AU2004202175A1 - HIV protease inhibitors - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant: Actual Inventors: Address for Service: Invention Title: The Scripps Research Institute WONG, Chi-Huey SLEE, Deborah H.

LASLO, Karen DAVIES COLLISON CAVE, Patent Attorneys, 1 Nicholson Street, Melbourne, 3000 HIV protease inhibitors The following statement is a full description of this invention, including the best method of performing it known to us;: Q:\OPER\PDB\DIVISIONALS\ 2452780 141.DOC 20/5/04 PAOPER\PDBMSp=Ai\12452780 div sp~.do-20O05/04

A-

HIV PROTEASE INHIBITORS This application is a divisional application derived from Australian Patent Application No. 18270/01, which in turn is a divisional application derived from Australian Patent Application No. 12844/97. The entire contents of both are incorporated herein by reference.

Field of the Invention: The invention relates to HIV and FIV protease inhibitors. More particularly, the invention is directed to combinatorial libraries of HIV and FIV protease inhibitors characterized by a-keto amide or hydroxyethylamine core structures flanked by on one side by substituted pyrrolidines, piperidines, or azasugars and on the other side by phenylalanine, tyrosine, or substituted tyrosines.

The invention is also directed to methods for making such libraries, to the disclosed compounds made by such method, and to a method for screening such libraries for identifying candidate drugs which have clinically useful activity and which are potentially resistive against loss of inhibitory activity due to development of resistant strains of HIV.

Background: Human irrmunodeficiency virus protease (HIV PR) is an important target for the inhibition of viral replication.

Though many potent in vitro inhibitors have been developed, most of them are either inactive -2or toxic in vivo, or mutant forms of the virus emerge which are resistant.(See J.H. Condra, et al., Nature (1995): vol. 374, 569-571; M. Markowitz, et al., J.

Virol. (1995): vol. 69, 701; D. J. Kempf, et al., S Proc. Natl. Acad. Sci. USA (1995): vol. 92, 2484- 2488; A.K. Ghosh, et al., J. Med. Chem. (1994): vol.37, 2506-2508; P.Y. Lam, et al., Science (1994): vol. 263, 380-384; N.A. Roberts, et al., Science (1990): vol. 248, 358; and E.E. Kim, et al., J. Am.

Chem. Soc. (1995): vol. 117, 1181-1182.) The lack of animal systems to test the efficacy of the inhibitors further slows down the drug development process. Recently a similar protease has been identified in the life cycle of feline immunodeficiency virus (FIV). Talbott, et al., Proc. Natl. Acad. Sci. USA (1989): vol. 86, 5743- 5747; and N.C.Pedersen, et al. Science (1987): vol.

235, 790-793.) FIV is a virus which leads to clinical symptoms comparable to those observed in human acquired immune deficiency syndrome (AIDS).

Studies have shown that up to 14% of the cats surveyed in the USA and Canada are infected with FIV.

Yamamoto, et al., JAVMA (1989): vol. 194, 213- 220.) In Japan, the figure is 28.9%. Ishida, et al., JAVMA (1989): vol. 194, 221-225.

In drug resistant mutants of HIV, there are at least six cases where HIV PR residues mutate to the structurally aligned residue found in FIV PR. The amino acid changes in HIV PR are V32I (I37-FIV), (M107-FIV), N88D (D105-FIV), I50V (V59'-FIV), -3and Q29K (K109-FIV). For a list of resistant HIV PR mutants is disclosed by J. W.

Mellors, e: al. (International Antiviral News (1995) vol. 3, 8-13.) The structure alignment was derived from the X-ray crystal structure of FIV PR solved by A. Wlodawer (reference 36). Superimposition of the two proteases based on their X-ray structures indicates similarities between the two proteases and the drug resistant HIV proteases.

HIV P: is a 99 amino acid aspartyl protease which functions as a homodimer.(M.A. Navia, et al., Nature (1989): vol. 337, 615-620; and D.D. Loeb, Virol.

(1989): vol.63, 111-121.) FIV PR is also a homodimeric aspartyl protease which consists of 116 amino acid residues.(J.H. Elder, Infectious Agentsand Disease (1994): vol. 2, 361-374.) Both HIV and FIV proteases are responsible for the processing of viral gag and gag-pol polyproteins into structural proteins and enzymes essential for the proper assembly and maturation of full infectious virions. (S.K.

Thompson, Bioorg. Med. Chem. Lett. (1994): vol. 4, 2441-2446.) In particular HIV and FIV proteases show high specificity for the selective cleavage of the Tyrosine/Phenylalanine-Proline amide bonds in the Matrix-Capsid domain of the gag-pol polyproteins, a specificity not exhibited by mammalian cellular proteases which are not known to efficiently hydrolyze peptide bonds involving the proline nitrogen. Debouck, Aids Research and Human Retroviruses (1992): vol. 8, 153-164; and J.H. Elder, J. Virol. (1993): vol. 67, 1869-1876.) It is this -4specificity that makes HIV PR an attractive target for inhibition. A comparison of the amino acid sequence about the matrix capsid cleavage site (Tyrosine Proline bond) in both HIV and FIV is provided below. As can be seen the residues about the cleavage site are the same at four positions, P3, Pi, and P 2 It is disclosed herein that, due to these similarities, certain HIV PR inhibitors also inhibit FIV PR.

P

4

P

3

P

2

P

1 PI P 2

P

3 P4' HIVPR Ser Gin Asn Tyr Pro Ile Val Gln FIVPR Pro Gln Ala Tyr Pro jlj Gin Thr Activated ketones, in general, have been shown to inhibit most kinds of proteases (Barrett et. al., Proteinase Inhibitors; Research monographs in cell and tissue physiology; Dingle, J. Gordon, J.L., General Eds.; Elsevier Science Publishers; Amsterdam, 1986). In particular, a recent study shows the design of three different classes of activated ketones which inhibit the aspartyl protease, renin. These potent analogs display IC 50 values from 4000 nM to 4.1 nM and include 1,1,1-trifluoromethyl ketones, a-keto esters, and a-diketones as the activated ketone functionalities (Patel et. al. J. Med Chem. 1993, 36, 2431).

The a-keto-amide core structure is isosterically analogous to the activated ketones but is more potent than the reported hydroxyethylamine or phosphinic acid HIV protease inhibitors that are mechanism-based isosteric core structures.

The a-keto-amide core structure has been used in inhibitors of various enzymes which include serine and cysteine proteases, hydrolases and aminopeptidases. As an example, a series of dipeptidyl and tripeptidyl a-keto amides have been synthesized and evaluated as potent inhibitors for cysteine proteases which include enzymes calpain I, calpain II, cathepsin B, and papain (Li et. al. J.

Med. Chem. 1993, 36, 3472). Another study has identified a-keto amide analogs as inhibitors of an epoxide hydrolase (Wong et. al. J. Med. Chem. 1993, 36, 211). Additionally, the inhibition of arginyl aminopeptidase (Ki 1.5 uM), cytosol aminopeptidase

(K

i 1.0 uM) and microsomal aminopeptidase (Ki uM) has been observed from a-keto amide analogs which can be derived from 3-amino-2-oxo-4-phenylbutanoic acid amides. (Rich et. al. J. Med Chem. 1992, 451).

The activity of dipeptide isosteres is often enhanced by addition of amino acid residues to either the N and C-terminus of the isostere to improve binding in the active site. The prior art provides examples of such inhibitors which include renin, aspartyl proteases and procine pepsin inhibitors (Rich et. al. J. Med Chem. 1992, 35, 451). The resulting inhibitors generally exhibit high binding affinity to HIV protease. They display, however, instability and/or poor oral bioavailability.

-6- The related art has provided examples of a-keto amide inhibitors of aminopeptidases which contain an unsubstituted proline moiety in the molecule. In particular, Gordon and co-workers have described aketo amide inhibitors with an unsubstituted proline ring for the themetalloprotease angiotensin converting enzyme (ACE) (Gordon et. al. Biochem.

Biophys. Res. Commun. 1984, 124, 141). Additionally, Arai et. al. (Chem. Pharm. Bull. 41, 9, 1583) have reported potent inhibitory activity by a prolyl endopepti.ase (PEP) inhibitor phenylbutanoyl)-L-prolyl]pyrrolidine).

What is needed are combinatorial libraries of HIV and FIV protease inhibitors and simple synthetic methods for making same.

What is needed is a class of HIV and FIV protease inhibitor having enhanced possibilities of variability at the P, and Pi' positions for improving the binding between the enzyme and its inhibitor.

What is needed are methods for screening combinatorial libraries of HIV and FIV protease inhibitors for identifying candidates having both clinically useful inhibitory activity and a potential resistivity to a loss of inhibitory activity due to development of resistant strains of HIV.

What is needed are new HIV and FIV protease inhibitors having clinically useful inhibitory activity and a resistivity to a loss of inhibitory activity due to development of resistant strains of -7-

HIV.

Summary It is disclosed herein that FIV PR provides a good model of drug resistance in retroviral proteases. Testing candidate drugs with respect to their inhibitory activity against both HIV and FIV proteases and determining which inhibitors are simultaneously efficacious against both of these mechanistically identical proteases, identifies inhibitors of HIV protease which are potentially'less prone to resistance development. Candidate drugs which are successfully screened in vitro may then be tested in cats as model systems on which to test HIV PR inhibitors in vivo.

Accordingly, s.e-sfs.t.-a--t-he-i-vne-i is directed to a method for identifying a drug candidate as an HIV protease inhibitor potentially resistive against loss of inhibitory activity due to development: of resistant strains of HIV. The method employs the following steps: Step A: determining whether the drug candidate has a binding activity with respect to HIV protease of less than 1 uM; Step B: determining whether the drug candidate has an inhibitory activity with respect to HIV protease of less than 1 uM; Step C: determining whether the drug candidate has a binding activity with respect to FIV protease of less than 1 uM; and Step D: determining whether the drug candidate has -8an inhibitory activity with respect to FIV protease of less than 1 pM.

If the drug candidate is determined to have binding and inhibitory activities with respect to both HIV protease and FIV protease of less than 1 M in each of the above steps, then the drug candidate is selected as an HIV protease inhibitor potentially resistive against loss of inhibitory activity due to development of resistant strains of HIV.

The above method may be applied either to individual drug candidates or to a library of drug candidates.

Another aspect of the invention is directed to methods for synthesizing drug candidates which potentially inhibit HIV protease. More particularly, the drug candidates are of a type which include an Nterminus, a C-terminus, and an a-keto amide core structure linking the N-terminus and the C-terminus.

The N-terminus incudes an aromatic amino acid residue selected from the group consisting of phenylalanine, tyrosine, and O-substituted tyrpsines. The aromatic amino acid includes a carbonyl group for linking to and incorporation into the a-keto amide core structure. The C-terminus includes a heterocyclic ring having a ring nitrogen and one or more substitutions. The ring nitrogen of the C-terminus is linked to and incorporated into the a-keto amide core structure. 'The synthetic method includes the following step: Step A: providing an N-terminus precursor identical to the N-terminus except that the carbonyl group is replaced by an a-hydroxyl acid group; Step B: providing a C-terminus precursor identical to the C-terminus except that the ring nitrogen forms a secondary amine; Step C: coupling the N-terminus precursor of step A to the C-terminus precursor of step B to form a drug candidate precursor identical to the drug candidate except that the a-keto amide core structure of the drug candidate is replaced by an a-hydroxyl amide core structure linking and incorporating the carbonyl group of the Nterminus and the ring nitrogen of the Cterminus; and then Step D: oxidizing the a-hydroxyl amide core structure of the drug candidate precursor of step C: for forming the a-keto amide core structure and the drug candidate.

The above synthetic method may also be adapted for synthesizing combinatorial libraries of HIV and FIV protease inhibitors containing nxm drug candidates.. The above synthetic method is modified by providing nxm reaction vessels, loading each of the n N-terminus precursors into m of the reaction vessels, and then loading each of the m C-terminus precursors into n of reaction vessels so as to form nxm admixtures of N-terminus precursor and C-terminus precursors. Then, each of the nxm admixtures is allowed to undergo a coupling reaction in which the N-terminus .precursor couples to the C-terminus precursor to form nxm drug candidate precursors. The drug precursor candidates are identical to the nxm drug candidates except that the a-keto amide core structure of the nxm drug candidates is replaced by an a-hydroxyl amide core structure linking and incorporating the carbonyl group of the N-terminus and the ring nitrogen of the C-terminus. Finally, the a-hydroxyl amide core structure of each of the nxm drug candidate precursors is oxidized so as to form the a-keto amide core structure of the desired drug candidate.

A further aspect of the invention is directed to combinatorial libraries nxm drug candidates characterized by having a a-keto amide core structure and a potential inhibitory activity with respect to HIV protease. The drug candidates are characterized by having an N-terminus selected from n N-termini where n is two or greater, a C-terminus selected from m C-termini where m is two or greater, and a a-keto amide core structure which links the Nterminus to the C-terminus. Each of the n N-termini incudes an aromatic amino acid residue selected from the group consisting of phenylalanine, tyrosine, and O-substituted tyrosine. The aromatic amino acid includes a hydroxyethyl group in lieu of a carbonyl group which is linked to and incorporated into the a-keto amide core structure. Each of the m C-termini includes a heterocyclic ring having a ring nitrogen and one or more substitutions. The ring nitrogen of the C-terminus is linked to and is incorporated into the a-keto amide core structure.

Another aspect of the invention is directed to combinatorial libraries of nxm drug candidates -11characterized by having a hydroxyethylamine core structure and a potential inhibitory activity with respect to HIV protease. The drug candidates are characterized by having an N-terminus selected from n N-termini where n is two or greater, a C-terminus selected from m C-termini where m is two or greater, and a hydroxyethylamine core structure which links the N-terminus to the C-terminus. Each of the n Ntermini incudes an aromatic amino acid residue selected from the group consisting of phenylalanine, tyrosine, and O-substituted tyrosine. The aromatic amino acid includes a hydroxyethyl group in lieu of a carbonyl group which is linked to and incorporated into the hydroxyethylamine core structure. Each of the m C-ter:nini includes a heterocyclic ring having a ring nitrogen and one or more substitutions. The ring nitrogen of the C-terminus is linked to and is incorporated into the hydroxyethylamine core structure.

Another aspect of the invention is directed to a series of improved mechanism based inhibitor of HIV or FIV aspartyl protease. Each of the improved mechanism based inhibitors is of a type having an Nterminus, a C-terminus, and a core structure for linking the N-terminus to the C-terminus. The Nterminus incudes an aromatic amino acid residue linked to the core structure. The C-terminus includes a heterocyclic ring having a ring nitrogen linked to the core structure. The core structure is isosteric with a scissile amide bond of an HIV or FIV aspartyl protease substrate.

-12- In the first embodiment of improved mechanism based inhibitor, the core structure is an a-keto amide and the heterocyclic ring of the N-terminus is a pyrrolidine having at least one substituant other than carboxylic acid and carboxymethyl ester.

Examples of this first embodiment of improved mechanism based inhibitors are provided as follows: 22 K Me d j^ OMe 24 Me N o OMe 23 Me OMe Me OMe 16 O 0 36 R=Me

R

37 R=Bn 0 0 0 HTOR O R N

R

52 R=H; 53 R=Bn, 54 R=Me 46 R=H; 47 R=Bn, 48 R=Me 58 R=H 59 R=B, 60 R=M (cis 58 R=H; 59 R=Bn, 60 R=Me (cis 64 R=H; 65 R= Bn, 66 R=Me (tr 0 0< Me N N OR M 1j HN 100 RO OR Bn BnO 101 R=H; 102 R=Bn, 103 R=Me 1050 13tBu-OGln-lie-- N 1055 Bn 1052 1053

H

2 N-Thr-Glri-lle- 1056 where: 1054 R

/H

1059 R

H

6 0R 0~N N H -14- In a second embodiment of improved mechanism based inhibitor, the core structure is an a-keto amide and the heterocyclic ring of the N-terminus is a piperadine or azasugar. Examples of piperadines and azasugars employed in this second embodiment of improved mechanism based inhibitors are provided as follows: o r o o I N N N N R "OR RO "OR OR OR R 85 H, 86 Me, 87 Bn R 88 H, 89 Me, 90 Bn based inhibitor, the core structure is an -keto N N N N HH H RO"' OR RO OR OR 94 OR R 91 H, 92 Me, 93 Bn R 95 H, 96 Me, 97 Bn In a third embodiment of improved mechanism based inhibitor, the core structure is an ca-keto amide and the aromatic amino acid of the C-terminus is selected from a group consisting of tyrosine having a protected amino, tyrosine having a protected amino and a substituted hydroxyl, and phenylalanine having a protected amino protected by carbobenzyloxy.

Examples of this third embodiment of improved mechanism based inhibitors are provided as follows:

R

2

R

2 0 0

R

1 -N N> -RN H f1- R \R 1 0 R-N IV> -HR RH N -R 0

R

1 0 wherein R is selected from the group consisting of hydrogen, hyjdroxy, benzyloxy, alkyl(C 1

-C

4 )-OXY, 0methoxy-benzyloxy, m-methoxy-benzyloxy, p-methoxybenzyloxy, o-methoxy-nitrobenzyloxy, m-rnethoxynitrobenzyloxy, p-methoxy-nitrobenzyloxy, acetoniie, benzylidene, 3-oxymethyl-catechol, 4-oxymethylcatechol; R, is selected from the group consisting of carbobenzyloxy (CBZ), tert-butoxycarbony. (t-BOC), acyl; R 2 is selected from the group consisting of hydrogen, benzyl, alkyl(CI-C 4 o-methoxy-benzyl, mmethoxy-benzyl, p-methoxy-benzyl, o-methoxynitrobenzyl, m-methoxy-nitrobenzyl, p-methoxynitrobenzyl, 3-methylene-catechol, 4-methylenecatechol.

In a fourth embodiment of improved mechanism based inhibitor, the core structure is an -16hydroxyethylamine and the heterocyclic ring of the Nterminus is a pyrrolidine having at least one substituant other than carboxylic acid and carboxymethyl ester. Examples of pyrrolidines employed in this fourth embodiment of improved mechanism based inhibitors are provided as follows: -17- Mecl 0

N

OMe 0-t 16 0

N

H

36 RMe bR 37 R=Bn 46 R=H; 47 R=Bn, 48 R=Me 52 R=H; 53 R=Bn, 54 R=Me 58 R=H; 59 R=Bn, 60 R=Me (cis) 64 R=H, 65 R= Bn, 66 R=Me (ira leH HN -rO A\ 1051 101 R=H; 102 R=Bn, 103 R=N~ and 1058 -18- In a fifth embodiment of improved mechanism based inhibitor, the core structure is hydroxyethylamine and the heterocyclic ring of the Nterminus is a piperadine or azasugar. Examples of piperadines and azasugars employed in this fifth embodiment of improved mechanism based inhibitors are provided as follows: 0 I I 0 N

N

H

RO "OR

OR

Bn R 88 H, 89 Me, 90 Bn

OR

R 85 H, 86 Me, 87 N N

SN

ROr Y OR H OR 94 R 91 H, 92 Me, 93 Bn

O

-N

N

H

RO OR

OR

R 95 H, 96 Me, 97 Bn

I

-19- In a sixth embodiment of improved mechanism based inhibitor, the core structure is hydroxyethylamine and the aromatic amino acid of the C-terminus is selected from a group consisting of tyrosine having a protected amino, tyrosine having a protected amino and a substituted hydroxyl, and phenylalanine having a protected amino protected by carbobenzyloxy. Examples of this sixth embodiment of improved mechanism based inhibitors are provided as follows:

R

2 0

R

2 0,O 0 0 -N

HN

R N R R HN" <N R-NN-R I -R H R 0O wherein R is selected from the group consisting of hydrogen, hydroxy, benzyloxy, alkylic-c4o-oxy, omethoxy-benzyloxy, m-methoxy-benzyloxy, p-methoxybenzyloxy, o-methoxy-nitrobenzyloxy, m-methoxynitrobenzyloxy, p-methoxy-nitrobenzyloxy, acetonide, benzylidene, 3-oxymethyl-catechol, 4-oxymethylcatechol; Ri is selected from the group consisting of carbobenzyloxy (CBZ), tert-butoxycarbonyl (t-BOC), acyl; R 2 is selected from the group consisting of hydrogen, benzyl, alkyl(c-c 4 o-methoxy-benzyl, mmethoxy-benzyl, p-methoxy-benzyl, o-methoxynitrobenzyl, m-methoxy-nitrobenzyl, p-methoxynitrobenzyl, 3-methylene-catechol, 4-methylenecatechol.

Des.criptiQL__Qf .Figures Figure 1 illustrates an approach to rapidly access a number of potential inhibitors of HIV and/or FIV proteases to determine the protecting group and ideal substitution pattern of the 'proline' moiety to provide maximum inhibition of the enzymes.

Figure 2 illustrates a comparison of the a keto amide core structure to other isosteric structures.

Compound activities are indicated.

Figure 3 illustrates hydrogen bond interactions between a hydroxyethylamine isostere and the active site of HIV PR as observed from X-ray crystallography.

Figure 4 illustrates a general acid base mechanism for inhibitor 2 interaction with HIV PR aspartate groups.

Figure 5 illustrates inhibitory activity of variously substituted pyrrolidine analogues against -21- HIV PR.

Figure 6 illustrates the synthesis of compound 11. The steps indicated are as follows: t-BuLi, ethyl vinyl: ether, MgBr 2 THF; (ii) 03, CH 2 C1 2 (iii) 0.17N LiOH, MeOH/H 2 0, 2:1, 98%.

Figure 7 illustrates the synthesis of compound 2. The steps indicated are as follows: NaBH 4 MeOH, 0 oC, 95%; (ii) 0.17 N LiOH, MeOH/H 2 0, 2:1, 98%; (iii) EDC, HOBT, DIEA, DMF/CH 3 CN, 84%; (iv) Dess--Martin periodinane, CH 2 C1 2 Figure 8 illustrates the synthesis of compound 3. The steps are indicated as follows: EDC, HOBT, DIEA, DMF/CH 3 CN, 80%; (ii) Dess-Martin periodinane, CH 2 Cl 2 quantitative.

Figure 9 illustrates the synthesis of compounds 31 and 32 with the indicated substrate and reagents.

Figure 10 illustrates the synthesis of compound with the indicated substrate and reagents.

Figure 11 illustrates a general coupling procedure to form hydroxyethyl amine compounds starting from epoxide 21 and using the indicated pyrolidines. The procedure is as follows: (i) Methanol, Et 3 N, reflux, 24 hours, 40-60%.

Figure 12 illustrates a general coupling procedure to form a-ketoamide compounds starting from -22carboxyacid 15 and using the indicated pyrolidines.

The two step procedure is as follows: EDC, HOBT, DIEA, DMF/CH 3 CN, (ii) Dess-Martin periodinane oxidation, CH 2 Cl 2 quantitative.

Figure 13 illustrates a general coupling procedure to form a-ketoamide compounds starting from carboxyacid 15 and using the indicated substituted piperidines. The two step procedure is as follows: EDC, HOBT, DIEA, DMF/CH 3 CN, (ii) Dess- Martin periodinane oxidation, CH 2 Cl 2 quantitative.

Figure 14 illustrates a general coupling procedure to form hydroxyethyl amine compounds starting from epoxide 21 and using the indicated substituted piperidines. The procedure is as follows: Methanol, Et 3 N, reflux, 24 hours, Figure 15 illustrates the synthesis of pyrolidines 46, 47 and 48 with the indicated substrate and reagents.

Figure 16 illustrates the synthesis of pyrolidines 52, 53 and 54 with the indicated substrate and reagents.

Figure '17 illustrates the synthesis of pyrolidine 44 with the indicated substrate and reagents.

Figure 18 illustrates the synthesis of -23pyrolidine 40 with the following steps: CBZ-C1, methylene chloride; (ii) TBDPSC1, Et 3 N, DMF; (iii) Methyl iodide, sodium hydride, DMF; (iv) TBAF, THF; 1.5 M H,S0 4 CrO,, acetone; (vi) H 2

N

t Bu, EDC, methylene chloride; (vii) H 2 Pd(OH) 2 on Carbon, MeOH.

Figure 19 illustrates the synthesis of pyrolidine 42 with the following steps: BOC-ON, methylene chloride; (ii) TBDPSC1, Et 3 N, DMF; (iii) Benzyl bromide, sodium hydride, DMF; (iv) TBAF, THF; 1.5 M H2SO 4 CrO 3 acetone; (vi) H 2

N

t Bu, EDC, methylene chloride; (vii) H 2 Pd(OH) 2 on Carbon, MeOH.

Figure 20 illustrates the synthesis of pyrolidines 58, 59, 60, 64, 65, and 66 with the following steps: For compounds 58, 59, 60: CBZ- Cl, methylene chloride; (ii) TBDMSC1, Et 3 N, DMF; (iii)

H

2

N

t Bu, EDC, methylene chloride; (iv) TBAF, THF; (v) "Mitsunobu" procedure: PPh 3

DEAD

(diethylaza.odicarboxylate),

CICH

2

CO

2 H, then water, pyridine to pH 6.7; (vi) Benzyl bromide for 59 or methyl iodide for 60, sodium hydride, DMF (omit this step for 58) (vii) H 2 Pd(OH) 2 on Carbon, MeOH. For compounds 64, 65, and 66 same conditions as above without step "Mitsunobu" procedure.

Figure 21 illustrates the synthesis of piperidines 86 and 87 with the following steps: (i) CBZ-C1 or BOC-ON, methylene chloride; (ii) TBDPSC1, Et 3 N, DMF; (iii) Benzyl bromide for 87 or methyl iodide for 86, sodium hydride, DMF (iv) TBAF, THF; 1.5 M H 2

SO

4 Cr0 3 acetone; (vi) H 2

N

t Bu, EDC, methylene chloride; (vii) H 2 Pd(OH) 2 on Carbon, MeOH.

-24- Figure 22 illustrates the synthesis of piperidines 85 with the following steps: CBZ-C1 or BOC-ON, methylene chloride; TBDPSC1, Et 3 N, DMF; (ii) acetic anhydride, triethylamine, DMF (iii) TBAF, THF; (iv) 1.5 M H 2

SO

4 Cr0 3 acetone;

H

2

N

t Bu, EDC, methylene chloride; (vi) NaOMe, MeOH; (vii) H,, Pd(OH) 2 on Carbon, MeOH.

Figure 23 illustrates tyrosine derivatives of pyrolidine or piperidine containing a-ketoamide and hydroxylethylamine core structures to probe for FIV and HIV protease selectivity.

Figure 24 illustrates targeted variable sites on the core structure which can be assessed using a combinatorial approach. Di-/tri-/tetrameric substrates for HIV-PR and FIV-PR are possible.

Candidate molecules display activity by cleavage of either HIV-PR or FIV-PR protease. The candidate molecules are subsequently rederivatized via replacement of the scissile amide bond to an a-keto amide or hydroxyethyl amine, affording further potential inhibitors. The synthetic strategy involves solution phase coupling of the added residues (standard coupling conditions, EDC HOBT,

CH

2 Cl 2 or DMF) to afford di-/tri-/tetra peptides.

Simple acidic and basic washes afford the desired peptides cleanly, with no purification necessary.

Compounds are then dissolved in a minimal amount of DMSO and added to buffer solution, arranged in 96well micro-titer plates. Addition of the enzyme and subsequent mass spectral analysis identifies the cleaved substrates as hits. These molecules are then rederivatized with an a-keto amide or hydroxyethyl amine core unit and tested for inhibitory activity.

Figure 25 illustrates the creation of non-linear protease inhibitors using the up and down search for new complementary binding sites attached to a designed mechanism-based core structure.

Figure 26 illustrates various a-ketoamide and hydroxyethyl HIV protease inhibitors.

Figure 27 illustrates the synthesis of aketoamide compound 1050 with the indicated substrate and reagents.

Figure 28 illustrates the synthesis of hydroxyethylamine compound 1051 with the indicated substrate and reagents.

Figure 29 illustrates the synthesis of aketoamide compound 1053 with the indicated substrate and reagents.

Figure 30 illustrates the synthesis of aketoamide cDmpounds 1054, 1059, and 1060 with the indicated substrates and reagents.

Figure 31 illustrates the synthesis of aketoamide compound 1055 with the indicated substrate and reagents.

-26- Figure 32 illustrates the synthesis of aketoamide compound 1056 with the indicated substrate and reagents.

Figure 33 illustrates the synthesis of aketoamide compound 1057 with the indicated substrate and reagents.

Figure 34 illustrates the synthesis of aketoamide compound 1058 with the indicated substrate and reagents.

-27- One aspect of the invention is directed to a new class of the HIV/FIV PR inhibitors, viz. a-keto amide inhibitors. The mode of action of the a-keto amide inhibitor is of particular interest as it represents a new type of mechanism-based enzyme inhibition which can lead to the development of tight-binding inhibitors :o overcome the problem of resistance.

When assayed against HIV PR, the novel a-keto amide 1 (Figure 3) was found to have a Ki of 6 pM.

Subsequent studies have shown that a simple modification of the N- and C-terminal protecting groups to give 2 (Figure 3) enhances the potency of this core isostere against HIV PR, to give a K i of 214 nM.

The increase in activity in compound 2 may be due to favorable hydrophobic interactions between the protecting groups the Cbz-protecting group and the t-Bu group) and the active site of HIV PR. The Boc-protecting group is also hydrophobic but is shorter and sterically more bulky, making it unable to extend effectively into the appropriate hydrophobic binding pocket. This result demonstrates how simple modifications of the core isostere can significantly improve its potency. It is disclosed that the pctency of the a-keto amide 2 can be improved by introduction of additional complementary groups to the proline ring moiety. Computer modeling (Insight/Discover) indicates that attachment of hydrophobic groups to the proline ring moiety will -28enhance binding. It is disclosed that addition of a cis-benzyl ether to C-4 of the proline moiety increased binding 3-fold (K i The activity of the isostere 2 can be then compared to that of the identically substituted ahydroxy amide precursors 23 and 24. The Sdiastereomer 24 (ICs 2pM) is disclosed to be more potent than the R-diastereomer 23 (IC50 300 pM), but less active than 2. The high potency of the ahydroxy amide 24 implies that the hydroxyl group is hydrogen bonding more effectively with the catalytic carboxylic: acid groups of HIV PR than in compound 23, similar to that observed in the X-ray structure of a hydroxyethylamine inhibitor enzyme complex (Figure The stereochemistry of the isosteres 23 and 24 was determined by "H NMR studies on the R- and S- Mosher esters derived from the S-a-hydroxy ester 19.

The ketone moiety of the a-keto amide 2 is disclosed to be hydrated and is hydrogen bonding in a similar manner to that of compound 23. However in this case, "1C NMR studies on compound 2 in deuterated

DMSO/D

2 0 indicates that, in the presence of water, the ketone moiety of the a-keto amide remains unhydrated even after incubation for 24 hours (Figure This indicates that the ketone moiety of 2 is stable in the presence of water and is therefore difficult to hydrate in the absence of a catalyst.

It is likely that hydration of the ketone moiety takes place within the active site of HIV PR as -29illustrated in Figure 6, and the resulting hydrate is then stabilized through hydrogen bonding interactions with the aspartate residues of the enzyme. The hydrated form of 2 is considered to be a good transition state mimic based on the model presented in Figure 6.

Time dependent assays do not exhibit time dependent inhibition, indicating that if the active form of the a-keto amide 2 is indeed the hydrate, the hydration step must be rapid or 2 itself is the active form. To further investigate the mode of action, the X-ray structure of the complex between the a-keto amide 2 and HIV PR was determined and the result indicated that the a-keto amide is hydrated, supporting the enzyme-assisted hydration mechanism (Figure 7).

As seen in the electron density map, the inhibitor is bound in the active site of HIV-PR in its hydrated state (Figure One of the two hydroxyls is located between two aspartates, making hydrogen bonds with both of them, while the other hydroxyl interacts with only one aspartate. The keto group is also hydrogen bonded to a catalytic aspartate. The phenylalanine and proline side chains occupy proper S 1 and SI' pockets making interactions similar to those observed in other structures. The carboxybenzyl group on the N-terminus and the tertbutyl group of the tert-butyl amide on the C-terminus of the inhibitor are roughly in the S 2 and S2'pockets, respectively. The conserved water (Wat-301), which is forming hydrogen bonds between the inhibitor and the two flaps of the enzyme, is clearly observed.

Its location is rather asymmetric, unlike the case of the majority of other inhibitor complexes. The distance between the carbonyl oxygen which mimics the

P

2 CO group and Wat-301 is 2.5 A, while the distance to the Pi'CO is 3.2 A.

Remarkable similarity is observed in the binding mode of the central and C-terminal parts of compound 2, and the similar parts of an inhibitor KNI-272. The differences in their binding constants are very significant (Ki for KNI-272 is almost five orders of magnitude lower than for despite the presence of an extra hydrogen bond in the central part of the complex of HIV PR and compound 2. Thus the increase in the potency of KNI-272 must be due to the elongation of its N-terminus, which has a bulky isoquinolyloxyacetyl (IQoa) moiety. The regions of the protein interacting with this group, including the Phe-53 in one monomer and Pro-81 in another shift significantly towards the inhibitor, making extensive hydrophobic contacts with the IQoa group.

When the a-keto amide 2 was tested against FIV PR, it was found to have no inhibitory effect when added in concentrations up to 70 mM. This result was surprising due to the similarity between the natural substrates for HIV and FIV proteases bordering the matrix/capsid cleavage site as illustrated earlier.

It appears that FIV PR may require additional specific residues between the P 4

P

4 'sites, than HIV PR, before it is able to recognize the core isostere -31- 2 as a substrate. This is also supported by the observation that HIV PR will cleave an Acetyl-(6 residue) peptide substrate of sequence Gln-Ala- Tyr-Pro-Ile-Gln whereas the smallest peptide FIV PR is known to cleave is an acetyl-(8 residue) peptide of sequence Pro-Gln-Ala-Tyr-Pro-Ile-Gln-Thr. The corresponding protected dipeptide (Cbz-Phe-Pro-NBu t was not cleaved by HIV or FIV proteases under normal assay conditions.

In an effort to develop inhibitors of the FIV PR, it was found that the addition of suitable residues to interact with just the Pz'and P 3 sites of FIV PR was sufficient for moderate inhibition.

Coupling of a side chain specific for FIV PR to the C-terminus of 2 gave 4 (Figure This extended isostere 4 was found to have an IC 50 of 25iM and a Ki of 29 pM against FIV PR and the activity against HIV PR was slightly enhanced (Ki of 154 nM). It appears that the isosteric core structure of HIV PR inhibitors do not bind tightly to the FIV PR, and additional complementary groups are needed to enhance the binding. This difference is also observed in the analysis of other known HIV PR inhibitors. The potent cyclic urea based HIV PR inhibitor DMP 323 (ICo 0 36n1, Ki 0.27nM) for example was also found to be a very poor inhibitor of FIV PR (IC 50 7.3mM) When the activity of the monomethylated derivative 7 is compared to that of the dimethylated derivative 5, it can be seen that a hydrophobic moiety at the C-5 position decreases the potency of the isostee, whereas the activity increases when -32similar hydrophobic substitutions are made at the C-3 and C-4 positions as in compound 6. The permethylated pyrrolidine derivative 6 was the most potent derivative of the series 5-7, but significant activity was lost due to the absence of the amide bond at C-1, as can be seen by comparison of the activity of the methylated derivatives 5-7 to that of compound 8. The C-4 substituted pyrrolidine derivatives 9-11 were shown to be more potent than 8, with the trans-C-4 methoxy derivative 10 being the best (ICs 2.9 SM).

The permethylated a-keto amide derivative 12 (Figure 10) was synthesized for comparison and was found to be more potent than the corresponding hydroxyethylamine isostere 6 but significantly less potent (K i 20~M) than the original isostere 2 against HIV PR. This result again illustrates the importance of an amide bond at C-l of the pyrrolidine derivative.

In summary, a new class of mechanism based inhibitors of the HIV and FIV proteases with pyrrolidine-containing a-keto amide and hydroxyethylamine core structures is disclosed.

These a-keto amide core structure 2 are disclosed to be approximately 300-fold better than the corresponding hydroxyethylamine isosteric structure and 1300-fold better than the corresponding phosphinic acid derivative as an inhibitor of the HIV protease. The a-keto amide is however not hydrated until it is bound to the HIV protease as indicated by -33the NMR stucy and the X-ray structural analysis.

Further analysis of the inhibition activities of hydroxyethylamine isosteres containing modified pyrrolidine derivatives disclosed that a cis-methoxy group at C-4 of the pyrrolidine improved the binding and 25-fold for the trans-isomer. When this strategy was applied to the a-keto amide isostere, a cis-benzyl ether at C-4 was found to enhance binding 3-fold. Of the core structures prepared as inhibitors of the HIV protease, none show significant inhibitory activity against the mechanistically identical FIV protease, and additional complementary groups are needed to improve inhibition.

Another aspect of the invention is directed to the use of FIV PR as a model for the development of HIV PR inhibitors and for the study of drug resistance. This disclosure is supported by the data reported herein and by several additional observations. First, both enzymes are mechanistically identical. Second, structure-based alignment of the two enzymes displays their structure similarity (Figures 11 and 12). Third, most potent non-covalent inhibitors of the HIV PR are not good inhibitors of the FIV PR, whereas FIV PR inhibitors are often better HIV PR inhibitors. Therefore, good low molecular weight inhibitors of the FIV PR are disclosed to be better for the HIV PR. Fourth, the HIV PR isolated from the inhibitor-resistant mutants contains mutations that are found in the corresponding positions of the FIV PR. As shown in Figure 12, the six highlights represent the changed amino acids found in the HIV PR isolated from mutants -34resistant to HIV PR inhibitors which are identical to those found in the FIV PR in the same position.

Accordingly, FIV PR is disclosed to be a good model for the development of HIV PR inhibitors with less resistance problem. For example, the HIV PR isolated from a mutant resistant to a synthetic protease inhibitor contains the I50V mutation, and the mutant protease is less inhibited by the same inhibitor by Table I. Inhibition activities against HIV and/or FIV PR. The inhibitory activities of various isosteres against the HIV and/or FIV PR illustrating a dual screening method against these related viral proteases are illustrated in Table I provided below: Table I. Inhibition activities against HIV and/or FIV PR.

HVPR____ f-FTV PR-_ Comipound IC: 3 IC50 K 1 1 6jiM 64M-- 2 700nM 214nM >70mM 2 a 1.57,uM 220nM 21) 1. 9gM 3l8nM 2c" 460nM 3 3 .18g M 405nM 4 54nM 254M 29gM 1.8mM 6 100gM 7 1.6mM 8 60gM 9 17gM 1( 2.9gM 1:1. 3.94M 12 20gM -36- Syntheit. Mehoda 1) Biological Assays For determination of IC 50 values for HIV protease, a backbone engineered HIV-1 protease, prepared by total chemical synthesis (Kent et. al.

Science 1992, 256, 221) 450 nM final concentration was added to a solution (152 uL final volume) containinc inhibitor, 28 M fluorogenic peptide substrate (sequence Abz-Thr-Ile-Nle-Phe-(p-NO 2 )-Gln- Arg-NH 2 (Toth et. al., Int. J. Peptide Res. 1990, 36, 544)) and 1.8 dimethylsulfoxide in assay buffer: 100mM MES buffer containing 0.5 mg/mL BSA (Bovine Serum Album, fatty acid, nuclease and protease free to stabilize enzyme) at pH 5.5. The solution was mixed and incubated over 5 minutes during which time the rate of substrate cleavage was monitored by continuously recording the change in fluorescence of the assay solution. An excitation filter of 325 nm, and an emission filter of 420 nm were used. This data was converted into M substrate cleaved per minute, using a predetermined standard calibration curve of change in fluorescence against concentration of substrate cleaved.

Determination of K i for HIV protease was performed similarly with the following modifications.

The substrate concentrations used were 57, 43, 28 and 14 uM. All other concentrations were as above. The curve fit for the data was determined and the subsequent K i derived using a computer program based on the equation of Morrison et. al. BioChim.

Biophys. ACTA 1969,185, 269, for tight binding -37inhibitors.

For determination of K i and IC 50 for FIV protease, 0.125 pg of the enzyme was added to a solution (lOOpL final volume) containing inhibitor, 560 pM peptide substrate (sequence Gly-Lys-Glu-Glu- Gly-Pro-Pro-Gln-Ala-Tyr-Pro-Ile-Gln-Thr-Val-Asn-Gly) and 2% dimethyl sulfoxide in a 1:3 mixture of assay buffer (as above) and 4M NaClaq. solution. The solution was mixed and incubated for 10 minutes at 37 oC and the reaction quenched by addition of 8M guanidine HC1 solution containing 0.2 M sodium acetate at pH 4.2 (100 pL). The cleavage products and substrate were separated by reverse phase HPLC.

Absorbance was measured at 215 nm, peak areas were determined and percent conversion to product was calculated using relative peak areas. The data were plotted as 1/V (V rate substrate is cleaved in nmol/min) against inhibitor concentration and the -K i determined as the point at which the resulting line intersects with 1/Vmax (Vmax 6.85 nmol/min). IC 50 was determined as the inhibitor concentration at inhibition. Vmax (6.85 0.7 nmol min 1 and Km (707 for FIV protease were determined from a plot of 1/V (V rate in nmol/min) against substrate concentration in nmol). The data used was generated similarly to that for K i with the following modifications. The substrate concentrations used were 560, 448, 336, 224, 111 and 56 pM, in the absence of inhibitor.

Purification of FIV protease: A 503 base pair Eco Ri-Bam HI fragment containing the coding sequence of FIV protease was cloned from FIV-34TF10 (Talbott et. al. Proc. Natl. Acad. Sci. USA 86 1989, 5743) -38into the pT7-7 vector (Tabor et. al. Proc. Natl.

Acad. Sci USA 82 1985, 1074). The 5' end of the insert was modified by the addition of an Ndel adaptor, which provided the proper reading frame with initiation of translation from the methionine encoded in the la:ter site. Translation resulted in production of an 18.6 kDa precursor, which autoprocessed to a 13.2 kDa FIV PR plus N- and Cterminal fragments of 3.6 kDa and 1.8 kDa, respectively. The construct was transformed into E.

coli strain BL21.DE3, lys S (Studier et. al. Meth.

Enzymol. 1990, 185, 60) and overnight cultures were used to inoculate 15 liter fermentations, performed using Circlegrow medium (Bio 101) plus 100 L ampicillin, 20 uM chloramphenicol, at 37 The cells were allowed to reach mid-log phase, then the temperature was reduced to 24 oC and IPTG (isopropyl3-thiogalactopyranoside) was added to a final concentration of 1 mM. The fermentation was allowed to proceed for 16 hours, at which time the cells were harvested by centrifugation and frozen at °C in 100 g aliquots for future use.

Cells (100 g) were lysed by addition of 600 mL, mM Tris-HC1, pH 8, 5 mM EDTA and 2 mM 2mercaptoethanol to the frozen pellet. The cells lysed upon thawing and the viscous mixture was homogenized at 4 OC for 2 min in a Waring blender.

The sample was centrifuged at 8,000 x g for 20 min and the pellet discarded. The sample was diluted to 1 liter, then subjected to tangential flow against a 300 K cut-off membrane (Filtron) and the PR was washed through the membrane using five liters of the same buffer. The retentate was discarded and the -39flow-through supernatant concentrated by tangential flow against a 10 K cut-off membrane. The retentate was passed over a DE52 anion exchange column (5 x cm) equilibrated in the same buffer. The flowthrough from this column was passed over an S- Sepharose Fast Flow matrix 2.5 x 20 cm column, Pharmacia), again equilibrated at pH 8 in the same buffer. The flow-through from S-Sepharose was made 1M with respect to ammonium sulfate and applied to a phenyl sepharose column (Pharmacia, 1.5 x 10 cm), washed with lysis buffer containing 1M ammonium sulfate, then eluted with a 100-0% linear ammonium sulfate gradient. Peak fractions containing PR were pooled, concentrated using Centripreps (Amicon), and dialyzed against 10 mM Tris-HCl, pH 8, 5 mM EDTA, 2 mM 2-mercaptoethanol. The sample was made 10 mM with respect to MOPS, adjusted to pH 5.5 with HC1, then applied to a Resource S column (Pharmacia) equilibrated in 10 mM Tris-MOPS, pH 5.5, 5 mM EDTA and 2 mM 2-mercaptoethanol. PR was eluted using a linear 0-300 mM NaCI gradient in the same buffer.

Peak fractions were pooled, concentrated, and stored as aliquots at -20 OC for further studies. The integrity cf the isolated FIV PR was confirmed by ion spray mass spectrometry.

2) Chemical__Synthesis All manipulations were conducted under an inert atmosphere (argon or nitrogen). All solvents were reagent grade. Anhydrous ether, tetrahydrofuran (THF), and toluene were distilled from sodium and/or benzophenone ketyl. Dichloromethane (CH 2 C12) was distilled from calcium hydride (CaH 2 N, N, Dimethylformamide (DMF) and acetonitrile were distilled from phosphorous pentoxide and calcium hydride. Methanol was distilled from magnesium and iodine. Organic acids and bases were reagent grade.

All other reagents were commercial compounds of the highest purity available. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel (60 F-254) plates (0.25 mm). Visualization was effected using standard procedures unless otherwise stated. Flash column chromatography was carried out on Merck silica gel 60 particle size (0.040-0.063 mm, 230-400 Mesh). Melting points were determined with a Thomas-Hcover capillary melting point apparatus and are uncorrected. Proton and carbon magnetic resonance spectra 1 H-NMR, 3 C-NMR) were recorded on either a Bruker AM-500, AMX-400 or AC250MHz Fourier transform spectrometer. Coupling constants are reported in hertz and chemical shifts are reported in parts per million relative to tetramethylsilane (TMS, 0 ppm), MeOH (3.30 ppm for H and 49.0 ppm for 13C) or CHC1 3 (7.24 ppm for H and 77.0 ppm for C) as internal reference. Infrared spectra (IR) were recorded on a Perkin-Elmer 1600 series FT-IR spectrophotometer. Absorptions are reported in wavenumbers (cm -41- Peptide fragments described herein were synthesized using traditional peptide coupling methodologies [EDC (3-dimethylaminopropyl)-3ethylcarbod:Limlde H1i), HOBt (1-hydroxybenzotriazole) and DIEA (d:isopropylethylam~ine) Esters were hydrolyzed either by base (LiOH for methyl esters) or acid (TFA for t-butyl esters).

-42- Synthesis of compound 10 as illustrated in figure 6 0 Cbz N OEt H 0 Compound 10: The a-keto ester 10 was synthesized according to the method described by Angelastro et al. J. Org. Chem. 1989, 54, 3913-3916. It is possible to synthesize a-keto esters via other methodologies, Wasserman, et al. J. Org. Chem. 1994, 59, 4364-4366(43); Wong et al. J. Med. Chem 1993, 36, 211-220, but the route employed was found to be most concise and high yielding. The methodology, shown in figure 1 and described by Angelastro, is as follows t-BuLi, ethyl vinyl ether, MgBr 2 THF; (ii) 03,

CH

2 C12.

Synthesis of compound 11 as illustrated in figure 6, step (iii) Cbz N OH H -43- Compound 11. To a 2:1 solution of compound 10 in methanol/water (.10 Molar) is added a 0.17 Normal solution of LiOH in water and the reaction is allowed to stir at 0 °C for 2 hours. The mixture is then purified by reverse phase chromatography and yielded compound 11 in 98% overall yield.

Synthesis of (2R, 3S) and (2S, 3S) N- (Benzyloxycarbonyl)-AHAP-(3-Amino-2-Hydroxy-4 Phenylbutanoic Acid) Ethyl Ester 12 and 13 as shown in figure 7.

0 Cbz-N OEt H H

OH

12-R 13-S As illustrated in figure 7, step i, the substrate (600 mg, 1.7 mmol) was dissolved in methanol (10 mL) and cooled to 0 Sodium Borohydride (70.3 mg, 1.9 mmol) was then added. After 20 minutes the reaction was quenched by addition of saturated ammonium chloride,,q.: (10 mL). The reaction mixture was concentrated in vacuo to remove most of the methanol.

The aqueous residue was then extracted with ethyl acetate (3 x 20 mL), washed with brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product as a mixture of diastereomers. The alcohols -44were separated by flash chromatography eluting with ethyl a~cetate in hexane to give the alcohols in a ratio of 3:4, 12 13 (590 ing, Rt 0.54 and 0.40 respectively (EtOAc/Hexane, 12 (2R, 3S colorless ciil); 1 H NI4R (500MHz, CDCl 3 67.34-7.18 in), 5.17 (1K, d, J 5.04 (2H, 4.43- 4.38 (1H, m)O, 4.33 (1H, dd, J 4.5, 4.15-4.08 (1H, in), 3.98-3.92 (1H, mn), 3.28 (1H, d, J IR (Na~l) Umax 3368, 3030, 2981, 1731, 1520, 1455, 1246, 1104, 1055, 748, 699 cm- 1 FABHRMS (NBA) m/e 358.1659

C

2

,)H

2 3 N0 5 requires 358. 1654) (Found: C, 67.30; H, E.50; N, 3.99. C 2 0

H

2 3 N0 5 requires C, 67.21; H, 6.49; N, 3.92%) 13 (2S, 3S crystalline); 1 H NI4R (500MHz, CEOCl 3 67.34-7.18 (10H, mn), 5.17 (1H, d, J 5.04 (2H, 4.43-4.38 (1H, mn), 4.33 (1K, dd, J 4.5, 4.15-4.08 (1K, mn), 3.98-3.92 (1H, mn), 3. 28 (1 H, dL, J IR (NaCl) 3368, 3030, 2980, 1731, 1520, 1455, 1246, 1104, 1055, 748, 699 cm- 1 FABHESMS (NBA) mle 358.1661 C 2 0

H

23 N0 requires 358.1654); (Found: C, 67.22; H, 6.57; N, 3.90. C 2 0

H

23 N0 5 requires C, 67.21; H, 6.49; N, 3.92%) in.p. 88-89 0

C.

Synthesis of (2R, 3S) and (2S, 3S) N- (Benzyloxyc-arbonyl) -3-Amino-2-Hydroxy-4- Phenylbutantoic Acid 14 15 respectively as illustrated in figure 7 3bz-N' Y OH

H

OH

14-R As illustrated in figure 7, step ii, the substrate (12 or 13) (250 mg, 0.70 mmol) was dissolved in 0.25 N LiOH in methanol/water, 2:1 (5 mL), and stirred at ambient temperature for 30 minutes. The pH of the reaction was adjusted to pH 7.0 with IN HCl and the methanol removed in vacuo. The aqueous residue was then acidified to pH 2.0 with IN HC1 and extracted with ethyl acetate (3 x 30 mL). The combined organic extracts were washed with water mL), brine (10 mL) and dried (MgSO 4 before concentration in vacuo to give the desired acid (14 or 15) as a white solid (212 mg, The acids were purified by recrystallization from hot ethanol.

14 (2R, 1H NMR (500MHz, CD30D) 6 7.31-7.18 6.87 (1H, d, J 5.00 (1H, d, J 10.0), 4.96 (1H, d, J 10.0), 4.31-4.23 (1H, 4.07 (1H, d, J 2.93 (1H, dd, J 13.5, 2.83 (1H, dd, J 13.5, 3C NMR (125MHz, CD30D) 6 176.6 158.5 139.8 138.5 130.7 (2 x CH), 129.8 (2 x CH), 129.7 (2 x CH), 129.7 129.1 -46- 128.8 127.8 67.6 57.1 (CR 2 57.0 (CR 2 39.3 EABRRMS (NBA) m/e 330.1353

C-_

8

H

19 N0 5 requires 330.1341); m.p. 209-210 (decomp.) 15 (2S, 3S); lH NNR (500MHz, CD 3 OD)6 7.28-7.18 (10H, mn), 7.09 (1H, d, J 12.5), 4.97 (1H, cd, J 12. 5) 4 .92 (iR, di, J 12. 5) 4. 26 (1H, di, T 4.25-4.20 (1H, mn), 2.81 (1H, dcl, J 14.0, 2.76 (1H, id., J 14.0, 1 3 C NMP. (125MHz, CD3OD) 8 175.9 158.5 140.0 138.6 130.6 (3 x CR), 129.6 129.5 (3 x CH), 129.0 128.3 127.6 74.3 67.4 (CH 2 57.1 36.5 (CR 2 FABHRMS (NBA/NaT) m/e 352.1174 [M+Na] C 18

H

19 N0 5 requires 352.1161); (Found: C, 65.34; H, 5.75; N, 4.33. C 18

H~

9

NO

requires C, 65.64; H, 5.82; N, rn.p. 173-174 0

C

(decorup.).

-47- Synthesis of L-Prolyl-tert-buty. amnide 16 (scheme 7)

HN

H

16 The substrate N-tert-Butoxycarbonyl-L-proline commercially available from Sigma, (3.0 g, 13.9 mmcl) was dissolved in dry CH 2 Cl 2 (20 mL). HOBT, 1hydroxyben2.Otriazole hydrate (2.07 g, 15.3 mmol), EDC, 1- 3 -climethylaminopropyl)-3-ethylcarbodiimide, (2.93 g, 15.3 mmcl), and tert-butylamine (1.6 mL, 15.3 mmol), were added and the mixture stirred for 18 hours at ambient temperature. The reaction was diluted wit~h ethyl acetate (100 mL), and washed with water (2 x 20 mL) 1 N HCl (10 mL saturated sodium bicarbonate solution (10 mL) water mL) brine (10 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Purification by flash chromatography, eluting with 33% EtOAc in Hexane gave N-tert-butoxycarbonyl-.Lprolyl-te-rt:-butyl amide as a colorless oil (1.53 mg, Rf =0.46 (EtOAc/Hexane, 1 H NNR signals broadened clue to rotamers: 'H NMR (500MHz, CDCl 3 )6 7.31-7.25 K 5H, in), 6.35 (1H, br 4.60-4.15 (3H-, in), 3.5S-3.22 (2H, in), 2.41-1.70 (4H, in), 1-.60-1.18 (9H, br IR (NaCl) Uma 3298, 3086, 2976, 1698, 1660, 1531, 1398, 1162 cm- 1 FA.BHRNS (NBA/NaI) m/e -48- 293.1832 ([M+Na] C 1 4

H

2 6

N

2 0 3 requires 293.1841). The N-tert-Butoxycarbonyl-L-prolyl-tert-butyl amide was carried on as follows: N-tert-Butoxycarbonyl-L-prolyl-tert-butyl amide (600 mg, 2.22 mmol) was dissolved in CH 2 C1 2 (10 mL) and cooled to 0 TFA, trifluoracetic acid, mL) was then added to the solution. After 1 hour at 0 °C the reaction was concentrated in vacuo (any remaining TFA was removed under high vacuum) to give the trifluoroacetic acid salt of the desired amine 16 as a colorless oil (800 mg, The amine was used without further purification in subsequent coupling steps. IH NMR (500MHz, CDCl 3 7.30 (1H, br s), 6.90 (1H, br 4.52 (1H, br 3.35 (2H, br s), 2.49-2.34 (1H, 2.08-1.97 (3H, 1.34 (9H, s); 13C NMR (125MHz, CDC1 3 167.4 59.6 (CH), 52.3 46.6 (CH 2 30.4 (CH 2 28.2 (3 x CH 3 24.6

(CH

2 FA13HRMS (NBA) m/e 171.1500

C

9

H

1 sN 2 0 requires 171.1497).

-49rQactL 3 as 1vstaecin Synthesis cf (2 S, 3R) and (2S, 3S) 3-(N- Benzyloxycarbofyl) amino-2 -hydroxcy-4 -phenylbutyryl -Lprolyl-tert-butyl amnide 17 and 18.

OH

Cbz-NN H -R

OHH

Cbz -NV N 18 -S As illustrated in figure 7, step iii, the substrate 14 or 15 (70 mg, 0.213 mrmol) was dissolved in dry DNF (3 mL) HOET, 1-hydroxybenzotriazole hydrate (31 mg, 0.22 mnxol), EDC, 1-(3-dimethylaminopropyl)-3ethyl ca rbc'di imide, (43 mg, 0.224 inmol), DIEA, di isopropyl ethyl amine, (122 pa1, 0.703 mmol) were added and the mixture stirred for 30 minutes at room temperature. The secondary amine 16 as its TFA salt (73 mg, 0.255 mmol) was added and the reaction stirred for 18 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL).

The combined organic phases were then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Flash chromatography eluting with ethyl acetate/hexane, 1:1 to give the desired coupled product 17 or 18 as a colorless oil (74 mg, Rf 0.33 and 0.28 respectively (EtOAc:Hexane, 17 (2R, 3S); 1 H NMR (500MHz, CDCI3) 8 7.37-7.23 6.44 ,1H, 5.16 (1H, d, J 5.03 (2H, s), 4.24-4.18 (1H, 4.11 (1H, d, J 3.97-3.90 (2H, 3.28-3.23 (1H, 3.12-3.06 (1H, m) 2.99- 2.90 (2H, 2.18-2.12 (1H, 2.01-1.96 (1H, m), 1.89-1.83 (1H, 1.83-1.76 (1H, 1.27 (9H, s); 13C NMR (100MHz, CDC3) 6 171.4 169.9 156.0 137.4 136.7 129.2 (2 x CH), 128.7 (2 x CH), 128.4 (2 x CH), 128.0 127.9 (2 x CH), 126.9 68.6 66.7 (CH 2 61.7 (CH), 52.8 51.1 46.1 (CH 2 38.4 (CH 2 28.6 (3 x

CH

3 27.4 (CH 2 24.7 (CH 2 IR (NaC1) Umax 3318, 2966, 1714, 1667, 1537, 1454, 1366, 1041 cm- 1

FABHRMS

(NBA) m/e 482.2677 C 2 7

H

35

N

3 0 5 requires 482.2655); (Found: C, 67.22; H, 7.32; N, 8.80.

C

2 7

H

3 5

N

3 0 5 requires C, 67.34; H, 7.33; N, 18 (2S, 3S); (major rotamer only) H NMR (400MHz, CDC1 3 -51- 6 7.33-7.17 (10H, 6.45 (1H, 5.21 (1H, d, J 4.99 (2H, 4.60 (1H, dd, J 6.8, 4.52- 4.47 (1H, 4.21-4.13 (1H, 3.81-3.66 (3H, m), 2.75-2.60 (2H, 2.41-2.30 (1H, 2.25-2.09 (1H, 2.05-1.90 (2H, 1.30 (9H, C NMR (100MHz, CDC13) 6 171.3 169.6 156.0 137.3 (2 x 129.1 (2 x CH), 128.5 (2 x CH), 128.4 (2 x CH), 128.0 127.8 (2 x CH), 126.5 71.3 66.6 (CH 2 61.1 54.3 51.3 47.5 (CH 2 33.8 (CH 2 28.6 (3 x CH 3 26.9 (CH 2 25.4 (CH 2 IR (NaC1) umax 3318, 2966, 1714, 1667, 1537, 1454, 1366, 1041 cm- 1 FABHRMS (NBA/CsI) m/e 614.1645 ([M+Cs] C 2 7

H

3 5

N

3 0 5 requires 614.1631); (Found: C, 67.20; H, 7.66; N, 8.54. C2 7

H

35

N

3 0 requires C, 67.34; H, 7.33; N, 8.73%).

General _Das-M artin oxidaij~n prffce Pr il l Ustrated in_figurg_71 Synthesis of (3S) and (3R) 3-(N- Benzyloxyc.arbonyl)amino-2-keto-4-phenylbutyryl-Lprolyl-tert-butyl amide 2.

20 3bz-N

N

2 3:1 mixture -52- As illustrated in figure 7, step iv, the substrate 17 (21 mg, 0.044 mmol) was dissolved in dry

CH

2 C12 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 mmol) added. The reaction mixture was stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and quenched by addition of saturated sodium bicarbonate (5 mL) and sodium thiosulfate. The aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts washed with water (10 mL), brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product. Flash chromatography eluting with ethyl acetate in hexane gave the desired product 2 as a 3:1 mixture of diastereomers (colorless oil) mg, Rf 0.47 (EtOAc/Hexane, Spectral data on mixture: 1H NMR (400MHz, DMSO) 6 7.86 (1H, d, J 7.71 (1H, d, J 7.64 (1H, 7.53 (1H, 7.37-7.10 (20 H, 5.10 (1H, ddd, J 11.0, 8.3, 5.01 (1H, d, J 12.6), 4.95 (1H, d, J 12.6), 4.95 (1H, d, J 16.3), 4.88 (1H, d, J 16.3), 4.79-4.73 (1H, 4.66 (1H, dd, J 7.8, 4.26 (1H, dd, J 7.8, 3.60-3.37 (3H, 3.33-3.24 (1H, 3.18 (1H, dd, J 14.7, 3.13 (1H, dd, J 10.2, 3.9), 2.79 (1H, dd, J 13.7, 10.2), 2.46 (1H, dd, J 14.7, 11.0), 2.23-2.17 (1H, 2.04-1.97 (1H, 1.90- 1.60 (6H, 1.24 (9H, 1.22 (9H, 13C NMR (100MHz, CDC1 3 6 198.91 196.7 170.7 169.9 162.6 162.2 156.1 155.9 138.5 137.6 136.9 136.9 129.0 (CH x 128.8 (CH x 128.4 (CH x 127.8 (CH x 127.6 (CH x 127.6 (CH x 2), -53- .2126. 5 (CH) 126. 4 (CH) 65. 6 (CH 2 65.3 (OH 2 60. 3 59.7 i1CH), 59.2 58.2 50.3 50.1 4 7 .6 (OH 2 4 7 .4 (CE 2 3 4 .8 (OH 2 3 4 .1 (OH 2 3 2 .5 (CH 2 2 9 .1 (CH 2 2 8.5 (CH 3 x 3) 2 8 .4 (OH 3

X

3) 24 .5 (C'1 2 21 .7 (CH 2 IR (NaCi) Vm,. 3325, 2966, 1715, 1670, 1634, 1531, 1454, 1258, 1051, 738, 699; FABHRMS (NBA/NaI) m/e 502.2295 C 2 7

H

3 3

N

3 0 requires 502.2318).; (Found: 0, 67.62; H, 7.05; N, 8.96. C 2 7

H

33

N

3 0 5 requires C, 67.62; H, 6.94; N, 8.76%).

Synthesis ofr L-Prolyl-L-isoleucyl-L-glutamnfe-tertbutyl amide 19 as illustrated in figure 8

HN

o NH-Ile.Gln..OBu 19 As illustrated in figure 8, the substrate N-tert- Butoxycarbc'nyl-L-proline commercially available from Sigma, (3.0 g, 13.9 mmol), was dissolved in dry 0H 2 01 2 (20 mL) HOBT, 1-hydroxybenzotriazole hydrate (2.07 g, 15.3 mmal), EDO, 1-(3-dimethylaminopropYl)- 3 ethylcarbociiimtide, (2.93 g, 15.3 mmol), and NH-TIe- Gln-OBu (1.6 mL, 15.3 mmol; pept ide fragments were synthesized using traditional peptide coupling methodologiLes: EDO, HOBT, DIEA, Ile, Gln, and -54protected as a tertButyl-ester), were added and the mixture stirred for 18 hours at ambient temperature.

The reaction was diluted with ethyl acetate (100 mL), and washed. with water (2 x 20 mL), 1 N HC1 mL saturated sodium bicarbonate solution mL), water (10 mL), brine (10 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Purification by flash chromatography, eluting with 33% EtOAc in Hexane gave NH-Ile-Gln-OBu- L-prolyl-t-ert-butyl amide 19 as a colorless oil (1.53 mg, Rf 0.46 (EtOAc/Hexane, 1 H NMR signals broadened due to rotamers: Spectral data: H NMR (403MHz, CD 3 0D) 6 4.35-4.30 (1H, 4.31 (1H, dd, J 9.3, 4.21 (1H, d, J 3.41-3.26 (2H, 2.48-2.36 (1H, 2.36-2.22 (2H, 2.20-1.79 (6H, 1.67-1.54 (1H, 1.46 (9H, 1.32-1.17 (1H, 0.99 (3H, d, J 0.93 (3H, t, J C 1 3 NMR (100MHz, CD 3 0D) 6 173.3 172.0 170.1 167.7 83.0 60.7 59.9 (CH), 54.0 47.4 (CH 2 37.9 32.5 (CH 2 31.2

(CH

2 28.4 (CH2), 28.2 (3 x CH 3 26.0 (CH2), 25.0

(CH

2 15.9 (CH 3 11.4 (CH3); FABHRMS (NBA) m/e 435.2575

C

20

H

36

N

4 0 5 requires 435.2583).

Synthesis of (2S, 3S) 3-(N-Benzyloxycarbonyl)amino-2hydroxy-4 -phenylbutyryl -L-prolyl -L-i soleucyl -Lglutamine-tert-butyl amide 20 as illustrated in figure 8 O NH-Ile-Gln-OBu Cbz-NH N H OH As illustrated in figure 8, step i, the coupling of the acid 15 to the amide 19 was carried out using the general peptide coupling procedure. A representative synthesis is as follows: the substrate 15(70 mg, 0.213 mmol), was dissolved in dry DMF (3 mL). HOBT, 1-hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol), DIEA, diisopropy-lethylamine, (122 pl, 0.703 mmol) were added and the mixture stirred for 30 minutes at room temperature. The secondary arine 19 as its TFA salt (73 mg, 0.255 mmol) was added and the reaction stirred for 18 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were then washed with water (2 x 5 mL), 1 N HC1(aq.) (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and -56dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography eluting with ethyl acetate/hexane, 1:1 to give the desired coupled product 20 as a colorless oil (74 mg, Rf 0.33 and 0.28 respectively (EtOAc:Hexane, 1:1) Flash chromatography eluting with 5% methanol in dichloromethane gave the desired product 20 as a colorless oil. H NMR (400MHz, CDC13) (major rotamer only) 6 7.42-7.12 (11H, 6.95 (1H, d, J 6.79 (1H, 6.07 (1H, 5.95 (1H, d, J 5.03 (1H, d, J 12.4), 4.96 (1H, d, J 12.4), 4.65 (1H, d, J 4.51 (1H, dd, J 8.0, 4.49-4.35 (2H, m), 4.32 (1H, d, J 3.99-3.94 (1H, 3.70-3.50 (2H, 2.91-2.83 (2H, 2.28-2.21 (1H, 2.21- 2.10 (3H, nm), 2.02-1.75 (5H, 1.43 (9H, 1.14- 1.06 (1H, 0.86 (3H, d, J 0.78 (3H, t, J

C

13 NMR (100MHz, CDC13) 6 175.5 172.2 171.5 (2 x 170.4 156.3 137.9 136.3 129.1 (2 x CH), 128.4 (4 x CH), 128.0 127.6 (2 x CH), 126.4 82.4 71.4 66.5 (CH 2 61.2 57.8 55.5 52.3 47.7 (2 x CH 2 37.0 33.8

(CH

2 31.48 (CH 2 28.76 (CH 2 27.9 (3 x CH 3 25.3

(CH

2 24.8 (CH 2 15.3 (CH 3 11.0 (CH 3

FABHRMS

(NBA/CsI) m/e 856.288 C3 8

H

5 3

N

5 0 9 requires 856.2898).

(3S) and (3R) 3 -(N-Benzyloxycarbonyl)amino-2-keto-4phenylbu ty2yl -L-prolyl-L- i soleucyl -L-glu tamine- tertbutyl amide 3 as illustrated in figure 8 -57- 0 0 NH-lle-Gln-OBut Cbz-N

N

H 3 3:1 mixture As illustrated in figure 8, oxidation of 20 was carried out using the general Dess-Martin oxidation procedure outlined above. Purification by flash chromatography eluting with 5% methanol in dichloromethane gave the desired a-keto amide 3(S) (2:1 mixture of isomers) as a colorless oil (37 mg, quantitative). A representative synthesis is as follows: the substrate 20 (21 mg, 0.044 mmol) was dissolved in dry CH 2 Cl 2 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 mmol) added. The reaction mixture was stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and quenched by addition of saturated sodium bicarbonate (5 mL) and sodium thiosulfate. The aqueous phase was extracted with ethyl acetate (3 x 20 mL).

The combined organic extracts washed with water mL), brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product. Flash chromatography eluting with 30% ethyl acetate in hexane gave the desired product 3 as a 3:1 mixture of diastereomers (colorless oil) (20 mg, (S) isomer: Rf 0.24 (EtOAc). H NMR (400MHz, CDC1 3 7.38-7.16 (11H, 6.98 (1H, d, J 6.80 (1H, 6.64 (1H, 5.64 (1H, d, J 5.09 (1H, d, J -58- 12.2), 5.02 (1H, d, J 12.2), 4.46 (1H, dd, J 8.2, 4.21 (1H, t, J 3.67-3.60 (1H, 3.59- 3.45 (1H, 3.70-3.50 (2H, 3.25 (1H, dd, J 14.1, 3.09 (1H, dd, J 14.1, 2.29-2.12 (4H, 2.12-1.78 (6H, 1.46 (9H, 1.20-1.05 (1H, 0.93-0.83 (6H, m, 2 x CH 3

C

13 NMR (100MHz, CDC1 3 6 197.1 175.1 171.1 170.5 (2 x 162.9 156.3 135.7 (2 x C), 129.1 (2 x CH), 128.7 (2 x CH), 128.5 (2 x CH), 128.5 127.5 (2 x CH), 126.9 82.4 77.2 67.3 (CH 2 61.0 (CH) 58.0 52.3 (CH), 48.0 (2 x CH 2 37.0 31.5 (CH 2 29.7 (CH 2 28.2 (CH 2 27.9 (3 x CH 3 25.0 (CH 2 24.8 (CH 2 15.5 (CH 3 10.8 (CH 3 (3R) isomer: 1 H NMR (400MHz, CDC1 3 5 7.38-7.16 (11H, 6.84 (1H, d, J 6.21 (1H, 5.54 (1H, 5.51 (1H, d, J 5.11-4.98 (1H, 5.09 (1H, d, J 12.5), 4.80 (1H, d, J 12.5), 4.75-4.61 (1H, 4.59 (1H, d, J 4.50-4.29 (2H, 3.59-3.45 (1H, 3.29 (1H, dd, J 14., 2.85 (1H, dd, J 14.0, 10.0), 2.29-2.12 (4H, m), 2.12-1.78 (6H, 1.46 (9H, 1.20-1.05 (1H, m), 0.93-0.83 (6H, m, 2 x CH 3 IR (NaCI) umax 3290, 2925, 1728, 1648, 1537, 1452, 1367, 1247, 1157; FABHRMS (NBA/CsI) m/e 854.2775 C 38

H

5 1 Ns0 9 requires 854.2741).

2 5 -Bis (methoxymethyl) -3 4(S)(dimethoxy)Npyrrolidine 23 as illustrated in figure 12 -59- OMe OMe

HN

OMe OMe 23 As illustrated in figures 11 and 12, a solution of 5(R)-bis(hydroxymethyl)-3(R),4(S)dihydroxypyrrolidine (806 mg, 4.95 mmol; Wong et. al J. Org. Chem. 1991, 56, 6280) in H 2 0 (25 mL) was cooled to 0 °C in an ice bath and the pH was adjusted to 9-10 with Na2CO 3 solution (0.3M).

Benzyloxycarbonyl chloride (1.4 mL, 9.9 mmol, 2 eq.) was added dropwise and the solution was stirred 1 hour at 0 and then 1 hour at ambient temperature.

Solvent was removed in vacuo, and the residue was taken up in EtOAc, filtered, and concentrated in vacuo. Flash chromatography eluting initially with 50% ethyl acetate in hexane then 100% ethyl acetate gave 5(R)-bis(hydroxymethyl)-3(R), 4(S)dihydroxy-N-(Benzyloxycarbonyl)-pyrrolidine product as a pale yellow oil. (l.lg, Rf 0.27 (EtOAc) 1H NMR (400MHz, CD 3 OD) 6 7.37-7.33 (5H, 5.13 (2H, 4.20-4.19 (1H, 4.13-4.10 (1H, 4.00 (1H, br 3.95-3.55 (5H, 1C NMR (100MHz, CD30D) (two rotamers) 6137.8, 129.6, 129.2, 129.0, 77.8, 77.1, 68.4, 67.4, 66.7, 63.2, 62.2, 61.7, 61.6, 61.1; FABHRMS (NBA) m/e 320.1121 C 1 4

H

1 9 N0 6 requires 320.1110).

To S(R)-bis(hydroxymethyl)-3(R), 4(S)dihydroxy--N- (Benzyloxycarbonyl) -pyrrolidine as synthesized above (35 mg, 0.117 mxnol) in dry THE (1 mL) was added CH 3 I (116 p.L, 1.86 mmol, 16 eq.) followed by NaH (60% dispersion in mineral oil) (28.1.

mg, 6 The reaction mixture was stirred at ambient temperature for 20 h. and concentrated in vacuo. Fl~ash chromatography eluting with 12% to ethyl acetate in hexane gave bis (methoxymethyl) 4 -dimethoxy-N- (Benzyloxyrcarbonyl)-pyrrolidine as a colorless oil.

975,). Rf 0.6 (50% EtOAc in Hexane) 1 NNR (400MH2Z, CD 3 OD) 8 7. 29-7. 23 (5H, in), 5. 04 (2H, br 4.10 'I1H, br 3.74 (3H, br, 3.44-3.42 (4H, in), 3.34 i'3H, br 3.31 OH, br 3.23 (6H, br FABHRMS (NBA) m/e 376.1722 C 1 8

H

2 7 N0 6 requires '376.1736).

To 2 5(R)-bis(methoxyrnethyl)-3(R), 4(S)dimethoxy--N- (Benzyloxycarbonyl) -pyrrolidine as synthesized above, (40 mng, 0.181 inmol), in methanol (2 mL) was added Pd/C (10 mg) The mixture was stirred under a balloon of H 2 for 3 h. Filtration through celite followed by concentration in vacuo yielded the desired product 23 as a pale yellow oil (25 mng, quant.). 1 H NMR (250MHz, CDCl 3 8 5.19 (1H, br s) 3.8--3.55 (8H, mn), 3.54-3.35 (12H, in); 13C NNR (62MHz, CD)Cl 3 84.7, 83.7, 71.9, 69.1, 62.4, 59.9, 59.1, 59.0, 57.6, 57.5; FABHR4S (NBA) m/e 220.1543 CoH 2 1NO 4 requires 220.1549).

-61- Synthesis of (3S) and (3R) 3-(N- Benzyloxycarbonyl)amino-2-keto-4-phenylbutyryl- 5'(.R)-bis(methoxymethyl)-3'(R) dimethoxypyrrolidine] 900 as illustrated in figure 12 OMe 0 OMe Cbz-N N 3:1 mixture H OMe 900 OMe As illustrated in figure 12, step i-ii, the substrate 15 (70 mg, 0.213 mmol), was dissolved in dry DMF (3 mL). HOBT, 1-hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, 1-(3dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol), DIEA, diisopropylethylamine, (122 al, 0.703 mmol) were added and the mixture stirred for minutes at room temperature. The secondary amine 23 as its TFA salt (73 mg, 0.255 mmol) was added and the reaction stirred for 18 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL).

The combined organic phases were then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Flash chromatography eluting with ethyl -62acetate/hexcane, 1:1 to give the desired coupled product 26 as a colorless oil. The reaction was followed by oxidation using the general Dess-Martin oxidation procedure as given supra for compound 2 to give the desired product 900. Flash chromatography eluting with 20% ethyl acetate in hexane gave the a~keto amide 900 (20 mg, quantitative). Rf 0.63 EtOAc in h~xane) All analysis performed on mixture of isomers: NMR major isomer: 1H NMR (400MHz, CDCl 3 8 7.32-7.1-7 (10H, in), 5.75 (1H, d, J 5.16-5.12 (1H, in), 5.09 (1H, d, J 12.4), S.04 (1H, d, J 12.4), 4.39 (1H, dt, J 3.9, 3.98-3.96 (1H, mn), 3.92 (1H, t, J 3.82-3.77 (1H, in), 3.73 (1H, dd, J 10.0, 3.55 (1H, dd, J 10.0, 3.47-3.17 (3H, in), 3.45 3.40 (3H, 3.26 (3H, s), 3.19 (3H, 3.11 (1H, dd, J 14.1, 6.92); C 13

NM

(100MHz, ODCd 3 8 197.62 164.7 155.7 136.4 129.7 (2 x CH), 128.4 (2 x CH), 128.3 (4 x CH), 127.9 126.7 84.'6 (OH), 8 3. 6 (OH) 7 0 .7 (OH 2 6 9 .7 (CH 2 6 6. 8 (OH 2 6 0 .7 (OH) 5 8 .9 (CR) 5 8. 7 (OCH 3 )1 5 8. 7 (OCR 3 58. 4

(OCR

3 58.4 (OCR 3 56.7 37.3 (OH 2 minor isomer: 1H NMR (400MHz, CDCl 3 8 7.32-7.17 (10H, 5.46 (1H, d, J 5.11-4.87 (3H, in), 4.56-3.17 (10H, in), 3.45 (3H, 3.38 (3H, 3.31 (3H, s), 3.21 (3H, s) IR (NaCl) Lunax 2930, 1717, 1635, 1506, 1456, 1110; FABHRNS (NEA/CsI) m/e 661.1550 ([M+Cs]l,

C

2 8

R

3 6

N

2 0 8 requires 661.1526) fe&ppcu1n~rccue~~~~pa k-etn-ade- f rom-PYro Wl2~ineasI s ~aedi-jnig -63- 12: As illustrated in figure 12, steps i-ii, the substrate 15 (70 mg, 0.213 mmol), is dissolved in dry DMF (3 mL). HOBT, l-hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide, (43 mg, 0.224 mmol), DIEA, diisopropylethylamine, (122 pl, 0.703 mmol) are added and the mix:ure is stirred for 30 minutes at room temperature. The secondary amine 22; 23; 24; 16; 36; 37; 40; 42; 44; 46; 47; 48; 52; 53; 54; 58; 59; 64; 65; 66; 100; 101; 102 or 103 as its TFA salt (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product which is directly carried on to the next step for oxidation of the secondary alcohol as follows. The secondary alcohol (21 mg, 0.044 mmol) is dissolved in dry CH 2 Cl 2 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 mmol) added. The reaction mixture is stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and quenched by addition of saturated sodium bicarbonate (5 mL) and sodium thiosulfate. The aqueous phase is extracted with ethyl acetate (3 x 20 mL).

-64- The combined organic extracts washed with water mL), brine (10 mL) dried (MgSO 4 and concentrated in vacuo to give the crude product. Flash chromatography eluting with 30% ethyl acetate in hexane give!s the respective product 70;900;71;2e;73;74;75;76;77;78;79a;80a;81a;79b;80b;81b ;82a;83a;84a;82b;83b;84b;lc;107;108 or 109 as a 3:1 mixture of diastereomers (colorless oil) (20 mg, as a colorl~ess oil.

Synthesis of epoxide 21 as shown in figures 11 and 14.

Cbz -N H 0 21 Compound 21: To a solution of compound 15 in methanol:THF (.10 M; at 0 oC, is added a solution boran-dimethyl sulfide (5 equivalents; 1.0 M) and allowed to stir for overnight. The solvent is then removed and the mixture is resuspended in methylene chloride (.10 M) and 1.2 equivalents of triethylamine is added at 0 OC and allowed to stir for 1 hour. Next, 1.1 equivlanents of tosylchloride is added and allowed to stir at 0 OC for an additional hour. The reaction mixture is then quenched with water, extracted with 100% ethylacetate, washed over bicarbonate, brine and dried over magnesium sulfate. The crude compound is purifed by flash chromatography. The compound is then resuspended in MeOH at 0 °C and 10 equivalents of KCO 3 is added and allowed to stir for one hour.

The reaction mixture is then filtered through celite, quenched with water, extracted with 100% -66ethylacetate, washed over bicarbonate, brine and dried over magnesium sulfate. The crude compound is purifed by flash chromatography give compound 21.

General procedure for coupling of epoxide to proline derivatives as illustrated in figure 11 To the pyrrolidine derivative 22; 23; 24; 16; 25; 36; 37; 40; 42; 44; 46; 47; 48; 52; 53; 54; 58; 59; 64; 65; 66; 100; 101; 102 or 103 (20 mg, 0.091 mmol) was added dry methanol (2 mL), Cbz-phenylalanyl epoxide 21 (27 mg, 0.091 mmol, 1.0 eq.) and triethylamine (14 pL, 0.100mmol, l.leq.). The solution was refluxed for 32 h, and then concentrated in vacuo. Flash chromatography, eluting with ethyl acetate provides the desired product as a clear oil to give respectively hydroxylethyl amine derivatives: 400; 500; 600; 700; 800; 38; 39; 41; 43; 45; 49; 51; 55; 56; 57; 61; 62; 63; 67; 68; 69; Ib; 104; 105; 106.

Synthesis of N-[l-Phenyl-2(S)- [(benzyloxycarbonyl)amino]-3 (R)-hydroxybutan-4-[2'(R) methoxymethyl]-pyrrolidine 600 as illustrated in figure 11

H

N

SR= MeON..

Cbz- -N R R= Me

H

OH 24 600 -67compound 600': The pyrrolidine derivative 24 was coupled to the epoxide 21 as described in the general.

procedure to provide 600. Flash chromatography eluting with ethyl acetate provided the desired product as a clear oil (40 mng, 56%) Rf= 0.15 (EtOAc) 'H NNPR (400MHz, CD 3 OD) 6 7.29-7.16 (10H, in), 4 .97 (1H, d, J 12. 7) 4. 92 (1H, d, J 12.7) 3. 85-3. 81 (1H, mn), 3.-7 3. 73 (1H, mn), 3. 42 25 (3H, in), 3. 31 (3H, 3.25-3.02 (2H, mn), 2.39 (1H, in), 2.64 (1K, dd, J 13.8, 10.5), 2.61-2.52 (2H, in), 1.95-1.37 (1H, in), 1.84-1.77 (2H, mn), 1.61-1.55 (1H, in); C 3NMR (100MHz, CDD 0D) 8 158.5 140.3 (2 x 130.5 (3 x OH), 129.4 (OH) 129.2 (3 x OH), 128.8 (OH) 128.5 127.2 7 3. 0 (OH) 67. 1 (2 x OH 2 59.9

(OH

2 59.3 (2 x OH 2 57.5 (00K 3 57.2 (OK 2 36.8

(OH

2 28.5 (OH 2 24 .2 (OH 2 IR (Na~l) Vmax 3330, 2939, 1699, 1538, 1454, 1252, 1203, 1134, 699; FABHRMS (NBA/NaI) m/e 413.2453 0 2 4

H

32

N

2 0 4 requires 413.2440).

Synthesis of N-[1-Phenyl-2(S)- (benzyloxycarboflyl) amino) -3 -hydroxybutan-4 [2 1(R) 5' bis(rethoxcymethyl)]PpyrrolidinG 400 as illustrated in figure 11 -68- 22 H R= MeO OMe Cbz- N

R

H

OH

400 Compound 4,00: The pyrrolidine derivative 22 (Cignarella, G. Nathansohn, G. J. Org. Chemn. 1960, 26, 1500-1,502) was coupled, according to the above conditions, to the epoxide 21 to provide 400. Flash chromatography eluting with 30% to 50% ethyl acetate in hexane provided the desired product as a clear oil mng, Rf= 0.47 (EtOAc/Hexane, 1:1) 1HNNR (400MHz, c:DCl 3 6 7 .33-7 .17 (10H, mn), 5. 02 (2H, dd, J 20.4, 3.89 (1H, in), 3.50 (1H, mn), 3.37 (3H, s) 3. 31 (2H, d, J 1. 1) 3.24 (3H, s) 3.20-3. 18 (2H, in), 2.95-2.89 (4H, in), 2.85-2.75 (2H, in), 1.87-1.83 (2H, mn), 1.54-1.52 (2H, mn); 1 3 C NMR (100MHz, CDC1 3 8 137.9, 129.6, 128.4, 128.2, 127.9, 127.8, 126.2, 77.5, 76.EII 71.2, 66.6, 66.3, 60.4, 59.0, 58.8, 54.9, 36.2, 29.6;; FABHRMS (NBA) m/e 457.2689 [M+H1+C 2 6 11 36

N

2 0 5 requires 457.2702) -69- Synthesis ofE N-[1-Phenyl-2(S)- (benzyloxy(-arboflyl) amino] -hyciroxcybutan-4- 51 -bis (iethoxyrnethyl) 4' dimethoxy I -yrrol idile 500 as illustrated in figure 511 23

H

N

Cbz- N RO=-Me e OH MeO 14 W~e 500 Compound 500: The pyrrolidine derivative 23 was coupled to the epoxide 21 as described above. Flash chromatography eluting with 50% ethyl acetate in hexane gave the desired product 500 as a pale yellow oil (20mg, 42%) Rf 0.37 (EtOAc/Hexane, 1:1).

1 MR (40GMAHz, CDCd 3 8 7.33-7.19 (10H, mn), 5.02 (2H-, dd, J 12.3, 20.6), 4.09 (1H, br s) 3.90 (1H, mn), 3.70 (1H, d, J 3.58 (1H, dd, J 6.4, 3.54 (2H, mn), 3.37 3.36 (3H, 3.35 3.40- 3.28 (5H, mn), 3.28 3.25-3.17 (l1H, mn), 2.95- 2.85 (2H, 2.82-2.77 (2H, m) 13 NNP. (100MHz, CDCd 3 8 155.9, 137.9, 129.7, 128.4, 128.3, 127.9, 127.8, 12.63, 84.8, 83.9, -75.1, 72.2, -70.6, 69.9, 66.9, 66.4, 61.0, 59.0, 58.9, 58.0, 57.1, 54.9, 29.7; FABHR4S (NBA) m/e 517.2932 C 2 8

H

4 0

N

2 0 7 requires 517.2914).

Synthesis of N-[1-Phenyl-2(S)f (benzyloxy'carbonyl)anino] -hycroxybutan-4-Lprolyl-tere-butyl amnide 700 as illustrated in figure 16

H

N. N

N

*H

Cbz--NR

HR

OH

700 Compound 700: L-Proline tert-butyl amide 16 (synthesized supra) was coupled according to the above conditions to the epoxide 21 to give 700.

Flash chromatography eluting with 100% ethyl acetate in hexane gave the desired product 700 as a colorless oil (70mg, Rf 17 (EtOAc/Hexane, 1: 1) 1 H NM'R (400MHz, ODCd 3 8 7.33-7.14 (10H, mn), 7.02 (1H, br 5.26 (1H, br 5.06 (2H, 3.93-3.86 (1H-, mn), 3.67-3.65 in), 3.29-3.16 (1H, mn), 2.90-2.75 (3H, mn), 2.67 (1H, d, J 2.48 (1H, dd, J 4.8, 2.2C-2.05 (1H, mn), 1.95-1.65 (3H, mn), 1.30 (9H1, -71- S) 13 NNR (63MHz, CDCl 3 6 175.7, 174.4, 137.5, 129.2, 128.3, 128.4, 128.0, 127.9, 126.5, 72.1, 68.9, 66.7, 59.8, 56.1, 55.5, 50.4, 35.4, 30.9, 29.6, 28.6, 24.3; FABHR14S (NBA) m/e 468.2810 C 2 7

H

3 7

N

3 0 4 requires 46'7.2862).

Synthesis of N-[1-Phenyl-2(S)- [(berizyloxcyz:arboflyl) amino] -hydroxybutan-4- (tert-butylami.do) (S)-methoxy]-pyrrolidifle 800 as illustrated in figure 11.

0

H

N

R=

N

H

Cbz-NR H R OH OWe 800 Compound 800O: the pyrrolidine derivative 25 (derived from cis-4-hydroxy-Lprolile) was coupled to the epoxidie 21 as described above. Flash chromatography eluting wit~h 50% ethyl acetate in hexane gave the desired product 800 as a pale yellow oil (40mg, Rf 0.23 (EtOAc) 1HNNR (250MHz, ODCd 3 8 7.34-7.14 (10H, mn), 7.04 (1H, br 5.01 (2H, br 4.81 (1H, d, J 3. 8) 3.9 8 85 (1 H, in), 3. 84 (1H, t, J 3.58 (1H, dd, J 6.0, 3.35-3.25 (1H, mn), 3.28 (3H, 3.20-3.12 (1H, in), 3.00-2.90 (2H, mn), -72- 2 .85 (1H, cid, J 13 8. 1) 2. 71 (1H, di, J 12. 4), 2 .6 6 (1H, di, J 12. 4) 2. 56 (1H, dcl, J 10.3, 3.1), 2.30-2.10 (1H, in), 2.05-1.95 (1H, in), 1.33 (9H, s); 13NIR (100MHz, ODCl3) 6 174.7 172.0 137.5 129.5 (2 x CH), 129.3 128.5 (3 x OH), 128.4 (2 x OH), 128.0 127.9 126.5 (OH), 79.9 71.4 68.3 66.6 60.6

(CH

2 5 9. 7 (OH 2 5S6. 0 (0H 3 0),1 55S. 0 (CH) 5 0 .3 (OH 2 8 (OH 2 29. 7 (CH- 2 28 .6 (3 x OH 3

FABHRMS

(NEA/OsI) m/e 630 .1970 ([IM+OS] I'0 2 8

H

3 9

N

3 0 5 requires 630.1944).

Synthesis of N-[1-Phenyl-2(S) (benzyoxycarbonyl) amino] -hydroxy-butan-4- -(tert-butyl-amido) -methoxyl-pyrrolidine 38 as illustr:ated in figure 11.

0 H

N

R=

N

H

Cbz-- NR H OH 36 X=Me Ox 38 or39 37 X=Bn Compound 38: The pyrrolidine derivative 36 (derived from trans 4-hydroxy-L-proline) was coupled to the epoxide 21 as described above. Flash chromatography eluting with 7S% ethyl acetate in hexanes gave the desired product 38 as a pale yellow oil (45mg, -73- Rf= 0.20 (EtOAc/Hexane, 4:1).

H NMR (400MHz, CDC1 3 6 7.33-7.13 (10H, 6.79 (1H, br 5.02 (2H, 4.89 (1H, d,J 3.88-3.85 (2H, 3.72-3.61 (1H, 3.49-3.33 (1H, 3.29 (3H, 3.20 (1H, t,J 2.97-2.57 2.29-2.22 (1H, 1.95-1.88 (1H, 1.67 (1H, br 1.31 (9H, C NMR (100MHz, CDC1 3 173.6, 156.5, 137.7, 136.3, 128.9, 128.3, 127.7, 127.6, 126.4, 79.7, 72.0, 68.0, 65.8, 60.2, 59.9, 56.6, 55.5, 50.5, 36.8, 35.3, 28.6; IR (NaCI) Umax 3307, 2968, 2932, 2357, 1749, 1713, 1652, 1531, 1455, 1365, 1258, 1230, 1095, 1027, 734, 698; FABHRMS (NBA) m/e 498.2955 C 28

H

39

N

3 0 5 requires 498.2968); Found: C, 67.36; H, 8.30; N 8.49. C 2 8

H

3 9

N

3 0 5 requires C, 67.57; H, 7.90; N, 8.45).

N-[l-Phenyl-2(S)-[(benzyoxycarbonyl)amino]-3(R)-hydro xybutan-4-[ 2 '(S)-(tert-butylamido-4'(R)-benzyloxy]pyrrolidine 39 as illustrated in figure 11.

0

H

N

R= N

H

Cbz--N R 3OX OH H H 36 X=Me Bn 38 or 39 37 X=Bn Compound 39: The pyrrolidine derivative 37 (derived from trans 4-hydroxy-L-proline) was coupled to the -74epoxide 21 as described above. Flash chromatography eluting with 50% ethyl acetate in hexanes gave the desired product 39 as colorless oil (28mg, 53%) Rf 0.20 (EtOAc/ Hexane, H NMR(400MHz, CDC1 3 7.34-7.16 (15H, 6.76 (1H, br 5.01 (2H, s), 4.85 (1H, d, J 4.51 (1H, d, J 11.7), 4.43 (1H, d, J 11.7), 4.13-4.07 (1H, 3.90-3.80 (1H, m), 3.70-3.62 (1H, 3.50-3.35 (2H, 3.24 (1H, t, J 2.92-2.68 (4H, 2.35-2.29 (1H, 1.99-1.93 (1H, 1.63 (1H, br 1.30 (9H, 1C NMR (100MHz, CDC1 3 137.5, 129.3, 128.6, 128.5, 128.4, 128.1, 127.9, 127.8, 127.7, 126.6, 80.1, 77.9, 71.1, 68.2, 60.8, 60.1, 55.7, 37.4, 35.9, 29.7, 28.6; IR (NaCi) ma x 3307, 2923, 1713, 1652, 1532, 1455, 1365, 1263, 1229, 1097, 1028, 733, 699; FABHRMS (NBA) m/e 706.2288 C 34

H

4 3

N

3 0 5 requires 706.2257).

Synthesis of compound 24 as shown in figure 11; 12

H

N

MeO 24 Compound 24: To a solution of L-proline in methanol:THF (.10 M; at 0 oC, is added a solution boran-dimethyl sulfide (5 equivalents; 1.0 M) and allowed to stir for overnight. The solvent is then removed and the mixture is resuspended in methylene chloride (.10 M) and 1.2 equivalents of NaH is added at 0 °C and allowed to stir for 1 hour. Next, 1.1 equivlanents of methyliodide is added and allowed to stir at 0 °C for an additional hour. The reaction mixture is then quenched with water, extracted with 100% ethylacetate, washed over bicarbonate, brine and dried over magnesium sulfate.

The crude compound is purifed by flash chromatography to give compound 24.

Synthesis of compound 25 as shown in figure 11; 12

SH

R

N N

H

OMe Compound 25: The substrate cis 4-hydroxy-L-proline commercially available from Sigma, (3.0 g, 13.9 mmol), was dissolved in dry CH2C12 (20 mL). HOBT, 1hydroxybenzotriazole hydrate (2.07 g, 15.3 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (2.93 g, 15.3 mmol), and tert-butylamine (1.6 mL, 15.3 mmol), were added and the mixture stirred for 18 hours at arbient temperature. The reaction was diluted with ethyl acetate (100 mL), and washed with water (2 x: 20 mL), 1 N HC1 (aq) (10 mL saturated sodium bicarbonate solution aq.) (10 mL), water mL), brine (10 mL) and dried (MgSO 4 before concentration in.vacuo to give the crude product.

Purification by flash chromatography, eluting with 33% EtOAc .n Hexane gave N-tert-butoxycarbonyl-cis-4hydoroxy-L-prolyl-tert-butyl amide as a colorless -76oil. The compound.was then resuspended in metylene chloride (.10 M) and 1.2 equivalents of NaH is added at 0 OC and allowed to stir for 1 hour. Next, 1.1 equivlanents of methyliodide is added and allowed to stir at 0 oC for an additional hour. The reaction mixture is then quenched with water, extracted with 100% ethylacetate, washed over bicarbonate, brine and dried over magnesium sulfate. The crude compound is purifed by flash chromatography to give compound Synthesis of compound 36 as shown in figure 11; 12

SH

R

NN

36 X=Me

OX

37 X=Bn Compound 36: The substrate trans- 4-hydroxy-L-proline commercially available from Sigma, (3.0 g, 13.9 mmol), was dissolved in dry CH2C1 2 (20 mL). HOBT, 1hydroxybenzotriazole hydrate (2.07 g, 15.3 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (2.93 g, 15.3 mmol), and tert-butylamine (1.6 mL, 15.3 mmol), were added and the mixture stirred for 18 hours at ambient temperature. The reaction was diluted with ethyl acetate (100 mL), and washed with water (2 x 20 mL), 1 N HC1 (aq) (10 mL saturated sodium bicarbonate solution (10 mL) water mL), brine (10 mL) and dried (MgSO 4 before -77concentration in vacuo to give the crude product.

Purification by flash chromatography, eluting with 33% EtOAc in Hexane gave N-tert-butoxycarbonyl-trans- 4-hydoroxy-L-prolyl-tert-butyl amide as a colorless oil. The compound was then resuspended in metylene chloride 10 M) and 1.2 equivalents of NaH is added at 0 OC and allowed to stir for 1 hour. Next, 1.1 equivlanents of methyliodide is added and allowed to stir at 0 °C for an additional hour. The reaction mixture is then quenched with water, extracted with 100% ethylacetate, washed over bicarbonate, brine and dried over magnesium sulfate. The crude compound is purifed by flash chromatography to give compound 36.

Synthesis of compound 37 as shown in figure 11; 12

H

R

N\N

H

36 X=Me

OX

37 X=Bn Compound 37: The substrate trans- 4-hydroxy-L-proline commercially available from Sigma, (3.0 g, 13.9 mmol), was dissolved in dry CH 2 Cl 2 (20 mL). HOBT, 1hydroxybenzotriazole hydrate (2.07 g, 15.3 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (2.93 g, 15.3 mmol), and tert-butylamine (1.6 mL, 15.3 mmol), were added and the mixture stirred for 18 hours at ambient temperature. The reaction was -78diluted with ethyl acetate (100 mL), and washed with water (2 x 20 mL), 1 N HC1 (10 mL saturated sodium bicarbonate solution (10 mL), water mL), brine (10 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Purification by flash chromatography, eluting with 33% EtOAc in Hexane gave N-tert-butoxycarbonyl-trans- 4-hydoroxy-L-prolyl-tert-butyl amide as a colorless oil. The compound was then resuspended in methylene chloride (.10 M) and 1.2 equivalents of NaH is added at 0 OC and allowed to stir for 1 hour. Next, 1.1 equivlanents of benzylbromide is added and allowed to stir at 0 °C for an additional hour. The reaction mixture is then quenched with water, extracted with 100% ethylacetate, washed over bicarbonate, brine and dried over magnesium sulfate.

The crude compound is purifed by flash chromatography to give compound 37.

-79- Synthesis of compound 27 (compound is shown in figures 9 and "W

"OH

NHBOC

Compound 27: To a solution of phenylalanine equiv.) in acetonitrile (.10 Molar) was added 1.2 equivalents of commercially available BOC-ON [2- (tert-butoxycarbonyloxyimino)-2-phenyl-acetonitrile] and the mixture was allowed to stir for 12 hours at The solvent was then evaporated and the crude compound 27 was carried on to the next step.

Synthesis of compound 28 as illustrated in figure 9 0 OMe

N

NHBOCMe 94% Compound 28: The substrate 27 (70 mg, 0.213 mmol), was dissolved in dry DMF (3 mL). HOBT, 1hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 immol), DIEA, diisopropyl-ethylamine, (122 pl, 0.703 mmol) were added and the mixture stirred for 30 minutes at room temperature. The commercially available N-methoxy-methyl amine hydrochloride salt (73 mg, 0.255 mmol; Aldrich) was added and the reaction stirred for 18 hours. The reaction mixture was diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL).

The combined organic phases were then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water mL), brine (5 mL) and dried (MgSO 4 before concentrat:.on in vacuo to give the crude product.

Flash chronatography eluting with ethyl acetate/hexane, 1:1 to give the desired coupled product 28 as a colorless oil Synthesis of 3(S)-1,4-Diphenyl-2-oxo-3-amino-N-Bocbutane (29) as illustrated in figure 9 NH3' CF3CO2- 29 3(S)-1,4-D:Lphenyl-2-oxo-3-amino-N-Boc-butane (29): To a stirred solution of N-Boc-L-phenylalanine-Nmethoxy-N-methylamide (28) (5.0 g, 14.5 mmol) in anhydrous THF (50 mL) under N 2 at 0°C was added 2.0 M -81benzyl magnesium chloride in THF (21.7 mL, 43.5 mmol). The mixture was gradually warmed at room temperature and stirred for an additional 3h. The reaction mixture was then poured onto 1 N HC1 mL). The organic layer was separated and the aqueous layer was extracted with ether (3 x 35 mL). The combined organic layers were dried (MgSO 4 and concentrated to give a crude product. Purification of the crude material by flash chromatography (EA:H;1:4) afforded Boc protected intermediate to 29 as a white solid (4.8 g, Rf 0.3 mp 86-87C; [a1 2 5 D +31.220 (c 2.21, CH 2 Cl 2 IR 3485, 2978, 1709, 1704, 1490, 1363, 1250 cm- 1 'H NMR (CDC13) 1.14 9H), 2.9-3.15 2H), 3.65 2H, J=11.6 Hz), 4.61 1H, J=6.9 Hz), 5.1 (bs, 1H), 7.0-7.2 (m, ppm; NMR (CDC1 3 28.3, 37.8, 47.8, 59.5, 79.9, 127.0, 127.1, 128.8, 129.2, 129.2, 129.6, 133.1, 135.2, 155.1, 206.5 ppm. HRMS: 472.0880, Calcd for C 21

H

25

NO

3 472.0889.

3(S)-1,4-Diphenyl-2-oxo-3-amino-butane HC1 To a solution of 3(S)-1,4-diphenyl-2-oxo-3-amino-N-Bocbutane (Boc protected intermediate to 29) (1.4 g, 4.12 mmol) in ether (10 mL) was added a saturated solution of HC1(g)/Ether (20 mL). After 3 h the precipitate was filtered to afford a crude white solid. Recrystallization (MeOH/Ether) gave 29 as a white solid (0.96 g, 'H NMR (300 MHz, 2.96 (dd, 1H, J=8.2 Hz), 3.24 (dd, 1H, J=6.2, 14.3 Hz), 3.73 2H, J=16.9 Hz), 4.41 (dd, 1H, 8.2 Hz) ppm. HRMS: 240.1388 Calcd. for C 16 H,,NO H': 240.1388.

Synthesis of Compound 30 as illustrated in figure 9 -82- NH-Asn-Leu-Ser.Bn

CF

3

CO

Compound 30: To a solution of 29 (0.032 g, 0.12 mmnd) in C&{2C12 (10 inL) was added O-benzyl-N-Boc-L- Ser-Leu-Asn (0.075 g, 0.15 rnmol), EDO (0.032 g, 0.17 Imnol), H-OBt (0.045 g, 0.33 mmol) and DMAP (cat.).

After 24 h the reaction mixture was washed with sat.

NaHCO 3 (2 x5 mL), 1 N Hl (2 x5mrL), sat. NaCi (2 x mL) dried (MgSO 4 and concentrated to give a crude solid. Recrystallization from MeOH/Ether afforded intermediate N-Boc protected compound to 30 as a white solid (0.05 g, yield) 1 H NMR (300 MHz, DMSOd 6 0.72-0D.85 (in, 6H), 1.35 9H), 1.50-1.72 (in, 1H), 2.30-2.45 (mn, 1H), 2.80-2.90 (mn, 1H), 3.05 (dd, 1H, J=5.l, 13.9 Hz), 3.5-3.63 (mn, 2H), 3.69 1H), J=17.0 Hz), 3.86 1H, J=17.3 Hz), 4.20-4.30 (mn, 1H), 4.30-4.45 (mn, 1H), 4.46 2H), 4.48-4.51 (mn, 1H), 6.80-7.42 (mn, 15H) ppm. HRMS: 876.2955 Calcd for C 4 1NS 3

NO

8

CS

4 876.2948. Anal Cacld for

C

41

H

53

N

5 0 8 C 60.20%, H 5.615, N 7.18%, S 9.41%. Found C 65.99%, H 7.22%, N 9.61%.

To a solutioni of intermediate N-Boc protected compound :0.12 g, 0.16 minol) in 0112012 was added a solution of 305 TFA/CH 2 C1 2 After 24 h the mixture was concentrated to afford a crude white solid which after recrystallization (MeOH/Ether) afforded the -83title compound 30 as a white solid (0.09 g, 75%) 1IH NNR (300 M~z, CD 3 OD) 0.71-0.85 (mn, 6H), 1.40-1.60 (in, 3H), 2.40-.262 (mn, 2H), 2.70-.291 (mn, 1H), 2.91-3.01 (mn, 3.50-3.58 (mn, 1H), 3.60-3.72 (mn, 3.75- 3.82 (mn, 3.88-4.00 (mn, 1H), 4.20-4.30 (mn, 1H-), 4.44-4.61 in, 2H), 6.93-7.45 (in, 15H) ppm. H-RNS: 644.3448 Calcd for C 3 6

H

4 6

N

5 0 6 H 4: 644.3448.

-84- Synthesis of Compound 31 as illustrated in figure 9 NH-Asn-Leu-Ser-'NI-

CF

3 02- 31 Compound 31.: A solution of 30 (80 mng, 0.11 inmol) in glacial acetic (10 inL) containing Pd(OH) 2 /C (cat.) was placed under a H 2 atmosphere at 50 psi. After 12 h the solution was filtered and the crude solid was recrystall--zed from MeOH/Ether to afford the title compound (15 mg, 22%) 'H NYR (300 MHz, CD 3 OD) 0.82 6H, J=15.6 Hz), 1.39 2H, J=7.2 Hz), 1.50-1.60 (in, 1H{) 2.36 (dd, 1H, J=8.1, 15. 6 Hz) 2.44-2. 50 (mn, 1H), 2.38 (dd, 1H, J=4.5, 13.9 Hz), 3.05 (dd, 1H, J=4.9, 13.9 Hz), 3.69 1H, J=17.3 Hz), 3.85 (dd, 1H, J=17.2 Hz), 4.28-4.32 (mn, 1H), 4.35-4.42 (mn, 1H), 4.48-4.53 (mn, 1H), 6.80-7.35 (in, 10H) ppm. HRMS: 686.1961 Calcd for C 31

H

43

N

5 0 8 CS*: 686.1955.

Synthesis of Compound 32 as illustrated in figure 9 H o

NH

NH2 32 Compound 32: To a solution of 29 (0.17 g, 0.62 mmcl) in DMF (5 iii) was added HO~t (0.17, 1.2 mmol), DMAP EDC (0.12 g, 0.62 mmol), Et 3 N (0.06 g, 0.09 mL, 0.62 mnvol) and quinolinic-Asn 0.52 inmol).

After 12 h the reaction mixture was taken up in EA mL) Thie organic layer was dried (MgS0 4 and concentrated to give a crude brown solid.

Recrystallization from MeOH/ether gave a white solid as 32 (0.12 g, 'H NNR (300 MHz, DMSO-d 6 2.62 (dd, 1H, J=4.9, 15.3 Hz), 2.75 (dd, 1H, J=6.9, 15.5 Hz), 2.84 (dd, 1H, J=14.3, 23.4 Hz), 3.06 (dd, 1H, J=4.6, 13.7 Hz),-3.76 1H, J=17.0 Hz), 3.91 (d, 1H, J=17.0 Hz), 4.51 1H, J=7.7 Hz), 4.82 1H, J=6.8 Hz), 6.95-7.32 (in, 11H), 7.48 1H), 7.73 (t, 1H, J=7.1 lIZ) 7.89 1H, J=8. 3 Hz) 8. 13 eH, J=8.7 Hz), 8.58 2H, J=6.7 Hz) ppm. HRMS: -86- 641.1166 Calcd for C 30

H

28 N0 4 CS+: 641.1165.

Synthesis of Compound 34 as illustrated in figure 0

N

O0H 34 Compound 34: To a solution of compound 33 (7:3 mixture) (0.21 g, 0.71 nunol), as previously prepared from 27 (Yuan, Munoz, Wong, Haeggstrom., Wetterholm, Samuelsson, B. J.

Med. Chem. 1993, 36, 211) in CH 2 Cl 2 (10 mL) was added L-proline mnethyl ester HCl (0.18 g, 1.1 minol), EDC (0.16 g, 0.85 nunol), HOBt (0.21 g, 1.56 inmol) and DMAP After 24 h the reaction mixture was washed wit'a sat. NaHCO 3 (2 x 2 mL) 1 N HCl (2 x 2 mL) and sat. NaCi (1 x 1 mL). The organic layer was dried (MgS2D 4 filtered and concentrated to give a crude solid. Purification by flash chromatography (EA:H; 1:4) gave the white solid compound 34 as a single (2R, 3S) (0.23 g, 80%) Rf=0.20 (EA:H;1:4) ;H NMP. (300 MHz, CDCl 3 1.34 Cs, 9H), 1.90-2.11 (mn, 4H), 2.88-2.91 (mn, 2H), 3.11-3.20 (mn, 1H), 3.36 1H, J=7. 6 Hz) 3. 65 Cs, 3H) 3. 89 1H, J=5. 1 Hz) 4.0 8 1H, J=5. 7 Hz) 4. 15 1H, J=9. 7 Hz) 4. 39 (d, 1W, J=7.4 Hz), 4.89 1H, J=9.9 Hz), 7.10-7.35 (mn, -87ppm. HRMS: 539.1169 Calcd for C 21

H

30

N

2 0, Cs': 539.1158.

Synthesis of Compound 35 as illustrated in figure 0 O

N

5

CO

2

CH

3 Compound 35: To a solution of compound 34 (0.062 g, 0.15 mmol) in anhydrous CH 2 Cl 2 under N 2 at room temperature was added the Dess-Martin reagent (0.078 g, 0.18 mmol). After 12 h the reaction was quenched with sat. NaHCO 3 (4 mL) and sat. Na 2

S

2 03 (4 mL). Ether mL) was added to the reaction and then stirred for 10 min. The reaction mixture was washed with sat. NaCl [2 x 10 mL), dried (MgS04), filtered and concentrated to give a crude oil. Purification by flash chronatography (EA:H; 1:3) afforded compound as a 1:0.8 mixture of an oil (0.030 g, 'H NMR (300 MHz, CDC1 3 1.35; 1.35 9H), 1.91-2.01 (m, 3H), 2.15-2.23 1H), 3.09-3.41 1H), 3.51-3.70 2H), 3.70 (m, 2H), 3.70 3H, J=6.3, 7.8 Hz), 4.40-4.49 1H), 4.72d-4.85 1H), 4.93-5.00 (m, 1H), 5.10-5.23 1H), 7.05-7.35 5H) ppm. HRMS: 537.1002 Calcd for C 2 1

H

28

N

2 0 6 Cs': 537.1002.

-88- CG-n ra pep.t ide.c o u pL i n pro.e.edur e__tf_o_rm_a k eto mide rom. p pedi ine__as-.-i.5_ ua..tate d__nf.ur As illustrated in figure 13, steps i-ii, the substrate 15 (70 mg, 0.213 mmol), is dissolved in dry DMF (3 mL). HOBT, 1-hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, l-(3-dimethylaminopropyl)-3ethylcarbodiimide, (43 mg, 0.224 mmol), DIEA, diisopropylethylamine, (122 pl, 0.703 mmol) are added and the mixture is stirred for 30 minutes at room temperature. The secondary amine 85;86;87;88;89;90;91;92;93;94;95;96 or 97 as its TFA salt (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL).

The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product which is directly carried on to the next step for oxidation of the secondary alcohol as follows. The secondary alcohol (21 mg, 0.044 mmol) is dissolved in dry CH 2 C12 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 rnmol) added. The reaction mixture is stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and quenched by addition of saturated sodium bicarbonate (5 mL) and sodium thiosulfate. The aqueous phase is -89extracted with ethyl acetate (3 x 20 mL). The combined organic extracts washed with waLer (10 mL), brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product. Flash chromatography eluting with 30% ethyl acetate in hexane gives the respective product 98; 99; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129 or 130 as a 3:1 mixture of diastereomers (colorless oil) (20 mg, as a colorless oil.

General procedure for coupling of epoxide to proline derivatives as illustrated in figure 14 To the pyrrolidine derivative 85;86;87;88;89;90;91;92;93;94;95;96 or 97 (20 mg, 0.091 mmol) was added dry methanol (2 mL), Cbzphenylalanyl epoxide 21 (27 mg, 0.091 mmol, 1.0 eq.) and triethylamine (14 uL, 0.100mmol, l.leq.). The solution was refluxed for 32 h, and then concentrated in vacuo. Flash chromatography, eluting with ethyl acetate provides the desired product as a clear oil to give respectively hydroxylethyl amine derivatives: 131; 132; 133; 134; 135; 136; 137; 138; 139; 140; 141; 142 or 143.

Synthesis of compound 1001 as illustrated in Figure A typical acylation is as follows: Compound 1000 (0.16 mmol; cis-dihydroxymethyl-pyrolidine from Aldrich followed by standard BOC protection; vida supra), the vinyl acetate (0.05 mL; Aldrich), DMF (0.2 mL; other solvents such as methylene chloride, chloroform, acetonitrile, tert-butyl alcohol, 3methyl-3-pentanol, DMSO and THF can also be used),

H

2 0 (0.005 mL), triethylamine (0.005 mL) and subtilisin BPN' or subtilisin Carlsberg (5 mg, is stirred at 370C for 36 h. The reaction is worked up by adding EtOAc and filtering the reaction mixture through celite. The filtrate is washed with sat.

NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. Some of the product, if possible, is crystallized out at this stage by addition of diisopropyl ether. The mother liquor is then chromatographed (SiO 2 hexane/EtOAc, 9/1-1/1) to give the product 1001.

Synthesis of compound 1002 as illustrated in Figure Compound 1002 was disolved in 0.10 Molar solution of acetone and then 1.5 M sulfuric acid was added at 0 Next chromium (VI) oxide (1.1 equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1002.

Synthesis of compound 1003 as illustrated in Figure The substrate 1002 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethy3.aminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for 30 minutes at room temperature. tert-butyl amine (73 mg, 0.255 rmmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium -91chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

Synthesis of compound 1004 as illustrated in Figure Compound 1003 was disolved in 0.10 Molar solution of methanol and then 0.10 equivalents sodium methoxide was added at 0 o C and the mixture was allowed to stir for 2 hours. The reaction is concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1004.

Synthesis of compound 1005 as illustrated in Figure Compound 1004 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (2 mL) at 0 OC under argon was added NaH (60% disp. in mineral oil) (96 mg, 2.40 mmol, 2.2 The mixture was allowed to stir for min., and then benzyl bromide or methyl iodide (0.14 mL, 1.20 mmol, 1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHC0 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with IN HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and -92concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product 1005.

Synthesis of compound 46; 47 or 48 as illustrated in Figure 15: Compound 46; 47 or 48: To a solution of CBZ-protected amine 0.092 mmol) in MeOH (2 mL) was added 20% palladium hydroxide on carbon (10 mg) and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product.

Synthesis of compound 1007 as illustrated in Figure 16: A typical acylation is as follows: Compound 1006 (0.16 mmol; trans-dihydroxymethyl-pyrolidine from Aldrich followed by standard BOC protection; vida supra), the vinyl acetate (0.05 mL; Aldrich), DMF (0.2 mL; other solvents such as methylene chloride, chloroform, acetonitrile, tert-butyl alcohol, 3-methyl-3-pentanol, DMSO and THF can also be used), H 2 0 (0.005 mL), triethylamine (0.005 mL) and subtilisin BPN' or subtilisin Carlsberg (5 mg, is stirred at 370C for 36 h. The reaction is worked up by adding EtOAc and filtering the reaction mixture through celite. The filtrate is washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. Some of the product, if possible, is crystallized out at this stage by addition of diisopropyl ether. The mother liquor is then chromatographed (SiO 2 -93hexane/EtOAc, 9/1-1/1) to give the product 1007.

Synthesis of compound 1008 as illustrated in Figure 16: Compound 1008 was disolved in 0.10 Molar solution of acetone and then 1.5 M sulfuric acid was added at 0 OC. Next chromium (VI) oxide (1.1 equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1008.

Synthesis of compound 1009 as illustrated in Figure 16: The substrate 1008 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for 30 minutes at room temperature. tert-butyl amine (73 mg, 0.255 mnol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

-94- Synthesis of compound 1010 as illustrated in Figure 16: Compound 1009 was disolved in 0.10 Molar solution of methanol and then 0.10 equivalents sodium methoxide was added at 0 0 C and the mixture was allowed to stir for 2 hours. The reaction is concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1010.

Synthesis of compound 1011 as illustrated in Figure 16: Compound 1010 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (2 mL) at 0 °C under argon was added NaH (60% disp. in mineral oil) (96 mg, 2.40 mmol, 2.2 The mixture was allowed to stir for 20 min., and then benzyl bromide or methyl iodide (0.14 mL, 1.20 mmol, 1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with lN HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product 1011.

Synthesis of compound 52; 53 or 54 as illustrated in Figure 16: Compound 52; 53 or 54: To a solution of CBZ-protected amine 0.092 mmol) in MeOH (2 mL) was added 20% palladium hydroxide on carbon (10 mg) and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product.

Synthesis of compound 1013 as illustrated in Figure 17: Compound 1012 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (0.10 Molar) at 0 °C under argon was added sodium hydroxide (0.10 equivalents), followed by CBZ-Cl (1.1 equivalents; Aldrich) and the mixture was stirred at 0 °C for 20 min. and then quenched with a few drops of water. Saturated ammonium chloride solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product 1013.

Synthesis of compound 1014 as illustrated in Figure 17: To a solution of substrate 1013 (0.5 mmol) in anhydrous DMF (5 mL) was added TBDPSC1 (1.05eq: Aldrich), Et 3 N (1.1 eq), and a catalytic amount of imidazole. The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and H 2 0 (30 mL). The organic layer was then washed with sat'd NH 4 solution (2 x 30 mL), water (30 mL), brine (30 mL), dried over MgSO 4 and concentrated in vacuo to yield product.

The mother liquor is then chromatographed to give the product 1014.

-96- Synthesis of compound 1015 as illustrated in Figure 17: To a solution of substrate 1014 (0.5 mmol) in anhydrous pyridine (5 mL) was added acetic anhydride (1.05eq: Aldrich). The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and H20 (30 mL) The organic layer was then washed with sat'd NH 4 Cl(aq.

solution (2 x 30 mL), water (30 mL), brine (30 mL), dried over MgSO 4 and concentrated in vacuo to yield product. The mother liquor is then chromatographed to give the product 1015.

Synthesis of compound 1016 as illustrated in Figure 17: The substrate was dissolved in THF and cooled to 0 C. TBAF (1.0 M solution in THF) was added (1.05 eq.) and the reaction monitored by TLC. After min. at 0°C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted to give the product.

Synthesis of compound 1017 as illustrated in Figure 17: Compound 1016 was disolved in 0.10 Molar solution of acetone and then 1.5 M sulfuric acid was added at 0 oC. Next chromium (VI) oxide (1.1 equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1017.

-97- Synthesis of compound 1018 as illustrated in Figure 17: The substrate 1017 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for minutes at room temperature. tert-butyl amine (73 mg, 0.255 nimol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HCl (5 mL), saturated sodium bicarbonate solution aq.) (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

Synthesis of compound 44 as illustrated in Figure 17: Step Compound 1018 was disolved in 0.10 Molar solution of methanol and then 0.10 equivalents sodium methoxide was added at 0 C and the mixture was allowed to stir for 2 hours. The reaction is concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the CBZprotected amine. Step To a solution of CBZprotected amine 0.092 mmol) in MeOH (2 mL) was added 20% palladium hydroxide on carbon (10 mg) and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The -98suspension was filtered through a pad of celite and concentrated in vacuo to yield product compound 44.

Synthesis of compound 1019 as illustrated in Figure 18: Compound 1012 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (0.10 Molar) at 0 oC under argon was added sodium hydroxide (0.10 equivalents), followed by CBZ-C1 (1.1 equivalents; Aldrich) and the mixture was stirred at 0 oC for 20 min. and then quenched with a few drops of water. Saturated ammonium chloride solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product 1019.

Synthesis of compound 1020 as illustrated in Figure 18: To a solution of substrate 1019 (0.5 mmol) in anhydrous DMF (5 mL) was added TBDPSC1 (1.05eq: Aldrich), Et3N (1.1 eq), and a catalytic amount of imidazole. The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and H 2 0 (30 mL). The organic layer was then washed with sat'd NHC1,,q.) solution (2 x 30 mL), water (30 mL), brine (30 mL), dried over MgSO4, and concentrated in vacuo to yield product.

The mother liquor is then chromatographed to give the product 1020.

-99- Synthesis of compound 1021 as illustrated in Figure 18: To an ice-cooled solution of substrate (0.2 mmol) in anhydrous DMF (2 mL) under argon was added NaH disp. in mineral oil) (2.2 The mixture was allowed to stir for 20 min., and then methyl iodide or benzyl bromide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with 1N HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford product. The mother liquor is then chromatographed to give the product 1021.

Synthesis of compound 1022 as illustrated in Figure 18: The substrate was dissolved in THF and cooled to 0°C. TBAF (1.0 M solution in THF) was added (1.05 eq.) and the reaction monitored by TLC. After min. at 00C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted to give the product.

Synthesis of compound 1023 as illustrated in Figure 18: Compound 1022 was disolved in 0.10 Molar solution of acetone and then 1.5 M sulfuric acid was added at 0 OC. Next chromium (VI) oxide (1.1 equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHC0 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother -100liquor is then chromatographed to give the product 1023.

Synthesis of compound 1024 as illustrated in Figure 18: The substrate 1023 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for 30 minutes at room temperature. tert-butyl amine (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL), saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

Synthesis of compound 40 as illustrated in Figure 18: CBZ-protected amine 1024 0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product compound -101- Synthesis of compound 1025 as illustrated in Figure 19: Compound 1025 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (0.10 Molar) at 0 °C under argon was added BOC-ON (1.1 equivalents; Aldrich) and the mixture was stirred at 0 °C for 20 min. and then quenched with a few drops of water. Saturated ammonium chloride solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product 1025.

Synthesis of compound 1026 as illustrated in Figure 19: To a solution of substrate 1025 (0.5 mmol) in anhydrous DMF (5 mL) was added TBDPSC1 (1.05eq: Aldrich), EItN (1.1 eq), and a catalytic amount of imidazole. The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and H 2 0 (30 mL). The organic layer was then washed with sat'd NH 4 Claq. solution (2 x 30 mL), water (30 mL), brine (30 mL), dried over MgSO 4 and concentrated in vacuo to yield product.

The mother liquor is then chromatographed to give the product 1025.

Synthesis of compound 1027 as illustrated in Figure 19: To an ice-cooled solution of substrate (0.2 mmol) in anhydrous DMF (2 mL) under argon was added NaH disp. in mineral oil) (2.2 The mixture -102was allowed to stir for 20 min., and then methyl iodide or benzyl bromide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with 1N HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford product. The mother liquor is then chromatographed to give the product 1027.

Synthesis of compound 1028 as illustrated in Figure 19: The substrate was dissolved in THF and cooled to 0°C. TBAF (1.0 M solution in THF) was added (1.05 eq.) and the reaction monitored by TLC. After min. at 0 0 C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted to give the product.

Synthesis of compound 1029 as illustrated in Figure 19: Compound 1028 was disolved in 0.10 Molar solution of acetone and then 1.5 M sulfuric acid was added at 0 OC. Next chromium (VI) oxide (1.1 equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1029.

-103- Synthes of compound 1030 as illustrated in Figure 19: The substrate 1029 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for minutes a, room temperature. tert-butyl amine (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL), saturated sodium bicarbonate solution (aq) (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

Synthesis of compound 42 as illustrated in Figure 19: CBZ-protected amine 1030 0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product compound 42.

Synthesis of compound 1032 as illustrated in Figure Compound 1031 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (0.10 Molar) at 0 °C under argon -104was added CBZ-Cl (1.1 equivalents; Aldrich) and the mixture was stirred at 0 oC for 20 min. and then quenched with a few drops of water. Saturated ammonium chloride solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product.

Next, to a solution of substrate (0.5 mmol) in anhydrous DMF (5 mL) was added TBDMSC1 Aldrich), :t 3 N (1.1 eq), and a catalytic amount of imidazole. The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and H 2 0 (30 mL). The organic layer was :hen washed with sat'd NH 4 solution (2 x 30 mL), water (30 mL), brine (30 mL), dried over MgSO 4 and concentrated in vacuo to yield product.

The mother liquor is then chromatographed to give the product 1032.

Synthesis of compound 1033 as illustrated in Figure The substrate 1032 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol; is added and the mixture is stirred for minutes at room temperature, tert-butyl amine (73 mg, 0.255 rnmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic -105phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL), saturated sodium bicarbonate solution (50 mL) water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

Synthesis of compound 1034 as illustrated in Figure 20: The substrate was dissolved in THF and cooled to 0°C. TBAF (1.0 M solution in THF) was added (1.05 eq.) and the reaction monitored by TLC. After min. at 0°C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted to give the product.

Synthesis of compound 1038 as illustrated in Figure Synthesis was carried out via a Mitsunobu inversion using identical conditions as published in Saiah et al. Tet. Lett. 1992, 33, 4317; Johnson et al J. Am. Chem. Soc. 1964, 86, 118. The reaction mixture is then worked up by dilution with ethyl acetate mL) and is added to saturated ammonium chloride mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 mL), saturated sodium bicarbonate solution mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

-106- Synthesis of compound 58 as illustrated in Figure To the CBZ-protected amine (0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product.

Synthesis of compound 59 as illustrated in Figure CBZ-protected amine 1038 0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product. Next, to an ice-cooled solution of substrate (0.2 mmol) in anhydrous DMF (2 mL) under argon was added NaH (60% disp. in mineral oil) (2.2 The mixture was allowed to stir for 20 min., and then benzyl bromide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0 C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with 1N HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford product. The mother liquor is then chromatographed to give the product to give 58.

Synthesis of compound 60 as illustrated in Figure -107- To an ice-cooled solution of substrate 1038 (0.2 mmol) in anhydrous DMF (2 mL) under argon was added NaH (60% disp. in mineral oil) (2.2 The mixture was allowed to stir for 20 min., and then methyl iodide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with IN HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO,, and concentrated to afford product. The mother liquor is then chromatographed to give the product to give intermediate product. Next, to the CBZ-protected amine (0.092 mmol) was dissolved in MeOH (2 mL) and palladium hydroxide on carbon (10 mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After min., TLC :Lndicated that the reaction was complete.

The suspension was filtered through a pad of celite and concentrated in vacuo to yield product.

Synthesis of compound 64, as illustrated in Figure To the CBZ-protected amine 1034 (0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon (10 mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product.

Synthesis of compound 65 as illustrated in Figure -108- CBZ-protected amine 1034 0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product. Next, to an ice-cooled solution of substrate (0.2 mmol) in anhydrous DMF (2 mL) under argon was added NaH (60% disp. in mineral oil) (2.2 The mixture was allowed to stir for 20 min., and then benzyl bromide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with 1N HCI, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford product. The mother liquor is then chromatographed to give the product to give Synthesis of compound 66 as illustrated in Figure To an ice-cooled solution of substrate 1034 (0.2 mmol) in anhydrous DMF (2 mL) under argon was added NaH (60% disp. in mineral oil) (2.2 The mixture was allowed to stir for 20 min., and then methyl iodide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with 1N HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO,, and -109concentrated to afford product. The mother liquor is then chromatographed to give the product to give intermediate product. Next, to the CBZ-protected amine (0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon (10 mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After min., TLC indicated that the reaction was complete.

The suspension was filtered through a pad of celite and concentrated in vacuo to yield product.

Synthesis of compound 1040 as illustrated in Figure 21: Piperidine compound 1039 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (0.10 Molar) at 0 °C under argon was added CBZ-C1 (1.1 equivalents; Aldrich) and the mixture was stirred at 0 °C for min. and then quenched with a few drops of water.

Saturated ammonium chloride solution was added mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product.

Synthesis of compound 1041 as illustrated in Figure 21: To a solution of substrate (0.5 mmol) in anhydrous DMF (5 mL) was added TBDPSC1 (1.05eq: Aldrich), Et 3

N

(1.1 eq), and a catalytic amount of imidazole. The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and HO mL). The organic layer was then washed with -110sat'd NH 4 C1(q.) solution (2 x 30 mL), water (30 mL), brine (30 mL), dried over MgSO 4 and concentrated in vacuo to yield product. The mother liquor is then chromatographed to give the product 1041.

Synthesis of compound 1042 as illustrated in Figure 21: To an ice-cooled solution of substrate (0.2 iuol) in anhydrcus DMF (2 mL) under argon was added NaH disp. in mineral oil) (2.2 The mixture was allowed to stir for 20 min., and then methyl iodide or benzyl bromide (1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with IN HC1, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford product. The mother liquor is then chromatographed to give the product 1042.

Synthesis of compound 1043 as illustrated in Figure 21: The substrate was dissolved in THF and cooled to 0°C. TBAF (1.0 M solution in THF) was added (1.05 eq.) and the reaction monitored by TLC. After min. at 0'C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted to give the product.

Synthesis of compound 1044 as illustrated in Figure 21: Compound 1043 was disolved in 0.10 Molar solution of acetone and then 1.5 M sulfuric acid was added at 0 OC. Next chromium (VI) oxide (1.1 -111equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product 1044.

Synthesis of compound 1045 as illustrated in Figure 21: The substrate 1044 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- 3 -dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for 30 minutes room temperature. tert-butyl amine (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 :nL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL), saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgS0 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product.

Synthesis o:f compounds 86 or 87 as illustrated in Figure 21: CBZ-protected amine 1045 0.092 mmol) was dissolvad in MeOH (2 mL) and 20% palladium hydroxide on carbon (10 mg) was added and the -112suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield product compound 86 or 87, depending on whether benzyl or methyl.

Synthesis of compounds 1046 as illustrated in Figure 22: Piperidine compound 1041 (290 mg, 1.09 mmol; Aldrich) in methylene chloride (0.10 Molar) at 0 °C under argor. was added acetic anhydride (3.3 equivalents; Aldrich), triethyl amine (3.3 equivalents) and the mixture was stirred at 0 °C for min. anc. then quenched with a few drops of water.

Saturated ammonium chloride solution was added mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. The mother liquor is then chromatographed to give the product.

Synthesis of compound 1047 as illustrated in Figure 22: The substrate was dissolved in THF'and cooled to 0°C. TBAF (1.0 M solution in THF) was added (1.05 eq.) and the reaction monitored by TLC. After min. at 0°C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted to give the product.

Synthesis of compound 1048 as illustrated in Figure 22: Compound 1047 was disolved in 0.10 Molar -113solution of acetone and then 1.5 M sulfuric acid was added at 0 Next chromium (VI) oxide (1.1 equivalents; Aldrich) was added and the mixture was allowed to stir for 2 hours. The reaction is worked up by adding EtOAc and washed with sat. NaHCO 3 solution, then with brine, dried over MgSO 4 and concentrated in vacuo to give an oil. The mother liquor is then chromatographed to give the product.

Synthesis of compound 1049 as illustrated in Figure 22: The substrate 1048 (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1- (3-dimethylaminopropyl) ethylcarbodiimide, (43 mg, 0.224 mmo3) is added and the mixture is stirred for minutes at room temperature. tert-butyl amine (73 mg, 0.255 mr.ol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HCI (5 mL), saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product. Compound is then redissolved in dry methanol (3 mL) and sodium methoxide (0.30 equivalents added and the mixture is stirred for 18 hours at reflux. The is concentrated in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the -114desired product 1049.

Synthesis of compounds 88 as illustrated in Figure 22: CBZ-protected amine 1049 0.092 mmol) was dissolved in MeOH (2 mL) and 20% palladium hydroxide on carbon (10 mg) was added and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to y:.eld product compound 88.

Synthesis of Compound 1050A as illustrated in Figure 27.

Step 1) To a solution of commerically available trans-3-hydroxyproline is added 1.1 equivalents BOC- ON (Aldrich) in a 1:1 v/v solution water/ dioxanes and NaOH (IN solution) and stirred at 0 oC for 12 hours. The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired protected product.

Step 2) As illustrated in figure 27 the substrate mg, 0.213 rnmol; vida supra), is dissolved in dry methylene chloride (3 mL). EDC, 1-(3dimethylam:.nopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol, is added and the mixture is stirred for minutes at room temperature. tert-butyl amine (73 mg, 0.255 rmmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl -115acetate (20 :nL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HCI (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgS04) before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired product 1050A.

Synthesis of Compound 1050B as illustrated in Figure 27.

Step 1) To an ice-cooled solution of N- (benzyloxycarbonyl)-cis-3'-hydroxyproline (290 mg, 1.09 mmol; vida supra) in anhydrous DMF (2 mL) under argon was added NaH (60% disp. in mineral oil) (96 mg, 2.40 mmol, 2.2 The mixture was allowed to stir for 20 min., and then benzyl bromide (0.14 mL, 1.20 mmol, 1.1 eq.) was added. The mixture was allowed to stir for 1 hr at 0°C and then quenched with a few drops of water. Saturated NaHCO 3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with 1N HCl, extracted with ethyl acetate (3 x 20 mL) dried over MgSO 4 and concentrated to afford a yellow oil. (205 mg, 53%) The crude material was carried on without purification.

Step 2) To the crude yellow oil (0.982 mmol)'was added TBAF (1M in THF) (1.9 mL, 1.9 mmol, 2 and the solution was stirred 2 hr at ambient temperature.

The solution was concentrated in vacuo and -116immediately applied to a short silica gel column, eluting with 33% ethyl acetate in hexanes. The product was isolated as a white foam (443 mg, Synthesis of Compound 1050 as illustrated in Figure 27.

As illustrated in figure 13, steps i-ii, the substrate 11 (70 mg, 0.213 mmol), is dissolved in dry DMF (3 mL). HOBT, l-hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide, (43 mg, 0.224 mmol), DIEA, diisopropylethylamine, (122 pl, 0.703 mmol) are added and the mixture is stirred for 30 minutes at room temperature. The secondary amine 1050B (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours.

The reaction mixture is diluted with ethyl acetate mL) and added to saturated ammonium chloride mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (aq) mL saturated sodium bicarbonate solution mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product which is directly carried on to the next step for oxidation of the secondary alcohol as follows.

The secondary alcohol (21 mg, 0.044 mmol) is dissolved in dry CH 2 C12 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 mmol) added. The reaction mixture is stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and -117quenched by addition of saturated sodium bicarbonate aq.) (5 mL) and sodium thiosulfate. The aqueous phase is extracted with ethyl acetate (3 x 20 mL).

The combined organic extracts washed with water mL), brine (10 mL), dried (MgS0 4 and concentrated in vacuo to give the crude product. Flash chromatogra:phy eluting with 30% ethyl acetate in hexane gives the respective product 1050 as a 3:1 mixture of diastereomers (colorless oil) (20 mg, as a colorless oil.

Synthesis of N-benzyl-cis-and methoxy-pyrrolidines Compound 1051A (Figure 28): Nbenzyl-cis-and were synthesized according to the procedure of Cignarella and Nathansohn (Cignarella, G. Nathansohn, JOC 1961, 26, 1500.) The two diastereomers (cis and trans) were separated by flash chromatography (1:9 EA/H to 1:4 EA/H). Analytical data matched that reported by Kemp and Curran (Kemp, Curran, T.P. JOC, 1988, 53, 5729) Synthesis of (Step 1, intermediate to compound 1051B (figure 28)): N-benzyl-cis-(2S, 5R)-dicarbmethoxypyrrolidine equivalents) was dissolved in MeOH (0.10 M) and a catalytic amount of 10% palladium on carbon was added. The mixture was stirred vigorously under a balloon of hydrogen until TLC indicated that the reaction was complete. Filtration and concentration afforded a clean product.

-118- Synthesis of N- (benzyloxycarbonyl) -dinmethyl (Step 2, intermediate to compound 1051B (figure To the crude substrate dimethyl dicarboxylate (869 mg, 4.06 mmol) was added water mL). The solution was cooled to 0 °C in an ice bath, and 0.3 M K 2

CO

3 was added dropwise until pH 9.

CbzCl (1.1 eq, 4.5 mmol) was added and the mixture was stirred 30 min at 0°C and 30 min at ambient temperature. The aqueous layer was extracted with EtOAc (3 x 20 mL) and was dried over MgSO, filtered, and concentrated under reduced pressure to yield a yellow oil, Purification by flash chromatography (1:3 EA/ H) yielded a clear oil. (1.2 g) Rf (1:1 EA/ H) 0.48.

Synthesis of dimethanol pyrrolidine compound 1051B: The substrate N-(benzyloxycarbonyl)-dimethyl pyrrolidine-(2S, dicarboxylate (766 mg, 2.3 mmol) was dissolved in anhydrous ':HF (15 mL) and the solution was cooled to -78 °C in a dry ice/acetone bath. To this solution was added DIBAL-H (1.5 M in toluene) (14.2 mL, 9.6 mmol, 4.2 The solution was allowed to stir at -780C, and gradually warmed to 0 OC during the course of 1 hr. The reaction was then quenched with 1N HC1 (a few drops), and THF removed in vacuo. The slurry was extracted with EtOAc (3 x 30 mL) and washed with brine (30 mL) After drying over MgSO 4 and concentratedin vacuo to yield a yellow oil. Flash column purification in 1:1 EA/ H gave the desired diol.

-119- Synthesis of N-(benzyloxycarbonyl)-cis-(2S)-methanol- (butyroxymethyl) pyrrolidine (Step 1, ate to compound 1051C (figure The substrate diol (800 mg, 3.02 mmol) was dissolved in vinyl butyrate (10 mL), and 553 mg of Lipase AK was added. The reaction was stirred slowly at ambient temperature until TLC indicated that starting material was gone. (60 The reaction mixture was diluted with CH2C12, filtered through Celite, and concentrated. After flash chromatography eluting with a gradient of 1:4 to 1:1 ethyl acetate in hexanes, the product was isolated as a colorless oil.

(675 mg, 67% yield, 85% ee) (The enantiomeric excess was determined by conversion to the Mosher ester followed by 19F NMR analysis).

Synthesis of (butyroxymieth)yl)-L-proline compound 1051C (figure 28). To an ice cooled solution of CrO3 (6.83 minol, 3.75 eq.) in 1.5M H2SO 4 (9 mL) was added substrate (611 mg, 1.82 mmol) in acetone (30 mL mL wash). The resulting orange solution was stirred vigorously at 0°C for 30 min., and then at ambient temperature for 7 hrs. Ethyl ether (40 mL) was then added and the organic layer was washed with brine solution (20 mL), dried over MgSO4, and concentrated in vacuo to afford crude product. This product which was subjected to a short column of silica eluting with 100% ethyl acetate to give pure product as a colorless oil (571 mg, 89%) 'H NMR (CD30D, 250 MHz) (peaks broadened by rotamers) 6 7.45-7.20 (5H, m), 5.25-4.90 (3H, 4.40-4.25 (1H, 4.25-4.00 (3H, 2.38-2.15 (2H, 2.15-1.73 (4H, 1.72-1.42 -120- (2H, 1.00-0.78 (3H, "C NMR (CD 3 OD) d 175.7, 174.9, 156.4, 156.0, 137.6, 129.5, 129.3, 129.2, 129.0, 128.8, 128.5, 128.4, 68.3, 68.1, 65.4, 65.0, 61.3, 60.9, 58.6, 58.1, 36.7, 29.9, 28.9, 28.1, 19.2, 13.9; FABHRMS (NBA) m/e 350.1593 C18H23N06 requires 350.1604.

Synthesis of (butyroxymethyl)-L-proline-tert-butyl amide compound 1051D (figure 28): The substrate i (13.9 mmol) was dissolved in dry CH 2 Cl 2 (20 mL) HOBT, 1hydroxybenzotriazole hydrate (2.07 g, 15.3 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (2.93 g, 15.3 mmol), and tert-butylamine (1.6 mL, 15.3 mmol), were added and the mixture stirred for 18 hours at ambient temperature. The reaction was diluted with ethyl acetate (100 mL), and washed with water (2 x 20 mL), 1 N HC1 (10 mL saturated sodium bicarbonate solution (10 mL), water mL), brine (10 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Purification by flash chromatography, eluting with 33% EtOAc in Hexane gave the title compound as a colorless oil.

Synthesis of hydroxymethyl-L-proline-tert-butyl amide compound 1052A (intermediate en route to compound 1051E (figures 23 and 29): The substrate was dissolved in MeOH: HO2 (8 mL: 4 mL) and cooled to 0°C. LiOH was added (128 mg, 5.0 eq.) and the mixture stirred for min. at 0°C. The mixture was neutralized with 1N HC1 and then extracted into EtOAc, dried over MgSO4, -121and filtered to afford product which was used in the next step without purification.

Synthesis of amide (compound 1051E (figure 28): Intermediate compound 1052A (1.0 equivalents; vida supra) was dissolved in MeOH (0.10 M) and a catalytic amount of palladium on carbon was added. The mixture was stirred vigorously under a balloon of hydrogen until TLC indicated that the reaction was complete.

Filtration and concentration afforded a clean product.

Synthesis of compound 1051 (figure 28) Using the general procedure as follows: To the pyrrolidine derivative 1051 E (20 mg, 0.091 mmol) was added dry methanol (2 mL), Cbz-phenylalanyl epoxide 21 (27 mg, 0.091 mmol, 1.0 eq; vida supra) and triethylamine (14 pL, 0.100mmol, l.leq.). The solution was refluxed for 32 h, and then concentrated in vacuo. Flash chromatography, eluting with ethyl acetate provides the desired product as a clear oil.

Synthesis of dimethyl-siyl-oxymethyl) -L-proline-tert-butyl amide (intermediate to compound 1052B; (figure 29) A solution of hydroxymethyl-L-proline-tert-butyl amide 1052A (81 mg, 0.24 mmol; vida supra) in CH2C12 (3 mL) was cooled to O°C under an argon bed. 2,6-lutidine (0.48 mL, 56 mL, 2.0eq.) and tertbutyldimethylsilyltriflate (88 mL, 0.38 mmol, 1.6 eq.) were added subsequently. After stirring for 1 -122hr at 0 0 C, the reaction was quenched by pouring the reaction mixture into an ice cold mixture of sat'd NaHCO3(aq)solution: H20 (5 mL: 25 mL) and extracting into CH2C12 (3 x 20 mL) The pooled organic layers were washed with 10% CuSO4(aq) solution (2 x dried over MgSO4, filtered, and evaporated under reduced pressure. Flash chromatography eluting with ethyl acetate in hexanes gave the intermediate compound as a colorless oil (85mg, Rf 0.58 (1:1 EtOAc/ Hexanes). 1H NMR (CDC13, 250 MHz) (peaks broadened by rotamers) d 7.33-7.26 (5H, 5.14 (2H, br 4.18-4.12 (1H, 4.08-3.90 (1H, m), 3.83-3.71 (2H, 2.39-2.00 (2H, 2.00-1.82 (2H, 1.24 (9H, 0.86 (9H, 0.03 (6H, br 13C NMR (CDC13, 62.5 MHz) 136.1, 128.4, 128.1, 127.9, 67.2, 64.3, 50.7, 28.4, 25.9; FABHRMS (NBA) m/e 581.1824 Cs]+ C24H40N204Si requires 581.1822.

Intermediate compound (1.0 equivalents; vida supra) was dissolved in MeOH (0.10 M) and a catalytic amount of 10% palladium on carbon was added. The mixture was stirred vigorously under a balloon of hydrogen until TLC indicated that the reaction was complete.

Filtration and concentration afforded a clean product to give 1052B.

Synthesis of 2S,3S)-3-(N-Benzyloxycarbonyl)amino-2butyldimethylsilyloxymethyl)-L-prolyl-tert-butyl amide (intermediate en route to compound 1052; figure 29, step 1) a-Hydroxy acid 11 (62.5 mg, 0.19 mmol, eq; vida supra) EDC (40 mg, 1.1 HOBT (28.2 mg, 1.1 eq.) were stirred in dry DMF (1 mL) at -123ambient temperature, under argon. After preactivaticfl for 30 min., the amine 1052B (59 mg, eq) dissolved in dry DMF (0.5 mL 2 x 0.5 ML washes) was cannulated into the activated acid mixture. After stirring for 12 hr, the reaction mixture was concentrated in vacuo, and WtOWc was added. The organic layer was washed with water (2 x dried over MgSO4, and solvent removed under reduced pressure. Flash chromatography eluting with 20% ethyl acetate in hexanes gave intemediate as a white crystalline solid (77mg, 65%) Rf= 0.59 (1:2 EtOAc! Hexanes) 1H NIAR (CDCl3, 400MHz): (mixture of two rotamers) d =7.32-7.09 (20H-, in), 6.86 s), 6.42 (1H, 5.63 (1H, d, J= 8.9 Hz), 5.42 d, J= 8.8 Hz), 5.07 (lH, d, J=12.0 Hz), 5.00 (1H, d, J= 12.0 Hz), 5.08-4.92 in), 4.53-4.38 (2H, in), 4.37- 4.19 (3H, in, 4.12-3.97 (3H, in), 3.89-3.71 (2H, in), 3.63-3.52 (1W 3.52-3.47 (1H, in, 2.90-2.62 (4H, in), 2.50O-2. 32 (1H, in), 2.20-1. 60 (9H, in), 1. 30 (9H, 1.27 (9H, 0.87 (9H, 0.86 (9H, 0.08 (3H, 0.06 (3H, 0.04 (3H, 0.01 (3H, s); 13C NNR (CDC13, 100MHz); FABHRMS (NBA) m/e 758.2618([M Cs3+ C34HS1N3O6Si requires 758.2601.

Synthesis of intermediate to compound 1052 (step 2, figure 29) General Dess-Martin Oxidation Procedure: (2S, 35)-3- (N-Benzyloxycarbonyl)amino- 2 (tertbutyldimethylsilyloxynethYl) -L-prolyl-tert-butYl amide The substrate X (28 mng, 0.044 initil) was dissolved in dry 0H2C12 (2 inL and Dess-Martin periodinane (26 mng, 0.088 minol, 2.0 eq.) was added. The reaction mixture -124was stirred at ambient temperature for 24 hr, then diluted with ethyl acetate (10 mL), and quenched by addition of sat'd sodium bicarbonate(aq.) (5 mL), and sodium thiosulfate. The aqueous phase was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed with water (10 mL), dried over MgSO4, anc. concentrated in vacuo to give the crude product. Flash chromatography eluting with 50% ethyl acetate in hexanes gave the desired product as a white solid (1:1 mixture of diastereomers). (26 mg, Rf 0.47 (1:2 EtOAc/ Hexanes); 1H NMR (CDC13, 400MHz); 7.36-7.13 (20 H, 6.40 (1H, 6.07 (1H, 5.36 (1H, d, J= 7.2 Hz), 5.18 (1H, d, J= 6.8 Hz), 5.11-4.97 (5h, 4.96-4.90 (1H, 4.50-4.47 (1H, 4.22-4.18 (2H, 3.99 (1H, dd, J= 9.6, 4.9 Hz), 3.96-3.87 (1H, 3.75 (1H, dd, J= 10.0, 4.8 Hz), 3.53-3.32 (3H, 3.27 (1H, dd, J= 14.2, Hz), 3.18 (1H, dd, J= 14.1, 8.1 Hz), 2.91 (1H, dd, J= 14.0, 9.7 Hz), 2.40-2.28 (1H, 2.25-2.16 (1H, m), 2.08-1.97 (1H, 1.96-1.78 (2H, 1.72-1.52 (3H, 1.34 (9H, 1.31 (9H, 0.88 (9H, 0.86 (9H, 0.09 (3H, 0.06 (3H, 0.04 (3H, s), 0.03 (3H, 13C (CDC13, 100MHz); IR (NaC1) vmax 3332.3, 2955.0, 2856.0, 2358.1, 1714.7, .1634.1, 1537.8, 1454.8, 1258.0, 1094.6; FABHRMS (NBA) m/e 756.2469 Cs]+ C34H49N306Si requires 756.2445.

Synthesis of compound 1052 (as illustrated in figure 29): To a 1.0 Molar solution of 1.0 equivalents substrate, TBAF (1.0 M solution in THF) was added (0.34 mL, 1.05 eq.) and the reaction monitored by TLC. After 30 min. at 0°C, the reaction was complete and the solvent was evaporated under reduced -125pressure. The crude material was applied to a short column of silica gel and eluted with 33% hexanes in ethyl acetate to give a yellow oil (162 mg, 94%) Synthesis of (methoxyinethyl) -L-proline-tert-butyl amide compound 1053A (figure 29). A solution of 1052A (156 mg, 0.467 mmol) in anhydrous DMF (3 mL) was cooled to 00C under argon. NaH, 60% disp. in mineral oil, (28.0 mg, 0.70 mmcl, 1.5eq.) was added and the suspension was allowed to stir for 20 min. Iodomethane (116 mL, 1.8 mmol, 4.0 eq.) was then added and the mixture stirred for 3 hr at 0°C. The reaction was quenched by addition of a few drops of 1N HCl(aq.) solution, and then diluted with H20 (10 mL). The aqueous layer was extracted with EtOAc (3 x 20 mL), dried over MgSO4, filtered, and evaporated under reduced pressure. Flash chromatography eluting with ethyl acetate in hexanes gave an intermediate as a colorless oil (146 mg, Rf 0.25 (1:1 EtOAc/ Hexanes). 1H NMR (CDC13) 13C NMR (CDC13, MHz) FABHRMS (NBA) m/e 349.2119 C19H28N204 requires 349.21270 To a solution of above intermediate (85 mg, 0.19 mmol) in MeOH (3 mL) was added a catalytic amount of Pd/C and the mixture stirred under a balloon of H2 at ambient temperature. After 20 min, TLC indicated that the reaction was complete and the mixture was filtered through a bed of Celite and washed with MeOH. Removal of solvent under reduced pressure gave a colorless oil (89mg, quant.) as compound 1053A which was used in the next step without fur:her purification. 1H NMR (CDC13, 400MHz) -126d 5. 64 (1H, br s) 4 .21 (1H, cid, J= 9. 2, 5. 0 Hz) 3.78-3.68 (1H, Qn, 3.53 (1H, dd, 9.8, 4.1Hz), 3.46- 3.37 Ql, Qn, 3.42 (3H, 2.41-2.31 (mn, 1H), 2.06- 1.89 (2H, Qn, 1.69-1.60 (1H, Qn, 1.36 (9H, 13C (CDCl3, 100 MHz) d 171.2, 73.4, 60.2, 59.2, 59.0, 51.0, 30.7, 28.6, 27.0. FABHRNS (NBA) Wie 215.1764 C11I-22N202 requires 215.1760.

Synthesis of (2S,3S) (N-Benzyloxycarbonyl)arnino-2hydroxy-4-phenylbutyryl-5 '-cis- (iethoxyinethyl) -Lprolyl-tert-butyl amride (intermediate to compound 1053, figure 29) Synthesized according to the General Coupling Procedure outlined above and as illustrated in synthesis of compound 1052 (figure 29) Rf= 0.30 (EtOAc: Hexanes 1:2) 1H NIAR (CDCl3, 400 MHz) (mixture cf rotamers) d =7.33-7.17 (20 H, Qn, 6.91 (2H, d, J= 5.6 Hz), 5.75 (1H, d, 8.8 Hz), 5.64 (1H, d, J= 8.8 Hz), 5.07 (1H, d, J=12.3 Hz), 5.03 (2H, 4.91 (lH, 4.61-4.55 (1H, mn), 4.53-4.38 (3H, mn), 4.28-4.15 (4H, mn), 4.02-3.95 (1H, Qn, 3.93- 3.86 (2H, mn), 3.81-3.75 (1H, in), 3.46 (1H, d, J= 8.7 Hz), 3.39-3.26 (3H, mn), 3.35 (3H, 3.20 (3H, s), 2.93-2.84 (3H, Qn, 2.80-2.73 (1H, Qn, 2.42-2.35 (1H, mn), 2.15-1.82 (3H, Qn, 1.80-1.74 (1H, Qn, 1.72-1.62 (1H, Qn, 1.30 (9H, 1.27 (9H, 13C NIAR (CDCl3, 100MHz) d =172.4, 171.6, 171.1, 169.9, 156.2, 156.0, 137.6, 132'.2, 136.4, 136.2, 129.5, 129.4, 129.1, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 126.9, 126.4, 73.2, 71.2, 71.0, 70.2, 66.6, 66.4, 61.8, 60.8, 60.7, 60.4, 59.5, 59.0, 58.8, 58.7 58.0, 55.3, 54.7, 54.5, 51.1, 50.8, 35.1, 29.8, 29.6, 28.5, 27.9, 26.2, 24.1, 21.0, 14.1; FABHRMS (NBA) Wie -127- 658.1879 ([Nj Cs]+ C29H39N306 requires 658.1893.

Synthesis 617 (2S,3S) (N-Benzyloxycarbonyl)a.Tll~ho- 2 keto-4 -phenylbu tyr-yl-5 '-ci s- (rethoxywniethyl) -L-prolyl tert-butyl amide (compound 1053; figure 29) Synthesized according to the General Dess-Martin procedure as noted above and as illustrated in the example for compound 1052 (figure 29) Rf 0.40 (1:1 EtOAc! H-exanes); 1H NMR (CDCl3, 400 MHz) (1:1 mixture of diastereomers) d 7.33-7.16 (20H, mn), 6.88 (1Hi, 6.79 5.70 d, J 8.6 Hz), 5.26 (1H, di, J 7.1 Hz), 5.18-5.12 (2H, mn), 5.08- 4.92 (4H, in), 4.48-4.43 (2H, in), 4.28-4.22 (1H, mn), 4.14-4.10 (1H, mn), 4.09-4.02 (1H, rm), 3.45 (1H, dd, J 9.5, 1.9 Hz), 3.37 (3H, 3.36-3.17 (6H, in), 3.15 (3H, 2.94 (1H, dci, J 13.9, 9.4 Hz), 2.42-2.30 (1H, in), 2.20-2.10 (1H, mn), 2.08-1.82 (SH, mn), 1.81- 1.72 (1H, mn), 1.34 (9H, 1.30 (9H, 13C (CDC13, 100MHz); 198.0, 197.0, 171.1, 169.4, 164.9, 163.9, 155.9, 155.6, 136.3, 136.1, 129.7, 129.3, 128.7, 128.5, 128.4, 128.3, 128.1, 127.0, 126.8, 74.3, 70.4, 67.1, 66.8, 63.6, 61.2, 59.8, 58.8, 58.6, 58.5, 57.9, 57.6, 51.0, 50.9, 37.2, 29.9, 28.6, 27.4, 25.7, 24.9; IR (NaCi) vrnax 3331.1, 2966.8, 2340.9, 1717.4, 1646.2, 1540.6, 1455.8, 1252.9, 1111.7, 1047.1; FABHRMS (NBA) in/e 656.1714 Cs]+ C29H37N306 requires 656.1737; (Found C, 66.24; H, 7.21; N, 7.78; 029H37N306 requires C, 66.50, H, 7.13; N, 8.03).

Synthesis of para- (orthc',-nitrobenzyloxy) hydroxybenzy1 bromide compound 1054A as illustrated -128in figure Step 1) To a solution of para-hydroxybenzyl alcohol g, 16.1 mmol; Aldrich) dissolved in dry acetone mL) was added K2CO3 (4.45 g, 32.2 mmol, 2.0 eq.) and ortho-nitrobenzylbromide (3.83 g, 17.7 mmol, 1.1 eq). The mixture was stirred at ambient temperature for 16 hr and then the solution was concentrated in vacuo. The residue was taken up in EtOAc (100 mL) and then the organic layer was washed with H20 (2 x 30 mL), brine (30 mL) and dried over MgSO4. The product was recrystallized from Et20/hexanes to give yellow needles (2.0 g, 60%) of para-(orthonitrobenzyl)-hydroxybenzyl alcohol. 1H NMR (CDC13, 400 MHz) d 8.16 (1H, dd, J 8.2, 1.3 Hz), 7.88 (1H, dd, 7.9, 1.0 Hz), 7.68 (1H, dt, J 7.5, 1.2 Hz), 7.51-7.46 (1H, 7.32-7.26 (2H, 6.98-6.95 (2H, 5.48 (2H, 4.61 (2H, 13C NMR (CDC13, 100 MHz) d 157.6, 133.9, 128.7, 128.5, 128.2, 124.9, 114.8, 66.8, 64.9; Step 2) To a suspension of Ph 3 P (1.03 g, 3.94 mmol, 1.02 eq.) in CH 3 CN (10 mL) cooled to 0°C. Bromine (0.20 mL, 3.86 mmol, 1.0 eq.) was added dropwise.

The ice bath was then removed and a solution of para- (ortho-nitrobenzyl)hydroxybenzyl alcohol in 5mL of CH3CN was added via cannulation. After stirring for min., the CH3CN was removed in vacuo and the residue was extracted with hexanes (5 x 30mL) and filtered through a pad of Celite. Concentration of the hexanes in vacuo yielded a fluffy white solid compound 1054A (l.lg, 1H NMR (CDC13, 400 MHz) d 8.17 (1H, d, J 8.2 Hz), 7.86 (1H, d, J 7.8 Hz), 7.68 (1H, t, J 7.4 Hz), 7.54-7.49 (1H, 7.34 (2H, d, J 8.4 Hz), 6.94 (2H, d, J 8.4 Hz), 5.49 -129- (2H, 4.50 (2H, s); Synthesi.s of trans-3'- (tert-butyldiriethYl silyloxcy)

I,-

proline tert-butyl amide compound 1054C as illustrated in Figure To a solution of N-(benzyloxycarboflyl)-trafls- 3 hydroxyprolile tert-butyl amide 1054B (739 mg, 2.31 mmol.; vida supra; synthesized as 1050A, figure 27) in anhydrous LMF (l0mL) was added TBDMSC1- (366 mg, 2.42 MMol, 1.05E:q), Et 3 N (0.35 mL, 2.54 mmcl, 1.1 eg), and a catalytic: amount of DMAP. The solution was stirred at ambient temperature for 15 hr and then was partitioned between EtOAc (60 mL) and H20 (30 mL).

The organic: layer was then washed with sat'd NH4Cl(aq.) solution (2 x 30 mL), water (30 brine dried over MgSO4, and concentrated in vacuc to yield the crude material, as a yellow oil. The yellow oil was then dissolved in MeO- (20 rnL) and a catalytic amount of 10% Pd/C was added. The mixture was stirred vigorously under a balloon of H 2 for 1 hr.

The mixture was then filtered through Celite and concentrat Ed in vacuo to yield a yellow oil (690 mg, 99 1H NNR (CDC13, 400 MHz) 6 6.97 (1H, s) 4.46 (1H, dd, J 1, 4. 6 Hz) 3.7 6 (1H, d, J 04 Hz) 3.42 dt, J 10.6, 7.6 Hz), 3.21 (1H, dcld, J 10.6, 7.5, 5.8 Hz), 1.34 (9H, 0.89 (9H, 0.12 (3H, 0.11 (3H, 130 NNR (CDCl3, 100 MHz) d 168.3, 75.5, 67.8, 51.4, 44.1, 33.8, 28.6, 25.7,- 4.6, FABHRMS (NBA) m/e 301.2317

H-

C15H32N202Si requires 301.2311); Synthesis of (2S,3S) (benzyloxycarbonyl)-amnfo- 2 hydroxy-4--phenylbutyryl-trals- 3 (tert- -130butyldimethylsilyloxy)-L-prolyl-tert-butyl amide compound 1054D as illustrated in figure Synthesized according to the general peptide coupling procedure outlined above as exemplified for compound 1052, figure 29. 'H NMR (CDC13, 400 MHz) d 7.28- 7.08 (10H, 6.48 (1H, 5.57 (1H, d, J 8.8 Hz), 5.08-4.93 (2H, 4.63-4.58 (1H, 4.52-4.38 (1H, 4.26 (1H, br 4.20-3.61 (4H, 1.24 (9H, 0.88 (9H, 0.09 (6H, 13C NMR (CDC13, 100 MHz) d 171.4, 170.0, 168.5, 156.6, 156.1, 137.4, 136.2, 135.9, 129.1, 128.8, 128.4, 128.0, 127.9, 127.4, 126.4, 73.1, 71.6, 71.2, 69.7, 69.2, 66.6, 66.5, 54.8, 54.5, 51.4, 51.3, 45.9, 45.5, 34.5, 33.6, 32.9, 31.8, 28.5, 28.4, 25.7, 17.9, 14.1; Synthesis of (2S,3S)-3-N-(benzyloxycarbonyl)-amino-2acetoxy-4-phenylbutyryl-trans-3'-(tertbutyldimethylsilyloxy) -L-prolyl-tert-butyl amide intermediate compound to 1054E as illustrated in figure 30. To a solution of (2S,3S)-3-N- (benzyloxycarbonyl)-amino-2-hydroxy-4-phenylbutyryltrans-3'-(tert-butyldimethylsilyloxy)-L-prolyl-tertbutyl amide 1054D (216 mg, 0.35 mmol) in anhydrous

CH

2 C1 2 was added acetic anhydride (67 mL, 0.71 mmol, 2.0 pyridine (71 mL, 0.882 mmol, 2.5 It was difficult to tell whether the reaction had gone to completion by monitoring by TLC, since the Rf values of the starting material and product were virtually identical. After stirring the reaction for 15 hr under argon, EtOAc (40 mL) was added and the solution was washed with sat'd NH4Cl(aq.) (20 mL), sat'd NaCC3 (20 mL), water (20 mL) and brine mL). The organic layer was dried over MgSO4, -131filtered, and concentrated to yield a white fluffy solid. (211 mg, 1H NMR (CDC13, 400 MHz) 8 7.33- 7.13 (10H, 6.47 (1H, 5.57 (1H, d), 5.36-5.30 (1H, 5.02 (2H, 4.69-4.58 (1H, m), 4.39-4.31 (1H, 4.20-4.09 (1H, 3.87-3.57 (2H, 2.98-2.79 (2H, 2.26-2.22 (1H, 2.08 (3H, 1.95-1.85 (1H, 1.31 (9H, 0.87 (9H, s), 0.099 (6H, 13C NMR (CDC13, 100 MHz) d 170.2, 169.9, 168.6, 167.8, 167.4, 166.5, 155.9, 136.9, 136.1, 128.9, 128.5, 128.4, 128.0, 127.8, 127.7, 126.6, 76.5, 73.3, 72.8, 71.3, 69.5, 69.4, 66.7, 66.5 52.7, 52.4, 51.4, 51.1, 45.5, 45.2, 35.5, 34.3, 33.8, 31.0, 28.5, 28.2, 25.6, 25.5,20.5, 20.3, 17.9, 17.6; FABHRMS (NBA) m/e 786.2571 Cs]+ C35H51N307Si requires 786.2551); Synthesis cf (2S,3S)-3-N-(benzyloxycarbonyl)-amino-2acetoxy-4-phenylbutyryl-trans-3'-hydroxy-L-prolyltert-butyl amide compound 1054E as illustrated in figure 30. The substrate (2S,3S)-3-N- (benzyloxycarbonyl)-amino-2-acetoxy-4-phenylbutyryltrans- 3 (tert-butyldimethylsilyloxy) -L-prolyl-tertbutyl amide (1.0 equivalents; vida supra) was dissolved in THF and cooled to 0°C. TBAF (1.0 M solution in THF) was added (0.34 mI,, 1.05 eq.) and the reaction monitored by TLC. After 30 min. at 0°C, the reaction was complete and the solvent was evaporated under reduced pressure. The crude material was applied to a short column of silica gel and eluted with 33% hexanes in ethyl acetate to give a yellow oil (162 mg, 94%) -132- Synthesis of (2S,3S)-3-N-(benzyloxycarbonyl) -aimino-2hydroxy-4--phenlbyluyyl-ans-3 (para- (or thonitrobenzyl)benzyloxy) -L-prolyl-tert-butyl amide compound 1054F, figure To a solution of (2S,3S)-3-N-(benzyloxycarbonyl)amino-2-acetoxy-4-phenylbutyryl-trans-3'-hydroxy-Lprolyl-tert-butyl amide dissolved in anhydrous THF (3 mL) was added para-(orthonitrobenzyl)hydroxybenzylbromide (106.4 mg, 0.331 mmol), 4A molecular sieves (spatula-full), and silver triflate (a spatula-full). The reaction was complete in 10 minutes at ambient temperature so it was immediately filtered through a pad of Celite and washed with Et20. After flash chromatography eluting with 50% ethyl acetate in hexanes, a colorless oil was isolated (165 mg, The subst!ate (2S,3S)-3-(N-Benzyloxycarbonyl)amino-2acetoxy-4-phenylbutyryl-3'-trans-[para-(orthonitrobenzyl)benzyloxy]-L-prolyl-tert-butyl amide was dissolved in MeOH: H20 (7 mL: 1 mL) and cooled to 0°C. K 2

CC

3 (146 mg, 1.06 mmol, 5.0 eq.) was added and the cloudy mixture was allowed to warm gradually to RT. Monitoring the reaction by TLC was difficult because the starting material and product had nearly identical Rf values. The reaction was stirred at RT for an additional 30 min., and then the MeOH was removed in vacuo. The resulting residue was partitioned between water (20 mL) and EtOAc (30 mL) and dried over MgSO4, filtered, and concentrated to yield a colorless oil (155 mg, 99%).

Synthesis of (2S,3S)-3-N-(benzyloxycarbonyl)-amino-2keto-4-phenylbutyryl-trans-3'-[para- -133hydroxybenzyloxy]-L-prolyl-tert-butyl amide compound 1054. Step 1) Synthesized according to the general Dess-Martin oxidation procedure as outlined above for compound 1052, figure 29.

Step 2) The substrate (2S,3S)-3-N- (benzyloxycarbonyl)-amino-2-keto-4-phenylbutyryltrans- 3 '-[para-(ortho-nitrobenzyloxy)benzyloxy]-Lprolyl-tert-butyl amide was dissolved in CHC1z (1 mL) and placed in the sunlight until TLC indicated that the reaction had gone to completion. The solution was concentrated in vacuo and purified by flash chromatography (eluent 33% ethyl acetate in hexanes) to yield a slightly yellow oil.

Alternative compounds 1059, 1060 and other hydroxybenzyloxyproline substituted a-keto amides The synthesis of the trans-3-meta-hydroxybenzyloxyproline substituted a-keto amides is done in an analogous fashion to that described above for the trans-3-parahydroxybenzyl-substituted syste; 1059 and 1060 can be synthesized using the bromides shown in figure Synthesis of N-(benzyloxycarbonyl)-cis-4'- (benzyloxy)-L-proline succinate ester compound 1055A as illustrated in figure 31. Step 1) To an icecooled solution of N-(benzyloxycarbonyl)-cis-4'hydroxyproline (290 mg, 1.09 mmol; Aldrich) in anhydrous DMF (2 mL) under argon was added NaH disp. in mineral oil) (96 mg, 2.40 mmol, 2.2 eq.).

The mixture was allowed to stir for 20 min., and then benzyl bromide (0.14 mL, 1.20 mmol, 1.1 eq.) was added. The mixture was allowed to stir for 1 hr at -134- 0°C and then quenched with a few drops of water.

Saturated NaHCO3 solution was added (10 mL) and the aqueous layer was washed with ethyl ether. The aqueous layer was then acidified to pH 2 with IN HCl, extracted with ethyl acetate (3 x 20 mL), dried over MgSO 4 and concentrated to afford a yellow oil. (205 mg, 53%) The crude material was carried on without purificat.on.

Step 2) To a solution of N-(benzyloxycarbonyl)-cis- 4'-(benzyloxy)-L-proline (41 mg, 0.115 mmol) in anhydrous CH 2 Cl 2 (2 mL) was added EDC (68 mg, 0.356 mmol, 3.1 eq.) and N-hydroxysuccinimide (40 mg, 0.345 mmol, 3.0 The solution was stirred for 2 hrs.

The solvent was removed, and the residue was partitioned between ethyl ether and water (10 mL/ mL). The water layer was extracted with ethyl ether and the combined organic layers were washed with brine (10 mL) and dried over MgS04. Concentration followed by flash chromatography eluting with 9% methanol in ethyl acetate yielded the desired product as a white solid (51 mg, 98%) IH NMR (CDC13, 400MHz) (two distinct rotamers, only major rotamer noted here) d 7.38-7.25 (10H, 5.23 (1H, d, J 12.4 Hz, ab-Cbz), 5.10 (1H, d, 12.4 Hz ab-Cbz), 4.75 (1H, dd, J 9.4, 3.0 Hz, 4.63 (1H, d J 12.5 Hz, ab- Bn), 4.48 (1H, d, J 12.5 Hz, ab-Bn), 4.15-4.09 (1H, m, CH-OBn), 3.75-3.62 (2H, m, NCH2CHOBn), 2.82 (4H, s, succ.CH2), 2.65-2.62 (1H, m, NCH2CHOBnCH2), 2.47- 2.34 (1H, m,NCH2CHOBnCH2); Synthesis of N-(benzyloxycarbonyl)-cis--4'- (benzyloxy) -L-proline-isoleucine-glutamine-tert-butyl ester compound 1055B. To a solution of N- -135- (benzyloxycarboflYl) -cis-4'- (benzyloxy) -L-proline succinate ester 1055A (28 mg, 0.0619 ramol) in anhydrous

CH-

2 C1 2 (2 mL) was added the dipeptide

H

2

N-

TIe-Gin- (OtBu ester) (39 mg, 0.123 mmol, 2 eq.) and the solution was stirred at ambient temperature for min The solution was diluted with CH 2 Cl 2 (5 ml) and then washed with sat'd NH 4 Cl solution (5 mL), sat'd NaHCO3 solution, brine (5 mL) and dried over MgSO4. Concentration followed by flash chromatography with 8% methanol in ethyl acetate afforded the desired product as a white solid (40 mg, 99) 1H NNR (CDCl3, 400 MHz) d =7.36-7.27 (10H, m), 6.99 (OH, br 6.84 (1H, d, J 8.6 Hz), 6.75 (1B, br 5.80 (1H, br 5.15 (2H, 4.53-4.32 (4H, in), 4.33-4.30 (1H, Wn, 4.12-4.09 (iN, mn), 3.71 (1H, d, J 11.9 Hz), 3.59-3.54 (1H, Qn, 2.64-2.61 (1H, in), 2. 30-2.15 (4H, in), 2. 00-1. 85 (1H, in), 1. 83-1. 69 (1H, in), 1.53-1.38 (2H, Qn, 1.45 (9H, 1.00-0.93 (1H, in), 0.81-0.71 (6H, mn); 13C NMR (CDCl3,100 MHz) d 175.2, 171.2, 170.5, 156.2, 137.4, 135.9, 81.9, 76.4, 70.5, 67.7, 60.6, 57.8, 53.4, 52.1, 36.7, 34.1, 31.7, 28.1, 24.5, 15.0, 11.2; FABHRMS (NBA) m/e 785.2520 Csl+ C35H48N408 requires 785.2526); Synthesis of cis-4- (benzyloxy) -L-proline-isoleucifleglutamine-tert-butyl ester compound 1055C as illustrated in figure 31.

To a solution of N-(benzyloxycarbonyl)-cis-- 4 (benzyloxy) -L-proline-isoleucine-glutainine-tert-butyl ester 1055B (60 mg, 0.092 mmxol) in MeOH (2 mL) was added 20% palladium hydroxide on carbon (10 ing) and the suspension was stirred vigorously under a balloon of hydrogen at ambient temperature. After 5 min., -136- TLC indicated that the reaction was complete. The suspension was filtered through a pad of celite and concentrated in vacuo to yield X (47 mg, quant.) 1H >TMR (CD3OD, 400 MHz) d 7.33-7.23 (5H, Qn, 4.47 (1H, d, J=11.8 4.41 (1H, d, J=11.8 Hz), 4.27-4.18 (2H, mn), 4.17-4.13 (1H, Qn, 3.88 (1H, dd, J=7.8, 4.3 Hz), 3.20 (1H, d, J=11.7 Hz), 3.12 (1H, dd, J=11.7, 4.6 Hz), 2.36-2.17 (2H, Qn, 2.12-2.07 (1H, in, 1.92- 1.75 (2H, Qn, 1.57-1.46 (1H, mn), 1.49 1.16- 1.05 (1H, mn), 0.92-0.88 (3H, in), 0.85-0.75 (3H, mn); 130 NMR (CD3OD, 100 MHz) d 177.4, 175.3, 173.3, 172.1, 139.6, 129.4, 128.9, 128.6, 82.9, 79.9, 71.9, 60.4, 59.0, 54.0, 53.0, 38.4, 37.1, 32.1, 32.5, 28.2, 25.8, 15.9, 11.5; Synthesis of (2S,3S) (benzyloxycarbonyl) -ainino-2keto-4-pheriylbutyryl-cis-4 '-benzyloxy-L-prolinei soleucine-glu tainine- tert-butyl ester compound 1055 as illustrated in Figure 31. The following substances were combined in a flask, under a bed of argon: a-hydroxy acid 11 (26 mg, 0.080 raiol, 1.2 amine 1055C (35 mg, 0.067 inmol), HBTU (30.5 mg, 0.080 inmol, 1.2 and HOBT (10.9 mng, 0.080 mmiol, 1.2 Anhydrous THE' (1 inL) was added, followed by DIEA (28 mL, 0.161 inmol, 2.4 eq.) and the solution was stirred for l8hr at ambient temperature. After removal of the solvent in vacuo, the resulting residue was taken up in EtOAc (10 mL) and washed with sat 'd NH 4 C L(aq) (2 x 5 mL) sat 'd NaHCO 3 (aq) (2 x mL), water (1 x 5 niL), and brine (1 x 5 inL). The organic layer was then dried over MgSO4, filtered, and concen'trated in vacuo to yield a crude yellow oil. After subjecting the crude oil to flash -137chromatography in 5% MeOH in CH 2 C1 2 a white solid was isolated (24 mg, 43% yield). Rf 0.33 (9:1 CH 2 C1 2 1H NMR(CDC13, 400 MHz) (major rotamer only) d 7.33-7.11 (15H, 7.11 (1H, d, J= 8.4Hz), 6.78 (1H, d, J=10.2 Hz), 6.71 (1H, 5.12 (1H, d, J=12.5Hz), 4.89 (1H, d, J=12.5 Hz), 4.67-4.55 (1H, 4.55 (1H, d J=12.5 Hzi, 4.50 (1H, d, J=12.5 Hz), 4.48-4.29 (3H, m) 4.19-4.03. (2H, 4.02-4.92 (1H, 3.74-3.66 (1H, 3.07 (2H, d, J=7.4 Hz), 2.52 (1H, d, J=14.3 Hz), 2.32-2.06 (4H, 2.05-1.86 (1H, 1.71-1.52 (1H, 1.42 (9H, 1.06-0.89 (1H, 0.83-0.59 (6H, 13C NMR (CDC13, 100 MHz) d 175.5, 173.4, 171.5, 170.5, 170.4, 156.6, 137.9, 137.7, 137.1, 136.3, 129.2, 129.1, 128.5, 128.4, 127.9, 127.8, 127.7, 126.5, 82.1, 76.5, 71.6, 70.7, 66.5, 60.8, 57.8, 56.2, 53.1, 52.2, 37.0, 34.4, 33.3, 31.7, 27.9, 25.0, 24.6, 14.9, 10.9.

Step 2) The final product was obtained using the general Dess-Martin oxidation procedure as outlined above for compound 1052, figure 29 to produce compound 1055.

Synthesis of Compound 1056 as illustrated in figure 32. The following extended peptide isostere was synthesized on solid phase resin (MBHA) using standard peptide coupling conditions. The peptide was synthesized starting from the C-terminal end. N- BOC-Threonine was coupled to the resin (DCC, HOBT, DIEA), followed by deprotection of the amino group (TFA), and thorough washings with DMF and CH2C1 2 This standard coupling/ deprotection/ washing procedure was repeated seven times with the following N-BOC amino acids: (Gln, lie, Pro, Phe (a-hydroxy acid, -138- Ala, Gin, Pro). Subsequent Dess-Martin oxidation was performed while the peptide was still bound to the solid support. The resin-bound peptide was suspended in CH 2 C12, and Dess-Martin periodinane was added eq). The resin was washed with copious amounts of water and MeOH through a fritted funnel. This procedure was repeated to ensure complete oxidation to the a-keto-amide.

Cleavage of the peptide from the solid support was done in the standard anhydrous HF apparatus, and purified by reverse phase HPLC Synthesis of 2 ,5(R)-Bis (hydroxyinethyl)-3 ,4 dihydroxy-N-(benzy yyloxyabonyl)-pyrrolidine-2,3benzylidene acetal compound 1057C Figure 33. To the substrate 5(R)-Bis(hydroxymethyl)-3(R),4 dihydroxy-N-(benzyloxycarbonyl)-pyrrolidine (267 mg, 0.90 mmol; synthesized according to the procedure in Slee et al. J. Am. Chem. Soc., 1995, 117, 11867) dissolved in anhydrous DMF (1 mL) was added benzaldehyde dimethyl acetal (242 mL, 1.62 mmol, 1.8 A catalytic amount of pTSA was added and the solution was allowed to stir under argon for 6 hr, after which the solution was partitioned between (30 mL)and EtOAc (30 mL), and the aqueous layer was extracted with EtOAc (2 x 30 mL). The pooled organic layers were washed with brine (20 mL), dried over MgSO4, filtered, and concentrated to yield a crude yellow oil. Subsequent flash column purification, eluting with 50% ethyl acetate in hexanes yielded a white foam 1057C (246 mg, 71%).

Synthesis of compound 1057D as illustrated in Figure -139- 33.

Step 1) To the 3(R),4(S)-dihydroxy-N- (benzyloxycarbonyl)pyrrolid.ine-2,3-benzylidene acetal compound 1057C (784 mg, 2.04 mmol) in anhydrous DMF (4 mL) was added TBDMSC1 (3:1.6 mg, 2.09 mmol, 1.02 triethylamine (310 mL, 2.24 mmol, 1.1 and a catalytic amount of DMAP. The solution was stirred under argon for 18 hr, and then the solution was partitioned between EtOAc (40 mL) and H20 (30 mL) The aqueous layer was extracted with EtOAc (2 x 40 mL), and the organic layers were washed with sat'd NH4C1 (30 ml,), brine (30 mL), dried over MgSO4, filtered, and concentrated in vacuo to yield a crude yellow oil.

(914 mg, Step 2) The crude yellow oil was redissolved in 5 mL of anhydrcus DMF (5 ml), and the solution was cooled to 0°C in an ice bath. NaH (52 mg, 2.16 mmol, 2.2 and benzyl bromide (0.128 mL, 1.08 mmol, 1.1 eq.) were added and the mixture was allowed to stir at 0°C for 1 hr. The reaction was quenched with sat'd NH4C1 (5 ml), and then EtOAc (40 mL) was added.

The organic layer was washed with sat'd NaHCO3(aq.) mL), water (2 x 30 mL), and brine (30 mL). After drying over MgSO4, filtration, and concentration in vacuo, the crude product was isolated as a yellow oil Synthesis of compound 1057E as illustrated in Figure 33.

Step 1) To the crude yellow oil (0.982 mmol) was added TBAF (1M in THF) (1.9 mL, 1.9 mmol, 2 and the solution was stirred 2 hr at ambient temperature.

-140- The solution was concentrated in vacuo and immediately applied to a short silica gel column, eluting with 33% ethyl acetate in hexanes. The product was isolated as a white foam (443 mg, Step 2) The white foam (0.191 mmol) was dissolved in CHC1 2 (1 mL) and Dess Martin periodinane (112 mg, mmol, 2.0 eq.) was added. After 1 hr, sodium thiosulfate (500 mg) and H20 (30 mL) were added, and the aqueous layer was extracted with EtOAc (3 x mL), dried over MgS04, filtered, and concentrated to yield a crude product which was dissolved in tertbutanol (2.5 mL). To this solution, 2-methyl-2butene (2M solution) (2 mL) was added and then a solution o:f sodium chlorite (199 mg, 1.75 mmol, 9.2 eq.) and NaH2P04 (354 mg, 1.32 mmol, 6.9 eq.) in (1 mL) was added dropwise to the stirring substrate solution. After 1 hr, TLC indicated that the reaction was complete and the volatiles were removed in vacuo, and then remaining aqueous layer was extracted with diethyl ether (3 x 20 mL).

Synthesis of Compound 1057 F as illustrated in Figure 33. As illustrated in figure33 the substrate 1057E (70 mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for 30 minutes at room temperature. tertbutyl amine (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate mL) and added to saturated ammonium chloride (30 mL).

The aqueous phase is extracted with ethyl acetate (3 -141x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HCl (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL) brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product Synthesis of Compound 1057G as illustrated in Figure 33. Reductive cleavage of the benzylidene ring is done using 1.1 equivalents DIBAL in 1.0 THF solution at 0 OC for 1 hour. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 1 N HCl (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgS04) before concentration in vacuo to give the crude product. Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desiredproduct Synthesis of Compound 1057H as illustrated in Figure 33. Deprotection of the Cbz group will be achieved selectively (in the presence of benzyl groups) using 0.10 equivalents palladium hydroxide on carbon, as described in the procedure above for compound 1055C, Figure 31.

Synthesis of Compound 1057 as illustrated in Figure 33.

-142- As illustrated in figure 33, CB-Phe(a-OH) 11 (70 mg, 0.213 mmol), is dissolved in dry DMF (3 mL). HOBT, 1-hydroxybenzotriazole hydrate (31 mg, 0.22 mmol), EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, (43 mg, 0.224 mmol), DIEA, diisopropylethylamine, (122 pl, 0.703 mmol) are added and the mixture is stirred for 30 minutes at room temperature. The secondary amine 1057H (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate (20 mL) and added to saturated ammonium chloride (30 mL). The aqueous phase is extracted with ethyl acetate (3 x mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentration in vacuo to give the crude product.

Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product which is directly carried on to the next step for oxidation of the secondary alcohol as follows. The secondary alcohol (21 mg, 0.044 mmol) is dissolved in dry CH 2 C12 (2 mL), and Dess-Martin periodinane (26 mg, 0.088 mmol) added. The reaction mixture is stirred at ambient temperature for 24 hours, then diluted with ethyl acetate (10 mL) and quenched by addition of saturated sodium bicarbonate (5 mL) and sodium thiosulfate. The aqueous phase is extracted with ethyl acetate (3 x 20 mL). The combined organic extracts washed with water (10 mL), brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product. Flash chromatography eluting with 30% ethyl acetate in -143hexane gives the respective product 1057 as a 3:1 mixture of diastereomers (colorless oil) (20 mg, as a colorless oil.

Synthesis o:f N-(benyloxycarbonyl) -3,4-didehydro-Lproline ethyl ester compound 1058C as illustrated in Figure 34. Synthesized according to the procedure from J. Org. Chem. 1994, 59, 5192.

Synthesis of N-(benzyloxycarbonyl)-(3R,4S)-dihydroxy- L-proline ethyl ester compound 1058D as illustrated in Figure 34.

To a solution of substrate (600 mg, 2.18 mmol) in tbutanol/ H 2 (10 mL: 10 mL) was added AD-mix-b and methanesulfonamide (290 mg, 3.05 mmol, 1.4 eq.), and reaction was stirred at 4°C for 48 hr. The aqueous phase is extracted with ethyl acetate (3 x mL). The combined organic extracts washed with water mL), brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product.

Flash chromatography eluting with 30% ethyl acetate in hexane gives the respective product 1058D as a colorless oil.

Synthesis of N-(benzyloxycarbonyl)-(3R,4S)dihydroacetal-L-proline ethyl ester compound 1058E as illustrated in Figure 34. To the crude mixture of cis and trans dihydroxy-L-proline ethyl ester was added acetone (10 mL), pTsA (a spatula-full), and CuSO 4 (spatula-full) and the mixture was refluxed and stirred for 24 h to give the corresponding acetonides. The aqueous phase is extracted with ethyl -144acetate (3 x 20 mL). The combined organic extracts washed with water (10 mL), brine (10 mL), dried (MgSO 4 and concentrated in vacuo to give the crude product. Flash chromatography eluting with 30% ethyl acetate in hexane gives the respective product 1058E as a colorless oil.

Synthesis of (3R,4S)-dihydroacetal-L-proline tertbutyl amide compound 1058F as illustrated in Figure 34 As illustrated in figure 34 the substrate 1058E mg, 0.213 mmol), is dissolved in dry methylene chloride (3 mL). EDC, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide, (43 mg, 0.224 mmol) is added and the mixture is stirred for 30 minutes at room temperature. tert-butyl amine (73 mg, 0.255 mmol) is added and the reaction stirred for 18 hours. The reaction mixture is diluted with ethyl acetate mL) and added to saturated ammonium chloride (30 mL).

The aqueous phase is extracted with ethyl acetate (3 x 10 mL). The combined organic phases are then washed with water (2 x 5 mL), 1 N HC1 aq.) (5 mL saturated sodium bicarbonate solution (50 mL), water (5 mL), brine (5 mL) and dried (MgSO 4 before concentra:ion in vacuo to give the crude product.

Flash chromatography, eluting with 1:1 ethyl acetate/hexane gives the desired coupled product Step 2) The product was dissolved in MeOH (0.10 M) and a catalytic amount of 10% palladium on carbon was added. The mixture was stirred vigorously under a balloon of hydrogen until TLC indicated that the reaction was complete. Filtration and concentration afforded a clean product 1058F.

-145- Synthesis of compound 1058 as illustrated in Figure 34 To the pyrrolidine derivative 1058F (20 mg, 0.091 mmol) was added dry methanol (2 ml), Cbz-phenylalanyl epoxide 21 (27 mg, 0.091 mmol, 1.0 eq.) and triethylamine (14 pI., 0.100mmol, l.leq.) The solution was refluxed for 32 h, and then concentrated in vacuo. Flash chromatography, eluting with ethyl acetate provides the desired product as a clear oil to give respectively hydroxylethyl amine derivative 1058.

Preparation, Assay and Inhibition Analysis of the HIV Protease Plasmid pLAC-PRO 6.5 which contains the lacZprotease fusion construct under the control of the rac promoter (composed of the E. coli ribosomal RNA promoter fused to lac operator) was prepared and transformed to E. coli JM103. To induce lacZ-pro synthesis, overnight culture of JM103 harboring pLAC- PRO 6.5 (PRO 6-5) was diluted 50 fold with LB broth containing 300 g/mL ampicillin. Cells were grown at 37°C for 1 h to As 00 0.2-0.3, lactose final concentration) was then added to induce the synthesis of lacZ-protease fusion protein. After overnight growth, cells were harvested by centrifugation in a Sorvall GSA rotor at 6,000 rpm for 7 minutes. Cell paste from 250 mL culture was suspended in 10 mL lysis buffer (50 mM Tris, 2 mM EDTA, 2 mM DTT, 100 mM NaC1, pH 7.5) containing 200 g/mL lysozyme and stood at 4°C overnight. The cell suspension was -146subsequently sonicated over a salted ice bath for times with 10-15 pulses each at the maximal power output of the sonifier (Branson 450). Care was taken during sonication to keep the temperature of cell suspension below 100C. The sonicates were then centrifuged at 6k rpm in a GSA rotor for 10 minutes.

The majority of the lacZ-pro fusion proteins were found in the pellet. The insoluble lacZ-pro proteins were solubilized and unfolded in an aqueous solution containing 8M urea, and 50 mM dithiothreitol (DTT) or 1% 2-mercaptoethanol at concentrations of 0.2 mg/mL to 0.4 mg/mL. To refold the lacZ-pro proteins, the protein suspension were diluted with 12 volumes of mM Tris at: pH 6.0 and incubated at room temperature overnight (final protein concentration at 15 g/mL to g/mL). Under these conditions, the lacZ-pro proteins underwent auto-proteolysis to yield the mature 10 kDa protease.

The renatured lacZ-protease fusion protein mixture was prepared as described above and freezedried in a lyophilizer overnight, resuspended in H 2 0, and dialy;zed against 10 mM Tris buffer at pH overnight to remove urea. Under these conditions the mature HIV protease forms an insoluble precipitate and can be readily purified by low speed centrifugation at 2,000xg for 20 min. The precipitated protease was redissolved in 8M urea, mM Tris, pH 8.0, 1 mM DTT and passed through a DEAE- Sephacel column or a Pharmacia monoQ column in the same buffer. The flow through fraction was collected and dialyzed against 10 mM Tris, pH 8.0, 1 mM DTT, and concentrated by lyophilization. Both SDS gel electrophoresis and immunoblot analysis and amino -147acid analysis showed homogeneity of the protease.

This procedure routinely gave 2-3 mg of the mature protease per gram of E. coli cells.

The synthetic HIV-protease containing a thioester linkage available from CalBiochem (San Diego) can also be used. The enzyme activity was assayed with the fluorogenic substrate Ac-Thr-Ile- NIe-Phe (P-NO 2 )-Gln-Arg-NH2 (from CalBiochem, San Diego) according to the procedure described in the literature. Inhibition analysis was performed in the presence of the inhibitor and expressed with IC itne concentration of inhibitor that causes 50% inhibi :1n of the enzyme activity).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a staled integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Unless the contxt indicates otherwise, the reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Claims (8)

1. A mechanism based inhibitor of HIV or FIV aspartyl protease with the following structure: 0N N H 0 R 3 wherein: R' is a radical selected from the group consisting of -OH, -O(CI-C 6 alkyl), -O13n, and -OP; R2 is a radical independently selected from the group consisting of -OH, -O(C I -C 4 alkyl), -O13n, -OC'H 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO2-P, -OCH 2 (2-N0 2 OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H3, OCI- 2 (2-NO: )(-OCH- 3 )C6H-3. -OCH 2 (2-OFI)(3 -OH)C 6 H 3 -OCH 2 (3-OH)(4-OH)C 6 H 3 and -OP; R 3 is a radical independently selected from the group consisting of -OH, -O(CI-C 4 alkyl), -OBn, -OC'H 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 OCH 3 )C 6 H 3 -OCH 2 (2-N0' 2 )(4-OCH 3 )C 6 H3, -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H3, -OCH 2 (2-NC' 2 )(6-OCH 3 )C 6 H3, -OCH 2 (2-OH)(3 -OH)C 6 H 3 -OCH 2 (3 -OH)(4- OH)C 6 H- 3 and -OP; R 4 is a radical selected from the group consisting of -CH 2 OH, -CH 2 O(C I -C 6 alkyl), and -CH 2 0P; where P is a 1,2.ydroxyl protecting group; and where p2 is a nitrogen protecting group; with the following prcovisos: R R2, R 3and p. 4cannot all be hydrogen. P:\OPER\PDB\Spci\l 2452780 div spec.doc-2/05/04 -149-

2. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim 1 with the following structure: 0 Cbz- NNOe

3. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim 1 with the following structure: CbzN H

4. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim 1 with the following structure: Cbz. N- PAOPERXPDBMSpecik2432780 div spov~do-20(05/04

150- A mechanism based inhibitor of HIV or FIV aspartyl protease with the following structure: Ri wherein: R' is a radical selected from the group consisting of -OH, -O(C I-C 6 alkyl), -O13n, and -OP; R2 is a radical selected from the group consisting of -CH 2 OH, -CH 2 O(C I-C 6 alkyl), -CH 2 OP, -C (O)NH-Ile-Gln-OBu-t, and -C(O)NH-Ile-Gln-Thr-NH 2 R 3 is a radical independently selected from the group consisting of -OH1, -O(C i-C 4 alkyl), -O13n, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -P, OCH 2 C 6 H 4 NO 2 -o, -OCH 2 C 6 H 4 N.O 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-NO 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-O]-J)(4-OH)C 6 H 3 and -OP; R 4 is a radical i.ndependently selected from the group consisting of -OH, -O(CI-C 4 alkyl), -OBn, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -P, OCH 2 C 6 H 4 NO0 2 -o, -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH- 2 (2-OH)(3-OH)C 6 H- 3 -OCH 2 (3-O1-I)(4-OH)C 6 H 3 and -OP; R 5 is a radical s:elected from the group consisting of -CH 2 OH, -CH 2 O(C I-C 6 alkyl), and -CH 2 OP; where P is a hydroxyl protecting group; and where p 2 is an amine protecting group; with the following proviso: R 2 R 3 W 4 and R' cannot all be hydrogen. P:\OPER\PDB\Spci\l 2452780 div spec.do-2005/04 151 6. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim with the following structure: Cbz, 7. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim with the following structure: Cbz-. F1 8. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim with the following structure: Cbz,, HOMe OMe P:\OPER\PDB\Spcmi\12452780 div sp=cdoc.2(/0S/04

152- 9. A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim with the following structure: 1OBn Ile-Gln-OBu-t A mechanism based inhibitor of HIV or FIV aspartyl protease as described in claim with the following structure: Cbz. 11. A mechanism based inhibitor of HIV or FIV aspartyl protease with the following structure: 0 HN-Pro-Gn-AI N N Hi 0 N-Ile-Gln-Thr-NH 2 H P:\OPER\PDB\SpviXI2432780 div spem.doc-20105/04 -153 12. A mechanism based inhibitor of HIV or FIV aspartyl protease with the following structure: Ri 0 ~R 3 54 wherein: R1 is a radical selected from the group consisting of -OH, -O(C 1 -C 4 alkyl), -OBn, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 NO 2 0 -OCH 2 C 6 H 4 N4 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-NO 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-OH)(4- OH)C 6 H 3 anid -OP; R 2is a radical Jindependently selected from the group consisting of -OH, -O(C 1 -C 4 alkyl), -OBn, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCHC 6 H 4 NO0 2 -o, -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-NO 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-NID 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-OH4)(4-OH)C 6 H 3 and -OP; R is a radical independently selected from the group consisting of -OH, -O(C I-C 4 alkyl), -OBn, -CCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCHC 6 H 4 O 2 -o, -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0D 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-NO 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0D 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH4)(3-OH)C 6 H 3 -OCH 2 (3-OH)(4- OH)C 6 H4 3 and -OP; R 4 is a radical independently selected from the group consisting of -OH, -O(C 1 -C 4 alkyl), -OBn, -CCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCH 2 C 6 I- 4 NqO 2 -0, -OCH 2 C 6 H- 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-NO 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N'D 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-NO 2 -OCH 3 )C 6 H 3 -OCH 2 (2-N0D 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 PAOPER\PDB\Sp=AI\2452780 div spev.doc.201)5/G4

154- -OCH 2 (3-OH-)(4-OH)C 6 H 3 and -OP; where P is a hydroxyl protecting group; and where p2 is a, nitrogen protecting group. 13. A mechanism based inhibitor of HIV or FIV aspartyl protease with the following structure: wherein: R' is a radical selected from the group consisting of -OH, -O(CI-C 4 alkyl), O13n, -OCH 2 C 6 H 4 'DCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 NO 2 0 -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3 -OH)C 6 H 3 -OCH 2 (3-OH1)(4-OH)C 6 H 3 and -OP; R~2isardclidpnetyslcefrmtegopcnitnof-,-HB; R is a radical independently selected from the group consisting of -CH 3 -Bn; R4 is a radical independently selected from the group consisting of -CH 3 -Bn; where P is a hydroxyl protecting group; and where p 2 is a nitrogen protecting group. P:\OPER\PDBWSpviNI 2452780 div spc=.doc-20/)5/G4 155 14. A mechanism based inhibitor of HIV or FIV aspartyl protease with the following structure: R 1 <NI 0 wherein: R' is a radical selected from the group consisting of -OH, -O(C 1 -C 4 alkyl), -OBn, -OCH 2 C 6 H 4 0CH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 NO 2 0, -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-OH)(4- OH)C 6 H 3 and -OP; R 2 is a radical independently selected from the group consisting of -OH, -O(C I-C 4 alkyl), -OBn, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCH 2 C 6 H 4 NO 2 -0, -OCH 2 C 6 H 4 'NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-OH)(4- OH)C 6 H 3 arid -OP; R 3 is a radical independently selected from the group consisting of -OH, -O(C i-C 4 alkyl), -OBn, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCH 2 C 6 H 4 NO 2 -o, -OCH 2 C 6 H 4 N0 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-N0 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(5-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-OHJ)(4-OH)C 6 H 3 and -OP; R4 is a radical :independently selected from the group consisting of -OH, -O(C I-C 4 alkyl), -OBn, -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 -OCH 2 C 6 H 4 OCH 3 OCH 2 C 6 H 4 NO 2 -o, -OCH 2 C 6 H 4 NO 2 -OCH 2 C 6 H 4 NO 2 -OCH 2 (2-NO 2 )(3-OCH 3 )C 6 H 3 -OCH 2 (2-N0 2 )(4-OCH 3 )C 6 H 3 -OCH 2 (2-NO 2 -OCH 3 )C 6 H 3 -OCH 2 (2-N'D 2 )(6-OCH 3 )C 6 H 3 -OCH 2 (2-OH)(3-OH)C 6 H 3 -OCH 2 (3-OH)(4- OH)C 6 H 3 and -OP; P:%OPER\PDMlSpmdXIZ452?80 div spmc.dom-2O05104

156- where P is a hydroxyl protecting group; and where p 2 is a nitrogen protecting group. DATED this 20th day of May, 2004 The Scripps Research Institute By DAVIES COLLISON CAVE Patent Attorneys for the Applicant