MANGANESE CHELATES WITH HIGH RELAXIVITY IN SERUM

Abstract: Compounds of formula (I) wherein: R1, R2 which are the same or different, are a hydrogen atom, or a -(CH2)m-O-(CH2)n-R3 group, wherein R3 is an aryl residue, m is an integer 1 to 5 and n is an integer 1 to 4, with the proviso that R1 and R2 cannot be at the same time hydrogen, as well as the optically active forms of the chelates thereof with Mn(II) and the salts thereof with physiologically compatible organic bases selected from primary, secondary, tertiary amines or basic amino acids, or with inorganic bases whose cations are sodium, potassium, magnesium, calcium, or mixtures thereof, are disclosed.

Description: MANGANESE CHELATES WITH HIGH RELAXIVITY IN SERUM The present invention relates to the field of the diagnostic technique known as Magnetic Resonance Imaging (M.R.I.) , a diagnostic procedure used in medical practice to detect anomalies in organs or tissues of the human or animal body (see: Rocklage S.M., Watson A.D. and Carvlin M.J., Magnetic Resonance Imaging, Chap. 14, Vol .1 , Second Ed., 1992; Stark D.D. and Bradley W.G. Eds.). Particularly, the invention relates to manganese chelated complexes of ethylenediaminotetraacetic acid derivatives, the physiologically compatible salts thereof and the use of these compounds as M.R.I, contrast agents. The potential usefulness of the Mn 2+ ion as an M.R.I, contrast agent has been taken in consideration since 1978 for myocardium imaging. In Magnetic Resonance in Medicine 1988, 8 , 293-313, the longitudinal proton relaxation times in rabbit tissues after intravenous administration of MnCl 2 and of Mn-PDTA (1,3- propylenediamino-N,N' ,N' ' ,N' ' ' -tetraacetate) have been evaluated. Manganese complexes of porphyrins and derivatives thereof (for example Mn (III) -meso-tetra (4- solfonatophenyDporphyrin, Mn-TPPS 4 ) were proposed as contrast agents specific for tumors (Investigative Radiology 1995, 30 (10 ) , 611-620). In a recent study, M.R.I. contrast agents were evaluated consisting of Mn-EDTA lipohilic derivatives (for example, manganese-EDTA-bis (hydroxypropylde- cylamine) , Mn-EDTA-DDP) bound to the membranes of small unilamellar liposomes ("memsomes"), which are potentially valuable for hepatic and cardiac perfusion imaging (Journal of Liposome Research 1994, 4 (2) , 811-834). The same compounds are object of International Patent 2 Application WO 92/21017 and of US 5,312,617, which disclose M.R.I. contrast agents based on manganese chelates (II) consisting of EDTA bis-amides where the amide nitrogens are substituted with long chain alkyl residues (C^-C^ Q ) . These chelates per se or, more preferably, in combination with lipids or liposomes, are reportedly particularly useful for imaging of liver and as blood-pool agents. M.R.I, contrast agents comprising manganese chelated complexes are also described in US patents 4,980,148 and 5,246,696. Said complexes, in which the ligand is an alkylenediaminotetraacetic acid in which the alkylene chain is interrupted by one or more substituents selected from 0, S, CHOH, CHSH, are stated to be particularly useful for imaging of liver, kidneys, pancreas and gastrointestinal tract. Recently, Teslascan (R) (manganese complex of N,N'- 1, 2-ethanediylbis [N- [ [3-hydroxy-2-methyl-5- [ (phosphono- oxy) methyl] -4 -pyridinyl] -methyl] lycine salified with Na + (1:3), mangafodipir trisodiu , was marketed in Europe and in the U.S.A. for use in M.R.I, of the liver. This compound was considered useful for M.R.I, diagnosis of pancreas adenocarcinoma and pancreatitis (Investigative Radiology 1995, 30 (10) , 611-620). Contrary to the Gd(III) ion (at present the M.R.I, contrast agent of choice) and, more generally, to the lanthanides ions, manganese is an essential oligoelement present in all mammal cells: this is undoubtedly an advantage as for the tolerability of the corresponding complexes. However, the manganese ion can cause toxicity when released from the corresponding chelates in vivo; this mainly applies when the manganese ion is administered intravascularly, as in this case the natural 3 regulation of its adsorption by the intestine fails. It is therefore necessary for it to be administered as stable complex, in order to prevent any toxicity due to the free metal and to improve its elimination. Some compounds of the above cited literature, for example Mn-DPDP, show some instability in vivo : a recent biodistribution study proved that this complex dissociates, releasing manganese which accumulates in liver, pancreas and kidneys, whereas the undissociated chelate is removed through glomerular filtration. The M.R.I. properties of Mn-DPDP would therefore be ascribable mainly to the manganese ion released by the complex, which accumulates in liver and pancreas (Investigative Radiology 1994, 29 (2) , S249-S250). Another recent study gave evidence of the dissociation of Mn-DPDP in liver homogenate, in the presence of calcium and magnesium ions (MRM 1996, 35, 14-19) . Besides the problem of the in vivo stability, a further problem of manganese chelated complexes is that of relaxivity, that is usually lower than the better studied and developed gadolinium contrast agents. At present, in the field of M.R.I, contrast agents, there are no valuable manganese contrast agents having good stability as well as high relaxivity. The compounds of the present invention solve this problem in the field of M.R.I, diagnostic imaging. A class of compounds selected from EP 230 893, more precisely a class of Mn(II) chelates, has, in fact, shown some unexpected properties which make them valuable for use in this field. The present invention relates to stable manganese chelated complexes in which said ion is in the oxidation state +2 (Mn(II) ) . 4 More particularly, the invention relates to Mn(II) chelates of compounds of formula (I) : COOH R. ( . ISL OOH HOOC N ' ) R, HOOC ( I ) wherein : R-^ , R 2 which are the same or different, are a hydrogen atom, or a - (CH 2 ) m ~0- (CH 2 ) n -R 3 group, wherein R 3 is an aryl residue, m is an integer 1 to 5 and n is an integer 1 to 4 , with the proviso that R-^ and R 2 cannot be at the same time hydrogen, as well as the optically active forms of the chelates thereof with Mn(II) and the salts thereof with physiologically compatible organic bases selected from primary, secondary, tertiary amines or basic amino acids, or with inorganic bases whose cations are sodium, potassium, magnesium, calcium, or mixtures thereof. Suitable substances for salifying the chelates of the invention are, for example: cations of inorganic bases such as alkali or alkaline-earth metals ions selected from sodium, potassium, magnesium, calcium, or mixtures thereof; cations of physiologically compatible organic bases selected from primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N, N-dimethylglucamine; - cations of amino acids such as those of lysine, arginine or ornithine. Particularly preferred are N-methylglucamine salts. In the compounds of formula (I) particularly preferred meanings for R^ and R 2 are the following: <^ H 3 C- ^ (CH 2 ) n -O-(CH 2 ) r (CH 2 ) π -O-(CH 2 ) m - ^^ <HO): (CD; (CH 2 ) n -O-(CH 2 ) r (CH 2 ) n -O-(CH 2 ) m - (CH 2 ) n -0-(CH 2 ) r (CH 2 ) n -0-(CH 2 ) r HOOC, HO- (CH 2 ) π -O-(CH 2 ) m - (CH 2 ) π -O-(CH 2 ) m - wherein: x = 1,2. Particularly preferred compounds within formula (I) , are those of formula (II) : R— (CH 2 ) n — 0-(CH 2 )— CH-COOH • N COOH COOH ( ii : COOH wherein R 4 is a phenyl, unsubstituted or substituted with one or more R^ groups, which are the same or different, wherein Rr- is halogen, -OH, -NH 2 , -COOH, -CONH 2 , -S0 3 H, -S0 3 NH 2 , or a C 1 -C 4 alkyl chain; and those of formula (III) R-(CH 2 )-0-(CH 2 )— CH-COOH COOH COOH " N R 4 — (CH 2 )-0-(CH 2 )-CH-COOH ( II I ) wherein the R 4 groups, which are the same or different, have the same meaning as defined for compounds of formula (II) . In compounds of formulae (II) and (III) , preferred meanings for R 4 are the following ones: rf"^ .^ (Br) wherein: x = 1,2. Within formula (I) , particularly preferred compounds are those of formula (IV) R 4 -N o .COOH N. .COOH HOOC N HOOC (IV) 7 wherein R 4 is as defined for compounds (II) , and (V) R, O COOH ,N^^,COOH HOOC N HOOC (V) wherein the R 4 groups, which are the same or different, have the same meanings as defined for compounds (II) . In compounds of formulae (IV) and (V) , preferred meanings for R 4 are as follows: Br Particularly preferred is the unsubstituted phenyl residue . Among the possible manganese chelates included within formula (I), particularly preferred are those having as ligands the compounds shown hereinbelow: 8 COMPOUND 1 (EXAMPLE 1) ,COOH ^^ ^N COOH COOH COOH COMPOUND 2 (EXAMPLE 2) ^ - Q - COOH ^ N^COOH N^COOH 'COOH The present invention also relates to the use of the Mn^+ complex chelates of the chelating compounds of formulae (I) to (V) and to the salts thereof, for the preparation of pharmaceutical compositions for diagnostic use, as well as the formulations themselves. The compounds of the invention can conveniently be used as they are or they can be conjugated with macromolecules or incorporated in structures which carry them to specific body regions. The compounds of the invention can be formulated with additives conventionally used for the pharmaceutical or veterinary formulations, for example stabilizers, antioxidants, osmolality and pH adjusters, buffers and the like. For the parenteral administration, they are preferably formulated as sterile aqueous solutions or suspensions, whose pH can range from 6.0 to 8.5. Said formulations can also be lyophilized and supplied as such for reconstitution before use. For the gastrointestinal use or for the injection into body 9 cavities, these agents can be formulated as a solution or suspension containing suitable additives in order to, for example, control viscosity. For the oral administration they can be formulated according to preparation methods routinely used in the pharmaceutical technique, optionally also as coated formulations to gain extra protection from the acid pH of stomach, thereby inhibiting the release of the chelated metal ion, which usually occurs at the typical pH values of gastric juices. Other excipients, such as sweeteners and/or flavours, can also be added according to known techniques of pharmaceutical technique. The solutions or suspensions of the compounds of this invention can also be formulated as aerosol for use in aerosol -bronchography. Contrary to the manganese chelated complexes of the prior art cited above, the compounds of the invention have shown characteristics such as good tolerability, good stability both in vi tro and in vivo and surprisingly high r-L and, particularly, r 2 relaxivities in human serum. The relevant data are summarized in Tables 1, 2 and 3. These characteristics make the compounds of the invention particularly interesting as M.R.I. contrast agents of general use. The chelates of the invention have also shown a good applicability in the imaging of the liver (Table 4) and of the biliary tracts, which organs are reached by said chelates without significant losses of the chelated metal, thus confirming their characteristics of stability. This is a further aspect of the present invention. Contrary to the manganese chelated complexes of the 10 prior art, which, as mentioned above, are generally considered particularly useful in the imaging of the liver, the pancreas and the gastrointestinal tract, the chelates of the present invention, thanks to their surprising relaxivity values in human serum, can advantageously improve the images obtainable using said compounds in formulations specific for the imaging of the cardiocirculatory system. This is a further aspect of the present invention. Furthermore, the chelated complexes of the present invention are characterized in having particularly high transversal relaxivity values (r 2 ) in human serum. This characteristic makes them unexpectedly unique compared with the general teaching of EP 230 893, that mainly focuses on the effects of the compounds on relaxation times T- j _ . This makes them most suitable, and this is a further aspect of the present invention, for use in M.R.I, diagnostic imaging in recording images of organs or tissues, wherein said images are acquired by combinations of T τ . / T 2 sequences or by T 2 weighed sequences . According to the present invention, said chelated complexes proved to be particularly suitable also in the preparation of pharmaceutical formulations for M.R.I. diagnostic imaging at low fields, i.e. fields ranging from 0.1 to 0.5 Tesla or, when measuring the intensity of the applied magnetic field in Hertz, fields below 20 MHz (An introduction to Magnetic Resonance in Medicine, Ed. Peter A. Rinck) . The compounds of the invention, thanks to their excellent relaxivity shown even at such low fields, can advantageously be used with apparatuses such as Artroscan (arthrographs which make use of the Magnetic resonance), open apparatuses, apparatuses intended for 11 diagnostic of single regions of the human body (e.g. the shoulder), and generally apparatuses less expensive, easier to handle and increasingly diffused. Furthermore, and this is a further aspect of the present invention, the considerable increase in relaxivity in human serum shown by the compounds of the invention allows the use thereof in diagnostic formulations requiring a low dosage of the contrast agent. This can be attained: a) by administering a reduced volume of contrastographic solution; b) by administering more diluted contrastographic compositions (low-dosage compositions) . Said compositions provide images of good quality, while assuring a significant improvement from the toxicologic point of view. The contrastographic formulations containing the chelated complexes of the invention can therefore be formulated with concentrations of contrast agent ranging from 0.001 to 1.0 mmol/mL, preferably from 0.01 to 0.5 mmol/mL; in particular, concentrations lower than 0.25 mmol/mL for the low-dosage formulations . Said formulations can be administered to the patient in doses ranging from 0.001 to 0.1 mmol/kg, preferably 0.1 mmol/kg; in particular, doses ranging from 5 to 50 μmol/kg when administered in low dosages . Among the possible synthetic routes which can be used for the preparation of the compounds of the invention, in addition to those already described in EP 230 893, some general schemes are reported in the following. 12 When only one of the substituents R- j _ or R 2 is different from hydrogen, a possible process is illustrated in the following Scheme 1: 13 SCHEME 1 NaNOj.H÷.X- R, COOH NH. -5°C - +5°C R« COOH H 2 N ' NH, a1 b1 (1A) (2A) R 1\/ C00H X COOH OH " , pH10,60° HN \ r COOH R. C .N_ .COOH HOOC N c1 NH, HOOC (3A) (4A) m- n 2+ , salifying R. agent N. .COO pB 2 OOC r .COO N ' d1 ) 2+ OOC Mn (5A) 14 wherein : R^ has the same meanings as in formula (I) ; X = halogen (Cl, Br, I); m = number of the total charges of the chelated complex; B = substance suitable for salifying the chelated complex (for example Na + , K + , Mg ++ , Ca ++ or mixtures thereof, meglumine, etc.); z = number of the charges of B; p is such an integer that the product: p • z = m. In step (al) an α-amino acid (1A) , natural or synthetic, is reacted with a suitable halide (e.g. KBr) in acid aqueous medium, in the presence of NaN0 2 and at a temperature which can range from -5 to +5°C. The resulting α-haloacid (2A) is reacted, in step (bl) , with 1, 2-diaminoethane in aqueous solution, at a temperature from 20 to 40°C. The resulting intermediate (3A) is carboxymethylated in step (cl) , in basic medium at pH 10 with an α-haloacetic acid (e.g. bromoacetic acid) at about 60°C, to give the free ligand (4A) . The latter is reacted in step (dl) with the stoichiometric amount of manganese, as salt, (for example chloride, carbonate), in the presence of the base amount necessary for the neutralization; the reaction is preferably carried out in water or in a suitable water-alcohol mixture, at 25 to 40°C; thereby obtaining the desired chelated complex (5A) . When both R, and R 2 are different from hydrogen and R- is the same as R 2 . the procedure summarized in the following Scheme 2 will be followed: 15 SCHEME 2 R 1V ^C00H J .X NH 2 a2 (1B) (2B) R, H R^ .COOH I .NH, a'2 . N ^ . COOH H 2 N HOOC N X I H R, (IB 1 ) (2B-) (3B) COOH Ri X " COOH N r Mn 2+ , salifying agent ^ .COOH HOOC X ^ . ' b2 c2 R< HOOC J (4B) m- COO R. r pB z OOC N + T R. OO J 2 C Mn (5B) 16 wherein : R 1 has the same meanings as in formula (I) ; X = halogen (Cl, Br, I); m = number of the total charges of the chelated complex; B = substance suitable for salifying the chelated complex (for example Na + , K + , Mg ++ , Ca ++ or mixtures thereof, meglumine, etc.); z = number of the charges of B; p is such an integer that the product: p • z = m. In step (a2) a suitable α-amino acid (IB) is reacted with a 1, 2-dihaloethane, such as 1 , 2 -dibromoethane (2B) in a suitable solvent (e.g. water/ethanol , water/methanol mixtures) , keeping pH at about 9 with a suitable buffer (preferably 2M borate buffer) at a temperature which can range from 60 to 90°C. Alternatively, (step a'2), an a- haloacid (IB 1 ) can be reacted with 1 , 2-diaminoethane (2B 1 ) in aqueous solution at 20 to 60°C. The resulting intermediate (3B) is carboxymethylated in step (b2) with an orhaloacetic acid (e.g. bromoacetic acid) at basic pH (about 10) and at a temperature from 40 to 70 °C, to give the free ligand (4B) . This is reacted in step (c2) with the stoichiometric amount of manganese, according to the general procedure already described for scheme 1, to obtain the desired chelated complex (5B) . Using a different stoichiometry, asymmetric ligands can be prepared, wherein R-^ and R 2 are both different from hydrogen and different from each other, as represented in the following Scheme 3: 17 SCHEME 3 R, ^^ COOH .NH, a3 . NH, H 2 N ' HOOC N X I H (1C) (2C) (3C) R 2 ^ . COOH H I . T .COOH HOOC 4.N I b3 H T *2 (4C) COOH X COOH I .N COOH ► HOOC N ' c3 J R„ HOOC (5C) m- salifying Mπ ' agent pB 2 ' d3 λ r COO OOC N Y R, OOC J Mn (6C) wherein : R- j _ , R 2 have the same meanings as in formula (I) ; X = halogen (Cl, Br, I); m = number of the total charges of the chelated complex; B = substance suitable for salifying the chelated 18 complex (for example Na + , K + , Mg ++ , Ca ++ or mixtures thereof, meglumine, etc.); z = number of the charges of B; p is such an integer that the product: p • z = m. An alternative process which provides disubstituted compounds is represented by the following Schemes 4A and 4B: 19 SCHEME 4 R, s ^ COOPg (1D) NH„ p-toluenesulfonic acid d2 pyridinium salt (cat.) (2D) Y .0. O OH + Y dl \ COOPg (3D) HN O O ' d3 OPg Br ^Nf L Λ \ COOPg p-toluenesulfonic acid R ι \ ^ COO P g l 4D) ' pyridinium salt (cat . ) I N _ N (5D) PgOOC EtOH / H 2 0 O O PgOOC d4 OH N-bromosuccinimide d5 triphenyl phosphine R, COOPg N . (6D) PgOOC r Br 2 0 wherein : R 1 has the same meanings as in formula (I) ; Pg = protective group (e.g. t-butyl); Y = Cl, Br or other leaving groups (I, -OMs, -OTf, -OTs) . Step (dl) comprises the protection of the alcoholic fraction of a 2-haloethanol (preferably 2 -bromoethanol) with dihydropyran, to give intermediate (2D) . The alcohol -protecting group can be, for example, benzyl and trityl . The reaction takes place in an organic solvent such as CH 2 C1 2 , CHC1 3 , CH 2 C1CH 2 C1, in the presence of 4- toluenesulfonic acid pyridinium salt or of another acid catalyst. In intermediate (2D), the leaving group is preferably Br . In step (d2) the ester (e.g. t-butyl ester) of a natural or synthetic α-amino acid (ID) , either in the race ic or optically active form, is reacted with the intermediate (2D) in the presence of a base, such as diisopropylethylamine, in a solvent such as CH 3 CN, DMF or a chlorinated solvent, to give intermediate (3D) . The latter is reacted, in step (d3), with a bromoacetic acid ester (e.g. t-butyl-bromoacetate) in the presence of a base, such as diisopropylethylamine, to give intermediate (4D) which is reacted, in the subsequent step (d4), with 4 -toluenesulfonic acid pyridinium salt or another acid catalyst, in an ethanol/water mixture, at 20-60°C, to give intermediate (5D) . In step (d5) , intermediate (5D) is brominated with N-bromosuccinimide in the presence of triphenylphosphine, to give intermediate (6D) , which is reacted, in step (d6) with the ester (e.g. t-butyl ester) of an α-amino acid (7D) , in a suitable solvent (e.g. CH 3 CN/buffer phosphate 21 pH 8) , to give intermediate (8D) . This is then reacted with a bromoacetic acid ester (e.g. t-butyl-bromoacetate) in the presence of a base, such as diisopropylethylamine, to give tetraester (9D) . The latter is deprotected in step (d8) with known methods (e.g. hydrolysis with CF 3 COOH or (CH 3 ) 3 SiI, when the protective group is t- butyl) to give the free ligand (10D) , which is subsequently complexed and salified according to the general procedure already described in the above schemes. 22 SCHEME 4B π F .COOPg R, COOPg ^ / b NH < 7D ) N ^ 2 . ^-\ ^ COOPg PgOOC N ^^ Y PgOOC d6 R, Br PPggOOOOCC " "2 (6D) (8D) X- 0P9 . L N^ R / COOP 9 0 ^ | 1 I ^ ^s .N. .. COOPg PgOOC N ^-^ Y d7 J 1 d8 R. PgOOC " (9D) ^ .COOH ^ ?' /\ .N r^ ^COOH > HOOC N ^^ Y R 2 HOOC (10D) 23 wherein: R- j _ , R 2 have the same meanings as in formula (I); Pg = protective group (e.g. t-butyl); X = halogen (Cl, Br, I) . The preferred synthetic procedure for the compounds of formula (IV) is the following: SCHEME 5 CH 3 OY R 4— CH 2,— 0-CH- £-. C jH — COOH R— CH.— 0— CH— CH— COOY X a5 X (1E) (2E) H,N 1. NH, X— CH— COOH R 4 — CH— 0— CH— CH— COOH 2. H + NH c5 b5 NH. (3E) R 4 — CH— O— CH— CH— COOH salifying Mn 2+ , agent * COOH d5 C rOOH COOH (4E) m- R— CH— O— CH— CH— COO -N. N' ^ COO pB z COO 2+ ~COO Mn (5E) 24 wherein : R 4 has the same meanings as in formula (IV) ; X = halogen (Cl, Br, I); Y is a cation of an alkali metal selected from Na, K; m = number of the total charges of the chelated complex; B = substance suitable for salifying the chelated complex (for example Na + , K + , Mg ++ , Ca ++ or mixtures thereof, meglumine, etc.); z = number of the charges of B; p is such an integer that the product: p • z = m. The starting product is a 3-benzyloxy-2-halo- propionic acid salt (2E) , optionally substituted at the benzene ring, wherein the halogen at the 2- position is selected from Cl , Br, I, preferably Cl . This compound can be prepared from the corresponding acid (IE) by salification with a suitable base (e.g. CH 3 OK) in a suitable solvent (such as methanol) . 3 -Benzyloxy-2- chloropropionic acid (optionally substituted) can be prepared, for example, according to Chem. Ber. 1958, 91 , 538. In step (b5) , intermediate (2E) is reacted with 1,2- diaminoethane in aqueous solution at a temperature from 25 to 80°C, preferably from 40 to 60°C. The subsequent acidification yields intermediate (3E) which, in step (c5) , is carboxymethylated with a 2-haloacetic acid (preferably 2-bromoacetic acid) at basic pH (about 10) and at 20 to 60°C. The subsequent acidification gives the free ligand (4E) , that is reacted, in subsequent step (d5) , with the stoichiometric amount of manganese, as salt (e.g. chloride, carbonate), according to the general 25 procedure already described in the above schemes, to obtain the desired chelated complex (5E) . As for, in particular, the compounds of formula (V) , when the R 4 groups are different from each other, the synthetic routes of Schemes 3 and 4 above will be followed; when the R 4 groups are the same, the preferred synthetic scheme is the following: 1.KOH;KCI 2. H + R 4 r— CH 2-O— CH 2r— C IH— COOY + H 2 N ^^ NH, X a6 (1F) ω iδ o (_n W M S M σ> &5 R 4 — CH— O— CH— CH— COOH R 4r- CH 2^-O— CHr-C IH— COOH i HN ^ 1.X-CH 2 -COOH,OH- 2. H + COOH I COOH ~NH b6 4 " N J S3 B R 4 — CH^-O— CH— CH— COOH R 4 — CH— O— CH -CH— COOH t (2F) (3F) m- R— CH -O— CH -CH— COO Mn 2+ , salifying agent _ COO 2+ COO n pB z+ c6 R— CH— O— CH^- 7 M CH— COO O (4F) vo vo CA O o 27 wherein : R 4 has the same meanings as in formula (V) ; X = halogen (Cl, Br, I) ; Y is a cation of an alkali metal selected from Na, K; m = number of the total charges of the chelated complex; B = substance suitable for salifying the chelated complex (for example Na + , K + , Mg ++ , Ca ++ or mixtures thereof, meglumine, etc.); z = number of the charges of B; p is such an integer that the product: p • z = m. In step (a6) , the salt (preferably the potassium salt) of a 3-benzyloxy-2-halopropionic acid (IF), optionally substituted at the benzene ring, wherein the halogen at the 2- position is selected from Cl , Br, I, preferably Cl , is reacted in aqueous solution at basic pH (preferably pH 12) by addition of inorganic bases (preferably KOH) with 1 , 2-diaminoethane in the presence of KC1 at a temperature from 50 to 80°C, preferably 65°C. The subsequent acidification yields intermediate (2F) which, in step (b6) , is carboxymethylated with a 2- haloacetic acid (preferably 2 -bromoacetic acid) at basic pH (about 10) and at 40 to 70°C, preferably 50 °C. The subsequent acidification gives the free ligand (3F) that is reacted, in the subsequent step, with the stoichiometric amount of manganese, according to the general procedure already described in the above schemes, to obtain the desired chelated complex (4F) . An innovative aspect of this process compared with what reported, for example, in EP 230893, and particularly applicable to the product described in the subsequent Example 2, consists in reacting, in step (a6) , 28 the potassium salt of acid (IE) , instead of the corresponding acid, with ethylenediamine in the presence of KCl . The use of the latter is the key to obtain the product in good yields. A further aspect is represented by temperature, which should be as near as possible to 65°C: lower temperatures decrease the reaction yield and temperatures higher then 65 °C involve undesired side- reactions. The process described above is a further aspect of the present invention. The following examples illustrate the best experimental conditions to prepare the compounds of the invention. EXAMPLE 1 Manganese complex (II) of N- [2 - [bis (carboxymethyl) amino] ethyl] -N- (carboxymethyl) -0- (phenylmethyl) -D, L-serine salified with 1-deoxy-l- (methylamino) -D-glucitol (1:2) CH 2 Ν + H 2 CH 3 H- -OH HO- -H COO " Mn + • 2 H- -OH H- -OH CH 2 OH A) N- (2-aminoethyl) -0- (phenylmethyl) -D, -serine hydrochloride A solution 3-benzyloxy-2-chloropropionic acid potassium salt (C.A.S. 138666-92-9; prepared according to Inorg. Chem. 1992, 31(6), 1100-3) (505 g; 2 mol) in H 2 0 (3 L) was dropped in ethylenediamine (commercial product) (1560 g; 26 mol), keeping temperature below 50°C. At the end of the addition the reaction mixture was heated at 29 50°C for 18 h. After evaporation of the ethylenediamine excess (60°C; 2 kPa) , the residue was dissolved in H 2 0 (2 L) and acidified (pH 1.5) with 37% HCl . The precipitate was filtered and the crude was crystallized from H 2 0 (2 L; under reflux, filtering off the insolubles) . After filtration and drying (P 2 0 5 , 50°C, 2 kPa) the desired product was obtained (350 g; 1,27 mol). Yield 64%. m.p. : > 230°C Acidic titer (0.1 N NaOH) : 101.3% Argentometric titer (0.1 N AgN0 3 ): 104.1% TLC: Rf 0.48 Silica gel plates 60 F 2 ^ 4 (E. Merck art. 5715) Eluent: EtOAc/AcOH/H 2 0 3:2:1 Detection: 1% KMn0 4 in 1 N NaOH HPLC: 99.2 % (% area) Loss of weight (120°C) : 0.21% The ^C-NMR anc j Ms spectra are consistent with the indicated structure. Elementary analysis (%) : C H Cl N Calculated 52.46 6.97 12.90 10.19 Found 52.20 6.88 13.00 10.09 B) [ [N- [2- [bis (carboxymethyl) amino] ethyl] -N- (carboxymethyl) -0- (phenylmethyl) -D, -serine ^^O ^ CO ° H ^/ ^^^N^COOH C00H COOH 30 A suspension of the product from the previous step (329 g; 1,2 mol) and bromoacetic acid (commercial product) (666 g; 4,8 mol) in H 2 0 (1 L) , was added with 10 N NaOH, keeping temperature below 50°C. The addition was continued to adjust pH to 10. The mixture was heated at 50°C for 2 hours then left at 20=j25°C for 18 h, always keeping pH 10 by a pH-stat. The solution was acidified (pH 1.5) with 37% HCl , to obtain a precipitate. After 18 h the solid precipitate was filtered and dissolved in H 2 0 (2 L) ; then 10 N NaOH was added to reach pH 8. The solution was acidified to pH 3 with 37% HCl, then Carbopuron ( ^ ) 4N (10 g) was added. After 30 minutes the solution was filtered and acidified (pH 1.5) with 37% HCl, to obtain a precipitate which, after 18 h, was filtered and washed with H 2 0 (300 mL) . After drying (P 2 0 5 ; 60°C; 2 kPa) , the desired product was obtained (345 g; 0.836 mol). Yield 70%. m.p. : 181 - 183°C Acidic titer (0.1 N NaOH): 98.8 % Complexometric titer (0.1 N ZnS0 4 ): 98 % TLC: Rf 0.52 Silica gel plates 60 F 254 (E. Merck art. 5715) Eluent: EtOAc/AcOH/H 2 0 3 : 2 : 1 Detection: 1% in KMn0 4 1 N NaOH HPLC : 97.6% (% area) K.F. : 0.30% The 13 C-NMR and MS spectra are consistent with the indicated structure. Elementary analysis (%) : C H N Calculated 52.49 5.87 6.79 Found 52.24 5.94 6.75 31 C) Manganese complex (II) of N- [2- [bis (carboxymethyl) amino] ethyl] -N- (carboxymethyl) -0- (phenylmethyl) -D, L-serine salified with 1-deoxy-l- (methylamino) -D-glucitol (1:2) A solution of MnCl 2 • 4 H 2 0 (commercial product) (29.7 g; 0.15 mol) in H 2 0 (50 mL) was dropped in 1 hour into a solution of the free ligand from the previous step (61.9 g; 0.15 mol) in H 2 0 (600 mL) neutralized (pH 6.5) by addition of meglumine 1 N (408 mL; 0.41 mol) by means of a pH-stat. The solution was filtered through Millipore HA 0.45 mm, diluted with H 2 0 (3 L) and nanofiltered. After adjusting pH to 6.9 with 1 N meglumine, the solution was concentrated. After drying (P 2 0 5 ; 50°C; 2 kPa) the desired product was obtained (110.9 g; 0.13 mol) . Yield 86 %. m.p. : 92 - 95°C Free ligand (HPLC) : < 0.20 % HPLC: 99.4 % (% area) K.F. : 2.74 % Elementary analysis (%) : C H Mn Ν Calculated 44.91 6 . 60 6 . 42 6 . 55 Found 45.25 6 . 63 6 . 11 6 . 65 on the anhydrous EXAMPLE 2 Manganese complex(II) of N, N ' -1 , 2-ethanediylbis [N- (carboxymethyl) -O- (phenylmethyl) -D, L-serine] salified with 1-deoxy-l- (methylamino) -D-glucitol (1:2) 32 COO " CH 2 NH 2 CH 3 O H- -OH N ^COO HO- -H Mn 2+ • 2 H- • OH N^COO " H- -OH ,0 COO " CH 2 OH A) N, N ' -1, 2-ethanediylbis [0- (phenylmethyl) -D, L-serine] A solution of 3-benzyloxy-2 -chloropropionic acid potassium salt (25.27 g; 0.1 mol), ethylenediamine (1.98 g; 0.033 mol) and KCl (12.37 g ; 0.166 mol) in H 2 0 (66 mL) , was heated at 65°C for 48 hours, adding 8 Ν KOH (12.1 mL; 0.097 mol) by means of a pH-stat to keep pH 12. The reaction mixture was cooled at r.t., diluted with H 2 0 (120 mL) and acidified (pH 6) with 1 Ν HCl (85 mL) under strong stirring. The precipitate was filtered, washed with H 2 0 and dried (P 2 0 5 , 50°C, 2 kPa) to give the desired product (8.3 g; 0.02 mol). Yield 61%. m.p. : 213°C (dec. ) Acidic titer (0.1 Ν ΝaOH) : 96% TLC: Rf 0.75 Silica gel plates 60 F ς4 (KGaA Merck art. 5715) Eluent = CHCl 3 /AcOH/H 2 0 5:5:1 Detection: 1% KMn0 4 in 1 Ν ΝaOH HPLC : 97% (% area) K.F. : 0.56 % The l-H-Ν R, 13 C-ΝMR, IR and MS spectra are consistent with the indicated structure. Elementary analysis (%) : 33 C H N Calculated 63.45 6.78 6.73 Found 63.53 6.81 6.75 on the. anhydrous B) N, N ' - l , 2 -ethanediylbis [N- (carboxymethyl) - 0- (phenylmethyl) -D, L-serine] A suspension of the product from the previous step (216 g; 0.518 mol) and bromoacetic acid (217 g; 1,56 mol) in H 2 0 (1.8 L) , was added with 10 Ν ΝaOH to pH 10. The mixture was heated at 50 °C for 22 hours, keeping pH at 10 by addition of 10 Ν ΝaOH by means of a pH-stat. After cooling at r.t., the reaction mixture was diluted with H 2 0 (0.5 L) and acidified (pH 1.5) with 6 Ν HCl (0.4 L) to obtain a precipitate which was separated by decantation and then suspended in H 2 0 (3 L) . After 18 hours the precipitate was again separated by decantation, powdered in a mortar, filtered, washed with H 2 0 and dissolved in 1 Ν ΝaOH (1,85 L) . After dilution with H 2 0 (2.45 L) , the solution was acidified (pH 3) with 2 Ν HCl, with strong stirring, filtered and then acidified to pH 1.5 with 2 Ν HCl. After 24 hours the precipitate was separated by filtration, powdered in a mortar, washed with H 2 0 (0.5 L) and dried (P 2 0 5 ; 50°C; 2 kPa) . The resulting solid was suspended in n-butanol saturated with water (3 L) and stirred at r.t. for 18 hours. The resulting suspension was filtered and the solid residue was washed with n-butanol saturated with water (1.5 L) and dried (P 0 5 ; 60°C; 2 kPa) , to give the desired product (178.4 g,- 0.335 mol). Yield 65%. .p. : 158-159°C Acidic titer (0.1 Ν ΝaOH): 99.4% Complexometric titer (0.1 Ν ZnS0 4 ) : 98.5% TLC : Rf 0.53 34 Silica gel plates 60 F 254 (KGaA Merck art. 5715) Eluent = CHCl 3 /AcOH/H 2 0 5:5:1 Detection: 1% KMn0 4 in 1 N NaOH HPLC : 97.3% (% area) K.F. : 3.10% The 1 H-NMR, 13 C-NMR, IR and MS spectra are consistent with the indicated structure. Elementary analysis (%) : C H N Calculated 58.64 6.06 5.26 Found 58.47 6.06 5.30 on the anhydrous C) Manganese complex ( II ) of N, N' - 1 , 2 -ethanediylbis [N- (carboxymethyl ) - O- (phenylmethyl ) -D , - serine] salif ied with 1 -deoxy- l - (methylamino) -D-glucitol ( 1 : 2 ) . A solution of MnCl 2 • 2 H 2 0 (11.33 g; 0.07 mol) in H 2 0 (50 mL) was dropped in 2 hours into a solution of the free ligand from the previous step (37.28 g; 0.07 mol) in H 2 0 (300 mL) , neutralized (pH 7) adding 1 Ν meglumine (273 mL; 0.273 mol) by means of a pH-stat. The reaction mixture was filtered through a Millipore HA 0.45 μm filter, diluted with H 2 0 (2 L) and nanofiltered. The retentate was evaporated under vacuum (2 kPa) to obtain a solid residue which was dried (P 2 0 5 , 50°C, 2 kPa) to give the desired product (52.9 g; 0.0542 mol). Yield 77%. m.p. : 95-100°C Free ligand (HPLC): < 0.01% HPLC: 99.7% (% area) K.F. : 4.71% The IR and MS spectra are consistent with the indicated structure. 35 Elementary analysis ( % ] C H Mn N Calculated 49.23 6.61 5.63 5.74 Found 49.18 6.56 5.13 5.64 on the anhydrous The relaxivity of the compounds of the invention was measured both in saline solution and in human serum, reconstituted from Seronorm (R ) Human (Nycomed Pharma), lyophilized human serum. The obtained data are summarised in the following Table 1. vo n vo 00 TABLE 1 C Compound Relaxivity (mM s 1 C Aqueous sol . Seronorm TMHuman ( v 1 )' I ay MnCl 2 6.59 (2) 39.18 (2i 53 (3) 75 (3) s 1*1 Mn-EDTA (4 ^ 2.58 (5) 3.39 ^ 10 < 3 > 13 (3) EXAMPLE 1 2.95 (5) 5.14 (5) 16.5 < 3 > 21.8 <3> EXAMPLE 2 3.21 (5) 4.48 (5) 36 < 3 > 50 < 3 > n n © 37 (1) Human serum freeze-dried and reconstituted prior to use; available from Nycomed Pharma AS Oslo, Norway. (2) Data measured on 0.15 M NaCl aqueous solutions; 20 MHz; 39°C; pH 4. (3) Data calculated between 0 and 1 mM; 20 MHz; 39°C; pH 7.0. (4) N-methylglucamme double salt. (5) Data measured on 0.15 M NaCl aqueous solutions; pH 7.3; 20 MHz; 39°C. The high r- j _ and, more specifically, r 2 relaxivity values measured in serum for the compounds of the invention, clearly show the remarkable potential of these compounds m M.R.I, diagnostic imaging, both for general use and, more particularly, for use in combinations of T- L /T 2 weighed or T 2 weighed sequences, as well as m the imaging of the cardiocirculatory system. Following Table 2 shows the stability constants in aqueous solution relating to the compounds of Examples 1 and 2, compared with Mn-EDTA. TABLE 2 Compound Log K j ^ <i l ' Mn-EDTA 13.88 (2) EXAMPLE 1 13.45 ± 0.07 EXAMPLE 2 13.8 + 0.1 (1) Thermodynamic stability constants, measured by the potentiometric method at 20°C m 0.1 M KCl aqueous solution . (2) Robert M. Smith and Arthur E. Martell : "Critical Stability Constants", Vol. 6, Second Supplement; Plenum Press, 1989. Following Table 3 shows the DL 5 Q m the mouse after 38 intravenous administration of the compounds of Examples 1 and 2, compared with the toxicity values reported in literature for MnCl 2 and Mn-EDTA. vo ≥ (Λ: 00 TABLE 3 C i Co Si Compound DL 5 Q (mmol/kg) Species Administration 1 MnCl 2 0.25 - 0.30 (1) mouse 1.v. GO VO Na 2 [Mn(EDTA) (H 2 0) ] 5.9 < 2 > mouse i.p. I EXAMPLE 1 4.79 mouse i . v. EXAMPLE 2 4.5 mouse i . v. v_ *nT3 IS o vo 40 (1) Investigative Radiology 1995, 30 (10 ) , 611-620. (2) Magnetic Resonance Quarterly 1990. 6(2 ) , 65- 84 As it is shown Dy the data of this table, the compounds of the invention have quite favourable acute toxicity values. The compound of example 1 was tested in a CC1 4 - mduced acute hepatitis model m the rat in comparison with Gd-BOPTA (one of the compounds claimed in EP 230893). The decrease m T^ m the pathological tissue, as well as the difference in the decrease m T-, between pathological and healthy tissues, obtained with the compound of example 1 of the invention are markedly higher than those of the control product. It can therefore by assumed that the compound of the invention s suitably effective m identifying the focal hepatic lesions related to hepatitis (Table 4). ≥: o\ 00 TABLE 4 Co Gd-BOPTA Compound Example 1 i Time Hepatitis liver Normal liver Hepatitis liver Normal liver Co 1 (min) (ms) (ms) (ms) (ms) 0 757±64 588±12 710±32 561+34 5 296+51 218±5 239±54 95±6 a 15 379±67 224+5 183+42 94 +4 t* j O H VO vo 42 T 1 s obtained with IR-SNAPSHOT FLASH 2.87 ms/1.74 ms/7° (TR/TE/α) after administration of 100 mmol/kg of the compound of example 1 and of Gd-BOPTA (gadobenate dimeglumme) to rats with acute hepatitis induced by CC1 4 per os (0.25 mL/Kg) .

Claims: 43 CLAIMS Compounds of formula (I) (li wherein: Ri R- which are the same or different, are a hyαrogen atom, or a - (CH 2 ) m -0- (CH 2 ) n -R 3 group, nerem R 3 s an aryl residue, m is an integer 1 to 5 and n is an integer 1 to 4 , with the proviso that R-^ and R 2 cannot be at the same time hydrogen, as well as the optically active forms of the chelates thereof with Mn(II) and the salts thereof with physiologically compatible organic bases selected from primary, secondary, tertiary amines or basic ammo acids, or with inorganic bases whose cations are sodium, potassium, magnesium, calcium, or mixtures thereof. 2. Compounds as claimed m claim 1 salified with cations selected from: cations of inorganic bases, in particular, ions of alkali or alkalme-earth metals selected from sodium, potassium, magnesium, calcium, or mixtures thereof; cations of physiologically compatible organic bases selected from primary, secondary and tertiary amines, m particular, ethanola ine, diethanelam e , orpholme, glucam e, N-methylglucamme , N, N-dimethylglucamme ; 44 - cations cf amino acids, in particular lysine, arginine or ornithine. 3. Compounds as claimed in claim 2 salified with N- methylglucamine . 4. Compounds according to any one of the above claims, in which -^ and R 2 are residues of formula: ^ H 3 C- ** (CH 2 ) n -0-(CH 2 ) m - (CH 2 ) n -0-(CH 2 ) r ^^ (HO); (ci) (CH 2 ) n -0-(CH 2 ) m - (CH 2 ) n -0-(CH 2 V ^ HO, (Br); (Dx- (CH 2 ) n -0-(CH 2 ) m - (CH 2 ) n -0-(CH 2 ) r HO, HOOC. (BO: HO- (CH 2 ) n -0-(CH 2 ) m - (CH 2 ) n -0-(CH 2 ) m - wherein : x = 1 , 2. 5. Compounds as claimed in claim 1, of formula (II) R— (CH 2 ) n — 0-(CH 2 2)/m— CH-COOH N^ " "COOH C 4OOH (ii, OOH 45 wherein R 4 is a phenyl, unsubstituted or substituted with one or more R^ groups, which are the same or different, wherein R 5 is halogen, -OH, -NH 2 , -COOH, -CONH 2 , -S0 3 H, -S0 3 NH 2 , or a C ] _-C 4 alkyl chain. 6. Compounds as claimed in claim 1, of formula (III) : R—(CH 2 )-0-(CH 2 )—CH—COOH COOH COOH N ' R 4 —(CH 2 )-0—(CH 2 )-CH-COOH (III) wherein the R 4 groups, which are the same or different, have the same meaning as claim 5. 7. Compounds as claimed in claim 5 or 6 , in which R 4 is one of the residues of formulae: r ^ ^^s (Br): wherein: x = 1,2. 8 . ComDounds as claimed m claim 1 , of formula ( IV) 46 (IV) wherein R 4 is as defined in claim 5. 9. Compounds as claimed in claim 1, of formula (V) (V) wherein the R 4 groups, which are the same or different, have same meanings as in claim 5. 10. Compounds as claimed in claim 8 or 9 , in which R 4 is one of the following groups: 47 11. Compounds as claimed in claim 10, in which R 4 is unsubstituted phenyl. 12. Compounds as claimed in claim 1, of formulae: COOH O N, ^ ^ N ' ^ COOH COOH COOH COOH 13. Contrastographic formulation for M.R.I, diagnostic imaging, comprising at least one of the chelated complexes according to claims 1 to 12, or a physiologically compatible salt thereof. 14. Compounds of claims 1 to 12, for M.R.I, diagnostic imaging of the liver and the biliary tract. 15. Compounds of claims 1 to 12, for M.R.I, diagnostic imaging of the cardiocirculatory system. 48 16. The use of chelated complexes according to any one of claims 1 to 12 for the preparation of M.R.I, diagnostic formulations wherein said formulations are used in the imaging of organs or tissues of the human or animal body. 17. The use of chelated complexes as claimed m claim 16 wherein the diagnostic formulations contain said chelated complexes m concentrations ranging from 0.001 to 1.0 mmol/ml . 18. The use of chelated complexes as claimed in claim 17 wherein the diagnostic formulations contain said chelated complexes in concentrations ranging from 0.01 to 0.5 mmol/ml . 19. The use of the chelated complexes as claimed in claim 17 wnerein the formulations are administered to the patient in doses ranging from 0.001 to 0.1 mmol/kg. 20. The use of the chelated complexes according to any one of claims 16 to 19, wherein the magnetic field used ranges from 0.5 to 2 Tesla. 21. The use of the chelated complexes according to any one of claims 16 to 19, wherein the magnetic field used ranges from 0.1 to 0.5 Tesla. 22. The use of the chelated complexes according to any one of claims 16 to 19 wherein said images are acquired by means of T^ weighed or T 2 weighed sequences or combinations thereof. 23. The use of the chelated complexes according to any one of claims 16 to 19, wherein said images are acquired T 2 weighed sequences .

Inventors: BROCCHETTA MARINO, CALABI LUISELLA, FEDELI FRANCO, MANFREDI GIUSEPPE, PALEARI LINO, UGGERI FULVIO, BROCCHETTA, MARINO, CALABI, LUISELLA, FEDELI, FRANCO, MANFREDI, GIUSEPPE, PALEARI, LINO, UGGERI, FULVIO
Applicants: BRACCO SPA, BROCCHETTA MARINO, CALABI LUISELLA, DIBRA SPA, FEDELI FRANCO, MANFREDI GIUSEPPE, PALEARI LINO, UGGERI FULVIO, BRACCO S.P.A., BROCCHETTA, MARINO, CALABI, LUISELLA, DIBRA S.P.A., FEDELI, FRANCO, MANFREDI, GIUSEPPE, PALEARI, LINO, UGGERI, FULVIO
Application year: 1999
Main classification: A61K49
IPC codes: A61K49, C07C229, C07D209