DE19817517A1 - New benzylguanidine derivatives useful for diagnosis or therapy, especially of neuroblastoma - Google Patents

Abstract

Benzylguanidine derivatives (I) comprising a therapeutic drug molecule, a therapeutically activatable drug molecule, and a radioactive or fluorescent label are new. Benzylguanidine derivatives of formula (I) and their salts are new: L = NH, O, S, CH2 or a stable, acid-labile and/or enzymatically cleavable linker; m, n = 0 or 1; either: m+n = 1 or 2; R<1> = Q1; and R<2>-R<7> = Q2; or m+n = 1 or 2; R<2> = Q1; and R<1>, R<3>-R<7> = Q2; or m+n = 0; and R<1> and/or R<2> plus the aromatic structure of (I) is part of a therapeutic drug molecule, a therapeutically activatable drug molecule, a radioactive label or a fluorescent label with at least one absorption maximum between 400 and 1000 nm; Q1 = a therapeutic drug molecule, a therapeutically activatable drug molecule, a radioactive label or a fluorescent label with at least one absorption maximum between 400 and 1000 nm; Q2 = H, halogen, OH, COOH, NH2, NO2, 1-10C alkyl, 1-10C alkoxy, 2-11C alkoxycarbonyl, 1-10C alkylamino or di(1-10C alkyl)amino, where the alkyl groups are optionally interrupted by 0-5 O atoms and/or substituted with 0-2 fluoro, 0-2 chloro, 0-5 OH, 0-3 COOH, 0-3 ester and/or 0-3 amino groups, provided that m = 0 and n = 1 if R<1> = H and m = 1 and n = 0 if R<2> = H.

Description

The invention relates to new benzylguanidine derivatives for Therapy, in-vivo and in-vitro diagnostics, the use This he connections as therapeutics and diagnostics and diagnostic agents containing these compounds.

The neuroblastoma is a tumor of the sympathetic nerve systems, which in 75% of all patients in metastasis form and a poor prognosis having. Numerous new therapeutic approaches, such as the Use of interleukins and monoclonal antibodies or autologous bone marrow transplants are cli tried and tested. Groundbreaking improvements remained however so far from; Pinkerton, C.R., Neuroblastoma in the 1990's-A clinical perspective. In: Human Neuroblastoma (Eds .: M. Schwab, G.P. Tonini, J. Bernard) Harwood Academic Publishers, pp. 3-10 (1993).

One substance with which diagnostic and therapeutic tests have already been carried out is meta-iodine benzylguanidine (MIBG). This connection can be taken up and enriched by new roblastoma cells via the noradrenaline transporter and therefore basically opens up the possibility of a specific neuroblastoma diagnosis and therapy. The results with MIBG are still unsatisfactory, especially with regard to the detection and treatment of small tumor metastases (minimum residual disease); J. Farahiti et al., Scintigraphy of neu roblastoma with radiolabeled m-iodo benzylguanidine. In:
ST Travis: Pedriatic Nuclear Medicine, Ser.Ed. Springer (1995);
J. Cancer Res. Clin. Oncol. 1989, 115, 269-75; Med. Pregl. 1993, 46, Suppl1, 74-75; J. Nucl. Med. 1996, 37, 1464-68; Eur. J. Pediatr. 1997, 156, 610-15.

The neuroblastoma is principally chemotherapeutic tradable. There are substances suitable for therapy primarily cytostatic and antibiotic same structures (see B. A. Chabner, Anticancer Drugs. In: Cancer - Principles & Practice of Oncology, 4th edi tion, Lippincott). Chemotherapy drugs used are e.g. B. cyclophosphamide, cisplatin, epipodophyllotoxins, Vincristine, dacarbazine, melphalan, ifosfamide. The problem in principle, when using such cytostatics systemic toxicity and the related side effects kungen.

The invention is therefore based on the object, new Ver to provide bonds that have the disadvantages overcome the state of the art.

The object is achieved in that the Benzylguanidine structure with substances or structures is coupled, which itself (i) has a therapeutic effect show speed and (ii) as a signaling molecule for imaging diagnostic tests are suitable.

The object is achieved by creating compounds of the general formula I.

solved in what

• a) R ¹ or R ² stand for a therapeutic active substance, for a therapeutically activatable active substance, for a signal molecule for radio diagnosis or for a signal molecule for fluorescence diagnosis with at least one absorption maximum between 400 and 1000 nm,
  L represents -NH-, -O-, -S-, -CH ₂ - or a stable, acid-labile and / or enzymatically cleavable linker structure,
  m and n each represent 0 to 1,
  in the event that R ^{1 has} the meaning given under a) and the sum of m and n is at least 1,
• b) R ² to R ⁷ are independently hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, carboxy, amino, Ni tro, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy, C ₁ -C ₁₀ Alkoxycarbonyl, C ₁ -C ₁₀ -alkylamino, C ₁ -C ₁₀ - (dialkyl) amino, the alkyl radicals being interrupted independently of one another by up to 5 oxygen atoms and / or with 0-2 fluorine, 0-2 chlorine, 0-5 hydroxyl, 0-3 carboxy, 0-3 esters and / or 0-3 amino groups are substituted, and in the event that R ^{2 has} the meaning given under a) and the sum of m and n at least 1 is
  R ¹ and R ³ to R ^{7 have} the meaning given under b),
  m is 0 and n is 1 when R ^{1 is} hydrogen and
  m is 1 and n is 0 when R ^{2 is} hydrogen,
  and if m and n stand for zero,
• c) the aromatic structure of the general formula I together with at least one of the radicals R ¹ to R ^2, a partial structure of a therapeutic active substance, a partial structure of a therapeutically activatable active substance, a partial structure for a signaling molecule for radio diagnostics or a partial structure of a signaling molecule for means fluorescence diagnostics with the aforementioned absorption maximum,
  and their physiologically tolerable salts.

Compounds in which R ³ to R ^{7 are} hydrogen are preferred.

The compounds of the invention are suitable for imaging in vivo diagnostics, in vitro diagnostics and the therapy of neuroblastomas.  

Surprisingly, there is a therapeutic benefit achieved by that therapeutically effective molecules to the Benzylguanidine round structure are coupled.

Therapeutic active substances in the general formula (I) are, for example, cytostatic or antibiotic molecules, in particular the compounds listed below:
Antibiotics: aclacinomycin, Actinomycin F _1, anthramycin, azaserine, bleomycins, cactinomycin, Carubicin, Carzino philin, chromomycins, dactinomycin, daunorubicin, Doxoru bicine, epirubicin, Mtiomycine, mycophenolic acid, Nogalamy cin, olivomycins, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin , Tubercidin, zorubicin and derivatives of the compounds mentioned.

Folic acid analogues: denopterin, methotrexate, pteropterin, trimetrexate,
Pyrimidine analogues: ancitabine, azacitidine, 6-azauridine, Carmofur, cytarabine, doxifluridine, enocitabine, floxuri din, 5-fluoro-uracil,
Purine analogs: fludarabine, 6-mercaptopurine, thiamiprine, thioguanine and derivatives of the compounds mentioned, alkylating substances: alkylsulfonates, aziridines, ethylene lenimines, methylmelamines, nitroureas and nitrogen-containing compounds, such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifethamine, ifosfamide, M Mel phalan, Novembichin, Phenesterine, Prednimustin, Trofos famid, furthermore hormonally active substances, such as androgens, anti-adrenals, anti-androgens, antiestrogens, estrogens, LH-RH analogues and progestogens,
as well as other cytostatically active substances such as taxol and taxol derivatives, endiin substances such as calicheamycin and its derivatives, alkaloids such as camptothecin, 10-hydroxycamtothecin, (alkylamino) camptothecine and its derivatives (topotecan, Camptorar CPT-11), podophyllotoxins, especially epipodophyllotoxins, such as etoposide, teniposide and their derivatives.

Therapeutically activatable compounds of the general Formula I are compounds, the elements with high new tron capture cross section included, and photodynamic active connections.

Photodynamically active molecules are compounds from the Class of porphyrins, expanded porphyrins, chlorins, Benzochlorine, Bacteriochlorine, Phthalocyanine, Naph thalocyanine or bacteriochlorophyll. These molecules have the ability, after excitation with light cytotoxic effects due to the generation of Singu letts oxygen or radical reactive molecules unfold. The compounds according to the invention are a can be used in photodynamic therapy.

Other therapeutic agents are substances with a high neutron capture cross section for neutron capture therapy, preferably compounds that contain the element boron. Particularly preferred are BSH and boron phenylalanine and molecules containing 1 to 12 boron atoms, preferably compounds of general formula I, wherein R ¹ and / or R ² for -B (OH) ₂ , ortho-carboranyl, para-carboranyl, ortho -Carboranylalkyl, para-carboranylalkyl, and for phenylboronic acid, the linkage being ortho, meta or para to the (HO) ₂ B group, and

stand.

For diagnostics using highly sensitive detection systems drive, such as radio diagnostics and fluorescence slide gnostics, appropriate signaling molecules are sent to the ben coupled cylguanidine structure. It is guaranteed that the resulting conjugates are a benzylguanidine or MIBG have comparable binding affinity.

Surprisingly, it was found that after coupling of fluorescein isothiocyanate (FITC) to 4-aminobenzyl Conjugate obtained guanidine binds to the Has noradrenaline transporter.

Radiodiagnostics is a highly sensitive, imaging method that is widely used in tumor diagnostics. It includes the detection of γ-radiation-emitting nuclides (γ-scintigraphy, SPECT) and positron-emitting nuclides (PET). Since the use of MIBG described above has disadvantages, there is a need for alternative radio diagnostics. Of particular interest are diagnostically usable nuclides, such as ¹²³ I, ¹²⁵ I, ^99m Tc, ¹¹¹ In, and therapeutically usable nuclides, such as ¹³¹ I, ⁹⁰ Y, ^186/188 Re. The nuclide ^99m Tc is widely used diagnostically due to its short half-life (6 h) and easy availability (Tc generators).

According to the invention, new means for scintigraphic Diagnostics based on the benzylguanidine structure for  Provided that are excellent for the Her position of radio diagnostics suitable and a simple Allow handling in the clinic.

Signal molecules for radio diagnostics in general Formula I are therefore preferably nuclide-binding chelators, Peptides and complexing agents, the nuclides mentioned above complex.

The invention relates to compounds of the general formula I in which the signal molecule for radiodiagnostics R ¹ and / or R ² for a nuclide-binding chelator which is a mono- or bifunctional chelating N ₄ -, N ₃ S-, N ₂ S ₂ -, NOS ₂ or S ₄ structure which forms stable complexes with ^99m Tc and / or ^186/188 Re,
or a nuklidbindenden complexing agent which has a Po-lyamino Strukur polycarboxylic acid which forms stable complexes with ¹¹¹ In,
or a nuclide-binding peptide of the amino acid sequence Cys-Gly-Gly or Cys-Gly-Cys, which forms stable complexes with ^99m Tc and / or ^186/188 Re,
stand.

Examples of chelators are in Bioconjugate Chem. 1997, 8, 407-415, Bioconjugate Chem. 1997, 8, 621-636; Nucl. Med. Biol. 1991, 18, 589-603. The chelators are particularly suitable for labeling with ^99m Tc for radio ^diagnostics and ^186/188 Re for radiotherapy.

The following structures are preferred:
4-carboxyethylphenylgloxal-bis-N-methylthiosemicarbazone-N-hydroxysuccinimide ester,
6- (4'-isothiocyanatobenzyl) -3,3,9,9-tetramethyl-4,8-dia zaundecan-2,10-dione-dioxime,
2-methyl-2- (4-isothiocyanatobenzyl) -N, N'-propylene-bis-sa lycidenamine,
N, N'-bis - [(2-mercaptopyridyl) methyl] -2-methyl-2- (4-isothiocyanatobenzyl) -1,3-propanediamine,
N, N'-bis (benzoylthioacetyl) propionic acid,
N, N'-bis (benzoylthioacetyl) -3,4-diaminobutyric acid,
N, N'-bis (benzoylthioacetyl) -4,5-diaminopentanoic acid,
N, N'-1,2-ethylenedi-yl-bis (2-mercapto-1-carboxyethyla min).

Thioacetylglycylglycylglycine are particularly preferred as well as thioacetylglycylglycine and derivatives, the Thiol group carries a suitable protective group. Ge Protected thiol groups are, for example, S-triphenylme thyl- (trityl), S-diphenylmethyl- (benzhydryl), S-benzy loxy-carbonyl, S-tert.butyloxycarbonyl, S-acetamidomes thyl- (Acm), as well as mixed disulfides (e.g. -S-S- tert-butyl).

Another subject is nuclide-binding, cysteine-rich Amino acid sequences such as Cys (Acm) GlyCys (Acm), Cys (Acm) GlyGly, CysGlyCys, CysGlyGly.

Examples of complexing agents based on carboxyl groups are in Nucl. Med. Biol. 1991, 18, 589-603 be wrote.

Preferred are ethylenediaminetetraacetic acid (EDTA), the ethylenediaminopentaacetic acid (DTPA),
trans-1,2-cyclohexanediaminetetraacetic acid,
1,4,7,10-tetraazacyclododecanetetraacetic acid,
1,4,7,10-tetraazacyclododecanetriacetic acid,
1,4,7-triazacyclononetriacetic acid,
1,4,8,11-tetraazatetradecanetetraacetic acid,
1,5,9-triazacyclododecane triacetic acid, where the vinegar acid residues can partially be present as an amide or ester. They are particularly suitable for complexing ¹¹¹ In for radio diagnostics, but also for complexing Gd, which has a high neutron capture cross section and is suitable for neutron capture therapy (Jpn. J. Cancer Res. 1993, 84, 841-43 ).

Other signaling molecules for radio diagnostics in the general formula I are substances for positrons mission tomography (PET). These are particularly preferred Structures containing isotope F-18, or those containing F-18 can be marked.

Preferred signaling molecules for fluorescence diagnostics are fluorescent dyes with an absorption maximum between 600 and 1000 nm for the in vivo near infrared diagnostics (NIR). With NIR diagnostics, the high Permeability of biological tissue to light the Wavelength 650-1000 nm exploited and a fluorescence zenzfarbstoff as contrast medium in vivo based on its Absorption or fluorescence detected.

Furthermore, fluorescent dyes with an absorber tion maximum between 400 and 800 nm preferred. Which he Compounds according to the invention are suitable for the In-vi Tro-diagnostics of components to which this one spe have specific binding (analysis of components from Body fluids).

Preferred compounds of the general formula I according to the invention are therefore characterized in that R ¹ and / or R ² for a xanthine, fluorescein, rhodamine, oxazine or phenoxazine, thiazine or phenothiazine, cyanine, oxonol, Merocyanine, styryl, Squarilium or Croconium dye is available.

L stands for -NH-, -O-, -S-, -CH ₂ - or for a carbon linker structure, the esters, ethers, amides, secondary or tertiary amines, ketones, thiourea. May contain urea, carbamate or maleimido groups. The invention further relates to compounds of the general formula (I) in which the benzylguanidine structure is linked to the radicals R ¹ and / or R ² via a physiologically cleavable bond. The chemical bond, which is contained in the linker structure L according to the general formula I, is structurally such that it is split at certain physiological parameters by which diseased tissue (tumors) are characterized and which differ from normal tissue areas. Preference is given to those compounds in which R ¹ and / or R ^{2 is} a therapeutically active molecule. After the compounds according to the invention have been taken up into the cells (lysosomes), they are exposed to reduced pH values (up to pH 5). A bond that is cleaved under acidic conditions causes the active ingredient to be released, with the development or increase in its cytostatic or antibiotic activity (“pro-drug” effect). According to the invention, L therefore stands for a linker structure which has an acid-labile bond, preferably an alkylhydrazone, acylhydrazone, arylhydrazone, sulfonylhydrazone, imine, oxime, acetal, ketal, orthoester corresponding to the fragments

or
where p represents a number between 2 and 4.

The cleavage of the compounds according to the invention can ne ben also the cleavage due to lower pH values through enzymes with which neuroblastomas in increased concentrations tration are carried out.

The invention therefore furthermore relates to compounds with linker structures L, which are cleaved enzymatically. Enzymatically cleavable linker structures are one example wise those caused by cathepsins, peptidases, carboxy peptidases, α- and β-glucosidases, lipases, oxidases, Phospholipases, phosphatases, phosphodiesterases, protea sen, elastases, sulfatases, reductases, transferases and bacterial enzymes, for example penicillin amidases, β-lactamases are cleaved.  

Peptides and short-chain amino acid sequences are preferred zen that are cleaved enzymatically. (P. D. Senter et al., Bioconjugate Chem. 6 (1995), 389-94). Preferred s zymatically cleavable structures are short-chain peptides sequences, such as sequences that encompass the amino acid contain sequence Val-Leu-Lys.

The synthesis of the compounds is preferably carried out by ge targeted representation of the radicals in the general formula I for R ¹ and R ² and coupling these to benzylguanidine. The structure L is optionally coupled either to the benzylguanidine structure or R ¹ or R ² . The compounds are particularly advantageously synthesized from 3- or 4-aminobenzylguanidine or N, N-di-Boc-3- or N, N-di-Boc-4-aminobenzylguanidine.

The invention therefore also relates to a method for producing
Compounds of the general formula I

wherein

• a) R ¹ or R ² stand for a therapeutic active substance, for a therapeutically activatable active substance, for a signal molecule for radio diagnosis or for a signal molecule for fluorescence diagnosis with at least one absorption maximum between 400 and 1000 nm,
  L represents -NH-, -O-, -S-, -CH ₂ - or a stable, acid-labile and / or enzymatically cleavable linker structure,
  m and n each represent 0 to 1,
  in the event that R ^{1 has} the meaning given under a) and the sum of m and n is at least 1,
• b) R ² to R ⁷ are independently hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, carboxy, amino, Ni tro, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy, C ₁ -C ₁₀ Alkoxycarbonyl, C ₁ -C ₁₀ -alkylamino, C ₁ -C ₁₀ - (dialkyl) amino, the alkyl radicals being interrupted independently of one another by up to 5 oxygen atoms and / or with 0-2 fluorine, 0-2 chlorine, 0-5 hydroxyl, 0-3 carboxy, 0-3 esters and / or 0-3 amino groups are substituted,
  and where R ^{2 has} the meaning given under a) and the sum of m and n is at least 1,
  R ¹ and R ³ to R ^{7 have} the meaning given under b),
  m is 0 and n is 1 when R ^{1 is} hydrogen and
  m is 1 and n is 0 when R ^{2 is} hydrogen,
  and if m and n stand for zero,
• c) the aromatic structure of the general formula I together with at least one of the radicals R ¹ to R ^2, a partial structure of a therapeutic active substance, a partial structure of a therapeutically activatable active substance, a partial structure for a signaling molecule for radio diagnostics or a partial structure of a signaling molecule for means fluorescence diagnostics with the aforementioned absorption maximum,
  and their physiologically tolerable salts,
  characterized in that
  • a) 3- or 4-aminobenzylamine is reacted with N, N'-di-Boc-S-methyl isothiourea to form N, N'-di-Boc-3- or N, N'-di-Boc-4-aminobenzylguanidine, then the 3- or 4-amino group with one of the structures mentioned for R ¹ and R ² , which is to contain a carboxylic acid group, is reacted by activating the carboxylic acid group to form an amide linkage, and the Boc protective groups are cleaved by acid to release the benzylguanidine structure , or
  • b) 3- or 4-aminobenzylamine is reacted with N, N'-di-Boc-S-methyl isothiourea to form N, N'-di-Boc-3- or N, N'-di-Boc-4-aminobenzylguanidine, then the 3- or 4-amino group is converted to the isothiocyanate, is reacted with one of the structures mentioned for R ¹ and R ² , which is intended to contain an amino group, and the Boc protective groups are cleaved by acid with the liberation of the benzylguanidine structure.

The therapeutic agents are synthesized according to the subject known methods.

literature

Chem. Rev. 1994, 94, 1553-1566 J. Med. Chem. 1986, 29, 1703-9 / 1988, 31, 1326-31 / 1990, 33, 1177-86 / 1991, 34, 98-107 / 1993, 36, 2689- 2700/1996, 38, 395-401 / 1996, 39, 713-19 / 1997, 40, 216-225.

The synthesis of chelators, complexing agents and peptides, and the labeling with radionuclides follows the methods known to the person skilled in the art.

literature

Bioconjugate Chem. 1997, 8, 407-415
Bioconjugate Chem. 1997, 8, 621-636
Nucl. Med. Biol. 1991, 18, 589-603
Nucl. Med. Biol. 1997, 24, 485-498
US 4897255, US 5011916, US 5225180, US 4444690, US 5620675;
EP 279417, EP 0248506, EP 0306168, EP 0417870, EP 0512661;
WO 91/17173.
The dyes are synthesized by the processes known to the person skilled in the art.

literature

FM Hamer in The Cyanine Dyes and Related Compounds, John Wiley and Sons, New York, 1964;
J. Fabian et al., Chem. Rev. 92 (1992) 1197;
LA Ernst et al., Cytometry 10 (1989) 3-10;
Pl. L. Southwick et al., Cytometry 11 (1990) 418-430;
RB Mujumdar et al., Bioconjugate Chem. 4 (1993) 105-11;
E. Terpetnig et al., Anal. Biochem. 217 (1994) 197-204;
JS Lindsey et al., Tetrahedron 45 (1989) 4845-66, EP-0591820 A1;
Strekowski, L. et al., J. Heterocycl. Chem. 33 (1996) 1685-1688;
SR Mujumdar et al., Bioconjugate Chem. 7 (1996) 356-362;
M. Lipowska et al., Synth. Commun. 23 (1993) 3087-94;
E. Terpetschnig et al., Anal. Chim. Acta 282 (1993) 633-641;
M. Matsuoka and T. Kitao, Dyes Pigm. 10: 13-22 (1988) and
N. Narayanan and G. Patonay, I. Org. Chem. 60 (1995) 2361-95.
Acid-labile bonds are generated based on the following examples of literature.
BM Mueller et al., Bioconjugate Chem. 1 (1990) 325-330;
K. Srinivasachar and DM Neville, Biochemistry 28 (1989) 2501-09;
DM Neville et al., J. Biol. Chem. 264 (1989) 14653-61;
T. Kaneko et al., Bioconjugate Chem. 2 (1991), 133-41;
BA Froesch et al., Cancer Immunol. Immunother. 42: 55-63 (1996) and
JV Crivello et al., J. Polymer Sci: Part A: Polymer Chem. 34 (1996) 3091-3102.

Another object of the invention is the use the verb containing the therapeutic agents for the therapy of neuroblastomas.

Subject is also the use of the described radioactive compounds as in vivo radio diagnostics and in vivo radiotherapeutics. The subject is also the Use of the dye-containing compounds for the In vivo diagnosis of diseased tissue using near infra red (NIR) radiation by detection of the non-absorbed Radiation and / or fluorescent radiation, as well as for the in vitro diagnostics of components from body fluids liquids such as blood, urine, cerebrospinal fluid, lymph by detection the radiation not absorbed and / or the fluorine escence radiation (inter alia by means of FACS = "fluorescence activated cell sorting ").

The compounds of the invention are also ge is suitable for the diagnosis and therapy of tumors and others their type and other origin.

Examples are neuroendocrine tumors (e.g. pheochromo cytomas; A. Lau et al., Clinical Nuclear Med. 1996, 21, 316-318, "Glomus jugulare tumors", E. Nilssen, J. Laryngol. Otol. 1996, 110, 373-375), as well as small ones intestinal carcinoids (S. Dresel et al., Nuclear Medicine 1994, 35, 53-58), tumors of the adrenal cortex (D.A. Sloan et al., Curr. Opin. Oncol. 1996, 8, 30-36), and secondary neuroendocrine tumors of the liver (J.K. Ramage et al., QLM 1996, 89, 539-542).

The special advantage of therapy and diagnostics with the Compounds according to the invention is that a Admission into the diseased cells takes place at the same time tiger faster excretion from the healthy residual body due to the hydrophilicity and the low molecular weight  weight of the connections. Especially compared to An antibodies as carrier molecules (e.g. EP 282057), the very systemic is long circulation times Reduced toxicity.

These agents are according to the Me methods, if necessary using conventional auxiliary and / or carriers as well as diluents and the same. These include physiologically compatible electr trolytes, buffers, detergents and substances for adaptation solution of the osmolarity and to improve the stabilization activity and solubility. Because of the use in pharmacy measures for the sterility of the preparations during manufacture and especially before application to care.

The following examples illustrate the invention.

Examples 1 and 2 Representation of fluorescent dye-benzylguanidine conjugates ( FIG. 1) example 1 Illustration of the fluorescein-benzylguanidine conjugate 3

1.1. Preparation of 4-aminobenzylguanidine sulfate 2
2.5 g (20.5 mmol) of 4-aminobenzylamine 1 and 6.0 g (21.5 mmol) of S-methylisothiourea sulfate are stirred in 10 ml of water for 48 h at room temperature. The reaction mixture is concentrated several times with repeated addition of water on a rotary evaporator until the methane thiol odor in the residue has largely subsided. The residue is stirred with 3 ml of water, the product crystallized out at 4 ° C. and recrystallized from water. After drying under high vacuum, 4.5 g (62%) of 4-aminobenzylguanidine sulfate 2 are obtained .

1.2 Coupling of 2 with fluorescein isothiocyanate (FITC) to conjugate 3
0.3 g (0.76 mmol) of fluorescein-5-isothiocyanate in 5 ml of DMF are added to a solution of 0.2 g (0.76 mmol) of 2 (see 1.1) in 5 ml of phosphate buffer (50 mM, pH 8) given. It gets 5 h at room temp. stirred and the reaction mixture lyophilized. Chromatographic purification (RP-18 Euro prep., Eluent H ₂ O + 5% CH ₃ COOH / MeOH) gives 0.15 g fluorescein-aminobenzylguanidine 3 .

Example 2 Coupling of 4-aminobenzylguanidine sulfate 1 with bis-1,1 '- (4-sulfobutyl) indotricarbocyanine-5-carboxylic acid-N-hydroxysuccinimidyl ester 4 to conjugate 5

The dye 4 is produced based on methods known from the literature.

0.1 g (0.12 mmol) 4 and 0.05 g (0.1 8 mmol) 1 are in 3 ml phosphate buffer (50 mM, pH 8) and 3 ml DMF for 5 h at room temp. coupled. After addition of diethyl ether, the precipitated crude product is purified by chromatography (RP-18, Europrep., Mobile phase H ₂ O + 1% CH ₃ COOH / 10% MeOH), yield 23 mg (20%) deep blue lyophilisate.

Examples 3 to 6 Preparation of conjugates from benzylguanidine and zyto structurally effective structures Example 3 Synthesis of p-di- (2-chloroethyl) aminobenzylguanidine 9

The connection is a structural mix of the therapeutically active, alkylating part of melphalan and the benzylguanidine.

The synthesis is carried out from p-di- (2-chloroethyl) aminobenzylamine 6 (J. Org. Chem. USSR (Engl. Transl.) 4 (1968) 578-581) and N, N'-di-Boc-S- methyl isothiourea 7 (EP 0 672 658 A1).

1.4 g (4.8 mmol) 7 are added to a suspension of 1.0 g (4.0 mmol) 6 in 30 ml DMF / water (1: 1) and the mixture is stirred at 40 ° C. for 24 h. After adding 100 ml of water, the aqueous phase is extracted three times with CH ₂ Cl ₂ . The combined organic phases are saturated with sat. Washed NaCl solution, dried over NaSO ₄ and concentrated. After chromatographic purification (Kieselgel 60, Merck, eluent CH ₂ Cl ₂ / hexane 1: 1 → 9: 1), 0.74 g (38%) of N, N'-di-Boc-p-Di- (2-chloroethyl) aminobenzylguanidine 8 as a white solid.

0.74 g (1.5 mmol) 8 are in 7 ml CH ₂ Cl ₂ and 0.5 ml trifluoroacetic acid for 20 h at room temperature. touched. After evaporation of the solvent, chromatographic purification is carried out (Kieselgel 60, Merck, mobile solvent CH ₂ Cl ₂ / methanol 9: 1). 0.45 g (74%) 9 is obtained as a white powder, mp. 120-123 ° C (dec.).

Elemental analysis:
calculated for C ₁₄ H ₁₉ N ₄ Cl ₂ O ₂ F ₃ : C 41.72; H 4.75; N 13.89;
found: C41.62; H 4.70; N 14.02.

Example 4 Synthesis of chlorambucil-benzylguanidine conjugate 12 p- [N- (5- (p-di- (2-chloroethyl) aminophenyl) pentyloxy) amino] benzylguanidine

The synthesis is carried out from chlorambucil and N, N'-di-Boc-p-aminobenzylguanidine 10 , which is obtained by reacting 1 with 7 (see Example 3).

1.0 g (8.2 mmol) of 4-aminobenzylamine 1 are placed in a mixture of 12 ml of DMF, 12 ml of water and 4 ml of CH ₂ Cl _{2 and} stirred with 3.6 g (12.3 mmol) fixed 7 ver sets. You leave 24 h at room temp. stir, dilute with 100 ml water and extract four times with CH ₂ Cl ₂ . The crude product obtained after washing, drying (NaSO ₄ ) and concentrating the organic phases is purified by chromatography as described in Example 3 (mobile phase CH ₂ Cl ₂ : hexane 1: 1 → CH ₂ Cl ₂ ), yield 1.4 g (47 %) 10 as a white powder.

0.1 g (0.33 mmol) chlorambucil in 10 ml DMF and 36 mg (0.36 mmol) triethylamine are mixed with (0.36 mmol) TBTU while cooling with ice. After stirring for 15 min, 0.14 g (0.39 mmol) of 10 in 2 ml of DMF is added dropwise and the mixture is stirred at room temperature for 18 h. After adding water (50 ml), the mixture is extracted three times with CH ₂ Cl ₂ , the organic phase with sat. Washed NaCl solution and dried over NaSO ₄ . The crude product 11 (0.18 g) is in 5 ml CH ₂ Cl ₂ and 0.5 ml trifluoroacetic acid for 18 h at room temperature. touched. After a narrow and chromatographic purification (Kieselgel 60, Merck, eluent CH ₂ Cl ₂ / methanol 9: 1), 0.15 g (78%) 12 is obtained as a white powder.

Elemental analysis:
calculated for C ₂₄ H ₃₀ N ₅ C ₁₂ O ₃ F ₃ Cl ₂ : C 51.07; H 5.36; N 12.41;
found: C 51.20; H 5.40; N 12.39.

Example 5 Synthesis of 9-guanidinomethyl camptothecin acecat 14

The compound combines the benzylguanidine structure and the Camptothecin structure.

The synthesis takes place from 9-aminomethylcamptothecin 13 (J. Med. Chem. 34 (1991) 98-107).

50 mg (0.13 mmol) 13 and 53 mg (0.18 mmol) 7 are stirred in 3 ml DMF / water (1: 1) at 40 ° C for 24 h. The processing is carried out as described in Example 2.1. The Rohpro is domestic product in 1 ml CH ₂ Cl _2/50 ul trifluoroacetic acid for 18 h at room temp. stirred, concentrated and purified by chromatography (RP-18 Europrep., mobile phase H ₂ O + 1% CH ₃ COOH / 10% MeOH), yield 42 mg (67%) 14 as a yellowish lyophilizate.

Elemental analysis:
calculated for C ₂₂ H ₂₁ N ₅ O ₄ .CH ₃ COOH: C 60.12; H 5.26; N 14.61;
found: C 59.81; H 5.20; N 14.33.

Example 6 Synthesis of the methotrexate γ-benzylguanidine derivative 18

The synthesis is carried out by reacting 4- (N- [2,4-diamino-6-pteridinylmethyl] -N-methylamino) benzoic acid 15 with HD-Lys (Boc) -OtBu (based on Biochem. 30 (1991) 5674 ), whose γ-amino group is released by cleaving the protective group to obtain 16 . The coupling then takes place to this amino group by reaction with N, N'-di-Boc-p-isothiocyanatobenzylguanidine 17 , which is obtained from 10 , to give product 18 .

6.1. Synthesis of 16

0.1 g (0.31 mmol) 15 , 0.24 g (0.70 mmol) HD-tys (Boc) - OtBu and 0.14 ml (1.0 mmol) triethylamine are dissolved in 5 ml anhydrous DMF under argon dissolved and at 0 ° C with a solution of 0.36 g (1.0 mmol) of diethyl phosphorus cyanidate in 1 ml of DMF. The mixture is 2 h at 0 ° C and 16 h at room temperature. touched. After removal of the solution by means of a vacuum, the mixture is stirred with water, the precipitated solid is filtered off and dried in vacuo. After chromatographic purification (Kieselgel 60, Merck, eluent CH ₂ Cl ₂ / methanol 95: 5 to 5: 1), 0.15 g of solid is obtained, which is dissolved in 10 ml of CH ₂ Cl ₂ and 2 ml of trifluoroacetic acid for 3 hours at room temperature . is stirred. After evaporation of the solvent, the product 16 is stirred with 20 ml of diethyl ether, filtered off and dried in vacuo; Yield 0.12 g (70%) 16 as a yellow solid.

6.2. Synthesis of N, N'-di-Boc-p-isothiocyanatobenzyl-guanidine 17 from 10

To a solution of 0.2 g (0.55 mmol) 10 and 0.15 ml (1.1 mmol) triethylamine in 5 ml CHCl ₃ 0.13 g (0.55 mmol) 1,1'-thiocarbonyl- 2,2'-pyridone added dropwise in 2 ml of CHCl ₃ . The reaction mixture is 16 h at room temperature. stirred, the solvent evaporated and the residue purified by chromatography (silica gel 60, Merck, mobile solvent CH ₂ Cl ₂ / methanol 99: 1), yield 0.13 g (58%) 17 as a brownish solid.

6.3. Synthesis of 18

0.1 g (0.24 mmol) of isothiocyanate 17 in 5 ml of DMF is added to a solution of 0.12 g (0.22 mmol) of 16 in 10 ml of DMF and 1 ml of triethylamine, for 5 hours at room temperature. stirred, the solvent evaporated in vacuo and the crude product precipitated by adding ether / hexane (1: 1) and filtered. To release the benzylguanidine group, the crude product in 10 ml of CH ₂ Cl ₂ and 2 ml of trifluoroacetic acid is at room temperature for 3 h. stirred and processed and cleaned analogously to Example 5; Yield 0.14 g (88%) 18 as yellow crystals.

Elemental analysis:
calculated for C ₃₀ H ₃₇ N ₁₃ O ₃ S.CH ₃ COOH: C 50.06; H 5.74; N, 25.30;
found: C 49.90; H 5.78; N 25.50.

Example 7 Presentation of acid labile conjugates from benzylguani din and cytostatic structures Synthesis of a Doxorubicin-hydrazono-isothiocyanato-benzylguanidine ( 22 )

The synthesis takes place by linking N, N'-di-Boc-p-isothiocyanatobenzylguanidin 17 with 4- (aminomethyl) - benzoic acid hydrazide 18 to the intermediate 20 and reaction of the hydrazide group with the keto group of 3'-deamino-3 '- (4- morpholinyl) doxorubicin 21 to product 22 to form an acid labile hydrazone bond.

7.1. Synthesis of 20

0.2 g (0.48 mmol) 17 in 5 ml DMF is added to a solution of 0.12 g (0.44 mmol) 18 in 10 ml DMF and 1 ml triethylamine, 5 h at room temperature. stirred, the solvent evaporated in vacuo, the crude product precipitated by adding ether / hexane (1: 1) and filtered off. To release the benzylguanidine group, the crude product in 10 ml of CH ₂ Cl ₂ and 2 ml of trifluoroacetic acid is kept at room temperature for 3 hours. stirred and worked up analogously to Example 6.1, from prey 0.17 g (80%) 20 as a brownish powder.

7.2. Generation of the acid-labile bond to the product 22 0.05 g (0.1 mmol) 20 and 0.05 g (0.08 mmol) of morpholino doxorubicin 21 are mixed in 1 ml of anhydrous methanol with 10 μl of a 5% methanolic trifluoroacetic acid solution and 48 h at room temp. touched. The product is crystallized by dropwise addition of diethyl ether until turbidity and storage at -20 ° C., yield 0.06 g (69%) 22 as red crystals, UV-VIS (H ₂ O): λ _max 530.492 nm .

Examples 8 to 11 Presentation of nuclide-binding chelator-benzylguanidine Conjugates Example 8 Synthesis of N- [3 - [(S-benzoyl-thioacetylglycylglycylglycylglycyl) amino] propyl] benzylguanidine-4-carboxamide 27

The synthesis takes place starting from 4-aminobenzoic acid 23 , which is nidinylated with 7 to N, N'-di-boc-4-carboxybenzylguanidine 24 . Linking with mono-Boc-diaminopropane, release of the amino group and reaction with S-benzoyl thioacetyl-Gly-Gly-Gly-COOH 26 gives the product 27 .

8.1. Synthesis of 24

The presentation is analogous to the synthesis of 8 as described in Example 3. The cleaning is carried out chromatographically (Kieselgel 60, Merck, eluent CH ₂ Cl ₂ / methanol 9: 1 → 8: 2). 1.1 g (42%) of N, N'-di-boc-4-carboxybenzylguanidine 24 are obtained from 1.0 g (6.6 mmol) of 23 .

8.2. Synthesis of 25

1.0 g (2.5 mmol) of 24 and 0.35 ml (2.5 mmol) of triethylamine are in 10 ml of DMF at room temperature. mixed with 0.8 g (2.5 mmol) of TBTU and 15 min. touched. After adding a solution of 0.5 g (2.9 mmol) of mono-N-boc-diaminopropane in 10 ml of DMF, the mixture is stirred at room temperature for 3 h. stirred, the mixture concentrated to about 5 ml, mixed with 30 ml of water and the aqueous phase extracted three times with CH ₂ Cl ₂ . After drying over MgSO ₄ and concentration, the Boc protective groups are cleaved directly by stirring in 5 ml of CH ₂ Cl ₂ / 2.5 ml of trifluoroacetic acid for 12 h. After evaporation of the solvents in vacuo and stirring with diethyl ether, a brown-yellow powder is obtained which is purified by chromatography (RP-18 Europrep, eluent H ₂ O + 2% CH ₃ COOH / 5% MeOH), yield 0.4 g (40% , calculated as diacetate) 25 as a yellowish lyophilisate.

8.3. Synthesis of the chelator-benzylguanidine derivative 27

A solution of 0.1 g (0.27 mmol) of 26 and 35 mg (0.3 mmol) of N-hydroxysuccinimide in 8 ml of DMF is at -10 ° C with 62 mg (0.3 mmol) of DCC in 2 ml of DMF transferred. After 4 h stirring at room temp. a solution of 0.1 g (0.27 mmol) of 25 and 80 µl (0.55 mmol) of triethylamine in 2 ml of DMF is added and the mixture is heated at room temperature for 24 h. touched. The mixture is cooled to -20 ° C., the precipitated dicyclohexylurea is filtered off, the mixture is concentrated to a minimum in a vacuum and the residue is stirred with diethyl ether. The resulting solid is filtered off and purified by preparative HPLC (Nucleosil C-18 7 μm, gradient H ₂ O + 2% CH ₃ COOH / MeOH). 27 is obtained as a yellowish powder.

Example 9 Synthesis of N- [3- (N, N'-bis (S-benzoylthioacetyl) -3,4-dia minobutyryl) aminopropyl] benzylguanidine-4-carboxamide 29

The synthesis is carried out analogously to the synthesis described in Example 8, using N, N'-bis (S-benzoylthioacetyl) -3,4-diaminobutyric acid 28 . The last stage is based on 0.1 g (0.22 mmol) 28 and 0.08 g (0.22 mmol) 25 . 29 is obtained as a yellowish powder.

Example 10 ^99m Tc complex of 27 and 29 respectively

0.5 mg 27 and 29 are dissolved in 0.5 ml phosphate buffer (100 mM Na ₂ HPO ₄ , pH 9.5) and with 50 µl trisodium citrate dihydrate solution (150 mM) and 2.5 µl tin (II) chloride dihydrate (200 mM in 0.05 N HCl) was added. Then 100 µl pertechnetate solution (from a ⁹⁹ Mo / ^99m Tc generator, 400-900 µCi) is added, 15 min at room temperature. in cubed and filtered (0.2 µ filter). The labeling is analyzed by HPLC (Nucleosil, 125 × 4 cm, 5 µm, eluent A: phosphate buffer (10 mM, pH 4), eluent B: acetonitrile / phosphate buffer (75: 25), 100% A after 100% B within of 10 min). The radiochemical purity for both compounds is <95%.

Example 11 Synthesis of N- [3- [Cys (Acm) -Gly-Cys (Acm)] aminopropyl] benzylguanidine-4-carboxamide 31

The synthesis is carried out as described in Example 8. The last stage is based on 0.16 g (0.25 mmol) of Fmoc-Cys (Acm) -Gly-Cys (Acm) 30 and 0.1 g (0.27 mmol) 25 . The crude product is split off in 2 ml of DMF and 0.1 ml of piperidine for 30 min at room temperature. stirred, then the solid precipitated with diethyl ether as in Example 8.3 purified. 31 is obtained as a yellowish powder.

Example 12 Binding of FITC-benzylguanidine 3 to neuroblastoma cells

FACS analysis of LS neuroblastoma cells.
Left: control; Right: after incubation with 3 .

Claims (13)

1. Compounds of the general formula I
wherein

• a) R ¹ or R ² stand for a therapeutic active substance, for a therapeutically activatable active substance, for a signal molecule for radio diagnosis or for a signal molecule for fluorescence diagnosis with at least one absorption maximum between 400 and 1000 nm,
  L represents -NH-, -O-, -S-, -CH ₂ - or a stable, acid-labile and / or enzymatically cleavable linker structure,
  m and n each represent 0 to 1,
  in the event that R ^{1 has} the meaning given under a) and the sum of m and n is at least 1,
• b) R ² to R ⁷ are independently hydrogen, fluorine, chlorine, bromine, iodine, hydroxy, carboxy, amino, Ni tro, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkoxy, C ₁ -C ₁₀ Alkoxycarbonyl, C ₁ -C ₁₀ -alkylamino, C ₁ -C ₁₀ - (dialkyl) amino, the alkyl radicals being interrupted independently of one another by up to 5 oxygen atoms and / or with 0-2 fluorine, 0-2 chlorine, 0-5 hydroxyl, 0-3 carboxy, 0-3 esters and / or 0-3 amino groups are substituted,
  and where R ^{2 has} the meaning given under a) and the sum of m and n is at least 1,
  R ¹ and R ³ to R ^{7 have} the meaning given under b),
  m is 0 and n is 1 when R ^{1 is} hydrogen and
  m is 1 and n is 0 when R is hydrogen, and in the event that m and n are zero,
• c) the aromatic structure of the general formula I together with at least one of the radicals R ¹ to R ^2, a partial structure of a therapeutic active substance, a partial structure of a therapeutically activatable active substance, a partial structure for a signaling molecule for radio diagnostics or a partial structure of a signaling molecule for means fluorescence diagnostics with the aforementioned absorption maximum,
  and their physiologically tolerable salts.

2. Compounds according to claim 1, characterized in that the therapeutic agent R ¹ and / or R ^{2 is} an antibiotic or cytostatic molecule or active fragments of these molecules. 3. Compounds according to any one of the preceding claims, characterized in that the therapeutic agent R ¹ and / or R ² for a cytostatically active molecule from the class of alkylating cytostatics, in particular compounds containing di- (2-chloroethyl) amino structures, the Folic acid analogs, in particular methotrexate and its derivatives, the alkaloids, in particular camptothecins or the podophyllotoxins, in particular epipodophyl lotoxine. 4. Compounds according to claim 1, characterized in that the therapeutically activatable active substance R ¹ and / or R ² for ortho-carboranyl, para-carboranyl, ortho-carboranylalkyl, para-carboranylalkyl, and for phenylboronic acid, the linkage being ortho-, meta - or para-to the (HO) ₂ B group, or stands for (HO) ₂ B-. 5. Compounds according to claim 1, characterized in that the therapeutically activatable active substance R ¹ and / or R ^{2 represents} a photodynamically active molecule from the class of the porphyrins, chlorines, Ben zochlorine, bacteriochlorins, phthalocyanines, naphthalocyanines or bacteriochlorophylls. 6. Compounds according to claim 1, characterized in that the signal molecule for radio diagnostics R ¹ and / or R ² for a nuclide-binding chelator, which is a mono- or bifunctional chelating N ₄ -, N ₃ S-, N ₂ S ₂ -, NOS ₂ or S ₄ structure which forms stable complexes with ^99m Tc and / or ^186/188 Re,
or a nuklidbindenden complexing agent which has a Po-lyamino Strukur polycarboxylic acid which forms stable complexes with ¹¹¹ In,
or a nuclide-binding peptide of the amino acid sequence Cys-Gly-Gly or Cys-Gly-Cys, which forms stable complexes with ^99m Tc and / or ^186/188 Re,
stands. 7. Compounds according to claim 1, characterized in that the signal molecule for radiodiagnostics R ¹ and / or R ^{2 stands} for a structural element containing fluorine-18. 8. Compounds according to claim 1, characterized in that the signal molecule for fluorescence diagnostics R ¹ and / or R ² for a xanthine, fluorescein, rhodamine, oxazine or phenoxazine, thiazine or phenothiazine, cyanine, oxonol -, Merocyanine, Sty ryl, Squarilium or Croconium dye. 9. Connections according to at least one of the preceding Claims, characterized in that L for a Hydrocarbon linker structure, the esters, ethers, Amides, secondary or tertiary amines, ketones, thio urea, urea, carbamate or maleimido group pen may contain. 10. Compounds according to at least one of the preceding claims, characterized in that in the general formula (I) L stands for a structure which is an acid-labile fragment
or
where p represents a number between 2 and 4. 11. Connections according to at least one of the previous ones Claims, characterized in that L for a Structure stands, which is an enzymatically cleavable contains chemical bond by Kathepsine, Pep tidases, carboxypeptidases, α- and β-glycosidases, Lipases, phospholipases, phosphatases, phosphody sterases, proteases, elastases, sulfatases, reducta and bacterial enzymes is broken down. 12. Use of compounds according to claim 1 for In vivo diagnosis of diseased tissue areas, for in vitro diagnostics of tissue components and for Therapy of diseased tissue areas. 13. Diagnostic agent for in vivo diagnosis of diseased Tissue areas, for the in vitro diagnosis of Tissue components as well as for therapy of diseased Tissue areas, characterized in that it at least one compound according to claim 1 together with the usual auxiliaries and / or carriers as well Contains diluents.