WO2008134289A2

WO2008134289A2 - High relaxivity coordinatively unsaturated lanthanide complexes

Info

Publication number: WO2008134289A2
Application number: PCT/US2008/061078
Authority: WO
Inventors: Dennis A. Moore
Original assignee: Mallinckrodt Inc.
Priority date: 2007-04-26
Filing date: 2008-04-22
Publication date: 2008-11-06
Also published as: WO2008134289A3

Abstract

The present invention is directed to tetraazamacrocyclic compounds and lanthanide complexes thereof useful as MRI contrast agents having enhanced relaxivity.

Description

HIGH RELAXIVITY COORDINATIVELY UNSATURATED LANTHANIDE COMPLEXES

FIELD OF THE INVENTION

[0001] The present invention generally relates to stable MRI contrast agents that exhibit enhanced relaxivity.

BACKGROUND OF THE INVENTION

[0002] Diagnostic imaging is an important non-invasive tool for the evaluation of pathology and physiology. Besides plain film x-ray and computerized tomography ("CT"), nuclear magnetic resonance imaging ("MRI") has become a widely used imaging modality. MRI uses non-ionizing strong magnetic fields and radiofrequency pulses to generate signals for producing images, and is best suited for imaging soft (non-calcified) tissue. Proton MRI is based on the acquisition of images underlining the physical properties of hydrogen nuclei, or "protons", in different environments. The most abundant proton source in biological tissues is water. It is the quantum mechanical "spin" of the water proton nuclei that ultimately gives rise to the signal in all imaging experiments. The differences in concentration and magnetic characteristics of protons (i.e., nuclear spin relaxation) in tissues and organs influence the intensities of the MRI signals from these sources to varying degrees. These differences form the basis for tissue differentiation on MR images, i.e., image contrast. Additionally, contrast agents can assist in delineating areas of interest by changing the nuclear spin relaxation characteristics of protons in vivo. The distinguishing feature of MRI agents is the presence of unpaired electrons within their atomic orbitals, which create local magnetic fields (paramagnetism) that interact with the nuclear spins of tissue water protons (hydrogen). In some instances, the atoms bearing unpaired electrons may be organized into groups (domains) that exert even greater local magnetic fields (superparamagnetism).

[0003] In an MRI experiment, the sample to be imaged is placed in a strong homogeneous static magnetic field (on the order of 1-12 Tesla). The presence of the magnetic field results in the redistribution of the proton spins into the two allowed quantum mechanical states, i.e., approximately one half aligned with the field (parallel; lower energy level) and the other half against the field (antiparallel; higher energy level). This establishes the ground state distribution in the presence of the magnetic field (net zero energy). An excited state distribution (net high energy) is achieved by an uneven population of the two levels, for example, when more of the hydrogen spins are aligned antiparallel than parallel. Such spin excitation is accomplished by introduction of energy from an external source, e.g., with a pulse of radiation in the radio frequency ("RF") region. The net effect of spin excitation is to produce a temporary net magnetization in the sample. The net magnetization decays or relaxes to the ground state by various relaxation processes (commonly longitudinal relaxation or T₁; transverse relaxation or T₂; see below) in the absence of continuous permanent application of pulses. It is the decay of magnetization that is converted to signals observed in MRI. Typically, the intensity of a signal arising from a decay process as described above is given by equation 1.

SI = [H]*k* {l-exp(-TR/Ti)} (exp(-TE/T₂)}, (1)

where [H] is the concentration of water hydrogens, k is a constant that includes instrument- specific- and motion-related factors, Ti and T₂ are the respective longitudinal and transverse proton relaxation times, TR is the pulse repetition time and TE is the echo delay time. TR and TE are extrinsic instrument selectable variables which are uniquely selected for the pulse sequence applied during the imaging experiment. According to equation (1), Ti and T₂ have inverse and reciprocal effects on image intensity; hence, image intensity may be increased either by shortening the longitudinal relaxation time Ti or lengthening the transverse relaxation time T₂.

[0004] In order to differentiate signals from different locations, various magnetic field gradients and other RF pulses are additionally applied on the spins to encode spatial information into the recorded signals. The relaxation of the spins is recorded as a function of time. MR images are typically displayed on a gray scale with the color black representing the lowest measured intensity and white representing the highest measured intensity.

[0005] Although variations in water concentration [H] can give rise to contrast in MR images, it is the dependence of the rate of change in the magnetization on local environment that is the major source of variation in MRI signal intensity (i.e., exponential terms in equation 1). The two characteristic relaxation times implicated in magnetic relaxation are the Ti and T₂ relaxation times. Ti is the longitudinal relaxation time, also known as the spin lattice relaxation time, and characterizes energy loss to the lattice or surroundings (the associated rate constant, Ri = 1/T₁, is the spin-lattice relaxation rate constant). T₂ is the transverse relaxation time, or spin-spin relaxation time, and is one of several contributions to T₂*. The associated rate constant, R₂ = 1/T₂, is the spin- spin relaxation rate constant. [0006] The signal amplitude recorded for any given scan is proportional to the number of spins that have decayed (or relaxed) back to equilibrium in the time period between successive scans. Thus, regions with rapidly relaxing spins (i.e. spins having short Ti values) will recover all of their signal amplitude between successive scans. The measured intensities of the regions with long T₂ and short Ti will reflect the spin density, i.e., that region's water content. Regions with long Ti values, as compared to the repetition time (time between scans, TR), will progressively lose signal (i.e., signal will broaden, "flatten out") until a steady state condition is reached when these regions will appear dark.

[0007] Clinical MRI takes advantage of the fact that water relaxation characteristics (e.g., Ti and T₂ values) vary between tissues. This inherent differential tissue relaxation provides image contrast which allows tissue identification. Additionally, the MRI experiment can be set up so that regions of a sample with short Ti values or long T₂ values are preferentially enhanced. Such designed MRI protocols are known as Ti-weighted and T₂- weighted imaging protocols. For a description of the MRI experimental process, see R. Hashemi, MRI: The Basics, 2nd ed., Lippincott, Williams & Wilkins, 2004 and V. Runge, The Physics of Clinical MR. Thieme, NY, 2005.

[0008] Paramagnetic contrast agents serve to modulate Ti and/or T₂ values. MRI contrast agents comprise compounds of both paramagnets and superparamagnets such as transition metal-chelate compounds and nitroxyl spin labeled compounds (paramagnets) and iron oxide suspensions (superparamagnets) which act as potent relaxation enhancement agents, i.e., they decrease the Ti and T₂ relaxation times of nearby proton spins. The relaxivity of a contrast agent is dominated by the selection of the metal atom. Gadolinium (Gd³⁺, f⁷), iron (Fe³⁺, d⁵) and manganese (Mn²⁺, d⁵) are the most commonly studied. The mechanism of Ti relaxation is generally a through-space dipole-dipole interaction between the unpaired electrons of the paramagnet and the bulk water molecules that are in fast exchange with inner sphere (IS) water molecules. In other words, the shortening of proton relaxation times by a metal ion (Gd(III), Gd³⁺) is mediated by adjacent (inner sphere, IS) water protons. The effectiveness of Gd(III)'s magnetic dipole drops off very rapidly as a function of its distance (r) from these protons, (r⁶ distance dependency). Therefore, bound water (IS) molecules are most affected.

[0009] The relaxation rate constant, R₁, has several components of which the inner sphere (IS) pathway to longitudinal relaxation rate enhancement is dominant. The longitudinal IS proton relaxation rate, X_\, is expressed as follows 55.5(7;. + r.)

where [M] is the concentration of paramagnetic metal ions, q is the number of inner sphere water molecules, i.e., water molecules directly bound to the metal ion, T_lm is longitudinal proton relaxation time, and T_M is the water exchange lifetime, i.e., lifetime of association of the water molecule with the metal center. For practical reasons, the relaxivity of a contrast agent is derived from the change in relaxation rate constant (ΔR) induced by a known concentration of the contrast agent ([M]), i.e., ΔRi/[M] (or AR₂Z[M]). Relaxivities are typically expressed in units of mM^sec^"1.

[0010] The water exchange lifetime, T_M, represents the lifetime of the water proton (or water molecule) in the gadolinium chelate complex (or the rate of exchange with the bulk water). It plays a dual role in its contribution to the relaxivity; hence, its contribution to relaxivity is more complicated and must be assessed with care. Generally, complexes with labile water molecules (T_M ~ 10^"8 sec) demonstrate high relaxivities (~4 mM^sec^'1). However, there are instances of "slow exchange" where the M-O bond remains intact but the MO-H bond breaking may be fast enough to yield significant relaxivities that approach the values typical for metal complexes containing rapidly exchanging water molecules (i.e., -1-2 mM^sec^"1). For a full description of the above relationships and their relevance to contrast agent design, see P. Caravan et al., Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications, Chem. Rev. 1999, 99, pp. 2293-2352 and M. F. Tweedle, Relaxation Agents in NMR Imaging, Lanthanide Probes in Life, Chemical and Earth Sciences, ed. by J. C. G. Bϋnzli and G. R. Choppin, (Amsterdam: Elsevier) pp 127-179 (1989).

[0011] Thus, although variations in tissue water concentration can give rise to MRI contrast, it is the strong dependence of the rate of change in the magnetization of the local water molecules that is the major source of variation in image contrast in an MRI experiment. The clinical effectiveness of paramagnetic contrast agents which serve to modulate Ti and/or T₂ values is now amply demonstrated in the literature. The capacity to differentiate between regions or tissues that can be magnetically similar but histologically different is a major impetus for the preparation of these contrast agents. Paramagnetic contrast agents provide additional image contrast by enhancing signal from those areas within which the contrast agent is localized.

[0012] When designing contrast agents for use in MRI experiments, attention must be given to a variety of properties that will ultimately affect the physiological applicability of the agent, as well as the ability of the agent to provide contrast enhancement in an MRI image. Two fundamental properties that must be considered are (i) biocompatibility, and (ii) proton relaxation enhancement. Biocompatability is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Amongst several other factors, proton relaxation enhancement (and relaxivity) is predominantly governed by the choice of metal employed in the agent and the accessibility of the metal to surrounding water molecules, which permits the rapid exchange of metal-associated water molecules with the bulk solvent. For a detailed discussion of contrast agent development, see Caravan et al. and Tweedle as noted above.

[0013] Gadolinium ion, Gd(III), has generally been chosen as the metal atom for contrast agents because it has a high magnetic moment (μ² =63 BM²), a symmetric electronic ground state (f⁷; S⁸), a large paramagnetic dipole and the largest paramagnetic relaxivity of any element. In order to render the Gd(III) complex nontoxic, Gd(III) can be chelated with any of a number of substances such as diethylenetriamine-pentaacetic acid ("DTPA"), 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid ("DOTA"), and derivatives thereof. See, U.S. Patent Nos. 5,155,215; 5,087,440; 5,219,553; 5,188,816; 4,885,363; 5,358,704; and 5,262,532; and D. Meyer et al., Advances in Macrocyclic Gadolinium Complexes as Magnetic Resonance Imaging Contrast Agents, Invest. Radiol. 25:S53 (1990). The stability constants (K) for the Gd-DTPA and Gd-DOTA complexes are very high (logK=22.4 and 28.5 respectively) which indicates that the fraction of Gd(III) ions that are in the unbound state will be quite small.

[0014] Several agents have been approved for clinical use, for example the dimeglumine salt of the chelate Gd-DTPA (Magnevist, Schering AG). Image enhancement achieved using Gd-DTPA is well documented for a variety of applications, see for example E.J. Russell et al., Multicenter Double-Blind Placebo-Controlled Study of Gadopentetate Dimeglumine as an MR Contrast Agent: Evaluation in Patients with Cerebral Lesions, (1989) Am. J. Roentgenol. 152:813. The macrocyclic analog, Gd-DOTA complex, based on the tetraazamacrocycle cyclen, is conformationally more rigid, and at physiological pH, possesses very slow dissociation kinetics and is therefore presumed safer. Regions associated with a metal ion (Gd(III)) having proximate water molecules appear bright in an MR image, while the normal aqueous solution appears as dark background when the time between successive scans in the experiment is short, i.e., in a Ti weighted image.

[0015] Cyclen, 1,4,7,10-tetraazacyclododecane, is the scaffold commonly used for generating several key ligands for formation of metal complexes. The tris-N- carboxymethylated tetraazamacrocycle, 4,7,10-triscarboxymethyl- 1 ,4,7, 10- tetraazacyclododecane, D03A, is a ligand by itself and also a key intermediate which can be variously derivatized, either at the free macrocyclic nitrogen or on the ethylene backbone, for formation of a range of ligands for several applications. Various metal complexes of D03A are useful for imaging and therapy. For example, in US Patent No. 4,877,600, Bonnemain et al. disclosed the gadolinium complex of DOTA for imaging/MRI. In US Patent No. 4,885,363, Tweedle et al. similarly disclosed a gadolinium complex of DO3A-HP, a DO3A-derivative in which the free macrocyclic N was alkylated with 2-hydroxypropyl, for imaging. Similarly, in US Patent No. 5,994,536, Petrov et al. disclosed another gadolinium complex for MRI, Gd- DO3A-butriol in which the alkyl substituent at the free macrocyclic N is 2,3-dihydroxy-l- (hydroxymethyl)propyl. Therefore, most new agents under development are based on the Gd- D03A motif. Several agents based on the Gd-DTPA and Gd-DO3A motif are extracellular agents, i.e., they accumulate in tissue by perfusion-dominated processes and perfuse the extracellular fluid spaces only.

[0016] The gadolinium complexes currently in use as MRI contrast agents are mono aqua complexes, i.e., q = 1 in equation (2) above. The relaxivities of the Gd-based agents described above are about 4 mM^sec^"1 in aqueous solution and in vivo. However, a newer class of q =1 agents are emerging which have relaxivities of about 4 mM^sec ^-1 in aqueous solutions but display significantly enhanced relaxivities upon interaction with serum albumin or other proteins. Such protein interaction in vivo results in an increase in rotational correlation time τ_r which translates to an increase in Rμ For a full discussion of relaxation enhancement pathways involving interaction with macromolecules, see Tweedle et al., and Lauffer et al., as cited herein. Examples of these "tunable" agents are Vasovist (EPIX Pharmaceuticals, Inc. Cambridge MA) and MultiHance (Bracco SpA, Milan). However, benefits from protein binding are not without risk. Binding to transport protein carries the risk of displacing drugs, and other transported endogenous species, from the binding site. Such a release may have a catastrophic outcome for a patient due to the sudden rise in concentration of a released drug in the blood stream, resulting in toxicity. Additional pitfalls upon protein binding include prolonged exposure of the patient to the contrast agent, increasing the likelihood of toxicity due to the contrast agent itself, as well as deposition of the agent in non-target sites due to non-specific protein binding. Unwanted venous brightening, for instance, in the typical MR angiography procedure, is also a risk of protein-bound blood persistent agents. The procedure is typically practiced dynamically, where imaging is performed at the moment the first-pass bolus rushes past the imaging area of interest. If there is an error in the procedure, then the clinician would have to wait for the agent to excrete before the procedure could be performed again, due to increased concentration of the agent in veins and the resulting complicated the field of view. Protein-bound agents typically have a much longer excretion time as compared to nonprotein bound agents.

[0017] From equation (2) above, diaqua (q = 2) and triaqua (q = 3) complexes would be expected to yield large increases in relaxivity. However, no diaqua or triaqua agents have been developed because of the expectation, based on data from other metal complexes with increased inner sphere water molecules, that they may be kinetically unstable with respect to acid or cation-mediated dissociation in vivo. There is a possibility that the interaction of diaqua or triaqua compound with physiologically endogenous anions such as phosphate, carbonate or lactate may result in displacement of the inner sphere water molecules; hence a decrease in relaxivity.

[0018] However, it has been shown recently that repulsions between such endogenous anions and appropriately designed highly anionic gadolinium analogs can be sufficient to prevent interactions that would displace the inner sphere water molecule(s) (see Messeri et al., Chem. Comm. 2001, 2742.). For example, Messeri reported that Gd-DO3A derivatives may give enhanced relaxivity if an appropriate bifurcated side arm is incorporated. For example, when a carboxylate of appropriate length, e.g., -CH₂(CH₂)D-CO₂H, a tris anionic complex is formed with Gd in which the pKa of the coordinated water is raised so that deprotonation would not occur. With the systematic increase in chain length, a significant increase in relaxivity was observed at n = 2, and it was subsequently confirmed that q = 2 was achieved for this complex and deprotonation of coordinated water does not occur due to the high anionic charge of the complex. Further, the n = 2 side arm carboxylate functionality is too long to effectively displace one of the inner sphere water molecules by binding to the gadolinium and yet the high negative charge repels other potential donors that would destabilize the Gd-complex by displacement of the bound water molecules. Thus the observation of Messeri et al. has established the basis for generating high relaxivity Gd complexes in water without protein binding.

[0019] The choice by Messeri et al. to use a strongly dissociated species, like the three non-coordinating carboxylate moieties in the chelate side arms, results in a highly charged, tris(anionic), complex. While this high negative charge is necessary to maintain a high relaxivity by preventing ionization of the IS water molecule (i.e., formation of Gd-OH^" from Gd-OH₂), and for repulsion of other potential ligands that would otherwise displace the IS water molecule, it nonetheless poses a clinical risk since high osmolality solutions are not desired as injectable contrast media due to safety concerns. The current invention addresses some of the persistent limitations of contrast agent design. SUMMARY OF THE INVENTION

[0020] Among the various aspects of the present invention is the provision of coordinatively unsaturated lanthanide complexes exhibiting enhanced relaxivity.

[0021] Briefly, the present invention is directed to a coordinated or uncoordinated tetraazamacrocycle having 12, 13 or 14 ring atoms. Generally, this macrocycle contains 4 ring nitrogen atoms and 8-10 ring carbon atoms where each ring nitrogen atom is separated from another ring nitrogen atom by at least two ring carbon atoms. The macrocycle is also substituted by at least three groups that are ionizable at physiological pH, provided that a di- aqua macrocyclic complex formed with a lanthanide metal having a +3 charge has an overall net charge of -1. The overall net charge of the heptacoordinate complexes of the present invention is determined from the formula: Overall Net Charge = ∑(charges of the three ionized coordinating groups) + valence of the lanthanide metal (e.g., +3) + ∑(charges of the ionized non-coordinating groups) = -1. Three of the ionizable groups are capable of coordinating with a single lanthanide metal when the macrocycle forms a lanthanide complex, and are independently carboxyl, phosphorous-oxo acid or sulfur-oxy acid groups; the three ionizable groups being bonded, directly or indirectly, to two or three ring atoms of the macrocycle. In addition, each of these three ionizable groups is separated from the ring atom to which it is bonded by no more than three atoms. In the embodiment where these three ionizable groups are attached to two atoms of the macrocycle, two ionizable groups are attached to the macrocycle via a single macrocycle atom. For example, two ionizable groups may both be substituents on a single chain connected to the macrocycle atom or, in the case of a macrocycle carbon atom having two bonds available for attachment of substituent groups, two ionizable groups may be substituents on separate chains connected to the macrocycle atom. Depending on whether the ionized coordinating groups are mono-anionic (e.g., -C(O)(OH) groups) or di- anionic (e.g., -P(OH)2, -P(O)(OH)2, and -S(O)(OH)2 groups), the number and charge of the ionized non-coordinating groups will be the number necessary to result in an overall net charge of -1.

[0022] Other features will be in part apparent and in part pointed out hereinafter.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The tetraazamacrocyclic compounds of the present invention (sometimes referred to herein as macrocyclic compounds or macrocycles) enable production of improved contrast agents for use in MR imaging due to increased relaxivity. The macrocyclic compounds tend to form stable di-aqua complexes with lanthanide metals, typically gadolinium. In particular, the macrocycles of the present invention comprise 12-, 13-, or 14- ring members and bear at least three groups that are ionizable at physiological pH, provided that a di-aqua macrocyclic complex formed with a lanthanide metal having a +3 charge has an overall net charge of -1. The overall net -1 charge of the heptacoordinate complexes of the present invention is determined from the formula: Overall Net Charge = ∑(charges of the three ionized coordinating groups) + valence of the lanthanide metal (e.g., +3) + ∑(charges of the ionized non-coordinating groups) = -1. Depending on whether the ionized coordinating groups are mono-anionic (e.g., -C(O)(OH) groups) or di-anionic (e.g., -P(OH)2, -P(O)(OH)2, and - S(O)(OH)2 groups), the number and charge of the ionized non-coordinating groups will be the number necessary to result in an overall net charge of -1. The sum of the charges of the three ionized coordinating groups may be -3, -4, -5, or -6 depending on the selection of the ionized coordinating groups. When the macrocycle is required to be substituted with one or more non- coordinating groups having a negative charge at physiological pH to produce a complex having an overall net charge of -1, the non-coordinating, ionizable group capable of generating the negative charge is selected from carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; wherein EWG is an electron withdrawing group. When the macrocycle is required to be substituted with one or more non- coordinating groups having a positive charge at physiological pH to produce a complex having an overall net charge of -1, the non-coordinating group capable of generating the positive charge is typically a group containing a primary, secondary or tertiary amine that can be protonated at physiological pH. The non-coordinating, ionizable group may either be a substituent on a single chain connected to a macrocycle atom or a substituent on a chain also containing one of the coordinating ionizable groups.

[0024] The following Table I provides an exemplary list of substituent combinations of coordinating and non-coordinating groups for the macrocycles of the present invention that are capable of producing a lanthanide complex of the present invention having an overall net charge of -1. Table I

[0025] In one embodiment, three of the ionizable groups function to coordinate a single lanthanide metal and are carboxyl groups while a fourth ionizable group imparts a single negative charge on the complex, at physiological pH, but does not participate in the coordination of the lanthanide.

[0026] In one aspect of the present invention, the macrocycle corresponds to Formula (I)

[0027] For the macrocycles corresponding to Formula (I), mi through m₄ are independently 1 or 2, provided the sum of mi through m₄ is 4-6. In one embodiment, the sum of mi through m₄ is 4 or 5; thus, the macrocyclic ring contains 12 or 13 ring atoms, respectively. [0028] Further, for macrocycles corresponding to Formula (I), n is 2 to 8, preferably 2, 3 or 4, where each R is independently substituted hydrocarbyl, optionally substituted by R^A, provided, in combination, the macrocycle is substituted by at least three groups that are ionizable at physiological pH. Three of the ionizable groups are capable of coordinating with a single lanthanide metal when the macrocycle forms a lanthanide complex, and are independently carboxyl, phosphorous-oxo acid or sulfur-oxy acid groups, the three ionizable groups being bonded, directly or indirectly, to two or three ring atoms of the macrocycle. In addition, each of these three ionizable groups is separated from the ring atom to which it is bonded by no more than three atoms. In the embodiment where these three ionizable groups are attached to two atoms of the macrocycle, two groups are attached to the macrocycle via a single macrocycle atom. For example, two ionizable groups may both be substituents on a single hydrocarbyl chain connected to the macrocycle atom, i.e., a single hydrocarbyl group is substituted with two ionizable groups, or, in the case of a macrocycle carbon atom having two bonds available for attachment of hydrocarbyl groups, two ionizable groups may be substituents on separate hydrocarbyl groups connected to a single macrocycle carbon atom. When required to obtain an overall net charge of -1 on the complex, the additional ionizable groups are non-coordinating groups that can form anions or cations at physiological pH. The non-coordinating groups that form anions are selected from carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; wherein EWG is an electron withdrawing group. The non-coordinating groups that form cations are non-coordinating amine groups, i.e. primary, secondary or tertiary amines, that can be protonated at physiological pH. In one embodiment, each of the at least four ionizable groups is a substituent of a separate hydrocarbyl group bonded to a macrocyclic ring carbon or nitrogen atom. In another embodiment, one of the three coordinating ionizable groups and a fourth non-coordinating ionizable group are both bonded to the same macrocyclic ring atom via a single hydrocarbyl group. In a further embodiment, one of the three coordinating ionizable groups and a fourth non-coordianting ionizable group are both bonded to the same macrocyclic ring atom via a single hydrocarbyl group, and the other two coordinating ionizable groups are both bonded to the same macrocyclic ring atom via a separate hydrocarbyl group, As used herein, R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_ 20 alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous- oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C4-20 carbohydrate, mercapto or thiol.

[0029] For the macrocycles corresponding to Formula (I), p is an integer from 0 to [(2x the number of ring carbon atoms + number of ring nitrogen atoms) - n]. Thus, each macrocyclic carbon atom may be substituted by up two substituents and each macrocyclic ring nitrogen atom may be substituted with one substituent. When p is greater than 0, each A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_₂o alkyl, or aryl, optionally substituted by one or more aryl, Ci_₂o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C4-20 carbohydrate, mercapto or thiol.

[0030] The at least three ionizable groups of macrocycles of Formula (I) can be categorized by their ability to coordinate a lanthanide metal thereby forming a metal complex (sometimes simply referred to as a "complex") at physiological pH. Whether a particular ionizable group of the present invention will coordinate a lanthanide metal is largely a function of (1) the identity of the ionizable group itself, and (2) the number of atoms separating the ionizable group from a macrocyclic ring atom.

Coordinating Ionizable Groups

[0031] Generally, to form a stable macrocyclic complex of the present invention, three ionizable groups coordinate with a single lanthanide metal. Typically, the three ionizable groups are separated from a macrocyclic ring atom by a number of atoms such that a 5-, 6- or 7-membered ring forms comprising the lanthanide metal and the macrocyclic ring atom. Accordingly, in order for these three ionizable groups to coordinate the metal, each group must have a -OH moiety that is (1) ionizable at physiological pH and (2) be part of a group, e.g., a carboxyl group, that is separated from a macrocyclic ring atom by no more than three atoms. When the ionizable group is attached via a macrocyclic nitrogen, there must be at least one atom, e.g., a methylene group, between the macrocyclic nitrogen and the ionizable group. For the compounds of Formula (I), the functional groups having an ionizable -OH moiety are selected from carboxyl, phosphorous-oxo acid and sulfur-oxy acid groups, and combinations thereof. Using a carboxyl group as an example, to coordinate the lanthanide, the carboxyl group is separated from the macrocyclic ring atom by no more than three atoms, i.e., the -O^" moiety of the carboxyl group is separated from a macrocyclic ring atom by no more than four atoms. An exemplary structure is illustrated below:

Coordinating -O^" moiety separated from the macrocycle by 3-atoms

[0032] In this example, the carboxyl group (-C(O)O^") is separated from the macrocyclic ring nitrogen by two carbon atoms and the -O^" moiety of the carboxyl group is separated from the macrocyclic ring nitrogen by three atoms. For ease of discussion, an ionizable group and the atoms through which the ionizable group is bonded to a macrocyclic ring atom, and any substituents thereof, are sometimes referred to as an "arm" of the macrocycle. Accordingly, the macrocycles of the present invention have at least three coordinating arms, each coordinating arm comprising one of the three coordinating ionizable groups.

[0033] While the ionizable -OH moiety of the carboxyl, phosphorous-oxo acid and sulfur-oxy acid groups is generally not separated from a macrocyclic ring atom by more than four atoms, it is typically separated by fewer than three atoms. For example, in one preferred embodiment, the ionizable -OH moiety of the carboxyl, phosphorous-oxo acid and sulfur-oxy acid group is separated by two atoms; e.g., a carboxyl moiety attached to a macrocyclic ring atom via a methylene group.

Preferred formation of a 5- membered chelate ring with the metal

Non-coordinating Ionizable Group

[0034] The non-coordinating ionizable group capable of forming an anionic group is preferably (1) ionizable at physiological pH and (2) separated from the macrocyclic ring atom to which it is attached, either directly or indirectly, by a sufficient number of atoms such that the ionizable group does not coordinate a metal in the macrocycle under conditions in which such ionizable group is in ionized form. For the macrocycles of the present invention, such as the compounds of Formula (I), the ionizable group capable of forming an anion is selected from carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the groups are separated from a macrocyclic ring atom by four to nine atoms. The ionizable moiety for the carboxyl, phosphorous-oxo acid, sulfur-oxy acid, and phenol groups is the -OH moiety, while the ionizable moiety for a thiophenol group is -SH. For the acetylacetonyl group, the ionizable -CH- moiety is alpha to both of the carbonyl carbon atoms and substituted by an electron withdrawing group (EWG). An example of an ionizable, non-coordinating substituted acetoacetonyl group is illustrated below:

Ionizable, non-coordinating -CH- moiety

[0035] For the acetylacetonyl group, typical electron withdrawing groups include, but are not limited to, hydroxyl, cyano (-CN), fluoro, -CF₃, -CF₂CF₃, nitro (-NO₂), alkylester (-CO₂R), and -CF₂CO₂R where R is Ci_6 alkyl. In one example of this embodiment, the electron withdrawing group is selected from cyano, nitro or -CF₃. In one preferred embodiment, the electron withdrawing group is -CF₃.

[0036] The non-coordinating ionizable group capable of forming a cationic group is a group that can be protonated at physiological pH. For the macrocycles of the present invention, such as the compounds of Formula (I), the ionizable group capable of forming a cation is selected from primary, secondary, or tertiary amine groups. Typically, the ionizable amine group is a substituent on a substituted hydrocarbyl moiety attached to the macrocycle, the hydrocarbyl group optionally comprising one or more hydrophilic imparting moieties, e.g., ethers, polyethers, ketones, alcohols, polyalcohols, carbohydrates and polypeptides.

[0037] While the three coordinating arms are each bonded to a separate macrocyclic ring atom in one preferred embodiment, the additional ionizable, non-coordinating groups may be bonded either directly to a separate macrocyclic ring atom or to a macrocyclic ring atom indirectly through one of the three coordinating arms. Thus, in one embodiment of compounds of Formula (I), n is 3 where a fourth ionizable group is bonded to one of the three coordinating arms. Stated another way, in some embodiments, one of the R substituents comprises one of the three coordinating ionizable groups and a fourth non-coordinating ionizable group. In an alternative embodiment, n is 4 and a fourth ionizable group is bonded to a macrocyclic ring atom, such ring atom not being bonded to any of the three coordinating arms. In this embodiment, the macrocycle will have at least four arms, three coordinating arms and at least one non-coordinating arm. In another alternative embodiment, n is 2 and one of the three coordinating ionizable groups and a fourth ionizable group are both bonded to the same macrocyclic ring atom via a single hydrocarbyl group, and the other two coordinating ionizable groups are both bonded to the same macrocyclic ring atom via a separate hydrocarbyl group,

Other Macrocyclic Ring Substituents

[0038] When the macrocycle corresponds to Formula (I) and p is greater than 0 and/or R is substituted by R^A, it is generally preferred that each A and/or R^A be a linker chemically linking one or more bio-directing modifiers to the macrocycle and/or be a substituent selected to positively impact stability and/or biodistribution. In one embodiment of macrocycles corresponding to Formula (I) having at least one A or R^A substituent, each A and/or R^A is selected from the group consisting of Ci_2o alkyl or aryl, optionally substituted by one or more aryl, Ci_2o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C₄_₂o carbohydrate, mercapto or thiol. For example, each A and/or R^A may be aryl or Ci_6 alkyl optionally substituted with one or more aryl, keto, amido and oxy. By way of further example, each A and/or R^A may be methyl.

[0039] In another aspect of this embodiment, one or more of the A and/or R^A substituents may be a linker chemically linking one or more bio-directing modifiers to the macrocycle. The linker may be modified or synthesized such that it bonds to multiple bio- directing modifiers and/or affects the biodistribution of the macrocycle. For example, the linker may comprise a C_4-C_2O carbohydrate moiety, the carbohydrate moiety having the capacity to bind one or more bio-directing modifiers through ether linkages. In addition, the carbohydrate moiety increases the water solubility of the macrocycle thereby affecting biodistribution.

[0040] In one embodiment, the linker is selected from the group consisting Of C_1-10 alkylene, oxygen, sulfur, keto (-C(O)-), amino (-NH-), amido (-C(O)NH-), urea (-NHC(O)NH-), thiourea (-NHC(S)NH-), ester (-C(O)O-), polyoxo (e.g., -0-CH₂CH₂-O- CH₂CH₂-O-), polyhydroxy (e.g., carbohydrates), and peptides, the alkylene, amino, amido, urea, and thiourea groups being optionally substituted with aryl, Ci_7 alkyl, Ci_7 hydroxyalkyl or Ci_₇ alkoxyalkyl. In one example of this embodiment, the linker is selected from the group consisting Of C_1-10 alkylene, oxygen, sulfur, keto, amino, amido, thiourea, ester, C₄-C₂O carbohydrate, the alkylene, amino, amido, and thiourea groups being optionally substituted with aryl, Ci_₇ alkyl, Ci_₇ hydroxyalkyl or Ci_₇ alkoxyalkyl. By way of further example, the linker may be selected from a more restrictive group, e.g., amido, thiourea, monosaccharides (e.g., hexoses and pentoses) and disaccharides (e.g., sucrose). In one alternative of this embodiment, the linker comprises other than a urea linkage.

[0041] When one or more of the A and/or R^A substituents is a linker chemically linking one or more bio-directing modifiers to the macrocycle, the bio-directing modifier(s) are selected from those moieties that restrict the complex to the vascular compartment. Generally, to achieve the restriction of the biodistribution of the complex to the vascular compartment, it is necessary to increase the molecular weight of the complex. Typically, the bio-directing modifiers are 10-30 KD in weight and are selected from PEG(s), carbohydrates, and polypeptides. In a preferred embodiment, the bio-directing modifier(s) are PEG.

[0042] In another aspect of the present invention, the macrocycle corresponds to Formula (II)

wherein:

[0043] mi through m₄ are independently 1 or 2, provided the sum of mi through m₄ is 4-6;

[0044] p is an integer from 0 to 14;

[0045] R1-R3 are independently substituted hydrocarbyl moieties, optionally substituted by R^A, in which the three hydrocarbyl moieties are substituted by first, second and third ionizable groups, respectively, each of the ionizable groups being carboxyl groups with each of the three ionizable groups being bonded, directly or indirectly, to different ring atoms of the macrocycle, provided each of the three ionizable groups are separated from the ring atoms to which they are bonded by no more than three atoms;

[0046] R₄ is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by a fourth ionizable group selected from carboxyl, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups separated from a ring nitrogen atom of the macrocycle by four to nine atoms, wherein EWG is an electron withdrawing group; and

[0047] when present, each A and R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_2o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C_4-2O carbohydrate, mercapto or thiol.

[0048] For the macrocycles of Formula (II), each of the four ionizable arms (R) is bonded to a separate macrocyclic ring atom. In a preferred embodiment for compounds of Formula (II), each of the ionizable arms is bonded to a separate ring nitrogen atom. Thus, each macrocyclic ring nitrogen atom is substituted by one of the ionizable arms. In one example of this embodiment, the macrocycle corresponds to Formula (III):

wherein

[0049] p is an integer from 0 to 8;

[0050] x is an integer from 0 to 4;

[0051] R1-R3 are independently substituted hydrocarbyl moieties, optionally substituted by R^A, in which the three moieties are substituted by first, second and third ionizable groups, respectively, each of the ionizable groups being independently selected from the group consisting of carboxyl, phosphorous- and sulfur-oxy acid groups with each of the three ionizable groups being bonded, directly or indirectly, to different ring atoms of the macrocycle, provided each of the three ionizable groups are separated from the ring atoms to which they are bonded by no more than three atoms; [0052] R₄ is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by a fourth ionizable group selected from carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups separated from a ring nitrogen atom of the macrocycle by four to nine atoms, wherein EWG is an electron withdrawing group;

[0053] R_x is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by additional ionizable, non-coordinating groups selected from: (iii) carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; where EWG is an electron withdrawing group, or (iv) amine groups; and,

[0054] when present, each A and R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_₂o alkyl, or aryl, optionally substituted by one or more aryl, Ci_2o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C4-20 carbohydrate, mercapto or thiol.

[0055] In another aspect of the invention, the macrocycles correspond to Formula (IV):

wherein:

[0056] p is an integer from 0 to 4;

[0057] x is an integer from 0 to 4;

[0058] X₁-X₃ are independently methylene, ethylene or propylene, optionally substituted by R^A;

[0059] X₄ is an alkylene group of length sufficient to separate the attached ring nitrogen atom from Z₄ by the required number of atoms, optionally substituted by R^A; [0060] Zi-Z₃ are independently carboxyl, phosphorous-oxo acid or sulfur-oxy acid groups;

[0061] Z₄ is a carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenol, thiophenol, or acetylacetonyl group; provided that if Z₄ is a phenol, thiophenol, or acetylacetonyl group, then the group may be optionally substituted by R^A;

[0062] R_x is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by additional ionizable, non-coordinating groups selected from: (i) carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; where EWG is an electron withdrawing group, or (ii) amine groups; and

[0063] when present, each A and R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_2o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C₄_₂o carbohydrate, mercapto or thiol. When Z₄ is carboxyl, phosphorous-oxo acid, or sulfur-oxy acid, it is currently preferred that X₄ is butylene or pentylene. When Z₄ is phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, it is currently preferred that X₄ is ethylene or propylene.

[0064] In one preferred embodiment of macrocycles of Formula (IV), X₁-X₃ are methylene or ethylene, optionally substituted by R^A. In one example of this embodiment, each OfZi-Z₃ is carboxyl.

Metal Complexes

[0065] Prior to use in a diagnostic procedure, e.g., MRI, the macrocycle is complexed with a lanthanide metal. Typical lanthanide metals include cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium. In a preferred embodiment, the lanthanide metal is gadolinium or dysprosium, more preferably, gadolinium.

[0066] At physiological pH, the four ionizable groups of the macrocycle deprotonate. The three coordinating ionizable groups coordinate the lanthanide metal, while the fourth ionizable group does not coordinate the metal. In addition to coordination by the three ionizable groups, the metal is coordinated by the macrocyclic ring nitrogen atoms and two inner sphere water molecules. The resulting complex possesses a high relaxivity in part because the pKa of the fourth ionizable group is more acidic than that of the coordinated inner sphere water molecules resulting in a bis-aqua complex bearing two exchangeable inner-sphere water molecules.

[0067] Any of the macrocycles of Formulae (I) through (IV) can coordinate a lanthanide metal thereby forming a complex. In one example, the complex has the structure:

wherein

[0068] M is a lanthanide metal; and

[0069] Z₄ is a carboxyl, phenol, thiophenol, or acetylacetonyl group.

[0070] In a preferred example, the complex has the following structure at physiological pH:

Synthesis of the Macrocycles

[0071] The macrocycles of the present invention can be synthesized as described below. Typically, coordinating and non-coordinating ionizable arms substituted on the macrocyclic nitrogen atoms are synthesized by controlled multistep alkylation of a polyazamacrocycle, such as cyclen, in a process as shown in General Scheme A:

1) Deprotection

2) Gd-complexation

Where X=Br

R=-CH₂CO2-Bu^f R^-CH₂CH₂CH₂CH₂CO₂-Et

[0072] The preparation of monodifferentiated species, such as D03-tris(tert-butyl) hydrobromide, are well known (see, for example, PCT application no. PCT/US07/06514). Thus, in an ordinary example of the chemistry, one might allow the reaction of the trisubstituted cyclen with ethyl 5 -bromo valerate. This yields a masked intermediate which may be sequentially deprotected. The free macrocycle (sometimes referred to herein as a "chelate") is complexed with gadolinium via the oxide in water. The choice of ester protecting group depends on the desired route. For instance, a similar sequence can be employed where all ester protecting groups are benzyl. In this case, deprotection can be accomplished across the entire molecule in one step:

[0073] Alternatively, chelate designs may be based on a macrocycle where the attachment of coordinating and non-coordinating ionizable side arms are not only attached via the macrocyclic nitrogen, but also attached at a macrocyclic carbon. Natural, or unnatural, amino acids make excellent building blocks for such species. For instance, a carbon-centered side-arm derived macrocycle could be prepared as shown below in General Scheme B.

Using synthetic techniques and reagents common to peptide chemistry, a base scaffold exhibiting three masked ionizable groups can be prepared. Ring closure with a suitable, masked ionizable non-coordinating group produces the protected macrocycle as shown below.

Reduction of the ring amide groups, followed by deprotection, gives the free chelate. Treatment with a lanthanide gives the desired di-aqua complex.

[0074] Macrocycles of the present invention having one or more bio-directing modifiers, e.g., PEG, may be synthesized, for example, as shown in General Scheme C.

Compositions

[0075] The complexes of the present invention can be formulated into diagnostic compositions for enteral or parenteral administration. These compositions contain an effective amount of the paramagnetic ion complex along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated. For example, parenteral formulations advantageously contain a sterile aqueous solution or suspension of from about 0.05 to 1.0M of a paramagnetic ion complex according to the present invention. Preferred parenteral formulations have a concentration of paramagnetic ion complex of 0.1M to 0.5M. Such solutions also may contain pharmaceutically acceptable buffers, such as sodium, calcium, or zinc salts of the macrocycle of the present invention, and, optionally, electrolytes such as sodium chloride. The compositions advantageously may contain one or more physiologically acceptable, non-toxic cations in the form of a gluconate, chloride or other suitable organic or inorganic salt. Such physiologically acceptable, non-toxic cations include sodium ions, calcium ions, magnesium ions, copper ions, zinc ions and the like. Calcium ions are preferred.

[0076] Parenteral compositions can be injected directly or mixed with a large volume parenteral composition for systemic administration.

[0077] Formulations for enteral administration may vary widely, as is well-known in the art. In general, such formulations are liquids which include an effective amount of the paramagnetic ion complex in aqueous solution or suspension. Such enteral compositions may optionally include buffers, surfactants, thixotropic agents, and the like.

Dosage

[0078] Dosage and regimens for the administration of the pharmaceutical compositions of the invention can be readily determined by those with ordinary skill in diagnosing or treating disease. It is understood that the dosage of the complex will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. For any mode of administration, the actual amount of complex delivered, as well as the dosing schedule necessary to achieve the advantageous effects described herein, will also depend, in part, on such factors as the bioavailability of the complex, the disorder being treated or diagnosed, the desired therapeutic or diagnostic dose, and other factors that will be apparent to those of skill in the art. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect the desired therapeutic or diagnostic response in the animal over a reasonable period of time.

[0079] In typical MRI-contrast enhanced procedures, employing conventional contrast agents, 0.1 mmole contrast agent/kg bodyweight is considered a single dose. An agent of higher relaxivity may be expected to use less (in comparable procedures), the same (to take advantage of the potential higher sensitivity for lesions a more potent agent may reveal), or even more (if safety testing proves the agent biocompatible at higher doses).

DEFINITIONS

[0080] The compounds described herein may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic form. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention.

[0081] The terms "oxo acid" or "oxy acid" as used herein utilizes the International Union of Pure and Applied Chemistry (IUPAC) nomenclature contained in the IUPAC Blue Book, Rigaudy, J. and Klesney, S.P. Nomenclature of Organic Chemistry; pages 234, 397; Pergammon: Oxford, 1979. The terms "oxo acid" or "oxy acid" are used in referring to the appropriate corresponding oxygenated phosphorus-, sulfur- and carbon-based acids, such as -P(O)(OH)₂ as phosphonic or -C(O)OH as carboxylic acids.

[0082] Unless otherwise indicated, the alkyl groups described herein are preferably lower alkyl containing from one to eight carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain or cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.

[0083] The term "amido" as used herein includes substituted amido moieties where the substituents include, but are not limited to, one or more of aryl and Ci_₂o alkyl, each of which may be optionally substituted by one or more aryl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, Ci_2o alkyl, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, mercapto, and thio substituents.

[0084] The term "amino" as used herein includes substituted amino moieties where the substituents include, but are not limited to, one or more of aryl and Ci_₂o alkyl, each of which may be optionally substituted by one or more aryl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, Ci_2o alkyl, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, mercapto, and thio substituents.

[0085] The terms "aryl" or "ar" as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic groups containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl. Phenyl and substituted phenyl are the more preferred aryl.

[0086] The term "chelate" as used herein refers to a macrocycle of the invention, e.g., Formula (I), which is capable of binding of complexing or coordinating a metal. Thus, the chelates of the present invention are not complexed with a metal.

[0087] The term "complex" as used herein refers to a macrocycle of the invention, e.g. Formula (I), complexed or coordinated with a metal. [0088] The terms "halogen" or "halo" as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.

[0089] The term "hydrocarbyl" as used herein describes an organic compound or radical consisting exclusively of the elements carbon and hydrogen. This moiety includes alkyl, alkenyl, alkynyl, and aryl moieties. This moiety also includes alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwise indicated, this moiety preferably contains 1 to 20 carbon atoms

[0090] The term "protecting group" as used herein denote a group capable of protecting a reactive group, e.g., a free hydroxyl group ("protected hydroxyl"), amine group ("protected amine"), sulfhydryl group ("protected sulfhydyl") etc., which, subsequent to the reaction for which protection is employed, may be removed without disturbing the remainder of the molecule. A variety of protecting groups and the synthesis thereof may be found in Protective Groups in Organic Synthesis by T. W. Greene, John Wiley and Sons, 1981, or Fieser & Fieser.

[0091] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.

EXAMPLES

[0092] The following non-limiting examples are provided to further illustrate the present invention.

[0093] Example 1. (Control) Gadolinium [l,4,7,10-tetraazacyclododecane-l-(3- propanato)-4,7, 10-triacetato(4-)

[0094] Sodium tert-butyl 2,2',2"-(10-(3-tert-butoxy-3-oxopropyl)-l,4,7,10- tetraazacyclododecane- 1 ,4,7-triyl)triacetate bromide.

A solution consisting of l,4,7-tris(tert-butyl ester)-l,4,7,10-tetraazacyclododecane (4.3g, 8.4mmoles) was treated with tert-butyl acrylate (6.5g, 50.7mmoles-total) in acetonitrile (25OmL) at 50⁰C. Over five days additional tert-butyl acrylate was added until the reaction was approximately 95% complete, by HPLC-MS. The product was isolated as a crude oil and treated with sodium bromide (1.28g, 12.5mmoles) in wet acetonitrile (73mL ACN:15mL H₂O) at 50⁰C. After one hour, all of the solid salt had dissolved. The mixture was filtered and the filtrate concentrated to an oil by rotary evaporation. The resulting oil was stirred with 10OmL diethyl ether. After 15 minutes, at room temperature, crystals began to form. The crystalline product was isolated by filtration. Yield 7. Ig, 84%. The product appears as the free chelate in TFA/ACN/Aq HPLC-MS with 643/665 present as M+H and M+Na. NMR Hl (300MHz, CDCl₃) 1.422 (s, 9H), 1.434 (s, 9H), 1.484 (s, 18H), 2.3-2.4 (br,m, 12H), 2.6-2.8 (br, 2H), 2.8- 3.3 (br, m, 12H); C13 28.189, 28.345, 28.383, 32.431, 50.109, 50.571 (br), 51.345 (v. br), 56.236, 57.143 (br), 81.043, 82.526, 82.849, 171.895, 172.901, 173.500.

[0095] 10-(2-Carboxy ethyl)- 1 ,4,7-tris(carboxymethyl)aza- 1 ,4,7- triazoniacyclododecane-l,4,7-triium tetra(2,2,2-trifluoroacetic acid salt))

A sample of the sodium complex (1.0Og, 1.3mmoles) was stirred in trifluoroacetic acid overnight. The resulting solution was evaporated to an oil. The oil was coevaporated with acetonitrile, 3x20mL, and water, 3x20mL, resulting in a solid foam. The foam was slurried in acetonitrile and ether and collected by filtration. Yield 0.7g (60%). HPLC-MS confirmed that deprotection was complete.

[0096] Gadolinium [l,4,7,10-tetraazacyclododecane-l-(3-propanato)-4,7,10- triacetato(4-)

A sample of the chelate (0.2g, 0.5mmole) was dissolved in water. To this was added gadolinium oxide (0.087g, 0.25mmole). The mixture was heated to 65°C overnight with stirring. The mixture was cooled to room temperature and pH of the solution was adjusted with NaOH (IM, just a few drops) to pH of 8. The resulting mixture was filtered, 0.2μm, and submitted for relaxivity and ICP testing.

[0097] Example 2. (Control) Gadolinium [l,4,7,10-tetraazacyclododecane-l-(4- butanato)-4,7,10-triacetato(4-)

[0098] Sodium tert-butyl 2,2',2"-(10-(4-ethoxy-4-oxobutyl)-l,4,7,10- tetraazacyclododecane- 1 ,4,7-triyl)triacetate bromide

A mixture of l,4,7-tris(tert-butyl ester)- 1, 4,7, 10-tetraazacyclododecane, (8.8g, 17.1mmoles) and sodium bicarbonate (1.41g 18.0mmoles) was stirred under argon. To this was added dropwise a solution of ethyl 4-bromobutanoate (4.08g, 18.0mmoles) in acetonitrile. When the addition was complete, the mixture was warmed to 35°C and stirred overnight. A small amount of solid, 0.14g, was removed by filtration and the filtrate was evaporated to an oil. Upon stirring with diethyl ether, crystals began to form. The product was isolated by filtration and vacuum dried. Yield 8.6g (74%). HPLC-MS shows trace amounts of starting DO3-tris(tert-butyl ester), M+H 515, and N-ethyl-DO3-tris(tert-butyl ester), M+H 543, a side product due to DO3-nucleophilic attack on the ethyl ester carbonyl-carbon, along with the desired product as the free chelate at M+H 629 and M+Na at 651. NMR (CDCl₃) Hl 1.233 (t, 3H), 1.325 (s, 9H), 1.376 (s, 18H), 1.645 (quintet, 2H), 2.148 (t, 2H), 2.278 (br, m, 8H), 2.51- 3.45 (br, m, 16H), 3.987 (q,2H); C13 14.638, 23.213, 26.263, 28.179, 28.327, 28.479, 34.394, 50.675, 51.0 (br), 54.279, 56.157, 56.867, 60.581, 82.500, 82.826, 172.864, 173.262, 173.756.

[0099] Disodium 4-(4,7, 10-tris(2-tert-butoxy-2-oxoethyl)-l ,4,7, 10- tetraazacyclododecan-l-yl)butanoate bromide

The ethyl ester (l .Og, 1.4mmoles) was saponified using aqueous sodium hydroxide (0.057g, 1.5mmoles dissolved in 5mL water) in methanol (5mL). After stirring overnight, under argon and at room temperature, the mixture was evaporated to a foam. Yield 0.9g (91%) HPLC-MS shows the material to be >95% desired sodium salt.

[00100] 2,2',2"-(10-(3-Carboxypropyl)-l,4,7,10-tetraazacyclododecane-l,4,7- triyl)triacetic acid tetra(2,2,2-trifluoroacetic acid salt)

The sodium salt was stirred with trifluoroacetic acid overnight at room temperature. The resulting mixture was evaporated and the residue co-evaporated with 3x20mL water followed by 3x20mL acetonitrile. The resulting oil crystallized on standing. It was slurried with diethyl ether and collected by filtration. Yield 1.2Og (93%) HPLC-MS confirms deprotection was complete.

[00101] Gadolinium [l,4,7,10-tetraazacyclododecane-l-(4-butanato)-4,7,10- triacetato(4-) A sample of the chelate (0.2g, 0.5mmole) was dissolved in water. To this was added gadolinium oxide (0.087g, 0.25mmole). The mixture was heated to 65°C overnight with stirring. The mixture was cooled to room temperature and pH of the solution was adjusted with NaOH (IM, just a few drops) to pH of 8. The resulting mixture was filtered, 0.2μm, and submitted for relaxivity and ICP testing.

[00102] Example 3. (Invention) Gadolinium [l,4,7,10-tetraazacyclododecane-l-(5- pentanato)-4,7,10-triacetato(4-)

[00103] Sodium tert-butyl 2,2',2"-(10-(5-ethoxy-5-oxopentyl)-l,4,7,10- tetraazacyclododecane- 1 ,4,7-triyl)triacetate bromide

A mixture of l,4,7-tris(tert-butyl ester)- 1, 4,7, 10-tetraazacyclododecane, (8.9g, 17.3mmoles) and sodium bicarbonate (1.53g 18.2mmoles) was stirred under argon. To this was added dropwise a solution of ethyl 5-bromopentanoate (5.75g, 18.2mmoles) in acetonitrile. When the addition was complete, the mixture was warmed to 35°C and stirred overnight. A small amount of solid, 0.18g, was removed by filtration and the filtrate was evaporated to an oil. Upon stirring with diethyl ether (10OmL), crystals began to form. The product was isolated by filtration and vacuum dried. Yield 7.2g (56%). HPLC-MS shows trace amounts of starting DO3-tris(tert-butyl ester), M+H 515, and N-ethyl-DO3-tris(tert-butyl ester), M+H 543, a side product due to DO3-nucleophilic attack on the ethyl ester carbonyl- carbon, along with the desired product as the free chelate at M+H 629 and M+Na at 651. NMR (CDCl₃) Hl 1.233 (t, 3H), 1.325 (s, 9H), 1.376 (s, 18H), 1.645 (quintet, 2H), 2.148 (t, 2H), 2.278 (br, m, 8H), 2.51-3.45 (br, m, 16H), 3.987 (q,2H); C13 14.638, 23.213, 26.263, 28.179, 28.327, 28.479, 34.394, 50.675, 51.0 (br), 54.279, 56.157, 56.867, 60.581, 82.500, 82.826, 172.864, 173.262, 173.756.

[00104] Disodium 5-(4,7,10-tris(2-tert-butoxy-2-oxoethyl)-l,4,7,10- tetraazacyclododecan-l-yl)pentanoate bromide

A methanol solution (ImL) of the sodium tert-butyl 2,2',2"-(10-(5-ethoxy-5-oxopentyl)- l,4,7,10-tetraazacyclododecane-l,4,7-triyl)triacetate bromide (1.0Og, 1.3mmoles) was treated with a water-methanol (5mL:5mL) of sodium hydroxide (0.056g, 1.4mmoles). The mixture was stirred overnight, under argon, at room temperature. HPLC-MS showed that the reaction was greater 95% complete. Acetone (2OmL) was added to precipitate the excess sodium hydroxide. The mixture was filtered and the filtrate evaporated to a solid. Yield 0.7g, 71%. HPLC-MS of the solid shows the material consists of approx. 10%(TIC) a di(tert-butyl ester) DO3A-pentanoate (M+H=559, M+Na=581) and approx. 90% tris(tert-butyl)DO3Apentanoate (M+H=615, M+Na=637). No other significant peaks in the TIC were visible.

[00105] 2,2',2"-(10-(4-Carboxybutyl)-l,4,7,10-tetraazacyclododecane-l,4,7- triyl)triacetic acid tetra(2,2,2-trifluoroacetic acid salt)

The sodium salt mixture was stirred with trifluoroacetic acid, 3mL, overnight at room temperature. The resulting mixture was evaporated and the residue co-evaporated with 3x20mL water followed by 3x20mL acetonitrile. The resulting oil crystallized on standing. It was slurried with diethyl ether and collected by filtration. Yield 0.74g (75%) HPLC-MS confirms deprotection was complete.

[00106] Gadolinium [l,4,7,10-tetraazacyclododecane-l-(5-pentanato)-4,7,10- triacetato(4-)

A sample of the chelate (0.2g, 0.4mmole) was dissolved in water. To this was added gadolinium oxide (0.087g, 0.25mmole). The mixture was heated to 65°C overnight with stirring. The mixture was cooled to room temperature and pH of the solution was adjusted with NaOH (IM, just a few drops) to pH of 8. The resulting mixture was filtered, 0.2μm, and submitted for relaxivity and ICP testing.

[00107] Example 4. Relaxivity and pH

The gadolinium complexes of DO3 A-propanoate, -butanoate and -pentanoate have been prepared in situ. Relaxivity measurements at low (pH 2), intermediate (pH 6 to 8) and high pH (pH 10) confirm our theory on the relationship between a pendant anionic charge and MRCM potency of di-aqua gadolinium complexes. The pH-dominated relaxivity behavior of our complexes is shown below:

8 2 8 2 3 8 sec^"1mM^"1

Example 3 (Invention) Gd-DO3-pentanoate

Example 2 (Control) Gd-DO3-butanoate

8 3 3 9

Example 1 (Control) Gd-DO3-propanoate

At low pH, at the left-hand side of the scheme, all of the complexes are fully protonated and exist as di-aqua complexes. The high relaxivity observed is the result of having twice as much complexed water available for exchange with bulk water as compared to ordinary mono- aqua complexes. As the pH increases, from left to right, all of the complexes deprotonate. The loss of H+ from the pentanoate (Example 3) merely results in the formation of a carboxylate group, leaving the di-aqua-Gd moiety intact, and a high relaxivity is still observed. However, a coordinated water is lost by the shorter-chained butanoate (Example 2) and propanoate (Example 1) complexes due to the side chains' ability to coordinate the metal center, resulting in a decreased relaxivity. This coordination of the pendant carboxylate to the metal cannot occur in the pentanoate structure because the chain is too long to accommodate coordination. Even higher pH levels again cause the loss of a proton from each of the complexes. This gives rise to a (hydroxo)(aqua) complex for the pentanoate (far right, Example 3), which exhibits a relaxivity typical for mono-aqua complexes. However, the relaxivity observed for the shorter- chained butanoate or propantoate decreases further, as the only remaining coordinated water is converted into a hydroxo ligand.

[00108] When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[00109] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

Claims

WHAT IS CLAIMED IS:

1. A tetraazamacrocycle comprising a macrocycle containing 4 ring nitrogen atoms and 8-10 ring carbon atoms wherein:

(a) each ring nitrogen atom is separated from other ring nitrogen atoms by at least two carbon atoms; and

(b) the macrocycle is substituted by at least three groups that are ionizable at physiological pH, three of the ionizable groups being capable of coordinating with a single lanthanide metal when the macrocycle forms a di-aqua, lanthanide complex and being independently selected from carboxyl, phosphorous-oxo acid or sulfur-oxy acid groups, the three ionizable groups being bonded, directly or indirectly, to two or three ring atoms of the macrocycle and where each of the three ionizable groups is separated from the ring atom to which it is bonded by no more than three atoms; provided that the di- aqua complex of the macrocycle with a +3 charge lanthanide metal has an overall net charge of -1, wherein the overall net charge is calculated by the formula: overall net charge = ∑(charges of the three ionized coordinating groups) + valence of the lanthanide metal + ∑(charges of any ionized non-coordinating groups); with the further proviso that if additional ionizable, non-coordinating groups are required to achieve a complex having an overall net charge of -1, the non-coordinating ionizable groups can form anions or cations at physiological pH, wherein non- coordinating groups that form anions are selected from carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the anion forming ionizable group is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; and EWG is an electron withdrawing group; and non-coordinating groups that form cations are non- coordinating amine groups.

2. The macrocycle of claim 1 wherein the ∑(charges of the three ionized coordinating groups) is -3, -4, -5 or -6 and the macrocycle is substituted with one or more non-coordinating ionizable groups selected to give an overall net charge of -1.

3. The macrocycle of claim 2 wherein the three ionizable coordinating groups are carboxyl groups and the macrocycle is further substituted with an ionizable non-coordinating group that forms a single anion at physiological pH.

4. The macrocycle of claim 1 wherein at least one of the three ionizable coordinating groups is selected from a phosphorous-oxo acid or sulfur-oxy acid group.

5. The macrocycle of claim 1 having the structure:

wherein mi through m₄ are independently 1 or 2, provided the sum of mi through m₄ is 4-6; n is an integer from 2 to 8; p is an integer from 0 to [(2x the number of ring carbon atoms + number of ring nitrogen atoms) - n]; each R is independently substituted hydrocarbyl, optionally substituted by R^A, provided, in combination, the macrocycle is substituted by at least three groups that are ionizable at physiological pH wherein:

(a) three of the ionizable, coordinating groups are independently selected from the group consisting of carboxyl, phosphorous-oxo acid and sulfur-oxy acid groups, the three ionizable groups being bonded, directly or indirectly, to two or three ring atoms of the macrocycle and where each of the three ionizable groups is separated from the ring atom to which it is bonded by no more than three atoms; and

(b) optionally additional ionizable, non-coordinating groups that form anions or cations at physiological pH, wherein:

(i) the non-coordinating groups that form anions are selected from carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; where EWG is an electron withdrawing group; or (ii) the non-coordinating groups that form cations are non-coordinating amine groups; and, when present, each A and R^A is independently a linker chemically linking one or more bio- directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_2o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C_4-2O carbohydrate, mercapto or thiol.

6. The macrocycle of claim 5 wherein mi through Hi₄ are each 1.

7. The macrocycle of claim 5 wherein one of the three ionizable, coordinating groups and an ionizable, non-coordinating group are bonded to a macrocyclic ring atom via a single R moiety.

8. The macrocycle of claims 5, 6 or 7 wherein two of the three ionizable, coordinating groups are bonded to a tetraazamacrocyclic ring atom via a single R moiety.

9. The macrocycle of claims 5, 6 or 7 wherein two of the three ionizable, coordinating groups are bonded to a tetraazamacrocyclic ring carbon atom, each ionizable group being bonded to the ring carbon atom via a separate R moiety.

10. The macrocycle of any of claims 1- 5 or 7 - 9 having the structure:

wherein mi through m₄ are independently 1 or 2, provided the sum of mi through m₄ is 4-6; p is an integer from O to 14;

R1-R3 are independently substituted hydrocarbyl moieties, optionally substituted by R^A, in which the three hydrocarbyl moieties are substituted by first, second and third ionizable groups, respectively, each of the ionizable groups being carboxyl groups with each of the three ionizable groups being bonded, directly or indirectly, to different ring atoms of the macrocycle, provided each of the three ionizable groups are separated from the ring atoms to which they are bonded by no more than three atoms;

R₄ is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by carboxyl, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms and where EWG is an electron withdrawing group; and when present, each A and R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_₂o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C_4-2O carbohydrate, mercapto or thiol.

11. The macrocycle of any of claims 1 - 5 or 7 - 9 having the structure:

wherein p is an integer from 0 to 8; x is an integer from 0 to 4;

R1-R3 are independently substituted hydrocarbyl moieties, optionally substituted by R^A, in which the three hydrocarbyl moieties are substituted by first, second and third ionizable groups, respectively, each of the ionizable groups being independently selected from carboxyl, phosphorous-oxo acid or sulfur-oxy acid groups with each of the three ionizable groups being bonded, directly or indirectly, to different ring atoms of the macrocycle, provided each of the three ionizable groups are separated from the ring atoms to which they are bonded by no more than three atoms;

R₄ is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by a fourth ionizable group selected from:

(i) carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; where EWG is an electron withdrawing group; or (ii) amine groups;

R_x is a substituted hydrocarbyl moiety, optionally substituted by R^A, in which the moiety is substituted by additional ionizable, non-coordinating groups selected from:

(iii) carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; where EWG is an electron withdrawing group; or (iv) amine groups; and, when present, each A and R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_2o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C_4-2O carbohydrate, mercapto or thiol.

12. The macrocycle of claim 11 wherein x is 0 and the ionizable groups Of Ri-R₄ are carboxyl groups.

13. The macrocycle of claim 10 or 11 wherein the fourth ionizable group is the acetylacetonyl -CH(EWG)- group and EWG is selected from the group consisting of hydroxyl, cyano, fluoro, -CF₃, -CF₂CF₃, -NO₂, alkylester, and -CF₂CO₂-Ci_-6 alkyl.

14. The macrocycle of claim 13 wherein EWG is selected from the group consisting of cyano, nitro and -CF₃.

15. The macrocycle of any of claims 1 - 5 or 7 - 9 having the structure:

wherein p is an integer from 0 to 4; x is an integer from 0 to 4; X1-X3 are independently methylene, ethylene or propylene, optionally substituted by

R^A;

X₄ is an alkylene group of length sufficient to separate the attached ring nitrogen atom from Z₄ by the required number of atoms, optionally substituted by R^A;

Z1-Z3 are independently carboxyl, phosphorous-oxo acid or sulfur-oxy acid groups; Z₄ is a carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenol, thiophenol, or acetylacetonyl group; provided that if Z₄ is a phenol, thiophenol, or acetylacetonyl group, then the group may be optionally substituted by R^A;

(i) carboxyl, phosphorous-oxo acid, sulfur-oxy acid, phenolic -OH, thiophenolic -SH, or acetylacetonyl -CH(EWG)- groups, where the ionizable group that forms an anion is separated from a ring carbon or nitrogen atom of the macrocycle by four to nine atoms; where EWG is an electron withdrawing group; or (ii) amine groups; and when present, each A and R^A is independently a linker chemically linking one or more bio-directing modifiers to the macrocycle, Ci_2o alkyl, or aryl, optionally substituted by one or more aryl, Ci_₂o alkyl, carbaldehyde, keto, carboxyl, cyano, halo, nitro, amido, polypeptides, phosphorous-oxo acid, sulfur-oxy acid, hydroxyl, oxy, ether, polyether, C₄_₂o carbohydrate, mercapto or thiol.

16. The macrocycle of claim 15 wherein X₁-X₃ are independently methylene or ethylene.

17. The macrocycle of claim 15 or 16 wherein each of Zi-Z₃ is carboxyl.

18. The macrocycle of any of claims 15 - 17 wherein Z₄ is carboxyl, phosphorous- oxo acid, or sulfur-oxy acid and X₄ is butylene or pentylene.

19. The macrocycle of claim 18 wherein each of Zi-Z₄ is carboxyl and x is 0.

20. The macrocycle of any of claims 15 - 17 wherein Z₄ is phenol, thiophenol, or acetylacetonyl and X₄ is ethylene or propylene.

21. A metal complex comprising the macrocycle of any of claims 1 - 20 and a coordinated lanthanide metal.

22. The complex of claim 21 wherein the metal is gadolinium or dysprosium.

23. The complex of claim 22 wherein the metal is gadolinium.

24. The complex of any of claims 21 - 23 wherein p is an integer from 1-4; and at least one A is a linker chemically linking one or more bio-directing modifiers to the macrocycle where the linker is selected from the group consisting Of C_1-10 alkylene, oxygen, sulfur, keto, amino, amido, thiourea, ester, C₄-C₂O carbohydrate, the alkylene, amino, amido, and thiourea groups being optionally substituted with aryl, Ci_₇ alkyl, Ci_₇ hydroxyalkyl or Ci_₇ alkoxyalkyl.

25. The complex of claim 24 wherein at least one A is a linker chemically linking a bio-directing modifier to the macrocycle and the bio-directing modifier is PEG.

26. A metal complex wherein the complex is lanthanide metal (M) 2,2',2"-(10-(4- carboxybutyl)- 1,4,7,10-tetraazacyclododecane- 1 ,4,7-triyl)triacetate.

27. A metal complex wherein the complex is gadolinium 2,2',2"-(10-(4- carboxybutyl)- 1,4,7,10-tetraazacyclododecane- 1 ,4,7-triyl)triacetate.

28. A composition for parenteral or enteral administration comprising the complex of any of claims 21 - 27 and at least one pharmaceutically acceptable carrier.

29. A metal complex of any of claims 21 to 28 for use in a medical diagnostic procedure.

30. The metal complex of claim 29 wherein the medical diagnostic procedure is MRI.