IE910162A1 - Macrocyclic tetraaza compounds containing a six-membered ring, processes for their production, and pharmaceutical agents containing the same - Google Patents

Abstract

6-Membered-ring-containing macrocyclic tetraaza compounds of the general formula I <IMAGE> in which ... represents a single or double bond, Q represents a nitrogen atom or the radical NH, X<1> represents a hydrogen atom, -(CH2)n-R<1>- or <IMAGE> group where n denotes the figures 1 to 5, m denotes the figures 0 to 2 and R<1> denotes a hydrogen atom or a hydroxyl group, X<2> represents X<1> or a -(CH2)n-(O)l-(CH2)k-(C6H4)q-R<2> group where k denotes the figures 0 to 4, l and q denote the figures 0 or 1 and R<2> denotes a hydrogen atom, a C1-C4-alkoxy group, a functional group or, bonded by means of this functional group, a bio- or macromolecule, A<1>, A<2>, B<1>, B<2>, C<1>, C<2>, D<1>, D<2>, E<1>, E<2>, F<1> and F<2> independently of one another each represent X<2> G represents R<2> or a second macrocycle, bonded by means of K, of the general formula II <IMAGE> where K denotes a direct bond, a bis(carbonylamino) group (-NH-CO-CO-NH-) or a C1-C14-alkylene group which optionally at the ends carries carbonyl( &rdurule& CO) or carbonylamino (-NH-CO-) groups or oxygen atoms and optionally contains one or more oxygen atom(s), hydroxymethylene (-CH-OH-), CH(X<2>)COOZ, acyl or hydroxyacyl- substituted imino groups or one or two C-C double and/or C-C triple bonds, Z represents a hydrogen atom and/or a metal ion equivalent of an element of atomic number 21-29, 31, 32, 37-39, 42-44, 49 or 57-83, with the proviso that the 12 ring substituents A<1> to F<2> represent at least 8 hydrogen atoms, that X<1> and X<2> only simultaneously represent hydrogen atoms when at least one of the ring substituents A<1> to F<2> does not represent a hydrogen atom and that the macrocycle of the general formula I no longer contains a bio- or macromolecule and that, if desired, the radical of the CO2H groups is present as ester or amide, and their salts with inorganic and/or organic bases, amino acids or amino acid amides are useful diagnostic and therapeutic agents.

Description

MACROCYCLIC TETRAAZA COMPOUNDS CONTAINING A SIX-MEMBERED RING, PROCESSES FOR THEIR PRODUCTION, AND PHARMACEUTICAL AGENTS CONTAINING SAME Summary of the. Invention The invention relates to macrocyclic tetraaza complexing compounds, complexes, and complex salts containing a six-membered ring. The invention further relates to agents containing these compounds, their use as diagnostic aids and therapeutic agents, as well as processes for the preparation of these compounds and agents.

Metallic complexes have been scrutinized as early as the beginning of the fifties as contrast media for radiology. The compounds then employed were, however, of such toxicity that utilization on human patients could not be considered. It was, therefore, entirely surprising to find that certain complex salts exhibited adequate compatibility for routine administration to human patients for diagnostic purposes.

The first recorded representative of this class of compounds is the dimeglumine salt of Gd DTPA [MAGNEVIST®, Schering A.G.; gadolinium (III) complex of diethylenetriaminepentaacetic acid] described in European Patent Application Publication No. 71564 as a contrast medium for nuclear magnetic resonance imaging or MRI.

Presently, the compound is primarily administered in case of diseases of the central nervous system.

.An essential reason for the satisfactory clinical applicability of Gd DTPA resides in its high efficacy in nuclear magnetic resonance imaging, particularly in connection with many brain tumors, on account of IE 91162 its high effectiveness, Gd DTPA, with 0.1 mmol/kg of body weight, can be utilized at a very much lower dosage than, for example. X-ray contrast media, in many X-ray examinations.

Another representative of the complex salts that has proven itself well for diagnostic purposes is the meglumine salt of Gd DOTA described in German Patent Application 3,401,052 (gadolinium(III, complex of 1,4,7,10-tetraazacyclododecanetetraacetic acid].

However, it is desirable to be able to use chelates also at a higher dosage. This is the case, in particular, for the detection of certain diseases ohtside of the central nervous system with the aid of magnetic resonance imaging (NMR diagnostics,, but quite especially in case of utilization of chelates as X-ray contrast media.

In order to keep the volume load on the body at a minimum, it is preferable to employ highly concentrated chelate solutions. The chelates known thus far show little suitability for this purpose, above all on account of their high osmolality.

Therefore, there is a need for chelates exhibit ing an osmolality which is lower than that of the previously known chelates. However, at the same time, the prerequisites must be met for the use of these compounds on human patients, as regards the spacing between the effective dose and the toxic dose in animal tests (the therapeutic range), organ specificity, stability, contrast-enhancing effect, compatibility, as well as solubility of the complex compounds.

IE 91162 - 3 Accordingly, an object of the invention is to make available such compounds and agents, and also to provide a maximally simple method for their production.

This object is met by the present invention.

Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art.

The complex compounds of this invention and the solutions prepared therefrom fulfill the aforementioned requirements in a surprising way. They possess reduced osmolality, as well as a more favorable therapeutic range and/or stability and shelf life of the chemical ingredients of the solution and/or organ specificity and/or contrast-enhancing effect (e.g., relaxivity) and/or compatibility (for example, reduced cardiovascular or allergy-type side effects) than the previously customary diagnostic aids.

Even without specific measures, the pharmacokinetics of the inventive compounds permit an improvement in the diagnosis of numerous diseases. The complexes, for the most part, are excreted again unchanged and rapidly, so that no damaging effects are observed in spite of a high dose, particularly when using even relatively toxic metallic ions.

The practical usage of the novel complexes and complexing compounds is also facilitated by their favorable chemical stability Another essential advantage of the described complexes and complexing compounds is their extraordinary chemical versatility. The properties can be adapted, not only by selection of the central atom, but also by the choice of variegated substituents in the macrocycle and/or by the selection of the salt-forming compounds, to the requirements regarding efficacy, pharmacokinetics, compatibility, solubility, handling ability, etc. Thus, it is possible for example, to achieve a specificity of the compounds, very much desirable in IE 91162 diagnostics and therapy, for structures in the organism, for certain biochemical substances, for metabolic processes, for conditions of the tissues or body fluids.

The macrocyclic compounds according to this 5 invention are characterized by general Formula I: cooz wherein is a single or double bond, is a nitrogen atom or the residue NH, is a hydrogen atom, a -(CH2)n-R1 group or a -(CH~) -(CH) -CH-OH group z m | η z OH wherein n means the numbers 1 to 5, m means the numbers 0 to 2, and means a hydrogen atom or a hydroxy group, means X1 or a - (CH2) n~ (Ο) (CH2) R- (CgH^ g-R2 group wherein k means the numbers 0 to 4, and and q mean the number 0 or 1, 2 R means a hydrogen atom, a C1-C4~alkoxy group, a functional group, IE 91162 - 5 or, bound via this functional group, a bio- or macromolecule, 12121212121 2 A , A , B , B , C , C , D , D , Ex, E, F and F mean in each case X , independently of one another 2 G means R or a second macrocycle, bound via K, of general Formula II cooz wherein K means a direct bond, a bis(carbonyl amino)group (-NH-CO-CO-NH-), or a C^-C^^10 alkylene group which optionally carries at the ends carbonyl (> CO) or carbonylamino (-NH-CO-) groups or oxygen atoms, and which contains optionally one or several oxygen atom(s), hydroxymethylene (-CH-OH-), CH(X )COOZ-, acyl-substituted or hydroxyacyl-substituted imino groups, or one to two C-C-double and/or C-C-triple bonds, is a hydrogen atom and/or a metal ion equivalent of an element of atomic numbers 21-29, 31, 32, 37-39, 42-44, 49 or 57-83, IE 91162 with the proviso that the 12 ring substituents A^ through F stand for at least 8 hydrogen atoms, 2 that X and X stand simultaneously for hydrogen only 1 2 if at least one of the ring substituents A through F 5 does not mean a hydrogen atom, and that the macrocycle of general Formula I contains no more than one bioor macromolecule, and that, if desired, the remainder of the CO2H groups is present as an ester or amide, as well as their salts with inorganic and/or organic 10 bases, amino acids or amino acid amides.

Preferred are tetraaza compounds of general Formula III cooz wherein IE 91162 --- is a single or double bond, Q is a nitrogen atom or the residue NH, χΐ is a hydrogen atom, a -(CH2)n-R1 group or a - (CH2)ni~ (CH) n-CH2OH group OH wherein n means the numbers 1 to m means the numbers 0 to R1 means a hydrogen atom hydroxy group, x2vi means X or a -(CH2)n-’l-(CH2)k-(C6H4>q-R2 10 group wherein k means the numbers 0 to 4, 1 and q mean the number 0 or 1, and 2 R means a hydrogen atom, a C^-C^-alkoxy group, a functional group, or, bound via this functional group, a bio15 or macromolecule, , B3, C1 and D1, independently of one another, mean X2 in each case, G means R or a second macrocycle, bound via K, of general Formula IV cooz IE 91162 wherein K means a direct bond, a bis(carbonylamino) group (-NH-CO-CO-NH-), or a C^-C^^alkylene group which optionally carries at the ends carbonyl (> CO) or carbonylamino (-NH-CO-) groups or oxygen atoms, and which contains optionally one or several oxygen atom(s), hydroxymethylene (-CH-OH-), CH(X )COOZ-, acyl-substituted or hydroxyacyl-substituted imino groups, or one to two C-C-double and/or C-C-triple bonds, Z is a hydrogen atom and/or a metal ion equivalent of an element of atomic numbers 21-29, 31, 32, 37-39, 42-44, 49 or 57-83, 2 with the proviso that X and X simultaneously mean 15 hydrogen atoms only if at least one of the 4 ring substituents A1, B1, C1 and D1 does not stand for a hydrogen atom, and that, if desired, the remainder of the CO2H groups is present as an ester or amide, as well as their salts with inorganic and/or organic 20 bases, amino acids or amino acid amides.

Compounds of general Formula I wherein Z means hydrogen are denoted as complexing compounds, and with at least two of the substituents Z meaning a metal ion equivalent, are called metal complexes.

The element of the above-mentioned atomic number forming the central ion of the physiologically compatible complex salt can, of course, also be radioactive for the intended purpose of using the diagnostic agent of this invention.

IE 91162 In case the agent of the invention is intended for use in NMR diagnostics, the central ion of the complex salt must be paramagnetic. These are, in particular, the divalent and trivalent ions of the elements of atomic numbers 21-29, 42, 44 and 58-70. Suitable ions are, for example, the chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III) and ytterbium(III) ions. On account of their very strong magnetic moment, the gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III) and iron(III) ions are especially preferred.

For using the agents of this invention in nuclear medicine, the central ion must be radioactive.

Suitable are, for example, radioisotopes of the elements copper, cobalt, gallium, germanium, yttrium, strontium, technetium, indium, ytterbium, gadolinium, samarium and iridium.

If the agent according to this invention is intended for use in X-ray diagnostics, the central ion must be derived from an element of a higher atomic number in order to attain adequate absorption of the X-rays. It has been found that diagnostic media containing a physiologically compatible complex salt with central ions of elements of the atomic numbers between 21-29, 42, 44, 57-83 are suitable for this purpose; these are, for example, the lanthanum(III) ion and the above-mentioned ions of the lanthanide series.

Preferred groups for Xx that can be mentioned are CH2OH, CI^CI^OH and CHOHCH^OH; and preferred groups for X2, A1, B1, C1 and/or D1 are CH2OH, CH2CH2OH, ch2och2c6h5, chohch2oh, ch2c6h4och3, ch2c6h5, CH2CgH4O(CH2)^COOH, CH2CgH4NCS, wherein the remaining 2 22121 2 ring substituents A,B,C,D,E,E,F and F preferably mean hydrogen.

IE 91162 - 10 The alkylene chain standing- for K, to which the second macrocycle II and IV, respectively, is linked, carries at the ends optionally carbonyl (CO), carbonylamino (NH-CO) groups or oxygen atoms and 5 contains 1-14 carbon atoms. This chain can be interrupted by one or several oxygen atom(s), hydroxymethy12 ene (-CHOH-), CH(X )COOZ, acyl- or hydroxyacyl-substituted imino groups or one to two C-C-double and/or C-C-triple bonds. The two macrocycles can, however, also be linked by a direct bond. Suitable optionally hydroxylated acyl groups are acyl residues of up to 10 carbon atoms. Examples in this connection are the acetyl, propionyl, butyryl, benzoyl and hydroxyacetyl residues.

The alkylene chain can be straight- or branched-chain, saturated or unsaturated, and can be interrupted, if desired, as described above. This chain can contain up to 4 oxygen atoms and/or up to carboxymethylimino groups.

IE 91162 Examples for the alkylene chain are: -ten >2~. -ch2-o-ch2-, -(ch2)4-, -(ch2-ch2-o-ch2-ch2j-, -(ch2-( -ch2).

(CH2-O-CH2)3-, -CH2-CH2-(O-CH2-CHz)3-, -CH2-CH2-(0-CH2-CH2), -CH-CH-, I I OH OH CH -CH-CH-CH - , -C=C-CEC-, -NH-C-(CH,)„ -C-NH > . ι f Z U * b 2 I I 2 OH OH CH -N-CH -CH -N-CH -CH -N-CH,-, M / * I / ?-C-(CH2>,-6 II C-, CHX I COOZ CHX I COOZ CHX I cooz -ch2-n-ch2- , CO-CH2OH -(CH2)2CH=CH-(CH2)291162 Preferably, the two macrocycles are linked by a direct bond or by the group -(O)q-(CH2)n-(CH)kOH (CH2) n- (0) r.

The functional groups are any moiety capable of reacting in a manner which will permit attachment of a biomolecule or macromolecule.

Preferred functional groups which R can represent arc, for example, the maleimidobenzoyl, 3-sulfomaleimidobcnzoyl, 4-(maleimidomethyl)eye]ohexylcarbonyl, 4-[3-sulfo-(maleimidomethyl) Jcyclohexylcarbonyl, 4-(p-maleimidophenyl)butyryl, 3-(2-pyridyldithio)propionyl, methacryloyl(pentamethylene)amido, bromoacetyl, iodoacetyl, 3-iodopropyl, 2-bromoethyl, 3-mercaptopropyl, 2-mercaptoethy1, phenyleneisothiocyanate, 3-aminopropy1, benzyl ester, ethyl ester, tert^butyl ester, amino, hydroxy, C^Cg-alkylamino, aminocarbonyl, hydrazino, hydrazinocarbonyi, maleimido, methacrylamido, methacryloylhydraz inocarbonyl, maleimidamidocarbony1, halogeno, mercapto, hydrazinotrimethylenehydrazinocarbonyl, aminodimethyleneamidocarbonyl, bromocarbonyl, phenylenediazonium, isothiocyanate, semicarbazide, thiosemicarbazide, isocyanate groups.

Several selected groups will be set forth for the sake of explanation: II — / C,H, -OlCH,K-N 6 t 2 J \ -CH2-CEHr01CH2!3NH~ . u □ -CHrC6HrOICH7liCOzCH2C5H5. - CH 2 - Cfi H < - 0 - C H ? - C02 CH ? Cfi H , . w -CHj-C^-OICH^CONHNH?, -Chz-C6h4-COHHNH-^Y , - CM - C g H t - 0 1 C H 2 ) - S H , π A CH2'CSMt’°(CH21-CH2’C5H4-OICK2I5-CONH-N V"CH2"C6H4"° ( CH2 ) JBr' -CO2’C6H4~NO2 ♦ IE 911®2 - 13 -CH2-C6H4-0(CH2)5CONHNH-(CH2)3-NHNH2, -CH2-NHNH2, -CH2-SH, -CH2CONHNH2 -fCH ) SH, -CH-CcH-Q-CH,COBr, -C,H.NHCOCH.Br, Z J c □ * £ 0¼ c -CH2-C6H4-OCH2-C-NH-(CH2)2NH2, -CH2-CgH4-NH2, -ΟθΗ^-Ν^ -CgH^NCS, -CH-CcH-NH-C-!CH)-S-SZ b s Z i.

-NHCO-NH-NH2, -NHCS-NH-NH2, /.\ 5 -ch2-c6h4-o-ch2-ch-ch2> -ch2-c6h4-o-ch2-c-nh-(ch2)10-c-nhnh21 0 CH II II I 3 -CH2-C5H4-0-CH2-CHOH-CH2-NHICH2)10-C-NHNH2, -0CH2-C-N-CH2-(CHOH)4-CH2OH, / \ -CH2-CH-CH2, -CH2-0-(CH2)3-N.

, -CH2-O-(CH2J4-SH, -CH2-0-(CH2)3-NHNH2, -CH2-O-CH2-C-NH-NH2, -CH2-0-CH2-CH2-NH2, -CH,-0-CH,-NH-C-ICH2) -S-S ^Νχ , CH , - 0 - CH , - C - NH-( CH , ) , n - C - N H - N Η , . 2Ί0 ~ . 2' -C-CH -N b L SO H II-../ 3 -C-C-H,-N b * -C=C-C=C-R, -C=C-CH = CRR', •CCH2Br, - C - C θ H 1 Q - C H 2 - Nv -NH-CO-CHj-Br, -NH-CO-CHjCl, ο II C-C6H10'CH2· Ο SO Η II- / 3 -c-(ch2)3-c6h4-n ο II C-CH2J, -(CH2)3SH, ο It -3)=CH2.

I -C=CRR CeH4CH2Br, OSifCH,), | 3 3 -C=CRR', -CH2Br, -CH2J, -CH = CH-CH2Br , -OSO2CgH;CH3. -SO2C1. -SOC1, II -C-Cl, 0 0 _ II II II /-N C-OR. -C'N-j. -C-N | . -CH = CH-CO2R, wherein R and R' are identical or different and in each case mean a hydrogen atom, a saturated or unsaturated C^C^-alkyl residue optionally substituted by a phenyl group, or a phenyl group. The -NCS, -NC>2, -OH, -NHNH2,-NHCOCH2Br, -NHCOCH2C1, -CO2H and -CON3 groups are especially preferred.

The residual acidic hydrogen atoms, i.e. those that have not been substituted by the central ion, can optionally be replaced entirely or in part by cations of inorganic and/or organic bases or amino acids.

The corresponding acid groups can also be entirely or partially converted into esters or amides.

IE 91162 Suitable inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion, the magnesium ion and especially the sodium ion.

Suitable cations of organic bases are, inter alia, those of primary, secondary or tertiary amines, such as, for example, ethanolamine, diethanolamine, morpholine, glucamine, Ν,Ν-dimethylglucamine, and, in particular, N-methylglucamine. Suitable cations of amino acids are, for example, those of lysine, of arginine, and of ornithine, as well as the amides of otherwise acidic or neutral amino acids.

Suitable esters are preferably those with a C^-Cg-alkyl residue; examples that can be mentioned are the methyl, ethyl and tert-butyl, benzyl and 4-methoxy15 benzyl residues.

If the carboxylic acid groups are to be present at least in part as amides, then suitable residues are saturated, unsaturated, straight- or branched-chain or cyclic hydrocarbons of up to 5 carbom atoms which are optionally substituted by 1-3 hydroxy or C1'-C4-alkoxy groups. Examples that can be mentioned are the methyl, ethyl, 2-hydroxyethyl, 2-hydroxy-l-(hydroxymethyl)ethyl, 1-(hydroxymethyl)ethyl, propyl, isopropenyl, 2-hydroxypropyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, butyl, isobutyl, isobutenyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-, 3- and 4-hydroxy-2-methylbutyl, 2and 3-hydroxyisobuty1, 2,3,4-trihydroxybutyl, 1,2,4trihydroxybutyl, pentyl, cyclopentyl and 2-methoxyethyl groups. The amide residue can also be a heterocyclic - or 6-membered ring formed with the inclusion of the amide nitrogen. Examples that can be cited are: the pyrrolidinyl, piperidyl, pyrazolidinyl, pyrrolinyl, pyrazolinyl, piperazinyl, morpholinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl rings.

IE 91162 The compounds of this invention exhibit the desired properties described in the foregoing. The complexes contain the metal ions, reguired for their usage, stably bound therein.

The value of the osmolality, responsible for side effects, such as pain, damage to the blood vessels, and cardiovascular disturbances, is markedly reduced as compared with MAGNEVIST· (compare Example lb: 0.55 (osmol/kg] with MAGNEVIST® 1.96 [osmol/kg], 0.5 mol/1 at 37 *C).

The value for the magnitude of relaxivity, representing a measure for imaging in MRI, is surprisingly high; the signal amplification in the plasma could be increased, for example in the case of the compound of Example lb, by twice the value as compared with MAGNEVIST®.

A further advantage of the present invention resides in that complexes with hydrophilic or lipophilic substituents have now become accessible. This affords the possibility of controlling the compatibility and pharmacokinetics of these complexes by chemical substitution.

By the choice of suitable bio- or macromolecules 2 (see farther below) in R f complexes of this invention are obtained which exhibit a surprisingly high tissue and organ specificity.

IE 91162 - 17 The compounds of this invention are produced by splitting off the blocking groups conventionally in compounds of general Formula I’ coor wherein * 21 12 G' , X and X in each case stand for G, X and X , the hydroxy groups and functional groups contained therein being present in the blocked form and, respectively, as a precursor, and Z' means a hydrogen atom or an acid blocking group,’ optionally generating the desired functional group; reacting, if desired, the thus-obtained complexing compounds of general Formula I wherein Z means hydrogen in a manner known per se with at. least one metal oxide or metal salt of an element of atomic numbers 21-29, 31, 32, 37-39, 42-44, 49 or 57-83; optionally binding the functional groups to a bioor macromolecule — wherein the complexing can take place before or after the splitting off of the blocking groups for the hydroxy groups and functional groups IE 91162 - 18 and/or generation of the functional groups and linkage to a macro- or biomolecule — and subsequently, if desired, substituting any still present acidic hydrogen atoms by cations of inorganic and/or organic bases, amino acids or amino acid amides or, respectively, converting the corresponding acid groups entirely or partially into esters or amides.

Suitable acid blocking groups Z’ are lower alkyl, aryl and aralkyl groups, e.g., the methyl, ethyl, 10 propyl, n-butyl, tertrbutyl, phenyl, benzyl, diphenylmethyl, triphenylmethyl, bis(p-nitrophenyl)methyl groups as well as trialkylsilyl groups.

Z' can also stand for an alkali metal.

The blocking groups are split off in accord15 ance with methods known to those skilled in the art, for example by hydrolysis, hydrogenolysis,, alkaline saponification of the esters with alkali in an aqueousalcoholic solution at temperatures of 0° to 50° C, acidic saponification with mineral acids or, in case of tert.-butyl esters, for example, with the aid of trifluoroacetic acid.

Suitable hydroxy blocking groups are, for example, the benzyl, 4-methoxybenzy1, 4-nitrobenzyl, trityl, diphenylmethyl, trimethylsilyl, dimethyl-tert. 25 butylsilyl, diphenyl-tert.-butylsilyl groups.

The hydroxy groups, can also be present, for example, as tetrahydrophyranyl (THP) ethers, oalkoxyethyl ethers, 2-methoxy-ethoxymethyl (MEM) ethers, or as esters with aromatic or aliphatic carboxylic acids such as, for example, acetic acid or benzoic acid. In case of polyols, the hydroxy groups can also be blocked in the form of ketals with, for example, acetone, acetaldehyde, cyclohexanone or benzaldehyde.

IE 91162 1' 2' The hydroxy groups present in X and X can also be present in blocked form by intramolecular esterification with the α-positioned carboxy groups to obtain the corresponding lactones.

The hydroxy blocking groups can be liberated in accordance with literature methods known to a person skilled in the art, for example by hydrogenolysis, reductive cleavage with lithium/ammonia, acid treatment of the ethers and ketals, or alkali treatment of the esters (see, for example, Protective Groups in Organic Synthesis, T.W. Greene, John Wiley and Sons 1981).

The synthesis of dimeric compounds, i.e. compounds containing a second macrocycle of general Formula II or IV, takes place according to methods dis15 closed in the literature, for example by way of an addition/elimination reaction of an amine with a carbonyl compound (e.g. acid chloride, mixed anhydride, activated ester, aldehyde); of two amine-substituted rings with a dicarbonyl compound (e.g. oxalyl chloride, glutaric dialdehyde); of two p-nitro-substituted nitroxides with bisalcoholates [cf. E. Klingsberg, The Chemistry of Heterocyclic Compounds, Interscience Publishers New York, p. 154 (1961) ] ; of two rings each exhibiting a nucleophilic group, with an alkylene compound carrying two leaving groups or, in case of terminal acetylenes, by oxidative coupling (Cadiot, Chodkiewicz in Viehe Acetylenes, 597-647, Marcel Dekker, New York, 1969). .

The ring-linking chain can subsequently be modified by secondary reactions (e.g. hydrogenation).

The synthesis of directly linked compounds (i.e. K meaning a direct bond, see Example 4) can be accomplished by cyclization of tetrahalogenomethyl-4,4'bispyridines (see further below).

IE 91162 - 20 The synthesis of the educts 1' takes place by alkylation of compounds of general Formula V wherein U U is hydrogen and V is an amino blocking group or represents amino blocking groups and V is hydrogen, wherein U and V can also be identical, with compounds of general Formula VI Nf-CH-COOZ (VI ) or with compounds of general Formula VII .,2' Nf-CH-COOZ (vii ), wherein Nf stands for a nucleofugal entity, such as, for example, Cl, Br, I, CH^-CgH^SO^, CH^SO^, 4-N°2-c6H4SO3, CF3SO3.

IE 91162 - 21 The hydroxy groups that may be contained in 1*2’ X and X can also form a lactone together with the 02' residue.

Examples for alkylating reagents &.re: bromo5 acetic acid, chloroacetic acid, bromoacetic acid methyl ester, bromoacetic acid tert,-butyl ester, fx chloroacetic acid benzyl ester, 2-chloro-3-benzyloxypropanoic acid sodium salt (EP 0,325,762), 2-bromo-3benzyloxypropanoic acid tert-butyl ester (J. Gen. Chem., USSR 36 : 52, 1966), 3,4-0-isopropylidene-2-p-tolylsulfonyl-3,4-dihydroxybutyric acid ethyl ester (Synth.

Comm. 19 : 3077, 1989), α-bromo-Y -butyrolactone.

Examples of amino blocking groups U and V, respectively, are: formyl, trifluoroacetate, benzoate, 4-nitrobenzoate, acetate, tosylate, mesylate, benzyl, 4-nitrobenzyl, 4-methoxybenzyl, trimethylsilyl, dimethyl-tert^butylsilyl. Γ Alkylation of the compounds of general Formula V to the educts of general Formula 1' with the compounds of general Formula VI or VII takes place in polar aprotic solvents, such as, for example, dimethylformamide, acetonitrile, dimethyl sulfoxide, aqueous tetrahydrofuran, dioxane, or hexamethyl phosphoric triamide in the presence of an acid captor, such as, for example, tertiary amine (e.g., triethylamine, trimethylamine, N,N-dimethylaminopyridine), 1,5diazabicyclo [. 4 . 3.0 ] nonene-5 (DBN), 1,5-diazabicyclo[5.4.o]undecene-5 (DBU), alkali, alkaline earth carbonate, bicarbonate or hydroxide (e.g., sodium, lithium, magnesium, calcium, barium, potassium carbonate, hydroxide and bicarbonate) at temperatures of between -10 °C and 120°C, preferably between O’C and 50°C, it being possible to add, if desired, catalytic amounts of iodide or bromide.

IE 91162 After splitting off the remaining amino blocking group(s) according to methods known to one skilled in the art (for example acidic or alkaline hydrolysis, hydrogenolysis, reductive cleavage with alkali metals in liquid ammonia, reaction with tetrabutylammonium fluoride), the remaining amino function(s) is or are reacted in a second alkylating reaction with VII and VI, respectively, so that compounds are obtained wherein φ X2.

The conversion of a precursor of the desired 6-membered ring contained in the final product takes place according to methods known to persons skilled in the art. Examples worth mentioning are the hydrogenation of pyridine [Advan. Catal. 14 : 203 (1963)], deoxygenation of nitroxide rings [E. Klingsberg, The Chemistry of Heterocyclic Compounds, vol. 14, part 2, Interscience Publishers New York, p. 120 (1961)], conversions and introduction of functional groups at the 6-membered ring, e.g. liberation of phenolic hydroxy groups [J. Org. Chem. 53 : 5 (1988)], introduction of halogen substituents [E. Klingsberg, The Chemistry of Heterocyclic Compounds, vol. 14, part 2, Interscience Publishers New York, p. 341 (1961); Houben-Weyl, Methoden der organischen Chemie, vo. V/3 : 651 (1962)].

Functionalization of 4-halopyridine derivatives (e.g. azide exchange) in the phase transfer process with the use of 18-crown-6 or tetrabutylammonium halogenide as the catalyst has been disclosed in Phase Transfer Reactions (Fluka Compendium vol. 2, Walter E. Keller, Georg Thieme publishers, Stuttgart, New York). A thus-obtained azide group can be converted into an amino function by using methods known to one skilled in the art (for example, catalytic IE 91162 hydrogenation, Houben-Weyl, Methoden der organischen Chemie vol. 11/1 : 539) or reaction with Raney nickel/ hydrazine (German Patent Application 3,150,917). This amino function can be transformed into an isothio5 cyanate group by means of methods known from the literature (e.g. with thiophosgene in a two-phase system, S. Scharma, Synthesis 1978 : 803; D.K. Johnson, J. Med. Chem. 1989, vol. 32, 236).

By reacting an amino function with a haloacetic 10 acid halogenide, an α-haloacetamide group can be generated (JACS 1969, vol. 90, 4508; Chem. Pharm. Bull. 29(1), 128, 1981), which is suitable, just as the isothiocyanate group, for example, for coupling to bioand macromolecules.

The synthesis of the compounds of general Formula V is conducted by cyclization according to methods known from the literature [for example, Org. Synth. 58 : 86 (1978), Macrocyclic Polyether Syntheses, Springer Publishers Berlin, Heidelberg, New York (1982); Coord. Chem. Rev. 2 : 3 (1968); Ann. Chem. 1976 : 916; J. Org. Chem. 49 : 110 (1984)]; one of the two reactants carries two leaving groups at the chain end, the other carries two nitrogen atoms, which displace these leaving groups in nucleophilic fashion.

An example that can be cited is the reaction of A -D -substituted diethylenetnammes, the terminalpositioned nitrogen atoms of which displace, in nucleophilic fashion, the leaving groups of, for. example, 2,6-dihalomethylpyridines, 2,6-ditosylmethylpyridines or 2,6-dimesylmethylpyridines. For the synthesis of directly linked dimers, 2,2’,6,6 *-tetrachloromethyl4,4 1-bispyridines (see, for example, Synthesis 552, 1989) are utilized in the cyclization reaction.

The nitrogen atoms are optionally blocked (for example as tosylates or trifluoroacetates) and are liberated prior to the subsequent alkylating reaction in accordance with methods known from the litera5 ture (the tosylates, for example, with mineral acids, alkali metals in liquid ammonia, hydrobrom|c acid and phenol, REDAL® (sodium-bis(2-methoxyethoxo) -aluminuradihydride), lithium aluminum hydride, sodium amalgam, compare, for example, Liebigs Ann. Chem. 1977. 1344; Tetrahedron Letters 1976; 3477; the trifluoroacetates, for example, with mineral acids or ammonia in methanol, compare, for example, Tetrahedron Letters 1967; 289) .

In order to prepare macrocycles with differing substitution on the nitrogen atoms, these atoms can be provided, in the educts, with different blocking groups, for example with tosylate and benzyl groups.

The latter are then likewise removed according to conventional methods disclosed in the literature (preferably by hydrogenation, e.g.^EP Patent Applica20 tion 232,751).

In case diesters are utilized in the cyclization reaction, the resultant diketo compounds must be reduced according to methods known to one skilled in the art, for example with diborane.

It is also possible to cyelize correspondingly substituted terminally positioned bisaldehydcs or bisketones, e.g.^2,6-bisacetylpyridines, with the respectively desired terminal-positioned bisamines; the reduction of the thus-obtained Schiff bases takes place according to methods known from the literature, e.g._ by catalytic hydrogenation IHelv. Chim. Acta 61 - 1376 (1978)).

The amines required as starting materials for the cyclization are produced in analogy to methods known from the literature (e.g., EP 299,795). Starting with an N-blocked amino acid, a triamine is obtained by reaction with a partially blocked diamine (for example according to the carbodiimide method), splitting off of the blocking groups, and diborane reduction.

Suitable substituents convertible into the functional group that can be linked to a macro- or bio10 molecule are, inter alia, hydroxy and nitrobenzyl, hydroxy and carboxyalkyl, as well as thioalkyl residues of up to 20 carbon atoms. They are converted, according to literature methods known to one skilled in the art [Chem. Pharm. Bull. 33 : 674 (1985); Compendium of Org. Synthesis vol. 1-5, Wiley and Sons, Inc.; HoubenWeyl, Methoden der organischen Chemie, vol. VIII, Georg Thieme publishers, Stuttgart; J. Biochem. 92 : 1413 (1982)], into the desired substituents (for example with the amino, hydrazino, hydrazinocarbonyl, epoxy, anhydride, methacryloylhydrazinocarbonyl, maleimidamidocarbonyl, halogeno, halogenocarbonyl, mercapto, isothiocyanate group as the functional group). In case of the nitrobenzyl residue, catalytic hydrogenation (e.g. according to P.N. Rylander, Catalytic Hydrogenation over Platinum Metals, Academic Press 1967) to the aminobenzyl derivative must first be performed.

Examples of conversion of hydroxy or amino groups linked to aromatic or aliphatic residues are the reactions performed in suitable solvents, such as tetrahydrofuran, dimethoxyethane or dimethyl sulfoxide, two-phase aqueous systems, such as, for example, water/dichloromethane, in the presence of an acid captor, such as, for example, sodium hydroxide, sodium hydride, or alkali or alkaline earth carbonates, such as, for example, sodium, magnesium, potassium, calcium carbonate or poly(4-vinylpyridine) REILLEX·, at temperatures between 0° C and the boiling point of the respective solvent, preferably, however, between 20° C and 60° C, with a substrate of general Formula VIII Nf - L - Fu (VIII) wherein L means an aliphatic, aromatic, arylaliphatic, branched, straight-chain or cyclic hydrocarbon residue of up to 20 carbon atoms, and Fu means the desired terminal-positioned functional group, optionally in the blocked form (DOS 3,417,413).

Examples of compounds of general Formula VIII 15 that can be cited are: Br(CHz)zNH2. Br{CH2)30H, BrCHjCOOCHj. BrCH2C02t-Bu. CICH2CONHNH2, Br(CH2);CO2CzHg, BrCH2COBr. BrCH2C0NH2. ClCl^COOCjHg , / \ BrCH2CONHNH2. BrCH2-CH-CH2. CFSOjtCH2IaBr. 8rCH2C=CH, BrCH2CH=CH?.

BrCH.C.II NCS . < o * Conversions of carboxy groups can be conducted, for example, according to the carbodiimide method (Fieser, Reagents for Organic Syntheses 10 : 142), by way of a mixed anhydride [Org. Prep. Proc. Int. Ί_ : 215 (1975)], or via an activated ester (Adv. Org. Chem. part B, 472).

The thus-obtained complexing ligands (as well as the complexes) can also be linked to bio- or macromolecules of which it is known that they are partic10 ularly accumulated in the organ or organ part to be examined. Such molecules are, for example, enzymes, hormones, polysaccharides, such as dextrans or amyloses, porphyrins, bleomycins, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides, such as polylysine, proteins (e.g. immunoglobulins, monoclonal antibodies, lectins), lipids (also in the form of liposomes), and nucleotides of the DNA or RNA type. Especially to be emphasized are conjugates with albumins, such as human serum albumin, antibodies, e.g. monoclonal antibodies specific for tumor-associated antigens, or antimyosin. In place of biological macromolecules, it is also possible to use suitable synthetic polymers for the linkage, such as polyethylenimines, polyamides, polyureas, poly25 ethers, such as polyethylene glycols and polythioureas. The pharmaceutical agents produced therefrom are suitable, for example, for use in tumor and infarction diagnostics as well as tumor therapy. Monoclonal antibodies (e.g. Nature 256 : 495, 1975) show the advantages over polyclonal antibodies that they are specific for an antigenic determinant, possess defined binding affinity, are homogeneous (thus substantially simplifying their preparation in pure form), and can be produced in cell cultures in large amounts.

Suitable as such are, for example for tumor imaging, monoclonal antibodies and/or their fragments Fab and F(ab')2 specific, for example, for human tumors of the gastrointestinal tract, the breast, the liver, the bladder, the gonads, and melanomas [Cancer Treatment Repts. 68 : 317 (1984); Bio. Sci. 34 : 150 (1984)], or act ing against carcinoembryonic antigen (CEA), human chorionic gonadotropin (8-HCG), or other tumorpositioned antigens, such as glycoproteins [New Engl. J. Med. 298 : 1384 (1973), U.S. Patent 4,331,647].

Suitable are also, inter alia, antimyosin, antiinsulin, and antifibrin antibodies (U.S. Patent 4,036,945).

Colon carcinomas can be detected with the aid of conjugates, complexed with gadolinium(III) ions, with the antibody 17-1A (Centocor, USA) by means of NMR diagnosis.

Suitable for liver examinations and, respectively, for tumor diagnosis are, for example, conjugates or inclusion compounds with liposomes which are used, for example, as unilamellar or multilamellar phosphatidylcholine-cholesterol vesicles .

In case of the antibody conjugates, binding of the antibody to the complex or ligand must not lead to loss or reduction of binding affinity and binding specificity of the antibody to the antigen. This can be accomplished either by binding to the carbohydrate portion in the Fc part of the glycoprotein and/or in the Fab or F(ab’)2 fragments, or by binding to sulfur atoms of the antibody or, respectively, the antibody fragments.

In the first instance, an oxidative cleavage of sugar units must first be performed for the generation of formyl groups capable of coupling. This oxidation can be carried out by chemical methods with oxidizing agents such as, for example, periodic acid, sodium metaperiodate, or potassium metaperiodate in accordance with methods known from the literature (e.g., J. Histochem. and Cytochem. 22 : 1084, 1974) in an aqueous solution in concentrations of 1 - 100 mg/ml, preferably 1-20 mg/ml, and with a concentration of the oxidizing agent of between 0.001 and 10 millimoles, preferably 1 to 10 millimoles, in a pH range of about 4 to 8 at a temperature of between 0° and 37° C and with a reaction period of between 15 minutes and 24 hours. The oxidation can also be performed by enzymatic methods, for example with the aid of galactose oxidase in an enzyme concentration of 10 - 100 units/ml, a substrate concentration of 1 - 20 mg/ml, at a pH of 5 to 8, a reaction period of 1 - 8 hours, and a temperature of between 20° and 40° C (for example, J. Biol. Chem. 234 : 445, 1959).

Complexes or ligands with suitable functional groups, such as, for example hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide, are bound to the aldehydes generated by oxidation; this is done by reacting between 0° and 37° C with a reaction period of 1-65 hours, a pH of between about 5.5 and 8, an antibody concentration of 0.5 - 20 mg/ml, and a molar ratio of the complexing compound to the antibody aldehyde of 1 : 1 to 1000 : 1. The subsequent stabilization of the conjugate takes place by reduction of the double bond, for example with sodium borohydride or sodium cyanoborohydride; the reducing agent is utilized herein with a 10- to 10035 fold excess (e.g. J. Biol. Chem. 254 : 4359, 1979).

•E 91162 The second possibility of forming antibody conjugates starts with a gentle reduction of the disulfide bridges of the immunoglobulin molecule; in this process, the more sensitive disulfide bridges between the H chains of the antibody molecule are cleaved whereas the S-S bonds of the antigen-binding region remain intact so that there is practically no reduction in binding affinity and specificity of the antibody (Biochem. 18 : 2226, 1979; Handbook of Experimental Immunology, vol. 1, 2nd ed., Blackwell Scientific Publications, London 1973, chapter 10). These free sulfhydryl groups of the inter-H-chain regions are then reacted with suitable functional groups.of complexing compounds or metal complexes at 0-37° C, a pH of about 4-7, and a reaction period of 3 - 72 hours with the formation of a covalent bond which does not affect the antigen binding region of the antibody. Suitable reactive groups are, for example: haloalkyl, haloacetyl, p-mercuribenzoate, isothiocyanate, thiol, epoxy groups, as well as groups to be subjected to a Michael addition reaction, such as, for example, maleinimides, methacrylo groups (e.g. J. Amer. Chem. Soc. 101 : 3097, 1979).

Additionally, for linking the antibody fragments with the polymer complexes or with the ligands, there is a number of suitable bifunctional linkers which are frequently also obtainable commercially (see, for example, Pierce, Handbook and General Catalogue 1986) which are reactive with respect to the SH groups of the fragments as well as with respect to the amino or hydrazino groups of the complexes.

Examples that can be cited are: m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N-sulfosuccinimide ester (Sulfo-MBS), N-succinimidyl-[4-(iodoacetyl)amino]benzoic acid ester (SIAB), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylic acid ester (SMCC), succinimidyl-4-(p-maleimidophenyl)butyric acid ester (SMPB), N-succinimidyl-3-(2-pyridyldithio)propionic acid ester (SDPD), 4-[3-(2,5-dioxo-3-pyrrolinyl)propionyloxy]-3-oxo2,5-diphenyl-2,3-dihydrothiophene-l,1-dioxide, acetylalanylleucylalanylaminobenzyl, acetamido-p-thioureidobenzy1.

It is also possible to utilize bonds not of the covalent type for coupling purposes wherein ionic as well as van der Waals and hydrogen bridge bonds can contribute toward the linkage in varying proportions and strengths (key and lock principle) (for example, avidin-biotin, antibody-antigen). Also inclusion compounds (host-guest) of relatively small complexes in relatively large cavities in the macromolecule are possible.

The coupling principle resides in first producing a bifunctional macromolecule by either fusing an antibody hybridoma directed against a tumor antigen with a second antibody hybridoma directed against a complex according to this invention, or linking the two antibodies chemically via a linker (e.g. in the way set forth in J. Amer. Chem. Soc. 101 : 3097, 1979) or binding the antibody directed against the tumor antigen to avidin (or biotin, respectively) , optionally via a linker [D.J. Hnatowich et al., J. Nucl. Med. 28 : 1294 (1987)]. In place of the antibodies, it is also possible to employ their corresponding F(ab) or F(ab')2 fragments.

For pharmaceutical usage, first the bifunctional macromolecule is injected which is accumulated at the target site, and then, at a time interval, the complex compound of this invention is injected [optionally bound to biotin (or avidin)] which is coupled on at the target site in vivo and there can deploy its diagnostic or therapeutic activity. Moreover, other coupling methods can likewise be utilized, such as, for example, reversible radiolabeling described in Protein Tailoring Food Med. Uses [Am. Chem. Soc. Symp. 349 (1985)].

A particularly simple method for the production of antibody conjugates or antibody fragment conjugates is available in the form of the so-called solid phase coupling procedure: The antibody is coupled to a stationary phase (e.g. an ion exchanger) located, for example, in a glass column. By successive flushing of the column with a solution suitable for generation of aldehyde groups, washing, rinsing with a solution of the functionalized complex, and finally elution, of the conjugate, very high yields of conjugate are obtained.

This procedure permits the automatic and continuous production of any desired quantities of conjugates.

Also other coupling steps can be performed in this way.

Thus, for example, fragment conjugates can be prepared by the sequence of papain reduction/bifunctional linker/functionalized complex or ligand.

The thus-formed compounds are subsequently purified preferably by chromatography by way of ion exchangers on a fast protein liquid chromatography unit.

The metal complexes of this invention are produced as disclosed in German Laid-Open Application 3,401,052 by dissolving or suspending the metal oxide or a metallic salt (e.g. the nitrate, acetate, carbonate, chloride or sulfate) of the element of atomic numbers 21-29, 42, 44, 57-83 in water and/or in a lower alcohol (such as methanol, ethanol or isopropanol), and reacting with a solution or suspension of the equivalent amount of the complexing ligand and subsequently, if desired, substituting any acidic hydrogen atoms present by cations of inorganic and/or organic bases or amino acids.

Introduction of the desired metal ions can take place in this process before as well as after the splitting off of the blocking groups for the hydroxy groups and functional groups, or, respectively, before or after the generation of the functional groups and linkage to a macro- or biomolecule.

Neutralization of any free carboxy groups still present takes place with the aid of inorganic bases (e.g. hydroxides, carbonates, or bicarbonates) of, for example, sodium, potassium, lithium, magnesium or calcium and/or organic bases, such as, inter alia, primary, secondary and tertiary amines, e.g. ethanolamine, morpholine, glucamine, N-methyl- and Ν,Ν-dimethylglucamine, as well as basic amino acids, such as, for example, lysine, arginine and ornithine, or of amides from originally neutral or acidic amino acids.

In order to prepare the neutral complex compounds, it is possible, for example, to add to the acidic complex salts in an aqueous solution or suspension such an amount of the desired bases that the neutral point is obtained. The resultant solution can then be concentrated to dryness under vacuum. It is frequently advantageous to precipitate the thusformed neutral salts by adding water-miscible solvents, e.g. lower alcohols (methanol, ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane and others) and to obtain in this way crystallized products which can be easily isolated and readily purified. It proved to be especially advantageous to add the desired base as early as during the complex formation to the reaction mixture and thereby to save a process step.

In case the acidic complex compounds contain several free acidic groups, it is frequently expedient to produce neutral mixed salts containing inorganic as well as organic cations as the counterions.

This can be done, for example, by reacting the complex forming ligand in an aqueous suspension or solution with the oxide or salt of the element yielding the central ion, and with half the amount of an organic base required for neutralization; isolating the thus-formed complex salt; purifying same if desired; and then combining, for complete neutralization, with the needed amount of inorganic base. The sequence of addition of the bases can also be reversed.

Another possibility of obtaining neutral complex compounds resides in converting the remaining acid groups in the complex entirely or partially into esters or amides, for example. This can be done by subsequent reaction at the finished complex (e.g. by exhaustive reaction of the free carboxy groups with dimethyl sulfate).

The conjugates of antibody and complex are dialyzed, prior to in vivo use, after incubation with a weak complexing agent, such as, for example, sodium citrate, sodium ethylenediaminetetraacetic acid, in order to remove weakly bound metal atoms.

The pharmaceutical agents of this invention are likewise produced in a manner known per se by suspending or dissolving the complex compounds of this invention — optionally combined with the additives customary in galenic pharmacy — in an aqueous medium and then optionally sterilizing the suspension or solution. Suitable additives are, for example, physiologically acceptable buffers (such as, for instance, tromethamine), additions of complexing agents (e.g.^ diethylenetriaminepentaacetic acid) or -if required — electrolytes, e.g.^sodium chloride or if necessary — antioxidants, such as ascorbic acid, for example.

If suspensions or solutions of the agents of this invention in water or physiological saline solution are desirable for enteral administration or other purposes, they are mixed with one or several of the auxiliary agents (e.g.) methylcellulose, lactose, mannitol) and/or tensides (e.g., lecithins, TWEEN*, -5 MYRJ*) and/or flavoring agents to improve taste (e.g., ethereal oils), as customary in galenic pharmacy.

In principle, it is also possible to produce the pharmaceutical agents of this invention without isolating the complex salts. In any event, special $0 care must be taken to effect chelate formation so that the salts and salt solutions according to this IE 91162 • - 37 invention are practically devoid of uncomplexed, toxically active metal ions.

This can be ensured, for example, with the aid of dye indicators, such as xylenol orange, by control titrations during the manufacturing process. Therefore, the invention also concerns processes for the production of the complex compounds and their salts. A final safety measure resides in purifying the isolated complex salt.

The pharmaceutical agents of this invention preferably contain 0.1 μmol - 1 mol/1 of the complex salt and are normally made into doses in amounts of 0.1 pmol- 5 mmol/kg. They are intended for enteral and parenteral administration. The complex compounds according to this invention are utilized (1) for NMR and X-ray diagnostics in the form of their complexes with the ions of the elements with atomic numbers 21-29, 42, 44 and 57-83; (2) for radiodiagnostics and radiotherapy in the form of their complexes with the radioisotopes of the elements with atomic numbers 27, 29, 31, 32, 37-39, 43, 49, 62, 64, 70, 75 and 77.

The agents of this invention meet the variegated requirements for being suitable as contrast media for nuclear spin tomography. Thus, they are excellently suited for improving the informative content of the image obtained with the aid of the NMR tomograph upon oral or parenteral administration, by increasing the signal intensity. Furthermore, they exhibit the high efficacy necessary to introduce into the body a minimum amount of burdening foreign substances, and they show the good compatibility required for maintaining the noninvasive character of the examinations.

The good water solubility and low osmolality 5 of the compounds of this invention make it possible to prepare highly concentrated solutions, thus maintaining the volume load on the circulation within tolerable limits and compensating for dilution by body fluid, i.e., NMR diagnostic aids should exhibit 100 - 1,000 times the water solubility of agents for NMR spectroscopy. Furthermore, the agents o£ this invention exhibit not - only a high in vitro stability but also a surprisingly high stability in vivo so that release or exchange of the ions — actually toxic — not bound in a covalent fashion in the complexes takes place only extremely gradually within the time period during which the , novel contrast media are again completely eliminated.

In general, the agents of this invention are used, for NMR diagnostic aids, in doses amounting to 0.0001 - 5 mmol/kg, preferably 0.005 - 0.5 mmol/kg. Details of use are discussed, for example, in H.J.

Weinmann et al., Am. J. of Roentgenology 142 : 619 (1984 ) .

Especially low doses (below 1 mg/kg body weight) of organ-specific NMR diagnostic aids are usable, for example, for the detection of tumors and of cardiac infarction.

Furthermore, the complex compounds according to this invention can be employed with advantage as susceptibility reagents and as shift reagents for in vivo NMR spectroscopy.

The agents of this invention, based on their favorable radioactive properties and good stability of the complex compounds contained therein, are also suited as radiodiagnostic agents. Details of their usage and dosage are described, for example, in ’’Radiotracers for Medical Applications", CRC Press, Boca Raton, Florida.

Another imaging method with radioisotopes is the positron emission tomography, using positron43 44 emitting isotopes, such as, for example, Sc, Sc, eo 55 6 fi Fe, . Co and Ga (Heiss, W.D.; Phelps, M.E. : Positron Emission Tomography of Brain, Springer publishers, Berlin, Heidelberg, New York 1983).

The compounds of this invention can also be utilized in radioimmuno- or radiation therapy. This process differs from the corresponding diagnostics only in the quantity and type of isotope employed.

The objective herein is the destruction of tumor cells by high-energy shortwave radiation with a minimum range 46 47 Suitable β-emittmg ions are, for example Sc, Sc, 48 72 73 90 Sc, Ga, Ga and Y. Suitable α-emitting ions exhibiting short half-life periods are, for example, 211Bi, 212Bi, 213Bi and 214Bi, wherein 212Bi is preferred. A suitable ion emitting photons and electrons is 158Gd which can be obtained from 157Gd by neutron capture.

If the agent of this invention is intended for use in the version of radiation therapy proposed by R.L. Mills et al. (Nature, vol. 336 : 787 (1988)(, then the central ion must be derived from a Mdssbauer isotope, such as, for example, 57Fe or 151Eu.

In the in vivo administration of the therapeutic agents according to this invention, they can be given together with a suitable carrier, e.g. serum or physiological sodium chloride solution and together with another protein, such as, for example, human serum albumin. The dosage herein is dependent on the type of cellular disorder, the metal ion used, and the type of imaging method, e.g. brachytherapy.

The therapeutic media of this invention are administered parenterally, preferably intravenously.

Details of usage of radiotherapeutic agents are discussed, for example, in R.W. Kozak et ai., TIBTEC, October 1986, 262.

The agents of this invention are excellently suited as X-ray contrast media; in this connection, it is to be especially emphasized that they reveal no indication of anaphylaxis-type reactions, known from iodine-containing contrast media, in biochemicalpharmacological studies. They are particularly valuable, on account of the favorable absorption properties in regions of higher tube voltages, for digital subtraction techniques.

In general, the agents of this invention are utilized, for administration as X-ray contrast media, analogously to, for example, meglumine diatrizoate, in doses amounting to 0.1 - 5 mmol/kg, preferably 0.25 - 1 mmol/kg.

Details of utilization of X-ray contrast media are discussed, for example, in Barke, Rflntgenkontrastmittel (X-Ray Contrast Media], G. Thieme, Leipzig (1970) and P. Thurn, E. Bilcheler, EinfCLhrung in die Rfintgendiagnostik [Introduction to X-Ray Diagnostics], G. Thieme, Stuttgart, New York (1977).

IE 91162 - 41 In summation, the synthesis has been accomplished of novel complexing compounds, metal complexes and methal complex salts, opening up new possibilities in diagnostic and therapeutic medicine.

This development appears to be desirable, above all in light of the evolution of novel imaging methods in medical diagnostics.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications, if any, cited above and below, and of corresponding application German P 40 01 655.2, filed January 18, 1990, are hereby incorporated by reference.

IE 91162 - 42 Example 1 (a) 3,6,9-Tris[dihydro-2(3H)-furanon-3-yl]-3, 6, 9, 15tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene g (242.38 millimoles) of α-bromo- y-butyro5 lactone is added to 10 g (48.48 mmol) of 3,6,9,15tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene, 33.5 g (242.38 mmol) of potassium carbonate and 805 mg (4.85 mmol) of potassium iodide in 200 ml of acetonitrile. This mixture is heated under reflux for 48 hours, evaporated to dryness under vacuum, and the residue is taken up in 500 ml of methylene chloride and extracted three times with 150 ml of water. The organic phase is dried over magnesium sulfate and evaporated under vacuum. The residue is purified by chromatography on silica gel. (Mobile phase: methylene chloride/methanol = 15:1) Yield: 7.11 g (32% of theory) of a slightly yellow-colored oil which solidifies when allowed to stand.

Analysis: C 60.25 H 6.59 N 12.22 (Calcd.) C 60.18 H 6.64 N 12.17 (Found) (b) Gadolinium Complex of 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-tris25 [a-(2-hydroxyethyl)acetic Acid] 6.7 g (14.61 mmol) of the title compound of Example 1(a) is dissolved in 50 ml of deionized water, and the pH is brought to 5.5 by adding 1-normal hydrochloric acid. To this mixture is added 2.65 g IE 91162 - 43 (7.3 mmol) of gadolinium oxide and the mixture is refluxed for 3 hours. The cooled solution is stirred for one hour with respectively 10 ml of acidic ion exchanger (IR 120) and 10 ml of alkaline ion exchanger (IRA 410). The mixture is filtered off from the exchanger, and the filtrate is boiled for one hour with active carbon. After filtration and freeze-drying, 9.25 g (95% of theory) of an amorphous, colorless powder is obtained (containing 8.3% of water per analysis).

Analysis (corrected for water): C 41.43 H 4.99 N 8.40 Gd 23.58 (Calcd.) C 41.35 H 5.09 N 8.34 Gd 23.50 (Found) (c) Europium Complex of 3,6,9,15-Tetraazabicyclo15 [9.3.1]pentadeca-1(15),ll,13-triene-3,6,9tris[a-(2-hydroxyethyl)acetic Acid] Analogously, the corresponding Europium complex is obtained with Analysis (corrected for water): C 41.76 H 5.03 N 8.47 Eu 22.97 (Calcd.) C 41.68 H 5.12 N 8.39 Eu 22.88 (Found) Example 2 (a) 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-tris[a-(benzyloxymethyl) acetic Acid] g (48.48 mmol) of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene and 114.71 g (484.8 mmol) of the sodium salt of 2-chloro-3-benzyloxy propionic acid in 200 ml of water are heated to 70° C for 48 hours. The solution is diluted with 400 ml of water and 300 ml of 2N hydrochloric acid is added thereto. The mixture is extracted 5 times with respectively 200 ml of methylene chloride. The aqueous phase is evaporated under vacuum. The residue is dissolved in 300 ml of ethanol and filtered off from the sodium chloride. Then the mixture is evaporated under vacuum and the remaining oil is chromatographed on silica gel (mobile phase: ethanol/water.= 20:1).

The main fractions are evaporated under vacuum and dissolved in 50 ml of 5% strength hydrochloric acid. The solution is passed over a column, filled with Reillex (= poly-4-vinylpyridine), and the product is eluted with a mixture of water/methanol 3:1.

After evaporation of the main fractions, 12.93 g (36% of theory) of a strongly hygroscopic solid is obtained (9.1% water per analysis).

Analysis (corrected for water): C 66.47 H 6.53 N 7.56 (Calcd.) C 66.38 H 6.60 N 7.48 (Found) methyl)acetic Acid] 12.6 g (17.01 mmol) of the title compound of 5 Example 2(a) is dissolved in a mixture of 200 ml of mefchanol/100 ml of water, and Λ ij of palladium catalyst is added (10% Pd on active carbon). The mixture is hydrogenated for 5 hours at 50° C, filtered off from the catalyst, and evaporated under vacuum.

Yield: 7.84 g (98% of theory) of a vitreous solid (6.9% water per analysis).

Analysis (corrected for water, : C 51.06 II 6.43 N 11.91 (Calcd.) C 50.97 H 6.51 N 11.81 (Found) 15 (c) Gadolinium Complex of 3,6,9,15-Tetraa2abicyclo- [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-trisla- (hydroxymethyl) acetic Acid] 7.5 g (15.94 mmol) of the title compound of Example 2(b) is dissolved in 50 ml of deionized water, and 2.89 g (7.97 mmol) of gadolinium oxide is added.

The mixture is heated for 3 hours at 90° C. The cooled solution is stirred for one hour at room temperature with respectively 2 ml of acidic ion exchanger (IR 120) and 2 ml of alkaline exchanger (IRA 410), filtered off from the exchanger, and the filtrate is briefly boiled with active carbon. After filtration and freezedrying, 9.56 g (96% of theory, of a colorless, amorphous powder is obtained (8.1% water per analysis,.

Analysis (corrected for water): C 38.45 H 4.36 N 8.97 Gd C 38.37 H 4.43 N 8.89 Gd .17 .06 (Calcd.) (Found) Example 3 (a) 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-tris[a-(1,2-O-isopropylidene-1,2-dihydroxyethyl)acetic Acid Ethyl Ester] A mixture of 15 g (72.71 mmol) of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene, 156.38 g (426.28 mmol) of 3,4-0-isopropylidene-2(p-tolylsulfonyl)-3,4-dihydroxybutyric acid ethyl ester, 60.3 g (436.28 mmol) of potassium carbonate, and 2.41 g (14.54 mmol) of potassium iodide in 400 ml of acetonitrile is heated under reflux for 48 hours.

The mixture is evaporated under vacuum and the residue taken up in 500 ml of methylene chloride. The mixture is extracted 3 times with 200 ml of water and the organic phase is dried over magnesium sulfate. After evaporation, the remaining oil is chromatographed on silica gel (mobile phase: methylene chloride/hexane/ methanol = 20:4:1).

Yield: 17.24 g (31% of theory) of a yellow, viscous oil.

Analysis: 59.67 59.59 7.91 7.98 7.32 7.27 (Calcd.) (Found) (b) 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-tris[a-(1,2-dihydroxyethyl)acetic Acid] 16.5 g (21.57 mmol) of the title compound of 5 Example 3(a) is dissolved in 100 ml of ethanol, and ml of 5N sodium hydroxide solution is added thereto. The mixture is heated under reflux for 10 hours and evaporated under vacuum. The residue is dissolved in 250 ml of methanol and filtered off from the sodium chloride. The filtrate is evaporated under vacuum and the residue purified on an ion exchanger as follows: The product is dissolved in 50 ml of water, and the solution is introduced into a cation exchange column (IR 120). After flushing with water, the ligand is eluted with 0.5N aqueous ammonia solution. The main fractions are evaporated, taken up in a small amount of water and passed over an ion exchange column (IRA 67). The product is first washed with water and then eluted with 0.5N formic acid. The mixture is evaporated under vacuum and the residue dissolved in a small quantity of hot methanol. By the gentle addition of acetone and cooling in an ice bath, the title compound is obtained in crystalline form.

Yield: 8.22 g (68% of theory) of a vitreous solid (9.2% water per analysis).

Analysis (corrected for water): C 49.28 C 49.17 H 6.47 H 6.56 N 9.99 N 9.88 (Calcd.) (Found) (c) Gadolinium Complex of 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-tris[a-(1,2-dihydroxyethyl)acetic Acid] g (14.27 mmol) of the title compound of 5 Example 3(b) is dissolved in 60 ml of deionized water, and 2.58 g (7.135 mmol) of gadolinium oxide is added. The mixture is heated for 3 hours at 90° C. The cooled solution is stirred for one hour at room temperature with respectively 2 ml of acidic ion exchanger (IR 120) and 2 ml of alkaline exchanger (IRA 410). The mixture is removed from the exchanger by filtration and the filtrate boiled with active carbon. After filtration and freeze-drying, 9.89 g (97% of theory) of a colorless, amorphous powder is obtained (containing 7.3% water per analysis).

Analysis (corrected for water): C 38.65 H 4.65 N 7.84 Gd 22.00 (Calcd.) C 38.54 H 4.74 N 7.78 Gd 21.92 (Found) Example 4 (a) 2,2’,6,6'-Tetra(hydroxymethyl)-4,4'-bipyridine g (128.77 mmol) of 2,2',6,6’-tetra(methoxycarbonyl) -4 , 4 ’ -bipyridine is dissolved in a mixture of 400 ml of dioxane/400 ml of water and, in portions, 48.71 g (1.28 mol) of sodium borohydride is added thereto. The mixture is stirred overnight at room temperature. The solution is acidified with 5N hydrochloric acid and evaporated to dryness. The residue is suspended in 1 liter of IN sodium hydroxide solution and extracted three times with 250 ml of chloroform. The organic phases are dried over magnesium sulfate and evaporated under vacuum. The residue is recrystallized from ethanol/ether.

Yield: 29.53 g (83% of theory) of colorless crystals Analysis : C 60.86 H 5.84 N 10.14 (Calcd.) C 60.77 H 5.93 N 10.06 (Found) (b) 2,21,6,6'-Tetra(chloromethyl)-4,4'-bipyridine 29 g (104.96 mmol) of the title compound of Example 4(a) is heated under reflux for 5 hours in 250 g (2.1 mol) of thionyl chloride. The mixture is evaporated to dryness and the residue taken up in 200 ml of concentrated soda solution. The mixture is extracted twice with 150 ml of methylene chloride.

The organic phase is dried over magnesium sulfate and evaporated under vacuum. The residue is crystallized from ether/hexane.

Yield: 35.54 g (94% of theory) of colorless crystals 20 Analysis: C 48.03 H 3.45 N 8.08 Gd 40.51 (Calcd.) C 48.10 H 3.40 N 7.96 Gd 40.59 (Found) IE 91162 - 50 (c) 13,13'-Bis[3,6,9-tris(p-tolylsulfonyl)-3,6,9,15tetraazabicyclo [9 . 3.1]pentadeca-1(15),11,13-triene] At 100° C, a solution of 34 g (97.12 mmol) of the title compound of Example 4(b) (dissolved in 700 ml of dimethylformamide) is addded dropwise within 4 hours to 118.43 g (194.25 mmol) of N,N',N-tris(p-tolylsulfonyl) diethylenetriamine-Ν,Ν'’-disodium salt in 1600 ml of dimethylformamide. The mixture is stirred overnight at 100° C. Two liters of water are added dropwise to the hot solution, and the latter is allowed to cool down to 0° C. The precipitate is suctioned off and washed with water. After drying under vacuum (60° C), the product is recrystallized from aceto- nitrile, thus obtaining 79.13 g (61% of theory) of a cream-colored powder. Analysis: C 57.55 H 5.28 N 8.39 S 14.40 (Calcd.) C 57.47 H 5.35 N 8.'13 S 14.32 (Found) id) 13,131-Bis[3,6,9,15-tetraazabicyclo(9.3.1]penta20 deca-1(15),11,13-triene] Octahydrosulfate g (59.15 mmol) of the title compound of Example 4(c) is introduced into 270 ml of concentrated sulfuric acid and stirred for 48 hours at 100° C, then cooled to 0° C, and 1.35 1 of absolute ether is added dropwise. The precipitate is suctioned off and extracted by stirring in 500 ml of methanol. After the product has been filtered off and dried under vacuum, 65.74 g (93% of theory) of a solid is obtained which deliquesces in the air.

Analysis: C 22.11 H 4.22 N 9.38 S 21.46 (Calcd.) C 22.04 H 4.33 N 9.29 S 21.38 (Found) (e) 13,13'-Bis[3,6,9,15-tetraazabicyclo[9.3.1]penta5 deca-1(15),11,13-triene] 65.5 g (54.80 mmol) of the title compound of Example 4(d) is dissolved in 100 ml of water and the pH is adjusted to 13 with 32% strength sodium hydroxide solution. The mixture is extracted three times with 250 ml of hot toluene. The combined toluene phases are heated under reflux for one hour with 20 g of finely pulverized sodium hydroxide. The mixture is filtered and the filtrate evaporated to dryness.

Yield: 21.6 g (96% of theory) of a solid having a slightly yellow color.

Analysis: C 64.36 H 8.35 N 27.29 (Calcd.) C 64.27 H 8.44 N 27.22 (Found) (f) 13,13'-Bis[3,6,9,15-tetraazabicyclo[9.3.1]penta20 deca-l(15),11,13-triene-3,6,9-tris[a-(benzyloxymethyl) acetic Acid]] 21.5 g (52.37 mmol) of the title compound of Example 4(e) and 247.8 g (1.05 mol) of the sodium salt of 2-chloro-3-benzyloxypropionic acid in 400 ml of water are heated for 48 hours to 70° C. The solution is diluted with 800 ml of water and combined with 600 ml of 2N hydrochloric acid, then extracted 5 times with respectively 300 ml of methylene chloride, and the aqueous phase is evaporated under vacuum. The residue is dissolved in 500 ml of ethanol and filtered off from the sodium chloride. The mixture is evaporated under vacuum and the residue chromatographed on silica gel (mobile phase: ethanol/water = 20:1).

The main fractions are evaporated under vacuum and dissolved in 100 ml of 5% strength hydrochloric acid.

The solution is passed over a column filled with Reillex (= poly-4-vinylpyridine), and the product is eluted with a mixture of water/methanol 2:1. After evaporation of the main fractions, 20.92 g (27% of theory) of a strongly hygroscopic solid is obtained (8.1% water per analysis).

Analysis (corrected for water): C 66.56 H 6.40 N 7.57 (Calcd.) C 66.47 H 6.51 N 7.48 (Found) (g) 13,13’-Bis[3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1 (15),11,13-triene-3,6,9-tris[a-(hydroxymethyl) acetic Acid]] .5 g (13.85 mmol) of the title compound of 20 Example 4(f) is dissolved in a mixture of 300 ml of methanol/150 ml of water, and 7 g of palladium catalyst (10% Pd on active carbon) is added thereto. The mixture is hydrogenated for 5 hours at 50° C. The product is removed from the catalyst by filtration and evaporat25 ed under vacuum.

Yield: 12.62 g (97% of theory) of a vitreous solid (8.5% water per analysis).

Analysis (corrected for water): C 51.17 H 6.23 N 11.93 (Calcd.) C 51.07 H 6.31 N 11.87 (Found) (h) Gadolinium Complex of 13,13’-Bis[3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene3,6,9-tris[a-(hydroxymethyl)acetic Acid]] g (12.78 mmol) of the title compound of 5 Example 4(g) is dissolved in 80 ml of deionized water, and 4.63 g (12.78 mmol) of gadolinium oxide is added.

The mixture is heated for 3 hours at 90° C. The drawnoff solution is stirred for one hour at room temperature with respectively 5 ml of acidic ion exchanger (IR 120) and 5 ml of alkaline exchanger (IRA 410). The product is filtered off from the exchanger, and the filtrate is briefly boiled with active carbon. After filtration and freeze-drying, 15.3 g (96% of theory) of a colorless, amorphous powder is obtained (containing 9.3% of water per analysis).

Analysis C 38.52 (corrected for N water) 8.98 Gd 25.21 (Calcd.) H 4.20 C 38.46 H 4.28 N 8.91 Gd 25.14 (Found) Example 5 (a) Trans-5-(p-tolylsulfonyl)amino-6-(p-tolylsulfonyloxy)-2,2-dimethyl-1,3-dioxepane Under agitation, 295.67 g of p-toluenesulfochloride is added in portions at -5° to 0° C to a solution of 100 g of trans-6-amino-2,3-dimethyl-l,325 dioxepan-5-ol in 903 ml of pyridine. The mixture is allowed to stand for 72 hours at +4° C and then stirred into 10 1 of ice water. After the precipitate has been suctioned off and washed with water, the residue is dried in a drying cabinet at 50° C and 200 torr for 48 hours. For purposes of purification, the crude product is recrystallized from 5 1 of dioxane, thus obtaining 196 g of the title compound as a white powder, mp 200-202° C. (b) Monosodium Salt of N-[2-(N-Tolylsulfonylamino)ethyl]-p-tolylsulfonamide 150 g of N-[2-(N-tolylsulfonylamino)ethyl]-ptolylsulfonylamide is suspended in 1.25 1 of ethanol, heated under reflux, and a solution of 10.3 g of sodium in 300 ml of ethanol is added dropwise thereto, thus forming a solution. During cooling, the title compound is precipitated, suctioned off, the precipitate washed with ethanol and dried at 50° C and 200 torr. Yield: 119 g of the title compound as a white powder. (c) cis-2,2-Dimethyl-5-[N-(p-tolylsulfonyl)amino]-6[N-(p-tolylsulfonyl)-Ν-(N’-2-p-tolylsulfonylaminomethyl)]-1,3-dioxepane 116 g of the monosodium salt of Example 5(b) is suspended in 2.66 1 of dimethylformamide. At 100° C, a solution of 141 g of trans-5-(p-tolylsulfonyl)amino6-(p-tolylsulfonyloxy)-2,2-dimethyl-l,3-dioxepane in 1.5 1 of dimethylformamide is added dropwise thereto and the mixture is stirred for 5 hours at a bath temperature of 120° C. The reaction solution is then concentrated under vacuum to 1 liter and diluted with 10 1 of ice water, suctioned off, the precipitate is washed with water and dried at 50° C and 200 torr, and the product is 182 g of the crude title compound. For purification, the product is extracted by boiling with 1.85 1 of ethanol. After suctioning off and drying, 125 g of the title compound is obtained as a white powder, mp 190-194° C. (d) Disodium Salt of cis-2,2-Dimethyl-5-[N-(p-tolylsulfonyl)amino]-6-[N-(p-tolylsulfonyl)-N-(N'-2p-tolylsulfonylaminomethyl)]-1,3-dioxepane 87.8 g of the compound obtained according to 5 Example 5(c) is suspended in 410 ml of ethanol, heated to boiling, and a solution of 6.67 g of sodium in 200 ml of ethanol is added dropwise thereto. The mixture is cooled in an ice bath, combined with 450 ml of ether, and suctioned off from the precipitate, which latter is dried at 80° C and 200 torr, thus producing 91 g of the title compound as a white powder. (e) Acetonide of 4,5-Bis(hydroxymethyl)-3,6,9-tritosylsulfonyl-3,5,9,15-tetraazabicyclo[9.3.1]pentadeca-1 (15) ,11,13-triene 78.75 g of the disodium salt of Example 5(d) is dissolved in 880 ml of dimethylformamide, heated to 100° C, and a solution of 19.53 g of bis (2,6-chloromethyl)pyridine in 360 ml of dimethylformamide is added dropwise thereto; the mxiture is heated for 5 hours to 120° C and concentrated under vacuum to 300 ml. The solution is stirred into 5 1 of ice water, the precipitate is suctioned off, washed with water, and dried. The crude product is recrystallized from 700 ml of dioxane, thus obtaining 45 g of the title compound as a white powder, mp 244-250° C. (f) Acetonide of 4,5-Bis(hydroxymethyl)-3,6,9,15-tetra azabicyclo[9.3.1]pentadeca-1(15),11,13-triene A suspension of 20 g of the compound obtained according to Example 5(e) in 140 ml of tetrahydrofuran is added to 260 ml of liquid ammonia; the mixture is stirred in a refrigerating bath at -50° C, and a total of 14.4 g of sodium is introduced in portions. The mixture is stirred for another 5 hours at -60° C, then the refrigerating bath is removed, and 50 ml of ethanol is added dropwise. The ammonia is allowed to evaporate the mixture is evaporated to dryness under vacuum, and the residue is purified by chromatography on silica gel. Elution with chloroform/ethanol/concentrated ammonia solution (3/1/0.5) yields 5.30 g of the title compound as an oil.

Analysis: C 62.72 H 8.55 N 18.29 (Calcd.) C 62.51 H 8.41 N 18.45 (Found) (g) Acetonide of 4,5-Bis(hydroxymethyl)-3,6,9,1520 tetraazabicyclo[9.3.1]pentadeca-1(15),11,13triene-3,6,9-tris(acetic Acid tert-Butyl Ester) .51 g of anhydrous sodium carbonate and 10.2 g of bromoacetic acid tert-butyl ester are added to a solution of 4 g of the compound produced according to Example 5(f) in 100 ml of tetrahydrofuran and 10 ml of water and the mixture is stirred for 5 hours at 50° C. The mixture is filtered, evaporated under vacuum, and the oily residue is stirred with 50 ml of hexane and decanted. The residue is purified by chromatography on 100 g of silica gel with dichloromethane (1-10% ethanol), thus obtaining 5.7 g of the title compound as a light-yellow oil. (h) 4,5-Bis(hydroxymethyl)-3,6,9,15-tetraaza5 bicyclo[9.3.1]pentadeca-1(15),11,13-triene3,6,9-tris-acetic Acid A mixture of 5.3 g of the ester prepared according to Example 5(g) and 50 ml of trifluoroacetic acid is stirred for 3 hours at 50° C. Then 10 ml of water is added, the mixture is stirred for another 2 hours at 50° C, and thereafter evaporated to dryness under vacuum. The residue is dissolved in 20 ml of water, and the solution is allowed to pass through a column with 100 ml of Reillex (poly-4-vinylpyridine), eluted with 100 ml of water, and the eluate is evap- orated under vacuum, thus obtaining an amorphous powder which still contains 8.5% water. Yield: 2.90 g· Analysis : C 51.81 H 6.41 N 12.72 (Calcd.) C 51.63 H 6.70 N 12.51 (Found) (i) Gadolinium Complex of 4,5-Bis(hydroxymethyl)3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-tris-acetic Acid g (water content 8.5%, corresponding to 1.83 g = 4.29 mmol) of 4,5-bis(hydroxymethyl)-3,6,9,15tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene3,6,9-tris-acetic acid (production see Example 5h) and 778 mg of gadolinium oxide are stirred for 5 hours with 50 ml of water at 90° C. After cooling, the mixture is stirred in succession with respectively 10 ml of anion exchanger IRA 410 and cation exchanger IRC 50 filtered, and the solution is subjected to freeze5 drying, thus obtaining 2.35 g of the title compound as a loose white powder, water content, according to K.-Fischer titration: 7.3%.

Analysis (after correction for water content): C19H25GdN4°8 38.38 38.51 4.24 4.31 9.42 9.36 Gd Gd 26.44 26.19 (Calcd.) (Found) Example 6 (a) Acetonide of 4,5-Bis(hydroxymethyl)-3,6,9-tris[dihydro-2-(3H)-furanon-3-yl]-3,6,9,15-tetraaza15 bicyclo[9.3.1]pentadeca-1(15),11,13-triene A solution of 5 g (16.34 mmol) of acetonide of 4,5-bis(hydroxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene in 100 ml of acetonitrile is combined with 12 g of potassium carbonate, 260 mg of potassium iodide and 13.50 g of a-bromo- V-butyro20 lactone and heated for 48 hours to boiling. The mixture is then evaporated under vacuum, the residue is dissolved in methylene chloride, shaken several times with water, the organic phase is dried over sodium sulfate, and evaporated to dryness. The oily residue is chromatographed with methylene chloride/methanol (15:1) on 150 g of silica gel, thus obtaining 5.3 g of the title compound as a light-yellow, viscous oil. (b) 4,5-Bis(hydroxymethyl)-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene3,6,9-tris[a-(2-hydroxyethyl)]acetic Acid g of the compound produced according to 5 Example 6(a) is dissolved in 50 ml of water and adjusted to a pH of 2 by addition of hydrochloric acid. The mixture is heated for 5 hours under reflux, cooled to room temperature, and the solution is allowed to pass over a column with 10 g of Reillex (poly-4-vinylpyridine). The column is rinsed with ml of water and the combined eluates are subjected to freeze-drying, thus obtaining 4.05 g of the title compound as a loose powder with a water content of 7.2%.

Analysis (after correction for the water content): C25H40N4°ll C 52.44 H 7.04 N 9.78 (Calcd.) C 52.61 H 7.33 N 9.62 (Found) (c) Gadolinium Complex of 4,5-Bis(hydroxymethyl)20 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-tris[a-(2-hydroxyethyl) ] acetic Acid 1.50 g (2.68 mmol) of 4,5-bis(hydroxymethyl)3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),11,1325 triene-3,6,9-tris[a-(2-hydroxyethyl)]acetic acid is stirred in 25 ml of water with 487 mg of gadolinium oxide for 4 hours at 90° C. After cooling, the solution is stirred in succession with 5 ml of anion exchanger IRA 410 and 5 ml of cation exchanger IRC 50, filtered, and subjected to freeze-drying, thus obtaining 1.69 g of the title compound as a white content): (Calcd.) (Found) Example 7 (a) 4-Hydroxymethyl-3,6,9-tritosyl-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene powder with a water content of 4.3%.

Analysis (after correction for the water C25H37GdN4°ll 41.31 41.07 .13 .33 7.71 7.61 Gd 21.63 Gd 21.89 A solution of 59.57 g of 3-aza-l-hydroxymethyl1,3,5-tritosylpentadiamine (preparation see International Patent Application PCT/DE 88/00200, WO 88/08422, page 45) in 500 ml of dimethylformamide is combined, in portions, with 9.60 g of a 50% strength suspension of sodium hydride in mineral oil, and the mixture is heated for one hour to 80° C. To this solution is added dropwise 17.61 g of 2,6-bis(chloromethyl) pyridine dissolved in 150 ml of dimethylformamide, and the mixture is heated for 6 hours to 110° C. The mixture is concentrated under vacuum to about 220 ml, and 1 liter of water is added dropwise. The precipitate is suctioned off, washed with water, and dried overnight at 50° C and 200 mbar. The crude product is recrystallized from 500 ml of ethanol and yields 45 g of the title compound as a yellow solid. (b) 4-Hydroxymethy1-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene g of the compound obtained according to Example 7(a) is heated with 120 ml of concentrated sulfuric acid for 48 hours to 100° C. The mixture is cooled to 0° C, and 350 ml of diethyl ether is added dropwise. The salt of the title compound is thus precipitated. The mixture is suctioned off, the residue is dissolved in 100 ml of water and combined with 40 g of sodium hydroxide and extracted repeatedly with dichloromethane. The combined organic phases are dried over sodium sulfate and evaporated under vacuum, thus obtaining 13.3 g of the title compound as a viscous oil. (c) 4-Hydroxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic Acid g of the amine prepared according to Example 7 (b) is dissolved in 100 ml of water and combined with 13.21 g of chloroacetic acid. The mixture is stirred for 5 hours at 60° C, maintaining the pH during this time at 9.0 by addition of 10N sodium hydroxide solution. The mixture is cooled to 0° C, combined with 100 ml of ethanol, and acidified with concentrated hydrochloric acid to pH 1. The thus25 formed precipitate is suctioned off, dissolved in 50 ml of water, and the solution is passed over a column with 30 ml of Reillex (poly-4-vinylpyridine), rinsed with 50 ml of water, the eluates are combined and subjected to freeze-drying. Yield: 16.8 g of the title compound as an amorphous powder with a water content of 9.3%.

IE 91162 Analysis (after correction for the water content): (Calcd.) (Found) (d) Gadolinium Complex of 4-Hydroxymethyl-3,6,9,15tetraazabicyclo [9.3.1]pentadeca-1(15),11,13triene-3,6,9-triacetic Acid C18H26N4°7 52.68 52.49 6.39 6.54 13.65 13.81 A solution of 2.3 g of 4-hydroxymethyl-3,6,9,15tetraazabicyclo [9 . 3 . 1] pentadeca-1 (15) ,11,13-triene in ml of water is combined with 1.015 g of gadolinium oxide and heated for one hour to 90° C. The product is removed from a small amount of unreacted oxide by filtration, and the solution is allowed to run in succession over respectively 10 ml of anion ex15 changer IRA 410 and cation exchanger IRC 50, rinsed with 30 ml of water, and the combined eluates are subjected to freeze-drying. Yield: 3.05 g of the title compound as a powder with a water content of 7.5%.

Analysis (after correction for the water content): C 38.29 H 4.11 N 9.92 Gd 27.85 (Calcd.) C 38.44 H 4.32 N 9.68 Gd 27.71 (Found) IE 91162 Example 8 (a) 4-Benzyloxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic Acid A solution of 10 g ( = 24.36 mmol) of 4-hydroxy5 methyl-3,6,9,15-tetraazabicyclo[9.3.ljpentadeca1(15),11,13-triene-3,6,9-triacetic acid (prepared according to Example 7b) in 150 ml of dimethylformamide is combined with 0.5 g of potassium iodide, 4.17 g of benzyl bromide, and 5 g of sodium carbonate. The mix10 ture is heated for 20 hours to 60° C, concentrated under vacuum, combined with 100 ml of water and 300 ml of ethanol, and the pH is set at 2 by addition of concentrated hydrochloric acid. The mixture is suctioned off from the precipitate, the precipitate is dissolved in 100 ml of water, and the solution is passed over a column with 50 g of Reillex (poly-4-vinylpyridine).

The column is washed with 50 ml of water and the combined aqueous phases are subjected to freeze-drying, thus obtaining 8 g of the title compound as an amorphous powder.

Analysis : C 59.99 H 6.44 N 11.19 C 59.71 H 6.49 N 11.38 (Calcd.) (Found) (b) Gadolinium Complex of 4-Benzyloxymethyl3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-triacetic Acid 1.81 g of gadolinium oxide is added to a solution 5 of 5 g of 4-benzyloxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid in 100 ml of water; the mixture is heated for 3 hours to 80-90° C, filtered, and the solution is allowed to pass in succession over columns with re10 spectively 15 ml of anion exchanger IRA 410 and cation exchanger IRC 50. The columns are rinsed with 75 ml of water, and the combined aqueous phases are subjected to freeze-drying, thus obtaining 5.85 g of the title compound as an amorphous powder, water content 7.4%.

Analysis (after correction for water content): C 45.86 H 4.46 N 8.56 Gd 24.02 (Calcd.) C 45.69 H 4.71 N 8.72 Gd 23.81 (Found) Example 9 Gadolinium Complex of 3,6,9-Tetraazabicyclo[9.3.1]- pentadecane-3,6,9-tris[a-(hydroxymethyl)acetic Acid] 4.5 g (7.2 mmol) of the title compound of Example 2(c) is dissolved in 150 ml of deionized water and hydrogenated in an autoclave over a rhodium catalyst (5% Rh/C) at 30 bar and 40° C. After 12 hours, the product is filtered off from the catalyst and the filtrate is stirred with respectively 3 ml of cation exchanger (IR 120) and 3 ml of anion exchanger (IRA 410) for one hour. The product is removed from the exchanger by filtration and freeze-dried.

Yield: 4.18 g (92% of theory) of a colorless, amorphous powder (containing 6.7% water per analysis). Analysis (corrected for water) : C 30.08 H 5.27 N 8.88 Gd 24.93 (Calcd.) 5 C 30.01 H 5.34 N 8.78 Gd 24.86 (Found) Example 10 Preparation of a Solution of the Gadolinium(III) Complex of 3,6,9 , 15-Tetraazabicyclo [9.3.1] pentadeca-1 (15) ,11,13triene-3,6,9-tris fa-(2-hydroxyethyl)acetic Acid (a) 361 g (0.5 mole, of the complex obtained according to Example 1(b) (water content: 8.3%) is dissolved in 500 ml of water pro injectione (p.i.) with slight heating. After adding 0.8 g of tromethamine, the solution is filled up with water p.i. to 1000 ml.

The solution is subjected to ultrafiltration and dispensed into bottles. After heat-sterilization, the solution is ready for use for diagnostic purposes by parenteral administration. (b) The solution obtained by ultrafiltra20 tion according to Example 10(a) is filled under sterile conditions into multivials and lyophilized. After adding the desired amount of water p.i., the administration dose suitable for intrastitial injection for radiation therapy is obtained.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and sdope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims (23)

1. WHAT IB CLAIMED IS: 1A macrocyclic compound of Formula I wherein Q X 1 n m R 1 X 2 k € and q R 2 A 1 , cooz is a single or double bond; is a N atom or NH, is H, -(CH 2 ) n -R 1 or - (CH) n -CH 2 OH; OH is 1, 2, 3, 4, or 5; is 0, 1 or 2; is H or OH; is X 1 or -(CH 2 ) n -(O) r (CH 2 ) k -(C 0 H 4 ) q -R 2 ; is 0, 1, 2, 3 or 4; are each independently 0 or 1; is H, C^C^-alkoxy, a functional group, or a bio- or macromolecule bound via a functional group; B 2 , C 1 , C 2 , D 1 , D 2 , Ε 1 , E 2 , F 1 and F 2 are each independently X 2 ; is R or a second macrocycle, bound via K, of general Formula II cooz K is a direct bond, a bis(carbonyl-amino)group (-NH—CO-CO-NH-), or a C 1 -C 14 -alkylene group which optionally carries at the ends carbonyl (-CO-) groups, carbonylamino (-NH-CO-) groups or oxygen atoms, and which optionally contains one or several oxygen atoms(s), hydroxymethylene (-CHOH-), CH(X 2 )COOZsubstituted imino, acyl-substituted imino and/or hydroxy-acyl-substituted imino groups, and or one to two C-C-double and/or C-C-triple bonds; and 2Is H and/or a metal ion equivalent of an element of atomic number 21-29, 31, 32, 37-39, 42-44, 49 or 57-83; wherein any or all remaining COjH groups can optionally be present as an ester or amide; with the provisos that the 12 ring substituents A 1 through F 2 stand for at least 8 hydrogen atoms, X 1 and X 2 stand simultaneously for hydrogen only if at least one of the ring substituents A 1 through F 2 is not H, and that the macrocycle of general Formula I contains no more than one bio- or micromolecule; and physiologically acceptable salts thereof with inorganic and/or organic bases, amino acids or amino acid amides.

2. A macrocyclic compound of claim 1, wherein said compound is of the Formula III cooz wherein --- is a single or double bond? Q is a N atom or NH, X 1 is Hr -(CH^-R 1 or - 2 ) m -(CH) n -CH 2 OH; OH n m R 1 X 2 k I and q R 2 A 1 , G C 1 is l, 2, 3, 4, or 5? is 0, 1 or 2; is H or OH? is x 1 or -(0^-(0)^-(0^,,-(0^)^: is 0, l, 2, 3 or 4; are each independently 0 or l? is H, ^-C^-alkoxy, a functional group, or a bio- or macromolecule bound via a functional group ? and D 1 , are each independently X 2 ; is R 2 or a second macrocycle, bound via K, of general Formula IV IE 91162 COOZ K is a direct bond, a bis(carbonyl-amino)group (-NH-CO-CO-NH-) , or a C 1 -C 14 -alkylene group which optionally carries at the ends carbonyl (-CO-) groups, carbonylamino (-NH-CO-) groups or oxygen atoms, and which optionally contains one or several oxygen atoms(s), hydroxymethylene (-CHOH-), CH(X z )COOZsubstituted imino, acyl-substituted imino and/or hydroxy-acyl-substituted imino groups, and or one to two C-C-double and/or C-C-triple bonds; and z is H and/or a metal ion equivalent of an element of atomic number 21-29, 31, 32, 37-39, 42-44, 49 or 57-83; wherein any or all remaining CO 2 H groups can optionally be present as an ester or amide; with the provisos that X 1 and X 2 stand simultaneously for hydrogen only if at least one of the ring substituents A 1 through D 1 is not H, and physiologically acceptable salts thereof with inorganic and/or organic bases, amino acids or amino acid amides. IE 91162

3. A compound according to claim 1, wherein Z is H.

4. A compound according to claim 1, wherein at least 2 of the substituents Z are metal ion equivalents of at least one element of atomic numbers 2129, 42, 44 or 57-83, or of at least one radionuclide of an element of atomic numbers 27, 29, 31, 32, 37-39, 43, 49, 62, 64, 70 ΟΓ 77.

5. A compound according to claim 1, wherein K is -(O) q -(CH 2 ) n - 2 ) k -(CH 2 ) n -(O)< or a direct bond. OH

6. A compound according to claim 1, wherein R 2 is a functional group and said functional group is NCS, NOj, OH, NHNH ? , NH 2 , NHCOCHjBr, NHCOCH 2 Cl, CO 2 H, or CONj.

7. A compound according to claim 1, wherein the bio- or macromolecule optionally contained in R 2 is an antibody or antibody fragment.

8. A compound according to claim 1, wherein the bio- or macromolecule optionally contained in R 2 is a protein.

9. A compound according to claim 8, wherein said protein is albumin, globulin or lectin.

10. A compound according to claim 1, wherein the bio- or macromolecule optionally contained in R 2 is a polysaccharide.

11. A compound according to claim 10, wherein said polysaccharide is amylose, dextran or dextrin. IE 91162 - 71

12. A compound according to claim 1, wherein X 1 is CH 2 OH, CH 2 CH 2 OH, or CHOHCH Z OH.

13. A compound according to claim 2, wherein X z , A 1 , B 1 , C 1 and/or D 1 each independently are CH 2 OH, CH 2 CH 2 OH, CH 2 OCH 2 C 6 H 5 , CHOHCH 2 OH, CH z C 6 H 4 OCH 3 , CH 2 C 6 H5, CH 2 C 6 H 4 O(CH 2 ) 3 COOH, or ch 2 c 6 h 4 ncs.

14. A method of NMR diagnostics, X-ray diagnostics, radiodiagnostics , radioimmunotherapy or I irradiation therapy comprising administering an agent wherein said agent contains at least one physiologically compatible compound of any of claims 1 to 13.

15. A process for the production of a macrocyclic compound of general Formula I wherein is a single or double bond; Q is a N atom or NH, X 1 is H, -(CH 2 ) n -R’ or - (CH Z ) m - (CH) n -CH 2 OH; I OH n is 1, 2 , 3, 4, or 5; m is 0, 1 or 2; R 1 * IS Η , or OH; X 2 is X 1 or -(CH 2 ) r -(O) e -(CH 2 ) k -(C 4 H 4 ) q -R 2 ; k is Ο, 1, 2, 3 or 4; t and g are each independently 0 or 1; R 2 is H, Cj-c^-alkoxy, a functional group, or a bio- or macromolecule bound via a functional group; A 1 , A 2 , Β 1 , B 2 , C 1 , C 2 , D 1 , D 2 , Ε 1 , E 2 , F 1 and F 2 are each independently X 2 ; G is R 2 or a second macrocycle, bound via K, of general Formula II K is a direct bond, a bis(carbonyl-amino) group (-NH-CO-CO-NH-) , or a C 1 -C 14 -alkylene group which optionally carries at the ends carbonyl (-CO-) groups, carbonylamino (-NH-CO-) groups or oxygen atoms, and which optionally contains one or several oxygen atoms(s), hydroxymethylene (-CHOH-), CH(X 2 )COOZsubstituted imino, acyl-substituted imino and/or hydroxy-acyl-substituted imino groups, and or one to two C-C-double and/or C-C-triple bonds; and Z is H and/or a metal ion equivalent of an element of atomic number 21-29, 31, 32, 37-39, 42-44, 49 or 57-83; wherein any or all remaining CO 2 H groups can optionally be present as an ester or amide; with the provisos that the 12 ring substituents A 1 through F 2 stand for at least 8 hydrogen atoms, X 1 and X 2 stand simultaneously for hydrogen only if at least one of the ring substituents A through F is not H, and that the macrocycle of general Formula I contains no more than one bio- or micromolecule; and physiologically acceptable salts thereof with inorganic and/or organic bases, amino acids or amino acid amides, said process comprising splitting off the blocking groups conventionally from a compound of general Formula 1' cooz wherein G', X 1 * and x 2 ' in each case stand for G, X* and x 2 , the hydroxy groups and functional groups contained therein being present in the blocked form and, respectively, as a precursor, and Z' means a hydrogen atom or an acid blocking group; optionally generating the desired functional group; optionally reacting the thus-obtained complexing compounds of general Formula I wherein z means hydrogen in a manner known per se with at least one metal oxide or metal salt of an element of atomic numbers 21-29, 31, 32, 37-39, 42-44, 49 or 57-83; optionally binding the functional groups to a bio- or macromolecule; and subsequent optionally substituting any still present acidic hydrogen atoms with cations of inorganic and/or organic bases, amino acids or amino acid amides; and/or converting corresponding acid groups entirely or partially into esters or amides; wherein the complexing can take place before or after the splitting off of the blocking groups for the hydroxy groups and functional groups and/or generation of the functional groups and linkage to a macro- or biomolecule.

16. A pharmaceutical agent comprising at least one compound according to any of claims 1 to 13 and a pharmaceutically acceptable carrier.

17. An agent according to claim 16, further comprising at least one additive customary in galenic pharmacy.

18. A pharmaceutical agent according to claim 16, wherein said at least one compound according to claim 1 is in the form of liposomes.

19. A process for the production of.a pharmaceutical agent according to claim 16, comprising dissolving or suspending the complex compound in water or a physiological saline solution, to bring it into a form suitable for enteral or parental administration, said agent optionally containing additives customary in galenic pharmacy. -7520.

20.A compound substantially as hereinbefore described with reference to the Examples.

21. A process substantially as hereinbefore described with reference to the Examples.

22. A method substantially as hereinbefore described with reference to the Examples.

23. A pharmaceutical agent substantially as hereinbefore described with reference to the Examples.