WO2004060916A2

WO2004060916A2 - Peptide compound containing boron

Info

Publication number: WO2004060916A2
Application number: PCT/DE2003/004216
Authority: WO
Inventors: Lutz Tietze; Ulrich Griesbach
Original assignee: Georg-August-Universität Göttingen
Priority date: 2003-01-06
Filing date: 2003-12-19
Publication date: 2004-07-22
Also published as: AU2003298079A1; EP1583775A2; DE10300361A1; WO2004060916A3

Abstract

Disclosed is a peptide compound comprising at least one derivative of a peptide ligand of a receptor that occurs naturally in the body and at least one component containing boron. Preferably, said derivative of a peptide ligand represents a gastrin derivative. In an advantageous embodiment of the invention, said derivative encompasses the amino acid sequence Trp-Met-Asp-Phe-NH2.

Description

Boron-containing peptide compound

The invention relates to processes for the preparation of peptide compounds with at least one boron-containing component, compounds obtainable therewith, and suitable uses of such peptide compounds and a pharmaceutical composition.

Despite intensive research in this field, the effective treatment of cancer or tumor diseases represents a frequently unsolved problem. An important therapeutic approach for the treatment of such diseases takes advantage of phenotypic and / or physiological peculiarities of cancer cells or tumor cells in order to selectively treat them in the presence of healthy cells to destroy. Boron neutron capture therapy (BNCT) is a promising approach in which the tumor cells are to be destroyed in a targeted manner. It is a binary system in which non-radioactive borosotopes ¹⁰ B are first built into the tumor or cancer cells or generally incorporated. Treatment with thermal neutrons triggers a so-called capture reaction, in which high-energy decay products - an α particle and a ⁷ Li ³⁺ ion - cause destruction of the tumor tissue. The trajectories of these decay products lie in the range of an average cell diameter, so that only those cells are destroyed that have incorporated or bound boron (RF Barth, AH Soloway, RG Fairchild, Cancer 1992, 70, 2995-3007; MF Hawthorne, Angew Chem. 1993, 105, 997-1033; Angew. Chem. Int. Ed. Eng /. 1993, 32, 950-984; C. Morin, Tetrahedron 1994, 50, 12521 -12569; D. Gabel, Chem Zeit 1997, 31, 235-240; AH Soloway, W. Tjarks, BA Barnum, F.-G. Rong, RF Barth, IM Codogni, JG Wilson, Chem Rev, 1998, 98, 1515-1562). The boron neutron capture therapy has the decisive advantage over conventional chemo-tumor therapies with the known serious side effects that the cytotoxic effect is only possible through the untreated tissue, i.e. for all cells and tissues except for the tumor or cancer cells with accumulated boron, harmless neutron radiation is generated.

The problem with the development of an efficient boron neutron capture therapy is the targeted accumulation of boron, for example by storage or by binding, in the tumor or cancer cells. It is crucial here that the boron is supplied to the organism in a form which ensures selective storage in the tumor or cancer cells in order to avoid destruction in healthy tissue which may be caused by boron being stored non-specifically. Furthermore, the boron-containing substances with which the boron is to be introduced into the organism may have only a low toxicity in order to largely avoid side effects of the treatment. Corresponding compounds should also have sufficient water solubility.

Various boron-containing substances for use in boron neutron capture therapy have already been tested. Conjugates of boron-containing substances with porphyrins, amino acids and peptides, nucleic acids, nucleosides and nucleotides were used. Furthermore, boron-containing glycosides and conjugates from carboranes and DNA-binding indole units were also synthesized for this purpose (LF Tietze, U. Bothe, Chem. Eur. J. 1998, 4, 1 179-1 183; LF Tietze, U. Bothe, I Schuberth, Chem. Eur. J. 2000, 6, 836-842; LF Tietze, U. Bothe, U. Griesbach, M. Nakaichi, T. Hasegawa, H. Nakamura, Y. Yamamoto, Bioorg. Med. Chem. 2001, 9, M ^ lM l; L F. Tietze, U. Bothe, U. Griesbach, M. Nakaichi, T. Hasegawa, H. Nakamura, Y. Yamamoto, ChemBioChem 2001, 2, 326-334; LF Tietze, U. Griesbach, U. Bothe, H. Nakamura, Y. Yamamoto, ChemBioChem 2002, 3, 219-225).

Clinical trials of boron neutron capture therapy in phase I are currently underway in the United States, Japan and Europe. The so-called "first generation" reagents Lp- Boronophenylalanine hydrochloride (BPA, first synthesized in 1958) as a water-soluble fructose complex and Na ₂ B _{l 2} H _{1 l} SH (BSH, first synthesized in 1965). However, these substances have an unclear specificity towards tumor or cancer cells. They therefore do not adequately meet the requirements for tumor selectivity, which is necessary for safe use of the therapy for the patient with correspondingly good chances of success.

A disadvantage of many boron-containing compounds is that the boron component is linked to other components via a disulfide bond. Disulfide bonds are very unstable in the body and are broken down very quickly there. Therefore, those compounds where the boron component is bound to the other components by disulfide bridges can only be used to a limited extent in therapy.

The invention is therefore based on the object of providing compounds which are particularly suitable for use in boron neutron capture therapy. These compounds are intended to ensure that boron is bound or embedded in tumor or cancer cells with high selectivity. The compounds should have the lowest possible cytotoxicity in order to

Avoid side effects of therapy.

This object is achieved by the peptide compounds according to the invention. Preferred embodiments of these peptide compounds gastrin derivatives, as shown below. The present invention further relates to a suitable method for producing the peptide compounds according to the invention. Finally, the invention also includes the use of peptide compounds according to the invention in tumor therapy or a corresponding pharmaceutical composition. The wording of all claims is hereby incorporated by reference into the content of the description. According to the invention, a peptide compound, in particular for use in boron neutron capture therapy, is provided, which comprises at least one oligopeptide and at least one component containing boron, which can be produced by a method in which the two components of the compound are directly linked via a peptide compound or via a linker are interconnected. In a preferred embodiment of the invention, the oligopeptide can be a ligand of an endogenous receptor, in particular a membrane-bound receptor, as well as a fragment or a derivative of such a ligand. In particular, a gastrin derivative is provided as a derivative of a peptide ligand of an endogenous receptor.

Recent developments in tumor research show that different receptors that are expressed in the human or animal organism under normal conditions according to a certain pattern in different tissues or organs are expressed in large numbers by certain cancer or tumor cells and for these cells as growth factors serve. For example, autoradiography studies have shown that the so-called CCK-B receptor, in addition to its natural presence in the stomach, blood, kidneys, gallbladder and brain, in various tumor cells, especially in thyroid carcinoma cells (92%), lung tumors

(Narrow cells, 57%), astrocytomas (65%) and stromal ovarian tumors (100%) occur in high density (J.C. Reubi, J.-C. Schaer, Cancer Bes. 1997, 57, 1377-1386).

These CCK-B receptors are high-affinity receptors that bind in particular the body's own regulator peptides or proteins cholecystokinin (CCK) and gastrin. These regulator peptides play an important role especially as hormones of the gastrointestinal tract and as neurotransmitters in the brain (J.-C. Schaer, J.C. Reubi, J.C / in. Endocrinol. Metah. 1999, 58, 1, 238-239). Both peptides or proteins have at their C-

Terminus is a matching primary structure of five amino acids. in this connection it is the amino acid sequence Gly-Trp-Met-Asp-Phe-NH ₂ . The biologically active part, which is responsible for the binding to the corresponding receptors, is formed by the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH ₂ (tetragastrin).

In addition to the high-affinity receptor CCK-B, the natural function of the regulator peptides is also controlled by another receptor (CCK-A), which has a low affinity, in particular for gastrin (JC Reubi, J.-C. Schaer, Cancer Res. 1997, 57, 1377-1386). Under natural conditions, the gastrin / CCK-B receptors endocytotically introduce the regulatory peptides into the cell. During a cycle that lasts about an hour, they return to the surface to bind new peptides (NI Tarasova, SA Wank, EA Hudson, VI Romanov, G. Czerwinsky, JH Resau, CJ Michejda, Cell Tissue Res. 1997, 287, 325-333).

The peptide compounds and especially oligopeptide compounds according to the invention therefore include, in addition to target peptides in particularly preferred embodiments according to the invention, peptide ligands of the body's own receptors, in particular membrane-bound receptors, as well as derivatives and fragments of such ligands. The use of a is very particularly preferred

Gastrin / CCK-B receptor binding oligopeptide. The peptide compounds can preferably bind specifically to the receptors which are expressed by cancer or tumor cells. As a result, the boron-containing component which is contained in the inventive peptide compounds is attached to the abnormal cells, so that these can be selectively and selectively destroyed as part of a boron neutron capture therapy.

The derivatives according to the invention contain oligopeptides which contain modified amino acids / peptides. Modifications include: glycosylated, phosphorylated, sulphated amino acids / peptides, as well as L-

Isomers and D isomers. Amino acid and peptide derivatives can by post-translational modifications, through chemical modifications, through enzymatic modifications or due to other mechanisms, or are specifically produced. The resulting peptides can contain modifications that can occur in all areas of the peptide molecule. For example, modifications can occur in the peptide backbone, in the amino acid side chains, at N-terminal ends of the peptide or at C-terminal ends of the peptide. The modifications can be for single amino acids, for several amino acids or for all amino acids and there may be none, one or more types of modifications in any combination in a peptide.

Furthermore, derivatives of the oligopeptides include peptides which, in addition to the oligopeptide according to the invention, have further amino acids N-terminal or C-terminal. These can also be modified, as described above.

Finally, the derivatives include in particular those which have increased water solubility due to modification. Such modifications include glycosylation

In a preferred embodiment, the peptide compound according to the invention is characterized in that the derivative of the peptide ligand is a gastrin derivative. This gastrin derivative advantageously comprises the amino acid sequence Trp-Met-Asp-Phe-NH ₂ (SEQ ID NO 1). This tetrapeptide sequence in particular is suitable for the specific attachment of the peptide compounds according to the invention to the cancer or tumor cells. The invention encompasses peptide compounds which only have these four amino acids as a peptide component, but also peptide or protein compounds which, in addition to the tetrapeptide sequence, have further amino acids or amino acid sequences. The tetrapeptide sequence (Trp-Met-Asp-Phe-NH ₂ ) in the compound C according to the invention is advantageously arranged terminally. However, it is also encompassed by the invention that this sequence is present in a connection at the amino terminal or also framed by further sequences. Further examples of a peptide sequence suitable as a gastrin derivative are Gly-Trp-Met-Asp-Phe-NH ₂ (SEQ ID NO 2) and Leu- (Glu) ₅ -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH ₂ (minigastrin) (SEQ ID NO 3).

Such gastrin derivatives are particularly suitable as part of the peptide compounds for the purposes of the invention, since they can be used to achieve a very high tumor / background selectivity. This means that the peptide compounds according to the invention bind very selectively to tumor or cancer cells. Furthermore, these compounds are subject to a very advantageous rapid "background clearance", since they do not largely bind to plasma proteins in human or animal serum.

In another advantageous embodiment, the peptide compound additionally has a core localization sequence. This allows the boron component to be placed in close proximity to the DNA and thus to achieve a more effective BNC therapy. Such sequences are well known to the person skilled in the art, for example the SV40 sequence can be mentioned here.

The peptide compounds according to the invention are further characterized by their hydrophilicity. This is very advantageous because it does not penetrate the blood-brain barrier so that the CCK-B receptors in the brain are not reached by the peptide compounds. Because of these properties, the peptide compounds according to the invention are particularly suitable for use in tumor therapy, in particular for a boron neutron capture therapy.

In a preferred embodiment of the peptide compounds according to the invention, the boron-containing component is linked to the

Derivative of the peptide ligand linked. Various suitable ones come for this Linkers, such as alkyl chains, in particular with a chain length of 2 to 10 carbon atoms, in question.

The boron-containing component is advantageously a borane, carborane and / or thiocarborane, which in an advantageous embodiment was obtained from a boronic acid, carborane carboxylic acid and / or thiocarborane carboxylic acid. In addition, it may be preferred that the boron-containing component is a carboranyl compound, in particular a carboranyl amino acid and / or a carboranyl glycoside. The water solubility of the peptide compounds according to the invention can advantageously be increased by using carboranyl glycosides as the boron-containing component. Examples of suitable sugar residues are glucose, mannose, maltose and / or lactose. In addition, the invention also includes other boron-containing components as part of the peptide compound according to the invention, which are familiar to a person skilled in the art.

Exemplary compounds are represented by the following structural formula (I):

where m and n are independently an integer from 1 to 20, preferably from 1 to 10 and particularly preferably from 1 to 6.

In the above compound (1) there is the oligopeptide component on one side, while on the other side there is another functionality, such as saccharides, for increasing the solubility in water. A preferred compound according to the invention is one of the formula (II):

where m and n are as defined above.

Other ways of increasing water solubility are to add alcohols or amino or carboxylic acid residues, which can then form salts.

The above Compounds, in particular the glycosylated compounds, make it possible to increase the water solubility of the compounds to be administered. This is particularly necessary if higher concentrations of peptide compounds are to be administered during therapy.

In combination with the water solubility considerations discussed above, or alone, the peptide compound can include components that bind to DNA. Such a possibility of binding to DNA molecules allows a higher toxicity of the boron component, since the DNA is a main target in the BNCT. These compounds can in particular also be used in combination with a core localization sequence. An example of a compound that allows DNA binding is the following compound

where m and n are as defined above.

In a further preferred embodiment of the peptide compound, the compound according to the invention has a further component in addition to the peptide and the boron-containing component. In particular, this further component can be a cytotoxic component, such as, for example, an alkylating component (for example a triazene derivative) or a DNA-binding component (for example indole). Other components, in particular cytotoxic components, which are suitable for this aspect of the invention will become apparent to the person skilled in the art. Such components as part of the peptide compound according to the invention can have a further cytotoxic effect on the tumor or cancer cells. The therapeutic effect of the peptide compounds according to the invention can thus be further enhanced and improved if necessary. The invention further comprises a method for producing the described boron-containing peptide compounds (eg FIG. 1). This method is characterized in particular in that the boron-containing component (3) is linked to the oligopeptide (1) via a carborane carboxylic acid. This can advantageously be achieved by exposing an amino group of the oligopeptide (1) by, for example, acidic cleavage of a protective group of the peptide or protein, and reacting this exposed amino group with the carboxyl group of a carborane carboxylic acid (3). A corresponding reaction can preferably be carried out with the reagents 1 - (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCΗCI) and 1 - hydroxybenzotriazole hydrate (HOBt-H ₂ O). The peptide bond between the boron component, either directly or via a linker, allows the peptide compound to be hardly degraded in the body, unlike compounds that contain a disulfide linkage.

In a preferred embodiment of this process, the carborane carboxylic acid (3) is obtained from a pentynecarboxylic acid, in particular a 4-pentynecarboxylic acid (2). This reaction takes place, for example, in three steps which are known to the person skilled in the art (WR Roush, K. Koyama, ML Curtin, KJ Moriarty, J. Am. Chem. Soc. 1996, 118, 7502-7512). In principle, any suitable alkynecarboxylic acid or a corresponding alkynecarboxylic acid derivative can be used instead of the pentynecarboxylic acid. The person skilled in the art can test suitable alkyne carboxylic acids or derivatives within the scope of his technical knowledge. Alkynecarboxylic acids with 3 to 20 carbon atoms are particularly suitable, alkynecarboxylic acids having alkyl groups with 1 to 10 carbon atoms being included. All suitable acid derivatives can be used as derivatives, in particular esters of alcohols having 1 to 6 carbon atoms and amides are considered suitable, but this does not preclude the use of other derivatives. The oligopeptide (1) can also be produced by customary methods. For example, the tetrapeptide sequence (Trp-Met-Asp-Phe-NH ₂ ) can be built up successively from the four individual amino acid building blocks by known methods (PJ Beishaw, S. Mzengeza, GA Lajoie, Synth. Commun. 1990, 20, 3157- 3160; SJF Macdonald, GDE Clarke, MD Dowle, LA Harrison, ST Hodgson, GGA Inglis, MR Johnson, P. Shah, RJ Upton, SB Walls, J. Org. Chem. 1999, 64, 5166-5175; AI Meyers, FX Tavares, J. Org. Chem. 1996, 61, 8207-8251; AG Myers, JL Gleason, T. Yoon, DW Kung, J. Am. Chem. Soc. 1997, 119, 656-673; A. Sutherland, CL Willis, J. Org. Chem. 1998, 63, 7764-7769). The oligopeptide, for example the said tetrapeptide, can have fer .'-butoxycarbonyl- (Boc) as a protective group, as is also known (A. Mehta, R. Jaouhari, TJ Benson, KT Douglas, Tetrahedron Lett. 1992, 33, 5441 -5444). Acid cleavage of this protective group exposes an amino group which can be reacted in accordance with the process of the invention. The peptide compound (5) according to the invention can then be obtained by palladium-catalyzed cleavage of the allyl (AII -) ester in the reaction product (4), for example an aspartic acid / 7allyl ester. This method according to the invention represents a very short and efficient synthetic route for the peptide compound according to the invention, which can be carried out in high yield (for example 72%). In addition to this synthetic route for the production of peptide compounds according to the invention, the invention also encompasses those production processes of these peptide compounds which are obvious to the person skilled in the art from the principle of synthesis.

The invention further comprises the use of the peptide compounds according to the invention for the treatment of tumors or cancer. The peptide compounds are used to great advantage in the context of boron neutron capture therapy. With regard to the particular advantages of this use according to the invention of the peptide compounds described, reference is made to the above description. Furthermore, the invention also relates to a Use of a peptide compound according to the description above for the manufacture of a medicament for tumor treatment, this medicament being particularly advantageously used in the context of boron neutron capture therapy. In addition, the invention also includes the treatment of tumor or cancer diseases, the peptide compounds described being used in the context of boron neutron capture therapy.

Finally, the invention comprises a pharmaceutical composition which is characterized in that it comprises a pharmaceutically effective amount of at least one of the described peptide compounds and at least one pharmaceutically acceptable carrier. The required dose of the peptide compound according to the invention, which can be administered with such a pharmaceutical composition, depends not only on the bioavailability of the peptide compound and the like, but also on the severity of the disease and the circumstances in the patient. The choice of pharmaceutically acceptable carrier (s) depends, among other things. depends on the form of administration and is readily apparent to the person skilled in the art.

As explained in more detail in the examples, the peptide compounds according to the invention have only a very slight influence on cell vitality. This means that the cytotoxic effect of the peptide compounds is very low even on cells and tissues. Therefore, by using the peptide compounds according to the invention in boron neutron capture therapy, the advantage of this form of therapy over conventional tumor radiation therapies can be optimally exploited. The illustrations show: FIG. 1 synthesis of the carborane linked to the tetrapeptide Trp-Met-Asp-Phe-NH ₂ ; Reagents: a) trifluoroacetic acid, Et ₃ SiH,

CH ₂ CI ₂ ; then DMF, NEt / Pr ₂ ; b) 3, EDC HCl, HOBt-H ₂ O, DMF, 72% starting from 1; c) Pd (PPh ₃ ) ₄ , PPh ₃ , pyrrolidine (10 eq.), CH ₃ CN,

72%;

Figure 2 Λ- (3C, 4C-dicarba-c / os -dodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl- / 7-allyl ester-L-phenylalaninamide 4;

Figure 3 / V- (3C, 4C-dicarba-c / σsr> dodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl-L-phenylalaninamide 5;

FIG. 4 tabular representation of the measurement results of an MTT

Cytotoxicity tests with the compound

dodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl-L-phenylalaninamide 5. The ED ₅₀ values determined indicate the concentration of the compound which causes cell death in different cell lines in 50% of the cells;

Figure 5 graphical representation of the influence of

dodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl-L-phenylalaninamide 5 on the cell vitality of different cell lines. Examples

1 General

¹ H NMR and ¹³ C NMR: Varian XL-500 and XL-300, Bruker AM-300; Signal multiplicities were determined using the APT pulse sequence. Mass spectrometry: Finnigan MAT 95. IR: Bruker Vector 22. UV: Models Lambda 2 and Lambda 9 from Perkin-E / mer. All solvents were dried using standard laboratory procedures. Commercially available reagents were used without further purification. As far as reasonable, the reactions were carried out in heated glass apparatus under a slight excess of argon and the progress of the reaction was monitored by thin layer chromatography (Macherey-Nagel & Co., DC ready-made films Alugram SIL G / UV ₂₅₄ ). The products obtained were purified by column chromatography on silica gel (Merck).

2) Synthesis of / \ (3C, 4g-dicarbag-g / osododecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl- / 7allylester-L-phenylalaninamide 4

The synthesis route is shown in FIG. 1, FIG. 2 shows the synthesis product 4. The / V-Boc-protected tetrapeptide Trp-Met-Asp-Phe-NH ₂ 1 (139 mg, 188 mol) was treated with trifluoroacetic acid (TFA, 3 ml, 38.9 mmol) and Et ₃ SiH (40 μ \, 248 μmol) in CH ₂ CI ₂ (30 ml) for 90 min at room temperature. The solvent was worked up i. Vak. removed and the residue i. Vak. dried. The TFA salt thus obtained was dissolved in DMF (8 ml) with the addition of NEt / Pr ₂ (35 μ \, 204 μmol) and the solution of the carboxylic acid 3 (45.2 mg, 209 μmol) stirred at room temperature for 30 min. , EDC-HCl (44.2 mg, 222 μmol) and HOBt-H ₂ O (37.2 mg, 243 μmol) in DMF (3 ml) and stirred for a further 16 h at room temperature. The crude product was adsorbed on silica gel and IV- (SC ^ C-Dicarba-c / σs-T-dodecaboranyK ^ JbutylcarbonyO-L-tryptophyl-L-methionyl-L-asparagyl- / 7-allylester-L-phenylalaninamide 4 (1 13 mg, 72%) Gradient column chromatography (P / EE = 1: 3, 1% AcOH Q EE / MeOH = 10: 1, 1% AcOH) was obtained as a yellowish foam.

R _f (P / EE = 1: 3, 1% HOAc) = 0.42. [α] ²⁰ _D = -19.3 ° (c = 0.5, MeOH).

IR (KBr): v = 3286 cm ^"1 (NH), 3059 (C _{Ar / l} -H), 2923 (CH), 2588 (BH), 1642 (amide-C = O, C = C), 1536, 1437, 1227, 1098.

UV (CH ₃ CN): λ _max (Ig ε) = 191.0 nm (5.041), 280.5 (3.717), 289.0 (3.631). ¹ H-NMR (300 MHz, CD ₃ OD): δ = 1.00-3.00 (s _br , 10 H, BH), 1.73-1.92 (m, 1 H, 2c-H _a ) , 1.93-2.08 (m, 1 H, 2c-H _b ), 1.97 (s, 4 H, 1e-H ₂ , 2e-H ₂ ), 1.98 (s, 3 H, SMe ), 2.08-3.28 (m, 8 H, 3c-H ₂ , 2b-H ₂ , 2a-H ₂ , 2d-H ₂ ), 4.23 (dd, = 8.7, 5.4 Hz, 1 H, 1c-H), 4.40 (s _br , 1 H, carborane-CH), 4.46-4.66 (m, 5 H, 1a-H, Cr ₂ CH = CH ₂ , 1b -H, 1d-H), 5.19 (dd, .7 = 10.5, 1.3 Hz, 1 H, CH ₂ CH = Cr), 5.27 (ddd, J = 17.2, 3, 2, 1.5 Hz, 1 H, CH ₂ CH = Cr _trans ), 5.88 (ddt, J = 17.2, 10.5, 5.8 Hz, 1 H, CH ₂ C / = CH ₂ ) , 6.99 (ddd, J = 6.9, 6.9, 1.0 Hz, 1H, 5d'-H), 7.08 (ddd, J = 7.1, 7.1, 1.2 Hz, 1 H, 6d'-H), 7.14 (s, 1 H, 2d'-H), 7.15-7.36 (m, 6 H, Ph-H, 7d'-H), 7 , 55 (d, J = 7.6 Hz, 1-H, 4d'-H); all NH not visible due to H / D exchange. ¹³ C-NMR (75 MHz, CD ₃ OD): δ = 15.21 (S6Η ₃ ) 28.42 (C-2d), 30.92 (C-3c), 31.64, 33.93, 35, 68, (C-2c, C-1e, C-2e), 36.43 (C-2b), 38.54 (C-2a), 51.46 (C-1c), 54.49, 56.09 , 56.40 (C-1a, C-1b, C-1d), 63.85 (CH-carborane), 66,70 (6Η ₂ CH = CH _2), 76.26 (C-carborane), 110, 6 (C-2d ', 127.8 (-Ph-C), 128.8 (C-3ad'), 129.5 (o-Ph-C), 130.3 (mP -C), 133.4 (CH = CH ₂ ), 138.0, 138.6 (/ -Ph-C, C-7ad '), 171.8, 171.9, 172.2, 173.4, 173.6, 174.7 (6 C = O). MS (ESI): m / z (%): 1709 (20) [2M + K] ⁺ , 1693 (35) [2 + Na] ⁺ , 988 (50) [M + TFA + K] ⁺ , 875

(100) [M + K] 858 (98) [M + a] ⁺ , 836 (42) [M + H] ⁺ . C ₃₇ H ₅₄ B ₁₀ N ₆ O ₇ S (835.0). 3) Synthesis of /V-(3C".4C-Dicarba-g/θ5.>dodecaboranyl(12)butylcarbonyl)-L- tryptophyl-L-methionyl-L-asparagyl-L-phenvIalaninamide 5

The synthesis route is shown in FIG. 1, FIG. 3 shows the synthesis product 5. To a solution of the allyl ester (20.0 mg, 24.0 mol), Pd (PPh ₃ ) ₄ (2.4 mg) stirred at 0 ° C. , 2.1 μmol) and triphenylphosphine (4.8 mg, 18.3 μmol) in dry and degassed CH ₃ CN (2 ml), pyrrolidine (30 μ \, 363 μmol) was added and the mixture was stirred for a further 45 min at this temperature. A white precipitate fell out. For working up, the reaction mixture was poured into HCl (1 M) and extracted with EA. The combined organic extracts were washed with sat. NaCl solution. washed, dried over Na ₂ SO ₄ , the solvent i. Vak. removed and the crude product adsorbed on silica gel. Gradient column chromatography (P / EE = 2: 1 → EE / MeOH = 4: 1, 0.5% HOAc) gave AV (3C, 4C-dicarba-c / θ5θdodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L- methionyl-L-asparagyl-L-phenylalaninamide 5 (13.7 mg, 72%) as a beige solid.

R _f (EE / MeOH = 4: 1.1% HOAc) = 0.27. ⁿ H-NMR (300 MHz, CD ₃ OD): δ = 1, 00-3.00 (s _br , 10 H, BH), 1, 62-3.28 (m, 10 H, 2c-H ₂ ,

3c-H ₂ , 2b-H ₂ , 2a-H ₂ , 2d-H ₂ ), 1.96 (s _br , 7 H, 1 eH ₂ , 2e-H ₂ , SMe), 4.20-4.33 (m, 1 H, 1 cH), 4.44 (s _br , 1 H, carborane-CH), 4.48-4.64 (m, 3 H, 1 aH, 1 bH, 1 d- H), 6.95-7.04 (m, 1H, 5d'-H), 7.04-7.30 (m, 7H, 6d'-H, 2d'-H, Ph-H), 7.33 (d _br , .7 = 7.5 Hz, 1H, 7d'-H), 7.50-7.61 (m, 1 -H, 4d'-H).

¹³ C NMR (125 MHz, CD ₃ OD): δ = 14.32 (SOH ₃ ), 28.33 (C-2d), 30.71 (C-3c), 33.88, 35.80, 36 , 43, 38.03, 38.65 (C-2c, C-1 e, C-2e, C-2b, C-2a), 50.03 (C-1 c), 56.05, 56.26 , 56.49 (C-1 a, C-1 b, C-1 d), 63.95 (carborane-CH), 76.30 (carborane-C), 1 10.5 (C-3d '), 1 12.6 (C-7d '), 1 19.4, 120.0 (C-4d', C-6d '), 122.6 (C-5d'), 125.1 (C-2d ') , 128.0 (-Ph-C), 128.2 (C-3ad '), 129.6 (o-Ph-C), 130.4 (r / 7-Ph-C), 135.7 (C-7ad '), 138.0 (/ -Ph-C), 168.4, 170.1, 172.8, 173.7, 174.4 .

174.5 (6 C = O).

MS (ESI ^" , in EtOH): m / z (%): 841 (85) [/ W + EtOH-H] ^" , 825 (100) [M + CH ₂ OH] \

C ₃₄ H ₅₀ B ₁₀ N ₆ O ₇ S (795.0).

4) Cytotoxicity test

The influence of the peptide compound II- (3C, 4-C-Oicarba-c / osododecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl-L-phenylalaninamide 5 on the cell vitality was determined with an MTT -Test (LM Greene, JL Reade, CF Ware, J. Immunol. Meth. 1984, 70, 257-268; T, Mosmann, J. Immunol. Meth. 1983, 65, 55-63) on various cell lines. This colorimetric test is based on the irreversible reduction of the yellow tetrazolium salt 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to a dark blue water-insoluble formazan derivative by mitochondrial dehydrogenases.

To carry out the MTT test, the cells of the lines A549 (human bronchial carcinoma, Institute for Cell Biology, Essen, ATCC CCL 185), B-16 (murine melanoma), PancTu 1 (human pancreatic carcinoma, not for sale,

Institute of Oncology at the Georg-August-Universität Göttingen) and LoVo (human colon rectal adenocarcinoma, ATCC CGI 229) in 96-well microtiter plates (TC Microwell 96F from Nunc) and sown for 24 h at 37 ° C and 7.5% CO ₂ -Fumigation cultivated in air. The mixture was then incubated with several concentrations of the toxin in serum-free medium (Ultra Culture) for 24 h. DMSO served as a solubilizer, so that ultimately there was a concentration of 1% DMSO in the wells. After the following 5 day cultivation period, MTT reagent was added for 4 h and overnight solubilization solution. The optical density of the dye released after cell lysis is the proportion of living, metabolically active

Cells are directly proportional and can be measured in the photometer (Thermo max microplate reader from Molecular devices) at a wavelength of 580 nm. The absorption was determined using a photometer at 580 vs 650 nm. 4 shows the results of a measurement in tabular form. 5 shows the influence of / V- (3C, 4C-dicarba-c / oso-dodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl-L-phenylalaninamide 5 on cell vitality of the different cell lines is shown graphically. The effective dose of ED _{50 is} the concentration required for an effect on 50% of the cells.

With the ED ₅₀ values found (FIGS. 4 and 5), / ^ SC ^ C-dicarba-c / σsσ-dodecaboranyl (12) butylcarbonyl) -L-tryptophyl-L-methionyl-L-asparagyl-L-phenylalaninamide is clear less cytotoxic than other carboranes linked, for example, to DNA-binding indole units. Examples from the literature show ED _εo values in the range from 7.5 to 42.5 μM (LF Tietze, U. Griesbach, U. Bothe, H. Nakamura, Y. Yamamoto, ChemBioChem 2002, 3, 219-225) , In contrast, the ED ₅₀ values of the peptide compound according to the invention are in the range between 161-641 μm.

Claims

claims:

1 . A process for the preparation of a peptide compound for use in boron neutron capture therapy, comprising at least one oligopeptide which is a ligand of an endogenous receptor, in particular a membrane-bound receptor, a fragment or a derivative of this ligand, and at least one boron-containing component, characterized in that that the boron-containing component is linked peptically to the oligopeptide via a linker via a carborane carboxylic acid, boronic acid and / or thiocarborane carboxylic acid.

2. The method according to claim 1, characterized in that a free or exposed amino group of the oligopeptide is reacted with the carboxyl group of the carborane carboxylic acid, preferably 1 - (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC HCl) and 1- Hydroxybenzotriazole hydrate (HOBt-H ₂ 0) is used.

3. The method according to claim 2, characterized in that the carborane carboxylic acid is obtained from an alkyne carboxylic acid or an associated derivative.

4. The method according to claim 3, characterized in that the alkyne carboxylic acid is one with 3 to 20 carbon atoms, including alkyne carboxylic acids which carry alkyl groups with 1 to 10 carbon atoms.

5. The method according to claim 4, characterized in that the alkynecarboxylic acid is pentincarboxylic acid.

6. The method according to any one of claims 3 to 5, characterized in that as a derivative of the alkyne carboxylic acid an ester of an alcohol with 1 to 6 C-

Atoms or an amide is used.

7. Peptide compound obtainable according to one of claims 1 to 6.

8. A peptide compound for use in boron neutron capture therapy, comprising at least one oligopeptide which has a ligand of an endogenous receptor, in particular a membrane-bound receptor, a fragment or a derivative of this ligand and at least one boron-containing component, characterized in that the derivative of the peptide ligand is a gastrin derivative.

9. Peptide compound according to claim 8, characterized in that the derivative of the peptide of the ligand at least one of the amino acid sequences Trp-Met-Asp-Phe-NH ₂ (SEQ ID NO 1), Gly-Trp-Met-Asp-Phe-NH ₂ (SEQ ID NO 2) and / or Leu- (Glu) ₅ -Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH ₂ (Minigastrin) (SEQ ID NO 3).

10. Peptide compound according to one of the preceding claims, characterized in that the boron-containing component is a borane, carborane, thiocarborane, boronic acid, carborane carboxylic acid and / or thiocarborane carboxylic acid.

1 1. Peptide compound according to one of the preceding claims, characterized in that the boron-containing component is a carboranyl compound, in particular a carboranyl amino acid and / or a carboranyl glycoside.

12. Peptide compound according to one of the preceding claims, characterized in that the boron-containing peptide compound has a further component, in particular a cytotoxic component.

13. Use of a peptide compound according to any one of claims 7 to 12 for tumor treatment, the peptide compound being used in the context of boron neutron capture therapy.

14. Pharmaceutical composition, characterized in that it comprises a pharmaceutically effective amount of at least one peptide compound according to one of claims 7 to 12 and at least one pharmaceutically acceptable carrier.