PL192786B1 - Hiv protease inhibitors - Google Patents

Abstract

HIV protease inhibitors, obtainable by chemical synthesis, inhibit or block the biological activity of the HIV protease enzyme, causing the replication of the HIV virus to terminate. These compounds, as well as pharmaceutical compositions that contain these compounds and optionally other anti-viral agents as active ingredients, are suitable for treating patients or hosts infected with the HIV virus, which is known to cause AIDS.

Description

Description of the invention

The present invention relates to a new chemical compound in the form of an isoquinoline carboxamide derivative, its pharmaceutical composition and use as an HIV protease inhibitor, and therefore the use of such a compound as an antiviral agent.

Acquired Immunodeficiency Syndrome (AIDS) is a relatively recent disease. AIDS causes a gradual collapse of the immune system of the organism, as well as progressive disturbances of the central and peripheral nervous systems. Since its discovery in the early 1980s, AIDS has spread rapidly and is now reaching epidemic proportions in a relatively limited segment of the population. As a result of intensive research, the causative agent of this disease has been discovered, namely human T-lymphotropic retrovirus III (HTLV-III), now more commonly referred to as human immunodeficiency virus (HIV).

HIV belongs to a class of viruses called retroviruses. The retroviral genome is made up of RNA that is converted to DNA by reverse transcriptase. This retroviral DNA is then stably integrated into the host cell chromosome and, through host cell proliferation processes, produces new retroviral particles and transfers the infection to other cells. HIV appears to have a special affinity for human T-4 lymphocytes, which play a key role in the body's immune system. Infection of these white blood cells with HIV reduces the levels of this white blood cell population. Finally, the immune system inactivates, which is no longer able to effectively defend itself against a variety of opportunistic diseases such as Pneumocystis carini pneumonia, Kaposi's sarcoma and cancer of the lymphatic system, among others.

Although the exact mechanism by which HIV makes and works is unknown, identifying the virus has resulted in some progress in controlling the disease. For example, the drug azidothymidine (AZT) has been found to be effective in inhibiting reverse transcription of the HIV retroviral genome, thereby controlling, but not curing, disease in people with AIDS. Drugs that could cure, or at least control, the lethal HIV virus are still being investigated.

Retrovirus replication is typically characterized by post-translational processing of polyproteins. This processing is carried out by the action of the virus-encoded enzyme protease. The result is mature polypeptides which then allow infectious virus to be produced and acted on.

When this molecular process is suppressed, normal HIV production is stopped. Thus, HIV protease inhibitors can act as anti-HIV drugs.

The HIV protease is one of the translation products of the pol gene of the HIV structural protein. This retroviral protease specifically cleaves other structural polypeptides at specific sites to release these newly activated proteins and structural enzymes, rendering the virion replicative. Mere inhibition of HIV protease by potent compounds can prevent proviral integration of infected T cells early in the HIV-1 life cycle, as well as inhibit viral proteolytic processing at a late stage. In addition, protease inhibitors are advantageous because of their easier availability, longer viral persistence, and lower toxicity than currently available drugs, possibly due to their specific targeting of retroviral protease.

The present invention relates to a chemical compound capable of inhibiting and / or blocking the activity of HIV protease, terminating the proliferation of HIV, a pharmaceutical composition containing this compound, and its use as HIV protease inhibitors.

The subject compound includes compound 21 and pharmaceutically acceptable salts thereof.

Η ι

PL 192 786 B1

The compound has a chemical name;

[3S- [2 (2S *, 3S *), 3alpha, 4abeta, 8abeta]] - N- (1,1-dimethyl-2-hydroxyethyl) decahydro-2- [2-hydroxy-3 - [(3-hydroxy -2-methylbenzoyl) amino] -4- (phenylthio) butyl] -3-isoquinolinecarboxamide.

The subject compound inhibits the protease encoded by human immunodeficiency virus (HIV) type 1 (HLV-1) or type 2 (HIV-2). The compound is beneficial in the treatment of HIV infection and in the treatment of acquired immune deficiency syndrome (AIDS). The subject compound, pharmaceutically acceptable salts thereof and pharmaceutical preparations of the present invention may be used alone or in combination with other antiviral, immunomodulating, antibiotic or vaccine agents. The compound of the present invention can also be used as a prodrug.

The compound of the present invention has at least five asymmetric centers, marked with an asterisk in formula 21 below.

Η ι

As a result of these asymmetric media, the compounds of the present invention may exist in any of the possible stereoisomeric forms and may be used in mixtures of stereoisomers which may be optically active or racemic, or they may be used alone as substantially pure stereoisomers for clean example at least 95%. All asymmetric forms, individual stereoisomers, and combinations thereof are within the scope of the present invention.

The individual stereoisomers can be prepared from their corresponding precursors by the methods described above, by cleavage of racemic mixtures or by separation of diastereoisomers. The cleavage can be performed in the presence of a cleaving agent, by chromatography or multiple crystallization, or by some combination of these methods known in the art. For further details on cleavage, see Jacques et al., Enantiomers, Racemates and Resolutions, John Wiley & Sons, 1981.

Preferably, the compound of the present invention is substantially pure, i.e. greater than 50% pure. More preferably, the compound is at least 75% pure. Even more preferably, the compound is at least 90% pure. Even more preferably, the compound is at least 95% pure, more preferably at least 97% pure, and most preferably at least 99% pure.

As mentioned above, the invention includes the pharmaceutically acceptable salts of the compounds defined by Formula 21.

The term "pharmaceutically acceptable salt" as used herein denotes the substantially non-toxic salts of the compounds of the above formula. Examples of pharmaceutically acceptable salts are those salts prepared by reacting the compounds of the present invention with a mineral or organic acid or an organic base.

The reagents are usually combined in one solvent such as diethylether or benzene to form acid addition salts, or water and alcohols to form basic addition salts. The salts normally precipitate out of solution in about one hour to about ten days, and can be isolated by filtration or other routine methods. Such salts are known as acid addition salts and base addition salts.

Acids that can be used in the preparation of acid addition salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, phosphoric, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic, oxalic, p-bromophenylsulfonic, carbonic acids. , amber, lemon, benzoic, acetic and the like.

Examples of pharmaceutically acceptable salts are sulfate, metabisulfite, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,

PL 192 786 B1 isobutyrate, caproesate, heptanoesane, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyno-1,4-dioate, hexine-1,6-dioesate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, dinitrobenzoate hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, g-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene and the like.

Preferred pharmaceutically acceptable acid addition salts are those prepared with inorganic acids such as hydrochloric and hydrobromic acid and prepared with organic acids such as maleic acid and methanesulfonic acid.

Basic addition salts include those derived from inorganic and organic bases such as ammonium or alkali bases or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases suitable for the preparation of the salts of the present invention, therefore, include: sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide and the like. The potassium and sodium salts are especially preferred.

The term "pharmaceutically acceptable prodrug" means a compound that can be converted under physiological conditions or by solvolysis to the compound of formula 21.

The term "pharmaceutically acceptable solvates" is intended to mean a solvate that retains the biological effectiveness and properties of the biologically active ingredients of the compounds of formula 21.

Examples of pharmaceutically acceptable solvates include, but are not limited to, compounds of formula 21 in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

It should be noted that the counterion forming part of any salt of the present invention is not critical so long as the salt as a whole is pharmaceutically acceptable and provided that the counterion does not impart undesirable properties to the salt as a whole.

The preparation of compound 21 is described below. Compound 21 is also prepared as a metabolite from the plasma of patients administered the acid salt (3R, 4aR *, 8aR *, 2'S *, 3'S *)] - 2-] 2'-hydroxy-3'- phenylthiomethyl-4'-aza-5'-oxo-5 '- (2' '-methyl-3' '-hydroxyphenyl) pentyl] decahydroisoquinoline-3-Nt-butylcarboxamidomethanesulfone, which is described in US Patent No. 5,484,926.

Scheme I. Synthesis of the amide of compound 21

The amine 17 was coupled with the chloroalcohol via an epoxide during an in situ procedure to give additive 18. Conventional removal of the protecting groups with a base and coupling of the free primary amine with acid chloride 20 resulted in the formation of the amide 21.

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Details of the reaction are described below in Examples 1A-F. A further use of the letters A through F in Scheme II corresponds to Examples 1A-F below.

The following examples illustrate aspects of the invention. These examples are for illustrative purposes only, and are not intended to limit the scope of the present invention.

Abbreviations of the terms: melting point, nuclear magnetic resonance spectrum, electron collision mass spectrum, field desorption mass spectrum, fast atom bombardment mass spectrum, infrared spectrum, ultraviolet spectrum, elemental analysis, high performance liquid chromatography and thin layer liquid chromatography, respectively, are mp, NMR , EIMS, MS (FD), MS (FAB), IR, UV, analysis, HPLC and TLC. In addition, the absorption maxima are listed for the IR spectra, which are relevant, not all the maxima observed.

With respect to NMR spectra, the following abbreviations are used: singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), multiplet (m), doublet of multiplets (dm), broad singlet (br.s.), broad doublet (br.d.), broad triplet (br.t.) and broad multiplet (br.m.). J is the coupling constant in hertz (Hz). Unless otherwise indicated, NMR data refer to the free base compound of the present invention.

NMR spectra were acquired on a General Instrument QE-300 300MHz instrument. Chemical shifts are expressed as δ values in ppm. Mass spectra were obtained on a VG ZAB-3 spectrometer at the Scripps Research Institute in La Jolla, CA. Infrared spectra were recorded on a Midac Corporation spectrometer. UV spectra were acquired on a Varian Cary 3E instrument. Thin layer chromatography was performed using silica plates purchased from E. Merck. The melting point was measured on a Mettler FP62 without correction.

P r z k ł a d 1

Method for the preparation of the amide of formula 21

[3S- [2 (2S *, 3S *), 3alpha, 4abeta, 8abeta]] - N- (1,1-dimethyl-2-hydroxyethyl) decahydro-2- [2-hydroxy-3 - [(3-hydroxy -2-methylbenzoyl) amino] -4- (phenylthio) butyl] -3-isoquinolinecarboxamide

Η ι

A. Perhydroisoquinoline (26.4 g, 111 mmol) (commercially available from NSC Technologies (Chicago, IL) or Procos SpA (Milan, Italy) was suspended in water (200 ml) and concentrated with aqueous HCl (200 ml). heated under reflux and stirred for 3 days during which time it became solution The solvents were removed under reduced pressure to give a light yellow solid, then it was suspended in 2-propanol (200 ml) and filtered. The filtrate was evaporated under reduced pressure to an oil EtOAc (100 mL) and water (100 mL) were added and the pH of the solution was adjusted to 8.0 by adding 2N aqueous KOH. Benzylchloroformate (15.8 mL, 111 mol) was added dropwise over 30 minutes, keeping the pH between 7 and 8 by adding 2N aq. KOH The mixture was stirred at room temperature for 18 hours EtOAc (200 ml) was added and the organic layer was washed with 1N aq. HCl (100 ml) and saline (100 ml). The organic layer was dried (MgSO4), filtered and evaporated under d low pressure to the oil. The product was purified by silica gel chromatography, eluting with 40-60 petroleum ether / EtOAC 1: 1 and then 100% EtOAc. Product fractions were collected and evaporated under reduced pressure to give compound 15 (11.3 g, 32%) as a colorless oil: ¹ H NMR (300 MHz, CDCl3) δ 7.43-7.28 (m, 5H) , 5.17 (br s, 2H), 4.76 (m, 1H), 3.79 (m, 1H), 3.33 (m, 1H), 2.19 (m, 1H), 1.96 (m, 1H), 1.88-1.15 (m, 10H).

B. 1-Hydroxybenzotriazole (4.2 g, 31.4 mmol) and EDC (6.0 g, 31.4 mmol) were added to a solution of acid 15 (8.3 g, 26.2 mmol) in DMF at ambient temperature. . The mixture was heated at 80 ° C for 10 minutes. 1,1-Dimethyl-2-trimethylsilyloxyethylamine (5.1 g, 31.4 mmol), prepared from 1,1-dimethyl-2-hydroxyethylamine (Aldrich Chemical Co.) and hexamethyldisilazane (Aldrich

PL 192 786 B1

Chemical Co.), by heating the untreated mixture under recirculation conditions for several hours and then evaporating the volatiles, and the solution was heated at 80 ° C for 17 hours. The yellow solution was poured into EtOAc (250 mL) and 2N aqueous HCl (250 mL). After stirring for 10 minutes, EtOAc (750 ml) was added and the mixture was washed with H 2 O (3 x 500 ml) and saline (1x250 ml). The combined aqueous layers were extracted with EtOAc (1x250 mL). The combined organic layers were dried (Na2SO4) and purified by flash chromatography (50/50 EtOAc / hexanes) to give compound 16 as a colorless oil (7.9 g, 78%): ¹ H NMR (300 MHz, CD3OD) δ 7. 36 (m, 5H), 5.20 (d, J = 8.1 Hz, 1H), 5.10 (m, 1H), 4.53 (m, 1H), 3.78 (dd, J = 13 , 2, 4.4 Hz, 1H), 3.60 (m, 2H), 3.48 (d, J = 10.7 Hz, 1H), 2.15-1.25 (m, 12H), 1 . 31 (s, 3H), 1.29 (s, 3H).

C. A mixture of carbamate 16 (7.9 g, 20.4 mmol) and 5% palladium on carbon (Pd / C) (1.6 g) was hydrogenated in 50 psi H2 in absolute EtOH (110 mL) at ambient temperature for 18 hours. The mixture was filtered through Celite and evaporated in vacuo to give the amine 17 as a white crystalline solid: ¹ H NMR (300 MHz, CD3OD) δ 3.63 (q, J = 7.0 Hz, 2H), 3.34 ( m, 1H), 3.27 (dd, J = 11.8, 3.3 Hz, 1H), 2.91 (m, 1H), 2.02-1.15 (m, 12H); 1.32 (s, 3H), 1.31 (s, 3H).

D. To a warm (27 ° C) suspension of chloroalcohol (purchased from Kanaka Industries, Japan) (10.4 g, 28.6 mmol) in isopropanol (IPA) (104 mL) was added aqueous 10.2 N NaOH ( 2.4 ml, 24.5 mmol). After 1 hour, 1N aqueous HCl in IPA (made by adding 1ml concentrated aqueous HCl to 12ml IPA) was added in an amount of about 1ml for neutralization (pH = 7). The amine 17 (5.2 g, 20.4 mmol) was added as a solution in IPA (50 mL) and the thin suspension was heated at 60 ° C for 10 hours. IPA was removed in vacuo. The residue was diluted with EtOAc (150 mL) and washed with H 2 O (2x50 mL), saturated aqueous NaHCO 3 (1x50 mL), and saline (1x50 mL). The combined organic layers were extracted with EtOAc (1x25 mL). The combined organic layers were dried (Na2SO4) and purified by flash chromatography (75/25 EtOAc / hexanes then EtOAc) to afford compound 18 as a white solid (8.98 g, 76%): ¹ H NMR (300 MHz, CD3OD ) δ

7.33 (m, 10H), 5.08 B, Jab = 12.2 Hz, ΔυΑΒ = 12.1 Hz, 2H), 3.96 (m, 2H), 3.56 (q, J = 7, 3 Hz, 2H), 3.50 (m, 1H), 3.20 (dd, J = 13.6, 9.2 Hz, 1H), 3.03 (m, 1H), 2.64 (m, 2H), 2.20-1.20 (m, 14H), 1.28 (s, 6H).

E. To a suspension of carbamate 18 (6.75 g, 11.6 mmol) in IPA (34 mL) at ambient temperature was added 50% aqueous NaOH (2.7 g, 1.8 mL, 33.6 mmol). The mixture was heated under reflux for 12 hours. After cooling to ambient temperature, the mixture was diluted with methyl t-butyl ether (MTBE) (600 mL) and washed with H 2 O (2x250 mL) and saline (1x125 mL). The combined organic layers were extracted with MTBE (1x150 ml). The combined organic layers were dried (Na2SO4) and evaporated in vacuo to give a mixture of compound 19 and benzyl alcohol as an oily white solid: ¹ H NMR (300 MHz, CD3OD) δ 7.34 (m, 10H), 4.63 ( s, 2H), 3.81 (m, 1H), 3.58 (m, 3H), 3.03-2.60 (m, 5H), 2.17 (m, 1H), 2.05 (m , 1H), 1.87-1.05 (m, 12H), 1.30 (s, 3H), 1.28 (s, 3H).

F. To a solution of a mixture of amine 19 (4.7 g, 10.4 mmol theoretically according to 18) and benzyl alcohol in EtOH (23 ml) at ambient temperature was added triethylamine (3.2 g, 4.3 ml, 31.2 mmol ). A solution of 3-acetoxy-2-methylbenzoyl chloride (20) (prepared by the method described in United States Patent Application 08/708411, filed September 5, 1996, which is hereby incorporated by reference) was added (2.4 g, 11.5 mmol) in THF (4 mL). After 2 hours, 50% aqueous NaOH (4.1 g, 2.8 mL, 52.2 mmol) was added and the mixture was refluxed for 1 hour. After cooling to ambient temperature, the mixture was neutralized to pH = 7 with 2N aqueous HCl (26 mL). This mixture was diluted with EtOAc (500 ml) and washed with H 2 O (1x250 ml), saturated aqueous NaHCO 3 (2x250 ml), H 2 O (1x250 ml) and saline (1x125 ml). The organic layer was dried (Na2SO4) and purified by flash chromatography (75/25 EtOAc / hexanes) ₁ to give the amide 21 as a white foam (1173-57A, 1.39 g, 23%). ¹ H NMR indicated the presence of 11% by weight of EtOAc, which could not be removed in vacuo.

Analysis:

¹ H NMR (300 MHz, CD3OD) d7,53 (d, J = 7.3 Hz, 2 H), 7.32 (t, J = 7.0 Hz, 2 H), 7.20 (t, J = 7.3 Hz, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.83 (d, J = 8 , 1 Hz, 1H), 4.42 (m, 1H), 4.08 (m, 1H), 3.61 (dd, J = 13.6, 4.0 Hz, 1H), 3.45 (AB, Jab = 11.0 Hz, ΔυΑΒ = 18.0 Hz, 2H), 3.29 (dd, J = 13.6, 10.3 Hz, 1H), 3.10 (m, 1H), 2.66 (m, 2H), 2.28 (s, 3H), 2.22 (m, 2H), 2.04 (m, 1H), 1.86-1.20 (m, 11 H), 1.19 (s, 3 H), 1.18 (s, 3 H), ¹³ C NMR (75.5 MHz, CD 3 OD) d 175.7, 172.5, 155.9, 138.8 , 136.7, 129.8, 128.9, 126.3, 126.0, 122.4, 118.4, 115.9, 70.3, 69.9, 68.2, 59.3, 58 , 8, 54.9, 53.0, 36.5, 34.2, 34.1, 31.1, 30.7, 26.4, 26.0, 23.1, 23.0, 20.8 , 12.1.

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P ryk l a d 2

HIV protease inhibitory activity and anti-HIV activity in cell cultures with Compound 21

Close binding kinetic analysis was used to determine the magnitude of the Ki value of compound 21.

Ki = 5.6 ± 0.91 nM.

Ways

HIV-1 protease expression

The HIV-1 protease gene was isolated from the virus strain IIIB (Ratner L. et al., Nature, 316, 227284 (1985). To increase the stability of the purified protease (Rose, JR et al., J. Biol. Chem. 268, 11939) -11945 (1993)), the glutamine residue at position 7 (Q7) was mutated to serine (S), replacing the 33 bp segment between the NdeI and BstEII sites of the protease gene sequence with synthetic oligonucleotides encoding the Q7S mutation. plasmid pGZ (Menge KL et al., Biochemistry, 34: 15934-15942 (1995) under the control of the phage T7 promoter. The resulting structure - pGZ / HP-19Q7S # 9, was transformed into E. coli BL21 (DE3) strain acquired at Novagen Lnc.

HIV-1 PR expression: cultures were grown in 2YT medium (1.6% tryptic peptone, 1% yeast extract, 0.5% NaCl at an initial pH of 7.5), containing 200 μg / L ampicillin in a 100 L fermentor (Biolafitte SA) at 37 ° C for 5 hours and then induced by adding 1 mM IPTG (isopropyl-eD-thiogalactopyranoside). The culture temperature during the induction period was raised to 42 ° C to enhance the accumulation of recombinant HIV-1 protease as insoluble inclusion bodies. After 2 hours at 42 ° C, cells were harvested by countercurrent filtration using Pellicon Cartridge # 10 0.1 µm WPP000C5 (Millipore) and cell paste was stored frozen at -70 ° C.

Purification of recombinant HIV-1 protease: Unless otherwise indicated, all steps were performed at 4 ° C. Protein concentration was determined with BioRad protein test solution with bovine serum albumin (BioRad, Richmond, CA) as standard. The chromatographic steps and the purity of the HIV PR were analyzed by didecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Final HIV PR purity was> 98%. Typical final yield from each 100 L of culture was about 120 mg.

Cell paste from 100 L of the culture was resuspended in 300 ml of lysis buffer (50 mM Tris-Cl, pH 8.0, 25 mM NaCl, 20 mM 2-mercaptoethanol) and microfluidized in a Microfluidics Corporation fluidizer at 22,000 psi. Crude cell lysate was clarified by centrifugation at 14,000 rpm. for 20 minutes. The HIV PR was mainly found in a clump in the form of inclusion bodies. The inclusion bodies were then washed repeatedly in lysis buffer, also containing 0.1% Trition-X100 and 1 M urea, and after each washing step, the inclusion bodies were granulated by centrifugation at 5000 rpm. for 20 minutes. The purified inclusion bodies were dissolved in a buffer containing 50 mM Tris-Cl, pH 8.0, 25 mM NaCl, 20 mM 2-mercaptoethanol and 8 M urea. The solution was clarified by centrifugation at 14,000 rpm. and placed at room temperature in a Fast Flow Q-Sepharose column (Pharmacia, Piscataway, NJ) equilibrated in the same buffer. Under these conditions, the HIV PR did not bind to the column and essentially pure enzyme was found in the flow-through fractions. For protein renaturation, fractions from the Fast Flow Q-Sepharose column were dialyzed against three changes of buffer containing 25 mM NaH2PO4, pH 7.0, 25 mM NaCl, 10 mM DTT and 10% glycerol. After reconstitution of the protein structure, small amounts of precipitates were removed by centrifugation and the resulting enzyme product was concentrated, dialyzed against 0.5 M NaCl, 50 mM MES, pH 5.6, 10 mM DTT, frozen in small aliquots of about 2 mg / ml and stored at temperature 70 ° C.

Tight binding kinetics test and analysis

The proteolytic activity of the purified HIV-1 protease was measured by a modified chromogenic assay by Richards et al. (Richards, AD et al., J. Biol. Chem., 256, 773-7736 (1990). The synthetic His-Lys peptide was used as a substrate. -Ala-Arg-Val-Leu-Phe- (paraNO2) -Glu-Ala-Nle-Ser-NH2 (American Peptide Company) (Nle is norleucine) The test was carried out in 0.5M NaCl, 50 mM MES pH 5.6, 5 mM DDT and 2% DMSO at 37 ° C. The cleavage of the cleavable bond between leucine and paranitrophenylalanine (Phe para-NO2) was investigated by spectrophotometric monitoring of the decrease in absorbance at 305 nm. The initial rate was determined as the rate of decrease in absorbance during the first 100 seconds. Under these conditions and using the HIV-1 Q7S protease, the Michaelis constant (Km) for this substrate is 59 17 µM.

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For the determination of compound 21 inhibition, a 200 µM saturating substrate concentration was used. From 13 to 20 inhibitor concentration values were evaluated, and the reaction rate was measured at each concentration as described above. The apparent Ki (Ki app), shown above, was determined by least-square computer non-linear fitting to the Morrison tight binding equation (Morrison, J.F., Biochem. Biophys. Acta, 185, 269-286 (1963)).

Example 3

Antiviral activity of compound 21 against HIV-1 in cell culture

Cells and viral strains:

The human CEM-SS and MT-2 cell lines and HIV-1 RF and IIIB strains were obtained from the AIDS Research and Reference Program (AIDS Research and Reference Program, Division of AIDS, NIAID and NIH) Department.

Cell Protection Tests:

The inhibitory effect of the individual agents on HIV-1 replication was measured by the reduction of MTT staining (Alley, MC et al., Cancer Res. 48: 589-601 (1988)). Compounds were dissolved in DMSO at a concentration of 40 mg / ml, then diluted 1: 200 in culture medium (RPMI supplemented with 10% fetal bovine serum). 100 µl of each dilution was taken and applied to a 96-well plate, and serial half-log dilutions were prepared. In separate tubes, MT-2 and CEM-SS cells were infected with HIV-1 IIB or RF at multiple infections (moi) 0.01 and 0.03, respectively. After a 4-hour adsorption period, 100 µl of infected or uninfected cells were added to the wells of the drug-containing plate to give a final concentration of 1x10 ⁴ cells, well. Six days (CEM-SS cells) or seven days later (MT-2 cells), MTT (5 mg / ml) was added to the assay plates and the amount of formazan produced was quantified spectrophotometrically at 570 nm. Data are expressed as the percentage of formazan produced in drug-treated cells compared to formazan produced in cavities in uninfected and untreated cells. The ED50 was calculated as the drug concentration increasing the percentage of formazan production in infected, drug-treated cells to 50% of that of uninfected and untreated cells. The cytotoxicity (TC50) was calculated as the drug concentration that reduced the percentage of formazan produced in drug-uninfected cells to 50% of the production value by untreated uninfected cells. The therapeutic index (TI) was calculated by dividing the cytotoxicity (TC50) by the antiviral efficacy (ED50).

Table 1

Assessment of antiviral activity and cytotoxicity of compound 21 in acute CEM-SS RF HIV-1 cell infection

Relationship ED50 (nM) ED95 (nM) TC50 (pM) Therapeutic index ³ 21 34.2 154.1 96.6 2825 Azidothymidine (AZT) 52.3 543.1 > 374.5 > 7161 Didesoxycytidine (ddC) 94.7 142.0 37.69 398

^a therapeutic index = cytotoxicity (TC50) + antiviral activity (ED50)

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T a b e l a 2

Evaluation of Antiviral Activity and Cytotoxicity of Compound 21 in Acute Infection of HIV-1 MT-2 IIIB Cells

Relationship ED50 (nM) ED95 (nM) TC50 (gM) Therapeutic index ³ 21 85.6 no done 92.6 1082 AZT 430.7 no done 109.4 254 ddC 5924 no done 176.3 thirty

^a therapeutic index = cytotoxicity (TC50) + antiviral activity (ED50)

As mentioned above, the compounds of the present invention are advantageous in inhibiting HIV protease, which is an enzyme associated with the production and assembly of viral components. One embodiment of the invention is a method of treating HIV infection which comprises administering to the host (patient), for example a Prime, an effective amount of a compound of formula (9) or a pharmaceutically acceptable salt thereof. Another embodiment of the invention is a method of inhibiting HIV protease, comprising administering to an HIV infected cell, an HIV susceptible cell or a host or patient, for example a primate, an effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof.

The term "effective amount" means an amount of a compound or a pharmaceutically acceptable salt thereof that is effective in inhibiting HIV protease dependent production and assembly of HIV protease components. The specific dose of a compound administered in the present invention to achieve a therapeutic or inhibitory effect will, of course, depend on the particular circumstances of the case, such as, for example, the compound administered, the route of administration, the condition being treated, and the host (patient) treated. An exemplary daily dosage (administered in a single dose or in several divided doses) is from about 0.01 mg / kg to about 50 mg / kg body weight of a compound of this invention. Preferably, the daily dose is from about 0.05 mg / kg to about 40 mg / kg, more preferably, from about 1.0 mg / kg to about 30 mg / kg.

The compounds of the present invention can be administered by a variety of routes, such as oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal. The compounds of the present invention are preferably formulated ready for administration prior to administration. Thus, another embodiment of a compound of the present invention is a pharmaceutical preparation comprising an effective amount of the compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof, for example a diluent or excipient.

The active ingredient preferably makes up from 0.1% to 99.9% by weight of the formulation. By "pharmaceutically acceptable" is meant a carrier, for example a diluent or excipient, compatible with the other ingredients of the formulation and not deleterious to the host (patient).

Pharmaceutical preparations can be prepared from the compounds of the present invention by known methods using known and readily available ingredients. In preparing the formulations of the present invention, the active ingredient is usually mixed with a carrier, or diluted with a carrier, or placed in a carrier, which may take the form of a capsule, bag, paper, or other suitable container. When the carrier serves as a diluent, it can be a solid, semi-solid or fluid material which acts as a carrier, excipient or medium for the active ingredient. Thus, the preparation can be produced in the form of tablets, pills, powders, lozenges, bags, wafer capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in a liquid medium), ointments (containing for example 10% by weight of active ingredient), soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders and the like. The following formulation examples are illustrative only and are not intended to limit the scope of the present invention. The term active ingredient refers to a compound or a pharmaceutically acceptable salt thereof.

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Preparation 1

The following ingredients are prepared into hard gelatin capsules: Amount (mg / capsule)

Active ingredient 250

Dried starch 200

Magnesium stearate 10

Total 460 mg

Preparation 2 A tablet is prepared from the following ingredients: Quantity (mg / tablet) Active ingredient 250 Microcrystalline cellulose 400 Silicon dioxide, smoked 10 Stearic acid 5 Together 665 mg The ingredients are mixed and compressed to produce tablets weighing 665 mg. Preparation 3 An aerosol is made from the following ingredients: Mass Active ingredient 0.25 Methanol 25.75 Propellant 22 (chlorodifluoromethane) 74.00 Together 100.00 The active ingredient is mixed with ethanol and the mixture is added to the propellant 22 portion and cooled to -30 ° C and transferred to the filling device. The appropriate amount is then placed in a stainless steel container and diluted with the rest of the propellant. Then onto the container valve is installed. Preparation 4 Tablets containing 60 mg of active ingredient are prepared as follows: Quantity (mg / tablet) Active ingredient 60 Starch 45 Microcrystalline cellulose 34 Polyvinylpyrrolidone (as 4 10% solution in water) Soda salt 4.5 carboxymethylcellulose Magnesium stearate 0.5 Talc 1 Together 150 The active ingredient, starch and cellulose are passed through a No. 45 (US) sieve and it mixes well. The aqueous solution, containing polyvinylpyrrolidone, is mixed with the resulting powder, and the mixture is then passed through a No. 14 (US) sieve. The granules so produced are dried at 50 ° C and sieved through a No. 18 (US) sieve. Sodium carboxymethylcellulose, magnesium stearate and talc, previously screened through a No. 60 sieve (US), are then added to the granules; after mixing, the granules are compressed on a tablet press to give tablets weighing 150 mg.

PL 192 786 B1

Preparation 5

Capsules containing 80 mg of active ingredient are prepared as follows:

Quantity (mg / tablet)

Active ingredient 80 mg

Starch 59 mg

Microcrystalline cellulose 59 mg

Magnesium stearate 2 mg

Total 200 mg

The active ingredient, cellulose, starch and magnesium stearate are mixed, sieved through a No. 45 screen (US) and the mixture filled into hard gelatin capsules, 200 mg of the mixture per capsule.

Preparation 6

Suppositories are prepared as follows, each containing 225 mg of the active ingredient:

Active ingredient 225 mg

Glycerides of saturated acids 2,000 mg of fatty acids

Total 2225 mg

The active ingredient is sieved through a No. 60 sieve (US) and suspended in saturated fatty acid glycerides, previously melted using as little heat as possible. The mixture is then filled into suppository molds of nominal 2 mg capacity and the mixture is allowed to cool.

Preparation 7

Suspensions containing 50 mg of active ingredient per 5 ml dose are prepared as follows:

Active ingredient 50 mg Soda salt 50 mg carboxymethyl cellulose Syrup 1.25 ml Benzoic acid solution 0.10 ml Flavoring substance q.v. Dye q.v. Purified water up to 5 ml The active ingredient is passed through a No. 45 (US) sieve and mixed with the sodium salt

carboxymethyl cellulose and syrup to a smooth paste. The benzoic acid solution, flavor, and color are diluted with water and added with stirring. Sufficient water is then added to make the desired volume.

Preparation 8

The preparation for intravenous administration is prepared as follows:

Active ingredient 100 mg

Isotonic saline 1000 ml of physiological

A solution of the above ingredients is usually administered intravenously to the patient at a rate of 1 ml per minute.

Preparation 9

A tablet is made using the following ingredients:

Quantity (mg / tablet)

Active ingredient 292 mg

Calcium silicate 146 mg

Crospovidone 146 mg

Magnesium stearate 5 mg

Total 589 mg

Claims (15)

Patent claims 1. Compound with formula Η ι or a pharmaceutically acceptable salt thereof. 2. A compound according to claim The process of claim 1, having a stereoisomeric purity greater than 90%. 3. The compound according to p. The process of claim 2, having a stereoisomeric purity of at least 95%. 4. The compound according to p. The process of claim 2, having a stereoisomeric purity of at least 97%. 5. The compound according to p. The process of claim 2, having a stereoisomeric purity of at least 99%. 6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an active ingredient, characterized in that the active ingredient is a compound of the formula 21 shown in claim 1. A pharmaceutical composition according to claim 1 6. A compound according to claim 6, characterized in that the compound has a stereoisomeric purity greater than 90%. 8. A pharmaceutical composition according to claim 1 8. A compound according to claim 7, characterized in that the compound has a stereoisomeric purity greater than 95%. 9. A pharmaceutical composition according to claim 1 8. A compound according to claim 7, characterized in that the compound has a stereoisomeric purity greater than 97%. 10. A pharmaceutical composition according to claim 1 8. A compound according to claim 7, characterized in that the compound has a stereoisomeric purity greater than 99%. 11. The use of an effective amount of a compound of formula 21 as set forth in claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a preparation for the inhibition of HIV protease. 12. The use of claims The compound of claim 11, wherein the compound has a stereoisomeric purity greater than 90%. 13. Use of claims The compound of claim 12, wherein the compound has a stereoisomeric purity greater than 95%. 14. Use of claims The compound of claim 12, wherein the compound has a stereoisomeric purity greater than 97%. 15. The use according to Claim The compound of claim 12, wherein the compound has a stereoisomeric purity greater than 99%.