SI8011695A8 - Process for obtaining novel tripeptide derivatives - Google Patents

Description

PROCEDURE FOR OBTAINING NEW TRIPEPTID DERIVATIVES

1. FIELD OF THE INVENTION

The invention is in the field of chemistry of natural organic compounds and more specifically the chemistry of peptides, and according to the International Patent Classification bears the following designations: C 07 C 103/52.

2. Technical problem

A technical problem to be solved in the present invention is to provide a chemical process for the production of novel tripeptides, which act on the central nervous system, and which have the following general formula (I):

X - Y - W - nh ₂ in which they represent

X L-pyroglutamyl-, D-pyroglutamyl-, L-2-ketoimidazolidine-4-carbonyl-., L-6-keto-pipecolyl-, L-thiazolidine-4-carbonyl-, L-prolyl-or orotyl group,

Y L-Leucyl-, L-norvalyl- or L-histidyl-group,

W is L-prolyl-, D-prolyl, L-thiazolidine-4-carbonyl-, L-homoprolyl-, L-leuyl-, L-isoleucyl-, L-methionyl-, L-pipecolyl-or D-pipecolyl-group, further -W-NH ₂ together denotes the pyrrolidyl- or piperidyl-group, but if X denotes L-pyrrolglutaminyl- and Y

- 2 denotes the L-hietidyl group, then '' and has a meaning different from

L-prolyl groups,

These new tripeptide derivatives, which correspond to the general formula I defined above, are analogues of L-pyroglutinyl-L-histidyl-L-prolinamide (Glp-His-Pro-RUg), which are known as thyrotropin-releasing hormone releasing hormones. ” TRH), and in which some amino acid groups have been replaced by certain other amino acid groups (given in the broader definition of general formula I), or in which -K-MIg is given <

case replaced by pyrrolidyl- or piperidyl- group.

3. State of the art

The TRH existences were still known in the Sixties, but its structure was only clarified in 1969 and 1970, respectively. · Rodins almost simultaneously from R. Guillemin and A. Schally, Research Group Parties (who did so independently of one another) (compared by Bovers , CY, etc., Endocrinology 86, 1143 (1970); R. Burgus etc., CR Aced. Sci. (Paris) 269. 1870 (1969) The tripeptide TEH was originally described as a sensation.Discovery of TSH (thyrotropic hormone) in the pituitary gland mammal a regulating factor, however, research on this hormone is headed in one new direction, when it has been found that the biological function of this tripeptide is not limited to regulating the release of thyrotropic hormone, but also has its effect on the central nervous system / compare * NP Plotnikoff et al., Science 178 _t 417 (197P), AJ Prange et oar ·, Loncot 2, 999 (197P) / · Metin J «found

- 3 that TRH, in addition to the aforementioned hormonal function, significantly reduces the duration of barbiturates or alcohol elicitation, further reduces and hypothermia caused by different drugs increases locomotor activity. A further significant factor in the effect of TRII on the central nervous system is the suppression of catalepsy ** caused by hal opera down. On the basis of this, it seems desirable for the therapeutic practice to produce such analogues of TRH that only have a weak effect on the pituitary gland, but, in contrast, show effects on the central nervous system that reach even exceed the effects of TRH. To this end, compounds of this type have been made described in DS-OS 2 W 055, 2 W 057, 2 449 16 ?, 2 514 581

609 154, and 2 659 395 as in BE-PS 819 198. The research to date (relating to these compounds) ', its results and experiences have been compiled by Λ. Of J. Prnnge and eax. / The Role of Hormones in Depression, Life Pcionce 20, 1305 (1977) / and A. V. Schally et al. /H.ypothalatiic Regulator? Hormones, Ann. Rev. »3iochem. 47, 89 (1978) /, but could not, however, lead to results which in all respects satisfy the requirements of therapeutic practice.

- 4 ...:

however, it could not produce results that satisfy the requirements of therapeutic practice in every respect. .

It has now been found that by eystematically replacing some amino acids of a TRS molecule (awarded with three amino acids), the hormonal action of the three "peptides can be completely abolished, or at least significantly reduced, but on the other hand maintained or even significantly increased by the central- system. Particularly suitable were those derivatives in which instead of the histidine group in position 2 there was an aliphatic amino acid group containing a bumpy or branched carbon sequence. In the interest of even more selective biological action, it has also proved appropriate to incorporate a 6-keto-pipecolic acid group into this molecule instead of the pyroglutamyl ring system.

Tripeptide derivatives corresponding to the general formula 1 defined above, according to the present invention, are obtained from the corresponding amino acids or amino acid derivatives according to the known peptide chemistry methods, such that

a. starting from compounds of general formula II

W - nh ₂ in which W has the meaning given above, (II) i

It »

4a i

Eat n article in J. Med. Cunm./ljh 484 (1971) / bsvi does not shake the action of the homonic str: kfcurno VRH-annlo ;; · ?, this action on the central nervous system. Another similar topic jo ubradjon · »by G. Π. Pettit · 'in 5ynthetic Poptides ”Vol. 4, p. 17 · 171. - Vedic patent file no. 564 518 touched upon the question. (by • »* right for the rite of Hgpothyroidis« ius-) pyrrolutamyl-hiiuidinpyrrolidide, whose action in the field of endocrinology is atped? .. It may also be stated that these literary tours treat one entirely different area • TRii-istr «is lively, who jr is opposite the area. these submissions.

In a publication of notes by Phana. Snec., 16, 95 (1979 / there is a document which speaks fluently of the availability of resuV-ιί, rijnve. This * lunak, namely, emphasizes, the basis of -, vu eats 1; r r - lelic intra * .iv. Hormonal ^ and DUS actions ··, JR7, and HIP “warrant-. ·, d” ·: with JRH analogs, the requirements are hotter. The structure is the same 23 reaching JRS and hormonal: ^ actions, 'ie j Mino or other action does not separate it In the sense of odor, it gives off stubborn targets, which is precisely what can be achieved, that is, an increase in the CMS of the hormone depletion factor ·· * (see Table I).

Table. I

Natural TRII

Hormone Action CrD Action 1 1

/Scientific patent application 42S ', 59

Kpc-iIis-Tca-KH ₂

KPc -tii s-Pro-NHp

») -Kpc-His-Pro-KHp

I »i c -Hi s-Pro-KHo

C.

Ki c -i! and with -Pro-OCH, __2_

Proposed Achilles Kpc - ^T 'va -Pro-PIio r.po -Leu-Pr <i-RH _o

f.

Glv-Leu-Tco-PHp Kpc-I ' ^r ~ -HPro-ulU f.

, Glp-Lou-Fip-KIL,

1 R 1 4 t-, 2 5 M 1 C, 001 0

G 8

0.027 3.5 0 1.6 0 1.3 o, v? z 14

In the comparison of javg bp. 425 6.59, crs · jOdinjčA- ·. poddejntvA;

he «trufc; -,» i! ie-citj * fw .ρχ / χη / Λ-Γ- the adjective of the proposed pr / JAtf.e, u. n Λ formerly selective j e.

s'i

-4b ~

4. Description of the solution to the technical problem

It has now been found that by systematically replacing some amino acids of the TRH molecule (rewarded with three amino acids), the hormone action of the tripeptide can be completely aborted, or at least significantly reduced, but on the other hand maintained or even significantly increased by the central nervous blue effect. Particularly suitable were those derivatives in which instead of the histidine group in position 2 there was an aliphatic amino acid group containing a bumpy or branched carbon sequence. In the interest of even more selective biological action, it has also been shown to be convenient to incorporate a group of "6-keto-pipecolic acid in this molecule instead of the pyroglutamyl ring system.

Tripeptide derivatives, corresponding to the general formula (I) defined above, according to the present invention are obtained from the corresponding amino acids or amino acid derivatives according to the known peptide chemistry methods, such that a. starting from the compound of the general formula til) w-nh ₂ (II) in which W has the meaning given above ^ of a molecule of the general formula I, in the steps, according to the coupling method, which are common in peptide chemistry, suitable with the use of active esters, mixed dicyclohexylcarbodiimide anhydride iii. iii

b. what are the compounds of general formula II,

W - nh ₂ (II) in which V name above deto meaning, <

are acylated by the azides derived from dipeptidhydrazides of general formula III

Z - X - T - nh-nh ₂ (III) in which Z denotes a benzyloxycarbonyl-β hole and X and

Y has the meanings given above, and then from the compounds of the general formula IV zxtw - nh ₂ (IV) in which Z, X, Y and W have the above detached meanings (which au obtained according to one of the above methods), cleave protective group Z primarily by ketalitic hydrogenation and the resultant product of general formula I is isolated from the reaction mixture and / or optionally converted into a therapeutically applicable complex.

- 6 In the process of stepwise construction of the molecule, the useful or starting compound of the general formula k-HHg used in excess reacts with the $ activated derivative, in particular the pentafluorophenyl ester of the protected amino acid of the general formula BOC-T-OH, in which BOC denotes a protective tert-butyloxycarbonyl group, wherein the corresponding BOC-TV-NHg dipeptide derivatives are obtained over a very short reaction time (within minutes), in a form which can be slightly hardened and which generally requires no further purification. However, these same dipeptide derivatives of the general formula BOC-T-Vf-NHg may also be rewarded by other coupling methods, e.g. aa mixed anhydrides iii aa alobodic acids in the presence of dicyclohexylcarbodiimide.

Dipeptides of the general formula HYW-NH ₂ , which are liberated by acidolysis from the obtained protected derivative of dipeptide B0C-YW-NH ₂ , are then conveniently reacted with the pentafluorophenyl ester of a protected amino acid of the general formula Χ-Χ-ΟΗ to give the above Suitable Method Described Suitable Tripeptide Derivatives of General Formula ZXYW-NH ₂ ·

The acylation of the starting compound W-NH ₂ with the azide, which can be obtained from the hydrazide of the general formula ZXY-NH-NH ₂ , has the advantage that the said hydrazides are intermediate products which can be very well crystallized and therefore tomeldolized in a very pure form.

-7 _

Compounds of general formula I containing the pyroglutamyl group at site X can be constructed by forming the pyroglutamyl ring only in the last synthesis stage from one glutamine group, which is fluid, which introduces the glutamine keo germic acid into the molecule and thus the resulting tripeptide of the general formula Gln-TW-NH2 was heated for a few minutes in acetic acid.

v '

From protected tripeptide derivatives of general formula

The Z-Z-T-V-NHg, * obtained by one of the methods described above, can be used to liberate the offended end products of general formula I, preferably by the application of catalytic hydrogenation. Purification of the obtained lime products can be accomplished by simple crystallization! "Or by precipitation, if necessary by check and column chromatography. In this case, the final product can be prepared by simple lyophilization after removal of the by-products."

Tripeptide derivatives of general formula I, obtained by the process of the present invention, have been tested for their pharmacological action by the biological methods described below.

- 9 1. Suppression of haloperidolma ketalppaya in rats

- i, (Compare: J. Delaj and P. Deniker: Coapt. Sand. Cong. Med. Alenietes Naurologistes, $ 1, 497, Luzemburg 1952) mg / kg Haloparidole, i. 4- (p-Chlorophenyl) -1- [5- (pfluorobenzoyl) -propyl-piperidin-4-ol, given animal pits s.c. and after 120 minutes, the occurrence of catrflexion was controlled; I harvest eu rats divided into groups of 10 animals each and treated intravenously with these doses of TRH or new TRH analogs of tripeptides. Control group animals were treated with saline ee. The effect of the individual compounds interrupting catalepeia was examined at an intervalue of 15, 5θ and 120 minutes after treatment.

Those animals were considered cataleptic, which, when affixed with their front paws to a 7 cm high pole, did not change their position for 50 seconds.

From the number of animals that do not show catalepsy, the corresponding ED ^ q values for individual active substances were determined by probit analysis.

These experiments were performed with male rats of strain V / istar of individual weight 160 to 180 g.

- 9 2. Potentiation of locomotor activity in L-dopa-elicited mice (Compare: The Thyroid Axis, Drug and Behavior, p. 116, A. J «Praga Jr., Raven Press, New lork, 197 *} '

Life in the pit was first intraperially tonally given at 40 mg / kg of N-methyl-N-propargyl-benzylamine (Pargillin), then at 20 mg / kg of TRH, at the ratio of latent doses of new tripeptides to be tested, and finally at 100 mg / kg. kg L-dopa · After 50, 60, and 90 minutes after the * upper treatment, the locomotor activity of these animals was measured / the values obtained are given in the following table as a percentage of the values obtained in the animal treated with aa TRH. For these experiments, 15 rye mice weighing 18 to 22 g each were used.

5 · The effect of reversible hypothermia in mice (Compare: B. M. Askew: Life Sci. 2, 725-750 (1965))

Mice in males, weighing 18 to 22 g each, were divided into groups of 10 animals each.

i.p. 5 mg / kg rezeppine each; after 16 hours, these animals were treated with 88 doses of 20 mg / kg TRH or tripeptide under test.

The rectal temperature of the aerated animals was treated with aer reserpino before treatment (aa in the following table), 16 hours after reserpine treatment (indicated in the table as res ·), and 1 and 2 hours after administration of the test tripeptide (in the table indicated as a post-stroke!} · The table shows the average values of rectal temperatures measured in mice.

* *

4. Effect on duration of hexobarbital-induced epithelium

House males, divided into groups of 10 animals, were intravenously detoxed at 60 mg / kg Na-hexobarbital p

(Evipan Bayer); after 10 minutesβ the animals were treated intraperitoneally with doses of 20 mg / kg TRH respectively. "

of the test compound. The duration of sleep is given in the following table in percentages relative to the values measured in the animals of the cohort group. The mean values obtained in 10 animals were taken from the animals.

- 11 5. Narcosis with ethanol (Compare: J. M. Cott et al., J. Bharmacol. Ezp. Ther.

196. 59 * (1976)

CPLP (LATI) mice, mixed sex, individual & weighing 18 to 22 g i.p. was given 4.5 g of ethanol t after 10 minutes of this animal and then treated intraperitoneally with doses of 20 mg / kg of tripeptide tested. In the following table, the detention time of the det eu in percent relative to the value measured in the control group animal. The mean values of measurements taken in 10 animals were taken from the sample.

6. Hormonal activity (action of TSH {at peco ^ a

Wister rats, males, each weighing approximately 200 g each, were divided into groups of 7 to 8 animals and treated intravenously with doses of 20 mg / kg TRH or tripeptide tested. The TSH response of these animals was determined 15 minutes after treatment with the TRH and TRH analogues from the plsma animal using radioimmunoassay. Relative effect sizes were calculated using a four-point method using a TPA 101 computer, whereby the TRH effect size (magnitude) was expanded to be 100.

- 12 The following table summarizes the biological values of the more important compounds of the general formula I, which were determined above by the pharmacological methods described.

cr fc »  *. Eph f- cu ca. ca. • fc • fc f-s Eph m O t> p-

%

O o

ft

Table o

β r4

O i

P

Φ i

h <6 O La β rf

1- »φ S pp p> tj p o © rf o β> P A O -H> TO X 44» N cn ko p ~ co m

ft in

In vo and tn rf • rs

P -p r ~ m

σι m

co <n tn io 'X) vo pcn en ko in and

OH Φ S TO rf S> TJ P O 44 p v «j o c> TO -H P ft o Ή XJ P> ra jg, 44> n

rf β o rf o e P φ • fc φ 44 rf tl TO p P β rf • H P Φ 3, rf r-1 P tl TO Φ Φ Oh TO A ft Φ • ε β φ φ ΠΡ • Φ • H TO Ό e d Φ R β β rf Φ ft

CM

P -P rf TO O P ί-t ΛΧ 44 -H TO O XJ k

A o

P *

O -H ® fiP t-3 o TO β 44 O d o P r — t

H

O rf P β Λ44 TO O rf P TJ o i to Aft β ο

σ>

it «it © a« ο

νο

TO β

rH φ Φ Ο '^> r-j'd -η

Φ Φ β Α (STOTO Η ftr-l Ο Ο rf N r-j Ρ rf rf rf TO X! 44 (M

I

N

I

H

I

H

Φ

P

Φ

P>

σι and CM Oh P- Oh CM kO co CM • fc • fc • k · »· · «K «Fc «K • fc • fc • fc tn CM CM r-1 o σι CJ and oo o tn tn m tn tn CM tn CM CM tn Oh r-4 p- and 00 r ~ ' and «K · » «K • fc «Fc • fc • fc • k • fc • k CM tn tn o r-1 o kO and c- and tn tn K \ ΚΛ tn tn CM CM CM tn and cn cn 10 00 kO P- ft σι fc «K «Fc «Fc «Fc «K «Fc «Fc • fc «Fc tn xtf · CM tn o P- and CM CM CM CM tn CM CM CM CM CJ tn m CM 4- and tn and and CM «H • k «Fc «Fc «H «B • fc • fc • k • fc kO kO kO 10 kO 10 10 kO and kO tn tn tn tn tn tn tn tn tn tn p- and CM CM σι o 00 and 00 Oh CM 10 and r-4 C— r- o ft cn Oh r-4 <—4 rH r-4 r — 1 2 and CM cn t- 00 o ao o o co «Σ $ - M- oo M ^- tn tn m P- o r-1 r-4 and — 1 cn kO ft r — 4 and tn m M 00 o H tn C * - o cn Oh kO and f - 4 o tn r-4 CM ft »—1 rH r-4 Oh Oh o Oh Oh o and m o and kO tn CM CM tn tn r — 4 r-1 tn t — 4

β «H ko r ~ tn h * * ft CM +1 +1 m- <n» k «k

10 m m in ft

W δβ δβ

C3

OJ> 4

and '0 tn tn «Fc «Fc • k «Fc • fc • fc tn o r-4 00 <O o and o o Q CJ rd tn tn m 10 tn P- cn o o rf ft rf rf  in P o o β P v ft o Oh ft P P ft ft El t-M El El n: i-M ft rf rf rf rf rf rf rf rf Φ eu ϊΐ Φ Φ Φ > > > ft ft Eph ft « ft ft l - · / ft .S tt ft 0 o ft ft ft Oh o o ft ft r-1 ft r-4 ft ft 1 ft w M o Oh o M P h4

W w this one σι +1 +1 P4 e-i 'χ. • rf • o β rf > rf ft ·· TO rf n Φ β ε rf a> > P rf > ft n o β TO p * fc TO P P ft ί » yes P Oh rf β , a rl o Oh TO β 44 rf TO 4J , β TO

From the values given in the table above, it can be concluded that these novel TRH analogues, in which two or three amino acids of the TRH molecule have been replaced with other amino acids, exhibit significant effects on the central nervous system.

In this aspect, derivatives are particularly suitable, in which the ηβ site of the His group at position 2 of the TRH molecule contains an aliphatic amino acid with a non-branched or branched carbon chain. Likewise, from that aspect, this one in

effects, suitable are those analogues of TRH, in which the Glp group is replaced by 6-keto-pipecolic acid. With these derivatives, the hormonal effect of TRH iii is completely discontinued or reduced to a minimum, while at the same time a very significant effect is exerted on the central nervous system, in some cases elevated even to eightfold.

The novel tripeptides obtainable according to the present invention, as well as their pharmaceutically applicable salts or complexes, can be masked in therapy in the form of the usual 1ek preparations. These preparations contain the medicaments by the invention of the given active substance together with inorganic or organic carriers, which are suitable for enteral or parenteral administration. Medicinal preparations can be obtained e.g. in the form of tablets, dragees, injectables, lyophilisates, etc. The preparation of these dosage forms can be accomplished by conventional pharmaceutical methods.

- 15 Practically performing the methods of the present invention, that is, chemical preparation, and finding new analogues of TRH will be more fully explained by the aid of the following examples. The abbreviations used ee in these examples correspond to abbreviations that are common and conventional in peptide chemistry (compare J. Biol Chem. 24-7 997 (1972). In the following examples, the following abbreviations are used:

HPro

Kic

Kpc

Oro

Pip

Tea

DCC

DOT

PPPOH

DMPA

L-homoprolin *

Zolidine-4-carboxylic acid l-2-kat oimide

L-6-ketopipecolic acid orotic acid L-pipecolic acid L-thiazolidine-4-carboxylic acid dicyclohexylcarbodiimide dicyclohexylurea p enta fluorf enol dimethylformamide.

The melting points of these compounds, which are inferred in the pneimers, were determined by the aid of a melting point apparatus according to Dr. Tottoli (Btichi). Optical quantities (polarized light rotation value) were measured using a Perkin-Slmer 141. type polarimeter. In the tests, thin layer chromatography and / or separation chromatography were performed.

- 16 thin films. Applied in silica gel plate “Kieselgel G naoh Stahl (E. Merck, Darmstadt). Solvent mixtures were used to develop the chromatograms:

Chloroform - methanol 9: 1

2. ethyl acetate: (pyridine: acetic acid: water 20: 6: 11) 95: 5

5 · ethyl acetate: (pyridintaracetic acid: water 20: 6: 11) 9: 1

4. ethyl acetate: (pyridine: acetic acid: water 20: 6: 11) 8: 2

5 · ethyl acetate: (pyridine: acetic acid: water 20: 6: 11) 5: 2

6. ethyl acetate: (pyridine: acetic acid 20: 6: 11) 2: 3

A ninhydrin solution was used to develop the mrl; after spraying, the plates were dried for about 5 minutes at 105 ° C. · Then, these chromatograms were placed in an atmosphere of chlorine gas and, after aeration, developed with o-tolidine-potassium iodide solution.

Purification of these products by column chromatography using Kieselgel G (E. Merck) (silica gel) by E. Merck, with a particle size of 0.062 to 0.2 mm.

For evaporation of the solution in vacuum, Rotevepor was applied

R iBttchi) vacuum evaporator; the pairing was performed at temperatures not exceeding 50 ° C.

The amino acid pentafluorophenyl esters protected by the BOC group were prepared according to the method of L. Kisfaludy-8 and aar., Ann. 1975 «1421.

- 17 Example 1:

L-Pyroglutamyl-L-leucyl-L-pipecolic acid amide

Degree ill:

1-Leucyl-L-pipecolic acid hydrochloride

1.54 g (12 mmol) of H-Bip-NH ₂ was suspended in 20 ml of 1MFA and this suspension was stirred with stirring with 5.16 g (13 mmol) of BOC-Leu-OPPP and 1.68 ml (12 mmol) of triethylamine . The mixture was stirred for another 6 hours, then the resulting solution was evaporated and the oil obtained as a residue was dissolved in chloroform (60 ml). This solution was mixed with 0.2 ml of 2- (dimethylamino) -ethylamine, allowed to stand for 5 minutes and then washed three times with 20 ml of 1 N hydrochloric acid each, three times with 20 ml of 1 N sodium bicarbonate solution each and once with 20 ml of water. It was then dried with anhydrous sodium sulfate and evaporated. The oily residue was dissolved in 3 ml of ethyl acetate and treated with 10 ml of 5 N hydrochloric acid in ethyl acetate. After standing for 1 hour, the reaction mixture was diluted with ether, filtered off the created taiog and dried under vacuum over anhydrous sodium hydroxide.

In this way, 3.12 g of H-Leu-Pip-NH ^ .HCl (94% of the theoretical yield calculated on the starting H-Pip-NH ₂ ) is obtained; R ^) = 0.46.

- 18 S tu pan ,,?

Benzyloxycarbonyl-L-pyroglutamyl-L-leucyl-1-pipecolic acid amide

5.13 g (11.2 mmol) of H-feu-Pip-NHg.HCl and 4.94 g (11.5 mmol) of Z-Glp-OPFP were dissolved in 35 ml of EMFA and treated with 1.57 ml (11 , 2 mmol) of triethylamine. A further 1.57 ml (11.2 mmol) of triethylamine was added to the stirring mixture for 5 minutes and after further stirring for 20 minutes the reaction mixture was evaporated in vacuo. The residue was dissolved in chloroform (90 ml), the aqueous solution was washed twice with 30 ml of 1 N hydrochloric acid each, three times with 30 ml of 1 N sodium hydroxide solution each and once with 30 ml of water. The organic phase was then dried with anhydrous sodium sulfur atom and evaporated. The amorphous residue is stirred with cold ether, the ether is decanted and the residual oil under n-hexane gives a solidification. The amorphous crude product (4.7 g) thus obtained was crystallized from ethyl acetate / ether. 3.32 g (61% of theory) of Z-Glp-Leu-Pip-NI2 are obtained;

Analysis for ΟοςΗ, .Οχ-Ν. (mol. wt. # 86.57)

34 6 4 calculated: C 61.71%, H 7.04%, N 11.51 found: C 61.67%, H 7.05%, N 11.40%.

Level 3:

I '· »,

L-Pyroglutamyl-L-leucyl-L-pipecolic acid amide

2.1 g (4.32 mmol) of Z-Glp-Leu-Pip-NH _{2 were} dissolved in 40 ml of methanol, 0.2 g of 10% palladium on activated carbon (as a catalyst) were added and hydrogen gas was introduced into this mixture. within 1 hour. Then the catalyst was filtered off, the filtrate was evaporated and the remaining three wells were evaporated. The crude product (1.48 g) thus obtained was dissolved in 20 ml of water, decolourised with charcoal and filtered. Dissolve 4 g of sodium chloride in the clear filtrate and wash the solution three times with 10 ml of chloroform each. The organic phase was dried with anhydrous sodium yurasulfate, and the amorphous residue of the three layers was evaporated from a mixture of ethyl acetate and ether. In this way 1.33 g (87.5% of theoretical yield) of GlP-I »eu-Pip-11H _{2 is} obtained; R ^^ * 0.60; / ^ ⁵ - -86.4 ° (c 1, in acetic acid).

- 20 Example 2i

L-plroglutarail-L-leucyl-I-vi indine-4-carboxylic acid amide

Level 1:

Amide hydrochloride

L-Leucyl-L-thiazolidine-4-carboxylic acids * ·

1.81 g (11 mmol) of H-Tca-NHg.HCl (compare S. Ratner and HT Clarke, J. Am. Chem. Soc. 2%, 200 (1937)) was suspended in 30 ml of DMFA and mixed with 3 , 97 g (10 mmol) of BOC-Ieu-OPFP, 1.49 g (11 mmol) of 1-hydroxy-benztriazole and 1.22 acre (11 mmol) of N-raethylmorpholine. The resulting solution was allowed to stand at room temperature overnight, then evaporated in vacuo and the residue dissolved in 80 ml of chloroform. The solution is washed three times with 20 acre solution of 1 N hydrochloric acid, three times with 20 acre each. N sodium hydroxide solution and finally with 10 ml water once. It was then dried with anhydrous sodium sulfate and evaporated in vacuo. The foamy, ararorphous residue was dissolved in 5 ml of ethyl ethoetate, mixed with 10 ml of 7 N hydrochloric acid solution and left to stand for 1 hour. Then the reaction mixture was diluted with ether, the resulting precipitate was filtered off and dried in vacuo over anhydrous sodium hydroxide. In this way 1.84 g of H-Leu-Tca-N ^ .HCl (65 J of the theoretical yield calculated on BOC-Leu-OPFP) is obtained? ^p f ⁵ ) = 0.25; mp 170-174 ° C. (breakup).

- 21 -

Level 2:

• · *.

Benzyloxycarbonyl-L-pyroglutamyl-L-leucyl-L-thiazolidine-4-carboxylic acid amide

1.69 g (6 ramol) of H-Leu-Tca-NH ^ .HCl was suspended in 20 ml of EMPA and mixed with 2.7 g (6.3 mmol) of Z-Glp-OPFP and 0.84 ml (6 mmol) ) of triethylamine. The mixture was stirred for 20 minutes at room temperature, then evaporated in vacuo. After evaporation, the residue is dissolved in 50 ml of chloroform and this solution is washed twice with 10 ml of 1 N hydrochloric acid twice, three times with 10 ml of 1 N sodium bicarbonate solution and finally with 10 ml of water once. The organic phase was then evaporated, the residue was triturated with ether and the crude product obtained was purified by repeated precipitation of ethyl acetate / ether. 1.64 g (56% of theory) of Z-Glp-leu-Tca-HH ₂ ·, mp 108-110%, 0.46 ·, M / p ⁵ = -130.30 ° (c »1 , in acetic acid). »

Level 3:

L-Pyroglutamyl-I-rleucyl-L-thiazolidine-4-carboxylic acid amide

1.62 g (3.3 mmol) of Z-Glp-Leu-Tca-NH _{2 were} dissolved in 6 ml of ice-cooled 3.5 N bromodonifac solution in glacial acetic acid and the solution was allowed to stand for 1.5 hours at 0 -4 ° C. The reaction mixture was then diluted with ether, the upper phase decanted and the remaining oil dissolved in 20 ml of water. The aqueous solution was neutralized with solid sodium bicarbonate and washed

- 22— three times with 10 acre ether. The aqueous phase was evaporated in vacuo, the residue dissolved in 20 acre of chloroform, the solution dried with anhydrous t.

sodium sulfate and evaporated. The amorphous residue was dried over 3 ether and dried. The crude product obtained (1.13 g) was dissolved in a solvent mixture (4) and applied to a column containing 20 g of silica gel. The column was eluted with the same solvent mixture. 0.78 g of an amorphous product is isolated from the fractions containing the pure product. This product is dissolved in 20 ml of water, the solution is discolored with active carbon and the resulting colorless aqueous solution is lyophilized. In this way 0.62 g is obtained

<

»-145.0 ° (c» 1, in eircic acid).

Prime r 3

L -Pi roglutamyl -L-leucyl -D-proi inamide

Level 1:

Benzyl oxycarbonyl-L-pyroglutamyl-L-leucyl-D-prolinamide

1.54 g (5.8 mraol) of H-Leu-D-Pro-HH ₂ .HC1 was dissolved in 20 ml of DMFA and the solution was mixed with 0.81 ml (5.8 romol) of triethylamine and 2.58 g (6 mmol) Z-Glp-OPFP. The mixture was stirred for 5 minutes, then a further 0.81 φ 5 (5.8 mmol) of triethylamine was added and after stirring for another 10 minutes the reaction mixture was evaporated in vacuo. The evaporation residue was dissolved in 60 ml of chloroform, the solution was washed three times with 15 ml of 1 N solution each

- 23 soups of acid, also three times with 15 ml of 1 N sodium hydroxide solution 1 each, at least once with 15 ml of water. Then 't.

is dried over anhydrous sodium sulfate and evaporated. The amorphous residue is rubbed with ether. The crude product obtained (2.1 g) was recrystallized from 5 ml of ethyl acetate; thus, 1.8 g (66% of theoretical yield) of Z-Glp-Leu-DPro-NHg are obtained, mp 153-157 ° C, 0.38-, M / p ⁵ - -24.5 ° ( c »1, in vinegar ki Belina).

*

Level 2:

L-Pi roglutarail-L-l eucyl-D-prol inarai d

1.5 g (3.25 ramol) of Z-Glp-Leu-D-> ro-NH _{2 were} dissolved in 70 ml of methanol, the solution was mixed with 0.3 g of 10% palladium on activated carbon (as a catalyst ) and hydrogen is passed through the solution over a period of 1 hour. After the catalyst was filtered off, the filtrate was evaporated and the amorphous residue was rubbed with ether. The crude product obtained (0.94 g) was dissolved in water, the solution was separated with activated charcoal and filtered. The colorless aqueous filtrate was lyophilized; thus 0.87 6 (79% of theory) of Glp-Iieu-D-Pro-NH ₂ is obtained;

Rp) »0.47; / oC / p + 8.4 ° (c «1, in acetic acid).

24-.

Example 4 • I.

L-Pyroglutamyl-L-norvalyl-L-thiazolidine- »4-carboxylic acid amide

Level 1:

k T *

L-norvalyl-L-thiazolidine-4 * carboxylic acid amide hydrochloride

8.0 g (20 mmol) of BOC-Nva-OH.DCHA was suspended in 60 ml of ether, mixed with 20 ml of 2 N sulfuric acid solution and stirred to dissolve the solid. Separate the ether phase with 20 ml of 2 N hydrochloric acid solution and once with 20 ml of water, dry with anhydrous sodium sulfate atom and evaporate.

As a residue, the resulting oil was dissolved in 40 acres of DMFA, the solution was mixed with 2.8 ml (20 mmol) of triethyl unin, cooled to -15 ° C and treated with stirring and dropwise with 2.5 ml (20 mmol) of pivaloyl chloride at a rate to keep the temperature of the reaction mixture below -5 ° C. The suspension thus obtained is stirred at the same temperature for another 10 minutes.

During this time period, 3.72 g (22 mmol) of H-Tca-NHg.HCl was suspended in 30 acres of DMFA, mixed with 3.1 ml (22 mmol) of triethylamine, filtered off the precipitate formed, and the filtrate was added dropwise to the resulting mixed solution. of anhydride. The addition is carried out by dropping at a temperature of -5 ° C. After the addition was complete, the reaction mixture was stirred for an additional 30 minutes at -10 ° C. Then he was left to stand

-25-.

in z

* t refrigerate overnight and evaporate the next day. The residue * after evaporation will be dissolved in 100 ml of chlorophyll, solution • 1.

Rinse three times with 20 ml of 1 N hydrochloric acid solution, then three times with 20 ml of 1 N sodium bicarbonate solution and finally once with 20 ml of water. It was then dried with anhydrous sodium sulfate and evaporated. The oily residue was dissolved in 20 ml of ethyl acetate, the solution cooled below 5 ° C and mixed with 20 ml of 5 N hydrochloric acid solution in ethyl acetate. The reaction mixture was kept in an ice bath for 1 hour and then diluted with ether. The resulting precipitate was filtered off and dried in a vacuum over anhydrous sodium hydroxide.

In this way 3.77 g of H-Nva-Tca-NHg.HCl (70% of the theoretical yield calculated on BOC-Nva-OH.DCHA) are obtained;

Rp) = 0.20.

Level 2:

J

Benzyloxycarbonyl-L-pyroglutamyl-L-norvalyl-L-thiazolidine-4-carboxylic acid amide

2.8 g (10.5 mmol) of H-Nva-Tca-NH ₂ .HC1 were co-precipitated in 50 ml of DMFA and 1.47 ml (10.5 mmol) of triethylamine and 5.15 g (12 mmol) were added with stirring. Z-Glp-OPFP. The reaction mixture was treated for a further 5 minutes with a further 1.47 ml (10.5 mmol) of triethylamine, stirred for a further 10 minutes and then evaporated in vacuo. The residue was dissolved in 100 ml of chloroform, the solution was washed three times with 20 ml of hydrochloric acid each, and three times with 20 acres of sodium carbonate solution each, and finally with 20 ml of water (one to one time). . It is then dried with anhydrous sodium hydroxide sulphide atom and evaporated. The amorphous trioxide residue with ether and the crude product obtained (4.6 g) was crystallized from a mixture of ethyl ether 1 ether. In this way 5.2 g (64% of theoretical yield) of Z-Glp-Nva-Tca-NH _{2 are} obtained; mp 116-118 ° C; R ^. ⁴ ^ 0.45; M / p ⁵ - 12.9.0 ° (c "1, in acetic acid).

Level 3:

1-Pyroglutamyl-L-norvalyl-L-thiazolidine-4-carboxylic acid amide t

4.76 g (10 mmol) of Z-Glp-Nva-Tca-NH _{2 were} dissolved in 20 ml of ice-cooled 3.5 H hydrobromic acid solution in glacial acetic acid and the resulting solution was left to stand for 1.5 hours at 0-5 ° C. The reaction mixture was triturated with ether and the separated oil separated by decantation from the solvent. The resulting oil was dissolved in 50 ml of water, the solution was neutralized by the addition of solid sodium bicarbonate and washed three times with 20 ml of ether each. The aqueous phase was evaporated, the residue was dissolved in 100 ml of chloroform, the solution was dried with anhydrous sodium sulfate, evaporated and the residue was rubbed with ether. The crude product obtained (3 g) was applied to a column of 80 g silica gel and eluted with a solvent mixture (4). 2.18 g of an amorphous product is isolated from the fractions containing the pure product, the solution is dissolved in 40 ml of water, the solution is decolorized with activated charcoal and the hectic aqueous solution is lyophilized. In this way, 1.84 g (54% of theory) of Glp-Nva-Tca-NH _{2 is} obtained; r |. ^^ = 0.50 | / ¼ ⁵ = -14.5.6 ° (c »1, in acetic acid).

Example 5

L-Pyrogiutamyl-L-nervalyl-L-leucinamide

Level 1:

Tert-butoxycarbonyl-L-norvalyl-L-leucine methyl ester

BOC-Nva-OH, which is liberated from 8.0 g (20 mmol) of DCHA-salt ¹ * (as described in Example 4, step 1) and 3.82 g (21 mmol) of H-Leu-OMe.HCl it was dissolved in 60 ml of chlorophyll and the solution was first mixed with 2.94 ml (22 mmol) of triethylamine, then ice-cold and stirred with a solution of 4.33 g (21 mmol) of DCC in 40 ml of chloroform. The reaction mixture was allowed to stand at 5 ° C overnight, then the separated DCU was filtered off, the filtrate was washed three times with 30 ml of 1 N hydrochloric acid each, three times with 30 ml of 1 N sodium bicarbonate solution and finally with 30 ml water (once). It was then dried with anhydrous sodium sulfate and evaporated. The crystalline residue was triturated with n-hexane, filtered off and the crude product obtained (6.65 g) was recrystallized from a mixture of 5 ml of ethyl acetate and 20 ml of light petroleum. In this way, 5.20 g of BOC-Nva-Leu-OMe (75% of theoretical yield) are obtained; mp 100-101 ° C; = 0.84j / «C / p5 = 47.5 ° (c» 1, in methanol).

-28 Level 2:

Tert-butyloxycarbonyl-L-norvalyl-L-leucinamide

5.0 g (14.5 mg) of BOC-Nva-Leu-OMe were dissolved in 50 ml of methanol, the solution was cooled with ice 1 and ammonia gases were introduced into such a cooled solution for 0.5 h. The solution was allowed to stand at room temperature overnight, then cooled again, saturated with Gaussian ammonia, allowed to stand for 4 hours and then evaporated. The crystalline residue was recrystallized from a mixture of ethyl acetate and ether. In this way, 4.57 g (91% of theory) of BCC-N <? - Leu-NH ₂ is obtained; mp 158-159 ° C;

Rp>»0.60; / o6 / p ⁵ - -48.7 ° (c = 1, in methanol).

rf

· T ·

Level 5:

z ·

-LzJ '

L-Norvalyl ^ Iuucinamide hydrochloride: 4

4.12 g (12.5 mmol) of BOC-Naa-Leu-NH? is suspended in 16 acres of ethyl acetate and mixed with 20 ml of 6 N hydrochloric acid solution, .3

The mixture was allowed to stand for 1 hour, then diluted with ether; The resulting precipitate was filtered off and the crude product (5.64 g) recrystallized from 25 ml of methanol. In this way it is obtained «

2.75 g (85% of theory) H-Nva-Leu-NH 2 .HCl; ·

mp 215-216 ° C _} r £ ⁵⁾ = 0.45; / ot /? ⁵ = -5.47 ° (c = 1, in methanol).

and i

J

- 29 St Hope 4:

Benzyloxycarbonyl-L-pyroglutamyl-L-norvalyl-L-leucinamide

2.67 g (10 mmol) of H-Nva-I »eu-NH ₂ .HC1 were dissolved in 30 ml of IMF A. To this solution was added first 1.4 ml (10 mmol) of triethylamine, then after filtration and the precipitate formed. , 4.72 g (11 mmol) of Z-Glp-OPFP was added. After 5 minutes. it is added. gave 1.4 np (10 mmol) of triethylamine, and after a further 10 minutes the reaction mixture was evaporated in vacuo. The residue was triturated with ether and the crude product (5.5 g) thus obtained was recrystallized from ethanol. 3.84 g (81% theoretical yield) of Z-Glp-Nva-Leu-RH _{2 are} obtained; mp 241-242 ° C; »0,55;

M / p ^{5 a} -52.6 ° (c · 1, in acetic acid).

Level 5:

»

L-Plroglutamyl-I »-norvalil-L-leucinamide

3.8 g (8 mmol) of Z-Glp-Nva-Leu-NH _{2 were} dissolved in 200 ml of acetic acid, the solution was mixed with 0.8 g of 10% palladium on activated carbon (as a catalyst) and introduced a gaseous mixture through the mixture. hydrogen for 1 hour. The catalyst was then filtered off and the gel residue was rubbed with ether. 2.7 g of Glp-Nva-Leu-NH ₂ (99% of theoretical yield) are thus obtained; mp 240 ° C (dec.); Rf) = 0.57; ⁼ “55.8 ° (c ^s 1, in acetic acid).

t

Example 6;

ί.

L-Pyroglutamyl-L-norvalyl-I-isoleucinamide

Level: 1:

'Tert-Butyloxycarbonyl-L-norvalyl-L-isoleucine-amide

3.7 g (19 mmol) of H-Ile-NH 4 .HCl was dissolved in 30 ml of BHPA, r &lt; 1 &gt; solution mixed with 2.7 ml (19 nunol) of triethylamine and 6.63 g (17.3 mmol). ) BOC-Nva-OPPP and after 5 minutes further 2.4 pl (17.3 mmol) of triethylamine were added. After a further 10 minutes, the reaction mixture was evaporated in vacuo, the residue was dissolved in 100 ml of chloroform, the solution was washed twice with 20 ml of 1 N hydrochloric acid each, then three times with 20 ml of 1 N sodium bicarbonate solution and finally once with 20 ml of water. It is then dried with anhydrous sodium sulfate

And it pairs. The crystalline residue was triturated with ether and dried. 4.7 g of B0C-Nva-Ile-NH ₂ (83% of theoretical yield) are thus obtained; mp 192-194 ° C; 0.60; »-43.7 ° (c - 1, in methanol).

Level 2:

L -No rval i 1 -1 -1 zol eucinami d -hi drohl ori d

4.5 g (13.7 mmol) of B0C-Nva-Ile-hH _{2 were} suspended in 20 ml of ethyl acetate and mixed with 1? ml of 6 M hydrochloric acid solution in ethyl acetate. The reaction mixture was allowed to stand for 1 hour, then diluted with ether. The precipitate formed was separated by filtration and the crude product (3.6 g) thus obtained was recrystallized from a mixture of methanol and ether. 3.5 g of H-Nva-Ile-NHg.HCl (96% of theory) are obtained, mp 254-255 ° C; Rp ^ ^e 0.45;

»+ 5.8 ° (c 1, in methanol).

Level 3:

Benzyloxycarbonyl-L-pyroglutarail-L-norvalyl-II-isoleucinamide

3.3 g (12.4 mmol) of H-Nva-Ishe-NH ₂ .HCl was dissolved in 40 ml of DMFA and the solution stirred with stirring with 1.74 ml (12.4 mmol) of triethylamine and 5.85 g (13 , 6 mmol) Z-Glp-OPPP. After 5 minutes, 1.74 ml (12.4 mmol) of triethylamine were added. The reaction mixture, which after a few seconds becomes gelled and solidified, is diluted with ether and stirred. It is then left to stand in the refrigerator for 2 hours and then filtered. In this way 5.1 g of Z-Glp-Nva-Ile-NH ₂ (86% of theoretical yield) are obtained; mp 252-253 ° C; R < ⁴ >»0.60; / oC / p ⁵ = -57.5 ° (c «1, in acetic acid).

Level 4:

L-Pi horn utamyl-L-norvalyl-L ~ isoleucinamide

4.75 g (10 mmol) of Z-Glp-Nva-Ile-NH _{2 are} dissolved in 200 ml of acetic acid and after the addition of 1 g of 10% -nofc palladium on activated carbon (as catalyst), hydrogen gas is passed through this solution 1 hour. After the hydrogenation is complete, the catalyst is separated by filtration, the filtrate is evaporated and the residue of the trio with ether. 3.44 g of Glp-Nva-Ile-NH _{2 is} obtained (98% of theory); tt

267-270 (dec.); = 0.61; [eta] - - 50.2 ° (c = 1, in siriacic acid). .

Example 7

L-Pyrogiutamyl-L-norvalyl-L-methioninamide

Level 1:

Tert-Butyloxycarbonyl-L-norvalyl-1-methionine methyl ester

BOC-NvaOH was released from 4.78 g (12 mmol) of DCHA salt (as described in Example 4, step l) and with 2.6 g (13 mmol) of H-Met-OMe.HCl dissolved in 40 ml of chloro form. To this solution was added 1.82 ml (13 mmol) of triethylamine first, then with ice-cooling and stirring, a solution of 2.58 g (12.5 mmol) of DCC in 20 ml of chloroform. The reaction mixture was allowed to stand

- 33 overnight at 5 ° 0, then filtered off the separated DCU, the filtrate • t, washed three times with 20 ml of 1 N hydrochloric acid each, three times with 20 ml of 1 N sodium bicarbonate solution and finally once with 20 ml of water . It was then dried with anhydrous sodium sulfate and evaporated. The oily residue is crystallized by the addition of light petroleum. The crude product (3.42 g) thus obtained was recrystallized from a mixture of ethyl acetate and petroleum ether. 3.08 g of BOC-Nva-Met-OMe (71% of theory) were obtained; m.p. 69-70 ° C; Rp) «0.81; / from / p ^ »-42.7 ° (c» 1, in methanol)

Level 2:

Tert-Butyloxycarbonyl-L-norvalyl-L-methioninamide

1.81 g (5 mmol) of BOC-Nva-Met-OMe were dissolved in 20 ml of methanol and ammonia gas was introduced into this solution in a gaseous state for 0.5 hours. The reaction mixture was allowed to stand over nights at room temperature, then kept in the refrigerator for several hours. The separated crystalline product (1.27 g) was filtered off; The filtrate was evaporated, the residue was crystallized from 5 ml of ethanol and combined with the main amount of this product, which was obtained as described above. In this way a total of 1.62 g of B0C-Nva-Met-NH ₂ (93% of theoretical yield) is obtained; mp 166-167 ° C; r ( ²⁾ = 0.57; / oirff «-40.9 ° (c = 1, in methanol).

- 34 -.

Level 3:

L-Norvali1-L-methioninamide hydrochloride

4.5 g (13 mmol) of BOC-Nva-Met-NH _? is suspended in 20 ml of ethyl acetate and mixed with 20 al 6 M hydrochloric acid solution in ethyl acetate. The reaction mixture was allowed to stand for 1 hour at room temperature; then dilute with ether; the resulting precipitate was filtered off and dried in vacuo over anhydrous sodium hydroxide. In this way, the crude product obtained (4 g) was further covered with a mixture of methanol and ether. 3.28 g (89% of theory) are obtained and mp 198-200 ° C; Rp = 0.40; / X // £ ⁵ - + 10.2 ° (c = 1, in methanol).

Grade 4i tert-Butyl oxycarbonyl-1-gl utamic ni 1-L-norvalyl-L methioninamide

2.84 έ (10 mmol) of H-Nva-Met-NI ^ .HCl was suspended in 40 ml of DMFA and stirring thereto was added 1.4 ml (10 mmol) of triethylamine and 4.53 g (11 mmol) of BOC- Gln-OPFP. After 5 minutes a further 1.4 ml (10 mmol) of triethylamine was added and after stirring for a further 10 minutes the resulting slurry was evaporated in vacuo. After evaporation, the solid residue was rubbed with ethanol and the crude product (4.82 g) thus obtained was boiled with 50 ml of ethanol. After cooling, this suspension is kept in the refrigerator for several hours and then filtered. On this one

- 35 mode yield 4.08 g (86% of theory) of BOC-ein-Nva-Met-HH ₂ ; mp 237-258 ° C; R ^ - 0,43; ' m / | ⁵ - -37.1 ° (c »1, in acetic acid).

Level 5:

L-Pi roglutamyl -L-ncrvalil «L-me thioninami d

3.6 g (8 mmol) of BOC-Gln-NavyMet-NH were dissolved in 100 ml of χ ^ά % formic acid, the solution was left to stand for two hours at room temperature and then evaporated in vacuo. The resulting oil was dissolved in 50 ml of acetic acid, the solution was boiled for 2 hours and then evaporated again in vacuo. The gel residue is rubbed with ether and the profile is three rays. 2.8 g (98% of theoretical yield) of Glp-Nva-Met-NH ₂ mp 249-250 ° C (decomposition.b Rf ⁵⁾ = 0.58 are thus obtained. / ot / p ⁵ ? -48.4 ° (c = 1, in acetic acid).

- 36 Example 8

N- (L-Pyroglutarail-L-leucyl) -pyrrolidine

Level 1:

N- (I '-Iieucyl) -pyrrolidine hydrochloride

3.46 g (15 mg) of BOC-Leu-OH was dissolved in 50 ml of ethyl acetate, the solution was mixed with 2.1 ml (15 mmol) of triethylamine and cooled to <

-4 ° C. Subsequently, 1.97 ml (16 mmol) of pivaloyl chloride was added with stirring at a temperature below -10 ° C. The resulting suspension was further stirred for another 10 minutes, then 1.37 ml (16.5 mmol) of pyrrolidine was added dropwise at the same temperature, and the mixture was stirred for half an hour at -10 ° C and then left to stand at 5 ° C for 3 hours. The resulting solution was washed three times with 10 ml of 1 N hydrochloric acid each, pouring three times with 10 ml of 1 N sodium bicarbonate solution each time and finally with 10 ml of water. It is then dried with anhydrous sodium sulfate atom and evaporated in vacuo. The oil, obtained as a residue, was dissolved in 5 ml of ethyl acetate and mixed with 10 ml of 5 N hydrochloric acid solution in ethyl acetate. The mixture was allowed to stand for 1 cae, then diluted with ether and washed with water. The aqueous phase was washed with ether, made alkaline with potassium carbonate and extracted with chloroform. The chloroform solution was separated, dried over anhydrous sodium sulfate and evaporated. The oily residue was dissolved in 10 ml of ether, adjusting the pH of this solution with a concentrated solution of hydrochloric acid in ethyl acetate.

- 37 3 and filter the isolated crystalline product. 2.41 g of H-Leu-pyrrolidine-hydrochloride (73% of theoretical yield) are thus obtained; m.p. 17O-174 ° C (dec.); Rp) ·

Level 2:

N - (Benzyloxycarbonyl-L-pyrrolglutamyl-L-leucyl) -pyrrolidine

1 »7 g (7.7 mmol) of H-ioucine-pyrrolidine-HCl, 3.0 6 (7 mmol) of Z-Glp-OPFP and 1.08 ml (7.7 mmol) of triethylamine were dissolved in 20 ml of chloroform and after 5 minutes, 0.98 ml (7 ramol) of triethylamine was added. After a further 5 min, the solution was diluted with chloroform, washed with 10 ml of 1 N hydrochloric acid each time, three times with 10 ml of 1 N sodium bicarbonate solution, and finally with 10 ml of water once. It is then dried with anhydrous sodium sulfate and

And pair it. The oily residue was crystallized using n-hexane and the crude product (2.85 g) thus obtained was recrystallized from a mixture of ethanol and ether. 2.14 g of Z-Glp-Leu-pyrrolidine (71% of theory) was obtained; mp 109-H0 ° C; Rp) - 0.55; / X / p ⁵ = -53.3 ° (c = 1, in acetic acid).

Analysis for ^C 23 ^H 31 ° 5 ^N 5 (mol. Weight 429.52):

calculated: C 64.32%, H 7.27%, N 9.78%;

Found: C 64.31%, H 7.47%, N 9.78%.

- 58 Level 3:

N- (L-Pyroglutamyl-L-leucyl) -pyrrolidine

2.0 g (4.66 mmol) of Z-Glp-Leu-perchlidine are dissolved in 40 ml of methanol, 0.4 g of palladium on activated carbon (as a catalyst) are added to this solution and hydrogen gas is bubbled through the solution for 1 hour . After filtration of the catalyst, the filtrate was evaporated and the resulting oil was dissolved in tt ether. The resulting solution was then left to stand overnight in the refrigerator and the separated crystals separated by filtration.

In this way 1.25 g of Glp-Leu-pyrrolidine (91% of theoretical yield) is obtained; m.p. 1O3-1O4 ° C; »0.65;

/ «Zz / ^ 5 ^β -31.6 ° (c» 1, in acetic acid).

Amino Acid Analysis: Glu 1.00 (1.0), Leu 1.00 (1.0).

t

Example 9

N- (H -roglutamyl-L-leucyl) -piperidine

Level 1:

N - (L-Leucyl) -piperidine hydrochloride

3.46 g (15 mmol) of BOC-Leu-OH was dissolved in 50 ml of ethyl ethoate, the solution was mixed with 2.1 ml (15 mmol) of triethylamine and cooled to -15 ° C. At this temperature, 1.97 ml (16 mmol) of pivaloyl chloride was added dropwise, followed by 1.65 ml (16.5 mmol) of piperidine after 10 min. After completion of the addition, the reaction mixture was stirred for a further half hour at -10 ° C, then allowed to stand overnight in the refrigerator. Then the precipitate is filtered off, the filtrate is washed three times with 10 ml of 1 N hydrochloric acid each, three times with 10 ml of 1 N sodium bicarbonate solution and finally with 10 acre of water.

It was then dried with anhydrous sodium sulfate and evaporated.

As a residue, the oil was dissolved in 15 ml and N solution of hydrochloric acid in ethyl acetate. The solution was allowed to stand for 1 hour, then diluted with ether and allowed to stand overnight in the refrigerator. The next day the product obtained as a precipitate was separated. This is done by filtration. In this way, 2.0 g of H-leu-piperidine-HCl (57% of the theoretical yield calculated on BOC-Leu-OH) are obtained, m.p. 123-125% R ^^ * 0.45.

Level 2: i

N- (Benzyloxycarbonyl-II-pyroglutamyl-L-leucyl) -piperidine

1.81 g (7.7 mmol) of H-leu-piperidine-HCl, 3.0 g (7 mmol)

Z-Glp-OPPP and 1.08 ml (7.7 mmol) of triethylamine were dissolved in 20 ml of chloroform, and a further 0.98 ml (7 mmol) of triethylamine was added after 5 minutes (standing). After a further 5 minutes, the solution was diluted with 30 ml of chloroform, washed twice with 10 ml of 1 N hydrochloric acid each, three times with 10 ml of 1 N sodium bicarbonate solution each and finally with 10 ml of water.

fcli *

- 40 /,

It is then dried with anhydrous sodium hydroxide sulphide atom and evaporated. The crystallizing oil, obtained as a residue, is rubbed with a mixture of ether and n-hexane. The crude product obtained in this way (2.75 g) was recrystallized from a mixture of ethanol and ether. 2.21 g of Z-Glp-Leu-piperidine (71% of theory) was obtained; m.p. 113-115% Rp ^ - 0.69;

M / * ⁵ »-42.5 ° (c« 1, in acetic acid).

Analysis for ^C 24 ^Ii 33 ° 5 ^ 3 (® ^o1 · wt. 443.55) izračunato: C 64.99%, H 7.50%, N 9.47%;

found: C 64.90%, H 7.72%, N 9.53%.

Level 3:

N- (L-Pi horn utamyl-L-leucyl) -pipe ridin

2.0 g (4.51 mmol) of Z-Glp-Leu-piperidine were dissolved in 40 ml of methanol, mixed with 0.4 g of 10% palladium on activated carbon (as catalyst) and introduced hydrogen gas in the stream period of 1 hour. After filtration of the catalyst, the filtrate was evaporated and the residue led to aa crystallization with ether. In this way 1.12 g of Glp-Leu-piperidine (81% of theoretical yield) is obtained; mp 99-100 ° C r (5) _a 0.71; * + 30.4 ° (c = 1, in acetic acid).

Amino Acid Analysis: Glu 0.95 (1.0), Leu 1.00 (1.0).

t.

Example 10

L-Thiazolidine-4-carbonyl-L-leuoyl-L-proIinamide

Level 1:

L -Ti azole and din-4-carbonyl -L -1 euc il -L -pro and us d «

5.13 g '(22 mmol) of BOC-Tca-OH and 4.05 g (22 mmol) of PPPOH rasF' were formed in 60 ml of ethyl acetate and the solution (stirred and ice-cold) was mixed with 4.12 g (20 mmol) ) DCC. The reaction mixture was stirred for 2 hours at 0-5 ° C, then filtered off with DCU, the filtrate was evaporated and the residue was dissolved in 50 ml of n-hexane. The solution is washed five times with 25 ml of 1 N sodium bicarbonate solution each and then once with 25 ml of water. The dried anhydrous sodium sulfate atom was then dried and evaporated. The thus obtained 7,> 2 µl of lobster BOC-Tca-OPPP was dissolved in 20 ml of DMPA and the solution added in a suspension of 5.23 g (20 mmol) of H-Leu-Pro-NH ₂ .HC1 in 30 ml. LMFA, then the resulting mixture (with stirring and ice-cooling) was mixed with 2.8 g (20 mmol) of triethylamine. After 5 minutes a further 2.8 ml (20 mmol) of triethylamine was added, the mixture was stirred for a further 20 minutes and then evaporated in vacuo.

The oil obtained as a residue was dissolved in chloroform, (100 ml), The resulting solution was washed three times with 30 ml of 1 N hydrochloric acid each, three times with 30 ml of 1 N sodium bicarbonate solution each and finally with 30 ml. ml of water.

It is then dried with anhydrous sodium sulfate atom and evaporated.

8.84 g of lobster BOC-Tca-Leu-Pro-NH ^ dissolved in 15 ml f

• s

S

- 42 -........

ethyl acetate and the resulting solution was mixed with 15 ml of 6 N hydrochloric acid solution in ethyl acetate. The reaction mixture was allowed to stand for 1 hour, then diluted with ethyl acetate, the resulting triol precipitate and filtered off. It is then dried in vacuo, above anhydrous sodium hydroxide. In this way, the 8.05 g of tripeptidamide hydrochloride thus obtained is dissolved in 80 ml of water, a solution. three times with 20 ml of ether each, then the pH of the aqueous sodium bicarbonate solution was adjusted to 8 and the alkaline solution was extracted five times with 20 ml of chloroform. The combined chlorophosm extracts were dried with anhydrous sodium sulfate and evaporated. After evaporation, the residue is triturated with ether and dried. In this way 5.20 g of H-Tca-Leu-Pro-NH ₂ (76% of theoretical yield) are obtained;

mp 159-16 ° C; Rp ^ - 0.63; / oC / p ⁵ - -162.9 ° (c - 1, in acetic acid).

Example 11

L-2-Ketoiraidazolidine-4-carbonyl-1, -prolineamide

Level 1:

Benzyloxycarbonyl-II-2-ketolmidazolidine Pentafluorophenyl ester

4-Carboxylic acids

10.56 g (40 mmol) of Z-Kic-OH and 9.09 g (44 mmol) of PFPOH were dissolved in 100 ml of a mixture of DMFA and dioxane (1: 2) and the resulting solution (cooled and stirred) was mixed with 9706 g (44 mmol) of DCC. The reaction mixture was stirred for 1.5 hours at 0-5 [deg.] C., the filter was filtered off with DCU, the filtrate was evaporated and the oil obtained as a residue was crystallized using n-hexane. The crude product (16.46 g) thus obtained was recrystallized from 50 ml ethyl acetate. 12.75 g of Z-Kic-OPFP (»74% of theory) was obtained; m.p. 146-148 ° C; »0,53;

-42.1 ° (c «1, ethyl acetate}.

Analysis for ^ο ΐ8 ^Η 11 ° 5 ^Ν 2 ^? 5 ( ^mo1 · ^te * · 430,29):

calculated: C 50.25%, H 2.58%, N 6.51%, F 22.08%; found: C 49.88%, H 2.35%, N 6.66%, F 21.81%.

Level 2:

Benzyloxycarl III -1, -2 ketoimidazolidine-4-carbonyl-L1 euc yl-L-prol and nam and d

-44-2.38 g (9 mmol) of H _r Leu-Pro-NH ₂ .HCl was suspended in 3θ ml of DMPA and mixed with 3.87 g (9 mmol) of Z-Kic-OPFP and 1.26 ml (9 mmol) of triethylamine. After stirring for 5 minutes, add further

1.26 ml (9 mmol) of triethylamine and, after stirring for an additional 20 minutes, the reaction mixture was evaporated in vacuo. After evaporation, the residue is dissolved in 50 ml of chloroform, the solution is washed twice with 10 ml of 1 N hydrochloric acid, three times with 10 ml of sodium bicarbonate solution 1 each, finally with 10 ml of J water. It was then dried with anhydrous sodium sulfate and evaporated. ζ

The oil, which was obtained as a residue, was crystallized with ether and filtered off the resulting precipitate. The crude product (3.31 g) thus obtained is boiled with 30 ml of ethyl acetate.

After cooling the suspensions it will stay standing for a few hours in the refrigerator 1 then profile three ra. 3.0 g is obtained

Z-Kic-Leu-Pro-NH ₂ (70% of theory); mp 172-174 ° C;

0.18; * -102.6 ° (c - 1, in acetic acid).

t

Analysis for ^C 23 ^H 31 ° 6 ^N 5 ( ^mo1 · ^wt · 473.50):

calculated: C 58.34%, H 6.60 N 14.79%;

found: C 57.52%, H 6.62%, N 14.62%.

Level 3:

L-2-Ketoimidazolidine-4-carbonyl-L-leucyl-L-prolinamide • t.

1.2 g (2.54 mmol) of Z-4tic-Leu-Pro-NH _{2 were} dissolved in 30 ml of water, the solution was mixed with 0.25 g of 10% palladium on coal (as a catalyst) and passed through this mixture. hydrogen gas over 4 hours. Then the catalyst was filtered off, the filtrate was evaporated and the amorphous residue was dried in vacuo over anhydrous phosphorpentoxide. To this k

the crude product obtained (0.75 g) is dissolved in water, the solution is discolored with activated carbon and the clear aqueous aqueous solution is lyophilized. 0.61 g of Kic-Leu-Pro-NHg (71% of theory) was obtained; Rp ^ »0.35; / e ^ / P * -90.4 ° (c «1, in acetic acid).

Example 12:

L-6-Ketopiperidine-2-carbonyl-L-leucyl-L-prolinamide

Degree: 1: · *

L-6-ketopipecolic acid pentafluorophenyl ester '4.3 g (30 mmol) of L-6-ketopipecolic acid and 6.07 g (33 mmol) of PPPOH are dissolved in 100 ml of chloroform and this solution (cooled with ice and water). mixing) was mixed with 6.8 g (33 mmol)

DCC. This mixture was stirred at 0 ° C for one hour, then allowed to stand

- 46: -:

standing overnight in the refrigerator. The next day ae filtered off the separated DCU, the filtrate evaporated and the crystalline residue of $ rust with n-hexane. In this way the crude product obtained (9.87 g) was dissolved in 20 ml of ethyl acetate, the solution was allowed to stand in the refrigerator for one hour, then discarded with activated charcoal, the profile was rubbed and evaporated. The resulting oil was dissolved in a mixture of 5 ml of ethyl acetate and 20 ml of M-hexane, the solution was left to stand overnight in the refrigerator and the next day the separated crystals were separated by filtration. 6.34 g of Kpc-OPFP (68.5% of theory) are obtained; m.p. 96-98 ° C; / d (»/ p5 * + 30.0 ° (c» 1, in acetic acid).

Analysis for θ ^ 2®8θ3 ^ 5 ( ^mQ ^ · Thesis. 309.20):

calculated: C 46.62%, H 2.61%, N 4.53%, F 30.72%; found: C 46.37%, H 2.88%, N 4.26%, F 30.51%.

Level 2: ¹

L-6rKetopiperidine-2-carbonyl-Lleucyl-L-prolinamide

1.32 g (5 mmol) of H-Leu-Pro-NHg.HCl was suspended in 20 ml of DMFA and cooled with ice and stirred with 1.61 g (5.2 mmol) of Kpc-OPFP and 0.7 ml ( 5 mmol) of triethylamine. After stirring for 5 minutes a further 0.7 ml (5 mmol) of triethylamine was added and after stirring for another 20 minutes the reaction mixture was evaporated in vacuo. The crystalline residue was triturated with ether, the crystals were filtered off, the filter was washed with ether and cold chloroform and then dried. This is how it gets

1.27 g of Kpc-Leu-Prp-NHg (72% of theory of theoretical bird); m.p. 214-216 ° C, Rp) 0.41, -83 ° (c »1, in acetic acid).

Amino acids analysis: ^ amino-adipic acid 0.99 (1.0), Leu 1.00 (1.0), Pro 0.98 (1.0).

Example 13 k

ar

L-6-Ketopiperidine-2-carbonyl-L-norvalyl-1-prolinamide

2.38 g (9.5 mmol) of H-Nva-Pro-NHg.HCl were suspended in 30 ml of DMFA and, under ice-cooling and stirring, ea 30.09 g (10 mmol) of Kpc-OPPP and 1.33 ml ( 9.5 mmol) of triethylamine.

After stirring for 5 minutes a further 1.3 ml (9.5 mmol) of triethylamine was added and after stirring for a further 20 minutes the reaction mixture was evaporated in vacuo. The crystalline residue was triturated with ether, filtered off, washed with cold ethanol on the filter and dried. In this way, the crude product obtained (3.66 g) was dissolved in water, the solution was discolored with activated charcoal, the colorless (clear) aqueous solution was evaporated and the crystalline residue of the trio with 10 ml of ethanol. This mixture was allowed to stand in the refrigerator and then filtered. 2.10 g of Kpc-Nva-Pro-NH ₂ (65% of theory) are obtained; mp 192-195 ° C; Rp ^ 0.31; / <Z / p ⁵ - -83.2 ° (c = 1, in acetic acid).

Example 14 · I.

D-Pi roglutamyl -L -1 eucll -L-prol inami d

Level 1:

Benzafluoxycarbonyl-D-pyroglutamic acid pentafluorophenyl ether

4.55 g (18 mmol) of ZD-Glp-OH and 3.68 g (20 mmol) of PPPOH were dissolved in 50 ml of ethyl acetate and this solution (with ice-cooling and stirring) was mixed with 4.12 g (20 mmol) ) DCC. The reaction mixture was stirred for 1 hour at 0 ° C, then filtered off with DCU, the filtrate evaporated and the oily residue crystallized with n-hexane. The crude product (7.3 g) thus obtained was dissolved in 20 ml of ethyl acetate, the solution was allowed to stand in the refrigerator for 1 hour, then discarded with activated charcoal and evaporated. The resulting oil was dissolved in 5 ml of ethyl acetate 1 by adding 20 ml of n-hexane to the product. ¢, 60 g of ZD-Glp-OPFP (85% of theory) was obtained; mp 81-82 ° C; Rp) «0.84; / ot / p ^{5 e} + 40.1 ° (c - 1, in ethyl acetate).

Analysis for θχ9 ^Η ^ 2 ° 5 ^Ν? 5 (mol. Weight 429.30):

calculated: C 53.16%, H 2.82%, N 3.26%, P 22.13%; found: C 53.28 Ji, H 3.04%, K 3.02%, P 21.86%.

Level 2:

Benzyloxycarbonyl-D-pyroglutamyl-L-leuyl-L-prolineoid • #.

5.24 g (12.5 mmol) of H-Leu-Pro-Ni ^ .HCl were suspended in 50 acre of IMPA both with ice-cooling and stirred with 5.6 g (15 mmol) of ZD-Glp-OPFP and 1. 72 ml (12.5 mmol) of triethylamine.

After stirring for 5 minutes a further 1.72 ml (12.5 mmol) of triethylamine was added and after stirring for another 20 minutes the reaction mixture was evaporated in vacuo. The residue was dissolved in 120 ml of chlorophyll, the solution was washed twice with 50 ml k each

N hydrochloric acid, three times each with 50 ml of 1 N sodium bicarbonate solution and finally with 50 ml of water (once). It was then dried with anhydrous sodium sulfate and evaporated. The oily residue from evaporation was led to crystallization with ether. The crude product (5.51 g) thus obtained was boiled with 50 acre ethyl acetate, and the resulting suspension was allowed to stand in the refrigerator for three hours and the separated product separated by filtration. In this way, 4.61 g of Z-D-Olp-Leu-Pro-NH ^ (80% of theoretical yield) is obtained; m.p. Mp 189-194 ° C; = 0.44; = -51.2 ° (c = 1, in acetic acid).

-5Γί? Level 3:

D-Pyrogutamyl-L-leucyl-L-prol inamide

4.48 g (9.5 mmol) of ZD-Glp-Leu-Pro-NH _{2 was} dissolved in 200 ml of methanol and the solution was mixed with 0.9 g of 10% palladium on activated carbon (as catalyst). It is passed through this mixture for 1 hour with you and you hydrogen. Then the catalyst was filtered off, the filtrate was evaporated and the araraffin residue was evaporated after evaporation with ether. The crude product (3.07 g) thus obtained was dissolved in water, the solution was discolored with activated charcoal and the resulting colorless aqueous solution was lyophilized. In this way, ee obtained 2.90 g (90.5% of theoretical yield) of D-Glp-Leu-Pro-NH ₂ (90.5%); r | ⁵⁾ = 0.40> / «i / p ⁵ « -23.6 ° (c «1, in acetic acid).

Example 15

Orotyl-L-histidyl-L-pipecolic acid amide

Level 1:

Okhotyl-L-histidine methyl ester

24.2 g (100 mmol) of H-His-0Mer2H01 was dissolved in a mixture of 120 acre (1: 1) BMPA-dioxane and the solution was mixed with 17.41 g (100 mmol) of oroic acid monohydrate. The solution is

- 51 cool to 0 ° C 1 at this temperature is added, J fe (100 »

mmol) of N-hydroxy-euccinimide, 11.1 ml (100 mmol) of N-methylraorfoline and finally 20.6 g (100 mmol) of DOC. The reaction mixture was stirred for one hour at 0 ° C and then overnight at ambient temperature. The next day the reaction mixture was allowed to stand for 2 hours in the refrigerator, then filtered DOU and the filtrate was evaporated. As a result, the resulting oil ae ktietalling with water, the crystals were filtered off 1 aa and the filter was washed with 5% aqueous solution of glacial acid and • x water. In this way 11.6 g is obtained. Oro-Hie-dle (38% of theoretical yield); mp 258-262 ° C> Rp ^ ^e 0.40.

Analysis for C ^ H ^ O ^ (mol. Wt. 307.27):

calculated: N 22.79%;

found: N 22.51%.

Level 2: t

Orotyl histldine hydrazide

9.21 g (SO mmol) of Oro-His-OMe was dissolved in 120 ml of DMFA and the solution was mixed with 7.33 ml (150 mmol) of hydrazinhydrate. The reaction mixture was allowed to stand at room temperature for 2 days, then diluted with 100 rolls of ethyl acetate! kept overnight in the refrigerator. The separated precipitate was filtered off and recrystallized from methanol. In this way 8.07 g of Oro-His- ^ H ^ (88% of theory) was obtained; mp 250-26 ° C; r £ ⁵⁾ «0.35.

Analysis for O ^^^ O ^ N? (ίηοϊ; tšx. 307,28):

calculated: N 31.91%;

found: N 30.75%. ·

- /,

Level 3:

Ototyl-1-histidyl-L-pipecolic acid amide

5.0 g (16.28 mmol) of Oro-His-N ^ H ^ was suspended in 135 ml of EMFA and mixed with 5.99 ml (48 * 4 mmol) of 8.1 N hydrochloric acid solution in dioxane. The resulting solution was cooled to ~ 15 ° ® and stirred dropwise with a mixture of 2.13 ml (17.9 mmol) of tert-butyl nitrite. The reaction mixture was then stirred at -10 ° β for 20 minutes, then mixed with 4.56 ml (32.56 fluppl) of triethylamine at -10 ° C, followed by a solution of 2.05 g (16, 28 mmol) of H-Pip-NH ₂ in 10 ml of EMPA and finally with a further 2.28 ml (16.26 mmol) of triethylamine. After the addition was complete, the reaction mixture was stirred for an additional 1 hour at -10 ° C, then allowed to stand overnight at 2 ° C. The next day the filtrate was filtered off, the filtrate was evaporated in vacuo and the residue was rubbed with ethyl acetate. The crude product thus obtained (4.6 g, 74% of theory) was separated by filtration and 1.3 g of this product was applied to a column made of a mixture of (1: 1) carboxymethylcellulose-23 and carboxymethylcellulose-52 and eluting with 0.005-0.1 M ammonium acetate (pH * 5).

Fractions containing pure product are combined and lyophilized. In this way 820 mg of amorphous is obtained: - 93 -. ...

Gro-HisuPip-NHg »R ^^ = O, 1O> / o £ / ^» -19.0 ° (8 = 1, in water).

Amyric Acid Analysis: His 1.00 (1.0), Pip 0.94 (1.0).

Example 16

Orotyl-L-histidyl-D-pipecolic acid amide

5.0 g (16.28 mmol) of Oro-His-NgH 2 was suspended in 135 ml of 1MFA and mixed with 5.99 ml (48.84 mmol) of 8.1 H hydrochloric acid solution in dioxane. The resulting precipitate was cooled to -15 [deg.] C. ⁽ and with stirring, dropwise mixed with 2.13 ml (17.9 mmol) of tert-butyl nitrite. The reaction mixture was stirred at -10 [deg.] C. for 20 minutes, then at the same dropwise temperature. mixed with 4.56 ml (32.56 mmol) of triethylamine, then with a solution of 2.05 g (16.28 mmol) of HD-Pip-N ^ and finally with another 2.28 ml (16.28 mmol) of triethylamine. After the addition was complete, the reaction mixture was stirred for an additional 1 hour at -10 [deg.] C., then left to stand in the refrigerator overnight, and the resulting precipitate was filtered off, the filtrate was evaporated in vacuo and the residue after evaporation of the trio with ethyl acetate. 4.1 g (66% of theory) of crude product 1.3 g of this product is applied to a column made of a mixture of (1: 1) carboxymethylcellulose-23 and carboxymethylcellulose-52 and eluting with 0.005-0.1 M ammonium acetate solution (pH «5) .fractions that keep pure '-> 4 - - · /.

the product is combined and lyophilized. 795 mg of amorphous Oro-Hic-D-Pip-NH ₂ is thus obtained; R4) ₃ 0.10; / <j6 / d ⁵ * ⁺ 43.5 ° (c - 1, in water).

Amino Acid Analysis: His 1.00 (1.0), Pip 0.96 (1.0).

Example 17

Orotyl-L-histidyl-L-homoprolinaraid

5.0 g (16.28 mmol) of Oro-His-S ^ was suspended in 135 ml of DMPA and mixed with 5.99 ml (40.84 nun) of 8.1 N hydrochloric acid solution in dioxane. The resulting solution was cooled to -15 [deg.] C. and stirred dropwise with 2.13 ml (17.9 mmol) of tert-butyl nitrite. The reaction mixture was stirred for 20 minutes at -10 ° C, then 4.56 ml (32.56 mmol) of triethylamine, a solution of 2.05 g (16.28 mmol), was added dropwise at -10 ° C. -Jro-NH ₂ in 10 ml DMPA and finally 2.28 ml (16.28 mmol) of triethylamine. After the addition was complete, the reaction mixture was stirred for an additional hour at -10 ° C, then allowed to stand in the refrigerator at 2 ° 0. The next day, the precipitate was filtered off, the filtrate was evaporated in vacuo and the residue of the trio with ethyl acetate.

Of the crude product obtained in this way (whose total weight is 4.2 g, which is 97% of theoretical yield), 1.3 g is applied to a column made from a mixture of (1: 1) carboxymethylcellulose-52 and carboxymethylcellulose-23 and eluting with 0.005-0.1 M solution - 55 -. · ·· • ammonium juraacetate. The fractions containing the Pure Product were combined and lyophilized. 802 mg of amorphous Oro-N, N is-HPro-NHg is obtained; Rp) = 0.10; / X / ^ ^{4 β} -12.8 ° (c »1, in water).

Amino Acid Analysis: His 1.00 (1.0), HPro 0.91 (1.0).

Example 18

L-6-ketopiperidine-2-carbonyl-L-norvalyl-1-thiazolidine-4-carboxylic acid amide

2.55 g (9.5 mmol) of H-Nva-Tca-NHg.HCl was suspended in 30 ml of IMFA and cooled and stirred with 3.09 g (10 mmol) of Kpc-OPFP and 1.33 ml (9 , 5 mmol) of triethylamine. After stirring for 5 minutes, a further 1.33 ml (9.5 mmol) of triethylamine was added and after 20 minutes (further stirring) the reaction mixture was evaporated in vacuo. As a residue, the resulting oil was allowed to stand overnight in the refrigerator and thus led to crystallization. The next day, the crystals were filtered off, washed with ether and cold ethanol. In this way the crude product obtained (3.25 g) was dissolved in water, the solution was discolored with activated charcoal, colorless (the clear aqueous solution was evaporated, the crystalline residue was rubbed with 20 acre of ethanol and left to stand overnight in the refrigerator. The product was filtered off and dried, yielding 2.0 g of Kpc-Nva-Tca-NH ₂ (59% of theory); mp, 183-185 ° C; Rp) = 0.47;

-136.0 ° (c = 1, in acetic acid).

Example 19

Ι · ι.ι. ^ '- /.

L-6-Ketopiperidine-2-carbonyl-L-norvalyl-L-homoprolin nid

Level 1:

hot) tert-Butyloxycarbonyl-L- ^ rolinamide

2.29 g (10 mmol) of BOC-HPro-OH was dissolved in 30 ml of ethyl acetate, the solution was mixed with 1.4 ml (10 mmol) of triethylamine, then the mixture was cooled to -10 ° C and 1.3 ml was added dropwise. 10 mmol) isobutyl ether of hydrochloric acid. P ^u messengers stirring for 15 minutes at -10 ° C in the CSO to the reaction mixture is introduced pole while gaseous ammonia, then the mixture is allowed to stand for 2 hours at 0-5 ° C. The precipitate was then filtered off, the filtrate was evaporated and the oil, obtained as a residue, was dissolved in 30 ml of chloroform. The solution is washed twice with 10 ml of 1 X hydrochloric acid each, twice with 10 ml of 1 N sodium bicarbonate solution each time, and finally with 10 ml of water once. It is then dried with anhydrous sodium sulfate, evaporated and the oil, obtained as a residue, results in crystallization with n-hexane. The crude product (1.95 g) thus obtained was crystallized from a mixture of ethyl acetate and ether; 1.78 g of B0C-HPro-NH ₂ (7θ% of theoretical yield) are thus obtained; mp 138-140 ° C; r | ²⁾ = 0.43; / << / p ⁵ = -24.85 ° (c = l, in acetic acid).

rf. *

- 57 - ·

Analysis for ( ^mo1 · wt. 228.29):

calculated: C 57.87%, H 8.83%, N 12.27%;

found: C 57.60%, H 8.89%, N 12.11%.

Level 2:

L -Homoprol Inami d-hi drohl o ri d

1.6 g (7 mmol) of BOC-HPro-NHg were dissolved warm in 10 ml of ethyl acetate, the solution cooled to room temperature and mixed with 10 ml of 6 N hydrochloric acid solution in ethyl acetate. The reaction mixture was allowed to stand for 1 hour at room temperature, then diluted with ether, the separated precipitate rubbed with ether and filtered off. In this way, 1.05 g of N, Pro-NHg.HCl (91% of theory) was obtained; m.p. 178-180 ° C; R ^) »0.32;

+ 26.2 ° (c · 1, in methanol).

>

Level B:

L-Norvalyl-L-homoprolinamide hydrochloride

0.99 g (6 mmol) of H-Pro-NH ₂ .HCl is suspended in 20 ml of EMKA and stirred with 0.84 ml (6 mmol) of triethylamine and

2.3 g (6 mmol) of BOC-Nva-OPFP. After standing for 5 minutes, an additional 0.84 ml (6 mmol) of triethylamine was added to the reaction mixture and, after stirring for 1 hour, the mixture was evaporated in vacuo. Dissolve the resulting residue in 50 ml of chlorophylls, rinse the resulting solution twice with 10 ml of 1 N solution each

-5 «hydrochloric acid, also twice a 10 ml 1 S solution of sodium bicarbonate. It is then dried with anhydrous sodium hydroxide, sulphate and evaporated. The oil, which was obtained as a residue, was dissolved in 6 ml of ethyl acetate and the solution was mixed with 10 ml of 6 N hydrochloric acid dissolved in ethyl acetate. After standing for 1 hour, the reaction mixture was diluted with ether, the amorphous residue was rubbed with ether, filtered off and dried in vacuo over anhydrous sodium hydroxide. This is how it gets

1.22 g of H-Nva-HPro-NHg.HCl (89.5 theory yield);

Rp) »0.12.

Grade 4: t

L-6-Ketopiperidine-2-carbonyl-L-norvalyl-L-4iomoprolinamide

1.22 g (5.37 mmol) of H-Nva-HJro-NH ^ .HCl was dissolved in 20 ml of DMFA and dissolved 0.75 ml (5.37 mmol) of triethylamine and 1.7 g (5.5 mmol) ) Kpc-OPFP mixed (with stirring). After stirring for 5 minutes a further 0.75 ml (5.37 mmol) of triethylamine was added and after further stirring for another 20 minutes the reaction mixture was evaporated in vacuo. The residue was triturated with ether and the thus obtained amorphous crude product (1.8 g) was applied to a column made of 40 g of silica gel and eluted with a solvent mixture (4). The fractions containing pure product were combined and evaporated. 1.1 g of pure product are obtained, this solution is dissolved in water, discolored with activated charcoal and the resulting colorless solution lyophilized. This gives 1.0 g

Kpc-Nva-Hpro-NHg (53% of theory); 1U ⁵⁾ · O, 35i / «4 / d ⁵ '* ⁴⁴ > 6 ° (c« 1, in acetic acid).

Claims (1)

Claim Ρ-Ϊ695 / 80 ia / c 1 · Procedure for the preparation of new tripeptide / opate derivatives of formula (I): X - Ϊ - W - NH ₂ (I) in which X is an L-pyroglutamyl, D-pyroglutamyl, L-thiazolidine4-carbonyl, L-2-ketoimidazoline-4-carbonyl, L-6-ketopiperidine2-carbonyl or an orotyl group. Y is L-leuyl, L-aorvaline, or L-hyathidyl group, V is L-plecolyl, D-pipecolyl, L-thiazolidine-4-carbonyl, L-leuyl, N-isoleucyl, D-prolyl, L-prolyl, L -methionyl, L-homoprolyl, or V-NB ₂ denotes the pyrrolidyl or piperidyl group indicated, i.e., e is the compounds of general formula (XI) t < « W - NHg (II) in which V, as previously defined in degrees, builds a molecule of the experimental formula (I) by the coupling procedure, in fact, so also the compounds of the general formula (II) in which V has the above compound is acylated in dimethylformamide. in the presence of triethylamine at room temperature with the protected pentafluorophenyl amino acid ester of the formula (III): BOC - Y - OPFP (III) * in which BOO denotes a tert-butyl oceicarbonyl group, OPPP stands for the pentafluorophenocell group, and the meaning given above, and from the resulting protected dipeptide of the formula (IV): BOC - Ϊ - W - NH2 (IV) in which BOO and Y have the meanings given above, in ethyl acetate via acidolysis with hydrochloric acid, the dipeptldaald of the general formula (V) is released: h - ϊ - to · - iiH ₂ (V) f, in which C, as defined above, H is hydrogen, and this with the protected pentafluorophenyl ester of an amino acid of general formula (VI): Z - X - OPFP (VI) in which Z denotes a benzyloxycarbonyl group and X and OFST have the foregoing meaning acylated in diethylformamide in the presence of triethylamine at room temperature · and from the obtained protected tripeptide of general formula (VII) " Z - X - Y - W - NH, (ra) in which Z, X, Ϊ and W have the above equations, the protecting group is cleaved off by catalytic hydrogenation in glacial eric acid at room temperature in the presence of palladium-10-palladium * on activated carbon · RICHTER GEDeOM VEGYEGZETI GYAR RT Dealer « DEAR BBOGRAO ^ I T M.ilSTIČ ara 7 ABSTRACT The invention provides a process for the preparation of novel peptides of the general formula U) χ - γ _ w - NH ₂ U) acting on the central nervous system, in which X is L-pyroglutamyl, D-pyroglutamyl, L-2-keto-iraidazoline-4 -carbonyl, L-6-keto-pipecolyl, L-Uazolidin-4-carbonyl, L-prolyl or otyl group, Y is L-leucyl, L-norvalyl or L-histidyl group, and V is L-prolyl, D- prolyl, L-thiazolidine-4-carbonyl, L-homoprolyl, L-leucyl, L-isoleucyl, L-methanyl, L-pipecolyl 111 D-pipecolyl group, or V-NH ,, denotes also the pilidylyl or piperidyl group, further their a pharmaceutically acceptable complex, characterized in that a) starting from a compound of the general formula (Π) v-nh ₂ * Ul) in which V is as previously defined, the desired tripeptide molecule is constructed stepwise, using couplet processes generally used in peptide chemistry, conveniently using active esters, mixed anhydrides iii dicyclohexylcarbodiimide, iii what b) a compound of general formula (II) w-nh ₂ Ul) in which V as defined above is acylated with an azide derived from dipeptide hydrazide of general formula UU) Z - X - Y - N / H - NH ₂ UlI) in which Z denotes benzyloxycarbonyl and X and Y are as previously defined, then if desired the Z-protecting group of the compounds of general formula (IV) obtained by any of the foregoing methods ZXYW - nh ₂ (IV) wherein Z, X, Y and V are as previously defined is deprotected, and the resulting tripeptide of general formula (I) is separated from the reaction mixture, arbitrarily after conversion to a pharmaceutically acceptable complex.