DE2723453A1 - ANTIDIURETIC DESAMINO-ASPARAGINE HIGH 4 -D-ARGININE HIGH 8 -VASOPRESSIN AND METHOD OF ITS MANUFACTURING - Google Patents

Description

GLAWE, DELFS, MOLL & PARTNER

PATENT LAWYERS

DR-ING. RICHARD GLAWE. MONKS DIPL-ING. KLAUS DELFS. HAMBURG DIPL.-PHYS. DR. WALTER MOLL. MÖNCHEN DIPL.-CHEM. DR. ULRICH MENGDEHL. HAMBURQ

8000 MÖNCHEN 26 POST BOX 37 LIEBHERRSTR. 20th TEL. (089) 22 65 48 TELEX 52 25 OS

2000 HAMBURG 13 POST BOX 2570 ROTHENBAUM-CHAUSSEE 58 TEL. (040) 41020 08 TELEX 21 29 21

MUNICH A 65

FERRING AB
Malmö / Sweden

L. Q

Antidiuretic desamino-asparagine -D-arginine -Vasopressin as well as process for its preparation

The invention relates to an antidiuretic

Λ Q

Desamino-asparagine -D-arginine -Vasopressin with prolonged effectiveness, which has no side effects from the general formula I.

Mep-Tyr-Phe-Asn-Asn-Cys-Pro-D-Arg-Gly-NH,
123 ^ 567 89

(D

wherein Mep is a 2-mercapto-propionyl radical (-S-CH ₂ CH ₂ CO-)

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BANK: DRESDNER BANK, HAMBURG, 4 030 448 (BLZ 200800 00) - POST CHECK: HAMBURG 147607-200 TELEGRAM: SPECHTZIES

^ 2723Λ53

and D-arginine is at the 8 position.

The duration of the antidiuretic activity of the vasopressin depends on the speed of the enzymatic breakdown of the intact peptides. Structural variations of the peptide, which reduce the rate of enzymatic degradation while maintaining the biological activity, are therefore very desirable, especially if there is an additional enhancement and expansion of the therapeutic efficacy. Replaced if one tion the cysteine group in the 1-position by Mep and the L-arg in the Posi in arginine vasopressin 8 by D-Arg, obtained Des amino-D-arginine vasopressin, DDAVP abbreviated which an analogue of vasopressin and has an expanded antidiuretic effectiveness and a significantly reduced effect on the smooth muscles of the vascular system and intestinal tract compared to vasopressin. Both effects are valuable in treating Diabetes Insipidus. In addition to these two valuable effects, uterotonic activity must also be considered in women suffering from diabetes insipidus and who are or are about to become pregnant. The older vasopressin derivatives in therapeutic use have too much uterotonic activity to be administered to pregnant women without creating the risk of miscarriage. There is therefore a great need for it for pregnant patients with diabetes

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Insipidus to create a drug which, in therapeutically effective doses, has a sufficiently low uterotonic Has activity.

In view of the fact that the disease Diabetes Insipidus requires constant, i.e. lifelong, drug treatment, there is a risk that the patient becomes either immune or hypersensitive after a certain period of treatment is against the drug. In order to be able to continue the treatment of the disease, another equivalent must be used A drug may be used which does not induce such immunity or allergy in the patient. Such an alternative However, drugs for vasopressin and DDAVP are not yet available. The object of the invention is therefore to remedy this need. The present invention provides a novel vasopressin derivative which has a antidiuretic effect of the same order of magnitude as has the previously known means, but at the same time has a significantly lower uterotonic effectiveness, so that the ratio of antidiuresis to uterotonic effectiveness is significantly improved. More precisely, it is uterotonic Effectiveness of the agent according to the invention to a Power of ten lower than with the previously known means, so that a ratio of Antidiuresis to uterotonic activity is preserved, which is considered a unique result. According to the invention

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ties can therefore be prescribed to women who have suffer from diabetes insipidus and who wish to conceive, and the prescription to such women can be throughout the period Pregnancy duration, which opens up completely new possibilities for this group of women.

According to the method of the invention, the amino acid sequence

Mep (SH) -Tyr-Phe-Asn-Asn-Cys (SH) -Pro-D-Arg-Gly-NH 1 23456 7 8 9

where Mep is the 2-mercaptopropionyl, in known per se Produced in stages, and these amino acid

4 sequence is then oxidized to form desamino-asparagine -

D-arginine -Vasopressin of the formula

ί '"Ί

Mep-Tyr-Phe-Asn-Asn-Cys-Pro-D-Arg-Gly-NH ₂ .

According to a preferred feature of the invention, the oxidation takes place by means of potassium ferricyanide in an aqueous solution with pH 6.5 to 7.0.

The polypeptide according to the present invention can be used both in the form of a free base and as a salt of inorganic or organic acid can be used,

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if with the addition of auxiliaries, stabilizers and Preservatives, sweeteners, flavoring substances, wetting agents and the like for the production of suitable forms of administration for parenteral, peroral, intranasal, subcutaneous, intramuscular and intravenous administration. Examples of suitable inorganic acids are hydrochloric acid and phosphoric acid, while organic acids can be used for example acetic acid, citric acid and tartaric acid.

Acid functional compounds such as tannin can also be used. Suitable additives are starch, Lactose, natural or hydrogenated oils, talc, glycerine, etc. A particular advantage of the new compound according to the invention is their good intranasal absorbability. As a result, the patient can take the drug in a simpler and easier way can be administered and dosed through the nose in an easily accessible manner. So he doesn't need an injection to use for intravenous administration, which would be much more cumbersome and uncomfortable.

The peptide according to the invention is produced using a protected pentapeptide amide (II) as a starting point

X-Asn-Cys (BzI) -Pro-D-Arg (Y) -GIy-NH ₂ . (II)

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Here, X is a protective group for the amino group (benzyl oxicarbonyl), BzI is a protective group for the mercapto group (benzyl) of cysteine and Y is a protective group for the guanidine nitrogen group (p-toluenesulphonyl) . The protecting group X is removed and the pentapeptide is coupled with (III),

X-Asn-ONp (III)

wherein ONp is a p-nitrophenyl ester group to give the protected hexapeptides (IV) :

X-Asn-Asn-Cys (BzI) -Pro-D-Arg (Y) -GIy-NH ₂ (IV)

Now the protective group X of the peptide (IV) is removed and the hexapeptide with the tripeptide hydrazide (V)

Mep (Bzl) -Tyr-Phe-NHNH ₂ (V)

coupled by means of the azide coupling method, whereby one the protected nonapeptide amide (VI) receives:

I4ep (Bzl) -Tyr-Phe-Asn-Asn-Cys (Bzl) -Pro-D-Arg (Y) -Gly-NH ₂ (VI)

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By treating this protected nonapeptide amide with an alkali metal in liquid ammonia solution, the protective groups are split off and the reduced nonapeptide amide (VII)

Mep (SH) -Tyr-Phe-Asn-Asn-Cys (SH) -Pro-D-Arg-Gly-WH ₂ (VII)

obtained, which is oxidized in an aqueous solution with potassium ferricyanide at a pH of 6.5 to 7.0, whereby the cyclic, biologically active peptide of the general formula (I) is obtained.

The peptide according to formula (I) differs from vasopressin in that the amino acid in position 4 is through Asparagine and at the same time the cysteine group in position 1 by Wep and the L-arginine in position 8 by D-arginine is replaced. Compared to vasopressin, this peptide according to formula (I) has good intranasal absorbability an improved antidiuretic effectiveness, a substantially reduced blood pressure increase effect, a very lasting effectiveness and a significantly reduced uterotonic activity (see Table I). As comparison substances are in Table I except for arginine vasopressin

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DDAVP and an analogous 4-val-DDAVP, which is known from the literature, are also given (Chemical Abstracts: 80 , page 347 ν (197A)). In the case of the antidiuretic peptide of the general formula (I), since the curve for the logarithm of the dose relative to the response is not linear, it is difficult to conventionally measure the effectiveness in units

to be indicated per milligram for the D-arginine analogue in comparison to vasopressin. Therefore, in Table I, the strengths were given as relative effectiveness values, the values for DDAVP as a comparison substance being arbitrarily set at 1.00 .

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J *

Table I.

Comparative values for antidiuretic effectiveness, blood pressure effectiveness and uterotonic effectiveness for ÜDAVP analogs compared to DDAVP

peptide anti- To activity Utero Antidiuresis Antidiuresis diuresis holding
speed blood tonic Blood pressure Uteroto-
niche pressure

DDAVP (known) 1.00

4-Val-DDAVP (known) 1.44

4-Asn- -DDAVP 1, 26

Arg-vasopressin (known) 0.10

1.00 1.00

<0 _f 15 0.9

0.9 0.09

1.00

> 9.60

1.4

1.0 0.00006

1.00

1.6

14.0

0.1

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Jl 2 7 2 3 Λ 5 3

It is evident from Table I that the invention Compound 4-Asn-DDAVP compared to the known compounds good values for antidiuresis, sustainability and Has Llutdruckffektffekt. In addition, it arises a much improved value for uterotonic activity and a much higher value for the ratio Antidiuresis to uterotonic activity. From this it follows that the agent at the same time has a good antidiuretic effectiveness, a lower increase in blood pressure, good sustainability of the effect and an extremely low uterotonic Has effect, which is particularly valuable for women suffering from Diabetes Insipidus after conception.

4 8 The desamino-asparagine -D-arginine -Vasopressin can be used as

therapeutic preparation in aqueous or non-aqueous solutions containing organic or inorganic salts, acids or bases, are prepared for oral, rectal or subcutaneous administration.

The invention is explained in more detail with reference to the following exemplary embodiments. Unless otherwise stated, the following information and definitions apply to these.

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The following abbreviations were used:

TLC = thin layer chromatography

AAA = analysis of amino acid composition

Cbz = carbobenzyloxy group

Tos = tosyl group (p-toluenesulphonyl group)

The evaporations were carried out with a water jet suction pump and at 35 ° C, unless otherwise specified is. All solvents were reagent grade. The pH of the non-aqueous solutions was moistened with Litmus paper for sure.

The optical rotation angle was measured with a Perkin-Elmer Polarimeter measured.

The thin-layer chromatogram was recorded on "Merck TLC plates Silica gel 60 "obtained in the following system:

A: butanol: acetic acid: water 4: 1: 1

B: butanol: pyridine: acetic acid: water 15: 10: 3: 6

C: cyclohexane: ethyl acetate: methanol 1: 1: 1

D: chloroform: methanol acetic acid 10: 2: 1

E: chloroform: methanol acetic acid: water 30:20: 4: 6

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For the analyzes of the amino acid compositions, samples were ^{hydrolyzed in 6 μl HCl in closed, evaporated tubes at 110 °} C. for 24 hours.

The analyzes were obtained by means of an automatic amino acid analyzer of the JEOL-5AH type with an accuracy of -1.2 % .

example 1
Cbz-D-Arg (Tos) -Gly-ÜKt (1)

A 0 ° solution of Cbz-D-Arg (Tos) (509 g, 1.10 mol), GIy-OEt.HCl (109 g, 1.21 mol) and 169 ml (1.21 mol) of triethylamine in 1 81 liters of chloroform was treated with a solution of dicyclohexylcarbodiiiid (227 g, 1.10 mol) in 600 ml of chloroform and left to stand at room temperature for 24 hours. The dicyclohexylurea was filtered off and washed with 3 parts of chloroform, and the filtrate and the washing product were evaporated under reduced pressure. The residue was dissolved in 6 liters of ethyl acetate and washed with 1 liter portions of 0.25 N HCl (6X), water (1X), 5% NaHCO ₃ (3X) and H ₂ O (2X). The EtOAc solution was dried (Na ₂ SO.) And evaporated (oil pump) to give 554 g (92 %) of the protected dipeptide ester 1.

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"'d + 3.1 ° (c 3.0, 95% AcOH)
AAA: Arg, 0.85; GIy, 1.00
TLC: Rf ^C : 0.62 Rf ¹ ^ O.67

Example 2
Cbz-Pro-D-Arg (Tos) -GIy-OEt (2)

The protected dipeptide ester 1 (425 g, 0.77 mol) was dissolved in a solution of HBr (478 g) in acetic acid (2800 ml) with shaking and the solution was heated to 50 ° for 10 minutes. The warm solution was poured into 18 liters of diethyl ether with stirring and the white precipitate collected on a filter, washed with 2 liter portions of diethyl ether (8X) and dried in vacuo over NaOH for 5 hours. The precipitate was dissolved in 1260 ml of chloroform, cooled to 0 ° and Et ₅ N was added to pH 7.5. Crystalline Cbz-Pro-ONp (280 g, 0.758 mol) was added and the solution was kept at room temperature for a week, the pH being readjusted to 7.5 with triethylamine if necessary. The solution was diluted with 5 liters of CHCl, and washed with 1 liter each of H ₂ O (1X), N NH ₃ (10X), H ₂ O (1X), N HCl (2X), and HpO (3X) . The CHCl, solution was dried (Na ₂ SO ^) and evaporated (oil pump) to give the protected tripeptin ester 2 as a yellowish-brown solid (486 g, 99%).

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- 'd-23.0 ° (c 1.80, 95 X> AcOH)

TLC: Kf ^C : 0.64 Rf ^D : O.74

Example 3 Cbz-Pro-D-Arg (Tos) -Gly NH ₂ (3)

In a solution of the protected Tripeptidesters (210 mol 0.328 g,) in 6.9 1 of methanol is allowed to speak Tillier tes NH _7, 7 hours at room temperature in bubbles bubbled. The NH and methanol are evaporated and the oil was diluted in 400 ml of chloroform. Ethyl acetate (2500 ml) was added to precipitate an oil which was pulverized with three 1 liter portions of diethyl ether with scratching. The solid was collected on a filter and dried, yielding 168 g (83%) of the protected tripeptide amide 3.

■ ^S 'D-22.6 ° (c 1.09, 95% AcOH) AAA: Pro, 1.03; Arg, 0.82: GIy, 1.00 TLC Kf ^C : 0.37 Rf ⁰ IO, 47

Example 4 Cbz-Cys (BzI) -Pro-D-Arg (Tos) -GIy-NH ₂

A slurry of the protected tripeptide amide

(168 g, 0.273 mol) in 455 ml of acetic acid was

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solution of Hbr (240 g) in acetic acid (700 ml) for 1 hour at room temperature and then poured into 9 l of diethyl ether with stirring. The white precipitate was collected on a filter and washed with 6 liters of diethyl ether. After drying in vacuo over WaOH for 6 hours, the precipitate was dissolved in 1070 ml of dimethylformamide and the solution was cooled to 0 °. The solution was adjusted to pH 8.0 with triethylamine and Cbz-Cys (BzI) -ONp (128 g, 0.27 inol) was added. After 3 days at room temperature, the dimethylformamide was evaporated (oil pump) and the remaining oil dissolved in 5 L of CHCl, and washed with 1 liter of N NH, (3X), N HCl (1X) and H ₂ O (2X). The CHCl ₃ solution was dried (Na ₂ SO 4), concentrated to 500 ml, and 1500 ml of diethyl ether was added to precipitate an oil, which was pulverized with 21 liter quantities of diethyl ether. The solid was collected on a filter and dried to give 194 g (88 %) of the protected tetrapeptide amide 4.

Ad -15.9 ° (c 0.91, dimethylformamide) AAAi Cys (Bzl), 0.81; Pro, 1.02; Arg, 0.79; GIy, 1.00

TLC: Rf ^C : 0.48, Rf ⁰ : 0.56

Example 5
Cbz-Asn-Cys (BzI) -Pro-D-Arg (Tos J-GIy-NH ₂ 5_

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A slurry of the protected tetrapeptide amide 4 (190 g, 0.235 mol) in 540 ml of acetic acid was treated with a solution of HBr (380 g) in acetic acid (1450 ml) for 1.25 hours treated for a long time and poured into 9 1 diethyl ether with stirring. The white precipitate was collected on a filter, washed with 7 l of diethyl ether, dried in vacuo over NaOH for 5 hours and dissolved in 4.5 l of methanol. A slurry of IUA-410 (0H ~) ion exchange resin (ÖOü ml bed) in methanol was added and the mixture was stirred for 10 minutes, the resin was filtered off and washed with Methanol washed. The filtrate and wash liquid were evaporated together to give an oil which was dissolved in 800 ml of dimethylformamide was dissolved. Cbz-Asn-ONp (100 g, 0.259 mol) was added, the pH adjusted to 7.5 with triethylamine and allowed to settle the solution at room temperature for 4 days. The solution was concentrated to 100 ml (oil pump) and the viscous oil obtained is diluted with 150 ml of warm methanol. The protected pentapeptide amide was by adding 1 1 Ethyl acetate precipitated, collected on a filter and treated with 2 l of a solution of ethyl acetate and methanol in the ratio 4: 1 and washed with 500 ml of ethyl acetate. The dried peptide 5 weighed 160 g (72%).

! ^ri '^; D-18.9 ° (c 1.10, dimethylformamide)

AAA: Asp, 0.91; Cys (Bzl), 0.72; Pro, 0.98; Arg, 0.80; GIy, 1.00

TLC Rf *: 0.5 °; Rf ^C : 0.19; Rf ^D : 0.16

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Example 6
Cbz-Asn-Asn-Cys (BzI) -Pro-D-Arg (Tos) -Gly-ΙΛίρ (6)

The protected pentapeptide amide 5 (923 mg, 1 mmol) was dissolved in 10 ml of 2.5 M HBr in acetic acid. After 1.25 hours at room temperature, the hydrobromide salt was precipitated with diethyl ether, filtered off, washed with several amounts of diethyl ether and dried in vacuo over NaOH. The precipitate was dissolved in 8 ml of dimethylformamide, cooled to 0 ° and Cbz-Asn-ONp (490 mg, 1.2 mmol) and triethylamine (0.58 ml) were added. After standing at room temperature for 24 hours, the dimethylformamide was evaporated (oil pump), the residue was diluted with ethanol and the solid formed was filtered off and washed with several amounts of ethanol. After drying over P ₂ ° 5, the protected hexapeptide amide 6 had a weight of 859 mg (83 %) .

mp 185-187 ° C

\ «R ²⁵

{_ "* - ·> D-18.0 (cl.O dimethylformamide)

TLC Rf * 0.43 Rf ° 0.11 Rf * ¹ 0.78

Example 7
Cbz-Tyr (Bzl) -Phe-OMe 2

A mixture of Cbz-Tyr (Bzl) -ONp (52.6 g, 0.10 mol), HCl'Phe-OMe (23.6 g, 0.11 mol) and triethylamine (15.3 ml,

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0.11 raol) in 170 ml DIIF was left to stand at room temperature for 19 hours . Ethanol (600 ml) was added to the solution and the crystalline material which had formed after 2.5 hours at 4 ° was collected on a filter and washed with ethanol (4 X 200 ml) and diethyl ether (2 X 200 ml ) washed. After air drying, the protected dipeptide ester 7 weighed 51.8 g (91 %) .

mp 179-181 ⁰ C

25 ι "^; D - 18.7 (c 1.05, dimethylformamide)

TLC: Hf ^C 0.84 Rf ^D 0.90

Example 8
HCLTyr-Phe-OMe 8

A suspension of Cbz-Tyr (Bzl) -Phe-OMe (20.4 g, 0.036 mol) and 1 g of palladium in 400 ml of methanol containing 7.2 ml of 5 N HCl (0.036 mol) were was hydrogenated at atmospheric pressure and Hydrogenated room temperature for 24 hours, an additional 1 g of palladium was added and hydrogenation continued for 10 hours. The palladium was filtered off and washed on the filter with methanol. The methanol film occurred and the washing liquid was evaporated together, and the oil obtained was diluted with diethyl ether and left to stand at 4 ° overnight. The crystalline hydrochloride salt 8 was collected on a filter with diethyl ether

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washed and dried to give 13.1 g (97 ^L / o). mp

^{L 1} D + 2.8 ° (c1.0, dimethylformamide) TLC: Rf ^C 0.54 Rf ⁰ 0.32

Example 9 Mep) Bzl) -Tyr-Phe-01> le (9)

A 0 ° solution of HCl'Tyr-Phe-OMe (5.0 g, 13.2 mmol), Mep (Bzl) -ONp (4.6 g, 13.2 mmol) and triethylamine (1.85 ml, 13 »2 mmol) in 40 ml of dimethylformamide was left to stand at room temperature for 2 days. The Di-IF was removed in vacuo and the residue dissolved in 100 ml of chloroform. The chloroform solution was washed with 25 ml amounts of N NH, (5X), W HCl (1X) and HPO (2X), dried (Na ^ SO ^) and evaporated to give 6.2 g (90%) of compound 9 .

mp 135-136 ° C

TLC: R ^ 0.92 Rf ^C 0.81 Rf ^D 0.80

example 10 Mep (Bzl) -Tyr-Phe-NHNH ₂ (10)

A solution of Mep (Bzl) -Tyr-Phe-OMe (3.0 g, 5.8 mmol)

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and IiH ₂ NH ₂ -K ₂ O (1.5 ml, 30 mmol) in a mixture of 50 ml of ethanol and 20 ml of DMF was allowed to stand at room temperature for 24 hours. The crystalline hydrazide 10 was collected on a filter, washed with 6 portions of methanol and _{dried in vacuo over H 2} SO to give 2.1 g (70 %).

rap 246 ° C

.. '"'. i _D -19.9 ° (c 1.0 dimethylformamide)

Example 11
Hep (BzI) -Tyr-Phe-Asn-Cys (BzI) -Pro-D-Arg (Tos) -GIy-NH ₂ (11)

The protected hexapeptide amide 6 (518 mg, 0.5 mmol) was dissolved in 10 ml of 2.5 M HBr in acetic acid. After 1.5 hours the hydrobromide salt was precipitated with diethyl ether, collected on a filter, washed on the filter with several portions of diethyl ether and dried in vacuo over NaOH. The salt was dissolved in 5 ml of dimethylformamide, the solution was made basic (pH 8.0) with triethylamine and cooled to -15 ° C. This solution was added to a solution, cooled to -15 ° C., of the azide prepared in situ from compound 10 (286 mg, 0.55 mmol) using isoamyl nitrite. The reaction was allowed to proceed with stirring at -15 ° C for 2 hours, the pH was adjusted with triethylamine to 8.0, and the reaction was held 24 hours at 4 ° C for. The solution was reduced to 1 ml in vacuo (oil pump)

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concentrated and diluted with 15 ml of ethanol. After standing overnight at 4 ° C., the precipitate was collected on a filter, washed with several portions of ethanol and dried, yielding 550 mg (79 %) of the protected nonapeptide amide 11.

mp 203-205 ° C

ιλιiJp-26.9 ° (c 0.5, acetic acid)
TLC Rf * 0.53 H ^ 0.74

Example 12

4 8

(Asn) -desamino-D-Arg -vasopressin (4-Asn-DDAVP) (12)

A solution of the protected Nonapeptidamids 11 (250 mg, 0.180 mmol) in 200 ml of NH, at -40 ⁰ C was added in intervals with Na (which was mounted in a narrow pipette) until in the solution a blue color after removal of the Na stayed behind. The permanent blue color was after 2 minutes by addition of 190 NH ^ Cl mg removed. The NH3 was evaporated in a slow stream of Np. The residue was extracted with two 20 ml portions of ethyl acetate, dissolved in 300 ml of water and the solution adjusted with acetic acid to pH 6.8. The oxidation was carried out at pH 6.8 by adding 3.6 ml of 0.1 MK, Fe (CN) ^. After stirring for 10 minutes, a 50 ml bed of IRA 400 (Ac ") ion exchange resin was added to the yellow solution,

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the suspension was stirred for 30 minutes and the resin was filtered out. The resin was washed with several portions of water, and so was the filtrate and wash together were acidified to pH 3> 9 and freeze-dried.

200 mg of the lyophilizate were dissolved in 10 ml of 50% acetic acid, applied to a 2.5 x 114.5 cm Sephadex G-15 column, which had been equilibrated with 50% acetic acid , and applied at a flow rate of 100 ml / Hours. eluted. The main peak, found at 280 nm and centered at 240 ml, was collected, diluted with one volume of HpO and freeze-dried to give 87 mg of the desalted peptide 12.

86 mg of desalted peptide 12 was dissolved in 10 ml of 0.2 M acetic acid and applied to a 2.5 x 112 cm column of Sephadex G-15 equilibrated with 0.2 M acetic acid and with the same solvent at a flow rate of 100 ml / hour. eluted. The the single peak (510 ml, 3.90 V _Q) corresponding fractions were collected and freeze-dried to give 43.5 mg of the compound 12th

Ί ♦ ■ "· '.: ■ jp-53.7 (c 0.2068 1 % acetic acid) AAA: Tyr 1.05 Phe 1.10 Asp 2.12 Per 0.99 Arg 1.04 GIy 1.00

TLC Rf ⁴ O ^ I Rf ³ 0.51 rA ^ o

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Example 13
Cbz-Gln-Cys (BzI) -Pro-D-Arg (Tos) -GIyNH ₂ (13)

A slurry of the protected tetrapeptide amide 4 (4.0 g »^» 95 mmol) in 15 ml of acetic acid was treated with a solution of 2.5 M HBr in acetic acid for 1 hour and poured into 300 ml of diethyl ether with stirring. The white precipitate was collected on a filter, washed with 200 ml of diethyl ether, dried in vacuo over NaOH for 5 hours and dissolved in 125 ml of methanol. A slurry of IRA 420 (OH) ion exchange resin (40 ml bed) in methanol was added and the mixture was stirred for 10 minutes. The resin was filtered off and washed with methanol. The filtrate and wash together were evaporated to an oil which was dissolved in 20 ml of dimethylformamide. Cbz-Gln-ONp (2.0 g, 4.95 mmol) was added, the pH was adjusted to 7.5 with triethylamine, and the solution was allowed to stand at room temperature for 24 hours. The solution was concentrated to 5 ml (oil pump) and the resulting viscous oil was diluted with 5 ml of methanol. The protected pentapeptide amide was precipitated by adding 50 ml of ethyl acetate, collected on a filter and washed with 50 ml of a solution of ethyl acetate and methanol in a ratio of 4: 1 and with 100 ml of ethyl acetate. The dried peptide 13 weighed 2.94 g (63 %) .

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• jp - 20.2 ° (c 0.960 dimethylformamide) TLC Rf ^A 0.60 Rf ^C 0.28 Rf ^D 0.27

Example 14
Cbz-Gln-Gln-Cys (BzI) -Pro-D-Arg- (Tos) -GIyNH ₂ (14)

The protected pentapeptide amide 13 (1 »05 g, 1.12 mmol) was dissolved in 14 ml of 2.5 M HBr in acetic acid. After one hour at room temperature, the hydrobromide salt was precipitated with diethyl ether, filtered off, washed on the filter with several portions of diethyl ether and dried in vacuo over NaOH. The precipitate was dissolved in 12 ml of dimethylformamide, cooled to 0 ° and Cbz-Gln-ONp (0.51 g, 1.25 mmol) and triethylamine (1.0 ml) were added. After 24 hours at room temperature, the dimethylformamide was evaporated (oil pump), the residue was diluted with ethanol and the resulting solid was filtered and washed on the filter with several portions of ethanol. After drying over PpO ₁ - the protected hexapeptide amide 14 had a weight of 1.04 g (87 %).

mp 155-160 ° C TLC: Rf ^A 0.45 Rf ¹⁵ 0.10

, · Jp - 26.8 (c 0.537 dimethylformamide)

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Example 15
lIep (Bzl) -Tyr-Phe-Gln-Üln-Cys (ß2l) -PrO-D-ArS (TOs) -UIyNH ₂ (15)

The protected hexapeptide amide 14 (40 mg, 0.33 mmol) was dissolved in 15 ml of 2.3 M HBr in acetic acid, after one hour the hydrobromide salt was precipitated with diethyl ether, collected on a filter, washed on the filter with several portions of diethyl ether and dried in vacuo over NaOH. The salt was dissolved in 5 ml of dimethylformamide, the solution made basic with triethylamine and cooled to -10 ° C. This solution was added to a solution, cooled to -15 °, of the azide prepared in situ from compound 10 (260 mg, 0.50 mmol) using isoamyl nitrite. The reaction was stirred at -15 ° C for 2 hours, the pH was adjusted to 8.0 with triethylamine and the reaction was kept at 4 ° C for 24 hours. The dimethylformamide was removed (oil pump) and the residue treated with ethanol. After standing overnight at 4 ° C., the precipitate was collected on a filter, washed with several portions of ethanol and diethyl ether and dried, yielding 285 mg (53 %) of the protected nonapeptide 15.

mp 190-195 ° C

1 * Iq ⁵ - 32.8 (c 0.47 95 % acetic acid) TLC Rjf * 0.51 Rf ^B 0.80 Rf ** 0.93

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Example 16
(Gln ⁵ ) -desamino-D-Arg ⁸ -vasopressin (5-Gln-DDAVP) (16)

A solution of the protected nonapeptide amide 15 (175 mg, 0.123 mmol) in 100 ml of NH at -40 ° C. was treated at intervals with Na (which was drawn into a narrow pipette ) until the solution turned blue after the Na was removed remained. The remaining blue color was decolorized again after 2 minutes by adding 150 mg of NH-Cl , and the NH

^H 3

was removed from Np in a slow stream; the residue was extracted with two 20 ml portions of ethyl acetate, dissolved in 250 ml of water and the solution was adjusted to pH 6.8 by adding acetic acid. The oxidation was carried out at pH 6.3 by addition of 2.5 ml 0, 1 M K ₅ Fe (CN). ₆ After 10 minutes a 50 ml bed of IRA-400 (Ac ") ion exchange resin was added to the yellow solution, the suspension stirred for 10 minutes, and the resin filtered off. The resin was washed with several portions of water and the filtrate and wash liquid together were acidified to pH 3.9 with acetic acid and freeze-dried. 150 mg of the lyophilizate were dissolved in 10 ml of 50% acetic acid and applied to a 2.5 x 114.5 cm Sephadex G-15 column , which was filled with 50% acetic acid had been balanced and eluted with the same solvent at a flow rate of 100 ml / h. The main peak, which was found at 280 nm and was centered at 220 ml , was collected.

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gen, diluted with a volume of H, 0 and frozen.r «>■■; · ■ resulting in 90 mg. The desalted peptide was ^ r-. 10 ml of 0.2 M acetic acid diluted, applied to a 2.5 x 105.0 era Sephadex G-15 column, which had been balanced with 0.2 M acetic acid, and with the same solvent at a flow rate of 100 ml / Hours. eluted. The fractions corresponding to the peak at 2.63 V ₀ were pooled and freeze-dried to give 62 mg of compound 16.

⁵ "^{59> 9} ° ( ^c ° ' ^{291 1} * acetic acid) AAA Tyr 1.03 Phe 1.06 GIu 2.01 Pro 1.06 Arg 0.96 GIy 1.00 TLC Rf * 0.14 Rf ³ 0, 48 R ^ 0.51

Example 17 Cbz-Asn-Gln-Cys (BzI) -Pro-D-Arg (Tos) -GIy-NH ₂ (17)

The protected pentapeptide amide 13 (1.05 g, 1.12 mmol) was dissolved in 14 ml of 2.5 M HBr in acetic acid. After one hour at room temperature, the hydrobromide salt was precipitated with diethyl ether, filtered off, washed on the filter with several portions of diethyl ether and dried in vacuo over NaOH. The precipitate was dissolved in 12 ml of dimethylformamide, ^{cooled to 0} ° C., and Cbz-Asn-ONp (0.51 g, 1.25 mmol) and triethylamine (1 ml) were added. After 24 hours at room temperature the dimethylformamide was evaporated (oil pump) and the residue became

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^'27 "BAD ORIGINAL

diluted with ethanol, the resulting solid filtered off and washed on the filter with several portions of ethanol. after drying over P ₀ Oc, the protected hexapeptiaamid 17 resulted in a weight of 0.87 g (74 %).

mp 175-180 ° C

· .- ■ Jp -24.9 ° (c 0.694 dimethylformamide) TLC Kf ^A 0.45 Kf ^D 0.10

Example 18
MepiBzD-Tyr-Phe-Asn-Gln-Cys (BzI) -Pro-D-Arg (Tos) -GIy-NH ₂ (18)

The protected hexapeptide amide 17 (400 mg, 0.38 mmol) was dissolved in 2.3 M HBr in acetic acid. After one hour the hydrobromide salt was precipitated in diethyl ether, collected on a filter and washed on the filter with several portions of diethyl ether and dried in vacuo over NaOH. The salt was dissolved in 5 ml of dimethylformamide, and the solution was basified with triethylamine (0.5 ml) and cooled to -10 ⁰ C. This solution was added to a solution, cooled to -15 ° C., of the azide prepared in situ from compound 10 (260 mg, 0.50 mmol) using isoamyl nitrite. The reaction was stirred at -15 ° C for 2 hours, the pH was adjusted to 8.0 with triethylamine , and the reaction was held at 4 ° C for 24 hours. The dimethylformamide was

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removed (oil pump) and the residue treated with ethanol. After standing overnight at A ⁰ C, the precipitate was collected on a filter, washed with several portions of ethanol and dried and gave 185 mg (35 %) of the protected nonapeptide 18.

mp 210-215 ° C

^5-28.3 (c 0.530 dimethylformamide) D

TLC Rf ^A 0.51 Rf ³ 0.80 Rf ^E 0.91

Example 19

(Asn -Gin) -desamino-D-Arg -vasopressin (4-Asn-5GIn, DDAVP)

A solution of the protected nonapeptide 18 (100 mg, 0.071 mmol) in 100 ml of NH at -40 ° C. was treated at intervals with Na (which had been drawn into a narrow pipette) until the solution turned blue when the Na was removed stayed behind. The constant blue color was removed again after 2 minutes by adding 140 mg of NH / Cl. The NH ^ was evaporated in a slow stream of N _2. The residue was extracted with two 20 ml portions of ethyl acetate, dissolved in 200 ml of water and the solution was adjusted to pH 6.8 with acetic acid. The oscillation was carried out at pH 6.8 by adding 1.5 ml of 0.1 IC ₅ Fe (CN) ₆ . After 10 minutes

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A 50 ml bed of IK.A-400 (Ac ") ion exchange resin was added to the yellow solution , the suspension was stirred for 10 minutes and the resin was filtered off. The resin was washed with several portions of water , and the 'iltrat and the v / aschflüssigkeit together were acidified to pH 3 »9 and 70 mg of the freeze-dried gefriergetrockne th wet were suspended in 10 al 50 ^ acetic acid and applied to a 2.5 ^ 114.5 cm -. column of Sephadex G-15 given up, which had been balanced with 50% acetic acid, and eluted at a flow rate of 100 inl / h. The main peak found at 200 mn, centered at 245 ml, was collected, diluted with one volume of water and freeze-dried and yielded 54 mg. The desalted peptide was dissolved in 10 ml of 0.2 M acetic acid and applied to a 2.5 × 105 cm Sephadex G-15 column which had been balanced with 0.2 μl acetic acid and with the same solvent eluted at a flow rate of 100 ml / h.. The be the peak i 2.84 V _Q appropriate fractions were collected and freeze-dried to give 30.0 mg of the compound 19th

. 25th
'D ~ 5 ^ * 3 (c 0.287 1 % acetic acid)

AAA Tyr 1.07 Phe 1.11 GIn 1.03 Per 1.03 Arg 0.99

GIy 1.00 Asp 0.98
TLC Rf ^A 0.18 Rf ^B 0.46 Rf ^E 0.53

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Claims (3)

Claims Θ 4 Anti-diuretic eesamino asparagine - D-arginine -Vasopressin of the formula (ι Mep-Tyr-Phe-Asn-Asn-Cys-Pro-D-Arg-Gly-Nl ·! ^ 123456 7 89 ^ 2. A process for the preparation of the antidiuretic active desamino-asparagine -D-arginine -Vasopressins according to claim 1, characterized in that the amino acid chain is gradually added in a manner known per se Mep (SH) -Tyr-Phe-Asn-Asn-Cys (SH) -Pro-D-Arg-GIy-NH 1 23456 7 8 9 builds up, in which Mep means 2-mercaptopropionyl, 4 and that this amino acid chain becomes desamino-asparagine D-arginine -Vasopressin's formula Mep-Tyr-Phe-Asn-Asn-Cys-Pro-D-Arg-Gly-INlH ₂ oxidized. 709850/0851 ORIGrtNAL INSPECTED 3. The method according to claim 2, characterized in that the oxidation is carried out by means of potassium ferricyanide in aqueous solution at a pH 6.5-7.0.