NZ263794A

NZ263794A - Compound suitable for forming tc and re chelates, radiopharmaceutical compositions thereof

Info

Publication number: NZ263794A
Application number: NZ263794A
Authority: NZ
Inventors: Christoph-Stephan Hilger; Ludger Dinkelborg; Wolfgang Kramp; Hans-Martin Schier
Original assignee: Diagnostikforschung Inst
Priority date: 1993-03-31
Filing date: 1994-03-29
Publication date: 1996-10-28
Also published as: DE59408142D1; WO1994022497A1; HUT73665A; EP0692976B1; NO953867L; NO953867D0; DE4311023A1; ATE179076T1; HU9502868D0; CA2156620A1; EP0692976A1; KR960700777A; AU691806B2; AU6501794A; JPH08508263A; DE4311023C2

Abstract

Novel bifunctional chalcogenic-atom-discontinuous chelating agents, pharmaceutical preparations containing them, their use in radio diagnosis and radio therapy, and methods of preparing them. The invention concerns compounds of the general formula (I): M-L, in which M is a radioisotope of Tc or Re, and L is a ligand of general formula (II). Surprisingly, it has been found that the bifunctional chalcogenic-atom-discontinuous chelating agents described, and their coupling products with specifically enriching compounds, are eminently suitable for use in the production of radiodiagnostic or radiotherapeutic compounds.

Description

<div class="application article clearfix" id="description"> New Zealand No. 263794 International No. PCT/DE94/00371 r Priority Dalsfc}: .3.ll3]q.3. Ccmplota Specification Filed: Publication Dnfa*'.-:.. 2. fl OCT fSQfi jj-.G. Journal No: ±1*93.. $ A * •* *»* jb ®. .'"'v & & «i S <f « i ^ NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Chelating agents of the type XN1S101 for radioactive isotopes, metal complexes thereof, and their use in diagnosis and therapy Name, address and nationality of applicant(s) as in international application form: INSTITUT FUR DIAGNOSTIKFORSCHUNG GMBH AN DER FREIEN UNIVERSITAT BERLIN, a German company of Spandauer Damm 130, D-14050 Berlin, Federal Republic of Germany d ID- L 3 3 i7 WO 263794 This invention relates to new bifunctional chelating agents with intermittent chalcogen atoms, pharmaceuticals containing these compounds, their use in radiodiagnostics and radiotherapy, and methods for the production of these compounds. It has been known for a long time that complexing agents for radioactive isotopes or their complexes with radioactive metals can be applied in radiodiagnostics and radiotherapy. Technetium-99m is thr. most frequently used radio-10 nuclide in radiodiagnostics because it is particularly well suited for in-vivo applications due to its favourable physical properties (no corpuscular radiation, low half-life of 6.02 h, good detectability by 140 keV y-radiation) as well as its low biological half-life and easy availabil-15 ity. The first step of forming complexes of technetium-99m is to gain pertechnetate from a nuclide generator; it is chan converted to a lower oxidation number using appropriate reductants (such as SnCl2, S2042", etc.). This oxidation number is stabilized by an appropriate chelating 20 agent. As technetium may have several oxidation numbers (+7 to -1) which may vehemently alter its pharmacological properties by changing the charge of the complex, it is necessary to provide chelating agents or complex ligands for technetium-99m that are capable of binding technetium 25 in a specific oxidaticna number safely, firmly and stably to prevent undesirable biodistribution due to in-vivo redox processes or release of technetium from the radiodiagnostic agent which would impede the safe diagnosis of the respective diseases. 30 For example, cyclic amines (Troutner, D.E. et al.r'j. Nucl. Med. 21, 443 (1980)) are regarded as suitable complexing agents for technetium and rhenium isotopes but their disadvantage is that they are only capable of binding technetium-99m in sufficient quantities from a pH value >9. 35 N2°2 systems (Pillai, M.R.A., Troutner, D.E. et al.: Inorg. ID-133L7WO 263794 Chem., 22., 1850 (1990)) are in'clinical use. Non-cyclic N4 systems such as HMPAO have the great disadvantage of low complex stability. Tc-99m-HMPAO has to be applied immediately after labelling due to its low stability 5 (Ballinger, J. R. et al., Appl. Radiat. Isot. A2., 315 (1991); Billinghurst, M. W. et al., Appl. Radiat. Isot. 4 2. 607 (1991)) to keep the portion of decomposition products low which have different pharmacokinetic and excretion properties. Such radiochemical impurities make detection of 10 the diseases to be diagnosed more difficult. Any coupling of these chelates or chelating agents with other substances that accumulate selectively in centres of diseases cannot be broken by simple means so that these normally spread unspecifically in the organism. 15 N2S2 chelating agents (Bormans, G. et al.: Nucl. Med. 3iol., 12, 499 (1990)) such as ethylene dicysteine (EC; Verbruggen, A.M. et al.; J. Nucl. Med. .13., 551 (1992)) meet the requirement of sufficient stability of their respective technetium-99m complex but form radiodiagnostic agents of a 20 purity greater than 69% only at pH values >9 of the complexing medium. N3S systems (Fritzburg, A.; EPA 0 173 424 and EPA 0 250 013) yield stable technetium-99m complexes but have to be heated up to temperatures of about 100°C to insert the radionuclide. 25 Another disadvantage of N2S2 and N3S systems is that they are discharged partially too rapidly and without specific accumulation by the organism. Thus they are only used clinically, though to a limited extent, in renal function diagnostics. In the last few years the demand has increased 30 for radiodiagnostics that accumulate specifically in diseased tissues. This can be accomplished if one manages to link complexing agents easily with selectively accumulating substances while the latter retain their favourable complexing properties. But as it happens quite frequently 35 that a certain reduction of complex stability is observed ID- 13 317W0 263794 after coupling the complexing agent to such a molecule by means of one of its functional groups, previous approaches to coupling chelating agents v/ith substances that accumulate selectively are hardly satisfactory because a quantity 5 of the isotope that is not tolerable with a view to diagnostics is released in vivo from the conjugate (Brechbiel, M. W. et al.; Inorg. Chem. 1986, 25., 2772). it is therefore necessary to produce bifunctional complexing agents that have functional groups to bind the desired 10 metallic ion and one (or several other) functional groups to bind the selectively accumulating molecule. Such bifunctional ligands allow specific, chemically defined bonding of technetium or rhenium isotopes to the most various biological materials even in cases in which pre-labelling is 15 applied. Some chelating agents coupled with monoclonal antibodies (e.g. EP Appl. 0 247 866 and EP Appl. 0 188 256) or fatty acids (EP Appl. 0 200 4.92) ha^e been described. But these chelating agents were based on the N2S2 systems mentioned above which are hardly appropriate due to their 20 low stability. As both the properties of the substances that accumulate selectively and the mechanisms of accumulation are quite varied, one should be able to vary the chelating agent meant for coupling to adapt it to the physiological requirements of its coupling partner with 25 regard to lipophilic or hydrophilic behaviour, membrane permeability or impermeability, etc. It is therefore an object of this invention to provide stable complex compounds coupled with or capable of coupling with various compounds that accumulate selectively, 30 and to provide such linkable chelating agents or complexes whose substituents show a wider range of chemical variation to be adaptable to the above requirements. It is another object of this invention to provide such compounds and pharmaceuticals containing these compounds, as well as 3 5 methods for their production. ■ ID-13317WO 263794 This problem is solved by the invention, surprisingly, in that the new, uncommon, bifunctional chelating agents with intermittent chalcogen atoms and their coupling products with compounds that accumulate selectively are excellently 5 suited for producing radiodiagnostic and radiotherapeutic agents. The subject matter of this invention are compounds of the general formula (I) M - L (I) 10 wherein M represents a radionuclide of Tc or Re and L represents a ligand of the general formula (II) B-CO-CR1R2-A-CR3R4-COOH (II) wherein 15 A represents an O, S, or Se chalcogen atom, R1, R2, R3, and R4 are same or different and represent a hydrogen atom and/or a branched or unbranched C^-Cg alkyl residue, B represents a residue 20 -NH-CR5R6-(CR7R8)n=1(2-S-R9, wherein R5 and R6 are same or different and represent a hydrogen atom or an unbranched, branched, cyclic, or polycyclic Cx-C60 alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, 25 aryl, alkylaryl, or arylalkyl residue which may optionally be carry an additional hydroxy, oxo, oxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, or. alkoxy groups containing up to 20 carbon atoms, and/or may optionally be interrupted, and/or replaced, by one or 30 several heteroatoms from the series of O, N, S, P, As, Se, ID-13 317WO 263 R7 and R8 are same or different and represent a hydrogen atom and/or a branched or unbranched C^-Cg alkyl residue, ^ R9 represents a hydrogen atom, a branched or unbranched C^-Cg alkyl residue, or a sulfur protecting group, 5 with R5 and R6, together with the groups that connect them, optionally forming a 4- to 8-membered ring which may ) optionally carry additional hydroxy, oxo, oxy, or alkoxy groups containing up to 6 carbon atoms. Preferred compounds of the general formula (I) are 10 characterized in that R1, R2, R3, and R4 are hydrogen atoms, and that A is a sulfur atom. Particularly preferred compounds according to the invention o£ the general formula (I) are characterized in that R5 and Re are different, and that R6, R7 and R8 each represent a 15 hydrogen atom. Another object of this invention is related to the new, bifunctional ligands with intermittent chalcogen atoms of the general formula (II) B-CO-CR1R2-A-CR3R4-COOH (II) 20 wherein R1, R2, R3, R4, A and B have the meanings specified above. Such ligands according to the invention of the general formula (II) are preferred in which A is a sulfur atom and R1, R2, R3 and R4 are hydrogen atoms. 25 Particularly preferred compounds according to the invention .of general formula (II) are characterized in that Rs and R6 are different, and that R6, R7 and R8 each represent a hydrogen atom. Yet another object of this invention are conjugates 30 containing compounds of the general formulae (I and/or II) ID-13 317WO 2*3794 and substances that accumulate selectively in diseased tissues, with a covalent bond existing between these substances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, 5 antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups. Particularly preferred conjugates according to the invention are characterized in that the substances that 10 accumulate in diseased tissue are peptides such as endo-thelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists. Other preferred embodiments of the conjugates according to 15 the invention are characterized in that the peptides comprise the following sequences or parts thereof: 20 30 I I cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr- 1 1 phe-cys-his-leu-asp-ile-ile-trp, I I cys- ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr- 25 I , phe-cys-his-leu-asp-ile-ile-trp, l I cys -1 hr - cys - phe -1 hr - tyr- lys - asp - lys - glu - cys - val - tyr - 1 1 tyr-cys-his-leu-asp-ile-ile-trp, cys -ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr- I 3 5 phe-cys-his-leu-asp-ile-ile-trp, ID-13 317W0 263794 J . —1 cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr- 1 1 5 phe-cys-his-leu-asp-ile-ile-trp. I 1 I cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn- 1 1 10 phe-cys-his-gin-asp-val-ile-trp, ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr -phe-ala-his-leu-asp-ile-ile-trp, 15 ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-20 phe-ala-his-leu-asp-ile-ile-trp, I 1 cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, 25 N-acetyl - leu-met-asp-lys-glu-ala-val- tyr-phe-ala-his-leu-asp-ile-ile-trp, the partial sequence 30 his-leu-asp-ile-ile-trp or the cyclic amino acid sequences Cyclo-(Dtrp-Dasp-pro-Dval-leu), 35 Cyclo-(Dglu-ala-alloDile-leu-Dtrp). ID-13 317WO 263794 The compounds according to the invention of the general formula (I) are produced by reacting technetium-99m in the form of pertechnetate or Re in the form of perrhenate in the presence of a reductant and, optionally, an auxiliary 5 ligand, with a compound of the general formula (II) B-CO-CR1R2-A-CR3R4-COOH (II) wherein R1, R2, R3, R4, A, and B, have the meanings specified above. The ligands according to the invention of the general for-10 mula (II) are produced by reacting compounds of the general formula (III) with compounds of the general formula (IV) according to the following reaction scheme: CR1R2-A-CR3R4 | | + NH2-CR5R6-(CR7R8)n=1/2-S-R9 15 CO O—CO (III) (IV) -» B-CO-CR1R2-A-CR3R4-COOH (II) wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, A, and B have the 20 meanings specified above. These reactions are carried out in polar and non-polar aprotic solvents such as dichlor. aethane, tetrahydrofurane, chloroform, 1,4-dioxane, DMF, or DMSO at temperatures between -30 and +100°C; an auxiliary base is added to trap 25 any acids that may be liberated. Among these bases could be, for example: tertiary amines, alkali and alkaline-earth hydroxides, alkali and alkaline-earth carbonates. .. Another object of the present invention is a kit for producing radiopharmaceuticals consisting of a compound of the 3 0 general formula (II) or a conjugate according to the invention containing compounds of the general formulae (I ID-13317WO 10 263794 and/or II) and substances that accumulate selectively in tissues, a reductant and, optionally, an auxiliary ligand, said agents being either dry or in solution, instructions for use including instructions for reacting the compounds 5 described with technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution. Another object of this invention is a radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or athero-10 sclerotic plaques. It contains a compound of the general formula (I) or a conjugate according to the invention containing compounds of the general formulae (I and/or II) and substances that accumulate selectively in tissues, optionally with the adjuvants common in galenics; the 15 compound is prepared in a kit using technetium-99m or rhenium in the form of a pertechnetate or perrhenate solution. Yen. another object of this invention is a method for carrying out radiodiagnostic examinations according to 20 which the radiopharmaceutical formulation is applied at doses from 0.1 to 30 mCi, preferably from 0.5 to 10 mCi, per 70 kg of a patient's body weight, and that radiation emitted by the patient is recorded. Many of the synthesized chelates that were labelled with 25 Tc-99m or Re surprisingly showed a greater stability than comparable N2S2 and N3S systems described in the literature. For example, no decomposition products were found of a substance according to the invention (Examples 2a, 2b) coupled with a fatty alcohol even after 23 hours. It was 30 also found in competition tests that the Tc-99m or Re chelating agents complex better than comparable N2S2, N3S and propylene aminoxium systems. The chelates and chelating agents described in the present invention are clearly better suited for diagnostic and therapeutic purposes than the 35 systems known so far. It is a specific advantage of the ID-13317KO 11 263794 chelating agents according to the invention that they may be synthesized without sulfur protecting groups. This makes synthesis very simple; in addition, the compounds according to the invention, when radiolabelled, do not contain any 5 other foreign molecules in the solutions used for radio-diagnostics or radiotherapy, for example, solutions to be administered intravenously. Biodistribution of the radiopharmaceutical and thus the value of diagnostic information are frequently diminished by such foreign molecules. More-10 over, such ligands or their coupling products with substances that accumulate selectively in diseased tissues can be labelled very gently. The ligands according to the invention and their coupling products with substances that accumulate selectively in diseased tissues can be labelled 15 at room temperature and at the physiological pH value without having to split off protecting groups using bases, acids, or other auxiliary substances known to a person skilled in the art. This guarantees that the very sensitive substances that accumulate selectively in diseased tissues 20 are not modified chemically by such auxiliary substances, as such modification frequently reduces selective accumulation in diseased tissue and diminishes the value of radiodiagnostic information. Sulfur protecting groups may be used here, of course, if 25 the disadvantages described can be accepted. The groups are attached to sulfur atoms and split off according to methods known to a person skilled in the art. The ways in which the substances that accumulate selectively in diseased tissues ar? bonded are also known to a person skilled in the art 30 (e.g. Fritzberg et al.; J. Nucl. Med. 2£, 7 (1987)), for example, by a reaction of electrophilic groups of the complex ligand with nucleophilic centres of the substances that accumulate selectively in diseased tissues. Otherwise, nucleophilic groups of the chelating agent are coupled with 35 electrophilic groups of the substances that accumulate selectively in diseased tissues. ID-13317WO _ ^ 26379 The partners for coupling are, among others, various bio-molecules, ligands that bond to specific receptors which are capable of detecting tissue showing a modified receptor density. This includes peptides, steroid hormones, growth 5 factors and neurotransmitters. Ways for improved diagnosis of carcinomas of the breast and the prostata were shown using ligands for steroid hormone receptors (S. J. Brandes and J. A. Katzenellenbogen, Nucl. Med. Biol. IS, 53, 1988). Tumour cells sometimes show a modified density of receptors 10 for peptide hormones or growth factors such as the epidermal growth factor (EGF). The differences in concentration could be utilized for selective accumulation of cytostatic agents in tumour cells (E. Aboud-Pirak et al., Proc. Natl. Acad. Sci. USA 86; 3778, 1989). Ligands for neuroreceptors 15 labelled with positron-emitting isotopes were successfully used for the diagnosis of various brain diseases (J. J. Forst, Trends in Pharmacol. Sci., Z, 490, 1989). Other biomolecules are metabolites that can be introduced into the metabolism of cells to make changes visible; this 20 includes fatty acids, saccharides, peptides, and amino acids. Fatty acids that were coupled with the more unstable N2S2 chelating agents have been described in EPA 0 200 492. Other metabolic products such as saccharides (desoxyglucose), lactate, pyruvate, and amino acids 25 (leucine, methylmethionine, glycine) were used in the PET technique for visualizing changes in metabolic processes (R. Weinreich, Swiss Med., £, 10, 1986). Likewise, non-biological substances such as misonidazol and its derivatives which bond irreversibly to cell components in 30 tissues or parts of tissues with a reduced oxygen concentration, can be used for specific accumulation of radioactive isotopes and thus for the visualization of tumours or ischaemic regions (M. E. Shelton, J. Nucl. Med. 3 0: 351, 1989). Finally, bifunctional chelating agents may 35 be coupled with monoclonal antibodies or their fragments. Coupling products of the chelating agents according to the ID-13317WO ^ ^ invention or their technetium-99m or Re complexes with fatty alcohols, fatty alcohol derivatives, or fatty amines I and their derivatives, or with endothelines, partial endotheline sequences, endotheline analogues, endotheline 5 derivatives, or endotheline antagonists have proved particularly favourable for the detection of atherosclerotic vascular diseases. These derivatives were I applied to WHHL rabbits that had high LDL concentrations in their blood - and thus atherosclerotic lesions - due to a 10 genetic defect of their LDL receptor. Concentration quotients from 3 to 40 were found in atheromatose plaques as compared with undamaged tissue about 4 to 5 hours after i.v. application of the derivatives to WHHL rabbits. This allowed detection of atherosclerotic areas of vessels using 15 the common methods of radiodiagnostics (e.g. a gamma scintillation camera). Only very late stages of acherogenesis could up to now be diagnosed by using more invasive methods (e.g. arteriography). The substances according to the invention provide the decisive advantage 20 of being able to diagnose much earlier stages of atherosclerosis using non-invasive methods. It is unimportant whether the chelating agent is labelled with Tc-99m or Re before or after coupling with the selectively accumulating molecule. But if coupling takes place 25 after complexing, it is a prerequisite that the reaction of the radioactive complex with the accumulating compound is rapid, gentle, and nearly quantitative, requiring no subsequent purification. The radiopharmaceuticals of the invention are produced in a 30 generally known way by dissolving the complexing agents according to the invention in an aqueous medium and adding a reductant, preferably tin(II) salts such as chloride or tartrate, optionally adding the adjuvants common in galenics, and subsequent sterile filtration. Among the 35 suitable additives are physiologically tolerable buffers ID-13317WO 14 (such as tromethamine), small quantities of electrolytes (e.g. sodium chloride) or stabilizers (e.g. gluconate, phosphate, or phosphorate). The pharmaceutical according to the invention is either available as a solution or as 5 lyophilizate and is mixed shortly before application with a solution of Tc-99m pertechnetate, eluated from commercial generators, or a perrhenate solution. For in-vivo applications in nuclear medicine, the agents according to the invention are administered at doses from 10 1 x 10~5 to 5 x 104 nmol/kg of body weight, preferably from 1 x 10"3 to 5 x 102 nmol/kg of body weight. The amount of radioactivity per application, based on an average body weight of 70 kg, is between 0.05 and 50 mCi, preferably between 5 and 30 mCi, for diagnostic applications. For 15 therapeutic applications, doses applied are between 5 and 500 mCi, preferably from 10 to 350 mCi. Normally, 0.1 to 2 ml of a solution of the agents according to the invention is applied by intravenous, intra-arterial, peritoneal or intra-tumoral injection. The intravenous injection is 20 preferred. The following examples shall explain the object of this invention in greater detail. 10-13317W0 15 2637; Example la N- (2-mercapto-l- (methoxycarbonyl) -ethyl) -thiodiglycolic acid monoamide 13.21 g (0.1 mol) of thiodiglycolic acid anhydride are 5 added by dropping, and in an argon atmosphere, to a solution of 17.16 g (0.1 mol) of cysteine methyl ester I hydrochloride and 20.24 g (0.2 mol) of triethyl amine in 500 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with 10 aqueous 2% citric acid. After drying above sodium sulfate, the solvent is evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether. Yield: 18.73 g (70.1%), white powder Analysis referring to the anhydrous substance: Calc.: C 35.95 H 4.90 N 5.24 O 29.93 S 23.99 Found: C 35.72 H 5.12 N 5.03 S 23.71 Example lb 20 Technetium-99m complex of N- (2-mercapto-l- (methoxycarbonyl) -ethyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example la are dissolved in 1.0 ml of 0.5 M phosphate buffer, pH 7.5. 50 /xl of this ligand solution are mixed with 250 fil of 25 phosphate buffer, pH 8.5, 50 /xl of a deoxygenated aqueous citrate solution (50 mg/ml) , 2.5 /xl of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and 100 /xl of a pertechnetate solution (400-900 iiCl) . After an incubation time of 10 minutes, the reaction mixture is 30 tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 /xm, 125 x 4.6 mm; gradient elution from 100% A to 100% E within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B: CD-13317WO 263 16 acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is >98V. Example 2a 5 N- (2 -aaercapto-1- (decyloxycarbonyl) -ethyl) - thiodiglycolic acid monoamide 1.32 g (10 mol) of thiodiglycolic acid anhydride dissolved in 50 ml of anhydrous dichloromethane are added by dropping, and in an argon atmosphere, to a solution of 10 2.98 g (10 mmol) of cysteine decyl ester hydrochloride and 2.02 g (20 mol) of triethyl amine in 250 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with aqueous 2% citric acid. After drying above sodium sulfate, the solvent is ^5 evaporated under reduced pressure, and the oily residue -ystallized by trituration with diethyl ether. Yield: 3.37 g (85.6%), white powder Analysis referring to the anhydrous substance: Calc.: C 51.88 H 7.94 N 3.56 0 20.33 20 Found: C 51.63 H 8.07 N 3.37 S 16.29 S 16.02 ID-13317WO ^ 17 Example 2b Technetium-99m complex of N-(2-mercapto-l-(decyloxycarbonyl) -ethyl-thiodiglycolic acid diamide 10 mg of the ligand produced according to Example 2a are j dissolved in 1.0 ml of 0.5 M phosphate buffer, pH 7.5. 50 (il of this ligand solution are mixed with 250 /xl of phosphate buffer, pH 7.5, 50 fil of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 fil of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl), and 10 100 /tl oil a pertechnetate solution (400-900 /iCi) . After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 /im, 125 x 4.6 mm; gradient eluation from 100% A to 100% B within 7.5 minutes (eluent 15 A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is >98%. 263794 120 Example 3a N- (2-oxo-l-tetrahydrothiophene-3-yl) -thiodiglycolic acid monoamide 13.21 g (0.1 mmol) of thiodiglycolic acid anhydride dissolved in 250 ml of anhydrous dichloromethane are added by dropping, and in an argon atmosphere, to a solution of 25 15.36 g (0.1 mol) of homocysteine thiolactone hydrochloride and 20.24 g (0.2 mol) of triethyl amine in 500 ml of anhydrous dichloromethane. This mixture is stirred for 16 hours at room temperature and washed with aqueous 2% citric acid. After drying above sodium sulfate, the solvent is 30 evaporated under reduced pressure, and the oily residue crystallized by trituration with diethyl ether. Yield: 22.73 g (91.2%), white powder Analysis referring to the anhydrous substance: ID-13317WO 18 263794 Calc.: C 38.54 H 4.45 N 5.62 O 25.67 S 25.72 Found: C 38.37 H 4.68 N 5.41 S 25.47 Example 3b 5 N- (3 -mercapto-1- (octylaminocarbonyl) -propyl) - thiodiglycolic monoamide 30 ml of octyl amine are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in Example 3a 10 in 30 ml of ethanol. The mixture is stirred at room temperature for 4 hours and evaporated in a medium high vacuum. The residue is mixed with 200 ml of aqueous 2% citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after 15 separation, is dried above sodium sulfate. The solvent is evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether. Yield: 878 mg (23.2%), white powder Analysis referring to the anhydrous substance: 20 Calc.: C 50.77 H 7.99 N 7.40 O 16.91 S 16.94 Found: C 50.48 H 8.13 N 7.15 S 16.71 Example 3c Technetium 99 -m complex of N- (3 -mercapto-1 - (octyl amino -25 carbonyl)-propyl)-thiodiglycolic acid monoamide 10 trig of the ligand produced according to Example 3b are dissolved in 1.0 ml of ethanol. 50 pil of this ligand solution are mixed with 250 /xl of phosphate buffer, pH 8.5, 50 Hi of a deoxygenated aqueous citrate solution (50 mg/ml) , 30 2.5 fxl of a deoxygenated aqueous tin(II) chloride solution (S mg/ml 0.05 N HCl), and 100 #tl of a pertechnetate solution (400-900 nCi) . After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex 263 formed using HPLC: Hamilton PRP-1 column, 5 nm, 125 x 4.6 mm; gradient eluation from 100% A to 100% B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 5 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is >95%. Example 4a N- (3-mercapto-1- (2-methoxyethylazaino carbonyl) -propyl) -10 thiodiglycolic acid monoamide 3 0 ml of 2-methoxy ethyl amine are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in Example 3a in 3 0 ml of ethanol. The mixture is 13 scirred at room temperature for 5 hours and evaporated under reduced pressure. The residue is mixed with 200 ml of aqueous 2% citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separation, is dried above sodium sulfate. The solvent is 20 evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether. Yield: 734 mg (22.6%), white powder ID-13 317WO 25 Analysis referring to the anhydrous substance: Calc.: C 40.73 H 6.21 N 8.64 O 24.66 Found: C 40.47 H6.49 N 8.38 S 19.76 S 19.51 ID-13 317WO 20 2637 Example 4b Technetium-99m complex of N-(3-mercapto-1-(2-methylethyl-amino carbonyl)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example 4a are 5 dissolved in 1.0 ml of ethanol. 50 /xl of this ligand solution are mixed with 250 /xl of phosphate buffer, pH 8.5, 50 [il of a decxygenated aqueous citrate solution (50 mg/ml) , 2.5 ill of a deoxygenated aqueous tin(II) chloride solution (5 mg/ml 0.05 N HCl) , and 100 /xl of a pertechnetate solu-10 tion (400-900 /xCi) . After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 /xm, 125 x 4.6 mm; gradient eluation from 100% A to 100% B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 15 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is >95%. Example 5a 20 N- (3-mercapto-1-(2-hydroxy-ethylamino carbonyl)-propyl)-thiodiglycolic acid monoamide 30 ml of 2-amino ethanol are added in an argon atmosphere to a solution of 2.49 g (10 mmol) of the thiolactone derivative of the thiodiglycolic acid monoamide produced in 25 Example 3a in 30 ml of ethanol. The mixture is stirred at room temperature for 4 hours and evaporated in a medium high vacuum. The residue is mixed with 200 ml of aqueous 2% citric acid and 200 ml of dichloromethane. The mixture is stirred thoroughly, and the organic phase, after separa-30 tion, is dried above sodium sulfate. The solvent is evaporated under reduced pressure, and the oily crude product is crystallized by trituration with diethyl ether. Yield: 435 mg (14.0%), white powder ID-13317WO 21 Analysis referring to the anhydrous substance: Calc.: C 38.70 H 5.85 N 9.03 O 25.77 S 20.66 Found: C 38.38 H 5.74 N 8.91 S 20.43 5 Example 5b Technetium-99m complex of N- (3-mercapto-1- (2-hydroxy-ethylaminocarbonyl)-propyl)-thiodiglycolic acid monoamide 10 mg of the ligand produced according to Example 5a are dissolved in 1.0 ml of ethanol. 50 fil of this ligand solu-10 tion are mixed with 250 fil of phosphate buffer, pH 8.5, 50 fil of a deoxygenated aqueous citrate solution (50 mg/ml) , 2.5 fil of a deoxygenated aqueous tin (II) chloride solution (5 mg/ml 0.05 N HCl), and 100 fil of a pertechnetate solution (400-900 fiCx) . After an incubation time of 10 minutes, 15 the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 /xm, 125 x 4.6 mm; gradient eluation from 1.00% A to 100% B within 7.5 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 20 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is >95%. Example 6a N- (3-mercapto-1- (carbonyl-gly-his-leu-asp-ile-ile-trp) -25 propyl)-thiodiglycolic acid monoamide 250 mg (1 mmol) of the N-(2-oxo-tetrahydrothiophene-3-yl)-thiodiglycolic acid monoamide produced in Example 3a are added in an argon atmosphere to a solution of 853 mg (1 mmol) of NH2-gly-his-leu-asp-ile-ile-trp (produced in a 30 similar way as described by Barany and Merrifield, The Peptides: Analysis, Biology, Academic Press, New York 1980; Stewart and Young, Solid Phase Peptides Syntheses, 2nd ed., Pierce Chemical W., Rockford, II, 1984) and 404 mg (4 mmol) 263794 ID-13 317W0 22 263794 of triethyl amine in 100 ml of anhydrous dimethyl formamide. The resulting reaction mixture is stirred at room temperature for 12 hours. When the reaction is finished, the solution is filtered and the solvent removed 5 under reduced pressure. The residual oil is mixed three times with 50 ml of dimethyl formamide and evaporated each time. The residue is stirred up with 200 ml of anhydrous diethyl ether. A white solid material settles down which is filtered off. The material is recrystallized from mixtures 10 of dimethyl formamide and diethyl ether for purification. Yield: 282 mg (25.6%), white powder Analysis referring to the anhydrous substance: Calc.: C 53.39 H 6.49 N 13.98 0 20.32 S 5.82 Found:' C 53.17 H 6.63 N 13.74 S 5.61 15 Example 6b Technetium-99m complex of N-(3-mercapto-1-(".arbonyl-gly-his-leu-asp-ile-ile-trp)-propyl)-thiodiglycolic acid monoamide 20 10 mg of the ligand produced according to Example 6a are dissolved in 1.0 ml of ethanol. 50 /xl of this ligand solution are mixed with 250 /xl of phosphate buffer, pH 8.5, 50 /xl of a deoxygenated aqueous citrate solution (50 mg/ml), 2.5 /xl of a deoxygenated aqueous tin(II) chloride solution 25 (5 mg/ml 0.05 N HCl) , and 100 /xl of a pertechnetate solution (400-900 /xCi) . After an incubation time of 10 minutes, the reaction mixture is tested for purity of the Tc complex formed using HPLC: Hamilton PRP-1 column, 5 /xm, 125 x 4.6 mm; gradient eluation from 100% A to 100% B within 7.5 30 minutes (eluent A: sodium hydrogenphosphate, 0.005 M, pH 7.4; eluent B: acetonitrile/sodium hydrogenphosphate, 0.005 M, pH 7.4 (75/25); 2.0 ml/min. Radiochemical purity is >97% . ID-13317WO 23 Example 7 Accumulation of N-(2-mercapto-l-(decyloxycarbonyl)-ethyl) -thiodiglycolic acid monoamide, technetium-99m complex, in atherosclerotic vascular lesions of WHHL rabbit N-(2-mercapto-l-(decyloxycarbonyl)-ethyl)-thiodiglycolic acid monoamide (produced according to Example 2a) is labelled as described in Example 3b. 99.9 GBq (2.7 mCi) of the substance labelled according to Example 3b were diluted to 1 ml with phosphate-buffered saline and administered via the ear vein to a narcotized WHHL rabbit, Rompun/Ketavet (1:2). The rabbit was killed 5 hoars after the application, and an autoradiogram of the aorta as well as a Sudan(III) staining were carried out to visualize the atherosclerotic plaques (Figure 1). The accumulation factor between normal and atherosclerotic walls was betv/een 3 and 8 depending on the thickness of the plaques (Sudan(III) staining). 26379 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> Claims l. Compounds of the general formula (I) B - CO- CRiR2 - A- CR3R4 - COOH (I) wherein A represents an 0, S, or Se chalcogen atom, R1, R2, R3, and R4 are same or different and represent a hydrogen atom and/or a branched or unbranched Ci-Cg alkyl residue, B represents a residue -NH-CR5R6- (CR7R8)n=1/2-S-R9, wherein R5 and R6 are same or different and represent a hydrogen atom or an unbranched, branched, cyclic, or polycyclic Ci-Cgo alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, aryl, alkylaryl, or arylalkyl residue which may optionally carry additional hydroxy, oxo, oxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, or alkoxy groups containing up to 20 carbon atoms, and/or may optionally be interrupted, and/or replaced, by one or several heteroatoms from the series of 0, N, S, P, As, Se, R7 and R8 are same or different and represent a hydrogen atom and/or a branched or unbranched Ci-Cg alkyl residue, R9 represents a hydrogen atom, a branched or unbranched C^Cg alkyl residue, or a sulj.^ protecting group, ^ N.Z. PATENT OFFICE 17 AUG 1S95 ID317-12.DOC *2S 263794 with R9 and R5, together with the groups that connect them, optionally forming a 4- to 8-membered ring which may optionally carry additional hydroxy, oxo, oxy, or alkoxy groups containing up to 6 carbon 5 atoms. their conjugates with substances that accumulate selectively in diseased tissues or tumours, with a covalent bond existing between these svibstances, said 10 bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain 15 aldehyde groups and their complexes with radioisotopes of Tc or Re.
2. Compounds according to Claim 1, characterized in that 20 R1, R2, R3 and R4 are hydrogen atoms and A is a sulfur atom.
3. Compounds according to Claims 1 or 2, characterized in that R5 and R6 are different, and that R6, R7 and R8 each represent a hydrogen atom. 25
4. Compounds according to Claims 1 to 3, characterized in that the substances that accumulate in diseased tissue are peptides such as endothelines, partial endotheline sequences, endotheline analogues, endotheline derivatives, or endotheline antagonists. 30
5. Compounds according to Claims 1 to 4, characterized in that the peptides comprise the following sequences or parts thereof: N.Z. PATENT OFFICE 17 AUG 1995 tOSn-13 .DOC 2«37fl 10 15 20 25 cys-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr- phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-cys-val-tyr- phe-cys-his-leu-asp-ile-ile-trp, cys-thr-cys-phe-thr-tyr-lys-asp-lys-glu-cys-val-tyr- tyr-cys-his-leu-asp-ile-ile-trp, cys-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr- phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-asn-ser-trp-leu-asp-lys-glu-cys-val-tyr- phe-cys-his-leu-asp-ile-ile-trp, cys-ser-cys-lys-asp-met-thr-asp-lys-glu-cys-leu-asn- 1 phe-cys-his-gin-asp-val-ile-trp, 30 I I ala-ser-cys-ser-ser-leu-met-asp-lys-glu-cys-val-tyr- phe-ala-his-leu-asp-ile-ile-trp, 35 ala-ser-ala-ser-ser-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, ID317-12.DOC K.'Z. PATENT OFFICE 17 AUS 1995 Ht-CSlVtv 2« 379 cys-ser-cys-ser-ser-trp-leu-asp-lys-glu-ala-val-tyr-phe-ala-his -leu-asp- ile-ile-trp , I 1 5 cys-val-tyr-phe-cys-his-leu-asp-ile-ile-trp, N-acetyl-leu-met-asp-lys-glu-ala-val-tyr-phe-ala-his-leu-asp-ile-ile-trp, 10 the partial sequence his-leu-asp-ile-ile-trp or the cyclic amino acid sequences 15 Cyclo-(Dtrp-Dasp-pro-Dval-leu), Cyclo-(Dglu-ala-alloDile-leu-Dtrp).
6. Method for the production of compounds according to 2 0 Claim 1, characterized in that compounds of the general formula (II) are reacted with compounds of the general formula (III) according to the following reaction scheme: CR^-R2 -A- CR3R4 25 | I + NH2-CR5R6-(CR7R8)n=1/2-S-R9 CO 0—CO (II) (III) B - CO - CR^-R2 - A- CR3R4 - COOH (I) 30 wherein R1, R2, R3, R4, R5/ R6, R7, R8, R9, A and B have the meanings specified in Claim 1 and that XD317-12.DOC HZ. P.AT3? SNT OFFICEj 17 AUG 1995 uCr.t'- ! v : 263794 optionally the compounds produced in this way are conjugated with substances that accumulate selectively in deseased tissues or tumours, with a covalent bond existing between these substances, said bond being amidic if the substances contain carboxy or amino groups such as peptides, proteins, antibodies or their fragments, ester-like if the substances contain hydroxy groups such as fatty alcohols, and imidic if the substances contain aldehyde groups and that optionally the compounds and conjugates produced in this way are reacted with technetium -99m or Re in the form of pertechnetate or perrhenate in the presence of a reductant and, optionally, an auxiliary ligand,
7. Radiopharmaceutical formulation for non-invasive in-vivo visualization of receptors and tissue containing receptors and/or atherosclerotic plaques characterized in that it contains a compound or a conjugate according to one of Claims 1 to 5. ; rvTiMT OFFICE 1 7 AUG 1S95 ID117-I2.DOC </div>