A method of treating retroviral infections in mammals (options)

 

(57) Abstract:

The invention relates to the field of biotechnology. The method involves the use of combinations of retroviral protease inhibitors, which are effective in preventing the replication of retroviruses in vitro or in vivo. Connection protease inhibitors are also used in combination therapy with other compounds, protease inhibitors, and combination therapy with antiviral agents other than protease inhibitors. The method allows to overcome the development of retroviral strains that are resistant to the inhibitor of retroviral protease. 2 C. and 18 h.p. f-crystals, 13 tables.

The invention relates to a method of treating retroviral infections in mammals, such as human immunodeficiency virus (HIV), using combinations of retroviral protease inhibitors, which are effective in preventing the replication of mammalian retroviruses like HIV, in vitro and in vivo. This invention particularly relates to compounds which are protease inhibitors (compounds inhibitors), used in combination therapy with other compounds protease inhibitors.

During the replication cycle of retroviruses p is Noah protease (or proteinase), producing viral enzymes and structural proteins bark of the virus. Often protein-gag precursors are processed cow's milk protein and protein-pol precursors are processed in viral enzymes such as reverse transcriptase and retroviral protease. It was shown that the correct protein processing predecessors retroviral protease required for Assembly of infectious virions. For example, it was shown that mutations with a bias by region pol gene of HIV prevent the processing of the protein of the gag precursor. It was also shown by site - directed mutagenesis of aspartic acid residue in the active site of HIV protease, which prevents the processing of the protein of the gag precursor. Therefore, attempts have been made to inhibition of viral replication, inhibition of retroviral proteases.

Inhibition of retroviral protease typically includes mimetic in a transitional state, whereby the retroviral protease is exposed mimetic compound that binds (usually reversible way) with the enzyme in competition with proteins gag and gag-pol, thereby inhibiting specific processing of the structural proteins and the release of the retroviral proposed several classes of mimetic compounds, in particular, for the inhibition of proteases, such as inhibition of HIV protease. Such mimetic compounds include isostere of hydroxyethylamine, isostere restored amides and ones isostere. See, for example, EP 0346847; EP 0342541; Roberts et al., "Rational Design of Peptide-Based Proteinase Inhibitors, and the 2.8 A Crystal Structure of C2Symmetric Inhibitor Complexed to HIV-I Protease", Science, 249, 527 (1990) and S. Thaisrivogs, "Structure-Based Design of Non-Peptide HIV Protease Inhibitors", 35th Annual Buffalo Medicinal Chemistry Meeting, State University of New York et Buffalo, NY, May 22 - 25, 1994.

The problem inhibitors of retroviral proteases such as HIV protease inhibitors, was the development of strains that are resistant to the inhibitor. For example, the inhibitor L-735524 HIV protease Merck and Co. effective against HIV infection in humans, but later L-735524 develops in patients resistant strains of HIV (Waldholz, The Wall Street Journal, February 25, 1994, page B3; and Condra et al., Nature 374: 569 - 571 (1995)). Other examples can be found in Vacca et al., Proc. Natl. Acad. Sci. USA 91: 4096 - 4100 (1994); But et al., J. Virol. 68: 2016 - 2020 (1994); and Sardana et al., Biochem. 33: 2004 - 2010 (1994).

This invention relates to a method of treating retroviral infections in mammals, such as human immunodeficiency virus (HIV), using combinations of retroviral protease inhibitors, which are effective in preventing replication retrom in combination therapy with other compounds protease inhibitors. In addition, this combination can be used in combination with other antiviral agents.

Retroviral protease is a critical enzyme in the process of retroviral replication. Propagation of retrovirus, such as HIV, can be prevent by exposure to the virus retroviral protease inhibitor. However, during prolonged exposure to protease inhibitors on retrovirus may occur variant selection of retroviruses, so there is a new dominant strain of retrovirus-resistant protease inhibitor. This new dominant strain of retrovirus can produce protease, which is no longer inhibited or, as happens more often, not enough is inhibited by protease inhibitors and may be freely reproduced even in the presence of protease inhibitor if the concentration of this inhibitor does not significantly increase. The present invention provides a method of overcoming development retroviral strains that are resistant to retroviral protease inhibitor.

The present method involves the administration to a mammal, such as human, monkey, cat, etc. effective amount of at least two inhibitors of retroviral protease, i.e. the introduction of two or more inhibitors of retroviral protease so that an effective amount of at least two inhibitors were present at this mammal at any time. Alternatively, the introduction you can perform sequential or alternating introduction of at least two inhibitors of retroviral proteases, i.e. the introduction of two or more inhibitors of retroviral protease so that this mammal at any time attended an effective amount of only one inhibitor. With proper selection of inhibitors of retroviral protease, this method can effectively regulate the reproduction of retroviruses, even in the presence of strains resistant to any one of the inhibitors.

Inhibitors of retroviral protease is chosen based on the profile of resistant strains (strains) of retrovirus, which appears in vivo or in vitro under the influence of inhibitor on breeding a culture of retroviruses. Inhibitors of retroviral protease is chosen by the absence of cross-resistance of at least one retroviral resistant strain. It is believed that retroviral strain is cross-resistant to two protease inhibitors, when retroviral strain so that there was no cross-resistance between the selected inhibitors of retroviral protease, when they are taken as a group. Thus, variant or mutant) strain of retrovirus that can develop after exposure of the first retroviral protease inhibitor, is still inhibited by the second retroviral protease inhibitor, or may develop as a result of the impact of both the first and second retroviral protease inhibitors, is still inhibited by a third retroviral protease inhibitor or fourth retroviral protease inhibitor, etc.

Conduct a comparison of the profiles of cross-resistance between protease inhibitors and combination therapies selected compounds, which preferably are weak or do not show any cross-resistance. The phenotype of resistance to the drug can be divided into the following: phenotype without resistance, a phenotype with a low resistance (less than about 10-fold shift in EC50or EC90), a phenotype with moderate levels of resistance (from about 10 - to about 100-fold shift in EC10or EC90or phenotype with high levels of resistance (more than about 100-fold shift in EC50or EC90). It is expected that resistance to drugs is E. in vivo concentrations of inhibitors have reduced protease inhibitory effect on the resistant virus. Thus, the preferred combinations of protease inhibitors will be the ones that have profiles minimal cross-resistance (i.e., preferably, the resistance is not more than the intermediate level; more preferably, the resistance is not more than a low level; and most preferably, no resistance) and the maximum specific activity for wild-type viruses and/or resistant viruses selected against another inhibitor. For example, preferred compounds for use in combination with the first compound will preferably be effective against strains of the virus that have intermediate level, more preferably a high level of resistance to the first connection. Pharmacology and toxicology of each inhibitor and combinations are also factors in the selection of inhibitors for combination therapy.

More preferably, choose inhibitors of retroviral protease, when at least one viral strains resistant to the first retroviral protease inhibitor, and at least one viral strain that is resistant to a second retroviral protease inhibitor having a different amino acid substitution in and contribute to the observed resistance to the inhibitor. Thus, the number of possible amino acid substitutions that can occur in the same site in the protease is limited. This is in particular true when the site is critical to the activity, efficiency and/or stability of the enzyme.

This case was observed in relation to HIV protease inhibitors examples 1 and 2. Retroviral resistance to the compound of example 1 was the result of a mutation site at amino acid 88 of HIV protease (substitution of asparagine 88 aspartic acid 88). Retroviral resistance to the compound of Example 2 also follows from the mutation site at amino acid 88 of HIV protease (substitution of asparagine 88 serine 88). It is known that some substitution at amino acid 88 cause the loss of enzymatic activity (Loeb et al. Nature 340: 387 - 400 (1989)). Thus, the introduction of both HIV protease inhibitors examples 1 and 2 significantly reduces the likelihood of further successful production of resistant virus strain, cross-resistant to both inhibitors. No resistance to both inhibitors used in combination, was not detected within 6 weeks of treatment compared with the appearance of a phenotype resistant to a single inhibitor, at the same time limit. In updat the Xia also other mutation sites, which does not significantly affect the enzymatic activity and/or stability.

An alternative, more preferably choose inhibitors of retroviral protease, when at least one viral strains resistant to the first inhibitor of retroviral protease, has an increased sensitivity to this second protease inhibitor, or when at least one viral strain that is resistant to a second inhibitor of viral protease, has an increased sensitivity to this first protease inhibitor.

Representative inhibitors of retroviral proteases, which are suitable for use in the present method include, but are not limited to, protease inhibitors, discovered and described in co-owned and jointly considered generic orders USA with numbers 08/152934 (filed November 15, 1993), 08/253531 (filed June 3, 1994), 08/109787 (filed August 20, 1993), 08/110911 (filed August 24, 1993), 08/110913 (filed August 24, 1993), 08/110912 (filed August 24, 1993), 08/204827 (filed March 2, 1994), 07/886556 (filed may 20, 1992), 07/886663 (filed may 20, 1992), 07/886531 (filed may 20, 1992), 08/148817 (filed November 8, 1993), 08/886700 (filed may 21, 1992) and 07/998187 (filed December 29, 1992) and the patent applications PCT N PCT/US 93/10552 (filed October 29, 1993), PCT/US the fullness. Additional inhibitors of retroviral proteases, which are suitable for use in the present method include, but are not limited to, protease inhibitors, is proposed and described in U.S. patent 5157041; EP 346847; generic application U.S. N 07/883825 (filed may 15, 1992); WO 93/09096; Tet. Lett. 35: 673 - 676 (1994); Proc. Natl. Acad. Sci. USA, 91: 4096 - 4100 (1994); Y. M. Wong et al., Biopharm. and Drug Dispos. 15: 535 - 544 (1994); M. L. West and D. P. Fairlie. Trends Pharmacol. Sci. 16: 67-75 (1995) and S. Thaisrivongs, "HIV Protease Inhibitors", Ann. Reports Med. Chem., Vol. 29, Chap. 14, pp. 133 - 144 (1994) (Academic Press, J. Bristol, Ed.), each of which is incorporated herein as reference in its entirety.

It is believed that no further development of any person skilled in the art can, using the preceding description, utilize the present invention to the full extent. The following preferred specific embodiments of the invention are not intended to provide an exhaustive account of all possible combinations of connections, but only to provide examples of combinations of drugs, which are expected effective. A similar test these and other protease inhibitors with the use of resistant viral isolates are not limited to those listed below, can help establish suitable combinations of medicinal cf the to as illustrative and in no way restrictive for the rest of the description.

Example 1

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[1S-[1R*(R*), 2S*] ] -N1-[3-[[[(1,1-dimethylethyl)amino]carbonyl] (2-methylpropyl)amino] -2-hydroxy-1-(phenylmethyl)propyl] - 2-[(2-chinainternational)amino]butadiene can be obtained in accordance with methods described in co-owned and jointly consider applications for U.S. patent numbers 08/152934 (filed November 15, 1993) and 08/156498 (filed November 23, 1993), which is incorporated herein as reference in its entirety.

Example 2

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(2R, 3S)-3-(N-Methylaminomethyl-L-tert-butylphenyl) amino-1-(N-isoamyl-N-(tert-butylcarbamoyl)amino-4-phenyl-2-butanol can be obtained in accordance with methods described in co-owned and jointly consider applications for U.S. patent numbers 08/109787 (filed August 20, 1993), Attorney docket N 27766/1 submitted together with this application, and 08/156498 (filed November 23, 1993), all three incorporated herein by reference in its entirety.

Example 3

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5-Pyrimidinethione ether [2R-hydroxy-3-[[(4-methoxyphenyl)-sulfonyl] (2-methylpropyl] amino] -1S-(phenylmethyl) propyl] carbamino acid can be obtained in accordance with methods described in co-owned and jointly consider applications for patchnote.

Example 4

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[1S-[1R*(R*),2S*]]-N-[2-Hydroxy-3-[N1-(2-methylpropyl)-N1-(4-methoxybenzenesulfonyl)amino]-1- (phenylmethyl) propyl]-2-methyl-3-(methylsulphonyl)propanamide can be obtained in accordance with methods described in co-owned and jointly consider applications for U.S. patent numbers 08/110913 (filed August 24, 1993) and 08/156498 (filed November 23, 1993), both incorporated herein by reference in its entirety.

Example 5

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N-(2-(R)-Hydroxy-1(S)-indanyl)-2-(R)-phenylmethyl-4(S)-hydroxy-5- (1-(4-(3-pyridylmethyl)-2(S)-N'-(tert-BUTYLCARBAMATE)piperazinil)) pentanone (L-735524) can be obtained in accordance with methods described in application for U.S. patent N 07/888825 (filed may 15, 1992), WO 93/09096, Tet. Lett. 35: 673 - 676 (1994) Proc. Natl. Acad. Sci. USA, 91: 4096-4100 (1994), each of which is incorporated herein by reference in its entirety.

Example 6

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N-tert-buildimage-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N- (2-hinolincarbonova)-L-asparaginyl] amino] butyl] -(4aR, 8aS) - isoquinoline-3(S)-carboxamide (Ro 31-8959) can be obtained in accordance with methods described in U.S. patent 5157041, incorporated herein by reference in its entirety.

Example 7

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[1S-[1R*(R*), 2S] ] -N1-[3-[[[(1,1-Dimethylethyl)amino]Carbo is arranged in accordance with the methods, described in jointly owned and jointly consider applications for U.S. patent numbers 08/152934 (filed November 15, 1993) and 08/156498 (filed November 23, 1993), both incorporated herein as reference in its entirety.

Example 8

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N-[3-[N2-[N1-(1,1-Dimethylethyl)aminosulfonyl]-N2- (2-methyl-propyl)amino] -2R-hydroxy-1S-(phenylmethyl)propyl] -2S- [(2-chinainternational)amino] butanamide can be obtained in accordance with methods described in co-owned and jointly consider applications PCT/US 93/10552 (29.10.93) and in the application of the U.S. 08/156498 from 23.11.93, incorporated herein as references.

Example 9

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(2S,3R,4S,5S)-2,5-bis-[N-[N-[N-Methyl-N-(2-pyridinylmethyl)amino]carbonyl] valinol] amino] -3,4-dihydroxy-1,6-diphenylhexane (A-77003) can be obtained according to methods described in J. Med. Chem. 36: 320 - 330 (1993), which is incorporated herein as reference in its entirety.

Example 10

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(2S, 3R, 4S,5S)-5-[N-[N-[N-Methyl-N-(2-isopropyl-4-thiazolyl) methyl)amino] carbonyl] valinol] amino] -3-hydroxy-1,6-diphenylhexane (A-84538, ABT-538) can be obtained in accordance with methods described in the patent application PCT N WO 94/14436 (filed December 16, 1993), which is incorporated herein as reference in its entirety.

-1S-(phenylmethyl)propyl] -carbamino acid (VX-478) can be obtained in accordance with methods, described in the patent application PCT N WO 94/05639 (filed 7 September 1993), which is incorporated herein as reference in its entirety.

Example 12

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N-tert-Buildimage-2-[2-(R)-hydroxy-4-(phenylthio)-3(S)- [[N-[(2-methyl-3-hydroxyphenyl)carbonyl] amino] butyl] -(4R,8aS)- isoquinoline-3(S)-carboxamide (AG-1343, AG-1350) can be obtained in accordance with methods described in Bioorg. and Med. Chem. Let. 5:715-720, 5:721-726 and 5:727-732 (1995), each of which is incorporated herein as reference in its entirety. In particular, NOT - active ester of 3-hydroxy-2-methyl-benzoic acid (Bioorg. and Med. Chem. Let. 5:727-732 (1995)) can be connected with N-tert - buildimage-2-[2(R)-hydroxy-4-(phenylthio)-3(S)-aminobutyl] - (4aR, 8aS)-isoquinoline-3(S)-carboxamide (Bioorg. and Med. Chem. Let. 5:715-720 (1995)).

Example 13

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[4R-(4, 5, 6, 7 ]-1,3-bis(3 - AMINOPHENYL)methyl]hexahydro-5,6-dihydroxy-4,7-bis(phenylmethyl)- 2H-1,3-diazepin-2-he (DMP-450, HMM-412) can be obtained in accordance with methods described in the patent application PCT WO 93/07128, which is incorporated herein as reference in its entirety. In particular, 3-nitrophenylacetylene, for example 3-nitrophenylarsonic or-bromide, process hydroxyamine derived [4R-((4 , 5 , 6 , 7 )] -hexahydro-5,6-dihydroxy-4,7-bis (phenylmethyl)-2H-1,3-diazepin-2-one, then remove the protective group (Osorno conduct, using standard techniques, well known to specialists in this field.

Example 14

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Obtaining N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl) sulfonyl]-(2-methylpropyl)amino]-1S-(phenylmethyl)propyl]-2S- [[(pyrrolidin-1-yl)acetyl]amino] -3,3-dimethylbutyramide

Part a: Obtaining 1,3-benzodioxol-5-sulphonylchloride

In a solution of 4.25 g of anhydrous N,N-dimethylformamide at 0oC in an atmosphere of nitrogen was added to 7.84 g sulfurylchloride, then formed a solid. After stirring for 15 min was added 6,45 g of 1,3-benzodioxole and the mixture was heated at 100oC for 2 h, the Reaction mixture was cooled, poured into ice-cold water, was extracted with methylene chloride, dried over magnesium sulfate, filtered and concentrated, getting to 7.32 g of the crude material in the form of a black oil. It was chromatographically on silica gel using a mixture of 20% methylene chloride/hexane, receiving of 1.9 g of (1,3-benzodioxol-5-yl)sulphonylchloride.

Alternatively, in a round bottom flask at 22 liters, equipped with a mechanical stirrer, a refrigerator, a heating jacket and a dropping funnel with a balanced pressure, poured a complex of sulfur trioxide - dimethylformamide (DMF) (2778 g, 18,1 mol). Then EXT is 1,3-benzodioxol (1905, 15.6 mol). The temperature was then raised to 75oC and maintained for 22 h (NMR showed that the reaction was ended after 9 h). The reaction mixture was cooled to 26oC was added oxalicacid (2290 g, 18,1 mol) at such a speed as to maintain the temperature below 40oC (1.5 h). The mixture was heated to 67oC for 5 h, then cooled to 16oC ice bath. The reaction was extinguished with water (5 l) at a rate that allowed to maintain the temperature below 20oC. After adding water, the mixture was stirred for 10 minutes, the Layers were separated and the organic layer was washed again twice with water (5 l). The organic layer was dried with magnesium sulfate (500 g) and filtered to remove the desiccant. The solvent was removed in vacuum at 50oC. Received a warm liquid was left to cool, during which began the formation of solids. After 1 h this solid was washed with hexane (400 ml), filtered and dried, obtaining the target sulphonylchloride (2823 g). Wash hexane was concentrated and formed solid substance was washed with 400 ml of hexane, gaining additional sulphonylchloride (464 g). The total yield was 3287 g (95.5% of considering 1.3-benzodioxol).

Part b: Getting 2S-[bi is the establishment of DIBAL phenylmethylene ether

Stage 1: a Solution of L-phenylalanine (50.0 g, 0,302 mol), sodium hydroxide (24.2 g, 0,605 mol) and potassium carbonate (83,6 g, 0,605 mol) in water (500 ml) was heated to 97oC. and Then slowly added benzylbromide (108,5 ml, 0,605 mol) (add - 25 min). The mixture was stirred at 97oC for 30 min in nitrogen atmosphere. The solution was cooled to room temperature and was extracted with toluene (2 x 250 ml). The combined organic layer was washed with water and with brine, dried over magnesium sulfate, filtered and concentrated to education oils. Fenilmetilovy ether N,N-bis(phenylmethyl)-L-phenylalanine can be cleared column chromatography (silica gel, 15% ethyl acetate/hexane). Usually the product is sufficiently pure to be used directly in the next stage without further purification. EIMS: m/z 434.

Stage 2: Antilibanus fenilmetilovy ester of phenylalanine (0,302 mol) from the previous reaction was dissolved in toluene (750 ml) and cooled to -55oC. a 1.5 M solution of DIBAL in toluene (443,9 ml, 0,666 mol) was added at a rate that permits to maintain the temperature between -55 ° C and -50oC (add - 1 h). The mixture was stirred for 20 min in nitrogen atmosphere and then extinguished when -55oC by slow addition of methanol (37 ml)listello 138 g) was separated by filtration and washed with toluene. The solid material is suspended in a mixture of toluene (400 ml) and water (100 ml). The mixture was cooled to 5oC and was treated with 2,5 N. NaOH (168 ml) and then stirred at room temperature to dissolve the solid material. The toluene layer was separated from the aqueous phase and washed with water and with brine, dried over magnesium sulfate, filtered and concentrated to a volume of 75 ml (89 g). To the residue was added ethyl acetate (25 ml) and hexane (25 ml), after which started to crystallize the target liquid product. After 30 min for promotion further crystallization was added 50 ml of hexane. The solid material was separated by filtration and washed with 50 ml of hexane, getting to 34.9 g of the product of the first portion. The second portion of the product (5.6 g) was isolated again by filtration mother liquor. Two portions were combined and were led from ethyl acetate (20 ml) and hexane (30 ml) to give 40 g of 2S-[bis(phenylmethyl)amino]-3-phenylpropanol, yield 40%, counting on L-phenylalanine. Analysis. Calculated for C23H25ON: C 83,34; H 7,60; N TO 4.23. Found C 83,43; H To 7.59; N 4,22.

METHOD 2: Obtain 2S-[bis(phenylmethyl)amino]benzotropine N,N-dibenzylamine L-phenylalaninol

L-Phenylalaninol (176,6 g 1,168 mol) was added to the mixed solution of potassium carbonate () in ethanol 3A (305 ml) was added with such speed, to maintain the temperature between 60-68oC. the Biphasic solution was stirred at 65oC for 55 min and then left for cooling to 10oC with vigorous stirring. The oil product was utverjdali in small granules. The product was diluted in 2.0 l of tap water and was stirred for 5 minutes to dissolve the inorganic by-products. The product was separated by filtration under reduced pressure and washed with water to pH 7. The crude product was recrystallized from 1.1 l of a mixture of ethyl acetate/heptane (1:10). The product was separated by filtration (-8oC), washed with 1.6 l of cold (-10oC) a mixture of ethyl acetate/hexane (1: 10) and dried in the air, getting 339 g (yield 88%) of 2S-[bis(phenylmethyl)amino] -3-phenylpropanol. So pl. = 71,5-73,0oC.

Part C: the Receipt of 2S-[bis(phenylmethyl)amino] -3-phenylpropionaldehyde (propane aldehyde)

METHOD 1: 2S-[bis(Phenylmethyl)amino]-3-phenylpropanol (200 g, 0,604 mol) was dissolved in triethylamine (300 ml, 2.15 g, 0,604 mol). The mixture was cooled to 12oC and was added to the solution of a complex of sulfur trioxide/pyridine (380 g, 2,39 mol) in dimethyl sulfoxide (DMSO) (1.6 l) at a rate that permits to maintain the temperature between 8 - 17oC. the Solution was stirred with a prevalence of 1.6 l of cold water (10-15oC) for 45 minutes Educated the solution was extracted with ethyl acetate (2.0 l), washed with 5% citric acid (2.0 l) and saline solution (2.2 l), dried over MgSO4(280 g) and filtered. The solvent was removed in vacuo and the residue was then dried in vacuum, obtaining 198,8 g of 2S-[bis(phenylmethyl)amino]-3-phenylpropionaldehyde in the form of a pale yellow oil (99.9 percent). The crude product was sufficiently pure to be used directly in the next stage without purification.

METHOD 2: a Solution of oxalicacid (8,4 ml, 0,096 mol) in dichloromethane (240 ml) was cooled to -74oC. and Then slowly added to the solution of MGSO (12.0 ml, 0,155 mol) in dichloromethane (50 ml) at a rate that permits to maintain the temperature at -74oC (add 1.25 hours). The mixture was stirred for 5 min, then was added a solution of 2S-[bis(phenylmethyl)amino]-3-phenylpropanol (0,074 mol) in 100 ml of dichloromethane (addition of 20 minutes, the temperature of -75oC to -68oC). The solution was stirred at -78oC in an atmosphere of nitrogen for 35 minutes and Then for 10 min (temperature from -78oC to -68oC) was added triethylamine (41,2 ml, 0,295 mol), after which the deposited ammonium salt. The cold mixture was stirred for 30 min and then was added to the atom of magnesium, was filtered and concentrated. The residue was diluted with ethyl acetate and hexane and then filtered to further remove the ammonium salt. The filtrate was concentrated, receiving 2S-[bis(phenylmethyl)amino] -3 - phenylpropionaldehyde. This aldehyde was transferred to the next stage without purification.

METHOD 3: TO a mixture of 1.0 g (3.0 mmol) of 2S-[bis(phenylmethyl)amino]-3 - phenylpropanol, mean HDI of 0.531 g (4,53 mol) N-methylmorpholine, and 2.27 g of molecular sieves (4A) and 9.1 ml of acetonitrile was added 53 mg (0.15 mol) of perruthenate of tetrapropylammonium (TRAR). The mixture was stirred for 40 min at room temperature and then concentrated under reduced pressure. The residue is suspended in 15 ml of ethyl acetate, filtered through a bed of silica gel. The filtrate was concentrated under reduced pressure, obtaining a product containing approximately 50% of 2S-[bis(phenylmethyl) amino]-3-phenylpropionaldehyde in the form of a pale yellow oil.

METHOD 4: TO a solution of 1.0 g (3,02 mole) of 2S-[bis(phenylmethyl) amino]-3-phenylpropanol 9.0 ml of toluene was added 4,69 mg (0.03 mmol) of 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), 0.32 g, (3.11 mmol) of sodium bromide, and 9.0 ml of ethyl acetate and 1.5 ml of water. The mixture was cooled to 0oC and slowly, over 25 min, was added an aqueous solution 2,87 ml of 5% bitor>C within 60 minutes After two more additives bleach (1,44 ml each) and the mixture was stirred for 10 minutes the Aqueous layer was extracted twice with 20 ml of ethyl acetate. The combined organic layer was washed 4,0 ml of a solution containing 25 mg of potassium iodide and water (4.0 ml), 20 ml of 10% aqueous sodium thiosulfate solution and then with brine. The organic solution was dried over magnesium sulfate, filtered and concentrated under reduced pressure, obtaining of 1.34 g of crude oils containing a small amount of the target aldehyde product, 2S-[bis(phenylmethyl)amino]-3 - phenylpropionaldehyde.

Part D: Obtaining N,N-dibenzyl-3(S)-amino-1,2-(S)-epoxy-4-phenylbutane

METHOD 1: a Solution of 2S-[bis(phenylmethyl)amino] -3 - phenylpropionaldehyde (191,7 g of 0.58 mol) and chloroiodomethane (of 56.4 ml, 0.77 mol) in tetrahydrofuran (1.8 l) was cooled to a temperature of from -30 to -35oC in the reactor of stainless steel in an atmosphere of nitrogen. Then the solution was added n-utility in hexane (1.6 M, 365 ml of 0.58 mol) at a rate that allows to maintain the temperature below -25oC. After addition the mixture was stirred at -30 to -35oC for 10 minutes Other added reagents was carried out as follows: (1) was added Dupli at a temperature of from -30 to -35oC for 10 minutes Is repeated once. (2) Added additional chloridometer (8.5 ml, 0.11 mol), then n - utility (55 ml, 0.88 mol) at < -25oC. After addition the mixture was stirred at -30 to -35oC for 10 minutes Is repeated 5 times. (3) Added additional chloridometer (8.5 ml, 0.11 mol), then n-utility (37 ml, 0,059 mol) at < -25oC. After addition the mixture was stirred at -30 to -35oC for 10 minutes Is repeated once. The external cooling was stopped and the mixture was heated to ambient temperature for 4 to 16 hours, when TLC (silica gel, 20% ethyl acetate/hexane) showed that the reaction had ended. The reaction mixture was cooled to 10oC and extinguished 1452 g of a 16% aqueous solution of ammonium chloride, keeping the temperature below 23oC. the Mixture was stirred for 10 min and the organic and aqueous layers were separated. The aqueous phase was extracted with ethyl acetate (2x500 stretch-forming press ml). The ethyl acetate layer was combined with a layer of tetrahydrofuran. The combined solution was dried over magnesium sulfate (220 g), filtered and concentrated in vacuum. The remainder in the form of a brown oil was dried at 70oC in vacuum (0,79 ATM) for 1 h, receiving 222,8 g of crude material. The crude is placed aldehyde (0,074 mol) and chloroiodomethane (7,0 ml, 0,096 mol) in tetrahydrofuran (285 ml) was cooled to -78oC in nitrogen atmosphere. Then was added a 1.6 M solution of n-utility in hexane (25 ml, 0.040 mol) at a rate that permits to maintain the temperature at -75oC. After the first add was again added additional chloridometer (1.6 ml, of 0.022 mol) and then n-utility (23 ml, 0,037 mol), keeping the temperature -75oC. the Mixture was stirred for 15 minutes Each of the reagents: chloromethane (0,70 ml 0,010 mol) and n-utility (5 ml 0,008 mol) was added 4 more times over 45 min at -75oC. the Cooling bath was then removed and the solution was heated to 22oC for 1.5 hours. The mixture was poured into 300 ml of a saturated aqueous solution of ammonium chloride. The layer of tetrahydrofuran was separated. The aqueous phase was extracted with ethyl acetate (CH ml). The combined organic layer was washed with brine, dried over magnesium sulfate, filtered and concentrated, obtaining a brown oil (27.4 g). The product can be used in the next stage without purification.

METHOD 3: a Solution of 2S-[bis(phenylmethyl)amino] -3-phenylpropionaldehyde (178,84 g, 0.54 mol) and bremgarten (46 ml, 0.71 mol) in tetrahydrofuran (1.8 l) was cooled to a temperature of from -30 to -35oC in the reactor stainless steel th to maintain the temperature below -25oC. After the addition the mixture is stirred at a temperature between -30 and -35oC for 10 minutes Other added reagents was carried out as follows: (1) added additional bromochloromethane (14 ml), then n-utility (102 ml) at < -25oC. After the addition the mixture is stirred at a temperature between -30 and -35oC for 10 minutes Is repeated once. (2) Added additional bromochloromethane (7 ml, 0.11 mol), then n-utility (51 ml, 0,082 mol) at < -25oC. After the addition the mixture is stirred at a temperature between -30 and -35oC for 10 minutes Is repeated 5 times. (3) Added additional bromochloromethane (7 ml, 0.11 mol), then n-utility (51 ml, 0,082 mol) at < -25oC. After the addition the mixture is stirred at a temperature between -30 and -35oC for 10 minutes Is repeated once. The external cooling was stopped and the mixture was heated to ambient temperature for 4 to 16 h, when TLC (silica gel, 20% ethyl acetate/hexane) showed that the reaction had ended. The reaction mixture was cooled to 10oC and extinguished 1452 g of a 16% aqueous solution of ammonium chloride, keeping the temperature below 23oC. the Mixture was stirred for 10 min and the organic and aqueous layers were separated. The aqueous phase of extraheavy over magnesium sulfate (220 g), was filtered and concentrated in a rotary evaporator at 65oC. the Residue in the form of a brown oil was dried at 70oC in vacuum (0,79 ATM) for 1 h, receiving 22.2,8 g of crude material.

Part E: obtaining a salt of N-[3(S)-[N,N-bis(phenylmethyl)amino]-2(R)- hydroxy-4-phenylbutyl]-N-isobutylamine oxalic acid

Stage 1: In the solution of the crude N,N-dibenzyl-3(S)-amino -1,2(S)-epoxy-4-phenylbutane (388,5 g 1.13 mol) in isopropanol (2.7 l) or ethyl acetate was added isobutylamine (1.7 kg, 23,1 mol) for 2 minutes, the Temperature was increased from 25oC to 30oC. the Solution was heated to 82oC and stirred at this temperature for 1.5 hours the Warm solution was concentrated in vacuum. The remainder in the form of a brown oil was dried in vacuo (0.8 mm RT.CT.) within 16 hours, getting 450 g of the product in the form of crude oil.

Stage 2: In a solution of oxalic acid (8,08 g, 89,72 mmol) in methanol (76 ml) was added a solution of the crude 3(S)-[N,N-bis(phenylmethyl)amino]-1-(2-methylpropyl)amino-4-phenylbutane-2(R)-ol in ethyl acetate (90 ml) for 15 min. the Mixture was stirred at room temperature for about 2 hours Solid material was isolated by filtration, washed with ethyl acetate (2x20 ml) and dried in vacuum for about 1 h, getting to 21%, N of 5.39%.

Alternatively, the crude 3(S)-[N, N-bis(phenylmethyl) amino]- 1-(2-methylpropyl)amino-4-phenylbutane-2(R)-ol (5 g) was dissolved in methyl tert-butyl ether (MTBE) (10 ml) was added oxalic acid (1 g) in methanol (4 ml). The mixture was stirred for about 2 hours resulting solid portion was separated by filtration, washed with cold MTBE and dried, obtaining 2.1 g of a white solid product a 98.9% diastereomeric purity (based on HPLC peak area).

Part F. Obtain 1-[N-[(1,3-benzodioxol-5-yl] sulfonyl] -N-(2-methylpropyl)amino]-3(S)-[N,N-bis(phenylmethyl)amino]-phenyl-2 (R)-butanol

In salt of N-[3(S)-[N,N-bis(phenylmethyl)amino]-2(R)-hydroxy-4-phenylbutyl]- N-isobutylamine oxalic acid (354,7 g, 0.7 mol) in 1,4-dioxane (2000 ml) solution was added potassium carbonate (2.41,9 g of 1.75 mol) in water (250 ml). The mixture was stirred for 2 h at room temperature, then for 15 min was added 1,3-benzodioxol-5-sulphonylchloride (162,2 g, 0,735 mol) in 1,4-dioxane (250 ml). The reaction mixture was stirred at room temperature for 18 hours was Added ethyl acetate (1000 ml) and water (500 ml) and stirring continued for a further 1 h the Aqueous layer was separated and further extracted with ethyl acetate (200 ml). The combined ethyl acetate layer was washed for 25% with the deposits with ethyl acetate (200 ml) the solvent was removed in vacuum, receiving target sulphonamide in the form of a viscous yellow foamy oil (440,2 g, exit 105%). HPLC/MS (elektrorazpredelenie) (m/z 601 [M+H]+).

Alternatively, a salt of N-[3(S)-[N,N-bis(phenylmethyl)amino]-2(R)- hydroxy-4-phenylbutyl] -N-isobutylamine oxalic acid (2800 g of 5.53 mol) and THF (4 l) was added into a round bottom flask at 22 liters, equipped with a mechanical stirrer. Potassium carbonate (1921, 13,9 mol) was dissolved in water (2.8 l) was added to the suspension in THF. The mixture then was stirred for 1 h 1,3-benzodioxol-5-sulphonylchloride (1281 g, 5.8 mol) was dissolved in THF (1.4 l) was added to the reaction mixture over 25 minutes of Additional 200 ml of THF was used to rinse the dropping funnel. The reaction mixture was stirred for 14 h and then added water (4 l). The mixture was stirred for 30 min and left to separate layers. The layers were removed and the aqueous layer was washed twice THF (500 ml). The combined THF layer was dried over magnesium sulfate (500 g) for 1 h, This solution is then filtered to remove the drying agent and was used in subsequent reactions.

Part G: Obtaining salt of 1-[N-[(1,3-benzodioxol-5-sulfonyl]-N- (2-methylpropyl)amino]-3(S)-amino-4-phenyl-2(R)-butanol methanesulfonate

The crude 1-[N-[(1,3-benzodioxol-5-yl)sulfonyl] -N-(2-mTOR then added methansulfonate (0,969 g, 0,010 mol) and water (5 ml). The mixture was placed in a Parr flask of 500 ml hydrogenation containing 20% Pd(OH)2coal (255 mg, water content 50%). The flask was placed in hydrogenator and was purged 5 times with nitrogen and 5 times with hydrogen. The reaction was carried out at 35oC under hydrogen pressure 4,430 ATM for 18 h was Added catalyst (125 mg) and after blowing the hydrogenation was continued for an additional 20 hours, the Mixture was filtered through celite, which was washed with methanol (2 x 10 ml). Approximately one third of the methanol was removed under reduced pressure. The remaining amount of the methanol was removed by azeotropic distillation with toluene at 80 mm RT.article Was added to the toluene in the form of portions 15, 10, 10 and 10 ml. of the Product has led from the mixture and was filtered and washed twice with toluene portions of 10 ml of the Solid product was dried at room temperature at 1 mm RT.article within 6 h, receiving an amine salt (4.5 g, 84%): m/z 421 [M+H]+.

Alternatively, in a solution of the crude 1-[N-[(1,3 - benzodioxol-5 - yl)sulfonyl] -N-(2-methylpropyl)amino] -3(S)-[bis(phenylmethyl)amino]-4-phenyl - 2(R)-butanol in TTF was added water (500 ml), then methansulfonate (531 g, 5.5 mol). The solution was stirred to ensure complete mixing and added to the autoclave 1 is whether four times with nitrogen and four times with hydrogen. The reactor was loaded hydrogen under a pressure of 4.2 MPa and mixed with the initial speed of 450 rpm After 16 h, the HPLC analysis showed that it was still a small amount monobenzylether intermediate product. Added additional catalyst (50 g) and the reaction was carried out for the night. The solution is then filtered

through celite (500 g) to remove the catalyst and concentrated in vacuum in five portions. To each portion was added toluene (500 ml) and was removed in vacuum for azeotropic removal of residual water. The obtained solid material was divided into three parts and each washed with methyl tert-butyl ether (2 l) and filtered. Residual solvent was removed at room temperature in a vacuum oven at less than 1 mm RT.art., getting 2714 g of the expected salt.

Part H: Obtaining N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino]-1S-(phenylmethyl)-propyl]-2S-[(phenylmethanesulfonyl)amino]- 3,3-dimethylbutyramide

In the solution of 118.8 g (0,776 mol) of N-hydroxybenzotriazole and 137,1 g (0.52 mol) of N-carbobenzoxy-L-tert-leucine in 750 ml of anhydrous DMF at 0oC in an atmosphere of nitrogen was added 109,1 g (or 0.57 mol) of EDC. After stirring at 0oC for 2 h EXT] -1S-(phenylmethyl)Propylamine, pre-neutralized 228 ml (210 g, 2,08 mol) 4-methylmorpholine, in 250 ml of anhydrous DMF. After stirring at 0oC for 30 min, the mixture was stirred 18 h at room temperature. The solvents were removed under reduced pressure at 45oC, was added 1.5 l of ethyl acetate, washed with 5% citric acid, saturated sodium bicarbonate solution, brine solution, dried over anhydrous magnesium sulfate, filtered and concentrated, gaining 400 g of the crude material. It was chromatographically in 3 portions on the chromatograph Prep 2000 on silica gel using as eluent 20-50% ethyl acetate/hexane, getting 320 g of purified material, m/e = 674 (M+Li), 98% by HPLC.

Part I: Obtaining N-[2R-hydroxy-3-[[(1,3-benzodioxol-5 - yl) sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl] - 2S-amino-3,3-dimethylbutyramide

A solution of 312 g of N-[2R-hydroxy-3-[[1,3-benzodioxol-5-yl) sulfonyl](2-methylpropyl)amino] -1S-(phenylmethyl)propyl]-2S- [(phenylmethanesulfonyl)amino] -3,3-dimethylbutyramide in 1 l of tetrahydrofuran was first made in the presence of 100 g of catalyst, 4% palladium on charcoal, under a hydrogen pressure of 4.2 MPa for 6 hours at room temperature. The catalyst was removed by filtration and the solvent was removed padikkal-5-yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl] -2S-[(chloroacetyl) amino]-3,3-dimethylbutyramide

In the solution 234,3 g (0,439 mol) of N - [2R-hydroxy-3-[[(1,3-benzodioxol-5-yl) sulfonyl] (2-methylpropyl)amino] - 1S-(phenylmethyl)propyl]-2S-amino-3,3-dimethylbutyramide in 1 l of methylene chloride was added 80 ml (59,5 g, 0.46 mol) of diisopropylethylamine, then slowly at room temperature was added 78.8 g (0.46 mol) Chloroacetic anhydride, keeping the temperature below 35oC. After stirring for an additional 1 h, HPLC analysis showed that it was still a small amount of the parent compound, was added 1.5 g of Chloroacetic anhydride. After 10 min the solvent was removed under reduced pressure, was added 1 l of ethyl acetate, washed with 5% citric acid, saturated sodium bicarbonate solution, brine solution, dried over anhydrous magnesium sulfate, filtered and concentrated, obtaining 314 g of crude material. It was chromatographically in 3 portions on the chromatograph Prep 2000 on silica gel using 20-50% ethyl acetate/hexane, getting 165 g of target compound, m/e = 616 (M+Li), 98% by HPLC.

Part K: Obtaining N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino]-1S-(phenylmethyl) propyl]-2S-[[(pyrrolidin-1-yl) acetyl] amino]-3,3-dimethylbutyramide

In 164,2 g (0.27 mol) of N-[2R-hydro is albuminemia was added 500 ml of tetrahydrofuran, the solvent was removed under reduced pressure to remove any quantity of ethyl acetate and then added 350 ml of tetrahydrofuran. In this solution at 10oC was then added 130 ml (1.56 mol) of pyrrolidine. After 1 h the solvents were removed under reduced pressure, was added 1 l of ethyl acetate, washed with saturated solution of sodium bicarbonate, saline, dried over anhydrous magnesium sulfate, filtered and concentrated, gaining 185 g of the crude material, the analysis of which HPLC showed him that 98.9% purity. It was divided into 3 portions and chromatographically on the chromatograph Prep 2000 using the first 50% ethyl acetate/hexane, then 5% methanol/ethyl acetate, receiving 160 g of purified material (99% by HPLC). It then recrystallize from 460 ml of diethyl ether and 70 ml of hexane, getting 121 g of the target product (> 99% by HPLC), m/e 651 (M+Li), so pl. = 112-114oC.

Example 15

< / BR>
Obtaining N-[2R-hydroxy-3-[(2-methylpropyl)[1,3-benzodioxol-5-yl) sulfonyl] amino] -1S-(phenylmethyl)propyl]-25-methyl-3-(methylsulphonyl) propanamide

Part a: Obtain 2(S)-methyl-3-(methylsulphonyl)propionic acid

Step 1: To a solution of 200 g (1,23 mol) of D-(-)-3-acetyl-- -mercaptoethanol acid in 1.0 l of methanol was added 161,0 g (2,47 mabana. After stirring an additional 20 min was added 117 ml (156 g of 1.23 mol) dimethylsulfate, keeping the temperature below 20oC. the Ice bath was removed and the mixture was stirred for additional 60 minutes Salt was removed by filtration, the solvent was removed under reduced pressure and added ethyl acetate. After separating the water layer was acidified with concentrated hydrochloric acid, was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered and concentrated, gaining 164 g (99%) target 2S-methyl-3-(methylthio)propionic acid, m/e 133 (M-H).

Stage 2: To a solution of 10.0 g (74,6 mmol) of 2S-methyl-3-(methylthio)propionic acid in 150 ml of acetone and 30 ml of water, cooled to 18oC in an ice bath, was added to 161.8 g (263 mmol) oxone. After adding about half of the material, the temperature rose to 24oC, the addition was stopped, the temperature was lowered to 18oC, then adding continued. After stirring at 15-20oC for 15 min, the bath was removed and the reaction mixture was stirred at room temperature for 1 h, the Solid portion was separated by filtration and washed with acetone, the filtrate was concentrated to approximately 40 ml and the residue was dissolved in 200 ml of ethyl acetate. When was dissolved in minimum amount of ethyl acetate and hexane was added to the sediment. It was collected, receiving of 6.95 g of the desired product, m/z = 167 (M+H).

Part of: Obtaining N-[2R-hydroxy-3-[(2-methylpropyl)-[1,3-benzodioxol - 5-yl)sulfonyl] amino] -1S-(phenylmethyl) propyl]-2S-methyl-3-(methylsulphonyl) propanamide

Into a solution of 5.0 g (30 mmol) of 2S-methyl-3-(methylsulphonyl) propionic acid and of 6.90 g (45 mmol) of N-hydroxybenzotriazole in 30 ml of anhydrous DMF at 0oC in an atmosphere of nitrogen was added 6,34 g (33 mmol) of EDC. After about 10 min the entire EDC was dissolved. After 60 min at 0oC was added a solution of 15.5 g (30 mmol) of methanesulfonate 2R-hydroxy-3-[[(1,3-benzodioxol-5 - yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)Propylamine in 30 ml of anhydrous DMF, previously neutralized with 3.4 ml (31,6 mmol) 4-methylmorpholine. After incubation for 3 hours at 0oC the mixture was then stirred at night for 17 hours DMF was removed under reduced pressure, was added ethyl acetate, washed with 5% citric acid, saturated sodium bicarbonate solution, water, saline solution, dried over anhydrous magnesium sulfate, filtered and concentrated, obtaining 16 g of the crude material, which had a purity of 88% by HPLC. The product was chromatographically on silica gel using 20-80% ethyl acetate/hexane, obtaining the pure product, to the SS="ptx2">

Alternatively, in a solution of 35.0 g (211 mmol) of 2S-methyl-3-(methylsulphonyl) propionic acid and 48.3 g (315 mmol) of N-hydroxybenzotriazole in 210 ml of anhydrous DMF at 0oC in an atmosphere of nitrogen was added 44.4 g (231 mmol) of EDC. After about 30 minutes the whole EDC was dissolved. After an additional 60 min at 0oC solution was added to 108.8 g (211 mmol) of methanesulfonate 2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl) amino] -1S-(phenylmethyl)Propylamine in 350 ml of anhydrous DMF, pre-neutralized 24 ml (22,3 g) 4-methylmorpholine. After incubation for 2 h at 0oC the mixture was then stirred at night for 18 hours DMF was removed under reduced pressure, was added 1 l of ethyl acetate, washed with 5% citric acid, saturated sodium bicarbonate solution, water, saline solution, dried over anhydrous magnesium sulfate, filtered and concentrated, getting to 120.4 g of the crude material, which had a purity of 90% by HPLC. The product has to recrystallize twice from 750-1000 ml of absolute ethanol, getting to 82.6 g of the target product.

Example 16

< / BR>
Receipt of 2S-[[(N-methylamino)acetyl] amino] -N-[2R-hydroxy-3- [[(1,3-benzodioxol-5-yl)sulfonyl] -(2-methylpropyl)-amino]-1S- (phenylmethyl)propyl] -3,3-dimethylbutyramide

Voracity)amino] -3,3-dimethylbutyramide was added 25 ml of tetrahydrofuran, the solvent was removed under reduced pressure to remove any quantity of ethyl acetate and then added 25 ml of tetrahydrofuran. In this solution at 10oC was added 19 ml (214 mmol) of 40% aqueous methylamine. After 2 h the solvent was removed under reduced pressure, was added 1 l of ethyl acetate, washed with saturated solution of sodium bicarbonate, saline, dried over anhydrous magnesium sulfate, filtered and concentrated, obtaining 6.0 g of product (98% purity).

Example 17

Connection-retroviral protease inhibitors of this invention are effective inhibitors of HIV protease. Enzymatic analysis, described below, can be used in the selection of retroviral protease inhibitors for use in combination therapy. Using this method it is possible to calculate IC50(the concentration at which the compound is an inhibitor reduces enzyme activity by 50%) for such compounds.

The enzymatic method is the following. The substrate is 2-aminobenzoyl-Ile-Nle-Phe(n-NO2)-Gln-Arg-NH2. Positive control is MVT-101 (Miller, M. et al., Science 246, 1149 (1989)). Buffer analysis is 20 mm sodium phosphate, pH of 6.4, 20% glycerol, 1 mm ethylenediaminetetraacetic acid, 1 I the concentration of the substrate in the analysis of about 80 μm. HIV protease was diluted in buffer for analysis to the final concentration of the enzyme is about 12.3 nm, considering the molecular weight 10780.

The final concentration of DMSO is about 14% and the final concentration of glycerol is about 18%. The test compound dissolved in DMSO and diluted in DMSO to about ten times (10 x), then add 10 ál of the substrate. The increase in fluorescence recorded at 4 time points (0, 8, 16 and 24 minutes) at room temperature. Each analysis is performed in duplicate holes.

Example 18

Efficacy of selected compounds-HIV protease inhibitors of the present invention can be determined using the above-described enzymatic analysis and further analysis of CD4+ cell line. Antiviral activity of protease inhibitors is expressed as the magnitude of the effective concentration 50 (EC50and/or effective concentration 90 (EC90). They are concentrations of inhibitors are required for inhibition of viral replication by 50% or 90%, respectively.

The analysis of the inhibition of acute HIV infected cells is essentially an automated colorimetric analysis on the basis of tetrazole described by Pauwels et al. , J. Virol. Methods 20, 309-321 (1988). service cell lines, grown in medium RPMI-1640 (Gibco), supplemented with 10% fetal calf serum, and then treated polybrene (2 μg/ml). Wednesday by volume of 80 μl, containing 1 to 104cells, place the dispenser into each well of the tissue culture plate. To each well was added 100 μl of the test compound dissolved in tissue culture medium (or medium without test compound as a control) to achieve the desired final concentration and the cells incubated at 37oC for 1 hour. A frozen culture of HIV-1 was diluted in culture medium to a concentration of 5 104TCID50per ml (TCID50= the dose of virus that infects 50% of cells in tissue culture) and 20 μl of virus sample (containing 1000 TXID50virus) is added to wells containing test compound and to wells containing only medium (infected control cells). Some wells receive culture medium without virus (uninfected control cells). Similarly, the inherent toxicity of the test compounds is determined by adding in some of the wells containing the test compound, medium without virus. In the amount of tissue culture tablets contain the following experiments (see tab. A to the 0 and 500 µg/ml As a positive drug control include azidothymidine (AZT) or deoxyinosine (ddI). Compound dissolved in DMSO and diluted with tissue culture medium to the final concentration of DMSO did not exceed 1.5% in any case. DMSO and added to all control wells at the appropriate concentration.

After addition of virus, cells incubated at 37oC in humidified containing 5% CO2the atmosphere within 7 days. Compound can be added at days 0, 2 and 5, if desired. On day 7 after infection, the cells in each well resuspended and 100 ál sample of each cell suspension is removed for analysis. In every ál of cell suspension, add 20 μl solution of 5 mg/ml bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) and the cells incubated for 4 h at 37oC in medium with 5% CO2. During this incubation the contents of the MTT metabolic rate is reduced by living cells, which leads to the production in the cell is painted formisano product. In each sample, add 100 ál of 10% sodium dodecyl sulphate 0.01 N. HCl for lizirovania cells and the samples incubated overnight. The absorption at 590 nm for each sample was determined using a microplate-mania values of absorption, received in the wells containing infected or uninfected cells incubated with compounds and uninfected, untreated control wells.

PROCEDURES TO CULTURE HIV STIMULATION OF LYMPHOCYTES OF DONOR

Leucocytes films were obtained from American Red Cross or Blood Bank et Washington University School of Medicine. These drugs are pre-tested for antibodies to HIV and CMV (cytomegalovirus) and surface antigen of HBV (hepatitis b virus) and ALT (activity albinterferon) as a marker for non-a, non-b" hepatitis. Enriched by leukocytes in the blood (30 ml) are removed from the plastic container and 15 ml carry the dispenser in two centrifuge tubes 50 ml with screw-on cap. Each sample was diluted with an equal volume of sterile PBS (phosphate buffered saline, SFR) and mix by pipetting. Ficoll-Pak (15 ml) or LSM placed below the diluted blood sample using a Pasteur pipette, and by allowing the solution to sink to the bottom of the tube. Each of the test tubes and then centrifuged at 1300 rpm (400 g) for 45 min at 20oC. After centrifugation limfocitna the area of the interface were removed and transferred into a test tube 50 ml Add SFR to dilute the Department is the W in SFR and again centrifuged. Final debris again suspended in 20 ml of SFR by pipetting and the total number of viable cells determined by exclusion of Trypan Blue.

ANALYSES OF ACUTE INFECTIVITY USING CLINICAL INSULATORS

Approximately 3107cells activated for 48 h with approximately 3-5 µg/ml RNA (phytohemagglutinin, PHA) in RPMI medium containing 10% fetal calf serum and IL-2 (10 u/ml). Quantitative viral solutions are added to a suspension of activated lymphocytes when the multiplicity of infection of about 0.001 to 0.01. Suspension cell-virus incubated at 37oC for 2 h to conduct viral absorption. Residual viral inoculum removed by centrifugation and cells resuspended in RPMI medium containing 10% FBS and 10 u/ml IL-2. These infected cells are added to the test compound, diluted in complete tissue culture medium from a source solution (10 mg/ml) in DMSO in titration microplate with 96 wells, receiving about 5105cells per well (200 μl). Infected, untreated cells and cells treated with only DMSO (0.1 per cent) or AZT or DDI, was used as a control. The culture was tested for the formation of syncytium at 7 and 11 days after infection or supernatant tested
CEM cells chronically infected with HXB2 (laboratory strain HIV-1), add in 6 holes tiralongo microplate with 12 holes, getting 5104cells per well. Half of the wells treated with the test compound at various concentrations and the same number of uninfected CEM cells retain without adding connections. A fresh environment with the test compound or without add every day for three consecutive days. The culture is then incubated for 48 hours without a change in the environment. Cells are harvested by centrifugation, washed 2 times in PBS and resuspended in 50 µl buffer 2x Laemmli, containing 0.125 M Tris, pH of 6.8, 4% SDS (sodium dodecyl sulphate), 20% glycerol, 10% beta-mercaptoethanol and 0.02% Bromophenol blue. Supernatant cultures passed through a 0.22 μm filter to remove debris and centrifuged at 50000 rpm for 90 min for the concentration of viral particles. Viral sediment resuspended in 50 µl buffer 2x Laemmli. Cellular or viral suspension is boiled for 5 min and then subjected to electrophoretic separation in 10 - 20% SDS-polyacrylamide gradient gel. The contents of the gel and then transferred to nitrocellulose by electroblotting. HIV-specific proteins detect, using monoclonal the consultative transformation of 4-chloro-1-naphthol was used to visualize specific proteins, recognized by monoclonal antibodies. In addition, we investigated the infectivity of the virus produced chronically infected CEM cells in the presence or in the absence of the test compound. Filtered supernatant serially diluted and used to infect uninfected CEM cells (about 1104/well). Culture was studied on the formation of the syncytium in days 7 and 11 after infection or supernatant was tested for reverse transcriptase activity or p24 antigen.

MICROANAL REVERSE TRANSCRIPTI (RT).

Microanalysis RT is an adaptation of several standard analyses of RT. It was developed for quantitative measurements of the activity of HIV RT in a small volume and facilitate the processing of various samples (see tab. B).

WAY:

1. Add 50 μl of RT cocktail per well in a microtiter 96-well U-shaped bottom.

2. Add 10 - 20 ál per well of a solution containing no cells supernatant.

3. Mix well with a mechanical rotor.

4. Incubated at 37oC for 2 h

5. Suck on DE81 filter paper or equivalent with TOMTEC.

6. CLASS="ptx2">

9. Prepared for counting by the counter Beta-plate (Pharmacia).

Example 19 (scheme I see the end of the description).

Multiple cycles ARE repeated with increasing concentrations of the compounds to the observation of a shift in EC50< / BR>
The following is the culture method used for selection of mutants resistant to inhibitors of HIV protease. Infected cells were grown continuously in the presence of protease inhibitor. Some cultures were subjected to on alternating weeks high and low concentrations of inhibitors. Others perseval at a constant concentration. The concentration of drug was increased periodically to monitor permanent shift in EC50. The shift in the curve, depending on the dose, usually revealed in drug concentration from 0.5 to 1 ál/ml or higher (5-10EC50and he depended on the processed viral isolate. Used as a laboratory-adapted and primary clinical isolates of HIV. The same viral isolates were perseval the same way in the absence of drugs, so it was possible to make a direct comparison nucleoside sequences of the treated and untreated isolates. Usually variants of HIV-1-resistant and is of ncentrate these protease inhibitors (see Markowitz et al. , Journal of Virology 69: 701 - 706 (1995)). Variants of HIV-1, listed below, show mutations that are present in the selected virus isolates and are not present in the control, untreated viral isolates.

RF represents a strain of HIV-1RFHIV-1 and RFR is a mixture of resistant strains obtained by selection against RF connection Example 1. RFR contains a mixture of viral strains with genotypes protease G48V (14/40 clones), G48V, V82A (18/40 clones), G48V, L90S (2/40 clones), G48V, 154T, V82A (1/40 clones), G48M (1/40 clones), G48V, Q61H (1/40 clones), V131, G48V (1/40 clones), G48V, F53L, V82A (1/40 clones) and G48V, V82A, C95Y (1/40 clones). RFR2 is a mixture of resistant strains obtained by cloning RFR three cycles of growth at limiting dilution. RFR2 contains a mixture of viral strains with genotypes protease G48V, V82A (13/15 clones), G17E, G48V, V82A (1/15 clones) and G48V, V82A, N88D, N88D (1/15 clones). RFRR is a mixture of resistant strains obtained by selection against RF compounds of Examples 1 and 2 and then cloning the three cycles of growth at limiting dilution. RFRR contains a mixture of viral strains with genotypes protease G48V, L63P, V82A (7/9 clones), G48V, 154T, L63P, V82A, N88S (1/9 clones) and G48V, 154T, L63P, G73M, V82A (1/9 clones). SF162 represents the strain SF-162 HIV-1 and SF162R is a mixture of resistant strains, obtained the L63P, A71V, N88D (2/3 clones) and M461, F53L, L63P, A71V, N88D, Q92R (1/3 clones). 89-959 represents the strain 89-959 HIV-1 and 89-959R is a mixture of resistant strains obtained by selection 89-959 against the compound of Example 2. 89-959R contains a mixture of viral strains with genotypes protease N88S (4/5 clones) and D25N, T26A, D30N, D37N, R41K, G73D, R87K, N88S (1/4 clones). NL4 is a strain of HIV-1NL4HIV-1. NL4 (G48V) represents the strain with synthetically generated site-directed mutation in the protease of glycine to valine at amino acid position number 48. NL4 (184V) represents the strain with synthetically generated site-directed mutation in the protease of isoleucine to valine at amino acid position 84. NL4 (R8Q, M46I) represents the strain with synthetically generated site-directed mutations in the protease arginine to glutamine at amino acid position number 8 and methionine to isoleucine at amino acid position number 46. NL4 (P22-538) is a mixture of resistant strains obtained by selection of HIV-1NL4-3against the compound of example 10 after 22 passages containing protease genotypes M46I, L63P, A71V, V82F, 184V, (4/10); M46I, L63P, V82F, 184V (3/10) and M46I, A71V, V82F, 184V (3/10). NL4(P37 - 538) is a mixture of resistant strains obtained by selection of HIV-1NL4-3against the compound of Example 10 after 37 passages, sod is otiv compound of Example 5 after 24 passages, containing protease genotype M46I, L63P, A71V, 184A. NL4(538/P7-AG) is a mixture of resistant strains obtained by selection NL4(P22-538) against compound of Example 12 after 7 passages containing protease genotypes M46I, L631, A71V, 184A and V32I, V82I. NL4(538/P24-AG) is a mixture of resistant strains obtained by selection NL4(P22 - 538) against compound of Example 12 after 24 passages containing protease genotype M46I, L63P, A71V, 184A. NLA(pI9-003) is resistant strains obtained by selection of HIV-1NL4-3against the compound of Example 9 after 19 passages containing protease genotype R8K, M46I. NL4 (P34-003) is resistant strains obtained by selection of HIV - 1NL4-3against the compound of Example 9 after 34 passages containing protease genotype R8K, M46I, L63P, A71V, L90M. Results stability of viral isolates are summarized in Tables 1 - 11.

Example 20

Results stability of viral isolates are summarized in Tables 1 - 3, was obtained in accordance with the following methods of analysis or minor modifications. Approximately 3107cells activate for approximately 46 hours with 3-5 μg/ml RNA in RPMI medium containing 10% fetal calf serum and IL-2 (10 u/ml). To a suspension of activated lymphocytes add quantitative viral source R is h for carrying out the absorption of the virus. Residual viral inoculum removed by centrifugation and cells resuspended in RPMI containing 10% FBS and 10 u/ml IL-2. These infected cells are added to the test compound, diluted in complete tissue culture medium from a source solution (10 mg/ml) in DMSO tablets for micrometrology with 96 holes, getting about 5 to 105cells per well in 200 μl. As control was used by the infected, untreated cells and cells treated with only DMSO (0.1 per cent) or AZT or DDI. Cultures were examined for the formation of the syncytium in days 7 and 11 after infection or supernatant was tested for reverse transcriptase activity or P24 antigen.

Example 21

Results stability of viral isolates are summarized in tables 4-6 were obtained in accordance with the following methods of analysis or small modifications. Analyses were performed in tissue culture plates at 96 wells. Cells CEM-T4 suspended in 90% RPMI medium (Gibco BRL Life Technologies, Inc. , Gaithsburg, MD), 10% heat-treated fetal calf serum (Gibco BRL Life Technologies, Inc., Gaithsburg, MD) to a final concentration of 5 105viable cells per ml Frozen aliquot sample of the culture of HIV (HIV-1 strainRFquickly thaw (water is the R units per cell. A suspension of virus-cell to rapidly mix the formation of turbulence and 100 μl immediately add 100 µl of each dilution of the test compound (obtained as a 2x concentrate in 90% RPMI, 10% FBS) to each well of the tissue culture plate. Each tablet contains control wells containing cells and virus, but do not contain the test compound. 3'-Azido-3 deoxythymidine (AZT) include as a positive control in all assays.

Tissue culture tablets incubated at 37oC in humidified containing 5% CO2the atmosphere within 7 days. The level of viral replication is then determined by measuring the activity of reverse transcriptase in supernatant using standard techniques as described previously and see, for example, Techniques in HIV Research, Aldovini and Walker, eds., 1990, Stockton Press, NY).

Example 22

Results stability of viral isolates are summarized in tables 7 - 11, received in accordance with the method of analysis described by Markowitz et al., Journal of Virology vol. 69, 701 - 706 (1995), which is incorporated herein by reference in its entirety.

Example 23

The protease inhibitors of examples 1 and 2, which contain unusual ISOStAR of gidroxiatilkrahmala, was used for the selection of drug-resistant sfericheskikh blood mononuclear cells (PBMCs) in the presence of increasing concentrations of the drug. Sustainable options are constantly shown the values EC50at least 10 times higher than in the control virus, preteenage during the identical period, but in the absence of inhibitor. Viral DNA was amplified by PCR and nucleotide sequence of the gene encoding the protease was determined using standard techniques. The virus resistant to protease inhibitors of examples 1 and 2, respectively, were constantly observed amino acid change at position 88 in many of the selected variants. The Asn residue 88 is located within a structurally conserved helical domain that is present in Monomeric and dimeric the aspartic proteinases. Corresponding carboxykinase the sequence Gly-Arg-Asp/Asn (residues 86-88) unusual for retroviral aspartic proteinases. Although any explanation of these results is only thinking, modeling studies based on template obtained on the basis of x-ray spectra of high-resolution structures of the prototypical gidroxiatilkrahmala inhibitors, United with recombinant HIV-1 protease, seems to confirm that Asn88 mutations can alter the conformation of the protease.

Compounds in which inanami and in particular, are effective inhibitors of retroviruses, including lentiviruses, as shown above. Thus, the compounds of the subject matter of this invention are effective inhibitors of HIV. It is assumed that the connection of the subject invention will also inhibit other strains of HIV, for example HIV-2 and other viruses, such as, for example, VISNA virus, and Simian immunodeficiency virus (SIV), HTLV-1 and HTLV-2. Thus, the compounds of the subject matter of this invention is effective in the treatment and/or prophylaxis of retroviral infections.

It is also understood that the invention includes, when possible, MES or hydrates of the compounds are inhibitors of retroviral protease, which receive and emit methods known in this field.

Connection-retroviral protease inhibitors can be applied in the form of salts derived from inorganic or organic acids. These salts include, but are not limited to, the following salts: acetate, adipate, alginate, citrate, aspartate, benzoate, bansilalpet, bisulfate, butyrate, comfort, camphorsulfonate, digluconate, cyclopentanepropionate, dodecyl sulphate, aconsultant, glucoheptonate, glycyrrhizinate, polysulfate, heptanoate, hexane otenet, 2-naphthalenesulfonate, oxalate, palmoate, luchinat, persulfate, 3 - phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesilate, undecanoate.

Examples of acids that can be used for the formation of pharmaceutically acceptable salts with acids, include such inorganic acids as hydrochloric acid, sulfuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid, the preferred cleaners containing hydrochloride salt. Other examples include salts with alkali metals or alkaline earth metals, for example sodium, potassium, calcium or magnesium, or with organic bases.

The total daily dose, administered to a patient in single or divided doses, may be, for example, from 0.01 to 50 mg/kg body weight daily and more usually from 0.1 to 30 mg Dosed unified compositions can contain such amounts of subcosta them to make up the daily dose.

The amount of active ingredient which can be combined with the material-carriers to obtain a single dosage form will vary depending on the patient, which it is introduced, Oceania compounds inhibitors of retroviral protease and/or compositions are chosen in accordance with many factors, including the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, route of administration, pharmacological value, such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular applicable connection use the system for delivery into the body of a medicinal product, enter whether the connection as part of combination medicines. So, actually used regimen medicines can vary widely and therefore may deviate from the preferred dosage regimen medicines suggested above.

The compounds of this invention can be administered orally, parenterally, by inhalation spray, rectally, or topically in a standardized pharmaceutical finished formulation, containing, if required, the conventional non-toxic pharmaceutically acceptable carriers and additives. Local administration can include the use of percutaneous injection, such as percutaneous patches or iontophoresis. The term parenteral as used here, includes subcutaneous injections, intravenous, intramuscular, intrasternally (nutrigrain is or oily suspensions, can be prepared according to known methods using suitable dispersing or wetting means or suspendresume funds. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable diluents or solvents, which can be used include water, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspendida environment usually apply sterile fatty oil. For this purpose you can use any fatty oil soft steps, including synthetic mono - or diglycerides. In addition, fatty acids such as oleic acid find use in injectable preparations.

Suppositories for rectal administration of medicinal remedies can be obtained by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycols which are solid at ordinary temperature but liquid at rectal temperature and therefore will melt in the rectum and release Lakers the tablet, pills, powders and granules. In such solid dosage forms the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms can also contain, as in normal practice, additional substances other than inert diluents, for example, lubricants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage form may also contain buffers. Tablets and pills can additionally be obtained intersolubility coverings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and Aleksiy containing inert diluents commonly used in this field, for example water. Such compositions can contain auxiliary agents, such wetting means, emulsifying and suspendresume tools, and sweetening means, corrigentov and fragrances.

Although the connection-retroviral protease inhibitors of the present invention can be entered as the sole active pharmaceutical drugs, they can also be used in combination with other protive limited to, other protease inhibitors HIV-1, various nucleoside analogues, non-nucleoside reverse transcriptase inhibitors, antagonists of tyrosine aminotransferase (tat) and glucosidase inhibitors.

Examples of protease inhibitors of HIV-1 include, but are not limited to, Po 31-859 (Roberts, N. A. et al. Science 1990, 248, 258 - 261, and Drugs of the Future 1991, 16(3), 210 - 212), INSTITUTE-272, (Kagayama, S., et al. Antimicrobial Agens and Chemotherapy 1993, 610 - 817), the number of cyclic urea (Lam, P. , et al., "De Novo Design and Discovery of Potent, Nonpeptidal HIV-1 Protease Inhibitors", paper 96 at the 205th American Ghemical Society National Meeting, Medicinal Chemistry Division, Denver, CO, March 28-April 2, 1993), L-735 524 (Dorsey, B. D., et al., "L-735 524: The Rational Design of Potent and Orally Bioavailable HIV Protease Inhibitor", paper 6 et the 206th American Chemical Society National Meeting, Medicinal Chemistry Division, Chicago, IL, August 22 - 27, 1993) and their analogues.

Examples of competing nucleoside analogues include, but are not limited to, azidothymidine (AZT), dideoxyinosine (DDI), DDC, ZTS, 4DT, RMEA. Examples of non-competing non-nucleoside reverse transcriptase inhibitors include, but are not limited to, class Spiridonov (Wei, J. S., et ai., J. Med. Chem. 1993, 36, 249 - 255; Hoffrnan, J. M., et al., J. Med. Chem. 1992, 35, 3784 - 3791; Saari et al., J. Med. Chem. 1992, 35, 3792 - 3802; Drugs of the Future 1992 17(4), 283 - 285) and their analogues; class bis(heteroaryl)piperazines (Romero, D. L., et al., J. Med. Chem. 1993, 36, 1505 - 1508; Romero, D. L., et al., Proc. Natl. Acad. Sci. USA 1991, 34, 746 - 751 and 3178 - 3198) and 990, 250, 1411 - 1413), and their analogs and 5-chloro-3-(phenylsulfonyl)indole-2-carboxamide and its analogues (Williams, T. M. et al., J. Med. Chem. 1993, 36, 1291 - 1294). Examples of tat antagonists include, but are not limited to, Ro 5-3335 and Ro 24-7429 (Hsu, M. C. et al., Proc. Natl. Acad. Sci. USA 1993, 909, 6395 - 6399; Tam, S. et al., "TAT INHIBITORS: A NEW CLASS OF ANT I-HIV AGENTS", paper 372, et the 204th American Chemical Society National Meeting, Organic Chemistry Division, Washington, DC, August 23 - 28, 1992) and their antagonists. Examples of glycosidase inhibitors include, but are not limited to, castanospermine, 6-botilony ether castanospermine, butyl - 1-deoxynojirimycin, per-botilony N-butyl-1-deoxynojirimycin and their analogues and prodrugs.

A therapeutic agent can be prepared as a separate composition, which give (give) essentially at the same time, or a therapeutic agent can be given as single tracks, so that all active agents were in the body of the patient in therapeutically effective amounts. Alternatively, therapeutic agents can enter the patient at different times, so that only one or two active agent at any time were in the body of the patient in therapeutically effective amount.

Compounds and methods of this invention are effective antiviral compounds of the et of this invention are effective inhibitors of HIV protease. It is assumed that the connection is the subject of this invention will also inhibit other retroviruses, such as other lentiviruses, in particular, other strains of HIV, for example HIV - 2 virus T-cell leukemia human, rous sarcoma virus, simian immunodeficiency virus, the virus leukemia cats, human immunodeficiency virus cats and similar viruses. Thus, the compounds of the subject matter of this invention is effective in the treatment and/or prophylaxis of retroviral infections.

Subject of the invention compounds and methods are also effective in preventing the growth of retroviruses in solution. Culture both human and animal cells, such as culture T-lymphocytes, is used for a number of well-known purposes, such as research and diagnostic methods, including bougie-measures and standards. Before and during growth and storage cell culture the compounds of this invention can be added to cell culture medium in an effective concentration to prevent unexpected and unwanted replication of retrovirus that can accidentally or unknown to be present in cell culture. The virus can be present in cell culture initially, for example known for the JVI, or because of exposure to culture the virus. This use of the compounds and methods of the present invention prevents unwanted or unexpected effects potentially lethal retrovirus for the researcher or Clinician.

The preceding description only illustrates the invention and is not intended to limit the invention described compounds. Implies that variations and changes which are obvious to the person skilled in the art, are included in the scope and essence of the present invention, which is defined in the attached claims.

From the preceding description specialist in this field can easily set the main characteristics of this invention without deviating from the essence and scope can make various changes and modifications of the invention to adapt it to various usages and conditions.

1. A method of treating retroviral infections in a mammal, containing the introduction to this mammal (a) an effective amount of a first retroviral protease inhibitor and (b) an effective amount of a second retroviral protease inhibitor, where this second retroviral protease inhibitor effective PI protease.

2. The method according to p. 1, characterized in that the first and second inhibitors injected so that an effective amount of both inhibitors were present in the mammal.

3. The method according to p. 1, characterized in that the introduction of the first and second inhibitors alternate to the mammal during the period of time attended an effective amount of one inhibitor.

4. The method according to p. 1, characterized in that the first retroviral protease inhibitor is N-(2-(R)-hydroxy-1(S)-indanyl-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-pyridylmethyl)-2(S)-N'-(tert-BUTYLCARBAMATE)piperazinil))pentanediol; N-tert-buildimage-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-hinolincarbonova)-L-asparaginyl]amino]butyl]-(4R, 8S)-isoquinoline-3(S)-carboxamide; (2S, 3R,4S,5S)-2,5-bis-[N-[N-[[N-methyl-N-(2-pyridinyl-methyl)amino] carbonyl] valinol]amino]-3,4-dihydroxy-1,6-diphenylhexane; (2S, 3S,5S)-5-[N-[N-[N-methyl-N-[(2-isopropyl-4-thiazolyl)methyl]amino] carbonyl]valinol]amino]-2-(N-[(5-thiazolyl)methoxycarbonyl]amino]-3-hydroxy-1,6-diphenylhexane; 3S-tetrahydropyranyloxy ester, [2R-hydroxy-3-[[(4-AMINOPHENYL)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)-propyl]carbamino acid; N-tert-buildimage-2-[2(R)-hydroxy-4-(phenylthio)-3(S)-[[N-[(2-methyl-3-hydroxyphenyl)carbon is 4,7-bis(phenylmethyl)-2H-1,3-diazepin-2-one; N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl] -2S-[[(pyrrolidin-1-yl)acetyl] amino]-3,3-dimethylbutyramide; N-[2R-hydroxy-3-[(2-methylpropyl)[(1,3-benzodioxol-5-yl)sulfonyl] amino] -1S-(phenylmethyl)propyl] -2S-methyl-3-(methylsulphonyl)propanamide; [1S-[1R*(R*), 2S*] ] -N-[2-hydroxy-3-[N1-(2-methylpropyl)-N1-(4-methoxybenzenesulfonyl)amino]-1-(phenylmethyl)propyl]-2-methyl-3-(methylsulphonyl)propanamide; 2S-[[(N-methylamino)acetyl]amino]-N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl](2-methylpropyl)amino]-1S(phenylmethyl)propyl] -3,3-dimethylbutyramide or (2R,3S)-3-(N-methylaminomethyl-L-tert-butylphenyl)-amino-1-(N-isoamyl-N-(tert-butylcarbamoyl))amino-4-phenyl-2-butanol

5. The method according to p. 1, characterized in that the second retroviral protease inhibitor is N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-pyridylmethyl)-2(S)-N'-(tert-BUTYLCARBAMATE)piperazinil))pentanediol; N-tert-buildimage-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-hinolincarbonova)-L-asparaginyl]amino]butyl]-(4aR, 8aS)-isoquinoline-3(S)-carboxamide; (2S, 3R,4S,5S)-2,5-bis-[N-[N-[[N-methyl-N-(2-pyridinylmethyl)amino] carbonyl] valinol] amino]-3,4-dihydroxy-1,6-diphenylhexane; (2S, 3S,5S)-5-[N-[N-[N-methyl-N-[(2-isopropyl-4-thiazo tetrahydrofuranyl ester, [2R-hydroxy-3-[[(4-AMINOPHENYL)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)-propyl]carbamino acid; N-tert-buildimage-2-[2(R)-hydroxy-4-(phenylthio)-3(S)-[[N-[(2-methyl-3-hydroxyphenyl)carbonyl] amino]butyl]-(4R, 8aS)-isoquinoline-3(S)-carboxamide; [4R-(4,5,6,7,)]-1,3-bis[(3-AMINOPHENYL)methyl]hexahydro-5,6-dihydroxy-4,7-bis(phenylmethyl)2H-1,3-diazepin-2-one; N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl] -2S-[[(pyrrolidin-1-yl)acetyl] amino]-3,3-dimethylbutyramide; N-[2R-hydroxy-3-[(2-methylpropyl)[(1,3-benzodioxol-5-yl)sulfonyl] amino] -1S-(phenylmethyl)propyl] -2S-methyl-3-(methylsulphonyl)propanamide; [1S-[1R*(R*), 2S*] ] -N-[2-hydroxy-3-[N1-(2-methylpropyl)-N1-(4-methoxybenzenesulfonyl)amino]-1-(phenylmethyl)propyl]-2-methyl-3-(methylsulphonyl)propanamide; 2S-[[(N-methylamino)acetyl]amino]-N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl] -3,3-dimethylbutyramide or (2R,3S)-3-(N-methylaminomethyl-L-tert-butylphenyl)-amino-1-(N-isoamyl-N-(tert-butylcarbamoyl)amino-4-phenyl-2-butanol.

6. The method according to p. 1, characterized in that the first retroviral protease inhibitor is N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-pyridylmethyl)-2(S)-N'-(tert-BUTYLCARBAMATE)piperazinil))pentanediol; N-tert-buildimage-2-[2(R)-hydroxy-4-phenyl-3 (is-N-(2-pyridinylmethyl)amino] carbonyl] valinol] amino]-3,4-dihydroxy-1,6-diphenylhexane; (2S, 3S,5S)-5-[N-[N-[N-methyl-N-[(2-isopropyl-4-thiazolyl)methyl]amino] carbonyl]valinol]amino]-2-(N-[(5-thiazolyl)methoxycarbonyl]amino]-3-hydroxy-1,6-diphenylhexane; 3S-tetrahydropyranyloxy ester, [2R-hydroxy-3-[[(4-AMINOPHENYL)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)-propyl]carbamino acid; N-tert-buildimage-2-[2(R)-hydroxy-4-(phenylthio)-3(S)-[[N-[(2-methyl-3-hydroxyphenyl)carbonyl] amino]butyl]-(4aR, 8aS)-isoquinoline-3(S)-carboxamide; [4R-(4,5,6,7]-1,3-bis[(3-AMINOPHENYL)methyl]hexahydro-5,6-dihydroxy-4,7-bis(phenylmethyl)-2H-1,3-diazepin-2-one; N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl](2-methylpropyl)amino]-1S-(phenylmethyl)propyl] -2S-[[(pyrrolidin-1-yl)acetyl] amino]-3,3-dimethylbutyramide; N-[2R-hydroxy-3-[(2-methylpropyl)[(1,3-benzodioxol-5-yl)sulfonyl] amino] -1S-(phenylmethyl)propyl] -2S-methyl-3-(methylsulphonyl)propanamide; [1S-[1R*(R*), 2S*] ] -N-[2-hydroxy-3-[N1-(2-methylpropyl)-N1-(4-methoxybenzenesulfonyl)amino]-1-(phenylmethyl)propyl]-2-methyl-3-(methylsulphonyl)propanamide or (2R, 3S)-3-(N-methylaminomethyl-L-tert-butylphenyl)-amino-1-(N-isoamyl-N-(tert-butylcarbamoyl))amino-4-phenyl-2-butanol and a second retroviral protease inhibitor is 2S-[[N-methylamino)acetyl] amino] -N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl](2-metery the retroviral protease inhibitor is N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-pyridylmethyl)-2(S)-N'-(tert-BUTYLCARBAMATE)piperazinil))pentanomial, and the second retroviral protease inhibitor is (2R, 3S)-3-(N-methylaminomethyl-L-tert-butylphenyl)-amino-1-(N-isoamyl-N-(tert-butylcarbamoyl))amino-4-phenyl-2-butanol; N-[2R-hydroxy-3-[(2-methylpropyl)[(1,3-benzodioxol-5-yl)sulfonyl]amino]-1S-(phenylmethyl)propyl] -2S-methyl-3-(methylsulphonyl)propanamide; [1S-[1R*(R*), 2S*]]-N-[2-hydroxy-3-[N1-(2-methylpropyl)-N1-(4-methoxybenzenesulfonyl)amino] -1-(phenylmethyl)propyl] -2-methyl-3-(methylsulphonyl)propanamide or 2S-[[(N-methylamino)acetyl]amino]-N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl]-3,3-dimethylbutyramide.

8. The method according to p. 1, characterized in that the first retroviral protease inhibitor is (2S, 3S, 5S)-5-[N-[N-[N-methyl-N-[(2-isopropyl-4-thiazolyl)methyl] amino] carbonyl]valinol]amino]-2-(N-[(5-thiazolyl)methoxycarbonyl] amino] -3-hydroxy-1,6-diphenylhexane, and the second retroviral protease inhibitor is (2R,3S)-3-(N-methylaminomethyl-L-tert-butylphenyl)-amino-1-(N-isoamyl-N-(tert-butylcarbamoyl))amino-4-phenyl-2-butanol; N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)propyl] -2S-[[(pyrrolidin-1-yl)acetyl] amino]-3,3-dimethylbutyramide or 2S-[[(N-methylamino)acetyl] amino]-N-[2R-g is S="ptx2">

9. The method according to p. 1, wherein the patient is administered a third retroviral protease inhibitor that is effective against at least one virus strain, which is resistant to both first and second retroviral protease inhibitors.

10. The method according to p. 9, characterized in that the third retroviral protease inhibitor is N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(3-pyridylmethyl)-2(S)-N'-(tert-BUTYLCARBAMATE)piperazinil))pentanediol; N-tert-buildimage-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-hinolincarbonova)-L-asparaginyl]amino]butyl]-(4aR, 8aS)-isoquinoline-3(S)-carboxamide; (2S, 3R,4S,5S)-2,5-bis-[N-[N-[[N-methyl-N-(2-pyridinylmethyl)amino] carbonyl] valinol] amino]-3,4-dihydroxy-1,6-diphenylhexane; (2S, 3S,5S)-5-[N-[N-[N-methyl-N-[(2-isopropyl-4-thiazolyl)methyl]amino] carbonyl]valinol]amino]-2-(N-[(5-thiazolyl)methoxycarbonyl]amino]-3-hydroxy-1,6-diphenylhexane; 3S-tetrahydropyranyloxy ester, [2R-hydroxy-3-[[(4-AMINOPHENYL)sulfonyl] (2-methylpropyl)amino] -1S-(phenylmethyl)-propyl]carbamino acid; N-tert-buildimage-2-[2(R)-hydroxy-4-(phenylthio)-3(S)-[[N-[(2-methyl-3-hydroxyphenyl)carbonyl] amino]butyl]-(4aR, 8aS)-isoquinoline-3(S)-carboxamide; [4R-(4,5,6,7)]-1,3-bis[(3-AMINOPHENYL)methyl]hexahydro-5,6-dihydroxy-4,7-bis(phenyl who drank] -2S-[[(pyrrolidin-1-yl)acetyl] amino]-3,3-dimethylbutyramide; N-[2R-hydroxy-3-[(2-methylpropyl)[(1,3-benzodioxol-5-yl)sulfonyl] amino] -1S-(phenylmethyl)propyl] -2S-methyl-3-(methylsulphonyl)propanamide; [1S-[1R*(R*), 2S*]]-N-[2-hydroxy-3-[N1-(2-methylpropyl)-N1-(4-methoxybenzenesulfonyl)amino]-1-(phenylmethyl)propyl]-2-methyl-3-(methylsulphonyl)propanamide; 2S-[[(N-methylamino)acetyl]amino]-N-[2R-hydroxy-3-[[(1,3-benzodioxol-5-yl)sulfonyl](2-methylpropyl)amino]-1S(phenylmethyl)propyl] -3,3-dimethylbutyramide or (2R,3S)-3-(N-methylaminomethyl-L-tert-butylphenyl)-amino-1-(N-isoamyl-N-(tert-butylcarbamoyl))amino-4-phenyl-2-butanol

11. The method according to p. 1, characterized in that at least one virus strain resistant to this first retroviral protease inhibitor and at least one viral strain resistant to this second retroviral protease inhibitor, each of which produces retroviral protease having at least one amino acid substitution in the peptide sequence of the protease, where this substitution affects the same region of the binding site of the protease substrate, and contributes to the observed resistance to the inhibitor.

12. The method according to p. 1, characterized in that it also includes the introduction of at least one antiviral agent is a nucleoside analogue, nucleoside analog reverse transcriptase inhibitor, an antagonist of tat (tyrosinekinase) or glycosidase inhibitor.

14. The method according to p. 13, wherein the nucleoside analogue is AZY, DDI, DDC, 3TC, D4T or RMAA and the glycosidase inhibitor is castanospermine or N-butyl-1-deoxynojirimycin.

15. The method according to p. 1, wherein the mammal is a human, a monkey or a cat.

16. The method according to p. 1, wherein the retrovirus is HIV or HTLV.

17. The method according to p. 16, wherein the retrovirus is HIV-1 or HIV-2.

18. A method of treating retroviral infections in a mammal that contains (a) a choice of two inhibitors of retroviral protease, where the second selected retroviral protease inhibitor is effective against at least one retroviral strain, which is resistant to the first inhibitor of retroviral protease, and (b) the introduction of this patient an effective amount of the first and second selected inhibitors.

19. The method according to p. 18, wherein the first and second inhibitors administered to this patient attended an effective amount of both inhibitors.

20. The method according to p. 18, atheiststhe effective amount of one inhibitor.

 

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