AU644627B2 - Chelating compounds - Google Patents

Description

WO 91/10669 PCT/EP91/00125 1 Chelatinq Compounds 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 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 chelating agents are nitrilotriacetic acid (NTA), N,N,N',N'-ethylenediamine-tetraacetic acid (EDTA), N-hydroxyethyl-N,N',N'-ethylenediamine-triacetic acid (HEDTA), N,N,N',N",N"-diethylenetriamine-pentaacetic acid (DTPA) and N-hydroxyethylimino-diacetic acid, as being suitable as contrast agents for MRI, contrast being achieved by the effect of the magnetic field of the paramagnetic species 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 WO 91/10669 PCT/EP91/00125 2 agent has recently received much attention. The Gd(III) chelate of 1,4,7,10-tetraazacyclododecanetetra-acetic acid (DOTA), referred to in DE-A-3401052 (Schering) and in US-A-4639365 (University of Texas), has 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 carry a fused aromatic ring 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.

However, all hitherto known APCA chelating agents and their metal chelates encounter problems of toxicity, stability or 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.

We now propose a novel class ot polyamine chelating agents which incorporate within their structure a 3 heterocyclic ring.

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

A

SCHR-CHR

2

-N-CHR

2

-CHR

2 I I A-N N-A CHR -CHR 2

-N-CHR

2

-CHR

2

A

(wherein each A independently represents a hydrogen atom, a C 1 3 alkyl group optionally substituted by a group Y or, where the R 2 groups on both a-carbons together form a bridging group, then A may also represent a group R 4 each Y independently represents a group -COZ,-POZ 2 or

CON(OH)R

2 each Z independently represents a group -OR 2 or -NR 2

R

2 cach R 2 independently represents a hydrogen atom or a C1.

8 alkyl group optionally mono- or poly-hydroxylated or a pair of R 2 groups represent a bridging group which together with the intervening two carbon and one nitrogen atoms form a 5 to 8 membered saturated fused ring containing one nitrogen heteroatom and where the ring is 6-membered optionally one further ring heteroatom selected from nitrogen, oxygen and sulphur, said ring optionally being substituted by a group R4; each R 4 independently represents a hydroxy group or an optionally mono- or poly-hydroxylated and/or alkoxylated

C

1 8 alkyl group; with the provisos that one or two pairs of R 2 groups form a said bridging group and with the further proviso that 4 where only one pair of R 2 groups together with the intervening 2 carbon and one nitrogen atoms form a 2,6piperidinyl group and three A groups each represent a carboxymethyl group then said piperdinyl ring must carry a substitute R 4 or a Y-carrying A group) or a chelate complex or salt thereof.

In the compounds of the invention, alkyl or alkylene moieties in groups R 2 and R unless otherwise stated, may be straight chained or branched and preferably contain from 1 to 8, especially preferably 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 R 2 or R' 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, e.g. R and R 4 the compounds of 5 the invention may thus include for example hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 1,2-dihydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3,4-trihydroxybutyl, 1-(hydroxymethyl)-2-hydroxy-ethyl, methoxymethyl, ethoxymethyl, 2-hydroxyethoxymethyl, methoxyethoxymethyl, (2-hydroxy-ethoxy)ethyl, etc, groups.

In the compounds of the invention, where two R 2 groups together with the intervening atoms form a fused cyclic group those fused groups preferably are pyrrolidine, piperidine, piperazine, morpholine or perhydropyrimidine.

N-membered fused rings attached to the macrocyclic skeleton of the molecule at the 2 and N-1 positions of the fused ring are especially preferred. Moreover, in the compounds according to the invention containing more than one fused ring, these are preferably separated by at least one macrocyclic ring nitrogen groups.

Particularly preferably, where there are two or more fused rings, these will be spaced approximately uniformly about the macrocyclic skeleton.

It is also especially preferred that groups NA comprising no ionizing group Y should be non-adjacent.

Particularly preferably such groups are hydrophilic groups such as are defined above.

Especially prefered chelants for use according to the invention include those having one or two fused rings formed by bridging groups L themselves formed by pairs of R 2 groups, eg. compounds of formula la 6 SI (Ia)

^NA

R2 R2

L

where L is (CHR 4 )2 (CHR 4 3 or CHR 4

OCHR

4 2 Where a group NR in a compound according to the invention is a nitrogen attached heterocyclic ring, it will conveniently be of formula

R

4 (cH R 4 -N

W

R

4

R

where q is 0,1 or 2, and W is CHR NR 4 0 or S, where q is zero W preferably being CHR 4 Particularly preferably such groups are of formula N 0 H 0

OH

-N or -NH -OH In the compounds of formula I, the groups Y preferably represent carboxylic acid or amide groups, for example groups of formula COOH, CONH 4C 4 4 4 2" R 2 CONCHRCHR W(CHR 4

CHR

4

CONHR

2 or CONR (where R 2 is an alky or mono or poly hydroxyaly group, for example an alkyl or mono or poly hydroxyalkyl group, for example 7 a C-6 alkyl group optionally carrying 1, 2, 3 or 4 hydroxyl groups).

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 equal 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 (or salt thereof) preferably contains three 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 wherein each R 2 other than those forming fused rings 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

NHR

2 and metal chelates and salt thereof.

8 Especially preferred compounds according to the invention include those of the following formulae Ib to Ii

A

NA NA A b) R2 R2 R4

N

A NA

N

10-2(Id) R4 R4 NA NA R2 R2 R2 R2 R4 rK N

AI

R A NA (C) R2 R2 0

A

A A A

N

O (1 h) (1 g) (i i) preferably where all or all but one group A is a CH 2

Y

group and/or where at least one group A is a non-ionizing hydrophilic group, and where some or all of 9 the R2 and R groups are hydrophilic groups, preferably for R 2 hydroxy- or alkoxy-alkyl groups and for R 4 hydroxyl or hydroxy- or alkoxy- alkyl groups, especially where each A is hydrogen, hydroxypropyl, CH 2

COOH,

CH2CON(CH 3

)-CH

2 CHOHCHO2H, or CH 2 CONHR7 (where R 7 represents CH CH 2 CHOHCH OH or CH(CH 2

OH)

2 or a group -CH2CONCH CHR W(CHR CH where W represents an oxygen S4" atom or a group CH 2 or CHOH, q is 0 or 1 and R is 4" hydrogen or where q is 1 and W is oxygen each R may also represent a C1-4 hydroxyalkyl group) and the metal chelates and the 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: reacting a compound of formula II [NA' (CR 21 2

(II)

(where each R 21 ir as defined for R 2 or is a protected R 2 group, and A'is a group A or a protected group A with the proviso that at least one A group is a hydrogen atom) with a compound of formula III Lv-alk-Y' (III) (where Y' is a group Y or a protected group Y, alk is a

C

1 .3 alkylene group 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 converting a compound of formula I into a chelate complex or salt thereof.

The compounds of formula II may be prepared in a number of ways using techniques known from the 10 literature or analogous to literature described techniques. In particular, starting materials as described in WO 90/08138 may be used.

Thus for example such compounds may be prepared by condensing bifunctional compounds of formula IV LvCO(CR 2 2 )[NA"(CR2') 2

]NA"(CR

2 )COLv (IV) (where R 2 is as defined above, Lv is as defined above or is an alkoxy leaving group,j is 0 or 1, and mid-chain NA" groups, if any, are groups NA' and end of chain NA" groups are NH groups) with a linking molecule of formula

V

H[NA"(CR

2 2) NA"H

(V)

(where i is 1 or 2 and R 2 and A" are as hereinbefore defined) 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 bifunctional compound of formula VI Lv(CR 2 2) 2[NA (CR 2 2 2 ]j+1Lv (VI) followed by removal of the tosyl and other protectin groups.

The compounds of formula II may also be prepared by cyclizing a linear compound of formu.l VII PrNHCO(CR 2 2

[NA"(CR

21 2 2 2 NA"(CR2' 2 )COLv (VII) 2' "2' (where R and NA are as defined above but where (CR 2) moieties adjacent NA" groups may represent carbonyl groups, Lv is a leaving group as defined above or is a hydroxyl group and Pr is a hydrogen atom or an amine 11 protecting group) by reduction, tosylation and base-catalysed cyclization or by carboxyl activation cyclization followed by reduction.

Where this procedure is followed, and where a polypeptide of formula VII is used, selection of the amino acid precursors, e.g. use of hydroxyproline, enables a bicyclo compound of formula II to be produced.

The compounds of formula II can also be prepared by reduction of cyclic imines of formula XIX [NA" (CR2 1 '2 (XIX) (wherein A" and R 2 are as defined above but wherein a 2' [NA"(CR 2 2 2 NA" moiety represents an aromatic 2' heterocyclic ring with two carbon attached -CR =Ngroups).

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

WO 91/1066) PCT'/EP91 /00125 ",oft 0 1 N H 14 N tj E t~ 0 N 0 1~1 tS.JH

H

"1 P.7

P,

-7

T~F

R7 This is similar to the procedurs described by Tabushi et al. in Tetrahedron Letters 4339(1976) and 1049 (1977).

In some cases the use of metal ions such as Ni K or Na as templates in the cyclization step may catalyse the reaction or improve the yield.

fL)

NH

0

N

NTr Tr

N

I. N (Ct, .or) LAim L0x i H N~j NH N

H

N

13 This is similar to the traditional cyclization procedure of Richmann et al. JACS 96:2268 (1974).

(CR

t R4' 1y^ flT Catal y t c 1N I)J hydagnation J

R

20

R

3 e.g. H, CH 3 The compounds of formula IX can be prepared from dicarbonyl derivatives in a Schiff's base condensation as described by Nelson in Pure and Applied Chemistry 52:461-476 (1980), e.g.

R4 o o Wit orwithout o o metal on template NK2 NH NH2 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), Hancock et al. JACS 110: 2788- 2794 (1988), Smith et al. TACS 111: 7437-7443 14 (1989) and the references listed therein.

To introduce an -alk-Y group onto a compound of formula II using the procedure of step may be effected in an aqueous, preferably basic medium, for example by using a halocarboxylic acid Hal-alk-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 an -alk-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.

R NH CH20 R1NCH2PO3H2 H3PO 3 (XIII) (XIV) (of formula II) (of formula I) (where R NCH2Y 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.

15 Z00 W I- 0

NH

(where R 18 N(CI-{COOIHDCH.;Y is a compound of formulaI) c) To introduce a suLfonic acid moiety, synthesis may be performed by alkylation of arn am-ino function for example with iodomethanesulfonic acid

ICHSOIH

(XI (XVI An-ide 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 chelants of formula I may be introduced by methods analogous to those of EP-A-199795.

Chelants of formula I may be used as the basis for bifunctiontal chelants or for polychelant compounds, that is compounds containing several independant chelant groups, by substituting for one Y or R2 or R 4group a bond or linkage to a macromolecule or polymer, e.g. a tissue specfic biomolecule or a backbone polymer such as polylysine or polyethylene--mine -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 and the metal chelates and salts thereof form 1G 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 WO 91/10669 PCT/EP91/00125 -1method, 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 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 Mn and Dy are particularly preferred.

Chelates of ions of these metals specifically listed above with chelants of for.ula I (defined as above with the exclusion of the second proviso) or their salts with physiologically tolerable counterions are particularly WO 91/10669 PCT/EP91/00125 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 MR.I, the parimagnetic 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 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 For use in detoxification of heavy metals, the chelating agent must be in 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 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 agent comprising a metal chelate, whereof the chelating entity is the WO 91/10669 PCT/EP91/00125 residue of a compound 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 agent of the present invention may be in a conventional pharmaceutical administration form such as a tablet, capsule, powder, solution, suspension, dispersion, syrup, suppository, 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 WO 91/10669 PCT/EP91/00125 solution or suspension then being sterilized. Suitable additives include, for example, physiologically biocompatible buffers (as for example, tromethamine hydrochloride), additions 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 or, optionally, additions 1 to 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, 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 prasent 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, intravenous solutions, should be sterile and free from physiologically unacceptable agents, and should have low osmolality to minimize irritation of 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., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: WO 91/10669 PCT/EP91/00125 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 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, ultrasnund or scintigraphic image of at least a part of said body.

Viewed from a further aspect, the present invention WO 91/10669 PCT/EP91/00125 provides a method of radiotherapy practised on the human or non-human animal body, which method comprises adninistering 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 its 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 4 nvention 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 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 yrt 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 wo 91/10669 PCT/EP91/00125 23 the form of a 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.

WO 91/10669 r/P]01.- Example 1 13-Oxa-3 .6,9.15-tetrakis (carboxvmethyl) -3,6,9,15tetraazabicyclo r9.3.1 iperitadecane (rl 121orh) 0 HOOC N COOH

COOH

COOH

Route 4,8-Dioxo-13-oxa-3,6,9,15-tetraazabicyclof9.3.11- ]2entadecane( F12J-dioxo-N 4(morph)) Iminodiacetic acid diethylester (10 mmol) and 2,6bis-aminomethyl-morpholine (10 mmol) (described in British patent aplication no. 89 00719.9) are dissolved in ethanol and ref luxed for 6 days. After stripping to dryness, the resulting oil is chromatogrphed on a silica column.

b) 13-Oxa-3,6,9.,15-tetraazabicyclof9.3.llpentadecane (r121N 4(morph)) The product from a) is disolved in 1 M borane in THF equiv.) at 0 0 C and stirred for 1 hour. The solution is refluxed overnight, cooled and excess methanol is added. The reaction mixture is stripped and treated with a mixture of methanol and 6 M HCl at ambient temperature. After evaporation of methanol, the pH of the solution is adjusted to 12 with NaOH, the reaction mixture is washed several times with chloroform and the WO 91/10669 PCT/EP91/00125 organic phase evaporated to dryness. Alternatively the alkaline water phase is stripped to dryness, the residue dried by repeated evaporation of ethanol and the title compound extracted from the residue with dry ethanol.

The resulting oil is purified on a silica column to yield the title compound.

c) 13-Oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6.9,15tetraazabicyclof9.3.1]pentadecane(r12]N 03(morph)) The product from b) is treated with bromoacetic acid lithium salt (5 equiv.) in water while the pH is kept between 9 and 11 with LiOH and the temperature is gradually increased to 80"C. After reaction overnight, cooling and adjustment of pH to 3, the reaction mixture is treated with a strong cation exchanger. The product is separated from the resin by treatment with saturated ammonia in water. The mixture is stripped to dryness to yield the title compound. The product is purified by precipitation at pH 3-3.5 in water/alcohol, or by reversed phase chromatography.

Route d) 13-Oxa-3,6,9,15-tetrakis(tert.butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclor9.3.11 pentadecane 0 tBu00C j 1 COOiBu COO Bu WO 91/10669 PCr/EP91 /00125 13-Oxa-3,6,9,15--tetraazabicyclo[9.3.1]pentadecane g; 2.3 mmol) was dissolved in dry DMF. Potassium carbonate (1.6 g; 11.5 mmol) and tert-butyl bromoacetate (2.3 g; 11.5 mmol) were added and the reaction mixture was stirred at 50*C overnight. The reaction mixture was cooled, evaporated to dryness, the residue was dissolved in chloroform (100 ml), washed with water (3 x 50 ml) and dried (M~gSO 4 The chloroform was removed and the crude title compound purified by flash chromatography (SiO 2 eluent chloroform:methanol Yield: 0.5 g; 32%. FAB MS: 671 13-Oxa-3,6,9,15-tetrakis(carboxethyl)-3,6,9,15tetraazabicyclor9-.3. lipentadecane 0 HOOCCN COON K H-"

COON

COON

13-Oxa-3 15-tetrakis (tert. -butoxycarbonylmethyl) 3,6,9, l5-tetraazabicyclo[9.3.l]pentadecane (0.4 g; 0.6 mmol) was dissolved in dichloromethane (3 ml) and trifluoroacetic acid (3 ml) was added. The reaction mixture was stirred at ambient temperature overnight, concentrated and treated with diethyl either to give the title compound in quantitative yield. FAB MS: 447 WO 91/10669PC/pIOl, Example 2 13-Hydrox--4 ,8-bis (hydroxymethyl) -3,6,9,15tetrakis (carboxymethyl) -3 .6,9.15-tetraazabicyclor9 .3.lllpentadecane( E12lDihydroxymethvl-N4 0 (hydroxvpip)) Route a) 2,10-Dioxo-4,8-bis(benzyloxymethvl)-13-hydroxy- 3.6,9. 15-tetraazabicvclof9.3.1lipentadecane (F121Dibenzvloxvmethvl-dioxo-N 4(hydroxypip) 2,6-Bis(benzyloxymnethyl)-1,4,7-triazaheptane (10 mnrol), (described in WO-A--89/00557, Example 6 d) and 2, 6-bis (ethyloxycarbonyl) -4-hydroxy-piperidine (10 mmol) (described in WO-A--90/08138) is reacted as described in Example 1 a) to yield the title compound.

b) 4,8-Bis(benzyloxymnethyl)-13-hydroxy-3,6,9,15tetraazabicvclo f9. 3.l11pentadecane 21Dibenzyloxymethvl1- N4 (hvdroxypip)) The product from a) is reduced with borane as described in Example 1 b) to yield the title compound.

WO 9110669PCT/EP91/00125 c) 4,8-Bis-(hydroxymethyl'j-13-hvdroxy-3,6,9,15tetraazabicyrclofr9. 3. 11 pentadecane (F121DihYdroxymethyl-N 4(hydroxvpip)l The product from b) is dissolved in methanol and treated with 10% palladium on carbon at 50 *C overnight. After filtration and evaporation, the title compound is purified on a silica column.

13-Hydroxy-4,8-bis(hydroxymethyl)-3,6,9,15tetrakis (carboxymethyl) -3 r9_.3. llpentadecane( Fl2lDihydroxymethvl-N 4 2 3 (hydroxypip)) The product from c) is alkylated and purified as described in Example 1 c) to yield the title compound.

The title compound is prepared using a scheme analogous to that described in Example 1, Route B.

Example 3 7-Hydroxy-15-oxa-3 11,17. 18-tetrakis-(carboxymethyl) 3 11, 17. 18-tetraazatricyclo Ell. 3. 1. 1.

5 9 1 octadecane 0 HOOC (N COOH -N COOH ~C (OH 1 WO 91/10669 PCT/EP91/00125 The title compound is scheme: prepared by the following reaction COOE I /CN

H

F 0 NH

NH

H

HII 0

H

El honol Ho a I I 8d ucf I o 2. A I ky IoIi o n ilh ticICHtCODH Ti1lIt P r oductI Example 4 13-Oxa-3 .6.9-tris (carboxymethyl) -3,9,15tetraazabicvclo r9. 3 .11ipentadecane

C,

HOOC N COOH N H _/1

N

NCO00H a) 4-Benzvl-3 P h WO 91/10669 PCT/EP91/00125 Tosyl chloride (5.5 g; 28 mmol) and triethylamine (2.9 g; 28 mmol) were dissolved in cry chloroform (100 ml) under nitrogen. 4-Benzyl-3,5-bis(aminomethyl)morpholine (prepared according to WO-A-90/08138) (6.8 g; 28 mmol), dissolved in dry chloroform (100 ml), was added dropwise during 1 hour. The reaction mixture was stirred at ambient temperature overnight, washed with water (3 x 100 ml), dried (MgSO and evaporated. The title compound was isolated after chromatography on SiO2, using ethyl acetate as eluent. Yield: 10.6 g (70 FAB MS: 544 b) 15-Benzyl-13-oxa-3,6,9-tris(tosyl)-3,6,9,15tetraazabicvclor9.3.llpentadecane 0

N

Ts-N h N-Ts

N

Is Ts 4-Benzyl-3,5-bis(tosylamid'methyl)-morpholine (2.3 g; 4.2 mmol) was dissolved in dry DMF (50 ml). Sodium hydride (50 in mineral oil, 1.1 g; 8.4 mmol) was added and the temperiture raised to 100"C after 30 min.

O,N,0'-Tris(tosyl)-diethanolamine (made according to the procedure described in Can. J. Chemistry 45: 1555 (1967)) (2.4 g; 4.2 mmol) dissolved in dry DMF (100 ml), was added dropwise during 1 hour. The reaction mixture was stirred at 100"C overnight, cooled on ice/water and 200 ml water was added dropwise. The precipitate was filtered off and dried. Yield: 8 g; 50%. FAB MS: 768 WO 91/10669 PCT/EP9I /00125 C) l3-Oxa-3,6,9,15-tetraazabicvcloF9.3.llthentadecane

N

H-N

N

H

l5-Benzyl-13-oxa-3 9-tris (tosyl) tetraazabicyclo[9.3.l]pentadecane (2 g; 2.6 mmol) was dissolved in concentrated sulfuric acid (10 mil) and heated to 100'C for 48 hours. The reaction mixture was cooled on ice/water and diethyl ether (30 ml) was added dropwise. The precipitated polyhydrosulfate salt was filtered under nitrogen, washed with small volumes of dry diethyl ether and dissolved in 50% sodium hydroxide solution. The resulting mixture was extracted continuously with chloroform for 48 hours. The chloroform was removed to yield the title compound.

Yield: 0.23 g, 42%. FAB MS: 215 13-Oxa-3 ,6,9-tris(tert.-butoxycarbonylmethyl) 3,6,9. 15-tetraazabicvclor9 lipentadecane NVO 91/10669 PCr/EP91/00125 IBU00C NCOOtBu H0 [COOI!Bu 13-Oxa-3,6,9,l5-tetraazabicyclo[9.3.13pentadecane (0.3 g; 1.4 mmol) was dissolved in dry DMF. Sodium hydrogen carbonate (0.6 g; 7 nimol) and tert-butyl bromoacetate (1.4 g; 7 mmol) were added and the reaction mixture was stirred at ambient temperature overnight. The reaction mixture was evaporated to dryness, the residue was dissolved in chloroform (100 nml), washed with water (3 x ml) and dried (MgSO 4 The chloroform was removed and the crude title compound purified by flash chromatography (SiO.; eluent chloroform: methanol 9:1).

Yield: 0.47 g; 60%. FAB MS: 557 e) 13-Oxa-3,6,9-tris(carboxmethylj-3,6,6,15tetraazabicycdo f9.3.llpentdecane HOOCI 1. COOH

H

COOH

13-Oxa-3 9-tris (tert. -butoxycarbonylmethyl) 6,9, tetraazabicyclo[9.3.1]pentadecane (0.4 g; 0.7 mlnol) was WO 91/10669 PCr/EP91/oo125 dissolved in dichioromethane (3 ml) and trifluoroacetic acid (3 ml) was added. The reaction mixture was stirred at ambient temperature overnight, concentrated and treated with diethyl ether to give the title compound in quantitative yield. FAB MS: 389 (IM1).

Example Gadolinium chelate of 13-oxa-3,6,9-tris(carboxymethyl)- 3,6,9. 15-tetraazabicyclor9.3. lloentadecane 13-Oxa-3,6, 9-tris(carboxymethyl)-3,6,9,15tetraazabicyclo[9.3.l]pentadecane (Example 4) (0.23 g; 0.6 mmol) was dissolved in water (2 ml) and the pH adjusted to 5 with 1 M NaOH. Gadolinium oxide (0.11 g; 0.3 mmol) was added and the resulting mixture stirred at for 24 hours. The reaction mixture was filtered and the filtrate evaporated to dryness to yield 0.3 g of a yellow 5olid. FAB MS: 543 Example 6 (2 '-Hvdrox ropvl) -13-oxa-3 9-tris (carboxymethyl) 3, 6,9. l5-tetraazabicyclo r9.3.l1 pentadecane, HO0O0C\ r C 0 0H -N0 H

COOH

13-Oxa-3,6,9-tris(carboxymethyl)-3,6,9,15- I tetraazabicyclo[9.3.1]pentadecane (Example 4) (0.23 g; WO 91/10669 PCT/EP91/00125 0.6 mmol) is dissolved in water (2 ml) and the pH adjusted to 12 with 1 M NaOH. The solution is cooled to ambient temperature, and propylene oxide (0.05 g; 0.9 mmol) is added. The reaction flask is stoppered, and left to stir at ambient temperature overnight. The reaction mixture is acidified and treated with a cation exchange resin to give the title compound.

Example 7 Sodium salt of the gadolinium chelate of 13-oxa- 3,6,9,-tetrakiscarboxmethl)-3,6,915-tetrakiseth15tetraazabicyclo 9.3.11pentadecane 13-Oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15tetraazabicyclo[9.3.1]pentadecane (Example 1) (0.22 g; mmol) was dissolved in water (2 ml) and the pH adjusted to 5 with 1 M NaOH. Gadolinium oxide (0.09 g; 0.25 mmol) was added and the resulting mixture stirred at 90°C for 24 hours. The reaction mixture was filtered and the filtrate evaporated to dryness to yield 0.3 g of a yellow solid. FAB MS: 622 (M+Na).

Claims (6)

1. A compound of formula I A CHR -CHR-N CHR 2 CHR 2 A-N N-A CHR -CHR 2 -N-CHR 2 -CHR 2 A (wherein each A independently represents a hydrogen atom, a C. 3 alkyl group optionally substituted by a group Y or, where the R 2 groups on both a-carbons immediately adjacent to the nitrogen atom bound to a particular group i together form a bridging group, then that group A may also represent a group R4; each Y independently represents a group COZ,-POZ, or CON(OH)R2; each Z independently represents a group -OR 2 or -NR 2 R 2 each R 2 independently represents a hydrogen atom or a C. l alkyl group optionally mono- or poly-hydroxylated or a pair of R 2 groups represent a bridging group which together with the intervening two carbon and one nitrogen atoms form a 5 to 8 membered saturated fused ring containing one nitrogen '-eteroatom and where the ring is 6-membered optionally one further ring heteroatom selected from nitrogen, oxygen and sulphur, said ring optionally being substituted by a group R4; each R 4 independently represents a hydroxy group or an optionally mono- or poly-hydroxylated and/or alkoxylated C 1 alkyl group; SUBSTITUTE SHEET 36 with the provisos that one or two pairs of R 2 groups form a said bridging group and with the further proviso that where only one pair of R 2 groups together with the intervening 2 carbon and one nitroge., atoms form a 2,6- piperidinyl group and three A groups each represent a carboxymethyl group then said piperdinyl ring must carry a substitute R 4 or a Y-carrying A group) or a chelate complex or salt thereof.

2. A compound as claimed in claim 1 being a compound of formula Ib 0 N 3 NA 9 NA (I b) R2 NA R

8-Y 6 R2 R2 R2 (where all or all but one A group is a group of formula CHY and R 2 and Y are as defined in claim 1) or a chelate ccmplex or salt thereof. 3. A compound as claimed in claim 2 being a compound o0 formula Ib wherein each R 2 is hydrogen and the A groups at the 6- and 9- positions are carboxymethyl groups, or a chelate complex or salt thereof. 4. A compound as claimed in any one of claims 1 to 3 being a chelate complex of a compound of formula I with 37 a heavy metal or paramagnetic metal ion, or a salt thereof. A compound as claimed in claim 4 wherein said metal is selected from Eu, Gd, Dy, Ho, Cr, Mn and Fe. 6. A process for the preparation of compounds as claimed in claim 1, said process comprising one or more of the following steps: reacting a compound of formula II 2 22 [NAI (CR '21 4 (II) (where each R 2 is a group R 2 as defined in claim 1 or a protected R 2 group, and NA' is a group NA as defined in claim 1 or a protected group NA with the proviso that at least one NA group is of formula NH) with a compound of formula III Lv-alk-Y' (III) (where Y' is a group Y as defined in claim 1 or a protected group Y, alk is a C 1 3 -alkylene group and Lv is a leaving group) and if necessary subsequently removing any protecting groups used; and converting a compound of formula I into a chelate complex or salt thereof. 7. A diagnostic or therapeutic agent comprising a metal chelate, whereof the che-ating entity is the residue of a compound of formula I as defined in claim 1, together with at least one pharmaceutical or veterinary carrier or excipient. 8. A detoxification agent comprising a chelating agent of formula I as defined in claim 1 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.

9. A method of generating enhanced images of the human or non-human animal body, which method comprises administecin% to said body a diagnostic agent as claimed in clair i. 7nd generating an X-ray, MR, ultrasound or scintigraphic image of at least a part of said body. 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 claim 1.

11. 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 of formula I as defined in claim 1 in the form of its weak complex or salt with a physiologically acceptable counterion.

12. A process for the preparation of the metal chelates as claimed in claim 1 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. DATED this 20th day of October 1993. NYCOMED AS By their Patent Attorneys: CALLINAN LAWRIE INTERNATIONAL SEARCH REPORT International Application No PCT/EP 91/00125 I. CLASSIFICATION OF SUBJECT MATTER (If several clasaiflcatlon symbols apply, indicate all) According to International Patent Classificallon (IPC) or to both National Classification and IPC C 07 D 498/08, 498/18, 471/08, A 61 K 31/535, 31/445, 49/00 49/02. 49/04 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols C 07 D; A 61 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in Fields Searched 8 III. DOCUMENTS CONSIDERED TO BE RELEVANT" Category* Citation of Document, 11 with Indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 13 P,X EP, A2, 0352218 (SCHERING AKTIENGESELLSCHAFT) 2-3 24 January 1990, see the whole document P,X WO, Al, 9011282 (GUERBET 4 October 1990, 2-3 see the claims X Tetrahedron, vol. 37, 1981, H. Stetter et 2-3 al.: "Darstellung und komplexbildung von polyazacycloalkan-n-essigsuren see page 767 page 772 Special categories of cited documents: 10 'T later.dopument published after he international filing date A dcundd d a e r eneral e of the art which is not prori date and not in Pcllict w thhe applcaion but consderetoenofpartculare ne rcited to understand the principle or theory underlying the cons ed to be o p a reevance invention E* earlrier document but published on or alter the international filing date X' document of particular relevance, the claimed, invention cannot be considered novel or cannot be co)nridered to 'L doqumpnt which may throw doubts pn priority claim(s) or involve an inventive step which is cited to establish the publication date of another Y' document of paiicular citation or other special reason (as specified) Y document of particular rdlevance, tlje claimed invention citation or other special reasn (as specified) cannot be considered to involve an inventive step when the 0' document referring to an oral disclosure, use, exhibiltin r document is comb ned with ne e or more her such docu- other means aiscosure, use, exiiion or ents, such combination being obvious to z person skilled doaument publlstie prior to tle international filing date but document member of the same patent family late t!han the priority date claimed the same patent amily IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing cf this International Search Report 24th April 1991 1 7. 05, 91 International Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE F.W. HECK norm PCT/ISA/210 (second sheet) (January 1985) lInternational Application No. PCT/EP 91/00125 Ill. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, with Indication, where appropriate, of the relevant passage$ Relevanlt to Claim No DE, Al, 3401052 (SCHERING AG) 26 July 1984, see th-i whole document EP, Al, 0071564 (SCHERING AKTIENGESELLSCHAFT) 9 February 1983, see the whole document 2-3 2-3 Form PCT/ISA/210 (extra sheet) (January 1985) International Application No, PCT/EP 91/00125 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V.0 OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE I This International search report has not been established In respect of certain claims under Article 17(2) for the following reasons: 1.r) Claim numbers because they relate to subject matter not required to be searched by this Authority, namely: Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods (PCT Rule 39.1(iv)). 2.Q- Claim because they relate to parts of the Internatinal application that do not comply with the preacrlbed require- ments to such an extent that no meaningful International search can be carried out, specrtfielly: *)The claims 1, 4-8, 12 and 13 are too broadly formulated to permit a meaningful search. The search has been limited to the compounds according to the claims 2 and 3. 3 Claim because they are dependent laims and are not drafted in accordance with the econd and third sentences of PCT Rule 6.4(a). VI. ORSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authority found multiple inventions In this International application as follows: 1. As all required additional search tees were timely paid by the applicant, this International search report covers all searchable claims of the international application, 2. As only some of the required additional search fees were timely paid by the applicant, this International search report covers only those claims of the International application for which fees were paid, specifically claims: No required additional search fees were timely paid by the applicant. Consequently, this International search report is restricted to the Invention first mentloned In the claims; it Is covered by claim numbers: 4.7 As all searchable claims could be searched without effort justifying an additional fee, the International Sarching Authority did not Invite payment of any additional tee. Remark on Protest The additional search lfes were accompanied by applicant's protest. E No protest accompanied the payment of additional search fees. Form PCTIISA210 (supplemental sheet (January 1985) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO.PCT/EP 91/00125 SA 43782 This annex lists the Patent family members relating to the patent documents cited In the above-mentvied i~ternationat search report. The members are as contained In the European Patent Office EDP file on 2303,91 The European Patent office Is In no way liable for theseparticulara which arm merely given for the purpose of Information. IPatent document I Publication IPatent family Publication I cited In search report date member~s) date EP-A2- 0352218 24/ 01/90 AU-D- DE-A- JP-A- 3827589 3825040 2104588 WO-Al- 9011282 04/10/90 EP-A- FR-A- 0391766 2644785 DE-Al- 3401052 26/07/84 AU-B- AU-D- AU-D- BE-A- CA-A- CH-A-B- FR-A-B- FR-A- GB-A- GB-A-B- GB-A-B- JP-A- LU-A- NL-A- SE-A- US-A- US-A- US-A- 574658 1018488 2355984 898708 1256249 660183 2539996 2590484 2M37612 2169598 2169599 59139390 85177 8400079 8400254 4647447 4957939 4963344 01/02/90 25/01/90 17/04/90 10/10/90 28/09/90 14/07/88 28/04/88 26/07/84 16/05/84 20/06/89 31/03/87 03/08/84 29/05/87 10/10/84 16/07/86 16/07/86 10/08/84 24/05/84 16/08/84 22/07/84 03/03/87 18/09/90 16/10/90 08/10/87 20/09/90 28/04/88 27/01/83 03/03/87 10/02/83 29/01/86 22/02/83 04/06/87 03/03/87 18/09/90 16/10/90 EP-Al- 0071564 09/02/83 AU-B- AU-B- AU-D- AU-D- CA-A- DE-A-C- EP-A-B- JP-A- .JP-A- US-A- US-A- US-A- 566007 601916 1018688 8633082 1218597 3129906 0169299 58029718 62123159 4647447 4957939 4963344 For more details about this annex see Official Journal of the European patent office, No0. 12)82 EPO FORM P0479