GB2100253A - N-[4-(3- aminopropyl)aminobutyl]-2,2- dihydroxyethanamide - Google Patents

Abstract

N-[4-(3- aminopropyl)aminobutyl]-2,2- dihydroxyethanamide of the formula <IMAGE> This compound is useful not only as an intermediate for the synthesis of a carcinostatic substance N[4-(3- aminopropyl)aminobutyl]-2-[(S)-7- guanidino-3-hydroxyheptanamido]-2- hydroxyethanamide or a derivative thereof, but also as an immunostimulant by itself.

Description

SPECIFICATION N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide and synthesis thereof This invention relates to N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide of the formula (I)

and acid addition salts thereof and to a process for their syhnthesis.

This novel compound is useful not only as an intermediate for the synthesis of a novel carcinostatic substance N-[4-(3-aminopropyl)aminobutyl]-2-[(S)-7-guanidino-3-hydroxyheptanamido]- 2-hydroxyethanamide or a derivative thereof, but also as an immunostimulant by itself.

In view of its stability, the novel compound of the present invention is prepared preferably in the form of acid addition salt. The acids to be added include 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, particularly preferred are hydrochloric acid, sulfuric acid and tartaric acid.

The physicochemical and biological properties of the present compound are shown below: (1) Physicochemical properties.

The hydrochloride of the present compound is a white powder which shows no distinct melting point. The elementary analysis coincides with the theoretical calculated for C5H21N3O3.2HCl (C 36.99%, H 7.93%, N 14.38%, Cl 24.27%). The proton NMR, as measured in heavy water, shows characteristic signals at ô = 2.1(2-, 3-CH3), 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 present compound (hydrochloride) on delayed hypersensitivity to sheep red blood corpuscle is as shown in Table 1. The test was performed according to the method of Mackaness et al.

[Lagrange, P. H., Mackaness, G.B. and Miller. T. E., J. Exp. Med., 139, 528-542 (1974)] in the following manner: Female CDF, mice, 6 weeks of age, were immunized by the intravenous injection of each 105 sheep red blood corpuscles and, at the same time, administered with varied amounts of the compound of formula (I) (hydrochloride) by peritoneal injection. After 4 days, each mouse was administered with 108 sheep red blood corpuscles by subcutaneous injection to induce the reaction. After 24 hours, the swelling of footpad of each mouse was measured by means of slide calipers.

TABLE 1

Compound of Increase of formula (I) footpad thickness Immunization (yg/mouse, i.p.) (x 0.1 mm + S.D.) T/C (%) 105SRBC none 8.0 + 0.7 100 lO5SRBC 1 13.0 + 1.7 163 " 4 13.0+0.5 163 16 13.4+0.6 168 63 14.9+0.9 186 " 250 11.8 + 0.6 148 1,000 11.7 + 1.0 146 As seen from Table 1, the reaction was enhanced by the administration of 1 g to 1 mg of the compound of formula (I).

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

is synthesized in the following manner: A compound represented by the formula (II)

(wherein R represents an amino-protective group such as benzyloxycarbonyl, chloroacetyl or a lower alkyloxycarbonyl group) is allowed to condense with a dialkylacetal of glyoxylic acid represented by the formula (III)

(wherein R' represents an alkyl group of 1 to 5 carbon atoms) or a reactive derivative of the carboxyl group of said dialkylacetal, and thereafter the amino-protective group and the group R' are removed to yield the compound of formula (I).

The condensation ofthe compound of formula (II) with the compound of formula (III) or its reactive derivative is effected by the method for forming an ordinary amide linkage in the peptide synthesis by utilizing, for example, an acyl halide, acid azide, reactive ester, or acid anhydride.Thus, the condensation is conducted in the presence of an activating agent for the carboxyl group of the compound of formula (III). Examples of the suitable reagents used for the activation of carboxyl group or the formation of a reactive derivative include reagents for forming active esters such as 6-chloro-1 -p-chlorobenzenesulfonyloxybenzotriazole (CCBT), N-ethyl-5-phenylisooxazolium-3'-sulfonate (NEPIS), N-tert-butyl-5 methylisooxazoli um perchlorate, N-ethoxycarbonyl-2-ethoxy- 1 ,2-dihydroquinoline, di-p-nitrophenyl sulfite, tri-p-nitrophenyl phosphite, p-nitrophenyl trichloroacetate, N-hydroxysuccinimide, p-nitrophenol, pentachlorophenol, and benzyl alcohol; carbodiimides such as dicyclohexylcarbodiimide (DDC), 1-ethyl- 3-(3-dimethyla minopropyl)carbodiimide, 1 -cyclohexyl-3-(2-morpholinoethyl)carbodiimide, di-p-toluylcarbodiimide, and diisopropylcarbodiimide; and reagents for azide synthesis.

The condensation reaction is carried out preferably in an organic solvent such as ethyl acetate and at a temperature of generally 0 to 1000C, preferably 100 to 4000. Although the reaction time varies with the reaction temperature, it is about 5 to about 30 hours at room temperature. The amount used of the carboxylic acid of formula (Ill) or a reactive derivative thereof is 0.5 to 10 moles, preferably 2 to 5 moles per mole of the amine of formula (II).

The removal of protective groups from the condensate formed as described above can be performed by hydrolysis or reduction according to the type of protective group, but those protective groups which are removable by acid hydrolysis are preferable. For instance, when the amino-protective group is tertbutoxycarbonyl group and the aldehyde-protective group is diethylacetal, the hydrolysis is conducted in an aqueous dioxane solution by adding 2 to 3 equivalents of dilute hydrochloric acid and heating at 10000 for 2 to 5 hours to obtain hydrochloride of the present compound of formula (I). When the amino-protective group is benzyloxycarbonyl group, the removal of the protective group is effected preferably by hydrogenolysis using palladium, platinum oxide, or the like.

The starting compound of the formula (II)

(where R is an amino-protective group) is difficult to obtain in a good yield by introducing selective two amino-protective group into spermidine. Accordingly, it is prepared by condensing it in a customary manner a monoamino-protected 1,4-butanediamine of the formula (IV) R"HN(CH2)4NH2 (lav) (where R" is an amino-protective group different from the above-noted R) with an amino-protected 3halopropanamine of the formula (V) X(CH2)2NHR (V) (where R is the same amino-protective group as noted above and X is a halogen atom) to form a compound of the formula (VI) R"HN(CH2)4NH(CH2)3NHR (Vl) (where R and R" are amino-protective groups different from each other), then protecting the remaining imino group with the same amino-protective group as R, and selectively removing other aminoprotective group R" to obtain the compound of formula (II).

Alternatively, the compound of the formula (VI) is obtained by condensing monoamino-protected 1,3-propanediamine of the formula (VII) RHN(CH2)2NH2 (ill) (wherein R is as defined above) with an amino-protected 4-halobutanamine of the formula (VIII) X(CH2)4NHR" (veil) (wherein R" and X are as defined above) in a manner as described above.

For the protection of amino groups, those amino-protective groups which are customarily used in the peptide synthesis can be utilized, but the amino-protective group of R" should be selectively removable, leaving behind the amino-protective group of R. Accordingly, a combination of benzyloxycarbonyl group removable by hydrogenolysis and a tert-butoxycarbonyl group removable by the weak acid treatment is a most preferable example. Either one of this pair may 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 conducted in an anhydrous solvent such as N,N-dimethylformamide at room temperatrue in the presence of triethylamine. The halogen in the compound of formula (VIII) is preferably bromine. The dialkylacetal of glyoxylic acid of formula (III) is easily formed by reacting glyoxylic acid with an alkanol using an acid catalyst in a customary manner. Suitable alkanols for use are methanol, ethanol, propanol, butanol and amyl alcohol. The dialkylacetal of formula (III) is also conveniently obtained by the alkaline hydrolysis of commercial ethyl 2,2-diethoxyacetate.

Starting from the present compound of formula (I), a useful carcinostatic substance N-[4-(3 aminopropyl)aminobutyl]-2-[(S)-7-guanidino-3-hydroxyheptanamido]-2-hydroxyethanamide (briefly GHA-GS) is synthesized by the condensation of the present compound of the formula (I) with (S)-7guanidino-3-hydroxyheptanamide of the formula

by heating both compounds in the presence of an inorganic or organic acid. As shown below, GHA-GS exhibits a carcinostatic activity against mouse ieukemia L1 210 and is useful as a carcinostatic.

Carcinostatic effect of GHA-GS on mouse leukemia L1210: A group of five male BDF1 strain mice (6 weeks of age) was inoculated intraperitoneally with 1 05 L1 210 cells, and immediately thereafter each mouse was administered intraperitoneally with a physiological saline solution of the sampie once a day for 6 consecutive days to determine the prolongation rate of survival period [(T/C) x 1 00;; T/C = (mean survival time of treated group)/(mean survival time of untreated group)].

prolongation rate of survival Number of Dose period mice survived Sample (mg/kg/day) (T/C x 100 (%) for 30 days 1.56 115 0 3.13 > 254 1 6.25 > 341 3 GHA#3S 12.5 > 405 4 25 > 351 2 50 > 365 2

The invention is illustrated below with reference to Example but the invention is not limited thereto.

EXAMPLE a) Synthesis of mono-N-benzyloxycarbonyl- 1,4-butanediamine: Into 30 ml of 50% aqueous methanol, was dissolved 1.76 g (20 mmoles) of 1,4-butanediamine followed by the addition of 5.48 g (20 mmoles) of S-benzyloxycarbonyl-4,6-dimethyl-2mercaptopyrimidine (a product of Kokusan Kagaku Co.). The mixture was stirred for 3 hours. Thereafter, the reaction mixture was filtered to remove the precipitate [2.08 g (29%) of di-N-benzyloxycarbonyl compound was recovered], and the filtrate was evaporated to dryness. The residue was dissolved in 250 ml of chloroform, washed 5 times with 100 ml of water. The chloroform layer was dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 1.0 g (23% yield) of mono-N benzyloxycarbonyl-1,4-butanediamine in colorless sirup form.

b) Synthesis of O-tosyl-3-tert-butoxycarbonylamino-1-propanol: Into 30 ml of methanol, was dissolved 1.5 g (20 mmoles) of 3-amino-1-propanol followed by the addition of 4.8 g (20 mmoles) of S-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine (a product of Kokusan Kagaku Co.). The mixture was stirred for 6 hours. The reaction mixture was evaporated to dryness, dissolved in 200 ml of chloroform, and washed with 200 ml of water. The chloroform layer was concentrated and subjected to column chromatography using 300 g of silica gel (Wako-Gel C--200) and a toluene-ethyl acetate (1 :1 by volume) mixture as developing solvent.Fraction Nos. 82 to 151 (each 1 5 ml in volume) were combined, evaporated to dryness to obtain 2.95 g (84% yield) of tertbutoxycarbonylamino-1 -propanol in colorless oily form.

Into 50 ml of pyridine, was dissolved 2.95 g (16.9 mmoles) of 3-tert-butoxycarbonylamino-1- propanol. To the solution, while being cooled in ice under an argon atmosphere, was added dropwise over a period of 40 minutes a solution of 3.36 g (17.7 mmoles) of p-toluenesulfonyl chloride in pyridine.

The mixture was left standing overnight at 70C, then admixed with a small volume of water, and evaporated to dryness. The residue was dissolved in 200 ml of chloroform, washed successively with 5% aqueous potassium hydrogensulfate solution, saturated aqueous sodium hydrogencarbonate solution, and water, then dried over anhydrous sodium sulfate, evaporated to dryness, and subjected to column chromatography using 120 g of silica gel (Wako-Gele C--200) and a toluene-ethyl acetate (8:1 by volume) mixture as developing solvent. Fraction Nos. 35 to 68 (each 15 ml in volume) were combined and evaporated to dryness to yield 3.06 g (55 /6 yield) of 0-tosyl-3-tert-butoxycarbonylamino-1 -propanol in colorless oily form.

c) Synthesis of N-tert-butoxycarbonyl-N'-(tertbutoxycarbonylaminopropyl)-1 ,4-butanediamine: Into 15 ml of dimethylformamide, was dissolved 800 mg (2.43 mmoles) of 0-tosyl-3-tertbutoxycarbonylamino-1 -propanol obtained in b) above. After addition of 510 mg (4.8 mmoles) of lithium bromide (LiBr.H20), the solution was stirred at room temperature for 24 hours. To the reaction mixture containing the bromo derivative, were added 540 mg (2.43 mmoles) of mono-N benzyloxycarbonyl-1 ,4-butanediamine obtained in a) above and 0.34 ml of triethylamine. The mixture was stirred at room temperature for 48 hours. The reaction mixture was admixed with 699 mg (2.9 mmoles) of S-tert-butoxycarbonyl-4,6-di methyl-2-mercaptopyrimidine and stirred at room temperature for 13 hours. The reaction mixture was 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 was dissolved in minimum volume of a toluene-ethyl acetate (4:1 by volume) mixture and subjected to column chromatography using 200 g of silica gel (Wako-Gels C--200) and a toluene-ethyl acetate (4:1 by volume) mixture as developing solvent. Fraction Nos. 134 to 1 65 (each 12 ml in volume) were combined and evaporated to dryness to obtain 608 mg (52% yield) of N benzyioxycarbonylN'-tert-butoxycarbonyl-N'-(tert-butoxycarbonylaminoprnpyl)-l ,4-butanediamine in colorless sirup form.

Into 5 ml of methanol, was dissolved 144 mg (0.3 mmole) of the above compound in colorless sirup form. After addition of 100 mg of 5% palladium-barium carbonate, the mixture was stirred at room temperature for 5 hours under a hydrogen stream, freed from the catalyst by filtration, and evaporated to dryness to yield 103 mg (100% yield) of Ntertbutoxycarbonyl-N-(tert-butoxycarbonylaminoprnpyI)- 1 ,4-butanediamine.

d) Synthesis of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxybutanamide: Into 2 ml of ethyl acetate, were dissolved 100 mg (0.29 mmole) of N-tert-butoxycarbonyl-N-(tertbutoxycarbonylaminopropyl)-1,4-butanediamine obtained inc) above and 148 mg (1 mmole) of 2,2diethoxyacetic acid. After addition of 135 mg (1 mmole) of 1 -hydroxybenzotriazole and 206 mg (1 mmole) of dicyclohexylcarbodiimide, the mixture was stirred at room temperature for 1 5 hours. The reaction mixture was filtered to remove the precipitate and the precipitate was washed with cold ethyl acetate. The filtrate and the washings were combined and washed successively with 1 M aqueous ammonia and water. The ethyl acetate layer was dried over anhydrous sodium sulfate, and evaporated to dryness.The residue was subjected to column chromatography using 20 g of silica gel (Wako-Gel C-200) and a toluene-ethyl acetate (1:2 by volume) mixture as developing solvent. The fraction Nos.

14 to 21 (each 3 ml in volume) were combined and evaporated to dryness to yield 109 mg (79% yield) of N-[4-(3-tert-butoxywarbonyla mi nopropyl)-4-tert-butoxycarbonylaminobutyl]-2,2-diethoxy- ethanamide as a colorless sirupy substance.

Into 1 ml of dioxane, was dissolved 44 mg (0.13 mmole) of the above sirupy substance. After addition of 2.5 ml of 0.1N hydrochloric acid, the mixture was stirred for 4 hours in an oil bath heated at 100 C. The reaction mixture was adjusted to pH 6 with 0.2N aqueous sodium hydroxide solution and evaporated to dryness. The residue was extracted with 1.5 ml of methanol. The extract was passed through a column (16.5 mm in inner diameter) packed with 100 ml of Sephadexs LH-20 and developed with methanol. Fraction Nos. 22 to 25 (each 2 ml in volume) positive to ninhydrin test were combined and evaporated to dryness to obtain 13 mg (46% yield) of N-[4-(3-aminopropyl)aminobutyl]2,2-dihydroxy ethanamide dihydrochloride in colorless sirup form.

REFERENCE EXAMPLE Synthesis of GHA-GS.

A mixture of 51 mg (0.214 mmole) of (S)-7-guanidino-3-hydroxyheptanamide hydrochloride, 112 mg (0.385 mmole) of N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide dihydrochloride, 70 mg (0.53 mmole) of glutaric acid, and 0.07 ml (3.9 mmoles) of water was stirred for 43 hours at 600C.

The reaction mixture was admixed with 20 ml of 0.4M sodium chloride solution, adjusted to pH 6.1 with 10% aqueous ammonia, and passed through a column (12 mm in inner diameter) packed with 20 ml of CM Sephadexe C--25 equilibrated with 0.4M sodium chloride solution. The column was then subjected to gradient elution using each 80 ml of 0.4M and 1.OM sodium chloride solutions. The fraction Nos. 41 to 50 (each 2 ml in volume) were combined, evaporated to dryness, and extracted three times with 10 ml of methanol. The methanol extract was passed through a column (20 mm in inner diameter) packed with 100 ml of Sephadexe LH-20, and developed with methanol.The fraction Nos. 30 to 42 (each 1 ml in volume) were combined and evaporated to dryness to obtain 38.4 mg (35% yield) of a white powder of N-[4-(3-am inopropyl)a minobutyl]-2-[(S)-7-guanidino-3-hydroxyheptana mido] -2-hydroxyethanamide (GHA-GS) trihydrochloride. The GHA-GS trihydrochloride thus obtained was a hygroscopic powder having no distinctly determinable melting point. It showed an optical rotation of [a]22 = --10 + 20 (C 2, water).The elementary analysis coincided with the theoretical calculated for C17H37NO.3HOl (C 39.81%, H 7.86%, N 9.12%, Cl 20.73%). The proton NMR, as measured in heavy water, showed characteristic signals at S = 1.8-2.3 (CH2 x 5), 2.57 (6"-0H2), 2.95 (2-OH2), 3.5-3.8 (NCH2 x 5), 4.55 (3-OH) and 5.98 (2'-OH).

(S)-7-Guanidino-3-hydroxyheptanamide used in the Reference Example was synthesized in the following way: Synthesis of (S)-7-guanidino-3-hydroxyheptanamide a) Synthesis of (S)-3,7-diaminoheptanoic acid: Into 1 50 ml of water, was dissolved 1 5 g (82.1 5 mmoles) of L-lysine hydrochloride followed by 8.7 g (82.1 5 mmoles) of sodium carbonate at 43.2 g (200 mmoles) of N-ethoxycarbonylphthalimide.

The mixture was stirred for 20 hours at room temperature and the reaction mixture was washed with 50 ml of ethyl acetate. The aqueous layer was adjusted to pH 3.0 with 6N hydrochloric acid, and extracted three times with 100 ml of toluene. The extract was 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 to obtain 27.95 g (84% yield) of a white powder of di-N-phthaloyl-L-lysine. Decomp. point, 710--720C; [aC]D232 (C 1, methanol).

To 27.0 g (66.4 mmoles) of di-N-phthaloyl-L-lysine, was added 40 ml of oxalyl chloride. To the mixture heated in an oil bath at 900C, was added 40 ml of 1 ,2-dimethoxyethane. The mixture was further heated under reflux for 2 hours. The reaction mixture was evaporated to dryness, dissolved again in 20 ml of 1 ,2-dimethoxyethane and added dropwise to 500 ml of an ether solution of diazomethane (330 mmoles) while cooling in ice. The mixture was further stirred for one hour. The reaction mixture was evaporated to dryness, dissolved in 250 ml of an hydros methanol, admixed with a solution of 3.4 g (14.8 mmoles) of silver benzoate in 50 ml of triethylamine, and stirred for 15 hours at room temperature.The precipitate was collected by filtration, dissolved in 100 ml of chloroform, freed from the insolubles by filtration, and evaporated to dryness to obtain 15.3 g (53 /0 yield) of methyl (S)-3,7 diphthaloylaminoheptanoate. Decomp. point, 1 180--1 190C; [a]22 --30, (C 2, chloroform).

To 15.0 g (34.5 mmoles) of methyl (S)-3,7-diphthaloylaminoheptanoate, were added 100 ml of a tM alcoholic hydrazine hydrate and 100 ml of 95% ethanol. The mixture was heated under reflux for one hour (at an oil bath temperature of 90 C). The reaction mixture was evaporated to dryness, dissolved in 250 ml of 5% hydrochloric acid, heated at 800C for one hour, adjusted to pH 7.1 with 17--0/0 aqueous ammonia, and passed through a column (27 mm in inner diameter) packed with 300 ml of Amberlites CG--50 (70% NH4-type). The column was washed with each 900 ml of water and 0.2M aqueous ammonia, and eluted with 0.5M aqueous ammonia.The fractions positive to ninhydrine test were collected and evaporated to dryness to obtain 3.15 g (57% yield) of (S)-3,7-diaminoheptanoic acid (C7H16N202-1/4H2CO3). [a]D21 +290(01, water) b) Synthesis of (S)-7-guanidino-3-hydroxyheptanamide.

Into 30 ml of a pyridine-water-triethylamine (10:10:1 by volume) mixture, was dissolved 3.1 g (19.3 mmoles) of (S)-3,7-diaminoheptanoic acid obtained in a) above. To the resulting solution, was added gradually 4.81 g (19.3 mmoles) of N-benzyloxycarbonyloxysuccinimide. The mixture was stirred at room temperature for 5 hours. The reaction mixture was evaporated to dryness, dissolved in 30 ml of water, adjusted to pH 6.4 with 6N hydrochloric acid, then passed through a column (16 mm in inner diameter) packed with 100 ml of Amberlites CG--50 (80% NH4-type), and developed with 300 ml of water. The effluent was collected and passes through a column (16 mm in inner diameter) packed with 100 ml of Dowexe 50 W-X4 (H-type).The column was washed with each 300 ml of water and 0.2M aqueous ammonia, and eluted with 0.5M aqueous ammonia (10 ml fractions). The fraction Nos. 16 to 33 were combined and evaporated to dryness to obtain 2.73 g (48% yield) of a white powder of (S)-3amino-7-benzyloxycarbonylaminoheptanoic acid (C1sH22N204-H20). Decomp. point 1430--1470C; [MD22 + 140 (C 1, methanol). The above-said column of Amberlite CG--50 was eluted with 0.5M aqueous ammonia to recover 746 mg (24% recovery) of (S)-3,7-diaminoheptanoic acid.

Into 50 ml of 33% aqueous acetic acid solution, was dissolved 2.7 g (9.17 mmoles) of (S)-3amino-7-benzyloxycarbonylaminoheptanoic acid. To the solution, while being cooled in ice, was added slowly over a period of one hour a solution of 1.9 g (27.51 mmoles) of sodium nitrite in 10 ml of water.

The mixture was stirred for one hour and left standing for 24 hours at 50C. The reaction mixture was diluted with 50 ml of water and extracted twice with 50 ml of ethyl acetate. The extract was dried with anhydrous sodium sulfate and evaporated to dryness to obtain 2.16 g of a crude powder. The crude powder was subjected to column chromatography using a column (28 mm in inner diameter) packed with 200 g of silica gel (Wako-Gels C--200) and a chloroform-methanol-concentrated ammonia (30:10:1 by volume) mixture as developing solvent. Fraction Nos. 51 to 60 (each 20 ml in volume) were combined and evaporated to dryness to obtain 460 mg (17% yield) of a white powder of (S)-7benzyloxycarbonylamino-3-hydroxyheptanoic acid.Decomposition point, 1150--1170C; [MD23 +30 (C 2, methanol).

Into 4 ml of 1 ,2-dimethoxyethane, was dissolved 450 mg (1.52 mmoles) of (S)-7benzyloxycarbonylamino-3-hydroxyheptanoic acid. To the solution, while being cooled in ice, was added dropwise 7 ml (4.56 mmoles) of an ether solution of diazomethane. The mixture was stirred for 30 minutes and then evaporated to dryness to yield 461 mg (98% yield) of methyl (S)-7benzyloxycarbonylamino-3-hydroxyheptanoate. [MD21 +10 jC 5, methanol).

Into 50 ml of anhydrous methanol, was dissolved 450 mg (1.45 mmoles) of methyl (S)-7 benzyloxycarbonylamino-3-hydroxyheptanoate. The solution, cooled art 1000, was saturated with gaseous ammonia and left standing in a sealed tube for 3 days at room temperature. The reaction mixture was evaporated to dryness and subjected to chromatography using a column (20 mm in inner diameter) packed with 50 g of silica gel (Wako-Gel6 C--200) and a chloroform-methanol (100:1 by volume) mixture as developing solvent. Fraction Nos. 82 to 106 (each 10 ml in volume) were combined and evaporated to dryness to yield 371 mg (87% yield) of a white powder of (S)-7-benzyloxycarbonylamino-3-hydroxyheptanamide.Decomp. point, 1 2601 2700; [MD22 30 (C 5, methanol).

Into a mixture of 10 ml of 90% aqueous methanol and 0.01 ml of acetic acid, was dissolved 350 mg (1.19 mmoles) of (S)-7-benzyloxycarbonylamino-3-hydroxyheptanamide. After addition of 50 mg of 5% palladium-carbon, the mixture was stirred under a hydrogen stream for 3 hours at room temperature. After removal of the catalyst by filtration, the filtrate was evaporated to dryness, then dissolved again in a small volume of water, and passed through a column (12 mm in inner diameter) packed with 30 ml of Dowex 50W-X4 (H-type). The column was then washed with 90 ml of water and eluted with 0.5M aqueous ammonia. Fraction Nos. 28 to 34 (each 3 ml in voiume) were combined and evaporated to dryness to obtain 201 mg (96% yield) of (S)-7-amino-3-hydroxyheptanamide. [MD22 20 (C 2, water).

Into 3 ml of water, was dissolved 190 mg (1.08 mmoles) of (S)-7-amino-3-hydroxyheptanamide followed by 0.54 mi of 2N aqueous sodium hydroxide solution. To the solution, while being cooled in ice, was added dropwise over a period of 30 minutes 1 ml of a methanol solution containing 129 mg (1.08 mmoles) of 2methyl1 -nitrosourea. The mixture was further stirred for 5 hours. The reaction mixture was adjusted to pH 6.0 with 6N hydrochloric acid, then evaporated to dryness, and purified by chromatography using a column (15 mm in inner diameter) packed with 30 g of silica gel (Wako-Gele C--200) and a mixture of chloroform-methanol-concentrated aqueous ammonia (60:10:1 by volume) as developing solvent.Fraction Nos. 67 to 90 (each 6 ml in volume) were combined and evaporated to dryness to obtain 1 87 mg (70% yield) of a white powder of (S)-7-nitroguanidino-3hydroxyheptanamide. Decomp. point, 148a1490O; [MD22 0 (C 2, methanol).

Into a mixture of 15 ml of water, 15 ml of methanol and 7.5 ml of acetic acid, was dissolved 1 70 mg (0.69 mmole) of (S)-7-nitroguanididino-3-hydroxyheptanamide. After addition of 50 mg of 5% palladium-carbon, the mixture was stirred under a hydrogen stream for one hour at room temperature.

After removal of the catalyst by filtration, the filtrate was evaporated to dryness to obtain 1 65 mg of a crude powder. This powder was dissolved in 10 ml of water, passed through a column (12 mm in inner diameter) packed with 20 ml of CM-Sephadex6 C--2 5 (Na-type), and eluted with 0.5M sodium chloride solution. Fraction Nows. 18 to 25 (each 2 ml in volume) were combined and evaporated to dryness. The dried substance was extracted three times with 10 ml of methanol. The methanol extracts were combined, passed through a column (20 mm in inner diameter) packed with 100 ml of Sephadexe LH-20, and developed with methanol. Fraction Nos. 28 to 46 (each 1 ml in volume) were combined and evaporated to dryness to obtain 149 mg (91% yield) of a white powder of (S)-7-guanidino-3hydroxyheptanamide hydrochloride (C H18N402. HOl). [MD22 ~ 20 (C 2, water).

Claims (8)

1. N-[4-(3-aminopropyl)aminobutyl]-2,2-dihydroxyethanamide of the formula (I)

or an acid addition salt thereof.

2. A compound according to claim 1, in the form of a hydrochloride salt.

3. A process for producing N-[4-(3-aminopropyl)-aminobutyl]-2,2-dihydroxyethanamide of the formula (I)

or an acid addition salt thereof, which comprises allowing a compound of the formula (II)

wherein R represents an amino-protecting group, to react with a dialkylacetal of glyoxylic acid of the formula (III)

wherein R' represents an alkyl group of 1 to 5 carbon atoms, in the presence of an activating reagent or with a reactive derivative of the carboxyl group of said acetal, and subsequently removing the aminoprotecting group and R'.

4. A process according to claim 3, wherein the activating reagent is an acyl halide forming reagent, acid azide forming reagent, reactive ester forming reagent, or acid anhydride forming reagent.

5. A process according to claim 3 or 4, wherein the reaction is conducted in an organic solvent, at 0 to 1000C, for 5 to 30 hours.

6. A process according to claim 3 substantially as hereinbefore described with reference to the Example.

7. A compound according to claim 1 or 2 for use in a method of therapy practised on the human or animal body.

8. A compound according to claim 1 or2 for use as an immunostimulant.