram (28* yield ) of the 4 (S) , 5 (R) epimer as a yellow oil. H NMR (500 MHz, CDC1 3 ) 5 2.42 (dd, J=17.2, 10.5Hz, 1H), 2.79 (dd, J=17.2, 8.4Hz, 1H), 2.99 (dd, J=16.7, 4.1Hz, 1H), 3.04 (dd, J=16.7, 4.1Hz, 1H), 3.22 (dd, J=17.2, 20 6.6Hz, 1H), 3.26 (dd, J=17.2, 6.6Hz, 1H), 4.53 (m, 3H), 4.70 (m, 1H), 5.20 (m, 2H), 5.60 (d, J=5.3Hz, 1H), 5.87 (m, 1H), 7.17 (m, 4H) ppm. LC-MS (ES+): m/e=318 (M+H). Analytical HPLC (C18 column): 17.094 min. 25 Also isolated was epimer mixture (0.75 gram, 12% yield), and the 4(S), 5(S) epimer (1.59 gram, 25%) as a white solid. 1 H-NMR (500MHz, CDC1 3 ) 62.38 (d, J=17.9Hz, 1H), 3.0 (m, 3H), 3.22 (i, 2H), 4.13 (m, 1H), 4.58 (m, 2H), 4.68 (m, 2H), 4.98 (br s, 1H), 5.26 (m, 1H), 5.57 3C is, 1H), 5.88 (ddt, J=18.0, 11.1, 5.4Hz, 1H), 7.20 (m, - 193 4H) ppm. LC-MS (ES+) : m/e=318 (M+H) . Analytical HPLC (CI8 column):17.025 (5.5%), 17.325 (94.5%) min. 1- [2- ( 4 -amino-3-chloro-benzoylamino) -propionyl] 5 pyrrolidine-2-carboxylic acid [2- (indan-2-yloxy) -5-oxo tetrahydro-furan-3-yll -amide (98at). Prepared from 97a and [2- (indanol-2-yloxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to 10 afford the title compound as a 71:29 mixture of epimers as an off-white solid (0.20g, 58% yield). 1 H-NMR (500 MHz, CDCl 3 ) 5 1.0-1.5 (m, 3H), 1.6-2.3 (m, 4H), 2.42 (m, 1H), 2.6-3.4 (m, 6H), 3.5-4.1 (m, 3H), 4.2-4.9 (m, 4H), 5.65 (d, J=5.oHz, 0.80H), 5.8 (m, 0.07H), 5.85 (d, 15 J=5.OHz, 0.13H), 6.8-7.3 (m, 6H), 7.4-7.9 (m, 3H) ppm. Analytical HPLC (CI8 column) 16.035 (71.4%), 16.476 (28.6%) min. LC-MS (ES+): m/e=555 (M+H). HJ N 00N 98au H 20 1- [2- (.
4 -Acetylamino-3-chloro-benzoylamino) -propionyl pyrrolidine-2-carboxylic acid [2- (indan-2-yloxy) -5-oxo tetrahydro-furan-3-y1] -amide (98au). Prepared from 97b and (2-(indanol-2 -yloxy-S-oxo 25 tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford the title compound as a 76:24 mixture of epimers as an off-white solid (0.22g, 57% yield). IH-NMR (500 MHZ, CDCl 3 ) 5 1.08 (d, J=6.9Hz, 0.4H), 1.26 (d, J=6.9Hz, - 194 0.6H), 1.35 (d, J=6.9Hz, 2H), 1.8-2.3 (m, 3H), 2.28 (s, 2H), 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=5.3Hz, 0.68H), 5.84 (d, J=5.3Hz, S 0.18H), 6.38 (br, 0.14H), 6.9-7.7 (m, 6H), 7.6-7.9 (m, 3H), 8.33 (br d, J=6.8Hz, 0.18H), 8.51 (br d, J=8.OHz, 0.82H) ppm. Analytical HPLC (C18 column) 15.596 (76.2%), 15.932 (23.8%) min. LC-MS (ES+): m/e=S97 (M+H). 10 0 HfH
H
2 N 0 a N 98av H O 1. 1- (2- ( 4 -amino-3 -chloro -benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid ( 2 -cyclopentyloxy-5-oxo 15 tetrahydro-furan -3-yl) -amide (98av). Prepared from 97a and syn-(2-cyclopentyloxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester according to the procedure used to prepare 98a to afford the title compound as an off-white solid (0.19g, 20 S9% yield). 1 H-NMR (500 MHz, CDC1 3 ) 6 1.2-2.4 (m, 15H), 2.4-3.1 (m, 2H), 3.6-3.9 (m, 2H), 4.2-4.4 (m, 2H), 4.5- 5.0 (m, 4H), 5.40 (d, J=5.OHz, 0.35H), 5.55 (d, J=5.OHz, 0.65H), 6.8-8.2 (m, 5H) ppm. Analytical HPLC (C18 column) 14.065 min. LC-MS (ES+): m/e=S07 (M+H). 25 C N HO C 9 T, CI Saw - 195 1- [2- (3, S-Dichloro-4-hydroxy-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl)-amide (98aw). Prepared from 1-[2-( 4 -allyloxy-3,5-dimethyl 5 benzoylamino)-propionyl)-pyrrolidine-2-carboxylic acid tert-butyl ester and syn-40 according to the procedure used to prepare 98a to afford the title compound as a pale yellow solid (1.087g, 64% yield). 1 H-NMR (500MHz, CDCl 3 ) 8 1-09 (d, J=6.9Hz, 0.6H), 1.33 (d, J=6.9Hz, 10 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, 10.2Hz, 0.8H), 2.56 (M, 0.2H), 2.85 (dd, J=17.3, 8.5Hz, 0.8H), 3.09 (dd, J=17.7, 10.2Hz, 0.2H), 3.57 (m, 1H), 3.73 (dt, J=9.2, 7.9Hz, 0.8H), 4.09 (m, 0.2H), 4.21 (d, J=7.9Hz, 0.2H), 15 4.44 (d, J=9.8Hz, 0.2H), 4.55 (dd, J=8.0, 3.0Hz, 0.88H), 4.62 (d, J=11.6Hz, 1H), 4.70 (m, 1H), 4.80 (m, 1H), 4.89 (d, J=11.6Hz, 0.8H), 5.52 (d, J=5.2Hz, 0.8H), 5.82 (d, J=5.2Hz, 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=1.0Hz, 20 1.6H) ppm. Analytical HPLC (C18 column) 10.135 min. LC MS (ES+): m/e= 564, 566 (6:4) (M+H). N 98ax 25 1- [2- ( 4 -Amino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid ( 2 -cyclopentyloy-5-oxo tetrahydro-furan-3-yl)-amide (98ax). Prepared according to the procedure used to prepare (98av) using anti- ( 2 -cyclopentylocy-5-oxo-tetrahydro 30 furan-3-yl)-amide to afford the title compound as an - 196 off-white solid (0.24 g, 74% yield). 1 H-NMR (500MHz, CDCl 3 ) 5 1.41 (d, J=6.5Hz, 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, 6.8Hz, 0.7H), 2.98 (dd, J=18.3, 8.2Hz, 5 0.3H), 3.60 (br, 1.4H), 3.77 (br, 0.6H), 4.1-4.6 (m, SH), 4.82 (m, 1H), 5.27 (m, 0.65H), 5,51 (d, J=5.3Hz, 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 -(C18 column) 9.756 min. LC-MS (ES+): 10 m/e=507 (M+H). 0 J, 0 N C1 H 98ay 1- [2 - ( 4 -Acetylamino-3 -chloro-benzoylamino) -propionyl) 15 pyrrolidine-2-carboxylic acid (2-ethoxy-5-oxo tetrahydro-furan-3-yl) -amide (98ay). Prepared from ( 2 -ethoxy-5-oxo-tetrahydro-furan-3-yl) carbamic acid allyl ester and 97b following the method used for 98a. The title compound was isolated as a 20 white solid (51 mg, 18% yield). 1 H-NMR (500MHz, 1:1 CDC1 3 :CD3OD) 6 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, 0.1H), 7.70 25 8.35 (m, 3H). Analytical HPLC 10.37, 10.54 min. LC-MS (ES*) m/e= 509.2 (M+H*).
- 197 H 2 N N N C1 H 0,N" 98az [2-(2-Chloro-ethoxy)-S-oxo-tetrahydro-furan-3-yl) carbamic acid allyl ester. 5 Prepared from 3 -allyloxycarbonylamino-4-hydroxy-butyric acid tert-butyl ester (5.2g, 20 mmol) as described for 40 using chloroethanol (4.05mL, 60 mmol) to afford 1.84g (35% yield) of the title compound as a mixture of epimers. For the anti-diastereomer: 'H-NMR (500 MHz, 10 CDCl 3 ) 5 2.42 (dd, J=18.lHz, 1H), 3.00 (dd, J=18.1, 7.8Hz, 1H), 3.63 (m, 2H), 3.85 (m, 1H), 4.02 (m, 1H), 4.23 (m, 1H), 4.57(br s, 2H), 5.17 (br s, 1H), 5.22 (d, H=11.5Hz, 1H), 5.29 (d, J=16.8Hz, 1H), 5.44 (s, 1H), 5.89 (m, 1H) ppm; LC-MS (ES+) n/e= 264 (M+H) . For the 15 syn-diastereomer: 'H-NMR (500 MHz, CDCl3) 6 2.47 (dd, J=17.3, 10.7Hz, 1H), 2.83 (dd, J=17.3, 8.4Hz, 1H), 3.65 (m, 2H), 3.83 (m, 1H), 4.11 (m, 1H), 4.57(m, 3H), 5.22 (d, H=10.4Hz, 1H), 5.30 (d, J=17.2Hz, 1H), 5.33, (m, 1H), 5.47 (d, J=5.2Hz, 1H), 5.89 (ddt, J=17.1, 11.0, 20 5.4Hz, 1H) ppm; LC-MS (ES+) m/e= 264 (M+H). [2-(2-Morpholin-4-yl-ethoxy)-S-oxo-tetrahydro-furan-3 yll-carbamic acid allyl ester. Is prepared from [2-(2-chloro-ethoxy)-S-oxo-tetrahydro 25 furan-3-yl]-carbamic acid allyl ester by reaction with morpholine (2 eq) and KI (1 eq) in DMF.
- 198 1- [2- ( 4 -Amino-3-chloro-benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid [2- (2-morpholin-4.-yl ethoxy) -5-oxo-tetrahydro- furan-3-ylI -amide (98az) . Is prepared from 97a and syn- 12- (2-morpholin-4-yl 5 ethoxy) -5-oxo-tetrahydro- furan-3-yl] -carbamic acid allyl ester following the method used for 98a. H 2N HIT H2N C 0 98ba 10 [2- ( 4 -Chloro-benzyloxy) -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 4 chlorobenzylalcohol to afford the title compound as a 15 white solid. Anti-diastereomer: HPLC (C18 column) 10.924 min; 1 H-NMR (500 MHz, CDC1 3 ) 8 2.41 (d, J=8.OHz, 1H), 3.02 (dd, J=18.1, 7.8Hz, 1H), 4.25 (br, IH), 4.56 (mn, 2H),4.58 (d, J=11.7Hz, 1H), 4.79 (d, J=11.7Hz, 1H), 4.99 (br, 1H), 5.22 (dd, J=10.4, 1.1Hz, IH), 5.28 (dd, 20 J=17.2, 1.3Hz, 1H), 5.44 (s, IH), 5.86 (m, 11), 7.25 (d, J=8.4Hz, 2H), 7.32 (d, J=8.4Hz, 2H) ppm; LC-MS (ES+) m/e=326 (M+H); Syn-diastereomer: EPLC (CI8 column) 10.780 min; :H-NMR (500 MHz, CDC1 3 ) 8 2.47 (dd, J=17.3, 10.5Hz, iH), 2.85 (dd, J=17.3, 8.4Hz, I), 4.55 25 (m, 3.H), 4.58 (d, J=11.7Hz, 1H), 4.84 (d, J=11.7Hz, 1H), 5.23 (dd, H=10.4, 1.1Hz, 1H), 5.30 (d, J=16.6Hz 1H), 5.49 (d, J=5.OHz, 1H), 5.89 (ddt, J=17.1, 11.0, 5.4Hz, 1H), 7.23 (d, J=8.3Hz, 2H), 7.31 (d, J=8.3Hz, 2H) ppm; LC-MS (ES+) m/e= 326 (M+H). 30 - 199 1- [2- (4-Amino-3-chloro-benzoylamino) -propionyl) pyrrolidine-2-carboxylic acid [2-(4-chloro-benzyloxy) 5-oxo-tetrahydro-furan-3-yll-amide (98ba). Prepared from 97a and syn- [2-(4-chloro-benzyloxy)-5 5 oxo-tetrahydro-furan-3-yl]-carbamic acid allyl ester following the method used for 98a to afford 154 mg (65% yield) of the title compound as a pale pink solid. HPLC (C18 column) 10.597 min; 2H-NMR (500 MHz, CDC1 3 ) 6 1.14 (d, J=6.SHz, 0.75H), 1.34, (d, J=6.8Hz, 2.25H), 10 1.6 (br, 0.25H), 1.91 (m, 1H), 2.03 (m, iR), 2.10 (m, 1H), 2.29 (m, 0.75H), 2.40 (dd, J=17.3, 10.3Hz, 0.75H), 2.51 (m, 0.25H), 2.82 (dd, J=17.3, 8.5Hz, 0.75H), 3.08 (dd, J=17.9, 10.9Hz, 0.25H), 3.58 (m, 1H), 3.72 (dd, J=16.5, 8.7Hz, 0.75H), 4.10 (m, 0.25H), 4.22 (d, 15 J=8.0Hz, 0.25H), 4.39 (d, J=10.BHz, 0.2SH), 4.54 (dd, J=9.1, 2.9Hz, 0.75H), 4.60 (d, J=11.9Hz, 0.75H), 4.68 (m, 1H), 4.85 (d, J=11.7Hz, 0.75H),'4.86 (m, 1H), 5.49 (d, J=5.2Hz, 0.75H), 5.81 (d, J=5.2Hz, 0.25H), 6.2 (br, 0.25H), 6.74 (m, 2H), 7.05 (d, J=8.5Hz, 0.SH), 7.17 (d, 20 J=8.4Hz, 0.5H), 7.30 (m, 3.25H), 7.48 (dd, J=8.4, 2.0Hz, 0.75H), 7.56 (d, J=1.9Hz, 0.25H), 7.73 (d, J=1.9Hz, 0.75H), 8.42 (d, J=5.7Hz, 0.25H) ppm; LC-MS (ES+) m/e=563, 565 (M+H). 25 A, N CI 98bb 200 1-12- ( 4 -Acetylamino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carboxylic acid [ 2 -(4-chloro-benzyloxy) 5-oxo-tetrahydro-furan-3-yll-amide (98bb). Prepared from 97b and syn-[ 2 -(4-chloro-benzyloxy)-5 5 oxo-tetrahydro-furan-3-y1]-carbamic acid allyl ester according to the procedure used to prepare 98a to afford 165mg (64i yield) of the title compound as pale yellow solid. HPLC (C18 column) 10.491 min; 1 H-NMR (500 MHz, CDCl 3 ) 6 1.16 (d, J=6.8Hz, 0.6H), 1.35, (d, 10 J=6.8Hz, 2.4H), 1.94 (m, 1H), 2.04 (m, 1H), 2.10 (m, 1H), 2.25 (s, 3H), 2.28 (m, 1H), 2.40 (dd, J=17.3, 10.4Hz, 0.8H), 2.53 (m, 0.2H), 2.84 (dd, J=17.3, 8.5Hz, 0.8H), 3.02 (dd, J=17.5, 10.5Hz, 0.2H), 3.58 (m, 1H), 3.72 (ddd, J=17.2, 8.3, 8.3Hz, 0.8H), 4.13 (m, 0.2H), 15 4.22 (d, J=8.2Hz, 0.2H), 4.40 (d, J=10.9Hz, 0.2H), 4.54 (dd, J=8.1, 3.0Hz, 0.8H), 4.60 (d, J=11.8H1z, 0.8H), 4.69 (m, 1H), 4.85 (d, J=11.8Hz, 0.8H), 4.87 (m, 1H), 5.49 (d, J=5.2Hz, 0.8H), 5.80 (d, J=5.2Hz, 0.2H), 6.47 (br, 0.2H), 6.95 (d, J=8.3Hz, 0.8H), 7.05 (d, J=8.3Hz, 20 0.4H), 7.18 (d, J=8.3Hz, 0.4H), 7.29 (m, 3.2H), 7.49 (dd, J=8.6, 1.9Hz, 0.2H), 7.63 (dd, J=8.6, 1.9Hz, C.8H, 7.74 (d, J=1.9Hz, 1H), 7.85 (d, J=1.9Hz, 0.8H), 8.25 (d, J=6.4Hz, 0.2H), 8.51 (m, 0.8H) ppm; LC-MS (ES+) m/e=605, 607 (M+H).
- 201 Scheme XVIII 40 O CO2H 99 O 100 0 "q yO- N Bo N1 ClH O LBc CIO 5 102a, Y=AcNH 101 102b, Y=NH 2 2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamoyl) piperidine-1-carboxylic acid-tert butyl ester (100) . 5 Prepared from piperidine-1,2-dicarboxylic acid 1-tert butyl ester 99 and 40 following the method used in the preparation of 75 to give the title compound as a yellow solid (2.63g, 57% yield) .
1 H-NMR (500MHz, CDCl 3 ) 5 1.15-1.79 (m, 15H), 2.12-2.50 (m, 2H), 2.56-2.83 (m, 10 1H), 2.89 (dd, 0.SH), 3.05 (dd, 0.5H), 3.81-4.15 (br s, 1H), 4.36-4.97 (m, 3H), 5.37-5.61 (m, 1H), 6.42-6.89 (br s, 1H), 7.17-7.51 (m, SH). LC-MS (ES*) m/e=419.4 (MH*). 15 {2-[ 2
-(
2 -Benzyloxy-5-oxo-tetrahydr-furan-3 ylcarbamoyl) -piperidin-1-yl -1-methyl-2-oxo-ethyl) carbamic acid tert-butyl ester (101). 2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamoyl) piperidine-1-carboxylic acid tert-butyl ester (100) was 20 dissolved in 20% TFA in CH 2 Cl 2 (25mL) and stirred at - 202 room temperature for 50min. The solvent was evaporated and the residual acid azeotroped with CH 2 C1 (4x). The resulting oil was dissolved in CH 2 Cl 2 (20mL) and DMF (SmL), cooled to O'C and treated with DIEA (4.7mL, 5 27.Ommol), Boc-alanine (970mg, 5.1mmol), HOBT (924mg, 6.8mmol) and EDC (1.31g, 6.8mmol) and the solution stirred under N 2 for 18hours. The solvent was concentrated in vacuo then dissolved in EtOAc and washed with 0.SN NaHSO 4 (2x), saturated NaHCO 3 (2x) and 10 brine. The organic layer was dried over anhydrous Na 2
SO
4 and evaporated to give an orange solid that was dissolved in CH 2 C1 2 and added dropwise to diethyl ether to afford a white precipitate. The title compound. as a white solid (1.21g, 73% yield)
.
1 H-NMR (500MHz, CDCl 3 ) 15 8 1.10-1.79 (m, 1SH), 1.98-2.19 (m, 0.SH), 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, O.4H), 5.80 (d, 0.3H), 7.24-7.40 (m, 5H). LC-MS (ES*) Tn/e=490.3 (MH+). 20 1-[2-( 4 -Acetylamino-3-chloro-benzoylaijno)-propionyl] piperidine-2-carboxylic acid ( 2 -benzyloxy-S-oxo tetrahydro-furan-3-yl)amide (102a). Prepared from{2- [2- (2 -benzyloxy-5-oxo-tetrahydro-furan 25 3-ylcarbamoyl)-piperidin-1-yl] -1-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 (71mg, 47% yield) . H-NMR (500MHz, CDCl 3 ) 6 1.10-1.97 (M, 30 1OH), 2.10-2.68 (m, 5H), 2.73-3.24 (m,.2H), 3.62-3.92 (m,. 1H), 4.24-5.27 (m, SH), 5.48-5.59 (m, 0.5H), 5.75 5.85 (m, 0.5H), 6.51-6.61 (d, 1H), 7..05-7.45 (m, 4H), - 203 7.52-8.12 (m, 4H). Analytical HPLC 8.30min. LC-MS (ES*) m/e=585.3 (MH+) 1- [2- ( 4 -Amino-3-chloro-benzoylamino) -propionyl) 5 piperidine-2-carboxylic acid (2-benzyloxy-5-oxo tetrahydro-furan-3-yl) -amide (102b). Prepared as above for 102a to give the title compound (0.06g, 27% yield) as a yellow solid. IH-NMR (500MHz, CDC1) 61.2-1.8 (m, 7H), 2.1-2.6 (m, 2H), 2.7-3.2 (m, 10 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 (cyano column) 14.559 (39.6%), 15.198 (60.4%).
LC-MS
(ES+): m/e=543 (M+H) 15 Scheme XIX N N? COH
CO
2 Bu-t 103 104 Cbz% N<
-B
C
2 CO2B-t 106 105 Alloc% NRa N O
H
2 N CO 2 Bu-t H 2 N C! 107 C08 'Re - 204 4-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-tert-butyl ester (104). Compound 104 was prepared according to the procedure 5 used to prepare compound 95. A suspension of Cbz-Hyp-OH (4.854g, 18mmol) in DMA (135ml), benzyltriethylammonium chloride (4.105g, lammol),
K
2
CO
3 (64g, 46mmol) and 2-bromo-2 10 methylpropane (99ml, 859mmol) was stirred at 55'C for 18hours. The mixture was diluted with iced water and extracted with EtOAc (3x). The organic phase washed with water, O.5N NaHSO 4 solution and brine dried and the solvent in vacuo to yield the title compound as a 15 yellow oil (5.368g, 98% yield) IH-NMR (500MHZ, CDCl 3 ) 8 1.33 (s, 5H), 1.47 (s, 4H), 2.01-2.14 (m, 1H), 2.22 2.38 (m, 1H), 3.50-3.72 (m, 2H), 4.34-4.45 (m, 1H), 4.45-4.53 (m, 1H), 5.04-5.20 (m, 2H), 7.22-7.42 (m, 5H). Analytical HPLC 10.14min. LC-MS (ES*) m/e=322.2 20 (M+) 4-Fluoro-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-tert-butyl ester (105). A solution of 104 (4.262, 13.96mmol) in CH 2 C1 2 (100ml) 25 at -78 0 C was treated with DAST (1.80ml, 13.6mmol), stirred for 10min then warmed to room temperature and stirred for 60h under N 2 . The mixture was poured into iced NaHCO 3 (10* solution, 350ml) and extracted with
CH
2 Cl 2 (2x). The organic phase was washed with water, 30 brine, dried over anhydrous Na 2
SO
4 and concentrated to give a brown oil (4.299g) which was purified by flash column chromatography on silica gel using hexanes/EtOAc (90/10 to 80 /20%) . The title compound was obtained as a - 205 yellow oil (2.805g, 64% yield). IH-NMR (500MHz, CDCl 3 ) 6 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, 0.51H), 4.49 (d, 0.SH), 5.03-5.21 (m, 3H), 7.23-7.44 (m, 5H). Analytical HPLC 5 12.15min. LC-MS (ES*) m/e=324.2 (MH*). 1- (2 -Benzyloxycarbonylamino -propionyl) -4-fluoro pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2 tert-butyl ester (106). 10 A solution of 105 (2.72g, 8.42mmol) in MeOH (SOml) and 10% Pd/C (1.27g) was stirred under H2 for 2hours then filtered through celite and the solvent evaporated to give a yellow oil (1.526g). This oil was dissolved in
CH
2 C1 2 (30ml) and treated with DIEA (1.5ml, 8.6mmol), 15 Cbz-ala-OH (2.34g, 10.5mmol) and EDC (2.32g, 12mmol) at o C. The mixture was stirred an additional 10min at O*C then allowed to warm to room temperature and stir for 18hours. The solvent was concentrated in vacuo and the residue dissolved in EtOAc then washed with O.SN 20 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 white solid which was purified -by flash column chromatography, eluting using hexanes/EtOAc (80/20 to 60/40%). The title compound 25 was isolated as a white solid (286g, 86% from 4-fluoro pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2 tert-butyl ester). 1 H-NMR (500MHz,
CD
3 0D) 81.26-1.59 (m, 12H), 2.20-2.-67 (m,.2H), 3.45-4.13 (m, 2H), 4.25 4.'47 (m, 1H), 4.58-4.71 (m, 1H),'4.96-5..17 (m, 2H), 30 5.19-5.45 (m, 1H); 7.23-7.48 (m, SH). Analytical HPLC 16.36min. LC-MS (ES*) m/e=395.3
(MH*).
- 206 1-[2-(4-Amino-3-chloro-benzoylamino)-propionyl)-4 fluoro-pyrrolidine-2-carboxylic acid-tert-butyl ester (107). A suspension of 106 (2.65g, 6.72mmol) in MeOH (40ml) 5 and 10% Pd/C (1.32g) was stirred under an atmosphere of
H
2 for 1.Shours, filtered through celite and the concentrated to give a waxy solid (1.694g). The solid was dissolved in CH 2 C1 2 (25ml) and treated with DIEA (3.4ml, 19.5mmol), 4-amino-3-chlorobenzoic acid 10 (1.362g, 7.9mmol), HOBT (1.164g, 8.62mmol) and EDC (1.645g, 8.S7mmol) at O'C under N 2 . The mixture was allowed to warm to room temperature and stirred for 18hours. The solvent was concentrated in vacuo. The residue was dissolved in EtOAc, washed with water (4x), 15 0.5N 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 white solid which was purified by flash column chromatography, using CH 2 Cl 2 /MeOH (99/1 to 98/2%). The product obtained as a white solid (2.705g, 20 97%). IH-NMR (500MHz, CD 3 0D) 81.33(s, 9H), 1.48 (d, 3H), 2.31-2.55 (m, 2H), 3.93 (d,d, 1H), 4.02-4.21 (m, 1H), 4.59-4.76 (m, 1H), 5.31 (br s, 0.5H), 5.41 (br s, 0.5H), 6.78 (d, 1H), 7.57 (d,d, 1H), 7.78 (s, 1H), 8.31 (d, 1H). Analytical HPLC 14.14mn. LC-MS (ES*) 25 m/e=414.2 (MH*). NYN H2N Cl 0O 1 08a - 207 1- [2- (4-Amino-3-chloro-benzoylamino)-propionyl] -4 fluoro-pyrrolidine-2-carboxylic acid (2-benzyloxy-5 oxo-tetrahydro-furan-3-yl)-amide (108a). Prepared from syn-(2-benzyloxy-5-oxo-tetrahydro-furan 5 3-yl)-carbamic acid allyl ester and 107a following the method used for the synthesis of 98a. The title compound was isolated as a white solid (41mg, 15% yield). 1 H-NMR (500MHz, CD 3 0D) 8 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 10 (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-MS (ES*) m/e= 547.1 (MH*) 15 F 0 0
H
2 N O N 108b 1- [2- (4-Amino-3-chloro-benzoylamino) -propionyl -4 fluoro-pyrrolidine-2-carboxylic acid (2 -benzyloxy-5 20 oxo-tetrahydro-furan-3-yl)-amide (108b). Prepared from (2-benzyloxy-5-oxo-tetrahydro-furan-3 yl)-carbamic acid allyl ester 107a following the method used for the synthesis of 98a. The title compound was isolated as a white solid (654mg, 54% yield). 1 H-NMR 25 (500MHz, CD30D) 6 1.07 (d, 0.5H), 1.25-1.56 (in, 2.5H), 2.21-2.65 (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, 1H), 5.45-5.50 (m, 0.3H), 5.54-5.58 (m, 0.2H), 5.61-5.67 (m, 0.3H), 5.77 (d, 0.2H), 6.72-6.84 (m, 1H), - 208 7.16-7.41 (M, 5H), 7.50-7.65 (M, 1H), 7.71-7.87 (M, 1H). Analytical HPLC 12.83min. LC-MS (ES*) m/e=547.1 (MH*). H2N CN 5 108c 1- [2- ( 4 -Amino-3-chloro-benzoylamino) -propionyll -4 fluoro-pyrrolidine-2 -carboxylic acid (2 -ethoxy-5-oxo tetrahydro-furan-3-yl)-amide (108c). 10 Prepared from syn- ( 2 -ethoxy-5-oxo-tetrahydro-furan-3 yl)-carbamic acid allyl ester and 107a following the method used for the synthesis of 98a to give the title compound (100.3mg, 38% yield) .
1 H-NMR (500MHz, CD30D) 6 1.09 (t, 1.2H), 1.25 (t, 1.8H), 1.40 (d, 1H), 1.49 (d, 15 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, 0.81), 7.79 (d, 0.55H), 8.06 (d, 0.55H), 9.00 (d, 0.3H). Analytical 20 HPLC 8.46min. LC-MS (ES+) m/e= 485.2 (MH+). H2N CO 1 08d - 209 1- [2- (4-Amino-3-chloro-benzoylanino) -propionyll -4 fluoro-pyrrolidine-2-carboxylic acid [2-(2-isopropyl-5 methyl-cyclohexyl) -5-oxo-tetrahydro-furan-3-yll -amide (108d) . 5 Prepared from {2-[1R-(2S-Isopropyl-5R-methyl cyclohexyloxy) I -5-oxo-tetrahydro-furan-3-yl}-carbamic acid allyl ester and 107a following the method used for the synthesis of 98a to give the title compound (95mg, 31% yield) . 1H-NMR (500MHz, CD30D) 6 0.42 (d, 2H) , 10 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), 2.61-2.88 (m, 1.5H), 3.02-3.23 (dd, 0.5H), 3.37-3.47 (m, 0.SH), 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), 15 6.77-6.84 (m, 1H), 7.53-7.67 (m, 1H), 7.78 (d, 0.4H), 7.84 (d, 1H) Analytical HPLC 8.34min. LC-MS (ES+) m/e= 595 (MH+). Scheme XX BoNJN 7 + AoG%,4 H C0 2 H0 74 109
K
2 H C ~CN HO 20 111 110 -210 (2- E2- ( 2 -Etboxyoxottrhyrof
---
y~rb PYrrolidin-1-y1] .IjethY12-O~cethY11Carb± acid Prepared from
(
2 -erhoxy..Oxo.tetrahydro-furn- 5 carbamic acid ally, ester 74 following the method used in the synthesis of 7S to give the title compound as a pale yellQw solid (660ing, 73t yield) . IH-NMR (SOOMHz, CD3 OD) S 1. 14 -1. 36 (in, 6H) , 1. 42 (8,. 9H) , 1. 75 -2. 29 (in, 4H-), 2.48 (dd, 0.58), 2.58 {dd, 0.5H), 2.72-2.85 (mn, 10 0.5H), 2.99 (dd, 0.SH-I, 3.339 (in, 4H), 4.07-4.52 (mn, 2.58), 4.S3-4.72 (mn, 0.5H), S..37 (s, 0.5H), S.57 (d, 0.SH)r. Analyti.cal HPLC (mixture of 2 diastereoners) 7.92, 8 .l'amin. LC-MS (ES*) rn/e=414.3 1- (2- ( 4 -Alayl xy3s.dchro-bzolamr -propionylj pyrrolidin-2-caroxlic acid (2 -ethoxy-5-ox 0 .. tatrahydro.turan-3.yl) -amide (110). Prepared from 2109 and 4 -allyloxy..3,5.dichloro-benzoic 20 acid following the method used in che synthesis of 82 to give the title compound as a white solid (228mg, 65%). 1H-NMR (500MHZ, CD3OD) 8 1. 10-1.30 (mn, 4H), 1.32-1.S2 (mn, 3H), 1.63-2.31 (m, 4H), 2.41-2.50 (d, 0.5H), 2.52-2.61 (dd, 0.58), 2.67-2.81 (m. 0.58), 2.94 25 3.05 (dd, 0.58j), 3.47-3.96 (m, 4H), 4.21-4.81 (mn, 58), 5.22-5.32 (mi, 1H), 5.3S-5.49 (in, 1.58), S.55-S.63 (m, 0.SH), 6.06-6.21 (mn, 18), 7.90 (8, 2H). Analytical RPLC (mixture of 2 diastereomrs) 1 2.S~min. LC-M~S (RS+) mfe=542.3(MH+ 4 . 30 1-[2- (3, S-Dichlor..4-ydroxy-be zyaino-propionyll pyrrolidine-2-carboxylic acid (2-ethx'..5..xo. tetabydofuran.3yl) -amide (111).
- 211 To a solution of 110 (194mg, 0.36mmol) in CH2Cl2 (5ml) was added DMBA (70.7mg, 0.45mmol) and Pd(PPh3)4 (50.3mg, 0.044mmol) at 0*C. The solution was warmed to room temperature after 15mins, stirred for 2hours, 5 diluted with CH2C12 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 Cl 2 /MeOH (99/1 to 95/5%) afforded the title compound (138.6mg, 77% yield). 10 NMR (500MHz, CD 3 0D) 6 1.13-1.31 (m, 3H), 1.35-1.49 (m, 3H), 1.84-2.35 (in, 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 8.70min. LC-MS (ES*) m/e=502.2 (MH*). 15 - 212 Scheme XXI O N?
C
2 Bu-t 103 112 F. CbzF F CO2Bu-t CO2Bu-t 114 113 Allo0c. X XF.JF N F R8 N N O CO2Bu- X H OIR8 115a, X=CI, Y=NH 2 , Z=H 116a- 116h 115b, X=CI, Y=AcNH, Z=H 115c, X=CI, Y=AcNH, Z=CH 3 0 Compounds 116a- 116h were prepared as described above for compounds 98 only substituting 1-(2 5 benzyloxycarbonylamino-propionyl) -4,4 -difluoro pyrrolidine-2-carboxylic acid tert-butyl ester (114) for 1- (2 -benzyloxycarbonylamino-propionyl) -pyrrolidine 2-carboxylic acid tert-butyl ester (95). 10 Preparation of 1- (2 -benzyloxycarbonylamino-propionyl) 4,4-difluoro-pyrrolidine-2-carboxylic acid tert-butyl ester (114). A solution of 4,4-difluoro-pyrrolidine-1,2-dicarboxylic acid-l-benzyl ester-2 - tert-butyl ester (113) - 213 (Karanewsky, et.al., J. Med. Chem, 33, pp. 1459-1469 (1990)) (0.42 g, 1.23 mmol) and 10% palladium on carbon (0.22g) in methanol (6 mL) was stirred at 1 atm hydrogen pressure for 3h. The mixture was filtered 5 through Celite and evaporated . The residue was dissolved in CH 2 C1 2 (4 mL) and DMF (2 mL) and cooled to 0 C. 2 -Benzyloxycarbonylamino-propionic acid (0.30 g, 1.35 mmol), EDC (0.30, 1.54 mmol), DIEA (0.65 mL) and HOBt (0.17 g, 1.23 mmol) was added and the reaction was 10 stirred 0.5h at O'C, then 16h at room temperature under nitrogen. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate, then was washed with 10% sodium bisulfate, saturated sodium bicarbonate, water and brine, was dried over sodium 15 sulfate and was evaporated. Purification by flash chromatography on silica, eluted with 25:75 ethyl acetate: hexanes provided 1- ( 2 -benzyloxycarbonylamino propionyl) -4, 4 -difluoro-pyrrolidine -2- carboxylic acid tert-butyl ester (0.39 g, 77t yield) as a colorless 20 oil. 1 H-NMR (500 MHz, CDC1 3 ) 6 1.3-1.6 (m, 12H), 2.5 (m, 0.8H), 2.7 (m, 1.2H), 3.9 (m, 1R), 4.1.(m, 1H), 4.4 (m, 1H), 4.7 (m, iH), 5.1 (m, 21H), 5.59 (br d, J=7.7Hz, 0.8H), 5.7 (br d, J=7.7Hz, 0.2H), 7.35 (m, SH) ppm. 25 Analytical HPLC (cyano column) 17.069 min. LC-MS (ES+): m/e=413 (M+H), 357 (M+H-tert-butyl), 313 [M+H-(CO 2 tert butyl)]. N N F
H
2 N C 1 30 - 214 1- [2- (4-Amino-3-chloro-benzoylamino) -propionyll -4,4 difluoro-pyrrolidine-2-carboxylic acid (2-benzyloxy-5 oxo-tetrahydro-furan-3-yl)-amide (116a). 5 Prepared from 115a and syn-40 to afford the title compound as an off-white solid (0.149, 73% yield) NMR (500 MHz, CD 3 0D) 5 1.0-1.5 (m, 3H), 2.0-3.5 (m, 4H+CH 3 0H), 3.5-5.5(M, 6H+H 2 0), 5.6-5.8 (m, 1H), 6.7-6.8 (m, IH), 7.1-7.8 (m, 8H), 8.2-8.6 (m, 1H) ppm. 10 Analytical HPLC (cyano column) 13.744 min. LC-MS (ES+) m/e=565 (M+H). O N j~_N F H 0N C1 116b N 15 1- [2- (4-Acetylamino-3-chloro-benzoylamino) -propionyl] 4,4-difluoro-pyrrolidine-2-carboxylic acid (2 benzyloxy-5-oxo-tetrahydro-furan-3-yl)-amide (116b). Prepared from 115b and syn-40 to afford the title compound as an off-white solid (0.08 g, 38% yield). 20 1 H-NMR (500 MHz, CDC1 3 ) 8 1.03 (d, J=6.9Hz, 0.4H), 1.30 (d, J=6.9Hz, 0.6H), 2.25 (d, J=2.9Hz, 3H), 2.4-3.2 (m, 4H),'3.6-4.4 (m, 4H), 4.6-4.9 (m 3H), 5.52 (d, J=S.2Hz, 0.6H), 5.78 (d, J=5.2Hz, 0.4H), 6.6 (br s, 1H), 6.9-7.9 (m, 8H), 8.39 (d, J=8.1 Hz, 0.4H), 8.44 (d, J= 8.3Hz, 25 0.6H), 8.74 (d, J=6.8Hz, 11H) ppm. Analytical HPLC (cyano column) 11.830 min. LC-MS (ES+): m/e= 607 (M+H) - 215 F NF ON C1 116c N 1- [2- ( 4 -Acetylamino-5-chloro-2-methoxy-benzoylamino) propionyl) -4, 4-difluoro-pyrrolidine-2-carboxylic acid 5 ( 2 -benzyloxy-5-oxo-tetrahydro-furan-3 -yl) -amide (116c). Prepared from 11Sc and syn-40 to afford the title compound as an off-white (O.07g, 29% yield). 1H-NMR (500 MHz, CDC1 3 ) 6 0.99 (d, J=6.9Hz, 1.35H), 1.32 (d, J=6.9Hz, 1.65H), 2.25 (s, 1.5H), 2.26 (s, 1.5H), 2.3 10 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.SH), 4.6-4.9 (m, 3H), 5.52 (d, J=5.3Hz, 0.5SH), 5.80 (d, J=5.3Hz, 0.45H), 7.0-7.4 (m, 4H), 7.7-7.9 (m, 2H), 8.0-8.4 (m, 2H), 8.49 (d, J=6.5Hz, 1H), 8.93 (d, J=6.7Hz, 1H) ppm. Analytical 15 HPLC (cyano column) 12.959 min. LC-MS (ES+): m/e=637 (M+H). F NF O Cl 116d 20 1- [2- ( 4 -Acetylamino-3-chloro-benzoylamino) -propionyl] 4, 4 -difluoro-pyrrolidine-2-carboxylic acid (2-ethoxy-S oxo-tetrahydro-furan-3-yl) -amide (116d) . Prepared from 115b and syn- ( 2 -ethoxy-5-oxo-tetrahydro furan-3-yl)-carbamic acid allyl ester to afford the 5 title compound as a 92:8 mixture of epimers. Off-white solid (0.27g, 66% yield) .
1 H-NMR (500 MHz, CDC1 3 ) 6 1.0- - 216 1.5 (m, 6H), 2.25 (s, 1.8H), 2.26 (s, 1.2H), 2.3-3.1 (m, 4H), 3.3-4.3 (M, 4H), 4.5-4.9 (m, 3H), 5.45 (d, J=5.3Hz, 0.75H), 5.59 (d, J=5.2Hz, 0.25H), 6.7-7.1 (m, 2H), 7.62 (dd, J=8.7, 2.0Hz, 1H), 7.76 (m, 1H), 7.85 5 (d, J=2.OHz, 1H), 8.48 (m, 1H) ppm. Analytical HPLC (C18 column) 13.300(91.8%), 14.046 (8.2%) min. LC-MS (ES+): m/e 545 (M+H). F N N F N H H 0Oqe HCl 116e 10 1- [2- (4-Acetylaino-3-chloro-benzoylanino) -propionyl] 4,4-difluoro-pyrrolidine-2-carboxylic acid (2 cyclohexyloxy-5-oxo-tetrahydro-furan-3-yl)-amide (116e). 15 Prepared from 115b and syn- (2-cyclohexyloxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester to afford the title compound as a 93:7 mixture of epimers. 1 H-NMR (500 MHz, CDC1 3 ) 6 1.0-2.0 (m, 13H), 2.25 (s, 2H), 2.26 (s, 1H), 2.40 (dd, J=17.3, 10.1Hz, 1H), 2.84 20 (dd, J=17.3, 8.5Hz,.1H), 2.5-3.0 (m, 2H), 3.5-4.3 (m. 3.5H), 4.5-4.9 (m. 2.5H), 5.59 (d, J=5.3Hz, 0.75H), 5.76 (d, J=5.2Hz, 0.25 H), 6.74 (br d, J= 5.7Hz, 0.25H), 6.93 (br d, J=7.1Hz, 1H), 7.06 (br d, J=7.8Hz, 0.75H), 7.62 (dd, J=8.6, 2.0Hz, 1H), 7.78 (m, 1H), 7.85 25 (d, J=2.OHz, 1H), 8.35 (br d, J=6.6Hz, 0.25H), 8.50 (br d, J=8.2Hz, 0.75H) ppm. Analytical HPLC (C18 column) 17.112 (93%), 17.433 (7%) min. LC-MS (ES+): m/e=599
(M+H).
- 217 N,__N F HO N C 1 1 1 6 f HO ' 1- [2 - (4 -Acetylamino- 3 - chloro-benzoylamino) -propionyll 4, 4-difluoro-pyrrolidine-2-carboxylic acid [2- (indanol 5 2-yl)oxy-S-oxo-tetrahydro-furan-3-yl]-amide (116f). Prepared from 115b and [2- (indanol-2-yl1] oxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester to afford the title compound as a 62:38 mixture of epimers. Off -white solid (0. 34g, 71% yield) . IH-NMR 10 (500 MHz, CDC1 3 ) 5 1.09 (d, J=6.9Hz, 0.6H), 1.21 (d, J=6.9Hz, 0.9H), 1.33 (d, J=6.9Hz, 0.9H),*1.42 (d, J=6.9Hz, 0.6H), 2.28 (s, 2H), 2.29 (s, 1H), 2.40 (dd, J=17.4, 10.3Hz, 1H), 2.4-3.3 (m, 7H), 3.6-4.2 (m, 2H), 4.5-4.8 (m, 4H), 5.66 (m, 0.6H), 5.84 (d, J=4.3Hz, 15 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), 7.8-8.0 (m, 2H), 8.52 (m, 0.6H), 8.62 (br d, J=6.SHz, 0.4H) ppm. Analytical HPLC (C18 column) 16.556 (62.0%), 16.824 (38.0%) min. LC-MS (ES+): m/e=633 (M+H). 20 N F C1 H CI116g C1N 1- [2- (4-Acetylamino-3-chloro-benzoylamino) -propionyl] 4, 4-difluoro-pyrrolidine-2-carboxylic acid (2- - 218 cyclopentylmethoxy-5-oxo- tetrahydro- furan-3 -yl) -amide (116g) . Prepared from 11Sb and syn- ( 2 -cyclopentylmethoxy-5-oxo tetrahydro-furan-3-yl)-carbamic acid allyl ester to 5 afford the title compound as an off-white solid (0.20g, 44% yield). 1 H-NMR (500 MHz, CDCl 3 ) 8 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), 10 5.44 (d, J=5.2Hz, 0.75H), 5.62 (d, J=5.2Hz, 0.25H), 6.7 (br, 0.25H), 6.91 (d, J=7.1Hz, 1H), 7.1 (m, 0.75H), 7.59 (d, J=8.5Hz, 0.25H), 7.63 (dd, J=8.5, 2.5Hz, 0.75H), 7.75 (m, 1H), 7.86 (d, J=1.8Hz, 1H), 8.33 (br d, J=6.5Hz, 0.25H), 8.49 (br d, J=8.4Hz, 0.75H) ppm. 15 Analytical HPLC ( C18 column) 17.705 min. LC-MS (ES+) m/e=599 (M+H) F Cl 116h H 20 1- [2- ( 4 -Acetylamino-3-chloro-benzoylanfo) -propionyll 4, 4 -difluoro-pyrrolidine-2- carboxylic- acid (2 phenylethoxy-5-oxo-tetrahydro-furan-3-yl) -amide (116h). Was prepared from 115b and syn- (S-oxo-2-phenethyloxy tetrahydro-furan-3-yl)-carbamic acid allyl ester to 25 afford the title compound as an off-white solid (0.15g, 24% yield). 1 H-NMR (500MHz, CDCl 3 ) 8 1.29 (d, J=6.9Hz, 0.75H), 1.40 (d, J=6.9Hz, -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, 3=5.3Hz, 0.75H), 5.67 (d, J=5.lHz, 0.25H), 6.65 (m, - 219 1H), 6.90 (d, J=7.OHz, 0.75H), 7.06 (d, J=7.6Hz, 0.25H), 7.1-7.3 (m, 5H), 7.57 (d, J=8.6Hz, 0.25H), 7.63 (d, J=8.6Hz, 0.75H), 7.75 (m, 1H), 7.86 (d, J=1.8Hz, 1H), 8.35 (d, J=6.2 Hz, 0.25H), 8.49 (d, J=8.3Hz, 5 0.75H) ppm. Analytical HPLC (C18 column) 17.265 min. LC-MS (ES+): m/e=621 (M+H). Scheme XXII BOCN'IQ + Alo 4 o!N? 4 N + N
CO
2 H H0 N 117 40 118 aN jyCO2H H 0 HH0 o NO N 118 H 119 - H OC6H5 121 120a= anti 120b= syn 10 2- (2-Benzyloxy-5-oxo-tetrahydro-furan-3-ylcarbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (118). Prepared from 40 (1.16 g, 4.0 mmol) and Boc-Pro-OH according to the procedure used to prepare 100 (Scheme XVIII) to afford 1.53 g (94% yield) of. the title 15 compound as a white solid. 1 H-NMR (500 MHz, CDCl 3 ): 6 1.61 (br, 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 (m, H), 7.26-7.45 (m, 5H), 7.60-7.80 (d, H); Analytical 20 HPLC: 11.32 min; LC-MS: m/e = 405 (M+H*).
- 220 2-Phenylaminopropionic acid (119). A mixture of alanine (356 mg, 4.0 mmol), iodobenzene (816 mg, 4.0 mmol), trans-dichlorobis(tri-o 5 tolylphosphine) palladium (II) (Pd[P(o-Tol) 3 12Cl2) (160 mg, 0.2 mmol), copper (I) iodide (40 mg, 0.2 mmol),
K
2
CO
3 (552 mg, 4.0 mmol), benzyltriethylammonium chloride (160 mg, 0.8 mmol), triethylamine (1.6 mL) and water (0.8 mL) in DMF (8 mL) was stirred under nitrogen 10 atmosphere at 100*C for 20 hours. The mixture was cooled to room temperature, diluted with ethyl acetate (50 mL) and water (50 mL), acidified with 6N HCl to the pH = 2 to 3. The aqueous layer was extracted with ethyl acetate (50 mL x 4). The combined organic layers 15 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.S) to afford 300 mg (45% yield) of the title compound as a pink 20 solid. 1 H-NMR (500 MHz, CDCl 3
/CD
3 0D = 0.5 ml/3 drops) 5 1.45 (d, 3H), 4.02-4.15 (m, H), 6.57-6.70 (m, 3H), 7.11-7.25 (m, 2H); Analytical HPLC: 6.10 min. LC-MS: m/e = 166 (M+H*). 25 1- (2 -Phenylamino-propionyl) -pyrrolidine-2 -carboxylic acid (2-benzyloxy-5-oxo-tetrahydro-furan-3-yl)-amide (120a and 120b). A solution of 118 (405 mg, 1.0.mmol) was treated with TFA (2 ML) in CH 2 Cl 2 (2 mL) for one hour. The reaction 30 solution was evaporated in vacuo and azeotrapped with
CH
2 C1 2 four times to give pyrrolidine-2-carboxylic acid
(
2 -benzyloxy-5-oxo-tetrahydro-furan-3-yl)-amide as a pale yellow solid. 1 H-NMR (500 MHZ, CDC1 3 ): 6 1.87-2.15 (m, 4H), 2.30-2.70 (m, 2H), 2.80-3.08 (m, H), 3.45 (br, - 221 2H), 4.35-4.98 (m, 3H), 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+H*). 5 2-Phenylaminopropionic acid (119) (300 mg, 1.8 mmol) in
CH
2 Cl 2 (10 mL) was treated with HOBT (270 mg, 2.0 mmol) and EDC (2.1 g, 11 mmol) at 0*C for 10 min. Diisopropylethylamine (2 mL) was added followed by a solution of pyrrolidine-2-carboxylic acid (2-benzyloxy 10 5-oxo-tetrahydro-furan-3-yl)-amide in CH 2 Cl 2 (10 ML). The mixture was stirred at room temperature for 4 hours, diluted with CH 2 Cl 2 (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 15 yellow solid. Flash chromatography using CH 2 C1 2 /methanol (99/1 to 98/2) afforded 151 mg (33% yield) of anti diastereomer of the title compound (120a) and 129 mg (29% yield) of syn diastereomer (120b) as a white solid. 1 H-NMR (500 MHz, CDC1 3 ) for the anti 20 diastereomer: 5 1.37-1.41 (m, 3H), 1.50-2.45 (m, 4H), 2.60-2.70 (m, 0.3H), 2.89-2.94 (m, 0.7H), 3.40-3.80 (m, 2H), 4.10-4.50 (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 25 7.70 (m, 0.7H), 7.82 (d, H). For the syn diastereomer: 5 0.86-0.89 (m, H), 1.20-1.40 (m, 4H), 1.80-2.45 (m, 4H), 2.80-2.86 (m, H), 3.58-3.65 (m, 2H), -4.20-4.40 (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), 7.10-7.20 (m, 2.3H), 30 7.30-72.50 (m, 5.7H); Analytical HPLC: 10.55 min for anti diastereomer and 10.62 uin for syn diastereomer; LC/MS: m/e = 452 (M+H*) for both diastereomers.
- 222 4-Oxo-3-( [1- (2 -phenylamino -propionyl) -pyrrolidine-2 carbonyl] -amino)-butyric acid (121). Prepared from 120 (151 mg, 0.33 mmol) using hydrolysis method A to afford 101 mg (83% yield) of the title 5 compound as a white solid. 'H-NMR (500 MHz, CDC1 3
/CD
3 OD = 1/1) : 6 1.20-1.65 (m, 2H) , 1. 65-2.35 (m, 3H) , 2.40 3.00 (m, H), 3.20-3.80 (m, 2H), 3.90-4.90 (m, 7H), 7.25-7.80 (m, SH); Analytical HPLC: 6.38 min.; LC-MS: m/e = 362 (M+H*). 10 GENERAL PROCEDURES FOR THE PREPARATION OF COMPOUNDS OF EMBODIMENT C FORMULA I (SCHEMES XXIII-XXV) Scheme XXIII RR
H
3 0 OH H4 orH2 0R1 RH R3R3 R R 0 15 Hydrolysis Method A: A 0.005-50 mmole sample of the alkylhemiacetal was dissolved in 2.5 N HC1/ CH 3 CN (10/ 1) and stirred at room temperature until the reaction was complete. The 20 resulting aqueous layer was washed with diethyl ether (2 x 20 mL) and lyophilized to afford the product. Hydrolysis Method B: - 223 A 0.005-50 mmole sample of alkylhemiacetal was taken into neat formic acid and stirred overnight at room temperature. The mixture was triturated with a 3:1 mixture of hexane/diethyl ether to give a precipitate. 5 The solvent was decanted and the precipitate washed with diethyl ether to afford the product. Hydrolysis Method C: A 0.005-50 mmole sample of the alkylhemiacetal was 10 dissolved in CH 3 0H and 20% Pd(OH) 2 /C and stirred under
H
2 until the reaction was complete. The resulting suspension was filtered and the solution concentrated in vacuo, then triturated with a 3:1 mixture of hexane/diethyl ether to give a precipitate. The 15 solvent was decanted and the precipitate washed with diethyl ether to afford the product. Hydrolysis Method D: A 0.005-50 mmole sample of the alkylhemiacetal in 20 CH 3 CN/ water (1/ 2) was shaken with acidic resin (Dowex 50w x 2, H* type) until the reaction was complete. - The solution was filtered and the resin washed with
CH
3 CN/water (1/4). The resulting water layer was washed with diethyl ether, concentrated to a smaller volume in 25 vacuo then lyophilized to afford the product. Scheme XXIV Ri, I N Hg+0R N 0H O O N O0 H 76-98 122 - 224 0N HH / \0 122a 4-oxo-3- [(1-(2- [9-oxo-9H-fluorene-4-carbonyl) - amino] propionyl)-pyrrolidine -2 -carbonyl) - amino] -butyric acid 5 (122a) A 109.0mg (0. 19mmol) sample of 91 was hydrolyzed according to method A to afford 88mg (96% yield) of the title compound: Analytical HPLC 7.15min. LC-MS (ES+) m/e=492 .2 (M+H) 10 N .H 00 CI H O 122b 3- ({1- [2- (4-Amino-3-chloro-benzoylamino) -propionyl] pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric acid 15 (122b). A 51.0mg (0.096mmole) sample of 76 was hydrolyzed according to method A to afford 43.0mg (100% yield) of the title compound: IH-NMR (500 MHz, CD 3 0D/D 2 0: 0.5 mL/10 drops): 8 1.37-1.52 (m, 3H), 1.80-2.20 (m, 3H), 20 2.20-2.37 (m, H), 2.49-2.60 (m, H), 2.60-2.75 (m, H), 3.70-3.80 (m, H), 3.80-3.95 (m, H), 4.20-4.35 (m, H), 4.40-4.50 (in, 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: m/z 25 = 439 (M+H*).
- 225 C N rN H 122c 3-({1- [2- (3,5-Dichloro-4-methoxy-benzoylamino) propionyll -pyrrolidine-2-carbonyl) -amino) -4-oxo-butyric 5 acid (122c). A 51.0mg (0.088mmole) sample of 92 was hydrolyzed according to method A to afford 24.0mg (56% yield) of the title compound: Analytical HPLC 6.41min. LC-MS (ES*) m/e=488.3 (M+H). 10 0 H H~ OH 0I 0 1 22d 3 - ({l- [2- (4-Methoxy-3, 5-dimethyl-benzoylamino) propionyl] -pyrrolidine-2-carbonyl} -amino) -4 -oxo-butyric 15 acid (122d). A 55.0mg (0.102mmole) sample of 77 was hydrolyzed according to method A to afford 44.0mg (96% yield) of the title compound: Analytical HPLC (C18) 8.70min, 'H NMR (dDCl 3 , 500Mhz): 8 1.23-1.70 (m, 3H), 1.80-2.70 (m, 20 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 (im, 4H); LC-MS (ES*) m/e=448.2 (M+H).
- 226 N rN H H~~ 0 Ok 00 122e 4-Oxo-3- [ (1-{2- [pyridine-2-carbonyl) -amino] -propionyl} pyrrolidine-2-carbonyl) -amino] -butyric acid (122e). 5 A 55.0mg (0.114mmole) sample of 88 was hydrolyzed according to method A to afford 30.0mg (67% yield) of the title compound: Analytical HPLC 4.60min. LC-MS (ES*) m/e=391.3 (M+H). 0 .~k K ~rY OH C1 H O 10 122f 3 -({- [2 - (4 -Acetylamino-3-chloro-benzoylamino) propionyl] -pyrrolidine-2-carbonyl) -amino) -4 -oxo-butyric acid (122f). 15 A 52mg (0.091mmole) sample of 78 was hydrolyzed according to method A to afford 40mg (91% yield) of the title compound: 1 H NMR (500MHz, CD 3 0D) 8 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, 1H), 3.81-3.94 20 (m, 1H), 4.18-4.34 (m, 1H), 4.42-4.64 (m, 1.7H), 4.77 (q, 1H), 7.79 (dd, 1H); Analytical HPLC 4.97min. LC MS (ES*) m/e=481.3 (M+H).
- 227 C N N C? H4O 122g 3- ((1- 2- (4-Amino-3, 5-dichloro-benzoylamino) propionyll -pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric 5 acid (122g). A 44.3mg (0.079mmole) sample of 89 was hydrolyzed according to method A to afford 30mg (81% yield) of the title compound: Analytical HPLC 5.40min. LC-MS (ES*) m/e=473.2 (M+H). 10 H H 122h 3- ({1- [2- (3-Isopropoxy-benzoylamuino) -propionyll pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric acid 15 (122h). A 52.0mg (0.097mmole) sample of 79 was hydrolyzed according to method A to afford 30.0mg (69% yield) of the title compound: Analytical HPLC 8.92min. LC-MS (ES*) m/e=448.3 (M+H). 20 - 228 ojrN H 0 O 122i 3 - ((1- (2 - (3 -benzyloxy-4 -methoxy-benzoylamino) propionyl] -pyrrolidine-2 - carbonyl) -amino) -4 -oxo-butyric 5 acid (1221). A 50.8mg (0.082mmole) sample of 81 was hydrolyzed according to method A to afford 22.4mg (52% yield) of the title compound: Analytical HPLC 6.72min. LC-MS (ES*) m/e=526.3 (M+H). 10 N N NH N O O , O H 122j 4-Oxo-3- [(1-{2- [ (quinoxaline-2-carbonyl) -amino] propionyl) -pyrrolidine-2 -carbonyl) -amino] -butyric acid 15 (122j). A 38.0mg (0.072mmole) sample of 80 was hydrolyzed according to method A to afford 32.0mg (100% yield) of the title compound: Analytical HPLC 5.95min. LC-MS (ES*) m/e=442.3 (M+H). 20 00 HO ON H HOO 1 0 122k - 229 3 - ((1- [2- (3, 5-Dichloro-4 -hydroxy-benzoylamino) propionyll -pyrrolidine-2-carbonyl) -amino) -4 -oxo-butyric acid (122k). 5 A 35mg (0.060mmole) sample of 83 was hydrolyzed according to method A to afford 29.4mg (75% yield) of the title compound: Analytical HPLC 7.91min. 1 H-NMR (500 MHz, CD 3 0D) 8 1.47 (m, 3H) . 1.8-2.3 (m, 4H), 2.49 (m, 1H), 2.61 (m, 1H), 3.5 (br m, 0.2H), 3.69 (br m, 10 0.9H), 3.84 (br m, 0.99H), 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) . 00
H
2 N OH F F . 1221 15 3- ({1- [2- (4-Amino-3-trifluoromethyl-benzoylamino) propionyll -pyrrolidine-2 -carbonyl) -amino) -4 -oxo-butyric acid (1221). A 10mg (0..021mmole) sample of 98w was hydrolyzed 20 according to method A to afford 7.9mg (94% yield) of the title compound: Analytical HPLC 6.64min. LC-MS (ES*) m/e=473.3 (M+H) . M N H C,0 0 122m 25 - 230 3 -({l- [2 - (3 -Chloro-4 -dimethylamino-benzoylamino) propionyll -pyrrolidine-2 - carbonyl)-amino) -4-oxo-butyric acid (122m). A 10.0mg (0.021mmole) sample of 98x was hydrolyzed 5 according to method A to afford 7.0mg (84% yield) of the title compound: Analytical HPLC 5.15min. LC-MS (ES-) m/e=467.3 (M+H). F rN H O H 00 122n 10 3- ({1- [2- (4-Dimethylamino-3,5-difluoro-benzoylamino) propionyl -pyrrolidine-2 -carbonyl)-amino) -4-oxo-butyric acid (122n). A 20.0mg (0.043mmole) sample of 98y was hydrolyzed 15 according to method A to afford 16.8mg (100% yield) of the title compound: Analytical HPLC 5.86min. LC-MS
(ES
4 ) m/e=469.3 (M+H). 40, H 00 122o 20 3- ({1- [2- (4-Amino-3-chloro-benzoylanino) -propionyll pyrrolidine-2-carbonyl}-amino) -4-oxo-butyric acid (122o). A 20.0mg (0.046mmole) sample of 98m was hydrolyzed 25 according to method A to afford 16.7mg (100% yield) of - 231 the title compound: Analytical HPLC 8.47min. LC-MS (ES*) m/e=439.2 (M+H). F N H 0Y OH
H
2 N F OTN H 00 122p 5 3- ((1- [2- ( 4 -Amino-2, 3, 5, 6 - tetrafluoro-benzoylamino) propionyll -pyrrolidine-2-carbonyl) -amino) -4-oxo-butyric acid (1 2 2p). A 20.0mg (0.042mmole) sample of 98z was hydrolyzed 10 according to method A to afford 15.3mg (91% yield) of the title compound: Analytical HPLC 7.90min. LC-MS (ES*) m/e=477.2 (M+H)'. 0 19~N H 0 T H 00 122q 15 4-Oxo-3- [ (1-(2- [ (quinoline-6-carbonyl) -amino) prop ionyl) -pyrrolidine -2- carbonyl) -amainol -butyric acid (122q). A 44mg (0.080mmole) sample of 93 was hydrolyzed 20 according to method A to afford 41mg (100% yield) of the title compound: 1 H NMR (500MHz, CD 3 0D) 5 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, IH), 9.27-9.39 (m, - 232 2H); Analytical HPLC 4.91min. LC-MS (ES+) m/e=441.2 (M+H). H OH INJO N T H ClH O 122r 5 3- ({1- [2- (4-Acetylanino-5-chloro-2-methoxy benzoylamnino) -propionyl) -pyrrolidine-2-carbonyl) amino)-4-oxo-butyric acid (122r). A 44.5mg (0.074mmole) sample of 87 was hydrolyzed 10 according to method A to afford 34.5mg (91% yield) of the title compound: Analytical HPLC 6.88min. LC-MS (ES*) m/e=511.2 (M+H). Nj N H H H C1 122s 15 3- [(l-{2- [3-Chloro-4- (2, 2-dimethyl-propionylamino) benzoylamaino] -propionyl) -pyrrolidine-2 -carbonyl) amino]-4-oxo-butyric acid (122s). A 19.0mg (0.036mmole) sample of 98aa was hydrolyzed 20 according to method A to afford 14.5mg (90% yield) of the title compound: Analytical HPLC 7.28min. LC-MS (ES*) m/e=523.3 (M+H) - 233 N N HH H C1 0 122t 3 - ({1- [2 - ( 3 - Chloro-4 -propionylamino -benzoylamino) propionyl] -pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric 5 acid (122t). A 21.0mg (0.042mmole) sample of 98ab was hydrolyzed according to method A to afford 17.5mg (97* yield) of the title compound: Analytical HPLC 5.72min. LC-MS (ES*) m/e=495.2 (M+H) . 10 0 H H 122u 3 - ({l- (2 - ( 3 -Chloro-4 -phenylacetylamino-benzoylamino) propionyl] -pyrrolidine-2-carbonyl)-amino) -4-oxo-butyric 15 acid (122u). A 10.0mg (0.017mmole) sample of 98ac was hydrolyzed according to method A to afford 7.9mg (85* yield) of the title compound: Analytical HPLC 7.52min. LC-MS (ES*) m/e=557.2 (M+H). 20 ' NJ NO ) H H HC1 H 122v - 234 3- [(1-{2- (3-Chloro-4- (3-methyl-butyrylamino) benzoylaminol -propionyl)-pyrrolidine-2 -carbonyl) amino]-4-oxo-butyric acid (122v). 5 An 8.0mg (0.015mmole) sample of 98ad was hydrolyzed according to method A to afford 6.5mg (96% yield) of the title compound: Analytical HPLC 6.92min. LC-MS (ES*) m/e=523.2 (M+H). Scheme XXV 0 Q20 2 O N N N 2H Y H H H 0 K 108a, X=CI, Y=NH 2 , Z=H, Q1=F, Q2=H 123a, X=CI, Y=NH 2 , Z=H, Qi=F. Q2=H 116b. X=CI, Y=AcNH, Z=H, Qi=Q2=F 123b, X=CI. Y=AcNH, Z=H, Qi=Q 2 =F 10 116c, X=CI, Y=AcNH, Z=CH3O,Q1=Q 2 =F 123c, X=CI, Y=AcNH, Z=CH 3
O,
1 =Q2=F 3- ((1- (2- (4-Amino-3-chloro-benzoylaimino) -propionyl) -4 fluoro-pyrrolidine-2-carbonyl}- amino) -4-oxo-butyric acid (123a). 15 A 12.4mg (0.022mmole) sample of 108b was hydrolyzed according to method A to afford 9.6mg (93% yield) of the title compound: Analytical HPLC 6.99min. LC-MS (ES*) m/e=473.2 (M+H). 20 3-({l- [2- (4-Acetylamino-3-chloro-benzoylaaino) propionyll -4, 4-difluoro-pyrrolidine-2-carbony )-aino) 4-oxo-butyric acid (123b). A 26.2mg (0.043mmole) sample of 116b was hydrolyzed according to method A to afford 10.8mg (49% yield) of 25 the title compound: Analytical HPLC 9.89min. LC-MS (ES*) m/e=517.2 - 235 3-((1- [2-(4-Acetylamino-3-chloro-2-methoxy benzoylamino) -propionyl] -4,4-difluoro-pyrrolidine-2 carbonyl)-amino)-4-oxo-butyric acid (123c). 5 A 23.1mg (0.036mmole) sample of 116c was hydrolyzed according to method A to afford 1.8mg (9% yield) of the title compound: Analytical HPLC 11.87min. LC-MS (ES*) m/e=547.1 (M+H). 10 BIOLOGICAL METHODS we obtained in vitro, ex vivo, and in vivo data for selected compounds of this invention using the methods described below. The results are shown in the Tables 2-8. The designation "ND" indicates that the 15 compound was not tested in the described assay. In the ICE Caspase assays, category "A" indicates <10 nM inhibition. Category "B" indicates 10-1000 nM inhibition. Category "C" indicates >1000 nm inhibition. See Tables 2 and 3. 20 In the PBMC assay, category "A" indicates <500 nM inhibition. Category "B" indicates 500-1000 nM inhibition. Category "C" indicates 1001-2000 nM inhibition. Category "D" indicates >2000 nM inhibition. See Table Table 4. 25 In the whole blood assay, category "A" indicates <2500 nM' inhibition. Category "B" indicates 2500-7500 nM inhibition. Category "C" indicates >7500 riM. See Table 5 In the in situ metabolism assay, values of 30 [f(g) X f(h)] are disclosed 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 biliary excretion measurement, - 236 category "A" indicates <5%. Category "B" indicates 5 10%. Category "C" indicates >10%. See Table 6. In the i.v. clearance assay, values are reported as follows: category "A" indicates <50. 5 Category "B" indicates 50-80. Category "C" indicates >80. See Table 7. In the bioavailability assay, the Cmax values (pg/ml) are disclosed as follows: category "A" indicates <2.5. Category "B" indicates 2.5-5.0. 10 Category "C" indicates >5.0. The AUC values (psg x hr/ml) are disclosed as follows: category "A" indicates <2.5. Category "B" indicates 2.5-5.0. Category "C" indicates >5.0. Half-life (hrs) ranges are disclosed as follows: category "A" indicates <1.5. 15 Category "B" indicates 1.5-2.0. Category "C" indicates >2.0. The F values (%) are disclosed as follows: category "A" indicates <33. Category "B" indicates 33 67. Category "C" indicates >67. See Table 8. In Vitro Assays 20 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 in 10 mM Tris (Sigma Corp, St Louis MO) 25 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, a solution of 8% glycerol was added to the assay buffer to improve enzyme stability. A 65 pL aliquot of the assay buffer and 5 AL aliquot of 30 the appropriate dilutions of inhibitor in DMSO where pipetted into a 96 well plate, treated with 10 AL of caspase, then diluted in assay buffer (0.5-40 nM active protein by active site titration). A control - 237 containing DMSO but no compound was included for each determination. The plates were then incubated for 15 minutes at 37 *C, before addition of the appropriate substrate (20 pL, final concentration 1-4 X KM, final 5 assay volume 100 pL) to initiate the reaction. Reaction rates were measured at 37 0 C either by following the time dependant increase in absorbance at 405 nM (for the pNA substrates) or in fluorescence (Ex 390, Em 460) (for the AMC substrates). The rates 10 obtained were plotted against inhibitor concentration and the data fit to the Morrison tight-binding equation for competitive inhibitors (Morrison, J.F., Biochem. Biophys. Acta, 185 pp. 269-286 (1969)). The substrates used for the individual assays were as follows: 15 Caspase-1 Suc-YVAD-pNA (Bachem, King of Prussia, PA) (final concentration in the assay 80 pM), Caspase-3 Ac-DEVD-pNA (Bachem, -King of Prussia, PA) (final concentration in assay, 60 uM) Caspase-4 Ac-WEHD-AMC (Synpep, Dublin, CA) (final 20 concentration in Assay 20 pM), Caspase-7 Ac-DEVD-AMC (Bachem, King of Prussia, PA) (final concentration in assay 50 pM), Caspase-8 Ac-DEVD-pNA (Bachem, King of Prussia, PA) (final concentration in assay 80 pM). 25 Table 2. Caspase-1 Inhibition Data Example Caspase-1 Ki (nM) 5a A 5b A Sc A 5d A Se B Sf
B
- 238 Example Caspase-1 Ki (nM) Sg B 5h A 5i A S j A 5k A 51 B Sm A Sn A So B 5p B 5q B Sr B 5s B 5t C 5u B 5v B Sw B 5x A Sy A 5z A Saa A Sab B Sac A Sad A 5ae B Saf B 5ag A Sah B Sai A Saj B Sak jB 239 Example Caspase-1 Ki (nM) Sal A Sam A San B Sao B Sap B Sag B Sar A Sas A Sat B Sau B Sav B Saw A Sax A 5ay A Saz A 5ba A 5bb A 5bc B 5bd A 7a A 7b B 7c A 7d A 7e B 7f B 7g A 7h B 7i B 7j C 7k B 71
B
- 240 Example Caspase-1 Ki (nM) 7m B 7n B 70 A 7p A 7q B 7r B 7s B 7t B 7u B 7v B 7w A 7x B 7y B 7Z B 7aa A 7ab B 7ac B 7ad B 7ae B 7af B 7ag B 7ah A 7ai A 7aj A 7ak A 7al B 7am A 7an A 7ao B 7ap B 7ag B - 241 Example Caspase-1 Ki (nM) 7ar A 7as A 7at A 9a B 9b A 9c A 9d A 9e A 9f A 99 A 15a A 15b A 15c A 15d B 15e B 1Sf B 16a B 16b A 17a B 17b B 17c A 17d B 17e B 18a B lb A 1sc B 18d B 18e A 18f B 20a A 20b
A
-242 Exml Caspase-I Ki (nM) 20c A 20d B 20e A 20f A 20g A 20h A 20i B -20Oj B 20k A 201 A 20m A 20n A 20o A 20p A 20q B 20r B 20s A 20t B 23a A 23bB 23c A 23d A 23e A 23f B 23q A 23h A 231 B 24a A 24b C 24c A 24d B - 243 Exml Caspase-I Ki (riM) 24e B 25a A 25b A 25c A 25d A 25e A 26a A 26b A 26c A 26d A 26e A 26f A 26g A 26h A 27a B 27b B 27c B 27d A 27e B 27f B 27g A 27h B 271 B 27J B 27k B 271 B 27m B 27n B 28a A 28b A 28c -A - 244 Example Caspase-1 Ki (nM) 29a A 29b A 29c A 29d A 29e A 29f A 29g A 29h A 29i A 29j A 29k A 291 B 29m A 29n B 29o B 29p A 29q A 29r A 29s B 32a C 32b C 32c C 32d C 32e B 34 C G1 C G2 B 42 B 46b A 46a C 57 A - 245 Example Caspase-1 Ki (nM) 65 A 61 A 69 A 73 A 121 C 122a A 122b A 122c A 122d A 122e B 122f A 122g A 122h B 122i A 122j B 122k A 1221 B 122m B 122n. B 122o C 122p A 122q B 122r B 122s B 122t B 122u A 122v B 123a B 123b B 123c
B
- 246 Table 3. Caspase-3, Caspase-4, and Caspase-8 Inhibition Data Example Caspase-3 Ki Caspase-4 Ki Caspase-8 Ki (M) (nM) (nM) 7c C ND C 7d C ND B 7f C ND C 24a C ND ND 29a C ND ND 29b C ND ND 32d B ND ND 46b B ND ND 69 C ND B 122b C A B 122d C A C 122f C ND B 122k C ND B 5 PBMC Cell Assay IL-1p Assay with a Mixed Population of Human Peripheral Blood Mononuclear Cells (PBMC) or Enriched Adherent Mononuclear Cells Processing of pre-IL-1p by ICE can be 10 measured in cell culture using a variety of cell sources. Human PBMC obtained from healthy donors provides a mixed population of lymphocyte subtypes and mononuclear cells that produce a spectrum of interleukins and cytokines in response to many classes 15 of physiological stimulators. Adherent mononuclear cells from PBMC provides an enriched source of normal monocytes for selective studies of cytokine production by activated cells.
- 247 Experimental Procedure: An initial dilution series of test compound in DMSO or ethanol is prepared, with a subsequent 5 dilution into RPMI-10% FBS media (containing 2 mM L glutamine, 10 mM HEPES, 50 U and 50 ug/ml pen/strep) respectively to yield drugs at 4x the final test concentration containing 0.4% DMSO or 0.4% ethanol. The final concentration of DMSO is 0.1% for all drug 10 dilutions. A concentration titration which brackets the apparent Ki for a test compound determined in an ICE inhibition assay is generally used for the primary compound screen. Generally 5-6 compound dilutions are tested 15 and the cellular component of the assay is performed in duplicate, with duplicate ELISA determinations on each cell culture supernatant. PBMC Isolation and IL-1 Assay: 20 Buffy coat cells isolated from one pint human blood (yielding 40-45 ml final volume plasma plus cells) are diluted with media to 80 ml and LeukoPREP separation tubes (Becton Dickinson) are each overlaid with .10 ml of cell suspension. After 15 min 25 centrifugation-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). Media is added to.bring the volume to 15 ml, gently mix the 30 cells by inversion and centrifuge at 300 xg.for 15 min. The PBMC pellet is resuspended in a small volume of media, the cells are counted and adjusted to 6 x 106 cells/ml.
- 248 For the cellular 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 test compound dilution and 0.5 ml LPS solution (Sigma #L 5 3012; 20 ng/ml solution prepared in complete RPMI media; final LPS concentration 5 ng/ml). The 0.5 ml additions of test compound and LPS are usually sufficient to mix the contents of the wells. Three control mixtures are run per experiment, with either 10 LPS alone, solvent vehicle control, and/or additional media to adjust the final culture volume to 2.0 ml. The cell cultures are incubated for 16-18 hr at 37 *C in the presence of 5% Co2 At the end of the incubation period, cells 15 are harvested and transferred to 15 ml conical centrifuge tubes. After centrifugation for 10 min at 200 xg, supernatants are harvested and transferred to 1.5 ml Eppendorf tubes. It may be noted that the cell pellet may be utilized for a biochemical evaluation of 20 pre-IL-1p and/or mature IL-i0 content in cytosol extracts by Western blotting or ELISA with pre-IL-1p specific antisera. Isolation of Adherent Mononuclear cells: 25 PBMC are isolated and prepared as described above. Media (1.0 ml) is first-added to wells followed by 0.5 ml of the PBMC suspension. After a one hour incubation, plates are gently shaken and nonadherent cells aspirated from each well. Wells are then gently 30 washed three times with 1.0 ml of media and final resuspended in 1.0 ml media. The enrichment for adherent cells generally yields 2.5-3.0 x 105 cells per well. The addition of test compounds, LPS, cell - 249 incubation conditions and processing of supernatants proceeds as described above. ELISA: 5 Quantikine kits (R&D Systems) may be used for the measurement of mature IL-10. Assays are performed according to the manufacturer's directions. Mature IL lp levels of about 1-3 ng/ml in both PBMC and adherent mononuclear cell positive controls are observed. ELISA 10 assays are performed on 1:5, 1:10 and 1:20 dilutions of supernatants from LPS-positive controls to select the optimal dilution for supernatants in the test panel. The inhibitory potency of the compounds can be represented by an IC 50 value, which is the 15 concentration of inhibitor at which 50% of mature IL-lP is detected in the supernatant as compared to the positive controls. The skilled practitioner realizes that values obtained in cell assays may depend on multiple factors. 20 The values may not necessarily represent fine quantitative results. Table 4. PBMC Cell Assay Data Example PBMC IC50 (nM) Sa D 5b B Sc B Sd B Se B 5f C Sg C. 5h
A
- 250 Example PBMC IC50 (nM) Si C 5j D 5k D 51 A 5m A 5n B Sr D Ss B 5u C 5v D Sw B 5x B Sy B 5z B Saa B Sab B Sac A Sad A Sag B Sai A Saj B Sak B Sal D Sam D Sao D Sag D Sar D Sas D Sat D 5au D Say D - 251 Ex l PBMC 1C50 (nM) Saw C Sax B Say B Saz B Sba C Sbb B 5bd C 7a D 7b B 7c A 7d A 7e D 7f D 7g A 7h B 7k B 71 B 7m B *7n B 70 A 7p D ?q D .7s___ B 7t D2 7u D 7v D 7w C 7x_ D2 7y1D 7z C 9a B -252 Example PBMC IC50 (riM) 9b B 9c A 9d B 9e C 9f B 9g C 15a D 15b C 15c B 15d C i~e D 15f D 16a A 16b C 17b C 17c D 17d D 17e B 18a B lab B 18c B 18d B 18e C 18f 12 20a B 20b. B 20c C 20d D 20e C 20f B 20g A -253 Example PBMC ICSO (nM) 20h A 20i B 20j D 20k A 201 B 20m B 20n B 20o A 20p A 20qB 20r B3 20s C 20t B 23a C 23b B 23c C 23d B 23eB 23f C 23g C 23h -C 23i A 24a B 24b D 24c A 24d B 24e C 25a A 25b B 25c B 26a C - 254 Example PBMC IC50 (nM) 26b B 26c B 26d B 26e A 26f A 26g A 26h A 27a A 28a B 28b B 28c B 29a A 29b C 29c B 29d B 29e A 29g B 29h A 29i A 29j B 29k A 291 B 29m B 42 D 46b D 57 B 65 B 61 B 69 A 73 B 122a D - 255 Example PBMC IC50 (nM) 122b B 122c D 122d B 122e C 122f B 122g B 122h C 122i C 122j D 122k A 1221 B Whole Blood Assay for IL-10 Production Whole blood assay IC 50 values for compounds 5 of this invention were obtained using the method described below: Purpose: The whole blood assay is a simple method for 10 measuring the production of IL-lp (or other cytokines) and the activity of potential inhibitors. The complexity of this assay system, with its full complement of lymphoid and inflammatory cell types, spectrum of plasma proteins and red blood cells is an 15 ideal in vitro representation of human in vivo physiologic conditions. Materials: Pyrogen-free syringes (~ 30 cc) 20 Pyrogen-free sterile vacuum tubes containing lyophilized Na 2 EDTA (4.5 mg/10 ml tube) - 256 Human whole blood sample ( 30-50 cc) 1.5 ml Eppendorf tubes Test compound stock solutions (~ 25mM in DMSO or other solvent) 5 Endotoxin -free sodium.chloride solution (0.9%) and HBSS Lipopolysaccharide (Sigma; Cat.# L-3012) stock solution at 1mg/ml in HBSS IL-1P ELISA Kit (R & D Systems; Cat # DLB5O) 10 TNFa ELISA Kit (R & D Systems; Cat # DTA50) Water bath or incubator Whole Blood Assay Experimental Procedure: Set incubator or water bath at 30 *C. 15 Aliquot 0.25ml of blood into 1.5 ml eppendorf tubes. Note: be sure to invert the whole blood sample tubes after every two aliquots. Differences in replicates may result if the. cells sediment and are not uniformly suspended. Use of a positive displacement pipette will 20 also minimize differences between replicate aliquots. Prepare drug dilutions in sterile pyrogen free saline by serial dilution. A dilution series which brackets the apparent Ki for a test compound determined in an ICE inhibition assay is generally used 25 for the primary compound screen. For extremely hydrophobic compounds, prepare compound dilutions in fresh plasma obtained from the same blood donor or in PBS-containing 5% DMSO to enhance solubility. Add 25 pl test compound dilution or vehicle 30 control and gently mix the sample. Then add 5.0 pl LPS solution (250 ng/ml stocked prepared fresh: 5.0 ng/ml final concentration LPS), and mix again. Incubate the tubes at 30 *C in a water bath for 16-18 hr with occasional mixing. Alternatively, the tubes can be - 257 placed in a rotator set at 4 rpm for the same incubation period. This assay should be set up-in duplicate or triplicate with the following controls: negative control - no LPS; positive control - no test 5 inhibitor; vehicle control - the highest concentration of DMSO or compound solvent used in the experiment. Additional saline is added to all control tubes to normalize volumes for both control and experimental whole blood test samples. 10 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 centrifuged at 1000 x g to pellet residual platelets if necessary. Plasma samples may be 15 stored frozen at -70 *C prior to assay for cytokine levels by ELISA. ELISA: R & D Systems (614 McKinley Place N.E. 20 Minneapolis, MN 55413) Quantikine kits may be used for measurement of IL-1p and TNF-a. The assays are performed according to the manufacturer's directions. IL-1p levels of - 1-5 ng/ml in positive controls among a range of individuals may be observed. A 1:200 25 dilution of plasma for all samples is usually sufficient for experiments for ELISA results to fall on the linear range of the ELISA standard curves. It may be necessary to optimize standard dilutions if you observe differences in the whole blood assay. Nerad, 30 J.L. et al., J. Leukocyte Biol., 52, pp. 687-692 (1992).
- 258 Table 5. Whole Blood Assay Data Example Whole Blood ICSO (nM) 5a A 5b A 5c A Sd B Se A Sf B Sh A Si C Sj C 5k B 51 C Sm A Sn B Sr C Ss C Su A 5w A 5x A 5y B 5z C 5aa A Sab B Sac A Sad A Sag B Sai C Saj C Sak C Sax B 5ay.
B
- 259 Example Whole Blood IC50 (rinm) 5az A 5bb B 7a B 7b A 7c A 7d B 7e C 7f B 7g B 7h A 7k A 71 A 7m B 7n A 70 A 7p B 7q A 7s B 7t B 7u B 7v A 7w A 7x c 7z A 7aa B 7ab A 7ac B 7ad B 7ah B 7ai
B
-260 Example Whole Blood ICSO (n4) 7aj C 7ak B 7am B 7 anB 7 ao __ _ _ _ _ _ _ _ _ _ _ _ 7 ap 9a 1 9bB 9c A 9d A 9e B 9f A 9g B 15a B 15b B 15c A 16b A 18a A 18b A lac A 18dA l8e A__ _ _ _ __ _ _ _ _ _ 18fB 20a A__ _ _ _ _ _ _ _ _ _ 20 Ob 20cB 20Od B__ _ _ _ __ _ _ _ _ 20 Be 20f A__ _ _ _ _ _ _ _ _ _ _ 20g A 20h
A
- 261 Example Whole Blood IC50 (nM) 20i A 20k A 201 A 20m A 20n A 20o A 20p A 20g c 20r B 20s A 20t A 23a B 23b B 23c B 23d A 23e B 23f A 23g A 23h A 23i B 24a B 24c B 24d B 24e B 25a B 25b B 25c B 25d A 25e C 26c B 26d
A
- 262 Example Whole Blood IC50 (iM) 26e A 26f B 26g B 26h A 27a A 27b A 27d A 27e A 27f B 27g B 27h B 27i B 271 B 27m B 27n A 28a B 28b B 28c c 29a A 29c A 29d A 29e A 29g A 29h A 291 A 29j A 29k A 291 A 29n B 29o A 29p
A
- 263 Example Whole Blood ICSO (nM) 29g A 29r A 29s A G2 B 42 B 46b B 57 c 65 A 61 A 69 A 73 A 122a A 122b A 122c B 122d A 122e B 122f A 122g A 122h A 122i A 122J B 122k A 1221 A 122m B 122p B 122q B 122r B 122s B 123a A 123b
B
- 264 Ex Vivo Assays Metabolism and Excretion Single pass perfusion studies in rat were performed to assess gastrointestinal (GI) wall 5 metabolism (f (g) ), liver metabolism (f (h)) , and biliary excretion. The method used has been described in Pang, C.S., J. Pharmacol. Exp. Therapeutics, 333, pp. 788-798 (1984). 10 Table 6. Metabolism and Excretion Data Example f(g)Xf(h) Biliary Excretion 5c A C Sk B A 5m B C 7d D A 7f C A 7ac C C 18f D A 20f B C 20g B A 23b B B 24a C A 24c A C 24e A C -25d C C 25e C 26c A C 26e B 26f A C 27a C A 27b B A 29b A
B
- 265 Example f (g)Xf (h) Biliary Excretion 299 B B 29n C A 29o C A 29p B C 29q C A 29r C B 46b, B C 57 B B 65 B C 69 C A 122a B A 122b C A 122c C B 122d C A 122r B A In Vivo Assays In vivo Rat Clearance Assay - Clearance Rates The rate of clearance in the rat (ml/min/kg) 5 for compounds of this invention may be obtained using the method described below: Representative Procedure Cannulations of the jugular and carotid vessels of rats under anesthesia are performed one day 10 prior to the pharmacokinetic study. M.J. Free, 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 (lOmg/mL) is administered via the jugular 15 vein in a vehicle usually consisting of: propylene glycol/saline, containing 1OOmM sodium bicarbonate in a 1:1 ratio. Animals are dosed with 10-20 mg drug/kg and - 266 blood samples are drawn at 0, 2, 5, 7, 10, 15, 20, 30, 60, and 90 minutes from an indwelling carotid catheter. The blood is centrifuged to plasma and stored at -20 "C until analysis. Pharmacokinetic analysis of data is 5 performed by non-linear regression using standard software such as RStrip (MicroMath Software, UT) and/or Pcnonlin (SCI Software, NC) to obtain clearance values. Representative Analytical: 10 Rat plasma is extracted with an equal volume of acetonitrile (containing 0.1% TFA) . Samples are then centrifuged at approximately 1,000 x g and the supernatant analyzed by gradient HPLC. A typical assay procedure is described below; 15 200 pL of plasma is precipitated with 200 AL of 0.1% trifluoroacetic acid (TFA) in acetonitrile and 10 yL of a 50% aqueous zinc chloride solution, vortexed then centrifuged at ~1000 x g and the supernatant collected and analyzed by HPLC. 20 HPLC procedure: Column: Zorbax SB-CN (4.6 x 150 mm) (SA particle size) Column temperature: 50 *C 25 Flow rate: 1.0 mL/min Injection volume: 75 pL. Mobile phase: A=0.1% TFA in water and B=100% acetonitrile Gradient employed: 100% A to 30% A in 15.5 min 30 0% A at 16 min 100% A at 19.2 min Wavelength: 214 .nm - 267 A standard curve is run at 20, 10, 5, 2 and 1 pg/mL concentrations. Table 7. Clearance Data Example Rat I.V. Clearance (ml/min/kg) 7d A 7f B 20h B 20m A 65 C 122b B 122c C 122d B 122f A 5 Bioavailability Oral pharmacokinetic studies Male Sprague-Dawley rats (Harlan, Indianapolis, IN, 300-350 g) were anesthetized by an intramuscular 10 injection of ketamine/rompun mixture. A PE-50 cannula was inserted in the right carotid artery for arterial blood sampling. The rats were allowed to recover from surgery overnight (.16 hours) prior to being used in the study. Test compounds were administered orally in 15 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 separated by centrifugation and stored at -20 0 C pending analysis. 20 Quantitation of the plasma samples was conducted using a gradient HPLC/MS/MS or enzymatic method detailed below: - 268 HPLC/MS/MS Method for the quantitation of ICE inhibitors in rat plasma Sample Preparation e 50pl of plasma are aliquotted into Ependorf 5 centrifuge vials. * An equal volume of Acetonitrile is added to the plasma to precipitate plasma proteins. * Samples are Votexed for 5 minutes, and centrifuged at 14,000 rpms for 5 minutes. 10 e 7 5pl of the supernatant is loaded into 12mm HPLC liquid sampler vials. e 50pl of sample is injected for analysis via the mass spectrometer. 15 HPLC Instrumental Parameters HPLC: Hewlett Packard HP1100 Binary Solvent Delivery System. HPLC Gradient Conditions 20 A = H 2 0 0.2% Formic Acid B = Acetonitrile 0.2% Formic Acid Mobile Phase Time %A %B 25 0 100 0 2 100 0 5 0 100 11 0 100 30 11.5 100 0 17 100 0 HPLC Analytical Column: Keystone Phenyl -2 Hypersil 2.Ox1OOmm, 5p 120A pore pore size, P/N# 105-39-2 - 269 Injection Volume: 50pl Flow Rate: 0.20 mL/min. Mass Spectrometry Instrumental Parameters 5 Instrument: P E Sciex API-365 Tandem Mass Spectrometer Ionization Technique: Turbo-Ionspray (ESI) Polarity: Positive Dwell Time: 300msec Pause Time: Smsec 10 Scan time: 0.9sec Scan Mode: MRM (Multiple Reaction Monitoring) ICE ENZYMATIC ASSAY FOR THE QUANTITATION OF ICE 15 INHIBITORS IN RAT PLASMA 50 pL of plasma was extracted with 150 pL acetonitrile,sonicated, vortexed, centrifuged at 10,000xg and 180 pL of the supernatant dried in a Sorvall vortex evaporator at room 20 temperature. Samples were reconstituted in 100 pL buffer (10 mM tris-HCl, pH 7.5 with 0.1%CHAPS, 1 mM DTT) with sonication. 10 AL of each sample was mixed with 10 pL ICE.(l.1 mg/mL) in a microtitre plate with 60 AL buffer. Samples were incubated for 15 min. at 25 room temperature then 20 pL Succ YVAD-pNA(400 pM, prewarmed to 37*C) added, and the plate monitored at 405 nm for 20 min. at 37 0 C used a SpectraMax reader. The data were fitted using a 4 parameter fit with the the SpectraMax software using an extracted standard 30 curve. The assay was linear from 0.15 to 2.0-3.0 pg/mL aldehyde.
- 270 Pharmacokinetic Parameters Pharmacokinetic analysis of these plasma concentration data was conducted using noncompartmental methods . The area under the curve (AUC(O-t)) was estimated from time 5 zero to the last measured time point using the linear trapezoidal rule. The rate of elimination (ke) was estimated by log-linear regression from the terminal phase of the plasma concentration-time curves. Area under the tail of the curve was estimated as the ratio 10 of the last measured concentration to ke. The area under the curve from time zero to infinity (AUC(O-oo)) was obtained by addition of the area under the tail to AUC(0-t). Elimination half-life was estimated as 0.693/ke. The observed values for the peak plasma 15 concentration (Cmax) were recorded. For prodrug studies: aldehyde availability (bioavailability) was calculated as: (AUCald/prodrug p.o.)/ (AUCald/ald i.v.) x (dose ald, ald i.v./dose prodrug, prodrug p.o.) x (MW prodrug/MW aldehyde). 20 Table 8. Bioavailability Data Example Cmax AUC t 1/2 (hrs) F (%) (pg/mL) (pgXh/mL) 56 A B A 45 A B 90 C C A C 85 A B B A 68 A C B 76 C C C 77 B B A A 78 A A B A 89 B C C 83 A C C - 271 Example Cmax AUC t 1/2 (hrs) F (%) (pg/mL) (pgXh/mL) 98d A A B 98h A C B 98e C C B 98c B C C 98k B C B 98ae -A A B 98af A A B 98b C C B 111 A A C 980 A A B 108a A A B 98ag C C B C 98a B B C 98am A A B 116a C C B 98an A A B 116g A A C 116h A A B 116e A A B 108b A A B Antiviral Assays The efficacy of the compounds of this invention at treating or preventing antiviral related 5 diseases, disorders, or conditions may be evaluated in various in vitro and in vivo assays. For example, assays may be preformed to determine the ability of these compounds to inhibit inflammatory responses associated with viral infections. In vitro assays may 10 employ whole cells or isolated cellular components. In vivo assays include animal models for viral diseases.
- 272 Examples of such animal models include, but are not limited to, rodent models for HBV or HCV infection, the Woodchuck model for HBV infection, and chimpanzee model for HCV infection. 5 Compounds of this invention may also be evaluated in animal models for dietary alcohol-induced disease. Other assays that may be used to evaluate the compounds of this invention are disclosed in PCT 10 application PCT/US96/20843, published 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, in vivo acute assay for anti-inflammatory efficacy, 15 measurement of blood levels of drugs, IGIF assays, mouse carrageenan peritoneal inflammation assay, and type II collagen-induced arthritis.. Insofar as the compounds of this invention are able to inhibit caspases, particlularly ICE, in 20 vitro and furthermore, may be delivered orally to mammals, they are of evident clinical utility for the treatment of IL-1-, apoptosis-, IGIF-, and IFN-y mediated diseases. While we have described a number of 25 embodiments of this invention, it is apparent that our basic constructions may be altered to provide other embodiments which utilize the products and processes of this invention. The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any - 273 other integers, unless in the context or usage an exclusive interpretation of the term is required. Any reference to publications cited in this specification is not an admission that the disclosures 5 constitute common general knowledge in Australia.

Claims (19)

1. A compound represented by formula I: 0 R R' R N N N R X N ~ 4 0 wherein: Y is: (a) R7 H 0 provided that when R 7 is -OH then Y can also be: (b) O 0 m H H ; X is -C(R 3 )2- or -N(R3). m is 0 or 1; R 1 is H, -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(H)-R 8 , -S(O)N(H)-R 8 , -C(O)C(O)N(H)R 8 , -C(0)CH=CHR 8 , -C(O)CH 2 OR 8 , -C(O)CH 2 N(H)R 8 , -C(O)N(R 8 ) 2 , -S(0)2N(R 8 ) 2 , -S(O)N(R 8 ) 2 , -C(O)C(O)N(R 8 ) 2 , -C(O)CH2N(R 8 ) 2 , ~ CH 2 R 8 , -CH 2 -alkenyl-R 8 , or -CH 2 -alkynyl-R 8 ; or R 1 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)aryl, -C(O)heteroaryl, or -C(O)heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally - 275 replaced by -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -CO2H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -N(H)alkyl, -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)N(H)alkyl, -N(H)C(O)N(alkyl)2, -S-alkyl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH 2 N(alkyl)2, or -alkyl, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by Ril, and a hydrogen atom bound to any nitrogen atom is optionally replaced by RI; 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 one R 3 together with the atoms to which they are bound, form a 3 to 7 membered cyclic or heterocyclic ring system, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by -Ril, a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -R 1 ; R 4 and one R 5 together with the atoms to which they are bound form a ring system: R10 N and the other R 5 is H; 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, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by Ril, and a - 276 hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 9 is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R10, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 10 is independently -OH, -SH, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -CO 2 H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-alkylaryl, -N(H)alkyl, N(H)aryl, -N(H)-alkylaryl, -N(alkyl)2, -C(O)N(H)alkyl, C(0)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(O)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH 2 NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 1 1 and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; and each R 11 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -CO2H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -alkyl, -cycloalkyl, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH 2 N(H)alkyl, or -CH2N(alkyl)2.
2. A compound represented by formula I: O R' R 5 R 2 R 6 N N XN Y R4 0 - 277 wherein: Y is: 0 OR 8 0 OR 8 m m R 8 0 H Ra , ,or H H OR 8 OR 1 2 N.R H (C) (d) (e) (t) R 6 is H or R 6 and Y, together with the nitrogen to which they are bound, form a ring (g): m NI R13 R13 (g) X is -C(R 3 )2- or -N(R3)_ m is 0 or 1; R 1 is H, -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)CH 2 OR 8 , -C(0)CH 2 N(H)R 8 , -C(O)N(R 8 ) 2 , -S(0)2N(R 8 ) 2 , -S(O)N(R 8 ) 2 , -C(O)C(O)N(R 8 ) 2 , -C(O)CH2N(R 8 ) 2 , CH 2 R 8 , -CH 2 -alkenyl-R 8 , or -CH 2 -alkynyl-R 8 ; or R 1 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)aryl, -C(0)heteroaryl, or -C(O)heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -C02H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -N(H)alkyl, -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)N(H)alkyl, - 278 -N(H)C(O)N(alkyl)2, -S-alkyl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH 2 N(alkyl)2, or -alkyl, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; 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 one R 3 together with the atoms to which they are bound, form a 3 to 7 membered cyclic or heterocyclic ring system, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by -R 11 , a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -R 1 ; R 4 and one R 5 together with the atoms to which they are bound form a ring system: R10 N and the other R 5 is H; each R 8 is independently -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by Ril, and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 9 is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R10, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; - 279 each R 10 is independently -OH, -SH, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -CO2H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH 2 , -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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(O)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH 2 N(H)alkyl, or -CH2N(alkyl)2, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; and each R 11 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -CO 2 H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -alkyl, -cycloalkyl, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2; R 12 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)alkylaryl, C(O)alkylheteroaryl, -C(O)heterocyclyl, or -C(O)alkylheterocyclyl; and R 13 is -H, -alkyl, -aryl, -alkylaryl or -alkylheteroaryl.
3. A compound represented by formula I: O 5 R5 R2 R6 Rl X N Y R4 0 wherein: Y is: (a) - 280 R7 m H 0 or (b) 0 m 0 HO H m is 0 or 1; X is -C(R3) 2 -' R 1 is H, -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) 2 N(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(R 8 )2, -C(O)C(O)N(R 8 )2, -C(O)CH2N(R 8 )2, CH2R 8 , -CH2-alkenyl-R 8 , or -CH 2 -alkynyl-R 8 ; or R 1 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)aryl, -C(O)heteroaryl, or -C(O)heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -CO2H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -N(H)alkyl, -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)N(H)alkyl, -N(H)C(O)N(alkyl)2, -S-alkyl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2, or -alkyl, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; 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 ,-wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -R 10 , a hydrogen atom bound to any -aryl - 281 or -heteroaryl carbon atom is optionally replaced by -R 11 , a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -Ri; R 4 and one R 5 together with the atoms to which they are bound form a ring system: R10 N and the other R 5 is H; 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, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 9 is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R10, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 10 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -C02H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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(O)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or - 282 -CH2N(alkyl)2, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R11 and a hydrogen atom bound to any nitrogen atom is optionally replaced by R1; and each R 11 is independently -OH, -SH, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -CO 2 H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -alkyl, -cycloalkyl, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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(O)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2; provided that if one R 3 is -H, then the other R 3 is not -H.
4. A compound represented by formula I: 0 R 5 R 5 R2 R6 R111NN- X) Y Y N IIY R4 wherein: Y is: 0 OR 8 0 OR 8 m m R 8 0 R 8 , ,or H H OR 8 OR 12 NR H (C) (d) (e) (M m is 0 or 1; X is -C(R 3 )2-; - 283 R 1 is H, -C(0)C(O)R 8 , -S(O) 2 R 8 , -S(O)R 8 , -C(O)OR 8 , -C(O)N(H)R 8 , -S(0)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(R 8 )2, -C(O)C(O)N(R 8 )2, -C(O)CH2N(R 8 )2, CH2R 8 , -CH2-alkenyl-R 8 , or -CH2-alkynyl-R 8 ; or R 1 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)aryl, -C(O)heteroaryl, or -C(O)heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -C02H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -N(H)alkyl, -N(alkyl)2, -C(O)N(H)alkyl, -C(O)N(alkyl)2, -N(H)C(O)alkyl, -N(H)C(O)Calkyl, -N(H)C(O)N(H)alkyl, -N(H)C(O)N(alkyl)2, -S-alkyl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2, or -alkyl, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; 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 ,-wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by -R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by -Rll, a hydrogen atom bound to any nitrogen atom of the ring system is optionally replaced by -R 1 ; R 4 and one R 5 together with the atoms to which they are bound form a ring system: R10 N and the other R 5 is H; R 6 is -H; - 284 each R 8 is independently -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 9 is independently -aryl, -heteroaryl, cycloalkyl, or -heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R10, a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; each R 10 is independently -OH, -SH, -F, -Cl, -Br, -I, -N02, -CN, -NH2, -CO2H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-alkylaryl, -N(H)alkyl, N(H)aryl, -N(H)-alkylaryl, -N(alkyl)2, -C(O)N(H)alkyl, C(0)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)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2, -alkyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl -alkylaryl, -alkylheteroaryl, or -alkylheterocyclyl, wherein a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by Rll and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1 ; and each R 11 is independently -OH, -SH, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -C02H, -C(O)NH2, -N(H)C(O)H, -N(H)C(O)NH2, -alkyl, -cycloalkyl, -perfluoroalkyl, -0-alkyl, -0-aryl, -0-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- - 285 alkylaryl, -S(0)2alkyl, -S(O)alkyl, -C(O)alkyl, -CH2NH2, -CH2N(H)alkyl, or -CH2N(alkyl)2; and R 12 is -C(O)alkyl, -C(O)cycloalkyl, -C(O)alkylaryl, -C (0) alkylheteroaryl, -C (O) heterocyclyl, or -C (O) alkylheterocyclyl.
5. The compound according to claim 2 or claim 4 wherein Y is: 0 m 0 H V and V is: CH 3 0, O0 00 0 0 0 00 0 00 "0 -1 0 -286 00e 0. 0 0. 0 0 N R 8 N 0 H 0i S R 8 0 H 2 Nl, N 0 I'N ~ (DN 0 N s N or 0 - 287
6. The compound according to any one of claims 1-4 wherein: one R 3 is -H and the other R 3 is methyl, isopropyl, or tert-butyl.
7. The compound according to claim 6 wherein one R 3 is -H and the other R 3 is methyl.
8. The compound according to claim 7 wherein R 1 is C(O)C(O)R 8 , -C(O)alkyl, -C(O)cycloalkyl, -C(O)aryl, -C(O)heteroaryl, or -C(O)heterocyclyl, wherein a hydrogen atom bound to any -alkyl or -cycloalkyl carbon atom is optionally replaced by R 10 , a hydrogen atom bound to any -aryl or -heteroaryl carbon atom is optionally replaced by R 11 , and a hydrogen atom bound to any nitrogen atom is optionally replaced by R 1
9. The compound according to claim 3 selected from the group consisting of: 25a-25e: N N C0 2 H N 02H 0 N f H2N 0 N H CI O 25a 25d NN N.4 N NO2H (N 020 N 02H 0NO N HCI 0 25b 25e N N N CO 2 H N HN H 25c 25c - 288
10. The compound according to claim 2 selected from the group Boc N 0 O N C H __C6H5 101 consisting of: 101, 102a and 102b: , and 0 H N N 4C YO c1 H 0 CH 5 102a, Y=AcNH 102b, Y=NH 2
11. A pharmaceutical composition comprising: a) a compound according to any one of claims 1-10; and b) a pharmaceutically acceptable carrier, adjuvant or vehicle.
12. A method for treating or preventing 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 excess dietary alcohol intake disease, a viral mediated disease, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave's 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 host disease, osteoporosis, leukemias and related disorders, myelodysplastic syndrome, multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer's disease, Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, - 289 neurological damage due to stroke, ulcerative colitis, traumatic brain injury, organ transplant rejection, hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, or Japanese encephalitis, in a patient comprising the step of administering to said patient a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11.
13. The method according to claim 12, wherein the disease is rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory peritonitis, septic shock, pancreatitis, traumatic brain injury, organ transplant rejection, osteoarthritis, asthma, psoriasis, Alzheimer's disease, atopic dermatitis, leukemias and related disorders, myelodysplastic syndrome, or multiple myeloma.
14. A method for inhibiting an ICE-mediated function in a patient comprising the step of administering to said patient a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11.
15. A method for decreasing IGIF or IFN-y production in a patient comprising the step of administering to said patient a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11.
16. The use of a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for treating or preventing 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 excess dietary alcohol intake disease, a viral mediated disease, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis, - 290 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 host disease, osteoporosis, leukemias and related disorders, myelodysplastic syndrome, multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma, sepsis, septic shock, Shigellosis, 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 stroke, ulcerative colitis, traumatic brain injury, organ transplant rejection, hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, or Japanese encephalitis, in a patient.
17. The use according to claim 16, wherein the disease is rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory peritonitis, septic shock, pancreatitis, traumatic brain injury, organ transplant rejection, osteoarthritis, asthma, psoriasis, Alzheimer's disease, atopic dermatitis, leukemias and related disorders, myelodysplastic syndrome, or multiple myeloma.
18. The use of a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for inhibiting an ICE-mediated function in a patient.
19. The use of a compound according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for decreasing IGIF or IFN-y production in a patient. Date: 22 September 2010
AU2010224367A 1998-03-19 2010-09-22 Inhibitors of Caspases Abandoned AU2010224367A1 (en)

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US60/078770 1998-03-19
AU2003255217A AU2003255217A1 (en) 1998-03-19 2003-10-22 Inhibitors of Caspases
AU2007200251A AU2007200251A1 (en) 1998-03-19 2007-01-22 Inhibitors of Caspases
AU2010224367A AU2010224367A1 (en) 1998-03-19 2010-09-22 Inhibitors of Caspases

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