Patent Application
20240216900
Nuclear magnetic resonance spectroscopy (NMR) and magnetic resonance imaging (MRI) methods are powerful tools widely used in biomedical, chemical, and materials science applications. These methods rely on the population difference of nuclear spin energy levels (called polarization) created after applying a strong magnetic field. Spins aligned with or against the applied field produce a net polarization, which is detected. Unfortunately, the nuclear polarization at thermal equilibrium (i.e., normal conditions) is inherently poor and remains a limitation to sensitivity and scope of the capabilities of magnetic resonance in general (Gunther, NMR Spectrosc. Basic Princ. Concepts i Appl. Chem., 13-28 (2013)).
Hyperpolarization techniques have been developed to overcome this problem and allow orders of magnitude NMR/MRI signal enhancement. The most widely used hyperpolarization techniques employ polarization transfer from electrons (dynamic nuclear polarization, DNP) (Hausser et al., Adv. Magn. Opt. Reson., 3, 79-139 (1968); Abagam et al., Reports Prog. Phys., 41, 395-467 (1978); and Ardenkjaer-Larsen et al., Proc. Natd. Acad. Sci. U.S.A, 100, 10158-10163 (2003)), photons (spin exchange optical pumping) (Bhaskar et al., Phys. Rev. Lett., 49, 25 (1982); Ebert et al., Lancet, 347, 1297 (1996); Albert et al., Nature, 370, 199-201 (1994); and Schroder et al., Science, 314, 446-449 (2006)), or parahydrogen (parahydrogen-induced polarization, PHIP) (Bowers et al., Phys. Rev. Lett., 57, 2645 (1986); Bowers et al., J Am. Chem. Soc., 109, 5541-5542 (1987); Eisenschmid et al., J Am. Chem. Soc., 109, 8089 (1987); Haake et al., J Am. Chem. Soc., 118, 8688 (1996); Goldman et al., C. R. Phys., 6, 575 (2005); and Chekmenev et al., J Am. Chem. Soc., 130, 4212 (2008)). Hyperpolarized magnetic resonance (MR) is an emerging molecular imaging method to monitor metabolism, enzymatic conversions, or biochemical pathways, previously inaccessible using MR.
Current hyperpolarized imaging with dissolution DNP and superconducting MRI scanners is very powerful because of its unique ability to track chemical transformations in vivo. However, DNP based experiments are relatively burdensome, slow, and expensive.
The PHIP approach and its subcategory SABRE (signal amplification by reversible exchange) allow the transfer of the 100% pure singlet spin order of parahydrogen (para-H2) into a target molecule. The PHIP method is a traditional hydrogenative method and relies on a catalytic hydrogenation reaction where a precursor, in the form of a hydrogen acceptor, is reduced by the parahydrogen and polarized. In contrast, the reversible exchange using SABRE leaves the hyperpolarized agent chemically unchanged. It is also not limited to one para-H2 molecule per molecule and therefore multiple spin transfer steps can lead to impressive levels of hyperpolarization. This effect has also been shown to transfer polarization to nucleis such as 1H, 13C, 19F, 31P and 15N and/or 29Si nuclei in a wide range of biologically relevant molecules (Barskiy et al., ChemPhysChem, 18, 1493-1498 (2017); Theis et al., J. Am. Chem. Soc., 137, 1404-1407 (2015); Shchepin et al., ChemPhysChem, 18, 1961-1965 (2017); Zhivonitko et al., Chem. Commun., 51, 2506-2509 (2015); Iali et al., Angew. Chemie—Int. Ed., 58, 10271-10275 (2019); and Gemeinhardt et al., Angew. Chemie Int. Ed., 59, 418-423 (2019)).
High polarization percentage, short signal build-up times, low cost, and scalability make SABRE a promising modality for studying metabolism in vivo using magnetic resonance spectroscopy technique.
The presently available hyperpolarized contrast agents come associated with a spin transfer catalyst component which contains a heavy metal, e.g., a transition metal atom, necessary to enable polarization transfer from para-H2 to the substrate. Toxicity concerns are raised when the hyperpolarization contrast agents are administered in vivo due to the presence of potentially toxic heavy metal-based complexes (e.g., catalysts are typically Ir-based organometallic compounds) in solution along with hyperpolarized contrast agents.
Another obstacle to the successful implementation of the SABRE process is the lower solubility of parahydrogen (H2 solubility in water is about 1.6 mg/L) in water compared to that in alcohol-based solvents. The SABRE hyperpolarization is mostly active in organic solvents, preferably methanol, which is not compatible with in vivo administration.
The foregoing shows that there exists a need for improved SABRE catalysts that are easily separable from a hyperpolarized substrate such that the hyperpolarized substrate is free of a heavy metal. There further exists a need for a method for separating a hyperpolarized substrate from the SABRE catalyst and/or hyperpolarized SABRE catalyst complex containing a heavy metal. There also exists a need for a method of administering a hyperpolarized substrate in a solvent medium that is suitable for in vivo administration.
The invention provides such SABRE catalysts and methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention provides a perfluorinated SABRE catalyst comprising a d-block element and a perfluorinated ligand, wherein the perfluorinated ligand is of Formula (I):
[Lm-(NHC)—(Y—Z)q] Formula (I),
The present invention also provides a method of preparing the perfluorinated SABRE catalyst described herein, comprising reacting a perfluorinated compound with a base to form a carbene, and reacting the carbene with [(d-block element)(COD)Cl]2, wherein COD stands for 1,4-cyclooctadienyl.
The present invention further provides a method of preparing a hyperpolarized substrate, the method comprising:
[Lm-(NHC)—(Y—Z)q] Formula (I),
The present invention further provides a hyperpolarized substrate obtained from a method described therein, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.
The present invention further provides a method of obtaining a magnetic resonance image of a tissue in a subject having or suspected to have a cancer or an adverse vascular condition comprising administering to the subject a hyperpolarized substrate described herein, or a pharmaceutical composition comprising the same, and imaging the subject by magnetic resonance imaging.
The present invention further provides a perfluorinated compound of Formula (III):
The present invention provides a perfluorinated SABRE catalyst comprising a d-block element and a perfluorinated ligand, wherein the perfluorinated ligand is of Formula (I):
[Lm-(NHC)—(Y—Z)q] Formula (I),
The perfluorinated SABRE catalyst comprises a d-block element such as, for example, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, La, Hf, Ta, W, Re, Os, Ir, Pt, Au, and/or Hg. In some embodiments, the d-block element is a transition metal such as, for example, Co, Rh, Ir, Ru, Pd, Pt, or Mt. In certain embodiments, the perfluorinated SABRE catalyst comprises an element of group 9 of the periodic table, i.e., Co, Rh, Ir, or Mt. In preferred embodiments, the perfluorinated SABRE catalyst comprises Ir or Co. For example, the perfluorinated SABRE catalyst can be prepared from [Ir(COD)(IMes)(Cl)].
The perfluorinated SABRE catalyst comprises a perfluorinated ligand of Formula (I):
[Lm-(NHC)—(Y—Z)q Formula (I),
In some aspects, NHC comprises an azolyl moiety, i.e., a five membered heterocyclic group having a nitrogen atom and at least one other hetero atom selected from nitrogen, sulfur, and oxygen. Thus, in some embodiments, NHC is a 5-membered N-heterocyclic carbenyl group. For example, the 5-membered N-heterocyclic carbenyl group can be imidazole-based, imidazoline-based, or thiazole-based. In other words, the 5-membered N-heterocyclic carbenyl group can be the resulting carbene formed from treatment of a perfluorinated ligand having an imidazole, an imidazoline, or a thiazole core.
In some embodiments, NHC is a 4,5-disubstituted, a 1,3-disubstituted, or a 1,3,4,5-tetrasubstituted imidazole-based or imidazoline-based 5-membered N-heterocyclic carbenyl group. For example, NHC can be a 4,5-disubstituted imidazolidinyl, a 1,3-disubstituted imidazolidinyl, a 1,3,4,5-tetrasubstituted imidazolidinyl, a 4,5-disubstituted 2,3-dihydro-imidazolyl, a 1,3-disubstituted 2,3-dihydro-imidazolyl, or a 1,3,4,5-tetrasubstituted 2,3-dihydro-imidazolyl. Examples of the imidazolylidinyl moiety include N,N′-di-(2,4,6-trimethylphenyl)-imidazolylidinyl moiety, N,N′-di-(2,6-diisopropylphenyl)-imidazolidinyl moiety, N,N′-di-(2,6-dicyclohexyl)-imidazolidinyl moiety, N,N′-di-(2,6-t-butyl)-imidazolidinyl moiety, and N,N′-di-(1-adamantyl)-imidazolidinyl moiety.
In some embodiments, the perfluorinated ligand is of Formula (Ia) or (Ib):
In some embodiments, the perfluorinated ligand is of Formula (Ic) or (Id):
In some embodiments, the perfluorinated ligand is of Formula (Ie) or (If):
In any of Formulae (I) and (Ia)-(If), each L is independently selected from hydrogen, adamantyl, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group.
As used herein, “substituted or unsubstituted aromatic” refers to a substituted (e.g., C1-6 alkyl substituted) or unsubstituted aromatic ring having 5 to 60 ring carbon atoms, e.g., phenyl, naphthyl, phenanthryl, and anthracenyl. As used herein, “substituted or unsubstituted heteroaromatic” refers to a substituted (e.g., C1-6 alkyl substituted) or unsubstituted aromatic ring having from 1 to 2 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon, or a stable bicyclic or tricyclic system containing at least one 5- to 7-membered aromatic ring which contains from 1 to 3, or in some aspects, from 1 to 2, heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms, in some aspects, bicyclic heteroaryl groups are 9- to 10-membered heteroaryl groups, that is, groups containing 9 or 10 ring atoms in which one 5- to 7-member aromatic ring is fused to a second aromatic or non-aromatic ring. When the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heteroaryl group is not more than 2. It is particularly preferred that the total number of S and O atoms in the aromatic heterocycle is not more than 1. Heteroaromatic groups include, but are not limited to, oxazolyl, piperazinyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl, pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzoisoxazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl, dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, isothiazolyl, and isoxazolyl.
In some embodiments, each L independently is hydrogen, adamantyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,4-dimethylphenyl, 2,5-dimethylphenyl, 2,6-dimethylphenyl, 3,5-dimethylphenyl, 2,4,6-trimethylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2,4-diethylphenyl, 2,5-diethylphenyl, 2,6-diethylphenyl, 3,5-diethylphenyl, 2,4,6-triethylphenyl, 2-npropylphenyl, 3-npropylphenyl, 4-npropylphenyl, 2,4-di-npropylphenyl, 2,5-di-npropylphenyl, 2,6-di-npropylphenyl, 3,5-di-npropylphenyl, 2,4,6-tri-npropylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2,4-di-isopropylphenyl, 2,5-di-isopropylphenyl, 2,6-di-isopropylphenyl, 3,5-di-isopropylphenyl, 2,4,6-tri-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2,4-di-isobutylphenyl, 2,5-di-isobutylphenyl, 2,6-di-isobutylphenyl, 3,5-di-isobutylphenyl, 2,4,6-tri-isobutylphenyl, 2-secbutylphenyl, 3-secbutylphenyl, 4-secbutylphenyl, 2,4-di-secbutylphenyl, 2,5-di-secbutylphenyl, 2,6-di-secbutylphenyl, 3,5-di-secbutylphenyl, 2,4,6-tri-secbutylphenyl, 2-tbutylphenyl, 3-tbutylphenyl, 4-tbutylphenyl, 2,4-di-tbutylphenyl, 2,5-di-tbutylphenyl, 2,6-di-tbutylphenyl, 3,5-di-tbutylphenyl, 2,4,6-tri-tbutylphenyl, 2-cyclohexylphenyl, 3-cyclohexylphenyl, 4-cyclohexylphenyl, 2,4-di-cyclohexylphenyl, 2,5-di-cyclohexylphenyl, 2,6-di-cyclohexylphenyl, 3,5-di-cyclohexylphenyl, or 2,4,6-tri-cyclohexylphenyl. In certain embodiments of Formulae (I) and (Ia)-(If), each L independently is hydrogen or 2,4,6-trimethylphenyl.
In any of Formulae (I) and (Ia)-(If), each Y independently is a bond or a spacer group. For example, Y can be a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted C2-10 alkynyl group, a substituted or unsubstituted C1-10 heteroalkyl group, a substituted or unsubstituted C3-6 cycloalkyl group, a substituted or unsubstituted C3-6 heterocycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted arylalkyl group, or a linear or branched alkyleneoxy group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).
In some embodiments, each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted C2-10 alkynyl group, a substituted or unsubstituted C1-10 heteroalkyl group, a substituted or unsubstituted C3-6 cycloalkyl group, a substituted or unsubstituted C3-6 heterocycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted arylalkyl group, or a linear or branched alkyleneoxy group. In certain embodiments, each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, or a substituted or unsubstituted arylalkyl group. In preferred embodiments, each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, or a substituted or unsubstituted arylalkyl group.
The perfluorinated ligand comprises a perfluorinated tag. For example, in any of Formulae (I) and (Ia)-(If), each Z is a perfluorinated tag. The perfluorinated tag can be any perfluorinated group such as, for example, a perfluorinated alkyl (e.g., linear or branched), aryl, alkyarl, or arylalkyl group containing up to 60 carbon atoms. In some embodiments, the perfluorinated tag is a perfluorinated C3-60 group comprising only carbon and fluorine atoms. In certain embodiments, the perfluorinated tag is a perfluorinated C3-40 group comprising only carbon and fluorine atoms. In other embodiments, the perfluorinated tag is a perfluorinated C3-20 group. For example, the perfluorinated tag can be selected from a C4F9 group, a C5F11 group, a C6F13 group, a C7F15 group, a C8F17 group, a C9F19 group, a C10F21 group, a C6F5 group, C4F7 group, a C5F9 group, a C6F11 group, a C7F13 group, a C8F15 group, a C9F17 group, and a C10F19 group, each of which can be a linear or branch alkyl, aryl, alkyarl, or arylalkyl group.
In an aspect, Z is a perfluoroalkyl chain, linear or branched, having a chain length of up to 60 or more carbon atoms, for example, the perfluoroalkyl chain has a chain length of 3-60, particularly, 3 to 40, more particularly 3 to 20, and even more particularly 3 to 10 or more, carbon atoms. For example, the perfluoroalkyl chain is selected from the group consisting of C4F9, C6F13, C7F15, C8F17, C9F19, and C10F21, preferably selected from the group consisting of C6F13, C8F17, and C10F21, each of which can be linear or branched and combinations thereof, wherein each of which can be linear or branched.
In an aspect, the perfluorinated ligand is of formula (Ig):
In an aspect, the perfluorinated ligand is of formula (Ih):
Exemplary perfluorinated ligands include:
Text use or a salt thereof, and wherein is a single bond or a double bond, and
represents the bond to the d-block element via the carbene.
As used herein, the symbol “” represents a single bond or a double bond. In some embodiments,
is a single bond. In other embodiments,
is a double bond. In embodiments where
is a single bond, the orientation of the two substituents stemming from
can have any suitable stereochemistry, i.e., can be cis or trans. In preferred embodiments, when
is a single bond, the stereochemistry of the substituents stemming from
is trans.
As used herein, the symbol “”represents the bond to the d-block element via the carbene. In other words,
represents the bond to the metal of the catalyst.
In some embodiments, the perfluorinated SABRE catalyst further comprises an additional ligand. For example, the perfluorinated SABRE catalyst may further comprise an additional ligand selected from phosphine ligands, carbene ligands, imidazole ligands, pincer chelating ligands, and compounds comprising a sulfoxide group. In certain embodiments, the perfluorinated SABRE catalyst comprises one or more phosphine ligands. Examples of phosphine ligands include, but are not limited to the following:
In some embodiments, the perfluorinated SABRE catalyst comprises a pincer chelating ligand. Generally, when the perfluorinated SABRE catalyst comprises a phosphine ligand or a pincer chelating ligand, the perfluorinated SABRE catalyst is in pre-catalyst form. In some embodiments, the perfluorinated SABRE catalyst comprises a ligand that is a compound comprising a sulfoxide group. Examples of compounds comprising a sulfoxide group can be selected from the group consisting of dimethylsulfoxide (DMSO), phenyl trifluoromethyl sulfoxide, phenyl methyl sulfoxide, phenyl chloromethyl sulfoxide, diphenyl sulfoxide, dibenzoyl sulfoxide, 2,2,2-trifluoroethyl methane sulfonate, di-2,2,2-trifluoroethyl sulfoxide, and dibutyl sulfoxide. Generally, when the perfluorinated SABRE catalyst comprises a compound comprising a sulfoxide group, the perfluorinated SABRE catalyst is in active form. As used herein, “the perfluorinated SABRE catalyst” can refer to the active perfluorinated SABRE catalyst or the perfluorinated SABRE precatalyst.
The active perfluorinated SABRE catalyst can be prepared by any suitable method. Generally, the active perfluorinated SABRE catalyst is prepared by combining the perfluorinated SABRE precatalyst with a substrate, parahydrogen, and optionally a co-ligand in a solvent to form a mixture comprising an active perfluorinated SABRE catalyst. In some embodiments, the active perfluorinated SABRE catalyst is prepared by combining the perfluorinated SABRE precatalyst with a substrate, parahydrogen, and a co-ligand in a solvent to form a mixture comprising an active perfluorinated SABRE catalyst. The co-ligand, when included in the preparation of the active perfluorinated SABRE catalyst, can be combined with the perfluorinated SABRE precatalyst in any order and by any suitable means. For example, the co-ligand, when included in the preparation of the active SABRE catalyst, can be provided first to interact with the transfer precatalyst to facilitate formation of the active perfluorinated SABRE catalyst. Alternatively, the co-ligand, when included in the preparation of the active perfluorinated SABRE catalyst, can be added together with the substrate to facilitate formation of the active perfluorinated SABRE catalyst. In some embodiments, the co-ligand, the substrate, and parahydrogen are essentially combined with the perfluorinated SABRE precatalyst in the solvent at the same time to facilitate formation of the active perfluorinated SABRE catalyst. In other embodiments, the substrate is provided first to interact with the perfluorinated SABRE precatalyst to facilitate formation of the active SABRE catalyst. In some embodiments, the co-ligand and the substrate are combined with the perfluorinated SABRE precatalyst in the solvent, and the parahydrogen is added to (e.g., bubbled through) the resulting mixture. In other embodiments, the substrate is combined with the perfluorinated SABRE precatalyst in the solvent, and the parahydrogen is added to (e.g., bubbled through) the resulting mixture. In some embodiments, the active perfluorinated SABRE catalyst is prepared by combining the perfluorinated SABRE precatalyst with a co-ligand in addition to the substrate and parahydrogen.
In some embodiments, the active perfluorinated SABRE catalyst is of formula
[Ir(H)2(F-IMes)(η2-SUBSTRATE)(Co-ligand)] or
[Ir(H)2(F-IMes)(η1-SUBSTRATE)(Co-ligand)2],
wherein SUBSTRATE is a target substrate to be hyperpolarized by the transfer of the pure singlet spin order of parahydrogen by the spin transfer catalyst, preferably a target substrate having enriched with an atom having 12 spin nuclei, for example, 1H, 13C, 15N, 19F, 31P and/or 29Si. Without wishing to be bound by any particular theory, the co-ligand interacts with the spin transfer pre-catalyst to form the activated polarization transfer catalyst and enhances the polarization transfer to the target substrate. F-IMes refers to a perfluorinated form of N-heterocyclic carbenyl (NHC) ligand such as a 1,3-Bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene group or a 1,3-Bis(2,4,6-trimethylphenyl)-1,3-dihydro-4H-imidazol-2-ylidene group described herein.
In some embodiments, the perfluorinated SABRE catalyst (e.g., the perfluorinated SABRE precatalyst) can be prepared by a method comprising reacting a perfluorinated compound with a base to form a carbene, and reacting the carbene with [(d-block element)(COD)Cl]2, wherein COD stands for 1,4-cyclooctadienyl. In certain embodiments, the method comprises reacting a perfluorinated compound with a base to form a carbene, and reacting the carbene with [Ir(COD)C]2. For example, a perfluorinated SABRE catalyst of formula:
can be prepared by reacting a carbene of formula:
with [Ir(COD)Cl]2.
Exemplary perfluorinated SABRE catalysts (e.g., the perfluorinated SABRE precatalyst) include:
or salts thereof. Any structure (e.g., catalyst) depicted herein can exist as the depicted enantiomer, the enantiomer of the depicted compound, or a racemic mixture thereof. Generally, the stereochemistry is depicted herein to show cis-trans relationships only. As a result, any structure (e.g., catalyst) provided herein can be a single enantiomer, a mixture of enantiomers, or a racemic mixture. In some embodiments, the catalysts described herein are racemic mixtures.
The present invention further provides a method of preparing a hyperpolarized substrate, the method comprising:
The methods of preparing a hyperpolarized substrate, described herein, comprise hyperpolarizing the mixture comprising the perfluorinated SABRE catalyst (e.g., the active perfluorinated SABRE catalyst) by exposing the mixture to a magnetic field or radiofrequency excitation to transfer the polarization from parahydrogen to the substrate to form the hyperpolarized substrate. Initially, the hyperpolarized substrate is complexed with the hyperpolarized perfluorinated SABRE catalyst; however, it will be understood by a person of ordinary skill in the art that the hyperpolarized substrate can be replaced by another substrate molecule such that the process can be repeated and the free hyperpolarized substrate bolus is produced.
The transfer of polarization from parahydrogen to the substrate to form the hyperpolarized substrate can occur under any suitable magnetic field or radiofrequency excitation. For example, the transfer of polarization from parahydrogen to the substrate can occur at a magnetic field below the magnetic field of earth. The suitable level of magnetic field or radiofrequency excitation necessary to transfer the polarization from parahydrogen to the substrate to form the hyperpolarized substrate will be readily apparent to a person of ordinary skill in the art.
In some embodiments, the method comprises replenishing the parahydrogen in the mixture during the step of hyperpolarizing the mixture comprising the perfluorinated SABRE catalyst (e.g., the active perfluorinated SABRE catalyst) by exposing the mixture to a magnetic field or radiofrequency excitation to transfer the polarization from parahydrogen to the substrate to form the hyperpolarized substrate. In other words, in some embodiments, the method comprises bubbling parahydrogen through the mixture comprising the perfluorinated SABRE catalyst (e.g., the active perfluorinated SABRE catalyst) during the step of hyperpolarizing the mixture comprising the perfluorinated SABRE catalyst (e.g., the active perfluorinated SABRE catalyst) by exposing the mixture to a magnetic field or radiofrequency excitation to transfer the polarization from parahydrogen to the substrate to form the hyperpolarized substrate.
The method of preparing a hyperpolarized substrate comprises providing a co-ligand to interact with the perfluorinated SABRE catalyst to facilitate formation of an active perfluorinated SABRE catalyst. The co-ligand can be any suitable compound containing one or more sulfoxide groups, thioester groups, phosphine groups, amine groups, CO groups, isonitrile groups, nitrogen-containing heterocyclic groups, or a combination thereof. In some embodiments, the co-ligand is a compound comprising a sulfoxide group. Examples of compounds comprising a sulfoxide group can be selected from the group consisting of DMSO, phenyl methyl sulfoxide, phenyl chloromethyl sulfoxide, diphenyl sulfoxide, dibenzoyl sulfoxide, phenyl trifluoromethyl sulfoxide, 2,2,2-trifluoroethyl methane sulfoxide, di-2,2,2-trifluoroethyl sulfoxide, and dibutyl sulfoxide. In certain embodiments, the co-ligand is dimethyl sulfoxide. In some embodiments, the co-ligand is fluorinated. For example, the co-ligand can be phenyl trifluoromethyl sulfoxide, 2,2,2-trifluoroethyl methane sulfoxide, or di-2,2,2-trifluoroethyl sulfoxide. Without wishing to be bound by any particular theory, it is believed that a fluorinated co-ligand may help facilitate separation of the hyperpolarized substrate and the perfluorinated SABRE catalyst when performing an aqueous phase extraction and/or a fluorinated phase extraction.
In embodiments of the method of preparing a hyperpolarized substrate, the magnetic field is an electro-magnetic field. For example, the strength of the electro-magnetic field can be in the range of 0-200 milliTeslas (mT). In some embodiments, the electro-magnetic field may be at least partially supplied by one or more permanent magnets in addition to or in lieu of the coil. In some embodiments, the electro-magnetic field may be an alternating magnetic field supplied at a frequency adapted to a particular nuclei. The alternating magnetic field can change directions (i.e., alternate between positive and negative relative to a positive direction). In some embodiments, the frequency can be a radio frequency, and preferably between 50 to 500 MHz, although other frequencies outside this range are contemplated as within the scope of the present disclosure.
The perfluorinated SABRE catalyst (e.g., active perfluorinated SABRE catalyst) is combined with parahydrogen and a substrate comprising a ½ spin nucleus or nuclei in a solvent to obtain a reaction mixture. The solvent can be any suitable solvent capable of forming a heterogeneous or homogeneous mixture. In some embodiments, the solvent comprises water, methanol, ethanol, a fluorous solvent, or a mixture thereof. For example, the solvent can be ethanolic or methanolic, i.e., comprising at least ethanol or methanol in combination with water. In certain embodiments, the solvent comprises a fluorous solvent. In some embodiments, the solvent is deuterated such that a deuterated solvent can be prepared without (i.e., with limited) deuterium-hydrogen exchange.
The fluorous solvent can be any organic solvent comprising at least one compound having a fluorine atom. Without wishing to be bound by any particular theory, it is believed that the fluorous solvent increases the solubility of the perfluorinated SABRE catalyst. In some embodiments, the solvent comprises a perfluoroalkane, diethyl ether, a nonafluorobutyl methyl ether, ethyl acetate, perfluorobutyl methyl ether, a fluorocarbon derivative of THF FC 75, a decafluoromethoxy trifluoromethyl pentane, a hexafluoro propanol, a perfluoromethyl cyclohexane, and a combination thereof. In some embodiments, the solvent (e.g., the fluorous solvent) is selected from a perfluorohexane/diethyl ether mixture, a methoxy nonafluorobutane and ethyl acetate mixture with a non-polar solvent, a perfluorohexane and ether mixture, a perfluorobutyl methyl ether and ethyl acetate mixture, an ether, a fluorocarbon derivative of THF FC 75, a decafluoromethoxy trifluoromethyl pentane, a hexafluoro propanol, a nonafluorobutyl methyl ether, a perfluoromethyl cyclohexane, a perfluoroalkane, a perfluorohexane, and a methoxy nonafluorobutane. In certain embodiments, the solvent comprises water, methanol, ethanol, a nonafluorobutyl methyl ether, deuterated variants thereof, or a mixture thereof.
In some embodiments, the method of preparing a hyperpolarized substrate further comprises isolating the hyperpolarized substrate. The hyperpolarized substrate can be isolated by any suitable method. For example, the hyperpolarized substrate can be isolated by extraction, filtration, column chromatography, distillation, crystallization, or a combination thereof.
In some embodiments, the hyperpolarized substrate is isolated by treating the reaction mixture with a solid phase adsorbent to adsorb the perfluorinated SABRE catalyst, and recovering a liquid containing the hyperpolarized substrate, wherein the liquid is free (i.e., undetectable) or substantially free (e.g., less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm) of the perfluorinated SABRE catalyst. See, for example,
In some embodiments, the hyperpolarized substrate is isolated by treating the reaction mixture with a solid phase adsorbent to adsorb the hyperpolarized substrate, and recovering a liquid containing the perfluorinated SABRE catalyst, wherein the liquid is free (i.e., undetectable) or substantially free (e.g., less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm) of the hyperpolarized substrate. The solid phase adsorbent can be any suitable adsorbent capable of preferentially adsorbing the hyperpolarized substrate over the perfluorinated SABRE catalyst. For example the solid phase adsorbent can be a normal phase adsorbent such as, for example silica, alumina, or the like. See, for example,
In any of the embodiments disclosed herein, the perfluorinated SABRE catalyst and/or the hyperpolarized substrate can exist in a monophasic or a biphasic mixture. The monophasic or biphasic mixture can comprise any combination of solvents described herein. For example, the biphasic mixture can comprise a polar solvent (e.g., water, methanol, and ethanol) in combination with a non-polar solvent (e.g., an organic solvent or a fluorous solvent). In embodiments, where the perfluorinated SABRE catalyst and/or the hyperpolarized substrate exists in a biphasic solvent, the perfluorinated SABRE catalyst and/or the hyperpolarized substrate can be isolated by a liquid/liquid extraction. See, for example,
In some embodiments, the hyperpolarized substrate is isolated by precipitating the perfluorinated SABRE catalyst and filtering and removing the precipitated perfluorinated SABRE catalyst from the hyperpolarized substrate. Typically, the perfluorinated SABRE catalyst is precipitated by addition of solvents in which the perfluorinated SABRE catalyst is not soluble (e.g., hexane, pentane, water, ethanol, or the like). In certain embodiments, the perfluorinated SABRE catalyst is precipitated by the addition of water.
The perfluorinated SABRE catalyst and/or the hyperpolarized substrate can be dried or concentrated (e.g., under reduced pressure, using a desiccant, heating, or a combination thereof). Alternatively, or additionally, the perfluorinated SABRE catalyst and/or the hyperpolarized substrate can be diluted or reconstituted with a solvent (e.g., water) to provide a desired concentration. For example, the perfluorinated SABRE catalyst can be isolated and re-used as a hyperpolarization catalyst. Similarly, the hyperpolarized substrates can be dried or concentrated to remove organic solvents and reconstituted in water for administration to a subject.
The substrate can be any compound comprising a 12 spin nucleus or nuclei. For example, the substrate can comprise 1H, 13C, 15N, 19F, 31p 29Si, or a combination thereof. In some embodiments, the substrate further comprises 2D. Thus, the methods described herein can be used to enhance the signal of 1H, 13C, 15N, 19F, 31P and/or 29Si response of a target substrate. Generally, the spin polarization transfer described herein is based on the SABRE effect; however the methods can be extended to parahydrogen—induced polarization (PHIP).
In some embodiments, the method further comprises recycling and reusing the perfluorinated SABRE catalyst more than one time (e.g., more than two times, more than three times, more than 4 times, more than 5 times, or more than 6 times). Thus, in some embodiments, the present invention provides a method of preparing a hyperpolarized substrate, the method comprising:
[Lm-(NHC)—(Y—Z)q] Formula (I),
In some embodiments, the substrate is selected from ketoglutarate, ketoisocaproate, pyruvate, N-acetyl cysteine, and salts or esters thereof. In certain embodiments, the substrate is selected from 1-13C-ketoglutarate, 1-13C-5-12C-ketoglutarate, 1-13C-pyruvate, 1-13C-N-acetyl cysteine, 15N2-isoniazid (or pyridyl-4-carbo-bis-15N2-hydrazide), 13C2,15N3-metronidazole, 15N2-1-aminoisoquinoline (1-AIQ), deuterated versions thereof, and salts thereof.
In some embodiments, the substrate is of Formula (II):
Each R1 may be independently selected from hydrogen, deuterium, a cation, C1-C6 alkyl, C3-C7 cycloalkyl, (C3-C7 cycloalkyl)C1-C6 alkyl, (heterocycloalkyl)C1-C6 alkyl, (heteroaryl)C1-C6 alkyl, and (aryl)C1-C6 alkyl. In some embodiments, each R1 is independently selected from a C1-C6 alkyl, for example, each R1 can be methyl, ethyl, propyl (e.g., isopropyl or n-propyl), butyl (e.g., isobutyl, n-butyl, tert-butyl, or sec-butyl), pentyl, or hexyl. In some embodiments, each R1 is independently selected from hydrogen, deuterium, and a cation. In embodiments where R1 is a cation, it will be readily understood by a person of ordinary skill in the art that the compound of Formula (II) is a salt (e.g., a pharmaceutically acceptable salt) where the negative charge on oxygen is balanced by the cation. In certain embodiments, each R1 independently is a cation or C1-C6 alkyl.
The present invention further provides a hyperpolarized substrate, or a pharmaceutically acceptable salt, obtained from any of the methods described herein, or a pharmaceutical composition comprising a hyperpolarized substrate, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier. In other words, the present invention provides imaging medium (e.g., an aqueous imaging composition) with enhanced sensitivity on a water-soluble compound comprising a hyperpolarizable nucleus or hyperpolarizable nuclei, which imaging medium is particularly well suited for nuclear magnetic resonance (NMR) spectroscopy and/or magnetic resonance imaging (MRI).
The present invention further provides a method of obtaining a magnetic resonance image of a tissue in a subject having or suspected to have a cancer or an adverse vascular condition comprising administering to the subject a hyperpolarized substrate described herein, or a pharmaceutical composition thereof, and imaging the subject by magnetic resonance imaging. In an aspect, the subject has a cancer such as, for example, a cancer is selected from breast cancer, colon cancer, rectal cancer, bladder cancer, endometrial cancer, kidney cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer. In another aspect, the subject has an adverse vascular condition such as, for example, a vascular condition selected from myocardial infarction, stroke, and pulmonary disease (e.g., COPD, lung fibrosis, long-term COVID-19 symptom, and combinations thereof).
In some embodiments, the invention provides a method of diagnosing or monitoring a patient having or suspected to have a cancer, the method comprising administering a hyperpolarized substrate or a pharmaceutical composition as described above and diagnosing or monitoring the patient by hyperpolarized 13C-MRI. For example, a hyperpolarized substrate can be used in the method of diagnosing or monitoring a patient having or suspected to have a cancer. In certain embodiments, the method or use comprises identifying a mutation or mutations responsible for the cancer. In certain embodiments, the method or use identifies an IDH1 mutation as being responsible for the cancer. In other words, the method or use can be used to identify whether the patient has a tumor, for example, an IDH1 mutation.
The present invention further provides a perfluorinated compound of Formula (III):
All aspects of the perfluorinated compound of Formula (III) are as described with respect to any of Formulae (I) and (Ia)-(If). For example, Arf is independently selected from a perfluorinated substituted or unsubstituted aromatic group or a perfluorinated substituted or unsubstituted heteroaromatic group, wherein substituted or unsubstituted aromatic group and substituted or unsubstituted heteroaromatic group are as described with respect to any of Formulae (I) and (Ia)-(If). In some embodiments of the perfluorinated compound of Formula (III), (i) each Ar is independently selected from a substituted or unsubstituted aromatic group, (ii) each Arf is independently selected from a perfluorinated substituted or unsubstituted aromatic group, and/or (iii) each Y is independently selected from a spacer group selected from C1-5 alkyl and C1-5 heteroalkyl.
In certain embodiments, the perfluorinated compound is of Formula (IIIa):
In Formulae (III) and (IIIa), X is an anion. X can be any suitable anion. For example, X can be a halide ion (e.g., fluoride chloride, bromide, or iodide) or a fluorate ion (e.g., tetrafluoroborate or hexafluorophosphate).
The perfluorinated compound of Formula (III) or (IIIa) can be prepared by any suitable means. For example, the perfluorinated compound can be prepared by a method comprising:
The perfluorinated compound of Formula (III) or (IIIa) can be used in any suitable application. For example, the perfluorinated compound of Formula (III) or (IIIa) can be used for catalysis (e.g., polymerization catalysis or SABRE catalysis). Thus, in some embodiments, the invention provides a catalyst (e.g., an olefin metathesis catalyst or a SABRE catalyst) comprising a d-block element and a perfluorinated compound of Formula (III) or (IIIa) as a ligand, and a method of using the same. For example, the olefin metathesis catalyst, comprising a perfluorinated compound of Formula (III) or (IIIa), can be used in a method of polymerizing an olefin, the method comprising combining the olefin metathesis catalyst and an olefin in a reaction mixture. Similarly, the SABRE catalyst, comprising a perfluorinated compound of Formula (III) or (IIIa), can be used in a method of hyperpolarizing a substrate, as described herein.
The invention further provides a kit comprising a perfluorinated SABRE catalyst (e.g., the precatalyst), a co-ligand, and a solvent as defined herein. In some embodiments of the kit, the perfluorinated SABRE catalyst (e.g., the precatalyst) is separated from the co-ligand and/or the solvent such that the perfluorinated SABRE catalyst (e.g., the precatalyst) can be added to the hyperpolarizing system separately from the co-ligand and/or the solvent. In certain embodiments, the kit further comprises a substrate. In embodiments where the kit further comprises a substrate, the substrate can be (i) combined with the co-ligand and/or the solvent, but separate from the perfluorinated SABRE catalyst (e.g., the precatalyst), (ii) combined with the perfluorinated SABRE catalyst (e.g., the precatalyst), a co-ligand, and a solvent, or (iii) separated from the perfluorinated SABRE catalyst (e.g., the precatalyst), a co-ligand, and a solvent.
Aspects, including embodiments, of the invention described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-81 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
(1) In aspect (1) is provided a method of preparing a hyperpolarized substrate, the method comprising:
[Lm-(NHC)—(Y—Z)q] Formula (I),
(2) In aspect (2) is provided the method of aspect 1, wherein the substrate comprises 1H, 13C, 15N, 19F, 31P 29Si, or a combination thereof.
(3) In aspect (3) is provided the method of aspect 2, wherein the substrate further comprises 2D.
(4) In aspect (4) is provided the method of any one of aspects 1-3, wherein the co-ligand is a compound containing one or more sulfoxide groups, thioester groups, phosphine groups, amine groups, CO groups, isonitrile groups, nitrogen-containing heterocyclic groups, or a combination thereof.
(5) In aspect (5) is provided the method of any one of aspects 1-4, wherein the solvent comprises water, methanol, ethanol, a fluorous solvent, or a mixture thereof.
(6) In aspect (6) is provided the method of any one of aspects 1-5, wherein the solvent comprises a perfluoroalkane, diethyl ether, a nonafluorobutyl methyl ether, ethyl acetate, perfluorobutyl methyl ether, a fluorocarbon derivative of THF FC 75, a decafluoromethoxy trifluoromethyl pentane, a hexafluoro propanol, a perfluoromethyl cyclohexane, and a combination thereof.
(7) In aspect (7) is provided the method of any one of aspects 1-4, wherein the solvent comprises water, methanol, ethanol, a nonafluorobutyl methyl ether, deuterated variants thereof, or a mixture thereof.
(8) In aspect (8) is provided the method of any one of aspects 1-7, wherein the solvent is deuterated.
(9) In aspect (9) is provided the method of any one of aspects 1-8, wherein the co-ligand is dimethyl sulfoxide.
(10) In aspect (10) is provided the p method of any one of aspects 1-8, wherein the co-ligand is fluorinated.
(11) In aspect (11) is provided the method of aspect 10, wherein the co-ligand is phenyl trifluoromethyl sulfoxide, 2,2,2-trifluoroethyl methane sulfonate, or di-2,2,2-trifluoroethyl sulfoxide.
(12) In aspect (12) is provided the method of any one of aspects 1-11, wherein the substrate is selected from ketoglutarate, ketoisocaproate, pyruvate, N-acetyl cysteine, and salts or esters thereof.
(13) In aspect (13) is provided the method of any one of aspects 1-12, wherein the substrate is selected from 1-13C-ketoglutarate, 1-13C-5-12C-ketoglutarate, 1-13C-pyruvate, 1-13C-N-acetyl cysteine, 15N2-isoniazid (or pyridyl-4-carbo-bis-15N2-hydrazide), 13C2,15N3-metronidazole, 15N2-1-aminoisoquinoline (1-AIQ), deuterated versions thereof, and salts thereof.
(14) In aspect (14) is provided the method of any one of aspects 1-12, wherein the substrate is of Formula (II):
(15) In aspect (15) is provided the method of any one of aspects 1-14, wherein NHC is a 5-membered N-heterocyclic carbenyl group.
(16) In aspect (16) is provided the method of aspect 15, wherein the 5-membered N-heterocyclic carbenyl group is imidazole-based, imidazoline-based, or thiazole-based.
(17) In aspect (17) is provided the method of any one of aspects 1-16, wherein NHC is a 4,5-disubstituted, a 1,3-disubstituted, or a 1,3,4,5-tetrasubstituted imidazole-based or imidazoline-based 5-membered N-heterocyclic carbenyl group.
(18) In aspect (18) is provided the method of any one of aspects 1-17, wherein NHC is a 4,5-disubstituted imidazolidinyl, a 1,3-disubstituted imidazolidinyl, a 1,3,4,5-tetrasubstituted imidazolidinyl, a 4,5-disubstituted 2,3-dihydro-imidazolyl, a 1,3-disubstituted 2,3-dihydro-imidazolyl, or a 1,3,4,5-tetrasubstituted 2,3-dihydro-imidazolyl.
(19) In aspect (19) is provided the method of any one of aspects 1-14, wherein the perfluorinated ligand is of Formula (Ia) or (Ib):
(20) In aspect (20) is provided the method of any one of aspects 1-14, wherein the perfluorinated ligand is of Formula (Ic) or (Id):
(21) In aspect (21) is provided the method of any one of aspects 1-14, wherein the perfluorinated ligand is of Formula (Ie) or (If):
(22) In aspect (22) is provided the method of aspect 20 or aspect 21, wherein a is 4 to 10.
(23) In aspect (23) is provided the method of any one of aspects 1-22, where each L independently is hydrogen, adamantyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,4-dimethylphenyl, 2,5-dimethylphenyl, 2,6-dimethylphenyl, 3,5-dimethylphenyl, 2,4,6-trimethylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2,4-diethylphenyl, 2,5-diethylphenyl, 2,6-diethylphenyl, 3,5-diethylphenyl, 2,4,6-triethylphenyl, 2-npropylphenyl, 3-npropylphenyl, 4-npropylphenyl, 2,4-di-npropylphenyl, 2,5-di-npropylphenyl, 2,6-di-npropylphenyl, 3,5-di-npropylphenyl, 2,4,6-tri-npropylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2,4-di-isopropylphenyl, 2,5-di-isopropylphenyl, 2,6-di-isopropylphenyl, 3,5-di-isopropylphenyl, 2,4,6-tri-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2,4-di-isobutylphenyl, 2,5-di-isobutylphenyl, 2,6-di-isobutylphenyl, 3,5-di-isobutylphenyl, 2,4,6-tri-isobutylphenyl, 2-secbutylphenyl, 3-secbutylphenyl, 4-secbutylphenyl, 2,4-di-secbutylphenyl, 2,5-di-secbutylphenyl, 2,6-di-secbutylphenyl, 3,5-di-secbutylphenyl, 2,4,6-tri-secbutylphenyl, 2-tbutylphenyl, 3-tbutylphenyl, 4-tbutylphenyl, 2,4-di-tbutylphenyl, 2,5-di-tbutylphenyl, 2,6-di-tbutylphenyl, 3,5-di-tbutylphenyl, 2,4,6-tri-tbutylphenyl, 2-cyclohexylphenyl, 3-cyclohexylphenyl, 4-cyclohexylphenyl, 2,4-di-cyclohexylphenyl, 2,5-di-cyclohexylphenyl, 2,6-di-cyclohexylphenyl, 3,5-di-cyclohexylphenyl, or 2,4,6-tri-cyclohexylphenyl.
(24) In aspect (24) is provided the method of any one of aspects 1-23, where each L independently is hydrogen or 2,4,6-trimethylphenyl.
(25) In aspect (25) is provided the method of any one of aspects 1-24, wherein each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted C2-10 alkynyl group, a substituted or unsubstituted C1-10 heteroalkyl group, a substituted or unsubstituted C3-6 cycloalkyl group, a substituted or unsubstituted C3-6 heterocycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted arylalkyl group, or a linear or branched alkyleneoxy group.
(26) In aspect (26) is provided the method of any one of aspects 1-24, wherein each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, or a substituted or unsubstituted arylalkyl group.
(27) In aspect (27) is provided the method of any one of aspects 1-24, wherein each Y independently is a bond or a substituted or unsubstituted C1-10 alkyl group.
(28) In aspect (28) is provided the method of any one of aspects 1-27, wherein the perfluorinated tag is a perfluorinated C3-60 group comprising only carbon and fluorine atoms.
(29) In aspect (29) is provided the method of any one of aspects 1-27, wherein the perfluorinated tag is a perfluorinated C3-40 group comprising only carbon and fluorine atoms.
(30) In aspect (30) is provided the method of any one of aspects 1-27, wherein the perfluorinated tag is a perfluorinated C3-20 group.
(31) In aspect (31) is provided the method of any one of aspects 1-27, wherein the perfluorinated tag is selected from a C4F9 group, a C5F11 group, a C6F13 group, a C7F15 group, a C8F17 group, a C9F19 group, a C10F21 group, a C6F5 group, C4F7 group, a C5F9 group, a C6F11 group, a C7F13 group, a C8F15 group, a C9F17 group, and a C10F19 group.
(32) In aspect (32) is provided the method of any one of aspects 1-14, wherein the perfluorinated ligand is
or a salt thereof, and wherein is a single bond or a double bond, and
represents the bond to the d-block element via the carbene.
(33) In aspect (33) is provided the method of any one of aspects 1-32, wherein the d-block element is a transition metal.
(34) In aspect (34) is provided the method of any one of aspects 1-33, wherein the d-block element is Co, Rh, Ir, Ru, Pd, Pt, or Mt.
(35) In aspect (35) is provided the method of any one of aspects 1-34, wherein the SABRE catalyst further comprises an additional ligand.
(36) In aspect (36) is provided the method of aspect 35, wherein the SABRE catalyst further comprises an additional ligand selected from phosphine ligands, carbene ligands, imidazole ligands, pincer chelating ligands, and compounds comprising a sulfoxide group.
(37) In aspect (37) is provided a hyperpolarized substrate obtained from the method of any one of aspects 1-36, or a pharmaceutically acceptable salt thereof.
(38) In aspect (38) is provided a pharmaceutical composition comprising a hyperpolarized substrate of aspect 37, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
(39) In aspect (39) is provided a method of obtaining a magnetic resonance image of a tissue in a subject having or suspected to have a cancer or an adverse vascular condition comprising administering to the subject a hyperpolarized substrate according to aspect 37 or a pharmaceutical composition according to aspect 38 and imaging the subject by magnetic resonance imaging.
(40) In aspect (40) is provided the method of aspect 39, wherein the subject has cancer.
(41) In aspect (41) is provided the method of aspect 40, wherein the cancer is selected from breast cancer, colon cancer, rectal cancer, bladder cancer, endometrial cancer, kidney cancer, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer.
(42) In aspect (42) is provided the method of aspect 39, wherein the adverse vascular condition is selected from myocardial infarction, stroke, and pulmonary disease.
(43) In aspect (43) is provided the method of aspect 42, wherein the pulmonary disease is selected from COPD, lung fibrosis, long-term COVID-19 symptom, and a combination thereof.
(44) In aspect (44) is provided a kit comprising:
[Lm-(NHC)—(Y—Z)q] Formula (I),
or a salt thereof, and wherein
(45) In aspect (45) is provided the kit of aspect 44, wherein NHC is a 5-membered N-heterocyclic carbenyl group.
(46) In aspect (46) is provided the kit of aspect 45, wherein the 5-membered N-heterocyclic carbenyl group is imidazole-based, imidazoline-based, or thiazole-based.
(47) In aspect (47) is provided the kit of any one of aspects 44-46, wherein NHC is a 4,5-disubstituted, a 1,3-disubstituted, or a 1,3,4,5-tetrasubstituted imidazole-based or imidazoline-based 5-membered N-heterocyclic carbenyl group.
(48) In aspect (48) is provided the kit of any one of aspects 44-47, wherein NHC is a 4,5-disubstituted imidazolidinyl, a 1,3-disubstituted imidazolidinyl, a 1,3,4,5-tetrasubstituted imidazolidinyl, a 4,5-disubstituted 2,3-dihydro-imidazolyl, a 1,3-disubstituted 2,3-dihydro-imidazolyl, or a 1,3,4,5-tetrasubstituted 2,3-dihydro-imidazolyl.
(49) In aspect (49) is provided the kit of any one of aspects 44-48, wherein the perfluorinated ligand is of Formula (Ia) or (Ib):
(50) In aspect (50) is provided the kit of any one of aspects 44-48, wherein the perfluorinated ligand is of Formula (Ic) or (Id):
(51) In aspect (51) is provided the kit of any one of aspects 44-48, wherein the perfluorinated ligand is of Formula (Ie) or (If):
(52) In aspect (52) is provided the kit of aspect 51 or aspect 52, wherein a is 4 to 10.
(53) In aspect (53) is provided the kit of any one of aspects 44-52, where each L independently is hydrogen, adamantyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,4-dimethylphenyl, 2,5-dimethylphenyl, 2,6-dimethylphenyl, 3,5-dimethylphenyl, 2,4,6-trimethylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2,4-diethylphenyl, 2,5-diethylphenyl, 2,6-diethylphenyl, 3,5-diethylphenyl, 2,4,6-triethylphenyl, 2-npropylphenyl, 3-npropylphenyl, 4-npropylphenyl, 2,4-di-npropylphenyl, 2,5-di-npropylphenyl, 2,6-di-npropylphenyl, 3,5-di-npropylphenyl, 2,4,6-tri-npropylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2,4-di-isopropylphenyl, 2,5-di-isopropylphenyl, 2,6-di-isopropylphenyl, 3,5-di-isopropylphenyl, 2,4,6-tri-isopropylphenyl, 2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2,4-di-isobutylphenyl, 2,5-di-isobutylphenyl, 2,6-di-isobutylphenyl, 3,5-di-isobutylphenyl, 2,4,6-tri-isobutylphenyl, 2-secbutylphenyl, 3-secbutylphenyl, 4-secbutylphenyl, 2,4-di-secbutylphenyl, 2,5-di-secbutylphenyl, 2,6-di-secbutylphenyl, 3,5-di-secbutylphenyl, 2,4,6-tri-secbutylphenyl, 2-tbutylphenyl, 3-tbutylphenyl, 4-tbutylphenyl, 2,4-di-tbutylphenyl, 2,5-di-tbutylphenyl, 2,6-di-tbutylphenyl, 3,5-di-tbutylphenyl, 2,4,6-tri-tbutylphenyl, 2-cyclohexylphenyl, 3-cyclohexylphenyl, 4-cyclohexylphenyl, 2,4-di-cyclohexylphenyl, 2,5-di-cyclohexylphenyl, 2,6-di-cyclohexylphenyl, 3,5-di-cyclohexylphenyl, or 2,4,6-tri-cyclohexylphenyl.
(54) In aspect (54) is provided the kit of any one of aspects 44-53, where each L independently is hydrogen or 2,4,6-trimethylphenyl.
(55) In aspect (55) is provided the kit of any one of aspects 44-54, wherein each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted C2-10 alkynyl group, a substituted or unsubstituted C1-10 heteroalkyl group, a substituted or unsubstituted C3-6 cycloalkyl group, a substituted or unsubstituted C3-6 heterocycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, a substituted or unsubstituted arylalkyl group, or a linear or branched alkyleneoxy group.
(56) In aspect (56) is provided the kit of any one of aspects 44-55, wherein each Y independently is a bond, a substituted or unsubstituted C1-10 alkyl group, a substituted or unsubstituted C2-10 alkenyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted alkaryl group, or a substituted or unsubstituted arylalkyl group.
(57) In aspect (57) is provided the kit of any one of aspects 44-52, wherein each Y independently is a bond or a substituted or unsubstituted C1-10 alkyl group.
(58) In aspect (58) is provided the kit of any one of aspects 44-57, wherein the perfluorinated tag is a perfluorinated C3-60 group comprising only carbon and fluorine atoms.
(59) In aspect (59) is provided the kit of any one of aspects 44-57, wherein the perfluorinated tag is a perfluorinated C3-40 group comprising only carbon and fluorine atoms.
(60) In aspect (60) is provided the kit of any one of aspects 44-57, wherein the perfluorinated tag is a perfluorinated C3-20 group.
(61) In aspect (61) is provided the kit of any one of aspects 44-60, wherein the perfluorinated tag is selected from a C4F9 group, a C5F11 group, a C6F13 group, a C7F15 group, a C8F17 group, a C9F19 group, a C10F21 group, a C6F5 group, C4F7 group, a C5F9 group, a C6F11 group, a C7F13 group, a C8F15 group, a C9F17 group, and a C10F19 group.
(62) In aspect (62) is provided the kit of any one of aspects 44-61, wherein the perfluorinated ligand is
or a salt thereof, and wherein is a single bond or a double bond, and
represents the bond to the d-block element via the carbene.
(63) In aspect (63) is provided the kit of any one of aspects 44-62, wherein the d-block element is a transition metal.
(64) In aspect (64) is provided the kit of any one of aspects 44-63, wherein the d-block element is Co, Rh, Ir, Ru, Pd, Pt, or Mt.
(65) In aspect (65) is provided the kit of any one of aspects 44-64, wherein the SABRE catalyst further comprises an additional ligand.
(66) In aspect (66) is provided the kit of aspect 65, wherein the SABRE catalyst further comprises an additional ligand selected from phosphine ligands, carbene ligands, imidazole ligands, pincer chelating ligands, and compounds comprising a sulfoxide group.
(67) In aspect (67) is provided the kit of any one of aspects 44-66, wherein the co-ligand is a compound containing one or more sulfoxide groups, thioester groups, phosphine groups, amine groups, CO groups, isonitrile groups, nitrogen-containing heterocyclic groups, or a combination thereof.
(68) In aspect (68) is provided the kit of any one of aspects 44-67, wherein the co-ligand is dimethyl sulfoxide.
(69) In aspect (69) is provided the kit of any one of aspects 44-67, wherein the co-ligand is fluorinated.
(70) In aspect (70) is provided the kit of aspect 69, wherein the co-ligand is phenyl trifluoromethyl sulfoxide, 2,2,2-trifluoroethyl methane sulfonate, or di-2,2,2-trifluoroethyl sulfoxide.
(71) In aspect (71) is provided the kit of any one of aspects 44-70, wherein the solvent comprises water, methanol, ethanol, a fluorous solvent, or a mixture thereof.
(72) In aspect (72) is provided the kit of any one of aspects 44-71, wherein the solvent comprises a perfluoroalkane, diethyl ether, a nonafluorobutyl methyl ether, ethyl acetate, perfluorobutyl methyl ether, a fluorocarbon derivative of THF FC 75, a decafluoromethoxy trifluoromethyl pentane, a hexafluoro propanol, a perfluoromethyl cyclohexane, and a combination thereof.
(73) In aspect (73) is provided the kit of any one of aspects 44-71, wherein the solvent comprises water, methanol, ethanol, a nonafluorobutyl methyl ether, deuterated variants thereof, or a mixture thereof.
(74) In aspect (74) is provided the kit of any one of aspects 44-73, wherein the solvent is deuterated.
(75) In aspect (75) is provided the kit of any one of aspects 44-74, wherein the kit further comprises a substrate.
(76) In aspect (76) is provided the kit of aspect 75, wherein the substrate comprises 1H, 13C, 15N, 19F, 31P 29Si, or a combination thereof.
(77) In aspect (77) is provided the kit of aspect 76, wherein the substrate further comprises 2D.
(78) In aspect (78) is provided the kit of any one of aspects 75-77, wherein the substrate is selected from ketoglutarate, ketoisocaproate, pyruvate, N-acetyl cysteine, and salts or esters thereof.
(79) In aspect (78) is provided the kit of any one of aspects 75-78, wherein the substrate is selected from 1-13C-ketoglutarate, 1-13C-5-12C-ketoglutarate, 1-13C-pyruvate, 1-13C-N-acetyl cysteine, 15N2-isoniazid (or pyridyl-4-carbo-bis-15N2-hydrazide), 13C2,15N3-metronidazole, 15N2-1-aminoisoquinoline (1-AIQ), deuterated versions thereof, and salts thereof.
(80) In aspect (80) is provided the kit of any one of aspects 75-79, wherein the substrate is of Formula (II):
(81) In aspect (81) is provided the kit of any one of aspect 44-80, wherein the perfluorinated SABRE catalyst is separated from the co-ligand and/or the solvent.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
This example illustrates a method of preparing 1H,1H,2H,2H-perfluorooctyl-N,N′-bis(2,4,6-trimethylphenyl)-9,10-diamine:
wherein x=6 and y=13.
A 1.7 M solution of tert-butyl lithium in pentane (5 mL, 9 mmol, 8 equiv.) was added to a solution of 1H,1H,2H,2H-perfluorooctyl iodide (2 g, 4 mmol, 4 equiv.) in dry Et2O (60 mL) at −78° C. After the mixture had been stirred for 20 min at −78° C., the solid N,N′-dimesitylethanediimine (0.30 g, 1.03 mmol, 1 equiv.) was added portion wise. The reaction mixture was stirred for 4 h. The reaction was slowly warmed to −30° C. and quenched with a saturated solution of ammonium chloride (0.6 mL).
Water (20 mL) was added, the organic layer was separated, and the aqueous layer was extracted with diethyl ether (3×15 mL). The combined organic layers were dried over anhydrous MgSO4, and concentrated. The crude product was purified by flash column chromatography (4:1 hexane/dichloromethane (DCM)) to yield diamine, diimine, and threo-diamine (0.6 g, 60% yield, white solid). 1H NMR (400 MHz, CDCl3) δ 1.64-1.82 (m, 2H, CH2CHHCH(NHAr)), 2.00 (s, 12H, o-CH3), 2.07˜2.27 (m, 4H,CHHCHHCH(NHAr)), 2.33 (s, 6H, p-CH3), 2.67˜2.91 (m, 2H, CHHCH2CH(NHAr)), 2.95 (br s, 2H, NH), 3.23 (br d, 3JH-H=10 Hz, 2H, CH), 6.82 (s, 4H, Ar—CH) ppm. 19F NMR (282.23 MHz, CDCl3) δ -83.8 (t, 3JF-F=10 Hz, 6F, CF3), −114.4 (m, 4F, CF2CH2), −121.9 (m, 4F, CF2CF2CH2), −122.8 (m, 4F, CF3CF2CF2CF2), −123.4 (m, 4F, CF3CF2CF2), −126.1 (m, 4F, CF3CF2) ppm. 13C NMR (400 MHz, CDCl3) δ 18.3 (o —CH3), 20.3 (p —CH3), 21.7 (m, CF2CH2CH2), 29.2 (t, 2JF-C=22.2 Hz, CF2CH2), 57.1 (CH), 107-115 (m, 8 CF2), 117.4 (qt, 1JF-C=288.3 Hz, 2JF-C=33.3 Hz, CF3), 118.7 (tt, 1JF-C=255.1 Hz, 2JF-C=31.0 Hz, CF2CH2), 128.6 (Ar—C), 129.9 (Ar—CH), 131.4 (Ar—C),140.4 (Ar—C) ppm. MS (ESI), m/z (%): 990 [M+H]+(100).
This example illustrates the synthesis of trans-4,5-bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium tetrafluoroborate:
A mixture of 1H,1H,2H,2H-Perfluorooctyl-N,N′-bis(2,4,6-trimethylphenyl)-9,10-diamine (0.250 g, 0.25 mmol) from Example 1, ammonium tetrafluoroborate (-10% molar excess) (0.030 g, 0.25 mmol), and triethyl orthoformate (0.5 mL) was heated to 125° C. and stirred for 15 h. After cooling to room temperature, the solution was evaporated and the solid was triturated with diethyl ether (6×3 mL). The residue was redissolved in acetone, filtered, and concentrated to yield to the dihydroimidazolium tetrafluoroborate. (0.2 g, 70% yield, yellowish solid). 1H NMR (400 MHz, acetone-d6) δ 2.15˜2.70 (m, 8H, CF2CH2CH2), 2.34 (s, 6H, p-CH3), 2.51 (s, 6H, o-CH3), 2.94 (br s, 2H, NH), 5.16 (m, 2H, CH2CH2CH), 7.17 (s, 4H, Ar—CH), 9.06 (s, 1H, N—CH═N) ppm. 19F NMR (400 MHz, acetone-d6) δ-81.6 (t, 4JF-F=10 Hz, 6F, CF3), −115.1 (m, 4F, CF2CF2CH2), −122.5 (m, 4F, CF2CF2CH2), −123.6 (m, 4F, CF3CF2CF2CF2), −124.5 (m, 4F, CF3CF2CF2), −126.9 (m, 4F, CF3CF2CF2), −150.9 (4F, BF4) ppm. 13C NMR (400 MHz, acetone-d6) δ 17.4 (o —CH3), δ 17.7 (o —CH3), 19.9 (p —CH3), 24.4 (m, CF2CH2CH2), 26.2 (t, 2JF-C=22.2 Hz, CF2CH2), 67.9 (CH), 105-118 (m, 8 CF2), 118 (qt, 1JF-C=288.3 Hz, 2JF-C=33.3 Hz, CF3), 118.9 (tt, 1JF-C=254 Hz, 2JF-C=31.0 Hz, CF2CH2), 129.5 (Ar—C), 130.1 (Ar—CH), 130.5 (Ar—CH), 135.8 (Ar—C), 136 (Ar—C),140.8 (Ar—C) ppm, 159.7 (N—C═N) ppm. MS (ESI) m/z (%): 999 [M+H]+(100).
This example illustrates the synthesis of trans-4,5-Bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-1,3bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium chloride:
Dihydroimidazolidium tetrafluoroborate salt (0.5 g, 0.46 mmol) was dissolved in MeOH (1.5 mL) and passed through a short column of ion exchange resin Amberlite 400. The column was washed with MeOH until no spot was visible via TLC under UV. The solvent was removed, and the resulting yellowish solid was dried with a vacuum pump to yield product (0.48 g, 99%). 1H NMR (400 MHz, acetone-d6) δ 2.15˜2.70 (m, 8H, CF2CH2CH2), 2.34 (s, 6H, p-CH3), 2.51 (s, 6H, o-CH3), 2.94 (br s, 2H, NH), 5.16 (m, 2H, CH2CH2CH), 7.17 (s, 4H, Ar—CH), 9.06 (s, 1H, N—CH═N) ppm. 19F NMR (400 MHz, acetone-d6) δ-81.8 (t, 4JF-F=10 Hz, 6F, CF3), −115.1 (m, 4F, CF2CF2CH2), −122.5 (m, 4F, CF2CF2CH2), −123.6 (m, 4F, CF3CF2CF2CF2), −124.5 (m, 4F, CF3CF2CF2), −127 (m, 4F,CF3CF2CF2) ppm. 13C NMR (400 MHz, acetone-d6) δ 17.4 (o —CH3), δ 17.7 (o —CH3), 19.9 (p —CH3), 24.4 (m, CF2CH2CH2), 26.2 (t, 2JF-C=22.2 Hz, CF2CH2), 67.9 (CH), 105-118 (m, 8 CF2), 118 (qt, 1JF-C=288.3 Hz, 2JF-C=33.3 Hz, CF3), 118.9 (tt, 1JF-C=254 Hz, 2JF-C=31.0 Hz, CF2CH2), 129.5 (Ar—C), 130.1 (Ar—CH), 130.5 (Ar—CH), 135.8 (Ar—C), 136 (Ar—C),140.8 (Ar—C) ppm, 159.7 (N—C═N) ppm. MS (ESI) m/z (%): 999 [M+H]+ (100).
This example illustrates a method of synthesis of a fluorinated SABRE catalyst containing a transition metal in accordance with an aspect of the invention.
Potassium tert-butoxide (112 mg, 1.00 mmol, 2.5 eq.) was added to a stirred solution of trans-4,5-bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazolium chloride (320 mg, 0.88 mmol, 2.2 eq.) from Example 3 in tetrahydrofuran (10 mL) at room temperature in a glove box. The resulting suspension was stirred for 30 minutes. A solution of [Ir(COD)Cl]2 (268 mg, 0.40 mmol, 1.0 eq.) was added and the resulting solution was stirred at room temperature overnight (Cowley et al., J Am. Chem. Soc., 133, 6134-6137 (2011)). The solvent was removed under reduced pressure to give the crude product and dried overnight under vacuum. This sample was dissolved in hexane and added to a 60 mL filter packed with SiO2 gel in hexane. The crude solution was absorbed on the top of the silica gel, then hexane was added, to elute the compound, to give 104 mg (40% yield). 1H NMR (400 MHz, CDCl3) δ 1.04, 1.18, 1.49, 1.76, 2.13, 2.22, 2.25, 2.4, 2.48, 2.66, 2.94, 3.73, 4.01, 4.2, 6.84, 6.91 6.97 ppm. 19F NMR (400 MHz, CDCl3) δ −81.08, −114.33, −122.05, −123.07, −124.02, −126.39 ppm. 13C NMR (100 MHz, CDCl3) δ 18.7, 20.95, 21.07, 21.34, 26.45, 27.21, 29.83, 30.74, 31.42, 49.93, 55.12, 68.11, 68.47, 83.26, 86.69, 108.32, 111.0, 112.95, 115.85, 117.75, 118.69, 129.02, 129.3, 130.36, 130.55, 134.71, 134.8, 135.32, 136.33, 137.92, 138.28, 138.45, 138.5, 206.72 ppm. MS (ESI) m/z (%): 1299 [M+H]+(100).
This example illustrates a method of hyperpolarizing a [1-13C]pyruvate in accordance with an aspect of the invention, as shown in Scheme 1.
wherein co-ligand=phenyl trifluoromethyl sulfoxide.
In the reaction scheme above, hyperpolarization of [1-13C]pyruvate was performed using SABRE in SHield Enabled Alignment Transfer to Heteronuclei (SABRE-SHEATH) (Theis et al., J Am. Chem. Soc., 137, 1404-1407 (2015) and Truong et al., J Phys. Chem. C, 119, 8786-8797 (2015)) tailored for the 13C nucleus (Barskiy et al., ChemPhysChem, 18, 1493-1498 (2017)) using the co-ligand approach developed by Duckett and co-workers (Iali et al., Angew. Chemie —Int. Ed., 58, 10271-10275 (2019)). Sodium [1-13C]-pyruvate and deuterated methanol-d4 solvent were purchased from Sigma-Aldrich and used without any further purification. The [IrCl(COD)(F-IMes)] SABRE catalyst used for this Example was prepared according to Example 4. The active catalyst used herein was prepared with a fixed ratio of substrate to Ir(F-IMes) SABRE catalyst of Example 4, and phenyl trifluoromethyl sulfoxide (PTFSO) in 0.6 mL of methanol-d4 in a 5 mm NMR tube.
Parahydrogen was generated using a Gas-Delivery Manifold. Ultra-high-purity hydrogen gas (Airgas) was fed into a ParaHydrogen flow cryostat (Xeus technology LTD) and enriched to about 50% parahydrogen in the presence of a spin-exchange catalyst (Fe2O3) at liquid nitrogen temperature (77K). The p-H2 flow was directed via PTFE tubing to a mass flow controller (MFC, Sierra Instruments SmartTrak 100 series) set at 90 scc/m and directed to a conventional 5 mm NMR tube (Norell) to allow bubbling through the sample. The entire pH2 line was pressurized to 100 psi.
The magnetic shield condition was as follows. Magnetic fields near or below ˜1 μT were achieved with an apparatus consisting of a solenoid coil placed inside a mu-metal shield (Magnetic Shield Corporation, model No. ZG-206). The shield was degaussed using internal homebuilt coils driven by a Variac when necessary. The solenoid had a 41 mm diameter (40 mm core, 20 cm long windings with 220 turns AWG20 (0.9 mm) Cu wire and with 220Ω resistor in series. The solenoid coil was driven by commercial 1.5V batteries with a variable-resistance decade box in series to provide finer control of the internal magnetic field inside the shield. Typical values of the field within the shield were between ±1.2μT, with SABRE SHEATH experiments typically between −0.7 μT and +0.8 μT in the sample region. The values were monitored between SABRE experiments using a Lakeshore Cryotronics Gaussmeter (Model No. 475 DSP with HMMA-2512-VR Hall Probe).
MR experiments were performed using a 1 T Magritek Spinsolve benchtop NMR spectrometer. All 13C NMR spectra were taken with 1H decoupling turned off throughout the duration of the experiment. Time required to manually transfer the sample from the shield region to the magnet for low-field NMR acquisition was usually <5 s.
The efficient hyperpolarization transfer from nascent p-H2-derived hydrides to the 13C nuclear spin of [1-13C]pyruvate was attained by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) using a solution mixture of [IrCl(H)2(PTFSO)2(F-IMes)], [1-13C]pyruvate) and p-H2 in deuterated methanol.
All experiments were performed with the solution containing fluorinated catalyst, co-ligand (phenyl trifluoromethyl sulfoxide) and [1-13C] Pyruvate in 0.6 mL CD3OD. The ratio and concentration can be further adjusted to attain better enhancement and polarization. The results were obtained for 8 mM fluorinated catalyst, 16 mM phenyl trifluoromethyl sulfoxide (PTFSO) and 30 mM [1-13C] Pyruvate in 0.6 mL CD3OD. The experiments were performed at room temperature, ˜100 scc/m p-H2 flow rate and 96 PSI p-H2 overpressure. The parahydrogen used in this example came from a low-cost 50% p-H2 generator. Each experiment, the p-H2 bubbling was applied for ˜1 min, the sample was quickly transferred to the 1 T NMR spectrometer for detection and the sample was then returned to the mu-metal shield to continue p-H2 bubbling for the next experiment. The 13C signal enhancement was computed by comparing HP signal area-undercurve (AUC) to external 13C signal thermal signal reference (4M sodium [1-13C]acetate) using Eq. 1:
where SHP and SREF are 13C signals from HP [1-13C] pyruvate and thermal signal reference [1-13C]acetate, CREF and CHP are concentrations of thermal signal reference [1-13C]acetate (4 M) and of HP [1-13C]pyruvate, respectively, and AREF and AHP are effective cross-sections of the NMR tubes for the thermal signal reference [1-13C]acetate and HP [1-13C]pyruvate samples.
This example illustrates a method of hyperpolarizing a [1-13C]pyruvate in accordance with an aspect of the invention, as shown in Scheme 2.
In the reaction scheme above, hyperpolarization of [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) (Theis et al., J. Am. Chem. Soc., 137, 1404-1407 (2015) and Truong et al., J. Phys. Chem. C, 119, 8786-8797 (2015)) tailored for the 13C nucleus (Barskiy et al., ChemPhysChem, 18, 1493-1498 (2017)) using the co-ligand approach developed by Duckett and co-workers (Iali et al., Angew. Chemie —Int. Ed., 58, 10271-10275 (2019)). Sodium [1-13C]-pyruvate and deuterated methanol-d4 solvent were purchased from Sigma-Aldrich and used without any further purification. The [IrCl(COD)(F-IMes)] SABRE catalyst used for this Example was prepared according to Example 4. The active catalyst used herein was prepared with a fixed ratio of substrate [1-13C]pyruvate, Ir(F-IMes) SABRE catalyst of Example 4, and co-ligand dimethyl sulfoxide (DMSO), in 0.6 mL of methanol-d4 in a 5 mm NMR tube.
Parahydrogen enriched to about 70 to 95% was used and directed via PTFE tubing to a mass flow controller (MFC, Sierra Instruments SmartTrak 100 series) set between 50 to 120 scc/m into a medium wall 5 mm NMR tube (Norell) to allow bubbling through the sample. The entire pH2line was pressurized values between 50 and 110 psi.
The polarization transfer magnetic field was established as follows. Magnetic fields near or below ˜1 μT were achieved with an apparatus consisting of a solenoid coil placed inside a three-layered mu-metal shield (6 in. ID & 15 in. in length, part number ZG-206, Magnetic Shield Corp., Bensenville, IL). The magnetic field was created using a custom-built solenoid coil and a triple independent channel DC power supply (KEITHLEY 2231A-30-3). The solenoid had a 41 mm diameter (40 mm core, 20 cm long windings with 220 turns AWG20 (0.9 mm) Cu wire and with 220Ω resistor in series. The solenoid coil was driven with a variable-resistance decade box in series to provide finer control of the internal magnetic field inside the shield. Typical values of the field within the shield were between ±1.2μT, with SABRE SHEATH experiments typically between −0.7 μT and +0.8 μT in the sample region.
MR experiments were performed using an 80 MHz Magritek Spinsolve benchtop NMR spectrometer. The following acquisition parameters were used:
All 13C NMR spectra were taken with 1H decoupling turned off throughout the duration of the experiment. Time required to manually transfer the sample from the shield region to the magnet for low-field NMR acquisition was usually <5 s.
The hyperpolarization transfer from p-H2-derived iridium hydrides to the 13C nuclear spin of [1-13C]pyruvate was attained by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) using a solution mixture of [IrCl(H)2(DMSO)2(F-IMes)], [1-13C]pyruvate, co-ligand (dimethyl sulfoxide) and p-H2 in 0.5 mL deuterated or non-deuterated methanol.
The 13C signal enhancement was computed by comparing HP signal area-undercurve (AUC) to external 13C signal thermal signal reference (4M sodium [1-13C]acetate) using Eq.:
where SHP and SREF are 13C signals from HP [1-13C] pyruvate and thermal signal reference [1-13C]acetate, CREF and CHP are concentrations of thermal signal reference [1-13C]acetate (4 M) and of HP [1-13C]pyruvate, respectively, and AREF and AHP are effective cross-sections of the NMR tubes for the thermal signal reference [1-13C]acetate and HP [1-13C]pyruvate samples. The percentage of 13C polarization (% P13c) was computed by multiplying the signal enhancement (ε13c) by thermal 13C nuclear spin polarization at 1.88 T (1.5681*10−4%) in accordance with Equation S2: (% P13c), =ε13c 1.56181*10−6 *100.
The fluorinated SABRE catalyst activation took less than 15 minutes, with the 13C polarization percentage shown in
This example demonstrates the effects on hyperpolarization of [1-13C]pyruvate, exhibited by changes in parahydrogen pressure and flow rate, as well as the effect of magnetic transfer field, temperature, and concentration of the fluorinated catalyst and DMSO. In addition, the relaxation dynamics of the [1-13C]pyruvate were also studied.
Hyperpolarization of [1-13C]pyruvate was repeated using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH), as described in Example 6 above, and the effects of parahydrogen pressure and flow rate, as well as the effect of magnetic transfer field, temperature, and concentration of the fluorinated catalyst and DMSO, were studied.
p-H2 parameters such as the pressure and flow rate were evaluated and the polarization percentage results are set forth in
The temperature and magnetic field in the micro Tesla regime were evaluated and the 13C polarization level and polarization transfer magnetic field at 0° C. are set forth in
The perfluorinated SABRE catalyst and DMSO concentrations were evaluated at a temperature of 0° C., a magnetic transfer field of 0.4 μT, a p-H2 flow of 90 scc/m, and a p-H2 pressure of 110 PSI, and the polarization percentages are set forth in
As is apparent from the results set forth in
The simultaneous exchange of p-H2 and [1-13C]pyruvate on activated Ir(F-IMes) catalyst leads to buildup of 13C hyperpolarization. In that respect,
Temperature has a profound effect on the exchange rates of [1-13C]pyruvate on Complex 3b of
As demonstrated by
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate, which includes extraction and filtration.
Hyperpolarization of sodium [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) tailored for the 13C nucleus using dimethyl sulfoxide (DMSO) as a co-ligand, [IrCl(COD)(F-IMes)] SABRE catalyst, and parahydrogen enriched to about 70 to 95% in deuterated methanol-d4 solvent, as described in Examples 6 and 7. The SABRE samples were prepared in 0.5 mL CD3OD, using 30 mM sodium [1-13C]pyruvate, 2.6 mM perfluorinated SABRE catalyst of Example 4, and 35 mM dimethyl sulfoxide (DMSO). The parahydrogen flow rate was established at 90 scc/m and pressurized to 8 bars, the mixing field was 0.4 μT, and the temperature was 0° C.
After the hyperpolarization procedure was completed, the sample was rapidly removed from the 0.40 μT field, depressurized, and 20% in volume (125 P L) of heavy water (D2O) was added to the solution to precipitate the perfluorinated SABRE catalyst. The resulting mixture was transferred into a 1 mL plastic syringe mounted to a Luer-locked filter (Waters Oasis Prime HLB Plus Light Cartridge (Part Number: 186008866), and the aqueous solution was guided through the filter into a 5 mm NMR tube, already located into the adjacent 1.8 T benchtop NMR spectrometer. The whole procedure took about 1.15 to 1.30 minutes and no HP 13C signal was observed in any sample. The relaxation study presented above indicates that [1-13C]-T1 relaxation time of pyruvate at earth field was substantially shorter (e.g., [1-13C]-T1=28.9±1.6 s at Earth's field). Automation and faster solution transfer should allow the observation of HP [1-13C]-pyruvate signal in aqueous solutions.
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate, which includes extraction by precipitation with organic solvent.
Precipitation and redissolution of HP [1-13C]pyruvate, which takes place in the same NMR tube where hyperpolarization, was performed (Schmidt et al., ACS Sensors, 7(11), 3430-3439 (2022)). The SABRE samples were prepared in CD3OD, using between 20 and 30 mM sodium [1-13C]pyruvate, 7.5 mM fluorinated SABRE catalyst, and 50 mM dimethyl sulfoxide (DMSO), as described in Examples 6 and 7. 100 μL of this solution was transferred into 5 mm NMR tube and exposed to the SABRE-SHEATH hyperpolarization conditions with the same set-up and optimum conditions described in Examples 6 and 7. The solution was located inside a 3-layer mu-metal of 3″ I.D. and 9″ depth to shield external magnetic fields, combined with a custom-made solenoid to generate a static magnetic field Bo of 0.4 μT. The NMR tubes were pressurized (110 psi, i.e., approximately 8 bar total pressure) with p-H2 bubbling through the solution at a flow of 90 scc/m to activate the catalyst and to 13C-hyperpolarize the sodium [1-13C]pyruvate solution. Activation of the catalyst took place for 15 min at ambient temperature and magnetic field. For polarization build-up, the sample was placed in the static magnetic field (typically about 0.4 μT) and a water bath to regulate the reaction temperature at 0° C. After polarization, the NMR tube was rapidly transferred inside the NMR spectrometer at 1.8 T and kept at room temperature. The precipitation of pyruvate is performed after depressurization by adding 400 μL of ethyl acetate (EtOAc) to the HP solution and redissolved by adding 300 μL heavy water (D2O) to reconstitute the pyruvate in water.
The NMR spectrum was acquired immediately after reconstitution in water using a 1.8 T benchtop NMR, and the results are set forth in the top spectrum of
In addition, the 13C-pyruvate concentrations were determined by LC-MS using a calibration curve for naturally occurring isotopic pyruvate. The pyruvate aqueous samples were further analyzed by ICP-MS (inductively coupled plasma Multi-Element Scan) for Iridium elemental content after the reconstitution SABRE-SHEATH methodology. The Iridium content was determined to be only about 150 ppb to about 300 ppm.
This example illustrates a method of the synthesis of 2,2,2-trifluoro-N-(4-iodo-2,6-dimethylphenyl)acetamide:
To a stirring solution of 2,6-diisopropylaniline (4.92 mL, 40.00 mmol, 1.0 equiv) in diethyl ether (50 mL) were added iodine (11.17 g, 44.00 mmol, 1.1 equiv) and a saturated sodium bicarbonate solution (30 mL). The solution was stirred at room temperature for 3 hours and gas evolution was observed. Excess iodine was destroyed by addition of sodium thiosulfate (1.33 g, 8.40 mmol). The phases were separated, and the aqueous phase was further extracted with diethyl ether (2×20 mL). The combined organic phases were washed with water (200 mL) and saturated solution of sodium thiosulfate. The organic phase was evaporated to dryness in vacuo to yield the product as a brown oil which slowly became solid under vacuum.
Hexane was added to dissolve product and the solution is filtered with Celite and evaporated and dried under vacuum. No further purification was necessary. 1H NMR (400 MHz, CDCl3): δ=7.14 (s, 2H), 4.45 (s, 2H), 2.04 (s, 6H).
This example illustrates a method of synthesis of N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooct-1-en-1-yl)phenyl)-2,2,2-trifluoroacetamide:
In a glove box, palladium(II) acetate (0.16 g, 0.051 equiv, 0.71 mmol), sodium acetate (1.72 g, 1.50 equiv, 21.0 mmol), tricyclohexylphosphane (660 mg, 0.168 equiv, 2.35 mmol), tetrabutylammonium bromide (30 mg, 0.0067 equiv, 93 μmol) and 2,2,2-trifluoro-N-(4-iodo-2,6-dimethylphenyl)acetamide (4 g, 0.8 equiv, 0.01 mol) were added to a Schlenk flask with 10 mL of dimethyl formamide. The Schlenk flask was warmed to 90° C., then 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooct-1-ene (4.84 g, 1 equiv, 14.0 mmol) was added to the reaction mixture and further heated to 120° C. The solution was stirred for 19 hours. After cooling, the mixture was filtered through Celite and washed with diethyl ether (50 mL). Water (50 mL) and diethyl ether (30 mL) were added, the organic phase was separated, and aqueous phase was extracted with Et2O (3×15 mL). The combined organic layers were dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the crude product. Purification by column chromatography (hexane/DCM 4:1) gave fluoroamide N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooct-1-en-1-yl)phenyl)-2,2,2-trifluoroacetamide, which was further crystallized (5 g, 70%, white needles).
This example illustrates a synthesis of N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl)-2,2,2-trifluoroacetamide:
To a solution of N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooct-1-en-1-yl)phenyl)-2,2,2-trifluoroacetamide (0.40 g, 0.70 mmol) in ethyl acetate (10 mL) in a glass autoclave, 10% Pd/C (0.08 g, 0.07 mmol) was added. The autoclave was evacuated, filled with hydrogen gas to 500 kPa and stirred for 5 hours at room temperature. The mixture was filtered through Celite and washed with EtOAc (30 mL). The solvent was removed on vacuum rotary evaporator to afford N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl)-2,2,2-trifluoroacetamide.
This example illustrates a synthesis of 2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)aniline:
To a solution of N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl)-2,2,2-trifluoroacetamide (0.14 g, 250 μmol) in n-butanol (1.5 mL), sodium hydroxide (0.10 g, 250 mmol) was added and the reaction mixture was heated to 110° C. for 21 hours. After cooling to room temperature, water (4 mL) and ethyl acetate (4 mL) were added and the organic phase was separated, washed with 1M solution of HCl (4 mL), saturated solution of NaHCO3(4 mL), and brine (4 mL). The aqueous phase was neutralized and extracted with ethyl acetate (3 times ×4 mL). The organic layers were combined and dried over anhydrous magnesium sulfate and concentrated under reduced pressure to afford 2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)aniline (0.50 g, 92%, brown crystals).
This example illustrates a synthesis of N,N′-Bis[2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl]ethane-1,2-diimine:
To the solution of 2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)aniline (0.5 g, 1 mmol) in ethanol (5 mL), 40% aqueous solution of glyoxal (0.1 mL, 1 mmol) and catalytic amount of formic acid (few drops) were added. The reaction mixture was stirred for 20 hours at room temperature, during which yellow precipitate was formed. The solid was filtered and washed with cold ethanol (3×5 mL) to give clean N,N-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl)ethane-1,2-diimine (0.2 g, 20%, yellow powder).
This example illustrates a synthesis of 1,3-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl)-1H-imidazol-3-ium-2-ide, chloride salt:
A flask was charged with N,N-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,10,10,10-tridecafluorooctyl)phenyl)ethane-1,2-diimine (2.40 g, 2.08 mmol) and tetrahydrofuran (80 mL). The mixture was cooled to 0° C. and suspension of paraformaldehyde (262 mg, 2.91 mmol) and conc. HCl (112 mg, 3.12 mmol) in dioxane (0.78 mL) was slowly added. The mixture was heated to reflux overnight. Purification was carried out by column chromatography. 1H NMR (400 MHz, CDCl3) δ 2.07, 2.55, 2.92, 7.37, 8.28, 9.65. MS (ESI) m/z (%): 1168.8 [M-TFA]+(100).
This example illustrates a synthesis of a fluorinated SABRE catalyst in accordance with an aspect of the invention.
Potassium tert-butoxide (2.5 eq.) was added to a stirred solution of 1,3-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-tridecafluorooctyl)phenyl)-1H-imidazol-3-ium-2-ide, chloride (2.2 equiv) in tetrahydrofuran at room temperature in a glove box. The resulting suspension was stirred for 30 min. A solution of [Ir(COD)Cl]+(1.0 eq.) was added and the resulting solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give the crude product and dried overnight under vacuum. This sample was purified with flash chromatography DCM/hexane (4:1) to obtain the fluorinated SABRE catalyst. MS (ESI) m/z (%): 1469 [M-Cl]+ (100).
This example illustrates the hyperpolarization of sodium pyruvate using the SABRE catalyst in accordance with an aspect of the invention, as shown in Scheme 3.
In the reaction scheme above, the efficient hyperpolarization transfer from p-H2-derived hydrides to the 13C nuclear spin of [1-13C]pyruvate was attained by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) using a solution mixture of [IrCl(H)2(DMSO)2(F-IMes)], [1-13C]pyruvate) and p-H2 in deuterated methanol.
The SABRE samples were prepared in CD3OD, using 40 mM sodium [1-13C]pyruvate, 6.6 mM perfluorinated SABRE catalyst of Example 16, and 50 mM dimethyl sulfoxide (DMSO), as described in Examples 6 and 7. The SABRE samples were exposed to the SABRE-SHEATH hyperpolarization conditions with the same set-up and optimum conditions described in Examples 6 and 7. The NMR tubes were pressurized (110 psi, i.e., approximately 8 bar total pressure) with p-H2 bubbling through the solution at a flow of 90 scc/m to activate the catalyst and to 13C-hyperpolarize the sodium [1-13C]pyruvate solution. Activation of the catalyst took place for about 15 minutes at ambient temperature and magnetic field. For polarization build-up, the sample was placed in the magnetic field (typically about 0.4 μT) and a water bath at 5° C. to regulate the reaction temperature. The spectrum was acquired immediately following manual sample transfer to a 1.8 T benchtop NMR after 5 seconds, and the results are set forth in the top spectrum of
This example illustrates a method of synthesis of N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)phenyl)-2,2,2-trifluoroacetamide:
In a glove box, palladium(II) acetate (0.16 g, 0.051 equiv, 0.71 mmol), sodium acetate (1.72 g, 1.50 equiv, 21.0 mmol), tricyclohexylphosphane (660 mg, 0.168 equiv, 2.35 mmol), tetrabutylammonium bromide (30 mg, 0.0067 equiv, 93 μmol) and 2,2,2-trifluoro-N-(4-iodo-2,6-dimethylphenyl)acetamide (4 g, 0.8 equiv, 0.01 mol) were added to a Schlenk flask with 10 mL of dimethyl formamide. The Schlenk flask was warmed to 90° C., then 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-ene (4.84 g, 1 equiv, 14.0 mmol) was added to the reaction mixture and further heated to 120° C. The solution was stirred for 19 hours. After cooling, the mixture was filtered through Celite and washed with diethyl ether (50 mL). Water (50 mL) and diethyl ether (30 mL) were added, the organic phase was separated, and aqueous phase was extracted with Et2O (3×15 mL). The combined organic layers were dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the crude product. Purification by column chromatography (hexane/DCM 4:1) gave fluoroamide N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)phenyl)-2,2,2-trifluoroacetamide, which was further crystallized (5 g, 70%, white needles).
This example illustrates a synthesis of 2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)aniline:
To a solution of N-(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)phenyl)-2,2,2-trifluoroacetamide (0.14 g, 250 μmol) in n-butanol (1.5 mL), sodium hydroxide (0.10 g, 250 mmol) was added and the reaction mixture was heated to 110° C. for 21 hours. After cooling to room temperature, water (4 mL) and ethyl acetate (4 mL) were added and the organic phase was separated, washed with 1M solution of HCl (4 mL), saturated solution of NaHCO3(4 mL), and brine (4 mL). The aqueous phase was neutralized and extracted with ethyl acetate (3 times×4 mL). The organic layers were combined and dried over anhydrous magnesium sulfate and concentrated under reduced pressure to afford 2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)aniline (0.50 g, 92%, brown crystals).
This example illustrates a synthesis of N,N′-Bis[2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)phenyl]ethane-1,2-diimine:
To the solution of 2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)aniline (0.5 g, 1 mmol) in ethanol (5 mL), 40% aqueous solution of glyoxal (0.1 mL, 1 mmol) and catalytic amount of formic acid (few drops) were added. The reaction mixture was stirred for 20 hours at room temperature, during which yellow precipitate was formed. The solid was filtered and washed with cold ethanol (3×5 mL) to give clean N,N-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)phenyl)ethane-1,2-diimine (0.2 g, 20%, yellow powder).
This example illustrates a synthesis of 1,3-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)-1H-imidazol-3-ium-2-ide, chloride salt:
A flask was charged with N,N-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)phenyl)ethane-1,2-diimine (2.40 g, 2.08 mmol) and tetrahydrofuran (80 mL). The mixture was cooled to 0° C. and suspension of paraformaldehyde (262 mg, 2.91 mmol) and conc. HCl (112 mg, 3.12 mmol) in dioxane (0.78 mL) was slowly added. The mixture was heated to reflux overnight. Purification was carried out by column chromatography. 1H NMR (400 MHz, CDCl3) δ 2.27, 6.71, 7.32, 7.36, 7.67, 8.16, 9.59 ppm. 19F NMR (400 MHz, CDCl3) δ-82.43, −112.52, −122. 59, −123.89, −124.17, −127.33 ppm. MS (ESI) m/z (%): 964.8 [M+H]+ (100).
This example illustrates a synthesis of a fluorinated SABRE catalyst in accordance with an aspect of the invention.
Potassium tert-butoxide (2.5 eq.) was added to a stirred solution of 1,3-bis(2,6-dimethyl-4-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-en-1-yl)phenyl)-1H-imidazol-3-ium-2-ide, chloride (2.2 equiv) in tetrahydrofuran at room temperature in a glove box. The resulting suspension was stirred for 30 min. A solution of [Ir(COD)Cl]2 (1.0 eq.) was added and the resulting solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give the crude product and dried overnight under vacuum. This sample was purified with flash chromatography DCM/hexane (4:1) to obtain the fluorinated SABRE catalyst. MS (ESI) m/z (%): 1266 [M-Cl]+ (100).
This example illustrates the hyperpolarization of sodium pyruvate using the SABRE catalyst in accordance with an aspect of the invention, as shown in Scheme 4.
In the reaction scheme above, the efficient hyperpolarization transfer from p-H2-derived hydrides to the 13C nuclear spin of [1-13C]pyruvate was attained by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) using a solution mixture of [IrCl(H)2(DMSO)2(F-IMes)], [1-13C]pyruvate) and p-H2 in deuterated methanol.
The SABRE samples were prepared in CD3OD, using 20 mM sodium [1-13C]pyruvate, 7.6 mM perfluorinated SABRE catalyst shown in Scheme 4, and 50 mM dimethyl sulfoxide (DMSO), as described in Examples 6 and 7. The SABRE samples were exposed to the SABRE-SHEATH hyperpolarization conditions with the same set-up and optimum conditions described in Examples 6 and 7. The NMR tubes were pressurized (110 psi, i.e., approximately 8 bar total pressure) with p-H2 bubbling through the solution at a flow of 90 scc/m to activate the catalyst and to 13C-hyperpolarize the sodium [1-13C]pyruvate solution. Activation of the catalyst took place for about 15 minutes at ambient temperature and magnetic field. For polarization build-up, the sample was placed in the magnetic field (typically about 0.4 μT) and a water bath at 5° C. to regulate the reaction temperature. The spectrum was acquired immediately following manual sample transfer to a 1.8 T benchtop NMR after 5 seconds, and the results are set forth in the top spectrum of
This example illustrates a method of hyperpolarizing a [1-13C]pyruvate in accordance with an aspect of the invention, using a fluorous mixture instead of only deuterated methanol.
The hyperpolarization procedure of Example 6 was repeated using a mixture of nonafluorobutyl methyl ether (NFBME) and deuterated methanol instead of only deuterated methanol.
The fluorinated SABRE catalyst activation took less than 25 minutes, with the 13C polarization percentage shown in
This example demonstrates the effects on hyperpolarization of [1-13C]pyruvate in a fluorous mixture, exhibited by changes in parahydrogen pressure and flow rate, as well as the effect of magnetic transfer field. In addition, the relaxation dynamics of the [1-13C]pyruvate were also studied.
Hyperpolarization of [1-13C]pyruvate was repeated using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH), as described in Example 24 above, and the effects of parahydrogen flow rate, as well as the effect of magnetic transfer field, were studied.
p-H2 flow rate was evaluated and the polarization percentage results are set forth in
The magnetic field in the micro Tesla regime was evaluated and the 13C polarization transfer magnetic field at 0° C. is set forth in
As is apparent from the results set forth in
The simultaneous exchange of p-H2 and [1-13C]pyruvate on activated Ir(F-IMes) catalyst leads to buildup of 13C hyperpolarization. In that respect, the top spectrum of
Temperature has a profound effect on the exchange rates of [1-13C]pyruvate on Complex 3b of
As demonstrated by
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate, which includes biphasic extraction with an aqueous phase and a fluorinated phase.
The hyperpolarization procedure of Example 6 was repeated using a mixture of nonafluorobutyl methyl ether (NFBME) and deuterated methanol instead of only deuterated methanol. The SABRE sample was prepared with 23 mM sodium [1-13C]pyruvate, 7.4 mM perfluorinated SABRE catalyst of Example 4, and 46 mM dimethyl sulfoxide (DMSO) in 0.2 mL NFBME and 0.2 mL MeOD, wherein the mixing field was at 0.4 μT, and the parahydrogen pressure and flow rate were set at 110 PSI and 90 scc/m, respectively.
After the hyperpolarization procedure was completed, the sample was rapidly removed from the 0.40 μT field and transferred inside the NMR spectrometer at 1.8 T at room temperature. The NMR sample was depressurized and 400 μL of heavy water (D2O) was added to the solution in order to drive the hyperpolarized sodium [1-13C]pyruvate into the aqueous phase. The pyruvate signal in the aqueous phase was collected, but the separation led to the formation of an emulsion, indicating that both bound and free pyruvate are present. See
The aqueous phase containing the sodium [1-13C]pyruvate was evacuated and tested for iridium content and pyruvate concentration. The ICP-MS study showed a content of 637 ppb of iridium and the LC-MS showed about 50-75% of the sodium [1-13C]pyruvate concentration was collected after filtration.
The fluorous mixture containing the perfluorinated SABRE catalyst in NFBME and MeOD was re-used for further sodium [1-13C]pyruvate hyperpolarization. After evacuation of the water phase, a solution containing 23 mM sodium [1-13C]pyruvate and 47 mM dimethyl sulfoxide in 0.2 mL CD3OD was added to the fluorous mixture containing the perfluorinated SABRE catalyst from 4 days earlier. The hyperpolarization of sodium [1-13C]pyruvate was repeated, and showed a polarization of the [1-13C] pyruvate with a signal enhancement ε of ˜3090 fold, corresponding to P13C of ˜0.48%, which was obtained via comparison of the NMR signal intensity to a reference sample, as shown in
This example illustrates a synthesis of 1,3-dimesityl-4,5-bis(2-(perfluorophenyl)propyl)-4,5-dihydro-1H-imidazol-3-ium, triflate salt:
which was prepared in accordance with the synthesis sequence set forth in
4-Pentafluorophenylbutanal (3). To a solution of the alcohol (2) (4.8 g, 20.0 mmol) in DCM (40 mL), was added Dess-Martin periodinane (16.96 g, 40.0 mmol) portionwise at 0° C. while being stirred under argon (5 minutes). The reaction was continued until the starting alcohol was consumed (2 hours). The resulting solution was concentrated to about 10.0 mL and adsorbed onto 10.0 g of silica and dried to a free flowing powder. The silica with the crude product was applied to a silica column (120.0 g), and elution with 5% ethyl acetate in hexanes yielded the product as colorless oil. Yield: 3.33 g (70%). 1H NMR (CDCl3): 1H NMR (400 MHz, CDCl3) δ 9.71 (t, J=1.3 Hz, 1H), 2.69 (tt, J=7.9, 1.8 Hz, 2H), 2.44 (td, J=7.3, 1.2 Hz, 2H), 1.86 (p, J=7.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 200.89, 42.81, 21.49.
1,2,3,4,5-Pentafluoro-6-(4-iodobutyl)benzene (4). An ice-cooled solution of the alcohol (2) (16.0 g, 66.67 mmol) was stirred with imidazole (5.89 g, 86.67 mmol) and triphenylphosphine (20.96 g, 80.0 mmol) in DCM (100 mL) under argon. Iodine (20.32 g, 80.0 mmol) was added portionwise over a period of 15 minutes at 0° C. while being vigorously stirred until a slight yellow color persisted. The reaction mixture was diluted with hexanes (300 mL) and filtered. The filtrate was washed with saturated sodium thiosulfate (3×50 mL), water (3×100 mL), and dried with sodium sulfate. The clear solution was filtered and concentrated to an oil that was chromatographed over silica gel (220 g). Elution with hexanes yielded compound (4) as colorless oil. Yield: 21.0 g (90%). 1H NMR (400 MHz, cdcl3) δ 3.20 (t, J=6.8 Hz, 1H), 2.73 (tt, J=7.5, 1.8 Hz, 1H), 1.86 (dq, J=8.6, 6.8 Hz, 1H), 1.76-1.66 (m, 1H). 13C NMR (101 MHz, cdcl3) δ 37.84, 30.01, 21.27, 5.39.
Iodo-(4-(pentafluorophenyl)butyl)triphenyl-λ5-phosphane (5). A solution of the iodobutane (4) (21.0 g, 60.0 mmol) and triphenylphosphine (17.29 g, 66.0 mmol) was refluxed under argon for 24 hours. The precipitated solid was filtered and washed with anhydrous ether and dried under high vacuum for 20 hours. Yield: 33.78 g (92%). 1H NMR (400 MHz, CDCl3) δ 7.73 (m, 15H), 3.92, 3.86 (m, 2H), 2.76, 2.74 (m, 2H), 2.07, 2.025 (m, 2H), 1.6 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 135.23, 135.20, 133.75, 133.65, 130.65, 130.53, 118.32, 117.46, 29.43, 29.27, 23.05, 22.55, 21.78, 21.74, 21.66.
Compound 6. To a solution of the phosphonium salt (5) (6.79 g, 11 mmol) in anhydrous THF (40 mL) was added potassium tert-butoxide in THF (2M, 12.5 mL, 25.0 mmol) dropwise and stirred under argon for 30 minutes. Aldehyde (3) (2.2 g, 9.24 mmol) in THF (5 mL) was added dropwise at room temperature and stirred for 3 hours. The solution was concentrated, and purified on flash silica (120 g). Elution with hexanes yielded the product as a colorless oil as mixture of cis/trans isomers (90:10). Yield: 3.2 g (72%). 1H NMR (400 MHz, CDCl3) δ 5.41 (m, 2H), 2.72, 2.68 (m, 4H), 2.12, 2.10, 2.06 (m, 4H), 1.64 (m, 4H).13C NMR (101 MHz, CDCl3) δ 129.46, 29.12, 26.76, 21.94
Compound 7. Olefin (6) (3.6 g, 8.1 mmol) in acetone (20 mL) and water (0.5 mL) was cooled to 0° C., and dibromamine-T (2.93 g, 8.91 mmol, Org. Biomol. Chem., 8, 1424-1430 (2010)) was added with vigorous stirring. After the addition, the solution was brought to room temperature and stirred until the starting material disappeared (30 minutes). The solution was quenched with solid sodium thiosulfate (2.0 g) and stirred until the color of bromine was completely discharged. The mixture was concentrated under reduced pressure, and the residue was diluted with water (50 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was chromatographed over flash silica (80 g) and eluted with 10% EtOAc in hexanes to yield the bromohydrin (7) as mixture of diastereomers and as colorless oil. Yield: 2.41 g (55%). 1H NMR (400 MHz), CDCl3) δ 4.02 (m, 1H), 3.48 (m, 1H), 2.73 (4H, CH2—C6F5), 1.6˜2.0 (m, 9H, —CH2— and OH)). 13C NMR (101 MHz, CDCl3) δ 143.76, 138.35, 129.62, 126.82, 59.74, 57.12, 35.29, 34.50, 27.39, 25.48, 21.74, 21.34.
Compound 8. The bromohydrin (7) (2.2 g, 4.06 mmol) was dissolved in DCM (10.0 mL) and stirred with 5.0 g of molecular sieves. Dess-Martin reagent (3.44 g, 8.12 mmol) was added and stirred until the starting material was completely consumed (30 minutes). The whole mixture was adsorbed onto flash silica (20.0 g) and dried to a free flowing powder. The silica with the crude product was loaded onto a flash silica (80.0 g) column and eluted with 0 to 20% EtOAc in hexanes over 20 minutes (80.0 mE/min; elution rate). Bromoketone (8) was eluted at 5% EtOAc in hexanes as a colorless syrup. Yield: 1.82 g (83%). 1H NMR (400 MHz, CDCl3) δ 4.25 (dd, J=8.3, 6.0 Hz, 1H), 2.94˜2.81 (m, 1H), 2.80-2.68 (m, 4H), 2.67˜2.52 (m, 1H), 2.09-1.86 (m, 4H), 1.86-1.59 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 202.53, 52.19, 38.18, 32.31, 26.87, 23.21, 21.62, 21.45.
Compound 9. Sodium bicarbonate (0.56 g, 6.68 mmol) was added to a stirred solution of bromoketone (8) (1.8 g, 3.34 mmol) in anhydrous acetonitrile (10.0 mL). Trimethylaniline formamidine (0.94 g, 3.34 mmol) was then added to the stirred solution as a solid, and the reaction mixture was heated to 60° C. with the exclusion of moisture. After the consumption of the bromoketone (70 hours), the reaction mixture was filtered, concentrated under reduced pressure, and chromatographed over flash silica (80 g). Elution with 20% DCM in hexanes yielded the ketoamidine (10) as a colorless syrup. Yield: 1.2 g (49%). 1H NMR 6 (CDCl3) δ.91 9s, 1H), 6.81 (s, 1H), 6.71 (s, 1H), 6.68 (s, 2H), 4.53 (m, 1H), 3.08 (m, 1H), 2.57 (m, 2 h), 2.43 (m, 2H), 2.35 (m, 2H), 2.36 (s, 3 h), 2.2 (s, 3H), 2.13 (s, 3H), 2.01 (s, 3H), 1.92 (s, 6H), 1.8 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 208.19, 151.90, 138.19, 131.45, 129.47, 128.74, 64.33, 60.38, 41.49, 28.39, 25.50, 22.96, 20.56, 18.44, 18.41, 18.23, 14.18. MS: 739.1 [M+H].
Compound 11 (triflate salt). To an ice-cooled solution of the keto amidine (0.994 g, 1.35 mmol) in ethanol, lithium borohydride (2M in THF, 0.675 ml, 1.35 mmol) was added and stirred under argon for 24 h at 0-5° C. The reaction mixture was poured into 50 ml of water and extracted with ethyl acetate (3×50 ml). The combined organic layers were dried (sodium sulfate), and filtered, and concentrated to a paste at room temperature. The above paste was dissolved in 5.0 ml of anhydrous benzene and cooled to 0° C. DIPEA (0.523 g, 4.05 mmol) was added and stirred under argon. Trifluoromethanesulfonic anhydride (1.104 g, 1.34 mmol) was then added dropwise and stirring was continued for 1 h. LC/MS indicated the complete consumption of peak corresponding to 741. The mixture was adsorbed onto 10.0 g of silica gel and dried to a free flowing powder. The silica with the crude product was loaded onto an empty loading column placed on the top of a 80.0 g silica column and chromatographed. Elution with 4% MeOH in DCM yielded the product as a mixture of cis/trans isomers and as brown paste. Yield: 0.338 g (33%). 1H NMR (CDCl3) δ 8.82 (s, 1H), 6.93 (s, 4H), 4.15 (m, 2H), 2.61 (m, 4H), 2.25, 2.23, 2.21 (3S, 18H), 1.77 (m, 4H), 1.41 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 159.41, 141.00, 135.66, 134.43, 130.60, 129.96, 128.58, 68.76, 66.17, 32.48, 26.91, 24.65, 21.71, 20.97, 18.38, 18.11. MS: 723.0 [M+H].
This example illustrates the synthesis of 1,3-dimesityl-4,5-bis(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-imidazol-3-ium triflate:
A flask was charged with Ph3P (7.64 g, 29.13 mmol), ICH2CH2C6F13 (1, 12.40 g, 26.16 mmol) and dimethylformamide (DMF) (15 ml). The mixture was stirred vigorously for 24 hours at 105° C. The DMF was removed by vacuum and the waxy solid was triturated with diethyl ether (100 ml), collected by filtration, washed with diethyl ether, and dried by vacuum to give phosphonium salt (2) as a white solid (16.95 g, 88%) [Rocaboy et al. (J. Phys. Org. Chem., 13: 596-603 (2000))].
A round-bottomed flask was charged with 2-benzyloxybenzaldehyde (3, 7.5 g, 50.0 mmol), phosphonium salt 2 (44.0 g, 60.0 mmol), K2CO3 (10.0 g, 72.4 mmol), reagent-grade 1,4-dioxane (100 ml), and water (2.0 ml) and fitted with a condenser (no inert atmosphere). The mixture was stirred at 95° C. for 20 hours. The volatiles were removed by reduced pressure, and CH2Cl2 (200 ml) and water (50 ml) were added to the orange residue. The layers were separated ad the aqueous layer was extracted with CH2Cl2 (3×50 ml) and the CH2Cl2 layers were combined and dried (MgSO4). The solvent was removed by reduced pressure and the oily solid was placed over 100.0 g of silica gel plug and eluted with hexanes. The solvent was removed by reduced pressure to give the olefin (4) as colorless oil (17.0 g, 88%, 90:10 Z/E). 1H NMR (400 MHz, CDCl3) δ 7.41-7.28 (m, 5H), 6.02-5.93 (m, 1H), 5.64 (dt, J=9.5, 7.3 Hz, 1H), 4.52 (s, 2H), 4.09 (dd, J=6.3, 1.6 Hz, 2H), 2.99˜2.82 (m, 2H).
Olefin 4 (17.0 g) was dissolved in methanol (100.0 ml), and palladium on carbon (10%, 50% by weight water, 3.4 g) was added and hydrogenated at 60 psi for 24 hours. The reaction mixture was filtered through a pad of celite and concentrated to yield the alcohol (5) as a colorless oil (14.0 g, 82%) [Tetrahedron Letters, 43, 6141-6143 (2002)].
The alcohol 5 (13.5 g, 34.4 mmol), triphenylphosphine (10.8 g, 41.3 mmol), and imidazole (3.52 g, 51.6 mmol) were dissolved in anhydrous dichloromethane (100.0 ml) and cooled in an ice bath. Iodine (10.5 g, 41.3 mmol) was added as a solid in portions with stirring over 30 minutes. Additional iodine (0.1 equiv.) was added until a pink color persisted. The solution was quenched with saturated sodium dithionite (100.0 ml) and the organic layer was separated. The aqueous layer was extracted with dichloromethane (3×50.0 ml), and the combined organic layer was washed with water, and dried (Na2SO4). The solution was filtered, concentrated under reduced pressure, and the residue was purified by silica gel column. Elution with hexanes yielded the iodide (6) as colorless oil (13.2 g, 76.4%). Loss of iodine was noticed while on standing at RT, and the iodide was taken to next step immediately. 1H NMR (400 MHz, CDCl3) δ 3.20 (t, J=6.8 Hz, 2H), 2.19-1.85 (m, 2H), 1.75 (ddd, J=15.6, 9.7, 6.0 Hz, 2H).
The iodide 6 (2.9, 5.78 mmol) and triphenylphosphine (1.57 g, 6.0 mmol) were dissolved in anhydrous toluene (25.0 ml) and refluxed for 48 hours under argon. The solution was cooled and concentration under reduced pressure yielded a gum that was triturated with 250 mL of anhydrous ether. The precipitated white solid was filtered and washed with ether and dried under vacuum to yield the phosphonium salt (7) as colorless solid (4.4 g, 99%). 1H NMR (400 MHz, CDCl3) δ 3.20 (t, J=6.8 Hz, 2H), 2.1 (m, 2H), 1.92 (dt, J=14.5, 7.0 Hz, 2H), 1.75 (tt, J=10.8, 6.2 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 135.23, 133.71, 130.65, 130.53, 118.32, 117.46, 114.10, 29.43, 29.26, 23.05, 22.55, 21.78, 21.74, 21.66.
A solution of the alcohol 5 (1.176 g, 3.0 mmol) and triethylamine (1.52 g, 15.0 mmol) was cooled to 0° C. DMSO (2.34 g, 30.0 mmol) was added and stirred, and pyridine-sulfur trioxide complex (1.52 g, 15.0 mmol) was then added in portions over 5 minutes. The resulting mixture was stirred at 0° C. until the alcohol was completely consumed (1 hour). The mixture was diluted with 30 ml of DCM and washed with 2×20 ml of water followed by saturated sodium chloride, dried (potassium carbonate), filtered, concentrated under reduced pressure at room temperature, and the residue was chromatographed over 40 g of flash silica. Elution with 10% ethyl acetate in hexanes yielded the aldehyde (8) as a colorless oil (0.72 g, 61.54%). 1H NMR (400 MHz, CDCl3) δ 9.80 (t, J=1.0 Hz, 1H), 2.61 (td, J=7.0, 1.0 Hz, 2H), 2.22˜2.03 (m, 4H), 2.03-1.90 (m, 2H).
A suspension of the phosphonium salt 7 (1.693 g, 2.215 mmol) in THF (10.0 ml) was cooled to −40° C., and potassium-t-butoxide in THF (1 M solution, 2.4 mL) was added dropwise and stirred until the solids went into solution to give an orange color. The solution was warmed to 0° C. and stirred for 30 minutes. The aldehyde 8 (0.72 g, 1.845 mmol) was then added through a cannula under a positive pressure of argon, and after the addition, the temperature was raised to 50° C., and the reaction was continued under argon for 24 hours. The reaction was quenched with 5.0 ml of saturated ammonium chloride and extracted with 3×30 ml of EtOAc. The combined organic layers were washed with water, saturated sodium chloride, and dried (MgSO4). The resulting residue was filtered, and concentrated under reduced pressure, and purified on a 40.0 g of flash column. Elution with hexanes yielded the olefin (9) as mixture of cis/trans isomers as colorless oil (0.828 g, 60%). 1H NMR (400 MHz, CDCl3) δ 5.4 (m, 2H), 2.61 (m, 2.09, 8H), 1.70 (m, 4H).
To a solution of the olefin 9 (804 mg, 1.074 mmol) in water and THF (6 ml, 1:3), N-Bromosuccinimide (crystallized, 385.2 mg, 2.15 mmol) was added in portions over 10 minutes and stirred at room temperature for 2 hours. TLC indicated the complete disappearance of olefin. The mixture was quenched with 5.0 ml of sodium bisulfite and extracted with 2×20 mL of EtOAc, washed with water, and dried (sodium sulfate). The solution was filtered, concentrated, and the residue was chromatographed over 24 g of silica. Elution with 1:1 hexanes/DCM yielded the bromohydrin (10) as a mixture of diastereomers (612.0 mg, 67.4%). 1H NMR (400 MHz, CDCl3) δ 4.03 (s, OH), 3.95 (dd, J=9.2, 4.0 Hz, 1H), 3.69 (s, OH), 3.47 (d, J=7.6 Hz, 1H), 2.13-1.53 (m, 11H). 19F NMR (376 MHz, CDCl3) δ-80.85 (t, J=10.0 Hz), −113.91-−114.73 (m), −121.95 (p, J=13.5 Hz), −122.74-−123.08 (m), −123.56 (dq, J=23.6, 11.3 Hz), −126.18 (td, J=14.7, 6.2 Hz). 13C NMR (101 MHz, CDCl3) δ 115.74, 77.18, 73.49, 63.16, 34.90, 34.86, 30.86, 30.64, 30.42, 30.21, 29.99, 18.92, 17.77.
To an ice-cooled solution of bromohydrin 10 (612.0 mg, 0.724 mmol) in DCM (10.0 ml), Dess-Martin reagent (460.7 mg, 1.086 mmol) was added in portions with stirring over 5 minutes. The solution was stirred at room temperature until all the alcohol was consumed (TLC, 1 hour). The reaction mixture was diluted with 25.0 mL of EtOAc and washed with 3×25 ml of saturated sodium bicarbonate. The organic layer was dried (sodium sulfate), filtered, and concentrated to a paste. The crude mixture was purified by flash column chromatography (40.0 g). Elution with 30% DCM in hexanes yielded bromoketone (11) as a colorless gum (440.0 mg, 70%). 1H NMR (400 MHz, CDCl3) δ 4.19 (dd, J=8.3, 5.9 Hz, 1H), 3.00-2.84 (m, 1H), 2.71˜2.58 (m, 1H), 2.15-1.86 (m, 8H), 1.78 (tdd, J=15.7, 8.4, 4.9 Hz, 1H), 1.68-1.52 (m, 1H). 19F NMR (376 MHz, cdcl3) δ-80.93, −114.31, −121.95, −122.88, −123.59, −126.26. 13C NMR (101 MHz, cdcl3) δ 202.15, 51.89, 37.92, 32.27, 30.48, 30.26, 30.03, 29.94, 29.72, 29.50, 18.41, 18.37, 14.86, 14.82, 14.77.
To a solution of the bromoketone 11 (440.0 mg, 0.522 mmol) in acetonitrile (5.0 mL), N,N′-dimesitylformamidine was added as a solid (176.0 mg, 0.63 mmol) followed by NaHCO3(132.0 mg, 1.57 mmol). The solution was heated with stirring at 60° C. under argon for 24 hours. The mixture was filtered, washed with 3×10 mL of EtOAc, and the filtrates were concentrated and purified by column chromatography (40.0 g of flash silica). Elution with 1:1 hexanes/DCM yielded amidino ketone (12) as a colorless gum. Yield: 138.2 mg (25.4%). 1H NMR (400 MHz, cdcl3) δ 7.06 (s, 1H), 6.94 (s, 1H), 4.65 (s, 1H), 3.30 (s, 1H), 2.66˜2.49 (m, 1H), 2.28 (s, 3H), 2.20 (s, 3H), 2.15 (s, 3H), 2.07 (s, 6H), 1.93 (d, J=8.1 Hz, 3H), 1.46 (d, J=17.0 Hz, 3H). 19F NMR (376 MHz, cdcl3) δ-80.64-−81.09 (m), −114.35, −114.72, −122.06, −122.98, −123.66 (d, J=107.9 Hz), −126.22. 13C NMR (101 MHz, cdcl3) δ 207.98, 152.08, 138.26, 131.57, 129.71, 128.86, 128.47, 77.31, 77.19, 76.99, 76.67, 64.30, 41.52, 29.68, 28.74, 20.52, 18.52, 18.50, 18.28, 16.95, 14.29. M>S.: [M+H] 1043.0.
To a solution of the amidino ketone 12 (100.0 mg, 0.1 mmol) in anhydrous benzene (0.5 ml) and diisopropylethylamine (65.0 mg, 0.5 mmol) was added and cooled in ice. Trifluoromethanesulfonic anhydride (142.0 mg, 0.5 mmol) was added and stirred under argon. The reaction mixture was allowed to warm to room temperature and stirred overnight (20 hours). The solution was adsorbed on to 5.0 g of flash silica and dried to a free-flowing powder under vacuum. The silica with the crude product was loaded on to a flash silica column (40.0 g) and eluted with 2% methanol in dichloromethane to yield the product as a brownish gum. The gum was triturated with isopropyl ether (5.0 mL) to yield an off-white solid that was filtered and washed with the same solvent (3×5 0.0 mL). The collected solid was dried under vacuum to yield the product (13) as an off-white powder (triflate salt) (26.4 mg, 25%). 1H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 7.11 (s, 4H), 2.70-2.62 (m, 4H), 2.39 (s, 6H), 2.37˜2.21 (m, 2H), 2.11 (s, 12H), 2.08-1.97 (m, 2H), 1.74-1.63 (m, 4H). 19F NMR (376 MHz, CDCl3) δ-78.85, −80.91, −114.11, −122.05, −122.99, −123.49, −126.33. 13C NMR (101 MHz, CDCl3) δ 142.03, 137.59, 134.28, 131.81, 130.28, 128.13, 30.26, 30.04, 29.82, 22.55, 21.13, 19.71, 17.58, 17.36. M. S: [M+H] 1025.0.
This example illustrates the synthesis of 1,3-dimesityl-4,5-bis(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-imidazol-3-ium chloride:
1,3-dimesityl-4,5-bis(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-imidazol-3-ium triflate (100 mg, 94.2 μmol) from Example 28 was dissolved in MeOH (1.5 mL) and passed through a short column of ion exchange resin Amberlite 400. The column was washed with MeOH until no spot was visible via TLC under UV. The solvent was removed, and the resulting yellowish solid was dried with a vacuum pump to yield product (0.1 g, 99%). MS (ESI) m/z (%): 999 [M+H]+(100).
This example illustrates a method of synthesis of a fluorinated SABRE catalyst containing a transition metal in accordance with an aspect of the invention.
Potassium tert-butoxide (112 mg, 1.00 mmol, 2.5 eq.) was added to a stirred solution of 1,3-dimesityl-4,5-bis(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)-1H-imidazol-3-ium chloride (100 mg, 94.2 μmol, 2.2 eq) from Example 29 in tetrahydrofuran (10 mL) at room temperature in a glove box. The resulting suspension was stirred for 30 minutes. A solution of [Ir(COD)C1]2 (28.8 mg, 42.8 μmol, 1.0 eq.) was added and the resulting solution was stirred at room temperature overnight (Cowley et al., J. Am. Chem. Soc., 133, 6134-6137 (2011)). The solvent was removed under reduced pressure to give the crude product and dried overnight under vacuum. The solvent was removed under reduced pressure to give the crude product and dried overnight under vacuum. This sample was purified with flash chromatography DCM/hexane (4:1). MS (ESI) m/z (%): 1325 [M-Cl]+(100).
This example illustrates a method of hyperpolarizing a [1-13C]pyruvate in a mixture of deuterated or non-deuterated methanol and fluorinated solvent (Novec™ 7100 fluid) in accordance with an aspect of the invention, as shown in Scheme 6.
In the reaction scheme above, hyperpolarization of [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) (Theis et al., J Am. Chem. Soc., 137, 1404-1407 (2015) and Truong et al., J Phys. Chem. C, 119, 8786-8797 (2015)) tailored for the 13C nucleus (Barskiy et al., ChemPhysChem, 18, 1493-1498 (2017)) using the co-ligand approach developed by Duckett and co-workers (Iali et al., Angew. Chemie —Int. Ed., 58, 10271-10275 (2019)) in combination with a fluorinated solvent. Sodium [1-13C]-pyruvate and deuterated methanol-d4 solvent were purchased from Sigma-Aldrich and the non-deuterated solvent 7100 fluid was purchased from Novec™. The compounds were used after being extensively purged from oxygen by bubbling Argon for 10 minutes and then further degassed by 3 cycles of freeze-thaw (FTC). The [IrCl(COD)(F-IMes)] SABRE catalyst precursor was synthesized from F-IMes, which was prepared according to Example 4. The active catalyst was prepared with a substrate [1-13C]-pyruvate in 0.2 mL of methanol-d4, Ir(F-IMes) SABRE pre-catalyst in 0.3 mL Novec™ 7100 fluid, and a dimethyl sulfoxide (DMSO) co-ligand in a 5 mm NMR tube.
The method of hyperpolarizing set forth in Example 6 was repeated except where indicated below. In particular, the hyperpolarization transfer from p-H2-derived Iridium hydrides to the 13C nuclear spin of [1-13C]pyruvate was attained by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) using a solution mixture of [IrCl(H)2(DMSO)2(F-IMes)], [1-13C]pyruvate, co-ligand (dimethyl sulfoxide) and p-H2 in a mixture of deuterated or non-deuterated methanol and Novec™ 7100 fluid.
The fluorinated SABRE catalyst activation took less than 15 minutes, with the 13C polarization percentage shown in
Optimization of p-H2 flow rate was evaluated and is presented in
Optimization of the temperature and magnetic field in micro-Tesla regime were evaluated and the results are set forth in
The relaxation dynamics are set forth in
Optimization of the co-ligand (i.e., dimethyl sulfoxide (DMSO)) concentration was evaluated and the results are set forth in
The exchange of p-H2 and [1-13C]pyruvate on activated Ir F-IMes catalyst leads to buildup of 13C hyperpolarization, as shown in
This example shows that the hyperpolarization of SABRE can be improved with fluorous solvents such as Novec™ 7100 fluid. Without wishing to be bound by any particular theory, it is believed that the increase in polarization may be due to improved parahydrogen hydrogen solubility in the fluorous solvent.
This example illustrates a method of hyperpolarizing a [1-13C]pyruvate in a mixture of deuterated or non-deuterated methanol and fluorinated solvent (Novec 7100 fluid) in combination with phenyl trifluoromethyl sulfoxide as a co-ligand in accordance with an aspect of the invention, as shown in Scheme 7.
In the reaction scheme above, hyperpolarization of [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) (Theis et al., J. Am. Chem. Soc., 137, 1404-1407 (2015) and Truong et al., J. Phys. Chem. C, 119, 8786-8797 (2015)) tailored for the 13C nucleus (Barskiy et al., ChemPhysChem, 18, 1493-1498 (2017)) using the co-ligand approach developed by Duckett and co-workers (Iali et al., Angew. Chemie —Int. Ed., 58, 10271-10275 (2019)) in combination with a fluorinated solvent. Sodium [1-13C]-pyruvate and deuterated methanol-d4 solvent were purchased from Sigma-Aldrich and the non-deuterated solvent 7100 fluid was purchased from Novec™. The compounds were used after being extensively purged from oxygen by 3 cycles of freeze-thaw (FTC) and then further degassed by bubbling Argon for 10 minutes. The [IrCl(COD)(F-IMes)] SABRE catalyst precursor was synthesized from F-IMes, which was prepared according to Example 4. The active catalyst was prepared with a substrate [1-13C]-pyruvate in 0.2 mL of methanol-d4, Ir(F-IMes) SABRE pre-catalyst in 0.3 mL Novec™ 710fid and a phenyl trifluoromethyl sulfoxide (PTMSO) co-ligand in a 5 mm NMR tube. The method of hyperpolarizing set forth in Example 6 was repeated except where indicated below. In particular, the hyperpolarization transfer from p-H2-derived Iridium hydrides to the 13C nuclear spin of [1_13C]pyruvate was attained by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) using a solution mixture of [IrCl(H)2(PTMSO)2(F-IMes)], [1-13C]pyruvate, co-ligand (phenyl trifluoromethyl sulfoxide) and p-H2 in a mixture of deuterated or non-deuterated methanol and Novec™ 7100 fluid.
The fluorinated SABRE catalyst activation took less than 15 minutes, with the 13C polarization percentage shown in
Optimization of p-H2 flow rate was evaluated and is presented in
Optimization of the temperature and was evaluated and the results are set forth in
The temperature sweep provided in
The relaxation dynamics are set forth in
Optimization of the co-ligand (i.e., phenyl trifluoromethyl sulfoxide (PTMSO)) concentration was evaluated and the results are set forth in
The exchange of p-H2 and [1-13C]pyruvate on activated Ir F-IMes catalyst leads to buildup of 13C hyperpolarization, as shown in
The PTMSO-based Ir F-IMes catalyst of this example increased solubility of the perfluorinated SABRE catalyst in fluorinated solvent leading to a reduction in the iridium content in the aqueous phase. Without wishing to be bound by any particular theory, it is believed that other fluorinated DMSO derivatives such as 2,2,2-trifluoroethyl methane sulfonate or di-2,2,2-trifluoroethyl sulfoxide, for example, could provide beneficial properties such as reduced iridium content in the aqueous phase.
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate using DMSO as a co-ligand, which includes extraction and filtration.
Hyperpolarization of sodium [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) tailored for the 13C nucleus using dimethyl sulfoxide (DMSO) as a co-ligand, [IrCl(COD)(F-IMes)] SABRE catalyst precursor, and parahydrogen enriched to about 70 to 95% in deuterated methanol-d4 solvent and Novec™ 7100, as described in Examples 31 and 32. The optimum parameters described previously in Examples 31 and 32, such as temperature, transfer field, para-hydrogen flow, and pressure, were used in this example. The SABRE samples consisted in 35 mM sodium [1-13C]pyruvate in 0.3 mL CD3OD, 6 mM fluorinated [IrCl(COD)(F-IMes)] SABRE catalyst precursor, which was prepared according to Example 4, and 56 mM dimethyl sulfoxide (DMSO) in 0.2 mL Novec™ 7100, the para-hydrogen flow was established at 90 ss/m and pressurized at 8 bars, and the mixing field was at 0.4 μT and a temperature of 0° C.
After the hyperpolarization procedure was completed, the pyruvate hyperpolarization was measured at 13.6%, and the sample was rapidly removed from the 0.40 μT field and depressurized. Heavy water (D2O) (200 μL) was added to the mixture, thereby resulting in the formation of an emulsion. The hyperpolarized pyruvate in solution was measure at 5.9%. The emulsion was transferred to a 1 mL plastic syringe held mounted to a Luer-locked filter (Waters Oasis Prime HLB Plus Light Cartridge (Part Number: 186008866) in order to break the emulsion. The aqueous solution was guided through the filter into a 5 mm NMR tube, already located into the adjacent 1.8 T benchtop NMR spectrometer, and the pyruvate hyperpolarization was measured at 0.34%. The whole procedure took about 1.30 minutes.
Inspired by these results, an alternative approach was explored which involved the rapid evaporation of methanol [Ding et al. (Chemistry-Methods, 2 (7), (2022)) and de Maissin et al. (Chemie Int. Ed., 62 (36), (2023))], as depicted in
As is apparent from the results set forth in Table 1, between 57 and 100% of the sodium [1-13C]pyruvate concentration was collected after filtration.
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate using phenyl trifluoromethyl sulfoxide (PTMSO) as a co-ligand, which includes extraction and filtration.
Hyperpolarization of sodium [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) tailored for the 13C nucleus using phenyl trifluoromethyl sulfoxide (PTMSO) as a co-ligand, [IrCl(COD)(F-IMes)] SABRE catalyst precursor, and parahydrogen enriched to about 70 to 95% in deuterated methanol-d4 solvent and Novec™ 7100, as described in Examples 31 and 32. The optimum parameters described previously in Examples 31 and 32, such as temperature, transfer field, para-hydrogen flow, and pressure, were used in this example. The SABRE samples consisted in 35 mM sodium [1-13C]pyruvate in 0.3 mL CD3OD, 6 mM fluorinated [IrCl(COD)(F-IMes)] SABRE catalyst precursor, which was prepared according to Example 4, and 56 mM phenyl trifluoromethyl sulfoxide (PTMSO) in 0.2 mL Novec™ 7100, the para-hydrogen flow was established at 90 ss/m and pressurized at 8 bars, and the mixing field was at 0.4 μT and a temperature of 0° C.
The alternative approach of Example 33, which included the rapid evaporation of methanol, was performed and the results are set forth in
As is apparent from the results set forth in Table 2, between 57 and 71% of the sodium [1-13C]pyruvate concentration was collected after filtration.
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate, which includes an extraction with an aqueous phase and a fluorinated phase.
Hyperpolarization of sodium [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) tailored for the 13C nucleus using dimethyl sulfoxide (DMSO) or phenyl trifluoromethyl sulfoxide (PTMSO) as a co-ligand, [IrCl(COD)(F-IMes)] SABRE catalyst precursor, and parahydrogen enriched to about 70 to 95% in deuterated methanol-d4 solvent and Novec™ 7100, as described in Examples 31 and 32. The optimum parameters described previously in Examples 31 and 32, such as temperature, transfer field, para-hydrogen flow, and pressure, were used in this example. The SABRE samples were prepared in a 9 inch NMR tube with 30 mM sodium [1-13C]pyruvate in 0.3 mL CD3OD, 5 mM fluorinated [IrCl(COD)(F-IMes)] SABRE catalyst precursor, which was prepared according to Example 4, and 25 mM dimethyl sulfoxide (DMSO) or 98 mM phenyl trifluoromethyl sulfoxide (PTMSO) in 0.3 mL Novec™ 7100, the para-hydrogen flow was pressurized at 8 bars, the para-hydrogen flow was set at 20 ss/m for activation and then at 90 ss/m for hyperpolarization, and the mixing field was at 0.4 μT and a temperature of 0° C. for DMSO or room temperature for PTMSO.
After the hyperpolarization procedure was completed, the sample was rapidly removed from the 0.4 μT field and transferred inside the NMR spectrometer at 1.8 T at room temperature. The NMR sample was depressurized and 300 μL of heavy water (D2O) was added to the solution in order to drive the hyperpolarized sodium [1-13C]pyruvate into the aqueous phase and keep the perfluorinated SABRE catalyst in the organic fluorinated phase. The procedure is depicted in
Signal enhancement in the water phase was measured at ε˜39300 fold and ˜25700 fold, corresponding to P13C of ˜6.13% (down from its initial level of 6.8%) and 4% (down from its initial level of 8.9%), for DMSO and PTMSO, respectively. Those values were obtained via comparison of the NMR signal intensity, as shown in
The aqueous phase containing the sodium [1-13C]pyruvate was evacuated and tested for iridium content (ICP-MS) and pyruvate concentration (LC-MS). The ICP-MS results showed a content of between 310 and 786 ppb iridium in all samples tested. The LC-MS and ICP-MS results for the concentration of pyruvate, methanol, DMSO or PTMSO, and Novec™ 7100, and iridium content in six separate samples are set forth in Table 3.
As is apparent from the results set forth in Table 3, between 66 and 10000 of the sodium [1-13C]pyruvate concentration was collected after filtration, with 100% of the sodium [1-13C]pyruvate concentration being collected from the DMSO sample.
This example illustrates an exemplary method for isolating hyperpolarized sodium [1-13C]pyruvate, which includes recycling and reusing the SABRE catalyst.
Hyperpolarization of sodium [1-13C]pyruvate was performed using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) tailored for the 13C nucleus using dimethyl sulfoxide (DMSO) or phenyl trifluoromethyl sulfoxide (PTMSO) as a co-ligand, [IrCl(COD)(F-IMes)] SABRE catalyst precursor, and parahydrogen enriched to about 70 to 95% in deuterated methanol-d4 solvent and Novec™ 7100, as described in Examples 31 and 32. The optimum parameters described previously in Examples 31 and 32, such as temperature, transfer field, para-hydrogen flow, and pressure, were used in this example. The SABRE samples were prepared in a 9 inch NMR tube with 30 mM sodium [1-13C]pyruvate in 0.3 mL CD3OD, 5 mM fluorinated [IrCl(COD)(F-IMes)] SABRE catalyst precursor, which was prepared according to Example 4, and 25 mM dimethyl sulfoxide (DMSO) or 98 mM phenyl trifluoromethyl sulfoxide (PTMSO) in 0.3 mL Novec™ 7100, the para-hydrogen flow was pressurized at 8 bars, the para-hydrogen flow was set at 20 ss/m for activation and then at 90 ss/m for hyperpolarization, and the mixing field was at 0.4 μT and a temperature of 0° C. for DMSO or room temperature for PTMSO.
After the hyperpolarization procedure was completed, the sample was rapidly removed from the 0.4 μT field and transferred inside the NMR spectrometer at 1.8 T at room temperature. The NMR sample was depressurized and 300 μL of heavy water (D2O) was added to the solution in order to drive the hyperpolarized sodium [1-13C]pyruvate into the aqueous phase and keep the perfluorinated SABRE catalyst in the organic fluorinated phase, as described in Example 35.
Given the distinct separation between the fluorous organic and aqueous phases, the aqueous phase was removed. In the case of the reactions containing DMSO, the organic fluorinated portion collected, about 100 μL, represents a small portion of the original volume. Without wishing to be bound by any particular theory, it is believed that in the case of DMSO, most of the methanol fraction is driven into the aqueous phase along with the DMSO excess as it is miscible in both the fluorinated phase and the aqueous phase. In contrast, when using PTMSO as a co-ligand, most (about 250 μL) of the organic phase was recovered.
The perfluorinated catalyst in the fluorous organic phase was kept in the NMR tube and 300 μL deuterated methanol was added and contained 30 mM sodium [1-13C]pyruvate and the co-ligand being either 25 mM DMSO or 98 mM PTMSO. This mixture was then subjected to a second round of polarization in the SABRE-SHEATH polarizer. The procedure is depicted in
The iterative process involving the hyperpolarization of [1-13C]pyruvate and its subsequent extraction in an aqueous phase, followed by the reutilization of the catalyst, was systematically conducted for a total of ten cycles. Impressively, no discernible diminishment in hyperpolarization activity was discerned after the second iteration of catalyst recycling. However, a noteworthy trend emerged during subsequent cycles. By the third recycle, a 39% diminution in [1-13C]pyruvate hyperpolarization in methanol was observed, marking the initial instance of a loss in polarization. Intriguingly, no further decline in hyperpolarization was noted until the sixth cycle, where a substantial 60.8% of the [1-13C]pyruvate polarization was lost.
Remarkably, the polarization remained relatively stable until the tenth cycle, which marked the conclusion of the experimental trials. It is imperative to highlight that the amount of co-ligand added in each cycle was not optimized, potentially contributing to the observed variations in hyperpolarization. Therefore, the potential for optimization persists, and the hyperpolarization for each cycle could be finely tuned by adjusting the precise amount of co-ligand added during the process.
Furthermore, the fluorinated catalyst exhibited a prolonged activity, retaining its efficacy for a duration of at least four days after the initial activation. This longevity suggests a robust and sustained catalytic performance. Consequently, this method presents a convenient and efficient approach, enabling the hyperpolarization of multiple batches of [1-13C]pyruvate using the same initial perfluorinated SABRE catalyst.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments can become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application is a continuation-in-part of International Patent Application No. PCT/US2023/017885, filed Apr. 7, 2023, which claims benefit to U.S. Provisional Patent Application No. 63/328,545, filed Apr. 7, 2022, which are hereby incorporated by reference in their entireties.
This invention was made with Government support and the Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63328545 | Apr 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/017885 | Apr 2023 | WO |
| Child | 18410773 | US |
