CH648289A5 - N- (4- (3-AMINOPROPYL) AMINOBUTYL) -2,2-DIHYDROXYAETHANAMIDE AND METHOD FOR ITS SYNTHESIS. - Google Patents

Description

The invention relates to N- [4- (3-aminopropyl) aminobutyl] -2,2-dihydroxyethanolamide of the formula (I)

ho.

ho

2H-JII -j chconhch-

2nd

, ch

3 4 2ch2ch2

1 '

no

2 '3'

2ch2ch2nh2

(I)

and its acid addition salts and a process for its synthesis.

The new compound is not only useful as an intermediate for the synthesis of the new carcinostatic compound N- [4- (3-aminopropyl) aminobutylI-2 - [(S) -7-guanodino-3-hydroxyheptanamido] -2-hydroxyethane amide or one of its derivatives, but also useful as an immunostimulant itself.

With regard to its stability, the new compound according to the invention is preferably produced in the form of the acid addition salt. The acids that are added are 45 inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and boric acid, and organic acids such as acetic acid, citric acid, tartaric acid and glutaric acid. Of these acids, hydrochloric acid, sulfuric acid and tartaric acid are particularly preferred, so

The physico-chemical and biological properties of the compounds according to the invention are as follows. (SRBC)

(1) Physico-chemical properties 55 105

The hydrochloride of compound 105 according to the invention is a white powder which has no pronounced melting point. The elementary analysis agrees with the theo-10S calculated on C9H2IN303 • 2HC1 (C 36.99%, H IO5 7.93%, N 14.38%, Cl 24.27%). The proton eo 105 NMR, determined in heavy water, shows characteristic 105 signals at 5 = 2.1 (2-, 3-CH2), 2.4-2.7 (2'-CH2), 3.5-3 , 8 (1-, 4-, 1 '-, 3'-CH2), 5.77 (2 "-CH, S).

(2) Biological properties

The effect of the compound according to the invention (Hy- 65

drochloride) on the delayed hypersensitivity to red blood cells in sheep is given in Table 1. The experiment is carried out according to the Macka

ness et al. [P.H. Lagrange, G.B. Mackaness and T.E. Miller, J. Exp. Med., 139, 528-542 (1974)] as follows.

6-week-old CDFj female mice are immunized by intravenous injection of 105 red sheep blood cells each, and at the same time, different amounts of the compound of formula (I) (hydrochloride) are injected peritoneally. After 4 days, 108 sheep red blood cells are injected subcutaneously into each mouse to induce the reaction. After 24 hours, the swelling of the foot cushion of each mouse is determined using a sliding thickness gauge.

Table 1

Compound of formula (I), (| ig / mouse i.p.)

Increase in foot T / C cushion thickness (%)

(x0, l mm + S.D)

none 1 4 16 63 250 1000

8.0 ± 0.7 13.0 ± 1.7 13.0 + 0.5 13.4 + 0.6 14.9 + 0.9 11.8 + 0.6 11.7 + 1.0

100 163 163 168 186 148 146

SRBC = red blood cells from sheep

From Table 1 it follows that the reaction was enhanced by administration of 1 ng to 1 mg of compound of formula (I).

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648 289

The compound N- [4- (3-aminopropyl) aminobutyl] -2,2-dihydroxyethanolamide of the formula (I)

H0 \ (I)

^ chc0nh (ch2) 4nh (ch2) 3nh2

ho is synthesized in the following way.

A compound of formula (II)

H2N (CH2) 4N (CH2) 3NHR (II)

I.

R

wherein R is an amino protecting group, such as a benzyloxycarbonyl, chloroacetyl or lower alkyloxycarbonyl group, is reacted with a dialkylacetal of glyoxylic acid of the following formula (III)

r'o

^ chcooh (iii)

r'o wherein R 'is an alkyl group having 1 to 5 carbon atoms, or a reactive derivative of the carboxyl group of the dialkylacetal is condensed, and then the amino protecting group and the group R' are removed, whereby the compound of formula (I) is obtained.

The condensation of the compound of formula (II) with the compound of formula (III) or its reactive derivative is carried out according to the process for the formation of a conventional amide bond in peptide synthesis, for example using an acyl halide, an acid azide, a reactive ester or an acid anhydride used. The condensation thus takes place in the presence of an activating agent for the carboxyl group of the compound of the formula (III). Examples of suitable reagents used to activate the carboxyl group or to form a reactive derivative include reagents for forming active esters, such as 6-chloro-lp-chlorobenzenesulfonyloxybenzotri-azole (CCBT), N-ethyl-5-phenylisoxazolium-3 '-sulfonate (NEPIS), N-tert-butyl-5-methylisoxazolium perchlorate, N-athoxycarbonyl-2-ethoxy-l, 2-dihydroquinoline, di-p-nitro-phenyl sulfite, tri-p-nitrophenyl phosphite, p-nitrophenyl trichloroacetate, N-hydroxysuccinimide, p-nitrophenyl, pentachlorophenol and benzyl alcohol; Carbodiimides such as dicyclohexylcarbodiimide (DCD), l-ethyl-3- (3-dimethylamino-propyl) -carbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide, di-p-toluylcarbodiimide and diisopropylcarbo-diimid; and reagents for azide synthesis.

The condensation reaction is preferably carried out in an organic solvent, such as ethyl acetate, and at a temperature of generally 0 to 100 ° C., preferably 10 to 40 ° C. Although the reaction time varies with the reaction temperature, it is about 5 to about 30 hours at room temperature. The amount of carboxylic acid of formula (III)

or a reactive derivative thereof which is used is 0.5 to 10 mol and preferably 2 to 5 mol / mol of amine of formula (II).

The protective groups can be removed from the condensate described above by hydrolysis or by reduction in accordance with the type of protective group. Protecting groups that can be removed by acid hydrolysis are preferred. For example, when the amino protecting group is a tert-butoxy carbonyl group and the

Aldehyde protective group is a diethylacetal, the hydrolysis is carried out in aqueous dioxane solution by adding 2 to 3 equiv. dilute hydrochloric acid and heating for 2 to 5 hours at 100 ° C., the hydrochloride of the compound of the formula (I) according to the invention being obtained. If the amino protecting group is a benzyloxycarbonyl group, the protecting group is preferably removed by hydrogenolysis using palladium, platinum oxide or a similar compound.

The starting compound of formula (II)

H2N (CH2) 4N (CH2) 3NHR (II)

I.

R

where R represents an amino protecting group is difficult to produce in good yield by selectively introducing two amino protecting groups into spermidine. Therefore, by condensation in a manner known per se, a monoamino-protected 1,4-butanediamine of the formula (IV)

R "HN (CH2) 4NH2 (IV)

wherein R "represents an amino protecting group which differs from the above group R with an amino protected 3-halopropane amide of the formula (V)

X (CH2) 2NHR (V)

wherein R represents the same amino protecting group as indicated above and X represents a halogen atom to form a compound of formula (VI)

R "HN (CH2) 4NH (CH2) 3NHR (VI)

wherein R and R "represent amino protecting groups which are different from each other, and then protecting the remaining imino group with the same amino protecting group as R and selectively removing the other amino protecting group R" to form the compound of formula (II).

Alternatively, the compound of formula (VI) can be obtained by condensation of a monoamino-protected 1,3-propane diamine of formula (VII)

RHN (CH2) 2NH2 (VII)

wherein R has the meaning given above, with an amino-protected 4-halobutanamine of the formula (VIII)

X (CH2) 4NHR "(VIII)

wherein R "and X have the meaning given above, are prepared in the manner described above.

For the protection of the amino groups, such amino protective groups as are usually used in peptide synthesis are used. However, the amino protecting group of R "should be selectively removable, leaving the amino protecting group R. Accordingly, a combination of a benzyloxycarbonyl group, which is removable by hydrogenolysis, and a tert.-butoxycarbonyl group, which can be removed by treatment with a weak acid is particularly preferred. Any of this pair can be R or R ".

The condensation of a compound of formula (IV) with a compound of formula (V) or the condensation of a compound of formula (VII) with a compound of formula (VIII) is easily carried out in an anhydrous solvent.

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tel, such as N, N-dimethylformamide, carried out at room temperature in the presence of triethylamine. The halogen in the compound of formula (VIII) is preferably bromine. The dialkyl acetal of glyoxylic acid of formula (III) is easily prepared by reacting glyoxylic acid with an alkanol using an acid catalyst in a manner known per se. Suitable alkanols for use are methanol, ethanol, propanol, butanol and amyl alcohol. The dialkylacetal of formula (III) is conveniently obtained by alkaline hydrolysis of commercially available ethyl 2,2-diethoxyacetate.

Starting from the compound of formula (I) according to the invention, a useful carcinostatic substance, N- [4- (3-aminopropyl) aminobutyl] -2 - [(S) -7-guanidino-3-hydroxyheptanamido] -2-hydroxyethane amide ( GHA-GS for short) by condensation of the compound of the formula (I) according to the invention with (S) -7-guanidino-3-hydroxyheptane amide of the formula

, N (S)

h2ncnh (ch2) 4chch2conh2

nh oh prepared by heating both compounds in the presence of an inorganic or organic acid. As indicated below, GHA-GS shows carcinostatic activity against L1210 mouse leukemia and is useful as an anti-tumor agent.

The carcinostatic effect of GHA-GS in mouse leukemia L1210:

A group of five male mice of the BDF2 strain (6 weeks old) are inoculated intraperitoneally with 105 L1210 cells and immediately afterwards each mouse is introperitoneally administered a physiological saline solution of the sample once a day for 6 consecutive days in order to increase the survival rate [( T / C) x 100; T / C = (mean survival of the treated group) / (mean survival of the untreated group)].

Sample dose extension rate d. Number of after

(mg / kg / day survival life 30 days-

(T / C) x 100 (%) the mice

1.56 115 0

3.13> 254 1

6.25> 341 3

GHA-GS 12.5> 405 4

25> 351 2

50> 365 2

The following examples illustrate the invention.

example

(a) Synthesis of Mono-N-benzyloxycarbonyl-

1,4-butanediamine 1.30 g (20 mmol) of 1,4-butanediamine are dissolved in 30 ml of 50% aqueous methanol, and 5.48 g (20 mmol) of S-benzyloxycarbonyl-4,6-dimethyl-2 are then added -mercapto-pyrimidine (product of Kokusan Kagaku Co.). The mixture is stirred for 3 hours. The reaction mixture is then filtered to remove the precipitate [2.08 g (29%) di-N-benzyloxycarbonyl compound are obtained] and the filtrate is evaporated to dryness. The residue is dissolved in 250 ml of chloroform, washed 5 times with 100 ml of water. The chloroform layer becomes over anhydrous

Dried sodium sulfate and evaporated to dryness. 1.0 g (23% yield) of mono-N-benzyloxycarbonyl-1,4-butanediamine are obtained in the form of a colorless syrup.

(b) Synthesis of O-TosyI-3-tert-butoxycarbonyl-

amino-1-propanol

1.5 g (20 mmol) of 3-amino-l-propanol are dissolved in 30 ml of methanol, and 4.8 g (20 mmol) of S-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine (are then added Product of Kokusan Kagaku Co.). The mixture is stirred for 6 hours. The reaction mixture is evaporated to dryness, dissolved in 200 ml of chloroform and washed with 200 ml of water. The chloroform layer is concentrated and subjected to column chromatography using 300 g silica gel (Wako-Gel "C-200) and a mixture of toluene and ethyl acetate (1: 1 by volume) as the developing solvent. Fractions 82 to 151 (15 ml volume each) are combined, evaporated to dryness and 2.95 g (84% yield) of tert-butoxycarbonylamino-l-propanol are obtained in the form of a colorless oil.

2.95 g (16.9 mmol) of 3-tert-butoxycarbonylamino-1-propanol are dissolved in 50 ml of pyridine. A solution of 3.36 g (17.7 mmol) of p-toluenesulfonyl chloride in pyridine is added dropwise to the solution with cooling with ice and under an argon atmosphere over the course of 40 min. The mixture is left to stand at 7 ° C. overnight, then a small volume of water is added and the mixture is evaporated to dryness. The residue is dissolved in 200 ml of chloroform, washed successively with 5% aqueous potassium hydrogen sulfate solution, saturated aqueous sodium hydrogen carbonate solution and water, then dried over anhydrous sodium sulfate, evaporated to dryness and column chromatography using 120 g silica gel (Wako- Gel® C-200) and a mixture of toluene and ethyl acetate (8: 1 by volume) as a developing solvent. Fractions No. 35 to 68 (each 15 ml volume) are combined and evaporated to dryness. 3.06 g (55% yield) of O-tosyl-3-tert-butoxycarbonyl-amino-1-propanol are obtained in the form of a colorless oil.

(c) Synthesis of N-tert-butoxycarbonyl-N '- (tert-butoxy-

carbonylaminopropyl) -1,4-butanediamine

800 mg (2.43 mmol) of the O-toxyl-3-tert-butoxycarbonylamino-l-propanol obtained under (b) above are dissolved in 15 ml of dimethylformamide. After adding 510 mg (4.8 mmol) of lithium bromide (LiBr • H2Ö), the mixture is stirred for 24 hours at room temperature. 540 mg (2.43 mmol) of the mono-N-benzyloxycarbonyl-l, 4-butanediamine obtained under (a) and 0.34 ml of triethylamine are added to the mixture containing the bromine derivative. The mixture is stirred for 48 hours at room temperature. The reaction mixture is mixed with 699 mg (2.9 mmol) of S-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine and stirred for 13 hours at room temperature. The reaction mixture is evaporated to dryness, dissolved in 100 ml of chloroform, washed with 50 ml of water, dried over anhydrous sodium sulfate and evaporated to dryness. The residue is dissolved in a small volume of a mixture of toluene and ethyl acetate (4: 1 by volume) and column chromatography using 200 g of silica gel (Wako-Gel® C-200) and a mixture of toluene and ethyl acetate ( 4: 1 expressed by volume) as a developing solvent. Fractions No. 134 to 165 (each 12 ml volume) are combined and evaporated to dryness. 608 mg (52% yield) of N-benzyloxycarbonyl

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N-tert-butoxycarbonyl-N '- (tert-butoxycarbonylaminopropyl) -l, 4-butanediamine in the form of a colorless syrup.

144 mg (0.3 mmol) of the above compound is dissolved in 5 ml of methanol in the form of a colorless syrup. After adding 100 mg of 5% palladium-barium carbonate, the mixture is stirred for 5 hours under a stream of hydrogen at room temperature, the catalyst is removed by filtration and the mixture is evaporated to dryness. 103 mg (100% yield) of N-tert-butoxycarbonyl-N '- (tert-butoxycarbonylaminopropyl) -1,4-butanediamine are obtained.

(d (Synthesis of N- [4- (3-aminopropyl) aminobutyl] -2,2-

dihydroxybutanamide

In 2 ml of ethyl acetate, 100 mg (0.29 mmol) of N-tert.-butoxycarbonyl-N '- (tert.-butoxycabonylaminopropyl) -1,4-butanediamine, obtained under (c) above, and 148 mg (1 mmol ) 2,2-diethoxyacetic acid. After adding 135 mg (1 mmol) of 1-hydroxybenzotriazole and 206 mg (1 mmol) of di-cyclohexylcarbodiimide, the mixture is stirred for 15 hours at room temperature. The reaction mixture is filtered to remove the precipitate and the precipitate is washed with cold ethyl acetate. The filtrate and the washing liquids are combined and washed successively with 1 M aqueous ammonia and water. The ethyl acetate layer is dried over anhydrous sodium sulfate and evaporated to dryness. The residue is subjected to column chromatography using 20 g of silica gel (Wako-Gel® C-200) and a mixture of toluene and ethyl acetate (1: 2 by volume) as a developing solvent. Fractions Nos. 14 to 21 (each 3 ml volume) are combined and evaporated to dryness. 109 mg (79% yield) of N- [4- (3-tert-butoxycarbonylaminopropyl) -4-tert-butoxycarbonylaminobutyl] -2,2-diethoxyethane amide are obtained in the form of a colorless syrup.

44 mg (0.13 mmol) of the above syrupy substance are dissolved in 1 ml of dioxane. After the addition of 2.5 ml of 0.1N hydrochloric acid, the mixture is stirred for 4 hours in an oil bath heated to 100.degree. The reaction mixture is adjusted to pH 6 by adding 0.2N aqueous sodium hydroxide solution and evaporated to dryness. The residue is extracted with 1.5 ml of methanol. The extract is passed through a column packed with 100 ml Sephadexv LH-20 (16.5 mm inside diameter) and developed with methanol. Fractions Nos. 22 to 25 (each 2 ml volume), which are positive for the ninhydrin test, are combined and evaporated to dryness. 13 mg (46% yield) of N- [4- (3-aminopropyl) aminobutyl] -2,2-dihydroxyethanolamide dihydrochloride are obtained in the form of a colorless syrup.

Reference example synthesis of GHA-GS

A mixture of 51 mg (0.214 mmol) of (S) -7-guanidino-3-hydroxyheptanamide hydrochloride, 112 mg (0.385 mmol) of N- [4- (3-aminopropyl) aminobutyl] -2,2-dihydroxyethane amide dihydrochloride, 70 mg (0.53 mmol) of glutaric acid and 0.07 ml (3.9 mmol) of water are stirred at 60 ° C. for 43 h. The reaction mixture is mixed with 20 ml of 0.4 M sodium chloride solution, adjusted to pH 6.1 with 10% aqueous ammonia and passed through a column (12 mm internal diameter), which is equilibrated with 20 ml of CM Sephadex "C-25 , 4 M sodium chloride solution. The column is then subjected to gradient elution using 80 ml of 0.4 M and 1.0 M sodium chloride solution, respectively. Fractions Nos. 41 to 50 (each 2 ml volume) are combined, evaporated to dryness and extracted three times with 10 ml of methanol, the Methanolex648 289

tract is passed through a column packed with 100 ml Sephadex® LH-20 (20 mm inner diameter) and developed with methanol. Fractions No. 30 to 42 (1 ml volume each) are combined and evaporated to dryness. 38.4 mg (35% yield) of a white powder of N- [4- (3-aminopropyl) aminobutyl] -2 - [(S) -7-guanidino-3-hydroxyheptanamido] -2-hydroxyethane amide (GHA -GS) trihydrochloride. The GHA-GS trihydrochloride obtained in this way is a hygroscopic powder with a melting point which cannot be determined exactly. It shows an optical rotation of [a] 22D = - 1 ° + 2 ° (C 2, water). The elementary analysis agrees with that theoretically calculated for C17H37N704 • 3HC1 (C 39.81%, H 7.86%, N 9.12%, Cl 20.73%). Proton NMR, determined in heavy water, shows characteristic signals at 5 = 1.8-2.3 (CH2 x 5), 2.57 (6 "-CH2), 2.95 (2-CH2), 3, 5-3.8 (NCH2 x 5), 4.55 (3-CH) and 5.98 (2'-CH).

The (S) -7-guanidino-3-hydroxyheptanamide used in the reference example is synthesized as follows.

Synthesis of (S) -7-guanidino-3-hydroxyheptanamide (a) Synthesis of (S) -3,7-diaminoheptanoic acid

15 g (82.15 mmol) of L-lysine hydrochloride are dissolved in 150 ml of water and 8.7 g (82.15 mmol) of sodium carbonate and 43.2 g (200 mmol) of N-ethoxycarbonylphthalimide are added. The mixture is stirred at room temperature for 20 hours and then the reaction mixture is washed with 50 ml of ethyl acetate. The aqueous layer is adjusted to a pH of 3.0 with 6N hydrochloric acid and extracted three times with 100 ml of toluene. The extract is washed twice with 100 ml of water which had been adjusted to pH 2.0, dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. 27.95 g (84% yield) of a white powder of di-N-phthaloyl-L-lysine are obtained; Decomposition point 71 to 72 ° C, fa] 22D = -32 ° (C 1, methanol).

40 ml of oxalyl chloride are added to 27.0 g (66.4 mmol) of di-N-phthaloyl-L-lysine. The mixture, heated on an oil bath at 90 ° C., is mixed with 40 ml of 1,2-dimethoxyethane. The mixture is refluxed for a further 2 h. The reaction mixture is evaporated to dryness, redissolved in 20 ml of 1,2-dimethoxyethane, and 500 ml of an ethereal solution of diazo-methane (330 mmol) are added dropwise while cooling with ice. The mixture is stirred for another hour. The reaction mixture is evaporated to dryness, dissolved in 250 ml of anhydrous methanol, mixed with a solution of 3.4 g (14.8 mmol) of silver benzoate in 50 ml of triethylamine and stirred at room temperature for 15 h. The precipitate is collected by filtration, dissolved in 100 ml of chloroform, insoluble materials are removed by filtration and evaporated to dryness. 15.3 g (53% yield) of methyl (S) -3,7-diphthaloylami-noheptanoate are obtained; Decomposition point 118 to 119 ° C,

[a] 22D = -3 ° (C 2, chloroform).

100 ml of 1 M alcoholic hydrazine hydrate and 100 ml of 95% ethanol are added to 15.0 g (34.5 mmol) of methyl (S) -3,7-diphthaloylamino heptanoate. The mixture is heated under reflux for 1 h (at an oil bath temperature of 90 ° C). The reaction mixture is evaporated to dryness, dissolved in 250 ml of 5% hydrochloric acid, heated at 80 ° C. for 1 h, adjusted to pH 7.1 by adding 17% aqueous ammonia and washed with 300 ml of Amberlite® CG-50 ( 70% NH4 type) packed column (27 mm inner diameter). The column is washed with 900 ml of water and 0.2 M aqueous ammonia and eluted with 0.5 M aqueous ammonia. The fractions positive compared to the Ninhy-drin test are collected and evaporated to dryness. 3.15 g (57% yield) are obtained.

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648289

(S (-3,7-diaminoheptanoic acid (C7Hl6N202 • 1 / 4H2C03);

[a] 21D = + 29 ° (C I, water).

(b) Synthesis of (S) -7-guanidino-3-hydroxyheptanamide

In 30 ml of a mixture of pyridine-water-triethyl

Amine (10: 10: 1, expressed by volume) is dissolved in 3.1 g (19.3 mmol) of (S) -3,7-diaminoheptanoic acid, obtained in step (a) above. 4.81 g (19.3 mmol) of N-benzyloxycarbonyloxysuccinimide are gradually added to the solution formed. The mixture is stirred for 5 hours at room temperature. The reaction mixture is evaporated to dryness, dissolved in 30 ml of water, adjusted to pH 6.4 with 6N hydrochloric acid, passed through a column (16 mm inner diameter) packed with 100 ml of Amberlite® CG-50 (80% NH4 type) and with Developed 300 ml of water. The effluent is collected and passed through a column (16 mm inner diameter) packed with 100 ml of Dowex® 50 W-X4 (H type). The column is washed with 300 ml of water and 0.2 M aqueous ammonia and eluted with 0.5 M aqueous ammonia (10 ml fractions). Fractions No. 16 to 33 are combined and evaporated to dryness. 2.73 g (48% yield) of a white powder of (S) -3-amino-7-benzyloxycarbonylamino-heptanoic acid '(C15H22N204 • H20) are obtained; Decomposition point 143 to 147 ° C, [a] 22o = 4-14 ° (C 1, methanol). The above Amberlite, r CG-50 column is eluted with 0.5 M aqueous ammonia. 746 mg (24% yield) of (S) -3,7-di-aminoheptanoic acid are obtained.

2.7 g (9.17 mmol) of (S) -3-amino-7-benzyloxycarbonylaminoheptanoic acid are dissolved in 50 ml of a 33% aqueous acetic acid solution. While cooling with ice, the solution is slowly added with a solution of 1.9 g (27.51 mmol) of sodium nitrite in 10 ml of water over the course of 1 h. The mixture is stirred for 1 and left to stand at 5 ° C. for 24 h. The reaction mixture is diluted with 50 ml of water and extracted twice with 50 ml of ethyl acetate. The extract is extracted over anhydrous sodium sulfate and evaporated to dryness. 2.16 g of a crude powder are obtained. The crude powder is subjected to column chromatography, whereby a column packed with 200 g silica gel (Wako-Gel® C-200) (28 mm inner diameter) and a mixture of chloroform, methanol and concentrated ammonia (30: 10: 1, expressed by the volume) is used as the developing solvent. Fractions Nos. 51 to 60 (each 20 ml volume) are combined and evaporated to dryness. 460 mg (17% yield) of a white powder of (S) -7-benzyloxycarbonylamino-3-hydroxy-heptanoic acid are obtained; Decomposition point 115 to 117 ° C,

[a] 23D = + 3 ° (C 2, methanol).

450 mg (1.52 mmol) of (S) -7-benzyloxycarbonylamino-3-hydroxyheptanoic acid are dissolved in 4 ml of 1,2-dimethoxyethane. While cooling with ice, 7 ml (4.56 mmol) of an ether solution of diazomethane is added dropwise to the solution. The mixture is stirred for 30 minutes and then evaporated to dryness. 461 mg (98% yield) of methyl (S) -7-benzyloxycarbonylamino-3-hydroxyheptanoate are obtained; [a] 21D = + 1 ° (C 5, methanol).

450 mg (1.45 mmol) of methyl (S) -7-benzyloxycarbonylamino-3-hydroxyheptanoate are dissolved in 50 ml of anhydrous methanol. The solution, cooled at -10 ° C, is saturated with gaseous ammonia and left to stand in a smelting tube at room temperature for 3 days. The reaction mixture is evaporated to dryness and subjected to chromatography, using a column packed with 50 g of silica gel (Wako-Gel "C-200) (20 mm inner diameter) and a mixture of chloroform and methanol (100: 1, expressed by the Fractions Nos. 82 to 106 (10 ml volume each) are combined and evaporated to dryness to give 371 mg (87% yield) of a white powder of (S) -7-benzyloxycarbonylamino-3-hydroxyheptanamide ; Decomposition point 126 to 127 ° C, [a] 22D = - 3 ° (C 5, methanol).

350 mg (1.19 mmol) of (S) -7-benzyloxycarbonylamino-3-hydroxyheptanamide are dissolved in a mixture of 10 ml of 90% aqueous methanol and 0.01 ml of acetic acid.

After adding 50 mg of 5% palladium-on-carbon, the mixture is stirred for 3 hours at room temperature under a stream of hydrogen. After filtering off the catalyst, the filtrate is evaporated to dryness, redissolved in a small volume of water and passed through a column (12 mm internal diameter) packed with 30 ml Dowex® 50W-X4 (H type). The column is then washed with 90 ml of water and eluted with 0.5 M aqueous ammonia. Fractions No. 28 to 34 (each 3 ml volume) are combined and evaporated to dryness. 201 mg (96% yield) of (S) -7-amino-3-hydroxyheptane amide are obtained; [a] 22D = -2 ° (C2, water).

190 mg (1.08 mmol (S) -7-amino-3-hydroxyheptanamide) is dissolved in 3 ml of water and 0.54 ml of 2N aqueous sodium hydroxide solution is added, and I ml is added dropwise to the solution over 30 minutes while cooling with ice a methanol solution containing 129 mg (1.08 mmol) of 2-methyl-1-nitrosourea. The mixture is stirred for a further 5 h. The reaction mixture is adjusted to a pH of 6.0 with 6N hydrochloric acid, then evaporated to dryness and purified by chromatography, a column packed with 30 g of silica gel (Wako-Gel® C-200) (15 mm inner diameter) and a mixture of chloroform, methanol and concentrated aqueous ammonia (60: 10: 1, expressed as fractions No. 67 to 90 (each 6 ml volume) are combined and evaporated to dryness to give 187 mg (70% yield) of a white powder of (S) -7-nitroguanidino-3-hydroxyheptanamide; Decomposition point 148 to 149 ° C, [a] 22D = -2 ° (C2, meth anol).

170 mg (0.69 mmol) of (S) -7-nitroguanidino-3-hydroxyheptanamide are dissolved in a mixture of 15 ml of water, 15 ml of methanol and 7.5 ml of acetic acid. After adding 50 mg of 5% palladium-on-carbon, the mixture is stirred for 1 hour at room temperature under a stream of hydrogen. After filtering off the catalyst, the filtrate is evaporated to dryness. 165 mg of raw powder are obtained. This powder is dissolved in 10 ml of water, passed through a column (12 mm inner diameter) packed with 20 ml of CM-Sephadex® C-25 (Na type) and eluted with 0.5 M sodium chloride solution. Fractions No. 18 to 25 (each 2 ml volume) are combined and evaporated to dryness. The dried substance is extracted three times with 10 ml of methanol. The methanol extracts are combined, passed through a column packed with 100 ml Sephadex® LH-20 (20 mm inner diameter) and developed with methanol. Fractions Nos. 28 to 46 (1 ml volume each) are combined and evaporated to dryness. 149 mg (91% yield) of a white powder of (S) -7-guanidino-3-hydroxyheptanamide hydrochloride (C8H18N402 • HCl) are obtained; [a] 22D = -2 ° (C 2, water).

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Claims (5)

648 289 2. Compound according to claim 1, characterized in that the acid addition salt is the hydrochloride. 2nd PATENT CLAIMS 1. N- [4- (3-aminopropyl) aminobutyl] -2,2-dihydroxyethanolamide of the formula (I) ho ho chconh (ch2) 4nh (ch2) 3nh2 (i) and its acid addition salts. 3. Process for the preparation of N- [4- (3-aminopropyl) aminobutyl] -2,2-dihydroxyethanolamide of the formula (I) ho. ho ' chconh (ch2) 4nh (ch2) 3nh2 (I) 20th characterized in that a compound of formula (II) H2N (CH,) 4N (CH2) 3NHR I. R (II) wherein R represents an amino protecting group with a di-alkylacetal of glyoxylic acid of formula (III) r'o 10th 15 r'o \ / chcooh (iii) wherein R 'is an alkyl group having 1 to 5 carbon atoms, in the presence of an activating agent or with a reactive derivative of the carboxyl group of the acetal and then the amino protective group and R' removed. 4. The method according to claim 3, characterized in that an acyl halide-forming reagent, an acid azide-forming reagent, a reactive ester-forming reagent or an acid anhydride-forming reagent is used as the activating agent. 5. The method according to claim 3, characterized in that the reaction is carried out in an organic solvent at 0 to 100 ° C for 5 to 30 h.