LT4619B - (4r,5s,6s,7r)-hexahydro-1-[5-(3-aminoinazole)methyl]-butyl-5,6-dihydroxy-4,7-bis[phenylmethyl]-2h-1,3-diazepin-2-one, its preparation and its use as hiv protease inhibitor - Google Patents

Abstract

This invention relates to formula (I) compounds, its pharmaceutically suitable salts and its pro-medicine forms are suitable to use as HIV protease inhibitors, to pharmaceutical compositions and diagnostic sets which include these compounds, and to its use for treatment of viral infections or to perform standard and reagent testing.<IMAGE>

Description

FIELD OF INVENTION

The present invention relates essentially to 1- (3-aminoindazol-5-yl) -3-butycyclic urea which is useful as an HIV protease inhibitor, to pharmaceutical compositions and diagnostic kits comprising this urea, and to methods of using it in viral for the treatment of infections or as standards and reagents for testing.

ORIGIN OF THE INVENTION

Two separate retroviruses, human immunodeficiency virus (HIV) type 1 (HIV-1) or type 2 (HIV-2), have been etiologically associated with immunosuppressive disease Acquired Immunodeficiency Syndrome (AIDS). HIV seropositive individuals initially show no evidence of disease, but generally develop AiDS-associated complex (ARO) followed by AiDS. Individuals affected by the disease are severely depressed of their immunity, leading to burnout and ultimately to fatal, timely infections.

AIDS disease is the end result of the action of HIV-1 or HIV-2 following the complex life cycle of the virus, the life cycle of the virus begins by attaching the virus to human host T-4 lymphocytic immune cells via a glycoprotein on the surface of the virus envelope CD4 glycoprotein on a lymphocyte cell. When attachment occurs, the virus dips its glycoprotein into its envelope, penetrates the membrane of the host cells and exposes its RNA. Viral enzyme reverse transcriptase - directs the RNA transcription process into single-stranded DNA, viral RNA is degraded and a second strand of DNA is created. This double-stranded DNA is integrated into the genes of a human cell, and these genes are used for cell reproduction.

In this case, the human cell undergoes a process of reproduction using its own RNA polymerase to transcribe integrated DNA into viral RNA. Viral RNA is translated into the precursor gag-pol fusion polyprotein. This polyprotein is then cleaved by the HIV protease enzyme to produce mature viral proteins. Thus, the HIV protease is responsible for regulating the cascade of cleavages that lead to the maturation of the viral particle into a virus that has full infection capacity.

The typical response of the human immune system, which kills the virus upon penetration, is complicated by the fact that most of the virus's life cycle goes through a latent state inside the immune cell. In addition, the viral reverse transcriptase, an enzyme used to form a new viral particle, is not very specific and causes transcription errors that yield ever-changing glycoproteins on the surface of the virus's protective envelope. This lack of specificity reduces the effectiveness of the immune system, since antibodies specifically produced against one glycoprotein may be useless against another glycoprotein, thus reducing the number of antibodies that can fight the virus. The virus continues to reproduce and the immune system's response is weakened. Finally, HIV completely overcomes the body's immune system, enabling timely infections, and death without the use of antiviral agents, immunomodulators, or both.

There are at least three critical points in the viral life cycle that have been identified as potential antiviral targets (1) initial viral binding to a T-4 lymphocyte or macrophage site, (2) transcription of viral RNA into viral DNA (reverse transcriptase, AT), and (3) ) assembly of a new viral particle during reproduction (e.g., HIV aspartic acid protease or HIV protease).

Retroviral genomes encode a protease that is responsible for the proteolytic maturation of one or more polyprotein precursors, such as po / and gag gene products. See also: Wellink, Arch. Virol. 98, 1 (1988). Retroviral proteases commonly mature the gag precursor to the parent protein, and also mature the pol precursor to the reverse transcriptase and the retroviral protease.

The formation of an infectious virus requires the precise maturation of its precursor polyproteins by a retroviral protease. It has been shown that in vitro protease deficient virus mutagenesis produces immature basic forms that are not infectious. See also: Cravford et al., J. Virol. 53: 899 (1985); Katoh et al., Virology 145, 280 (1985), therefore, inhibition of retroviral protease is an attractive target for antiviral therapy. See also: Mitsuya, Native Re 325, 775 (1987).

The ability to inhibit viral protease creates a way to block viral replication, and thus a treatment for viral diseases such as AIDS, which may have fewer side effects, be more effective and less drug resistant than current treatments. As a result, three HIV protease inhibitors are currently on the market, Roche saquinavir, Abbott ritonavir and MerckO indinavir, and clinical trials of several potential protease inhibitors, e.g. Vertex VX-478, Agouron nelfinavir, Japan Energy KNI-272 and Ciba-Geigy CGP 61755.

Based on data from commercially available and clinically tested protease inhibitors, a variety of compounds that may be HIV protease inhibitors have been investigated. One structure, cyclic ureas, has received special attention. For example, in PCT Application Nos. WO94 / 19329 and WO93 / 07128, Lam et al. generally describes cyclic ureas having the formula:

HO OH and methods for preparing such ureas. Although the compounds of the present invention fall within the scope of Lam and Irt. description, they are not specific.

Other cyclic ureas are described in Lam et al., J. Med. Chem. 1996, 39. 3514-3525. Although various cyclic ureas are described in this article, compounds having an imidazolomethyl group are not described.

Even with relatively successful current protease inhibitors, it has been found that HIV patients can become resistant to a single protease inhibitor. Thus, further protease inhibitors are desirable for further combating HIV infection.

SUMMARY OF THE INVENTION

Thus, one object of the present invention is to provide novel protease inhibitors.

Another object of the present invention is to provide pharmaceutical compositions having a 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 or a prodrug thereof.

Yet another object of the present invention is to provide a novel treatment for HIV infection which comprises administering to a subject in need of such treatment a therapeutically effective amount of at least one of the compounds of the present invention, or a pharmaceutically acceptable salt thereof, or prodrug thereof.

Yet another object of the present invention is to provide a novel method of treating HIV infection which comprises a therapeutically effective combination of (a) one of the compounds of the present invention and (b) one or more compounds selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors. to a subject in need of such treatment.

Yet another object of the present invention is to provide a method of inhibiting HIV in a body fluid sample which comprises treating the body fluid sample with an effective amount of a compound of the invention.

Another object of the present invention is to provide a kit wherein the amount of at least one of the compounds of the present invention is effective as a standard or reagent in a test or assay to identify a potential drug that can inhibit HIV protease, HIV growth, or both.

These and other objects, which will be apparent from the following detailed description, have been achieved by the inventors, having discovered that the formula:

(b) administering to a subject in need of such treatment a therapeutically effective amount of at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors.

In another most preferred embodiment, the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor.

In a more preferred embodiment, the nucleoside reverse transcriptase inhibitor is selected from AZT, 3TC, ddl, ddC and d4T, and the protease innibitor is selected from saquinavir, ritonavir, indinavir, VX478, nelfinavir, KNI-2717, CGP-61755.

In another more 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 more preferred embodiment, the nucleoside reverse transcriptase inhibitor is AZT.

In another more preferred embodiment, the protease inhibitor is indinavir.

A fifth embodiment of the present invention provides a pharmaceutical kit for treating HIV infection comprising a therapeutically effective amount of:

(a) a compound of formula i; and (b) at least one compound 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 of inhibiting HIV in a bodily fluid sample which comprises treating the bodily fluid sample with an effective amount of a compound of the formulas.

A seventh embodiment of the present invention provides a novel kit or container containing an amount of a compound of Formula I that is effective for use as a standard or as a reagent in a test or assay for determining the ability of a potential drug to inhibit HIV protease, HIV growth, or both.

DEFINITIONS

The following terms and expressions have the meanings given below. It will be appreciated that the compounds of the present invention have an asymmetrically substituted carbon atom and may be isolated as optically active or as racemic forms. It is known to those skilled in the art how to prepare optically active forms, for example, by resolving racemic forms or by synthesis from optically active starting materials. Unless a particular stereochemistry or isomeric form is specified, all chiral, diastereomeric, racemic, and geometric isomeric forms are meant.

As used herein, the term "HIV reverse transcriptase inhibitor" refers to both nucleoside and non-nucleoside HIV reverse transcriptase (AT) inhibitors. Examples of nucleoside AT inhibitors include, but are not limited to, AZT, ddC, ddl d4T, and 3TC. Examples of non-nucleoside AT inhibitors include, but are not limited to, viviradine (Pharmacia and Upjohn U90152S), TIBO derivatives, BI-RG-587, nevirapine, L-697,661, LY 73497, and Ro 18,893 (Roche).

As used herein, the term "HIV protease inhibitor" refers to compounds that 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-272 (Japan Energy), CGP-61755 (Ciba-Geigy) and U-103017 (Pharmacia and Upjohn). Preferred examples include cyclic protease inhibitors described in WO 93/07123, WO 94/19329, WO 94/22840 and PCT Application No. US96 / 03426.

As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the compounds of the invention where the parent compound is modified to form its salts with acids or bases. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as an amino group: acidic residues such as a carboxylic acid group, alkali metal or organic salts; and etc. Pharmaceutically acceptable salts include the usual non-toxic salts or the resulting quaternary ammonium salts of the parent compound, for example, from non-toxic inorganic or organic acids. For example, such common non-toxic salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, sulfate, sulfamic, phosphate, nitrate and the like; and salts derived from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamo, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanyl, 2-acetoxybenzoic, fumar, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxal, isethion and the like. of acids.

Pharmaceutically acceptable salts of the compounds of the present invention may be synthesized from the parent compound containing a basic or acidic residue by conventional chemical means. Generally, such salts may be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the corresponding base or acid in water or in an organic solvent, or in a mixture of both; non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are usually preferred. A list of suitable salts is found in Remington's Pharmaceutical Sciences, 17th ed., Mack-Publishing Company, Easton, PA, 1985, p. 1418, which is given here as a source of literature.

As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms which, by medical judgment, are suitable for use in contact with human or animal tissues and do not show excessive toxicity, irritation, allergic response, or other problems and complications. acceptable ratio of benefits, physics,

It is considered that ^; Prodrugs include any covalently attached carrier which will release the active parent compound of formula I or other compounds of the present invention or compounds in vivo when such a prodrug is administered to a mammal. Prodrugs of a compound of the invention, such as a compound of formula (I), are prepared by modifying the functional groups present in the compound such that such modifications are cleaved either by conventional manipulation or in vivo to the parent compound. Prodrugs are compounds of the present invention wherein the hydroxyl or amino group is attached to a group such that when such a prodrug is obtained in a mammal, it is cleaved to form a free hydroxyl or amino group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, or benzoate derivatives of alcohol and amino functional groups of a compound of Formula I; Phosphate esters, dimethylglycine esters, aminoalkyl benzyl esters, aminoalkyl esters and carboxyalkyl esters of alcohol functional groups of a compound of formula I; and etc. Further examples are compounds which combine two hydroxyl groups of compounds of formula I to form an epoxide; -OCH ₂ SCH ₂ O-:

-OC (= O) O-, -OCH 2 O-; -OC (= S) O-; -00 (= 0) 0 (= 0) 0-: -OC (CH ₃ ) ₂ O: -00 ((ΟΗ ₂ ) ₃ ΝΗ ₂ ) (ΟΗ ₃ ) 0-; -00 (ΟΟΗ3) (ΟΗ ₂ ΟΗ ₂ ΟΗ3) 0-; or-0S (= 0) 0-.

"Stable compound" and "stable structure" are intended to mean a compound which is sufficiently resistant to withstand isolation from the reaction mixture to an appropriate degree of purity and preparation of an effective therapeutic agent composition. The present invention includes only stable compounds.

The term "substituted" means that one or more hydrogens at the atom specified in the expression "substituted" are substituted in any set of the specified groups, provided that the normal valence of the indicated atom is not exceeded and that such substitution produces a stable compound. (ie, = 0) then 2 hydrogens are substituted at that atom.

"Therapeutically effective crack" means an amount of a compound or combination of compounds of this invention that is defined as effectively inhibiting a subject's ZiV infection or treating a subject's HIV infection symptoms. Preferably, the combination of compounds would be a synergistic combination. Synergistic effect as described, for example, in Chou and Talalay, Adv. Enzyme Reaul. 22: 27-55 (1934), is when the effect of a compound (in this case inhibition of HIV replication) when used in combination is greater than the additive effect of the compounds when used alone. Generally, synergistic effects are most clearly demonstrated at sub-optimal concentrations of compounds. The synergistic effect may be due to the combination's reduced cytotoxicity, enhanced antiviral activity, or any other beneficial effect compared to the individual components.

Other features of the invention will become apparent from the following description of exemplary embodiments, which are given by way of illustration of the invention.

EXAMPLES

The following abbreviations are used in the examples: ° C - degrees Celsius, - d - doublet, - dd - double doublet, - eq - equivalent or equivalents, g - gram or grams, - mg - milligram or milligrams, - ml - milliliter or milliliters, "H is hydrogen or hydrogen,". - hour or hours, "m - multiplet," M - molar concentration, "min. - minute or minutes, "MHz - megahertz," MS - mass spectroscopy, "NMR nuclear magnetic resonance spectroscopy," t - triplet, and "PSC thin layer chromatography.

EXAMPLE (4R, 5S, 6S, 7R) -Hexahydro-1- [5- (3-aminoindazole) methyl] -3-butyl-5,6-dihydroxy-4,7-bis [phenylmethyl] -2H-1,3-diazapine Obtaining -2-one (I)

Compound A can be prepared by known methods. For example, the preparation of compound A is illustrated in Scheme 1 'by Rossano et al., (Tetr. Lett. 1995, 36 (28), 4967, 4968), which is given as a source of literature. An additional process for preparing Compound A is shown in U.S. Pat. 5,530,124, which is given as a reference in Example 6

Part A: Methyl triflate (3.4 mL, 30 mmol) was added to a suspension of compound A (10.0 g, 27.3 mmol) in 1,2-dichloroethane (100 mL). After refluxing overnight, the reaction mixture was washed with saturated NaHCO ₃ , saturated NaCl, dried (Na ₂ SCu), and basified with 12.5 g of a yellow oil, column chromatography (SiO ₂ flash chromatography, 25% EtOAc / hexane). 7.86 g of compound 1 is obtained which is a yellowish oil which liberates on standing (75% yield). Lyd. temp. = 97-100 ° C. MH ⁺ = 381,

OMe

O.O.

Part B: To a solution of compound 1 (10.0 g, 26.3 mmol) in anhydrous DMF (30 mL) was added sodium hydride (1.58 g, 65.8 mmol), and the reaction mixture was stirred at room temperature for 45 min. A solution of 1-iodobutane (9.68 g, 52.6 mmol) in anhydrous DMF (10 mL) was added dropwise. After the addition is complete, stir overnight at room temperature. The reaction mixture was cooled to 0 ° C and methanol (5 mL) was added to quench the excess sodium hydride. The mixture was partitioned between ethyl acetate (200 mL) and water (150 mL). The organic phase was separated and washed with water (4 x 100 mL), brine (100 mL) and dried over sodium sulfate. Purification by flash chromatography (25% EtOAc / hex) afforded n-butyl isocarbamide 2 (10.5 g, 92% yield): MS (NH ₃ -Cl / DDIP) (M + H ⁺ ) 437.2 (100%). ): Ή NMR (300 MHz, CDCl ₃ , 25 ° C) δ 7.23 (m, 10H), 4.19 (m, 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-fluorenyl) methyl] -5,6O-isopropylidene-4,7-bis- (4-phenylmethyl) - 3-Butyl-2H-1,3-diazapin-2-one (3).

To a solution of compound 2 (5.0 g, 11.5 mmol) in acetonitrile (40 mL) was added 4-fluoro-3-cyanobenzyl bromide (3.68 g, 17.25 mmol). The reaction mixture was refluxed overnight. After evaporation of the solvent under reduced pressure, the residue was purified by flash chromatography (35% EtOAc / hex.) To give the cyclic urea 3 as a white solid (4.5 g, 71% yield): MS (NH3-CI / DDIP) ( M + H ⁺ ) 556.3 (100%); ¹ H NMR (300 MHz, CDCl ₃ , 25 ° C) δ 7.41 (m, 1H), 7.28 (m, 7H), 7.13 (d, J = 9.2 Hz, 2H), δ , 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 -Diazapin-2-one (I).

To a solution of compound 3 (4.5 g, 8.11 mmol) in n-butanol (20 mL) was added hydrazine hydrate (0.81 g, 16.2 mmol), and the mixture was refluxed for 6 hours. The solvent and excess hydrazine are evaporated off under reduced pressure. The residue was dissolved in anhydrous methanol (20 mL), followed by the addition of 4M HCl in dioxane (2 mL), and the reaction mixture was stirred at room temperature for 2 hours. Methanol was evaporated and the residue partitioned between ethyl acetate (80 mL) and acidic sodium carbonate (saturated solution) (50 mL). The organic phase was separated, washed with water (2 x 50 mL) and dried over sodium sulfate (anhydrous). Purification by flash chromatography afforded I (3.0 g, 72% yield) as a white solid: m.p. temp. 129-131 ° C; MS (NH ₃ Cl / DDIP) (M + H ⁺ ) 528.3 (100 To); HRMS calculated for C ₃₁ H ₅ O ₃ 7N ₃ +1

528.29 / 5, found 528.2958;

Ή NMR (300 MHz, CD ₃ OD, 25 ° C) δ 7.19 (m, 12H)

6.98 (d, Hz, 1H) 1.37 (m

J = 1.5 Hz, 2 Hz, 4.74 (d, J = 13.9 Hz, 1H), 3.85 (dd, J = 10.25, 4.76 3.65 fm, 1H), 3, 56 (m, 4H), 3.15 (m, 2H), 2.96 (m, 3H), 2.07 (m, 2H), 2H), 1.22 (m, 2H), 0.84 (r, J = 7.0 Hz, 3H),

OPPORTUNITIES

The compounds of formula I exhibit HIV protease inhibitory activity and are therefore useful as antiviral agents for the treatment of HIV infection and related diseases. The compounds of formula I exhibit HIV protease inhibitory activity and are effective as HIV growth inhibitors. The ability of the compounds of the present invention to inhibit viral growth or infectivity is demonstrated in a standard assay for viral growth or infectivity, for example, using the assay described below.

The compounds of formula I of the present invention are also suitable for inhibiting HIV in an ex vivo HIV-positive sample or in contact with HIV. Thus, the compounds of the present invention may be used to: inhibit HIV in a body fluid sample (e.g., a serum or seminal sample) containing HIV that is suspected of having or has been exposed to HIV.

The compounds of the present invention are also useful as standard or reference compounds in assays or assays for determining the ability of any agent to inhibit viral clone replication and / or HIV protease, e.g., in drug discovery programs. Thus, 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 may be provided in commercial kits or containers for use as such standard or reference compound.

Because the compounds of the present invention have specificity for HIV protease, the compounds of the present invention may also be useful as diagnostic reagents in diagnostic assays for HIV protease. Thus, the inhibition of this protease activity by a compound of the present invention (as described herein) will indicate the presence of HIV protease and HIV virus.

The abbreviations "ug" used herein mean microgram, mg milligram, "g" gram, "μ," microliter. "Ml" - milliliter, "i" - liter, "nM" ·· nanomolar, "uM" - micromolar, "mM" - millimolar, "M" - molar concentrations, and nm "- nanometer. "Sigma" means SigmaAldrich Corporation of St, Louis, MO.

HIV RNA TESTING

DNA plasmids and in vitro RNA transcripts:

Plasmid pDAB 72 containing both gag and pol BH10 (bp 113-1816) sequences cloned into PTZ 19R was prepared according to Erickson-Viitanen et al. AIDS Research and Human Retroviruses 1989, 5, 577, Prior to generation of in vitro RNA transcripts, this plasmid was linearized in Bam HI using Riboprobe Gemini System Set II (Promega) with T7 RNA polymerase. The synthesized RNA was purified by treatment with RNAse-free DNAse (Promega), phenol-chloroform extraction and ethanol precipitation. RNA transcripts were dissolved in water and stored at −70 ° C. RNA concentration is determined from A250.

Samples:

After synthesis using Applied Biosystems (Foster City, CA) DNA synthesizer, binding biotin to the 5 'end of the oligonucleotide using Cocuzza, Tet. Lett. 1989, 30, 6287 Biotin-phosphoramidite reagent, biotinylated test samples were purified by high performance liquid chromatography (HPLC). gag biotinylated test sample (5-biotinCTAGCTCCCTGCTTGCCCATACTA 3 ') was complementary to ΗΧΒ2889-912 nucleotides, whereas pol biotinylated test sample (5-biotinCCCTATCAi I I IGGTTTCCAT 3') was complementary to ΗΧΒ2394-239. Alkaline phosphatase-conjugated oligonucleotides used as control samples were made using Syngene (San Diego, CA), a pol conirole sample (5 'CTGTCTTACTTTGATAAAACCTC 3') was complementary to ΗΧΒ2 2403-2425 nucleotides. gag control sample (5

CCCAGTATTTGTCTACAGCCTTCT 3y was complementary to ΗΧΒ2 950-973 nucleotides. All nucleotide positions are as in Genbank's Genetic Seguence Data Bank as determined by Genetics Computer

Group Seguence Analysis software package (Devereau Nucleic Acids Research 1984, 72, 387). Control samples were prepared as 0.5 μΜ blanks in 2 x SSC (0.3 M NaCi, 0.03 M sodium citrate). 0.05 M Tris pH 8.8, 1 mg / ml BSA. Biotinylated test samples were prepared as 100 μΜ preforms in water.

Streptavidin-coated plates:

Streptavidin-coated plates were obtained from Du Pont Biotechnology Systems (Boston, MA).

Cells and virus blanks:

MT-2 and MT-4 cells were maintained in RPMI 1640 supplemented with 5% fetal calf serum (FCS) for MT-2 cells or 10% FCS for MT-4 cells, 2 mM L-glutamine and 50 µg / ml gentamicin. (all reagents from Gibco). HIV-1 RF was propagated in MT-4 cells in the same medium.

Virus blanks were prepared approximately 10 days after acute infection of MT-4 cells and stored as aliquots at -70 ° C. Infection titers for HIV-1 (RF) blanks were 1-3x10 ⁷ PFU (plaque forming units / ml as measured using a plaque assay with MT-2 cells (see below). Each aliquot of virus used for infection was thawed only generation.

Antiviral efficacy For evaluation, the cells to be infected were recultivated one day before infection. On the day of infection, cells were resuspended in 5x10 ³ cells / ml RPMI 1640, 5% FCS for volume infection or 2x10 ⁶ cells / ml in Dulbecco's modified Eagies medium with 5% FCS for microtitre plate infection. Virus is added and cultured for 3 days at 37 ° C.

HIV RNA test:

Cell lysates or purified RNA in 3M or 5M GED were mixed with 5M GED and the test sample to give 3M final guanidine isothiocyanate concentration and 30 nM final biotic oligonucleotide concentration. Hybridization was performed in closed U-bottom 96-well tissue culture plates (Nunc or Costar) for 16-20 h. At 37 ° C, mixtures of RNA hybridization reactions were diluted twice with deionized water to a final concentration of 1M guanidinium isothiocyanate and aliquoted (150 μΙ) were added to streptavidin-coated microtiter wells. Binding of the test sample and test sample-RNA hybrid to immobilized streptavidin was allowed for 2 hours. at room temperature, and then the plates were washed 6 times with DuPont ELISA plate wash buffer (phosphate buffer with NaCl (PBS), 0.05% Tween 20). The second hybridization of the control sample with immobilized test sample and hybridized test RNA complex was performed in a washed, straptavidin-coated well by addition of 120 μΙ 4xSSC hybridization mixture, 0.66% Tritor X 100, 6.66% deionized formamide, BSA and 5 nM control. After 1 or. the plates were washed 6 times again at 37 ° C hybridization. Immobilized alkaline phosphatase activity was determined by adding 100 μΙ of 0.2 mM 4-methylumbeliferyl phosphate (MUBP, JBL Scientific) in buffer 5 (2.5 M diethanolamine, pH 8.9 (JBL Scientific), 10 mM MgCl 2, 5 mM zinc acetate dihydrate and 5 mM N-hydroxyethyl ethylenediaminetetriacetic acid). The plates were incubated at 37 ° C. Fluorescence is measured at 450 nm with a microplate fluorimeter (Dynateck), excited at 365 nm.

Microplate Assay-Based Compound Evaluation in HIV-1-Infected MT-2 Cells

Test compounds were dissolved in DMSO and diluted in culture medium to twice the concentration tested (maximum DMSO concentration 2%). Following this triple serial serial dilutions in culture medium are made directly in microtiter plates (Nuncj with u-bottomed wells. After dilution, MT-2 cells (50 μl) are added to give a final concentration of 5x10 ³ cells / ml (1x10 ° cells / well). Cells are incubated with compounds for 30 minutes at 37 ° C in a CO ₂ incubator Anti-viral Assay To culture wells containing cells and diluted test compounds, respectively, diluted HIV-1 (RF) virus blank (50 μΙ), Final volume in each well is 200 μΙ Eight wells in each plate were non-infectious by adding 50 μΙ medium instead of virus, and eight wells were inoculated without the addition of an antiviral compound.

After 3 days of culturing at 37 ° C in a humidified chamber inside a CO ₂ incubator, the HIV-infected plates are removed by leaving 25 μΙ in well. 37 μΙ 5M GED-containing biotinylated test sample is added to the cells in each well and the remaining medium to give final concentrations of 3M GED and 30 nm test sample. The hybridization of the test sample with HIV RNA in the cell lysate was performed in the same microplate well used for virus culturing by sealing the plate with a plate sealer (Costar) and incubating for 16-20 hours. In a 37 ° C incubator, distilled water is added to each well, diluting the hybridization reaction mixture three times, and 150 μΙ of this dilution mixture is added to a streptavidin-coated microtiter plate. HIV RNA levels are determined by the method described above. To determine the amount of viral RNA obtained during infection, a standard curve for each microtiter plate is obtained by adding known amounts of pDAB 72 in vitro RNA transcript to wells containing uninfected cells.

To standardize the viral inoculum used in the evaluation of the antiviral activity of the compound, virus dilutions were selected which give an ICgO (the concentration of compound required to achieve 90% reduction in HIV RNA) value of 0.2 ug / m! dideoxycytidine (ddC). IC ₉ values for other antiviral compounds that are more or less effective than ddC were obtained using several ZIV-1 (RF) blanks according to this procedure. The concentration of this virus corresponded to% 3x10 'PFU (measured by plaque assay with MT-2 cells) in the assay well and generally gave about 75% of the maximum amount of viral RNA obtained with any viral inoculum. For the HIV RNA assay, IC ₉₀ values were determined from the percentage reduction in pure signal (signal from infected cells minus signal from uninfected samples) in the RNA assay compared to the pure signal from uninfected cells in the same culture plate (8 wells average). The significance of individual infections and the realization of RNA tests were judged based on three criteria. The viral infection required an RNA test signal equal to or greater than that obtained with 2 ng of pDAB 72 in vitro RNA transcript. The value of ddC l ₉ o determined in each test shall be between 0,1 and 0,3 pg / ml. Finally, the amount of viral RNA produced by an effective protease inhibitor should be less than 10% of the amount produced by non-inhibited infection. A compound was considered active if its IC90 was found to be less than 1 μΜ.

In antiviral assays, all manipulations in microtitre plates, initially adding 2x concentrations of compound solution to a single well, were performed using Perkin Elmer / Cetus ProPette.

DOSAGE AND PHARMACEUTICAL FORM

The antiviral compounds of the present invention can be used to treat viral infections by any means that brings the active agent into contact with the site of action of the agent, i.e. viral protease in the mammalian body. They may be administered by any conventional route of administration, either as individual therapeutic agents or in combination of therapeutic agents. They may be used alone, but are preferably administered with a pharmaceutical carrier selected on the basis of the route of administration chosen and the usual pharmaceutical oactics.

It will be appreciated that the dosage administered should depend on known factors such as

As the pharmacodynamic characteristics of the particular agent and the route of administration, age, health and weight of the recipient, acceptance and degree of symptoms; the type of competition treatment; frequency of treatment and desired effect. The daily dose of the active ingredient can be expected to be in the range of about 0.001 mg to about 1000 mg / kg body weight, with doses in the range of 0.1-30 mg / kg being most appropriate.

Formulations suitable for administration in unit dosage form contain from about 1 mg to about 100 mg of active ingredient per unit dosage form. In these pharmaceutical compositions, the active ingredient is generally present in an amount of about 0.5-95% by weight of the total composition. The active ingredient may be administered orally in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. It can also be administered in parenterally sterile liquid dosage forms.

Gelatin capsules contain the active ingredient and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products, allowing the active ingredient to be released over an hour. Compressed tablets may be sugar-coated or film-coated to mask the unpleasant taste and weathering, or enteric-coated to provide selective gastrointestinal disintegration. Liquid dosage forms for oral administration may contain coloring and flavoring agents to enhance patient acceptability.

Suitable carriers for parenteral solutions are usually water, suitable oils, saline, aqueous dextrose (glucose) and related sugars, and glycols such as propylene glycol or polyethylene glycols. Solutions for parenteral administration generally contain a water-soluble salt of the active ingredient, suitable stabilizing agents and, if necessary, buffering agents. Suitable stabilizing agents include antioxidants, such as acidic sodium sulfite, sodium sulfite or ascorbic acid, alone or in combination. Citric acid and its salts and EDTA sodium are also used.

In addition, parenteral solutions may contain safeners such as benzalkonium chloride, methyl- or propyl-paraben and chiorbutanoyl. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, supra, which is a standard reference in the art.

Suitable pharmaceutical dosage forms of the compounds of the present invention are shown below:

Capsules

Multiple unit capsules can be made by filling standard double-sided hard gelatin capsules with 100 mg of the active ingredient powder, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesium stearate in each.

Soft gelatine capsules

A mixture of the active ingredient in edible oil, such as soybean oil, cottonseed oil or olive oil, can be prepared and injected into a gelatin ejection pump to form soft gelatin capsules containing 100 mg of the active ingredient. Thereafter, such capsules should be washed and dried.

Pills

Many tablets may be formulated according to conventional techniques so that the unit dose contains 100 mg of the active ingredient, 0.2 mg of colloidal silica, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 mg of lactose. The tablets may be coated accordingly to increase acceptability or to provide delayed absorption.

Suspensions

Aqueous oral suspensions may be formulated to contain 25 mg of finely divided active ingredient, 200 mg of sodium carboxymethylcellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution (U.S.P), and 0.025 g of vanillin per 5 ml volume.

Solution for injection

Injectable parenteral compositions may be prepared by mixing 1.5% by weight of the active ingredient in 10% by volume propylene glycol and water. This solution is sterilized according to the usual procedures.

COMBINATION OF COMPONENTS (a) AND (b)

Each agent of the present invention may independently be in any dosage form as described above, and may also be administered in a variety of ways as described above. In the following description, component (b) is considered to correspond to one or more of the agents described above. Thus, if components (a) and (b) are to be treated as the same component or independently of each other, each agent of component (b) can also be treated as the same or independently of one another.

Components (a) and (b) of the present invention may be formulated together in one dosage unit (i.e., one capsule, tablet, powder, liquid and 1.1) as a product combination. If components (a) and (b) are not mixed together in one dosage unit, component (a) may be administered at the same time as component (b) or in any order: for example, component (a) of the present invention may be used first, and then component (b) is used, or they can be used in reverse order. If component (b) contains more than one agent, such as one AT inhibitor and one protease inhibitor, these agents may be administered together or in any order. If administered simultaneously, it is best to administer components (a) and (b) within less than one hour of each other. The preferred route of administration of Components (a) and (b) is oral, as used herein, the term oral agent, oral inhibitor, oral compound, or the like, refers to compounds that may be used orally. Although it is desirable that components (a) and (b) be administered in the same way (i.e., for example, both orally) or in a dosage form, if desired, they may be administered in different ways (ie, one product-combination component may be used). orally, and the second component may be administered intravenously) or in dosage forms.

It will be apparent to the skilled practitioner that dosages in such combination therapies of the present invention may vary depending on a variety of factors such as the pharmacodynamic characteristics of the particular agent and its mode and form of administration, age, health and weight of the recipient, nature and degree of competition. , frequency of treatment and desired effect as described above.

Suitable dosages of components (a) and (b) of the present invention will be readily determined by one of ordinary skill in the art, based upon the disclosure herein. According to general instructions, a typical daily dose of each component may be from about 100 mg to about 1.5 g. If component (b) is more than one compound, a typical daily dose of each agent of component (b) may be from about 100 mg to about 1.5 g. As a general guideline, when the compounds of component (a) and component (b) are used in combination, the dose of each component may be reduced by about 70-80% compared to the standard dose of the component when used alone as a single agent for HIV infection.

The product combinations of the present invention may be formulated so that while the active ingredients are formulated in a single dosage unit, physical contact between the active ingredients is minimized. To minimize contact, for example when the product is administered orally, one active ingredient may be enteric-coated. Not only can one of the active ingredients be coated with an enteric coating, it is possible to minimize contact between the active ingredients of the combination but also to control the release of one of these components into the gastrointestinal tract so that one of these components is not excreted in the stomach.

In another embodiment of the present invention, where oral administration is desirable, there is provided a product-denny having one active ingredient coated with a sustained-release material which provides sustained-release gastrointestinal delivery and also serves to minimize physical contact between the active ingredients in the combination,

Additionally, the sustained release component may also be enteric coated so that the release of this component is limited to the intestine. Another strategy could include a pharmaceutical formulation of the product combination wherein one component is coated with a sustained release material and / or an enteric release polymer and the second component is also coated with a polymer such as low viscosity hydroxypropylmethylcellulose or other appropriate material known in the art. it is better to distinguish between the active components. Polymer coating creates an additional barrier to interaction with another component. Each pharmaceutical form that protects against contact between components (a) and (b) due to coating or any other material may also protect against contact of individual agents in component (b).

Dosage forms of the product combinations of the present invention, wherein one active ingredient is enteric coated, may be in the form of a tablet in which the enteric coated component and the other active ingredient are mixed into a tablet, or in which the enteric coated component is compressed in a single tablet layer. , and the second active ingredient is compressed in another layer. If necessary, one or more layers of piacebo may be present to further separate the two layers so that there is a layer of piaceo between the active ingredient layers. In addition, the dosage forms of the present invention may be in the form of capsules containing one active ingredient compressed into a tablet or in the form of a plurality of granules, granules, granules, or protected particles which are enteric-coated. Such enteric-coated microtablets, particles, granules or protected particles are encapsulated or encapsulated by granulation of the second active ingredient.

According to this disclosure, these and other methods of minimizing contact between product-combination components, when used as a single dosage form or as separate forms, but at the same time or in the same combination, are readily apparent to those skilled in the art.

The present invention also encompasses pharmaceutical kits for the treatment of HIV infection, comprising a pharmaceutical composition comprising a compound of (a) and one or more component (b) in a therapeutically effective amount in one or more sterile containers. These containers may be sterilized using a conventional sterilization technique well known to those skilled in the art. Component (a) and component (b) may be in the same sterile container or in separate sterile containers. The sterile containers of the materials may be individual containers or, if desired, one or more containers comprising several compartments. Component (a) and component (b) may be separate or physically combined in a single dosage form or unit as described above. Such kits may, if desired, include one or more components of a variety of Conventional Pharmaceutical Kits, such as one or more pharmaceutically acceptable carriers, admixture components and 1.1, which are readily understood by those skilled in the art. The kit may also include instructions (either as inserts or labels) indicating the quantities of components to be used, how to use the medicine, and / or instructions on how to mix the components.

It will be understood that various modifications of the present invention are possible based on the above advice. It is to be understood, therefore, that within the scope of the following, the present invention may be practiced otherwise than as specifically described herein.

Claims (12)

DEFINITION OF INVENTION 1. A compound of formula I: a pharmaceutically acceptable salt thereof, or a prodrug thereof, wherein the prodrug of Formula I is a compound wherein both hydroxyl groups are bonded to form an epoxy, -OCH ₂ SCH ₂ O-, -OC (= O) O-, -OCH ₂ O-, -OC (= S). ) O-, -OC (= O) C (= O) O-, -OC (CH ₃ ) ₂ O-, -OC ((CH ₂ ) ₃ NH ₂ ) (CH ₃ ) O-, -OC (OCH ₃ ) (CH ₂ CH ₂ CH ₃ ) O -, or -OS (= O) O -. 2. A compound according to claim 1 having the formula i. 3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of claim 1, a pharmaceutically acceptable salt thereof, or a prodrug thereof. Composition according to Claim 3, characterized in that the compound has the formula i. 5. Compound of Formula I: a pharmaceutically acceptable salt thereof, or a prodrug thereof, wherein the prodrug of Formula I is a compound wherein the two hydroxyl groups are bonded together to form an epoxy, -OCH ₂ SCH ₂ O-, -OC (= O) O-, -OCH ₂ O-, - OCf = S) O-, -OC (= O) C (= O) O-, OC (CH ₃ ) ₂ O-. -OC ((CH ₂ ) ₃ NH ₂ ) (CH ₃ ) O-, -OC (OCH 3) (CH ₂ CH ₂ CH ₃ ) O-, or -O S (= 0) 0 - for use in the manufacture of a medicament for the treatment of HIV infection. Use according to claim 5, characterized in that the compound has the formula I. 7. A combination of (a) and (b), wherein: (a) is a compound of formula I: a pharmaceutically acceptable salt thereof, or a prodrug thereof, wherein the prodrug of formula i is a compound wherein the two hydroxyl groups are bonded together to form an epoxy, -OCH ₂ SCH ₂ O-, -OC (= O) O-, -OCH ₂ O-, - OC (= S) O-, -OC (= O) C (= O) O-, -OC (CH ₃ ) ₂ O-, -OC ((CH ₂ ) ₃ NH ₂ ) (CH ₃ ) O-, -OC (OCH ₃ ) (CH ₂ CH ₂ CH ₃ ) O-, or -OS (= O) O-, and (b) at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and HIV proteases inhibitors, in the manufacture of a medicament for the treatment of HIV infection. Use according to claim 7, Come on distinguishing s that that the formula of the compound is I. 9. Use according to claim T, Come on distinguishing s that that reverse transcriptase inhibitor is nucleoside the reverse transcriptase inhibitor, Use according to claim 3, bes i s k i r I a n t i s that that nucleosidic reverse transcriptase inhibitor chooses also Az. ), w 1, ddi, ddC and d4T, and select protease inhibitor from saquinavir, rkonavir, indinavir, VX-478, neifinavir, KNl-272, CGP-61755 and U-103017. Use according to claim 10, other than that. that the nucleosidic reverse transcriptase inhibitor is selected from AZT and 3TC and that the protease inhibitor is selected from saquinavir, ntonavir and indinavir. 12. Use according to claim 11, wherein the nucleoside reverse transcriptase inhibitor is AZT. 13. The use of claim 11, wherein the protease inhibitor is indinavir. 14. A pharmaceutical kit for treating HIV infection, characterized in that it comprises a therapeutically effective amount: (a) a compound of claim 1; and (b) at least one compound selected from the group consisting of an HIV reverse transcriptase inhibitor and an HIV protease inhibitor in one or more sterile containers. 15. The kit of claim 14, wherein component (a) is a compound of formula i.