Synthetic Approaches for ¹⁵N-labeled Hyperpolarized Heterocyclic Molecular Imaging Agents for ¹⁵N NMR Signal Amplification By Reversible Exchange in Microtesla Magnetic Fields

Abstract

NMR hyperpolarization techniques enhance nuclear spin polarization by several orders of magnitude resulting in corresponding sensitivity gains. This massive sensitivity gain enables new applications ranging from studies of small molecules using high-resolution NMR spectroscopy to real-time metabolic imaging in vivo. Several hyperpolarization techniques exist for hyperpolarization of a large repertoire of nuclear spins, although ¹³C- and ¹⁵N- sites of biocompatible agents are the key targets due to their widespread in biochemical pathways. Moreover, their long T₁ allows retaining hyperpolarized states for up to tens of minutes. Signal Amplification by Reversible Exchange (SABRE) is a low-cost and ultrafast hyperpolarization technique that has been shown to be versatile for hyperpolarization of ¹⁵N nuclei. While large sensitivity gains are enabled by hyperpolarization, ¹⁵N natural abundance is only ~0.4%, so that isotopic labeling of the to-be-hyperpolarized molecules is required in order to take full advantage of the hyperpolarized state. Here, we describe selected advances in the preparation of ¹⁵N-labeled compounds with the primary emphasis on using these compounds for their SABRE polarization in microtesla magnetic fields via spontaneous polarization transfer from parahydrogen. Also, these principles can certainly be applied for hyperpolarization of these emerging contrast agents using Dynamic Nuclear Polarization and other techniques.

Frontis Graphics Accompanying Text

Signal Amplification by Reversible Exchange enables efficient nitrogen-15 hyperpolarization of a wide range of bio-compatible substrates using microtesla magnetic fields. This process is enabled by simulatenous exchange of parahydrogen (the source of polarization) and to-be-hyperpolarized N-containing biocompatible molecule. Here, we review selected advances in nitrogen-15N enrichment of pyridine and imidazole derivatives that can potentially be employed as novel molecular contrast agents.

Graphical Abstract

This minireview provides a selective overview of ¹⁵N-labeling approaches for the preparation of contrast agents hyperpolarized using the SABRE-SHEATH technique. This technique employs parahydrogen as a source of hyperpolarization for fast and inexpensive means to produce hyperpolarized biocompatible contrast agents for molecular imaging applications.

Introduction

Conventional NMR techniques have substantial sensitivity limitations due to low nuclear spin polarization (P), i.e., the degree of nuclear spin alignment with the applied static magnetic field. For example, P_H, P_13C and P_15N are only 1*10⁻³%, 2.6*10⁻⁴% and 1*10⁻⁴% at 3 T respectively at physiologically relevant conditions. The detected NMR signal in spectroscopy and imaging applications is directly proportional to P. As a result, detection of metabolites in vivo at physiological conditions is very challenging using proton and especially ¹³C NMR spectroscopy. There have been no reports of in vivo ¹⁵N detection of metabolism to the best of our knowledge. Yet, ¹³C and ¹⁵N nucleus in biological and biocompatible molecules offer a wide dynamic range of chemical shifts (~250 ppm and ~1,000 ppm respectively) making them potentially useful for detecting metabolic transformation. NMR hyperpolarization techniques offer a revolutionary approach, which allow to enhance the sensitivity of NMR by several orders of magnitude through a large increase in P.^[1-7] The process of hyperpolarization frequently (but not always) occurs outside the NMR detector.^[8-13] As a result, the delays associated with the transfer of hyperpolarized (HP) biocompatible agents and also frequently with their biochemical conversion require using molecular carrier capable of retaining HP state for at least a few tens of seconds.^{[14-16] 13}C nucleus can certainly fulfill this requirement—indeed a large number of ¹³C HP biocompatible contrast agents have been developed for their biomedical use.^{[9, 17]} However, ¹⁵N nucleus has an even lower gyromagnetic ratio (γ) than ¹³C and correspondingly can retain a HP state even longer. Many examples have been demonstrated with HP T₁ exponential decay constant of 10 minutes or more.^[18-21] Therefore, ¹⁵N HP compounds offer a substantially longer time window for their utilization. Moreover, ¹⁵N isotope is substantially less expensive than ¹³C offering critical translational advantages.

The key shortcoming for the use of ¹⁵N HP compounds as metabolic contrast agents is the low sensitivity of ¹⁵N NMR due to low γ and low natural abundance. As a result, only one published in vivo study has been reported to date.^[22] However, this limitation can be remedied through the use of polarization transfer approaches allowing to perform the NMR signal readout using spin-spin coupled protons.^{[19, 23-25]} Protons have 10 times higher γ thereby offering a 10¹-10² sensitivity boost.^[26]

The second reason for the slow development of ¹⁵N HP compounds in the context of biomedical applications relates to the first-generation dissolution Dynamic Nuclear Polarization (d-DNP) hardware, which relied on a very slow process of polarization transfer from HP electrons to ¹⁵N nuclei. As a result, the pioneering efforts required long (>2 h) hyperpolarization times and yielded low P_15N < 4%,^[22] although recent d-DNP studies have demonstrated P_15N of up to 8%.^[27] The new-generation d-DNP hardware circumvents this limitation by a more rapid polarization transfer to lattice protons followed by cross-polarization to ¹⁵N^{[28, 29]}--it should also be noted that d-DNP can hyperpolarize a broad range of compounds in general including ¹⁵N-labeled biocompatible molecules.^{[8, 27]} Moreover, Signal Amplification by Reversible Exchange (SABRE), a hyperpolarization technique introduced by Duckett and co-workers in 2009, has been shown to be very effective for spontaneous polarization transfer from parahydrogen (p-H₂) to the to-be-hyperpolarized molecules using polarization transfer catalyst.^[30-33] Biocompatible contrast agent molecule and p-H₂ can simultaneously exchange in Ir hexacoordinate complex leading to spontaneous polarization transfer from parahydrogen-derived hydrides to nuclear spins of the biocompatible substrate, Figure 1a.^{[6, 34, 35]} The spontaneous polarization transfer occurs when the spin mixing at level anti-crossing (LAC) occurs for a system of spin-spin coupled nuclear spins.^[35] In SABRE, the LAC condition is met for spontaneous polarization transfer to substrate protons in millitesla (mT) magnetic fields.^[35] Theis and co-workers have shown in 2014 that the LAC condition is met for spontaneous polarization transfer to substrate ¹⁵N nuclei in microtesla (μT) magnetic fields.^[36-38] The method was termed SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH).^{[37, 38]} It allows for ultrafast (in 1 min or less) and efficient (30% or more) ¹⁵N hyperpolarization of biocompatible molecules.^{[39, 40]} Alternatively, radio frequency (RF) pulses can also be employed in order to create LAC for polarization transfer to ¹⁵N heteronucleus—work is in progress to improve attainable ¹⁵N polarization using RF-based SABRE approaches.^[41-44]

Figure 1.

a) Schematic of SABRE hyperpolarization technique. b) ¹H-¹H spin-relays in a millitesla magnetic field enable spontaneous polarization transfer of p-H₂-derived hyperpolarization through all proton sites of pyridine substrate molecule via 3- and 4-bond ¹H-¹H spin-spin couplings. c) ¹⁵N-¹⁵N spin-relays in a μT magnetic field enable spontaneous polarization transfer of p-H₂-derived hyperpolarization through all ¹⁵N sites of [¹⁵N₃]nimorazole molecule via 2-bond ¹⁵N-¹⁵N spin-spin couplings (note, since ¹⁵N spin-spin coupling are an order of magnitude lower than those of protons, spin-relayed polarization transfer via three-bond spin-spin couplings is inefficient).

To summarize, ¹⁵N-containing biocompatible agents can now be readily hyperpolarized. They can retain the HP state for tens of minutes and can be monitored via the indirect but more sensitive proton detection. All-in-all, ¹⁵N HP compounds offer an outstanding biomedical platform as efficient carriers of HP state.

The range of molecular structural motifs amenable to ¹⁵N hyperpolarization via SABRE-SHEATH has been growing rapidly and includes nicotinamide (vitamin B₃),^[45] imidazole (for pH sensing),^[46] metronidazole (for hypoxia sensing),^{[47, 48]} nimorazole (for radiosensitization sensing)^[49] and many others.^{[9, 50-52]} In order to take full advantage of the HP state, ¹⁵N enrichment from its natural abundance (~0.4%) to ~99% must be implemented, which in turn boosts the hyperpolarization payload by ~250 fold. ¹⁵N enrichment requires specialized ¹⁵N-labeling approaches, which are discussed in this focused Minireview. Moreover, the new advances in SABRE-SHEATH (e.g., spin-relayed mechanism^{[48, 53]} and quadrupolar relaxation sinks^[54]) require special attention for the ¹⁵N labeling patterns of the to-be-hyperpolarized substrates. The key approaches are discussed in this minireview, which would be of interest to those working in this rapidly emerging field of SABRE, as well as those working with other hyperpolarization techniques (e.g., d-DNP).

Spin-relayed SABRE-SHEATH

SABRE exploits the spin-spin couplings for polarization transfer of the p-H₂-derived spin order to the target nucleus.^{[30, 31]} In a canonical SABRE experiment performed in the mT magnetic field range, the LAC condition is fulfilled between p-H₂-derived hydrides and to-be-polarized spin-spin coupled ¹H spin,^{[30, 31, 37, 55]} for example using the four-bond ¹H-¹H spin-spin coupling between hydrides and Ha in pyridine molecule as shown in Figure 1b. Of note, the pioneering experiments revealed that polarization then spreads through the network of spin-spin couplings within the pyridine molecule itself.^{[30, 31]} This means that Hb and Hc protons are not hyperpolarized directly from p-H₂ derived hydrides; indeed, the corresponding five and six-bond spin-spin couplings are too weak.^[56] Instead, the polarization is spin-relayed from Ha to Hb and to Hc. This mechanism was additionally validated via re-polarization studies.^[53] Thus, the mT magnetic field range allows for both “canonical” SABRE polarization transfer and the spin-relayed polarization transfer at the same time.

Theis and co-workers (ca. 2015)^[36-38] have shown that LAC for SABRE polarization transfer to ¹⁵N nucleus is fulfilled in magnetic fields on the order of 1 μT or less via two-bond ¹H-¹⁵N spin-spin couplings, Figure 1c. SABRE-SHEATH hyperpolarization in μT magnetic fields yields substantially higher P_15N than comparable efforts in millitesla magnetic fields. Moreover, the efforts to transfer polarization from p-H₂-derived hydrides to ¹⁵N sites via four- and five- chemical bonds yielded P_15N at least one order of magnitude lower than those obtained via two-bond spin-spin couplings.^[47] This is not surprising, because γ_15N is 10 times lower than that of protons resulting in correspondingly diminished spin-spin couplings.

Later, Shchepin and co-workers have demonstrated that ¹⁵N polarization gained in a SABRE-SHEATH experiment can be spin-relayed to other ¹⁵N-¹⁵N spin-spin coupled sites,^[53] and the spin-relaying occurs in a wide range of μT magnetic fields (including the Earth’s magnetic field).^[48] In a series of systematic measurements, it was shown that ¹⁵N-¹⁵N spin-relayed transfer is very efficient up to two chemical bonds.^{[48, 49]} Three-bond interactions are too weak (<0.1 Hz as compared to >1 Hz two-bond interactions).^{[48, 49]} The network of two-bond ¹⁵N-¹⁵N spin-spin coupling may be extensive.^{[48, 49]} For example, the spin system of [¹⁵N₃]nimorazole (Figure 1c) and [¹⁵N₃]metronidazole have exhibited spin-relayed polarization transfer from ¹⁵N3 to ¹⁵N1 and from ¹⁵N1 to ¹⁵NO₂ sites.^{[48, 49] 15}N polarization of all three sites can be enhanced to substantial levels: 16% and more.^{[48, 54]}

The discovery of ¹⁵N-¹⁵N SABRE-SHEATH spin-relayed mechanism of polarization transfer posed the question about HP ¹⁵N site interaction with ¹⁴N nuclei. In high magnetic fields, the quadrupolar ¹⁴N nucleus is self-decoupled and effectively shows no interactions with other nearby spins. In μT magnetic fields, it has been shown that the nearby ¹⁴N nucleus effectively acts as a quadrupolar sink, because ¹⁵N polarization is spin-relayed from ¹⁵N to ¹⁴N via two-bond spin-spin couplings. Because ¹⁴N is quadrupolar, the net effect of these interactions is a substantially reduced ¹⁵N T₁ in μT magnetic fields due to two-bond interactions with ¹⁴N nucleus.^[54] Because ¹⁵N polarization levels linearly correlate with their microtesla T₁, the presence of ¹⁴N quadrupolar sinks results in substantially diminished P_15N.^[54] Therefore, the practical solution is to ensure that all nitrogen sites within two chemical bonds from each other are labeled with ¹⁵N.^[54] This approach was effectively demonstrated for [¹⁵N₃]metronidazole and [¹⁵N₃]nimorazole.^{[48, 49]} On the other hand, there is no practical benefit to isotopically enrich distant nitrogen sites, which are separated by at least three chemical bonds from the spin-relayed SABRE-SHEATH ¹⁵N-¹⁵N transfer network, as demonstrated for [¹⁵N₃]nimorazole.^[49]

With respect to the choice of ¹⁵N site in the context of biomedical applications, other factors influencing ¹⁵N T₁ relaxation should also be considered. For example, chemical shift anisotropy (CSA) may dominate the relaxation mechanism in high magnetic field as CSA in units of Hz scales linearly with magnetic field. Non-aromatic compounds such as aliphatic tertiary amines have relatively small ¹⁵N CSA, and therefore, enjoy long T₁ values even at high magnetic fields. Indeed, this class of compounds have been successfully hyperpolarized by d-DNP^{[19, 22, 27, 57]} and PHIP,^{[58, 59]} and this class of compounds could also be of interest for future SABRE studies to develop functional and metabolic contrast agents. More importantly, the presence of directly attached protons should be avoided to to-be-hyperpolarized ¹⁵N biocompatible molecules, because strong ¹⁵N-¹H dipolar interactions may cause very efficient ¹⁵N relaxation thus resulting in low ¹⁵N T₁ values. Therefore, the presence of nearby protons should be generally avoided in the context of development of long-lasting ¹⁵N HP contrast agents.

Synthesis of ¹⁵N-labeled pyridine derivatives

Pyridine derivatives are the first compounds that have shown NMR signal enhancement by the SABRE method.^[30] They contain a sp²-hybridized nitrogen atom capable of reversibly binding to the SABRE catalyst. Moreover, the synthesis of ¹⁵N-labeled pyridine and its derivatives is very promising due to the biological activity of numerous compounds based on this heterocycle.

Synthesis of [¹⁵N]pyridine

Pyridine itself is a convenient substrate for fundamental investigations regarding SABRE. Its ¹⁵N-labeled isotopomer allows to observe hyperpolarization on the heteronucleus, which is directly coordinated (i.e., bound) to the Ir complex. An appropriate method of [¹⁵N]pyridine synthesis could be rather useful for such experiments. The most readily available source of the ¹⁵N isotope is labeled ammonium chloride (¹⁵NH₄Cl), Sigma-Aldrich 299251. [¹⁵N]pyridine is obtained by interacting 2-ethoxy-3,4-dihydro-2H-pyran with ¹⁵NH₄Cl in an acidic medium in the presence of methylene blue (Figure 2).^[60] Pyran in this case is a latent form of pentanedial readily closing cycle with ammonium chloride. Methylene blue dye is required for subsequent oxidation of intermediate to an aromatic ring.

Figure 2.

Pyran-based synthesis of ¹⁵N-labeled pyridine.^[60]

This method, in addition to the good yield (up to 60%) and an excellent ¹⁵N isotopic enrichment (>98%), does not require a large excess of ¹⁵NH₄Cl.^[61] The product can be easily separated from the resulting impurities due to the ability of pyridine to form salts in an acidic medium and its volatility in a free base form. However, the key drawback of this method is that it is applicable to the synthesis of only pyridine itself. This is due to limitations in the structure of the starting pyran. The synthesis of 4-methylpyridine can be cited as the only exception so far.^[62]

In our opinion, the approach via Zincke salt intermediate^[63] is potentially more universal for the preparation of ¹⁵N-enriched pyridine derivatives (Figure 3).^{[45, 64]} This method involves the interaction of pyridine with 2,4-dinitrochlorobenzene, followed by the reaction of the intermediate with ¹⁵NH₄Cl.^[61] The disadvantages of this method include moderate yields (30-55%), reduced ¹⁵N enrichment (60-98%), and the need to use a large excess of ¹⁵NH₄Cl.^[61] However, this sequence of steps makes it possible to expand the range of derivatives that can be synthesized, see the corresponding Sections below for details.

Figure 3.

Zincke-salt-intermediate-based approach for the synthesis of ¹⁵N-labeled pyridine.^[61]

The utility of the synthesized ¹⁵N-labeled pyridine can be employed in SABRE as a model compound in a number of ways. For example, the enrichment of pyridine by a ¹⁵N nucleus boosts the observed NMR signal of the HP state, and therefore, it provides an opportunity to detect the catalyst-bound substrates (which present in small concentrations – down to below 1 mM), which is challenging to detect in case of pyridine substrate at natural ¹⁵N abundance. This is important, because it becomes possible to optimize the parameters for SABRE polarization transfer, especially when using RF pulses, for example, SLIC-SABRE.^[65] For example, Pravdivtsev et al. explored the effects of various physical parameters such as the amplitude and frequency of the RF field, and the effects of chemical parameters such as the exchange rate constants, the effective lifetime of the SABRE complex was estimated, as well as the entropy and enthalpy of the complex dissociation reaction.^[65] It was shown that the SLIC-SABRE approach can be useful for the evaluation of the chemical exchange parameters that are very important for the production of highly polarized contrast agents via SABRE.^[65]

Synthesis of [¹⁵N]nicotinamide

Nicotinamide is a well-known molecule, vitamin B₃, which has low in vivo toxicity.^[66] Nicotinamide enrichment with ¹⁵N isotope can lead to the preparation of a potentially useful HP contrast agent for metabolic MRI studies. The presence of a substituent in the pyridine ring makes the Zincke salt intermediate approach more useful for the preparation of this isotopically enriched heterocycle. During the first reaction step, the Zincke salt is formed, followed by ring opening and displacement by ammonia in the second step (Figure 4).^[67] Shchepin et al. improved this procedure to obtain [1-¹⁵N]nicotinamide with 55% overall yield and high isotopic purity (98%).^[45]

Figure 4.

Zincke-salt-intermediate-based approach for the synthesis of ¹⁵N-labeled nicotinamide.^[45]

This ¹⁵N-labeled molecule was employed by Svyatova et al. demonstrating that the SLIC-SABRE hyperpolarization technique can be used to hyperpolarize [1-¹⁵N]nicotinamide and obtain ultrafast ¹⁵N MRI.^[44] This study demonstrated the possibility of high-field SABRE implementation for biomedical applications.

Synthesis of 3-fluoro[¹⁵N]pyridine

There is a wide range of FDA-approved drugs containing ¹⁹F nucleus.^[68] Therefore, it is advantageous to employ ¹⁹F nucleus for hyperpolarized bioimaging due to high γ_19F and a wide dynamic range of chemical shifts sensitive to local environment. Moreover, ¹⁹F nucleus has nearly 100% natural abundance, so additional isotopic enrichment is not required. For instance, 3-fluoro[¹⁵N]pyridine is a useful model substrate to investigate hyperpolarization transfer from ¹⁵N site to ¹⁹F.

One approach for obtaining 3-fluoro[¹⁵N]pyridine is the reaction of the corresponding Zincke salt with the isotopically enriched ¹⁵NH₄Cl (Figure 5).^[64] It is important to note that fluorine atom has electron-withdrawing effect so the reaction with 2,4-dinitrochlorobenzene is slow. Moreover, 3,5-difluoropyridine does not react with 2,4-dinitrochlorobenzene even upon heating. Volatile pyridine derivatives such as pyridine or 3-fluoropyridine can be isolated from the reaction mixture through the usual distillation. At the same time, basic properties of pyridines make it possible to separate them from volatile solvents. Thus, the reaction of Zincke salt with ¹⁵NH₄Cl occurs with 84% ¹⁵N isotopic purity and 35% overall yield of 3-fluoro[¹⁵N]pyridine.^[64]

Figure 5.

Zincke-salt-intermediate-based approach for the synthesis of ¹⁵N-labeled 3-fluoropyridine.^[64]

3-fluoro[¹⁵N]pyridine was successfully employed for mechanistic SABRE and SABRE-SHEATH hyperpolarization studies demonstrating that the polarization transfer from p-H₂-derived hydrides to ¹⁹F nucleus occurs in mT and μT static magnetic-field regimes.^[64] Moreover, it was shown that fluorine hyperpolarization can be generated directly through five chemical bonds using RF pulses via the QUASR-SABRE experiment.^[69]

Synthesis of [¹⁵N]fampridine

4-Aminopyridine (fampridine) is used as a research tool for characterizing subtypes of the potassium channels.^[70] It has also been employed as a medication, which allows management of some of the symptoms of multiple sclerosis.^{[71, 72]} Moreover, 4-aminopyridine was efficiently hyperpolarized by SABRE with ~8% ¹⁵N polarization at ¹⁵N natural abundance. Thus, ¹⁵N-labeled 4-aminopyridine could be a promising biocompatible molecule for MRI as an ultrafast-imaging contrast agent.

A three-step procedure was applied to synthesize 4-amino[¹⁵N]pyridine, Figure 6, starting from [¹⁵N]pyridine.^[73] Pyridine itself is an unreactive compound especially for an electrophilic substitution reaction. Therefore, in the first step, [¹⁵N]pyridine was converted to [¹⁵N]pyridine-N-oxide, which is more reactive in the subsequent step. This reaction occurs with H₂O₂ as an oxidant with an 80% yield. The N-oxide group in pyridine allows carrying out nitration reaction under mild conditions in the second step to achieve a 90% yield of 4-nitro[¹⁵N]pyridine-N-oxide. The reaction with iron in acetic acid in the third step reduces both the nitro group and the N-oxide group, producing 4-amino[¹⁵N]pyridine with 35% yield. The moderate yield is due to high reaction sensitivity to iron quality. Apparently, iron should be active, but not too active as reduced iron. Optimization of reaction conditions including the reducing agent may potentially increase the yield substantially.

Figure 6.

The scheme of ¹⁵N-labeled 4-aminopyridine synthesis using [¹⁵N]pyridine as a starting source.^[73]

Future ¹⁵N SABRE-SHEATH hyperpolarization and MRI studies can demonstrate the potential of this promising compound as a prospective MRI contrast agent.

Synthesis of ¹⁵N-labeled imidazole derivatives

Imidazole derivatives also represent heterocycles with sp²-hybridized nitrogen atom. Many biomolecules include imidazole moiety, e.g., amino acid histidine and hormone histamine. Moreover, many classes of drugs contain imidazole heterocycle. Thus, imidazole derivatives represent a promising class of substrates amenable for ¹⁵N hyperpolarization by the SABRE-SHEATH method.

Synthesis of [¹⁵N₂]imidazole

Imidazole itself is a basic compound with pKa ~ 7.0. Therefore, ¹⁵N isotopically enriched HP version of this compound can be potentially employed for in vivo applications of pH imaging of cancer^{[46, 74]} and other diseases. Indeed, [¹⁵N₂]imidazole has been successfully hyperpolarized via the SABRE-SHEATH method resulting in MR signal enhancement of up to 146000-fold.^[46] The proton binding to a ¹⁵N site of imidazole occurs at physiological pH (pKa ~ 7.0), and changes the ¹⁵N isotropic chemical shift by ~30 ppm. These properties make this molecule suitable for in vivo pH sensing.^[46]

The one-step synthesis of [¹⁵N₂]imidazole is shown in Figure 7. Glyoxal, formaldehyde, and ¹⁵NH₄Cl react in the presence of hydrochloric acid forming the heterocycle through the imine derivatives intermediates.^[75] It was shown that the basic medium facilitates an undesirable side reaction reducing the yield of [¹⁵N₂]imidazole.^{[76, 77]} A catalytic amount of acid can be added to the reaction mixture to avoid the formation of large amounts of polymer impurities, resulting in an overall product yield of 50%.^[75]

Figure 7.

The [¹⁵N₂]imidazole synthesis scheme.^[75]

Synthesis of [¹⁵N₃]nimorazole

Nitroimidazoles is a class of antibiotics developed against anaerobic bacterial infections. Nimorazole is a representative nitroimidazole-based antibiotic. Moreover, nimorazole has been investigated as a hypoxia radiosensitizer for the treatment of head and neck cancers.^[78] [¹⁵N₃]Nimorazole was successfully hyperpolarized via SABRE-SHEATH with P_15N of more than 3%, and ¹⁵N T₁ of over 5 min at a clinically relevant field of 1.4 T. Moreover, the feasibility of ultrafast high-resolution 2D ¹⁵N MRI (0.5×0.5 mm² pixel size) has been demonstrated, indicating the great potential of this biologically active molecule as a metabolic contrast agent.^[49] Furthermore, ab initio calculations predict that all three ¹⁵N-labeled sites will experience large changes in their chemical shifts (by up to 700 ppm) due to step-wise reduction in a hypoxic environment.^[49] As a result, ¹⁵N HP [¹⁵N₃]nimorazole can be potentially employed to visualize hypoxic tumors, which is important in the context of personalized cancer treatment planning.

[¹⁵N₃]Nimorazole was synthesized in two steps (Figure 8) using [¹⁵N₂]imidazole as a starting material, see the corresponding Section below for details.^[49] Nitration reaction with labeled H¹⁵NO₃ forms4(5)-[¹⁵N]nitro[¹⁵N₂]imidazole with 30% yield. Due to the tautomerism of this compound, both nitrogen atoms of the imidazole ring can undergo an alkylation reaction. Thus, the second reaction with 4-(2-chloroethyl)morpholine yields two isomers. One of them is nimorazole, the other is the alkylation product of the N3 nitrogen atom. The yield of nimorazole, which is the minor isomer, was 20%. The two isomers were separated from each other by means of column chromatography, which made possible subsequent SABRE experiments with each of them.^[49]

Figure 8.

The ¹⁵N-labeled nimorazole synthesis scheme.^[49]

Synthesis of [¹⁵N₃]metronidazole

Another representative example of nitroimidazoles is metronidazole. This drug is an FDA-approved antibiotic that can be safely administered orally and intravenously in multi-gram doses.^[79] Metronidazole has been hyperpolarized by SABRE-SHEATH in a non-labeled form^[47] and several ¹⁵N-labeled forms.^{[48, 53, 80]} It has been employed for a wide range of studies to date: demonstration of record P_15N levels exceeding 50%,^[40] preparation of catalyst-free solutions,^[39] ultralong ¹⁵N T₁ (10 min),^[54] feasibility of high-resolution ¹⁵N MRI^[54] and others.^{[53, 54]} All-in-all, HP metronidazole could be potential hypoxia sensing metabolic contrast agent for HP MRI in a manner similar to that described for HP [¹⁵N₃]nimorazole above.

The first step of the synthesis of [¹⁵N₃]metronidazole is the preparation of 2-methyl[¹⁵N₂]imidazole. Using a modified approach for [¹⁵N₂]imidazole (see the corresponding Section), this substance was obtained by the reaction of glyoxal, acetaldehyde, ¹⁵NH₄Cl, and potassium carbonate with 90% yield, Figure 9.^[48] In the second step, the mixture of Na¹⁵NO₃ and H₂SO₄ was employed for the nitration reaction. Note that although H¹⁵NO₃ can be utilized for nitration reaction, Na¹⁵NO₃ is approximately 2 times cheaper than H¹⁵NO₃ per mol of ¹⁵N isotope. The yield of this reaction was about 45%. In the last step, the obtained 2-methyl-4(5)-nitroimidazole was alkylated by ethylene oxide in acidic media introducing hydroxyethyl moiety with 40% yield.^[48]

Figure 9.

The ¹⁵N-labeled metronidazole synthesis scheme.^[48]

Synthesis of other ¹⁵N-labeled compounds

There are several examples of other substrates that attract interest from the perspective of their suitability for ¹⁵N hyperpolarization by the SABRE technique. SABRE requires transient binding of a to-be-hyperpolarized compound to the Ir complex, and therefore, biocompatible molecules with the structural motif of an sp²-hybridized nitrogen atom(s) are the natural choice of compounds for SABRE hyperpolarization.^[50]

Diazirine derivatives are applied in photoaffinity labeling, and some of them were synthesized to characterize an unstable diazo-compound formed via photoisomerization.^[21] Several ¹⁵N hyperpolarization SABRE studies have been reported with diazirines.^{[24, 81, 82]} 3-(3-methyl-3H-[¹⁵N₂]diazirin-3-yl)propionic acid and 3-(2’-((tert-butyldimethylsilyl)oxy)ethyl)-3-methyl-3H-[¹⁵N₂]diazirine were prepared from levulinic acid and 4-(tert-butyldimethylsilyloxy)butan-2-one respectively by using hydroxylamine ¹⁵NH₂OH as the source of ¹⁵N isotopic label.^[83] Also there is an example of ¹⁵N-labeled diazirine, which has shown P_15N = 4.4% and T₁ of over 4 min.^[50]

Diazenes. 1,2-diphenyl[¹⁵N₂]diazene could be prepared from [¹⁵N]aniline by oxidative dimerization.^[84] Some diazenes have already been hyperpolarized by the SABRE method.^{[50, 85]}

Nitriles represent another potent class of compounds amenable to SABRE-SHEATH hyperpolarization.^{[43, 50, 51]} Nitrile group is present in a wide range of FDA-approved drugs.

¹⁵N nuclei in Schiff bases, which are known for their metabolic relevance,^[86] have been shown to be hyperpolarized via SABRE-SHEATH as well.^[86] Moreover, Schiff bases can be found in FDA-approved drugs, e.g., nifurtimox.

Summary and Outlook.

In summary, SABRE hyperpolarization is a vibrant and rapidly expanding area of hyperpolarized NMR research. SABRE-SHEATH approach in particular has been very effective in delivering strong P_15N (in excess of 50%) for a wide range of nitrogen-containing biocompatible molecules. In this minireview, we covered selected advances for ¹⁵N enrichment of relevant compounds employed in SABRE-SHEATH hyperpolarization. ¹⁵N labeling is critical for boosting the payload of HP state. Moreover, in some cases (most notably metronidazole), extensive ¹⁵N labeling is required in order to minimize ¹⁵N polarization losses due to otherwise present ¹⁴N quadrupolar sinks. The synthetic approaches described here will benefit those working in the field of SABRE and d-DNP hyperpolarization and other hyperpolarization techniques. As the field of SABRE continues to develop, we expect that a broader scope of biocompatible contrast agents will become amendable to SABRE-SHEATH hyperpolarization, thereby requiring new synthetic advances for the development of ¹⁵N-labeled drugs and biomolecules that can be employed as contrast agents to probe a wide range of real-time metabolic processes in vivo. Moreover, as demonstrated in this minireview, many prospective ¹⁵N HP contrast agents can be isotopically enriched with ¹⁵N taking advantage of inexpensive ¹⁵N sources such as ¹⁵NH₄Cl, H¹⁵NO_3, and Na¹⁵NO₃. Combined with the low-cost and high-throughput nature of the SABRE-SHEATH technique, these advances bode well for the future biomedical transition of this revolutionary hyperpolarization method.

Biography

Dr. Nikita V. Chukanov graduated from the Novosibirsk State University in 2006 with a master’s degree in chemistry. He received his PhD diploma in 2009 from Novosibirsk Institute of Organic Chemistry. Since 2017 Dr, Chukanov works at the International Tomography Center (ITC) SB RAS. The main area of his research is the synthesis of isotopically labeled compounds for hyperpolarization.

Prof. Roman V. Shchepin, PhD in Chemistry with Prof. Patrick H. Dussault, 2006. Postdoc, Vanderbilt University with Ned A. Porter, 2007-10. Research Fellow to Research Professor, Vanderbilt University Institute of Imaging Science with Eduard Y. Chekmenev, 2010-18. He is a tenure-track Assistant Professor at the South Dakota School of Mines & Technology since 2019. Research interests include organic synthesis and low-cost instrumentation development for hyperpolarization.

Dr. Sameer Joshi finished his M.Sc. in Organic Chemistry from the University of Pune, India, and then he spent 1.5 years in NCL, Pune, and 2 years in a private company. Later, he completed his Ph.D. in organic chemistry as a Marie Curie fellow with Dr. Llop at CIC biomaGUNE, Spain in 2017 by working on the synthesis of ¹³N-labelled compounds. Now, he is working as a Postdoctoral fellow at Wayne State University, USA since 2018 by working on heterocyclic compounds.

Mohammad Shah Hafez Kabir is a 3rd year Ph.D. student from Chekmenev Lab, Department of Chemistry, Wayne State University, USA. He completed his B.Pharm from IIUC, Bangladesh in 2015. Earlier he worked on drug discovery from natural resources and rational drug design. He is now working on the polarization of ¹⁵N-metronidazole for in vivo hypoxia imaging. He is also a founder of GUSTO A Research Group.

Dr. Oleg G. Salnikov completed his M.Sc. studies at Novosibirsk State University in 2014. In 2018, he obtained PhD degree in physical chemistry under the supervision of Dr. Kirill Kovtunov at the International Tomography Center SB RAS, the group of Prof. Igor Koptyug. He is continuing there as a post-doctoral researcher. His research is devoted to the development of parahydrogen-based NMR hyperpolarization methods – PHIP and SABRE.

Alexandra Svyatova finished her M.Sc. in Chemical and Biological Physics at Novosibirsk State University in 2019. Now she is a PhD student at Novosibirsk State University. Since 2017, she works at the International Tomography Center SB RAS in the group of Prof. Igor Koptyug. Her main research interest is magnetic resonance imaging of hyperpolarized compounds.

Prof. Igor V. Koptyug received his Ph.D. degree in 1991; in 1992–1995 he was a PD researcher in the photochemistry group of Professor N. J. Turro (Columbia University, NY). He earned his Dr. Sci. (Habilitation) degree in catalysis in 2003 and a title of Professor in 2006; currently, he is the deputy director of International Tomography Center, SB RAS, Novosibirsk. His research interests include combination of catalysis and signal enhancement in NMR.

Prof. Juri G. Gelovani received his M.D. in 1986 and his Ph.D. in 1990 from the University of Tartu, Estonia. 1991-1996, post-doc Fellow, Memorial Sloan Kettering Cancer Center (New York, NY). 2003-2012, Professor & Director, Center for Advanced Biomedical Imaging Research, MD Anderson Cancer Center (Houston, TX). 2012-2019, Professor & Leader, Molecular Imaging Program at Karmanos Cancer Institute, Wayne State University (Detroit, MI). 2008-2009 President, Society for Molecular Imaging; 2010-2011 President, Academy of Molecular Imaging; 2011-2012 President, World Molecular Imaging Society.

Prof. Eduard Y. Chekmenev, PhD in Physical Chemistry with Prof. R. J. Wittebort, 2003, University of Louisville, KY, USA. PD fellow at NHMFL in Tallahassee, FL (Prof. T. A. Cross), Caltech (Prof. D. P. Weitekamp) and HMRI (Dr. B. D. Ross). He serves as a tenured Associate Professor of Chemistry at Wayne State University since 2018. Research interests include development of methods of hyperpolarization and their biomedical and industrial use.