WO1995001346A1

WO1995001346A1 - Chelating compounds

Info

Publication number: WO1995001346A1
Application number: PCT/EP1994/002126
Authority: WO
Inventors: Franciscus Michael Kaspersen; David N. Reinhoudt; Willem Verboom; Catherina Joanna Van Staveren
Original assignee: Akzo Nobel N.V.
Priority date: 1993-06-30
Filing date: 1994-06-28
Publication date: 1995-01-12
Also published as: AU7384394A

Abstract

The invention provides chelating comounds having nine ligand sites capable of simultaneously chelating a single metal cation as well as their metal chelates, the compound having formula (1), wherein A?1, A2, A3, A4 and A5¿ represent optionally substituted organic chains; Q?1, Q2, Q3 and Q4¿ each represent a nitrogen atom, or Q?1, Q2, Q3 and Q4¿ represent C-Z?1, C-Z2, C-Z3¿ and C-Z4 respectively, in which Z?1, Z2, Z3 and Z4¿ are exocyclic and represent O, S or NR?11; Z5, Z6 and Z7¿ preferably represent carboxyl; Z8 represents O, S or NR12; Z9 represents O, S or NR13, to which a hydrogen atom or an organic substituent may be attached; R?1, R2, R3, R4, R5, R6, R7, R8, R9 and R10¿ independently represent hydrogen, C¿1?-C6 alkyl or C2-C6 alk(adi)enyl, or any pair of R?1 and R5, R2 and R6, R3 and R7, R4 and R8, and R9 and R10¿ may also represent oxo, or R?8 and R9 or R9¿ and a substituent at A5 may be connected to form a ring; R11 represents hydrogen, C¿1?-C6 alkyl, C2-C6 alkenyl or C1-C6 hydroxyalkyl; R?12¿ represents hydrogen, C¿1?-C6 alkyl, C2-C6 alkenyl, C1-C6 hydroxyalkyl or a second bond together with one of the neighbouring chain atoms of Z?8; R13¿ represents hydrogen, C¿1?-C6 alkyl, C2-C6 alkenyl, C1-C6 hydroxyalkyl, ar(alk)yl or a second bond with the neighbouring chain atom of Z?9¿; wherein one of R?1, R2, R3, R4 and R9¿ or one of the susbtituents on A?1, A2, A3, A4, A5 or Z9¿ may represent A6-X, wherein A6 represents a direct bond or an organic linking group, and X represents a functional group capable of binding to a targeting molecule; or a mono- or polyanion thereof derived by abstraction of one or more acidic hydrogens.

Description

Chelating compounds

The present invention relates to chelating compounds, metal chelates and conjugates of these chelates with targeting biomolecules for use as targeted (radio)diagnostic and (radio)therapeutic agents. 5 Chelating compounds are useful in medicine as diagnostic agents and therapeutic agents for the controlled supply of metals, e.g. in nuclear magnetic imaging, radioimmunoscintigraphy and radioimmunotherapy. Metals to be used can be stable or radioactive, depending on the type of usage. Chelating compounds that are covalently bound to targeting

10 molecules, such as antibodies, can be regarded as bifunctional chelators capable of conveying a metal to a specific site in the body, for therapeutic or diagnostic purposes, e.g. to bring a radionuclide to a tumour cell for locating or treating the tumour.

US 5t053.503 discloses bifunctional chelating agents which are or

15 l,4,7,10-tetraaza-cyclododecane-l,4,7.10-tetraacetic acid (DOTA) or l.'l^.lO-tetraaza-cyclododecane-l.'J^-triacetic acid (D03A) , containing a function capable of reacting with a protein site, as well as radiodia- gnostic and radiotherapeutic agents containing such a chelating agent, a metallic radionuclide and an antibody. Similarly, WO 89/01^76 describes

20. DOTA having a functional group, in particular a 6-vinyl-2-pyridylmethoxy- acetamido-butyl group, linked at the 2-position of DOTA. EP-A-292,689 discloses D03A derivatives having a substituent with a terminal functional group at N-10; as an example 10-(N-hydroxyethylcarbamoyl- methyl)-D03A is described. EP-A-374,947 describes DOTA derivatives having

25 a aminophenyl or isothiocyanatophenyl groups as coupling functions attached at one of the cyclododecane carbon atoms or at the α-position of one of the acetic acid residues.

EP-A-43^.3^6 discloses 10-(3~Polyethyleneoxy-2-hydroxypropyl)- D03A and its metal complexes as diagnostic contrast agents. Similarly,

30 10-poly(oxyethylene)-D03A derivatives are disclosed in EP-A-466,200 and 10-trihydroxybutyl-D03A derivatives are described in EP-A-448,191. EP-A- 287,465 discloses, inter alia, 2-hydroxyethy1-DOTA and 1,4,7,10.13-penta- aza-cyclopentadecane-1,4,7.10,13-pentaacetic acid as well as their metal complexes for use in radiodiagnostics. EP-A-325.762 discloses, inter

35 alia, α-hydroxymethyl-l,ⁱ.,7,10-tetraaza-cyclododecane-l,4,7,10-tetra- acetic acid, its mono(hydroxyalkyl)amides and their metal chelates as radiodiagnostic agents. Chelating compounds wherein one or two 1,4,7.10- tetraaza-cyclododecane-l,4,7-triacetic acid groups are bound at their 10-position to a heterocycle are described in W092/12978.

Calix[4]arenes, which are phenol-formaldehyde cyclic tetramers, having four carbamoylmethoxy substituents at the 2-positions of the phenyl rings, and their complexes with lanthanide ions have been described by Sabbatini et al., J. Chem. Soc. Chem. Comm. 1990, 878-9.

There is a need for improved (radio)diagnostic and radio- therapeutic agents comprising a (radioactive) metal and a targeting agent (e.g. an antibody), wherein the metal is tightly bound with the targeting agent. This requires a chelating compound which complexes the metal ions very rapidly under mild, ambient conditions (such as temperatures from 20 to 40^*0 and a pH between 4 and 8) , even at very low concentrations of the metal ions, in order to allow an effective and economical use of the reagents. Also, decomplexation must be very slow so as to reduce the release of metal ions, e.g. in the human body, at the acceptable minimum, preferably well below 1% per day. The chelating agents known e.g. from the references cited above, although satisfactory in some applications, do not meet these high standards.

The object of the present invention is thus to provide chelating (complexing) compounds that can rapidly form highly stable complexes with metal ions, in particular trivalent radioactive metal ions. A special object of the invention is to provide such chelating compounds which also have a function capable of binding to a targeting agent.

The object is achieved according to the invention by means of chelating compounds which in one and the same molecule have nine ligand sites preorganised in the molecule in such a way that they are capable of simultaneously chelating a single metal cation. The nine ligand sites of these chelating compounds are selected from nitrogen, oxygen and sulphur atoms having a free pair of electrons. The compounds preferably contain a functional group capable of binding to a targeting agent.

In particular the chelating compounds according to the invention are represented by formula 1, wherein:

A¹, A², A³ and A* represent optionally substituted organic chains; A⁵ represents an optionally substituted organic chain; Q¹, Q², Q³ and Q¹ represent Z¹, Z², Z³ and Z* respectively, in which case each of these is a nitrogen atom, or Q¹, Q², Q³ and Q* represent C-Z¹, C-Z², C-Z³ and C-Z⁴ respectively, in which case Z¹, Z², Z³ and Z⁴ are exocyclic and independently represent 0, S or NR¹¹; wherein the spatial distances between Z¹ and Z², Z² and Z³, Z³ and Z^b, and Z^h and Z¹ each can be 3.15 * 0.25 A, and the spatial distances between Z¹ and Z³, Z² and Z^k each can be 4.45 ± 0.35 A;

Y_

Z⁵, Z and Z⁷ independently represent carboxyl, thiocarboxyl, carbox- imidoyl, phosphono, thiophosphono, phosphorimidoyl, sulpho, thiosulpho, sulfa oyl, preferably carboxyl or thiocarboxyl; Z⁸ represents 0. S or MR¹²;

Z⁹ represents 0, S or NR¹³, to which a hydrogen atom or an organic substituent may be attached; R¹, R², R³, R", R⁵, R⁶, R⁷, R⁸. R⁹ and R¹⁰ independently represent hydrogen or C_x-C₆ alkyl or alk(adi)enyl, or any pair of R¹ and R⁵, R² and R⁶, R³ and R⁷, E and R⁸, and R⁹ and R¹⁰ may also represent oxo, or R⁸ and R⁹ or R⁹ and a substituent at A⁵ may be connected to form a ring together with the carbon atoms to which they are bound; R¹ represents hydrogen, C_j-C₆ alkyl, C₂-C₆ alkenyl or Ci-C₆ hydroxyalkyl; R¹² represents hydrogen, C_j-C₆ alkyl, C₂-C₆ alkenyl, C^C^ hydroxyalkyl or a second bond together with one of the neighbouring chain atoms of Z⁸; R¹³ represents hydrogen, C--C₆ alkyl, C₂-C₆ alkenyl, C^Cg hydroxyalkyl, optionally substituted C₅-C₁₀ aryl or C^-C^ aralkyl, or a second bond together with the neighbouring chain atom of Z⁹; wherein one of R¹, R², R³, R⁴ and R⁹ or one of the substituents on A¹, A², A³, A'', A⁵ or Z⁹ may represent A⁶-X, wherein A represents a direct bond or an organic linking group, and X represents a functional group capable of binding to a targetting molecule; or a mono- or polyanion thereof derived by abstraction of one or more acidic hydrogens; with the proviso that if Q¹, Q², Q³ and Q* are each nitrogen and Z⁹ is hydroxy, C-R⁹R¹⁰-Z⁸ does not represent methyleneoxy or carbonylimino.

In a variant of the compounds according to the invention, the symbols Q¹, Q², Q³ and Q^ή each represent a nitrogen atom as Z¹, Z², Z³ and Z*. and together with the organic chains A¹, A², A³ and A* they constitute a tetraaza-cycloalkane system, as shown in formula 3-

3.

In the compounds having formula 3_t the organic chains A¹, A², A³ and A^ή preferably represent two-carbon or three-carbon chains, most preferably two-carbon chains. The corresponding ring systems include 1,4,7.10-tetraaza-cyclododecane, l,4,7ιlO-tetraaza-cyclotridecane and 1,4,8,11-tetraaza-cyclotetradecane. The organic chains may also be unsaturated and/or substituted, e.g. with C_j-Cg alkyl groups, resulting in e.g. the cyclododecene, cyclotetradecadiene, 2,5.8,11-tetramethyl- cyclododecane, and dibenzo[i>,h]cyclododecadiene analogues of these tetra- azacycloalkane systems. One of the four organic chains, e.g. A¹ or A³, may advantageously substituted with A⁶-X, i.e. with the group providing a bonding function for a targeting agent. The preferred ring system is 1,4,7,10-tetraaza-cyclododecane.

In another variant of the compounds according to the invention, the symbols Q¹, Q², Q³ and Q^ή represent C-Z¹, C-Z², C-Z³ and C-Z^ή respectively, wherein the C-atoms are inserted between A¹, A², A³ and A*, and each have a double bond with one of the neighbouring ring atoms. In particular, each C-atom together with both its neighbouring ring atoms forms a benzene ring thus constituting a tetrabenzo[α_ ,e/, ij , mn_cyt_-.o- hexadecatetraene. Z¹, Z², Z³ and Z^h represent 0, S or NR¹¹, preferably 0, attached at positions 2, 6, 10 and 14 of the cyclohexadecatetraene as shown in formula 2.

The benzo groups may contain a p-substituent R 16 such as C^-Cg alkyl, in particular tert-butyl, whereas one of the four symbols R¹⁶ may also represent the bonding group A⁶-X as described above.

In the formulae 1, 2 and 3t the R¹, R^2, R³ and R* preferably represent hydrogen or

alkyl, whereas one of the three may also represent the group A⁶-X, and R⁵, R⁶, R⁷ and R⁸ preferably represent hydrogen. Most preferably each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ represents hydrogen.

R⁹ and R¹⁰ may advantageously together represent an oxo group, especially when Z⁸ is a nitrogen group NR¹². Also, each one of these symbols R⁹ and R¹⁰ may represent hydrogen or one hydrogen and one C_j-C^ alkyl or C₂-C₆ alk(adi)enyl, in which cases R* and R⁸ may be hydrogen or together be oxo. As another useful alternative, if Z⁸ is a preferred nitrogen group NR¹², R⁹ may be connected with R¹², or with the first atom of the chain A⁵ to form a ring such as pyridine, in which latter case R¹² represents a double bond with one of the neighbouring carbon atoms.

The group A⁵ is preferably a two-carbon or three carbon chain that may be substituted. The substitution may be so as to form a ring. In particular (Z⁸)-A⁵-(Z⁹) may represent a group (Z⁸)-CR¹⁷R^l8-(CHR²¹)„-CR¹⁹R²⁰- (Z⁹), wherein n equals 0 or 1, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ independently represent hydrogen, Cι-C₆ alkyl or C₂-C₆ alk(adi)enyl, and R¹⁷ and R¹⁸, or R¹⁹ and R²⁰ may also together represent oxo, and whe- -n R⁹ and R¹⁷, R¹² and R¹⁷, R¹⁷ and R¹⁹, R¹⁷ and R²¹, R¹⁹ and R²¹. R¹⁹ and R¹³. or R¹⁹ and the substituent attached to Z⁹ may also be connected to form a ring. One of R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ may also represent the group A⁶-X.

The group Z⁹ may e.g. be hydroxy, alkoxy, mercapto, alkylthio, --.---^■inn, (di)alkylamino, acylamino, or may be a part of a heterocycle which does or does not include the neighbouring atom of A⁵. Z⁹ may advantageous¬ ly carry the group A⁶-X. Examples of the combined group A⁵-Z⁹ are carboxy- methyl, 3^_(2-oxo-l-pyrrolidinyl)propyl, acetylaminoethyl, 2-pyridyl- methyl, N-(2-mercaptoethyl)carbamoylethyl, and 2-pyridylacetyl; examples of the combined groups CR⁹R¹⁰-Z⁸-A⁵-Z⁹ are 6-hydroxymethyl-2-pyridyl, 6-carboxy-2-pyridyl and 6-(benzylcarbamoyl)-2-pyridyl.

In the group A⁶-X, X may e.g. represent isocyanato, isothio- cyanato, mercapto, amino, haloacetyl, diazo, succinimido, maleimido, a reactive ester group or an anhydride. A may be any organic chain which may incorporate alkylene, alkenylene, cycloalkylene, arylene, carbonyl groups, heteroatoms; A⁶ may for example be or contain ethylene, butylene, methylenecarbonyl, polyoxyethylene, phenylen^'e, phenylenemethylene etc. The combined groups A⁶-X may be e.g. be ω-mercaptoalkyl, p-aminophenyl, p-isothiocyanatobenzyl, p-aminophenylalkyl, chloroacetyl ethyl, p-di- azoniobenzyl, ω-maleimidoalkyl, etc. The linker A⁶ may be stable or metabolically labile (see Y. Arano et al, Biocon ug. Chem. 7 -76 (1991) and L. Yuanfang et al, Pure Appl. Chem. 63. 427-463 (1991))- A labile linker may e.g. contain a hydrolysable ester group.

The chelating compounds according to the invention are suitable for complexing trivalent metal ions, in particular heavy metal ions, such as Y, Ru, Rh, In, La, the lanthanides, Bi, Ac and the actinides. For therapeutic and diagnostic purposes, radioisotopes of such metals, e.g. ⁹⁰Y. ^99mTc. ^U1ln, ¹⁵²Eu. ¹⁵³Sm. ^2uBi, ²¹²Bi. ²¹³Bi or ²²⁵Ac are preferably used. The invention also concerns the chelates of the chelating compounds described above with such metal ions.

Coupling of the chelating compounds and chelates described above, which have a functional link A⁶-X, with targeting agents can be performed in a way known per se, e.g. by reaction of the functional group X with an amino group, hydroxy group or mercapto group of a targeting protein, by reaction of functional group X with a hydroxy group of a targeting polysaccharide (as such or a glycosyl side chain of a protein) or by reaction of functional group X with an amino group or hydroxy group of a targeting poly- or oligo-nucleotide. Methods of coupling chelators to targeting agents have been reviewed by L. Yuanfang and . Chuanchu in Pure & Appl. Chem. 63(3). 427-463 (1991)- The targeting agents are site- specific or receptor-specific. Preferred targeting agents are antibodies or fragments thereof, site-specific proteins, hormones or other receptor ligands and poly- or oligonucleotides. Coupling may be -ichieved directly or through the intermediacy of further coupling agents such as dialdehydes or dithiols as is known in the art. Coupling of the chelating compound to the targeting molecule may be performed before or after the complexation with the metal ion in the chelating compound. Coupling car be accomplished e.g. by incubating the targeting protein and the chelating agent in a buffer and purifying the protein-chelate conjugate by gel filtration chromatography. The invention also relates to the conjugates obtained by coupling of a chelating compound or a chelate as described above. The conjugates are useful in cases where tightly bound metal ions are required on specific sites, such as in therapy, in vitro diagnostics, as MRI contrast agents, etc.

The invention furthermore relates to pharmaceutical compositions containing a chelating compound, a chelate or a conjugate as described above, together with a pharmacologically acceptable carrier, such as an injection fluid or an excipient, possibly with additives, adjuvants etc.

The chelating compounds may be prepared by methods known per se.

For example, the compounds of the DOTA-type and the cyclotridecane and cyclotetradecane homologues can be preprared starting for the appropriate N-unsubtituted tetraaza ring system, to which first the group -CR⁴R⁸- CR⁹R¹⁰-Z⁸-A⁵-Z⁹ is attached, whereafter the three groups -CR^-Z⁵, -CRR⁶- Z⁶ and -CR³R⁷-Z⁷ are introduced. Especially when the latter three groups are identical, e.g. carboxy-methyl, it may be advantageous to start with the appropriate tri-substituted tetraaza system, such as triprotected D03A, to which the side chain -CRV-CR⁹R¹⁰-Z⁸-A⁵-Z⁹ is then attached.

The compounds of the calix[4]arene type may start with the appro¬ priate tetrabenzo[αb, ef, ϊ;/,mπ]cyclohexadecatetraene-2,6,10,l4-tetraol. To the tetraol, which is taken as an example, the groups -CR^-Z⁵, -CR²R⁶- Z⁶, -CR³R⁷-Z⁷, and -CRV-CR⁹R¹⁰-Z⁸-"^*'-Z⁹ or a precursor of the latter such as -CR⁴R⁸-CR⁹R¹⁰-Z⁸Y (wherein Y may e.g. be hydrogen, alkyl or a protecting group), may than be attached, either simultaneously, e.g. when they are all carboxymethyl,or consecutively if they are different. Preferably Z⁵, Z⁶, and Z⁷ are attached in a protected form. The group -CR*R⁸-CR⁹R¹⁰-Z⁸- A⁵-Z⁹ is then introduced as such or by coupling of A⁵-Z⁹ (optionally protected) to Z⁸ (deprotected if necessary). Finally the protecting groups are removed. Examples of these approaches are given here below.

The synthesis of 9^_coordinating acrocycles fitted with a handle A⁶-X for conjugation can be carried out via several routes, depending on the attachment position of the handle. Examples are given below.

Synthesis of Q-coordinating tetra-aza macrocvcles

Example I

Synthesis of 1-(6-hvdroxymethvl-2-pvridvlmethvl)-1.4.7.10-tetraazacvclo- dodecane-4.7.10-triacetic acid (compound with formula 4)

2-Bτomomethyl-6-hydτoxymethylpyτidine

2,6-Bis-hydroxymethyl-pyridine (10.0 gram, 71-8 mmol) was dissolved in 100 mL 482 HBr in H₂0. The mixture was heated at bath temperature l4θ°C for 2 hours, and subsequently cooled to -5^*C. The solution was neutralized by dropwise addition of 0% aqueous NaOH at -5^βC until pH=7- The resulting slurry was extracted with CH₂C1₂ (5 x 100 mL) .

The organic layers were combined, dried over Na₂S0j,, and the solvent was evaporated to give an oil. The crude products was separated on silica.

Upon elution with CH₂C1₂ 0.40 gram of bis(bromomethyl)pyridine was obtained. Further elution with ether gave 4.50 gram of 2-bromomethyl-

6-hydroxymethylpyridine as a white crystalline product. Yield 31 ; ¹H-NMR

(CDC1₃/CD₃0D) δ: 7-75 (t, J=7Hz, 1H. PyrH) , 7-38 (d. J=7Hz, 2H, PyrH) ,

4.72 (s, 2H, CH2Br). 4.53 (s, 2H, CH20H).

2-Bτomomethyl-6-tetτahydτopyτanyloxymethyl-pyτidine To a solution of 2.00 gram (9-90 mmol) of 2-bromomethyl-6-hydroxymethyl- pyridine in 10 ml CH₂C1₂ was added 0.14 gram (1.98 mmol) p-toluene- sulphonic acid and 3-34 gram (40 mmol) of dihydropyran. The mixture was stirred for 3 days. The reaction mixture was washed with 1% aqueous NaHC0₃ and dried over Na₂S0i,. The solvent was evaporated, and the excess of dihydropyran was removed by distillation under reduced pressure. The residue was then purified by chromatography on silica with toluene/- EtOH 9/1 as the eluent to give 1.20 gram of the 2-bromomethyl-6-hydroxy- methyl-pyridine tetrahydropyranyl (THP) ether. Yield 3%. H NMR (CDC1₃) δ: 7-72 (t, J=7Hz,lH, PyrH), 7.41 (d, J=7Hz, 1H, PyrH), 7.32 (d, J=7Hz, 1H, PyrH), 4.90, (d, 1H, PyrCH) 4.62 (d, 1H, PyrCH) 4.77 (t, J=lHz, 1H, CH-0). 4.55 (s, 2H, CH2Br), 3-72-3.98 and 3- 7-3- 2 (m, 2H, CH₂) , 1.45-2.00 (m, 6H, 3 x CH2), MS: M+ 285 (calcd: C₁₂H_l6Nθ₂Br 285.036)

Tvi-t-butyl l-(6-tetτahydτopyτanyloxymethyl-2-pyτidylmethyl)-l,4, 7,10- tetτaazacyclododecane-4 , 7 , 10- triacetate (protected macrocycle) To a solution of 0.5 gram (0.97 mmol) of l,4,7-tricarboxymethyl-l,4,7,10- tetraazacyclododecane tris-t-butyl ester in 10 mL CH₃CN was added 0.60 gram (O.85 mmol) Cs₂C0₃ and 0.257 gram (O.96 mmol) 2-bromomethyl- 6-tetrahydropyranyloxymethyl-pyridine. The mixture was refluxed overnight and subsequently cooled. The salts were removed by filtration and the solvent was evaporated to give the product as a white powder. *H NMR (CDC1₃) δ: 7.65 (t. J= 7Hz. 1H, PyrH), 7-34 (d, J=7Hz, 1H, PyrH). 7.36 (d, J=7Hz, 1H, PyrH), 4.88 (d, J=l4Hz, lH, OCH) , 4.60 (d, J=l4Hz, lH. OCH), 4.78 (t. J=lHz. 1H. CH-0), 3-99-3-85 and 3-62-3.50 (m, 2H, CH₂) , 3.75 (s, CH₂), 3-35 (s, 2H, CH₂C0₂). 3-22 (s. 4H, 2 x CH₂C0₂) , 2.85 (bs, 12H, CH₂N), 2.75-2.65 (m. 4H, CH₂N) , 2.0-1.5 (m. 6H. 3 x CH₂ THP), 1.45 (s, 9H. tBu), 1.43 (s. 18H. tBu).

Synthesis of chelating macrocycle 4

A solution of 0.20 gram (0.28 mmol) of tri-t-butyl l-(6-tetrahydro- pyranyloxymethyl-2-pyridylmethyl)-1,4,7,10-tetraazacyclododecane-4,7,10- triacetate in 0 L EtOAc containing an excess of HC1 was stirred at room temperature for 1 hour. After addition of 0.5 mL of water the solvent was evaporated and the residue was triturated with EtOAc. The resulting solid product was filtered off to give 0.15 gram (98%) of the product as the dihydrochloride salt as a white powder. The product was recrystallised from MeOH/acetone. ^XH NMR (D₂0) δ 8.55 (t. J=7Hz. 1H, PyrH), 8.05 (d. J=7Hz. 1H, PyrH). 8.00 (d. J=7Hz. 1H. PyrH). 5-07 (s. 2H, CH₂0H) . 4.2-3.0 (m. 24H, NCH₂). MS (FAB) M+H 468 (Calculated C₂₁H₃„N₅0₇= 467). Example II

Synthesis of 1-(6-benzylcarbamoyl-2-pyridylmethvl)-1.4.7.10-tetraaza- cvclododecane-4.7.10-triacetic acid (compound with formula 5) 2-Methoxycarbonyl-6-benzylaminocaτbonyl-pyridine

To a solution of 19-5 gram (0.1 mol) 2,6-pyridinedicarboxylic acid di¬ methyl ester in 100 mL MeOH was added 10.7 gram (0.1 mol) of benzylamine. The solution was refluxed for 60 h. The solvent was evaporated, and the resulting product was purified by chromatography (silica, EtOAc/heptane 2/3) . The yield of the monoamide monoester was 11 gram (4l ) . *H NMR (CDC1₃) δ 8.45 (d, J=7Hz, IH, PyrH). 8.23 (d, J=7Hz. IH, PyrH), 8.00 (t, J=7Hz, IH, PyrH). 7-4-7-2 (m, 5H. ArH), 4.73 (d, J=5Hz, 2H, CH₂N) , 3-98 (s, 3H, OCH₃). MS 270 (M+, Calculated: C₁₅H_l4N₂0₃: 270)

2-Hydτoxymethyl-6-benzylaminocarbonyl-pyridine

To a solution of 10.0 gram (37 mmol) of 2-methoxycarbonyl-6-benzylamino- carbonylpyridine in 100 L MeOH at 30^βC was added 2.85 gram (75 mmol) NaBH, in small portions over a period of 2 hours. The mixture was stirred overnight. Water was added (50mL) and the mixture was concentrated to 25 mL. Another portion of 100 mL water was added and the mixture was extracted with 3x100 mL of CH₂C1₂. The organic layers were combined and dried over Na₂S0ι,. The solvent was evaporated to give an oil which solidi¬ fied upon standing. Yield 8.5 gram (95?!). Mp 89.5"92.0^eC, *H NMR (CDC1₃) δ 8.4-8.2 (bs, IH. NH), 8.15 (d. J=7Hz, IH, PyrH), 7-85 (t, J=7Hz, IH. PyrH). 7-48 (d. J=7Hz, IH, PyrH), 7-4-7-2 (m. 5H, ArH). 4.78 (d, J=5Hz. 2H. CH₂0). 4.68 (d, J=6Hz. 2H. CH₂N) . 3-16 (bt, IH, OH). MS 242 (M*. calcd C_l4H_lJkN₂0₂: 242) 2-Chloromethyl-6-benzylaminocarbonyl-pyridine

A solution of 8.5 gram (35 mmol) of 2-hydroxymethyl-6-benzylamino- carbonylpyridine in 150 mL CH₂C1₂ was treated with 6.75 gram (35 mmol) tosyl chloride and 7-07 gram (70 mmol) Et₃N at room temperature. The mixture was stirred overnight. The solution was then washed with H20, and dried over MgS04. After evaporation of the solvent, the product was purified by flash chromatography (silica, EtOAc/heptane 1/1), to give the product as an oil, which solidified upon standing. Yield 3-0 gram. H NMR (CDC1₃) δ 8.3 (bs, IH, NH), 8.18 (d, J=7Hz, IH, PyrH), 7-89 (t, J=7Hz, IH, PyrH), 7-6 (d, J=7Hz, IH, PyrH), 7-4-7-2 (m, 5H, ArH), 4.67 (d, J=5Hz, 2H, CH₂N), 4.65 (s, 2H, CH₂C1).

Tri-t-butyll- (6-benzylaminocarbonyl-2-pyτidyϊmeth l)-l ,4 , 7,10-tetraaza- cyclododecane-4 , 7 , 10-triacetate (protected macrocycle)

To a solution of 0.23 gram (0.45 mmol) l,4,7-tricarboxymethyl-l,4,7,10- tetraazacyclododecane tris-t-but- ^ster in 10 mL EtOH was added 0.12 gram 2-chloromethyl-6-benzylamir- -'bonylpyridine and 0.25 gram (2.35 mmol) Na₂C0₃. After addition of 5 mg Nal the mixture was refluxed for 70 hours. The solvent was evaporated and the product was dissolved in CH₂C1₂/H₂0 and this was extracted with CH₂C1₂. The organic layers were combined and dried over Na₂S0i,. After evaporation of the solvent, the product was purified by chromatography to give 90 mg of product. H NMR (CDC1₃) δ 8.95 (bt. IH, NH). 8.25 (d, J=7Hz, IH, PyrH), 7-90 (t, J=7Hz. IH, PyrH). 7-35-7-20 ( . 5H. ArH), 4.7 (bs. 2H. NCH₂) , 3-7 (m, 2H, PyrCH₂). 3-3-2.0 (b . 24H, NCH₂) , 1.5 (s. 9H. tBu). 1.35 (s, l8H. 2x tBu).

Synthesis of chelating macrocycle 5

A solution of 80 mg (0.1 mmol) of tri-t-butyl l-(6-benzylaminocarbonyl-2- pyridylmethyl)-l,4,7.10-tetraazacyclododecane-4,7,10-triacetate in 50 mL CH₂C1₂ was saturated with HC1 gas and the mixture was stirred overnight at room temperature. The solvent was evaporated and the product was d -solved in 0.5 mL of MeOH. Upon addition of EtOAc the product pre¬ cipitated and this was filtered off, to i"e a white powder. Yield: 65 mg (100?;). ^XH NMR (CD₃OD) δ 8.05-7-75 (*. 3H, PyrH), 7-45-7-10 (m. 5H. ArH). 4.60 (s. 2H. ArCH₂) . 4.5~3-0 (m. 24H. NCH₂) . FAB MS 571 (M+H: Calculated for C₂₈H₃₈N₆0₇: 570) Example III

Synthesis of l-(6-methoxycarbonyl-2-pyridylmethyl)-l A7,10-tet-raaza-cyclodcκiecane-4,7,10-tri- acetic acid (compound with formula 6a) and

Synthesis of l-(6-carboxyl-2-pyridylmethyl)-l,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid

(compound with formula 6b)

2-Hydroxymethyl-6-methoxycarbonyl-pyridine

A solution of 2,6-pyridinedicarboxylate dimethyl ester (10.0 gram, 43 mmol) in 500 mL MeOH at

40°C was treated with NaBH₄ (2.47 gram, 65 mmol), which was added in portions over 1 hour. The mixture was stirred overnight. Water (100 mL) was added, and the product was extracted with

CH₂C1₂. The organic layer was dried (Na₂SO₄, and concentrated in vacuo. The product was purified by chromatography (silica, EtOAc/heptane 1/3). Yield: 34%. *H NMR (CDC1₃) δ 8.15 (d, J=7Hz,

IH, PyrH, 7.87 (t, J=7Hz, IH, PyrH), 7.55 (d, J=7Hz, IH, PyrH), 4.88, (bs, 2H, CH₂OH), 4.00 (s,

3H, OCH₃).

6a. 6b.

2-Methanesulphonyloxymethyl-6-methoxycarbonyl-pyridine

A solution of 2-hydroxymethyl-6-methoxycarbonyl-pyridine (1.67 gram, 10 mmol) in 100 mL CH₂Cl₂ was cooled to 0°C. Et₃N (2.02 gram, 20 mmol) was added, followed by methanesulfonyl chloride (1.35 gram, 11 mmol). The mixture was stirred for 1 hour, diluted with 200 mL CH₂C1₂, and washed with H₂O. The organic layer was dried (MgSO₄) and concentrated in vacuo. The product was purified by chromatography (silica, CH₂Cl₂/EtOH 98/2). Yield: 41%. 1H NMR (CDC1₃) δ 8.15 (d, J=7Hz, IH, PyrH), 7.92 (t, J=7Hz, IH, PyrH), 7.68 (d, J=7Hz, IH, PyrH), 5.45, (s, 2H, CH₂O), 4.00 (s, 3H, OCH₃), 3.15 (s, 3H, SCH₃).

Tή-t-B tyl 1 -(6 -methoxycarbonyl-2 -pyridylmethyl) -1,4,7,10- tetraaza - cyclododecane-4, 7, 10-triacetic acid (protected macrocycle)

To a solution of 2.05 gram (4.0 mmol) of tri-t-butyl-l,4,7,10-tetraaza-cyclododecane-l,4,7-triacetate in 50 mL CH₃CN was added 0.98 gram (4.0 mmol) 2-methanesulfonyloxymethyl-6-methoxy- carbonyl-pyridine and 1.30 gram (4.0 mmol) CS₂CO₃. The mixture was stirred at 50°C for 5 hours. After cooling to room temperature the salts were filtered off, and the organic layer was concentrated. The residue was taken up in CH₂C1₂ and washed with H₂O. The organic layer was dried (Na₂SO₄) and concentrated. The product was purified by chromatography (silica, CH₂Q₂/E-OH 95/5) Yield 50%. 1H NMR (CDCI₃) δ 8.04 (d, J=7Hz, IH, PyrH), 7.95 (t, J=7Hz, IH, PyrH), 7.58 (d, J=7Hz, IH, PyrH), 3.92 (s, 3H, OCH₃), 3.4-2.2 (m, 24H, N H₂), 1.50 (s, 9H, tBu), 1.32 (s, 18H, 2 x tBu). FAB MS: 664 (M+H)⁺, 686 (M+Na)⁺ (Calcd for C₃₄H₅₇N₅O₈: 663).

Synthesis of chelating macrocycle 6a

A solution of 1.0 gram of tri-t-butyl l-(6-methoxycarbonyl-2-pyridylmethyl)-l,4,7,10-tetraaza- cyclododecane-4,7,10-triacetic acid in 100 mL CH₂Cl₂ was saturated with HC1 gas, and the solution was kept overnight. The mixture was concentrated to 10 mL. The product precipitated and was filtered off, and was washed with CH₂C1₂, EtOAc, and ether, and was dried in vacuo. Yield 70%. ^lH NMR (D₂O) δ 8.20 (d, J=7Hz, IH, PyrH), 8.05 (t, J=7Hz, IH, PyrH), 7.95 (d, J=7Hz, IH, PyrH), 4.00 (s, 3H, OCH₃) 3.9-2.9 (m, 24H, NCH₂). FAB MS: 496 (M+H)⁺ (Calculated for C₂₂H₃₃N₅O_g: 495).

Synthesis of chelating macrocycle 6b

A solution of compound 6t (0.25 gram, 0.5 mmol) in 10 mL dioxane/H₂O 7/3 was treated with excess LiOH (pH=l 1) at room temperature overnight. After evaporation of dioxane, the product was acidified with HC1 to pH=l, and precipitated with AtOH/ether. FAB-MS: 482 (M+H)⁺ (Calculated for C₂ιH₃₁N₅O₈: 481).

Example IV

Synthesis of l-(6-carboxyl-2-pyridylmethyl)-5-(4-aminobenzyl)- 1 ,4,7,10-tetraaza-cvclododecane-

4,7,10-triacetic acid (compound with formula 7)

2-Aminobenzyl-3-oxo-l ,4 ,7-triaza-heptane

A solution of 8.07 gram (36 mmol) DL-p-nitro-phenylalanine methyl ester was dissolved in 10 mL

MeOH, and added dropwise to 75 mL ethylene diamine at room temperature. The mixture was stirred overnight at room temperature. The solvents were removed in vacuo, and the residue solidified upon stirring with EtOAc. The product was filtered off. Yield: 99%. *H NMR (D₂O) 8.15

(d, 2H, ArH), 7.35 (d, 2H, ArH), 3.62 (dd, IH, αH), 3.4-2.7 (m, 5H, CH₂NH, CHNH, ArCH₂),

2.40-2.65 (m, 2H, CH₂NH).

N-(t-Butoxycarbonyl)-iminodiacetic acid

A suspension of 20 gram (0.15 mol) iminodiacetic acid in 500 mL tBuOH/H₂O (2/1) was treated with 4N NaOH until pH 8.5. The starting material dissolved. Boc₂O (36 gram ,0.16 mol) was dissolved in tBuOH/H₂O (2/1) and this was added rapidly throough a dropping funnel, while keeping the pH at 8 by addition of 4N NaOH. The mixture was stirred overnight at room temperature. The mixture was diluted with 200 mL water, and was washed with heptane. The pH was adjusted to pH=3 with KHSO₄, and the product was isolated by extensive extraction with EtOAc. The organic layers were combined, and dried over Na₂SO₄, and the solvent was evaporated to give an oil. Yield 72%. ^!H NMR (CDC1₃) δ 1.45 (s, 9H, tBu), 4.00 (s, 2H, NCH₂), 4.10 (s, 2H, NCHi), 4.5 (bs, 2H, COOH).

N-(t-Butoxycarbony I) -iminodiacetic acid bis N-hydroxysuccinimide ester.

A solution of 15 gram (64 mmol) N-(t-butoxycarbonyl)-iminodiacetic acid in 1500 mL CH₂C1₂ was cooled to 0°C. 26.6 gram (128 mmol) dicyclohexyl-carbodiimide and 14.8 gram (128 mmol) N-hydroxysuccinimide were added, and the mixture was stirred at room temperature overnight. The precipitated dicyclohexylurea was removed by filtration. The solvent was evaporated to give 26.7 gram (97%) of the product. ^lH NMR (CDCl₃/CD₃OD)) δ 1.50 (s, 9H, tBu), 2.86 (s, 4H, CH₂CH₂), 2.89 (s, 4H, CH₂CH₂), 4.45 (s, 2H, NCH₂), 4.58 (s, 2H, NCH₂). 1 -(t-Butc r. :rbonyl)-5-(4-nitrobenzyl)-3 ,6,11 -trioxo-1 ,4,7 ,10-tetraaza-cyclododecane. A solutio-. 2-aminobenzyl-3-oxo-l,4,7-triaza-heptane (9.3 gram, 37 mmol) and 6 mL Et₃N in 200 mL of dry DMF/dioxane (1/1) and a solution of 15.8 gram of N-(t-butoxycarbonyl)-iminodiacetic acid bis N-hydroxysuccinimide ester in 200 mL of dry dioxane were added dropwise over 1 hour to a solution of 500 mL of refluxing dioxane. The mixture was refluxed overnight. After cooling to room temperature the solvent was evaporated. Acetone was added and the product precipitated, and was filtered off. Yield: 10%. 1H NMR (DMSO-d₆) δ 8.65 (bd, IH, NH), 8.18 (d, 2H, ArH), 7.75 (bt, IH, NH), 7.55 (d, 2H, ArH), 7.15 (b, IH, NH). 4.4 (bq, IH, αH), 4.2-2.9 (m, 8H, 4 x CH^, 1.45 (s, 9H, tBu). FAB-MS 450 (M+H)⁺ (calcd for C₂₀H₂₇O₇N₅: 449).

l-(t-Butoxycarbonyl)-5-(4-nitrobenzyl)-l ,4 ,7 ,10-tetraaza-cyclododecane. A solution of 1.0 gram (2.2 mmol) of l-(t-butoxycarbonyl)-5-(4-nitrobenzyl)-3,6,l 1-trioxo- 1,4,7,10-tetraaza-cyclododecane in 20 mL of dry THF under N₂ was cooled to -15°C . A solution of BH₃.THF (1 M, 30 mL) was added and the mixture was stirred at -15°C for 1 hour and subsequently heated to reflux for 18 hours. After cooling to room temperature 10 mL of MeOH was added carefully, and the solvent was evaporated. The residue was taken up in EtOAc and the product was precipitated with ether. Yield 56%. NMR (DMSO-d₆) δ 8.1 (bd, 2H, ArH), 7.5 (bd, 2H, ArH), 7.5 (bm, IH, NH), 6.7 (bm, IH, NH), 6.4 (bm, IH, NH), 4.9 (bm, IH, αH), 3.7-2.1 ( m, 16 H, CH₂), 1.4 (s, 9H, tBu). FAB-MS 408 (M+H)⁺ (calcd for

407).

l-(t-Butoxycarbonyl)-5-(4-nitrobenzyl)-l ,4 ,7 ,10-tetraaza-cyclododecane-4 ,7 ,10-triacetic acid triethyi ester.

To a solution of 0, 5 gram (0.36 mmol) of l-(t-butoxycarbonyl)-5-(4-nitrobenzyl)- 1,4,7, 10- tetraaza-cyclodode- ane in 20 mL CH₃CN was added 0.50 gram (1.53 mmol) Cs CO₃ and the mixture was heated to reflux temperature. Subsequently 0.4 gram (2.39 mmol) of ethyl bromoacetate was added. The mixture was refluxed for 1 hour. The solvent was evaporated. The residue was dissolved in H₂O/CH₂Cl₂. The organic layer was separated off, dried over Na₂SO₄, and the solvent was evaporated. Yield: 62%. ^!H NMR (CDC1₃) δ 8.15 (d, 2H, ArH), 7.32 (d, 2H, ArH), 4.3-4.1 (m, 6H, 3 x OCH₂), 4.1-4.0 (m, IH, αH), 3.9-2.4 (m, 22H, 11 x CH₂), 1.48 (s, 9H, tBu), 1.35-1.05 (m, 9H, 3 x CH₃). FAB-MS 666 (M+H)⁺, calcd for C₃₂H₅₁N₅O₁₀665.

2-(4-Nitrobenzyl)-l ,4 ,7 ^' ,10-tetraaza-cyclododecane-l ,4 ,7-triacetic acid triethyi ester. A solution of 0.14 gram (0.21 mmol) of l-(t-butoxycarbonyl)-5-(4-nitrobenzyl)-l,4,7,10-tetraaza- cyclododecane-4,7,10-triacetic acid triethyi ester in 50 mL CH₂C1₂ was treated with HC1 gas for 30 minutes. The mixture was stirred at room temperature for 1 hour. The solvent was evaporated and the residue was dissolved in H₂O, and extracted with CH₂C1₂. The organic layer was dried on Na₂SO₄ and the solvent was evaporated. FAB-MS: 566 (M+H)⁺ (Calcd for C₂₇H₄₃N₅O₈: 565) l-(6-Methoxycarbonyl-2-pyridylmethyl)-5-(4-nitrobenzyl)-l ,4 ,7 ,10-tetraaza-cyclododecane- 4 ,7 ,10-triacetic acid triethyi ester

To a solution of 0.11 gram (0.19 mmol) of 2-(4-nitrobenzyl)-l, 4,7,10-tetraaza-cyclododecane- 1,4,7-triacetic acid triethyi ester in 20 mL CH₃CN was added 0.25 gram (0.95 mmol) Cs₂CO₃ The mixture was heated to reflux for 2 hours, and 0.048 gram (0.19 mmol) of

2-methanesulphonyloxymethyl-6-methoxycarbonyl-pyridine was added. The mixture was heated for an additional 2 hours. After cooling to room temperature the salts were removed by filtration. The filtrate was concentrated and dissolved in EtOAc, washed with water, and dried over Na₂SO₄. Yield: 91%. ^!H NMR (CDC1₃) δ 8.15 (d, 2H, ArH), 8.10-7.85 (m, 2H, PyrH), 7.80-7.65 (m, IH, PyrH),7.20 (d, 2H, ArH), 4.3-4.05 (m, 7H, 3 x OCH₂ + αH), 3.9-2.2 (m, 24H, CH₂N), 1.4-1.1 (m, 9H, 3 x CH₃).

l-(Carboxyl-2-pyridylmethyl)-5-(4-nitrobenzyl)-l ,4 ,7 ,10-tetraaza-cyclododecane-4 ,7 ,10-triacetic acid

To a solution of 0.15 gram (0.17 mmol) of l-(6-methoxycarbonyl-2-pyridylmethyl)-5-(4-nitro- benzyl)-l,4,7,10-tetraaza-cyclododecane-4,7,10-triacetic acid triethyi ester in 20 mL of dioxane H₂O (7/3) was added LiOH.H₂O to pH=l 1. The mixture was stirred a t room temperature overnight. 2M HC1 was added to pH=l, and the product was precipitated with EtOH/ether. *H NMR (D₂O) δ 8.40 (d, 2H, ArH), 8.35-8.1 (m, 2H, PyrH), 7.50 (d, IH, PyrH), 7.50 (d, 2H, ArH), 4.3-2.6 (m, 25H, CH₂, CH). FAB-MS: 617 (Calcd for C₂₈H₃₆N₆O₁₀: 616).

Synthesis of chelating macrocycle 7

A solution of 21.6 mg (0.07 mmol) of SnCl₂ in 5 mL of concentrated HC1 was heated to 110°C. To this solution 30 mg (0.05 mmol) of i-(carboxyl-2-pyridylmethyl)-5-(4-nitrobenzyl)-

1,4,7, 10- tetraaza-cyclododecane-4,7,10-triacetic acid in 5 mL HC1 was added and heating was continued for 2 hrs. After cooling to room temperature, the solvent was evaporated and the product was purified by HPLC (fosfate buffer/water/acetonitrile). The product was desalted by the HPLC with HCl/water/acetonitrile. Yield: 16%. 1H NMR (D₂O) δ 8.1-7.9 (m, 3H, PyrH), 7.4-6.9 (m, 4H,

ArH), 4.0-2.6 (m, 25H, CH, CH₂). FAB-MS 587 (M+H)⁺ (Calcd for C^gNeOg 586).

Example V ) Results of complexation experiments of 1-.6-hvdroxymethyl-2-pyridyl- methvl1-1.4.7.10-tetraazacvclododecane-4.7.10-triacetic acid (macrocycle 4) ²²⁵Ac^3* complexation temperature 25°C 50^βC 70^*C 2²²⁵Ac^3* complex 712 782 862 ¹⁵²Eu^3* complexation temperature 25°C 50^*C 70^βC 2 ¹⁵²Eu^3* complex 932 902 1002

^24lAm³* complexation temperature 25^*C 50^βC 70^*C

2^24lAm^3* complex 822 802 892

Competition experiment with Eu^3*, starting with the ²²⁵Ac complex; Excess Eu^3*, lOx relatieve to chelator. temperature 2 ^βC 50"C 70^βC

2²²⁵Ac^3* complex 832 832 892-

Comparative example

Synthesis of tetra-p-t-butyl-tetrabenzofab.ef.ii.mnlcvclohexadecatetra- ene-2.6.10.l4-tetra(oxyacetic acid, mono(propylamide) (calix[4]arene- tetraoxyacetic acid mono(propylamide) ) (compound with formula 8, R = H) Calix[4]arene-tetraoxyacetic acid triethyi ester monopropylamide (compound with formula 8, E = ethyl) . Monoacid calix[4]arene-tetraoxyacetic acid triethyi ester with formula 9 (Bohmer, V. et al. J. Chem. Soc , Perkin Trans. 1. 1990. 431.) (1.5 g, I.56 mmol) was refluxed in S0C1₂ (10 mL) for 3 hours. The solvent was evaporated in vacuo, and residual solid was dissolved in CH₂C1₂ (10 mL) . N-Propylamine (0.92 g, 15.6 mmol) in CH₂C1₂ (10 mL) was then added. The reaction mixture was stirred overnight at room temperature, evaporated, dissolved in CH₂C1₂ (lOmL), washed with H₂0, dried (MgS0₄) , evaporated and recrystallized from EtOH-H₂0, 4:1. Yield 772; H NMR (CDC1₃) δ 8.46 (br s, IH). 6.89 (s, 2H) 6.81 (s. 4H). 6.70 (s. 2H) . 5-0-4.5 (m, 16H) . 4.15 (q. J = 7.0 Hz. 6H). 3-30 (m. 2H) . 1.68 (m. 2H) . 1.10 (s, 9H) , 1.04 (s. 18H). 1.00 (s, 9H). 0.97 (t, J = 7.0 Hz. 3H). Mass spectrum (FAB), m/z 1006.8 ((M+H)^*, calcd 1006.6). Example V

b)

Results of complexation experiments of l-(6-methoxycarbonyI-2-pyridylmethyl)-l,4,7,10-tetraaza- cvclododecane-4,7,10-triacetic acid (macrocycle 6a)

²²⁵Ac³⁺complexation

Temperature 25 °C 50 °C 70 °C

% ²²⁵Ac³⁺complex 8% 79% 100%

Competition experiment with Eu³⁺, starting with the ²⁵Ac³⁺ complex;

Excess Eu³⁺, lOx relative to chelator.

Temperature 25 °C 50 °C 70 °C

% ²²⁵Ac³⁺complex 96% 97% 61%

c)

Results of complexation experiments of l-(6-carboxyl-2-pyridylmethyl)-l,4,7,10-tetraaza- cvclododecane-4,7,10-triacetic acid (macrocycle 6b)

²²⁵Ac³⁺complexation

Temperature 25 °C 50 °C 70 °C

% ^{2 5}Ac³⁺complex 20% 92% 100%

Competition experiment with Eu³⁺, starting with the ²²⁵Ac³⁺ complex;

Excess Eu³⁺, lOx relative to chelator.

Temperature 25 °C 50 °C 70 °C

% ²²⁵Ac³⁺complex 96% 91% 38%

8.

Calix[4jarene-tetτaoxyacetic ac id monopτopylamide (compound with formula 8, R = H).

The triester monoamide (0.5 g. 0.49 mmol) and K₂C0₃ (1.38 g, 10 mmol) were refluxed in MeOH-H₂0 (20 mL. 5:1) for 1 hour, acidified (pH 4). The product was extracted with EtOAc (2 x 2 mL) , dried (MgS0_Λ) , the solvent was evaporated. Yield 5 2; *H NMR (CDC1₃) δ 7-19 (s. 4H) 6.76 (s, 2H) . 6.38 (s. 2H). 5-0-3-0 (m, 18H). I.85 (m, 2H) . 1.4l (s. 18H) . 0.97 (t, J = 7.0 Hz. 3H), 0.95 is , 9H). 0.81 (s. 9H).

11.

10.

Complex of 8 with Eu^3*- (formula 10).

EuCl₃.6H₂0 (0.032 g, 0.09 mmol) and trimethyl orthoformate (excess) were refluxed in dried MeCN (10 mL) for 1 hour. The triacid with formula 8 (R = H) (0.09 mmol) and equimolar Et₃N in MeOH (5 mL) were added, and the mixture was refluxed for 3 hours, evaporated, washed with H₂0 (2x15 mL) and dried. Mass spectrum (FAB), m/z 1070.9 (M+H)„, calcd 1070.6). Anal. Calcd. for C₅₅H₇₁N0_uEu:0.5CHCl₃ (M_r ll69.6): C, 58.54; H, 6.32; N, 1.23. Found: C.58.89;H,6.62;N,1.20.

Example VI Synthesis of calixl^"41arene compounds (compounds with formula 11, R' = t-butyl) .

Triester mono(3-(2-oxo-l-pyrrolidinyl)propyl)amide (compoundwith formula

11, R = Et, R' = t-Bu).

This amide was obtained in a way analogous to the procedure for the mono- amide with formula 8 (R = Et), starting with monoacid of formula 9- *H

NMR (CDCI₃) δ 8.50 (br s, IH). 6.89 (s, 6H) , 6.71 (s. 2H) , 5.0-1.8 (m,

34H), 1.31 (t.J = 7.0 Hz, 9H). 1.11 (s. 9H), 1.09 (s. l8H), O.96 (s. 9H) .

Mass spectrum (FAB), m/z 1089-7 ((M+H)^*, calcd 1088.6).

Triacid mono ( 3- (2-oxo-l-pyτrolidinyl)pτopyl) amide (compound with formula 11, R = H, R' = t-Bu)λ

This was obtained analogous to the procedure for the triacid with formula 8 (R = H). Yield 932. H NMR (CDC1₃) δ 7-90 (br s, IH) , 7.11 (s, 4H, AΓ H), 6.83 (s, 2H). 6.51 (s. 2H). 6.0 (br s. 3H) . 5-0-3.0 (m, 22H) , 2.4l (t.J - 7.0 Hz. 2H), 2.00 (m, 4H) . 1.37 (s, I8H, 1.00 (s. 9H) , 0.91 (s, 9H). Mass spectrum (FAB), m/z 1005-3 ((M+H)^*, calcd 1005-6. Anal. Calcd. for C₆₅H₇₆N₂0₁₂ (M_r 1005-2): C, 72.47; H. 7.11: N.2.60. Found: C, 72.61; H. 7.41: N. 2.51.

Complex with Eu^3* (formula 12. R' = t-Bu) .

This was obtained analogous to the procedure for the complex with formula 10. Mass spectrum (FAB), m/z 1155-5 ((M+H)^*, calcd 1155-6). Anal. Calcd. for C₅₉H₇₆N₂0₁₂Eu.CH₂Cl₂ (M_r 1154.6) :C, 57-12; H. 6.27; N.2.25- Found: C. 57-30; H. 6.46; N. 2.36. Example VII

Synthesis of calixf41arene compounds (compounds with formula 11, R' = hydrogen) .

Triester mono(3-(2-oxo-l -pyrrol idinyl)propyl) amide (compoundwith formula 11. R = Et. R' = H).

This was obtained in a way analogous to example VI. Mass spectrum (FAB), m/z 865.4 ((M+H)^*, calcd 865-4).

Triacid mono(3-(2-oxo-l -pyτrolidinyl)propyl) amide (compound with formula 11. R = R* = H). Triester (formula 11. R = Et. R' = H) (0.83 g. O.96 mmol) and K₂C0₃ (1-32 g, 9.6 mmol) were refluxed in MeOH-H₂0 (5:1. 12 mL) for 1 hour, acidified (pH 4). The product was extracted with CH₂C1₂ (2 x 25 mL) , dried (MgSO , the solvent was evaporated. The product was recrystallized from EtOH-H₂0, 4:1. Mass spectrum (FAB), m/z 781.4 (M+H)^*, calcd (781.3)- Anal. Calcd. for C₄₃H_/tilN₂0₁₂»0.5CH₂Cl₂ (M_r 780.3): C: 62.80; H: 5-39; N: 3.40. Found: C: 63.13; H: 5.67; N: 3-71

Complex wi th Eu^3* (formula 12. R^* = H) .

This was obtained in the same way as in example VI. Mass spectrum (FAB), m/z 931-2 ((M+H)^*, calcd (931-2). Anal. Calcd. for C^H^O^Eu (M_r 931-2): C. 55-46; H, 4.76; N. 3-00. Found: C, 55-32; H. 4.82; N. 2.89.

12.

Example VIII

Synthesis of calixr41arene compounds (compounds with formula 13) Triester mono ( -acetylamidopτopyl) amide (compound of formula 13, R = Et) This was obtained analogous to the procedure for the monoamide of the comparative example (formula 8). Yield 82*. *H NMR (CDC1₃) δ 8.63 (br s, IH), 7-14 (br s, IH). 6.84 (s. 2H) 6.76 (s. 26H) . 5-0-3-0 (m, 26H) , 1.97 (s, 3H). 1.25 (t.J = 7-0 Hz. 9H). 1.11 (s. 9H). 1.06 (s, 9H) , 1.04 (s, 18H). Mass spectrum (FAB), m/z 1049-5 ((M+H)^*, calcd. 1049-0). Anal. Calcd. for C₆₂H₈₄N₂0₁₂ ^«CH₃CN (M_r IO89.O): C, 69.08; H, 8.00; N, 3.00. Found: C, 68.77; H. 7-86; N. 3-35-

Triacid mono (3- acetylamidopropyl) amide (formula 13, R « H) . This was obtained analogous to procedure for the triacid of example VII. Yield 902. *H NMR (CDC1₃) δ 8.32 (br s, 3H) . 8.01 (br s. 2H) , 7.14 (s, 4H), 6.66 (s. 2H), 6.49 (s. 2H), 5-1-3-1 (m, 20H) . 2.16 (s, 3H) . 1.31 (s. 18H), 0.91 (s. 9H. CH₃). 0.77 (s. 9H). Mass spectrum (FAB), m/z 965.6 ((M+H)^*. calcd. 965-5).

Complex with Eu^3* (formula 14).

This was obtained in the same way as the complex with formula 12 (example

VII). Mass spectrum (FAB), m/z 1115-5 ((M+H)^*. calcd 1115-4).

13.

Exaaple IX

Fluorescence of the calix[4]arene triacid aono(3-(2-oxo-l-pyrrolidinyl)propyl)aaide coaplex with Eu³⁺ (foraula 12, E = t-Bu).

The fluorescence aeasureaents of the Eu³⁺ coaplex with foraula 12 (K = t-Bu) aade in situ (dissolving the ligand with foraula 11, R = H, _.' = t-Bu) and EuCl₃.6H₂0 in KeOH and adding a saall excess of Et-.N) showed a luainescence lifetiae 0.900 as (for coaplex 12 higher than 0.900 as). These luainescence lifetiaes are larger than reported by ϋngaro et al, (J. Chea. Soc., Chea. Coaaun. 1990, 878) for an analogous coapound. Correspondingly the luainescence quantua yield are also high with the coaplexes containing ninth coordination centre. The solid coaplexes with foraula 12 (£' = H or t-Bu) also showed high luainescence upon excitation. These results show that the Eu³⁺ is efficiently shielded froa solvent aolecules, like MeOH, H₂0.

Exaaple X

Using siailar aethods as described before were prepared

Coapound 15c was converted into the coapound with foraula 16 using tributylphosphine:

Claims

1. Chelating compound having nine ligand sites capable of simultaneously chelating a single metal cation, the compound having formula 1:

wherein:

A¹, A², A³ and A* represent optionally substituted organic chains;

A⁵ represents an optionally substituted organic chain;

Q¹, Q². Q³ and Q* represent Z¹, Z², Z³ and Z^ή respectively, in which case each of these is a nitrogen atom, or Q¹, Q², Q³ and Q* represent C-Z¹, C-Z², C-Z³ and C-Z* respectively, in which case Z¹, Z², Z³ and Z* are exocyclic and independently represent 0, S or NR¹¹; wherein the spatial distances between Z¹ and Z², Z² and Z³, Z³ and Z*. and Z and Z¹ each can be 3.15 ± 0.25 A, and the spatial distances between Z¹ and Z³, Z² and Z* each can be 4.45 ± O.35 A; Z⁵, Z⁶ and Z⁷ independently represent carboxyl, thiocarboxyl, carbox- imidoyl, phosphono, thiophosphono, phosphorimidoyl, sulpho, thiosulpho, sulfamoyl;

Z⁸ represents 0, S or NR¹²;

Z⁹ represents 0, S or NR¹³, to which a hydrogen atom or an organic substituent may be attached;

R¹, R², R³, R*. R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ independently represent hydrogen,

C^C_f, alkyl or C₂-C₆ alk(adi)enyl, or any pair of R¹ and R⁵, R² and R⁶, R³ and R⁷, R* and R⁸, and R⁹ and R¹⁰ - also represent oxo, or R⁸ and R⁹ or R⁹ and a substituent at A⁵ may be connected to form a ring together with the carbon atoms to which they are bound;

R¹¹ represents hydrogen, C^Ce alkyl, C₂-C₆ alkenyl or C_x-C₆ hydroxyalkyl; R¹² represents hydrogen, C_j-Ce alkyl, C₂-C₆ alkenyl, C₁-C₆ hydroxyalkyl or a second bond together with one of the neighbouring chain atoms of Z⁸;

R¹³ represents hydrogen, C_j-Cg alkyl, C₂-C₆ alkenyl, C^Cg hydroxyalkyl, opt;, - ly substituted C₅-C₁₀ aryl or C₆-C_lή aralkyl, or a second bond toge ;r with the neighbouring chain atom of Z⁹; wherean one of R¹, R², R³, R^ή and R⁹ or one of the substituents on A¹, A²,

A³, A⁴, A⁵ or Z⁹ may represent A⁶-X, wherein A represents a direct bond or an organic linking group, and X represents a functional group capable of binding to a targeting molecule; or a mono- or polyanion thereof derived by abstraction of one or more acidic hydrogens; with the proviso that if Q¹, Q², Q³ and Q^ή are each nitrogen and Z⁹ is hydroxy, C-R⁹R¹⁰-Z⁸ does not represent methyleneoxy or carbonylimino.

2. Chelating compound according to claim 1, wherein A¹, A², A³ and A⁴ each represent -CHR^l4-CHR¹⁵-, and Q¹, Q², Q³ and Q⁴ each represent an endo- cyclic nitrogen atom, wherein R¹* and R¹⁵ independently represent hydrogen or C_j-C_fc alkyl, and one of the four R¹* may represent the group A⁶-X.

3. Chelating compound according to claim 2, wherein A¹, A², A³ and

and Q¹, Q², Q³ and Q^u together represent a 1,4,7.10-tetraaza-cyclo- dodecane.

4. Chelating compound containing a tetrabenzo[ai ,e/, f^, π]cyclohexa- decatetraene (calix[4]arene) group, having nine ligand sites capable of simultaneously chelating a single metal cation, said ligand sites being selected from n trogen, oxygen and sulphur atoms, the compound optionally containing a f -.ctional group capable of binding to a targeting agent.

5. Chelating compound according to claim 1 or 4, wherein A¹, A², A³ and A⁴, and Q¹, Q², Q³ and Q^ή together form a tetrabenzo[α_b,e/, ij, mn - cyclohexadecatetraene, and Z¹, Z², Z³ and Z^k represent 0, attached at positions 2, 6, 10 and 13 of the cyclohexadecatetraene according to formula 2:

wherein R^l6 represents hydrogen or C^C^ alkyl, and one of the four R¹⁶ may represent the group A⁶-X.

6. Chelating compound according to one of the preceding claims, wherein (Z⁸)-A⁵-(Z⁹) represents (Z⁸)-CR¹⁷R^l8-(CHR²¹)_n-CR¹⁹R²⁰-(Z⁹) , wherein R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ independently represent hydrogen or C_j-C₆ alkyl, and R¹⁷ and R¹⁸, or R¹⁹ and R²⁰ may also together represent oxo, and wherein R⁹ and R¹⁷, R¹² and R¹⁷, R¹⁷ and R¹⁹, R¹⁷ and R²¹, R¹⁹ and R²¹, R¹⁹ and R¹³, or R¹⁹ and the substituent attached to Z⁹ may also be connected to form a ring, and n equals 0 or 1.

7. Chelating compound according to claim 6, wherein -CR⁹R¹⁰-Z⁸-CR¹⁷R¹⁸- represents a heterocyclic group, in particular 2,6-pyridinediyl.

8. Chelating compound according to any one of the preceding claims, wherein Z⁵, Z⁶ and Z⁷ each represent carboxyl and R¹, R², R³, ^u , R⁵, R⁶, R⁷ and R⁸ are hydrogen.

9« Chelating compound according to one of the preceding claims, wherein Z⁸ represents 0 or NR¹², and Z⁹ represents NR¹³, wherein R¹³ preferably comprises the group A -X.

10. Chelating compound according to one of the preceding claims, containing the group A⁶-X, wherein X represents isocyanato, isothio- cyanato, mercapto, amino, haloaceta ido, diazo, succinimido, maleimido, a reactive ester group or an anhydride.

11. Chelate of a chelating compound according to one of the preceding claims and a trivalent metal ion, in particular a radioactive metal.

12. Target-specific conjugate comprising a chelating compound according to one of claims 1-11 or a chelate according to claim 10, bound through functional group X to a targeting agent, such as a protein, in particular an antibody.

13. Pharmaceutical composition containing a compound, a chelate, a conjugate according to one of the preceding claims together with a pharmacologically acceptable carrier.