WO1991010645A2 - Chelants - Google Patents

Abstract

There are provided novel chelating agents useful in the preparation of contrast media for diagnostic imaging or of radiotherapeutic or detoxification compositions.

Description

Chelants

The present invention relates to certain novel chelating agents, in particular polyamines, and to their uses, especially their medical uses.

The medical use of chelating agents is well

established, for example as stabilizers for

pharmaceutical preparations, as antidotes for poisonous heavy metal species and as diagnostic agents for the administration of metal species (e.g. ions or atoms) for diagnostic techniques such as X-ray, magnetic resonance imaging (MRI) or ultrasound imaging or scintigraphy.

Polyamine chelating agents, for example aminopoly- (carboxylic acid or carboxylic acid derivative)

(hereinafter APCA) chelating agents and their metal chelates, are well known and are described for example in US-A-2407645(Bersworth), US-A-2387735 (Bersworth), EP-A-71564 (Schering), EP-A-130934 (Schering),

EP-A-165728 (Nycomed AS), DE-A-2918842 (Rexolin

Chemicals AB), DE-A-3401052 (Schering), EP-A-258616 (Salutar), DE-A-3633245 (Schering), EP-A-263059

(Schering), EP-A-277088 (Schering) and DE-A-3633243 (IDF).

Thus, for example, EP-A-71564 describes

paramagnetic metal chelates, for which the chelatingagents are nitrilotriacetic acid (NTA),

N,N,N',N'-ethylenediamine-tetraacetic acid (EDTA), N-hydroxyethyl-N,N',N'-ethylenediaminetriacetic acid (HEDTA), N,N,N',N",N"- diethylenetriaminepentaacetic acid (DTPA) and N-hydroxyethyl-iminodiacetic acid, as being suitable as contrast agents for MRI, contrast being achieved by the effect of the magnetic field of the paramagnetic species (e.g. Gd(III)) with the chelating agents serving to reduce the toxicity and to assist administration of that paramagnetic species.

Amongst the particular metal chelates disclosed by EP-A-71564 was Gd DTPA, the use of which as an MRI contrast agent has recently received much attention. The Gd(III) chelate of 1,4,7,10- tetraazacyclododecanetetraacetic acid (DOTA) referred to in DE-A-3401052 (Schering) and in US-A-4639365

(University of Texas) and of l-hydroxypropyl-4,7,10- triscarboxymethyl-1,4,7,10-tetraazacyclododecane (HP- D03A) have also recently received attention in this regard.

To improve stability, water solubility and

selectivity, relative to the APCA chelating agents described in EP-A-71564, Schering, in EP-A-130934, have proposed the partial substitution for the N-attached carboxyalkyl groups of alkyl, alkoxyalkyl,

alkoxycarbonylalkyl or alkylaminocarbonylalkyl groups, where any amide nitrogens may themselves carry

polyhydroxyalkyl groups. More recently, to improve compatibility, stability, solubility and selectivity, in EP-A-250358 Schering have proposed a narrow range of compounds having a DTPA-like structure including a bridging alkylene chain.

In the field of hepatobiliary MRI contrast agents, where lipophilicity rather than hydrophilicity is desired, Nycomed in EP-A-165728, have proposed the use of paramagnetic chelates of certain anilide

group-containing iminodiacetic acids and Lauffer in WO-A-86/06605 has suggested the use of paramagnetic chelates of triaza and tetraaza macrocycles which have fused aryl group on one of the alkylene chains linking the ring nitrogens but are otherwise unsubstituted.

Nycomed, in EP-A-299795, suggest that the toxicity of certain APCA chelating agents and their chelates may be reduced by introducing at least one hydrophilic moiety as a substituent on one or more of the alkylene bridges between the amine nitrogens. stability selectivity and there is thus a general and continuing need for such polyamine chelating agents which form metal chelates of reduced toxicity, improved stability or improved water solubility or having

improved biodistribution characteristics.

We now propose a novel class of polyamine chelating agents which incorporate within their structure at least one 5- or 6-membered heterocyclic ring.

Thus viewed from one aspect the present invention provides a compound of formula I

R¹(CR²R³)_n[X(CR²R³)_n]_mR¹ (I )

(wherein each X independently represents an oxygen or sulphur atom or a group of formula NA, or

(CR²R³)_n X(CR²R³) represents a group of formula

E represents COH, NR² , O or S;

each A independently represents a hydrogen atom or a group (CR²R³)_pY, (CR²R³)_nN[(CR²R³)_pY]₂ or

where two (CR²R³ ) Y groups on different nitrogens, preferably in adjacent groups, may together represent a group -(CR²R³) _n -;

each Y independently represents a group COZ, SO₂Z, POZ₂ ,

CON(OH)R², CH₂ SR², CS₂ R² or CSZ;

each Z independently represents a group OR ² or NR²R²; each G is a 3 or 4 membered chain of carbon atoms and optionally a nitrogen, oxygen or sulphur atom; each J is a 2 or 3 membered chain of carbon atoms and optionally a nitrogen, oxygen or sulphur atom; each n is an integer of 2 to 4, preferably 2 or 3 or in a group (CR²R³)_n attached to a moiety R¹ which represents a hydrogen atom or a group R⁴ n may also be zero or 1;

m is an integer of 3 to 8 , preferably 3 to 6;

p is an integer of 1 to 3, preferably 1;

each R ¹ represents a hydrogen atom or a group R⁴ or together both groups R¹ represent a carbon-carbon bond; each R ² independently represents a hydrogen atom or a C_1-8 alkyl group optionally mono- or poly-substituted by hydroxyl or C_1-8 alkoxy groups or NR ²R² may together represent a nitrogen-attached 5 to 7 membered saturated heterocyclic ring optionally containing as a further ring heteroatom a nitrogen, oxygen or sulphur atom and optionally substituted by a group R⁴ ;

each R³ independently represents a hydrogen atom or a C_1-8 alkyl or C_1-8 alkoxy group optionally mono or poly substituted by hydroxy or C_1-8 alkoxy groups; and

each R⁴ independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an optionally mono- or poly-hydroxylated C_1-8 alkyl, C_1-8 alkoxy, (C_1-8 alkoxy)-C_1-8 alkyl or poly(C_1-8 alkoxy)-C_1-8 alkyl group, a sulphonate group or a group (CR²R³) Y or two groups R⁴ on the same ring represent a (CR²R³)_n-1 [X(CR²R³)_n]_m-1 (CR²R³)_n-1 group in which case the said ring may be saturated; with the provisos that at least 2 Y groups, preferably at least 3, are present, that where both groups R¹ together form a bond, m is 4 or 5, all n are 2, one X is

2,6-pyridindiyl and the remainder are NCH COOR², then at least one R ², R³ or R⁴ is other than hydrogen, and that either at least one X group comprises an aromatic

heterocyclic group or both R groups together represent a bond and two (CR²R³)_pY groups together represent a

-(CR²R³ ) - group or both R¹ groups represent a bond, m is 6 or greater and two X groups separated by at least two other X groups are oxygen or sulphur atoms, and preferably that where m is 3 or 4, all n are 2, one X is

4-substituted-2,6-pyridindiyl, 2,5-furandiyl, 2,5- pyrroldiyl, 2,5-thiophendiyl or 1-hydroxy-2,6-phenylen- and the remainder are NCH₂ COOR² or NH then at least one

R², R³ or R⁴ is other than hydrogen) or a chelate complex or salt thereof.

In the compounds of the invention, alkyl or

alkylene moieties in groups R ¹ to R⁴, unless otherwise stated, may be straight chained or branched and

preferably contain from 1 to 6 and most preferably 1 to

4 , carbon atoms. Where substituents may themselves optionally be substituted by hydroxyl or alkoxy groups, this may be monosubstitution or polysubstitution and, in the case of polysubstitution, alkoxy or hydroxyl

substituents may be carried by alkoxy substituents.

Where, as is particularly preferred, the compounds of the invention incorporate one or more hydrophilic R¹ to R⁴ groups, these are preferably straight-chained or branched moieties having a carbon atom content of from 1 to 8, especially preferably 1 to 6, carbon atoms. The hydrophilic groups may be alkoxy, polyalkoxy,

hydroxyalkoxy, hydroxypolyalkoxy, polyhydroxyalkoxy, polyhydroxylated polyalkoxy, hydroxyalkyl,

polyhydroxyalkyl, alkoxyalkyl, polyalkoxyalkyl,

hydroxylated alkoxyalkyl, polyhydroxylated alkoxyalkyl, hydroxylated polyalkoxyalkyl, or polyhydroxylated polyalkoxyalkyl groups. More preferably however they will be monohydroxyalkyl or polyhydroxyalkyl groups.

The hydrophilic groups serve to increase the

hydrophilicity and reduce the lipophilicity of the metal chelates formed with the chelating agents of the

invention and it is preferred that the compounds of formula I should contain at least 1, conveniently from 1 to 4, and preferably 1, 2 or 3 such hydrophilic groups. As hydrophilic groups, the compounds of the invention may thus include for example hydroxymethyl,

2-hydroxyethyl, 1,2-dihydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3,4-trihydroxybutyl,

1-(hydroxymethyl)-2-hydroxy-ethyl, methoxymethyl, ethoxymethy1, 2-hydroxyethoxymethy1,

methoxyethoxymethyl, (2-hydroxy-ethoxy)ethyl, etc, groups.

Particularly preferred compounds of formula I according to the invention include those of monocyclic structure containing at least 6 ring heteroatoms, those of fused bicyclic structure containing at least two ring heteroatoms in the smaller ring, and those of fused tricyclic or higher polycyclic structure.

In the compounds of the invention, the aromatic groups comprised by X groups preferably are pyridine, pyrazine, pyrrole, furan, phenol, pyrimidine or

thiophene rings, especially pyridine rings. Where in a group X, E is COH, e.g. where X comprises a phenol group, it is especially preferred that an electron withdrawing R ⁴ substituent (e.g. a lower alkyl or halogen such as chlorine or methyl) should be present on the ring, preferably at the para position to the hydroxyl group. "N"-membered aromatic rings attached to the linear or cyclic skeleton of the molecule at the 2 and/or "N-1" positions are especially preferred.

Moreover in the compounds according to the invention, adjacent X groups preferably do not both comprise such aromatic groups. Where the chelants of the invention contain fused saturated heterocyclic rings these

preferably are piperazine, or 1,4-diazacycloheptane rings, especially piperazin-1,4-diyl groups.

Particularly preferably one, two or three X groups should comprise such aromatic groups, the remaining X groups, or all but one remaining X group being

N(CR²R³)_p Y groups. It is especially preferred that groups X comprising no ionizing group Y should be non-adjacent particularly that they should adopt opposed positions in macrocyclic chelants, e.g. as the 1st and 5th X groups in an 8 X ring. Particularly preferred compounds of formula I include those of formulae lb and Ic

and in particular those of formulae Id to II.

(where A' is CHR²Y or hydrogen, z is 1 or 2, t is 1 or

2, v is 0,1,2,3 or 4 and X ² is O or S).

Where a group NR in a compound according to the

invention is a nitrogen attached heterocyclic ring, it will conveniently be of formula

where q is 0,1 or 2, and W is CHR4_{, NR}4 _{, O or S, where q} is zero W preferably being CHR ⁴. Particularly

preferably such groups are of formula

In the compounds of formula I, the groups Y

preferably represent carboxylic acid or amide groups, for example groups of formula COOH, CONH₂,

CONCHR⁴CHR⁴W(CHR⁴) CHR⁴, CONHR^2" or CONR^2" ₂(where R^2" is an

alkyl or mono or poly hydroxyalkyl group, for example a C _{1 -6}alkyl group optionally carrying 1, 2, 3 or 4 hydroxyl groups). Particularly preferably, in the case where R ¹ are not bonds terminal X groups will comprise an aromatic heterocyclic group.

Where Y is a carboxyl group, the compounds of formula I can conveniently form salts or chelates in which Y represents -COOM (wherein M⁺ is a monovalent cation or a fraction of a polyvalent cation, for example an ammonium or substituted ammonium ion or a metal ion, for example an alkali metal or alkaline earth metal ion). Particularly preferably, M⁺ is a cation deriving from an organic base, for example meglumine or lysine. In such salts or chelates one or more (but not

necessarily all) of the carboxyl groups are transformed into COOM groups.

It is particularly preferred that the number of the ion-forming groups Y in the compounds of formula I be chosen to egual the valency of the metal species to be chelated by the compound formula I. Thus, for example, where Gd(III) is to be chelated, the compound of formula I (oo salt thereof) preferably contains three or six ion-forming Y groups, for example -COOH (or -COOM). In this way, the metal chelate will be formed as a neutral species, a form preferred since the osmolalities in concentrated solutions of such compounds are low and since their toxicities relative to their ionic analogues are significantly reduced.

Compounds of formula I in which all the Y groups are -COOH groups or salts or amides of such compounds are especially preferred since compositions containing metal chelates of such compounds can readily be

sterilized, for example by autoclaving.

Included amongst the particularly preferred

compounds according to the invention are those of formulae lb to Ii wherein each R² represents a hydrogen atom or a mono- or poly-hydroxylated alkyl group, Y represents a group of formula COZ and Z represents a hydroxyl group or a group NHR²) and metal chelates and salt thereof.

Especially preferred compounds according to the invention include those of the following formulae Im to

Iw

\

(where R³⁰ is (CH₂)₂ or (CH₂)₃, each r is 1 or 2, t is 1 or

2, R ²" is hydrogen or methyl, and R⁶ is a carboxymethyl group or derivative thereof, e.g. CH₂ COOH,

CH₂CON(CH₃)CH₂CHOHCH₂OH, or CH₂CONHR (where R⁷

represents CH₃ , CH₂ CHOHCH₂ OH or CH(CH₂ OH) ₂ or a group -CH₂CONCH₂CHR^{4 "} W(CHR^{4 "}) _q CH₂ where representsan oxygen

atom or a group CH₂ or CHOH, g is 0 or 1 and R4"

hydrogen or where q is 1 and W is oxygen each R ^4" may also represent a C_1-4 hydroxyalkyl group) and the metal chelates and the salts thereof.

Particularly preferred compounds according to the invention include those of formulae Ij to lo wherein R⁶ is CH₂ COOH and the chelates, e.g. with Gd³⁺, and salts thereof.

Viewed from a further aspect, the invention also provides a process for the preparation of the compounds of the invention, said process comprising one or more of the following steps:

(a) reacting a compound of formula II

R^1' (CR^2' R^3' )_n[X (CR^2' R^3')_n]mR^1' (II)

(where R ^1' to R^3' are as defined for R¹ to R³ or are protected R¹ to R³ groups, and X' is a group X or a protected group X with the proviso that at least one X^' group is of formula NH or (CR ^2'R^{3 '})_pNH₂) with a compound of formula III

LV-(CR^2'R^{3 '})_p-Y' (III) (where Y' is a group Y or a protected group Y, R^2' and R^3' are as hereinbefore defined and Lv is a leaving group for example a halogen atom, e.g. bromine or chlorine or a tosylate group) and if necessary

subsequently removing any protecting groups used; and

(b) converting a compound of formula I into a chelate complex or salt thereof.

The compounds of formula II are known from the literature or may be prepared in a number of ways using techniques known from the literature or analogous to literature described techniques. Thus for example such compounds may be prepared by condensing mono or

bifunctional heterocyclic compounds of formula IVa or IVb

(where R ^{2 ,} R ^{3 ,} and R^4' are as defined for R² to R⁴ or are protected R ² to R⁴ groups and R¹² is hydrogen, an amine protecting group or a group (CR ^2'R^3')_n-1 COR^3') with a linking molecule of formula V

H[X" (CR^2'R^3')_n]_i.X"H (V)

(where i is 1 to 5, R^2' and R^3' are as hereinbefore defined, mid-chain X" groups, if any are groups X' and end of chain X" groups are oxygen, sulphur or ring nitrogen atoms or, preferably, NH groups) followed if necessary by removal of any protecting groups and if necessary by reduction.

The compounds of formula II may also be prepared by activating starting compounds of formula V, e.g. by tosylation, and condensing the product with a mono or bifunctional heterocyclic compound of formulae VI or VII

(where R¹³ is hydrogen, an amine protecting group or a group (CR ^2'R^3,) Lv)), followed by removal of the tosyl and other protecting groups.

Thus for example linear or cyclic compounds of formula II can be prepared using the following reaction schemes.

R¹⁶ = H or CH₃ z = 1 or 2

Compounds of formula II containing a

group may be prepared by condensing a linear compound having active groups at each end with a compound of formula

for example as follows

The active linear compound of formula XII may of course be prepared by other routes, e.g.

Compounds of formula II containing a

^X

group can also be prepared by condensing the

corresponding compound in which one of these (CR²R³ ) bridges is missing and the nitrogens carry hydrogens instead with a compound of formula Lv-(CR^2'R^3')_n Lv, for example as follows

This is described for example in J.Chem. Soc. Chem. Commun. 1982,277.

In the reactions mentioned above, a starting material containing more than one heterocyclic ring may of course be used in place of the compound of formulae IVa,IVb,V,VI and VII. This is exemplified by the following scheme:

Combinations and extensions of these procedures may be used to prepare further compounds of formula II, e.g. by condensing a compound of formula VIIIB

H[X" (CR^{2 '}R^{3 '})n]_m-1 X"H (VIIIB) with a compound of formula IX

Lv-(CR^{2 '}R^{3 '})_n-X'-(CR^{2 '}R^{3 '})_n-Lv (IX) or a compound of formula IXB

Ts-[X"(CR^{2 '}R^{3 '})_n]_m-1X" Ts (IXB) with a compound of formula XI

Lv-(CR^{2 '}R^{3 ' '})_n-Lv (XI) followed by removal of the tosyl groups.

This may be illustrated by the reaction schemes:

The precursors of formula II for dimeric

macrocyclic chelants - i.e. compounds wherein two R⁴ groups on a cyclic X group together represent a group (CR²R³)_n-1 [X(CR²R³)_n]_m-1(CR²R³)_n-1 - can be prepared for example by a condensation equivalent to those of the schemes above using a tetrafunctional precursor for the cyclic X group and bifunctional co-reagents. It may be desirable to utilize aromatic tetrafunctional precursors and subsequently to reduce the product, e.g. by high pressure catalytic hydrogenation. Thus compounds of formula II may be prepared by the following scheme:

Further reaction schemes for the production of compounds of formula II will be evident to the skilled chemist from the literature, e.g. Tabushi et al. Tetr. Lett. 4339 (1976) and 1049 (1977), Richmann et al. JACS 96: 2268 (1974), Nelson, Pure and Applied Chemistry 52: 461-476 (1980). Moi et al. JACS 110: 6266 (1988), - EP-A-287465 (Guerbet), Stetter et al. Tetrahedron 37 : 767 (1981), EP- A-232751 (Squibb), J. Chem. Soc. Commun. 277 (1982). Hancock et al. JACS 110: 2788-2794 (1988), Smith et al. JACS 111: 7437-7443 (1989) and the

references listed therein.

To introduce a (CR²R³) Y group onto a compound of formula II using the precedure of step (a) may be effected in an aqueous, preferably basic, medium, for example by using a halocarboxylic acid Hal(CR^2'R^3')_p-

COOH or a metal, e.g. Li, salt thereof (where Hal is bromine or chlorine) followed by amidation or

esterification of the carboxyl group.

The introduction of (CR²R³) Y moiety other than a carboxylic acid residue may for example be performed as follows:

a) To introduce a phosphonic acid moiety, the general method for synthesis of alpha-aminophosphonic acids described by K.Moedritzer et al. in J.Org.Chem 31 : 1603:

(1966) may be used.

R¹⁷NH R ¹⁷ NCH₂PO₃H₂

(XVII) (XVIII)

(of formula II) (of formula I)

(where R¹⁷NCH₂Y is a compound of formula I). b) To introduce a hydroxamic acid moiety, the general method for transformation of an activated acid

derivative into hydroxamic acid described by

P.N.Turowski et al. in Inorg. Chem. 27: 474 (1988) may be used.

(where R¹⁸N(CH₂COOH)CH₂Y is a compound of formula I). c) To introduce a sulfonic acid moiety, synthesis may be performed by alkylation of an amino function for example with iodomethanesulfonic acid

R¹⁷NH R¹⁷NCH₂SO₂H

(XVII) (XXI)

Amide derivatives of formula I may be produced from the oligo acids by methods analogous to those of

EP-A-250358 or of EP-A-299795. Furthermore hydrophilic substituents on the skeleton of the linear or cyclic chelants of formula I may be introduced by methods analogous to those of EP-A-299795.

Chelants of formula I may be used as the basis for bifunctional chelants or for polychelant compounds, that is compounds containing several independant chelant groups, by substituting for one Y or R¹ to R⁴ group a bond or linkage to a macromolecule or polymer, e.g. a tissue specific biomolecule or a backbone polymer such as polylysine or polyethyleneimine which may carry several chelant groups and may itself be attached to a macromolecule to produce a bifunctional-polychelant.

Such macromolecular derivatives of the compounds of formula I and the metal chelates and salts thereof form a further aspect of the present invention.

The linkage of a compound of formula I to a

macromolecule or backbone polymer may be effected by any of the conventional methods such as the carbodiimide method, the mixed anhydride procedure of Krejcarek et al. (see Biochemical and Biophysical Research

Communications 77: 581 (1977)), the cyclic anhydride method of Hnatowich et al. (see Science 220: 613 (1983) and elsewhere), the backbone conjugation techniques of Meares et al. (see Anal. Biochem. 142: 68. (1984) and elsewhere) and Schering (see EP-A-331616 for example) and by the use of linker molecules as described for example by Nycomed in WO-A-89/06979.

Salt and chelate formation may be performed in a conventional manner.

The chelating agents of the formula I (as defined above but with the deletion of the second proviso) are particularly suitable for use in detoxification or in the formation of metal chelates, chelates which may be used for example in or as contrast agents for in vivo or in vitro magnetic resonance (MR), X-ray or ultrasound diagnostics (e.g. MR imaging and MR spectroscopy), or scintigraphy or in or as therapeutic agents for

radiotherapy, and such uses of these metal chelates form a further aspect of the present invention. Salts or chelate complexes of the compounds of the invention containing a heavy metal atom or ion are particularly useful in diagnostic imaging or therapy. Especially preferred are salts or complexes with metals of atomic numbers 20-32,42-44,49 and 57 to 83,

especially Gd, Dy and Yb. For use as an MR-diagnostics contrast agent, the chelated metal species is

particularly suitably a paramagnetic species, the metal conveniently being a transition metal or a lanthanide, preferably having an atomic number of 21-29, 42, 44 or 57-71. Metal chelates in which the metal species is Eu,

Gd, Dy, Ho, Cr, Mn or Fe are especially preferred and Gd ³⁺, Mn²⁺ and Dy³⁺ are particularly preferred.

Chelates of ions of these metals specifically listed above with chelants of formula I (defined as above with the exclusion of the second proviso) or their salts with physiologically tolerable counterions are particularly useful for the diagnostic imaging procedures mentioned herein and they and their use are deemed to fall within the scope of the invention and references to chelates of compounds of formula I herein are consequently to be taken to include such chelates.

For use as contrast agents in MRI, the paramagnetic metal species is conveniently non-radioactive as

radioactivity is a characteristic which is neither required nor desirable for MR-diagnostics contrast agents. For use as X-ray or ultrasound contrast agents, the chelated metal species is preferably a heavy metal species, for example a non-radioactive metal with an atomic number greater than 37, preferably greater than 50, e.g. Dy ³⁺.

For use in scintigraphy and radiotherapy, the chelated metal species must of course be radioactive and any conventional complexable radioactive metal isotope, such as 99mTc or ¹¹¹In for example, may be used. For radiotherapy, the chelating agent may be in the form of a metal chelate with for example ¹⁵³Sm, ⁶⁷Cu or ⁹⁰Y. For use in detoxification of heavy metals, the chelating agent must be in weak complex or salt form with a physiologically acceptable counterion, e.g.

sodium, calcium, ammonium, zinc or meglumine, e.g. as the sodium salt of the chelate of the compound of formula I with zinc or calcium.

Where the metal chelate carries an overall charge, such as is the case with the prior art Gd DTPA, it will conveniently be used in the form of a salt with a physiologically acceptable counterion, for example an ammonium, substituted ammonium, alkali metal or alkaline earth metal (e.g. calcium) cation or an anion deriving from an inorganic or organic acid. In this regard, meglumine salts are particularly preferred.

Viewed from a further aspect, the present invention provides a diagnostic or therapeutic composition

comprising a metal chelate, whereof the chelating entity is the residue of a compound of formula I according to the present invention, together with at least one pharmaceutical or veterinary carrier or excipient, or adapted for formulation therewith or for inclusion in a pharmaceutical formulation for human or veterinary use.

Viewed from another aspect, the present invention provides a detoxification agent comprising a chelating agent according to the invention in the form of a weak complex or salt with a physiologically acceptable counterion, together with at least one pharmaceutical or veterinary carrier or excipient, or adapted for

formulation therewith or for inclusion in a

pharmaceutical formulation for human or veterinary use.

The diagnostic and therapeutic agents of the present invention may be formulated with conventional pharmaceutical or veterinary formulation aids, for example stablizers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, etc. and may be in a form suitable for parenteral or enteral

administration, for example injection or infusion or administration directly into a body cavity having an external escape duct, for example the gastrointestinal tract, the bladder or the uterus. Thus the compositions of the present invention may be in conventional

pharmaceutical administration forms such as tablets, capsules, powders, solutions, suspensions, dispersions, syrups, suppositories, etc; however, solutions,

suspensions and dispersions in physiologically

acceptable carrier media, for example water for

injections, will generally be preferred.

The compounds according to the invention may therefore be formulated for administration using

physiologically acceptable carriers or excipients in a manner fully within the skill of the art. For example, the compounds, optionally with the addition of

pharmaceutically acceptable excipients, may be suspended or dissolved in an aqueous medium, with the resulting solution or suspension then being sterilized. Suitable additives include, for example, physiologically

biocompatible buffers (as for example, tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole

percent) of chelants (such as, for example,. DTPA,

DTPA-bisamide or non-complexed chelants of formula I) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide, calcium salts or chelates of chelants of formula I), or, optionally, additions (e.g., 1 to 50 mole percent) of calcium of sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate combined with metal chelate complexes of chelants formula I and the like).

If the compounds are to be formulated in suspension form, e.g., in water or physiological saline for oral administration, a small amount of soluble chelate may be mixed with one or more of the inactive ingredients traditionally present in oral solutions and/or

surfactants and/or aromatics for flavouring. For MRI and for X-ray imaging of some portions of the body the most preferred mode for administering metal chelates as contrast agents is parentral, e.g.,

intravenous administration. Parenterally administrable forms, e.g., intravenous solutions, should be sterile and free from physiologically unacceptable agents, and should have low osmolality to minimize irritation or other adverse effects upon administration, and thus the contrast medium should preferably be isotonic or

slightly hypertonic. Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium

Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack

Publishing Co., pages. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington:

American Pharmaceutical Association (1975). The

solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the chelates and which will not interfere with the manufacture, storage or use of products.

Where the diagnostic or therapeutic agent comprises a chelate or salt of a toxic metal species, e.g. a heavy metal ion, it may be desirable to include within the formulation a slight excess of the chelating agent, e.g. as discussed by Schering in DE-A-3640708, or more preferably a slight excess of the calcium salt of such a chelating agent. For MR-diagnostic examination, the diagnostic agent of the present invention, if in

solution, suspension or dispersion form, will generally contain the metal chelate at concentration in the range 1 micromole to 1.5 mole per litre, preferably 0.1 to 700mM. The diagnostic agent may however be supplied in a more concentrated form for dilution prior to

administration. The diagnostic agent of the invention may conveniently be administered in amounts of from 10 ^-3 to 3 mmol of the metal species per kilogram of body weight, e.g. about 1 mmol Dy/kg bodyweight.

For X-ray examination, the dose of the contrast agent should generally be higher and for scintigraphic examination the dose should generally be lower than for MR examination. For radiotherapy and detoxification, conventional dosages may be used.

Viewed from a further aspect, the present invention provides a method of generating enhanced images of the human or non-human animal body, which method comprises administering to said body a diagnostic agent according to the present invention and generating an X-ray, MR, ultrasound or scintigraphic image of at least a part said body.

Viewed from a further aspect, the present invention provides a method of radiotherapy practised on the human or non-human animal body, which method comprises

administering to said body a chelate of a radioactive metal species with a chelating agent according to the invention.

Viewed from a further aspect, the present invention provides a method of heavy metal detoxification

practised on the human or non-human animal body, which method comprises administering to said body a chelating agent according to the invention in the form of a weak complex or salt with a physiologically acceptable counterion.

Viewed from a yet further aspect, the present invention also provides the use of the compounds, especially the metal chelates, according to the

invention for the manufacture of diagnostic or

therapeutic agents for use in methods of image

generation, detoxification or radiotherapy practised on the human or non-human animal body. Viewed from a still further aspect, the present invention provides a process for the preparation of the metal chelates of the invention which process comprises admixing in a solvent a compound of formula I or a salt (e.g. the sodium salt) or chelate thereof together with an at least sparingly soluble compound of said metal, for example a chloride, oxide, acetate or carbonate.

Viewed from a yet still further aspect, the present invention provides a process for the preparation of the diagnostic or therapeutic agent of the present

invention, which comprises admixing a metal chelate according to the invention, or a physiologically

acceptable salt thereof, together with at least one pharmaceutical or veterinary carrier or excipient.

Viewed from a yet still further aspect, the present invention provides a process for the preparation of the detoxification agent of the invention, which comprises admixing a chelating agent according to the invention in the form of a weak complex or salt with a

physiologically acceptable counterion, together with at least one pharmaceutical or veterinary carrier or excipient.

The disclosures of all of the documents mentioned herein are incorporated by reference.

The present invention will now be illustrated further by the following non-limiting Examples. All ratios and percentages given herein are by weight and all temperatures are in degrees Celsius unless otherwise indicated. Example 1 [ 18 ]N₆H₈O₄ (py) ₂

[Ca[18]N₆H₄(py)₂]Cl₂.3H₂O

To a stired solution of 2,6-diformyl-pyridine (1.306 g, 9.662 mmol) and calcium chloride dihydrate (0.71 g, 4.83 mmol) in 80 mL of methanol was added ethylenediamine (0.646 mL, 9.662 mmol). The solution was refluxed for 3 hours, stirred at ambient temperature for 15 hours, and stripped to. dryness. The resulting solid was taken up in 10 mL of ethanol, and ethyl acetate added until precipitation ceased, giving 2.214 g (94%) of the title product as the trihydrate.

¹H NMR(MeOD) : δ 3.9(s, 8H), 7.85-8.23(m, 6H), 8.67(s,

4H).

(b)

[18]N₆H₈(py)₂

To a stirred solution of [Ca[18]N D₆H₄ (py)₂]Cl₂. 3H₂O

(500mg, 0.93 mmol) in 50 ml of ethanol at ambient temperature was added sodium borohydride (0.2184 mg, 5.58 mmol). The mixture was refluxed for 3 hours, stirred at ambient temperature overnight, stripped to dryness, and diluted with 100 mL of water. The water layer was extracted with methylene chloride (2 x 250 mL) and the combined organic layers dried over K₂CO₂.

Evaporation of the solvent gave 162 mg (52%) of the title product as a white solid.

¹H NMR (MeOD) :δ 2.73(s, 8H), 2.80-3.0 (br s, 4H),

3.78(s, 8H), 6.90-7.60(m, 6H). (c) [18[N₆H₈O₄(py) ₂

[18]N₆H₆(py)₂ (162 mg, 0.49 mmol) in 50 mL of

ethanol/water (1/1) is placed in a 100 mL three neck round-bottom flask equipped with two addition funnels, pH electrode, thermometer, and stir bar. NaOH (199 mg, 4.98 mmol) and BrCH₂CO₂H (346 mg, 2.49 mmol) are each dissolved in 10 mL of water and the resulting solutions placed in the two addition funnels. The solution of NaOH is added to the amine solution to bring the pH to 10.5. The temperature is raised to 50°C and the

BrCH₂CO₂H and NaOH solutions are added concurrently over a 24 hour period maintaining the pH at 11 during the addition. When the pH no longer drops, the reaction mixture is stirred at 75°C for 4 hours, cooled to ambient temperature and stripped to dyness. The solid is taken up in 5 mL of water, the pH adjusted to 3 and the solution applied to AG50-X8 (200-400 mesh, H⁺) resin and the title product eluted with 1N NH₄OH.

Example 2

[18](Me)₄N₆H₄O₄(py)₂

(a) [Ca [ 18 ] (Me) ₄N₆ (py) ₂ ] Cl₂ . 6H₂O

To a stirred solution of 2,6-diacetyl-pyridine (50.0 g, 0.306 mol) and calcium chloride dihydrate (22.49 g, 0.153 mol) in 600 mL of methanol was added

ethylenediamine (20.42 mL, 0.306 mol). The solution was heated at 55°C for 3 hours and then stirred at 25°C for 15 hours and stripped to dryness to leave a pale

yellow-orange solid. The addition of ethanol (200 ml) followed by methylene chloride (200 mL) produced 60 g (66%) of a pale yellow solid isolated as the

hexahydrate. ¹H NMR (MeOD) : δ 2.56(s, 12H), 3.98(s, 8H), 8.18-8.26 (m, 6H). FAB Mass Spectrum, m/z : 449

(MH⁺-Cl), 413 (MH⁺-2Cl) b) [18](Me)₄N₆H₄(py)₂. 2H₂O

To a stirred solution of [Ca[18](Me)₄N₆(py)₂]Cl₂.6H₂O (20 g, 33.9 mmol) in 600 mL of ethanol- at ambient temperature was added sodium borohydride (9.2 g, 242- mmol). The mixture was refluxed for 6 hours, cooled to ambient temperature, stripped to dryness and diluted with 100 mL of water. After adjusting the pH to 10, the water layer was extracted with methylene chloride (3 x 200 mL) and the combined organic layer dried over K₂CO₃. Evaporation of the solvent gave a pale yellow solid which was triturated with ether and collected by suction filtration to give 8.61 g (58%) of the title product as the dihydrate. ¹H NMR (CDCl₃) :δ 1.24-1.52 (m, 12H), 2.1-2.8 (m, 12H), 2.7-2.9 (m, 4H), 6.9-7.7 (m, 6H). FAB Mass Spectrum, m/z : 383 (MH ).

(c) [ 18](Me)₄N₆H₄O₄(py)₂

[18](Me)₄N₆H₄O₄(py)₂.2H₂O (8.00 g, 19 mmol) in 250 ml of ethanol/water (1/1) was placed in a 500 ml three neck round-bottom flask equipped with two addition funnels, pH electrode, thermometer, and stir bar. NaOH (7.66 g, 191 mmol) and BrCH₂CO₂H (13.28 g, 95.6 mmol) were each dissolved in 30 ml of water and the resulting solutions placed in the two addition funnels. The solution of NaOH was added to the amine solution to bring the pH to 10.5. The temperature was raised to 50°C and the

BrCH₂CO₂H and NaOH solutions were added concurrently over a 24 hour period maintaining the pH at 11 during the addition. When the pH no longer dropped the

reaction mixture was stirred at 75°C for 4 hours, cooled to ambient temperature and stripped to dryness. The solid was dissolved in 30 ml of water and adjusted to pH 3 using 1N NaOH. The solution was applied to AG50-X8 (200-400 mesh, H⁺) resin and the product eluted with 1N NH₄OH to yield 10.93 g of the title product as the ammonium salt. ¹³C NMR (D₂O) :δ (ppm) 15.58, 48.83, 54.26, 60.97,

121.24, 137.5, 159.80, 172.52.

Example 3 [12]N₄O₃(py)

a) 2,6-Di(bromomethyl)pyridine

2,6-Di(hydroxymethyl)pyridine (10.0 g; 71.8 mmol) and hydrobromic acid (42% solution in water; 100 ml, 70 mmol) were heated under reflux for 2 hours. The

resulting solution was cooled to 0°C, neutralized by slow addition of a 40% w/w solution of sodium hydroxide in water (87 ml), diluted with water (200 ml) and extracted with dichloromethane (5x100 ml). The combined organic phases were concentrated in vacuo to yield a red solid (about 10 g). This was chromatographed on silica gel (170 g) and eluted with dichloromethane to yield 3.75 g (20%) of the title product.

1H NMR(CDCl₃) :δ 7.69(t, 1H), 7.35(d, 2H), 4.51(s, 4H). b) [ 12 ] N₄ (py) (Ts) ₃

Route A

To a solution of the tritosylate of diethylenetriamine (4.59 g; 8.113 mmol) in dimethylformamide (100 mL) heated at 110°C, was added NaH (60% dispersion; 0.75 g) under a nitrogen atompshere. The resultant, white suspension, and also a solution of 2,6-di(biomo- methyl) pyridine (1.5 g; 8.113 mmol) in DMF (80 ml) at 110°C were dripped separately and simultaneously into DMF (100 mL) also maintained at 110°C. After 2 1/2 hours of vigorous stirring at this temperature, the solution was cooled and then concentrated to dryness in vacuo. Water (150 ml) was added and the beige

precipitate collected by suction filtration.

Dichloromethane (250 mL) was added to the solid,

followed by water (200 mL), and after shaking the organic phase collected via a separating funnel. The aqueous phase was extracted with dichloromethane (2X100 mL) and the combined organic fractions dried (MgSO₄) and concentrated in vacuo to a solid (about 5.6g).

Purification on silica gel (200 g) eluting with chloroform/acetone (95:5) afforded the title product as a white crystalline solid (2.43 g; 45%).

1H NMR(CDCl₃) : δ 7.24-7.74(m, 15H); 4.26 (s, 4H);

3.3(t, 4H); 2.71(s, 4H); 2.4(d, 9H).

Route B

2,6-Di(bromomethyl) pyridine (5.0 g; 19 mmol; 0.86 eq) in dimethylformamide (200 ml) was dripped over a period of 10 hours into a mixture of the tritosylate of

diethylenetriamine (12.72 g; 22 mmol) and potassium carbonate (7.154 g; 58 mmol) in DMF (375 mL), stirring at 25°C. After stirring for 15 hours at 25ºC the excess potassium carbonate was removed by suction filtration and the filtrate concentrated to about 120 mL. Water (250 mL) was added and the white precipitate removed by suction filtration and washed with water until the ensuing filtrate was neutral pH. Excess tritosylate was precipitated by adding chloroform to the solid residue, the precipitate was removed by suction filtration and the filtrate concentrated in vacuo to a white solid (16 g). This solid was chromatographed on silica gel (600 g) and eluted with chloroform to yield 12.3 g of the title product (97% with respect to

2,6-dibromomethylpyridine; 82% with respect to

diethylenetriamine tritosylate).

¹Η NMR(CDCl₃) : δ 7.24-7.74(m, 15H); 4.26(s, 4H); 3.30(t, 4H); 2.71(s, 4H); 2.40(d, 9H). (c) [ 12 ] N₄ (py)

A mixture of [12]N₄ (py) (Ts)₃ (56.6 g; 84 mmol) and phenol (90 g) in a solution of HBr in acetic acid (30% w/w; 1000 mL) was stirred at 80ºC for 48 hours. The reaction mixture was allowed to cool and was then poured into ice-water (1000 mL). With vigorous stirring, diethylether (1500 mL) was added, followed by ethanol (1500 mL). After 5 minutes, the mixture was allowed to stand, whereupon a white precipitate began to settle. The solid was removed by suction filtration, washed with ethanol and dried under vacuum. The off-white solid (about 38 g) was dissolved in water (100 mL) and passed through a bed of AG 1X-8 anion-exchange resin (hydroxide form, 560 g). The water was then removed by

roto-evaporation to yield the title product as a white solid (15 g; 85%).

1H NMR(D₂O) : δ 7.5(t, 1H); 7.0(d, 2H); 3.76(s, 4H);

2.60(s, 4H); 1.8(s, 4H)

Mass Spectrum : (M+H⁺) 207.3.

(d) [12]N₄O₃(py)

Chloroacetic acid (24 g; 254 mmol) in water (500 ml) was adjusted to pH 7 with NaOH solution. This was added dropwise to a solution of [12]N ₄ (py) (15.00 g; 727 mmol) in water maintained at a temperature of 95°C. During the addition, the pH is maintained at pH 9-10 by the addition of 1N NaOH solution. When addition was

complete, the reaction mixture was adjusted to pH 3 using 1N HCl solution and concentrated to a solid.

Water (150 mL) was added, the pH readjusted to pH 7 and the sample applied to a bed of AG 1X-8 anion-exchange resin (100-200 mesh, acetate form, 1.5 L). The column was eluted with an aqueous acetic acid solution to yield 10.2 g (37%) of the title product.

¹H NMR(D₂O) : δ 7.72(t, 1H); 7.2(d, 2H); 4.56(s, 4H); 3.78(s, 4H); 2.76(s, 4H) .

Example 4

1,4,7,10,13-Pentaazabicyclo[11.2.2]heptadecane- 4,7,10-tris(acetic acid) ([15]N₅O₃(pip))

a) N,O-Bis(tosyl)ethanolamine

To a solution of tosyl chloride (100 g, 520 mmol) in 56 ml of dry pyridine, was added dropwise ethanolamine (15 ml, 250 mmol) in 65 ml of pyridine. The temperature was held below 5°C, and vigorous stirring was maintained together with a steady flow of nitrogen gas. After the addition was completed, the mixture was allowed to stir at ambient temperature overnight. The solution was then cooled to 5°C, and 500 mL of ice-water was added slowly. Precipitated solid was separated by filtration and washed with water. Crystallization from methanol (150 ml) gave 56.4 g (62%) of the title product.

1H NMR(CDCl₃) : δ 2.45(s, 3H), 2.6(s, 3H), 3.2(m, 2H), 4.1(t, 2H), 5.0(t, 1H), 7.3-7.7 (m, 8H). b) N-Tosylaziridine

To a vigorously stirred suspension of N,0-bis (tosyl)- ethanolamine (40 g, 110 mmol) in 500 ml of toluene was added KOH (20% solution in water, 140 ml) over a period of 0.5 hour. After stirring for an additional 2 hours, the water phase was removed, and the organic layer was washed with water (3 x 150 ml) and dried over MgSO₄. After filtration, toluene was evaporated to give 20.1 g (92.5%) of the title product.

¹H NMR(CDCl₃): δ 2.4(s, 4H), 2.5(S, 3H), 7.4(d, 2H), 7.8(d, 2H). c) N,N'-Bis(N-tosylaminoethyl)piperazine

Piperazine (22.0 g, 255 mmol) and N-tosylaziridine (105.5 g, 530 mmol) were refluxed in 650 ml of

acetonitrile for 18 hours. After the solution had been cooled to ambient temperature, the precipitated product was separated by filtration, washed with cold acetonitrile and dried in vacuo to yield 83.7 g (68%) of the title product.

¹H NMR(CDCl₃) : δ 2.5(s, 6H), 2.4(t, 4H), 2.9(t, 4H)-, 2.3(s, 8H). d) 4,7,10-Tris(tosyl)-1,4,7,10,13-pentaazabicyclo- [11.2.2]-heptadecane

N,N'-Bis(N-tosylaminoethyl)piperazine (4.0 g, 8.7 mmol) was dissolved in 200 ml of DMF, and Cs CO (6.0 g, 18.4 mmol) was added. The mixture was stirred under a nitrogen atmosphere at 110°C for 2 hours before

N,N-bis(tosyloxyethyl) amine (4.9 g, 8.6 mmol - prepared as described by Guerbet in EP-A-287465) in 80 ml of DMF was added dropwise over 0.5 hour. Stirring at 110°C was maintained for an additional 3 hours. The reaction mixture was evaporated to dryness. The resulting solid was stirred in CH₂Cl₂ overnight. Undissolved material was filtered, and the solution was evaporated to

dryness. The resulting solid was stirred in acetone for 19 hours. The precipitated product was separated by filtration to yield 1.3 g (21%) of the title product. ¹³C NMR(CDCl₃) : δ 21.8, 47.8, 49.6, 50.3, 57.9, 127.6, 130.1, 136.0, 143.8. e) 1,4,7,10,13-Pentaazabicyclo[11.2.2]heptadecane

HBr in acetic acid (32% w/w, 82 ml) was added to a mixture of phenol (4.3 g, 46 mmol) and 4,7,10- tris(tosyl)-1,4,7,10,13-pentaazabicyclo[11.2.2]heptadecane (2.06 g, 3 mmol). The solution was stirred at 70°C for 24 hours. The temperature was raised to 85°C, and stirring was continued for 5.5 hours. The solution was allowed to cool to ambient temperature.

Precipitated solid was collected and triturated with ether and cold ethanol. The solid was then dissolved in water and passed down a Dowex AGI-8X column. Water was removed. Drying of the solid overnight yielded 0.32 g

(50%) of the title product.

¹H NMR(CDCl₃) :δ 2.4(br s, 12H), 2.7(br s,12H) f) 1,4,7,10,13-Pentaazabicyclo[11.2.2]heptadcane- 4.7,10-tris(acetic acid) ([15]N₅O₃(pip))

Bromoacetic acid (2.57 g, 19 mmol) is disolved in water, and LiOH (0.77 g, 19 mmol) is carefully added at 5°C. This solution is added to a solution of

1,4,7,10,13-pentaazabicyclo[11.2.2]heptadecane (1.3 g, 5.4 mmol) in water (2.5 ml). The mixture is heated to 60°C, while the pH is held between 9 and 10 with

addition of 4 M LiOH. After 2 hours, the temperature is increased to 80°C. Stirring is maintained at this temperature for 45 minutes following the addition of LiOH. The mixture is then cooled to ambient temperature and neutralized with HBr. The volume is reduced to 2 ml, and then the mixture is loaded onto a Dowex 1-X8 column (acetate, 50-100 mesh). The material is eluted with deionized water, then 1N, 2N, 3N and 4N acetic acid. Fractions containing product are concentrated by rotary evaporation, and repeatedly reconcentrated with several portions of deionized water until the title product is obtained as an acetate-free solid.

Example 5 [15]N₅O₃(py)₂

The title compound is prepared from

2,6-di(hydroxymethyl)-pyridine, reacted with HBr to form 2-bromomethyl-6-hydroxy-methylpyridine, condensed with 2-aminomethyl-6-hydroxy-methylpyridine, then

pertosylated and cyclized with the sodium salt of bistosyl- ethylenediamine, detosylated with HBr and acetic acid and alkylated with chloroacetic acid (see Scheme (I)).

Example 6

N.N"-Bis(pyrid-2-yl-methyl)-diethylenetriamine-N,N',N"- triacetic acid(Bis(py)DTTA)

a) N ,N''-Bis(pyrid-2-yl-methyl)diethylenetriamine

Diethylenetriamine (76 g, 0.75 mol) and pyridine-2- carboxaldehyde (174 g, 1.62 mol) in 2.5 L of absolute ethanol were heated for 2 hours at 50 °C with stirring. After the reaction mixture was cooled to ambient

temperature, 25 g of 10% palladium on charcoal was added and the schiff base hydrogenated at slightly greater than 1 atmosphere of hydrogen, over a 48-hour period. The catalyst was removed by filtration, the filtrate adjusted to pH 4 with HCl gas and then lowered to pH 1 using 12 N HCL. The resulting precipitate was removed by suction filtration, washed with absolute ethanol until the washings were colourless, and recrystallized from 95% ethanol. This hydrochloride salt was then dissolved in 500 mL of water and neutralized with 5 N NaOH, then raised to pH 12.5, and the free base, the title compound, was extracted with methylene chloride (4 x 500 mL). The methylene chloride solution was dried to give 136 g (65%) of a pale yellow oil. NMR (D₂O) : δ 2.46 (s, 8H), 3.55 (S, 4H), 7.10 (m, 4H) , 7.55 (t, J=12.5 Hz, 2H), 8.25 (d, J=10 Hz, 2H), 7.50 (t, J=10 Hz, 2H), 8.40 (d, J=10 Hz, 2H). (Preparation of the trihydrochloride salt is also described in Inorg. Chem. 17: 889 (1978)). b) N.N''-Bis(pyrid-2-yl-methyl)-N,N',N''-tris(t- butylcarboxymethyl)diethylenetriamine

To a solution of N, N''-bis(pyrid-2-yl- methyl) diethylenetriamine (23.6 g, 82.6 mmol) and diisopropylethylamine (53.4 g, 0.4 mol) in 1.2 L of methylene chloride at ambient temperature was added dropwise t-butylbromoacetate (50 g, 0.2 mol) in 300 mL of methylene chloride. After being stirred for 24 hours, the solution was evaporated to dryness and placed under vacuum for 2 hours to remove excess

diisopropylethylamine. The crude solid was taken up in 1.5 L of methylene chloride, washed with 0.2 N NaOH, water (2 x 250 mL), brine (200 mL) and dried (MgSO₄).

The methylene chloride was removed, 200 mL of ethyl acetate added and this solution passed through 300 g of silica gel in a Bύchner funnel using EtOAc to elute the product. The pure fractions (TLC: methanol : CH₂Cl₂ (3:7)) were combined to give 40.6 g (79.5%) of the title

compound. NMR (CDCl₃) S 1.26 (s, 9H), δ 1.31 (s, 18H), 2.62 (s, 8H), 3.12 (s, 2H), 3.17 (s, 4H), 3.78 (s, 4H), 7.02 (t, J=10 Hz, 2H), 7.35 (d, J=10 Hz, 2H). c) N,N''-Bis(pyrid-2-yl-methyl)diethylenetriamine- N,N',N''-triacetic acid

The tris(t-butylcarboxymethyl)ester (24.89g, 0.1 mol) of step (b) was dissolved in a solution of 600 mL of methylene chloride containing 380 mL of trifluoroacetic acid. The solution was stirred for 48 hours, evaporated under reduced pressure and diluted with 50 mL of water. This solution was applied to 200 mL of AG50-X8 (H⁺ form, 100-200 mesh) and after washing with water until

neutral, the product was eluted with 1N NH₄OH. After removal of NH₄OH solution, the product was taken up in 24 mL of water, and the solution was adjusted to pH 10 and then applied to AG1-X8 (acetate, 100-200 mesh). The column was washed with three bed volumes of water and the product eluted with 2 N HOAc to give 12.0 g (69%) of the title product after several lyophilizations. NMR (D₂O) 5 3.02 (t, J=6 Hz, 4H), 3.08 (t, J=6 Hz, 4H), 3.14 (s, 4H), 3.41 (s, 2H), 4.08 (s, 4H), 7.52 (m, 4H), 8.05 (t, J=10 Hz, 4H), 8.40 (d, J=10 Hz, 2H). Example 7 [ 17 ] N₅O₃ (pip)

The title compound is prepared from

1,4-di(3-aminopropyl)-piperazine and

di(hydroxyethyl) amine by the method of Scheme (J) followed by alkylation with bromoacetic acid.

Example 8

[12]N₄O₂(pip)

A) The title compound is prepared by reaction of

1,4,7,10-tetraazacyclododecane and 1,2-dibromoethane (according to the method of J. Chem. Soc. Chem. Commun. 227 (1982)), followed by alkylation with bromoacetic acid.

B) The title compound is prepared by reaction of the bis-tosylate of 1,4-di(hydroxyethyl)piperazine with the sodium salt of bistosyl- ethylenediamine by the method of Scheme (F), followed by alkylation with bromoacetic acid.

Example 9

[ 14 ] N₄O₂ (pip)

The title compound is prepared by reaction of the sodium salt of the bistosylate of 1,4-di(3-aminopropyl)- piperazine with mesylated ethan-1,2-diol by the method of scheme (K), followed by alkylation with bromoacetic acid. Example 10

[15]N₅O₂(py)(pip)

The title compound is prepared by reaction of 2,6- diformyl-pyridine with 1,4-di(2-aminoethyl) piperazine by the method of scheme (G), followed by alkylation with bromoacetic acid.

Example 11

[15](Me)₂N₅O₂(py) (pip)

The title compound is prepared by reaction of 2,6- diacetyl-pyridine with 1,4-di(2-aminoethyl)pipperazine by the method of scheme (G), followed by alkylation with bromoacetic acid.

Example 12

[17]N₅O₂(py) (pip)

The title compound is prepared by reaction of 2,6- diformyl-pyridine with 1,4-di(3-aminopropyl)piperazine by the method of scheme (G), followed by alkylation with bromoacetic acid.

Example 13

[17] (Me)₂N₅O₂(py) (pip)

The title compound is prepared by reaction of 2,6- diacetyl-pyridine with 1,4-di(3-aminopropyl)piperazine by the method of scheme (G), followed by alkylation with bromoacetic acid.

Example 14

[24]N₈H₈O₆(py)₂

Tetraimine and bisimine/bisimidazolidine

To a solution of diethylenetriamine (0.76 g, 7.40 mmol) in 200 mL of acetonitrile at ambient temperature

diformylpyridine (1.00 g, 7.40 mmol) in 130 mL of acetonitrile was added dropwise over 4 hours. The solution was stirred overnight and the white precipitate formed was removed by filtration, and washed with acetonitrile to yield 1.21 g (81%) of the tetraimine and bisimine/bisimidazolidine title products as a mixture (about 1:1). ¹H NMR (CD₃OD) : δ 2.5 - 3.75 (m, 17 H); 4.25 (s, 3 H); 7.25 - 7.75 (m, 6 H) . FAB mass spectrum, m/z: 405. (b) [24]N₈H₈(py)

The tetraimine:bisimine/bisimidazolidine (3.00 g, 7.42 mmol) was added to a suspension of sodium borohydride (1.17 g, 31 mmol) in 100 mL of ethanol. The mixture was stirred for 1 hour at ambient temperature, refluxed for 0.5 hours, and then stirred overnight at ambient

temperature. The reaction mixture was then stripped to dryness, 20 mL of water was added and the product was extracted with chloroform (6 x 100 mL), and dried (MgSO₄) to give 2.70 g (88% yield) of the title compound as a pale yellow oil. ¹H NMR (CD₃OD) : δ 2.55 (br s, 16H,); δ 3.71 (s, 8H); 7.10 (d, J = 10 Hz, 4H); δ 7.55 (t, J = 10 Hz, 2H). (c) [24 ]N₈H₈O₆ (py) ₂

The amine (1.34 g, 3.24 mmol) of step (b) and

diisopropylethylamine (3.55 g, 25.9 mmol) were dissolved in 125 mL of methylene chloride and to this was added neat t-butylbromoacetate (4.11 g, 3.40 mL, 21.06 mmol) all at once. The reaction mixture was stirred overnight at ambient temperature, refluxed for 0.5 hour, cooled, and stripped to dryness. The resulting oil. was taken up in 400 mL of methylene chloride, washed with water (2 x 100 mL), brine (100 mL) and dried (MgSO₄). The product was purified by silica gel chromatography, eluting with methamol: CH₂Cl₂ (5:95) to yield the hexa-t-butyl ester. ¹H NMR (CDCl₃): 1.36 (br s, 54H); δ 2.61 (br s, 16H); δ 3.20 (br s, 12H); δ 3.75 (br s, 8H); δ 7.15-7.25 (m, 6H). The material was taken up in 100 mL of methylene chloride and 100 mL of trifluoroacetic acid, stirred overnight and stripped to dryness. The resulting thick oil was taken up in 20 mL of water, the pH adjusted to 10.9 using 5N NaOH and the solution applied to AG1-X8,

(OAc- form) resin and the product eluted with 0.1 N HOAc to yield, after recrystallization from water, 194 mg of the title product. ¹H NMR (D₂O): δ 2.90 (s, 8H); δ 3.15

(s, 4H); δ 3.25 (s, 8H); δ 3.55 (s, 8H), δ 4.35 (s, 8H); δ 7.15 (d, J = 10 Hz, 4H); δ 7.55 (t, J = 10 Hz, 2H). FAB mass spectrum, m/z: 761.

Example 15

[Gd[18]N₆O₄(py)₂]Na

5 mL of a 100 mM aqueous solution of [18]N₆O₄ (py)

(Example 1) and 5 mL of a 100 mM aqueous solution of GdCl₃ are mixed thoroughly and the pH is adjusted to 6.9 with 1N NaOH to yield the title product.

Example 16

[Gd[18](Me)₄N₆H₄O₄(py)₂]Na

5 mL of a 100 mM aqueous solution of [18] (Me) ₄N₆O₄(py) (Example 2) and 5 mL of a 100 mM aqueous solution of GdCl are mixed thoroughly and the pH is adjusted to pH 6.9 with 1N NaOH to yield the title product.

The T₁ relaxivity in mM^-1s^-1, measured in water at 10 MHz and 37°C was 1.7.

Example 17

Gd[12]N₄(py)O₃

1.902 g (5 mmol) of [12]N₄O₃ (py) (Example 3) was dissolved in water (3 ml). Gd₂ O₃ (915 mg; 1.01 eq) was added and the volume made up to 8 ml with water. This was then stirred for 2 hours at 100°C. Using Xylenol Orange as an indicator, further [12]N₄O₃ (py) was added in small aliquots until a negative test result was achieved. A titration of excess ligand with GdCl was used to check for less than 0.05% ligand excess. The solution was then diluted to 10 ml with water to produce a 350 mM solution of Gd[12]N₄O₃(py). Finally the pH was adjusted to 6.2 with 1N NaOH.

The T₁ and T₂ relaxivities of the chelate, in mM^-1s^-1 measured in water at 10MHz and 37 °C were respectively 6.99 and 6.23.

Example 18

Gd[15]N₅O₂(pip)

To a suspension of 1,4,7,10,13-pentaazabicyclo[11.2.2]- heptadecane-4, 7, 10-tris (acetic acid) (1.0 g, 2.4 mmol) (Example 4) in water (1 ml) is added Gd₂O₃ (0.24 g, 1.2 mmol) with stirring. The mixture is heated at 75°C overnight. Evaporation to dryness gives the title product.

Example 19

(Mn[18](Me)₄N₆H₄O₄(py)₂)Na₂

The title compound is prepared analogously to that of Example 16 by reaction of the chelant of Example 2 with MnCl₂. Example 20

GdBis(py)DTTA

N,N"-Bis(pyrid-2-yl-methyl)diethylenetriamine-N,N'N"- triacetic acid (2,8719 g, 6.25 mmol) (the compound of Example 6) and gadolinium oxide (1.1328 g, 3.125 mmol) were combined in 15 mL of water and heated at 95°C for 4 hours to provide a solution of the desired complex.

Relaxivity (water) at 10 MHz, 37°C : R₁ = 3.65 mM^-1sec^-1 and R₂ = 3.57 mM^-1 sec^-1.

Example 21

(CaBis(py)DTTA) Na

The title compound is prepared analogously to that of Example 16 by reaction of the chelant of Example 6 with CaCl₂.

Example 22

Composition comprising GdBis(py) DTTA and CaNa

Bis(py)DTTA

The compounds of Examples 20 and 21 are admixed in a 95:5 (by weight) ratio and dispersed in water for injections to a Gd content of 400 mM. Example 23

Gd [17] N ₅O₃ (pip)

The title chelate is prepared analogously to that of Example 16 by reaction of the chelant of Example 7 with GdCl₃.

Example 24

Mn[12]N₄O₂

The title chelate is prepared analogously to that of Example 16 by reaction of the chelant of Example 8 with MnCl₂.

Example 25

Mn[14]N₄O₂(pip)

The title chelate is prepared analogously to that of Example 16 by reaction of the chelant of Example 9 with MnCl₂.

Example 26

Mn[15]N₅O₂(py)(pip)

The title chelate is prepared analogously to that of Example 16 by reaction of the chelant of Example 10 with MnCl₂. Example 27

Mn[15](Me)₂N₅O₂(py)(pip)

The title chelate is prepared analogously to that of Example 16 by reaction of the chelant of Example 11 with MnCl₂.

Claims

Claims

1. A compound of formula I

R¹(CR²R³)_n[X(CR²R³)_n]_mR¹ (I)

(wherein each X independently represents an oxygen or sulphur atom or a group of formula NA, or

(CR²R³)_nX(CR²R³)_n represents a group of formula

E represents COH,NR² ,O or S;

each A independently represents a hydrogen atom or a group (CR²R³)_pY, (CR²R³)_nN[(CR²R³)_pY]₂ or

where two (CR²R³)_nY groups on different nitrogens, may together represent a group -(CR²R³)_n-;

each Y independently represents a group COZ, SO₂Z, POZ₂,

CON(OH) R², CH₂SR², CS₂R² or CSZ;

each Z independently represents a group OR ² or NR²R²; each G is a 3 or 4 membered chain of carbon atoms and optionally a nitrogen, oxygen or sulphur atom;

each J is a 2 or 3 membered chain of carbon atoms and optionally a nitrogen, oxygen or sulphur atom;

each n is an integer of 2 to 4, or in a group (CR²R³)_n attached to a moiety R¹ which represents a hydrogen atom or a group R⁴ n may also be zero or 1;

m is an integer of 3 to 8;

p is an integer of 1 to 3;

each R¹ represents a hydrogen atom or a group R⁴ or together both groups R¹ represent a carbon-carbon bond; each R² independently represents a hydrogen atom or a

C_{1- 8} alkyl group optionally mono- or poly-substituted by hydroxyl or C_{1- 8} alkoxy groups or NR ²R² may together represent a nitrogen-attached 5 to 7 membered saturated heterocyclic ring optionally containing as a further ring heteroatom a nitrogen, oxygen or sulphur atom and optionally substituted by a group R⁴;

each R³ independently represents a hydrogen atom or a

C_{1- 8} alkyl or C_{1- 8} alkoxy group optionally mono or poly substituted by hydroxy or C_{1- 8} alkoxy groups; and

each R 4 i.ndependently represents a hydrogen atom, a halogen atom, a hydroxyl group, an optionally mono- or poly-hydroxylated C_{1- 8} alkyl, C_{1- 8} alkoxy, (C_{1- 8} alkoxy)-C_{1- 8} alkyl or poly(C_{1- 8} alkoxy)-C_{1- 8} alkyl group, a sulphonate group or a group (CR²R³ )_pY or two groups R⁴ on the same ring represent a (CR²R ³)_n-1 [X(CR²R³)_n]_m-1 (CR²R³)_n-1 group in which case the said ring may be saturated; with the provisos that at least 2 Y groups, are present, that where both groups R¹ together form a bond, m is 4 or 5, all n are 2, one X is 2,6-pyridindiyl and the remainder are NCH COOR², then at least one R², R³ or R⁴ is other than hydrogen, and that either at least one X group comprises an aromatic heterocyclic group or both R¹ groups together represent a bond and two (CR²R³)_pY groups together represent a -(CR²R³) _n- group or both R¹ represent a bond, m is 6 or greater and two X groups separated by at least two other X groups are oxygen or sulphur atoms) or a chelate complex or salt thereofo

2. A compound of formula I as claimed in claim 1 wherein both R¹ groups together represent a bond and at least one pair of -(CR²R³)_P Y groups together represent a -(CR²R³)_n- group, or a chelate complex or salt thereof.

3. A compound of formula I as claimed in claim 1 wherein both R¹ groups together represent a bond and at least two moieties (CR²R³)_n X (CR²R³)_n represent

heteroaromatic groups of formula

which incorporate at least one ring heteroatom, or a chelate complex or salt thereof.

4. A compound of formula I as claimed in claim 1 wherein both R¹ groups together represent a bond, m is at least 6 and two X groups separated by at least two other X groups are oxygen or sulphur atoms, or a chelate complex or salt thereof.

5. A compound of formula I as claimed in any one of claims 1 to 4 wherein each n is 2 or 3, each p is 1, and at least 3 Y groups are present, or a chelate complex or salt thereof.

6. A compound of formula I as claimed in any one of claims 1 to 5 comprising at least one hydrophilic R¹, R² R³ or R⁴ group, or a chelate complex or salt thereof.

7. A compound of formula I as claimed in any one of claims 1 to 6 of formula le. If, Ig, Ih, Ii, Ij, Ik or Il

(where A' is CHR²Y or hydrogen, z is 1 or 2, t is 1 or 2, v is 0, 1, 2, 3 or 4 and each X² is oxygen or sulphur and R², R³, R⁴, X, n, m and Y are as defined in claims 1 to 6), or a chelate complex or salt thereof.

8. A compound of formula I as claimed in any one of claims 1 to 7 of formula In, lo, Ip, Iq, Ir, Is, It, lu, lv or Iw

(where R³⁰ is trimethylene or ethylene, each r is 1 or 2, t is 1 or 2, R^2" is hydrogen or methyl and each R⁶ is a group CH₂COZ or CH₂CON(OH)R², and each X² is oxygen or sulphur), or a chelate complex or salt thereof.

9. A chelate complex as claimed in any one of claims 1 to 8 wherein the chelated species is a paramagnetic or heavy metal ion, or a salt thereof.

10. A diagnostic or therapeutic agent comprising a metal chelate, whereof the chelating entity is the residue of a compound of formula I

R¹(CR²R³)_n[X(CR²R³)_n]_mR¹

(wherein each X independently represents an oxygen or sulphur atom or a group of formula NA, or

(CR²R³)_n X(CR²R³)_n represents a group of formula

E represents COH, NR², O or S;

each A independently represents a hydrogen atom or a group (CR²R³)_pY, (CR²R³)_nN[(CR²R³)_pY]₂ or

where two (CR²R³)_n Y groups on different nitrogens, may together represent a group -(CR²R³)_n-; each Y

independently represents a group COZ, SO₂Z, POZ₂ ,

CON (OH) R², CH₂SR², CS₂R² or CSZ; each Z independently represents a group OR² or NR²R²; each G is a 3 or 4 membered chain of carbon atoms and optionally a nitrogen, oxygen or sulphur atom; each J is a 2 or 3 membered chain of carbon atoms and optionally a nitrogen, oxygen or sulphur atom; n is an integer of 2 to 4, or in a group (CR²R³)_n attached to a moiety R¹ which represents a hydrogen atom or a group R⁴ n may also be zero or 1; m is an integer of 3 to 8; p is an integer of 1 to 3; each R¹ represents a hydrogen atom or a group

R 4 or together both groups R¹ represent a carbon-carbon bond; each R² independently represents a hydrogen atom or a C_1-8 alkyl group optionally mono- or poly- substituted by hydroxyl or C_1-8 alkoxy groups or NR ²R² may together represent a nitrogen-attached 5 to 7 membered saturated heterocyclic ring optionally

containing as a further ring heteroatom a nitrogen, oxygen or sulphur atom and optionally substituted by a group R ⁴; each R³ independently represents a hydrogen atom or a C_1-8 alkyl orC_1-8 alkoxy group optionally mono or poly substituted by hydroxy or C_1-8 alkoxy groups; and each R⁴ independently represents a hydrogen atom, a halogen atom, a hydroxyl group, an optionally mono- or poly-hydroxylated C_1-8 alkyl, C_1-8 alkoxy, (C_1-8 alkoxy) C_1-8 alkyl or poly (C_1-8 alkoxy) C_1-8 alkyl group, a sulphonate group or a group (CR²R³) Y or two groups R4 on the same ring represent a (CR²R³)_n-1 [X(CR²R³)_n]_m-1 (CR²R³)_n-1 group in which case the said ring may be saturated; with the provisos that at least 2 Y groups, are present, and that either at least one X group comprises an aromatic heterocyclic group or both R groups together represent a bond and two (CR ²R³)_pY groups together represent a

-(CR ²R³)_n - group or both R¹ represent a bond, m is 6 or greater and two X groups separated by at least two other

X groups are oxygen or sulphur atoms.

11. A composition as claimed in claim 10 wherein the chelating entity is the residue of a compound of formula I as defined in any one of claims 1 to 8.

12. A magnetic resonance imaging contrast enhancing composition as claimed in either of claims 10 and 11 wherein the chelated metal species is selected from the paramagnetic ions of Eu, Gd, Dy, Ho, Cr, Mn and Fe.

13. A detoxification agent comprising a weak complex or salt of a compound of formula I (as defined in any one of claims 1 to 8 and 10) with a physiologically

acceptable counterion, together with at least one pharmaceutical carrier or excipient.

14. A process for the preparation of compounds as claimed in any one of claims 1 to 9 said process

comprising one or more of the following steps:

(a) reacting a compound of formula II

R^1, (CR^2' R^3' )_n[X (CR^2' R^3' )_n]_mR^1' (II)

(where R^1, to R^3, are as defined for R¹ to R³ in claim 1 or are protected R ¹ to R³ groups, and X' is a group X as defined in claim 1 or a protected group X, with the proviso that at least one X' group is of formula NH or (CR^2' R^3' )_pNH₂) With a compound of formula III

LV- (CR^2' R^3' )_p-Y (III)

(where Y' is a group Y as defined in claim 1 or a protected group Y, p is as defined in claim 1, R ^2' andR^3' are as hereinbefore defined and Lv is a leaving group) and if necessary subsequently removing any protecting groups used; and

(b) converting a compound of formula I into a chelate complex or salt thereof.

15. A process as claimed in claim 14 for the

preparation of metal chelate complexes of compounds of formula I which process comprises admixing in a solvent a compound of formula I or a salt or chelate thereof together with an at least sparingly soluble compound of said metal.

16. A method of generating enhanced images of the human or non-human animal body, which method comprises

administering to said body a diagnostic agent as claimed in any one of claims 10 to 12 and generating an X-ray, MR, ultrasound or scintigraphic image of at least a part of said body.

17. A method of radiotherapy practised on the human or non-human animal body, which method comprises

administering to said body a chelate of a radioactive metal species with a chelating agent of formula I as defined in any one of claims 1 to 8 and 10.

18. A method of heavy metal detoxification practised on the human or non-human animal body, which method

comprises administering to said body a weak complex or salt with a physiologically acceptable counterion of a compound of formula I as defined in any one of claims 1 to 8 and 10.

19. The use of compounds of formula I and chelates and salts as defined in any one of claims 1 to 10 for the manufacture of diagnostic or therapeutic agents for use in methods of image generation, detoxification or radiotherapy practised on the human or non-human animal body.