TITLE OF THE INVENTION NEW HIV PROTEASE INHIBITORS

This application is related to 18882, 18882IA, 18996, 18996IA.

The present invention is concerned with compounds which inhibit the protease encoded by human immunodeficiency virus (HIV). The compounds, or pharmaceutically acceptable salts thereof, are of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS).

The present invention also relates to pharmaceutical compositions containing the compounds and to a method of use of the present compounds and other agents for the treatment of AIDS & viral infection by HIV.

BACKGROUND OF THE INVENTION

A retrovirus designated human immunodeficiency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the extensive post-translational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Inhibition of this processing prevents the production of normally infectious virus. For example, Kohl, N.E., et. al, Proc. Natl. Acad. Sci. USA, 85, 4686 (1988), demonstrated that genetic inactivation of the HIV encoded protease resulted in the production of immature, non-infectious virus particles. These results indicate that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV.

Nucleotide sequencing of HIV shows the presence of a rjol gene in one open reading frame [Ratner, L. et ah, Nature, 313. 277(1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an endonuclease and an HTV protease [Toh, H. et al., EMBO J. 4, 1267 (1985); Power, M.D. et ah, Science, 231, 1567 (1986); Pearl, L.H. et aL, Nature 329, 351 (1987)]. Applicants demonstrate that the compounds of this invention are inhibitors of HIV protease.

The compounds of the present invention contain sulfonamide, urea or carbamate linkages in the peptide analog backbone.

BRIEF DESCRIPTION OF THE INVENTION

Compounds of Formula I, as herein defined, are disclosed. These compounds are useful in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS, either as compounds, pharmaceutically acceptable salts, hydrates or esters, pharmaceutical composition ingredients, whether or not in combination with other antivirals, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS This invention is concerned with the compounds of

Formula I, combinations thereof, or pharmaceutically acceptable salts thereof, in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS). Compounds of formula I are defined as follows:

Formula I

Wherein

R is a) -V-R3; wherein V is -C(0)-Q-, or -S02-Q-;

Q is a) absent, -0-, or -NH-,

Rl is a) hydrogen, or

b) -Cl-5alkyl unsubstituted or substituted with one or more of i) halo, ii) hydroxy, iii) Cl -3 alkoxy, iv) aryl unsubstituted or substituted with one or more of Cl_4alkyl, Cl-4alkoxy, nitro, amino, amido, carboxy, hydroxy, halo or aryl; v) -W-aryl or W-benzyl, wherein W is -0-, -S-, or

-NH-; or vi) heterocycle, unsubstituted or substituted with one or more of Cl_4alkyl, hydroxy or halo; viii) carboxyl;

c) -C3_5cycloalkyl, unsubstituted or substituted at the 3- position with Cl-4alkyl; or

d) aryl unsubstituted or substituted with one or more of Cι_ 4alkyl, Cl -4alkoxy, nitro, amino, amido, carboxy, hydroxy, halo or aryl; and

R2 is a) phenyl unsubstituted or substituted with one or more of -OH or Cι_3 alkoxy; or

b) C5_7 cycloalkyl, unsubstituted or substituted with one or more of -OH or Cj _3 alkoxy;

R is a) a 5- to 7- membered heterocycle, which heterocycle is unsubstituted or substituted with one or more of -Cl_4alkyl, oxo, amino or halo;

b) aryl unsubstituted or substituted with one or more of -Cl -4alkyl, oxo, amino or halo;

c) Cl_4alkyl, unsubstituted or substituted once with aryl or 5- to 7-membered heterocycle;

d) C3_5cycloalkyl, unsubstituted or substituted at the 3- position with Cl-4alkyl;

J is

or a pharmaceutically acceptable salt thereof.

The compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures and as individual diastereomers or enantiomers, with all isomeric forms being included in the present invention. When any variable (e.g., heterocycle, R* or R2, etc.) occurs more than one time in any constituent or in Formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. As used herein except where noted, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl); "alkoxy" represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge. C3 -5 cycloalkyl is a saturated cyclic hydrocarbon ring with the indicated number of carbons. "Halo", as used herein, means fluoro, chloro, bromo or iodo.

As used herein, with exceptions as noted, "aryl" is intended to mean phenyl (Ph) or naphthyl. The term heterocycle or heterocyclic, as used herein except where noted, represents a stable 5- to 7-membered mono- or bicyclic or stable 7- to 10-membered bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include piperidinyl, piperazinyl, 2-oxoρiρerazinyl, 2-oxoρiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl,

isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiopyranyl, tetrahydrofuryl, tetrahydropyranyl, and tetrahydrothienyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulf oxide, thiamo holinyl sulfone and isobenzothiopyranyl.

The pharmaceutically-acceptable salts of the compounds of Formula I (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts or the quaternary ammonium salts of these compounds, which are formed, e.g., from inorganic or organic acids. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, bisulfate, citrate, digluconate, dodecylsulfate, fumarate, glycerophosphate, hemisulfate, hydrochloride, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, succinate and tartrate.

One preferred embodiment of this invention is compounds of Formula I, wherein

R is a) -V-R3; wherein V is -C(0)-Q-, or -S02-Q-;

Q is a) absent, or -0-,

R! is -Cl-5alkyl unsubstituted or substituted with one or more of i) aryl unsubstituted or substituted with one or more of Ci-4alkyl, Ci-4alkoxy, nitro, amino, amido, carboxy, hydroxy, halo or aryl; or ii) heterocycle, unsubstituted or substituted with one or more of Cl _4alkyl, hydroxy or halo;

R2 is phenyl unsubstituted or substituted with one or more of

-OH or Cι_3 alkoxy; or

a) aryl unsubstituted or substituted with one or more of

-Cl-4alkyl, oxo, amino or halo;

b) Ci-4alkyl, unsubstituted or substituted once with aryl or 5- to 7-membered heterocycle;

J is

or a pharmaceutically acceptable salt thereof.

Most preferred compounds of this invention include the following:

Compound A:

named N-(2'(R)-hydroxy- 1 '(S)-indanyl)-2(R)-phenylmethyl-4(S)- hydroxy-5-[((2'"-methyl)propyl)-benzyloxycarbonyl]-amino- pentaneamide.

or pharmaceutically acceptable salt thereof;

Compound B:

named N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)-phenylmethyl-4(S)- hydroxy-5-[((2'"-methyl)butyl)-3'(S)-(tetrahydrofuranyloxy)- carbonyl]amino-pentaneamide,

or pharmaceutically acceptable salt thereof.

The compounds of the present invention include but are not limited to those of the following Tables 1 -4:

TABLE 1

TABLE 1 (CONT'D^

-F ¹ R ^J

TABLE 1 (CONT'D)

R R^

TABLE 1 (CONT'D)

¹ R^

X CH3CH2

X CH3CH2CH2CH2

TABLE 1 (CONT'D)

R ¹ R^

TABLE 2 (CONT'D)

R ¹ R^

R ¹

TABLE 3 (CONT'D)

R ¹ R°

__L_ R' _____ H tertbutyl isobutyl

isobutyl H benzyl

isobutyl H n-butyl

isobutyl H cyclopentyl isobutyl H phenyl

isobutyl H 3-pyridylmethyl

H benzyl

H tertbutyl

i ~ CH ₂ H 3-pyridylmethyl

/-CH ₂ N isobutyl Q N — = R' R-N

CH 2

The compounds of the present invention are prepared in accordance with Schemes I-II.

SCHEME1

Synthesis of

Compounds of Formula 1 are synthesized by the general method outlined in Scheme I. Briefly, the epoxide compound A is synthesized according to EP 0541 168, herein incorporated by references for these purposes. It is reacted at refluxing temperature with an excess of a primary amine, e.g., isobutyl amine, in an alcoholic solvent such as isopropanol. The isolated amine B is then reacted with the appropriate acylating agent. For the synthesis of Formula I compounds with urea linkages, t-butyl isocyanate is the appropriate acylating agent. For sulfonamide linkages, toluene-sulfonyl chloride. For amides, the appropriate acylating agent is a peptide coupling reagent such as cyclopentane acetic acid. The isolated compound C is then treated with acid in isopropanol to cleave the acetonide group to afford the desired product. The acetal cleavage step can also be performed after the amine epoxide opening and the resulting amine then directly reacted with the desired acylating agent to give the product as seen in Scheme 2.

SCHEME 2

Altemate synthesis of

Ηie compounds of the present invention are useful in the inhibition of HIV protease, the prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS. Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection: AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compounds of this invention are useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, accidental needle stick, or exposure to patient blood during surgery.

The compounds of this invention are also useful in the preparation and execution of screening assays for antiviral compounds. For example, the compounds of this invention are useful for isolating enzyme mutants, which are excellent screening tools for more powerful antiviral compounds. Furthermore, the compounds of this invention are useful in establishing or determining the binding site of other antivirals to HIV protease, e.g., by competitive inhibition. Thus the compounds of this invention are commercial products to be sold for these purposes. For these purposes, the compounds of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.

Thus, in accordance with the present invention there is further provided a method of treating and a pharmaceutical composition for treating HIV infection and AIDS. The treatment involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically- effective amount of a compound of the present invention, or a pharmaceutically-acceptable salt thereof.

These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile

injectable preparations, for example, as sterile injectable aqueous or oleagenous suspensions or suppositories.

When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally- acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.

Dosage levels of the order of 0.02 to 5.0 or 10.0 grams- per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher. For example, infection by HIV is effectively treated by the administration of from 10

to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. The present invention is also directed to combinations of the HIV protease inhibitory compounds with one or more agents useful in the treatment of AIDS. For example, the compounds of this invention may be effectively administered, whether at periods of pre- exposure and/or post-exposure, in combination with effective amounts of the AIDS antivirals, immunomodulators, anti-infectives, or vaccines known to those of ordinary skill in the art.

TABLE C

ANTIVIRALS

Drug Name Manufacturer Indication

Trisodium Astra Pharm. CMV retinitis, HIV

Phosphonoformate Products, Inc infection, other CMV (Westborough, MA) infections

Dideoxycytidine; Hoffman-La Roche AIDS, ARC ddC (Nutley, NJ)

Novapren Novaferon Labs, Inc. HIV inhibitor (Akron, OH) Diapren, Inc. (Ro Seville, MN, marketer)

Peptide T Peninsula Labs AIDS

Octapeptide (Belmont, CA)

Sequence

Zidovudine; AZT Burroughs Wellcome AIDS, adv, ARC AIDS, adv, ARC (Rsch. Triangle Park, pediatric AIDS, NC) Kaposi's sarcoma, asymptomatic HIV infection, less severe HIV disease, neurological involvement, in combination with other therapies.

Ansamycin LM 427 Adria Laboratories ARC (Dublin, OH) Erbamont (Stamford, CT)

(Rsch. Triangle HIV in combination

Park, NC) w/Retrovir

Acyclovir Burroughs Wellcome AIDS, ARC, asymptomatic HIV positive, in combination with AZT.

Antibody which Advanced Biotherapy AIDS, ARC neutralizes pH Concepts labile alpha aberrant (Rockville, MD) Interferon in an immuno-adsoφtion column

Drug Name Manufacturer Indication L-697,661 Merck AIDS, ARC, (Rahway, NJ) asymptomatic HTV positive, also in combination with AZT.

L-735,524 Merck AIDS, ARC, (Rahway, NJ) asymptomatic HTV positive, also in combination with AZT.

RO-31-8959 Hoffmann-LaRoche AIDS, ARC, asymptomatic HIV positive, also in combination with AZT.

IMMUNO-MODULATORS

(Irving, TX) anti-virals)

Drug Name Manufacturer Indication CL246.738 American Cyanamid AIDS, Kaposi's (Pearl River, NY) sarcoma Lederle Labs

(Wayne, NJ)

EL10 Elan Corp, PLC HIV infection (Gainesville, GA) (See also anti¬ virals)

Gamma Interferon Genentech ARC, in combination

(S. San Francisco, w/TNF (tumor

CA) necrosis factor)

Granulocyte Genetics Institute AIDS Macrophage Colony (Cambridge, MA) Stimulating Sandoz Factor (East Hanover, NJ)

Granulocyte Hoeschst-Roussel AIDS Macrophage Colony (Sommerville, NJ) Stimulating Immunex Factor (Seattle, WA)

Granulocyte Schering-Plough AIDS Macrophage Colony (Madison, NJ)

Stimulating Factor AIDS, in combination w/AZT

Drug Name Manufacturer Indication Methionine- TNI Pharmaceutical AIDS, ARC Enkephalin (Chicago, IL)

MTP-PE Ciba-Geigy Corp. Kaposi's sarcoma

Muramyl- (Summit, NJ)

Tripeptide

Granulocyte Amgen AIDS, in combination Colony Stimulating (Thousand Oaks, CA) w/AZT Factor

rCD4 Genentech AIDS, ARC

Recombinant (S. San Francisco,CA)

Soluble Human CD4

Drug Name Manufacturer Indication SK&F106528 Smith, Kline & HIV infection Soluble T4 French Laboratories (Philadelphia, PA)

Thymopentin Immunobiology HIV infection Research Institute (Annandale, NJ)

Tumor Necrosis Genentech ARC, in combination Factor; TNF (S. San Francisco, w/gamma Interferon

CA)

ANTI-INFECTIVES

Trimetrexate Warner-Lambert PCP

OTHER

Drug Name Manufacturer Indication Recombinant Human Ortho Pharm. Coφ. severe anemia Erythropoietin (Raritan, NJ) assoc. with AZT therapy

Megestrol Acetate Bristol-Myers treatment of (New York, NY) anorexia assoc. w/AIDS

Total Enteral Norwich Eaton diarrhea and Nutrition Pharmaceuticals malabsoφtion (Norwich, NY) related to AIDS

It will be understood that the scope of combinations of the compounds of this invention with AIDS antivirals, immunomodulators, anti-infectives or vaccines is not limited to the list in the above Table, but includes in principle any combination with any pharmaceutical composition useful for the treatment of AIDS. Certain compounds of Table C are the following: L-

697,661 or '661' is 3-([4,7-dichloro-l ,3-benzoxazol-2-yl)methyl]- amino)-5-ethyl-6-methyl-pyridin-2(lH)-one. The synthesis of L- 697,661 is described in EPO 484071, and EPO 462800, both herein incoφorated by reference. The synthesis of ddC, ddl and AZT are also described in EPO 484071. L-735,524 is

N-(2(R)-hydroxy-l(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy -5-(l - (4-(3-pyridylmethyl)-2(S)-N'-(t-butylcarbamoyl)-piperazinyl) )- pentaneamide; the synthesis of which is described in EP 0541168. RO 31-8959 which is compound XVII as described in Roberts, N.A. et al., Science 248. 358 (1990) is synthesized according to EP 0346847.

Preferred combinations are simultaneous or alternating treatments of an inhibitor of HIV protease and a non-nucleoside inhibitor of HIV reverse transcriptase. An optional third component in the combination is a nucleoside inhibitor of HIV reverse transcriptase, such as AZT, ddC or ddl.

EXAMPLE 1

N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5- r3'"-(methyl)butyl-(4"-(amino)phenylsulfonyl)1amino-pentanea mide

Step l : Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl 4(S)-hydroxy-5-[3"-(methyl)butyl]amj_no- pentaneamide N'.O'-dimethyl acetal

A mixture of epoxide 1 (2.0 g, 5.30 mmol) and isoamylamine (0.57 g, 6.52 mmol) was refluxed in isopropanol (20 mL) for 16 h. The solution was concentrated and purified by flash chromatography (3% methanol/chloroform saturated with ammonia) to yield 2 (2.3 g, 4.95 mmol) as a clear liquid.

isopropanol

Step 2: Preparation of N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[3"-(methyl)butyl]amino- pentaneamide hydrochloride

A solution of 2 (2.3 g, 5.0 mmol) in isopropanol (50 mL) was cooled to 0°C and saturated with anhydrous HCl. The solution was stirred and allowed to gradually warm to room temperature over 4 h, at which time it was concentrated. The resulting white hydrochloride salt was then diluted with ethyl acetate (20 mL), poured into saturated NaHC03 (100 mL), and washed with ethyl acetate(l x 150 mL). The organic layer was dried (MgSθ4), and concentrated to give a crude white solid which was purified by flash chromatography (5% methanol/ethyl acetate). The resulting solid was diluted with ethyl acetate, cooled to 0°C, and treated with anhydrous HCl to yield 3 (0.75 g, 1.63 mmol) as a white solid.

Step 3: Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[(3"'-(methyl)butyl)-(4"-

(nitro)phenylsulfonyl)1amino-pentaneamide

To a stirred solution of 3 (100 mg, 0.217 mmol) in dichloromethane (3 mL) at 0°C was added triethylamine (48.6 mg, 0.067 mL, 0.477 mmol) and 4-nitrobenzenesulfonyl chloride (45 mg, 0.236 mmol). The solution was warmed to room temperature and stirred for 48 h at which time it was diluted with dichloromethane and washed with saturated NaHC03 (2 x 25 mL). The organic layer was dried (MgS04), concentrated, and recrystallized (ethyl acetate) to give 4 (74 mg, 0.121 mmol) as a white solid m.p. 217-219°C.

Analysis calculated for C32H39N3O7S 0.10 CHCI3 C, 62.02; H, 6.34; N, 6.76; MWT, 621.69; Found: C, 63.26; H, 6.34; N, 6.89.

Step 4: Preparation of N-(2'<R)-hydroxy-l '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[(3'"-(methyl)butyl)-(4"-

(amino)phenylsulfonyDlamino-pentaneamide A solution of 4 (40 mgs, 0.066 mmol) in tetrahydrofuran

(5 mL) was stirred under Argon for 15 minutes. Palladium (10%) on activated carbon (6 mg) was added and the mixture was stirred for 15 minutes under Argon. The reaction vessel was then placed under a hydrogen filled balloon and stirred for 16 h. The mixture was diluted with tetrahydrofuran and filtered through celite. The filtrate was

concentated and recrystallized (ethyl acetate) to yield 5 (33 mg, 0.057 mmol) as a white solid m.p. 205-207°C.

Analysis calculated for C32H41N3O5S 0.10 CHCI3 C, 65.16; H, 7.00; N, 7.10; MWT, 591.70;

Found: C, 65.25; H, 6.98; N, 7.02.

EXAMPLE 2

Preparation of N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)-phenylmethyl- 4(S)-hydroxy-5-[((2 ^* ",2-'"dimethyl)propyl)-(4"-(methyl)- phenylsulfonvDIamino-pentaneamide

Step l : Preparation of N-(2'(R)-hydroxy-l"(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-((2",2''-dimethyl)propyl)- amino-pentaneamide-N.O-dimethyl acetal

isopropanol

A mixture of epoxide 1 (1.5 g, 3.97 mmol) and neopentyl amine (0.42 g, 0.56 mL, 4.77 mmol) were refluxed in isopropanol (8

mL) for 16 h. The solution was concentrated to yield crude product 6 (1.93 g, 4.15 mmol) as a white solid. This material was used without further purification.

Step 2: Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-((2",2''-dimethyl)- propyPamino-pentaneamide

anhyd. HCl isopropanol

A solution of 6 (1.84 g, 3.97 mmol) in isopropanol (5 mL) was cooled to 0°C and saturated with anhydrous HCl. The solution was stirred and allowed to gradually warm to room temperature over 3 h, at which time it was concentrated. The resulting white hydrochloride salt was then diluted with ethyl acetate, and washed with saturated NaHCθ3 (2 x 30 mL). The organic layer was dried (MgS04) and concentrated to give a crude white solid which was purified by flash chromatography

(5% methanol/ethyl acetate) to yield 7 (1.45 g, 3.41 mmol, 86%) as a white solid.

Step 3: Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2'",2"'-dimethyl)propyl)- (4"-(methyl)-phenylsulfonyl)1amino-pentaneamide

To a stirred solution of 8 (100 mg, 0.236 mmol) in dichloromethane (2 mL) at 0°C was added triethylamine (0.039 mL, 0.283 mmol) and p-toluenesulfonyl chloride (45 mg, 0.236 mmol). The solution was warmed to room temperature and stirred for 16 h at which time it was diluted with dichloromethane and washed with saturated NaHC03 (2 x 25 mL). The organic layer was dried (MgS04), concentrated, and purified by flash chromatography (30% ethyl acetate/

hexanes) to give the title compound (123 mg, 0.213 mmol, 90%) as a white solid m.p. 70-73°C.

Analysis calculated for C33H42N2O5S C, 68.48; H, 7.31 ; N, 4.84; MWT, 578.78;

Found: C, 68.29; H, 7.50; N, 4.86.

EXAMPLE 3

Using the same methods as described in Example 1, Steps

1-3, the sulfonamides listed in Table 1 were prepared.

EXAMPLE 4

Preparation of N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)-phenylmethyl- 4(S)-hydroxy-5-[((2"(RS)-tetrahydrofuryl)methyl)-(phenylmeth yl)- aminocarbonyllamino-pentaneamide

Step l : Preparation of N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2"(RS)-tetrahydro- futyl)methyl)-(phenylmethyl)aminocarbonyl]amj_αo- pentaneamide N'.O'-dimethylacetal

The protected indan epoxide 1 (100 mg, 0.265 mmol.) was refluxed with (RS)-tetrahydrofurfurylamine (500 microliters) in isopropanol overnight. After 16 h the reaction was evaporated and the residual oil azeotroped with toluene (3X 10 mL). The yellow oil that remained was dissolved in methylenechloride followed by the addition of benzyl isocyanate (1.1 eq.). The reaction was stirred at room temperature overnight. After evaporation of the crude reaction left an

oil that was dissolved in chloroform and placed on a 1 mm. prep TLC plate. The plated was developed in a 95/5 chloroform/methanol solvent system. The major product band was collected, extracted from the silica with 90/10 chloroform/methanol, filtered and evaporated to give a solid.

Step 2: Preparation of N-(2'(R)-hydroxy- 1 '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2"(R,S)-tetrahydrofuryl)- methyl)-(phenylmethyl)aminocarbonyllamino-pentaneamide

10

1 1

The purified product from above was dissolved in 15 mL of isopropanol and treated with 12M HCl (3 mL)and stirred until HPLC indicated no starting material. Yield 42.8 mg (m.p. 144-147°C)

Analysis calculated for C34H41 N3O5 0.15 CHCI3

C, 69.56; H, 7.03; N, 7.13; MWT, 557.67; Found: C, 69.47; H, 7.09; N, 7.29.

EXAMPLE 5

Preparation of N-(2'(R)-hydroxy- 1 '(S)-indanyl)-2(R)-phenylmethyl- 4(S)-hydroxy-5-[((2'"-methyl)propyl)-(3"-(pyridyl)methyl)- aminocarbonyllamino-pentaneamide

Step 1 : Preparation of N-(2'(R)-hydroxy- 1 '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2"-tetrahydro- furyl)methyl)-(phenylmethyl)aminocarbonyl]amino- pentaneamide N'.O'-dimethylacetal

NH ₂ isopropanol

The protected indane epoxide 1 (100 mg, 0.265 mmol) was refluxed with isobutylamine (0.50 mL) in isopropanol overnight. The reaction was evaporated and the residue azeotroped with toluene (3X 10 mL). A portion of the yellow oil (50 mg) that remained was dissolved in methylenechloride and stored in a stoppered round bottom flask. In a seperate flask 3-aminomethylpyridine was dissolved in methylene¬ chloride and cooled to 0°C. Carbonyldiimidazole (CDI) was added and

the reaction stirred for 30 min. The CDI reaction mixture was then added to the flask containing the indanamine 12. The coupling reaction was allowed to proceed at room temperature overnight. After evaporation the crude reaction product was dissolved in chloroform and placed on a 1 mm. prep TLC plate. The plated was developed in a 90/10 chloroform/methanol solvent system. The major product band was collected, extracted from the silica with 85/15 chloroform/- methanol, filtered and evaporated to give a solid which was used directly in the next step.

Step 2: Preparation of N-(2 ^, (R)-hydroxy-l '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2'"-methyl)propyl)-(3"- (pyridyDmethyDaminocarbonyllamino-pentaneamide

The purified product from above 13 was dissolved in 15 mL of isopropanol to which 12M HCl (3 mL) was added. The reaction was monitored by HPLC. The desired product 14 was purified by prep TLC as described above. Yield 32.2 mg (m. p. 75-79°C).

Analysis calculated for C32H40N4O4 0.60 CHCI3

C, 63.53; H, 6.64; N, 9.09; MWT, 616.327; Found: C, 63.76; H, 6.68; N, 9.09.

EXAMPLE 6

Using the same methods as described in Examples 2 and 3, the ureas listed in Table 4 were prepared.

EXAMPLE 7

Preparation of N-(2'(R)-hydroxy-r(S)-indanyl)-2(R)-phenylmethyl- 4(S)-hydroxy-5-[((2'"-methyl)propyl)-benzyloxycarbonyl]amino - pentaneamide

Step l: Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2'"-methyl)propyl)- benzyloxycarbonyl]amino-pentaneamide N',0'- dimethylacetal

A mixture of 12 (100 mg, 0.232 mmol) prepared as described above, diisopropylethylamine (37.1 mg, 0.050 mL, 0.287 mmol) and benzyl chloroformate (39.4 mg, 0.033 mL, 0.231 mmol) were stirred in methylene chloride (6 mL) at room temperature for 16 h. The solution was concentrated and purified by preparative thin layer chromatography (5% methanol/chloroform). The product was used directly in the next step.

Step 2: Preparation of N-(2'(R)-hydroxy- 1 '(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2'"-methyl)propyl)- benzyloxycarbonyllamino-pentaneamide

The acetonide 15 and camphorsulfonic acid were stirred in 10:1 methanol/water (13 mL) for 16 h. The solution was concentrated, diluted with ethyl acetate (15 mL), and washed with saturated NaHCθ3 (2 x 5 mL) and saturated NaCl (2 x 5 mL). The organic layer was dried (Na2Sθ4), concentrated, and purified by preparative thin layer chromatography (10% methanol/chloroform) to give 77 mg of a white solid (m.p. 119-122°C).

Analysis calculated for C33H40N2O5 C, 72.77; H, 7.40; N, 5.14; MWT, 544.69;

Found: C, 72.58; H, 7.42; N, 5.19.

EXAMPLE 8

Preparation of N-(2'(R)-hydroxy- 1 '(S)-indanyl)-2(R)-phenylmethyl- 4(S)-hydroxy-5-[((2'"-methyl)butyl)-3'(S)-(tetrahydrofuranyl oxy)- carbonyllamino-pentaneamide

17 O

A solution of amine 3 prepared as described above, (30 mg, 0.067 mmol) in dichloromethane (3 mL) at 0°C was treated with triethylamine (17 mg, 0.021 mL, 0.168 mmol) and then 17 (16 mg, 0.067 mL). The resulting solution was allowed to warm to room temperature and stirred for 16 h, at which time it was poured into saturated NaHC03 (20 mL). The biphasic system was then washed with dichloromethane (25 mL) and the organic extracts were dried (MgSθ4), and concentrated under reduced pressure to give a crude white solid. The solid was purified by recrystallization (CH2Cl2/hexanes) to give pure 18 (16 mg, 0.030 mmol) as white needles m.p. 153-155°C.

EXAMPLE 9

Using the same methods as described in Examples 6 and 7, the carbamates listed in Table 2 were prepared.

EXAMPLE 10

Preparation of N-(2'(R)-hydroxy-l '(S)-indanyl)-2(R)-phenylmethyl- 4(S)-hydroxy-5-[((2"'-methyl)propyl)-(cyclopentylmethyl)- carbonyllamino-pentaneamide

Step 1: Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2'"-methyl)propyl)- (cyclopentylmethyl)carbonyl]amino-pentaneamide NO' dimethylacetal

A mixture of 1 prepared as described above (100 mg, 0.232 mmol), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide»HCl (49 mg, 0.255 mmol), 1 -hydroxybenzotriazole hydrate (35 mg, 0.255 mmol), triethylamine (73 mg, 0.100 mL, 0.717 mmol), and cyclopentylacetic acid (102 mg, 0.100 mL, 0.797 mmol) were stirred in methylene chloride (8 mL) at room temperature. After 24 hours the solution was diluted with methylene chloride and washed with 10% citric acid (3 x 25 mL), saturated NaHCθ3 (3 x 25 mL), and saturated NaCl (1 x 25 mL). The organic layer was dried (Na2S04) and

concentrated to an oil which was purified by preparative thin layer chromatography (5% methanol/chloroform).

Step 2: Preparation of N-(2'(R)-hydroxy-l'(S)-indanyl)-2(R)- phenylmethyl-4(S)-hydroxy-5-[((2"'-methyl)propyl)- (cyclopentylmethyl)carbonyllamino-pentaneamide

The acetonide 19 prepared from above was dissolved in isopropanol (10 mL) and treated with 12M HCl (3 mL). After 4 hours the solution was neutralized with 4N NaOH, concentated, and the product was extracted into ethyl acetate. The organic layer was dried (Na2Sθ4) and concentrated. The crude product was purified by preparative thin layer chromatography (8% methanol/chloroform) to yield 20 (115 mg, 0.220 mmol) as a white solid (m.p.l41-145°C).

Analysis calculated for C32H44N2O4O.OI CHCI3

C, 73.66; H, 8.50; N, 5.37; MWT, 521.88; Found: C, 73.29; H, 8.42; N, 5.19.

EXAMPLE 11

Using the same methods as described in Example 9 the amides listed in Table 4 were prepared.

EXAMPLE 12

Preparation of Amide 21

A solution of (-)-cis-l-aminoindan-2-ol (884 g, 5.93 mol) in 17.8 L of dry THF (KF = 55 mg/mL) (KF stands for Karl Fisher titration for water) and triethylamine (868 mL, 6.22 mol) in a 50 L round bottom flask equipped with a thermocouple probe, mechanical stirrer, and a nitrogen inlet adapter and bubbler, was cooled to 15°C. Then, 3-phenylpropionyl chloride (1000 g, 5.93 mol) was added over 75 minutes, while the internal temperature between 14-24°C with an ice-water cooling batch. After addition, the mixture was aged at 18 to 20°C for 30 minutes and checked by HPLC analysis for the disappearance of (-)-cis-l -aminoindan-2-ol. Progress of the reaction is monitored by high performance liquid chromatography (HPLC) analysis: 25 cm Dupont C8-RX column, 60:40 acetonitrile/10 mM (KH2PO4/K2HPO4), 1.0 mL/min., injection volume = 20 mL, detection = 200 nm, sample preparation = 500 X dilution. Approximate retention times:

retention time (min.) identity

6.3 cis-aminoindanol

The reaction was treated with pyridinium p-toluene- sulfonate (241 g, 0.96 mol, 0.16 equiv.) and stirred for 10 minutes (the pH of the mixture after diluting 1 mL sample with an equal volume of water is between 4.3-4.6). Then, 2-methoxypropene (1.27 L, 13.24 mol, 2.2 equiv.) was added and reaction was heated to 38-40°C for 2 h. The reaction mixture was cooled to 20°C and partitioned with ethyl acetate (12 L) and 5% aqueous NaHC03 (10 L). The mixture was agitated and the layers were separated. The ethyl acetate extract was washed with 5% aqueous NaHCθ3 (10 L) and water (4 L). The ethyl acetate extract was dried by atmospheric distillation and solvent switched to cyclohexane (total volume of ~30 L). At the end of the distillation and concentration (20 volume % of ethyl acetate extraction volume), the hot cyclohexane solution was allowed to slowly cool to 25°C to crystallize the product. The resulting slurry was further cooled to 10°C and aged for 1 h. The product was isolated by filtration and the wet cake was washed with cold (10°C) cyclohexane (2 X 800 mL). The washed cake was dried under vacuum (26" of Hg) at 40°C to afford 1.65 kg of acetonide 21 (86.4%, 98 area % by HPLC). H NMR (300.13 MHz, CDC13, major rotamer) δ 7.36-7.14 (m, 9 H), 5.03 (d, J=4.4, 1 H), 4.66 (m, 1 H), 3.15 (m, 2 H), 3.06 (br s, 2 H), 2.97 (m, 2 H), 1.62 (s, 3 H), 1.37 (s, 3 H); 13c NMR (75.5 MHz, CDCI3, major rotamer) δ _c 168.8, 140.9, 140.8, 140.6, 128.6, 128.5, 128.4, 127.1 , 126.3, 125.8, 124.1 , 96.5, 78.6, 65.9, 38.4, 36.2, 31.9, 26.5, 24.1. Anal. Calcd for C21H23NO2:

C, 78.47; H, 7.21 ; N, 4.36. Found: C, 78.65; H, 7.24; N, 4.40.

EX AMPLE 13

Preparation of Epoxide 1 Tosylate Method

A solution of acetonide 21 (1000 g, 3.1 1 mol) and 2(S)- glycidyl tosylate 22 (853 g, 3.74 mol, 1.2 equiv.) in 15.6 L of THF (KF = 22 mg/mL) in a 50 L 4-neck round bottom flask, equipped with a thermocouple, mechanical stirrer, addition funnel and nitrogen inlet adapter was degassed 3 times via vacuum-nitrogen purge and cooled to -56°C. Then, lithium hexamethyldisilazide (LiN[(CH3)3Si]2)(2.6 L, 1.38 M, 1.15 equiv.) was added over 2 h, while keeping the internal temperature between -50 to -45 °C. The reaction mixture was stirred at -45 to -40°C for 1 h and then allowed to warm to -25°C over 1 h. The mixture is stirred between -25 to -22°C for 4 h (or until the starting acetonide is 3.0 area %).

Progress of the reaction is monitored by HPLC analysis: 25 cm X 4.6 nm Zorbax Silica column, 20% ethyl acetate in hexane, 2.0 mL/min, injection volume = 20 mL, detection = 254 nm, sample preparation = 100 X dilution. Approximate retention times:

The reaction mixture was quenched with DI water (6.7 L) at -15°C and partitioned with ethyl acetate (10 L). The mixture was agitated and the layers were separated. The ethyl acetate extract was washed with a mixture of 1 % aqueous NaHCθ3 (5 L) and saturated NaCl (0.5 L). The ethyl acetate extract (28.3 L) was concentrated by vacuum distillation (28" of Hg) and additional ethyl acetate was added to complete the solvent switch to ethyl acetate (final volume = 11.7 L). The ethyl acetate concentrate was further solvent switched to MeOH to crystallize the product and concentrated to a final volume of 3.2 L. The residual ethyl acetate solvent was removed by charging 10 L of methanol and collecting 10 L of distillate. The resulting slurry was stirred at 22°C for 1 h, then cooled to 5°C and aged for 0.5 h. The product was isolated by filtration and the wet cake was washed with cold methanol (2 X 250 mL). The washed cake was dried under vacuum (26" of Hg) at 25°C to afford 727 g of epoxide 1 (61.2%, 98.7 area % of the major epoxide by HPLC). ¹ C NMR (300 MHz, CDCI3) δ 171.1, 140.6, 140.5, 139.6, 129.6, 128.8, 128.2, 127.2, 126.8, 125.6, 124.1, 96.8, 79.2, 65.8, 50.0, 48.0, 44.8, 39.2, 37.4, 36.2, 26.6, 24.1.

EXAMPLE 14

Assay for Inhibition of Microbial Expressed HIV Protease

Inhibition studies of the reaction of the protease expressed in Escherichia coli with a peptide substrate [Val-Ser-Gln-Asn- (betanaphthyl)Ala-Pro-Ile-Val, 0.5 mg/mL at the time the reaction is initiated] were in 50 mM Na acetate, pH 5.5, at 30°C for 1 hour. Various concentrations of inhibitor in 1.0 ul DMSO were added to 25 ul of the peptide solution in water. The reaction is initiated by the addition of 15 ul of 0.33 nM protease (0.1 1 ng) in a solution of 0.133 M Na acetate pH 5.5 and 0.26% bovine serum albumin. The reaction was quenched with 160 ul of 5% phosphoric acid. Products of the reaction were separated by HPLC (VYDAC wide pore 5 cm C- 18 reverse phase, acetonitrile gradient, 0.1 % phosphoric acid). The extent of inhibition of the reaction was determined from the peak heights of the products. HPLC of the products, independently synthesized, proved quantitation

standards and confirmation of the product composition. Compound A showed IC50 value of about 8-9 nM.

While the foregoing specification teaches the principles of the present invention, with examples provided for the puφose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, or modifications, as come within the scope of the following claims and its equivalents.