SK110996A3 - Heterocyclicly substituted pseudo-peptides, producing method thereof and drugs containing these substances - Google Patents

Abstract

Heterocyclic pseudo-peptides of formula (I) are new where R<1> = a gp. of formula (a) or (b) R<3>, R<4> = a gp. of formula (c), (d) or (e) and R<2> = H or 1-20C acyl opt. substd. with carboxy or 1-6C alkoxycarbonyl.

Description

HETEROCYCLICALLY SUBSTITUTED PSEUDOPEPTIDES, METHOD OF MANUFACTURING THEREOF AND ACTIVE SUBSTANCES CONTAINING

Technical field

The invention relates to novel heterocyclically substituted pseudopeptides, to a process for their preparation and to antiretroviral agents containing them.

BACKGROUND OF THE INVENTION

EP 528 242 discloses trifluoromethyl groups containing pseudopeptides as antiretroviral agents.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides novel high-performance heterocyclically substituted pseudopeptides of the formula I r'-nh

H (I) wherein

R ¹ is a radical of formula

or R 4 -O-CO wherein r 3 and R 6 are the same or different and represent a heterocyclic radical of the formula

r 2 is a hydrogen atom or a straight or branched acyl group of up to 20 carbon atoms optionally substituted by a straight or branched alkoxycarbonyl group of up to 6 carbon atoms; i

and salts thereof.

The compounds of the formula I according to the invention have a plurality of asymmetric carbon atoms.

Representative radical of formula (Ia)

(Ia) has 5 asymmetric carbon atoms (*), which independently of one another occur in the R- or S- configuration. Preferred are configurations 1 (R), 2 (R), 3 (S), 4 (S) 5 (S), and 6 (S).

The amino acids used in the compounds of the formula I according to the invention can be present in both the L-form and the D-form.

Physiologically acceptable salts of heterocyclically substituted pseudopeptides may be salts of the compounds of the present invention with mineral acids, carboxylic acids or sulfonic acids. Particularly preferred are, for example, salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, citric acid. , maleic acid or benzoic acid.

Preferred are compounds of the present invention of the general formula I in which:

R '' is a radical of the formula

or R ⁴ - O - CO -, wherein

R 5 and R ⁴ are the same or different and represent a heterocyclic radical of the formula

r 2 represents a hydrogen atom or a straight or branched acyl group of up to 6 carbon atoms, and salts thereof.

Particularly preferred are the compounds of the present invention of the general formula I in which

R 1 represents a radical of formula r ³ -o

or R ⁴ - O - CO wherein

R 5a are the same or different and represent a heterocyclic radical of the formula

or

R ² represents hydrogen or straight-chain or branched acyl having up to 5 carbon atoms, and salts thereof.

In addition, the present invention provides a process for the preparation of compounds of the formula (I), which comprises reacting compounds of the formulas (II) or (III).

with compounds of formula IV

R ¹ - OH (IV) wherein the R as defined above, if appropriate in the prior activation of the carboxylic acid, in inert solvents and in the presence of a base and / or of an auxiliary, and in a second step, if R ² does not represent hydrogen, is performed acylation in inert solvents, optionally in the presence of a base and / or adjuvant.

The process of the present invention can be illustrated by way of example by the following reaction scheme.

Suitable solvents for all process steps are customary inert solvents which do not change under the reaction conditions. These preferably include organic solvents such as ethers, for example diethyl ether, glycol monomethyl ether, glycol dimethyl ether, dioxane or tetrahydrofuran, hydrocarbons such as benzene, toluene, xylene, cyclohexane or petroleum fractions, halogenated hydrocarbons such as methylene chloride, chloroform or carbon tetrachloride dimethylsulfoxide, dimethylformamide, hexamethylphosphoric triamide, ethyl acetate, pyridine, triethylamine or picoline. It is also possible to use mixtures of the solvents mentioned. Particularly preferred is dichloromethane, dimethylformamide or tetrahydrofuran.

Suitable bases are the customary inorganic or organic bases, depending on the individual process steps. These preferably include alkali metal hydroxides such as sodium hydroxide or potassium hydroxide, alkali metal carbonates such as sodium carbonate or potassium carbonate, alkali metal alcoholates such as sodium or potassium ethanolate or sodium or potassium methoxide, organic amines such as is, for example, ethyldiisopropylamine, triethylamine, picoline, pyridine, dimethylaminopyridine or N-methylpiperidine, amides, such as sodium amide or lithium diisopropylamide, lithium-N-silylalkyl amides, such as lithium N- (bis) triphenylsilylamides, or lithium amides; for example n-butyllithium.

The bases are used in an amount of 1 mol to 10 mol, preferably 1 mol to 3 mol, based on 1 mol of the compound of the formula II or III.

Suitable adjuvants are preferably condensing agents, which may also be bases, especially when the carboxyl group is activated in the anhydride form. Preferably, conventional condensing agents such as carbodiimides are used, for example Ν, Ν'-diethylcarbodiimide, N, N'diisopropylcarbodiimide, N, N'-dicyclohexylcarbodiimide, N- (3-dimethylaminoisopropyl) -N'-ethylcarbodiimide hydrochloride, N- cyclohexyl-N '- (2-morpholinoethyl) -carbodiimide-meto-p-toluenesulfonate (CMCT or morpho CDI), carbonyl compounds such as carbonyldiimidazole, 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2 -oxazolium-3-sulfate or 2-tert-butyl-5-methyl-isoxazolium perchlorate, acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline and propanephosphonic anhydride, isobutyl chloroformate, benzotriazolyloxy-tri (dimethylamino) phosphonium hexafluorophosphate or 1-hydroxybenzotriazole or dimethylaminopyridine.

The reactions can be carried out both at normal pressure and at elevated or reduced pressure, for example in the range of 0.5 to 5 bar, but preferably at normal pressure.

The reactions are generally carried out at a temperature in the range of -20 to 40 ° C, preferably 0 ° C to room temperature.

The acylation is generally carried out in one of the abovementioned ethers or halogenated hydrocarbons, preferably tetrahydrofuran or methylene chloride, at a temperature ranging from -30 ° C to 50 ° C, preferably -10 ° C to room temperature.

Suitable bases and / or auxiliaries for the acylation are the abovementioned organic amines and pyridines. Triethylamine and dimethylaminopyridine are preferred.

The bases are used in an amount of 1 mol to 10 mol, preferably 1 mol to 3 mol, based on 1 mol of the corresponding alcohol.

The compounds of formula II are known.

The compounds of formula III are novel and can be prepared, for example, by the preparation of compounds of formula V

CO-NH

NH (V) in which

W is an amino protecting group, preferably a tert-butoxycarbonyl group, first converted by means of an epoxidation reaction, optionally using a base or phase transfer catalysis, to the compounds of formula VI

wherein W is as defined above and then reacted with 3-trifluoromethyl-2-azabicyclo [3.3.0] octane of formula VII

in a solvent and the protecting group is cleaved by conventional methods.

Suitable solvents are customary organic solvents which do not change under the reaction conditions. These include organic solvents such as alcohols such as methanol, ethyl or n-propyl alcohol, ethers such as diethyl ether, glycol monomethyl ether, glycol dimethyl ether, dioxane or tetrahydrofuran, hydrocarbons such as benzene, toluene, xylene, cyclohexane or petroleum fractions, halogenated hydrocarbons such as methylene chloride, dichloroethane (DCE), chloroform or carbon tetrachloride, and dimethylsulfoxide, dimethylformamide, hexamethylphosphoric triamide, ethyl acetate, pyridine, triethylamine or picoline. It is also possible to use mixtures of the solvents mentioned. Dichloromethane, dichloroethane, dimethylformamide and n-propyl alcohol are particularly preferred.

Suitable epoxidation reagents are those known from the literature, such as chloroperbenzoic acid, magnesium monoperoxyphthalate, dimethyloxirane, or methyl (trifluoromethyl) dioxirane. Preference is given to chloroperbenzoic acid and magnesium monoperoxyphthalate (see P. Brongham et al., Synthesis (1987), 1015, W. Adam et al., J. Org. Chem., 52, 2800 (1987) and R. Curci et al. , J. Org. Chem., 53, 3890 (1988)).

When the epoxidation is carried out by phase transfer catalysis, for example, organic ammonium chlorides or ammonium bromides such as benzyltriethylammonium chloride or benzyltriethylammonium bromide, methyltrioctylammonium chloride, tetrabutylammonium bromide, or tricumatrylmethylamide are used as adjuvants. Benzyltriethylammonium chloride and benzyltriethylammonium bromide are preferred.

The epoxidation is carried out at a temperature in the range of -10 ° C to 90 ° C, preferably 0 ° C to 60 ° C.

The reactions can be carried out at both normal pressure and i

increased or decreased, for example in the range 0.05 to 0.5 MPa. Preferably, it is operated at normal pressure.

The compounds of formula (IV) are known or can be prepared by conventional methods.

The compounds of formulas V and VI are for the most part known (EP 528 242).

The compounds of formula VII are novel and can be prepared, for example, by fluorination of 2-azabicyclo [3.3.0] octane-3-carboxylic acid in the corresponding configuration with HF or SF4.

Surprisingly, it has been found that the compounds of formula I have a markedly potent action against retroviruses. This is demonstrated by the HIV-specific protease-enzyme assay

The results of the examples below were determined using the HIV test system described by Hansen J., Billich S., Schulze T., Sukrow S. and Mollig K., (1988), EMBO Journal, 7, no. 6, p. The purified HIV proteases were incubated with a synthetic peptide that mimics the gag precursor protein cleavage site and represents the in vivo HIV protease cleavage site. The resulting synthetic peptide cleavage products are analyzed by RP-HPLC (reverse phase high performance liquid chromatography). The IC50 values reported refer to the concentration of the substance that is caused by 50% inhibition of protease activity under the test conditions.

HIV-1 enzyme assay

Table I

Example Inhibition of HIV-1 protease IC ₅₀ (mol / l) IC95 (mol / l) 1 3,0x10- ⁸ 3,8x10 ^-7 2 2,7x10-10 3.6 x ΙΟ ' ⁹ 3 9.5 x 10 ⁹ Not tested 4 2.1 x10- ⁹ 3,9x10- ⁹ 5 4.1 x ΙΟ ' ⁹ 4,7x10-8 6 2,5x10'8 5.0 x ΙΟ ' ⁸ 7 2,5x10- ⁸ Not tested 8 3.2 x ΙΟ ' ⁹ 4.1 x 10-8

In addition, the compounds of the present invention exhibit activity in lentivirus-infected cell cultures. This can be shown in the example of HIV-virus.

HIV-infection in cell culture

The HIV test is performed with slight modifications according to the method of Pauwels et al. (See Journal of Virological Methods 20, (1988), 309-321).

Normal human blood lymphocytes (PBL) are enriched in Ficol-Hypaque and stimulated in RPMI 1640 with 20% fetal calf serum with phytohemagglutinin (90 pg / ml) and interleukin-2 (40 U / ml). For infection with infectious HIV, PBLs are pelleted and the cell pellets are then suspended in 1 ml HIV-virus adsorption solution and incubated for 1 hour at 37 ° C.

The viral adsorption solution is centrifuged and the infected cell pellets are introduced into the growth medium to contain 1 x 10 6 cells per ml. The cells so infected are pipetted into 96-well microtiter plate dishes at 1 x 10 ⁴ cells / dish.

The first vertical row of the microtiter plate contains only growth medium and cells that were not infected but were treated as above (cell control). The second vertical row of the microtiter plate contains only HIV-infected cells (virus control) in the growth medium. The other dishes contain the compounds of the present invention in different concentrations, starting with the third vertical row of microtiter plate dishes, with the test substances diluted 2-fold times in two steps.

The test substances are incubated at 37 ° C until HIV syncytic formation (between days 3 and 6 post infection) occurs in the unprocessed viral control, which is then evaluated microscopically. In the untreated virus control, about 20 syncytes result under these test conditions, whereas the untreated cell control contains no syncytes.

IC 50 values were determined as the concentration of processed and infected cells at which 50% (about 10 syncytes) of virus-induced syncytes were suppressed by treatment with a compound of the present invention.

It has been found that the compounds of the present invention protect HIV-infected cells from virus-induced disruption.

PBL Cell Culture Assay

Table II

Example PBL IC50 (mol / l) IC95 (mol / l) 1 2,2x10-6 3,5x10-6 2 2,0x10-6 3,5x10-6 3 1.2 x 10-6 1,9x10-6 4 > 1,6x10-5 Not tested 5 1.4 x 10-5 2,2x10-5 6 2.8 x 10-6 1,3x10-5 7 6.2 x 10-θ Not tested 8 1,7x10'5 > 2 x 10-5

The compounds of the present invention are valuable active substances for the treatment and prophylaxis of diseases caused by retroviruses in human and veterinary medicine.

Indicative areas in human medicine include:

1. Treatment and prophylaxis of human retroviral infections.

2. Treatment or prophylaxis of diseases caused by HIV I (human immunodeficiency virus, first called HTLV III / LAV and HIV II (AIDS) and their associated stages such as ARC (AIDS related complex) and LAS (Lymphadenophathy - Syndrome), as well as this virus-induced immune deficiency and encephalopathy.

3. Treatment or prophylaxis of HTLV-I or HTLV-I I infection.

4. Treatment or prophylaxis of the AIDS carrier stage (AIDS carrier status).

Indications in veterinary medicine include, for example:

(a) Maedi-visna infection (in sheep and goats).

b) Progressive pneumoniavirus (PPV) infection (in sheep and goats).

(c) Infection with caprine arthritis encephalitis virus (in sheep and goats).

d) Virus infection (in sheep).

(e) Anemia virus infection (in horses).

f) Infections caused by feline leukemia virus.

g) Infections caused by feline immunodeficiency virus (FIV).

h) Infections caused by simian immunodeficiency virus (SIV).

Of the indication areas in human medicine, the areas listed in points 2, 3 and 4 are preferred.

The present invention also relates to pharmaceutical compositions which, in addition to non-toxic, inert, pharmaceutically acceptable carriers, contain one or more compounds of the formula I or consist of one or more active compounds of the formula I as well as a process for the preparation of such compositions.

The active compounds of the formula I should be present in the pharmaceutical compositions in a concentration of about 0.1 to 99.5% by weight, preferably about 0.5 to 95% by weight of the total mixture.

In addition to the compounds of formula (I), the above pharmaceutical compositions may also contain other pharmaceutical active ingredients.

The above pharmaceutical compositions are prepared in a conventional manner by known methods, for example by mixing the active ingredient (s) with the carrier or carriers.

In general, both in human and veterinary medicine, it has proven advantageous to administer the active compound or compounds in a total amount of about 0.5 to about 500 mg / kg, preferably 1 to 100 mg / kg of body weight per 24 hours to achieve the desired results. , optionally in the form of multiple individual doses. One unit dose contains the active compound (s) preferably in an amount of about 1 to 80 mg / kg, in particular 1 to 30 mg / kg body weight. Depending on the type and body weight of the object being treated, the type and severity of the disease, the type of preparation and the administration of the medicament, as well as the period or interval at which it is administered, it may be necessary to deviate from said doses.

The compounds of the present invention are enzyme inhibitors and as such can be used for all purposes for which enzyme inhibitors are useful. For example, use in affinity chromatography, use as an aid to elucidate enzyme structures and reaction mechanisms, and use as reagents for diagnostics may be mentioned.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 (2S, 3S) -3- (tetrahydrofuran-3-yl-oxycarbonyl-L-asparginyl) -amino-1 - [(S, S, S) 3-trifluoromethyl-2-azabicyclo [3.3.0] octane- 2-yl] -2-hydroxy-4-phenylbutane

0.1 g (0.22 mmol) of (2R, 3S) -3- (L-asparginyl) -amino-1 - [(S, S, S) -3-trifluoromethyl-2-azabicyclo [3.3.0] octane-2 -yl] -2-hydroxy-4-phenylbutane and 0.11 g (0.44 mmol) of tetrahydrofuran-3-yl-4-nitrophenyl carbonate (according to Daniel F. Veber et al., J. Org. Chem., Part 42, No. 20, 1977, pp. 3286-3288) was dissolved in 3 mL of tetrahydrofuran and stirred at room temperature overnight under argon. The reaction mixture was then evaporated. to dryness and separated by column chromatography (silica gel 60, dichloromethane / methanol = 10: 5, R f = 0.4).

Yield: 120 mg (96% of theory) of a colorless foam.

The compounds listed in Table 1 are prepared analogously to Example 1.

Table 1

NH ₂

Example r3 Rf / tt (° ^C ) yield

2 Γ ° Ί x Dichloromethane / MeOH = 100/5 0.4 (safe foam) 89.6 Sat ₂ Dichloromethane / MeOH 3 = 100/5 92.3 - x 0,2 (safe foam) with Dichloromethane / MeOH A 2 \ = 100/5 74.0 0.4 (safe foam)

Example 5 (2R, 3S) -3- (Tetrahydro-furan-3-yl-oxycarbonyl) -amino-1 - [(S, S, S) -3-trifluoromethyl-2-azabicyclo [3.3.0] octan-2-yl] - 2-hydroxy-4-phenylbutane

0.1 g (0.29 mmol) of (2R, 3S) -3-amino-1 - [(S, S, S) -3-trifluoromethyl-2-azabicyclo [3.3.0] octan-2-yl] - 2-hydroxy-4-phenylbutane and 0.11 g (0.44 mmol) of tetrahydrofuran-3-yl-4-nitrophenyl carbonate (according to Daniel F. Veber et al., J. Org. Chem., Part 42, No. 20) , 1977, pp. 3286-3288) was dissolved in 3 mL of tetrahydrofuran and stirred at room temperature overnight under argon. The reaction mixture was then evaporated to dryness and separated by column chromatography (silica gel 60, dichloromethane / ethyl acetate = 4: 1).

Rf = 0.5 (dichloromethane / ethyl acetate = 1: 1)

Yield: 50 mg (37.5% of theory) of a colorless foam.

Analogously to Example 5, the compounds listed in the following Table 2 were prepared.

Table 2

Example R3 RfA.t. (° C) yield (% of theory) Dichloromethane / ethyl 6 © acetate = 9/1 0.4 (non-crystalline) 125 ° C 75.0 so, Dichloromethane / ethyl 67.8 7 Q acetate = 4/1 0.5 (safe foam) SO Dichloromethane / ethyl 95.4 8 U acetate = 4/1 0,5 (bezf.sklo)

Example 9 (2S, 3S) -3- (tetrahydrofuran-3-yl-oxycarbonyl-L-asparaginyl) amino-1 - {(S, S, S) 3-trifluoromethyl-2-azabicyclo [3.3.0] octane-2 yl} -acetoxyfenylbután

mg (0.0876 mmoles) of the compound of Example 1, 25 mg (0.035 ml) of triethylamine and 13 mg (0.13 mmoles) of acetic anhydride are dissolved in 2 ml of dried tetrahydrofuran (THF), mixed with a small amount of dimethylaminopyridine (DMAP). and stirred overnight under argon.

The mixture is evaporated to dryness and stirred with 5 ml of water and 15 ml of ethyl acetate. The organic phase is separated, dried over sodium sulphate and separated by column chromatography (silica gel 60, dichloromethane / ethyl acetate = 4: 1) F _p = 191 ° C

Rf = 0.4 (dichloromethane / ethyl acetate = 4: 1) Yield: 35 mg of colorless crystals (65.2% of theory)

Example 10 (2S, 3S) -3- (Tetrahydrofuran- (3S) -3-yl-oxycarbonyl) -amino-1 - [(S, S, S) -3-trifluoromethyl-2-azabicyclo [3.3.0] octane-2 yl] -2-isobutyryloxy-4-phenylbutane

Analogously to Example 9, the title compound is obtained from 50 mg (0.11 mmol) of the compound of Example 6, 20 mg (0.027 mL, 0.2 mmol) of triethylamine and 15 mg (0.14 mmol) of anhydride. butyric acid with a small amount of dimethylaminopyridine. The product is obtained in the form of a colorless foam.

Rf = 0.6 (dichloromethane / ethyl acetate = 4: 1) Yield: 40 mg (69.3% of theory).

Claims (7)

Heterocyclic-substituted pseudopeptides of the general formula I in which R ¹ represents a radical of formula or R ⁴ -O-CO wherein R 5 and R ⁴ are the same or different and represent a heterocyclic radical of formula R 2 is a hydrogen atom or a straight or branched acyl group of up to 20 carbon atoms optionally substituted by a straight or branched alkoxycarbonyl group of up to 6 carbon atoms and salts thereof. Heterocyclically substituted pseudopeptides according to claim 1 of the general formula I, in which R ¹ represents a radical of formula r ³ - ° nh - Π T or R ⁴ -O-CO-, wherein r 3 and R ⁴ are the same or different and represent a heterocyclic radical of formula or R ² represents hydrogen or straight-chain or branched acyl having up to 6 carbon atoms, and salts thereof. The heterocyclic substituted pseudopeptides according to claim 1 of the general formula I, in which R 1 represents the remainder of the formula R ³ -C5 NH * \ CO-- Ύ T or R ⁴ - O - CO where R ³ and R ⁴ are the same or different and represent a heterocyclic radical of the formula R ² represents hydrogen or straight-chain or branched acyl having up to 5 carbon atoms, and salts thereof. 4. A compound according to claim 1, wherein: EMI112.0 R2 represents a hydrogen atom or a straight or branched acyl group of up to 4 carbon atoms. 5. A process for the preparation of heterocyclic substituted pseudopeptides of the formula I according to claim 1, characterized in that the compounds of the formulas II or III are reacted with the compounds of the formula IV. R1-OH (IV) in which R &apos; as previously defined, optionally with prior activation of the carboxylic acid, in inert solvents and in the presence of a base and / or adjuvant, and in a second step if it is not a hydrogen atom, acylation is performed in inert solvents, optionally in the presence of a base and / or adjuvant . A medicament comprising one or more compounds according to claims 1 to 4. Use of the compounds according to claims 1 to 4 for the manufacture of antiretroviral medicaments.