PT98227A - PROCESS FOR PREPARING SUBSTITUTED 1,4-DIAMINO-2-3-DIHYDROXY-BUTANES AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM - Google Patents

Description

72 875 SB CASE 14514 -2-

DESCRIPTIVE MEMORY

BACKGROUND OF THE INVENTION The present invention relates to the process of preparing compounds which are inhibitors of aspartic proteases, particularly retroviruses.

Retroviruses, that is, viruses of the Retroviridae family, are a class of viruses that carry their genetic material as ribonucleic acid instead of deoxyribonucleic acid. Also known as tumor RNA viruses, their presence has been associated with a wide variety of diseases in humans and animals. It is believed that they are the causative agents of pathological conditions associated with infections of 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 immune deficiency syndrome virus (SIDAS), human T-lymphotropic virus ( HTLV-I, -II) and human immunodeficiency virus (HIV-1, HIV-2) which is the etiological agent of AIDS (acquired immune deficiency syndrome) and AIDS-related complexes, and many others . Although pathogens have been isolated in many of these cases, no satisfactory method for the treatment of this type of infection has yet been developed. Among these viruses, HTLV and HIV have been especially well characterized. The production of functional viral proteins is critical for retrovirus replication. Protein synthesis is performed by translating the open reading frames into poly-lipoprotein constructs, corresponding to the gag, pol and env reading structures. Gag and pol precursor proteins are processed by a viral protease into the functional proteins. HIV-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 the processing of the polyproteins can not be provided by the host and is essential to the life cycle of the retrovirus. In fact, there was shown to be 72 875 SB CASE 14514-3-

that infectivity is lacking to retroviruses that lack the protease or which contain a mutated form of protease. See Katoh et al., Viroloav. 145, 280-92 (1985), Crawford, et al., J. Virol., 53, 899-907 (1985), Debouck, et al., Proc. Natl. Acad. Know. USA. 84, 8903-6 (1987). Thus, inhibition of retroviral protease appears to be a therapy method for retroviral diseases. Processes for expressing retroviral proteases in E-coli have been described (Debouck, et al., Proc Natl Acad. Sci USA 8903-06 (1987) and Tomasselli et al., Biochemistry, 29, 264-9 (1990 ), and references cited therein). Recombinant HIV protease inhibitors have been described (Dreyer et al., Proc Natl Acad Sci USA 86, 9752-56 (1989), Tomasselli et al., Supra, Roberts et al. , Et al., European Patent Application No. 337714, Dreyer et al., European Patent Application No. 352,000). In addition, some of these inhibitors have been shown to be potent inhibitors of viral proteolytic processing in HIV-1 infected T lymphocyte cultures (Meek et al., Nature (London), 343, 90 (1990) and Roberts et al., Supra ).

The limitations of current strategies for inhibition of aspartic protease include (1) oral bioavailability, (2) plasma clearance (" clearance ") (e.g., by biliary excretion or degradation); (3) selectivity of inhibition; and (4) in the case of intracellular targets, membrane permeability or incorporation into cells. The present invention relates to novel inhibitors of aspartic proteases and retroviral proteases. Unlike the inhibitors previously described, the compounds of the present invention are not analogues of peptidic substrates having a fissile mimetic dipeptide. They also deviate substantially from a peptide substrate-like structure in that they do not have a conventional amino-terminal-to-carboxyl-terminal orientation. SUMMARY OF THE INVENTION The present invention relates to the process for the preparation of compounds having the structures particularly indicated in the claims and which are described hereinbelow, which bind to retroviral proteases. These compounds are viral protease inhibitors and are useful in the treatment of diseases related to virus infection.

The present invention also relates to the process for the preparation of a pharmaceutical composition comprising one of the abovementioned compounds and a pharmaceutically acceptable carrier thereof. The present invention further describes a method for the treatment of viral diseases comprising administering to a mammal in need thereof an effective amount of one of the abovementioned inhibitor compounds. DETAILED DESCRIPTION OF THE INVENTION The compounds of the invention have the structure of formula I:

in which X * is A- (B) n -, where n = 0-2; and B is independently an α-amino acid selected from the group: Ala, Asn, Cys, Trp, Gly, Gin, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr, Val, His or trifluoroalanine, in which the amino group of B is attached to A or the carboxyl group of the adjacent B residue, as appropriate, and the carboxyl group of B is attached to the amino group of the adjacent B residue or to the structure, as appropriate; and A is covalently linked to the amino group of adjacent residue B or to the amino group of the structure if n = 0, and is: 1) trityl, 2) hydrogen, 3) C1-6 alkyl,

Wherein: R 3 is: a) hydrogen, b) unsubstituted or substituted C 1 -C 6 -alkyl with one or more hydroxyl groups, chlorine atoms or fluorine atoms, and C 1 -C 6 -alkyl, ) unsubstituted or substituted phenyl or naphthyl, with one or more substituents R 4, where R 4 is:

i) C1-3 alkyl, halogen, where halogen is F, Cl, Br or I, (iii) hydroxy, iv) nitro, v) C1-4 alkoxy, or vi) R1 is independently H or C1 -C4 alkyl or d) a 5-7 membered heterocycle, such as pyridyl, furyl or benzisoxazolyl; 5) phthaloyl in which the aromatic ring is unsubstituted or substituted with one or more substituents R4; 6) R 5 (R 6 R 7 C) m -CO-, wherein m = 1-3 and R 5, R 6 and R 7 are independently a) hydrogen, b) chlorine or fluorine, c) unsubstituted or substituted C 1 -C 3 alkyl, with one or more chlorine or fluorine atoms or hydroxyl groups, d) hydroxyl, e) unsubstituted or substituted phenyl or naphthyl, with one or more substituents R4, f) C1-C3 alkoxy, g) a 5-7 membered heterocycle , or h) R 3, R 6 and R 7 may independently be attached to form a monocyclic, bicyclic or tricyclic ring system, each of which is C 3 -C 6 cycloalkyl ring; 7) R 5 (R 6 R 7 C) mW-, where m = 1-3 and W is OCO or SO 2, and R 5, R 6 and R 7 are as defined above, except for R 1, R 2 and R 3 are not chlorine, fluorine or hydroxyl , if they are adjacent to W; 8) R 8 -W-, where R 8 is a 5-7 membered heterocycle, such as pyridyl, furyl or benzisoxazolyl; 9) R 9 -W-, where R 9 is unsubstituted or substituted phenyl or naphthyl, with one or more substituents R 4;

10 8 R 5 - (R 6 R 7 C) m-P (O) (O R n) -, wherein R 11 is C 1-4 alkyl or phenyl; 11) R8-P (O) (OR11) -; or 12) R < - P (0) (0R11) -; R1 is: 1) -CH2R12, where R12 is: a) NH-A, where A is as defined above, b) R5- (R6R7C) m-, c) R5- (R6R7C) mV-, where V is O or NH, with the exception of R5, R6 and R7 are not hydroxyl, chlorine or fluorine if they are adjacent to V, d) R5 - (R7 R7 C) m S (O) n, where m = and R 5, R 6 and R 7 are as previously defined, except that R 8, R 9 and R 7 are not hydroxyl, chlorine or fluorine if they are adjacent to the sulfur, e) R 8 -S (O) n -, f) R 9 -S 0) n - (g) (R130) P (O) (OR14) -, where R3 * and R14 are independently: i) C1-C6 alkyl, ii) C3-6 cycloalkyl, iii) H, iv) R9 , or (v) R8, h) R13P (O) (OR14) -, i) N (R10) 2, j) NR1 R2 where R1 and R2 are attached to form a saturated 4- (I) azetidinyl, (ii) pyrrolidinyl, (iii) piperidinyl, or (iv) morpholinyl, (k) R170CH20, where R17 is i) C1-C6alkyl, ii) R9, or iii) CH2Ar, where Ar is phenyl, naphthyl or a 5-7 membered heterocycle, D177202ch2och2, m) N-imidazolyl, wherein the imidazole ring is not 72 875 SB CASE 14514 -7-

substituted or substituted with a R4 substituent, n) N-benzimidazolyl, wherein the condensed benzene ring is unsubstituted or substituted with one or more substituents

2) hydrogen,

3) C 1 -C 6 alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C 3 -C 6 cycloalkyl; and pharmaceutically acceptable salts thereof.

Preferred are the peptidic compounds of the foregoing description which are symmetrical at C-2, wherein X1 = X2 and R1 = R2.

Suitably, R 1 is C 1 -C 4 alkyl, benzyloxymethyl, 3-phenyl-propyl or benzyloxy. In particular, R1 may be benzyloxymethyl or 3-phenylpropyl. Preferably, R 1 is benzyl.

Suitably, X 1 is CbzAla, AlaAla, Val, CbzVal, Cbz or hydrogen.

The compounds of the present invention are useful in the preparation of a medicament, in particular, of a medicament for the treatment of infections caused by retroviruses.

C-2 symmetrical peptidic compounds are also preferred, in which R1 and R2 are C1-C6 alkyl or arylalkyl, and X1 and X2 are single amino acids or mono- or dipeptides; these groups may be terminally substituted by common acyl groups or by blocking groups commonly used in peptide synthesis, such as t-Boc or Cbz.

Also included in the present invention are pharmaceutically acceptable acid addition salts, complexes or prodrugs of the compounds of the invention. Pro-drugs are considered to be

which release covalently attached original drug carriers.

As used herein, and except where indicated, the term " alkyl " refers to a straight-chain or branched alkyl radical having 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 with the indicated number of carbon atoms attached through a bridging oxygen atom; it is intended that " cycloalkyl " includes saturated ring groups, such as, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl; it is understood that " alkenyl " includes both linear and branched hydrocarbon chains containing one or more carbon-carbon double bonds, which may occur at any stable point along the chain, such as ethenyl, propenyl, butenyl, pentenyl, 2-methylpropenyl and the like; the term " alcinyl " refers to a straight or branched hydrocarbon chain with 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, and except where indicated, the term " heterocycle " represents a stable 5- to 7-membered mono- or bicyclic heterocyclic ring which is either saturated or unsaturated and consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O, and S, and wherein the nitrogen or sulfur heteroatoms may be optionally oxidized and the nitrogen atom may be optionally quaternized, and including any bicyclic group in which any of the heterocyclic rings defined above is condensed with a benzene ring. The heterocyclic ring may be attached to any heteroatom or to any carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolylidyl, imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydropyranyl, thienyl, thiamorpholinylsulfoxide, thiamorpholinylsulfoxide, and oxadiazolyl.

When any variable (eg, A, B, R 1, R 2, R 3, R 4, heterocycle, substituted phenyl, etc.) occurs more than once in any constituent or in formula I, each case is independent of its definition in each of the other cases. Combinations of substituents and / or variables are also permissible only if these combinations result in stable compounds. By the convention used herein, it is intended that a geminal diol, for example when R 6 and R 7 are both hydroxyl, is equivalent to a carbon-oxygen double bond.

Other commonly used abbreviations and symbols in the art used herein to describe peptides include the following:

Amino Acids Three-Letter Code Alanine Ala Arginine Arg Asparagine Asn Aspartic Acid Asp Cysteine Cys Glutamine Gin Glu Acid Glu Glycine Gly Histidine His Isoleucine Ile Asparagine or Aspartic Acid Glutamine or Glutamic Acid Amino Acid Code Three Letters Leucine Leu Lysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine Ser T reonine Thr T riptophane Trp Tyrosine Tyr Valine Go Asx Glx

72 875 SB CASE 14514 -10-

According to the conventional embodiment, the amino terminus is on the left and the carboxyl terminus is on the right. All chiral amino acids (AA) can occur as racemates, racemic mixtures, or individual enantiomers or diastereomers, all the isomeric forms being included in the present invention. β-Ala is re-fused to 3-aminopropanoic acid. -Boc refers to the t-butyloxycarbonyl radical, Cbz refers to the carbobenzyloxy radical, i-Bu refers to iso -butyl, Ac refers to acetyl, Ph refers to phenyl, DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, HOBT refers to 1-hydroxybenzotriazole, NMM is N-methylmorpholine, DTT is dithiothreitol, EDTA is ethylenediaminetetraacetic acid, DIEA is diisopropylethylamine, DBU is 1, 8-diazobicyclo [5.4.0] undec-7-ene, DMSO is dimethylsulfoxide, DMF is dimethylformamide and THF is tetrahydrofuran. HF refers to hydrofluoric acid and TFA refers to trifluoroacetic acid.

The peptide moieties represented by X 1 and X 2 are generally dipeptides or smaller. However, it is believed that longer peptides, which include the residues defined herein, are also useful and are considered within the scope of the invention. The selection of residues or end groups can be used to give the compound favorable biochemical or physicochemical properties. Hydrophilic residues may be used to impart desirable solubility properties or D-amino acids at the carboxyl terminus may be used to impart resistance to exopeptidases.

The synthesis of the compounds represented by structure I can be carried out by pinacol coupling of alpha-amino aldehydes, with the protected N, P 2 NHCH (R 1) CHO. A representative procedure involves the addition of the aldehyde to a mixture of TiCl 4 and magnesium-mercury amalgam in an inert solvent such as THF under an inert atmosphere as described in EJ Corey, RL Da-nheiser and S. Chandrasekaran , J. Oro. Chem. 41, 260-266 (1976). The N-protected, N-protected alpha-aldehydes required are readily preparable from the alpha-amino acids, with N 72875 SB CASE 14514-11-

for example, by reduction of the corresponding esters with di-isobutylaluminum hydride, by reduction of the N-methyl-N-methoxy-amides derived, P2NHCH (R3 -) COOH (0Me), P2NHCH (R1) COOH, with L1AIH4 (Fehrentz and Castro, Svnthesis 676 (1983)), or by reduction of the protected N alpha-amino alcohol followed by oxidation with DMSO- (C0Cl) 2 or SC3-pyridine (Revision: Jurczak and Golebiowski, Chem. Rev. 89, 149 (1989)).

J

Generally, the amino protecting group, P2, is t-Boc-, Cbz-, p-toluenesulfonyl or other conventional protecting group, chosen as is well known in the art of the peptides. Subsequent to the coupling of pinacol, the protecting group P2 can be removed and X1 groups can be introduced. The above procedures may be used to prepare all stereoisomers of the compounds represented by the structure.

Alternatively, the compounds represented by structure I may be prepared from D - (+) - mannitol by conversion to diaziridine (Compound B, Scheme 1). The resulting diaziridine is reacted with the appropriate nucleophiles, such as (CH3) 3 CuCli, to introduce the side chain groups R *, as illustrated in Scheme 1 and is described in detail in the Examples. described by Y. Le Merrer et al., Heterocycles 25, 541-548 (1987) and by A. Dureault, C. Greck and JC Depezay, Tet. Letters 27, 4157-4160 (1986). the preparation of compounds having the structure wherein R 1 = CH 2 R 12, where R 12 is hydrogen or a stable and reactive cuprate ion group such as methyl, butyl, isopropyl, or other alkyl, alkenyl or aryl which is optionally substituted, for example , with fluorine, alkoxy or protected hydroxyl.

Alternatively, the compounds represented by structure I can be prepared from D - (+) - mannitol by conversion to 1,6-ditosyl-3,4- (O-isopropylidene) mannitol, as shown in Scheme 1, followed by treatment with a base such as KH or KOt-Bu in an inert solvent such as THF or ether to give the di-epoxide, 1,2: 5,6-dianhydro-3,4- (O-isopropylidene) mannitol (compound C, Scheme 2). This di-epoxide is reacted with the

Suitable carbon atoms, such as cuprate (R12) 2 CuCl reagents or alkynyl aluminum reactants, are used to introduce the side chain groups (Scheme 2). This procedure is also especially suitable for the preparation of compounds I wherein R1 is a group which forms a stable and reactive cuprate reagent, as previously described, the resulting diol product is converted in the corresponding diamine with inversion of configuration in the alcohol carbons. One way of achieving this is by conversion of the diol into the dimesylate by reaction with methanesulfonyl chloride and triethylamine followed by displacement with NaN 3 in DMF to give 2,5-diazido-3,4- (O-isopropylidene diol) substituted hexane; the conversion to the 2,5-diamino derivative proceeds by reduction with a hydride reagent such as L1AIH4 or by catalytic hydrogenation with a catalyst such as Pd (O) or Raney-Ni to give the core structure I ( in the form of the isopropylidene derivative) (Scheme 2). The introduction of the groups X1 is carried out by conventional condensation reactions, as is well known in the art.

The compounds of structure I wherein NH-A may be prepared from the diepoxide ou or the ditosylate (compound A, Scheme 2) by reaction with NaN 3 in DMF to give the resulting dihydroxyl terminal diazide, which is converted to the corresponding tetra-azide, with inversion of the configuration in the alcohol carbons, as described above, and subsequently in the corresponding tetraamine. After the selective reaction of terminal amines with A groups or protecting groups, such as Boc or Cbz, the introduction of X 1 = X 2 groups follows. Similarly, R1 = R2 groups can be introduced into compounds in which R1 is N (R10) 2, NR15 R16, R5 - (R6 R7 C) mV- or R5 - (R6 R7 C) m S (O) n -, by reaction of the diepoxide com with the appropriate oxygen, nitrogen or thiol nucleophile, with subsequent oxidation of the thiol as required. Reaction of the diepoxide com with the appropriate phosphorus nucleophile in a Arbuzov or Michaelis-Arbu-zov reaction allows the introduction of R1 = R2 groups which are (R130) P (O) - (OR14) - or R13P (O) ) (OR14) -.

By the procedures shown in Schemes 1-2, one can

Prepare any enantiomer of the compounds having the structure, from the respective enantiomer of mannitol.

Scheme 1

Synthesis of General Structure I

HO R1

I

J 72 875 SB CASE 14514

Scheme 2 Second approach to I

base -14-

R'-

1) MsCl, Et 3 N 2) NaN 3, DMF -1 3) H 2, cat., Or LiAIH 4

NH2 R '

R1 OH

HO R1

I

Thus, in a further aspect thereof, the present invention relates to a process for the preparation of a compound having the formula

in which R 'is 1) a) NH-A wherein A is as defined for formula I; b) R 5 - (R 6 R 7 C) m -; c) R8- (R7 R7 C) mV-, where V is O or NH, except that R8, R6 and R7 are not hydroxyl, chlorine or fluorine, if they are adjacent to V, d) R5- (R6R7C) m) -S-. where m = 1-3 and R5, R6 and R7 are as previously defined, except that R5, R6 and R7 are not hydroxyl, chlorine or fluorine, if they are adjacent to the sulfur, e)

(R 4): wherein R 13 and R 24 are independently: (i) C 1 -C 8 alkyl, (Ii) C3 -C6 cycloalkyl, iii) H, iv) R9, or v) R8, h) R13 P (O) (O R14) -, i) N (R10) 2, j) , wherein R8 and R6 are attached to form a 4-6 membered saturated nitrogen heterocycle, including: i) azetidinyl, ii) pyrrolidinyl, iii) piperidinyl, or iv) morpholinyl, k) R170CH20, wherein R17 is i C1-4 alkyl, ii) R9, or iii) CH2 Ar, where Ar is phenyl, naphthyl or a 5-7 membered heterocycle, 1) m is 2 or 2, m) N-imidazolyl, wherein the imidazole ring is unsubstituted or substituted with a R4, n) N-benzimidazolyl substituent. wherein the fused benzene ring is unsubstituted or substituted with one or more R4 substituents, (o) C2 -C6 alkynyl, optionally substituted with one or more R9 groups; or p) C2 -C10 alkenyl, optionally substituted with one or more R9 groups; 2) hydrogen, 3) C 1 -C 6 alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C 1 -C 6 cycloalkyl, and R " and R 2 'are hydrogen, an amino protecting group or together are N 2, which comprises

72 875 SB CASE 14514 1) reacting a compound of the formula:

with a compound R'-Z, where Z is a moiety which renders R 'nucleophilic, 2) converting the resulting hydroxyl groups into displaceable groups, 3) reacting the displaceable groups with a nitrogen nucleophile.

Typically, Z is hydrogen, an alkali metal such as Li, Na or K, an alkaline earth metal, such as magnesium, or a transition metal, such as copper, aluminum, titanium, zinc or cadmium, or a species thereof derivative. Representative R'-Z compounds include optionally substituted alkyl-, aryl- or heteroaryl-lithium, alkyl-, aryl- or heteroarylmagnesium halides (eg, Grignard reagents), dialkyl lithium cuprate, diaryl lithium cuprate, or the alkali metal salts of optionally substituted alkyl alcohols, phenols or benzyl alcohols. Diphenyl lithium cuprate is especially useful.

Hydroxyls are converted to suitable displaceable groups, such as mesylate, tosylate, brosylate, benzoate, acetate and halide, by processes common in the art. The tosyl group is especially suitable and is formed by reacting the hydroxyl groups with, for example, tosyl chloride.

Suitable nitrogen nucleophiles are those which are capable of reacting with a suitable displaceable group. Unhindered organic amines or heterocycles, amine metal salts, heterocycles or azide are useful. Generally, a nitrogen-containing group of the formula R " 'R'-N-Z, where Z is as defined above and R' and R'v are hydrogen, an amine protecting group or together are N2 (eg, azide). Preferred is a metal azide, such as sodium or potassium azide. Subsequent reduction of the azido groups yields the amino groups. 72 875 SB CASE 14514 -17-

The particularly useful intermediates of the invention are:

(III) wherein R 'is 1) R 12 is defined as for formula I, (2) hydrogen, (3) C 1 -C 4 alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups or 4) C3-Cy cycloalkyl.

The compounds of the present invention are prepared by the Merrifield solid phase technique (J. Am. Chem. Soc., 85, 2149 (1964), or, preferably, by solution processes known in the art. synthesis in solution and in solid phase, such as in a convergent synthesis in which di-, tri- or tetra-peptide fragments can be prepared by solid phase synthesis, and are either coupled or further modified by synthesis in solution. Peptide synthesis processes are generally described in JM Stewart and JD Young, "Solid Phase Peptide Synthesis", Pierce Chemical Company, Rockford, II (1984), in M. Bodonsky, YA Klauser and Μ (1977), or in The Peptides " Gross and Meienhoffer, eds., Acad. Press, 1979, Vol. I, III, to produce the peptides of the invention and such processes are incorporated herein by reference.

Each amino acid or peptide is suitably protected, as is known in the art of peptides. For example, the Boc- or carbobenzyloxy group is preferred for protecting the amino group, especially in the α-position. A suitable benzyl or benzyl substituted group is used to protect the mercapto group from cysteine or other thiol-containing amino acids; or the hydroxyl of the serine or threonine. Tosyl or the nitro group may be used for protection of the guanidine of Arg or the imidazole of His, and a carbobenzyloxy or benzyl group may suitably be used,

for the hydroxyl group of Tyr, Ser or Thr, or for the ε-amino group of lysine. Suitable substitution of the carbobenzyloxy or benzyl protecting groups is an ortho and / or chloro, bromo, nitro or methyl substitution, and is used to modify the reactivity of the protecting group. Cysteine and other sulfur containing amino acids may also be protected by the formation of a disulfide having a thioalkyl or thioaryl group. Except for the Boc group, the protecting groups are most conveniently those which are not removed by treatment with mild acid. These protecting groups are removed by methods such as catalytic hydrogenation or treatment with sodium in liquid ammonia or with HF, such as is well known in the art.

If solid phase processes are used, the peptide is constructed sequentially from the carboxy terminus towards the amino terminus of the peptide. Solid-phase synthesis is initiated by covalently linking the C-terminus of a protected amino acid to a suitable resin, such as benzhydrylamine (BHA) resin, methylbenzhydrylamine (MBHA) resin or chloromethyl resin (CMR), such as generally describes in U.S. Patent 4,244,946. The carrier resins BHA or MBHA are used if the carboxyl terminus of the product peptide is intended to be a carboxamide. Generally, a CMR support is used if the carboxyl terminus of the peptide produced is intended to be a carboxyl group, although this resin may also be used to produce a carboxamid or an ester. Modification of the terminal amino group of the peptide is accomplished by alkylation or acetylation, as is generally known in the art. Such modifications may be made to the amino acid prior to incorporation into the peptide or peptide after it has been synthesized and the terminal amino group has been released but prior to removal of the protecting groups.

Typically, the acetylation is carried out on the free amino group using an acyl halide, anhydride or activated ester, of the corresponding α-alkyl acid, in the presence of a tertiary amine. The monoalkylation is most conveniently carried out by reductive alkylation of the amino group with an appropriate aliphatic aldehyde or ketone in the presence of a moderate reducing agent such as lithium or sodium. Dialkylation as well as quaternization can be carried out by treatment of the amino group with an excess of alkyl halide in the presence of a base.

J

Synthesis in peptide solution is performed using standard procedures to form amide linkages. Typically, a Boc-protected amino acid having a free carboxyl group is coupled to a protected amino acid having a free amino group using a suitable carbodiimide coupling agent such as N, N'-dicyclohexylcarbodiimide ( DCC), optionally in the presence of catalysts, such as 1-hydroxybenzotriazole (HOBT) and dimethylaminopyridine (DMAP). Other processes, such as the formation of activated acid esters, anhydrides and acid halides, of the free carboxyl of a Boc-protected amino acid, and subsequent reaction with the free amine of a protected amino acid, optionally in the presence of a base . For example, a Boc-protected amino acid or peptide is treated in an anhydrous solvent such as dichloromethane or tetrahydrofuran (THF) in the presence of a base such as N-methylmorpholine or a trialkylamine 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 processes can be selectively deprotected, using conventional techniques, at the amino or carboxyl terminus, and coupled to other peptides or amino acids using similar techniques. After the peptide is complete, the protecting groups may be removed, as hereinbefore described, for example, by hydrogenation in the presence of a palladium or platinum catalyst, or by treatment with sodium in liquid ammonia, hydrofluoric acid, trifluoroacetic acid or with an alkali.

Often, the esters are used to protect the terminal carboxyl group of peptides in the solution synthesis. These 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 in the other esters may be converted via an activated acyl intermediate as described above.

The substituted amides and amides of the invention are prepared from carboxylic acids of the peptides in a similar manner. Thus, ammonia or an amine substituted with an activated acyl intermediate of an α-amino acid or a protected amino acid oligopeptide can be reacted to produce the amide. It is convenient to use coupling reagents, such as DCC, to form substituted amides from the carboxylic acid itself and a suitable amine.

In addition, the methyl esters of the invention may be converted to the amides or substituted amides either directly by treatment with ammonia or with a substituted amine in methanol solution. A methanolic solution of the methyl ester of the peptide is saturated with ammonia and stirred in a pressurized reactor to provide the simple carboxamide of the peptides.

Methods for determination of inhibition constant (Ki) are described in the art by Dixon analysis, e.g. in Dreyer, et al. Proc. Natl. Acad. Know. U.S.A. 86, 9752-9756 (1989). A peptidolytic assay is used using the substrate Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and recombinant HI1-protease, as described in Stricker, et al., Proteins. 6, 134-154 (1989). A lower Ki value indicates a higher binding affinity.

Pharmaceutical compositions of the compounds of the invention, or derivatives thereof, may be formulated as solutions or post-opticized powders for parenteral administration. The powders may be reconstituted, prior to use, by addition of a suitable diluent or other pharmaceutically acceptable carrier. The liquid formulation is generally an aqueous, isotonic, buffered solution. Examples of suitable diluents include normal isotonic saline, dextrose, 5% standard in water or 72 875 SB solution CASE 14514-

sodium acetate or buffered ammonium acetate. This formulation is especially suitable for parenteral administration but may also be used for oral administration or may be contained in a controlled dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxycellulose, gum arabic, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

A preferred composition for parenteral administration may additionally contain an amount of compound capped in a liposomal carrier. The liposome can be formed by dispersing the compounds in an aqueous phase with phospholipids, with or without cholesterol, using a variety of techniques, including conventional manual agitation, high pressure extrusion, reverse phase evaporation and microfluidization. A suitable method for preparing these compositions is described in more detail in co-pending copending application Ser. 06/763484 which is hereby incorporated by reference. Such a carrier may be optionally directed to its site of action by an immunoglobulin or a reactive protein, the viral particle or the infected cells. Of course, the choice of these proteins will depend on the antigenic determinants of the infecting virus. An example of such a protein is the T-cell glycoprotein, CD-4, or a derivative thereof, such as sCD-4 (soluble CD-4), which is reactive with the human immunodeficiency virus glycoprotein coating HIV). These proteins are described in copending U.S. 07/160463 which is hereby incorporated by reference. Similar bleeding proteins can be designed by methods known in the art for other viruses and these are also contemplated within the scope of the invention.

Alternatively, these compounds may be capsulated, prepared as tablets or in the form of an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to improve or stabilize the composition, or to facilitate the preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline

72 875 SB CASE 14514 and water. Solid carriers include starch, lactose, calcium sulfate dihydrate, kaolin, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar or gelatin. The carrier may also include a sustained release material, such as glycerol monostearate or glycerol distearate, alone or together with a wax. The amount of solid carrier will vary, but will preferably be from about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are prepared following conventional pharmacy techniques involving grinding, blending, granulating and pressing, as required, for tablet forms; or grinding, blending and filling, for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of an aqueous or non-aqueous syrup, elixir, emulsion or suspension. Such a liquid formulation may be administered directly p.o. or it may be used for filling a soft gelatin capsule.

For rectal administration, a powdered powder of the compounds of the invention may be combined with an excipient, such as cocoa butter, glycerin, gelatin or polyethylene glycols, and molded into a suppository. Powdered powders may also be prepared with an oily preparation, gel, ointment or emulsion, buffered or unbuffered, and administered through a transdermal patch.

Also disclosed in the present invention is a method for the treatment of viral infections, particularly retrovirus infections, which comprises administering a compound of formula I to a patient infected with a susceptible virus. The method is particularly applicable to human immunodeficiency virus type 1 infection. When the compounds of the invention are used to induce antiviral activity in patients who are infected with susceptible viruses and requiring such treatment, the treatment method comprises orally, parenterally, buccally, transdermally, intravenously, intramuscularly, rectally or by insufflation, of an effective amount of the chosen compound, preferably, dispersed in a pharmaceutical carrier. Dosage units of the active ingredient are selected in the range of 0.05 to 50 mg / kg body weight. Typically, the dosage units will be from 50 to 1000 mg. For acute or chronic infection, these dosage units may be administered from one to ten times a day. The dosage will be readily determined by one skilled in the art and will depend upon the age, weight and condition of the patient, and the route of administration. Combination therapy is herein included, as described in European Patent Application No. 337714, on pages 42-47.

The following Examples serve to illustrate the present invention. The Examples are not intended to limit the scope of the invention in any way and are presented only to show how to prepare and use the compounds of the invention,

In the Examples all temperatures are in degrees Celsius. Amino acid analyzes were performed on a Dionex Autoion 100. Peptide content analysis is based on amino acid analysis. FAB mass spectra were run on a Vab Aab mass spectrometer using fast atom bombardment. NMR spectra were recorded at 250 MHz using a Bruker Am 250 Spectrotron. The indicated multiplicities are: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br denotes a broad signal.

Purification of recombinant HIv-protease

In Debouck, et al., Proc. Natl. Acad. Know. USA, 84, 8903-6 (1987), methods for expressing recombinant HI1-protease were described in E. coli. The enzyme used to test the compounds of the invention was thus produced and purified from the cell pellet, as previously described. described by Stickler et al. Proteins. 6, 139-154 (1989).

EXAMPLES

The following compounds were prepared from D-mannitol as described in LeMerrer et al., Heterocycles. 25, 541-548 (1987): 1) 1,6-di-O-tosyl-3,4-O-isopropylidene-D-mannitol (Compound A)

72 875 SB CASE 14514 2) (2S, 3R, 4R, 5S) -1,2,5,6-di- (N-carbobenzyloxyimino) -3,4- (O-iso-propylidene) -hexanediol (Compound B). 3) 1,2: 5,6-dianhydro-3,4-O-isopropylidene-D-mannitol (Compound c).

Example 1

Preparation of (1S, 2R, 3R, 4S) -carbobenzyloxyalanylalanyl-N- (1-ethyl-2,3-dihydroxy-4- (carbobenzyloxyalanylalanylamino) hexylamide 2a) (1S, 2R, 3R, 4S) -carbobenzyloxy-N- - (1-ethyl-2,3- (β-isopropylidene diol) -4-carbobenzyloxyamino) hexylamide 3

Lithium (18.9 mL, 23.6 mmol, 1.25 M in ether) was slowly added to a suspension of Cul (2.24 g, 11.8 mmol) in THF (25 mL), at -78 ° C for 15 minutes and then cooled to -78 ° C. A solution of compound B (265 mg, 0.59 mmol) in THF (2 mL) was added and the mixture was maintained at -25 ° C overnight, without stirring. The mixture was diluted was treated with saturated NH 4 Cl (100 ml) and concentrated aqueous NH 3 (10 ml) and extracted with ethyl acetate (3 x 25 ml). By concentration and flash chromatography " of the residue (gradient elution, 0% to 16% ethyl acetate in hexanes) provided the title compound (143 mg, 50% yield). b) (1S, 2R, 3R, 4S) -carbobenzyloxyalanylalanyl-N- (1-ethyl-2,5- (Q-iso-propylidenediol) -4- (carbobenzyloxyalanylalanylamino) hexylamide 4

A solution of 3 (135 mg, 0.28 mmol) in methanol (10 mL) was stirred with 10% Pd on carbon (70 mg) under a H 2 atmosphere for 4 h. The mixture was filtered and concentrated, and the residue was combined with DMF (5 mL), carbobenzyloxyalanylalanine (165 mg, 0.56 mmol), HBOT (76 mg, 0.56 mmol) and DCC (116 mg, 0.56 mmol). The solution was stirred for 24 h, then filtered, diluted with ethyl acetate (50 ml) and washed with water (2 x 20 ml). By concentration and flash chromatography " of the residue (gradient, 0% to 8% methanol in CH2 Cl2) provided the title compound (196 mg, 91%). c) (1S, 2R, 3R, 4S) -carbobenzyloxyalanylalanyl-N- (1-ethyl-2,3-dihydroxy-4- (carbobenzyloxyalanylalanyl) amino) hexylamide 2

A solution of 4 (191 mg, 0.25 mmol) in 70% acetic acid (20 mL) was stirred at 50 ° C for 24 h and then concentrated,

72 875 SB CASE 14514 under vacuum. The residue was dissolved in ethyl acetate and washed successively with 5% NaHCO 3, water and brine, then dried over Na 2 SO 4 and concentrated to a solid. The residue was triturated with CH 2 Cl 2 and filtered to give the title compound 2 (105 mg, 58%) in which R 2 is ethyl β X * and CbzAlaAla. Calc'd for C 36 H 52 N 6 O 10: C, H, 7.19; N, 11.53. Found: C, 59.11; H, 7.40; N, 11.19.

Example 2

Preparation of (1S, 2R, 4S) -alanylalanyl-N- (1-ethyl-2,3-dihydroxy-4- (alanylalanyl) amino) hexylamide dihydrochloride

A solution of compound 2 (24.3 mg) in methanol (3 ml) with 10% Pd on carbon (13 mg) was stirred under an atmosphere of H 2 for 2 h. The mixture was filtered and the filtrate was concentrated. The residue was dissolved in 0.1 N HCl (10 ml), washed with ethyl acetate and lyophilized to give the title compound 5 (26 mg) in which R @ 3 is ethyl and X @ 2 and AlaAla. MS (DCI): m / z 461 (M + H) +

Example 3

Preparation of (1S, 2R, 3R, 4S) -carbobenzyloxyalanylalanyl-N- (1- (2-methyl) propyl-2,3-dihydroxy-4- (carbobenzyloxyalanylalanyl) amino-6-methyl-) heptylamide a) (2-methoxypropyl) -2,3- (4-iso-propylidenediol) -4-carbobenzyloxyamino-6-methyl) heptylamide To a mixture of anhydrous LiBr ( 0.989 g, 11.5 mmol) and CuBr (1.65 g, 11.5 mmol) in THF (10 mL) at -60 ° C under Ar was added isopropylmagnesium chloride (12.7 mL, A solution of compound B (0.52 g, 1.15 mmol) in THF (6 ml) was added. After 20 minutes a solution of compound B (0.52 g, 1.15 mmole) in THF (6 ml) was added. The mixture was diluted with saturated aqueous NH4 Cl (100 ml) and with 14% aqueous ammonia solution (50 ml) The combined organic extracts were washed with water (50 ml) and concentrated, and the residue was purified by flash chromatography (quot; Chromatography " flash " (gra ethyl acetate in hexanes) to give the title compound (0.27 g, 27%). 72 875 SB CASE 14514 -26-

b) (1S, 2R, 3R, 4S) -carbobenzyloxyalanylalanyl-N- (1- (2-methyl) propyl-2,3- (Q-isopropylidenediol) -4- (carbobenzyloxyalanylalanyl) heptylamide The title compound was prepared from compound 7 in 46% yield by the procedure described in Example 1 (b). MS (FAB): m / z 825.4 (M + H) +. c) (1S, 2R, 3R, 4S) -carbobenzyloxyalanylalanyl-N- (1- (2-methyl) propyl-2,3-dihydroxy-4- (carbobenzyloxyalanylalanyl) amino-6-methyl) heptylamide

Compound 8 (0.22 g, 0.27 mmol) was deprotected by the procedure described in Example 1, Part (b) to give the title compound (89 mg, 0.113 mmol, 42% yield) , wherein R1 is isobutyl and X * is Cbz-AlaAla. Federal Police. 220-221 ° C. Calc'd for: 40-60Â ° C 60: 61.21; H, 7.70; N, 10.71. Found: C, 60.91; H, 7.61; N, 10.58.

Example 4

Preparation of (1S, 2R, 5R, 4S) -alanylalanyl N- (1- (2-methylpropyl) -2,3-dihydroxy-4- (alanylalanylamino) -6- The title compound (17 mg) in which R3 is isobutyl and X1 is Ala Ala was prepared from compound 9 (44 mg) by the procedure of Example 2.

Example 5

Preparation of (1S, 2R, 3R, 4S) -carbobenzyloxyalanyl-N- (1- (2-methyl-propyl) -2,5-dihydroxy-4- (carbobenzyloxyalanyl) amino-6-methyl) heptylamide wherein R1 is isobutyl and X1 is Cbz-Al was prepared from compound 7 as described in Example 3, Parts (b) - (c), except that carbobenzyloxyalanylalanine was replaced with carbobenzyloxyalanine MS (DCI / NH 3): m / z 643 (M + H) + Analysis calculated for C 25 H 50 N 4 O 8: C, 63.53; H, 7.84; N, 8.72 Found: C, 64.01, H, 8.21, N, 9.11, 72.875, SB CASE 14514

-27-

Example 6

Preparation of (2S, 3R, 5S) -1,6-diphenyl-2,5-bis (benzyloxycarbonyl-nylvalinylamino) hexane-3,4-diol a) (2R, 3R, 4R, 5R) -1,6- (isopropylidenedioxy) hexane-2,5-diol To a suspension of 865 mg (4.5 mmol) of copper (I) iodide in 10 ml of ether at -78 ° C was added a solution of 5 ml of phenyl lithium in cyclohexane (9 mmol). The reaction mixture was slowly warmed to -40 ° C and cooled again to -78 ° C. To this dark brown mixture was added a solution of 435 mg (2.33 mmol) of bis-epoxide em in 10 ml of ether using a syringe. After stirring at -78 ° C for 2 h, the reaction mixture was allowed to warm to room temperature and stirred overnight. It was quenched with a 1: 1 mixture of saturated ammonium chloride solution and 30% ammonium hydroxide solution, extracted with ether and washed with ammonium chloride until complete disappearance of the blue color. The solvents were dried over anhydrous sodium sulfate and removed in vacuo. The residual oil was flash chromatographed " flash " (silica, ethyl acetate: hexane 1: 8) to give 691 mg (87%) of 2 as a yellowish oil. NMR (CDCl 3, 250 MHz) δ 7.4 (m, 10 H), 3.7 (m, 4H), 3.1 (dd, 2H, J = 2.14 Hz), 3.0 (s , 2H), 2.7 (dd, 2 H, J = 7.14 Hz), 1.4 (s, 6 H). To a solution of 300 mg (0.8 mmol) of the 12-diol in 5 (b) (2R, 3R, 4R, 5S) -6-diphenyl-3,4- (isopropylidenedioxy) -2,5-bis (tosyloxy) ml pyridine at 0 ° C was added p-toluenesulfonyl chloride (1.5 g, 7.8 mmol). After stirring at 0 ° C for 2 h, the reaction mixture was warmed to room temperature and The reaction mixture was added to 15 ml of ice-cold 6N hydrochloric acid, extracted with methylene chloride, washed with 3% sodium bicarbonate solution and the (DCI, NH 3) (Μ + ΝΗ)): 668 (M + H +), 668 (M + H +) (CDCl3, 250 MHz) δ 7.5-7.0 (m, 10 H), 4.9 (m, 2 H), 4.3 (s, 2 H), 3 , 0.72 (s, 6H), 1.5 (s, 6H), 0.72 (s, 6H), 0.72 ) -1,6-diphenyl-3,4- (isopropylidenedioxy) -2,5-bis-azidohexane

To a solution of 355 mg (0.54 mmol) of the bis-tosylate 3 in 5 ml of dimethylformamide was added 975 mg (15 mmol) of sodium azide. The reaction mixture was heated at 90 ° C for 8 h , then at 105 ° C for 6 h. The reaction mixture was cooled and the unreacted sodium azide was removed by filtration, washed with ether and the solvents removed in vacuo. The residue was chromatographed on silica gel (1:20 ethyl acetate: hexane) to give 180 mg (89%) of an oil, as a mixture of the title product 14 and an elimination product, (3R, 4R, 5S) -1,6-diphenyl-3,4- (isopropylidenedioxy) -5-azidohex-1-ene in the ratio of 4.6: 1 as evidenced by H-NMR *. This mixture was found to be inseparable by chromatography. MS (DCI, NH 3) of 14 [M + NH 4 +] 410.5; (Typical peaks for bis-azide 14)) δ 7.4-7.2 (m, 10H), 4.1 (s, 2H), 2.8-3.4 (m , 6H), 1.5 (s, 6H), and characteristic peaks for the elimination product 15 δ 6.72 (d, 1 H, J = 14 Hz), 6.05 (dd, 1H, J = 6 J = 7 Hz), 3.75 (dd, 1H, J = 2.7 Hz), 3.43 (m, 5 H), 1.4 (s, 3H), 1.6 (s, 3H).

d) (2S, 3R, 4R, 5S) -1,6-diphenyl-3-4- (isopropylidenedioxy) hexane-2, 5-diamine

180 mg of a mixture of azides, prepared in the previous experiment, was hydrogenated at 1 atm over 100 mg of Pearlman's catalyst (10% Pd (OH) 2 over C). After 10 h, the catalyst was filtered through celite and washed with ether, and the solvents were removed in vacuo to give 124 mg (73%) of amine mixtures. The pure diamine 16 was obtained by chromatography in Florisil (ether followed by methanol). MS (DCI, NH 3) (M + H) + 341.5; 1 H-NMR (CD 3 OD, 250 MHz) δ 7.4-7.0 (m, 10 H), 3.9 (bs, 2H), 3.0 (bt, 2 H), 2.6 (m, 4 H ), 1.4 (s, 6 H). ε) (2S, 3R, 4R, 5S) -1,6-diphenyl-2,5-bis (benzyloxycarbonylvalinylamino) -3,4- (isopropylidenedioxy) hexane To 100 mg (0.4 mmol) of Cbz-Val, in 2 ml of tetrahydrofuran-

72 875 SB CASE 14514 -29-NO, at -40 ° C was added 65 μΐ (0.59 mmol) of N-methylmorpholine followed by 53 μΐ (0.4 mmol) of isobutyl chloroformate. After stirring at -40 ° C for 15 minutes, a solution of 40 mg (0.12 mmol) of diamine in 4 ml of dimethylformamide was added. The reaction mixture was warmed to room temperature over a period of 2 h and stirred overnight. Dilute with ethyl acetate (100 mL), wash with 2 x 20 mL of hydrochloric acid 5% with 50 ml of 5% sodium bicarbonate solution and dried over anhydrous sodium sulfate. Removal of the solvents followed by " flash " (silica gel, 10% MeOH / CH2 Cl2) gave an oil which, on trituration with ether / hexane gave 67 mg (70%) of the adduct 17 as a solid. Calc'd for: 47: 53: 03: 0 (69.95), H (7.24), N (6.94); Found: (69.79), H (7.20), N (6.88); 1 H-NMR (CDCl 3, 250 MHz) δ 7.5-7.0 (m, 20 H), 6.1 (br d, 2H), 5.1 (br s, 4 H), 4.9 d, 2 H), 4.4 (q, 2 H, J = 7 Hz), 3.75 (dd, 2 H, J = 7 Hz), 3.6 (br s, 2 H), 2.8 (br d, 4 H), 2.0 (m, 2 H), 1.4 (s, 6 H), 0.75 (d, 6 H, J = 7 Hz), 0.6 (d, 6 H, J = 7Hz). f) (2S, 3R, 4R, 5S) -1,6-Diphenyl-2,5-bis (benzyloxycarbonylvalinylamino) hexane-3,4-diol 18

33 mg of the acetonide was suspended in 4 ml of 70% acetic acid and heated in an oil bath maintained at 75 ° C for 12 h. During the reaction a solid product forms on the walls of the flask. Cool and 15 ml of methylene chloride were added. The methylene chloride phase was carefully removed using a pipette, and the solvents were removed in vacuo to give 29 mg (88%) of a solid. Trituration with ether / hexane mixture gave 23 mg of pure diol. Anal. Calc'd for C 44 H 54 N 4 O 8: C (68.91), H (7.09), N (7.31); Found C (68.83), H (7.07), N (7.08); 1 H-NMR (CDCl 3, 250 MHz) δ 7.5-7.1 (m, 20 H), 6.25 (d, 2H, J = 7 Hz), 5.1 (s, 4 H) 9 (bs, 2 H), 4.1 (q, 2H, J = 7 Hz), 3.8 (two doublets, 4 H, J = 7 Hz), 3.4 (bs, 2H), 2.9 (m, 4H), 2.0 (m, 2 H), 0.8 (d, 6 H, J = 7 Hz), 0.6 (d, 6 H, J = 7).

Hz).

72 875 SB CASE 14514

Example 7

Preparation of (4S, 5R, 6S, 7S) -10-diphenyl-4,7-bis-N-benzyloxycarbonylvalinylamino decane-5,6-diol a) (4R, 5R, 6R, 7R) -1,10-diphenyl-4,7- To a solution of phenylacetylene (1.8 g, 18 mmol) in tetrahydrofuran (20 ml) at -78 ° C was added 10 ml (10 ml) 16 mmol) of a 1.6 M solution of n-BuLi in hexane over a period of 10 minutes. After stirring for 15 minutes at -78 ° C, 2 ml of boron trifluoride etherate was added and stirred for another 15 minutes. To this mixture was added 650 mg (3.5 mmol) of the diepoxide in 8 ml of tetrahydrofuran. It was stirred for 2 h and quenched with saturated ammonium chloride solution. Extract with ethyl acetate, wash with water, dry over anhydrous sodium sulfate, and remove the solvents in vacuo. The residual oil was flash chromatographed " flash " was dissolved in 50 g of silica gel affording 1.36 g (100%) of 19 as a yellowish oil. 1 H-NMR (CDCl 3, 250 MHz) δ 7.5-7.1 (m, 10 H), 3.95 (m, 2H), 3.8 (br s, 2H), 3.35 (br s, 2H), 2.9 (dd, 2H, J = 4, 14 Hz), 2.65 (dd, 2 H, J = 7.14 Hz), 1.4 (s, 6H). b) (4R, 5R, 6R, 7R) -1,10-diphenyl-4,7-dihydroxy-5,6-fisopropylidene dioxy) decane 20

295 mg of the diol obtained above was dissolved in 5 ml of methanol and hydrogenated over 50 mg of 10% Pd / C. After stirring overnight, the catalyst was filtered and washed with ether. Removal of the solvents gave 290 mg of 10 as a colorless oil. MS (DCI, NH 3) (M + H) + 399.4; 1 H-NMR (CDCl 3, 250 MHz) δ 7.5-7.1 (m, 10 H), 3.6 (m, 4 H), 3.25 (s, 2 H), 2.6 (m , 4H), 1.4-1.9 (m, 8H), 1.35 (s, 6H). To a solution of 250 mg (0.628 mmol) of the diol in 2,6-dichloroethane (2: 1) 5 ml of pyridine, 900 mg (4.8 mmol) of p-toluenesulfonyl chloride was added at 0 ° C and stirred for 1 h. It was warmed to room temperature and stirred overnight. The reaction mixture was worked up as usual. By MPLC (silica,

72 875 SB CASE 14514-ethyl acetate: hexane, 1: 8) 220 mg of the bistosylate 21 were obtained as a colorless oil. 1 H-NMR (CDCl 3, 250 MHz) δ 7.7-7.0 (m, 18 H), 4.6 (m, 2H), 3.9 (q, 2 H, J = 4 Hz), 2.5 (m, 4H), 2.4 (s, 6H), 1.8-1.3 (m, 8H), 1.25 (s, 6H). d) C 4 S 5 R 6 R 7 S) -? 10-diphenyl-4,7-bis-azido-5,6- (isopropylidene-dioxy) -decane

220 mg of the 1-ditosylate and 500 mg of sodium azide in 3 ml of DMF were suspended and heated in an oil bath maintained at 95 ° C for 10 h. It was cooled and diluted with ether. Unreacted sodium azide was removed by filtration and the solvents were removed in vacuo. The residual oil (85 mg) was found to be sufficiently pure to be used in the next experiment. MS (DCI, NH 3) (M + NH 4) + 466; NMR (DMSO-d 6) δ 7.4-7.1 (m, 10H), 4.0 (s, 2 H), 2.95 (m, 2 H), 2.6 (m, 4 H), 2.0- 1.4 (m, 8 H), 1.4 (s, 6 H). e) (4S, 5R, 6R, 7S) - 10-diphenyl-4,7-bis-amino-5,6- (isopropylidene dioxy) decane

85 mg of bis-azide were hydrogenated over 40 mg of Pearlman's catalyst in 5 ml of ethyl acetate until TLC showed complete disappearance of the starting material. The catalyst was filtered through celite, washed with methanol and the solvents removed in vacuo to give 65 mg (80%) of diamine 23 as an oil. 1 H-NMR (CD 3 OD, 250 MHz) δ 7.4-7.0 (m, 10 H), 3.7 (m, 2H), 2.6 (m, 6H) Δ (m, 8 H), 1.35 (s, 6H). f) (4S, 5R, 6R, 7S) -Î ±, 10-diphenyl-5,6- (isopropylidenedioxy) -4,7-bis-β-benzyloxycarbonylaminovalinylaminoldecane To 100 mg (0.4 mmol) of Cbz-Val in 2 ml of THF at -40 ° C, 65 μΐ of N-methylmorpholine and 53 μΐ of isobutyl chloroformate were added. The reaction mixture was stirred for 15 minutes and a solution of diamine 23 (51 mg, 0.128 mmol) in DMF (2.5 mL) was added. The mixture was slowly warmed to room temperature and stirred overnight. It was diluted with 75 ml of ethyl acetate, washed with 2 x 25 ml of 5% hydrochloric acid and 50 ml of 3% sodium bicarbonate solution, and the solvents were removed in vacuo. The residual oil was subjected to

72 875 SB CASE 14514 -32 flash chromatography " (ethyl acetate: hexane 1: 4) to give 69 mg of 24 as a colorless solid. 1 H NMR (CDCl 3, 250 MHz) δ 7.4-7.0 (m, 20 H), 6.2 (d, 2H, J = 7 Hz), 5.2 (d, 2 H, J = 7Hz), 5.1 (m, 4H), 4.1 (m, 2H), 3.9 (m, 2H), 3.5 (bs, 2H), 2.6 (m, 4H (S, 6H), 1.0 (d, 6H, J = 7Hz), 0.9 (d, 6H) 6H, J = 7Hz). 9) (4S, 5R, 6R, 7S) -10,10-diphenyl-4,7-bis-N-benzyloxycarbonylvalinylamino] decane-5,6-diol

A 23 mg suspension of acetonide 24 from the previous experiment was heated in 3.2 mL of 70% acetic acid in an oil bath maintained at 90 ° C for 6 h. The reaction mixture was cooled and the solvents were removed in vacuo to give 20 mg of 25 as a colorless solid. MS (FAB) (M + H) + 823.3; 1 H-NMR (CD 3 SOCD 3) δ 7.4-7.0 (m, 20 H), 5.0 (q, 4 H, J = 7 Hz), 4.6 (bs, 2H), 3.8 (M, 2H), 2.5 (m, 4H), 2.0 (m, 2H), 1.5 (m, 8H) 8 (two overlapping doublets, 12 H, J = 7 Hz).

Example 8

Preparation of (5S, 6R, 7R, 8S) -5,8-bisbenzyloxycarbonylaminovalaminolino1-dodecane-6,7-diol a) (5R, 6R, 7R, 8R) -5,8-dihydroxy-6.7- (isopropylidenedioxy) dodeca -deca-1,4-diene To a suspension of 400 mg of copper (I) iodide in 6 ml of ether at -60 ° C was added 4 mmol of allylmagnesium bromide through a syringe , over a period of 3 minutes. It was stirred at -60 ° C for 15 minutes, cooled to -78 ° C and 140 mg (0.75 mmol) of bis-epoxide in 4 ml of ether was added. The cooling bath was removed at -78 ° C for 1 h, the reaction was allowed to warm to room temperature and stirred for 4 h. It was quenched with 5 ml of saturated ammonium chloride solution and 10 ml of 30% ammonium hydroxide solution. Extract with ether, wash with ammonium chloride solution and remove the solvents in vacuo. yielding 216 mg (> 100%) of the diol which was found to be sufficiently pure for the next step. 1 H-NMR (CDCl 3, 250 MHz) δ 5.3 (m, 2H), 5.0 (m, 4H), 3.5-3.8 (m, 6H), 2.0-2 , 4 (m, 4 H), 1.95-1.7 (m, 2H), 1.5-1.6 (m, 2H), 1.3 (s, 6H). 72 875 SB CASE 14514

B) (5R, 6R, 7R, 8R) -5,8-dihydroxy-6,7- (isopropylidenedioxy) dodeca-decane 27

220 mg of the diol 26 was dissolved in 5 ml of methanol and hydrolyzed over 20 mg of 10% Pd / C as usual to give the title product (210 mg), MS (DCI, NH 3) (M + NH 4) + 292.4; 1 H-NMR (CDCl 3, 250 MHz) δ 3.95 (s, 2H), 3.2-3.6 (m, 4 H), 1.2-1.8 (m, 12 H), 1.3 (s, 6 H), 0.8 (t, 6 H, J = 7 Hz). c) (5R, 6R, 7R, 8R) -5,8-diamino-6,7- (isopropylidenedioxy) dodecadeca-

215 mg of the bisdiol were dissolved in pyridine and 0.5 ml of methanesulfonyl chloride was added at 0 ° C. The reaction mixture was stirred at 0 ° C for 3 h and at room temperature overnight. By usual work up 410 mg of a tan solid was obtained which was used directly for the formation of the azide. Bismesylate 28 had the following spectral characteristics: 1 H NMR (CDCl3, 250 MHz) δ 4.7 ( 1.8 (m, 4H), 1.2-1.6 (m, 8H), 1.3 (m, 2H) (s, 6 H), 0.9 (t, 6H, J = 7 Hz).

The mesylate 28 was dissolved in 5 ml of DMF and 650 mg of sodium azide were added. The reaction mixture was heated in an oil bath maintained at 90 ° C for 8 h, cooled and the solid removed by filtration. Removal of the solvents gave compound 29 as a yellowish oil (185 mg). MS (DCI, NH 3) (M + NH 4) + 466.

The bis-azide 29 was hydrogenated, as usual, over 25 mg of Pearlman's catalyst in 10 ml of ethyl acetate to give 172 mg of the diamine 30. An analytical sample was obtained by chromatography on Florisil ( hexane and then ethyl acetate: hexane). 1 H-NMR (CD 3 OD, 250 MHz) δ 3.7 (dd, 2H, J = 2.4 Hz), 2.7 (bs, 2H), 1.2-1.6 (m, 12 H), 1. , 4 (s, 6H), 0.85 (t, 6H, J = 7Hz). d) (5S, 6R, 7R, 8S) -5,8-bisbenzyloxycarbonylaminovalinylaminol-6,7- (isopropylidenedioxy) -dodecane

50 mg of the diamine was coupled using the anhydride method

Mixed with 100 mg of Cbz-Val, 65 μl of N-methylmorpholine and 53 μl of isobutyl chloroformate in 2 ml of THF and 3 ml of DMF. By " flash chromatography " (silica gel, 1: 2 ethyl acetate: hexane) gave 42 mg of the title product. 1 H NMR (CDCl 3, 250 MHz) d 7.4-7.3 (s, 10H), 6.1 (d, 2H, J = 7Hz), 5.2 (d, 2H, J = 7 Hz), 5.1 (two doublets, 4 H, J = 12 Hz), 4.0 (m, 4 H), 3.7 (s, 2 H), 2.2 (m, 2H), 1.2 -1.6 (m, 12 H), 1.3 (s, 6 H), 1.0 (d, 6 H, J = 7 Hz), 0.9 (d, 6 H, J = 7 Hz) , 0.8 (t, 6 H, J = 7 Hz). e) (5S, 6R, 7R, 8S) -5,8-bisbenzyloxycarbonylaminobutylamino] -1-decane-6,7-diol

25 mg of the acetonide was suspended in 2 ml of 70% acetic acid and heated in an oil bath at 75 ° C for 8 h. The reaction mixture was cooled and the solvents were removed in vacuo. The residue was triturated with ether / hexane to give 20 mg of a colorless solid. MS (FAB) (M + H) + 699.5; NMR (CDCl 3, 250 MHz) δ 7.5 (s, 10 H), 6.3 (d, 2H, J = 7 Hz), 5.2 (d, 2H, J = 7 Hz) 8.4 (m, 4 H), 3.4 (s, 2 H), 2.2 (m, 2 H), 1.1-1.9 (m, 12 H), 1.0 d, 6 H, J = 7Hz), 0.9 (d, 6H, J = 7Hz), 0.8 (t, 6H, J = 7Hz).

Example 9

Preparation of (4S, 5R, 6S, 4S) -4,7-bisbenzyloxycarbonylaminovalaminino) -5,6-dihydroxy-2,9-dimethyldecane a) (4R, 5R, 6R, 7R) -4,7-dihydroxy-5,6- isopropylidenedioxy) -2,9-dimethyldeca-2,9-diene 35

A standard solution of isopropenyl lithium was prepared by reacting 700 mg of lithium and 7 ml (75 mmol) of 2-bromopropene at 0 ° C for 1 h (in 75 ml of diethyl ether). 16 mmol of this solution was added at -78 ° C to a suspension of 1.5 g (7.77 mmol) of copper (I) iodide and heated to -45 ° C for half an hour. The dark brown mixture was re-cooled to -78 ° C and a solution of 2.5 g (13.5 mmol) of bis-epoxide em in 50 ml of diethyl ether was added. The reaction mixture was stirred at -78 ° C and slowly warmed to room temperature. It was quenched with saturated ammonium chloride and ammonium hydroxide solution, extracted with ether, washed with ammonium hydroxide solution, dried over anhydrous sodium sulfate and the solvents were removed in vacuo to give 3.20 g (88%) of 33,

72 875 SB CASE 14514 a colorless oil. 1 H-NMR (CDCl 3, 250 MHz) δ 4.9 (s, 2H), 4.82 (s, 2 H), 3.7 (bs, 4H), 2.5 (d, 2H, J = 13 Hz ), 2.1 (m, 2 H), 1.8 (s, 6 H), 1.3 (s, 6 H). b) (4R, 5R, 6R, 7R) -4,7-dihydroxy-5,6- (isopropylidenedioxy) -2,9-di-methyldecane 34

2.10 g (7.75 mmol) of the diol were dissolved in 10 ml of methylene chloride and mixed with 0.5 g of Crabtree's catalyst. The reaction mixture was subjected to hydrogenation at 1 atm using a flask filled with hydrogen. After stirring for 24 h the catalyst was precipitated by the addition of ether, filtered through silica gel and washed with ether. Removal of the solvents afforded 1.95 g (93%) of 34 as a colorless oil. 1 H-NMR (CDCl 3, 250 MHz) δ 3.8-3.5 (m, 4H), 1.8 (m, 2 H), 1.6-1.4 (m, 4 H), 1.35 (s, 6 H), 0.9 (d, 6 H, J = 7 Hz), 0.85 (d, 6 H, J = 7 Hz). c) (4R, 5R, 6R, 7R) -4,7-bis (methanesulfonyloxy) -5,6- (isopropylidene dioxy) -2,9-dimethyldecane To 1.88 g (6.87 mmol) of the diol 24 in 15 ml of pyridine, 4 ml of methanesulfonyl chloride was added at 0 ° C and stirred for 2 h. The reaction mixture was warmed to room temperature and stirred overnight. To ice cold 3N hydrochloric acid (50 ml) was added, extracted with methylene chloride, washed with 3% sodium bicarbonate solution, dried over anhydrous sodium sulfate and the solvents in vacuo to give a tan oil. By " flash chromatography " there was obtained 1.65 g (55%) of bismesylate 35. MS (DCI, NH 3) (M + NH 4) + 448.3; 1 H-NMR (CDCl 3, 250 MHz) δ 4.85 (m, 2 H), 4.1 (bs, 2 H), 3.1 (s, 6 H), 1.4-2.0 (m, 6 H), 1.4 (s, 6 H), 1.0 (d, 6 H, J = 7 Hz), 0.9 (d, 6 H, J = 7 Hz). d) (4S, 5R, 6R, 7S) -4,7-bis (azido) -5,6- (isopropylidenedioxy) -2,9-dimethyldecane 36 and (4S, 5R, 6R, 7S) -4,7- -5,6- (isopropylidenedioxy) -2,9-dimethyldecane To a solution of 1.19 g of bismesylate 35 in 10 ml of DMSO was added sodium azide (1.1 g, 17 mmol) and 100 ° C in an oil bath for 10 h. The reaction mixture was cooled to -78 ° C.

The sodium azide was removed by filtration, washed with ether and the combined solvents were removed in vacuo. The combined organic extracts were diluted with ether, washed with 50 ml of water, dried over anhydrous sodium sulfate and the solvents removed in vacuo to give 810 mg of bis-azide 36 which was used directly reduction without further purification.

790 mg of the crude azide obtained in the previous step were hydrogenated over 400 mg of Pearlman's catalyst at 1 atm of hydrogen. The catalyst was filtered through Florisil, washed with 75 ml of ether and the solvents were removed in vacuo. The residual oil was chromatographed on 10 g of Florisil (hexane and then 1: 4 ethyl acetate: hexane and finally methanol) to give 620 mg of the di-amine 37 as a colorless oil. 1 H-NMR (CDCl 3, 250 MHz) δ 3.7 (q, 2 H, J = 4 Hz), 2.75 (m, 4 H), 1.75 (m, 2 H), 1. 1 H NMR (CDCl 3): δ (CDCl3): Î'(s, 6H), 1-1.3 (m, 6H), 0.9 (d, 6H, . e) (4S5R.6R.7S) -4,7-bis (benzyloxycarbonylaminovalinylamino) -5,6-fisopropylidenedioxy) -2,9-dimethyldecane To 200 mg (0.8 mmol) of Cbz-Val in 5 ml of THF a- 40 ° C, 130 μΐ of N-methylmorpholine followed by 106 μΐ (0.8 mmol) of isobutyl chloroformate were added. The reaction mixture was stirred for 30 minutes and a solution of 75 mg (0.276 mmol) of diamine 37 in 5 mL of THF was added. It was stirred at -40 ° C for 2 h, warmed to room temperature and stirred for 16 h. It was diluted with 100 ml of ethyl acetate, washed with 2 x 50 ml of 5% hydrochloric acid and 2 x 50 ml of 5% sodium bicarbonate solution, dried over anhydrous magnesium sulfate and the solvents in vacuo yielding 300 mg of a crude product, from which, by trituration with 20 ml of hexane and 4 ml of ether, 190 mg of 38 were obtained as a colorless solid. MS (ESMS) (M-H) + 737.2; 1 H-NMR (CDCl 3, 250 MHz) δ 7.3 (s, 10 H), 5.8 (bd, 2 H, J = 7 Hz), 5.1 (d, 2 H, J = 7 Hz), 5.0 (bs, 4 H), 4.1 (m, 2 H), 3.9 (dd, 2 H, J = 7.9 Hz), 2.2 (m, 2 H), 1.1 J = 7 Hz), 0.75 (two overlapping doublets, 12H, J = 7 Hz), 1.2 (s, 6 H), 0.9 (d, 6 H, ).

72 875 SB CASE 14514 (f) (4S, 5R, 6R, 7S) -4,7-bis (benzyloxycarbonylaminovalinylamino) -5,6-dihydroxy-2,9-dimethyldecane

45 mg of the coupled product 38 was suspended in 5 ml of 70% acetic acid and heated in an oil bath maintained at 85 ° C for 10 h. The reaction mixture was cooled and the solvents were removed in vacuo to give a white solid. Calc'd for C18 H58 N4 O2 · 0.1 H2 O: C (64.47), H (8.40), N (7.91); Found C (64.36), H (8.39), N (7.82); MS (ESMS) (M + H) + 699.2: (M + Na) + 721.2; 1 H-NMR (CDCl 3, 250 MHz) δ 7.35 (s, 10 H), 6.25 (d, 2 H, J = 7 Hz), 5.2 (d, 2 H, J = 7 Hz), 5.0 (s, 4 H), 4.0 (m, 2 H), 3.9 (dd, 2 H, J = 7.9 Hz), 3.35 (s, 2 H), 2.2 (m, 2 H), 1.1-1.6 (m, 6 H), 1.0 (d, 6 H, J = 7 Hz), 0.9 (d, 6 H, J = 7 Hz) , 0.85 (b, 12 H).

Example 10

Preparation of (4S, 5R, 6R, 7S) -4,7-bis (benzyloxycarbonylamino) anilinoyl) -5,6-dihydroxy-2,9-dimethyldecane a) (4S, 5R, 6R, 7S) -4 ', 7-bis (benzyloxycarbonylaminoalanylamino ) -5.6 - (isopropylidenedioxy) -2,9-dimethyldecane 40

A mixture of 51 mg (0.1875 mmol) of the diamine, 109.3 mg (0.4125 mmol) of AlaCbz, 85 mg (0.4125 mmol) of DCC and 68 mg (0.5 mmol) of HOBT from one day to the next. The precipitated dicyclohexylurea was removed by filtration and the solvents were removed in vacuo to give 72 mg of 40 as a colorless solid. 1 H-NMR (CDCl 3, 250 MHz) δ 7.3 (s, 10 H), 6.1 (d, 2 H, J = 7 Hz), 5.3 (d, 2 H, J = 7 Hz), 5.1 (m, 4 H), 4.1 (m, 4 H), 3.5 (s, 2 H), 1.2-1.6 (m, 6 H), 1.4 (d, 6 H, J = 7 Hz), 1.2 (s, 6H), 0.8 (two overlapping doublets, 12 H, J = 7 Hz). b) (4S, 5R, 6R, 7S) -4,7-bis (benzyloxycarbonylaminoalanylamino) -5,6-dihydroxy-2,9-dimethyldecane

10 mg of the coupled product 40 was suspended in 2 ml of 70% acetic acid and heated in an oil bath at 80 ° C for 10 h. The reaction mixture was cooled and the solvents were removed in vacuo and the residue was triturated with hexane / ether to give 8 mg of the title compound 41. 1 H-NMR (CDCl3, 250 MHz) δ 7.3 ( (bs, 2 H), 5.1 (bs, 4 H), 4.1 (m, 4 H), 3.4 (bs, 2 H), 3.3 bs, 2H), 1.5 (m, 6 H), 1.3 (d, 6 H, J = 7 Hz), 0.8 (bs, 12 H). 72 875 SB CASE 14514 -38-

Example 11

Preparation of (2S, 3R, 4R, 5S) -dibenzyloxy-2,5-bis-carboxybenzyloxy-valinyl) -amino-3,4-hexanediol a) 1,6-dibenzyloxy-3.4-0-isopropylidene-D-mannitol

Sodium hydride (~ 50% oil dispersion) (1.2 g ca. 25 mmol) was washed with dry THF (2 x 10 mL), a THF (20 mL) phase was added again, cooled to 4 ° C and anhydrous benzyl alcohol (5.2 mL, ca. 50 mmol) was added dropwise. To the resulting solution at 4 ° C was added a solution of 1,2: 5,6-dianhydro-3,4-O-isopropylidene-D-mannitol in THF (2 ml). The cooling bath was removed and the reaction mixture was stirred at 23 ° C for 18 h, 1N HCl (50 mL) was added and extracted with EtOAc (3 x 50 mL). The extract was washed with H 2 O (50 mL) and saturated aqueous NaCl solution (50 mL), dried (Na 2 SO 4), concentrated and flash chromatographed with hexane / EtOAc to give 1 H-NMR (CDCl 3) 7.3-7.2 (m, 10), 4.54 (dd, 4), 3.96 (m, 2) , 3.87 (m, 2), 3.75 (m, 4), 3.54 (dd, 2), 1.32 (s, 6). b) 1,6-dibenzyloxy-2,5-ditosyl-3,4-O-isopropylidene-D-mannitol

1,6-Dibenzyloxy-3,4-Q-isopropylidene-D-mannitol 42 (402 mg, 1.0 mmol) tosyl chloride (764 mg, 4.0 mmol) and pyridine (10 ml) were stirred for 3 days, the mixture was added to ice-cooled 3 N HCl (30 mL), and extracted with EtOAc (3 x 25 mL). The extracts were washed with 3N HCl (3 x 25 mL), 5% NaHCO 3 solution (25 mL) and saturated aqueous NaCl solution (5 mL), dried (Na 2 SO 4), concentrated, if " flash " with hexane / EtOAc to give 550 mg (77%) of the title compound 43.1 H-NMR (CDCl3) 7.66 (m, 4), 7.40-7.15 (m, 16) 83 (m, 2), 4.37 (m, 6), 3.82-3.60 (m, 4), 2.37 (s, 6), 1.34 (s, 6). c) (2e, 3R, 4R, 5S) -dibenzyloxy-2,5-diazido-3,4-O-isopropylidenehexanediol

Compound 43 (1.00 g, 1.41 mmol), DMF (20 mL) and NaN 3 (0.917 g, 14.1 mmol) were combined and heated at 85 ° C for 24 h, filtered, washed The insoluble NaN 3 was dissolved in DMF and the filtrate was concentrated in vacuo. The residue was combined with H2 O (30 mL) and extracted with EtOAc (3 x 50 mL). The extract was washed with H2O (2x25

Ajf

72 875 SB CASE 14514 -39-ml) and saturated aqueous NaCl solution (25 ml), dried (Na2 SO4) and flash chromatographed. (CDCl 3) 7.33 (m, 10), 4.66 (dd, 4), 4.18 (m, 10H) m), 3.87-3.68 (m, 4), 3.46 (m, 2), 1.44 (s, 6). d) (2S, 3R, 4R, 5S) -dibenzyloxy-2,5-diamino-3,4-O-isopropylidenehexanediol

A solution of 44 (203 mg, 0.45 mmol) in THF (1 mL) was added to a 1 M solution of LiAlH 4 in THF (4.5 mL, 4.5 mmol). The reaction mixture was stirred at 23 ° C for 15 minutes, and then the excess LiAlH 4 was quenched by the slow addition of EtOAc (2 mL) and 10% NaOH solution (20 mL). Extraction with EtOAc (3 x 50 ml) and washing the extract with saturated aqueous NaCl solution (10 ml), drying (Na 2 SO 4), concentration and flash chromatography the title compound 45 (0.146 g, 81%) was obtained.1 H-NMR (CDCl3) 7.32 (m, 10), 4.51 (s, (S, 2), 3.50 (m, 2), 3.43 (m, 2), 2.94 (m, 2), 1.65 (br m, 4), 1.39 , 6). e) (2S, 3R, 4R, 5S) -dibenzyloxy-2,5-bis (carboxybenzyloxyvaleryl) ami-3,4-O-isopropylidenehexanediol

Cbz-Valine (166 mg, 0.66 mmol) and THF (3 mL) were cooled to -15 ° C and NMM (72 μ, 0.66 mmol) was added followed by i-BuOCOCl (94 μ, 73 mmol). The resulting mixture was stirred for 1 minute and then the diol 45 (120 mg, 0.3 mmol) in THF (1 mL) was added. The mixture was warmed to 23 ° C, stirred for 16 h, diluted with THF (5 mL) and filtered. The insoluble NMM-HCl was washed with additional THF and the filtrate was concentrated, redissolved in EtOAc (50 mL), washed with H2 O (10 mL) and saturated aqueous NaCl solution (10 mL) , dried (Na 2 SO 4), concentrated and flash chromatographed. with CH2 Cl2 / CH3 OH to elute the expected product as a solid and an oil. Trituration with Et 2 O and filtration afforded 0.121 g (47%) of a pure solid 46. 1 H-NMR (CDCl 3) 7.32 (m, 20), 4.48 (m, 6), 4.00 , 2.92 (m, 2), 1.93 (m, 2), 1.32 (m, 6), 0.92 (d, 6), , 85 (d, 6).

72 875 SB CASE 14514 -40-f) (2S, 3R, 4R, 5S) -dibenzyloxy-2,5-bis (carboxybenzyloxyvalinyl) amino-3,4-hexanediol

(2S, 3R, 4R, 5S) -dibenzyloxy-2,5-bis (carboxybenzyloxyvalinyl) amino-3,4-O-isopropylidenehexanediol 46 (121 mg, 0.14 mmol) was combined with acetic acid to 70% (12 mL) and heated at 90 ° C for 14 h, cooled and concentrated in vacuo. The residue was dissolved in EtOAc (50 ml) and washed with 5% NaHCO 3 solution (2 x 20 ml), H 2 O (20 ml) and saturated aqueous NaCl solution (20 ml), dried (Na 2 O ), concentrated and flash chromatographed, " with CH 2 Cl 2 / CH 3 OH to elute 39 mg (34%) of (2S, 3R, 4R, 5S) -dibenzyloxy-2,5-bis (carboxybenzyloxyvalinyl) amino-3,4-hexanediol 47. M.p. (methanol): 223 ° C; Calc'd for C 46 H 58 N 4 O 4 ú1.22 HoO: C, 66.09; H, 7.11; N, 6.70. Found: C, 66.23; H, 6.93; N, 6.68; NMR (CDCl3) δ 7.32 (m, 20), 5.07 (m, 4), 4.48 (m, 4), 4.35 (m, 2), 4.06 (m, 2 ), 3.63 (m, 4), 2.10 (m, 4), 0.91 (d, 6), 0.85 (d, 6); MS (FAB): [M + H +] 827.3.

PE ENZYME INHIBITION

Inhibition of HIv-protease activity The inhibition assay was previously described in Dreyer et al. Proc. Natl. Acad. Know. USA, 86, 9752-9756 (1989) and Moore et al. Bioch. Bioph. Res. Com .. 159, 420 (1989). A typical assay contained 10 ml of 50 mM MENDT buffer (pH 6.0; 2- (N-morpholino) ethanesulfonic acid) 1 mM EDTA, 1 mM dithiothreitol, 200 mM NaCl, 0.1 Triton X-100 %); N-acetyl-L-arginyl-L-alanyl-L-seryl-L-glutaminyl-L-asparaginyl-L-tyrosyl-L-prolyl-L-valyl-L-valinamide (Ac -Arg-Ala-Se 1-Gln-Asn-Tyr-Pro-Val-Val-NH 2; Km = 7mM); and micromolar and submicromolar concentrations of the synthetic compounds. After incubation at 37 ° C for several minutes, the reaction was started with 0.001-0.10 mg of purified HIv-protease. The reaction mixtures were quenched (37øC) 10-20 minutes later with an equal volume of cold trichloroacetic acid 0.6 N and, after centrifugation to remove the precipitated material, the peptidolysis products were analyzed by reverse phase HPLC (Beckman Ultrasphere 0DS, 4.5 mm x 25 mm, mobile phase, 5-20% acetonitrile / H2 O - 0.1% TFA (915 minutes), 20% acetonitrile / H2 O - 0.1% TFA (5 minutes ) at 1.5 ml / min, detection at 220 nm). The elution positions 72 375 SB CASE 14514 -41-

of Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH 2 (17-18 min) and Ac-Arg-Ala-Ser-Gln-Asn-Tyr (10-11 min. ) were confirmed with authentic material. The initial rates of formation of Ac-Arg-Ala-Ser-Gln-Asn-Tyr were determined by integration of these peaks and, typically, the inhibitory properties of the synthetic compounds were determined from the slope / in the origin of a 1 / v vs. [inhibitor] (Dixon analysis). The resulting values of this type of primary analysis are accurate, only for competitive inhibitors, and under conditions in which the Michaelis constant of the substrate used is well determined. It is desirable for the compounds of the invention to have values of less than 50 μΜ, preferably less than 10 μΜ and more preferably less than 1 μΜ.

Inhibition of HIV-1-protease [IDOS to Kc (ÂμM) 2 5.8 5 39.0 9 0.58 10 0.80 11 2.4

Following the procedures described herein and the teachings of the previous examples, the compounds listed in the following table, with the structure and substituent groups indicated therein, may be prepared. (Table below) 14514

Table of Compounds

R1 OH X1HN

Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala- Bu-Bu-Bu-Ac-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu-Bu- -Bu Ac Ac-Bu-Bu-Bu-Butoxy-Butoxy-Bu-Buyryl-Bu Ph (CH2) 2 -O-Bu-BuSi-Val-i-Bu Phenyl-lactoyl i-Bu Phenyl- Cbz-Val 2-Propenyl Cbz-Val 3-Butenyl Cbz-Val n-Pentyl Cbz-Val Ph (CH 2) 2-Cbz-Val-Phenyl Cbz-Val- Val-cyclohexyl-CH2-Cbz-Val 2-Naphthyl-CH2-Cbz-Val 3-Naphthyl-CH2-Cbz-Val 2-Butynyl Cbz-Val 3-Indoylmethyl Cbz-Val trans-3-phenyl-3-propenyl H ethyl Cbz-Val (ch3) 2n-ch2 Cbz-Val t-ButylNH-CH-2Cbz-Val N-Imidazoyl-CH2-Cbz-Val CH 2 Cbz-Val t-ButylCNH-CH 2 Cbz-Val BocNHCH 2 Cbz- Val (CH2) 2 -CH2 CH2 -CH2 CH3 -CH2 -Cbz-Val (CH3) 2 -CH2 -Cbz-Val t-Butyl- 0-CH2 Cbz-Val (ch3) 2chch2o-ch2 Cbz-Val ch3ch2 (ch3) cho-ch2 Cbz-Val Cyclohexyl-0-CH2 Cbz-Val P hCH 20CH 20 -CH 2 Cbz-Val ch3och2o-ch2 Cbz-Val ch3och2ch2och2och2 Cbz-Val ch3s-ch2

(CH 3) 2 CH 2 -CH 2 CH 2 -CH 2 CH 2 -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 (0) 2-CH 2 Cbz-Val i-Propiopyl-S (O) 2-CH Cbz-Val n-Propyl-S (O) (O) -CH 2 Cbz-Val (n-Butyl) 2 P (O) -CH 2 Cbz-Val (CH 3) 2 P (O) (O) -CH 2 Cbz-Ala (CH 3) 2 P (O) -CH 2 Cbz-Val (EtO) 2 P (O) -CH 2

Claims (10)

72 875 SB CASE 14514 45- CLAIMS A process for the preparation of a compound of the formula: NHX1 B is, independently of the other, an α-amino acid selected from the group: Ala, Asn, Cys, Trp, Gly, Gin, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr, Val, His, or trifluoroalanine, wherein the amino group of B is attached to A or to the carboxy group of adjacent residue B, where appropriate, and the carboxy group of B is attached to the amino group of adjacent residue B or to the structure, appropriate; and A is covalently bonded to the amino group of the adjacent residue B or to the amino group of the structure if n = 0, and is: 1) trityl, 2) hydrogen, 3) C1-4 alkyl, 4) R3 -CO- in wherein R 3 is: a) hydrogen, b) C 1 -C 6 alkyl, unsubstituted or substituted with one or more hydroxyl groups, chlorine atoms or fluorine atoms, c) phenyl or naphthyl, unsubstituted or substituted by one or more substituents R 4 , wherein R4 is: (i) alkyl (i) halogen, wherein halogen is F, Cl, Br or I, iii) hydroxyl, iv) nitro, v) C1-4 alkoxy, or vi) ) 2, wherein R 10 is, independently of the other, H or C 1-4 alkyl; or d) a 5-7 membered heterocycle, such as pyridyl, furyl or benzisoxazolyl, 5) phthaloyl, wherein the aromatic ring is unsubstituted or substituted with one or more R 4 substituents; 6) R8 (R7 R7 C) m -CO-, wherein m = 1-3 and R8, R4 and R7 are independently of the others: a) hydrogen, b) chlorine or fluorine, c) unsubstituted alkyl or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, d) hydroxyl, e) phenyl or naphthyl, unsubstituted or substituted by one or more substituents R4, f) C1 -C3 alkoxy, g) a 5-7 membered rings, or h) R 5, R 6 and R 7 may be independently of one another joined together to form a monocyclic, bicyclic or tricyclic ring system, each of which is cycloalkyl-1 ° C 3 -C 6 R 6 and R 7 are as defined above, except that R 5, R 6 and R 7 are not chlorine, fluorine or hydroxyl if they are adjacent to W; 8) R8 -W- wherein R8 is a 5-7 membered heterocycle, such as pyridyl, furyl or benzisoxazolyl; 9) R 9 -W- wherein R 9 is phenyl or naphthyl, unsubstituted or substituted by one or more substituents R 4; 10) phenyl; 11) wherein R1 is C1-4 alkyl or R8-P (O) (OR1): R8 is selected from the group consisting of: - or R 9 -P (O) (OR 11) -; R6 and R7 -CH2 R12 wherein R12 is a) NH-A wherein A is as defined above, b) R5 - (R6 R7 C) m -; c) R5- (R6R7C) mV-, wherein V is 0 or NH, except that R5 is not chlorine, fluorine or hydroxyl if they are adjacent to V, d) R4 (R6R7C) m -S (O) n, R6 and R7 are as defined above, except that R5, R6 and R7 are not chlorine, fluorine or hydroxyl if they are adjacent to the sulfur, e) R8 -S (O) ) n-, an independently of the other: i) C1-4 alkyl, - (C1-6 alkyl), - (C1-6 alkyl) (I) n (r 10) 2, j) NR 15 R 16, wherein R 15 and R 16 are attached in such a manner as to form a compound of the formula wherein R 15, R 16, (i) azetidinyl, (ii) pyrrolidinyl, (iii) piperidinyl, or (iv) morpholinyl, (i) R17QCH20 wherein R17 is: (i) R 9, or iii) CH 2 Ar where Ar is phenyl, naphthyl or a 5-7 membered heterocycle, (1) wherein R 2, n) N-imidazolyl wherein the imidazole ring is unsubstituted or substituted by a substituent R 4, n) N-benzimidazolyl, wherein the condensed benzene ring is unsubstituted or substituted by one or more R4 substituents, (o) C2 -C6 alkynyl, optionally substituted with one or more R9 groups; or p) C2 -C8 alkenyl, optionally substituted with one or more R9 groups; 2) hydrogen, (3) C 1 -C 4 alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C 1 -C 3 cycloalkyl, characterized in that 1) formula: 72 875 SB CASE 14514 in which R 1, R 2, X 1 and X 2 are as defined above, with an acid and 2) optionally removing any protecting groups. J A process according to claim 1, wherein R 1 is C 1 -C 6 alkyl, benzyloxymethyl, 3-phenylpropyl or benzyloxy, A process as claimed in claim 2, wherein X is CbzAla, AlaAla, Val, CbzVal, Cbz or hydrogen. A process as claimed in claim 1, wherein R * is benzyloxymethyl. 5. A process as claimed in claim 1, wherein R 1 is 3-phenylpropyl. A process as claimed in claim 1, wherein the acid is aqueous acetic acid. 7. A compound according to claim 1, wherein the compound prepared exhibits a protease activity inhibition constant of less than about 10 μg. 8. A pharmaceutical composition according to claim 1, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier. A process for the preparation of a compound of the formula: 875 SB CASE 14514 -49- > 4λ in which R * is: 1) a) NH-A wherein A and R1-2 are as defined in claim 1; b) R 5 - (R 6 R 7 C) m - (c) R 5 - (R 6 R 7 C) mV-, wherein V is O or NH, except that R 5, R 5 and R 7 are not hydroxyl, chlorine or fluorine if they are adjacent to V, d R8 and R7 are as defined above, except that R8, R6 and R7 are not hydroxyl, chlorine or fluorine if they are adjacent to the sulfur, e) R8 -R8 (R6R7C) mS, where m = 1-3 and R8, R9 and R7 are as defined above, , wherein R 13 and R 14 are each independently of the other: i) C 1 -C 6 alkyl, ii) C 3 -C 6 cycloalkyl, iii) H, iv) R9, or v) R8, h) i) j) form one comprising: R13P (O) (OR14) -, n (r10) 2, NR18R * 6, where R18 and R16 are heterocyclically linked to 4 (Iii) piperidinyl, or iv) morpholinyl, where R17 is: (i) C1-6 alkyl, or (ii) R9, or Iii) CH 2 Ar where Ar is phenyl, naphthyl or a 5-7 membered heterocycle, 72 875 SB CASE 14514 -50- 1) r17och2ch2och2 m) N-imidazolyl wherein the imidazole ring is unsubstituted or substituted with a substituent R4, n) N-benzimidazolyl, wherein the fused benzene ring is unsubstituted or substituted with one or more C2 -C6 o) alkynyl, optionally substituted with one or more R 9 groups; or p) C2-C6 alkenyl, optionally substituted with one or more R9 groups; 2) hydrogen, 3) C 1 -C 6 alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C 3 -C 7 cycloalkyl, and R 9 and R 9 are hydrogen, an amino protecting group or together are N2, characterized in that it comprises 1) reacting a compound of the formula: O with a compound R-Z, wherein Z is the moiety which renders R nucleophilic, 2) converting the resulting hydroxy groups into displaceable groups, 3) reacting the displaceable groups with a nucleophile with nitrogen. A process for the preparation of a compound of the formula: in which R 'is: 1) a) NH-A wherein A and R 5 -R 10 are as defined in claim 1; b) R 5 - (R 6 R 7 C) m -; Wherein R is 0 or NH, except that R 5, R 6 and R 7 are not hydroxyl, chlorine or fluorine if they are adjacent to V, d) R6 and R7 are as defined above, except that R5, R6 and R7 are not hydroxyl, chlorine or fluorine if they are adjacent to the sulfur, e) R8 -S (O) n -, f) R9 -S (O) n -, g) (R130) P (O) (OR14) - wherein R13 and R14 are each independently of the other : (i) C 1 -C 4 alkyl, (ii) C 3 -C 6 cycloalkyl, iii) H, iv) R 9, or V) R 8, h) R 13 P (O) (I) azetidinyl, ii) pyrrolidinyl, iii) piperidinyl, or iv) morpholinyl, k) wherein R17 is: i) alkyl ii) R9, or iii) CH2 Ar wherein Ar is phenyl, naphthyl or a 5-7 membered heterocycle, (1) wherein n is imidazolyl wherein the imidazole ring is unsubstituted or substituted with a substituent R4, n) N-benzimidazolyl, where the benzene ring the condensate is unsubstituted or substituted with one or more R4 substituents, (o) C2 -C6 alkynyl, optionally substituted with one or more R9 groups; or (b) C2 -C6 alkenyl, optionally substituted with one or more R9 groups; 2) hydrogen, 3) C 1 -C 6 alkyl, unsubstituted or substituted with one or more chlorine or fluorine atoms or hydroxyl groups, or 4) C 3 -C 7 cycloalkyl, characterized in that it comprises 1) reacting a compound of the formula: in which R is defined as in claim 10 and T is a displaceable group, with azide. Lisbon, -JUL 1991 By SMITHKLINE BEECHAM CORPORATION = 0 OFFICIAL AGENT =