MXPA99004294A - (4r,5s,6s,7r)-hexahydro-1- [5-(3-aminoinazole)methyl]-3-butyl-5,6-dihydroxy-4,7-bis [phaenylmethyl]-2h-1,3-diazepin-2-one, its preparation and its use as hiv protease inhibitor - Google Patents

Abstract

The present invention relates to compounds of formula (I) or pharmaceutically acceptable salt forms or prodrugs thereof, which are useful as inhibitors of HIV protease, and to pharmaceutical compositions and diagnostic kits comprising the same, and methods of using the same for treating viral infection or as an assay standard or reagent.

Description

(4R, 5S, 6S, 7R) -HEXAHIDRO-1- C5- (3-AMIN0INAZ0L) METHYL 3 -3-BUTIL-5, β-DIHYDROXY-4,7-BIS TFENYLMETHYL] -2H-1, 3-DIAZEPIN- 2 -ONA, Sü PREPARATION AND Sü USE AS AN HIV PROTEASE INHIBITOR FIELD OF THE INVENTION This invention relates generally to urea 1- (3-aminoindazol-5-yl) -3-butilcyclic compounds which are useful as inhibitors of HIV protease, with pharmaceutical compositions and with diagnostic equipment comprising the same, and with methods for use them to treat viral infections or as standards or assay reagents.

BACKGROUND OF THE INVENTION Etiologically, two different retroviruses have been linked, the human immunodeficiency virus (HIV) type-1 (HIV-1) or type-2 (HIV-2), with immunosuppressive diseases, the acquired immunodeficiency syndrome (AIDS). Individuals seropositive to HIV are generally asymptomatic but typically develop an AIDS-related complex (ARC) followed by AIDS. Affected individuals show severe immunosuppression what REF .: 29939 that predisposes them to weakening and finally to deadly opportunistic infections. The AIDS disease is the final result of an HIV-1 or HIV-2 virus after its complex life cycle. The life cycle of the virion begins when the virion binds itself to immune cells host T4 human lymphocytes through the binding of a glycoprotein on the surface of the protective coat of the virion with the CD4 protein on the lymphocytic cell. Once bound, the virion removes its coat of glycoprotein, penetrates the membrane of the host cell and eliminates the coating of its RNA. The virion enzyme, a reverse transcriptase, directs the process of transcribing the RNA into single-stranded DNA. Viral RNA is degraded and a second strand of DNA is generated. The new double-stranded DNA is now integrated into the genes of human cells and those genes are used for cell reproduction. At this point, the human cell carries out its reproduction process using its own RNA polymerases to transcribe the integrated DNA in viral RNA. The viral RNA is translated into the precursor gag-pol fusion polyprotein. The polyprotein is then separated by the HIV protease enzyme to provide the mature viral proteins. Therefore, the protease is HIV is responsible for regulating the cascade of rupture events that lead to the maturation of the virus particle in a virus that is capable of complete infectivity. The typical response of the human immune system, 'to destroy the invading virion, is expensive because a large portion of the virion's life cycle develin a latent state within the immune cell. In addition, the viral reverse transcriptase, the enzyme used to make the new virion particle, is not very specific and causes transcription errors that result in glycoproteins that change continuously on the surface of the viral protective coat. This lack of specificity diminishes the effectiveness of the immune system because the antibodies produced specifically against one glycoprotein may be useless against another, and therefore reduce the number of antibodies available to fight against the virus. The virus continues to reproduce as the response of the immune system continues to weaken. Finally, HIV largely maintains a free environment over the body's immune system, which allows opportunistic infections to be established, and without the administration of antiviral agents, immunomodulators or both, death can occur.

There are at least three critical points in the life cycle of the virus which have been identified as possible targets for antiviral drugs: (1) the initial binding of the virion to the T4 lymphocyte or site of the macrophage, (2) the transcription of the RNA viral viral DNA (reverse transcriptase, RT) and (3) the assembly of the new virus particle during reproduction (for example HIV aspartic acid protease or HIV protease). The retrovirus genomes encode a protease that is responsible for the proteolytic processing of one or more polyprotein precursors such as the pol and gag gene products. See Wellink, Arch. Virol. 9.8 1 (1988). Retroviral proteases most commonly process the gag precursor within the core proteins, and also process the pol precursor within the reverse transcriptase and the retroviral protease. The correct processing of the precursor polyproteins by the retroviral protease is necessary for the assembly of the infectious virions. It has been shown that in vitro mutagenesis that produces protease-defective virus leads to the production of immature nucleus forms which lack infectivity. See Crawford et al., J. Virol. 53 899 (1985); Katoh et al., Virology 145 280 (1985). Therefore, the inhibition of retroviral protease provides an attractive target for antiviral therapy. See Mitsuya, Nature 325 775 (1987). The ability to inhibit the viral protease provides a method to block viral replication and therefore a treatment for viral diseases, such as AIDS, which may have minor side effects, be more effective and be less susceptible to drug resistance when compare with current treatments. As a result, three HIV protease inhibitors, Roche's saquinavir, Abbott's ritonavir and Merck's indinavir, are currently sold in the market and numerous potential protease inhibitors are in clinical trials, eg, vertex VX-478, Agouron's nelfinavir. , KNI-272 from Japan energy and CGP-61755 from Ciba Geigy. As evidenced by currently sold protease inhibitors and their clinical trials, a wide variety of compounds have been studied as potential inhibitors of HIV protease. They have received significant attention cyclic ureas of a nucleus. For example, in PCT application number 094/19329, Lam et al., Genetically describe cyclic ureas of the formula: and methods to prepare these ureas. Although the present compounds are within the description of Lam et al., They are not specifically described. Additional cyclic ureas are described in Lam et al, J. Med. Chem. 1996, 39, 3514-3525. Although various cyclic ureas are described in this publication, compounds containing undazolmethyl are not disclosed. Even with the current success of protease inhibitors, it has been found that patients with HIV become resistant to a single protease inhibitor. Therefore, it is desirable to develop additional protease inhibitors to further combat an HIV infection.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, an object of the present invention is to provide novel protease inhibitors. Another objective of the present invention is to provide pharmaceutical compositions with protease inhibitory activity comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof. Another object of the present invention is to provide a novel method for treating an HIV infection which comprises administering to a host in need of such treatment a therapeutically effective amount of at least one of the compounds of the present invention or a salt or a form thereof. of pharmaceutically acceptable prodrug thereof. Another objective of the present invention is to provide a novel method for treating HIV infection which comprises administering to a host in need thereof a therapeutically effective combination of (a) one of the compounds of the present invention, and (b) one or more compounds that are selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors. Another objective of the present invention is to provide a method for inhibiting HIV present in a body fluid sample, which comprises treating the body fluid sample with an effective amount of a compound of the present invention. Another objective of the present invention is to provide a kit or container containing at least one of the compounds of the present invention in an amount effective for use as a standard or reagent in a test or assay to determine the capacity of a potential pharmaceutical substance to inhibit HIV protease, HIV growth, or both. These and other objects will become apparent during the following detailed description, which have been obtained based on the discovery of the inventors that the compounds of formula I: or pharmaceutically acceptable salts or prodrug forms thereof are effective protease inhibitors.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Therefore, in a first embodiment, the present invention provides a novel compound of formula I: or a pharmaceutically acceptable salt or averaged form thereof. In a second embodiment, the present invention provides a novel pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or prodrug form thereof. In a third embodiment, the present invention provides a novel method for treating an HIV infection which comprises administering to a host in need of such treatment a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or prodrug form thereof. same. In a fourth embodiment, the present invention provides a novel method for treating an HIV infection which comprises, in combination, administering to a host in need thereof a therapeutically effective amount of: (a) a compound of formula I; and, (b) at least one compound that is selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors. In another preferred embodiment, the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor. In another preferred embodiment, the nucleoside reverse transcriptase inhibitor is selected from AZT, 3TC, ddI, ddC and d4T and the protease inhibitor is selected from saquinavir, ritonavir, indinavir, VX-478, nelfinavir, KNI-272, CGP- 61755 and U-103017. In a further preferred embodiment, the nucleoside reverse transcriptase inhibitor is selected from AZT and 3TC and the protease inhibitor is selected from saquinavir, ritonavir and indinavir. In a further preferred embodiment, the nucleoside reverse transcriptase inhibitor is AZT.

In another additional preferred embodiment, the protease inhibitor is indinavir. In a fifth embodiment, the present invention provides a pharmaceutical kit useful for treatment of HIV infection, which comprises a therapeutically effective amount of: (a) a compound of formula I; and (b) at least one compound that is selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors, in one or more sterile containers. In a sixth embodiment, the present invention provides a novel method for inhibiting HIV present in a body fluid sample, which comprises treating the body fluid sample with an effective amount of a compound of formula I. In a seventh embodiment, the present invention provides a novel device or container comprising a compound of formula I or II in an amount effective for use as a standard or reagent in a test or assay to determine the ability of a potential pharmaceutical substance to inhibit HIV protease, HIV growth or both.

DEFINITIONS As used herein, the following terms and expressions have the indicated meanings. It will be appreciated that the compounds of the present invention contain an asymmetrically substituted carbon atom, and can be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, for example by separation of racemic forms or by synthesis, or starting with optically active starting materials. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are understood, unless a stereochemistry or specific isomeric form is specifically indicated. As used herein, "HIV reverse transcriptase inhibitor" is intended to refer to both nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT). Examples of nucleoside RT inhibitors include, but are not limited to, AZT, ddC, ddl, d4T and 3TC. Examples of non-nucleoside RT inhibitors, include, but are not limited to, viviradine (Pharmacia and Upjohn U90152S), derivatives of TI30, BI-RG-587, nevirapine, L-697,661, LY 73497 and Ro 18,893 (Roche; As used herein, it is intended that "HIV protease inhibitor" refers to compounds which inhibit HIV protease. Examples include, but are not limited to saquinavir (Roche, Ro31-8959), ritonavir (Abbott, ABT-538), indinavir (Merck, MK-639), VX-478 (Vertex / Glaxo Wellcome), nelfinavir (Agouron, AG-1343), KNI-72 (Japan Energy), CGP-61755 (Ciba-Geigy), and U-103017 (Pharmacia and Upjohn). Additional examples include the cyclic protease inhibitors described in WO93 / 07128, WO 94/19329, WO 94/22840 and PCT Application US96 / 03426. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by producing acid or basic salts thereof.

Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids of basic residues such as amines; alkaline or organic salts of acidic residues such as carboxylic acids; and similar. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the prepared salts of organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroximic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic and the like. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acid portion by conventional chemical methods. Generally such salts can be prepared by reacting free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.; generally a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile as preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Marek Pubiishing Company, Easton, PA, 1985, p. 1418, the description of which is incorporated herein by reference. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals. without excessive toxicity, irritation, allergic responses or other problems or commensurate complications with a reasonable risk-benefit ratio. The term "prodrug" is intended to include any covalent linked carrier that releases the active parent drug according to formula I or other formulas or compounds of the present invention in vivo when such a prodrug is administered to a mammalian subject. The prodrugs of a compound of the present invention, for example, formula (I) are prepared by modifying functional groups present in the compound such that the modification is separated, either by routine manipulation or in vivo, to the original compound. The prodrugs include compounds of the present invention wherein the hydroxy or amino group is attached to any group which, when the prodrug is administered to a mammalian subject, is separated to form a free hydroxyl or free amino, respectively. Examples of prodrugs include, but are not limited to, acetate, formate or benzoate derivatives of alcohol and amine functional groups in the compounds of formula I; phosphate esters, dimethylglycine esters, aminoalkylbenzyl esters, aminoalkyl esters and carboxyalkyl esters of alcohol functional groups in the compounds of formula I; and similar. Additional examples include compounds wherein two hydroxy groups of formula I are joined to form an epoxide; -OCH2SCH20-; -0C (= 0) 0-; -OCH20-; -OC (= S) 0-; -OC (= 0) C (= 0) 0; -OC (CH3) 20-; -0C ((CH2) 3NH2) (CH3) 0-; -OC (0CH3) (CH2CH2CH3) O-; or -OS (= 0) 0-. The terms "stable compound" and "" stable structure "are meant to indicate a compound that is sufficiently strong to survive isolation to a useful degree of purity from a reaction mixture and its formulation into an effective therapeutic agent. Only stable compounds are contemplated by the present invention The term "substituted" is intended to indicate that one or more hydrogen atoms indicated in the expression using "substituted" terms are replaced with a selection of the indicated group or groups, with the proviso that the normal valence of the indicated atom is not exceeded, and that the substitution results in a stable compound.When the substituent is the keto group "ie, = 0)", then 2 hydrogens on the atom are substituted. the term "(therapeutically effective amount") includes an amount of a compound of the present invention or an amount of the combination of claimed effective to inhibit HIV infection or treat the symptoms of HIV infection in a host. The combination of compounds is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme Regul. 22: 27-55 (1984), occurs when the effect (in this case, the inhibition of HIV replication) of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a only agent. In general, the synergistic effect is demonstrated more clearly at suboptimal concentrations of the compounds. The synergy can be presented in terms of lower cytotoxicity, increased antiviral effect or some other beneficial effect of the combination compared to the individual components. Other features of the invention will become apparent in the course of the following descriptions of the exemplary embodiments which are provided for illustration of the invention.

Examples The abbreviations used in the examples are as defined below "° C" for degrees Celsius, "d" for doublet, "dd" for doublet of doublet, "eq" for equivalent or equivalents, "g" for gram or grams " mg "for milligram or milligrams," ml "for milliliter or milliliters," H "for hydrogen or hydrogens," h "for hour or hours," m "for multiplet," M "for molar," min "for minute or minutes , "MHz" for megahertz, "EM" for mass spectroscopy, "nmr" or "NMR" for nuclear magnetic resonance spectroscopy, "t" for triplet, and "CCD" thin layer chromatography.

EXAMPLE 1 Preparation of (4R, 5S, 6S, 7R) -h.exahidro-1- [5- (3-aminoindazole) methyl] -3-butyl-5,6-dihydroxy-4,7-bis [phenylmethyl] -2H- 1,3-diazepin-2 -one (I) Compound A can be prepared by known methods. For example, in scheme 1 of Rossano et al. { Tetr. Lett, 1995, 36 (28), 4967, 4968), (the preparation of compound A is shown), the content of which is incorporated herein by reference. A further method of preparing compound A is shown in example 6 of U.S. Patent No. 5,530,124, the content of which is incorporated herein by reference.

PART A: To a suspension of compound 1 (10.0 g; 27.3 mmol) in 1,2-dichloroethane (100 ml) is added methyltriflate (3.4 ml, 30 mmol). After refluxing overnight, the reaction is washed with saturated NaHCO3, saturated NaCl, se. dry (Na2SO4) and evaporate which gives 12.5 g of a yellow oil. Column chromatography (Instantaneous Si02, EtOAc / hexane 25%) gives 7.86 g of compound 2 as a light yellow oil which crystallizes upon standing (yield 75%), m.p. = 97-100 ° C. MH * = 381.

PART B: To a solution of 1 (10.0 g, 26.3 mmol) in anhydrous DMF (30 mL) is added sodium hydride (1.58 g, 65.8 mmol). The reaction mixture is stirred at room temperature for 45 minutes followed by dropwise addition to a solution of 1-iodobutane (9.68 g, 52.6 mmol) in anhydrous DMF (10 ml). After the addition, stirring is continued at room temperature overnight. The reaction mixture is cooled to 0 ° C and methanol (5 ml) is added to remove excess sodium hydride. The mixture is partitioned between ethyl acetate (200 ml) and water (150 ml). The organic phase is separated and washed with water (4 x 100 ml), brine (100 ml) and dried over sodium sulfate. Flash chromatographic purification (25% EtOAc / Hex.) Provides n-butylisourea 2 (10.5 g, 92% yield):? M (NH, -CI / DDIP) (M + H *) 437.2 (100%); XU-NMR (300 MHz, CDCl 3, 25 ° C): d 7.23 (m, 10H), 4.19 (, 3H), 3.64 (m, 1H), 3.44 (s, 3H), 3.36 (m, 1H), 3.02 ( m, 2H), 2.76 (m, 2H), 2.04 (m, 1H), 1.52 (s, 3H), 1.49 (s, 3H), 1.21 (m, 4H), 0.82 (t, J = 7.0 Hz, 3H ). PART C: (4R, 5S, 6S, 7R) -hexahydro-1- [3-cyano-4-fluorophenyl) methyl] -5,6-isopropylidene-4,7-bis (4-phenylmethyl) -3- phenylmethyl-2H-l, 3-diazepin-2-one (3).

To a solution of 2 (5.0 g, 11.5 mmol) in acetonitrile (40 ml) is added 4-fluoro-3-cyanobenzyl bromide (3.68 g, 17.25 mmol). The reaction mixture is refluxed overnight. After the solvent is removed under reduced pressure, the residue is purified using flash chromatography (35%? TOAc / Hex.) To provide the cyclic urea 3 as a white solid (4.5 g, 71% yield): E ( NH, -CI / DDIP) (M + H *) 556.3 (100%); NMR: H (300 MHz, CDC13, 25 ° C): d 7.41 (m, 1H), 7.28 (m, 7H), 7.13 (d, J = 9.2Hz, 2H), 7.05 (t, J = 8.8 Hz, 1H), 6.95 (d, J = 9.2 Hz, 2H), 4.50 (d, J = 14.0 Hz, 1H), 4.07 (m, 2H), 3.70 (m, 3H), 3.44 (t, J = 7.7 Hz, 1H), 2 ^ 90 (m, 4H), 2.12 (m, 1H), 1.50 (s, 6H), 1.26 (m, 4H), 0.83 (t, J = 7.0 Hz, 3H).

PART D: (4R, 5S, 6S, 7R) -hexahydro-1- [5- (3-aminoindazole) methyl] -3-butyl-5,6-dihydroxy-4,7-bis [phenylmethyl] -2H-1 , 3-diazepin-2-one (I) To a solution of 3 (4.5 g, 8.11 mmol) in n-butanol (20 ml) is added hydrazine hydrate (0.81 g), 16.2 mmoles). The mixture is refluxed for 6 h. The solvent and excess hydrazine are removed under reduced pressure. The residue is dissolved in anhydrous methanol (20 ml) followed by the addition of 4 M HCl in dioxane (2 ml). The reaction mixture is stirred at room temperature for 2 h. The methanol is removed and the residue is partitioned between ethyl acetate (80 ml) and sodium bicarbonate (saturated) (50 ml). The organic phase is separated, washed with water (2x 50 ml) and dried over sodium sulphate. (anhydrous). Flash chromatographic purification affords I (3.0 g, 72% yield) as a white solid: m.p. 129-131 ° C; MS (NH3-CI / DDIP) (M + H *) 528.3 (100%) calculated HRMS for CflH37NE0 -. + 1 528.2975, found 528.2958; XH NMR (300 MHz, CD30D, 25 ° C): d 7.19 (m, 12H), 6.98 (d, J = 1.5 Hz, 2H), 4.74 (d, J = 13.9 Hz, 1H), 3.85 (dd, J = 10.25, 4.76 Hz, 1H), 3.65 (m, 1H), 3.56 (m, 4H), 3.15 (m, 2H), 2.96 (m, 3H), 2.07 (m, 2H), 1.37 (m, 2H) , 1.22 (m, 2H), 0.84 (t, J = 7.0, 3H).

Utility The compounds of formula I possess HIV protease inhibitory activity and are therefore useful as antiviral agents for the treatment of HIV infection as antiviral agents for the treatment of HIV infection and associated diseases. The compounds of formula I possess HIV protease inhibitory activity and are effective as inhibitors of HIV growth. The ability of the compounds of the present invention to inhibit viral growth or infectivity is demonstrated by standard viral growth or infectivity assays, for example, using the assay described below. The compounds of formula I of the present invention are also useful for the inhibition of HIV in an ex vivo sample that contains HIV or expects it to be exposed to HIV. Therefore, the compounds of the present invention can be used to inhibit HIV present in a body fluid sample (e.g., a serum or semen sample), which contains or is suspected to contain or be exposed to HIV. The compounds provided by this invention are also useful as standard or reference compounds for use in tests or assays to determine the ability of an agent to inhibit replication of a viral clone and / or HIV protease, for example, in a program of pharmaceutical research. Therefore, the compounds of the present invention can be used as a control or reference compound in such assays and as a quality control standard. The compounds of the present invention can be provided in a commercial kit or container for use as such standard or reference compound. Since the compounds of the present invention show specificity for HIV protease, the compounds of the present invention may also be useful in diagnostic reagents in diagnostic assays for the detection of HIV protease. Therefore, the inhibition of protease activity in an assay (such as the assays described herein) by a compound of the present invention would be indicative of the presence of HIV protease and HIV virus. As used herein "μg" indicates micrograms, "mg" indicates milligrams, "g" indicates grams, "μl" indicates microliters, "ml" indicates milliliters, "1" indicates liters, "nM" indicates nanomolar, "μM "indicates micromolar," mM "indicates millimolar," M "indicates molar, and" nm "indicates nanometers. "Sigma" indicates Sigma-Aldrich Corp. of St. Louis, MO.

HIV RNA assay DNA plasmids and RNA transcripts in vi tro Plasmid pDAB 72 containing both gag and pol sequences of BH10 (pb 113-1816) cloned in PTZ 19R is prepared, according to Erickson-Viitanen et al. AIDS Research and Human Retroviruses 1989, 5, 577. The plasmid is linearized with Bam Hl prior to generation of AN in vi tro transcript using the Riboprobe Gemini System II (Promega) kit with T7 RNA polymerase. The synthesized RNA is purified by treatment with DNase-free RNase (Promega), extraction with phenol-chloroform and precipitation with ethanol. The RNA transcripts dissolve in water and are stored at -70 ° C. The concentration of RNA is determined from A260.

Probes: Biotinylated capture probes are purified by CLAP after synthesis in a DNA synthesizer Applid Biosistems (Foster City, CA) by adding biotin to the 5 'terminal end of the oligonucleotide, using Cocuzza's biotin-phosphoramidite reagent, Tet. Lett. 1989 30, 6287. The biotinylated capture probe gag (5-biotin-CTAGCTCCCTGCTTGCCCATACTA 3 ') is complementary to nucleotides 889-912 of HXB2 and the biotinylated capture probe pol (5'-biotin-CCCTATCATTTTTGGTTTCCAT 3') is complementary to nucleotides 2374-2395 of HXB2. Conjugated alkaline phosphatase oligonucleotides are used as reporter probes and are prepared by Syngene (San Diego, CA.) (5'-CT6TCTTACTTT6ATAAAACCTC 3 '). The pol indicator probe is complementary to nucleotides 2403-2425 of HXB2. The gag indicator probe (5 'CCCAGTATTTGTCTACAGCCTTCT 3') is complementary to nucleotides 950-973 of HXB2. All nucleotide positions are those from the GenBank gene sequence databank accessed through the sequence analysis software package from Genetics Computer Group (Devereau Nucleic Acids Research 1984, 12, 387). The indicator probes are prepared as 0.5 μM concentrates in 2 x SSC (0.3 M NaCl, 0.03 M sodium citrate), 0.05 M Tris, pH 8.8, 1 mg / ml BSA. The biotinylated capture probes are prepared as 100 μM concentrates in water.

Plates coated with streptavidin Streptavidin coated plates are obtained from Du Pont Biotchnology Systems (Boston, MA).

Cell and virus concentrates MT-2 and MT-4 cells are maintained in RPMI 1640 supplemented with 5% fetal bovine serum (FCS) for MT-2 cell or 10% FCS for MT-4, L-glutamine-2 mM, and 50 μg / ml cells of gentamicin, all from Gibco. HIV-1 RF is propagated in MT-4 cells in the same medium. Virus concentrates are prepared approximately 10 days after acute infection of MT-4 cells and stored as aliquots at -70 ° C. The infectious titers of HIV-1 (RF) concentrates are 1-3 x 107 PFU (plaque-forming units / ml), as measured by plaque assay in MT-2 cells (see below). Each aliquot of virus concentrate used for infection was heated only once. For evaluation of antiviral efficacy, the cells to be infected are subcultured one day before infection. On the day of infection, cells are resuspended at 5 x 10 5 cells / ml in RMPI 1640, 5% FCS for volume infections or 2 x 106 ml in Dulbecco's modified Eagle's medium with 5% FCS for plaque infection of microtitration. Virus was added and the culture continued for 3 days at 37 ° C.

HIV RNA assay: Cell lysates or RNA purified in 3 M or 5 M GED was mixed with 5 M GED and a capture probe to a final concentration of 3 M guanidinium isothiocyanate and a final concentration of biotin oligonucleotide of 30 nM. Hybridization was carried out in tissue culture plates of 96 wells with U-shaped bottom, sealed (Nunc or Costar) for 16-20 hours at 37 ° C. The RNA hybridization reactions were diluted 3 times with deionized water to a final concentration of 1 M guanidino isothiocyanate and aliquots (150 μl) were transferred to streptavidin-coated microtiter plate wells. The binding of the capture probe and the capture-RNA probe hybrid to the immobilized streptavidin was allowed to proceed for 2 hours at room temperature, after which the plates were washed six times with DuPont ELISA plate wash buffer (solution phosphate buffered saline (PBS), 0.05% Tween 20. A second hybridization of indicator probe to the immobilized complex of capture probe and hybridized target RNA was carried out in a well coated with streptavidin washed by the addition of 120 μl of a mixture. hybridization containing 4 x SSC, triton X100 0.66%, deionized formamide 6. 66 ^, 1 mg / ml BSA and 5 nM indicator probe.After hybridization for one hour at 37 ° C, the plate is washed again six The immobilized alkaline phosphatase activity is detected by the addition of 100 μl of 0.2 mM 4-methylumbelliferyl phosphate.

(MUBP, JBL Scientific) in buffer d (diethanolamine 2.5 M, pH 8.9) (JBL Scientific), MgCl; 10 mM, zinc acetate 5 mM dihydrate and 5 mM N-hydroxyethylethylenediamine tetraacetic acid). The plates are incubated at 37 ° C. Fluorescence was measured at 450 nM using a microplate fluorometer (Dynateck) of excitation at 365 nM.

Evaluation of the microplate-based compound in MT-2 cells infected with HIV-1: The compounds to be evaluated are dissolved in DMSO and diluted in culture medium at two times the highest concentration to be tested and a maximum DMSO concentration of 2%. Subsequently serial triple dilutions of the compound in culture medium are made directly in microtiter plates with U-bottom (Nunc). After dilution of the compound, MT-2 cells (50 μl) are added to a final concentration of 5 x 10S per ml (1 x 105 per well). The cells are incubated with compounds for 30 minutes at 37 ° C in a C02 incubator. For evaluation of the antiviral potency, an appropriate dilution of virus concentrate (50 μl) (RF) HIV-1, culture wells which contain cells and dilutions of the test compounds are added to the culture wells. The final volume in each of the wells is 200 μl. Eight wells were left uninfected per plate with 50 μl of added medium instead of virus, while eight wells were left infected in the absence of any antiviral compound. For the evaluation of the compound's toxicity, plaques were grown in parallel without virus infection. After 3 days of culture at 37 ° C in a humidified chamber inside a C02 incubator, all except the 25 μl of medium / well of the HIV-infected plates was removed. 37 μl of 5 M GED containing biotinylated capture probe was added to the pelleted cells and the remaining medium in each well to a final concentration of 3 M GED and 30 nM capture probe. Hybridization of the capture probe to HIV RNA in the cell lysate was carried out in the same microplate well used for virus culture by sealing the plate with a plate sealer (Costar) and incubating for 16-20 h in an incubator at 37 ° C. Distilled water is then added to each well to dilute the hybridization reaction three times and 150 μl of this diluted mixture is transferred to a microtiter plate coated with streptavidin. The HIV RNA is quantified as described above. A standard curve, prepared by adding known quantities of RNA transcript pDAB 72 in vi tro to wells containing lysed uninfected cells, runs in each microtiter plate in order to determine the amount of viral RNA made during infection. In order to standardize the virus inoculum used in the evaluation of compounds for antiviral activity, dilutions of virus were selected which resulted in an IC90 value (concentration of compound necessary to reduce the level of HIV RNA in 90%) for dideoxycytidine (ddC) of 0.2 μg / ml. The IC90 values of other antiviral compounds, both more and less potent than ddC, were reproducible using several concentrates of HIV-1 (RF) when this procedure was followed. This concentration of virus corresponds to -3 x 10s PFU (measured by plaque assay in MT-2 cells) per test well and typically produces approximately 75% of the maximum level of viral RNA available to any virus inoculum. For the HIV RNA assay, IC90 values were determined from the percent reduction of the net signal (signal from infected cell samples minus the signal from uninfected cell samples) in the RNA assay relative to the net signal of untreated infected cells in the same culture plate (average of 8 wells). A judgment of the valid functioning of the individual infection and the RNA assay tests was performed according to three criteria. It was required that the virus infection result in an RNA test signal equal to or greater than the signal generated from 2 ng of the transcript of AFNpDAB 72 in vi tro. The CI90 for CDD, determined in each test run, should be between 0.1 and 0.3 μg / ml. Finally, the level of meceta or stable viral RNA produced by an effective protease inhibitor should be less than 10% of the level reached in an uninhibited infection. A compound is considered active if CI90 is found to be less than 1 μM. For the antiviral potency tests, all manipulations in the microtiter plates, followed by the initial addition of a 2X concentrated compound solution to a single row of wells, was performed using a ProPette Perkin Elmer / Cetus kit.

Dosage and formulation The antiviral compounds of this invention can be administered as a treatment for viral infections by any means that produces contact of the active agent with the site of action of the agent, i.e., the viral protease, in the body of a mammal. It can be administered by any conventional means available for use together with pharmaceutical substances, either individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but are preferably administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. Of course, the dosage administered will vary based on known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and extent of the symptoms; the class of concurrent treatment; the frequency of treatment; and the desired effect. It can be expected that a daily dosage of active ingredient is from about 0.001 to about 1000 milligrams per kilogram of body weight, with a preferred dose being between about 0.1 and about 30 mg / kg. Dosage forms of compositions suitable for administration contain from about 1 mg to about 100 mg of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will usually be present in an amount from about 0.5-95% by weight based on the total weight of the composition. The active ingredient can be administered orally in solid dosage form, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. It can also be administered parenterally, in the form of a sterile liquid dosage. Gelatin capsules contain the active ingredient and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. Diluents and the like can be used to make compressed tablets. Both the tablets and the capsules can be manufactured as sustained release products to provide continuous release of medication during a period of hours. Compressed tablets can be coated with sugar or a coating film to mask any unpleasant taste and to protect the tablet from the atmosphere, or they can be coated with an enteric layer for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorants and flavors to increase patient acceptance. In general, water, a suitable oil, saline solution, aqueous dextrose (glucose) and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering substances. Suitable stabilizing agents are antioxidant agents such as sodium bisulfite, sodium sulfite or ascorbic acid, either alone or in combination. Citric acid and its salts, and sodium EDTA are also used. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceu t? ca.1 Sciences, supra, a standard reference text in this field.

The pharmaceutical dosage forms useful for administration of the compounds of this invention can be illustrated as follows: Capsules A large number of capsule units can be prepared by filling standard hard gelatin capsules of two pieces each with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesium stearate.

Soft gelatin capsules A mixture of active ingredient can be prepared in a digestible oil such as soybean oil, cottonseed oil or olive oil and can be injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 mg of the active ingredient. The capsules should then be washed and dried.

Tablets A large number of tablets can be prepared by conventional procedures so that the dosage unit is 100 mg of active ingredient, 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 mg of lactose. Appropriate coatings can be applied to increase palatability or to recharge absorption.

Suspension An aqueous suspension can be prepared for oral administration so that each 5 ml contains 25 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, USP and 0.025 mg of vanillin Injectable solution A parenteral composition suitable for administration by injection can be prepared by stirring 1.5% by weight of active ingredient and 10% by volume of propylene glycol and water. The solution is sterilized by commonly used techniques.

Combination of components (a) and (b) Each therapeutic agent component of this invention can be independently in any dosage form, such as those described above, and can also be administered in various ways, as described above. In the following description, it should be understood that the component (bi represents one or more agents as previously described.) Therefore, if components (a) and (b) are not treated equally or independently, each component agent ( b) can also be treated equally or independently The components (a) and (b) of the present invention can be formulated together, in a single dosage unit (that is, combined together in a capsule, tablet, powder, liquid, etc.), as a combination product When component (a) and (b) are not formulated together in a single dosage unit, component (a) can be administered at the same time as component (b) or in any order, for example, component (a) of this invention may be administered first, followed by administration of component (b) or may be administered in reverse order If component (b) contains more than one agent, for example a RT inhibitor and a protease inhibitor , these agents can be administered together or in any order. When not administered at the same time, preferably the administration of component (a) and (b) occurs with a difference of less than one hour. Preferably, the route of administration of component (a) and (b) is oral. The terms oral agent, oral inhibitor, oral compound or the like, as used herein, denote compounds which can be administered orally.

Although it is preferred that component (a) and the component (b) both are administered by the same route (that is, for example, both orally) or by dosage form, if desired, each can be administered by different routes (ie, for example, a component of the product of combination can be administered orally, and another component can be administered intravenously) or dosage forms.

As appreciated by a person familiar with medical technology, the dosage of the combination therapy of the invention may vary based on various factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the age, health and weight of the recipient, the nature and extent of the symptoms , the type of concurrent treatment, the frequency of treatment and the vectorized, as described above. The proper dosage of components (a) and (b) of the present invention will be readily determined by a person familiar in medical practice based on the present disclosure. As a general guide, typically a daily dosage may be from about 100 milligrams to about 1.5 grams of each component. If component (b) represents more than one compound, then typically a daily dosage will be from about 100 milligrams to about 1.5 grams of each component agent (b). By means of a general guide, the compounds of component (a) and component (b) are administered in combination, the dosage amount of each component can be reduced by approximately 70-80% relative to the usual dosage of the component when it is administered alone as a single agent for the treatment of HIV infection. The combination products of this invention can be formulated so that, although the active ingredients are combined in a single dosage unit, physical contact between the active ingredients is minimized. To minimize contact, for example, when the product is administered orally, an active ingredient may be enteric coated. By enterically coating one of the active ingredients, it is possible not only to minimize the contact between the combined active ingredients, but it is also possible to control the release of these components in the gastrointestinal tract so that one of these components is not released in the stomach but rather be released in the intestines. Another embodiment of this invention where oral administration is desired provides a combination product wherein one of the active ingredients is coated with a sustained release material which carries out a sustained release through the gastrointestinal tract and also serves to minimize the physical contact between the combined active ingredients. In addition, the sustained release component can be enteric coated additionally so that the release of this component occurs only in the intestine. Another additional approach would involve the formulation of a combination product in which one component is coated with a sustained release and / or enteric polymer, and the other component is also coated with a polymer such as a low viscosity grade of hydroxypropylmethylcellulose or other suitable materials known in the art, in order to separate the active components. The polymer coating serves to form an additional barrier for interaction with other components. In each formulation, where contact between components (a) and (b) is avoided via a coating or some other material, contact between individual agents of component (b) can also be avoided. Dosage forms of the combination products of the present invention wherein an active ingredient is enteric coated, may be in the form of tablets such as an enteric coated component and the other active ingredient is mixed together and then compressed into a tablet. so that the enteric coated component is compressed into a tablet layer and the other active ingredient is compressed into an additional layer. Optionally, in order to further separate the two layers, one or more placebo layers may be present so that the placebo plate is between the layers of active ingredients. In addition, the dosage forms of the present invention may be in the form of capsules wherein an active ingredient is compressed from a tablet or in the form of a plurality of microtablets, particles, granules or innocuous materials, which are then encapsulated enterically. These enteric coated microtablets, particles, granules or innocuous materials are then placed in a capsule or compressed into a capsule together with a granulation of the other active ingredient. These as well as other ways of minimizing contact between the components of combination products of the present invention, when administered in a single dosage form or when administered in separate forms but at the same time or concurrently in the same manner, will be evident. easily for those familiar with the art, based on the present description. Pharmaceutical kits useful for the treatment of HIV infection which comprise a therapeutically effective amount of a pharmaceutical composition comprising a compound of component (a) in one or more compounds of component (b) in one or more sterile containers, is also within the scope of the present invention. Sterilization of the container can be carried out using conventional sterilization methodology well known to those familiar with the art. Component (a) and component (b) can be in the same sterile container or in separate sterile containers. The sterile containers of the materials may comprise separate containers, or one or more containers with multiple parts, as desired. Component (a) and component (b) can be separated, or physically combined in a single dosage form or unit as described above. Such equipment may also include, if desired, one or more components of conventional pharmaceutical equipment, such as, for example, one or more pharmaceutically acceptable carriers, additional bottles for mixing the components, etc., as will be readily apparent to those familiar with The technique. Instructions such as inserts or labels can also be included in the equipment, indicating quantities of the "components to be administered, the guidelines for administration and / or the guide lines for mixing the components. numerous modifications or variations of the present invention in light of the foregoing teachings, it should therefore be understood that within the scope of the appended claims, the invention may be practiced in other ways in addition to that specifically described herein. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers.

Claims (15)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1 . A compound of formula I:

Or a pharmaceutically acceptable salt or prodrug form, wherein a prodrug of formula 1 is a compound in which two hydroxy groups are joined to form an epoxide group; -OCH2SCHO-; -OC (= 0) 0-; -OCH20-; -OC (= S) 0-; -OC (= O) C (= 0) 0; -0C (CH,). 0-; -OC ((CH,) 3 NH?) (CH3) O-; -OC (OCH3) (CH2CH2CH3) 0-; O -OS (= 0) 0-. 2. The compound according to claim 1, characterized in that the compound is of formula I.

3. A pharmaceutical composition characterized in that it comprises a pharmaceutically acceptable carrier and • a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt or prodrug form thereof.

4. The composition according to claim 3, characterized in that the compound is of formula I.

5. The use of a compound of formula I or a pharmaceutically acceptable salt or prodrug form thereof, for the manufacture of a medicament for treating HIV infection: wherein a prodrug of formula I is a compound in which two hydroxy groups join to form an epoxide group; -OCH2SCH20-; -OC (= 0) 0-; -0CH20-; -OC (= S) 0-; -OC (= 0) C (= 0) 0; -OC (CH3) 20-; -OC ((CH2) 3NH2) (CH3) O-; -OC (OCH3) (CH2CH2CH3) 0-; or -OS (= 0) 0-.

6. The use according to claim 5, characterized in that the compound is of formula I.

7. The use of a combination of (a) and (b) for the manufacture of a medicament for treating HIV infection, wherein: (a) is a compound of formula I or a pharmaceutically acceptable salt or prodrug form thereof : wherein a prodrug of formula I is a compound in which two hydroxy groups join to form an epoxide group; -OCH2SCH20-; -0C (= 0) 0-; -0CH20-; -0C (= S) 0-; -0C (= 0) C (= 0) 0; -OC (CH3) 20-; -OC ((CH2) 3NH2) (CH3) O-; -OC (OCH3) (CH2CH2CH3) 0-; or -OS (= 0) 0- and (b) is at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and protease inhibitors. HIV

8. The use according to claim 7, characterized in that the compound is of formula I.

9. The use according to claim 7, characterized in that the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor.

10. The use according to claim 9, characterized in that the nucleoside reverse transcriptase inhibitor is selected from AZT, 3TC, ddl, ddC and d4T and the protease inhibitor is selected from saquinavir, ritonavir, indinavir, VX-478, nelfinavir, KNI-272, CGP-61755 and U-103017.

11. The use according to claim 10, characterized in that the nucleoside reverse transcriptase inhibitor is selected from AZT, and 3TC and the protease inhibitor is selected from saquinavir, ritonavir and mdinavir.

12. The use according to claim 11, characterized in that the inhibitor of nucleoside reverse transcriptase is AZT.

13. The use according to claim 11, characterized in that the transcriptase inhibitor is indinavir.

14. A pharmaceutical equipment, useful for the treatment of HIV infection, characterized in that it comprises a therapeutically effective amount of: (a) a compound according to claim 1; and (b) at least one compound that is selected from the group consisting of. HIV reverse transcriptase inhibitors and HIV protease inhibitors, in one or more sterile containers.

15. The equipment according to claim 14, characterized in that component (a) is a compound of formula I.