N1-[2,2-dimethyl-1s-(pyridin-2-ylcarbonyl)propyl]-n4- hydroc c-2r-isobutyl-3s-methoxycinnamic or its pharmaceutically acceptable salt, hydrate or mes

 

(57) Abstract:

The invention relates to new N1-[2,2-dimethyl-1S-(pyridin-2-ylcarbonyl)propyl] -N4-hydroxy-2R-isobutyl-3S-methoxybenzamido or its pharmaceutically acceptable salt, hydrate or solvate. These compounds possess biological activity and can be used in medicine for the treatment of rheumatoid arthritis, cancer, multiple sclerosis or psoriasis. table 2.

BACKGROUND OF THE INVENTION

In the international application WO 95/19956 (British Biotech Pharmaceuticals Ltd) is described and claimed a class of compounds which are inhibitors of the matrix metalloprotease (MMP) inhibitors and allocation factor that causes necrosis of tumor cells (TNFa) from cells. Specifically, the application discloses compounds of General formula (I)

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in which X denotes a group-CO2H or -- CONHOH;

R1denotes hydrogen; and (C1WITH6)alkyl, (C2-C6)alkenyl; phenyl; substituted phenyl;

phenyl(C1-C6)alkyl; a substituted phenyl(C1-C6)alkyl; heterocyclic residue; substituted heterocyclic residue; heterocycle(C1-C6)alkyl; substituted heterocycle (alkyl, phenyl, substituted phenyl, the rest of the heterocycle, (C1-C6)acyl, Venizelou or substituted Venizelou group and a is a (C1-C6)alkyl; amino; protected amino group; acylamino; HE; SH; and (C1-C6)alkoxy; a (C1-C6)alkylamino; di(C1-C6)alkylamino; and (C1-C6)alkylthio; aryl(C1-C6)alkyl; amino(C1- C6)alkyl; hydroxy(C1-C6)alkyl; mercapto(C1-C6)alkyl or carboxy(C1-C6)alkyl, where the amino-, hydroxy-, mercapto - or carboxyl group optionally is protected or the carboxyl group is in the form amide; lower carbamoylmethyl alkyl, mono(lower alkyl)carbarnoyl, di(lower alkyl)carbarnoyl, di(lower alkyl)amino or carboxy-lower-alkanolamine;

R2means (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)quinil, phenyl(C1-C6)alkyl, heteroaryl(C1-C6)alkyl, cycloalkyl(C1-C6)alkyl or cycloalkenyl(C1-C6)alkyl group, each of which optionally can have one or more substituents selected from (C1WITH6alkyl, -O(C1
R4denotes phenyl or 5 - or 6-membered heteroaryl cycle, where any nitrogen atom in the cycle can be oxidized to N-oxide, which if necessary can connect with the benzene ring or 5-, 6 - or 7-membered heterocyclic ring, where any of the loops can optionally be substituted with:

(a) one or more Deputy independently selected from groups: hydroxyl, halogen, -CN, -CO2H, -CO2(C1-C6)alkyl, -(C1-C6)alkyl-CO2(C1-C6)alkyl,

-CONH2, -N(C1-C6)alkyl, -SOP(C1-C6)alkyl)2, -CHO, -CH2OH, -(C1-C4)perfluoroalkyl, -O(C1-C6)alkyl, -SO(C1-C6)alkyl, -S(C1-C6)alkyl,

SO2(C1-C6)alkyl, -NO2, NH, -NH(C1-C6)alkyl, -N(C1-C6)alkyl)2-N(C1-C6)alkyl, or

(b) a group selected from (C1-C6)alkyl, (C2-C6)alkenylphenol, (C2-C6)alkenylphenol, (C3-C8)cycloalkyl, (C4-C6)cycloalkenyl, phenyl, benzyl, heteroaryl or heteroarylboronic, pridratsya, amino, carboxyl, (C1-C4)-perforaciones, (C1-C6)alkyl, -O(C1-C6)alkyl or S(C1-C6)alkyl group;

R5denotes hydrogen or (C1-C6)alkyl group,

or its salt, hydrate or MES.

THE INVENTION

N1-[2,2-Dimethyl-1S-pyridin-2-ylcarbonyl)propyl] -N4-hydroxy-2R-isobutyl-3S-methoxycinnamic is representative of the class described and claimed in General in the international application WO 95/19956, but neither he nor his property not specifically described.

In the international application WO 95/19956 argues that in the described class of compounds, aromatic or heteroaryl substituent R4causes in General unexpected and desired effect of increasing activity in relation to stromelysin compared with the known compounds is almost the same structure, but with other (usually metal) substituents R4, while maintaining activity against collagenase and gelatinase. As a representative of this class that is selected in this case, the compound N1-[2,2-dimethyl-1S-(pyridin-2-ylcarbonyl)propyl]- N4-hydroxy-2R-isobutyl-3S-methoxycinnamic oblad ecou pain (sometimes also called tendonitis) in the joints, for example, in the shoulder, some animals and people after high and/or prolonged doses. Although it is believed that this effect is practically reversible upon discontinuation, it is, however, undesirable. The mechanism of pain hitherto incomprehensible, and not seems possible to correlate the susceptibility of a compound to cause pain with the specific characteristics of the structure of the molecule or its profile inhibition of the enzyme. Accordingly, it is impossible to predict in advance whether any given inhibitor of MMP cause side effect and, in case of its occurrence, the severity of this effect, and it must be determined empirically.

Currently, it is found that the selected N1-[2,2-dimethyl-1S-(pyridin-2-ylcarbonyl)propyl] -N4-hydroxy-2R-isobutyl-3S-methoxycinnamic has a very low tendency to cause side effects muscle pain. In this respect it differs from the similar structure analogues, such as N1-[2,2-dimethyl-1S-(pyridin-3-ylcarbonyl)-propyl] -N4-hydroxy-2R-isobutyl-3S-hydroxysuccinimide are more likely to cause this side effect.

In the application WO 95/19956 also argues that the described class arylamine of MMP inhibitors in what avcoi WO 95/19956, are bioavailable to the extent feasible by oral administration, as evidenced, for example, the peak level of MMP-inhibitory activity, or level of activity over time (area under the curve"), attributed to drug drug blood of animals after oral administration. It was found that the compound selected in accordance with this invention, N1-[2,2-dimethyl-1S-(pyridin-2-ylcarbonyl)propyl] -N4-hydroxy-2R-isobutyl-3S-methoxycinnamic when a dose is bioavailable in the human body and in the body of other mammals.

This combination of oral availability and reduced propensity to cause tendonitis as a side effect assumes that the connection according to the invention should have a relatively wide range of application in therapy for the treatment of diseases requiring systemic injections of MMP inhibitor for moderate or long time. Therefore, the connection is indicated for the treatment of, for example, rheumatoid arthritis, malignancies, multiple sclerosis (PC), Guillain-Barre syndrome-Strole (GBS) and psoriasis.

DETAILED DESCRIPTION OF THE INVENTION

Therefore,denneysrestaurants.html the II

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and its pharmaceutically acceptable salt, hydrate and solvate.

In connection according to the invention has three asymmetric carbon atoms, the stereochemical configuration of which is shown in the title compound illustrated by formula (II). However, it should be understood that the invention includes a mixture of enantiomers of the compound (II), provided that the named enantiomer predominates. Preferably, in such an enantiomeric mixture of 90 wt.% or more was named enantiomer.

Salts of the compounds according to the invention include physiologically acceptable salts, resulting in accession acids, such as hydrochloride, hydrobromide, sulfate, methanesulfonate (mesilate), n-toluensulfonate (toilet), phosphate, acetate, citrate, succinate, lactate, tartrate, fumarate and maleate. Salts can be formed with bases, for example sodium, potassium, magnesium and calcium salts.

Selected compounds according to the invention can be obtained is illustrated in the example of this description way and you can enter in any way, consistent with its pharmacokinetic properties. Introduce oral compositions may be in the form of tablets, capsules, powders, granules, pellets, liquid, or Geleos the works, or suspension for parenteral administration. Tablets and capsules for oral administration may be in the form of a standard dose and can contain suitable excipients, such as binders, fillers, lubricant for tableting, splitting the substance or acceptable moisturizers. Tablets can be covered by membrane methods well known in normal pharmaceutical practice. Liquid preparations for oral administration can be, for example, in the form of a suspension in water or in oil, solutions, emulsions, syrups or elixirs, or may be in dry form and before use, mix ("recreated") with water or other suitable media. Such liquid preparations may contain appropriate additives, such as suspendresume agents; emulsifying agents; non-aqueous vehicles (which may include edible oils), preservatives, and if necessary, flavorings and dyes.

The active ingredients can also be administered parenterally in a sterile environment. Depending on the vehicle and concentration of the drug can suspendibility or dissolved in the carrier. Adjuvants such as local anesthetics local anesthetics, preservatives and superstory, can be dissolved in wear is edenia is determined by clinical trials in accordance with usual practice, subject to the approval of the relevant authorities. In General, currently believe that the connection should be introduced to the person orally at a dose of 5-100 mg two or three times a day.

The following example describes how to obtain the compounds according to the invention. Educt - 2R-(2,2-dimethyl-5-oxo[1,3]dioxolane-4S-yl)-4-methylpentanol acid and L-tert-leucine-N-(2-pyridyl)amide - get as described in international application WO 95/19961.

The example uses the following abbreviations:

DMF - N,N-dimethylformamide,

EDC hydrochloride, N-ethyl-N-(3 - dimethylaminopropyl)carbodiimide,

HOBt is 1-hydroxybenzotriazole,

NMM is N-methylmorpholine,

THF is tetrahydrofuran.

EXAMPLE

N1-[2,2-Dimethyl-1S-pyridin-2-ylcarbonyl)propyl]-N4-hydroxy-2R-isobutyl-3S-methoxycinnamic

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Stage A. Dimethyl ether 2S-hydroxy-3R-isobutylamino acid.

2R-(2,2-Dimethyl-5-oxo[1,3] dioxolane-4S-yl)-4-methylpentanol acid (75.0 g, 0.326 mmol) dissolved in methanol (500 ml) and cooled to 0oC and the resulting solution is saturated with gaseous hydrogen chloride. The reaction mixture is brought to room temperature and stirred over night. The solvent is removed under reduced pressure, the residue is dissolved in chloride meiland anhydrous magnesium sulfate, filtered and evaporated to dryness under reduced pressure to obtain the above compound as a yellow oil (53 g, 75%).

1H-NMR, (D13): 4.10 (1H, d, J=4.0 Hz), 3.60 (3H, s), 3.50 (3H, s), 3.18 (sh.C.), 2.78 (1H, m), 1.61-1.40 (2H, m), 1.28 (1H, m), and 0.76-0.73 (6N, m).

Stage C. Dimethyl ether 2R-isobutyl-3S-methoxybutanol acid.

Dimethyl ether 2S-hydroxy-3R-isobutylamino acid (23.9 g, 110 mmol) dissolved in DMF (200 ml) and add distilled modesty methyl (8.2 ml, 132 mmol), and then the oxide of silver (I) (27.95 g, 121 mmol). The reaction mass is stirred for 7 days at room temperature in the dark. The solvent is removed under reduced pressure and the residue purified by column chromatography (silica gel, methylene chloride as solvent) to give the above compound as a viscous yellow oil (19.16 g, 75%).

1H-NMR, (CDCl3): 3.83 (1H, d, J=7.5 Hz), 3.71 (3H, s), 3.62 (3H, s), 3.30 (3H, s), 2.85 (1H, m), 1.65-1.39 (2H, m), 1.10 (1H, m) and 0.83-0.81(6N, m).

Stage C. Deliciosa salt 2R-isobutyl-3S-methoxybutanol acid.

The lithium hydroxide (1.76 g, 42.0 mmol) are added to a solution of dimethyl ether 2R-isobutyl-3S-methoxybutanol acid (4.70 g, 20.0 mmol) in methanol (3 is storytell under reduced pressure to obtain the above-mentioned compounds in a solid yellow color (4.40 g, quantitative yield).

1H-NMR, (CD3D): 3.52 (1H, d, J=5.1 Hz), 3.27 (3H, s), 2.65 (1H, m), 1.56-1.53 (2H, m), 1.31 (1H, m) and 0.82-0.78 (6N, m).

Stage D. 4-Methyl ether 2R-isobutyl-3S-methoxybutanol acid.

Deliciious salt 2R-isobutyl-3S-methoxybutanol acid (25.14 g, 116 mmol) dissolved in dry THF (150 ml) and cool the solution to 0oC. Add triperoxonane anhydride (30 ml) and the mixture was stirred at 0oWith an additional 4 hours. The solvent is removed under reduced pressure, the residue is dissolved in anhydrous methanol (200 ml) at 0oAnd the solution is stirred over night at room temperature. The solvent is removed under reduced pressure to obtain the above compound as a yellow oil (54.3 g, including 2 equivalent lithium salt triperoxonane acid), which is used in stage E without further purification.

1H-NMR, (CD3OD): 7.71 (1H, d, J=7.5 Hz), 3.65 (3H, s), 3.24 (3H, s), 2.72 (1H, m), 1.56-1.42 (2H, m), 1.06 (1H, m), and 0.81-0.79 (6N, m).

Stage I.e. Methyl ether 3R-[2,2-dimethyl-1S-(pyridylcarbonyl)propellerblades]-2S-methoxy-5-methylhexanoic acid.

The product from stage D (25.06 g, the equivalent of 53.7 mmol 4-methyl ester 2R-isobutyl-3S-methoxybutanol cisal). The mixture is stirred and after 2 hours, brought to room temperature. The solution of active ester thus obtained, cooled to 0oTo add L-tert-leucine-N-(2-pyridyl)amide (11.11 g, 53.7 mmol) and the mixture is stirred over night at room temperature. The solvent is removed under reduced pressure, the residue is dissolved in ethyl acetate. Wash solution consistently 1M sodium carbonate solution and brine, dried over anhydrous magnesium sulfate, filtered and evaporated to dryness. The residue is purified by column chromatography (silica gel, 0 to 5% methanol in methylene chloride) to give the above compound as a white solid (13.41 g, 61%).

1H-NMR, (CDCl3): 9.61 (1H, s), 8.24 (1H, d, J=8.4 Hz), 7.74 (1H, m), 7.07 (1H, m), 6.97 (1H, d, J=8.9 Hz), 4.64 (1H, d, J=8.9 Hz), 4.01 (1H, d, J=7.6 Hz), 3.76 (3H, s), 3.41 (3H, s), 2.75 (1H, m), 1.79 (1H, m), 1.51 (1H, m), 1.11(1H, m), 1.02 (S, s), 0.84 (3H, d, J=6.3 Hz) and 0.82 (3H, d, J=6.3 Hz).

Stage F. Lithium salt 3R-[2,2-dimethyl-1S-(pyridin-2-ylcarbonyl)propellerblades]-2S-methoxy-5-methylhexanoic acid.

Methyl ether 3R-[2,2-dimethyl-1S-(pyridylcarbonyl)propellerblades]-2S-methoxy-5-methylhexanoic acid (13.4 g, 32.9 mmol) dissolved in THF (265 ml) and water (65 ml) and add monohydrate guide the tion under reduced pressure and receives a yellow oil, which is dried via azeotropic distillation with toluene. Product (13.4 g, containing residual solvent) immediately used without further purification.

1H-NMR, (CD3OD, mixture of diastereomers 3.5:1): 8.31 (1H, m), 7.99 (1H, d, J=8.3 Hz), 7.66 (1H, m), 7.00 (1H, m), 4.49 (0.23 H, C; minor diastereoisomer), 4.37 (0.77 H, C; major diastereoisomer), 3.68 (0.23 H, d, J=6.7 Hz; minor diastereoisomer), 3.52 (0.77 H, d, J=6.7 Hz; major diastereoisomer), 3.21 (0.69 H, C; minor diastereoisomer), 3.20 (2.31 H, C; major diastereoisomer), 2.64 (1H, m), 1.53 (2H, sh.C.), 1.18 (1H, m), 1.01 (S, s), 0.85 (3H, d, J= 6.4 Hz) and 0.81 (3H, d, J=6.3 Hz).

Stage G. N4-Benzyloxy-N1-[2,2-dimethyl-1S-pyridin-2-ylcarbonyl]propyl-2R-isobutyl-3S-methoxycinnamic.

The product of stage (F (13.4 g, 33 mmol) dissolved in dry DMF (250 ml), placed in an argon atmosphere and cooled to -10oWith stirring. Add dropwise ethylchloride (3.47 g, 36 mmol) and then NMM (1.8 ml, 16.5 mmol). The mixture is stirred for 30 minutes and then added dropwise O-benzylhydroxylamine (6 g, 49 mmol) in DMF (10 ml). The reaction mixture is brought to room temperature and stirred over night. The solvent is removed under reduced pressure and the residue is distributed between ethyl acetate and brine. The organic layer is Lesh-chromatography (silica gel, 5% methanol in methylene chloride). The fractions containing the desired product are collected and evaporated. The product is saturated with diethyl ether to remove weakly colored impurities. Output: 9.44 g (58%, mixture of diastereomers >10:1).

1H-NMR, (D13the main diastereoisomer): 10.26 (1H, s), 9.93 (1H, s), 8.32 (1H, m), 8.23 (1H, d, J=8.2 Hz), 7.63 (1H, m), 7.25 (5H, m), 7.12 (1H, d, J=9.2 Hz), 7.02 (1H, m), 4.94 (1H, d, J=10.8 Hz), 4.76 (2H, d, J=3.8 Hz), 3.88 (1H, d, J=5.5 Hz), 3.32 (3H, s), 2.91 (1H, m), 1.72 (1H, m), 1.55 (1H, m), 1.35 (1H, m), 1.02 (S, s), 0.89 (3H, d, J=6.5 Hz) and 0.85 (3H, d, J=6.5 Hz).

Stage N. N1-[2,2-Dimethyl-1S-(pyridin-2-ylcarbonyl)propyl]-N4hydroxy-2R-isobutyl-3S-methoxycinnamic.

N4-Benzyloxy-N1-[2,2-dimethyl-1S-(pyridin-2-ylcarbonyl)propyl]-2R-isobutyl-3S-methoxycinnamic dissolved in a mixture of methanol (75 ml) and ethanol (75 ml) and placed in an atmosphere of argon. Add 10% palladium on coal and passed through a solution of hydrogen for 2 hours, after which TLC analysis shows that all of the original product reacted. The system is rinsed with argon and the catalyst removed by filtration. The solvent is removed under reduced pressure to obtain the above compound as a white solid (8.8 g, quantitative yield; mixture of diastereomers 12:1). Hz), 7.81 (1H, d, J=8.8 Hz), 7.60 (1H, m), 6.93 (1H, m), 4.53 (0.12 H, d, J=9.4 Hz), 4.43 (0.88 H, d, J=8.8 Hz), 3.74 (0.12 H, d, J= 10.0 Hz), 3.32 (0.88 H, d, J=9.8 Hz), 2.98 (0.3 H, s), 2.96 (2.64 H, s), 2.78 (1H, m), 1.23 (2H, m), 0.84 (10H, m), 0.65 (3H, d, J=6.4 Hz) and 0.56 (3H, d, J= 6.3 Hz). 13C-NMR, (CD3OD): 172.6, 170.0, 166.0, 151.5, 147.9, 136.0, 119.5, 113.6, 61.2, 60.6, 56.7, 46.2, 37.0, 34.0, 26.5, 25.2, 23.7 and 21.7. IR, nmax(KBr): 3255, 2957, 1700, 1645, 1524, 1467, 1435, 1370, 1301, 1213 and 1152 cm-1.

Found, %: C At 58.40; N, 7.92; N 13,61. WITH20H32N4O50.2 H2O. Found,%: C 58.29; N, 7.92; N, 13.60.

BIOLOGICAL EXAMPLE AND

The effectiveness of the compounds according to the invention as an inhibitor of interstitial collagenase determined by the method of Cawston and Barrett (Anal. Biochem. , 99, 340-345, 1979), whereby a 1 mm solution of the test compound or its breeding thermostatic at 37oC for 16 hours with collagen and collagenase (buferizovannyiy 25 mm Hepes, pH 7.5, containing 5 mm CaCL2, 0.05% Brij 35 and 0.02% NN3). Collagen is an acetylated14With collagen prepared by the method of Cawston and Murphy (Methods in Enzvmology, 80, 711, 1981), entered into this description by reference. The samples are centrifuged for sedimentation of non-hydrolyzed collagen and selected aliquot of the supernatant liquid to determine the number scintil the AI 1 mm of the test compound or its cultivation compared with the activity of the control (sample), without inhibitor, and the results presented below are given as the concentration of inhibitor which causes 50% inhibition of collagenase activity (IC50).

The effectiveness of the compounds according to the invention as an inhibitor stromelysin-1 determined by the method of Cawston et al. (Biochem. J., 195, 159-165, 1981), whereby a 1 mm solution of the test compound or its breeding thermostatic at 37oC for 16 hours with stromelysin and14With-acetylated casein (buferizovannyiy 25 mm Hepes, pH 7.5, containing 5 mm CAC2, 0.05% Brij 35 and 0.02% NaN3). Casein is an acetylated14C-casein, prepared by the method of Cawston et al (ibid). Activity stromelysin in the presence of 1 mm of the test compound or its cultivation is compared to activity in a control sample containing no inhibitor, and the results presented below, expressed as the concentration of inhibitor which causes 50% inhibition activity stromelysin (IC50).

The effectiveness of the compounds according to the invention as an inhibitor of 72 kDa-gelatinase determined by the method based on the method Sllers et al., Biochem. J. , 171, 493-496 (1979). kDa-gelatinase obtained from cells RPMI-7951, purified using chromatography on g thermostatic with 50 μg labeled [14S]-gelatin in the appropriate buffer for 16 hours at 37oC. At the end of the incubation, add 50 µg of bovine serum albumin with trichloroacetic acid (final concentration 16%) to terminate the reaction and precipitation of undigested substrate. The reaction tube was placed in ice for 15 minutes before centrifuged for 15 minutes at 10 000 rpm for sedimentation deposited substrate. Take an aliquot part 200 μl of the supernatant of the reaction liquid and determine radioactively using a scintillation counter for liquids. The effect of the inhibitor is determined by the curve of the effect of dose. IC50(concentration of inhibitor required for 50% reduction of enzyme activity) is determined by drawing a curve through the data, and calculating the concentration of inhibitor required to achieve 50% inhibition of the enzyme. For each definition IC50determine the effect of the inhibitor on the activity of gelatinase at about 8 concentrations of inhibitor. Inhibitors dissolved and diluted (diluted) in DMSO.

The results of the above tests, the compounds according to the invention are the following:

Enzyme - IC50(nm)

Collagenase - 10

kDa-gelatinase in the blood of laboratory animals in time after administration of the test compounds. The compound is injected through the probe group of 6 Wistar male rats (weight 300 g). Blood samples are taken by venipuncture from the tail vein after 0.5, 1.0, 2.0, 6.0 and 24 hours after injection. 0.4 ml of blood is placed in a test tube 4.5 ml, containing 0.1 ml of acid citrate dextrose (ACD) as an anticoagulant. For extraction add 3 ml of methanol and precipitated blood granulated by centrifugation (30 min, 3000 rpm). Selected aliquot part 2 ml of supernatant and concentrated by lyophilization. The extract is again dissolved in 200 μl DMSO and take an aliquot of 10 ál for analysis of the activity of inhibiting collagenase. The inhibitory activity of the extracts determined using the method of analysis of collagenase, as described above, in the Biological Sample and the concentration of inhibitor (i.e., drug plus any active metabolites) produced by comparison with a standard curve. The results are expressed as maximum concentrations in ng/ml x hours in 0.5-24 hours and AUC among the IC50x watch. The results are shown in table.1.

The connection according to the invention also have, introducing his oral igranka and people-volunteers, while there is a high degree of inhibitory activity in the blood of subjects on the public tumors tested on two species of experimental animals, it found its activity.

Model: melanoma in mice B16

In this case, the test mice C57/BL6J injected subcutaneously with 105cells of mouse melanoma B16. The tumor grows in a period of 18 days and the tumor weight is calculated using a micrometer (Tangenziale) according to the following formula: weight (mg) length (mm) x width (mm) 4. A group of mice (n=19) the connection gets entered using osmotic mini-pump implanted subcutaneously on the side opposite to that where the B16-tumor. The pump is implanted the day before injection (seeding) of tumor cells, and the dose of injected compound is 360 mg/day. Control group (n=17) receives the appropriate amount of media using the same implanted pump.

The average weight of the control tumors in 18 days is 114597 mg. Weight of the tumor in mice treated with the compound according to the invention is 77450 mg Is the weight loss (32% in the case of the test compound) is significant (p<0.005). The evaluation of the samples at the end of the experiment shows that the plasma is 26.83.2 ng/ml in the case of the test compounds.

BIOLOGICAL EXAMPLE D

The connection according to the invention have at its disposition to cause ViDi is"tendonitis-effect") is compared with that of similar structure isomer, namely, the N1-[2,2-dimethyl-1S-pyridin-3-ylcarbonyl)propyl]N4hydroxy-2R-isobutyl-3S-hydroxysuccinimide (Connection B).

Take male Lewis rats. In each experiment, 30 rats are weighed and randomly distributed into groups, regardless of body weight, n=6/group. In each experiment, one group give the appropriate media for comparison with each group receiving the drug.

Connection And take in the views of nelfinavir (salt methanesulfonic acid), whereas Compound B is in the form of free base. In the case of Compound And @ 15 mg/ml of media is a: 50% DMSO, 37% 0.1 M methansulfonate and 13% sterilized water; and @ 30 mg/ml carrier: 50% DMSO, 37% of 0.2 M methansulfonate and 13% of sterilized water. To connect B @ 15 mg/ml of media is a 50% DMSO/water.

Alzet (trade mark) osmotic Minnesota (2ML2; 14 days, 5 μl/hour from Akza Corp., Palo Alto CA 94303) is weighed empty and full, to ensure correct fill volume. Before implantation filled pumps placed in a thermostat at 37oC. All rats anaesthetize with halothane gas. After anesthesia the scruff of the neck vypivaut and disinfected. On podgotovlen is Jay doing the subcutaneous pocket. Minnesota placed so that the outlet of the pump was drawn out from the wound. The incision is stitched up again and disinfect the area around the wound. Immediately after surgery, all rats give anesthetic (Temgesic-trade mark-Reckitt and Colman), 0.1 mg/kg subcutaneously in the flank. After the end of the anesthetic, the animals return to the cell with dry litter, to ensure that the wound remains clean to heal. The next day after surgery, all rats are placed back on the standard litter and placed in groups of 3 so that you can more clearly be observed for signs of tendinitis.

After implantation of mini-pumps rats are weighed and control the possible beginning of tendinitis. The beginning and the severity of the tendonitis is measured using observation-based estimates (see below). Rats observed daily for 16 days. Averages >2 are considered to be significant. Applicable grading system is as follows:

The prone position

Normal - 0

Based on one paw - 1

Not resting on legs 2

By stimulating the movement of animals

Move normal - 0

Reluctant to move - 1

Move with moderate reluctance - 2

Very reluctant to move - 3 is about being both hind legs - 2

The results show that rats receiving a dose of a Compound A (15 mg/ml and 30 mg/ml) and only the media do not show visible signs of tendinitis, whereas rats receiving a dose of a Compound B, show noticeable signs, starting from the 8th day onwards (averages >2 on 8th day increase to 6 to 15 and 16 days).

It was confirmed that the rats receiving the compounds a and B in the above tests, are exposed to compounds of the mini-pumps on the plasma. Take blood samples at 3 and 10 days after implantation of mini-pumps weak after anesthesia, rats were Goltana from the tail vein. Blood samples (0.5 ml) was placed in tubes containing 3.0 ml of methanol for the extraction of free and bound compounds. Blood concentration of Compound a and Compound B is determined by the method of fluorometry using coumarin-peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2(Mca=(7-methoxycoumarin-4-yl)acetyl, Dpa= N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropan) (see Knight et al., FEBS Lett., 1992, 296, 263-266). On 16 or 17 days after implantation of the experiment, rats finally euthanized and take blood samples (0.5 ml) heart puccia and determine the concentration of Compound a and Compound B.

BIOLOGICAL EXAMPLE D

the th neuritis (EAP, EAN) is mediated T-cell autoimmune disorder of the peripheral nervous system. In experimental animals manifested many of the pathological features of GBS (GBS) with symptoms of ataxia, weakness and paralysis. EAN can cause the animals, introducing them an injection of myelin of peripheral nerves or protein components of myelin, such as protein 0, Freund. EAN-lesions occur in the spinal roots and peripheral nerves and are characterized by perivascular infiltration of mononuclear cells, demyelination of axons and impaired nerve conduction. In the pathology of GBS and EAN TNFa is involved; the levels of TNFain the blood of patients with GBS rise and antibodies to TNFareduce the severity of the disease when EAN (Hartung HP. Annals of Neurology, 1993, 33, 563-567).

The connection according to the invention have on rats with EAN, symptoms caused by injection of myelin of peripheral nerves in Freund. The connection is entered using surgically implanted mini-pumps with a concentration of 15 or 30 mg/ml at 5 μl/hour (see Biological example D), the delivery of respectively 7 and 14 mg/kg / day. therapy connection during the entire experiment, from 1 to 15 days, significantly reduces the occurrence is the development of symptoms in the case of EAN-model with therapeutic introduction of from 8 to 15 days at a concentration of 15 or 30 mg/ml at a rate of 10 μl/hour and the delivery of 14 and 28 mg/kg / day, respectively.

BIOLOGICAL EXAMPLE E

The active compounds according to the invention in the case of experimental models of PC (MS).

Method.

Model PC with delayed-type hypersensitivity described Matyszak and Perry (M. K. Matyszak and Perry, V. H., 1995. Demyelination in the central nervous system following a delayed type hypersensitivity response to bacillus Calmette-Guerin. Neuroscience 64, 967-977). Anaesthetize male rats Lewis, introducing the injection of 1 µl of physiological solution with fosfatnym buffer containing 105killed thermally bacilli Calmette Guerin (BCG) in the striatum of the left hemisphere. BCG is administered stereotaxically with a syringe 27G 10 µl. The coordinates for injection: bregma +1.2 mm, lateral (side)+3.0 mm and a depth of 4.5 mm After 4 weeks injected intradermally (intradermal) in the rear paw 200 μl of a solution containing 107 heat-killed BCG organisms in complete Freund's adjuvant. Even after 15 days the rats injected injection 2750 Units. horseradish peroxidase type II (HRP). After 30 minutes, the rats more deeply anaesthetize with pentobarbital sodium and implement transcardial perfusion with 100 ml 0.9% (W/V) NaCI containing 5000 Units. /l heparin, and then 200 ml of paraformaldehyde-lysine-controllable periodic destruction latch (PLP) containing 2% paraformaldehyde and 0.05% glutaraldehyde. The brain in the night at 4oWith before to be placed in Tissue-Tek O. S. T. (miles inc, Elkhart, USA) and freeze in liquid nitrogen. For HRP-localization method Hanker-Yates (Perry V. H. et al. , 1992) make moving coronal slices with a thickness of 50 μm. This procedure causes the delayed-type hypersensitivity in place stereotactic injection of BCG, characterized by local disturbance of the blood-brain barrier, as indicated by staining due to extravascular presence of HRP; infiltration of lymphocytes, as indicated by staining with antibodies OH-22 specific for high molecular weight forms total leukocyte antigens, activation of leukocytes, as indicated by staining with antibodies OH-6, specific for MHC antigens and the breakdown of myelin, as indicated by staining with antibodies to myelin basic protein. All these features are signs of active pathological changes or plaques observed in the Central nervous system of patients with PC (MS). The number of cells is determined by counting the number of OH-22-positive cells at the site where the most intense staining. The number of cells per unit area in the field of view recorded and expressed as cells/mm2. The field of expression of MHC class II leakage through hematoencephalic and output in mm2.

The action of the compounds according to the invention is evaluated, giving his rat oral 30 mg/kg twice daily, starting 5 days after the second injection of BCG and continuing to provide up to 15 days. The connection according to the invention give in physiological solution with fosfatnym buffer as the media containing 0.01% Tween 80. The control animals given only the media.

The area of leakage through the blood-brain barrier, the expression of MHC class II, demyelination and the number of T cells in animals receiving the compound according to the invention, compared with the control, the receiving medium by applying the criterion of T student.

Results. DTH response (delayed hypersensitivity) in animals treated with the carrier, characterized by the destruction of the blood-brain barrier, infiltration of lymphocytes, the expression of MHC class II and demyelination. In animals treated with the compound according to the invention, there is a noticeable decline in the area of leakage through the blood-brain barrier (p<0.05) and the rate of infiltration of T cells (p<0.0001). The decrease in demyelination and expression of MHC class II was observed, but did not reach statistically significant values.

The action of the compounds according to the invention on DTH-model PC, see table.2.amide of the formula (II)

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or its pharmaceutically acceptable salt, hydrate or MES.

 

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