DK159654B - METHOD OF ANALOGUE FOR THE PREPARATION OF AMINO ACID DERIVATIVES OR PHYSIOLOGICALLY ACCEPTABLE SALTS THEREOF - Google Patents

Description

DK 159654 B

The present invention relates to an analogous process for the preparation of novel amino acid derivatives with valuable biological and pharmaceutical effects.

The amino acid derivatives prepared by the process according to the invention have the general formula

R1-NH-CH-COOH

(CH 2) n I

10 io-NH-CH- [CEO) -B1 lo Δ

R

Wherein R, R, B, n and t have the meanings set forth in the preamble of the claim, or are physiologically acceptable salts or optically active antipodes thereof.

These compounds have valuable biological or pharmacological effects. All the compounds of the invention are novel.

Among the compounds of the invention, due to its biological effect, particular mention is made of γ-L-glutamyltaurine corresponding to the formula

HoN-CH-C00H 1 I CH0 I 1

CH2 XXIV

co-nh-ch2-ch2-so2oh and which has a broad therapeutic and preventive spectrum of action against morbid changes that can be reversed to damage by "AGAS" (the aerobiospheric genetic adaptation system).

To illustrate the concept of "AGAS", the following are the main tissues and organs that form this system.

(a) all biological interfaces in contact with the external air such as the biosphere (skin and skin formation, eye cornea and conjunctiva, oral and pharyngeal cavities, respiratory tract and lung);

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2 (b) skeleton and joints (tubular and spongy bones, joints, synovial membranes, skeletal muscle); (c) the organs involved in the regulation of the ion household (transepithelial transport system: intestinal tracts and renal duct); (d) the toothpaste (rooted in the tooth anaolobes with roots) needed to decompose the food; (e) hearing, smell and voice organs.

Thus, the compounds of this invention exert a favorable biological or therapeutic effect on the organs or tissues of the AGAS system enumerated herein.

The compounds of the process according to the invention additionally act on the following functions which are related to the AGAS system: radiation protection, favoring wound healing, general activating effect on mesenchymal, protection against the ever growing infection and soiling danger of skin and mucosa (the moist mucosal lysozyme production, activation of gut epithelium in the respiratory tract, etc.), increased protection against infections caused by viruses and fungi.

The compounds produced by the process according to the invention are effective against the ever-increasing stress effects associated with mainland life (e.g. meteorological influence, large differences between day and night temperature, increased risk of injury) as they stabilize. adaptation syndrome and at the same time avert the peripheral tissue damage of the glucocorticoids (such as damage to the connective tissue, injuries to bone matrix components, etc.). Development of immune homeostasis (increasing body recognition of which cells are suitable and which are not).

30 The compounds prepared by the process according to the invention exert their effect, both directly and partly through the regulation of vitamin A metabolism, in the production of vitamin A metabolites of a stronger polar nature. This effect is comparable to the effect of the parathormone on the 25-hydroxycholecalciferol-35a-hydroxylase enzyme in the renal tract. The directions of action of the compounds are as follows:

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3 A) Vitamin A grade effects: a) Pharmacological and biochemical effects: increasing effect on chondroitin sulfate synthesis; beneficial effect on the wound healing or on the experimentally impaired wound healing in cortisone in rats and dogs; potentiating effect on vitamin A's effect in rats and chickens in experimentally induced hypo- or hypervitaminosis; attenuating effect on the ulceration-related stress effects in rats; beneficial effect on the degranulation of mastocytes; increasing effect on the production of lysozyme; effect on household traceability (silicon, zinc, copper, manganese, fluorine); promoting effect on epithelial formation; promoting effect on the alkaline phosphatase activity; the effect on the granuloma bag formation (Granulomsackbildung) caused by local action of vitamin A; the extremely flat course of the dose-effect curve or the change of effect sign at large doses; activating effect on the Golgi apparatus; beneficial effect on the formation of mucus or goblet cells; increasing effect on the concentration of vitamin A.

B) Clinical-therapeutic effects: keratoconjunctivis sicca; Sjogren's syndrome; rhino-laryngo-pharingitis sicca; ozæna; chronic bronchitis; sinobronchitis; mucoviscidosis; constitutional lung disease in young children; periodontal disease; skin and mucosal susceptibility to viruses and fungi; cortisone antagonistic action; beneficial effect on healing of operative and mucosal wounds; erosio colli; pruritus-like disorders; reducing the sense of smell and taste.

30 B) Vitamin A-grade effects (a) Pharmacological and biochemical effects: effects on blood glucose levels with a transient decrease; increasing effect on phosphaturia ·, lowering effect on phosphate level 35 in serum; radiation protective effect; diminishing effect on

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4 the time needed to reach the goal of maze trials in inactivated animals; diminishing effect on experimentally induced fluorine and cadmium toxicosis; increasing effect on the cyclic adenosine monophosphate depletion of the kidneys; attenuating effect 5 on the symptoms of experimentally induced lathyrism; decrease in histamine sensitivity; enhancing effect on liver enzyme tyrosine aminotransferase activity.

b) Therapeutic effects: weak radiation damage; vitiligo; muscle hypotonia; psychoenergetic effect; beneficial effect on involutional and gerontological conditions as well as on the mnestic functions; keloid inclinations; spondylosis ankylo-poetics; diseases of the movement organs due to wear and tear; scle-rotic fundus; amyloidosis; morphea; fibrocytic mastopathy.

In the veterinary medicine, the compounds prepared according to the invention have similar fields of application as in human medicine, ie. eg skin damage (peeling), wound healing and bone fractures.

The process according to the invention is characterized by the characterizing part of the claim.

Compounds of general formula I are thus prepared by forming the ω-amides by reaction of a R N -substituted pyrrolidic acid with, for example, taurine, homotaurine or other amines of formula III containing a strong acidic functional group or their alkali metal salts or with a tertiary base, salts formed at the opening of the lactam ring.

The process according to the invention is illustrated in the following by way of example.

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5

Example 4.43 g (10 mmol) of N-carbobenzyloxy-L-pyroglutamic acid di-cyclohexylamine salt (Liebigs Annalen 640, 145, 1961), 1.25 g (10 mmol) of taurine and 0.84 g (10 mmol) dissolved sodium bicarbonate in 50 ml of water. The solution is warmed for four hours (or left at room temperature for 24 hours) and then evaporated under vacuum. The residue is dissolved in water, transferred to a column of "Dowex" 50 and eluted with water. The eluate is evaporated. The recovered substance is catalytically hydrogenated while shaking in the presence of 0.5 10 g of 10% Pd / C as catalyst. After cessation of hydrogen consumption, the solution is filtered and evaporated at 30 ° C in vacuo to an oily residue which is dried in a phosphorus pentoxide desiccator. The obtained γ-glutamyltaurine is readily dissolved in water but not in ethanol. a little crystalline crude product is obtained, mp 202-204 ° C. After recrystallization of the crude product from a mixture of ethanol and water, 2.03 g of γ-L-glutamyltaurine are obtained, mp 219-220 ° C .

= + 14 ° (water, C = 1.02). Relative motility to cysteic acid by paper electrophoresis at pH 6.5 is 0.73, at pH 1.8 0.53. Rf (n-butanol / pyridine / glacial acetic acid / water 15: 10: 3: 12) = 0.19.

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6

Biological Test Report A.

Study of γ-L-glutamyltaurine as in this report is called litoralon.

In the following Tables 1-6, the experimental results 5 are shown as means + standard deviation with the number of brackets. Determination of whether there is a significant difference between control trials and trials in treated animals was done with Student's t-test.

Table 1

The effect of lltoralon on vitamin A concentration in serum.

Group of litoralone vitamin A concentration in serum _Ug / day_ 15 I. Control - 28.3 + 0.7 (20) II. 0.1 41.9 + 1.0X (20) III. 0.3 32.9 + 1.1XX (20) IV. 1.0 27.4 + 0.6 (20) 20 x P <0.001 xx P <0.01 20 Sprague Dawley rats (10 males and 10 females) weighing 180-200 g were orally treated with the daily doses listed in Table 1 -25 sees litoralon in the form of an aqueous solution for a period of 8 days. On the 9th test day, the animals were sacrificed by decapitation and the blood was collected. The vitamin A concentration in the serum was determined by Neeld and Pearson's method.

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7

Table 2

The Effect of Litoralone and Vitamin A on Granule Formation Caused by Implanted Cotton Balls_ '' ~ '____

Group Dose Dry weight of 5 Vitamin Ax litoralon granuloma local local oral _mg_yg_yg / day_ I. Control - - - 52 + 1.0 (24) II. Control +

Solvent - - - 54 + 3.1 (8) III. 2 - - 64 + 2.5 (8) IV. 2 0.1 - 65 + 2.7 (8) V. - 0.1 73 + 2.9 (8) VI. 2 - 0.1 90 + 4.1 (8) 15 x Hoffmann La Roche

The difference is significant: between groups II and III at P <0.05, between II and V at P <0.001, and between V and VI at 20 P <0.01. The granule conversion was determined according to Lee et al

Male Sprague-Dawley rats weighing 110-120 g. The tampons were removed from the dorsolateral subcutaneous implants after 10 days and weighed after drying to constant weight at 65 ° C.

25

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Η Μ · H HH di H -H dl Η -P

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Table 5

The effect of Litoraion on the metamorphosis of Rana dalmatina

Group Body length Tail length _mm_mm_

Control 45.9 + 0.2 31.4 + 0.2 (60) 5 Treated Animals 40.2 + 0.2X 26.7 + 0.2X (60) x Significance: P <0.001

The experiment was conducted with larvae of Rana dalmatia with a 30 day age, a body length of 20-25 mm, and with 10 visible projecting hind legs already. Over a period of 30 days, the test animals were placed in tap water containing 0.5 µg / ml litoralon for two hours each day. The control group was placed in tap water without the addition of litoralone. The nipples were measured on the 3rd day.

15

Table 6

Litoralone's effect on blood sugar levels

Group I. Study II. Study mg% mg%. 2Q -

Control 94 + 3.6 (10) 94 + 4.09 (10)

Treated Animals 82.4 + 3.8 (10) 81 + 1.32 (10)

Significance at I. study: P <0.05 Significance at II. study: P <0.01 25 Male CGY white rats of body weight 160-180 g were used for the experiment. The animals were kept on a standard diet. Blood samples were taken on the 5th day of testing after 18 hours of fasting. The blood glucose assays were performed according to the method of E. Hultman. Litoralon was given orally at daily doses of 1 µg / kg.

3 "

u DK 159654B

Biological Test Report B.

Study of synthetic β-aspartyl-N-methyltaurine, β-aspartyl homotaurine and γ-glutamyl-colamine phosphate.

1. β-Aspartyl-N-methyltaurine 5 a) Effect on blood sugar level Control 107 mg%

Treated animals 93 mg%

Significance: P <0f05

20-20 rats were used for the samples. Measurements were carried out -10 after 18 hours of fasting. The dose used was 1 µg / kg body weight for 4 days in the form of a solution by oral administration.

b) Effect to increase serum vitamin A level

Oral dose Evoked vitamin Ά yg% 15 µg / 200 g body weight 0 (control) 8.5 5 11.0 1 11.5 0.3 12.5 20 0.1 16.1 0.05 14.8 0.01 12.5 Q, 005 10.5 Significance: P <0.01

For the experiments, 20-20 Wistar male rats weighing 200-220 g were used.

Trial period: 6 days.

C) Effect on blood silicon level:

Silicon (mg / g blood) 0 hours 20 days 40 days

Control group 0.110 + 0.004 0.120 + 0.010 0.154 + 0.015

Treatment group 35 µg / day 0.100 + 0.005 0.315 + 0.0141 0.345 + 0.0151

treatment Group

II 10 µg / day 0.107 + 0.009 0.370 + 0.119XX 0.360 + 0.017XX

Significance: P <0.001

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12

The results are significant from the 13th day at the significance level P <0.01 and from the 20th day at the level P <0.001. The experiments were performed on inbred male rabbits of body weight 2.5-3 kg. The active ingredient was administered orally in the 5 daily doses shown in the table. The determination of silicon was performed according to Gaubatz's method (Gaubatz E., Clin. Wschrft. 14, 1753, 1935) in 5 ml of blood samples taken from the animals' ears.

d) The effect of β-aspartyl-N-methyl-taurine and vitamin A on the 10 granulomatous effects caused by the implantation of cotton wool:

Group Dose Weight of dry

Vitamin Ax β-aspartyl-N- rf 9ranalom methyltaurine local local oral mg yg yg / day 15 --- ~

Control I. - - - 54 + 1.7

Control II. + solvent - - - 55 + 3.4

Treatment Group III. 2 - - 66 + 12.5

Treatment Group IV. 2 0.1 - 67 + 2.6 on

Treatment group V. - - 0.1 79 + 2.8

Treatment Group VI. 2 - 0.1 96 + 4.4 x Hoffmann la Roche

The differences are significant as follows: 25 Between groups II and III P <0.05, between groups II and V P <0.001 and between groups V and VI P <0.01.

The determination of granuloma was performed on male Sprague-Dawley body weights 110-120 g by the method of Lee et al (Lee K.H., Fu Ch.Ch., Spencer M.R. Tong T.G. and Poon R.J., Pharm.

30 Sci., 62, 895, 1973). The dorsolaterally subcutaneously implanted tampons were removed after 10 days and measured after drying at 65 ° C to constant weight.

2. β-Asparfeyl homotaurin.

35 a) Effect on blood sugar level

Control group 105 mg%

Treated group 94 mg%

Significance, number of test animals and test method were as indicated in section. 1 a) above.

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13 b) Increasing effect on serum vitamin A level

Oral dose Induced vitamin A

yg / 200 g body weight yg% 0 (control) 8.6 5 5 12.0 1 13.5 0.3 14.0 0.1 15.8 0.05 15.0 10 0.01 12.0 _0, 005_10,0_

Significance, number of test animals and test method were as indicated above under section. Ib).

C) Effect on blood silicon level;

Silicon (mg / g blood) _0 hours_20 days_40 days_

Control group 0.104 + 0.009 0.134 + 0.015 0.157 + 0.020 2n Treatment group I. 5yg / day 0.094 + 0.007 0.309 + 0.014 0.340 + 0.014

Treatment Group II. 10 µg / day 0.109 + 0.010 0.372 + 0.120 0.363 + 0.018 x

Significance, number of test animals, arrangement and method of determination as indicated above under cl. 1 c).

(D) The effect of β-aspartyl homotaurine and vitamin A on the granulomatous effect of cotton implantation;

Dose Weight of dried 3Q Vitamin Ax {3-aspartyl granuloma local homotaurin 'mg local oral __y g yg / day ___

Control group I - - 52 + 1.5

Control group II - - - 53 + 3.2 + 25 + solvent

Treatment Group III. 2 - - 64 + 12.3

Treatment Group IV. 2 0.1 - 65 + 2.4

Treatment group V. - - 0.1 77 + 2.6

Treatment group VI._2_- 0.1_94 + 4.2_

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14 x Hoffmann la Roche

Significance, number of test animals, arrangement and method of determination were as indicated above under point (ld).

3. γ-Glutamyl-Colamine Phosphate.

a) Effect on blood sugar level

Control group 104 mg%

Treatment group 93 mg%

Significance, number of test animals, arrangement and dosage 10 were as indicated above under point 1a).

b) Increasing effect on serum vitamin A level

Oral dose Vitamin A action yg / 200 g body weight yg% 15 ----- 0 (control) 8.8 5 12.3 1 13.4 0.3 14.3 20 0.1 16.0 0.05 15.5 0.01 11.2 0.005_9 ^ 2_

Significance, number of test animals, arrangement and method of determination were as indicated above under point Ib).

c) Effect on blood silicon level:

Silicon (mg / g blood) 0 hours 20 days 40 days 30 -

Control group 0.106 + 0.011 0.136 + 0.017 0.159 + 0.022

Treatment group I 5 tg / day 0.096 + 0.009 0.311 + 0.016xx 0.340 + 0.017

Treatment group II 10 µg / day 0.111 + 0.011 0.374 + 0.121xx 0.365 + 0.019 35 Significance, number of test animals, arrangement and method of determination were as given above under item 1 (c).

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(D) The effect of γ-glutamyl-colamine phosphate and vitamin A on the granuloma-inducing effect of water implantation:

Dose Weight of dried granuloma

Vitamin Ax γ-glutamyl mg 5 Local Colamine Phosphate mg Local Oral _yg µg / day_

Control group I. - - - 49 + 1.2

Control group II. + solvent - - - 50 + 2/9 10 Treatment group III. 2 - - 61 + 12.0

Treatment Group IV. 2 0.1 - 62 + 2.1

Treatment group V. - - 0.1 74 + 2.3

Treatment Group VI. 2 - 0.1 91 + 3.9 x Hoffmann la Roche 15

Significance, number of test animals, arrangement and method of determination were as indicated under 1 d) above.

Claims (2)

5 R 1 -NH-CH-COOH (CH9) II 2 n -i CO-NH-CH- (CHO). -BI 9 2 t R 2 where 10. is hydrogen, C 1-4 alkoxycarbonyl or phenyl-C 2 is hydrogen or carboxyl, B is a group of the formula -SC 2 OH or -OPCHOH 5 is 1 or 2, 15. is 1 or 2, or a physiologically acceptable salt or an optically active antipode thereof, characterized in that an optically active or racemic compound of the general formula 20 is reacted with R4-N-CH-C00H II I \ O = C- (CH0) 2 n 4 1 where R has the same meaning as given above for R 25 except hydrogen and n is as defined above, or a salt thereof having a compound of the general formula H "N-CH- (CHO) -B1 III 2. o 2 t R 2 1 or a salt thereof, wherein R, B and t have the above meanings, and if desired, the protecting group R 4 and / or, if desired, forms a compound thus obtained DK 159 6 b 4 B into a physiological acceptable salt thereof or release it from its salt and / or prepare the compound in optically active form by digestion of a won racemic product.