The derived peptide, a method of treating cancer in a patient

 

(57) Abstract:

The derived peptide containing the remainder of somatostatin, or bombezin, or derivative, fragment, or analog, and the Deputy of the formula I, II or III (values radicals, see p. 1 claims) has a higher and prolonged biological activity compared to the unmodified somatostatin. 2 C. and 15 C.p. f-crystals, 4 tab., 1 Il.

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The invention relates to therapeutic peptides.

Several attempts were made to extend the activity of biologically active peptides. For example, the peptides were chemically modified by synthetic add sugar environment to increase the period during which the peptide is active (Sandoz, WO 88/02756; Sandoz, WO 89/09786; DE 3910667 Al, EP 374089 A2 (1990); and Breipohl, U.S. patent N 4861755 (1989)). Adding cationic anchors (EP 0363589 A2 (1990)) and the lipid environment (Whittaker, WO 91/09837; Zhang, US patent N 4837303 (1989)) was also used to increase the lifetime of the peptide.

In a General sense, the present invention allows to derive biologically active peptides that contain one or more substituents, separately connected to the amino group, once the have a higher and prolonged biological activity, than the corresponding unmodified peptide.

Derivatives of peptides are advantageous because they are inexpensive, highly biocompatible, devoid of harmful side effects and is compatible with different types of therapeutic treatment. In particular, many derivatives having somatostatin as a peptide environment has significantly improved selenocystine and selectivity compared to the unmodified somatostatin.

In one aspect of the invention is characterized derived peptide containing biologically active peptide environment and at least one Deputy, attached to the peptide environment; Deputy is selected from the group comprising compounds I, II and III, where compound I is:

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where ROis O, S or NR5where R5is H or (C1-C6)alkyl; each R1and R2, independently, is H, (CH2)mOR6or CH(OR7)CH2OR8where R6is H or (C2-C7)acyl, and each R7and R8, independently, is H, (C2-C7)acyl or C(R9)(R10), where each R9and R10, independently, is H or (C1-C6)alkyl; or each R1and R2is =CHCH2OR11where R11part; and one of R3or R4is (CH2)nR12or (CH2)nCH(OH)R12where R12is CO, CH2or SO2and n is an integer between 1 and 5 inclusive; and the remaining R3or R4is H, (C1- C6)hydroxyalkyl or (C2-C7)acyl; and

compound II is;

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where each R13, R14and R15, independently, is H or (C2-C24)acyl;

R16is NH or is absent;

R17is CO, O, or is absent;

R18is CO, CH2, SO2or absent; and

m is an integer between 1 and 5 inclusive; and n is an integer between 0 and 5 inclusive; and

compound III is

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where R19is H, NH2, an aromatic functional group, OH, (C1-C6)hydroxyalkyl, H(R27)(R28), SO3H or absent, where each R27and R28, independently, is H or (C2-C6)alkyl;

R20is O or is absent;

R21is (C1-C6)alkyl or is absent;

-R22is N, CH, O, or C;

-R23is (C1-C6)alkyl or is absent;

R24is N, CH or C;

R25is NH, O, or is absent;

R26- it's SO2, 5, inclusive;

p is an integer between 0 and 5 inclusive; and

q is an integer between 0 and 5 inclusive.

In compounds I, II and III peptide environment attached to each of the Vice-through communication CO-N, CH2-N or SO2-N between the substituent and the nitrogen atom of the N-end or side chain of this peptide environment.

In preferred embodiments of the invention, R23- (C1-C6)alkyl; R22is N, C or CH; and R24is C.

Alternatively, R22is About; R19, R20, R21, -R23is absent; and the sum of m and n is 3, 4 or 5.

In other preferred embodiments of the invention, the Deputy is the connection I; in this example, R12is preferably CH2or SO2. Alternatively, the Deputy may be compound II, and in this case, R18is preferably CH2or SO2; R13, R14, R15is H; and R17no. In particularly preferred embodiments, the Deputy is (HOCH2)3C-NH-(CH)2-SO2or (HOCH2)2C-CH2.

In still other embodiments of the invention, the Deputy is the connection I is the number of alternatives, both R22and R24can be n

In other examples, the Deputy is

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or

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Preferably the peptide environment is selected from a group comprising: somatostatin, bombesin, calcitonin, a peptide associated with the gene calcitonin (CGRP), Amylin, a hormone parathyroid (PTH), gastrin releasing peptide (GRP), melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH) associated with parathyroid peptide (PTHrP), luteinizing releasing hormone hormone (LHRH), releasing the growth hormone factor (GHRF), releasing the growth hormone peptide (GHRP), cholecystokinin (CCK), glucagon, Bradykinin, glucagon-like peptide (GLP), gastrin, enkephalin, neuromedin, endothelin, substance P, neuropeptide Y (NPY), peptide YY (PYY), vasoactive intestinal peptide (VIP), guanylin, a polypeptide that activates the mucous adenylate the cyclase (PACAP), beta-cell according to adrenomedullin and their derivatives, fragments and analogs.

Peptide environment is preferably simastatin or its derivative, fragment or analog. The preferred analog somastatin is one of: H-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2H-D-Phe-c[Cys-Tyr-D-Trp-Lys-Thr-Cys] -Nal-NH2and H-D-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2. Alternatively, peptidebased implementation derivative peptide is

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or

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In another aspect of the invention involves a dimeric derivative of a peptide that contains two biologically active peptide environment and at least one Deputy, attached to each of these peptide environments.

The Deputy is selected from the group consisting of compounds IV and V, where the compound IV is generic ("generic") structure, equivalent to a compound I and compound V has a common structure, equivalent to compound III. In each of the dimer peptide environments attached to the deputies by a liaison CO-N, CH2-N or SO2-N between the substituent and the nitrogen atom of the N-end or side chain of one of the peptide environments.

In another aspect of the invention includes a method of treating diseases, such as cancer, in a patient; the method includes the step of making the patient a therapeutic amount described here is derived peptide. In preferred embodiments the peptide environment used in the treatment is somatostatin.

Under used herein the term "biologically active" refers to naturally occurring recombinant and synthetic peptide having physiological or therapeutic activity. In General the which is qualitatively similar or opposite effect effect produced by the unmodified peptide.

The invention is illustrated by the drawing, which shows a graph of the two growth curves of AR42J cells in the presence of different derivatives of somatostatin.

Derived peptides

In General, derivatives of the peptides according to this invention contain two separate components: 1) a biologically active peptide; and 2) at least one substitute having the structure of compounds I, II and III. Derivatives of peptides obtained according to methods described here include the following substances.

Derivative works based on the connection I

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where R0, R1, R2, R3, R4, R12and n are those that were defined, and NH-P' is a biologically active peptide environment.

In these examples, the implementation of the NH group is placed on the N-terminal end or a side chain of the peptide and P' represents a residue of the peptide.

Derivative works based on the compound (II)

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where R13, R14, R15, R16, R17, R18, m, n and NH-P' are those that are defined here.

Derivative-based compound III

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where R19, R20, R21, R22, R23above the structures of compounds obtained according to the invention include peptide derivatives containing two or more of the substituents attached to one of the peptide environment. These embodiments of the invention are derivatives of biologically active peptides having more than one free amino group, for example the radical Lisin.

This invention also involves the presence of dimeric derivatives of peptides containing two peptide environment associated with one Deputy, for example, two similar Bradykinin associated with Deputy connection V.

Derivatives of the peptides of this invention are derivatives of biologically active peptides selected from the following group: somatostatin, bombesin, calcitonin, a peptide associated with the gene calcitonin (CGRP), Amylin, a hormone parathyroid (PTH), gastrin releasing peptide (GRP), melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH) associated with parathyroid peptide (PTHrP), luteinizing releasing hormone hormone (LHRH), releasing the growth hormone factor (GRF), releasing the growth hormone peptide (GHRP), cholecystokinin (CCK), glucagon, bradykinin, glucagon-like peptide (GLP), gastrin, enkephalin, neuromedin, endothelin, a substance P, Naropa the polypeptide (PACAP), beta-cell according to adrenomedullin or derivatives, fragments or analogs of the above.

In especially preferred embodiments the peptide is a somatostatin or a derivative, fragment or analog of somatostatin. Analogs of somatostatin, which can be used in connection with this invention include the following substances is not limited to (see the end of the description).

The above peptide compounds described in the following links:

Application EP N P5 164 EU; van Binst, and other "Research peptides 5:8 (1992): Horvath A. and other Abstract Structures of analogs of somatostatin with antitumor activity", 22nd European Symposium on peptides, 13 - 19 September 1992, Interlaken, Switzerland; PCT application WO 91-09056 (1991); application EP 0363589 A2 (1990); application EP 0203031 A2 (1986); U.S. patent NN 4904642; 4871717; 4853371; 4725577; 4684620; 4650787; 4603120; 4585755; 4522813; 4486415; 4485101; 4435385; 4395403; 43691794; 4360516; 4358439; 4328214; 4316890; 4310518; 42910224; 4238481; 4235886; 4224190; 4211693; 4190648; 4146612 and 4133782.

In the above analogs of somatostatin each the radical of the amino acid has the structure NH-C(R)H-CO-, in which R is the side chain; dash between the radicals of amino acids are peptide bonds that link amino acids. When naznaceno not specified form D. When two radical Cys present in the peptide, disulfide bridge is formed between the two environments. However, this relationship is not shown in these radicals.

Additionally preferred somatostatin analogues according to the invention have the following formula:

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where A1is the D - or L-isomer - Nal, Trp, - pyridyl-Ala, Phe, substituted Phe or absent; and each A2and A7regardless, is Cys, Asp, or Lys. These environments are covalently bound to each other through a disulfide bridge or amide bridge. In addition, A3is - Nal, Phe, or o-, m - or p-substituted X-Phe, where X is a halogen, OH, NH2NO2or (C1- C3alkyl; A6is Val, Thr, Ser, Ala, Phe, - Nal, Abu, Ile, Nle, or Nva; and A8is Phe, Thr, Tyr, Trp, Ser, - Nal, or group of alcohols, or absent; each R1and R2independently is H, lower acyl or lower alkyl; and R3is OH, NH2or missing. Preferably, when one of the A2and A7is Cys, the other too Cys; when A8is alpha amerosport, R3is absent; and when neither A2and neither A7is not Cys, A2different from A7.

Particularly preferred analogs of somatostatin in this example embodiment of the invention NH2;

H-D-Phe-Cys-Tyr-D-Trp-Lys-Abu-Cys-Thr-NH2;

H-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Nal-NH2;

H-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-ol.

In other embodiments the linear somatostatin analogues of this invention have the following structure:

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where A1is the D - or L - isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser, - Nal, - pyridyl-Ala, Trp, Phe, 2,4-d sodium dichloro-Phe, PENTAFLUORO-Phe, p-X-Phe, or o-X-Phe, where X is CH3, Cl, Br, F, OH, OCH3or NO2;

A2is Ala, Leu, Ile, Val, Nle, Phe, - Nal, pyridyl - Ala, Trp, 2,4-d sodium dichloro-Phe, PENTAFLUORO-Phe, o-X-Phe, or p-X-Phe, where X is CH3, Cl, Br, F, OH, OCH3or NO2;

A3is pyridyl-Ala, Trp, Phe, -Nal, 2,4-d sodium dichloro-Phe, PENTAFLUORO-Phe, o-X-Phe, or p-X-Phe, where X is CH3, Cl, Br, F, OH, OCH3or NO2;

A6is Val, Ala, Leu, Ile, Nle, Thr, Abu, or Ser;

A7is Ala, Leu, Ile, Val, Nle, Phe, -Nal, pyridyl-Ala, Trp, 2,4-d sodium dichloro-Phe, PENTAFLUORO-Phe, p-X-Phe, or o-X-Phe, where X is CH3, Cl, Br, F, OH, OCH3or NO2;

A8is the D - or L-isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser, Phe, -Nal, pyridyl-Ala, Trp, 2,4-d sodium dichloro-Phe, PENTAFLUORO-Phe, p-X-Phe, or o-X-Phe, where X is CH3, Cl, Br, F, OH, OCH3or NO2or alcohol; and

each R1and R2, independently, is H, lower acyl or lower alkyl; and R3is OH, NH2or expunged. Preferably, as mini8is the alcohol, R3expunged. In addition, A1, A2, A7and A8can't all be aromatic amino acids. Among the particularly preferred analogs in this aspect of the invention include:

H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Thr-Phe-Thr-NH2;

H-D-Phe-p-NO2-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2;

H-D-Nal-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2;

H-D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2;

H-D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2;

H-D-Phe-p-chloro-Phe-Tyr-D-Trp-Lys-Val-Phe-Thr-NH2;

and

H-D-Phe-Ala-Tyr-D-Trp-Lys-Val-Ala-D -- Nal-NH2.

In still other preferred embodiments the peptide environment is bombesin or its derivative, fragment or analog. Among the analogues bombesin that can be used to implement this invention include, without limitation, Neuromedin C, Neuromedin B, winkles and gastrin releasing peptide (GRP), having the following amino acid sequence:

H-Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-;

Ala-Lys-Met-Tyr-Pro-Arg-Cly-Asn-His-;

Trp-Ala-Val-Gly-His-Leu-Met-NH2.

Other analogues bombesin that you can use in this invention include compounds described in the following links:

Koi and other "Peptides. Proceedings of the 11th American Symposium on pept is aka", 51:1798 (1991);

Ueng and other "Biochemistry", 29:616 (1990); Heimbrock and other "Synthetic peptides. Approaches to biological problems, "UCLA Symposium on Mol. and Cell. Biol. New Series, vol. 86, under the editorship of TEM and Kaiser; Martinez and others J. Med. Chem. 28: 1874 (1985); Gaworski and other Biochem. J. 247:427 (1987): Dubra and other Design and delivery of drugs", volume 2:49, Harwood Academic Publishers, GB (1987); Heikkila and other J. Biol. Chem. 262:16456 (1987); Karanikas and other J. Med. Chem. 25:1313 (1982); Sayyid and other "Peptides" 10:597 (1989); Rosell and others, "Trends in pharmacological Sciences", 3:211 (1982); Landberg and other "Proc. Nat. The Aca. Sci. " 80:1120, (1983); Engberg and other Nature 293:222 (1984); Mirachi and other "Euro.J. Pharma." 82:101 (1982); Linder and other Nature 294:467 (1981); wall and other "Biochem. Biophys. Res. Comm." 155: 359 (1988); Riviere and other "Biochem." 17:1766 (1978); Cuttitta and other Reviews cancer" 4:707 (1985); Omelas etc. "Int.J. Peptide Res." 30:596 (1987); Szepeshazi and other "cancer Research" 51:5980 (1991); Jensen and others, "Trends Pharmacol. Sci". 12:13 (1991); U.S. patent NN 5028692; 4943561; 4207311; 5068222; 5081107; 5084555; application EP NN 0315367 A2 (1989); 0434979 A2 (1991); 0468497 A2 (1992); 0313158 A2 (1989); 0339193 A1 (1989); PCT application NN WO 90/01037 (1990); 90/02545 (1992); the proposal England GB 1231051 A (1990).

The peptides of this invention can be in the form of pharmaceutically acceptable salts. Examples of preferred salts are those salts which are therapeutically acceptable organic acids, such as excelto, toluensulfonate or Mamonovo acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalide acid, including hydrochloric acid, sulfuric acid and phosphoric acid.

Now is described the synthesis of compounds I, II and III.

When describing the synthesis of compounds according to this invention, the following abbreviations are used:

Nal - nafcillin (1 or 2),

Abu - alpha-aminobutyric acid,

D - programalso,

L - levogyrate,

HOAC is acetic acid,

BOP - benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-phosphate

BOC - tert-butyloxycarbonyl,

DCC - DICYCLOHEXYL carbodiimide,

EDC - 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,

DEPC - diethylthiophosphate,

DMF - dimethylformamide,

CH2Cl2- dichloromethane,

MeOH is methanol,

EtOH - ethanol,

DIEA IS N,N-diisopropylethylamine,

HOBT is 1-hydroxybenzotriazole,

HBTU - O-benzotriazol-1-yl,N,N,N',N'-tetramethyluronium hexafluorophosphate,

THF is tetrahydrofuran,

TFA - triperoxonane acid.

The source and intermediate materials for the production of compounds I, II and III are available) which are found in literature. For example, the chemistry of derivatives associated with ascorbic acid can be found in "J. Chem. Soc. ". Perkin Trans. 1:1220 (1974); "Carbohyd. Res." 67:127 (1978); Yakugaku Zasshi, 86:376 (1966); U.S. patent N 4552888; "J. Med. Chem", 31: 793 (1988); ibid 34:2152 (1991); and 35:1618 (1992). The chemistry of these Tris-related derivatives can be found in "Arch. Biochem. Biophy.", 96, 653 (1962), "Biochem.", 5, 467 (1996).

Synthesis of derivatives of peptides

In a General sense, the accession of compounds I, II or III to the corresponding free amino group of the protected amino acid or peptide can be achieved according to well known methods used for the synthesis of peptides (for example, DCC, DCC-HOBT, DIC-HOBT PPA, EDC-HOBT, DEPT, BOP, HBTU) using a base (for example, DIEA) in an inert solvent (such as DMF, THF or CH2Cl2ethyl acetate, or a combination). Release of protected groups can also be well-known methods (for example, removing the group by adding acid or base, TFA, dioxane-HCl, ammonia, NaOMe, piperidine). In most cases, the reaction temperature should be in the range from -30oC to room temperature.

Basically, the first step in the synthesis involves the reaction between the epoxide and the free amino group of a protected amino acid or peptide; complexitiy and unlocks the can is. the donkey synthesis purification of intermediates and products can be achieved by conventional methods such as chromatography or HPLC. Compounds can be identified by conventional means, such as NMR, analysis of amino acids and mass spectrometry.

The following examples illustrate the preferred methods for the formation of compounds of this invention.

Example 1. Synthesis of derivatives of somatostatin.

The following is the derivative of somatostatin, here called BIM-23118, was synthesized in accordance with this invention

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An example of 1.1. 3-O-(Benzyloxycarbonylamino)-2,5,6-triacetylcellulose acid.

Acetic anhydride (6 ml) was added dropwise to a solution of 3-O-(benzyloxycarbonylamino)-ascorbic acid (2.2 g) in pyridine (30 ml); the mixture is then mixed half days at room temperature. The pyridine was evaporated under reduced pressure, leaving a residue, which was then divided between ethyl acetate and 1 N. HCl. The ethyl acetate layer was washed 1 N. HCl and then with water. After drying (MgSO4) the ethyl acetate was evaporated under reduced pressure; the traces of pyridine and acetic anhydride, which still remained, removed multiple joint evaporation with toluene. The floor is th gel, remaining in the residue (2.4 g). TLC (silica gel: CHCl3/acetone [9:1], Rf=0,52).

The example of 1.2. 3-O-(Carboxymethyl)-2,5,6-triacetylcellulose acid.

A suspension of Pd-C (100 mg) in water (2 ml) was added to a solution of 3-O-(benzyloxycarbonylamino)-2,5,6-triacetylcellulose acid (2.4 g) in ethanol (30 mg) and the suspension was shaken under hydrogen (17 psi) for 6 hours.

Then the catalyst was removed by filtration through zletovo pad and the filtrate was evaporated under reduced pressure to obtain 3-O-(carboxymethyl)-2,5,6-triacetylcellulose acid. TLC (silica gel: CHCl3/MeOH/HOAC [9:1:0,1], Rf=0,2).

Example 1,3. 5,6-O-Isopropylideneuridine acid.

Acetylchloride (of 0.67 ml) was added to a rapidly stirred suspension of ascorbic acid (8.0 g) in acetone (80 ml) and the mixture was mixed half days at room temperature. The precipitate was collected by filtration, rinsed with ethyl acetate and dried under reduced pressure to get 8,29 g of 5,6-O-isopropylidenediphenol acid as colorless solid. TLC (silica gel: CHCl3/MeOH/HOAC [3:1:0,1], Rf=0,54).

An example of 1.4. 3-O-(Ethoxycarbonylmethyl)-5,6-isopropylidenediphenol acid.

An example of 1.5. 3-O-(Carboxypropyl)-5,6-isopropylidenediphenol acid.

4,6 ml 2 N. NaOH was added to a solution of 3-O-(ethoxycarbonylmethyl)-5,6-isopropylidenediphenol acid (1,02 g) in 15 ml EtOH. 1 hour later a large part of the ethanol was removed under reduced pressure and the residue was diluted with water (10 ml) and pagkilala DIL-HCl (pH 3). Then the solution was saturated with NaCl and extracted several times with ethyl acetate; the besieged extracts were then dried using MgSO4. The solvent was evaporated under reduced pressure to obtain a viscous residue containing 3-O-(carboxypropyl)-5,6-isopropylidenediphenol acid (0.84 g). TLC: (si/P> A solution of di-terbutyl of dicarbonate (0.36 g) in 10 ml DMF was added dropwise into a solution of D-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys] -Thr-NH2acetate (2 g, BIM-23014) in 45 ml of DMF. After 2 hours at room temperature the solvent was removed under reduced pressure giving a residue, which was then chromatographically on silica gel (150 g) using CHCl3/MeOH (9 : 1) as eluent. The appropriate fractions were collected and the solvents were removed under reduced pressure to obtain a residue containing D-Nal-c[Cys-Tyr-D-Trp-Lys(BOC)-Val-Cys] -Thr-NH2(1.45 g). TLC (silica gel: CHCl3/MeOH [3:1], Rf=0,52).

An example of 1.7.

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0.2 ml of Diisopropylethylamine was added to a solution of D-Nal-cyclo- [Cys-Tyr-D-Trp-Lys(BOC)-Val-Cys] -Thr-NH2(300 mg), 3-O-(carboxypropyl)-5,6-isopropylidenediphenol acid (56 mg) in 5 ml of DMF. The mixture is then stirred at room temperature for half a day and the solvent was removed under reduced pressure. The residue was divided between a mixture of ethyl acetate/MeOH and saturated aqueous NaCl, and the ethyl acetate layer was washed with saturated aqueous NaCl, then with saturated aqueous NaHCO3and dried (MgSO4). The solvent was evaporated under reduced pressure, and the residue was subjected to preparatory TLC using a mixture of CHCl33/MeOH. The solvents were removed under reduced pressure to provide the above product (0.20 g). TLC (silica gel: CHCl3/MeOH[5: 1], Rf=0,54).

An example of 1.8. Deleting groups VOS.

A derivative of ascorbic acid, D-Nal-c [Cys-Tyr-D-Trp-Lys(BOC)-Val-Cys]-Thr-NH2(95 mg), shown above, was treated with 25% TFA in CHCl3for 45 min at room temperature. Volatiles were removed under reduced pressure to get the dried residue, which was purified using a Vydac C18HPLC and CH3CN/0.1% aqueous TFA. The final yield was 90 mg (FAB-MS (m/e) 1341).

An example of 1.9. Other examples of implementation.

The following derivatives of somatostatin was synthesized in a similar manner:

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Example 2. Synthesis of BIM-23107.

The following is the derivative of somatostatin, called BIM-23107, was synthesized in accordance with this invention. (AcO-CH2)3-C-NH-CO-(CH2)2-CO-D-Nal-c [Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2.

An example of 2.1. (AcO-CH2)3-C-NH-CO-(CH2)2-CO-D-Nal-c [Cys-Tyr-D-Trp-Lys(BOC)-Val-Cys]-Thr-NH2.

0,03 ml of DIEA was added to chilled in ice to a solution of 2-N-(succinyl)amino-2-(acetoxymethyl)-1,3-propandiol-diacetate (83 mg) and HBTU (92 NH2(100 mg) in 2 ml of DMF containing 0.03 ml of DIEA. The mixture is first stirred at 0 - 5oC for 1 hour and then stirred at room temperature for half a day. The solvent was removed under reduced pressure, giving the dried residue, which was divided between ethyl acetate and aqueous saturated NaCl, and the EtOAc layer was washed with 5% aqueous NaHCO, and finally water saturated NaCl; then the resulting solution was dried using MgSO4. The solvent was evaporated under reduced pressure, leaving a residue containing (AcO-CH2)3-C-NH-CO-(CH2)2-CO-D-Nal-c [Cys-Tyr-D-Trp-Lys(BOC)-Val-Cys] -Thr-NH2(0.14 mg). TLC (silica gel: CHCl3/MeOH/HOAC = 4:1:0,1, Rf=0,82).

An example of 2.2. Removal of the BOC group.

30 mg of the Above compound was treated with 50% TFA in CHCl345 minutes at room temperature; then volatiles were removed under reduced pressure giving a residue. Traces of TFA were co-evaporated with ethanol several times, and the residue was titrated with ether and then dried to obtain 30 mg of the product. TLC (silica gel: CHCl3/MeOH/HOAc = 3:1:1. Rf= 0,24).

Example 2,3. Other examples of implementation.

The following derivatives of somatostatin was synthesized in a similar manner.

The following is the derivative of somatostatin, called (BIM-23201), was synthesized in accordance with this invention.

(HO-CH2)3-C-CH2-D-Phe-c [Cys-Tyr-D-Trp-Lys-Thr-Cys]-Nal-NH2.

An example of 3.1. (HO-CH2)3-C-CH2-D-Phe-c [Cys-Tyr-D-Trp-Lys-Thr-Cys]-Nal-NH2.

2 g of the Last molecular sieve (filter) NaCNBH3(36 mg) was added in portions with periods of 15 minutes in a solution of D-Phe-c[Cys-Tyr(OBt)-D-Trp-Lys(BOC)-Thr(OBt) Cys] -Nal-NH2(250 mg) and Tris(acetoxymethyl)acetaldehyde (120 mg) obtained by oxidation of triacetyl-Penta-erythriol the pyridinium dichromate or DMCO/oxalyl chloride/triethylamine) in methanol (10 ml) containing 10% acetic acid. The mixture is then stirred at room temperature for 30 minutes and was heated 4 hours.

After filtration, the residue was separated between ethyl acetate and water. The ethyl acetate layer was washed with water, is the group of residue (0.4 g), which was then dissolved in methanol (5 ml) was treated with a solution of NaOMe/MeOH (pH 10), stirred for 1 hour and finally was kind of balanced out 1 N. HCl to pH 5-6. After evaporation of the solvent the residue was dissolved in 90% aqueous TFA (5 ml) and stirred 30 minutes. Volatiles were removed under reduced pressure and traces of TFA and water in the resulting residue was removed by joint evaporation with ethanol (2x). The residue was dried, then titrated with ether, and finally purified HPLC using conditions similar to those described previously, allowing 41 mg (HO-CH2)3-C-CH2-D-Phe-c [Cys-Tyr-D-Trp-Lys-Thr-Cys]-Nal-NH2as a colorless solid. MS (m/e) 1262,8.

An example of a 3.2. Other examples of implementation.

The following is the derivative of somatostatin, called BIM-23195, was synthesized in a similar way

(BUT-CH2)3-C-CH2-D-Phe-c [Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2< / BR>
BIM-23195

Example 4. Synthesis of BIM-23197.

The following is the derivative of somatostatin, called BIM-23197, was synthesized in accordance with this invention.

< / BR>
An example of 4.1. 2-Bromoethanesulfonate chloride

Na 2-bromoethanesulfonate (4.0 g) was treated PCl5(11,8 g) under cooling in an ice bath. After reaching the liquid phase is in 50 g of crushed ice and then stirred 15 minutes The mixture was extracted CH2Cl2(G ml) and the combined extracts were washed H2O (2x), 5% NaHCO3(2) and again H2O (2). Drying over anhydrous MgSO4and distillation under reduced pressure gave 2-bromoethanesulfonate chloride as a colorless liquid (1,95 g, 42-44oC/1 mm Hg).

An example of 4.2. Br-(CH2)2-SO2-D-Phe-c[Cys - Tyr(tBu)-D-Trp-Lys(BOC)-Abu-Cys] -Thr(tBu)-NH(1-cyclopropyl-1-methyl)-ethyl.

A solution of 2-bromatan-sulfonyl-chloride (30 mg) in DMF (1 ml) was added dropwise into a solution of H-D-Phe-c[Cys-Tyr(tBu)-D-Trp-Lys(Boc)- Abu-Cys]-Thr(tBu)-(1-cyclopropyl-1-methyl)-ethyl (150 mg) and DIEA (55 mg) in DMF (2 ml) and N2and 0oC. the Reaction mixture was stirred at 0-5oC 3 hours; then the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate and washed with 5% citric acid (2x), 5% NaHCO3(2x) and brine (2x). Then the solution was dried over anhydrous MgSO4was filtered and condensed to a degree of dryness under reduced pressure. Then the product was purified on a short column of silica gel, lirovannomu with ethyl acetate. The fractions containing the product were collected and the solvent was removed under reduced pressure, giving 105 mg Br-(CH2)2-SO2-D-Phe-c[Cys-Tyr(tBu)-D-Trp - Lys(BOC)-Abu-Cys]-Thr(tBu)-NH(1-SS="ptx2">

Example 4.3.

< / BR>
A solution of Br-(CH2)2-SO2-D-Phe-c[Cys-Tyr(tBu)- D-Trp-Lys(Boc)-Abu-Cys] -Thr(tBu)-NH(1-cyclopropyl-1-methyl)-ethyl (100 mg) and 2-hydroxyethylpiperazine (55 mg) in 2 ml of 1-propanol was watered with N2for 2.5 hours. Then the solution was cooled to room temperature and the solvent was removed under reduced pressure. Then the residue was dissolved in ethyl acetate containing 5% MeOH, and were washed with brine (3x). Finally, the solution was dried over anhydrous MgSO4was filtered and condensed to dryness under reduced pressure to obtain 110 mg of the above solid. Without further purification, this compound was used directly in the next step.

Example 4.4.

< / BR>
110 mg of the Protected derivative of somatostatin obtained in the previous step, was dissolved in 10 ml of an aqueous solution of 90% TFA and stirred at room temperature for N2within 1 hour. TFA and H2O was removed under reduced pressure, and the residue was titrated with cold ether (3x10 ml). Turned out slightly yellowish solid; this material was further purified by preparatory reverse phase HPLC, algirus: 1) aqueous solution of NH4OAc; and (2) aqueous solution of HOAc.

the)/e) 1252,7).

Example 4.5. Other examples of implementation

The following derivatives of somatostatin was synthesized in a similar manner:

< / BR>
< / BR>
< / BR>
< / BR>
Example 5. Synthesis of derivatives bombesin.

The following derivative bombesin, also called BIM-26333, was synthesized in a manner analogous to that described above:

< / BR>
Other derivatives of the peptides according to this invention can be synthesized in a similar manner using known in the art synthetic modifications.

The results of the analysis of the tested peptides.

Example 6. Analysis of education links.

To demonstrate binding of the chemical affinity of the analogues of somatostatin (SRIF) receptor somatostatin described above purified compounds were tested in the analysis of education relations somatostatin, comprising measuring in vitro inhibition of education relations [125I-Tyr11SRIF-14] the membranes of the pancreas of the rat AR42J. As shown in table 1, purified analogs of somatostatin according to this invention showed high binding chemical affinity to these receptors. In addition, molecular weight, specific mass spectrometry and evaluated from molecular structure purified similar bombesin was tested in the experience of education relations bombesin. Analysis of education relations consisted of measurements in vitro inhibition of education relations [125I-Tyr11] bombesin with membranes of the pancreas of rats AR34J; from the analysis determined that the binder chemical affinity similar bombesin with GRP receptor was about 21 nm.

Example 7. Analysis of the inhibition of growth hormone (GH).

Groups of 5 male rats Sprague Dawley (each had a weight of 250-300 g) were inheritability in normal conditions derived somatostatin or saline. 30 minutes before the selected time periods after administration of the drugs shown in table 2 (2 hours, 4 hours, 6 hours, 8 hours) rats were anesthesiologis i Nembutal.p. (50 mg/kg).

15 minutes after anesthesia a multiple of the number of blood out of the puncture in the heart over heparin for measuring primary GH. In addition, were injection in normal conditions D-Ala2-GRF (10 g/kg). After 15 minutes the blood was taken to kanterovich stimulated GH, which was measured in plasma using radioimmunoassay provided NIADDKD. The percentage inhibition of GH was calculated from the difference obtained between the values of the primary and stimulated GH.

Table 2 shows the Nal-NH2(BIM-23060) in the inhibition of growth hormone in rats compared with the effectiveness of other derivatives of somatostatin (BIM-23167, BIM-23179 and BIM-23181) this invention. All derivatives show an amazing extended duration, which decreases depending on the time.

Additional experiments were carried out on D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys] -Thr-NH2, the analogue of somatostatin, and BIM-23190, BIM-23195 and BIM-23197 to determine the ED50(i.e., the concentration of each compound required to inhibit the release of 50% of growth hormone after a certain period of time) of the corresponding connection. The experiments were carried out in the range of doses from 25 to 0.25 g/kg table 3 shows amazing improvement actions derived somatostatin compared with non-modifiable peptide at different time intervals, which shows time-dependent inhibition of stimulated GH release compounds of this invention.

Example 8. Analysis of activism against reproduction.

Two described above purified analog of somatostatin was also tested for activity against proliferating cells. Table 4 shows the effect of these peptides on the growth of tumor cells podjeludochnuyu activity against reproduction. As can be seen from the drawing, as BIM 23014C (somatostatin analogue), and BIM-23118 (a derivative of BIM-23014) inhibit the growth of tumor cells in the pancreas of the rat AR42J depending on the concentration, and BIM-23118 is the more effective of the two connections. Both compounds inhibit the growth of tumor cells to a greater extent than unmodified analogs of somatostatin at equivalent concentrations.

Example 9. Analysis of the uptake of thymidine.

In this experience the culture of the Scion Swiss cells ZTZ grown in modified eagle's medium Dulbecco (Dulbecco''s Modified Eagles Medium - DMEM) and supplemented with 10% fetal serum fetal calf in a humidified atmosphere of 10% CO2and 90% air at 37oC. Then cells were sowed in group trays with 24 tanks and used 4 days after the last change of environment. To arrest cells in phase G1/G0 cell cycle, was used free of serum DMEM for 24 hours before analysis of the uptake of thymidine; then cells were washed twice with 1 ml increments of DMEM (serum, 0.5 m) and [methyl-3H] thymidine (20 Ci/mmol, New England Nuclear). Derivatives bombesin initially tested at 0,001, 0,01, 0,1, 1, 10, 100, 100 nm. After 28 hours at 37oC analyzed the incorporation of [methyl-3H] townsman tima ice-cold 0.9% NaCl (multiples of 1 ml); then acid-soluble radioactivity was removed by exposure to thermostat for 30 minutes at 40oC with 5% trichlorosucrose acid (TCA). Then the cultures were washed once (1 ml) of 95% ethanol was done soluble 30-minute delay in thermostat with 1 ml of 0.1 G. of NaOH. Become soluble material was transferred to vials containing 10 ml ScintA (Packard), and radioactively was determined by liquid scintillation spectrometry. This analysis has shown the ability derivatives bombesin to stimulate uptake of thymidine by the cells. EC50was calculated and amounted to 0.48 nm, which showed that derivatives bombesin of this invention are strong simulations uptake of thymidine.

Methods of use.

Derivatives of the peptides according to this invention may be a mammal, especially man, one of the traditional ways (e.g., through the mouth, parentino, through the skin or through the mucous in part supported by the release with the use of bio-degrading, bio-compatible polymer or the introduction on the site (for example, in the case of derived anticancer bombesin or somatostatin in the lungs) using micelles, gels and liposomes. Doses are usually those who water the peptides of this invention are suitable for the improved treatment of diseases, treatable corresponding unmodified peptide. In particular, the above-described derivatives of somatostatin suitable for the treatment of cancer, acromegaly, pancreatitis, caused by trauma reproduction, diabetes, diabetic retinopathy, restenosis following angioplasty, AIDS, neurogenic inflammation, arteritis and gastrointestinal problems, including diarrhea.

1. The derived peptide containing the remainder of somatostatin, or bombezin, or derivative, fragment or analog and Deputy selected from the group consisting of compounds I, II or III, where the connection I

< / BR>
where R0oxygen;

R1and R2each independently - H, (CH2)mOR6; CH2(OR7)CH2OR8where R6Is H and R7and R8each independently - H;

m = 1 to 5, an integer,

one of R3and R4- (CH2)nR12where R12- CO, n = 1 to 5, an integer, and the other of R3and R4- H, (C1- C6)hydroxyalkyl;

compound II

< / BR>
where R13- R15each independently - H or (C2- C4)acyl;

R16- NH or is absent;

R17- CO or missing;

R18- CO, SO2or missing;

m = lcyl;

R20is oxygen or absent;

R21- (C1- C6)alkyl or is absent;

R22- N or CH;

R23- no;

R24IS N, CH, C;

R25- no;

R26- CH2, SO2or missing;

m = 0 to 5, an integer;

n = 0 to 5, an integer;

p = 0 to 5, an integer;

q = 0 to 5, an integer,

while the remainder of somatostatin, or bombezin, or derivative, fragment or analog is attached to each of the alternate communication CO-N, CH2-N between the substituent and the nitrogen atom of the N - end or side chain of the peptide residue.

2. Derived under item 1, characterized in that the Deputy is a connection I.

3. Derived under item 1, characterized in that the Deputy is compound II.

4. Derived under item 3, wherein R18- SO2.

5. Derived under item 4, wherein R13- R15- H, and R17is missing.

6. Derived under item 5, characterized in that the Deputy is (HOCH2)3C-NH-(CH2)2-SO2or (HOCH2)3C-CH2.

7. Derived under item 1, characterized in that the Deputy is compound III.

8. Production is inimum one of R22and R24is n

10. Derived by p. 9, wherein both R22and R24is n

11. Derived by p. 10, where the Deputy is

< / BR>
< / BR>
12. Derived by p. 10, characterized in that it contains the remainder of somatostatin or its derivative, fragment or analogue.

13. Derived under item 12, characterized in that the analogue of somatostatin is one of:

H-D-Phe-c[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2,

H-D-Phe-c[Cys-Tyr-D-Trp-Lys-Thr-Cys]-Nal-NH2< / BR>
or

H-D-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2.

14. Derived on p. 11, characterized in that it contains the remainder of bombezin or its derivative, fragment or analogue.

15. Derived under item 1, characterized in that it is

< / BR>
or

< / BR>
16. A method of treating cancer in a patient, comprising making the patient a therapeutic amount of a derivative of a peptide, characterized in that the quality of the derived peptide use a connection on p. 1.

17. The method according to p. 16, characterized in that a derivative of the peptide is derived somatostatin or its analogues.

 

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SUBSTANCE: invention relates to a method based on phage display for preparing peptides interacting specifically with mammary Ehrlich tumor and can be used in therapy and diagnosis of malignant neoplasm. Peptides are prepared by affinity selection from phage peptide libraries comprising ten millions of different peptides of size 15 amino acid residues, the group of nine peptides wherein each peptide shows ability for accumulation in Ehrlich tumor. For practice using mimetic-peptides selected by such manner can be prepared by chemical synthesis and to use for preparing conjugates on their basis with the known cytotoxic preparations, radioactive isotopes and they can be incorporated in the composition of liposomal preparations for visualization of tumor neoplasm also.

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2 dwg, 2 ex

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