CA2144588A1

CA2144588A1 - Acyclic chelating agents based on aminodialkylphosphoric oxides for the preparation of technetium or rhenium complexes

Info

Publication number: CA2144588A1
Application number: CA 2144588
Authority: CA
Inventors: Wilhelm Stahl; Axel Walch; Wilfried Doll; Ludwig Kuhlmann; Dietrich Putter
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1994-03-15
Filing date: 1995-03-14
Publication date: 1995-09-16
Also published as: NO950971D0; DE4408729A1; NO950971L; JPH07278166A; EP0672673A1

Abstract

Acyclic chelating agents based on aminodialkylphosphoric oxides for the preparation of technetium or rhenium complexes The invention relates to acyclic chelating agents based on aminodialkylphosphoric oxides of the formula (I) (I).

The compounds according to the invention are used for the labeling of substances, in particular with radioactive technetium or rhenium isotopes. These compounds are also used as a diagnostic or therapeutic, and in the form of a test kit for the detection of tumors.

Description

214~588 HOECHST A~TIENGESELLSCHAFT HOE 94/F 066 Dr.FI/As Description Acyclic chelating agents based on aminodialkylphosphoric oxides for the preparation of technetium or rhenium complexes The invention relates to acyclic chelating agents based on aminodialkylphosphoric oxides, their use for the labeling of substances, in particular with radioactive technetium or rhenium isotopes, and the use of the chelates in diagnostic and therapeutic processes.

In the past, the demand to label chemical compounds with radioactive nuclides grew. Especially in the field of medical diagnosis, where pathological conditions can already be indicated by substances which occur in the body only in ppm or even in still lower concentrations, radiolabeled substances can today no longer be dispensed with.

Technetium-99m, in particular, because of its favorable physical properties (absence of a corpuscular radiation, ~ energy of 140 keV and half-life of 6 hours) and the low radiation burden associated therewith has become the most important radionuclide in nuclear medical diagnosis.

Technetium-99m, which can be obtained from nuclide generators, is initially present as pertechnetate, which is suitable in this form, e.g. for thyroid and brain scintigraphy. The scintigraphy of other organs by means of technetium-99m is carried out with the aid of specific "transport substances" which on the one hand are able to bind technetium and on the other hand to enrich the radionuclide in the target organ with high selectivity.
To label the organ-specific transport substance with ~echnetium-99m, the pertechnetate eluted from the nuclide generator must first be converted to a lower oxidation state. In this reduced form, technetium forms compounds which are stable to a greater or les~er extent with the organ-specific substance. For bone scintigraphy, e.g.
Tc-99m-phosphoric acid derivatives, especially organic phoæphonic acids, are employed. Thus in the labeling unit described in European Patent 002485 the sodium salt of 3,3-diphosphono-1,2-propanedicarboxylic acid is present as the organ-specific transport substance. European Patent 108253 describes Tc-99m tri- and tetraphosphonic acids for the scintigraphic demonstration of the reticuloendothelial system (RES), in particular of the liver. The Tc-99m complex with diethylenetriaminepenta-acetic acid (DTPA) is used for the diagnosis of kidney disorders or pathological brain processes.

To label specific substances with technetium-99m and to prepare test kits suitable for routine clinical needs, special processes were developed and described. To prepare labeling kits for macromolecules of biological interest, in particular porphyrins, dextrans, cytochromes and myoglobin, a method is described (G.D. Zanelli, D. Ellison, M.P. Barrowcliffe, Nucl. Med. Commun. 8, 199 to 206, 1987) in which the substance to be labeled is lyophilized together with p-aminobenzoic acid and a hydrochloric acid solution of SnCl2. To reconstitute and label this kit, Tc-99m generator eluate which has pre-viously been diluted with sufficient buffer solution, e.g. citrate/sodium chloride buffer pH 9.5, is added.
This method i8 not suitable, however, for acid-sensitive substances.

In another process (E.K.J. Pauweis, R.I.J. Feitsma, International Patent Application WO 86/03010), Tc-99m pertechnetate is first reduced by heating to 140C for four hours in strong hydrochloric acid solution and bound to a co:~pound which contains an amino group, e.g.
dimethyllormamide. The reactive Tc-99m-labeled intermediate, which precipitates as a sparingly soluble crystalline substance, is reacted in a buffer solution, e.g. sodium carbonate solution, by incubation for one hour at room temperature with the compound to be labeled.

The method does work in the absence of tin, but because of the complicated process steps is hardly suitable for routine use.

To label proteins, in particular antibodies, two dif-ferent routes are known. In the direct method the reducedtechnetium-99m is bound by donor groups (amino, amide, thiol, etc.) of the protein.

Such proces~es are described in European Patent 005638 and US Patent 4,478,815. Tin(II) salts are used there in excess for the simultaneous reductive cleavage of disulfide bridges and for the reduction of the Tc-99m pertechnetate added. In general, longer incubation times (24 hours) are needed for the cleavage of the -S-S- bond, F(ab') 2 fragments being partially cleaved to give Fab' fragments. More recent literature references (e.g.
Journal of Nuclear Medicine 27 (1986), pages 685 to 693 and 1315 to 1320, and International Journal of Nuclear Medicine Biology 12 (1985) page~ 3 to 8) show that the ratio of the two fragments is dependent on the "tinning reaction" and that the ratio of the two components after Tc-99m labeling no longer changes to a noticeable extent, the main component being Tc-99m-labeled F(ab'). In all cases, the labeled F(ab') fragment had to be repurified, as despite a reaction time of at least 30 minutes a quantitative reaction of the pertechnetate was not achieved.

In a rapid chemical method for the Tc-99m labeling of human plasma proteins (D.W. Wong, F. Mishkin, T. Lee, J.
Nucl. Med. 20, 967 to 972, 1979), pertechnetate is first reduced in acidic solution by tin(II) ions and the reduced technetium is then reacted with the protein.

With the aid of bifunctional complexing agents, a stable labeling of substances with radioisotopes can be achieved. In US Patent 4,479,930, the cyclic anhydrides of DTPA and EDTA are indicated as chelating agents not 21~588 -only for In-lll and Ga-67, but also for Tc-99m. In European Patent 35765, the use of deferox~m;ne as a complexing agent for technetium-99m to proteins is mentioned. In International Patent Application WO
85/3063, the partially reduced disulfide bridges in the antibody are reacted with the sodium salt of tetrachloro-nitridotechnetate, which has to be prepared beforehand by reaction of pertechnetate with sodium azide. In European Patent Application 194853, free thiol groups likewise produced by reduction in antibody fragments are used for the b;n~;ng of [(7-maleimidoheptyl);~;nohis(ethylene-nitrilo)]tetraacetic acid as a chelate complex. The coupling of the complex to the antibody is carried out via the reaction of the SH groups with the double bond in the maleimide moiety of the complex compound, while the radioactive metal ion is complexed via the nitrilodi-acetic acid radicals.

Metallothionein, a metal-b;n~;ng protein having a molecular weight of 6000 and a high cysteine content in the molecule was introduced into antibodies as a complex-ing agent (G.L. Toman, R.J. Hadjian, M.M. Morelock et al., J. Nucl. Med. 25, 20, 1984). By means of exchange with Tc-99m glucoheptonate, it was possible to label the antibody-metallothionein conjugate with technetium. The exchange, however, remained incomplete, 80 that a subse-quent purification was necessary. Several bisthiosemi-carbazone ligands were likewise described aR bifunctional chelating agents (Y. Arano, A. Yokoyama, H. Magat et al., Int. J. Nucl. Med. Biol. 12, 425 to 430, 1986).
p-Carboxyethylphenylglyoxal di(N-methylthioæemicarbazone) was conjugated with human serum albumin. The Tc-99m-labeled 1:1 complex showed a certain instability, while complexes having a higher ratio than 1:1 had an increased liver storage. The couplirg of a diamide-dimercaptide-N2S2 ligand to proteins (A.R.Fritzberg, S. Rasina,J.M. Reno et al., J. Nucl. Med. 27, 957 to 958, 1986) is carried out via an additional function group. ThuR, e.g.
4,5-di(S-ethylcarbonylmercaptoacetamide)pentanoyl-N-hydroxysuccinimide was reacted with an anti-melanoma antibody. The resultant conjugate was incubated at pH 8 and 50C with Tc-99m tartrate solution. After one hour, 78% of the technetium were transferred from the tartrate to the antibody.

In order to be able to use technetium-99m widely in diagnosis, it is necessary to transport this nuclide selectively into the organ to be examined. The technetium-99m should be rapidly eliminated again from other organs or organ systems or not taken there at all in order to avoid any unnecessary radiation burden for the patient. For this purpose, until now substances have mainly been employed which can be labeled directly with technetium-99m and have a high organ specificity. More-over, there are, however, a number of substances which do have a high organ specificity, but cannot be directly labeled. These can be proteins (fibrinogen, human serum albumin), monoclonal antibodies, antibody fragments, diagnostic peptides (amidinophenylalanine derivatives;
EP 0508220, EP 0513543), enzymes (streptokinase, lactate dehydrogenase), sugars (dextran, glucose) or even poly-mers. Low-molecular weight substances such as, for example, fatty acids are likewise included therein, which as a result of the high energy requirement of the heart concentrate in the myocardial tissue. In order to be able to label these substances, they are coupled with complex-ing agents which for their part can firmly bind technetium-99m.

Macrocyclic amines, inter alia also cyclams, are known as suitable complexing agents for the complexation of metal ions. The complexation yield for the technetium-cyclam complex under suitable conditions is 99%. Details of technetium-amine complexes are mentioned in D.E. Troutner, J. Simon, A.R. Retrin, W.A. Volkert, R.A. Holmes, J. Nucl. Med. 21 (1980), 443 or S.A. Zuckmann, G.M. Freeman, D.E. Troutner, W.A. Volkert, R.A. Holmes, D.G. van der Reer, E.K. Barefiled, Inorg.

21~4588 Ch. 20 (1981), 3386 or J. Simon, D. Troutner, W.A. Volkert, R.A. Holmes, Radiochem. Radioanal. Lett. 47 (1981), 111. Substituted cyclams, substituted both on the 1-nitrogen and the 6-carbon, are also known (A.R. Ketrin, D.E. Troutner et al., Int. J. Nucl. Med. Biol. 11 (1984), 113 or J. Simon. Diss. Abstr. Int. B42 (1981), 645 or M. Struden, T.A. Raden, Helv. Chim. Acta 69 (1986), 2081 or E. Kimura, R. Machida, M. Rodama, J. Am. Chem. Soc.
106 (1984), 5497).

A number of attempts to conjugate amines and also other ligands to proteins (see Fritzberg et al., J. Nucl. Med.
27 (1986), 957 or Tolman et al., J. Nucl. Med. 25 (1984), 20 or Arano et al., Int. J. Nucl. Med. Biol. 12 (1986) 425) led to products which did not fulfil or only par-tially fulfilled the high in vivo stability dem~n~.

The present invention was therefore based on the objectof making available suitable chelating agents for the preparation of extremely stable, in particular radio-active, technetium and rhenium complexes, which can be prepared in a simple manner and can be employed for the labeling of substances.

This object was surprisingly achieved using substituted aminodialkylphosphonic acids of the formula (I) R 11 1 ~ R

R2~o N_R4_RS_R6 ( I ).
R ~11 wherein Rl i8 hydroxyl, amino or thiol, R2 i B hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C4-alkyl, phenyl or benzyl, unbranched or branched Cl- to C4-alkoxy, phenoxy or benzyloxy, unbranched or branched C1- to C4-alkyl-amino, phenylamino or benzylamino, unbranched or branched C1- to C4-mercaptoalkyl, thiophenyl or mercaptobenzyl, R3 and R3 are identical or different and are hydrogen or (Cl-C4)-alkyl, R4 is unbranched or branched C0- to C6-alkylene or ortho-, meta-, or para-C7-Cl5-aralkylene, 0 R5 i8 amino, hydroxyl, thiol, an ester or an amide group, -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-and R6 is -COOH if R5 is -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-, or a group of the formula II or III if R5 is amino, hydroxyl or thio, or a group of the formula IV or V if R5 is amino, )~Ha I ( I I I ) O

( IV) o = C ~ S (V) or is -N3-, -Hal or -SH, Hal being fluorine, chlorine, bromine or iodine and R7 is the side chain of a proteinogenic amino acid.

In particular, the in~ention relates to compounds acco:rd-ing to formula (I), wherein Rl is hydroxyl, amino or thiol, 21~588 R2 iB hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C4-alkyl, phenyl or benzyl, un-branched or branched C1- to C4-alkoxy, phenoxy or benzyloxy, R3 and R3 are hydrogen or (C1-C4)-alkyl, R4 iB unbranched or branched C0- to C6-alkylene or ortho-, meta- or para-C7-C15-aralkylene, R5 iB amino, hydroxyl, thiol, an ester or an amide group, -CH2-, -CH (R7)-, -CH(NH2)- or -CH(OH)-and R6 iB -COOH if R5 iB -CH2-, -CH (R7)-, -CH(NH2)- or -CH(OH)-, or a group of the formula II or III if R5 i8 amino, hydroxyl or thio, or a group of the formula IV or V if R5 i8 amino, ~`N~/ ( I I ) ~I~HaI (I I 1) o ,1~
b~ (IV) o = C = S (V) or is -N3, -Hal or -SH, Hal being chlorine, bromine or iodine and R7 i8 the side chain of a proteinogenic amino acid.

Preferred compounds according to the formula (I) are those wherein Rl is hydroxyl, amino or thiol, R2 is hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C3- alkyl, phenyl or benzyl, g unbranched or branched C1- to C3 - alkoxy, phenoxy or benzyloxy, R3 and R3 are hydrogen or (C1-C2)-alkyl, R4 is unbranched or branched C0- to C5-alkylene, ortho-, meta- or para-C7-C12-aralkylene, R5 is amino, hydroxyl, thiol, an ester or an amide group, -CH2-, -CH (R7) -, -CH (NH2) - or -CH (OH) -, R6 is -COOH if R5 i8 -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-, or is a group of the formula II or III if R5 is amino, hydroxyl or thio, or a group of the formula IV or V if R5 is amino, ~3 ~ (Il) ~Ho I ( I I I ) o ~ ( I V ) = C = S (V) or is Hal, Hal being chlorine, bromine or iodine and R7 is the side chain of a proteinogenic amino acid.

Very particularly preferred compounds according to formula (I) are finally thoæe wherein R1 is hydroxyl, amino or thiol, R2 is hydroxyl, amino or l~nhranched or branched C1- to C2-alkyl, 2 0 R3 and R3 are hydrogen, R4 is unbr~nche~ or branched C0- to C5-alkylene, ortho-, meta- or para-C7-C12-aralkylene, R5 is amino, hydroxyl, an e~ter or an amide group, -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-and R6 i 8 - COOH if R5 is -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)- or a group of the formula II or III if R5 is amino or hydroxyl or a group of the formula IV if R5 is amino, ~1`N~// ( I I ) al (I I I) o ~U~
( I V ) or Hal, Hal being chlorine, bromine or iodine and R7 is the side chain of a proteinogenic amino acid.

Proteinogenic amino acid i8 understood as me~n;ng a natural amino acid, in particular an L-amino acid.

The present invention furthermore relates to a chelate complex cont~;n;ng one or two compounds of the formula (I) and at least one technetium or rhenium ion. Chelate complexes cont~;n;ng a technetium or rhenium ion are preferred.

The present invention furthermore relates to a conjugate containing a compound according to formula I and, bonded via the ~ide chain R6, a "substance of diagnostic and/or therapeutic interest". "Substances of diagnostic and/or therapeutic interest" are primarily understood as r-An;ng those compounds which can be employed in medical diag-nosis as "transport substances", i.e. usually organ-specific substances, such as antibodies, antibody fragments [e.g. F(ab') 2 or Fab' fragments], proteins (e.g. fibrinogen), peptides (e.g. somatostatin), oligonucleotides, sugars (e.g. dextran, glucose) or even polymers. On the other hand, however, those substances can also generally be labeled with the acyclic chelating agents according to the invention or chelate complexes prepared therefrom, which with their functional group in the side chain react with the formation of a chemical bond. Intended here is e.g. the "monitoring" of chemical substances in production plants, the determination of their concentration, flow rate and residence time.

The preparation of the chelating agents according to the invention, based on aminodialkylphosphoric oxides, is carried out by two different routes.

a) Starting from compounds having a free terminal amino group of the formula VI, e.g. commercially available amino acids, all functional groups present, apart from the amino group, are blocked with protective groups as disclosed in the literature (Protective Groups in Organic Synthesis, Second Edition, T.W. Greene, P.G.M. Wuts, John Wiley & Sons, Inc., New York, 1991). The amino group which is then free can be converted, e.g. for the preparation of the aminodialkylphosphoric oxide derivatives, by reaction of a carbonyl compound of the formula VII and of a phosphorus compound of the formula VIII. The reaction principle iB described in Houben-Weyl, Methoden der Organischen Chemie (Methods of organic chemistry), Georg Thieme Verlag, Stuttgart 1982, E2, pp. 130 ff. and p. 302 ff. After removal of the protective groups a compound of the formula I results.

Scheme I illustrates the reaction se~uence.

Scheme I R 3 H2N-R~ -RS _Rt + ~C~X + RR R R
R~
Yl Vl I Vl l I

N- 4 - R 5 ' - R 6 ' ~P--C/ 3 o R 3 In Scheme I, R4 , R5 and R6 are the groups R4, R5 and R6 provided with protective groups if R4, R5 or R6 is a group which can and must be protected. X is oxygen or nitrogen which is substituted by the nitrogen or the phosphorus of the compounds of the formulae VI and VIII.

R', R" and R'l' are halogen, in particular chlorine, bromine, fluorine, hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C4-alkyl, phenyl or benzyl, unbranched or branched C1- to C4-alkoxy, phenoxy or benzyloxy, unbranched or branched C1- to C4-alkylamino, phenylamino or benzylamino, unbranched or branched C1- to C4-mercaptoalkyl, thiophenyl or mercaptobenzyl. If R' and R" are one of the abovementioned groups - apart from hydroxyl, amino or thiol - in the conversion of a compound of the formula I' to a compound of the formula I R' and R" are either hydrolyzed such that R1 and R2 are hydroxyl, or R' and R" are converted to R1 and R2, which are amino, hydroxyl or thiol, according to methods known to the person skilled in the art. The protective groups of the radicals R4 , R5 and R6 are then removed such that groups R4, R5 and R6 are formed.

21~4588 b) Starting from benzylamine of the formula IX, the aminodialkylphosphoric oxide derivatives of the formula I are prepared by reaction with a carbonyl compound of the formula VII and of a phosphorus compound of the formula VIII. The reaction principle i8 described in "Houben-Weyl, Methoden der Organischen Chemie" (Methods of Organic Chemistry), Georg Thieme Verlag, Stuttgart 1982, p. 130 ff. and p. 302 ff. After removal of the benzyl protective group, e.g. by hydrogenation, the compound with the free terminal amino group of the formula X is linked to the R4 terminus of the unit Y-R4-R5-R6 by alkylation with a halide or tosylate or by reductive amination with a carbonyl compound of the formula XI. During the reductive amination, the carbonyl group forms an imide with the amine via an amide. The carbonyl group is converted in this process to a methylene group. The reaction principles are described in "J. March, Advanced Organic Chemistry", 4th edition, John Wiley & Sons, New York 1992. After removal of the remain-ing protective groups the target compound is formed.Scheme II illustrates the reaction sequence.

214~8~

Scheme II
~ CH2 NH2 ~ /C. X ~ PR R R

lX vl I vl l I

R 11 ¦~ R ~ R 11 1~ R

,r- C ;3 1 \

+ YR~-R5-R6 .

2 P--C~ R 11 ~R3 N-R4 RS R~ ~ . N-R -R3 R6 2--11 ~--R 3 1 .

In Scheme II, the radicals R', R", R"' and R4, R5 and R6 have the same meaning as in Scheme I. Y here is halogen, tosylate or a carbonyl group. It also applies here that if R' and R" denote one of the abovementioned groups -apart from hydroxyl, amino or thiol - during the con-version of a compound of the formula I" to a compound of the formula I, R' and R" are either hydrolyzed such that R1 and R2 are hydroxyl, or R' and R" are converted into Rl and R2, which are amino, hydroxyl or thiol, according to a method known to the person skilled in the art.

2I4~588 In the process for the preparation of the conjugate, the acyclic chelating agent of the formula I, which carries a functional group at the end of the side chain (R6 in formula (I)), is finally bound with the aid of this S functional group to the substance to be labeled (organ-specific substance). The substance to be labeled for this purpose itself carries at least one reactive group, in particular an amino, carboxyl or thiol group, such that the coupling or the preparation of the conjugate is to be carried out under gentle conditions. Optionally after appropriate purification of the conjugate (e.g. by ultrafiltration or dialysis in the case of proteins or polymers, by column chromatography in the case of low-molecular weight substances such as steroids or lipids), technetium in the form of pertechnetate or rhenium in the form of perrhenate and a suitable reductant for the reduction of the pertechnetate or perrhenate to the oxidation state required for complexation are added in any desired sequence or together. The labeled substrate is optionally purified again. Alternatively, the technetium or rhenium complex of the acyclic chelating agent based on aminodialkylphosphoric oxides can also be produced first and this can then be reacted with the substance to give the conjugate. In this case, the complexation and reduction proceeds as described abo~e.
Preferably the complexation reaction is carried out at basic pH (4 to 10).

The reduction of the pertechnetate or of the perrhenate can be carried out by processeE3 known from the lite-rature, preferably u~3ing a tin(II) compound.

Particularly preferably, the reduction is carried outwith a complex-stabilized tin(II) salt, by a "labeling process" as has been proposed in German Offenlegungs-schrift DE-A :3728599. In this process the tin(II) compound is first treated with a complexing agent, preferably a phosphorus compound such as a phosphonate or pyrophosphate or citric acid which makes sure that the 21~588 tin compound remains in solution, especially in the physiological pH range. This complex-stabilized tin(II) salt solution can then be added either to the substance to be labeled, after which the addition of the pertechne-tate or perrhenate solution is carried out, or else to amixture of the substance to be labeled and the per-technetate or perrhenate solution.

The present invention furthermore relates to a diagnostic cont~;n;ng a chelate complex, as defined above, with technetium-99m and an organ-specific substance corres-po~;ng to the above definition.

Moreover, the present invention relates to a therapeutic containing a chelate complex correspo~;ng to the above definition with rhenium-186 or -188 and an organ-specific substance, as indicated above.

The present invention further relates to a test kit for the determination of tumors, comprising two separate, lyophilized components, of which one contains an antibody which is directed against tumor-associated antigens, or whose F(ab')2 fragment is linked with an acyclic chelat-ing agent based on aminodialkylphosphoric oxides of the formula (I) as claimed in claim 1, and the other contains a reductant which is required for the reduction and b; n~; ng of the technetium to the antibody component.

In a preferred embodiment, a tin(II) salt is used as the reductant. The tin(II) salt of methanediphosphonic acid, aminomethanediphosphonic acid, 3,3-diphosphonopropionic acid, 3,3-dipho8phono-1,2-propanedicarboxylic acid, citric acid or propane-1,1,3,3-tetraphosphonic acid is particularly preferably employed in this case.

In the following, the invention is illustrated in greater detail with the aid of examples.

1) Di[N-methyl(phosphonyl)]glycine Di[N-methyl(diethylphosphonyl)]glycine methyl ester 0.5 g of glycine methyl ester hydrochloride is suspended in 100 ml of acetonitrile (abs.) and, after addition of 3 ml of diethyl phosphite, the mixture is brought to reflux under protective gas in the presence of pulverized molecular sieve. After adding 0.9 g of paraformaldehyde the mixture is heated at reflux until reaction is com-plete (about 1 h). The precipitate is filtered off and the filtrate is concentrated. The excess of diethyl phosphite is distilled off at 50C on an oil pump. The residue is chromatographed on silica gel (eluent: ethyl acetate/methanol 90:10).
Yield: 1.25 g (80%) 1H-NMR (CDCl3): 4.1 (POCH2), 3.3 (PCH2), 3.85 (NCH2) 3.7 (-CH3), 1.3 (POCH2CH3) ppm.

Di[N-methyl(phosphonyl)]glycine 1 g of di[N-methyl(diethylphosphonyl)]glycine methyl ester is heated to reflux for about 2 h in 30 ml of concentrated hydrochloric acid. After concentrating, 900 mg of residue are obtained.
Yield: 900 mg (94%) 1H-NMR (D2O): 3.3 (PCH2), 3.95 (NCH2) ppm.

2) ~-N-Di[N-methyl(phosphonyl)]lysine ~-N-Di[N-methyl(diethylphosphonyl)]-~-N-carbonyloxy-benzyllysine methyl ester 992 g of ~-N-carbonyloxybenzyllysine methyl ester hydrochloride are su~pended in 50 ml of acetonitrile (abs.) and, after addition oE 1.66 g of diethyl phosphite, the mixture is brought to reflux under pro-tective gas in the presence of pulverized molecular sieve. After adding 1.2 g of paraformaldehyde the mixture is heated at reflux until reaction i8 complete (about 3 h). The precipitate is filtered off and the filtrate is concentrated. The exces~ of diethyl phosphite is dis-tilled off at 50C on an oil pump. The residue is chroma-tographed on silica gel (eluent: ethyl acetate/methanol95:5)-Yield: 1.5 g (84%) H-NMR (CDCl3): 7.4 (benzyl-H), 5.84 (-NH), 5.1 (benzyl-CH2), 4.3 (-CH-), 4.1 (POCH2), 3.1 (PCH2), 2.78 (NCH2), 1.9-1.4 (-CH2-), 1.3 (POCH2CH3) ppm.

~-N-Di[N-methyl(phosphonyl)~lysine 200 g of ~-N-Di[N-methyl(diethylphosphonyl)]-~-N-carbonyloxybenzyllysine methyl ester are heated at reflux for about 6 h in 10 ml of concentrated hydrochloric acid.
After concentrating, 140 mg of residue are obtained.
Yield: 140 mg (97%) 1H-NMR (D2O): 3.98 (-CH-), 3.4 (PCH2), 2.0-1.3 (-CH2-) ppm.

3) Synthesis of Di[N-methyl(diethylphosphonyl)]glycine Di[N-methyl(diethylphosphonyl)]benzylamine 1.07 g of benzylamine are dissolved in 30 ml of aceto-nitrile (abs.) and, after addition of 5.5 g of diethyl phosphite, the mixture is brought to reflux under pro-tective gas in the presence of pulverized molecular sieve. After ~;ng 1.2 g of paraformaldehyde the mixture is heated at reflux until reaction is complete (about l h). The precipitate is filtered off and the filtrate is concentrated. The excess of diethyl phosphite is dis-tilled off at 50C on an oil pump. The residue is chro-matographed on silica gel (eluent: ethyl acetate/methanol90: 10) .
Yield: 1.49 g (37%) H-NMR (CDCl3): 7.2 (benzyl-H), 4.1 (POCH2), 3.95 (benzyl-CH2), 3.1 (PCH2), 1.3 (POCH2CH3) ppm.

214~588 Di[N-methyl(phosphonyl)]benzylamine 1 g of di[N-methyl(diethylphosphonyl)]benzylamine is heated to reflux for about 2 h with 30 ml of concentrated hydrochloric acid. After concentrating, the residue is stirred with 10 ml of methanol. The precipitate is filtered off with suction, rendered alkaline with ammonium hydroxide solution and concentrated. The residue is chromatographed on silica gel (eluent:
methanol/ammonium hydroxide solution 90:10).
Yield: 300 mg (34%) H-NMR (D20): 7.6 (benzyl-H), 4.7 (benzyl-CH2), 3.55-3.35 (PCH2) ppm.

Di[N-methyl(phosphonyl)]amine 300 mg of di[N-methyl(phosphonyl)~benzylamine are dis-solved in 5 ml of methanol and hydrogenated with palla-dium hydroxide on active carbon in a hydrogen atmosphere.
After 4 houræ, the palladium catalyæt iB filtered off and the solution is concentrated.
Yield: 210 mg (95%) 1H-NMR (D20): 3.61-3.40 (PCH2) ppm.

Di[N-methyl(diethylphosphonyl)]glycine 210 mg of di[N-methyl(diethylphosphonyl)]amine are dissolved in liquid ammonia and treated with 234 mg of sodium amide and 416 mg of sodium iodoacetate. After 8 h, 25- the mixture i8 neutralized with a~monium chloride and the ammonia is evaporated. The residue iæ chromatographed on silica gel (eluent: methanol/ammonium hydroxide æolution 100:O-1:1).
Yield: 193 mg (72%).
lH-NMR (D20): 3.3 (PCH2), 3.95 (NCH2) ppm.

Claims

1. A substituted aminodialkylphosphonic acid of the formula (I) (I), wherein R1 is hydroxyl, amino or thiol, R2 is hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C4-alkyl, phenyl or benzyl, unbranched or branched C1- to C4-alkoxy, phenoxy or benzyloxy, unbranched or branched C1- to C4-alkylamino, phenylamino or benzylamino, unbranched or branched C1- to C4-mercaptoalkyl, thiophenyl or mercaptobenzyl, R3 and R3' are identical or different and are hydrogen or (C1-C4)-alkyl, R4 is unbranched or branched C0- to C6-alkylene or ortho-, meta-, or para-C7-C15-aralkylene, R5 is amino, hydroxyl, thiol, an ester or an amide group, -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-and R6 is -COOH if R5 is -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-, or a group of the formula II or III if R5 is amino, hydroxyl or thio, or a group of the formula IV or V if R5 is amino, (II) (III) (IV) = C = S (V) or is -N3-, -Hal or -SH, Hal being fluorine, chlorine, bromine or iodine and R7 is the Side chain of a proteinogenic amino acid.

2. A compound according to formula I as claimed in claim 1, wherein R1 is hydroxyl, amino or thiol, R2 is hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C4-alkyl, phenyl or benzyl, unbranched or branched C1- to C4-alkoxy, phenoxy or benzyloxy, R3 and R3' are hydrogen or (C1-C4)-alkyl, R4 is unbranched or branched C0- to C6-alkylene or ortho-, meta- or para-C7-C15-aralkylene, R5 is amino, hydroxyl, thiol, an ester or an amide group, -CH2-, -CH (R7)-, - CH(NH2)- or -CH(OH)-and R6 is -COOH if R5 is -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-, or a group of the formula II or III if R5 is amino, hydroxyl or thio, or a group of the formula IV or V if R5 is amino, (II) (III) (IV) = C = S (V) or is -N3, -Hal or -SH, Hal being chlorine, bromine or iodine and R7 is the side chain of a proteinogenic amino acid.

3. A compound according to formula (I) as claimed in claim 1 or 2, wherein R1 is hydroxyl, amino or thiol, R2 is hydrogen, hydroxyl, amino, thiol, unbranched or branched C1- to C3- alkyl, phenyl or benzyl, unbranched or branched C1- to C3-alkoxy, phenoxy or benzyloxy, R3 and R3 are hydrogen or (C1-C2)-alkyl, R4 is unbranched or branched C0- to C5-alkylene, ortho-, meta- or para-C7-C12-aralkylene, R5 is amino, hydroxyl, thiol, an ester or an amide group, -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-, R6 is -COOH if R5 is -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-, or is a group of the formula II or III
if R5 is amino, hydroxyl or thio, or a group of the formula IV or V if R5 is amino, (II) (III) (IV) = C = S (V) or is Hal, Hal being chlorine, bromine or iodine and R7 is the side chain of a proteinogenic amino acid.

4. A compound according to formula (I) as claimed in claims 1 to 3, wherein R1 is hydroxyl, amino or thiol, R2 is hydroxyl, amino or unbranched or branched C1-to C2-alkyl, R3 and R3' are hydrogen, R4 is unbranched or branched C0- to C5-alkylene, ortho-, meta- or para-C7-C12-aralkylene, R5 is amino, hydroxyl, an ester or an amide group, -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)-and R6 is -COOH if R5 is -CH2-, -CH(R7)-, -CH(NH2)- or -CH(OH)- or a group of the formula II or III if R5 is amino or hydroxyl or a group of the formula IV if R5 is amino, (II) (II) (IV) or Hal, Hal being chlorine, bromine or iodine and R7 is the side chain of a proteinogenic amino acid.

5. A chelate complex containing one or two compounds of the formula I as claimed in claims 1 to 4 and at least one technetium or rhenium ion.

6. A conjugate containing a chelate complex as claimed in claim 5 and an organ-specific substance which is bound to a compound according to formula I via R6.

7. A process for the preparation of a compound of the formula I as claimed in claims 1 to 4, which com-prises reacting compounds of the formulae VI, VII
and VIII to give a compound of the formula I' H2N-R4'-R5'-R6' + + PR'R"R"' ?
VI VII VIII

and converting these in turn with removal of the protective groups on R4', R5' and R6' to a compound of the formula I

I' I

where R4', R5' and R6' are the groups R4, R5 and R6 pro-vided with protective groups, X is oxygen or nitrogen and R', R" and R"' are halogen, in particular chlorine, bromine, fluorine or a radical which applies to R2.

8. A process for the preparation of a compound of the formula I as claimed in claims 1 to 4, which comprises converting compounds of the formulae IX, VII and VIII with removal of the benzyl protective group to a compound of the formula X

+ + PR'R"R"' ?
IX VII VIII

? X

and converting this with Y-R4-R5-R6 (XI) via a compound of the formula I" to a compound of the formula I

? where R', R", R"' and R4, R5 and R6 apply as defined in claim 7 and Y is halogen or tosylate, and also a carbonyl group which after coupling with the amine is converted via the imine formed to a methylene group.

9. A diagnostic containing a compound as claimed in claim 5 and an organ-specific substance.

10. A therapeutic containing a compound as claimed in claim 5 and an organ-specific substance.

11. A test kit for the detection of tumors, containing, spatially separated from one another, a compound as claimed in claim 4 bound to an organ-specific substance and a reductant which is capable of the reduction of the technetium from the pertechnetate to technetium(II).