RU2137768C1 - Inhibitors of hiv-protease - Google Patents

Abstract

FIELD: organic chemistry, biochemistry, pharmacy. SUBSTANCE: invention describes new compounds of the formula (I) or their pharmaceutically acceptable salts where

means a stable 8-10-membered bicyclic heterocycle each ring of that can be saturated or unsaturated and indicated heterocycle consists of carbon atoms and 1-3 heteroatoms taken among the group including atoms N, S or O. Indicated heterocycle can be unsubstituted or substituted with halogen atom or lower C_1-4-alkyl at condition that

Description

 The invention is a partial continuation of the application Merck 191391A, filed December 15, 1993. This application relates to the application Merck 18996 with U. S-S-N. 08/059038 dated May 7, 1993, which is a partially continued application for Merck 185971B, which in turn is a partially continued application for Merck 185971A, which is a partial continued application for US application serial number 07/7895 08 dated November 8, 1991 This application relates to the following patent documents: US application serial number 595913 dated October 11, 1990 (Merck case 18236); US application serial number 746460 of August 16, 1991 (Merck case 18466); Merck application filed October 23, 1991, and Merck application 18416.

 The invention relates to compounds that inhibit the protease encoded by the human immunodeficiency virus, or their pharmaceutically acceptable salts, and such compounds are used to prevent HIV infection, treat HIV infection and treat acquired as a result of immunodeficiency syndrome (AIDS). The present invention also relates to pharmaceutical compositions containing such compounds, and to a method for using the present compounds and other agents for the treatment of AIDS and viral infection of HIV.

 The technical field to which the invention relates.

 Retrovirus, designated as human immunodeficiency virus (HIV), is an etiological agent of a complex disease, which consists in the progressive destruction of the immune system (acquired immunodeficiency syndrome) and degeneration of the central and peripheral nervous system. Such a virus was previously known as LAV, HTLV-111 or ARY. A common feature of retroviral replication is the extensive post-translational treatment of viral-encoded protease precursor polyproteins, resulting in the production of mature viral proteins required for the assembly and functioning of the virus. Inhibition of this process prevents the formation of an infectious virus under normal conditions. For example, Kohl, N.E. with al., in Proc. Natl Acad. Sci .. 85, 4686 (1988) showed that genetic inactivation of HIV-encoded protease results in the formation of immature, non-infectious viral particles. These results indicate that inhibition of HIV protease is a viable method of treating AIDS and preventing or treating HIV infection.

 The nucleotide sequence of HIV detects the presence of a ro1 gene in one open reading frame (Rather, Z, et al., Nature, 313, 277 (1985)). The amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, endonuclease, and HIV protease (Toh, H. et al., EMBO J., 4, 1267 (1985); Power MD, et al., Science, 231, 1567 (1986); Pearl, LH et al., Nature, 329, 351, (1987)). Applicants have demonstrated that the compounds of the present invention are HIV protease inhibitors.

 Summary of the invention.

 The compounds of formula I indicated in the description can be used to inhibit HIV protease, prevent HIV infection, treat HIV infection and treat AlDS, and they can be used as such, in the form of pharmaceutically acceptable salts, ingredients of the pharmaceutical composition, optionally in combination with other antiviral agents, immunomodulators, antibiotics or vaccines. Methods for treating AIDS, methods for preventing HIV infection and methods for treating HIV infection are also disclosed.

 The application may use the abbreviations presented at the end of the description.

 Description of the invention and preferred implementations.

или их фармацевтически приемлемые соли
где

представляет собой устойчивый 8-10-членный бициклический гетероцикл, каждое из колец которого может быть насыщенным или ненасыщенным, причем указанный гетероцикл состоит из атомов углерода и 1-3 гетероатомов, выбранных из группы, состоящей из N, S или O, и указанный гетероцикл может быть незамещенным или замещенным OH, галогеном, C_1-4алкилом, оксогруппой;
при условии, что

не является следующими группами:

Одна из реализаций настоящего изобретения представляет собой соединения формулы I или их фармацевтически приемлемые соли, в которых

представляет собой устойчивый 8-10-членный бициклический гетероцикл, любое из колец которого может быть насыщенным или ненасыщенным, и указанный гетероцикл состоит из атомов углерода и 2-х гетероатомов, выбранных из группы, состоящей из N или O, причем гетероатомы находятся в различных кольцах.The invention relates to compounds of formula I, their combinations or their pharmaceutically acceptable salts, intended for inhibiting HIV protease, preventing or treating HIV infection, and treating acquired as a result of immunodeficiency syndrome (AIDS). The compounds of formula I are defined as follows:

or their pharmaceutically acceptable salts
Where

represents a stable 8-10 membered bicyclic heterocycle, each of the rings of which may be saturated or unsaturated, said heterocycle consisting of carbon atoms and 1-3 heteroatoms selected from the group consisting of N, S or O, and said heterocycle may be unsubstituted or substituted by OH, halogen, C _1-4 alkyl, oxo;
provided that

not the following groups:

One of the implementations of the present invention are compounds of formula I or their pharmaceutically acceptable salts, in which

represents a stable 8-10 membered bicyclic heterocycle, any of whose rings may be saturated or unsaturated, and said heterocycle consists of carbon atoms and 2 heteroatoms selected from the group consisting of N or O, and the heteroatoms are in different rings .

ограничен значениями:

X представляет собой O или S,
или их фармацевтически приемлемые соли.The second implementation of the invention is a compound of formula I, in which

limited by values:

X represents O or S,
or their pharmaceutically acceptable salts.

ограничен значением:

или их фармацевтически приемлемые соли.A third embodiment of the invention is a compound of formula I, in which

limited to:

or their pharmaceutically acceptable salts.

которое представляет собой N-(2(R)-гидрокси-1(S)-инданил)- 2(R)-фенилметил-4(S)-гидрокси-5(1-(4-(3-фуро[2.3-b] пиридил- метил)-2(S)-N'-(трет. -бутилкарбоксамидо)пиперазинил)пентанамид, или его фармацевтически приемлемую соль.Another embodiment of the present invention is a compound A:

which is N- (2 (R) -hydroxy-1 (S) -indanyl) - 2 (R) -phenylmethyl-4 (S) -hydroxy-5 (1- (4- (3-furo [2.3-b ] pyridylmethyl) -2 (S) -N '- (tert.-butylcarboxamido) piperazinyl) pentanamide, or a pharmaceutically acceptable salt thereof.

 The compounds of the present invention have chiral centers and exist as racemates, racemic mixtures and individual diastereomers or enantiomers, all of the isomeric forms being included in the present invention. The racemic mixture encompasses mixtures of stereoisomers in a ratio of 50:50 and in other ratios.

) встречается более одного раза в любой составляющей части соединения или в формуле I, ее обозначение в каждом случае не зависит от обозначения в любом другом случае. Кроме того, комбинации заместителей и/или переменных фрагментов допустимы лишь в тех случаях, когда такие комбинации обеспечивают в результате устойчивые соединения.In the case when any variable (for example,

) occurs more than once in any component of the compound or in formula I, its designation in each case does not depend on the designation in any other case. In addition, combinations of substituents and / or variable moieties are permissible only when such combinations result in stable compounds.

 Used in the text of the description, the term "alkyl", unless otherwise indicated, includes both branched and straight chain (normal structure) saturated aliphatic hydrocarbon groups containing the indicated number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl); as used herein, the term “halogen” means fluoro, chloro, bromo and iodo.

 Pharmaceutically acceptable salts of the compounds of formula I (as water or oil soluble or dispersible products) include conventional non-toxic salts or quaternary ammonium salts, which are obtained, for example, from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dihydrochloride, diphenosulfate, heptamisulfonate, gumetansulfonate, gumetansulfonate, gemfansulfonate, gemansulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, ikrat, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine, N-methyl-D-glucamine salts, and salts with amino acids such as arginine, lysine, and the like. In addition, basic nitrogen-containing groups can be quaternary with agents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, such as aralkyl halides such as benzyl and phenethyl bromides, etc. Other pharmaceutically acceptable salts include ethanolate sulfate and sulfate salts.

 Schemes I-II (see end of description) show the preparation of new compounds of the invention. The examples specifically illustrate the application of the following schemes to specific compounds.

 The amide coupling reactions used to prepare the compounds of the present invention are usually carried out by the carbodiimide method using reagents such as dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Other methods for producing an amide or peptide bond include; but are not limited to synthetic routes through an acid chloride, azide, mixed anhydride or activated ester. A liquid-phase amide coupling reaction is usually carried out, however, solid-phase synthesis can be used instead of it using the classical Merrifield method. Attaching or removing one or more protecting groups is a common procedure.

 Further related information on the nature of synthetic methods is contained in EPO 0337714 and EPO 0541168.

 One of the methods for preparing compounds of formula I is represented by Scheme I. Dihydro-5 (S) - (tert-butyldimethylsilyloxymethyl) - 3 (2H) -furanone (compound 1, below) is prepared by standard methods known in the art from commercially available dihydro-5 (S) - (hydroxymethyl) -2 (3H) -furanone. After alkylation of compound 1 to form compound 2, the protective group of lactone 2 is removed with an aqueous HF solution to give compound 3.

 The alcohol group of compound 3 is activated by conversion to a leaving group such as mesylate, tosylate or triflate, by treating the alcohol with sulfonyl chloride or (preferably) sulfuric anhydride, for example trifluoromethanesulfonic anhydride, in the presence of a hindered amine base such as triethylamine, diethylisopropylamine or 2.6 β-lutidine to give a compound such as Compound 4. The leaving group of Compound 4 is replaced with amine 5, for example 4- (1,1-dimethylethoxycarbonylamino) piperazine-2 (S) -carboxamide, in such a solution a reactor such as DMF or xylene, to give a compound such as compound 6. The trifluoromethanesulfonyloxy group can be replaced with an amine at room temperature in a solvent such as isopropanol or methylene chloride by treatment with N, N-diisopropylethylamine.

 Compound 6 is hydrolyzed with an aqueous solution of lithium or sodium hydroxide, and the resulting hydroxy acid 7 is converted to the protected hydroxy acid 8. The hydroxyl group is conveniently protected with a standard silyl protecting group such as tert-butyldimethylsilyl or tert-butyl diphenylsilyl.

The protected hydroxy acid 8 is then combined with the desired R ¹² amine to form compound 9, and the silyl protecting group is removed with a fluoride ion to give compound 10.

One of the preferred methods is the synthesis of epoxide 12 by reaction 11 in the presence of a strong base. A metal-containing base can be used as a strong base, the reaction being carried out in an inert anhydrous organic solvent such as cyclic or acyclic hydrocarbons, including hexane, pentane, cyclohexane and the like. Suitable strong bases include: LiN / (CH ₃ ) ₃ Si / 2, KN (CH ₃ ) ₃ Si / 2, NaN / (CH ₃ ) ₃ Si / 2, n-butyllithium (n-BuLi), sec-BuLi , tert-BuLi, potassium tert-butylate, lithium diisopropylamide (LDA), lithium isopropylcyclohexylamide, lithium pyrrolidide, lithium tetramethylpiperidide, phenyl lithium, isopropyl magnesium chloride, isobutyl magnesium chloride, and other similar strong bases known in the art. Preferred strong bases are n-BuLi, sec. -BuLi, LiN / (CH ₃ ) ₃ Si / 2 and LDA, wherein n-BuLi and LiN / (CH ₃ ) ₃ Si / 2 are most preferred.

 Preferably, 1 to 2 molar equivalents of a strong base are used per 1 molar equivalent of compound 11.

 Compound 13 is prepared by the reaction of compound 12 with N-tert.-butyl-4- (1, 1-dimethylethoxycarbonylamino) piperazin-2 (S) carboxamide (5). Preferably, 1-3 molar equivalents of amine (5) are used per molar equivalent of epoxide 12, with a V: IV ratio of 1.05: 1 molar equivalent being more preferred.

This reaction can be carried out in any suitable solvent, for example, in a solvent selected from hydrocarbons such as toluene, ethers such as diethyl ether, alcohols such as methanol, ethanol or isopropanol, nitriles such as acetonitrile, and such esters, as ethyl acetate, or combinations thereof, with alcohols being the most preferred solvents and isopropanol the most preferred solvent. The reaction temperature can be maintained in the range from ambient to reflux temperature of the solvent used, however, it is preferable to carry out the reaction at an elevated temperature, for example in the range of 80-90 ^o C, most preferably at 83-85 ^o C.

 Activated glycidols can be prepared by methods known in the art, as described, for example, in J. Klunder et al., J. Org. Chem., 1989, 54, 1295-1304, and in the references cited in this work.

 Amide compounds, such as compound 11, can be prepared according to standard procedures known to those skilled in the art, for example, as described in Example 10, using appropriate starting materials.

 Protecting groups, for example nitrogen protecting groups, can be used, when necessary, for the practical implementation of the present invention. For example, nitrogen at position 4 of piperazine 2-tert.-butylcarboxamide can be protected by a group such as BOC, CBZ, benzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, trifluoroacetamide, trialkylsilyl, and other groups known in the art.

в которой P представляет собой такую азот-защищающую группу, как -BOC или -CBZ,
также получают согласно способу, описанному в Схеме I, предпочтительно используя 5-трифторметансульфонилоксиметиловый аналог лактона 4.The compound of formula 15

in which P represents a nitrogen protecting group such as —BOC or —CBZ,
also prepared according to the method described in Scheme I, preferably using the 5-trifluoromethanesulfonyloxymethyl analogue of lactone 4.

может быть получено различными путями из соединения 14

которое получают после удаления азот-защищающей группы из соединения 15 с использованием способов, хорошо известных в данной области, например путем каталитического гидрирования с целью удаления CBZ группы или обработки триметилсилилтрифлатом и 2,6- лутидином при 0^oC в таком растворителе, как CH₂Cl₂, или обработкой с помощью 6 N HCl в изопропаноле с целью удаления группы BOC.The compound of formula 16

can be obtained in various ways from compound 14

which is obtained after removal of the nitrogen protecting group from compound 15 using methods well known in the art, for example by catalytic hydrogenation to remove the CBZ group or treatment with trimethylsilyl triflate and 2,6-lutidine at 0 ^° C. in a solvent such as CH ₂ Cl ₂ , or by treatment with 6 N HCl in isopropanol to remove the BOC group.

The nitrogen at position 4 of piperazinyl in compound 14 can be alkylated with a compound of formula R ¹ —X in a solvent such as DMF in the presence of Et ₃ N at room temperature when X is Cl, Br or I. Procedures for such procedures well known to the person skilled in the art.

 The compounds of the present invention are also illustrated in the table of Example 3 below.

 The compounds of the present invention are useful for preparing and conducting screening assays for antiviral compounds. For example, the compounds of the present invention are very useful for isolating enzyme mutants, which are excellent screening tools for more powerful antiviral compounds. In addition, the compounds of the present invention can be used to establish or determine the binding site of other antiviral agents with an HIV protease, for example, a competitive inhibition method. Thus, the compounds of the present invention are commercial products sold for such purposes.

 The compounds of the present invention can be used to inhibit HIV protease, prevent or treat infection with human immunodeficiency virus (HIV), and treat subsequent pathological conditions such as AIDS. Treating AIDS or preventing or treating HIV infection includes, but is not limited to treating a large number of HIV conditions: AIDS ARC (AIDS related complex), both symptomatic and asymptomatic, as well as acute or potential exposure to HIV. For example, the compounds of the present invention can be used to treat HIV infection after suspicion of subsequent exposure to HIV, for example, blood transfusion, organ transplantation, replacing the body fluid, the presence of bite marks, an accidental injection with a needle, or exposure to the patient’s blood during surgery.

 To this end, the compounds of the present invention can be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intravenous injections or infusions), by inhalation spray or rectally in unit dosage formulations comprising conventional non-toxic pharmaceutically acceptable carriers, stimulants and auxiliary agents.

 Thus, in accordance with the present invention, a treatment method and pharmaceutical compositions for treating HIV and AIDS infection are further provided. Such treatment includes the use, on a patient in need of such treatment, of a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

 Such pharmaceutical compositions may be formulated as orally administered suspensions or tablets; nasal sprays; sterile injectable preparations, for example in the form of sterile injectable aqueous or oily suspensions, or suppositories.

 When administered orally in the form of suspensions, such compositions are prepared according to methods well known in the pharmaceutical art and may contain microcrystalline cellulose to provide mass, alginic acid or sodium alginate as a suspending agent, methyl cellulose as a viscosity enhancer and sweetening agents and / or perfumes known in the art. In the form of immediate release tablets, such compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

 When used in the form of nasal aerosols or by inhalation, such compositions are prepared by methods well known in the field of pharmaceutical formulations, and they can be produced as solutions in physiological saline, using benzyl alcohol or other suitable preservatives, adsorption promoters to enhance bioavailability, fluorocarbons and / or other solubilizing or dispersing agents known in the art.

 Injectable solutions or suspensions may be formulated according to known methods using non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution or suitable dispersing or wetting and suspending agents, such as sterile, soft, stable oils, including synthetic mono- or diglycerides, or fatty acids, including oleic acid.

 For rectal administration in the form of suppositories, such compositions can be prepared by mixing the drug with a non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solids at ordinary temperatures, but liquefy and / or dissolve in the rectal cavity with the release of the drug.

 Dosage levels of the order of 0.02-5.0 or 10.0 g / day can be used to treat or prevent the above conditions, with oral dosages being 2-5 times higher. For example, HIV infection is effectively treated by using 1.0-50 mg of the compound per kilogram of body weight when taking the drug 1-4 times a day. According to one preferred treatment regimen, dosages of 100-400 mg every 6 hours were administered orally in each patient. However, it should be borne in mind that the specific dosage level and frequency of drug administration for each particular patient may vary and will depend on a large number of factors, including the activity of a particular compound, metabolic stability and duration of action of such a compound, patient age, body weight, general health , gender, type and time of application, rate of excretion, drug combination, severity of the particular disease state and the host being treated.

 The present invention also relates to a combination of HIV protease inhibiting compounds with one or more agents used to treat AIDS. For example, the compounds of the present invention can be effectively used both before and after exposure, in combination with effective amounts of AIDS antiviral agents, immunomodulators, disinfecting agents or vaccines known to those skilled in the art.

 It should be borne in mind that the scope of the combinations of the compounds of the present invention with AIDS antiviral agents, immunomodulators, anti-infective agents or vaccines is not limited to the list presented in the table at the end of the description, but, in principle, includes any combination with any pharmaceutical composition intended for treatment AIDS

Some compounds shown in the table are as follows:
Compound B is 6-chloro-4- (S) -cyclopropyl-3,4-dihydro-4 - ((2-pyridyl) ethynyl) quinazolin-2 (1H) -one;
Compound C is (-) - 6-chloro-4 (S) -trifluoromethyl-1,2-dihydro-4 (H) -3,1-benzoxazin-2-one;
nevirapine is N-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido / 3,2-b /: 2 ', 3'-e // 1,4 / diazepin-6-one.

 Compounds B and C were synthesized according to the methods of EP 0569083, the contents of which are referred to in this description. Nevirapine synthesized by Klunder J. M. et al., Med. Chem., 35, 1887 (1992); Hargrave K.D. et al., J. Med. Chem. 34, 2231 (1991); Cohen K.A. et al., J. Biol. Chem., 260, 14670 (1991), with all three of these works referenced in the present description.

Preferred combinations are simultaneously and alternately used in the treatment of an HIV protease inhibitor and a non-nucleoside HIV reverse transcriptase inhibitor. An optional third component in combination is a nucleoside HIV reverse transcriptase inhibitor such as AZT, ddC or ddI. A preferred HIV protease inhibitor is compound A. Preferred non-nucleoside HIV reverse transcriptase inhibitors include compound B, compound C or nevirapine. Such combinations may have synergistic effects on limiting the spread of HIV. Preferred combinations include:
(1) Compound A, together with a preferred non-nucleoside HIV reverse transcriptase inhibitor, and optionally AZT or ddI or ddC;
(2) Compound A and any compound selected from AZT, ddI or ddC.

Microbially Expressed HIV Protease Inhibition Assay
A study of the inhibition of the protease expression reaction in Eschericia coli in the presence of a peptide substrate (Val-Ser-Gln-Asn- (betanaphthyl) Ala-Pro-Ile-Val, 0.5 mg / ml during reaction initiation) was carried out in 50 mM Na acetate, pH 5.5 at 30 ^o C for 1 hour. Different concentrations of the inhibitor in 1.0 μl DMSO were added to 25 μl of the peptide solution in water. The reaction was initiated by adding 15 μl of 0.33 nM protease (0.11 ng) in a solution of 0.133 M Na acetate, pH 5.5 and 0.1% bovine serum albumin. The reaction was terminated with 160 μl of 5% phosphoric acid. The reaction products were separated by HPLC (VVDAC, broad-porous, 5 cm C-18 reversible phase, acetonitrile gradient, 0.1% phosphoric acid). The degree of inhibition of the reaction was determined from the heights of the product peaks. HPLC independently synthesized products that served as quantitative standards confirmed the composition of the products. Compound A had an IC ₅₀ value of the order of 0.27 nM.

Cell Spread Analysis
Inhibition of the spread of HIV in cell culture was measured according to the method of Nunberg JH, al., J. Virol. 65, 4887 (1991). In this assay, MT-4 T-lymphoid cells infected with HIV-1 (wild type, unless otherwise indicated) using a predetermined inoculum and culture was incubated for 24 hours. By this time, ≤ 1% of the cells were positive according to indirect immunofluorescence. Then the cells were washed thoroughly and distributed in culture dishes with 96 recesses. Serial twofold dilutions of the inhibitor were added to the wells and the cultures were kept for another 3 days. 4 days after infection, 100% of the cells in the control culture were infected. The accumulation of HIV p24 was directly correlated with the spread of the virus. The inhibitory concentration of cell culture was defined as the concentration of the inhibitor, expressed in nanomoles / liter, which reduces the spread of infection by at least 95%, or it was designated as CIC ₉₅ . The CIC ₉₅ value for compound A was 25 nM.

 Inhibition of the spread of the virus.

 A. Preparation of a suspension of MT-4 cells infected with HIV.

 MT cells were infected on day 0 with a concentration of 250,000 / ml 1: 1000 by diluting the HIV-1 strain of line IIIb (final concentration of 125 pg p24 / ml, which is sufficient for the formation of ≤ 1% of infected cells on day 1 and 25-100% on day 4 ) Cells were infected and grown in the following medium: RPMI (Wittaker Bio-Products), 10% inactivated fetal calf serum, 4 mM glutamine (Flexo Labs.) And 1: 100 Penicillin-Streptomycin (Flexo Labs).

The mixture was incubated overnight at 37 ^° C. in an atmosphere containing 5% CO ₂ .

 B. Treatment with inhibitors.

A matrix of the nanomolar concentration range of pairwise combinations was prepared. On day 1, aliquots of 125 μl of inhibitors were added to equal volumes of HIV-infected MT-4 cells (50,000 per well) in a 96-well cell microtiter plate. Incubation was continued for 3 days at 37 ^° C. in an atmosphere containing 5% CO ₂ .

 C. Measuring the spread of the virus.

 Using a multichannel pipette, the precipitated cells were resuspended and 125 μl was collected on a separate microtiter plate. The supernatant was analyzed for the presence of HIV p24 antigen.

 The concentration of HIV p24 antigen was measured using enzyme immunoassay described below. Aliquots of p24 to be measured were added to microcavities coated with a monoclonal antibody specific for the HIV envelope antigen. At this and other subsequent corresponding stages, microdeeps were washed. Then a biotinylated HIV-specific antibody was added, followed by the addition of streptavidin-horseradish peroxidase conjugate. When hydrogen peroxide and tetramethylbenzidine substrate were added, a color reaction proceeded. Color intensity was proportional to the concentration of HIV p24 antigen.

 Calculation of the degree of synergy or enhanced inhibition.

 In the presence of a synergistic effect, paired combinations of inhibitors, as it was found, exhibit distinctly enhanced inhibition of the spread of the virus compared with the action of each inhibitor individually or in comparison with a simple additive inhibition from each inhibitor.

 The data obtained were processed as follows: fractional inhibitory concentration ratios (FIC) were calculated according to Elion et al. J. Biol, Chem., 208, 477 (1954). The minimum amount of FIC, showing the maximum synergistic effect, was determined for various pairwise combinations. The smaller the number, the higher the synergistic effect.

Example 1. Obtaining N- (2 (R) -hydroxy-1 (S) -indanyl) - 2 (R) -phenylmethyl-4 (S) -hydroxy) -5- (1- (2 (S) -N- (tert.-butyl-carboxamido) piperazinyl) -pentanamide, Compound 14
Step 1: Preparation of Dihydro-5- (S) - ((tert.-butyldiphenylsilyl) - hydroxymethyl) -3 (R) phenylmethyl-3 (2H) -furanone.

A solution of lithium diisopropylamide (LDA) was prepared by adding 1.55 ml of n-BuLi (2.5 M in hexane) to 0.55 ml (39 mmol) of diisopropylamine in 10 ml of THF at -78 ^° C. After 30 minutes, dihydro-5 was added. - (S) - ((tert.-butyldiphenylsilyl) -oxymethyl) - 3 (2H) -furanone (1.38 g, 3.89 mmol) in 5 ml THF. After another 30 minutes of stirring, benzyl bromide (0.68 g, 3.9 mmol) was added and stirring was continued for 3 hours, after which the reaction was stopped by adding a 10% aqueous citric acid solution. The solution was extracted with ethyl acetate (2 x 50 ml), which was backwashed with brine, dried, filtered and concentrated to give an oil. The product was purified by chromatography (SiO ₂ , 20% EtOAc / hexane) to give the desired compound.

 Step 2: Preparation of Dihydro-5 (S) - (hydroxymethyl) -3 (R) -phenylmethyl-3 (2H) -furanone

To 5.26 g of dihydro-5 (S) - ((tert-butyl diphenylsilyl) oxymethyl-3 (R) phenylmethyl-3 (2H) -furanone in 40 ml of acetonitrile, 1.34 ml of a 49% aqueous solution of HF was added. After 18 hours at room temperature, the reaction mixture was concentrated to dryness and the residue was partitioned between water (50 ml) and ethyl acetate (50 ml). The organic layer was washed with brine, dried, filtered and concentrated to give the product as a tan solid (mp. 69 -72 ^o C).

 Step 3: Preparation of Dihydro-5 (S) - ((trifluoromethanesulfonyl) - hydroxymethyl) -3 (R) -phenylmethyl-3 (2H) -furanone

To a solution of 18.4 g (89.2 mmol) of dihydro-5 (S) - (hydroxymethyl) -3 (R) -phenylmethyl-3 (2H) -furanone in 350 ml of methylene chloride, cooled to 0 ^° C, 13 was added 51 ml of 2,6-lutidine (115.98 mmol), after which 16.51 ml of trifluoromethanesulfonic anhydride (98.1 mmol) were added dropwise. After 1.5 hours at 0 ^° C., the reaction mixture was poured into a mixture of 300 ml of ice with brine and stirred for 0.5 hour. Then the aqueous layer was extracted with methylene chloride (3 x 150 ml), the organic layers were washed with 10% HCl (2 x 75 ml), saturated with NaHCO ₃ (100 ml), water (100 ml), dried over MgSO ₄ , filtered and concentrated to obtain solid residue. As a result of purification by flash evaporation chromatography (column 120 x 150 mm, gradient elution with hexanes: EtOAc, 4: 1-3: 1), the target compound was obtained, mp. 53-54 ^o C.

Step 4: Preparation of 4- (1,1-Dimethylethoxycarbonylamino) -1- (phenylmethylcarbonylamino) piperazine-2S-carboxylic acid
The target compound was obtained following the procedure described by Bigge CF, Hays SJ, Novak PM, Drummond JT, Johnson Y., Bobovski T., Tetrahedron Lett, 1989, 30, 5193; using 2 (S) -piperazine carboxylic acid as the starting compound (see Felder E., Maffei S., Pietra S., Pitre D., Helv, Chim. Acta, 1960, 117, 888.

 Stage 5: Getting N-tert. -butyl-4- (1,1-dimethylethoxycarbonylamino) -1- (phenylmethylcarbonylamino) piperazin-2 (S) -carboxamide.

To 9.90 g (27.16 mmol) of the product from step 4 dissolved in 75 ml of DMF and cooled to 0 ^° C, 5.73 g (29.88 mmol) of EDC, 4.03 g (29.88 mmol) of HOBt were added 3.14 ml (29.88 mmol) of tert.-butylamine; and finally 4.16 ml (29.88 mmol) of triethylamine. The reaction mixture was stirred for 18 hours and the reaction volume was concentrated in half. The mixture was then diluted with 600 ml EtOAc and washed with 10% HCl (2 x 75 ml), saturated NaHCO ₃ (1 x 75 ml), water (3 x 75 ml) and brine (1 x 50 ml), dried over MgSO ₄ and concentrated to obtain a solid. Such a solid was treated with EtOAc: hexane (1: 2) and filtered to give the title compound as a white solid; so pl. 134-135 ^o C.

 Stage 6: Getting N-tert. -butyl-4- (1,1-dimethylethoxycarbonylamino) piperazin-2 (S) -carboxamide.

 To 1.20 g (2.86 mmol) N-tert-butyl-4- (1,1-dimethylethoxycarbonylamino) -1- (phenylmethylcarbonylamino) piperazine-2- (S) -carboxamide and 1.1 g (0.086 mmol) ) 10% Pd / C was added 15 ml of methanol. Hydrogen was introduced into the reaction vessel and the reaction mixture was stirred for 2 hours, filtered through celite and washed with ethanol. Solvents were removed in vacuo to give the desired product as a foam.

Spectrum ¹ H-NMR (300 MHz, CDCl ₃ ) δ: 6.65 (broad band, 1H), 4.10 (multiplet, 1H), 3.81 (wide band, 1H), 3.21 (doublet of doublets, J = 18 and 7 Hz ), 3.02-2.70 (multiplet, 4H), 2.10-2.0 (broad, 1H), 1.50 (singlet, 9H), 1.41 (singlet, 9H).

 Step 7: Preparation of Dihydro-5 (S) - (4- (1,1-dimethylethoxycarbonylamino) -2 (S) -N- (tert-butylcarboxamido) piperazinyl) methyl) -3 (R) -phenylmethyl-3 ( 2H) -furanone.

 To a solution of 22.40 g (0.0662 mol) of dihydro-5 (S) - ((trifluoromethanesulfonyl) oxymethyl) -3- (R) -phenylmethyl-3 (2H) -furanone (obtained in step 3) and 18.0 g (0.063 mol) N-tert-butyl-4- (1,1-dimethylethoxycarbonylamino) piperazin-2 (S) -carboxamide dissolved in 180 ml of isopropanol, 11.53 ml (0.062 mol) of N, N-diisopropylethylamine was added . After 2.5 hours, another 1.2 g of dihydro-5 (S) - (trifluoromethanesulfonyl) oxymethyl) -3 (R) -phenylmethyl-3 (2H) -furanone was added. The reaction mixture was analyzed by thin layer chromatography (TLC) after 3.5 hours and concentrated to a thick oil. Treatment with EtOAc / hexanes (1: 2, 200 ml) provided a white solid, which was filtered and discarded. The oil was purified by flash flash chromatography (120 x 150 mm column, gradient elution with 1: 1, 2: 1, 3: 1 EtOAc / hexanes to 100% EtOAc) to obtain the desired compound.

Spectrum ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 7.34-7.17 (multiplet, 5H), 6.31 (wide singlet, 1H), 4.38 (wide multiplet, 1H), 3.96-3.92 (multiplet, 1H), 3.79 ( wide multiplet, 1H), 3.16 (doublet of doublets, J = 13.6 and 4.4 Hz, 1H), 3.08-2.99 (multiplet, 3H), 2.90-2.82 (multiplet, 1H), 2.80 (doublet of doublet, J = 13.5 and 8.9 Hz, 1H), 2.78 (multiplet, 1H), 2.67-2.61 (multiplet, 1H), 2.58-2.49 (multiplet, 1H), 2.38-2.32 (multiplet, 1H), 2.32-2.04 (multiplet, 1H) , 1.99-1.92 (multiplet, 1H), 1.45 (singlet, 9H), 1.29 (singlet, 9H).

 Step 8: Preparation of 2 (R) -phenylmethyl-4 (S) - (tert.-butyldimethylsilyloxy) -5- (1- (4- (1,1-dimethylethoxycarbonylamino))) - 2 (S) -N- (tert. butylcarboxamido) piperazinyl) pentanamide.

To 25.50 g (52.50 mmol) of dihydro-5 (S) - (4- (1,1-dimethyl-ethoxycarbonylamino)) - 2 (S) -N- (tert-butylcarboxamido) piperazanil) methyl 3 (R) -phenylmethyl-3 (2H) -furanone dissolved in 120 ml of DMF, cooled to 0 ^° C., a solution of 60 ml of water and 1.512 g (63.01 mmol) of lithium hydroxide were added. After 0.5 hours, the reaction was stopped by adding 10% HCl to pH 6 and the solution was concentrated in vacuo. The residue was dissolved in 50 ml of water and extracted with EtOAc (4 x 75 ml) and the organic layers were washed with water (1 x 20 ml), brine (1 x 20 ml). The aqueous layer was back extracted with EtOAc (2 x 75 ml) and the combined organic layers were dried over MgSO ₄ and concentrated to give a yellow solid. Such a crude product was dissolved in 100 ml of DMF and 17.87 g (0.262 mol) of imidazole was added, cooled to 0 ^° C and then 31.50 g (0.21 mol) of tert-butyl dimethylsilyl chloride was added. The resulting mixture was stirred for one hour at 0 ^° C. and then warmed to room temperature. After 20 hours, the reaction was stopped with 10 ml of methanol and the reaction mixture was concentrated to half the volume. Added 100 ml of buffered water with a pH of 7 and the aqueous layer was extracted with EtOAc (4 x 100 ml), the combined organic layers were washed with 10% HCl (2 x 50 ml), water (3 x 75 ml) and brine (1 x 50 ml), dried over MgSO ₄ and concentrated to obtain the target compound. The resulting material was directly used in the next step.

 Step 9: Preparation of N- (2 (R) -hydroxy-1 (S) -indanyl) -2 (R) - phenylmethyl-4 (S) - (tert-butyldimethylsilyloxy) -5- (1- (4- ( 1,1-dimethylethoxycarbonylamino))) - 2 (S) -N- (tert-butylcarboxamido) piperazinyl) pentanamide.

To 27.0 g (0.0446 mol) of the crude material from step 6, dissolved in 180 ml of DMF and cooled to 0 ^° C., 8.98 g (0.0468 mol) of EDC, 6.32 g (0.0468 mol) of HOBt and 7.31 g (0.049 mol) of amino hydroxyindane. Triethylamine (6.52 ml, 0.0468 mol) was added and the reaction mixture was stirred for 2 hours at 0 ^° C., at room temperature for 16 hours, and the reaction was stopped by dilution with 500 ml EtOAc. The organic layer was washed with 10% HCl (2 x 100 ml), saturated NaHCO ₃ (1 x 100 ml), water (3 x 150 ml), brine (1 x 75 ml), dried over MgSO ₄ and concentrated to obtain the target compound in in the form of white foam.

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 7.4-7.17 (multiplet, 9H), 6.51 (wide singlet, 1H), 5.79 (wide singlet, 1H), 5.23 (multiplet, 1H), 4.23 (wide singlet, 1H), 4.06 (multiplet, 1H), 3.96-3.84 (multiplet, 2H), 3.07-2.78 (multiplet, 8H), 3.65 (doublet of doublets, J = 9.6 and 4.1 Hz, 1H), 2.56-2.44 (multiplet, 2H ), 2.29 (doublet of doublets, J = 12.0 and 4.5 Hz, 1H), 2.17-2.09 (multiplet, 1H), 1.79 (wide singlet, 1H), 1.44 (singlet, 9H), 1.35 (singlet, 9H), 1.10 ( singlet, 2H), 0.84 (singlet, 9H), 0.12 (singlet, 3H), 0.08 singlet, 3H).

 Step 10: Preparation of N- (2 (R) -hydroxy-1 (S) -indanyl) - 2 (R) -phenylmethyl-4 (S) - (hydroxy) -5- (1- (4- (1,1 -dimethyl-ethoxycarbonylamino))) - 2 (S) -N- (tert-butylcarboxamido) piperazinyl) pentanamide.

K 32.20 g (0.0437 mol) N- (2 (R) -hydroxy-1 (S) -indanyl) -2- (R) -phenylmethyl-4 (S) - (tert-butyldimethylsilyloxy) -5 - (1- (4- (1,1-dimethylethoxycarbonylamino)) - 2 (S) -N- (tert-butylcarboxamido) piperazinyl)) pentanamide was added 437 ml (0.437 mol) of tetrabutylammonium fluoride (1.0 M solution at THF, Aldrich). The reaction mixture was stirred for 18 hours and then concentrated to a volume of 200 ml and diluted with 700 ml EtOAc. The resulting solution was washed with water (2 x 100 ml), brine (1 x 50 ml) and the aqueous layers were back extracted with EtOAc (2 x 200 ml). The combined organic layers were dried over MgSO ₄ and concentrated to an oil. As a result of purification by flash chromatography (column 120x150 mm, gradient elution with a mixture of CH ₂ Cl ₂ : CHCl ₃ saturated with a mixture of NH ₃ : methanol, with an increase in the amount of methanol in the order of 1%, 1.5%, 2%), the target compound was obtained in the form of a white foam.

Spectrum ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 7.31-7.11 (multiplet, 9H), 6.41 (wide singlet, 1H), 6.23 (doublet, J = 8.6 Hz, 1H), 5.25 (doublet of doublets, J = 8.6 and 4.7 Hz, 1H), 4.21 (multiplet, 1H), 3.83-3.82 (multiplet, 2H), 3.78-3.61 (multiplet, 2H), 3.22-3.19 (multiplet, 2H), 3.03-2.78 (multiplet, 8H) , 2.62-2.58 (multiplet, 1H), 2.41-2.35 (multiplet, 2H), 2.04-2.02 (multiplet, 1H), 1.57-1.50 (multiplet, 1H), 1.45 (singlet, 9H), 1.32 (singlet, 9H) .

 Step 11: Preparation of N- (2 (R) -hydroxy-1 (S) -indanyl) - 2 (R) -phenylmethyl-4 (S) - (hydroxy) -5- (1- (2 (S) -N - (tert-butylcarboxamido) piperazinyl) pentanamide Compound 14.

K 21.15 g (0.034 mmol) N- (2 (R) -hydroxy-1 (S) -indanyl) -2 (R) -phenylmethyl-4 (S) - (hydroxy) -5- (1- (4 - (1,1-dimethylethoxycarbonylamino)) - 2 (S) -N- (tert.-butylcarboxamido) piperazinyl)) - pentanamide dissolved in 350 ml of methylene chloride and cooled to 0 ^o C, was added 22.43 ml (0.204 mol) of 2,6-lutidine and then 32.85 ml (0.170 mol) of trimethylsilyl triflate for 5 minutes. After 0.5 hours, the reaction was stopped with 10% HCl (80 ml) and the resulting mixture was stirred for 0.5 hours. To the resulting mixture was added 100 ml of saturated NaHCO ₃ and then solid NaHCO ₃ to pH 8. Then the aqueous layer was extracted with EtOAc (4 x 100 ml) and the combined organic layers were washed with water (1 x 50 ml), brine (1 x 75 ml). dried over MgSO ₄ and concentrated. The residue was purified by column chromatography (120x150 mm, gradient elution with a mixture of CH ₂ Cl ₂ : CHCl ₃ saturated with a mixture of NH ₃ : MeOH, slowly increasing the concentration of methanol in the order of 2%, 3%, 4%, 5%, 6% and up to 10 %). As a result, the desired product was obtained in the form of a white foam.

Spectrum ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 7.53 (singlet, 1H), 7.29-7.09 (multiplet, 9H), 6.52 (doublet, J = 1.83 Hz, 1H), 5.24 (doublet of doublets, J = 1.82 and 4.9 Hz, 1H), 4.23 (doublet of doublets, J = 4.7 and 4.03 Hz, 1H), 4.25-4.00 (wide singlet, 1H), 3.83-3.81 (multiplet, 1H), 3.03-2.88 (multiplet, 4H), 2.82-2.73 (multiplet, 7H), 2.50-1.60 (wide singlet, 2H), 2.45 (doublet, J = 6.2 Hz, 2H), 2.32-2.29 (multiplet, 1H), 1.98 (multiplet, 1H), 1.51 (multiplet , 1H), 1.33 (singlet, 9H).

К раствору известного (Snyder H. R., Ebetino F.F., J. Het. Chem., 1, 202-205 (1966) диэтил-(фуро-[2,3-b] пиридин-2,5-дикарбоксилата (1,22 г, 4,923 ммоля) в 10 мл 95%-го этанола добавляли раствор гидроксида калия (0,66 г, 11,81 ммоля) в 10 мл воды. Реакционную смесь нагревали в течение 3 часов до 80^oC, охлаждали до комнатной температуры и фильтровали. Двухкалиевую соль растворяли в воде и подкисляли 10% HCl до pH 2. Осадок отфильтровывали и сушили в вакууме с получением 850 мг белого твердого вещества.Example 2. Obtaining N- (2 (R) -hydroxy-1 (R) -indanyl) - 2 (R) -phenylmethyl-4 (R) -hydroxy-5- (1- (4- (3-furo [2 , 3-b] pyridylmethyl) -2 (R) -N- (tert.-butylcarboxamido) -piperazinyl)) -pentanamide
Step 1: Preparation of (furo [2,3-b] bipyridin-2,5-dicarboxylic acid

To a solution of the well-known (Snyder HR, Ebetino FF, J. Het. Chem., 1, 202-205 (1966) diethyl- (furo- [2,3-b] pyridine-2,5-dicarboxylate (1.22 g, 4.923 mmol) in 10 ml of 95% ethanol, a solution of potassium hydroxide (0.66 g, 11.81 mmol) in 10 ml of water was added.The reaction mixture was heated for 3 hours to 80 ^° C, cooled to room temperature and filtered. The two-potassium salt was dissolved in water and acidified with 10% HCl to pH 2. The precipitate was filtered off and dried in vacuo to give 850 mg of a white solid.

Spectrum ¹ H-NMR (400 MHz, (CD ₃ ) ₂ SO) δ: 8.98 (doublet, J = 2.2 Hz), 8.76 (doublet, J = 2.2 Hz), 7.69 (singlet, 1H), 4.25 (wide singlet, 3H).

К суспензии фуро[2,3-b]пиридин-2,5-дикарбоновой кислоты (0,36 г, 1,484 ммоля) в 3 мл хинолина, в атмосфере Ar, добавляли порошкообразную медь (180 мг, 2,82 ммоля) и реакционную смесь нагревали в течение 1,5 часов до 210^oC. Реакционную смесь охлаждали до комнатной температуры и разбавляли 50 мл хлористого метилена и фильтровали через целит. Органический слой экстрагировали насыщенным раствором (2 х 40 мл) Na₂CO₃ подкисляли до pH 3 с помощью 3 N HCl и фильтровали с получением 80 мг желтовато-коричневого твердого вещества. Водный слой экстрагировали смесью эфир/метанол (85/15) (3х50 мл) и промывали рассолом (1 х 10 мл), сушили над MgSO₄ фильтровали и концентрировали с получением еще 35 мг продукта.Stage 2: Obtaining furo [2,3-b] pyridine-5-carboxylic acid

To a suspension of furo [2,3-b] pyridine-2,5-dicarboxylic acid (0.36 g, 1.484 mmol) in 3 ml of quinoline, in an Ar atmosphere, powdered copper (180 mg, 2.82 mmol) was added and the reaction the mixture was heated for 1.5 hours to 210 ^° C. The reaction mixture was cooled to room temperature and diluted with 50 ml of methylene chloride and filtered through celite. The organic layer was extracted with a saturated solution (2 x 40 ml) of Na ₂ CO ₃ acidified to pH 3 with 3 N HCl and filtered to obtain 80 mg of a tan solid. The aqueous layer was extracted with ether / methanol (85/15) (3 x 50 ml) and washed with brine (1 x 10 ml), dried over MgSO _4, filtered and concentrated to give another 35 mg of product.

Spectrum ¹ H-NMR (400 MHz, CD ₃ OD) δ 8.89 (singlet, 1H), 8.67 (doublet, J = 2.0 Hz, 1H), 7.97 (doublet, J = 2.5 Hz, 1H), 7.01 (doublet, J = 2.4 Hz, 1H).

 Stage 3: Preparation of methyl furo- [2,3-b] pyridine-5-carboxylate.

К фуро-[2,3-b] пиридин-5-карбоновой кислоте (3,0 г, 18,40 ммоля), растворенной в 40 мл метанола, добавляли 160 мл хлороформа и затем медленно добавляли триметилсилилдиазометан (42 мл, 10% раствор в гексенах). Через 0,5 часа добавляли 4 капли ледяной уксусной кислоты и реакционную смесь концентрировали. В результате получали 3,20 г белого твердого вещества.

To furo [2,3-b] pyridine-5-carboxylic acid (3.0 g, 18.40 mmol) dissolved in 40 ml of methanol was added 160 ml of chloroform, and then trimethylsilyldiazomethane (42 ml, 10% solution) was slowly added. in hexenes). After 0.5 hours, 4 drops of glacial acetic acid were added and the reaction mixture was concentrated. The result was 3.20 g of a white solid.

Spectrum ¹ H-NMR (400 MHz, CDCl ₃ ) δ /: 9.02 (doublet, J = 2.0 Hz, 1H), 8.60 (doublet, J = 2.0 Hz, 1H), 7.9 (doublet, J = 2.5 Hz, 1H) 6.87 (doublet, J = 2.5 Hz, 1H); 3.98 (singlet, 3H).

 Stage 4: Obtaining 5-hydroxymethyl-furo [2.3-b] pyridine.

В высушенную на пламени 500 мл круглодонную колбу загружали метил-фуро-[2,3-b] пиридин-5-карбоксилат (3,20 г, 18,08 ммоля), растворенный в ТГФ и охлажденный до 0^oC. К полученному раствору добавляли диизобутилалюминий гидрид (46 мл 46,1 ммоля, 1 М раствор в гексанах) в течение 10 минут и охлаждающую баню снимали. Через 4 часа реакционную смесь охлаждали до 0^oC и медленно тушили реакцию с помощью солей Рошель (100 мл). Еще через 18 часов слои разделяли и водный слой экстрагировали этилацетатом (4 х 40 мл). Объединенные органические слои промывали рассолом (1 х 20 мл), сушили над MgSO₄, фильтровали и концентрировали. Остаток очищали методом хроматографии мгновенного испарения (колонка 40х150 мм, градиент элюирования CH₂Cl₂:CHCl₃, насыщенный смесью NH₃:MeOH, в соотношении 60-39-1,0 (1000 мл), 60:38:2 (1000 мл), 60-37-3 (1000 мл), 60-36-4 (1000 мл). В результате получали 2,16 г белого твердого вещества.

In a flame-dried 500 ml round-bottom flask, methyl furo- [2,3-b] pyridine-5-carboxylate (3.20 g, 18.08 mmol) was dissolved in THF and cooled to 0 ^° C. To the resulting solution diisobutylaluminum hydride (46 ml of 46.1 mmol, 1 M solution in hexanes) was added over 10 minutes, and the cooling bath was removed. After 4 hours, the reaction mixture was cooled to 0 ^° C. and the reaction was slowly quenched with Rochelle salts (100 ml). After another 18 hours, the layers were separated and the aqueous layer was extracted with ethyl acetate (4 x 40 ml). The combined organic layers were washed with brine (1 x 20 ml), dried over MgSO ₄ , filtered and concentrated. The residue was purified by flash chromatography (column 40x150 mm, elution gradient CH ₂ Cl ₂ : CHCl ₃ saturated with a mixture of NH ₃ : MeOH, in a ratio of 60-39-1.0 (1000 ml), 60: 38: 2 (1000 ml ), 60-37-3 (1000 ml), 60-36-4 (1000 ml), and 2.16 g of a white solid are obtained.

¹ H-NMR spectrum (400 MHz, CDCl ₃ ) δ: 8.19 (doublet, J = 2.0 Hz, 1H), 7.92 (doublet, J = 2.0 Hz, 1H), 7.64 (doublet, J = 2.5 Hz, 1H), 6.69 (doublet, J = 2.4 Hz, 1H), 4.78 (doublet, J = 3.8 Hz, 2H), 4.69 (wide singlet, 1H).

 Step 5: Preparation of 3-chloromethyl-furo [2,3-b] pyridine hydrochloride.

К раствору 5-гидроксиметил-фуро[2,3-b] пиридина, растворенного в 9 мл хлористого метилена, охлажденного до 0^oC, добавляли хлористый тионил (4,23 мл, 57,99 ммоля). Ледяную баню снимали и через 1 час реакционную смесь концентрировали с получением 2,86 г белого твердого вещества.

To a solution of 5-hydroxymethyl furo [2,3-b] pyridine dissolved in 9 ml of methylene chloride, cooled to 0 ^° C., thionyl chloride (4.23 ml, 57.99 mmol) was added. The ice bath was removed and after 1 hour the reaction mixture was concentrated to obtain 2.86 g of a white solid.

Spectrum ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 8.40 (doublet, J = 2.0 Hz, 1H), 8.13 Doublet, J = 2.2 Hz, 1E), 7.80 (doublet, J = 2.4 Hz, 1H), 6.86 (doublet, J = 2.4 Hz, 1H), 4.74 (singlet, 2H).

 Step 6: Preparation of N- (2 (R) -hydroxy-1 (S) -indanyl) -2 (R) -phenylmethyl-4 (S) -hydroxy-5- (1- (4- (3-furo [2 , 3-b] pyridylmethyl) - 2 (S) -N '- (tert.-butylcarboxamido) piperazinyl)) - pentanamide.

К раствору N-(2(R)-гидрокси-1(S)-инданил)-2(R)-фенилметил- 4(S)-гидрокси-5(-2(S)-N'-(трет.-бутилкарбоксамидо)-пиперазинил- пентанамида (6,50 г, 12,48 ммоля), растворенному в 12 мл диметилформамида, в атмосфере аргона, добавляли 3-хлорметилфуро [2.3-b]пиридин гидрохлорид (2,80 г, 13,72 ммоля) и триэтиламин (5,21 мл, 38.44 ммоля). Через 18 часов реакционную смесь разбавляли 400 мл этилацетата и промывали насыщенным NaHCO₃ (1 x 25 мл), водой (5 х 20 мл) и рассолом (1 х 25 мл).

To a solution of N- (2 (R) -hydroxy-1 (S) -indanyl) -2 (R) -phenylmethyl-4 (S) -hydroxy-5 (-2 (S) -N '- (tert-butylcarboxamido ) -piperazinyl pentanamide (6.50 g, 12.48 mmol) dissolved in 12 ml of dimethylformamide under argon atmosphere was added 3-chloromethylfuro [2.3-b] pyridine hydrochloride (2.80 g, 13.72 mmol) and triethylamine (5.21 ml, 38.44 mmol). After 18 hours, the reaction mixture was diluted with 400 ml of ethyl acetate and washed with saturated NaHCO ₃ (1 x 25 ml), water (5 x 20 ml) and brine (1 x 25 ml).

The resulting solution was dried over MgSO ₄ , filtered and concentrated to an oil. The residue was purified by flash chromatography (column 60x150 ml, gradient elution with a mixture of CH ₂ Cl ₂ : CH ₂ Cl ₂ saturated with a mixture of NH ₃ : MeOH in a ratio of 60: 39: 1 (1000 ml), 60: 38: 2 (1000 ml ), 60: 37: 3 (1500 ml), 60: 36: 4 (1500 ml). The resulting foam was treated with ethyl acetate and the desired product was filtered and dried overnight in high vacuum at 65 ^° C. to obtain 5.30 g white crystalline solid Mixed fractions from the chromatographic column can be combined and subjected to re-purification to obtain additional coli ETS, m.p. 183,5-184,5 ^o C.

Spectrum ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 8.25 (doublet, J = 2.2 Hz, 1H), 7.85 (doublet, J = 2.0 Hz, 1H), 7.5 (singlet, 1H), 7.73 (doublet, J = 2.4 Hz, 1H), 7.32-7.10 (multiplet, 9H), 6.75 (doublet, J = 2.4 Hz, 1H), 5.95 (doublet, J = 8.6 Hz, 1H), 5.27 (double double, J = 8.5 and 4.8 Hz, 1H), 4.27-4.26 (multiplet, 1H), 4.12 (wide singlet, 1H), 3.89-3.83 (multiplet, 1H), 3.51 (singlet, 2H), 3.29 (doublet of doublets, J = 17.5 and 4.0 Hz, 1H), 3.16 (doublet of doublets, J = 3.36 and 3.48 Hz, 1H), 3.15 (doublets of doublets, J = 6.6 and 5.1 Hz, 1H), 2.94-2.50 (multiplet, 11H), 2.36-2.34 (multiplet, 1H) , 1.66 (singlet, 1H), 1.62-1.47 (multiplet, 1H), 1.35 (singlet, 9H).

Elemental analysis: calculated for C ₃₈ H ₄₇ N ₅ O ₅
C 69.81, H 7.25. N, 10.71;
Found: C, 69.46; H, 7.22; N, 10.69.

 Example 3

 Using almost the same methodology as described in example 2, but carrying out the reaction between N- (2 (R) -hydroxy-1 (S) -indanyl) -2 (R) -phenylmethyl-4 (S) -hydroxy-5- (1- (2 (S) -N '- (tert. Butyl carboxamido) piperazinyl)) - pentanamide used in this example (compound (1), see below) and an alkylating agent (11) below instead of an alkylating agent the agent used in stage 6, received the following products of formula (III), see the end of the description.

 Example 4. Obtaining amide I (see the end of the description).

A solution of (-) - cis-1-aminoindan-2-ol (884 g, 5.93 mol) in 17.8 L dry THF (KF = 55 mg / ml) (KF - Karl Fischer titration of water) and triethylamine (868 ml, 6.22 mol) in a 50 L round bottom flask equipped with a thermocouple pocket, a mechanical stirrer, a nitrogen inlet device and a bubbler, cooled to 15 ^° C. Then 3-phenylpropyl chloride (1000 g, 5 , 93 moles), maintaining the temperature inside the flask in the range of 14-24 ^o C using a cooling bath containing a mixture of ice with water. After complete addition, the mixture was allowed to age at 18-20 ^° C. for 30 minutes, and the disappearance of (2) -cis-1-aminoindan-2-ol was checked by HPLC analysis.

Реакционную смесь обрабатывали п-толуолсульфонатом пиридиния (241 г, 0,96 моля, 0,16 эквивалентов) и перемешивали в течение 10 минут (pH смеси после разбавления 1 мл образца равным объемом воды составляло 4,3- 4,6). Затем добавляли 2-метоксипропен (1,27 л, 13,24 моля, 2,2 эквивалента) и реакционную смесь нагревали до 38-40^oC в течение 2 часов. Реакционную смесь охлаждали до 20^oC и распределяли между этилацетатом (12 л) и 5% водным раствором NaHCO₃ (10 л). Смесь перемешивали и слои разделялись. Этилацетатный экстракт промывали 5% водным раствором NaHCO₃ (10 л) и водой (4 л). Этилацетатный экстракт сушили дистилляцией при атмосферном давлении, и растворитель меняли на циклогексан (общий объем ~ 30 л). После окончания дистилляции и концентрирования (объем экстракции этилацетатом 20% об.) горячий циклогексановый раствор медленно охлаждали до 25^oC с кристаллизацией продукта реакции. Полученную в результате суспензию дополнительно охлаждали до 10^oC и давали ей стареть. Продукт реакции выделяли фильтрацией и влажный осадок на фильтре промывали холодным (10^oC) циклогексаном (2 х 800 мл). Промытый осадок на фильтре сушили в вакууме (26" Н; примерно 66 мм рт.ст.) при 40^oC с получением 1,65 кг ацетонида 1 (86,4%, 98% площади, согласно HPLC).The progress of the reaction was monitored by high-resolution chromatography: 25 cm DuPont C8-RX column, 60-40 acetonitrile / 10 mM (KH ₂ PO ₄ / K ₂ HPO ₄ ), 1.0 ml / min, injection volume = 20 ml, detection = 200 nm, sample preparation: 500-fold dilution. Approximate retention times:

The reaction mixture was treated with pyridinium p-toluenesulfonate (241 g, 0.96 mol, 0.16 equivalents) and stirred for 10 minutes (the pH of the mixture after diluting 1 ml of the sample with an equal volume of water was 4.3-4.6). Then 2-methoxypropene (1.27 L, 13.24 mol, 2.2 equivalents) was added and the reaction mixture was heated to 38-40 ^° C. for 2 hours. The reaction mixture was cooled to 20 ^° C. and partitioned between ethyl acetate (12 L) and 5% aqueous NaHCO ₃ solution (10 L). The mixture was stirred and the layers were separated. The ethyl acetate extract was washed with 5% aqueous NaHCO ₃ solution (10 L) and water (4 L). The ethyl acetate extract was dried by atmospheric distillation, and the solvent was changed to cyclohexane (total volume ~ 30 L). After distillation and concentration (volume of extraction with ethyl acetate 20% vol.), The hot cyclohexane solution was slowly cooled to 25 ^o C with crystallization of the reaction product. The resulting suspension was further cooled to 10 ^° C. and allowed to age. The reaction product was isolated by filtration and the wet cake was washed with cold (10 ^° C.) cyclohexane (2 x 800 ml). The washed filter cake was dried in vacuo (26 "N; approximately 66 mm Hg) at 40 ^° C to give 1.65 kg of acetonide 1 (86.4%, 98% area, according to HPLC).

¹ H-NMR spectrum (300.13 MHz, CDCl ₃ main rotamer) δ: 7.36-7.14 (multiplet, 9H), 5.03 (doublet, J = 4.4 Hz, 1H), 4.66 multiplet, 1H), 3.15 (multiplet, 2H), 3.06 (wide singlet, 2H), 2.97 (multiplet, 2H): 1.62 (singlet, 3H), 1.37 (singlet, 3H);
¹³ C-NMR (75.5 MHz, CDCl ₃ main rotamer) δ _c : 168.8, 140.9, 140.8, 140.6,128.6, 128.5, 128.4,127.1, 126.3, 125.8, 124.1, 96.5, 78.6, 65.9, 38.4, 36.2, 31.9, 26.5, 24.1.

Elemental analysis: calculated for C ₂₁ H ₂₃ NO ₂
C 78.47; H 7.21; N 4.36;
Found; C 78.65, H 7.24, N 4.40.

Раствор ацетонида 1 (1000 г, 3,11 моля) и 2(S)-глицидилтозилата 2 (853 г, 3,74 моля, 1,2 эквивалента) в 15,6 л ТГФ (KF=22 мг/мл) в 50 л четырехгорлой круглодонной колбе, снабженной термопарой, механической мешалкой, капельной воронкой и адаптером для ввода азота, трижды дегазировали, создавая вакуум и осуществляя продувку азотом, и полученную смесь охлаждали до -56^oC. Затем в течение 2 часов добавляли гексаметилдисилазид лития (LiN/(CH₃)₃ Si/2) (2,6 л, 1,38 моля, 1,15 эквивалентов), поддерживая при добавлении температуру внутри колбы в интервале -50 - -45^oC. Затем реакционную смесь перемешивали в течение 1 часа при -45 - -40^oC и давали ей нагреваться до -25^oC в течение 1 часа. Смесь перемешивали в течение 4 часов при -25 - -22^oC (или до тех пор, пока исходный ацетонид не занимал 3,0% площади).Example 5. Obtaining epoxide 3

A solution of acetonide 1 (1000 g, 3.11 mol) and 2 (S) -glycidyltosylate 2 (853 g, 3.74 mol, 1.2 equivalents) in 15.6 L of THF (KF = 22 mg / ml) in 50 A four-necked round-bottom flask equipped with a thermocouple, a mechanical stirrer, a dropping funnel and an adapter for introducing nitrogen was degassed three times, creating a vacuum and purging with nitrogen, and the resulting mixture was cooled to -56 ^° C. Then, lithium hexamethyldisilazide (LiN / (CH ₃ ) ₃ Si / 2) (2.6 L, 1.38 mol, 1.15 equivalents), maintaining, when added, the temperature inside the flask in the range of -50 - -45 ^o C. Then the reaction mixture stirred for 1 hour at -45 - -40 ^o C and allowed to warm to -25 ^o C for 1 hour. The mixture was stirred for 4 hours at -25 - -22 ^° C (or until the starting acetonide occupied 3.0% of the area).

The progress of the reaction was monitored by HPLC analysis: Zorbax silica column, 25 cm x 4.6 nm in size, 20% ethyl acetate in hexane, 2.0 ml / min, injection volume = 20 ml, detection = 254 nm, sample preparation = 100-fold dilution. Approximate retention times:
Retention Time (min) - Identification
5.5 - amide 1
6.5 - glycidyl tosylate 2
13.5 - epoxy 3
The reaction mixture was treated with deionized water (6.7 L) at -15 ^° C and treated with ethyl acetate (10 L). The mixture was stirred and the layers were separated. The ethyl acetate extract was washed with a mixture of 1% aqueous NaHCO ₃ solution (5 L) and saturated NaCl solution (0.5 L). An ethyl acetate extract (28.3 L) was concentrated by vacuum distillation (28 "Hg; 71.1 mm Hg) and additional ethyl acetate was added to replace the solvent with ethyl acetate (final volume = 11.7 L). In ethyl acetate concentrate, a change was made. solvent on MeOH to crystallize the product and the system was concentrated to a final volume of 3.2 L. The remaining ethyl acetate solvent was removed by loading 10 L of methanol and collecting 10 L of distillate. The resulting suspension was stirred for 1 hour at 22 ^° C, then cooled to 5 ^o C and wasps fected aging for 0.5 hr The product was isolated by filtration and the wet cake was washed with cold methanol (2 x 250 mL) The washed filter cake was dried in vacuo (26 "Hg; about 66 mm Hg).. at 25 ^o C to give 727 g of epoxide 3 (61.2%, 98.7% of the area of the main epoxide, according to HPLC).

¹³ C NMR (300 MHz, CDCl ₃ ) δ: 171.1, 140.6, 140.5, 139.6, 129.6, 128.8, 128.2, 127.2, 126.8, 125.6, 124.1, 96.8, 79.2, 65.8, 50.0, 48.0, 44.8, 39.2, 37.4, 36.2, 26.6, 24.1
Example 6. Obtaining penultimate 6 (see the end of the description).

Suspension 2 (S) -tret. -butylcarboxamido-4-N-Boc-piperazine 4 (1950 g, 6.83 mol,> 99.5% ee) (ee = enantiomeric excess) and epoxide 3 (2456 g, mixture in a ratio of 97.5: 2, 5 epoxides 4 S / R, 6.51 mol) in isopropanol (2-propanol, 18.6 L), placed in a 72-liter round bottom flask with four inlets, equipped with a mechanical stirrer, a reflux condenser, a steam bath coated with a Teflon thermocouple and an inlet for nitrogen, was heated until reflux (internal temperature 84-85 ^o C). After 40 minutes, a homogeneous solution formed. The mixture was heated under reflux for 28 hours.

The internal temperature during reflux was 84-85 ^o C. The progress of the reaction was monitored by HPLC analysis: 25 cm DuPont C8-PX column, ratio acetonitrile / 10 mM (KH ₂ PO ₄ / K ₂ HPO ₄ 60:40, 1, 0 ml / min, detection = 220 nm, sample preparation: 2 μl, the reaction mixture was diluted to a volume of 1 ml with acetonitrile.
Retention Time (min) - Assignment
4.8 - piperazine 4
8.9 - epoxy 3
15.2 - product combination 5
After 28 hours, the remaining epoxide 3 and combination product 5 (according to HPLC data) accounted for 1.5% of the area and 91-93% of the area, respectively. The mixture was cooled to 0-5 ^° C. and 20.9 L of 6 NHCl was added, keeping the temperature below 15 ^° C. After the addition was complete, the mixture was heated to 22 ^° C. At this point, gas evolution (isobutylene) was observed. The mixture was allowed to age at 20-22 ^o C for 6 hours.

2-Пиразинкарбоновая кислота (8) - 3,35 кг (27 ммолей)
Хлористый оксалил - 3,46 кг (27,2 моля)
Трет.-бутиламин (KF = 460 мкг/мл) - 9,36 л (89 молей)
EtOAc (KF = 56 мкг/мл) - 27 л
ДМФ - 120 мл
1-Пропанол - 30 л
Карбоновую кислоту 8 суспендировали в 27 л EtOAc и 120 мл ДМФ в 72-литровой трехгорлой колбе, снабженной механической мешалкой, в атмосфере N₂ и суспензию охлаждали до 2^oC. Хлористый оксалил вводили в систему, поддерживая температуру в интервале 5-8^oC.The progress of the reaction was monitored by the HPLC method: using the same conditions as described above. Exemplary retention times were as follows:
Retention Time (min) - Assignment
7.0 - cis aminoindanol
11.9 - penultimate 6
15.1 - combination product 5
The mixture was cooled to 0 ^° C. and 7.5 L of a 50% NaOH solution was slowly added to adjust the pH of the mixture to a pH of 11.6, keeping the temperature below 25 ^° C. during the addition. The mixture was partitioned between ethyl acetate (40 L) and water (3 L). The mixture was stirred and the layers were separated. The organic phase (60 L) was concentrated under reduced pressure (29 "Hg; 73.7 mm Hg) and the solvent was replaced with DMF and the resulting mixture was concentrated to a final volume of 10.5 L (KF = 1.8 mg / ml) According to HPLC analysis, the yield of compound 6 in ethyl acetate was 86.5%. Compound 6 in DMF was directly used in the next step without further purification.
¹³ C NMR (75.4 MHz, CDCl ₃ ) δ: 175.2, 170.5, 140.8, 140.5, 139.9, 129.1, 128.5, 127.9, 126.8, 126.5, 125.2, 124.2, 73.0, 66.0, 64.8, 62.2, 57.5, 49.5, 47.9, 46.4, 45.3, 39.6, 39.3, 38.2, 28.9
Example 7. Pyrazine-2-tert-butylcarboxamide 9

2-Pyrazinecarboxylic acid (8) - 3.35 kg (27 mmol)
Oxalyl chloride - 3.46 kg (27.2 mol)
Tert.-Butylamine (KF = 460 mcg / ml) - 9.36 L (89 moles)
EtOAc (KF = 56 μg / ml) - 27 L
DMF - 120 ml
1-Propanol - 30 L
Carboxylic acid 8 was suspended in 27 L of EtOAc and 120 ml of DMF in a 72-liter three-necked flask equipped with a mechanical stirrer in an atmosphere of N ₂ and the suspension was cooled to 2 ^o C. Oxalyl chloride was introduced into the system, maintaining the temperature in the range of 5-8 ^o C .

Addition was completed in 5 hours. During exothermic addition, CO and CO _{2 were released} . The formation of HCl occurred, which mainly remained in solution. A precipitate was present in the system, probably representing the HCl salt of pyrazinic acid chloride. Analysis of the formation of acid chloride was carried out by quenching an anhydrous sample of the reaction mixture with tert-butylamine. After completion of the reaction, less than 0.7% acid 8 remained.

 Analysis of the completeness of the formation of acid chloride is important, since incomplete reaction leads to the formation of impurities of bis-tert.-butyloxamide.

The progress of the reaction can be monitored by the HPLC method: DuPont Zorbax RXC8 column 25 cm in size, flow rate 1 ml / min and detection at 250 nm; linear gradient with 98% 0.1% aqueous solution of H ₃ PO ₄ and 2% CH ₃ CN to 5% aqueous solution of H ₃ PO ₄ and 50% CH ₃ CN for 30 minutes. Retention times: acid 8 = 10.7 min, amide 9 = 28.1 min.

The reaction mixture was allowed to age for 1 hour at 5 ^° C. The resulting suspension was cooled to 0 ^° C., tert-butylamine was added at such a rate as to maintain the temperature inside the reactor below 20 ^° C.

 Addition requires 6 hours if the reaction was highly exothermic. A small portion of the resulting tert.-butylammonium hydrochloride was removed from the reaction system as a fluffy white solid.

The mixture was allowed to age for another 30 minutes at 18 ^° C. Precipitated ammonium salts were removed by filtration. The filter cake was washed with 12 L EtOAc. The combined organic phases were washed with 6 L of 3% NaHCO ₃ and 2 x 2 L of a saturated aqueous NaCl solution. The organic phase was treated with 200 g of Darko G 60 carbon and filtered through Zolka Flock, and the filter cake was washed with 4 L EtOAc.

 Carbon treatment effectively removes the magenta color of the product.

 A solution of compound 9 in EtOAc was concentrated at a pressure of 10 mbar to 25% of the original volume. 30 L of 1-propanol was added and distillation was continued to a final volume of 20 L.

At this point, the amount of EtOAc was below the detection limit by ¹ H-NMR (<1%). The internal temperature of this solvent change was <30 ^° C. The solution of 1-propanol / EtOAc of compound 3 was stable until reflux for several days.

Evaporation of an aliquot afforded a tan solid, mp. 87-88 ^o C.

¹³ C NMR (75 MHz, CDCl ₃ , ppm): 161.8, 146.8, 154.0, 143.8, 142.1, 51.0, 28.5
Example 8. rac-2-tert.-butylcarboxamide piperazine 10.

Материалы:
Пиразин-2-трет. бутилкарбоксамид 9 2,4 кг (23,4 моля), в растворе 1-пропанола 12 л 20% Pd(OH)₂/C 16% вес., вода 144 г.

Materials:
Pyrazin-2-tert. butylcarboxamide 9 2.4 kg (23.4 mol), in a solution of 1-propanol 12 l 20% Pd (OH) ₂ / C 16% by weight, water 144 g.

A solution of pyrazine-2-tert-butylcarboxamide / 1-propanol was placed in an autoclave with a capacity of 5 gallons (18.3 L). The catalyst was added in and the mixture was hydrogenated at 65 ^o C and a pressure of H ₂ 40 lb / ⁱⁿ² (3 atm).

After 24 hours, the reaction mixture absorbed a theoretical amount of hydrogen and GC showed the presence of <1% of compound 9. The mixture was cooled, N ₂ was purged, and the catalyst was removed by filtration through Zolka Flock. The catalyst was washed with 2 L of warm 1-propanol.

 It was found that the use of warm 1-propanol during the washing of the filter cake improves filtration and reduces product loss from the filter cake.

The progress of the reaction was monitored by GC: a Megabor column 30 m long, programming the temperature in the range of 100-160 ^o C at a speed of 10 ^o C / min, holding for 5 minutes, then raising the temperature to 250 ^o C at a speed of 10 ^o C / min, times retention: compound 9 = 7.0 minutes, compound 10 = 9.4 minutes. The progress of the reaction can also be monitored by TLC using the EtOAc / MeOH system (50:50) as a solvent and ninhydrin as a developing agent.

 Evaporation of an aliquot showed that the yield during amidation and hydrogenation was 88% and that the concentration of compound 10 was 133 g / L.

Evaporation of an aliquot afforded compound 10 as a white solid, mp. 150-151 ^o C;
¹³ C NMR (75 MHz, D ₂ O, ppm): 173.5, 59.8, 52.0, 48.7, 45.0, 44.8, 28.7
Example 9. (S) Salt (S) -2-tert-butylcarboxamide piperazine bis (S) camphorosulfonic acid 11.

Материалы:
рац-2-трет. -бутилкарбоксамид-пиперазин 10 в растворе 1-пропанола - 4,10 кг (22,12 моля) в 25,5 кг растворителя
(S)-(+)-10-камфоросульфокислота - 10,0 кг (43,2 моля)
1-пропанол - 12 л
Ацетонитрил - 39 л
Вода - 2,4 л
Раствор амина 10 в 1-пропаноле загружали в 100 л колбу, соединенную с центрифугой (концентратором) периодического действия. Раствор концентрировали при давлении 10 миллибар и при температуре < 25^oC до объема около 12 л.

Materials:
rat-2-tert. -butylcarboxamide-piperazine 10 in a solution of 1-propanol - 4.10 kg (22.12 mol) in 25.5 kg of solvent
(S) - (+) - 10-camphorosulfonic acid - 10.0 kg (43.2 mol)
1-propanol - 12 l
Acetonitrile - 39 L
Water - 2.4 L
A solution of amine 10 in 1-propanol was charged into a 100 L flask connected to a batch centrifuge (concentrator). The solution was concentrated at a pressure of 10 mbar and at a temperature of <25 ^o C to a volume of about 12 L.

At this point, the product precipitated from the solution, but returned back to the solution when the mixture was heated to 50 ^o C.

Analysis of a homogeneous aliquot showed that the concentration of compound 10 was 341 g / L. The concentration was determined by HPLC: DuPont Zorbax PXC8 column 25 cm long, feed rate 1.5 ml / min, detection at a wavelength of 210 nm, isocratic (98/2) CH ₃ CN / 0.1% aqueous solution of H ₃ PO ₄ . Compound Retention Time 10: 2.5 minutes.

 Acetonitrile (39 L) and water (2.4 L) were added to form a clear, slightly brownish solution.

Determination of the water content of KF by titration and the ratio of CH ₃ CN / 1-propanol by integration of ¹ H-NMR data showed that the ratio of CH ₃ CN / 1-propanol / H ₂ O is 26/8 / 1.6. The concentration in the solution was 72.2 g / l.

(S) -10-camphorosulfonic acid was charged in 4 portions for 30 minutes at 20 ^° C. Before adding CSA, the temperature rose to 40 ^° C. After a few minutes, a viscous white precipitate formed. The white suspension was heated to 76 ^° C. to dissolve all solids, then the slightly brownish solution was cooled to 21 ^° C. for 8 hours.

The product precipitated at 62 ^° C. The product was filtered without aging at 21 ^° C. and the filter cake was washed with 5 L of a solvent mixture of CH ₃ CN / 1-propanol / H ₂ O in a ratio of 26/8 / 1.6. The resulting solid was dried at 35 ^o C in a vacuum oven with a feed of N ₂ with the formation of 5.6 kg (39%) of compound 11 as a white crystalline solid, so pl. 288-290 ^o C (decomposition).

[α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = 18.9 ^o (c = 0.37, H ₂ O).

¹³ C NMR (75 MHz, D ₂ O, ppm): 222.0, 164.0, 59.3, 54.9, 53.3, 49.0, 48.1, 43.6, 43.5, 43.1, 40.6, 40.4, 28.5, 27.2, 25.4, 19.9, 19.8
The ee value of the material was 95% according to the following chiral HPLC analysis: an aliquot of compound 11 (33 mg) was suspended in 4 ml EtOH and 1 ml Et ₃ N. Boc ₂ O (11 mg) was added and the reaction mixture was allowed to age for 1 hour . The solvent was completely removed in vacuo and the residue was dissolved in about 1 ml of EtOAc and filtered using a Pasteur pipette through SiO ₂ using EtOAc as eluent. The evaporated product fractions were redissolved in hexanes in an amount of 1 mg / ml. Enantiomers were separated on a Dysel Hirasell AS column using a hexane / IPA (97: 3) dissolution system at a flow rate of 1 ml / min, detecting at 228 nm. Retention times: S antipode = 7.4 minutes, R = 9.7 minutes.

 Example 10. (S) -2-tert.-Butylcarboxamide-4-tert.-butoxycarbonyl-piperazine 4 from salt 11.

Материалы;
(S)-2-трет. -бутилкарбоксамид-пиперазиновая Бис(S)- (+)-CSA соль 11, 95% ее - 5,54 кг (8,53 моля)
Ди-трет.-бутилдикарбонат - 1,86 кг (8,53 моля)
Et₃N - 5,95 л (42,6 моля)
EtOHПунктилиус, крепость 200 - 55 л
EtOAc - 2 л
К (S)-CSA соли 11 в трехгорлой колбе емкостью 110 л, снабженной капельной воронкой, в атмосфере N₂ добавляли EtOH, после чего при 25^oC добавляли триэтиламин. Твердое вещество легко растворялось при добавлении Et₃N. Boc₂O растворяли в EtOAc и загружали в капельную воронку. Раствор Boc₂O в EtOAc добавляли с такой скоростью, чтобы поддерживать температуру ниже 25^oC. Добавление продолжали в течение 3 часов. Реакционной смеси давали стареть в течение 1 часа после завершения добавления раствора Boc₂O.

Materials
(S) -2-tert. -butylcarboxamide-piperazine Bis (S) - (+) - CSA salt 11, 95% of it - 5.54 kg (8.53 mol)
Di-tert.-butyl dicarbonate - 1.86 kg (8.53 mol)
Et ₃ N - 5.95 L (42.6 mol)
EtOHPunktius, strength 200 - 55 L
EtOAc - 2 L
To (S) -CSA salt 11 in a 110 L three-necked flask equipped with a dropping funnel, EtOH was added under N ₂ atmosphere, after which triethylamine was added at 25 ^° C. The solid was easily dissolved by adding Et ₃ N. Boc ₂ O was dissolved in EtOAc and loaded into a dropping funnel. A solution of Boc ₂ O in EtOAc was added at such a rate as to maintain a temperature below 25 ^° C. Addition was continued for 3 hours. The reaction mixture was allowed to age for 1 hour after complete addition of the Boc ₂ O solution.

The progress of the reaction can be monitored by the HPLC method: DuPont Zorbax RXC8 column at a feed rate of 1 ml / min, detection at. 228 nm, isocratic mixture (50/50) CH ₃ CN / 0.1 M KH ₂ PO ₄ , at pH 6.8 with NaOH. Retention time of compound 4 = 7.2 minutes. Chiral analysis was performed using the same system as in the previous step. The progress of the reaction can also be monitored using TLC, using 100% EtOAc (R _f = 0.7) as a solvent.

Then the solution was concentrated to a volume of about 10 l at an internal temperature of <20 ^o C in a centrifuge (concentrator) of periodic action, in vacuum with a pressure of 10 mbar. The solvent change was completed by adding EtOAc in 20 L and reconcentrating to a volume of 10 L. The reaction mixture was washed into the extractor with 60 L EtOAc. The organic phase was washed with 16 L of a 5% aqueous solution of Na ₂ CO ₃ , 2 x 10 L of deionized water and 2 x 6 L of a saturated aqueous solution of sodium chloride. The combined aqueous washes were back extracted with 20 L EtOAc and the organic phase was washed with 2 x 3 L water and 2 x 4 L saturated aqueous sodium chloride. The combined EtOAc extracts were concentrated in vacuo at a pressure of 10 mbar, at a temperature inside the reaction vessel> 20 ^o C in a batch centrifuge with a capacity of 100 l, to a volume of about 8 l. The solvent was replaced by cyclohexane by slowly adding about 20 L of cyclohexane and re-concentrating to a volume of 8 L. 5 L of cyclohexane and 280 ml of EtOAc were added to the suspension, and the mixture was heated to reflux, when all contents had passed into solution. The solution was cooled and seed (10 g) was added at 58 ^° C. The suspension was cooled to 22 ^° C. over 4 hours and the product was isolated by filtration after 1 hour of aging at 22 ^° C. The filter cake was washed with 1.8 L of cyclohexane and dried in a vacuum oven at 35 ^° C. with a supply of N ₂ , whereby 1.87 kg (77%,> 99.9% of the area, according to HPLC R-isomer was present in an amount below the detection level) of compound 4 as a slightly tan powder.

[α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = 22.0 (s = 0.20, MeOH), mp 107 ^o C;
¹³ C NMR (75 MHz, CDCl ₃ ppm): 170.1, 154.5, 79.8, 58.7, 50.6; 46.6, 43.6, 43.4, 28.6, 38.3.

 Although the principles of the present invention are set forth in the present description, and examples are presented for purposes of illustration, it should be borne in mind that the practical implementation of the invention covers all the usual changes, adaptations or modifications falling within the scope of the following claims and equivalents of the claims.

 Appendix 1. Obtaining the compound of claim 13 (in the refined formula of claim 7) (hereinafter referred to as compound 13).

2-Иодфенол (500 мг, 2,27 ммоль), пропаргиловый спирт (265 мкл, 4,54 ммоль), Pd(OAc)₂ (5,1 мг, 0,03 ммоль), трифенилфосфин (12 мг, 0,046 ммоль), н-бутиламин (450 мкл, 4,5 ммоль) и CuI (8,6 мг, 0,045 ммоль) объединяют в 4,5 мл ТТФ и смесь нагревают при 40^oC в атмосфере N₂ в течение 36 ч. Смесь охлаждают до комнатной температуры и растворители удаляют в вакууме и остаток очищают SiO₂ колоночной хроматографией на 100 г силикагеля, элюируя 20% этилацетатом в гексанах. 2-(Гидроксиметил) бензофуран получают путем концентрирования фракций, содержащих продукт. [Kudu, N. G.; Pal, М.; Nahanty, J.S.; Dasgupta, F.K.; JCS Chem. Com. 1992, 41].The first stage - Getting benzofuran-2-carbinol

2-Iodophenol (500 mg, 2.27 mmol), propargyl alcohol (265 μl, 4.54 mmol), Pd (OAc) ₂ (5.1 mg, 0.03 mmol), triphenylphosphine (12 mg, 0.046 mmol) , n-butylamine (450 μl, 4.5 mmol) and CuI (8.6 mg, 0.045 mmol) are combined in 4.5 ml of TTF and the mixture is heated at 40 ^° C. under N ₂ for 36 hours. The mixture is cooled to room temperature and the solvents were removed in vacuo and the residue was purified by SiO ₂ column chromatography on 100 g of silica gel, eluting with 20% ethyl acetate in hexanes. 2- (Hydroxymethyl) benzofuran is obtained by concentration of fractions containing the product. [Kudu, NG; Pal, M .; Nahanty, JS; Dasgupta, FK; JCS Chem. Com. 1992, 41].

Бензофуран-2-карбинол (10,38 г, 68,4 ммоль) растворяют в метиленхлориде (100 мл) и охлаждают до 5^oC. Через 5 минут загружают тионилхлорид (5,49 мл, 75,2 ммоль) и реакционную смесь выдерживают при 5^oC в течение 30 минут и нагревают до 22^oC. Реакционную смесь выдерживают при 22^oC в течение 4 часов. Метиленхлоридную загрузку промывают ДИ водой (4 х 60 мл) и фильтруют через силикагель. Раствор концентрируют в вакууме, чтобы получить твердое вещество при охлаждении. Сырое твердое вещество растворяют в гексанах(120 мл) и обрабатывают Darco G-60 (1,0 г). Суспензию фильтруют и раствор концентрируют в вакууме, чтобы получить соединение бензофуран-2-хлорметил в виде твердого вещества.The second stage - Obtaining benzofuran-2-chloromethyl

Benzofuran-2-carbinol (10.38 g, 68.4 mmol) was dissolved in methylene chloride (100 ml) and cooled to 5 ^° C. After 5 minutes, thionyl chloride (5.49 ml, 75.2 mmol) was charged and the reaction mixture was allowed to stand at 5 ^° C. for 30 minutes and heated to 22 ^° C. The reaction mixture was kept at 22 ^° C. for 4 hours. The methylene chloride charge is washed with DI water (4 x 60 ml) and filtered through silica gel. The solution was concentrated in vacuo to give a solid by cooling. The crude solid was dissolved in hexanes (120 ml) and treated with Darco G-60 (1.0 g). The suspension is filtered and the solution concentrated in vacuo to give the benzofuran-2-chloromethyl compound as a solid.

Выделенное предпоследнее твердое вещество (13,1 г, 25 ммоль) объединяют с IPAc (60 мл), водой (20 мл), KHCO₃ (4,25 г, 42,5 ммоль), иодидом натрия (1,88 г, 12,5 ммоль) и бромидом тетрабутиламмония (600 мг, 1,86 ммоль) и смесь нагревают до 45^oC под атмосферой азота. Добавляют 2- (хлорметил)бензофуран (4,6 г, 27,5 ммоль) и полученную смесь нагревают до 59-61^oC в течение 5 ч. Смеси позволяют остыть до комнатной температуры и разбавляют IPAc (100 мл) и отделяют водный слой. Органический слой промывают 3 х 50 мл водой, затем 50 мл солевого раствора и сушат (MgSO₄) и фильтрат концентрируют в вакууме и промывают 100 мл IPAc и концентрируют в атмосферных условиях до 80 мл, охлаждают до 25^oC, вводят затравку и выдерживают при перемешивании 2 ч. Твердое вещество отфильтровывают и промывают холодным IPAc (2 х 15 мл), чтобы получить Соединение 13 (температура плавления = 152-153,5^oC).Third Stage - Alkylation with benzofuran-2-chloromethyl

The isolated penultimate solid (13.1 g, 25 mmol) was combined with IPAc (60 ml), water (20 ml), KHCO ₃ (4.25 g, 42.5 mmol), sodium iodide (1.88 g, 12 5 mmol) and tetrabutylammonium bromide (600 mg, 1.86 mmol) and the mixture is heated to 45 ^° C. under nitrogen. 2- (Chloromethyl) benzofuran (4.6 g, 27.5 mmol) was added and the resulting mixture was heated to 59-61 ^° C for 5 hours. The mixture was allowed to cool to room temperature and diluted with IPAc (100 ml) and the aqueous layer was separated . The organic layer was washed with 3 x 50 ml of water, then 50 ml of brine and dried (MgSO ₄ ) and the filtrate was concentrated in vacuo and washed with 100 ml of IPAc and concentrated under atmospheric conditions to 80 ml, cooled to 25 ^° C., seeded and incubated stirring for 2 hours. The solid was filtered off and washed with cold IPAc (2 x 15 ml) to give Compound 13 (melting point = 152-153.5 ^° C.).

 Appendix 2. Biological data for compound 13.

Inhibition assay of microbial expressed HIV protease
Inhibition studies of the reaction of a protease expressed in Eschericia coli with a peptide structure of [Val-Ser-Gln-Asn- (beta-naphthyl) Ala-Pro-Ile-Val, 0.5 mg / ml, at the initiation of the reaction is carried out in 50 mm Na acetate, pH 5.5, at 30 ^o C for 1 hour. Different concentrations of the inhibitor in 1.0 μl DMSO are added to 25 μl of the peptide solution in water. The reaction is initiated by adding 15 μl of 0.33 nM protease (0.11 ng) in a solution of 0.133 M Na acetate pH 5.5 and 0.1% bovine serum albumin. The reaction is quenched with 160 μl of 5% phosphoric acid. The reaction products are separated by HPLC (VYDAC wide pore 5 cm C-18 reverse phase, acetonitrile gradient, 0.1% phosphoric acid). The degree of inhibition of the reaction is determined by the heights of the product peaks. The HPLC of products independently synthesized confirms compliance with the quality standards of analysis and confirms the composition of the product. Compound 13 shows IC ₅₀ values of about 0.17 nM.

 Cell distribution analysis.

Inhibition of the spread of HIV in cell culture is measured according to Nunberg et al. J. Virol. 65, 4887 (1991). In this assay, MT-4 T-lymphoid cells infect HIV-1 (wild type other than otherwise indicated) using a predetermined inoculum, and cultures are incubated for 24 hours. At this time, ≤ 1% of positive cells are detected by indirect immunofluorescence. The cells are then extensively washed and dispensed into 96-well culture plates. A sequentially twice diluted inhibitor is added to the wells and cultivation is continued for 3 additional days. 4 days after infection, 100% of the cells in the control cultures are infected. The accumulation of HIV-1 p24 is directly correlated with the spread of the virus. Inhibitory concentration for cell culture is defined as the concentration of inhibitor in nanomoles / liter, which reduces the spread of infection by at least 95% or CIC ₉₅ . CIC ₉₅ for Compound 13 - 25 nM.

 Appendix 3. Pharmaceutical compositions containing compound 13.

Свободное основание Соединения 13 (25 г, 38,3 ммоль) растворяют в абсолютном этаноле (150 мл) при 22^oC. Эту порцию фильтруют через 5 мкм фильтр и фильтр промывают абсолютным спиртом (50 мл). Раствор серная кислота/этанол готовят при < 5^oC путем введения концентрированной серной кислоты (3,91 г, 38,3 ммоль) в охлажденный раствор < 5^oC) абсолютного этанола (50 мл) с такой скоростью, чтобы температура оставалась < 5^oC. Часть кислотного раствора (10 мл, 20 об.%) загружают в раствор порции Соединения 13 при 22^oC. В этот момент в порцию Соединения 13 необязательно может быть внесена затравка. Соединение 13^*сульфат^*этанолят (500 мг), при 22^oC. Идеально вносить затравку в порцию Соединения 13, когда затравка помогает пересыщению во время кристаллизации. Суспензию выдерживают при 20-25^oC в течение 30 минут. Остальной раствор кислоты загружают в порцию через канюлю свыше 60 минут. Во время добавления температура порции остается 20-25^oC (примечание: раствор кислоты держат при < 5^oC).Stage One - Salt Formation

The free base of Compound 13 (25 g, 38.3 mmol) was dissolved in absolute ethanol (150 ml) at 22 ^° C. This portion was filtered through a 5 μm filter and the filter was washed with absolute alcohol (50 ml). A sulfuric acid / ethanol solution is prepared at <5 ^° C by introducing concentrated sulfuric acid (3.91 g, 38.3 mmol) in a cooled solution of <5 ^° C) absolute ethanol (50 ml) at such a rate that the temperature remains <5 ^o C. A portion of the acid solution (10 ml, 20 vol.%) was loaded into the solution of a portion of Compound 13 at 22 ^o C. At this point, a portion of Compound 13 could optionally be seeded. Compound 13 ^* sulfate ^* ethanolate (500 mg), at 22 ^° C. It is ideal to seed a portion of Compound 13 when the seed helps supersaturation during crystallization. The suspension is maintained at 20-25 ^o C for 30 minutes. The remaining acid solution is loaded into the portion through the cannula over 60 minutes. During the addition, the portion temperature remains 20-25 ^o C (note: the acid solution is kept at <5 ^o C).

The final portion suspension is maintained at 20-25 ^° C. for 60 minutes and filtered. The filter cake was washed with absolute ethanol (2 x 25 ml) and dried in vacuo (635 mm Hg, 20 ^o C) for 18 hours in a nitrogen stream to obtain Compound 13 monoethanolate sulfate. The differential scanning calorimetry (DSC) curve was characterized at a heating rate of 10 ^° C./min in an open vessel under current nitrogen, detecting an endotherm with an extrapolated initial temperature of about 190 ^° C., a peak temperature of about 193 ^° C., and associated heat of about 120 J / g. Based on the results of TG and TG-FTIR, the endotherm is the result of a combination of ethanol loss and melting with decomposition. The X-ray powder diffraction pattern is characterized by d-intervals of 11.72, 5.56, 5.20, 5.00, 4.60, 4.50, 4.40, 4.26, 4.17, 4.08, 3.90, 3.81, 3.69, 3.24 and 3.33 Angstroms.

The second stage - Preparation A
100 mg of the sulfate salt of Compound 13 from the First Stage is mixed with sufficiently finely divided lactose to obtain a total amount of 580-590 mg to fill the capsule from an O-type solid gel.

 The third stage is Preparation B.

 An oral suspension is prepared by suspending 400 mg of the sulfate salt of Compound 13 in 20 ml of water.

 Appendix 4. Characteristics of the compounds of example 3.

Melting points (mp) of typical compounds:

Claims (13)

1. The compound of formula I

-
or a pharmaceutically acceptable salt of such a compound,
Where

represents a stable 8-10 membered bicyclic heterocycle, each of the rings of which may be saturated or unsaturated, and said heterocycle consists of carbon atoms and 1-3 heteroatoms selected from the group consisting of N, S or O, wherein said heterocycle may be unsubstituted or substituted by halogen or lower C _1-4 alkyl;
provided that

not groups;

or

2. The compound according to claim 1, in which

represents a stable 8-10 membered bicyclic heterocycle, any ring of which may be saturated or unsaturated and said heterocycle consists of carbon atoms and 2 heteroatoms selected from the group consisting of N or O, and the heteroatoms are present in different rings, or pharmaceutically an acceptable salt of such a compound. 3. The compound according to claim 1, in which

represents:

X represents O or S,
or a pharmaceutically acceptable salt of such a compound. 4. The compound according to claim 1, in which

limited to fragment

or a pharmaceutically acceptable salt of such a compound. 5. The compound according to claim 1, which represents

called N- (2 (R) -hydroxy-1 (S) -indanyl) -2 (R) -phenylmethyl-4 (S) -hydroxy-5- (1- (4- (3-furo [2,3- b] pyridylmethyl) -2 (S) -N '- (tert-butylcarboxamido) piperazinyl)) pentanamide, or a pharmaceutically acceptable salt of such a compound. 6. The compound according to claim 1 of the formula

or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 6, represented by the formula

or a pharmaceutically acceptable salt thereof.  8. A pharmaceutical composition, characterized in that the active ingredient comprises an effective amount of a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.  9. The pharmaceutical composition of claim 8, inhibiting HIV protease.  10. A method of inhibiting HIV protease, characterized in that it is prescribed to a mammal in need of such treatment an effective amount of a compound according to any one of claims 1 to 7.  11. The method according to claim 10, characterized in that an effective amount of a compound according to claim 7 is prescribed.  12. The pharmaceutical composition of claim 8, characterized in that as the active ingredient contains a compound of claim 7.  13. The pharmaceutical composition according to p. 12, characterized in that it further comprises a non-nucleoside HIV reverse transcriptase inhibitor selected from compound B, compound C and nevirapine, where compound B is 6-chloro-4 (S) -cyclopropyl-3,4 -dihydro-4 - ((2-pyridyl) ethynyl) quinazolin-2 (IH) -one, Compound C is (-) 6-chloro-4 (S) -trifluoromethyl-1,2-dihydro-4 (H) -3,1-benzoxazin-2-one.  14. The composition according to p. 13, characterized in that, in addition, further comprises a nucleoside inhibitor of HIV reverse transcriptase selected from AZT, ddi and ddC, where AZT is Zidovudine, ddi is dideoxyninosine and ddC is dideoxyimitidine .

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