HUT74168A - Bicyclopolyazamacrocyclophosphonic acids, their complexes and conjugates, for use as contrast agents, and process for their preparation - Google Patents

Abstract

The present invention relates to bicyclopropazamacrocyclophosphonic acid derivatives of the formula (I) and pharmaceutically acceptable salts thereof, wherein R is a group of the formula wherein X and Y are independently hydrogen, hydroxy, alkyl or carboxyl, n is n. 1, 2 or 3; provided that when n is 2, then X and Y together represent two or more hydrogen atoms; and when n is 3, then X and Y together represent three or more hydrogen atoms; T is hydrogen, alkyl, carboxyl, hydroxyl, SO3H, (b), (c) or (d) wherein R1 is hydroxy, alkyl or -O-alkyl; R4 is hydrogen, NO2, NH2, isothiocyanate, semicarbazido-, thiosemicarbazido-, maleimido, bromoacetamido or carboxyl; R2 is hydrogen or hydroxy; with the proviso that when R 2 is hydroxy, the R substituent R 2 in each case comprises X and Y is hydrogen;

Description

Bicyclopoly azamacrocyclic of phosphoric acids, their complexes and their conjugates, their use as diagnostic contrast agents and their preparation

The present invention relates to bicyclopolyazamacrocyclophosphonic acid ligands, their pharmaceutically acceptable salts, complexes and conjugates. These compounds can be successfully used as contrast agents in magnetic resonance imaging (MRI) imaging. Some ligands and complexes can be used effectively as oral protective agents and to inhibit dandruff treatment in aqueous systems.

In order to better understand the essence of the invention, the basics of the MRI system are briefly described.

The MRI method is a non-aggressive diagnostic technique that provides good resolution cross-sectional images of soft tissues of animal bodies, preferably human bodies. This test method is based on the property of some nuclei to have magnetic moments (such as water protons) [as determined by mathematical equations, see Barrow, GM: Physical Chemistry, 3rd edition, McGraw-Hill, NY (1973). ], which is the magnetic moments of the applied magnet

Case No. 82788-TF / KmO

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · This directed equilibrium can be disturbed by the use of an external radio frequency (RF) pulse so that the protons in the magnetic field are deflected from the ordered sequence. By interrupting the radio frequency pulse, the nuclei return to their equilibrium state; this is called relaxation time. The relaxation time consists of two parameters, one is the spin-lattice (T1) and the other is the spin-spin (T2) relaxation, these relaxation measurements provide information on the degree of molecular organization and the interaction of protons with the environment.

Since the water content of living tissues is very important and different types of tissues show differences in water content and in the environment, a diagnostic picture of biological organs can be obtained, showing proton density and relaxation time. The greater the difference in proton relaxation time (T1 and T2) in the test tissue, the greater the contrast of the resulting image (see, e.g., J. Magnetic Resonance 33, 83-106 (1979)).

It is known that paramagnetic chelates in which the electron ground state is symmetric have a great influence on the relaxation values of T1 and T2 sequentially aligned water protons, and in this respect the efficiency of the chelate depends in part on the number of unpaired electrons providing the magnetic moment. Annual, 231-266, Raven Press, NY (1985). In addition, it has been found that when this type of paramagnetic chelate is given to living organisms, the chelate has a variety of woven fabrics. ···· ···· · · · · · · · · ·

- In 3 cases, the effect on T1 and T2 can be directly observed in magnetic resonance (MR) images, as there is an increased contrast in the area around the east © location. Accordingly, it has been suggested that test animals be provided with non-toxic paramagnetic chelates to enhance diagnostic information obtained by MRI [see, e.g., Frontiers of Bioi. Energetics I, 752-759. (1978); J. Nucl. Med 25, 506-513. (1984); Proc. of NMR Imaging Symp. (October 26-27, 1980); Cotton, F.A., et al., Adv. Inorg. Chem. 634-639 (1996). Paramagnetic metal chelates used in this sense are called contrast enhancers or contrast agents.

Many paramagnetic metal ions can be considered when designing an MRI contrast agent. However, in practice, gadolinium (Gd ³ *), iron (Fe ³ *), manganese (Mn ² *) and (Mn ³ *) and chromium (Cr ³ ) ions are considered to be the most useful paramagnetic metal ions, since these ions are high magnetic because of their momentum, they have the greatest effect on water protons. However, in their non-complex form (such as GdCl ₃ ), these metal ions exert toxic effects on animals, which precludes the use of these ions in the form of simple salts, so that organic chelating agents (also called ligands) have the essential role of paramagnetic metal ions. to render it non-toxic to animals while maintaining the beneficial effect of ions on T1 and T2 relaxation values in protons of water surrounding the chelate.

• ·

The state of the art in the field of MRI is very extensive and, therefore, the following summary is not intended to be exhaustive, only to review this field and to describe compounds which may have similar structures. U.S. Pat. No. 4,899,755 discloses a method that affects the proton NMR relaxation time of an animal in the liver or bile duct, for which Fe ³⁺ -ethylene-bis (2-hydroxyphenylglycine) complexes and their and suggest the use of a pyridine macrocyclomethylene carboxylic acid among other compounds. U.S. Pat. No. 4,880,008 (CIP of U.S. Pat. No. 4,899,755) discloses further liver tissue data for rats without disclosing the use of any other complex. U.S. Patent No. 4,980,148 discloses gadolinium complexes which are useful for MRI, but are not cyclic compounds. Broan, CJ et al., J. Med. Chem. Soc., Chem. Commun, 1739-1741 (1990)] discloses some bifunctional macrocyclic phosphinic acid compounds. Broan, CJ et al., J. Med. Chem. Soc. Chem. Commun., 1738-1739 (1990)] describes triazabicyclo compounds. Adzamli IK et al., J. Med. Chem. 32, 139-144 (1989)] discloses acid phosphonate derivatives of gadolinium complexes useful in NMR imaging.

· · · ♦ «♦« ♦ ♦ ♦ ♦ ♦

Currently, only the gadolinium complex of diethylene triamine pentaacetic acid (DTPA-Gd ³ * -Magnevist ™, manufactured by Schering) and a DO ₃ A derivative [1,4,7-tris (carboxy- methyl) -10- (2-hydroxypropyl) -1,4,7,10-tetraazacyclododecanatan gadolinium (Prohance ™, manufactured by Squibb). Magnevist ™ and Prohance ™ are considered to be non-specific / perfusion agents since they are freely distributed in the extracellular fluid and then completely cleared by the renal system. Magnevist ™ is very valuable in the diagnosis of brain injuries, because the perfusion contrast medium administered during perfusion of the blood / brain barrier following brain injury may reach the affected parts. In addition to Magnevist ™, Guerbet has launched a macrocyclic perfusion agent (Dotarem ™)] which is currently only available in Europe. Prohance ™ shows fewer side effects than Magnevist ™. There are currently several contrasts at different stages of development.

Surprisingly, it has been found that various bicyclopolyazamacrocyclophosphonic acid ligands are useful as contrast agents. In addition, modifying the loading of these ligands, such as by appropriate selection of the structure of the ligand and the metal used, results in agents that are more specific to a given tissue region than previously known. More particularly, the present invention relates to novel bicyclo-poly-azamacrocyclophosphinic acid ligands of the formula I wherein

- β R represents a group of the formula (a) in which

X and Y are each independently hydrogen, hydroxy, (C 1 -C 3) alkyl or carboxyl; n is 1, 2 or 3;

with the proviso that when n is 2, X and Y together represent two or more hydrogen atoms; and when n is 3, X and Y together represent three or more hydrogen atoms;

T is hydrogen, C 1 -C 18 alkyl, carboxyl, hydroxy, SO ₃ H, (b), (c) or (d), wherein

R ¹ is hydroxy, C 1-5 alkyl or -O- (C 1-5 alkyl);

R ⁴ is hydrogen, NO ₂ , NH ₂ , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;

R ² is hydrogen or hydroxy; with the proviso that when R ² is hydroxy, the R substituent on R ^{2 is in} each case X and Y are hydrogen;

with the proviso that at least one of the substituents T is P (O) R ¹ OH, and provided that if one of T is a group of the formula (b), one of X and Y in the R substituent is is a carboxyl group and all other X and Y in R are hydrogen;

A is CH, N, C-Br, C-Cl, C-OR ³ , C-OR ⁸ , N ⁺ -R ⁵ X '; or a group of the formula (e);

R ³ is hydrogen, C 1-5 alkyl, benzyl or at least one R ⁴ substituted benzyl;

R ⁴ is as defined above;

R ⁵ is C 1 -C 16 alkyl or benzyl optionally substituted with at least one R ⁴ group;

R ⁸ is C 1 -C 16 alkylamino;

X 'is Cl ·' Br '· Γ or H ₃ CCO ₂ ';

Q and Z are each independently CH, N, N ⁺ -R ⁵ X 1 · C-CH ₂ -OR ³ or CC (O) -R ⁶ ;

^R5 is as defined above;

R ⁶ is -O- (C 13 -alkyl) -, -OH or -NHR ⁷ ;

R ⁷ is C 1 -C 5 alkyl or a biologically active substance;

X 'has the same meaning as given above:

with the condition that

a) when Q, A or Z is N or N ⁺ -R ⁵ X '' then the other two groups are CH;

b) when A is C-Br, C-Cl, C-OR ³ or C-OR ⁸ , then Q and Z are both -CH;

(c) the sum of the substituents R ⁴ , R ⁷ and R ⁸ , if present, must not exceed 1; and

d) Only one of Q and Ζ can be -CC (O) -R ⁶ , and if one of Q or Z is -CC (O) -R ⁶ then A is -CH.

In the case where at least two R groups of the above ligands of formula (I), T is PO ₃ H ₂ [a P (O) R ¹ OH group wherein R ¹ is hydroxy] and the third T is hydrogen, carboxyl - or C 1 -C 18 alkyl; A, Q and Z are CH; n is 1; and X and Y are independently hydrogen or C 1-3 alkyl, the ligands may also be used for oral treatment. Particularly preferred are ligands in which, in the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is hydroxy; n is 1; and X and Y are hydrogen. The use of these ligands is detailed in another dependent application

In the case where the ligands of the formula I have at least two T substituents in the R group are P (O) R ¹ OH, where R ¹ is a hydroxy group, and at the other end of R, T is -COOH or P (O) R ^1. OH, and n, ^R1, X, Y, A, Q and Z are as defined above; and when in at least one R group, T is P (O) R ¹ OH, wherein R ¹ is -OH, wherein in the other two R substituents T is -COOH or P (O) R ¹ OH, and n, R ¹ , X, Y, A, Q and Z are as defined above; or if • ·

9 in R represents three T substituents selected from the group consisting of P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl), and n, R ¹ , X, Y , A, Q and Z are as defined above;

then these ligands can be used as contrast agents.

Particularly preferred are the ligands of the formula I in which

X and Y are hydrogen;

n is 1; obsession

A, Q and Z are -CH.

The ligands and complexes of Formula (I) are the same preferably contain not all three T moieties place of PO3H2 group [a P (0) ^R1 OH, where ^R1 is OH], in the case where A, Q and Z is - CH; although these complexes can be used effectively as contrast agents or oral agents.

Thus, for the ligands and complexes of formula (I), it is understood that not all T is PO ₃ H ₂ (P (O) R ¹ OH where R ¹ is hydroxy) when A, Q and Z are -CH unless they are used as contrast agents or oral agents.

Preferably, the conjugates of the present invention use bifunctional ligands of formula (I), wherein these ligands must be a R substituent wherein T is (b) or a group of the formula wherein R ² and R ⁴ are as defined above; particularly preferred are those wherein, in each R substituent not containing R ⁴ , both T are P (O) R ¹ OH, wherein R ¹ is as defined above, or wherein in the two R groups which do not contain R ⁴ , one of the substituents T is -COOH; wherein the other T is P (O) R ¹ OH, wherein R ¹ is as defined above; preferred is a ligand wherein one of X or Y is -COOH in addition to T as defined above, and preferred are ligands wherein n is 1 and / or the remaining X and Y are hydrogen; obsession

A is C-OR ³ or C-OR ⁸ , wherein R ³ and R ⁸ are as defined above; or a group of formula (e) wherein

R ⁴ is as defined above; obsession

A is CH and Q or Z is CH and the other is CC (O) -R ⁶ , wherein R ⁶ is as defined above;

particularly preferred are ligands wherein R ⁶ is -NHR ⁷ , wherein R ⁷ is a biologically active agent.

• · · ·· ·· ·· • ···· · ·· <

• ···· ·· ··

Ε · · «« • • · • ««

The ligands of the formula I can be complexed with various metal ions, of which the gadolinium (Gd ³ *), iron (Fe ³⁺ ) and manganese (Mn ²⁺ ) ions are preferred, with Gd ³ * being preferred. . The complexes thus prepared may be used alone or covalently linked to a larger molecule such as dextran, polypeptide or biologically active molecule; biologically active molecules include antibodies or fragments thereof; these complexes or conjugates may be used for diagnostic purposes. These conjugates and complexes can be used effectively as contrast agents.

The complexes and conjugates of Formula (I) may be formulated to have a specific surface charge which will favorably affect the in vivo distribution of the administered dose and the contrast of the image.

In the case where the metal ion has a value of 3+, we can form the following:

(A) a total charge of 2 or more when, in three R groups, T is P (O) R ¹ OH, wherein R ¹ is hydroxy and n is 1; or in two R groups, T is P (O) R ¹ OH, wherein R ¹ is hydroxy and in the third R group, T is -COOH and n is 1; or in two R groups, T is P (O) R ¹ OH, wherein R ¹ is hydroxy, and in the third R group, T is

- 12 P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl and n is 1; or in two R groups, T is P (O) R ¹ OH, wherein R ¹ is hydroxy, and in the third R group, T is P (O) R ¹ OH, wherein R ¹ is -O- (C 1 -C 5 alkyl) - group; and n is 1; or (B) the total charge is -1 in the case where one of the R groups is T is P (O) R ¹ OH where R ¹ is hydroxy and the other two R groups are T is P (O) R ¹ OH where R ¹ is -O- (C1-C5 alkyl) and n is 1; or in one of the R groups, T is P (O) R ¹ OH, wherein R ¹ is -OH, and in the other two R groups, T is P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl and n is 1. ; or in one of the R groups, T is P (O) R ¹ OH, wherein R ¹ is hydroxy and in the other two R groups, T is COOH and n is 1; or (C) providing a partially neutral charge when T in the three R groups is P (O) R ¹ OH, wherein R ¹ is -O- (C 1 -C 5 alkyl) and n is 1; or in the three R groups, T is P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl and n is 1; or (D) the total charge is 1+ if

One of A, Q or Z is N ⁺ -R ⁵ X '; wherein R ⁵ and X 'are as defined above; and in one R group, T is P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl); and in the other two R groups, T is carboxyl or P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl, -O- (C 1 -C 5 alkyl) tube powder; and each X and Y is hydrogen.

The complexes and conjugates may be formulated into a pharmaceutically acceptable formulation for administration to animals.

The ligands of formula (I) may be used in conjunction with a metal ion to diagnose various diseases such as cancer.

The numbering of compounds of formula (I) for the purpose of determining the nomenclature is represented by formula (I ').

According to one aspect of the present invention, it is an object of the present invention to provide a contrast agent which synthesizes paramagnetic modifications to provide a contrast agent that can be delivered to a particular site at a desired tissue type. The contrast agents of the present invention have the advantage of providing enhanced contrast on the affected surface due to their affinity for tissue, unlike contrast agents which, after nonspecific perfusion, may or may not produce an image in an extracellular matrix. The compound of formula (I)

The specific affinity of 14 ligands can be influenced by regulating the charge and lipophilic properties of the complex. As mentioned above, the total charge of the complex can range from 3 to 0. For example, complexes having 3 PO ₃ H ₂ groups are strongly negative in overall charge and expected to be absorbed by bone; in contrast, if the total charge of the complex is 0 (i.e., neutral), the complexes may have the ability to cross the blood / brain boundary and normal brain cells can absorb these compounds.

Specific affinity for a particular tissue can be achieved by ionic or covalent attachment of the chelate, wherein the chelate is linked to a naturally occurring molecule or a synthetic compound (e.g. via the R ² group) which has specific affinity for the target tissue. One possible application of this approach is the use of chelated conjugated monoclonal antibodies, which deliver the paramagnetic chelate to the diseased tissue, which allows visualization by MRI. In addition, attachment of a paramagnetic chelate to a macromolecule may enhance the effectiveness of the contrast agent if it improves the contrast relative to the contrast created by the unbound chelate. Lauffer (U.S. Patent Nos. 4,880,008 and 4,899,755) discloses that differences in lipophilic properties allow the production of tissue-specific agents, and that enhanced lipophilicity promotes the non-action of blood proteins.

«· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 15 covalent interactions, resulting in an increase in relaxivity.

In addition, the contrast agents of formula (I) according to the invention, which are neutral charged, are particularly suitable for forming the conjugates of the invention, since only undesirable ionic interactions between the chelate and a protein occur, ensuring that the immunological reactivity of the antibody is unchanged. left. In addition, the neutral complexes of the present invention have a lower osmotic pressure relative to DTPA-Gd ³ *, which reduces the discomfort when injecting.

While not wishing to be hypothesized, it is believed that the charged complexes of the present invention (such as -2 or 3- for bone scans, -1 for liver scans, or +1 for myocardial scans), changes in the chelation ion charge can influence the biological localization of the complexes. . Thus, when an antibody or other targeting moiety is also specific for that particular biological site, the conjugate of the present invention promotes site-specific delivery.

The terms used in Formula I are defined as follows:

C 1-3 -alkyl, C 1-5 -alkyl, C 1-18 -alkyl refer to both straight and branched chain alkyl groups.

- 16 • «··· · ··························································•

The term animal refers to warm-blooded mammals, preferably human beings.

The term biologically active agent refers to dextran, a peptide, or molecules that have a specific affinity for a receptor, or preferably an antibody or antibody fragment.

The term antibody refers to any polyclonal, monoclonal chimeric antibody or hetero-antibody, preferably monoclonal antibody; the term antibody fragment includes Fab fragments and F (ab ') ₂ fragments, as well as any portion of an antibody that has a specific binding ability to the epitope or epitopes of interest. Where the terms radioactive metal chelate / antibody conjugate or conjugate or antibody are used, it is intended to include whole antibodies and / or antibody fragments, including semi-synthetic or genetically engineered variants.

Eligible antibodies include: 1116-NS-19-9 (anti-cancer carcinoma), 1116-NS3-d (anti-CEA), 703D4 (anti-human lung cancer), 704A1 (anti-human lung cancer), CC49 (Tag) -72), CC83 (anti-Tag-72) and B72.3. The hybridoma cell lines: 1116-NS-19-9, 1116-NS3-δ, 703D4, 704A1, CC49, CC83 and B72.3 are deposited with the American Type Culture Collection under numbers ATCC HB 8059, ATCC CRL 8019, ATCC HB 8301, ATCC HB 8302, ATCC HB 9459, ATCC HB 9453 and ATCC HB 8108.

The term "complex" as used hereinabove refers to a metal ion complex of a compound of formula (I) wherein at least one metal atom is bonded or separated; the term conjugate refers to a metal ion-containing chelate covalently bound to an antibody or antibody fragment. The terms bifunctional coordinator, bifunctional chelating agent, and functional group chelate, used interchangeably herein, refer to compounds containing a chelate moiety capable of covalently bonding metal ions in the form of a chelate which can then be covalently attached to an antibody or antibody fragment.

The bifunctional chelating agent of formula (I) described herein is capable of binding metal ions in the form of chelates or of separating the metal ions by forming chelates with the metal ions (also referred to herein as complexes). Due to the presence of functional groups (represented by R ⁴ or R ^{8 in} Formula I), the complexes may be covalently bonded to biologically active substances such as dextran, as well as molecules that have specific affinity for a receptor, or preferably they may be covalently linked to antibodies or antibody fragments. Thus, the complexes mentioned herein may be covalently bound to an antibody or antibody fragment, or exhibit specific affinity for a receptor, also referred to herein as a conjugate.

- 18 ·· · ······ · ···· · ···

As used herein, the pharmaceutically acceptable salts are any salts of the compounds of formula (I) or mixtures thereof which are non-toxic enough to be useful in the diagnosis of animals, preferably mammals. These salts are useful in the practice of the present invention. Salts may be prepared by organic or inorganic compounds using conventional techniques such as sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, palmitic acid, glutamic acid, colamic acid, with salts of glutaric acid, glycolic acid, phthalic acid, tartaric acid, formic acid, lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid, cinnamic acid and the like. Also included are salts formed by conventional reactions using organic or inorganic sources such as ammonium or 1-deoxy-1- (methylamino) -D-glucitol, alkali metal ions, alkaline earth metal ions, and the like. Particularly preferred are the salts of the compounds of formula (I) which are formed using potassium, sodium or ammonium ions. The invention also relates to mixtures of the above salts.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula (I) of the present invention may be prepared by a variety of methods. The general methods which can be used in the preparation are shown in the reaction schemes.

Scheme 1 illustrates the preparation of compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used if n is 2 or 3 by selecting the appropriate reagent),

T is a PO ₃ H _2, and Q, A and Z is CH.

In Scheme 2, compounds of formula I are prepared wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent). is a group of formula (d) in which

R ¹ is -O- (C 1 -C 5) alkyl; and Q, A and Z are CH.

Scheme 3 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent),

T is a group of formula (d) wherein R ¹ is C 1 -C 5 alkyl, and

Q, A and Z are -CH.

In Scheme 4, compounds of formula I are prepared wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent). · • ·

- 20 T is a group of formula (d) in which

R ¹ is -O- (C 1 -C 5 alkyl) - or (C 1 -C 5) alkyl; A is C-Br and Q and Z are -CH.

Scheme 5 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent), T is a group of the general formula (d) in which

R ¹ is -O- (C 1 -C 5 alkyl) - or (C 1 -C 5) alkyl;

A is a group of formula (e ') in which

R ⁴ is hydrogen, NO ₂ , NH ₂ or SCN; and

Q and Z are -CH.

Scheme 6 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent),

T is a group of formula (d) in which

R ¹ is -O- (C 1 -C 5 alkyl) - or (C 1 -C 5) alkyl;

A is C-OR ⁸ wherein R ⁸ is C 1 -C 5 alkylamino; and • ·

- Q and Z are -CH.

Scheme 7 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent),

T is a group of formula (d) in which

R ¹ is hydroxy, -O- (C 1 -C 5 alkyl), or

C 1-5 alkyl;

Z is CC (O) -R ⁶ wherein R ⁶ is hydroxy and

Q and A are -CH.

Scheme 8 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent),

T is a group of formula (d) in which

R ¹ is hydroxy, -O- (C 1 -C 5 alkyl), or C 1 -C 5 alkyl;

Z is -C-CH ₂ -OR ³ , wherein R ³ is benzyl and

Q and A are -CH.

Scheme 9 illustrates the preparation of compounds of formula I wherein X and Y are hydrogen, n is 1 (but this method can be used to provide a · ·

- in 22 cases, where n is 2 or 3, by choice of the appropriate reagent),

T is a group of formula (d) in which

R ¹ is hydroxy, -O- (C 1 -C 5) alkyl, or (C 1 -C 5) alkyl;

A is N or NR ⁵ wherein R ⁵ is C 1 -C 16 alkyl halide; and

Q and Z are -CH.

Scheme 10 illustrates the preparation of compounds of formula (I) wherein

X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent),

T is a group of formula (d) wherein

R ¹ is hydroxy, -O- (C 1 -C 5 alkyl), or C 1 -C 5 alkyl;

Q is -NR ⁵ ; wherein R ⁵ is C 1 -C 16 alkyl halide; and

A and Z are -CH.

Scheme 11 provides compounds of formula I wherein X and Y are hydrogen, n is 1 (but this method can be used when n is 2 or 3 by selecting the appropriate reagent). ·

- 23 T is a group of formula (d) in which

R ¹ is hydroxy, -O- (C 1 -C 5 alkyl), or

C 1-5 alkyl;

Q is N or NR ⁵ wherein R ⁵ is C 1 -C 16 alkyl halide; and

A and Z are -CH.

Alternatively, the phosphonate group can be introduced into the Ν-6 position by another synthetic method. The phosphonate addition is carried out starting from the compound of formula (4) using formaldehyde sodium bisulfite to give a quantitative conversion of the 4,9-substituted sulfonate to the corresponding nitrile. Subsequently, the resulting compound is subjected to phosphonomethylation followed by hydrolysis to give the desired product.

Scheme 12 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent). In the 3-position of the R substituent

T is a group of the formula wherein d

R ¹ is hydroxy or -O- (C 1 -C 5 alkyl), wherein in the other two R groups T is -COOH; and • ·

- 24 A, Q and Z are -CH.

Scheme 13 provides compounds of formula (I) wherein X and Y are hydrogen, n is 1 (but this method can also be used when n is 2 or 3 by selecting the appropriate reagent). In substituents R 3 and 6, T is a group of formula (d):

R ¹ is hydroxy or -O- (C 1 -C 5 alkyl); and, in the third substituent R at position 9, T is -COOH; and

A, Q and Z are -CH.

Scheme 14 provides compounds of formula I wherein X and Y are hydrogen, n is 1 (but this method can be used when n is 2 or 3 by selecting the appropriate reagent). The R substituents at the 3- and 9-positions are represented by the formula: wherein R ¹ is hydroxy or -O- (C 1 -C 5) alkyl; in the third R substituent at position 6, T is -COOH, and

A, Q and Z are -CH.

Scheme 15 provides compounds of formula (I) wherein n is 1 (but this method can be used when n is 2 or 3 by selecting the appropriate reagent), 3 and 9. in the R substituents at position T is (d)

- a group of Formula 25 wherein R ¹ is hydroxy or -O- (C 1 -C 5 alkyl) and X and Y are hydrogen;

at the 6-position of the R substituent, T is a group of formula (b) wherein R ⁴ is -NO ₂ or NH ₂ ; One of X and Y is hydrogen and the other is -COOH; and

A, Q and Z are CH.

Scheme 16 provides compounds of formula I wherein n is 1 (but can be used when n is 2 or 3 by selecting the appropriate reagent), 3 and 6. in the R position of the substituent R, T is a group of the formula (d) in which

R ¹ is hydroxy or -O- (C 1 -C 5 alkyl), and X and Y are hydrogen;

at position 9 of the R substituent, T is a group of formula (b) in which

R ⁴ represents N0 ₂ or NH _2, X and Y are one hydrogen and the other is -COOH,

A, Q and Z are -CH.

Scheme 17 illustrates the preparation of compounds of Formula I wherein n is (but can be used when n is or 3 by selecting the appropriate reagent), and T at position 6 is a group of formula (d) wherein:

R ¹ is hydroxy, and X and Y are hydrogen;

at substituents R 3 and 9, T is -COOH, and

A, Q and Z are -CH.

The general operations described in the above Schemes illustrate specific steps for performing the desired reaction. A general description of these steps is given below.

In Scheme 1, as a first step, the commercially available bis (pyridyl alcohol) bis (pyridyl alcohol) is halogenated with thionyl chloride. Other methods of converting an alcohol to an electrophilic compound, such as treatment with toluene sulfonyl chloride, hydrobromic acid or hydrochloric acid, also provide a reactive product which is subsequently cyclized. Numerous macrocyclization processes are known in the literature. The tetraazamacrocyclic compound of formula (3) may be prepared by the method of Stetter et al., Tetrahedron 37: 767-772 (1981). Since then, other general methods have been described for the preparation of similar macrocyclic compounds under mild reaction conditions [Sherry A.D. et al., J. Org. Chem. 54: 2990-2992 (1989). Detosylation of the cyclic compound obtained as an intermediate (from compound (3) to compound (4)) can be carried out under favorable acidic conditions. Reductive detosylation methods are well known in the literature and can be found in • ·

- 27 solutions.

Phosphonomethylation can be performed under typical Mannich base conditions using phosphorous acid and formaldehyde to give the trisaminophosphonic acid derivative (5, PCTMP).

In addition to phosphonic acid derivatives, phosphonate esters (such as 6) may be prepared in organic media in the presence of an alcohol or aprotic solvent (e.g. using acetonitrile, benzene, toluene or tetrahydrofuran) using the appropriate dialkyl phosphite as nucleophilic reagent. Scheme 2). Depending on the reactivity of the amine compound used in the reaction, this operation may be carried out at a temperature of about -10 to about 100 ° C. Similarly, under the conditions of the Mannich reaction, using trialkyl phosphite derivatives, phosphonate esters can be prepared by oxidation of trivalent phosphorus derivatives to pentavalent phosphorus derivatives by simultaneous removal of one mole of alcohol (Arbuzov reaction). This operation may be carried out in the presence or absence of a solvent. When alcohols are used as solvents in the reaction for the preparation of dialyl or trialkyl phosphites, it is preferable to choose the alcohol from which the corresponding phosphonate ester is derived, thereby avoiding the formation of other products during transesterification. Esters of this type may also be prepared by N-alkylation of an alpha-halo dialkyl phosphonate in a solvent such as acetonitrile, chloroform, dimethylformamide, tetrahydrofuran or 1,4-dioxane.

The reaction temperature is generally at or above room temperature. The reaction is carried out by addition of a non-nucleophilic base such as potassium carbonate, optionally. The reaction ester intermediate is then readily hydrolyzed under basic conditions (aqueous hydroxide may be used, pH 8-14, 30-110 ° C) to give the corresponding half-acid derivative.

In Scheme 3, the macrocyclic methylphosphinic acids (10) and (11) are prepared under reaction conditions as described in Scheme 2. Diethoxymethylphosphinic acid is used as the nucleophilic derivative and para-formaldehyde is condensed in a solvent such as tetrahydrofuran, dimethylformamide, dioxane, acetonitrile or an alcoholic medium. The resulting phosphinate ester is then bridged under acidic conditions (6N HCl, 80-100 ° C) or basic conditions (stoichiometric amount of alkali, 40-100 ° C) to give the corresponding methyl phosphinic acid. Alternatively, the method of Sherry, A.D., et al., Inorg. Chem. (1991)], according to this process, produces ethylphosphinic acid under in situ conditions which are used to prepare phosphinate derivatives having enhanced lipophilic properties.

Scheme 4 illustrates our efforts to introduce an additional functional group into the pyridine moiety of the 12-membered tetraazamacrocyclic compound. Thus, chelidamic acid (manufactured by Sigma Chemical Company, ve. 12)

- 29 compounds] can be converted to the bis (halomethyl) derivative (13) which has a suitable substituent at the 4-position of the pyridyl. Conversions for the preparation of intermediates of this structure are known in the art. Such solutions are described by Takalo et al., Acta Chemic Scandinavica B 42, 3733-77 (1988). This intermediate (15) is then subjected to macrocyclization using a conventional reaction in DMF at 100 ° C using sodium tritosylated triamine as reactant or at room temperature tritosylated free base and potassium carbonate or sodium carbonate. carbonate is used as the base to give compounds similar to those mentioned above. The subsequent reaction to obtain compounds containing a phosphonate half-acid and a phosphinate function is the same as the conversions described in the preceding schemes.

Scheme 4 illustrates 4,4-halo-pyridyl substituted macrocyclic compounds of formula (16) which are subjected to a substitution reaction at the 4-position of the pyridyl group as described in Scheme 5. Thus, organometallic Pd (II) complexes can be used to facilitate the coupling reaction wherein the coupling takes place between phenylacetylene and phenylacetylene derivatives and a pyridyl macrocyclic compound. This reaction is carried out under standard reaction conditions using anhydrous conditions, the solvent being triethylamine and the reaction temperature being from about 10 to about 30 ° C.

- 30 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The same product can also be prepared by using Cu (1) -phenylacetylide as a starting material in anhydrous pyridine at a temperature of about 80 to about 100 ° C. In addition, conventional anionic alkylation can include substituents on the pyridine ring, such as sodium alkoxides in dimethylformamide or dioxane at temperatures between about 80 ° C and about 100 ° C using potassium carbonate or sodium hydroxide. Proceeding as described above, the macrocyclic tetraazamacrocyclic derivatives of formulas (24), (25), (26), (27) and (28) may be prepared and analogous phosphonate chelates can be prepared as described above.

Another embodiment of the substitution at the 4-position of the pyridyl group is illustrated in Scheme 6 wherein the 4-hydroxypyridyl group of formula (29) is alkylated with a bromoalkyl nitrile, thereby providing the nitrile group (31) attached via an ether group. yielding a support intermediate which is next incorporated into the macrocyclic structure. This type of alkylation operation is preferably carried out under anhydrous conditions in an aprotic solvent such as tetrahydrofuran (THF) using a non-nucleophilic base such as sodium hydride or butyllithium at a temperature of about 3-0 to +80 ° C. This procedure is generally described by Chaubet et al., Tetrahedron Letters 31 (40), 5729-5739 (1990), which is a non-cyclic analogue.

- 31 are described. The macrocyclic nitrile compound prepared as described above can then be reduced to the primary amine derivative of formula (36), the reaction being carried out in the usual manner and the resulting primary amine moiety protected with 2- (tert-butoxycarbonyloxyimino). -2-phenylacetonitrile (BOC-ON; 37) is included. The macrocyclic secondary amine derivatives of formulas (38), (39), (40), (41), (42), (43) can then be converted to a functional group as described above, together with the removal of the BOC protecting group, to remove the protecting group. acetic acid was used according to Scheme 6.

Various functional groups may also be introduced at the 3-position of the pyridine ring in the macrocyclic structure as illustrated in Scheme 7. Newkome et al. (Tetrahedron 39 (12), 2001-2008 (1983)) described the synthesis of 2,6-halomethyl-nicotinic acid ethyl ester (45) as a starting material for the present invention, followed by ( The tri-tosylated macrocyclic intermediate 46 can be detosylated under acidic conditions (HBr / AcOH, 25-115 ° C) to simultaneously hydrolyze the nicotinic acid derivative 48 or reflux with the ester derivative prior to detosylation. medium to give 3-hydroxymethyl intermediate (47). The macrocyclic derivative of nicotinic acid can then be converted to a secondary amine functional group according to the general methods, from which various types of • ·

32 phosphonate chelates of formula (I) are obtained (49), (50), (51), (52), (53).

In contrast, before converting the macrocyclic amine compounds to a functional derivative, it is desirable to protect the 3-hydroxymethyl analog. Scheme 8 illustrates the application of a benzyl (Bz) protecting group. This protecting group must be resistant to the strongly acidic conditions used in the detosylation step. As mentioned in the previous Schemes, after conversion of the secondary amines to the appropriate functional group, the benzyl protecting group is removed by mild catalytic hydrogenation (58).

Macrocyclic derivatives can be prepared as described in Examples 12-14. wherein both carboxylate and phosphonate functional groups are present within a given molecule. Thus, under standard aqueous alkylation conditions, carboxylate functionality can be introduced to varying degrees using bromoacetic acid. Subsequently, the remaining amine groups are subjected to phosphonomethylation, which is detailed in the previous schemes, for the conversion of formaldehyde and phosphoric acid, dialkyl phosphonates or tri-I-I-phosphate.

With reference to Schemes 15 and 16, a synthetic solution is described which involves the introduction of an aromatic nitrobenzyl substituent on a nitrogen of the macrocyclic compound. Typically, the macrocyclic amine is in an organic solvent such as acetonitrile or dimethyl

33-formamide can be converted to a mono-N function using a non-nucleophilic base such as potassium carbonate. Additional functional groups may be added to the remaining nitrogen atoms, and the functional groups may be introduced using the methods described in the previous schemes. After the addition of the appropriate chelating group, the nitro group is reduced using platinum oxide and water in an aqueous medium. The chelating agent thus obtained is capable of forming conjugates which allow coupling with larger synthetic or natural molecules.

Scheme 17 illustrates the synthesis of macrocyclic compounds of formula 4 wherein the amine groups at the 3- and 9-positions are reacted with at least 2 moles of the sodium salt of hydroxymethanesulfonic acid in an aqueous medium at about pH 9 to give the corresponding macrocyclic compound. positions 3 and 9 contain the methanesulfonic acid sodium salt (119). The use of sodium cyanide removes the sulfonic acid group to give the corresponding cyanomethane derivative (120). Hydrolysis of the cyano group yields a carboxyl group; this conversion may be accomplished by the simultaneous addition of phosphoric acid and formaldehyde, or successively by treatment with a phosphorous acid derivative followed by formaldehyde to give a compound containing the phosphonic acid group at the 6-position (121) followed by the cyano groups and the higher phosphonic acid derivatives. and subjected to acidic hydrolysis at ambient temperature. The operation originates

As a result 34, macrocyclic compounds having two carboxyl groups at the 3 and 9 positions and a phosphonic acid group at the 6 position are obtained. The phosphonomethylation operation described above can also be carried out in this case.

GD ³ *, Mn ² *, Fe ³ * ions used as metal ions for the complexes of the present invention are commercially available, such as from Aldrich Chemical Company. The anion present as an anion is a halide, preferably chloride, or salt-free (metal oxide).

The term paramagnetic nucleus as used in the present invention refers to a metal ion having a spin angle moment and / or an orbital angle moment. The combination of these two types of moments results in the observed paramagnetic momentum, in that it is highly dependent on the atom carrying the odd electron and to a lesser extent on the environment of such atoms.

The gadolinium (Gd ³ *), iron (Fe ³⁺ ) and manganese (Mn ² *) ions are preferred paramagnetic nuclei, of which Gd ³⁺ is most preferred.

The preparation of the complexes of the invention is carried out in a known manner, for example, the description of the complexation can be found in the Chelating Agents and Metallic Chelates monograph (Dwyer & Mellor, Academic Press, Chapter 7, 1964). Reference is also made to the literature on amino acid production, Synthetic Production and Utilization of Amino Acids [edited by Kameko et al., Published by John Wiley

35 & Sons (1974)]. An example of a complex is the reaction of a bicyclopolyazamacrocyclophosphonic acid with a metal ion carried out under aqueous conditions at a pH of 5 to 7. The complex thus formed is chemically bonded and forms a stable paramagnetic nucleus, e.g. stable against dissociation of the paramagnetic nucleus in the ligand.

In the present invention, the molar ratio of ligand to metal in complex formation is at least about 1: 1, preferably 1: 1 to 3: 1, more preferably 1: 1 to 1.5: 1. It is not advantageous to use a large excess of ligands because the non-complexed ligand may be toxic to the treated animal, causing cardiac arrest or hypocalcemic seizures.

Antibodies or antibody fragments useful in the preparation of the conjugates of the invention may be prepared by methods well known in the art. High specificity monoclonal antibodies can be prepared by hybridization according to methods well known in the art (see, for example, Kohler and Milstein, Naturre, 256, 495-497 (1975) and Eur. J. Immunol., 6, 511-519 (1976)). . These antibodies generally show very high specific reactivity. Any of the antibodies directed against the antigen or the hapten can be used for the conjugates of the invention. Preferably, the conjugates utilize monoclonal antibodies or fragments thereof that have a high specificity for a desired binding site.

- 36 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··· · show. Antibodies directed against, for example, tumors, bacteria, fungi, viruses, parasites, microplasmas, other cell membrane antigens, pathogenic surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecule are used in the present invention. Examples of antibodies and antibody fragments include 1116-NS-19-9, 1116-NS3-d, 703D4, 704A1, CC49, CC83, and B72.3. All of these antibodies are deposited in the ATCC collection. A more detailed list of antigens can be found in U.S. Patent No. 4,193,983. The conjugates of the present invention are particularly useful for the diagnosis of various cancers.

The composition of the invention is administered together with a physiologically acceptable excipient or carrier. Methods for preparing these compositions are well known. The formulations may be formulated as suspensions, injectable solutions or other suitable formulations. Physiologically acceptable suspending media may be used, optionally with an adjuvant.

An effective amount of the composition is used to make the diagnosis. The dose used will depend on the disease being studied and the physical parameters of the subject being tested, such as weight. In vivo diagnostic use of the compositions of the invention is also contemplated.

·· · ·· · 4 · β · »··· ···· • · ··· ·················· ·

The chelating agents of the present invention may also be used in other applications, including the removal of undesired metals (such as iron), which may be bonded to polymeric supports for a variety of applications, such as diagnostic agents, and selective extraction of metal ions. Ligands of formula (I) wherein at least two of the R groups in the T substituent have the meaning of -P (O) R ¹ OH may be used as inhibitors of metal ions. Some ligands may be used in amounts less than stoichiometric. Compounds useful for similar purposes are described in U.S. Patent Nos. 2,609,390, 3,331,773, 3,336,221 and 3,434,969.

The present invention is illustrated by the following non-limiting examples.

The meaning of some of the terms used in the following examples is defined as follows:

LC = Liquid Chromatography, purified by low pressure using a Dionex 2010I system using a hand-filled Q-Sepharose ™ anion exchange column (23 x 2 cm).

DMF = dimethylformamide,

AcOH = acetic acid,

ICP = inductively coupled plasma g = gram mg = milligram • ·

- 38 kg = kilogram ml = milliliter μΙ = microliter pH stability; prepared by the general method GdCI3 ¹⁵⁹ (or ¹⁵³ SmCI3) stock solution, such that two μΙ 3 x 10 ⁴ mol / L ¹⁵⁹ does GdCI3 in 0.1N HCl in 2 ml of 3 X 10 'a solution ^{of 4} mol / L GdCI3 carrier solution . Appropriate ligand solutions are prepared with deionized water. The ligand / metal 1: 1 complexes were prepared by mixing the ligands (dissolved in 100 - 500 μΙ deionized water) with 2 ml of ¹⁵⁹ GdCl ₃ stock solution and mixing thoroughly to obtain an acidic solution (pH 2). . The solution was then adjusted to pH 7.0 with 0.1 N NaOH. The percentage of complexed metal bound is determined by passing a sample of the complex solution through a Sephadex ™ G-50 column eluting with 4: 1 saline (85%).

NaCl / NH ₄ OH) was used and a 2x3 ml fraction was collected. The amount of radioactivity in the pooled eluate solution was determined and compared with the radioactivity of the metal remaining on the resin (the metal not bound in the complex is retained by the resin). The pH stability profile is examined by adjusting the pH of an aliquot of the complex solution with n sodium hydroxide solution or n hydrochloric acid solution and determining the percentage of complexed metal bound by the ion exchange method described above. The S _m values can be determined experimentally by comparison,

They have the same value for complexation and biodistribution for the ligands of the invention.

Starting compounds

The example

Preparation of 2,6-bis (chloromethyl) pyridine

Thionyl chloride (100 mL) was cooled in an ice bath and 24 g (0.17 mol) of 2,6-bis (hydroxymethyl) pyridine was added. After 30 minutes, the reaction mixture was warmed to room temperature and refluxed for 1.5 hours. The reaction mixture was cooled to room temperature, and the resulting solid was collected by filtration, washed with benzene, and dried in vacuo. The resulting solid was neutralized with saturated sodium bicarbonate solution, filtered and dried to give 23.1 g of the title compound as an off-white crystalline solid (71.5% yield); mp 74.5-75.5 ° C; Characteristics of the resulting material:

¹ H-NMR (CDCl ₃ ) δ 4.88 (s, 4H); 7.25-7.95 (m, 3H).

Example B

Preparation of 3,6,9-tris (p-tolylsulfonyl) -3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene

A solution of the sodium salt of 1,4,7-tris (p-tolylsulfonyl) -diethylene-triamine disodium salt (6.9 g, 11.4 mmol) in dimethylformamide (92 mL) was stirred at 100 ° C under nitrogen. To the solution was added 2 g (11.4 mmol) over about 45 minutes.

A solution of 2,6-bis (chloromethyl) pyridine (prepared as in Example A) in 37 ml of dimethylformamide is added dropwise. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · After completion, the reaction mixture was stirred at 40 ° C for 12 hours. Water (50-75 mL) was added to the reaction mixture, and the sodium chloride dissolved immediately and the title compound precipitated. The resulting slurry was then filtered, the solid washed with water and dried in vacuo. The title compound is obtained as a light brown powder; 6.5 g (86% yield), mp: 168-170 ° C (dec), further characteristic data: ¹ H NMR _(CDCl3) δ 2.40 (s, 3H); 2.44 (s, 6H); 2.75 (m, 4H); 3.30 (m, 4H); 4.28 (s, 4H); 7.27 (d, 2H); 7.34 (d, 4H); 7.43 (d, 2H); 7.65 (d, 4H);

7.75 (t, 1H); and ¹³ C NMR δ 21.48, 47.29, 50.37, 54.86, 124.19, 127.00, 127.11, 129.73, 135.04, 138.95, 143.42, 143.73, 155, 15.

Example C

Preparation of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene

A solution of hydrobromic acid in acetic acid was prepared by mixing 48% hydrobromic acid and glacial acetic acid in a ratio of 64:35. To 112 mL of HBr / AcOH, 5.5 g (8.2 mmol) of 3,6,9-tris (p-tolylsulfonyl) 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15 ), 11,13-triene (prepared as described in Example B) is added and the reaction mixture is refluxed gently while stirring for 72 hours. The reaction mixture was then cooled to room temperature and about 1/10 of its original volume.

- 41 concentrated. The resulting solution was stirred vigorously and then diethyl ether (15-20 mL) was added. An off-white solid precipitated, which was collected by filtration, washed with diethyl ether and dried in vacuo. The dry tetrahydrobromide salt was then dissolved in water (10 mL), adjusted to pH 9.5 with 50% aqueous sodium hydroxide solution, and extracted continuously for 4 hours. The extract was dried over anhydrous sodium sulfate and the chloroform was evaporated to give a pale brown oily product which crystallized gradually at room temperature to give 1.2 g (71% yield) of the title compound; mp 86-88 ° C; additional characteristics:

¹ H-NMR (CDCl ₃ ) δ 2.21 (m, 4H); 2.59 (m, 4H); 3.06 (s, 3H); 3.85 (s, 4H); 6.89 (d, 2H); 7.44 (t, 1H); and ¹³ C NMR δ 48.73, 49.01, 53.63, 119.67, 136.29, 159.54.

Example D

Preparation of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 9-dimethylenesulfonic acid

500 mg (2.4 mmol) of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (prepared as described in Example C) are mixed with 6 ml of water to give adjust the pH of the suspension to 3 with 6N hydrochloric acid. To the resulting mixture was added 682 mg (5.1 mmol) of the sodium salt of hydroxymethanesulfonic acid and the pH was adjusted to 9 with 50% aqueous sodium hydroxide solution. The mixture was stirred for 3 hours · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··

After stirring at room temperature for 42 hours, ¹³ C-NMR showed complete conversion to give the title bis-methylenesulfonic acid product.

This is an example

Preparation of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 9-dimethylenenitrile

To the reaction mixture of Example D containing 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,11-triene-3-, 9-dimethylenesulfonic acid, 47 mg (9.6 mmol) of sodium cyanide is added. The reaction mixture was stirred at room temperature for 24 hours. The ¹³ C NMR showed complete conversion to the bis-nitrile. The reaction mixture was filtered, extracted with chloroform (3 x 25 mL), dried over anhydrous magnesium sulfate, and the filtrate concentrated to give a viscous oil. The resulting oily substance was dissolved in chloroform, triturated with cyclohexane and concentrated to give 530 mg (78% yield) of the title dimethylenenitrile as a white powder.

Example F

Preparation of 3,9-bis (sodium methylene sulfonate) 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (PC2S)

A solution of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11.13-triene (prepared according to Example C) (1.03 g, 5.0 mmol) in water (10 ml) was added. 0.5 ml of concentrated hydrochloric acid is added and the mixture is stirred for 10 minutes so that the triene is completely

- In 43, it dissolves. The resulting solution had a pH of 8.6. Then the solution

A solution of 1.37 g (10.2 mmol) of HOCH ₂ SO ₃ Na in 5 mL of deionized water was added. The resulting solution was heated at 60 ° C for 10 minutes and then the pH was lowered to 5.6. After cooling, the pH was adjusted to 9.0 using n aqueous sodium hydroxide. Lyophilization of the solution afforded the title compound in quantitative yield as a white solid; characteristic data;

¹ H-NMR (D ₂ O):

δ 2.87 (t, 4H); 3.18 (t, 4H); 3.85 (s, 4H); 4.11 (s, 4H); 7.03 (d, 2H); 7.55 (t. 1H); and ¹³ C NMR (D ₂ O)

48.52, 54.04, 58.92, 79.09, 123.90, 141.37, 161.89.

Example G

Preparation of 3,9-bis (methylene nitrile) 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene

3,9-bis (sodium methylene sulfonate) 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (2.26 g, 5 mmol) To a solution of 10.0 mL of water (0.6 g, 12.24 mmol) was added sodium cyanide (10.0 mL). The mixture was then stirred at room temperature for 3 hours. The pH of the reaction mixture is about 10. The pH of the reaction mixture was adjusted to above 13 with concentrated aqueous sodium hydroxide. The precipitated product was extracted with chloroform (3 x 20 mL), dried over anhydrous magnesium sulfate and filtered. The solvent is then evaporated and the residue is concentrated in vacuo to give the desired product.

- isolated in the form of 44 white waxy products; 1.0 g (71% yield) of the title compound are obtained; Characteristics: ¹ H-NMR (CDCl 3) δ 2.03 (bs, 4H), 2.64 (m, 4H); 3.82 (s, 4H); 3.90 (s, 4H);

7.14 (d, 2H); 7.62 (t, 1H); and ¹³ C-NMR (CDCl 3):

δ 46.08, 46.64, 52.89, 60.78, 115.31, 122.02, 137.57, 157.33.

Example H

3,9-bis (methylene nltril) -6- (dimethylamino methylene phosphonate) 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-trién3-, 9- Preparation of dimethylene nitrile

289 g (1.0 mmol) of 3,9-bis (methylenenitrile) 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (as in Example G). 60 mg (2.0 mmol, excess) of paraformaldehyde and 0.354 mL (372 mg, 3.0 mmol, excess) of trimethyl phosphite are added. The reaction mixture was stirred gently for 10 minutes to give a suspension and then held at 90 ° C for 1 hour. Excess reagent and by-products were removed in vacuo (125 ° C / 0.01 mm Hg for 1 hour), and the resulting dark brown residue was dissolved in 20 mL of chloroform and washed with 5 x 15 mL of deionized water. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the excess solvent evaporated in vacuo to give the title compound (168 mg, 41% yield) as a yellow waxy solid; Characteristic data: ¹ H-NMR (CDCI3) • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · «« |

- 45 δ 2.61 (broad s, 8 Η), 2.73 (d, 2H); 3.62 and 3.68 (s, 6H); 3.73 (s, 4H); 3.84 (s, 4H); 7.06 (d, 2H); 7.57 (t, 1H); and ¹³ C NMR (CDCl 3) δ 44.44, 50.74, 51.03, 51.85, 52.51, 60.28, 115.61, 122.27, 137.24, 156.61.

Example I

Preparation of 3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene diethylphosphonate g (4.8 mmol) 3,6,9 , 15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (prepared according to Example C), 4.8 g (28.8 mmol) of triethyl phosphite and 864 mg (28.8 mmol) Paraformaldehyde was added and the mixture was heated at 90 ° C for 45 minutes with constant stirring. The reaction mixture was then concentrated in vacuo and the resulting viscous oil was chromatographed on a column of basic alumina using chloroform as eluent. The organic eluate was concentrated to give 2.0 g (64% yield) of the title compound as a colorless oily product. Characteristics:

¹ H-NMR (CDCl 3) δ 1.23 (m, 18H); 2.77 (m, 12H); 3.04 (d, 6H); 4.13 (m, 12H);

7.17 (d, 2H); 7.60 (t, 1H); and ¹³ C NMR (CDCl 3) δ 16.43, 50.03, 50.31, 50.43, 50.77, 51.23, 51.38, 52.63, 53.30, 60.86, 60, 92, 61.63, 61.74, 61.83, 61.93, 62.32, 76.46, 76.97, 77.18, 77.48, 122.50, 137.10, 157.18; and ³¹ P NMR δ 24.92 (s, 2P), 24.97 (s, 1P).

Example J

Preparation of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene di (n-propyl) phosphonate

To a mixture of chloroform and dioxane (3 mL, 1: 1) was added 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11.13-triene (100 mg, 0.48 mmol) (prepared according to Example C). ), Tripropyl phosphite (318 mg, 1.53 mmol) and paraformaldehyde (46 mg, 1.53 mmol) were added. The reaction mixture was stirred at 90 ° C for 1 hour. The resulting homogeneous solution is concentrated in vacuo and the resulting viscous oil is chromatographed on a column of neutral alumina using chloroform as eluent. Concentration of the organic eluate gave the desired product as a colorless oily substance; 320 mg (90% yield) of the title compound are obtained. Characteristics:

¹ H-NMR (CDCl 3) δ 0.88 (m, 18H); 1.61 (m, 12H); 2.72 (m, 12H); 3.03 (d, 6H); 3.97 (m, 12H); 7.13 (d, 2H); 7.55 (t, 1H); and ¹³ C NMR (CDCl 3) δ 9.96, 23.73, 49.84, 50.14, 50.26, 50.57, 51.11, 51.23, 52.43, 53.01, 60, 78, 60.84, 67.27, 67.40, 122.48, 137.04, 157.16; and ³¹ P NMR δ 24.98 (3P).

• · · ·

- 47 Example K

Preparation of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene di (n-butyl) phosphonate

500 mg (2.4 mmol) of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (prepared according to Example C), 2.0 g (8 mmol) Tributyl phosphite and 240 mg (8 mmol) of paraformaldehyde were added and the mixture was stirred at 100 ° C for 1 hour. The resulting viscous solution was concentrated in vacuo and the oily residue was chromatographed on a column of basic alumina using chloroform as eluent. The organic eluate is concentrated and the desired product is isolated as a colorless oily substance; 1.25 g (65% yield) of the title compound are obtained. Characteristics:

1 H-NMR (CDCl ₃ ) δ 0.84 (m, 18H); 1.27 (m, 12H); 1.58 (m, 12H); 2.57 (m, 12H); 3.01 (d, 6H); 3.99 (m, 12H); 7.12 (d, 2H); 7.54 (t, 1H); and ¹³ C NMR (CDCl ₃ ) δ 13.42, 13.46, 18.50, 18.59, 32.16, 32.43, 49.88, 50.03, 50.16, 50.63, 51 , 11, 51.27, 52.48, 53.16, 60.71, 60.78, 65.38, 65.48, 65.58, 122.46, 136.96, 157.14; and ³¹ P NMR δ 24.88 (2P), 24.93 (1P).

Example L

Preparation of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene 3 - [(4-nitrophenyl) methyl acetate]

3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11.13-triene (prepared according to Example C) (200 mg, 0.97 mmol) in 2.5 ml

A solution of 48 chloroform was stirred vigorously and a solution of bromo (4-nitrophenyl) methyl acetate (266 mg, 0.97 mmol) in chloroform (2.5 ml) was added in one portion. The reaction mixture was then stirred at room temperature for 24 hours. The solution was concentrated in vacuo to give a semi-solid which was chromatographed on a silica gel column eluting with chloroform / methanol / ammonium hydroxide (16: 4: 1). The organic eluate is concentrated and the desired product is isolated as a pale yellow solid. 250 mg (64% yield) of the title compound are obtained; Characteristics: ¹³ C NMR (CDCl ₃ ) δ 45.67, 45.90, 45.97, 51.65, 52.08, 52.28, 53.78, 69.54, 119.03, 119.23 , 122.85, 130.30, 137.06, 143.27, 147.05, 159.59, 160.41, 171.70.

end products

Example 1

Preparation of 3,6,9,15-etraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-trimethylene-phosphonic acid (PCTMP) 2.06 g (10 mmol) 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (prepared according to Example C), 11.3 g (138 mmol) of phosphoric acid and 15 g (152 mmol). Concentrated hydrochloric acid was added and the mixture was heated to gentle reflux (103 ° C) with constant stirring, followed by dropwise addition of 12.2 g (150 mmol, 15 mL) of a 37% aqueous formaldehyde solution at 2 mL / min. . After the addition was complete, the reaction mixture was refluxed for 16 hours with stirring, then cooled to room temperature and concentrated to give a thick viscous oily substance. The product thus obtained was purified by LC column anion exchange column chromatography (03-0% formic acid, 3 ml / min, retention time = 32 min). The combined fractions were freeze-dried to give 4.8 g (99% yield) of the title compound as a white solid; 275-280 ° C; additional characteristics:

¹ H-NMR (D ₂ O):

δ 2.83 (m, 6H); 3.46 (m, 10H); 7.28 (d, 2H); 7.78 (t, 1H); and ¹³ C NMR δ 53.61, 53.81, 55.27, 57.93, 62.20, 125.48, 143.08, 152.31; and ³¹ P NMR δ 8.12 (2P), 19.81 (1P).

Example 2 Preparation of ¹⁵³ Sm3, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3-, 6,9-trimethylene phosphonic acid ( ¹⁵³ Sm-PCTMP) complex

A solution of the ligand of Example 1 was prepared by dissolving 3.8 mg of the ligand in 0.517 ml of deionized water (pH 2). The ligand / metal complex (1: 1 mixture) was then prepared by diluting 40 μ 40 of the ligand solution with 2 ml of an aqueous solution of SmCl ₃ .H ₂ O (3 x 10 ' ⁴ M / L containing ¹⁵³ SmCl ₃ tracers). 1 N hydrochloric acid solution). The mixture is thoroughly mixed and the metal cured in the complex is determined so that · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··········· ··

A sample of 50 complex solutions was passed through a column packed with Sephadex, eluted with 4: 1 brine (0.85% NaCl / NH ₄ OH), and a 2 x 3 mL fraction was collected. The amount of radioactivity in the pooled eluates is determined and compared with the radioactivity of the resin. Under the conditions described above, the complex is incorporated into the eluate, and the non-complexed metal remains on the resin. By the above method, the formation of the complex bond is found to be 98%. The resin permeate solution is sampled and its pH is determined. The pH stability test is carried out as described in the general methods.

Example 3

Preparation of 3,9-Diacetic acid-6-methylene-phosphonic acid 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene (PC2A1P)

3,9-Bis (methylenenitrile) -6- (methylene dimethylphosphonate) 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 168 mg (1.0 mmol). 13-Triene (prepared as in Example H) was dissolved in 5 ml of 37% concentrated hydrochloric acid and heated under reflux for 16 hours. After cooling, the solution is concentrated to dryness, and then 2 x 10 mL of deionized water is added to remove the excess hydrochloric acid by evaporation. The concentrated aqueous solution was lyophilized to give the title compound as a dark brown solid; Characteristics:

¹ H NMR (D ₂ O)

51 δ 2.68 (br s, 4H), 3.31 (br s, 4H), 4.08 (s, 4H); 4.55 (s, 4H); 7.16 (d, 2H); 7.68 (t, 1H); and ¹³ C NMR (D ₂ O) δ 52.35, 54.04, 57.02, 59.24, 62.26, 125.52, 143.64, 152.36, 171.54; and ³¹ P NMR (D ₂ O) δ 20.03.

Example 4

Preparation of 3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene-ethylphosphonate tris (potassium salt) (PMEHE)

3,6,9,15-Tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3-, 6,9-methylene diethyl phosphonate (250 mg, 0.38 mmol) (Example 1). prepared as described in Example 1) is added to 2 ml of 0.1 N aqueous potassium hydroxide solution. The solution was heated at 90 ° C for 5 hours. The reaction mixture was cooled to room temperature, filtered and freeze-dried. The desired product is obtained in the form of an off-white solid. 252 mg (97% yield) of the title compound are obtained; Characteristics:

¹³ C NMR (D ₂ O) δ 18.98, 19.82, 51.78, 52.06, 53.08, 54.46, 54.68, 57.01, 58.22, 60.24, 63 , 19, 63.25, 63.36, 63.49, 63.59, 63.95, 64.18, 64.25, 66.80, 126.62, 141.63, 159.40; and ³¹ P NMR δ 20.58 (s, 2P), 20.78 (s, 1P).

·· · ·

Example 5

Preparation of 3.6.9.15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene (n-propyl) phosphonate tris (potassium salt) (PMPHE) mg (0.108) mmol) 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylenedi (n-propyl) phosphate (a)

Prepared as in Example J), to an aqueous solution of potassium hydroxide (0.5 mL of n / dioxane (0.5 mL)) was heated under reflux for 24 hours, cooled to room temperature and extracted with diethyl ether. The ether extract was then concentrated in vacuo to give the desired product as an off-white solid to give 48.6 mg (60% yield) of the title compound;

³¹ P NMR δ 20.49 (s, 3P).

Example 6

Preparation of 3.6.9.15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene (n-butyl) phosphonate tris (potassium salt) (PMBHE)

3.21 g (3.88 mmol) of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene di (n-butyl) -phosphate (prepared as in Example K) is added to 35 ml of aqueous potassium hydroxide solution; the solution is refluxed for 5 days. The reaction mixture was cooled to room temperature, filtered and the filtrate was freeze-dried to give a buttery solid. The resulting solid was slurried in 150 mL of methanol and stirred for 12 hours at room temperature. The slurry was then filtered and the filtrate concentrated to give a semi-solid. The resulting solid was taken up in 150 ml of chloroform, dried over anhydrous sodium sulfate and filtered. The chloroform solution was concentrated in vacuo to give the title compound as an off-white solid; 1.86 g (62% yield) are obtained; Characteristics:

¹ H-NMR (D ₂ O):

δ 0.68 (m, 9H); 1.14 (m, 6H); 1.37 (m, 6H); 2.76 (d, 6H); 3.41 (m, 12H); 3.73 (m, 6H); 7.24 (d, 2H); 7.76 (t, 1H); and ¹³ C NMR (D ₂ O) δ 15.76, 15.80, 21.12, 21.20, 34.96, 35.06, 35.14, 52.08,

52.53, 53.38, 53.48, 54.49, 54.75, 57.70, 57.76, 61.86, 67.65, 67.75, 67.98, 68.08, 125, 15, 142.93, 152.25; and ³¹ P NMR δ 9.73 (s, 2P), 21.00 (s, 1P).

Example 7

3,6,9,15-tetraazabicyclo (9.3.1) pentadeca-1 (15), 11,13-trien-3 - [(4-nitrophenyl) methyl acetate] -6,9-methylenedioxy-diethyl- preparation of phosphonate

250 mg (0.62 mmol) of 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene 3 - [(4-nitrophenyl) methyl acetate] (prepared in Example L), triethyl phosphite (624 mg, 3.7 mmol) and paraformaldehyde (111 mg, 3.7 mmol) were added and the resulting solution was stirred at 100 ° C for 1 hour. The resulting homogeneous solution was concentrated in vacuo to give a viscous oily residue. The resulting oily substance was dissolved in chloroform (ml) and washed with water (3 x 5 ml). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated in vacuo; 326 mg (96% yield) of a viscous oily product are obtained; Characteristics:

³¹ P NMR (CDCl ₃ ) δ 24.67 (s, 2P), 24.88 (s, 1P).

Biodegradation General method

Sprague-Dawley rats were acclimatized for five days and then the animals were injected with 100 µl of complex solution by injection into the tail vein. At the time of treatment, the animals weigh between 150 and 200 g. After 30 minutes, the rats are sacrificed by unrolling the neck of the animals, and the animals are dissected. The amount of radioactivity in each tissue is determined using a Nal scintillation counter and a multi-channel analyzer. The measured values are compared with those obtained with a standard solution of 100 μΙ. The percentage of dose in each tissue or organ is then determined.

In determining the blood dose, it is assumed that the blood represents 7% of body weight. The dose in the bone is determined by taking 25 times the value in the femur. For the purpose of determining the dose for the muscles, it is assumed that the muscles represent 43% of the total body weight.

- 55 «··· • · · · · · · · · · · · · · ···

In addition to the biological distribution (biodistribution) studied in organs, chelates containing compounds of Formula I are tested for their ability to localize to bone, as phosphonates are known to bind to hydroxyapatite. The results of these assays are shown below.

Example I

The complex of Example 2 ( ¹⁵³ Sm-PCTMP) is administered by injection, and the percentage distribution of the administered dose in the various tissues is shown in Table 1. The data in the table represent the average of at least three rats, 2 hours after injection.

Table I% distribution of dose of ^1S3 Sm-PCTMP injected into various tissues

Organ Average Bone 34.87 Liver 0.99 Kidney 1.42 spleen 0.07 muscles 4.77 Blood 6.27

II. example

The complex prepared according to Example 5 ( ¹⁵³ Sm-PMPHE) is administered by injection, the percentage distribution of the administered dose in the various tissues being shown in Table II. is shown in Table. The values in the table represent the mean of at least three rats; studies were performed 2 hours after injection.

II. Table ¹⁶³ Distribution of% ¹⁶³ Sm-PMPHE Injection After 2 Hours

Designation of tissues average Bone 10.86 Liver 4.14 Kidney 1.55 spleen 0.05 muscles 1.19 Blood 0.25 Heart 0.08 Lungs 0.12 Brain 0.00 Gastric 0.44 Small Intestine 10.71 Colon 2.17

III. example

The complex prepared according to Example 6 ( ¹⁵³ Sm-PMBHE) is administered by injection at a dose of various doses. · ·

The percent distribution in 57 tissues is shown in Table 3. The values in the table represent the mean of at least three rats; studies were performed 2 hours after injection.

III. % distribution of dose of ¹⁶³ Sm-PMBHE injected after 2 hours

Designation of tissues average Bone 3.73 Liver 2.70 Kidney 0.43 spleen 0.05 muscles 1.09 Blood 0.14 Heart 0.02 Lungs 0.04 Brain 0.00 Gastric 0.08 Small Intestine 57.89 Colon 0.77

ARC. example

The complex prepared as in Example 3 ( ¹⁵³ Sm-PC2A1) was administered by injection, the percentage distribution of the administered dose in the various tissues being shown in Table IV. is shown in Table. The values in the table are at least three times.

Represent the mean value of a measurement in 58 rat rats; studies were performed 2 hours after injection.

ARC. % distribution of dose of ¹⁶³ Sm-PC2A1P injected after 2 hours

Designation of tissues average Bone 47.98 Liver 1.46 Kidney 0.93 spleen 0.02 muscles 1.00 Blood 0.36 Heart 0.04 Lungs 0.06 Brain 0.01 Gastric 0.25 Small Intestine 13.10 Colon 0.12

Experiments relating to image training

General methods

A solution for injection is obtained by dissolving the appropriate amount of the complex in 0.5 ml deionized water to make a 0.5 mol / l solution. Adjust the pH of the solution to 7.4 with n hydrochloric acid or n sodium hydroxide, as needed. Each solution

- 59 ·· · ···················································· · · · · · · · · · · · · · · · · Determining the total Gd content by ICP analysis.

Anesthetized Sprague-Dawley rats are given one of the metal solutions described above by intramuscular injection at a dose of 0.05 to 0.1 mmol Gd / kg body weight. Thereafter, at different times, recordings were made and compared with those obtained at 0 control times before injection.

II. example

The Gd-PCTMP complex prepared according to Example 2 showed a good contrast in kidney images, with bone localization in the shoulder, spine and sternum.

Other embodiments of the present invention will be apparent to those skilled in the art from those set forth herein. The description and examples are provided by way of non-limiting illustration of the present invention. The invention is summarized in the following claims.

Claims (55)

Claims CLAIMS 1. A bicyclopolyazamacrocyclophosphonic acid compound of formula (I) and pharmaceutically acceptable salts thereof, wherein: R is a group of formula (a) wherein X and Y are each independently hydrogen, hydroxy, (C 1 -C 3) alkyl or carboxyl; n is 1, 2 or 3; with the proviso that when n is 2, X and Y together represent two or more hydrogen atoms; and when n is 3, X and Y together represent three or more hydrogen atoms; T is hydrogen, C 1 -C 18 alkyl, carboxyl, hydroxy, SO ₃ H, (b), (c) or (d) wherein R ¹ is hydroxy, C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl); R ⁴ is hydrogen, NO ₂ , NH ₂ , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl; • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · exp metalö | - 61 R ² is hydrogen or hydroxy; with the proviso that when R ² is hydroxy, the R substituent on R ^{2 is in} each case X and Y are hydrogen; with the proviso that at least one of the substituents T is P (O) R ¹ OH, and provided that if one of T is a group of the formula (b), one of X and Y in the R substituent is is a carboxyl group and all other X and Y in R are hydrogen; A is CH, N, C-Br, C-Cl, C-OR ³ , C-OR ⁸ , N ⁺ -R ⁵ X '; or a group of the formula (e); R ³ is hydrogen, C 1-5 alkyl, benzyl, or at least one R ⁴ substituted benzyl; R ⁴ is as defined above; R ⁵ is C 1 -C 16 alkyl or benzyl optionally substituted with at least one R ⁴ group; R ⁸ is C 1 -C 16 alkylamino; X 'is Cl' · Br '· Γ or H ₃ CCO ₂ '; Q and Z are each independently CH, N, N ⁺ -R ⁵ X '' C-CH ₂ -OR ³ or CC (O) -R ⁶ ; ^R5 is as defined above; R ⁶ is -O- (C 13 -alkyl) -, hydroxyl or -NHR ⁷ : R ⁷ is C 1 -C 5 alkyl or a biologically active substance; - 62 X 'has the same meaning as given above; with the condition that a) when Q, A or Z is N or N ⁺ -R ⁵ X '' then the other two groups are CH; b) when A is C-Br, C-Cl, C-OR ³ or C-OR ⁸ , then Q and Z are both -CH; (c) the sum of R ⁴ , R ⁷ and R ⁸ , if present, must not exceed 1; and d) Only one of Q and Z can be -CC (O) -R ⁶ , and if one of Q or Z is -CC (O) -R ⁶ then A is -CH. Compounds according to claim 1, wherein in at least two R substituents T is P (O) R ¹ OH, wherein R ¹ is hydroxy and the third T is hydrogen, -COOH or C 1 -C 18 alkyl; A, Q and Z are -CH; n is 1 and X and Y are each independently hydrogen or C 1-3 alkyl. A compound according to claim 1, wherein in each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is OH; and X and Y are hydrogen, which is named 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3-, 6,9-trimethylene phosphonic acid, or a pharmaceutically acceptable salt thereof. salts. • · Compounds according to claim 2, wherein in two R substituents T is P (O) R ¹ OH, in which R ¹ is hydroxy, and in the third R substituent T is -COOH and n is 1. Compounds according to claim 2, wherein in two R substituents T is P (O) R ¹⁰ OH, wherein R ¹ is hydroxy and in the third R substituent T is P (O) R ¹⁰ OH, wherein R ¹ is C 1 -C 5 alkyl and n is 1. 6. A compound according to claim 2, wherein in two R substituents T is P (O) R ¹ OH, wherein R ¹ is hydroxy and in the third R substituent T is P (O) R ¹ OH, wherein R ¹ is -O- (C1-C5 alkyl) and n is 1. Compounds according to claim 1, wherein at least one T in the R substituent is P (O) R ¹ OH, wherein R ¹ is as defined in claim 1 and in the other two R substituents T is COOH or P (O) R ¹ OH group, and n, ^R1, X, Y, A, Q and Z are as defined in claim 1. 8. A compound according to claim 7 wherein one of R is T is P (O) R ¹ OH, wherein R ¹ is hydroxy and the other two R are T is P (O) R ¹ OH. group in which ^R1 is -0- (C1-5 alkyl), and n first Compounds according to claim 7, wherein in one of the R substituents T is P (O) R ¹ OH, wherein R ¹ is hydroxy and in the other two R substituents T is P (O) R ¹ OH. wherein R ¹ is C 1 -C 5 alkyl and n is 1. Compounds according to claim 7, wherein in one of the R substituents T is P (O) R ¹ OH wherein R ¹ is hydroxy and in the other two R substituents T is -COOH and n is 1. . A compound according to claim 7, wherein in one R substituent T is P (O) R ¹ OH, wherein R ¹ is hydroxy; in the other two R substituents, T is -COOH; n is 1; X and Y are each hydrogen; 3,9-Diacetic acid-6- (methylene phosphonic acid) 3-, 6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene, and pharmaceutically acceptable salts thereof. Compounds according to claim 1, wherein all three T in the R substituents are each P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl). ), n, R ¹ , X, Y, A, Q and Z are as defined in claim 1. 13. A compound according to claim 12, wherein T for each of the three R substituents P (O) ^R1 OH, wherein ^R1 is -0- (C1-5 alkyl) group and n is the first The compound of claim 13, wherein for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is -OC ₂ H ₅ ; which is named 3,6,9,1 5-tetraazabicyl o [9.3.1] pentadeca-1 (15), 11,13-triene-3-, 6,9-methylene ethyl phosphonate. A compound according to claim 13, wherein for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is -OC ₃ H ₇ ; which is named 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3-, 6,9-methylene (n-propyl) phosphonate. A compound according to claim 13, wherein, for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is -O-C 4 H 9; which is named 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-methylene (n-butyl) phosphonate. 17. A compound according to claim 12 wherein, for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is C 1-5 alkyl and n is 1. 18. A compound according to claim 1 wherein X and Y are hydrogen. 19. A compound according to claim 1 wherein n is 1. 20. A compound according to claim 1 wherein A, Q and Z are -CH. Compounds according to claim 1, wherein A, Q and Z are -CH, in at least one of the R substituents being different from P (O) R ¹ OH, wherein R ¹ is hydroxy. Compounds according to claim 1, wherein Q, A and Z are -CH, X, Y and n in the three R substituents are as defined in claim 1, and T is (b) or (c). wherein R ² and R ⁴ are as defined in claim 1 and the other two T are as defined in claim 1. 23. The compound of claim 22, wherein n is 1. A compound according to claim 22, wherein Q, A and Z are -CH, and two R substituents X and Y are hydrogen, one R substituent X is -CO 2 CH 3 and Y is hydrogen; n is 1; one of T is a group of formula (b) which is named 3,6,9,1 5-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,1 3-triene 3 - [(4-nitrophenyl) ) methyl acetate] -6,9-methylenedioxy-diethyl phosphonate. 25. A compound according to claim 22 wherein one of T is a group having R ⁴ and one of X or Y is -COOH. 26. The compound according to claim 22, wherein the two R substituents, not containing a group R ^4, hydrogen atoms in X and Y. 27. A compound according to claim 26, wherein the two R substituents that do not contain R ⁴ , wherein each T is P (O) R ¹ OH, wherein R ¹ is the same as defined in claim 1. 28. A compound according to claim 26, wherein the two R substituents that do not contain R ⁴ , one of which is T-COOH and the other T is P (O) R ¹ OH, wherein R ¹ has the same meaning as in claim 1. 29. A compound according to claim 1 wherein X and Y are hydrogen, T is -COOH, or a group of formula (d) wherein R ¹ is -OH, C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl). 30. The compound of claim 29, wherein Q and Z are -CH. 31. A compound according to claim 30, wherein A is C-OR ³ or C-OR®, wherein R ³ and R® are as defined in claim 1, or A is a group of formula (e) wherein R ⁴ is as defined in claim 1. 32. A compound according to claim 29, wherein A is -CH and one of Q or Z is -CH and the other is CC (O) -R ⁶ , wherein R ⁶ is The same as in claim 1. 33. The compound according to claim 32, wherein R ⁶ is NHR ^7, where R ⁷ is a biologically active agent. 34. A compound according to claim 1 wherein A, Q or Z is N ⁺ -R ⁵ X ', wherein R ⁵ and X' are Identical to that given in claim 1; and in one of R and T is P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl); and in the other two R substituents, T is P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl, -O- (C 1 -C 5 alkyl) -, or -COOH; and each X and Y is hydrogen. 35. A compound according to claim 34 wherein, for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is C 1 -C 5 alkyl or -O- (C 1 -C 5 alkyl) -. group. Complexes comprising a bicyclopolyazamacrocyclophosphonic acid according to any one of claims 1 to 35 to form a complex with Gd ³⁺ , Mn ²⁺ or Fe ³ *. 37. Complexes according to claim 36, wherein for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is hydroxy, and X and Y are hydrogen, which is designated 3,6,9, 15-Tetrazazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3, 6,9-trimethylene phosphonic acid. 38. Complexes according to claims 36 and 37, wherein Gd ³⁺ is present as metal ^ions . 39. A conjugate of any one of claims 1-35. A bicyclopolyazamacrocyclophosphonic acid compound according to claim 1, provided that at least one of R ⁴ , R ⁷ and R ⁸ is present and wherein said compound is complexed with a metal ion Gd ³⁺ , Mn ² * or Fe ³⁺ , and complex is covalently bonded to a biologically active substance. 40. The conjugate of claim 39, wherein the biologically active agent is dextran, peptide or polypeptide, a molecule having specific affinity for a receptor or an antibody or antibody fragment. 41. The conjugate of claim 40, wherein the antibody or antibody fragment is a monoclonal antibody or fragment thereof. 42. The conjugate of claim 39, wherein A is -CH and one of Q or Z is -CH and the other is a tube of the formula CC (O) -R ⁶ wherein R ⁶ is NHR ^7. , wherein R ⁷ is a biologically active substance. 43. The conjugate of claim 42, or pharmaceutically acceptable salts thereof, wherein, for each of the three R substituents, T is P (O) R ¹ OH, wherein R ¹ is OH and X and Y are hydrogen. 44. 30-43. The conjugates of claim 1, wherein Gd ³⁺ is present as the metal ion. 45. Pharmaceutical compositions comprising a complex according to claim 36 and a pharmaceutically acceptable excipient or carrier. 46. Pharmaceutical compositions comprising the conjugate of claim 39 and a pharmaceutically acceptable excipient or carrier. 47. A method for diagnosing a disease in an animal comprising administering to the test animal an effective amount of a composition according to claim 45. 48. A method of diagnosing a disease in an animal comprising administering to the test animals an effective amount of a composition according to claim 46. 49. Use of complexes according to claim 36 as a di-agnostic agent. Use of the conjugate of claim 39 as a diagnostic agent. * · · · · ··························································································· · 51. A process for preparing a complex according to claim 36, wherein the bicyclopolyazamacrocyclophosphonic acid as defined in claim 1 is reacted under aqueous conditions with a metal ion of pH 5-7 Gd ³ *, Mn ²⁺ or Fe ³⁺ . 52. The process of claim 51 wherein the bicyclopolyazamacrocyclophosphonic acid is 3,6,9,15-tetraazabicyclo [9.3.1] pentadeca-1 (15), 11,13-triene-3-, 6,9-trimethylene phosphonic acid. . 53. A process for the preparation of a bicyclopolyazamacrocyclophosphonic acid as defined in claim 1, wherein (A) a compound of formula (I) wherein at least one R is hydrogen is treated with a phosphonating agent; or (B) reacting a compound of formula (I) wherein Q, A or Z is a protecting group, according to (A), followed by deprotection by catalytic hydrogenation or acid treatment. 54. The process of claim 53, wherein the phosphonating agent is P (OR) ₃ , wherein R is as defined in claim 1. 55. The process of claim 53, wherein the phosphonating agent is a compound of formula P (OR) ₃ , wherein R is as defined in claim 1, and formaldehyde is used in a solvent.