HRP930283A2 - Antiviral peptides - Google Patents

Description

This invention relates to certain peptide derivatives containing a heterocyclic group which are useful in the treatment or prophylaxis of infections caused by retroviruses in humans.

The human immunodeficiency virus (HIV), a retrovirus, is the cause of various clinical conditions, the most serious of which is commonly called AIDS (acquired immunodeficiency syndrome) and ARC (AIDS - associated complex). Infection caused by HIV is characterized by progressive deterioration of the immune system and dysfunction of the CNS. Severely immunodeficient patients suffer from many opportunistic infections (eg, Pneumocystis carinii, human cytomegalovirus, or Candida), and neoplasms such as Kaposi's sarcoma. The loss of cells, especially CD4+ lymphocytes, that follows HIV infection is an important factor in the progressive weakening of immune function. Infection of cells of the monocyte/macrophage lineage with HIV also contributes to the established pathology. Thus, the successful infection of CD4+ cells by HIV is a key step in the development of the disease.

HIV is a retrovirus; it encodes its genetic information into RNA, which is converted into DNA after the virus enters the host cell. The basic step in the retrovirus replication cycle is the production of the initial polypeptide precursor into mature structural and replicative proteins. This process is carried out by means of a virus-encoded protease and, in the absence of the activity of this enzyme, viral replication is blocked.

We have found that certain peptide derivatives attached to the heterocyclic group are potent inhibitors of retrovirus proteases, both in free cell and infected cell assays, and also exhibit antiviral activity in tissue culture assays. This activity makes such compounds useful for the treatment and prophylaxis of retroviral infections, particularly those caused by HIV.

Thus, the present invention encompasses compounds having the formula

[image]

and their pharmaceutically acceptable salts and their bioprecursors, where:

R1 is C1-C6 alkyl, C3-C8 cycloalkyl, aryl, heterocyclyl or CONR9R10;

R2 is C1-C6 alkyl, C3-C8 cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl or heterocyclyl(C1-C4)alkyl;

R3 is C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl, aryl(C2-C4)alkenyl, heterocyclyl(C1-C4)alkyl or heterocyclyl(C2 -C4)alkenyl;

R 4 is C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, aryl or heterocyclyl;

each of R 5 , R 6 , R 7 and R 8 is independently H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl; or R 5 and R 6 , or R 7 and R 8 may be bonded together to form a three to eight membered carbocyclic ring;

X is a 4- to 10-membered mono- or bicyclic heterocyclic group containing carbon ring atoms and one nitrogen ring atom through which the group is attached to an adjacent carbonyl group; the group may be saturated or partially unsaturated and, in addition to the -(CR7R8)m-Het substituent, may be substituted with up to 4 further substituents each independently selected from F, C1-C6 alkyl, C3-C8 cycloalkyl, OR11 or NR9R10;

Het is an imidazolyl or triazolyl group, each of which may be optionally substituted with C1-C6 alkyl, C3-C8 cycloalkyl, NR9R10 or CONR9R10,

Each of R9 and R10 is independently H, C1-C6 alkyl or C3-C8 cycloalkyl, or R9 and R10 may be bonded together and form, with the nitrogen to which they are bonded, a 4- to 8-membered nitrogen- containing heterocyclic group,

R 11 is H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl; and n and m are each independently 0, 1 or 2; wherein each alkyl or cycloalkyl group included in the aforementioned definitions may optionally be fully or partially substituted with fluorine.

In the above definition of R 1 , R 2 , R 3 and R 4 , heterocyclyl means a 4 to 6-membered heterocyclic group containing as heteroatoms up to four nitrogen atoms, or an oxygen or sulfur atom optionally with one or two nitrogen atoms. The ring may be aromatic, or fully or partially saturated and may optionally be benzo-fused or substituted with C1-C6 alkyl, C3-C8 cycloalkyl, C2-C5 alkanoyl, C1-C4 alkoxy, halo, hydroxy, oxo or aryl. Preferred heterocyclyl groups are pyridyl, pyrimidinyl, thienyl, isoquinolyl and tetrazolyl.

In the above definitions of R1, R2, R3 and R4, aryl means phenyl optionally substituted with from 1 to 3 substituents each independently selected from C1-C6 alkyl, C3-C8 cycloalkyl, C1-C4 alkoxy, C2-C5 alkanoyl, hydroxy, halo, C1 -C4 alkyl fully or partially substituted with fluorine, C1-C4 alkoxy fully or partially substituted with fluorine, phenyl, phenoxy, benzyl, benzoyl, phenylSO2-, pyridyl, tetrazolyl, phenyltetrazolyl, NR9R10 or CONR9R10; where R9 and R10 are as defined earlier. Halo means fluoro, chloro, bromo or iodo.

An imidazolyl or triazolyl group, Het, may be attached to either a ring carbon or a ring nitrogen and may be unsubstituted or mono-, di-, or tri-substituted. Triazolyl includes both 1,2,3 and 1,2,4-triazolyl groups.

Alkyl and alkoxy groups containing 3 or more carbon atoms can be branched or straight chain. Any alkyl, alkoxy or cycloalkyl group included in the above definitions may optionally be fully or partially substituted with fluorine.

The term bioprecursor in the above definition means a pharmaceutically acceptable derivative of a compound of formula (I) that can be biologically degraded, which, when applied to an animal or a human, is converted in the organism to produce a compound of formula (I). Examples include ester derivatives formed between a free hydroxy group in a compound of formula (I) and, for example, an amino acid (such as L-valine).

It is preferred that the compound of formula (I) has numerous asymmetric carbon atoms and the invention includes all possible stereoisomers whether separated or not.

In one particular and preferred aspect of the invention there are compounds having the following stereochemistry-:

[image]

In the formula above, heavy bonds are used to show that the group lies above the plane of the molecule while broken bonds are used to show that the group lies below.

In the definition of R1, aryl is preferably phenyl and heterocyclyl is preferably oxetan-3-yl or 1,1-dioxothiethane-3-yl. R1 is preferably t-butyl, isopropyl, oxetan-3-yl or 1,1-dioxothiethan-3-yl and (CR5R6)n is absent; or R1 is phenyl and (CR5R6)n is CH2; or R1 is H2NCO-, CH3NHCO- or (CH3)2NCO- and (CR5R6)n is CH2 or CH(CH3). Especially preferred are compounds where R1 is t-butyl, isopropyl or oxetan-3-yl and n is at most where R1(CR5R6)n- is t-butyl.

In the definition of R2, aryl is preferably phenyl and heterocyclyl is e.g. pyridyl, pyrimidinyl or thienyl. R 2 is preferably aryl(C 1 -C 4 )alkyl; benzyl is particularly preferred.

In the definition of R4, aryl is preferably phenyl and heterocyclyl is preferably pyridyl, pyrimidinyl or thienyl. R4 is preferably C1-C6 alkyl; isopropyl and sec-butyl (valine or isoleucine derivatives) are particularly preferred.

The heterocyclic group X is preferably a 4-6-membered saturated or monounsaturated group and is most preferably an azetidine, pyrrolidine, tetrahydropyridine or piperidine group; piperidine is particularly preferred.

R7 and R8 are preferably H and m is preferably 0 or 1.

In the definition of R3, aryl is phenyl, unsubstituted or substituted as the term aryl is defined above, and heterocyclyl is e.g. pyridyl, pyrimidinyl, isoquinolyl or thienyl. R 3 is preferably aryl(C 1 -C 4 )alkyl or aryl(C 2 -C 4 )alkenyl; R 3 is most preferably benzyl optionally substituted in the phenyl ring with fluoro, chloro, iodo, methyl, trifluoromethyl or trifluoromethoxy, or R is 3-phenyl-prop-2-enyl or 3-phenylpropyl.

Special and desirable individual compounds include:

1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazole -1-yl)azetidine,

1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazole -1-yl) piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(3-phenylprop-2-en-1-yl)hexanoyl )-(S)-valyl]-4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl] -4-(imidazol-1-yl)piperidine,

1-[N-((S)-5-(t-butoxycarbonylamino)-(R)-2-(4-chlorobenzyl)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]- 3-(imidazol-1-yl)azetidine and

1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl] -4-(imidazol-1-yl)piperidine.

In another aspect of the present invention, there is a compound of formula (I), or a pharmaceutically acceptable salt thereof or a bioprecursor thereof, for use as a medicament, particularly for use in the treatment and prophylaxis of human retroviral infections, particularly HIV infections. The invention also includes the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof or a bioprecursor thereof, for the production of a medicament used in the prophylaxis and treatment of retroviral infections.

The invention further includes a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof or a bioprecursor thereof, and a pharmaceutically acceptable diluent or carrier.

The antiviral activity of compounds of the general formula (I) was determined using an in vitro test system. For example compounds of formula (I) are able to fully protect human T-cells of the H9 line for 7 days against the progressive effects of HIV infection. Untreated, virus-infected cells show typical cytopathic effects such as syncytial formation and cell death. In addition, virus particles produced from virus-infected cells treated with a compound of formula (I) are non-infectious.

Examples of infections that can be treated or prevented with compounds of formula (I) include those caused by human or animal retroviruses, particularly HIV-1. Clinical conditions that can thus be treated or prevented include AIDS, ARC, and HIV-associated dementia. The compounds can also be used to stop the progression of the disease in infected individuals without symptoms.

Compounds of formula (I) can be prepared using coupling and protection techniques known to those skilled in the art of peptide chemistry.

The procedure is illustrated by a series of examples using the following methods for the preparation of compounds of formula (Ia):

The procedure highlighted in Scheme A starts with the protected lactone (II). It is alkylated, using e.g. n-butyllithium or lithium hexamethyldisilazide followed by addition of a compound of formula R3 Br and separation of the desired isomer to give product (III). The lactone ring is then opened by treatment with dilute alkali to give the corresponding hydroxy acid and the hydroxy group is consequently protected by e.g. as a t-butyldimethylsilyl derivative, by reaction with t-butyldimethylsilyl chloride in N,N-dimethylformamide, followed by hydrolysis of the protected ester to give intermediate (IV).

Coupling of this intermediate with another fragment (VIII) to give a compound of formula (Ia) is illustrated in Scheme B. In this procedure, the protecting group on intermediate (V) is removed (in the case of t-butoxycarbonyl by treatment with HCl) and the resulting compound (VI) is reacted with the N-protected amino acid Boc-NHCR(R4)CO2H, using an amide as the coupling compound, to give intermediate (VII). The N-protecting group from (VII) is then removed and the amine product (VIII) is coupled with intermediate (IV) to give intermediate (IX). The hydroxy protecting group X1 is then removed from (IX) to give the final product of formula (Ia). In an alternative process, intermediate (IX) can be produced by coupling intermediate (X) with intermediate (VI). Intermediate (X) can be prepared from intermediate (IV) by reaction with the carboxyl-protected amino acid H2NHC(R4)CO2 R followed by basic hydrolysis.

[image]

Protecting group, X': t-Butyldimethylsilyl is preferred R1-(CR5R6)n: t-Butyl or benzyl is preferred

[image]

Amide coupling compounds:-eg, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

Protective groups: - P; t-butoxycarbonyl or benzyloxycarbonyl are preferred

X'; t-butyldimethylsilyl is preferred

R; methyl or ethyl are preferred

In another alternative process illustrated in Scheme C, removal of the t-butoxycarbonyl group from intermediate (XI) by treatment with an acid, e.g. with trifluoroacetic acid, and then reacting with a carbon derivative of formula (XII) gives a product of formula (XIII) in which the group R1(CR5R6)n is something other than t-butyl. For example reaction of the product of formula (XI) with 3-oxetanyloxy-carbonyloxysuccinimide gives the product (XIII) where R1 - is 3-oxetanyl and n is 0.

[image]

X' is either H or a protecting group, eg, t-butyldimethylsilyl.

Y is a group that is susceptible to nucleophilic displacement - succinimidooxy is preferred.

The starting materials of formula (V) required for the process described earlier are in some cases compounds from the literature or can be prepared by routine synthetic procedures from already available starting materials. So, for example reaction of 1(N-t-butoxycarbonyl)-3-hydroxy-azetidine with methanesulfonyl chloride followed by reaction with a compound of the formula Het-H in the presence of a base gives the desired compounds of the formula (V) where X is an azetidine ring. Alternatively, e.g. 1-(N-Benzyloxycarbonyl)-4-ketopiperidine is converted to the 4-amino derivative by reaction with sodium cyanoborohydride in the presence of ammonium acetate. This is followed by a reaction with dimethylformamide azine, which gives the 4-(1,2,4-triazol-4-yl) derivative.

Reaction of 1-(N-t-butoxycarbonyl)-4-keto-piperidine with the anion obtained by the reaction of 1-diethoxymethyl)imidazole with n-butyllithium followed by treatment of the product with methanesulfonyl chloride in the presence of base gives N-protected-4-imidazole-2 -yl(1,2,5,6-tetrahydropyridine) intermediate. Catalytic hydrogenation gives the corresponding N-protected-4-(imidazol-2-yl)-piperidine. Reaction of the above keto compound directly with a heterocyclic compound, e.g. with imidazole, in the presence of thionyl chloride gives compounds of formula (V) where X is a tetrahydropyridine group, reduction again gives a piperidine derivative.

Intermediates of formula (V) in Scheme B where m is not equal to 0 can be prepared by standard transformation from the appropriate precursors using, e.g., nucleophilic opening of the epoxide group to introduce a Het group. So, for example reaction of 1-(N-t-butoxycarbonyl)-4-ketopyridine with trimethylsulfonium iodide in the presence of base gives 4-spiro-2-oxirane. Reaction of this product with imidazole followed by removal of the 4-hydroxy group gives 1-(N-t-butoxycarbonyl)-4-(imidazol-1-yl)methyl-1,2,5,6-tetrahydropyridine; reduction gives the corresponding piperidine derivative.

The naturally occurring N-t-butoxycarbonyl (BOC-) derivatives of amino acids used in the synthesis of compounds of formula (VII) are commercially available as are their hydroxysuccinimido esters. The corresponding intermediates derived from unnatural amino acids can be prepared by standard procedures (see, eg, M. J. O'Donnell et al. J. Amer. Chem. Soc., 1989, 111, 2353). Compounds of formula (II) can be prepared from the corresponding t-butyloxycarbonyl-protected amino aldehydes (see D. H. Rich et al. J. Org. Chem., 1978, 43, 3624 and Y. Hamada et al, Chem. Pharm. Bull., 1982, 30(5), 1921) by reaction with ethyl propiolate (see A. H. Fray et al, J. Org. Chem:, 1986. 51, 4828), followed by reduction to give 5-t-butyloxycarbonylamino-4-hydroxy -6-phenylhexanoate. Cyclization by means of reflux in toluene then gives the lactones of formula (II), as a mixture of diastereomers which can be separated by standard procedures.

In the above methods and the specific examples presented herein, certain hydroxy and amino-protecting and carboxy-activating groups are required. Those skilled in the art will appreciate that the coupling and protection procedures described herein can be performed using any standard method for peptide synthesis, and these procedures are therefore included within the scope of this invention. The choice of an individual protective group depends largely on the availability of the necessary reagent, its effect on the solubility of the protected compound, the ease of its removal in the presence of other groups that may be damaged by its use. For example, it is necessary in the above procedure to protect and deprotect a specific amino group to enable further reaction on the restored amino group and the choice of protecting group for a given amino group will depend on the role of the said amino group in the overall reaction scheme. Amino protecting groups having different levels of instability can be used. Such groups are known in the art and attention is directed to the works of: Bodansky et al., "Peptide Synthesis", 2nd Ed., John Wiley and Sons, N.Y. (1976); Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, N.Y. (1981); McOmie, "Protective Groups in Organic Chemistry", Plenum Press, N.Y. (1973); and to Sheppard in "Comprehensive Organic Chemistry, The Synthesis and Reactions of Organic Compounds", Pergamon Press, N.Y. (1979), edited by E. Haslam, Part 23.6, pages 321-339.

Representative amino groups include, but are not limited to, aryloxycarbonyl such as benzyloxycarbonyl; substituted or unsubstituted aralkyl such as benzyl, trityl, benzhydryl and 4-nitrobenzyl; benzylidene; arylthio such as phenylthio, nitrophenylthio and trichlorophenylthio; phosphoryl derivatives such as dimethylphosphoryl and 0,0-dibenzylphosphoryl; trialkylsilyl derivatives such as trimethylsilyl; and others as described in U.S. Pat. But. 4, 322, 341. A preferred amino protecting group for use in the above sequence is t-butoxy-carbonyl. Procedures for the substitution of said groups on a given amino group are well known. In general, they involve acylation of the appropriate amino compound with the appropriate carbonyl chloride or anhydride in a reaction-inert solvent, e.g. water, methylene chloride or tetrahydrofuran, in the presence of a base (acid acceptor) eg sodium or potassium hydroxide when water is the solvent; and when an organic solvent is used, in the presence of a tertiary amine such as triethylamine or pyridine. When using an aqueous solvent system, the pH of the reaction is typically kept at around pH 8-10, and preferably pH 9.

Protected amino groups are converted to unprotected amino groups by methods known to those skilled in the art, as appropriate for the particular groups represented. The t-butoxycarbonyl group is, for example, easily removed by treatment with dichloromethane saturated with hydrogen chloride gas.

Various hydroxy-protecting groups are also known and described in the literature sources mentioned earlier. A preferred hydroxy protecting group is t-butyldimethylsilyl. It is introduced as described earlier and is easily removed by treatment with tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature.

Activation of the carboxy group as a means of promoting a given acylation reaction is also a methodology known to those skilled in the art. Particularly useful in the reactions described here is the use of anhydrides and activated esters, especially those esters obtained from N-hydroxyphthalimide, N-hydroxysuccinimide or 1-hydroxybenzotriazole, all of which are used in peptide synthesis.

A dehydrated coupling agent is used to form the activated ester. Representatives of such coupling agents are 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide, N,N'dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, ethoxyacetylene, diphenylketene and N -ethyl-5-phenylisoxazoline-3'-sulfonate. Reaction conditions for the use of such coupling agents are well described in the literature. In general, they involve the use of a reaction-inert solvent and temperatures ranging from ambient to . The carbodiimide reagents mentioned above are the most valued because they allow the use of ambient reaction temperatures and provide satisfactory amounts of the desired esters.

Upon completion of the coupling reactions leading to the final product, the various protecting groups can be removed using the appropriate techniques discussed earlier, and the compounds of formula (I) isolated and purified using conventional procedures such as recrystallization or column chromatography.

The above sequences can be adapted as appropriate to perform any of the required variants for R1 to R8, X and Het by appropriate choice of starting materials.

Thus, in accordance with the following aspect, the invention includes a process for the preparation of a compound of formula (I) which comprises the removal of a protective group from a compound whose formula is:

[image]

wherein X 1 is a selectively movable hydroxy-protecting group, and isolating a compound of formula (I) and optionally forming a pharmaceutically acceptable salt thereof.

A preferred protecting group for X1 is t-butyldimethylsilyl; it is removed by treatment with tetra-n-butylammonium fluoride in an organic solvent, preferably tetrahydrofuran.

Novel intermediates of formulas (VIII), (IX), and (X) also form part of this invention.

Examples of pharmaceutically acceptable salts of compounds (I) are salts with an acidic addition, e.g. sulfates, bisulfates, phosphates, lactates, mesylates, fumarates, citrates, succinates and gluconates.

In the treatment of patients infected with a retrovirus, especially HIV, the compounds (I) will be administered by any suitable route, e.g. by oral, parenteral (eg subcutaneous, intravenous, intramuscular or intradermal), rectal, nasal, topical (including buccal and sublingual) or vaginal routes. Formulations, which will contain an antiviral agent of the present invention together with one or more pharmaceutically acceptable carriers and optionally other therapeutic agents, may be prepared according to conventional techniques well known in the art of pharmacy. Forms for oral administration include, in particular, syrups, tablets and capsules which may contain flavor enhancers in addition to some inert carrier. Tablets can be produced using conventional compression or molding techniques by pressing powders of the appropriate ingredients, e.g. an antiviral substance combined with a binder, a solvent, a fat agent and a surfactant. Preparations for rectal use will be in the form of suppositories, and vaginal as, for example, tampons, creams or foams. Parenteral preparations will be in sterile form, e.g. as vials for injections containing aqueous or non-aqueous solvents, buffers and antioxidants so that the preparation will be isotonic with blood. In general, an appropriate dosage of the anti-retroviral agent of formula (I) will be from 1-50 mg/kg/day, preferably 1-25 mg/kg/day administered in up to six divided doses per day. There may also be cases where a higher or lower dose is determined according to age, body weight, degree of disease and response of the patient, and the appropriate therapy will be determined by the medical practitioner.

Compounds of formula (I) can be used in combination with other drugs, some of which can enhance their action. Such substances include the following:

(a) Reverse transcriptase inhibitors such as AZT, ddI, ddC, foscarnet, TIBO compounds, dipyridodiazepinones or 6-substituted acyclopyrimidine derivatives (HFPT);

(b) gp120/CD4 blockers such as dextran sulfate and soluble CD4, including its combinations with toxicants such as Pseudomonas toxin

(c) tat antagonists, such as D-penicillamine

(d) other retroviral protease inhibitors, such as Ro 31-8959; and

(e) substances that modify the biological response including interferons, interleukins or colony-stimulating factors, e.g. GM-CSF.

Compounds of the present invention were evaluated for antiviral activity by dissolving the test compound in 50 µl of DMSO and diluting in RPMI 1640, a complex salt solution with a pH of 7.2 to 1 mg/ml. Testing was performed at 0.001, 0.01, 0.1, 1 and 10 µg/ml against HIV 1 (strain IIIB) on a human T-cell line (H9). Untreated control infections were initiated at the same time.

Seven days after infection, tissue culture supernatants were titrated for the presence of infectious virus on C8166 cells (a human T-cell line). 3,5, and 7 days of culture were examined for the appearance of syncytia. Controlled infections untreated with the drug show typical viral cytopathic effects, including syncytial formation and cell death. IC100 is the smallest test concentration that allows complete protection of the culture. Using this test method, the compounds had IC100 values ranging from 0.1 to 10.0 µg/ml.

The preparation of certain starting materials and compounds of formula (I) will now be illustrated in more detail by means of the following experimental examples. The purity of the compounds was routinely monitored by thin-layer chromatography using Merck Kieselgel 60 F254 plates. 1H-NMR spectra were determined using a Nicolet QE-300 or Bruker AC-300 spectrometer and were in all cases consistent with the proposed structures. Chemical shifts are expressed in parts-per-million lower than tetramethylsilane using common abbreviations to designate major peaks: s, singlet; d, double; t, triple; m, multiple and b, broad. Optical rotations were read at a concentration of 0.1% in methanol until otherwise specified. All temperatures are in degrees Celsius.

Preparation of starting materials and intermediate products

Preparation 1

(S)-5-[(S)-1-t-Butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone)

a) Ethyl (4S, 5S)- and (4R, 5S)-5-t-butoxycarbonylamino-4-hydroxy-6-phenylhex-2-inoate

A solution of diisopropylamine (6.4 ml) in dry tetrahydrofuran (25 ml) was stirred under nitrogen and a 1.6 molar solution of n-butyllithium in hexane (24.4 ml) was added over 5 minutes, keeping the temperature below . After the next 15 minutes, the solution is cooled to and ethyl propiolate () is added dropwise over 10 minutes, keeping the temperature below . The resulting yellow suspension is stirred for a further 20 minutes and then treated dropwise, over 10 minutes, with a solution of N-t-butoxycarbonyl-L-phenylalaninal (see J.R. Luly et al, J. Org. Chem. 1987, 52, 1487) in with dry tetrahydrofuran (15 ml), again keeping the temperature below . The clear yellow solution was stirred for 2 hours and then treated with acetic acid (4 ml). The cooling bath was removed and the mixture was allowed to warm at which point water (100 ml) and ethyl acetate (100 ml) were added with vigorous stirring. Separation of the organic layer, followed by washing with 1 M hydrochloric acid (50 ml), saturated aqueous sodium bicarbonate (50 ml) and saturated brine (50 ml), gave the crude product as an oil after drying (Na2SO4) and evaporation of the solvent. The oil was purified by silica gel chromatography using ethyl acetate-hexane (1:4) as eluent. Evaporation of the fractions containing the product yields an oil which solidifies on standing overnight. Recrystallization from ether-hexane affords the title compound as an approximately 2:1 mixture of (4S, 5S:4R, 5S) diastereomers, (), mp 98-99°. Found: C,65.62; H, 7.42; N, 4.33. C19H25NO5 required C,65,70; H, 7.20; N, 4.03%.

N.M.R. (CDCl3)δ = 1.30-1.39 (m, 3H); 1.43 (s, 9H); 2.90-3.11 (m, 2H); 3.37-3.38 and 4.16-4.19 (2x brm, 1H); 3.93-4.04 (m, 1H); 4.22-4.33 (m, 2H); 4.51-4.56 (m, 1H); 4.77-4.79 and 4.87-4.90 (2x brm, 1H); 7.24-7.35 (m, 5H).

b) Ethyl (4S, 5S) - and (4R, 5S)-5-t-butoxycarbonylamino-4-hydroxy-6-phenyhexanoate

The above product () is dissolved in ethanol (50 mg) and 5% Pd-BaSO4 catalyst is added. The mixture is then hydrogenated at 50 psi (344.7 kPa) for 2 hours. Filtration, followed by evaporation of the solvent under vacuum, gave the title compounds (approximately 2:1 mixture of diastereomers) as a white solid, (), m.p. 125-126°. Found: C,64.91; H, 8.40; N,3.98, C19H29NO5 requires C,64.95; H, 8.26; N, 3.98%.

N.M.R. (CDCl3)δ = 1.24-1.33 (m, 3H); 1.39 and 1.42 (2x S, 9H); 1.72-1.95 (m, 2H); 2.38-2.62 (m, 2H), 2.77-2.98 (m, 2H); 3.02-3.04 and 3.40-3.42 (2 x m, 1H exch. D2O); 3.59-3.91 (m, 2H); 4.08-4.22 (m, 2H); 4.58-4.61 and 4.86-4.89 (2 x m, 1H); 7.22-7.37 (m, 5H).

c) (S)-5-[(S)-1-t-Butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone

The gamma-hydroxyester from (b) above is dissolved in 2.5% acetic acid-toluene (35 ml) and the solution is heated at reflux for 2 hours. After cooling and evaporation to dryness under vacuum, the residue was purified by silica gel chromatography, eluting with diethyl ether-hexane (40:60), to give the title compound (), m.p. 98-99°. Found: C,66.77; H, 7.78; N, 4.38. C17H23NO4 requires: C,66.88; H, 7.54; N, 4.59%. m/e = 306 (MH+).

N.M.R. (CDCl3)δ = 1.42 (s, 9H); 2.11-2.19 (m, 2H); 2.51-2.58 (m, 2H); 2.87-3.02 (m, 2H); 4.00-4.07 (m, 1H); 4.47-4.52 (m, 1H); 4.63 (d, J=10, NH); 7.26-7.36 (m, 5H); [image] -22.60 (C=1, MeOH).

IR (KBr) 1775, 1690, 1525 cm-1.

Preparation 2

(R)-2-Benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoic acid

a) (R)-3-Benzyl-(S)-5-[(S)-1-t-butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone.

A cold solution (-10°) of hexamethyldisilazane (7.9 ml) in tetrahydrofuran (15 ml) is treated over 3 minutes with n-butyllithium in hexane (23 ml), maintaining the temperature below . After the next 5 minutes at 0°, the solution is cooled to -70° and a solution of (S)-5-[(S)-1-t-butoxycarbonylamino-2-phenylethyl]-gamma-butyrolactone (5g) in tetrahydrofuran (38) is added ml), by keeping the temperature below -65°. The solution was stirred at -70° for 15 minutes before benzyl bromide (1.95 ml) in tetrahydrofuran (12.5 ml) was added over 1 minute and the solution was stirred for an additional 10 minutes at -70° before being treated with acetic acid ( 6.5 ml) and remove the cooling bath. Water (50 ml) and ethyl acetate (50 ml) were added and the mixture was allowed to warm to room temperature. Separation of the organic layer, followed by drying (MgSO4) and evaporation of the solvent under vacuum gives the crude product in the form of an oil. Chromatography on silica gel, eluting with diethyl ether hexane (50:50=, gives the title compound as a pure oil (). Found: C,73.22; H,7.50; N,3.50. C24H29NO4 requires C,72.91; H,7.34; N ,3.54%.

[image] -14° (C=0.1%, MeOH)

N.M.R. (CDCl3)δ = 1.37 (s, 9H); 1.95-2.30 (m, 2H); 2.79-3.20 (m, 5H); 3.92-4.01 (m, 1H); 4.21-4.25 (m, 1H); 4.52-4.56 (m, 1H); 7.21-7.36 (m, 10H).

b) (R)-2-Benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoic acid

A suspension of the product from (a) () in dioxane (120 ml) and water (60 ml) is treated with sodium hydroxide (1N, 53.5 ml) at room temperature. The reaction was stirred for 23 hours before starting to acidify to pH 5 by adding acetic acid. After standing for the next 30 minutes, the precipitate is removed by filtration and washed with water. This solid was dissolved in ethyl acetate, dried (MgSO4) and evaporated under vacuum to a white solid which was triturated with hexane, filtered and dried to give the intermediate hydroxy acid (). A solution of this hydroxy acid in N,N-dimethylformamide is treated with imidazole () and t-butyldimethylsilyl chloride () at room temperature. After stirring for 18 hours, the solvent is evaporated under vacuum, the residue is treated with ice/water, 10% citric acid to pH 4, and extracted using 2 x 400 ml of ethyl acetate. The combined extracts are dried (MgSO4) and evaporated under vacuum to a pale oil (). A solution of this oil in tetrahydrofuran (240 ml) is treated with acetic acid (240 ml) and water (80 ml) at room temperature. After stirring for 2 hours at room temperature and for 18 hours, the solution is evaporated under vacuum and the residue is partitioned between water (400 ml) and ethyl acetate (400 ml). The separated organic layer was washed with water (2 x 400 ml), saturated brine (100 ml), dried (MgSO4) and evaporated under vacuum to a pale oil. Chromatography on silica gel, eluting with diethyl ether hexane (70:30) gives the title compound in the form of a white glass ().

m/e 528 (MH)+

N.M.R. (DMSO-d6)δ = 0.10 (s, 6H); 0.95 (s, 9H); 1.30 (m, 10H); 1.35 (m, 1H); 1.95 (m, 1H); 2.40 (m, 1H); 2.72 (m, 2H); 2.85 (m, 1H); 3.60 (m, 1H); 3.75 (m, 1H); 6.88 (d, 1H); 7.22 (m, 10H).

Preparation 3

5-Bromomethylisoquinoline

A solution of an isomeric mixture of 5- and 7-bromoisoquinoline (5:7, 40:60; ; see Glyde and Taylor, J. Chem. Soc. Perkin Trans 2,.1975, 1783), in dry tetrahydrofuran (15 ml) is treated with 1.6M n-butyllithium in hexane (3.3 ml) at . The reaction was maintained at this temperature for 30 minutes and then a solution of dry dimethylformamide (0.74 ml) in dry tetrahydrofuran (5 ml) was added. After a further 15 minutes, the reaction was quenched with ethanol (5 ml) and allowed to warm to room temperature. Saturated ammonium chloride solution (10 ml) and diethyl ether (15 ml) were then added one after the other and the organic phase separated, washed with saturated brine, dried (MgSO4) and evaporated under vacuum. Purification by chromatography on silica gel eluting with hexane - ethyl acetate (50:50) and isolation of higher aldehydes gives 5-formylisoquinoline as an unstable yellow solid (). Rf 0.3 (hexane ethyl acetate 50:50).

A solution of this product () in dry methanol (20 ml) is treated with sodium borohydride () at and the resulting mixture is allowed to warm to room temperature and kept at that temperature for 1 hour. The solution was diluted with diethyl ether (20 ml) and then water (10 ml) was added. The two phases are separated and the aqueous phase is extracted with diethyl ether (20 ml). The two organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried (MgSO4), and evaporated under vacuum to give 5-hydroxymethyl-isoquinoline as a pale yellow powder, (), m.p. 71-. Found: C,75.06; H, 5.72; N, 8.66. C10H9NO requires C,75.45; H, 5.70; N, 8.80%.

A solution of the above product () in glacial acetic acid (15 ml) is treated with 49% aqueous hydrobromic acid (30 ml) and the resulting mixture is heated to reflux for 2 hours. The reaction is then concentrated under vacuum and the residue suspended in methylene chloride and basified with saturated aqueous sodium bicarbonate. The two phases are separated and the aqueous phase is extracted with methylene chloride. The combined organic phases were washed with saturated aqueous sodium bicarbonate, dried (MgSO4) for 0.5 hours and evaporated under vacuum at room temperature to give the title product as a colorless solid. The solid is azeotroped with toluene and then used directly ().

N.M.R. (CDCl 3 )δ = 4.90 (s, 2H); 7.50 (t, 1H); 7.70 (d, 1H); 7.90 (m, 2H); 8.65 (d 1H); 9.25 (s, 1H).

Preparation 4

7-Bromomethylisoquinoline

7- Formylisoquinoline is obtained from the initial step Preparation 3 isolated as a yellow solid (). Rf 0.25 (hexane - ethyl acetate 50:50). Reaction with sodium borohydride as described above gives 7-hydroxymethylisoquinoline, m.p. 129-.

A solution of the hydrochloride salt of the above product () in thionyl bromide (0.5 ml) is heated to and maintained for 45 minutes. The reaction was then cooled with ice and excess water carefully added, followed by diethyl ether (15 mL). Then, concentrated aqueous ammonia is added to pH 9 and the ether phase is separated, washed with water and dried (MgSO4). Then a solution of hydrogen chloride in isopropyl alcohol (0.06 ml, 5.9 N) is added and the resulting cloudy suspension is evaporated under vacuum at room temperature. The residue is azeotroped with toluene to give the product hydrochloride as a colorless solid ().

m/e (MH)+ 222

N.M.R. (DMSO-d6)δ = 5.05 (s, 2H); 8.10 (d, 1H); 8.25 (d, 1H); 8.30 (d, 1H); 8.45 (s, 1H); 8.65 (d, 1H); 9.70 (s, 1H).

Preparations 5-13

The following compounds of formula (IV) are prepared by the procedures described in Preparation 2 but using the appropriate substituted benzyl bromide or bromomethylisoquinoline to alkylate the gamma-butyrolactone in step (a) followed by ring opening and reaction with t-butyldimethylsilyl chloride as described in step (b).

[image]

[image]

Preparation 14

1-(N-t-Butoxycarbonyl)-3-methanesulfonyloxy-azetidine

a) Azetidin-3-ol hydrochloride () is stirred in methylene chloride (40 ml) and diisopropylethylamine () is added followed by di-t-butyldicarbonate (). The mixture is stirred at room temperature for 4 hours and the solvent is evaporated under vacuum. The residue was dissolved in ethyl acetate (200 ml) and washed with 1.5 M hydrochloric acid (50 ml), saturated sodium bicarbonate solution (25 ml) and brine (25 ml). The organic layer is dried (MgSO4), filtered and evaporated under vacuum.

Chromatography on silica gel, elution with ethyl acetate - hexane (50:50), gives 1-(N-t-butoxycarbonyl)-3-hydroxy-azetidine in the form of a white solid (), m.p. 51-. Found: C,55.29; H, 8.70; N, 7.98. C8H15NO3 requires C,55.47; H, 8.73; N, 8.09%.

b) A solution of the above product () in methylene chloride (35 ml) is treated with methanesulfonyl chloride (0.75 ml) and pyridine (1.5 ml) and the mixture is stirred for 3 days at room temperature. The solution was diluted with methylene chloride (75 ml), washed with aqueous citric acid (5%, 100 ml), saturated aqueous sodium bicarbonate (100 ml), dried (MgSO 4 ) and evaporated under vacuum to give the title product in in the form of a colorless oil (). Found: C,43.00; H, 6.80; N, 5.50. C9H17NO5S requires C,43.03; H, 6.77; N, 5.58%. m/e 269 (MNH4)+.

N.M.R. (DMSO-d6)δ = 1.37 (s, H); 3.24 (s, 3H); 3.88-3.96 (m, 2H); 4.17-4.28 (m, 2H); 5.25 (m, 1H).

Preparation 15

1-(N-Benzyloxycarbonyl)-(R)-3-methanesulfonyl-oxypyrrolidine

The title compound is prepared from 1-(N-benzyloxycarbonyl)-(R)-3-hydroxypyrrolidine (J.Med. Chem., 1992, 35, 1764), using the procedure described earlier in Preparation 14, except that triethylamine is used as the base instead of pyridine, to obtain the product in the form of an oil. Found: C,51.60; H, 5.80; N, 4.30. C13H17NO5S 1/6 H2O requires C,51.63; H, 5.78; N, 4.63%. m/e MH+ 300.

N.M.R. (CDCl3)δ = 2.15 (m, 1H); 2.55 (m, 1H); 3.0 (s, 3H); 3.45-3.80 (m, 4H); 5.10 (s, 2H); 5.25 (m, 1H); 7.30 (m, 5H).

Preparation 16

1-(N-Benzyloxycarbonyl)-(S)-3-(paratoluene)sulfonyl-oxypyrrolidine

To a solution of 1-(N-benzyloxycarbonyl)-(R)-3-hydroxypyrrolidine () in dry tetrahydrofuran (100 ml) under nitrogen was added triphenylphosphine (). The resulting solution was cooled to and methyl tosylate () was added, followed by diethyl azodicarboxylate () over a period of over 0.5 hours. 1 hour later, the reaction is allowed to warm to room temperature and maintained for 65 hours. The reaction is evaporated under vacuum, the residue is dissolved in dichloromethane, washed with water, dried (MgSO4) and evaporated under vacuum to an oil. Purification by silica gel chromatography eluting with methylene chloride - methanol (98:2 to 96:4), followed by a second chromatography eluting with hexane - ethyl acetate (80:20 to 50:50) gives the product as a golden colored oil ( ). Found: 0.60.59; H, 5.68; N, 3.67. C19H21NO5S requires C,60.78; H, 5.64; N, 3.73%

[image] + 9° (C=0.1%, MeOH)

N.M.R. (CDCl 3 )δ = 1.85-2.25 (m, 2H); 2.45 (s, 3H); 3.40-3.65 (m, 4H); 4.95-5.15 (m, 3H); 7.20-7.35 (m, 7H); 7.75 (d, 2H).

Preparation 17

1-(N-t-Butoxycarbonyl)-4-methanesulfonyloxypiperidine

The title compound was prepared from 1-(N-t-butoxycarbonyl)-4-hydroxypiperidine, according to the procedure described in preparation .p. 85-86° C. Found: C, 47.2; H, 7.66; N, 4.91. C11H21NO5S requires C,47.3; H, 7.58; N, 5.02%.

N.M.R. (CDCl3)δ = 1.5 (s, 9H); 1.85 (m, 2H); 2.0 (m, 2H); 3.08 (s, 3H); 3.35 (m, 2H); 3.75 (m, 2H); 4.9 (m, 1H).

Preparation 18

1-(N-t-Butoxycarbonyl)-3-(imidazol-1-yl)azetidine

A solution of imidazole () in N,N-dimethylformamide (30 ml) is treated with sodium hydride (60%, ) and the mixture is stirred at room temperature for 1 hour. 1-(N-t-Butoxycarbonyl)-3-methanesulfonyloxy-azetidine (preparation 14) () was added and the mixture was heated for 3 days. The solvent was then removed under vacuum and the residue was dissolved in ethyl acetate (50 ml), washed with water (2x50 ml), dried (MgSO4) and evaporated under vacuum to give a colorless oil. Purification by chromatography on silica gel eluting with methylene chloride-methanol-concentrated aqueous ammonia (93:7:1) gave the title compound as an oil ().

m/e 224 (MH)+

N.M.R. (DMSO-d6)δ = 1.40 (s, 9H); 3.95-4.06 (m, 2H); 4.26-4.35 (m, 2H); 5.12 (m.1H); 6.96 (s. 1H); 7.43 (s, 1H); 7.79

(s. 1H).

Preparations 19-25

The following compounds of formula (V) are prepared following the procedure described above in Preparation 18 but using the appropriate starting material from Preparations 14 to 17 and reacting with the appropriate substituted or supersubstituted imidazole or triazole.

[image]

Preparation 26

(a) 1-(N-t-Butoxycarbonyl)-4-(4-methylimidazol-1-yl)piperidine

The reaction of 4-methylimidazole with 1-(N-t-butoxy-carbonyl)-4-methanesulfonyloxypiperidine gives 2 regioisomeric products, which are separated by chromatography on silica gel, eluting with dichloromethane:methanol:concentrated aqueous ammonia (96:3.5:0.5). Larger isomer, Rf 0.47. m/e 265.9 (MH)+

N.M.R. (DMSO-d6) δ = 1.43 (s, 9H); 1.7 (dq, 2H); 1.93 (bd, 2H); 2.07 (s, 3H); 2.83 (m, 2H); 4.05 (m, 3H); 6.95 (s, 1H); 7.55 (s, 1H).

(b) 1-(N-t-Butoxycarbonyl)-4-(5-methylimidazol-1-yl)piperidine

Minor isomer, Rf 0.52. m/e 265.9 (MH)+

N.M.R. (DMSO-d6) δ = 1.43 (s, 9H); 1.7 (dq, 2H); 1.9 (bd, 2H); 2.05 (s, 3H); 2.88 (m, 2H); 4.05 (m, 3H); 6.6 (s, 1H); 7.65 (s, 1H).

Preparation 27

1-N-t-Butoxycarbonyl)-4-(imidazol-2-yl)-(1,2,5,6-tetrahydropyridine)

(a) 1-(Diethoxymethyl)imidazole () was stirred in dry tetrahydrofuran (50 ml) under nitrogen at . n-Butyllithium (25 ml, 1.6N in hexane) is added in such an amount that the temperature remains below . 1-(N-t-Butoxycarbonyl)-4-keto-piperidine () in dry tetrahydrofuran (10 ml) was added dropwise over 10 minutes, maintaining the temperature below , and stirring the resulting mixture for 2 hours. The reaction mixture was mixed with hydrochloric acid (50 ml, 0.1N) for 15 minutes, then ethyl acetate (50 ml) was added and the resulting mixture was stirred for 5 minutes. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (1x50 ml). The combined organic extracts are washed with a saturated solution of sodium bicarbonate (1x50 ml), then with a saturated solution of sodium chloride. The organic layer is then dried (MgSO4) and evaporated to a yellow oil, . Chromatography on silica gel, eluting with ethyl acetate:methanol:concentrated aqueous ammonia (90:10:1), gave 1-(N-t-butoxycarbonyl)-4-hydroxy-4-imidazol-2-ylpiperidine as a cream solid, m /e 268.0 (MH)+

(b) The above product (267 mg) was stirred with diisopropylamine (350 ml) in dry dimethylformamide (2 ml) at . Methanesulfonyl chloride (155 ml) was added in one portion, and the reaction mixture was stirred for 2 hours at . Second portions of diisopropylamine (350 ml) and methanesulfonyl chloride (155 ml) are then added and the reaction mixture is stirred for 16 hours at room temperature. The resulting mixture is diluted with water (4 ml), adjusted to pH 9 with 1M sodium hydroxide solution, and extracted with ethyl acetate (3x10 ml). The combined organic extract was dried (MgSO4), and evaporated to a resin, 260 mg. Chromatography on silica gel, eluting with dichloromethane: methanol: concentrated aqueous ammonia (95:5:1) gave the title product as a yellow resin, 144 mg. m/e 250.1 (MH)+.

N.M.R. (CDCl 3 ) δ = 1.46 (s, 9H); 2.6 (bs, 2H); 3.53 (t, 2H); 4.0 (bs, 2H); 6.3 (m, 1H); 7.0 (s, 2H); 9.4 (bs, 1H).

Preparation 28

1-(N-t-Butoxycarbonyl)-4-imidazol-2-ylpiperidine

The product from the intermediate preparation 27 () is dissolved in ethanol (30 ml) and hydrogenated at 30 p.s.i. (2.0 bar) with palladium on carbon catalyst (200 mg, 10%). Filtration of the catalyst and removal of the solvent gives foam, . m/e 252. 1 (MH)+.

N.M.R. (CDCl3) δ = 1.4 (s, 9H); 1.68 (qd, 2H); 1.95 (bd, 2H); 2.75 (bt, 2H); 2.92 (bt, 1H); 4.1 (bd, 2H); 6.9 (s, 2H); 8.77 (bs, 1H).

Preparation 29

1-(N-t-Butoxycarbonyl)-4-(imidazol-1-yl)methyl-1,2,5,6-tetrahydropyridine

(a) A 60% oil dispersion of sodium hydride () in dry dimethylsulfoxide (100 ml) is washed from the oil with hexane and then heated under stirring for 1 hour. Dry tetrahydrofuran (100 ml) was added and the reaction was cooled to . A solution of trimethylsulfonium iodide () in dimethylsulfoxide (80 ml) is then added, followed by 1-(N-t-butoxycarbonyl)-4-ketopiperidine () in dry tetrahydrofuran (100 ml) and the reaction is stirred for 0.5 hours at and then for 1 hour at room temperature. Water (500 ml) was then added and the mixture was extracted with ethyl acetate (3x250 ml). The combined extracts are then washed with brine, dried (MgSO4) and evaporated under vacuum. Purification by chromatography on silica gel eluting with cyclohexane-ether-isopropyl alcohol (60:40:1) gives 1-(N-t-butoxycarbonyl)-piperidine-4-spiro-2-oxirane as a colorless solid, (), m.p. 65-. Found: C,62.11; H, 9.06; N, 6.55. C11H19NO3 requires C,61.97; H, 8.92; N, 6.57%.

(b) A stirred solution of imidazole () in dry acetonitrile (30 ml) is treated under nitrogen with an 80% oil dispersion of sodium hydride () and the resulting mixture is heated until solution appears. After 15 minutes, the product from step (a) () is added and the reaction is maintained for 5 hours. After standing the reaction at room temperature overnight, the solution is evaporated under vacuum and the oily residue is partitioned between methylene chloride (40 ml) and water (20 ml). The organic phase is separated and washed with water, dried (MgSO4) and evaporated under vacuum. Purification by chromatography on silica gel, solution with methylene chloride-methanol-conc. 880 aqueous ammonia (95:4:1) gives 1-(N-t-butoxycarbonyl)-4-hydroxy-4-(imidazol-1-yl)methylpiperidine as a colorless powder (). Found: C, 58.35; H, 8.30; N, 14.52. C13H23N303 1/10 CH2Cl2 requires C, 58.43; H, 8.07; N, 14.50%.

m/e 282 (MH)+.

(c) A stirred solution of the product from step (b) () and triethylamine (5.44 ml) in dry methylene chloride (80 ml) at 0 to is treated with a solution of methanesulfonyl chloride (2.20 ml) in dry methylene chloride (10 ml) and a mixture that is formed is allowed to warm up to room temperature and maintained for 14 hours. The reaction mixture is then washed with water, dried (MgSO4) and evaporated under vacuum. Purification of the residue by chromatography on silica gel eluting with methylene chloride-methanol-conc. 880 aqueous ammonia (96:4:0 to 95:4:1) gives the product in the form of a golden colored oil (1.36 g). Found: C, 59.93; H, 7.60; N, 14.79. C13H21N3O2 1/4 CH2Cl2 requires C, 60.14; H, 7.61; N, 14.77%.

m/e (MH)+ 264

N.M.R. (CDCl3) δ = 1.45 (s, 9H); 1.97 (m, 2H); 3.50 (t, 2H); 3.90 (s, 2H); 4.50 (s, 2H); 5.50 (s, 1H); 6.90 (s, 1H); 7.10 (s, 1H); 7.55 (s, 1H).

Preparation 30

1-(N-t-Butoxycarbonyl)-4-(imidazol-1-yl)methyl-piperidine

A solution of the product from preparation 29 () in absolute ethanol (25 ml) is hydrogenated with stirring over 10% palladium on charcoal () at 50 p.s.i. (3.5 bar), room temperature, for 4 hours. The reaction mixture is then filtered and evaporated under vacuum, azeotroped with methylene chloride. Purification by chromatography on silica gel with methylene chloride-methanol solution (96:4) gives the product in the form of an oil (). Found: C, 63.08; H, 8.60; N, 15.50. C13H23N3O2 1/4 CH2Cl2 requires C, 63.36; H, 8.74; N, 15.84%. m/e (MH)+ 266

N.M.R. (CDCl3) δ = 1.15 (m, 2H); 1.45 (s, 9H); 1.58 (m, 2H); 1.85 (m, 1H); 2.65 (t, 2H); 3.80 (d, 2H); 4.10 (m, 2H); 6.90 (s, 1H); 7.10 (s, 1H); 7.45 (s, 1H).

Preparation 31

1-(N-t-Butoxycarbonyl)-4-(imidazol-1-yl)-1,2,5,6-tetrahydropyridine

Imidazole () was stirred in dry methylene chloride (30 ml) and thionyl chloride (4.8 ml) in dry methylene chloride (30 ml) was added. The resulting mixture is allowed to warm to room temperature and after 2 hours a solution of 1-(N-t-butoxycarbonyl)-4-keto-piperidine () in dry methylene chloride (50 ml) is added dropwise. The reaction mixture was stirred overnight and then evaporated under vacuum. Potassium carbonate () in water (30 ml) was added to the oily residue and the product was extracted with methylene chloride (2x60 ml). The combined extracts are washed with water (40 ml), dried (MgSO4) and evaporated under vacuum. Purification by chromatography on silica gel eluting with ethyl acetate-methanol (100:0 to 90:10) gives the product in the form of an oil ().

N.M.R. (CDCl 3 ) δ =1.50 (s, 9H); 2.55 (s, 2H); 3.70 (t, 2H); 4.05 (s, 1H); 7.10 (s, 2H); 7.65 (s, 1H).

Preparation 32

1-(N-Benzyloxycarbonyl)-4-(1,2,4-triazol-4-yl)piperidine

Ammonium acetate () and sodium cyanoborohydride () were added to a solution of 1-(N-benzyloxycarbonyl)-4-keto-piperidine () in methanol (25 ml) and the mixture was stirred at room temperature for 24 hours. The solvent was then removed under reduced pressure and the residue was partitioned between ethyl acetate and 1M sodium hydroxide solution. The ethyl acetate layer was separated, dried over magnesium sulfate, and evaporated under reduced pressure to give a yellow oil. Chromatography of this residue on silica gel, eluting with methylene chloride-methanol-conc. aqueous ammonia (95:5:1) gives 4-amino-1-(N-benzyloxycarbonyl)-piperidine as a yellow oil. A solution of this product () in toluene (20 ml) is treated with dimethylformamide azine () and p-toluenesulfonic acid (), and the mixture is heated under reflux for 24 hours. The solvent was then removed under reduced pressure and the residue by chromatography on silica gel, eluting with methylene chloride-methanol-conc. aqueous ammonia (93:7:1), gives the title compound as a colorless oil. m/e (MH+) 287

N.M.R. (DMSO-d6) δ = 1.82 (m, 2H); 2.01 (m, 2H); 2.83-3.09 (m, 2H); 4.12 (m, 2H); 4.40 (m, 1H); 5.11 (s, 2H); 7.28-7.45 (m, 5H); 8.65 (s, 2H).

Preparation 33

1-(N-t-Butoxycarbonyl-(S)-valyl)-3-(imidazol-1-yl)azetidine

A solution of 1-(N-t-butoxycarbonyl)-3-(imidazol-1-yl)azetidine (from preparation 18) () in methylene chloride (30 ml) was saturated with hydrogen chloride at and kept at that temperature for the next hour. The solvent was removed under vacuum to give the amine hydrochloride which was dissolved in N,N-dimethylformamide (25 ml) and the solution was treated at room temperature with N-t-butoxycarbonyl-(S)-valine N-hydroxysuccinimide ester () and N ,N-diisopropylethylamine (1.7 ml). The reaction mixture was stirred for 18 hours at room temperature and the solvent was then removed under vacuum. Purification of the residue using chromatography on silica gel, eluting with methylene chloride-methanol-conc. ammonia (97:7:1) gives the title compound as a colorless foam (). m/e 323 (MH)+

N.M.R. (DMSO-d6) δ = 0.89 (m, 6H); 1.38 (s, 9H); 1.90 (m, 1H); 3.61-3.78 (m, 1H); 3.92-4.10 (m, 1H); 4.24-4.80 (m, 3H); 5.19 (m, 1H); 6.98 (s, 1H); 7.06 (dd, 1H); 7.38 (s, 1H), 7.77 (s, 1H).

Preparation 34-43

The following compounds of formula (VII) where R4 is (S)-isopropyl and R7 and R8 are hydrogen are prepared according to the procedure of preparation 33 using the appropriate intermediates from preparations 18 to 32 and coupling to N-t-butoxycarbonyl-(S)-valine N-hydroxysuccinimide ester .

[image]

[image]

Preparations 44-48

The following compounds of formula (VII) where R4 is sec-butyl and m is 0 are prepared following the preparation of preparation 33 using N-t-butoxycarbonyl-(S)-isoleucine N-hydroxy-succinimide ester in the coupling step.

[image]

[image]

Preparation 49

1-(N-t-Butoxycarbonyl-(S)-isoleucyl)-4-ketopiperidine

The title compound was prepared using the same procedure as described for preparation 33 but using 4-ketopiperidine hydrochloride hydrate instead of 3-(imidazol-1-yl)azetidine hydrochloride and (S)-isoleucine N-hydroxysuccinimide ester. m/e 313 (MH)+

[image] -16° (C=0.34%, MeOH)

N.M.R. (CDCl3) δ = 0.9 (m, 6H); 1.2 (m, 1H); 1.4 (s, 9H); 1.6 (m, 1H); 1.75 (m, 1H); 2.5 (m, 4H); 3.7 (m, 2H); 4.15 (m, 2H); 4.55 (m, 1H); 5.2 (d, 2H).

Preparation 50

N-((R)-2-Benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoyl)-(S)-valine

The title compound is prepared using the method described in S. J. deSolms et al., J. Med. Chem., 1991, 34, 2852.

Preparation 51

1-Isocyano-3-methyl-1-(p-toluenesulfonyl)-but-1-ene

The title compound was prepared from p-toluenesulfonylmethylisocyanide and isobutyraldehyde by the method of Van Leusen, Schaart and Van Leusen Recueil, 98, No. 5, 258 (1979). i. r. (Nujol) 2100 cm-1

m/e 267 (M+NH3)+

N.M.R. (CDCl3) δ = 1.15 (d, 6H); 2.5 (s, 3H); 2.84 (m, 1H); 6.88 (d, 1H); 7.4 (d, 2H); 7.85 (d, 2H).

Preparation 52

3-Oxetanyloxycarbonyloxysuccinimide

Oxetan-3-ol () and N,N-diisopropylethylamine () were dissolved in methylene chloride (50 ml) and the solution was added dropwise to a solution of bis-trichloromethyl carbonate () in methylene chloride (100 ml) by maintaining at -20°C over for a period of 15 minutes under a nitrogen atmosphere. The solution is then stirred for the next 15 minutes at and N-hydroxy-succinimide () is added in one portion. The solution was allowed to warm to room temperature over a period of 2 hours and then washed with water (50 ml), saturated aqueous sodium bicarbonate (50 ml) and brine (25 ml). The organic layer was then dried (MgSO 4 ), filtered and the solvent was removed under vacuum to give the title compound as a light brown oil, (). N.M.R. (CDCl 3 ) δ = 2.83 (s, 4H); 4.75 (m, 2H); 4.90 (m, 2H); 5.58 (m, 1H).

Example 1

1-[N-((R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazole -1-yl)azetidine

a)1-[N-((R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-t-butyldimethylsilyloxy-6-phenylhexanoyl)-(S)-valyl]- 3-(imidazol-1-yl)azetidine

A solution of 1-(N-t-butyloxycarbonyl-(S)-valyl)-3-(imidazol-1-yl)azetidine (from preparation 33, ) in methylene chloride (50 ml) is saturated with hydrogen chloride at 0° and kept at that temperature for another hour. The solvent is evaporated under vacuum to give the amine as a colorless solid. A solution of this product in dimethylformamide (20 ml) was added to a solution of the active ester prepared earlier by stirring together (R)-2-benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy)-6 -phenylhexanoic acid (from preparation 2, ), 1-hydroxy-benzotriazole (), 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride () and N,N-diisopropylethylamine (1.75 ml) in dimethylformamide (75 ml) over 20 minutes. After stirring for a further 24 hours, the solvent was removed under vacuum and the residue was partitioned between ethyl acetate and water. The ethyl acetate layer was separated, dried (MgSO4) and stripped in vacuo to give a colorless oil. Purification by chromatography on silica gel eluting with ethyl acetate-methanol-conc. aqueous ammonia (90:10:1) gives the title compound as a colorless foam (). Found: C,66.04; H; 8.26; N, 9.61. C41H61N5O9 Si. 1/2 H2O requires C,66.39; H, 8.36; N, 9.44%.

m/e 733 (MH)+

[image] - 6° (C=0.29%. MeOH)

N.M.R. (DMSO-d6) δ = 0.12 (m, 6H); 0.90 (m, 15H); 1.25-1.29 (2 x s, 9H); 1.96 (m, 1H); 2.32-2.77 (m, 4H); 2.89 (m, 1H); 3.48-3.74 (m, 2H); 3.88-4.06 (m, 3H); 4.18-4.66 (m, 4H); 5.12 (m, 1H); 6.77 (t, 1H); 6.98 (s, 1H); 7.07-7.32 (m, 10H); 7.40-7.82 (2 x s, 1H); 7.76-7.96 (2 x s, 1H); 8.0-8.20 (2 x d.1H).

b)1-[N-((R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3- (imidazol-1-yl)azetidine

The product from step (a) () is dissolved in tetrahydrofuran and treated with a 1M solution of tetra-n-butylammonium fluoride in tetrahydrofuran at room temperature. After 48 hours, the solvent is removed under vacuum, the product is taken up in ethyl acetate, washed with saturated aqueous sodium bicarbonate and water, dried over MgSO4 and the solvent is evaporated under vacuum. Purification by chromatography on silica gel eluting with methylene chloride-methanol-conc. aqueous ammonia (93:7:1) followed by recrystallization from ethyl acetate/hexane gives the product as a colorless solid, (), m.p. 124-126°. Found: C,67.99; H, 8.01; N, 11.06. C35H47N5O5 requires C,68.05; H, 7.67; N, 11.34%. m/e 618 (MH)+

[image] -3° (C = 0.14%, MeOH)

N.M.R. (DMSO-d6) δ = 0.80 (m, 6H); 1.21-1.47 (m, 1H); 1.27 (s, 9H); 1.58-1.71 (m, 1H); 1.81-1.98 (m, 1H); 2.44-2.62 (m, 2H); 2.68-2.92 (m, 3H); 3.37-3.61 (m, 2H); 3.85-4.02 (m, 2H); 4.13-4.36 (m, 2H); 4.41-4.56 (m, 2H); 5.18 (m, 1H); 6.43 (d, 1H); 6.98 (s, 1H); 7.05-7.30 (m, 10H); 7.26-7.77 (s x s, 1H); 7.38-7.82 (2 x s, 1H); 7.88-7.98 (2 x d, 1H).

Examples 2-10

The following compounds are prepared according to the general procedure described in Example 1 but using the appropriate protected carboxylic acid intermediates of formula (IV) and the appropriate amines of formula (VIII) in coupling step (a) followed by removal of the t-butyldimethylsilyl protecting group as described in step (b).

[image]

[image]

[image]

Example 11

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-(trifluoromethoxy)benzyl)hexanoyl)-(S) -valyl]-(S)-3-(imidazol-1-yl)pyrrolidine

The procedure of Example 1 was followed using 1-(N-t-butoxycarbonyl-(S)-valyl)-3(S)-imidazol-1-yl)pyrrolidine (preparation 36) in the coupling step followed by removal of the t-butyldimethylsilyl group as was described in Example 1(b) to give the title product. m. p. 111-. Found: C,61.90; H, 6.85; N, 9.61. C37H48F3N5O6 requires C, 62.08; H, 6.76; N, 9.78%.

m/e (MH)+ 716

[image] + 2° (C = 0.1% MeOH) [image] + 14° (C = 0.1% MeOH)

N.M.R. (DMSO-d6) δ = 0.65-0.90 (m, 6H); 1.15-1.35 (m, 10H); 1.60 (m, 1H); 1.85 (m, 1H); 2.05-2.90 (m, 7H); 3.20-4.10 (m, 6H); 4.20 (m, 1H); 4.55 (m, 1H); 4.85 (m, 1H); 6.40 (d, 1H); 6.90-6.95 (2 x s, 1H); 7.10-7.30 (m, 10H); 7.70-7.80 (2 x s, 1H); 7.90-8.0 (2 x d, 1H).

Example 12

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-(trifluoromethoxy)benzyl)hexanoyl)-(S) -valyl]-(R)-

3-(imidazol-1-yl)pyrrolidine

The above procedure was followed but starting with 1-(N-t-butoxycarbonyl-(S)-valyl-3(R)-imidazol-1-yl)pyrrolidine to give the title compound, m.p. Found: C,61.44; H, 7.05; N.9.71. C37H48F3N5O6 2/5 H2O requires C,61.47; H, 6.80; N, 9.69%. m/e (MH)+ 716

[image] -23° (C = 0.1%, MeOH)

N.M.R. (DMSO-d6) δ = 0.70-0.85 (m, 6H); 1.15-1.40 (m, 10H); 1.60 (m, 1H); 1.85 (m, 1H); 2.00-2.90 (m, 7H); 3.25-3.70 (m, 5H); 3.95 (m, 1H); 4.20 (m, 1H); 4.45-4.65 (m, 1H); 4.75-4.95 (2xm, 1H); 6.40 (m, 1H); 6.90 (s, 1H); 7.05-7.25 (m, 10H); 7.70-7.75 (2xs, 1H); 7.90-8.05 (2xd, 1H).

Example 13

1-[N-(R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazole- 1-yl) piperidine

a) 1-[N-((R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy-6-phenylhexanoyl)-S-valyl-4-(imidazole) -1-yl) piperidine

The title compound was prepared from 1-(N-t-butoxycarbonyl-(S)-valyl-4-imidazol-1-yl piperidine and (S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethyl-silyloxy-( R)-2-benzyl-6-phenylhexanoic acid using the same procedure as described in Example 1 step (a) Purification by chromatography on silica gel eluting with ethyl acetate-methanol-conc ammonia (90:10:1) gives product in the form of a colorless foam (). Found: C, 67.32; H, 8.44, N, 9.22. C43H65N5O5Si.

1/4 H2O requires C,67.54; H, 8.56; N, 9.15%.

m/e 760 (MH)+

N.M.R. (DMSO-d6) δ=0.12 (m, 6H); 0.91 (m, 15H); 1.13-1.30 (m, 2H); 1.26 (s, 9H); 1.47-1.76 (m, 2H); 1.89-2.08 (m, 4H); 2.36-2.97 (m, 6H); 3.50-3.75 (m, 2H); 3.99-4.61 (m, 4H); 6.79 (m, 1H); 6.90 (d, 1H); 7.07-7.29 (m, 11H); 7.68-7.73 (2xs, 1H); 7.93-7.99 (2xd, 1H).

b) 1-[N-(R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-( imidazol-1-yl)piperidine

The title compound is prepared from the product of step a) above using the same procedure as described for example 1 step b). Purification by chromatography on silica gel eluting with methylene chloride-methanol-conc. aqueous ammonia followed by recrystallization from ethyl acetate/hexane gives the product as a colorless solid, m.p. 121-123° (). Found: C,68.38; H, 7.96; N, 10.59. C37H51N5O5. 1/4 H2O requires C,68.30; H, 7.92; N, 10.77%. m/e 646 (MH)+

[image] -10° (C=0.1 MeOH)

N.M.R. (DMSO-d6) δ = 0.77 (m, 6H); 1.19-1.37 (m, 1H); 1.28 (s, 9H); 1.48-1.72 (m, 3H); 1.82-2.04 (m, 3H); 2.42-2.94 (m, 7H); 3.38-3.60 (m, 2H); 3.96-4.15 (m, 1H); 4.28 (m, 1H); 4.37-4.58 (m, 3H); 6.43 (d, 1H); 6.88 (s, 1H); 7.04-7.29 (m, 11H); 7.65-7.69 (2xs, 1H); 7.84-7.88 (2xd, 1H).

Examples 14-37

The following compounds are prepared according to the procedure described in Example 13 but using the appropriate protected carboxylic acid intermediates of formula (IV) and the appropriate valine or isoleucine derivative of formula (VIII) in coupling step (a) followed by removal of the t-butyldimethylsilyl protecting group as described in step (b).

[image]

[image]

[image]

[image]

Example 31

1-[N-((R)-2-Benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(1 ,2,4-triazol-4-yl)piperidine

The benzyloxycarbonyl protecting group was removed from 1-(N-benzyloxycarbonyl)-4-(1,2,4-triazol-4-yl)-piperidine (preparation 32) by catalytic hydrogenation and the resulting amine product was coupled with N-( R)-2-benzyl-(S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy)-6-phenylhexanoyl)-(S)-valine followed by the procedure described for Example 1 step (a) , to give 1-N-((R)-2-benzyl-(S)-5-(t-butoxy-carbonylamino)-(S)-4-(t-butyldimethylsilyloxy)-6-phenylhexanoyl)-(S )-valyl-4-(1,2,4-triazol-4-yl)piperidine, m/e 761 (MH + ).

The above product is treated with tetra-n-butylammonium fluoride followed by the procedure of Example 1 step (b) to give the title product. Found: C,65.90; H.7.93; N, 12.48. C36H50N6O5. H2O requires C,65.06; H, 7.83; N, 12.65%. m/e 647 (MH)+

[image] -9° (C = 0.12 MeOH)

N.M.R. (DMSO-d6) δ = 0.70-0.92 (m, 6H); 1.15-1.41 (m, 10H); 1.46-1.76 (m, 3H); 1.81-2.16 (m, 3H); 2.53-2.96 (m, 7H); 3.38-3.64 (m, 2H); 3.95-4.15 (m, 1H); 4.27-4.61 (m, 4H); 6.36-6.44 (m, 1H); 7.01-7.12 (m, 10H); 7.78-7.91 (m, 1H); 8.54 (s, 1H); 8.60 (s, 1H).

Example 32

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl] -4-(imidazol-1-yl)1,2,5,6-tetrahydropyridine:tartrate

a) Reaction of (S)-5-t-butoxycarbonylamino-(S)-4-t-butyldimethylsilyloxy)-(R)-2-(4-trifluoromethoxy-benzyl)-6 phenylhexanoic acid and amine prepared by deprotection of 1-(N-t- butoxycarbonyl)-4-imidazol-2-yl(1,2,5,6-tetrahydropyridine) following the procedure of Example 1 (a) gives 1-N-((S)-5-t-butoxycarbonyl-amino)-(S )-4-t-butyldimethylsilyloxy)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl-(S)-valyl-4-(imidazol-1-yl)-1,2,5,6- tetrahydropyridine. Found: C,62.19; H, 7.39; N, 8.38. C33H62F3N5O6 Si 1/10 CH2Cl2 requires C,62.27; H, 7.37; N, 8.23%. m/e 842 (M)+

[image] -10° (C = 0.1%, MeOH)

N.M.R. (CDCl3) δ = 0.10 (m, 6H); 0.80 (d, 6H); 0.90 (s, 9H); 1.35 (s, 9H); 1.20-1.95 (m, 5H); 2.35-2.55 (m, 3H); 2.70 (m, 2H); 3.41-4.10 (m, 6H); 4.55 (m, 1H); 4.65 (d, 1H); 5.60-5.80 (2xs, 1H); 6.25 (d, 1H); 6.90-7.30 (m, 11H); 7.65 (s, 1H).

b) Deprotection by reaction with tetra-n-butylammonium fluoride following the procedure of Example 1 (b) gives the title compound, the free base. This is dissolved in absolute ethanol and treated with a solution of 1-tartaric acid () in absolute ethanol. Addition of ether gives a precipitate which is filtered and dried to give the 1-tartrate salt as a colorless solid, m.p. 92- (). Found: C,57.40; H, 6.64; N, 7.51. C38H38F5N5O6. CH16O6 1/3 H2O requires C,57.11; H, 6.23, N, 7.93%.

m/e (MH)+ 728

[image] +6.7° (C = 0.1%, MeOH)

[image] -6.7° (C = 0.1%, MeOH)

N.M.R. (DMSO-d6) δ = 0.75 (m, 6H); 1.25 (s, 9H); 0.95-1.40 (m, 1H); 1.50 (m, 1H); 1.90 (m, 1H); 2.35-2.95 (m, 7H); 3.0-3.85 (m, 4H); 4.0 (m, 1H); 4.10 (m, 1H); 4.25 (s, 2H); 4.40-4.60 (m, 2H); 6.0 (m, 1H); 6.40 (d, 1H); 7.0 (s, 1H); 7.10-7.30 (m, 9H); 7.50 (s, 1H); 7.95 (m, 2H).

Example 33

1-[N-((S)-5-(t-Butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl] -4-(5-isopropylimidazol-1-yl)piperidine

a) Deprotection of 1-(N-t-butoxycarbonyl-(S)-isoleucyl)-4-ketopiperidine (preparation 49) followed by reaction with (S)-5-t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy) -6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoic acid (preparation 10) gives 1-[N-((S)-5-(t-butoxycarbonylamino-(S)-4-(t-butyldimethylsilyloxy )-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl-(S)-isoleucyl]-4-ketopiperidine. Found: C, 62.50; H, 8.00; N, 5.20. C42H62F33O7Si requires C, 62.60; H, 7.80, N, 5.20%, m/e 806.5 (MH)+.

[image] -5.30 (C=0.1% MeOH).

b) The above product () and aqueous ammonia (specific gravity 0.88), in ethanol (25 ml) are hydrogenated with palladium on carbon (5 100 mg) at 30 p.s.i. (2.0 bar) over 4 hours. Filtration of the catalyst and evaporation of the solvent gives a colorless foam, which is chromatographed on silica gel, eluting with dichloromethane:methanol:conc. aqueous ammonia (90:10:1), to obtain, after concentration of the appropriate fractions, 1-[N-((S)-5-t-butoxycarbonyl-amino-(S)-4-t-butyldimethylsilyloxy-6-phenyl- (R)-2-(4-trifluoromethoxy)benzyl)hexanoyl)-(S)-isoleucyl]-4-aminopiperidine in the form of a colorless glass, .

c) The product from step b) above (404 mg) and 1-isocyano-3-methyl-1-(p-toluenesulfonyl)-but-1-ene, (from preparation 51, 150 mg) are mixed together in methanol (15 ml) with diisopropylethylamine (100 mg) over 16 hours. The solvent was removed and the residue was purified by chromatography on silica gel, eluting with dichloromethane:methanol:conc. aqueous ammonia (98:2:0.4), to give 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-t-butyldimethylsilyloxy-6-phenyl-(R)-2 -(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl]-4-(5-isopropylimidazolyl-1-yl)piperidine in the form of a colorless glass (380 mg). m/e 900 (MH+).

d) The product from step c) above is deprotected by treatment with tetra-n-butylammonium fluoride followed by the procedure from example (1b) to obtain the title product.

Found: C,63.5; H, 7.70; N, 8.70. C42H58F3N5O6. 1/2 H2O requires C,63.5; H, 7.48; N, 8.81%.

m/e 785.9 (MH)+

N.M.R. (DMSO-d6) δ = 0.8 (m, 6H); 1.05 (m, 1H); 1.2 (m, 6H); 1.3 (s, 9H); 1.3-2.0 (m, 7H); 2.5-3.0 (m, 6.5H); 3.2 (bt, 0.5H); 3.3 (m, 1H); 3.58 (m, 1H); 4.13 (m, 3H); 4.4-4.65 (m, 4H); 6.43 (m, 1H); 6.63 (s, 1H); 7.1-7.3 (m, 9H); 7.47, 7.6 (s, s, 1H); 7.95 (bt, 1H).

Example 34

1-[N-((S)-4-Hydroxy-(S)-5-(oxetan-3-yloxycarbonyl-amino)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)-hexanoyl)-( S)-Valyl]-4-(imidazol-1-yl)piperidine

The product from example 16 () is dissolved in methylene chloride (40 ml) and cooled in an ice bath. Trifluoroacetic acid (10 ml) was added dropwise over a period of 5 minutes and the solution was stirred for 1.5 hours. The solvent was evaporated under vacuum and the residue was taken up in ethyl acetate (250 ml) and washed with 1M sodium hydroxide (50 ml) and brine (50 ml). The organic solution is dried (MgSO4), filtered and the solvent is evaporated under vacuum. Chromatography on silica gel, elution with ethyl acetate-methanol-conc. aqueous ammonia (90:10:1) gives 1-[N-(S)-5-amino-(S)-4-hydroxy-6-phenyl--(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)- (S)-Valyl]-4-(imidazol-1-yl)piperidine as a white solid (). m/e 630 (MH)+

N.M.R. (DMSO-d6) δ = 0.78 (m, 6H); 1.34-1.75 (m, 4H); 1.81-2.07 (m, 3H); 2.37-2.93 (m, 8H); 3.05-3.29 (m, 3H); 4.05-4.56 (m, 5H); 6.88 (s, 1H); 7.10-7.27 (m, 10H); 7.67 (d, 1H); 7.95 (m, 1H);

b) The product from step a) () is dissolved in methylene chloride (30 ml). 3-oxetanylcarbonyloxy-succinimide () is added and the solution is stirred at room temperature for one hour. Then the solution is washed with 0.5 M sodium hydroxide (15 ml) and brine (15 ml), dried (MgSO4), filtered and evaporated under vacuum. The resulting solid was recrystallized from ethyl acetate to give the title compound as a white solid, m.p. 201-. Found: C,60.32; H, 6.22; N, 9.15. C37H46F3N5O7 1/2 H2O requires C,60.15; H, 6.41; N, 9.48%.

m/e 730 (MH)+

[image] +3° (C=0.1%, MeOH), [image] +32° (C=0.1, MeOH)

N.M.R. (DMSO-d6) δ = 0.77 (m, 6H); 1.22-2.05 (m, 7H); 2.50-2.93 (m, 8H); 3.42-3.60 (m, 2H); 4.08 (d, 1H); 4.22-4.73 (m, 7H); 5.12 (m, 1H); 6.89 (s, 1H); 7.11-7.28 (m, 11H); 7.67 (d, 1H); 7.86 (d, 1H).

Example 35

1-[N-(S)-4-Hydroxy-(S)-5-(oxetan-3-yloxycarbonylamino)-6-phenyl-(R)-2-(3-phenylprop-2-enyl)hexanoyl)-( S)-isoleudyl]-4-(imidazol-1-yl)piperidine

A solution of the product from Example 18 () in methylene chloride (20 ml) is treated with anhydrous trifluoroacetic acid (4 ml) at 0- for 5 hours. The reaction is then concentrated under vacuum and the resin that remains is azeotroped with toluene (x3) and dried. A solution of the product of the crude amine and diisopropylethylamine (0.82 ml) in methylene chloride (25 ml) was then cooled to and a solution of 3-oxetanyl-oxycarbonyloxysuccinimide () in methylene chloride (5 ml) was added dropwise. The reaction is allowed to warm to room temperature, maintained for 17 hours and then washed with water, dried (MgSO4), and evaporated under vacuum. Purification by chromatography on silica gel eluting with methylene chloride-methanol-conc. aqueous ammonia (95:4:1) gives the product in the form of a colorless foam, m.p. 157- ().

Found: C,66.45; H, 7.49; N, 10.22. C38H49N5O6 3/4 H2O requires C,66.60; H, 7.43; N; 10.22%.

[image] +32° (C=0.1% MeOH)

N.M.R. (DMSO-d6) δ = 0.75 (m, 6H); 1.25-2.95 (m, 12H); 3.05-3.65 (m, 4H); 3.95-4.75 (m, 10H); 5.05 (m, 1H); 6.10 (m, 1H); 6.30 (d, 1H); 6.80-6.85 (2xs, 1H); 6.95-7.35 (m, 11H); 7.55-7.65 (2xs, 1H); 7.95 (d, 1H).

Example 36

1-[N-(S)-4-Hydroxy-(S)-5-(isopropyloxycarbonylamino)-6-phenyl-(R)-2-(3-phenylprop-2-enyl)hexanoyl)-(S)-isoleucyl ]-4-(imidazol-1-yl)piperidine:tartrate

The title compound was prepared using the procedure described earlier for Example 35, except that isopropylchloroformate was used to react with the amine intermediate. Purification by chromatography on silica gel eluting with methylene chloride-methanol-conc. aqueous ammonia (97:2:1 to 95:4:1) gives the product as a colorless powder, which is recrystallized from ethyl acetate-hexane. The free base is dissolved in ethanol and treated with a solution of 1-tartaric acid. Addition of diethyl ether gives the tartrate salt as a colorless powder, m.p. 156-.

Found: C,61.60; H, 6.87; N, 8.38. C38H51N5O5 C4H6O6 1/2 H2O requires C,61.75; H,7.16; N, 8.57%.

m/e 658 (MH)+

[image] +26° (C=0.1% MeOH)

N.M.R. (DMSO-d6) δ = 0.75 (m, 6H); 1.0 (m, 6H); 1.25-3.65 (m, 14H); 3.95-4.70 (m, 10H); 6.10 (m, 1H); 6.30 (d, 1H); 6.65 (d, 1H); 6.85-6.90 (2xs, 1H); 7.05-7.35 (m, 11H); 7.65-7.75 (2xs, 1H); 7.90 (d, 1H).

Example 37

1-[N-(S)-5-(t-Butoxycarbonylamino)-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)-4-((S)-valyloxy)hexanoyl)-(S)-valyl ]-4-(imidazol-1-yl)piperidine;tartrate

a) N-Benzyloxycarbonyl-L-valine () and dicyclohexylcarbodiimide () were dissolved in dichloromethane (25 ml) and the mixture was stirred for 3 hours. The precipitated dicyclohexylurea is removed by filtration and the filtrate is evaporated to a white foam. This white foam was combined with the product of Example 16 in N,N-dimethylformamide (20 ml) and 4-dimethylaminopyridine () was added. After stirring at room temperature for 5 days, the mixture was partitioned between ethyl acetate and water. Drying of the organic extract (MgSO4) and evaporation of the solvent followed by silica gel chromatography, eluting with ethyl acetate:methanol (0-10%) gives 1-[N-(S)-4-(N-benzyloxycarbonyl-(S)- valyloxy)-(S)-5-(t-butoxy-carbonylamino)-6-phenyl-(R)-2-(4-(trifluoromethoxy)-benzyl)hexanoyl-(S)-valyl]-4-(imidazole- 1-yl)piperidine in the form of white foam. Found: C,62.63; H, 6.73; n, 8.37. C51H65F3N6O9. H2O requires C,62.43; H, 6.88; N, 8.56%.

m/e 963 (MH)+

b) The product from above step a) () is dissolved in absolute ethanol (50 ml), and the solution is treated with 10% palladium on carbon () and hydrogenated at 60 p.s. i., (4.1 bar) at room temperature for 4 hours. After removing the catalyst, the filtrate is evaporated to dryness. Purification by means of silica gel chromatography eluting with dichloromethane-methanol-conc. aqueous ammonia (97:3:0.5) gives the product in the form of a white foam. This foam was dissolved in ethyl acetate (6 ml) and a solution of tartaric acid () in 10% methanol in ethyl acetate (10 ml) was added. Evaporation of the solvent and trituration with diethyl ether gives the product in the form of a white glass (), m.p. Found: C,56.74; H, 6.78; N, 8.56.

C43H59F3N6O7 : C4H6O6: H2O requires C,56.61; H, 6.77; N, 8.43%.

N.M.R. (DMSO-d6) δ = 0.8-0.95 (m, 12H); 1.2 (s, 11H); 1.35-1.77 (m, 2H); 1.8-2.15 (m, 4H); 2.4-3.2 (m, 8H); 3.51 (m, 1H); 3.8 (m, 1H); 4.1 (m, 1H); 4.3 (m, 1H); 4.4-4.65 (m, 2H), 4.81 (m, 1H); 6.83 (s, 1H); 7.0-7.32 (m, 12H); 7.66 (d, 1H); 7.73-8.0 (m, 1H)

Claims (24)

1. A compound having the formula: [image] or its pharmaceutically acceptable salt or bioprecursor, where-: R 1 is C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, aryl, heterocyclyl or CONR 9 R 10 ; R2 is C1-C6 alkyl, C3-C8 cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl or heterocyclyl(C1-C4)alkyl; R3 is C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl (C1-C4)alkyl, aryl(C1-C4)alkyl, aryl(C2-C4)-alkenyl, heterocyclyl(C1-C4)alkyl or heterocyclyl ( (C2-C4)-alkenyl; R 4 is C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, aryl or heterocyclyl; each of R 5 , R 6 , R 7 and R 8 is independently H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl; or R 5 and R 6 , or R 7 and R 8 may be bonded together forming a 3 to 8 membered carbocyclic ring; X is a 4-10-membered mono or bicyclic heterocyclic group containing carbon ring atoms and one ring nitrogen atom through which the group is attached to the adjacent carbonyl group; group may be saturated or partially unsaturated and, in addition to the -(CR7R8)m-Het substituent, may be substituted with up to 4 subsequent substituents each independently selected from F, C1-C6 alkyl, C3-C8 cycloalkyl, OR11 or NR9R10; Het is an imidazolyl or triazolyl group, each of which may be optionally substituted with C1-C6 alkyl, C3-C8 cycloalkyl, NR9R10 or CONR9R10, each of R9 and R10 is independently H, C1-C6 alkyl or C3-C8 cycloalkyl, or R9 and R10 may be bonded together forming, with the nitrogen to which they are bonded, a 4-8 membered nitrogen-containing heterocyclic group, R 11 is H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl; n and m are each independently 0, 1 or 2; wherein each alkyl or cycloalkyl group included in the aforementioned definitions may be optionally fully or partially substituted with fluorine. 2. A compound having the stereochemistry as claimed in claim 1: [image] wherein R1 to R8, n, m, X and Het are as defined in claim 1 above. 3. A compound as claimed in claim 1 or claim 2 wherein R1 is t-butyl, isopropyl, or oxetanyl and n is 0. 4. A compound as claimed in claim 3 wherein R 2 is benzyl. 5. A compound as claimed in claim 3 or claim 4 wherein R4 is isopropyl or sec-butyl. 6. A compound as claimed in any of claims 3 to 5 wherein R 3 is benzyl optionally substituted in the phenyl ring with methyl, fluoro, chloro, iodo, CF 3 , or OCF 3 , or R 3 is 3-phenylpropyl or 3-phenyl-prop -2-enyl. 7. A compound as claimed in any one of claims 3 to 6 wherein each of R5, R6, R7 and R8 is H. 8. A compound as claimed in any one of claims 3 to 7 where m is 0 or 1. 9. A compound as claimed in claim 1 wherein said compound is: 1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazole -1-yl)azetidine, 1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazole -1-yl) piperidine, 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(3-phenylprop-2-en-1-yl)hexanoyl )-(S)-valyl]-4-(imidazol-1-yl)piperidine, 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl] -4-(imidazol-1-yl)piperidine, 1-[N-((S)-5-(t-butoxycarbonylamino)-R)-2-(4-chlorobenzyl)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3 -(imidazol-1-yl)azetidine i 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl] -4-(imidazol-1-yl)piperidine. 10. Process for the preparation of the compound of formula (I) as required in claim 1, which includes the removal of protective groups from the compound whose formula is: [image] wherein X1 is a hydroxy protecting group which can optionally be removed from R1 to R8, X and Het as defined in claim 1, and by isolating a compound of formula (I) and optionally forming a pharmaceutically acceptable salt thereof. 11. A pharmaceutical ingredient comprising a compound of formula (I) or (Ia) or a pharmaceutically acceptable salt or bioprecursor thereof as claimed in any one of claims 1 to 9, together with a pharmaceutically acceptable diluent or carrier. 12. A compound of formula (I), or (Ia) or a pharmaceutically acceptable salt or bioprecursor thereof, as claimed in any of claims 1 to 9 for use in medicine, in particular for use in the treatment or prophylaxis of retroviral infections in humans. 13. Use of a compound of formula (I) or (Ia) as claimed in any of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for the manufacture of medicaments for use in the treatment or prophylaxis of retroviral infections in humans. 14. A method of treating retroviral infections in humans comprising administering an effective amount of a compound of formula (I) or (Ia) as claimed in any one of claims 1 to 9. 15. Process for the production of a compound having the formula: [image] or its pharmaceutically acceptable salts, where-: R 1 is C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, aryl, heterocyclyl or CONR 9 R 10 ; R2 is C1-C6 alkyl, C3-C8 cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl or heterocyclyl(C1-C4)alkyl; R3 is C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8cycloalkyl(C1-C4)alkyl, aryl(C1-C4)alkyl, aryl(C2-C4)-alkenyl, heterocyclyl(C1-C4)alkyl or heterocyclyl(C2 -C4)-alkenyl; R 4 is C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, aryl or heterocyclyl; each of R 5 , R 6 , R 7 and R 8 is independently H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl; or R 5 and R 6 , or R 7 and R 8 may be bonded together to form a 3 to 6 membered carbocyclic ring; X is a 4-10-membered mono or bicyclic heterocyclic group containing carbon ring atoms and one nitrogen ring atom through which the group is attached to an adjacent carbonyl group; the group may be saturated or partially unsaturated, and in addition to the -(CR7R8)rn -Het substituent, may be substituted with up to 4 subsequent substituents each independently selected from F, C1-C6 alkyl, C3-C8 cycloalkyl, OR11 or NR9R10; Het is an imidazolyl or triazolyl group, each of which may be optionally substituted with C1-C6 alkyl, C3-C8 cycloalkyl, NR9R10 or CONR9R10, each of R9 and R10 is independently H, C1-C6 alkyl or C3-C8 cycloalkyl, or R9 and R10 may be bonded together to form, with the nitrogen to which they are bonded, a 4-8 membered nitrogen-containing heterocyclic group, R 11 is H, C 1 -C 6 alkyl or C 3 -C 8 cycloalkyl; n and m are each independently 0, 1 or 2; wherein each alkyl or cycloalkyl group included in the aforementioned definitions may optionally be fully or partially saturated with fluorine; which includes the removal of the protective group from the compound whose formula is: [image] wherein X is an optionally removable hydroxy protecting group and R1 to R8, X and Het are as defined in claim 1, and isolation of a compound of formula (I) and optionally formation of a pharmaceutically acceptable salt thereof. 16. A process as claimed in claim 1 wherein the optionally removable hydroxy protecting group X1 is t-butyldimethylsilyl and it is removed by reaction with tetra-n-butylammonium fluoride in an organic solvent. 17. The process as claimed in claim 1 wherein the compound of formula (I) has the stereochemistry-: [image] where R1 to R8, n, m, X and Het are as defined in claim 1 above. 18. A process as claimed in claim 1, claim 2 or claim 3 wherein R1 is t-butyl, isopropyl, or oxetanyl and n is 0. 19. A process as claimed in claim 4 wherein R 2 is benzyl. 20. A process as claimed in claim 4 or 5 wherein R4 is isopropyl or sec-butyl. 21. The process claimed in any of claims 4 to 6 wherein R3 is benzyl optionally substituted in the phenyl ring with methyl, fluoro, chloro, iodo, CF3, or OCF3, or R3 is 3-phenylpropyl or 3-phenyl-prop- 2-enyl. 22. The process as claimed in any one of claims 4 to 7 wherein each of R5, R6, R7 and R8 is H. 23. A process as claimed in any one of claims 4 to 8 where m is 0 or 1. 24. The process as claimed in claim 1 wherein said manufactured compound of formula (I) is-: 1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-3-(imidazole -1-yl)azetidine, 1-[N-((R)-2-benzyl-(S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]-4-(imidazole -1-yl) piperidine 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(3-phenylprop-2-en-1-yl)hexanoyl )-(S)-valyl]-4-(imidazol-1-yl)piperidine, 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-valyl] -4-(imidazol-1-yl)piperidine, 1-[N-((S)-5-(t-butoxycarbonylamino)-(R)-2-(4-chlorobenzyl)-(S)-4-hydroxy-6-phenylhexanoyl)-(S)-valyl]- 3-(imidazol-1-yl)azetidine or 1-[N-((S)-5-(t-butoxycarbonylamino)-(S)-4-hydroxy-6-phenyl-(R)-2-(4-trifluoromethoxybenzyl)hexanoyl)-(S)-isoleucyl] -4-(imidazol-1-yl)piperidine.