CN1336934A - Bis-amino acid sulfonamides containing N-terminally a substituted benzyl group as HIV protease inhibitors - Google Patents

Abstract

This invention relates generally to bis-amino acid sulfonamides containing substituted benzyl amines of formula (I) or stereoisomeric forms, stereoisomeric mixtures, or pharmaceutically acceptable salt forms thereof, which are useful as HIV protease inhibitors, pharmaceutical compositions and diagnostic kits comprising the same, methods of using the same for treating viral infection or as assay standards or reagents, and intermediates and processes for making the same.

Description

Diamino acid sulfonamides containing N-terminal substituted benzyl groups as HIV protease inhibitors

  Field of Invention

The present invention generally relates to bisamino acid sulfonamide substances containing substituted benzylamines for use as HIV protease inhibitors, pharmaceutical compositions and diagnostic kits containing said substances, methods of using said substances for the treatment of viral infections or using said substances for As analytical standards or analytical reagents.

Background of the invention

Two different retroviruses, human immunodeficiency virus type 1 (HIV-1) or type 2 (HIV-2), are known to share etiology with the immunosuppressive disease acquired immunodeficiency syndrome (AIDS). ))related. HIV seropositive individuals are initially asymptomatic but typically develop AIDS-related syndrome and then AIDS. Infected individuals display severe immunosuppression, thereby debilitating their bodies and ultimately making them susceptible to fatal opportunistic infections.

AIDS is the end result of the complex life cycle of the HIV-1 or HIV-2 virus itself. At the beginning of the virion life cycle, the virion itself adheres to the host human T-4 lymphoid immune cells through the combination of the virus expansive coat surface glycoprotein and the lymphocyte CD4 glycoprotein. Once attached, the virion's glycoprotein coat is shed, penetrates the host cell's membrane, and sheds its RNA. The virion enzyme (reverse transcriptase) is involved in the process of transcription from RNA to single-stranded DNA. The viral RNA is degraded, producing another strand of DNA. This double-stranded DNA then integrates into human cell genes, which are used for viral replication.

At this point, RNA polymerase transcribes the integrated DNA into viral RNA. The viral RNA is translated into a precursor gal-pol fusion polyprotein. The polyprotein is then cleaved by HIV protease to yield mature viral proteins. Thus, HIV proteases are involved in regulating a series of cleavage reactions that lead to the maturation of virus particles into fully infectious viruses.

The typical human immune system response to kill invading virions is burdened by the virus infecting and killing the immune system's T cells. In addition, the enzyme used to make new virion particles, viral reverse transcriptase, is non-specific and can cause transcription errors, resulting in persistent changes in the glycoproteins on the surface of the virus' protective coat. The lack of specificity reduces the effectiveness of the immune system because antibodies are produced specifically against one glycoprotein and not others, reducing the number of antibodies available to fight the virus. The virus continues to replicate while the immune system continues to weaken. Finally, this free area of HIV mass control overruns the body's immune system, thereby incurring various opportunistic infections that can lead to death when antiviral agents or immunomodulators or both are not administered.

In the virus life cycle, there are at least three key points identified as possible targets for antiviral drugs, which are: (1) virions start to adhere to T-4 lymphocytes or macrophages; (2) viral RNA Transcription into viral DNA (reverse transcriptase, RT), and (3) processing of gag-pol protein by HIV protease.

These retroviral genomes encode a protease involved in the proteolysis of one or more polyprotein precursors such as pol and gag gene products. See Wellink, Arch. Virol. 981 (1988). Retroviral proteases most commonly process gag precursors to nucleoproteins and also process pol precursors to reverse transcriptase and retroviral proteases.

For assembly of infected virions, retroviral proteases are essential for correct processing of precursor polyproteins. It was shown that in vitro mutagenesis to produce protease-deficient virus results in an immature nuclear form that lacks infectivity. See Crawford et al., J. Virol. 53899 (1985); Katoh et al., Virology 145 280 (1985). Therefore, retroviral protease inhibition provides an attractive target for antiviral therapy. See Mitsuya, Nature 325 775 (1987).

A large number of compounds have been investigated as potential HIV protease inhibitors, as currently commercially available protease inhibitors and clinical trials demonstrate. One core, sulfamate has attracted intense attention. For example, PCT applications WO94/05639, WO94/04492, WO95/06030 and WO96/28464 disclose sulfonamides of the formula:

Although there are some compounds of the present invention that fall within the general disclosure of the above publications, these compounds are not specifically disclosed, suggested or claimed.

Even though some successful protease inhibitors are currently available, it has been found that HIV patients may develop resistance to one protease inhibitor. Therefore, there is a need to develop additional protease inhibitors to further combat HIV infection.

Invention Summary

It is therefore an object of the present invention to provide novel protease inhibitors.

Another object of the present invention is to provide novel methods of treating HIV infection comprising: administering to a patient in need of such treatment a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof.

Still another object of the present invention is to provide a new method for the treatment of HIV infection, comprising: combining (a) a compound of the present invention and (b) one or more therapeutic agents selected from HIV reverse transcriptase inhibitors and HIV protease inhibitors An effective amount of the combination is administered to a patient in need of such treatment.

Still another object of the present invention is to provide a pharmaceutical composition having protease inhibitory activity, which comprises: a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof.

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

Yet another object of the present invention is to provide a kit or container containing at least one compound of the present invention in an amount suitable for use in assays and assays for determining potential drug inhibition of HIV protease, HIV growth, or both. An effective amount as a standard or reagent.

I(其中R¹、R²和R³定义如下)、其立体异构体、其立体异构体混合物或其可药用盐属于有效的蛋白酶抑制剂。These and other objects of the invention, which will be more apparent in the following detailed description of the invention, are achieved by the discovery of compounds of formula (I) by the present inventors:

I (wherein R ¹ , R ² and R ³ are defined below), its stereoisomers, its stereoisomer mixtures or pharmaceutically acceptable salts thereof are among the effective protease inhibitors.

            Invention Details

IR¹为F；R²为F或H；和R³选自4-氨基苯基、3-氨基苯基、2，3-二氢苯并呋喃-5-基和1，3-苯并间二氧杂环戊烯-5-基。[1] Accordingly, in a first embodiment, the present invention provides a novel compound of formula I or a pharmaceutically acceptable salt thereof:

IR ¹ is F; R ² is F or H; and R ³ is selected from 4-aminophenyl, 3-aminophenyl, 2,3-dihydrobenzofuran-5-yl and 1,3-benzomethylene Dioxol-5-yl.

II.[3]在更优选的实施方案中，本发明提供式IIa新化合物：

IIa.[2] In a preferred embodiment, the present invention provides a new compound of formula II:

II. [3] In a more preferred embodiment, the present invention provides a new compound of formula IIa:

IIa.

[4] In still more preferred embodiments, the present invention provides novel compounds of formula IIa wherein R ³ is 3-aminophenyl.

[5] In another more preferred embodiment, the present invention provides a novel compound of formula IIa wherein R ³ is 4-aminophenyl.

[6] In another more preferred embodiment, the present invention provides that R is ^2,3 -dihydrobenzofuran-5-yl or 1,3-benzodioxol-5-yl Novel compound of formula IIa.

IIb.[7] In another more preferred embodiment, the present invention provides a novel compound of formula IIb:

IIb.

[8] In another more preferred embodiment, the present invention provides a novel compound of formula IIb wherein R ³ is 3-aminophenyl.

[9] In another still more preferred embodiment, the present invention provides a novel compound of formula IIb wherein R ³ is 4-aminophenyl.

[10] In another still more preferred embodiment, the present invention provides that R ³ is 2,3-dihydrobenzofuran-5-yl or 1,3-benzodioxol-5- The new compound of formula IIb based.

[11] In another preferred embodiment, the present invention provides a new compound of formula IIc: IIc.

[12] In another more preferred embodiment, the present invention provides a novel compound of formula IIc wherein R ³ is 3-aminophenyl.

[13] In another yet more preferred embodiment, the present invention provides novel compounds of formula IIc wherein R ³ is 4-aminophenyl.

[14] In another still more preferred embodiment, the present invention provides that R ³ is 2,3-dihydrobenzofuran-5-yl or 1,3-benzodioxol-5- The new compound of formula IIc based.

III.[15] In another preferred embodiment, the present invention provides a new compound of formula III:

III.

[16] In another more preferred embodiment, the present invention provides a new compound of formula IIIa: IIIa.

IV.[17] In another more preferred embodiment, the present invention provides a novel compound of formula IV:

IV.

IVa.[18] In another more preferred embodiment, the present invention provides a new compound of formula IVa:

IVa.

In another embodiment, the present invention provides novel pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a novel method of treating HIV infection comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to a patient in need of such treatment.

In another embodiment, the present invention provides a novel method of treating HIV infection comprising: administering the combination of (a) and (b) below in a therapeutically effective amount to a patient in need of said treatment:

(a) a compound of formula I; and,

(b) At least one compound selected from HIV reverse transcriptase inhibitors and HIV protease inhibitors.

In another preferred embodiment, the reverse transcriptase inhibitor is selected from azidodeoxythymidine (A2T), dideoxycytidine (ddC), dideoxyinosine (ddI), d4T, 3TC, delavirdine, efavirenz , nevirapine, Ro18,893, trovirdine, MKC-442, HBY097, ACT, UC-781, UC-782, RD4-2025 and MEN10979; protease inhibitors selected from saquinavir, ritonavir, indinavir , amprenavir, nelfinavir, palinavir, BMS-232623, GS3333, KNI-413, KNI-272, LG-71350, CGP-61755, PD173606, PD177298, PD178390, PD178392, U-140690 and ABT- 378.

In a more preferred embodiment, the reverse transcriptase inhibitor is selected from AZT, efavirenz and 3TC; the protease inhibitor is selected from saquinavir, ritonavir, nelfinavir and indinavir.

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

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

In another preferred embodiment, component (b) is an HIV reverse transcriptase inhibitor and HIV protease inhibitor.

In another preferred embodiment, component (b) is two different HIV reverse transcriptase inhibitors.

In another embodiment, the present invention provides a pharmaceutical composition for the treatment of HIV infection comprising a therapeutically effective amount of:

(a) a compound of formula I; and,

(b) at least one compound selected from HIV reverse transcriptase inhibitors and HIV protease inhibitors placed in one or more sterile containers.

In another embodiment, the present invention provides a novel method of inhibiting the presence of HIV in a sample of bodily fluid comprising: treating the bodily fluid with an effective amount of a compound of formula I.

In a ninth embodiment, the present invention provides novel kits or containers containing a compound of formula I in an amount for use in assays and assays for determining inhibition of HIV protease, HIV growth, or both by potential drugs An effective amount as a standard or reagent.

Definition

In this application, the following terms and expressions have the indicated meanings. It should be understood that the compounds of the present invention contain asymmetrically substituted carbon atoms which can be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, for example by resolution of racemic forms or by synthesis from optically active starting materials. Unless a specific stereochemical or isomeric form is specifically indicated, said compounds refer to all chiral, diastereomeric, racemic and all geometric isomeric forms.

It is contemplated that the method of the invention may be carried out at least on a gram scale, a kilogram scale, a kilogram scale or an industrial scale. A "gram scale" as used herein is preferably a scale in which at least one starting material is 10 grams or more, more preferably at least 50 grams or more, even more preferably at least 100 grams or more. As used herein, "kilogram scale" refers to a scale in which at least one starting material is used in an amount exceeding 1 kg. "Industrial scale" as used herein refers to a scale that is not laboratory scale and that is sufficient to provide a product sufficient for clinical trials or distribution to consumers.

In the compounds of the present invention, all isotopic forms of atoms are also included. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen atoms include deuterium and tritium; isotopes of carbon atoms include C-13 and C-14.

As used herein, "HIV reverse transcriptase inhibitor" refers to nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT). Examples of nucleoside RT inhibitors include, but are not limited to, azideoxythymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddI), d4T, and 3TC. Examples of non-nucleoside RT inhibitors include, but are not limited to, delavirdine (Pharmacia and Upjohn, U90152S), efavirenz (Dupont), nevirapine (Boehringer Ingelheim), Ro18,893 (Roche), trovirdine (Lilly), MKC-442 (Triangle) , HBY097 (Hoechst), HBY1293 (Hoechst), ACT (Korean Research Institute), UC-781 (Rega Institute), UC-782 (Regainstitute), RD4-2025 (Tosoh Co. Ltd) and MEN10979 (Menarini Farmaceutici).

Herein, "HIV protease inhibitor" refers to a compound that inhibits HIV protease, examples of which include but are not limited to saquinavir (Roche, Ro31-8959), ritonavir (Abbott, ABT-538), indinavir Wei (Merck, MK-639), amprenavir (Vertex/GlaxoWellcome), nelfinavir (Agouron, AG1343), palinavir (Boehringer Ingelheim), BMS-232623 (Bristol-Myers Squibb), GS3333 (GileadSciences), KNI -413 (Japan Energy), KNI-272 (Japan Energy), LG-71350 (LG Chemical), CGP-61755 (Ciba-Geigy), PD173606 (Parke Davis), PD177298 (Park Davis), PD178390 (Parke Davis), PD178392 (Parke Davis), tipranavir (Pharmacia and Upjohn U-140690) and DMP-450 (Dupont) and ABT-378.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, wherein the parent compound is modified into acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of base residues such as amines; alkali or organic salts of acid residues such as carboxylic acids; and similar salts. Pharmaceutically acceptable salts include conventional non-toxic or quaternary ammonium salts of the parent compound prepared, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include: those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; and from organic acids such as acetic, propionic, succinic, glycolic, hard Fatty acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxy Salts prepared from benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid and isethionic acid.

The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing basic or acidic residues by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a chemical equivalent of the appropriate base or acid in water or in an organic solvent, or in a mixture of both, which reaction is usually preferably In water-insoluble coals such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of suitable salts can be found in Remington Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is incorporated herein by reference.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment, and do not cause undue toxicity, Irritation, allergic reaction, or other problem or complication with a reasonable benefit/risk ratio.

"Prodrug" is meant to include any covalently linked carrier capable of releasing the active parent drug of formula (I) or other structural formulas of the present invention in vivo when such prodrug is administered to a mammalian patient or compound. Prodrugs of compounds of the present invention, such as compounds of formula (I), are prepared by modifying functional groups in the compound in such a way that the modified product can be cleaved into the parent compound by routine manipulation or in vivo. Prodrugs include compounds of the invention in which the hydroxy or amino group is bonded to any group that is capable of cleavage to a free hydroxy or free amino group, respectively, when the prodrug is administered to a mammal. Examples of prodrugs include, but are not limited to, acetic, formic and benzoic acid derivatives of alcohol and amine functional groups in the compounds of the present invention, and the like.

"Stable compound" and "stable structure" refer to a compound that is sufficiently stable to survive isolation to a useful degree of purity from a reaction mixture and to withstand formulation into an effective therapeutic agent. Only stable compounds are contemplated by the present invention.

"Substituted" means that one or more hydrogen atoms on the atom specified in the expression "substituted" is used to replace with a group selected from the specified group, provided that the normal valency of the specified atom is not exceeded, and the substitution results in stable compound. When the substituent is keto (ie =0), then 2 hydrogen atoms are replaced.

"Therapeutically effective amount" refers to the amount of the compound of the present invention or the amount of the compound administered in combination that can effectively inhibit HIV infection in the host or treat the symptoms of HIV infection in the host. The combination of compounds is preferably a synergistic combination. Synergy, as in Chou and Talalay, Adv. As described in Enzyme Regul. 22:27-55 (1984), synergy occurs when the effect of compounds administered in combination (here, inhibition of HIV replication) is greater than the sum of the effects of these compounds administered individually as single agents. Typically, synergistic effects are most pronounced at suboptimal concentrations of the compounds. A synergistic effect can be manifested as lower cytotoxicity, higher antiviral effect, or other beneficial effects of the combination compared to the individual components.

A certain diastereoisomer of a compound of formula I may exhibit superior activity over the other isomer. When desired, the racemic compound can be separated by HPLC using a chiral column or by resolution using a resolution reagent such as camphonic chloride (see: Thomas J. Tucker et al., J.Med.Chem 1994, 37 , 2437-2444). The chiral compound of formula I can also be directly synthesized by using a chiral catalyst or a chiral ligand (for example, see: Mark A. Huffman et al., J. Org. Chem. 1995, 60, 1590-1594).

Other features of the present invention will be more apparent through the description of the following embodiments, which are only for illustrating the present invention, but not for limiting the present invention.

Example

The abbreviations used in these examples are defined as follows: "°C" is degrees Celsius, "d" is doublet, "dd" is double doublet, "eq" is equivalent weight, "g" is gram, "mg" is milligram, "mL" is milliliters, "H" is hydrogen or hydrogen atoms, "hr" is hours, "m" is multimodal, "M" is moles, "min" is minutes, "MHz" is megahertz, "MS" is Mass spectrum, "nmr" or "NMR" is nuclear magnetic resonance spectrum, "t" is triplet, and "TLC" is thin layer chromatography.

Example 1

1B Add NaOH (50% aqueous solution, 44.5g) to N-[3(S)-[N,N-bis(phenylmethyl)amino]-2(R)-hydroxy-4-phenylbutyl]- N-Isobutylamine·oxalate 1A (127.6 g, 251 mmol) in a mixture of toluene (1 L), water (500 mL) and dichloromethane (400 mL). After stirring for 10 minutes, the reaction mixture was extracted with toluene. The combined organic layers were washed with brine, dried (magnesium sulfate) and the solvent was removed under reduced pressure. The residue was dissolved in THF (1 L), cooled to 0°C, and treated with triethylamine (28.15 g, 278 mmol) and di-tert-butyldicarbonate (55.23 g, 253 mmol). The solution was warmed to room temperature and stirred overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (1 L), washed successively with water, 5% citric acid, water, saturated sodium bicarbonate and brine, and dried (magnesium sulfate). Removal of solvent under reduced pressure gave carbamate IB which was used without further purification. CIMS ( _NH3 ) m/z: 517 (M+H ⁺ , 100%).

1C Palladium hydroxide on carbon (20%, 10 g) was added to a solution of crude 1B (ca. 251 mmol) in methanol (500 mL). The suspension was placed in a Parr bottle and flushed with hydrogen (55 psi). After shaking overnight, the reaction mixture was filtered through celite and the solvent was removed under reduced pressure. The resulting solid was recrystallized (ethyl acetate/hexanes) to afford amine 1C as a white solid (56.6 g, 67% yield (2 steps)): CIMS (NH ₃ ) m/z: 337 (M+H ⁺ , 100%).

1D At 0°C, N-hydroxybenzotriazole (38.6g, 285mmol) and EDC (35.7g, 186mmol) were added to N-carbonylbenzyloxy-L-tert-leucine (47.5g, 179mmol) DMF (250mL) solution. After stirring for 1.5 hours, the solution was added to a suspension of 1C (56.6 g, 167 mmol) and 4-methylmorpholine (52.9 g, 521 mmol) in DMF (200 mL). The reaction mixture was warmed to room temperature. After stirring overnight, N,N-dimethylethylenediamine (4 mL) was added, the solution was stirred for 1.5 hours, and the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (1 L), washed sequentially with water, 5% citric acid, water, saturated sodium bicarbonate and brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure to afford 1D (97.5 g, 100%) which was used without further purification. CIMS ( _NH3 ) m/z: 584 (M+H ⁺ , 100%).

1E Palladium hydroxide on carbon (20%, 10 g) was added to a solution of 1D (97.5 g, 167 mmol) in methanol (300 mL). The suspension was placed in a parr bottle and flushed with hydrogen (55 psi). After shaking overnight, the reaction mixture was filtered through celite and the solvent was removed under reduced pressure. The resulting solid was recrystallized (ethyl acetate/hexane) to give white solid 1E (72.8 g, 97%): CIMS (NH ₃ ) m/z: 450 (M+H ⁺ , 100%).

1F KHCO ₃ (27.7 g, 276 mmol) and chloroacetyl chloride (12.4 g, 111 mmol) were added to a solution of amine 1E (43.8 g, 97.6 mmol) in ethyl acetate (400 mL) and water (270 mL). After stirring for 3 hours, ethyl acetate (1 L) was added and the resulting solution was washed successively with water, 5% citric acid, water, saturated sodium bicarbonate and brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure to give white solid 1F (51.0 g, 99%): CIMS (NH ₃ ) m/z: 526 (M+H ⁺ , 100%).

1G A solution of 4N HCl in dioxane (80 mL, 320 mmol) was added to a solution of 1F (33.8 g, 64.2 mmol) in ethyl acetate (600 mL). The reaction mixture was stirred for 6 hours. The solvent was removed under reduced pressure and the resulting solid was triturated with cold diethyl ether to give 1G hydrochloride (28.75 g, 97%): CIMS (NH ₃ ) m/z: 426 (M+H ⁺ , 100%).

1H Potassium carbonate (56.7 g, 411 mmol) and 4-nitrobenzenesulfonyl chloride (16.9 g, 76.0 mmol) were added to a solution of salt 1G (32.0 g, 69.2 mmol) in THF (350 mL) and water (450 mL). After stirring for 4 hours, water was added and the suspension was extracted with ethyl acetate. The organic layers were combined, washed sequentially with brine, 5% citric acid, water, saturated sodium bicarbonate, and brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure and the resulting solid was recrystallized (ethyl acetate/hexanes) to give Sulfa 1H as a white solid (35.8 g, 85%). CIMS ( _NH3 ) m/z: 611 (M+H ⁺ , 100%).

1I 3-Fluorobenzylamine (20.0 g, 160 mmol) was added to a solution of chloride 1H (16.0 g, 26.1 mmol) in THF (200 mL), and the reaction mixture was refluxed overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed successively with water and brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure and the residue was chromatographed (silica gel, 4% methanol/dichloromethane) to afford amine 1I as a white solid (16.3 g, 89%). CIMS (NH ₃ ) m/z: 700 (M+H ⁺ , 100%).

1 Add palladium hydroxide on carbon (20%, 1.5 g) to a solution of 1I (14.6 g, 20.8 mmol) in methanol (500 mL). Hydrogen was flushed into the reaction mixture. After stirring for 3 hours, the mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (13.2 g, 95%). 1 N HCl in ether (37 mL, 37 mmol) was added to a solution of the free base (11.68 g, 17.4 mmol) in ether (300 mL) and ethyl acetate (100 mL). The resulting mixture was stirred for 15 minutes and filtered to give white solid dihydrochloride 1 (12.5 g, 96%): CIMS (NH ₃ ) m/z: 670 (M+H ⁺ , 100%).

Example 2

2A 3,5-Difluorobenzylamine (25.0 g, 174 mmol) was added to a solution of chloride 1H (16.0 g, 26.1 mmol) in THF (200 mL), and the reaction mixture was refluxed overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed successively with water and brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure and the residue chromatographed (silica gel, 4% methanol/chloroform) to afford amine 2A as a white solid (15.2 g, 81%). CIMS (NH ₃ ) m/z: 718 (M+H ⁺ , 100%).

2 Palladium hydroxide/carbon (20%, 1.5 g) was added to a solution of 2A (15.2 g, 21.2 mmol) in methanol (500 mL), and the reaction mixture was bubbled with hydrogen. After stirring for 4 hours, the reaction mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (10.3 g, 71%). 1 N HCl in ether (32 mL, 32 mmol) was added to a solution of the free base in ether (300 mL) and ethyl acetate (100 mL). The resulting suspension was stirred for 15 minutes and filtered to give dihydrochloride 2 as a white solid: CIMS (NH ₃ ) m/z: 688 (M+H ⁺ , 100%).

Example 3

3A 2,5-Difluorobenzylamine (1.2 g, 8.5 mmol) was added to a solution of chloride 1H (300 mg, 0.49 mmol) in THF (4 mL), and the reaction mixture was refluxed for 4 hours. The reaction mixture was diluted with ethyl acetate, washed with water and brine, dried (magnesium sulfate). The solvent was removed under reduced pressure and the residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford amine 3A as a white solid (270 mg, 77%). CIMS (NH ₃ ) m/z: 718 (M+H ⁺ , 100%).

3 Palladium hydroxide on carbon (20%, 50 mg) was added to a solution of 3A (260 mg, 0.36 mmol) in methanol (25 mL). Hydrogen was bubbled into the reaction mixture. After stirring for 1 hour, the mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (226 mg, 91%). 4N HCl in dioxane (0.2 mL, 0.8 mmol) was added to a solution of the free base in ether (30 mL) and ethyl acetate (10 mL). The resulting suspension was stirred for 15 minutes and filtered to give dihydrochloride 3 as a white solid: CIMS (NH ₃ ) m/z: 688 (M+H ⁺ , 100%).

Example 4

4A 2,6-Difluorobenzylamine (1.2 g, 8.5 mmol) was added to a solution of chloride 1H (300 mg, 0.49 mmol) in THF (4 mL). The reaction mixture was refluxed for 4 hours. The reaction mixture was then diluted with ethyl acetate, washed with water and brine, dried (magnesium sulfate). The solvent was removed under reduced pressure and the residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford amine 4A as a white solid (306 mg, 87%). CIMS (NH ₃ ) m/z: 718 (M+H ⁺ , 100%).

4 Add palladium hydroxide on carbon (20%, 50 mg) to a solution of 4A (295 mg, 0.41 mmol) in methanol (25 mL). Hydrogen was bubbled into the reaction mixture. After stirring for 1 hour, the mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (228 mg, 81%). A solution of 4N HCl in dioxane (0.2 mL, 0.8 mmol) was added to a solution of the free base in diethyl ether (30 mL) and ethyl acetate (10 mL). The resulting suspension was stirred for 15 minutes and filtered to give dihydrochloride 4 as a white solid: CIMS (NH ₃ ) m/z: 688 (M+H ⁺ , 100%).

Example 5

5A Potassium carbonate (51.4g, 370mmol) and 3-nitrobenzenesulfonyl chloride (15.14g, 68.3mmol) and 3-nitrobenzenesulfonyl chloride (15.14g, 68.3mmol) were added to salt 1G (28.8g, 62.1mmol ) in THF (300ml) and water (400mL). After stirring for 4 hours, water was added and the suspension was extracted with ethyl acetate. The organic layers were combined, washed with brine, 5% citric acid, water, saturated sodium bicarbonate, brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The resulting solid was triturated with ethyl acetate and hexanes to afford sulfonamide 5A as a white solid (32.1 g, 85%). CIMS ( _NH3 ) m/z: 611 (M+H ⁺ , 100%).

5B 3-Fluorobenzylamine (17.0 g, 135 mmol) was added to a solution of chloride 5A (16.0 g, 26.1 mmol) in THF (200 mL). The reaction mixture was refluxed overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed with water and brine, dried (magnesium sulfate). The solvent was removed under reduced pressure and the residue was chromatographed (silica gel, 4% methanol/dichloromethane) to afford amine 5B as a white solid (16.0 g, 87%). CIMS (NH ₃ ) m/z: 700 (M+H ⁺ , 100%).

5 Palladium hydroxide on carbon (20%, 1.25g) was added to a solution of 5B (12.0g, 17.22mmol) in methanol (400ml). Hydrogen was bubbled into the reaction mixture. After stirring for 3 hours, the mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (11.2 g, 97%). 1 N HCl in ether (36 mL, 36 mmol) was added to a solution of the free base in ether (400 mL) and ethyl acetate (75 mL). The resulting suspension was stirred for 15 minutes and filtered to give dihydrochloride 5 as a white solid: CIMS (NH ₃ ) m/z: 670 (M+H ⁺ , 100%).

Example 6

6A 3,5-Difluorobenzylamine (25.0 g, 174 mmol) was added to a solution of chloride 5A (16.0 g, 26.1 mmol) in THF (200 mL). The reaction mixture was stirred for 2 hours and refluxed overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed with water and brine, dried (magnesium sulfate). The solvent was removed under reduced pressure and the residue was chromatographed (silica gel, 3.5% methanol/dichloromethane) to afford amine 6A as a white solid (15.6 g, 83%). CIMS (NH ₃ ) m/z: 718 (M+H ⁺ , 100%).

6 Palladium hydroxide on carbon (20%, 1.5 g) was added to a solution of 6A (14.4 g, 20.0 mmol) in methanol (500 mL). Hydrogen was bubbled into the reaction mixture. After stirring for 4 hours, the mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (12.5 g, 91%). 1N HCl in diethyl ether (31 mL, 31 mmol) was added to a solution of the free base (9.57 g, 13.9 mmol) in diethyl ether (300 mL). The resulting suspension was stirred for 20 minutes and filtered to give dihydrochloride 6 as a white solid (9.9 g, 94%): CIMS (NH ₃ ) m/z: 688 (M+H ⁺ , 100%).

Example 7

7 Add 3-fluorobenzylamine (550mg, 4.4mmol) to N-[2R-hydroxy-3-[[(2,3-dihydro-2,3-dihydrobenzofuran-5-yl)sulfonyl ](2-methylpropyl)amino]-1S-(phenylmethyl)propyl]-2S-[(chloroacetyl)amino]-3,3-dimethylbutanamide 7A (100mg, 0.16mmol ) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was then diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (91 mg, 79%). A solution of 4N HCl in dioxane (0.05 mL, 0.20 mmol) was added to a solution of the free base (91 mg, 0.13 mmol) in diethyl ether (25 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 7 as a white solid (72 mg, 65%): CIMS (NH ₃ ) m/z: 697 (M+H ⁺ , 100%).

Example 8

8 3,5-Difluorobenzylamine (605 mg, 4.2 mmol) was added to a solution of 7A (100 mg, 0.16 mmol) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (83 mg, 70%). A solution of 4N HCl in dioxane (0.05 mL, 0.20 mmol) was added to a solution of the free base (83 mg, 0.11 mmol) in ether (25 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 8 as a white solid (65 mg, 75%): elemental analysis (C ₃₇ H ₄₉ N ₄ O ₆ S ₁ F _{2C 11} ): Calculated: C, 59.15; H, 6.45; N, 7.47. Calculated: C, 58.90; H 6.51; N, 7.2l.

Example 9

9 Add 2,5-difluorobenzylamine (610 mg, 4.3 mmol) to a solution of 7A (100 mg, 0.16 mmol) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (110 mg, 93%). A solution of 4N HCl in dioxane (0.05 mL, 0.20 mmol) was added to a solution of the free base (110 mg, 0.15 mmol) in ether (25 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 9 as a white solid (76 mg, 66%): CIMS (NH ₃ ) m/z: 715 (M+H ⁺ , 100%).

Example 10

10 2,6-Difluorobenzylamine (600 mg, 4.2 mmol) was added to a solution of 7A (100 mg, 0.16 mmol) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (103 mg, 87%). A solution of 4N HCl in dioxane (0.05 mL, 0.20 mmol) was added to a solution of the free base (103 mg, 0.14 mmol) in ether (25 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 10 as a white solid (82 mg, 76%): CIMS (NH ₃ ) m/z: 715 (M+H ⁺ , 100%).

Example 11

11 Add 3-fluorobenzylamine (1.1g, 8.8mmol) to N-[2R-hydroxyl-3-[[(1,3-benzodioxol-5-yl)sulfonyl]( 2-methylpropyl)amino]-1S-(phenylmethyl)propyl]-2S-[(chloroacetyl)amino]-3,3-dimethylbutanamide 11A (750mg, 1.23mmol) THF (2mL) solution. The reaction mixture was stirred overnight. The reaction mixture was then diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (532 mg, 62%). A solution of 4N HCl in dioxane (0.22 mL, 0.88 mmol) was added to a solution of the free base (532 mg, 0.76 mmol) in ether (100 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 11 as a white solid (417 mg, 75%): CIMS (NH ₃ ) m/z: 699 (M+H ⁺ , 100%).

Example 12

12 3,5-Difluorobenzylamine (2.42 g, 16.9 mmol) was added to a solution of 11A (2.0 g, 3.27 mmol) in THF (7 mL). The reaction mixture was refluxed for 5 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (2.26 g, 97%). A solution of 4N HCl in dioxane (0.66 mL, 2.67 mmol) was added to a solution of the free base (1.8 g, 2.51 mmol) in diethyl ether (100 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure, the resulting solid was triturated with diethyl ether, and filtered to obtain a white solid benzylamine salt 12 (1.65 mg, 87%): CIMS (NH ₃ ) m/z: 717 (M+H ⁺ , 100%). Elemental analysis ( _{C36H47N4O7S1F2Cl1} ) _{: Calculated} : C _, 57.40; _{H ,} 6.17; _N , 7.45. Calculated: C, 57.25; H 6.25; N, 7.24.

Example 13

13 2,5-Difluorobenzylamine (1.2 g, 8.5 mmol) was added to a solution of 11A (750 mg, 1.23 mmol) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (702 mg, 80%). A solution of 4N HCl in dioxane (0.3 mL, 1.2 mmol) was added to a solution of the free base (702 mg, 0.98 mmol) in diethyl ether (100 mL) and ethyl acetate (25 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 13 as a white solid (586 mg, 79%): CIMS (NH ₃ ) m/z: 717 (M+H ⁺ , 100%).

Example 14

14 2,6-Difluorobenzylamine (1.2 g, 8.3 mmol) was added to a solution of 11A (750 mg, 1.23 mmol) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (717 mg, 81%). A solution of 4N HCl in dioxane (0.3 mL, 1.2 mmol) was added to a solution of the free base (717 mg, 1.00 mmol) in diethyl ether (100 mL). After stirring for 10 minutes, the solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether and filtered to give the hydrochloride 14 as a white solid (663 mg, 88%): CIMS (NH ₃ ) m/z: 717 (M+H ⁺ , 100%).

Example 15

15 3,4-Difluorobenzylamine (1.2 g, 8.3 mmol) was added to a solution of 11A (750 mg, 1.23 mmol) in THF (2 mL). The reaction mixture was refluxed for 3 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (760 mg, 86%). A solution of 4N HCl in dioxane (0.3 mL, 1.2 mmol) was added to a solution of the free base (760 mg, 1.06 mmol) in ether (100 mL). After stirring for 10 minutes, the resulting solid was filtered to give hydrochloride 15 as a white solid (730 mg, 91%): CIMS (NH ₃ ) m/z: 717 (M+H ⁺ , 100%).

Example 16

16 2,4-Difluorobenzylamine (1.2 g, 8.3 mmol) was added to a solution of 11A (750 mg, 1.23 mmol) in THF (2 mL). The reaction mixture was refluxed for 6 hours. The reaction mixture was diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (693 mg, 79%). A solution of 4N HCl in dioxane (0.3 mL, 1.2 mmol) was added to a solution of the free base (693 mg, 0.97 mmol) in ether (100 mL). After stirring for 10 minutes, the resulting solid was filtered to give hydrochloride 16 as a white solid (638 mg, 88%): CIMS (NH ₃ ) m/z: 717 (M+H ⁺ , 100%).

Example 17

17 Add 4-fluorobenzylamine (1.0 g, 8.0 mmol) to a solution of 11A (500 mg, 0.82 mmol) in THF (2 mL). The reaction mixture was stirred overnight. The reaction mixture was then diluted with ethyl acetate, washed with water (4x), brine, and dried (magnesium sulfate). The solvent was removed under reduced pressure. The residue was chromatographed (silica gel, 5% methanol/dichloromethane) to afford the amine as a white solid (470 mg, 82%). A solution of 4N HCl in dioxane (0.18 mL, 0.7 mmol) was added to a solution of the free base (400 mg, 0.57 mmol) in diethyl ether (30 mL). After stirring for 15 minutes, the resulting solid was filtered to give hydrochloride 17 as a white solid (413 mg, 98%): CIMS (NH ₃ ) m/z: 699 (M+H ⁺ , 100%).

Application

Compounds of formula I have HIV protease inhibitory activity and can therefore be used as antiviral agents for the treatment of HIV infection and related diseases. Also because the compound of formula I has HIV protease inhibitory activity, it can effectively inhibit the growth of HIV. The ability of the compounds of the invention to inhibit viral growth and infectivity is demonstrated in standard assays for viral growth and infectivity, for example using the test methods described below.

The compounds of formula I according to the invention can also be used for inhibiting HIV in vitro in HIV-containing samples or in samples which may be exposed to HIV in vitro. Accordingly, the compounds of the present invention are useful for inhibiting HIV in bodily fluid samples containing or suspected of containing or potentially exposed to HIV.

The compounds provided herein can also be used as standard or reference compounds in tests or assays for determining the ability of agents to inhibit viral clone replication and/or HIV protease, eg, in pharmaceutical research programs. Accordingly, the compounds of the invention can be used as control or reference compounds for these assays and as quality control standards. When used as such a standard or reference compound, the compound of the present invention may be provided in the form of a commercially available kit or container.

Since the compounds of the invention have been shown to be specific for HIV protease, the compounds of the invention can also be used as diagnostic reagents in diagnostic assays for the detection of HIV protease. Thus, in assays such as those described herein, inhibition of protease activity by compounds of the invention indicates the presence of HIV protease and HIV virus.

In the symbols described here, "μg" means microgram, "mg" means milligram, "g" means gram, "μL" means microliter, "L" means liter, "nM" means nanomole, "μM" means Micromole, "mM" means millimole, "M" means mole, and "nM" means nanomole. "Sigma" stands for Sigma-Aldrich Company of St. Louis, MO.

HIV RNA Analysis DNA Plasmids and In Vitro RNA Transcription:

Following Erickson-Viitanen et al. AIDS Research and Human Retroviruses (AIDS Research and Human Retroviruses) 1989,5,577 disclosed method, prepare the pDAB72 plasmid containing the BH10 gag and pol sequence (bp113-1816) cloned into PTZ 19R. This plasmid was linearized with Bam HI and RNA transcripts were generated in vitro using the Riboprobe Gemini System II kit (Promega) containing T7 RNA polymerase. Synthetic RNA was purified by treatment with DNase-free RNase (Promega), phenol-chloroform extraction and ethanol precipitation. Dissolve RNA transcriptase in water and store at -70°C. RNA concentration was determined from A260. Probe:

Using the biotin-phosphoramidite reagent described in Cocuzza, Tet. City, CA) DNA synthesizer after synthesis of biotinylated capture probes, the biotinylated capture probes were purified by HPLC. The gag biotinylated capture probe (5-biotin-CTAGCTCCCTGCTTGCCCATACTA 3′) complements nucleotides 889-912 of HXB2; the pol biotinylated capture probe (5′-biotin-CCCTATTCATTTTTGGTTTCCAT3′) complements In nucleotides 2374-2395 of HXB2; alkaline phosphatase-binding oligonucleotides used as reporter probes were prepared by Syngene (San Diego, CA). The pol reporter probe (5'-CTGTCTTACTTTGATAAAACCTC3') complements nucleotides 2403-2425 of HXB2. The gal reporter probe (5'CCCAGTATTTGTCTACAGCCTTCT3') complements nucleotides 950-973 of HXB2. All nucleotide positions exist in the GenBank gene sequence database, obtained by the Gnentics Computer Group Sequence Analysis Software Package (Devereau Nucleic Acids Research 1984, 12, 387). The reporter probe was prepared as: 0.5 μM stock solution (in 2×SSC (0.3M sodium chloride, 0.03M sodium citrate), 0.05M Tris pH8.8, 1 mg/mL BSA). The biotin capture probe was prepared as: 100 μM stock solution in water. Streptavidin-coated dishes:

Streptavidin-coated dishes were obtained from Du Pont Biotechnology Systems (Boston, MA). Cell and virus stocks:

MT-2 and MT-4 cells were cultured in RPMI 1640 supplemented with 5% (for MT-2 cells) or 10% (for MT-4 cells) of fetal calf serum (FCS), 2 mM L- Glutamine and 50 μg/mL gentamicin, both from Gibco. In the same medium, HIV-1RF was propagated in MT-4 cells. Approximately 10 days after acute infection of MT-4 cells, virus stocks were prepared and stored in aliquots at -70°C. The infectious titer of HIV-1(RF) stock solution was 1-3×10 ⁷ PFU (plaque forming unit)/mL by MT-2 cell surface plaque assay (described below). Each aliquot of virus stock used for infection was thawed only once.

To evaluate the antiviral effect, the cells to be infected were cultured again the day before infection. On the day of infection, resuspend cells in RPMI 1640 with 5% FCS at a concentration of 5 x ¹⁰⁵ cells/mL for bulk infections and 2 x ¹⁰⁶ cells/ml for infections in microtiter plates. Virus was resuspended in Dulbecco's modified Eagle's medium containing 5% FCS. The virus was added and the culture continued for 3 days at 37°C. Analysis of HIV RNA:

Cell lysates or purified RNA in 3M or 5M GED were mixed with 5M GED and capture probes to give a final concentration of 3M guanidinium isothiocyanate and 30nM biotin oligonucleotide. Hybridization was performed in sealed U-bottom 96-well tissue culture dishes (Nunc or Costar) for 16-20 hours at 37°C. The RNA hybridization reaction was diluted three-fold with deionized water to a final concentration of 1 M guanidine isothiocyanate, and aliquots (150 μL) were transferred to streptavidin-coated microtiter plates. Capture probes and capture probe-RNA hybridized to immobilized streptavidin were allowed to bind for 2 hours at room temperature, then washed with DuPont ELISA wash buffer (phosphate buffered saline (PBS), 0.05% Tween 20) Plate the titer 6 times. Add 120 μl hybridization cocktail containing 4 × SSC, 0.66% Triton × 100, 6.66% deionized formamide, 1 mg/mL BSA and 5 nM reporter probe to the washed streptavidin-coated wells to make the reporter probe The needle hybridizes a second time to the capture probe immobilization complex and hybridizes to the target RNA. After hybridization for 1 hour at 37°C, the plate was washed again 6 times. Add 100 μL of 0.2 mM 4-methyl-4-methyl in buffer delta (2.5 M diethanolamine pH 8.9 (JBL Scientific), 10 mM magnesium chloride, 5 mM zinc acetate dihydrate, and 5 mM N-hydroxyethyl-ethylenediamine-triacetic acid) umbelliferyl phosphate (MUBP, JBLScientific), detection of immobilized alkaline phosphatase activity. The plate was incubated at 37°C and applied to a microtiter plate fluorometer (Dynateck) excited at 365nM to measure fluorescence at 450nM. Evaluation of microtiter plate-based compounds in HIV-1-infected MT-2 cells:

Dissolve the compound to be evaluated in DMSO, dilute to 2 times the highest concentration to be tested in the culture medium, and make the maximum concentration of DMSO reach 2%. Compounds were then serially diluted 3-fold in culture medium directly in U-bottom microtiter plates (Nunc). After compound dilution, MT-2 cells (50 μl) were added to a final concentration of 5×10 ⁵ /ml (1×10 ⁵ /well). Cells were incubated with compounds for 30 minutes at 37°C in a carbon dioxide incubator. To assess antiviral activity, appropriately diluted HIV-1 (RF) virus stocks (50 [mu]L) were added to wells containing cells and diluted test compounds. The final volume of each well was 200 μL. In each titer plate, 8 wells were left uninfected, that is, 50 μL of medium was added to replace the virus, and no antiviral compound was added to the 8 infected wells. To assess compound toxicity, parallel plates were grown in the absence of virus infection.

After 3 days of incubation at 37°C in a humid chamber in a carbon dioxide incubator, remove all material from the HIV-infected titer plates, leaving only 25 μL of medium/well. Add 37 μL of 5M GED containing biotinylated capture probes to the fixed cells, leaving a certain amount of culture medium in each well, so that the final concentration of GED is 3M, and the final concentration of capture probes is 30nM. In the same microtiter well used for virus culture, seal the microtiter plate with a microtiter plate sealer to allow the capture probe to hybridize with HIV RNA in the cell lysate, and then incubate in a 37°C incubator for 16-20 Hour. Distilled water was then added to each well to dilute the hybridization reaction 3-fold, and 150 µL of this diluted mixture was transferred to a streptavidin-coated microtiter plate. HIV RNA was quantified as described above. A standard curve was prepared by spiking known amounts of pDNA 72 in vitro RNA transcripts into wells containing lysates of uninfected cells and applied to individual microtiter plates in order to determine the amount of viral RNA prepared during infection.

To standardize the virus inoculum used to evaluate the antiviral activity of compounds, those dilutions of virus that resulted in dideoxycytidine (ddC) _IC90 values (concentration of compound required to reduce HIV RNA levels by 90%) of 0.2 μg/mL were selected liquid. _IC90 values for other antiviral compounds, including those stronger or weaker than dideoxycytidine, were reproducible when the method was performed using several HIV-1 (RF) stock solutions. Virus concentrations corresponded to ~3 x ¹⁰⁵ PFU (determined by plaque assay in MT-2 cells) per assay well, typically yielding approximately 75% of the maximum viral RNA content achievable upon inoculation with any virus. For HIV RNA analysis, the percent reduction in net signal from RNA analysis relative to the net signal produced by infected untreated cells (infected cell sample signal minus uninfected cell sample signal) on the surface of the same culture plate (average 8 wells), Determine _IC90 values. The validity of each infection and RNA analysis was judged according to three criteria. It should be: viral infection can result in an RNA assay signal equal to or greater than that produced by in vitro RNA transcription of 2 ng pDNA 72; the _IC90 of dideoxycytidine (ddC) determined by each assay should be in the range of 0.1-0.3 μg/ml; Inhibitors should produce stable levels of virus RNA in plates less than 10% of the levels achieved in uninhibited infection. A compound was considered potent if it was found to have an _IC90 of less than 1 μM.

For viral efficacy testing, Perkin Elmer/Cetus Propettes were used for all microtiter plate runs after initially adding 2X concentrated compound solutions to a row of wells. Dosage and Form

As a treatment for viral infection, the antiviral compound of the present invention can be administered by any means that can bring the active ingredient into contact with its site of action, ie viral protease, in mammals. The compounds, whether as individual therapeutic agents or in combination, may be administered by any conventional means of administration of medicines. They can be administered alone, but are preferably administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

Obviously, the dosage administered may also vary and is determined by known factors such as: the pharmacokinetic properties of the particular drug; the mode and route of administration; the age, health and weight of the recipient; the nature and nature of the condition and Severity; type of concurrent treatment; frequency of treatment; desired effect to be achieved. Daily doses of active ingredient can be expected to range from about 0.001 to about 1000 mg/kg body weight, with preferred doses ranging from about 0.1 to about 30 mg/kg.

Dosage forms of compositions suitable for administration contain from about 1 mg to about 100 mg of active ingredient per unit. In these pharmaceutical compositions, the content of the active ingredient is usually about 0.5-95% of the total weight of the composition. The active ingredient can 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 parenterally in 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. Tablets can also be compressed using similar diluents. Both tablets and capsules can be prepared as extended release products, whereby the drug is released continuously over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the environment, or enteric coated to provide selective disintegration in the gastrointestinal tract. Oral liquid dosage forms can contain coloring and flavoring to increase patient acceptance.

In general, water, a suitable oil, saline, water-soluble dextrose (glucose) and related sugar solutions and glycols such as propylene glycol or polyethylene glycol are among the suitable carriers for use in formulations for parenteral administration. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid alone or in combination may be suitable stabilizers. Citric acid and its salts and sodium EDTA can also be used. In addition, formulations for parenteral administration may contain preservatives such as benzalkonium chloride, methyl or propyl p-hydroxybenzoate and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, supra, a standard reference text in the field.

Examples of useful pharmaceutical dosage forms for administering the compounds of the invention are: Capsules

A number of unit capsules may be prepared as follows: Each standard two-segmented hard gelatin capsule is filled with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesium stearate. soft gelatin capsules

Soft gelatin capsules containing 100 mg of the active ingredient are prepared by preparing a mixture of the active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil and injecting the mixture into gelatin by means of a displacement pump. The capsules should then be washed and dried. tablet

A number of tablets can be prepared according to conventional methods so that the dosage form contains 100 mg of active ingredient, 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 mg of lactose. It may also be suitably coated to improve mouthfeel or delay absorption. Suspension

An aqueous suspension prepared for oral administration contains 25 mg finely ground active ingredient, 200 mg sodium carboxymethylcellulose, 5 mg sodium benzoate, 1.0 g sorbitol solution (U.S.P.) and 0.025 mg vanillin per 5 mL dose. injection

Compositions for parenteral administration by injection can be prepared by stirring 1.5% by weight of the active ingredient in 10% by volume polyethylene glycol and water and sterilizing the solution using conventional techniques. Combination of components (a) and (b)

Each therapeutic agent component of the present invention can exist independently in any dosage form, such as the above-mentioned dosage forms, and they can also be administered by various routes as described above. In the following description, ingredient (b) should be understood to represent one or more of the aforementioned therapeutic agents. Thus, if components (a) and (b) are treated the same or applied independently, each therapeutic agent of component (b) can also be treated the same or applied independently.

Components (a) and (b) of the present invention may be formulated together in a single dosage unit form (ie, so that they are combined in the same dosage form as capsule, tablet, powder or liquid) as a combination product. When components (a) and (b) are not formulated together in a single 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 administered first administration followed by component (b), or they may be administered in the reverse order. If component (b) contains more than one therapeutic agent, eg, an RT inhibitor and a protease inhibitor, these therapeutic agents may be administered together or in any order. When not administered at the same time, preferably components (a) and (b) are administered less than about 1 hour apart. Preferably, components (a) and (b) are administered orally. The terms "oral therapeutic agent", "oral inhibitor" or "oral compound" and the like as used herein represent compounds capable of oral administration. Whilst it is preferred that ingredients (a) and (b) are administered by the same route (e.g. both orally) or dosage form, they may be administered by different routes if desired (i.e. e.g. in a combination product where one ingredient may be administered orally and the other One component may be administered intravenously) or in different dosage forms.

Those skilled in the art of medicine will readily understand that the dosage of the combination therapy of the present invention may vary depending on various factors such as the pharmacokinetic properties of the specific therapeutic agent, its mode of administration and route of administration, age and health status of the user. and body weight, the nature and severity of the condition, the type of concomitant therapy, the frequency of treatment and the expected therapeutic effect, these factors are as mentioned above.

Appropriate doses of components (a) and (b) of the present invention can be readily determined by those skilled in the art of medicine based on the disclosure of the present invention. In principle, the daily dosage of the individual ingredients is generally about 100 mg to about 1.5 g. If component (b) represents more than one compound, the daily dosage of each therapeutic agent of component (b) will generally be from about 100 mg to about 1.5 g. In principle, when component (a) and component (b) are administered in combination, due to the synergistic effect of the combined drug, the dosage of each component can be reduced by about 70- 80%.

The combination products of the present invention may be prepared in such a dosage form that, although the active ingredients are combined in a single dosage unit, physical contact of the active ingredients is minimized as much as possible. To reduce exposure, for example, when the product is orally administered, one of the active ingredients may be enteric-coated. By applying an enteric coating to one of the active ingredients it is possible not only to minimize contact between the combined active ingredients but also to control the release of one of these active ingredients in the gastrointestinal tract so that one of the active ingredients is not in the in the stomach but in the intestines. In another embodiment requiring oral administration, the present invention provides a combination product in which one active ingredient is coated with a sustained-release material, which enables sustained release of the active ingredient throughout the gastrointestinal tract and reduces the risk of combination. contact between the active ingredients. Furthermore, the sustained-release composition may be enteric-coated so that the composition is released only in the intestinal tract. Yet another way, in combination products, one ingredient is coated with sustained release and/or enteric release polymers and the other ingredient is also coated with polymers such as low viscosity hydroxypropyl methylcellulose or other Coatings with suitable materials are known in the art to further isolate these active ingredients. These polymer coatings form an additional barrier to avoid interaction with other ingredients. In each formulation, when contact between ingredients (a) and (b) is prevented by a coating or some other material, contact between the individual agents of ingredient (b) may also be prevented.

The dosage form of the combination product of the present invention in which one of the active ingredients is enteric-coated may be in the form of a tablet, such that the enteric-coated ingredient is mixed with the other active ingredient and then compressed into a tablet; or the enteric-coated Pressed into one sheet, and other active ingredients pressed into another sheet. Optionally, to further separate the two layers, one or more placebo layers may be present such that the placebo layer is intermediate to the active ingredient layer. Alternatively, the dosage form of the present invention may be in the form of a capsule in which an active ingredient is compressed into microtablets, granules, granules or pellets which are then enteric-coated and these enteric-coated microtablets, granules, granules Or pellets are placed in capsules or put into capsules together with other active ingredient granules.

Whether the combination of the invention is administered in a single dosage form or in separate dosage forms but administered at the same time or simultaneously in the same manner, these and other ways of reducing contact between the components of the combination of the invention are important to readers of the present disclosure. The content will be obvious to those skilled in the art.

Also within the scope of the present invention is a pharmaceutical kit for treating HIV infection comprising a therapeutically effective amount of a composition in one or more sterile containers, wherein said composition comprises the components ( a) compound and one or more component (b) compounds. The containers can be sterilized according to conventional aseptic techniques well known to those skilled in the art. Component (a) and component (b) may be present in the same sterile container, or in separate containers. The sterile container may comprise self-contained containers or comprise one or more multi-part containers, as desired. Components (a) and (b) may be separated or physically mixed into a single dosage form or unit as described above. Such a kit may also contain, if desired, one or more of the various conventional pharmaceutical kit components, such as one or more pharmaceutically acceptable carriers, additional vials for mixing the achieved components, etc., which are within the skill of the art. It is obvious to the personnel. The kit may also include instructions, either in the form of an enclosure or on a label, stating the amounts of the components to be administered, the directions for administration and/or the method of mixing the components.

Obviously many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as herein described.

Claims (24)

1. formula I compound or pharmaceutically acceptable salt thereof,

I is R wherein ¹Be F; R ²Be F or H; And R ³Be selected from the 4-aminophenyl, 3-aminophenyl, 2,3-Dihydrobenzofuranes-5-base and 1,3-benzo dioxole-5-base.

2. the compound of claim 1, wherein said compound is a formula II compound: II.

3. the compound of claim 2, wherein said compound is a formula IIa compound:

IIa.

4. the compound of claim 3, wherein R ³Be the 3-aminophenyl.

5. the compound of claim 3, wherein R ³Be the 4-aminophenyl.

6. the compound of claim 3, wherein R ³Be 2,3-Dihydrobenzofuranes-5-base or 1,3-benzo dioxole-5-base.

7. the compound of claim 2, wherein said compound is a formula IIb compound:

IIb.

8. the compound of claim 7, wherein R ³Be the 3-aminophenyl.

9. the compound of claim 7, wherein R ³Be the 4-aminophenyl.

10. the compound of claim 7, wherein R ³Be 2,3-Dihydrobenzofuranes-5-base or 1,3-benzo dioxole-5-base.

11. the compound of claim 2, wherein said compound are the IIc compound:

IIc.

12. the compound of claim 11, wherein R ³Be the 3-aminophenyl.

13. the compound of claim 11, wherein R ³Be the 4-aminophenyl.

14. the compound of claim 11, wherein R ³Be 2,3-Dihydrobenzofuranes-5-base or 1,3-benzo dioxole-5-base.

15. the compound of claim 1, wherein said compound are the formula III compound:

III.

16. the compound of claim 15, wherein said compound are formula III a compound:

IIIa.

17. the compound of claim 1, wherein said compound are formula IV compound: IV.

18. the compound of claim 17, wherein said compound are formula IVa compound:

IVa.

19. a pharmaceutical composition comprises: any described compound of claim 1-19 of pharmaceutically acceptable carrier and treatment significant quantity.

20. treat the method that HIV infects, comprising for one kind: to any described compound or pharmaceutically acceptable salt thereof of claim 1-19 of host's drug treatment significant quantity of this treatment of needs.

21. treat the method that HIV infects, comprising for one kind: to host's Combined Preparation treatment significant quantity of this treatment of needs:

(a) any described compound of claim 1-19 or its steric isomer, its stereoisomer mixture or its pharmacologically acceptable salt; With,

(b) at least a compound that is selected from hiv reverse transcriptase inhibitor and hiv protease inhibitor.

22. the method for claim 21, wherein reverse transcriptase inhibitors is selected from zidovudine, dideoxycytidine, dideoxyinosine, d4T, 3TC, U-90152, efavirenz, nevirapine, Ro18, and 893, trovirdine, MKC-442, HBY097, ACT, UC-781, UC-782, RD4-2025 and MEN10979; Proteinase inhibitor is selected from Saquinavir, ritonavir, indinavir, amprenavir, nelfinavir, palinavir, BMS-232623, GS3333, KNI-413, KNI-272, LG-71350, CGP-61755, PD173606, PD177298, PD178390, PD178392, U-140690 and ABT-378.

23. the method for claim 22, wherein reverse transcriptase inhibitors is selected from AZT, efavirenz and 3TC, and proteinase inhibitor is selected from Saquinavir, ritonavir, nelfinavir and indinavir.

24. the method for claim 21, wherein compound (b) is a ritonavir.