Peptides, or their pharmaceutically acceptable acid, or a salt, a pharmaceutical composition having antagonistic activity against bombezin, a method of treating cancer in mammals

 

(57) Abstract:

The invention relates to peptides of formula (I): X - A1- A2- Thr - Ala - Val - Gly - His - Leu - psi - A9- Q, where X represents a hydrogen, a simple relationship linking the alpha-amino group of A1with gamma-carboxyl part 3-propionyloxy part of A2if A2is Glu[-], or a group of the formula R1CO-, where R1selected from the group comprising: hydrogen, C1- C10- alkyl, or phenyl C1- C10- alkyl, A1is a D - or L-amino acid residue selected from the group consisting of: Phe, p - Hl - Phe, pGlu, Nal, Pal, Tpi, unsubstituted Trp or Trp substituted in the benzene ring by one or more substituents from the group comprising C1- C3- alkyl, or A1represents a peptide bond linking the acyl part of R1CO with alpha aminocyclo A2if A2represents Gln, Glu/-/ Glu (Y) or His, where /-/ is a simple relationship linking the gamma-carboxyl group of A2with the alpha-amino group of A1if A2is Glu, where X represents a simple bond, Y represents - or SIG5where R5is hydrogen, C1- C3the alkyl is so, that connection is specified - CH2- part C the alpha-amino group of A neighboring9- balance is pseudopeptides communication; A9is a TAS, Ista, or D; and Q represents NH2or CQ1where Q1is hydrogen, and pharmaceutically acceptable acids or salts, and pharmaceutical compositions, which has antagonistic activity against bombezin and to a method of treating cancer in mammals on the basis of the peptides of formula (I). 4 C. and 11 C.p. f-crystals, 11 tab., 4 Il.

The invention relates to new peptides that affect the growth of cancerous tumors in humans. More specifically, the invention relates to antagonists of bombezin, which are 8-9- pseudopeptide containing Tac-, MTac or OMTac - residue C-terminal position; their salts and pharmaceutical compositions, and to methods of synthesis of these peptides and to methods of their use. These peptides are antagonists against bombezin or babesymptom peptides.

The invention relates to polypeptide compounds, which have antagonistic activity against bombezin or babesymptom peptides such as gastrin-vysvobozhdayushchii used, for example, for the treatment of malignant tumors in warm-blooded animals, including humans. The invention relates to new polypeptide compounds and methods for their preparation and to new pharmaceutical compositions containing the polypeptide compounds, and to methods for producing medicines, is able to produce bombezin-antagonistic effect when the introduction of warm-blooded animals, including humans.

The bombezin is an amide of tetradecapeptide, which was first isolated from the skin of the frog Bombina bombina (fire-bellied toad) (Anastasi, Erspamer and Bucci, Experientia, 1971, 27, 166). It is known that the bombezin is a strong mitogen for fibroblastic cells of Swiss mice T (Rozengurt and Sinnett-Smith, Proc. Nat. Acad. Sci. USA, 1983, 80, 2936) and stimulates the secretion of amylase from acinosa pancreas of Guinea pigs (Jensen, Jones, Folkers and Gardner, Nature, 1984, 309, 61). In addition, it is known that jamesindubai peptides are produced and are secreted by the cells of small cell lung cancer (SCL C) man (Moody, Pert, Gazdar, Garney and Minna, Science, 1981, 214, 1246). It is also known that exogenously added bromoindoline peptides can stimulate in vitro growth of SCLC cells human (Garney, Cuttita, Moody and Minna, Canser Research, 1987, 47, 821), and that monoethylene fiberglass with its receptor and to inhibit the growth of SCLC-human cells as in vitro, and in vivo (Cuttita, Carney, Mulshine, Moody, Fedorko, Fischler and Minna, Nature, 1985, 316, 823).

GRP (gastrin-releasing peptide), which has jamesindubai properties is a common emitirovannykh peptide containing 27 amino acids, isolated from the intestine of the pig (McDonald, Jornvall, Nilsson, Vagne, Ghatei, Bloom and Mutt, Biochem. Biophys. Res. Commun., 1979, 90, 227), where C - terminal amino acid sequence is almost identical to the C-terminal sequence of bombezin. Neiromidin C represents aminirovanie Decapeptide, which has a structure identical to the last ten amino acids in the C-terminal region CPP, and which has been isolated from the small intestine of the dog (Reeve, Walsh, Chew, Clark, Hawke and Shively, J. Biol. Chem. , 1983, 258, 5582). GRP stimulates a number of biological reactions, including the release of gastrin in the big circle of blood circulation, and in fibroblastic mouse cells ZTZ and in the cells of small cell lung cancer (SCLC), he also plays the role of a growth factor. Therefore, it was suggested that GRP plays a direct pathophysiological role in the development of SCLC by means of autocrine growth mechanism.

The bombezin, neuromedin C and C-terminal nonapeptide GRP have the following structure:

The bombezin: pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2.

The study of other bambuterodrug peptides derived from amphibians, from the skin of frogs that live in Papua New Guinea, was found to be of winkles, which is a nonapeptide ( pGlu-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2and which, as it turns out, is exceptionally strong analog bombezin (Yasukara et al, Chem. Pharm. Bull., 1979, 27, 492). Studies of analogues of bombezin showed that the segment consisting of at least 9 amino acid residues in the 6-14 provisions have full range babusikova activity.

It was characterized by several types of antagonists bombezin. Substance P (Arg-Pro-Lys-Pro-Gln-Gen-Phe-Phe-Gly-Leu-Met-NH2), which has a very weak homology with the amino acid sequence of bombezin not able to inhibit the binding of bombezin and bambuterodrug peptides, however, as has been discovered, the analogues of substance P, modified by replacing several L-amino acids, D-amino acids, such as D-Arg1D-Pro2, D-Trp7,9, Leu11), substance P and (D-Arg1, D-Phe5, D-Trp7,9, Leu11), substance P (Moody and others, Fed. Proceedings, 1987, 46, 2201), can block the secretion of bombezin in cells acinosa pancreas and poets created the bombezin, originating from bombezin, for example (D-Phe6, D-Phe12), bombezin and [Leu13-psi-Leu14] bombezin (Coy and others, J. Biol. Chem., 1988, 263, 5056 and peptides, 1989, 10, 587), are strong in vitro and in vivo inhibitors babusikova answer.

Another type of antagonist bombezin found Heimbrook, and others, (Bio. Chem., 1989, 264, 11258) represents N-acetyl-GRP (20-26) and its analogues, which are formed from fiberglass (20-27) - analogues by deletion of the C-terminal methioninamide balance. Coy [J. Biol. Chem., 264, 1989, 25, 14691)] showed that some short-chain antagonists bombezin obtained on the basis of the sequence litorina, for example, [D-Phe6, Leu13-psi-Phe16] bombezin (6-14) and [D-Phe6, Leu13-psi-Leu14] bombezin (6-14), have much higher activity than their respective related peptides [Leu13-psi-Leu14] bombezin.

Linear (acyclic) bombazine analogues fiberglass and bombezin amphibians with, but not necessarily, in CH2-NH ones relationship described in the patent application PCTWO 90/03980 (a related analogs are described in WO 91/02746). As indicated in this application, these analogues, which act as natural inhibitors bumbasirevic peptides have the following formula:

< / BR>
where

R12can be a Gln; A4can represent Ala; A5can be a Val; A6can be a Gly; A7can represent His, a W is a:

< / BR>
where R4= CH2-NH; in some cases, Z1can be a identifying side chain of Leu, i.e., - CH2OH (CH3)2; Z2can be a identifying side chain of Cys or Met, i.e., - CH2-SH or - (CH2)2- S-CH3; V = N(R6R7where R6, R7and R8can represent H; and R1and R2can represent H or COE1where E1may be C1C20-alkyl.

Linear peptide analogs of bombezin also described in EP 0309297. These peptides may have a C - terminal Met residue and [CH2- NH] pseudopeptide the relationship between C - end and the adjacent residue.

The invention relates to new polypeptides that are strong antagonists of bombezin, to methods for their synthesis and to new medicines, including pharmaceutical compositions, containing these polypeptides, and the use of these compositions as the pharmaceutically Alenia invention relates to pseudopeptides, possessing a strong bombezin-antagonistic activity and having the formula I;

X-A1-A2-Trp-Ala-Val-Gly-His-Leu-psi-A9-Q (1)

where

X represents hydrogen; a simple relationship linking the alpha-amino group of A1with gamma-carboxyl part 3-propionyloxy group A2if A2is Glu or a group of formula R1CO-, where R1choose from a group that includes:

a) hydrogen, C1-C10-alkyl, phenyl, phenyl-C1-C10-alkyl, P-H1-phenyl, p-H1-phenyl-C1-C10-alkyl, naphthalene - C1-C10-alkyl, indolyl, indolyl - C1-C10-alkyl, pyridyl, pyridyl - C1-C10-alkyl, thienyl, thienyl - C1-C10-alkyl, cyclohexyl, or cyclohexyl - C1-C10-alkyl, where H1, for example, is F, Cl, Br, OH, CH3or OCH3;

b)

< / BR>
where

R2represents hydrogen, C1-C10-alkyl, phenyl, or phenyl-C1-C10-alkyl;

R3represents hydrogen or C1-C10-alkyl;

c) R4- O, where R4represents a C1-C10-alkyl, phenyl, or phenyl-C1-C10-alkyl;

A1represents the D-, L - or DL - amino acid residue selected from the group is likemy substituents, selected from F, Cl, Br, NH2or C1-C3-alkyl; or a peptide bond linking the acyl part of R1CO with alpha-amino-part A2;

A2represents Gln, Glu[-], Glu (Y), or His, where [-] denotes a simple link, if X is a simple bond, and A2is Glu, and this connection binds to the gamma-carboxyl piece or 3-propionyloxy part of A2with the alpha-amino group of A1and Y represents: (a) - OR5where R5is hydrogen, C1-C3-alkyl or phenyl; or (b) where R6is hydrogen or C1-C3-alkyl; and R7is hydrogen, C1-C3-alkyl, or-NHCONH2; Leu-psi is a reduced form Leu, where instead of-CH2- there is a part of C=O, so that the link specified-CH2- part C the alpha-amino group of A neighboring9- balance is pseudopeptides communication;

A9is a Tac MTac, or DMTac;

Q represents NH2or OQ1where Q1is hydrogen, C1-C10- alkyl, phenyl, or phenyl - C1-C10- alkyl, and, in addition, the invention relates to pharmaceutically acceptable acids or salts described pseudopeptides.

Etweda by oxidation of the side chain residue of A9at a certain stage of synthesis of the nonapeptide of the formula I (preferably conducted using formaldehyde or acetaldehyde), resulting in the cyclization of the side chain with the alpha-amino group of residue A9. Identity obtained cyklinowanie balance A9depends on the identity of the original (i.e., deoxidising A9and from the developer.

So if, for example,- CH2-SH group of Cys9cyclized with the alpha-amino group of Cys9related through Leu-psi-pseudopeptides communication, in the reaction with formaldehyde, the resulting ring has the structure of formula IIA below shows how-Leu8-psi-Tac9-NH2-fragment of a nonapeptide of the formula (I):

< / BR>
These pseudopeptide have a higher biological activity and greater stability than their acyclic options, where A9is Cys or Ren. However, in some preferred embodiments of the invention in the case in the peptides of formula I-A9is recyclebank, and X, A2and Q are as defined above, the A9the remainder represents Cys or Ren, and A1is not a naturally occurring amino acid, is Oh, R1CO., R1represents hydrogen or C1C10-alkyl (preferably methyl); A1= D - Cpa, D - Nal, D - Rhe, D - or D-Tpi or D-Trp; A2= Gln; A9= Tac or DMTac, and Q = NH2.

However, in another preferred embodiment of the invention, if A1is a peptide bond linking the acyl part of R1- CO-group to the alpha-amino group of residue A2then A2is Gln or His; A9is Tac M-Tac, or DM-Tac, and Q is NH2. In the preferred form of these peptides X is Hca, Hna, Paa, Mpp, Hpp or Naa; A2is Cln; and A9is TAS.

B. synthesis Methods.

Antagonists bombezin formula I can be synthesized by solid-phase synthesis. In the first scheme of the synthesis of all amino acids sequentially attached to each other after the C-terminal residue to be associated with the solid-phase polymer carrier. Then after binding amino acid residues of the polymer carry out the reaction of the side chain C-terminal residue with the oxidant, in which is formed a 5 membered heterocyclic ring pseudopeptide. The resulting pseudopeptide subjected to HF treatment for separation from the solid phase polymer carrier. Eptide formula I can be obtained in the form of two fragments, which are synthesized by solid-phase synthesis, or by synthesis in the liquid phase. The Tripeptide containing the C-end, associated with the Oligopeptide, resulting in a gain full antagonist bombezin formula I.

5-membered heterocyclic ring formed by reaction of the side chain A9with the oxidant. Then the peptide is subjected to HF treatment, which removes all protective groups of the side chain of amino acid residue and the peptide cleaved from the polymer carrier.

C. Medicines.

Pseudopeptide formula I can be used in pharmaceutical compositions intended for the treatment of cancer or other diseases in mammals by introduction of this mammal an effective dose of pseudopeptide or its therapeutically acceptable acid or salt in combination with a pharmaceutically acceptable carrier. These pseudopeptide in combination with a pharmaceutically acceptable carrier can be introduced in a dose comprising from about 1 to 1000 mg per 1 kg of body weight per day. These compositions can be introduced parenterally, intravenously, subcutaneously, intramuscularly, intranasally, via pulmonary aerosols and slow injection vsasyvanie depending on the introduction of some antagonists bombezin and derived from the data presented in table.7 example 7; Fig. 2 is a graph illustrating the magnitude of SCLC tumor volume in "Nude" mice, depending on the introduction of some antagonists bombezin and obtained on the basis of the data presented in table. 9 example 8; Fig. 3 is a graph illustrating the volume of the tumor pancreatic cancer (MIA PACA-2) in "Nude" mice, depending on the introduction of some antagonists bombezin and obtained on the basis of the data presented in table. 10 example 9; Fig. 4 is a graph illustrating the volume of the tumor pancreatic cancer CAPAN-2 in "Nude" mice, depending on the introduction of some antagonists bombezin and obtained on the basis of the data presented in table.11 example 10.

Description of the preferred embodiments of the present invention.

A. Synthetic peptides.

1. Item.

In this specification, for convenience, used amino acids, peptides and their derivatives are designated in accordance with standard abbreviations generally accepted in the peptide chemistry and recommended by the Commission on biochemical nomenclature of the International Union for pure and applied chemistry (IUPAC - IUB) [European J. Biochem., 1984, 138, 9-37].

Abbrevia, la means alanine, Cys - cysteine, Gln is glutamine, Glu is glutamic acid, pGlu - pyroglutamyl acid, Gly is glycine, His is histidine, Leu is leucine, Phe is phenylalanine, Trp is tryptophan, and Val is valine. Clu may have a functional group associated with the gamma-carboxyl side chain.

[-] or Y is the same as defined above, Dpa represents a 2,3-diaminopropionic acid. In that case, if the amino acid residue has isomeric forms, if there are any indications, means L-shape, otherwise, before the name of the amino acids are D - or DL.

Used in this description rarely occurring amino acids have the following notation:

Cpa - p-chlorophenylalanine

Dpa - 2,3-diaminopropionic acid

PGlu - pyroglutamyl acid

Nal - 3-(2-naphthyl)-alanine

Pal - 3-(3-pyridyl)-alanine

Pen - penicillamine

Tpi - 2,3,4,9-tetrahydro-1H-pyrido-[3,4-b]indole-3-carboxylic acid

Tac - thiazolidin-4-carboxylic acid

DMTac - 5,5-dimethyl-thiazolidin-4-carboxylic acid

MTac - 2-methyl-thiazolidin-4-carboxylic acid

Similar amino acids have the following notation:

Hca - hydracarina acid or para-amino-phenylalanine

Hna - 3-gidromolot

Naa - naphthyloxy acid

Paa - phenylacetic acid

In addition, we used the following notation:

AC - acyl

Ac - acetyl

AcOH - acetic acid

BHA - benzhydrylamine

Boc - tert-butoxycarbonyl

(BOC)2O - di-tert-BUTYLCARBAMATE

Bom - benzyloxyethyl

But - butyl

Bzl is benzyl

BSA - bovine serum albumin

DIC - 1,3-diisopropylcarbodiimide

DMEM - modified according to the method of Dulbecco, Wednesday Needle

DMF (DMF - dimethylformamide

Et - ethyl

EDTA - ethylenediaminetetraacetic acid

FCBS - amniotic calf serum

Fmoc - fluorenylmethoxycarbonyl

For - formyl

HITES - medium RPMI 1640 + 10-8M hydrocortisone, 5 μl/ml bovine insulin, 10 μg/ml human transferrin, 10-8M - estradiol and 3 of 10-8M Na2SeO3< / BR>
HOBt is 1-hydroxybenzotriazole

HPLC (URGH) - high resolution liquid chromatography

Leu - psi reduced form of Leu, where instead of - CH2- there is C = 0 - part, so that the link specified - CH2- part amino-part of the remainder of the A9is pseudopeptides communication

Me - methyl

MeCN is acetonitrile

MeOH methyl alcohol

PBS - phosphate buffer restoredeviceobjects conducted in accordance with generally accepted norm, i.e., the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. However, it should be noted that in the description, if it is not specifically mentioned, the standard numbering of amino acid residues in a fragmented peptide is not observed and do not correspond to their true positions in full tetradecapeptide the antagonist bombezin. If you follow the standard numbering the residues in the nonapeptide of the formula I must be numbered from 6 to 14, and the core Trp - Ala - Val - Gly - His - Leu - antagonists bombezin should be numbered in A8- A9- A10- A11- A12- A13. Instead, to avoid confusion peptide antagonists bombezin used in this description, were numbered as follows:

N-terminal amino acid residue (or residual analogue) was designated A1; C-terminal amino acid residue (or residual analogue) was designated A9; and the intermediate residues were numbered in order, from A2(the residue adjacent to the N-terminal residue of A1) to A8(the residue adjacent to the C-terminal residue of A9).

2. Preferred embodiments of the invention.

In preferred embodiments, swaeg the X - A1- A2- Trp - Al - Val - Gly - His - Leu - - A9- Q I

where

X, A1, A2, Leu - , A9and Q are the same as defined above.

These pseudopeptide, antagonists bombezin, differ in their amino acid sequence, especially in the remains of A1, A2and A9but pseudopeptides communication between A8and A9and not necessarily the presence of 5-membered heterocyclic ring in A9. The ring structure is determined by the identity of the remainder of the A9and the connection used for its oxidation. For example, if formaldehyde is used, and A9represents Cys, the resulting ring has the structure of a Tac of formula IIA (shown below in the form of Leu8- Tac9- Q - fragment peptide of formula I, where Q = NH2) :

< / BR>
If you are using acetaldehyde, and A9is Cys, the resulting 5-membered heterocyclic ring has the structure MTac formula IIB (shown below in the form of Leu8-MTac9- Q - fragment peptide of formula I, where Q = NH2) :

< / BR>
If formaldehyde is used, and A9is the Pen, the resulting ring has the structure DMTac formula IIC ( shown below in the form - Leu8- - DMTac9Q - the effect of the invention preferably to X=H or Ac, A1=D-Phe, A2=Gln and Q=NH2.

Below are the most preferred pseudopeptide antagonists bombezin of the present invention:

Peptide - Structure

1. - D-pGlu-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
2. - D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
3. - D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
4. - Ac-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
5. - D-Cpa-Gln-Trp-Ala-Gly-His-Leu - Tac-NH2< / BR>
6. - D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
7. - D-Nal-Glh-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
8. - Pal-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
9. - D-Pal-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
10. - D-Trp-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
11. - Ac-D-Trp-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
12. - Tpi-Gin-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
13. - D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
14. - Hca-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
15. - D-Phe-His-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
16. - D-Phe-Glu(OMe)-Trp-Ala-Val-Gly-His - Leu - Tac-NH2< / BR>
17. - D-Phe-Glu[-]-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
18. - D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
19. - Ac-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
20. - D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
21. - Tpi-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
22. - D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
Particularly preferred variants are the following peptides

2. - D-Phe-Gln-Trp-Ala-Gly-His-Leu - Tac-NH2< / BR>
Pseudopeptide formula I can be obtained by any method commonly used in peptide chemistry. Such methods are described, for example, in the book of M. Bodanszky, Principees of Peptide Synthesis, Springer-Verlag, Heidelberg, 1984.

All pseudopeptide formula I can be obtained in accordance with the technique of solid-phase synthesis. This method is most preferable to obtain these pseudopeptides and intermediate peptides. The technique of solid-phase synthesis is described in the manual J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, Pierce Chem. Co., Rocrford, IL, 1984 (2nd ed. ), and in the image of G. Barany and others, Imt. J. Peptide Protein Res., 30, 705 - 739, 1987. Additional oxidation step C-terminal residue of A9held for cyclization specific side chain of this residue with its alpha-amino group can be accomplished in one of several stages of the synthesis of the peptides of formula I.

To obtain pseudopeptide antagonists bombezin formula I can be used at least two schemes of synthesis. In the first scheme, all amino acids are sequentially attached to each other, starting from the C-terminal residue of A9pre-attached to the solid phase carrier. In the second scheme, the first polymer carrier synthesize the trip the formation of the desired peptide. In both schemes the synthesis starts with the C-terminal residue of A9to which sequentially add the appropriate amino acids up to the N-terminal residue.

1 (a) the First scheme. As the polymer solid-phase carrier for the synthesis of pseudopeptides can be used benzhydrylamine (BHA) resin or chloromethylpyridine polystyrene resin, crosslinked (1%) with divinylbenzene, and the two resins are commercially available. C-terminal residue of A9attached to one of the above resins through its carboxyl group. To prevent the reaction between these two residues before attaching this residue (A9) to the resin-media, the alpha amino group of each A9- residue block using chemical protective group. Protecting group for the alpha amino group of the A9-balance, and each successively joining residue can be either a 9 - fluorenylmethoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc) group. Fmoc preferable to use when linking C-terminal residue of A9with the remains to A2because the reaction of Boc removal also facilitates the removal of the protective groups of the side chain from the C-terminal Akislotnyh residues can also be protected from undesirable chemical reactions by binding with a chemical protecting group (matching band for A9is a But) prior to reaction of the merger.

After joining the C-terminal Fmoc-A9(But)-residue to a solid carrier alpha-amino group of this residue is subjected to unblock, and then it added Fmoc-Leu-CHO, thereby forming pseudopeptide link.

The remaining residues, A5, A4, A3and A2protected preferably oc group, and A1protected preferably Boc group, Paladino add in reverse order (A7, A6and so on), resulting in getting the desired pseudopeptide.

If the specified pseudopeptide present His or Clu in the process of the reactions of their accession or addition reactions of other residues side chains of these residues can be protected from undesirable chemical effects. In accordance with this before carrying out the reactions of addition of these amino acids in their side chains can be associated with a chemical protecting group.

After all amino acid residues are attached to the BHA-resin, Boc group at the N-Terminus of pseudopeptide can be removed. 5-membered heterocyclic ring with Tac MTac or DMTac can be formed using reaction - CH2- SH-h>and A9(i.e., alpha-amino group of A9), with an oxidant, such as formaldehyde or acetaldehyde. Intermediate pseudopeptide, still bearing protective groups of the side chains, is subjected to HF treatment for its removal from the solid-phase carrier, as well as for the removal of the protective groups of the side chain.

1. The second scheme.

In the second scheme in accordance with the procedure of solid-phase synthesis design Tripeptide using BHA-resin as a carrier, resulting in receiving the following protected Tripeptide on the media: Boc-His (Bom)7-Leu8- -Cys(But)9-BHA-resin

Boc1- the group But the group is removed, and-CH2-SH-part Cys subjected to cyclization with a secondary amine of the reduced connection connecting the remainder of the A8with the rest of the A9resulting in getting His(Bom)7-Leu8-psi-Tac9-BHA-resin. After HF treatment, receive a free Tripeptide: His7-Leu8-psi-Tac9-NH2. In accordance with the standard method of solid-phase synthesis, Gly-OCH2the resin Paladino design peptide on the media:

"Boc A1-A2-Trp-Ala-Val-Gly-OCH2the resin, after which the design process 95% methanol containing 1% KCN (within 12 h), in denaut to free His7-Leu8- Tac9-NH2the Tripeptide using BOP-reagent. Then Boc-group is removed and you get the desired peptide.

Below a detailed description of the synthesis of antagonists bombezin formula I contains some General procedures for the synthesis characteristic of one or both of these schemes.

2. General procedures for polypeptide synthesis.

Procedure 1. Getting some synthetic residues for reagents.

a) L-and D-Tpi: 2,04 g (10 mm) L-Trp was dissolved in 25 ml of boiling water containing 2.1 g of citric acid. Then add 0.5 ml of 40% aqueous formaldehyde, and then immediately began to form solids. The resulting mixture was cooled in an ice bath, and the precipitate was collected, washed with cold water, dried by air and then dried at room temperature, resulting in a received 2.14 g of a solid (99%), so square (decomposition) of about 310oC. D - isomer, which was obtained in the same way from D-Trp, also had so square (Razlog.) about 310oC.

b) L - and D-Boc-Tpi. To a stirred suspension of 10.8 g (50 mm)-Tpi 250 ml of 0.2 N. NaOH and 7.5 ml of triethylamine was added 10 g of Di-tert-BUTYLCARBAMATE, the resulting mixture was stirred 4 h, and then, stirring was added chem is stirred over night and were extracted (2100 ml) ether, and the ether layer was separated and discarded. To the aqueous layer was added citric acid (in order to bring the pH to 3-5). The solid residue was collected, washed with water and dried by air during the night.

Solids suspended in 100 ml of tetrahydrofuran. Almost all solids were dissolved. Insoluble substances were removed by filtration and the THF was removed in vacuum. The residue is triturated with ether and received 9,20 g (or 58%) of the desired material. This material had the same so square as the original material, but different from the source material in its solubility. TLC on silica gel was performed using a mixture of CHCl3: MeOH : HOAC = 85:15:0.5 in.

Repeating the same procedure using 2.55 g - Tpi, got 2,22 g or 59% Boc-Tp.

(C) Fmoc-Leu-CHO. Methyl ester Fmoc-leucine (35 g, 134 mm) in anhydrous toluene (250 ml) under nitrogen atmosphere was cooled with a mixture of dry ice and acetone, and then for 30 min was added 150 ml of 25% hydride, di-isobutylamine in toluene. After that, the mixture stirred 20 min in a bath of dry ice/acetone, and then carefully added methanol (15 ml). The resulting mixture was poured into 1000 ml of ice water, stirred and filtered. Then the toluene was separated and wodna. The obtained oily substance was rapidly passed through a column of silica gel (h cm) in 1500 ml of 15% EtOAc/ petrol. Finally got Fmoc-Leu-CHO in the form of solids (27,6 g).

d) Synthetic amino acids or analogs of amino acids that are introduced into pseudopeptide of the present invention are generally commercially available. For example, Hca is supplied by the company Aldrich Co., 1001 St. Paul Avenue, Milwaukee, W1 53233. Boc - or Fmoc-protected amino acids are delivered by the company Advanced Chem. Tech., 5609 Fern Valley Road, Louiville, KV 40228; or Bachem California, 3132 Kashiwa Street, Torrance, CA UNO 90505.

Procedure 2. Obtaining resin and attaching A9-balance.

BHA-resin was obtained by treatment with 10% TEA in CH2Cl2(for neutralization) (two times for 3 min), and after processing it 6 times washed with methylene chloride. Fmoc-A9(But) - the remainder was associated with resin by the addition of 1.35 mm Fmoc-A9(But) and 1.50 mm 1-hydroxy-benzotriazole (HOBt) in DMF, followed by stirring the resulting mixture for 3 minutes Then the mixture was stirred by shaking for 60 min at room temperature. The obtained Fmoc-A9(But)-BHA-resin was washed with methylene chloride, methanol each time (twice) and three times again with methylene chloride, after which the resin was analyzed using tx2">

Procedure 3. Education pseudopeptides connection.

Cleavage (i.e., unlocking) Fmoc group from A9carried out by adding 50% pyridine in DMF and subsequent 30-minute stirring, washing with DMF (h min), and then (i) the addition of Fmoc-Leu-CHO (3 copies) in DMF containing 1% AcOH; (ii) the addition of NaBH3CN(3.5 EQ.) in DMF, and stirring (by shaking) for 60 minutes the resulting mixture was sequentially washed with 50% MeOH (h min); 100% MeOH (h min) and DMF (h min).

Procedure 4. The joining of amino acids and the formation of the peptide bond

As a protective group for the alpha amino group of the A9-balance and each successively attached amino acid residue can be used either 9-fluorenylmethoxycarbonyl (Fmoc) group, or tert-butyloxycarbonyl (Boc) group. When linking A9subsequent amino acid residues up to the N-terminal amino acid residue is preferred Fmoc-group, since the reaction of Boc removal allows you to simultaneously remove the protective group of the side chain A9-balance.

A) Using Fmoc-amino acids.

For the introduction of Fmoc-amino acids in Fm-intermediate peptide was carried out by following metilenhloride (h min) and DMF (h min)

(2) Fm - amino acid was attached to an unlocked intermediate peptide by (i) adding Fm-amino acids (3 EQ.) and HOBt (3.3 EQ.) in DMF (3 minutes) to intermediate peptide; (ii) addition of 3 equiv. DIC (in the form of a 20% aqueous solution of CH2Cl2); and a 90-minute shaking of the mixture.

(3) the mixture is Then washed with ethanol (g min) and dimethylformamide (h min).

For accession by other residues newly attached Fm-amino acid was subjected to unlock 50% piperidine for 30 min, and then washed with dimethylformamide (h min). Subsequent entry procedures carried out in accordance with the description in stage (2).

Each time after the accession of the new amino acid to the resin or peptide conducted test Kaizer and in case of detection of incomplete binding reaction was repeated.

To attach Fm-Gly and Fm-Gln stage (2) describes the procedure was modified as follows: to a mixture of DMF solution Fm - amino acid (3.0 EQ.) and HOBt (3.3 EQ.) was added for 15 min at 0oC and for 15 min at room temperature) 3 EQ. IC (in the form of a 20% aqueous solution of CH2Cl2). Then the reaction mixture was added to the peptide resin in the case Fm-Gly size is s Boc-amino acid Fmoc-intermediate peptide was carried out by the following procedures:

(1) Boc-group was removed by adding 50% TFA in CH2Cl2;

(2) and then add 50% TFA in CH2Cl2containing 5% mercaptoethanol and 5% anisole for 25 min and

(3) washing with methylene chloride (2x1 min), methanol (2x1 min) and dimethylformamide (h min);

(4) coupling of Boc-amino acid residue is carried out by adding 3 equivalents of Boc-amino acids and 3.3 equivalents of HOBt in DMF and subsequent stirring for 3 minutes Then add 3 equivalent of 20% of diisopropylcarbodiimide in CH2Cl2and stirred by shaking for 90 minutes then the reaction mixture was washed with methanol (h min) and then with methylene chloride (h min).

(5) Boc-group was removed (i.e., carried out unlocking) by washing the product obtained in stage (5), 50% TFA in CH2Cl2containing 5% mercaptoethanol (5 min) and 5% anisole (25 min). Then washed in CH2Cl2(2x1 min), MeOH (2x1 min) and DMF (h min).

It should be borne in mind that the removal of the Boc-group also entails the removal of the protective But-groups, suitably connected with the side chain of A9- balance. Therefore, the removal of the Boc-group should be carried out only immediately before cycles the>The formation of the ring structure of formula IIA was performed as follows: an intermediate peptide having unlocked Cys9the residue in a mixture of 50% AcOH, and 3.7% HCHO, and DMF (1:1:8) stirred for 3 min at room temperature, and then filtered. The obtained product was washed with dimethylformamide, methanol and methylene chloride (each 3 times).

The formation of the ring structure of formula IIB carried out as follows: an intermediate peptide having unlocked Cys9the residue in a mixture of 50% AcOH, 10% CH3CHO and DMF (1:5 : 0:58) stirred 10 min at room temperature, and then filtered. The obtained product was washed with dimethylformamide, methanol and methylene chloride (each 3 times).

The formation of the ring structure of formula IIC was carried out as follows: an intermediate peptide having unlocked Pen9the residue in a mixture of 50% AcOH, and 3.7% HCHO and DMF (1:1:8) stirred 10 min at room temperature, and then filtered. The obtained product was washed with dimethylformamide, methanol and methylene chloride (each 3 times).

Procedure 6. The separation of the peptide from the resin.

After joining all the necessary amino acid residues of the intermediate peptide to the carrier ebsites. The result of this reaction also removes the protective groups of the side chains. In the case of BHA-resin obtained intermediate peptide is Q = NH2.

If X is not H, then X - group is placed at the N-end or by the introduction of amino acids, already bearing X - group (e.g., Ac-D-Phe), or by reaction unlocked the alpha-amino group at the N-terminal A1the remainder of the intermediate pseudopeptide with the appropriate reagent. For example, after separation from the resin that is used as a solid-phase carrier, full peptide may be subjected to reaction with KOC obtaining X=NH2CO.

Procedure 7. Purification of peptides.

Pseudopeptide formula I cleared mainly by using reversed-phase high-performance liquid chromatography (URGH) on the speaker system Rainin (Rainin Inc., Co., Woburn, Ma), consisting of three pumps Rainin Rabbit HP, HPLC, controlled by a computer (Apple Macintosh Plus), dispenser (Pheocyne) and monitor AC UV radiation (Knauer Model 87). The crude peptides (10-40 mg) was loaded into the column Dynamax Marco (21,2x250 mm), Packed spherical C18-silica gel (pore size - particle size of 12 μm) (Rainin Inc. Co.) and suirable linear gradient, using the solvent system, sostoyashchie and retention time using analytical URGH, described below.

The quality and characteristics of elution of the crude and purified peptide was determined using the analytical URGH using the installation for liquid chromatography (model Hewlett).

Packard 1090), equipped with a diode array detector set at 220 and 280 nm, and reversed-phase W-Porex-C18-column (4,h mm) (pore size: the particle size 5 μm). The flow rate of solvent system (A) and (B) described above, was maintained at a level of 1.2 ml/min, and the separation was performed at room temperature.

In most cases pseudopeptide antagonists bombezin was purified by re-chromatography on the same column, but with slight changes in the conditions for gradient elution. As shown by analytical HPLC, the obtained homogeneous purified peptides had a purity of more than 97%. If necessary, can be performed amino acid analysis of pseudopeptides formula I using amino acid analyzer, Beckman 630 and samples hydrolyzed for 20 h at 110oC, in sealed obesogenic tubes with 4 M methanesulfonic acid containing 0.2% 3-2(2-amino-ethyl)-indole.

C. Synthetic intermediate peptides.

When choosing a particular protective group of the side chain, which can be used in the synthesis of peptides, usually guided by the following rules: (a) it is preferable that the protective group maintained its protective properties under the reaction conditions of accession; (b) the protective group must be stable relative to the binding reagent, and preferably stable under the reaction conditions attaching selected for deletion alpha aminosidine group in each stage of the synthesis; (c) a protective group of the side chain must be removed after completion of the synthesis of the desired amino acid sequence, and in such reaction conditions, which did not lead to undesirable changes in the peptide chain.

Protective groups of the side chain is associated with amino acid residues stepwise sposabella But.

2. Synthetic intermediate peptides.

It should be noted that the scope of the invention includes an intermediate peptides at appropriate stages of the synthesis. Below is illustrated the intermediate peptide 2 in three stages:

Stage A: after joining all the amino acids and resins:

Boc-D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)-BHA-resin ("1/02/A")

Stage B: after removal - terminal Boc-group:

D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys-BHA-resin ("1/02/B").

Stage C: after cyclization of the A9:

D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Tac-BHA-resin ("1/02/C").

And there is still one more stage to stage D, which is illustrated in the synthesis of peptide N 17 (example 2). Intermediate phase peptide D is separated from the media, but he still does not have a simple relation X connecting A1c A2.

Used in medicine.

Antagonists bombezin can be used to treat diseases such as hypergastrinemia, for example, perriniana anemia, chronic atrophic gastritis syndrome Zollinger-Ellison and vitiligo, associated with diffuse hyperplasia enterochromaffin cells of the stomach and an increased risk of developing primary multiple gastric cancer. In addition, hyperplasia of these of this invention have advantages over existing drugs, since H2antagonists like cimetidine cause hypergastrinemia and can lead to the formation of carcinoid tumors in humans. In addition, therapy using H2-antagonists immediately leads to recurrent ulcer disease due to the presence of hypergastrinemia.

Since the compounds according to the invention are antagonists of bombezin/GRP receptors, they can be used to treat lung cancer, colon cancer and stomach cancer.

Antagonists bombezin formula I of this invention can be introduced in the form of a pharmaceutically acceptable non-toxic acids or salts, such as acid additive salt. Examples of such acid additive salts can serve as the hydrochloride, hydrobromide, sulfate, phosphate, fumarate, gluconate, tannat, maleate, acetate, citrate, benzoate, succinate, alginate, pamoate, malate, ascorbate, tartrate etc.

Basically, the pharmaceutical compositions contain pseudopeptide formula I in combination with standard pharmaceutically acceptable carriers, of which the most suitable is a physiological solution, although you may be used and other known carriers.

Treatment using the clinical treatment with other opioid agonists and LHRH antagonists or analogues of somatostatin. For example, antagonists of bombezin formula I can be administered intravenously, subcutaneously, intramuscularly, intranasally, via pulmonary aerosols or using forms of delayed absorption (e.g., microcapsules, microspheres or cylindrical rod implants made of biodegradable polymer (such as DL-lacticacid). The invention also includes other methods of administration of the compositions, for example, by drip infusion, infusion using an infusion pump and injection using dosage forms for sustained release, for example, microcapsules, etc.

The effective dose of the peptides of formula I can vary depending on the method of administration and the type of mammal undergoing treatment. When injecting the dose of injected peptides can be from about 1 to 1000 mg per 1 kg of body weight per day. This dose range is preferred, however, in some cases, there may be used higher doses. Physiological solution containing the indicated peptide can be introduced in such a quantity, which would provide a dose of about 0.01 - 0.20 mg/kg of body weight per day, except t is ivalsa approximately 15-30 days or more.

In the examples to identify intermediate peptides at certain stages of the synthesis, there are three code symbols. The peptide under code 1/01/A" represents an intermediate peptide to peptide "01" obtained in example 1, which was synthesized in A stage of synthesis. Similarly codes "2/08/B" and "4/19/C" refers to the intermediate peptides for peptides "08" and "19", obtained in examples 2 and 4, which were synthesized in stages B and C, respectively.

Example 1.

Peptide #

1. D-pGlu-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
2. D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
4. Ac-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
5. D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
7. D-Nal-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
8. Pal-Gln-Trp-Ala-Gly-His-Leu - Tac-NH2< / BR>
9. D-Pal-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
10. D-Trp-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
11. Ac-D-Trp-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
12. Tpi-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
13. D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
14. Hea-Gln-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
These peptides can be synthesized from a common intermediate peptide 1-1 (Fmoc-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)-BHA-resin in the following way:

Fmoc-Leu-Cys (But)-BHA-resin was prepared as follows: 2.0 g BHA-resin (0.55 mmol H2/g) amrabat the e within 3 min were mixed with 3.3 mm Fmoc-Cys(But) and 3.6 mm HOBt in DMF. Then added 3 EQ. DIC (in the form of a 20% aqueous solution of CH2Cl2). The mixture was shaken for 90 min at room temperature. The obtained Fmoc-Cys(But)-BHA-resin was washed twice with methylene chloride, twice with methanol and three times again with methylene chloride and then subjected to the test Kaizer.

Join Fmoc-Leu-CHO with education pseudopeptides communication was carried out as follows. The removal of Fmoc group from A9carried out by adding 15 ml of 50% piperidine in DMF and subsequent 30-minute stirring and washing (h min) 15 ml DMF. After this was added 3.3 mm Fmoc-Leu-CHO (3 EQ.) in DMF containing 1% AcOH, and then NaBH3CN(equ.) in DMF. The mixture was shaken for 1 h, and then washed with 15 ml of 50% aqueous MeOH (3x1 min); 15 ml 100% MeOH (3x1 min); and 15 ml of DMF (h min).

Removal of Fmoc groups (unlocking) of Fmoc-Leu - Cys (Bom)-BHA - resin was carried out in accordance with the procedure 4A.

After removal of Fmoc group from Fmoc-Leu - Cys (But)- BHA-resin and neutralizing the spent reaction accession Fmoc-His(Bom) in accordance with the procedure of 4A (see above).

Join Fmoc-GlY was carried out as described in procedure 4. For this to DMF - solution (0oC) 3.3 mm Fmoc-GLy and 3.6 mm HOBt was added 20% 1,3 - diisopropylcarbodiimide, was added to the resin and stirred for 60 minutes Then the same way consistently attached amino acid residues Fmoc-Val, Fmoc-Ala and Fmoc-Trp, resulting in received 3.80 g of the intermediate peptide is 1-1, i.e., the protected peptide on the carrier with the structure:

Fmoc-Trp-Ala-Vol-Gly-His(Bom)Leu - Cys (But)- BHA-resin.

In the subsequent addition of Fmoc-Cln and Boc-D-pGlu to intermediate peptide 1-1, received Boc-D-pGlu - Gln-Trp-Ala-Val-Cly-His(Bom)-Leu-Cys (But)-BHA-resin ("1/01/A").

In the subsequent addition of Fmoc-Gln and Boc-D-Phe to intermediate peptide 1-1 received Boc-D-Phe-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu - Cys(But)-BHA-resin ("1/02/A").

In the subsequent addition of Fmoc Gln and Ac-D-Phe to intermediate peptide 1-1, received Ac-D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys (But) - BHA-resin ("1/04/A").

In the subsequent addition of Fmoc-Gln and Boc-D-Cpa to intermediate peptide 1-1, received Boc-D-Cpa-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(But)-BHA-resin ("1/05/A").

In the subsequent addition of Fmoc-Gln and Boc-D-Nal to the intermediate peptide 1-1, received Boc-D-Nal-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(But)-BHA-resin (1/07/A").

In the subsequent addition of Fmoc-Gln and Boc-Pal to intermediate peptide 1-1, received Boc-Pal-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)-BHA-resin ("1/08/A").

The result is-BHA-resin ("1/09/A").

In the subsequent addition of Fmoc - Cln and Boc-D-Trp to intermediate peptide 1-1 received Boc-D-Trp-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys-(But)-BHA-resin ("1/10").

In the subsequent addition of Fmoc-Gln and Ac-D-Trp to intermediate peptide 1-1 received Ac-D-Trp-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)-BHA-resin "1/11/A").

In the subsequent addition of Fmoc-Gln and Boc-Tpi to intermediate peptide 1-1 received Boc-Tpi-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(But)-BHA-resin ("1/12").

In the subsequent addition of Fmoc-Gln and Boc-D-Tpi to intermediate peptide 1-1 received Boc-D-Tpi-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(But)-BHA-resin ("1/13").

In the subsequent addition of Fmoc - Gln and Hca to intermediate peptide 1-1 received Hca-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(But)-BHA-resin ("1/14/A").

Then, the N-terminal Boc group of intermediate peptide 1/01/A was removed by treatment with 50% TFA in CH2Cl2containing 5% mercaptoethanol and 5% anisole (twice: first 5 min, and then 25 min). The obtained intermediate peptide was washed with methylene chloride ( 2x1 min), methanol (2x1 min) and DMF (h min). Then from Cys was also tsalala protective But-group, resulting in a received intermediate peptide D-pGlu-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys-BHA-resin" ("1/01/B"), which is then washed methylenchloride peptide 1/01/B was subjected to cyclization by oxidation with the formation of 5-membered heterocyclic ring, shown in formula IIA (procedure 5). To do this, to the specified peptide was added 10 ml of a mixture of AcOH, and 3.7% HCHO and DMF (1:1:8). The resulting reaction mixture was shaken for 3 min at room temperature, washed with water, DMF and CH2Cl2(3 times each), resulting in a received intermediate peptide 1/01/C (D-pGln-Gln-Trp-Ala-Gly-His(Bom)-Leu - Tac-BHA-resin).

Then, the intermediate peptide 1/01/C was treated with hydrogen fluoride and anisole according to the procedure 6 for separating the protective Bom group from His and simultaneous removal of the nonapeptide resin carrier, which formed the C-terminal Q-group, which represents-NH2. The resulting peptide was purified using URGH, as described in procedure 7. Thus was obtained a peptide antagonist bombezin, referred to as peptide 1.

Similar stage to remove the Boc-group and But group and subsequent cyclization - CH2-SH group of Cys-residue with a secondary amine of the reduced communication was performed using intermediate compounds 1/02/A, 1/05/A, 1/07, 1/08, 1/09, 1/10 and 1/12/A, which received the following intermediate peptides:

1/02/B D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)- Leu - Tac-BHA-resin

1/04/B Ac-D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu - Tac-BHA-resin

1/05/B D-Cpa-Gln-Trp-

1/09/B D-Pal-Gln-Trp-Ala-Val-Gly-His(Bom)- Leu - Tac-BHA-resin

1/10/B D-Trp-Gln-Trp-Ala-Val-Gly-His(Bom)- Leu - Tac-BHA-resin

1/11/B Ac-D-Trp-Gln-Trp-Ala-Val-Gly-His(Bom)- Leu - Tac-BHA-resin

1/12/B Tpi-Gln-Trp-Ala-Val-Gly-His(Bom)- Leu - Tac-BHA-resin

1/13/B D-Tpi-Gln-Trp-Ala-Val-Gly-His(Bom)- Leu - Tac-BHA resin

1/14/B Hca-Gln-Trp-Ala-Gly-His(Bom)- Leu - Tac-BHA resin

The crude peptides were tsalala from BHA-resin, and then purified in accordance with the procedures 6 and 7, the result of which has been purified peptides 2, 4, 5 and 7-14.

In table. 1 provides data relating to the retention time in the HPLC.

Alternative 5-membered heterocyclic ring can be formed in the solution in accordance with the following procedure:

25 mg of the intermediate peptide, including the structure of Leu-psi Cys-NH2obtained by solid-phase synthesis, 0.8 glacial acetic acid was mixed with 100 μl of 1% formaldehyde (wt.%, the solution in water) for 30 s at room temperature, and then added 20 μl of a 50% aqueous solution of ammonium acetate. After purification of the reaction mixture was obtained the desired peptide, which contained structure - Leu-psi-Tac-NH2.

Example 2. 15. D-Phe-His-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
16. D-Phe-Glu(OMe)-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
17. D-Phe-Glu[-]-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
In this example, the peptides Paladino constructed on the basis of 0.5 g BHA - resin (0.55 mm NH2/g). Intermediate peptide 1-2 was obtained in accordance with the described procedures, and the procedures of example 1. Part (150 mg) intermediate peptide 1-2 was divided into 3 aliquots and subjected to two subsequent reactions of accession in accordance with the procedure 4, which received final nonapeptide on the media.

As a result of successive accession Fmoc-Glu (OMe) and Boc-D-Phe to intermediate peptide 1-2 received BOC - D-Phe-Glu(OMe)-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)- BHA-resin ("2/15/A").

In the serial connection (OMe) and Boc-D-Phe to intermediate peptide 1-2 received Boc-D-Phe-Glu(But)-Trp-Ala-Val - Gly-His(Bom)-Leu-Cys(But)-BHA-resin ("2/16/A").

In the sequential joining of Fmoc-Glu-(But) and Boc-D-Phe to intermediate peptide 1-2 received Boc-D-Phe-Glu(But)-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)-BHA-resin ("2/17/A")/

Removal of Boc group of intermediate peptide 2/15/A was carried out in accordance with procedure 4 using 50% TFA in CH2Cl2containing 5% mercaptoethanol and 5% anisole. In this reaction was also removed But the group from the remainder of the A9, resulting in the received intermediate peptide 2/15/B:

"D-Phe-His(Bom)-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys-BHA-resin".

At this stage side Cys-group promezhutochnogo in formula IIA), as described in procedure 5. To do this, to the specified peptide was added 10 ml of a mixture of AcOH, and 3.7% HCHO and DMF (1:1:8). The reaction mixture was shaken for 3 min at room temperature, and then washed with water, dimethylformamide and methylene chloride (3 times each), resulting in a received intermediate peptide 2/15/C:

D-Phe-His(Bom)-Trp-Ala-Val-Gly-His(Bom)-Leu - Tac-BHA-resin.

Then, the intermediate peptide 2/15/C was treated with the freshly distilled hydrogen fluoride (5 ml) and anisole (0.25 m) for 1 h at 0oC, resulting from His was useplease protective Bom group. After that, the solvent is evaporated under vacuum, washed with ethyl acetate, was extracted with 70 - 80% aqueous solution of acetic acid and liofilizovane. After purification was obtained peptide antagonist bombezin N 15.

Similar stage to remove the N-terminal Boc-group, cyclization Cys9, the elimination of intermediate peptide from BHA-resin and purification using URGH was performed using the intermediate peptides 2/16 and 2/17/A, which received the peptide N 16, and an intermediate peptide 2/17/D:

D-Phe-Glu-Trp-Ala-Val-Gly-His-Leu - Tac-NH2.

A mixture of 15 mg of the intermediate peptide 2/17/1 and 15 mg of HOBt 0.8 DMF at 0oC was added to 50 μl of 25% of diisopropylcarbodiimide in CHI am the alpha amino group of residue D-Phe1with gamma-carboxyl part 3-propionyloxy part of the residue Glu2:

D-Phe-Glu[-]-Trp-Ala-Val-Gly-His-Leu - Tac-NH2< / BR>
Then the reaction mixture was subjected to purification using VRGH in accordance with procedure 7.

In table. 2 shows the retention times for these peptides.

Example 3. Peptide N

3. D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-MTac-NH2< / BR>
6. D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu - MTac-NH2< / BR>
These peptide antagonists bombezin can be synthesized from a common intermediate peptide 1 - 3 (i.e., Fmoc-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Cys(But)-BHA-resin). This peptide was derived from BHA-resin (1.0 g, 0.55 mm NH2/g) by successive reactions of the merger, as described in example 1, starting from Fmoc-Cys(But) and subsequent merger of Fmoc-Leu-CHO (NaBH3CN); Fmoc-His(Bom), etc., in accordance with the procedure described in example 1. Fmoc-Gln was added to the N-end in accordance with the procedure 4 and received intermediate peptide 1 - 3. After the final annexation of Boc-D-Phe or Boc-D-Cpa to intermediate peptide 1 - 3, conducted in accordance with the procedure 4, received intermediate peptides "3/3/A", or "3/6/A", respectively.

Removal of Boc-group was performed using 50% TFA in CH2Cl2containing 5% mercaptoethyl 3/3/B was subjected to cyclization by oxidation with the formation of 5-membered heterocyclic ring shown in formula IIB), as described in procedure 5. To do this, to the specified peptide was added 10 ml of a mixture of 50% AcOH, 10 CH3CHO and DMF (1,5:0,5: 8). The reaction mixture was stirred in a shaker for 10 min, and then washed with water, dimethylformamide and methylene chloride (3 times each), resulting in a received intermediate peptide 3/3/C : D-Phe-Trp-Ala-Val-Gly-His-Leu-MTac-BHA-resin.

Then the intermediate peptides 3/3/C and 3/6/C tsalala from resin carrier, as described in procedure 6, i.e. by treatment (1 h at 0o(C) fluoride (5 ml) and anisole (0.25 ml). This procedure was also useplease from His protective Bo-group. The result has been the target nonapeptide 3 and 6.

These peptides were purified using VRGH in accordance with the procedure 5; their retention times are presented in table. 3.

Alternative 5-membered heterocyclic ring can be formed in solution by adding 25 mg of intermediate peptide containing the structure of Leu-Cys-NH20.8 ml of glacial acetic acid, mixed with 100 ál of 10% of acetaldehyde at room temperature. The mixture is stirred 5 min, and then was added 100 μl of ammonium acetate. The reaction mixture was subjected to purification and got the desired peptide, which contained the structure of Leu - -MTac-NH2< / BR>
Example 4.ac-NH2< / BR>
21. Tpi-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
22. D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu-DMTac-NH2< / BR>
These polypeptides can be synthesized from a common intermediate peptide 1-4 (Fmoc-Trp-Ala-Val-Gly-His(Bom)-Leu-Pen(But)-BHA-resin). This intermediate peptide Paladino designed in accordance with standard methods of solid-phase synthesis (described in examples 1 and 2 using benzhydrylamine (BHA) resin.

So, for example, 1.0 g BHA-resin (0.55 mm NH2/g), obtained in accordance with procedure 2, was treated with 10 ml of 10% TEA in CH2Cl2(neutralization) (two times for 3 min) and washed 6 times with 10 ml of methylene chloride, and then for 3 min was mixed with 1.6 mm Fmoc - Pen (But) and 1.8 mm 1-hydroxybenzotriazole (HOBt) in DMF. Then add 20% 1,3-diisopropylcarbodiimide (DLC) (1.6 mm) in CH2CI2. The mixture is stirred in a shaker for 90 min at room temperature. The resulting peptide "Fmoc-Pen (But)-BHA-resin was washed with methylene chloride, methanol (each 2 times) and again with methylene chloride (three times), and then subjected to the test Kaizer.

The removal of Fmoc groups (unlock) from peptide "Fmoc-Pen(But)-BHA-resin was carried out in accordance with the procedure 4A.

Join Fmoc-Leu-CHO carried out in accordance with the procedure 3.Les this 1.8 mm NaBH3CN in DMF. The reaction mixture was stirred for 60 min in a shaker, and then twice washed with 50% methanol in water, twice with 100% methanol, three times with methylene chloride each time for 1 min).

After removing Fmoc group from the peptide "Fmoc-Leu-Pen (But)-BHA-resin, and neutralization was carried out by attaching Fmoc-His (Bom) in accordance with the procedure 4.

Join Fmoc-Gly was carried out as described in procedure 4. For this to DMF-solution (0oC) containing 1.5 mm Fmoc-Gly and 1,65 mm HOBt, add 20% 1,3-diisopropylcarbodiimide (1.5 mm) in CH2CI2and stirred for 15 minutes while cooling and for 15 min at room temperature, after which the precipitate was filtered, added to the resin and stirred for 60 min in a shaker. Then in the same way consistently attached amino acid residues Fmoc-Val, Fmoc-Ala and Fmoc-Trp, resulting in received 1.9 grams of the intermediate peptide 1-4, i.e., the protected peptide on the media having the structure:

Fmoc-Trp-Ala-Val-Gly-His(Bom)-Leu-Pen(But)-BHA-resin.

0.91 g of the obtained intermediate peptides 1-4 were divided into five aliquot (200 mg each) were used for the synthesis of protected polypeptides on the media in accordance with the procedure 4.

In the sequential joining of Fmoc-Gln and >/P>As a result of successive accession Fmoc-Cln and Ac-D-Phe to intermediate peptide 1-4 received peptide:

Ac-D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Pen(But)-BHA-resin" ("4/19/A").

In the sequential joining of Fmoc-Gln and Boc-D-Cpa to intermediate peptide 1-4 received peptide:

"Boc-D-Cpa-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Pen(But)-BHA-resin" ("4/20/A").

In the sequential joining of Fmoc-Gln and Boc-Tpi to intermediate peptide 1-4 received peptide:

Boc-Tpi-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu - Pen(But)-BHA-resin" ("4/21/A").

In the sequential joining of Fmoc - Gln and Boc-Tpi to intermediate peptide 1-4 received peptide:

Boc-D-Tpi-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Pen(But)-BHA-resin" ("4/22/A").

Then from the intermediate peptide 4/18/And deleted the Boc group by treatment with 50% TFA in CH2Cl2containing 5% mercaptoethanol and 5% anisole, resulting in a received intermediate peptide 4/18/B:

D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu-Pen-BHA-resin.

At this stage side Pen-group intermediate peptide 4/18/B was subjected to cyclization by oxidation with the formation of 5-membered heterocyclic ring (shown in the formula IIC) as described in procedure 5. To this was added 10 ml of a mixture of AcOH, and 3.7% HCHO and DMF (1:1:8) and the resulting reaction mixture razmera 3 times). The result of this procedure received intermediate peptide 4/18/C: D-Phe-Gln-Trp-Ala-Val-Gly-His(Bom)-Leu- -DMTac-BHA-resin.

Then, the intermediate peptide 4/18/C was washed in 5 ml of CH2Cl2, methanol and CH2Cl2(each three times), and was treated with the freshly distilled hydrogen fluoride (5 ml) and anisole (0.25 ml) at OoC for 1 h the Solvent is evaporated in vacuo, washed with ether or ethyl acetate, was extracted with 70-80% acetic acid and was liofilizovane, resulting received untreated nonapeptide resin. The reaction mixture was purified and obtained peptide antagonist bombezin N 18.

Similar stage of removal of the protective groups, cyclization unlocked A9-balance and cleavage of the peptide from the BHA-resin was performed using the intermediate peptides 4/19, 4/20, 4/21, and 4/22/A, resulting in the obtained peptides 19, 20, 21 and 22.

Purification was performed using VRGH in accordance with procedure 7, using a solvent system consisting of (A) with 0.1%TFA and (B) 1% TFA in 70% acetonitrile. As shown by analytical URGH, purified peptides had a purity of over 97%. The retention times for these peptides are shown in table.4.

Alternative A9-the remainder can be cycles the ml glacial acetic acid, mixed with 100 ál of 10% formaldehyde, followed by stirring for 30 min at 0oC. the result of this procedure was given peptide having a 5-membered ring structure Leu - - DMTac-NH2.

Example 5.

Pal-Gln-Trp-Ala-Val-Gly-His-Leu-Cys-NH2< / BR>
D-Pal-Gln-Trp-Ala-Val-Gly-His-Leu-Cys-NH2< / BR>
Tpi-Gln-Trp-Ala-Val-Gly-His-Leu - Cys-NH2< / BR>
D-Tpi-Gln-Trp-Ala-Val-Gly-His-Leu-Cys-NH2< / BR>
Hea-Gln-Trp-Ala-Val-Gly-His-Leu-Cys-NH2< / BR>
These peptides can be synthesized from intermediate peptide: "Fmoc-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu-Cys (But)-BHA-resin.

Fmoc-Leu-Cys(But)-BHA-resin was prepared as follows:

1.0 g BHA-resin (0.55 mm H2/g) was consistently associated with Fmoc-Cys (But), and Fmoc-Leu-CHO in accordance with procedures 2 and 3 (see above).

As a result of successive accession Fmoc-His (Bom),

Fmoc-Gly, Fmoc-Val, Fmoc-Ala and Fmoc-Trp and Fmoc-Gln received intermediate peptide 1-5 (Fmoc-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu-Cys (But)-BHA-resin).

150 mg aliquot of this (common to all peptides in this example) of the intermediate peptide 1-5 were subjected to additional reactions accession conducted in accordance with the procedure 4, which received the peptides on the resin carrier.

After joining Boc-Pal to intermediate peptide 1-5 received pedo 1-5 received the peptides: Boc-D-Pal-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu-Cys( But)-BHA-resin ("5"09/A").

After joining Boc-Tpi to intermediate peptide 1-5 received the peptides: Boc-Tpi-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu-Cys (But)-BHA-resin ("5/12/A").

After joining Boc-D-Tpi to intermediate peptide 1-5 received the peptides: Boc-D-Tpi-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu-Cys( But)-BHA-resin ("5/13/A").

After joining Hca to intermediate peptide 1-5 received peptide: Hca-Gln-Trp-Ala-Gly-His (Bom)-Leu-Cys (But)-BHA-resin ("5/14/A").

N-terminal Boc-group was removed from intermediate peptide 5/01/A by treatment with 50% TFA in CH2Cl2containing 5% mercaptoethanol and 5% anisole, which was performed twice: first for 5 min, and then for 25 minutes In the process was removed protective group them Cys. Then the peptide was washed with methylene chloride and dimethylformamide in accordance with the description in the procedure 4 and received in the intermediate peptide 5/08/B: Pal-Gln-Trp-Ala-Val-Gly-His (Bom)-Leu-Cys-BHA-resin.

Then, the intermediate peptide 5/08/B were treated for 1 h (at 0oC) the freshly distilled hydrogen fluoride (95 ml) and analysis (0.25 ml), resulting in useplease (His) protective Bo-group. After that, the solvent is evaporated under vacuum, washed with ethyl acetate, was extracted with 70-80% acetic acid and was liofilizovane, resulting on the A, 5/13 and 5/14/A remove the protective group and BHA-resin and received the peptides mentioned in the beginning of this example. The resulting peptides were tested for purity according to the description in the procedure 7. The data are given in table. 5.

Alternative these peptides can be Paladino synthesized according to standard solid-phase synthesis technique based on the BHA-resin with Boc-protected amino acids and using Bz for the protection of the-SH group of Cys residue9. The result can be obtained following intermediate peptides:

Boc-Pal-Gin-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(Bz)-BHA resin ("5/08/A").

Boc-D-Pal-Gin-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(Bz)-BHA resin ("5/09/A").

Boc-Tpi-Gin-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(Bz)-BHA resin("5/12/A").

Boc-D-Tpi-Gin-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(Bz)-BHA resin("5/13/A").

Hca-Gin-Trp-Ala-Val-Gly-His(Bom)-Leu - Cys(Bz)-BHA resin("5/14/A").

Boc-group was removed by treatment with 50% TGA in CH2Cl2containing 5% mercaptoethanol and 5% anisole, and the peptide resin was separated by treatment with HF and anisole, the result was also derived protective Bom group (from His) and Bz-group (Cys). In the described procedures received the peptides mentioned in the beginning of this example.

Example 6. The analysis procedure.

And Analysis on linking with PE and was assessed using 24-well plates for culturing tissues (CIBCO) and cells of SWiss ZTZ. Murine fibroblasts Swiss ZTZ maintained by weekly passage in DMEM containing 10% FCB and antifungal agents. Cultures were incubated at 37oC in air with 5% CO2. The wells were seeded with cells (105cells/well, viability > 95%), grown to the condition and continuity of the rest phase. The procedure of binding was carried out 7 days after seeding cells. Cells were washed 2 times with 0.5 ml buffer for binding (modified by way of Dulbecco, Wednesday Needle containing 20 nm HEPES-NaOH, pH of 7.4; 0.2% BSA; and 100 μg/ml bacitracin). Then cells were incubated with 0.2 nm125I-Tyr4-bombezin in the presence (or absence) of different concentrations of antagonists (610-11-610-6M, total volume 0.4 ml).

Cells were incubated for 30 min at 37oC, since binding125I-GRP at 37oC reaches its maximum for 30 min, and then decreases, as indicated Zachary and Rozengurt (1985) and Layton and others, (1988). After that, the cells twice washed with ice (4oC) buffer and twice - ice of a phosphate buffer solution (PBS, mm): NaCl 138, KCl 2,8; Na2HPO48; KH2PO41,45; CaCl20,91; MgCl20,49. The washed culture was extracted with 0.5 ml of 0.5 M NaOH and transferred in p the corresponding tubes. The radioactivity of the sample was counted in an automatic gamma counter (Micromedic System, Inc. Huntsville, Ala.).

To determine the types of the receptor binding dissociation constants (Kd), Association constants (Ka) and the maximum binding capacity of the receptor (Bmax) used the program Munson and Rodbard for computerized selection "ligand-RS" - curve.

Data related to the binding polypeptides of this invention are presented in table. 6. To assess the ability of the competitive specific binding125I-Tyr4-bombezin used different doses of its peptide.

Data are the average of 2-3 independent tests conducted for each peptide in 3 duplicates.

Example 7. The test is to assess the impact of treatment with antagonists bombezin on the size (volume) of tumor estrogen-independent breast cancer mouse Mat was carried out as follows.

40 female mice B61D2F1received from the National cancer Institute, Frederick Cancer Research Facility (Frederick, Maryland) and put them in condition 55+5% humidity at 211oC. Animals were kept in an automatic light mode: 12 h light/12 h darkness, and gave laboratory diet to bite the SHL Mat (3,2/0 Ovex, received from Dr.A.E. Bogden (Biomeasare Inc.,Hopkinton, MA) was inoculable subcutaneously Mature mouse-female with tumor volume Mat (3,2)/Ovex - 1 mm3.

Two days after transplantation of tumor mice were randomly divided into 4 groups and started to therapy using prolonged release (microcapsules) peptides. These four groups were as follows:

1) Control, injection of only one filler;

2) [D-procedures defined in the TPI6, Leu13- - Leu14]Bn (6-14);

3) [D-Phe6, Leu13- Tac14]Bn (6-14);

4) Bilateral surgical oophorectomy.

With the reduction in the names of the antagonists bombezin above used the standard numbering of residues of the peptide fragment, i.e., each residue is numbered according to its position in the full fragment. However, these antagonists bombezin can be easily compared with other peptides, the numbering starts with "1" from the N-Terminus. So, for example, [D-Tpi6-Leu13- - Leu14]Bn(6-14) represents a D-Tpi1-Gln2-Trp3-Ala4-Val5-Gly6- His7-Leu8- - Leu9-NH2(also called "B1", and "[D-Phe6-Leu13- Tac14] Bn (6-14)" represents the method of solid-phase synthesis. Mouse group 2 received composition sustained release of "B1", and mouse group 3 received composition with peptide N 2 (see above). The composition of extended release provided a slow release of B1 or peptide N 2 at the rate of 25 mcg/day for 15 days.

For prolonged delivery used osmotic Alzet pumps (Alzo Co., Pa 10 Alto, CA). Pump Alzet model 2002 c the rate of release of 0.48 mg/h implanted subcutaneously in the inferolateral region of the back. For filling osmotic minnasota peptides were dissolved in 50% (vol. /about.) an aqueous solution of propylene glycol.

After all the tumors became visible and palpable, their volumes were measured and perform calculations in accordance with the description Szende B., and others in the "Growth inhibition of MXT Mammary Carcinoma by Enhancing Programmed Cell Depth" (apoptosis with analogrus of LH-RH and Somatostatin (apoptosis analogues of Lh-PH and somatostatinoma). Breast Cancer Res. Treat, 307-314 (1989).

Experiments were completed at the exponential growth phase. First measurement of the volume of the tumor was performed after 10 days. Differences in tumor volumes between control groups and groups treated with antagonists bombezin, as well as between the two groups treated with antagonists bombezin were statistically the ass end of the experiments, mice were killed by exsanguination under Metofane anesthesia, was determined the mass of tumors and the obtained data were subjected to statistical analysis using the criterion Duncan's t-test. The results of the analysis are presented in table. 8.

Example 8. The influence of one of the analogues of somatostatin and three antagonists bombezin on small cell carcinoma of the human lung in "Nude" mice was evaluated as follows. From the National cancer Institute (Bethesda, MD) received male "Nude" mice (Nude mice with a mutation in the gene nude) at the age of 6 weeks., and contained in the conditions close to sterile.

Cells small cell human lung cancer (SCLC line H69) were cultured as monolayer in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% albumin bovine serum, antibiotics and antifungal agents at 37oC in humidified atmosphere containing 5% CO2. The xenografts were initiated by subcutaneous injection of 1 to 107cells taken from exponentially growing cells in tissue culture (right side) five "Nude" mice. After three weeks, the resulting tumors were dissected under aseptic conditions and mechanically crushed. Then the pieces of tumor tissue 3 mm3transplanted subcutaneously (using needle Trocar) 60 animals. Through what were randomly divided into 5 experimental groups to handle five different compounds (starting with the first day after two weeks after transplantation). Tumors were measured weekly for 5 weeks. Tumor volume was calculated by the formula: (length width height)/6. Then 8 animals from each group were killed to measure the mass of tumors.

As the first drug for the introduction of the "Nude" mice used similar somatostatin D-Phe-Cys Tyr-D-Trp-Lys-Val-Cys-Trp-NH2(denoted by "SI").

The first of the three antagonists bombezin was a peptide-D-Tpi1-Gln2-Trp3-Ala4-Val5- Gly6-His7-Leu8-psi-Leu9-NH2, i.e., BI. The second antagonist bombezin were:

[Tpi6-Leu13-psi-Tpi14] Bn (6-14), i.e. D-Tpi1Gln2-Trp3-Ala4-Val5- Gly6-His7-Leu8-psi-Tpi9-NH2marked "B2", and the third antagonist peptide was 2.

Microspheres of pamoate salt SI into poly (DL-actigraphically) received from A Cytotech and was so for 2 weeks. from 16 mg aliquots were released about 100 mcg per day. These microspheres were injected with every 15 days on the side opposite the tumor location. Each of the antagonists bombezin was dissolved in saline containing 0.1% dimethyl sulfoxide, and twice a day under the L. 9 and illustrated in Fig. 2.

Example 9. Effects of antagonists bombezin on tumor pancreatic cancer MIA PACA-2 were evaluated as follows.

"Nude" mice, the same mice described in example 8, were subcutaneously injected with cancer cells of the pancreas human MIA PACA-2, taken from tissue culture, cultivated in the same way that SCLC in example 8. The volumes of the tumors were measured for the two experimental groups in the same way as in example 8.

These two experimental groups were: group of mice treated as antagonist bombezin peptide 2, and a control group who received only an injection of filler. 50 µg of the indicated peptide 2 and excipient solution was administered to each mouse subcutaneously twice a day.

The results of measurement of the volume of the tumors are shown in table. 10 and illustrated in Fig. 3.

Example 10. Effects of antagonists bombezin on tumor pancreatic cancer human CAPAN-2, transplanted "Nude" mice, was evaluated as follows.

"Nude" mice, similar to those described in example 7, was introduced xenografts tumors of the pancreas human CAPAN-2, derived from tissue group, receiving only a solution of a filler, and a group receiving an injection of the antagonist bombezin (peptide 2). 50 ml of the indicated solution filler or peptide 2 was administered to each mouse subcutaneously twice a day.

The results of measurement of the volume of the tumors are shown in table. 11 and illustrated in Fig. 4.

Although the invention is described in preferred examples of its implementation, it can be modified and the modification does not extend, however, beyond the scope and substance of the invention stated in the claims. This can be made some substitutions known in the art, and does not adversely impact on the efficiency of the invention.

Example 11. Composition of tablets for transbukkalno (for example, sublingual) administration.

1. Peptide N 14 10.0 mg; presswise sugar, USP, 86,0 mg; calcium stearate to 4.0 mg.

2. Peptide N 14 10.0 mg; presswise sugar, USP, and 88.5 mg; magnesium stearate and 1.5 mg.

3. Peptide N 14 5.0 mg; mannitol, USP, to 83.5 mg; starch-magnesium, USP, to 1.5 mg.

4. Peptide N 14 10.0 mg; pregelatinized starch, USP, 10.0 mg; lactose, USP, to 74.5 mg; pregelatinized in water in a quantity sufficient for the formation of wet granulation mass when mixed with sugar component of the fillers. Upon completion of mixing, granulation mass dried in tray drier liquid layer. Dry granulation mass is then screened to remove any large aggregates, and then mixed with other components. Granulation mass is then pressed on standard equipment for the manufacture of tablets to attain the desired weight of tablets.

Method B. this method of making all of the recipes contain 0.01% of gelatin, USP. Gelatin is first dissolved in an aqueous solvent granulation mass, then dissolve the peptide. The remaining steps are the same as in method A, described above.

Example 12. Composition for intramuscular injection of long-lasting action.

Gel sesame oil for a/m introduction prolonged.

Peptide N 14 10,0 mg

The aluminum monostearate, USP, 20,0 mg

Sesame oil to 1.0 ml.

The aluminum monostearate is combined with sesame oil and heated to 125oC under stirring to obtain a clear yellow solution. This mixture is then placed in avima abrasion.

Example 13. Microcapsules of the biodegradable polymer for intramuscular long-acting.

Peptide No. 14 - 1%

The copolymer of glycolide/lactide 25/75 (characteristic viscosity of 0.5) - 99%.

Microcapsules (0o- 150odescribed formulations suspended in the following components:

Dextrose 5,0%

SMS, sodium 0,5%

Benzyl alcohol 0.9% of

Tween 80 at 0.1%

Purified water up to 100.0%

25 mg of microcapsules suspended in 1.0 ml of media.

Example 14. Aqueous solution for intramuscular injection.

Peptide N 14 500 mg.

Neoantigenic gelatin 5 mg

Water for injection to 100 ml.

Gelatin and the peptide is dissolved in water for injection and the resulting solution is filtered under sterile conditions.

Example 15. The composition for rectal administration.

The suppository vehicle for rectal administration.

Peptide No. 14 of 5.0 mg.

Witepsol H15 (Witepsol H15) 20,0 mg

The peptide is combined with molten Witepsol H15, stirred and poured into molds with a capacity of 2,

The list of sequences.

(i) General information.

(i) Applicant: Schally, Andrew V. Cai, Renzhi.

(ii) the Name R>
(A) Address: OMPI M. BEHR, ESQ

(B) Street: 325 PIERSON AVENUE

(C) City: EDISON

(D) State: NEW JERSEY

(E) Country: USA

(F) Postal code: 08837

(v) the Type of machine reading.

(A) medium Type: floppy disk

(B) Computer: PC compatible with IBM

(C) Operating system: PC-DOS/MS-DOS

(D) Software: Patentin Release # 1.0, Version # 1.25 times

(vi) the Data of this application.

(A) application Number: US 08/031, 325

(B) filing date: March 15, 1993

(C) Classification:

(vii) Data of the previous application.

(A) application Number: US07/619, 747

(B) filing date: November 29, 1990

(viii) Information about the attorney/agent:

(A) the Name, surname: BEHR, OMPI M.

(B) Registration number: 22940

(C) the Number of document/dossier: SHAL3, 0-014

(ix) Telecommunication information.

(A) Phone: (908) 494-5240.

(B) Telefax: (908) 494-0428

(C) telex: 511642 BEPANEDIN.

(2) Data sequence SEQ 1D N1:

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/key: various. the pH is over.

(A) Name/key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note.= "The rest 8 = reduced isostere leucine

(ix) a Distinctive feature.

(A) Name/key: various. distinguish. features

(B) Localization: 9

(D) Additional information: /note. = "The residue 9 = Tac-NH2"

(xi) sequence Description EO 1 N 1.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID N 2:

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Phe"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B)Localization: 8

(D)Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A)Name/key: various. distinguish. features

(B) Localization: 9

(D) Additional informa 5

(2) the sequences SEQ ID No. 3.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Phe

(ix) a Distinctive feature.

(A) Name/Key: various. excellent. features

(B) Localization: 8

(D) Additional information: /Note = "the Residue 8 = reduced isostere leucine"

(ix) feature:

(A) Name/Key: various. distinguish. features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 8 = MTac-NH2

(xi) sequence Description SEQ ID B3.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID No. 4.

(i) sequence Characteristics.

(B) Type: amino acid

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) the Distinctive feature of the traveler information: /Note. = "Residue 1 = Ac-D-Phe

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N4.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N5.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Cpa"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key Is 9 = Tac-NH2"

(xi) sequence Description SEQ ID N5.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N6.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Cpa"

(ix) feature:

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = MTac-NH2

(xi) sequence Description SEQ ID N6.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N7.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: Lin is otlichitelnye features

(B) Location: 1

(D) Additional information. /Approx. = "Residue 1 = D-Nal"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N7:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N8:

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded (D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = Pal"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) Illicitencounters information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N8:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N9.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Pal

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N9.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N10.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Trp"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N10.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID no 11.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = Ac-D-Trp"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reducely features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N11:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N2.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N12.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N13.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N13:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 14.

(I) sequence Characteristics.

(A) Length: 8 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = Hca-Gln"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localize is a recreational feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = Tac-NH2"

(xi) sequence Description SEQ ID N14.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 15.

(i) sequence Data.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Phe"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N15:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data posledovatelnosti.html.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Phe"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 2

(D) Additional information: /Note. = Remainder 2 = Glu(OMe)

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N16:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 17.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: Lin is otlichitelnye features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Phe"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 2

(D) Additional information: /Note. = Remainder 2 = Glu [-]

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID N17:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N18.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Phe"

(ix) a Distinctive feature.


(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = MTac-NH2

(xi) sequence Description SEQ ID N18:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N19.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = Ac-D-Phe

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = MTac-NH2

(xi) sequence Description SEQ ID N19:

Xaa Gln Trp Ala Val Gly His Xaptx2">

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(x) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Cpa"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = MTac-NH2

(xi) sequence Description SEQ ID N20:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 21.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional uchitelya features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) feature:

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = MTac-NH2

(xi) sequence Description SEQ ID N21:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SEQ ID N22.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = D-Tpi"

(ix) a Distinctive feature/

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information: /Note. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = MTac-NH2

(xi) a Description of the CLASS="ptx2">

(i) sequence Characteristics.

(A) Length: 14 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = pGlu

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 14

(D) Additional information: /Note. = "The residue 14 = Met-NH2

(xi) sequence Description SEQ ID N23:

Xaa Gln Arg Leu Gly Asn Gln Trp Ala Val Gly His Leu Xaa

1 5 10

(2) the sequences SEQ ID No. 24.

(i) sequence Characteristics

(A) Length: 10 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) feature:

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information: /Note. = "Residue 1 = H-Gly"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 10
aa Asn His Trp Ala Val Gly His Leu Xaa

1 5 10

(2) Data sequence SEQ ID N25.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information: /Note. = "The residue 9 = Met-NH2

(x) a Description of the sequence SEQ ID N25:

Asn His Trp Ala Val Gly His Leu Xaa

1 5

(2) Data sequence SEQ ID N26.

(i) sequence Characteristics.

(A) Length: 11 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 11

(D) Additional information. /Approx. = "The residue 11 = Met-NH2

(x) a Description of the sequence SEQ ID N26:

Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Xaa

1 5 10

(2) Data sequence SEQ ID N27.

(i) sequence Characteristics.

(A) Length: 8 amino acids

(B) Type: amino acid

(C is the th feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information./Approx.="The remainder 1= (R1) (R2)-AO-Al, where AO = deleterow; Al=D-Phe, D-Trp or D=Nal; R1 and R2 = H

(ix) a Distinctive feature.

(A) Name/Key different. distinguish. features

(B) Localization: 8

(D) Additional information./Approx.="The rest 8=A8-W, where W=-N(R8)-CH(Z1)-R4CH(Z2)-CO-V, where R4= CH2NH; Z1=-CH2CH(CH3)2; Z2=-CH2SH or (CH2)2-S-CH3; V=N(R6R7where R6, R7and R8can be H; R1and R2can be H or COEI, where EI=CI-C20-alkyl"

( ix) a Description of the sequence SEQ ID No. 27:

Xaa Gln Trp Ala Val GIy His Xaa

1 5

(2) Data sequence SEQ N 28.

(i) Characteristics posledovatelnosti.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information. Approx. ="Residue 1 = X-Al, X is H, a simple connection between the alpha-amino group , the de R1represents H, C1-C10-alkyl, phenyl, phenyl-C1-C10-alkyl, p-HI-phenyl, p-1-phenyl-C1-C10-alkyl, naphthyl, naphthyl-C1-C10-alkyl, indolyl, indolyl-C1-C10-alkyl, pyridyl, pyridyl-C1-C10-alkyl; thienyl, thienyl-C1-C10-alkyl, cyclohexyl or cyclohexyl-C1-C10-alkyl, where HI represents F, Cl, Br, OH, CH3or OCH3; or R1 represents N(R2) (R3) if R2represents H, C1-C1-alkyl, phenyl or phenyl-C1-C10-alkyl; R3represents H or C1-C10-alkyl; or R1is an R4O, if R4represents a C1-C10-alkyl, phenyl or phenyl-C1C10-alkyl; and Al is a D-, L - or DL-amino acid residue selected from Phe, p-HI-Phe, pGlu, Nal, Pal, Tpi, unsubstituted Trp or Trp substituted in the benzene ring of 1 or fluorine, chlorine, bromine, NH2or C1-C3-alkyl; or a peptide bond linking the acyl portion of the remainder R1CO with alpha aminocyclo residue 2".

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 2

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information./Approx.="The rest 8= reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 9

(D) Additional information. /Approx. ="The residue 9=A9-Q, if A9 = Tac MTac or DMTac; and Q = NH2or OQl, where Ql = H, phenyl or phenyl-C1-C10-alkyl"

(x) a Description of the sequence SEQ ID No. 28:

Xaa Xaa Trp Ala Val Gly His Xaa Xaa

1 5

(2) Data sequence SED ID No. 29.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information. / Approx.="The remainder 1 = D-Phe"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Localization: 8

(D) Additional information./Approx.="The rest 8 = reduced isostere leucine".

(ix) a Distinctive feature.

(ix) the s: 9

(D) Additional information. /Approx. = "The residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID No. 29:

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 30.

(i) sequence Characteristics

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information. /Approx. = "Residue 1 = D-Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information. /Approx. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 9

(D) Additional information. /Approx. = "the residue 9 = Leu-NH2

(xi) sequence Description SEQ 1D N 30.

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 31.

(i) sequence Characteristics.

(A) Length: 8 amino acids

(B) Type: amino acid

(C) Nepochetnoe.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information. /Approx. = "residue 1 = D-Phe"

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 4

(D) Additional information. /Approx. = "The remainder 4 = D-Trp"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information. /Approx. = "The residue 8 = Trp-NH2

(xi) sequence Description SEQ 1D N 31:

Xaa Cys Xaa Lys Val Cys Xaa

1 5

(2) the sequences SEQ ID N 32.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinctive features

(B) Location: 1

(D) Additional information. /Approx. = "Residue 1 = D-Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information. /Approx. = "The residue 8 = reduced isostere latinitate: 9

(D) Additional information. /Approx. = "The residue 9 = Tpi-NH2

(xi) sequence Description SEQ ID no 32

Xaa Gln Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID N 32.

(2) Data sequence SEQ ID No. 33.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Location: 1

(D) Additional information. /Approx. = "Residue 1 = X-Al, where X = H or Ac; and Al = D-Phe, L - or D-Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information. /Approx. = "The residue 8 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 9

(D) Additional information. /Approx. = "the residue 9 = Tac-NH2"

(xi) sequence Description SEQ ID No. 33:

Xaa Gln Trp Ala Vla Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID No. 34.

(i) sequence Characteristics.

(A) Name/Key: various. distinguish. features

(B) Location: 1

(D) Additional information. /Approx.="The remainder 1 = X-Al, where X = H or Ac, and Al = D-Phe, L - or D-Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information. /Approx.="The remainder of 1 reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 9

(D) Additional information. /Approx.="The remainder 9 = MTac-NH2

(xi) sequence Description SEQ ID N34:

Xaa Gln Trp Ala Val Gly Hic Xaa Xaa

1 5

(2) Data sequence SEQ ID N35.

(i) sequence Characteristics.

(B) Length: 9 amino acids

(B) Type: amino acid

(C) Apachectl: odnozadachnaya

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Location: 1

(D) Additional information. /Approx. ="Residue 1 = X-A1, where X represents a hydrogen, a simple connection between the alpha amino group of residue A1 c galaida hydrogen, C1-C10-alkyl, phenyl, phenyl-C1-C10-alkyl; or R1is N(R2)(R3), where R2represents hydrogen, C1-C10-alkyl, phenyl or phenyl-C1-C10-alkyl; R3represents H or C1-C10-alkyl; or R1is an R40where R4is1-C10-alkyl, phenyl or phenyl-C1-C10-alkyl; and A1 is an L-, D - Pal, L - or D-Tpi"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Location: 2

(D) Additional information./Approx.="Remainder 2 = Gln, Glu [-], Giu (Y) or His

(x) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information./Approx.="The rest 8 = reduced isostere leucine"

(x) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 9

(D) Additional information. /Approx. = "The residue 9 = A9-0, where A9 is a Cys or Pen, and Q represents NH2or OQ1, where Q1 is hydrogen, C1-C10-alkyl, phenyl or phenyl-C1-C10-alkyl"

(xi) a Description posledovatelnyi sequence.

(A) Length: 8 amino acids

(B) Type: amino acid

(C) Apachetest: single-stranded

(D) Topology: linear

(ii) molecule Type: peptide

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Location: 1

(D) Additional information. /Approx. = "Residue 1 = X-A1-Gln, where X represents the Hca, Hna, Paa, Mpp, Hpp or Naa; and A1 represents a peptide bond linking the acyl portion of the residue X with alpha-amino-part Gln"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 7

(D) Additional information. /Approx. = "The remainder 7 = reduced isostere leucine"

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 8

(D) Additional information. /Approx.="The rest 8 = Tac-NH2"

(x) a Description of the sequence SEQ ID No. 36.

Xaa Trp Ala Val Gly His Xaa Xaa

1 5

(2) the sequences SEQ ID N 37.

(i) sequence Characteristics.

(A) Length: 9 amino acids

(B) Type: amino acid

(C) Topology: linear

(D) Apachetest: single-stranded

(ii) molecule Type: peptide

(ix) the Profile for More information /Approx.="The remainder of 1 = pGlu

(ix) a Distinctive feature.

(A) Name/Key: various. distinguish. features

(B) Localization: 9

(D) Additional information /Approx.="The remainder 9 = Met-NH2

(xi) sequence Description SEQ ID N 37.

Xaa Gln Trp Ala Gly His Leu Xaa

1 5n

1. Peptides having the formula

X-A1-A2-Trp-Ala-Val-Gly-His-Leu-psi-A9-Q

where X represents a hydrogen, a simple relationship linking the alpha-amino group of A1with gamma-carboxyl part 3-propionyloxy group A2if A2is Clu[-],

or a group of the formula

R1CO.,

where R1selected from the group comprising hydrogen, C1-C10-alkyl, phenyl - C1-C10-alkyl;

A1represents the D-, L - or DL-amino acid residue selected from the group comprising Phe, p-HI-Phe, pGlu, Nal, Pal, Tpi, unsubstituted Trp or Trp substituted in the benzene ring by one or more substituents, a represents C1-C3-alkyl, or a peptide bond linking the acyl part of R1CO with alpha aminocyclo A2provided that X - R1CO;

A2represents Gln, Glu[-] , Glu(Y) or His, where [-] denotes a simple link, if X is a simple bond, and A2userupp A1and Y is a OR5where R5is hydrogen, C1-C3-alkyl or phenyl;

Leu-psi is a reduced form Leu, where instead of - CH2there is C= O, so that the link specified-CH2-part C the alpha-amino group of A neighboring9-balance is pseudopeptides communication;

A9is a Tac MTac or DMTac;

Q represents NH2or CQ1I, where QIis hydrogen,

or their pharmaceutically acceptable acid or salt.

2. The peptide under item 1, where X represents hydrogen or R1CO., where R1is hydrogen or C1-C10-alkyl; A1is D-Cpa, D-Nal, L - or D-Pal, D-Phe, L-or D-Tpi, or D-Trp; A2is Gln or HiS, and Q is NH2.

3. The peptide under item 2, where A9is Tac.

4. The peptide under item 2, where A9is DMTac.

5. The peptide under item 3, where X is H or Ac, A1is D-Phe, L - or D-Tpi, and A2is Gln.

6. The peptide under item 4, where X is H or Ac, A1is Phe, L - or D-Tpi, and A2is Gln.

7. The peptide under item 3, having the formula

P-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-psi-Tac-NH2.

8. The peptide under item 3, having the formula

D-Tpi-Gln-Trp-Ala-Va"ptx2">

10. Peptides having the formula

X-A1-A2-Trp-Ala-Val-Gly-His-Leu-psi-A9-Q

where X represents hydrogen or a group of the formula

R1CO-,

where R1represents hydrogen;

A1is an unnatural amino acid selected from the group including L-, D-Pal, L - or D-Tpi;

A2represents Gln;

Leu-psi is a reduced form Leu, where instead of-CH2there is a part of C= O, so that the link specified-CH2-part C the alpha-amino group of A neighboring9-balance is pseudopeptides communication;

A9represents Cys;

Q represents NH2,

or their pharmaceutically acceptable acid, or salt.

11. The peptide under item 1, where X is an R1CO-; A1represents a peptide bond linking the acyl part of R1CO-alpha-aminocyclo A9; A2represents Gln or His, and Q represents NH2.

12. The peptide according to p. 11, where X represents the Hca; A2represents Gln, and A9is a Tac.

13. The peptide according to p. 12, having the formula

Hca-Gln-Trp-Ala-Val-Gly-His-Leu-psi-Tac-NH2.

14. Pharmaceutical composition, oblad Lemley media characterized in that the active agent it contains the polypeptide under item 1 or a therapeutically acceptable salt as an effective dose.

15. A method of treating cancer in mammals by introduction of an active agent to a mammal, wherein the active agent is administered polypeptide under item 1, or a therapeutically acceptable acid, or salt in an effective dose.

 

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