AP1280A - Inhibitors of interleukin-1 converting enzyme. - Google Patents

Abstract

This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are particularly well suited for inhibiting ICE activity and consequently, may be advantageously used as agents against IL-1-, apoptosis-, IGIF-, and IFN-y-mediated diseases, inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, degenerative diseases, and necrotic diseases. This invention also relates to methods for inhibiting ICE activity, for treating interleukin-1-, apoptosis-, IGIF- and IFN-y-mediated diseases and decreasing IGIF and IFN-y production using the compounds and compositions of this invention.

Description

VPI96-01 CIP2

INHIBITORS OF INTERLEUKIN-1β CONVERTING ENZYME

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel 5 classes of compounds which are inhibitors of interleukin-ΐβ converting enzyme ("ICE"). Thisinvention also relates to pharmaceutical compositionscomprising these compounds. The compounds andpharmaceutical compositions of this invention are 10 particularly well suited for inhibiting ICE activity and consequently, may be advantageously used as agentsagainst interleukin-1- ("IL-1"), apoptosis-, interferongamma inducing factor- ("IGIF") and interferon-y-("IFN-γ") mediated diseases, including inflammatory 15 diseases, autoimmune diseases, destructive bone,proliferative disorders, infectious diseases anddegenerative diseases. This invention also relates tomethods for inhibiting ICE activity, and decreasingIGIF production and IFN-γ production and methods for 20 treating interleukin-Ι-, apoptosis-, IGIF- and IFN-y-mediated diseases using the compounds and compositionsof this invention. This invention also relates tomethods of preparing N-acylamino compounds.

BACKGROUND OF THE INVENTION 25 Interleukin 1 ("IL-1") is a major pro- inflammatory and immunoregulatory protein thatstimulates fibroblast differentiation andproliferation, the production of prostaglandins,collagenase and phospholipase by synovial cells and 30 chondrocytes, basophil and eosinophil degranulation andneutrophil activation. Oppenheim, J.H. et al,

Immunology Today, 7, pp. 45-56 (1986). As such, it isinvolved in the pathogenesis of chronic and acuteinflammatory and autoimmune diseases. For example, in 35 rheumatoid arthritis, IL-1 is both a mediator of sr

CM fit. cn

U

Cl· < inflammatory symptoms and of the destruction of thecartilage proteoglycan in afflicted joints. Wood, DID.et al., Arthritis Rheum. 26, 975, (1983); Pettipher,

E.6. et al., Proc. Natl. Acad. Sci. UNITED STATES OF 5 AMERICA 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., 6., OralPath 17, 145 (1988); Dewhirst, F.E. et al., J. Immunol.8, 2562 1985). It is alternately referred to as 10 "osteoclast activating factor" in destructive bone diseases such as osteoarthritis and multiple myeloma.Bataiile, R, et al., Int. J. Clin. Lab, Res. 21(2), 283(1392). In certain proliferative disorders, such asacute myelogenous leukemia and multiple myeloma, IL-1 15 can promote tumor cell growth and adhesion. Bam, M.R., J. Natl. Cancer Inst. 83, 123 (1991); Vidal-Vanaclocha, F., Cancer Res. 54, 2667 (1994). In thesedisorders, IL-1 also stimulates production of othercytokines such as IL--6, which can modulate tumor 20 development (Tartour et al., Cancer Res. 54, 6243 (1994). IL-1 is predominantly produced by peripheralblood monocytes as part of the inflammatory responseand exists in two distinct agonist forms, IL-Ια and IL-1 β . Mo s e 1 y, B. S . e t. a I., Proc. Nat. Acad. Sci. , 84, 25 pp. 4572-4576 (1987); Lonnemann, G. et al., Eur. J.

ImmuneI ., 19, pp. 1531-1536 (1989). IL-Ιβ is synthesized as a biologicallyinactive precursor, pIL-Ιβ. pIL-Ιβ lacks aconventional leader sequence and is not processed by a 30 signal peptidase. March, C.J., Nature, 315, ρρ. 641-647 (1985). instead, pIL-Ιβ is cleaved byinterleukin-ΐβ converting' enzyme ("ICE") between Asp-116 and Ala-117 to produce the biologically active

V 6 I 10-36 /J/dV - 3 - C-terminal fragment found in human serum and synovialfluid. 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

5 protease localized primarily in monocytes. It convertsprecursor IL-Ιβ 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 UNITED STATES OF

) AMERICA, 86, pp. 5227-5231 (1989). Processing by ICE 10 is also necessary for the transport of mature IL-Ιβ through the cell membrane. ICE, or its homologs, also appears to be involved in the regulation of programmed cell death orapoptosis. Yuan, J. et al., Cell, 75, pp. 641-652 15 (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 particular, ICE or ICE homologs arethought to be associated with the regulation ofapoptosis in neurodegenerative diseases, such as 20 Alzheimer's and Parkinson's disease. Marx, J. and M. •j Baringa, Science, 259, pp. 760-762 (1993); Gagliardini, V. et al., Science, 263, pp. 826-828 (1994).

Therapeutic applications for inhibition of apoptosismay include treatment of Alzheimer's disease, 25 Parkinson's disease, stroke, myocardial infarction,spinal atrophy, and aging. ICE has been demonstrated to mediate ΑΡ/Γ/ 9 6.-01294 apoptosis (programmed cell death) in certain tissuetypes. Steller, H., Science, 267, p. 1445 (1995); 30 Whvte, M. and Evan, G., Nature, 376, p. 17 (1995); Martin, S.J. and Green, D.R., Cell, 82, p. 349 (1995); Alnemri, E.S., et al., J. Biol. Chem., 270, p. 4312 (1995); Yuan, J. Curr. Ooin. Cell Biol., 7, D . 211 ..... 4 - (1995). A transgenic mouse with a disruption of theICE gene is deficient in Fas-mediated apoptosis (Kuida,ft. et al., Science 267, 2000 (1995)). This activity ofICE is distinct from its role as the processing enzyme 5 for pro-ILip. It is conceivable that in certain tissuetypes, inhibition of ICE may not affect secretion ofmature IL-Ιβ, but may inhibit apoptosis.

Enzymatically active ICE has been previouslydescribed as a hetercdimer composed of two subunits, 10 ρ20 and plO (20kDa and 10kPa molecular weight, respectively). These subunits are derived from a 45kDaproenzyme (p4 5) by way cf a p30 form, through anactivation mechanism that is autocatalytic.

Thornberry, N.A. et ai ., Nature, 356, pp. 768-774 15 (1992). The ICE proenzyme has been divided into several functional domains: a prodomain (pl4), ap22/20 subunit, a polypeptide linker and a plO subunit.Thornberry et al., supra; Casano et al., Genomics, 20,pp. 474-481 (1994). 20 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 plO cDNA andamino acid sequences are also known. Thornberryet al., supra. Murine and rat ICE have also been 25 sequenced and cloned. They have high amino acid andnucleic acid sequence homology to human ICE. Miller,D.K. et al., Arm,. Nhf_.„,., Acad. Sci . , 696, pp. 133-148(1993); Molineaux, S.M. et al., Proc. Nat. Acad. Sci..,,90, pp. 1809-1813 (1953). The three-dimensional 30 structure of ICE has been determined at atomic

resolution by X-ray crystallography. Wilson, K.P., etal., Nature, 370, pp. 270-275 (1994). The activeenzyme exists as a tetramer of two p20 and two plO subunits .

Additionally, there exist human homologs ofICE with sequence similarities in the active siteregions of the enzymes. Such homologs include TX (or 5 IC£rel-ll or ICH-2) (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_jjj) (Nicholson et al., j Biol. Chem., 270, p. 15870 (1995); ICH-1 (orNedd-2) 10 (Wang, L. et al., Cell, 78, p. 739 (1994)), MCH-2, (Fernandes-Alnemri, T. et al., Cancer Res., 55, p. 2737(1995), CPP32 (or YAMA or apopain) (Fernandes-Alnemri,T. et al., J. Biol. Chem., 269, p. 30761 (1994);Nicholson, D.W. et al., Nature, 376, p. 37 (1995)), and 15 CMH-1 (or MCH-3) (Lippke, et al., J. Biol. Chem., (1996); Fernandes-Alnemri, T. et al., Cancer Res.,(1995)). Each of these ICE homologs, as well as ICEitself, is capable of inducing apoptosis whenoverexpressed in transfected cell lines. Inhibition of

20 one or more of these homologs with the peptidyl ICE inhibitor Tyr-Val-Ala-Asp-chloromethylketone results ininhibition of apoptosis in primary cells or cell lines.Lazebnik et al., Nature, 371, p. 346 (1994). Thecompounds described herein are also capable of 25 inhibiting one or more homologs of ICE (see Example 5).Therefore, these compounds may be used to inhibitapoptosis in tissue types that contain ICE homologs,but which do not contain active ICE or produce matureIL-Ιβ.

Interferon-gamma inducing factor (IGIF) is anapproximately 18-kDa polypeptide that stimulates T-cellproduction of interferon-gamma (IFN-γ). IGIF isproduced by activated Kupffer cells and macrophages in 30 vivo and is exported out of such cells upon endotoxinstimulation. Thus, a compound that decreases IGIFproduction would be useful as an inhibitor of such T-cell stimulation which in turn would reduce the levels 5 of IFN-γ production, by these cells. IFN-γ is a cytokine with immunomodulatory effects on a variety of immune cells. In particular,IFN-y is involved in macrophage activation and Thl cellselection (F. Belardelli, APMIS, 103, p. 161 (1995)). 10 IFN-γ 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.OncoK., 121, p. 516 (1995)). 15 Mice lacking IFN-γ or its receptor have multiple defects in immune cell function and areresistant to endotoxic shock (S. Huang et al., Science,259, p. 1742 (1993); D. Dalton et al. , Science, 259,p. 1739 (1993); B.D. Car et al., J. Exp. Med., 179, 20 p. 1437 (1994)). Along with IL-12, IGIF appears to bea potent inducer of IFN-γ 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 al., J. . Immunol., 156, p. 4274 ¢1996(). 25 IFN-γ 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-γ productionwcuic be useful to ameliorate the effects of IFN-y 30 related pathologies.

The biological regulation of IGIF and thus IFN-y has not been elucidated. It is known that IGIFis synthesised as a precursor protein, called "pro-IGIF", It has been unclear, however, how pro-IGIF is cleaved and whether its processing has biologicalimportance.

Accordingly, compositions and methods capableof regulating the conversion of pro-IGIF to IGIF would 5 be useful for decreasing IGIF and IFN-γ production in vivo, and thus for ameliorating the detrimental effectsof these proteins which contribute to human disordersand diseases.

However, ICE and other members of the10 ICE/CED-3 family have not previously been linked to the conversion of pro-IGIF to IGIF or to IFN-γ productionin vivo. ICE inhibitors represent a class of compoundsuseful for the control of inflammation or apoptosis or 15 both. Peptide and peptidyl inhibitors of ICE have beendescribed. PCT patent applications WO 91/15577; WO93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; andEuropean patent application 0 547 699. Such peptidylinhibitors of ICE has been observed to block the 20 production of mature IL-Ιβ in a mouse model of inflammation (vide infra) and to suppress growth ofleukemia cells in vitro (Estrov et al., Blood 84, 380a(1994)). However, due to their peptidic nature, suchinhibitors are typically characterized by undesirable 25 pharmacologic properties, such as poor cellularpenetration and cellular activity, poor oralabsorption, poor stability and rapid metabolism.Plattner, J.J. and D.W. Norbeck, in Drug DiscoveryTechnologies, C.R. Clark and W.H. Moos, Eds. (Ellis 30 Horwood, Chichester, England, 1990), pp. 92-126. Thishas hampered their development into effective drugs.

Non-peptidyl compounds have also beenreported to inhibit ICE in vitro. PCT patent ΑΡ/Γ/ 9 8/01294 application WO 95/26958; US Patents 5,552,400; Dclleet «1., J. Med. Chen;, , 39, pp. 2438-2440 (1996);However, it is not clear whether these compounds havethe appropriate pharmacological profile to be 5 therapeutically useful.

Additionally, current methods for the preparation of such compounds are not advantageous.These methods use tributyltin hydride, a toxic,moisture sensitive reagent. Thus, these methods are 10 inconvenient to carry out, pose a health risk and create toxic-waste disposal problems. Furthermore, itis difficult to purify compounds prepared by thesemethods.

Accordingly, the need exists for compounds 15 that can effectively inhibit the action of ICE in vivo,for use as agents for preventing and treating chronicand acute forms of IL-1-mediated diseases, apoptosis-,IG1F-, or IFN-Y-mediated diseases, as well asinflammatory, autoimmune, destructive bone, 20 proliferative, infectious, or degenerative diseases.

The need also exists for methods of preparing suchcompounds. SUMWiY OF THE INVENTIONThe present invention provides novel classes 25 of compounds, and pharmaceutically acceptable derivatives thereof, that are useful as inhibitors ofICE. These compounds can be used alone or incombination with other therapeutic or prophylacticagents, such as antibiotics, immunomodulators or other 30 ant;-inflammatory agents, for the treatment or prophylaxis of diseases mediated by IL-1, apoptosis,IGIF or IFN-y. According to a preferred embodiment,the compounds of this invention are capable of bindingto the active site of ICE and inhibiting the activity

C*J v—

O <30 Ο» a < AP V u 1 2 8 0 - 9 - of that enzyme. Additionally, they have improvedcellular potency, improved pharmacokinetics, and/orimproved oral bioavailability compared to peptidyl ICEinhibitors . 5 It is a principal object of this invention to provide novel classes of compounds which are inhibitorsof ICE represented by formulas: (i) , (VI) R-j_-N-R2 ; and

wherein the various substituents are describedherein. 15 It is a further object of this invention to provide a process of preparing N-acylamino compounds bycoupling a carboxylic acid with an alloc-protectedamine.

BRIEF DESCRIPTION OF THE DRAWINGS 20 Fig. 1A ICE cleaves pro-IGIF in vivo. Cell lysatesfrom Cos cells transfected with the various indicatedexpression plasmids or controls were analyzed for thepresence of IGIF by separating proteins by SDS-PAGE andimmunoblotting with anti-IGIF antisera (lane 1, mock 25 transfected cells; lane 2, pro-IGIF alone; lanes 3-12,pro-IGIF in combination with ICE, ICE-C285S, CPP32,CPP32-C163S, CMH-1, CMH-1-C186S, Tx, Tx-C258S,respectively). Mobilities of pro-IGIF and the 18-kDamature IGIF are indicated on the right. Molecular 30 weight markers in kDa are shown on the left (Example23) .

Fig. IB ICE cleaves pro-IGIF at the authentic ΑΡ/Γ7 98.01294 processing site in_ vitro as shown by Coomassie bluestaining of proteolytic reaction products separated bySDS-PAGE (Example 23) . The proteases and inhibitorsused were* lane 1, buffer control; lane 2, 0.1 nK ICE;

5 lane 3, 1 nM ICE; lanes 4 and 5, 1 nM ICE with 10 nM

Cbz-Val-Ala-Asp-( (2,6-dichlorobenzoyl)oxy]methyl ketoneand 100 nM Ac-Tyr-Val-Ala-Asp-aldehyde, respectively;lanes 6 and Ί, 15 nM CPP32 with and without 400 nMAc-Asp-Glu-Val-Asp-aidehyde (D. W. Nicholson et ,ai ., 10 Nature, 376, p. 37 (1995)), respectively; lane 8, 100nM CMH-1; lane 9, 10 units/ml granzyme B; and M,molecular weight markers in kDa.

Fig. 1C ICE cleavage converts inactive pro-IGIF toactive IGIF which induces IFN-γ production in Thl 15 helper cells. Uncleaved (Pro-IGIF), ICE-cleaved (Pro-IGIF’/ICE), CPP32-cieaved (Pro-IGIF/CPP32) , andrecombinant mature IGIF (rIGIF) were incubated withA.E'’ Thl cells at 12 ng/ml (open bar) and 120 ng/ml(hatched bar) for eighteen hours and the levels or IFN- 20 V released into the culture medium assayed by ELISA(Example 23). A.E7 cells cultured with buffer, ICEalone (ICE) or CPP32 alone (CPP32) were assayedsimilarly for negative controls. The numbers representthe average of three determinations. 25 Fig. 2A Mature IGIF >;18-kDa) is produced by Cos cellsco-transfected with pro-IGIF and ICE-expressingplasmids. Cell lysates (left) and conditioned medium(right) from Cos cells transfected with a pro-IGIFexpression plasmid in the absence (-) or presence of an 30 expression plasmid encoding wild type (ICE) or inactivemutant (ICE-C285S) ICE. Transfected cells weremetaboli.cally labeled with ';5S-methionine, proteins fromcell lysates and conditioned medium immunoprecipitated APG 01280 - 11 - with anti-IGIF antisera and separated by SDS-PAGE(Example 24). Mobilities of pro-IGIF and the 18-kDamature IGIF are indicated on the right. Molecularweight markers in kDa are shown on the left. 5 Fig. 2B IFN-γ inducing activity is detected in Coscells co-transfected with pro-IGIF and ICE-expressingplasmids. Cell lysates (hatched bar) and conditionedmedium (open bar) from Cos cells transfected with apro-IGIF expression plasmid in the absence (Pro-IGIF) 10 or presence (Pro-IGIF/ICE) of an expression plasmid encoding wild type (ICE) were assayed for IFN-γ levels(ng/ml) by ELISA. Cos cells transfected with buffer(Mock) or an ICE-expressing plasmid alone (ICE) servedas negative controls (Example 24) . 15 Fig. 3A Kupffer cells from mice lacking ICE are defective in the export of IGIF. Kupffer cells fromwild type mice (ICE +/+) or ICE-deficient micehomozygous for an ICE mutation (ICE-/-) were isolatedand primed with LPS for three hours. The levels of 20 immunoreactive IGIF polypeptides in the conditioned media (ng/ml) of wild type cells were measured by ELISA(Example 25). N.D. (not detectable) indicates that theIGIF concentration was less than 0.1 ng/ml.

Fig. 3B Kupffer cells from mice lacking ICE are 25 defective in the export of mature IGIF. Kupffer cellsfrom wild type mice (ICE +/+) or ICE deficient micehomozygous for an ICE mutation (ICE -/-) were isolatedand primed with LPS for three hours. Primed cells weremetabolically labeled with 35S-methionine, proteins from 30 cell lysates and conditioned medium immunoprecipitatedwith anti-IGIF antisera and separated by SDS-PAGE(Example 25). Mobilities of pro-IGIF and the 18-kDamature IGIF are indicated on the right. Molecular mass

V β I V o / 8 β /J/dV ο Δ. markers in kDa are shown on the left.

Fig. 3C Serum from ICE-deficient mice containsreduced levels of IGIF. Serum samples from wild typemice (ICE + /+) or ICE deficient mice homozygous for an 5 ICE mutation (ICE -/-) were assayed for IGIF levels(ng/ml) by ELISA (Example 25).

Fig. 3D Serum from ICE-deficient mice containsreduced levels of IFN-y. Serum samples from wild typemice (ICE +/+) or ICE deficient mice homozygous for an 10 ICE mutation (ICE -/-) were assayed for IFN-γ levels(ng/ml) by ELISA (Example 25) .

Fig. 4 3 erum IFN-y levels are significantly reduced in ICE-deficient mice after an acute challenge with LPS(Example 26). Serum samples from wild type mice 15 (filled squares) or ICE-deficient mice (filled circles)were assayed for IFN-γ levels (ng/ml) by ELISA as afunction of time (hours) after LPS challenge.Temperatures of the animals during the time course indegrees Celcius is shown for wild type mice (open 20 squares) or ICE-deficient mice (open circles).

Fig.. 5 The ICE inhibitor, AcYVAD-aldehyde (AcYVAD-

I CEO), inhibits LPS-stimulated IL-Ιβ and IFN-γ synthesisby human peripheral blood mononuclear cells (PBMC)1Percent >%) inhibition as a function of inhibitor: 25 concentration (μΜ) is shown for IL-Ιβ (open squares)and IFN-y (open diamonds) synthesis.

Fig. 6 Compound 214e inhibits IL-Ιβ production inLPS-chailenged mice. Serum samples from CD1 mice wereassayed for IL-Ιβ levers (pg/ml) by ELISA after LPS 30 challenge. Compound 214e was administered by intraperitoneai (IP) injection one hour after LPSchallenge. Blood was collected seven hours after LPSchallenge (see Examole

/J/dV APt01280 - 13 -

Fig. 7 Compound 217e inhibits IL-Ιβ production inLPS-challenged mice. Serum samples from CD1 mice wereassayed for IL-Ιβ levels (pg/ml) by ELISA after LPSchallenge. Compound 217e was administered by

5 intraperitoneal (IP) injection one hour after LPS challenge. Blood was collected seven hours after LPSchallenge (see Example 7).

Fig. 8 Compound 214e, but not compound 217e, ) inhibits IL-Ιβ production in LPS-challenged mice when 10 administered by oral gavage. This assay measures oralabsorption under similar conditions as those describedfor Figs. 6 and 7. These results indicates that 214eis potentially orally active as an ICE inhibitor (seeExample 7). 15 Fig. 9 Compound 214e and analogs of 214e alsoinhibit IL-Ιβ production after IP administration.

These results were obtained in the assay described forFigs. 6 and 7 and Example 7.

Fig. 10 Compound 214e, and analogs of 214e, also 20 inhibit IL-Ιβ production after oral (PO)j administration. These results were obtained in the assay described for Figs. 6 and 7 and Example 7.

Figs. 11A/B Compounds 302 and 304a show detectableblood levels when administered orally (50mg/kg, in 25 0.5 % carboxymethylcellulose) to mice. Blood samples were collected at 1 and 7 hours after dosing.

Compounds 302 and 304a are prodrugs of 214e and aremetabolized to 214e in vivo. Compound 214e shows noblood levels above 0.10 pg/ml when administered orally 30 (Example 8).

Fig. 12 Compound 412f blocks the progression of typeII collagen-induced arthritis in male DBA/1J mice(Wooley, P.H., Methods in Enzymologv, 162, pp. 361-373 14 (1988) and Geiger, T., Clinical and Experimental

Rheumatology, 11, pp. 515-522 (1993)). Compound 412fwas administered twice a day (10, 25 and 50mg/kcij ,,approximately 7n apart, by oral gavage. Inflammation 5 was measured on the Arthritis Severity Score on s 1 to 4 scale of increasing severity. The scores of the twofront paws were added to give the final score (seeExample 21).

Fig. 13 Compound 412d blocks the progression cf type10 II collagen-induced arthritis in male DBA/1J mice. The results were obtained as described for Fig. 12 and inExample 21.

Fig. 14 Compound 696a blocks the progression of typeII collagen-induced arthritis in male DBA/U mice. The 15 results were obtained as described for Fig. 12 and in

Example 21.

ABBREVIATIONS AND DEFINITIONS

Abbreviations

Designation Reagent or Fragment

Ala alanine Arg arginine Asn asparagine Asp aspartic acid Cys cysteine Gin glutamine Gin glutamic acid Gly glycine His histidine lie isoleucine .L 6 11 leucine Lys 1ysine Met methionine Phe ρ h e n ylalanine ΑΡ/Γ/ 9 8/01294 10 15 20 25 APC01280 - 15 -

Pro

Ser

Thr

Trp

Tyr

Val

Ac2O n-Bu

DMF

DIEA

EDC

Et2O

EtOAc

Emoc

HBTU

HOBT

Me OH

TFA

Alloc proline serine threonine tryptophan tyrosine valine acetic anhydride normal-butyl dimethylformamide N, W-diisopropylethylamine1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloridediethyl ether ethyl acetate 9-fluorenylmethyoxycarbonylO-benzotriazol-l-yl-W, N, Ν', N' -tetramethyluronium hexafluorophosphate 1-hydroxybenzotriazole hydratemethanol trifluoroacetic acid allyloxycarbonyl

Definitions

δ Z ·, 0 8 6 /J/dV

The following terms are employed herein:

The term "interferon gamma inducing factor"or "IGIF" refers to a factor which is capable ofstimulating the endogenous production of IFN-y.

The term "ICE inhibitor" refers to a compoundwhich is capable of inhibiting the ICE enzyme. ICEinhibition may be determined using the methodsdescribed and incorporated by reference herein. Theskilled practitioner realizes that an in vivo ICEinhibitor is not necessarily an in vitro ICE inhibitor. 16

For example, a prodrug form of a compound typicallydemonstrates little or no activity in in vitro assays.Such prodrug forms may be altered by metabolic or otherbiochemical processes in the patient to provide an in 5 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. Cellsmay be in any form, including but not limited to cells 15 retained in tissue, cell clusters, immortalized cells,transfected or transformed cells, and ceils derivedfrom an animal that have been physically orphenotypically altered.

The term "active site" refers to any or all 20 of the following sites in ICE: the substrate binding'site, the site where an inhibitor binds and the sitewhere the cleavage of substrate occurs.

The term "heterocycle" or "heterocyclic"refers to a stable mono- or polycyclic compound which 25 mav optionally contain one or two double bonds or mayoptionally contain one or more aromatic rings. Eachheterocycle consists of carbon atoms and from one tofour heteroatoms independently selected from a groupincluding nitrogen, oxygen, and sulfur. As used 30 herein, the terms "nitrogen heteroatoms" and "sulDhurheteroatoms" include any oxidized form of nitrogen orsulfur arid the quaternized form of any basic nitrogen.Heterocycles defined above include, for example,p y r i n i d t n y 1, t e t r a h y d r o q u 1 η ο 1 y 1, APC 0 1280 - 17 - tetrahydroisoquinonlinyl, .purinyl, pyrimidyl,indolinyl, benzimidazolyl, imidazolyl, imidazolinoyl,imidazolidinyl, quinolyl, isoquinolyl, indolyl,pyridyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyrazinyl, 5 quinoxolyl, piperidinyl, morpholinyl, thiamorpholinyl,furyl, thienyl, triazolyl, thiazolyl, β-carbolinyl,tetrazolyl, thiazolidinyl, benzofuranoyl, thiamorpholinyl sulfone, benzoxazolyl, oxopiperidinyl,oxopyrroldinyl, oxoazepinyl, azepinyl, isoxazolyl, 10 tetrahydropyranyl, tetrahydrofuranyl, thiadiazolyl, benzodioxolyl, benzothienyl, tetrahydrothiophenyl andsulfolanyl. Further heterocycles are described in A.R.Katritzky and C.W. Rees, eds., Comprehensive

Heterocyclic Chemistry: The Structure, Reactions, 15 Synthesis and Use of Heterocyclic Compounds, Vol. 1-8,Pergamon Press, NY (1984).

The term "cycloalkyl" refers to a mono- orpolycyclic group which contains 3 to 15 carbons and mayoptionally contain one or two double bonds. Examples 20 include cyclohexyl, adamantyl and norbornyl.

The term "aryl" refers to a mono- orpolycyclic group which contains 6, 10, 12, or 14carbons in which at least one ring is aromatic.

Examples include phenyl, naphthyl, and 25 tetrahydronaphthalene.

The term, "heteroaromatic" refers to a mono-or polycyclic group which contains 1 to 15 carbon atomsand from 1 to 4 heteroatoms, each of which is selectedindependently from a group including sulphur, nitrogen 30 and oxygen, and which additionally contains from 1 to 3five or six membered rings, at least one of which isaromatic .

The term "alpha-amino acid" ία-amino acid)refers to both the naturally occurring amino acids and 18 other "non-protein." α-amino acids commonly utilized bythose in the peptide chemistry arts when preparingsynthetic analogues of naturally occurring peptides,including D and L forms. The naturally occurring amino 5 acids are glycine, alanine, valine, leucine, iso-leucine, serine, methionine, threonine, phenylalanine,tyrosine, tryptophan, cysteine, proline, histidine,aspartic acid, asparagine, glutamic acid, glutamine, y-carhoxyglutamic acid, arginine, ornithine and lysine. 10 Examples of "non-protein" alpha-amino acids includehydroxylysine, homoserme, homotyrosine, homo-phenylalanine, citruiline, kynurenine, 4-amino-phenylalanine, 3-(2-naphthyl)-alanine, 3-(1-naphthyl)-alanine, methionine sulfone, t-butyl-alanine, 15 t-butviglycine, 4-hydroxyphenylglycine, aminoalanine, phenvlglycine, vinylalanine, propargyl-glycine, 1,2,i-triazolo-3-alanine, 4,4,4-trifluoro-threonine,thyronine, 6-hydroxytryptophan, 5-hydro-xytryptophan, 3-hydroxykynurenine, 3-aminotyrosine, trifuoromethyl- 20 alanine, 2-thienylalanine, (2-(4-pyridyl)ethyl) - cysteine, 3, 4-dimet.hoxy-phenylalanine, 3-(2-thiazoiyl}-alanine, ibotenic acid, 1-amino-l-cyclopentane-carboxyiic acid, l-amino-l.-cyclohexanecarboxylic acid,quisqualic acid, 3-trif'luoromethylphenylalanine, 25 4-trifluoro-methylphenylalanine, cyclohexylalanine, cyclo-hexylglycine, thiohistidine, 3-methoxytyrosine,elastatinal, norleucine, norvaline, alloisoleucine,homo.arginine, thioproline, dehydroprcline, hydroxy-proline, isonipectotic acid, homoproline, cvclohexyl- 30 glycine, a-amino-n-butyric acid, cyclohexylalanine, amiiiophenylbutyric acid, phenylalanines substituted atthe ortho, meta, or para position of the phenyl moietywith one or two of the following: a (C1-C4) alkyl, a ΑΡ δ Ο 12 8 Ο - 19 - (C^—C4) alkoxy, halogen or nirro groups or substitutedwith a methylenedioxy group; β-2- and 3-thienyl-alanine, β-2- and 3-furanylalanine, β-2-, 3- and 4- pyridylalanine, β-(benzothienyl-2- and 3-yl) alanine, 5 ' β-(1- and 2-naphthyl) alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, 5- alkylated homocysteine, O-sulfate, O-phosphate and 0-carboxylate esters of tyrosine, 3-sulfo-tyrosine, 3- i carboxy-tyrosine, 3-phospho-tyrosine, 4-methane 10 sulfonic acid ester of tyrosine, 4-methane phosphonic acid ester of tyrosine, 3,5-diiodotyrosine, 3-nitro-tyrosine, ε-alkyl lysine, and delta-alkyl ornithine.

Any of these α-amino acids may be substituted with amethyl group at the alpha position, a halogen at any 15 aromatic residue on the oi-amino side chain, or an appropriate protective group at the 0, N, or S atoms ofthe side chain residues. Appropriate protective groupsare disclosed in "Protective Groups In Organic

Synthesis," T.W. Greene and P.G.M. Wuts, J. Wiley &amp; 20 Sons, NY, NY, 1991.

The term "substitute" refers to the ) replacement of a hydrogen atom in a compound with asubstituent group. In the present invention, thosehydrogen atoms which form a part of a hydrogen bonding 25 moiety which is capable of forming a hydrogen bond withthe carbonyl oxygen of Arg-341 of ICE or the carbonyloxygen of Ser-339 of ICE are excluded from substitution. These excluded hydrogen atoms includethose which comprise an -NH- group which is alpha to a 30 -CO- group and are depicted as -NH- rather than an Xgroup or some other designation in the followingdiagrams: (a) through (t), (v) through (z).

The term "straight chain" refers to acontiguous unbranching string of covalently bound ΑΡ/Γ/98/0 1 29 4 20 atoms. The straight chain may be substituted, butthese substituents are not a part of the straightchain.

The term "Kf refers to a numerical measure5- of the effectiveness of a compound in inhibiting the activity of a target enzyme such as ICE. Lower valuesof Kg reflect higher effectiveness. The Pf value is aderived by fitting experimentally determined rate datato standard enzyme kinetic equations (see I. K. Segel, 10 Enzyme Kinetics, Wiley-Interscience, 1975).

The term "patient" as used in this ..cation refers to any mammal, especially humans.

The term "pharmaceutically effective amount" refers to an amount effective in treating or 15 ameliorating an 1L-1-, apoptosis-, IGIF- or IFN-γ™mediated disease in a patient. The term"prophyiactically effective amount" refers to an amounteffective in preventing or substantially lesseningIL-I-, apoptosis-, "GIF or IFN-γ mediated diseases in a 20 patient.

The term "pharmaceutically acceptable carrieror adjuvant" refers to a non-toxic carrier or adjuvantthat may be administered to a patient, together with acompound of this invention, and which does not destroy 25 the pharmacological activity thereof.

The term "pharmaceutically acceptablederivative" means any pharmaceutically acceptable salt,ester, or salt of such ester, of a compound of thisinvention or any o'.; -· compound which, upon 30 administration to a recipient, is capable of providing•directly or indirectly) a compound of this inventionor an anti-ICE active metabolite or residue thereof.

Pharmaceutically acceptable salts of toe ΑΡ/Γ/98/0 1294 ΑΡ υ Ο 1 2 8 Ο - 21 - compounds of this invention include, for example, thosederived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acidsinclude hydrochloric, hydrobromic, sulfuric, nitric, 5 perchloric, fumaric, maleic, phosphoric, glycolic,lactic, salicylic, succinic, toluene-p-sulfonic,tartaric, acetic, citric, methanesulfonic, formic,benzoic, malonic, naphthalene-2-sulfonic andbenzenesulfonic acids. Other acids, such as oxalic, 10 while not in themselves pharmaceutically acceptable,.may be employed in the preparation of salts useful asintermediates in obtaining the compounds of theinvention and their pharmaceutically acceptable acidaddition salts. Salts derived from appropriate bases 15 include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(Ο1-4 alkyl)4+salts .

This invention also envisions the"quaternization" of any basic nitrogen-containing 20 groups of the compounds disclosed herein. The basicnitrogen can be quaternized with any agents known tothose of ordinary skill in the art including, forexample, lower alkyl halides, such as methyl, ethyl,propyl and butyl chloride, bromides and iodides; 25 dialkyl sulfates including dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides andiodides; and aralkyl halides including benzyl andphenethyl bromides. Water or oil-soluble or 30 dispersible products may be obtained by suchquaternization.

The ICE inhibitors of this invention maycontain one or more "asymmetric" carbon atoms and thusmay occur as racemates and racemic mixtures, single ΑΡ/ΓΖ 9 8 / Q 1 2 9 4 2 9 _ enantiomers, diastereomeric mixtures and individualdiastereomers. All such isomeric forms of thesecompounds are expressly included in the presentinvention, Each stereogenic carbon may be of the R or 5 S configuration. Although specific compounds andscaffolds exemplified in this application may bedepicted in a particular stereochemical configuration,compounds and scaffolds having either the oppositestereochemistry at any given chiral center or mixtures 10 thereof are also envisioned.

The ICE inhibitors of this invention may comprise ring structures which may optionally besubstituted at carbon, nitrogen or other atoms byvarious substituents. Such ring structures may be 15 singly or multiply substituted. Preferably, the ringstructures contain between 0 and 3 substituents. Whenmultiply substituted, each substituent may be pickedindependently of any other substituent as long as thecombination of substituents results in the formation of 20 a stable compound.

Combinations of substituents and variables envisioned by this invention are only those that resultin the formation of stable compounds. The term’’stable", as used herein, refers to compounds which 25 possess stability sufficient to allow manufacture and administration to a mammal by methods known in the art.Typically, such compounds are stable at a temperatureof 4 0°C or less, in the absence of moisture or otherchemically reactive conditions, for at least a week. 30 Substituents may be represented in various forms. These various forms are known to the skilledpractitioner and may be used interchangeably,example, a methyl substituent on a phenyl ring may berepresented in any of the following forms: ΑΡ/Γ7 9 8 / a i 2 8 4 ε. 10 15 20 25 ΑΡ δ Ο 128 Ο

Various forms of substituents such as methylare used herein interchangeably. DETAILED DESCRIPTION OF THE INVENTIONIn order that the invention herein described may be more fully understood, the following detaileddescription is set forth.

The ICE inhibitors of another embodiment (B)of this invention are those of formula (J): (I) R1-N-R2

H wherein: (elO) R2

Ri

o

m is 1 or 2; R5 is -C(O)-R10, -C(O)O-R9, -C(O)-N(R10) (R10) ,-S(O)2-R9, -C(O)-CH2-O-R9, -C(O)C(O)-R10/ -R9, -H, or-C(0)C(0)-OR10; X5 is -CH-; Y2 is H2 or 0; ΑΡ/Γ/ 9 6/01294 24 each R9 is independently -Ar3 or a -C2_6 straightor branched alkyl group optionally substituted withArp, wherein the ---C j alkyl group is optionally unsaturated; 5 each R10 is independently -H, -Ar3, a C3_g cycloalkyl group, or a -ci-6 straight or branched alkylgroup optionally substituted with Ar3, wherein the -C3_galkyl group is optionally unsaturated;

Ri3 is H, Ar3, or a C-L_g straight or branched alkyl10 group optionally substituted with Ar3, -CONH2, -ORg,, -OK, -OR9, or ~C02K; each R51 is independently R9, -C(O)-R9, -C(0)~N(H)-R9, or each R51 taken together forms a saturated 4-8 member carbocyclic ring or heterocyclic ring containing15 -O-, -S-, or -NH-; each R2i is independently -H or a -C2„6 straight orbranched alkyl group; each Ar3 is a cyclic group independently selectedfrom the set consisting of an aryl group which contains 20 6, 10, 12, or 14 carbon atoms and between 1 and 3 rings and an aromatic heterocycle group containing between 5and 15 ring atoms and between 1 and 3 rings, saidheterocyclic group containing at least one heteroatomgroup selected from ......0-, -S-, -SO-, S02, =N-, and .....NH-, 25 said heterocycle group optionally containing one ormore double bonds, said heterocycle group optionallycomprising one or more aromatic rings, and said cyclicgroup optionally being singly or multiply substitutedoy .....id ; each Cy is independently -NH2, -CO2H, -Cl, ~F, -3r,-I, -'.NCy, -CN, =0, “OH, -perfluoro C2_3 alkyl, R5, -0R6,Alkrp, ORq, -NHRg, Ko, -C(O)-Rt_q, or

* δ 7, I 0 I 8 6 ZJ/dV 30 - 25 - Ο / \ CH2, \ / 5 Ο provided that when -Ar3 is substituted with a Qjgroup which comprises one or more additional -Ar3groups, said additional -Ar3 groups are not substituted 10 with another -Ar3.

Preferably, R5 is -C(O)-R10, -C(O)O-R9, or -C(0)-NH-R10. Alternatively, R5 is -S(O)2-R9, —S(O)2—NH-R10, -C(0)-C(0)-R10, -R9, or -C(0)-C(0)-OR10 .

More preferably: 15 m i s 1; R13 is H or a -C3_4 straight or branched alkylgroup optionally substituted with -Ar3 -OH, -0R9, or-CO2H, wherein the R9 is a -Ο2_4 branched or straightalkyl group, wherein Ar3 is morpholinyl or phenyl, 20 wherein the phenyl is optionally substituted with Q^· R21 is -H or -CH3; R51 is a C]__6 straight or branched alkyl group) optionally substituted with Ar3, wherein Ar3 is phenyl, optionally substituted by -Q3; 25 Ar3 is phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl,benzotriazolyl, benzimidazolyl, thienothienyl,imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridylbenzofuranyl, and indolyl; 30 each Q4 is independently -NH2, -Cl, -F, -Br, -OH, -R9, -NH-R5 wherein R5 is -C(O)-R10 or -S(O)2-R9, -OR5wherein R5 is -C(O)-R10, -0R9, -NHR9, or AP/F/ »8/0^294 26 ch2, wherein each Rg and Biq are independently astraight or branched alkyl group optionally substwith Ar3, wherein Ar3 is phenyl; provided that when -Ar3 is substituted withgroup which comprises one or more additional -Amgroups, said additional -Ar3 groups are not substwith another -Ar3.

The ICE inhibitors of another embodimeof this invention are those of formula (II):

O wherein 13 m is i or 2; X2 / h

—\ L

Rs-rrW' |W"' is p, -C(O)-CH2' J)-OR10, -C (0) -C^-Tp-R-J -1, -C (G) -- (0)0-Rg, -C(O;-N(R10) iR10! ho, -R9, uted iteo ΑΡ/Γ/ 9 8/0^294 27 Χ5 is -CH- i [ Y2 is H2 or 0; 5 each Τχ is independently -0-, -S-, -S(0)-, or -S(0)2-; each Rg is independently -Ar3 or a straight or branched alkyl group optionally substituted withAr3, wherein the -C^g alkyl group is optionally 10 unsaturated; each R10 is independently -H, -Ar3, a C3_gcycloalkyl group, or a -Cj.g straight or branched alkylgroup optionally substituted with Ar3, wherein the -C3_galkyl group is optionally unsaturated; 15 each Ri;l is independently -Ar4, - (CH2) 3_3-Ar4, -H, or -C(0)-Ar4; R13 is H, Ar3, or a Cj__g straight or branched alkylgroup optionally substituted with Ar3, -CONH2, -0R5, -OH, -ORg, or -CO2H; 20 -OR13 is optionally -N(H)-OH; each R2i is independently -H or a -Cj__g straight or branched alkyl group;

Ar2 is independently selected from the followinggroup, in which any ring may optionally be singly or 25 multiply substituted by -Qj:

Μ Z I 0 / 8 6 /JZdV , and Ν' wherein each Y is independently O or S; each Ar3 is a cyclic group independently selected from the set consisting of an aryl group which contains 30 28 - 6, 10, 12, or 14 carbon atoms and between 1 and 3 ringsand an aromatic heterocycle group containing between 5and 15 ring atoms and between 1 and 3 rings, saidheterocyclic group containing at least one heteroatom 5 group selected from -O-, -S-, -SO-, S02, =N-, ana -NH-,-NiRj)-, and -N(Rg)- said heterocycle group optionallycontaining one or more double bonds, said heterocyclegroup optionally comprising one or more aromatic rings,and said cyclic group optionally being singly or 10 multiply substituted by each Ar4 is a cyclic group independently selected from the set consisting of an aryl group which contains6, 10, 12, or 14 carbon atoms and between 1 and 3rings, and a heterocycle group containing between 5 and 15 15 ring atoms and between 1 and 3 rings, said heterocyclic group containing at least one heteroatomgroup selected from—Ό-, -S-, -S0-, S02/ =Ν-, -NH-,-NiR5)-? and -N(Rg)~ said heterocycle group optionallycontaining one or more double bonds, said heterocycle 20 group optionally comprising one or more aromatic rings,ano. said cyclic group optionally being singly ormultiply substituted by -Qj ; each Q]_ is independently -NH2, ~CO2H, -Ci, -F, -Br,-I, -NO2, ~CN, ==0, -OH, -perfluoro 0^3 alkyl, R5, -ORg, 25 --NHRg, ORg, -NHRg, Rq, -C(O)-R10, or0 / \ CH?,; \ i 30 0 provided that \ -Arp is substituted with 5 Q,group which comprises one or more additional -Arpwith another -Arg.

Preferred compounds of this embodiment

* 6 2 I 0 I 8 6 /JZdV AP ti o 1 2 8 0 - 29 - include, but are not limited to compounds 220b, 223b,223e, 226e, 227e, 307a, 307b, and 429.

In more preferred compounds of embodiment C,R3 is CO-Ar2. Alternatively, R3 is -C(0)-CH2-T1-R11 and 5 R13 is - (CH2)i_3~Ar4. Alternatively, R3 is -C(O)-CH2-

Ti-Riv T1 and R11 is -C(O)-Ar4. Alternatively, R3 is -C(O)-H. Alternatively, R3 is -CO-CH2-T1-R11 andR12 is -Ar4.

More preferably, in these more preferred10 compounds, R5 is -C(O)-R10, -C(O)O-Rg, or -C(0)-NH-R10.

Alternatively, in these more preferred compounds, R5 is-S(O)2-R9, -S(O)2-NH-R10, -C(0)-C(0)-R10, -R9, or -C(0)-C(O)-OR10.

Most preferably, in these more preferred 15 compounds: m is 1; T-j. is 0 or S; r13 is H or a -C3_4 straight or branched alkylgroup optionally substituted with -Ar3 -OH, -0R9, or 20 -CO2H, wherein the R9 is a -Ο3_4 branched or straightalkyl group, wherein Ar3 is morpholinyl or phenyl,wherein the phenyl is optionally substituted with Q·^; R21 is -H or -CH3; R51 is a C3_g straight or branched alkyl group 25 optionally substituted with Ar3, wherein Ar3 is phenyl,optionally substituted by -Q3;

Ar2 is (hh) ; Y is 0; and

Ar3 is phenyl, naphthyl, thienyl, quinolinyl, 30 isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl,benzotriazolyl, benzimidazolyl, thienothienvl,imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridylbenzofuranyl, or indolyl;

Ar4 is phenyl, tetrazolyl, pvridinyl, oxazolyl, V 6 2 1. 0 / e 6 Ζ^,'ςίν naphthyl, pyrimidinyi, or thienyl; each is independently -NH2, -Cl, -F, -Br, ~OH, “Ro, -NH~R5 wherein Re. is ~C(O)-R10 or -S(O)2_Rq« -OR5 wherein Rs is -C(O)-Rin, -OR9, -NHP.g, or 5 0 / \ CHo, \ / 0 10 wherein each Rg and Pc θ are independently a -Ci„q straight or branched alkyl group optionally substitutedwith Arp wherein Ar3 is phenyl; provided that when -Ar3 is substituted with a ζρgroup which comprises one or more additional -Arp 15 groups, said additional -Ar3 groups are not substitutedwith another -Ar3,

Preferred compounds of embodiment B include,but are not limited to 213e, 302, and 304a.

Preferred compounds of embodiment C include, 20 but are not limited to 214c, 214e, 217c, 217d, 217e, 24 6, 257, 280, 281, 282, 283, 284, 285, 286, 287, 204, 405, 406, 407, 408, 409, 410, 411, 412, 413, 415, 416, ill’, 218, 419, 420, 422, 423, 424, 425, 426, 430, 431, 2 32, 433, 434, 435, 236, 437, 438, 4 39, 440, 441, 242, 25 24 3, 444, 445, 446, 2 4 7, 4 4 8 , 449, 4 50, 451, 452, 453, 4 52, 455, 456, 457, 258, 459, 4 60, 4 61, 462, 463, 464, 2 65, 4 66, 4 6 7, 4 6 8, 269, 470, 471, 472, 473, 472, 475, 27 6, 477, 478, 479, 2 8 0, 4 81, 4 81s , 282 , 4 82s, 4 8 3, 4 82 , 485, 486, 487, 2 8 8, 4 8 9, 4 90, 4 91, 493, 492, 295, 30 496, 49 p 4 98, 4 99, 421, 427, and 42 8. The ICE 1 nr; 1 ii i tors of another embodiment (D) of this invention are those of formula (I): ΑΡ/Γ/ 9 8/Q1284 APt 0 1280 - 31 - (i) r1-n-r2

H wherein each R5 is independently -C(O)-R10, -C(O)O-5 Rg, -C(0)-N(R10)(R10), -S(O)2-R9, -S(O)2-NH-R10, -C(O)- CH2-O-R9, -C(0)C(0)-R10/ -R9, -H, -C(0)C(0)-OR10, or-C(O)C(O)-N(R9) (R10) ; each is independently -NH2, -CO2H, -Cl, -F, -Br,-I, -NO2, -CN, =0, -OH, -perfluoro C4_3 alkyl, R5, -OR5, 10 -NHR5, OR9, -N(R9)(R1o), Rg, -C(O)-R10, or

O / \ ch2, \ /

15 O provided that when -Ar3 is substituted with a Q2group which comprises one or more additional -Ar3groups, said additional -Ar3 groups are not substitutedwith another -Ar3; 20 and the other substituents are as defined above in embodiment (B).

Preferably, R5 is -C(O)-R10, -C(O)O-R9, or) -C(0)-NH-R10· Alternatively, R5 is -S(O)2-R9, -S(0)2- NH-R10, -C(O)-C(O)-R10, -R9, or -C (0)-C (0)-OR10 . 25 More preferably: m is 1; R13 is H or a -C2_4 straight or branched alkylgroup optionally substituted with -Ar3 -OH, -ORg, or-CO2H, wherein the R9 is a -C4_4 branched or straight 30 alkyl group, wherein Ar3 is morpholinyl or phenyl, wherein the phenyl is optionally substituted with Q2; R21 is -H or -CH3; R51 is a C2_g straight or branched alkyl groupoptionally substituted with Ar3, wherein Ar3 is phenyl, 35 optionally substituted by -Q-p ΑΡ/Γ7 9 8 /01294 - 32 - each Ar3 cyclic group is independently phenyl,naphthyl, thienyl, quinolinyl, isoquinolinyl,pvrazolyl, thiazolyl, isoxazolyl, benzotriazolyl,benzimidazolyl, thienothienyl, imidazolyl, 5 thiadiazolyl, benzo[bjthiophenyl, pyridyl, benzofuranyl, or indolyl, and said cyclic groupoptionally being singly or multiply substituted by -Qt ; each is independently -NH2, -Cl, -F, -Br. -OH,-R9, -NB-R5 wherein R5 is -C(O)-R10 or _S(°^2_R9' “0R5 10 wherein R5 is -C(O)-R10, -OR9, -N(R9)(R10), or0 / \ CHo, \ / 15 0 wherein each R9 and R-t 2 are independently a -C2_gstraight or branched alkyl group optionally substitutedwith Ar3 wherein Ar3 is phenyl; provided that when -Ar3 is substituted with a Q220 group which comprises one or more additional -Ar3 groups, said additional -Ar3 groups are not substitutedwith another -Ar3.

The ICE inhibitors of another embodiment (E)of this invention are those of formula (II) :

wherein: each R5 is -CiC)~R10, -C(O)O-R9, -C (Ο)-N (Rj 0 URj 0) , -~S (C)2~R9, ~S(O)2-NH......Rl0, -C (0)-CH2-O-Rq, -c (0) C (0! ~R10f .....Ry, -H, “C(O)C(O)-CR1C, or -C (0) C (0)-N (R9) (R10) ; R25 is -OH, -OAiy, -N(H)-OH, or a -OC2_g straightcr branched alkyl group optionally substituted wish APS 0 1280 - 33 - -Ar3, -CONH2, -OR5, -OH, -ORg, or -CO2H; each Q2 is independently -NH2, -CO2H, -Cl, -F, -Br, -I, -NO2, -CN, =0, -OH, -perfluoro alkyl, R5, -OR5, -NHR5, ORg, -N(Rg)(Rjq), Rg, -C(O)-Rjq, Or 5 0 / \ CH2; \ / 0 10 provided that when -Ar3 is substituted with a Q]_ ) group which comprises one or more additional -Ar3 groups, said additional -Ar3 groups are not substitutedwith another -Ar3; and the other substituents are as described above15 in embodiment (C).

In more preferred compounds of embodiment E, R3 is C0-Ar2. Alternatively, R3 is -C(0)-CH2-T1-R11 andR11 is - (CH2)i_3-Ar4. Alternatively, R3 is -C(O)-CH2-T1“R11' Tl 0, and R12 is -C(0)-Ar4. Alternatively, 20 R3 is -C(O)-H. Alternatively, R3 is -CO-CH2-T1-R11 andR11 is -Ar4.

More preferably, in these more preferred) compounds, R5 is -C(O)-R10, -C(O)O-Rg, or -C(0)-NH-R10.

Alternatively, in these more preferred compounds, R5 is25 -S(O)2-R9, -S (0) 2-NH-R10, -C(O)-C(O)-Rlo, -Rg, or -C (0)- C(O)-OR10.

Most preferably, in these more preferred compounds : m is 1; 30 T2 is 0 or S (except as defined above); R25 is -OH or -OC2_4 straight or branched alkyl group optionally substituted with -Ar3 -OH, -ORg, or-CO2H, wherein the Rg is a -0]__4 branched or straightalkyl group, wherein Ar3 is morpholinyl or phenyl, 35 wherein the phenyl is optionally substituted with Qp ΑΡ/Γ/ββ / 0 1294

It R2i is -H or -CH-;

Ar2 is ihh); Y is 0; and each Ar3 cyclic group is independently phenyl, 5 naphthyl, thienyl, quinolinyl, isoquinolinyl, pyratolyl, thiazolyl, isoxazolyl, benzotriazolyl,benzimidazolyl, thiencthienyl, imidazolyl,thiadiazolyl, benzo[b]thiopnenyl, pyridyl,benzofuranyl, or indolyl, and said cyclic group 10 optionally being singly or multiply substituted by -Gy; each A.r4 cyclic group is independently phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, pyrimidinyl,or truenyl, said cyclic group being singly or multiplysubstituted by -Q-] ; 15 each Q2 is independently -NH2, -Cl, -F, -Br, -OH, -Rq, -NK-R5 wherein R5 is -C(O)-R10 or -S(O)2-Rsm ......OR5 wherein R5 is -C(O)--R10, -0R9, -N(R9) (R10), or 0 / \ 20 CH2, \ / 0 wherein each R9 and R1C are independently a -C]__estraight or branched alkyl group optionally substituted 25 with Ary wherein Ar3 is phenyl; provided that when -Ar3 is substituted with a Q-> group which comprises one or more additional ~Ar-groups, said additional -Ar3 groups are not substitutedwith another -Ar·?. 30 The ICE inhibitors of another embodiment {F) of this invention are those of formula (III): ΑΡ/Γ/98/0 1284 ΑΡθΟ 128 0 - 35 - (III) r1-n-r2

H wherein R2 and R2 are as described above in embodiment5 (D) .

Preferably, R5 is -C(O)-R10, -C(O)O-R9, or-C(0)-NH-R10. Alternatively, R5 is -S(O)2-R9, -S(O)2-NH-R10, -C(O)-C(O)-R10, -Rg, -C(0)-C(0)-OR10, or-C(O)C(O)-N(R9) (R10) · 10 More preferably, R5 is R-C(0)-C(0)-R10.

Alternatively, R5 is -C(0)-C(0)-OR10 .

More preferably,m is 1; R21 is -H or -CH3; 15 R51 is a Cj_-g straight or branched alkyl group optionally substituted with Ar3, wherein the Ar3 cyclicgroup is phenyl, said cyclic group optionally beingmultiply or singly substituted by -Q-p each Ar3 cyclic group is phenyl, naphthyl, 20 thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl,thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, benzofuranyl, or indolyl,and said cyclic group optionally being singly or 25 multiply substituted by -Qlz· each Q-l is independently -NH2, -Cl, -F, -Br, -OH, -R9, -NH-R5 wherein R5 is -C(O)-R10 or -S(0)2-Rg, -0R5 wherein R5 is -C(0)-R10, -0R9, -N(R9)(R10)> and0 30 / \ CH2, \ / 0 wherein each R9 and R10 are independently a -Cj.gstraight or branched alkyl group optionally substitutedwith Ar3, wherein the Ar3 cyclic group is phenyl, and AP/P/ 9 8/01294 35 saia cyclic group optionally being singly or multiplysubstituted by -Qy; provided that when -Ar3 is substituted with a -tbgroup which comprises one or more additional -Ar3 5 groups, said additional ~Ar3 groups are not substitutedwith another -Ar3.

More preferably, in these more preferredcompounds, the Ar3 cyclic group is phenyl, naphthyl,thienyl, quinolinyi, isoquinolinyl, pyrazolyl, 10 thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl,thienothienyl, imidazoiyl, thiadiazolyl,bento[bjthiophenyl, benzofuranyl, or indolyl, and saidcyclic group optionally being singly or multiplysubstituted by -Qy, 15 Compounds m a preferred form of this embodiment F are those wherein: R5 is ~C{O)-Rt0, wherein:

Rj0 is Ar3, wherein, the Ar3 cyclic group is phenyl,said cyclic group optionally being singly or multiply 20 substituted by: -F, -Cl, -NfHl-Rj, wherein ~R3 is -H or -C(O)-R10, whereinRyn is a ~-Cy_g straight or branched alkyl group25 optionally substituted with Ar3, wherein the Ar3 cyclicgroup is phenyl, said cyclic group optionally beingsingly or multiply substituted by -Qy, -N(Piq) iRyob wherein R9 and R10 are independently a“t'-_4 straight or branched alkyl group, or -O-Rr, wherein Ry is H or a -Cy_, straight orbranched alkyl group.

More preferably the Arp cyclic group isphenyl optionally being singly or multiply substitutedat the 3- or 5-position by -Cl or at the 4-positlon by AP/F/ »8/0 1294 30 AP C Ο 12 8 Ο - 37 - -NH-R5, -N(R9)(R10), or -O-R5.

Other preferred compounds of embodiment Finclude those wherein R5 is -C(O)-R10, wherein R10 isAr3 and the Ar3 cyclic group is indolyl, 5 benzimidazolyl, thienyl, and benzo[b]thiophenyl, or said cyclic group optionally being singl-y or multiplysubstituted by -Qj.

Other preferred compounds of embodiment Finclude those wherein R5 is -C(O)-R10, wherein R10 is 10 Ar3 and the Ar3 cyclic group is quinolyl or isoquinolyl, and said cyclic group optionally beingsingly or multiply substituted by -0χ.

Other preferred compounds of embodiment F arethose wherein R5 is -C(O)-R10, wherein R10 is Ar3, 15 wherein the Ar3 cyclic group is phenyl, substituted by0 / \ CH2 \ / 20 0

In another form of embodiment F the compoundsare as described above, further provided that when:m is 1; R 5 is - OH ; 25 R21 is -H; and Y2 is 0 and R3 is -C(O)-H, then R3 cannot be:-C(O)-R10, wherein R10 is -Ar3 and the Ar3 cyclic group is phenyl, unsubstituted by -Q2, 4-(carboxymethoxy)phenyl, 2-fluorophenyl, 2-pyridyl, N- 30 (4-methylpiperazino)methylphenyl, or -C(O)-OR9, wherein R9 is -CH2-Ar3, and the Ar3 cyclic group is phenyl, unsubstituted by -Ch; and when Y2 is 0, R3 is -C (0)-CH2-T1-R11, T3 is 0, and Rnis Ar4, wherein the Ar4 cyclic group is 5-(1-(4- ΑΡ/Γ; &amp; 8 / a 1 2 8 4 chlorophenyl)-3-trifIuoromethyl)pyrazolyl} , then R5 cannot be : --CiOJ-R^g, wherein R10 is -Ar3 and the Am cyclicgroup is 4-(dimethyiaminomethyl)phenyl, phenyl, 4~ 5 (carboxyinethyl thio) phenyl, 4- (carboxyethyl thio) phenyl, 4--.carboxyethyl) phenyl, 4-(carboxypropyl)phenyl, 2-fluorophenyl, 2-pyr. : · i, N-(4- methylriperazino)methyiphenyl, or -C(O)-ORg, wherein Rg is -CH2-Ar3 and the Ar3 10 cyclic group is phenyl; and when R31 is Am, wherein the Ar4 cyclic group is S~(l-phenyi-3-trifIuoromethyl)pyrazolyl', then 5ncannot be: -C£O)-OR9, wherein Rg is -CH2-Ar3, and the Am15 cyclic group is phenyl; and when R-q is Ar4, wherein the Ar4 cyclic groupis 5- (1-- (2-pyridyl) -is-tri fluoromethyl) pyrazolyl) , then cannot be: -C(O)-R10, wherein R1C is -Ar3 and the Ar3 cyclic20 group is 4-(dimethyiaminomethyl)phenyl, or -C(O)-ORg, wherein Rg is -CH2-Ar3, and the Arycyclic group is phenyl, unsubstituted by -Q3; and when Y2 is 0, R3 is ™C JO)-CH2-T1-R11, T]_ is 0, and R31is -C(0)-Ar4, wherein the Ar4 cyclic group is 2,5- 25 dicnlorophenyl, then Fy cannot be: -C'iCn-RjQ, wherein R10 is -Ar3 and the Am group is 4-(dimethyiaminomethyl) phenyl, 4-(N-niorpholinomethyl) phenyl, 4- (N-niethylpiperazinojmethyl) phenyl, 4- (N- (2- 30 methyl)imidazolylmethyi) phenyl, 5-benzimidazolyl, 5..... ben z t r i a zο 1 y 1, N - c a mo e thoxy-5-benz t r ia zo1yi, N-carboethoxy-5-benzinudazolyl, or AP c ο 12 β ο - 39 - -C(O)-ORg, wherein Rg is -CH2-Ar3, and the Ar3cyclic group is phenyl, unsubstituted by -Qj; and when Y2 is H2, R3 is —C (0) —CH2—T2—R]_i, is 0' and R]_l is -C(O)-Ar4, wherein the Ar4 cyclic group is 2,5- 5 dichlorophenyl, then R5 cannot be: -C(O)-ORg, wherein Rg is -CH2-Ar3 and the Ar3 cyclic group is phenyl.

In another form of embodiment F, preferred compounds are those wherein R21 is -H. Alternatively, 10 R21 is -CH3.

More preferably, in these more preferredcompounds, R5 is -C(O)-R10, -C(O)O-Rg, or -C(0)-NH-R10.Alternatively, R5 is -S(O)2-R9, -S(0)2-NH-R10, -C(0)-C(O)-R10, -Rg, -C (0) -C (0) -OR10, or -C(O)C(O)-N(Rg) (R10) . 15 Most preferably, in these more preferred compounds, m is 1; R]_3 is H or a -C;j__4 straight or branched alkylgroup optionally substituted with -Ar3 -OH, -ORg, or 20 -CO2H, wherein the Rg is a -C;]__4 branched or straightalkyl group, wherein Ar3 is morpholinyl or phenyl,wherein the phenyl is optionally substituted with Q^; R21 is -H or -CH3; R51 is a C3_6 straight or branched alkyl group 25 optionally substituted with Ar3, wherein Ar3 is phenyl,optionally substituted by -Q-p· each Ar3 cyclic group is independently phenyl,naphthyl, thienyl, quinolinyl, isoquinolinyl,pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, 30 benzimidazolyl, thienothienyl, imidazolyl,thiadiazolyl, benzo[b]thiophenyl, pyridyl,benzofuranyl, or indolyl, and said cyclic groupoptionally being singly or multiply substituted by -Qj; ΑΡ/Γ/ 9 8 / 0 1 2 9 4 each Q]_ is independently -NH2, -Cl, -F, -Br, -OH,--Hq, -NH-R5 wherein Rq is -C(O)-R3q or -S(O)2-Rcw -OR5wherein R5 is -C (0}--R-, r,, -ORg, -N(Rg) (R^q) , or 0 5 / \ CH?, \ / 0 wherein each Rg and R!0 are independently a -Ci__q10 straight or branched alkyl group optionally substituted wirh Ar~, wherein Are is phenyl; provided that when -Ars is substituted with a ζρ group which comprises one or more additional -Am,groups, said additional -Ar3 groups are not substituted 15 with another -Ar3.

Preferred compounds of embodiment (F) include, but are not limited to 2001, 2100a, 2100b,2100c, 2100d, and 2100e.

The ICE inhibitors of another embodiment (H)20 of this invention are those of formula (V), wherein R?1 is -CH3 and the other substituents are as defined abovein embodiment (G).

Compounds of another form of embodiment (I)(form 1; are those of formula (V) wherein each Rq is 25 -C(Ο)C(Os-OR10; Y2 is H3 or 0; and the other substituents are as defined above in embodiment (0).Alternatively, compounds of this form of embodiment I(form 2} are those wherein R21 is -CH-.

Compounds of another form of embodiment of) 30 (fcrni 10 are those of formula (V) , provided that when:w 1. s 1;h15 is -OK;

Rq-i is -H; and Y2 is 0 and R-j is ~C(Q)-H, then R3 cannot be: AP c ο 12 8 Ο - 41 - -C(O)-R10, wherein R10 is -Ar3 and the Ar3 cyclicgroup is phenyl, unsubstituted by -Q1; 4-(carboxymethoxy)phenyl, 2-fluorophenyl, 2-pyridyl, N-(4-methylpiperazino)methylphenyl, or 5 -C(O)-OR9, wherein R9 is -CH2-Ar3, and the Ar3 cyclic group is phenyl, unsubstituted by -Qj; and when Y2 is 0, R3 is -C (0) -CH2 — T2—R]_2, T2 is 0, and R]_2is Ar4, wherein the Ar4 cyclic group is 5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazolyl), then R5 10 cannot be: -C(O)-R10, wherein R10 is -Ar3 and the Ar3 cyclicgroup is 4-(dimethylaminomethyl) phenyl, phenyl, 4-(carboxymethvlthio)phenyl, 4-(carboxyethylthio)phenyl,4-(carboxyethyl)phenyl, 4-(carboxypropyl)phenyl, 2- 15 fluorophenyl, 2-pyridyl, N-(4- methylpiperazino)methylphenyl, or -C(0)-0R9, wherein R9 is -CH2-Ar3 and the Ar3cyclic group is phenyl; and when R14 is Ar4, wherein the Ar4 cyclic group20 is 5-(l-phenyl-3-trifluoromethyl)pyrazolyl), then R5 cannot be : -C(0)-0R9, wherein R9 is -CH2-Ar3, and the Ar3cyclic group is phenyl; and when R1;L is Ar4, wherein the Ar4 cyclic group25 is 5-(1-(2-pyridyl)-3-trifluoromethyl)pyrazolyl), then R3 cannot be: -C(O)-R2q, wherein R10 is -Ar3 and the Ar3 cyclicgroup is 4-(dimethylaminomethyl) phenyl, or -C(O)-OR9, wherein R9 is -CH2-Ar3, and the Ar330 cyclic group is phenyl, unsubstituted by -Q-p· and when Y2 is O, R3 is — C (0) — CH2—T4—R2]_, T2 is 0, and R]_i ...... 42 is -C(O)~Ar4, wherein the Ar4 cyclic group is 2,5..... dichlorophenyl, then Rs cannot be: -C(Q)-R|q, wherein R10 is -Ar3 and the Ar3 cyclicgroup is 4-(dimethylami nomethyl)phenyl, 4-(N- 5 morpholinomethyl) phenyl, 4-(N- methylpiperazino)methyl;phenyl, 4-(N-(2- methyl) imidazolylmethyl} phenyl, 5-benzimidazolyl, 5..... benztriazolyl, N-carboethoxy-5-benztriazolyl, N-carboethoxy-5-benzimidazolyl, or 10 —C (0)-ORg, wherein Rg is -CH2-Ar3, and the Ary cyclic croup is phenyl, unsubstituted by -Cp; and when ϊ2 is ^2' s 10) ~CH2~T“Rgi, T3 is 0, and M] is ~C(0)-Ar4, wherein the Ar4 cyclic group is 2,5-15 dichlorophenyl, then R3 cannot be: -C(0)-ORg, wherein Rg is -CH2-Ar3 and the Ar3 cyclic group is phenyl; and the other substituents are as described abovein embodiment (G). 20 Compounds of another form of embodiment 1 (form 2) are those wherein R21 is -CH-j. Compounds ofanother form of embodiment J (form 3) are those wherein.Rr, is -C (0)-C (0)-OR] o . Compounds of another form ofembodiment J (form 4) are those wherein R5 is ~C (Ch- ic c i 01 ~O’K” q and R2i is άιά ·

Preferred compounds of embodiments Η, I, and J employ formula (V;, wherein R3 is -CO-Ary. Morepreferably, R3 is -CO.....Ary and Y is 0.

Preferred compounds of embodiments H, 1, and 30 J employ formula (V) , wherein R3 is -C (0) -CH^-T-i~R-< Ί andRpi is ~(CH?) j__3-Arύ. More preferably, when R3 is•~C (0} "CH2-Ti“R11 and Rp is - (CH2 ) i-3~Ar4, T2 is 0.

Preferred compounds of embodiments Η, I, and. ΑΡ/Γ7 98/01294 ) 10 15 20

J 25 AP u Ο 12 8 Ο J employ formula (V), wherein R3 is -C (0)-CH2-T1-R-L-l, Tjis 0, and Rl:1 is -C(O)-Ar4.

Preferred compounds of embodiments Η, I, andJ employ formula (V), wherein R3 is -C(O)-H.

Preferred compounds of embodiments Η, I, andJ employ formula (V), wherein R3 is -CO-CH2-T1-R11 andRj! is -Ar4. More preferably, when R3 is -CO-CH2-T1-R11and Rj_]_ is -Ar4, T]_ is 0 or S.

More preferred compounds of embodiments H andJ (forms 1 and 2) are those wherein R5 is -C(O)-R10,-C(0)0-Rg, or -C(0)-NH-R10 . Alternatively, R5 is-S(O)2-R9, -S(O)2-NH-R10, -C(0)-C (0)-R10, -Rg, -C(0)-C(O)-OR10, or -C(0)-C(Ο)-N(Rg) (R10) .

Most preferably, R5 is -C(0)-C(0)-R10.Alternatively, R5 is -C(0)-C(0)-OR10.

More preferred compounds of embodiments Η, I(form 2), and J (forms 2 and 4) are those wherein: m is 1 ; Y2 is 0;

Rl5 is -OH or -0C]__4 straight or branched alkylgroup optionally substituted with Ar3, -OH, -ORg, -CO2H,wherein the Rg is a C]__4 branched or straight chainalkyl group; wherein Ar3 is morpholinyl or phenyl,wherein the phenyl is optionally substituted with Q4;

Ar2 is (hh); Y is 0, and each Ar3 cyclic group is independently phenyl,naphthyl, thienyl, quinolinyl, isoquinolinyl,pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl,benzimidazolyl, thienothienyl, imidazolyl,thiadiazolyl, benzo[b]thiophenyl, pyridyl,benzofuranyl, or indolyl, and said cyclic groupoptionally being singly or multiply substituted by -Qi; each Ar4 cyclic group is independently phenyl, AP/F7 9 8 I 0 129 4 30 tetrazolyl, pyridyl, oxazolyl, naphthyl, pyrimidinyl,or thienyl, and said cyclic group optionally beingsingly or multiply substituted by -Qjj each Gy is independently -NH2, -Cl, -F, -Br, -OH, 5 -R9, -NH-R5, wherein ΕΡ is -C(Q)-R10 or -S(O)2"Rcp -OR5 wherein R5 is -C{O) -Rin, -OR9, -N(Rq) (R10) , and . 0 / \ ch2' 10 \ / 0 wherein each Re and R10 are independently a ~Cb_gstraight or branched alkyl group optionally substitutedwitl Ar3 wherein the Ar3 cyclic group is phenyl, and, 15 said cyclic group optionally being singly or multiplysubstituted by -Q]_; provided that when -Ar3 is substituted with a ζρgroup which comprises one or more additional -Ar3groups, said additional -Ar3 groups are not substituted 20 with another -Ar3.

More preferred compounds of embodiments 1 (foi’m 1) , and J (form, 3) are those wherein:m i s 1; R2~ is --H or -CH-y 25 is a C]__g straight or branched alkyl group optionally substituted with Ar3, wherein the Ar3 cyclicgroup is phenyl, said cyclic group optionally beingmult iply or singly substituted by -Qi ; each Ary cyclic group is independently phenyl, 30 naphthyl, thienyl, quinoiinyl, isoquinolinyl, pyrazclyl, thiazolyl, 1soxazolyl, benzotriazolvl,be n z imidazoiyl, t h1e n 01tlenyl, imidazoiyl,thiadiazoiyl, benzo lb ·thiophenvl, pyridyl,benzofuranyl, or indclyl, and said cyclic group 35 optionally being singly or multiply substitute’.: ......lb; ΑΡ/Γ/ 9 8*01294 - 45 - each Q2 is independently -NH2, -Cl, -F, -Br, -OH, -Rg, -NH-R5 wherein R5 is -C(O)-R]_q or -S(O)2-Rg, -OR5 wherein R5 is -C(O)-R10, -ORg, -N(Rg)(R10), or0 5 / \ CH2, \ / 0 10 wherein each Rg and R10 are independently a straight or branched alkyl group optionally substitutedwith Ar3, wherein the Ar3 cyclic group is phenyl, andsaid cyclic group optionally being singly or multiplysubstituted by -Qlz· 15 provided that when -Ar3 is substituted with a -Q3 group which comprises one or more additional -Ar3groups, said additional -Ar3 groups are not substitutedwith another -Ar3.

Preferably, in these more preferred compounds 20 the Ar3 cyclic group is phenyl, naphthyl, thienyl,quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl,isoxazolyl, benzotriazolyl, benzimidazolyl,thienothienyl, imidazolyl, thiadiazolyl,benzo[b]thiophenyl, benzofuranyl, or indolyl, and said 25 cyclic group optionally being singly or multiplysubstituted by -Q]_.

Preferred compounds of embodiments H, and J(forms 1 and 1) are those wherein: R3 is —C(0)-CH2-T1-R11; 30 Ti is O; and

Rll is -C(0)-Ar4, wherein the Ar4 cyclic group istetrazolyl, pyridyl, oxazolyl, pyrimidinyl, or thienyl,and said cyclic group optionally being singly ormultiply substituted by -Qi_. 35 Preferred compounds of embodiments Η, I, and J employ formula (V), wherein R3 is -CO-CH2-T1-R11 and

<r 6 Z 1· 0 / θ 6 ZJ/dV

Rn ~Ar4, wherein the Ar4 cyclic group is pyridyl,and said cyclic group optionally being singly ormultiply substituted by -Qj .

Preferred compounds of embodiment J (form 1) 5 are those wherein: R3 is -C(0)-H, and. R3 is -C(O)-Ri0, wherein: R10 is Ar3, wherein the Ar3 cyclic group is phenyloptionally being singly or multiply substituted by: 10 -F, -Cl, -N(K)-R5, wherein -R5 is -H or -C(O)-R10, whereinRiq is a "Cj-g straight or branched alkyl groupoptionally substituted with Ar3, wherein Ar3 is phenyl, 15 -N(Rcj) (R10), wherein R9 and R10 are independently a ~C:1„4 straight or branched alkyl group, or “O-Rg, wherein R5 is H or a -Ci_4 straight orbranched alkyl group.

More preferably, Ar3 is phenyl being20 optionally singly or multiply substituted at the 3- or 5-position by -Cl or at the 4-position by -NH-R5, -N(Rg) (RpR, or -O-Ry -

Other more preferred compounds of embodimentJ (form ly are those wherein R3 is -C(O)-H and Rr is 25 -C(O!-R1q, wherein R10 is Ar3 and the Ar3 cyclic group is indolyl, benzimidazolyl, thienyl, orbenzo[bltniophenyl, and said cyclic group optionallybeing singly or multiply substituted by ~Qi;

Other more preferred compounds of embodiment 30 J (form 1) are those wherein R3 is -C(O)-H and R3 is -CPIP-Rtq, wherein R-jfi is Ar3 and the Ar3 cyclic nroupis quinolyl or isoqumolyl, and said cyclic groupoptionally being singly or multiply substituted by -Q-> .

Other more preferred compounds of emboaiment

* 8 Z I 0 / 8 6 /J/dV AP V Ο 12 8 Ο - 47 - J (form 1) are those wherein R3 is -C(O)-H and R5 is-C(0)-R]_o/ wherein R10 is Ar3 and the Ar3 cyclic groupis phenyl, substituted by 0 5 / \ CH2 \ / 0

The ICE inhibitors of another embodiment (K)10 of this invention are those of formula: (VI) R1-N-R2 wherein:

m is 1 or 2; each R3 is independently -C(O)-R10, -C(O)O-R9,-C(O)-N(R10) (R10) , -S(O)2-R9, -S (0) 2-NH-R1o, -C(O)-CH2-0-R9, -C (0) C (0) -R10, -Rg, -H, -C(O)C(O)-OR10f or 25 -C(O)C(O)-N(R9) (R10) ; X5 is CH; Y2 is H2 or 0; each R9 is independently -Ar3 or a -C1_g straight or branched alkyl group optionally substituted withArc, wherein the -C~1„6 alkyl group is optionallyuns a t ura t e d; each R10 is independently -H, -Ar3, a -C3_65 cycloalkyl group, or a -C]._g straight or branched alkyl group optionally substituted with Ar3, wherein the -Cj.galkyl group is optionally unsaturated; F03 is H, Ar3, or a -Cy-g straight or branchedalkyl group optionally substituted with Ar3, ~CONH?, 10 -OR5, -OH, -OR9, or ~CO2H; each R51 is independently Rg, -C(O)-Rg, or -C (O}-~ Ν{Η)~Β.ς, or each R51 taken together forms a saturated 4-8 member carbocyclic. ring or heterocyclic ringcontaining -0-, -S-, or -NH-; 15 each R2i is independently -H or a -C^g straight or branched alkyl group; each Ar3 is a cyclic group independently selectedfrom the set consisting of an aryl group which contains6, 10, 12, or 14 carbon atoms and between 1 and 3 rings 20 and an aromatic heterocycle group containing between 5and 15 ring atoms and between 1 and 3 rings, saidheterocyclic group containing at least one heteroatomgroup selected from -0-, -S-, -SO-, SO2, =N-, and --NH-,said heterocycle group, optionally containing one or 25 more double bonds, said heterocycle group optionally comprising one or more aromatic rings, and said cyclicgroup optionally being singly or multiply substitutedby -dig each Q2 is independently -NH2, -CO2H, -Cl, ~F, -Er, 30 -I, -NCg, -CN, =0, -OK, -perfluoro C-_3 alkyl, Ph, ™CRr,, ΑΡ/Γ/ 9 8 / a 1 2 S 4 APCO1280 - 49 - -NHR5, -0R9, -N(R9)(R1o), -R9, -C(O)-R10, and 0 / \ CH2, 5 \ / 0 provided that when -Ar3 is substituted with a Q2group which comprises one or more additional -Ar3 10 groups, said additional -Ar3 groups are not substituted with another -Ar-s. ) 3

Preferred compounds of this embodiment are thosewherein: m is 1; 15 R13 is H or a C]__4 straight or branched alkyl group optionally substituted with Ar3, -OH, -OR9, -CO2H,wherein the R9 is a C2_4 branched or straight chainalkyl group; wherein Ar3 is morpholinyl or phenyl,wherein the phenyl is optionally substituted with Q-j_; 20 R21 is -H or -CH3; R51 is a C]__6 straight or branched alkyl group optionally substituted with Ar3, wherein Ar3 is phenyl,optionally substituted by -Q]_; ) each Ar3 is independently phenyl, naphthyl, 25 thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl,thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, benzofuranyl, or indolyl,and said cyclic group optionally being singly or 30 multiply substituted by -Qp each Q]_ is independently -NH2, -Cl, -F, -Br, -OH, -R9, -NH-R5 wherein R5 is -C(O)-R10 or -S(O)2-R9, -OR5wherein R5 is -C(O)-R10, -OR9, -NHR9, or

f 6 Z i- 0 / β 6 /J/dV 50 Ο / \ <-Η2 r \ / Γ Ζ"\ 5 ν wherein each R9 and R10 are independently a -Cb_gstraight or branched alkyl group optionally substitutedwith Ar3 wherein Ar3 is phenyl; provided that when -Ar3 is substituted with a O3 0 group which comprises one or more additional -Ar3 groups, said additional -Ar3 groups are not substitutedwith another -Ar3.

Preferably, in any of the above compounds ofembodiment (K) , R5 is --C(O)-R10 or -C(0)-C (0)-R30 and 5 the other substituents are as defined above.

More preferably, R10 is -Ar3 and the other substituents are as defined above.

More preferably, in these more preferred compounds R5 is -C(O)-R10 and R10 is Ar3, 0 wherein the Ar3 cyclic group is phenyl optionally being singly or multiply substituted by: -Ro (wherein Rq is a Cj_4 straight or branchedalkyl group), -F, -Cl, -N(H)-R5 (wherein -R5 is --H or-CiO5-R10, wherein R10 is a -C^-q straight or branched 5 alkyl group optionally substituted with Ar3, whereinAr3 is phenyl), -N(Rg)(Rjo) (wherein R9 and R10 areindependently a -0-..4 straight or branched alkyl group)or -0-R=, (wherein Rg is H or a straight or branched alkyl group), 0 Most pre - -.., Ar3 is phenyl being singly or multiply subst; Ci. - : at the 3- or 5-position by -Clor at: the 4-positicn by -NK-Rq, -N(R3) (R10), or -0 Rs.

Other preferred compounds of this most•' . -mbodiment include, but are not limited to

V 6 Z I 0 / 8 6 /d/dV APC 0 1280 - 51 - 213k, 213m, 550k, and 550m.

Alternatively, Ar3 is phenyl being singly or multiply substituted at the 3- or 5-position by -R9,wherein R9 is a straight or branched alkyl group; 5 and at the 4-position by -O-R5.

Other preferred compounds of this most preferred embodiment include, but are not limited to214w-l, 214w-2, 214w-3, 214w-4, 214w-5, 214w-6, and214W-7. 10 Alternatively, in this more preferred embodiment, R5 is -C(O)-R10, wherein R10 is Ar3 and theAr3 cyclic group is selected from the group consistingof is indolyl, benzimidazolyl, thienyl, quinolyl,isoquinolyl and benzo[b]thiophenyl, and said cyclic 15 group optionally being singly or multiply substitutedby -03.

Most preferably, the Ar3 cyclic group isisoquinolyl.

Other preferred compounds of this most 20 preferred embodiment include, but are not limited to213y, 412a, 412b, 412c, 412d, 412e, 412f, 412g, 412h,and 550q.

Alternatively, in this more preferredembodiment, R5 is -C(O)-R10, wherein R10 is Ar3 and the 25 Ar3 cyclic group is phenyl, substituted by0 / \ CH2 \ / 30 0

Preferred compounds of this more preferred embodiment include, but are not limited to 213n, 415a,415b, and 415c.

Other compounds of embodiment (K) include, 35 but are not limited to 213f, 213g, 213h, 213i, 213j,

V β I I 0 / 8 6 /J/dV 1 3v, 2131, 213o, 213p, 213q, 213r, 213s, 213t, 213u, 21 3w, 213x, 245b, 5501, 550g, 550h, 550i, 550j, 5501,550n, 550o, 550p, 21001, 2100g, 2100h, 2100k, 21001,2100m, 2100n, and 2100©.

The ICE inhibitors of another embodiment (L)of this invention are those of formula :

O (v; wherein: m is 1;

Ri is: ielO-B}

Yb is H2 or 0; each ϊχ is independently -O- or -S-; R21 is -H or -CH-5 ;

Ar? is:

ΑΡ/Γ7 9 6/01294 wherein. Y is 0; each Ary is independently phenyl, naphthyl,thienyl, quinolinyl, isoquinolinyl, pyrazolyl,thiazolylf isoxazolyl, benzotriazolyl, benzimidazolyl,thienothienyl, imidacolyl, thiadiazolyl,benzo[b>thiophenyl, pyridyl benzofuranyl, or indoiyl,and said cyclic group optionally being singly ormultiply substituted by -Qj; APCΟ 128 0 - 53 - each Ar4 is independently phenyl, tetrazolyl,pyridinyl, oxazolyl, naphthyl, pyrimidinyl, or thienyl,and said cyclic group optionally being singly ormultiply substituted by -Qp 5 each Q]_ is independently -NH2, -Cl, -F, -Br, -OH, -Rg, -NH-R5 wherein R5 is -CfOJ-R^Q or -S(O)2-Rg, -OR5wherein R5 is -C(O)-R10, -ORg, -NHRg, or 0 / \ 10 CH2, \ / 0 provided that when -Ar3 is substituted with a Qjgroup which comprises one or more additional -Ar3 15 groups, said additional -Ar3 groups are not substitutedwith another -Ar3; and the other substituents are as defined above inembodiment (L); provided that when: 20 m is 1; R15 is -OH; R21 is -H; and Y2 is 0 and R3 is -C(O)-H, then R5 cannot be: -C(0)-Rio' wherein R10 is -Ar3 anci the Ar3 cyclic 25 group is phenyl, unsubstituted by -Q]_, 4- (carboxymethoxy)phenyl, 2-fluorophenyl, 2-pyridyl, N-(4-methvlpiperazino)methylphenyl, or —C(0)-ORg, wherein Rg is -CH2-Ar3, and the Ar3cyclic group is phenyl, unsubstituted by -Q^; and when 30 Y2 is O, R3 is -C (0)-CH2-T1-R11, Tx is 0, and R12_ is Ar4, wherein the Ar4 cyclic group is 5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazolyl), then R5cannot be: -H;

*6210/86 /J/rfV - 54 - ~C(O)-R10, wherein R10 is -Ar3 and the Ar3 cyclicgroup is 4-(dimethylaminomethyl) phenyl, phenyl, 1-(carboxymethvlthio)phenyl,4-(carboxyethylthio)phenyl, 4-(carboxyethyl)phenyl, 4-(carboxypropyl)phenyl, 2- 5 fluorophenyl, 2-pyridyl, N-(4- methylpiperazino)methylphenyl, or —C(0)—0R9, wherein Rg is isobutyl or -CH2-Ar3 andthe Ar3 cyclic group is phenyl; and when R13 is Ar4, wherein the Ar4 cyclic group10 is 5-(l-phenyl-3-trifluoromethyl)pyrazolyl or 5-(1-(4- ckloro-2-pyriciinyl) -3-trifluoromethyl) pyrazolyl, thenR3 cannot be: -C(C)-ORg, wherein Rg is -CH2-Ar3, and the Ar3 cyclic group is ph- 15 and when R1;L is Ar4, wherein the Ar4 cyclic group is 5-(1-(2-pyridyl]-3--trifluoromethyl)pyrazolyl) , thenR5 cannot be: -C(O)-R10, wherein R10 is -Ar3 and the Ar3 cyclicgroup is 4-(dimethyiaminomethyl) phenyl, or 20 -C(O)-ORg, wherein Rg is -CH2-Ar3, and the Ar3 cyclic group is phenyl, unsubstituted by -Q3; and when ϊ2 is 0, R3 is — c (o} ~0-H2-T2-R2i, Tis O, ano Ryyis ~C(Oj~Ar4, wherein the Ar4 cyclic group is 2,5--di chlorophenyl, then Re, cannot be: 25 ~C(O)~R10, wherein R10 is ~Ar3 and the Ar3 cyclic group is 4-(dimethylaminomethyl)phenyl, 4-(N-rocrpholinomethyl) pheny l, 4-- (N- methylpiperazino)me- . ( phenyl, 4-(N-(2- methyl) imidazolylmethyl}phenyl, 5-benzimidazolyl, 5- 30 benztriazolyl, N-c - axy-5-benztriazolyl, N- carboethoxy-5-benz iyl, or -CUO(-ORq, wherein Rg is -CH2-Ar3, and the incyclic group is pi .isubstituted by -Qi,; - mu.

»62)0/86 /J/dV ΑΡ δ Ο 12 8 Ο - 55 - Υ3 is Η3, R3 is — C (Ο) —CH3~Τ2_—Rj_ 1' Ίχ i® and R^iis -C(O)-Ar4, wherein the Ar4 cyclic group is 2,5-dichlorophenyl, then R5 cannot be: -C(O)-ORg, wherein Rg is -CH2-Ar3 and the Ar35 cyclic group is phenyl.

Preferably, R3 is -C(O)-Ar2. Alternatively,R3 is -C (0)CH2-T1_R11. Alternatively, R3 is -C(O)-H.

Preferably, in any of the above compounds ofembodiment (L), R3 is -C(O)-H and R5 is -C(O)-R10 or 10 -C(0)-C(0)-R10 and the other substituents are asdefined above.

More preferably R10 is -Ar3.

More preferably in these more preferred compounds: R5 is -C(O)-R10 and R10 is Ar3, wherein the15 Ar3 cyclic group is phenyl optionally being singly or multiply substituted by: -Rg (wherein Rg is a C4_4straight or branched alkyl group), -F, -Cl, -N(H)-R5(wherein -R5 is -H or -C(O)-R10, wherein R10 is a -cl-6straight or branched alkyl group optionally substituted 20 with Ar3, wherein Ar3 is phenyl), -N(Rg) (Riq) (whereinRg and R10 are independently a -C]__4 straight orbranched alkyl group) , or -O-R5 (wherein R3 is H or a-C]__4 straight or branched alkyl group) .

Most preferably, Ar3 is phenyl being singly 25 or multiply substituted at the 3- or 5-position by -Clor at the 4-position by -NH-R5, -N(Rg) (R10) , or -O-R5.

Other preferred compounds of this mostpreferred embodiment include, but are not limited to214k and 214m. 30 Alternatively, Ar3 is phenyl being singly or multiply substituted at the 3- or 5-position by -Rg,wherein Rg is a C3_4 straight or branched alkyl group;and at the 4-position by -0-R5.

* 6 Z !· 0 ' 8 6 /J/dV

Another preferred compound of this mostpreferred embodiment includes, but is not limited to 214c,

Alternatively, in this more preferred5 embodiment: R5 is ~C(0)—R10, wherein R10 is Ar3 and the

Arp cyclic group is selected from the group consistingof is indolyl, benzxnddazolyl, thienyl, quinolyl,isoquinolyl and benzo[bjthiophenyl, and said cyclic 1 group optionally being singly or multiply substituted 10 by .....Qi_»

Most preferably, the Ar3 cyclic group isisoquinolyl, and said cyclic group optionally beingsingly or multiply substituted by -Q]_.

Another preferred compound of this most15 preferred embodiment includes, but is not limited to 412, .Alternatively, in this more preferredembodiment R5 is -C(O)-R10, wherein R10 is -Ar3 and theAr3 cyclic group is phenyl, substituted by ΑΡ/Ϊ7 9 8.01294 25 A preferred compound of this more preferred embodiment includes, but is not limited to115 .

Other compounds of embodiment (L) include,but are not limited to 214f, 214g, 214h, 2141, 214j, 30 2141, 246b, 280b, 282c, 280d, 283b, 283c, 283d, 282, 285, 286, 308c, 308d, 500, 501, 505b, 505c, 505d, 505e, , 510a, 510b, 510c/ 510d, 511c, 2100i, 2100j.

The most preferred compounds of embodiments arid (L) are those wherein the Ar-a cyclic ai APC01280 - 57 -

Compounds of this invention are described inco-pending United States Application Serial Nos.08/575,641 and 08/598,332 the disclosures of which areherein incorporated by reference. 5 The compounds of this invention have a molecular weight of less than or equal to about 700Daltons, and more preferably between about 400 and 600Daltons. These preferred compounds may be readilyabsorbed by the bloodstream of patients upon oral 10 administration. This oral availability makes suchcompounds excellent agents for orally-administeredtreatment and prevention regimens against IL-1-,apoptosis-, IGIF- or IFN-γ mediated diseases.

It should be understood that the compounds of 15 this invention may exist in various equilibrium forms,depending on conditions including choice of solvent,pH, and others known to the practitioner skilled in theart. All such forms of these compounds are expresslyincluded in the present invention. In particular, many 20 of the compounds of this invention, especially thosewhich contain aldehyde or ketone groups in R3 andcarboxylic acid groups in T, may take hemi-ketal (orhemi-acetal) or hydrated forms. For example, compoundsof embodiment (A) may take the forms depicted below: 25 EQ1

Ri-N—Xi

(CJ2)m—C ./

OH

OH H (CH2)g_o_Rl3

Ah

Hydrated Form (CJ2)m—c

/ XOH R,-N—X, H \cH2)g_c—Ro 1 R·]—" N.....Xi

(CJ2)m—C (CH2)g.

OH

Hemi-ketal orHemi-acetal Form

»62 I 0 ! 86 /d/dV

Depending on the choice of solvent and other ..... 58 conditions known to the practitioner skilled in theart, compounds of this invention may also take acyloxyketal, acyloxy acetal, ketal or acetal form: O- -ht—Xi K Xi->ti£L_Z-Re h

Acyioxy Ketal orAcyloxy Acetal Form R,~

-N—X

OH

-N—X (CJOro—c

OH (CH2)S_O_Rb ϋ,

OR

ν-Λ,'θ—Ο—RbI

OR

Ketal cr Acetal Form

In addition, it should be understood that the5 equilibrium forms of the compounds of this invention may include tautomeric forms. All such forms of thesecompounds are expressly included in the present invention.

It should oe understood that the compounds of 10 this invention may be modified by appropriate functionalities to enhance selective biologicalproperties. Such modifications are known in the artand include those which increase biological penetrationinto a given biological system (e.g., blood, lymphatic 15 system, central nervous system), increase oralavailability, increase solubility to allowadministration by .injection, alter metabolism arid alterrate of excretion. In addition, the compounds may bealtered to pro-drug form such that the desired compound 20 is created in the body of the patient as the result ofthe action of metabolic or other biochemical processeson the pro-drug. Such pro-drug forms typicallydemonstrate little or no activity in in vitro assays.Some examples of pro-drug forms include ketal, acetal, ΑΡ/Γ/ 9 8/01294 APCO1280 - 59 - oxime, imine, and hydrazone forms of compounds whichcontain ketone or aldehyde groups, especially wherethey occur in the R3 group of the compounds of thisinvention. Other examples of pro-drug forms include 5 the hemi-ketal, hemi-acetal, acyloxy ketal, acyloxy acetal, ketal, and acetal forms that are described inEQ1 and EQ2.

ICE and TX Cleave and Thereby Activate Pro-IGIF

The ICE protease was identified previously by 10 virtue of its ability to process inactive pro-IL-lfi tomature active IL-lfi, a pro-inflammatory molecule, invitro and in vivo . Here we show that ICE and itsclose homologue TX (Caspase-4, C. Faucheu et al., EMBO,14, p. 1914 (1995)) can proteolytically cleave inactive 15 pro-IGIF. This processing step is required to convertpro-IGIF to its active mature form, IGIF. Cleavage ofpro-IGIF by ICE, and presumably by TX, also facilitatesthe export of IGIF out of cells.

We first used transient co-expression of 20 plasmids transfected into Cos cells to determine whether any known members of the ICE/CED-3 proteasefamily can process pro-IGIF to IGIF in cultured cells(Example 23) (Fig. 1A).

Fig. 1A demonstrates that ICE cleaves 25 pro-IGIF in Cos cells co-transfected with plasmids thatexpress pro-IGIF in the presence of active ICE. Coscells were transfected with an expression plasmid forpro-IGIF alone (lane 2) or in combination with theindicated expression plasmids encoding wild type or 30 inactive mutants of ICE/CED-3 family of proteases (lanes 3-12) . Cell lysates were prepared and analyzedfor the presence of IGIF protein by immunoblotting withan anti-IGIF antiserum. Lane 1 contained lysates from AP/F/ 9 8 / 0 1 28 4 60 mock transfected ceils.

Co-expression of pro-IGIF with ICE or TX resulted in the cleavage of pro-IGIF into a polypeptidesimilar in size to the naturally-occurring 18-kDa 5 mature IGIF. This processing event is blocked bysingle point mutations that alter the catalyticcysteine residues and thus inactivate ICE and TX (F. Guet al., EMBO, 14, p. 1923 (1995)).

Co-expression with CPP32 (Caspase-3), o10 protease involved in programmed cell death (T.

Fernandas-Alnemri et al., J. Biol. Chem., 269, p. 30761(1994); D. W. Nicholson et al., Nature, 376, p. 37(1995)), resulted in the cleavage of pro-IGIF into asmaller polypeptide, while co-expression with CMH-1 15 (Caspase-7), a close homolog of CPP32 (J. A. Lippke etal., J, Biol, Chem., 271, p. 1825 ¢1996)), failed tocleave pro-IGIF to any significant extent. Thus, ICEano. TX appear to be capable of cleaving pro-IGIF into a.polypeptide similar in size to the naturally-occurring 20 18-kDa IGIF.

We next examined the ability of thesecysteine proteases to cleave pro-IGIF in vitro using apurified, recombinant (His)g-tagged pro-IGIF as asubstrate (Example 23). 25 Fig. IB demonstrates that pro-IGIF is cleaved in. vitro by ICE. Purified recombinant (His) 6-taggedpro-IGIF (2 μα) was incubated with the indicatedcysteine protease in the presence or absence of ICE orCPP32 inhibitors as described in Example 23. The 30 cleavage products were analyzed by SDS-PAGE andCoorassBlue staiηinq. ICE cleaved the 24 kDa pro-IGIF into twopolypeptides of approximate!v 18-kDa and 6-kDa. APC01280 - 61 - N-terminal amino acid sequencing of the ICE cleavageproducts indicated that the 18-kDa polypeptide containsthe same N-terminal amino acid residues (Asn-Phe-Gly-Arg-Leu) as the naturally occurring IGIF. 5 This shows that ICE cleaves pro-IGIF at the authentic processing site (Asp35-Asn36) (H. Okamura et al.,

Infection and Immunity, 63, p. 3966 (1995); H. Okamuraet al., Nature, 378, p. 88 (1995)). N-terminal aminoacid sequencing of the CPP32 cleavage products 10 indicated that CPP32 cleaved pro-IGIF at Asp69-Ile7O..

The cleavage by ICE of pro-IGIF is highly specific with a catalytic efficiency of 1.4 x 107 M_1 s"1 (KM= 0.6 ± 0.1 μΜ; kcat = 8.6 ± 0.3 s-1) andis inhibited by specific ICE inhibitors 15 (Ac-Tyr-Val-Ala-Asp-aldehyde) and Cbz-Val-Ala-Asp-[(2, 6-dichlorobenzoyl)oxy]methylketone, (N.A.

Thornberry et al., Nature, 356, p. 768 (1992); R. E.Dolle et al., J. Med. Chem., 37, p. 563 (1994)).

Fig. 1C demonstrates that ICE cleavage in 20 vitro activates pro-IGIF. Uncleaved pro-IGIF, ICE- orCPP32-cleaved products of pro-IGIF, or recombinantmature IGIF (rIGIF) were each added to A.E7 cellcultures to a final concentration of 12 ng/ml or 120ng/ml (see, Example 23). Eighteen hours later, IFN-γ 25 in the cultural medium was quantified by ELISA. Whilethe uncleaved pro-IGIF had no detectable IFN-γ inducingactivity, ICE-cleaved pro-IGIF was active in inducingIFN-γ production in Thl cells.

Like ICE, the ICE homolog TX also cleaved 30 pro-IGIF into similarly sized polypeptides. However,its catalytic efficiency was about two orders ofmagnitude lower than that shown for ICE. ΑΡ/Γ/ 9 8 / 0 1 2 9 4 62

Consistent with the observations from the Coscell experiments above, CPP32 cleaved pro-'IGIF at. adifferent site (Asp69-Ile7O) and the resultingpolypeptides had little IFN-γ inducing activity (Fig. 5 let . CMH-1 and granzyme B each failed to cleavepro-IGIF to any significant extent.

Together, these results demonstrate that,both in Cos cells and in vitro, ICE and TX are capableof processing the inactive pro-IGIF precursor at the 10 authentic maturation site to generate a biologicallyactive IGIF molecule.

Processing of Pro-IGIF by ICE Facilitates Its Export IGIF is produced by activated Kupffer cellsand macrophages in vivo and is exported out of the

15 cells upon stimulation by endotoxin (H. Okamura et al.,Infection and Immunity, 63, p. 3966 (1995); H. Okamuraet al., Nature, 378, p. 88 (1995). We used the Coscell co-expression system (Example 23) to examinewhether the intracellular cleavage of pro-IGIF by ICE 20 would facilitate the export of mature IGIF from thecell. Such is the case for pro-IL-Ιβ when it iscleaved by ICE into active IL-Ιβ (N.A. Thornberry etal., Nature, 356, p. 768 (1992)).

In Fig. 2A, Cos cells transfected with an 25 expression plasmid for pro-IGIF alone (lanes 2 and 6)or in combination with art expression plasmid encodingwild type (lanes 3 and 7) or inactive mutant ICE (lanes4 and 8} were metaboiically labeled with ~ S-methiornne(see, Example 24). Cell lysates (left) and conditioned 30 medium (right) were immunoprecipitated with an anti-IGlF antiserum. The immunoprecipitated proteinswere analyzed by SDS-PAGE and fluorography (Fig. 2A). APC 01280 - 63 -

An 18-kDa polypeptide corresponding in sizeto mature IGIF was detected in the conditioned mediumof Cos cells co-expressing pro-IGIF and ICE, while Coscells co-expressing pro-IGIF and an inactive ICE mutant 5 (ICE-C285S), or pro-IGIF alone (-) exported only very low levels of pro-IGIF and no detectable mature IGIF.

We estimate that about 10% of the mature IGIF wasexported from co-transfected cells, while greater than99% of pro-IGIF was retained within the cells. 10 We also measured the presence of IFN-γ inducing activity in cell lysates and in theconditioned medium of the above transfected cells (see,Example 24). IFN-γ inducing activity was detected inboth cell lysates and the conditioned medium of Cos 15 cells co-expressing pro-IGIF and ICE, but not in cellsexpressing either pro-IGIF or ICE alone (Fig. 2B).

These results indicate that ICE cleavage ofpro-IGIF facilitates the export of mature, active IGIFfrom cells. 20 Pro-IGIF is a Physiological Substrate of ICE In Vivo

To study the role of ICE in the proteolyticactivation and export of IGIF under physiologicalconditions, we examined the processing of pro-IGIF andexport of mature IGIF from lipopolysaccharide 25 (LPS)-activated Kupffer cells harvested from

Propiobacterium acnes-elicited wild type and ICEdeficient (ICE-/-) mice (Example 25).

As shown in Fig. 3A, Kupffer cells fromICE-/- mice are defective in the export of IGIF. 30 Kupffer cell lysates of wild type and ICE-/- micecontained similar amounts of IGIF as determined byELISA. IGIF, however, could be detected only in theconditioned medium of wild type but not of the ICE-/- 64 cells. Thus, ICE-defxcient (ICE-/-) mice synthesizepro-IGIF, but fail to export it as extracellular pro-ormature IGIF.

To determine whether ICE-deficient (ICE-/-) 5 mice process intracellular pro-IGIF but fail to export IGIF, Kupffer cells from wild type and ICE-/- mice weremetabolically labeled with 35S-methionine and IGIFimmunoprecipitation experiments were performed on celllysates and conditioned media as described in Example 10 25,. These experiments demonstrated that unprocessed· pro-IGIF was present in both wild type and ICE-/-Kupffer cells. However, the 18-kDa mature IGIF waspresent, only in the conditioned medium of wild type andnot ICE-/- Kupffer cells (Fig. 3B). This shows that 15 active ICE is required in cells for the export ofprocessed IGIF out of the cell.

In addition, conditioned medium from wildtype but not from ICE-/- Kupffer cells contained IFN-γinducing activity that was not attributed to the action 20 of IL-12 because it was insensitive to a neutralizinganti-IL-12 antibody. The absence of IGIF in theconditioned medium of ICE-/- Kupffer cells isconsistent with the finding in Cos cells that theprocessing of pro-IGIF by ICE is required for the 25 export of active IGIF.

Figs. 3.C and 3D show that, in vivo, ICE-/- · mice have reduced serum levels of IGIF and IFN-γ,respectively. Wild type (ICE+/+) and ICE-/- mice (n=3)primed with heat-inactivated P. acnes were challenged 30 with EPS (Example 26), and the levels of IGIF (Fig. 3C5and IFN-γ (Fig. 3D) in the sera of challenged mice weremeasured by ELISA three hours after LPS chsllenae - 65 - (Example 25).

The sera of ICE-/- mice stimulated byP. acnes and LPS contained reduced levels of IGIF(Fig. 3C) and no detectable IFN-γ inducing activity in 5 the presence of an anti-IL-12 antibody. The reducedserum levels of IGIF likely accounts for thesignificantly lower levels of IFN-γ in the sera ofICE-/- mice (Fig. 3D), because we have observed nosignificant difference in the production of IL-12 in 10 ICE-/- mice under these conditions. Consistent withthis interpretation is the finding that non-adherentsplenocytes from wild type and ICE-/- mice producedsimilar amounts of IFN-γ when stimulated withrecombinant active IGIF in vitro. Thus the impaired 15 production of IFN-γ is not due to any apparent defectin the T cells of the ICE-/- mice.

Taken together, these results establish acritical role for ICE in processing the IGIF precursorand in the export of active IGIF both in vitro and in 2 0 vivo.

To examine in more detail the relationshipbetween serum levels of IFN-γ and ICE activity in vivo,a time course after challenge of wild type andICE-deficient mice with LPS was performed (Example 26) 25 (Fig. 4) .

Fig. 4 shows a time course increase of serumIFN-γ in wild type mice, with sustained levels of>17 ng/ml occurring from 9-18 hrs after LPS challenge.As predicted by the experiments discussed above, serum 30 IFN-γ levels were significantly lower in ICE-/- mice,wirh a maximum of 2 ng/ml achieved over the same timeperiod, which is approximately 15% of the level 66 - observed in wild type mice (Fig. 4).

Animals were also observed for clinical signsof sepsis and body temperature was measured at 4-hourintervals in wild type and ICE-/- mice challenged, with 5 30 mg/kg or 100 mg/kg LPS (ICE-/-only). Results in

Fig. 4 show that wild type mice experienced asignificant decrease in body temperature (from 36’C to26'O within 12 hours of LPS challenge. Signs ofclinical sepsis were evident and all animals expired 10 within 24-23 hours.

In contrast, ICE-/- mice challenged with 30 mg/kg LPS experienced only a 3'-4*C decrease in bodytemperature with minimal signs of distress and with noobserved lethality. ICE-/- mice challenged with 15 100 mg/kg LPS experienced clinical symptoms, a decrease in body temperature, and mortality similar to wild typemice at the 30 mg/kg LPS dose.

The ICE Inhibitor Ac-YVAD-CHO is an EquipotentInhibitor of IL-Ιβ and IFN-y Production_ 20 Since the processing and secretion of biologically active 1GIF is mediated by ICE, wecompared the activity of a reversible ICE inhibitor(Ac-YVAD--CHO) on IL-ίβ and IFN-γ production in aperipheral blood mononuclear cell (PBMC) assay 25 (Examples 27).

Results in Fig. 5 show a similar potency forthe ability of the Ac-YvAD-CHO ICE inhibitor todecrease IL-ίβ and IFN-γ production in human PBMCs,with an 1C5Q of 2.5 μΜ for each. Similar results.were 30 obtained in studies with wild type mouse splenocytes.

These findings provide additional evidencethat pro-IGIF is a physiological substrate for ICE andsuggest that ICE inhibitors will be useful tools for APCΟ 128 0 - 67 - controlling physiological levels of IGIF and IFN-γ.

In summary, we have found that ICE controls IGIF and IFN-γ levels in vivo and in vitro and that ICEinhibitors can decrease levels of IGIF and IFN-γ in 5 human cells. These results have been described in co-pending United States Application Serial No. 08/712, 878, the disclosure of which is hereinincorporated by reference. 1 Compositions and Methods 10 The pharmaceutical compositions and methods of this invention will be useful for controlling IL-1,IGIF and IFN-γ levels in vivo. The methods andcompositions of this invention will thus be useful fortreating or reducing the advancement, severity of 15 effects of IL-1, IGIF- and IFN-y-mediated conditions.

The compounds of this invention are effective ligands for ICE. Accordingly, these compounds arecapable of targeting and inhibiting events in IL-1-,apoptosis-, IGIF-, and IFN-y-mediated diseases, and, 20 thus, the ultimate activity of that protein in) inflammatory diseases, autoimmune diseases, destructive bone, proliferative disorders, infectious diseases, anddegenerative diseases. For example, the compounds ofthis invention inhibit the conversion of precursor IL- 25 1β to mature IL-Ιβ by inhibiting ICE. Because ICE is essential for the production of mature IL-1, inhibitionof that enzyme effectively blocks initiation of IL-1-mediated physiological effects and symptoms, such asinflammation, by inhibiting the production of mature 30 IL-1. Thus, by inhibiting IL-Ιβ precursor activity, the compounds of this invention effectively function asIL-1 inhibitors. 68 - 10 15 25

Similarly, compounds of this inventioninhibit the conversion of precursor IGIF to matureIGIFi Thus, by inhibiting IGIF production, thecompounds of this invention effectively function asinhibitors of IFN-y production.

Accordingly, one embodiment of this inventionprovides a method for decreasing IGIF production in asubject comprising the step of administering to thesubject a pharmaceutical composition comprising atherapeutically effective amount of an ICE inhibitor,and a pharmaceutically acceptable carrier.

Another embodiment of this invention providesa method for decreasing IFN-γ production in a subjectcomprising the step of administering to the subject apharmaceutical composition comprising a therapeuticallyeffective amount of an ICE inhibitor and a pharmaceutically acceptable carrier.

In another embodiment, the methods of thisinvention comprise the step of administering to asubject a pharmaceutical composition comprising aninhibitor of an ICE-related protease that is capable ofcleaving pro-IGIF to active IGIF, and a pharmaceutically ac? .t;ole carrier. One such ·. Μ., as described above. Thismethods and pharmaceutical1 mg IGIF and IFN-γ levels bymor . md proteases capable of n active IGIF form may alsocloned that inhibitors ofvified by those of skill inwithin the scope of this I1E-related pr o t e a s-invention thus prcvcompositions for c\administering a TTOther Idprocessing pro-IGIFbe found. Thus itthose enzymes may :the art and will a.invention . APCO1280 - 69 -

The compounds of this invention may beemployed in a conventional manner for the treatment ofdiseases which are mediated by IL-1, apoptosis, IGIF orIFN-γ. Such methods of treatment, their dosage levels 5 and requirements may be selected by those of ordinary skill in the art from available methods and techniques.For example, a compound of this invention may becombined with a pharmaceutically acceptable adjuvantfor administration to a patient suffering from an 10 IL-1-, apoptosis-, IGIF- or IFN-y-mediated disease in a pharmaceutically acceptable manner and in an amounteffective to lessen the severity of that disease.

Alternatively, the compounds of thisinvention may be used in compositions and methods for 15 treating or protecting individuals against IL-1-,apoptosis-, IGIF- or IFN-y-mediated diseases overextended periods of time. The compounds may beemployed in such compositions either alone or togetherwith other compounds of this invention in a manner 20 consistent with the conventional utilization of ICEinhibitors in pharmaceutical compositions. Forexample, a compound of this invention may be combinedwith pharmaceutically acceptable adjuvantsconventionally employed in vaccines and administered in 25 prophylactically effective amounts to protect individuals over an extended period of time againstIL-1-, apoptosis-, IGIF- or IFN-γ- mediated diseases.

The compounds of this invention may also beco-administered with other ICE inhibitors to increase 30 the effect of therapy or prophylaxis against variousIL-1-, apoptosis, IGIF- or IFN-y-mediated diseases.

In addition, the compounds of this invention 70 may be used in combination either conventional anti-inflammatory agents or with matrix metalioprotease;inhibitors, lipoxygenase inhibitors and antagonists ofcytokines other than IL- 1β. 5 The compounds of this invention can aiso be acted mistered in combination with immunomodulators(e.g., bropirimine, anti-human alpha interferonanribody, IL-2, GM-CSP, methionine enkephalin,interferon alpha, diethyldithiocarbamate, tumor 10 necrosis factor, naltrexone and rEPO) or with prostaglandins, to prevent or combat IL-l-mediateddisease symptoms such as inflammation.

When the compounds of this invention areadministered in combination therapies with other IS agents, they may be administered sequentially orconcurrently to the patient. Alternatively,pharmaceutical or prophylactic compositions accordingto this invention comprise a combination of an ICEinhibitor of this invention and another therapeutic or 2.0 prophylactic agent.

Pharmaceutical compositions of this invention comprise any of the compounds of the present invention,and pharmaceutically acceptable salts thereof, with anypharmaceutically acceptable carrier, adjuvant or 25 vehicle. Pharmaceutically acceptable carriers,adjuvants and vehicles that may be used in thepharmaceutical compositions of this invention include,but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug 30 delivery systems (SEIODt! such as da-tocopherol poiyethyieneglycoi 1000 succinate, or other similarpolymeric delivery matrices, serum proteins, such ashuman serum albumin, ouffer substances such as AP/r/ 9 8 i a 1 2 8 4 APCO1280 - 71 - phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fattyacids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium 5 hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethyleneglycol, sodium carboxymethylcellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, 10 polyethylene glycol and wool fat. Cyclodextrins suchas Οί-, β- and γ-cyclodextrin, or chemically modifiedderivatives such as hydroxyalkylcyclodextrins,including 2-and 3-hydroxypropyl~3-cyclodextrines, orother solubiliezed derivatives may also be 15 advantageeously used to enhanve delivery of compoundsof this invention.

The pharmaceutical compositions of thisinvention may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally,

20 buccally, vaginally or via an implanted reservoir. Weprefer oral administration. The pharmaceuticalcompositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptablecarriers, adjuvants or vehicles. In some cases, the pH 25 of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers toenhance the stability of the formulated compounds orits delivery form. The term parenteral as used hereinincludes subcutaneous, intracutaneous, intravenous, 30 intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intralesional andintracranial injection or infusion techniques.

The pharmaceutical compositions may be in the 72 form of a sterile injectable preparation, for example,as a sterile injectable aqueous or oleaginoussuspension. This suspension may be formulatedaccording to techniques known in the art using suitable 5 dispersing or wetting agents (such as, for example,Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenteral1y-acceptable diluent cr solvent, for example, as a 10 solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed aremannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending 15 medium. For this purpose, any bland fixed oil may beemployed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation ofingectables, as are natural pharmaceutically-acc.eptable 20 oils, such as olive oil or castor oil, especially intheir polyoxyethvlated versions. These oil solutionsor suspensions may also contain a long-chain alcoholdiluent or dispersant such as Ph. Helv or a similaralcohol. 25 The pharmaceutical compositions of this invention may be orally administered in any orallyacceptable dosage form including, but not limited to,capsules, tablets, and aqueous suspensions andsolutions. In the esse of tablets for oral use, 30 carriers which are commonly used include lactose andcorn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oraladministration in a capsule form, useful diluents

* β Z I 0 / 8 6 /J/dV APV01280 - 73 - include lactose and dried corn starch. When aqueoussuspensions are administered orally, the activeingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening and/or 5 flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also be administered in the form ofsuppositories for rectal administration. Thesecompositions can be prepared by mixing a compound of 10 this invention with a suitable non-irritating excipientwhich is solid at room temperature but liquid at therectal temperature and therefore will melt in therectum to release the active components. Suchmaterials include, but are not limited to, cocoa 15 butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of this invention is especially usefulwhen the desired treatment involves areas or organsreadily accessible by topical application. For 20 application topically to the skin, the pharmaceuticalcomposition should be formulated with a suitableointment containing the active components suspended ordissolved in a carrier. Carriers for topicaladministration of the compounds of this invention 25 include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxy-ethylene polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositioncan be formulated with a suitable lotion or cream 30 containing the active compound suspended or dissolvedin a carrier. Suitable carriers include, but are notlimited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol,

'«F 09 fxl w

OO 4' Λ · flk - 74 - 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may alsobe topically applied to the lower intestinal tract byrectal suppository formulation or in a suitable enema 5 formulation. Topically-administered transdermaipatches are also included in this invention.

The pharmaceutical compositions of thisinvention may be administered by nasal aerosol crinhalation. Such compositions are prepared according 10 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 enhancebioavailability, fluorocarbons, and/or other 15 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 about1 and 50 mg/kg body weight per day of the activeingredient compound are useful in the prevention and 20 treatment of IL-1-, apoptosis, IGIF and IFN-y-mediateddiseases, including inflammatory diseases, autoimmunediseases, destructive bone disorders, proliferativedisorders, infectious diseases, degenerative diseases,necrotic diseases, osteoarthritis, acute pancreatitis, 25 chronic pancreatitis, asthma, adult respiratory distress syndrome, glomeralonephritis, rheumatoidarthritis, systemic lupus erythematosus, scleroderma,chronic thyroiditis, Graves' disease, autoimmunegastritis, insulin-dependent diabetes mellitus (Type 30 I), autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic activehepatitis, myasthenia gravis, inflammatory boweldisease, Crohn's disease, psoriasis, graft vs. host ΑΡΟΟ 128 0 - 15 - disease, osteoporosis, multiple myeloma-related bonedisorder, acute myelogenous leukemia, chronicmyelogenous leukemia, metastatic melanoma, Kaposi'ssarcoma, multiple myeloma sepsis, septic shock, 5 Shigellosis, Alzheimer's disease, Parkinson's disease,cerebral ischemia, myocardial ischemia, spinal muscularatrophy, multiple sclerosis, AIDS-related encephalitis,HIV-related encephalitis, aging, alopecia, and neurological damage due to stroke. Typically, the10 pharmaceutical compositions of this invention will be administered from about 1 to 5 times per day oralternatively, as a continuous infusion. Suchadministration can be used as a chronic or acutetherapy. The amount of active ingredient that may be 15 combined with the carrier materials to produce a singledosage form will vary depending upon the host treatedand the particular mode of administration. A typicalpreparation will contain from about 5% to about 95%active compound (w/w). Preferably, such preparations 20 contain from about 20% to about 80% active compound.

Upon improvement of a patient's condition, a maintenance dose of a compound, composition orcombination of this invention may be administered, ifnecessary. Subsequently, the dosage or frequency of 25 administration, or both, may be reduced, as a functionof the symptoms, to a level at which the improvedcondition is retained when the symptoms have beenalleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent 30 treatment on a long-term basis upon any recurrence ordisease symptoms.

As the skilled artisan will appreciate, loweror higher doses than those recited above may be ΑΡ/Γ/ 9 8.01294 required. Specific dosage and treatment regimens forany particular patient, will depend upon a variety offactors, including the activity of the specificcompound employed, the age, body weight, general healthstatus, sex, diet, time of administration, rate ofexcretion, drug combination, the severity and course ofthe disease, and the patient's disposition to thedisease and the judgment of the treating physician,

The IL-1 mediated diseases which may betreated or prevented by the compounds of this inventioninclude, but are not limited to, inflammatory diseases,autoimmune diseases, destructive bone disorders,proliferative disorders, infectious diseases, anddegenerative diseases, The apoptosis-mediated diseaseswhich may be treated or prevented by the compounds ofthis invention include degenerative diseases.

Inflammatory diseases which may be treated orprevented include, but are not limited toosteoarthritis, acute pancreatitis, chronicpancreatitis, asthma, and adult respiratory distresssyndrome. Preferably the inflammatory disease isosteoarthritis or acute pancreatitis.

Autoimmune diseases which may be treated orprevented include, but are not limited to,glomerulonephritis, rheumatoid arthritis, systemiclupus erythematosus, scleroderma, chronic thyroiditis,Graves' disease, autoimmune gastritis, insulin-dependent diabetes meliitus (Type I), autoimmunehemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myastheniagravis, multiple sclerosis, inflammatory bowel disease,Crohn's disease, psoriasis, and graft vs. host disease.Preferably the autoimmune disease is rheumatoid ΑΡ/Γ/ 9 8 / 0 1 2 9 4 ΑΡΓΌ 1 28 0 - 77 - arthritis, inflammatory bowel disease, Crohn's disease,or psoriasis.

Destructive bone disorders which may betreated or prevented include, but are not limited to, 5 osteoporosis and multiple myeloma-related bonedisorder.

Proliferative diseases which may be treatedor prevented include, but are not limited to, acutemyelogenous leukemia, chronic myelogenous leukemia, 10 metastatic melanoma, Kaposi's sarcoma, and multiplemyeloma.

Infectious diseases which may be treated orprevented include, but are not limited to, sepsis,septic shock, and Shigellosis. 15 The IL-l-mediated degenerative or necrotic diseases which may be treated or prevented by thecompounds of this invention include, but are notlimited to, Alzheimer's disease, Parkinson's disease,cerebral ischemia, and myocardial ischemia. 20 Preferably, the degenerative disease is Alzheimer'sdisease .

The apoptosis-mediated degenerative diseaseswhich may be treated or prevented by the compounds ofthis invention include, but are not limited to, 25 Alzheimer's disease, Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy,multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, and neurologicaldamage due to stroke. 30 The methods of this invention may be used for treating, or reducing the advancement, severity oreffects of an IGIF-or IFN-y-mediated inflammatory,autoimmune, infectious, proliferative, destructive bene, necrotic, and degenerative conditions, includingdiseases, disorders or effects, wherein the conditionsare characterized by increased levels of IGIF or l'FN-γ production. 5 Examples of such inflammatory conditions include, but are not limited to, osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, rheumatoidarthritis, inflammatory bowel disease, Crohn's aisease,ulcerative collitis, cerebral ischemia, myocardial 10 ischemia and adult respiratory distress syndrome.

Preferably, the inflammatory condition is rheumatoid arthritis, ulcerative collitis, Crohn’sdisease, hepatitis and adult respiratory distresssyndrome . 15 Examples of such infectious conditions include, but are not limited to, infectious hepatitis,sepsis, septic shock and Shigellosis.

Examples of such autoimmune conditionsinclude, but are not limited to, glomerulonephritis, 20 systemic lupus erythematosus, scleroderma, chronicthyroiditis, Graves’ disease, autoimmune gastritis,insulin-dependent diabetes mellitus (Type I), juvenilediabetes, autoimmune hemolytic anemia, autoimmuneneutropenia, thrombocytopenia, myasthenia gravis, 25 multiple sclerosis, psoriasis, lichenplanus, graft vs.host disease, acute dermatomyositis, eczema, primarycirrnosis, hepatitis, uveitis, Behcet's disease, acutedermatomyositis, atopic skin disease, pure red ceilaplasia, aplastic anemia, amyotrophic lateral sclerosis 30 and nephrotic syndrome.

Preferably the autoimmune condition is glomerulonephritis, insulin-dependent diabetes mellitus(Type 1;, juvenile diabetes, psoriasis, graft vs. host

*821086 / J/dV AP C012 8 0 - 79 - disease, including transplant rejection, and hepatitis.

Examples of such destructive bone disorders include, but are not limited to, osteoporosis andmultiple myeloma-related bone disorder. 5 Examples of such proliferative conditions include, but are not limited to, acute myelogenousleukemia, chronic myelogenous leukemia, metastaticmelanoma, Kaposi's sarcoma, and multiple myeloma.

Examples of such neurodegenerative conditions10 include, but are not limited to, Alzheimer's disease,

Parkinson's·disease and Huntington's disease.

Although this invention focuses on the use of the compounds disclosed herein for preventing andtreating IL-1, apoptosis, IGIF- and ΙΕΝ-γ-mediated 15 diseases, the compounds of this invention can also be used as inhibitory agents for other cysteine proteases.

The compounds of this invention are alsouseful as commercial reagents which effectively bind toICE or other cysteine proteases. As commercial 20 reagents, the compounds of this invention, and theirderivatives, may be used to block proteolysis of atarget peptide in biochemical or cellular assays forICE and ICE homologs or may be derivatized to bind to astable resin as a tethered substrate for affinity 25 chromatography applications. These and other useswhich characterize commercial cysteine proteaseinhibitors will be evident to those of ordinary skillin the art.

Process of Preparing N-Acylamino Compounds 30 The ICE inhibitors of this invention may be synthesized using conventional techniques.Advantageously, these compounds are convenientlysynthesized from readily available starting materials. AP/P/ 9 8/01294 so

The compounds of this invention are among themost readily synthesized ICE inhibitors known.Previously described ICE inhibitors often contain fouror more chiral centers and numerous peptide linkages. 5 The relative ease with which the compounds of this invention can be synthesized represents an advantage inthe large scale production of these compounds.

For example, compounds of this invention maybe prepared using the processes described herein. As 10 can be appreciated by the skilled practitioner, these processes are not the only means by which the compoundsdescribed and claimed in this application may besynthesized. Further methods will be evident to thoseof ordinary skill in the art. Additionally, the 15 various synthetic: steps described herein may be performed in an alternate sequence or order to give thedesired compounds.

This invention also provides a preferredmethod for preparing the compounds of this invention. 20 Accordingly, in another embodiment (M) is provided aprocess for preparing an N-acylamino compoundcomprising the steps of: a) mixing a carboxylic acid with an N~alioc-protected amino in the presence of an inert 25 solvent, triphenylphoshine, a nucleophilic scavenger,and tetrakis-triphenyl phosphine palladium(0) atambient temperature under an inert atmosphere; and b) adding to the step a) mixture, HOST andEDC; and optionally comprising the further step of: c) hydrolyzing the step b) mixture in thepresence of a solution comprising an acid and H2Cuwherein the step bj mixture is optionally concentrated,prior to hydrolyzing. 30 AP c012 8 0 - 81 -

Preferably, the inert solvent is CH2C12, DMF,or a mixture of CH2C12 and DMF.

Preferably, the nucleophilic scavenger isdimedone, morpholine, trimethylsilyl dimethylamine, or 5 dimethyl barbituric acid. More preferably, the nucleophilic scavenger is trimethylsilyl dimethylamineor dimethyl barbituric acid.

Preferably, the solution comprisestrifluoroacetic acid in about 1-90% by weight. More 10 preferably, the solution comprises trifluoroacetic acidin about 20-50% by weight.

Alternatively, the solution compriseshydrochloric acid in about 0.1-30% by weight. Morepreferably, the solution comprises hydrochloric acid in 15 about 5-15% by weight.

More preferably, in the above process, theinert solvent is CH2C12, DMF, or a mixture of CH2C12 andDMF and the nucleophilic scavenger is dimedone,morpholine, trimethylsilyl dimethylamine, or dimethyl 20 barbituric acid.

Most preferably, in the above process theinert solvent is CH2C12, DMF, or a mixture of CH2C12 andDMF and the nucleophilic scavenger is trimethylsilyldimethylamine or dimethyl barbituric acid. 25 Preferably, the N-acyclamino compound is represented by formula (VIII):

Rl~N-R2

H 30 wherein: R1 is as defined above in embodiment (A); R2 is: ΑΡ/ΓΖ 90/01294 APS 0 1 2 a 0 0 ' ϊ R51

H /herein R=,i is as defined above in embodiment (B)

, J

'fK

OH rt

r^OH preferably, the N-alloc-protected amine

wherein is as defined above

10 In order that this invention be more fully understood, the following examples are set forth.

These examples are for the purpose of illustration onlyand are not to be construed as limiting the scope ofthe invention in anv wav. ΑΡ ε ο 12 8 ο - 83 -

Example 1Inhibition of ICE

We obtained inhibition constants (KjJ and IC50values for compounds of this invention using the three 5 methods described below: 1. Enzyme assay with UV-visible substrate

This assay is run using an Succinyl-Tyr-Val-Ala-Asp-pNitroanilide substrate. Synthesis ofanalogous substrates is described by L. A. Reiter (Int. 10 J. Peptide Protein Res. .43., 87-96 (1994)). The assaymixture contains: 65 μΐ buffer (lOmM Tris, 1 inM DTT, 0.1% CHAPS @pH 8.1)10 μΐ ICE (50 nM final concentration to give a rate of~lmOD/min) 15 5 μΐ DMSO/Inhibitor mixture 20 ul 400μΜ Substrate (80 μΜ final concentration) 100μ1 total reaction volume

The visible ICE assay is run in a 96-wellmicrotiter plate. Buffer, ICE and DMSO (if inhibitor 20 is present) are added to the wells in the order listed.The components are left to incubate at room temperaturefor 15 minutes starting at the time that all componentsare present in all wells. The microtiter plate readeris set to incubate at 37 °C. After the 15 minute 25 incubation, substrate is added directly to the wellsand the reaction is monitored by following the releaseof the chromophore (pNA) at 405 - 603 nm at 37 °C for20 minutes. A linear fit of the data is performed andthe rate is calculated in mOD/min. DMSO is only 30 present during experiments involving inhibitors, bufferis used to make up the volume to 100 μΐ in the otherexperiments . 2 . Enzyme Assay with Fluorescent Substrate

This assay is run essentially according to 35 Thornberry et al. (Nature 356: 768-774 (1992)), using substrate 17 referenced in that article. The substrate A.P v Ο ί 2 6 Ο - 84 - is : Acetyl-Tyr-Val-Aj a-Asp-amino-4-methylcoumai:in (AMC;, The following components are mixed: 65 pi buffer(lOmM Tris,lmM DTT, 0.1% CHAPS @pHS.110 ul ICE (2 - 10 nI-5 final concentration) 5 ul DMSO/inhibitor solution20 ul 150 μΜ Substrate (30 μΜ final) lOOul total reaction volume

The assay is run in a 96 well microtiterplate. Buffer and ICE are added to the wells. The 10 components are left to incubate at 37 °C for 15 minutesin a temperature-controlled wellplate. After the 15minute incubation, the reaction is started by addingsubstrate directly to the wells and the reaction ismonitored @37 °C for 30 minutes by following the 15 release of the AMC fluorophore using an excitation wavelength for 380 nm and an emission, wavelength of 460run. A linear fit of the data for each well is performed and a rate is determined in fluorescenceunits per second. 20 For determination of enzyme inhibition constants (Kj.) or the mode of inhibition (competitive,uncompetitive or noncompetitive), the rate datadetermined in the enzyme assays at varying inhibitorconcentrations are computer-fit to standard enzyme

*62 1.0/86 /J/dV 25 kinetic equations (s;W11 e y -1 n t e r s c i e n c ,

The determ:constants for irreve.fitting the fiuor· - 30 equations of Morr.:Sioohys^^ 2, pp.have published a imeasurement of ra'inhibitors of ICE. 35 fbnyjaerumtny, 33, pp . H. Segel, Enzyme,,Kinetics., ation of second order rateidle inhibitors was performed byce vs time data to the progress

Morrison, J.F., MoK__CelJ^(1985). Thornberry et al. iption of these methods forrants of irreversible ’tuberry, N.A., et al . 33-3940 (1994). For - 85 - where no prior complex formation can be observedkinetically, the second order rate constants (kj_riact)are derived directly from the slope of the linear plotsof kobs vs. [I]. For compounds where prior complex 5 formation to the enzyme can be detected, the hyperbolicplots of kobs vs. [I] are fit to the equation forsaturation kinetics to first generate and k' . Thesecond order rate constant kj_nact is then given byk' /Ki. 10 3. PBMC Cell assay IL-Ιβ Assay with a Mixed Population of HumanPeripheral Blood Mononuclear Cells (PBMC) or Enriched Adherent Mononuclear CellsProcessing of pre-IL-Ιβ by ICE can be 15 measured in cell culture using a variety of cellsources. Human PBMC obtained from healthy donorsprovides a mixed population of lymphocyte subtypes andmononuclear cells that produce a spectrum ofinterleukins and cytokines in response to many classes 20 of physiological stimulators. Adherent mononuclearcells from PBMC provides an enriched source of normalmonocytes for selective studies of cytokine productionby activated cells. ) Experimental Procedure: 25 An initial dilution series of test compound in DMSO or ethanol is prepared, with a subsequentdilution into RPMI-10% FBS media (containing 2 mML-glutamine, 10 mM HEPES, 50 U and 50 ug/ml pen/strep)respectively to yield drugs at 4x the final test 30 concentration containing 0.4% DMSO or 0.4% ethanol.

The final concentration of DMSO is 0.1% for all drugdilutions. A concentration titration which bracketsthe apparent for a test compound determined in anICE inhibition assay is generally used for the primary 35 compound screen. ΑΡ/Γ/ 98/9 1294 ;a- :01250 - 86 -

Generally 5-1 compound dilutions are testedand the cellular component of the assay is performed induplicate, with duplicate ELISA determinations on eachcell culture supernatant, 5 PBMC Isolation and IL-1 Assay:

Buffy coat cells isolated from one pint humanblood (yielding 40-45 nil final volume plasma pluscells) are diluted with media to 80 ml and LeukoPREPseparation tubes (Becton Dickinson) are each overlaid 10 with 10 ml of ceil suspension. After 15 min centrifugation at 1500-1800 xg, the plasma/media layeris aspirated and then the mononuclear cell layer iscollected with a Pasteur pipette and transferred to a15 ml conical centrifuge tube (Corning). Media is 15 added to bring the volume to 15 ml, gently mix the cells by inversion and centrifuge at 300 xg for 15 min.Resuspend the PBMC pellet in a small volume of media,count cells and adjust to 6 x 106 cells/ml.

For the cellular assay, 1.0 ml of the cell 20 suspension is added to each well of a 24-well flatbottom tissue culture plate (Corning), 0.5 ml testcompound dilution and 0.5 ml LPS solution (Sigma#L~3012; 20 ng/ml solution prepared in complete RPMImedia; final LPS concentration 5 ng/ml). The 0.5 ml 25 additions of test compound and LPS are usually sufficient to mix the contents of the wells. Threecontrol mixtures, are run per experiment, with eitherLPS alone, solvent vehicle control, and/or additionalmedia to adjust the final culture volume to 2.0 ml. 30 The cell cultures are incubated for 16-18 hr at 37 "Cin the presence of 51 CO;,.

At the end of the incubation period, cellsare harvested and transferred to 15 ml conicalcentrifuge tubes. After centrifugation for 10 min at ΑΡ/Γ/ 88/81284 AP c 0 1 2 8 0 i

.J - 87 - 200 xg, supernatants are harvested and transferred to1.5 ml Eppendorf tubes. It may be noted that the cellpellet may be utilized for a biochemical evaluation ofpre-IL-Ιβ and/or mature IL-Ιβ content in cytosol 5 extracts by western blotting or ELISA with pre-IL-Ιβspecific antisera.

Isolation of Adherent Mononuclear cells: PBMC are isolated and prepared as described above. Media (1.0 ml) is first added to wells followed 10 by 0.5 ml of the PBMC suspension. After a one hourincubation, plates are gently shaken and nonadherentcells aspirated from each well. Wells are then gentlywashed three times with 1.0 ml of media and finalresuspended in 1.0 ml media. The enrichment for 15 adherent cells generally yields 2.5-3.0 x 10^ cells perwell. The addition of test compounds, LPS, cellincubation conditions and processing of supernatantsproceeds as described above. ELISA: 20 We have used Quantikine kits (R&amp;D Systems) for measurement of mature IL-Ιβ. Assays are performedaccording to the manufacturer's directions. MatureIL-Ιβ levels of about 1-3 ng/ml in both PBMC andadherent mononuclear cell positive controls are 25 observed. ELISA assays are performed on 1:5, 1:10 and1:20 dilutions of supernatants from LPS-positivecontrols to select the optimal dilution forsupernatants in the test panel.

The inhibitory potency of the compounds can 30 be represented by an IC5C value, which is the concentration of inhibitor at which 50% of mature IL-Ιβis detected in the supernatant as compared to thepositive controls. ΑΡ/Γ/ 9 8,-01294 AP C012 3 0 - 88 -

The skilled practitioner realizes that valuesobtained in cell assays, such as those describedherein, can depend on multiple factors, such as celltype, cell source, growth conditions and the like. 5 Example 2

Pharmacokinetic Studies in the Mouse

Peptidyl ICE inhibitors are cleared rapidlywith clearance rates greater than 100 μ/min/kg.Compounds with lower clearance rates have improved 10 pharmacokinetic properties relative to peptidyl ICEinhibitors.

We obtained the rate of clearance in themouse (μ/min/kg) for several compounds of thisinvention using the method described below: 15 Sample Preparation and Dosing

Compounds were dissolved in sterile TRL3solution (0.02M or 0.05M) at a concentration of2.5mg/mi. Where necessary to ensure a completesolution, the sample was first dissolved in a minimum 20 of dimethylacetamide (maximum of 5% of total solutionvolume) then diluted with the TRIS solution.

The drug solution was administered to CD-Imice (Charles River Laboratories - 26-31g) via thetailvein at a dose volume of 10ml/kg giving a drug dose 25 of 25mg/kg,

Mice were dosed in groups of 5 for eachtimepoint (generally from 2 minutes to 2 hours) then atthe appropriate time one animals were anaesthetisedwith halothane and the blood collected into indi’·i dual 30 heparinized tubes by jugular severance. The bloou samples were cooled to 0 °C then the plasma separaredand stored at -20 cC until assayed. AP £01280 - 89 -

Bioassav

Drug concentration in the plasma samples weredetermined by HPLC analysis with UV or MS (ESP)detection. Reverse phase chromatography was employed 5 using a variety of bonded phases from Cl to Cl8 witheluents composed of aqueous buffer/acetonitrilemixtures run under isocratic conditions.

Quantitation was by external standard methodswith calibration curves constructed by spiking plasma 10 with drug solutions to give concentrations in the rangeof 0.5 to 50pg/ml.

Prior to analysis the plasma samples weredeproteinated by the addition of acetonitrile,methanol, trichloroacetic acid or perchloric acid 15 followed by centrifugation at 10,000g for 10 minutes.Sample volumes of 20μ1 to 50μ1 were injected foranalysis.

Compound 214eDosing and sampling 20 The drug was dissolved in sterile 0.02M Tris to give a 2.5mg/ml solution which was administered to11 groups of 5 male CD-I mice via the tail vein at adose of 25mg/kg. At each of the following timepoints: 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes a 25 group of animals was anaesthetised and the blood collected into heparinized tubes. After separation theplasma was stored at -20 °C until assayed.

Assay

Aliquots of plasma (150μ1) were treated with 30 5% perchloric acid (5μ1) then mixed by vortexing and allowed to stand for 90 minutes prior tocentrifugation. The resulting supernatant wasseparated and 20μ1 was injected for HPLC analysis.

*6210/86 /d/dV - 90 - HPLC Conditions

Column

Mobile Phase

Flowrate 100 x &amp; .6mm0.1m Tris pH" . 5Acetonitrile1ml/mw

Kromasil KR 100 5C4 86% 14%

Detection UV at llOnm

Retention Time 3.1 mins

The results of the analysis indicated a decrease in the mean plasma level of the drug from -70pg/'ml at 2 minutes to < 2pg/ml at 90 and 120 minutes.

Compound 217 e

Dosing and sampling

The drug was dissolved in sterile 0.02M Tristo give a 2.5mg/ml solution which was administered to11 groups of 5 male CD-I mice via the tail vein at adose of 25mg/kg. At each of the following timepoints:2, 5, 10, 15, 20,-30, 45, 60, 90 and 120 minutes sgroup of animals was anaesthetised and the bloodcollected into heparinized tubes. After separation theplasma was stored at -20 °C until assayed.

Assay

Aliquots of plasma (ΙΟΟμΙ) were diluted withacetonitrile (ΙΟΟμΙ) then mixed by vortexing for’ 20seconds before centrifugation for 10 minutes. Theresulting supernatant was separated and 20μ1 was

•r 8 1 t 0 I 8 6 Zd/cfV injected for HPLC analysis.HPLC ConditionsColumn 150 x 4.6mm

Mobile Pnase 0.05M Phosphate buffer ph7.1Acetonitrile

Flowrate 1 . iml/nun

De t e c t i o n UV a t 2 1 0 ran

Zorbax S3C872% 28%

Retention Time 6 mins (diasteromers) - 91 -

The results of the analysis indicated adecrease in mean plasma concentrations from ~ 55ug/mlat 2 minutes to < 0.2pg/ml at 60-120 minutes.

Example 3 5 Peptidyl ICE inhibitors are cleared rapidly with clearance rates greater than 80 ml/min/kg.Compounds with lower clearance rates have improvedpharmacokinetic properties relative to peptidyl ICEinhibitors . 10 We obtained the rate of clearance in the rat ) (ml/min/kg) for several compounds of this inventionusing the method described below:

In vivo Rat Clearance AssayCannulations of the jugular and carotid 15 vessels of rats under anesthesia were performed one dayprior to the pharmacokinetic study. M.J. Free, R.A.Jaffee; 'Cannulation techniques for the collectionblood and other bodily fluids'; in: Animal Models;p. 480-495; N.J. Alexander, Ed.; Academic Press; 20 (1978). Drug (lOmg/mL) was administered via the jugular vein in a vehicle usually consisting of:propylene glycol/saline, containing lOOmM sodium ) bicarbonate in a 1:1 ratio. Animals were dosed with 10-20 mg drug/kg and blood samples were drawn at 0, 2, 25 5, 7, 10, 15, 20, 30, 60, and 90 minutes from an indwelling carotid catheter. The blood was centrifugedto plasma and stored at -20 °C until analysis.

Pharmacokinetic analysis of data was performed by non-linear regression using standard software such as 30 RStrip (MicroMath Software, UT) and/or Pcnonlin (SCISoftware, NC) to obtain clearance values. AP/F/ 98/01294 ΑΡ ε ο 1 2 8 0 - 92 -

Rat plasma was extracted with an equal volumeof acetonitrile (containing 0.1% TEA). Samples werethen centrifuged at approximately 1,000 x g and the 5 supernatant analyzed by gradient HPLC. A typical assayprocedure is described below. 200 pL of plasma was precipitated with 200 pLof 0.1% trifluoroacetic acid (TFA) in acetonitrile and10 uh of a 50% aqueous zinc chloride solution, v rtexed 10 then centrifuged at -1000 x g and the supernatantcollected and analyzed by HPLC. HPLC procedure: Column: Zorbax SB-CN (4.6 x 150particle size) mm) 15 Column temperature: 50 °C Flow rate: 1. 0 mL/min Injection volume: 7 5 pL . Mobile phase: A-G.l% TFA in water andacetonitrile B=1 20 Gradient employed: 100% A to 30% A in 15.50% A at 16 min 100% A at 19.2 min min Wavelength: 214 nm A standard curve was run at 20, 10, 5, 2 and25 1 yg/mL concentrations..

Example 4

Whple_,Blood_ Assay for Ih-1L ProductionWe obtained IC5Q values for several compounds of this invention usinq the method described below: 30 Purpose:

The whole blcmeasuring the prodf’and the activity c: rcomplexity of thi 35 complement of lymph. 1spectrum of plasm. . _ideal ip · pfrp repr ··physiol condii _ ' od assay is a simple method forof IL-lb (or other cytokines) >.-ntial inhibitors. Thesystem, with its full hid inflammatory cell types, .ns and red blood cells is an u.tion of human in vivo ΑΡ ί 0 1 2 8 0 -93-

Materials:

Pyrogen-free syringes (~ 30 cc)

Pyrogen-free sterile vacuum tubes containing lyophilized Na2EDTA (4.5 mg/10 ml tube)

Human whole blood sample (~ 30-50 cc) 1.5 ml eppendorf tubes

Test compound stock solutions (~ 25mM in DMSO or othersolvent)

Endotoxin-free sodium chloride solution (0.9%) and HBSSLipopolysaccharide (Sigma; Cat.# L-3012) stock solutionat lmg/ml in HBSS IL-Ιβ ELISA Kit (R &amp; D Systems; Cat #DLB50) TNFa ELISA Kit (R &amp; D Systems; Cat # DTA50)

Water bath or incubator

Whole Blood Assay Experimental Procedure:

Set incubator or water bath at 30 °C.

Aliquot 0.25ml of blood into 1.5 ml eppendorf tubes.Note: be sure to invert the whole blood sample tubesafter every two aliquots. Differences in replicatesmay result if the cells sediment and are not uniformlysuspended. Use of a positive displacement pipette willalso minimize differences between replicate aliquots.

Prepare drug dilutions in sterile pyrogen-free saline by serial dilution. A dilution serieswhich brackets the apparent Kj_ for a test compounddetermined in an ICE inhibition assay is generally usedfor the primary compound screen. For extremelyhydrophobic compounds, we have prepared compounddilutions in fresh plasma obtained from the same blooddonor or in PBS-containing 5% DMSO to enhancesolubility.

Add 25 μΐ test compound dilution or vehiclecontrol and gently mix the sample. Then add 5.0 μΐ LPSsolution (250 ng/ml stocked prepared fresh: 5.0 ng/ml

*8210/86 ZJ/dV - 94 - final concentration .IPS), and mix again. Incubate thetubes at 30 °C in a water bath for 16-18 hr withoccasional mixing. Alternatively, the tubes can beplaced in a rotator set at 4 rpm for the same 5 incubation period. This assay should be set up induplicate or triplicate with the following controls:negative control- no L?S; positive control- no testinhibitor; vehicle control- the highest concentrationof DMSO or compound solvent used in the experiment, 10 Additional saline is added to all control tubes tonormalise volumes for both control and experimentalwhole blood test samples

After the incubation period, whole bloodsamples are centrifuged for 10 minutes at ~ 2000 rpm in 15 the microfuge, plasma is transferred to a fresh microfuge tube and centrifuged at 1000 x g to pelletresidual platelets if necessary. Plasma samples may bestored frozen at -70 °C prior to assay for cytokinelevels by ELISA. 20 ELISA:

We nave used R &amp; D Systems (614 McKinleyPlace N.E. Minneapolis, MN 55413) Quantikine kits formeasurement of IL-Ιβ and TNF-α. The assays areperformed according to the manufacturer's directions. 25 We have observed IL-Ιβ levels of ~ 1-5 ng/ml in positive controls among a range of individuals. A1:200 dilution of plasma for all samples has beensufficient in our experiments for ELISA results to fallon the linear range of the ELISA standard curves, It 30 may be necessary to optimize standard dilutions if youobserve differences in one whole blood assay. Nerad,J.L. et at,,, J, Leukocyte Biol., 52, pp. 687-692

V 6 2 I· C t 8 6 Zd/dV ΛΓν 012 δ 0 - 95 -

Example 5

Inhibition of ICE homologs 1. Isolation of ICE homologs

Expression of TX in insect cells using a baculovirusexpression system. We have subcloned Tx cDNA. (Faucheuet al., supra 1995) into a modified pVL1393 transfervector, co-transfected the resultant plasmid (pVL1393/TX) into insect cells with viral DNA andidentified the recombinant baculovirus. After thegeneration of high titer recombinant virus stock, themedium was examined for TX activity using the visibleICE assay. Typically, infection of Spodopterafrugiperda (Sf9) insect cells at an MOI of 5 withrecombinant virus stock resulted in a maximumexpression after 48 hours of 4.7pg/ml. ICE was used asa standard in the assay.

Amino terminal T7 tagged versions of ICE orTX were also expressed. Designed originally to assistthe identification and purification of the recombinantproteins, the various constructs have also allowedexamination of different levels of expression and ofthe relative levels of apoptosis experienced by thedifferent homologs. Apoptosis in the infected Sf9cells (examined using a Trypan Blue exclusion assay)was increased in the lines expressing ICE or TXrelative to cells infected with the viral DNA alone.Expression and purification of N-terminally (His)g-tagged CPP32 in E. coli. A cDNA encoding a CPP32(Fernandes-Alnemri et al, supra 1994) polypeptidestarting at Ser (29) was PCR amplified with primersthat add in frame Xhol sites to both the 5' and 3' endsof the cDNA and the resulting Xhol fragment ligatedinto a Xho I-cut pET-15b expression vector to create anin frame fusion with (his)g tag at the n-terminus of V 6 c I 0 I 8 6 /d/dv .01280 - 96 - the fusion protein. The predicted recombinant proteinstarts with the amino acid sequence ofMGSSHHHHHHS S GLVPRGSHMLE, where LVPRGS represents athrombin cleavage site, followed by CPP32 starting at 5 Ser (29). E. coli BL21(DE3) carrying the plasmid weregrown to log phase at 30 °C and were then induced with0.8 mM IPTG. Cells were harvested two hours after IPTGaddition. Lysates were prepared and soluble proteinswere purified by Ni-agarose chromatography. All of the : 10 expressed CPP32 protein was in the processed form. N- terminal sequencing analysis indicated that theprocessing occurred at the authentic site between Asp(175) and Ser (176). Approximately 50 yg of CPP32protein from 200 ml culture. As determined by active 15 site titration, the purified proteins were fully active. The protease preparation were also very activein vitro in cleaving PARP as well as the syntheticDEVD-AMC substrate (Nicholson et al, supra 1995). 2. Inhibition of ICS homologs 20 The selectivity of a panel of reversible inhibitors forICE homologs is depicted in Table 1. ICE enzyme assayswere performed according to Wilson et al (supra 1994) ) using a YVAD-AMC substrate (Thornberry et al, supra 1992). Assay of TX activity was performed using the 2 5 ICE) substrate under identical conditions to ICE. Assayof CPP32 was performed using a DEVD-AMC substrate(Nicholson et al., supra 1995). In general, there islow selectivity between ICE and TX for a wide range ofscaffolds. None of the synthetic ICS compounds nested 30 are effective inhibitors of CPP32. Assay of the reversible compounds at the highest concentration (1yM) revealed no inhibition. ΑΡ/Γ7 9 8 / 0 1 2 9 4

Table 1

Compound K± ICE (nM) Ki TX (nM) K± CPP32 (nM) 214e 7.5 7. 0 ± 1.1 > 1000 135a 90 55 + 9 >1000 125b 60 57 + 13 > 1000 137 40 40 + 7 > 1000 Second-order rate constants for inactivation of ICE and ICE homologs with selected irreversible inhibitors are presented below (Table 2). The 10 irreversible compounds studied are broad spectrum inhibitors of ICE and its homologs. Some selectivity,however, is observed with the irreversible compoundscomparing inhibition of ICE and CPP32.

Table 2

Compound ^mact (ICE) M"1 s’1 ^inact (TX) kj_nact ,.-1 „-l M s (CPP32) W-1 M S 138 120,000 150,000 550,000 217d 475,000 250,000 150,000

Example 6

Inhibition of apoptosis 20 Fas-Induced Apoptosis in U937 cells. Compounds wereevaluated for their ability to block anti-Fas-inducedapopotosis. In a preliminary experiment using RT-PCR,we detected mRNA encoding ICE, TX, ICH-1, CPP32 andCMH-1 in unstimulated U937 cells. We used this cell 25 line for apoptosis studies. U937 cells were seeded in 5 6 culture at 1 x 10 cells/ml and grown to ~5 x 10cells/ml. For apoptosis experiments, 2 x 10 cellswere plated in 24-well tissue culture plates in 1 mlRPMI-1640-10% FBS and stimulated with 100 ng/ml anti- 30 Fas antigen antibody (Medical and Biological

Laboratories, Ltd.). After a 24 hr incubation at - 96 - 37 C'c, the percentage of apoptotic cells was determinedby FACS analysis using ApoTag reagents.

All compounds were tested initially at 20 μΜ andtitrations were performed with active compounds to 5 determine IC5Q values. Inhibition of apoptosis (> 75% go 20 μΜ) was observed for 136 and 138. An IC5Qof 0.6 μΜ was determined for 217e compared to noinhibition of anti-Fas-induced apoptosis by 214e at 20μΜ. 10 Example 7

An vivo acute assay for efficacy as anti-inflammatory agentLPS-Induced IL-Ιβ Production.

Efficacy of 214e and 217e was evaluated m15 CD1 mice (n=6 per condition) challenged with LPS <20 mg/kg IF) . The test compounds were prepared in oliveoil:DMSO:ethanol (90:5:5) and administered by IPinjection one hour after LPS. Blood was collectedseven hours after LPS challenge. Serum IL-Ιβ levels 20 were measure by ELISA. Results in Fig. 6 show a dosedependent inhibition cf IL-Ιβ secretion by 214e, withan EDjq of approximately 15 mg/kg. Similar resultswere obtained in a second experiment. A significantinhibition of IL-Ιβ secretion was also observed in 217© 25 treated, mice (Fig, 7} . However, a clear dose responsewas not apparent.

Compounds 214e and 217e (50 mg/kg) were alsoadministered by oral gavage to assess absorption.Results in Fig. 8 show that 214e, but not 217e when 30 administered orally inhibited IL-Ιβ secretion,suggesting potential fox oral efficacy of ICEinhibitors as anti-inflarrmatory agents. 0 1 2 3

A

A Ο Γ·Mr V - 99 -

The efficacy of analogs of 214e were alsoevaluated in LPS challenged mice after IPadministration (Fig. 9) and PO administration(Fig. 10) . 5 Table 3 % Inhibition of IL-β production by analogs of214e in LPs-chellenged mice after PO and IPadministration (50 mg/kg).

Table 3

Compound PO% Inhibition IP% Inhibition 214e 75 78 416 52 39 434 80 74 438 13 40 442 10 0 2002 - 78

Table 4

Comparison of 214e Prodrugs forEfficacy in LPS Challenged Mice:

Time Course Inhibition of IL-Ιβ Production_

Time of Compound Administration(relative to time of LPS challenge, PO, 50 mg/kg

Compound -2 hr -1 hr 0 hr + 1 hr 214e 39* _dr 80* 55% 75* 43* 44* 48* 11* _ dr _ "k _ dr 47* 304a 30 33 68 37 2100e 49 54 94 66 2100a 8 71 67 58 213e 0 48 41 89 302 0 27 21 26 2100c 0 0 85 4 0 2100d 42 35 52 26 2100b 0 0 47 26 ΑΡ εο 1 2 8 ο - 100 -

2001 -63 -62 -57 -54 1 64* 62* 58* c, ς * I * Values obtained in subsequent assays

Example 8

Measurement of blood levels of prodruqs of 214e. 5 Mice were administered a p.o. dose of compounds 302 and 304a (50 mg/kg) prepared in 0.5 0carboxymethylcellule.se. Blood samples were collectedat 1 and '7 hours after dosing. Serum was extracted byprecipitation with an equal volume of acetonitrile 10 containing 2 % formic acid followed by centrifugation.The supernatant was analyzed by liquid chromatography-mass spectrometry (ESI-MS) with a detection level of0.03 to 3 pg/ml. Compounds 302 and 304a showeddetectable blood levels when administered orally,. 214e 15 itself shows no blood levels above 0..10 pg/mL whenadministered orally. Compounds 302 and 304a areprodrugs of 214e and are metabolized to 214e in vivo(see Fig. 11) .

Example 9 20 We obtained the following data (see Tames 5 and 6) for compounds of this invention using themethods described in Examples 1-8. The structures ofthe compounds of Example 9 are shown in Example 10-12.Table 5 ί UV- Cell PBMC Whole human Clearance clearance Rat, i.v. 25 Compound (VisibleίKi (nM) avg. IC50 crM) blood IC50 (nM) Mouse,i . v. ml/min,/kg mi/min/kg 47b .. IS 00 <600 338 47a —J.......... 19^. 2 600 5100 79 135a ( 90 . : o 5000 >100 ; 135b 320 1700 30 j 125b 60 3 0 0 4500 ' 137 ! 40 10-00 14 000 139 350 ( 5 ) 10 15 J 20 25 - 101 - i Compound UV- VisibleKi (nM) Cell PBMC avg. IC50 (nM) Whole human blood IC50 (nM) Clearance Mouse, i . v. ml/min/kg ClearanceRat, i.v.ml/min/kg 213e 130 900 600 400* 214c 1200 5000 214e 7.5 1600 1300 23 12 217c 1700 7000 70 217e 175 2000 >50 220b 600 2125 223b 99 5000 >100 223e 1.6 3000 >20000 89 226e 15 1100 1800 109 227e 7 234 550 230e 325 300 67 232e 1100 4500 22 26 235e 510 4750 36 i 238e 500 4250 246 12 950 10000 31 1 257 13 11000 6600* 281 50 600 2500* 302 4500 >20000 >20000 304a 200 1,400 2400 14000* 307a 55 14500 16000 ί 307b 165 14000 1 E 404 2.9 1650 1800* 1100 64 24 S i 405 6.5 1700 2100 ί ί 406 4 1650 2300 1 407 0.4 540 1700 408 0.5 1100 1000 41 23 i 409 3.7 2500 410 17 2000 2800 32 20 411 0.9 540 1900 412 1.3 580 660* 700 1000* 25 413 750 6200 415 2.5 i 990 1000* 450 3500* 26 ί i 18 i ΑΡ/Γ/ 98/01294 30 APV01290 - 102 - ......................~ ~.........r i UV- Compound {Visible iKi (nMi •21'v«lC-.· g. 50 32, Whole human blood IC50 (nM) ClearanceMouse,i . v. ml/min/kg Clearance Rat, a.v.ml/min/kg 416 12 -00 3400 4 ! 417 8 . 0 0 6000 33 .4 Z 418 "Ί Z. , έ. . TO 1 . 0 * 7800 1800* 13 5,9 ~— “ ~ ” 419 280 •-VU0 420 1200 ' 1 0’ t . >0* 421 200 ng - ; · 422 50 ' 2 00 1200 423 424 10 OO- f c * 1500 4 5 4 5 . : 11.1 4000 425 0.8 - 0 i r 650 426 90 4 - .)0 1 O' 427 180 4 ; 0 0 428 280 429 7000 430 60 >8000 431 8 >8000 8000 432 1.6 “ 0 2000 433 2.9 1000 1100* 1100 434 4.9 1600 12 00* 1800 1300* 2 0 4 35 8 4 1 -)0 436 7.5 2'0 * 437 12 ' > 5000 438 28 700 2900* .:. . 439 3.7 3 1C· 32 0 0 34 00* 440 2.3 2000 44 , 1 4 50 0 442 «2 Zl 200 0 ' - 4 4 3 6 150 0 444 1 5 3500 445 135 4000 ΑΡ/Γ, δ b ,Ό 1 2 8 4 - 103 -

Compound UV- VisibleKi (nM) CellPBMCavg.IC50(nM) Whole human blood IC50 (nM) Clearance Mouse, i . v. ml/min/kg ClearanceRat, i.v.ml/min/kg 446 62 3000 447 5.8 2500 1500 448 130 4000 449 12 1500 3200 13000* 450 5 800 2200 1700* 18 12 451 4 1800 1500 9000* 452 4.5 600 800* 650 1600* 27.3 453 0.65 1300 1900 1600* ί 454 45 2500 455 1.2 400 2800 2600* 54 456 4.5 600 1300* 600 1400* 12.7 457 6.2 2000 3500 458 20 2900 459 5 1800 460 115 400 2400 1 461 47 462 40 463 14 2400 2800* 1 ί ! 4 64 2.5 1000 >1000 2500* ! 465 3 1000 800 466 0.8 1400 600 467 11 1900 i 468 4.5 850 2500 470 5 500 360 500* 63 471 i 750 400 17 472 140 473 1 1000 400 _j_[ 4 50* ! 474 ί 85

V 6 2 I δ / 8 6 /J/dV 10 lb AP e ο 12 8 ο - 104 - 5 Comoound uv- Visibl·-Ki (nM PBMC (nM) Whole human blood IC50 (nM) ClearanceMouse,i . v. ml/min/kg ClearanceRat, i.v. ml/mm/kg i 475 i 5.5 h Q 0 400 350* 31 / 476 7 : r,Q 2500 477 60 ------------ . S 478 380 479 i 15 900 700 2400* i 480 25 481 1.2 390 9 3 0 * 600 500’ ! 482 i....................................... <0.2 340 380 2 60* i 483 1.7 9 0 Ci 700 ί 484 ! . ........ .. 1550!. 0’ 5000 i 485 1 0 900 S 486 2.3 480' ·, * 500 cn 487 2.4 6 50960» 500 400* ..... 5. ' 488 1.5 940 750 —....................... i 489 6 2250 1700* 15000 490 4.3 930 1000* 700 1900* 491 5 2500 4 93 25 1200 800 850* 494 15 . ....... 13 50 1 500* 7000 495 43 496 16 : . 6000 497 3.5 '7 4 0 350 700* 498 1.5 500 500 400’ 499 3.5 12 00 - 9000 2100e 250 600 2100a ί 100 8 50 ...... '

V 8 Z i- Ο / 8 ? ZJ/dV AP vΟ 12 8 Ο - 105 -

Compound uv- VisibleKi (nM) CellPBMCavg .IC50(nM) Whole human blood IC50 (nM) Clearance Mouse, i . v. ml/min/kg ClearanceRat, i.v.ml/min/kg 2002 4 810 860* 70 1400* 32 2100d >100000 >20000 >20000 2100c 7400 >20000 >20000 2100b 8000 >20000 >20000 2001 135 1800 3500

Table 6

Compound Fluorescent Assay ^inact „-i M s CellPBMCavg.IC50(nM) Whole human blood IC50 (nM) Clearance Mouse, i .v. ml/min/kg ClearanceRat, i.v.ml/min/kg 136 5.4x10s 870 2800 93 138 1.2xl05 900 2900 116 217d 4.7xl05 340 4000 280 4xl0S 650 >1000 187 283 lxlO5 <200 450 104 284 3.5xl05 470 550 77 100 285 4.3xl0S 810 1000 130 50 15 * Values obtained upon reassay. J Example 10

Compound 139 was synthesized by a methodsimilar to the method used to synthesize 47a. ΑΡ/Γ/ 90/01294

O

20 Compounds 136 and 138 were synthesized by a methodsimilar to the method used to synthesize 57b. AP co 12 3 Ο - 106 - Ο

Compounds 135a, 135b, and 137 were5 synthesized by a method similar to the method used to synthesize 69a.

si net

Compounds 830e, 832e, 835e, 838e, 846, 85/,865, 907a, 907b, 1015-1045, and 1070-1091 were f”.’ 't-sozed by methods similar to those used tosynthesize compound 264 and the corresponding .ids a. η E x amp i e s 10 and 11. AP C Ο 12 3 Ο - 107 -

Compounds 47a, 47b, 108a, 108b, 125b, 213e,214c, 217c, 217d, 217e, 220b, 223b, 223e, 226e, 227e,230e, 232e, 235e, 238e, 246, 257, 280-287, 302, 304a,307a, and 307b were synthesized as described below.

(a) X = O (b) X = H2 10 (44a). To a solution of (IS,9S)t-butyl 9-amino-6,10- dioxo-octahydro-6H-pyridazino [1,2-a][1,2]diazepine-1-carboxylate (690mg; 2.32mmol; GB 2128984) in dioxane(16ml) and water (4ml) at 0 °C was added solid sodiumbicarbonate (292mg; 3.48mmol) followed by dropwise 15 addition of 3-phenylpropionyl chloride (470mg; 2.78mmol). The mixture was stirred at room temperaturefor 2h then more sodium bicarbonate (200mg; 2.38mmol)and 3-phenylpropionyl chloride (lOOmg; 0.6mmol) wereadded. The mixture was stirred for a further 2h at 20 room temperature, diluted with ethyl acetate (50ml),washed with saturated sodium bicarbonate (2 x 25ml)then dried (MgSO^) and concentrated. The residue waspurified by flash chromatography (0—50% ethylacetate/chloroform) and finally crystallized by ΑΡ δ Ο 12 8 Ο - 108 - trituration with ether to afford 8 6.0mg (86%) of a whitesolid: mp. 137-138 °C; [a]D23 -95.1° (c 0.549, CH2CI2); IR (KBr) 3327, 1736, 1677, 1664, 1536, 1422, 1156; LH NMR (CDC13) δ 7.24 (5H, m) , 6.50 (1H, d, 7=7,0), 5 5.24 (IH, m) , 4.90 (Id, m) , 4.60 (1H, m) , 3.44 i.IE, m) , 2.93 (2H, m) , 2.84 (IK, m) , 2.64 (1H, m) , 2.54 (IK, m) , 2.26 (2K, m) , 1.70 i 4d, m) , 1.7 0 (9H, s). MS (FAB, m/ z) : 130 (M+ + 1) , 3'?i, 242, 105 , 91. (44b) w a s prepared from (IS, 9S) t -butyl 9-amino-

10 octahydro-10-oxo-6H-pyridazino [ 1,2-a] [1,2] diazepme-l-carboxylate (Attwood et al., J. Chent. Soc. Perkin 1,pp, 1011-19 (1986)) as for 44a, to afford 810mg (81%)of a colorless oil: ioyD23 - 33.5° (c 0.545, CHoCi-d ; 3R (film) 3334, 2935, 1737, 1728, 1659, 1642; 2Η NMR (CDCI3) δ 7. 2 4 (5H, is). 6.75 (IH, d, 7=6 . 7), !f. '3 ’·· -.1 7.. ' (IH, ml, 4.92 (1H, m) , 3 ., 39 (IH, m) , 3.03 (4H, m) , 2.55 (3H, m) i 2.33 (IH, m) , 2.17 (IH, m) , 1.80 (SH, m) , 1. 47 (9H, s) , 1.39 (1H, m). MS(FAB, m/z) : 416 (M+ + 1) , 3 6 0 , 211, , 143, 97. 20 (45a). To a solution of (IS, 9S) t-butyl 6,10-dioxc- octahydro-9-(3-phenyIpropiony1amino) -6H-pyridazinof1,2-a] (1,2] diaze.pine-1-carboxylate (44a) (SOOmg; 1.863mmol) in dry dichloromethane (5ml) at 0 °Cwas added triflucroacetic acid (5ml). The solution was 25 stirred at room temperature for 3h then concentrated.Dry ether (10ml) was added to the residue then removedunder vacuum. This process was repeated three times toafford a crystalline solid. The solid was tr.wit.n ether and filtered to afford 590mg (85%) mt

CM w—

O 00 a* u a < white crystalline sol d I mp . 196-197.5 °C; ta]D-° -129.5° (c 0.2, CH3OH 1; IR (KBr) 3237, 1729, 1-: 1 6 6 0, 1 6 3 3, 1 5 7 4 , 1 4 3 2, 1285, 1205; 1H NMR (CDqOt 0 8.18 (IH, d, 7=7.4),- 7.22 (5H, m), 5.32 (IH, dd, = .5 2,9;, 4,75 (IH, m), 1 ,51 (IH, m), 3.50 (IH, m), ) i . 01 10 15 20 25 ΑΡ Ο Ο 1 2 8 Ο ;1Η, m) , 2.91(2Η, κι), 1.71 - 109 - (2Η, κι), 2.55 (2Η, κι), 2.29 (3Η, κι), 1.95(2Η, κι) . Anal. Calcd for 0^9^23^3¾: C, 61.12; Η, 6.21; Ν, 11.25. Found: C, 60.80; Η, 6.28; Ν,10.97. MS(FAB, m/z) 374 (Μ+ + 1), 242, 105, 91. (45b) was prepared from (IS, 9S) t-butyl octahydro-10-oxo-9-(3-phenylpropionylamino)-6H- pyridazino[1,2-a] [1,2]diazepine-l-carboxylate (44b) bythe method described for compound 45a to afford 657mg -,-F A Ck -, - -, —— ino_om°z

[a]D m) ,m) ,m/z) ) of 45b as a crystalline solid: mp. 198 23 -86.2° (c 0.5, CH3OH); IR (KBr ) 3294, , 1620, 1574, 1453, 1214; 1H NMR (CD3OD) d, J=7.9) , 7.20 (5H, m), 5.29 (1H, m ) , 3.47 (1H, m), 3.08 (2H, m) , 2.90 (2H, m) 2.36 (1H, m), 1.81 (5H, m) , 1.43 (2H, m) 360 (M+ +1), 211,143,91. .90 (1H, 2.55 (3H,MS (FAB, (46a). To a solution of (IS,9S) 6,1O-dioxo-octahydro-9-(3-phenyl-propionylamino)-6H-pyridazino [1,2-a] [1,2]diazepine-l-carboxylic acid (45a) (662mg; 1.773mmol) in dry dichloromethane (9ml) and drydimethyl formamide (3ml) at room temperature was addedbis (triphenylphosphine)palladium chloride (30mg) and(3S, 2R,S) -3-allyloxycarbonylamino-2-benzyloxy-5-oxotetrahydrofuran (Chapman, Bioorg. Med. Chem. Lett.,2, pp. 613-18 (1992)) (568mg; 1.95mmol) followed by dropwise addition of tri-n-butyltin hydride (1.19g;4.09mmol). 1-Hydroxy-benzotriazole (479mg; 3.546mmol)was added to the mixture and the mixture was cooled to0 °C before addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (408mg; 2.128mmol).

The mixture was stirred at room temperature for 3.25hthen diluted with ethyl acetate (50ml), washed twicewith dilute hydrochloric acid (20ml), twice withsaturated sodium bicarbonate (20ml), once with brinethen dried (MgSO4) and concentrated. The resulting oil AP/F/ 9 8/01294 30 10 15 20 25 AP c Ο 1 2 8 Ο 110 was purified by flash chromatography (0-100% ethylacetate/'chloroform) to afford 810mg (81%) of 46a as a IR (KBr) 3311 1659, 1651, 1536; ~H NMR(CDC13) δ 7.49, 6.56 ( 2=6,7, 7,8), 7.29 (10H, m), 6.37, 6.18 (1H, 2d,0-7.7,7.6), 5.56, 5,.34 (1H, d, s, J=5.2), 5.08-4(6H), 3.18-2.80 (5H), 2.62-2.28 (5H), 2.04-1.53MS (FAB, m/z), 563 (if + 1), 328, 149, 91. (46b) was prepared from 45b by the method described for46a to yield 7 90mg (96%) of a glass: m.p. 58-60cC: IR(KBr) 3316, 2940, 1793, 1678, 1641, 1523, 1453, ; "E NMR (CDC13) δ 7.28 (10H, m), 6.52, 6.42 (1H, 2d,5=7.2, 7.1), 5.53, 5.44 (1H, d, s, 5=-5.2), 5.35 (ill,m) , 4.6-4.9, 4.34 (4H, m), 3.1-2.8 (6H, m) , 2.6-2,7PHO, 1.95-1.05 (5H) . MS (FAB, m/z), 549 (M+ + 1), 400,310, 279, 91. (47a). A mixture of [3S, 2R,S, (IS, 9S) ] N-(2- benzyloxy-5-oxotetrahydrofuran-3-yl)-6,10-dioxo-octahydro-9-(3-phenylpropionylamino)-6H-pyridazino[1,2-a] [1,2]diazepine-l-carboxamide (46a) (205mg; 0.364mmol), 10% palladium on carbon (200mg) andmetnanol (20ml) was stirred under hydrogen at mixture of anomers: mp. 92-94i. ’C; 791, 2d, ΈΗ) ΑΡ/ΓΖ 98/91294 atmospheric pressurethen concentrated tt 11 6--118 cC; ία]β23 -.(br), Π33, 1731, 1«(CD-jOD) δ 7.21 (oE, 4.50 (2 E, m!, 4.22 ( ή h r m) , 2.9 a (2 H,- m) , 2.7'- I . 6H, 30 Anal . baled for C s + 11,4 2 . Fo und: C, s H, w'z) 473 (if + 1), 17 p. 149, (47b) was prepared f r om 4 6b 5h. The mixture was filteredrid 154mg (90%) of a glass: mp. c 0.1, CH3OH); IR (KBr) u323 .539, 1455, 1425; 1H NMR, 5.17 (1H, m), 4.73 (1H, m) , 38 (1H, m), 3.06 pH, m),) and 2.01-1.59 (5H, m).0: C, 56.32; H, 6,16; N,11; N, 11.25. MS (FAB, 47a, The residue was purified by flash chromaf ' spy AP c ο 12 8 Ο - Ill - (0-10% methanol/chloroform) to afford 65mg (52%) of aglass; m.p. 87-90°C; [a]D23 -167.0° (c 0.1, methanol); IR (KBr) 3329, 2936, 1786, 1727, 1637; 1H NMR (CD3OD) δ7.23 (5H, m), 5.29 (1H, m), 4.83 (1H, m), 4.59 (1H, d, 5 J=3.6), 4.29 (1H, m), 3.3-3.0 (3H, m), 2.91 (2H, m), 2.70-2.34 (5H, m) , 2.19 (2H, m) , 1.75 (4H, m), 1.36(2H, m) . Anal. Calcd for C23H3QN4O6 + 0.5H20: C, 59.09;H, 6.68; N, 11.98. Found: C, 58.97; H, 6.68; N, 11.73.MS(FAB, m/z) 459 (M+ + 1), 310, 149, 105, 91.

10 (99). A solution of 5-(2,6-Dichlorophenyl) oxazole (2.71g, 12.7mmol; prepared by a similar methoddescribed in Tet. Lett. 23, p. 2369 (1972)) intetrahydrofuran (65mL) was cooled to -78 °C under anitrogen atmosphere. To this solution was added n- 15 butyl lithium (1.5M solution in hexanes, 8.5mL, 13.3mmol) and stirred at -78 °C for 30min. Magnesiumbromide etherate (3.6g, 13.9mmol) was added and thesolution was allowed to warm to -45 °C for 15min. Thereaction was cooled to -78 °C and aldehyde 58 (3.26g, 20 12.7mmol; Graybill et al., Int. J. Protein Res., 44, pp. 173-182 (1993)) in tetrahydrofuran (65mL) was addeddropwise. The reaction was stirred for 25min., thenallowed to warm to -40 °C and stirred for 3h, and thenat room temperature for Ih. The reaction was quenched 25 with 5% NaHCC>3 (12mL) and stirred for 3h. The tetrahydrofuran was removed in vacuo and the resultingresidue was extracted with dichloromethane. Theorganic layer was washed with saturated sodium chloridesolution and dried over magnesium sulfate, filtered,

V 6 Z 1C ! 8 6 ZJ/dV ΑΡ ε ο 1 2 8 ο — 112 - and concentrated to yield 6.14g of the title compound.

Purification gave 4." ’9g (80%) of 99: 1H NMR (CL Cl3) δ 1, (s, 9H) , 2.7-2.5(1 2H), 2.8 (dd, 1H), 4.2, 4(2 x kA / .i K), 4.7-4.5(m, 3H) , 5.35-5.1(m, 2H), 5.6, 5,~ (2 x df 1 H), 6.0-5.8(m, 1H; . 7.2 (d, 1H) , 7.3 (m, 1H) , ( . 4 (m, 2H5 , ^CO-2-tBu H 0 no2-tBu Of C! ...... —·— 122 123 CQBBu

O 124

„CO2H

(123), Potassium fluoride (273mg, 4.70mmol) and then 2-chlorophenylmethyl thiol (373mg, 2.35mmol) were addedto a stirred solution of (3S) t-butyl N- 10 (a1lyloxycarbonyl)- 3amino-5-bromo-4-oxo-pentanoste (122; 749mg, 2.14mmol; WO 93 16710) in dimethyl formamide (20ml). The mixture was stirred for3.5n, quenched with water (50ml) and extracted withethyl acetate (2 . The combined organic 15 extracts were washed with water (4 x 50ml) then brine 0ml:

They were dried (MgSO4) and concentrated to afford an oil whicn was purified by flash chromatography (10-35% ethyl acetate/hexane) to afford832 mg (Slid of a colourless solid: mp. 45-6 c'C;-19.0° (c 1.0, CK?C.L-d ; IR (film) 3340, 2980, 2935, ΑΡ ν Ο 1 2 8 Ο - 113 - 1725, 1712, 1511, 1503, 1474, 1446, 1421, 1393, 1368,1281, 1244, 1157, 1052, 1040, 995, 764, 739; 1Η NMR(CDC13) δ 7.36 (2Η, m) , 7.21 (2Η, m), 5.91 (2Η, m) , 5.27(2Η, m), 4.76 (1Η, m), 4.59 (2Η, d) , 3.78 (2Η, s), 3.36 5 (2Η, m) , 2.91 (1Η, dd) , 2.74 (1Η, dd), 1.43 (9Η, s).

Anal. Calcd for C20H26ClNO5S: C, 56.13; H, 6.12; N,3.27; S, 7.49. Found: C, 56.08; H, 6.11; N, 3.26; S,7.54. MS (C.I.) 430/28 (M+ + 1, 3%), 374/2 (100). (124a). 6-Benzyl-l,2-dihydro-2-oxo-3- (3- 10 phenylpropionylamino)-pyridyl acetic acid (52b; 300mg,0.76mmol) in THF (7ml) was stirred with 1-hydroxybenzotriazole (205mg, 1.52mmol) and 1-(3-dimethylaminopropy-3-ethylcarbodiimide hydrochloride).After 3 min, water (12 drops) was added and the mixture 15 stirred lOmin then treated with t-butyl (3S) N-(allyloxycarbonyl)-3-amino-5-(2- chlorophenylmethylthio)-4-oxopentanoate (123) (325mg, 0.76mmol), bis (triphenylphosphine) palladium IIchloride (20mg) and tributyltin hydride (0.6ml, 20 2.28mmol). The mixture was stirred for 5h at room temperature, poured into ethyl acetate and washed withaqueous 1M HC1 (x2), aqueous sodium bicarbonate, brine,dried (MgSO4) and concentrated. The residue wastriturated with pentane and the supernatant discarded. 25 Chromatography (silica gel, 50% ethyl acetate/hexane)afforded a colourless foam (439mg, 81%): [a]D21 -18.3° (c 0.5, CH2C12); IR (KBr) 3356, 3311, 1722, 1689, 1646,1599, 1567, 1513, 1367, 1154; 1H NMR (CDC13) δ 8.39 (1H, d) , 8.23 (1H, s) , 7.24 ( 14H, m) , 6.16 (1H, d) , 4.95 30 (1H, m) , 4.63 (2H, m) , 4 .02 (2H, s), 3.74 (2H, s), 3.27 (2H, s), 2.85 (6H, m) , 1 .40 (9H, s) . Anal . Calcd for c39h 42CIN3O5S: c, 65.39; H, 5.91 ; N, 5.8 7 . Found: C, 65.51; H, 5.99; N,5.77. AP C Ο 12 8 Ο - 114 - (124b) was prepared by a similar method as 124a fromthe thioether 123 and 3S(1S,9S)-3-(6,10-dioxo-1,2,3,4,7,8,9,10-cctahydro)-9-(3-phenylpropionylamino}-6K-pyridazino[1,2~aj [1,2]diazepine-l-carboxylic acid 5 (45a) to afford 4 52mg (50%) of colourless foam: mp δο- ν °C; i«iD22 -94.0° (c 0.12, CH2C12); IR (KBr) 2934, 1741, 1722, 1636, 1666, 1644, 1523, 1433, 1260,1225, 1146, 757; X NMR (CDC13) δ 7.35 (3H, m) , (.20

(75., mi , 6.46 (IH, di. 5.21 (IH, m) , 4.97 (2H, ry , 4.56 10 (IK, m), 3.75 (2H, si. 3.25 (3H, mi, 2.93 (5H, ro, 2.71 (IK, dd) , 2.55 (2H, by 2.30 (1H, m) , 1.92 (3H, m) } 1.66 (2H, m), 1.42 (9H, s) . Anal. Calcd for *4* c35h43cin4o7s. 0.2 55X0: ; c, 59.73; E, 6.23; Cl, 5. 04 ; O N, 7.96; S, 4. 56. Found: C , 59.73; H, 6.19; CX 5.10; Γ4 15 N, 7.79; 5, 4. 58. MS ( -FAB) 697 (M-l, 100). (125a). t-Butyl-3(2(6- benzyl-1,2-dihydro-2-oxo-3~ (3- O phenylpropionylamino)-1-pyridyl)acetyl-amino-5-(2-chiorophenylmethylthio)-4-oxopentanoate (124a) (400mg, 0.56mmol) in dichloromethane (3ml) at 0 °C was treated j 20 with trifluoroacetic acid (3ml) and stirred at 0 'X fcx·ih and room temperature for 0.5h. The solution wasconcentrated then redissolved in dichloromethane andreconcentrated. This procedure was repeated threetimes. The residue was stirred in ether for lhr and 25 filtered to yield a colourless solid (364mg, 99 mp. 165-7 °C; [a]D22 -27.7° (c 0.2, CH2C12); IR (KBr) 3289, 17(12 , 168 2 , 1657, 1645, 1593 , 156 2, 15 27, 14 97, 41 6, 12 0 2 , 118 2 ; 1H NMR (XX13) d 8.47 (IH, d) , 8.21 XH, s ( , 7X0 ( IH, d), 7.22 (14H, m) , 6.24 (IH, d) , 5 , 0 3 30 (IH, nh , 4 .65 (2H, mi, 4.06 (2H, s) , 3 . 69 (2H, r 1 , 3.23 (2H, ml , 2 .88 (6H, m.i . (125b) was prepared by a similar method as 125a fromthe t-butyl ester 124b to afford 362mg (93%) of AP C Ο 1 2 9 Ο - 115 - colourless powder: mp 76-80 °C; r π 21 [odo -134° ( c 0.10, MeOH); IR (KBr) 330 9, 2935, 1725, 1658, 1528, 1445, 1417, 1277, 1219, 1175; 1H NMR (D 6-DMSO) δ 8.80 (1H, d) 8.19 (1H, d), 7.31 (9H, m), 5.09 (1H, m) , 4.74 (1H, m), 5 4.63 (1H, m), 4.35 (1H, m), 3.76 (2H, m) , 3.28 (3H, m) , 2.80 (5H, m), 2.52 (4H, m), 2.16 (2H, m) , 1.90 (3H, m) .

Anal. Calcd for C31H35C12N4O7S. 0.25H2O: C, 57.49; H,5.53; N, 8.65; S, 4.95. Found: C, 57.35; H, 5.43; N,8.45; S, 4.88. MS (-EAB) 641 (M-l, 100).

MT

CM 10 2-ChIorophenylmethyliodide. A mixture of 2- chlorophenylmethylbromide (4g, 19.47mmol) and Nal (14g,97.33mmol) in acetone (40ml) was stirred under refluxfor 1 hour. The reaction mixture was cooled, filteredand concentrated in vacuo. The residue was triturated 15 with hexane and filtered. The solution was concentrated in vacuo, and the resulting oil purifiedby flash chromatography (silica, hexane) to afford thetitle compound (4.67g, 63%) as an oil: 1H NMR (CDC13)δ 7.34 (4H, m), 4.54 (2H, s). 20 (201). (3S) t-Butyl N-(allyloxycarbonyl)-3-amino-5- hydroxy-4-oxopentanoate (81, Chapman, et al., Bioorq. &amp;Med. Chem. Lett., 2, pp. 613-618 (1992) 0.144g, 0.5mmol) and 2-chlorophenylmethyliodide (0.569g, 1.5mmol) in CH2C12 (4ml) were stirred vigorously with 25 silver oxide (0.231g, lmmol) and heated at 38 °C for 40hours. The reaction mixture was cooled, filtered andthe filtrate evaporated. The residue was purified byflash chromatography (silica, 0-20% ethylacetate inhexane) to afford the product as a colourless oil <X> σ»

Qa < ΑΡ Γ012 8 0 - 116 -

.Cl

a

* Alloc—

H OH 202 203 204 (203) . A solution of 2,4-dichloro-6-nitrophenol (202,40g containing 20% moisture) in EtOAc (500ml) was driedusing MgSO4, filtered and the filter cake washed with alittle _Et.OAc. Platinum on carbon (5% sulphided - 2g)was added and the mixture hydrogenated until uptake ofH? ceased. Triethyl orthoformate (160ml) and p-·toluenesulphcnic acid (IbOrngi were added and the mixturerefluxed for 4h. After cooling and removal of spentcatalyst by filtration the solution was washed withsat. NaHCC>3 solution, water and brine, dried with MgSO4and evaporated to dryness. Trituration with hexanegave a solid which was collected by filtration, washedwith hexane and dried to give the title compound (25. Sg, 83%) as a crystalline solid: mp 98-99 °C; IP.(K3r) 3119, 1610, 1590, 1510, 1452, 1393, 1296, 2067,850; ~K NMR (CDC13) 0 8.16 (1H, s), 7.69 (1H, d, J-1.9i, 7.42 (1H, d, J = 1.9); Anal. Calcd for C7.H3C.ipNO:C, 44.72; H, 1.61; N, 7.45; Cl, 37.70. Found: Ci44.Si; E, 1.69; N, "’,31; Cl, 37.71. (204) . Magnesium bromide was prepared by reaction ofMg (7.45g, O.SOmole) in THF (516ml) with 1? (5ur i and.1, 2-dibromoethane (26.3ml, 57.3g, 0.30mole) at refluxfor 2h and then cooling- to -40 °C. To the above wasadded rapidly via cannula a solution of 2-lith.io-5f 7- AP ΐ Ο 12 8 0 - 117 - dichlorobenzoxazole at 70 °C (prepared from 5,7-dichlorobenzoxazole (203, 28.9g, 0.154mole) and butyllithium (100ml 1.52M in hexane) in THF (150ml) at-70 °C). The mixture was stirred at -40 °C for lh and 5 then cooled to -70 °C before adding a solution of (3S)t-butyl N-(allyloxycarbonyl)-3-amino-4-oxo-butanoate(Chapman, et al., Bioorcr. &amp; Med. Chem. Lett., 2, pp.613-618 (1992)) (20.3g, 0.078mole) in THF (160ml) at less than -60 °C. The reaction was allowed to warm to 10 ambient temperature and was stirred for 16h beforequenching with ammonium chloride solution andextracting with 1:1 hexane:ethylacetate 600ml. Theorganic solution was washed with water and brine, driedwith MgSO4 and evaporated to a syrup (52.9g). Flash 15 chromatography (SiO2 250g -11 aliquots of 1:1 hexane:CH2C12 x 2, CH2C12, 5% EtOAc in CH2C12, 10% EtOAc inCH2C12, 20% EtOAc in CH2C12) gave impure product 24.6gwhich on further chromatography (SiO2 1:1 hexane: ether)give the title compound as a golden-brown glass (22.7g, 20 64%); IR (film) 3343, 2980, 1723, 1712, 1520, 1456, 1398, 1369, 1254, 1158, 993; 1H NMR (CDC13) δ 7.60 (1H,m), 7.37 (1H, m), 5.72 (1H, m), 5.64 (0.5H, d), 5.10 J (2.5H, m), 4.7-4.3 (4H, m), 2.9-2.6 (2H, m), 1.46 and 1.42 (9H combined, 2 x s). MS ES+ Da/e 445 (M + 1)+ Cl 25 35 62%, 447 (M + 1)+ Cl 37 40%, 389 100%. ΑΡ/Γ/ 9 8/01294 ?-BuO2C. ΑΡ ν Ο 12 δ Ο 118

€Ο2Η i-BuO2Cx_x^Ss^,CO2H

ΝΗ2 Alloc-NH * = S 205a - R 205b •Bu°2c-^X;z N'

H -K,NH2

Alloc-NH O 208a 208b i-BuO2C,

’''ψ^ΟΗAlloc-NH 206a 206b f'Bu°2C^-X^At0

Alloc-NH 207a 207b (205a). To a mixture of THF (200ml) and water (100ml)containing NaHCO3 (16.6g, 0.2mol) was added glutericacid t-butyl ester (lOg, 49.2mmol) and then dropwiseover 20 minutes allyl chloroformate (6.8ml, 64mmei).The mixture was stirred for 2 hours, extracted wishEtOAc, washed with a sat. hydrogenocarbonate solution,water and a sat. salt solution, dried and evaporated tcan oil 205a (9.5g, J'K NMR (D6-DMSO) δ 6.15.31-5.12 (2H, mi, 2.18 (2 H, m), 1.85(205b) was prepared by an analogous method to 205a toafford a colourless oil (6.27g, 88%) : [αίρ*-0 +16' (c 0,095, MeOH); IR (KBr) 3678, 3332, 3088, 2980, . 1721, 1530, 1453, 1393, 1370, 1331, 1255, 1155, 7.2% i ; I η 2 0 © , - a]D -6 ic 1. 5, MeOH) . 1 0 (1H, d), 5.96-5.88 (1H, m( , .43 (2H, m) , 3.90-3.84 (1H, tj , . / b (2K, mi, 1.36 (9H, si . 995, 935, 845, 778,9.29 (IE, broad si,m) , r? (2H, 95 (4H, I'M 636, 583; ±H NMR (CDCi394-5.79 (1H, m), 5.58 (IK,ο? (2K, di, 4.38-4.31 (1H, ;A (9H, s); Anal. Calcd for

I 6 2 V 0 i 8 6 /J/dV APV 012 8 0

Found: C, - 119 - C13H21NO6: C, 54.35; H, 7.37; N, 4.88. 54.4; H, 7.5; N, 4.8. (206a). To a solution of 205a (3.6g, 12.5mmol) in THF(100ml) at 0 °C was added N-methyl morpholine (1.5ml, 5 13mmol) followed by isobutyl chloroformate, (1.1ml, 13mmol). After 15 minutes, this mixture was added to asuspension of NaBH4 (0.95g, 25mmol) in THF (100ml) andMeOH (25ml) at -78 °C. After 2 hours at -70 °C, themixture was quenched with acetic acid, diluted with 10 EtOAc, washed with a sat. hydrogenocarbonate solution 3 times, water and a sat. solution of salt, dried and evaporated. Flash chromatography (2% MeOH in CH2C12) 20 afforded 206a as a colourless oil (2.4g, 70%): [a]D-10° (c 3.88, CH2C12); 1H NMR (CDC13) δ 5.84 (1H, m), 15 5.34-5.17 (3H, m), 4.56-4.53 (2H, m) , 3.68-3.59 (2H, m), 2.98 (1H, m), 2.40-2.30 (2H, t) , 1.84-1.78 (2H, m) ,1.43 (9H, s); Anal. Calcd for C13H23NO5: C, 57.13; H,8.48; N, 5.12. Found: C, 57.1; H, 8.6; N, 6.0(206b) was prepared by an analogous method to 206a 20 which afforded the title compound as a light yellow oil(3.42g, 57%) : [ce]D20 +14 (c 0.166, MeOH); IR (KBr) 3341, 3083, 2976, 2936, 2880, 1724, 1533, 1454, 1419,1369, 1332, 1251, 1156, 1062, 997, 933, 846, 777, 647;1H NMR (CDC13) δ 5.98-5.81 (1H, m), 5.35-5.10 (3H, m) , 25 4.55 (2H, d), 3.70-3.56 (3H, m), 2.50-2.47 (1H, broad s), 2.37-2.30 (2H, m) , 1.89-1.74 (2H, m), 1.44 (9H, s);Anal. Calcd for C13H23NO5: C, 57.13; H, 8.48; N, 5.12.Found: C, 56.9; H, 8.6; N, 5.6 (207a). To a solution of DMSO (1.51g, 19.3mmol) in 30 CH2C12 (25ml) at -70 °C was added oxalyl chloride(1.34g, 19.3mmol). After 10 minutes at -70 °C, asolution of (206a) (2.4g, 8.8mmol) in CH2C12 (10ml) was added dropwise and the mixture stirred for 15 minutesat -70 °C. Diisopropylethylamine (3.4g, 26.3mmoi) was ΑΡ/Γ/ 98/01294 A? ε ο 1 2 8 ο - 120 - added and the mixture stirred at -25 °C for 15 minutesthen diluting with EtOAc (50ml) washed with a solutionof sodium hydrogen sulfate 2M, concentrated to give anoil which was used immediately without purification: 5 1H NMR (CDC13) δ 9.5 (1H, s), 6.0-5.5 (2H, m) , 5.5-5.1(2H, m) , 4.5 (2H, m) , 4.2 (1H, m), 2.4-2.10 (2H, m) ,2.05 {2H, m), 1.36 (9K, s). (207b) was prepared by an analogous method to 207awhich afforded an oil (2.95g, 96%) which was used 10 without further purification in the next step: 1^}^'' + 21" (c 0.942, MeOH); ΧΗ NMR (CDCI3) δ 9.58 (IK, s) ,6.05-5.80 (1H, m), 5,57 (1H, broads), 5.35-5.18 . (2H,m), 4.56 (2H, d) , 4.34-4.24 (1H, m) , 2.38-2.16 <3H, m) ,1,96-1.73 (1H, m) , 1.43 (9H, s). 15. (208a). To a solution of 207a (2.39g, 8.8mmol), in

MeOH (20ml) was added sodium acetate (0.72g, 8.8mmol)and semicarbazide (0.98g, 8.8mmol) stirred overnight,concentrated and diluted with CH2CI2 (100ml), washedwith water, dried and concentrated. Flash 20 chromatography (2% MeOH in CH2CI2) afforded 208a (2.10g, 73%) as an oil: [a]D20 -21 (c 2.55°, CH2C12);"H NMR (CDCI3) δ 9.98 (1H, s), 7.27 (1H, d), 5.8 (1H,m), 5.5 (1H, d), 5.35-5.19 (2H, m), 4.58 (2H, m), 4.14(1H, m), 2.37 (2H, t < , 2.09 (1H, m), 2.0-1.75 (2H, m) ; 25 Anal. Calcd for C14H24N4O5: C, 51.21; H, 7.37; N, 17,06. Found: C, 50.2; K, 7.3; N, 16.1(208b) was prepared by an analogous method to 208awhich afforded a glassy oil (2.37g, 66%) : [ajp"'"’ +30 (c 0.26, CHCI3); IB. (KBr) 3476, 3360, 2979, 2923, 1700, 30 1586, 1527, 1427, 1394, 1369, 1338, 1253, 1156, 1060, 997, 329, 846, 775; 1H NMR (CDC13) δ 9.87 (1H, s), 7.09(1H, d), 6.05-5.75 (3H, m), 5.58 (1H, d) , 5.32-5.16(2H, m), 4.54 (2K, d), 4.35 (IK, m) , 2.32-2.26 (2H, m) ,2.15-1.55 (2H, m), 1.41 (9H, s); Anal. Calcd for

* 6 U K5 / 8 6 /J/dV AP C Ο 12 8 Ο - 121 -

Cj^H^N^Og: C, 51.21; H, 7 . j 7; N, 17,06. Found: 51.0; H, 7.5; N, 16.7. C, R1— Ν'

R1— I

211 H °O^O-M3u H θΟ^ΟΗ (b) R1 = MeSO2 212 (b) R1 = MeSO2 (c) R1 = MeCO (c) R1 = MeCO (d) R1 = PhCH2OCO (d) R1 = PhCH2OCO (e) R1 = PhCO (e) R1 = PhCO (f) R1 = Fmoc (f) R1 = Fmoc solution of t-butyl 9- amino- -6,10-dioxo- 8,9, 10-octahydro-6H- (211b). 1,2,3,4,

10 pyridazino[1,2-a][1,2]diazepine-l-carboxylate (GB 2,128,984; 831mg, 2.79mmol) and diisopropylethylamine(1.22ml, 6.99mmol, 2.5 equiv) in CH2Cl2 (10ml) underdry nitrogen was treated with methanesulphonyl chloride(237μ1, 3.07mmol 1.1 equiv). The mixture was stirred 15 for lh, diluted with EtOAc (75ml) and washed with saturated NaHCO3 (50ml) and saturated aqueous sodiumchloride (30ml), dried (MgSC^) and concentrated. Flashchromatography (10-35% EtOAc in CH2CI2) afforded 211b(806mg, 77%) as a colourless solid: mp 68-70 °C; ΑΡ/ΓΖ 98,01294 20 [a] 23 c 1.09, CH2C12); IR (KBr) 3270, 2980, 2939,1735, 1677, 1458, 1447, 1418, 1396, 1370, 1328, 1272,1252, 1232, 1222, 1156, 1131, 991; 1H NMR (CDC13) δ6.15 (1H, d), 5.31 (1H, m), 4.65-4.11 (2H, m), 3.47(1H, m) 2.99 (3H, s), 2.89 (1H, m), 2.72-2.51 (2H, m),2.34 (1H, m), 2.26 (1H, m), 2.05-1.62 (4H, m) , 1.47(9H, s); Anal. Calcd for C15H23N3O6S: C, 47.97; H, 6.71; N, 11.19; S, 8.54. Found: C, 48.28; H, 6.68; N, -109 25

Zap V Ο 1 2 8 ο - 122 - 10,86; S, 8.28. MS H FAB) 376 (Μ" + 1, 66%) , :..: (1. 00) , (2lie) . Acetic anhydride (307mg, 3.01mmol) was addedto a stirred mixture of t-butyl 9-amino-6,1D-dioro- 5 1,2,3,4,7,8,9,10-octahydro-6H- pyridazino[1,2-a][1,2]diazepine-l-carboxylate(GB 2,128,984; 813.7mg, 2.74xnmol), diisopropvlethylamine (884mg, 6.84mmol) and CH2CI7(20ml). The mixture was kept for Ih then diluted with 10 EtGAc, washed with NaHCO3 solution then brine, dried(MGSO4) and concentrated to yield a colourless 01.;,

The product was purified by flash chromatography »,0.5—8% MeOK/CHoCl^) to afford 211c (804mg, 71%) ofcolourless powder: rap 162-3 °C; [«Ιι/3 (c 1.03, 15 CK2CI2); IR(KBr) 3358, 2974, 1733, 1693, 1668, 1528,1462, 1431, 1406, 1501, 1278, 1271, 1250, 1233, 1217,1154, 1124; δ XH NMR (CDC13) d 6.32 UH, d), 5.29-5.25 (IE, m) , 4 . 98-4.85 (IH, m), 4.68-4.58 (IH, m), 3.55- 3.39 (IH, m ), 2.91-2.66 (2H, m), 2.39-2.18 (2H, ho , 20 2.03 ί 3.H, s ), 1.88-1.64 (4H, m) , 1.47 (9H, s); Ana1. Calc d for C 16H25N3°5: C , 56.62; H, 7.43; N , 12.38.

Found; C, 56.62; H, 7.43; N,12.36; MS (+ FAB) 340 (M+ + 1, 40%), 284 (100). (211d). Benzyl chloroformate (1.07g) was added 25 dropwise to a stirred ice cold mixture of the (1.5,95)t-butyl 9-amino-6,10-dicxo-l,2,3,4,7,8,9,10-octahvdro-oK-pyridasino[1,2-a] i1,2]diazepine-l-carboxylate (GB2,128,984; 1.55g, 5.21mmol), NaHCO3 (0.66g, 7.82mmoi),dioxan (32ml) and water (8ml). The mixture was kept at 30 6) "C for 15mm then for 2h at room temperature. The mixture was diluted with EtOAc (50ml), washed tvm.eewith sat. NaHCO3 solution, dried (MgSO4) andconcentrated. The oily residue was purified by flashchromatography to afford 211d (1.98g, 88%) of a

* β Z 0 ' 8 6 ZJ/dV AP V Ο 1 2 8 Ο - 123 - 24 [a]D"" -56.4 (c 1.0, CH2C12); IR(thin2946, 1728, 1677, 1528, 1456, 1422, colourless oil:film) 3325, .29791370, 1340, 1272, 1245, 1156, 1122, 1056, 916, 734, (CDC13) δ 7.29 (5H, m) , 5.81-5.72 (1H, m)s), 4.69-4.51 (2H, m),(2H, m) , 2.34-2.19 (2H,;9H, s); Anal. Calcd for6.92; N, 9.35. Found:

699; H NMR

5.26-5.20 (1H, m) , 5.05 (2H, 3.48-3.36 (1H, m) , 2.81-2.51 m), 1.90-1.54 (4H, m) , 1.41 C22H2 9N3 0 g*H2 θ · c, 58.79; H, 59.10; H, 6.57 ; N, 9.25; MS C, 10 432 (M +1, 100). (211e). A solution of benzoyl chloride (1.61g,11.47mmol) in CH2C12 (15ml) was added dropwise to astirred ice cold mixture of (IS, 9S) t-butyl 9-amino-6,10-dioxo-l,2,3,4,7,8,9,10-octahydro-6H- 15 pyridazino[1,2-a] [1,2]diazepine-l-carboxylate (GB 2,128,984; 3.1g, 10.43mmol), dry CH2C12 (20ml) anddiisopropylethvlamine (4.54ml, 26.06mmol). The mixturewas kept cold for lh then left at room temperature for0.5h. The mixture was diluted with CH2C12, washed 20 twice with brine, dried (MgSO4) and concentrated. Theresidue was purified by flash chromatography (0-5%methanol in CH2C12) to afford 211e (4.0g, 96%) of a 75.0° (c 0.12,

CM ao 25 colourless glass: mp 74 -76 °C; r π 3 [a]D CH2C1 2) . IR (KBr)' 3350, 2979, 2938, 1536, 1422, 1276, 1250, 1155; 1H NMR (2H, m) , 7.53-7.40 (3H, m), 7.07 (1H, (1H, dd, J - 3.0, 5.8) , 5.12 (1H , m) , 3.51 (1H, m), 2.90 (2H, m), 2.38 (1H, 2.25 (1H, m), 1.9 (2H, m), 1.70 (1H, 5.30 :iH, m) , for C21H27N3O5 0.5H2O: C, 61.45; H, 6.88; N, 10.24.Found C, 61.69; H, 6.71; N, 10.18. (211f) was prepared in a similar manner to 211e, except9-fluorenylmethylchloroformate was used instead ofbenzoylchloride to give a white glassy solid 211f 30 ΑΡΐ0 1 2 3 0 ~ 124 - (2.14g, 89%) : mp 190-192 °C; (a]D25 -81.5" (c 0.1,CH2C12) IR (KBr) . 2977, 1731, 1678, 1450, 1121, 1246, 1156, 742; “H !«R (CDC13) δ 7.60 (2H, m) , 7.57 mj , 7.50-7.26 (4H, m) , 5.60 (1H, d, J = 7.8; m) , 4.67 (2H, ltd , 4.38 (2H, m) , 4.23 (1H , ro ; --3.41 (1H, m) , 2.92-2.65 (2H, m) , 2.41-2. 21 ( 1.95- 1.58 (4H, m) , 1.47 (9H, s). MS(ES~, m/ c 5 1, 97%), 179 (- ) .

5tU (212b) was synthesised by the same method as compound212e (635mg, 85%) as a colourless powder: mp 209...... I a] )4 -132 (c 0.12, MeOH); IR (KBr) 3308, 2940, 1717, 1707, 1699, 1619, 1469, 1456, 1442, 1417, 1391, 1348, 1339, 1330, 1310, 1271, 1247, 1222, 1175,1133, 993, 976; "H NMR (CD3OD) 6 5.35 (1H, m) , 4.58-4.48(IH, m), 4.46-4.36 (IE, m) , 3.60-3.42 (1H, m) , 3,01-2.87 (in, m), 2.95 (3H, s), 2.55-2.39 (1H, m), 2,32-2.20 (2H, m), 2.09-1.89 (2H, m) , 1.78-1.62 (2H, cn ;

Anal. Calcd for C11Hi7K3O6S: C, 41.37; H, 5.37; 2513,16; S, 10.04. Found: C, 41.59; K, 5.32; N, 12,75; S, 9.76; MS(ES -). Accurate Mass calculated forC11H18N3O6S (MH+): 320.0916. Found: 320.0943. (212c) was prepared from 211e the same method ascompound 212e as a white glassy solid (595mg, 77%); mp >250 °C; !. a J 24 D -153 (c 0.10, 2942, 17 42, 1697 , 1675, 1650, 12 81, .. ? 49, 12 02 , 1187, 1171; £, '2 1 (IK , m) , 4. 81-4 271 (1H, b NMK (LJUjUlJ) O 5 . .5 0“)1-4 27% (1H, m) , 4.61-4.46 (1H, nd , 3.59-3.44 (2H, m) , 3.11-2.94 (1H, m) , 2.58-2.39 ilH,m) , 2.36-2.19 (2H, m; , 2.11-1.83 (3H, m), 1.99 )35, S),"8-1.56 (2H, m) ; Anal. Calcd for C^H-ryNgQ^:

Found: C, 5 0.82; H, 6,02 ; j. 50.68; H, 6.05; N,

55 14.58; MS (ES - . (M-l, 100%): Accurate V calculated for C12u- , L (MH+): 284.1246. Found; 284.1256, 19,83,

V 6 Z )0/96 /J/dV AP &amp; ο 12 8 0 - 125 - (212d) was prepared from 211d by the same method ascompound 212e as colourless crystals (170mg, 97%) : mp 60-100 °C; [a]D22 -103 (c 0.10, MeOH); IR (KBr) 3341, 2947, 1728, 1675, 1531, 1456, 1422, 1339, 1272, 1248, 1221, 1174, 1122, 1056, 982, 699; 1H NMR (CDC13) δ 7.35 (5H, s), 5.65 (1H, d) , 5.48-5.40 (1H, m), 5.10 ( 2H, s), 4.76-4.57 (2H, m), 3.49-3.30 (2H, m), 2.92-2.59 (2H, m), 2.40-2.27 (2H, m) , 1.97-1.67 (4H, m); MS (ES 1 -) 374 (M - 1, 100%). Accurate mass calculated for C18 H22N3°6 (MH+): 376.1509. Found: 376.1483. Accurate mass calculated for C^gH^NgOgNa (MNa+) : 398.1328. Found: 398.1315. (212e). TFA (20ml) was added to an ice cold stirredsolution of the t-butyl ester 211e (4.l5g, 10.34mmol)in dry CH2Cl2 (20ml). The mixture was kept cold for1.5h then left for 2.5h at rt, concentrated. TFA wasremoved by repeated concentrations of CH2Cl2\ether andether solutions of the residue. Finally trituration ofthe residue with ether afforded 212e 3.05g (85%) of a 74 white glassy solid: mp 118-126 °C; [a]D -70.5 (c0.1, CH2C12). IR (KBr) 3361, 2943, 1737, 1659, 1537,1426, 1220, 1174; 1H NMR (CDC13) δ 7.80 (2H, m) , 7.54-7.33 (4H, m), 8.83 (brs), 5.44 (1H, m), 5.26-5.13 (1H,m), 4.66 (1H, m), 3.59-3.41 (1H, m), 2.97, 2.76 (2H,2m), 2.36 (2H, m), 1.98 (2H, m), 1.75 (2H, m). MS(ES~,m/z) 344 (M - 1, 100%) . (212f) was prepared from 211f in 96% yield by the samemethod as for 212e: mp 120-126 °C; [a]D25 -72.5° (c 0.1, CH2C12). IR (KBr) 3406, 2950, 1725, 1670, 1526, 1449, 1421, 1272, 1248, 1223, 1175, 761, 741; H NMR (CDC13) δ 7 .76 (2H, m) , 7.62-7.26 (4H, m) , 6 5.76 (2H, brs, d, d, J = 2.9) , 5.46, 5.36 (1H, 2m) , 4.79-4.54 (2K, m) , 4.77 (2H, m) , 4.21 (1H , m) , 3.41 (1H, m), 2.89 (1H, m) , 2.69 ( 1H, m), 2.35 (2H, m) , 2 ΠΟ AP C 0 1 2 8 0 126 MSiES , m/z) 462 (M' 1.98, 1,73 (4 H,240 (100*5 ,

R

PhCO

(214) ie) Pl

OH -13

Me CO- PhCO (213c) was synthesized from 212c by the same method ascompound 213e to afford a mixture of diastereomers(193mg, 36%) as colour less crystals: IP (KBr) 1799, 1701, 1682, 1650, 1555, 1424, 1412, 1278, 1258, 112( 937; H NMR (CDC13) δ 7.41-7.28 (5E, in.) , 38 i (0.5K, d), 5.36 (0.5K, s) 5.10-5.05 (1H, m) , 5.00-4.45(5.5H, ®), 3.19-2.84 (3H, m) , 2.72-2.56 (1H, m) , 2.51-2.25 (2H, m), 2.02 (3K, s), 1.98-1.70 (3H, m), 1.66-1.56 (3H, m) ; Anal, Calcd for C23H28N4O7: C, 58.47; H,5.97; N, 11.86. Pound; C, 58.37; H, 6.09; N, 13..47,MS (ES -) 471 (M-l, 100%). Accurate mass calculated 6.52 (0.5H, d), 6.38 (C.5H, d), 6.22 (0.5H, d), 473.2012 +. for C23H29N4O7 (MH3 : 473.2036. Found:

Accurate mass calculated for C23H2gN4O7Na (Mna’495,1856. Found: 495,1853. (213e). Tributyltin hydride (2.2ml, 8.18mmol) wasadded dropwise to a solution of acid 212e (1.95g,d.cmmol), (3S, 2AS) 5--ailyIoxycarbonylamino-2-b e r.: t y 10 x y - 5 - 0 x 01 e t r a lit d rofuran (Chapman, Bioorq. &amp; Med.Chen. Left., 2, pp. · *-618 (1992); 1.80g, 6.16mmol) and (ph.,?) 2pdCl2 (50nq) in dry CH2C1? (36ml), withstirring, under dry nitrogen. After 5 min 1- z 1 a.: ’triazoie (l.Slg, li.2mmol 6.72mmoi) waradded followed after cooling (ice/H+O) by AP C Ο 12 8 Ο - 127 - ethyldimethylaminopropyl carbodiimide hydrochloride(1.29g, 6.72mmol). After 5 mins the cooling bath wasremoved and the mixture was kept at room temperaturefor 4h, diluted with EtOAc, washed with 1M HC1, brine, 5 sat. aq. NaHCO3 and brine, dried (MgSO4) and concentrated. Flash chromatography (silica gel, 0-90%EtOAc in ΟΗ2Ο12) gave the product as a white solid(2.34g, 78%); IR (KBr) 3499, 1792, 1658, 1536, 1421,1279, 1257, 1123, 977, 699; 1H NMR (CDCl3) δ 7.81 (2H, 10 m) , 7.54-7.34 (8H, m), 7.1, 6.97, 6.89, 6.48 (2H, m, d,J 7.7, d, J = 7.5, d, J = 7.6), 5.57, 5.28 (1H, d, J=5.2, s), 5.23-5.07 (2H, m) , 4.93-4.42, 3.22-2.70, 2.51-2.26, 2.08-1.69, 1.22 (15H, 5m). Anal. Calcd forc28H30N4°7 0.5H2O: C, 61.87; H, 5.75; N, 10.32. Found 15 C, 62.02; H, 5.65; N, 10.25. (214c) was synthesized from 213c by a method similar tothe method used to synthesize 214e from 213e to provide 9 9 colourless crystals (140mg, 99%): mp 90-180 °C; [a]D-114 (c 0.10, MeOH); IR (KBr) 3334, 3070, 2946, 1787, 1658 , 1543, 1422, 1277, 1258; H NMR (d -: DMSO) δ 8.66 (1H, m) , 8.18 (1H, d), 6.76 (1H, s), 5.08 (1H, m), 4.68 (1H, m) , 4.30 (1H, m), 2.92-2.70 (2H, m), 2.27- -2.06 (3H, m) , 1.95-1.72 (4H, m), 1.85 (3H, s), 1.58 (2H, m); cvf 0 co k a < MS(ES -) 381 (M-l, 100%); Accurate mass calculated for 25 C16H23N4O7 (MH+): 383.1567. Found: 383.1548. (214e). A mixture of 213e (2.29g, 4.28mmol), 10%palladium on carbon (1.8g) and MeOH (160ml) wasstirred under H2 at atmospheric pressure for 6.3h.

After filtering and concentrating the hydrogenation was 30 repeated with fresh catalyst (1.8g) for 5h. After filtering and concentrating the residue was trituratedwith diethyl ether, filtered and washed well with etherto give 214e as a white solid (1.67g, 88%): mp 143-147 °C; [aa]D23 -125° (c 0.2, CH3OH). IR (KBr) 3391, ΑΡ ν ΰ 1 2 8 0 - 128 - 1657, 1651, 1538, 1471, 1280, 1258; "Ή NMR (CD3OD; δ 7.90 (2H, mi, 7.63-7.46 (3H, m) , 5.25 (1H, m), 4.65 (1H, ml, 4.68-4.53 (2H, m), 4.33-4.24 (1H, m) ,3.62-3.44, 3.22-3.11, 2.75-2.21, 2.15-1.92, 1.73-1.66illH, 5mi . Anal. Calcd for C21H24N4O7 H2O: C, 54.54;H, 5.67; N, 12.11. Found C, 54.48; H, 5.63; N, 11,92. 0

(C) = MeCO (d) Ri = PhCH2OCO 10 (e) = PhCO

15 (215c) 'was synthesized from 214c by the same method ascompound 215e, to afford a mixture of diastereomers as ' *_e glassy solid )398mg, 84%) : 1R (KBr) 3335, 2577, 1738, 1658, 1562. 1541, 1433, 1368, 1277, 1120; "H NMR (CDCI3) δ 7.36-7.32 (3H, m), 6.91 (IE, d) , 6.30 112, d) , 5.15-5.09 (12, in) 5.01-4.88 (1H, m) , 4.::1-4.44 12 K, m), 4.37-4.08 i2.25 i 1H, in i , 2.8 2 - 2 2.7 2-1 . 64 i 4 H, m) 2, :22 .2 C, 52.44; H, 5.87; N, m) , 3.32-3.18 (1H, m), 3.:1 (4H, m) , 2.39-2.29 (1H, mi.02 (3H, s); Anal. Calcd for2.26; H, 5.64; N, 8.71. 216. MS (ES -') 645/3/1

Foundi(M~i. 1 ΑΡ Ε Ο 12 8 Ο - 129 - 189 (81), 134 (100). Accurate mass calculated forc28H37N4cl2°9 (MH+): 643.1938. Found: 643.1924.

Accurate mass calculated for Ο28Η36Ν4Ο12Ο9Ν3 (MNa+)665.1757. Found: 665.1756. 5 (215d) was synthesized from 214d by the same method as compound 215e to afford a mixture of diastereomers(657mg, 70%) as a glassy white solid: IR (KBr) 3420,3361, 2975, 2931, 1716, 1658, 1529, 1434, 1367, 1348,1250, 1157, 1083, 1055; 1H NMR (CDC13) δ 7.32 (8H, m), 10 7.14 (1H, d), 5.81 (1H, d), 5.15 (1H, m), 5.07 (2H, s) ,4.74-4.65 (1H, m) , 4.58-4.22 (4H, m), 4.15-4.06 (1H,m), 3.72 (1H, m), 3.32-3.21 (1H, m), 3.04-2.94 (1H, m),2.69-2.52 (3H, m) , 2.33-2.27 (1H, m), 1.95-1.59 (4H,m) , 1.28 (9H, s); Anal. Calcd for C34H40N4Cl2O10.0.5 15 H2O: C, 54.70; H, 5.54; N, 7.50. Found: C, 54.98; H,5.59; N, 7.24. MS (ES -) 737/5/3 (M-l, 22%), 193/1/89(100). Accurate mass calculated for C34H41N4C12°10(MH+) 735.2120. Found: 735.2181. (215e). Tributyltin hydride (4.6ml; 11.4mmol) was 20 added dropwise to a stirred mixture of (3S, 4RS) t-Butyl(N-allyloxycarbonyl)-3-amino-5-(2,6- dichlorobenzoyloxy)-4-hydroxypentanoate (prepared by amethod similar to the method described in Revesz etal., Tetrahedron. Lett., 35, pp. 9693-9696 (1994)) 25 (2.64g; 5.7mmol), (Ph3P)2PdCl2 (50mg), CH2C12 (100ml) and DMF (20ml) at room temperature. The mixture wasstirred for a further lOmin was then 1- hydroxybenzotriazole (1.54g, 11.4mmol)was added. Themixture was cooled to 0 °C then 30 ethyldimethylaminopropyl carbodiimide hydrochloride (1.31g; 6.84mmol) added. The mixture was kept at thistemperature for 15min then at room temperature for 17h.The mixture was diluted with EtOAc (300ml), washed with1M HC1 (2x100ml), sat. aq. NaHCO3 (3x100ml) and brine a>

CM <3» ex? t i «

CL < - 130 - i2xl00ir,l), dried (MciSGA and concentrated. The residuewas purified by fir- Chromatography (2-5% CMeOH/CHoCl2) to afford 3.24g (81%) of 215e as a glassysolid: mp 106-110 °C; IR (KBr) 3354, 1737, 1655, 1531, 1433, 1276, 1150; 'Ή NMR (CDC13) δ 7.80 (2K, dd, J-7,5 and 1.5), 7.75-7.26 (6H, m) , 7.14-6.76 (2H, ms, 5.30-5.02 (2H, m) , 4.63-4.11 (5H, m) , 3.44-3.26 (2H,m) , 3.10-2,30 (5H, mi, 2.10-1.60 (5H, m), 1.44 (95, s);Anal. Calcd for C33H->oCl2N4C>9. 0.75H2O: C, 55.12; K,5.54; N, 7.79; Cl, 9.86. Found: C, 55.04; H, 5034; N,7.80; CI, 10.24. MS (ES +) 709/7/5 (M + 1), 378 (59),,324 (64), 322 (100) . (216c) was synthesized from 215c by the same method ascompound 216e as a glassy white solid (300mg, 83*}: mp60-125 °C; [a]Dz3 -89.1 (c 1.08, CH2C12) ; IR (K3r) 3356,2979, 2935, 1740, 1659, 1532, 1434, 1369, 1276, 1260, 1151; NMR (CDCI3) δ 7.39-7.32 (3H, m), 7.13 (lip < 6.3 4 (1H, d), 5.22-5.17 (1H, m), 5.11 (1H, d) , 5.04 (1H, d) ,. 4.99-4.88 (25, m), 4.64 -4.52 (1H, m) , 3.2 9- 3.11 (1H, m), 3.05-2,67 (4H, m), 2.39-2.29 (1H, HO, 2.02 (3H, s), 1.98-1,.75 (4H, m), 1.46 (9H, s) ; Anal. Calcd for C2gH^N^Ci: C, 52.42; H, 5.34; N, 8 553. Found : C, 52.53; K, 5. 70; N, 7. 85. MS (ES -)

Ch

«mJ

O

aD i, « a < 643/41/39 (M-l, 1005). Accurate mass calculated forC28H35N4C12O9 (MH+): 611.1781. Found: 641.1735.

Accurate mass calculated for C^I^^C^OgNa (Mna 5 ;663.1601. Found: 663,1542. <216d) was synthesized from 215d by the same method ascompound 216e to afford 216d as a white glassy solid(-:--00, 68%) : mp 90-170 °C; [a]D25 -83.4 (c 1.01,CH,C12(; IR (KBr) 3338, 2933, 1736, 1670, 1525, . 1417, 1368, 1258, 1151, 1056, 1031; NMR (CDC? ' 67.3}; (855 ms, 7.18 (15, d) , 5.65 (1H, d) , 5.19 . m 5 , 5.09 (25, s), 4.So- - (1H, m), 4.82-4.49 (2H, d>, AP i0 1 2 8 0 - 131 - 3.30- 3.07 (1H, m), 3.05-2.59 (4H, m), 2.42-2.27 (1H,m) , 2.18-1.59 (5H, m) , 1.42 (9H, s) ; MS (ES-) 737/5/3(M, 13%), 185 (100). (216e). Dess-Martin reagent (3.82g; 9.0mmol) was added5 to a stirred solution of the alcohol 215e (3.17g; 4.5mmol) in CH2Cl2 (100ml). The mixture was sirred forlh, diluted with EtOAc (300ml), then washed with a 1:1mixture of sat. Na2S2O3 and sat. NaHCO3 (100ml) followedby brine (100ml) . The mixture was dried (MgSO4) then 10 concentrated. The residue was purified by flashchromatography to afford 2.2g (70%) of 216e as acolourless solid: mp 102-107 °C; [ce]D32 -82.5 (c 0.1,CH2C12); IR (KBr) 3374, 2937, 1739, 1661, 1525, 1433,1275, 1260, 1152; 1H NMR (CDC13) δ 7.85-7.78 (2H, m) , 15 7.57-7.32 (6H, m), 7.09 (1H, d, J = 7.9), 7.01 (1H, d, J 7.3), 5.25-5.16 (1H, m) , 5.16-5.05 (1H, m), 5.15 (1H,d) , 5.03 (1H, d), 4.99-4.90 (1H, m) , 4.68-4.54 (1H, m), 3.31- 3.17 (1H, m), 3.17-2.72 (4H, m) , 2.45-2.35 (1H,m) , 2.30-1.66 (5H, m), 1.44 (9H, s); Anal. Calcd for 20 C33H36C12N4O9. 0.5H20: C, 55.62; H, 5.23; N, 7.86; Cl,9.95. Found: C, 55.79; H, 5.15; N, 7.80; Cl 9.81. MS(ES +) 729/7/5 (M + Na), 707/5/3 (M + 1), 163 (100%).(217c) was synthesized from 216c by the same method as 25 85-175 °C; compound 217e as a glassy white solid (166mg, 66%): mpa]D25 -156 (c 0.13, MeOH); IR (KBr) 3373, 2929, 1742, 1659, 1562, 1533, 1433, 1412, 1274, 1266,1223, 1197, 1145, 1138; 1H NMR (CD3OD) δ 7.38 (3H, s),5.14-5.03 (1H, m), m), 3.11-2.92 (1H, (2H, m), 2.05-1.46 4.49-4 .32 (2H, m) , 3.5C 1-3.27 (1H, m) , 2 . 84-2.62 (2H, m) , 2.46-2.11 (5K, m ), 1.92 (3H, s) ; Anal. Calcd 2O: C, 47.77; H, 4 . 68; N, 9.29. N, 9.07. MS (ES +) 627/5/3

Found: C, 47.75; N, 4.59; (M+K, 21%), 611/9/7 (M+Na, 87), 589/7/5 (M +1, 71), 30 AP e 012 8 0 132 2 66 (100); Accurst (ME ): 535.1155. (217d) was synthescompound 217e to a(Slung, 96%) : mp

js calculated for C24H27N4CI2CC id: 585.1134. from 216d by the same mr as ; 217d as a white glassy solid,.., 24 C; [o(]D"" -85.9 (c 0.15, 2945, 1738, 1669, 1524, 1433,1258, 1147, 1057; Ml NMR (CD3OD) δ 7.56 (4H, m) , 7.45(5H, m) , 5.32 (2H, rn) , 5.20 (2H, s) , 4.76-4.48 O/E, m) ,3.65-3.38 (3H, m) , 3.27-3.09 (2H, m) , 3.03-2.89 GoH, 1. mj , 65-2.24 (3H, mt, 2.19-1.62 (5H, m) ; MS (E( 679/7/5 (Μ-·1, 100%) .Accurate mass calculated forc30h31n4c12°10 (ΜΗ") : 677.1417 . Found: 677.1430, (217e) TFA (25ml) was added dropwise to an ice col' stirred solution of the ester 216e (2.11g, 3.0mmoi·.The mixture was stirred at 0 °C for 20min then at roomtemperature for Ih. The mixture was evaporated todryness then coevaporated with ether three times.Addition of dry ether (50 ml) and filtration afforded1,9g (98%) of 217e as a colourless solid: mp 126-· f) 130 ~'C; [o]D -122.0 <c 0.1, MeOH) ; IR (KBr) 3322,1740, 1658, 1651, 1532, 1433, 1277, 1150; XH NMR (Dg-DMSO) δ 8.87 (IH, d, J - 7.4), 8.61 ilH, d, J = 7 ,8) ,7.92-7,86 (2H, m) , 7,05--7.43 (6H, m) , 5.25-5.12 (3H,m) , 4.94-4.60 (2H, m), 4.44-4.22 (IH, m), 3.43-3,10(IE, m) , 3.00-2.52 (3H, m) , 2.45-2.10 (3H, m) , 2.ΙΟ-Ι, 75 (2H, m), 1.75-1,50 (2H, m); Anal. Calcd forC2qH28Cl2N4O9. 1H2O: C, 52.34; H, 4.54; N, 8.42; Cl,10.66. Found: C, 52.02; H, 4.36; N, 8.12; Cl, 10.36.MS OES -) 649/7/5 (m - 1;, 411 (100%). AP C Ο 12 8 Ο - 133 -

218b Rx = MeSO2 219b

O

(218b) was prepared from the acid 212b and 99 in ananalogous way to compound 215e to afford a mixture of 5 diastereomers (865mg, 80%) as a colourless solid: IR(KBr) 3298, 2974, 1723, 1659, 1544, 1518, 1430, 1394,1370, 1328, 1273, 1256, 1156, 1134; 1H NMR (CDCl3)δ 7.45-7.28 (4H, m) , 7.26-7.15 (2H, m), 5.26-5.10 (2H,m), 4.80-4.67 (1H, m) , 4.59-4.42 (2H, m), 3.32-3.17 10 (1H, m), 2.96 (3H, 2xs), 2.93-2.79 (1H, m), 2.71-2.53 (4H, m), 2.38-2.28 (1H, m) , 2.07-1.81 (4H, m); Anal.Calcd for C28H35N5C12O9S. 0.5 H2O: C, 48.21; H, 5.20; N, 10.03. Found: C,48.35; H, 5.26; N, 9.48. MS (ES+) 714/2/0 (M + Na, 25%), 692/90/88 (M+ + 1, 51), 15 636/4/2 (38), 246 (100). Accurate mass calculated forc28h36n5c12°9s ): 688.1611. Found: 688.1615. (219b) was prepared from 218b in an analogous way to off-white powder ( 675mg, 81%) mp compound 216e as an 100-200 °C; r ·, 24 -84.9 3336, 2978, 2936, 1719, 1329, 1274, 1257, 1155, 7.47-7.38 (4H, m), 7.245.48 (1H, d), 5.38-5.30 (c 1.01, CH2C12); IR (KBr) 1674, 1510, 1433, 1421, 1369, 991, 789; 1.. H NMR (CDCI3) δ (1H, d) , 5.6 1-5. 53 (1H, m) , (1H, m) , 4.6 7-4 . 45 (2H, m) , 20 - 134 - 3.48-3.18 (2H, m) , 3.04-2.90 (2H, m), 2.97 (3H, s< .2.69-2.54 (IK, m) , 2.42-2.32 (IK, m), 2.22-2.15 (1H,m) , 2.07-1.S3 (3H, mi, 1.71-1.65 (2H, m), 1.36 (9K2 s);final. Calcd for C28K33N3Cl2O9S: C, 48.98; H, 4.84; N, 5 10.20; S, 4.67. Found: C, 48.73; H, 4.95; N, 5.65- S, 4.54. MS (ES +} 692/90/88 (M+ + 1, 100%), 636/4/2(71), Accurate mass calculated for C;28H34N5C12°9S(MH J : 686.1454. Found: 686.1474. (220b) was prepared from 219b in an analogous way to10 compound 217e as a pale cream powder (3 96mg, 87%) : rap

100-2 00 °C; Md27 - 129 (c 0.12, MeOH); IR (KBr ) 3310, 3153, i / 13, 1667, 1557, 1510, 1432, 1421, 1329, 12 73, 1253, 12 21, 1193, 1153» 1134, 992, 789; NMR { 0 0 DMSO) δ '7.88 (1H, s) » 7 , 81-7.60 (4H, m), 5.49-5, 28 (1H

15 m) , 5.24-5.14 (IK, m) , 4.46-4.22 (2H, m) , 3.30-3.03(2H, m$ , 2.97-2.76 (3K, m), 2.96 (3H, s) , 2.46-2.24(1H, m) , 2.16-2.05 (1H, in), 2.03-1.78 (3H, m) , 1.28-1.46 (2H, m) ; MS (ES-} 632/30/28 (M - 1, 68%), 149/7/5(103) . Accurate mass calculated for C24H26N5CI2O9S 20 (Mlf) : 630.0828. Found: 630.0852.

I ΑΡ C Ο 12 3 Ο - 135 -

223b R-l = MeSO2 Cl

223e RT = PhCO 5 (221b) was prepared from the acid 212b and (3S,4RS) t-

butyl N-(allyloxycarbonyl)-3-amino-4-hydroxy-4-(5, 7-dichlorobenzoxazol-2-yl)butanoate (204) by an analogousmethod as that used for compound 215e to afford amixture of diastereomers (460mg, 70%) as a glass: IR 10 (film) 3325, 1725, 1664, 1453, 1399, 1373, 1327, 1274,1256, 1155; XH NMR (CDC13) δ 7.57 (1H, m) , 7.36 (2H,m) , 6.06 (1H, t), 5.29 (2Ή, m), 4.79 (1H, m), 4.47 (1H,m), 3.23 (1H, m), 2.97 and 2.94(3H combined, 2 x s),2.9-2.4 (4H, m), 2.30 (1H, m), 1.96 (4H, m), 1.41 and 15 1.37 (9H combined, 2 x s). MS ES Da/e 660 (M - 1)" Cl35 100%, 662 (M - 1)" C1J7. (221e) was prepared from the acid (212e) and (3S,4RS)t-butyl N-(allyloxycarbonyl)-3-amino-4-hydroxy-4-(5, 7-dichlorobenzoxazol-2-yl)butanoate (204) by an analogous 20 method as that used for compound 215e to afford a

mixture of diastereomers (613mg, 87%) as a glass: IR ΑΡ/Γ/ 98/01294 A? C 012 8 0 - 138 - chromatography (lv- . (35%) of a white glassy--82,5° (c 0.02, CK2C12)1534, 1524, 1422, 1237,7.83-7.78 (2H, m) , 7 ,77,

EtOAc in CH2C12) to afford 0.2asolid: mp 70-72 °C;

IR (KBr) 3404, 1726, 1660,1254, 1154; 'LH NMR (CDC13) ό7.75-7.32, 7.26-7.20 (7H 7.12 (1H, d, U ' = 8.2), 7.01 (1H, d, J = 7.3), 5,23 (2H, m) , 5.03- 4.94 (IE, m), 4.62 (1H, dt, J = 14.5 3,78 (2H, m) , 3.38-3.29 (1H , m), 3.26 (2H, s), 3.0 2.62 (4K, m) , 2.71 (IR, dd, J = 17.2, 4.5), 2.33 ( dd, J 1 3,2, 6.5), 2.15-1. 83, 1.73-1.63 (5H, m), ( 9H, S· Anal . Calcd for C33H39C1N4O7S: C, 59.05; .5 . 8 6 ; N, 8.35. Found: C, 59.00; K, 5.80; N, 7.32 1.4! 3 m) , .....5.08 (225e) was prepared from acid 212e and (35) t-buryj. N~ (ally1oxycarbonyl)-3.......amino-5- (2-chlorophenylmethyloxy) - 4-oxopentanoate (201) using a method similar to thatused for compound 224e, to afford 4Ong (23%) of aglassy solid: 1H NMR (CDC13) δ 7.83-7.73 (2H, is),7.67-7.10 (9K, m), 5..23-5.09 (2H, m) , 4.59 (1H, m) ,4.45-4.22 (2H, m) , 3.7-3.19, 3.08-2.72, 2.71-2.4’·,2.05-1.85, 1.72-1.61, 1.45-1.26 (20H, 6m). (226e) was prepared from 224e by an analogous method asthsc used for compound 217e which afforded 0.22g (81%) mp 95-100 °C; [a]D23 0.2, CH2C12) · IR (KBr) 3393, 172C, 1658, 1525, 1, of an off-white sol -95.6' 1422, 1.279; "H NMR (D6-DMSO) δ 8.80 (1H, d, J =7.89 (2K, m), 7.7 (IK, d, J- 7.7), 7.56-7.28 (7R5.10 (1I-I, m) , 4.87-4,73 (2H, m) , 4.39 (1H, m) , 3.)2K, m) , 3.44, 3.3; « R;, +H2O, 2m), 2.97-2.56, 2. 1.92, 1.61 (11H, 4m). Anal. Calcd for C29K31C1N5· • 5.10; N, 8.85. Found: C, πυ , 3: C, 55.02; Ή, 5.09; N, 8 571. (227e) was prepared from 225e by an analogous merhodthat used for compouna 217e. The product was furtherpurified by flash chromatography (0-5% MeOH/CHoCi-n f 55.00; AP/7,' 98.01294 AP - Ο 12 8 0 - 139 - afford 19mg (81%) of a glassy solid: 1H NMR (CDC13) δ 7.79 (2H, m), 7.66-7.18 (9H, m) , 5.30-5. 10 (2H, m) , 4.85 (1H, m), 4.65 (2H, m) , 4.53 (1H, m) , 4.28 (2H, m), 3.28, 3.01, 2.72, 2.33, 1.94 , 1.60 (11H, 6m) . MS (ES~, 5 m/z) 597 (M+ - 1, 100%).

O

(228e). 1-Hydroxybenzotriazole (0.23g, 1.68mmol) 10 followed by ethyldimethylaminopropyl carbodiimide hydrochloride (0.21g, 1.09mmol) were added to a stirredsolution of the acid 212e (0.29g, 0.84mmol) in CH2CI2(3ml) at rt. The mixture was kept for lOmin then asolution of (3RS, 4RS) t-butyl 3-amino-5-fluoro-4- 15 hydroxypentanoate (Revesz, L. et al. Tetrahedron Lett.,52, pp. 9693-9696 (1994); 0.29g, 1.40mmol) in CH2C12(3ml) was added followed by 4-dimethylaminopyridine(lOmg). The solution was stirred for 17h, diluted withEtOAc, washed with 1M HC1, brine, sat. aq. NaHCO3 and AP c ο 1 2 8 0 - 140 - brine again, dried (MgSO,*) and concentrated. The residue was purified by flash chromatography (50.......100%

EtQAc/CHoClj and 5% MeOH/EtOAc) to afford 0.25g :56%)of a white glassy solid: IR (KBr) 3343, 1726, 1618,1536, 1426, 1279, 1257, 1157; 1H NMR (CDC13) δ 7.84-7.7? (2H, hi), 7.57-7..40 (3H, m) , 7.05-6.92, 6.73 (2H,2m), 5.17-5.04 (2H, no, 4.56, 4.35-4.21, 4.04 (511 3m),3.36, 3.09--2.34, 2.00 (11H, 3m), 1.46 (9H, s). Anal.Cared for C26H35FN4O7 0,5H2O: C, 57.45; H, 6.65; N,,10.81. Found: C, 577.64; H, 6.56; N, 10.15. (229e) was prepared from 228c by an analogous method tothat used for compound 216e. After purification byflash chromatography. (30-50% EtOAc/CH2Cl2) the productwas obtained as a white glassy solid (0.194g, 893d : IR(KBr) 3376, 1728, 1659, 1529, 1424, 1279, 1256, 1156.(230e) was prepared from 229e by an analogous method to that used for compound 217e to afford 230e as a whiteglassy solid (100%); mp 105-125 °C; -91.9° (c 0,72, CH3OH). IR (KBr) 3336, 1789, 1737, 1659, 1535, 1526, 1279, 1258, 1186; 1H NMR (CD3OD) δ 7.71-7.68 (2H, m) , 7.37-7,.23 (3H, m) , 5.02, 4.88-4.63, 4.37--4.0 (6H, 3m), 3.30, 2.97, 2.68-2.60, 2.37-1.54 (1 IK,4m). MS (ES~, m/z) 475 (M+ - 1, 100%).

O

It A. C231g)

°cAA:

H (232e) (2 31e) . N-Fi uor eny1me thy1oxy-carbony1-3-ami no- 3..... cyanopropionic acid methyl ester (EPC547699A1, 3· w,r . mmol? was treated with 17ml of diethylamine. 1. on snoring at room temperature the solution was AP C Ο 1 2 9 Ο - 141 -

concentrated. The residue was chromatographed onsilica gel (3% methanol in CH2C12) and gave the freeamine as a pale yellow oil. To a solution of this oiland hydroxybenzotriazole (297mg, 2.19mmol) in DMF 5 (5ml), was added at 0 °C ethyldimethylaminopropyl carbodiimide (232mg, 1.21mmol, 1.1 equiv) followed by(IS, 9S) 9- (benzoylamino)-[6,10-dioxo-l,2,3,4,7,8,9,10-octahydro-6H-pyridazino[1,2 — a] [1,2]diazepine-1-carboxylic acid (212e). After stirring at 0 °C for 5 10 min and then at room temperature overnight, the mixturewas diluted with CH2C12 (50ml) and the resultingsolution washed successively with 1M HCl (2 x 30ml), H2O (30ml), 10% NaHCO3 (2 x 30ml) and sat. aq.

NaCl,dried (MgSO4) and concentrated. Purification by 15 flash chromatography on silica gel (3% methanol inCH2C12) afforded the compound 231e (404mg, 83%) as a solid: [ . 20a]D -121° (c 0.14, ch2 Cl2) ; 1H NMR ( CDC13) δ 7.40-7 . 83 (5H, m) , 7.38 (1H, d) , 6.96 (1H, d) , 5.27- 5.07 ( 2H, m) , 4.66-4.50 (1H, m) , 3.79 (3H, s) , 3.23- 20 2.73 ( 6H, m) , 2.47-2.33 (1H, m) , 2.15- 1.82 (4H , m) ;

Anal. Calcd for C22H25N5O6: C, 58.0; H, 5.53; N, 15.38.Found: C, 57.6; H, 5.6; N, 15.0. ) (232e). A solution of methyl ester 231e (400mg, 0.88mmol) in methanol (30ml) and water (30ml) was 25 cooled at 0 °C and treated with diisopropylethylamine.The solution was stirred at 0 °C for lOmin and then atroom temperature overnight. The heterogeneous mixturewas concentrated and the solid obtained was chromatographed on silica gel (5% methanol/1% formic 30 acid in CH2C12) affording the free acid 232e (170mg, ΑΡ/Γ/ 9 8/01294 44%) as a white solid: mp 155 °C (dec); [ . . 20 a]D -117° (c 0.1, MeOH); IR (KBr) 3343, 3061 , 2955, 1733, 1656, 1577, 1533, 1490, 1421, 1342, 1279 , 1256, 1222, 1185, 708; ΤΗ NMR (D4-MeOH) δ 7.88-7.28 (5H, m) , 5.20- 5.03 APC Ο 1 28 0 - 142 - ΠΗ, m) , 4.98-4.84 £2H, m) , 4.75-4.53 (1H, m), 4.51-4.34 (1H, m), 3.45-3.22 (1H, m) , 3.14-2.94 (1H, .raj,3.14-2.94 (1H, nt), 2.86-2.61 £2H, nt), 2.53-1.50 [8K,m) : Anal. Calcd for CAiHiqNcOc. 1.5H«?O: C, 53.84; H,5.59; N, 14.95; 0, 25.61. Found: C, 54.3; H, 5.4; N,

Ph" o // -5

/ NN Η O, R p Ο 'K N'^NH2

Η H COW-Bu

233e R 234e R =

CO2-/-Bu

:36e R co2-abu 237eR =

co2h AP/.'/ 9 8/01294

235eR 238eR =

CO?H

P (233e). A solution of (IS, 9S) 6,10-dioxo-1,2,3,4,7,8,9,10-octabydro-9-(benzoylamino)-6H-pyridazmo [1,2-aj [1,2] diazepine-l-carboxylic acre APC Ο 1 2 8 Ο - 143 - (212e) (345mg, l.Ommol), (208a) (361mg, l.lmmol, 1.1 equiv) and (Ph3P)2PdCl2 (20mg) in CH2C12 (5ml), wastreated dropwise with n-Bu3SnH (0.621ml, 2.3mmol, 2.1equiv). The resulting orange brown solution was 5 stirred at 25 °C for lOmin and then 1- hydroxybenzotriazole (297mg, 2.2mmol, 2 equiv) wasadded. The mixture was cooled to 0 °C andethyldimethylaminopropyl carbodiimide (253mg, 1.3iranol,1.2 equiv) added. After stirring at 0 °C for lOmin and 10 then at room temperature overnight, the mixture wasdiluted with EtOAc (50ml) and the resulting solutionwashed successively with 1M HC1 (3 x 25ml), 10% NaHCO3(3 x 25ml) and sat. aq. NaCl, dried (MgSO,}) andconcentrated. Flash chromatography on silica gel (2- 15 10% methanol in CH2C12) afforded compound 233e (280mg, 49%) as a tan solid: [a]D20 -95 (c 0.09, MeOH); IR(KBr) 3477, 3333, 2968, 2932, 1633, 1580, 1535, 1423,1378, 1335, 1259, 1156, 1085, 709; 1H NMR (CDCl3) δ9.32 (1H, s), 7.83-7.39 (6H, m) , 7.11-7.09 (1H, m) , 20 6.30-5.30 (2H, brs), 5.17-5.05 (2H, m), 4.62-4.38 (2H, m) , 3.30-3.15 (1H, m), 3.13-2.65 (2H, m), 2.46-2.19(3H, m) , 2.15-1.54 (8H, m) , 1.42 (9H, s). ) (236e) was prepared by an analogous method to that used for 233e using (4R) t-butyl N-allyloxycarbonyl-4-amino- 25 5-oxo-pentanoate semicarbazone (208b, 435mg, 1.33mmol).The product was obtained as a foam (542mg, 71%): ο n [a]D -99° (c 0.19, CHC13); IR (KBr) 3473, 3331, 3065,2932, 2872, 1660, 1580, 1533, 1488, 1423, 1370, 1337,1278, 1254, 1223, 1155, 1080, 1024, 983, 925, 877, 846, 30 801, 770, 705; 1H NMR (CDC13) δ 9.42 (1H, s), 7.81 (2H,d), 7.51-7.40 (4H, m), 7.06 (1H, d), 6.50-5.50 (2H,broad s), 5.25-5.00 (2H, m) , 4.60-4.45 (2H, m) , 3.15-2.85 (2H, m) , 2.75-2.35 (1H, m), 2.30-1.23 (11H, m), 1.42 (9H, s) . v— * co r § - 14 4 - <234e). A solution of semicarbazone 233e (390mg,0.68mmol) in methanol (10ml) was cooled at 0 °C andthen treated with a 38% aq. solution of formaldehyde(2ml) and 1M HC1 (2ml). The reaction mixture was then 5 stirred overnight at room temperature. The solutionwas concentrated to remove the methanol. The aq.solution was extracted with EtOAc (30ml). The organicsolution was successively washed with 10% NaHCCR (30ml)and sat. aq. NaCl (30ml!, dried (MgSO^) and 10 concentrated. Purification by flash chromatography onsilica gel ¢2-5% methanol in CH2CI2) afforded 234e(I79mg, 51%} as a white foam: ία]ο^° -101° (c 0. 064,MeOH); IR (KBr)3346, 2976, 2934, 1730, 1657, 1535, 1456, 1425, 1278, 1255, 1156, 708; "H NMR (CDC135 0 9.56 (1H, s), 7.88-7.38 (5H, m), 7.01 and 6.92 (2E, 2d) , 5.27-5.08 (2H, it) , 4.69-4.46 (1H, m), 3.50-3.27 <2H, m) , 3.15-2.73 (2K, m), 2.46-1.83 (10H, m), 1.45 (9E, s) , <237e) was prepared from 236e by an analogous method to20 234e to afford a white foam (390mg, 85%) : [cdcT''' -113° (c 0.242, CHCI3); IR (K3r) 3352, 3065, 2974, 1725, 1657, 1536, 1489, 1454, 1423, 1369, 1338, 1278, 1255,1223, 1156, 1078, 1026, 981, 846, 709. (235e). A solution cf t-butyl ester 234e (179mg, 25 0.35mmoi) in dry CHpCl;· (3ml) was cooled to 0 °C and treated with trifluoroacetic acid (2ml). The resultingsolution was stirred at 0 °C for 30min and then at roomThe solution was concentrated, theiry CH9CI2 (5ml) and the mixture30 again concentrated. This process was repeated once again with more CH-Clo (5ml). The residue obtained wascrystallized in diethyl ether. The precipitate wascollected and purified on silica gel column (5%methanol in CH2CI9) which afforded compound 235e as a temperature for 2h.residue taken up inagain concentrated. «<· C‘-4

AP C O 12 8 Ο - 145 - 20

D white solid (lllmg, 70%): mp 142 °C (dec); [a] -85.5 (c 0.062, MeOH); IR (KBr) 3409, 3075, 2952, 1651, 1541, 1424 , 1280, 1198, 1136, 717; 1H NMR (D6-DMSO) δ 9.40 (1H, s), 8.62 (2H, m) , 7 .96-7.38 (5H, m), 5.19- 5 5.02 (1H, m), 4.98-4.79 (1H, m) , 4.48- -4.19 (1H, m), 3.51-3.11 (2H, m) , 3.04· -2.90 (2H, m), 2.38- -1.46 (10H, m) . (238e) was prepared from 237e by an analogous route to20 235e which afforded a beige foam (190mg, 60%): [a]D10 -78 (c 0.145, MeOH); IR (KBr) 3400, 3070, 2955, 2925, 2855, 1653, 1576, 1541, 1490, 1445, 1427, 1342, 1280,1258, 1205, 1189, 1137, 1075, 1023, 983, 930, 878, 843,801, 777, 722; d NMR (D6-DMSO) δ 9.40 (1H, s), 8.72- 8 . 60 (2H, m) , 7.89 (2H, d) , 7.56-7.44 (3H, m), 5.17 15 ( 1H, m) , 4.90-4.83 (1H, m) , 4.46-4.36 (1H, m) , 4.20 4 .15 (1H, m), 3.40- 3.30 (1H, m) , 2.98-2.90 (2H, m), 2 .50· -1.60 (10H, m).

O-f-Bu (243)

(244)

CJ 4» α> ο> fit < 20

(246) (243) was prepared from

H OBn (245) :iS,9S) t-butyl 9-amino-octahydro-10-oxo-6H-pyridazino[1,2-a][1,2]diazepine-1-carboxylate (Attwood, et al. J. Chem. Soc., Perkin 1, pp. 1011-19 (1986)),to afford 2.03g (86i by the method described for 211e, ?5

i of a colourless foam: [a]D - 14 6 - -15.9° (c 0,5, CH2C1;>); IR (KBr) 3400, 2976, 2937, 1740, 1644, 1537, 1448, 1425, 1367, 1154; 1H NMR(CDCI3) δ 7.88-7.82 (2H, m) , 7.60-7.38 (4H, m) , 5,48ilH, m) , 4.98 (1H, in), 3.45 (1H, m) , 3.22-2.96 (2H, m) ,2.64 (1H, m) , 2.43-2.27 (2E, m), 1.95 (2H, m) , 1.82-1.36 (4H, m) , 1.50 (SH, s) ; Anal. Calcd for C21H-QN-5O4.0.25K2O: C, 64.35; H, 7.59; N, 10.72. Found: C, 64.57; K, 7.43; N, 10.62. MS (ES +, m/z) 388 (100%, M+ (244) was prepared from (IS,95} t-butyl 9-benzoylaminc- octahydro-10-oxo-6H-pyridazino- i1,2-a3 [1,2jdiazepine-1-carboxylate (243), by the method described for 212e, to afford 1.52g (89%) of awhite powder: mp. 166-169 °C (dec); [a]D25 -56.4s (c0.5, CH3OH); IR (KBr) 3361, 2963, 2851, 1737, 166o, 1620, 1534, 1195, 1179; 1H NMR (D6-DMSO) δ 12.93 (1H,brs), 8,44 (1H, d, J = 8.4), 7.93 (2H, m), 7.54 (3H,m) , 5.4 6 (1H, m) , 4 .S'7 (1H, m) , 3.12 (2H, m) , 2.64 (1H,m), 2.64 (1H, m) , 2.27 (1H, m) , 1.98-1.68 (7H, m) , 1.40(IK, m) ; Anal. Calcd for C17H21N3O4. 0.25H2O: C, 60.79;K, 6,45; N, 12.51. Found: C, 61.07; H, 6.35; N, 12.55. MS (ES+, m/z) 332 (58%, M+ + 1), 211 (100). (245) was prepared from (IS,9S) 9-benzoylamino-octahydro-10-oxo-6H-pyridazino [ 1,2-a] [1,2] -diazepme-1-carboxyiic acid (244), by the method described for213e, to afford 601mg :76%) of a colourless foam: IR(KBr) 3401, 2945, 1794, 1685, 1638, 1521, 1451, 1120; 1H NMR (CDCI3) δ 7.87-7.77 (2H, m) , 7.57-7.14 (10H, m),5,59.....S.4'7 (2H, m) , 4.9)^-4.32 (4H, m) , 3.27-1.35 (14H, m) ; Anal, Calcd fox' C2gH32N40g. 0.5H2O: C, 63.50; H,6.28; N, 10.58. Found: C, 63.48; H, 6.14; N, 10,52. MS (ES +, m/z) 521 (100%, M* + 1). (246) was prepared from [35, 2RS (1S,9S) ] N-(2- benzyloxy-5-oxotetrahydrofuran-3-yl) -9-benzoylamwo·- AP/."/ 9 8.01284 AP C Ο 12 8 Ο - 147 - octahydro-10-oxo-6H-pyridazino[1,2-a][1,2]diazepine-1-carboxamide (245), by the method described for 214e, toafford 396mg (84%) of a white powder: mp. 110-115 °C;[O]D26 -126.3° (c 0.2, CH3OH); IR (KBr) 3345, 2943, 1787, 1730, 1635, 1578, 1528, 1488, 1450, 1429; 1H NMR(CD3OD) δ 7.88 (2H, m) , 7.48 (3H, m) , 5.55 (1H, m), 1H, m), 4.29 (1H, m), 3,41-3.053H, m), 2.28-2.01 (3H, m) , 1.86- 4.91 (1H, m), 4.56(3H, m), 2.76-2.411.65 (4H, m), 1.36 I1H, m) ; Anal. Calcd for C2iH26N4O6. 10 1.25H2O: C, 55.68; H, 6.34; N, 12.37. Found: C, .} 55.68; H, 6.14; N, 12.16.- 1) .

MS (ES -, m/z) 429 (100%, M

ΑΡ/Γ/98 / 0 1 2« 4 (249) (250) (247). n-Butyllithium (1.6M in hexane) (22.3ml,35.7mmol) was added dropwise over 20min to a solutionof (2R)- (-)-2,5-dihydro-3, 6-dimethoxy-2-(1- 15 λ η 0 12 8 0 - 148 - metnylethyl)pyrazine (5.8ml, 6.0g, 32.4mmol) in THF(250ml) cooled to "C at a rate such that the temperature was maintained below -72 °C. The reactionmixture was stirred for lh at -75 °C and a solution of 5 2,6-di~t-butyl~4-methoxyphenyl-2-butenoate (Suzuck et al. Liebigs...Ann. Chem. pp. 51-61 (1992) ) (9.9g, 32.5mmol) in THF (60nsl .) was added over 30 minutesmaintaining the temperature below -72 °C during theaddition. The reaction mixture was kept at -75 '"'C for

10 1.5h and a solution of glacial acetic acid (6ml) in THF (25ml) was added at -75 °C and the solution warmed toroom temperature. The solution was poured onto 101NH/.C1 (300ml) and extracted with diethyl ether (3 x250ml). The combined organic phases were washed with 15 brine (2 x 200ml), dried over Na2SO4 and evaporated to dryness under reduced pressure. The residual oil was purified by flash chromatography on silica gel (201 heptane in CH2Cl2) which afforded the title compound aso f) a light yellow oil (13.Sg, 85%): [ajD -64° (c 0.22, 20 MeOH); 1R (KBr) 2S62, 2873, 2840, 1757, 1697, 1593, 1460, 1433 , 1366, 1306, 1269, 1236, 1187, 1157 , 1126, 1063, 1038 , 1011, 9T0, 924, 892, 867, 846, 831 , 7 97, 773, 754; ΧΗ NMR (CDCI3) δ 6.85 (2H, s), 4.21 (IH, t, J = 3.5) , 3.98 (1H, t, J= 3.5), 3.79 (3H, s), 3.71 25 (3H, s) , 3 .69 (3H, s), 3.15 (IH, dd, J 17.8, 7 .9) , 2.86-2.81 (1H, m), 2.58 (1H, dd, J= 17.8, 5.9 ) , a .. u 8 2.19 (IB, ffi) , 1.33 ( '· :, S) , 1.02 (3H, d, J = 6.6) , 0 270 (6H, dd, J = 1.3, 6 . 8 ) . (248). A solution of (247) (22.4g, 45.8mmol) in 30 acetonitrile (300ml) and 0.25N HC1 (366ml, 2 e was stirred, at room temperature under nitrogen atm re for 4 days. The acetonitrile was evaporated underreduced pressure and diethylether (250ml) was added tothe aq. phase. The nil of the aq. phase was ad d to AP S Ο 12 8 Ο - 149 - 10 15 pH8-9 with concentrated ammonia solution (32%) and thephases separated. The aq. phase was extracted withdiethylether (2 x 250ml). The combined organic phaseswere dried over NajSC^ and evaporated to dryness underreduced pressure. The residual oil was purified byflash chromatography on silica gel (2% methanol inCH2CI2) which afforded the required product as a lightyellow oil (8.2g, 45%) : [c<]D20 +20 (c 0.26, MeOH);IR(KBr) 3394, 3332, 3000, 2962, 2915, 2877, 2838, 1738,1697, 1593, 1453, 1430, 1419, 1398, 1367, 1304, 1273,1251, 1221, 1203, 1183, 1126, 1063, 1025, 996, 932, 891, 866, 847, 800, 772, 745; 1H NMR (CDCI3) δ 6.85 (2H, s), 3.79 (3H, s), 3.74 (3H, s), 3.72-3.69 (1H, m) , 3.05-2.85 (1H, m), 2.67-2.50 (2H, m), 1.32 (18H, s),0.93 (3H, d, J = 7); Anal. Calcd for C22H35NO5: C, 67.15; H, 8.96; N, 3.56. Found: C, 67.20; H, 9.20; N, 3.70. (249). A solution of (2S, 3S)-5-[2,6-di-t-butyl-4-

MT o

CM V“

OO methoxyphenyl]3-methylglutamate (248) (8.0g, 20.3mmol) 20 in 5N HCl (200ml) was heated at reflux for 2h. The reaction mixture was evaporated to dryness under i «

reduced pressure. The residue was dissolved in cyclohexane (x4) and evaporated to dryness (x4) which afforded a white solid (7.9g, 93%) : mp 230 °C; r i 2( [a]D +22° (c 0.27, MeOH); IR (KBr) 3423, 2964, 1755, 1593, 1514, 1456, 1421, 1371, 1303, 1259, 1201, 1179, 1138, 1106, 1060, 966, 926, 861, 790, 710; 1H NMR (MeOD) δ6.76 (2H, s), 4.02 (1H, d, J = 3.7), 3.67 (3H, s),3.05-2.85 (1H, m), 2.80-2.55 (2H, m), 1.22 (18H, s), 30 1.09 (3H, d, J = 6.3); 13C NMR (MeOD) δ 174.5, 171.4, 158.6, 145.2, 143.1, 113.2, 58.3, 56.3, 39.8, 36.9,32.5, 16.6; Anal. Calcd for C2iH34C1NO5: C, 60.64; H,8.24; N, 3.37. Found: C, 60.80; H, 8.40; N, 3.40. A? ϊ Ο 1 2 8 Ο - 150 -- (250) Diisopropylethvlamine (4.1ml, 3.04g, 23.5mmol,1.25 equiv) and phthalic anhydride (3.5g, 23.6mmol, 1.25 equiv) were added to a solution of (25, 3S) ~5~ [2, 6--di-t-buryl-4-methoxypnenyl]3-methylglutamate (249) 5 (7.8g, 18.6mmol) in toluene (300ml). and the resulting mixture was heated at reflux for 3 hours. Aftercooling to room temperature, the reaction mixture wasevaporated to dryness and the resulting oil purified byflash chromatography on silica gel (2% methanol in 10 CH2CI2) which afforded the required product as a white foam (8.35g, 87%): [g JD20 -20° (c 1.04, MeOH); Id (KBr) 3480, 2968, 2860 , 1753, 1721, 1594, 1462, 1422, 1388, 1303, 1263, 1216 , 1183, 1148, 1062, 1003, 933, 899, 755, 723; *H NMR (CDCI3) δ 7.92-7.87 (2H, m?, 7.78-7.7 3 (2H, m) , 6.8 4 (2H, s), 4.95 (1H, d), 3.78 (3K, s), 3.30-3.05 (2H , m), 2.85-2.65 (1H, m), 1.30 (18H, s), 1.13 (3H, d) txj u

CO ,n ΑΡ ε ο 1 2 8 ο - 151 -

(250) (251)

(251). A solution of the amino acid (250) (1.2g, 2.35mmol) in dry diethylether (10ml) was treated with APz;7 9 8,0 1 29 4 10 phosphorus pentachloride (0.52g, 2.5mmol) at room temperature for 2h. The mixture was concentrated andtreated several times with toluene and again evaporatedto dryness. The resulting acid chloride was dissolved APV 0 1 2 8 0 - 152 - in dry THF (5ml) and C'H?Cl2 ί) and cooled to 0 °C.t-Butyl-1- (benzyloxycarbonyi)-hexahydro-3-pyridazine-carboxylate (0.753g, 2.35mmol, 1 equiv) and N-ethylmorpholine (3ml) were added to the solution.. Thereaction mixture was stirred for 30min at 0 °C and. thenovernight at room temperature. The mixture wasevaporated and the resulting residue taken up withCH2CI2 (30ml). The solution was washed with 1M HC1,water, 10% NaHCO3, dried (MgSO4) and evaporated. Theresulting white foam was purified on silica gel (0—2%methanol in CH2CI2) which afforded the requiredcompound 251 as a pale yellow glassy solid (740mg. 39%) : (ct]D20 -22 (c 0.12, Me OH) ; IR (KBr) 3441, 2966, 1725, 1693, 1386, 1255, 1221, 1186, 1154, 1123, 1063,721; XH NMR (CDC13) 6 7,94-7.89 (4H, m), 7.56-7.28 (5H,mi, 6.84 (2H, 2s), 5.29-5.20 (2H, A3), 4.91-4.81 (1H,m) , 1.05-3.88 (1H, m) , 3.78 (3H, s), 3.75-3.80 ?1H, m) ,3.28-2.95 (2H, m), 2.23-1.51 (6H, m) , 1.45 (9H, si, 1.31 (9H, s) , 1.28 (9H, s), 1.27 (3H, d) . (254). A solution of the protected acid (251) (715mg, 0.893mmol) in acetonitrile was treated with Cerium (IV)ammonium nitrate (1.8g, 3.3mmol, 3.7 equiv) in water(3ml) for 4h at room temperature. Mannitol (600mg,3.3mmol, 3.7 equiv) was added and the mixture wasstirred for Ih. Diethylether (50ml) and water (30ml)were added to the mixture. After decantation, the aq.phase was extracted with diethylether (4 x 50ml). Thecombined organic phase was washed with water, dried T·. _ and concentrated. Chromatography on silica gel (10% methanol in CKpCI^) afforded 5-(1- cyearbonyl-3-%-butoxycarbonyl-hex ahydropyrida2in-2-yr 5 carbonyl-3-methyl-4-phthalimidopentanoic acid (252) (360mg, 64%): bh!n~v 0.118, MeOH). This product was used without ΑΡ ε ο 1 2 8 ο - 153 - further purification (360mg, 0.609mmcl), and was hydrogenated in methanol (30ml) using 10% Pd/carbon (36mg) for 3h. The reaction mixture was filtered and the resulting solution concentrated to afford the amine20 5 (253) as a foam (270mg, 96%) [Oi]D -56.1 (c 0.18

MeOH). The amine (253) was dissolved in dry THF (10ml)and phosphorous pentachloride (305mg, 1.47mmol, 2.5equiv) was added. The mixture was then cooled to -5 °Cand N-ethylmorpholine was added under nitrogen. The 10 reaction mixture was stirred overnight at room temperature. The mixture was concentrated and theresidue taken up with CH2Cl2 (20ml), cold H2O (20ml), 1MHC1 (20ml). After decantation, the aq. phase wasreextracted with CH2C12 (2 x 20ml). The combined 15 organic phase was washed with 10% NaHCC>3 and water, dried (MgSO4) and concentrated. The resulting oil waspurified on silica gel (1% methanol in CH2C12)affording the bicyclic compound (254) as a solid (65mg,25%): [P]D2° -77 (c 0.208, MeOH); IR (KBr) 3471, 3434, 20 2975, 2928, 1767, 1723, 1443, 1389, 1284, 1243, 1151, 1112, 720; 1H NMR (CDC13) δ 7.94-7.69 (4H, m) , 5.34-5.27 (1H, m), 4.89-4.66 (2H, m) , 3.94-3.64 (2H, m),3.02-2.84 (1H, m), 2.34-2.19 (2H, m), 1.94-1.61 (3H,m) , 1.47 (9H, s) , 1.14 (3H, d); Anal. Calcd for 25 C23H27N3O6: C, 62.57; H, 6.17; N, 9.52. Found: C, 62.60; H, 6.40; N, 9.10. (255). A solution of the bicyclic compound (254) (70mg, 0.16mmol) in ethanol was treated with hydrazinehydrate (0.02ml, 4mmol, 2.5 equiv). After 5h stirring 30 at room temperature, the mixture was concentrated andthe resulting residue taken up in toluene andreevaporated. The residue was treated with 2M aceticacid (2ml) for 16h. The resulting precipitate wasfiltered and washed with 2M acetic acid (10ml). The AP/.'7 98.01294 AP e ο 1 2 8 ο - 154 - filtrate was basified with solid NaHCO3 and thenextracted with EtOAc. The organic solution was washedwith water, dried (MgSO4) and concentrated.

Purification by flash chromatography on silica gel (2% 5 methanol in CH2C12) afforded the free amine as a foam (Sumg, 100%)- The amine (50mg, 0.16mmol) was dissolvedin dioxane (1ml) and water (0.25ml) and treated withNaHCO3 (C.034g, 0.04mmol) followed by benzoylchloride(0.047ml, 0.40mmol, 2.8 equiv). The mixture was 10 stirred overnight at room temperature, then dilutedwith EtOAc (15ml). The organic solution was washedwith 10% NaHCO3 and sat. aq. NaCl, dried (MgSO4) andconcentrated. Purification by flash chromatography onsilica gel (2% methancl in CH2C12) afforded the 15 benzamide 255 as a foam (67mg, 100%): NMR (CDC13) δ 7.89-7.39 (5H, m), 6,79 (1H, d), 5.32-5.20 (1H, m) ,4.98-4.82 (IK, m) , 4,.75-4.64 (1H, m) , 3.84-3.65 (1H,m) , 3.OS-2.89 (1H, m), 2.45-2.18 (2H, m), 2.00-1.61(4H, m), 1.48 (9H, s) , 1.28 (3H, d) . 20 (257). A solution of t-butyl ester 255 (67mg, 0.16mmol) in CH2C12 (1ml) was treated at 0 °C withtrifluoroacetic acid (1ml). The resulting solution wasstirred at 0 °C for Irmin and then at room temperaturefor lh. The solution was concentrated, the residue 25 taken up in dry CH2C12 (2 x 2ml) and the mixture againconcentrated (x2) . The residue was crystallized fromdiethylether. Filtration of the precipitate affordedthe free acid of 255 as a grey solid (40mg, 70%). Asolution of acid (40mcj, O.llmmol), N-allyloxycarbonvl- 30 4-amino-5-benzyloxy-2-oxotetrahydrofuran (Chapman, 2, pp. 615-18 (1992) : 39mg, O.'iSmmol, 1.2equiv) and (Ph3P)2PdCl2 (3mg) in a mixtureof dry CH2C12 (lml) and dry DMF (0.2ml) was treateddropwise with η-ΒυβίηΗ (0.089ml, 0.33mmol, 3 equiv), AP V Ο 1 2 8 Ο - 155 -

The resulting solution was stirred at 25 °C for lOminand then 1-hydroxybenzotriazole (36mg, 0.266mmol, 2.4equiv) was added. The mixture was cooled to 0 °C andethyldimethylaminopropyl carbodiimide (31mg, 0.16mmol, 5 1.5equiv) was added. After stirring at 0 °C for lOminand then at room temperature overnight, the mixture wasdiluted with EtOAc (20ml) and the resulting solutionwashed successively with 1M HC1 (2 x 5ml), 10% NaHCO3(2 x 5ml) and sat. aq. NaCl (5ml), dried (MgSO4) and 10 concentrated. Flash chromatography on silica gel (2% J methanol in CH2Cl2) afforded a mixture of diastereoisomers (256) as a grey solid (50mg, 82%).

This product (256) was used without furtherpurification (50mg, 0.091mmol) and was hydrogenated in 15 methanol (5ml) using 10% Pd/carbon (30mg) for 24h. Thereaction mixture was filtered and the resultingsolution concentrated. Flash chromatography on silicagel (2-20% methanol in CH2C12) afforded compound 257(9mg, 21%) as a white solid: 1H NMR (D4-MeOH) δ 7.88- 20 7.29 (5H, m), 5.18-4.99 (1H, m) , 4.59-4.35 (3H, m) , 4.26-4.11 (1H, m) , 3.65-3.41 (2H, m) , 3.18-2.91 (1H,m) , 2.62-1.47 (8H, m), 1.29-1.00 (3H, 2d) (mixture of ) acetal and hemiacetal). MS (ES -) 457.

Compounds 280-283 were prepared from 212b by 25 a method similar to the method used to prepare 226e.Compounds 284-287 were prepared by a method similar tothe method used to prepare 217e.

CM

ΑΡ ν Ο 1 2 3 Ο - 156 -

compound 280 281 282 283 284 285 286 287

M 2 i. 0 : 8 6 Z-/dV AP C Ο 1 2 8 Ο - 157 - ϊ Η

Alloo-Ν^γ CO^i-Bu CO2-i-Bu

Η2Ν—OR

Alloc- Κ.,

OR 306

AP/."; 9 8.01294 (306a) was prepared by a similar procedure as 208aexcept that 2,6-dichlorophenylmethoxyamine (prepared bya similar method as 306b) was used instead of semicarbazide to give 870mg (quant.) as a clear oil. 5 (306b) was prepared by a similar procedure as 208a except that 2-(phenyl) ethoxyamine (US 5 346 911) wasused instead of semicarbazide to give 395mg (quant.) asa clear oil. (307a) was prepared by a procedure similar to 233e10 except 306a was used instead of 207a to give 23 mg(23%) of 307a as a white solid. AP V Ο 1 2 8 0 10 - 158 - (307b) was prepared by a procedure similar to 233eexcept 306b was usee instead of 207a to give 43 mg(48%)of 307b as a white solid. (308a) was prepared by from 307a a procedure simdar tothe preparation of 235e from 234e to give 15.2 mg (74%)as white solid: NER(CD3OD) δ 0.9im), 1,3{s), 1.7 (m), 1.8 (m) , 2.0 (mW, 2.1-2.2(m), 2.3 (dd), 2.4-- 2.6 (m) , 2.7-2.8(m), 3.1(m), 3.3 (m), 3.4-3.5 (m),4.9 (m) , 5.1(ra), 5.3(d), 5.4(s), 6.8(d), 2 2~ 7.8(dd), 8.4 fad). (308b) was prepared by from 307b a procedure similar tothe preparation of 235e from 234e to give 25.2 me (68%)as white solid: 21 NMR(CD30D) δ 1.2 (m) , 1.6-1.7 art,.2.0-2.1(m), 2.2(m), 2.3 (m), 2.5(m), 2.6-2.7(dd), 5 (in) ,5 as) ,5 (m) , 15 2.9(: 0(t), 3.1.(:2, 3.3-3.5(m), 4.2(t) , 4.2 >., 4.5(m), 5.2(t), 5.3(d), 6.7(d), 7.1-7.2(m), 7.35(dd),7.4(m), 7.5 (m) , 7.8(da), 8.3(dd).

Ί 8 z 0 · 8 $ ,'2/dV - 159 - Ο Ο

5 (304a) R=CH3 (302) .

Step A: 301 was prepared by procedure similar to 605a(Step A), except 212e was used instead of 603a to give540 mg (34%) to give a white solid. 10 Step B: 302. A solution of 301 (50.7 mg; 0.091 mmol)in 2.8 ml of MeOH/HOAc/37% aq. formaldehyde (5:1:1) wasstirred at rt for 5.5 h. and the reaction was concentrated to 0.7 ml in vacuo. The residue wasdissolved in 3 ml of CH3CN and concentrated to 0.7 ml 15 (3x), dissolved in toluene and concentrated to 0.7 ml in vacuo (2x) , and concentrated to dryness.Chromatography (flash, SiO2, 5% isopropanol/CH2Cl2) gave302 (45.5 mg, 78%) as a white solid: 1H NMR(DMSO-dg) δ *621686 /J/d* AP V ο 1 2 8 - 160 - 1 . 0-1.15(m. 2H) , 1.4is , 9H) , 1.65( m, 2H), 1.9- -2.2 ? m, 2Kh 2.15--2 . 4 (m, 3H) , 2.55( m, 1H), 2.7-3.0(m, 2H1 , 4.3 4, hip 2R) , 4.9(m, Uh ,. 5.2 (m, 1H) , 7.4-7.6(m, 2R) , "d 8-3.0 (m, 2H) , 8.6 (in, 1H) , 8.8 (m, 1H), 9.4(5, 1H) . (304a). Step A: A solution of 302 (90 mg; 0.18 mmol) in 10 ml of MeOH was treated wish trimethylorthoforisste (Imi)and p-toluene sulfonic acid hydrate (5 mg; 0.026 mmol) the reaction was stirred for 20 h. The rear: s i on created with 3 ml of aq. sat . NaHCO3 and entrated in vacuo. The residue was taken up ο n ,c and washed with dilute aq. NaHCO3, dried . ver a ana concentrated in vacuo to give 80 mg o f 303a.

Step B: 303a was dissolved in 2 mi of TEA and stirredat rt for 15 min. The reaction was dissolved in CH3C12and concentrated in vacuo (3x). Chromatography (flash,SiCh, 1% co 3% MeOH/CK2Cl2 gave 43 mg (64%) of 304a as awhite solid: 1HNMR(CDC13) δ 1.55-1.8(m, 2H) , 1,.9- 15 (m, 4H) , 2.2 5-2 ,. - 2H) , 2.7-3.3 (m, 4H) 6 ) s, s , 3H) , 4.4, 4.75(2m, 1H), 4.6(m, 1H) 4 it, d. 'LH) , 5.1-5. 1H), 6.45, 7.05(2d, 9 5 (m, IK) , 7.45(m, 2H), 7.5(m, 1H), 7.85(m

♦βΖ10/86 /J/dV

Example......11

Compounds 214e, 404-413, 415-445, 446-468,470-491, and 493-499 were synthesized as described inExamole 11 and Table 7h ΑΡ ν Ο 1 2 3 ο - 161 -

ο - 162 -

Step A. Synthesis of 401. TentaGel s'" NH-> resin(0,16 inmol/g, 10.0 g< was placed in a sintered glassfunnel and washed with DMF (3 x 50 mL), 10% (v/v) DIEAin DMF (2 x 50 mL) and finally with DMF (4 x 50 mL).Sufficient DMF was added to the resin to obtain aslurry followed by 400 (1.42 g, 2.4 mmol, prepared from(353 -3-Cfluorenylmethyloxycarbonyl)-4-oxobutryrc acidc-butyl ester according to A.M. Murphy et. al. 5. Ain.Chert, See., 114, 3156-3157 (1992)), 1- hydroxybenzotriazoie hydrate (HQBT-H2O; 0.367 g, 2,4 mmol) , O-benzotriazcI......l-yl-77, N, 77, 77' -tetramethyiaronium hexafluorophosphate (KBTU; 0.91 g, 2.4 mmol), and DIEA(0.55 siL, 3.2'mmol) . The reaction mixture was agitatedovernight at rt using a wrist arm shaker. The resinwas isolated on a sintered glass funnel by suctionfiltration and washed with DMF (3 x 50 mL). Unreactedamine groups were then capped by reacting the resinwith 20% (v/v) Ac2O/DMB" (2 x 25 mL) directly in thefunnel (10 min/wash). The resin was washed with DMF (3x 50 mL) and CH2C12 (3 x 50 mL) prior to dryingovernight in vacuo to yield 401 (11.0 g, quantitativeyield).

Step B. Synthesis of 402. Resin 401 ¢6.0 g, 0.16mmol/g, 0.96 mmol) was swelled in a sintered glassfunnel by washing with DMF (3 x 25 mL). The Fmocprotecting group was then cleaved with 25% (v/v)piperidine/DMF (25 mh) for 10 min (intermittentstirring) and then for 20 min with fresh piperreagent (25 mi.) . The resin was then washed with DMF (3x. 25 ml) , followed by 77-methypyrrolidone (2 x 25 rth) .After transferring the resin to a 100 mL flask,methypyrrolidone was added to obtain a slurry followedby 212f (0.725 g, 1,57 mmol), ΗΟΒΤ·Η2Ο (0.25 g, 1.6nmol), KBTU (0.61 g, 1.6 mmol) and DIEA (0.84 nh, 4.8 AP/r; 9 8 2 i 4 - 163 - mmol). The reaction mixture was agitated overnight atrt using a wrist arm shaker. The resin work-up andcapping with 20% (v/v) Ac2O in DMF were performed asdescribed for 401 to yield 402 (6.21 g, quantitative 5 yield).

Step C. Synthesis of 403. This compound wasprepared from resin 402 (0.24 g, 0.038 mmol) using anAdvanced ChemTech 396 Multiple Peptide synthesizer.

The automated cycles consisted of a resin wash with DMF 10 (3x1 mL), deprotection with 25% (v/v) piperidine in DMF (1 mL) for 3 min followed by fresh reagent (1 mL)for 10 min to yield resin 403. The resin was washedwith DMF (3x1 mL) and M-methypyrrolidone (3x1 mL).

Step D. Method 1. (409). Resin 403 was acylated 15 with a solution of 0.4M thiophene-3-carboxylic acid and0.4M HOBT in A7-methypyrrolidone (1 mL) , a solution of0.4M HBTU in N-methylpyrrolidone (0.5 mL) and asolution of 1.6M DIEA in AT-methypyrrolidone (0.35 mL)and the reaction was shaken for 2 hr at rt. The 20 acylation step was repeated. Finally, the resin was washed with DMF (3x1 mL), CH2Cl2 (3x1 mL) and driedin vacuo. The aldehyde was cleaved from the resin andglobally deprotected by treatment with 95% TFA/5% H2O(v/v, 1.5 mL) for 30 min at rt. After washing the 25 resin with cleavage reagent (1 mL), the combined filtrates were added to cold 1:1 Et2O:pentane (12 Ml)and the resulting precipitate was isolated bycentrifugation and decantation. The resulting pelletwas dissolved in 10% CK3CN/90% H2O/0.1% TFA (15 mL) and 30 lyophilized to obtain crude 409 as a white powder. Thecompound was purified by semi-prep RP-HPLC with aRainin Microsorb™ CIS column (5 μ, 21.4 x 250 mm)eluting with a linear CK3CN gradient (5% - 45%)containing 0.1% TFA (v/v) over 45 min at 12 mL/min.

AP C Ο 12 8 Ο - 164 -

Fractions containing the desired product were pooledand lyophilized to provide 409 (10.8 mg, 63%).

Step D. Method 1A. Synthesis of 418. Following asimilar procedure as method 1, resin 403 was acylated 5 with 4-(l-fluorenylmethoxycarbonylamino)benzoic acidand repeated. The fncrc group was removed as describedin Step C and the tree amine was acetylated with 20%(v/v) Ac2O in DMF (1 mL) and 1.6M DIEA in N- methylpyrrolidone (0.35 mL) for 2 hr at rt. The10 acetylation step was repeated. Cleavage of the aldehyde from the resin gave 418 (3.2 mg).

Step D. Method IB. Synthesis of 447. Following 2 similar procedure at method 1A, resin 403 wasacylated with 0.4M 4-(1- 15 fluorenylmethoxycarbonylamino)benzoic acid. The acylation step was repeated once. The Fmoc group wasremoved as before and the free amine was reacted with1M methanesulfonyl chloride in CH2Ci2 (0.5 mL) and 1Mpyridine in CH2C12 (0.60 mL) for 4 hr at rt. Cleavage 20 of the aldehyde from the resin gave 447 (10.0 mg?.

Step D. Method 2. Synthesis of 214e. Following a similar procedure as method 1, resin 403 was acylatedwi?.h 0.5M benzoyl chloride in N-methypyrrolidone (1 mL)and 1.6M DIEA in W-methypyrrolidone (0.35 mL) for 2 hr 25 at rt. The acylation step was repeated. Cleavage ofthe aldehyde from the resin gave 214e (5.1 mg, 30%).

Step D. Method 3. Synthesis of 427. Following asimilar procedure as method 1, resin 403 was reactedwith 1.0M benzenesuifonyl chloride in CH?C12 (0.5 mL) 30 and 1M pyridine in . (0.60 mL) for 4 hr at rt.

The reaction was repea ted. Cleavage of the aid·- m-from the resin aave 427 (7.2 ma, 40%). AP C Ο 1 2 8 0 - 165 -

Step D. Method 4. Synthesis of 420. Following asimilar procedure as method 1, resin 403 was reactedwith 0.5M methylisocyanate in N-methypyrrolidone (1mL) and 1.6M DIEA in W-methypyrrolidone (0.35 mL) for 2 5 hr at rt. The reaction was repeated. Cleavage of thealdehyde from the resin gave 420 (8.3 mg, 55%).

Step D. Method 5. Synthesis of 445. Following asimilar procedure at method 1, resin 403 was acylatedwith 0.27M imidazole-2-carboxylic acid (1 mL) in 2:1. 10 DMF:H2O (with 1 eq. DIEA) and 1M 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC) in 2:1 N-methypyrrolidone/H20 (0.35 mL) for 3 hrat rt. Cleavage of the aldehyde from the resin gave445 (9.5 mg). 15 Analytical HPLC methods: (1) Waters DeltaPak 08, 300A (5μ, 3.9 x 150 mm).Linear CH3CN gradient (5% - 45%) containing 0.1% TFA(v/v) over 14 min at 1 mL/min. (2) Waters DeltaPak C18, 300A (5μ, 3.9 x 150 mm).

20 Linear CH3CN gradient (0% - 25%) containing 0.1% TFA (v/v) over 14 min at 1 mL/min. (3) Waters DeltaPak 08, 300A (5μ, 3.9 x 150 mm).Isocratic elution with 0.1% TFA/water (v/v) at 1mL/min. 25 (4) Waters DeltaPak 08, 300A (5μ, 3.9 x 150 mm).

Linear CH3CN gradient (0% - 30%) containing 0.1% TFA(v/v) over 14 min at 1 mL/min. (5) Waters DeltaPak 08, 300A (5μ, 3.9 x 150 mm).

Linear CH3CN gradient (0% - 35%) containing 0.1% TFA 30 (v/v) over 14 min at 1 mL/min. ΑΡ/Γ7 9 8 ; )1 1 28 4 AP i 012 3 Ο - 166

Table

ΑΡ/Γ/ 9 8 / a 1 2 β 4 AP C 012 8 0 - 167 -

ΑΡ/Γ/9 8/S 1 28 4 AP vΟ 1 2 8 Ο 168 -

ΑΡ/Γ7 9 8 / Δ 1 2 8 4 ΑΡ Ο Ο 12 8 0 - 169 -

Syn. Method T—} r—j i—1 + <D σ σ o 68 W X S +s *vT <sT OS r—t C\] r—1 PLC min o\o r~ ra 21 98? s “ X • • • co r- o r—1 lO s • • • S to CO co sr sT ST Γ* σι o o o x X X LO sr X X X co CsJ u o U X OH H tructure O X °-w° O X °<>o Q-s i r° osbr °=\ ω h Ό CJ tn in X ϊ-1 r“l r-H G U ΑΡ/.”/ 9 8 . Μ 1 2 9 4 ΑΡ ί Ο 1 2 8 Ο - 170 -

ΑΡ/Γ/ δ 8 ί Δ 1 2 9 4 .) AP C012 8 0 - 171 -

Syn. Method CM CO MS (M+H) + 399 398 433 OS CM CM CM Γ ................ HPLC min o\- CO s 5.25 98? m ro Γ" s s 398.38 397.39 432.46 tc-J 2 C16H22N4O8 C16H23N5O7 C16H24N4O8S Structure X ~ O j-. o=^ )= a o - - ---”*2X o=< o / X O X °=Q° °<x o=< ZX / *T" O X °=q° ο-ξ8 0-^0° Ο·ώ·Ο k Cmpd. 419 420 421 AP/.",' 9 8 i Jl 1 2 9 4 AP C 0 1 2 3 0 io *·' φ - r—! r~i Γ-- o co ‘c-’ r-«. co m '—i £ m r£ r-< 1—f rH H-i £ te ..,, or* CM £ .28 450 ( Γ) ^-- « J ^r., CM i-J-« CD CO cr, «· CM r-i * . * λΑι Ολ O if) C.0 CO 00 z>, o tO <<^Jf fcj £ S CM CM CM .£ K σϊ lO r j £ u u Φ 0° 0X=< \=0 "φν-ζ χ''·''· δχ 0 =^ >= O o=T £ tructur /—~~·\ 2XC < >—c O 2 -2 no 0 ,u ?ζχ —11 ί ϊ Cl IN \ 0 j \ zO.'.J J o ££"§=- vX Ο 'Z.-'Z. r'· ^·=ύ γ *" o=t· \ ,£“~ LO o=< \ CM "sT m 1 Q- CM CM CM CM 0 ’t? _ ΑΡ/Γ7 8 8 / fl I Z 8 4 AP e012 8 0 - 173 -

Syn. Method CsJ ro n + MS (M+H) <—I 1-0 481 CO *3* OS r—4 i—1 rH PLC min CO ο σ\ Γ- fO 00 a: 00 r- in o o LO to 2 o o r* ’sT Uj 2 C21H30N4O7 C20H24N4O8S C16H25N5O8S tructure 2 O X °^o °<χ δ 2 °=v^° °\!r° 2 O 2 °=Q° °cc ω °ί-, O-cb-O °<r o ό °T Ό VO r- co a CM CM CM u AP/."/ 98/81284 AP V Ο 1 2 8 Ο 174 - > 1 i L J ύ <’ t fc,-~ 1 «·- r-f -—— - O '"~i to «ς- rv; i.n to £Xi .......: C\l c*C' S.' -,— »—4 C' XJ σι r,,.. co , 66 98' ~J σϊ γ-ί t-·'· to lO ,, ςΓ 5? to x; r„. σ lO lO CO 1 t0 ") ό 0 S' lO »x« s i“ lO co •xT :x X r—1 3 Γ ) CM O CM 1 O : 1 J i f 51 r~! δ κ °Q° r~\ ZX ( >< y·^ o^. \κ»ς z—v zi { h··^ H *£“«£ <«sO x..^4> 'g.'JL 5x 0=^^=0 r-\ CZ , Q“*O °V° 0=/ o=< >0 cy-C z z n'-'1 XX o=< to V 1 -- ’>--—\ ^J/ \ Q z. w / iz ,zr "Z. j CXi O r—1 CM sr xr *3* i

CM

QQ Ο» I . a < AP V Ο 1 2 8 Ο - 175 -

Syn. Method 1—i γ“Η γΗ -- co LO kO CO σι co σ σ + s *?Γ Η — χ — τ~4 ^τ PLC mir Γ* >ο σι m 03 Ζ2 σ'» 10 98? 20 98? X γΗ τ—Η •ςτ LD CO rH Γ— ο ο LO •χΓ LO LD s • σ CO σ σ ’sT «χΤ Γ~- Γ~- r- Γ*· ο ο ο ο 2 2 2 2 s CM CM CM CM X X X X CM CM CM CM υ υ ο υ tructure I Ο X ο=^^=ο ο 2·^ο° ο=< 4> 0 X Ο X 0=^^^=0 0^0° ^2Χ O=Q° ο-ζ1 Ο^'^γΟ0 V'-"^2x °=<_ ω Ο=< /=\ Cz W //ν_/ t=2 Ό m in co a. η σι σι σι β •q· *3* ο AP/." 9 8 / Δ 1 2 9 4 AP101280 tii ---------------------- tV 1 S j .... r—i rH to χ CO o CM σι r-4 £ M; lO __, --------_._η H Cu lu ιΩ u *"*4 £ CO CO c- cn CO jo; cr. o\o ~ CO £ O' i Ό 20 σ> 1Cn co σ\ | CM o ! CM 5 iCt U ) LO to »?> ,.’ • CM X σ, Ο Lf) r- r- i O o o ο a X s iX4 !.j'' ID ,.,2.4 CN •0-,· CM CM lO Uf) a co CM CM u o As X O X 5 2 a) Ui Q=< )= o ill ζ ( >*£ Γ> r>O o=< \=o W( SO-C O 2-2 qO n< ο=ζ_><) ο-ίτ Ο. 2-2 pa-’ | -Struct ---V <O ' Ho H X O t / O==C // M £""0 \_=a n xx no VT XX O=^ x-^ VC aSO-2, o=z <5 O [ I 1 CH f'·· σ- σ> i O j 0., D cn m •g* sr *3· ? s ΑΡ/Γ/ 9 8 i ΰ 1 2 9 4 ΑΡ ν Ο 1 2 8 Ο - 177 -

Syn. Method t-) r-H r-f + W X CO ΓΟ r*· o Γ- ω 2 ±2 lD lD Lf) Η O£ t-H 1-1 t-H u S "" c?P o\o 0\3 x e X ld co rH c-H ΟΛ «Ή 0Λ X co o co co <O Lf) IO tO Lf) S . . • 2 Γ- kO MO LO lD to CM o Γ*- o Cxj 2 2 2 ΓΌ 04 CM re CM X Γ" X CM u 1-4 CM u I o x X δ x O X °M_/° o-c °v° ure n cP o a rX= r> 2-2 rP truct XX VV MJ «^MJ o=< co \ // \\ o=< 'y—\ /^y=/ o o X- / Sir Ό CM co e <3* <3· u ΑΡ/.”,'S 8 /1 29 4 AP e Ο ί 2 8 Ο 178 -

ΑΡ/Γ/ 98/91294 AF V ΰ 1 2 s Ο - 179 -

Syn. Method CQ r—i <r—i T-1 CO X CO co r—t rH 2 + 2 n in in H X 1“”! r-H rH PLC mil] CO s c\° IO £ kO COco σϊ X • • co kO lO CO *“f lO m m s . . • 2 r* lO o LO ID m [X4 fN5O9S CO O lO 2 σ\ CO O sr £ kO 2 CM CM CM U C24H2 C25H2 Structure *T δ i °V° 51 °=VO Sx °-w° vX o o-£ °O° 2Σ o=c O 2-2 QO ο=ς o Wo„ o z~£ \ . og-07· o >Λ Ό Γ" co σι a *3* M· ST £ *0· U

001280 • 180 -

c\i

Oj

'"•'W co 1 «

(X AF C Ο 12 8 Ο - 181 -

Syn. Method CM CM CM + MS (M+H) 501 M) σι r- (M+Na 483 — OS CM r~) PLC mir c*p t-H CO t-1 O'» CAC £ “ _ CO X t-i r—1 l£> σ> Lf) co σι m IO ς? co s • • • • 2 o LO co o lD sr ω r- r* O co O o o X i—1 X Uj co u s CM CM X co CM CM K CO o X rH CM U u CM O u X O X X δ x o °=ζ^=° Z—X ZZ U X CV= O=W=° Z7*T M 3 o 0^0° /'W; Γκ.: truct vX o=< °<X °O° 21 °=< ΙΌ o=$_ °=ς § o €>c S Ό m ST in kD a in to in in c U ΗΡΰί 9 8 iΰ ί 29 4 18: >«* ω Method rM rH t—1 IS fo r—H co co o r—i co X U-! J, IO CO Cd e· 'xT £± — ,... — sx “ ,o — u CM CO .-, co r—i CO O.j *3“ CM >o r-M Cd o 9 O r-M .............. r-4 Ό re CO uQ c? s o <0 i~O 'S’ o o 407 o S Z r. z CM CX4 CM |Z_| *£* H2 8 22c C28 Hi-. rH CM u C21H ΣΞΞΖ dj^n X o X 0==(^^=0 0 XX n 5x O=^^:O UJ Λ, J" Ο X-X oU w 0'·=/ o 21 0=/ XX O=< Q y~x Q W O o- C o >< Lu LL □ i r* GO Cd ££.. m ΙΠ If) 5; •g· ΑΡ/Γ7 9 8 i ,0 1 2 9 4 AP C Ο 1 2 8 0 - 192 -

Syn . Method CM r“d r—H CM rH + r-1 CO — CO ω x2 + 5—I Γ" Π3 Z o 2 I ) Lf) Lf) s 10 H X 5—I t—1 ί-H IPLC min .13 98? . 89 98? .27 98? CO X to CM o in m lO s • . • 2 r- m m m CO CO ΟΊ o o o 2 2 2 U-i f—( CO Γ* X X X LO m m CM CM CM u u u 5x O=<^>=o o 2-2 O° Sx 5x °=w=° O-o 2 4-J u □ \JT ΖΓ °=\ o=3\ o=< Sh 4-J o w \=/ ΖΣ 22 °=^2 % £ < o 2 Ό 00 σϊ o a 00 00 0) e u

6 2 1<S ' θ 6 /2W AP 0 Ο 1 2 8 0 r Syn. ! f o ρ-Ή r—i i—( Ό co CO CO H m C RT O'·'·' O\O rH HPL 6 cn 0-. Lf> ’«cr lO 462. CO ‘0 *sT tO O', J-—. b r- O 2 X g 3 co X CO X rH O υ u 5 X O X Γ'—/ \— o ( Wo ·ίλπ&amp; *"« V»** /“> /*>, M-' O k\/i 2Ξ 5i O =\^>= O _ 'U ~/^O 1 ; <£b- -SS* -J5· -r 0 x o=Mc=o rvC °cr° o °< °< ΛΛ o 3=./ 0 >=- <Λ ϋ''θ rc _L T; O rH co <5* αι

CM <3|

CO οι f i « <£ AP C Ο 1 2 8 0 - 184 -

Syn. Method t—< rH + m σι CO X co £ + rH σ> *sT H — oS τ—1 rH PLC mir co s σ> colo σ» σ kO u’ ίΕ Γ"~ r—4 r-d σι CM s £ CM co CM ’g’ m r- O N4O7 r* O txj £ H23FN 22C12 r-H u Lf) CM ffi CM CM U C21 C21H X X O I O X 5 ΣΕ °^° °^=° aaL. °=<^=O CD ο-ξ1 °*s°° -P u 2 o 22 o vr °ϋ° zx zx co o=< O=S-x M w XD Ll. o o Ό n m e <3* u ΑΡ/Γ7 9 8 / fl 1 2 9 185 -

ΑΡ/Γ/ 9 8 / fl 1 2 8 4 AP C Ο 12 3 Ο - 186 -

Syn . Method rH T—( MS (Ml Η) i 489.5 488.2 r- ’χΓ HPLC RT min 4.57 (1) 98% 5.74 (1) 98% r—1 o'° o “ § ST S 487.52 487.52 r-H r- [xj C23H29N5O7 C23H29N5O7 C22H25N5O7 Structure X O X sv° °vr° XX o=< x" X 5i °Cr° °s Z-CJ I X 5 x °=Q=° O° XX τ Cmpd. 470 471 472 ΑΡ ν ΰ12 8 ο -- 187 -

Syn. Method r—( -»· CD r~H X fO CO m ^.,L, O co CO — Li'} Op *^r Η „ cC ·_. I—i o k-A a. ’d CO t',·' σ> . z- CO £ s ►-A·-· r- r cr, σ. fT’ σ> ^r 5· CN ',.0 CO ς"*'; co co '-Γ σ·ι co c o o X Li'iC s *ςΜ VO CN Lf) C M CM t ) ( ) U A X X ό X O X 0--=(^^^=0 ,. — τ?·"“ °=w=° Structure M „ Zl-r" o=<3 co <z Yu -- VO Q «Ζ Q yzz. '—S b O 2-Z ou zx O=K. 5 ft \ Yb >o X) o o \X rl /V', vr m c *5* *3· ΑΡ/Γ/ &amp; 8 Ζΰ 1 29 4 PP V Ο 1 2 3 ο - 188 -

ΑΡ/Γϊ 9 8 ί ΰ 1 2 9 4 AP V Ο 1 2 8 Ο 13 9 ~

Syn , Method 1 <ζ t—I 4._ CO r~; f—I co 5 lO Γ”1 CO if) m σ» ............ if) *cr V ,-4 cC CO r-1 "" cC CO tx XC :...« 0 <* O; Γ" rt cn 2 0 CO co L,o Q.’< σ>, r·*- ό 0 z ‘00 o u CM cm CM rH L) Λ — δ x 5 x O X ο=ς^>=ο °\_f ° ,—. 22 ο υ /“J. ό, d^oo^-^o ZE < >~<O Q 2-2 r-y cy~T~ °Cr° ZT »“4 r .-/ °=< υ0 3 o 1 3 fVu vr=y o y=d o o X ......2Γ Z'—ty d. x o 2 X?' ............................. r~-< σ> o T—( co Γ'"' 03 00 fed «3· *CP ·- ο*

CM ο; ο* η α < AP C Ο 1 2 8 0 - 190 -

Syn. Method r—I 1,2 CM r-H + CO <0 CO 2 cn Lf) Γ0 CM kO Lf) Lf) Lf) H _u o', r—i r—1 - £ ε s - r-4 00 ο σ) s ” X σι o rH r~- Γ' lO LO lO s s co CM bO CO kD Lf) cn CM Lf) o LO 2 00 O Lf) co O lO CM<—4 s σ) 2 Γ—( 2 o CO CM CM X CO n 2 LQ r-H CM CM u CM O u ructure X o x °=Q° 0-¾ °v° XX 5 τ °Mf° 0-¾ o XJT XX o=k St °Mf=° °O° XX 4-» °K /y\\ ω )—< \=/ fVo —C XX x—C XX _?—< O X CM °$u„ X o I X Ό CM m *ζΓ a GO CD co u ΑΡ/Γ/ 9 8 / a 1 2 9 4

AP/r; 98/91294 /\p v Ο 1 2 8 0 - 193 -

Syn. Method | r—1 CM + co σι CM co Κ LQ ΡΊ i—1 2 + ΙΟ σ r* £ LO Η OS ί—I i—-1 PLC min . 9 98% 31 98% cKO _ 03 Z κ CM • • r—! co σ kD σ\ S » 2 ST CM ο m σι ο F4O8 407 2 [*4 2 CM X co Ή25 CM w η CM U CM CM O CM u H HO OH H X jcture o=V^-° 0-¾ °\JT ZX o=< 0=^^^=0 0-¾ °ϋ° ο X o-Q=° 0-¾ °Cr° Μ o=/ 2X -U o=( ω w o to t O=$-x o <n> d m w \=y 2 Ό r-H CO α σ> σ σ Cm •O’ ο» αο α < APC Ο 1 28 0 194 -

APt01280 - 195 -

AP/7/9 8/fi 1 29 4 AP v ύ 1 2 8 Ο - 196 -

Example 12

The preparation of compounds 2001, 2002,2100a-e, and 2201 is described below.

2002 2001 (2000). To a solution of t-butyl 9-amino-6,10-dj.oxo-5 1,2,3,4,7,8,9,10-octahydro-6H-pyridazino [ 1, 2-a] 11,2]diazepin?.—1-carboxylate (GB 2, 128, 964; 340nig, 1.15 mmol) in CH-sCl2 was added benzoyl formic acid(260 mg, 1.7 mmol), HOBT (230 mg, 1.7 mmol) and EDC(340 mg, 1.7 mmol). The resulting mixture was stirred. 10 at ambient temperature for 16 hours, poured into IN KC1and extracted with CTE-Cl^. The organic extracts werefurther washed with saturated NaHCCu, dried over MgSO4and concentrated to afford 1999 as a pale yellow solid.The solid was dissolved in CH2CI2 (25 ml) and TEA (25 15 ml) and stirred overnight and concentrated in vacuo togive 560 mg of 2000 as an oil. AP/."/ 9 8 fl 1 2 9 4 AP V Ο 12 8 Ο - 197 - (2001) was synthesized from 2000 by methods similar tocompound 213e to afford 410 mg (63%) of 2001 as a whitesolid: 1H NMR (CDC13; mixture of diastereomers) δ 8.25 (1H, d) , 8.23 (1H, d), 7.78 (1H, dd), 7.65 (1H, bm) , 5 7.50 (2H, m), 7.40-7.25 (4H, m), 6.55 (1H, d) , 5.57 (1H, d), 5.10 (1H, t), 5.05-4.95 (2H, m), 4 .90, (1H, d) , 4.80 (1H, d), 4.72 (1H, bm), 4.65 (1H, m) , 4.55 (1H, m) , 4.45 (1H, t), 3.25 (1H, m), 3.15 ( IK, m) , 3.00 (2H, bm) , 2,90 (1H, dd) , 2.70 (1H, m) , 2.47 (1H , dd), 10 2.45 (1H, (1H, m) , bm) . 2.35 (1H, m) , 2.00-1.75 (4H, m) , 1.60 (2002). Compound 2001 (58.6 mg, 0.10 mmol) was treated with 15 ml of TFA/MeCN/water (1:2:3) and stirred atroom temperature for 6.5 h. The reaction was extracted 15 with ether. The aqueous layer was concentrated withazeotropic removal of the water using MeCN. Theproduct was suspended in CH2Cl2z concentrated in vacuoand precipitated with ether to give 46.8 mg (99%) of 2002 as a white solid: 1H NMR (CD3OD) δ 9.05 (0.25H, 20 d) , 8.15 (1H, d) , 7.68 ( 1H, t), 7.64 ( 0.25H, d), 7.55 (3H, t) , 7.35 (0.5H, m) , 5.22 (1H, t), 4.90 (1H, m) , 4.58 (1H, dd) , 4.50 (1H, m) , 4.28 (1H, bm), 3.45 (1H, m) , 3.10 (1H, bt), 2.68 (1H, ddd), 2.60-2.45 (2H, m) , 2.30 (1H, dd) , 2.15-2.05 (2H, m), 1.90 (2H, bm), 1.68 25 (1H, bm). ΑΡ/Γ/9 8/a 1 28 4 ΑΡ Ο Ο 12 8 Ο 198 -

,-COOEt ^COOiPr OEt 2100d Rl„ >Lt)iPr OiPr ci 214e (101 mg, 0.23 mmol ί 2100b Rl= JL, (2100a). A solution isopropanol (10 ml) was stirred at room temperaturewith a catalytic amount of p-toluenesulfonic acid {10mg). After 75 minutes, the reaction mixture was pouredinto saturated NaHCO-? and extracted with CH2Cl2, Thecombined extracts were dried over Na2SO4 andconcentrated. Flash chromatography (SiC^, CH2C]p toEtOAc) afforded 56 mg (51%) of 2100a as a white solid: “H NMR (C r-.p ί.UL· X 3 r mixture of diastereomers) δ 7.9-7.S 10 (2H, in) , 7 , 6-7 . 5 (1H, in .), 7.5-7.4 (2H, m) , 7.1 (CASH, d '* - V'1 ( 0.5H, d), 6.4 (0.5H,d), 5.6 (0.5H, d), 5.3 (0,5H, s) , 5.2 - 5.1 (1H , m), 4.95 (0.5H, m), 4.75-4.5 (1.5H, m) , 4.3 Ο \ ύ s -Ο.Ρί t), 4.1 (0.5H, m) , 3.98 (0.5H, m)? 3.3-2 .75 { 4H, m), 2.5-2.4 (2H,m), 2.25 (1H, no , Ϊ a 1 — Ί Q s 3H, m) 1.7 5 ··* 1 55 (2H,m). ΑΡ/Γ7 98/81284 (2100b). A solution of 214e (16 mg, 0.036 mmol) .1ethanol (2 ml) was stirred at room temperature wincatalytic amount of ρ - toluenesulfonic acid (2 mg) .After 5 days, the reaction mixture was poured into - 199 - saturated NaHCO3 and extracted with CH2C12. Thecombined extracts were dried over Na2SO4 andconcentrated. Flash chromatography (SiO2, CH2Cl2:EtOAc95:5 v/v) afforded 16 mg (81%) of 2100b as a white 5 solid: 1H NMR (CDC13) d 7 .85-7.74 1 :2H,m) , 7.55-7.38 (3H,m) , 7.04-6.95 (lH,d) , 6.61-6.48 (lH,d) , 5.15-5.08 (lH,m) , 4.63-4.53 (lH,m) , 4.52-4.45 (lH,m) , 4.42-4.35 (lH,m) , 4.15-4.05 (2H,m), 3.74-3.60 (2H,m) , 3.57-3.42 (2H,m), 3.39-3.28 dH,m) , 3.03-2.93 (lH,m) , 2.92-2.82 10 (lH,m) , 2.65-2.52 (2H,m) , 2.42-2.25 (lH,m) , 2.20-1.88 (4H,m) , 1.76-1.50 (2H,m) , 1.35-1.10 (9H,m) (2100c). A solution of 214e ¢165 mg, 0.37 mmol) inmethanol (5 ml) was stirred at room temperature with acatalytic amount of p-toluenesulfonic acid (17.5 mg). 15 After 4 days, the reaction mixture was diluted with

EtOAc and washed with 10% NaHCO3 (3x) and brine. Thecombined extracts were dried over Na2SO4 andconcentrated. Flash chromatography (SiO2, EtOAc)afforded 127 mg (68%) of 2100c as a white solid: 20 NMR (CDC13) δ 7.82 (2H, d), 7 .55- 7.50 (1H, m) , 7.47- •7.43 (2H, m), 7.02 (1H, d), 6.53 (1H, d) , 5.20-5.10 (1H, m) , 4.56-4.50 (1H, m) , 4.45-4.50 (1H each, two m), 3.69 (3H, s), 3.41 (3H, s), 3.43 (3H, s), 3.35-3.25 (1H, m) , 3.06-2.98 (1H, m) , 2.94-2.83 (1H, m), 2.65-2.53 (2H, 25 m), 2.35-2.32 (1H, m), 2.15-2.07 (lH,m), 2.00-1.89 (3H, m), 1.75-1.56 (2H, m) . ΑΡ/«7·β<ΰ,1 29 4 (2100d). A solution of 214e (53 mg, 0.12 mmol) in isopropanol (5 ml) was stirred at 50 °C with acatalytic amount of p-toluenesulfonic acid (5 mg). 30 After 3 days the reaction mixture was poured intosaturated NaHCO3 and extracted with CH2C12. Thecombined extracts were dried over Na2SO4 andconcentrated. Flash chromatography (SiO2, CH2C12:EtOAc AF ί Ο 1 2 8 Ο - 200 - (4:1 to 1:1 ν/ν)) afforded 49 mg (68%) of 2100d as awhite solid: XH NMR (CDC13) δ 7.85 (2H, d) , 7.50-7.43(IK, m), 7.41-7.35 (2R, m) , 7.02 (1H, d), 6.47 (1H, d) , 5.13--5.07 (1H, m) 5.00-4.9 (1H, m) , 4.61-4.55 (2H, m) , 5 4.37-4.30(1H, m) , 3.80-3.70 (1H, m) , 3.90-3.80 5., m) , 3.42-3.35 (1H, m) , 3.03-2.93 (1H, m) , 2.91-2.81 ΠΗ,in) , 2.62-2.50 (2H, m) , 2.38-2.33 (1H, m) , 2.12-2.06(IH.m), 1.97-1.81 (3H, m) , 1.70-1.60 (2H, m) , 1.28-1.05(18H,m). 10 (2100e> was synthesized from 302 via methods used tc synthesize 304a to afford 2100e, except ethanol andtriethylorthoformate were used instead of methanol andtrimethylorthoformate, Chromatography (SiO2, 5*ethanol/CH2Cl2) afforded 92 mg (68%) of a white solid: 15 3'H NMR (CDC13; mixture of diastereomers) δ 7.30-7,80 (2E, m) » 7.60-7.50 (IB, m) , 7.50-7.4 0 (2H, m) , 7,30(0.5H, d) , 7.00 (0,. 5H, d) , 6.50 (0.5H, d) , 5.50 (0.5H,d) , 5.20-5.10 (1.5H, m) , 4.95 (0.5H, m), 4.75-4.65 (C.5H, in), 4.65-4.5G slH, m) , 4.38 (0.05H, t) , 4,00- 20 3.90 (0.5H, m), 3.85-3.75 (0.5H, m) , 3.75-3.65 iO.5H, mi, 3.65-3.55 (0.5H, lit), 3.30-2.70 (4H, m) , 2.50-2.35(2H, nt), 2.30 (1H, c.) , 2.15-1.90 (3H, m) , 1.80-1.60(2K, m) , 1.25-1.20 (3B, two t) . AP/r/9e.fl 1 29 4 - 201 -

Example 13

We obtained the following data for selected compounds of this invention using the methods describedherein (Table 8, see Example 7; Tables 9 and 10, see 5 Examples 1-4). The structures and preparations ofcompounds of this invention are described in Examples23-24.

Table 8 Comparison of Prodrugs for Efficacy inLPS Challenged Mice: Inhibition of IL-Ιβ Production. 10 The percent inhibition of IL-Ιβ production after treatment with a compound of the invention is shown asa function of time after LPS challenge ("-" indicatesthat no value was obtained at that relative time). 15 20 25 30

Time of Compound Administration

214m ; 0 214w j 11 404 ! 55 AP/J7 8 8 ·'fi 4 28 4 91 56 6 412 Ϊ0- 0 ; 11 37 35 ) 10 15 25 AP V Ο 1 2 8 0 - 202 -

Compound ~2h -lh Oh +lh 418 ... - - 64 .·». Ο - 52 - 434 - - 80 0 - 63 450 - 35 - 452 - - - 70 2 8 - 8 9 - 456 .... - 56 41 - 69 - 470 U - 3 6 - 471 u - 34 - 475 0 - 15 - 481 ά ‘ - 0 - 486 19 - 15 - 487 17 2 0 .. 550f 0 - 50 550h i.” 21 - 7 3 550i (-10) - 2 3 550k 36 - 34 - 5501 Q - 3 8 550m 45 - 52 550n 19 - 65 - 550o 19 - 64 550p 30 - 60 2001 64 62 58 55 2001a 10 - 16 i 2002 q - 87 j 2100h 34 - 32 - 2100i 19 7 4 i 2100j 46 41 0 33 2100k 30 50 32 72 , 21001 52 - 28 - 2100m 4 0 4 2 ) 2100n 21 9 64 73 i 2100o 31 44 68 64 : AF/Γ/ββ.'ΰ 1 28 4 30 '«j» - 203 - 5 10 15 20 25 30

Table 9 Data for selected compounds of this invention obtained using the methods described in Examples 1-4.

Compound uv- VisibleKi (nM) Cell PBMCavg. IC50(nM) WholehumanbloodIC50(nM) ClearanceMouse, i . v. ml/min/kg ClearanceRat, i.v.ml/min/kg 213f 3000 213g 2200 213h 1500 213i 1100 213j _ 213k 2000 2131 2000 213m 2500 213o 5000 3300 213p <300 213q <300 213r <300 213v 0.5 1,100 1100 41 23 213x 4500 2500 213y 930 214j 4.2 2500 6000 214k 0.2 500 580 22 2141 6 1900 1100 12 214m 1.5 530 2200 33.4 214w 0.6 620 370 15 246b 30000 >30000 87 280b 13000 280c 10000 86 280d 25000 283b 1750 41 283c 4000 50 283d >8000 10000 308c 3000 308d 3000 i 500 25 1800 1800 i 1 i 501 2.5 1800 1600 ! 505c 1500 ““—-1 i 505d >20000 - i 505f 550 510a j 65 j 200 j 267 510d 1 2300 | >20000 j j ΑΡ/Γ/ίβ/Λ 1 29 4 35 i : uv- Cell PBMC WholehumanbloodIC50(nM) ClearanceMouse,i . v. ml/min/kg Clearance Compound Visible Ki (nM) ί avg, 1C50 (nM) Rat. i.v. ml/min/kg 511c ( 730 >20000 78 4 0 550f i 1100 550h ! i 1800 5501 ( ! 1400 550k ( 3000 5501 ( i 750 550m i 2000 55Qn ! <300 550o 4 50 3000 550p ! | 2900 550q ( ! 7 00 2001a ( 3000 s 21G0f § I 21Q0g ’ i J 2100h 1 ----------------- ' 2000 21001 i ' 21005 ! 30000 i 12000 2100k 520 4 00 0 600 21001 5 7 50 2200 2100m ( 2100n ( 670 770 4000 2100ο 670 1150 1500

We obtained the following data for selected AP/."/ 3 8 i Δ 1 2.9 4 compounds of this invention (Table 10) using the25 methods described herein (see Examples 1-4). The structures and preparations of compounds of thisinvention are described in Examples 23-24.

Table 10

Cmpd. (Fluorescent Assay Kinact ~ - . . PBMC avg. IC50(nM) Whole human blood IC50 (nM) ClearanceMouse,i . v. ml/min/kg ClearanceRa z, ml/min/kg 286 370000 300 1600 a 505b ( 190000 too 2100 161 505e 420000 < oo 1000 - 205 -

Example 14

In vivo acute assay for efficacy as anti-inflammatory agentResults in the Table 11 show that 412f and 412d inhibit IL-Ιβ production in LPS-challenged mice after oral adminstration using ethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/water as

vehicles. The compound was dosed at time of LPS challenge. The protocol is described in Example 7.

Table 11 Inhibition (%) of IL-Ιβ production in LPS-challenged mice.

Compound 10 mg/kgdose 25 mg/kgdose 50 mg/kgdose 412f 17% 25% 32% 412e 5% 17% 61%

Example 15

Mouse Carrageenan Peritoneal Inflammation

Inflammation was induced in mice with anintraperitoneal (IP) injection of 10 mg carrageenan in0.5 ml of saline (Griswold et al., Inflammation, 13,pp. 727-739 (1989)). Drugs are administered by oralgavage in ethanol/PEG/water, β-cyclodextrin, labrosol/water or cremophor/water vehicle. The mice aresacrificed at 4 hours post carrageenan administration,then injected IP with 2 ml of saline containing 5U/mlheparin. After gentle massage of the peritoneum, asmall incision is made, the contents collected andvolume recorded. Samples are kept on ice untilcentrifuged (130 x g, 8 mins at 4 °C) to removecellular material, and the resultant supernatant storedat -20 °C. IL-Ιβ levels in the peritoneal fluid aredetermined by ELISA.

Results in the Table 12 show prodrug 412finhibits IL-Ιβ production in carrageenan-challengedmice after oral adminstration of drug. Compound 214e ΑΡ/Γ, 9 8 fi 1 2 9 4 ΑΡ ν Ο 1 2 8 Ο - 206 - did not inhibit IL-10 production when dosed orally at50 mg/kg.

Table 12 Inhibition (%) of IL-Ιβ production by 412fand 412d in carrageenan-challenged irtice ,

Dose (mg/kg) Compound 412f Compound 412d 1 30% 0 10 54% 32% 25 49% 31% 50 73% 36% 100 75% 53%

Example 16

Type II Collagen-induced Arthritis

Type II collagen-induced arthritis was 15 established in male DBA/1J mice at described Wooley andGeiger (Wooley, P.H., Methods in Enzvmoloav, 162,pp. 361-373 (1988) and Geiger, T., Clinical andExperimental Rheumatology, 11, pp. 515-522 (1993¾ : ,Chick sternum Type II collagen (4 mg/kg in 10 mil acetic 20 acid) was emulsified with an equal volume of Freund'scomplete adjuvant (FCA) by repeated passages (400,·between two 10 ml glass syringes with a gauge 16double-hub needle. Mice were immunized by intradermalinjection (50 μΐ; lOOpl CII per mouse) of collagen 25 emulsion 21 days later at the contra-lateral side ofthe tail base. Drugs were administered twice a day(10, 25 and 50 mg/kg) by oral gavage approximately 7 hapart. Vehicles used included ethanol/PEG/water,β-cyclodextrin, labrosol/water or cremophor/water. 30 Drug treatments were initiated within 2 h of the CIIbooster immunization. Inflammation was scored on a 1to 4 scale of increasing severity on the two front pawsand the scores are added to give the final score.

Results m the Figs. 12 and 13 show prodrugs 35 412f ano' 412d inhibit inflammation in collagen-induced arthritits in mice after oral adminstration. " : n.no AP/.A 9 8 . Λ 1 2 9 4 10 15 20 25 ΑΡ ϊ Ο 1 2 8 0 207 - 214e did not inhibit inflammation when dosed (50 mg/kg)once a day by oral gavage.

Example 17

In vivo bioavailabilitv determination

The drugs (10-100 mg/kg) were dosed orally torats (10 mL/kg) in ethanol/PEG/water, β-cyclodextrin,labrosol/water or cremophor/water. Blood samples weredrawn from the carotid artery at 0.25, 0.50, 1, 1.5, 2,3, 4, 6, and 8 hours after dosing, centrifuged toplasma and stored at -70°C until analysis. Aldehydeconcentrations were determined using an enzymaticassay. Pharmacokinetic analysis of data was performedby non-linear regression using RStrip (MicroMathSoftware, UT). Drug availability values weredetermined as follows: (AUC of drug after oral prodrugdosing/AUC of drug after i.v. dosing of drug)x(dosei.v./dose p.o.) xl00%.

Results in Table 13 show that prodrugs 412f and 412d give significant blood levels of drug and have good drug availability when dosed orally. Blood levels of 214e were not detected when it was dosed orally.

Table 13 Oral Bioavailability of 412f, 412d, and 214ein Rat. ΑΡ/Γ,'9 8 / ΰ 1 29 4

Compound Dose (mg/kg) Cmax (pg/ml) Drug Availability (%) 412f 25 2.4 32 412d 25 2.6 35 214e 45 0.2 0.9%

Example 18 ICE cleaves and activates pro-IGIF30 ICE and ICE homolop expression plasmids A 0.6 kb cDNA encoding full length murineprc-IGIF (H. Okamura et al., Nature, 378, p. 88 (1995)was ligated into the mammalian expression vectorpCDLSRa (Y. Takebe et al., Mol. Cell Biol., 8, p. 466 35 (1988) ) . AP t· (I 1 2 8 0 - 208 -

Generally, plasmids (3 pg) encoding activeICE (above), or the three ICE-related enzymes TX, CPP32, and CMH-1 in the pCDLSRa expression vector(C. Faucheu et al,, EMBO, 14, p. 1914 (1995); Y, Gu 5 et al,, EMBO, 14, p. 1923 (1995); J. A, Lippke et al.,J. Biol. Chem.» 271, p. 1825 (1996)), were transfectedinto subconfluent monolayers of Cos cells in 35~mmdishes using the DEAE-dextran method (Y. Gu et al.,,

1 4, p. 1923 (1995)). Twenty-four hours later,10 cells were lysed and the lysates subjected to SDS-PAGE and immunoblotting using an antiserum specific for IGIF(H, Okamura et al,, Nature, 378, p. 88 (1995).

Polymerase chain reaction was used tointroduce Nde I sites at the 5' and 3' ends of the 15 murine pro-IGIF cDNA using the following primers;GGAATTCCA.TATGGCTGCCATGTCAGAAGAC (forward) andGGTTAACCATATGCTAACTTTGATGTAAGTTAGTGAG (reverse) . Theresulting NdeI fragment was ligated into E. colrexpression vector pET-15B(Novagen) at the Ndel site to

20 create a plasmid that directs the synthesis of apolypeptide of 213 amino acids consisting of a 21-residue peptide (MGSSKHHHHHSSGLVPRGSHM, where LVPRGSrepresents a thrombin cleavage site) fused in-;. tothe N-terminus of pro--IGIF at Ala2, as confirmed by DNA 25 sequencing of the plasmid and by N-terminal sequencingof the expressed proteins. E, coli strain BL21 (1213)carrying the plasmid was induced with 0.8 mM isopropyl--l-thio-β-D-galactopyrsnoside for 1.5 hours at 3'X'C,harvested, and lysed by microfluidization 30 (Mrcrofruidic, Watertown, MA) in Buffer A (20 mM sodiumphosphate, pH 7.0, 300 mM NaCl, 2 mM dithiothreifol, 101 glycerol, 1. mM , ; yimethylsulfonyi fluoride, and2.5 ug/rnl leupeptin! . Lysates were cleared bycentrifugation at 100,000 x g for 30 min. (His.) c- AP/.7 S 8 . a 1 2 8 4 ΑΡ ί Ο 1 2 8 Ο - 209 - tagged pro-IGIF protein was then purified from thesupernatant by Ni-NTA-agarose (Qiagen) chromatographyunder conditions recommended by the manufacturer.

In Vitro pro-IGIF Cleavage Reactions 5 In vitro cleavage reactions (30 μΐ) contained 2 pg of purified pro-IGIF and various concentrations ofthe purified proteases in a buffer containing 20 mMHepes, pH 7.2, 0.1% Triton X-100, 2 mM DTT, 1 mM PMSFand 2.5 pg/ml leupeptin and were incubated for 1 hour 10 at 37'C. Conditions for cleavage by granzyme B were asdescribed previously (Y. Gu et al., J. Biol. Chem., 271, p. 10816 (1996)). Cleavage products were analyzedby SDS-PAGE on 16% gels and Coomassie Blue staining,and were subjected to N-terminal amino acid sequencing 15 using an ABI automated peptide sequencer underconditions recommended by the manufacturer.

Kinetic Parameters of IGIF Cleavage by ICE

The kinetic parameters (kcat/KM, KM, and kcat) for IGIF cleavage by ICE were determined as follows. 35 20 S-methionine-labeled pro-IGIF (3000 cpm, prepared byin vitro transcription and translation using, the TNTT7-coupled reticulocyte lysate system (Promega) andpro-IGIF cDNA in a pSP73 vector as template) wereincubated in reaction mixtures of 60 μΐ containing 0.1 25 to 1 nM recombinant ICE and 190 nM to 12 μΜ of unlabeled pro-IGIF for 8-10 min at 37'C. Cleavageproduct concentrations were determined by SDS-PAGE andPhospholmager analyses. The kinetic parameters werecalculated by nonlinear regression fitting of the rate 30 vs. concentration data to the Michaelis-Menten equationusing the program Enzfitter (Biosoft). IFN-y Induction Assays A.E7 Thl cells (H. Quill and R. H. Schwartz, J. Immunol., 138, p. 3704 (1987)) (1.3 x 105 cells in α»

CM Οζ «

C3O t hP V ύ 1 2 8 0 - 210 -

0,15 ml Click's medium supplemented with 10% F33, 50 μΜ2-mercaptoethanol and 50 units/ml IL-2) in 96-wel.iplates were treated with IGIF for 18-20 hours and theculture supernatant were assayed for IFN-γ by ELISA 5 (Endogen, Cambridge, MA).

Example 19

Processing of pro-IGIF by ICE In Cos Cells

Cos cells were transfected with variousexpression plasmid combinations as described in Example 10 18, Transfected Cos cells (3.5 x 10° cells in a 35-mm dish) were labeled for 7 hours with 1 ml of methionine- free DMEM containing 2,5% normal DMEM, 1%. dialyzed35 ί =. retai bovine serum and 300 pCi/ml S-methiomne > "S-Express Protein Laoe.1 ing-Mix, New England Nuclear.) . 15 Cell lysates (prepared in 20 mM Hepes, pH 7.2, 150 mMNaCl, 0,1% Triton X-100, 5 mM N-ethylmaleimide, 1 aMPMSF, 2,5 pg/nil leupeptine) or conditioned medium wereimmunoprecipitated with an antiIGIF antibody thatrecognises both the precursor and the mature forms of 20 IGIF (H. Okamura et al., Nature, 378, p. 88 (1995)),Immunoprecipitated proteins were analyzed by SDS-PAGE(polyacrylamide gel electrophoresis) and fluorography(Fig. 2A) ,

We also measured the presence of IFN-v 25 inducing activity in the cell lysates and the conditioned media of transfected cells (Fig. 2B).Transfected Cos cells (3.5 x 105 cells in a 35-mm dish)were grown in 1 ml medium for 18 hours. Media washarvested and used at 1:10 final dilution in the IFN-v 30 induction assay (Example 18). Cos cell pellets fromthe same transfection were lysed in 100 μΐ of 20 mMHepes, pH 7.0, by freeze-thawing 3 times. Lysates werecleared by centrifugation as described above and wereused at a 1:10 diluti.cn in the assay.

Ap/i; 9 8 . Λ 1 2 8 4 ΑΡ ε o12 8 ο - 211 -

Example 20

IGIF is a physiological substrate of ICE

Wild type (ICE+/+) and ICE-/- mice wereprimed with heat-inactivated P. acnes, and Kupffer 5 cells were isolated from these mice 7 days after priming and were then challenged with 1 pg/ml LPS for3 hours. The amounts of IGIF in the conditioned mediawere measured by ELISA.

Wild type or ICE-deficient mice were injected10 intraperitoneally with heat-killed p. acnes as described (H. Okamura et al., Infection and Immunity, 63, p. 3966 (1995)). Kupffer cells were prepared sevendays later according to Tsutsui et al. (H. Tsutsui etal., Hepato-Gastroenterol., 39, p. 553 (1992)) except a 15 nycodenz gradient was used instead of metrizamide. For each experiment, Kupffer cells from 2-3 animals were pooled and cultured in RPMI 1640 supplemented with 10% fetal calf serum and 1 pg/ml LPS. Cell lysates and conditioned medium were prepared 3 hours later. 20 Kupffer cells from wild type and ICE-/- mice 35 were metabolically labeled with S-methionine as forCos cells (described above in Example 19) except thatmethionine-free RPMI 1640 was used in place of DMEM.IGIF immunoprecipitation experiments were performed on 25 cell lysates and conditioned media and immuno- precipitates were analyzed by SDS-PAGE and fluorographyas described in Example 18. See Fig. 3.

Example 21

Induction of IFN-y Production In Vivo 30 LPS mixed with 0.5% carboxymethyl cellulose in PBS, pH 7.4, was administered to mice byintraperitoneal injection (30 mg/kg LPS) in a dosevolume of 10 ml/kg. Blood was collected every 3 h for24 h from groups of three ICE-deficient or wild type

CXI o 00 > «a - 212 - mice. Serum IFN-y levels were determined by ELISA(Endogen).

Example 22 IGIF and ΙΓΝ-γ Inhibition Assays

Inhibition oi IGIF processing by ICEinhibitors was measured in ICE inhibition assays asdescribed herein (see Example 1 and Table 14).

Human PBMC Assays

Human buffy coat cells were obtained fromblood donors and peripheral blood mononuclear cells(PBMC) were isolated by centrifugation -in LeukoPreptubes (Becton-Dickinson, Lincoln Park, NJ). PBMC wereadded (3 x 106/welii to 24 well Corning tissue cultureplates and after 1 hr incubation at 37°C, non-adherentceils were removed by gently washing. Adherentmononuclear cells were stimulated with LPS (1 pg/ml)with or without ICE inhibitor in 2 ml RPMI-1640-ΙΰΐFBS. After 16-18 hr incubation at 37°C, IGIF and IFN-ywere quantitated in culture supernatants by ELISA.

For example, we obtained the following datafor compound 412 of this invention using the methodsdescribed herein. The structure of compound 412 isshown below. ΑΡ/Γ/8 8/fi 128 4 compound UV-Visible Fq (nM) Cell PBMCavg. IC50 (nM) 412 Ί * 3 580

Example 23

Compounds of this invention may be prepared via various methods. The following illustrates apreferred method:

To a solution of A (1.1 equivalent) in CH2C12(or DMF, or CH2C12:DMF (1:1)) is added triphenyl-phosphine (0-0.5 equivalent), a nucleophilic scavenger(2-50 equivalents) and tetrakis-triphenylphosphine 5 palladium(O) (0.05-0.1 equivalent) at ambient temperature under inert atmosphere (nitrogen or argon).After 10 minutes, the above reaction mixture isoptionally concentrated, then a solution of acid A-I inCH2C12 (or DMF, or CH2C12:DMF (1:1)) is added followed 10 by addition of HOBT (1.1 equivalent) and EDC (1.1 equivalent). The resulting reaction mixture is allowedto stir at ambient temperature 1 hour-48 hours toprovide coupled product C-I.

Various nucleophilic scavengers may be used 15 in the above process. Merzouk and Guibe, TetrahedronLetters, 33, pp. 477-480 (1992); Guibe and Balavoine,Journal of Organic Chemistry, 52, pp. 4984-4993(1987)). Preferred nucleophilic scavengers that may beused include: dimedone, morpholine, trimethylsilyl 20 dimethylamine and dimethyl barbituric acid. Morepreferred nuclophilic scavengers are trimethylsilyldimethylamine (2-5 equivalents) and dimethyl barbituric(5-50 equivalents). When the nucleophilic scavenger istrimethylsilyl dimethylamine, the above reaction 25 mixture must be concentrated prior to addition of A-I.

Other compounds of this invention may be prepared by hydrolyzing compounds represented by C-I to AP/F/ 9 8 . A 1 2 9 4 AP CO 1 2 3 Ο - 214 - compounds represented by H-I as described in thefc11owiησ scheme:

Π

The hydrolysis may be carried out under variousconditions, provided that the conditions include an 5 acid and H2O. Acids t.nat may be used include

p-toluensulfonic, methanesulfonic acid, sulfuric,perchloric, trifluoroacetic, and hydrochloric. Forexample, trifluoroacetic acid (1-90% by weight) crhydrochloric acid (0.1-30% by weight) in CHgCN/H^-O 10 (1-90% K2O bv weight) at between 0-50 °C may be used.

Example 24

Compounds 213f, 213g, 213h, 213i, 213jf 213k,2131, 213m, 214f, 214g, 214h, 214i, 214j, 214k, 2141,214m, 550f, 550g, 550h, 550i, 550j, 550k, 5501 and 550m 15 were prepared as follows. ΑΡ/Γ/9 8'Λ 1 29 4 AP c ο 12 θ ο - 215 -

550f-m, R1 = Et

0 o

(213f) was synthesized from 212f by the methods used toprepare 213e from 212e to afford 504 mg of 213f as ayellow solid, 1H NMR (CD3OD) δ 1.10 (br. m, 0.25H),1.30(br. m, 2H), 1.50(br. m, 1H), 1.65(br. m, 1.5H), 5 1.80(br. m, 0.25H), 1.90(br. m, 0.25H), 1.95(br. m, 0.5H), 2.05(br. m, 0.25H), 2.15(m, 1H) , 2.3(m, 1H),2.5(br. m, 1H), 2.6(dd, 1H), 2.8 (m, 1H), 3.1(br. s, 3H), 3.15(br. m, 1H), 3.32(br. s, 3H), 3.5(m, 1H),4.5(br. m, 1H), 4.62(d, 0.25H), 4.72{m, 3H), 4.95(m, 10 1H), 5.1(br. t, 0.25H), 5.15(br. t, 0.75H), 5.7(d, 1H), 6.75(d, 2H), 7.35(br. s, 5H), 7.75(d, 2H). (213g) was synthesized from 212g by the methods used toprepare 213e from 212e to afford 400 mg of 213g, 1H NMR(CD3OD) δ 1.5(br. m, 1H), 1.65(br. m, 2H), 1.70(br. m, 15 0.25H), 1.90(br. m, 1H), 1.95(br. m, 1H), 2.05(br. m, ΑΡ/Γ7 9 8 . A) 1 29 4 AP v 01 2 8 0 - 216 - 0.25H), 2,. 10 (m, 1H) , 2.3 (in, 1H) , 2.5 (m, 2H) , 2.5S(d,1H), 2,6(d, 1H), 2,78 (d, 1H) , 2.8(d, 1H) , 2.93 (its, s,4H) , 3.05(br. m, 1H) , 3.15 (br. in, 0.25H), 3.3 (bn s,3E), 3.5(m, 2K) , 4.5(br. m, 2H), 4.65(d, 1H), 4,7(br. m, :H), 4.95{br. m, IK), 5.15(br. t, 0.25H), 5.2(br. t, 0.75H), 5.2(d, 1H), 6.95(d, 1H), 7.15(d, 1H), 7,.- br.s, 1H), 7.3(br. t, 2H) , 7.45(br. s, 6H). (213h) was synthesized from 212h by the methods used toprepare 213e from 212e to afford 296 mg of 213h, Ή NMR(CDC13) 5 1.55-1.68(m, 1H) , 1.7-2.05(m, 3H) , 2.3-2..5 (m,2H), 2.65-2.8 (m, 1H) , 2.85-2.93(m, 1H) , 2.95-3.25(m,· 3H) , 4.44-4.65 (m, 2K), 4.68-4.82(m, 1H), 4.9-4.95(0,-0.18 (m, 2K) , 5.28 (s, 0.5H), 5.55-5.58(5,, 52-6.58 (d, 0.5H), 6.7-6.76(m, 2H) , 6.82--6.85(d, 0.5H), 7,3-7,. 4 (m, 5H), 7.52-7.58(m, 1H) , 7.75 (s, 0.5H) , 7.8 (s, 0.5H) . (213i) was synthesized from 212i by the methods used toprepare 213e from 212e to afford 1.1 g of 213i, "Ή NMR(CDC13) δ 1.55-2.05 cm, 6H), 2.26-2.5(m, 2H), 2.68-2.82(m, 1H), 2.85-2.92(m, 1H), 2.95-3.25(m, 2H), 3.62 is, 1.5H), 3.85(s, 1.5H), 4.4-4.65(m, 2H), 4.7-4.78(m, 1H), 4.88-4.95(m, 1H), 5.05-5.23(m, 1H), 5.28(s, 0.5H), 5.55-5.58(d, 0.5H), 6.6-6.65(m, IHi ,6.8-6.84(m, 1H), 6.9~6.95(m, 3H) , 7.3-7.45(m, 4K), -7.85 <m, 2H) , 1H) , 5 O.SHi, ? 7 ί (213j) was synthesi from 212j by the methods used toid. prepare 213e from 212e to afford 367 mg of 213j, JH NMR(CDCI3) δ 1.55-2.05 (in, 12H) , 2.25 (d, 1H) , 2.35(m, IHi,

iP

Hi, 2.75 (m, 2H) , 2.9 (m, 1H) , 2.95-3.: 5H) , 4.45(.5, IHi, 4.5-4.6 in, 4H) , 4.7 (m, 1H) , 4.75 (d, IHi,4.88(m, IHi, 5.05(m, 2H), 5.15(q, 1H), 5.3(s, IK),5.58 id, IHi, 6.5 id, lib, 6.9(d, 1H) , 7.05(d, IK), 7.2"Kilim, 5H- , 7.6(s, 2Hi , 7.7 (s, 2H) . AP/17 9 8 . .0 t 2 9 4 10 15 20 25 ΑΡ ΐ Ο 12 8 Ο 217 - (213k) was synthesized from 212k by the methods used toprepare 213e from 212e to afford 593 mg of 213k, NMR(CD3OD) δ 1.5(m, 1H), 1.6-1.7(m, 2H) , 1.75-1.95(m,4H),2.15(m, 2H), 2.3(m, 1H), 2.6(m, 1H) , 2.7(m, 1H), 3.05(m, 2H) , 3.15(m, 1H), 3.5(m, 2H), 4.45(m, 2H),4.65(d, 1H), 4.7(m, 1H), 4.95(m, 1H), 5.15(m, 1H),5.4(s, 1H), 5.7(d, 1H), 7.3(m, 5H) , 7.85(s, 2H) . (2131) was synthesized from 2121 by the methods used toprepare 213e from 212e to afford 133 mg of 2131, 1H NMR(CDCI3) δ 1.55-1.7(m, 1H), 1.75-2.05(m, 3H), 2.25(s,1.5H), 2.27(s, 1.5H), 2.3-2.48(m, 2H), 2.7-2.83(m, 1H) ,2.85-2.94(dd, 1H), 2.95-3.25(m, 2H), 4.42-4.65(m, 2H) ,4.68-4.85(m, 1H), 4.88-4.95(m, 1H), 5.05-5.18(m, 2H),5.32(s, 0.5H), 5.55-5.6(d, 0.5H), 6.48-6.55(d, 1H),6.88-6.92(d, 1H), 7.0-7.04(d, 0.5H), 7.15-7.2(d, 0.5H),7.3-7.4(m, 4H), 7.64-7.78(m, 2H), 7.88-7.94(m, 1H),8.45-8.56(m, 1H). (213m) was synthesized from 212m by the methods used toprepare 213e from 212e to afford 991 mg of 213m, 1H NMR(CDCI3) δ 1.5-2.15(m, 5H), 2.2-2.55(m, 3H), 2.6-3.3(m,4H) , 3.95(2s, 3H) , 4.45-4.7(m, 2H) , 4.7-4.85(m, 1H),4.8504.95(m, 1H), 5.05-5.25(m, 1H), 5.3(s, 0.5H), 5.6(d, 0.5H), 6.55(d, 0.5H), 6.85(d, 0.5H), 7.0(d, 0.5H), 7.25-7.6(m, 5.5H), 7.75(s, 1H) , 7.85(s, 1H) .(550f) was synthesized from 212f by the methods used toprepare 213e from 212e to afford 420 mg of 550f as anoff white solid, 1H NMR (CDC13) δ 1.2-1.25(br. t, 3H),

fr β 2 Iff8 6 /J/dV 35 (m, 1H) , 1.55 (br. m, 1H), 1.88-2 .02 (br. m , 4H), 3 (d, 1H) , 2.35(m, 1H) , 2.45(m, 1H) , 2.55-2. 75(m, 3H) 0 (s, 6H) , 3.25(m, 1H) , 3.55(m, 1H) , 3.65(m, 1H) , 75 (m, 1H) , 3.9(m, 1H), 4.3(t, 1H), 4.55(m, 2H) , 68 (br . m, 1H) , 3.9 (m, 1H), 4.3(t, 1H) , 4.55 (m, 2H), 30 ΑΡ ο ο 1 2 θ 0 - 218 - 4,68 ibr. m, 1H) , 4-9Si.br. m, 1H) , 5.1 (br. m, 2Ki , 5.45(d, 1H) , 6.5(m, 2H), 7.7 (m, 2H). (550h) was synthesized from 212h by the methods tsed to prepare 213e from 212e to afford 195 mg of 550h as awhite solid, 1H NMR (DMSO-d6) δ 1.1-1.18 (2t, 3H), 1.6-1.7 (m, 2H), 1.88-2.05 (m, 2H) , 2.1-2.35(m, 3H) , 2.48-2.56(m, 1H) , 2.75-2.8cm, 0.75H), 2.88-3.08(m, 1.25H),3.25-3.4(m, 1H) , 3.55-3.8 (m, 2H) , 4.35-4.45(m, lib, 4.55-4.62(m, 1H) , 4.8.....4.88(m, 1H), 4.98-5.03(m, 0.25H), 5.1-5.13(m, 0.75H), 5.33(s, 0.25H), 5.58-5.6(d, 0..75H) , 5.9-6.0 ibr. s, 2H) , 6.8-6.85 (d, 1H) , 7.58-7.62 (d, 1H.) ,7.82(5, 1H) , 8.22-8.28 id, 1H) , 8.48-8.52(d, 0.7537,'8.72-8.76(d, 0.25H). (550i) was synthesized from 212x by the methods tsed to prepare 213e from 212e to afford 135 mg of 550i, '7H NMR(CDC13) δ 1.18-1.26(2t, 3H), 1.6-1.75(m, 1.5H), 1.9-2,1 (a, 3.5H), 2.22-2.3 (d, 0.5H), 2.38-2.47 (m, l.SH), im, 0.5H), 2.8-2.93 (m, 1H) , 2 ., 94-3.15 (m, 5H) , 3.15-3.28(m, 1H), 3.S5-3.62(q, 0.5H), 3.62-3.73(q,Q.5H7, 3.78-3.88 (q, 0.5H), 3.88(s, 3H) , 3.9-3.95cq,0.5H), 4.33-4.4(m, 0.SK), 4.5-4.55(m, 1H), 4.68-4.76(mf0.5H), 4.9-4.95(m, 0.5H), 5.1-5.2(m, 1.5H), 5.18(s,0.SH), S . 48--5.52 id, 0.5H), 6.48-6.55>d, 0.5H), 6,85- ί. 9 Cm, . 85(m, 1H), 6.9-6.35 (m 2H( . 2H) , 7.34-7.38(d, 0.5H), 7,.78- ( .

(550k) was synthesized from 212k by the methods used toprepare 213e from 212e to afford 174 mg of 550k as a white solid, 1H NMR (DMSO-d6) δ 1.15 (2t, 3H) , 1.6 i.7S(m, 2Kj , 1.9-2.05(m, 2H) , 2.1-2.4(m, 5H) , 2.5-2.55im, IK), 2.7-2,8(m, 0.5H), 2.85-3.0 (m, 1H) , 3.0-3.1 cm, 0.5H), 3.55-3.7(m, 1H), 3.7-3.8(m, 1H), 4.2(0,0.5K), 4.35-4.45(m, 0.SH), 4.55-4.65(m, 0.5H), 1.1-9.97m, 0.5H), 5.05(0, 0..5H), 5.15(t, 0.5H), 5.35is, AP 0 Ο 12 8 Ο - 219 - 0.5Η), 5.6(d, 0.5Η), 7.95(s, 2H), 8.5(d, 0.5H), 8.65(d,1H), 8.75(d, 0.5H), 10.9(br. s, 1H).

(5501) was synthesized from 2121 by the methods used toprepare 213e from 212e to afford 151 mg of 5501, 1H NMR 5 (CDC13) δ 1.2-1.28 (2t, 3H), 1.6-1.72(m, 1.5H), 1.88- 2.15(m, 3.5H), 2.22-2.28(m, 0.5H), 2.28(s, 3H) , 2.38-2.48(m, 1.5H), 2.66-2.92(m, 1.5H), 2.95-3.14(m, 1.5H),3.2~3.34(m, 1H), 3.56-3.63(q, 0.5H), 3.63-3.72(q, 0.5H), 3.8-3.85(q, 0.5H), 3.9-3.95(q, 0.5H), 4.32- 10 4.38(m, 0.5H), 4.5-4.62(m, 1H), 4.68-4.75(m, 0.5H), 4.88-4.92(m, 0.5H), 5.08-5.2(m, 1.5H), 5.18(s, 0.5H),5.46-5.5(d, 0.5H), 6.5-6.55(d, 0.5H), 6.98-7.05(m, 1H) ,7.42-7.48(d, 0.5H), 7.63-7.78(m, 2.5H), 7.9-7.94(d, 0.5H), 8.44-8.52(m, 1H). 15 (550m) was synthesized from 212m by the methods used toprepare 213e from 212e to afford 301 mg of 550m as awhite solid, 1H NMR (CDC13) δ 1.2-1.35 (2t, 3H) , 1.5-1.8 (m, 2H), 1.9-2.15(5H), 2.25(d, 0.5H), 2.4-2.5(m, 2H), 2.65-2.8(m, 0.5H), 2..8-3.0(m, 0.5H), 3.0-3.2(m, 20 1H), 3.2-3.35(m, 0.5H), 3.55-3.65(m, 0.5H), 3.65- 3.75(m, 0.5H), 3.8-3.9(m, 0.5H), 3.9-4.0(m, 0.5H), 4.4-4.45(m, 0.5H), 4.55-4.65(m, 0.5H), 4.7-4.8(m, 0.5H),4.85-4.95(m, 0.5H), 5.05-5.2(m, 0.5H), 5.2(s, 0.5H),5.5(d, 0.5H), 6.5(d, 0.5H), 6.9(d, 0.5H), 6.95(d, 25 0.5H), 7.35(d, 0.5H), 7.75(s, 1H), 7.85(s, 1H). (214j) was synthesized from 213j by the method used toprepare 2002 from 2001 to afford 62 mg of 214j as awhite solid, ΤΗ NMR (CD3OD) δ 0.9 (t, 1H) , 1.3(br. s,1H), 1.7(br. m, 1H), 1.9(br. m, 1H), 2.1(br. s, 1H), 30 2.25(q, 1H), 2.35(m, 1H), 2.48(m, 2H) , 2.65(t, 1H), 3.15(br. t, 1H), 3.5(br. m, 1H), 4.3(br. s, 1H), 4.55(m, 2H), 4.95(t, 1H), 5.25(br. s, 1H), 7.6(br. s, 1H), 7.85(br. s, 1H). AP c 012 8 0 - 220 - (214k) was synthesized from 213k by the method used toprepare 2002 from 2001 to afford 80 mg of 214k as awhite solid, 1H NMR (CD3OD) δ 1.6-1.7(m, 1H), 1.8-2.0(m,2H) , 2.0-2.1(m, 2H), 2.15-2.25(m, 1H), 2.3-2.4(m, 1H) , 2.4-2.55(m, 2H) , 2.6-2.75(m,1H), 3.05-3.2(m, 1H) , 3.4-3.6im, 2H) , 4.2-4.3(m. 1H) , 4.45-4.6(m, 1H) , 4.8-5.0{m,IE), 5. .1-5.2 (m, 1H) , 7.85(s, 2H) . (2141) was synthesized from 2131 by the method csed toprepare 2002 from 2001 to afford 91 mg of 2141 a?, awhite solid, 1H NMR (DMSO-dg) δ 1.65(br. m, 6H) ? 1.9(br.m, 6H), 2.15 (s, 3H) , 2.3(m, 3H), 2.6-2.85(m, 3H). 2 . 9 (m, 2H), 3.0(m, 1H5, 4.15(br. q, 1H), 4.4(m, 3H) ,5.0im, IK), 5.15(m, IK), 5.45(s, IE), 7.8(d, 2H), 7.S5(d, 1H), 8.05 (s, IB), 8.65{m, 2H), 9.65(s, IK),(214m) was synthesized from 213m by the method used toprepare 2002 from 2001 to afford 105 mg of 214m as awhite solid, 1H NMR iCD3OD) δ 1.6-1.75(m, IK), 1.85-1.95(m, IHj, 2.0-2.1 (η, 2H) , 2.15-2.25(m, 1H), 2.3-2.1(in, 1H), 2.45-2.55(m, 2H), 2,65-2,75(m, 1H), 3,4-3.55(m, 2H), 3.95(s, 3K), 4.2-4.3(m, 1H) , 4 .45-4,6(m, 1H), 4.S-5.0(m, IB), 5.15-5.2(m, 1H), 7.9(s, 2H),

Compounds 308c and 308d were prepared as

AP/." 9 8 , ύ 1 2 9 4 c R1= Me d R1= (308c) was synthesized from 212e via the methods usedto prepare 308b from 212e to afford 266 mg of 308c “HNMR (,CDC13) δ 1.6-1.7(01, 1H) , 1.88-1.98 (m, 3H> , 2.02- AP i’ ο 1 2 θ ο - 221 - 2.15(m, 1Η), 2.3-2.4(m, 1H) , 2.65-2.95(m, 3H) , 3.04-3.09(m, 1H) , 3.12-3.25 (in, 1H) , 3.84(s, 3H) , 3.86(s, 3H) , 4.5-4.58(m, 1H), 4.88-4.95(m, 1H), 5.1-5.25(m,2H), 6.86-6.9(d, 2H), 7.15-7.25(m, 2H), 7.36-7.4(m, 5 1H), 7.75-7.8(d, 2H).

(308d) was synthesized from 212e via the methods usedto prepare 308b from 212e to afford 270 mg of 308d, 1H NMR (CDC13) δ 1.55-1.65(m, 1H), 1.8-2.1(m, 4H), 2.3-2.4(m, 1H), 2.65-2.88(m, 3H), 2.9-3.3(m, 3H), 4.5- 10 4.58(m, 1H), 4.88-4.95(m, 1H), 5.05(s, 2H), 5.1-5.2(m, 1H), 6.82-6.95(m, 2H), 7.02-7.15(m, 2H), 7.28(m, 5H) ,7.45(m, 1H), 7.72(d, 2H) .

Compounds 2100f, 2100g, 2100h, 2100i and2100j were prepared as described below.

OtBu

AllocN

H

2101a

AllocN'^n'OHΗ O

H 212e 2100f, g ΑΡ/Γ/9 8fi 1 2 9 4 f, R1= g. R1=

- 222 - (2101a) was synthesized from allyloxycarbonylamino-β-te.rt-butyl aspartate by the methods employed by Chapman(Bioorq. &amp; Med. Chem. Lett., 2, pp.615-618 (1992;’ toprepare (35, 2RS) 3-allyloxycarbonylamino-2-benzy.'.oxy-5- 5 oxocetrahydrofuran using 4-chlorobenzyl alcohol insteadof benzyl alcohol to afford 1.84 g of 2101a as acrystalline solid. (2100f) was synthesized from 212e by the methods usedto prepare 213e from 212e using 2101a to afford ISO mg 10 of 2100f, 3"B NMR (CPC 7,; δ 1.8-2.0 (in, 10H) , 2 .30 id, 1H), 2.31--2.5 (m, 3H) , 2.7- 2.9 (m, 3H) , 3.05(m, 2H), 3.1...... 3.2(m, 43) , 4.45( q? 1 EE 4.5-4.6(m, 3H), 4.7( d, . - , 4.85yd, 1H), 4.9( t, 1 .3) , 5.2 (t, 1H), 5.15(m, 23) , 5.25(s, 1H 1 , 5.55 id, 1H;, 6.5(d, 1H) , 6.9(d, 1H) , 15 6.95(d, 1H), 7.25 (m, ί, 7.35(t, 2H), 7.45(m 2H) , 7.55(135, 7. 8 (m, 33) . (2101b) was synthesized from (3S,2RS) 3-allyloxy·-carbonylamino-2-benzyloxy-5-oxotetrahydrofuran vis themethod used to prepare 2100d from 214e using H2SO4 20 instead of pTSA to afford 2101b. (2100g) was synthesized from 212e by the methods usedto prepare 213e from 212e using 2101b to afford 31 mgof 2100g, 1H NMR (CDCljj δ 1.19 (d) , 1.94 (br 2.00-2.12 (my, 2.24 (d) , 2,42 <dd), 2.71-2.83 (m) , 3. . 25 (dec, 3.12-3.27 (overlapping m), 3.93 (m), 4.32-4,37 im, ), 4.52-4.63 (m$, 4.90-4.95 (m), 5.12-5.20 (my, 5.28is), 6.93 (d), 7.10 (di, 7.41-7.50 (m), 7.51-7.58 (m), AP, 7, 98/91294 214e ΑΡ ο Ο 12 8 Ο - 223 -

(2100h). A solution of 214e (287 mg, 0.65 mmol) inpyridine (5 mL) was treated with Ac2O (0.4 mL, 3.62mmol). After 6 hours, the reaction mixture was pouredinto 5% NaHSO4 and extracted 3 times with EtOAc. The 5 combined organics were washed with brine, dried over

Na2SO4 and concentrated in vacuo. Chromatography (SiO2,EtOAc) afforded 119 mg of 2100h, 1HNMR (CDC13, mixtureof four diastereoisomers) δ 1.80-2.05(m), 2.12 (s),2.13(s), 2.19(s), 2.22(d), 2.67-2.75(m), 2.80-2.95(m), 10 3.00-3.20(m), 3.21-3.33(m), 3.50-3.95(four discrete multiplets), 4.19(m), 4.55(m), 4.57-4.65(m), 4.69(m),4.85-4.95(m), 5.04(m), 5.10{s), 5.10-5.22(m), 6.46(d),6.03(s), 6.50(d), 6.58(d), 6.75(d), 6.95-7.05(m),7.22(m), 7.30(m), 7.71(d), 7.75-7.83(m). 2100b

α»

CM v-~

CP o I-

CL < 15 (2100i). To a solution of 2100b (1.5 g, 2.7 mmol) in CH3CN (10 mL) was added IN HC1 at ambient temperature.After 6 hours solid NaHCO3 was added and the productextracted with EtOAc, dried over MgSO4 and concentratedin vacuo. Chromatography (SiO2, 30-100* CH2C12 in 20 EtOAc) afforded 123 mg of 2100i, ΣΗ NMR (CDC13) δ 1.25(t, 3H), 1.6-1.8(m, 1H), 1.9-2.2(m, 5H), 2.4-2.5(m, AP C Ο 1 2 8 Ο - 224 - .Η), 2.75-2.9(m, 2Η), 3.0-3.1(m, 2Η), 3.2-3.25(m, h05-4.2(m, IH), 4.5......4.7 (m, 1H), 5.1-5.25(m, IH) , ’.2(m, 2H), 7.4-7.45 Cm, 2H), 7.5(t, 1H), 7.8 (t, i».S(s, IH) . . 0- 2100Ϊ

{2100j) was synthesized from 2100i via the method usedto prepare 2100h from 214e to afford 347 mg of 2100j, NMR (CDC13) δ 1.3 it, 3H) , 1.6-1.8 (m, 2H) , 1.9-2.25 (κι., 10 )!H) , 7.5(t, 1H) , 7.8 it, 45(m, 1H), 2.8-3 . 0 (m, IH), 3.0 2H) , 4.1-4 . 2 (m, 2H) , 4.55- IH), 6.8(s, IH) , 7.0-7 .1 ffi. 2H), 9.5(s, IH) . and 501 are described in Table 25. These compounds were prepared by methods similarto the methods used to prepare compounds 404-449 ''see, 15 Example 11}. ΑΡ/Γ/9 8/fi 1 29 4 APC01280

Table 15 - 225 - -r C“t MS (M+H) 523. 533 d Γ- RT mi thod) rity 48 (A) 991 .3 0.9 ,, 0) 3 J o o Oj X r—i 5—1 CM τ-1 cn m s • • 2 τΉ CM CM m ID in CO O o lD 2 O Eid rH u 2 2 CM C22H2 X sr CM U X O X 9 \ °=Q=° <D iq °=Q=o ___ Z X CU; Structu ο \jr 2 X O -°\ η JX’ x k' \o M x Ό 3 3 <3 i—1 c O o Q< e in m o u ΑΡ/Γ7 8 8 I a 1 2 8 4 ΑΡ ΐ Ο 12 θ Ο - 226 -

The compounds described below (213m, 213n,213o, 213p, 213q, 213r, 213s, 213t, 213u, 213v, 213w,213x, and 214w), were prepared by methods similar, tothe methods used to prepare compounds 213b-f. 5 Compounds 419, 415, 450, 456, 475, 404, 486, 487, 417, 408 and 418 may also be prepared as describedbelow.

AP/P/ 9 8 . ft 1 2 9 4 213m-?: 214w, 404, 408, 415,10 417, 418, 419, 450, 456, 475, 486, 487 compound R1 213m, 419 MeOC(0)- 213n, 415 i I o o f 213o, 450 ..... ............................................... qA HN Me Π O 213p, 456 .,ol AP t Ο 12 3 Ο - 227 - 213q, 475 o 213r, 404 Me O 213s, 486 H 213t, 487 Αα,χΛ H 213u, 417 M'°rA OMe 213v, 408 213w, 214w Me ΑΡ/Γ, δ 8 ί Λ 1 2 9 4 (213n) AP C 0 1 2 8 0 - 228 -

213x, 418 0 O H was isolated as a mixture of diastereomers (syn:anti isomer ratio 6:4) (1.43g, 82%) as a white solid.: mp. 206-10’C; JR (KBr) 3288, 1787, 1680, 1657,1651, 1619, 1548, 1440, 1256, 1135; 1H NMR (D6~DMSO) δ 8.75 (0.4H, d) dd 6H, d) , 8.45 and 8.43 (IH, d) , 7.50 (IH, d), 7.42 (IH, s), 7.40-7.27 (5H, m- (IE, d) , 6.11 (2H, sf , 5.67 (0.6H, d) .43 (0.4H, 5.10-5.00 (IH, m), 4.90-4.59 (3.5H, m), 4.45-4.25(1.5K, if, 3.47-3.20 (IH, m) , 3.20-2.70 (2H, m), 2.2.35 (IE, m), 2.35-2.00 (3H, m) , 2.00-1.75 (2H, m) , 1.65-1.40 (2H, m) . Arial 60.20; H, 5.2 3; N, S , 63 . x 01 65-

Calcd for C29H30K4®9: of

Found: C, 60.08; H, 5.32; N,,+ 9.50. MS (ES ) 580 (id + 2, 35%), 579 (M ‘ + 1, 100),401 (5), 367 (5), 236 (7), 107 (5). (213o) anti-isomer as a white foamy solid (0.73g, 59%) :: mp. 135-40 °C; [a] 21 -37 (c 0.1, CH2C12); IR OKBri o

CM ||«ιιχ» C3| ** 4» ao α» i « a < 345) 3310, 1790, 1664, 1659, 1650, 1549, 1258, 1121; di NMR (D6-DMSCt S 10.11 (IH, s) , 8.77 (IH, a), 8.57 (IE, d) , 8.01 (ltd S), 7.76 (IH, d), 7.55 (ltd d). 7 . IS- --7.25 ί 6H, m) , 5.43 (IH, s), 5.08 -5.00 (IH, if. 4.95- -4 d?3 (IH, m) , 4 ,, f b and 4.68 (2H, dd) , 3.40—3.2 0 (1E, m) , 3.09 (IH, dd) , 3.02-2.75 (IH , m) , 2.45-2.06 ( 4 K, m) , 2.06 (3H, s . , 2 . 00-1.75 (2H, m) , 1.7 0-1 . 40 (2H, m) . Anal. Calcd for C30H33N5O8·0.75H20: C, 59,54; H, 5.75; N, 11.57. Found: C, 59.40; H, 5.62; N, 11.50.MS iSS*: 593 (5d + 2, 33%), 592 (M+ + 1, 100), 57% ¢7), 10 15 j 20 25 AP i Ο 12 8 Ο - 229 - 487 (7), 475 (6), 385 (9), 373 (26), 318 (14), 296(11), 266 (10), 221 (22). ο ο (213ρ) was isolated as a foam (1.2g, 77%): [a]D -115°(c 0.20, CH2C12); IR (KBr) 3368, 2946, 1794, 1654, 1609, 1540, 1505, 1421, 1277, 1175, 1119, 980; 1H NMR(D6-DMSO) δ 10.1 (1H, s), 8.80 (0.5H, d, J = 6.6), 8.60(0.5H, d, J = 7.2), 8.40-8.36 (1H, 2d), 7.82 (2H, d, J= 8.0), 7.41 (5H, bs), 6.86 (2H, d, J 8.6), 5.72 (0.5H,d, J = 5.0), 5.49 (0.5H, bs), 5.13-5.07 (1H, m), 4.95-4.65 (2.5H, m), 4.49-4.38 (2.5H, m), 3.49-3.30 (2H, m),3.21, 2.79 (2H, m), 2.40-1.41 (7H, m). MS (ES+) 551.(213q) was isolated as a white glassy solid (80%): mp.145-149 °C; [a]D23 -56.0° (c 0.05, CH2C12); IR (KBr) 3399-3319, 1791, 1657, 1543, 1420, 1253, 1119; 1H NMR(CDC13) 69.54 (1H, s), 7.65 (1H, d, J = 7.9), 7.51 (1H,d, J = 6.9), 7.44-7.25 (7H, m), 7.18-7.06 (3H, m) ,5.30-5.20 (1H, m), 5.27 (1H, s) , 4.84 (1H, m), 4.79(1H, d, J = 11.4), 4.56 (1H, d, J = 11.3), 4.47 (2H,m) , 3.28 (1H, m), 3.10-2.97 (2H, m), 2.71 (1H, m),2.47-2.37 (1H, m), 2.26 (1H, d, J = 17.9), 2.09 (1H,m) , 1.83, 1.70, 1.51 (4H, 3m) . (213r) was isolated as a mixture of diastereomers(syn:anti isomer ratio 55:45) as a white foamy solid(1.46g, 89%): mp. 106-10'C; IR (KBr) 3306, 2947, 1791,1659, 1650, 1535, 1421, 1256, 1122; 1H NMR (Dg-DMSO) δ8.76 (0.45H, d), 8.56 (0.55H, d), 8.49 and 8.47 (1H, 2x d), 7.41-7.19 (9H, m), 5.67 (0.55H, d), 5.43 (0.45H,s), 5.11-5.02 (1H, m) , 4.86-4.55 (3.5H, m) , 4.45-4.25(1.5H, m) , 3.40-3.20 (1H, m) , 3.20-2.70 (2H, m), 2.65- 2.40 (1H, m), 2.34 (3H, s), 2.30-1.70 (5H, m), 1.65-

1.40 (2H, m) . Anal. Calcd for C^l^N^C^: C, 62.66; H,5.95; N, 10.08. Found: C, 62.91; H, 6.00; N, 9.70. MS

V 6 2 Iff8 6 /2/sSV 30 AP v Ο 1 2 8 0 - 230 - (ES · 550 (M+ +2, 4 3%), 549 (M+ + 1, 100), 374 -3 ), 280 ¢4-, 2 ί F ί <i 0 ) , 118 (213s) Wcs isolated as the anti-isomer as a white foamy solid (0 « 64g, 77%) : mp. 137-41 °C; [a]D21 -48.2° « c 5 >J » V -.7 / CE3OH); IR (KB r) 3477, 3314, 1791, 1659, 155 j 9* I o. 9 q 1499, 1406, 12 56, 1122; 1H NMR (D6-DMSO) δ 1C 1,45 ), 8.76 (1H, d ), 8.50 (1H, d), 7.86 (2H, a , 7 , 5 9 (2 H, d ), 7.41-7.20 1 10H, m), 5.43 (1H, s), 5.08-4. 9 8 (IE, m .), 4.90-4.73 i IK, m) , 4.76 and 4.68 (2H, da) i IQ 3.67 ( 2H, s), 3.40-3 ..20 (1H, m) , 3.09 (1H, dd) , s. 02- 2 * z o { IB, m), 2.39 ( in, dd), 2.30-2.00 (3H, m) , 2, 00- 1 ./3 ( 2B, m) , 1.70-1 ..40 (2H, m) . Anal. Calcd for Ίε,Οθ * 0.5H2O: C, 63.90; H, 5.66; N, 10.35. 11 ?und: f" 9 9 e -- s · 68; K, 5.67; N , 10.24. MS (ES+) 669 (M* + 2 15 4 U ί; j ,.- 668 (M + 1, 1 00), 640 (12), 435 (18), 425 ( 23 1 , 103 (3 3), 328 (17), 302, (32), 274 (22), 197 (16), 138 (213t) was isolated as a white foamy solid (0.63g, 8 01) t mp. 159-64 °C; ia]D21 -37.0°(c 0.05, CH3OH); IR 20 (KBr ) 3463, 3321, 17 90, 1680, 1658, 1650, 1644, )5 . O M -· —· f Ί 7 _L K,· 4,., ·„' f 15C1, 1408, 12 51, 1113, 933; 1H NMR (Dg-DMSO ) δ .1 kJ p Λ 2 (IB, s), 8.76 (.IB, d), 8.48 (1H, d), 7.85 (2 H, d) , 7 . 68 (2H, d), 7. 40-7.25 (5H, m), 5.43 (IB, s). 9 . ij 8 4 . 95 (1H, m.) , 4 .92-4.73 (1H, m), 4.76 and 4.6 8 25 (2H, d d) , 3.4 0-3.20 (IR, m) , 3.09 (1H, dd), 3.02-2 ... Ί 5 {1H, m. }, 2.39 (1H, d d«, 2.35-2.00 (6H, m), 2.00-1. 75 :2H, m ), 1.70-1.40 ( 1H, m) , 0.93 (6H, d) . Anal. C n 1 C ύ r o r C 3 3Β39Κ508·0.5Η27 d C, 61.67; H, 6.27; N, 10.90. Found: C, 61.49; K, 5.24; N, 10.86. MS (ES+) 635 'll’ + 30 9 / -.I in '0 ), 634 (M+ + 1 , 100), 484 (10), 427 (9), 274 (16;, 268 (37), 204 (19), 117 (13). 117 (13). AP 0 0 1 2 8 0 - 231 - <213u) was isolated as a white solid (81%): mp. 120-132’C; IR (KBr) 3361-3334, 1792, 1659, 1585, 1536, 1499, 1457, 1416, 1340, 1236, 1126, 989; 1H NMR (CDC13)δ 7.39-7.29 (6H, m), 7.12 (1H, s), 7.03 (1H, s), 6.92,6.83, 6.48 (approx 3H, 3d, J - 8.1, 7.5, 8.1), 5.57 (d,J =5.3), 5.27 (1H, s), 5.23-5.06, 4.91-4.71, 4.64-4.43, (6H, 3m), 3.92, 3.91, 3.89, 3.88 (9H, 4s), 3.32- 2.70, 2.52-2.08, 1.91, 1.63 (1H, 4m). (213v) was isolated as a white solid (78%): mp. 121-7'C; IR (KBr) 3534-3331, 1791, 1659, 1528, 1420, 1256,1122; 1H NMR (CDC13) δ 8.34-8.29 (1H, m), 7.98-7.87 (2H,m), 7.68-7.45 (4H, m), 7.34-7.24 (5H, m) , 7.04 (d, J =6.8), 6.78 (d, J = 7.8), 6.66 (d, J = 7.7), 6.48 (2H,d, J = 7.5)5.56 (d, J = 5.4), 5.15 (1H, s), 5.30-5.14,5.0, 4.89 (d, J= 11.2), 4.71-4.41 (6H), 3.18-2.80,2.50-2.27, 2.08-1.60 (11H, 3m). (213w) (65/35) as(decomp.);1486, 1420, was isolated as a mixture of diastereoisomersa white solid (0.9g, 65%): mp. 110-115'CIR (KBr) 3409, 2945, 1792, 1658, 1606, 1534,1330, 1276, 1209, 1122, 980, 960; 1H NMRJ = 6.9), 7.46-7.20 (7H, m), (CDC13)6 7.66 (0.35H, d,

6.93 (0.35H, d, J = 7.7) 6.73 (0.65H, d, J = 7.6) (0.65H, bs), 5.56 (0.65H bs), 5.20-4.98 (2H, m),(3H, (7H, 6.85 (0.65H, d, J = 7.6) , 5.96 (0.35H, bs), 5.85 d, J = 5.2), 5.28 (0.35H, 96-4 . .40 (4H, m) , 3.28-2.55 4 m), 2.53-2.32 (1H, m), 2.23 (6H, 2s), 2.03-1.40m). MS (ES~) 577, (ES+) 579. (213x) was isolated as a colourless poweder (691mg, 8 6%) : mp. 150-70 (KBr) 3313, 1791 1407, 1371, 1315 DMSO) δ 8.75 (1H,

°C; [a]D22 -10.1° (c 0.10, Me2CO); IR , 1679, 1654, 1597, 1528, 1501, 1457, , 1255, 1184, 1122, 933; 1H NMR (d6-d), 8.47 (1H, d), 7.84 (2H, d) , 7.66 AP,?, S 8 . ΰ 1 2 9 4 £Ρ ϊ· 0 1 2 3 0 - 232 - (2H, d) , 7.35 (5H, m ( , 5.43 (1H, s) , 5.06-5.00 (IE, m.) ,4.90-4,64 i3H, mi, 4,.46-4.26 (2H, m) , 3.16-2.86 (2H,m) , 2.45-2.05 (5H, m) , 2.07 (3H, s), 2.00-1.84 {2K, roj , I. 68-1.56 (2H, mi; Anal. Calcd for CgQHggNgGg · H2O: C,59,11; H, 5.79; N, 11,49. Found: C, 59.38; H, 5.66; N, II. 31; M.S. (ESA 614 (100%), 592 (M++1.66j. (415) was prepared by a similar method as compound 214eto afford a white solid (297mg, 84%) : mp. 158-62 ,JC;[ajnz4 "109.5° (c 0.1, CH3OH); IR (KEr) 3700-2500 (br) ,1783,1659, 1650, 1538, 1486, 1439, 1257, 1037; "HNMR(CD3OD) 5 7.48 (1H, ad), 7.35 (1H, d) , 6.88 (1H, do, 6.03 (2K, s), 5.25-5.15 (1H, m), 5.02-4.90 (1H, Kb,4.63-4.45 (2H, m), 4.30-4.20 (1H, m) , 3.57-3.30 . 1, in) , 3.20-3.05 (1H, m) , 2.75-2.10 (5H, m) , 2.10-1,60(4H, m) . MS (ES~) 488 (M+, 25%), 487 (M+ - 1, 100), 443 (8), 387 ¢3), 315 (5), 150 (6), 127 (5), 113 )8).Accurate mass calculated for C22H25N4O9 (MH+): 489.1621.Found 489.1648.

(450) was prepared py a similar method as compound 214eto afford a white foamy solid (378mg, 94%) : mp. .:.'75~9°C; ia]D22 -91.7° (c 0,1, CH3OH); IR (KBr) 3700-2500ibri, 3319, 1659, 1590, 1553, 1427, 1260; 1H NMR ! CD3OD) δ 8.01 (1H, ci;, 7.74 (1H, dd) , 7.58 (1H, di. "'· . 45-7.35 (1H, m) , 5.25-5.15 (1H, m) , 5.05-4.90 (IK, in) , 4.60-4.45 (2H, no , 4.30-4.20 (1H, m) , 3.55-3.30 JIK, m), 3.20-3.00 (IK, in), 2.75-2.20 (5H, m), 2.14 ¢35, si, 2.20-1.60 ' 411; . Anal. Calcd for C23K2-N5O8'·! .5H2O: 52 52.27; H, 5.72; N, 13.25. round C, :5 2.31; H, 5.86; N, 12,85. MS (ES + i 501 (M+, 2 Idd , 500 (M+ - 1, 100), 328 ¢2), 149 (3), 113 (3). (456) was prepared by a similar method as compound 214eto afford a white · . (0.73g, 72%): mp. >260 °C. AP V Ο 12 8 Ο - 233 - [a]D20 -66° (c 0.34, MeOH); IR (KBr) 3401, 2946, 1651, 1609, 1584, 1506, 1426, 1277, 1257, 1177; 1H NMR (Dg-DMSO)6l0.2 (1H, verybs), 9.17 (1H, bs), 8.65 (1H, s),8.37 (1H, d, J 5.4), 7.81 (2H, d, J = 8.2), 6.87 (2H, 5 d, J = 8.4), 5.24 (1H, m) , 4.92-4.86 (1H, m) , 4.41-4.32(2H, m), 3.68-3.21 (3H, m), 3.12-2.79 (1H, m), 2.50-1.42 (7H, m). MS (ES+) 459. (475) was prepared by a similar method to thatdescribed for compound 214e to afford a white solid 10 (79%): mp. 150 °C (softens) 190-210 °C; [a]D23 -97.5° (c 0.1, CH3OH); IR (KBr) 3319, 1658, 1650, 1549, 1421,1256; 1K NMR (CD3OD)57.61 (1H, d, J= 8.0), 7.43 (1H,d, J = 8.1), 7.21 (2H, m), 7.05 (1H, m), 5.21 (1H, m),5.07-4.77 (1H, m), 4.54 (2H, m), 4.23 (1H, m), 3.46 15 (1H, m), 3.14 (1H, m), 2.66-1.71 (9H, m). MS (ES+, m/z), 482 (M+ - 1, 100%). (404) was prepared by a similar method as compound 214eto afford a white solid (0.79g, 86%): mp. 156-9 °C;[a]D25 -119.7° (c 0.1, CH3OH); IR (KBr) 3700-2500 (br), 20 3387, 3309, 2956, 1785, 1659, 1650, 1535, 1422, 1278; 1H NMR (CD3OD) δ 7.46-7.15 (4H, m), 5.25-5.15 (1H, m),5.02-4.90 (1H, m) , 4.58-4.45 (2H, m), 4.30-4.20 (1H, ΑΡ/Γ/ 96.01294 m) , 3.55-3 .30 (1H, m) , 3.20-3.05 (1H, m) , 2.80-2.20 (4H, m) , 2 .41 (3H, s) , 2.20-1.60 (5H, m). MS (ES+) 458 25 (M-, 27%), 457 (M+ - 1, 100), 413 (13 ), 339 (8), 285 (5), 134 ( 6) , 127 (11). Accurate mas s calculated for C22h27n4°7 (ΜΗΧ): 459.1880. Found 459.1854. (486) was prepared by a similar method as compound 214eto afford a white solid (325mg, 89%): mp. 165-9 °C; 30 [a]D22 -69.1° (c 0.1, CH3OH); IR (KBr) 3700-2500 (br),

3318, 1658, 1599, 1530, 1505, 1407, 1258; 1H NMR 10 - 234 - (CD3OD) δ 7 .85 (2K, d)f 7.69 (2H, d) , 5.25-5.15 (1H, m) , 5.05-4.90 (1H, m) m) , 4.30-4 .20 (1H, m), 3.70 (2H, s), 3.20--3,00 (1H, m), 2.75-1.60 (9H, m) ^29^31^5^8‘ <1.5H2O: C, 57.61; H, 5.67; C, 57.81; H, 5.74; N, 11.47. MS (ES 57 6 £M+ - 1, 100), 502 (2) . 7.38-7.20 (5H, m),

Found: (487) was prepared by a similar method as compound 214eto afford a white foamy solid (335mg, 93%): mp. 176-80°C; [α]0"·Ζ -88.0° (cO.l, CH3 no? ) ; IR (KBr) 3700-2500 1597, 1529, 1407, 125 , d) , 7.69 £2H, di, 5 25- H NMR (CD3OD) δ 7.86 (2H, d) 5..15 (1H, m) , 5.05-4.90 (1H, m) , 4.60-4.45 (2H, m) ,4.30-4.20 (1H, m) , 3.57-3.30 (1H, m) , 3.20-3.00 (IB, 15 m), 2.75-1.60 (12H, m), 1.00 (6H, d). Anal. Calcd for C2 ’ ^'r 61; H, 6.28; N, 12.45.

Pound: C, 56.00; H, 6.37; N, 12.15. MS (ES ) 543 (M+, 31%), 542 (M - 100), 49c (2;, 468 (3) 20 25 (417) was prepared by a similar method to thatdescribed for compound 214e to afford a white solid(0.63g, 92%): mp. 145-155 °C (approx., not sharp); [a]D2/ -114.6° (c 0.11, CH3OH); IR (KBr) 3327, 1658,1586, 1548, 1501, 1416, 1341, 1238, 1126; XH NMR(CD3OD) 5 7.22 (2H, s), 5.21 (1H, m) , 5.00 (1H, m> , 4.56,4.49 (2H, 2m), 4.25 (1H, m) , 3.88 (6H, s) , 3.80 (3H,s), 3.55-3.43 (1H, ml, 3.12 (1H, m), 2.71-1.70 (9H, m)..Anal. Calcd for θ24Η30Ν4Ο10·2Η2Ο: C, 50.52; H, 6.01; N,9.82. Found: C, 50.49; H, 6.05; N, 9.68. MS (ES+,m/p 533 (M' -1, ) „ 30 (408) was prepared by a similar method to that described for compound 214e to afford a white solid27 , .., , (not sharp); [a] mp. 157-16( ΑΡ Ο Ο 12 8 Ο 10 15 - 235 - 0.1, CH3OH); IR (KBr) 3325, 1658, 1531, 1420, 1278, 1257; 1H NMR (CD3OD) δ 8.33-8.28 (1H, m), 8.01-7.78 (2H,m) , 7.71 (1H, d, J = 6.0), 7.59-7.52 (3H, m) , 5.27 (1H,m) , 5.12-5.03 (1H, m) , 4.55 (2H, in) , 4.25 (1H, m) ,3.64-3.43 (1H, m) , 3.24-3.12 (1H, m) , 2.80-1.67 (9H,m) . Anal. Calcd for C25H26N4O7·2Η2θ: C, 56.60; H, 5.70;N, 10.56. Found: C, 56.70; H, 5.80; N, 10.33. MS(ES+, m/z), 493 (M+ - 1, 100%). (214w) was prepared by a similar method as compound214e to afford 210mg (62%) of a white solid: mp. >260°C; [a]D20 -93° (c 0.20, MeOH); IR (KBr) 3401, 2948, 1651, 1604, 1559, 1486, 1421, 1325, 1276, 1210; 1H NMR(D6-DMSO) δ 9.39 (1H, bs) , 8.29 (1H, d, J = 5.9), 7.55(2H, s), 6.64 (1H, d, J = 6.1), 5.79 (1H, s), 5.25-5.21(1H, m) , 1.90-1.82 (1H, m) , 4.41-3.69 (2H, m), 3.47-3.20 (3H, m), 2.97-2.91 (1H, m), 2.23 (6H, s), 2.25-1.60 (7H, m).

213y R= Bn (550q) was synthesized via methods used to prepare 213eto afford 550q. (213y) was synthesized via methods used to prepare 213eto afford 213y. 20 AP V Ο 1 2 8 Ο 236 -

'Ό Χ> d 'or 'ZO-— c '°O f (412a) was synthesized via methods used to prepare 550q using 513a-l to afford 412a. (412b) was synthesized via methods used to prepare 550q using 513a-2 to afford 412b. (412c) was synthesized via methods used to prepare 550q using 513b-l to afford 412c. (412d) was synthesized via methods used to prepare 550q using 513b-2 to afford 412d: 1H NMR (CDC13 )59.5 { IH, ΑΡ/Γ7 9 8 . Λ 1 2 9 4 ci) , 8.9 (IB, d), 8. 5 (IH, d) , 7.9-7.8 (2H, m) , 7.,3-7.6510 (2H, m) , 6.55 (IH, d) , 5.55 (1H, d) , 5.25-5.1 (2E, m) , 4.75-4.65 (IK, m) , 4.65-4.6 (IH, m) , 4.4-4.3 (IH, m),3.25--3.15 (IH, m) , 3.15-3.05 (IH, m) , 2.95-2.8 (2K,. m) t2.55-2.4 (2H, m) , 2.15-1.5 (14H, m) . (412e) was synthesized via methods used to prepare 550q 15 using 513f-l to afford 412e. (412f) was synthesized via methods used to prepare 550qusing 513f-2 to afford 412f.

Compounds 410 and 412 were prepared via methods used to prepare 605 . . 604. ΑΡ ν Ο 12 8 ο - 237 -

502y, 502ζ 410, 412 compound R1 502y, 410 0 502z, 412 10 (410) was purified by flash chromatography (5-25%methanol in dichloromethane) to give 296mg (94%) of acolourless solid: mp. 90-200'C; IR (KBr) 3338, 3096,2950, 1787, 1726, 1657, 1546, 1420, 1279, 1258, 1125,1092, 984, 933; 1H NMR (CD3OD) 68.41 (1H, d) , 8.13 (1H,d), 7.54-7.41 (3H, m), 7.20 (1H, d), 5.19-5.11 (1H, m) ,4.54-4.30 (1H, m), 3.27 (1H, m), 3.18-3.03 (1H, m),2.81-2.64 (2H, m), 2.56-1.59 (7H, m). Anal. Calcd forC19H22N4O7S·2.5H2O: C, 46.05; H, 5.49; N, 11.31. Found:C, 46.36; H, 5.25; N, 11.10. MS (ES+) 449 (M - 1, 80%), 113 (100). Accurate mass calculated forc19h23n4°7s (MH+): 451.1287. Found: 451.1295. ΑΡ/Γ7 9 8 . ' J5 1 2 9 4 (412) was prepared by a similar method to that described for compound 605 to afford a white glassy 15 AP v 012 θ 0 - 238 - solid (69%) : mp. 138-141 °C; [a]D23 --105.5° (c 0.5, CH2C12); IR (KBr) 3375, 1787, 1659, 1515, 1421, 1278,1256; 1H NMR (CDC135 δ 9.32 (1H, m) , 8.79 (1H, m), 8.47(IK, in) , 7.86-7.64 (4K, xn) , 5.31, 5.18, 4.59, 4.3"? (4or 5H, Ed , 3.55-2.76, 2.49-2.39, 2.05, 1.65 (HE, 4m).Anal. Calcd for C24H25.N5O7· 1.5H2O: C, 55.17; H, 5.40; K,13.40. Found: C, 54.87; H, 5.22; N, 13.15. MS (E5\m/r) 494 (M+ - 1, 100%). (502y) was synthesized via methods used to prepare 604from 603 to afford a pale cream powder: mp. 120-180 °C; [ajD --109° (c 0.18, CH2C12); IR (KBr) 3478, 3327,1670, 1582, 1543, 1421, 1279, 1257, 1155; 1K NMR(CDC13, CD3OD) 58.04 (1H, rod, 7.49 (1H, m) , 7.38 (IK, m) , 7.17 (1H, m) , 5.1 4 . 61-4.5 0 (1H, m), 3. m) , 2.79 -2.54 (3H, m? (5 K, ml , 1.44 (9H, Sj Z~' k». f 49.56 ; H, 6.07; N, H, 5.93; N, 16.31; S,

Anal. Calcd for C24H33N7O7S»H2C-: 5.17. MS (ES ) 586 (100%), 564 (M^ + 1, 1.59). Accurate mass calculated forC24H34N7O7S (MH+): 564.2240. Found: 564.2267.(502z) was prepared by a similar method to that ΑΡ/Γ7 9 8 . .0 1 2 9 4 described for compound 604 to afford a pale yellowsolid (90%) : mp. 142-145 °C; [a]D24 -136.5° (c 0.06, CH2C12); hi NMR (CDCI3) δ 9.51-9.46 (1H, m) , 9.11 ( 1H, 3} , 8.83 (1H, d, J - 7.8), 8.53 (1H, d, J = 5.5), 7.89- "?.83 (2H, m) , 7.77-7.65 (2H, m) , 7.55 (1H, d, J -= 7.2) ,7.18 (In, d, J = 2.7'd 5.26-5.12 (2H, m) , 4.87 (1H, m) ,4.59 (1H, m), 3.25-3.12 (2H, xn) , 2.95-2.76 (2H, ml,2.59-2.38, 2.18-1.94, 1.70 (5H, 3m), 1.44 (9H, s(, AP V Ο 1 2 8 Ο - 239 -

compound R4 R1 415a o o /ΊΟ 415b 415c o=b o o 'no 214w-l 0 Chb-y-vJL. HO'V ch3 'no 214w-2 0 ho γch3 'no 214w-3 0 ho γch3 nX) 214w-4 0 ho γch3 ZO—O 214w-5 o ΟΗ3γ\Χ HoV ch3 AP c Ο 1 2 8 Ο - 240 - compound „4 a R1 214w-6 0 J? ch3 Z°O 214w-7 ‘yb ch3 zO'O 412g z°~O 412h ° 5 (415a), (415b), (415c), (214w-l), (214w-2), (214w-3), (214w-4) , (214w-5), £214w-6), (214w-7) , (412g) and (412h) were synthesised via methods used to prepare55 Oq.

(415) was synthesized by the method used to prepare0 2002 from 2001 to afford 415. (214w) was synthesized by the method used to prepare2002 from 2001 to afford 214w.

• '?·_ 'Zr.

AP c 012 8 0 - 241 -

2100k-o compound R 2100k 21001 X°O 2100m 2100n 2100ο 98.01294 (2100k) was prepared by a similar method as compound213e to afford a mixture of diastereoisomers (75/25) as 10 a white solid (258mg, 83%) : mp. 101 °C; [a]D25 -96° (c

0.2, CH2C12); IR (KBr) 3328, 2935, 2978, 1732, 1669,1603, 1483, 1450, 1414, 1237, 1155, 1082, 989, 755; ΧΗNMR (CDC13) δ.7.84-7.80 (2H, m) , 7.54-7.17 (8H, m) , 7.06-6.99 (1H, m), 6.25 (1H, d, J = 7.9H), 5.41 (0.75H, d, J 15 = 5.4H), 5.31 (0.25H, bs), 5.23-5.09 (1H, m) , 4.93-4.87 (1H, m), 4.68-4.51 (2H, m), 4.40-4.33 (0.25H, m), 4.24-4.14 (0.75H, m), 3.95-3.70 (1H, m), 3.30-3.13 (1H, m), - 242 - 3.14-2.78 (5Η, m), 2.47-2.21 (2Η, m) , 2.05-1.50 (SH,m) . Anal. Calcd for C29H32N4O7 · 0 · 5Η2Ο: C, 62.47; Η,5.97; Ν, 10.05. Found: C, 62.17; Η, 5.83; Ν, 9.97. ms(Erb 54 9. 5 (21001) was prepared by a similar method as 213e, (74%.1 as a colourless solid: mp. 172-80 °C; [a]D‘"'' -91 bb’ (c 0.1, CH2C12); IR (KBr) 3290, 1792, 1677, 1657, 1642,1544, 1425, 1280, 1259, 1124, 977; NMR (CDCI3) 87.80 (2K, m) , 7.46 (3.5H, m), 7.00 ilH, d, J = 6.7), 6.48 10 (0.5H, d, J = 7.9), 5.55 (0.5H, d, J = 5.3), 5..19 (2H, s + m) , 4.93 (0.5H, in), 4.62 (1.5H, m) , 4.34 (1H, in),4.18 (0.5H, m), 3.28-2.70 (4H, m), 2.49-2.29 (2H, m),205-1.48 (15H, m) . (2100m) was prepared by a similar method as 213e, (76% 1 15 as a colourless solid: mp. ~140'C, remelts 187-9 bb[air/·3 -96.9° (c 0.11, CH2C12); IR (KBr) 3507, ... 3251, 1772, 1660, 1651, 1566, 1545, 1457, 1424, 1346,1326, 1302, 1275, 1258, 1136, 1085, 1018, 981; NMR(CDCI3) δ 7.78 (2H, mi, 7.53 (3H, m) , 7.19 (4H, mb 6.91

W Ci 07· a 6 ,b7dV (1H, d, J = 7.4), 6.27 (1H, d, J = 7. 6), 5.66 (IE, d, .j . .1) , 5.10 (1H, mj, 4.96 (1H, m) , 4 .75 (2H, m), 4.52 (IE, mi , 3.08 (3H, mi, 3.03-2.71 (5H, m) , 2.4 8 -- 2 bb. (2H, nd , 1.90-1.40 (4H, m), 1.22 (1H, m) . (2100n) was prepared by a similar method to that25 described for compound 213e to afford a white glassy solid (76%) : mp. 112-5 °C; [a]D23 -62.0° (c 0.1, CK2ib2s; IR (KBr) 3305, 1789, 1677, 1665, 1535, 1422, 127 9, 1256, 1119, 94 2, 7 0 0; 1H NMR (CDCI3) 5 7.84 (.-, m) , 7 .58-7.27 (9H, ire , 6.99 (1H, d, J = 7.8), 5.2 3 (1H, 30 3), 5 .23-5.11 (IH, mi , 4 . 89 (1H, m) , 4.76 (IH, d, 3 = 11.3 i . , 4.55 (1H, d ? = 11. 4) , 4.5S-4.43 (2H, m), 3 . ΙΟ- 2.9 6, 2..81-2..69, 2 , 4 6 -- 2.3 7, 2.16-1.66 (10H, 4m}, ι .. 2 7 ΑΡ Ο Ο 12 8 Ο - 243 - (1Η, d, J = 17.8). Anal. Calcd for Ο28Η30Ν4θ7 · 0.5H2O: C, 61.87; H, 5.75; N, 10.32. Found: C, 61.88; H, 5.70;N, 10.33. MS (ES+, m/z) 535 (M+ + 1, 100%). (2100o) (containing about 7% of (2S)) was prepared by asimilar method to that described for compound 213e to 10 afford a white glassy solid (81%): mp. 115-7 °C; [a]D23-121.8° (c 0.11, CH2C12); IR (KBr) 3326, 1792, 1659,1535, 1421, 1278, 1257, 1124, 978; 1H NMR (CDC13) δ 7.82(2H, m) , 7.58-7.24 (8H, m), 6.90 (1H, d, J =7.3), 6.49(1H, d, J = 7.7), 5.57 (1H, d, J = 5.5), 5.11 (2H, m) , 15 4.91 (1H, d, J = 11.4) , 4.57 (1H, d, J = 11.1), 4.81- 4.68 (1H, m), 4.65-4.54 (1H, m) , 3.18-2.71 2.52-2.30, o /λ» 1 2.05-1.62 (11H, 3m). Anal. Calcd for 028Η30Ν407·0.5H2O: V\l C, 61.87; H, 5.75; N, 10.32. Found: C, 61.70; H, 5.71; 4- N, 10.15. MS (ES , m/z) 535 (M + 1, 94.3%), 557 (100%). co α»

(550n) was prepared by a similar method as compound213e to afford a mixture of diastereoisomers (65/35) as 20 a tan powder (390mg, 28%): mp. 139-145 °C; [a]D23 -104°(c 0.2, MeOH); IR (KBr) 3318, 2405, 2369, 1792, 1660,1591, 1549, 1484, 1422, 1257, 1117; 1K NMR (D6-DMSO) δ AP C Ο 1 2 8 Ο - 244 - i 0 . -3. 1 31 , s), 8.80 J - 6.6), 8.59 UH 7.79 (1H, m), 7.61 5.61 (Ο .3 5H, d, J = {Ο . 35Η, κι) , 5.08-5.04.67-4.61 {0.35Η, κι)

2nd, 3.80-3.59(7Η, κι) , 2.10 { MS (ES+) 528. Ο,65Η, ά, J = 6.6) , 8.58 (0.5, 0=7.0), 8.06 (1Η, bs) ,.57 (1Η, m), 7.47-7.39 ί1Η,5.0), 5.37 (0.65Η, bs), 5.176 (0.65Η, m), 4.92-4.86 (1Η,ί 4.47-4.41 (0.65Η, κι), 4.28(2Η, κι), 3.23-2.75 (3Η, κι),3Η, s), 1.25 and 1.17 (3Η, 2 d, -5,14 ϋί. } , "4.11

10 550ο (550ο) was synthesized by a similar method as compound213e to afford a colourless solid (1.071g, 80%) : rap.155-70 S'C; [a]D22 -75.3° (c 0.26, CH2C12) ; IR (KBr)2941, 1791, 1658, 1545, 1420, 1341, 1312, Έ NMR (CDC13) 6 9.45 (0.5H, s) ,.62 (1H, m), 7.49-7.39 (2H, m), r ο i 118) O '3 . 1118,)0.SH, ysy, /43) , 7.6-7.2 6 (1H, nt), (0.5 Η, s) , 5.4.76-4.49 (1H, mI, 3.72-3.5 3 ilH, m), 2.20 -1 ,1 9 (3H, ml! . 7.18-7.03 (3H, m), 5.49 (0.5H, 26......5.13 (1H, κι), 4.90-4.83 (0. κι) , 4.42-4.35 (0.5H, m) , 3.97-, m) , 3.35-2.64 (4H, m) , 2 ..2 (5H, m) , 1.69-1.50 (2H, rn

*6210/86 /y/dV ΑΡ ε ο 1 2 8 ο - 245 - Ο

550ρ (550ρ) was prepared by a similar method as compound 213e to afford a mixture of diastereoisomers as a white 94 foam (820mg, 47%) : [ot]D _75 <c °·16' CH2C12); IR5 (KBr) 3401, 2937, 1791, 1657, 1609, 1539, 1505, 1423, 1277, 1177, 1118; 1H NMR (CDCl3) δ 8.07-8.05 (1H, m) , 7.67 (2H, d, J = 7.9), 7.38-7.29 (2H, m), 6.80 (2H, d, J = 8.5), 5.49 (0.5H, d, J = 4.6), 5.23 (0.5H, bs),5.24-5.20 (1H, m), 5.12-5.08 (1H, m), 4.68-4.29 (2H, 10 m) , 3.92-3.45 (3H, m), 3.32-2.30 (2H, m), 2.80-1.56(11H, m), 1.21 (3H, t, J = 7.OH).

MeSO2—NH

O

286, 505b-e

OH O

Η O ΑΡ/Γ/ 9 8 : a 1 2 8 4 AP C Ο 1 2 3 0 - 246 - compound R 503a 504 a 286 5O3.b Me 504b \ Y 505b Y° Pt/ "N OPh 503c I 504 c A » rn^ «» i) j o \ .OPh 5 0 3d Y 504d J f) R/4 *->* t»/ *4# 50.3e 504e y 50 5e Me / ΑΡ/Γ/ 9 8 .D 1 2 9 4 (503a) was prepared from 212b and (35,4/?) t-butyi (N- allyloxycarbonyl)-3~amino~4-hydroxy-5-(1-naphthoyloxy) pentanoate by the method described ie 0 (213e) to afford 533mg (81%) of an off-white foam: ialD22 -31.4° (c 0.5, CH,Clo); IR(KBr) 3342, 2976, 1664, 1323, 1278, 123 ... , d, u 8 ... (3K, m( , 7.23 (1H, d5,30 (1H, m) , 4.59--4(IK, brcO, 3.29 (IK,2 .. 6 9--2.50 ( 3K, m) , 2 1153, 1137. KI NMR (CDCS.21 (1H, dd, J = 1.3, m,7.88 (1H, d, J = 8. 6), 7. = 8.6), 5.96 (1H, d, J == (5H, m), 4.24 (1H, m) , 3, 2.95 (1H, m) , 2.93 (3H, (1H, m), 1.96 (4K, m), 1 APC 0 1280 - 247 - (1H, m), 1.41 (9H, s). Anal. Calcd forc31h40n4°10s,°-25h2° : c> 55.97; H, 6.14; N, 8.42.Found: C, 55.90; H, 6.11; N, 8.23. M.S. (ES+) 683(M+Na, 100%), 661 (M+1,39), 605 (78). 5 (504a) was synthesized from 503a via method used to 10 15 20 prepare 216e from 215e to afford 446mg (91%) of acolourless foam: [a]D21 -111.6° (c 0.5, CH2C12); IR(KBr) 3319, 2978, 2936, 1723, 1670, 1413, 1370, 1329,1278, 1246, 1153. 1HNMR (CDC13) δ8.87 (1H, d, J = 8.9), 8.29 (1H, d, J = 7.2), 8.06 (1H, d, J = 8.3), 7.90 (1H, d, J = 8.2), 7.66-7.48 (3H, m), 7.37 (1H, d, J = 8.1), 5.61 (1H, d, J = 9.0), 5.31 (1H, m), 5.22(1H, AB, J = 16.9), 5.09 (1H, AB, J = 16.92), 4.99 (1H,m) , 4.65-4.43 (2H, m), 3.28 (1H, m), 2.96 (3H, s), 2.86(2H, m), 2.59 (1H, m) 2.38 (1H, dd, J= 6.8, 13.2),2.21-1.70 (6H, m), 1.45 (9H, s). Anal. Calcd forc31h38n4°10S,°-25h2°· C' 56.14; H, 5.85; N, 8.45.

Found: C, 56.11; H, 5.83; N, 8.29. M.S. (ES+) 657 (Μ-Ι, 100%) . (286) was prepared from 504a by the method described for 217 to afford 356mg (93%) of a white powder: mp120-123 °C; [a]D23 -121° (c 0.194, CH2C12); IR (KBr)

3314, 2937, 1722, 1663, 1412, 1328, 1278, 1245, 1195,1132. Ti NMR (d6-DMSO) δ 12.63 (1H, brs), 8.94 (1H, d, J 25 = 7.4), 8.78 (1H, d, J = 8.6), 8.26 (2H, m), 8.11 (1H, d, J = 8.0), 7.77-7.62 (4H, m), 5.28 (2H, s), 5.21 (1H,m) , 4.82 (1H, m), 4.44-4.29 (2H, m), 3.31 (1H, m) , 2.98(3H, s), 2.98-2.86 (2H, m), 2.72 (1H, dd, J = 7.3, 16.9), 2.40 (1H, m), 2.24-1.84 (4H, m), 1.69 (2H, m). 30 Anal. Calcd for C27H30N4O10S·Η2Ο : C, 52.25; H, 5.20; N,9.03. Found: C, 52.11; H, 4.97; N, 8.89. M.S. (ES+) 601 (M-l, 100%). AP V 012 8 0 - 248 - (503b) was synthesized by a similar method as compound213e, to afford an off-white powder (67ling, 88%} : mp.90-120‘C; IR (KBr) 3345, 2977, 1727, 1664, 1532, 1450,1423, 1369, 1323, 1310, 1276, 1257, 1154, 1101, 290, 766; XH NMR (CDC13) δ 7.61-7.55 (2H, m) , 7.51-7,42 (3H,mi, 6.86 (IK, d), 5.69 (1H, d), 5.21 (1H, m), 4.64-4.38 (2H, m) , 4.15-4.05 {3H, m), 3.84 (1H, s), 3.31-3.14 (2K, mi, 2.97-2.87 (IK, m), 2.94 (3H, s), 2.76 (3H, s), 2.64-2.48 (3HZ m) , 2.39-2.29 (1H, m), 2.04-1.61 (5H,mi. Anal. Calcd for ·Η2Ο: C, 52.46; K, 6.11; N, 9,87; S, 4.52. Found: C, 52.34; H, 5.92; N,9.56; S, 4.44. MS (ES+) 714 (47%), 692 (M+ + 1, Si), 636 (10 0 i . (504b) was synthesized by a similar method as compound216b to afford a colourless powder (601mg, 93%): mp.75-115 ’:C; [Ot] D23 -134° (c 0.26, CH2C12); IR (KBr) 3324,2977, 2935, 1730, 1676, 1525, 1452, 1422, 1369, 2317,1276, 1256, 1222, 1155, 1107, 990, 766; 1H NMR (CDC13) δ7.68-7.61 (2H, mi, 7,47-7.38 (3H, m), 7.32-7.24 PH, mi , ί 3.56 (1H, d), 5. 3 6 - 5.24 (1H, m) , 3 5.04 (1H, di , 4 . (IE, d), 4.86-4.77 ( 1H, m) , 4.64-4.39 (2H, m), 3.32- 3.17 (IE, m), 2.97-2 .65 (1H, m), 2.93 (3H, s), 2 .. ’ ' b (3H, s), 2.80-2.71 ( IK, m), 2.65-2.49 (1H, m), 2 ., 41 - 2.30 (1H, m) , 2.12-1 . 61 (6H, m), 1.42 (9H, s) . Anal . AP/-"/ 9 8 / ϋ 1 2 9 4

Calcd for C31H39N5OliS’H2O: C, 52.61; H, 5.84; N, 9.90;S, 4.53. Found: C, 52.94; H, 5.69; N, 9.72; S, 4.51. MS (ES*i 712 (31%), 707 (100), 690 (M+ + 1, 41), 634(55 ) , (505b) was synthesized by a similar method as compound217 to afford a colourless powder (499mg, 96%): mp. 95-145 °C; Eoc]d22 ~137c (c 0.12, MeOH) ; IR (KBr) 3323, 2936, 1732, 1665, 1529, 1452, 1421, 1312, 1275, 1256, AP C Ο 12 8 Ο - 249 - ) 10 5 1221, 1183, 1153, 1135, 1101, 990; 1HNMR (CD3OD) 6 7.67- 7.56 (2H, m) , 7.49-7.38 (4H, m), 5.23- 5.12 (1H, m) , 5.02 (1H, d) , 4.79-4.73 (1H, m), 4.52- 4.34 (3H, m) , 3.48-3.25 (2H, m), 3.03-2.85 (2H, m), 2.94 (3H, s) , 2.74 (3H, s), 2.79-2.66 (1H, m), 2.52- 2.38 (1H, m) , 2.29-2.14 (1H, m), 2.04-1.70 (4H, m). Anal . Calcd for C27H31N5°1 1S»H2O: C, 49.77; H , 5.18; N, 10.75; S, 4.92. Found: C, 49.83; H, 5.01; N, 10.27; S, 4.84 . MS (ES+) 746 (42%), 632 (M - 1, 100), 386 (60). Accurate mass calculated for C27H32N5O11S (MH+): 634.1819. Found:634.1807.

(503c) was synthesized by a similar method as compound213e to afford a colourless solid (446mg, 84%): IR ,'9 8. 1 29 4 15 (KBr) 3345, 2976, 2935, 1727, 1664, 1603, 1535, 1483, 1451, 1416 , 1395, 1369, 1328, 1297, 1277, 1237, 1155, 1135, 1076 , 990, 755; NMR (CDC13) 6 7.98-7.89 (1H, m), 7.55-7.45 (1H, m), 7.39-7.18 (3H, m) , 7.14-7.07 (1H, m), 7.00-6 .90 (3H, m), 6.75 ( 1H, d), 5.57-5.50 (1H, m), 5.21-5.09 (1H, m), 4.64-4.42 (2H, m) , 4.36-4.12 (3H, m), 3.95-3.87 (1H, m) , 3.39-3.18 (1H, m) , 3.00-2.82(1H, m), 2.95 (3H, s), 2.69-2.48 (3H, m) , 2.42-2.28(1H, m), 2.07-1.62 (6H, m), 1.42 (9H, s) . Anal. Calcdfor C33H42N4OnS-H2O: C, 54.99; H, 6.15; N, 7.77; S,4.45. Found: C, 54.95; H, 5.95; N, 7.34; S, 4.20. MS(ES+) 725 (26%), 720 (47), 703 (M+ + 1, 34), 433 (100),403 (89) .

(504c) was synthesized by a similar method as compound 216e to afford a colourless powder: mp. 85-100 °C; [a]D22 -91.3° (c 0.52, CH2C12); IR (KBr) 3328, 2978, 30 2935, 1732, 1669, 1603, 1524, 1483, 1450, 1396, 1369,

1296, 1276, 1237, 1155, 1132, 1082, 989, 755; 1H NMR (CDC13) 5 8.03-7.98 (1H, m) , 7.52-7.44 (1H, m), 7.37-7.07 ΑΡ Ο Ο 1 2 8 Ο 20 - 250 - CSH, m) ,· 7.01-6.92 (3Η, m), 5.52 (IK, d) , 5.28-5,20 (1Η, m) , 5.06-4.84 (3Ε, m) , 4.64-4.39 (2Η, π»), 3.32- 3.14 £1Η, m) , 2.99-2.88 (1Η, m) , 2.94 (3Κ, s), 2.65- 2.45 (2Η, ηι) , 2.39-2.29 (ΙΗ, m) , 2.12-1.58 {6Η, ιη.) , 1.40 (9Η, sj . Anal. Calcd for C33H4QN4O], ]S: C, 56,56; H, 5.75; N, 8.00; S, 4.58. Found: C, 56.37; H, 5,84; N, 7,69; S, 4.37. MS (ES+) 723 (30%), 718 (100),, 701(M+ + 1, 23), 645 (59), (505c) was synthesized by a similar method as compound217 to afford a colourless foam (252mg, 72%): mp. 30-125 °C; [ajD23 -133° (c 0.11, MeOH) ; IR (KBr) 3314,2938, 1792, 1734, 1663, 1604, 1535, 1483, 1448, 1415,1250, 1132, 756; “H NMR (Dg-DMSO) δ 8,81-8.76 (IB, m) ,7.92 (IB, d) , 7.68-7,.54 (2H, m) , 7.41-7.25 (3H, mj ,7.16-6.91 (4H, m), 5.13-4.98 (2H, m) , 4.72-4.63 (1H,m) , 4.37-4.21 (2H, m) , 2.92 (3H, s), 2.90-2.60 (3H, m),2.35-2.26 (1H, m), 2.17-2.05 (2H, m), 1.99-1.80 (2H,m) , 1.61-1.50 (1H, np.Anal. Calcd for C29H32N4°11S*0· 5H2O: C, 53.29; H, 5,09; N, 8.57; 5, 4.90. Found: C, 53.57; h, 5.18; N, 8.32; S, 4.75. MS(ES+) 643 (M - 1, 100%) . AP/.~,'9 8/fi 1 28 4 (503d) was synthesized by a similar method as compound2i3e to afford a colourless solid (563mg, 90%) : IR(KBr) 3349, 2978, 2935, 1724, 1664, 1583, 1536, 1489,1443, 1370, 1327, 1271, 1226, 1189, 1155, 1073, 755; XH NMR (CDCI3) 57.77 (1H, d) , 7.67 (1H, m) , .45- 7.10 (6K, m) , 7.00 025, d) , 5.93-5.80 (1H, m) , 5, 5.30 (1H, m) , 4.63- - 4 , 2 4 (5H, m), 4.15-4.09 (1H, m ; ; η... _ ο p (1H, m) , 2.98 - 2.74 (1H, m), 2.94 (3H, s 2 . "0-2.4 7 ( 3K, m) , 2 . 4 0 - -2.30 (1H, m) , 2.15-1.60 ( m), 1.42 (SH, s) . Anal . Calcd for 033Η42Ν4θιτ.5Ήρ 54.99; K, 6.15 ; S, ? "0 · S, 4.45. Found: C, 54.4. 30 ΑΡ Ο Ο 1 2 8 Ο - 251 - 5.88; Ν, 7.49; S, 4.50. MS (ES+) 725 (19%), 720 (91),703 (Μ+ + 1, 74), 647 (76), 629 (100), 433 (78). (504d) was synthesized by a similar method as compound216e to afford a colourless powder (466mg, 85%): mp. 5 75-100 °C; Γ , 22 [a]D -99.3 ° (c 0. 60, ch2ci2); IR (KBr) 3335, 2978, 2937, 1728, 1669, 1584, 1525, 1487, 1444, 1416, 1369, 1328, 1272, 1227, 1188, 1155, 989, 754; 1H NMR (CDCI3) δ 7.82· -7.77 (1H, m) , 7.66-7.65 (1H, m), 7.46-7.32 (4H, m), 7.26-7.10 (2H, m), 7.04-6.98 (2H, 10 m), 5.68 (1H, d), 5.37-5.31 (1H, m), 5.11 (1H, d) ,5.02-4.88 (2H, m), 4.66-4.42 (2H, m), 3.35-3.17 (1H,m), 2.98-2.89 (1H, m), 2.96 (3H, s), 2.84-2.78 (1H, m) ,2.72-2.47 (1H, m), 2.42-2.32 (1H, m), 2.14-1.58 (6H,m) , 1.43 (9H. s) . Anal. Calcd for C33H4QN4OHS: C, 15 56.56; H, 5.75; N, 8.00. Found: C, 56.36; H, 5.82; N, 7.71. MS (ES+) 723 (56%), 718 (90), 701 (M+ + 1, 36),645 (100). (505d) was synthesized by a similar method as compound217 to afford a colourless foam (353mg, 73%): mp. 80- 20 115 °C; [a]D -138° (c 0.11, MeOH); IR (KBr) 3327, 2937, 1728, 1666, 1584, 1529, 1487, 1443, 1413, 1328,1273, 1227, 1189, 1155, 1134, 989, 754; 1H NMR (Dg- 9 8 / J) 1 2 9 4 DMSO) δ 8.82 (1H, d) , 7. 76-7.72 (1H, m) , 7.61-7.53 (2H m) , 7 .48-7.32 (4H, m) , 7.24-7.17 (1H, m), 7.11-7.06 25 (2H, m), 5.14-5.06 (3H, m), 4.73-4.64 (1H, m), 4.38- 4.24 (2H, m), 2.92 (3H, s), 2.89-2.61 (3H, m), 2.38- 2.27 (1H, m), 2.19- 2.06 (2H, m), 2.02- 1.79 (3H, m), 1.63-1.52 (1H, m) . Anal. Calcd for C29H32N4O11S· 0.5H2O:C, 53.29; H, 5.09; N, 8.57; S, 4.90. Found: C, 53.24; 30 H, 5.14; N, 8.34; S, 4.86. MS (ES+) 643 (M - 1, 100%),385 (62) . 10 15 ) 20 25 - 252 - (503e) was prepared by a similar method to thatdescribed for compound 213e, to afford an off whitesolid (70%): mp. 100-103 °C; [a]D25 -84.0° (c 0.05,CH2C12); IR (KBr) 3455-3359, 1722, 1664, 1514, 1368,1328, 1278, 1247, 1155; 1H NMR (CDC13) δ7.52 (IK, m) ,7.06-6.99 (2H, m) , 5.69 (1H, d, J = 9.0), 5.23 UH, m) ,4.61-4.16 <6H, m) , 3.36-3.19 (1H, m) , 2.96 (3H, s) ,2.67-2.49, 2.42-2.32, 2.06-1.89, 1.69 (10H, 4m), 1.43(9H, s) . (504e) was prepared by a similar method to thatdescribed for compound 216e, to afford a white solid (98%) : mp. 91-98 °C; fot]D25 -112.5°C (c 0.06, CK2Ci2);IR (KBr) 3453-3364, 1727, 1668, 1513, 1420, 1368, 1245,1155; NMR (CDC13) 67.54 (1H, d, J= 5.3), 7.IS (1H, d, J = 7.13), 7.05 (1Ή, d, J = 5.4), 5.42 (1H, d, J =8.9), 5.25 (1H, m), 5,02 (2K, m) , 4.96-4.87 (1H, m),4.65-4.42 (2H, m) , 3.34-3.17 (1H, m) , 2.97-2.93 (1H,m), 2.97 (3H, s), 2.87-2.78, 2.73-2.50, 2.38-2.32,2.13-1,88, 1.69-1.60 (9H, 5m), 1.44 (9H, s) . (505e). A solution of 217 (0.33g, 0.51mmol) in drydichloromethane (3mi) was cooled (ice/water) withprotection from moisture. Trifluoroacetic acid (2ml)was added with stirring. The solution was kept at roomtemperature for 2h after removal of the cooling bath,then concentrated in vacuo. The residue was evaporatedthree times from di chloromethane, triturated withdiethyl ether and filtered. The solid was purified byflash chromatography (silica gel, 0-6% methanol inaichloromethane) to give the product as a white glassysolid (0.296g, 98%): mp 110-122 °C; ia]D22 -163.1° (c0.1, CH3OK); IR (KBr) 3514-3337, 1726, 1664, 1512, 1420, 1245, 1152, 1134, 990; 1H NMR (CD3OD) δ7.79 (1H, AP/.U9 e.'JH 28 4 30 Μ?-,· ΑΡ ε ο 1 2 8 Ο - 253 - J = 5.2 ), 7.12 (1H, d, J = 5.2), 5. 20 (1H, m), 5.02- 72 (2H, : m, masked by H2O), 4.59-4 .32 (3H, m), 3. .48- 29, 3.08 -2.75, 2.50-2.41, 2.31-2. 22, 2.08-1.89, 1.72- 63 (UH, 6m) , 2.95 (3H, s) . 10

507a-c,g 506a-c,g compound R1 506a 507a PhC(O)- 506b 507b MeS(0)2- 506c 507c MeOC(0)- 506g 507g CH3C(0)- 15 (506a). A solution of 212e (321mg, 0.929mmol) and (3S) t-butyl 3-amino-5-diazo-4-oxopentanoate (198mg,0.929mmol) in dichloromethane (3ml) was cooled to 0'and N,N-diisopropylethylamine (0.16ml, 1.86mmol) and[2-(lH-benzotriazol-l-yl)-1,1,3,3-tetramethyl-uronium 20 tetrafluoroborate (328mg, 1.02mmol) were added. Thesolution was stirred overnight at room temperature,diluted with ethyl acetate and washed with 1M NaHSC>4(x2), aqueous NaHCC>3 (x2), brine, dried over magnesiumsulphate and evaporated. Chromatography on silica gel 25 eluting with ethyl acetate gave 506a (425mg, 85%) as a AP V Ο 1 2 8 0 - 254 -

colourless foam: [<x] p.....' -124.9° (c 0.2, CH2C12) ; IR (KBr) 3332, 2111, 1728, 1658, 1532, 1421, 1392, )5367,1279, 1256, 1155; 1H NMR (CDC13 )5 7.82 (2H, m) , 7.49 (3H, m) , 7.28 (IH, d, J = 9.3), 7.05 (IH, d, J ’7.3) , 5 5.06 (IH, s), 5.18 (2H, m) , 4.78 (1H, m) , 4.62 f H, m) , 3.2 9 (IH, m), 3.08- -2.79 (3H, m) , 2.58 (IH, dd, J = 16.8, 5.6) , 2 . .20-1. .85 (4H, m), 1 .70 (IH, m) , 1. . 4 5 (9H, s). MS (ES+) 539.58 )M - 1, 97.9%) 529.59 (100). (506b) was prepared by a similar method as compound 10 506a. 74% as yellow orange solid: mp. 75 °C (decomp.); [ajD20 -92.0° (c 0.036, CH2C12); IR iKBr) 3438, "4, 2113, 1728, 1669, 1523, 1368, 1328, 1155; XH NMR(CDCI3) 67.48 (IH, d, J= 8.1), 5.83--5.68 (1H, m, } ,5.55--5.50 (IH, m) , 5.43-5.14 (1H, m) , 4.83-4.45 f.3H, 15 m), 3.40-3.19 (1H, m) , 2.98 (3H, s), 2.92-2.30 (4H, m),2.24-1.70 (6H, m), 1.43 (9B, s).

(506c) was prepared by a similar method as compound506a to afford a pale yellow foam (405mg, 82%) : (a]D 20 -144° (c 0.2, CH2C12);1728, 1674, 1530, 1459,1063; NMR (CDC13) δ 7(2H, m), 5.21-5.16 (IK,(3H, s ) ,- 3,35-3.18 RIH,2.30 (IH, m) , 2.09-1.66493. (506g) was prepared by IR (KBr) 3339, 2978, 2958, 21121415, 1367, 1274, 1252, 1.154, 23 (IH, d, J = = 8.2), 5.51-5.31 m) , 4.77-4.55 (3H, m), 3..68 m) , 3.04-2.51 (4H, m), 2.40- (5H , m), 1.45 (9H,s). MS (ES" = similar method as compound 506a. 81%: [a]D“S -246.7° (c 0.4, CH2C12); IR (KBr)3439, 2904, 2113, 172S, 1669, 1523, 1368, 1328, 1155; XH NMR (CDCI3) 5 7.32 (1H, d) , 6.43 (1H, d) , 5.50 (IH,s', , 5.22 (IH, in) , 4.94 (IK, m) , 4 ,ΊΊ (1H, m) , 4.65 (1H,πϋ , 3.24 (IH, ml, 3.03-2.52 (4H, m) , 2.36 (1H, ra>. AF/7.’S 8 Λ i 29 4 30 APt Ο 1280 - 255 - 2.10-1.64 (5H, m) , 2.02 (3H, s), 1.45 (9H, s). Anal.Calcd for C21H20N6O7: C, 52.69; H, 6.32; N, 17.05.

Found: C, 52.51; H, 6.27; N, 17.36. MS (ES+) 477(M+ -1, 100%). 5 (507a). 506a (3.0g, 5.55mmol) in dry dichloromethane (40ml) was cooled to 0’ and 30% hydrobromic acid inacetic acid (1.1ml, 5.55mmol) was added dropwise over4min. The mixture was stirred at 0' for 9min andquenched with aqueous sodium bicarbonate. The product 10 was extracted into ethyl acetate, washed with aqueoussodium bicarbonate, brine, dried (MgSO4) and evaporatedto give 2.97g (92%) of a colourless foam: [a]D23 -82.3°(c 0.23, CH2C12); IR (KBr) 3333, 1726, 1659, 1530, 1458, 1447, 1422, 1395, 1368, 1279, 1256, 1222, 1155, 15 728; 1H NMR (CDC13) 57.81 (2H, m) , 7.50 (3H, m) , 7.11 (1H, d, J = 8.0), 7.01 (1H, d, J = 7.4), 5.20 (2H, m),5.00 (1H, m), 4.06 (2H, s) , 3.28 (1H, m), 3.20-2.70(4H, m), 2.42 (1H, m), 2.10-1.85 (4H, m), 1.72 (1H, m) ,1.44 (9H, s) . Anal. Calcd for C26H33N4O7Br· 0.7H2O: C, 20 51.53; H, 5.72 N, 9.24. Found: C, 51.55; H, 5.52; N, 9.09. MS (ES+) 595, 593 (M+ + 1). (507b) was prepared by a similar method as compound507a. (68%) as an orange foam: [a]D20 -135° (c 0.053, ΑΡ/Γ/ 9 8 : Δ 1 2 S 4 ch2ci2 ) ; IR (KBr) 3429, 2944, 2935, 1723, 1670, 1458, 25 1408, 1327, 1225, 1154, 991; 1H NMR (CDC13) 5 7.38 (1H, d, J = 8.2) , 5.69 (1H, d, J = ; 9.3), 5,43-5.34 ( 1H, m), 5.07-4 .97 ( 1H, m) , 4.70-4.42 (2H, m) , 4.12 (2H, s) , 3.35-3 .17 ( 1H, m) , 3.10-2.69 (4H, m) , 2.98 (3H, s) , 2.43-2 .33 (1H, m) , 2.15-1.65 (5H, m) , 1.43 (9H, s) . 30 Anal. Calcd for C 20H31BrN4°8s : C, 42 .33; H, 5.51; N, 9.87. Found: C, 42.69; H, 5.52; N, 9.97. ΑΡ ί Ο 1 2 8 Ο - 256 - (507c) was prepared by a similar method as compound 2 0 507a to afford a pale yellow foam (320mg, 78*): :a]D-107° (c 0.2, CH2C12;.· IR (KBr) 3401, 2956, 1726, 1670,1526, 1452, 1415, 1395, 1368, 1276, 1251, 1155, 1064; 5 XK NMR (CDC13) δ 7.07 (1H, d, J = 7.6), 5.47 (1H, d, J =8.1), 5..21-5.16 (1H, nil, 5.03-4.94 (1H, m) , 4.75-4.56 (2H, m), 4.06 <2H, s) , 3.69 (3H, S) , 3.31-3.13 OS, m) , 3.03-2.92 (2H, m) , ώΐ . 81-2.58 (2H, m), 2.41-2.31 : IK, a), 2.10-1.66 (5H, a) , 1.4 4 (9H, s) . 10 (507g) was prepared by a similar method as compound 507a to afford a pale veliow foam (84%): [a]D-109.6° (c 0.1, CH2Ci2); IR (KBr) 3324, 1727, l<bb\ 1535, 1458, 1444, 1423, 1369, 1279, 1256, 1223, 1155; 1K NMR (CDC13) δ 7.12 (1H, d, J =7.8), 6.33 (1H, d, J = 15 7.5), 5.19 (1H, m,), 4.97 (2H, m) , 4.58 (1H, m), 4.06(211, s), 3.20 (1H, El, 3.05-2.69 (4H, m), 2.35 (IB, m) , 2.14-1.68 (5H, m), 2.03 (3H, s), 1.44 (9H, s) . Anal.Calcd for C21H31BrN4O7·0.3H2O: C, 46.99; H, 5.93; N,10.44. Found: C, 46.97; H, 5.90; N, 10.35.

O

508a, b

284, 285 ΑΡ/ΓΖ Si 8 .'ID 1 2 9 4 compound — R Cl 508a 284 | |i ΑΡ εΟ 12 8 ο

(508a). To a solution of 506c (547mg, lmmol) in DMF(4ml) was added potassium fluoride (145mg, 2.5mmol, 2.5 5 equiv). After lOmin stirring at room temperature, 2,6- dichlorobenzoic acid (229mg, 1.2mmol, 1.2 equiv) was added. After 3h reaction at room temperature, ethyl acetate (30ml) was added. The solution was washed with a saturated solution of sodium bicarbonate (30ml), 10 brine, dried over MgSO4 and concentrated in vacuo to2 2 afford 590mg (90%) of a pale yellow foam: [a]D -85° (c 0.20, CH2C12); IR (KBr) 3400, 2956, 1737, 1675, 1528, 1434, 1414, 1368, 1344, 1272, 1197, 1152, 1061; XH NMR (CDC13) δ 7.36-7.33 (3H, m), 7.04 (1H, d, J = 15 8.0), 5.46 (1H, d, J = 7.8), 5.19-5.16 (1H, m), 5.08 (2H, AB) , 4.97 - 4.55 (1H, m), 4.69-4. .55 (2H, m), 3.68 (3H, s) , 3.30-3.10 (1H, m), 3. 01-2.50 (4H, m), 2.40- 2.33 (1H, m) , 2.15-1.60 (5H, m ), 1.44 (9H, s) . Anal. Calcd for C28H34Cl2N4O10: C, 51 .15; H, 5.21; N, 8.52. α»

CM 4» co u 20 Found: C, 51.35; H, 5.32; N, 8.56. (284) was synthesized from 508a via method used toprepare 505 from 504 which afforded 330mg (65%) of a 2 ft white solid: mp. 115 °C (decomp.); [a]D -107° (c 0.2,CH2C12); IR (KBr) 3340, 2954, 1738, 1664, 1530, 1434, 25 1272, 1198, 1148, 1060; 1H NMR (D6-DMSO) δ 8.91 (1H, d, J = 7.2H), 7.67-7.63 (3H, m), 7.54 (1H, d, J = 8.0),5.24 (2H, s), 5.20-5.15 (1H, m), 4.79-4.70 (1H, m),4.46-4.37 (2H, m), 3.58 (3H, s), 3.33-3.20 (1H, m),2.94-2.55 (4H, m), 2.30-1.60 (6H, m). Anal. Calcd for APC 0 1280 - 258 - C24h26l-12n4°10*h2O: 46.54; H, 4.56; N, 9.05. Found: C, 4 6.3 c>' K, 4.14 ; N» b . 8 8 . (508b) was synthesized by a similar method as compound 22 508a to afford a pale yellow foam (460mg, 82%): ,ajp -115® (c 0.20, CH2C121675, 1528, 1514, 146 IR (KBr) 3413, 2960, 1729,1421, 1368, 1265, 1116, 1096;~E NMR iCDCl3) δ 7.27-7,03 <4H, m) , 5.48 (IK, d, 0 ==8.2), 5..20-5.14 (1H, in.}, 5.04 (2H, AB), 4.93-4.86 (1.H, m) , 4.60-4,56 (2H, m) , 3.77 (3H,3.00-2.56 (4H, m) , 2..37 (6H, s),1.95 (9K, s), 2.41-2,25 (1H, m) . s), 3.32-3.15 (1H, m.) ,2.19-1.77 (5H, m), MS (ES+) 617., (285) was synthesized by a similar method as compound284 to afford a while solid (3Q3mg, 78%): mp. 110'C(decomp.); ia]D -128° (c 0.10, CH2Cl2); IR (KBr) 3339,2953, 1731, 1666, 1529, 1420, 1266, 1248, 1115, 1070; 1K. NMR (Dg-DMSO) δ 8.90 (1H, d, J = 7.4), 7.54 (152 d, 0 = 7 » 9) , 7.36-7.28 (IK, m) , 7 .17-7.14 (2H, m), 5.19-5.15 (3H, m) , 4.84-4.74 (IE, m) / 4.45-4.37 (2H, m) , 3,59 ί 3H, s) , 3.45-3.25 (IE, m) ? 2.95-2.64 (4H, m) O 2. it / d. , (6H, S) , 2.30-1.60 (6H, m) - Anal. Calcd for c26H32N4°10’H2O: C' 53.98; H, 5.92; N, 9.68. Fot. C, :: o r~ <. -J 0; H , 5.52; N, 9,49 MS (ES+) 559. ΑΡ/Γ; 9 G ΰ 1 2 8 4

‘01 Bu

''R 509a-d

510a, 280, 283, 510d ΑΡΰ 0 12 3 0 - 259 - compound R 509a s—/"jl 510a o 509b 280 509c ZY| 283 0 It 509d vs 510d v (510a). A solution.of 506a (2.27g, 4.2mmol) in drydichloromethane (50ml) was treated with 30% hydrobromicacid in acetic acid (1.84ml, 9.2mmol, 2.2equiv) at 0’C,under nitrogen. After lOmin stirring at 0'C thereaction was complete and a white solid crystallised inthe medium. The solid was filtered and washed withethylacetate and diethylether to afford 2.20g (100%) of[3S(IS,9S)] 5-bromo-3-(9-benzoylamino-6,10-dioxo- 1,2,3,4,7,8,9,10-octahydro-6H- pyridazino[1,2-a][1,2]diazepine-l-carboxamido)-4-oxopentanoic acid which was used without furtherpurification: 1H NMR (Dg-DMSO) 58.87 (1H, d, J = 7.3), ΑΡ/ΓΖ δ 8 . β 1 2 9 4

8.63 (1H, d, J = 7 .6) , 7.91-7.87 (2H, m) , 7.60-7.44 (3H, m) , 6.92 (1H, bs) , 5.14-5.09 (1H, m) , 4.92-4.65 (2H, m) , 4.43 (2H, AB) , 4.41-4.35 (1H, m) , 3.33-3.22 (1H, m) , 2.98 -2.90 (1H, m) , 2.89-2.57 (2H, m), 2.35- 2.15 (3H, m) , 1.99- -1.91 (2H, m), 1.75- 1.60 (2H, m) . A AP v 0 f 2 8 0 - 260 -

solution of the bromoketone (535mg, lmmol) in dry DMF (10ml) was treated with potassium fluoride (150mgy 2.5inmol, 2.5 equivi , under nitrogen. After 5minstirring at room temperature, 2-mercaptothiazole(liOmg, I.2mmol, l.requiv) was added. After overnightreaction ethylacetate (150ml) was added and the organicsolution was washed with brine, dried over magnesiumsulphate and reduced in vacuo. The residue wascrystallised in diethyl ether, filtered and purified onsilica gel using a gradient of MeOK ¢0% to 5%) indichloromethane. Evaporation afforded 344mg (60%) of awhite solid: mp. 90-95 eC (decomp.); [a]Dz° -82 e (c0.2, CK2CI2); IR (KBr) 3328, 2941, 1745, 1659, 1422, 1276, 1255, 1223, 1072; 1H NMR (D6-DMSO) δ δ. 92(IE, d, 0 = 7.6), S.6&amp; (1H„ d, J = 7..6), 7.98-7.90 (2E,m), 7.75-7.67 (1H, m), 7,64-7.50 (4H, m), 5.22-5.13(IE, m), 4.95-4.74 (2H, m) , 4.58-4.38 (3H, m) , 3,,52-3.19 (1.R, m), 3.05-2.65 (4H, m) , 2.40-1.50 (6H, mi.Anal. Calcd for C25H2-?N5O4S2*H2O: C, 50.75; H, 4.94 N,11.84. Found: C, 51,34; H, 4.70; N, 11.58. MS (ES+)572 . (509b) . 507a (lOOrags, 0.17mmol) in dry dimethyl formamide (1,5ml) was treated with 1-phenyl-lH-tetrazoIe-5-thiol (33mg, 0.187mmol) and potassiumfluoride (15mg, Q.34mmci). The mixture was stirred atroom temperature for 2h, diluted with ethyl acetate,washed with aqueous sodium bicarbonate (x2), brine,dived (MgSO4) and evaporated. The product was ptirifiedby flash chromatography on silica gel eluting withethyl acetate to give 103mg (88%) as a colourless foam:2.2° (c 0.1,, yw2Ci2) ; IR (KBr) 3334, 172fc, : ex - t1660, 1501, 141 , 1394, 1368, 1279, 1253, :3 AP t Ο 1 2 8 Ο - 261 - 1H NMR (CDCl3)67.82 (2H, m) , 7.60-7.40 (8H, nt), 7.39(1H, d, J = 8.1), 7.05 (1H, d, J = 7.3), 5.26 (1H, m) ,5.15 (1H, nt), 4.99 (1H, m) , 4.60 (2H, m), 4.30 (1H, d,J= 17.2H), 3.32 (1H, m), 3.10-2.75 (4H, m) , 2.40 (1H, 5 m), 2.24 (1H, m) , 1.90 (3H, m) , 1.75 (1H, m), 1.44 (9H, s). MS (ES+) 691.47 (M+ + 1). (280) was synthesized via method used to prepare 505from 504. 509b (98mg, 0.142mmol) in dichloromethane (lml) was cooled to 0' and trifluoroacetic acid (1ml) 10 was added. The mixture was stirred at 0' for 15min andat room temperature for 30min before evaporation underreduced pressure. The residue was triturated with drytoluene and evaporated. Chromatography on silica geleluting with 10% methanol in dichloromethane gave a 15 colourless glass which was crystallised from dichloromethane/diethyl ether to give 62mg (69%) ofcolourless solid: mp. 145 °C (decomp.);[a]D22 -80.9° (c0.1, CH2C12); IR (KBr) 3400, 1727, 1658, 1530, 1501,1460, 1445, 1416, 1280, 1254; 1H NMR (CDCl3)68.00 (1H, ) 20 m) , 7.79 (2H, d, J = 6.7), 7.58-7.30 (9H, m), 5.25 (2H, m) , 4.94 (1H, m), 4.53 (2H, m), 4.35 (1H, m), 3.35 (1H, m) , 3.01 (3H, m), 2.73 (1H, m), 2.38 (1H, m), 1.98 (4H, m) , 1.64 (1H, m) . Anal. Calcd for C^I^NgC^S· 0.2TFA: C, ΑΡ/Γ/9 8/ΰ 1 29 4 53.71; H, 4.63 N, 17.04. Found: C, 53.97; H, 4.92; N, 25 16.77. MS (ES+) 633.55 (M+ - 1). (509c) was prepared by a similar method as compound2 2 509b to afford a colourless glass (34%) : [a]D -77.1°(c 0.25, CH2C12); IR (film) 3311, 1724, 1658, 1603, 1578, 1536, 1488, 1458, 1426, 1368, 1340, 1279, 1256, 30 1231, 1155, 707; 1H NMR (CDCl3)5 8.29 (2H, m), 7.84 (2H, m), 7.48 (4H, m), 7.22 (3H, m), 5.20 (2H, m), 4.90(2H, m), 4.58 (1H, m), 3.29 (1H, m), 3.20-2.70 (4H, m), APC 0 1230 - 262 - 2.38 (2K, m) , 1.96 ί,4Η» ία) , 1.68 (1H, m) , 1.42 UH, s) .MS (ES+) 608.54 (M + 1). (283) was prepared by a similar method as compound 280 to afford a colourless foam (100%) : nip. ~125 °C; ία] 19 -84,1° (c 0.1, 20% MeOK/CH2Cl2)/ IR (KBr) 3401, 1736,1663, 1538, 1489, 1·1 1425, 1281, 1258, 1200, 1134; £H NMR (CD3OD/CDC13) δ 8.38 (2H, m) , 7.84-7.40 (8H, m),5.16 (4H, m), 4.80 (IB, m), 4.56 (1H, m), 3.50 . m), 3.12 (2H, mi, 2.82 (2K. m), 2.37 (1H, m) , 2.10-1.6535H, m) . Anal. Calcd for ^27^29^5^8 ’ 0 · 4H2O: C, 51.717; H,4.61; N, 10.41. Found: C, 52.19; H, 4.93; N, 9.99.(509d) was synthesized by a similar method as compound509b to afford a colourless solid (49.6mg, 82%): “Ή NMR(CDC13) δ 8.02 (1H, si , 7.95-7.86 (IK, m), 7.84-7.7615 (255 mi , 7.62-7.35 (4K, rn.) , 7.22-7.07 (1H, m) , 6.43UK, di, 5.26-5.08 UH, m) , 5.03-4.72 (3H, ro) , 4,.66-4.50 <1H, m), 3.43-3.19 (1H, m), 3.15-2.97 (1H, mi,2.86-2.72 (3H, m) , 2..48-2.31 (1H, m) , 2.18-1,60 (SB,mi, 1.4 3 (9H, s) . 20 (510d) was synthesized by a similar method as compound 280 to afford a colourless solid (25.7mg, 57%): mp. ΑΡ/Γ7 9 8 ,'fl 1 2 9 4 140-8 0' C; IR (KBr) 3391, 2945, 1733, 1664, 1530, 1422, 13 63, 12 7 7, 1259, 1 a 6 a ; "Ή NMR (CD3OD) δ 8.23 (1H, si , " . 94 (IB, d) , 7.87 UH, d), 7.54-7.42 (3H, m), 6.48 UK, di , 5.22-5.15 UK, m), 4.57-4.46 (1H, m), 3.62- 3.41 UK, m) , 3.22 - 3.1 3 UH, m) , 3.02- 2.81 (2H, mt, 2.7 ο- 1.8 G (6H, ml . An 5.1 . Calcd for C2 gHigNgOg’1·5H2O: ι:, 54 '3, \ '· « K, 5.35; Nt 1 4 .61. Found: C , 54.14; H, 5,35; N, 13 . 04 . MS (ES" ) 551 (M - 1, 100%). Accurate mass caiculatea for C2eU . (MH ) : 553.2047. Found:553,5080, 30 AP V Ο 12 8 Ο - 263 -

504f-h 505f, 280b, 283b

compound R 504f 505f o 504g 280b Q N-N A > 504h 283b V ΑΡ/Γ/8 8.Λ 1 28 4 (505f) was prepared by a similar method as compound 10 508a using 507b and 3-chloro-2-hydroxy-4H- pyrido[1,2-a]pyrimidin-4-one and directly followed by the hydrolysis of 504f with trifluoroacetic to afford a7 Π

tan powder (65mg, 30%): [ a 3 d -128° (c 0.10, MeOH); IR (KBr) 3414, 2928, 1667, 1527, 2459, 1407, 1328, 1274, 1153, 1134; 1H NMR (MeOD) δ 9.35 (1H, d, J = 6.6H) , (1H, t, J = 7.2H), 7.99-7.95 (1H, m), 7.76-7.69 (1H m), 5.85-5.45 (3H, m) , 5.30-5.21 (1H, m), 4.93-4.66 (2E, m), 3.81-3.65 (1H, m), 3.66 (3H, m), 3.45-2.52 (4H, m), 2.52-1.71 ( 6H, m) . D. J. Hlasta et al., J. Med. Chem. 1995, 38 , 4687-4692. 15 AP C Ο 12 θ Ο - 2 64 - (504g) was prepared by a similar method as compound23 -112.7'" 509b, <&amp;3%) as a colourless foam: [a] 0.2, CH?C1?); IR (KBr) 3312, 1726, 1668, 1501, lob 1395 , 1369, 13 28, 1 .27 6, 1254 , 1155; 1H NMR (CDCl-u δ 7.59 ;5H, m) , 7.4 3 (1.K, d, J = 8. 0), 5.68 (IH, d, J »=»· 9.0), 5.37 (IE, m) , 4.95 (IH, m), 4.62-4.31 (4H, m) , 2 , 3 6 (IH, m) , 2.98 (3H, ε) , 2.88 (4H, m), 2.66 (IH, m), 2.42 (2K, m), 1.98 (IH, m j , 1.7 5 (IH, m), 1.43 (9H,s) . (280b) was prepared by a similar method as compound 10 280, (100%) as a colourless foam: mp. 120-5 °C; ία 25

i D -112.4° (c 0.1, CH2C1,); IR (KBr) 3328, 1730, 1664, 1529, 1501, 1410, 1328, 1277, 1219, 1153, 1134, 991; '4 NMR (CDCI3) 6 8.07 (IH, d, J = 7.8), 7 .58 (5K, s ; , i ;.4i (IH, d, J = 9.5), 5.32 (IH, m) , 5.04 (IH, m) , 4,10 (IH, 1 5 t 7 = 1 7,5) , 4.60 (35, m) , 3.50-2.9 (3H, m), 2, S3 (3H, S / > 2.45 (2H, m), 2.06 (4H, m) , 1.68 (IH, m) . (504h) was prepared by a similar method as compound23 509b (24%) as a colourless foam: [a] -101.01 0.2, CH2C12); IR (KBr) 3330, 1727, 1669, 1425, 1396,1369, 1328, 1276, 1256, 1231, 1155, 1137, 991; AH NMR(CDCI3) 58.28 (2H, hr d, J = 9.4), 7.71 (IH, d, J =7.9», 7.22 (2H, s), 6.03 (1H, d, J- 9.4), 5.36in), 4.95 (2H, m) , 4.52 (2H, m) , 3.29 (1H, m) , 3.07 (3H,3), 3.23-2.75 (3H, ini , 2.66-2.35 (2H, m) , 2.30-1.00»5H, ml , 1.42 (9H, s) . (283b) was prepared fay a similar method as compound. 280, (100%) as a co ±ou - 8 5 , 2° (c 0.1, 10 % CEu lewd , 1360, 1457, 1421 11S 9 , 1150, 1133, 991; m) , Ό.54 (2H, m), d 7 d dess foam: mo. 120 _ ° - .j

U. J D .· u c ,00, di NMR (CDCI3/CD3OD) δ δ.35 (2H,(2H, m) , 4.83 (2H, m), 4.in (2H, 30 ΑΡ ε· Ο 1 2 8 ο - 265 - m) , 3.43-2.77 (4Η, m), 2.97 (3Η, s), 2.42 (2Η, m) ,2.C5-1.72 (5Η, m).

compound R 508c 511c 508d 280c 0 ^N-N A > 508e 283c /°YiXN V—· <*·».'

CO

Cft

I c; <c 10 (508c) was prepared by a similar method as compound 509b to afford 544mg (97%) of a pale yellow foam: [ajD20 -86° (c 0.19, CH2C12); IR (KBr) 3426, 2947, 1725,1669, 1551, 1418, 1383, 1253, 1155, 1064; 2Η NMR(CDC13) δ 8.4 9 (2H, d, J = 4.8), 7.13 (1H, d, J = 7.9) , 15 7.03-6.98 (1H, m), 5.47 (1H, d, J = 7.9), 5.23-5.19 (1H, m), 5.09-5.01 (1H, m) , 4.84-4.51 (2H, m) , 4.04 (2H, AB) , 3.69 (3H, s) , 3. 38-3.19 (1H, m), 3.06-2.64 (4H, m) , 2.40-1.76 (6H, m) , 1.43 (9H, s). Anal. Calcd AP001230 - 266 - for C25E34N6O8S: C, 51.89; H, 5.92; N, 14.52. Found; C,51.49; K, 6.04; N, 13.87. MS (ES+) 579. (511c) was prepared by a similar method as compound 280to afford 370mg ¢79%} of a white powder: rap. 105 Ή 5 (deed; iajDz2 -94° (c 0.20, CH2C12); IR (KBr) / 0v: , 2957, 1724, 1664, 1252, 1416, 1384, 1254, 1 ί :,. -.Λ 1063; "K NMR (Dg- DM30) 6 8.85 (1H, d, J = 7.8 ) , ϊ . .· f 2H, d, J = 4.7) , 7.53 (1H, d, J = 8. 0) , 7.28 - * 3 (1H m) , 5.21-5.17 (IE, si), 4.87-4.79 (1H, m) , 4.47-4. 10 (2H, m), 4.23 (2H, 223;» 3.58 (3H, s), 3.30-3.21 ( 1Η» m) , 2,95-2,50 (4H, rc), 2.35-1.60 (6H, m) . Anal. _'d for c21h26n6°8s,h2O: C, 46.66; H, 5.22 ; N, 15.55.

Found: C, 46.66; H, 5,13; N, 15.07. MS (ES+) 523, <E5~') 521. 15 (508d) was synthesized by a similar method as ;;r. -.; and 509b to afford a colourless solid (269ntg, 87%) : nip. SO-HO °C; [ajD2j -108 (c 0.60 CH2C12); IR (KBr) 3315, 297", 1727, 1688, 152", 1501, 1458, 1418, 1368, 1279, 1250, 1155, 1064; Ή NMR . (CDC13) δ 7.70 (1H, d) , id 63 20 7.53 (5H, m) , 5.84 (1H, d), 5.34-5.27 (1H, m) , 5.05- 4.92 (1H, m) , 4.7 8- •4.54 (3H, m), 4.38 (1H, d) , Id 66 (3d, s) , 3,37-3.19 (1H, m) , 3.07-2.94 (1H, m) , 2.51- Δ . c z (2 id, m), 2.71- •2.56 (1H, m), 2.40-2.30 (1H, nr, 2 , 1 9- 2.13 (1H, m) , 2.08- 1.68 (4H, m), 1.42 (9H, 3) . 2 5 (ES+) 667 (31%), 64 5 NO + 1, 100), 589 ¢62). (280c) was synthesized by a similar method as compound280 to afford a pale cream solid (203mg, 88%); inp, 105-130 c'C; ica^'2 -235" (c 0.11 MeOH) ; IR (KBr) 3342, 2 951, 1727, 1667, I'd·, 1501, 1459, 1416, 1276, 1252, 1192, 1062; "d~ NMR (Dg-DMSO) δ 8.89 (1H, di. 7.69(5h, s), 7.50 (1H, d), 5.18-5.11 (1H, m) , 4,79-4.69(lid nd , 4.57 (2H, sp 4.42-4.32 (1H, m) , 3.54 ddd 5),, - 267 - 2.92-2.63 (3Η, m), 2.21-1.82 (5Η, m), 1.65-1.57 (1Η,m) . MS (ES+) 587 (M- 1, 100%). (508e) was synthesized by a similar method as compound509b to afford a pale orange solid (199mg, 25%): mp. 5 80-120 °C; [a]D23 -89° (c 0.51 CH2C12); IR (KBr) 3333, 2978, 1726, 1669, 1578, 1536, 1478, 1426, 1368, 1277, 1253, 1232, 1155, 1064; 1H NMR (CDC13)5 8. 41-8.18 (2H, m) , 7 . 81 (1H, d), 7.26-7.20 (2H, s), 5.91 (1H, d), 5.24-5.16 (1H, m), 5.07-4.86 (3H, m), 4.81-4.51 (2H, 10 m) , 3.67 (3H, s) , 3.34-3.16 (1H, m) , 3.10-2.81 (3H, m) , 2.72-2.54 (1H, m), 2.41-2.31 (1H, m), 2.07-1.62 (5H,m), 1.47 (9H s). MS (ES+) 562 (M+ + 1, 100%), 506(38) . (283c) was synthesized by a similar method as compound15 280 to afford an off-white powder (167mg, 98%): mp. 90- 105 °C; [a]D22 -106° (c 0.11 MeOH); IR (KBr) 3325, 3070, 2956, 1669, 1544, 1423, 1256, 1199, 1133, 1062; 1H NMR (D6-DMSO) δ 8.95 (1H, d), 8.45-8.20 (2H, m) ,

7.53-7.45 (3H, m) , 5.19-5.08 (3H, m), 4.70-4.62 (1H m) , 4.41-4 .30 (2H, m) , 3.53 (3H, s), 2.92-2.68 (3H, 2.22-2.06 (2H, m) , 1.95-1.82 (2H, m) , 1.63-1.53 (1H m) . MS (ES+) 506 (M+ + 1, 100%) .

MT Ο»

CM *

CD *"* w 1 i «

Cu

512a, 512b

280d, 283d 10 15 ΑΡ ν ο 1 2 8 Ο - 2 68 ~ compound R n J. di ci n-n 280d 512b 283d (512a) was prepared by a similar method as compound y ·" < 509b, to afford (831) as a colourless foam: [a]r3-129.6° (c 0.1, CH2C12>; IR (KBr) 3323, 1726, 1664, 1531, 1501, 1444, 1415, 1394, 1369, 1279, 1254, 1156; Ί1 NMR (CDC13) δ7.59 <5H, s) , 7.37 (1H, d, J = 7.2), 6.38 UH, d, J = 7,4?, 5.27 (1H, m), 4.98 (2H, ml, 4.58(2n, a + m), 4.28 (1H, d, J = 17.2), 3.28 (1H, m),3.10-2.65 (4H, m) , 2.31 (2H, m) , 2.03 (3H, s) , 2.,10-1.72 (4H, m), 1.48 (9R, s). (280d) was prepared by a similar method as compound

CM 00 ·« «

CL

«X 280, to afford (77%) as a colourless foam: [a]D'-95.3° (c 0.1, CH2C12); IR (KBr) 3316, 1728, 1659,

1531, 1501, 1415, 1341, 1278, 1253, 1222, 1185; Ή NMR (CDC13)6 3.Q5 (1H, d, J == 7.9), 7.57 (5H, br s) , 5.30 UH, m), 5.01 (2H, no , 4.70-4.10 (4H, m), 3.40-2 , 6 5 (4K, m) , 2.62 (1H, nd , 2.33 (1H, m), 2.27-1.65 , S.H, m) 2.01 ί 3H, s ) . (512b) was prepared by a similar method as compound 509b, to afford (9%) as a colourless foam: IR (KBr) 5333, 1727, 1661, 1542, 1427, 1369, 1279, 1257, 1232, 3156; J'H NMR (CDC13 ) δ 3 , ,30 (2H, m), 7.20 (3H, m), 6 ., 4 5 )1K, d, 5 = 7.4), 5.17 UH, m), 4.91 (3H, m), 4. 55 (IB

- 269 - m) , 3.27 (1H, m) , 3.14-2.70 (4H, m) , 2.41 (1H, m), 2.04(3H, s), 2.10-1.65 (6H, m), 1.44 (9H, s). (283d) was prepared by a similar method as compound280. (100%) as a colourless foam: [a]D22 -106.0° (c 0.2, 10% CH3OH/CH2Cl2) ; IR (KBr) 3312, 1735, 1664, 1549, 1426, 1279, 1258, 1200, 1135; 1H NMR (CDC13) 58.27 (2H, m), 7.46 (2H, m), 5.09 (1H, m), 4.79 (3H, m), 4.47 (1H, m), 3.40 (1H, m), 3.30-2.70 (3H, m) , 2.54 (1H, m), 2.30 (1H, m), 1.98 (3H, s), 2.05-1.65 (4H, m).

10 245b 246b (245b) was prepared from (IS, 91?) 9-Benzoylamino- 1,2,3,4,7,8,9,10-octahydro-10-oxo-6H- pyridazino[1,2-a][1,2]diazepine-l-carboxylic acid bythe method described for 245 to afford 416mg (85%) of a 15 colourless foam (~1:1 mixture of diastereoisomers): IR(KBr) 3392, 3302, 2942, 1792, 1642, 1529, 1520, 1454,1119; 1H NMR (CDC13) δ7.79 (2H, m) , 7.51-7.09 (10H, m) ,5.52 (0.5H, d, J = 5.3), 5.51 (0.5H, s), 5.36 (1H, m),4.84 (1H, m), 4.74-4.59 (1.5H, m), 4.51 (1H, m), 4.38 20 (0.5H, m), 3.22-2.83 (5H, m), 2.51 (1H, m) , 2.25 (2H, m) , 2.01-1.46 (6H, m) . Anal. Calcd for c28H32N4°6,0-75H2O: C' 62.97; H, 6.32; N, 10.49. Found: AP/77 9 8 . fl 1 2 9 4 APC 01 2 9 0 - 270 - C, 63,10; H, 6.16; N, 10.21. MS (ES+) 521 (M + 1, 100%). (246b) was prepared from 245b by the method describedfor 246 to afford 104iag (33%) of a white powder; rnp. 5 115-119 °C; [a]D24 -19.8° (c 0.2 MeOH); IR (KBr) 3293,2944, 1786, 1639, 1576, 1537, 1489, 1450, 1329, 1162,1121; NKR (CD3GD) 6 7.35 (2H, d, J= 7.0), 7.49 (3H,mb 5.49 (1H, m) , 4.55 (1H, m) , 4.30 (2H, m) , 3-, (1H,m), 3.19-2.89 (3H, m) , 2.63 (2H, in), 2.16-1.81 (5K, mb 10 1.60 (3H, m) . Anal. Calcd for C21H26N4O6’H2O: C, 56.24;H, 6.29; N, 12.49, Found: C, 56.54; H, 6.05; K, 12.29.MS (ES + ) 429 (M - 1, 100%) .

Compounds 513a-j were prepared as descr....-a below. o

15 513a-f compound R 513a 513a-1 1’’ j^jj 513a~2 513b 513b-l σ'" ό AP/.b 9 8 1 ΰ 1 2 9 4 - 271 - 513b-2 °O 513c KO 513d 513e >OX3 513f 513f-l 513f-2

513h AP/."/ 9 8 / a 1 2 9 4 APC 0 1230 - 2 72 --

5131

513j (513a) was prepared by a similar method as compound 513d/e to afford a mixture of diastereoisomers ) 67Qrag, 5 501) as an oil: IR (KBr) 3331, 2946, 1790, 1723, 1713, 1531, 1329, 1257, 1164, 1120, 1060, 977, 937, 701; J'HNMR (CDC13) δ 7.36-7.18 (5H, m), 5.99-5.83 (1H, m), 5.415.34 (2K, m) , 5.28-5.18 (2H, m) , 4.59-4.56 (2H, , 4.32-3.96 (2H, m) , 3.85-3.73 (1H, m) , 3.02-2.76 (3R, 10 m), 2.4 9-2.34 (1H, mi . (513b) was prepared as 513d/e to afford 8g (511) of amixture of diastereoisomers as a clear oil: -13 (c 0.25,, CH2C12); IR (KBr) 3325, 2959, 2875, llMCy1723, 1535, 1420, . 5 , 1257, 1120, 1049, 973, 83^ 15 NMR (CDC1 1 )25, m) , 4.584.34-4.25 (IK, , m) , 1.85-1.50 (8H, in). Anal S0,1.0..1.3) 6 6.02-5.8 0 (1H, m) , 5.53-5.4 6 (2H, m) 5.21 )25, m), 4.58 (25 m), 4.34-1.25 (IK, m) , (15, in 5 . 2.53- 2,35 ) 15 ΑΡ/Γ/9 8/ΰ 1 2 9 4 AP V Ο 12 8 Ο - 273 -

Calcd for C13H19NO5: C, 57.98; H, 7.11; N, 5.20. Found:C, 56.62; H, 7.22; N, 4.95. MS (ES+) 270. (513c) was synthesized by a similar method as compound513d/e to afford a single isomer (20%) as a pale yellow 5 oil: [a]D24 -63.1° (c 0.2, CH2C12); IR (film) 3338, 2948, 1791, 1723, 1529, 1421, 1330, 1253, 1122, 984, 929, 746; 1H NMR (CDC13) 67.20 (4H, m) , 5.87 (1H, m) , 5.61 (1H, d, J = 5.4), 5.33-5.10 (2H, m) , 4. 70 (1H, m) , 4.56 (3H, m), 3.33-3.19 (2H, m), 3.10-2.94 ( :2H, m), 10 2.81 (1H, dd, J = 8.3, 17.3), 2.43 (1H, dd, J = 10.5, 17.3) (513d) and (513d/e) were prepared [via method describedby Chapman Biora. &amp; Med. Chem. Lett., 2, pp. 615-618(1992)]. Following work-up by extraction with 15 ethylacetate and washing with NaHCO3, the product wasdried (MgSO4), filtered and evaporated to yield an oilwhich contained product and benzyl alcohol. Hexane(200ml) (200ml hexane for every 56g of

AllocAsp(CO2tBu)CH2OH used) was added and the mixture 20 stirred and cooled overnight. This afforded an oilysolid. The liquors were decanted and retained forchromatography. The oily residue was dissolved inethyl acetate and evaporated to afford an oil which wascrystallised from 10% ethyl acetate in hexane (~500ml). 25 The solid was filtered to afford 513d (12.2g, 19%): mp. AP/F,' 98/0 1234 108-110 °C; [a] 24 D +75.72° ( c 0.25, CH2C12); IR (KBr)3361, 1778 , 1720, 1517, 1262, 1236, 1222, 1135, 1121, 944, 930, 760; 1H NMR (CDC13) 5 7.38 (5H, m) , 5.90 (1H, m), 5.50 ( 1H, s), 5.37 (0.5H, m) , 5.26 (2.5H, m), 4.87 (1H, ABq), 4 . 63 (3H, m) , 4.31 (1H, m) , 3.07 (1H, dd), 2.46 (1H, dd) . Anal. Calcd for C15H17NO5: C, 61.85; H, 5.88; N, 4.81. Found: C, 61.85; H, 5.89; N, 4.80. APC01280 274 -

The liquors were combined and evaporated toyield an oil (-2000} containing benzyl alcohol.Hexane/ethyl acetate (9:1, 100ml) was added and theproduct purified by chromatography eluting with 10% 5 ethyl acetate in hexane to remove the excess benzylalcohol, and then dichloromethane/hexane (1:1containing 10% ethyl acetate). This afforded 513e /4 containing some 513d (20.5g, 32%): mp. 45-48 C; (ajp'-71.26° (c 0.25, CH2CI2); IR (KBr) 3332, 1804, 1S91, 10 1535, 1279, 1252, 1125,976. 1H NMR (CDC13) 57.38 (SH, m) , 5.91 (1H, m) , 5.54 (1H, d, J == 5.2), 5.38 (35, m) ;4.30 (1H, ABq); 4.60 (4H, m), 2.86 (1H, dd); 2.52 (1H,dd) . Anal. Calcd fox ΰ'^Η^ΝΟς · Q . 1H2O C, 61.47; 5, 5.91;N, 4.78. Found: C, 61.42; H, 5.88; N, 4.81. 15 (5l3f) was synthesized by a similar method as 513d/e toafford a colourless (152mg, 79%): IR (film) 3334, 2983, 2941, 1783, 1727, m3, 1547, 1529, 1422, 1378, 1331, 151 1164, 1122, 1060, 938; XH NMR (CDC1-, j δ 6.09- 20 5.82 (2K, m), 5.50-5.18 (3H, m), 4.64-4.54 (2H, ah,4.27-4.16 (1H, m) , 3.S5-3.78 (1H, m), 3.73-3.56 (iH,m), 3.05-2.77 (1H, m) , 2.56-2.37 (1H, m) , 1.35-1.17 ΑΡ/Γ/9 8/fi 1 2# 4 (4E, nt) N, 6,11 A-

Anal. Calcd for C10H15NO5: C, 52.40; H, 6.60;Found: C, 52.16; H, 6.62; N, 5.99. MS (SS+) 229 (M‘ + 1, 100%). (513g). 4-Dimethylamxno-pyridine ¢76.Omg, 622mmol) wasadded tc a solution of 2-phenoxybenzoyl chlorideif/mg, 2.49mmol) and 517 (600mg, 2.07mmol) in pvridineas stirred at room temperature; ' ’ Ine (25ml) and extracting with-). The combined organic1 IM hydrochloric acid if xsodium hydrogen carbonate (2 x ilOini) . The mixtcfor ISh before adc.ethyl acetate (30mextracts were wash25ml), saturated r 30 AP tO12 8 0 - 275 - 25ml) and brine (25ml), dried (MgSO4) and concentrated.The pale orange oil was purified by flash columnchromatography (1—10% acetone in dichloromethane) toafford 447mg (44%) of colourless oil: IR (film) 3375, 5 2980, 1721, 1712, 1602, 1579, 1514, 1484, 1451, 1368,1294, 1250, 1234, 1161, 1137, 1081, 754; 1H NMR (CDC13)5 7.98-7.93 (1H, m) , 7.50-7.41 (1H, m) , 7.35-7.25 (2H,m), 7.22-7.03 (3H, m) , 6.95 (3H, d), 5.95-5.76 (1H, m),5.57 (1H, d), 5.30-5.13 (2H, m), 4.51 (2H, d), 4.25 10 (2H, d) , 4.18-4.04 (1H, m) , 3.88 (1H, m), 3.50 (1H, m), 2.51 (2H, m), 1.41 (9H, s). MS (ES+) 508 (57%), 503(76), 486 (M+ + 1, 45), 468 (27), 412 (100). Accuratemass calculated for C26H32NO8 (MH+): 486.2128. Found:486.2158. 15 (513h) was prepared from (3S, 4R) t-butyl (N- allyloxycarbonyl)-3-amino-4,5-dihydroxypentanoate bythe method described for 513g to afford 562mg (85%) ofa colourless oil: IR(film) 3418, 2980, 1722, 1711,

1512, 1368, 1278, 1245, 1198, 1157, 1139; 1H NMR 20 (CDCI3) 5 8.90 (1H, d, J = 8.6), 8.21 (1H, dd, J = 1.2,7.3), 8.04 (1H, d, J = 8.2), 7.89 (1H, dd, J = 1.5, 7.9), 7.67-7.46 (3H, m) , 5.88 (1H, m) , 5.49 (1H, d, J =9.0), 5.35-5.18 (2H, m), 4.57-4.46 (4H, m), 4.19 (2H,m) , 2.67 (2H, m), 1.40 (9H, s). Anal. Calcd for 25 C24H29NO7: C, 65.00; H, 6.59; N, 3.16. Found: C, 64.74;H, 6.56; N, 3.09. M.S. (ES+) 466 (M+Na, 100%), 444(M+l, 39), 388 (44). (513i) was synthesized by a similar method as compound513g to afford a colourless oil (569mg, 85%): IR (film) 30 3400, 1723, 1712, 1584, 1528, 1489, 1443, 1367, 1276,1232, 1190, 1161, 1098, 1074, 995, 755; 1H NMR (CDCl3) 58.65-8.59 (1H, d), 7.84-7.66 (2H, m), 7.45-711 (5H, m) , ΑΡ/Γ/ 9 8 i J) 1 2 9 4 - 276 - 7.05-6.97 (2H, m) , 6.00-5.78 (1H, m) , 5.54-5.14 (2H, m) , 4.62-4.52 (2H, m) , 4.42-4 .32 (2H, m) , 4.08-4.22 (2K, in), 2.78-2.47 (2R, m), 1 .44 (9H, s). MS (£ST) 508 (100%), 486 (M4 + 1, 33. Accurate mass calculated forC2SK32NO8 (MH+) : 486.2128. Found: 486.2121., {513j) was synthesized, by a similar method as compound

513g to afford a pale orange oil (905mg, 91%): IF (film) 3418, 3383, 2980, 1722, 1711, 1601, 1517, 1450, 1424, 1368, 1308, 1252, 1154, 1100, 994, 767, 658; H NMR (CDCI3) δ 7.62-7.55 (2H, m), 7.51-7.42 (3H, m) , 5.9 8 - 5,7 6 (1H, m), 5.33-5,1? 3 (2H, m), 4.53 (2H, d), 4.18 (2H, d), 3.91 (1H, si. 3.80 (1H, m), 2.76 (3H, s), 2.50 (2H, m1 , 1.43 (9H, s) . Anal. Calcd for C-Mh^QNnOg · 0.5H2O: C, 0 9.62; H, 6.46; N, 5.79. Found; C, 59.46; H, 6.24; N, 5.72. MS (ES+) 497 (100%), 475(hf + 1, 15), 419 (48). o

514

516 AP/17 0 8 / fi 1 2 8 4 (514) was prepared by the method described in H.Matsunaga, et al. Tetrahedron Letters 24, pp. 3009-3012(1983) as a pure diastereomer (60%) as an oil: (a"jrf‘"‘-36.9" ic 0.5, dichloromethane) ; IR (film) 2982, 2S34, - 277 -

1726, 1455, 1369, 1257, 1214, 1157, 1068; 1Η NMR (CDC13) δ 7.31 (5H, m) , 4.10 (1H, q, J = 6.0), 4.05-3.75 (4H, m), 3.10 (1H, q, J = 6 .0) , 2.40 (2H, m), 1.42 (9H, s), 1.40 (3H, s) , 1.34 (3H, s) . 5 (516). 514 (3.02g, 9.00mmol) and 10% palladium on carbon (300mg) in ethanol (30ml) were stirred underhydrogen for 2h. The suspension was filtered throughcelite and a 0.45mm membrane and the filtrateconcentrated to give a colourless oil 515 (2.106g, 95%) 10 which was used without purification. The oil (1.93g,7.88mmol) was dissolved in water (10ml) and 1,4-dioxanand sodium hydrogen carbonate added (695mg, 8.27mmol).The mixture was cooled to 0'C and allyl chloroformate(1.04g, 919ml, 8.66mmol) added dropwise. After 3h the 15 mixture was extracted with ether (2 x 50ml). Thecombined ether extracts were washed with water (2 x25ml) and brine (25ml), dried (MgSO4) and concentratedto give a colourless oil. Flash column chromatography(10-35% ethylacetate in hexane) afforded a colourless 20 solid (2.69g, 95%): mp. 64-5 °C; [a]D23 -21° (c 1.00, CH2C12); IR (KBr) 3329, 1735, 1702; 1H NMR (CDC13) 5 6.00-5.82 (1H, m) , 5.36-5.14 (2H, m), 542 (1H, s), 4.56 (1H, d), 4.40-4.08 (2H, m), 4.03 (1H, m) 3 .70 (1H, m), 2.52 (2H, m) , 1.44 (12H, 2 x s), 1.33 (3H, s); Anal. Calcd for C16H27NO6 : C, 58.34; H, 8.26; N, 4.2 5. Found: C, 58.12; H, 8.16; N, 4.19; MS ( + FAB) 320 (M++l, 41%), 274 (70), 216 (100) (517). A solution 516 (2.44g, 7.41mmol) in 80% aqueousacetic acid (25ml) was stirred at room temperature for 30 24h then concentrated and azeotroped with toluene (2 x 25ml). The residue was treated with brine (25ml) andextracted with ethylacetate (2 x 25ml). The organic APC 0 12 8 0 - 278 - fractions were dried (MgSO4) and concentrated to afforda colourless oil. Flash chromatography (20—80% ethylacetate in dichloromethane) gave a colourless solid(1.,.990, 90%) : mp. 74-5 °C; [a}D25 -1.3° (c 1.0, 5 CH2C12); IR (KBr . . , 1691; XH NMR (CDC13) δ 6.02-5.78 (2K, m), 5.35-5.16 (2H, m) , 4.55 (2H, d) , 4.16-4,04(2E, mi, 2.76 (2H, s) , 3.56 (2H, in), 2.56 (2H, mt, 1.43(9H, s); Anal. Calcd for C13H23NO6 : C, 53.97; H, 8.01;N, 4.84. Found : C, 53.79; H, 7.88; N, 4.81; MSB-EAB) 10 290 (M+el, 44%), 234 (100).

The data of the examples above demonstrate that compounds according to this invention displayinhibitory activity towards IL-Ιβ Converting Enzyme.,

Insofar as hhe compounds of this invention are15 able to inhibit ICE in vitro and furthermore, may be delivered orally to mammals, they are of evidentclinical utility for the treatment of IL-1-,apoptosis-, IGIF-, and IFN-γ mediated diseases. Thesetests are predictive of the compounds ability tc 2 0 inhibit ICE in vivo.

While we have described a number of embodiments of this invention, it is apparent that our basic con-structions may be altered to provide other embodimentswhich utilize the products and processes of this inven- 25 tion. Therefore, it will be appreciated that the scopeof this invention is to be defined by the appendedclaims, rather than oy the specific embodiments whichhave been presented 5y’ way of example. ΑΡ,'Γ/ 9 8 . Λ 1 2 9 4

Claims (30)

1. - 279 -

   1. A compound represented by the formula:(VI) R-L-N-R2 wherein: *1 (elO)

   10 o

   15 m is 1 or 2; each R5 is -C(O)-R10, -C(O)O-R9, -C(0)-N(R10) (R10) ,-S(O)2~R9, -S(O)2-NH-R10, -C(0)-CH2-O-R9, -C(O)C(0)-R10/-R9f -H, -C(O)C(O)-OR10/ or -C (0) C (0)-N (R9) (R10) ; X5 is CH; Y2 is H2 or 0; each R9 is -Ar3 or a -C^g straight or branchedalkyl group optionally substituted with -Ar3, whereinthe -C2_6 alkyl group is optionally unsaturated; each R10 is -H, -Ar3, a -C3_6 cycloalkyl group, ora -C1_6 straight or branched alkyl group optionallysubstituted with -Ar3, wherein the -C]__g alkyl group isoptionally unsaturated; 20

   AP I Ο 1 2 8 0 280 - R13 is H, Ar3, or a straight or branched alkyl group optionally substituted with -Ar3, -CONH2,-ORr, -OK, —OR 9, or -CO2H; each R31 is Rg, -0(0)-Rg, -C(Ο)-N(H)-Rg, or each5 R31 taker, together forms a saturated 4-8 member carbocyclic ring or heterocyclic ring containing -O-,-S-, or -NH-; 1 each R2l h or 5 ’"'-'1-6 straight or branched alkyl group; 10 each Ar3 is a cyclic group independently selected from the set consisting of an aryl group which contains6, 10, 12, or 14 carbon atoms and between 1 and 3 ringsand an aromatic heterocycle group containing between 5and 15 ring atoms and between 1 and 3 rings, said 15 heterocyclic group containing at least one heteroatom group selected front ~o~, -S-, -SO-, S02, -N-, and ......NH-, said heterocycle group optionally containing one ormore double bonds, said heterocycle group optionallycomprising one or more aromatic rings, and said cyclic 20 group optionally being singly or multiply substitutedby -Q2; eacn is -NH2r -CO2H, —Ci, —F, -Br, -I, -NCy·? -CN, =0, -OH, -perfluoro C3_3 alkyl, R5, -OR5, -UHRr, -ORa, -N(Rg) (R-,θ) , -E.q, -C(O)-R10, or 0 25 " / \ CHo, \ / 0 provided that when -Ar3 is substituted with a Q-, 30 group which comprises one or more additional -Ax - groups, said additional -Ar3 groups are not substitutedwith another -Ar3; provided that when : R2 is AP i 0 1280 - 281 -

   R21 is H; and Y2 is 0, then R5 cannot be -C(O)R10, wherein R10 is-CH2CH2Ar3 and Ar3 is unsubstituted phenyl; or -SO2R9,wherein R9 is methyl; and when Y2 is H2, then R5 cannot be -C(O)R10, wherein R10is -CH2CH2Ar3 and Ar3 is unsubstituted phenyl.
2. 2. The compound according to claim 110 selected from the group consisting of: O 213e 302

   O X /CH3 y <Ά-^-οη3°Ο^Ν^γΗ CH3

   304a

   'CH3 23 0 282 - Ο

   - 283 - Ο

   Λ

   0 1 2 S ο ο

   η; ο R / 3 1 2 β 4 213w '' \ 4 213χ 245 245b 5 256 - 285 -

   ΛΪ3/Ρ/ <18/012 94 AP t Ο 12 3 Ο

   e r ϋ* € ( I ί 5501 550η 1 550ο 550ρ ) 5 2100f ΑΡ 0 01 2 3 0 - 287 - Ο 2100g

   Cl ΑΡ ν Ο 1 2 8 0

   - 289 -

   αο α α

   OMe - 2 90 -
3. 4. The compound according to claim 1, wherein : 5 s is 1; R-L-i is H or a C-;„4 straight or branched alkyl groupoptionally substituted with ~Ar3, -OH, -ORg, -Cwherein the Rg is a οΊ_4 branched or straight chainalkyl group; wherein Ar3 is morpholinyl or phei 10 wherein the phenyl is optionally substituted by -Qj; R2i is -H or -Ciby R51 is a Ci_6 straight or branched alkyl groupoptionally substituted with -Ar3, wherein Ar3 isphenyl, optionally substituted by -Qj.; 15 each Ar3 cyclic group is phenyl, naphthyl, thienyl,- quinolinyl, isoquinolinyl, pyrazolyl,thiazolyi, isoxazolyl, benzotriazolyl, benzimidazole!,thienotdiienyl, imidazolyl, thiadiazolyl,benzo '.it) thiophenyl, pyridyl, benzofuranyl, or L·. : ./1,and said cyclic group optionally being singly ormultiply substituted by -Q1; <31 *r- cc Γ'. Π 20 - 291 - each Q-l is -NH2, -Cl, -F, -Br, -OH, -R9, -NH-R5wherein R5 is -C(O)-R10 °r -S(O)2-R9/ -OR5 wherein R5 is-C(O)-R10, -OR5, -NHR9, or 0 5 / \ CH2, \ / 0 wherein each R9 and R10 are independently a -C-i-qJ 10 straight or branched alkyl group optionally substituted with -Ar3 wherein Ar3 is phenyl; provided that when -Ar3 is substituted with a Qq_ group which comprises one or more additional -Ar3groups, said additional -Ar3 groups are not substituted 15 with another -Ar3.
4. 5. The compound according to claim 1 or 4,wherein R5 is -C(O)-R10 or -C(0)-C(0)-R10.
5. 6. The compound according to claim 5,wherein R10 is Ar3. O
6. 7. The compound according to claim 6, wherein R5 is -C(O)-R10 and R10 is Ar3, wherein the Ar3cyclic group is phenyl optionally being singly ormultiply substituted by: -R9, wherein R9 is a C]__4 straight or branched 25 alkyl group; -F, -Cl, -N(H)-RS, wherein -R3 is -H or -C(O)-R10, whereinR10 is a -C^g straight or branched alkyl group 30 optionally substituted with -Ar3, wherein Ar3 isphenyl, -N(R9) (R10) , wherein R9 and R10 are independently a

   CO Oi AP C Ο 1 2 S Ο -C;.....4 straight or branched alkyl group, or -O-R5, wherein R5 is E or 3 -C1_4 straight or branched alkyl group.
7. 8. The compound according to claim 7,wherein Ar3 is phenyl being singly or multiplysubstituted at the 3-· or 5-position by -Cl or at the 4- positior. by -NH-R5, ™R(R9) (R10) , or -O-R5 .
8. 9. The compound according toselected from the group consisting of: claim 8,

   G* CM *“#·· Γ. M r'\ v.7 c

   a AP £ Ο 128 Ο 550m

   CH3
9. 10. The compound according to claim 7,wherein Ar3 is phenyl being singly or multiplysubstituted at the 3- or 5-position by -Rg, wherein Rg 5 is a C1_4 straight or branched alkyl group;and at the 4-position by -0-R5. 11. selected from The compound according tothe group consisting of: claim 10, 10

   O

   HO' API 0 1230 2 94 -

   σ r Τ' C" W #*·. Q
10. 12. The compound according to claim 6., wherein Py is -C(0)-lyQf wherein R10 is Ar3 and the Arcyclic group is indolyl, benzimidazolyl, thienyl,quinolyl, isoquinolyl or benzo[b]thiophenyl, and sandcyclic croup optionally being singly or multiply 10 substituted by -Qy.
11. 13. The compound acccrdirKf to claim 11,wherein the Ar3 cyclic group is isoquinolyl, and said

    •it* AP Ο Ο 12 8 Ο - 295 - cyclic group optionally being singly or multiplysubstituted by -Qj.
12. 14. The compound according to claim 13,selected from the group consisting of: O

    Oi 04 V" C5 OS &amp; i £ c AP ο 1 2 3 Ο - 29 6 - Ο

    Η 6^,CH3 ο

    Η O^,CH3

    c Γ <« C V. C Γ ί ί * - 297 -
13. 15. The compound according to claim 6,wherein R5 is -C(O)-R10, wherein R10 is Ar3 and the Ar3cyclic group is phenyl, substituted by 0 / \ CH2 \ / 0 10
14. 16. The compound according to claim 15,selected from the group consisting of:

    AP/Pz Q fi / λ 10 wh. e r i

    m i s Ri i:
15. 17. A compound represented by the fori (V) or 2; K’i is -u\l, or -CO-Ar2 < ί ς -· C ί O \-S(0)2-NK~ ’ (0)C(0)-0?Η-, or Or

    -C (0) -C^-Tx-R!!, ~C(G) 0, -C(O)O-R9, -C(0)-N{Ri, -c (0)-ch2-o-r9, -c(o;or -C(O)C(O)-N(R9) (R10; , '· -S (0) ~, or -S (0) •tf Oft CM AP»ύ12 8 Ο - 299 - each R9 is -Ar3 or a -C^.g straight or branchedalkyl group optionally substituted with -Ar3, whereinthe -Ci_6 alkyl group is optionally unsaturated; each R10 is -H, -Ar3, a -C3_g cycloalkyl group, or5 a -Ο2_6 straight or branched alkyl group optionally substituted with -Ar3, wherein the “Cj-g alkyl group isoptionally unsaturated; each Rn is -Ar4, - (CH2) i-3_Ar4, -H, or -C(O)-Ar4; R15 is -OH, -OAr3, -N(H)-OH, or -OC-j__6, wherein10 C3.-6 is a straight or branched alkyl group optionally substituted with -Ar3, -C0NH2, -OR5, -OH, -ORg, or-CO2H; each R21 is -H or a -C^g straight or branchedalkyl group; 15 Ar2 is independently selected from the following group, in which any ring may optionally be singly ormultiply substituted by -Qj or phenyl, optionallysubstituted by Q]_:

    a 0 L c < wherein each Y is independently 0 or S; each Ar3 is a cyclic group independently selected from the set consisting of an aryl group which contains25 6, 10, 12, or 14 carbon atoms and between 1 and 3 rings and an aromatic heterocycle group containing between 5and 15 ring atoms and between 1 and 3 rings, saidheterocyclic group containing at least one heteroatomgroup selected from -0-, -S-, -SO-, SO2, =N-, and -NH-, 30 —N(R5)—, and -N(R9)- said heterocycle group optionally APt012 8 0 ..... 3 00 - containing one or more double bonds, said heterocyclegroup optionally comprising one or more aromatic rings,and said cyclic group optionally being singly ormultiply substituted by -Q]_; 5 each Ar4 is a cyclic group independently selected from the set consisting of an aryl group which contains6, 10, 12, or 14 carbon atoms and between 1 and 3rings, and a heterocycle group containing between 5 and15 ring atoms and between 1 and 3 rings, said 10 heterocyclic group containing at least one heteroatoingroup selected from --0-, -S-, -SO-, SO2, -N-, -NK-, and -N(Rc)~ said heterocycie group optionallycontaining one or mere double bonds, said heterocyciegroup optionally comprising one or more aromatic rings, 15 and said cyclic group optionally being singly ormultiply substituted by -Qj; each Q2 is independently -NH2, -CO2H, -Cl, -S', -Er, -I, -NO2, -CN, =0, -OH, -perfluoro Cj__3 alkyl, R2, -OR^, ~NHR5, ~ORq, -N (Rq) (R·! o) , -R9, ~C(O)--R10, or20 " ' ' “ 0 / \ ch2; \ / 0 25 provided that when -Ar3 is substituted witn a 0Ί group which comprises one or more additional -Ar-.>groups, said additional -Ar3 groups are not substitutedw i t h another - Ar ; provided that when: 3 0 m ,s 1 ; R1,, is -OH;ip- is -H; Ό, is 0; and € C B < 10 15 20 AP J u 1 2 8 Ο - 301 - R3 is -C(O)-H; then R5 is not: -S (0) 2-CH3; -C(0)-OCH2phenyl; or-CiO-Rxo' wherein R10 is: -CH2CH2phenyl; phenyl unsubstituted by -Qi, 4-(carboxymethoxy)phenyl, 2-fluorophenyl, 2-pyridyl, or N-(4-methylpiperazino)methylphenyl, and provided that when: m is 1; R15 is -OH; R2j is —H; Y2 is H2; and R3 is -C(O)-H; then R5 is not -C (0)-CH2CH2phenyl; and provided that when: m is 1 ; R15 is -OH or -OC(CH3)3; O C t· C a c c c < R21 is -H; Y2 is 0; andR3 is —CO—Ar2z

    R5 is not -C(0)-CH2CH2phenyl; and provided that when: 25 10 15 20 25 128 0 κι ι.ε 1; R1Fl is -OH or ......OC(CH3)3; R.-< is -H; is 0; and R3 is -C(0)-CH2-S-CH2-2-chlorophenyl; then R5 js not —C {O', ~CH2CK2phenyl; and when R3 is -C (0)-GHh—O-C (0)-2, 6-dichlorophenyl; men R5 is not: -S(O)2“CH3, or~C(0)-O-Cmphenyl, -C(O)“Rlo, wherein R10 is: -CH2C.H2phenyl, 4- (dimethylaminomethyl) phenyl, 4-(K-aorpholinomethyl}phenyl, 4- (N-rnethylpiperazino) methyl) phenya., 4- (N-(2-methyl)imidazolylmethyl}phenyl, 5- benzimidazolyl, 5-benztmazolyl, N-carboethoxy-5-benztriazolyl, oxN-carboethoxy-5-benzinidazolyl; and when P-3 is -C (0) -CH?-0-5- (1- (4-chlorophenyl}-3-trifluoromethyl)pyrazoiyl); then R3 is not: r *· c V c i. f -H, -C(O)-R10, wherein R10 is 4- idimethylaminomethyl) phenyl, phenyl, 4-{carboxymethylthio)phenyl, 4-(carboxyethylthio)phenyl, (carbcxyethyl) phenyl, or 4-(carboxypropyl)phenyl, or-C(O)-ORg, wherein Rg is isobutyl or phenyl; and when Ry is -C (0)wherein R·^ is 5-(1-:3-trifluoromethyl) pyrazoiyl or 5-(1-(4-chloro-h-pyrrarnyi ;< - J--tri f luor oraethyl) pyrazoiyl; then 30 'ϊν.*'.'·· - ,i >-ί··

    AP v 012 Θ 0 - 303 - R5 is not -C(0)-O-CH2phenyl; and whenR3 is -C(O)-CH2-O-5-(l-(2-pyridyl)-3- trifluoromethyl)pyrazolyl), thenR5 is not: -C(0) -4-(dimethylaminomethyl)phenyl, or-C(0)-O-CH2phenyl; and provided that when:m is 1; 10 R15 is -OH or -OC(CH3)3; R22 is —H; Y2 is H2; and R3 is -C (0)-CH2-O-C(0)-2,6-dichlorophenyl, then R5is not -C(0)-O-CH2phenyl. 15
16. 18. The compound according to claim 17,wherein the compound is: 2002

    ήΠ/η/ g

    H AP· υ12 8 Ο 304 - Ο

    Ο AP"01280 - 305 - 280c 280d 283b 283c 5 283d 308c o

    MeO' ο υ /wav AP- Ο 12 8 Ο 306 -

    xs =, n Cl ,-4 o x V, ,/i y~-f N—AΗ Ό

    -/ Ύ f 'OHN O 1 ’ Ή H Η O p ( <Ί 3M"U ! °H <r- x ·· 501 Λ-/Ύ <"OH o MeSO2—hi vk 1 J Η ύΟίί!Κ''Ν'^χη/χ'ΟΗ O V /?

    MeSO2-NZ I Ϊ h Υ·χ« x "o· MeSpz-hf 1 i Η υζΤ·'Ν' AP v Ο 1 2 8 Ο - 307 - 505e 505f 510a

    510d 511c 2100j

    © v C M, <x a g fi < 30 8 -

    <'· V1* P < u. <

    Cl 307b 20

    The compound according to selected from the group consisting of: 214c 217c 217e

    claim 17, *4 σι 0 00 Oi a a 246 0 ^p„01280 Π

    c r <· c d- c £ £ c ΑΡ.υ ί 2 3 Ο - 311 - 286 287 404 405 406 'J 407 ο

    Ο Ο C ·>·» α ο £ £ < 408 312 -

    Ο c w c ** a © C •k*. &amp; C

    419 420 422 423 - 313 -

    5 424 425 Ο

    ΑΡΖΡΖ 9 8/01294 Ο

    426 431 434 AP 314 - 0

    o £58 O ci ©ή U < 431

    APZD/ Ofi / ft 1 9 ft I AP- ύ1 2 3 Ο 31 6

    ο Η fs £3 ex α C'

    - 317 - 451 452 453 454 5 455 J 456 Ο

    * 6 I V 1) Ζ 8 6 /d/dv 457

    .01280 318 -

    OH H Ο fsi -r— •**5 cs fi. rs c~" »<;·

    AP * 0 1 2 8 0 466 467 3 468 469 - 319 -

    5 470 G 471

    ί 6 3 V Ο / β 6 M/dV 472 320 -

    AP/P/ 9 8/01294

    480 481 ") 481s 482 5 482s G 483 ΑΡ Ο 0 1 2 8 Ο - 321 -

    oft CO CL c'

    484 0 AP - ΰ 1 2 8 Ο 4 87 322 - Ο

    ΑΡ/Ρ/ 9 8/01294 493 AP - Ο 1 2 8 Ο - 323 -

    494 ) 495 5 497

    498 499 21

    The compound according to claim 17, Oft' £ selected from the group consisting of: AP 01280 32 4 - Ο
17. 22. The compound according to claim 17, 5 wherein; E i s 1; Ti is 0 or S; R·-!·] is -H or -Ctb/ Arc is (hh); 10 ¥ is 0; each Ar3 cyclic group is independently phenyl,naphthyl, thienyl, qumolinyl, isoquinolinyl,pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyi,benzimidazolyl, thiencthienyl, imidazolyl, 15 thiadiazolyl, ben; .ophenyl, pyridyl, benzofuranyl, or j and said cyclic group x- singly or multiply _tuted by ~Q-; each Ar4 cyclic group is phenyl, tetrazolyi,pyrrdmyl, oxazolyl, naphthyl, pyrimcdinvl, or : >y: 20 and sail, cyclic group being singly or multiplysubstituted by -Q-j; AP/?/ »8/01294

    AP.ύ12 8 Ο - 325 - each £>! is -NH2, -Cl, -F, -Br, -OH, -Rg, -NH-R5wherein R5 is -C(O)-R10 or -S(O)2-Rg, -OR5 wherein R5 is—C(O)—R^q, —ORg, —NHRg, or 0 5 / \ ch2, \ / 0 wherein each Rg and R10 are independently a -C^.g10 straight or branched alkyl group optionally substituted with -Ar3 wherein Ar3 is phenyl; provided that when -Ar3 is substituted with a Qj group which comprises one or more additional -Ar3groups, said additional -Ar3 groups are not substituted 15 with another -Ar3. 23. The compound according to claim 22, T? wherein R3 is -C(O)-Ar2. os 24. The compound according to claim 22, , *> wherein R3 is -C(0)CH2-T1-R11. 20 25. The compound according to claim 22, wherein R3 is -C(O)-H. 26. claims 17, or -C(0)C(0)-R10. The compound according to any one of 22-25, whereii i R5 is -CtO-Rio or 25 27. The compound according to claim 26, wherein R10 is Ar3. 28. The compound according to claim 27, wherein R5 is -C(O)-R10 and R10 is Ar3, wherein the Ar3 AP? ύ 12 5 Ο ..... 326 - cyclic group is phenyl optionally being singly ormultiply substituted by: -Rq, wherein Rq is a C]__4 straight or branched alkyl group; 5 -F, -Cl, -Nifij-Rq, wherein ~R5 is -H or -C(O)-R]_q, whereinR10 is a straight or branched alkyl group optionally substituted with -Ar3, wherein Ar3 is 10 phenyl, -NCRq) (Riq) / wherein R9 and R10 are independently a~Ci__4 straight or branched alkyl group, or -O-R5, wherein R5 is H or a -C]_„4 straight cr branched alkyl group.
18. 29. The compound according to claim 29, wherein Ar3 is phenyl being singly or multiplysubstituted at the 3- or 5-position by -Cl or at the 4-position by -NH-R5, ~N(R9) (R10), or -O-R5.
19. 30. The compound according to claim 29, 20 selected from the group consisting of: AP/P/88/01 284

    AP '01290 - 327 - 10 214m
20. 31. The compound according to claim 28,wherein Ar3 is phenyl being singly or multiplysubstituted at the 3- or 5-position by -R9, wherein R9is a C]__4 straight or branched alkyl group;and at the 4-position by -O-R5.
21. 32. The compound according to claim 31,wherein the compound is: 214w
22. 33. The compound according to claim 27,wherein R5 is -C(O)-R1q, wherein R10 Ar3 anc* the Ar3cyclic group is indolyl, benzimidazolyl, thienyl,quinolyl, isoquinolyl or benzo[b]thiophenyl, and said 15 cyclic group optionally being singly or multiplysubstituted by -Qj.
23. 34. The compound according to claim 33,wherein the Ar3 cyclic group is isoquinolyl, and saidcyclic group optionally being singly or multiplysubstituted by -Qi- «e d* *—

    20
24. 35. The compound according to claim 34wherein the compound is: OH B
25. 36. The compound according to claim 27, wherein R5 is -C{0)-JOo? wherein R10 is Ar3 and the Ar3cyclic group is phenyl, substituted by / \ 10 ch2 1 / 0
26. 37. The compound according to claim 36, wherein the compound is: 15 ) 0
27. 38. A pharmaceutical composition comprisinga compound according to any one of claims..1-37 and apharmaceutically acceptable carrier.

    33. The use of a compound according to any20 one of claims 1-37 for the manufacture of a medicam.net for use in treating or preventing a disease selectedfrom osteoarthritis, acute pancreatitis, chronicpanceatitis, asthma, adult respiratory distress AP-υ12 8 0 ) 10 15 - 329 - syndrome, infectious hepatitis, glomeralonephritis,rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Grave's disease,autoimmune gastritis, insulin-dependent diabetesmellitus (Type I), autoimmune hemolytic anemia,autoimmune neutropenia, thrombocytopenia, chronicactive hepatitis, myasthenia gravis, inflammatory boweldisease, Crohn's disease, ulcerative collitis, multiplesclerosis, psoriasis, lichenplanus, graft vs hostdisease, acute dermatomyositis, eczema, primarycirrhosis, uveitis, Behcet’s disease, acute aplasia,aplastic anemia, amyotrophic lateral sclerosis,nephrotic syndrome, osteoporosis, multiple myeloma-related bone disorder, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma, sepsis, septicshock, Shigellosis, Alzheimer's disease, Parkinson'sdisease, cerebral ischemia, or myocardial ischemia. t «·
28. 40. The use according to claim 39, wherein f*· »· the disease is selected from osteoarthritis, acute r' pancreatitis, rheumatoid arthritis, inflammatory bowel disease, ulcerative collitis, Crohn's disease, hepatitis, adult respiratory distress syndrome, glomerulonephritis, insulin-dependent diabetes mellitus (Type I), juvenile diabetes, psoriasis, graft vs. host disease, hepatitis, or Alzheimer's disease.
29. 41. The use of a compound according to anyone of claims 1-37 for the manufacture of a medicamentfor use in treating or preventing a disease selectedfrom Alzheimer's disease, Parkinson's disease, cerebralischemia, myocardial ischemia, spinal muscular atrophy, J 20 8P ( Q 128 4 25 rtf' · υ ι I 3 Ο ~ 330 - multiple sclerosis, MDS-related encephalitis, HIV™relaced encephalitis, aging, alopecia, or neurologicaldamage due to stroke.
30. 42. The use according to claim 41, wherein5 the disease is Alzeheimer's disease. I