IE912838A1 - Reducing chelating agents, their technetium and rhenium¹complexes, process for their production as well as their use¹in diagnosis and treatment - Google Patents

Abstract

Compounds of the general formula I are capable of reducing pertechnetate to an oxidation level of less than +7 without an addition of reducing agents, forming, via the groups Y and Z, stable complexes with the technetium or rhenium isotopes which have been reduced in this manner, and, if appropriate, accumulating selectively in certain tissues or lesions due to a compound which is coupled with the aid of a functional group which is part of R<1>. The technetium and rhenium complexes of the compounds of the general formula I, and their salts with organic and inorganic acids, can be employed in in-vivo diagnostics and in therapy, preferably in tumour therapy.

Description

REDUCING CHELATING AGENTS, THEIR TECHNETIUM AND RHENIUM COMPLEXES, PROCESS POR THEIR PRODUCTION AS NELL AS THEIR USE IN DIAGNOSIS AND TREATMENT S Background of the invention Radioactive metal ions, usually bound to a complexing agent, have been used for in vivo diagnosis for some time.

Of these, technetium-99m (Tc-99m), because of its almost ideal physical properties for these purposes — good absorption of radiation in corresponding detection devices (gamma camera, spect devices) relative to a low absorption in the human organism and easy availability with a molybdenum/technetium generator — is the radionuclide most often used in clinical nuclear medicine. Its short half15 life of 6.02 hours guarantees an only slight exposure of the patient to gamma radiation, particularly since also the secondary product technetium-99 has only an insignificant residual radiation. But a drawback of the technetium is its complicated and not yet completely known complex chemistry.

Technetium can be present in a number of oxidation states (+7 to -1), and the pharmacological properties can be greatly changed by changing the charge of a complex. It is therefore necessary to use complexes which bind the technetium in a defined oxidation state and to prevent redox - 2 reactions, which could lead to a redistribution of the pharmaceutical agent.

For organ- or tissue-specific diagnosis, it is necessary that the radiopharmaceutical agents be selectively concentrated in the desired target organs or tissues and remain there for a while. This selectivity can be achieved, on the one hand, by the formation of complexes, which on their own show a specificity for certain tissues, or by coupling the technetium complexes to selective substances, such as, e.g., monoclonal antibodies.

For labeling organ-specific substances with Tc-99m, the pertechnetate eluted from the nuclide generator first has to be converted to a lower oxidation state. In this reduced form, technetium forms more or less stable compounds with the selectively concentrated substances. The special problem of labeling with Tc-99m consists in the fact that normally tin(II) ions are present in the reaction solution as reducing agents. Tin(II) is thus far the only reducing agent which makes possible a quick and quantitative conversion of the pertechnetate at room temperature to a lower and thus reactive oxidation state. There, the added tin(II) salts have to be used in a high excess (about 100:1) relative to the pertechnetate. But the tin(II) and tin(IV) ions present after the reduction has been completed, in addition to the reduced Tc-99m, compete for the binding sites of the ligands, so that either the complexing agent again has to be used in excess relative to the tin, by which the specific activity is greatly reduced or unbound Tc-99m and tin as common colloid results in undesirable storage of radioactivity in other organs. In both cases, the diagnostic informative value is reduced.

This problem can be avoided by the use of reducing ligands. In the production of diagnostic agents according - 3 to this principle, a part of the added ligand excess acts as reducing agent for pertechnetate, which reduces technetium in an oxidation state lower than +7. In this way, reduced technetium species are then complexed by the excess of the unoxidized chelating agent. In this case, it is important to obtain stable complexes in a defined oxidation state for the technetium.

DeLearie et al. (L. A. deLearie, R. C. Haltiwanger, C.

G. Pierpont; J. Am. Chem. Soc. Ill: 4324, 1989) showed that 3,5-di-tert-butylcatechols are suitable for reduction and chelation of Tc-99 (half-life: 212,000 years). The reduction took place by 24 hours of boiling in methanol.

For the labeling with the short-lived Isotope of technetium (Tc-99m; half-life: 6 hours), however, only substances are usable as radiopharmaceutical agents which can be labeled quickly and gently also in the clinic and for which no subsequent purification after the labeling with, e.g., Tc99m is necessary. The compounds described by DeLearie are thus unsuitable for the production of a clinically usable radiopharmaceutical agent.

Summary of the invention The present invention provides new reducing and tissuespecific chelating agents, as well as their stable technetium and rhenium complexes. Surprisingly, substances 25 were found which reduce and completely complex Tc-99m under mild conditions as well as quickly. Moreover, they also can be used, in contrast to the above-described compounds, for coupling per se to selectively concentrated substances in foci of disease or certain tissues.

Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art. - 4 According to the invention, compounds of general formula I, this object is achieved by <0 in which X stands for -0-, -S-, -NR2- with R2 meaning a hydrogen atom or a CV6 alkyl radical, Y and Z are the same or different and stand for the radicals -OH, -NHR3 or -SR3 with R3 meaning a hydrogen atom or a C,.6 alkyl radical and U stands for a hydrogen atom, a branched or unbranched CV6 alkyl, a Cw alkoxy, a hydroxyl or a carboxyl radical, n means the numbers 2 to 6 and m means the numbers 2 or 3 and R1 is present only if m stands for the number 2, r’ stands for a hydrogen atom, a benzyl radical or a branched or unbranched C0.A alkyl radical optionally substituted with one to three hydroxyl, carboxyl or amino groups; wherein said radicals optionally contain (i) a functional group B, (ii) a compound T which is selectively concentrated in lesions or certain tissues; or (iii) a compound T bound to the radical through a functional group; and B, if Y and/or Z stand for -NHR3, stands for an amino, a hydrazino or hydrazide, a carboxyl, a Cv6-alkynyl or alkenyl, a hydroxyl, an aminophenyl, an oxiranyl, a fluorinated phenoxycarbonyl or a biotin radical; or, if Y and Z stand for -OH or -SR3, B in addition also stands for a halogen, a formyl, a nitrile, a phenylisothiocyanate or a succinimidoxycarbonyl radical - 5 optionally substituted with a sodium sulfate radical; and T stands for monoclonal antibodies or their fragments, hormones, growth factors, ligands for cell membrane receptors, steroids, neurotransmitters, fatty acids, saccharides, amino acids and oligopeptides, biotin, as well as radiosensitizers, such as, e.g., misonidazole, and optionally present functional groups optionally in protected form or their precursors are present in R1, with the exception of compound N{CH2-CH2-CH2-O-C6H3-2,310 (OH)2)3, their technetium and rhenium complexes, as well as their salts with inorganic and organic acids.

The excluded compound N{CH2-CH2-CH2-0-c6H3-2,3-(0H)2}3 is known (B. Wolff, Angew. Chem. [Appl. Chem.] 98: 173, 1986) and was used for complexing germanium and silicon.

Physiologically compatible inorganic acids, such as, e.g., hydrochloric or sulfuric acid, and organic acids, such as, e.g., acetic or citric acid, are used as acids for salt formation.

A Co alkyl radical is to be understood to mean a direct bond from the nitrogen atom in Formula I to one of the optional substituents -Β, -T or -B-T.

According to the invention, preferred are those compounds in whose general formula I>U represents a hydrogen atom and X represents an oxygen atom, n is the number 3, Y and Z are the same and stand for OH- or NH2- and R1 represents a hydrogen atom, a benzyl radical, an unbranched C0.3 alkyl radical optionally substituted with a hydroxyl or amino group, wherein said radicals optionally contain a functional group B or antibodies or their fragments, steroids or misonidazole bound to the radical through a functional group B.

The substances according to the invention surprisingly have the advantages that they - 6 1. can reduce pertechnetate from a nuclide generator under mild conditions, quickly and without adding reducing agents to an oxidation state lower than +7, 2. form stable complexes with the thus reduced techne5 tium without further adding reducing agents at neutral pH, 3. are concentrated selectively in certain tissues or lesions because of compounds T, which are coupled with the help of functional group B, such as monoclonal antibodies or their fragments, hormones, growth factors, ligands for cell membrane receptors, steroids, neurotransmitters, fatty acids, saccharides, amino acids and oligopeptides, biotin as well as radiosensitizors, such as, e.g., misonidazole or, without containing a group T, are concentrated in certain tissues or lesions.

The formation of the technetium complexes of the abovedescribed chelates takes place with TcO4- from a nuclide generator with neutral pH without adding reducing agents in aqueous solution.

This property of the chelating agent described here offers significant advantages in comparison with previously known ligands. The incorporation of tin, which has to be added as reducing agent in the known ligand systems, is avoided in the chelate. The formation of the Tc-99m species, which are not bound by the above-described chelates (examples 6 and 7), was not observed. The chelates according to the invention are thus definitely better suited for diagnostic purposes than the previously known chelates.

Their production takes place in that amines of general formula II R1*— N-(-(CH2)n—Nu)e in which Nu stands for a nucleofuge, I, CH3C6H4SO3-, CH3SOj- or CFjSOj-; (ID such as, e.g., Cl, Br, - 7 and R1' stands for a substituent R1, whose optionally present functional groups are present in protected fora or as their precursors, and which contains no selectively concentrated compound T, and aromatic substances of general formula Ill in which U' stands for a substituent U, whose hydroxy or carboxyl radical is present in protected form, and Y* and z· stand for Y and Z or their precursors or in protected form, are reacted under base catalysis in polar solvents at temperatures of 50-200°C within 6 hours to 6 days, preferably 2 hours to 4 days, and then functional group B optionally contained in R1 or desired aromatic substance substituents Y and Z are generated, optionally the thus obtained couplable or complexable compounds are coupled with the respective desired selectively concentrating compound T or complexed with the respective desired technetium or rhenium isotope — and the sequence of the steps coupling on T and complexing with the technetium or rhenium isotope can be interchanged — and then the still present protective groups are removed or the precursors are converted to the finally desired substituents.

As hydroxy protective groups, e.g., the benzyl, 4-methoxybenzyl, 4-nitrobenzyl, trityl, diphenylmethyl, trimethylsilyl, dimethyl-t-butylsilyl and diphenyl-tbutylsilyl groups are suitable. In the case of polyols, the hydroxy groups can also be protected in the form of ketals with, e.g., acetone, acetaldehyde, cyclohexanone or benzaldehyde. Further, the hydroxy groups also can be present, e.g., as THP ether, α-alkoxyethyl ether, MEM ether or as esters with aromatic or aliphatic carboxylic acids, such as, e.g., acetic acid or benzoic acid.

The hydroxy protective groupe can be released according to the methods in the literature known to one skilled in the art, e.g., by hydrogenolysis, reductive cleavage with lithium/ammonia, acid treatment of ethers and ketals or alkali treatment of the esters (see, e.g., Protective Groups in Organic Synthesis, T. W. Greene, John Wiley and sons 1981).

As acid protective groups, lower alkyl, aryl and aralkyl groups, for example, the methyl, ethyl, propyl, n-butyl, t-butyl, phenyl, benzyl, diphenylmethyl, triphenylmethyl, bis(p-nitrophenyl)-methyl group, as well as trialkylsilyl groups, are suitable.

The cleavage of the protective groups takes place according to processes known to one skilled in the art, for example, by hydrolysis, hydrogenolysis, alkaline saponification of the esters with alkali in aqueousalcoholic solution at temperatures of 0 to 50°C, acidic saponification with mineral acids or in the case of, e.g., tert-butyl esters with the help of trifluoroacetic acid.

The starting materials for the production of the compounds of this invention are all either commercially available or routinely synthesizable by one of ordinary skill in the art using conventional synthetic methods. For the production of compounds of general formula I with Y and Z meaning SH groups, the starting materials are ligand precursors of general formula III with Y' and 2' meaning SR3 radicals. The cleavage of the protective groups after the reaction with the amines of general formula II takes place, - 9 alternatively with alkali alkylthiolates, alkali alcoholates or alkali metals, preferably with sodium methylthiolate in a polar solvent, preferably in HMPT, DMF or dimethylacetamide♦ As amino protective groups, e.g., trifluoroacetyl, t-butoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzoxycarbonyl and acetyl groups are suitable. The amino protective groups can be cleaved according to methods known in the literature, e.g., by basic or acidic hydrolysis, rpAtr-fWft cleavage with zinc in acetic acid or hydrogenolysis.

If the ligand precursor of formula III contains functionalized aromatic substances with Y' and Z‘ meaning NO2 groups, then the production of the chelating agent according to claim 1 takes place by reduction, preferably with tin in hydrochloric acid solution.

Radical R1' can be modified in compounds according to general formula I, in which m means the number 2. If R , for example, is a benzyl group, it can be removed by reaction with hydrogen under increased pressure and increased temperature in the presence of a palladium catalyst. Radical R1 is then a hydrogen atom.

Rhenium 186 has a physical half-life of 3.7 days and emits beta particles with an energy of l.l MeV suitable for the treatment of, e.g., tumors, and, at the same time, gamma radiation with an energy of 137 keV (9% frequency). Rhenium is found in periodic systems in group VII A directly under technetium and exhibits a practically identical structural and chelate chemistry as technetium. These properties make rhenium 186 into an ideal isotope for therapeutic uses (damage of diseased tissue by particular beta radiation) with the possibility at the same time for diagnostic study of a concentration with the help of the portion of gamma radiation. Rhenium 188, which also can be used for - 10 treatment of tumors, has a significantly shorter half-life of 17 hours and a beta energy of 2.1 MeV. Also, rhenium 188 has a gamma radiation portion (155 keV; 15%) and can thus also be used for treatment and, at the same time, detection with a gamma camera, e.g., according to the methods disclosed in Fritzburg et al., N. Nucl. Med. 30. 743 (1989).

If the chelating agents complexed with a radioactive isotope show no selectivity for lesions or certain tissues, it is necessary that they be coupled to a selective substance. Radical R1 is suitable for this purpose, e.g., with the help of functional group B, to produce a stable connection to proteins or other selectively accumulating molecules. By the corresponding selection of the functional group, the coupling is possible under mild reaction conditions, which do not influence the biological function and/or selectivity.

The coupling to the described compounds also takes place according to methods known in the art (e.g., Fritzberg et al.; J. Nucl. Med.: 26. 7 [1987]), for example, by reaction of group B with nucleophilic groups of the selectively accumulating molecule or, if a nucleophile is involved in the case of group B itself, with activated groups of the selectively accumulating molecule.

Group B represents every substituent which, on the one hand, represents a functional group, which makes possible a coupling to a selectively accumulating molecule under mild conditions (e.g., by acylation or amidation) as well as every activated group, which can react with nucleophilic groups of proteins, antibodies, hormones or other biomolecules, such as the amino, phenol, sulfhydryl, aldehyde or imidazole group. By an activated group is to be understood a function which is capable of reacting with the formation of a conjugate with a nucleophilic substituent of - 11 a selective molecule or of the complex ligand itself in aqueous solution within a suitably short time, under reaction conditions which, result in neither denaturing nor loss of the biological activity or selectivity. Examples in this respect are imide esters, alkylimide esters, aroidoalkylimide esters, succinimide esters, acylsuccinimides, phenol esters, substituted phenol esters, tetrafluorophenol esters, anhydrides, hydrazides, alkyl halides and Michael acceptors. B is preferably a monoanhydride, acid chloride, acid hydrazide, mixed anhydride, activated ester (such as phenol or imide ester), nitrene or isothiocyanate, in particular for the coupling with nucleophilic groups of amino acids or an aliphatic or aromatic primary amine for the coupling to carbohydrate radicals of proteins.

If a nucleophile is involved in the case of group B itself, it can react with activated groups of a selectively accumulating molecule, and also reacted groups of the selective molecule are enclosed with so-called crosslinking reagents. These can be homofunctional crosslinkers having two identical functional groups, e.g., imidoester groups or N-hydroxysuccinimide ester (NHS) groups. Alternatively, these crosslinking reagents can be, for example, heterobifunctional crosslinkers, which contain two different functional groups, for example, two of an NHS ester, a pyridyl disulfide and an activated halogen, such as an α-keto-halide. Such crosslinkers can be obtained commercially.

Compounds which selectively accumulate compounds in certain tissues or lesions are used as coupling partners.

Often, the selective accumulating of these substances could already be shown by labeling with positron-emitting isotopes (PET technique), iodoisotopes or other coupling partners. Compounds labeled with Tc-99m have already partially come - 12 into use. See, e.g., Chiton, H.M. and Witcofski, R.L., Nuclear Pharmacy: An Introduction to the Clinical Application of Radiopharmaceuticals; Lea & Febiger, Philadelphia, PA (1986) . But technetium-labeled compounds in such a way have the drawback that tin (II) ions have to be added as reducing agent, which results in the abovedescribed consequences (reduced specific radioactivity and the possibility that unbound Tc-99m together with tin as colloid results in an undesirable storage of radioactivity in other organs and a reduced diagnostic informative value).

Ligands which bind to specific receptors can recognize a changed tissue in their receptor density; they include, i.a., peptide and steroid hormones, growth factors and neurotransmitters. With ligands for steroid hormone is receptors, the possibility of an improved diagnosis of breast and prostate cancers was demonstrated (S. J. Brandes & J. A. Katzenellenbogen, Nucl. Med. Biol. 15:53, 1988). Often, ligands labeled with positron-emitting isotopes could be used for neuroreceptors for the diagnosis of various brain diseases (J. J. Forst, Trends in Pharmacol. Sci. 7: 490, 1987). occasionally, tumor cells exhibit a changed density of receptors for peptide hormones or growth factors, such as, e.g., the epidermal growth factor” (EGF). The concentration differences could be used for selective concentration of cytostatic agents in tumor cells (E. AboudPirak et al., Proc. Natl. Acad. Sci. USA 86: 3778, 1989).

Other biomolecules are metabolites that can be put in the metabolism of cells which make a changed metabolism recognizable; they include, for example, lipids (also in the form of liposomes), saccharides, porphyrins, peptides and amino acids. Fatty acids coupled with Tc chelating agents were described in EPA 0 200 492. other metabolic products such as saccharides (deoxyglucose), lactate, pyruvate and - 13 amino acids (leucine, methylmethionine, glycine) were used with the help of the PET technique for graphic display of changed metabolic processes (R. Weinreich, Swiss Med. 8, 10, 1986). Certain porphyrins showed a concentration in tumors (P. A. Scourides, Cancer Res. 47: 3439, 1987).

Also, nonbiological substances such as misonidazole and its derivatives, which are bound irreversibly to cell components in tissues or tissue parts with reduced oxygen concentration, can be used for specific concentration of radioactive isotopes and thus graphic display of tumors or ischemic regions (Μ. E. Shelton, J. Nucl. Med. 30: 351, 1989) . Other suitable nonbiological substances include cytostatic agents, such as bleomycin, which accumulate in tumors. Also, suitable polymers such as dextrans, polyethylenimines, polyamides, polyureas, polyethers and polythioureas are suitable as coupling partners.

The compounds according to the invention containing biotin make possible the binding of radioactive conjugates to substances containing avidin or streptavidin. This can be used to concentrate antibody-streptavidin conjugates on the tumor and only later to apply the radioactive component containing biotin, which results in a reduced exposure of the patient to radiation (0, J. Hnatowich et al., J. Nucl. Med. 28: 1294, 1987). Finally, the direct coupling of the bifunctional chelating agents to proteins, such as, e.g., monoclonal antibodies or their fragments, albumin, enzymes (e.g., urokinase, streptokinase), fibrin, fibrinogen or myosin, is also possible.

By complexing the conjugates with Tc-99m or rhenium isotopes, a diagnosis and treatment of tumors or other diseases is made possible. In this case, it is unimportant whether a labeling of the chelating agents with Tc-99m or a rhenium isotope is performed before or after the coupling to - 14 the selectively accumulating molecule. But for a coupling to the selectively accumulating molecule after a complexing, the requirement is that the reaction of the radioactive complex with the accumulating compound occurs quickly and almost quantitatively under mild conditions, and that no subsequent purification is necessary.

The production of the pharmaceutical agents according to the invention takes place in a way known in the art, in which the complexing agents according to the invention are dissolved --optionally by adding the additives usual in galenicals — in aqueous medium and then sterilized by filtration. Suitable additives are, for example, physiologically harmless buffers (e.g., tromethamine), small additions of electrolytes (e.g., sodium chloride), stabilizers (e.g., gluconate or phosphonate) and small amounts of oxidizing or reducing agents (10-500 micrograms/dose). The pharmaceutical agent according to the invention is present in the form of a solution or in freezedried form and is mixed shortly before the administration with a Tc-99m-pertechnetate solution, eluted from commercially obtainable generators, or a perrhenate solution.

In the case of the nuclear medicinal in vivo use, the agents according to the invention are administered in amounts of 1 · 10 to 5 · 10 nmol/kg of body weight, preferably in amounts between 1 10'3 and 5 · 102 nmol/kg of body weight. Starting from an average body weight of 70 kg, the amount of radioactivity for diagnostic uses is between 0.05 and 50 mCi, preferably 5 to 30 mCi per administration.

For therapeutic uses, between 5 and 500 mCi, preferably 10350 mCi, is administered. The administration is normally performed by intravenous, intraarterial, peritoneal or intratumoral injection of o.l to 2 ml of a solution of the agents according to the invention. The intravenous administration is preferred.

The following examples are used to explain the object of the invention in more detail.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications, cited above and below, and of corresponding application Federal Republic of German P 40 25 788.6, filed August 10, 1990, are hereby incorporated by reference. - 16 EXAMPLES Example 1: Tri(β-Carbethoxyethyl)amine, l and di(Boarbethoxyethyl) amine, 2 300 ml of freshly distilled ethyl acrylate is brought to reaction with 300 ml of liquid ammonia for one day in a sealing tube and, after removal of the final ammonia residues from the water bath, the resulting product mixture is fractionated in a vacuum. The first fraction forms bis10 (β-carbethoxyethyl)amine, 2, (boiling point 97-110°C/0.05 mbar), the main fraction consists of the desired product, 1, (boiling point 120-133°C/0.05 mbar).

Yield: 144 g (49%) of tri(β-carbethoxyethyl)amine, 1 ’H-NMR (CDClj, i, ppm): 4.10 (q, 2H, C (0) OCH2CH3) ? 2.74 (t, 2H, NCH2CH2) ; 2.41 (t, 2H, CH2CH2C(O)O) ; 1.23 (t, 3H, OCH2CH3) 13C-NMR (CDC13, 6, ppm): 172.2 (CH2C(O)O); 60.1 (C(O)OCH2CH3) ; 49.1 (NCH2CH3) ; 32.8 (CH2CH2C(O)O) ; 14.1 (OCH2CH3) For di(β-carbethoxyethyl)amine 2, it was found: ’H-NMR (CDClj, £, ppm): 4.12 (q, 4H, C(O}0CH2CHj) ; 2.88 (t, 4H, NCH2CH2) ; 2.47 (t, 4H, CH2CH2C(O)O) ; 1.60 (S, br, 1H, HN(CH2)2); 1.24 (t, 6H, OCHzCH3) Tris(3-Hydroxypropyl) amine, 3 g (0.7 mol) of lithium aluminum hydride in 900 ml of absolute ether is suspended in a 2-liter three-necked flask with dropping funnel and reflux condenser. A solution of 77 g (0.24 mol) of ester 1 in 200 ml of absolute ether is instilled in it within one hour so that the solution boils moderately. After five hours of stirring at 25°C and - 17 careful hydrolysis of excess hydride with water, the product is separated by a Buchner funnel from precipitated hydroxides. After removal of the solvent in a vacuum, the residue is briefly boiled up in ethanol, additional LiAl(OH)4 is filtered off by suction by a frit (D3), the alcohol is drawn off and the remaining liquid is taken up in methylene chloride. Pressureless filtering yields a honey yellow, highly viscous liquid by another frit (D4) and removal of the solvent in a vacuum.

Yield: 27.3 g (59%) 1H-NMR (CDC13, S, ppm): 3.78 (t, 2H, CH2CH2OH) ; 2.62 (t, 2H, NCH2CH2) 7 1.80 (q, 2H, ch2ch2ch2) ; UC-NMR (CDC13, 5, ppm): 60.6 (CH2CH2OH) ; 51.3 (NCHjCHz) 7 28.1 (CH2CH2CH2) Tria(3-Chloropropyl)amine, 4 18.9 g (160 mmol) of thionyl chloride is added to 8.6 g (45 mmol of tris(3-hydroxypropyl)amine, 3, dissolved in 80 ml of chloroform. Thus, an insoluble white mass results, which slowly dissolves again. After the reaction mixture has been refluxed for three hours, excess SOC12 is hydrolyzed with water after cooling off. The organic phase is shaken out four times with 50 ml of hot water, the combined aqueous phases are made strongly alkaline with 40% sodium hydroxide solution and then are extracted four times with 80 ml of ether each. After drying on Na2SO4 and removal of the ether in a vacuum, the yellowish residue is fractionated, and the product goes over as colorless liquid and crystallizes out after prolonged standing at room temperature. Tris(3-chloropropyl)amine can be recrystallized from ethanol (3 g of 4 for 7 ml of ethanol). - 18 Yield 9.6 g (87%) Melting point: 35°C Boiling point: 120°c/0.05 mbar 1H-NMR (CDClj, S, ppm): 3.60 (t, 2H, CH2CH2C1); 2.52 (t, 2H, NCH2CH2) ? 1.88 ppm (q, 2H, CH2CH2CH2) 13C-NMR (CDC13, 6, ppm): 50.5 (CH2CH2C1) } 43.0 (NCH2CH2) ? 30.1 (CH2CH2CH2) 2,2-Dimethyl-l,3-benzodioxol-4-ol, 5 77 g (0.61 mol) of pyrogallol in 250 ml of absolute toluene is suspended and heated in a 500-ml two-necked flask with dropping funnel and a Widmer spiral column. When the solvent begins to boil, 75 ml (0.61 mol) of 2,2dimethoxypropane is added. After that, distillate continuously passes over at the column head at about 60°C.

After two hours, another 75 ml of dimethoxypropane is added. After the temperature at the column head drops (about six hours), the reaction mixture is refluxed overnight for completion of the reaction. The cooled solution is freed in a vacuum from toluene and the viscous residue is distilled over a bridge with large cross section. As a result, the product already crystallizes out on the bridge wall and has to be transferred to the receiver by heating. The distillation temperature is selected so that existing yellowish impurities distill over only to a limited extent. The slightly yellowish product can be sublimated at 75°C, 0.2 mbar. The acetal is readily soluble in acetone and methanol, slightly soluble in chloroform.

Variants for working up: After removal of the toluene, the crude product is taken up in so much hot carbon tetrachloride, that it just about dissolves. With cooling off to room temperature, white product 5 crystallizes out from the solvent. It is sublimated according to the same conditions as described. A significant difference in the yields is not observed.

Yield: 50 g (50%) Melting point: 90qC 1H-NMR (CDC13, S, ppm): 6.68 (t, 1H, Ar-H); 6.46 (d, 1H, Ar-H); 6.40 (d, 1H, Ar-H); 5.13 (s, 1H, Ar-OH); 1.69 (s, 6H, CH3) 13C-NMR (MeOD, S, ppm): 149.7, 141.8, 135.3, 122.1 (Ar) ; 118.6 (C(CH3)2) ; 111.3 (Ar) ; 25.8 (C(CH3)2) Tris(3-(2,2-Dimethyl-l,3-benzodioxol-4-yloxy)-propyl]amine, « .1 g (121 mmol) of dry pyrogallol acetal 5 is intro15 duced in a stung-out 250-ml two-necked schlenk flask with a reflux condenser and dissolved in 100 ml of absolute (99%) ethanol. For removal of the final oxygen residues, the flask is alternately degassed five times and aerated with argon. Then, 4.75 g (121 mmol) of potassium metal is added in small pieces. First, white potassium phenolate precipitates from the solution, which is at once dissolved again. Now, a likewise degassed solution of 9.6 g (39 mmol) of tri(3-chloropropyl)amine and 20 ml of ethanol is sprayed in the now extremely oxygen-sensitive solution. After the solution has been refluxed for four days, 4 ml of glacial acetic acid is added, precipitated KCl is filtered off from the still hot but no longer air-sensitive solution and the filter cake is rewashed with a little hot ethanol. By allowing it to stand at room temperature, the ligand precursor crystallizes out slowly. The crystals are filtered off and rewashed with ice-cold ethanol. A second fraction can be obtained by concentrating the mother liquor by evaporation. - 20 Yield: 11.5 g (46%) Melting point: 65°C ’H-NMR (CDC13, S, ppm): 6.66 (t, IH, Ar-H); 6.42 (d, IH, Ar-H); 6.26 (d, IH, Ar-H); 3.95 (t, 2H, CH2CH2O) ; 2.59 (t, 2H, NCH2CH2) i 1.91 (q, 2H, CHjCHjCHj); 1.69 (s, 6H, C(CH3)2) 13C-NMR (CDC13, S, ppm): 148.3 (Ar); 143.2 (Ar) ; 135.2 (Ar) ; 121. (Ar) ; 117.9 (C(CH3)2); 107.9 (Ar); 102.0 (Ar) ; 66.8 (CH2CH2O) ; 49.8 (NCHjCHj) ; 26.9 (CH2CH2CH2) ; 25.7 (C(CH3)2 Tris- (3- (2,3-Dihydroxyphanoxy) propyl} aminohydrochloride t 7 39.6 g (62 mmol) 6 is dissolved under argon in 250 ml of glacial acetic acid and heated to boiling. 200 ml of a mixture of 50% glacial acetic acid, 20% water and 30% fuming hydrochloric acid is instilled in it within two hours. The solvent is distilled off, so that about 200 ml remains in the flask. The solution is slowly cooled off. The precipitated yellowish crystals are filtered off and recrystallized in a little hot glacial acetic acid. The white powder thus obtained is dried at 90°C, 10J mbar for two days on an oil pump vacuum.

Yield: 31.7 g (92%) Melting point: 190°C ’h-NMR ( [DJ-DMSO, 6, ppm): .47 (s, br, 1/3H, NH)? 8.93 (s, br, IH, OH); 8.19 (s, br, IH, OH); 6.42-6.54 (m, 3H, Ar-H); 4.02 (m, br, undissolved, 2H, CH2CH2O) ; 3.40 (m, br, undissolved, 2H, NCH2CH2) ; 2.17 (m, br, undissolved, 2H, CH2CH2CH2) Uc-NMR ((DJ-DMSO, i, ppm): 147.5, 146.2, 134.7, 118.5, 109.6, 105.2 (Ar); 66.2 (CH2CH2O) ; 49.9 (NCH2CH2) ; 23.4 (CH2CH2CH2) - 21 Example 2: Benzyl-di(β-carbethoxyethy1)amine, β 172 g (1.60 mol) of benzylamine is introduced in 500 ml of ethanol and mixed under ice cooling with 384 g (3.84) mol of acrylate. The reaction mixture is stirred for 5 days at room temperature. Solvents and excess feedstocks are drawn off in a rotary evaporator. The remaining solution is fractionated in a vacuum.

Fraction 1: less than 140 degrees/0.05 mbar 10 Fraction 2: 140-145 degrees/0.05 mbar Fraction 3: 145-148 degrees/0.05 mbar Fraction 4: 145-150 degrees/0.05 mbar Yield: 380 g (77%) 8 from fraction 4 ’h-NMR (CDCIj, S, ppm): 7.27 (m, 5H, Ar-H); 4.10 (q, 4H, C (0) OCH2CH3) 7 3.59 (S, 2H, C6H5CH2N) ; 2.80 (t, 4H, NCH2CH2) 7 2.46 (t, 4H, CH2CH2C(O) ) ; 1.23 (t, 6H, OCH2CH3) UC-NMR (Ct^OD, &, ppm): 173.7 (CH2C(O)O)7 140.2, 129.7, 129.0, 127.9 (ar); 61.1 (OCH2CH3) 7 59.1 (C^CHjN) 7 51.1 (NCH2CH2) 7 33.5 (CHZCH2C(O) ) ; 14.5 (OCH2CH3) Benzyl-bis(3-hydroxypropyl)amine, 9 g (0.5 mol) of lithium aluminum hydride is introduced in 900 ml of ether and 92 g (0.3 mol) of ester β is instilled slowly under ice cooling. The solution is stirred for 12 hours at room temperature and then carefully hydrolyzed with water. The ether and the aqueous phase are decanted from precipitated LiAl(OH)4. The solid is washed several times with ether. The combined organic phases (ether and ethanol) are separated from water in a separating funnel, dried on MgSO4 and filtered. After removal of the - 22 solvent in a vacuum, the product remains as colorless liquid.

Yield: 60.5 g (90%) 1H-NMR (CdCl3, S, ppm): 7.30 (m, 5H, Ar-H); 4.11 (s, br, 2H, CHZOH) ; 3.67 (t, 4H, CH2CH2OH) ; 3.56 (s, 2H, C^CHjN) ; 2.61 (t, 4H, NCH2CH2) ; 1.75 (q, 4H, CH2CH2CH213C-NMR (CDjOD, i, ppm): 137.8, 130.1, 129.2, 128.0 (Ar) ? 61.9 (CH2CH2O) ; 59.5 10 (C6H5CH2N) ; 52.3 (NCH2CH2) ; 30.2 (CH2CH2CH2) Benzyl-bis(3-chloropropyl)amine, io 143 g (0.64 mol) of 9 is introduced in 600 ml of chloroform. 182 g (1.53 mol) of thionyl chloride, dissolved in 100 ml of chloroform, is instilled in it at room temperature. The addition has to take place so that the solvent boils moderately. After completion of the addition, it is refluxed for 3 hours. The cooled solution is carefully hydrolyzed with water and washed twice with 300 ml of hot water. Now, the organic phase is strongly concentrated by evaporation and further shaken out twice with 250 ml of hot water each. After combining the aqueous phases, the latter are made strongly alkaline with sodium hydroxide solution (40%) and extracted twice with 400 ml of ether each. The combined ether extracts are dried on sodium sulfate. Then the solvent is drawn off on a rotary evaporator. The crude product is fractionated.

Yield: 140 g (84%) Boiling point: 114-125°C ’h-NMR (CDClj, 6, ppm): 7.30 (m, 5H, Ar-H); 3.58 (t, 4H, CH2CH2C1) ; 3.55 (S, 2H, C6H5CH2N) ; 2.58 (t, 4H, NCH2CH2) ; 1.92 (q, 4H, CH2CH2CH2) AUG-06-’91 18:22 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P24 - 23 13C-NMR (CDjOD, δ, ppm): 140.3, 129.9, 129.2, 128.0 (Ar)ί 59.8 (C6H5CH2N) ; 51.9 (NCH2CH2) ; 43.8 (CH2CH2C1) i 31.3 (CH2CH2CH2) Benzyl-bis[(3-(2,2-dimethyl-1,3-benzodioxol-45 yloxy]propyl)amine, n 13.3 g (80 mmol) of ketal 5 is dried for a half hour in a high vacuum at 50 degrees in a stung-out 250 ml two-necked Schlenk flask. Then 5 is dissolved in 100 ml of absolute (99%) ethanol. The solution is evacuated several times 10 until boiling and aerated with argon. Potassium (3.1 g, 80 mmol), which is cut until clear, is added and oxygen-free chloride 10 (9.7 g, 37.3 mmol) is added to the now oxygensensitive solution. The batch is refluxed for 3 days. 3 ml of glacial acetic acid is added for working up and the 15 precipitated potassium chloride is filtered off still hot.

The filtrate is drawn dry and dissolved in a mixture of about 30 ml of pentane/ether 1:1. The brown solution is eluted on a short column (about 50 g of silica gel) with ether/pentane 1:1. Here, attention must be paid that dark20 colored products are not coeluted. After the removal of the mobile solvent, a yellow oil remains, which is recrystallized from ethanol (16 g in 200 ml of ethanol). In doing so, the product accumulates at room temperature as colorless crystals.

Yield: 16 g (80%) Melting point: 46-47°C 1H-NMR( [D6] -acetone, «5, ppm): 7.30 (dd, 2H, benzyl aromatic substance); 7.20 (ro, 3H, benzyl aromatic substance); 6.67 (dd, 2H, catechol); 6.41 (dd, 4H, catechol); 4.07 (t, 4H, CH2CH2O) ; 3.58 (s, 2H, C6H5CH2N) ; 2.61 (t, 4H, NCH2CH2) ; 1.91 (q, 4H, CH2CH2CH2) ; 1.60 (s, 12H, C(CH3)2) - 24 ,3C-NMR (CDjOD, &, ppm): 149.3, 144.1 (catechol); 140.7 (benzyl aromatic substance); 136.1 (catechol); 129.4, 128.8, 127.4 (benzyl aromatic substance); 121.9 (catechol); 118.4 (C(CHj)2); 109.4, 102.6 (catechol); 67.8 (CH2CH2O) ; 59.3 (C6HjCH2N) ; 50.7 (NCH2CH2) ; 27.9 (CH2CH2CH2) ; 25.8 (C(CH3)2) Benzyl-bis[(3-(2,3dihydroxyphenoxy)propyl]aminohydrochloride, 12 10 14 g (27 mmol) of ligand precursor ll is dissolved in 100 ml of glacial acetic acid and heated to boiling, loo ml of an acid mixture (50% glacial acetic acid, 20% water, 30% fuming hydrochloric acid) is added to it within two hours, and the liquid loss resulting from distilled-off solvent is compensated for by acetone that is being liberated. After completion of the addition, so much solvent is distilled off that about 50 ml remains in the flask. The hot solution is slowly cooled off. But the product cannot be precipitated in this way. If all solvent is removed, the ligand accumulates as voluminous residue. This crude product is liberated from the final acetic acid residues by washing with ether and is dried in a high vacuum.

Yield: 12.4 g (96%) ’h-NMR( [DJ-DMSO, S, ppm): .85 (s, br, 1H, NH); 8.98 (S, 2H, OH); 8.17 (s, 2H, OH); 7,65 (d, 2H, benzyl aromatic compound); 7.43 (m, 3H, benzyl aromatic compound); 6.53 (t, 2H, catechol); 6.43-6.38 (m, 4H, catechol); 4.39 (s, br, 2H, C6HSCH2N); 3.97 (t, br, CH2CH2O) ; 3.27 (t, br, NCH2CH2) ; 1.92 (q, br, 4H, CH2CH2CH2) AUG-06-’91 13:24 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 8266 P26 - 25 nC-NMR (CDjOD, S, ppm): 148.3, 147.0, 135.7 (catechol)? 132.2, 131.1, 130.4, 130.4 (benzyl aromatic compound); 120.3, 110.6, 106.6 (catechol); 67.9 (CH2CH2O); 58.5 (C6H5CH2N); 52.5 (NCH2CH2) ; 24.8 (CH2CH2CH2) bia [3- (213-Dihydroxyphenoxy) propyl) aminohydrochloride, 13 g (21 mmol) of 12, is dissolved in 300 ml of absolute methanol and mixed with 2 g of Pd(OH)2/C (20%). In a hydrogenation unit, the mixture was shaken with a hydrogen pressure of 3 bars for six hours at room temperature. The catalyst is filtered off and the solvent is drawn off. The oily residue is dried for 24 hours at 50°C in a high vacuum.

Yield: 7 g (70%) 1H-NMR ([Ds]-pyridine, S, ppm): 8.96 (s, 6H, OH and NHZ) ; 6.90-6.30 (m, 6H, catechol); 3.97 (t, 4H, CH2CH2O) ; 3.17 (t, 4H, NCH2CH2) ? 2.28 (q, 4H, CH2CH2CH2) 13C-NMR ([Ds] -pyridine, 6, ppm): 148.8, 148.2, 136.5, 119.7, 111.0, 106.0 (catechol); 67.6 (CH2CH2O) ; 46.5 (NCH2CH2) ; 26.8 (CH2CH2CH2) Example 3: bis [3-(2,2-Dimethyl-l,3-benzodioxol-4-yloxy) propyl] amine, 14 17.9 g (34.5 mmol) of 11 is dissolved in a hydrogenation flask in 300 ml of absolute methanol, mixed with 2.0 g (2.8 mmol) of catalyst (Pd(OH)2/C) and shaken for four hours in a hydrogen hydrogenation apparatus at 3 bars of H2 pressure and 25°C. Then, the catalyst is filtered off and the solvent as well as resulting toluene are removed in a water jet vacuum. The resulting oil is dried at 60°C/0.05 mbar for six hours.

HUd-tlO- yi ia: ItL MJ: r03-^4.j-b4ltd S2bb P2Y Yield: 12.9 g (87%) ’H-NMRiCDClj. 8, ppm): 6.69 (dd, 2H, catechol)} 6.46 (d, 2H, catechol); 6.43 (d, 2H, catechol); 4.14 (t, 4H, CH2CH2O) ; 2.83 (t, 4H, NCH2CH2); 1.99 (q, 4H, CH2CH2CHZ) ; 1.68 (S, 12H, C(CH3)2) n-NMR([D6]-benzene, 8, ppm): 149.3, 143.9, 136.2, 121.6 (catechol); 117.9 (C(CH3)2); 109.1, 102.6 (catechol); 67.9 (CH2CH2O) ; 46.6 (NCl^CHz) ; 29.8 (CH2CH2CH2) ; 25.7 (C(CH3)2 bis[3-(2,3-Dihydroxyphenoxy)propyl)aaiiaohydroehloride, 13 g (18.6 mmol) of 14 is dissolved in 80 ml of glacial acetic acid and mixed in boiling heat within two hours with 80 ml of an acid mixture (50% glacial acetic acid, 30% water, 20% fuming hydrochloric acid). In this case, the solvent distills off with acetone that is liberated as azeotrope. After completion of the addition, it is distilled for another half hour. Then, the residual solvent is drawn off on a rotary evaporator. The remaining residue is dried at 60°c/0.05 mbar for six hours in a high vacuum.

The powdery product is washed with ether. Ether residues are then removed on the oil pump.

Yield: 6.25 g (87%) The analytical data is identical with that of 13 from 12.

Example 4: 2,3-Dinitrophenol, 15 .0 g (108 mmol) of 3-nitrophenol is dissolved in 150 ml of ethanol. 30 g (124.5 mmol) of Cu(NO3)2-(3 H2O) is added to it. The reaction mixture is then refluxed to boiling for 20 hours. The solvent is drawn off at a rotary evaporator. The solid residue is dissolved in 2 M HCl and - AUG-06-’91 13:25 ID:MILLEN WHITE 2ELAN0 TEL NO:703-243-6410 «266 P2S - 27 extracted four times with 50 ml of ether each. The combined ether extracts are dried on Na2SO4 and liberated from solvent. The orange solid (21 g) is chromatographed on a short column on about 60 g of silica gel with petroleum ether/ethyl. First, 3,6-dinitrophenol is eluted, followed by 3-nitrophenol and 3,4-dinitrophenol. Finally, the desired 2,3-dinitrophenol is obtained. The desired product can be recrystallized from benzene/petroleum ether (7:93, v:v) .

Yield: 2.9 g (14.6%) Melting point: 146°C ^H-NMR (CDCl3, 5, ppm): 7,18-7.79 (m, 3H, ArH) ; 9.90 (s, br, 1H, Ar-OH) UC-NMR (CDjOD, £, ppm): 151.87 (C-OH); 142.17 (C-NO2(m); 134.22 (C-N02(o))? 132.27 (C-HO; 124.54 (C-H); 116.12 (C-H) Benzyl-bis[(3-(2,3-dinitrophenoxy)propyl]amine, 16 0.92 g (5 mmol) of 2,3-dinitrophenol is dissolved in 10 ml of ethanol. 0.28 g (5 mmol) of KOH in 25 ml of ethanol 20 is added to it under argon. In doing so, the potassium salt precipitates as red solid. 0.65 g (2.5 mmol) of benzylbis(3-chloropropyl)amine, 10 in 5 ml of ethanol is added to it, and the solid is partially dissolved. With subsequent heating to the boiling temperature of the ethanol, the solid is completely dissolved. The red solution is now refluxed for 24 hours. A precipitate of KCl forms. The suspension is filtered hot. With cooling, 16 crystallizes out in the form of colorless feathers. The product is recrystallized from ethanol.

Yield: 0.76 g (55%) Melting point: 98-104°C 1H-NMR ( CDC13, 6, ppm) : 1.91 (q, 4H, CH2CH2CH2) ; 2.60 (t, 4H, NCH2) 7 3.58 (s, 2H, C6H5CH2); 4.15 2) 7 7.21 (s, 5H, benzylH); 7.22-7.81 (m, 6H, catechol-H) 13C-NMR (CDC13, $, ppm)i 26.70 (CH2CH2CH2) 7 49.81 (NCH2) ,’ 58.77 (^8,0¾) 7 68.57 (00¾) 7 116.08, 119.56 (CH(phenyl)): 126.89, 128.20, 128.74 (CH(benzyl)): 131.04 (CH(phenyl)): 134.99 (C(N02-o)): 139.06 (C(benzyl)): 140.59 (C(N02-m)), 151.37 (C(O-phenyl)) Benzyl-bis[3-(2,3-<5iaminophenoxy) propyl Janine, 17 1.06 g of tin (8.93 mmol) is added to 5 ml of concentrated hydrochloric acid. A solution of 0.5 g (0.9 mmol) of 16 in 5 ml of methanol is sprayed to it. The reaction mixture is heated for 30 minutes to 50°C, and its color becomes brown. Then the reaction mixture is poured into a solution of 2.5 g of NaOH in 50 ml of water. This mixture is extracted five times with 15 ml of ether each. The ether extracts are washed with water and dried on Na2SO4. After the removal of the ether, a brownish oil remains, which is not further purified.

Yield: 0.275 g (70%) ’H-NMR (CDC13, S, ppm): 1.99 (q, 4H, CH2CH2CH2) 7 2.69 (t, 4H, NCH2) 7 3.32 (s, 8H, NH2) 7 3.65 (S, 2H, benz-CHz) 7 4.04 (t, 4H, OCH2) ; 6.28-6.80 (m, 6H, phenol-H); 7.31 (s, 5H, benz-H) 13C-NMR (CDjCI, S, ppm): 27.14 (CH2CH2CH2) 7 50.24 (NCH2) 7 58.69 (C6H5CH2) 7 62.23 (OCH2) 7 103.38, 109.50, 118.99 (CH(phenol) ; 123.67 (C(NH2-o)); 126.69, 128.06, 128.59 (CH(benzyl))7 135.32 (C(NH2-m)),* 139.59 (C(benzyl)); 147.71 (C(O-phenol) - 29 bis[3-(2,3-Diaminophanoxy)propyl]amine, 18 846 mg (1.66 mmol) of ligand 17 is dissolved in 50 ml of methanol. 0.09 g of Pd(QH)2 (on carbon, 10%) and 5 ml of hydrazine hydrate (80% in water) are added to it. The reaction mixture is heated to boiling for 10 hours. The resulting suspension is filtered and evaporated to dryness. The product is soluble in methanol and, after adding diethyl ether, precipitates as light greenish oil. This oil is liberated from solvent residues on the oil pump, and a gray powder is obtained.

Yield: 400 mg (69%) ’Η-NMR (CD3OD, 6, ppm): 1.99 (q, 4H, CH2CHZCH2) ; 2.89 (t, 4H, NCH2) 7 4.05 (t, 4H, OCH2) ; 6.24-6.68 (m, 6H, Ar-H) 13C-NMR (CE^OD, 6, ppm) : 29.77 (CH2CH2CH2) ; 47.06 (NCH2) ? 67.70 (OCH2) ; 104.39, 111.13, 120.08 (CH(phenol))7 124.47 (C(NH2-o)); 136.70 (C(NHz-m)), 148.87 (C(O-phenol)) Example 5? tris[3-(2,3-Dinitrophenoxy)propyl]amine, 19 g uf 2,3-dinitrophenol 15 (5.43 mmol) is stirred under reflux with 0.446 g of tris(3-chloropropyl)amine, 4 (1.81 mmol) and 0.305 g of KOH (5.43 mmol) in 50 ml of ethanol for 10 hours. Then, the reaction mixture is allowed to cool off and the precipitated solid is filtered off. The solid is taken up in acetone. In this case, organic components are dissolved while the formed KC1 remains. Xnsolubl? c<'’Tr'npn+'c avo by f i 1 i on . The acetone solution is concentrated by evaporation to 20 ml and cooled to 4°C. After 24 hours, 19 can be isolated in the form of colorless needles. - 30 Yield: 638 mg (51%) Melting point: 145-147°C ’h-NMR([D6]-acetone, &, ppm): 1.93 (q, 6M, CH2CH2CH2) ; 2.59 (t, 6H, NCH2) ,’ 4.31 (t, 6H, OCH2) ; 7.63-7.88 (m, 9H, Ar-H) tris[3-(2,3-Diaminophenoxy,propyl]amine, 20 638 mg of 19 (0.925 mmol) is added to a mixture of 5 ml of HCl (cone.), 5 ml of methanol and 1.64 g (13.8 mmol) of tin. The reaction mixture is stirred under reflux for 2 hours. The reaction is completed when the tin has completely dissolved. After the cooling, the green reaction solution is made strongly alkaline (pH 13) with an excess of KOH and shaken out twice with 15 ml of acetone each. The combined acetone extracts are mixed with 5 ml of water and shaken out twice with 30 ml of ether each. The ether extract is dried on sodium sulfate. The solvent is removed in a pump vacuum. A yellowish oil is obtained which solidifies with drying in a pump vacuum.

Yield: 638 mg (51%) 1H-NMR (CDC13, 5, ppm): 1.92 (q, 6H, CH2CH2CH2) ; 2.65 (t, 6H, NCH2) ; 3.36 (s, 12H, NH2) ; 3.97 (t, 6H, OCH2) ; 6.23-6.74 (m, 9H, Ar-H) Example 6: Production of the technetium complexes of 12 and 13 Methanol solutions of ligands 12 and 13 (30 mmol/1) are produced. 8 microliters of such a solution is mixed with 10-40 microliters of a saline solution made from a 'Tc/^MO reactor. The resulting solution is immediately examined by thin-layer chromatography. This examination (mobile solvent THF) shows a complete incorporation of *Tc in the ligands.

The R, values are 0.3 for *TcO4, 0.65 for 9ft'Tc’12 and 0.60 for 'Tc 13. Residues of pertechnetate can be easily discovered in these various R, values, but are not found.

Example 7: Production of the technetium complex of 17 5 A methanol solution of 17 with the concentration of 50 mmol/1 is produced. One microliter of this solution is mixed with 50 microliters of an eluate solution of a ’Tc/99!!© generator. 10 microliters of a 0.1 N NaOH and 10 microliters of phosphate buffer (ionic strength of 0.1, pH 10 7) are added to it. The reaction mixture is allowed to stand for 5 minutes at room temperature and then is characterized by thin-layer chromatography (mobile solvent THF). This analysis shows the complete incorporation of ^Tc in the ligands. The Rf values are 0.15 for tcO^ and 0.24 for r''Tc‘17.

Example 8? Biotin NHS 1.72 g (8.19 mmol) of DCC is added to a solution of 2.0 g (8.19 mmol) of D(+)biotin and 1.23 g (10.66 mmol) of N20 hydroxysuccinimide in 25 ml of DMF. The resulting suspension is stirred for 24 hours at room temperature. The solid is filtered off and the solution is cooled for 4 hours to -16eC. It is filtered off from the precipitated solid and the solvent of the filtrate is drawn off in a vacuum.

The colorless residue is washed several times with ether and finally dried.

Yield: 2.47 g (89%) ’h-NMR([DJ-DMSO, S, ppm 400 MHz) : 6.43, 6.37 (s, br, 2H, b and g); 4.29 (m, br, IH, c) ; 4.13 (m, br, IH, f); 3.09 (m, br, IH, e); 2.82 (dd, J—12 Hz, 6 Hz, lh, d) ; 2.80 (s, 4H, n) ,’ 2.66 (t, J-7 HZ, 2H, k) ; 2.57 (d, J-13 Hz, IH, d); 1.63-1.50 (m, 6H, h-j); UC-NMR ([DJ-DMSO, δ, ppm): 170.1 (2C, m); 168.8 (1); 162.6 (a); 60.9 (f) , 59.1 (c); 55.1 (e); 39.8 (d)) 29.9 (k) ; 27.7, 27.5 (i and j); 25.4 (2C, n); 24.2 (h) Diagram for the allocation of the NMR signals for biotin NHS.

Coupling of biotin NHS to bis[3-(2,2-dimethyl-i,3benzodioxol-4-yloxy)propyl]amine 860 mg (2.0 mmol) of ligand precursor 14 is stirred in ml of degassed DMF with 670 mg (2 mmol) of biotin NHS and 860 mg (8 mmol) of triethylamine for 80 hours at room temperature. Then, the reaction mixture is mixed with 80 ml of degassed water and cooled for 1 hour to 4°C. The precipitated solid is isolated by filtration and taken up in 50 ml of acetone. After the removal of the solvent, the TEL NO:703-243-6410 «266 P34 AUG-06-’91 18:31 ID:MILLEN WHITE ZELANO conjugate is obtained as viscous oil. A voluminous white powder is obtained by drying on an oil pump vacuum.

Yield: 1.0 g (76%) ’h-NMR( [D0]-acetone, S, ppm, 400 MHz): 6.70 and 6.69 (t, 2H, r and r'); 6.54 and 6.52 (dd, 2H, s and s'); 6.43 and 6.41 (d, 2H, g and q'); 6.23 (s, 1H, b) ; 5.96 (s, 1H, g): 4.46 (m, 1H, c) 4.27 (m, 1H, f) ; 4.11 and 4.07 (t, 4H o and o’); 3.57 and 3.51 (t, 4H, m andm'); 3.15 (m, 1H, e); 2.89 (dd, 1H, d); 2.67 (d, 1H, d); 2.38 (t, 2H, k); 1.90 (m, 6H, n, n' and h) ; Diagram for the allocation of the NMR signals for the biotin-14 conjugate 15 13c-nmr(cd3od, s, ppm): 175.6 (1); 165.0 (a) ,· 150.0 and 150.1 (p and p·); 144.2 and 143.9 (t and t*); 136.8 (2C, u and u·) 1226 and 1225 (r and r'); 110.2 and 119.1 (v and v'); 110.3 (2C, s and s'); 103.6 and 103.4 (q and q') 68.6 and 67.4 (o and o'); 63.3 (f); 61.5 (c, ; 56.9 (e); 46.9 and 44.3 (m and m'); 41.0 (d); 33.7 (k); 22.9, 29-7 and 29.5 (h, i and j); 28.8 and 26.5 (n and n'); 25.9 (4C, w, x, V and x') AUG-06-’91 13:32 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P35 - 34 Cleavage of the protective groups of conjugate biotiu-14 g of the biotin-14 conjugate is dissolved in 20 ml of methanol. 4 ml of fuming HCl is added and it is stirred at room temperature for 3 days. The solvents are removed in a vacuum and the solid residue is dissolved in methanol. The purification takes place chromatographically on Sio2 with methanol/THF (1:1) as mobile solvent. The cleavage of the protective groups in the absence of the signals for the acetal unit is detected in the 1H-NMR spectrum. This io observation is also confirmed by the 13C-NMR spectrum.

Yield: 53% Because of the problems in the cleavage of the protective group, an alternative method of synthesis was also examined.

Example 8a: Tatra[dimethyl(tert-butyl)]silylether of 13 3.85 g (10 mmol) of 13 and 6.55 g (96 mmol) of imidazole in 35 ml of DMF are introduced in a 100 ml Schlenk flask and placed under protective gas. Then, solid TBDMSCI (7.5 g, 50 mmol) is added, and heating takes place. It is stirred for 20 hours at room temperature. Then, the solution is mixed with 200 ml of ether. This mixture is washed three tiroes with water (removal of the DMF) and dried on sodium sulfate. After the filtering off of the drying agent, the ether is drawn off on a rotary evaporator. A bright green liquid remains. From this, the product can be isolated on Sio2 by column chromatography (pentane:ether 1:2) .

Yield: 7 g (86%) TEL NO:703-243-6410 «266 P36 ALIG-06-’91 13:33 ID:MILLEN WHITE ZELANO - 35 1H-NMR (CDClj, 6, ppm): 6.71 (t, 1H, catechol); 6.50 (d, 1H, catechol); 6.40 (d, 1H, catechol); 3.99 (t, 2H, CH2CH2O) ; 2.81 (t, 2H, NCH2CH2) ; 2.01 (q, 2H, CH2CH2CH2) ; 1.01 and 0.97 (s, 18H, ((CH3)3C-SiO) ; 0.21 and 0.14 (s, 12H, (CH3)2-SiO) 13C-NMR (CDClj, 6, ppm): 151.9, 148.0, 136.3, 120.2, 113.7, 105.9 (catechol); 66.8 (CH2CH2O) ; 46.9 (NCH2CH2) ; 30.0 (CH2CH2CH2) ; 26.1 and 26.0 (6C, (CHj)jCSiO); 18.6 (2C, (CHj)jCSiO) ; -3.8 and -4.0 (4C(CH3)2SiO) MS (70 eV): 806 (2) M*; 749 (23) M*-C(CH3)3; 452 (100) M+-C6H3(OH) (OSi(CH3)2C(CH3)3)2 Coupling of tetra[dimethyl(tert-butyl)silylether of 13 to biotin 1.64 g (4.84 mmol) of biotin NHS and 3.90 g (4.84 mmol) of tetra[dimethyl(tert-butyl)]silylether of 13 are stirred with 2.02 g (20 mmol) of triethylamine in 70 ml of DMF for 3 days under argon. The solvent is drawn off in an oil pump vacuum and the remaining oil is washed several times with water. The thus obtained crude product is chromatographically (SiO2, methanol) purified. The identity of the product could be shown by nuclear resonance spectroscopy.

Yield: 43% Cleavage of the protective groups of conjugate biotintetra[dimethyl(tert-butyl)]silylether of 13 1.03 g (1 mmol) of (TBDMS)-13-biotin conjugate is stirred for 24 hours in a mixture of 2 ml of HCl (cone.) and ml of thf. Then, it is mixed with 20 ml of water and the THF is removed in a vacuum. The conjugate precipitates in AUG-06-’91 18:34 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 8266 P37 - 36 doing so. The hydrochloric water solution is decanted and the residue is washed several times with water. The residue can be recrystallized from methanol.

Yield: 45% ’H-NMR (CDClj, ¢, ppm): 6.70-6.20 (m, 6H, catechol) ; 4.25 (m, 1H, NH-CH-CH2) ; 4.15 (m, 1H, CHj-CH-CH) ; 3.92 (m, undissolved, 4H, 0CHj-CHj) ; 3.48 (m, undissolved, 4H, N-CH2-CH2) ; 3.10 (m, 1H, CH-CH(R)-S); 2.75 (d, 1H, CH-CH(H)-S); 2.61 (sbr, 1H, CH-CH(H)-S); 2.28 (tbr, 2H, NC(O)-CH2-CH2) ; 1.95 (m, undissolved, 4H, CH2-CH2-CH2-O); 1.55-1.30 (m, 6H, biotin alkyl chain) Example 9; tert-Butyl ethyl acetate of 14 2.7 g (6.3 mmol) of 14 is dissolved in 40 ml of THF/H2O (9:1) and mixed with 0.67 g (1 eq.) of N&2CO3. 2.4 6 g (2 eq.) of bromoacetic acid-tert-butyl ester is added and stirred at room temperature for 24 hours. Then, 80 ml of CH2C12 is added and dried on MgSO4. After the filtering off of the drying agent, the solution is rigorously concentrated by evaporation. The residue is taken up several times in hexane and decanted. The crude product is purified by column chromatography (70 g of SiOg/pentane/ether 3:1).

Yield: 3.3 g (95%) ’H-NMR (CDC13, 6, ppm): 6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCH2CH2) ; 3.26 (s, 2H, OC(O)CH2N); 2.81 (t, 4H, NCH2CH2) ; 1.93 (q, 4H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH3)2) ; 1.45 (s, 9H, (CHj) jCOC(0) ) aiy-13 3.10 g (5.7 mmol) of the product of the preceding reaction is treated in 50 ml of methanol in boiling heat with 50 ml of acid mixture analogously to the production of 13 from 12. After completion of the reaction, the solvent is drawn off on a rotary evaporator. In this case, the product precipitates so that the aqueous phase can be decanted. The product is dissolved in acetone and liberated from the solvent in a vacuum. In this case, it accumulates as white powder.

Yield: 1.90 g (75%) ’h-NMR (CDClj, 8, ppm): 6.90-6.30 (m, 6H, catechol) ; 4.17 (t, 4H, O-CH2-CH2) ; 3.80 (t, 4H, NCH2CH2) ; 3.60 (s, 2H, HOOCCH2N, ; 2.5 (q, 4H, CH2CH2CH2) Production of the NHS ester of gly-13 0.464 g (2.25 mmol) of DCC is added to a solution of 1 g (2.5 mmol) of gly-13 and 0.388 g (2.4 mmol, of Nhydroxysucc in imide in 8 ml of DMF. After 24 hours of stirring at room temperature, the precipitated solid is filtered off and filtrate is cooled for 4 hours to 4°C. The additional precipitated solid is again filtered off and the filtrate is liberated from the solvent in a vacuum. The residue is rewashed several times with ether and dried in a high vacuum.

Yield: 0.95 g (78%) ’h-NMR (CDCIj, 8, ppm, : 6.80-6.20 (m, 6H, catechol); 4.13 (t, 4H, OCH2CH2) ; 3.80 (S, 2H, OC(O)CH2N); 3.58 (t, 4H, NCH2CH2) ! 2.80 (S, 4H, C(O)CH2CH2C(O) ) ; 2.07 (q, 4H, CH2CH2CH2) ' AUG-06-'91 13:35 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P39 - 38 Example 10; 4-Nitrophenyl-bia((3- (2,2-dimethyl-l,3-benzodIoxol-4yloxy)propyl]amine, 21 2.15 g (5 mmol) of 14 and 0.5 g (5 mmol) of 5 triethylamine are introduced in 10 ml of ethanol and mixed with 1.41 g (10 mmol) of 4-fluoronitrobenzene. The mixture is stirred for 3 days. Then, the solvent is removed and the crude product is purified chromatographically.

Yield: 1.50 g (55%) 1H-NMR (CDClj, ί, ppm): 8.06 (d, 2H, nitrophenyl); 6.72 (t, 2H, catechol); 6.71 (d, 2H, nitrophenyl); 6.47 (d, 2H, catechol); 6.46 (d, 2H, catechol); 4.13 (t, 4H, OCHjCHj) ; 3.67 (t, 4H, NCH2CH2) ; 2.11 (q, 4H, CH2CH2CH2) 7 1.71 (s, 12H, C(CH3)2) 4-Aminophenyl-bis( (3-(2,2-dimethyl-l,3-benzodioxol-4yloxy)propyl]amine, 22 1.50 g (2.75 mmol) of 4-nitrophenyl-14 is dissolved in 30 ml of methanol and stirred with 150 mg of Pd/C (10%) under a hydrogen atmosphere for 4 hours at room temperature.

The catalyst is filtered off and the solvent is removed. A greenish oil is obtained.

Yield: 1.27 g (89%) 4-Aminophenyl-bis[3-(2,3dihydroxyphenoxy)propyl]aminohydrochloride, 23 2 5 1.04 g (2 mmol) of 22, DIPACE is reacted under the conditions for the production of 13 from 12. A white powder is obtained: Yield: 0.965 g (94%) AUG-06-’91 18:36 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P40 - 39 4-Isothiooyanato-bis[3-(2,3dihydroxyphenoxy, propyl ] aminohydrochloride , 24 0.51 g (1 sunol) of 23 of the preceding reaction is reacted with 0.267 g (1.5 mmol) of n,n'5 thiocarbonyldiimidazole. After completion of the reaction, imidazole is washed out with water. The product is obtained as yellow oil.

Yield: 360 mg (70%) Example 11: N,N-bis[3-(2,2-Dimethyl-1,3-benzodioxol—4— yloxy) propyl]-N-(2,3-epoxypropyl) amine, 2 5 1.15 g (48 mmol) of sodium hydride is suspended in 40 ml of DMF at room temperature under nitrogen. A solution of 18.0 g of amine 14 (42 mmol) in 20 ml of DMF is slowly in15 stilled in it and, after completion of the addition, it is stirred for 1 hour. Then, a solution of 5.48 g of epibromohydrin (40 mmol) in 20 ml of DMF is instilled and stirred for another 24 hours. The mixture is diluted with 70 ml of ice water, extracted several times with ethyl acetate and the combined organic extracts are dried on potassium carbonate. After removal of the solvent, a yellow oil remains.

Yield: 71% ’h-NMR (CDClj) 6.90-6.30 (ra, 6H, catechol),* 4.12 (t, 4H, OCH2CH2) ; 3.24 (m, 1H, epoxide)7 2.80-2.60 (m, 8H, NCH2, epoxide) ,· 1.93 (q, 4H, CH2CH2CH2) ,* 1.69 (s, 12H, C(CH3)2) N,N-bis(3-(2,2-dimethyl-l,3-benzodioxol-4-yloxy) propyl]-N(2-hydroxy-3-(2-nitroimidazolyl)propyl]amine, 26 A mixture of 250 mg of 2-nitroimidazole (2.2 mmol), 237 mg of l,8-bis-(dimethylamino)naphthalene (l.l mmol), 2.12 g of 25 (4.4 mmol) and 5 ml of DMSO is heated with exclusion AUG-06-’91 13:37 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P41 - 40 of moisture and stirring for 6 hours to 80°C, the solvent is drawn off in a vacuum and the residue is chromatographed (silica gel, 230-400 mesh, 3 x 15 cm column, 20% CH3CN/CHC13 to 80% CH3CH/CHC1j) . The individual fractions are examined by thin-layer chromatography and thin-layer chromatography analogous fractions are combined. The solvent is drawn off and the residue is dried in a vacuum.

Yield: 27% 1H-NMR (CDC13) 8.40 (s, 1H, imidazole); 8.34 (s, 1H, imidazole); 6.906.30 (m, 6H, catechol); 4.20-4.00 (m, 5H, OCH2CH2, CHOH); 2.90-2.60 (m, 8H, NCH2CH2, ; 1.93 (q, 4H, CH2CH2CH2) ; 1.69 (S, 12H, C(CHj)2) EyafflFlfi 1¾¾ 3-(Ν,Ν-bis[3-(2,2-Dimethyl-l,3-benaodioxol-4yloxy)propyl])aminopropanoio acid ethyl eater, 27 0.898 g of potassium-tert-butylate (8.0 mmol) is dissolved in 150 ml of anhydrous tert-butanol and a solution of 27.24 g of 14 (110 mmol) in 30 ml of tert-butanol and 400 ml of ether are added. With stirring, 33.04 g of freshly distilled ethyl acrylate (330 mmol) is slowly distilled and the reaction mixture is left for 3 days at room temperature. After removal of the solvent, the remaining oil is taken up in ether. The ether phase is washed neutral with water and dried on magnesium sulfate. After the concentration by evaporation, a pale yellow oil remains.

Yield: 48% 1H-NMR (CDClj) 6.90-6.30 (m, 6H, catechol,; 4.25-4.10 (m, 6H, OCH2CH2, CO2CH2CHj) ; 2.80-2.50 (m, 8H, NCH2CH2, CH2N, COCH2) ; 1.93 (q, 4H, CH2CH2CH2, ? 1.69 (s, 12H, C(CH3)2) ; 1.25 (t, 3H, OCH2CH2) AUG-06-'91 13:33 ID:MILLEN WHITE ZELANO TEL NO :703-243-6410 8266 P42 - 41 30 3-(N,N-bis(3-(2,2-Diaethyl-l,3-benzodioxol-4yloxy)propyl])aminopropanoia acid hydrazide, 28 g of anhydrous hydrazine (624 mmol) is added to a solution of 10.0 g of ester 27 (18.0 mmol) in 200 ml of anhydrous pyridine and refluxed for 3 days. It is concentrated by evaporation to 50 ml and then mixed with 200 ml of water, extracted several times with ethyl acetate, the combined organic phases are washed with water, dried on sodium sulfate and concentrated by evaporation. A white residue remains.

Yield: 69% ’h-NMR (CDClj) 6.90-6.30 (m, 6H, catechol); 4.08 (t, 4H, OCH2CH2) ί 2.80-2.50 (m, 8H, NCH2CH2, CH2N, COCH2) } 1.93 (q, 4H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH3)2) 3-{N,N-bis[3-(2,2-Dimethyl-l,3-benzodioxol-4yloxy)propyl]}aminopropanoic acid, 29 14.5 g of ester 27 (27.4 mmol) is refluxed in a 20 solution of 5.00 g of potassium hydroxide (90.0 mmol) in 75 ml of 95% ethanol for 2 hours. The ethanol is drawn off in a vacuum and the remaining residue is taken up in 100 ml of water. After shaking out with 50 ml of ether, the aqueous phase Is carefully acidified with dilute hydrochloric acid. 25 The free acid is extracted by shaking out several times with 50 ml of ether each. The combined ether phases are washed with saturated common salt solution and dried on magnesium sulfate. After removal of the solvent, a colorless oil remains.

Yield: 85% AUG-06-’91 18:39 ID:MILLEN WHITE 2ELAN0 TEL NO:703-243-6410 »266 P43 - 42 ’h-NMR (CDClj) 6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCH2CH2) ? 2.80-2.50 (m, 8H, NCH2CH2, C^N, COCH2) , 1.93 (q, 4H, CH2CH2CHj) 7 1.69 (S, 12H, C(CH3)2) 3—(N,N-bis[3-(2,2-Dimethyl-1,3-benzodiozol-4yloxy)propyl]) aminosuecinimidopropionate, 30 The solution of 12.38 q of dicyclohexylcarbodiimide (60 mmol) in 50 ml of tetrahydrofuran is instilled in a solution, cooled to -5°C, of 25.1 9 of carboxylic acid 29 (50 mmol) and 5.75 g of N-hydroxysuccinimide (50 mmol) in 100 ml of anhydrous tetrahydrofuran within 20 minutes and is stirred for another 2 hours at this temperature and then for another 15 hours at room temperature. After adding 200 microliters of acetic acid, it is stirred for another hour, then filtered and the residue is extracted twice with hot tetrahydrofuran. The combined filtrates are evaporated to dryness and the residue is recrystallized from ethyl acetate.

Yield: 65% 1H-NMR (CDClj) 6.90-6.30 (m, 6H, catechol); 4.11 (t, 4H, OCH2CH2) 7 2.80-2.50 (m, 8H, NCH2CH2, CHjN, COCHj) 7 2.76 (s, 4H, COCH2CH2CO) ; 1.93 (q, 4H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH3)2) Example 14: 3-(N,N-bis[3-(2,2-dimethy1-1,3-benzodioxol-4yloxy)propyl]}aminosuecinimidopropionate, 30 The solution of 10.37 g of l-(3-dimethylaminopropyl)-3ethylcarbodiimide (54 mmol) in 100 ml of acetonitrile is instilled in a solution, cooled to 0°C, of 25.1 g of carboxylic acid 29 (50 mmol) and 8.30 g of 2,3,5,6ACJG-06-’91 18:40 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P44 - 43 tetrafluorophenol (50 mmol) within 5 minutes and heated for 2 hours to 75°C. After adding 200 microliters of acetic acid, it is stirred for another hour, then filtered and the residue is extracted twice with hot acetonitrile. The combined filtrates are evaporated to dryness and the residue is recrystallized from ethyl acetate.

Yield: 65% 1H-NMR (CDClj) 6.90- 6.30 (m, 7H, catechol, tetrafluorophenol); 4.11 (t, 4H, OCH2CH2) ; 2.80-2.50 (m, 8H, NCH2CH2, CH2N, COCH2) ; 1.93 (g, 4H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH3)2 Example IS: 3-{Ν,Ν-bis[3- <2,2-Dimethyl-l, 3-benzodiojeol-4-yloacy)propyl]}aminopropanol, 32 The solution of 20 g of ester 27 (38 mmol) in 50 ml of anhydrous ether is instilled in a suspension of 2.88 g of lithium aluminum hydride (76 mmol) in 150 ml of anhydrous ether within one hour so that the solution boils moderately. Then, it is refluxed for another 5 hours, cooled to room temperature and excess hydride is carefully hydrolyzed with water. It is filtered off from precipitated hydroxide and the filtrate is washed several times with warm ether. After removal of the solvent in a vacuum, the residue is boiled up briefly in ethanol, filtered again and the solvent is drawn off. A highly viscous liquid remains.

Yield: 61% 1H-NMR (CDClj) 6.90- 6,30 (rn, 6H, catechol); 4.12 (t, 4H, OCH2CH2) ; 3.54 (t, 2H, CH2OH) ; 2.80-2.60 (m, 6H, NCH2) ; 2.00-1.80 (m, 6H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH5)2) HCJb-Ub- bl ia:41 lUH'IILLbN WMlIt ^tLHNU ill nu: rdJ-^4J-b4ia 02bb H45 - 44 Example 16: bis [3- (2,2-Dimethyl-l,3-benzodioxol-4-yloacy) propyl] -N- (3chloropropyl) amine, 33 A solution of 10.1 g of alcohol 32 (20.7 mmol) in 60 ml 5 of anhydrous carbon tetrachloride is mixed under a nitrogen atmosphere with 7.86 g of triphenylphosphine (30 mmol). It is refluxed for several hours. After cooling off, it is diluted with half the volume of petroleum ether and stored for some time at -20°C. The precipitate is suctioned off and washed with petroleum ether, after drying on sodium sulfate and removal of the solvent, a yellow oil remains. yield: 78% 1H-NMR (CDC13) 6.90-6.30 (ro, 6H, catechol); 4.11 (t, 4H, OCH2CH2) ; 3.62 (t, 2H, CH2C1) 7 2.80-2.60 (m, 6H, NCH2) i 2.00-1.80 (m, 6H, CH2CH2CH2) 7 1.66 (s, 12H, C(CH3)2) Example 17: 3-{N,N-bis[3-(2,2-Dimethyl-l,3-benzodioxol-4yloxy)propyl]}aminopropanol, 34 4.88 g of alcohol 32 (10 mmol), dissolved in 20 ml of dichloromethane, is added all at once to a well-stirred suspension of 3.23 g of PCC in 25 ml of anhydrous dichloromethane and the mixture is stirred for 90 minutes at room temperature. After adding 50 ml of anhydrous ether, it is decanted and the residue is washed three times with 20 ml each, the combined ether solutions are filtered on 20 g of silica gel. After removal of the solvent, a yellow oil remains.

Yield: 70% - 45 ’Η-NMR (CDClj) 9.74 (s, 1H, aldehyde); 6.90-6.30 (m, 6H, catechol); 4.12 (t, 4H, OCH2CH2) ? 2.80-2.60 (m, 6H, NCH2) ; 2.42 (t, 2H, CH2CH0) ; 2.00-1.70 (m, 6H, CH2CH2CH2) } 1.66 (S, 12H, 5 C(CH3)2 Example 18t 3-{N,H-bis[3-(2-2-Dimethyl-l,3-benzodioxol-4yloxy)propyl]}aminopropionic acid nitrile, 35 0.90 g of potassium-tert-butylate (8.0 mmol) in 150 ml 10 of anhydrous tert-butanol is dissolved and a solution of 27.2 g of 14 (110 mmol) in 30 ml of tert-butanol and 400 ml of ether is added. With stirring, 17.5 g of freshly distilled acrylonitrile (330 mmol) is slowly instilled and the reaction mixture is refluxed for 12 hours. After removal of the solvent, the remaining oil is taken up in ether. The ether phase is washed neutral with water and dried on magnesium sulfate. After the concentration by evaporation, a pale yellow oil remains.

Yield: 62% 1H-NMR (CDClj) 6.90-6.30 (m, 6H, catechol); 4.11 (5, 4H, OCH2CH2) ; 2.81 (t, 4H, NCH2CH2) ; 2.50-2.30 (m, 4H, NCH2CH2CN) ; 1.97 (q, 4H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH3)2) Example 19t N,N-bis[3-(2,2-Dimethyl-l,3-benzodioxol-4yloxy)propyl]propylenediamine, 36 39.0 g of 100% sulfuric acid (0.40 mol) is slowly instilled in a suspension of 30.6 g of lithium aluminum hydride (0.81 mol) in 500 ml of anhydrous ether under ice cooling. Then, it is stirred for one hour at room AUG-06-'91 18:43 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 8266 P47 - 46 temperature, then the solution of 12.55 g of nitrile 35 (0.26 mmol) in 50 ml of anhydrous ether is instilled so that the solution boils moderately. Then, it is refluxed for another 8 hours, cooled to room temperature and excess hydride is carefully hydrolyzed with water. A solution of 40 g of NaOH in 360 ml of water is added, filtered off from the precipitated hydroxide and the filtrate is washed several times with warm ether. The combined ether extracts are dried on potassium carbonate and the solvent is drawn off. A yellow oil remains.

Yield: 47% ’h-NMR (CDClj) 6.90-6.30 (m, 6H, catechol); 4.12 (t, 4H, OCH2CH2) ; 2.80-2.50 (m, 8H, NCH2) ; 2.00-1.80 (m, 6H, CH2CH2CH2) ; 1.69 (s, 12H, C(CH3)2) Example 201 Ν,Ν-bis[3-(2,2-Dimethyl-l,3-benzodioxol-4-yloxy)propyl]-N[4-(nitrobenzyl)]amine, 37 1.15 g (48 mmol) of sodium hydride is suspended in 40 20 ml of DMF at room temperature under nitrogen. A solution of 18.0 g of amine 14 (42 mmol) in 20 ml of DMF is slowly instilled in it and after completion of the addition, it is stirred for another hour. Then, a solution of 8.64 g of 4nitrobenzyl bromide (40 mmol) in 20 ml of DMF is instilled 25 and it is stirred for another 24 hours. The mixture is diluted with 70 ml of ice water, extracted several times with ethyl acetate and the combined organic extracts are dried on potassium carbonate. After removal of the solvent, a yellow oil remains.

Yield: 71% AUG-06-’91 18:44 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P48 - 47 ’h-NMR ([D6]-acetone) 8.21 (d, 2H, nitroaryl); 7.53 (d, 2H, nitroaryl); 6.606.40 (m, 6H, catechol); 4.10 (t, 4H, OCH2CH2) ; 3.63 (s, 2H, C6H5CH2N) ; 2.61 (t, 4H, NCH2CH2) i 1.91 (q, 4H, CH2CHjCH2) ; 1.60 (s, 12H, C(CH3)2 Ν,Ν-bis[3-(2,2-Dimethyl-1,3-benzodioxol-4-yloxy,propyl]-N(4-aminobenzyl)amine, 38 200 mg of 10% Pd/C in 250 ml of methanol is suspended in a 500 ml two-necked flask, cooled to -20°C and saturated with water. Then, the solution of 5.0 g of 37 (8.8 mmol) in 50 ml of methanol is quickly instilled and stirred at -20°C. After completion of the absorption of hydrogen, it is separated from the catalyst and the solvent is drawn off in a vacuum. Pale yellow crystals remain.

Yield: 85% ’h-NMR ([D6]-acetone) 7.04 (d, 2H, aminoaryl); 6.60-6.40 (m, 8H, aminoaryl, catechol); 4.10 (t, 4H, OCH2CH2) ; 3.36 (s, 2H, CAH5CH2N) ; 2.63 (t, 4H, NCH2CH2) ; 1.89 (q, 4H, CH2CH2CH2) ; 1.59 (s, 12H, C(CH3)2 N,N-bis[3-(2,3-Dihydroxyphenoxy) propyl]-N-(4aminobenzyl)aminohydrochloride, 39 .7 g (20 mmol) of 37 is dissolved in 80 ml of glacial acetic acid and mixed in boiling heat within two hours with 80 ml of an acid mixture (50% glacial acetic acid, 30% water, 20% fuming hydrochloric acid). In doing so, solvent distills off with acetone that is being liberated as azeotrope. After completion of the addition, it is distilled for another 30 minutes. Then, residual solvent is drawn off on a rotary evaporator. The remaining residue is dried for 6 hours in a high vacuum.

AUG-06-’91 18:45 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P49 - 48 Yield: 79% ’h-NMR ( [Dj]-pyridine) 8.93 (s, 6H, OH and NH2) i 7.10-6.40 (m, 10H, aminoaryl, catechol); 3.92 (t, 4H, OCH2CH2) ; 3.47 (s, 2H, C6H5CH2N) j 3.12 (t, 4H, NCH2CH2) ; 2.25 (q, 4H, CH2CH2CH2) N,N-bis (3-(2,2-Dimethyl-i,3-benzodioxol-4-yloxy) propyl] —N— (4-isothiooyanatobeazyl)aminohydrochloride, 40 1.15 g of thiocarbonyldichlori.de (10 mmol) is added to a solution of 1.10 g of aniline 39 (2.23 mmol) in 50 ml of 3M hydrochloric acid and 50 ml of chloroform under a nitrogen atmosphere with a one-way spray and is intensively stirred for 6 hours at room temperature. Then, it is evaporated to dryness in a vacuum.

Yield: 78% ’-NMR ([D5]-pyridine) 8.96 (s, 4H, OH); 7.10-6.40 (m, 10H, aryl, catechol); 3.92 (t, 4H, OCH2CH2) ; 3.61 (s, 2H, C&H5CH2N) ; 3.21 (t, 4H, NCH2CH2) ; 2.27 (q, 4H, CH2CH2CH2) Bxample 21: N,N-bis(3-(2,2-Dimethy1-1,3-benzodioxol-4-yloxy) propyl]-N(2-propenyl)amine, 41 1.20 g (50 mmol) of sodium hydride is suspended in 50 ml of DMF at room temperature under nitrogen. A solution of .6 g of amine 14 (48 mmol) in 40 ml of DMF is slowly instilled in it and, after completion of the addition, it is stirred for another hour. Then, a solution of 6.04 g of allyl bromide (50 mmol) in 25 ml of DMF is instilled and stirred for another 24 hours. The mixture is diluted with 70 ml of ice water, extracted several times with ethyl acetate and the combined organic extracts are dried on - AIJG-06-’91 13:46 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 8266 P50 - 49 potassium carbonate. After removal of the solvent, a yellow oil remains.

Yield: 75% ’h-NMR (CDC13) 6.90-6.30 (m, 6H, catechol); 5.62 (a, 1H, CH=CH2) ; 4.93 (m, 2H, CH-CH2); 4.09 (t, 4H, OCH2CH2) ; 2.80-2.60 (m, 6H, NCH2) ; 2.32 (m, 2H, CH2CH=CH2) ; 2.00-1.80 (ro, 6H, CH2CH2CH2) ; 1.66 (s, 12H, C(CH3)2) example 22: bis(3-(2,2-Dimethyl-l,3-benzodioxol-4-yloxy)propyl](2propinyl) amine, 42 0.24 g (10 mmol) of sodium hydride is suspended in 50 ml of DMF at room temperature under nitrogen. A solution of 4.29 g of amine 14 (10 mmol) in 20 ml of DMF is slowly instilled in it and, after completion of the addition, it is stirred for another hour. Then, a solution of 1.43 g of propargyl bromide (12 mmol) in 10 ml of DMF is instilled and stirred for another 24 hours at 50°C. After the cooling off, the mixture is diluted with 70 ml of ice water, extracted several tiroes with ethyl acetate and the combined organic extracts are dried on potassium carbonate. After removal of the solvent, a yellow oil remains.

Yield: 66% ’H-NMR (CDClj) 6.90-6.30 (m, 6H, catechol); 4.15 (t, 4H, OCH2CH2) ; 2.80-2.60 (m, 8H, NCH2, CH2CCH) ; 2.00-1.80 (m, 7H, CH2CH2CH2, CH2CCH) ; 1.65 (s, 12H, C(CH3)2) AUG-06-’91 13:47 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P51 - 50 Example 23; Coupling of a Tc-99m complex, containing isothiocyanates, to proteins The coupling of Tc-99m complexes containing 5 isothiocyanate (example 10) to proteins is to be described by the example of F(ab‘)2 fragments of monoclonal antibody 17-IA. Instead of the antibody fragments, any other protein or a substance containing amino groups can be used.

Monoclonal antibody 17-IA is obtained corresponding to methods known in the literature after administration of 107 of the corresponding hybridoma cells in the abdominal cavity of a Balb/c-mouse and aspiration of the ascitic liquid after 7-10 days. The purification takes place according to methods also known in the literature by ammonium sulfate precipitation and affinity chromatography on protein Asepharose. The purified antibody (10 mg/ml) is treated at pH 3.5 for 2 hours with 25 micrograras/ml of pepsin and the F(ab·)2-fragments are then isolated by FPLC. Before coupling with the chelating agent, the fragments are dialyzed at 4 °C for 12-24 hours from 0.1 Μ KH2PO4/0.1 M NaHCOj, pH 8.5. The protein concentration is adjusted to 10 mg/ml. The complex containing NCS labeled analogously to example 6 is added in a molar ratio of 1:10 (complex: protein) to the protein solution. For conjugate formation, the mixture is incubated for 1 hour at 37°C.

Example 24: Biodiatribution of a Tc-99m complex coupled to fragments of monoclonal antibody 17-ia The biodistribution of protein-bound Tc-99m complexes is to be described by the example of a conjugate with F(ab')2 fragments of monoclonal antibody 17-IA. The antibody, from which the fragments are obtained, recognizes HUb-db— yl lb· 4b ILbl'IlLLtN IdHilt btLHNU I EL NU: r(dJ-24J-b41U »2bb l-OL - 51 an antigen, which is expressed by the human carcinomic cell line MHT29”. A control cell line, which also was obtained from a human carcinoma (MX-1), does not express this antigen. Isolated cells of both lines are administered subcutaneously to immunodeficient nude mice. After the tumors have grown to a size of 300-800 mg, the mice are intravenously administered 20 micrograms of the complex, labeled with 200 microCi of Tc-99m, coupled to F(ab’)2fragments (example 11). The immune reactivity of the conjugates is determined in a parallel manner by the binding to an excess of intracellular antigen and is 75-80%. The biodistribution is determined 24 hours after administration of the conjugate by killing the animals, removing the organs and measuring the radioactivity in the organs. The following table represents the found amounts of radioactivity and shows a marked concentration of chelate in the antigen-positive tumor.

Organ Spleen Liver Kidneys Lung Muscle Blood MX-1 HT29 % of the administered dose per gram of tissue 0.4 1.1 2.8 0.9 0.1 0.6 1.9 8.8 Example 25: Biodistribution of a Tc-99m complex containing biotin 20 microliters of a commercially available streptavidin-coupled sepharose gel (corresponding to 20 AUG-06-’91 18:49 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P53 - 52 micrograms of streptavidin) is administered to a 200 g rat in the muscle of the left hind leg. Then, about 30 minutes later, the intravenous administration of 5 micrograms of the complex containing biotin (example 8) labeled with 200 microci of Tc-99m takes place according to example 6. The determination of radioactivity in the individual organs of the rat takes place after 4 hours. A 16-times higher radioactivity, which is found in the left hind leg muscle in comparison with the right hind leg muscle, shows a clear spe10 cific concentration of the Tc complex by binding to streptavidin-sepharose. In all other organs, no activities over 1.4% of the administered dose per gram of tissue are detected after 4 hums,. The highest concentration after the left hind leg muscle (1.4% of the administered dose per gram of tissue) is found in the kidneys with 0.6% of the administered dose per gram of tissue. About 89% of the administered radioactivity is found in the urine after 4 hours.

The example shows that the complexes containing biotin in the organism can bind to streptavidin conjugates. In20 stead of a streptavidin-sepharose conjugate, selective substances, such as, e.g., monoclonal antibodies, enzymes or hormones, can be used, which — coupled to streptavidin — can be detected after selective concentration in lesions or certain tissues by Tc-99m complexes containing biotin.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims (25)

1. WHAT IB CLAIMED IS:

1. A compound of formula I, (I) wherein X is -O-; -S-; -NR 2 -, wherein R 2 is H; or a C,. 0 -alkylene radical, Y and Z are the same or different and are an -OH, -NHR 3 or -SR 3 radical, wherein R 3 is H or a C V6 -alkyl radical, U is H or a branched or unbranched C V6 -alkyl, C V6 -alkoxy, hydroxyl or carboxyl radical, n is 2, 3, 4, 5 or 6, m is 2 or 3, and R 1 is only present if ra is 2, R 1 is H, a benzyl radical or a branched or unbranched C 0 . 6 -alkyl radical, which alkyl radical is optionally substituted with l, 2 or 3 hydroxyl, carboxyl or amino groups, and wherein said benzyl or alkyl radical optionally contains (i) a functional group -B, (ii) a compound -T which is capable of selectively concentrating in lesions or certain tissues, AUG-06-’91 13:51 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P55 - 54 or (iii) a compound —T which is capable of selectively concentrating in lesions or certain tissues bound to the radical through a functional group -B-, wherein any optionally present functional group or a precursor thereof in R* is optionally in protected form, when Y and/or 2 are -NHR 3 , B is an amino, a hydrazino or hydrazide, a carboxyl, a C,. 4 -alkynyl or alkenyl, a hydroxyl, an aminophenyl, an oxiranyl, a fluorinated phenoxycarbonyl or a biotin radical, or, when Y and Z are -OH or -SR 3 , B is as defined above, or a halogen, a formyl, a nitrile, a phenylisothiocyanate or a succinimidoxycarbonyl radical optionally substituted with a sodium sulfate radical, and T is a monoclonal antibody or fragment thereof, a hormone, a growth factor, a ligand for a cell membrane receptor, a steroid, a neurotransmitter, a fatty acid, a saccharide, an amino acid or oligopeptide, a biotin, or a radiosensitizer; and with the proviso that the compound is not N(CH 2 -CH 2 -CH 2 -O-C A H 3 -2,3-(OH) 2 ) 3 ; or a technetium or rhenium complex thereof, or a salt of said compound or complex with an inorganic or organic acid.

2. A compound of claim l, wherein U is H.

3. A compound of one of claims 1 or 2 wherein X is -0-. ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P56 - AUG-06-’91 IS:ΞΙ

4. A compound of one of claims 1 to 3, wherein n is 3.

5. A compound of one of claims 1 to 4, wherein Y and Z are the same and are -NH 2 or -OH.

6. A compound of any preceding claim, wherein m is 2 and R is H, a benzyl radical, or an unbranched C 0 . 5 -alkyl radical which alkyl radical is optionally substituted with a hydroxyl or amino group, and wherein said benzyl or alkyl radical optionally contains a functional group -B, or a monoclonal antibody or fragment thereof, a steroid or misonidazole bound to the radical through a functional group -B-.

7. A compound of claim 1, wherein ra and n are 3, X is -0-, U is H and Y and Z are the same and are NH 2 - or 0H-.

8. A pharmaceutical preparation comprising an effective amount of a technetium or rhenium complex of a compound of any preceding claim, and a pharmaceutically acceptable excipient.

9. A method of treating diseased tissue in a patient, comprising administering an effective amount of a technetium or rhenium complex of a compound of any preceding claim.

10. A method of claim 9, wherein the amount of the technetium or rhenium complex administered is 1 · 10 5 to 5 · 10 4 nmol/kg of body weight.

11. A method of claim 9, wherein the amount of the technetium or rhenium complex administered is 5 to 500 mCi.

12. A method of claims 9 to 11, wherein the diseased tissue is a tumor. AUG-06-'91 13:52 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 «266 P57

13. A method of one of claims 9 to 12, further comprising first ad ministering to the patient a diseased-tissue-apecific agent coupled to streptavidin, and wherein the compound of any of claims 1 to 7 contains a compound -T which is biotin.

14. In a radiopharmaceutical diagnostic method, the improvement comprising administering to a patient an effective amount of a technetium or rhenium complex of a compound of claim 1.

15. A method of claim 14, wherein the amount of the technetium or rhenium complex is 1 · 10 5 to 5 · 10 4 nmol/kg of body weight.

16. A method of claim 14, wherein the amount of the technetium or rhenium complex administered is about 0.05 to 50 mCi.

17. A method of claim 14, further comprising first administering to the patient a tissue-specific agent coupled to streptavidin, and wherein the compound of any of claims 1 to 7 contains a compound -T which is biotin.

18. A process for the production of a compound of formula I 8 1 -M TEL NO :703-243-6410 «266 P58 AUG-06-’91 13:53 ID:MILLEN WHITE ZELANO - 57 wherein 2 2 X is -O-; -S-; -NR -, wherein R is H; or a Cj.^-alkylene radical, Y and Z are the same or different and are an -OH, -NHR 3 or -SR 3 radical, wherein R 3 is H or a C V6 -alkyl radical, U is H or a branched or unbranched C^-alkyl, C^-alkoxy, hydroxyl or carboxyl radical, n is 2, 3, 4, 5 or 6, m is 2 or 3, and R 1 is only present if m is 2, r 1 is H, a benzyl radical or a branched or unbranched C 0 . 4 -alkyl radical, which alkyl radical is optionally substituted with i, 2 or 3 hydroxyl, carboxyl or amino groups, and wherein said benzyl or alkyl radical optionally contains (i) a functional group -B, (ii) a compound -T which is capable of selectively concentrating in lesions or certain tissues, or (iii) a compound -T which is capable of selectively concentrating in lesions or certain tissues bound to the radical through a functional group -B-, wherein any optionally present functional group or a precursor thereof in R 1 is optionally in protected form, when Y and/or Z are -NHR 3 , B is an amino, a hydrazino or hydrazide, a carboxyl, a c V6 -alkynyl or alkenyl, a hydroxyl, an aminophenyl, an oxiranyl, a fluorinated phenoxycarbonyl or a biotin radical, or, AUG-06-’91 13:54 ID:MILLEN WHITE ZELANO TEL NO:703-243-6410 8266 P59 - 58 when Y and Z are -OH or -SR 3 , B is as defined above, or a halogen, a formyl, a nitrile, a phenylisothiocyanato or a succinimidoxycarbonyl radical optionally substituted with a sodium sulfate radical, and T is a monoclonal antibody or fragment thereof, a hormone, a growth factor, a ligand for a cell membrane receptor, a steroid, a neurotransmitter, a fatty acid, a saccharide, an amino acid or oligopeptide, a biotin, or a radiosensitizer; and with the proviso that the compound is not N{CH 2 -CH 2 -CH 2 -O-C & H 3 -2,3-(OH) 2 ) 3 ; or a technetium or rhenium complex thereof, or a salt of said compound or complex with an inorganic or organic acid, comprising (a) reacting an amine of formula II, R 1 --N-{-(CH 2 ) n —Nu) m (II) wherein Nu is a nucleofuge and R 1 ' is a substituent R 1 , wherein any optionally present functional group or a precursor thereof in R 1 is present in protected form, and R 1 contains no compound T, with an aromatic compound of formula III U ’ Y ' 2 wherein U’ is a substituent U wherein, when U is a hydroxyl or carboxyl radical, said radical is present in protected form, and — HUb-Ub-'91 ia:03 1 U 'fl i LLfcN WHI It 4hLHNU TfcL Νϋ: '703-243-6410 «266 HbU - 59 Y· and Z* are Y and Z, or a precursor thereof, or a protected form thereof, under base catalysis in polar solvents at temperatures of 50-200’C for about 2 hours to 6 days, (b) generating a functional group B optionally contained in R 1 or a functional groups Y and Z, optionally, (c) coupling the thus obtained couplable or complexable compound with a compound T which is capable of selectively concentrating in lesions or certain tissues, and/or (d) complexing said compound with a technetium or rhenium isotope, or optionally, steps (c) and (d) can be performed in reverse order; and (e) removing any remaining protective group or (f) converting any remaining precursor to the final product. -6019 . 20. 21. 22. 23. 24. 25.

19. .

20.

21.

22.

23.

24.

25. Use of a compound of formula I in the treatment of diseased tissue wherein the components are as defined in any of the preceding claims. A compound of formula I substantially as hereinbefore described with reference to the Examples. A pharmaceutical preparation substantially as hereinbefore described with reference to the Examples. A method of treating diseased tissue in a patient substantially as hereinbefore described with reference to the Examples. A radiopharmaceutical diagnostic method substantially as hereinbefore described with reference to the Examples. A process substantially as hereinbefore described with reference to the Examples. Use of a compound of formula 1 substantially as hereinbefore described with reference to the Examples.