IE912381A1

IE912381A1 - Inhibitors of aspartic proteases

Info

Publication number: IE912381A1
Application number: IE238191A
Authority: IE
Original assignee: Smithkline Beecham Corp
Priority date: 1990-07-06
Filing date: 1991-07-08
Publication date: 1992-01-15
Also published as: WO1992000956A1; AU8231391A; EP0538383A1; EP0538383A4; MX9100121A; ZA915270B; JPH05508851A; PT98229A

Abstract

Compounds useful as inhibitors of retroviral proteases characterized by the structure (I), wherein the X<1> and X<2> groups may consist of 0 to 2 alpha -amino acid groups terminally substituted by hydrogen or one of a number of end groups, and the R<1> and R<2> group can be selected from a wide variety of hydrocarbon radicals. Compounds which exhibit a protease activity inhibition constant Ki of less than 50 mu M are desired.

Description

BACKGROUND OF THE INVENTION This invention relates to compounds which are inhibitors of aspartic proteases, particularly of retroviruses.

Retroviruses, that is, viruses within the family of Retroviridae, are a class of viruses 10 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 (MLV), mouse mammary tumor virus (MMTV), feline leukemia virus (FeLV), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (MPMV), 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 satisfactory method for treating this type of infection has been developed. Among these viruses, the HTLV and HIV have been especially well characterized.

Critical to the replication of retroviruses is the production of functional viral proteins. Protein synthesis is accomplished by translation of the open reading frames into polyprotein constructs, corresponding to the gag, poi and env reading frames. The gag and poi precursor proteins, are processed by a viral protease into the functional proteins. The SB 14516 -2HIV-1 protease has been classified as an aspartic acid protease (Meek et al., Proc. Natl. Acad. Sci. USA. 88. 1841 (1989)). The proteolytic activity provided by the viral protease in processing the polyproteins cannot be provided by the host and is essential to the life cycle of the retrovirus. In fact, it has been demonstrated that retroviruses which lack the protease or contain a mutated form of it, lack infectivity. See Katoh et al., Virology. 145, 280-92(1985), Crawford, et al., J. Virol.. 53, 899-907(1985), Debouck, et al., Proc. Natl. Acad. Sci. USA. 84, 8903-6(1987). Inhibition of retroviral protease, therefore, presents a method of therapy for retroviral disease.

Methods to express retroviral proteases in E. coli have been disclosed (Debouck, et al., Proc, Natl, Acad. Sci. USA. 8903-06(1987) and Tomasselli et al., Biochemistry. 29. 264-9 (1990) and refs, therein).

Inhibitors of recombinant HIV protease have been reported (Dreyer et al., Proc, Natl. Acad. Sci. USA. 86, 9752-56 (1989); Tomasselli et al. supra: Roberts et al., Science. 248, 358 (1990); Rich et al., J. Med, Chem.. 33. 1285-88 (1990); Sigal et al., Eur. Pat.

Appl. No. 337 714; Dreyer et al. Eur. Pat. Appl. No. 352 000). Moreover, certain of these inhibitors have been shown to be potent inhibitors of viral proteolytic processing in cultures of HIV-l infected T-lymphocytes (Meek et al., Nature (London). 343. 90 (1990) and by Roberts et al. supra).

The limitations of current strategies for aspartic protease inhibition include (1) oral bioavailability; (2) plasma clearance lifetimes (e.g., through biliary excretion or degradation); (3) selectivity of inhibition; and (4) in the case of intracellular targets, membrane permeability or cellular uptake. The present invention relates to a new inhibitors of retroviral and aspartic proteases. Unlike previously described inhibitors , the compounds of this invention are not analogues of peptide substrates possessing a scissile dipeptide mimetic. They also deviate substantially from peptide substrate-like structure in that they do not possess a conventional amino-to-carboxyl terminus orientation.

SUMMARY OF THE INVENTION This invention comprises compounds having the structures particularly pointed out in the claims and described hereinafter which bind to retroviral proteases. These compounds are inhibitors of viral protease and are useful for treating disease related to infection by viruses.

This invention is also a pharmaceutical composition, which comprises an aforementioned compound and a pharmaceutically acceptable carrier therefor.

This invention further constitutes a method for treating viral diseases, which comprises administering to a mammal in need thereof an effective amount of an aforementioned inhibitor compound. -3SB 14516 DETAILED DESCRIPTION OF THE INVENTION The compounds of this invention have the structure: R1 R2 X1HN NHX2 HO OH wherein X’ and X2 are the same or different and are A-(B)n- where n = 0-2; and B is, independently, an α-amino acid chosen from the group: Ala, Asn, Cys, Trp, Gly, Gin, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr, Val, His, or trifluoroalanine, wherein the amino group of B is bonded to A or ±e carboxy group of the adjacent residue B, whichever is appropriate, and the carboxy group of B is bonded to the amino group of the adjacent residue B or the structure, whichever is appropriate; and A is covalently attached to the amine group of the adjacent residue B or to the amine group of the structure if n=0, and is: 1) trityl, 2) hydrogen, 3) Ci-C6 alkyl, 4) R3-CO- wherein R3 is: a) hydrogen, b) Cj-C^ alkyl, unsubstituted or substituted with one or more hydroxyl groups, chlorine atoms, or fluorine atoms, c) phenyl or naphthyl unsubstituted or substituted with one or more substituents R4, wherein R4 is: i) C1-C4 alkyl, ii) halogen, where halogen is F, Cl, Br or I, iii) hydroxyl, iv) nitro, v) C1-C3 alkoxy, or vi) -CO-N(rO)2 wherein R’0 is, independently, H or C1-C4 alkyl; or d) a 5-7 member heterocycle such as pyridyl, furyl, or benzisoxazolyl; ) phthaloyl wherein the aromatic ring is unsubstituted or substituted with one or more substituents R4; 6) R3(RbR7c)m-CO- wherein m = 1-3 and R3, Rb, and R2 are independently: a) hydrogen, b) chlorine or fluorine, SB 14516 -4c) C4-C3 alkyl unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, d) hydroxyl, e) phenyl or naphthyl unsubstituted or substituted with one or more substituents R4, f) Cj - C3 alkoxy, g) a 5-7 member heterocycle, or h) r5, r6; and r7 may be independently joined to form a monocyclic, bicyclic, or tricycle ring system each ring of which is C3-C6 cycloalkyl; 7) R5(R6R7Qm \y_ wherein m = 1-3 and W is OCO or SO2 and R^, r6, and R7 are as defined above, except R3, R^, and R7 are not chlorine, fluorine or hydroxyl if they are adjacent to W; 8) R8-W- wherein R^ is a 5-7 member heterocycle such as pyridyl, furyl, or benzisoxazolyl; 9) R9-W- wherein R9 is phenyl or naphthyl unsubstituted or substituted with one or more substituents R4; ) R5-(R6R7C)m-P(O)(ORl b- wherein Rl 1 is Cj - C4 alkyl or phenyl; 11) R8-P(O)(ORn)-;or 12) R9-P(O)(ORH)-; Rl and R2 are the same or different and are: 1) -CH2RI2 wherein R^2 is a) NH-A wherein A is defined as above; b) R5-(R6R7Qm-; c) R5-(R6R7Qm γ- wherein V is O or NH, except R^, R^ and R7 are not hydroxyl, chlorine or fluorine if they are adjacent to V, d) R5-(R6R7Qm.s(O)n- wherein m = 1-3 and n = 0-2 and R^, r6 and R7 are as defined above except R3, r6, and R7 are not hydroxyl, chlorine or fluorine if they are adjacent to sulfur, e) R8-S(O)n-, f) R9-S(O)n-, g) N(r10)2, h) NR15r16 wherein Rl5 and Rl 6 are joined to form a 4-6 membered saturated nitrogens heterocycle including: i) azetidinyl, ii) pyrrolidinyl, iii) piperidinyl, or iv) morpholinyl, SB 14516 i) R’7oCH2O wherein r17 jS: i) C’-C^ alkyl, ii) R^, or iii) CH2Ar wherein Ar is phenyl, naphthyl or a 5-7 membered heterocycle, j) R17OCH2CH2OCH2, k) N-imidazolyl where the imidazole ring is unsubstituted or substituted by a substituent R^, l) N-benzimidazolyl where the fused benzene ring is unsubstituted or substituted by one or more substituents R^; m) C2-C6 alkynyl, optionally substituted with one or more groups R^; or n) C2-C6 alkenyl, optionally substituted with one or more groups R^; 2) hydrogen, 3) Cj-C^ alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C3-C7 cycloalkyl; and pharmaceutically acceptable salts thereof.

Ordinarily, X’ = and R’ = R^ to produce truly symmetric compounds; however, the procedures allow for preparation of pseudosymmetric compounds as well where X’ is different from and R’ is different from R^.

Peptide compounds of the foregoing description are preferred which are C2 symmetric wherein Χ’=χ2, and R’=r2.

C2 symmetric peptide compounds wherein R’ and R^ are CpC^ alkyl or aralkyl and χΐ and X- are single amino acids or mono- or dipeptides; these groups may be terminally substituted by common acyl groups or blocking groups commonly used in peptide synthesis, such as t-Boc or Cbz, are also preferred.

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 parent drug.

As used herein except where noted, the term alkyl refers to a straight or branched chain alkyl radical of the indicated number of carbon atoms including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl, and the like; alkoxy represents an alkyl group of the indicated number of carbon atoms attached through a bridging oxygen atom; cycloalkyl is intended to include saturated ring groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl; alkenyl is meant to include either straight or branched hydrocarbon chains containing one or more carbonIE 912381 SB 14516 -6carbon double bonds which may occur at any stable point along the chain, such as ethenyl, propenyl, butenyl, pentenyl, 2-methyl-propenyl, and the like; alkynyl refers to either a straight or branched hydrocarbon chain of the indicated number of carbon atoms which contains a carbon-carbon triple bond which may occur at any stable point along the chain, such as ethynyl, 2-propynyl, 2-butynyl, 4-pentynyl, 2-methyl-3-propynyl, and the like.

As used herein except where noted, the term heterocycle represents a stable 5- to 7-membered mono- or bicyclic heterocyclic ring, which is either 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 the nitrogen heteroatom may optionally be quaternized, 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-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 215 oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, furyl, tetrahydrofuryl,tetrahydropyranyl, thienyl, thiamorpholinyl sulfoxide, thiamorpholinyl sufone, and oxadiazolyl.

When any variable (e.g., A, B, R1, R3,..., R17, heterocycle, substituted phenyl, 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. By convention used herein, a geminal diol, for example when R6 and R7 are simultaneously hydroxyl, is meant to be equivalent with a carbon-oxygen double bond.

Other abbreviations and symbols commonly used in the art used herein to describe the peptides include the following: Amino acid three letter code Amino acid three letter code Alanine Ala Leucine Leu Arginine Arg Lysine Lys Asparagine Asn Methionine Met Aspartic Acid Asp Phenylalanine Phe Cysteine Cys Proline Pro Glutamine Gin Serine Ser Glutamic Acid Glu Threonine Thr Glycine Gly Tryptophan Trp Histidine His Tyrosine Tyr Isoleucine Ile Valine Val Asparagine or Aspartic Acid Asx Glutamine or Glutamic Acid Glx SB 14516 -7In accordance with conventional representation, the amino terminus is on the left and the carboxy terminus is on the right. All chiral amino acids (AA) can occur as racemates, racemic mixtures, or individual enantiomers or diastereomers, with all isomeric forms being included in the present invention. β-Ala refers to 3-amino propanoic acid. Boc refers to the t-butyloxycarbonyl radical, Cbz or Z refers to the carbobenzyloxy radical, i-Bu refers to isobutyl, Ac refers to the acetyl, Ph refers to phenyl, DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, HOBT refers to 1hydroxybenzotriazole, NMM is N-methylmorpholine, DTT is dithiothreitol, 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. HF refers to hydrofluoric acid and TFA refers to trifluoroacetic acid.

The peptide moieties denoted by and X2 are generally dipeptides or smaller. However, longer peptides which encompass the residues defined herein are also believed to be active and are considered within the scope of this invention.

The selection of residues or end groups may be used to confer favorable biochemical or physico-chemical properties to the compound. The use of hydrophilic residues may be used to confer desirable solubility properties or D-amino acids at the carboxy terminus may be used to confer resistance to exopeptidases.

Synthesis of compounds of the structure is achieved by aldol condensation of an amino-protected alpha-amino aldehyde, P2NHCH(Rl)CHO, with an amino-protected alpha-amino ketone, P3NHCH(R2)COCH3, wherein P2 and P3 are amino protecting groups,using lithium diisopropylamine (LDA) at low temperature as detailed in the Examples. The resulting 3-hydroxyketone is reduced to the 1,3-diol by use of a hydride reducing agent such as Me4NHB(OAc)3 in CH3CN/HOAC (Evans and Chapman, Tetrahedron Let.. 5939-42 (1986)) or NaBH4; the former procedure having the advantage that anti 1,3-diols are produced with high diastereoselectivity. The amino-protected alphaamino ketone, P3NHCH(R2)COCH3, is prepared by addition of MeLi or MeMgBr to the N-methyl, N-methoxy amides P3NHCH(R2)CONMe(OMe) as described by Fehrentz and Castro, Synthesis 676 (1983), and Nahm and Weinreb, Tetrahedron Let.. 3815-18 (1981).

This route is also versatile in that it allows access to all stereoisomers of compounds of the structure and to unsymmetrical compounds, in which Rl, R2 are different and XL X2 are different.

The compounds of this invention are prepared by the solid phase technique of Merrifield (J. Am. Chem, Soc.. 85, 2149 (1964)), or preferably by solution methods known to the art. A combination of solid phase and solution synthesis may be used, as in a convergent synthesis in which di-, tri-, or tetra-peptide fragments may be prepared by solid phase synthesis and either coupled or further modified by solution synthesis. The methods SB 14516 -8of peptide synthesis generally set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Il (1984) or M. Bodansky, Y.A. Klauser and M. A. Ondetti, Peptide Synthesis. John Wiley & Sons, Inc., New York, N.Y. (1976), or The Peptides Gross and Meienhoffer, eds.; Acad. Press, 1979, Vols I5 III, may be used to produce the peptides of this invention and are incorporated herein by reference.

Each amino acid or peptide is suitably protected as known in the peptide art. For example, the Boc- or carbobenzyloxy (Cbz) group is preferred for protection of the amino group, especially at the a position. A benzyl group or suitable substituted benzyl group is used to protect the mercapto group of cysteine, or other thiol containing amino acids; or the hydroxyl of serine or threonine. The tosyl or nitro group may be used for protection of the guanidine of Arg or the imidazole of His, and a suitably substituted carbobenzyloxy group or benzyl group may be used for the hydroxyl group of Tyr, Ser or Thr, or the ε-amino group of lysine. Suitable substitution of the carbobenzyloxy or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group. Cysteine and other sulfur-containing amino acids may also be protected by formation of a disulfide with a thioalkyl or thioaryl group. Except for the Boc group, the protective groups are, most conveniently, those which are not removed by mild acid treatment. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment as known in the art.

If solid phase methods are used, the peptide is built up sequentially starting from the carboxy terminus and working toward the amino terminus of the peptide. Solid phase synthesis is begun by covalently attaching the C terminus of a protected amino acid to a suitable resin, such as a benzhydrylamine resin (BHA), methylbenzhydrylamine resin (MBHA) or chloromethyl resin (CMR), as is generally set forth in U.S. Patent No. 4,244,946. A BHA or MBHA support resin is used for the carboxy terminus of the product peptide is to be a carboxamide. A CMR support is generally used for the carboxy terminus if the produced peptide is to be a carboxyl group, although this may also be used to produce a carboxamide or ester.

Modification of the terminal amino group of the peptide is accomplished by alkylation or acetylation as is generally known in the art. These modifications may be carried out upon the amino acid prior to incorporation into the peptide, or upon the peptide after it has been synthesized and the terminal amino group liberated, but before the protecting groups have been removed.

Typically, acetylation is carried out upon the free amino group using the acyl halide, anhydride or activated ester, of the corresponding alkyl acid, in the presence of a tertiary amine. Mono-alkylation is carried out most conveniently by reductive alkylation of the SB 14516 -9amino group with an appropriate aliphatic aldehyde or ketone in the presence of a mild reducing agent, such as lithium or sodium cyanoborohydride. Dialkylation as well as quaternization may be carried by treating the amino group with an excess of an alkyl halide in the presence of a base.

Solution synthesis of peptides is accomplished using conventional methods used to form amide bonds. Typically, a protected Boc-amino acid which has a free carboxyl group is coupled to a protected amino acid which has a free amino group using a suitable carbodiimide coupling agent, such as N, Ν' dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole (HOBT) and dimethylamino pyridine (DMAP). Other methods, such as the formation of activated esters, anhydrides or acid halides, of the free carboxyl of a protected Boc-amino acid, and subsequent reaction with the free amine of a protected amino acid, optionally in the presence of a base, are also suitable. For example, a protected Boc-amino acid or peptide is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran (THF), in the presence of a base, such as N-methyl morpholine, or a trialkyl amine, with isobutyl chloroformate to form the mixed anhydride, which is subsequently reacted with the free amine of a second protected amino acid or peptide. The peptide formed by these methods may be deprotected selectively, using conventional techniques, at the amino or carboxy terminus and coupled to other peptides or amino acids using similar techniques. After the peptide has been completed, the protecting groups may be removed as hereinbefore described, such as by hydrogenation in the presence of a palladium or platinum catalyst, treatment with sodium in liquid ammonia, hydrofluoric acid, trifluoroacetic acid or alkali.

Esters are often used to protect the terminal carboxyl group of peptides in solution synthesis. They may be converted to carboxylic acids by treatment with an alkali metal hydroxide or carbonate, such as potassium hydroxide or sodium carbonate, in an aqueous alcoholic solution. The acids may be converted to other esters via an activated acyl intermediate as previously described.

The amides and substituted amides of this invention are prepared from carboxylic acids of the peptides in much the same manner. Thus, ammonia or a substituted amine may be reacted with an activated acyl intermediate of an amino-protected α-amino acid or oligopeptide to produce the amide. Use of coupling reagents, such as DCC, is convenient for forming substituted amides from the carboxylic acid itself and a suitable amine.

In addition, the methyl esters of this invention may be converted to the amides, or substituted-amides, directly by treatment with ammonia, or a substituted amine, in methanol solution. A methanol solution of the methyl ester of the peptide is saturated with ammonia and stirred in a pressurized reactor to yield the simple carboxamide of the peptides. Procedures for the determination of the inhibition constant (Ki) by Dixon analysis are SB 14516 - 10described in the art, e.g., in Dreyer, et al. Proc. Natl. Acad, Sci. U.S.A.. 86, 9752-9756 (1989). A peptidolytic assay is employed using the substrate Ac-Arg-Ala-Ser-Gln-AsnTyr-Pro-Val-Val-NH2 and recombinant HIV protease as in Strickler, et al., Proteins. 6, 134-154 (1989). The lower Ki value indicates a higher binding affinity.

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 excipient such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

A preferred composition for parenteral administration may additionally be comprised of a quantity of the compound encapsulated in a liposomal carrier. The liposome may be formed by dispersion of the compounds in an aqueous phase with phospholipids, with or without cholesterol, using a variety of techniques, including conventional handshaking, high pressure extrusion, reverse phase evaporation and microfluidization. A suitable method of making such compositions is more fully disclosed in copending Application Serial No. 06/763,484 and is incorporated herein by reference. Such a carrier may be optionally directed toward its site of action by an immunoglobulin or protein reactive with the viral particle or infected cells. The choice of such proteins would of course be dependent upon the antigenic determinants of the infecting virus. An example of such a protein is the CD-4 T-cell glycoprotein, or a derivative thereof, such as sCD-4 (soluble CD4), which is reactive with the glycoprotein coat of the human immunodeficiency virus (HIV). Such proteins are disclosed in copending Application Serial No. 07/160,463, which is incorporated herein by reference. Similar targeting proteins could be devised, by methods known to the art, for other viruses and are considered within the scope of this invention.

Alternatively, these compounds may be encapsulated, tableted or prepared in a emulsion or syrup or 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 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 glycerol monostearate or glycerol distearate, SB 14516 - 11 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 rectal administration, a pulverized powder of the compounds of this invention may be combined with excipient 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.

When the compounds of this invention 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, bucally, trans-dermally, intravenously, intramuscularly, 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 selected from the range of 0.05 to 15 mg/kg. These dosage units may be administered one to ten times daily for acute or chronic infection. Combination therapy as described in Eur. Pat. Appl. No. 337 714 at pages 42-47 are included herein.

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.

In the Examples, all temperatures are in degrees Celsius. Amino acid analyses were performed upon a Dionex Autoion 100. Analysis for peptide content is based upon Amino Acid Analysis. FAB mass spectra were performed upon a VG mass spectrometer using fast atom bombardment. NMR spectra were recorded at 250 MHz using a Bruker Am 250 spectrometer. Multiplicities indicated are: s=singlet, d-doublet, t-triplet, q-quartet, mmultiplet and br indicates a broad signal.

Purification of Recombinant HIV Protease Methods for expressing recombinant HIV protease in E.coli have bee described by Debouck, et al., Proc. Natl. Acad. Sci. USA, 84, 8903-6 (1987). The enzyme used to assay the compounds of this invention was produced in this manner and purified from the cell pellet as previously described by Stickler et al. Proteins. 6, 139-154 (1989).

SB 14516 - 12EXAMPLE 1 2.6-Di(t-butoxycarbonylamino)-3.5-dihydroxy-1.7-diphenylheptane (a.) (2S,3RS,6S)-2,6-Di-(i-butoxycarbonylamino)-3-hydroxy-l,7-diphenyl-5-heptanone To 1.19 mL of diispropylamine (8.49 mmol) in 10 mL of the THF was added 3.38 5 mL of 2.5 M n-butyllithium in hexane (8.45 mmol) at - 21 °C and the resulting mixture was stirred for 1 hour followed by cooling to - 78°C. To the solution of LDA was added 1.00 g (3.80 mmol) of (2S)-l-phenyl-2-i-butoxycarbonylamino-3-butanone in 4 mL of THF. The mixture was stirred for 15 min followed by the addition of 900 mg (3.61 mmol) of (2S)-1phenyl-2-t-butoxycarbonylamino-3-propanal in 4 mL of the THF. The resulting mixture 10 was stirred for 10 min and quenched by addition of 20 mL of IN hydrochloric acid. After separation of organic layer, the aqueous layer was extracted with ethyl acetate three times. The combined organic layer was dried (Na2SO4) and concentrated in vacuo to give 1.89 g of off-white solid. The residue was separated by flash chromatography to afford 1.21 g (65.4 %) of the hydroxyketone. NMR (250 MHz, CDC13) δ 1.35, 1.39, 1.42 (3s, 18H), 2.46 (d, 1H), 2.41 - 3.15 (m, 6H), 3.59 - 4.04 (m, 2H), 4.46 (m, 1H), 4.87 - 5.09 (m, 2H), 7.09 - 7.38 (m,10H). Mass spectrum mle 513.2 (M++H). (b.) (2S,3R,5R,6S)-2,6-Di(r-butoxycarbonylamino)-3,5-dihydroxy-1,7-diphenylheptane and (2S,2S,5S,6S)-2,6-Di(f-butoxycarbonylamino-3,5-dihydroxy-l,7-dipenylheptane (Compound 1) To 3.37 g (12.8 mmol) of tetramethylammonium triacetoxyborohydride in 20 mL of acetic acid - acetonitrile (1:1) was added 1.31 g (2.56 mmol) of the mixture of hydroxyketone prepared in (a) above in 12 mL of acetonitrile - methylene chloride - acetic acid (6:5:1) was added at 5°C and the resulting mixture was stirred for 3 hours with cooling in an ice - water bath. The reaction was quenched with 40 mL of 0.5 N potassium hydrogen tartarate. The mixture was extracted with five 20-mL portions of methylene chloride. The combined organic layer was washed with saturated sodium bicarbonate solution until neutral, dried (Na2SO4), filtered and concentrated in vacuo. The residue was separated by flash chromatography into two fractions. Dihydroxydiamine A: 434 mg (33 %), NMR (250 MHz, CDCI3) δ 1.38 (s, 18H), 1.71 (m, 2H), 2.86 (d, 4H), 3.78 (m, 4H), 4.79 (m, 2H), 7.15 (m, 10H); Mass spectrum mle 515.2 (M+ + H). Dihydroxydiamine B : 410 mg (31 %), NMR (250 MHz, CDCI3) δ 1.35, (s, 18H), 2.78(dd, 1H), 2.99 (dd, 2H), 3.88 (m,2H), 4.01 (Μ, 2H), 4.53 (m, 2H), 7.28 (m, 10H); Mass spectrum mle 515.2 (M+ + H). Also 123 mg (9.4 %) of a mixture of the above two compounds was obtained. Compound 1 of this example is a compound of the structure in the table wherein Rl and R2 are PhCH2 and X^ and X2 are Boc.

SB 14516 - 13 EXAMPLE 2 (2S. 3S, 5S. 6S)-2,6-bisi(N-benzyloxylcarbonyl)alanylaminol-3.5-dihydroxy-1.7diphenvlheptane A mixture of 20 mg (39 μιηοΐ) of the compound from Example 1 (b) in 0.2 mL of 5 trifluoroacetic acid was stirred at RT for 1 hour and evaporated to dryness in vacuo. The residue was dissolved in 2 mL of dry CH2CI2 followed by addition of 29 mg (84 pmol) of M-benyloxylcarbonylalanine fl-nitrophenyl ester and 27 pL (194 pmol) of Et3N. The resulting solution was stirred at RT overnight. The mixture separated by flash chromatography (1 g silica gel, ethyl acetate/hexane) NMR δ 1.20 (d, 6H), 1.57(m, 2H), 1.78(br s, 2H), 2.85(m, 4H), 3.82(m, 2H), 4.02(q, 2H), 4.1 l(q, 2H), 5.05(s, 4H), .3l(d, 2H), 6.48((d, 2H), 7.20(m,10H), 7.33(s, 10H). The product was a compound of the structure in the table wherein R’ and R^ are PhCH2 and X’ and X^ are ZAla.

EXAMPLE 3 (2S, 3S, 5S, 6S)-2,6-bis-iN-ri-butoxycarbonyl)alanylaminol-3,5-dihydroxy-l,7diphenvlheptane A mixture of 15 mg (29 pmol) of the compound from Example 1(b) in 0.2 mL of trifluoroacetic acid was stirred at RT for 1 hour and evaporated to dryness in vacuo. The residue was dissolved in 2 mL of dry methylene chloride followed by addition of 18 mg (63 pmol) of N-t-butoxylcarbonylalanine N-hydroxysuccinimide ester and 20 pL (143 pmol) of triethylamine. The resulting mixture was stirred at RT overnight and then separated by flash chromatography (1 g silica gel, E/H = 3) to afford 14 mg (73 %) of desired product as white solid. NMR (250 MHz, CDC13) δ 1.20(d, 6H), 1.44(s,18H), 1.61(r, 2H), 2.00(s, 2H), 2.87(m, 4H), 3.85(s, 2H), 4.06(m, 4H), 5.03(d,2H), 6.56(d,2H), 7.20(m, 4H)„ The product was a structure of the table wherein R’ and R^ are PhCH2 and x’ and X" are BocAla.

ENZYME INHIBITION Inhibition of HIV protease activity The inhibition assay has been previously described in Dreyer et al. Proc. Natl.

Acad. Sci. USA. 86. 9752-9756 (1989) and Moore et al. Bioch. Bioph. Res. Com.. 159. 420 (1989). A typical assay contained 10 mL MENDT buffer (50 mM Mes (pH 6.0; 2-(Nmorpholino) ethanesulfonic acid), 1 mM EDTA, ImM dithiothreitol, 200 mM NaCl, 0.1% Triton X-100); 2, 3, or 6 mM N-acetyl-L-arginyl-L-alanyl-L-seryl-L-glutaminyl-Lasparaginyl-L-tyrosyl-L-proIyl-L-valyl-L-valinamide (Ac-Arg-Ala-Ser-Gln-Asn-Tyr-ProVal-Val-NH2; Km = 7 mM); and micromolar and submicromolar concentrations of SB 14516 - 14synthetic compounds. Following incubation at 37°C for several minutes, the reaction was initiated with 0.001-0. lOmg purified 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/H2O - .1% TFA 915 min.), 20% acetonitrile/H2O - .1% TFA (5 min) at 1.5 mL/min, detection at 220 nm. The elution positions of Ac-Arg-Ala-Ser-GlnAsn-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.

It is desirable for the compounds of this invention to have Ki values less than 50 μΜ, preferably less than 10 μΜ and more preferably less than ΙμΜ.

Following the procedures set forth herein and the teachings of the foregoing examples the compounds set forth in the following Table can be prepared having the structure and the substituent groups as designated therein.

TABLE OF COMPOUNDS R1 R2 X1 HN NHX2 HO OH No, X1 R2 E2 X2 101 Cbz-Ala i-Bu i-Bu Cbz-Ala 102 AlaAla i-Bu i-Bu AlaAla 103 CbzVal i-Bu i-Bu CbzVal 104 β-Ala i-Bu i-Bu β-Ala 105 β-AlaVal I-Bu I-Bu β-AlaVal 106 H i-Bu i-Bu H 107 Cbz-AlaAla i-Bu i-Bu Cbz-AlaAla 108 Boc-Ala i-Bu i-Bu Boc-Ala 109 Ac-AlaAsn i-Bu i-Bu Ac-AlaAsn 110 Ac-GlnAsn i-Bu i-Bu Ac-GlnAsn 111 Cbz-PheAla i-Bu i-Bu Cbz-PheAla SB 14516 trifluoroAlaAla i-Bu i-Bu trifluoroAlaAla Cbz-trifluoroAlaAla i-Bu i-Bu Cbz-trifluoroAlaAla trifluoroAla i-Bu i-Bu trifluoroAla Cbz-trifluoroAla i-Bu i-Bu Cbz-trifluoroAla Ph(CH2)2CO i-Bu i-Bu Ph(CH2)2CO Boc i-Bu i-Bu Boc Boc PhCH2 PhCH2 Boc Cbz i-Bu i-Bu Cbz Ac i-Bu i-Bu Ac PhSO2 i-Bu i-Bu PhSO2 HCO i-Bu i-Bu HCO Propionyl i-Bu i-Bu Propionyl i-Butyryl i-Bu i-Bu i-Butyryl Ph(CH2)2CO i-Bu i-Bu Ph(CH2)2CO PhSO2Val i-Bu i-Bu PhSO2Val Phenyllactoyl i-Bu i-Bu Phenyllactoyl Phenyllactoyl-Val i-Bu i-Bu Phenyllactoyl-Val Cbz-Ala PhCH2 PhCH2 Cbz-Ala Boc-Ala PhCH2 PhCH2 Boc-Ala Cbz-Val i-Butenyl i-Butenyl Cbz-Val Cbz-Val 2-Propenyl 2-Propenyl Cbz-Val Cbz-Val 3-Butenyl 3-Butenyl Cbz-Val Cbz-Val n-Pentyl n-Pentyl Cbz-Val Cbz-Val Ph(CH2)2 Ph(CH2)2 Cbz-Val Cbz-Val Cyclohexyl-CH2 Cyclohexyl-CH2 Cbz-Val Cbz-Val PhCH2 i-Butenyl Cbz-Val Boc PhCH2 i-Butenyl Cbz-Val Ac 2-Propenyl PhCH2 Cbz-Val Boc PhCH2 3-Butenyl Cbz-Val Boc PhCH2 n-Pentyl Cbz-Val Boc PhCH2 Ph(CH2)2 Cbz-Val Boc PhCH2 Cyclohexyl-CH2 Cbz-Val Cbz-Val 2-Naphthyl-CH2 2-Naphthyl-CH2 Cbz-Val Cbz-Val 3-Naphthyl-CH2 3-Naphthyl-CH2 Cbz-Val Cbz-Val 2-Butynyl 2-Butynyl Cbz-Val Cbz-Val 3-Indoylmethyl 3-Indoylmethyl Cbz-Val Cbz-Val trans-3-phenyl-3- propenyl trans- 3-pheny 1-3propenyl Cbz-Val SB 14516 Cbz-Val n-butyl n-butyl Cbz-Val Cbz-Val N-Piperidinyl-CH2 N-Piperidinyl-CH2 Cbz-Val Cbz-Val N-Morpholinyl-CH2 N-Morpholinyl-CH2 Cbz-Val Cbz-Val (CH3)2N-CH2 (CH3)2N-CH2 Cbz-Val Cbz-Val t-butyl-NH-CH2 t-butyl-NH-CH2 Cbz-Val Cbz-Val N-Imidazoyl-CH2 N-Imidazoyl-CH2 Cbz-Val Boc PhCH2 2-Butynyl Cbz-Val Boc PhCH2 3-Indoylmethyl Cbz-Val Boc PhCH2 trans-3-phenyl-3- propenyl Cbz-Val Boc PhCH2 N-butyl Cbz-Val Boc PhCH2 N-Piperidinyl-CH2 Cbz-Val Boc PhCH2 N-Morpholinyl-CH2 Cbz-Val Boc PhCH2 (CH3)2N-CH2 Cbz-Val Boc PhCH2 t-butyl-NH-CH2 Cbz-Val Boc PhCH2 N-Imidazoyl-CH2 Cbz-Val Boc PhCH2 4-Pyridyl-CH2 Cbz-Val Cbz-Val PhCH2 4-Pyridyl-CH2 Cbz-Val Boc PhCH2 4-Pyridyl-CH2 Boc Cbz-Val PhCONHCH2 PhCONHCH2 Cbz-Val Cbz-Val N-Indoyl-CH2 N-Indoyl-CH2 Cbz-Val Cbz-Val t-ButylCONH-CH2 t-ButylCONH-CH2 Cbz-Val Cbz-Val BocNHCH2 BocNHCH2 Cbz-Val Cbz-Val nh2ch2 NH2CH2 Cbz-Val Cbz-Val N-Benzimidazoyl N-Benzimidazoyl Cbz-Val Cbz-Val PhCH2O-CH2 PhCH2O-CH2 Cbz-Val Cbz-Val PhO-CH2 PhO-CH2 Cbz-Val Cbz-Val CH3(CH2)2OCH2 CH3(CH2)2OCH2 Cbz-Val Cbz-Val CH2O-CH2 Cbz-Val Cbz-Ala (CH3)2CHOCH2 (CH3)2CHOCH2 Cbz-Ala Cbz-Val t-Butyl-O-CH2 t-Butyl-O-CH2 Cbz-Val Cbz-Val (CH3)2CHCH2OCH2 (CH3)2CHCH2OCH2 Cbz-Val Cbz-Val CH3CH2(CH3)CHO- CH3CH2(CH3)CHO- Cbz-Val ch2 ch2 Cbz-Val Cyclohexyl-O-CH2 Cyclohexyl-O-CH2 Cbz-Val Cbz-Val PhCH2OCH2O-CH2 PhCH2OCH2O-CH2 Cbz-Val Cbz-Val CH3OCH2O-CH2 CH3OCH2O-CH2 Cbz-Val SB 14516 184 Cbz-Val CH3OCH2CH2OCH2O- ch2 CH3OCH2CH2OCH2O- CH2 Cbz-Val 185 Cbz-Val CH3S-CH2 CH3S-CH2 Cbz-Val 186 Cbz-Val PhS-CH2 PhS-CH2 Cbz-Val 187 Cbz-Val (CH3)2CHS-CH2 (CH3)2CHS-CH2 Cbz-Val 188 Cbz-Val CH3(CH2)2S-CH2 CH3(CH2)2S-CH2 Cbz-Val 189 Cbz-Val CH3(CH2)3S-CH2 CH3(CH2)3S-CH2 Cbz-Val 190 Cbz-Val CH3S(O)-CH2 CH3S(O)-CH2 Cbz-Val 191 Cbz-Val CH3S(O)2-CH2 CH3S(O)2-CH2 Cbz-Val 192 Cbz-Val PhS(O)2-CH2 PhS(O)2-CH2 Cbz-Val 193 Cbz-Val i-PropylS(O)2-CH2 i-PropylS(O)2-CH2 Cbz-Val 194 Cbz-Val n-Propyl-S(O)2-CH2 n-Propyl-S(O)2-CH2 Cbz-Val 195 Cbz-Val n-ButylS(O)2-CH2 n-ButylS(O)2-CH2 Cbz-Val SB 14516

Claims

Claims: 1. ) -CH2RI2 wherein R^ is a) NH-A wherein A is defined as above; b) R5-(R6R7Qm-; c) R5-(R6R7Q m V- wherein V is O or NH, except R^, r6 and R^ are not hydroxyl, chlorine or fluorine if they are adjacent to V, d) R5-(R6R7Q m -S(O)n- wherein m = 1-3 and n = 0-2 and R^, r6 and R^ are as defined above except R^, r6, and R^ are not hydroxyl, chlorine or fluorine if they are adjacent to sulfur, e) R 8 -S(O)n-, f) R9-S(O)n-, g) N(r10) 2 , h) NR’5r16 wherein R’5 and R’6 are joined to form a 4-6 membered saturated nitrogens heterocycle including: i) azetidinyl, ii) pyrrolidinyl, iii) piperidinyl, or SB 14516 -20iv) morpholinyl, i) R^ 7 OCH2O wherein R^ 7 is: i) C^C 6 alkyl, ii) R 9 , or 1) trityl,

1. A compound having the structure: R 1 R 2

2. ) hydrogen, 15 3) CpCg alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C3-C7 cycloalkyl; and pharmaceutically acceptable salts thereof. 20 2. The compound as defined in claim 1 wherein X^ = X 2 and R^ = R 2 . 2) hydrogen,

3. The compound as defined in claim 1 wherein X^ and X 2 are selected from Cj and Cg alkyl, phenyl Cj-Cg alkyl, and Cj-Cg alkenyl. 25 4. The compound as defined in claim 1 wherein R^ and R 2 are selected from Boc, BocAla, ZAla, ZVal and AlaAla. 3) Ci-C 6 alkyl,

4. ) R 3 -CO- wherein R 3 is: 20 a) hydrogen, b) C]-C6 alkyl, unsubstituted or substituted with one or more hydroxyl groups, chlorine atoms, or fluorine atoms, c) phenyl or naphthyl unsubstituted or substituted with one or more substituents R 4 , wherein R 4 is: 25 i) C1-C4 alkyl, ii) halogen, where halogen is F, Cl, Br or I, iii) hydroxyl, iv) nitro, v) C1-C3 alkoxy, or 30 vi) -CO-N(Rl0)2 wherein R^O is, independently, H or C1-C4 alkyl; or d) a 5-7 member heterocycle such as pyridyl, furyl, or benzisoxazolyl; 5. Virus type 1.

5. The compound as defined in claim 1 wherein Rl and R 2 are benzyl and X^ and X 2 are Boc. 5 iii) CH2Ar wherein Ar is phenyl, naphthyl or a 5-7 membered heterocycle, j) R 17 OCH2CH 2 OCH2, k) N-imidazolyl where the imidazole ring is unsubstituted or substituted by a substituent R 4 , 5) phthaloyl wherein the aromatic ring is unsubstituted or substituted with one or more substituents R 4 ; 35 5 HO OH wherein χΐ and X 2 are the same or different and are A-(B) n - where n = 0-2; and B is, independently, an α-amino acid chosen from the group: Ala, Asn, Cys, Trp, Gly, Gin, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr, Val, His, or trifluoroalanine, wherein the 10 amino group of B is bonded to A or the carboxy group of the adjacent residue B, whichever is appropriate, and the carboxy group of B is bonded to the amino group of the adjacent residue B or the structure, whichever is appropriate; and A is covalently attached to the amine group of the adjacent residue B or to the amine group of the structure if n=0, and is:

6. The compound as defined in claim 1 having a protease activity inhibition constant Ki less than about 10 μΜ. 6) R 3 (R^R 7 C)m-CO- wherein m - 1-3 and R 3 , R^, and R 7 are independently: a) hydrogen. SB 14516 - 19 10 b) chlorine or fluorine, c) C1-C3 alkyl unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, d) hydroxyl, e) phenyl or naphthyl unsubstituted or substituted with one or more substituents R.4, f) Cj - C 3 alkoxy, g) a 5-7 member heterocycle, or h) R5, r6, a nd R? may be independently joined to form a monocyclic, bicyclic, or tricycle ring system each ring of which is C3-C6 cycloalkyl;

7. A compound according to claim 1 for use in a medicament. 35 8. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier. SB 14516 -21 9. A method of treating infection by a retrovirus which comprises administering a compound according to claim 1. 7) RAR^R^Qm W- wherein m = 1-3 and W is OCO or SO 2 and rA r6, an d R^ are as defined above, except R^, r6, and R? are not chlorine, fluorine or hydroxyl if they are adjacent to W;

8. ) R8-W- wherein R 8 is a 5-7 member heterocycle such as pyridyl, furyl, or benzisoxazolyl;

9. ) R^-W- wherein R^ is phenyl or naphthyl unsubstituted or substituted with one or more substituents R^;

10. A method according to claim 9 wherein the retrovirus is the Human Immunodeficiency 10 1) N-benzimidazolyl where the fused benzene ring is unsubstituted or substituted by one or more substituents R 4 ; m) C2-C5 alkynyl, optionally substituted with one or more groups R 9 ; or n) C2-C6 alkenyl, optionally substituted with one or more groups R 9 ; 10) R5-(R6R7Qm-P(O)(ORl ’)- wherein R” is C( - C4 alkyl or phenyl;

11. A process for preparing a compound as defined by general formula in claim 1, substantially as described herein by way of Example. 11) R 8 -P(O)(OR n )-; or

12. A compound whenever prepared by a process according to claim 11. 12) R^-PiOjiOR 1 ’)-; R1 and R^ are the same or different and are:

13. A Pharmaceutical composition comprising a compound as claimed in any of claims 1 to 7 or 12.