Systems and methods for producing hyperpolarized materials - Patents.com

Abstract

This disclosure describes hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications.This disclosure describes methods for producing hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications.This disclosure describes precursor compounds for use in producing hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 202,709, filed June 22, 2021, U.S. Provisional Patent Application No. 63 / 260,631, filed August 27, 2021, and U.S. Provisional Patent Application No. 63 / 260,934, filed September 07, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] The disclosed embodiments relate generally to the production and purification of hyperpolarized materials for use in nuclear magnetic resonance, magnetic resonance imaging, or similar applications. [Background technology]

[0003] Parahydrogen-induced polarization (PHIP) is a low-cost, high-throughput method for polarizing metabolites for hyperpolarized (HP) magnetic resonance imaging (MRI). Parahydrogen-induced polarization with side-arm hydrogenation (PHIP-SAH) can be used to polarize metabolites, such as acetate molecules. However, existing PHIP-SAH polarization approaches may not be suitable for preclinical or clinical HP MRI applications. Summary of the Invention

[0004] In some embodiments, the present disclosure describes a method for producing an administration composition comprising a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof. In some embodiments, the present disclosure describes a method for producing an administration composition comprising a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, the method comprising: obtaining a solution in a container, the solution comprising a first organic solvent, an aqueous mixture, the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm; one or more washing steps, where the solution is washed with a second organic solvent, thereby forming (i) an organic mixture phase comprising the first organic solvent, the second organic solvent, and the optional unbound side arm, and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof; one or more separation steps, where the organic mixture phase is separated from the aqueous mixture phase, and either the organic mixture phase or the aqueous mixture phase is transferred to a separate container; and obtaining the administration composition from the aqueous mixture comprising the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof. In some embodiments, the method further comprises one or more evaporation steps, in which the aqueous mixture phase is subjected to organic vapor extraction conditions to evaporate at least a portion of the organic solvent remaining in the aqueous mixture, hi some embodiments, the organic vapor extraction conditions comprise bubbling with an inert gas, such as nitrogen gas.

[0005] In some embodiments, the present disclosure describes a method for producing an administration composition comprising a hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof, the method comprising: obtaining a solution in a container, the solution comprising a first organic solvent, an aqueous mixture, the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm; one or more evaporation steps, in which the solution is subjected to organic vapor extraction conditions to evaporate a portion of the organic solvent and the optional unbound side arm from the solution, thereby providing an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof; and obtaining the administration composition from the aqueous mixture comprising the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof. In some embodiments, the organic vapor extraction conditions comprise bubbling with an inert gas, such as nitrogen gas. In some embodiments, the method further comprises: one or more washing steps, where the solution or mixture is washed with a second organic solvent, thereby forming (i) an organic mixture phase comprising the first organic solvent, the second organic solvent, and any unbound side arms, and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof; and one or more separation steps, where the organic mixture phase is separated from the aqueous mixture phase, and either the organic mixture phase or the aqueous mixture phase is transferred to a separate container.

[0006] In some embodiments, the disclosed method includes obtaining a solution in a container, the obtaining a solution in the container includes the steps of: obtaining a solution comprising a first organic solvent and a biorelevant imaging agent precursor dissolved in the first organic solvent, the biorelevant imaging agent precursor comprising (i) a biorelevant imaging agent and (ii) a side arm comprising an unsaturated carbon-carbon double bond (-C=C-) or a carbon-carbon triple bond (-C≡C-), hydrogenating the unsaturated carbon-carbon double bond (-C=C-) or the carbon-carbon triple bond (-C≡C-) of the side arm with para-hydrogen in the first organic solvent, thereby forming a para-hydrogenated derivative of the biorelevant imaging agent precursor, and applying a polarization transfer waveform to the biorelevant imaging agent precursor. transferring nuclear spin order from the para-hydrogen on the side arm to the non-hydrogen nuclear spins on the biorelevant imaging agent; optionally hydrolyzing the para-hydrogenated biorelevant imaging agent precursor by adding an aqueous hydrolysis agent to the solution to produce a hyperpolarized biorelevant imaging agent and an unbound side arm; and optionally neutralizing the solution with a buffer to slow or terminate the hydrolysis reaction, thereby producing a solution comprising the first organic solvent, the aqueous mixture, the hyperpolarized biorelevant imaging agent or a pharma- ceutically acceptable salt thereof, and the optional unbound side arm (if the optional hydrolysis step is complete). In some embodiments, obtaining the solution in the container further comprises a catalyst collection step prior to the optional addition of the aqueous hydrolysis agent, the catalyst collection step comprising filtering the solution to remove residual catalyst atoms (e.g., catalyst atoms remaining from the hydrogenation step), such as rhodium atoms, iridium atoms, and / or any other catalyst atoms, from the solution. In some embodiments, the solution is concentrated (eg, by evaporation) prior to the catalyst collection step.

[0007] In some embodiments, the biorelevant imaging agent precursor is a precursor comprising a compound of formula Ia or formula Ib. In some embodiments, the biorelevant imaging agent is selected from pyruvate, glutamate, glutamine, lactate, acetate, acetoacetate, zymonate, alanine, fructose, fumarate, bicarbonate, urea, dehydroascorbate, alpha-ketoglutarate, dihydroxyacetone, glucose, ascorbate, and their conjugate acids. In some embodiments, the biorelevant imaging agent has a solubility in the first organic solvent of less than 50 millimolar (mM). In some embodiments, the biorelevant imaging agent has a solubility in water of more than 50 millimolar (mM).

[0008] In some embodiments, the aqueous mixture comprises water, sodium hydroxide, potassium hydroxide, or any mixture thereof.

[0009] In some embodiments, the first organic solvent comprises acetone, ethanol, methanol, chloroform, ethyl acetate, methyl ethyl ketone (MEK), acetophenone, hexone, cyclohexanone, cyclopentanone, or a combination thereof. In some embodiments, the first organic solvent comprises acetone. In some embodiments, the first organic solvent comprises methyl ethyl ketone (MEK). In some embodiments, the first organic solvent has a solubility in water at 20° C. that is greater than 75 millimolar (mM). In some embodiments, the first organic solvent has a solubility in water at 20° C. that is greater than the solubility of chloroform in water at 20° C. In some embodiments, the first organic solvent does not comprise chloroform.

[0010] In some embodiments, the second organic solvent comprises one or more ICH Class 2 solvents selected from acetonitrile, chlorobenzene, chloroform, cyclohexane, dibromomethane (DBM), 1,2-dichloroethene, dichloromethane (DCM), 1,2-dimethoxyethane, n,n-dimethylacetamide, n,n-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2-methoxyethanol, methylbutylketone, methylcyclohexane, n-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene, 1,1,2-trichloroethene, or xylene. In some embodiments, the second organic solvent comprises one or more ICH class 3 solvents selected from acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, methyl tert-butyl ether (MTBE), cumene, diethyl ether, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate. In some embodiments, the second organic solvent comprises methyl tert-butyl ether (MTBE). In some embodiments, the second organic solvent comprises dibromomethane (DBM). In some embodiments, the second organic solvent comprises dichloromethane (DCM).

[0011] In some embodiments, the washing and / or evaporation steps are repeated until the first organic solvent has a concentration in solution of 5000 ppm or less, hi some embodiments, the washing and / or evaporation steps are repeated until the second organic solvent has a concentration in solution below the ICH toxicity limit (see, e.g., Table 1).

[0012] In some embodiments, the present disclosure describes an administration composition comprising a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof. In some embodiments, the present disclosure describes an administration composition comprising a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, the administration composition being produced by a method of the present disclosure. In some embodiments, the administration composition comprises no more than 20 mM, 10 mM, 5 mM, 2 mM, or 1 mM of a first organic solvent. In some embodiments, the administration composition comprises no more than 20 mM, 10 mM, 5 mM, 2 mM, or 1 mM of a second organic solvent.

[0013] In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container and a second container fluidically connected to the first container. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container and a second container fluidically connected to the first container through a first fluid transfer element. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container, a second container fluidically connected to the first container and a third container fluidically connected to the second container. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container, a second container fluidically connected to the first container through a first fluid transfer element and a third container fluidically connected to the second container through a second fluid transfer element.

[0014] In some embodiments, the system further comprises a magnetic guide system. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over at least a portion of the system. In some embodiments, the system further comprises a magnetic guide system comprising one or more solenoid valves providing the defined magnetic field. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over the first vessel, the second vessel, and / or the optional third vessel. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over the first fluid transfer element and / or the optional second fluid transfer element. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over the entire system. In some embodiments, the defined magnetic field prevents undesired hyperpolarization loss in the solution during implementation of the disclosed method. [Brief description of the drawings]

[0015] The accompanying drawings, which form a part of this specification, illustrate several embodiments and, together with the description, serve to explain certain principles and features of the disclosed embodiments. [Figure 1] 1 illustrates an embodiment 100 of a method for producing an administration composition comprising a hyperpolarized biologically relevant imaging agent according to the present disclosure. [Diagram 2] 2 illustrates a method embodiment 200 for producing an administration composition including a hyperpolarized biologically relevant imaging agent according to the present disclosure. [Diagram 3] 1 illustrates an embodiment 300 of a system for generating an administration composition including a hyperpolarized biologically relevant imaging agent according to the present disclosure. [Figure 4] 4 illustrates an embodiment 400 of a system for generating an administration composition including a hyperpolarized biologically relevant imaging agent according to the present disclosure. [Diagram 5] 5 illustrates an embodiment 500 of a system for generating an administration composition including a hyperpolarized biologically relevant imaging agent according to the present disclosure. [Figure 6]1 shows NMR spectrum results of SABRE catalyst in an acetone extraction test, according to certain embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Exemplary embodiments will now be described in detail and discussed with reference to the accompanying drawings. Unless otherwise defined, technical and / or scientific terms have the meanings commonly understood by those skilled in the art. The disclosed embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosed embodiments. It will be understood that other embodiments may be utilized and changes may be made without departing from the scope of the disclosed embodiments. Thus, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0017] Recent research in the fields of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) has demonstrated that NMR and MRI signals associated with various biorelevant contrast agents can be enhanced by orders of magnitude using various so-called hyperpolarization techniques. This signal enhancement allows for improved spectroscopic analysis of the biorelevant contrast agents as they are metabolized by various tissues at different locations in the body. Analysis of metabolic information determined by such spectroscopic imaging can allow for non-invasive determination of the health of tissues in the body. For example, abnormal metabolism of a biorelevant contrast agent can indicate disease, such as cancer, at several locations in the body.

[0018] Existing techniques for hyperpolarized biorelevant contrast agents include dissolution dynamic nuclear polarization (DNP), parahydrogen induced polarization (PHIP), PHIP sidearm hydrogenation (PHIP-SAH), and signal amplification by reversible exchange (SABRE). In PHIP and PHIP-SAH, a precursor of the biorelevant contrast agent is reacted with parahydrogen to form a parahydrogenated derivative of the precursor. Spin order is then transferred from the added proton via the parahydrogenation reaction to the nucleus of interest (such as the carbon-13 nucleus) contained within the biorelevant contrast agent. In PHIP-SAH, the parahydrogenated derivative of the precursor is cleaved (e.g., hydrolyzed) to obtain a hyperpolarized biorelevant contrast agent. The biorelevant contrast agent is then purified and used in an NMR or MRI procedure. In PHIP-SAH, the precursor can include a biorelevant contrast agent attached to a sidearm that contains at least one unsaturated bond (e.g., at least one carbon-carbon double bond or at least one carbon-carbon triple bond) suitable for reaction with parahydrogen. In SABRE, the biorelevant contrast agent itself is directly hyperpolarized (i.e., without the need for a parahydrogenation reaction) via the transient formation of a complex between the biorelevant contrast agent, parahydrogen, and a polarization transfer catalyst. However, previous methods for producing biorelevant contrast agents have resulted in polarization levels or concentrations that are insufficient for clinical relevance (e.g., when performing all or part of the SABRE, PHIP, and / or PHIP-SAH processes in aqueous solutions) or place the biorelevant contrast agent in a mixture that contains excessive concentrations of harmful organic solvents (e.g., when performing all or part of the SABRE, PHIP, and / or PHIP-SAH processes in organic solvents). Thus, there is a need for new methods for producing biorelevant contrast agents that achieve clinically relevant polarization, concentration, volume, and purity.

[0019] The disclosed embodiments include systems and methods for producing biologically relevant imaging agents with clinically relevant polarization, concentration, volume, and purity. The disclosed embodiments provide technical improvements in polarizing biologically relevant imaging agents in solution. These technical improvements support increased biologically relevant imaging agent concentrations and degrees of biologically relevant polarization.

[0020] Hyperpolarization and Parahydrogen As used in this disclosure, hyperpolarization describes a state in which the absolute value of the difference between a population of spin states (e.g., nuclear spin states, proton spin states, etc.) in one state (e.g., spin up) and a population of spin states in another state (e.g., spin down) exceeds the absolute value of the corresponding difference at thermal equilibrium.

[0021] Parahydrogen may be used as a polarization source consistent with disclosed embodiments. Parahydrogen is a form of molecular hydrogen in which two proton spins are in a singlet state, as described herein. The disclosed embodiments are not limited to a particular method of generating parahydrogen. Parahydrogen may be formed in gaseous or liquid form. In some embodiments, parahydrogen is generated in gaseous form by flowing hydrogen gas at low temperature through a chamber with a catalyst (e.g., iron oxide or another suitable catalyst). The hydrogen gas may contain both parahydrogen and orthohydrogen. The low temperature may bring the hydrogen gas to thermodynamic equilibrium in the chamber, increasing the population of parahydrogen.

[0022] The disclosed embodiments are not limited to a particular parahydrogen production or use location. Parahydrogen may be produced at a first location and then transported to a second location for use. In some embodiments, the first location is a chamber that may be part of a container, bottle, holder, or other area that may hold a gas or liquid. Such a chamber may be maintained at a suitable pressure or temperature. In some embodiments, the first location is a physical location such as a room, a laboratory, a particular warehouse, a hospital, or other location where parahydrogen is produced.

[0023] The disclosed embodiments are not limited to a particular parahydrogen transport method. The parahydrogen produced may be transported in a chamber that may be different from the chamber in which the parahydrogen was produced. The chamber that transports the parahydrogen gas may be maintained at a suitable pressure or temperature that allows it to be transported by vehicle or person. Transporting the parahydrogen may involve moving the parahydrogen from one container to a different container. Transporting the parahydrogen may involve moving the parahydrogen within the same location, such as from one part of a room to another part of a room. Transporting the parahydrogen may involve moving the parahydrogen from one room of a building to a different room of the same building, or to a nearby building. Transporting the parahydrogen may involve moving the parahydrogen to a different location in another part of the same city, or to a different city. Transporting the parahydrogen may involve bringing the parahydrogen near a polarizer, an NMR device, or an MRI device. Transporting the parahydrogen may involve packaging or transporting the parahydrogen in a suitable container.

[0024] In some embodiments, the population difference between two spin states is the difference between the populations of the two spin states divided by the total population of the two spin states. The population difference can be expressed as a fractional population difference or a percentage population difference. In some embodiments, the fractional population difference is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more, up to about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less, or within a range defined by any two of the foregoing values.

[0025] Hydrogen gas may exhibit a population difference between proton spin states at thermal equilibrium that significantly exceeds the population difference between the proton spin states. Parahydrogen may have a large population difference between either the singlet spin state and the triplet spin state. In the case of Iz1Iz2, for example, there is a large population difference between the spin states |↑>|↓> and the spin states |↑>|↑>. The population difference of the proton spin states may be at least about 0.1 (e.g., a 10% difference in spin states - 55% of the parahydrogen molecules in a sample are in the singlet state and 45% are in the triplet state), 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more, up to about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less, or within a range defined by any two of the foregoing values.

[0026] Bio-related contrast agents Disclosed embodiments include systems and methods for producing and utilizing biorelevant imaging agents with clinically relevant polarization, concentration, volume, or purity. In some embodiments, the method is for preparing an NMR material. In some embodiments, the NMR material is suitable for use in NMR or MRI operations. In some embodiments, the NMR material increases the NMR or MRI signal and signal-to-noise ratio (SNR). In some embodiments, the NMR material is suitable for use in solution NMR spectroscopy. In some embodiments, the NMR material is a chemical compound. In some embodiments, the NMR material is a metabolite (e.g., a molecule with biorelevance, such as an amino acid, sugar, derivatives thereof), such as a metabolite suitable for use in NMR metabolomics applications. In some embodiments, the NMR material is suitable for in vitro probing of the metabolism of cell cultures or other biological tissues. In some embodiments, the NMR material is used in an NMR probe to investigate transient effects where high signal enhancement due to hyperpolarization is required, such as proton exchange between water and biomolecules. In some embodiments, the NMR material is a small molecule or metabolite suitable for injection into cells, tissues, or organisms for detection in MRI scans. In some embodiments, the NMR material is introduced into the chamber for further analysis by NMR or MRI operations. In some embodiments, the NMR material is introduced into the chamber for further analysis by NMR or MRI operations. 2 H) or carbon-13 ( 13 C) concentrated in atoms.

[0027] Consistent with the disclosed embodiments, the NMR material may include a bio-relevant contrast agent. In some embodiments, the bio-relevant contrast agent may be suitable for use in NMR or MRI operations. In some embodiments, the bio-relevant contrast agent may increase the NMR or MRI signal or signal-to-noise ratio (SNR). In some embodiments, the bio-relevant contrast agent may be suitable for use in solution NMR spectroscopy. In some embodiments, the bio-relevant contrast agent may be a metabolite (e.g., a molecule with bio-relevance, such as an amino acid, sugar, derivatives thereof, etc.), such as a metabolite suitable for use in NMR metabolomics applications. In some embodiments, the bio-relevant contrast agent is used for perfusion or contrast-enhanced imaging in MRI scans. In some embodiments, the bio-relevant contrast agent may be suitable for in vitro probing of the metabolism of cell cultures or other biological tissues. In some embodiments, the bio-relevant contrast agent is used for in vitro probing of the metabolism of cell cultures or other biological tissues. In some embodiments, the bio-relevant contrast agent may be used in NMR probes to investigate transient effects where high signal enhancement due to hyperpolarization is required, such as proton exchange between water and biomolecules. In some embodiments, the biorelevant imaging agent may be a small molecule or metabolite suitable for injection into a cell, tissue, or organism for detection in an MRI scan. In some embodiments, the biorelevant imaging agent may be introduced into the chamber for further analysis by NMR or MRI operation. In some embodiments, the biorelevant imaging agent may be one or more 2 H or 13 It is enriched in C atoms.

[0028] In some embodiments, the biorelevant imaging agent is pyruvate, lactate, alpha-ketoglutarate, bicarbonate, fumarate, urea, dehydroascorbate, glutamic acid, glutamine, acetate, dihydroxyacetone, acetoacetate, glucose, ascorbate, zymonate, alanine, fructose, imidazole, nicotinamide, nitroimidazole, pyrazinamide, isoniazid, conjugate acids of any of the above, natural and unnatural amino acids, esters thereof, or any of the foregoing. 2 H, 13C, or nitrogen-15 ( 15 N)-enriched versions. In some embodiments, the biologically relevant imaging agent includes pyruvate, lactate, alpha-ketoglutarate. In some embodiments, the biologically relevant imaging agent includes pyruvate. In some embodiments, the biologically relevant imaging agent includes lactate. In some embodiments, the biologically relevant imaging agent includes alpha-ketoglutarate (e.g., ethyl alpha-ketoglutarate).

[0029] In some embodiments, the biologically relevant imaging agent comprises at least one non-hydrogen nuclear spin. In some embodiments, the non-hydrogen nucleus comprises at least one spin 1 / 2 atom. In some embodiments, the non-hydrogen nuclear spin is 13 C or 15 N. In some embodiments, the biologically relevant imaging agent is at least partially isotopically labeled with non-hydrogen nuclear spins. In some embodiments, the biologically relevant imaging agent is at least partially enriched in non-hydrogen nuclear spins compared to an analog of the biologically relevant imaging agent that characterizes the non-hydrogen nuclear spins at their natural abundance. In some embodiments, the biologically relevant imaging agent is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to about 99%, 98%, 97%, 96%, The enrichment may be such that the non-hydrogen nuclear spins are characterized by an abundance of 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or an abundance within a range defined by any two of the foregoing values.

[0030] In some embodiments, the non-hydrogen nuclear spins are NMR inactive (i.e., spin 0) nuclei of an analog of a biologically relevant imaging agent that characterizes the non-hydrogen nuclear spins at their natural abundance (e.g., 12C or quadrupolar (i.e., spin >1 / 2) nuclei (e.g., nitrogen-14, 14 N) at their natural abundance. 13 The pyruvate analogue that characterizes C is either C of the structure H3C-C*(=O)-C*OOH, with approximately 98.9% 12 C and about 1.1% 13 As a biorelevant imaging agent, pyruvate may contain one or both C* at any abundance as described herein. 13 To include C, instead 13 As used herein, *C and C* refer to 12 C or 13 As another example, carbon can be any of the C carbon isotopes at its natural abundance: 15 The analogue of urea that features N has the structure H2N*-C(=O)-*NH2 with approximately 99.6% N*. 14 N and about 0.4% 15 As a biorelevant imaging agent, urea may contain one or both N* in any abundance as described herein. 15 To include N, instead 15 As used herein, *N and N* refer to 14 N or 15 Describes nitrogen, which can be any of the nitrogen isotopes of N.

[0031] Bio-relevant contrast agent precursors In some embodiments, the present disclosure describes a precursor (i.e., precursor compound) that includes a biologically relevant imaging agent and a side arm. In some embodiments, the biologically relevant imaging agent is covalently attached to the side arm. In some embodiments, the biologically relevant imaging agent is attached to the side arm via a transfer moiety, such as a PHIP transfer moiety that is part of the side arm.

[0032] In some embodiments, the present disclosure describes a precursor (i.e., precursor compound) that includes an acyl derivative of a biorelevant imaging agent (i.e., RC=O)-) and a side arm. As used herein, the term "acyl derivative of a biorelevant imaging agent" refers to a covalent derivative of a biorelevant imaging agent in which the terminal acid moiety [RC(=O)OH)] of an unbound biorelevant imaging agent is modified to an acyl group and a covalent bond [RC(=O)-)] in a bound biorelevant imaging agent. In some embodiments, the acyl derivative of a biorelevant imaging agent is covalently attached to the side arm. In some embodiments, the acyl derivative of a biorelevant imaging agent is attached to the side arm via a transfer moiety, such as a PHIP transfer moiety, that is part of the side arm.

[0033] The side arms can be para-hydrogenated using para-hydrogen (e.g., by mixing the precursor and para-hydrogen). In some embodiments, the hydrogenation produces Iz1Iz2 order, which is the lower energy state between |↑>|↓>, |↓>|↑>, or singlet spin order of two hydrogen spins, depending on whether the hydrogenation is performed in a low or high magnetic field.

[0034] In some embodiments, the precursor is selected such that after hydrogenation and other optional chemical reactions, the biorelevant contrast agent is suitable for use in hyperpolarized NMR or MRI applications. In some embodiments, an additional chemical reaction after hydrogenation can be used to separate the biorelevant contrast agent from the precursor. Such additional chemical reactions can include, for example, cleavage of the side arm of the precursor by hydrolysis. For example, the biorelevant contrast agent can be a metabolite molecule such that the precursor can be a derivative of a metabolite molecule, the derivative having the general chemical structure of Formula Ia or Formula Ib. The biorelevant contrast agent can be polarized using the PHIP-SAH method (i.e., para-hydrogenation of the side arm and subsequent polarization transfer to the biorelevant contrast agent). After hydrogenation and polarization transfer, the bond in the precursor (e.g., an ester bond) can be hydrolyzed to produce a polarized biorelevant contrast agent and a separate side arm element.

[0035] As used herein, hydrolysis is defined as the cleavage of a molecule via a nucleophilic substitution reaction involving the addition of the element water. Hydrolysis can also be carried out under anhydrous conditions in the presence of hydroxide ions.

[0036] Consistent with the disclosed embodiments, precursors of the general chemical form presented in Formula Ia or Formula Ib can be used as precursors for PHIP-SAH. After hydrogenation of such precursors, two PHIP-SAHs exhibiting spin order are obtained. 1 The H spin is close (e.g., no more than 3, 4, or 5 bonds away) from the target carbon or nitrogen on the metabolite, as described herein. 13 C concentrated or 15 In some embodiments, the 13 C or 1 5 Originating from N spin and parahydrogen 1A high J-coupling between at least one of the H spins is achieved. In some embodiments, the J-coupling is achieved at least about 0.1 Hertz (Hz), 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, or more, up to about 10 Hz, 9 Hz, 8 Hz, 7 Hz, 6 Hz, 5 Hz, 4 Hz, 3 Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz, or less, or within a range defined by any two of the aforementioned values. For example, in some embodiments, the J-coupling may be from 1 Hz to 2 Hz, 1 Hz to 3 Hz, 1 Hz to 4 Hz, 1 Hz to 5 Hz, 1 Hz to 6 Hz, 1 Hz to 7 Hz, 1 Hz to 8 Hz, 1 Hz to 9 Hz, 1 Hz to 10 Hz, 2 Hz to 3 Hz, 2 Hz to 4 Hz, 2 Hz to 5 Hz, 2 Hz to 6 Hz, 2 Hz to 7 Hz, 2 Hz to 8 Hz, 2 Hz to 9 Hz, 2 Hz to 10 Hz, 3 Hz to 4 Hz, 3 Hz to 5 Hz, 3 Hz to 6 Hz, 3 Hz to 7 Hz, 3 Hz to 8 Hz, Hz, 3Hz-9Hz, 3Hz-10Hz, 4Hz-5Hz, 4Hz-6Hz, 4Hz-7Hz, 4Hz-8Hz, 4Hz-9Hz, 4Hz-10Hz, 5Hz-6Hz, 5Hz-7Hz, 5Hz-8Hz, 5Hz-9Hz, 5Hz-10Hz, 6Hz-7Hz, 6Hz-8Hz, 6Hz-9Hz, 6Hz-10Hz, 7Hz-8Hz, 7Hz-9Hz, 7Hz-10Hz, 8Hz-9Hz, 8Hz-10Hz, or 9Hz-10Hz. Such J-couplings are 13 This may enable efficient polarization of the C spins.

[0037] The present invention relates to compounds of formula Ia, Ib, IIa, IIb, IIIa, IIIb, IVa and IVb, their tautomers, deuterated derivatives of these compounds, and their tautomers, salts thereof, and to compounds having a tautomer structure at one or more sites within the molecule. 13 C or 15Novel precursors are disclosed, including N-enriched derivatives (which can then be subjected to hyperpolarization), as well as the subsequent generation of precursors given by the general formulae Ia, Ib, IIa, IIb, IIIa, IIIb, IVa and IVb.

[0038] Precursors of formula Ia and formula Ib In some embodiments, the precursor comprises a compound of formula Ia, which includes the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z in formula Ia is (i) 1 H (proton), 2 H (deuterium), or combinations thereof (e.g., -C 1 H=C 1 H-, -C 1 H=C 2 H-, -C 2 H=C 2 H-), or (ii) a carbon-carbon triple bond (-C≡C-). In some embodiments, R1 of formula Ia comprises a parahydrogen induced polarization (PHIP) transfer moiety as described herein. In some embodiments, R2 of formula Ia comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine as described herein. In some embodiments, R3 of formula Ia comprises a biorelevant imaging agent as described herein. In formula Ia, all moieties to the right of the R3-R1 bond (i.e., -R1-Z-(C═O)-R2) may be collectively referred to as a side arm.

[0039] In some embodiments, the precursor comprises a compound of formula Ib, which includes the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 In some embodiments, Z in formula Ib represents an ethynyl (-C≡C-) group, an optionally substituted prop-2-ynyl (-CC≡C-) group, an optionally substituted ethenyl (-C=C-) group, an optionally substituted prop-2-enyl (-CC=C-) group, or an optionally substituted but-3-enyl (-CCC=C-) group. In some embodiments, R2 in formula Ib comprises an optionally substituted hydrocarbon group, alkyl group, cyclic alkyl group, aryl group, carboxyl group, keto group, or alkoxy group, as described herein. In some embodiments, R3 in formula Ib comprises a biorelevant imaging agent, as described herein. In formula Ib, all moieties to the right of R3 (i.e., -SZ-R2) may be collectively referred to as a side arm.

[0040] In some embodiments, the compound of Formula Ia or Formula Ib has a solubility in water of at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more, and up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in water within a range defined by any two of the aforesaid values.

[0041] In some embodiments, the compound of Formula Ia or Formula Ib is dissolved in at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more of an organic solvent (e.g., acetone, ethanol, chloroform, toluenza, toluene ... In some embodiments, the compound has a solubility in an organic solvent of up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in an organic solvent within a range defined by any two of the aforesaid values.

[0042] In some embodiments, the compound of formula Ia comprises methyl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises methyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((4-methoxy-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0043] In some embodiments, the compound of formula Ia comprises isopropyl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises isopropyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((4-isopropoxy-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0044] In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((4-(tert-butoxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0045] In some embodiments, the compound of formula Ia is tert-butyl 4-((2-oxopropanoyl-1- 13 C) oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-hydroxypropanoyl-1- 13 C l)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia is 5-((4-(tert-butoxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid 13 Contains C acid.

[0046] In some embodiments, the compound of formula Ia comprises 2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-oxopropanoyl-1-13C)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6 4-((2-hydroxypropanoyl-1-13C)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((4-((2-(methyl-d3)propan-2-yl-1,1,1,3,3,3-d6)oxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0047] In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-oxopropanoyl)oxy)but-2-ynoate-4-d. In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate-4-d. In some embodiments, the compound of formula Ia comprises 5-((4-(tert-butoxy)-4-oxobut-2-yn-1-yl-1-d)oxy)-4,5-dioxopentanoic acid.

[0048] In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-oxopropanoyl)oxy)pent-2-ynoate. In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)pent-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((5-(tert-butoxy)-5-oxopent-3-yn-2-yl)oxy)-4,5-dioxopentanoic acid.

[0049] In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-oxopropanoyl)oxy)-4-phenylbut-2-ynoate. In some embodiments, the compound of formula Ia comprises tert-butyl 4-((2-hydroxypropanoyl)oxy)-4-phenylbut-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((4-(tert-butoxy)-4-oxo-1-phenylbut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0050] In some embodiments, the compound of formula Ia comprises benzhydryl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises benzhydryl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 5-((4-(benzhydryloxy)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0051] In some embodiments, the compound of formula Ia comprises 4-oxo-4-phenylbut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-oxo-4-phenylbut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 4,5-dioxo-5-((4-oxo-4-phenylbut-2-yn-1-yl)oxy)pentanoic acid.

[0052] In some embodiments, the compound of formula Ia comprises 4-oxo-4-(phenyl-d5)but-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-oxo-4-(phenyl-d5)but-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 4,5-dioxo-5-((4-oxo-4-(phenyl-d5)but-2-yn-1-yl)oxy)pentanoic acid.

[0053] In some embodiments, the compound of formula Ia comprises tert-butyl 4-acetoxybut-2-ynoate. In some embodiments, the compound of formula Ia comprises 1-(4-(Tert-butoxy)-4-oxobut-2-yn-1-yl) 5-ethyl 2-oxopentanedioate.

[0054] In some embodiments, the compound of formula Ia comprises methyl 4-(2,2-dichloroacetoxy)but-2-ynoate.

[0055] In some embodiments, the compound of formula Ia comprises trityl 4-((2-oxopropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises trityl 4-((2-hydroxypropanoyl)oxy)but-2-ynoate. In some embodiments, the compound of formula Ia comprises 4,5-dioxo-5-((4-oxo-4-(trityloxy)but-2-yn-1-yl)oxy)pentanoic acid.

[0056] In some embodiments, the compound of formula Ia comprises 4-(diphenylamino)-4-oxobut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-(diphenylamino)-4-oxobut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 5-((4-(diphenylamino)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0057] In some embodiments, the compound of formula Ia comprises 4-(diisopropylamino)-4-oxobut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-(diisopropylamino)-4-oxobut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 5-((4-(diisopropylamino)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0058] In some embodiments, the compound of formula Ia comprises 4-oxopent-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-oxopent-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 4,5-dioxo-5-((4-oxopent-2-yn-1-yl)oxy)pentanoic acid.

[0059] In some embodiments, the compound of formula Ia comprises 4-oxo-4-(pyridin-2-yl)but-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-oxo-4-(pyridin-2-yl)but-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 4,5-dioxo-5-((4-oxo-4-(pyridin-2-yl)but-2-yn-1-yl)oxy)pentanoic acid.

[0060] In some embodiments, the compound of formula Ia comprises 4-(1-methyl-1H-imidazol-2-yl)-4-oxobut-2-yn-1-yl 2-oxopropanoate. In some embodiments, the compound of formula Ia comprises 4-(1-methyl-1H-imidazol-2-yl)-4-oxobut-2-yn-1-yl 2-hydroxypropanoate. In some embodiments, the compound of formula Ia comprises 5-((4-(1-methyl-1H-imidazol-2-yl)-4-oxobut-2-yn-1-yl)oxy)-4,5-dioxopentanoic acid.

[0061] In some embodiments, the composition of formula Ib comprises S-(phenylethynyl)ethanethioate. In some embodiments, the composition of formula Ib comprises S-(3-phenylprop-2-yn-1-yl)ethanethioate. In some embodiments, the composition of formula Ib comprises S-(3-(4-methoxyphenyl)prop-2-yn-1-yl)ethanethioate. In some embodiments, the composition of formula Ib comprises S-(3-(3,4,5-trimethoxyphenyl)prop-2-yn-1-yl)ethanethioate. In some embodiments, the composition of formula Ib) comprises S-(3-(benzo[d][1,3]dioxol-5-yl)prop-2-yn-1-yl)ethanethioate. In some embodiments, the composition of formula Ib) comprises S-(3-(4-nitrophenyl)prop-2-yn-1-yl)ethanethioate. In some embodiments, the compound of formula Ib comprises tert-butyl 4-(acetylthio)but-2-ynoate. In some embodiments, the compound of formula Ib comprises tert-butyl 4-((2-oxopropanoyl)thio)but-2-ynoate. In some embodiments, the composition of formula Ib comprises S-(3-phenylprop-2-yn-1-yl)2-oxopropanethioate. In some embodiments, the composition of formula Ib comprises S-(3-(4-methoxyphenyl)prop-2-yn-1-yl)2-oxopropanethioate. In some embodiments, the composition of formula Ib comprises S-(3-(3,4,5-trimethoxyphenyl)prop-2-yn-1-yl)2-oxopropanethioate. In some embodiments, the composition of Formula Ib) comprises S-(3-(benzo[d][1,3]dioxol-5-yl)prop-2-yn-1-yl) 2-oxopropanethioate.

[0062] Para-hydrogenated precursors of formula IIa and formula IIb In some embodiments, the compound of formula Ia is para-hydrogenated (i.e., modified via addition of a para-hydrogenation proton across Z via a hydrogenation reaction between formula Ia and para-hydrogen) as described herein. In some embodiments, para-hydrogenation of the compound of formula Ia gives rise to a compound of formula IIa. Formula IIa encompasses the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z′ of formula IIa is (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that has been substituted to include H (deuterium), or combinations thereof (e.g., -CH2H*-CH2H*-, -CHDH*-CH2H*-, -CD2H*-CH2H*-); or (ii) 1 H (proton), 2 H (deuterium), or a combination thereof. In some embodiments, H* represents a hydrogen with spin order derived from para-hydrogen (i.e., a hydrogen atom or a proton added across the carbon-carbon double bond or carbon-carbon triple bond Z via a hydrogenation reaction between a compound of formula Ia as described herein and para-hydrogen). In some embodiments, H* represents a hydrogen with spin order derived from para-hydrogen (e.g., prior to polarization transfer). In some embodiments, R1 of formula IIa comprises a PHIP transfer moiety as described herein. In some embodiments, R2 of formula IIa comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine as described herein. In some embodiments, R3 of formula IIa comprises a biorelevant imaging agent as described herein. In Formula IIa, all moieties to the right of the R3-R1 bond (ie, -R1-Z'-(C=O)-R2) can be collectively referred to as the para-hydrogenated side arm.

[0063] In some embodiments, the compound of formula Ib is para-hydrogenated (i.e., modified via addition of a para-hydrogenation proton across Z via a hydrogenation reaction between formula Ib and para-hydrogen) as described herein. In some embodiments, para-hydrogenation of the compound of formula Ib gives rise to a compound of formula IIb. Formula IIb encompasses the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 and C-enriched derivatives. In some embodiments, Z' in formula IIb represents a para-hydrogenated ethenyl (-CH*=CH*-) group, an optionally substituted para-hydrogenated prop-2-enyl (-C-CH*=CH*-) group, an optionally substituted para-hydrogenated ethanyl (-CH*-CH*-) group, an optionally substituted para-hydrogenated propanyl (-C-CH*-CH*-) group, or an optionally substituted para-hydrogenated butanyl (-CC-CH*=CH*-) group. In some embodiments, H* represents a hydrogen with spin order derived from para-hydrogen (i.e., a hydrogen atom or proton added across the carbon-carbon double bond or carbon-carbon triple bond Z via a hydrogenation reaction between a compound of formula I and para-hydrogen as described herein). In some embodiments, H* represents a hydrogen with spin order derived from para-hydrogen (e.g., prior to polarization transfer). In some embodiments, R2 of formula IIb comprises an optionally substituted hydrocarbon group, alkyl group, cyclic alkyl group, aryl group, carboxyl group, keto group, or alkoxy group, as described herein. In some embodiments, R3 of formula IIb comprises an acyl derivative of a biorelevant imaging agent, as described herein. In formula IIb, all moieties to the right of R3 (i.e., S-Z'-R2) may be collectively referred to as the para-hydrogenated side arm.

[0064] In some embodiments, the compound of Formula IIa or Formula IIb has a solubility in water of at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more. has a solubility in water of about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in water within a range defined by any two of the foregoing values.

[0065] In some embodiments, the compound of Formula IIa or Formula IIb is dissolved in at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more of an organic solvent (e.g., acetone, ethanol, chloroform, toluene ... In some embodiments, the compound has a solubility in an organic solvent of up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in an organic solvent within a range defined by any two of the aforesaid values.

[0066] In some embodiments, when a composition of Formula Ia or Formula IIb is reacted with para-hydrogen, the chemical yield (e.g., the chemical yield of a compound of Formula IIa or Formula IIb) is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, up to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, or less, or within a range defined by any two of the foregoing values.For example, in some embodiments, when a composition of Formula Ia or Formula Ib is reacted with parahydrogen, the chemical yield is between 30% and 35%, 30% and 40%, 30% and 45%, 30% and 50%, 30% and 55%, 30% and 60%, 30% and 65%, 30% and 70%, 30% and 75%, 30% and 80%, 30% and 85%, 30% and 90%, 30% and 95%, 35% and 40%, 35% and 45%, 35% and 50%, 35% and 55%, 35% and 60%, 35% and 65%, 35%~70%, 35%~75%, 35%~80%, 35%~85%, 35%~90%, 35%~95%, 40%~45%, 40%~50%, 40%~55%, 40%~60%, 40%~65%, 40%~70%, 40%~75%, 40%~80%, 40%~85%, 40%~90%, 40%~95%, 45%~50%, 45%~55%, 45%~60%, 45%~65%, 45%~70%, 45%~75%, 45%~80% , 45%~85%, 45%~90%, 45%~95%, 50%~55%, 50%~60%, 50%~65%, 50%~70%, 50%~75%, 50%~80%, 50%~85%, 50%~90%, 50%~95%, 55%~60%, 55%~65%, 55%~70%, 55%~75%, 55%~80%, 55%~85%, 55%~90%, 55%~95%, 60%~65%, 60%~70%, 60%~75%, 60%~80%, 60 %~85%, 60%~90%, 60%~95%, 65%~70%, 65%~75%, 65%~80%, 65%~85%, 65%~90%, 65%~95%, 70%~75%, 70%~80%, 70%~85%, 70%~90%, 70%~95%, 75%~80%, 75%~85%, 75%~90%, 75%~95%, 80%~85%, 80%~90%, 80%~95%, 85%~90%, 85%~95%, or 90%~95%.

[0067] Cleaved precursors of formula IIIa and IIIb In some embodiments, the compound of formula IIa is cleaved (e.g., hydrolyzed) as described herein. In some embodiments, the compound of formula IIa is cleaved (e.g., hydrolyzed) as described herein to provide a side arm compound and a corresponding biorelevant imaging agent. In some embodiments, cleavage of the compound of formula IIa results in a compound of formula IIIa and a corresponding biorelevant imaging agent as described herein. Formula IIIa encompasses the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z″ of formula IIIa is (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that has been substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 H (deuterium), or a combination thereof. In some embodiments, R1' of formula IIIa comprises a PHIP transfer moiety as described herein. In some embodiments, R2 of formula IIIa comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine as described herein. In formula IIIa, all of the moieties R1-Z''-(C=O)-R2 may be collectively referred to as a cleaved sidearm or a hydrolyzed sidearm.

[0068] In some embodiments, the compound of formula IIb is cleaved (e.g., hydrolyzed) as described herein. In some embodiments, the compound of formula IIb is cleaved (e.g., hydrolyzed) as described herein to provide a side arm compound and a corresponding biorelevant imaging agent. In some embodiments, cleavage of the compound of formula IIb results in a compound of formula IIIb and a corresponding biorelevant imaging agent as described herein. Formula IIIb encompasses the following structure: [ka]

[0069] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 and C-enriched derivatives. In some embodiments, Z″ of formula IIIb represents a para-hydrogenated ethenyl (—CH*═CH*—) group, an optionally substituted para-hydrogenated prop-2-enyl (—C—CH*═CH*—) group, an optionally substituted para-hydrogenated ethanyl (—CH*—CH*—) group, an optionally substituted para-hydrogenated propanyl (—C—CH*—CH*—) group, or an optionally substituted para-hydrogenated butanyl (—CC—CH*═CH*—) group. R2 of formula IIIb includes an optionally substituted hydrocarbon group, alkyl group, cyclic alkyl group, aryl group, carboxyl group, keto group, or alkoxy group, as described herein. In formula IIIb, all of the moieties HS-Z′-R2 may be collectively referred to as a cleaved side arm or a hydrolyzed side arm.

[0070] In some embodiments, the compound of Formula IIIa or Formula IIIb has a solubility in water of at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more. In some embodiments, the compound has a solubility in water of about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in water within a range defined by any two of the aforesaid values.

[0071] In some embodiments, the compound of Formula IIIa or Formula IIIb is dissolved in at least about 1 millimolar (mM), 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, 1,000 mM, or more of an organic solvent (e.g., acetone, ethanol, chloroform, toluene), a solubility in an organic solvent of up to about 1,000 mM, 950 mM, 900 mM, 850 mM, 800 mM, 750 mM, 700 mM, 650 mM, 600 mM, 550 mM, 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less, or a solubility in an organic solvent within a range defined by any two of the aforesaid values.

[0072] Side arm of formula IVa In some embodiments, a biorelevant imaging agent and a side arm, such as a side arm compound of formula IVa, are conjugated as described herein to form a precursor compound, such as a compound of formula Ia, which includes the following structure: [ka] tautomers thereof, deuterated derivatives of these compounds and their tautomers, pharma- ceutically acceptable salts thereof, and 13 C or 15 In some embodiments, Z in formula IVa is (i) 1 H (proton), 2 H (deuterium), or combinations thereof (e.g., -C 1 H=C1 H-, -C 1 H=C 2 H-, -C 2 H=C 2 In some embodiments, R1 of formula IVa comprises a parahydrogen induced polarization (PHIP) transfer moiety as described herein. In some embodiments, R2 of formula IVa comprises a solubilizing moiety as described herein. In some embodiments, R2 of formula IVa comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine. In some embodiments, complexation of a compound of formula IVa with a biologically relevant imaging agent provides a compound of formula Ia, as described herein.

[0073] PHIP moving part In some embodiments, the compositions of the present disclosure include a PHIP transfer moiety. In some embodiments, the compositions of the present disclosure include a PHIP transfer moiety between the Z, Z', or Z'' moiety and the sulfur atom of Formula Ib, Formula IIb, or Formula IIIb. In some embodiments, the PHIP transfer moiety described herein transfers one or more para-hydrogenated protons H* (e.g., side-arm H*) to one or more non-hydrogen nuclear spins of the biologically relevant imaging agent (e.g., one or more non-hydrogen nuclear spins of the biologically relevant imaging agent as described herein). 13 C or 15 In some embodiments, the PHIP transfer moiety includes a chemical moiety configured to enable or enhance polarization transfer from the para-hydrogenated proton H* in the side arm of the compound of Formula IIa or Formula IIb to the non-hydrogen nuclear spins of the corresponding biologically relevant imaging agent of Formula IIa or Formula IIb after the para-hydrogenation reaction between para-hydrogen and Formula Ia or Formula IIa. In some embodiments, the PHIP transfer moiety enables or enhances polarization transfer from the para-hydrogenated proton H* in the side arm of the compound of Formula IIa or Formula IIb to the non-hydrogen nuclear spins of the corresponding biologically relevant imaging agent of Formula IIa or Formula IIb after the para-hydrogenation reaction between para-hydrogen and Formula Ia or Formula IIa.

[0074] In some embodiments, the PHIP transfer moiety comprises an optionally substituted C1 hydrocarbon or an optionally substituted C2 hydrocarbon.

[0075] In some embodiments, the PHIP transfer moiety comprises a chemical moiety in the form of *CR4R5, *CR4Y, *C=Y, or any deuterated version thereof. 12 C or 13 C is a carbon isotope. In some embodiments, R and R are each independently selected from: 1 H, 2 H, 3 H, linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups. In some embodiments, Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups. In some embodiments, the spin 1 / 2 atoms are selected from: 1 H, 13 C. 15 N, 19 F, and 31 P. In some embodiments, 15 N may be substituted with a nitro group, an amine group, an amide group, or an imine group. 31 P may be substituted with one or more keto groups, one or more nitro groups, one or more amine groups, one or more amide groups, or one or more imine groups.

[0076] In some embodiments, the PHIP transfer moiety comprises a chemical moiety of the form *CR6R7-*CR8R9, or any deuterated version thereof. 12 C or 13C is a carbon isotope. In some embodiments, R6, R7, R8, and R9 are each independently selected from: 1 H, 2 H, 3 H, straight chain, branched chain, or cyclic C1-C10 alkyl hydrocarbon, C6 aryl, benzyl, phenyl, heteroaryl, and haloalkyl groups.

[0077] In some embodiments, the PHIP transfer moiety comprises a chemical moiety in the form of *CH2, *CH2-*CH2, *CHY, *C=Y, or any deuterated version thereof. 12 C or 13 is a carbon isotope of C. In some embodiments, Y is selected from spin 1 / 2 atoms covalently bonded to one or more chemical moieties selected from linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups, or heteroatoms such as N, O, S, optionally substituted with linear, branched, or cyclic C1-C10 alkyl hydrocarbons, C6 aryl, benzyl, phenyl, heteroaryl, halogen, or haloalkyl groups. In some embodiments, the spin 1 / 2 atoms are selected from: 1 H, 13 C. 15 N, 19 F, and 31 P.

[0078] In some embodiments, the compositions described herein include a first J-coupling, J, between a spin ½ atom described herein and a non-hydrogen nuclear spin described herein. 12 In some embodiments, the compositions described herein include a second J-coupling J between a spin 1 / 2 atom described herein and a para-hydrogenated proton H* described herein. 13 In some embodiments, the compositions described herein include a third J-coupling J between a non-hydrogen nuclear spin as described herein and a para-hydrogenated proton H* as described herein. 23 In some embodiments, J 12and / or J 13 J 23 In such cases, the PHIP transfer moiety may enable or enhance polarization transfer.

[0079] In some embodiments, the PHIP transfer moiety induces J-coupling between one or both of *H nuclear spins having non-hydrogen nuclear spins of at least about 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz or more, up to about 10 Hz, 9 Hz, 8 Hz, 7 Hz, 6 Hz, 5 Hz, 4 Hz, 3 Hz, 2 Hz, 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz or less, or induces J-coupling with non-hydrogen nuclear spins within a range defined by any two of the foregoing values. For example, in some embodiments, the J-coupling may be from 1 Hz to 2 Hz, 1 Hz to 3 Hz, 1 Hz to 4 Hz, 1 Hz to 5 Hz, 1 Hz to 6 Hz, 1 Hz to 7 Hz, 1 Hz to 8 Hz, 1 Hz to 9 Hz, 1 Hz to 10 Hz, 2 Hz to 3 Hz, 2 Hz to 4 Hz, 2 Hz to 5 Hz, 2 Hz to 6 Hz, 2 Hz to 7 Hz, 2 Hz to 8 Hz, 2 Hz to 9 Hz, 2 Hz to 10 Hz, 3 Hz to 4 Hz, 3 Hz to 5 Hz, 3 Hz to 6 Hz, 3 Hz to 7 Hz, 3 Hz to 8 Hz, Hz, 3Hz to 9Hz, 3Hz to 10Hz, 4Hz to 5Hz, 4Hz to 6Hz, 4Hz to 7Hz, 4Hz to 8Hz, 4Hz to 9Hz, 4Hz to 10Hz, 5Hz to 6Hz, 5Hz to 7Hz, 5Hz to 8Hz, 5Hz to 9Hz, 5Hz to 10Hz, 6Hz to 7Hz, 6Hz to 8Hz, 6Hz to 9Hz, 6Hz to 10Hz, 7Hz to 8Hz, 7Hz to 9Hz, 7Hz to 10Hz, 8Hz to 9Hz, 8Hz to 10Hz, or 9Hz to 10Hz.

[0080] R2 units In some embodiments, the R2 groups described herein include optionally substituted hydrocarbons, alkoxy groups, primary amines, secondary amines, or tertiary amines. In some embodiments, the R2 groups described herein include optionally substituted hydrocarbons, alkoxy groups, primary amines, secondary amines, or tertiary amines that function as solubilizing moieties. In some embodiments, the R2 groups described herein include solubilizing moieties. In some embodiments, the solubilizing moieties include any chemical moiety configured to enable or enhance the solubility of a compound, such as any of the compounds of formula Ia, Ib, IIa, IIb, IIIa, IIIb, and / or IVa, in the solution in which the parahydrogenation reaction or the cleavage (e.g., hydrolysis) reaction is carried out. In some embodiments, the enhanced solubility is measured with respect to variants of the compounds of formula Ia, Ib, IIa, IIb, IIIa, IIIb, and / or IVa that utilize one or more protons in place of the R2 group. In some embodiments, the solubility enhancement is measured for variants of compounds of formula Ia, Ib, IIa, IIb, IIIa, IIIb, and / or IVa that utilize a methyl group as the R2 group.

[0081] In some embodiments, the solubilizing moiety comprises a hydrophobic moiety or an organophilic moiety. In some embodiments, the solubilizing moiety comprises an organic solubilizing moiety. For example, in some embodiments, the solubilizing moiety comprises a hydrophobic moiety, an organophilic moiety, or an organic solubilizing moiety. In some embodiments, the solubilizing moiety comprises a hydrophilic moiety or an organophilic moiety.

[0082] In some embodiments, the R2 group comprises or is selected from methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, hydroxy, methyl alcohol, ethyl alcohol, n-propanol, isopropyl alcohol, propionic acid, n-butyl alcohol, s-butyl alcohol, t-butyl alcohol, isobutyl alcohol, methoxy, ethoxy, propoxy, isopropoxy, propionic acid, butoxy, t-butoxy, s-butoxy, ester, phenyl, substituted phenyl, primary amine, secondary amine, tertiary amine, primary amide, secondary amide, and tertiary amide. In some embodiments, the substituted phenyl group is selected from fluorobenzene, chlorobenzene, bromobenzene, iodobenzene, toluene, cumene, ethylbenzene, styrene, ortho-xylene, meta-xylene, para-xylene, phenol, benzoic acid, benzaldehyde, acetophenone, methyl benzoate, anisole, aniline, nitrobenzene, benzonitrile, benzamide, benzenesulfonic acid, naphthalene, and anthracene.

[0083] R3 groups In some embodiments, the R3 group described herein comprises a biorelevant imaging agent. In some embodiments, the biorelevant imaging agent has the formula R4C(=O)X-. In some embodiments, R4 is selected from a linear, branched, or cyclic C1-C10 alkyl group, where one or more C atoms are optionally substituted with CO, COOH, CH2COOH, CONH2, OH, amino (NR'R''), one or more halogen atoms, one or more haloalkyl groups, or one or more carbocycles, where the carbocycles are optionally substituted with one or more aliphatic or aromatic rings optionally substituted with one or more functional groups. In some embodiments, X is selected from NR''' and O. In some embodiments, R', R'', and R''' are each independently 1 H, 2 H, 3H and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzyl, tert-butylcarbonate, and benzyl. In some embodiments, the R3 group comprises any biologically relevant imaging agent described herein.

[0084] In some embodiments, the R3 group described herein comprises an acyl derivative of a biorelevant imaging agent. In some embodiments, the biorelevant imaging agent has the formula R9C(=O)O-. In some embodiments, R9 is selected from a linear, branched, or cyclic C1-C10 alkyl group, where one or more C atoms are optionally substituted with CO, COOH, CH2COOH, CONH2, OH, amino (NR'R''), one or more halogen atoms, one or more haloalkyl groups, or one or more carbocycles, where the carbocycles are optionally substituted with one or more aliphatic or aromatic rings, optionally substituted with one or more functional groups. In some embodiments, R' and R'' are each independently selected from 1 H, 2 H, 3 H and an amino protecting group optionally selected from trifluoroacetyl, acetyl, benzoyl, carbobenzyl, tert-butylcarbonate, and benzyl. In some embodiments, the R group comprises an acyl derivative of any of the biorelevant imaging agents described herein.

[0085] In some embodiments, the R3 group comprises at least one non-hydrogen nuclear spin. In some embodiments, the non-hydrogen nucleus comprises at least one spin 1 / 2 atom. In some embodiments, the non-hydrogen nuclear spin is 13 C or 15N. In some embodiments, the R3 group is at least partially isotopically labeled with non-hydrogen nuclear spins. In some embodiments, the R3 group is at least partially enriched in non-hydrogen nuclear spins compared to an analog of the R3 group that characterizes the non-hydrogen nuclear spins at their natural abundance. In some embodiments, the R3 group is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to about 99%, 98%, 97%, 96%, 95 .... %, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less abundance, or an abundance within a range defined by any two of the aforesaid values.

[0086] In some embodiments, the non-hydrogen nuclear spins are characterized as NMR inactive (i.e., spin 0) nuclei of an analog of an R3 group that characterizes the non-hydrogen nuclear spins at their natural abundance, as described herein (e.g., 12 C or quadrupolar (i.e., spin >1 / 2) nuclei (e.g., 14 In some embodiments, the non-hydrogen nuclear spin is located no more than about one or two chemical bonds from the carbonyl (C=O) carbon in the R3 group.

[0087] Parahydrogenation In some embodiments, the non-hydrogen nuclear spins are characterized as NMR inactive (i.e., spin 0) nuclei of an analog of an R3 group that characterizes the non-hydrogen nuclear spins at their natural abundance, as described herein (e.g., 12 C or quadrupolar (i.e., spin >1 / 2) nuclei (e.g., 14 In some embodiments, the non-hydrogen nuclear spin is located no more than about one or two chemical bonds from the carbonyl (C=O) carbon in the R3 group.

[0088] Consistent with the disclosed embodiments, a precursor of a biorelevant imaging agent (such as a compound of Formula Ia or Formula Ib as described herein) may be para-hydrogenated by combining the precursor, para-hydrogen, and a hydrogenation catalyst. The disclosed embodiments are not limited to a particular method of producing the para-hydrogenated precursor. In some embodiments, the precursor is added to a mixture containing para-hydrogen. In some embodiments, para-hydrogen gas is added to a solution containing the precursor (e.g., para-hydrogen gas may be bubbled into such a solution). In hydrogenating the precursor, para-hydrogen may create Iz1Iz2 order, a preferred population of lower energy states between |↑>|↓>, |↓>|↑>, or singlet spin order on the two hydrogen spins in the precursor.

[0089] The precursor can have unsaturated bonds (such as unsaturated carbon-carbon double bonds or unsaturated carbon-carbon triple bonds) that can be hydrogenated by parahydrogen gas. After combination of the precursor with parahydrogen, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the precursor, up to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the precursor, or a percentage of the precursor that is within a range defined by any two of the foregoing values, can be hydrogenated.

[0090] In some embodiments, the para-hydrogenated precursor has a population difference in the para-hydrogenated proton spin state of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or within a range defined by any two of the foregoing values. For example, in some embodiments, the population difference is between 10% and 15%, 10% and 20%, 10% and 25%, 10% and 30%, 10% and 35%, 10% and 40%, 10% and 45%, 10% and 50%, 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, 15% and 45%, 15% and 50%, 20% and 25%, 20% and 30%, 20% and 35 ... 0%-35%, 20%-40%, 20%-45%, 20%-50%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 35%-40%, 35%-45%, 35%-50%, 40%-45%, 40%-50%, or 45%-50%. In some embodiments, the population difference is between a spin state that includes a para-hydrogenated proton and another nuclear spin, e.g., a spin state that includes an additional proton on the compound. In some embodiments, the para-hydrogenated precursor includes a side arm, and the para-hydrogenated spin may be located on the side arm.

[0091] In some embodiments, the concentration of the hydrogenation catalyst during hydrogenation is at least about 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or more, up to at about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.2 mM, 0.1 mM, or less, or within a range defined by any two of the aforesaid values.

[0092] Disclosed embodiments may include methods implemented by the disclosed systems for producing a hyperpolarized biologically relevant contrast agent. The disclosed methods may include mixing (e.g., by a mixing mechanism) a solution including a precursor to the biologically relevant contrast agent and a hydrogenation catalyst. The mixing mechanism may be a device for introducing, holding, and facilitating a blend, mixture, or solution of two or more materials. In some embodiments, the mixing mechanism is disposed within a chamber and mixing occurs within the chamber. In some embodiments, the solutions are mixed at a location remote from the chamber. The solution can be at least about 1 milliliter (ml), 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, or more in volume, up to 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml, or less in volume, or within a volume range defined by any two of the foregoing values.

[0093] In some embodiments, the mixing mechanism is a gas-liquid exchange mechanism. For example, the gas-liquid exchange mechanism can be a bubbler or a diffusion system. In some embodiments, the mixing mechanism includes a membrane adapted to allow diffusion of molecular hydrogen. In some embodiments, the mixing can be performed using a spray chamber, where the solution is sprayed into a chamber filled with pressurized parahydrogen.

[0094] In some embodiments, the catalyst is a molecule, complex, or particulate system that catalyzes hydrogenation. In some embodiments, the catalyst comprises a homogeneous metal catalyst, such as a rhodium complex or a ruthenium complex. The rhodium complex may be used to prepare and activate the precursor molecule and parahydrogen. In some embodiments, the heterogeneous metal catalyst is attached to a nanoparticle.

[0095] Various embodiments of the present disclosure describe introducing a solution including a precursor of a biorelevant imaging agent and a hydrogenation catalyst into a chamber configured to hold the solution during polarization transfer. In some embodiments, the solution is mixed in the chamber. In some embodiments, the solution is hydrogenated in the chamber. In some embodiments, the chamber is within a magnetic shield (e.g., a mu metal shield). The magnetic shield can reduce the effects of the Earth's magnetic field (or other external magnetic fields) and allows for modulation of the amplitude of a low-level magnetic field applied to the solution. Thus, placing the solution in the chamber can include placing the solution in a magnetic shield.

[0096] As described herein, in some embodiments, para-hydrogenation occurs prior to polarization transfer (e.g., before modulating the amplitude of a magnetic field applied to a solution, etc.). In some embodiments, para-hydrogenation occurs during polarization transfer. For example, para-hydrogen may be combined with the solution (e.g., flowed or bubbled through the solution) during modulation of the amplitude of the magnetic field.

[0097] In some embodiments, the parahydrogen gas is combined with the solution in the hydrogenation chamber at a pressure. The pressure may be at least about 10 bar, 15 bar, 20 bar, 30 bar, 50 bar, or more, and may be up to about 50 bar, 30 bar, 20 bar, 15 bar, 10 bar, or less, or within a range defined by any two of the preceding values. In some embodiments, the parahydrogen is combined with the solution in a metal chamber that can withstand the pressure. The parahydrogen may be combined with the solution over a time interval (or dissolution of the parahydrogen may occur in less than a time interval). The time interval can be up to about 90 seconds, 60 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, or less, at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, or more, or within a range defined by any two of the foregoing values. In some embodiments, hydrogenation is performed or occurs within the time interval.

[0098] Polarization transfer using radio frequency waveforms In some embodiments, the concentration of the precursor or target molecule in solution prior to polarization transfer is at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1,000 mM, or more. up to about 1,000 mM, 900 mM, 800 mM, 700 mM, 600 mM, 500 mM, 400 mM, 300 mM, 200 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, or less, or within a range defined by any two of the foregoing values. The volume of the solution may be at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, 1000 ml, 2000 ml, or more, up to about 2000 ml, 1 The volume may be 000ml, 900ml, 800ml, 700ml, 600ml, 500ml, 400ml, 300ml, 200ml, 100ml, 90ml, 80ml, 70ml, 60ml, 50ml, 40ml, 30ml, 20ml, 10ml, 9ml, 8ml, 7ml, 6ml, 5ml, 4ml, 3ml, 2ml, 1ml or less, or within a range defined by any two of the aforesaid values.

[0099] Various embodiments of the present disclosure describe applying a polarization transfer magnetic perturbation intended to generate a magnetic field around a solution (e.g., around a solution comprising Formula IIa or Formula IIb as described herein). In some embodiments, the magnetic field is at least about 0.1 Gauss (G), 0.2G, 0.3G, 0.4G, 0.5G, 0.6G, 0.7G, 0.8G, 0.9G, 1G, 2G, 3G, 4G, 5G, 6G, 7G, 8G, 9G, 10G, 20G, 30G, 40G, 50G, 60G, 70G, 80G, 90G, 100G, 200G, 300G, 400G, 500G, 600G, 700G, 800G, 900G, 1,000G, 2,000G, 3,000G, 4,000G, 5,000G, 6,000G, 7,000G, 8,000G, 9,000G, 10,000G, 20,000G, 30,000G, 40,000G, 50,000G, 60,000G, 70,000G, 80,000G, 90,000G, 100,000G, 200,000G or more, up to a maximum of approximately 200 ,000G, 100,000G, 90,000G, 80,000G, 70,000G, 60,000G, 50,000G, 40,000G, 30,000G, 20,000G, 10,000G, 9,000G, 8,000G, 7,000G, 6,000G, 5,000G, 4,000G, 3,000G, 2,000G, 1,000G, 900G, 800G, 700G, 600G, 500G , 400G, 300G, 200G, 100G, 90G, 80G, 70G, 60G, 50G, 40G, 30G, 20G, 10G, 9G, 8G, 7G, 6G, 5G, 4G, 3G, 2G, 1G, 0.9G, 0.8G, 0.7G, 0.6G, 0.5G, 0.4G, 0.3G, 0.2G, 0.1G, or less, or within a range defined by any two of the preceding values. In some embodiments, the magnetic field has a strength of 0.1G to 200,000G around the solution. The magnetic perturbation may be generated by an electromagnet or a permanent magnet. The magnetic field may be applied to the sample in a pulse or continuous wave (CW). The magnetic perturbation may be static or time-varying.

[0100] The signal generator may be configured to generate one or more radio frequency (RF) waveforms that may be applied to the sample to transfer polarization. The signal generator may include another computing unit, processor, controller, associated memory, PC, computer services, or any device that can perform computational operations using inputs and generate outputs. In some embodiments, the RF coil may emit or "apply" a pulse sequence that includes the first RF waveform. In some embodiments, the RF coil may have one or more channels. A channel may be a path for an RF signal. At least one channel may be provided for each different type of NMR spectroscopy. In some embodiments, 1 H has at least one channel, 2 H, 13 C. 15 N, 19 F, and 31 P has at least one channel. For example, the first RF waveform is generated by one or more radio frequency coils (RF coils) disposed around the sample. 1 In some embodiments, the second RF waveform may be applied to the H channel of the RF coil. 13 In some embodiments, 1 H channel and 13 The RF waveform on the C channel is configured to apply a polarization transfer sequence such as PH-INEPT, Goldman's sequence, S2M, S2hM, SLIC, ADAPT, or ESOTERIC.

[0101] In some embodiments, the RF waveform is configured to support polarization transfer even in the presence of large proton full width at half maximum (FWHM). Such RF waveforms can include pulse sequences that can include tens to hundreds of RF pulses. The sequences can be configured such that the pulses protect against the deleterious effects of magnetic field inhomogeneities on polarization transfer.

[0102] In some embodiments, the polarization pulse sequence can be used to separate two non-equivalent ions, e.g., when the chemical shift difference is greater than the J-coupling between them. 1H. ESOTHERIC may, for example, be a pulse sequence suitable for polarization transfer in this regime.

[0103] In some embodiments, the pulse sequences are equivalent when, for example, the chemical shift difference is smaller than the J-coupling between them. 1H hydrogen spins. Such pulse sequences are configured to transfer spin order from the H hydrogen spins at least about 0.01 millitesla (mT), 0.02 mT, 0.03 mT, 0.04 mT, 0.05 mT, 0.06 mT, 0.07 mT, 0.08 mT, 0.09 mT, 0.1 mT, 0.2 mT, 0.3 mT, 0.4 mT, 0.5 mT, 0.6 mT, 0.7 mT, 0.8 mT, 0.9 mT, 1 mT, 2 mT, 3 mT, 4 mT, 5 mT, 6 mT, 7 mT, 8 mT, 9 mT, 10 mT, 11 mT, 12 mT, 13 mT, 14 mT, 15 mT, 16 mT, 17 mT, 18 mT, 19 mT, 20 mT, 21 mT, 22 mT, 23 mT, 24 mT, 25 mT, 26 mT, 27 mT, 28 mT, 29 mT, 30 mT, 31 mT, 32 mT, 33 mT, 34 mT, 35 mT, 36 mT, 37 mT, 38 mT, 39 mT, 40 mT, 41 mT, 42 mT, 43 mT, 44 mT, 45 mT, 46 mT, 47 mT, 48 mT, 49 mT, 50 mT, 51 mT, 52 mT, 53 mT, 54 mT, 55 mT, 56 mT, 57 mT, 58 mT, 59 mT, 60 m T, 9mT, 10mT, 20mT, 30mT, 40mT, 50mT, 60mT, 70mT, 80mT, 90mT, 100mT, 200mT, 300mT, 400mT, 500mT, 600mT, 700mT, 800mT, 900mT, 1,000mT, 2,000mT, 3,000mT, 4,000mT, 5,000mT, 6,000mT or more, up to about 6,000mT, 5,000mT, 4,000mT, 3,000mT, 2,000mT, 1,000mT, 900mT, 800mT, 700mT, 600mT, 500mT, 400mT, 300mT, 200mT , 100mT, 90mT, 80mT, 70mT, 60mT, 50mT, 40mT, 30mT, 20mT, 10mT, 9mT, 8mT, 7mT, 6mT, 5mT, 4mT, 3mT, 2mT, 1mT , 0.9mT, 0.8mT, 0.7mT, 0.6mT, 0.5mT, 0.4mT, 0.3mT, 0.2mT, 0.1mT, 0.09mT, 0.08mT, 0.07mT, 0.06mT, 0.05mT, 0.04mT, 0.03mT, 0.02mT, 0.01mT, or less, or within a range defined by any two of the preceding values. Examples of such sequences may be Goldman's sequence (M. Goldman, H. Johannesson, CR Phys. 2005, 6, 575-581, which is incorporated herein by reference in connection with pulse sequence configurations for transferring spin order), singlet to heteronuclear magnetization (S2hM) sequences, or other sequences used in singlet NMR (e.g., ADAPT, SLIC, etc.).

[0104] In some embodiments, the magnetic shielding can be configured to withstand a magnetic field applied to the solution of at least about 0mG, 0.1mG, 0.2mG, 0.3mG, 0.4mG, 0.5mG, 0.6mG, 0.7mG, 0.8mG, 0.9mG, 1mG, 2mG, 3mG, 4mG, 5mG, 6mG, 7mG, 8mG, 9mG, 10mG, 20mG, 30mG, 40mG, 50mG, 60mG, 70mG, 80mG, 90mG, 100mG, or more, up to about 100mG. The magnetic shield is configured to maintain a magnetic field applied to the solution of less than or equal to 0.5 G, 90 mG, 80 mG, 70 mG, 60 mG, 50 mG, 40 mG, 30 mG, 20 mG, 10 mG, 9 mG, 8 mG, 7 mG, 6 mG, 5 mG, 4 mG, 3 mG, 2 mG, 1 mG, 0.9 mG, 0.8 mG, 0.7 mG, 0.6 mG, 0.5 mG, 0.4 mG, 0.3 mG, 0.2 mG, 0.1 mG, or a magnetic field within a range defined by any two of the preceding values. The magnetic shield may maintain the magnetic field strength in the polarization chamber at such amplitudes during application of the polarizing waveforms to the one or more radio frequency coils.

[0105] Consistent with disclosed embodiments, an RF waveform may be applied to a solution containing a para-hydrogenated precursor.

[0106] Polarization transfer using magnetic field modulation In some embodiments, the polarization transfer magnetic perturbation is performed within a magnetic shield (e.g., a mu-shield, etc.) to achieve a homogeneous low magnetic field. The magnetic shield is designed to provide a low magnetic field in the microtesla (μT) range, below the Earth's magnetic field. 13The low magnetic field may be at least about 0 mG, 0.1 mG, 0.2 mG, 0.3 mG, 0.4 mG, 0.5 mG, 0.6 mG, 0.7 mG, 0.8 mG, 0.9 mG, 1 mG, 2 mG, 3 mG, 4 mG, 5 mG, 6 mG, 7 mG, 8 mG, 9 mG, 10 mG, 20 mG, 30 mG, 40 mG, 50 mG, 60 mG, 70 mG, 80 mG, 90 mG, 100 mG, or more, up to about 100 mG, 90 mG, 100 mG, or more. The value may be 0mG, 80mG, 70mG, 60mG, 50mG, 40mG, 30mG, 20mG, 10mG, 9mG, 8mG, 7mG, 6mG, 5mG, 4mG, 3mG, 2mG, 1mG, 0.9mG, 0.8mG, 0.7mG, 0.6mG, 0.5mG, 0.4mG, 0.3mG, 0.2mG, 0.1mG, or less, or within a range defined by any two of the aforesaid values.

[0107] In such a magnetic field, the polarization is determined by the proton spin and 2 H, 13 C. 15 N, 19 F, and 31 The polarization transfer is performed by utilizing a level pseudocrossing (LAC) between the spin species of interest, including P. In some embodiments, the magnetic field can be tuned to a specific field strength for LAC, for example, as implemented in the SABRE-SHEATH experiment. In various embodiments, the magnetic field strength can be modulated in time to enable robust polarization transfer in larger volumes of samples. For example, the magnetic field strength can be swept through LAC conditions. Alternatively or additionally, the sample can be physically moved within the magnetic field. Such modulation can relax constraints on magnetic field homogeneity and magnetic field offset. Thus, robust polarization transfer can be performed in larger volumes and with greater efficiency. Furthermore, relaxing constraints on magnetic field homogeneity and magnetic field offset can enable the use of less complex, accurate, or expensive polarization systems.

[0108] The lower limit of the magnetic field modulation may be at least about -10μT, -9μT, -8μT, -7μT, -6μT, -5μT, -4μT, -3μT, -2μT, -1μT, -0.9μT, -0.8μT, -0.7μT, -0.6μT, -0.5μT, -0.4μT, -0.3μT, -0.2μT, -0.1μT, or more, up to about -0.1μT, The value may be -0.2μT, -0.3μT, -0.4μT, -0.5μT, -0.6μT, -0.7μT, -0.8μT, -0.9μT, -1μT, -2μT, -3μT, -4μT, -5μT, -6μT, -7μT, -8μT, -9μT, -10μT, or less, or within a range defined by any two of the aforesaid values. The upper modulation limit can be at least about 0.1 μT, 0.2 μT, 0.3 μT, 0.4 μT, 0.5 μT, 0.6 μT, 0.7 μT, 0.8 μT, 0.9 μT, 1 μT, 2 μT, 3 μT, 4 μT, 5 μT, 6 μT, 7 μT, 8 μT, 9 μT, 10 μT, or more, up to but not including about 10 μT, 9 μT, 8 μT, 7 μT, 6 μT, 5 μT, 4 μT, 3 μT, 2 μT, 1 μT, 0.9 μT, 0.8 μT, 0.7 μT, 0.6 μT, 0.5 μT, 0.4 μT, 0.3 μT, 0.2 μT, 0.1 μT, or less, or within a range defined by any two of the foregoing values.

[0109] The magnetic field may be applied to a volume of at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, 1,000 ml, 2,000 ml, or more, and up to about 2,000 ml, 1,000 ml, 90 The modulation may have such amplitude over a volume of 0 ml, 800 ml, 700 ml, 600 ml, 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml or less, or a volume within a range defined by any two of the foregoing values. The modulation may be performed over a duration. The duration may be at least about 100 milliseconds (ms), 200 ms, 300 ms, 400 ms, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, 1,000 ms, 2,000 ms, 3,000 ms, 4,000 ms, 5,000 ms, 6,000 ms, 7,000 ms, 8,000 ms, 9,000 ms, 10,000 ms, 20,000 ms, 30,000 ms, 40,000 ms, 50,000 ms, or more, up to about 50,000 ms. The time may be within a range defined by any two of the foregoing values, or less than or equal to 40,000 milliseconds, 30,000 milliseconds, 20,000 milliseconds, 10,000 milliseconds, 9,000 milliseconds, 8,000 milliseconds, 7,000 milliseconds, 6,000 milliseconds, 5,000 milliseconds, 4,000 milliseconds, 3,000 milliseconds, 2,000 milliseconds, 1,000 milliseconds, 900 milliseconds, 800 milliseconds, 700 milliseconds, 600 milliseconds, 500 milliseconds, 400 milliseconds, 300 milliseconds, 200 milliseconds, 100 milliseconds, or less, or less than or equal to 40,000 milliseconds, 30,0 ...

[0110] Thus, the rate of change of the amplitude of the magnetic field is at least about 0.01 μT / s, 0.02 μT / s, 0.03 μT / s, 0.04 μT / s, 0.05 μT / s, 0.06 μT / s, 0.07 μT / s, 0.08 μT / s, 0.09 μT / s, 0.1 μT / s, 0.2 μT / s, 0.3 μT / s, 0.4 μT / s, 0.5 μT / s, 0.6 μT / s, 0.7 μT / s, sec, 0.8 μT / sec, 0.9 μT / sec, 1 μT / sec, or more, up to about 1 μT / sec, 0.9 μT / sec, 0.8 μT / sec, 0.7 μT / sec, 0.6 μT / sec, 0.5 μT / sec, 0.4 μT / sec, 0.3 μT / sec, 0.2 μT / sec, 0.1 μT / sec, or less, or within a range defined by any two of the foregoing values. The upper limit of the rate of change of the amplitude of the magnetic field may be determined by the capabilities of the equipment used to perform the sweep.

[0111] In some embodiments, when the magnetic field is within the upper and lower limits disclosed above, the spatial deviation of the magnetic field across the volume during modulation is less than about half (or a quarter, or an eighth, or a tenth) of the amplitude of the magnetic field. For example, when the magnetic field strength is less than 2 μT (or greater than −2 μT), the spatial deviation of the magnetic field across the volume during modulation can be less than 1 μT. As an additional example, when the magnetic field strength is less than 10 μT (or greater than −10 μT), the spatial deviation of the magnetic field across the volume during modulation can be less than 5 μT. The spatial deviation can be measured, for example, by taking at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more spatially randomly sampled or equally spatially distributed measurements of the magnetic field within the volume and calculating the standard deviation of the sampled magnetic field measurements. Such homogeneity can be achieved within a large homogeneous magnetic shield, for example, by having a large puncture solenoid valve through the magnetic shield, or by using a large Helmholtz coil with a large homogeneous area to generate the magnetic field amplitude modulation. In some embodiments, the modulation is a sweep of the magnetic field. In some embodiments, the magnetic field amplitude modulation includes a non-adiabatic jump, a monotonic amplitude change, or a combination thereof.

[0112] In some embodiments, following the polarization transfer step, a biologically relevant imaging agent (e.g., 13 C or 15 The non-hydrogen nuclear spins of N) have a nuclear spin polarization of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or a polarization that is within a range defined by any two of the foregoing values. For example, in some embodiments, following the polarization transfer step, the non-hydrogen nuclear spins of the biologically relevant contrast agent are polarized to 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 10% to 35%, 10% to 40%, 10% to 45%, 10% to 50%, 15% to 20%, 15% to 25%, 15% to 30%, 15% to 35%, 15% to 40%, 15% to 45%, 15% to 50%, 20% to 25%, having a nuclear spin polarization of 20% to 30%, 20% to 35%, 20% to 40%, 20% to 45%, 20% to 50%, 25% to 30%, 25% to 35%, 25% to 40%, 25% to 45%, 25% to 50%, 30% to 35%, 30% to 40%, 30% to 45%, 30% to 50%, 35% to 40%, 35% to 45%, 35% to 50%, 40% to 45%, 40% to 50%, or 45% to 50%.

[0113] In some embodiments, this polarization is achieved for a solution volume of at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml or more, up to about 500 ml, 400 ml, 300 ml, 200 ml, 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml or less, or for a volume within a range defined by any two of the foregoing values.

[0114] In some embodiments, after polarization transfer, a portion of the population difference in the para-hydrogenated proton spin state is determined by the target of the biologically relevant imaging agent (e.g., 13 C or 15 N) is transferred to the polarization of the nuclear spins, which may be at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, or within a range defined by any two of the foregoing values. For example, in some embodiments, this portion is between 10% and 15%, between 10% and 20%, between 10% and 25%, between 10% and 30%, between 10% and 35%, between 10% and 40%, between 10% and 45%, between 10% and 50%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 15% and 35%, between 15% and 40%, between 15% and 45%, between 15% and 50%, between 20% and 25%, between 20% and 30%, 20%-35%, 20%-40%, 20%-45%, 20%-50%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 35%-40%, 35%-45%, 35%-50%, 40%-45%, 40%-50%, or 45%-50%.

[0115] In some embodiments, the magnetic field modulation includes a non-adiabatic jump of the magnetic field. The non-adiabatic jump may be performed for a magnetic field where a level pseudo-crossing occurs involving proton spins and non-proton spins. Considering the J-coupling between the nuclear spins in the system, this value may be analytically calculated or identified by plotting the energy levels of the Hamiltonian for different magnetic fields and identifying the LAC. In some embodiments, the duration for which the magnetic field amplitude is in the LAC condition is at most about 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, 0.9 seconds, 0.8 seconds, 0.7 seconds, 0.6 seconds, 0.5 seconds, 0.4 seconds, 0.3 seconds, 0.2 seconds, 0.1 seconds, or less, at least about 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, or more, or within a range defined by any two of the aforementioned values.

[0116] In some embodiments, modulating the amplitude of the magnetic field comprises varying the magnetic field amplitude monotonically (or monotonically over each of a limited number of intervals, such as 1-10 increasing intervals and / or 1-10 decreasing intervals). In some embodiments, modulating the amplitude of the magnetic field comprises linearly varying the amplitude of the magnetic field. The initial magnetic field amplitude of the sweep, the end magnetic field amplitude, and the total duration of the sweep may be optimized for the target molecule. In some embodiments, the magnetic field amplitude during the sweep is within a lower and upper limit. The lower limit can be at least about -2μT, -1μT, -0.9μT, -0.8μT, -0.7μT, -0.6μT, -0.5μT, -0.4μT, -0.3μT, -0.2μT, -0.1μT or more, up to about -0.1μT, -0.2μT, -0.3μT, -0.4μT, -0.5μT, -0.6μT, -0.7μT, -0.8μT, -0.9μT, -1μT, -2μT or less, or within a range defined by any two of the foregoing values. The upper limit can be at least about 0.1 μT, 0.2 μT, 0.3 μT, 0.4 μT, 0.5 μT, 0.6 μT, 0.7 μT, 0.8 μT, 0.9 μT, 1 μT, 2 μT, or more, up to about 2 μT, 1 μT, 0.9 μT, 0.8 μT, 0.7 μT, 0.6 μT, 0.5 μT, 0.4 μT, 0.3 μT, 0.2 μT, 0.1 μT, or less, or within a range defined by any two of the foregoing values. In some embodiments, the duration of the modulation is at least about 100 ms, 200 ms, 300 ms, 400 ms, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, 1,000 ms, 2,000 ms, 3,000 ms, 4,000 ms, 5,000 ms, 6,000 ms, 7,000 ms, 8,000 ms, 9,000 ms, 10,000 ms, or more, up to about 10 ,000 ms, 9,000 ms, 8,000 ms, 7,000 ms, 6,000 ms, 5,000 ms, 4,000 ms, 3,000 ms, 2,000 ms, 1,000 ms, 900 ms, 800 ms, 700 ms, 600 ms, 500 ms, 400 ms, 300 ms, 200 ms, 100 ms, or less, or within a range defined by any two of the foregoing values. In some embodiments, the rate of amplitude change varies along the amplitude profile.In some embodiments, a constant adiabatic sweep is calculated by selecting a specific subpopulation of level pseudocrossings of the spin system. In some embodiments, the magnetic amplitude modulation includes a combination of non-adiabatic jumps, monotonic amplitude modulation, and rate of change sign reversals. In some embodiments, the precursors may be selected or designed such that, following hydrogenation and other potential chemical reactions, one of the products is a biorelevant imaging agent that can be used in hyperpolarized NMR or MRI applications.

[0117] Hydrolysis, purification, and separation The present disclosure provides methods and systems for producing compositions (e.g., clinical dose compositions) comprising a hyperpolarized biologically relevant imaging agent (or a pharma- ceutically acceptable salt thereof) in a solvent. In some embodiments, the biologically relevant imaging agent is produced by additional chemical reactions and / or processing steps after hydrogenation and polarization transfer according to the present disclosure. Such additional chemical reactions and / or processing steps may include, but are not limited to, (i) catalyst filtration and collection (e.g., filtration collecting rhodium and / or iridium atoms), (ii) cleavage of the side arm of the bio-relevant imaging agent precursor molecule (e.g., cleavage of a compound of Formula IIa or Formula IIb, as described herein) by, for example, hydrolysis with aqueous sodium hydroxide, to form a bio-relevant imaging agent and a side arm (e.g., a compound of Formula IIIa or Formula IIIb, as described herein), (iii) washing the solution with an organic solvent and separating any resulting aqueous mixture phase from the organic mixture phase, (iv) evaporative extraction of volatile organics from the aqueous mixture (e.g., using nitrogen gas bubbling), and (v) additional filtration / purification / concentration / polishing steps known in the art.

[0118] The volume of solution containing the biologically relevant contrast agent (e.g., after cleavage) and / or the concentration of biologically relevant contrast agent produced may depend on the volume of solution used for polarization transfer and the concentration of precursors in that solution. Exemplary ranges of solution volumes and precursor concentrations are described herein. As a further specific example, at least about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml or more of solution, up to about 100 ml, 90 ml, 80 ml, 70 ml, 60 ml, 50 ml, 40 ml, 30 ml, 20 ml, 10 ml, 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml or less of solution, or an amount of solution that is within a range defined by any two of the aforementioned values, may be produced. In some embodiments, the solution may contain at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or more of a biologically relevant imaging agent, up to about 500 mM, 400 mM, 300 mM, 200 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, or less of a biologically relevant imaging agent, or an amount of a biologically relevant imaging agent that is within a range defined by any two of the foregoing values.

[0119] In some embodiments, the present disclosure describes a multi-step liquid-liquid separation and purification process for producing a dose (e.g., a clinical dose) of an administration composition comprising a biologically relevant imaging agent (e.g., a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof).

[0120] In some embodiments, the polarization step is followed by the addition of an aqueous mixture (e.g., water) to the solution comprising the first organic solvent and the biologically relevant contrast agent. In some embodiments, the first organic solvent and the aqueous mixture (e.g., water) generate a biphasic solution comprising (i) an organic mixture phase comprising the first organic solvent, and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof. In some embodiments, the first organic solvent and the aqueous mixture (e.g., water) generate a biphasic solution, and a portion of the organic solvent is retained in the aqueous mixture. In some embodiments, the first organic solvent and the aqueous mixture (e.g., water) generate a partial mixture. In some embodiments, the aqueous mixture phase is separated from the organic mixture phase to provide an administration composition. In some embodiments, the aqueous mixture phase is separated from the organic mixture phase for further processing (e.g., one or more washing steps with a second organic solvent, one or more additional separation steps, one or more evaporation steps).

[0121] In some embodiments (i.e., for PHIP-SAH procedures), following the polarization step, the side arm is cleaved (e.g., via hydrolysis with an aqueous mixture) from the target molecule precursor (e.g., biorelevant imaging agent precursor) to generate the target molecule (e.g., biorelevant imaging agent) and an unbound side arm (such as a compound of formula IIIa described herein). In some embodiments, following the polarization step, the side arm is cleaved by mixing a solution (comprising the first organic solvent and the polarized product, e.g., hyperpolarized biorelevant imaging agent or a pharma- ceutically acceptable salt thereof) with a hydrolysis agent, such as a base (e.g., sodium hydroxide) in an aqueous solution. In some embodiments, the first organic solvent and the aqueous mixture (e.g., water) generate a biphasic solution. In some embodiments, the first organic solvent and the aqueous mixture (e.g., water) generate a biphasic solution, with a portion of the organic solvent being retained in the aqueous mixture. In some embodiments, the first organic solvent and the aqueous mixture (e.g., water) generate a partial mixture.

[0122] FIG. 1 presents an embodiment 100 of the multi-step liquid-liquid separation and purification process of the present disclosure. In step 110, a solution is obtained and provided in a container, the solution comprising a first organic solvent, an aqueous mixture, a hyperpolarized bio-relevant contrast agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm. In some embodiments, the solution may be obtained by a method of producing a hyperpolarized bio-relevant contrast agent according to the present disclosure. In step 120, the solution from step 110 is processed through one or more washing steps, the solution being washed with a second organic solvent. Step 120 forms (i) an organic mixture phase comprising the first organic solvent, the second organic solvent, and an unbound side arm (if present), and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized bio-relevant contrast agent or a pharma- ceutically acceptable salt thereof. In step 130, the mixture from step 120 is processed through one or more separation steps, where an organic mixture phase is separated from an aqueous mixture phase, such as by transferring either the organic mixture phase or the aqueous mixture phase to a separate vessel. Following step 130, the resulting aqueous mixture phase may be recycled through step 120 and / or step 130 one or more times to further process the aqueous mixture. In optional step 140, the aqueous mixture from step 120 and step 130 is optionally processed through one or more evaporation steps, where the aqueous mixture is subjected to organic vapor extraction conditions to evaporate at least a portion of the organic solvent remaining in the aqueous mixture. In some embodiments, the organic vapor extraction conditions include bubbling with an inert gas, such as nitrogen gas (e.g., nitrogen gas bubbling using a glass frit at 60° C. and 150 mbar absolute pressure with vacuum). Following optional step 140, the resulting aqueous mixture phase may be recirculated one or more times through steps 120, 130, and / or 140 to further process the aqueous mixture. In step 150, an administration composition is obtained from the aqueous mixture from steps 120 / 130 / 140, comprising the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof.

[0123] FIG. 2 presents an embodiment 200 of a multi-step liquid-liquid separation and purification process of the present disclosure. In step 210, a solution is obtained and provided in a container, the solution comprising a first organic solvent, an aqueous mixture, a hyperpolarized bio-relevant contrast agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm. In some embodiments, the solution may be obtained by a method of producing a hyperpolarized bio-relevant contrast agent according to the present disclosure. In step 220, the solution from step 210 is processed through one or more evaporation steps, the solution being subjected to organic vapor extraction conditions to evaporate at least a portion of the organic solvent remaining in the solution. In some embodiments, the organic vapor extraction conditions include bubbling with an inert gas, such as nitrogen gas (e.g., nitrogen gas bubbling using a glass frit at 60° C. and 150 mbar absolute pressure with vacuum). In optional step 230, the solution resulting from step 220 (including the aqueous mixture and the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof) is processed through one or more washing steps, where the solution is washed with a second organic solvent. Step 230 forms (i) an organic mixture phase comprising the remaining first organic solvent, the second organic solvent, and at least a portion of the unbound side arm (if present), and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof. In optional step 240, the mixture from step 230 is processed through one or more separation steps, where the organic mixture phase is separated from the aqueous mixture phase, such as by transferring either the organic mixture phase or the aqueous mixture phase to separate containers. Following step 240, the resulting aqueous mixture phase may be recirculated through steps 220, 230, and / or 240 one or more times to further process the aqueous mixture. In step 250, an administration composition is obtained from the aqueous mixture from steps 220 / 230 / 240, comprising the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof.

[0124] In some embodiments, a target molecule precursor having a side arm (e.g., a biorelevant imaging agent precursor) is polarized (e.g., hydrogenated via a PHIP-SAH process) in a solution containing a first organic solvent. In some embodiments, the first organic solvent is selected such that hydrogenation of the precursor is efficient and facilitates high spin order on the para-hydrogenated proton. In some embodiments, the first solvent is selected from acetone, ethanol, methanol, chloroform, ethyl acetate, methyl ethyl ketone, acetophenone, hexone, cyclohexanone, or cyclopentanone. In some embodiments, the first solvent is acetone. In some embodiments, the first organic solvent has a solubility in water of greater than 50 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water of greater than 55 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water of greater than 60 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 65 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 70 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 75 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 80 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 85 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 90 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 95 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 100 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 125 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 150 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 200 millimolar (mM) at 20° C.In some embodiments, the first organic solvent has a solubility in water greater than 300 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 400 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water greater than 500 millimolar (mM) at 20° C. In some embodiments, the first organic solvent has a solubility in water at 20° C. that is greater than the solubility of chloroform in water at 20° C. In some embodiments, the first organic solvent does not include chloroform.

[0125] In some embodiments, the solution containing an aqueous solvent (e.g., water) and a first organic solvent is subjected to one or more washing steps with a second organic solvent. In some embodiments, the second organic solvent is a biocompatible solvent. In some embodiments, the second organic solvent is a Class 2 solvent according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. In some embodiments, the second organic solvent comprises one or more Class 2 solvents selected from acetonitrile, chlorobenzene, chloroform, cyclohexane, dibromomethane, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, N,N-dimethylacetamide, N,N-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2-methoxyethanol, methylbutylketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene, 1,1,2-trichloroethene, or xylene. In some embodiments, the second organic solvent is a Class 3 solvent according to the ICH guidelines. In some embodiments, the second organic solvent comprises one or more Class 3 solvents selected from acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, diethyl ether, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate. In some embodiments, the second organic solvent is a Class 2 or Class 3 solvent according to the ICH guidelines. In some embodiments, the second organic solvent facilitates separation and / or extraction of one or more of the cleaved sidearm residue, the first organic solvent, and any catalyst residue (e.g., rhodium atom, iridium atom) from the aqueous solvent.

[0126] In some embodiments, the second organic solvent has a solubility in water greater than 50 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 75 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 100 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 150 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 200 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 250 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 300 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 350 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 400 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 450 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water greater than 500 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water less than 500 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water less than 450 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water less than 400 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water less than 350 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water less than 300 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water of less than 250 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water of less than 200 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water of less than 150 millimolar (mM) at 20° C.In some embodiments, the second organic solvent has a solubility in water of less than 100 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water of less than 75 millimolar (mM) at 20° C. In some embodiments, the second organic solvent has a solubility in water of less than 50 millimolar (mM) at 20° C.

[0127] In some embodiments, a solution containing an aqueous solvent (e.g., water) and a first organic solvent is subjected to one or more evaporation steps. In some embodiments, a solution containing an aqueous solvent (e.g., water) and a first organic solvent is subjected to one or more evaporation steps to reduce or remove an amount of volatile organic material from the solution. In some embodiments, the evaporation step includes flushing and / or bubbling a gas (e.g., nitrogen gas) through the solution. In some embodiments, the evaporation step reduces the amount of organic solvent in the solution.

[0128] In some embodiments, a solution containing an aqueous solvent (e.g., water) and a first organic solvent is subjected to (i) one or more washing steps with a second organic solvent, (ii) one or more evaporation steps, and (iii) optionally, one or more additional processing steps (e.g., filtration, filling / finishing, dilution) to result in the production of an administration composition comprising a biologically relevant imaging agent (i.e., a composition that is pharma- ceutically acceptable for administration to a subject).

[0129] In some embodiments, the present disclosure provides an administration composition (i.e., a composition that is pharma- ceutically acceptable for administration to a subject) that includes a biorelevant imaging agent and a pharma- ceutically acceptable carrier (e.g., a solvent). In some embodiments, the administration composition includes a level of residual organic solvent that is below the toxicity limit based on ICH Q3C guidance. In some embodiments, the administration composition includes a level of residual ICH Class 1, Class 2, or Class 3 organic solvent that is below the toxicity limit provided in Table 1. As used herein, "ppm" refers to the parts per million concentration of residual organic solvent in a pharma- ceutically acceptable carrier. [Table 1-1] [Table 1-2]

[0130] system In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container and a second container fluidically connected to the first container. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container and a second container fluidically connected to the first container through a first fluid transfer element. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container, a second container fluidically connected to the first container and a third container fluidically connected to the second container. In some embodiments, the present disclosure describes a system for implementing the method of the present disclosure, the system comprising a first container, a second container fluidically connected to the first container through a first fluid transfer element and a third container fluidically connected to the second container through a second fluid transfer element.

[0131] In some embodiments, the system further comprises a magnetic guide system. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over at least a portion of the system. In some embodiments, the system further comprises a magnetic guide system comprising one or more solenoid valves providing the defined magnetic field. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over the first vessel, the second vessel, and / or the optional third vessel. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over the first fluid transfer element and / or the optional second fluid transfer element. In some embodiments, the system further comprises a magnetic guide system providing a defined magnetic field over the entire system. In some embodiments, the defined magnetic field prevents undesired hyperpolarization loss in the solution during implementation of the disclosed method.

[0132] 3 presents an embodiment 300 of a multi-step liquid-liquid separation and purification system of the present disclosure. System 300 comprises a first vessel 310, a second vessel 320, and a third vessel 330. First vessel 310 is fluidly connected to second vessel 320 through a first fluid transfer element 315. Second vessel 320 is fluidly connected to third vessel 330 through a second fluid transfer element 325.

[0133] The system 300 optionally comprises a magnetic guiding system 350 that provides a defined magnetic field across at least a portion of the system 300. In some embodiments, the magnetic guiding system 350 comprises one or more solenoid valves that provide the defined magnetic field. In some embodiments, the magnetic guiding system 350 provides a defined magnetic field across the first vessel 310, the second vessel 320, and / or the third vessel 330. In some embodiments, the magnetic guiding system 350 provides a defined magnetic field across the first fluid transfer element 315 and / or the second fluid transfer element 325. In some embodiments, the magnetic guiding system 350 provides a defined magnetic field across the entire system 300. In some embodiments, the defined magnetic field prevents undesired hyperpolarization loss in the solution during implementation of the methods of the present disclosure.

[0134] In some embodiments, system 300 may be used in implementing the multi-step liquid-liquid separation and purification process of the present disclosure. In some embodiments, a solution is provided in vessel 310, the solution comprising a first organic solvent, an aqueous mixture, a hyperpolarized bio-relevant contrast agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm. In some embodiments, the solution may be obtained by a method of producing a hyperpolarized bio-relevant contrast agent according to the present disclosure, including embodiments in which the method of producing a hyperpolarized bio-relevant contrast agent is completed in part or in whole in vessel 310. The solution in vessel 310 is processed through one or more washing steps, in which the solution is washed with a second organic solvent, which forms in vessel 310 (i) an organic mixture phase comprising the first organic solvent, the second organic solvent, and an unbound side arm (if present), and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized bio-relevant contrast agent or a pharma- ceutically acceptable salt thereof. The resulting mixture in vessel 310 is processed through one or more separation steps, where the aqueous mixture phase is separated from the organic mixture phase by using a first fluid transfer element 315 to transfer the aqueous mixture phase into vessel 320. The resulting aqueous solution in vessel 320 is processed through one or more additional washing steps, where the solution is washed again with a second organic solvent, which forms in vessel 320 (i) an organic mixture phase comprising the remaining first organic solvent and at least a portion of the second organic solvent, and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof. The resulting mixture in vessel 320 is processed through one or more additional separation steps, where the aqueous mixture phase is separated from the organic mixture phase by using a second fluid transfer element 325 to transfer the aqueous mixture phase into vessel 330. The resulting aqueous mixture in vessel 330 is then processed through one or more evaporation steps, where the aqueous mixture is subjected to organic vapor extraction conditions to evaporate at least a portion of the organic solvent remaining in the aqueous mixture. In some embodiments, the organic vapor extraction conditions include bubbling with an inert gas such as nitrogen gas (eg, nitrogen gas bubbling using a glass frit at 60° C. and 150 mbar absolute pressure with vacuum).In some embodiments, the administration composition is obtained from the aqueous mixture in the container 330 after one or more evaporation steps, and the administration composition comprises a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof.

[0135] In some embodiments, the first container 310, the second container 320, and / or the third container 330 each independently comprise a commercially available or homemade container made from a suitable chemically and physically (temperature, pressure) material, such as plastic (e.g., polyetheretherketone [PEEK], polytetrafluoroethylene [PTFE], fluorinated ethylenepropylene [FEP], ethylenetetrafluoroethylene [ETFE], perfluoroelastomer [FFKM], polypropylene [PP], polyethylene [PE]), glass, and non-magnetic metals / alloys. In some embodiments, the first container 310, the second container 320, and / or the third container 330 each independently comprise a centrifuge tube, optionally between 10 ml and 1000 ml, and may optionally be made of polypropylene (PP). In some embodiments, the first container 310 and / or the second container 320 may comprise two chambers in a single piece produced in an injection molding process using plastic (PP, PEEK, or other suitable molded plastic).

[0136] In some embodiments, the first fluid movement element 315 and / or the second fluid movement element 325 may independently include (a) at least one valve component, (b) at least one tubing component, (c) at least one tubing connector component, or (d) any combination of (a), (b), and (c). In some embodiments, the valve component is manually actuated (e.g., stopcock valve), electrically actuated (e.g., solenoid valve, motor), pressure actuated (e.g., pneumatic), or combinations thereof. In some embodiments, the valve component includes a 3 / 2-way Burkert type 6724 solenoid valve. In some embodiments, the tubing component includes microfluidic tubing (e.g., standard tubing for laboratory use). In some embodiments, the tubing dimensions are 0.5 mm to 4 mm outer diameter (e.g., 1.5 mm) and 0.25 to 1 mm inner diameter (e.g., 0.5 mm). In some embodiments, the tubing components include commercially available or homemade tubing made from chemically and physically (temperature, pressure) suitable materials such as plastics (e.g., polyetheretherketone [PEEK], polytetrafluoroethylene [PTFE], fluorinated ethylenepropylene [FEP], ethylenetetrafluoroethylene [ETFE], perfluoroelastomers [FFKM], polypropylene [PP], polyethylene [PE]), glass, and non-magnetic metals / alloys. In some embodiments, the tubing connector components include microfluidic fittings (e.g., standard fittings for laboratory use). In some embodiments, the tubing connector components include 1 / 4"-28 fittings with flat-bottom ferrules as seals. In some embodiments, the tubing connector components include molded plastic tubing connections or orifices between two chambers of the same part.

[0137] 4 presents an embodiment 400 of a multi-step liquid-liquid separation and purification system of the present disclosure. System 400 comprises a first vessel 410, a second vessel 420, and a third vessel 430. First vessel 410 is fluidly connected to second vessel 420 through a first fluid transfer element 415. Second vessel 420 is fluidly connected to third vessel 430 through a second fluid transfer element 425.

[0138] 5 presents an embodiment 500 of a multi-step liquid-liquid separation and purification system of the present disclosure. The system 500 comprises a first vessel 510, a second vessel 520, and a third vessel 530. The first vessel 510 is fluidly connected to the second vessel 520 through a first fluid transfer element 515. The second vessel 520 is fluidly connected to the third vessel 530 through a second fluid transfer element 525. The system 500 optionally comprises a magnetic guiding system 550 that provides a defined magnetic field across at least a portion of the system 500. In some embodiments, the magnetic guiding system 550 may include one or more of a permanent magnet, an electromagnet, a ferromagnetic core (e.g., iron core) electromagnet, a ferrimagnetic core (e.g., ferrite core) electromagnet, a superconducting magnet, a solenoid valve, a Helmholtz coil, a Maxwell coil, a birdcage coil, a cross coil, a saddle coil, a pair of saddle coils, a bipolar magnet, or any combination thereof. In some embodiments, the magnetic guide system 550 is configured to generate a magnetic field or magnetic field gradient in the vicinity of all or a portion of the system 500. For example, in some embodiments, the magnetic guide system 550 is configured to generate a magnetic field or magnetic field gradient in the vicinity of any one, two, three, four, five, or six of the first vessel 510, the first fluid movement element 515, the second vessel 520, the second fluid movement element 525, and the third vessel 530. In some embodiments, the magnetic field guide 550 is configured to mitigate loss of polarization due to spurious magnetic fields associated with any one, two, three, four, five, or six of the first vessel 510, the first fluid movement element 515, the second vessel 520, the second fluid movement element 525, and the third vessel 530, or spurious magnetic fields associated with the environment surrounding the system 500.

[0139] In some embodiments, the magnetic field is at least about 10 nT, 20 nT, 30 nT, 40 nT, 50 nT, 60 nT, 70 nT, 80 nT, 90 nT, 100 nT, 200 nT, 300 nT, 400 nT, 500 nT, 600 nT, 700 nT, 800 nT, 90 nT, 1 μT, 2 μT, 3 μT, 4 μT, 5 μT, 6 μT, 7 μT, 8 μT, 9 μT, 10 μT, 20 μT, 30 μT, 40 μT, 50 μT, 60 μT, 70 μT, 80 μT, 90 μT, 100 μT, 200 μT T, 300μT, 400μT, 500μT, 600μT, 700μT, 800μT, 900μT, 1mT, 2mT, 3mT, 4mT, 5mT, 6mT, 7mT, 8mT, 9mT, 10mT, 20mT, 30mT, 40mT, 50mT, 60mT, 70mT, 80mT, 90mT, 100mT, 200mT, 300mT, 400mT, 500mT, 600mT, 700mT, 800mT, 900mT, 1,000mT or more, up to about 1,000mT, 9 00mT, 800mT, 700mT, 600mT, 500mT, 400mT, 300mT, 200mT, 100mT, 90mT, 80mT, 70mT, 60mT, 50mT, 40mT, 30mT, 20mT, 10mT, 9mT, 8m T, 7mT, 6mT, 5mT, 4mT, 3mT, 2mT, 1mT, 900μT, 800μT, 700μT, 600μT, 500μT, 400μT, 300μT, 200μT, 100μT, 90μT, 80μT, 70μT, 60μT, 5 The magnetic field strength may be 0 μT, 40 μT, 30 μT, 20 μT, 10 μT, 9 μT, 8 μT, 7 μT, 6 μT, 5 μT, 4 μT, 3 μT, 2 μT, 1 μT, 900 nT, 800 nT, 700 nT, 600 nT, 500 nT, 400 nT, 300 nT, 200 nT, 100 nT, 90 nT, 80 nT, 70 nT, 60 nT, 50 nT, 40 nT, 30 nT, 20 nT, 100 nT, or an average magnetic field strength that is within a range defined by any two of the preceding values.

[0140] Clinically relevant purity Consistent with the disclosed embodiments, steps, methods, and systems described herein, the hyperpolarized biologically relevant imaging agent may be separated from other substances in the original solution (e.g., catalyst, original solvent, reaction products, etc.) For example, most of the hydrogenation catalyst present in the original solution may be removed from the administered composition. In some embodiments, the administered composition contains up to about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, or less of a hydrogenation catalyst, at least about 0.001%, 0.002%, 0.003%, or less. In some embodiments, the hydrogenation catalyst may be present in a concentration range of 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more of the hydrogenation catalyst, or an amount of the hydrogenation catalyst that is within a range defined by any two of the foregoing values.Similarly, the administered compositions should contain up to about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, or less of cleavage by-products (e.g., side arms or other residues of cleavage), at least about 0.5%, 0.7%, 0.8%, 0.9%, 0.1%, 0.0%, 0.1%, 0.0%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 0.1%, 0.0 ... 001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or more of the cleavage byproducts, or an amount of the cleavage byproducts that is within a range defined by any two of the foregoing values, may be retained.

[0141] In some embodiments, the methods and systems described herein produce administered compositions having a concentration of hyperpolarized biologically relevant imaging agent of at least about 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more, up to about 500 mM, 450 mM, 400 mM, 350 mM, 300 mM, 250 mM, 200 mM, 150 mM, 100 mM, or less, or within a range defined by any two of the foregoing values.

[0142] In some embodiments, the methods and systems described herein produce administered compositions that have a polarization of hyperpolarized biologically relevant contrast of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or within a range defined by any two of the foregoing values. For example, in some embodiments, the methods and systems described herein may be configured to provide a method for determining whether the polarization of the hyperpolarized biologically relevant contrast is between 10% and 15%, 10% and 20%, 10% and 25%, 10% and 30%, 10% and 35%, 10% and 40%, 10% and 45%, 10% and 50%, 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, 15% and 45%, 15% and 50%, 20% and 25%. %, 20%-30%, 20%-35%, 20%-40%, 20%-45%, 20%-50%, 25%-30%, 25%-35%, 25%-40%, 25%-45%, 25%-50%, 30%-35%, 30%-40%, 30%-45%, 30%-50%, 35%-40%, 35%-45%, 35%-50%, 40%-45%, 40%-50%, or 45%-50%.

[0143] In some embodiments, the methods and systems described herein provide for a method for determining whether or not a concentration of a catalyst, precursor, or cleavage by-product is at most about 1 μM, 900 nanomolar (nM), 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or In some embodiments, the present invention produces a dosing composition that may be at or below about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, or more, or within a range defined by any two of the preceding values. The methods and systems described herein produce administered compositions having a hyperpolarized biologically relevant imaging purity of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, or less, or within a range defined by any two of the preceding values. In some embodiments, at least a fraction of the hyperpolarized compound is separated from the cleaved side arms or other reaction by-products, if present.

[0144] transportation Consistent with the disclosed embodiments, polarization transfer and use of the biorelevant contrast agent can occur at different locations. In some embodiments, the administered composition is transported to a different location. In some embodiments, the administered composition is transported to a different location. The disclosed embodiments are not necessarily limited to any particular transport distance or duration. Instead, the maximum distance or duration can be determined based on the target molecule, the original degree or polarization, the final degree of polarization required, and the transport conditions. In some embodiments, the administered composition is transported at least 1 meter with a suitable transport device.

[0145] Consistent with disclosed embodiments, the transport device may be configured to transport a sample of a precursor or a biologically relevant contrast agent. The transport device may be arranged and configured to simultaneously transport one or more samples (e.g., one or more administration compositions). The transport device may include a transport chamber configured to receive one or more samples. The transport device may be configured to maintain the transport chamber within a predetermined temperature range and a predetermined magnetic field strength. The transport device may be configured to maintain one or more samples in a magnetic field of at least about 10G, 20G, 30G, 40G, 50G, 60G, 70G, 80G, 90G, 100G, 200G, 300G, 400G, 500G, 600G, 700G, 800G, 900G, 1,000G, or more, up to about 1,000G, 900G, 800G, 700G, 600G, 500G, 400G, 300G, 200G, 100G, 90G, 80G, 70G, 60G, 50G, 40G, 30G, 20G, 10G, or less, or in a magnetic field within a range defined by any two of the foregoing values.

[0146] A permanent magnet or electromagnet included in the transport device can provide the magnetic field. In some embodiments, the permanent magnet or electromagnet is shielded to reduce the strength of the magnetic field outside the transport device. The transport device can also include a cooling system. The cooling system can be configured to maintain the sample at a predetermined temperature or within a predetermined temperature range during transport. For example, the cooling system can be configured to maintain the sample at a temperature below 270K, below 80K, or below 4K. In some embodiments, the transport device is configured to maintain the sample at approximately liquid nitrogen temperature. The transport device can include thermal insulation between the cooling system and the exterior of the transport device to minimize heat exchange with the external environment. In some embodiments, the cooling system is configured to maintain the temperature of the sample using a cold gas flow. In some embodiments, the cooling system is configured to maintain the temperature of the sample using a cooling liquid. In some embodiments, the transport device includes a dewar that provides cooling for the sample. To distribute the hyperpolarized sample over long distances, the container can be transported by standard transport vehicles such as planes, trains, trucks, cars, and ships.

[0147] In some embodiments, the administered composition containing the hyperpolarized biologically relevant contrast agent is transported in a transport device. In some embodiments, the relaxation time of the hyperpolarized biologically relevant contrast agent in the transport device is at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or more, up to about 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, or less, or within a range defined by any two of the foregoing values.

[0148] Generation of polarized biorelevant contrast agents Hereinafter, a first exemplary process for producing a polarized biologically relevant imaging agent according to various embodiments will be described. In some embodiments, the first process includes providing a composition comprising a compound of formula Ia. In some embodiments, the compound of formula Ia is a compound selected from the group consisting of: (i) a cyclic amine; 1 H (proton), 2 a Z group comprising a carbon-carbon double bond (-C=C-) substituted to include H (deuterium), or a combination thereof; (ii) a carbon-carbon triple bond (-C≡C-); an R group comprising a PHIP transfer moiety as described herein; an R group comprising an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety as described herein; and an R group comprising a biorelevant imaging agent as described herein.

[0149] In some embodiments, the double or triple bond in the compound of formula Ia is hydrogenated with para-hydrogen to form a para-hydrogenated derivative of the compound of formula Ia, the para-hydrogenated derivative being a compound having the structure of formula IIa. In some embodiments, the compound of formula IIa is a compound having the structure of (i) 1 H (proton), 2 Para-hydrogenated carbon-carbon single bonds (-CH*-CH*-) substituted to contain H (deuterium), or combinations thereof; or (ii) 1 H (proton), 2 The compound of formula Ia comprises Z', a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or a combination thereof, where H* is hydrogen with spin order derived from para-hydrogen, an R1 group comprising a PHIP transfer moiety as described herein, an R2 group comprising an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety as described herein, and an R3 group comprising a biorelevant imaging agent as described herein. In some embodiments, the compound of formula Ia is hydrogenated with para-hydrogen using a hydrogenation process as described herein.

[0150] In some embodiments, a polarization transfer waveform is applied to transfer nuclear spin order from at least one H* in the side arm of the compound of formula IIa to any non-hydrogen nuclear spins in the biologically relevant imaging agent of the compound of formula IIa, as described herein, thereby forming a derivative of the compound of formula IIa with a hyperpolarized biologically relevant imaging agent. In some embodiments, the nuclear spin order is transferred using any polarization transfer process described herein.

[0151] A second exemplary process for producing a polarized biologically relevant imaging agent according to various embodiments of the present disclosure is now described. In some embodiments, the second process comprises providing a composition comprising a compound of formula IIa. In some embodiments, formula IIa is as described herein: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that has been substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 a Z' group that is a para-hydrogenated carbon-carbon double bond (-CH*=CH*-) substituted to include H (deuterium), or a combination thereof, where H* is a hydrogen with spin order derived from para-hydrogen; an R1 group that comprises a PHIP transfer moiety as described herein; an R2 group that comprises an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety as described herein; and an R3 group that comprises a biorelevant imaging agent as described herein.

[0152] In some embodiments, a polarization transfer waveform is applied to transfer nuclear spin order from at least one H* in the side arm of the compound of formula IIa to any non-hydrogen nuclear spins in the biologically relevant imaging agent of the compound of formula IIa, as described herein, thereby forming a derivative of the compound of formula IIa having a hyperpolarized biologically relevant imaging agent.

[0153] In some embodiments, the derivative compound of formula IIa is hydrolyzed to form a composition comprising a hyperpolarized biologically relevant imaging agent and a separate side arm compound of formula IIIa. In some embodiments, the compound of formula IIIa is a compound of formula IIa, as described herein: (i) 1 H (proton), 2 a para-hydrogenated carbon-carbon single bond (-CH*-CH*-) that has been substituted to contain H (deuterium), or a combination thereof; or (ii) 1 H (proton), 2 H, or a combination thereof; an R1' group that includes a parahydrogen induced polarization (PHIP) transfer moiety as described herein; and an R2 group that includes an optionally substituted hydrocarbon, alkoxy group, primary amine, secondary amine, or tertiary amine, or a solubilizing moiety as described herein.

[0154] In some embodiments, the hyperpolarized biologically relevant imaging agent is washed one or more times with an organic solvent. In some embodiments, the non-hydrogen nuclear spins in the biologically relevant imaging agent have a non-hydrogen spin polarization after the washing step of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or within a range defined by any two of the foregoing values.

[0155] In some embodiments, the first or second process includes one or more additional steps or operations. In some embodiments, the first or second process omits one or more steps or operations. In some embodiments, one or more steps or operations of the first or second process are combined. In some embodiments, all steps or operations of the first or second process are combined to obtain a complete process for producing a hyperpolarized imaging agent from a precursor having the structure of formula Ia.

[0156] A third exemplary process for producing a polarized biologically relevant imaging agent according to various embodiments is now described. In the example shown, the process comprises providing a composition comprising a compound of formula Ib. In some embodiments, the compound of formula In comprises a Z group comprising an ethynyl (-C≡C-) group, an optionally substituted prop-2-ynyl (-CC≡C-) group, an optionally substituted ethenyl (-C=C-) group, an optionally substituted prop-2-enyl (-CC=C-) group, or an optionally substituted but-3-enyl (-CCC=C-) group as described herein; an R2 group comprising an optionally substituted hydrocarbon group, alkyl group, cyclic alkyl group, aryl group, carboxyl group, keto group, or solubilizing moiety as described herein; and an R3 group comprising an acyl derivative of a biologically relevant imaging agent as described herein.

[0157] In some embodiments, the double bond or triple bond in the compound of formula Ib is hydrogenated with para-hydrogen to form a para-hydrogenated derivative of the compound of formula Ib, the para-hydrogenated derivative being a compound having the structure of formula IIb. In some embodiments, the compound of formula IIb comprises a Z' group comprising a para-hydrogenated ethenyl (-CH*=CH*-), an optionally substituted para-hydrogenated prop-2-enyl (-C-CH*=CH*-), an optionally substituted para-hydrogenated ethanyl (-CH*-CH*-), an optionally substituted para-hydrogenated propanyl (-C-CH*-CH*-), or an optionally substituted para-hydrogenated butanyl (-CC-CH*=CH*-) group, where H* is a hydrogen with spin order derived from para-hydrogen, an R2 group comprising an optionally substituted hydrocarbon group, alkyl group, cyclic alkyl group, aryl group, carboxyl group, keto group, or alkoxy group or solubilizing moiety, as described herein, and an R3 group comprising an acyl derivative of a biologically relevant imaging agent as described herein. In some embodiments, the compound of formula Ib is hydrogenated with para-hydrogen using a hydrogenation process as described herein.

[0158] In some embodiments, a polarization transfer waveform is applied to transfer nuclear spin order from at least one H* in the side arm of the compound of formula IIb to any non-hydrogen nuclear spins in the acyl derivative of the biologically relevant imaging agent of the compound of formula IIb, as described herein, thereby forming a derivative of the compound of formula IIb with the acyl derivative of the biologically relevant imaging agent hyperpolarized. In some embodiments, the nuclear spin order is transferred using any polarization transfer process described herein.

[0159] A fourth exemplary process for producing a polarized biologically relevant imaging agent according to various embodiments of the present disclosure is now described. In the example shown, the process includes providing a composition comprising a compound of formula IIb. In some embodiments, Formula IIb comprises a Z′ group that comprises a para-hydrogenated ethenyl (—CH*═CH*—) group, an optionally substituted para-hydrogenated prop-2-enyl (—C—CH*═CH*—) group, an optionally substituted para-hydrogenated ethanyl (—CH*—CH*—) group, an optionally substituted para-hydrogenated propanyl (—C—CH*—CH*—) group, or an optionally substituted para-hydrogenated butanyl (—CC-CH*═CH*—) group, where H* is a hydrogen with spin order derived from para-hydrogen; an R2 group that comprises an optionally substituted hydrocarbon group, alkyl group, cyclic alkyl group, aryl group, carboxyl group, keto group, or alkoxy group or solubilizing moiety, as described herein; and an R3 group that comprises an acyl derivative of a biologically relevant imaging agent, as described herein.

[0160] In some embodiments, a polarization transfer waveform is applied to transfer nuclear spin order from at least one H* in the side arm of the compound of formula IIb to any non-hydrogen nuclear spins in the acyl derivative of the biologically relevant imaging agent of the compound of formula IIb, as described herein, thereby forming a derivative of the compound of formula IIb with a hyperpolarized acyl derivative of the biologically relevant imaging agent.

[0161] In some embodiments, the derivative compound of formula IIb is hydrolyzed to form a composition comprising a hyperpolarized biologically relevant imaging agent and a separate side arm compound of formula IIIb. In some embodiments, the compound of formula IIIb comprises a Z' group comprising a parahydrogenated ethenyl (-CH*=CH*-), an optionally substituted parahydrogenated prop-2-enyl (-C-CH*=CH*-), an optionally substituted parahydrogenated ethanyl (-CH*-CH*-), an optionally substituted parahydrogenated propanyl (-C-CH*-CH*-), or an optionally substituted parahydrogenated butanyl (-CC-CH*=CH*-) group, as described herein, and an R2 group comprising an optionally substituted hydrocarbon, alkyl, cyclic alkyl, aryl, carboxyl, keto, or alkoxy group or solubilizing moiety, as described herein.

[0162] In some embodiments, the hyperpolarized biologically relevant imaging agent is washed one or more times with an organic solvent. In some embodiments, the non-hydrogen nuclear spins in the biologically relevant imaging agent have a non-hydrogen spin polarization after the washing step of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or within a range defined by any two of the foregoing values.

[0163] In some embodiments, the third or fourth process includes one or more additional steps or operations. In some embodiments, the third or fourth process omits one or more steps or operations. In some embodiments, one or more steps or operations of the third or fourth process are combined. In some embodiments, all steps or operations of the third or fourth process are combined to obtain a complete process for producing a hyperpolarized imaging agent from a precursor having the structure of formula Ib.

[0164] Enumerated Embodiments The foregoing non-limiting embodiments disclosed herein include the following:

[0165] Enumerated embodiments 1. A method for producing an administration composition comprising a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, the method comprising: obtaining a solution in a container, the solution comprising a first organic solvent, an aqueous mixture, the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm; one or more washing steps, wherein the solution is washed with a second organic solvent, thereby forming (i) an organic mixture phase comprising the first organic solvent, the second organic solvent, and the optional unbound side arm, and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof; one or more separation steps, wherein the organic mixture phase is separated from the aqueous mixture phase, and either the organic mixture phase or the aqueous mixture phase is transferred to a separate container; and obtaining the administration composition from the aqueous mixture comprising the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof.

[0166] Enumerated embodiment 2. The method of embodiment 1, wherein the method further comprises one or more evaporation steps, wherein the aqueous mixture phase is subjected to organic vapor extraction conditions to evaporate at least a portion of the organic solvent remaining in the aqueous mixture.

[0167] Enumerated embodiment 3. A method for producing an administration composition comprising a hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof, the method comprising: obtaining a solution in a container, the solution comprising a first organic solvent, an aqueous mixture, the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof, and optionally an unbound side arm; one or more evaporation steps, wherein the solution is subjected to organic vapor extraction conditions to evaporate a portion of the organic solvent, and the optional unbound side arm, from the solution, thereby providing an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof; and obtaining the administration composition from the aqueous mixture comprising the hyperpolarized biologically relevant contrast agent or a pharma- ceutically acceptable salt thereof.

[0168] Enumerated embodiment 4. The method of embodiment 3, further comprising: one or more washing steps, wherein the solution or aqueous mixture is washed with a second organic solvent, thereby forming (i) an organic mixture phase comprising the first organic solvent, the second organic solvent, and optional unbound side arms, and (ii) an aqueous mixture phase comprising the aqueous mixture and the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof; and one or more separation steps, wherein the organic mixture phase is separated from the aqueous mixture phase, and either the organic mixture phase or the aqueous mixture phase is transferred to a separate container.

[0169] Enumerated embodiment 5. The method of obtaining a solution in a container, comprising: obtaining a solution comprising a first organic solvent and a biorelevant imaging agent precursor dissolved in the first organic solvent, the biorelevant imaging agent precursor comprising (i) a biorelevant imaging agent and (ii) a side arm comprising an unsaturated carbon-carbon double bond (-C=C-) or a carbon-carbon triple bond (-C≡C-); hydrogenating the unsaturated carbon-carbon double bond (-C=C-) or the carbon-carbon triple bond (-C≡C-) of the side arm with para-hydrogen in the first organic solvent, thereby forming a para-hydrogenated derivative of the biorelevant imaging agent precursor; applying a polarization transfer waveform to hydrogenate the biorelevant imaging agent precursor on the side arm. 5. The method of any one of embodiments 1 to 4, comprising the steps of transferring nuclear spin order from para-hydrogen to non-hydrogen nuclear spins on the biologically relevant imaging agent; optionally hydrolyzing the para-hydrogenated biologically relevant imaging agent precursor by adding an aqueous hydrolysis agent to the solution to produce a hyperpolarized biologically relevant imaging agent and an unbound side arm; and optionally neutralizing the solution with a buffer to slow down or terminate the hydrolysis reaction, thereby producing a solution comprising the first organic solvent, an aqueous mixture optionally comprising the aqueous hydrolysis agent, the hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, and the optional unbound side arm.

[0170] Enumerated embodiment 6. The method of embodiment 5, wherein obtaining a solution in a vessel further comprises a catalyst trapping step prior to the addition of the aqueous hydrolysis agent, the catalyst trapping step comprising filtering the solution to remove rhodium atoms, iridium atoms, and / or any other catalyst atoms from the solution, and optionally, the solution is concentrated prior to the catalyst trapping step.

[0171] Enumerated embodiment 7. The method of any one of embodiments 1 to 6, wherein the biologically relevant imaging agent is selected from pyruvate, glutamate, glutamine, lactate, acetate, acetoacetate, zymonate, alanine, fructose, fumarate, bicarbonate, urea, dehydroascorbate, alpha-ketoglutarate, dihydroxyacetone, glucose, ascorbate, and their conjugate acids.

[0172] Enumerated embodiment 8. The method of embodiment 7, wherein the biologically relevant imaging agent has a solubility in the first organic solvent of less than 50 millimolar (mM).

[0173] Enumerated embodiment 9. The method of embodiment 7 or claim 8, wherein the biologically relevant imaging agent has a solubility in water of greater than 50 millimolar (mM).

[0174] Enumerated embodiment 10. The method of any one of embodiments 1-9, wherein the aqueous mixture comprises water, sodium hydroxide, potassium hydroxide, or any mixture thereof.

[0175] Enumerated embodiment 11. The method of any one of claims 1 to 10, wherein the first organic solvent comprises acetone, ethanol, methanol, chloroform, ethyl acetate, methyl ethyl ketone (MEK), acetophenone, hexone, cyclohexanone, cyclopentanone, or a combination thereof.

[0176] Enumerated embodiment 12. The method of embodiment 11, wherein the first organic solvent comprises acetone.

[0177] Enumerated embodiment 13. The method of embodiment 11, wherein the first organic solvent comprises methyl ethyl ketone (MEK).

[0178] Enumerated embodiment 14. The method of any one of embodiments 1 to 13, wherein the first organic solvent has a solubility in water of greater than 75 millimolar (mM) at 20° C.

[0179] Enumerated embodiment 15. The method of any one of embodiments 1-14, wherein the second organic solvent comprises one or more ICH class 2 solvents selected from acetonitrile, chlorobenzene, chloroform, cyclohexane, dibromomethane (DBM), 1,2-dichloroethene, dichloromethane (DCM), 1,2-dimethoxyethane, n,n-dimethylacetamide, n,n-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2-methoxyethanol, methylbutylketone, methylcyclohexane, n-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene, 1,1,2-trichloroethene, or xylene.

[0180] Enumerated embodiment 16. The method of any one of embodiments 1-14, wherein the second organic solvent comprises one or more ICH class 3 solvents selected from acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, methyl tert-butyl ether (MTBE), cumene, diethyl ether, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, or propyl acetate.

[0181] Enumerated embodiment 17. The method of any one of embodiments 1 to 16, wherein the second organic solvent comprises methyl tert-butyl ether (MTBE).

[0182] Enumerated embodiment 18. The method of any one of embodiments 1 to 16, wherein the second organic solvent comprises dibromomethane (DBM).

[0183] Enumerated embodiment 19. The method of any one of embodiments 1 to 16, wherein the second organic solvent comprises dichloromethane (DCM).

[0184] Enumerated embodiment 20. The method of any one of embodiments 1 to 19, wherein the washing step and / or the evaporation step are repeated until the first organic solvent has a concentration of 5000 ppm or less in the solution.

[0185] Enumerated embodiment 21. The method of any one of embodiments 1 to 20, wherein the washing step and / or the evaporation step are repeated until the second organic solvent has a concentration in the solution below the ICH toxicity limit.

[0186] Enumerated embodiment 22. The method of any one of embodiments 1 to 22, wherein the evaporating step comprises bubbling with an inert gas, optionally wherein the inert gas is nitrogen gas.

[0187] Enumerated embodiment 23. An administration composition comprising a hyperpolarized biologically relevant imaging agent or a pharma- ceutically acceptable salt thereof, wherein the administration composition is produced by a method according to any one of embodiments 1 to 22.

[0188] Recited embodiment 24. The administration composition of embodiment 23, comprising no more than 20 mM, 10 mM, 5 mM, 2 mM, or 1 mM of the first organic solvent.

[0189] Recited embodiment 25. The administration composition of embodiment 23, comprising no more than 20 mM, 10 mM, 5 mM, 2 mM, or 1 mM of a second organic solvent.

[0190] Enumerated embodiment 26. A system for implementing the method according to any one of embodiments 1 to 22.

[0191] Enumerated embodiment 27. The system of embodiment 26, comprising a first container, a second container fluidly connected to the first container through a first fluid transfer element, and optionally a third container fluidly connected to the second container through a second fluid transfer element.

[0192] Enumerated embodiment 28. A system as described in embodiment 26 or 27, further comprising a magnetic guide system providing a defined magnetic field across at least a portion of the system, and optionally, the magnetic guide system providing a defined magnetic field across the entire system.

[0193] Enumerated embodiment 29. The system of embodiment 27, further comprising a magnetic guide system providing a defined magnetic field across the first fluid movement element and the optional second fluid movement element.

[0194] Enumerated embodiment 30. The system of embodiment 28 or 29, wherein the defined magnetic field prevents undesired hyperpolarization loss in the solution as the solution is processed through the system. EXAMPLES

[0195] Example 1 - System for liquid-liquid purification A system was provided that included a first container, a second container fluidly connected to the first container, and a third container fluidly connected to the second container, wherein the containers and flow path were covered by a defined magnetic field provided by a solenoid valve to prevent undesired hyperpolarization loss during processing of the solution through the system.

[0196] container 1 A 2.3-2.7 mL mixture containing parahydrogenated and hyperpolarized pyruvate esters (with side arms) in acetone (concentration of 180-250 mM) was injected into the first vessel. The mixture was bubbled with nitrogen gas and 1.0 mL of base (400 mM NaOD in DO) was injected into vessel 1 to cleave the pyruvate esters into sodium pyruvate and the corresponding unbound side arms. Then, 3 mL of phosphate buffer (18 mM NaH2PO4, 9 mM Na2HPO4, 52 mM NaCl in DO) was added to the mixture (while bubbling with nitrogen gas). The sample was extracted and analyzed and found to contain a mixture of acetone, DO, and sodium pyruvate (dissolved at a pH of 6-9).

[0197] The solution in vessel 1 was then subjected to a first washing step. 20 mL of an organic washing solvent (e.g., diethyl ether or tert-butyl methyl ether) was injected into the solution, which was then stirred using nitrogen gas bubbling. The mixture was then allowed to settle for approximately 10 seconds until a clear phase separation between the aqueous phase (lower phase) and the organic phase (upper phase) was formed. Samples of each phase were extracted and analyzed: (i) acetone, diethyl ether, and most of the non-polar by-products (including unbound side arms) were contained in the organic phase, and (ii) sodium pyruvate remained in the aqueous phase (70-100 mM). The aqueous phase was also found to contain residual concentrations of acetone (2-3.5 M) and diethyl ether (0.8-1 M).

[0198] container 2 The aqueous phase from vessel 1 was pumped into vessel 2, which had been pre-filled with 20 mL of the same organic wash solvent (e.g., diethyl ether or tert-butyl methyl ether). The mixture was stirred again using nitrogen gas bubbling, and the mixture was allowed to settle for approximately 10 seconds until a clear phase separation between the aqueous (lower) and organic (upper) phases was formed. Samples of each phase were extracted and analyzed: (i) most of the acetone and diethyl ether were in the organic phase, and (ii) sodium pyruvate remained in the aqueous phase, reducing the residual concentrations of acetone (300-700 mM) and diethyl ether (700-900 mM).

[0199] container 3 0.3-3 mL of the aqueous phase from vessel 2 was pumped into vessel 3. The solution was flushed with nitrogen gas through the frit to evaporate any remaining volatile organics in the solution. As the solution continued to be exposed to volatile organic evaporation conditions with nitrogen gas flushing through the frit, the aqueous phase from vessel 2 continued to be pumped into vessel 3. The final sample was extracted and analyzed and found to contain (i) sodium pyruvate (70-100 mM), acetone (200-400 mM), and diethyl ether (10-50 mM), and had a pH between 6 and 9.

[0200] Example 2 - Liquid-Liquid Purification Test The liquid-liquid purification process was completed using a parahydrogenated and hyperpolarized pyruvate ester with a side arm (starting concentration of 200 mM) and the general system and processing steps of Example 1. Hydrolysis was completed using 200 mM base in a T-mixer followed by in-line saponification with phosphate buffer to quench the hydrolysis reaction. Two washing steps were completed in a 50 ml Falcon tube (hand mixed). Wash step 1 contained an organic to aqueous ratio of 4:1. Wash step 2 contained an organic to aqueous ratio of 6:1. Evaporation in vessel 3 was completed by bubbling nitrogen gas at 1.5 L / min using a glass frit at 60° C. and 150 mbar absolute pressure with vacuum.

[0201] The solvent combinations, specific processing conditions, and test results are shown in Table 2. [Table 2]

[0202] Test results showed that the liquid-liquid purification system and processing steps were effective in producing hyperpolarized pyruvate dosage compositions containing ICH-accepted concentrations of organic solvents using either acetone or MEK as the first organic solvent and MTBE or DCM as the second organic solvent.

[0203] Example 3 - Acetone Extraction Test Tests were completed to analyze the effectiveness of extracting acetone from the aqueous phase using five different organic wash solvents: MTBE (methyl-tert-butyl ether), DCM (dichloromethane), DBM (dibromoethane), Et2O (diethyl ether), and DCB (dichlorobenzene). The extraction efficiency of each solvent was tested at two different acetone concentrations (9000 mM and 2000 mM) with and without pyruvate added to each concentration, and at an organic wash:aqueous volume ratio of 4:1. Extraction efficiency was calculated as the output acetone concentration in water (after wash) divided by the input acetone concentration in water (before wash), where acetone concentration was measured by NMR.

[0204] The test results (extraction efficiency) are shown in Table 3. [Table 3]

[0205] The test results showed that DCM (dichloromethane) and DBM (dibromoethane) had the best extraction efficiency (lowest power / input ratio) both with and without pyruvate in solution. MTBE, EtO, and DCB were also shown to have effective extraction efficiencies, although they were less effective (higher power / input ratios) compared to DCM and DBM.

[0206] Example 4 - Evaporation Test A study was completed to analyze the effectiveness of using evaporation to remove residual organic solvent material from water. The evaporation conditions were similar to those in Example 2 (1.5 L / min nitrogen gas bubbling with a glass frit at 60° C. and 150 mbar absolute pressure with vacuum). The following solutions were included in the study:

[0207] The test results (extraction efficiency) are shown in Table 3: (i) Mixture 1--466 mM acetone + 230 mM MTBE in water, (ii) Mixture 2--466 mM acetone + 150 mM DCM in water, (ii) Mixture 3--277 mM acetone + 332 mM MTBE in water. Extraction efficiency was calculated as the output acetone concentration in water (after evaporation) divided by the input acetone concentration in water (before evaporation), where the acetone concentration was measured by NMR.

[0208] Evaporation of Mixture 1 resulted in an acetone extraction efficiency of 0.070.

[0209] Evaporation of Mixture 2 resulted in an acetone extraction efficiency of 0.040.

[0210] Evaporation of Mixture 3 resulted in an acetone extraction efficiency of 0.086.

[0211] The test results showed that the evaporation process and parameters of the liquid-liquid purification system were effective in extracting residual organic solvents from aqueous mixtures with various concentrations of acetone and MTBE / DCM.

[0212] Example 5 - SABRE Catalyst in Acetone Extraction Test 5 mg of IrIMes(COD)PF6 precatalyst was dissolved in 1 ml acetone-d6. The solution was then mixed with 6 ml MTBE and 1 ml DO. The aqueous and organic phases were separated within seconds and both were extracted for proton NMR measurements. The concentrations of both acetone and precatalyst were low in the aqueous phase after washing. The NMR spectra of both phases are shown in Figure 6. As can be seen in the NMR spectra, the precatalyst was only observable in one phase and not the other.

Claims

1. A method for generating an administration composition comprising a hyperpolarized bio-related contrast agent or a pharmaceutically acceptable salt thereof, said method comprising: - obtaining a solution in a container, said solution comprising a first organic solvent, an aqueous mixture, said hyperpolarized bio-related contrast agent or a pharmaceutically acceptable salt thereof, and optionally an unbound side arm; - one or more treatment steps selected from the group consisting of the following (a) and (b): (a) one or more washing steps, wherein said solution is washed with a second organic solvent, thereby forming (i) an organic mixture phase comprising said first organic solvent, said second organic solvent, and said optionally unbound side arm, and (ii) an aqueous mixture phase comprising said aqueous mixture and said hyperpolarized bio-related contrast agent or a pharmaceutically acceptable salt thereof; and one or more separation steps, wherein said organic mixture phase is separated from said aqueous mixture phase, and either said organic mixture phase or said aqueous mixture phase is transferred to a separate container; (b) one or more evaporation steps, wherein said solution is subjected to organic vapor extraction conditions to evaporate a portion of said first organic solvent or said second organic solvent and optionally an unbound side arm from said solution, thereby providing an aqueous mixture phase comprising said aqueous mixture and said hyperpolarized bio-related contrast agent or a pharmaceutically acceptable salt thereof; - obtaining an administration composition from said aqueous mixture comprising said hyperpolarized bio-related contrast agent or a pharmaceutically acceptable salt thereof.

2. The step of obtaining a solution in a container comprises: - obtaining a solution comprising said first organic solvent and a bio-related contrast agent precursor dissolved in said first organic solvent, said bio-related contrast agent precursor comprising (i) a bio-related contrast agent and (ii) a side arm comprising an unsaturated carbon-carbon double bond (-C=C-) or a carbon-carbon triple bond (-C≡C-); - hydrogenating said unsaturated carbon-carbon double bond (-C=C-) or carbon-carbon triple bond (-C≡C-) of said side arm with parahydrogen in said first organic solvent, thereby forming a parahydrogenated derivative of said bio-related contrast agent precursor. - Applying a polarization transfer waveform to transfer nuclear spin order from the para-hydrogen on the side arm to the non-hydrogen nuclear spins on the bio-related contrast agent; - Optionally, hydrolyzing the para-hydrogenated bio-related contrast agent precursor by adding an aqueous hydrolyzing agent to the solution to produce the hyperpolarized bio-related contrast agent and the unbound side arm; - Optionally, neutralizing the solution with a buffer to slow down or terminate the hydrolysis reaction, and including: Thereby producing the solution comprising the first organic solvent, the aqueous mixture optionally containing the aqueous hydrolyzing agent, the hyperpolarized bio-related contrast agent or a pharmaceutically acceptable salt thereof, and the optional unbound side arm, according to the method of claim 1.

3. The step of obtaining a solution in a container further includes a catalyst collection step before the addition of the aqueous hydrolyzing agent, the catalyst collection step including filtering the solution to remove rhodium atoms, iridium atoms, and / or any other catalyst atoms from the solution, and optionally, the solution is concentrated before the catalyst collection step, according to the method of claim 2.

4. The bio-related contrast agent is selected from pyruvate, glutamate, glutamine, lactate, acetate, acetoacetate, zymonate, alanine, fructose, fumarate, bicarbonate, urea, dehydroascorbate, alpha-ketoglutarate, dihydroxyacetone, glucose, ascorbate, and their conjugate acids, according to the method of claim 1.

5. The bio-related contrast agent has a solubility in the first organic solvent of less than 50 millimolar concentration (mM), according to the method of claim 4.

6. The bio-related contrast agent has a solubility in water of more than 50 millimolar concentration (mM), according to the method of claim 4.

7. The aqueous mixture includes water, sodium hydroxide, potassium hydroxide, or any mixture thereof, according to the method of claim 1.

8. The first organic solvent includes acetone, ethanol, methanol, chloroform, ethyl acetate, methyl ethyl ketone (MEK), acetophenone, hexone, cyclohexanone, cyclopentanone, or a combination thereof, according to the method of claim 1.

9. The first organic solvent includes acetone, according to the method of claim 8. **Claim 10**: The method according to claim 8, wherein the first organic solvent comprises methyl ethyl ketone (MEK). **Claim 11** The method according to claim 1, wherein the first organic solvent has a solubility in water exceeding 75 millimolar concentration (mM) at 20°C. **Claim 12** The method according to claim 1, wherein the second organic solvent comprises one or more ICH class 2 solvents selected from acetonitrile, chlorobenzene, chloroform, cyclohexane, dibromomethane (DBM), 1,2 - dichloroethene, dichloromethane (DCM), 1,2 - dimethoxyethane, n,n - dimethylacetamide, n,n - dimethylformamide, 1,4 - dioxane, 2 - ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2 - methoxyethanol, methyl butyl ketone, methyl cyclohexane, n - methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydrofuran, tetralin, toluene, 1,1,2 - trichloroethene, and xylene. **Claim 13** The method according to claim 1, wherein the second organic solvent comprises one or more ICH class 3 solvents selected from acetic acid, acetone, anisole, 1 - butanol, 2 - butanol, butyl acetate, methyl - tert - butyl ether (MTBE), cumene, diethyl ether, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3 - methyl - 1 - butanol, methyl ethyl ketone, methyl isobutyl ketone, 2 - methyl - 1 - propanol, pentane, 1 - pentanol, 1 - propanol, 2 - propanol, and propyl acetate. **Claim 14**: The method according to claim 1, wherein the second organic solvent comprises methyl - tert - butyl ether (MTBE). **Claim 15**: The method according to claim 1, wherein the second organic solvent comprises dibromomethane (DBM). **Claim 16**: The method according to claim 1, wherein the second organic solvent comprises dichloromethane (DCM). **Claim 17** The method according to claim 1, wherein the washing step is repeated until the first organic solvent has a concentration of 5000 ppm or less in the solution. **Claim 18** The method according to claim 1, wherein the washing step is repeated until the second organic solvent has a concentration in the solution below the ICH toxicity limit.

19. The method according to claim 1, wherein the evaporation step comprises bubbling with an inert gas, and optionally, the inert gas is nitrogen gas.

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