WO1999052861A1

WO1999052861A1 - Novel benzyl guanidine derivatives for therapy and in-vivo and in-vitro diagnosis

Info

Publication number: WO1999052861A1
Application number: PCT/DE1999/000377
Authority: WO
Inventors: Kai Licha; Wolfgang Wrasidlo
Original assignee: Institut für Diagnostikforschung GmbH an der Freien Universität Berlin
Priority date: 1998-04-09
Filing date: 1999-02-05
Publication date: 1999-10-21
Also published as: AU3406599A

Abstract

The invention relates to novel benzyl guanidine derivatives for therapy and in-vivo and in-vitro diagnosis. The invention also relates to the use of these compounds as therapeutic agents and diagnostic agents, especially for treating and diagnosing neuroblastomas. In addition, the invention relates to the diagnostic agents which contain the inventive compounds.

Description

description

New benzylguanidine derivatives for therapy, in vivo and in vitro diagnostics

The invention relates to new benzylguanidine derivatives for therapy, in vivo and in vitro diagnostics, the use of these compounds as therapeutic agents and diagnostic agents and diagnostic agents containing these compounds.

The neuroblastoma is a tumor of the sympathetic nervous system that is found in 75% of all patients in the metastatic form and has a poor prognosis. Numerous new therapeutic approaches, such as the

The use of interleukins and monoclonal antibodies or autologous bone marrow transplants are being clinically tested. However, pioneering improvements have so far not been made; Pinkerton, C.R., Neuroblasto a 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-iodobenzylguanidine (MIBG). This connection can be taken up and enriched by neuroblastoma cells via the noradrenaline transporter and therefore basically opens up the possibility of specific neuroblastoma diagnosis and therapy. The results with MIBG are particularly unsatisfactory with regard to the detection and treatment of small tumor metastases (minimum residual disease); J. Farahiti et al. , scintigraphy of new roblasto a 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, Suppll, 74-75; J. Nucl. Med. 1996, 37, 1464-68; Eur. J. Pediatr. 1997, 156, 610-15.

In principle, the neuroblastoma can be treated chemotherapy. Substances suitable for therapy are primarily cytostatic and antibiotic structures (see B. A. Chabner, Anticancer Drugs. In: Cancer - Principles & Practice of Oncology, 4th edition, Lippincott). Applied chemotherapy drugs are e.g. B. cyclophosphamide, cisplatin, epipodophyllotoxins,

Vincristine, dacarbazine, melphalan, ifosfamide. The problem with the use of such cytostatics is, in principle, the systemic toxicity and the associated side effects.

The object of the invention is therefore to provide new compounds which overcome the disadvantages of the prior art.

The object is achieved in that the benzylguanidine structure is coupled with substances or structures which themselves (i) have a therapeutic activity and (ii) are suitable as a signaling molecule for imaging diagnostics.

The object is achieved by creating compounds of general formula I. R5 R6 _R7 NH Λ NH,

solved in what

R ¹ or R ² stand for a therapeutic agent, for a therapeutically activatable agent, for a signal molecule for radio diagnostics or for a signal molecule for fluorescence diagnostics with at least one absorption maximum between 400 and 1000 nm,

L stands for -NH-, -0-, -S-, -CH ₂ - or for 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,

R to R ⁷ independently of one another for hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, carboxy, amino, nitro, Cι-Cιo-alkyl, Cι-Cι ₀ -alkoxy, Cι-Cιo-alkoxycarbonyl, Cι-Cιo- Alkylamino, Ci-Cio- (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 chloro, 0-5 hydroxy, 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 if 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 radiodiagnostics or a partial structure of a signaling molecule means for fluorescence diagnostics with the aforementioned absorption maximum,

and their physiologically tolerable salts.

Compounds in which R to R are hydrogen are preferred. The compounds according to the invention are suitable for imaging in vivo diagnostics, in vitro diagnostics and the therapy of neuroblastomas.

Surprisingly, a therapeutic benefit is achieved in that therapeutically active molecules are coupled to the basic benzylguanidine structure.

Therapeutic active ingredients in the general formula (I) are, for example, cytostatic or antibiotic molecules, in particular the compounds listed below:

Antibiotics: aclacinomycin, actinomycin Fi, 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 analogs: ancitabine, azacitidine, 6-azauridine, Carmofur, cytarabine, doxifluridine, enocitabine, floxuridine, 5-fluoro-uracil, purine analogs: fludarabine, 6-mercaptopurine, thiamiprine, thioguanine and derivatives of the compounds mentioned, alkylating Substances: alkylsulfonates, aziridines, ethyleneimines, methylmelamines, nitroureas and nitrogen-leach compounds, such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mel ^¬ phalan, novembichin, phenesterols, prednimustine, troferenfamide, desweiters substances having hormone activity, such as androgens, antiadrenals, antiandrogens, antiestrogens, estrogens, LH-RH analogues and progestogens, and further cytostatic 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 its derivatives.

Compounds of the general formula I which can be activated therapeutically are compounds which contain elements with a high neutron capture cross section and photodynamically active compounds.

Photodynamically active molecules are compounds from the class of porphyrins, expanded porphyrins, chlorines, benzochlorines, bacteriochlorins, phthalocyanines, naphthalocyanines or bacteriochlorophylls. After excitation with light, these molecules have the ability to develop a cytotoxic effect through the generation of singlet oxygen or radical reactive molecules. The compounds according to the invention 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 as well as molecules that 1 to 12 boron atoms ent ^¬ hold, preferably compounds of the general formula I wherein R ¹ and / or R ² is -B (OH) _2, ortho-carboranyl, para- 7

Carboranyl, ortho-carboranylalkyl, para-carboranylalkyl, and for phenylboronic acid, the linkage being ortho, meta or para to the (HO) ₂ B group, and

(HO) ₂ B-

stand .

Appropriate signaling molecules are coupled to the benzylguanidine structure for diagnostics using highly sensitive detection methods, such as radio diagnostics and fluorescence diagnostics. This ensures that the resulting conjugates have a binding affinity comparable to that of benzylguanidine or MIBG.

Surprisingly, it was found that the conjugate obtained after coupling fluorescein isothiocyanate (FITC) to 4-aminobenzylguanidine has a bond to the noradrenaline transporter.

Radiodiagnostics is a highly sensitive, imaging method that is widely used in tumor diagnostics. It includes u. a. 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, as well as therapeutically usable nuclides, such as ¹³¹ I, ⁹⁰ Y, ^186/188 re. The nuclide ^9m Tc is widely used diagnostically due to its short half-life (6 h) and easy availability (Tc generators).

According to the invention, new agents for scintigraphic diagnostics based on the benzylguanidine structure are made available which are outstandingly suitable for the production of radiodiagnostics and which allow easy handling in the clinic.

Signal molecules for radiodiagnostics in the general formula I are therefore preferably nuclide-binding chelators, peptides and complexing agents which complex the above-mentioned nuclides.

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 having ₄ structure which forms with ^99m Tc and / or ^186/188 R _Θ stable complexes or nuklidbindenden complexing agent which forms a polyvinyl lyamino polycarboxylic acid has Strukur, the stable complexes with 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 ⁱ⁸⁶ / ⁱ⁸⁸ _Re .

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 radiodiagnosis and ^186/188 R _Θ f _ur _e di 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-zaundecane-2, 10-dione-dioxime, 2-methyl-2- (4-isothiocyanatobenzyl) -N, N '-propylene-bis-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-l-carboxyethylamine).

Thioacetylglycylglycylglycine and thioacetylglycylglycine and derivatives are particularly preferred, the thiol group bearing a suitable protective group. Protected thiol groups are, for example, S-triphenylmethyl (trityl), S-diphenylmethyl (benzhydryl), S-benzyloxycarbonyl, S-tert. Butyloxycarbonyl, S-acetamidomethyl (Acm) and mixed Disulfides (e.g. -SS- 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 10

wrote.

Preferred are ethylenediaminetetraacetic acid (EDTA), diethylenediaminepentaacetic acid (DTPA), trans-1, 2-cyclohexanediamine tetraacetic acid, 1,4,7, 10-tetraazacyclododecanetetraacetic acid, 1,4,7, 10-tetraazacyclododecane triacetic acid, 1,4, 7-triazacyclic acid 1,4,8, 11-tetraazatetradecanetetraacetic acid, 1, 5, 9-triazacyclododecane triacetic acid, where some of the acetic acid residues may be present as an amide or ester. They are particularly suitable for complexing In 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 general formula I are substances for positron emission tomography (PET). Structures containing the isotope F-18 or those which can be labeled with F-18 are particularly preferred.

Preferred signal molecules for fluorescence diagnostics are fluorescent dyes with an absorption maximum between 600 and 1000 nm for in-vivo near infrared diagnostics (NIR). In NIR diagnostics, the high permeability of biological tissue for light of the wavelength 650-1000 nm is used and a fluorescent dye as a contrast medium is detected in vivo based on its absorption or fluorescence. 11

Furthermore, fluorescent dyes with an absorption maximum between 400 and 800 nm are preferred. The compounds according to the invention are suitable for the in vitro diagnosis of components to which they have a 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-, -0-, -S-, -CH ₂ - or for one

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 are linked via a physiologically cleavable bond to the radicals R ¹ and / or R ² . 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. Preferred compounds are those 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 under acidic conditions 12

is split, causes a release of the active ingredient with unfolding or increasing its cytostatic or antibiotic activity ("pro-drug" effect). Therefore, according to the invention, L 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

"NC-" N -C _N— S -. ^H MN —— S S »- / /// \ vy.

N 'N' ^ II W "II II II 0 II

, c. -C.

H - _.o--

N 'N' N 'II II II II

HH. , CH ₃ CH ₃

-oc ι -o -o— c ι -o - (/ '/ \ --- --- oc' -o oc '-o - / ⁽ /' \ ^N v

I I \ _ / I I

CH ₃ CH ₃ CH ₃ CH ₃

(CH _{^ 2} ) _P C, ^ (CH - IC or

where p represents a number between 2 and 4.

In addition to the cleavage due to lowered pH values, the compounds according to the invention can also be cleaved by enzymes with which neuroblastomas are endowed in an increased concentration. 13

The invention therefore furthermore relates to compounds having linker structures L which are cleaved enzymatically. Enzymatically cleavable linker structures are, for example, those which are cathepsins, peptidases, carboxypeptidases, α- and β-glucosidases, lipases, oxidases, phospholipases, phosphatases, phosphodiesterases, proteases, elastases, sulfatases, reductases, transferases and bacterial enzymes, for example Penicillin amidases, ß-lactamases, are cleaved.

Preferred are peptides and short-chain amino acid sequences that are cleaved enzymatically. (P. D. Senter et al., Bioconjugate Chem. 6 (1995), 389-94). Preferred enzymatically cleavable structures are short-chain peptide sequences, such as, for example, sequences which contain the amino acid sequence Val-Leu-Lys.

The compounds are preferably synthesized by deliberately representing the radicals in the general formula I for R ¹ and R ² and coupling them 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 NN-di-Boc-4-aminobenzylguanidine.

The invention therefore also relates to a method for producing

Compounds of the general formula I 14

R ⁵ R6 _R 7 NH ΛNH,

wherein

m and n each represent 0 to 1,

in the event that R ^{1 has} the meaning given under a) and the sum of and n is at least 1,

b) R ² to R ⁷ independently of one another for hydrogen,

Fluorine, chlorine, bromine, iodine, hydroxy, carboxy, amino, nitro, Cι-Cιo-alkyl, Cι-Cι ₀ -alkoxy, Cι-Cι ₀ -alkoxycarbonyl, Ci-Cirj-alkylamino, Cι-Cι ₀ - ( Dialkyl) amino stand, the alkyl radicals being interrupted independently of one another by up to 5 oxygen atoms and / or with 0-2 fluorine, 0-2 chloro, 0-5 hydroxy, 0-3 carboxy, 0-3 ester 15

and / or 0-3 amino groups are substituted,

R ¹ and R ³ to R ^{7 have} the meaning given under b),

m is 0 and n is 1 if 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, includes 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 radiodiagnostics or a partial structure of a signaling molecule for fluorescence diagnosis the aforementioned absorption maximum means

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 , subsequently 16

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 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 methods known to the person skilled in the art.

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 the chelators, complexing agents and peptides, and the labeling with radionuclides is carried out according to the methods known to the person skilled in the art. 17

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:

F. M. Hamer in The Cyanine Dyes and Related Compounds,

John Wiley and Sons, New York, 1964;

J. Fabian et al. , Chem. Rev. 92 (1992) 1197;

L.A. Ernst et al. , Cytometrie 10 (1989) 3-10; Pl. L. Southwick et al. , Cytometry 11 (1990) 418-430;

R. B. Mujumdar et al. , Bioconjugate Chem. 4 (1993) 105-11;

E. Terpetnig et al. , Anal. Biochem. 217 (1994) 197-204;

J. S. Lindsey et al. , Tetrahedron 45 (1989) 4845-66, EP-

0591820 AI; L. Strekowski et al. , J. Heterocycl. Chem. 33 (1996)

1685-1688;

S.R. 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 (1988) 13-22 and N. Narayanan and G. Patonay, I. Org. Chem. 60 (1995) 2361-95.

Acid-labile bonds are generated based on the following literature examples.

B. M. Mueller et al. , Bioconjugate Chem. 1 (1990) 325-

330;

K. Srinivasachar and D. M. Neville, Biochemistry 28 (1989) 2501-09;

D. M. Neville et al. , J. Biol. Chem. 264 (1989) 14653-61;

T. Kaneko et al. , Bioconjugate Chem. 2 (1991), 133-41;

B. A. Froesch et al. , Cancer Immuno1. Immunother. 42

(1996), 55-63 and J.V. Crivello et al. , J. Polymer Sei: Part A: Polymer

Chem. 34 (1996) 3091-3102.

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

The subject is also the use of the radioactive compounds described as in vivo radio diagnostics and in vivo radiotherapeutics. The use of the dye-containing compounds for the in vivo diagnosis of diseased tissue by means of near infrared (NIR) radiation by detection of the unabsorbed radiation and / or the fluorescent radiation, and for the in vitro diagnosis of constituents from body fluids, such as Blood, urine, cerebrospinal fluid, lymph by detection of the non-absorbed radiation and / or the fluorescent radiation (inter alia by means of FACS = "fluorescence activated cell sorting"). 19

The compounds according to the invention are also suitable for the diagnosis and therapy of tumors of other types and other origins. Examples are neuroendocrine tumors (e.g. pheochromocytoma; A. Lau et al., Clinical Nuclear Med. 1996, 21, 316-318, "glomus jugulare tumors", E. Nussen, J. Laryngol. Otol. 1996, 110, 373-375), and small intestinal carcinoids (S. Dresel et al., Nuclear Medicine 1994, 35, 53-58), tumors of the adrenal cortex

(D.A. Sloan et al., Cur. Opin. Oncol. 1996, 8, 30-36), and secondary neuroendocrine tumors of the liver (J.K. Ramage et al., QLM 1996, 89, 539-542).

The particular advantage of therapy and diagnostics with the compounds according to the invention is that they are taken up into the diseased cells with simultaneous rapid elimination from the healthy residual body due to the hydrophilicity and the low molecular weight of the compounds. The systemic toxicity is reduced in particular in comparison to antibodies as carrier molecules (eg EP 282057) which have very long circulation times.

These agents are prepared by methods known to those skilled in the art, optionally using customary auxiliaries and / or carriers as well as diluents and the like. These include physiologically tolerable electrolytes, buffers, detergents and substances for adaptation of Os olarität and to improve the Stabili ^¬ ty and solubility. The measures used in pharmacy are responsible for the sterility of the preparations 20th

to take care of during production and especially before application.

The following examples illustrate the invention.

Examples 1 and 2

Illustration 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 described in

10 ml of water stirred 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

Product at 4 ° C and auskristalliert lisiert of water umkristal ^¬. 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 To a solution of 0.2 g (0.76 mmol) 2 (see 1.1) in 5 ml of phosphate buffer (50 mM, pH 8), 0.3 g (0, 76 mmol) of fluorescein-5-isothiocyanate in 5 ml of DMF. It gets 5 h at room temp. stirred and the reaction mixture lyophilized. Chromatographic purification (RP-18 euro 21

prep., mobile phase H ₂ 0 + 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) indotricarbocyanin-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 (0.18 mmol) 1 are in 3 ml phosphate buffer (50 mM, pH 8) and 3 ml DMF for 5 h at room temp. coupled. After adding diethyl ether, the precipitated crude product is purified by chromatography (RP-18, Europrep., Mobile phase H ₂ 0 + 1% CH ₃ COOH /

10% MeOH), yield 23 mg (20%) deep blue lyophilisate.

Examples 3 to 6:

Preparation of conjugates from benzylguanidine and cytostatic structures

Example 3:

Synthesis of p-di- (2-chloroethyl) aminobenzylguanidine 9

The compound is a structural mixture of the therapeutically active, alkylating part of the melphalan and the benzylguanidine.

The synthesis takes place 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 Al). 22

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 C1 ₂ . The combined organic phases are saturated with sat. Washed NaCl solution, dried over NaS0 ₄ and concentrated. After chromatographic purification (Kieselgel 60, Merck, eluent CH ₂ C1 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 C1 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, eluent 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 ₄ H ₁₉ N Cl ₂ 0 ₂ F ₃ : C 4 1, 72 H 4, 75 N 13, 8 9 found: C 41, 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 takes place from chlorambucil and N, '-di-Boc-p-aminobenzylguanidine 10, which is obtained by reacting 1 with 7 (cf. Ex. 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 C1 ₂ and stirred with 3.6 g (12.3 mmol) of solid 7 ver 23

puts. You leave 24 h at room temp. stir, dilute with 100 ml of water and extract four times with CH ₂ C1 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 C1 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 C1 ₂ , the organic phase with sat. Washed NaCl solution and dried over NaS0 ₄ . The crude product 11 (0.18 g) is in 5 ml CH ₂ C1 ₂ and 0.5 ml trifluoroacetic acid for 18 h at room temperature. touched. After concentration 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 ₃ oN ₅ Cι ₂ 0 ₃ F ₃ Cl ₂ : C 51, 07 H 5, 36 N 12, 4 1 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). 24

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 takes place as described in Example 2.1. The crude product is dissolved in 1 ml CH ₂ C1 _2/50 ul trifluoroacetic acid for 18 h at room temp. stirred, concentrated and purified by chromatography (RP-18 Europrep., mobile phase H ₂ 0 + 1% CH ₃ COOH / 10% MeOH), yield 42 mg (67%) 14 as a yellowish lyophilizate.

Elemental analysis: calculated for C ₂₂ H ₂ ιN ₅ 0 ₄ ^» CH ₃ COOH: C 60, 12 H 5, 2 6 N 14, 61 found: C 59, 81 H 5, 20 N 14, 33

Example 6: Synthesis of 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), the γ-amino group of which is released by cleavage of the protective group to give 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-Lys (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 25th

added in 1 ml DMF. The mixture is 2 h at 0 ° C and

16 h at room temp. touched. After removal of the solvent in vacuo, 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 C1 ₂ / methanol 95: 5 to 5: 1), 0.15 g of solid is obtained, which is dissolved in 10 ml of CH ₂ C1 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 CHC1 ₃ 0.13 g (0.55 mmol) 1, 1'-thiocarbonyl 2, 2 '-pyridone added dropwise in 2 ml of CHC1 ₃ . The reaction mixture is 16 h at room temperature. stirred, the solvent evaporated and the residue purified by chromatography (Kieselgel 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) 16 in 10 ml DMF and 1 ml triethylamine, 5 h at room temperature. stirred, the solvent evaporated in vacuo and the crude product precipitated by adding ether / hexane (1: 1) and filtered off. To release the benzylguanidine group, the crude product is dissolved in 10 ml CH ₂ C1 ₂ and 2 ml trifluoroacetic acid 26

3 h at room temp. stirred and worked up and cleaned analogously to Example 5; Yield 0.14 g (88%) 18 as yellow crystals.

Elemental analysis: Calculated for C ₃₀ H ₃₇ O ₃ S Nι ₃ ^"CH ₃ COOH: C 50.06 H 5.74 N 25.30 found: C 49.90 H 5.78 N 25.50

Example 7: Preparation of acid-labile conjugates from benzylguanidine and cytostatic structures

Synthesis of a Doxorubicin-hydrazono-isothiocyanato-benzylguanidine (22)

The synthesis is carried out 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 becomes a solution of 0.12 g (0.44 mmol) 18 in 10 ml DMF and 1 ml triethylamine

DMF given, 5 h at room temp. stirred, the solvent evaporated in vacuo, the crude product precipitated by adding ether / hexane (1: 1) and filtered off. For free ^¬ Benzylguanidingruppe the reduction, the crude product in 10 ml CH ₂ C1 ₂ and 2 ml of trifluoroacetic acid is 3 h at room temp ^¬. stirred and worked up analogously to Example 6.1, yield 0.17 g (80%) 20 as a brownish powder. 27

7.2. Generation of the acid-labile bond to product 22 0.05 g (0.1 mmol) 20 and 0.05 g (0.08 mmol) morpholino-doxorubicin 21 are dissolved in 1 ml of anhydrous methanol with 10 μl of a 5% methanolic trifluoroacetic acid solution offset 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 0): λ _max 530, 492 nm.

Examples 8 to 11

Preparation 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 guanidinylated 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, mobile solvent

CH ₂ Cl ₂ / methanol 9: 1 → 8: 2). From 1.0 g (6.6 mmol) 23 28

hold 1.1 g (42%) of N, N '-Di-boc-4-carboxybenzylguanidine 24.

8.2. Synthesis of 25 1.0 g (2.5 mmol) of 24 and 0.35 ml (2.5 mmol) of triethylamine 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 C1 ₂ . After drying over MgS0 ₄ and concentration, the Boc protective groups are cleaved directly by stirring in 5 ml of CH ₂ C1 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 0 + 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) DCC in 2 ml DMF. 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. stirred. 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 29

C-18 7 µm, gradient H ₂ 0 + 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-diaminobutyryl) 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 step is carried out starting from 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 HP0 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. incubated and filtered (0.2 μ filter). The analysis of the label is carried out by means of HPLC (Nucleosil, 125x4cm, 5 μm, eluent A: phosphate buffer (10 mM, pH 4), eluent B: acetonitrile / phosphate buffer (75:25), 100% A after 100% B within 10 min ). The radiochemical purity is> 95% for both compounds. 30th

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 takes place starting from 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 used to split off the protective group in 2 ml DMF and 0.1 ml piperidine 30 min at room temp. stirred, then the solid precipitated with diethyl ether as in Example 8.3 purified. 31 is obtained as a ₀ yellowish powder.

Example 12:

Binding of FITC-benzylguanidine 3 to neuroblastoma cells

FACS analysis of LS neuroblastoma cells.

The results are shown in Fig. 6.

6a: Incubation of control cells with 3.

6b: Incubation of LS neuroblastoma cells with 3.

Claims

31 Patent claims

1. Compounds of the general formula I

R ⁵ R6 _R 7 NH

N NH, H

wherein

m and n each represent 0 to 1,

R ² to R ⁷ independently of one another for hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, carboxy, amino, nitro, Ci-Cirj-alkyl, Cι-Cι ₀ -alkoxy, Cι-Cι ₀ -alkoxycarbonyl, Cι- Cιo-Alkylamino, -Cι-Cι ₀ - (dialkyl) amino, where 32

the alkyl radicals are interrupted independently of one another by up to 5 oxygen atoms and / or substituted with 0-2 fluorine, 0-2 chlorine, 0-5 hydroxyl, 0-3 carboxy, 0-3 ester and / or 0-3 amino groups are,

R ¹ and R ³ to R ^{7 have} the meaning given under b),

m is 0 and n is 1 if 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 agent, 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 Fluorescence diagnostics with the aforementioned absorption maximum means

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. 33

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-lotoxins.

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, benzochlorines, 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 -, N ₃ S-, N ₂ S ₂ -, NOS ₂ - or S ₄ structure, which forms stable complexes with ^99m Tc and / or ^186/188 R _Θ , or a nuclide-binding complexing agent, which has a polyamino-polycarboxylic acid structure which with ^{11: L} In 34

forms stable complexes, 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 ^{i86 / i88} _Re .

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 penothiazine, cyanine, Oxonol, merocyanine, styryl, Squarilium or Croconium dye is available.

9. Compounds according to at least one of the preceding claims, characterized in that L for one

Hydrocarbon linker structure, which may contain esters, ethers, amides, secondary or tertiary amines, ketones, thiourea, urea, carbamate or maleimido groups.

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 35

o o

H H |

, N-C ---,, NN - CC _ -o- ^. |

_N— S - J ^H i — S »

N ^ II

O II o

. £ *, c.

N ^ y \ - / ^ N '

II II ^λ - 'II II

HH,. CH ₃ CH ₃

--- oc ¹ -o --- --- o— c ^! -o - (/ 'l' ^ \ s --- - --- no —— cr "—- no - - no —— cr> —- no

I I \ _ ./ I I

CH ₃ CH ₃ CH ₃ CH

or

where p represents a number between 2 and 4.

11. Compounds according to at least one of the preceding claims, characterized in that L stands for a structure which contains an enzymatically cleavable chemical bond by cathepsins, peptides, carboxypeptidases, α- and β-glycosidases, lipases, phospholipases, phosphatases , Phosphodiesterases, proteases, elastases, sulfatases, reductases and bacterial enzymes.

12. Use of compounds according to claim 1 for the in vivo diagnosis of diseased tissue areas, for the in vitro diagnosis of tissue components and for the therapy of diseased tissue areas.

13. Diagnostic agent for the in vivo diagnosis of diseased tissue areas, for the in vitro diagnosis of 36

Tissue components and for the therapy of diseased tissue areas, characterized in that it contains at least one compound according to claim 1 together with the usual auxiliaries and / or carriers and diluents.