EA001154B1

EA001154B1 - (4r,5s,6s,7r)-hexahydro-1[5-(3-aminoinazole)methyl]-3-butyl-5,6-dihydroxy-4,7-bis[phenylmethyl]-2h-1,3-diazepin-2-one and its useas hiv protease inhibitor

Info

Publication number: EA001154B1
Application number: EA199900451A
Authority: EA
Inventors: Джеймс Дэвид Роджерс; Патрик Юк-Сун Лэм
Original assignee: Дюпон Фармасьютикалз Компани
Priority date: 1996-11-08
Filing date: 1997-11-04
Publication date: 2000-10-30
Also published as: CN1235601A; PL333412A1; SK59599A3; LV12374A; EP0937066A1; KR20000053123A; NO992229L; IL129726A0; JP2001503429A; SI9720071A; WO1998020009A1; CZ157499A3; LV12374B; AU722489B2; EA199900451A1; AU5103398A; BR9712755A; HUP9904143A2; NZ335919A; EE9900187A

Abstract

1. A compound of formula I: or a pharmaceutically acceptable salt or prodrug form thereof, wherein a prodrug of formula I is a compound wherein the two hydroxy groups join to form an epoxide, -OCH2SCH2O-; -OC (=O)O-; -OCH2O-; -OC(=S)O-; -OC(=O)C(=O)O-; -OC(CH3)2O-; -OC((CH2)3NH2) (CH3)O-; -OC(OCH3) (CH2CH2CH3)O-; or -OS(=O)O- group. 2. A compound according to claim 1, wherein the compound is of formula I. 3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt or prodrug form thereof. 4. A composition according to claim 3, wherein the compound is of formula I. 5. Use of a compound of formula I or a pharmaceutically acceptable salt or prodrug form thereof for the manufacture of a medicament for treating HIV infection: or a pharmaceutically acceptable salt or prodrug form thereof, wherein a prodrug of formula I is a compound wherein the two hydroxy groups join to form an epoxide, -OCH2SCH2O-; -OC (=O)O-; -OCH2O-; -OC(=S)O-; -OC(=O)C(=O)O-; -OC(CH3)2O-; -OC((CH2)3NH2) (CH3)O-; -OC(OCH3) (CH2CH2CH3)O-; or -OS(=O)O- group. 6. A use according to claim 5, wherein the compound is of formula I. 7. Use of a combination of (a) and (b) for the manufacture of a medicament for treating HIV infection, wherein: (a) is a compound of formula I or a pharmaceutically acceptable salt or prodrug form thereof: wherein a prodrug of formula I is a compound wherein the two hydroxy groups join to form an epoxide, - OCH2SCH2O-; -OC (=O)O-; -OCH2O-; -OC(=S)O-; -OC(=O)C(=O)O-; -OC(CH3)2O-; -OC((CH2)3NH2) (CH3)O-; -OC(OCH3) (CH2CH2CH3)O-; or -OS(=O)O- group.and (b) is at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors. 8. A use according to claim 7, wherein the compound is of formula I. 9. A use according to claim 7, wherein the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor. 10. A use according to claim 9, wherein the nucleoside reverse transcriptase inhibitor is selected from AZT, 3TC, ddI, ddC, and d4T and the protease inhibitor is selected from saquinavir, ritonavir, indinavir, VX-478, nelfinavir, KNI-272, CGP-61755, and U-103017. 11. A use according to claim 10, wherein the nucleoside reverse transcriptase inhibitor is selected from AZT and 3TC and the protease inhibitor is selected from saquinavir, ritonavir, and indinavir. 12. A use according to claim 11, wherein the nucleoside reverse transcriptase inhibitor is AZT. 13. a use according to claim 11, wherein the protease inhibitor is indinavir. 14. A pharmaceutical kit useful for the treatment of HIV infection, which comprises a therapeutically effective amount of: (a) a compound of claim 1; and, (b) at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors, in one or more sterile containers. 15. A kit according to claim 14, wherein component (a) is a compound of formula I.

Description

The present invention relates to 1- (3-amino-indazol-5-yl) -3-butylcyclic ureas, which are used as HIV protease inhibitors, to containing their pharmaceutical compositions and diagnostic kits, and to methods of their use for treating a viral infection or as test standards or reagents.

The level of technology

Two different retroviruses, human immunodeficiency viruses (HIV) type 1 (HIV-1) or type 2 (HIV-2) are etiologically related to the immunosuppressive disease, acquired immunodeficiency syndrome (AIDS). HIV-seropositive subjects initially asymptotically, but usually develop AIDS-related complex (SSC), and then AIDS. Patients manifest strong suppression of immunity, which makes them weak and prone to extremely dangerous opportunistic infections.

AIDS is the end result of the HIV-1 virus or HIV-2 developing in its own life cycle. The life cycle of a virion begins with the virion attaching to the host human T-4 lymphocyte immune cell by binding the glycoprotein on the surface of the virion's protective membrane with the glycoprotein C'.P4 on the lymphocyte cell. After attachment, the virion sheds its glycoprotein membrane, enters the cell membrane of the host cell and opens its RNA. The virion enzyme, revertase (reverse transcriptase) controls the process of RNA transcription into single-stranded DNA. Viral RNA disintegrates and a second DNA helix is created. New double-stranded DNA is integrated into the genes of human cells, and these genes are involved in the reproduction of cells.

From this point on, the human cell carries out its reproductive process, using its own RNA polymerase to transcribe the integrated DNA into viral RNA. Viral RNA is translated into the polyprotein precursor fusion insert dad-ροΐ. The polyprotein is then degraded by the HIV protease enzyme, forming mature viral proteins. Thus, HIV protease is responsible for the regulation of the splitting cascade, which leads to the maturation of viral particles into a virus that has full infectious ability.

The normal human immune system response that kills invading virions is weakened, since most of the virion's life cycle is latent inside the immune cell. In addition, viral revertase — an enzyme used in the formation of new virion particles — is not very specific and causes transcription errors that lead to continuous changes in glycoproteins on the surface of the viral protective membrane. This loss of specificity reduces the effectiveness of the immune system, since antibodies specifically developed against one glycoprotein may be useless against another, thereby reducing the number of antibodies capable of fighting the virus. The virus continues to reproduce while the response of the immune system continues to weaken. Gradually, HIV keeps the body's immune system at full power, allowing opportunistic infections to develop, which can lead to death without the intervention of antiviral agents, immunomodulators or both.

There are at least three critical points in the viral life cycle that have been identified as possible targets for antiviral drugs: (1) initial virion attachment to T-4 lymphocyte or a macrophage site, (2) viral RNA transcription into viral DNA (revertase , PT), and (3) the formation of a new viral particle during reproduction (for example, HIV protease aspartic acid or HIV protease).

The genomes of retroviruses encode a protease that is responsible for the proteolytic transformation of one or more polyprotein precursors, such as poli and dan gene products (see \ Us11. Ags. Νίτοί. 98 1 (1988)). Retroviral proteases most often convert dad precursor into nuclear proteins, and also convert po1 precursor into revertase and retroviral protease.

Proper processing of polyprotein precursors by the retroviral protease is essential for the assembly of infectious virions. Mutagenesis of ίη νίΐΓο, which produces a protease-defective virus, has been shown to lead to the production of immature nuclear forms that do not have the infectious ability [see Sga \\ Tog6 c1 a!., 1. νίτοί. 53,899 (1985); KAYU e! A1., Carving 145 280 (1985)]. Therefore, inhibition of retroviral protease provides an attractive target for antiviral therapy [see Myyiua, Wing 325 775 (1987)].

The ability to inhibit viral protease provides a method of blocking viral replication and thus a method of treating viral diseases, such as AIDS, which may have fewer side effects, be more effective and cause less drug resistance than modern methods of treatment. As a result, three HIV protease inhibitors - saquinavir (Κο ^), ritonavir (ΑΠΠοΙΙ) and indinavir (Megsk) were put on sale, and a number of potential protease inhibitors are in clinical trials, for example, νΧ-478 ^ eeeh), nelfinavir (ΑβουΓοη ), ΚΝΙ-272 (1 Epegdu) and CcP 61755 (С1Ба-Се1Гу).

As is evident from clinical trials of commercially available protease inhibitors, a wide variety of compounds have been studied as possible HIV protease inhibitors. Mononuclear cyclic ureas attracted special attention, for example, in PCT applications 94/19329 and 93/07128 ba e! a1. described in detail cyclic urea formula

and methods for producing these ureas. Although the compounds shown fall under the description of a bash e! A1., they are not specifically described.

Additional cyclic ureas are described by bash e! a1. I. Syesh. Meb. 1996, 39, 35143525. Although this publication describes a wide range of cyclic ureas, it does not describe the indazolmethyl-containing compounds.

Despite the current success of protease inhibitors, it has been found that HIV patients can become resistant to one protease inhibitor. Therefore, it is desirable to develop additional protease inhibitors to further fight HIV infection.

Brief description of the invention

Accordingly, one object of the present invention is new protease inhibitors.

Another object of the present invention are pharmaceutical compositions having protease inhibiting activity, comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

Another object of the present invention is a new method of treating HIV infection, which comprises administering to a patient in need of such treatment a therapeutically effective amount of at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

Another object of the present invention is a new method of treating HIV infection, which comprises administering to a patient in need of such treatment a therapeutically effective combination of (a) one of the compounds of the present invention and (b) one or more compounds selected from the group including Hivrevertazy and HIV protease inhibitors.

Another object of the present invention is a method of inhibiting HIV present in a sample of body fluid, which comprises treating a sample of body fluid with an effective amount of a compound of the present invention.

Another object of the present invention is a kit or container containing at least one of the compounds of the present invention in an amount effective for use as a standard or reagent in a test or analysis to determine the ability of a potential pharmaceutical agent to inhibit HIV protease, HIV growth or both.

These and other objects that will become clear from the following detailed description have been discovered due to the discovery by the inventors that the compounds of formula I

I or their pharmaceutically acceptable salts, or their prodrug forms are effective protease inhibitors.

Detailed description of preferred implementations. Thus, in the first embodiment, the present invention provides a new compound of formula I

I or its pharmaceutically acceptable salt, or its prodrug form.

In the second embodiment, the present invention relates to a novel pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In a third embodiment, the present invention relates to a new method of treating HIV infection, which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

In a fourth embodiment, the present invention relates to a new method of treating HIV infection, which comprises administering to a patient in need thereof a combination of a therapeutically effective amount (a) of a compound of formula I, and (b) of at least one compound selected from the group comprising HIV inhibitors -revertase and HIV protease inhibitors.

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

In another more preferred embodiment, the nucleoside revertase inhibitor is selected from ΑΖΤ, 3TC 66Ι, 66C and 64T. and the protease inhibitor is selected from saquinavir, ritonavir, indinazira, nelfinavir UH-478, ΚΝΙ-272, CCP-61755 and I-103017.

In an even more preferred embodiment, the nucleoside reverse transcriptase inhibitor is selected from 3 and 3TC, and the protease inhibitor is selected from saquinavir, ritonavir and indinavir.

In an even more preferred embodiment, the nucleoside revertase inhibitor is ΑΖΤ.

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

In the fifth embodiment, the present invention relates to a pharmaceutical kit used to treat HIV infection, which contains a therapeutically effective amount of:

(a) a compound of formula I, and (b) at least one compound selected from the group consisting of HIV revertase inhibitors and HIV protease inhibitors, in one or more sterile containers.

In the sixth embodiment, the present invention relates to a new method of inhibiting HIV present in a sample of body fluid, which involves treating a sample of body fluid with an effective amount of a compound of formula I.

In the seventh embodiment, the present invention relates to a new kit or container containing a compound of formula I in an amount effective for use as a standard or reagent in a test or analysis to determine the ability of a potential pharmaceutical agent to inhibit HIV protease, HIV growth, or both.

Definitions

The terms and expressions used herein have the meanings indicated below. It should be understood that the compounds of the present invention contain an asymmetrically substituted carbon atom and can be isolated in optically active or racemic form. Methods for preparing optically active forms, such as separating racemic forms or synthesizing from optically active starting materials, are well known in the art. The scope of the invention includes all chiral, diastereomeric, racemic forms and all geometrically isomeric forms of the structure, with the exception of those cases when specific stereochemistry or isomeric form is specifically indicated.

The term HIV reverse transcriptase inhibitor, as used herein, refers to both nucleoside and non-nucleoside HIV reverse transcriptase (PT) inhibitors. Examples of nucleoside PT inhibitors include, but are not limited to, ΑΖΤ, 66C, 66Ι, 64T and 3TC. Examples of non-nucleoside PT inhibitors include, but are not limited to, viviradine (Ryattasla a6 Ir_) uip I901528), derivatives, VIVS-587, nevirapine, L-697 661, LY 73497, and Bo 18,893 (Voye).

The term HIV protease inhibitor, as used herein, refers to compounds that inhibit HIV protease. Examples include, but are not limited to, saquinavir (Cosier, Wo 31-8959), ritonavir (Aboy, ABT538), indinavir (Megsk, MK-639), UH-478 (Weieh / S1aho Ae 11cete), nelfinavir (Adoigop, AC- 1343), ΚΝΙ-272 Zarap Epegdu), SSR61755 (С1Ьа-Се1ду) and и-103017 (Ryattaaa6 Arshcheyp). Additional examples include the cyclic protease inhibitors described in AO 93/07128, AO 94/19329, AO 94/22840 and PCT application I8 96/03426.

As used herein, the term pharmaceutically acceptable salts refers to derivatives of the described compounds in which the parent compound is modified by preparing its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids and basic residues, such as amines; salts of inorganic or organic alkalis and acid residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the usual non-toxic salts or quaternary ammonium salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and salts derived from organic acids, such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamine, benzoic, salicylic, sulfanic, 2, and phenylacetic, glutamine, benzoic, salicylic, sulfanic, 2, and phenylacetic, glutamic, benzoic, salicylic, sulfanic, 2, and phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2 toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic and the like.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains a basic or acidic group, by conventional chemical methods. Typically, such salts can be obtained by reacting the free acidic or basic forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or in a mixture of them; typically non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Ket1p§1op'8 Rigatsei1tsa1 8c1sepse8, 171Iu her., Mask RiI18Ip§ Sotrapu, Ye81op, RA, 1985, p. 1418, which is incorporated herein by reference.

The term pharmaceutically acceptable refers to those compounds, materials, compositions and / or dosage forms that are medically suitable for contact with human and animal tissues without causing a toxic effect of irritation, an allergic reaction or other problems or complications, taking into account a reasonable ratio of benefits / risk.

The term prodrugs includes any covalently bonded carriers that release the active parent drug in accordance with Formula I or other formulas of the compounds of the present invention, w νΐνο, when such prodrugs are administered to a mammal. Prodrugs of a compound of the present invention, for example, the compounds of formula (I), are obtained by modifying the functional groups present in the compound in such a way that the modifications decompose either by normal manipulations or ΐη νΐνο, to form the parent compound. Prodrugs include compounds of the present invention in which the hydroxy or amino group is bonded to any group, so that when the prodrug is administered to a mammal, it decomposes to form a free hydroxyl or free amino group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate or benzoate derivatives of alcohol or amine functional groups in the compounds of formula I; phosphate esters, dimethylglycine esters, aminoalkylbenzyl esters, aminoalkyl esters and carboxylic acid esters of alcohol functional groups in the compounds of formula I; etc. Additional examples include compounds in which two hydroxy groups of a compound of formula I are linked to form an epoxide; -OCH28CH2O-; -OC (= O) O-; -OCH2O-;

-OC (= 8) O-; -OC (= O) C (= O) O-; -OC (CH3DO-; -OC (OCH2) zNH2) (CH3) O-; -OC (OCH3) (CH2CH2C-DHO- or -O8 (= O) O-.

The terms stable compound or stable structure are used to indicate that the compound is sufficiently strong to withstand the isolation process with the required degree of purity from the reaction mixture and formulation to produce an effective therapeutic agent. The present invention offers only stable compounds.

The term substituted indicates that one or more of the hydrogens on the atom to which the expression is substituted is substituted by any of the specified group (s), provided that the normal valence of the specified atom is not exceeded and that the substitution results in a stable compound. If the substituent is a keto group (i.e., = O), then two hydrogen at one atom are replaced.

The expression a therapeutically effective amount refers to that amount of a compound of the present invention or that amount of a combination of compounds that is effective for inhibiting HIV infection or for treating the symptoms of HIV infection in a carrier. The combination of compounds is preferably a synergistic combination. Synergism, as described, for example, Sioi apy Taulaus, Αάν. Eu / ute ke§i1. 22: 27-55 (1984), occurs when the effect (in this case inhibition of HIV replication) of the compounds administered in combination is greater than the total effect of the compounds administered separately. In general, the synergistic effect is most pronounced with suboptimal concentrations of the compounds. Synergism can be expressed in lower cytotoxicity, increased antiviral effect, or some other beneficial effects when using the combination compared with the use of individual components.

Other features of the invention will be clear from the following examples, which are given to illustrate the invention and not to limit its scope.

Examples

The abbreviations used in the examples are defined as follows: ° C means degrees Celsius, d means doublet; yy is a double doublet; eq - equivalent, g - grams or grams, mg - milligram or milligrams, ml - milliliter or milliliters, H - hydrogen or hydrogen, h - hours, t - multiplet, M - molarity, min - minutes, MHz - megahertz, MS - mass spectroscopy, NMR spectroscopy of nuclear magnetic resonance, 1 - triplet, TLC - thin layer chromatography.

Example 1. Preparation of (4K, 58.68.7K) hexahydro-1 - [5 - (3-aminoindazole) methyl] -3 butyl-5,6-dihydroxy-4,7-bis [phenylmethyl] -2H1,3- diazapin-2-one (1).

A 1

Compound A can be obtained by known methods. For example, the acquisition of compound A is shown in the diagram in the Coszapo e1 a1 article. (Tegr. 1.e11. 1995, 36 (28), 4967, 4968), which is incorporated here by reference. Another method for preparing compound A is shown in Example 6 of US Pat. No. 5,530,124, which is hereby incorporated by reference.

Part A. To the suspension of compound 1 (10.0 g;

27.3 mmol) in 1,2-dichloroethane (100 ml) was added methyl triflate (3.4 ml, 30 mmol). After boiling under reflux overnight, the reaction mixture was washed with a saturated solution No. 11CO ₃ , a saturated solution of IaC1, dried (Ia ₂ 8О _four ) and evaporated, leaving 12.5 g of yellow oil. Column Chromatography (Flash 8U ₂ , 25% E1OAc / hexane) gave 7.86 g of compound 2 as a pale yellow oil that crystallized upon standing (yield 75%), mp. 97-100 ° C, MN ⁺ = 381.

° x °

Part B. To a solution of compound 1 (10.0 g,

26.3 mmol) in anhydrous DMF (30 ml) was added sodium hydride (1.58 g, 65.8 mmol). The reaction mixture was stirred at room temperature for 45 minutes, after which a solution of 1-iodobutane (9.68 g, 52.6 mmol) in anhydrous DMF (10 ml) was added dropwise. After the addition, stirring was continued at room temperature overnight. The reaction mixture was cooled to 0 ° C and methanol (5 ml) was added to quench the excess sodium hydride. The mixture was partitioned between ethyl acetate (200 ml) and water (150 ml). The organic phase was separated and washed with water (4 x 100 ml), brine (1 00 ml) and dried over sodium sulfate. Purification by flash chromatography (25% EYLE / hexane mixture) gives nbutilisourea 2 (10.5 g, yield 92%): M8 (INGSYUR) (M + H ⁺ ) 437.2 (100%); ^one H NMR (300 MHz, ATP1 ₃ , 25 ° C) δ 7.23 (t, 10H), 4.19 (t, 3H), 3.64 (t, 1H), 3.44 (s, 3H), 3.36 (t, 1H) , 3.02 (t, 2H), 2.76 (t, 2H), 2.04 (t, 1H), 1.52 (z, 3H), 1.49 (z, 3H), 1.21 ( t, 4H), 0.82 (1, 1 = 7.0 Hz, 3H).

Part C. (4K, 58,68,7K) -hexahydro-1 - [(3 cyano-4-fluorophenyl) methyl] -5,6-O-isopropylidene-4,7-bis- (4-phenylmethyl) -3- phenylmethyl-2H1, 3-diazapin-2-one (3).

To a solution of compound 2 (5.0 g, 11.5 mmol) in acetonitrile (40 ml) was added 4-fluoro-3-cyanbenzyl bromide (3.68 g, 1.15 mmol). The reaction mixture was refluxed overnight. After removal of the solvent under reduced pressure, the residue was purified using column chromatography (35% EYLE / hexane mixture) to obtain cyclic urea 3 as a white solid (4.5 g, 71% yield): M8 (IN ₃ α / ϋϋΙΡ) (M + H ⁺ ) 556.3 (100%); ^one H NMR (300 MHz, ATP1 ₃ , 25 ° C) δ 7.41 (t, 1H), 7.28 (t, 7H), 7.13 (, 1 = 9.2 Hz, 2H), 7.05 (1, 1 = 8, 8 Hz, 1H), 6.95 (, 1 = 9.2 Hz, 2H), 4.50 (, 1 = 14.0 Hz, 1H), 4.07 (t, 2H), 3.70 (t, 3H), 3.44 (1, 1 = 7.7 Hz, 1H), 2.90 (t, 4H), 2.12 (t, 1H), 1.50 (s, 6H), 1 , 26 (t, 4H), 0.83 (1, 1 = 7.0 Hz, 3H).

Part. (4K, 58.68.7K) -hexahydro-1- [5- (3-aminoindazole) methyl] -3-butyl-5,6-dihydroxy4,7-bis [phenylmethyl] -2H-1,3-diazapine-2- he (i)

To a solution of compound 3 (4.5 g, 8.11 mmol) in n-butanol (20 ml) was added hydrazine hydrate (0.81 g, 1 6.2 mmol). The mixture was heated under reflux for 6 hours. The solvent and excess hydrazine were removed under reduced pressure. The residue was dissolved in anhydrous methanol (20 ml), followed by dissolving in 4M HCl in dioxane (2 ml). The reaction mixture was stirred at room temperature for 2 hours. Methanol was removed and the residue was partitioned between ethyl acetate (80 ml) and saturated sodium bicarbonate (50 ml). The organic phase was separated, washed with water (2 x 50 ml) and dried over anhydrous sodium sulfate. Purification using flash chromatography gave compound I (3.0 g, yield 72%) as a white solid: mp. 129-131 ° C; M8 (IN3-C1 / PP1R) (M + H ⁺ ) 528.3 (100%); NKM8 (calc. For C31H32I3O3) 528.2975, found 528.2958; ^one H NMR (300 MHz, SP3OP, 25 ° C) δ 7.19 (t, 12H), 6.98 (ά, 1 = 1.5 Hz, 2H), 4.74 (, 1 = 13.9 Hz , 1H), 3.85 (άά, 1 = 10.25, 4.76 Hz, 1H), 3.65 (t, 1H), 3.56 (t, 4H), 3.15 (t, 2H) , 2.96 (t, 3H), 2.07 (t, 2H), 1.37 (t, 2H), 1.22 (t, 2H), 0.84 (1, 1 = 7.0, 3H ).

Application

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

The compounds of formula I of the present invention are also useful for inhibiting HIV in an ex ν νο sample containing HIV or suspected of being infected with HIV. Therefore, the compounds of the present invention can be used to inhibit HIV present in a sample of body fluid (for example, in serum or seminal fluid), which contains or is assumed to contain or be infected with HIV.

The compounds of the invention are also useful as standard or reference compounds for use in tests or assays for determining the ability of an agent to inhibit the replication of a viral clone and / or HIV protease, for example, in pharmaceutical research programs. Therefore, the compounds of the present invention can be used as control or reference compounds in such analyzes and as a quality control standard. The compounds of the present invention may be provided in a commercial kit or container for use as such a standard or reference compound.

Since the compounds of the present invention exhibit specificity for the HIV protease, the compounds of the present invention can also be used as diagnostic reagents in a diagnostic assay for the detection of HIV protease. Thus, inhibition of protease activity in a test (such as described here) by a compound of the invention will be indicative of the presence of HIV protease and the HIV virus.

As used herein, μg means micrograms, mg means milligrams, g means grams, μl means microliter, ml means milliliter, l means liter, nM means nanomolar, μM means micromolar, mmM means millimolar, M means molar and nm means nanometer. 8 | dta means 8 | dta-A1bps11 Sogr., 81 Bosh, MO.

HIV RNA Analysis

DNA plasmids and RNA transcripts ίη νίΐΓΟ.

Plasmid ρΌΛΒ 72, containing both dad- and po1-sequences of BH10 (lp 1131816), cloned into ΡΤΖ 19B, was obtained according to Epkkop-USCHep e1 a1. ΆΙΌ8 Kekeagsk apit Nitap VeChouikek 1989, 5, 577. Plasmid was linearized with Wat ΗΙ before the formation of η νίίτο RNA transcripts using the set B1GorOeTeIr with T7 RNA polymerase. The synthesized RNA was purified by treatment of RNA-free containing DNA-ase (Rgheda), extraction with phenol-chloroform and precipitation from ethanol. RNA transcripts were dissolved in water and stored at -70 ° C. The concentration of RNA was determined by A ₂₆₀ .

Probes.

Biotinylated catching probes were purified by HPLC after synthesis on Arrieb Vyuk81et (Eocher Syu, SA) DNA synthesizer by adding biotin to the 5 'terminal end of the oligonucleotide using the Biotinphosphoramidite reagent according to Coxicha, Te1. Be11. 1989, 30, 6287. DBP-catching biotinylated probe (5biotin-PT AT AOSTSSSTOSTTOSSS ACT A 3 ') was complementary to nucleotides 889-912 of HXB2 and PO1-catching biotinylated probe (5'-biotin-SSSTATSATTTTTOOTTTSSAT 3') complementary to nucleotides 2374 bl -2395 HHB2. Alkaline phosphatase-conjugated oligonucleotides used as reporter probes were prepared in a superdelay (Grade O1edo, CA). The po1 reporter probe (5 'STOTSTTSTTTOATAAAAASSTS 3') was complementary to nucleotides 2403-2425 HXB2. The dad-reporter probe (5 'СССАТАТТТТСТСТСАССТТСТТ 3') was complementary to nucleotides 950-973 НХВ2. All nucleotide positions were positions on the OepVapk genetic sequence data bank, available through the Opeisk Souther Ogoir software package for 8 hectares | Apses Ap118 (Aegegeai and Asyke Veiagsi 1984, 12, 387). Reporter probes were prepared in the form of 0.5 μM stock solutions in 2 × 88 ° C (0.3 M Iac1, 0.03 M sodium citrate), 0.05 M Tris pH 8.8, 1 mg / ml B8A (bovine serum albumin). Biotinylated catching probes were prepared as 1 00 μM stock solutions in water.

Streptavidin-coated tablets.

Streptavidin-coated plates were obtained from Ei Rop1 Vulnecodipa 8u81etk (Wokloop, MA).

The original solutions of cells and viruses.

The MT-2 and MT-4 cells were stored in a BPM1 1640 medium supplemented with 5% fetal calf serum (PTS) for MT-2 cells or 10% PTS for MT-4 cells, 2 mM L-glutamine and 50 μg / ml gentamicin ( all drugs from 01). BE (replicative form) HIV-1 was cultured in MT-4 cells in the same medium. Virus stock solutions were prepared approximately 10 days after acute infection of MT4 cells and stored in aliquots at -70 ° C. Infectious titers of initial solutions of HIV-1 (BE) were 1-3 x 10 ⁷ PFU (plaque-forming units) / ml according to the analysis of plaque formation on MT-2 cells (see below). Each aliquot of virus stock used for infection was thawed only once.

To assess antiviral efficacy, infected cells were re-cultured one day before infection. On the day of infection, the cells were resuspended to a concentration of 5 x 10 ^five cells / ml in BPM1 1640, 5% PTS for volume infection in volume and up to a concentration of 2 x 1 0 ⁶ cells / ml in Dulbecco's modified Eagle medium with 5% PTS for infection in a microtiter plate. The virus was added and culture continued for 3 days at 37 ° C.

Analysis of HIV RNA.

Cell lysates or purified RNA in 3M or 5M ΟΕΌ were mixed with 5M лов and a catching probe to a final concentration of 3M isothiocyanatuganidium and a final concentration of biotinoligonucleotide 30nM. Hybridization was carried out in sealed 96-well tissue culture plates with a-shaped bottom (Yips or Co gil) for 16–20 h at 37 ° C. After hybridization, the RNA was diluted three times with deionized water to a final concentration of 1 M isothiocyanatine guanidium and aliquots (150 μl) were transferred to cells of a streptavidin-coated microtiter plate. Binding of the catching probe and the hybrid catching probe-RNA with immobilized streptavidin was allowed to proceed for 2 hours at room temperature, after which the plates were washed 6 times with EnRop1 Erythrophystery Buffer (PBS), 0.05% Tween- 20). A second hybridization of the reporter probe to the immobilized complex of the catching probe and hybridized RNA targets was performed in a washed streptavidin-coated cell by adding 120 μl of a hybridization cocktail containing 4 x 88 C, 0.66% Tyop x 100, 6.66% deionized formamide, 1 mg / ml albumin bovine serum and 5nM reporter probe. After hybridization for one hour at 37 ° C, the plate was washed again 6 times. The activity of the immobilized alkaline phosphatase was determined by adding 100 μl of 0.2 mM 4-methylumbelliferyl phosphate (MibR, 1Bb Dyed) in δ buffer (2.5M diethanolamine, pH 8.9 (1Bb 8c1eptCPS). 10mM MdCl ₂ , 5 mM zinc acetate dihydrate and 5 mM Ν-hydroxyethylethylenediamine triacetic acid). The plates were incubated at 37 ° C. Fluorescence at 450 nm was measured using a microplate fluorometer (Loupe-ec) excited at 365 nm.

Evaluation of the effect of the compound on HIV-1-infected MT-2 cells using a microplate.

The evaluated compounds were dissolved in DMSO and diluted in culture medium to a concentration two times higher than the tested one, and the maximum concentration of DMSO 2%. Further threefold serial dilutions of the compound in the culture medium were performed directly in a microtiter plate with an I-shaped bottom (Νιικ). After diluting the compound, MT-2 cells (50 μl) were added to a final concentration of 5 x 10 ^five per ml (1 x 10 ^five per cell). Cells were incubated with the compound for 30 min at 37 ° C in a CO2 incubator. To assess the antiviral capacity, the appropriate dilution of the initial solution of the HIV-1 virus (RF) (50 μl) was injected into culture cells containing cells and dilutions of the tested compounds. The final volume in each cell was 200 μl. Eight cells per tablet were left uninfected by adding 50 μl of medium instead of virus, while eight cells were infected in the absence of any antiviral compound. To assess the toxicity of the compound, parallel plates were cultured without viral infection.

After 3 days of cultivation at 37 ° C in a humidified chamber inside an incubator with CO ₂ all but 25 μl of medium per well were removed from HIV-infected plates. To the deposited cells and the remaining medium in each cell, 37 μl of 5M конеч, containing a biotinylated catching probe, were added to a final concentration of 3M and 30nM of the catching probe. Hybridization of the catch probe with HIV RNA in the cell lysate was performed in the same well microplate used to cultivate the virus by sticking the tablet with a device for sticking the plates (Co81ag) and incubating for 16-20 hours in an incubator at 37 ° C. Then distilled water was added to each well to dilute the hybridization reaction mixture three times and 150 μl of this diluted mixture was transferred to a streptavidin-coated microtiter plate. The amount of HIV RNA was determined as described above. For each microtiter plate, a standard curve was prepared, obtained by adding known amounts of the transcript ρΌΑΒ 72 w νίΙΐΌ RNA into cells containing lysed infected cells, in order to determine the amount of viral RNA obtained during infection.

In order to standardize the viral inoculum used in evaluating compounds for antiviral activity, a sample of virus dilutions was sampled, which resulted in a value of 1C ₉₀ (the concentration of the compound required to reduce the concentration of HIV RNA by 90%) for dideoxycytidine (ddC) at a concentration of 0.2 μg / ml. When following this technique, the value of 1C ₉₀ for other antiviral compounds, and more and less active than ddC, were reproduced using several initial solutions of HIV-1 (RF). This virus concentration was ~ 3 x 10 ^five REI (measured by plaque assay on MT-2 cells) per cell to be analyzed and typically yielded about 75% of the maximum concentration of viral RNA attainable for any viral inoculum. In the analysis of HIV RNA values 1C ₉₀ was determined as the percentage reduction of the net signal (signal from samples with infected cells minus the signal from samples with uninfected cells) when analyzing RNA from a pure signal from infected untreated cells of the same culture plate (average for eight cells). The good results of individual experiments on infection and RNA analysis were judged by three criteria. It was required that the viral infection gave a signal in the analysis of RNA that was equal to or exceeded the signal generated from 2 ng of the transcript of ρΌΛΒ 72 Ιη νίίτο RNA. 1C value ₉₀ for ddC, determined in each experiment, should be between 0.1 and 0.3 µg / ml. Finally, the plateau level of viral RNA obtained with an effective protease inhibitor should be lower than 10% of the level obtained with uninhibited infection. A compound is considered active if it is found that its 1C ₉₀ lower than 1 μM.

When testing antiviral activity, all manipulations with microtiter plates, following the initial addition of a 2x concentrated solution of the compound to one row of cells, were performed using Regix E1tc / Ch1i Pro-Raye.

Dose and formulation

The antiviral compounds of the invention may be administered to treat viral infections by any means that ensure contact of the active agent with the site of exposure of the agent, i.e. with viral protease in the body of a mammal. They can be administered by any conventional methods used for the administration of pharmaceuticals, either as individual therapeutic agents or in combination with therapeutic agents. They may be administered in pure form, but are preferably administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The dose administered must, of course, vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent, the method and route of administration; the age, health and weight of the recipient; the nature and severity of the symptoms; type of concomitant treatment; frequency of treatment; desired effect. It can be expected that the daily dose of the active ingredient should be from about 0.001 to about 1000 mg per kilogram of body weight, and the preferred dose should be from about 0.1 to about 30 mg / kg.

Dosage forms suitable for administration contain from about 1 mg to about 1 00 mg of active ingredient per unit. In these pharmaceutical compositions, the active ingredient should generally be present in an amount of 0.5-95% by weight, calculated on the total weight of the composition. The active ingredient may be administered orally in the form of solid dosage forms, such as capsules, tablets and powders, or in the form of liquid dosage forms, such as elixirs, syrups and suspensions. It may also be administered parenterally in the form of sterile liquid dosage forms.

Gelatin capsules contain the active ingredient and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. The same diluents can be used to make compressed tablets. Both tablets and capsules can be produced in a sustained release form to ensure continuous release of the drug for several hours. Extruded tablets may be coated with sugar or a film in order to mask any unpleasant taste and to protect the tablet from the effects of the atmosphere, or the intestinal sheath for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorants and flavors to improve patient perception.

In general, water, suitable oil, saline, brine, aqueous dextrose (glucose), and related solutions of sugars and glycols, such as propylene glycol or polyethylene glycols, are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents and, if required, buffering agents. Suitable stabilizing agents are antioxidants, such as sodium bisulfite, sodium sulfite or ascorbic acid, either alone or in combination. Citric acid and its salts and the sodium salt of EDTA are also used. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in RettDuy'Riagthasei1 8c1epse5. hprga. standard reference in this area.

Pharmaceutical dosage forms used to administer the compounds of the invention are illustrated.

Capsules

A large number of single capsules can be obtained by filling each of the two-piece standard hard gelatin capsules with 1 00 mg of the active ingredient in the powder, 150 mg of lactose, 50 mg of cellulose and 6 mg of magnesium stearate.

Soft gelatin capsules

The mixture of active ingredient in edible oil, such as soybean oil, cottonseed oil or olive oil, can be prepared and injected into gelatin using a piston injection pump to form soft gelatin capsules containing 1,00 mg of active ingredient. Then the capsules should be washed and dried.

Pills

A large number of tablets can be prepared by conventional means so that the dosage unit 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. Suitable coatings may be used to improve the taste or eliminate absorption.

Suspension

An aqueous suspension for oral administration can be prepared in such a way that each 5 ml contains 25 mg of finely ground active ingredient, 200 mg of sodium carboxymethylcellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution I.8.P. and 0.025 mg vanillin.

Injection solution

A parenteral composition suitable for administration by injection can be prepared by mixing 1.5% by weight of the active ingredient in a 10% by volume solution of propylene glycol in water. The solution is sterilized by conventional methods.

The combination of components (a) and (b)

Each component of the therapeutic agent of the invention may be independently presented in any dosage form described above, and may also be administered in various ways, as described above. In the following description, it should be understood that component (b) represents one or more of the agents described above. Thus, if components (a) and (b) are to be administered for treatment simultaneously or independently, each agent of component (b) can also be administered simultaneously or independently.

Components (a) and (b) of the present invention can be formulated together as a single dosage unit (i.e. combined in one capsule, tablet, powder, liquid, etc.), i.e. as a combined product . When components (a) and (b) are not formulated together as a single dosage unit, component (a) can be entered simultaneously with component (b) or in any other order, for example, component (a) according to the invention can be administered first followed by the introduction component (b), or they can be entered in the reverse order. If component (b) contains more than one agent, for example, one PT inhibitor and one protease inhibitor, these agents can be administered together or in any other order. If components are not administered at the same time, it is preferable that component (a) and (b) be administered at intervals of not more than one hour. The preferred route of administration of components (a) and (b) is oral. The terms oral agent, oral inhibitor, oral compound, or the like, as used herein, mean compounds that can be administered orally. Although it is preferable that both component (a) and component (b) are administered in the same way (i.e., for example, both orally) or in the same dosage form, if required, they can be administered in different ways (t. that is, for example, one component of the combined product may be administered orally, and the other component may be administered intravenously) or in various dosage forms.

As will be understood by the person skilled in the art, the dosage for the combination therapy of the invention may vary depending on various factors, such as the pharmacodynamic characterization of a particular agent and the method and route of administration; the age, health and weight of the recipient; the nature and severity of the symptoms; type of concomitant treatment; frequency of treatment and the desired effect, as described above.

The proper dose of components (a) and (b) of the invention should be easily determined by a qualified physician based on the present description. As a general guideline, a typical daily dose is from about 100 mg to about 1.5 g of each component. If component (b) represents more than one compound, then a typical daily dose may be from about 1 00 mg to about 1.5 g of each agent of component (b). As a general indication, if the compounds of component (a) and component (b) are administered in combination, then the amount of each component in a dose can be reduced to about 70-80% relative to the usual dose of the component when it is administered alone as the only agent for treating HIV .

Combined products according to the invention can be formed in such a way that although the active ingredients are combined into a single dosage unit, physical contact between them is minimized. In order to minimize contact, for example, by the oral administration of products, one active ingredient may be formulated with an intestinal (cr) coating. Using the intestinal coating of one of the active ingredients, it is possible not only to minimize the contact between the combined active ingredients, but also to control the release of one of these components in the gastrointestinal tract so that one of these components is not released in the stomach, but released in the intestine.

In another embodiment of the invention in which oral administration is desired, a combination product is proposed, where one of the active ingredients is coated with a sustained-release material, which serves for sustained release in the gastrointestinal tract and also serves to minimize the physical contact between the combined active ingredients. In addition, the release-retarding component can additionally have an intestinal coating so that the release of this component occurs only in the intestine. Another option includes the preparative form of a combined product, in which one component is coated with a polymer, slowing down the release and / or providing release in the intestine, and the other component is also coated with a polymer, such as low viscosity hydroxypropylmethylcellulose or other suitable materials known in the art. to further separate the active ingredients. The polymer coating serves to form an additional barrier against interaction with another component. In each formulation where contact between components (a) and (b) is prevented by coating or some other material, contact between the individual agents of component (b) can also be prevented.

The dosage forms of the combination products of the present invention, in which one active ingredient has an intestinal coating, may be in the form of tablets, where the component with the intestinal coating and the other active ingredient are mixed together and then compressed into a tablet, or those where the component with the intestinal coating is pressed in one layer of the tablet, and the other active ingredient is pressed into an additional layer. Optionally, for the additional separation of two layers, one or more placebo layers may be present between the layers of active ingredients. In addition, the dosage forms of the present invention may be in the form of capsules, in which one active ingredient is compressed into a tablet or prepared in the form of a variety of microtablets, particles, granules or other non-cancerous forms (pop-ups) that have an intestinal coating. These microtablets, particles, granules or non-damaging forms (pop-ups) with an intestinal coating are then placed in a capsule or compressed in a capsule together with a granulate of another active ingredient.

These, as well as other ways to minimize contact between the components of the combined products of the present invention, regardless of whether they are administered in the same dosage form or are administered in separate forms, but at the same time or together in the same way, will be easily understood by specialists in this area based on the present description.

The subject of the present invention also includes pharmaceutical kits for treating HIV infection, which include a therapeutically effective amount of a pharmaceutical composition containing a compound of component (a) and one or more compounds of component (b) in one or more sterile containers. Sterilization of the container can be accomplished using conventional sterilization methodology well known to those skilled in the art. Component (a) and component (b) may be in the same sterile container or in separate sterile containers. Sterile containers of materials may include, if desired, separate containers or one or more containers with many compartments. Component (a) and component (b) can be separated or physically combined into a single dosage form or unit, as described above. Such kits may additionally include, if required, one or more of the various conventional components of pharmaceutical kits, such as, for example, one or more pharmaceutically acceptable carriers, additional vessels for mixing the components, etc., which should be easily understood by those skilled in the art. Instructions, either in the form of inserts or in the form of labels, indicating the quantities of the components that need to be entered, directions for administration and / or instructions for mixing the components may also be included in the kit.

It is clear that numerous modifications and variations of the present invention are possible in the light of the foregoing. Therefore, it should be clear that within the framework of the following claims, the invention may be implemented more broadly than specifically described in the examples.

Claims

CLAIM

1. The compound of formula I

I or a pharmaceutically acceptable salt thereof, or a prodrug thereof, wherein the prodrug of formula I is a compound in which two hydroxy groups are joined to form an epoxide, —OCH ₂ 8CH ₂ O—; -OS (= O) O-; -OSH ₂ O-; -OS (= 8) O-; -OS (= O) C (= O) O-; -OS (CH3) ₂ O-;

-OS ((CH2) zPN2) (CH3) O-; -OS (OCH3) (CH ₂ CH ₂ CH ₃ ) O-; or —O8 (= O) O— groups.

2. The compound according to claim 1, where the compound is a compound of formula I.

3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a prodrug thereof.

4. The composition according to claim 3, where the compound is a compound of formula I.

5. The use of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a prodrug thereof for the manufacture of a medicament for the treatment of HIV infection, wherein a prodrug of formula I is a compound in which two hydroxy groups are connected to form an epoxide, —OCH ₂ 8CH ₂ O—; -OS (= O) O-; -OS1GO-; -OS (= 8) O-; -OS (= O)

C (= O) O-; -OC (CH ₃ ) ₂ O-; -OS ((CH ₂ ) ₃ XH ₂ ) (CH ₃ ) O-; -OS (OCH ₃ ) (CH2CH2CH ₃ ) O-; or -O8 (= O) of the Group.

6. The use according to claim 5, where the compound is a compound of formula I.

7. The use of combination (a) and (b) for the preparation of a medicament for the treatment of HIV infection, where (a) is a compound of formula I, or a pharmaceutically acceptable salt thereof, or a prodrug thereof

I where a prodrug of formula I is a compound in which two hydroxy groups are connected to form an epoxide, —OCH ₂ 8CH ₂ O—; -OS (= O) O-; -OSH2O-; -OS (= 8) O-; -OS (= O) C (= O) O-; -OC (CH ₃ ) ₂ O-; -OS ((CH ₂ ) ₃ XH ₂ ) (CH ₃ ) O-;

-OS (OCH3) (CH2CH2CH3) O-; or —O8 (═O) Ogroup, and (b) represents at least one compound selected from the group consisting of HIV revertase inhibitors and HIV protease inhibitors.

8. The use according to claim 7, where the compound is a compound of formula I.

9. The use of claim 7, wherein the invertase inhibitor is a nucleoside invertase inhibitor.

10. The use according to claim 9, where the nucleoside inhibitor of revertase is selected from ΑΖΤ, 3TC, άάΣ, 00C and ά4Τ, and the protease inhibitor is selected from saquinavir, ritonavir, indinavir, UX478, nelfinavir, ΚΝΣ-272, COP-61755 and ϋ103017.

eleven . The use of claim 1, wherein the nucleoside revertase inhibitor is selected from ΑΖΤ and 3TC, and the protease inhibitor is selected from saquinavir, ritonavir and indinavir.

1 2. The use of claim 11, wherein the nucleoside revertase inhibitor is ΑΖΤ.

1 3. The use of claim 11, wherein the protease inhibitor is indinavir.

14. A pharmaceutical kit for treating HIV infection, which comprises a therapeutically effective amount of (a) a compound according to claim 1; and (b) at least one compound selected from the group consisting of HIV revertase inhibitors and HIV protease inhibitors in one or more sterile containers.

15. The kit according to 14, in which component (a) is a compound of formula I.

Eurasian Patent Organization, EAPO

Russia, Moscow, GSP 103621, M. Cherkassky per., 2/6