HIV PROTEASE INHIBITORS

Field of the Invention

This invention relates to non-peptide inhibitors of proteases encoded in retroviruses, in particular, to inhibitors of the virally encoded protease of the Human Immunodeficiency Virus.

BACKGROUND

Retroviruses, that is, viruses within the family of Retroviridae, are a class of viruses which transport their genetic material as ribonucleic acid rather than deoxyribonucleic acid. Also known as RNA-tumor viruses, their presence has been associated with a wide range of diseases in humans and animals. They are believed to be the causative agents in pathological states associated with infection by Rous sarcoma virus (RSV) , murine leukemia virus ( LV) , mouse mammary tumor virus (MMTV) , feline leukemia virus (FeLV) , bovine leukemia virus (BLV) , ason-Pfizer monkey virus ( P V) , simian sarcoma virus (SSV) , simian acquired immunodeficiency syndrome (SAIDS) , human T- lymphotropic virus (HTLV-I, -II) and human immunodeficiency virus (HIV-1, HIV-2), which is the etiologic agent of AIDS (acquired immunodeficiency syndrome) and AIDS related complexes, and many others. Although the pathogens have, in

many of these cases, been isolated, no effective method for treating this type of infection has been developed.

Current treatments for viral diseases generally involve administration of compounds which inhibit reverse transcriptase, such as 3'-azido-3'-deoxythymidine and 2 ^I _/ 3'- dideoxycytidine. These treatments have not proven effective to arrest or reverse the disease, they may have adverse side effects, and they may lose their efficacy over time. Accordingly, new treatments for viral disease are needed. Virally-encoded proteases function in many of these viruses to hydrolyze viral polyprotein precursors and to yield functional viral proteins. The proteolytic activity provided by the virally-encoded protease in processing the polyproteins cannot be provided by the host and is essential to the life cycle of the retrovirus. It has been demonstrated that retroviruses which lack a protease or contain a mutated form of it, lack infectivity. See Katoh et al.. Virology. 145, 280-92(1985), Crawford, et al., J _. Vjrol. _r 53, 899-907(1985) and Debouck, et al., Proc. Naf.l. Afiari. Sc-i . USΑ _r 84, 8903-6(1987). Inhibiton of retroviral protease, therefore, presents a method of therapy for retroviral disease.

Methods to express retroviral proteases in E. coli have been disclosed by Debouck, et al., Proc. Natl. Acad. Sci. USA, 8903-06(1987) and Graves, et al., Proc. Nat] . Acad. Sc-i . USA. 85, 2449-53(1988) for the HIV-1 virus. The crystal structure of an HIV-1 protease has been disclosed by Miller _f ital. _f Sci nce. 246, 1149 (1989).

The method of isosteric replacement has been disclosed as a strategy for the development of protease inhibitors for HIV-1. Published European Patent applications EP-A 337 714, EP-A 352 000 and EP-A 357 332, EP-A 346 847, EP-A 342 541 and EP-A 393 445 are representative. Similar strategies have also been reported for inhibition of renin in U.S. Patents 4,713,445 and 4,661,473. Other inhibitors of the HIV protease, which contain a symmetrical isostere are reported in EP-A 402 646. There remains a need for protease-

inhibiting compounds which have a favorable balance of potency and pharmacokinetic properties.

SUMMARY OF THE INVENTION

This invention comprises compounds, hereinafter, of the formula (I) , which inhibit the retroviral protease of HIV-1, and are useful for treating infection by the human immunodeficiency virus and Acquired Immunodeficiency Syndrome (AIDS) .

This invention is also a pharmaceutical composition, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier.

This invention further constitutes a method for treating retroviral disease, which comprises administering to a mammal in need thereof an effective amount of a compound of formula (I) •

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are given by formula (I) :

^' ( I )

R ¹ , R ¹ ', R ⁴ , R ⁵ are independently H, Cι- ₆ alkyl, C ₂ - ₆ alkenyl,

C ₃ - ₇ Cycloalkyl, Ar, Het, T-Ci-βalkyl, T-C ₂ -βalkenyl;

T is Ar, Het or C ₃ _ ₇ cycloalkyl

R ² is Y, (CHR ) _n -Yf =CR ⁵ (CHR ⁴ ) _n -Y; R ³ is H or OH;

Q is OH or NH ₂ ; is R ⁶ , R ⁶ CO, R ⁶ OCO, R ⁶ OCH(R ⁷ )CO, R ⁶ NHCH(R ⁷ )CO,

R ⁶ SCH(R ⁷ )CO, R ⁶ S0 ₂ , R ⁶ SO or an amino acid with a blocked or unblocked amino terminus; Re and R ₇ are independently H, Ci-βalkyl, C ₃ - ₇ cycloalkyl,

Ar, Het, T-Cι- ₆ alkyl, T-(CH ₂ ) _n CH(T) (CH ₂ ) _n .

X is (OH,H) or =0;

Y is H; OH, NR'R ⁴ , Ar, Het or CO-Z;

Z is OH, NR'R ⁴ , OR ⁴ or an amino acid with a blocked or unblocked carboxy terminus; R* is H, Cι_ ₆ alkyl, Ar-Ci-galkyl; n is 1 to 4; and pharmaceutically acceptable salts thereof. Suitably R ¹ and R ¹ ' are benzyl. Suitably R ³ is hydrogen. Suitably R ² is CH(isopropyl)-Y, CH ₂ -phenyl, CHR ⁴ (2- imidazolyl) or CH(i-propyl)CO-Z.

One subgeneric group of compounds is given by formula

(II)

Representative compounds of this invention are: Δ ³ ' ⁴ trans-(6R,8S,9S)-10-phenyl-9-t-butoxycarbonylamino-8- hydroxy-2-methyl-6-phenylmethyl-dec-3-ene-5-one; 9S,8S,6R)-10-phenyl-9-t-butoxycarbonylamino-8-hydroxy-2- methyl—6-phenylmethyl-decan-5-one;

Δ^trans-(4R,6S,7S)-7-t-butoxycarbonylamino-6-hydroxy-l,8 - diphenyl-4-phenylmethyl-octene-3-one; adn

(4R,6S,7S)-7-t-butoxycarbonylamino-6-hydroxy-l,8-diphenyl -4- phenylmethyl-octan-3-one. Also included in this invention are pharmaceutically acceptable addition salts, complexes or prodrugs of the compounds of this invention. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo. The compounds of this invention have favorable pharmacokinetic properties, and are useful, in particular, for the treatment of infections by the human immunodeficiency virus.

The definition of any substituent moiety which may occur more than once in formula (I) is independent of any other occurrence. Formula (I) is intended to encompass all unique

— o —

nonracemic stereoisomers which may occur due to the presence of asymmetric carbon atoms in the molecule.

Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three Cι- ₄ alkyl, Cι-4alkoxy, Cι- ₄ alkthio, trifluoroalkyl, guanidino, amidino, HetCι-4alkoxy, HetCι-4alkyl, OH, Cl, Br or I.

Het, or heteroaryl, indicates a five or six membered aromatic ring, or a nine or ten-membered aromatic ring, containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis. Illustrative heterocycles are morpholine, tetrazole, imidazole, benzimidazole, pyrrole, indole, pyridine, pyrimidine, pyri idone, quinoline, benzofuran, furan, benzothiophene or thiophene. The Het ring may optionally be substituted by a Cι- ₄ alkyl or Cι-4alkenyl or Cι-4alkoxy group.

Any accessible combination of up to three substituents on the phenyl, naphthyl or Het ring which is available by chemical synthesis and is stable are within the scope of this invention.

Boc refers to the t-butyloxycarbonyl radical, Cbz refers to the benzyloxycarbonyl radical, Bzl refers to the benzyl radical, Ac refers to acetyl, Ph refers to phenyl, EDTA is ethylenediamine tetraacetic acid, DIEA is diisopropyl ethylamine, DBU is 1,8 diazobicyclo[5.4.0]undec-7-ene, DMSO is dimethylsulfoxide, DMF is dimethyl formamide and THF is tetrahydrofuran. as applied herein is meant to include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl and hexyl, isohexyl, 3- methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, and 1-ethylbutyl. C2-galkenyl as applied herein means C2-6alkyl wherein one carbon-carbon single bond is replaced by a carbon-carbon double bond. Ar-C**__galkyl and Ar- C2-6alkenyl mean C^-galkyl or C2-6al ^k «Ξnyl wherein a carbon- hydrogen bond is replaced by a carbon-Ar bond. Het-Ci-galkyl and Het-C2-6 ^a - ^L * ^fcen y* ^L mean Cι_galkyl or C2-galkenyl wherein a carbon-hydrogen bond is replaced by a carbon-Het bond.

AA as used herein indicates one to three amino acids, which may be:

Asparagine or Aspartic Acid Glutamine or Glutamic Acid

When Y is a CO-Z and Z is an amino acid, the amino acid is joined by an amide bond via its amino terminus to the carbonyl group, and the carboxy terminus of the amino acid is blocked or unblocked. An unblocked carboxy terminus is a free carboxyl group. Typical blocking groups are esters and amides, such as NR ^T R ⁴ or OR ⁴ ., wherein R ⁶ and R ⁷ are as defined in formula (I) .

When W is an amino acid, the amino acid is joined via its carboxy terminus to the amino group, and the amino terminus of the amino acid may be blocked or unblocked. Valine and alanine are useful amino acids, Cbz-Val and 2- quinolinylcarbonyl-Val are illustrative blocked amino acids. An unblocked amino terminus is an unsubstituted amino group. Typical blocking groups for the amino terminus are R ⁶ , R ⁶ CO, R ⁶ OCO, R ⁶ OCH(R ⁷ )CO, R HCH(R ⁷ )CO, R ⁶ SCH(R ⁷ )CO, R ⁶ S0 ₂ or R ⁶ SO, wherein R ⁶ and R ⁷ are as defined in formula (I) . Illustrative substituents are acetyl, Boc, Cbz, pyridinylmethyloxycarbonyl and 3-quinolinylmethyloxycarbonyl.

When compounds of formula (I) are administered to an animal infected or potentially infected with a virus, which is dependent upon a virally encoded protease for processing of viral polyproteins, viral replication is inhibited, hence, disease progression is retarded.

The compounds of this invention are prepared by conventional methods of organic chemistry. Representative

methods for preparing the compounds of formula (I) are illustrated in Schemes 1-3.

Scheme 1

(2)

H ₂ , Pd/C

R ¹ O R ⁴ R ¹ O R ⁴

Boc-NH ^{' "} y ^f ^^ -* Boc-NH

HO R ^1' TBD S-0 R

Scheme 2

Scheme 3

Scheme 3 f continued)

R ¹ O R ⁴ R R ⁴

Boc-NH CONH, Boc-NH ^{* *} C0 ₂ H OH R OH R ¹ '

Compounds of the structure . are prepared by reacting an appropriately protected hydroxyethylene isostere, such as methyl 5-t-butoxycarbonylamino-4-t-butyldimethylsilyloxy-6- phenyl-2-phenylmethyl-hexanoate or 5-[(l'-N-t- butoxycarbonylamino-2'-phenyl)-ethyl]-3-phenylmethyl-γ- but rolactone, with dimethyl methylphosphonate in the presence of a strong base, such as n-butyllithium or lithium diisopropylamide, in an inert solvent, such as tetrahydrofuran. Compounds of the structure 2. are prepared by reacting the carboxylic acid of an appropriately protected hydroxyethylene isostere, such as methyl 5-t- butoxycarbonylamino-4-t-butyldimethylsilyloxy-6-phenyl-2- phenylmeth l-hexanoic acid, with 0,N-dimethylhydroxylamine in the presence of a suitable coupling reagent, such as a carbodiimide or BOP reagent (benzotriazol-1- yloxytris (dimeth lamino)phosphonium hexafluorophosphate. Methods for preparing protected 5-amino-4-hydroxy-2,5- disubstituted-pentanoate esters and acids, and the corresponding γ-lactones, are well known and are disclosed, for instance, in Szelke et al., U.S. Patent 4,713,455, Boger et al., U.S. Patent 4,661,473, EP-A 0 352 000, Evans et al., J. Org. Chem., 50, 4615 (1985), Kempf, J. Org. Chem., 51, 3921 (1986), Fray et al., J. Org. Chem., 51, 4828 (1986), Halladay et al., Tett. Lett., 24, 4401 (1983), Wuts et al.,

— y —

J. Org. Chem., 53, 4503 (1988) and Szelke et al., WO 84/03044, all of which are incorporated herein by reference.

In accordance with Scheme 1, an appropriate acyl intermediate, such as 2,5-disubstituted-4-hydroxy-5-amino- pentanoic(N-methyl,N-methoxy)amide, wherein the hydroxyl and amino groups are suitably protected as is common in the art, is treated with an appropriate nucleophile, such as a substituted lithium acetylide, a Grignard reagent, an organocopper reagent or other suitable carbon nucleophile, in an inert solvent, such as THF, to yield a ketone. If lithium acetylide is used as the nucleophile an OC,β-acetylenic ketone is prepared. Subsequent reduction of the triple bond to a double bond, such as with Lindlar's catalyst or 5% palladium on BaSθ ₄ in the presence of quinoline, yields the corresponding α,β-alkenyl ketone, while reduction with palladium on carbon yields the corresponding keto-alkane. Deprotection of the hydroxyl group is accomplished by treatment with a reagent appropriate for the hydroxyl protecting group chosen. For instance if a silyl protecting group is chosen, a fluoride reagent, such as tetrabutylammonium fluoride is suitable.

In accordance with Scheme 2, in order to introduce the substituent R ² , wherein R ² is (CHR ) _n -Y or =CR ⁵ (CHR ) _n -Y, a β- keto-phosphonate is reacted with an appropriate aldehyde or ketone to yield an α,β-unsaturated ketone. For instance, compound JL may be reacted in an appropriate solvent, such as THF, with an aldehyde or ketone, such as benzaldehyde, isobutyraldehyde or l-benzyloxymethyl-2-formyl-imidazole or glyoxylamide to provide the corresponding β-substituted ketones. The unsaturated ketone may be reduced, as described for Scheme 1, or further reacted, as in Scheme 3 or by other methods common in the art, to introduce functionality into the molecule.

According to Scheme 3, hydrocyanation provides a nitrile, which may be further converted to an amide or acid by methods common to the art . Carboxylic acids may, of course be converted to esters, amides and alcohols.

Other typical reactions to introduce functionality into the α,β-unsaturated ketone are epoxidation of the double bond and conjugate addition of nucleophiles to the double bond, as illustrated in Scheme 4.

Scheme 4

i.e. Y=CONH ₂ , CO-jCH*-,,

CH-2-OH, CH-Hrridazote R ⁴ = CH ₂ -Ph, CH ₂ -iPr

For example, treatment of the α,β-unsaturated ketone 2. with an organocuprate, such as lithium divinyl cuprate or lithium di-(1-methoxy-vinyl) cuprate, yields the corresponding β-akyl or -alkenyl substituted ketone.

Ozonolysis of a methoxyvinyl substituent or a vinyl substituent yields the corresponding ester or aldehyde. Reaction of an aldehyde, for instance with sodium borohydride in methanol, yields an alcohol. Reaction of the aldehyde with glyoxal and ammonia yields an imidazole.

For a further example, treatment of α,β unsaturated ketone 3_ with alkaline hydrogen peroxide in an alcoholic solvent, or m-chloroperbenzoic acid or trifluoroperoxyacetic acid in a halocarbon solvent, such as methylene chloride, yields an α,β-epoxy ketone. The epoxide may be further reacted with diethylaluminum cyanide to provide an α- hydroxy,β-cyano ketone. The cyano group may be hydrolyzed by methods common to the art to give an acid or an amide, and further converted to derivatives thereof, such as an ester.

As another illustration, the epoxide may be reacted with an organo cuprate reagent, such as lithium divinyl cuprate or lithium di-(1-methoxy-vinyl) cuprate, to yield the corresponding α-hydroxy,β-vinyl ketone or α-hydroxy,β- (1- methoxy-vinyl) ketone. Hydrolysis of a methoxyvinyl adduct

_ _χl _

yields a methyl ketone; ozonolysis of a methoxyvinyl adduct yields an ester. Similarly, ozonolysis of a vinyl adduct yields an aldehyde. Reduction of an aldehyde, with sodium borohydride, or an ester, with borane-methyl sulfide, yields an alcohol. Treatment of an aldehyde with glyoxal and ammonia in methanol yields an α-hydroxy, β-imidazolyl ketone.

Reaction of the α,β-unsaturated ketone with lithium tris(thiophenyl)methylide in THF yields an β-tris-

(thiophenyl)methyl ketone. Subsequent alcoholysis in the presence of mercuric chloride yields the β-carboalkoxy ketone.

Suitable protective groups for functional groups and intermediates are disclosed in Greene et al., Protective Groups .-in Organic Synthesis. Second Edition, John Wiley and Sons, New York, 1991.

If a group other than a protecting group is desired for the substituent W, then the protecting group is removed and the amino group is reacted with an appropriate alkylating or acylating reagent. Alkyl halides, acyl halide, sulfonyl halides, anhydrides, activated esters, and the like, of the appropriate group W are useful for this purpose.

If the final compound, after it has been deprotected, contains a basic group, an acid addition salt may be prepared. Acid addition salts of the compounds are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic. The acetate salt form is especially useful. If the final compound contains an acidic group, cationic salts may be prepared. Typically the parent compound is treated with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation. Cations such as Na ⁺ , K ⁺ , Ca ⁺⁺ and NH ⁺ are examples of cations present in pharmaceutically acceptable salts. Certain of the compounds form inner salts or zwitterions which may also be acceptable.

The compounds of formula (I) are used to induce anti¬ viral activity in patients which are infected with

susceptible viruses and require such treatment. The method of treatment comprises the administration orally, parenterally, buccally, trans-dermally, intra-vaginally, rectally or by insufflation, of an effective quantity of the chosen compound, preferably dispersed in a pharmaceutical carrier. Dosage units of the active ingredient are generally selected from the range of 0.1 to 25 mg/kg, but will be readily determined by one skilled in the art depending upon the route of administration, age and condition of the patient. These dosage units may be administered one to ten times daily for acute or chronic infection. The compounds of this invention are particularly useful for the treatment of HIV-1. No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.

Pharmaceutical compositions of the compounds of this invention, or derivatives thereof, may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

Alternately, these compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include

starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

For intra-vaginal or rectal administration, a pulverized powder of the compounds of this invention may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository. The pulverized powders may also be compounded with an oily preparation, gel, cream or emulsion, buffered or unbuffered, and administered through a transdermal patch. These and other pharmaceutically acceptable formulation are found in Remington's Pharmaceutical Sciences, 18th Edition, Alfonso R. Gennaro (ed.). Mack Publishing Company, Easton, Pennsylvania (1990) .

Beneficial effects may be realized by co-administering, individually or in combination, other anti-viral agents with the protease inhibiting compounds of this invention. Examples of anti-viral agents include nucleoside analogues, phosphonoformate, rifabutin, ri aviran, phosphonothioate oligodeoxynucleotides, castanospermine, dextran sulfate, alpha interferon and ampligen. Nucleoside analogues, which include 2',3'-dideoxycytidine (ddC) , 2' ,3 '-dideoxyadenine (ddA) and 3'-azido-2' ,3 '-dideoxythymide (AZT) , are especially useful. AZT is one preferred agent. Suitably pharmaceutical compositions comprise an anti-viral agent, a protease

inhibiting compound of this invention and a pharmaceutically acceptable carrier.

The protease inhibiting properties of the compounds of this invention, are demonstrated by their ability to inhibit the hydrolysis of a peptide substrate by rHIV protease in the range of about 0.5 μM to about 2 mM.

The Examples which follow serve to illustrate this invention. The Examples are intended to in no way limit the scope of this invention, but are provided to show how to make and use the compounds of this invention.

Purification of Recombinant HIV Protease

Methods for expressing recombinant HIV protease in £_.. coli have been described by Debouck, et al., Proc. Natl.

Acad. Sci. USA, 84, 8903-6(1987). The enzyme used to assay the compound of this invention was produced in this manner and purified from the cell pellet as follows. The £_,. coli cell pellet was resuspended in a buffer consisting of 50 mM Tris-HCl, pH 7.5; 1.0 mM each DTT, EDTA and PMSF

(phenylmethyl-sulfonyl fluoride) . The cells were lysed by sonication and insoluble material was removed by centrifugation at 15,000 x g av, for 15 min. The clarified supernatant was then brought to 40% of saturation with ammonium sulfate. This suspension was stirred at room temperature for 30 min. and then centrifuged as above. The resulting precipitate was redissolved/resuspended in a minimal volume of 20 mM Tris-HCl, pH 7.5; 200 mM NaCl; 0.1 mM each DTT and EDTA. The sample was centrifuged again before application (in 5 mL aliquots) to a Beckman TSK G2000SW preparative HPLC gel filtration column (2.1 cm x 60 cm.) . The column was equilibrated in the same buffer at a flow rate of 4 mL/min. The effluent of the column was monitored at 280 n and 1 min. fractions collected. Typically, the rHIVPRT (recombinant HIV protease) eluted 45-46 min. into the run. At this stage, the protease was 85-95% pure. By immunoblot analysis >90% of the immunoreactive material was precipitated at the ammonium sulfate step. By activity assay, the highest

peak of activity was found in the fractions collected at 45 and 46 minutes. Analysis of the TSK column fractions by RP- HPLC and SDS-PAGE indicated that the majority of the 11,000 Mr protein is also found in fractions 45 and 46. The activity itself cannot be used to obtain reliable recovery data as it is influenced by high salt, i.e., with increasing salt, increasing levels of activity were obtained. Thus, with each step in the purification, more total activity was recovered than was started with. The overall yield of rHIVPRT was 1 mg from a 50 g E. coli cell pellet.

Inhibition of HIV protease activity

A typical assay contained 10 mL MENDT buffer (50 mM Mes (pH 6.0; 2-(N-morpholino)ethanesulfonic acid), 1 mM EDTA, 1 mM dithiothreitol, 200 mM NaCl, 0.1% Triton X-100) ; 2, 3, or 6 mM N-acetyl-L-arginyl-L-alanyl-L-seryl-L-glutaminyl-L- asparaginyl-L-tyrosyl-L-prolyl-L-valyl-L-valinamide (Ac-Arg- Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 K _m =7 mM) ; and micromolar and sub-micromolar concentrations of synthetic compounds. Following incubation at 37°C for several minutes, the reaction was initiated with purified 0.01-1 mg HIV protease. Reaction mixtures (37°C) were quenched after 10-20 minutes with an equal volume of cold 0.6 N trichloroacetic acid, and, following centrifugation to remove precipitated material, peptidolysis products were analyzed by reverse phase HPLC (Beckman Ultrasphere ODS, 4.5 mm x 25 mm; mobile phase: 5-20% acetonitrile/H2θ - 0.1% TFA (15 min), 20% acetonitrile/H2θ -

.1% TFA (5 min) at 1.5 mL/min, detection at 220 nm. The elution positions of Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val- NH2 (17-18 min) and Ac-Arg-Ala-Ser-Gln-Asn-Tyr (10-11 min) were confirmed with authentic material. Initial rates of Ac- Arg-Ala-Ser-Gln-Asn-Tyr formation were determined from integration of these peaks, and typically, the inhibitory properties of the synthetic compounds were determined from slope/intercept analysis of a plot of 1/v vs. [inhibitor] (Dixon analysis) . Kj_ values resulting from this type of primary analysis are accurate for competitive inhibitors

only, and under conditions in which the Michaelis constant of the substrate used is well-determined.

The Examples which follow serve to further illustrate this invention. The Examples are intended to in no way limit the scope of this invention, but are provided to show how to make and use the compounds of this invention.

In the Examples, all temperatures are in degrees Centigrade. FAB indicates fast atom bombardment mass spectrometry. FAB mass spectra were performed upon a VG Zab mass spectrometer using fast atom bombardment. ESMS indicates electrospray ionization mass spectrometry. NMR were recorded at 250 MHz using a Bruker AM 250 spectrometer. Multiplicities indicated are: s=singlet, d=doublet, t=triplet, q=quartet, m=muitiplet, dd=doublet of doublets, dt=doublet of triplets etc.and br indicates a broad signal.

Example 1

Preparation of Δ ³ _' ⁴ trans-(6R,8S_ 9S)-10-Phenyl-9-t-butoxy- carbonylamino-8-hydroxy-2-methyl-6-phenylmethyl-dec-3-ene-5- one. and A-^cis-(6R.8S.9S)-10-phenyl-9-t-butoxycarhonylamino- 8-hydroxy-2-methyl-6-phenylmethyl-dec-3-ene-5-one.

a) (l'S,3R,5S)-5-[(1'-N-t-butoxycarbonylamino-2'-phenyl)- ethyl]-3-phenylmethyl-γ-butyrolactone

A solution of lithium diisopropylamide (1.5 M in hexane, 3.9 mL, 0.0058 mol) and THF (4 mL) was cooled to -78°C. (1•S,3R,5S)-5-[(1'-N-t-butoxycarbonylamino-2*-phenyl)-ethy l]- γ-butyrolactone (0.81 g, 0.0027mol) in THF (6 mL) was slowly added and the mixture stirred for 10 min. HMPA (0.93 mL, 0.0053 mol) and benzyl bromide (0.63 mL, 0.0053 mol) were added. After 3 h the reaction was treated with 10% hydrochloric acid (25 mL) and extracted with methylene chloride (3 x 35 mL) . The combined extracts were dried

(sodium sulphate) , filtered, and concentrated in vacuo to afford a yellow oil. Vacuum assisted chromatography (silica; 15% ethyl acetate/hexane) of the crude material yielded the

pure benzyl lactone (0.47 g, 45%) as a colourless oil. A further 0.32 g (31%) of the benzyl lactone was isolated as a colourless oil which was slightly contaminated by the undesired isomer: ---HNMR (CDCI3, 400 MHz) δ 1.41 (s, 9 H) , 2.0-2.2 (m, 2 H) , 2.6-3.0 (m, 4 H) , 3.11 (m, 2 H) , 3.93 (br q, 1 H), 4.30 (m, 1 H) , 4.59 (br d, 1 H, J=8) , 7.05-7.35 (m, 10 H) .

b) (2R,4S,5S)-5-t-butoxycarbonylamino-4-t-butyldimethyl- silyloxy-6-phenyl-2-phenylmethyl-hexanoic acid

The benzyl lactone of step 1(a) (1.01 g, 0.0026 mol) in dioxane (2.8 mL) and water (1.4 mL) was treated with sodium hydroxide (1 M, 4.25 mL, 0.00425 mol). The solution was stirred for 15 min after which time citric acid (5% aqueous, 80 mL) was added and the mixture extracted with diethyl ether (3 x 75 mL) . The combined extracts were dried (sodium sulphate) , filtered, and the solvents removed in vac o. The residue was re-dissolved in DMF (10 mL) and treated with imidazole (1.77 g, 0.026 mol) and t-butyldimethylsilyl chloride (1.96 g, 0.013 mol). This solution was stirred for 16 h after which time citric acid (10% aqueous, 50 mL) was added and the resultant mixture extracted with diethyl ether (3 x 75 mL) . The combined extracts were concentrated in vacuo to yield an oily residue which was dissolved in a mixture of THF, acetic acid and water (2:2:1, 30 mL) . This solution was stirred for 2 h, water (50 mL) was added and the solution extracted with diethyl ether (3 x 75 mL) . The combined extracts were dried (sodium sulphate) , filtered, and the solvents removed in vacuo to yield a yellow oil. Vacuum assisted chromatography (silica; 10% ethyl acetate/hexane) furnished the silyl ether (0.82 g, 61%) homogeneous by t.l.c (silica; 10% ethyl acetate/hexane) and a further sample (0.41 g, 30%) which was slightly impure as shown by t.l.c (silica; 10% ethyl acetate/hexane) .

c) methyl (2R,4S,5S)-5-t-butoxycarbonylamino-4-t- butyldimethylsilyloxy-6-phenyl-2-phenylmethyl-hexanoate

An ethereal solution of diazomethane was prepared from N-methyl-N*-nitro-N-nitrosoguanidine (0.345 g, 0.0023 mol) . A solution of the silyl ether of step 1(b) (0.82 g, 0.0016 mol) in diethyl ether (10 mL) was cooled to 0°C and ethereal diazomethane added until the yellow colouration persisted. The reaction mixture was quenched by the addition of a small amount of glacial acetic acid. The ethereal solution was then washed with water (10 mL) , 1% sodium hydrogen carbonate solution (10 mL) and saturated sodium chloride solution (10 mL) , dried (sodium sulphate) , filtered, and concentrated in vacuo . Preparative HPLC (silica; 10% ethyl acetate/hexane) yielded the starting acid (0.605 g) and the pure methyl ester (0.210 g, 95%) . The reclaimed acid was subsequently re- submitted to the same procedure, to yield the methyl ester (0.491 g, 84% overall) : ---HNMR (CDC1 ₃ , 400 MHz) δ 0.06 (s, 3

H), 0.09 (s, 3 H), 0.91 (s, 9 H) , 1.37 (s, 9 H) , 1.53 (m, 1 H), 1.91 (in, 1 H), 2.55-2.93 (m, 6 H) , 3.57 (s, 3 H) , 3.67 (br t, 1 H), 4.69 (br d, 1 H, J=8) , 6.95-7.35 (m, 10 H) ; •^C M (CDCl _3j, 62.89) δ 175.27, 155.36, 138.87, 138.70,

129.11, 128.82, 128.82, 126.36, 126.18, 79.18, 71.16, 54.99, 51.24, 43.80, 38.61, 37.78, 36.05, 28.23, 25.95, 18.06.

d) dimethyl [ (3R,5S, 6S)-6-t-butoxycarbonylamino-5-t- butyldimethylsilyloxy-3, 6-bisphenylmethyl-2-oxo]hexyl phosphonate

A solution of dimethyl methylphosphonate (0.298 g, 0.0024 mol) was dissolved in THF (4 mL) and cooled to -78°C. Butyl lithium (1.6 M in hexane, 1.5 mL, 0.0024 mol) was added and the mixture stirred for 10 min. After this time the methyl ester of step (c) (0.325 g, 0.0006 mol) in THF (5 mL) was slowly added and the mixture stirred at -78°C for 2 h. Citric acid (10% aqueous, 10 mL) was added and the solution extracted with diethyl ether (3 x 30 mL) . The combined extracts were dried (sodium sulphate) , filtered, and concentrated in vacuo to yield a yellow oil. Vacuum assisted chromatography (silica; 1.5% methanol/methylene chloride)

furnished the phosphonate (0.365 g, 96%) upon concentration of the appropriate fractions: ---HNMR (CDCI3, 250 MHz) δ 0.00

(s, 3 H), 0.05 (s, 3 H) , 0.91 (s, 9 H) , 1.32 (s, 9 H) , 1.82 (m, 1 H) , 2.31-3.18 (m, 9 H) , 3.58 (m, 6 H) , 3.87 (m, 1 H) , 4.71 (br d, 1 H) , 6.87-7.41 (m, 10 H) ; --^CNMR (CDCl ₃ 62.89) δ 175.1, 155.7, 139.1, 138.6, 129.2, 129.1, 128.9, 128.5,

126.4, 79.1, 70.9, 54.1, 52.7, 51.2, 41.7, 39.6, 38.9, 38.7, 36.9, 35.5, 28.3, 25.9, 18.1.

e) Δ ³ ' ⁴ trans- (6R, 8S, 9S) -10-phenyl-9-t-butoxycarbonylamino-8- t-butyl-dimethylsilyloxy-2-methyl-6-phenylmethyl-dec-3-ene-5 - one, and

Δ ^{3 4} cis- (6R, 8S, 9S) -10-phenyl-9-t-butoxycarbonylamino-8-t- butyl-di-methylsilyloxy-2-methyl-6-phenylmethyl-dec-3-ene-5- one

The phosphonate of step 1(d) (0.128 g, 0.0002 mol) was dissolved in THF (3 mL) . Potassium carbonate (0.300 g, 0.002 mol) was added followed by isobutyraldehyde (183 μL, 0.002 mol) and the mixture stirred for 48 h. After this time water (5 mL) was added and the mixture was extracted with diethyl ether (3 x 50 mL) . The combined extracts were re-extracted with 20% sodium bisulphite solution (20 mL) , dried (sodium sulphate), filtered, and concentrated in vacuo . Vacuum chromatography (silica; 100% methylene chloride) furnished the cis ,β-unsaturated ketone(0.005 g, 4 %) and the trans α,β-unsaturated ketone (0.051 g, 44%), homogeneous by t.l.c (silica; methylene chloride) : trans -"-HNMR (CDCI3, 250 MHz) δ

-0.06 (s, 3 H), 0.03 (s, 3 H) , 0.89 (s, 9 H) , 0.90 (d, 6 H, J=7.7), 1.31 (s, 9 H), 1.81 (m, 1 H) , 2.14-3.09 (m, 7 H) , 3.63 (ddd, 1 H, 3=1 .1 , 6.9, 1.2), 3.89 (m, 1 H) , 4.68 (br d, 1 H), 5.93 (dd, 1 H, J=15.7, 1.2), 6.67 (dd, 1 H, J=15.7, 6.9), 6.95-7.30 (m, 10 H) ; ¹³ CNMR (CDCl3 _f 62.89) δ 203.1,

156.0, 154.1, 139.5, 138.7, 129.2, 128.9, 128.4, 126.8, 79.1, 71.1, 54.1, 47.8, 38.5, 37.7, 36.2, 31.1, 28.3, 25.9, 21.2, 18.1, -4.0, -4.4.

Cis -J-HNMR (CDCI3, 250 MHz) δ -0.06 (s, 3 H) , 0.03 (s, 3 H) ,

0.89 (m, 15 H), 1.31 (s, 9 H) , 1.80 (m, 1 H) , 2.32-2.97 (m, 6 H), 3.45 (m, 1 H) , 3.58 (m, 1 H) , 3.89 (m, 1 H) , 4.64 (br d,

93/02674 _ _2Q _

1 H) , 5 .75 (dd, 1 H, J=11.3, 9.7) , 5 . 95 (d, 1 H, J=11.3) , 6.92-7 .30 (m, 10 H) .

f) Δ ³ _' ⁴ cis-(6R,8S,9S)-10-phenyl-9-t-butoxycarbonylamino-8- hydroxy-2-methyl-6-phenylmethyl-dec-3-ene-5-one

The cis α,β-unsaturated ketone of step 1(e) (0.005 g, 8.6 μmol) was dissolved in THF (0.5 mL) . Tetrabutylammonium fluoride (1M in THF, 52 μL, 52 μmol) was added and the mixture stirred for 16 h. Hydrochloric acid (3M, 200 μL) was added and the solution extracted with methylene chloride (2 x 1 mL) . The combined extracts were concentrated in vacuo. Vacuum chromatography (silica; methylene chloride) furnished the titled alcohol (1.65 mg, 41%): ^" --HNMR (CDCI3, 250 MHz) δ

1.04 (br d, 6 H), 1.43 (s, 9 H) , 2.50 ( , 1 H) , 2.67-3.54 (m, 7 H), 3.82 (m, 1 H), 4.14 (m, IH) , 4.50 (br s, 1 H) , 4.66 (br s, 1 H), 5.01 (m, IH), 6.05 (m, 1 H) , 6.95-7.42 (m, 10 H)

g) Δ ³ _' ⁴ trans-(6R,8S,9S)-10-phenyl-9-t-butoxycarbonylamino-8- hydroxy-2-methyl-6-phenylmethyl-dec-3-ene-5-one The trans α,β-unsaturated ketone of example 6 (10 mg,

17.2 μmol) was dissolved in THF (0.4 mL) . Tetrabutylammonium fluoride (1M in THF, 103 μL, 103 μmol) was added and the mixture stirred for 16 h. Hydrochloric acid (3M, 200 μL) was added and the solution extracted with methylene chloride (2 x 1 mL) . The combined extracts were concentrated in vacuo.

Vacuum chromatography (silica; methylene chloride) furnished the titled alcohol (1.1 mg, 14%): 1H NMR (CDCI3, 250 MHz) δ

1.28 (d, 3 H, J=6.5), 1.34 (d, 3 H, J=6.7), 1.49 (s, 9 H) , 1.87 (m, 1 H), 2.0-3.2 (m, 7 H), 3.81 (m, 1 H) , 3.96 (m, 1 H), 4.64 (br s, 1 H) , 4.76 (br s, 1 H) , 6.11 (m, 1 H) , 6.42 (m, 1 H), 7.05-7.45 (m, 10 H) .

Example 2

Preparation of f9S. AS.6R. -10-phenyl-9-t-.-hut-.oxγcarbonylamino- 8-hydroxy-2-methγl-6-phenylmethγl-decan-5-one

a) • (9S, 8S, 46R)-10-phenyl-9-t-butoxycarbonylamino-8-t- butyldimethyl-silyloxy-2-methyl-6-bisphenylmethyl-decan-5-on e The trans α,β-unsaturated ketone of example 1(e) (10 mg, 17.2 μmol) was dissolved in anhydrous methanol (0.5mL) . 10% palladium on charcoal (1 mg) was added and the mixture stirred under an atmosphere of hydrogen for 1.5 h. The mixture was filtered through a 0.2 μm filter and the palladium on charcoal washed with diethyl ether (0.5 mL) . Removal of the solvents yielded the titled alkane (8 mg, 80%) homogeneous by t.l.c (silica; methylene chloride) : **-HNMR

(CDC1 ₃ , 250 MHz) δ -0.09 (s, 3 H) , 0.10 (s, 3 H) , 0.68 (d, 6

H, J=6.5), 0.81 (s, 9 H), 1.19 (m, 3 H) , 1.24 (s, 9 H) , 1.81 (m, 2 H) , 2.24 (m, 2 H) , 2.65 (m, 2 H) , 3.63 (m, 1 H) , 3.89 (m, 1 H) , 4.63 (br d, 1 H) , 6.90-7.30 (m, 10 H) .

b) (9S, 8S, 6R)-10-phenyl-9-t-butoxycarbonylamino-8-hydroxy-2- methyl-6-phenylmethyl-decan-5-one

The alkane of example 2(a) (8 mg, 13.7 μmol) was dissolved tetrabutylammonium fluoride (1M in THF, 83 μL, 83 μmol) and the mixture stirred for 16 h. Hydrochloric acid (3M, 400 μL) was added and the solution extracted with methylene chloride (2 x 2.5 mL) . The combined extracts were concentrated in vacuo . Vacuum chromatography (silica; methylene chloride) furnished the titled alcohol (3 mg, 47%) : ---HNMR (CDCI3, 250 MHz) δ 0.86 (d, 3 H, J=6.5), 1.45 (s, 9 H) ,

1.56 (m, 3 H) , 2.30 (m, 2 H) , 2.55-2.95 (m, 5 H) , 3.6-3.9 (m, 2 H), 4.95 (br d, 1 H) , 7.10-7.35 (m, 10 H) .

Example 3

Preparation of Δ^ ² trans- R.6S.7S. -7-t-butoxvcarbonylamino-6- hydroxy-1.8-diphenvl-4-phenγlmethvl-octene-3-one.

a) dimethyl [ (3R,5S, 6S)-6-t-butoxycarbonylamino-5-t- butyldimethylsilyloxy-3, 6-bisphenylmethyl-2-oxo]hexyl phosphonate

A solution of dimethyl methylphosphonate (0.088 g, 0.00071 mol) was dissolved in THF (1 mL) and cooled to -78°C.

Butyl lithium (1.6 M in hexane, 0.45 mL, 0.00071 mol ) was added and the mixture stirred for 15 min. The benzyl lactone of example 1(a) (0.070 g, 0.00018 mol) in THF (0.5 mL) was slowly added and the mixture stirred at -78°C for 2 h. Hydrochloric acid (10% aqueous, 1 mL) was added and the solution extracted with methylene chloride (2 x 10 mL) followed by ethyl acetate (1 x 10 mL) . The combined extracts were dried (sodium sulphate) , filtered, and concentrated in vacuo to yield a yellow oil. Vacuum assisted chromatography (silica; 0.75% methanol/methylene chloride, 5 x 10 mL then 1.5% methanol/methylene chloride, 10 x 10 mL) furnished the starting lactone (8 mg) and the phosphonate (0.78 g, 85%) upon concentration of the appropriate fractions: ^■ '-HNMR (CDC1 ₃ , 250 MHz) δ 1.39 (s, 9 H) , 1.72-2.38 (m, 6 H) , 2.51- 3.00 (m, 4 H), 3.50-4.00 ( , 6 H) , 4.24 (m, 1 H) , 4.81 (s, 1 H), 5.04 (d, 1 H, J=7.6), 7.04-7.41 (m, 10 H) ; ¹³ CNMR (CDCl ₃

62.89) δ 170.1, 153.2, 134.2, 133.6, 129.1, 128.7, 128.2, 126.2, 126.0, 79.9, 55.4, 53.7, 52.4, 52.1, 50.3, 50.1, 40.9, 35.5, 33.2, 27.1.

b) Δ ^1,2 trans-(4R,6S, 7S)-7-t-butoxycarbonylamino-6-t-butyl— dimethylsilyloxy-1,8-diphenyl-4-phenylmethyl-octene-3-one

The phosphonate of step 3(a) (25.5 mg, 0.05 mmol) was dissolved in THF (1 mL) and water (0.5 mL) . Potassium carbonate (50 mg, 0.4 mmol) was added followed by benzaldehyde (12 mg, 0.12 mmol) and the mixture stirred for 24 h. After this time the mixture was extracted with ethyl acetate (3 x 10 mL) and the combined extracts dried (sodium sulphate), filtered, and concentrated in vacuo . Vacuum chromatography (silica; 1.5% methanol/methylene chloride) furnished the starting phosphonate (10 mg) and the desired trans α,β-unsaturated ketone (14.5 mg, 59%) : -'-HNMR (CDCI3, 250

MHz) δ 1.36 (s, 9 H), 2.51 (m, 1 H) , 2.78-3.04 (m, 3 H) ,

3.44-4.02 (m, 4 H) , 4.52 (dt, 1 H, J=7.8, 1.2), 4.74 (br d, 1 H, J=7.6), 6.83 (2 d, 2 H, J=16.8), 7.01-7.50 (m, 15 H) .

c) ^• Δ ¹ _' ² trans- (4R, 6S, 7S) -7-t-butoxycarbonylamino-6-hydroxy-

1, 8-diphenyl-4-phenylmethyl-octene-3-one

The alkane of example 3(b) (29 mg, 47 μmol) was dissolved tetrabutylammonium fluoride (1M in THF, 156 μL, 156 μmol) and the mixture stirred for 16 h. Hydrochloric acid (100 μL ) was added and the solution extracted with methylene chloride. The combined extracts were concentrated in vacuo. Vacuum chromatography (silica; methylene chloride) furnished the titled alcohol (14 mg: iH MR (CDC1 ₃ , 250 MHz) δ 1.39 (s, 9 H), 2.50 (br d, 1 H) , 2.82 (m, 2 H) , 2.94 (d, 2 H, J=7.4), 3.52 (s, 2 H), 3.89- (m, 1 H) , 4.52 (dt, 1 H, J=7.2, 1.1), 4.7 (br d, 1 H) , 6.72 (dd, 2 H, J=17, 7.25 (m, 15 H) ) .

Example 4

Preparation of (4R.6S, 7S)-7-t-butoxycarbonylamino-6-t- butyldimethγlsilyloxy-1.8-diphenγl-4-phenγlmethyl-octan-3 -one

a) (4R, 6S,7S)-7-t-butoxycarbonylamino-6-t-butyldimethyl- silyloxy-1, 8-diphenyl-4-phenylmethyl-octan-3-one

The compound of Example 3 (b) was dissolved in ethanol and hydrogenated at 40 psi for 4 h using 5% palladium on carbon. Filtration and evaporation of the solvent yields the titled compound: ---HNMR (CDCI3, 250 MHz) δ 7.29-6.89 (m, 15 H),4.67 (br d, 1 H, J=9.7), 3.87 (q, 1 H, J=8.5), 3.59 (m, 1 H), 2.79-2.29 (m, 8 H) , 1.75-1.48 (m, 4 H) , 1.26 (s, 9 H) , 0.88 (s, 9 H) , 0.06 (s, 3H) , -0.009 (s, 3 H)

b) (4R, 6S,7S)-7-t-butoxycarbonylamino-6-hydroxy-l,8-diphenyl- 4-phenylmethyl-octan-3-one

The alkane of Example 4(a) (8 mg, 13.7 μmol) was dissolved tetrabutylammonium fluoride (1M in THF, 83 μL, 83 μmol) and the mixture stirred for 16 h. Hydrochloric acid (3M, 100 μL) was added and the solution extracted with methylene chloride. The combined extracts were concentrated in vacuo. Vacuum chromatography (silica; methylene chloride) furnished the titled alcohol (6.4 mg) : ---HNMR (CDCI3, 250 MHz) δ 7.40-6.90 (m, 15 H) , 4,77-4.67 (d, 1), 4.05-3.85 (m, 1 H) ,

3.70-3 .55 (dd, 1 H) , 2 . 95-2.55 (m, 7 H) , 2. 50-2 .25 (m, 3 H) , 1, 85-1. 65 (m, 1 H) , 1.45-1.50 (m, 1 H) , 1 .38 (s, 9 H) .

Example 5

Preparation of _8S.7S.5R.2RS.-9-phenyl-8-t-hut-.oxyr.arbonyl- amino-7-hydroxy-5-phenylmethyl-4-oxo-2-isopropyl-nonanoic acid amide.

(a) (9S,8S,6R,3RS)-10-phenyl-9-t-butoxycarbonylamino-8-t- butyldimethysilyloxy-3-cyano-2-methyl-6-phenylmethyl-decan-5 - one

The trans α,β-unsaturated ketone of Example 1(e) (0.030 g, 51.8 μmol) was dissolved in anhydrous toluene (0.5 mL) . The mixture was cooled to 0°C and diethylaluminum cyanide (1M in toluene, 155 μL, 155 μmol) was added. After

45 min no product formation was observed. A further three equivalents of diethylaluminum cyanide (155 μL, 155 μmol) was added and the mixture warmed to room temperature. After 1.15 h hydrochloric acid (3M, 500 μL) was added and the mixture extracted with ethyl acetate (3 x 2.5 mL) . The combined extracts were dried (sodium sulphate) , filtered, and concentrated in vacuo to yield a colourless oil. Chromatography (silica; 10% ethyl acetate/hexane) furnished a diastereomeric mixture of the two nitriles which were readily separable by HPLC (silica; 10% ethyl acetate/hexane) . Overall yield 13.1 mg, 42%, individual yields of 7.1 mg (23%) and 6.0 mg (19%) were obtained; ¹ HNMR (CDCI3, 250 MHz) δ

-0.09 (s, 3 H), 0.10 (s, 3 H), 0.68 (d, 6 H, J=6.4), 0.81 (s, 9 H), 1.25 (s, 9 H) , 1.34 (m, 1 H) , 1.6-2.8 ( , 10 H), 3.63 (m, 1 H), 3.89 (m, 1 H) , 4.69 (br d, 1 H) , 6.79-7.30 (m, 10 H) .

b) (9S,8S,6R,3RS)-10-phenyl-9-benzyloxycarbonylamino-8-t- butyldimethysilyloxy-3-cyano-2-methyl-6-phenylmethyl-decan-5 - one

The compound of Example 5(a) (30 μmol) is dissolved in trifluoroacetic acid (1 mL) and stirred at room temperature

— Δ O —

for 10 min. The trifluoroacetic acid is evaporated and the residue is dissolved in THF (0.5 mL) and triethylamine is added (150 μmol) followed by benzyl chloroformate (36 μmol) .

The reaction is stirred overnight. The reaction mixture is diluted with methylene chloride, the organic solution is washed with 3N hydrochloric acid, water, 5% sodium carbonate, and brine. Evaporation of the solvent and chromatography (silica gel) yields the title compound.

c) methyl (8S,7S,5R,2RS)-9-phenyl-8-benzyloxycarbonylamino-

7-t-butyldimethysilyloxy-5-phenylmethyl-4-oxo-2-isoproρy l- nonanoate

The compound of Example 5(b) (25 μmol) is dissolved in methanol (0.5 mL) at 0°C and treated with saturated methanolic HC1 (0.5 mL) for 1 h. The solvents are evaporated at 0°C and the residue is dissolved in methanol:water (1 mL, 1:1) and stirred overnight. The mixture is extracted with ethyl acetate and the organic extract is washed with 5% sodium bicarbonate and water, and dried over sodium sulphate. Filtration and evaporation of the solvent yields a crude residue. Chromatography of the residue yields the title compound.

d) methyl (8S,7S,5R,2RS)-9-phenyl-8-benzyloxycarbonylamino- 7-hydroxy-5-phenylmethyl-4-oxo-2-isopropyl-nonanoate

The compound of Example 5(c) (30 μmol) was treated with tetrabutyl ammonium fluoride in THF (1M in THF, .18 mmol) for 4 h. The reaction mixture is diluted with methylene chloride, washed with water (3X) and dried (Na ₂ S0 ₄ ) . Filtration and evaporation of the solvent, and chromatography (silica gel) yields the title compound.

Example 6

Preparat i on of (7S . 6S . 4R . -8-phenyl-7-t-butoxycarbonγ1 -ami no- 6-hydroxy-4-phenylmethγl-3-oxo-_l - . 2-iτnidazolyl . -octane .

a) - (7S, 6S,4R)-8-phenyl-7-t-butoxycarbonylamino-6-t- butyldimethylsilyloxy-4-phenylmethyl-3-oxo-l-[2-(1- benzyloxymethyl)-imidazolyl]-oct-1-ene

The phosphonate of Example 1(d) (0.5 mmol) is dissolved in THF (10 mL) and DBU (1 mmol) is added. 1-Benzyloxymethyl- 2-formyl-imidazole (1 mmol) is added and the reaction is stirred for two days at room temperature. The reaction mixture is diluted with methylene chloride and the organic phase is washed with 5% HC1, water and brine. The organic extract is concentrated to a crude residue, which is chromatographed (silica gel) to yield the title compound.

b) (7S, 6S,4R)-8-phenyl-7-t-butoxycarbonylamino-6-t- butyldimethylsilyloxy-4-phenylmethyl-3-oxo-l-(2-imidazolyl)- octane

The compound of Example 6(a) (0.1 mmol) is dissolved in methanol (5 mL) and hydrogenated at 60 psi over 5% palladium on carbon (5 mg) overnight. The catalyst is removed by filtration. The reaction mixture is treated with Triton-B according to the procedure of Andersen et al. , Tet. Lett. , 34, 3165 (1971) , to complete removal of the benzyloxymethyl protecting group and yield the title compound.

c) (7S, 6S,4R)-8-phenyl-7-t-butoxycarbonylamino-6-hydroxy-4- phenylmethyl-3-oxo-l-(2-imidazolyl)-octane

The compound of Example 6(b) is desilylated according to the procedure of Example 4(b) to yield the title compound.

Example 7

Preparation of (9S.8S.6R.2RS)-10-phenvl-9-t-butoxγcarbonγl- amino-8-hydroxy-6-phenvlmethγl-5-oxo-3- .2-imidazolyl. -2- me hyl-dp.cane.

a) (9S,8S, 6R,2RS)-10-phenyl-9-t-butoxycarbonylamino-8-t- butyldimethylsilyloxy-6-phenylmethyl-5-oxo-3-[2-(1- benzyloxymethyl)-imidazolyl]-2-methyl-dec-l-ene

The compound of Example 6(a) (0.2 mmol) is dissolved in dry THF (5 mL) and reacted with lithium bis- (2-propenyl) cuprate (1 mmol, prepared from 2-proρenyl lithium and cuprous iodide) at -78°C under an Ar atmosphere for 2 h. The reaction is quenched by the addition of 5% aqueous ammonium chloride, diluted with ethyl acetate and washed with aqueous ammonium chloride/ammonium hydroxide solution (9:1 5% aq. NH ₄ Cl:conc. NH ₄ OH) . The organic phase is washed with water and brine, and dried over sodium sulphate. Filtration and evaporation of the ethyl acetate solution, and chromatography (silica) yields the title compound.

b) (9S, 8S, 6R,2RS)-10-phenyl-9-t-butoxycarbonylamino-8-t- butyldimethylsilyloxy-6-phenylmethyl-5-oxo-3- (2-imidazolyl)- 2-methyl-decane

The compound of Example 7(a) (0.15 mmol) is hydrogenated and the benzyloxymethyl protecting group is removed according to the procedure of Example 6(b) to yield the title product.

c) (9S, 8S, 6R,2RS)-10-phenyl-9-t-butoxycarbonylamino-8- hydroxy-6-phenylmethyl-5-oxo-3-(2-imidazolyl)-2-methyl-decan e

The compound of Example 7(b) (0.1 mmol) is desilylated according to the procedure of Example 4 (b) to yield the title compound.

Example 8

Parenteral Dosage Unit Composition

A preparation which contains 25 mg of a compound of this invention is prepared as follows:

25 mg of the compound is dissolved in 15 mL of distilled water. The solution is fiϊι:ered under sterile conditions into a 25 mL multi-dose ampoule and lyophilized. The powder is reconstituted by addition of 20 mL of 5% dextrose in water(D5W) for intravenous or intramuscular injection. The dosage is thereby determined by the injection volume. This solution is also suitable for use in other methods for

administration, such as addition to a bottle or bag for IV drip infusion.

Example 9

Oral Dosage Unit Composition

A capsule for oral administration is prepared by mixing and milling 200 mg of the compound with 450 mg of lactose and 30 mg of magnesium stearate. The resulting powder is screened and filled into a hard gelatin capsule.

The above description fully discloses how to make and use this invention. This invention, however, is not limited to the precise embodiments described herein, but encompasses all modifications within the scope of the claims which follow.