Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D255/00—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00
  • C07D255/02—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00 not condensed with other rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
  • A61K49/106—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being cyclic, e.g. DOTA

Definitions

• the invention relates to novel compounds able to complex Fe(III), and to the complexes with Fe(III) thereof. These complexes are particularly suitable as contrast agents for magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• Magnetic resonance imaging (MRI) contrast agents currently used in clinical practice are small and hydrophilic paramagnetic Gd(III) complexes, or chelates, that accelerate the relaxation rates (R 1 and R 2 ) of proximate tissue water protons in regions of agent accumulation.
• Gd (III) complexes are generally very safe and very well tolerated by patients, there has been recently some concerns related to contraindications in patients with severely compromised kidney function (nephrogenic systemic fibrosis), to the retention of small amounts of Gd(III) in the tissues of patients exposed to multiple MRI scans (although without any evidence that this is associated with clinical harm), and possibly to the environment (due to difficulties in the removal of gadolinium-based contrast agents, or GBCAs, in wastewater treatment plants). Therefore, alternative contrast agents based on chemical species different than Gd(III) have been sought.
• iron complexes chelating endogenous paramagnetic metals, such as iron, are possible candidates. Indeed, iron complexes, and in particular Fe(III) complexes, have been studied for their use in MRI. Fe(II) complexes are generally less suitable for providing MRI contrast, as they are characterized by lower relaxivity compared to Fe(III) complexes.
• the ideal Fe(III) complex possesses high relaxivity to obtain high contrast in vivo, high thermodynamic stability and kinetic inertness to minimalize and possibly to avoid the hydrolyzation, transmetallation and transchelation reactions with the challenging endogenous metal ions and ligands, and stability to reduction to avoid triggering the Fenton reaction, that is the reduction of Fe(III) to Fe(II) (Baranyai et al. (Chem. Sci. 2021, 12, 11138)) triggered in vivo e.g. by anti-oxidants such as ascorbic acid. This reduction would indeed lower the relaxivity of the administered iron complex (due to the generation of the Fe(II)-complex) and might generate in vivo OH- radicals, which are toxic.
• Schellenberger etal. discloses low molecular weight Fe(III) complexes such as the Fe(III) chelates of pentetic acid (Fe(DTPA)) and of trans-cyclohexane diamine tetraacetic acid (Fe(CDTA)), which provide image contrast in vivo and exhibit enhancement kinetics very similar to that of Gd(DTPA) 2- (Magnevist®), a clinically used GBCA.
• Fe(DTPA) pentetic acid
• Fe(CDTA) trans-cyclohexane diamine tetraacetic acid
• Gd(DTPA) 2- Magnnevist®
• These complexes are however characterized by unsatisfying redox stability and relaxivity (as reported in Baranyai et al., Chem. Sci. 2021, 12, 11138)).
• Gale etal. J. Am. Chem. Soc., 2019, 141, 5916 discloses the redox-active iron complex Fe-PyC3A, characterized by relatively high relaxivity at the imaging fields and by Fe 3+/2+ interchange (mediated by biochemical processes; e.g. Fe 3+ is rapidly reduced to Fe 2+ by L- cysteine). Accordingly, this complex is purposely characterized by very low redox stability to allow the Fe 3+/2+ interchange, which is a very specific application for imaging of acute inflammations and is not ideal for a wide range of MRI applications.
• Morrow et al. discloses a series of substituted macrocyclic ligands used for Fe(III) complexation and their use as MRI contrast agents.
• the ligands of the invention when complexed to Fe(III) ions, thus forming the complexes of the invention, have surprisingly and advantageous properties.
• said ligands have been found to possess a balanced profile of high relaxivity, kinetic inertness, thermodynamic stability and stability to reduction. Accordingly, the complexes of the invention can be advantageously used as contrast agents for MRI.
• the present invention relates to a compound of formula (I), or an ion, a stereoisomer, a tautomer, a hydrate, a solvate, or a salt thereof, or a mixture of same, as set out in the claims.
• the compound of formula (I) is a ligand that, when complexed to Fe(III) ions, forms a Fe(III) complex (also object of the present invention, as set out in the claims) that possess a balanced profile of high relaxivity, kinetic inertness, thermodynamic stability and stability to reduction.
• the present invention further relates to the methods of preparation of the compound and of the complex of the invention, as well as their use as MRI contrast agents and in diagnostic (MRI) imaging, as set out in the claims.
• MRI diagnostic
• the present invention relates to a compound of Formula (I):
• n, m, and o are integer numbers independently selected between 1 and 2;
• Y 1 and Y 2 are independently selected from the group consisting of hydrogen and a C 1 -C 4 -alkyl
• R 1 and R 2 are independently selected from the group consisting of hydrogen and C 1 -C 4 -alkyl
• L 1 and L 2 are independently selected from the group consisting of C 1 -C 4 -alkylaminyl, C 1 -C 4 -alkylamidyl, and C 1 -C 4 -alkylether; preferably of C 1 -C 4 -alkylaminyl, and C 1 -C 4 - alkylamidyl;
• Z 1 and Z 2 are independently selected from the group consisting of hydrogen and a C 1 -C 6 -alkyl, preferably a C 1 -C 4 -alkyl, said C 1 -C 6 -alkyl (preferably C 1 -C 4 -alkyl) being optionally substituted by one or more groups selected from the group consisting of hydroxyl (-OH), carboxyl (-COOH), and phosphonate (-PO3H2);
• R is selected from the group consisting of hydrogen, C 1 -C 4 alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), and the moiety of Formula (IA) : Formula (IA) wherein : the asterisk (*) indicates the point of attachment of said moiety of Formula (IA) to the nitrogen bearing the R group;
• Y 3 has the same meaning provided above for Y 1 and Y 2 ;
• R 3 has the same meaning provided above for R 1 and R 2 ;
• L 3 has the same meaning provided above for L 1 and L 2 ; and Z 3 has the same meaning provided above for Z 1 and Z 2 ; or an ion, or a stereoisomer, or a tautomer, or a hydrate, or a solvate, or a pharmaceutically acceptable salt thereof, or a mixture of the same.
• alkyl refers to any linear or branched hydrocarbon chain.
• C 1 -C 4 -alkyl comprises within its meaning a linear or branched chain comprising from 1 to 4 carbon atoms such as: methyl, ethyl, propyl, isopropyl, butyl, /so-butyl, tert-butyl, and the like.
• C 1 -C 3 -alkyl refers to a linear or branched chain comprising from 1 to 3 carbon atoms such as, for instance, methyl, ethyl, propyl and /so-propyl;
• C 1 -C 2 -alkyl refers to a linear or branched chain comprising from 1 to 2 carbon atoms such as methyl and ethyl;
• C 1 -alkyl refers to a methyl (-CH 3 ) group;
• C 1 -C 6 -alkyl refers to methyl, ethyl, propyl, iso-propyl, butyl, /so-butyl, tert-butyl, and the like, as well as n-penthyl and isomers thereof (such as isopentane, neopentane, and the like), and n-hexyl and isomers thereof (such as isopentane, neopentan
• alkylaminyl refers to an alkyl as above defined wherein one of the hydrogen atoms is substituted by an amine group, said alkylaminyl being attached to both (i) the phenolic moiety of the compound of the invention and (ii) the Z 1 , Z 2 , or Z 3 group (as the case may be).
• C x -C y -alkylaminyl wherein x and y denote two integer numbers, refers to an alkylaminyl as above defined with a number of carbons comprised between x and y.
• C x -C y -alkylamidyl wherein x and y denote two integer numbers, refers to an alkylamidyl as above defined with a number of carbons comprised between x and y.
• alkylether refers to an alkyl as above defined wherein one of the hydrogen atoms is substituted by an ether group (-O-), said alkylether being attached to both (i) the phenolic moiety of the compound of the invention and (ii) the Z 1 , Z 2 , or Z 3 group (as the case may be).
• C x -C y -alkylether wherein x and y denote two integer numbers, refers to an alkylether as above defined with a number of carbons comprised between x and y.
• aryl refers to an aromatic hydrocarbon and, preferably, to a phenyl ring.
• aryls according to the invention can be either unsubstituted or substituted with one or more substituent groups that, simultaneously with or independently of each other, are selected from hydroxyl (-OH), halogen, and C 1 -C 4 -alkyl optionally substituted by one or more hydroxyl (-OH); preferably, the term “aryl” refers to unsubstituted aromatic hydrocarbons, such as unsubstituted phenyl.
• the term "L-Z" when referring to substituent groups generally refers to any or all of the L 1 -Z 1 , L 2 -Z 2 , and L 3 -Z 3 (if present) substituent groups.
• macrocycle or macrocyclic cage when referring to triazacyclononane (or [9]-membered cycle), triazacyclodecane (or [10]-membered cycle), triazacycloundecane (or [ll]-membered cycle), or triazacyclododecane (or [12]-membered cycle), refers to the macrocycles having the following structures:
• protecting group designates a protective group adapted for preserving the function of the group and/or atom to which it is bonded.
• protective groups can be used to preserve amino, hydroxyl or carboxyl functions.
• Appropriate carboxyl protective groups may thus include, for example, benzyl, alkyl e.g. tert- butyl or benzyl esters, or other substituents commonly used for the protection of such functions, which are all well known to those skilled in the art (e.g. from T. W. Greene and P. G. M. Wuts; "Protective Groups in Organic Synthesis", Wiley, N.Y. 1999, third edition).
• the compounds of the above formula (I) may have one or more asymmetric carbon atom, otherwise referred to as a chiral carbon atom, and may thus give rise to diastereomers, optical isomers and enantiomers.
• the present invention further includes all such possible diastereomers, optical isomers and enantiomers, as well as their racemic mixtures, their substantially pure resolved enantiomers. All possible geometric isomers are included as well.
• the individual stereoisomer of a compound of formula (I), e.g. a particular diastereomer may be isolated by any conventional means, such as for example chromatography, possibly chiral chromatography.
• pharmaceutically acceptable salt refers to derivatives of the compounds of the invention wherein the parent compound is suitably modified by converting any of the free acid or basic groups, if present, into the corresponding addition salt with any base or acid conventionally intended as being pharmaceutically acceptable, for example as disclosed in S. M. Berge, et al., J. Pharm. Sci. 1977, 66, 1-19.
• Y 1 and Y 2 , and Y 3 if present are independently selected from the group consisting of hydrogen and a C 1 -C 3 -alkyl, preferably of hydrogen and a C1-C 2 - alkyl, and more preferably of hydrogen and a C 1 -alkyl (that is, methyl).
• Y 1 and Y 2 , and Y 3 if present are simultaneously the same group, most preferably hydrogen.
• R 1 , R 2 , and R 3 if present are independently selected from the group consisting of hydrogen and C 1 -C 3 -alkyl, preferably of hydrogen and C 1 -C 2 -alkyl, more preferably of hydrogen and C 1 -alkyl, and even more preferably C 1 -alkyl (that is, methyl (-CH 3 )).
• R 1 , R 2 , and R 3 if present are simultaneously the same group.
• L 1 , L 2 , and L 3 are independently selected from the group consisting of C 1 -C 3 -alkylaminyl, C 1 -C 3 -alkylamidyl, and C 1 -C 3 -alkylether; preferably of C 1 -C 2 -alkylaminyl, C 1 -C 2 -alkylamidyl, and C 1 -C 2 -alkylether; and more preferably of C 1 - alkylaminyl, C 1 -alkylamidyl, and C 1 -alkylether; according to this latter more preferred embodiment, L 1 , L 2 , and L 3 if present, are independently selected from *-CH 2 -NH- ⁇ , *-C(O)-NH- ⁇ , *-NHC(O)- ⁇ , and *-CH 2 -O- ⁇ , preferably from *-CH 2 -NH- ⁇ , *-C(O)-NH- ⁇ , and *- CH
• the nitrogen atoms of the alkylaminyl and of the alkylamidyl groups L 1 , L 2 , and L 3 if present, preferably bear at least one hydrogen; in other words, the amines of the alkylaminyl groups and/or the amides of the alkylamidyl groups L 1 , L 2 , and L 3 if present, are preferably primary or secondary; more preferably secondary.
• Z 1 , Z 2 , and Z 3 if present are independently selected from the group consisting of hydrogen, C4-C6-alkyl substituted by two or more hydroxyl (-OH) groups, and C 1 -C 3 -alkyl substituted by at least one group selected from the group consisting of hydroxyl (-OH), carboxyl (-COOH), and phosphonate (-PO3H2).
• Z 1 , Z 2 , and Z 3 if present, are independently selected from the group consisting of hydrogen, C 6 -alkyl substituted by two or more, such as two to five (preferably five), hydroxyl (-OH) group, C 1 -C3-alkyl substituted by at least one, such as two, hydroxyl (-OH) groups, and C 1 -alkyl substituted by carboxyl (-COOH) or phosphonate (-PO 3 H 2 ).
• R when R is C 1 -C 4 alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), R is preferably a C 1 -C 3 -alkyl or a C 1 -C 3 -alkyl substituted by an aryl (such as a substituted or unsubstituted aryl), preferably phenyl, more preferably C 1 -C 2 -alkyl or a C 1 -C 2 -alkyl substituted by an aryl (such as a substituted or unsubstituted aryl), preferably phenyl, and even more preferably a C 1 -alkyl or a C 1 -alkyl substituted by an aryl (such as a substituted or unsubstituted aryl), preferably phenyl (thereby providing the benzyl group -CH 2 -C6H5).
• aryl such as a substituted or unsubstit
• R is selected from the group consisting of hydrogen, C 1 -alkyl, C 1 -alkyl substituted by an aryl (such as a substituted or unsubstituted aryl), preferably phenyl (thereby providing the benzyl group -CH 2 -C6H5), and the moiety of Formula (IA), wherein Y 3 , R 3 , L 3 , and Z 3 have the same meanings provided above for, respectively, Y 1 , R 1 , L 1 , and Z 1 of formula (I) and any embodiments thereof.
• n, m, and 0 of formula (I) are 1, whereby the compound of the invention has a triazacyclononane macrocyclic cage and has the following formula (II) Formula (II) wherein R, R 1 , R 2 , Y 1 , Y 2 , L 1 , L 2 , Z 1 , and Z 2 are as above defined for formula (I) or for any embodiment thereof.
• n, m, and 0 of formula (I) is 2, and the other two are 1, whereby the compound of the invention has a triazacyclodecane macrocyclic cage and has one of the formulae (IIIA), (IIIB) or (IIIC) Formula (IIIA)
• R 1 , R 2 , R 3 , Y 1 , Y 2 , Y 3 , L 1 , L 2 , L 3 , Z 1 , Z 2 , and Z 3 are as above defined for formula (I) or for any embodiment thereof, and R' is hydrogen or C 1 -C 4 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), preferably is hydrogen or C 1 -C 3 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), more preferably is hydrogen or C 1 -C 2 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), and even more preferably is hydrogen or C 1 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), and even more preferably is hydrogen or C 1 -alkyl optional
• n, m, and o of formula (I) are 1, and the other two are 2, whereby the compound of the invention has a triazacycloundecane macrocyclic cage and has one of the formulae (IVA), (IVB), or (IVC) Formula (IVA)
• R 1 , R 2 , R 3 , Y 1 , Y 2 , Y 3 , L 1 , L 2 , L 3 , Z 1 , Z 2 , and Z 3 are as above defined for formula (I) or for any embodiment thereof, and R' is hydrogen or C 1 -C 4 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), preferably is hydrogen or C 1 -C 3 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), more preferably is hydrogen or C 1 -C 2 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), and even more preferably is hydrogen or C 1 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), and even more preferably is hydrogen or C 1 -alkyl optional
• n, m, and o of formula (I) are 2, whereby the compound of the invention has a triazacyclododecane macrocyclic cage and has the following formula (V) Formula (V) wherein R, R 1 , R 2 , Y 1 , Y 2 , L 1 , L 2 , Z 1 , and Z 2 are as above defined for formula (I) or for any embodiment thereof.
• the compound of the invention is selected from the group consisting of:
• Compound 110 ( ⁇ 1,5,9-triazacyclododecane-1,5-diylbis[methylene(2-hydroxy-5-methyl- 3,1-phenylene)azanediyl(2-oxoethane-2,l-diyl)] ⁇ bis(phosphonic acid)),
• Compound 150 (2,2'-[(4-benzyl-1,4,8-triazacycloundecane-1,8-diyl)bis(methylene)]bis[6-
• Compound 165 ( ⁇ (8-benzyl-1,4,8-triazacycloundecane-1,4-diyl)bis[methylene(2-hydroxy-5- methyl-3,1-phenylene)azanediyl(2-oxoethane-2,l-diyl)] ⁇ bis(phosphonic acid)), Compound 166 (3,3'-[(9-benzyl-1,5,9-triazacyclododecane-1,5-diyl)bis(methylene)]bis[N-
• the invention refers to a complex of a compound of formula (I) as above defined in any of its embodiments, hence encompassing compounds of formulae (II) to (V) as above defined, with Fe 3+ , or a physiologically acceptable salt thereof.
• the complex of the invention possesses a balanced profile of high relaxivity, kinetic inertness, thermodynamic stability and stability to reduction, making it very suitable for its use in the diagnosis field, in particular as a contrast agent for magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• the invention refers to a complex as defined above or a physiologically acceptable salt thereof for use in a method of diagnosis preferably in vivo; more preferably, the complex or a physiologically acceptable salt thereof is for use in a method of diagnosis e.g. in vivo of a pathology by magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• a further aspect of the invention is the use of the complex of the invention as defined above, or of a salt thereof, as a contrast agent, preferably for MRI.
• the invention refers to the use of the complex as defined above or of a physiologically acceptable salt thereof for the manufacture of diagnostic agents, such as contrast agents, preferably of contrast agents for magnetic resonance imaging (MRI) e.g. for in vivo applications.
• diagnostic agents such as contrast agents, preferably of contrast agents for magnetic resonance imaging (MRI) e.g. for in vivo applications.
• MRI magnetic resonance imaging
• the invention refers to a method of imaging of a body tissue in a patient comprising the steps of administering to the patient an effective amount of a complex as defined above or of a physiologically acceptable salt thereof in a pharmaceutically acceptable carrier, and subjecting the patient to magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• the invention refers to a pharmaceutical composition
• a pharmaceutical composition comprising a complex as defined above or a physiologically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
• a process for manufacturing a compound of the invention as above defined in any of its embodiment, hence encompassing compounds of formulae (I) to (V) as above defined, represents a further aspect of the present invention.
• This process can be generally carried out by coupling a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane, possibly suitably protected with one or more protecting groups on one or more nitrogen atoms, with one or more suitable moieties to obtain the compound of the invention, or to obtain an intermediate thereof that can be then converted (e.g. by further coupling and/or reduction reactions) to the compound of the invention.
• the compound of the invention can be prepared according to the following general synthesis steps: a) providing a phenol substituted at least in its orto positions (i) with a C 1 -C 5 -alkyl- bonded to a suitable leaving group, such as mesylate (MsO) or halo, e.g. thus providing a halo-C 1 -C 5 -alkyl, such as chloro-C 1 -C 5 -alkyl or bromo-C 1 -C 5 -alkyl; and (ii) with a L-Z group or a suitable substituent group that can be later converted to the L-Z groups, e.g.
• a suitable leaving group such as mesylate (MsO) or halo, e.g. thus providing a halo-C 1 -C 5 -alkyl, such as chloro-C 1 -C 5 -alkyl or bromo-C 1 -C 5 -alkyl
• the phenol can be the compound of formula (VI) Formula (VI) wherein Y 1 and R 1 have the same meaning provided for formula (I) or any embodiment thereof;
• X is a leaving group, such as mesylate (MsO) or halogen, preferably Cl or Br, and
• L 4 is a group selected from the group consisting of C 1 -C 4 -alkyl-aldehyde, C 1 -C 4 -alkyl-ester, and C 1 -C 4 -alkyl-carboxyl; b) providing a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane, optionally suitably protected with one or more protecting groups on one or more of the nitrogen atoms thereof, and/or optionally bearing on one of its nitrogen atoms a C1-C4 alkyl group optionally substituted by an aryl (such as a substituted or unsubstituted aryl); c) reacting the phenol provided in step a), e.g.
• suitable substituent groups are e.g. at least two L 4 moieties, which can advantageously be converted in the subsequent step(s) to the groups L 1 -Z 1 , L 2 -Z 2 , and eventually L 3 -Z 3 (if present).
• Such intermediate is for example the compound of formula (VII)
• L 4 is a group selected from the group consisting of C 1 -C 4 -alkyl-aldehyde, C 1 -C 4 -alkyl-ester, and C 1 -C 4 -alkyl-carboxyl;
• R" is selected from the group consisting of hydrogen, C1-C4 alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl), and the moiety of Formula (VIIA): Formula (VIIA) wherein: the asterisk (*) indicates the point of attachment of said moiety of formula (VIIA) to the nitrogen bearing the R' group;
• Y 3 has the same meaning provided above for Y 1 and Y 2 ;
• R 3 has the same meaning provided above for R 1 and R 2 ;
• L 4 is a group selected from the group consisting of C 1 -C 4 -alkyl-aldehyde, C 1 -C 4 -alkyl-ester, and C 1 -C 4 -alkyl-carboxyl; and d) converting the intermediate of step c) to the compound of the invention, e.g. converting the L 4 moieties to the groups L 1 -Z 1 , L 2 -Z 2 , and L 3 -Z 3 (if present), whereby the compound of the invention is obtained, e.g. by reacting the intermediate obtained in step c), such as the compound of formula (VII), with one or more suitable substrates, and optionally by reducing the so-obtained compound.
• C 1 -C 4 -alkyl-aldehyde refers to an alkyl group as above defined comprising from 1 to 4 carbons, one of which is an aldehyde group. Accordingly, the group C 1 -C 4 -alkyl-aldehyde comprises up to four carbons.
• C 1 -C 4 -alkyl-ester refers to an alkyl group as above defined comprising from 1 to 4 carbons, one of which is a carboxylate bound to an alkyl group (/.e. one of which is -C(O)O-R 0 , wherein R° is an alkyl group, preferably a C 1 -C 2 - alkyl group). Accordingly, the group C 1 -C 4 -alkyl-ester comprises up to four carbons (not counting the alkyl group R° bound to the oxygen).
• C 1 -C 4 -alkyl-carboxyl refers to an alkyl group as above defined comprising from 1 to 4 carbons, one of which is a carboxyl (-COOH) group. Accordingly, the group C 1 -C 4 -alkyl-carboxyl comprises up to four carbons.
• Step a) involves providing a phenol that is advantageously substituted in one of its orto positions with a leaving group-C 1 -C 5 -alkyl group, preferably a halomethyl group such as chloromethyl, or a MsO-methyl group.
• This group allows to couple the phenol to one or more, possibly two or three, of the nitrogen atoms of the macrocycle in step c).
• the phenol further comprises, in its orto position, a group that can be advantageously converted in the subsequent steps in the L 1 -Z 1 , L 2 -Z 2 , and eventually the L 3 -Z 3 (if present) groups, such as a group selected from the group consisting of C 1 -C 4 -alkyl-aldehyde, C 1 -C 4 -alkyl-ester, and C 1 - C 4 -a I kyl -carboxyl.
• the phenol may further comprise in its orto positions the L-Z group, e.g.
• the phenol of step a), e.g. the compound of formula (VI), can be obtained by reacting the correspondent non- methylene-halogenated compound with paraformaldehyde in concentrated hydrohalic acids, preferably in hydrochloric or hydrobromic acid, at a temperature comprised in the range of 30 to 70 °C, preferably of 40 to 60 °C, more preferably at 50 °C.
• Step b) involves providing a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane, optionally suitably protected with one or more protecting groups on one or more of the nitrogen atoms thereof, and/or optionally bearing on one of its nitrogen atoms a C 1 -C 4 alkyl group optionally substituted by an aryl (such as a substituted or unsubstituted aryl).
• Such macrocycle will be coupled in step c) with the phenol of step a) in order to provide an intermediate of the compound of the invention (later to be converted to the compound of the invention), or to directly provide the compound of the invention.
• the macrocycle may be unprotected.
• the nitrogen atoms of the macrocycle may be unprotected.
• the coupling reaction of step c) could involve all three unprotected nitrogen atoms of the macrocycle, whereby the compound of the invention, or an intermediate such as the one of formula (VII), with R" being the moiety of formula (VIIA), could be obtained; this intermediate can be later converted in the subsequent step(s) to a compound of the invention wherein R is the group of formula (IA).
• the nitrogen atoms of the macrocycle may be unprotected also in case the compound to be obtained has only two nitrogen atoms of the macrocycle alkylated by the phenols bearing the L-Z groups, i.e. when the compound to be obtained has R or R' being hydrogen or C 1 -C 4 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl) (or the preferred embodiments thereof disclosed above).
• step c) the reaction between the phenol and the macrocycle (step c)) could involve only two unprotected nitrogen atoms of the macrocycle instead of all three unprotected nitrogen atoms, whereby the product of such reaction is a macrocycle bonding only two (and not three) phenols bearing the L-Z groups, for example as set out in Example 6.
• step c) in particular for the macrocycles triazacyclodecane and triazacycloundecane, either all three or two out of three nitrogen atoms of the macrocycle can be de-protonated by suitably modulating the basicity of the reaction mixture of step c), whereby all three or only two out of three nitrogen atoms are alkylated during step c); e.g.
• step c when a base such as DIPEA is added to the reaction mixture of step c), all three nitrogen atoms of the macrocycle triazacycloundecane or triazacyclododecane are de-protonated, and thus a tri-alkylated macrocycle is obtained in step c); whereas if only carbonate is used in the reaction mixture, one nitrogen atom of the macrocycle triazacycloundecane or triazacyclododecane remains protonated and will not participate in the alkylation step c), thereby providing a di-alkylated macrocycle (see e.g. Example 6).
• a base such as DIPEA
• the macrocycle can also possibly be suitably protected with one or more protecting groups on one or more of the nitrogen atoms thereof.
• R or R' being hydrogen or C 1 -C 4 -alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl) (or the preferred embodiments thereof disclosed above)
• one or more of the nitrogen atoms of the macrocycle may be suitably protected with one or more protecting groups.
• step b) a macrocycle suitably protected with one or more protecting groups on one or more of the nitrogen atoms thereof, allows selecting which and how many nitrogen atoms of the macrocycle will react with the orto-substituted phenols in the coupling reaction of step c).
• any macrocycle described herein suitably protected with one or more protecting groups on one or more of the nitrogen atoms thereof can be synthesized for example starting from a suitable dialkylene triamine (e.g. diethylene triamine, dipropylene triamine, (2-aminoethyl)-1,3-propanediamine, etc.) that can be suitably protected (e.g.
• the two nosyl groups (Ns) can be removed by using thiophenol in the presence of carbonate, whereby the two deprotected amines of the macrocycle can be alkylated with the suitable pendant arms e.g. as disclosed in step c), eventually by converting the groups on the pendant arms e.g. as disclosed in step d); Boc can finally be removed e.g. as disclosed in Scheme 1 above, thus obtaining the compound of the invention wherein R or R' is hydrogen, or by eventually further alkylating the deprotected nitrogen that once was bonded Boc.
• R or R' is C 1 -C 4 alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl)
• an aryl such as a substituted or unsubstituted aryl
• R or R' is C 1 -C 4 alkyl optionally substituted by an aryl (such as a substituted or unsubstituted aryl) can also be obtained starting from commercially available mono-alkylated macrocycles, for example as illustrated in Scheme 3 below for manufacturing Compound 112:
• Step c) provides for reacting the phenols of step a) with the macrocycle of step b) to directly obtain the compound of the invention, or to obtain an intermediate of the compound of the invention, said intermediate being indeed reacted in the next step d) to obtain the final product.
• Step c) can be carried out in an organic solvent, such as toluene or acetonitrile.
• Salts such as potassium salts, for example KI, KOH, and K2CO3, can be comprised within such organic solvent, preferably in an amount of four times molar equivalents compared to the macrocycle provided in step b).
• KI might also be used in an amount of 0.05 to 0.4 molar equivalents, e.g. 0.1 to 0.2 molar equivalents, compared to the macrocycle provided in step b).
• Step c) can be advantageously carried out without heating the reaction mixture.
• step c) can be carried out at temperatures equal or lower than room temperature, that is, lower than 25 °C), for example for a temperature comprised in the range of 0 °C to 25 °C.
• Step d) is optional, and it may be optionally carried out when step c) provides an intermediate of the compound of the invention.
• Step d) involves converting the group groups of the intermediate, e.g. group L 4 , to the L 1 -Z 1 , L 2 -Z 2 , and eventually L 3 -Z 3 (if present), groups. This can be done e.g. by reacting the L 4 groups with one or more suitable substrates.
• L 4 of formula (VII) and (VIIA) could be a C 1 -C 4 -alkyl-aldehyde
• the suitable substrate could be a C 1 -C 6 -alkyl-amine substituted by two or more hydroxyl groups, such as serinol or glucamine, whereby the coupling of the two provides an imide that is later reduced to obtain the intended L 1 -Z 1 moiety (such as disclosed e.g. in Example 2 below).
• L 4 of formula (VII) and (VIIA) could be a C 1 -C 4 alkyl-aldehyde, and the suitable substrate could be e.g. diethyl-2-aminomethylphosphonate, for example as illustrated in Scheme 4 below for obtaining Compound 11.
• L 4 of formula (VII) and (VIIA) could be a C 1 -C 4 alkyl-ester, and the suitable substrate could be e.g. ammonia, for example as illustrated in Scheme 5 below for obtaining Compound 5.
• L 4 of formula (VII) and (VIIA) could be a C 1 -C 4 - alkyl-aldehyde, which can be reduced to obtain an hydroxyl group that can be in turn converted to an alkoxide; such alkoxide can take part in the well-known Williamson synthesis of ethers, whereby it is reacted with a suitable substrate such as e.g. an alkyl-hydroxyl-halide with its hydroxyl groups suitably protected, for example 2-chloro-1,3-propanediol or 2- bromo-1,3-propanediol with their hydroxyl groups suitably protected.
• a suitable substrate such as e.g. an alkyl-hydroxyl-halide with its hydroxyl groups suitably protected, for example 2-chloro-1,3-propanediol or 2- bromo-1,3-propanediol with their hydroxyl groups suitably protected.
• the compound of the invention might also be prepared by providing in step a) a phenol as defined above and bearing i.a. in its orto position the L-Z group.
• the compound of the invention can be directly obtained by reacting in step c) such orto-subtituted phenol with the macrocycle provided in step b).
• This embodiment can be illustrated for example according to the Scheme 6 below, which provides for obtaining a compound of the invention having as L 1 the following group *-NHC(O)- ⁇ , with the asterisk (*) representing the phenolic moiety and the middle dot ( ⁇ ) representing Z 1 group, and as Z 1 hydrogen (namely, Compound 1
• the compound of the invention can also be obtained by: providing a phenol according to step a) in any of its embodiment, and by: b') providing an orthoamide derivative, i.e. a tricyclic trisaminomethane derivative, of a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane, for example an orthoamide derivative of formula (VIII): wherein m, n, and o have the same meaning provided for formula (I) or any embodiment thereof; c') reacting one or two phenols provided in step a), e.g.
• an orthoamide derivative i.e. a tricyclic trisaminomethane derivative, of a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane, for example an orthoamide derivative of formula (VIII
• the compound of formula (VI), such as 2-hydroxy-3-bromometyl-5-methylbenzaldehyde with the orthoamide derivative, e.g. the orthoamide derivative of formula (VIII), to obtain an orthoamide derivative coupled with one or two phenols provided in step a), e.g. with the compound of formula (VI), such as 2-hydroxy-3-bromometyl-5- methylbenzaldehyde; d') hydrolysing the orthoamide derivative obtained in step c'), e.g.
• a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane coupled with one or two phenols provided in step a), e.g. with the compound of formula (VI), such as 2-hydroxy-3-bromometyl-5-methylbenzaldehyde, and with a formyl (-C(O)H) group; e') optionally reacting a further phenol provided in step a), e.g.
• step d' a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane coupled with two phenols provided in step a), e.g.
• step f' hydrolysing the macrocycle obtained in step d') or e'), and optionally convert the possible C 1 -C 4 -alkyl-aldehyde, C 1 -C 4 -alkyl-ester, or C 1 -C 4 -alkyl-carboxyl, wherein the hydrolysis and the optional conversion of this step f') are carried out in any order, to obtain a compound of the invention (wherein two nitrogen atoms of the macrocycle are bonded to the phenols bearing the L-Z groups, and one is bonded to a hydrogen); and g') optionally reacting a further phenol provided in step a), e.g.
• step c' provides for reacting one phenol with the orthoamide derivative
• step e' is carried out (and thus provides for reacting a further phenol with the macrocycle obtained in step d'))
• step f' provides for first carrying out the step of converting the possible C 1 -C 4 -alkyl-aldehyde and then to carry out the hydrolysis step to obtain the compound of the invention
• step g' is not carried out:
• the orthoamide derivative provided in step b') can be obtained according to standard means in the art, e.g. by reacting a macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane, with formaldehyde dimethylacetal, or as reported in T. Atkins, J. Am. Chem. Soc. 1980, 102, 6364- 6365; R. W. Alder et al. J. Chem. Soc. Chem. Commun. 1992, 507-508, e.g. starting from 1,4,6-triazabicyclo[3.3.0]oct-4-ene 1,5,7-triazabicyclo[4.4.0]dec-5-ene.
• step d' allows converting the orthoamide derivative to the macrocycle bonding a formyl group (as well as bonding the phenol(s) previously reacted in step c')).
• the formyl group seems to predominantly bond the nitrogen atom of the macrocycle that is less sterically hindered, such as e.g. one of the two nitrogen atoms in-between the ethylene and propylene moieties (and not the one in-between the two propylene moieties) of the macrocycle triazacycloundecane.
• step d') can be carried out before step c'); in this case, step c') is carried out by reacting one or two phenols provided in step a) with the macrocycle obtained by the hydrolyzation step d'), i.e. with the macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane coupled with a formyl (-C(O)H) group.
• the macrocycle obtained by the hydrolyzation step d' i.e. with the macrocycle selected from the group consisting of triazacyclononane, triazacyclodecane, triazacycloundecane, and triazacyclododecane coupled with a formyl (-C(O)H) group.
• a step providing for complexing the compound of the invention with Fe(III) may be carried out, e.g. after step c) or step d), or after step f') or g'); said complexation step can be carried out for example according to the following step: e) reacting the compound of the invention, for example as obtained in step c), step d), step f') or step g'), with a Fe(III) salt, such as with FeCl 3 , Fe(NO 3 )3, Fe(OH) 3 , and FeO(OH) to obtain the Fe(III) complex of the compound of the invention.
• a Fe(III) salt such as with FeCl 3 , Fe(NO 3 )3, Fe(OH) 3 , and FeO(OH
• Step e) can be carried out in a non-aqueous polar solvent, such as a lower alcohol, e.g. methanol, ethanol, n-propanol, i-propanol, and mixtures thereof.
• a non-aqueous polar solvent such as a lower alcohol, e.g. methanol, ethanol, n-propanol, i-propanol, and mixtures thereof.
• Reactants and/or solvents employed in the following examples that are not specifically synthesized in the following Examples are known and readily available. If they are not commercially available per se, they may be prepared according to known methods in literature.
• Triazacyclononane.3HCI (0.095 g, 0.4 mmol, TACN) was dissolved in a small amount of toluene. KI (0.007 g, 0.04 mmol) and KOH (0.067g, 1.2 mmol) were added and the solution was cooled to 0°C. (2-hydroxy-5-metyl-3-chlorometyl)methyl benzoate (0.258 g, 1.2 mmol) obtained in the previous step was dissolved in 2 ml of toluene and added dropwise in ca. 15 min to avoid as much as possible polyalkylation reactions. Then, the reaction mixture was stirred at room temperature for 2 h.
• Serinol (2-Amino-1,3-propanediol, 0.045 g, 0.5 mmol) was dissolved in DMF (1 mL) and added to a solution of 1,4,7-tris-(3-carboxymethyl-2-hydroxy-5- methylbenzyl)-1,4,7-triazacyclononane (0.066 g, 0.1 mmol, obtained in the previous step) in DMF (2 mL). The reaction mixture was heated to 50°C and stirred overnight.
• Triazacyclononane (0.042 g, 0.18 mmol, TACN) was dissolved in 3 ml of acetonitrile and KI (0.003 g, 0.018 mmol) and KOH (0.030g, 0.54 mmol) were added.
• 2-hydroxy-3-chlorometyl-5-methylbenzaldehyde (0.21 g, 0.9 mmol) obtained in the previous step, dissolved in 1 ml of acetonitrile, was added dropwise to the solution. The reaction mixture was heated to 60 °C and stirred overnight.
• the product was purified using semi-preparative HPLC-MS and obtained after lyophilization as a white monotrifluoroacetate salt (0.16 g, 53%).
• the reducing agent was quenched by adding dropwise 1 ml of water and then the solvent was removed under reduced pressure.
• the product was redissolved in 20 ml of ethanol, the precipitate was removed by filtration and finally the solvent was removed under reduced pressure.
• ESI-MS (m/z) 604.5 (M+H + ) (calculated for C 32 H 53 N 5 O 6 : 603.8).
• Fe(III) chelates were prepared through complexation reaction, by dissolving the ligands obtained in Examples 1, 2, 7 and 8 in ethanol, and in Examples 3 to 6 in water, and then by adding FeCH or Fe(NO 3 ) 3 with equimolar Fe 3+ .
• the reaction was carried out at 298 K for 18 h.
• the solvent was then removed, the product dissolved in water, and (for all the complexes) the pH of the solution was corrected to 8.5 with diluted NaOH in order to promote the precipitation of any excess of free Fe 3+ .
• the relaxivity values of some the complexes of the invention were evaluated by measuring them at 298 K at the magnetic fields that are relevant in clinical practice, namely at 1.5 and 3.0 T as follows.
• 1 H NMRD profiles were measured in aqueous solution by using a variable field relaxometer equipped with an HTS-110 3T Metrology Cryogen-free Superconducting Magnet (Mede, Italy), operating in the overall range of proton Larmor frequencies of 20-120 MHz (0.47-3.00 T).
• the measurements were performed using the standard inversion recovery sequence (20 experiments, 2 scans) with a typical 90° pulse width of 3.5 ps and the reproducibility of the data was within ⁇ 0.5%.
• the temperature was controlled with a Stelar VTC-91 heater airflow.
• the complexes of the invention do not substantially interact with proteins of the human serum, such as e.g. human serum albumin, because the relaxivity values in SeronormTM (lyophilized human serum) are similar to the values obtained in the two other conditions, i.e. NaCI 0.15 M and NaCI 0.15 M (HCO 3 - 25 mM).
• the complexes of the invention could have a faster clearance and a homogeneous distribution into the organs and textiles, and thus could have a broader spectrum of MRI applications, compared to complexes interacting with proteins of the human serum.
• thermodynamic stability constants of the respective ligands were determined by pH-potentiometry and/or by UV spectrophotometry.
• thermodynamic stability constant of the Fe(III) complexes of the invention were determined by observing the competition reaction between the Fe(III) complexes and the N,N'-bis(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid (HBED) ligand using capillary zone electrophoresis (CZE) for Compound 2 and Vis spectrophotometry for Compound 4.
• HBED is the ligand with the following formula that forms a complex with Fe(III) having very low relaxivity (rl of 0.49 mM -1 ⁇ s -1 at 60 MHz, 40°C in PBS buffer as mentioned in Bales et al., Contrast Media & Molecular Imaging, Volume 2019, Article ID 8356931)
• Solid Fe(NO 3 )3 was dissolved in 0.1 M HNO3 solution.
• the concentration of Fe(NO3)3 solution was determined by using standardized Na 2 H 2 EDTA in excess.
• the excess of the Na 2 H 2 EDTA was measured with a standardized ZnCI 2 solution and xylenol orange as indicator.
• the H + concentration of the Fe(NO3)3 solution was determined by pH potentiometric titration in the presence of Na 2 H 2 EDTA excess.
• the concentration of the ligands Compound 2, Compound 4, H3NOTA (comparative - see formula below) and H4HBED (comp.) was determined by pH-potentiometric titrations in the presence and absence of a 40-fold excess of Ca 2+ .
• pH-potentiometric titrations were made with standardized 0.2 M NaOH (concentration of the ligands was generally 0.002 M).
• concentration of the ligands was generally 0.002 M.
• Metrohm 888 Titrando titration workstation Metrohm- 6.0234.110 combined electrode was used.
• the deprotonation of the phenol-OH and isoserinol-NH2 + groups of Compound 2 was investigated by spectrophotometry on the absorption band of the aromatic group of the ligand, following the absorbance values at 243 and 303 nm.
• the absorbance of the ligand is a combination of the absorption of each protonated species and expressed by the following equation (Beck, M. T.
• the protonation constants of Compound 4 have been determined by 1 H-NMR spectroscopy with Bruker Avance III (9.4 T) spectrometer, equipped with Bruker Variable Temperature Unit (BVT), Bruker Cooling Unit (BCU) and a BB inverse z gradient probe (5 mm) by recording the chemical shift variations of the non-labile protons as a function of pH at 25°C in 0.15 M NaNO 3 solution. Since the protonation/deprotonation is fast on the NMR time scale, the chemical shifts of the observed signals represent a weighted average of the shifts of the different species with different protonation states and expressed by the following equation (Pagado, J. M.; Goldberg, D.
• Metrohm 888 Titrando titration workstation Metrohm-6.0234.110 combined electrode was used for the pH measurements and titrations. Equilibrium measurements were carried out at a constant ionic strength (0.15 M NaNO 3 or NaCI04) in 6 ml samples at 25 °C. The solutions were stirred, and N2 was bubbled through them. The titrations were made in the pH range of 1.7- 12.0.
• thermodynamic stability constants ([MLigand] I ([M] x [Ligand]) of the Fe(III) complexes were determined as follows.
• the concentrations of Fe 3+ and NOTA were 0.002 M.
• the samples were kept at 25°C for two weeks.
• the absorbance values of the samples were determined at 11 wavelengths (370, 380, 390, 395, 400, 405, 410, 415, 420, 425 and 430 nm).
• the molar absorptivities of Fe 3+ and Fe(NOTA) were determined by recording the spectra of l.OxlO' 3 , 1.5xl0' 3 , 2.0xl0' 3 and 2.5xl0 -3 M solutions of Fe 3+ and Fe(NOTA) solutions.
• the absorption spectra of the Fe(NOTA) solutions were recorded in the pH range of 1.7 - 7.5. All spectrophotometric measurements were performed at 25°C in 0.15 M NaNO 3 solution.
• the pH was adjusted by stepwise addition of concentrated NaOH or HNO3 solutions.
• the stability constants of the Fe(HBED) complex was determined by following the competition reaction between HBED and NOTA ligands for Fe 3+ -ion with spectrophotometry at the absorption band of Fe(HBED) complex in the wavelength range of 400-700 nm.
• the time needed to reach the equilibria was determined by spectrophotometry.
• thermodynamic stability constants were calculated with the PSEQUAD program. (L. Zekany et al., Computational Method for Determination of Formation Constants, Ed. Legett D J, Plenum, New York, 1985, p. 291.)
• thermodynamic stability constants of the Fe(Compound 2) complex was determined by following the competition reaction between Compound 2 and HBED ligands for Fe 3+ -ion with Capillary Zone Electrophoresis (CZE) at the signal of Fe(Compound 2) complex.
• CZE Separations were performed with Agilent 7100 Capillary Electrophoresis system using bare fused-silica capillaries of 64 cm x 50 pm i.d. (Agilent). Before the first use of the capillary, the latter was washed with 1.0 M NaOH (15 min), with 0.1 M NaOH (30 min) and with the buffer electrolyte (30 min).
• the individual linear regression equation (response-concentration) for Fe(Compound 2) was determined according to three concentrations.
• the peak areas were found to be linear (R2>0.998) in a 22 - 87 ⁇ M concentration range with a precision better than 4%.
• the stability constant of the Fe(Compound 4) complex was determined by following the competition reaction between Compound 4 and HBED ligands for Fe 3+ -ion with spectrophotometry at the absorption band of Fe(Compound 4) and Fe(HBED) complexes in the wavelength range of 400-700 nm.
• thermodynamic stability constants of the Fe(Compound 4) complexes For the calculations of the thermodynamic stability constants of the Fe(Compound 4) complexes, the molar absorptivities of [Fe(Compound 4)H-i], [Fe(HBED)]- and [Fe(HBED)H -1 ] 2- species were determined by recording the spectra of 0.5, 0.1 and 0.2 mM solutions of [Fe(Compound 4)] and [Fe(HBED)]- in the pH range 7.0 - 12.5 in the presence of 0.15 M NaNO 3 . The spectrophotometric measurements were made with the use of PerkinElmer Lambda 365 UV-Vis spectrophotometer, using 1.0 cm cells. The thermodynamic stability constants were calculated with the PSEQUAD program. (L. Zékány et al., Computational Method for Determination of Formation Constants, Ed. Legett D J, Plenum, New York, 1985, p. 291.)
• thermodynamic stability and protonation constants of Fe(III) -complexes of Compound 2 and Compound 4 as well as of comparative complexes NOTA, and HBED are summarized in Table 2 below.
• Fe(Compound 2) has the highest thermodynamic stability, confirming the in vivo safety of the Fe(III) complex of the invention; such high thermodynamic stability is even higher than that of the comparative Fe(NOTA) complex.
• Fe(Compound 4) shows a very high thermodynamic stability, even higher than that of the comparative Fe(NOTA) complex.
• the transchelation reactions of Fe(Compound 4) and Fe(NOTA) (comparative) were studied by spectrophotometry, following the formation of the resulting Fe(HBED) complex at 472 and 470 nm with PerkinElmer Lambda 365 UV-Vis spectrophotometer.
• the concentration of the Fe(Compound 4) and Fe(NOTA) complex was 0.1 and 0.2 mM, while the concentration of the HBED was 10 - 200 times higher, in order to guarantee pseudo-first-order conditions.
• the temperature was maintained at 25 °C and the ionic strength of the solutions was kept constant, 0.15 M for NaNO 3 .
• the exchange rates were studied in the pH range about 9.5 - 14.0.
• the ligand exchange reactions in Fe(Compound 2) - HBED reacting systems have been studied by Capillary Zone Electrophoresis (CZE) in the pH range 9.5 - 11.5.
• CZE Capillary Zone Electrophoresis
• the transchelation reactions of Fe(Compound 2) were studied by following the dissociation of the Fe(Compound 2) complex with Agilent 7100 Capillary Electrophoresis system (using the same conditions of the CZE experiments of Example 11).
• the concentration of the Fe(Compound 2) complex was 87 ⁇ M, while that of HBED was 0.1 and 0.2 M in order to guarantee pseudo-first-order conditions.
• the temperature was maintained at 25 °C and the ionic strength of the solutions was kept constant, 1.0 M for NaCIO 4 .
• the pseudo-first-order rate constants (kd) were calculated by fitting the area-time data pairs to Equation 2 above, where A t , A o and A p are the area values at time t, the start of the reaction and
• Transferrins are Fe 3+ -binding transport proteins present in the body fluids.
• the concentration of the Fe(III)-Compound 2, Fe(III)-Compound 4, Fe(III)-Compound 57, Fe(III)-Compound 60 and Fe(III)-Compound 61 complexes and 22.6% Fe 3+ saturated human serum transferrin was 0.1 mM.
• the temperature was maintained at 25 °C and the ionic strength of the solutions was kept constant (0.15 M of NaCI).
• the pH of the sample was adjusted by stepwise addition of the concentrate NaOH and HCI solution.
• Fe(III) such as of Fe(III)-Compound 2, Fe(III)- Compound 4, Fe(III)-Compound 57, Fe(III)-Compound 60 and Fe(III)-Compound 61.
• the redox stability of the Fe(III)(NOTA), Fe(III)-Compound 2, Fe(III)-Compound 4, Fe(III)-Compound 57, Fe(III)-Compound 60 and Fe(III)-Compound 61 was characterized by assessing the rates of their reduction with ascorbic acid, observing the reduction reactions by spectrophotometry, following the formation of the Fe(II)-Ligand complexes at 375 nm for Fe(III)(NOTA), 480 nm for Fe(III)-Compound 2 and Fe(III)- Compound 57, 471 nm for Fe(III)-Compound 4 and 500 nm for Fe(III)-Compound 60 and Fe(III)-Compound 61 with PerkinElmer Lambda 365 UV-Vis spectrophotometer.

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