Compounds for use as iron(III) MRI contrast agents, including anionic pendants and auxiliary groups.

Abstract

To provide macrocyclic compounds that can be complexed with Fe(III).SOLUTION: The macrocyclic complexes or macrocyclic compounds have a 1,4,7-triazacyclononane (TACN) moiety with one or more amine group(s) or an O- or S- substituted TACN moiety. The macrocyclic complexes have a high-spin Fe(III) atom coordinated to the TACN moiety. The macrocyclic complexes can be used in imaging methods.SELECTED DRAWING: None

Description

Cross-reference with related applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 768,823, filed on 16 November 2018, the disclosure of which is incorporated herein by reference.

[0002] [Statement regarding federally funded research] This invention was made with government support under contract number 1710224 granted by the National Science Foundation and contract number EB025369 granted by the National Institutes of Health. The government has certain rights to this invention. [Technical Field]

[0003] This disclosure generally relates to macrocyclic compounds. When these compounds form complexes with iron(III), they can be used as MRI contrast agents. [Overview of the project]

[0004] The object of this disclosure is to provide macrocyclic compounds that can form complexes with Fe(III). The object of this disclosure is also to provide compositions and methods for producing and using the compounds and complexes.

[0005] An Fe(III) coordination complex for use as a T1 MRI contrast agent is disclosed. The disclosed Fe(III) T1 MRI contrast agent contains a macrocyclic ligand. The Fe(III) complex can also be used as a T2 MRI contrast agent.

[0006] This disclosure provides a macrocyclic compound or macrocyclic ring (macrocyclic ring) having i) a macrocyclic core containing at least two heteroatoms as ligand donors, and ii) at least one pendant donor (sometimes referred to as an anionic pendant) as a substituent of the macrocyclic core. The macrocyclic core has a ring structure containing carbon atoms and at least one heteroatom (e.g., an N atom, an O atom, or an S atom). The macrocyclic core may be a TACN moiety. The macrocyclic core may be a TACN moiety in which one or more N atoms are substituted with O atoms or S atoms.

[0007] For example, the pendant donor may be an oxygen-containing group (e.g., alcohol, oxide (e.g., alcoholic acid, or phenoxide), sulfonate, phosphinate, phosphonate, etc.). Some pendant donors, such as alcohol, phosphinic acid, phosphonic acid, or sulfonic acid, can be deprotonated when they form a complex with Fe(III) or at a specific pH.

[0008] Macrocyclic compounds may contain one or more auxiliary pendant groups. These auxiliary pendant groups may be one or more coordinating auxiliary pendant groups and / or one or more non-coordinating auxiliary pendant groups.

[0009] In one embodiment, the compounds of the present disclosure have two or more macrocyclic cores linked to one another via aromatic groups (e.g., aryl groups), macrocyclic rings, polymers, dendrimers, proteins, or peptides.

[0010] For use in the methods of this disclosure, the compounds or complexes described herein may be administered as pharmaceutical preparations. Therefore, they may be provided in various compositions and may be combined with one or more pharmaceutically acceptable carriers.

[0011] In one aspect, this disclosure provides an imaging method using the macrocyclic compounds described herein. The imaging method uses magnetic resonance imaging. Examples of such methods include, but are not limited to, magnetic resonance imaging (MRI). Specifically, the macrocyclic compounds of the Disclosure may be used as T1 MRI contrast agents when they form complexes with Fe(III). The imaging methods of the Disclosure may be used to image cells, tissues, organs, vascular systems (vascular structures), or parts thereof. Cells, tissues, organs, and vascular systems may be parts of an organism.

[0012] [Background of Disclosure] Contrast agents containing Fe(III) as trivalent iron offer an alternative to Gd(III) contrast agents. Despite the fact that a significant proportion of patients in the US population (approximately 10%) are considered at risk regarding being administered Gd(III) contrast agents, almost all clinically used contrast agents to date contain gadolinium (Gd as trivalent Gd(III)). There is a growing concern that Gd(III)-based MRI contrast agents are leading to Gd(III) deposition in the brain, bone, and skin of all patients. Alternatives to Gd(III) contrast agents containing biologically relevant transition metal ions, such as high-spin Fe(III) complexes, are expensive. Alternatives to Gd(III) contrast agents containing biologically relevant transition metal ions include high-spin Mn(II) and high-spin Fe(III) complexes. Potential advantages of using Fe(III) include the extensive mechanisms in the human body for recycling and storing iron as the most abundant transition metal ion. Also, Fe(III) complexes tend to dissociate less than Mn(II). Notably, the redox potentials of both Mn(II) and Fe(III) complexes can be tuned to prevent the generation of reactive oxygen species (ROS). For example, certain ligands form redox-inactive Fe(III) complexes that do not produce hydroxyl radicals, even under harsh conditions. The Fe-based MRI contrast agents described herein (as trivalent Fe(III)) generate contrast by the same paramagnetic mechanism as Gd(III) agents and are in small molecule form as coordination complexes, i.e., not nanoparticles.

[0013] Most Fe(III) MRI contrast agents reported to date contain simple linear chelating agents, including those with an ethylenediamine skeleton having a combination of phenol and carboxylate pendants, such as EHBG(NN'-ethylenebis[(2-hydroxybenzyl)glycine]). Another type contains polyaminocarboxylate ligands, such as the Fe(III) complex of EDTA. A third type includes microbial siderophores, desferrioxamine (DFO). All of these complexes lack an exchangeable water ligand, have a reduction potential susceptible to ROS generation, and / or It has drawbacks, including the difficulty of synthetic modification. Furthermore, the chemical properties of aqueous solutions of Fe(III) complexes are governed by the formation of insoluble complexes with hydroxides and bridging oxide ligands. Improvements are needed to obtain Fe(III) complexes that are desirable T1 relaxants rather than effective catalysts for generating ROS by altering the redox potential to stabilize Fe(III). Another consideration is the overall charge of the complex. Anionic groups, either bonded to Fe(III) as pendants or attached more detachably as auxiliary groups, are important for regulating the pharmacokinetics of contrast agents and their elimination from the body.

[0014] Based at least on the foregoing, there is an unmet need in the art for Fe(III) MRI contrast agents with improved properties. [Brief explanation of the drawing]

[0015] Figure 1 shows the synthesis scheme of TASO ligands.

[0016] Figure 2 shows the synthesis scheme for the TRAP-Ph macrocyclic ring with a phosphine pendant and the corresponding Fe(III) complex.

[0017] Figure 3 shows the synthesis of a macrocyclic compound having a phosphinate pendant.

[0018] Figure 4 shows the synthesis of macrocyclic compounds having a phosphinate pendant or phosphonic acid pendant, and the corresponding Fe(III) complex.

[0019] Figure 5 shows the synthesis scheme of the triazole pendant having an anionic group, its attachment to the macrocyclic ring, and the formation of the Fe(III) complex.

[0020] Figure 6 shows Fe(TASO) 1 The 1H NMR spectrum shows a broadening within the baseline, which is consistent with high-spin Fe(III). A shift is observed in the t-butanol proton resonance.

[0021] Figure 7 shows that, unlike Fe(III) complexes of EDTA or CDTA, Fe(III) contrast agents such as Fe(TASO) do not oxidize benzoates in the presence of peroxides and ascorbates. Oxidation of benzoates with 50 μM complex, 50 μM H2O2, and 50 μM ascorbate (pH 7.2). The oxidation rate of [Fe(EDTA)] is set to 100%.

[0022] Figure 8 shows the temperature at various temperatures. 17 The data obtained from 1O NMR studies is plotted. Regarding the experiment involving 20 mM Fe(TASO) at pH 4.5, 17 The natural logarithm of the reciprocal of the transverse relaxation rate of the O NMR resonance is shown as a function of temperature.

[0023] Figure 9 shows a plot of the time course of T1 relaxation in mice after Fe-TASO injection for various tissues, including the liver and kidney (compared to Gd(DTPA) and Fe(TOB)).

[0024] Figure 10 shows a plot of the time course of T1 relaxation in mice after Fe(TASO) injection in the blood (compared to Gd(DTPA) and Fe(TOB)).

[0025] Figure 11 shows the relaxation data obtained at 4.7 Tesla and 37 °C for an example of the macrocyclic complex of the present disclosure. Fe-NOTP shows an r1 of 0.66 ± 0.01 mM -1 s -1 and Fe-NOTP with HSA shows an r1 of 1.04 ± 0.06 mM -1 s -1 .

[0026] Figure 12 shows the cyclic voltammograms of a 1.0 mM solution of Fe(TASO) at various pH values in water containing potassium chloride (100 mM) as the supporting electrolyte and HEPES buffer. The maximum sweep width was between -1.5 V and 1.5 V, the scan rate for Fe(TASO) was 100 mV / s, and E 1 / 2 was found to be -204 mV at pH 7 and 513 V at pH 3, corrected with respect to NHE.

[0027] Figure 13 shows the transverse 17 O NMR relaxation of Fe(TASO) measured over a range of pH values and compared to Fe(DTPA), Fe(TOB), and Fe(CDTA) (as a function of temperature, ln(1 / T 2r )). Fe(CDTA) has exchangeable water ligands while Fe(DTPA) does not have exchangeable inner-sphere water ligands.

[0028] [[ID=*]] Figure 14 shows the UV-visible absorption spectrum of Fe(TASO) obtained over 72 hours at **37 °C**. The aqueous solution contained 0.2 mM of Fe(TASO) dissolved in 0.1 M HCl. ε(250 nm) = 6097 M- cm-1, ε(325 nm) = 3557 M -1 cm -1 . The dissociation after **24 hours** was 18.1%. The dissociation after **72 hours** was 53.6%.

[0029] **Note**: There seems to be a mistake in the original text where the temperature value of 37 °C was missing in Figure 14 description, and the time values of 24 hours and 72 hours were not clearly associated with the dissociation description. I've added the missing information in the translation for better understanding. If this is not what you intended, please correct the original text.Figure 15 shows the ultraviolet-visible absorption spectrum of Fe(TASO) obtained over 72 hours at 37°C. An aqueous solution containing 0.2 mM Fe(TASO) dissolved in 25 mM NaHCO, 0.50 mM Na2HPO4, and 10 mM HEPES buffer at pH 7.1 was used. ε(245nm)=6960 M- cm-, ε(325nm)=3571 M -1 cm -1 .

[0030] Figure 16 shows the ultraviolet-visible absorption spectrum of Fe(TASO) obtained over 72 hours at 37°C. The aqueous solution contained 0.2 mM Fe(TASO) dissolved in 10 mM HEPES buffer at pH 7.1. ε(245nm)=6762 M- cm-, ε(325nm)=3687 M -1 cm -1 .

[0031] Figure 17 shows T1-weighted MRI images of healthy Balb / C mice at 4.7T with a dose of 0.2 mmol / kg of Fe(TASO). Top panel: Before injection (a), 5 minutes after injection (b), 40 minutes after injection (c). Post-injection images show kidney enhancement (arrows). Bottom panel: Bladder images (arrows). Before injection (d), 5 minutes after injection, and 40 minutes after injection (f).

[0032] Figure 18 shows T1-weighted MRI images of healthy Balb / C mice at 4.7T with a dose of 0.05 mmol / kg of Fe(TASO). Top panel: Before injection (a), 30 minutes (b), 4 hours (c), and 24 hours (d). Post-injection images show kidney enlargement (arrows). Stomach is labeled S. Bottom panel: Images of the liver (L) and gallbladder (arrows), before injection (e), 30 minutes (f), 4 hours (g), and 24 hours (h).

[0033] Figure 19 shows mouse pharmacokinetic data for Fe(TASO).

[0034] Figure 20 shows mouse pharmacokinetic data for Fe(TASO) compared with Gd complexes used clinically.

[0035] Figure 21 shows T1-weighted MR images of healthy Balb / C mice at 3, 7, and 12 minutes with 4.7T, dose of 0.05 mmol / kg Fe(L4). Enhancement is shown in the bladder. Detailed description of the invention

[0036] The object of this disclosure is to provide macrocyclic compounds that can form complexes with Fe(III). The object of this disclosure is also to provide compositions and methods for producing and using the compounds and complexes.

[0037] High-spin Fe(III) complexes having macrocyclic ligands may be used as T1 MRI contrast agents. In particular, high-spin Fe(III) has desirable paramagnetic properties that shorten the T1 relaxation time of water protons for MRI contrast through both in-sphere and out-sphere interactions with water molecules.

[0038] Fe(III) coordination complexes for use as T1 MRI contrast agents are disclosed. The disclosed Fe(III) T1 MRI contrast agents contain macrocyclic ligands. While not intended to be bound by any particular theory, it is thought that the macrocyclic ligands control the spin and oxidation states, and the anionic groups control the overall charge of the complex. The Fe(III) complexes can also be used as T2 MRI contrast agents.

[0039] When complexing with Fe(III), the macrocyclic compounds (as ligands) disclosed herein are advantageous for achieving control over spin and oxidation states, as well as interactions with introsphere and extrosphere water. The cavities of these macrocyclic ligands are suitable for stabilizing high-spin Fe(III). Furthermore, control of the chemical properties of aqueous solutions can be achieved using these macrocyclic compounds. The macrocyclic complexes described herein almost encapsulate Fe(III), but in some cases have coordination sites for water ligands that enhance their effectiveness as T1 MRI contrast agents. In some cases, the introsphere water ligand can generate T1 relaxation of bulk water protons through second-sphere interactions. The Fe-based MRI contrast agents described herein (high-spin, macrocyclic rings complexed with trivalent Fe(III)) generate contrast by the same paramagnetic mechanism as Gd(III) agents. Also, they are small molecule forms as coordination complexes, i.e., they are not nanoparticles.

[0040] As used herein, unless otherwise specified, the terms “group” or “part” refer to a chemical substance that is monovalent (i.e., having one end that can covalently bond to another chemical species), divalent, or polyvalent (i.e., having two or more ends that can covalently bond to another chemical species). Examples of groups include, but are not limited to: [ka]

[0041] As used herein, unless otherwise specified, the terms “alkyl” or “alkyl group” refer to branched or unbranched saturated hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n- and isopropyl groups, and n-, iso-, sec- and tert-butyl groups. For example, alkyl groups are C1 to C 12The alkyl group may be an alkyl group (including all integer carbon numbers and the range of carbon numbers between them). The alkyl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups), aryl groups, alkoxide groups, thioalkoxide groups, carboxylate groups, carboxylic acids, ether groups, etc., and combinations thereof.

[0042] As used herein, unless otherwise indicated, the terms “aryl” or “aryl group” refer to C5 to C5. 12 This refers to aromatic or partially aromatic carbocyclic groups (including all integer carbon numbers and the range of carbon numbers between them). Aryl groups may also be called aromatic groups. Aryl groups can include polyaryl groups such as fused rings or biaryl groups. Aryl groups may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkenes, alkynes, etc.), aryl groups, alkoxides, thioalkoxides, carboxylates, carboxylic acids, ether groups, etc., and combinations thereof. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups, etc.), and fused ring groups (e.g., naphthyl groups, etc.).

[0043] As used herein, unless otherwise specified, the term “aralkyl” means any group derived from an alkyl group by substituting one or more hydrogen atoms on the alkyl group with one or more aryl groups.

[0044] As used herein, unless otherwise specified, the term “heterocyclic group” refers to a C3-C ring containing one or more heteroatoms (e.g., N, O, S, etc.) as part of its ring structure. 20 (For example, C3, C4, C5, C6, C7, C8, C9, C 10, C 11 , C 12、 C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 This refers to a cyclic group. Heterocyclic groups may be substituted or unsubstituted, and / or may have additional degrees of unsaturation. Heterocyclic groups may be fused rings (e.g., pyrrolidinyl groups). Non-restrictive examples of heterocyclic groups include furanyl groups, oxazolyl groups, isothiazolyl groups, thiazolyl groups, tetrahydropyranyl groups, piperazinyl groups, dioxanyl groups, pyrrolidinyl groups, tetrahydrothiophenyl groups, tetrahydrofuranyl groups, quinuclidinyl groups, azaadamantanyl groups, and decahydroquinolinyl groups.

[0045] This disclosure provides i) macrocyclic cores comprising at least two heteroatoms as ligand donors, and ii) macrocyclic compounds or macrocyclic rings (macrocyclic rings) having at least one pendant donor as substituents on the macrocyclic core. The macrocyclic core has a ring structure comprising a carbon atom and at least one heteroatom (e.g., a N atom, an O atom, or an S atom). As used herein, “macrocyclic donor” means a heteroatom that, when present in the macrocyclic core of a compound, has a lone pair of electrons available to donate to the Fe(III) center. For example, a macrocyclic donor may be a nitrogen atom (e.g., a tertiary amine, a secondary amine) or an oxygen atom (e.g., an ether). As used herein, “pendant donor,” including anionic pendants and auxiliary pendants, means a heteroatom that, when present in a substituent on the macrocyclic core of a compound, has a lone pair of electrons available to donate to the Fe(III) center. For example, the pendant donor may be an oxygen-containing group (e.g., alcohol, oxide (e.g., alkoxide or phenoxide), sulfonate, phosphinate, phosphonate, etc.). For example, some pendant donors, such as alcohols, phosphinic acids, phosphonic acids, or sulfonic acids, can be deprotonated when they form a complex with Fe(III) or at a specific pH. Such forms of protonation and deprotonation are within the scope of this disclosure. In some embodiments, macrocyclic compounds form a complex with Fe(III) to provide a stabilized trivalent state (E0<0mV vs. NHE). In some embodiments of schemes I and II, R1 may be an auxiliary group that does not bond to Fe(III). Anionic groups may be added to the auxiliary group that does not bond to Fe(III), and these include sulfonate, phosphinate, phosphate, phosphonate, or carboxylate groups.

[0046] The macrocyclic core may be a TACN portion. The macrocyclic core may be a TACN portion in which one or more N atoms are substituted with O atoms or S atoms.

[0047] In one embodiment, the macrocyclic ring has the following structure (Scheme 1) [ka] Scheme I Here, X1, X2, and X3 are N; W1 is either O or S; Y1, Y2, and Y3 are each independently i) pendant donors containing O, where O has at least one lone pair of electrons, preferably two or three lone pairs (e.g., ketones, alcohols, alkoxides, phenols or phenoxides, sulfonic acids, phosphinic acids, or phosphonic acids, or their deprotonated forms, such as oxides of alkoxides or phenoxides); or ii) A pendant donor containing N, where N has at least one lone pair of electrons (e.g., triazole); m1, m2, and m3 are each independently 1, 2, or 3; n1, n2, and n3 are each independently 1, 2, or 3; R1 is a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted alkyl group, where the alkyl segments of the alkyl-Y chain (alkyl-Y1, alkyl-Y2, and / or alkyl-Y3) may each be independently substituted or unsubstituted. In another embodiment, any or all of alkyl-Y1, alkyl-Y2, and alkyl-Y3 may each be independently any of structures 1 to 9 as defined in Scheme III.

[0048] In one embodiment, R1 is not replaced by a pendant donor.

[0049] In some embodiments, the macrocyclic ring may have the following structure (Scheme II). [ka] Scheme II Here, R1 is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted alkyl, where if the macrocyclic ring has structure I, Z1 is H or one of the pendant groups of scheme III, and Z2 and Z3 are each independently one of the pendant groups of scheme III; if the macrocyclic ring has structure II or III, Z1 and Z2 are each independently one of the pendant groups of scheme III; and here, for all structures I to III, if Z1, Z2, and Z3 are applicable to each other, they are selected independently of each other. This paragraph will hereafter be referred to as "scheme II".

[0050] In some embodiments, when the macrocyclic ring of scheme II forms a complex with Fe(III), R1 does not coordinate to Fe(III).

[0051] In one embodiment, a macroannular ring, as defined in paragraph

[0045] or according to scheme I or II, has at least one pendant donor on the macroannular core. For example, the pendant donor may have the following structure (scheme III): [ka] Scheme III Here, R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group (which may be an aryl group), or a substituted ether; R3 is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group; and R4 is a substituted alkyl group (e.g., substituted with a hydroxyl or carboxylate group), an unsubstituted alkyl group, or a substituted or unsubstituted aryl group. For example, some pendant donors, such as alcohols, phosphinic acids, phosphonic acids, or sulfonic acids, can be deprotonated when forming complexes with Fe(III) or at specific pH values. Such forms of protonation and deprotonation are within the scope of this disclosure. For example, the pendant donor may be an alkoxide, phosphinate, phosphonate, or sulfonate (as shown in Scheme IV). TIFF0007864792000005.tif35169 Scheme IV - Ionized Group

[0052] In one embodiment, the R1 group of the macrocyclic ring in schemes I and II (which may be a coordination auxiliary or a non-coordination auxiliary) may be the structure according to scheme V. [ka] Scheme V Here, A and A' are, independently, substituted or unsubstituted C1-C in a linear or branched structure. 12Q1 is an alkyl group or a proton, where Q1 is an aryl group substituted with an anionic group (e.g., carboxylate, sulfonate, phosphonate, phosphate ester, or phosphinate), an alkyl group substituted with an anionic group (e.g., carboxylate, sulfonate, phosphonate, phosphate ester, or phosphinate), or an aralkyl group substituted with an anionic group (e.g., carboxylate, sulfonate, phosphonate, phosphate ester, or phosphinate), where at least one of A or A' is an alkyl group substituted with an anionic group (e.g., an amino acid, particularly glycine, serine, or aspartic acid). Aminophosphinic acids and phosphate esters are preferred.

[0053] In one embodiment, Q1 is aralkyl, and the alkyl portion of the aralkyl group is methyl (C1).

[0054] In some embodiments, the pendant donor of scheme V (e.g., R1 in schemes I and II) does not coordinate to the Fe(III) center. While not intended to be bound by any particular theory, these pendants are thought to help modulate the charge on the complex and facilitate binding to serum albumin. In other embodiments, the pendant donor of scheme V (e.g., R1 in schemes I and II) coordinates to the Fe(III) center.

[0055] As previously mentioned, some pendant donors, such as alcohols, sulfonic acids, phosphinic acids, or phosphonic acids, can be deprotonated when they form complexes with Fe(III) or in solutions with a more basic pH. These corresponding alkoxides, sulfonates, phosphinates, and phosphonates are within the scope of this disclosure.

[0056] A particular pendant may have two or more N or O donor atoms (e.g., triazole, phosphonate, or phosphinate), but generally only one is coordinated to the metal ion.

[0057] If there are two or more pendant donors, they may be the same or different. In various examples, if there are two or more pendant donors, the individual pendants may be the same or different.

[0058] Macrocyclic compounds may contain one or more auxiliary pendant groups. These auxiliary pendant groups may consist of one or more coordinating auxiliary pendant groups and / or one or more non-coordinating auxiliary pendant groups.

[0059] Non-coordinating auxiliary pendant groups do not have heteroatoms that can bond to Fe(III) metal ions to form five-membered or six-membered chelates. Non-limiting examples of non-coordinating auxiliary pendant groups include benzyl groups, phenyl groups, and other aromatic (e.g., aryl) groups having one or more methylene groups bonded to an aromatic group or not having a methylene group, alkyl groups (both branched and linear). Other non-limiting examples of non-coordinating auxiliary pendant groups include biphenyl, naphthyl, anthracenyl, pyridyl, quinolyl, methyl, ethyl, isopropyl, n-propyl, ethyl methoxy ether, and PEG derivatives (polyethylene glycol).

[0060] Non-limiting examples of coordinating pendant groups (e.g., a third pendant group when two are hydroxypropyl, phosphinate, or phosphonate) include oxygen or nitrogen donors that form five- or six-membered chelates, such as amides, carboxylates, alcohols or phenols, or derivatives of triazoles, imidazoles, pyrazoles, picolyls, pyridines, alkylamines, aminopyridines, aminophenols, aniline, etc. Some of these groups can be deprotonated when bonded to Fe(III).

[0061] A macrocyclic complex containing one or more non-coordinating auxiliary pendant groups may have one or more open coordination sites (having open coordination), which may be one or more inspheric water molecules, one or more hydroxides, or a combination thereof. A macrocyclic complex containing one or more coordinating auxiliary pendant groups may not have open coordination sites (having closed coordination). In the case of closed coordination or open coordination, the macrocyclic complex may have one or more second-spheric water molecules.

[0062] In various examples, the macrocyclic core may have two or three nitrogen atoms, zero or one oxygen atom, and / or zero or one sulfur atom. For example, the macrocyclic core may have six, seven, eight, or nine carbon atoms. For example, the macrocyclic core may have nine to twelve atoms (including all ranges and integers in between), where at least one of the atoms in the macrocyclic core is a heteroatom, such as N. In another embodiment, at least two of the atoms in the macrocyclic core are heteroatoms, such as N. In various examples, there are two or three carbon atoms separating the heteroatoms in the macrocyclic core. One or more carbon atoms in the macrocyclic core may be unsubstituted (e.g., -CH2-) or substituted (e.g., -CHR- or -CHRR-), provided that at least one carbon atom in the macrocyclic core is substituted with a pendant donor. For example, they may be substituted with substituents disclosed herein. In another embodiment, the macrocyclic core comprises at least two heteroatoms, each independently of N or O, and they are separated from each other by at least two carbon atoms.

[0063] In some embodiments, the pendant group is covalently bonded to a macrocyclic core (e.g., to nitrogen): in particular, TACN(I).

[0064] Non-restrictive examples of macrocyclic compounds are shown in schemes VI and VII below: [ka] [ka] [ka] [ka]

[0065] In some embodiments, if the macrocyclic ring has the structure (I) of scheme II, and Z1 and Z2 are the structure 6 of scheme III where R3 is an unsubstituted ethyl group, then Z3 is not the structure 6 of scheme III where R3 is an unsubstituted ethyl group. In further embodiments, if the macrocyclic ring has the structure (I) of scheme II, and Z1 and Z2 are the structure 6 of scheme III where R3 is an unsubstituted or substituted ethyl group, then Z3 is not the structure 6 of scheme III where R3 is an unsubstituted or substituted ethyl group. In additional embodiments, if the macrocyclic ring has the structure (I) of scheme II, and Z1 and Z2 are the structure 6 of scheme III where R3 is an unsubstituted alkyl group, then Z3 is not the structure 6 of scheme III where R3 is an unsubstituted alkyl group. In yet another embodiment, if the macrocyclic ring has the structure (I) of scheme II, and Z1 and Z2 are the structure 6 of scheme III where R3 is an unsubstituted or substituted alkyl group, then Z3 is not the structure 6 of scheme III where R3 is an unsubstituted or substituted alkyl group.

[0066] In a particular embodiment, if the macrocyclic ring has a scheme II structure (I) and Z1 and Z2 have a scheme III structure 7, then Z3 does not have a scheme III structure 7.

[0067] In certain embodiments, if the macrocyclic ring has the structure of scheme II (I) and Z1 and Z2 are structure 8 of scheme III where R3 is an alkyl with a terminal hydroxyl substitution, then Z3 is not structure 8 of scheme III where R3 is an alkyl with a terminal hydroxyl substitution. In further embodiments, if the macrocyclic ring has the structure of scheme II (I) and Z1 and Z2 are structure 8 of scheme III where R3 is a substituted alkyl, then Z3 is not structure 8 of scheme III where R3 is a substituted alkyl.

[0068] In some embodiments, if the macrocyclic ring has the structure of scheme II (II) and Z1 and Z2 are structure 8 of scheme III, where R4 is an alkyl with a terminal hydroxyl substitution, then R1 is not an alkyl with a terminal aryl group. In additional embodiments, if the macrocyclic ring has the structure of scheme II (II) and Z1 and Z2 are structure 8 of scheme III, where R4 is an alkyl with a terminal hydroxyl substitution, then R1 is not a substituted alkyl.

[0069] In certain embodiments, Fe(III) forms complexes with macrocyclic rings. In other embodiments, Fe(III) does not form complexes with macrocyclic rings. Fe(III) can form complexes with macrocyclic rings as shown herein.

[0070] While not intended to be bound by any particular theory, the macrocyclic rings described herein are thought to be able to stabilize the trivalent iron (Fe(III)) state when bonded to Fe(III). For example, coordination forms are designed for the desirable bonding of Fe(III) compared to Fe(II) to maintain the Fe(III) oxidation state under biologically relevant conditions. The stabilization of the Fe(III) state (E0 < 0 mV vs. NHE) also plays a role in suppressing the generation of reactive oxygen species that result from the reduction of the complex to the Fe(II) state.

[0071] The Fe(III) center is preferably stabilized with respect to Fe(III) so as not to react with biological reducing agents to produce reactive oxygen species (ROS). Such a redox-inactive Fe(III) center has a negative redox potential with respect to NHE. Examples of macrocyclic complexes of the present invention having a macrocyclic core and pendant group that produce stabilized Fe(III) include, but are not limited to, a 1,4,7-triazacyclononane macrocyclic core and an alcohol pendant group that is deprotonated upon Fe(III) binding.

[0072] In various examples, the macrocyclic compounds or compounds of the present disclosure exhibit a reduction potential (E0) of less than 0 mV relative to a standard hydrogen electrode (NHE) in aqueous solution at biologically relevant pH (e.g., pH 6.5–7.5 or 7.2–7.4). In various other examples, the macrocyclic compounds or compounds of the present disclosure exhibit a reduction potential (E0) of at least -100, at least -150, at least -200, at least -300, at least -400, at least -500, or at least -600 mV relative to a standard hydrogen electrode (NHE) in aqueous solution at biologically relevant pH (e.g., pH 6.5–7.5 or 7.2–7.4). In various other examples, the macrocyclic compounds or compounds of the present disclosure exhibit a reduction potential (E0) of less than 0, greater than -100, greater than -150, greater than -200, greater than -300, greater than -400, greater than -500, or greater than -600 mV relative to a standard hydrogen electrode (NHE) in aqueous solution at biologically relevant pH (e.g., pH 6.5–7.5 or 7.2–7.4).

[0073] In various other examples, the macrocyclic compounds or compounds of the present disclosure exhibit a reduction potential (E0) of less than 0 to -600 mV relative to a standard hydrogen electrode (NHE) in aqueous solution at biologically relevant pH (e.g., pH 6.5 to 7.5 or 7.2 to 7.4).

[0074] The shortening of the T1 relaxation time of water protons, and T1 relaxation, are facilitated by both introspheric and extrospheric water. That is, in various examples, the macrocyclic complexes and compounds of the present disclosure contain one or more pendant donor groups that can hydrogen-bond to water via heteroatoms such as oxygen or nitrogen. Non-limiting examples of such pendant donor groups include pendant alcohol groups, phosphinates, and phosphonates that deprotonate to alkoxide groups (forming hydrogen bonds to secondary sphere water). Furthermore, in various examples, the macrocyclic compounds and compounds of the present disclosure contain open coordination sites that bond to water. These water ligands can ionize to form hydroxide ligands at neutral pH, as demonstrated, for example, by pH potentiometric titration. Rapid exchange of water ligands is desirable. Evidence for rapid exchange of water ligands is found at various temperatures. 17 This has been shown by NMR spectroscopy studies. Reduced transverse relaxation time (T 2r )teeth 17 This is approximated by measuring the linewidth of the O resonance.

[0075] The coordination chemical properties of Fe(III) depend on the coordination number. The compounds of this disclosure have donor groups that may be part of a macrocyclic core, also called macrocyclic donors, and the donor groups may also be part of substituents on the macrocyclic core, also called pendant donors. In one embodiment, when Fe(III) is complexed with the compounds of this disclosure, 4 to 7 donors are complexed around the metal ion center. In one embodiment, the macrocyclic core may have 2 to 4 donors and 1 to 4 (e.g., 2 to 4) pendant donors, and this includes all combinations. In various embodiments, there are 2 macrocyclic donors and 3 pendant donors, 2 macrocyclic donors and 4 pendant donors, 3 macrocyclic donors and 1 pendant donor, 3 macrocyclic donors and 2 pendant donors, and 3 macrocyclic donors and 3 pendant donors.

[0076] Scheme VIII presents several macrocyclic complexes (with Fe(III)) that fall within the scope of this disclosure. [ka]

[0077] As used herein, macrocyclic complex refers to a macrocyclic compound that forms a complex with Fe(III). In some embodiments of the macrocyclic complex, the macrocyclic ring has a scheme II structure (I), and if Z1 and Z2 are scheme III structure 5, then Z3 is not scheme III structure 5.

[0078] In some embodiments of the macrocyclic complex, the macrocyclic ring has a scheme II structure (I), and Z1 and Z2 are scheme III structure 8 where R4 is an unsubstituted aryl, while Z3 is not scheme III structure 8 where R4 is an unsubstituted aryl.

[0079] In one embodiment, the compound of the present invention may have two or more macrocyclic cores linked together via aromatic (e.g., aryl) groups, macrocyclic rings, polymers, dendrimers, proteins, or peptides. [ka]

[0080] For tumor uptake and retention, the size of the contrast agent-containing molecules is important. Furthermore, the degree of T1 relaxation increases in proportion to the number of iron complexes, and also the size of the molecules, more precisely the rotational correlation time (τ). c Assuming that it increases with τ, the use of multiple linked macrocyclic complexes should enhance contrast. c An increase in binding can also be achieved by binding to blood proteins, particularly human serum albumin (HSA). Certain ligand functional groups promote binding to human serum albumin (HSA). Typically, these include anionic groups and aromatic groups (e.g., aryl groups).

[0081] In various embodiments, the compounds of the Disclosure may be salts, partial salts, hydrates, polymorphs, or stereoisomers, or compounds of the Disclosure, or mixtures thereof. For example, the compounds may exist as racemic mixtures, single enantiomers, single diastereomers, or mixtures of diastereomers. In certain embodiments, after metal complexation, the compounds may exist as mixtures of diastereomers and / or conformational isomers that can be measured by NMR. Diastereomers arise from the conformation of the macrocyclic core and the orientation of substituents on the macrocyclic core.

[0082] The compounds of this disclosure may have intrinsic water or hydroxide ligands. In one embodiment, the compound has one intrinsic ligand (q) that contributes to relaxation, as shown in Formula 1.

number

[0083] Equation 1 shows that the relaxation is due to contributions from bound water (inner sphere, IS) and second sphere (SS) and (outer sphere) water. Equation 2 predicts that a larger number of bound water molecules and a faster ligand exchange rate constant (short lifetime of bound water (τm)) are favorable. In particular, r1, a parameter used to evaluate the characterization of relaxation, is mM -1 s -1 It has units of T relative to contrast agent concentration. lobs (s -1 This is obtained from the plot of ). The number and residence time are not clearly defined, but a similar relationship exists for water in the second circle. The r1 and r2 relaxation properties (from the T1 and T2 relaxation rate constants at 37°C and 4.7T) are summarized in Table 1. [Table 1]

[0084] The structures of the specific complexes in Table 1 are shown below. [ka]

[0085] In particular, the ratio of T1 relaxation (r1 / r2) to T2 for the macrocyclic complexes or compounds of the present disclosure is preferably as close as possible to 1 (identical). By definition, r2, i.e., transverse relaxation, is always greater than r1, i.e., longitudinal relaxation. In various preferred examples, the Fe(III) contrast agents of the present disclosure have a low r2, giving an r1 / r2 ratio close to 1, as shown, for example, in Table 1. In various examples, the macrocyclic complexes or compounds of the present disclosure have an r1 / r2 ratio of 0.3 to 1.0. These data demonstrate the suitability of the TACN ligand, alcohol pendant, and open coordination site in Fe(TASO), for example, compared to complexes without an open coordination site for mononuclear complexes such as Fe(TOP) (see, for example, Table 1). However, pendant groups such as phosphonates and phosphinates are expected to have strong second-circumferential interactions that increase r1, as shown for Fe(TRAP-Ph) in Table 1.

[0086] Table 1 shows that the interaction between Fe(III) complexes and water molecules can enhance the relaxation of water protons. While not bound by any particular theory, the exchange of intrinsic water and bulk water is considered an important mechanism for proton relaxation. However, Fe(III) is a much smaller metal ion than Gd(III) (0.78 Å and 1.25 Å, respectively). The shorter MH distance of Fe(III) in bound water compared to Gd(III) suggests that the relative efficiency of contributions from the second sphere and the intrinsic sphere may differ for the two metal ion complexes.

[0087] Related paramagnetic relaxation of water (1 / T 1m There are three mechanisms that contribute to longitudinal relaxation: the scalar (contact) contribution, the dipole-dipole contribution, and the Curie spin relaxation. Of these, the most important for longitudinal relaxation considered here is the dipole-dipole contribution (1 / T1DD). For magnetic field strengths greater than 1.5T, 1 / T1DD is defined as shown in Equation 3, where S is the spin quantum number, ω H is the Larmor frequency of protons, r MH γ is the metal ion-proton distance,H is the gyromagnetic ratio of protons, ge is the g factor of electrons, μ B This is the Bohr magneton, μ o is the permittivity of vacuum. In particular, 1 / T1DD increases at larger total spins (S) that are more favorable to Gd(III) than to Fe(III) (higher relaxation). However, the distance (r) from the paramagnetic Fe(III) center to the water proton is different. MH The fact that ) is shorter is especially true for 1 / r 6 Considering the dependence, it is favorable for proton relaxation of Fe(III).

number

[0088] Correlation time of the dipole relaxation mechanism (τ c ) is the lifetime of bound water (1 / τ m ), rotational motion of contrast agent (1 / τ R ) and longitudinal relaxation of unpaired electrons (1 / T 1e It is influenced by various processes, including ). Any of these three processes can contribute, but their importance depends on the magnetic field strength. Much of the literature focuses on the importance of these processes at low magnetic field strengths (<1T). Under these conditions, rotational processes or electron relaxation times may be limited, and τ m It should be in a narrow range close to 10 ns (k ex =10 8 s -1 However, at higher magnetic field strengths (≧1.5T), the optimal τ m Simulations show that it has a larger range (1-100 ns), and the rotational motion should be an intermediate value between that of small molecules and proteins. 1e Therefore, the electron relaxation time is an important parameter. The long T of Fe(III) 1e These arise from complexes with high symmetry, rarely cause zero-field splitting, and can lead to slow relaxation of the electronic state. Furthermore, the coordination sphere must be favorable to high spin (S=5 / 2) and not favorable to low spin S=1 / 2Fe(III). The definition for Fe(TASO) 1The defect in the H NMR spectrum is also consistent with high-spin Fe(III) that efficiently relaxes water protons (Figure 6). 5.8 Solution effective magnetic moment μ eff This also matches the state S=5 / 2.

[0089] The test to determine whether a metal complex contains bound water is temperature-variable. 17 This may include the collection of NMR data. The data in Figure 8 were obtained using a Varian 400 MHz NMR spectrometer with a broadband probe. 17 Because the natural abundance of O isotopes is low, each complex is H2 17 The compound was dissolved in an O-rich aqueous solution to make the peak measured by NMR larger and therefore easier to detect visually. NMR tests were performed at various temperatures. The temperature range for Fe(TASO) was 298K to 340K. Temperature-dependent transverse relaxation data were fitted to various equations by least-squares fitting analysis using Scientist for Windows version 3.0. First, complexes with open coordination sites are known to follow the Swift-Connick equation shown in equation 5a:

number

[0090] In complexes tested within the temperature range where data was recorded and analyzed, T 2m -1 and 1 / T 2os This can be ignored, and the Swift-Connick equation is reduced to equation 5b. In relation to this, since the reduced transverse relaxation rate is often very large, taking the natural logarithm on both sides of the equation allows for better scaling and a simpler representation of the data, as shown in equation 5c:

number

[0091] The reciprocal of the residence time of bound water, and the chemical shift difference between bound water and bulk water, are expressed by equations 6a and 6b, respectively:

number

[0092] In equation 6a, k ex k is the water exchange rate constant at the coordination site and is the reciprocal of the bound water residence time. b is the Boltzmann constant, h is the Planck constant, T represents absolute temperature, and ΔS ‡ and ΔH ‡ θ represents activation entropy and enthalpy, respectively. In equation 6b, g L μ is the isentropic Lande factor. bis the magnetic moment, S represents the total spin states, B represents the applied magnetic field, and (A / h [H-bar]) represents the hyperfine coupling constant. In equation 6b, the isentropic Lande factor, magnetic moment, spin states, magnetic field, and hyperfine coupling constant terms are integrated into a single parameter, which is solved by processing the data. This integration limits equation 6a to a simple inverse temperature dependence, and the simplified constant is represented by the constant C. Using this method, the exchange rate constant of bound water in Fe(TASO) was tested, as shown in Figure 8. Additional studies over a range of pH values ​​confirmed the absence of inner-sphere ligands at neutral pH (Figure 13). This data compared Fe(CDTA), i.e., the complex with bound water, and Fe(DTPA), i.e., the complex without inner-sphere water, and revealed that Fe(TASO) lacks inner-sphere water that is directly bound. Thus, the T1 relaxation arises from the water molecules in the second and outer spheres.

[0093] The compounds of this disclosure are thermodynamically stable and / or kinetically inert to dissociation. In one embodiment, the compounds are thermodynamically stable and kinetically inert to dissociation. In one embodiment, the kinetic inertness of the compounds of this disclosure can be described using the rate constant of dissociation. In one embodiment, the macrocyclic donor and pendant donor do not significantly dissociate from the metal center for up to 24 hours at neutral pH in the presence of 1) 25 mM carbonate, 0.40 mM phosphate, and 100 mM NaCl (pH 7.2).

[0094] In one embodiment, Fe(III) is high-spin S=5 / 2. A paramagnetic spin state is required for effective T1 (longitudinal) relaxation. To maintain Fe(III) in a high-spin state, ligand (or crystal) field splitting should not be too large. If the crystal field splitting is greater than the pair formation energy, a low-spin (S=1 / 2) state will occur. Fe(III) is readily maintained in a high-spin paramagnetic state with certain ligand donor groups, particularly those containing anionic oxygen or nitrogen donors.

[0095] It is desirable that the Fe(III) complex remains in a trivalent oxidation state and is not reduced by peroxides, superoxide, ascorbic acid, or glutathione at concentrations present in extracellular media such as mammalian cells (e.g., human cells). Typically, a redox potential of less than zero mV relative to NHE is sufficient. If the complex is reduced to Fe(II) and the Fe(II) complex, and the complex has a positive redox potential relative to NHE, reactive oxygen species can be generated. For example, [Fe(EDTA)] has a redox potential of approximately 300 mV and generates ROS as shown in the assay in Figure 7.

[0096] For use in the methods of this disclosure, the compounds or complexes described herein may be administered as pharmaceutical preparations. Accordingly, they may be provided in various compositions and may be combined with one or more pharmaceutically acceptable carriers. Some examples of pharmaceutically acceptable carriers can be found in Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins. The compositions may be provided as liquids, solutions, or solids and may be provided in combination with any suitable delivery form or vehicle, including, but not limited to, caplets, capsules, tablets, inhalants, or aerosols.

[0097] The compositions of this disclosure can be introduced into an organism using various methods known to those skilled in the art. These methods include, but are not limited to, intravenous, intramuscular, intracranial, intrathecal, intradermal, subcutaneous, and oral routes. In one embodiment, the composition is administered intravenously.

[0098] In one embodiment, the complex used in the method of the present disclosure may have the following structure: [ka] [ka]

[0099] The required solubility of the complex depends on its effectiveness in generating contrast. For Fe(III)T1 contrast agents with good relaxability, the complex should be 100 μL. -2 A solubility of mM is required. However, solubility or resilience may be increased by using other additives, such as human serum albumin (HSA) or meglumine. In a non-limiting example, at 4.7 Tesla, at least 1.5 mM is required. -1 s -1 Contrast agents with moderate relaxation properties are considered to exhibit good relaxation properties when conjugated to human serum albumin in vitro. The addition of HSA (35 mg / mL) to some iron(III) complexes can give higher T1 relaxation properties, as shown in Table 1. However, in vitro, contrast agents with moderate relaxation properties (0.6~1.4 mM) are considered to exhibit good relaxation properties. -1 s -1 ) can produce good contrast in vivo when the contrast agent interacts with organs such as the kidneys or liver. Solubility is generally measured at 37°C in an aqueous solution with a nearly neutral pH (6.5-7.5) in 100 mM NaCl containing 25 mM carbonate and 0.4 mM phosphate. The dose of the composition used necessarily depends on the needs of the individual to whom the composition of this disclosure is administered. These factors include, but are not limited to, the individual's body weight, age, sex, and medical history. Figures 9 and 10 show data from in vivo MRI studies performed on mice. The contrast agent Fe(TASO) was injected as 0.20 mL of 6.3 mM Fe(TASO) (10.5 mg of HSA or 2 equivalents of meglumine).

[0100] In one aspect, this disclosure provides an imaging method using macrocyclic compounds described herein. The imaging method uses magnetic resonance imaging. Examples of such methods include, but are not limited to, magnetic resonance imaging (MRI).

[0101] Specifically, when complexed with Fe(III), the macrocyclic compounds of this disclosure can be used as T1 MRI contrast agents. These complexes may have properties that change with pH. Such properties make these complexes useful for pH mapping, which enables better therapeutic management of diseases such as cancer, stroke, and heart disease. When complexed with Fe(III), the macrocyclic rings can also be used as T2 MRI contrast agents.

[0102] The imaging methods of this disclosure may be used to image cells, tissues, organs, vascular systems (vascular structures), or parts thereof. Cells, tissues, organs, and vascular systems may be parts of an organism. "Organism" means human or animal. In one embodiment, this disclosure provides a method for obtaining an image of cells, tissues, organs, or at least part of a vascular system, comprising the steps of contacting cells, tissues, organs, or a vascular system with a compound of this disclosure, and imaging at least part of the cells, tissues, organs, or vascular system to obtain an image of the cell, tissue, organ, or vascular system portion. At least part of the cells, tissues, or organs may be alive or dead. Similarly, an organism may be alive or dead.

[0103] In one embodiment, a macrocyclic compound can form a complex with Fe(III) and be used as a T1 MRI contrast agent. This contrast is generated by T1-weighted imaging, which provides positive contrast to the region where the iron complex accumulates. The complex is high-spin Fe(III) under biological reducing conditions involving inspheric or exospheric water interactions that reduce the T1 relaxation time of bulk water protons.

[0104] The following statements provide non-limiting examples of macrocyclic complexes, macrocyclic compounds, and imaging methods of the present disclosure: statement 1 1,4,7-Triazacyclononane (TACN) moieties or substituted TACN moieties (e.g., O-substituted TACN moieties or S-substituted TACN moieties); A substituent on the TACN moiety, one or more anionic pendant groups independently selected from the following: [ka] Here, R2 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, and may be an aryl group or a substituted ether; R3 is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group, and R4 is a substituted alkyl group (e.g., substituted with a hydroxyl or carboxylate group or similar), or an unsubstituted alkyl group, or a substituted or unsubstituted aryl group; and Q1 is an aryl group substituted with an anionic group (e.g., carboxylate, sulfonate, phosphonate, phosphate ester, or phosphine), an alkyl group substituted with an anionic group (e.g., carboxylate, sulfonate, phosphonate, phosphate ester, or phosphine), or an aralkyl group substituted with an anionic group (e.g., carboxylate, sulfonate, phosphonate, phosphate ester, or phosphine); and i) a TACN moiety or a substituted TACN moiety, and ii) a high-spin Fe(III) cation complexed to at least one anionic pendant group substituent of a macrocyclic compound (e.g., a TACN moiety or a substituted TACN moiety). Macrocyclic complexes containing these compounds, or their salts, partial salts, hydrates, polymorphs, and stereoisomers. statement 2 If the macro-ring has a scheme II structure (I) and Z1 and Z2 have a scheme III structure 5, then Z3 does not have a scheme III structure 5; and / or If the macrocyclic ring has a scheme II structure (I), and Z1 and Z2 are scheme III structure 8 where R4 is an unsubstituted aryl, then Z3 is not scheme III structure 8 where R4 is an unsubstituted aryl. The macrocyclic complex described in Statement 1. statement 3 The macrocyclic complex according to statement 1 or 2, wherein at least one or all of the one or more pendant groups are covalently bonded to a nitrogen atom on the TACN moiety. statement 4 The macrocyclic complex according to any one of the preceding statements, wherein the macrocyclic complex has at least one open coordination site. statement 5 The macrocyclic complex according to any one of the preceding statements, wherein the macrocyclic complex has at least one water and / or at least one hydroxide complexed to a high-spin Fe(III) cation. statement 6 At least one of the pendant groups is substituted at the benzyl position or at any carbon of an alkyl group attached to a heteroatom of the pendant group, the macrocyclic complex according to any one of the preceding statements. statement 7 The macrocyclic complex according to any one of the preceding statements, wherein the macrocyclic complex further comprises one or more auxiliary groups (e.g., one or more coordinating pendant groups, one or more non-coordinating pendant groups, or a combination thereof). statement 8 The macrocyclic complex according to statement 7, wherein the coordinating pendant group or the non-coordinating pendant group has the following structure:

Chemical formula

[0105] In this application, the use of the singular form includes the plural form, and vice versa.

[0106] The macrocyclic compounds of this disclosure may be prepared, for example, as described in the details of the experiment. The following examples are provided to illustrate this disclosure and are not intended to limit it in any way. Those skilled in the art will recognize that customary changes and modifications may be made to these embodiments which are intended to be within the scope of this disclosure.

[0107] The steps of the methods described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of this disclosure. Therefore, in one embodiment, the method essentially consists of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of only such steps.

[0108] Experiment details Measurement means Using a Varian Inova 500 MHz NMR spectrometer equipped with an FTS Systems TC-84 Kinetics Air Jet Temperature Controller, 1 1H NMR data was collected. 13 The 13C NMR spectrum was acquired using a Varian Mercury 300 MHz NMR spectrometer operating at 75 MHz. 17NMR spectra were recorded using a Varian Inova 400 MHz spectrometer with a 5 mm broadband probe operating at a resonant frequency of 54.24 MHz. All pH measurements were obtained by using an Orion 8115BNUWP Ross Ultra Semi Micro pH electrode connected to a 702 SM Titrino pH. Mass spectral data were collected using a ThermoFinnigan LCQ Advantage IonTrap LC / MS with a Surveyor HPLC system. Absorbance spectra were collected using a Beckman-Coulter DU 800 UV-vis spectrophotometer with a Peltier temperature controller. 1% H2 17 Samples were prepared in water containing 0. Chemical shifts and the linewidth at half maximum of the symmetric water peak were measured at various temperatures and pH 6, both in the presence and absence of the metal complex. Transverse relaxation times were calculated using the Swift-Connick equations (Equations 5-6) with the signal linewidths at variable temperatures in the presence and absence of the metal complex. All fits originate from the simultaneous least-squares fit of the measured data.

[0109] TASO 0.100 g of TACN (1,4,7-triazacyclononane) (0.774 mmol) in a 4 mL toluene / 1 mL chloroform solution was added to a 25 mL round-bottom flask equipped with a gas inlet and a stirring rod. 0.0920 g of N,N-dimethylformamide dimethylacetal (0.774 mmol) was added to the flask. The solution was stirred at room temperature for 24 hours. ESI-MS (m / z) of 1,4,7-triazatricyclo[5.2.1.04,10]decane (TACN-orthamide), calculated value: 140.1 [M+H +(100%). The solution was dried by placing the flask on a rotary evaporator. The dried TACN-orthoamide and 15 mL of dry tetrahydrofuran (THF) were added to a 50 mL three-neck round-bottom flask equipped with a magnetic stir bar, reflux condenser, gas injection tube and addition funnel. 92.2 μL of benzyl bromide (0.774 mmol) was added to the flask and the solution was stirred at room temperature overnight. The white-beige precipitate was collected by suction filtration and washed with dry THF (10 mL) and diethyl ether (10 mL). 7 mL of methanol and 7 mL of 12 M HCl were added to the precipitate in the flask for deprotection treatment. The solution was heated to reflux for 4 hours. After cooling the solution to room temperature, NaOH pellets were added to adjust the pH of the solution to 8. Next, the solution was filtered to remove the NaCl salt precipitate and extracted with chloroform (3 × 60 mL). ESI-MS (m / z), calculated: 220.3 [M+H + (100%). The solution was rotary evaporated, dissolved in 15 mL of ethanol in a 25 mL round-bottom flask containing 0.225 g of S-propylene oxide (3.870 mmol) and stirred at room temperature for 24 hours. The solution was rotary evaporated, dried under vacuum on a Schlenk line and dissolved in 10 mL of methanol. %ESI-MS (m / z), calculated: 336.3 [M+H + (100%). Benzyl deprotection was carried out with a Pd / C (10%) catalyst. 30 mg of Pd / C (10%) in 5 mL of methanol and 1 mL of water was added to the ligand solution. Air was removed from the solution for 30 minutes under argon gas. Catalytic hydrogenation was carried out for 3 days at room temperature with vigorous stirring under a hydrogen atmosphere. The mixture was filtered through celite to remove the catalyst from the reaction solution and (1,4,7-triazonane-1,4-diyl)bis(propan-2-ol) (DACO ligand) was obtained. ESI-MS (m / z), calculated: 246.3 [M+H +(100%). The solution was dried by placing the flask on a rotary evaporator. The DACO ligand was dissolved in 15 mL of acetonitrile in a 25 mL round-bottom flask. One equivalent of 1,3-propanesultone in 3 mL of acetonitrile and N,N-diisopropylethylamine (DIEA) were added to the flask. The solution was stirred under reflux for 3 days. The solution was evaporated by rotary evaporation and dried under vacuum on a Schlenk line. The purification process was carried out by basic alumina column with MeOH / DCM solvent. The yield was calculated to be 37%. ESI-MS (m / z) (0.1% formic acid method), calculated value: 368.3 [M+H + ](100%) and 390.3[M+Na + (27%).

[0110] Fe(TASO) (See Figure 1, for example) TASO ligand (0.0589 g, 0.160 mmol) and 8 mL of ethanol were added to a 25 mL round-bottom flask equipped with a stirring bar. Then, FeCl2·4H2O (0.0318 g, 0.160 mmol) was dissolved in 2 mL of ethanol and added to the flask. The solution was stirred at room temperature for 2 days. A yellow precipitate was obtained. The precipitate was obtained by centrifugation and washed three times with diethyl ether. A yellow powder was obtained by removing the solvent using a rotary evaporator. ESI-MS (m / z), calculated value: 421.1 [M+H + ](100%) and 443.2[M+Na + ](55%). Here, M is neutral Fe(TASO). The magnetic susceptibility of Fe(TASO) was measured in aqueous solution using the Evans method and was 5.67 μeff.

[0111] NOTPMe (see, for example, Figure 4) 1,4,7-Triazacyclononane (3.1 mmol) was added to a two-necked flask. Paraformaldehyde (15.4 mmol, 5 equivalents) and triethyl phosphite (31 mmol, 10 equivalents) were added under an argon atmosphere. The reaction mixture was stirred at 60°C for 12 hours. The excess reaction mixture was removed under reduced pressure. 0.50 g of the resulting substance (hexaethyl((1,4,7-triazonane-1,4,7-triyl)tris(methylene))tris(phosphonate)) was weighed into a flask containing 2.5 mL of H2O. 1.25 g of sodium hydroxide pellet was added, and the mixture was stirred until all the sodium hydroxide was dissolved. Once dissolved, the mixture was refluxed for 15 minutes, then the flask was removed from the apparatus, 3 mL of H2O was added immediately after removal, and then the mixture was cooled to room temperature. After reaching room temperature, the flask was cooled further in ice for 15 minutes, during which time crystals began to form. The solvent was removed, and then the crystals were washed with water and a 1:1 mixture of ethanol and ethyl ether. ESI-MS: 494[MH] + ], 516[M+Na + Here, M is a neutral ligand (NOTPMe).

[0112] TRAP-H synthesis (see, for example, Figure 3) Triazacyclononane (500 mg, 3.87 mmol) and paraformaldehyde (527 mg, 17.5 mmol) were dissolved in 2.5 mL of 50 wt% aqueous hypophosphorous acid (23.2 mmol) in 18.75 mL of water, and the mixture was stirred at room temperature for 48 hours. The reaction mixture was evaporated under vacuum while being gently heated to below 40°C. The resulting oily substance was purified by water elution using a strong cation exchanger, DOWEX 50H+ foam. The fractions containing the pure ligand (confirmed by ESI mass spectrometry using LCQ) were combined, and the solvent was removed while being gently heated below 40°C to obtain a clear oily substance (492 mg, 35%). MS (ESI, positive): m / z 364[(TRAP-H)+H + ].

[0113] TRAP-Ph synthesis (see, for example, Figure 2) 1,4,7-Triazacyclononane (0.30 mmol) was added to a two-necked flask and placed in a reflux apparatus under an argon atmosphere. Dimethylphenylphosphonite (1.35 mmol, 4.5 equivalents) and paraformaldehyde (1.8 mmol, 6 equivalents) were then added to a flask containing 15 mL of dry tetrahydrofuran. The solution was refluxed for 16 hours. The excess solvent was then removed under reduced pressure. ESI-MS:634[M+H + ].

[0114] Next, the obtained TRAP-Ph-methoxy was transferred to a double flask with 4 mL of 50% w / v sodium carbonate-free hydroxide. The flask was placed in a reflux apparatus and refluxed for 30 minutes. 4 mL of distilled water was added, and the solution was refluxed for another 30 minutes. After the second reflux, the reflux apparatus was removed, and the solvent was evaporated until the two-layer system was rearranged. The flask was then removed from the heat source and allowed to cool to room temperature. The supernatant oil was removed and dissolved in ethanol. The ethanol was evaporated to leave TRAP-Ph. The obtained product was purified by recrystallization in methanol, followed by acetone, and then dichloromethane. ESI-MS: 590[M+H + Here, M is the TRAP-pH in neutral form.

[0115] Fe(TRAP-Ph) (see Figure 2, for example) TRAP-Ph was complexed with iron by mixing it with 1 equivalent of 0.01 M iron(III) nitrate in methanol. The solution was stirred at room temperature for 4 days. Then, the methanol was evaporated to obtain the resulting product. ESI-MS: 645, 667[M+Na + ].

[0116] 1,4,7-Triazacyclononane (0.15 mmol) was measured in a two-necked flask and set up on a reflux apparatus. Phosphorous acid (0.9 mmol, 6 equivalents) was further added to the flask along with 0.35 mL of hydrochloric acid and 1.0 mL of distilled water. Immediately after refluxing this solution, paraformaldehyde (0.675 mmol, 4.5 equivalents) was added to the solution over 1 hour. After the addition of paraformaldehyde was complete, the solution was refluxed for another 1 hour. The solution was cooled to room temperature and then slowly added dropwise to ethanol in an ice bath. After all the solution had been added, the mixture was stirred for another 1 hour, after which the solid was collected and washed with ether and ethanol. This solid was recrystallized in a small amount of hot water to obtain pure NOTP as a white solid. Negative mode ESI-MS: 410.2[MH] + Here, M is a neutral NOTP.

[0117] NOTP was complexed with iron by combining it with 1 equivalent of 0.01 M iron(II) bromide aqueous solution. The solution was stirred at room temperature for 4 days, after which the water was evaporated. ESI-MS (negative mode): 465[MH] + Here, M is the neutral form of Fe(NOTP).

[0118] Synthesis of DRAP-H (see, for example, Figure 3) 1-Benzyl-1,4,7-Triazacyclononanedihydrobromide (1.0 g, 2.64 mmol) and paraformaldehyde (0.24 g, 7.92 mmol) were dissolved in a mixture of 50% aqueous H3PO2 (1.3 mL, 11.9 mmol) and water (5 mL). The reaction mixture was stirred at room temperature for 24 hours, then co-evaporated three times with water to obtain a strong cation exchanger (DOWEX50, H + - Chromatography was performed on a foam. The column was washed with deionized water, and then the NaCl gradient (5% to 10% by weight) was increased to collect the fraction containing benzyl TRAP-H (monitored by MS). The eluate was evaporated to obtain a pale brown oily substance. 1H NMR (300MHz, D2O): δ = 3.02-3.52 (m, ring-CH2, 12H and N-CH2-P, 4H), 4.35 (s, N-CH2-C6H5, 2H), 7.30-7.50 (m, -C6H5, 5H). MS (ESI, positive): m / z=376[M+H + Here, M is a neutral ligand.

[0119] Synthesis of DRAP-OH (Figure 3) DRAP-H was dissolved in 6M HCl (20 mL). Paraformaldehyde (0.24 g, 7.92 mmol) was added, and the solution was refluxed for 5 hours (monitored by MS), then evaporated to obtain crude benzyl DRAP-OH. MS (ESI, positive): m / z] = 436[M+H] + ]. M is a neutral ligand. Fe(NO3)3·9H2O and benzyl DRAP-OH were dissolved in 1:1 ratio in 10 ml of deionized water and stirred for 4 days (monitored using MS). MS (ESI, positive): m / z = 489 [M + Here, M is a neutral Fe(III) complex.

[0120] General method for "clicked" triazole pendants and their attachment to macrocyclic rings (see, for example, Figure 5). Two equivalents of sodium azide were added to a methanol solution of bromo / chloro reagent using a suitably sized round-bottom flask. The solution was heated to 60°C and stirred for 4 hours. The next reaction step was carried out without isolation or purification of the previous product. 1.1 equivalents of propargyl alcohol were added to the stirred solution obtained from the previous reaction. In a separate flask, 0.1 equivalents of copper sulfate hexahydrate and 0.2 equivalents of sodium ascorbate (in 2 mL of water) were combined. The mixture was stirred for 1 minute and then added to the previous reaction flask. The reaction mixture was stirred at 60°C for 8–12 hours, or until complete (monitored by TLC). The reaction mixture was evaporated until dry, water was added, and the product was extracted with RINKAN (3x). The organic layers were combined and dried over anhydrous sodium sulfate. RINKAN was removed under vacuum to obtain the crude product. Purification was performed by recrystallization in RINKAN / DCM solution at 0°C or on silica gel (25–50% RINKAN in hexane). ESI-MS (m / z): 262.3 (product 1), 186.2 (product 2). Three equivalents of PBr3 were added to the DCM solution of the previous product under Ar(g). After 12 hours, the reaction mixture was quenched with water, and the product was extracted with DCM. The organic layers were combined and dried over anhydrous sodium sulfate. DCM was removed under vacuum to obtain the crude product. Purification was performed by recrystallization in EtOAC / DCM solution at 0°C, or on silica gel (25-50% Â in hexane). ESI-MS (m / z): 323.1 / 325.1 (product 1), 247.1 / 249.1 (product 2).

[0121] The starting reagent was dissolved in acetonitrile, and 1.5 equivalents of DIPEA were added. The mixture was stirred for 8 hours or until complete (monitored by ESI-MS). The solvent was removed under vacuum, and the crude product was purified with basic alumina (1-10% MeOH in DCM). ESI-MS (m / z): 489.6 (product 1), 413.5 (product 2)

[0122] The ligand from the previous reaction was dissolved in ethanol, and 2 equivalents of KOH dissolved in water were added. Stirring was carried out for about 4 hours or until completion (monitored by ESI-MS). The solvent was removed under vacuum. An equal amount of 1M HCl and DCM were added. The product was extracted with DCM (3×), the layers were combined, and dried over anhydrous sodium sulfate.

[0123] The ligand was dissolved in ethanol, and 1 equivalent of FeCl2 was added while stirring at 60 °C. After completion of the metallation (monitored by ESI-MS), diethyl ether was added until the product precipitated. The product was filtered, washed with diethyl ether, and dried under vacuum.

[0124] The ICP-MS iron concentration was measured using a Thermo X-Series 2 ICP-MS. All samples were diluted with 2% nitric acid (to 1 μM) to a total of 10 mL aqueous solution and decomposed by heating (90 °C) for 24 hours. A linear calibration curve of iron metal in the range of 0.1 ppb - 250 ppb was prepared daily for quantification. Samples were decomposed in nitric acid over 4 days, and the iron concentration was measured.

[0125] Magnetic moment Samples for magnetic moment tests were prepared using a coaxial NMR insert containing a diamagnetic standard of 5% t-butanol in D2O using the Evans method. The outer 5 mm NMR tube contained 5 mM paramagnetic complexes with fixed concentrations of 4 mM, 8 mM, 40 mM, and 70 mM in the presence of 5% t-butanol. The effective magnetic moment (μ eff , BM) was calculated at 298 K (T) using the modified Evans method for small molecules.

[0126] Preparation of samples for phantom MR imaging: Samples for phantom imaging experiments contained 50 - 500 μM complex, 20 mM HEPES, and 100 mM NaCl. For samples containing human serum albumin (HSA), 35 mg of HSA was added to these solutions. The pH of the entire solution was adjusted to 7.0.

[0127] Phantom (in vitro) imaging at 4.7T MRI acquisition was performed using a General Electric 4.7T / 33 cm horizontal bore magnet (GE NMR instruments, Fremont, CA) incorporating AVANCE digital electronics (Bruker BioSpec platform with ParaVision v 3.0.2 acquisition software, Bruker Medical, Billerica, MA). Each complex was diluted to concentrations ranging from 0.05 mM to 400 mM in HEPES in 100 mM NaCl (pH 7.4) and imaged at 25°C. The T1 relaxation rate (r1) was obtained using saturation recovery and spin echo (SE) sequencing (using fixed echo time [TE] and repetition times [TR] ranging from 10 ms to 75–8000 ms). Using commercially available image processing software (Analyze 7.0, AnalyzeDirect, Overland, KS), signal intensity at each iteration time was sampled by taking the average intensity within the region of interest (ROI). R1 ​​and SMAX were then calculated by nonlinear fitting of the equations using Matlab's Curve Fitting Toolbox (Matlab 7.0, MathWorks Inc, Natick, MA). Next, the T1 relaxation property for each complex was determined by obtaining the gradient of molar concentration versus -r1 of the compound through linear regression fitting of the data. Similarly, the T2 relaxation rate (R2) was obtained using multiple echoes and a Carr-Purcell-Meiboom Gill (CPMG)SE sequence with a fixed TR of 2500 ms and TE times of 15-300 ms. R2 and SMAX were then calculated using the equations described above. As stated above, T2 relaxation property was determined by obtaining the gradient of concentration versus -r2 through linear regression fitting of the data.

[0128] In vivo imaging in mice The efficacy of Fe(III) complexes for in vivo contrast enhancement was tested in a mouse model (BABC / cJ, Jackson Laboratory) using 4.7 T Bruker preclinical MRI. Sealed phantoms were included for imaging sessions to standardize the signal. Scans were acquired before contrast agent administration to serve as baseline values ​​for enhancement. Two scan protocols were used: (1) T1 weighted, 3D, spoiled gradient echo scan covering the mouse thoracic cavity to tail to measure signal enhancement; and (2) inversion-recovery, steady-state free precession scan (IR-SSFP) to measure T1 velocity in the blood (inferior vena cava), kidneys, liver, gallbladder, and spinal muscles. The compounds were administered intravenously via the tail vein at doses of 50–200 μmol[Fe] / kg, and MR data were acquired continuously up to 1 hour post-injection to examine distribution and clearance kinetics. Specifically, mice were injected with 0.2 mL of 6 mM stock solution at 0.05 mmol / kg, 0.100 mmol / kg, or 0.200 mmol / kg. Further scans were taken at 3 and 6 hours post-injection to identify slower clearance rates by the biliary system. FDA-approved MRI contrast agent: gadopentetate dimeglumine (Gd-DTPA, Magnevist) [登録商標] For comparison, ) or Dotarem (Gd(DOTA)) was injected into a separate cohort of mice at 50 μmol [Gd] / kg. The data are shown in Figures 17 and 18. For the SPGR dataset, signal intensity was normalized to a phantom, and signal enhancement for each organ, as well as enhancement of the contrast-to-noise ratio compared to the back muscle, was measured. Fe(III) concentration was estimated by calculating the increase in T1 rate and dividing by the relaxation value of the compound measured in vitro.

[0129] IPP synthesis scheme [ka]

[0130] NOTP iron complex formation [ka]

[0131] Figure 11 shows relaxation data for examples of the macrocyclic complexes of the present disclosure. Fe-NOTP is 0.66±0.01 mM -1 s -1 The r1 value is shown, and the Fe-NOTP with HSA is 1.04±0.06 mM. -1 s -1 The value of r1 was shown.

[0132] NOTP was synthesized using a procedure similar to those described in the literature. The off-white precipitate was recovered by vacuum filtration and then recrystallized multiple times from a 50:50 hot water:ethanol mixture until a pure white solid was formed. MS (ESI negative mode) m / z: 410.2 (MH) + ). 1 ¹H NMR (D2O): 3.19 (6H, doublet, J 11.30), 3.42 (12H, singlet). 31 P NMR (D2O) 11.95. 13 ¹³C NMR (D₂O): 51.02 (6C, singlet), 53.03 (3C, doublet, J 141.61).

[0133] A purified NOTP ligand (0.33 mmol) was dissolved in water (20 mL) with sodium hydroxide (1.01 mmol, 3 equivalents). This solution was heated at 55°C for 5 minutes. After 5 minutes, an aqueous solution of FeBr2 (0.33 mmol, 1 equivalent) (10 mL) was added to the heated solution. The combined solution was heated for a further 1 hour. After cooling to room temperature, the solvent was evaporated under reduced pressure to obtain a yellow oily substance. The yellow oily substance was dissolved in water (2 mL), and ethanol (20 mL) was added to precipitate a yellow solid. The solid was collected and dried. MS (ESI negative mode) m / z: 463.2 (MH) + ). Here, M is a neutral Fe(NOTP) complex. μ eff 5.85±0.14 ICP-MS purity: 95% Logarithm of Fe-NOTP P Octanol water: -2.02±0.30

[0134] DAPO synthesis DAPO was synthesized as follows: [ka] 1,4,7-Triazonan-1,4-diylbis(propan-2-ol) was synthesized as previously published. 0.301 mmol of 1,4,7-Triazonan-1,4-diylbis(propan-2-ol) was dissolved in 500 μL of 3.3 M HCl solution. To this solution, 1.80 mmol (6 equivalents) of phosphorous acid was added, and the mixture was brought under reflux. Once reflux was achieved, 1.35 mmol (4.5 equivalents) of paraformaldehyde was added over 1 hour, and the solution was then refluxed for a further 24 hours.

[0135] TRAP-POP synthesis TRAP-POP was synthesized as follows: [ka] Di-tert-butyl-1,4,7-triazacyclonane-1,4-dicarboxylate was synthesized as described in the literature. 0.566 mmol was dissolved in 1.1 mL of 3.3 M HCl with phosphorous acid (1.98 mmol, 3.5 equivalents). This solution was set to reflux, and once reflux was achieved, paraformaldehyde (1.13 mmol, 3 equivalents) was added over 1 hour. After all of the paraformaldehyde had been added, the solution was refluxed for another hour. MS-ESI: 224.1[M+H + The product from step 1 (1.52 mmol) was dissolved in 30 mL of MeOH, and an inert atmosphere was created using argon. Dimethylphenylphosphonite (3.04 mmol, 2 equivalents) and paraformaldehyde (1.52 mmol, 1 equivalent) were added, and the solution was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure. MS-ESI (negative mode): 557.7 [MH] + Here, M is a neutral ligand.

[0136] Synthesis of NOTPMe NOTPMe was synthesized as follows. [Chemical formula] The synthesis was carried out as reported in the literature. ESI-MS: 494 [M+H + , 516 [M+Na + Here, M is the neutral ligand.

[0137] Synthesis of PhOTO TPhOTO was synthesized as follows. [Chemical formula] PhOTO-methoxy was synthesized by dissolving TACN-benzyl (0.74 mmol) in 30 mL of methanol. Dimethylphenylphosphonite (3.68 mmol, 5 equivalents) was added to the solution together with paraformaldehyde (2.94 mmol, 4 equivalents). The solution was refluxed for 16 hours under an inert atmosphere, and then the solvent was removed under reduced pressure. MS (ESI) + m / z: 556.2 (M+H + ) Here, M is the neutral ligand. The dried PhOTO-methoxy was dissolved in 4 mL of 30% w / v carbonate-free sodium hydroxide. The solution was refluxed for 30 minutes with vigorous stirring, and at that point 4 mL of carbonate-free water was added. After adding water, the solution was refluxed for an additional 30 minutes. At the end of the second reflux, the solution was heated to reduce the solvent volume until a two-phase system appeared. The flask was cooled to room temperature. The upper layer (oil) was collected, dissolved in ethanol, and then evaporated to dryness. MS (ESI) - m / z: 526.4 (M-H + ) Here, M is the neutral ligand.

[0138] Synthesis of TOPA TOPA was synthesized as follows. [Chemical formula] 1-Benzyl-1,4,7-triazacyclononane was synthesized according to a previously published procedure. 1-Benzyl-1,4,7-triazacyclononane (0.576 mmol) was measured in a two-necked flask and set up in a reflux apparatus. Phosphorous acid (6.91 mmol, 12 equivalents) was further added to the flask along with 0.200 mL of hydrobromic acid and 0.538 mL of distilled water. After refluxing the solution, paraformaldehyde (1.15 mmol, 2 equivalents) was added to the solution over 1 hour. After the addition of paraformaldehyde was complete, the solution was refluxed for 24 hours. The solution was then reduced to its minimum volume, and Dowex H + The ion exchange column was packed. First, the column was eluted with water to remove impurities, and then the product was eluted with a 5% ammonium hydroxide solution. The ligand-containing fraction was dried under reduced pressure and dissolved in water. The product was obtained using an Amberlite CG-50 type 1 ion exchange column and eluted with water to obtain a white solid. MS(ESI) - m / z: 406.18 (MH) + ) Here, M is a neutral ligand. δ H( D2O ) 3.05 (4H, doublet, J 10.96) 3.32 (12H, multiplet) 4.34 (2H, singlet) 7.38 (5H, multiplet) δ P (D2O) 14.93

[0139] Bis(phosphonate)biphenyl TACN (TOBP) Starting with biphenyl TACN derivatives, the same procedure as for the TOPA ligand was used. δ H( D2O ) (500MHz): Doublet (2H, 7.704-7.687), Doublet (2H, 7.644-7.628), Doublet (2H, 7.601-7.585), Triplet (2H, 7.452-7.421), Triplet (1H, center 7.354), Singlet (4.369, 2H). The broad resonance range between 3.365 and 2.973 roughly represents 21H. MS(ESI) m / z: 484.2(MH) +The Fe(III) complex was prepared by adding Fe(NO3)3 and refluxing overnight. MS(ESI) - m / z:535.17(M - Here, M is a neutral Fe(TOBP) complex. [ka]

[0140] Synthesis of TRAP-OPO TRAP-OPO was synthesized as follows: [ka] Dissolve 1,4,7-triazabicyclo[5.2.1]decane (0.475 mmol) in 10 mL of methanol. Create an inert atmosphere and add dimethylphenylphosphonite (0.095 mmol, 0.2 equivalents) together with paraformaldehyde (0.118 mmol, 0.25 equivalents). Stir the solution overnight at room temperature and remove the solvent under reduced pressure. MS-ESI: 310.2[M+H + Here, M is a neutral ligand. Dry methyl ((1,4,7-triazabicyclo[5.2.1]decane-4-yl)methyl)(phenyl)phosphinate (0.221 mmol) was dissolved in 400 μL of 3.3 M HCl, and phosphorous acid (1.11 mmol, 5 equivalents) was added. This solution was set to reflux, and once reflux was achieved, paraformaldehyde (0.442 mmol, 2 equivalents) was added over 1 hour. After the addition of paraformaldehyde was complete, the solution was refluxed for another 1 hour. MS-ESI (negative mode): 470.5 [MH] + Here, M is a neutral ligand.

[0141] Synthesis of TRAP-Ph TRAP-Ph was synthesized as follows: [ka] TRAP-Ph-methoxy was synthesized as previously reported. Dried TRAP-Ph-methoxy was dissolved in 4 mL of 50% w / v carbonate-free sodium hydroxide. This solution was refluxed for 30 minutes with vigorous stirring, at which point 4 mL of carbonate-free water was added. After adding the water, the solution was refluxed for another 30 minutes. At the end of the second reflux, the solution was heated to reduce the solvent volume until two phases appeared. The solution was cooled to room temperature, the upper layer (oil) was removed, dissolved in ethanol, and then evaporated to dryness. TRAP-Ph was then purified by precipitation. Excess salt was removed by precipitation using methanol. Methanol was removed under reduced pressure, and acetone was added to form another solid, which was recrystallized with dichloromethane. .MS(ESI) - m / z: 590.7 (MH) + ). 1 ¹H NMR (D2O, pD ~ 0.9): 3.02 (12H, multiplet), 3.09 (6H, doublet, J 7.12), 7.31 (9H, multiplet), 7.48 (6H, multiplet) 31 NMR (D2O, pD ~ 0.9) 29.27 The iron complex was synthesized according to a previously published procedure. MS(ESI) + m / z:645.1(M+H + ), 667.1(M+Na + Here, M is a neutral ligand. [ka] Synthesis of 1,3-bis((1,4,7-triazonan-1-yl)methyl)benzene 0.100 g of TACN (0.774 mmol) in 4 mL of toluene and 1 mL of chloroform solution was added to a 25 mL round-bottom flask equipped with a gas inlet and a stirring rod. 0.0920 g of N,N-dimethylformamide dimethylacetal (0.774 mmol) was added to the flask. The solution was stirred at room temperature for 24 hours. 1,4,7-Triazatricyclo[5.2.1.0 4,10ESI-MS (m / z) of decane (TACN orthoamide), calculated value: 140.1 [M+H + (100%). The solution was dried by placing the flask on a rotary evaporator. The dried TACN orthoamide and 15 mL of dried acetonitrile were added to a 50 mL three-necked round-bottom flask equipped with a magnetic stirring rod, reflux condenser, gas injection tube, and addition funnel. 0.100 g (0.384 mmol) of α,α'-dibromo-m-xylene in 10 mL of dried acetonitrile solution was added to the flask dropwise over 30 minutes using the addition funnel. The solution was heated under reflux for 2 hours and stirred overnight at room temperature. The white-beige precipitate was collected by suction filtration and washed with dried acetonitrile (5 mL) and diethyl ether (5 mL). 6 mL of methanol and 6 mL of 12 M HCl were added to the precipitate in the flask for deprotection. The solution was heated under reflux for 4 hours. After the solution cooled to room temperature, NaOH pellets were added to adjust the pH of the solution to 8. The solution was then filtered to remove the NaCl salt precipitate and extracted with chloroform (3 × 60 mL). ESI-MS(m / z), calculated value: 361.4[M+H + ](100%). Here, M is a neutral ligand.

[0142] Synthesis of m-diTOPA 1,3-Bis((1,4,7-Triazonan-1-yl)methyl)benzene (0.155 mmol) was dissolved in 500 μL of 3.3 M HCl, and phosphorous acid (1.86 mmol, 12 equivalents) was added. The solution was placed in a reflux apparatus, and once reflux conditions were reached, paraformaldehyde (0.930 mmol, 6 equivalents) was added over 1 hour. After all of the paraformaldehyde had been added, the solution was refluxed for another 1 hour. MS-ESI - :735.3 [MH + Here, M is a neutral ligand. [ka]

[0143] Fe(TASO) complexes were synthesized using iron(II) chloride tetrahydrate. The product was isolated as a yellow solid by washing with diethyl ether in ethanol solvent. The Fe content of NaFe(L1)Cl was calculated by ICP-MS: 11.29%, experimental value: 11.13% ± 0.17%. LCQ-MS: experimental value m / z 421.2(M+H + ,70%), m / z 443.2(M+Na + ,30%), m / z 841.0 (2M+H + , 100%), and m / z 863.0 (2M+Na + ,18%). Here, M is neutral Fe (TASO complex). The effective magnetic moment was 5.68.

[0144] Fe(TASO) was characterized by cyclic voltammetry as shown in Figure 12, and cyclic voltammograms of a 1.0 mM solution of Fe(TASO) were obtained in water at various pH levels using potassium chloride (100 mM) as a supporting electrolyte and HEPES buffer.

[0145] Fe(TASO) was characterized by ultraviolet-visible absorbance. The data are shown in Figures 14-16.

[0146] TBzC ester Bis(2-hydroxypropyl)-1,4,7-triazacyclononane (0.245 g, 1.0 mmol) was dissolved in acetonitrile (5.0 mL) and heated to 60°C. Next, anhydrous potassium carbonate (0.207 g, 1.5 mmol) was added to the solution, followed by ethyl 4-(bromomethyl)benzoate (0.304 g, 1.2 mmol). The reaction was stirred for 8 to 12 hours until complete (monitored by ESI-MS). The solvent was then removed under vacuum, and the crude product was purified on alumina with methanol / dichloromethane in a ratio of 0 / 100 to 5 / 95. The product was eluted as a pale yellow oily substance with methanol / dichloromethane in a ratio of approximately 1 / 99 (0.288 g, 71%). 1H NMR (500MHz, DCM-d2) δ7.98(d, J=10Hz, 2H), 7.49(d, J=10Hz, 2H), 4.35(dd, J=15Hz, 2H), 3.89(s, 2H), 3.72-3.68(m, 2H), 2. 88-2.76(m, 6H), 2.67-2.45(m, 10H), 2.25(dd, J=10Hz, 2H), 1.39(t, J=5Hz, 3H), 1.05(d, J=10Hz, 6H). 13 C NMR (75MHz, CHCl3-d) δ 167.0, 144.8, 129.5, 129.4, 128.9, 66.5, 63.9, 62.5, 60.8, 55.7, 55.4, 54.6, 19.8, 14.3. ESI-MS:m / z 408.3(M + , 100%), 430.3(M+Na + , 10%).

[0147] TBZC synthesis TBZC ester (0.408 g, 1.0 mmol) was dissolved in ethanol (10.0 mL) and heated to 90°C. Then, NaOH (0.120 g, 3.0 mmol) was added to the solution, and the reaction was stirred for 12-24 hours until completion was monitored by ESI-MS. The solution was washed with dichloromethane (3 × 25 mL), and the aqueous layer was collected. The solvent was removed under vacuum. 12 M HCl was added to the solution until the pH became 3, and the ligand was obtained by extracting product (7) with dichloromethane (3 × 25 mL). The organic layers were then combined and dried over anhydrous sodium sulfate, and the solvent was removed under vacuum. The product was isolated as a pale yellow oil (0.278 g, 73%). 1 H NMR (500MHz, D2O) δ7.77(d, J=5Hz, 2H), 7.40(d, J=5Hz, 2H), 3.82(q, J=15Hz, 2H), 3.00(d, J=10Hz, 2H), 2.87-2.58(m, 16H), 1.04(d, J=5Hz, 6H). 13 C NMR (75MHz, D2O) δ 171.0, 140.4, 135.8, 129.7, 129.0, 63.6, 63.0, 60.2, 51.0, 49.7, 48.8, 19.7. ESI-MS: m / z 380.3(M+ , 100%).

[0148] Fe(TBZC) This complex was synthesized using ferrous chloride tetrahydrate, and the product was isolated as a yellow solid (45.7 mg, 52%). The Fe content calculated by ICP-MS for [Fe(H2-L4)Cl]Cl was 9.56%, measured value: 9.52% ± 0.10%. FT-ICR-MS: calculated value m / z 514.198541, measured value m / z 514.198394(M + ,100%) Here, M is a neutral Fe(TZBC) complex.

[0149] beside 17 O NMR relaxation properties, ln(1 / T) as a function of temperature for Fe(L2) measured at pH 3.5. 2r The coefficients were measured for various examples of macrocyclic compounds in this disclosure. The data are shown in Figure 13.

[0150] Relaxation values ​​of Fe(TASO) with and without HSA, measured at 4.7T and 9.4T, pH 7.2, 37°C. Compared with Gd(DTPA). Data are shown in Tables 2 and 3. [Table 2] [Table 3]

[0151] Fe(TASO) was used as the T1 contrast agent. Figure 17 shows the T1-weighted MRI of healthy Balb / C mice at 4.7T with a Fe(TASO) dose of 0.2 mmol / kg. Figure 18 shows the T1-weighted MRI of healthy Balb / C mice at 4.7T with a Fe(TASO) dose of 0.05 mmol / kg. Figure 19 shows the T1-weighted MRI using Fe(TBZC) as the contrast agent.

[0152] Pharmacokinetic studies of Fe(TASO) were conducted. Pharmacokinetic data for Fe(TASO) in mice are shown in Figures 19 and 20. T1-weighted MR images were obtained in healthy Balb / C mice at 4.7T with a dose of 0.05 mmol / kg Fe(L3). Time-dependent changes in the T1 rate constants for Fe(TASO), Gd(DOTA), and Gd-DTPA were examined in the kidneys, livers, and blood of healthy Balb / C mice at 0.05 mmol / kg·4.7T. Signal intensity was normalized to the phantom, and the increase in signal in each organ was measured. The increase in contrast-to-noise ratio was compared with that of muscle.

[0153] While this disclosure has been described with reference to one or more specific embodiments and / or examples, it will be understood that other embodiments and / or examples of this disclosure are possible without departing from the scope of this disclosure.

Claims

1. i) A 1,4,7-triazacyclononane (TACN) moiety having the following structure: 【Chemistry 1】 Here, Z1 and Z ２ is an anionic pendant group independently selected from the following: 【Chemistry 2】 R １ is an anionic pendant group independently selected from the following: 【Transformation 3】 Here, R ２ is a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aromatic group; A and A' are each independently a linear or branched substituted or unsubstituted C 1 ~C 12 A alkyl group, or -H, and at least one of A or A' is an alkyl group substituted with an anionic group; and Q 1 is an aryl group substituted with an anionic group, an alkyl group substituted with an anionic group, or an aralkyl group substituted with an anionic group; and ii) The TACN moiety and the high-spin Fe(III) cation complexed to at least one anionic pendant group substituent of the TACN moiety. A macrocyclic complex containing this compound.

2. The macrocyclic complex according to claim 1, wherein the macrocyclic complex has at least one open coordination site for a water ligand or a hydroxide ligand.

3. The macrocyclic complex according to claim 1, wherein the macrocyclic complex has at least one water and / or at least one hydroxide complexed with a high-spin Fe(III) cation.

4. The macrocyclic complex according to claim 1, wherein the TACN portion has the following structure: 【Chemistry 4】 【Transformation 5】

5. i) A 1,4,7-triazacyclononane (TACN) moiety having the following structure: 【Transformation 6】 and ii) The TACN moiety and the high-spin Fe(III) cation complexed to at least one anionic pendant group substituent of the TACN moiety. A macrocyclic complex containing this compound.

Images