Alpha-amino-1-(phosphonomethyl)-1h-benzimidazole-2-propanoic acid and its preparation

 

(57) Abstract:

Usage: in medicine as a competing NM YES - antagonists. The inventive product-amino 1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid f-ly I, where R1and R2is hydrogen, lower alkyl or halogen, or a pharmacologically acceptable salt. Reagent I. the joint f-crystals 2, where R3- R5- protecting group. Reaction conditions: perform unprotect connection f-crystals 2 using acidic or alkaline hydrolysis or hydrogenolysis.

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connection 1 connection 2 tabl.

-glutamate and a-aspartate, endogenous acidic amino acids, were clearly established as the main excitatory neurotransmitters. The effect of these excitatory amino acids is passed through several different subtypes of receptors, the most studied of which is the receptor N-methyl-D-aspartate (NMDA). Excessive activation of the complex of NMDA receptors may lead to neutron overstimulation with pathological consequences. Experimental studies show that prolonged agonist induced conductivity blocked the NMDA ion channel dopuske kletochnogo calcium are the most important and adverse role in excitotoxic neural violation, neurodegeneration and delayed neuronal death.

Excitatory amino acids are formed when neuropathology traumatic, endogenous genetic and environmental origin. Violation of the brain associated with hypoxia, hypoglycemia, traumatic disorders, stroke, epilepsy, specific metabolic disorders and certain chronic neurodegenerative diseases, largely due excitotoxicity mechanisms.

Some of these studies showed that blockade of the NMDA receptor - subclass significantly reduces neural violations and losses that occur in animal models simulating a variety neuropathologically situation. These observations clearly indicate that the NMDA antagonists provide effective neuroprotection in several clinical cases. Thus, agents antagonizers excitotoxic effects transmitted by the NMDA receptor are effective in the treatment of ischemic diseases, shock, disorders of the brain or spinal cord, and, in General, patients with increasing concentrations of excitatory transmitters. Specific p the unit of Huntington, and other dominant or recessive spinal and cerebellar degenerations, when the NMDA-antagonists prevent or slow the progression of the disease.

Compounds of the present invention are competing the NMDA - antagonists, which have the following formula:

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in which

R is hydrogen, lower alkyl, benzyl or pivaloyloxymethyl,

R1and R2are, independently of each other, hydrogen, lower alkyl, lower alkoxy, trifluoromethyl, triptoreline, methanesulfonamido, acetylamino, or halo, or taken together R1and R2are methylenedioxy group; and acceptable from a pharmacological point of view of salt.

The term "lower alkyl" and "lower alkoxy" refers to the constituents containing from 1 to 4 carbon atoms in the carbon chain. The term "halo" refers to fluorine, chlorine, bromine and iodine.

Compounds of the present invention exhibit chirality, and thus, the connection of the present invention include not only the racemic mixture, but also separate the enantiomers. Enantiomers are built in accordance with the R/S system, using a rule sequence.

Join this izobrajenii, protected prior connection benzimidazolyl-D-alanine interacts with a complex ester alkylphosphonate with subsequent removal of the protection, in order to obtain the target final products:

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where in the above sequence R3and R4are known for protecting amino groups such as lower alkyl and benzyl for R3and tration.-butyloxycarbonyl or benzyloxycarbonyl for R4. Protecting groups alkylphosphines acid R5this is lower alkyl, benzyl or 4-nitrobenzyl.

Substituted by a group X reagent, alkyl alkylphosphonates acid, can be halo, methylsulphonyl, tamilselvan, or triptorelin where triptorelin is especially preferred. Protected prior connection benzimidazolidinone can be obtained in optically pure form by using enantioselective synthesis of R or S derived N-protected complex ester of aspartic acid using the procedure of Nestor and others J. Med. Chem. so 270 320 S. (1984). Removing protection from alkylphosphocholines intermediate compounds can be carried out using acidic or alkaline hydrolysis, hydrogenolysis, and/or processing trimethyl is connected, when it is necessary or appropriate. When R3is benzyl, R4is benzyloxycarbonyl, and R5is a 4-nitrobenzyl, you can make one General gidroenergetichesky deprotection.

In alternative sequences ortho-phenylenediamine can interact with the reagent complex air alkylphosphonate to obtain the intermediate compound diamino, which then interacts in accordance with the procedure Nestor, etc. see above, to obtain the protected intermediate compound which is then subjected to deprotection as described above:

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In those cases, when the structure of substitution for R1and R2on the benzimidazole ring are asymmetric, in the alkylation by means of ester alkylphosphonate in the above reaction sequence would diastereometric a mixture of products, therefore, should be required fractional crystallization or chromatography to separate the products. This can be avoided by using appropriate starting materials, which give the target preceding materials, suitably protected ortho-nitroaniline or otvetstvenno, in order to obtain an intermediate compound, which then reacts in accordance with the procedure Nestor and others further protects shoot and get the target final products:

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Starting materials in the above sequence is either manufactured by the industry, or can be obtained by means known in this technical field techniques and procedures.

Compounds that are the subject of the present invention can form pharmacologically acceptable salts of the acceptable from a pharmacological point of view of organic and inorganic acids such as hydrochloric, Hydrobromic, (mono) sulfo-, sulfuric, phosphoric, nitric, maleic, fumaric, benzoic, ascorbic, Mamonova, amber, methane (mono) sulfo-, ukusna, propionic, tartaric, citric, benzoic, lactic, malic, almond, cinnamic, palmitic, taconova and benzene (mono) acid. Compounds of the present invention such as phosphonocarboxylate acid, ways to form carboxylates of alkali and alkaline-earth metals, and carboxylates acceptable from a pharmacological point of view of the cations derived from ammonia or alkaline amine. Palimony, mono-, di - and triethylammonium, mono-, di and Tripropylamine (ISO and normal), ethyldimethylammonium, benzyldimethylammonium, cyclohexylammonium, benzylammonium, dibenzylammonium, piperidine, morpholine, pyrrolidine, piperazine, 1 methylpiperidine, 4-ethylmorpholine, 1 isopropylpyrimidine, 10,4-dimethylpiperazine, 1-n-butyl of piperidine, 2-methylpiperidine, 1-ethyl-2-methylpiperidine, mono-, di and triethanolamine, the ethyl diethanolamine, n-butylanthraquinone, Tris(oxymethyl)methylammonium, phenyleneethynylene etc.

Connection, which is the subject of the present invention are competing the NMDA-antagonists used in the treatment of convulsions, cerebral ischemia, stroke, lesions of the brain or spinal cord disorders of the CNS (Central nervous system), transl.) such as senile dementia, a disease of Alzheimer, Huntington's chorea and other dominant or recessive spinal and cerebellar degenerations. The above compounds may be particularly effective as a pre-anesthetic and neuroprotective agents during surgical interventions with high risk such as brain surgery and surgery of the spinal cord, or in resultvalue cessation of blood current to the brain. Additional advantages consist in the use of the compounds of the present invention as a pre-anesthetic agents, since they have a weak trankvilizatoram (sedative properties, property, short term memory loss) short-term amnesia) and the ability to potentiate the effect of anesthetic agents so that the latter can be used in a lower dose.

Therefore, in addition to the new compounds is proposed a method of preventing disorders caused by overstimulation excitatory amino acid receptors in the brain and spinal cord, which contains the application to a mammal suffering from such disease, the NMDA - antagonist of the formula given above.

As such, the compounds of the present invention can be applied pure or with a pharmaceutical carrier, and therefore they can be made in the form somaticheskih doses such as tablets, capsules, etc. of the Proposed connection can be used by combining them with well known carriers such as magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragakant, methylcellulose, sodium to the thinner, flavouring agents, solubilization, lubricating agents, suspendresume agents, binding materials, agents, loosening tablets, etc., These compounds may also be injected parenterally, in this case they are used in the form of a sterile solution containing other dissolved substances, for example, enough saline or glucose to make the solution isotone.

Dosage will vary depending on the specific composition, route of administration, severity of symptoms and the patient's condition to be treated. The treatment in the General case, start with small dosages less than the optimum dose of the compound. Further, to increase the dosage up until when the circumstances will not achieve an optimal effect. In General, the compounds of the present invention in the most preferred embodiment is used in a concentration that will generally provide effective results without any harmful or adverse side effects, and they can be applied either as a single dose, or, if necessary, this dose can be divided into several podos, which are used at certain points of time in techinicality, can be confirmed using standard pharmacological procedures, which Illuminous their in vitro inhibition (3H)SSR-binding in the brain tissue of the rat and their in vitro antagonism of convulsions in mice induced by NMDA.

The examples below show the receipt and pharmacological testing of compounds that are the subject of the present invention.

Examples

The enantiomeric purity of the examples of the present invention was determined using a modification of the procedure proposed Tapuh, Y. Miler, N. Karger, B. Journal of Chromatography, 1981, T. 205, page 325 337.

Getting [bis-(4-nitrobenzyloxy)phosphinyl] methyl ether complex triptorelin(mono)sulfonic acids

A solution of di-4-nitrobenzyl oxymetholone (3,82 g, 10.0 mmol) [Hoffmonn, M. Synthesis, 1988 62] and pyridine (0.87 g, 11.0 mmol) in dichloromethane (50 ml) was treated at -10 ° -20oC anhydride triptorelin(mono)sulfonic acids (3.1 g, 11.0 mmol) and stirred at -10oC for 1 hour. The solution was washed with cold 1N HCl solution (2 x 50 ml), cold water (3 x 50 ml) and dried over magnesium sulfate. The solution was filtered, the solvent evaporated and the remaining oil was utverjdali when defending. Output 4,18 g (80%). The material was pure enough, h is adopted on a dry column on silica gel Grade II-III with ethyl acetate as eluent. The product fractions evaporated and the residue was led from dichloromethane/hexane and dried: melting point 73 and 75oC1H-NMR (400 MHz, CDCl3): 8,2 (doublet, 4H), 7.5 (a doublet, 4H), and 5.2 (multiplet, 4H), 4,8 (doublet, 2H); MS (+ FAB) 515 (M + H)+.

Elemental analysis for C16H14N2O10PSF3.

Calculated: C 37,36, H 2,74 N Of 5.45.

Found: C 37,27, H 3,03, N 5.52.

P R I m e R 1.

R-a-amino-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

Benzyl N-(benzyloxycarbonyl)-3-(2-benzimidazolyl)-D-alaninate (30 g, of 0.07 mol) (obtained in accordance with the procedure Nestor and others J. Med, Chem. 1984, 27 so, S. 320), (diethoxyphosphoryl)methyl ester triptorelin(mono)sulfonic acids (23,04 g, 0,076 moles) and powdered anhydrous potassium carbonate (37 g, 0,268 moles) was stirred in acetonitrile (500 ml) at room temperature for 20 hours the Mixture was filtered, the filtrate evaporated, and the residue was dissolved in dichloromethane. Dichloromethane was washed with water (500 ml), 5% NHCO3(2 x 300 ml), water (300 ml) and dried over magnesium sulfate. The solution was filtered and the solvent evaporated to a resin. The output 43, the Resin was dissolved in dichloromethane (300 ml) and subjected to purification by dry column with silicagel zitat 4:1) 0,17] was collected and evaporated to a resin. Yield 10.6 g (26%).1H-NMR (400 MHz, CDCl3): d 7.7 (doublet, 1H), 7,35 (singlet, 5H), 7,3 (singlet, 5H), 7,2 (singlet, 2H)= 6,9 (doublet, 1H), 5,2 5,0 (5H multiplet), 4,4 4,2 (multiplet, 2H), 4.1 (multiplet, 1H), 3.9 (multiplet, 4H), 3,6 (double doublet, 1H), 3,4 (double doublet, 1H), 1,2 (triplet, 3H), 1,1 (triplet, 3H), MS (+ FAB) 580 (M + N)+. Material (10.6 g, 0,0183 moles) was dissolved in acetic acid (300 ml) and shaken with 10% Pd/C (1 g) at a pressure of 1 ATM H2at room temperature, as long as you do not stop the absorption of H2the filtrate is evaporated and the residue together evaporated with dioxane (4 x 100 ml), IR, NMR and mass spectrum of the residue (8 g) indicated that removal of the benzyl protecting groups. 7.5 g of residue and trimethylsilyl bromide (15 g, 0.1 moles) was delegirovali in N2in dichloromethane (110 ml) for 1.5 hours, the Solvent evaporated and the residue was stirred in water (50 ml) and common air. Received solid, which was filtered and stored. The filtrate was separated, the aqueous layer was diluted with ethanol (50 l) and was treated with propylene oxide (10 ml) with stirring for 0.5 hours the Solution is evaporated to remove ethanol, and the solid substance thus formed was combined with the solid obtained previously. The product was dissolved in 1 vN HCl solution (100 ml) and Phil is face-to-face connection. The product has led warm water (50 ml) and ethanol (100 ml), and dried under vacuum. The output of 2.27 g (37%). The compound did not melt at temperatures below 310oC.1H-NMR (400 DMSO-d6): d 7,5 (triplet, 2H), and 7.3 (multiplet, 2H), 4,35 (doublet, 2H), 4,25 (triplet, 1H), 3.5 (multiplet, 2H); MS (-FAB) 298 (M-H)-; [a]2D5-49,5o(from 1.01 1 N HCl); Ehud analysis: the enantiomeric purity: 1S 99R.

Elemental analysis for C11H14N3O5P 2H2O:

Calculated: C 38,88, H 5,41, N 12,37

Found: 38,95, N 5,13, N To 12.52.

The dihydrate hydrochloride was obtained by dissolving the free acid in 2 N HCl, evaporated until dry, and then dried under vacuum.1H-NMR (400 MHz, DMSO-d6: 9,2 8,2 (multiplet, 2H), 7,9 (doublet, 1H), 7.7 (doublet, 1H), 7,5 (multiplet, 2H), 4,8 (multiplet, 3H), 3.8 (doublet, 2H); [a]2D5-41,6o(c 1.0 in 1 N HCl).

Elemental analysis for C11H14N3O5P HCl 2H2O:

Calculated: C 32,37, H is 4.93, N 10,29

Found: C 32,57, H 4,87, N 10,53

P R I m m e R 2.

R--amino-5,6-dichloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

Using the same procedure as used in Example 1, but using benzyl N-(benzyloxycarbonyl)-3-[(5,6-�), received connection from header example (Output is 15.3%), the melting-point was not determined.1H-NMR (400 MHz, DMSO-d6): d 8,55 (singlet, 3H), 8,95 (singlet, 1H), cent to 8.85 (singlet, 1H), 4,6 (doublet, 2H), 4,55 (triplet, 1H), 3.8 (multiplet, 2H); MS (-FAB) 366 (M-H)-[a]2D5-50,0o(c 1,01; 1 N HCl); Ehud analysis (free acid) enantiomeric purity: 2,6 S 97,4 R.

Elemental analysis for C11H12N3O5PCl22 HCl

Calculated: C 29.99 is; H 3,20, N AT 9.53

Found: C 29,78, H 3,31, N 9,65.

P R I m e R 3.

R--amino-5,6-dimethyl-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

Benzyl N-(benzyloxycarbonyl)-3-[/5,6-dimethyl)-2-benzimidazolyl] -D-alaninate (Nestor and others J. Med. Chem, 1984, T. 27, S. 320 (3,7 n, 0.8 mmol), [bis-(4-nitrophenylacetic/phosphinyl] methyl ester triptorelin(mono)sulfonic acids (5.5 g, 10 mmol) and powdered anhydrous K2CO3(5.5 g, 40 mmol) was stirred in acetonitrile (100 ml) at room temperature in an atmosphere of N2(20 hours). The acetonitrile is evaporated and the residue was dissolved in dichloromethane (100 ml) and water (2 x 100 ml). The dichloromethane layer was washed with 5% NaHCO3(2 x 100 ml), brine solution (100 ml) and dried over magnesium sulfate, filtered solution Varra with 10% Pd/C (1 g) and N2[38 psig (2,67 kg/cm2in the beginning] up until the absorption of H2don't stop (3 h). The filtered solution is evaporated and co evaporated with dioxane (3 x 50 ml) and the residue was diluted with water (350 ml). the pH of the mixture provided at level 3 using 6 N HCl solution was rapidly cooled and filtered. Air-dried solid (2.8 g) was dissolved in water (50 ml) with 1 N Hcl solution (1 ml) and precipitated by adding 1 N NaOH solution (1 ml). The product was washed with water, ethanol and simple ether. IR, NMR and mass spectra were consistent with phosphonomethyl amino acid. This material was dissolved with 1 N Hcl solution (20 ml), treated with activated charcoal and quickly cooled. Salt cordigera was filtered, washed with ice water and dried under vacuum. The output of 1.23 g (37,4%1P-NMR (400 MHz, DMSO-d6+ 20% DCl (D2(2 drops): d 7,75 (singlet, 1H), 5,7 (singlet, 1H), 5 4,75 (multiplet, 3H), 3,85 (multiplet, 1H), 2,35 (singlet, 3H); MS (-FAB) 326 (M-H)-; [a]2D554,4o(c 1,01, 1 N HCl); Ehud analysis: the enantiomeric purity: 1S 99R

Elemental analysis for C13H18N3O5P 2 HCI:

Calculated: C 39,02, H 5,04 N 10,50.

Found: C 38,62, H 5,27, N 10,32.

P R I m e R 4.

R-(-)(--amino-1-(phosphonomethyl the l-N-(benzyloxycarbonyl)-3-(2-benzimidazolyl)-D-alaninate, received connection from the header of the example. As a result of crystallization from hot water and prolonged drying under vacuum received hydrate with 3.5 moles of water output (61%).

1H-NMR (400 Hz, DMSO-d6+ DCl): d 7,95 (Quartet, 1H), 7,8 (Quartet, 1H), 7,55 (2N), 4,8 (multiplet, 3H), 3.8 (doublet, 2H); [a]2D553,1o(c 1,03 I NHCl); Ehud analysis, enantiomeric purity: 1,3 S 98,7 R.

Elemental analysis of C11H14N3O5P 3,5 H2O

Calculated: C 36,46, H Of 5.84, N 11,59.

Found: C 36,67, H 5,64, N 11,74.

Cordigera received by dissolution in 2 N HCl (5 ml) and water (15 ml), and evaporation until dry. [a]2D5-40,2 (c 1,0, 1 N HCl).

Elemental analysis for C11H14N3O5P HCl H2O.

Calculated: C 33,86, H 4,65, N 10,77.

Found: C 33,77, H 4,59, N 10,67.

P R I m e R 5.

2-Amino-2-(oxymethyl)-1,3-propandiol, R--amino-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid (2:1).

The connection of the free acid of example 4 (1.86 g, 5 mmol) tromethamine base (1,33 g, 11 mmol), was heated in water (10 ml) and precipitated with ethanol (100 ml). The material was filtered, washed with ethanol, then simple with ether, and air dried in technolog and dried under vacuum over P2O5. Yield 2.58 g (86.6 per cent).

Elemental analysis for C11H14N3O5P 2 C4H11NO33H2O.

Calculated: C 38,35, H 7,11, N 11,77.

Found: C 38,39, H 6,76, N Up 11,86.

P R I m e R 6.

S-a-amino-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

Benzyl N-(benzyloxycarbonyl)-3-(2-benzimidazolyl)-a-alanine (2.85 g, 6.3 mmol), [bis-(4-nitrobenzyloxy)-phosphonyl]methyl ester triptorelin(mono)sulfonic acids (3,67 g, 8.9 mmol) and powdered anhydrous potassium carbonate (4 g, 28.9 mmol) was stirred in acetonitrile (100 ml) at room temperature over night. Then continued the procedure of Example 1 and was obtained derivatives tetrabenzyl. The crude product (4.3 g) was subjected to purification on jhud type waters prep 500 using a gradient elution from hexane (100% ) to ethyl acetate (100%). The output of 1.0, the Product was dissolved in acetic acid with 10% Bd/C (0.5 g) and subjected to hydrogenation at a pressure of 1 ATM. The mixture was filtered, evaporated to install Rotavapor (Rotovapor) was rapidly diluted twice with dioxane and stirred in water (10 ml). The product was filtered and dried. Yield 0.35 g (17,6%); NMR identical to the product from example 1 MC (+FAB) 300 (M + H)B+[a]2D is C11H14N3O5P H2O.

Calculated: C 41,65, H 5,08, N 13,25.

Found: C 41,60, H 5,04, N To 13.09.

P R I m e R 7.

R--amino-6-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid

A) In a dry nitrogen atmosphere a-benzyl ester of N-Boc-D-aspartic acid (13.4 mmol, 4,34 g) was dissolved in dry tetrahydrofuran (67 ml) and cooled to -10oC. was Sequentially added triethylamine (13.4 mmol, 1,28 ml) and the reaction mixture was stirred for 10 min at a temperature of -10oC, and then slowly added to the solution produced by industry 4-chloro-1,2-phenylenediamine (14.7 mmol, 2.1 g) in dry tetrahydrofuran (27 ml). The mixture gave the opportunity to slowly warm to ambient temperature. Then it was poured into ice-cold brine (150 ml), was extracted with ethyl acetate (2 x 100 ml). Connected the organic layer was washed sequentially with saturated ice NaHCO3(100 ml), then with brine (100 ml) and then dried over magnesium sulfate, filtered and evaporated until dry under vacuum. The residue was subjected to chromatography (ghvd). In the elution ethyl acetate (hexane were obtained 2.9 g of N4-(2-amino-5-chlorophenyl)-N2-[(dimethylmethoxy/th, 2.15 g) in glacial acetic acid (70 ml) was heated to 70oC for 5 h without access of humidity. The mixture is then evaporated under vacuum and the residue was promptly subjected to chromatography on silica gel (60 g). In the elution with 20% ethyl acetate/hexane was obtained 1.6 g phenylmethylene of ester 6-chloro-a-[1,1-dimethylmethoxy/carbonyl]amino]-1H-benzimidazole-2-propanoic acid in the form of butter.

C) a Solution of the oil from the Stage In (3.7 mmol, 1.6 g) in acetonitrile (50 ml) was treated at a temperature of 25oC in a dry nitrogen [dimethoxyphosphinyl] methyl ether complex triptorelin(mono)sulfonic acids (4.1 mmol, 1,115 g) and anhydrous powdered potassium carbonate (10 mmol, 1.38 in). The reaction mixture was stirred at a temperature of 25oC overnight, was filtered, washed with methylene chloride (20 ml), the United filtrates evaporated under vacuum, and the residue was divided between methylene chloride and water. The organic layer was separated, dried over magnesium sulfate, filtered and evaporated under vacuum. The residue was subjected to rapid chromatography on silica gel (60 g). In the elution with chloroform/ethyl acetate was obtained 1.4 g phenylmethylene of ester 6-chloro-1-[(dimethoxyphosphinyl)methyl] - a-[[(1,1-dimethylmethoxy)ka is her 1.4 g) in glacial acetic acid (20 ml) was treated with 10% palladium on charcoal (140 mg) and subjected to hydrogenation for 3 hours at 25oC. the Reaction mixture was purged with nitrogen, filtered through filter Salk-flock (Solka-floc), the cake was washed with acetic acid (10 ml) and the filtrate evaporated until dry under vacuum. The rest drove with toluene (2 x 10 ml) and evaporated under deep vacuum to get to 1.15 g of 6-chloro-1- [(dimethoxyphosphinyl)-methyl] -a-[[1,1-dimethylmethoxy/carbonyl]amino]- 1H-benzimidazole-2-propanoic acid in the form of butter.

E) Oil on Stage D (1.9 mmol, 0.9 g) was subjected to reflux distilled 6 N HCl solution (20 ml for 45 minutes the Reaction mixture is then evaporated until dry under vacuum, the residue drove with toluene (2 x 20 ml), and then subjected to crystallization from hot water/acetonitrile to obtain 330 mg of the compound of header example; melting point 198 - 200oC.1H-NMR (DMSO-d61 drop of DCl, 400 MHz: d a 3.87 (double doublet, J1and 5.5 Hz, J27.2 Hz, 2H, ) 4,86 (triplet, J 7.2 Hz, 1H ), 4,96 (double doublet, J112 Hz, J232,7 Hz, 2H, CH2-P), to 7.59 (double doublet, Jo8,7 Hz, Jm2 Hz, 1H, H-5), 7,83 (doublet, Jo8,8 Hz, 1H, H-4), 8,13 (doublet, Jm1,9 Accitane: C 36,62, H 4,47, N 11,64

Found: C 36,32, H 4,55 N 11,35

P R I m e R 8.

R--amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

A) Used the procedure as in example 7A), however, the obtained residue was not purified and used in the next stage.

B) Used the procedure as in example), however, the obtained residue was not purified, but used in the next stage.

C) the Oil obtained from Stage (21.8 mmol, 9.4 g, a mixture of 5 - and 6-chlororesorcinol) in acetonitrile (250 ml) was treated once dry nitrogen and under stirring [dimethoxyphosphinyl]methyl ether complex triptorelin/mono(sulfonic acids) 24 mmol, 6,528 g) and anhydrous powdered K2CO3(65 mmol, 8,97 g). The reaction mixture was stirred at 25oC overnight, was filtered and washed with acetonitrile. The filtrate is evaporated and the residue is divided between water and methylene chloride. The separated organic layer was dried, and then evaporated under vacuum until dry. The residue was subjected to chromatography (ghvd), was suirable ethyl acetate/hexane, resulting in received 4 g phenylmethylene of ester 5-chloro-1-[(dimethoxyphosphinyl/methyl] - alpha-[[(1,1-dimethoxyethoxy/carbonyl]-amino]-1H-by acetic acid (60 ml) was treated with 10% palladium (charcoal (400 mg) and subjected to hydrogenation at a temperature of 25oC at atmospheric pressure for 4 h Then the mixture was purged with nitrogen, filtered through filter Solka-floc, washed with acetic acid (20 ml) and the filtrate evaporated until dry under vacuum. The rest drove with toluene (2 x 20 ml), and finally evaporated under a deep vacuum to get 3,43 g of 5-chloro-1-[(dimethoxyphosphinyl)methyl] -a-[[(1,1 - dimethylmethoxy/carbonyl]amino]-1H-benzimidazol-2-propanoic acid in the form of butter.

E) Oil on Stage D (7.2 mmol, 3,43 g) was subjected to reflux distilled 6 N hydrochloric acid (60 ml) for 50 minutes Then the mixture is evaporated until dry under vacuum and the residue is again evaporated with water (15 ml). The residue was dried under high vacuum, and then were led out of hot water (acetonitrile; the Connection was filtered, washed with simple ether (10 ml) and was dried at a pressure of 1 mm RT.article 60oC (above the P2O5to obtain 1.4 g of the target product. The melting point 113 116oC (Razlog.).1H-NMR (DMSO-d6+ 1 drop of DCl, 400 MHz): d 3,86 (triplet, J 6.4 Hz, 2H, a 4.83 (triplet, J 6.8 Hz, 1H ), 4.95 points (multiplet, 2H, CH2-P), to 7.61 (double doublet (Jothe 8.9 Hz, Jm1.9 Hz, 1H, H-6), to $ 7.91 (doublet, Jmthe 1.7 Hz, 1H, H-4), of 7.96 (doublet, Jothe 8.9 Hz, 1H, H-7).

Found: C, 35,87, H 3,94, N OF 11.26

P R I m e R 9.

S--Amino-5,6-dichloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

The compound was obtained in accordance with the procedure of Example 7; melting point 230oC (Razlog.).1H-NMR (DMSO-d6+ drop DCl/ d of 3.78 (doublet, J 7 Hz, 2H, ), 4,72 (triplet, J 7 Hz, 1H ), 4,86 (multiplet, 2H, CH2-P), 8,06 (singlet, 1H, Ar-H), 8,23 (singlet, 1H, Ar-H); MS: (-FAB) m/e 366 (M-H); [a]2D5+ 58,8o(1N HCl).

Elemental analysis for C11H12N3O5P 2H2O

Calculated: C, 32,69, H 3,99, N accounted for 10.39

Found: C, 32,66, H 4,13, N 10,34

P R I m e R 10.

Compounds that are the subject of the present invention, were tested on their NMDA-competing antagonistic activity through their ability to displace saturated with tritium 3-(2-carboxypeptidase-4-yl)propyl-1-alkylphosphines acid (CPR), a known competitive antagonist of NMDA in the homogenates of the anterior frontal lobe of the rat in vitro analysis 3H/CAT-linking.

This analysis was carried out as follows.

Rats were decapitated and the brain was immediately removed, weighed and placed in approximately 15 volumes cooled with ice to 10% sucrose. CA rpm), equipped with a Teflon pestle. Eaten the homogenate was subjected to centrifugation at 1 000 x g for 10 minutes the resulting tablet was dropped and the upper layer was centrifuged at 20 000 x g for 20 min. Crude mitochondrial tablet again suspended in a cooled ice distilled water and was dispersively using device type Polytron Brinkmann (RT-10 with mode 6 for 30). The suspension was centrifuged with a speed of 8000 x g for 20 minutes the resulting upper layer and a dark yellow floor was centrifuged with a speed of 48 000 x g for 20 minutes Final crude pill sinapinic membranes again suspended in ice distilled water and centrifuged with a speed of 48 000 x g for 20 min.

In order to facilitate the removal of endogenous glutamate, the membrane is again suspended in 15 volumes of cooled ice 50 mm TRIS/pH to 7.6) containing 0.04% of Triton X-100. The suspension is incubated at a temperature of 37oC for 15 minutes and then centrifuged at 20 000 x g for 20 minutes the Tablet was washed (i.e., again suspended in a cooled ice TRIS buffer and centrifuged at 20 000 x g is, especilaly several Pribram to a centrifuge and subjected to centrifugation at 20 000 x g for 20 min, and tablets frozen (-70oC) for later use to analyze the binding.

For the analysis of binding tablets membranes were thawed and again suspended in 15 volumes of cooled ice 50 mm TRIS (pH of 7.6) buffer. Only three samples (1000L) suspension of membranes containing from 0.2 to 0.4 mg protein/ml, were incubated at a temperature of 23oC for 15 min with 8 nm (3H/PPC) company New England Nuclear), one of the various test solutions, and buffer in a final volume of incubation 2 ml using plastic miniprobe (firm Skatron). Then the samples were centrifuged with a speed of 48 000 x g for 20 min, and the upper layers were dropped. Tablets were digested tissue solubilizer (firm Amersham, NCS; 500 l sample) for 1 hour In each sample was added hydrochloric acid /100 l, 4N/ to restore chemoluminiscence during subsequent counting. In each of miniprobe was added to the scintillation cocktail /Agasol, the firm Du Pont; 3.2 ml/, then covered and shaken for 15 minutes before counting. The tubes were placed in a counter type, Packard 460 CD /or equivalent/ c a is minus binding in the presence of 1 mm NMDA. Specific binding in the presence of the test drug was expressed as a percentage of specific binding, when such drug is not. When the test compounds were analyzed for the ratio of dose-response, the results were plotted on a plane in the view log link relative to the log concentration of the test drug. Analysis of linear regression then gives a straight line, from which you can calculate the IC50with 95% confidence limits.

Standard connections:

Ligand IC50+ senior Osh. M/mm/ i

L Glutamic acid and 64.3 4,4 (3)

AP7 639,2 OF 128.6 (3)

THE NMDA 1 882,6 612,2 (5)

When tested in this assay compounds that are the subject of the present invention gave the following results.

The compound of Example N IC50nm

1 59

2 18,2

3 123

6 63% c 10 M

7 21,7

8 7

9 1010

P R I m e R 11.

Compounds of the present invention further tested for their in vivo antagonistic ability against the NMDA in mice in a convulsive condition caused by NMDA.

This analysis was carried out as follows:

Male mice Swiss-albino (strain CD-I, Charles River) weighing 18 to 22 g, after 18 h of food deprivation, were placed in Nablus thirty minutes with the help of the NMDA, dose of 195 mg/kg intraperitoneal way, with this dose, in General, in 90% of cases leads to death, coming as a result of paralysis of motor function, including nekontroliruemoe scratching hind legs or twitching of the muscles of the limbs and/or spiral muscles with loss of right reflex and subsequent death of the animal during the observation period of 30 min after application of NMDA. From the last determined the ED50for live mice.

Data were analyzed using an independent analysis of PS-NONLIN (Version Natural Reaction Rate). The output from this program contains the statistical significance of the slope of the dose-response with ED with 50% and 95% confidence limits for live mice.

The control connections are shown in the table.

When tested in this assay compounds that are the subject of the present invention gave the following results:

The compound of Example N ED50mg/kg, nutriplus. or alive

1 2,0

2 2,7

3 10% at 3 mg/kg

7 <5
9 > 10

1. Alpha-amino-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid of General formula I

< / BR>
where R1and R2hydrogen, lower alkyl or Galia-amino-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

3. Connection on p. 1, representing R-alpha-amino-5,6-dichloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

4. Connection on p. 1, representing R-alpha-amino-5,6-dimethyl-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

5. Connection on p. 1, representing R-alpha-amino-6-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

6. Connection on p. 1, representing R-alpha-amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid.

7. The method of obtaining compounds of formula I on p. 1 or its pharmacologically acceptable salts, characterized in that it includes removing protection from compounds of formula II

< / BR>
where R1and R2hydrogen, lower alkyl or halogen,

R3and R4protecting the amino group, R5- protecting alkylphosphonic acid group,

with subsequent isolation of the desired product in the form of the acid or its pharmacologically acceptable salt.

8. The method according to p. 1, characterized in that the removal of protection carried out using acidic or alkaline hydrolysis, hydrogenolysis and/or treatment trimethylsilylpropyne.

9. The method according to p. 7, characterized in that benzil, removing protection carried out by hydrogenolysis.

10. The method according to p. 7, characterized in that the use of the compounds of formula II in which R3lower alkyl or benzyl.

11. The method according to p. 7, characterized in that the use of the compounds of formula II in which R4tert-butyloxycarbonyl or benzyloxycarbonyl.

12. The method according to p. 7, characterized in that the use of the compounds of formula II in which R5lower alkyl, benzyl, or 4-nitrobenzyl.

 

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< / BR>
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FIELD: medicine, oncology.

SUBSTANCE: the present innovation deals with treating patients with uterine cervix cancer with relapses in parametral fiber and in case of no possibility for radical operative interference and effect of previous radiation therapy. During the 1st d of therapy one should intravenously inject 30 mg platidiam incubated for 1 h at 37 C with 150 ml autoblood, during the next 3 d comes external irradiation per 2.6 G-r. During the 5th d of therapy one should introduce the following composition into presacral space: 60 ml 0.5%-novocaine solution, 1 ml hydrocortisone suspension, 2 ml 50%-analgin solution, 1 ml 0.01%-vitamin B12 solution, 1.6 g gentamycine, 800 mg cyclophosphan, 10 mg metothrexate. These curative impacts should be repeated at mentioned sequence four times. The method enables to decrease radiation loading and toxic manifestations of anti-tumor therapy at achieving increased percent of tumor regression.

EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine, oncohematology.

SUBSTANCE: the present innovation deals with treating elderly patients with chronic lympholeukosis accompanied with cardiovascular failure. The method deals with applying chemopreparations and cytoprotector. Moreover, 1 wk before the onset of chemotherapeutic therapy one should prescribe preductal at the dosage of 105 mg daily. At this background one should sample blood out of elbow vein at the volume of 200 ml into a vial with glugicir to centrifuge it, isolate plasma, divide into two portions, add into the 1st vial - cyclophosphan 600-800 mg/sq. m, vincristin 1.4 mg/sq. m, into the 2nd vial - adriamycin 50 mg/sq. m to be incubated for 30 min at 37 C and intravenously injected by drops for patients. Simultaneously, the intake of prednisolone should be prescribed at the dosage of 60 mg/sq. m since the 1st d and during the next 5 d and preductal at the dosage of 105 mg daily during a week, and then 2 wk more at the dosage of 60 mg daily. All the procedures should be repeated in above-mentioned sequence 4-6 times. The method enables to decrease toxic manifestations of chemotherapy while applying adequate dosages of cytostatics, anthracycline antibiotics, among them, at no great manifestations of their toxicity due to preductal's cardioprotective action.

EFFECT: higher efficiency of therapy.

1 ex, 5 tbl

FIELD: medicine, oncology, gynecology.

SUBSTANCE: method involves external irradiation and using chemopreparations combined with autoplasma. External irradiation in the dose 2 Gr is carried out from 1 to 4 day of treatment followed by intravenous administration of cisplatin on 5 day in the dose 10 mg with 250 ml of physiological solution. On the following day 10 ml of autoplasma incubated with 10 mg of cisplatin is administrated by paratumoral route by right and left of tumor into paravaginal cellular tissue and by intratumoral route into projection of cervical channel. Blood cellular elements are incubated with 600 mg of cyclophosphane and reinfused by intravenous drop route. Effects are repeated in indicated sequence once per a week, 4 times per treatment course. Method provides increasing percent of tumor regression due to the complex effect on tumor, enhancing the topical concentration of cytostatic drug and prolongation of its contact in malignant growth zone, and providing radiosensibilizing effect in diminishing the general toxic effect of therapy. Invention can be used in treatment of patients with topically disseminated forms of uterus cervix cancer at the stage T3NxM0.

EFFECT: improved and enhanced treatment method.

1 ex

FIELD: medicine.

SUBSTANCE: method involves incubating flasks containing 70-80 ml of blood and 40 ml of preservative agent for 20 min at 37°C together with chemotherapeutical preparations. Next to it, treatment with alternating magnetic field of 50 mTesla units intensity and frequency of 50 Hz is applied at continuous rotation about its axis at 20 rpm speed. 30 mg/m2 of Doxorubicin is added to the first flask contents. 500 mg/m2 of cyclophosphane are added to the second flask and 20 mg/m2 of methotrexate are added to the third flask. Incubation and treatment with alternating magnetic field is started with the first flask at the first and the eighth day. Total chemopreparations quantity is equal to 60-80 mg, cyclophosphan is applied in the amount of 1200-1600 mg, methotrexate - 40 mg.

EFFECT: enhanced effectiveness of treatment.

FIELD: medicine, oncology.

SUBSTANCE: the present innovation deals with treating malignant tumors of mammary gland in case no tumor penetration into thoracic fasciae. The method suggested includes autohemochemotherapy. Moreover, on the 1st d of therapy it is necessary to sample 30 ml blood out of patient's peripheral vein into the 1st vial to combine its content with 500 mg 5-fluorouracil and 600 mg cyclophosphan; then one should sample 20 ml blood into the 2nd vial to combine its content with 40 mg doxorubicin; both vials should be incubated at 37-37.5 C for 20-30 min, afterwards its necessary to inject the mixtures out of both vials under the tumor and along its circumference. On the 8th d of therapy one should repeat impact procedures at the same dosages of anti-tumor preparations and at the same order. The innovation suggested enables to develop the largest concentration of anti-tumor preparations in lesion focus, divide into fragments, decrease tumor's size and its inflammatory component and border it against surrounding tissues for performing radical surgical treatment.

EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine.

SUBSTANCE: method involves introducing antitumor chemo preparations with blood components. To do it, 300 ml of patient autoblood is subjected to centrifuging during 20 min at 2200 rpm. The produced 150-200 ml of autoplasma and 100 ml of packed red blood cells are placed into separate reservoirs. Cys-platinum as single dose of 100 mg is incubated with the autoplasma and cyclophosphane as single dose of 1 g is incubated with the packed red blood cells. Single doxorubicin dose of 30-50 mg is concurrently introduced with one of the preparations. When combined with cis-platinum, doxorubicin is incubated with the packed red blood cells. When combined with cyclophosphane, doxorubicin is incubated with the autoplasma. Reinfusion is carried out to bring total dose of the preparations to 150 mg of doxorubicin, 3-5 g of cyclophosphane and 200 mg of cis-platinum. Pause between the procedures is 3-4 days long.

EFFECT: avoided risk of adverse side effects; increased preparation activity; accelerated treatment course.

FIELD: experimental medicine and oncology.

SUBSTANCE: claimed method includes intraperitoneum cyclophosphan administration in dose of 120 mg/kg and intravenous transplantation of fetal liver steam cells to experimental animal. Fetal liver steam cells are transplanted for 3 days before cyclophosphan administration in dose of 25x106 cells/kg of mass.

EFFECT: decreased tumor growth, reduced frequency and area of metastasis due to optimization of cyclophosphan and steam cells administration.

2 ex

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