Verdazyl-based polarizing agents and uses in dynamic nuclear polarization

Abstract

The syntheses of a series of verdazyl mono- and biradicals, as well as verdazyl-nitroxide biradicals, are described for high-field dynamic nuclear polarization nuclear magnetic resonance (DNP NMR) spectroscopy. These radicals were examined in high-field DNP NMR experiments (600 MHz / 395 GHz), by measuring 1H signal enhancements directly and through 13C{1H} cross- polarization experiments. X-band EPR, 1H DNP field profiles, and experiments to determine the nuclear build-up times were performed for verdazyl-nitroxide biradicals VerTEMPol and VerTEKol. These hybrid biradicals provide enhancements of up to 100-fold increased signal intensities, representing >104-fold time savings, and approximately 4 times as high as that of the nitroxide biradical TEKPol, a commonly used polarizing agent in the field.

Description

[0001] VERDAZYL-BASED POLARIZING AGENTS AND USES IN DYNAMIC NUCLEAR POLARIZATION

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 720,827, filed November 15, 2024, which is incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Solid-state nuclear magnetic resonance (NMR) spectroscopy is a widely used technique for characterizing a range of (bio-)molecules and materials. However, low sensitivities can restrict these measurements, especially when dealing with NMR-active isotopes with low natural abundance (e.g.,13C,15N,29Si) and / or small gyromagnetic ratios (e.g.,67Zn,15N,89Y). High field dynamic nuclear polarization (DNP) NMR has emerged as an effective way to enhance NMR signal intensities, relying on the spin polarization transfer from unpaired electrons in a polarizing agent to nuclei of interest in the sample. Based on the ratio of the electronic and nuclear gyromagnetic ratios, DNP can produce a maximum theoretical1H signal enhancement (e) of 658. This enhancement greatly improves NMR sensitivity and results in significant time-savings for the experiments (e.g., e = 20 represents a 400-fold (e2) timesavings). DNP enhancement can occur via several mechanisms such as the solid effect, cross effect, Overhauser effect, and thermal mixing. At high magnetic fields (Bo > 5T), the cross effect (CE) is the preferred polarization mechanism, as the DNP enhancement scales with Bo1(whereas the solid effect scales with Bo2). In these cases, sample irradiation with high-frequency microwaves induces a spin-flip of two coupled electrons, which produces a polarization transfer to a nucleus through a hyperfine interaction. For polarization transfer to occur via the cross effect, a given spin-system must be able to satisfy the condition

[0006] | u)e2 ~ coei| = o>n, where o)eand o>nare the electronic and nuclear Larmor frequencies, respectively. Therefore, the breadth of the polarizing agent’s EPR spectrum (|coe2>mei|) must be on the order of, or larger than, the relevant nuclear Larmor frequency (mn), to allow individual electron-electron-nucleus systems to match this condition.

[0007] Accordingly, there is a need for versatile polarizing agents for use in NMR spectroscopy.

[0008] SUMMARY

[0009] Described herein are verdazyl-containing mono- and biradicals that have been successfully synthesized. When solubility permitted (e.g., VerTEMPol, and VerTEKol), these radicals were evaluated in DNP NMR experiments and compared to established nitroxide radicals

[0010] 515.026WO1 1

[0011] UA 2025-035-02 (bTbK and TEKPol), to determine how well they perform as polarizing agents at high magnetic fields (600 MHz / 395 GHz). Verdazyl -nitroxi de hybrid biradicals VerTEMPol and VerTEKol are the most promising under these conditions, providing13C{JH} CP enhancements of s = 40 and 113, respectively. VerTEMPol matched and VerTEKol surpassed enhancements provided by TEKPol and were significantly higher than verdazyl / nitroxide monoradicals. Nuclear spin relaxation times and1H DNP field profiles of the new radicals were obtained, with the relaxation measurements demonstrating that the biradical VerTEKol has slower relaxation than biradical VerTEMPol, likely because of its greater molecular weight and more rigid structure.

[0012] Accordingly, this disclosure provides a compound of Formula I:

[0013]

[0014] wherein,

[0015] each R1is independently radical, H, or (Ci-Ce)alkyl;

[0016] R2and R3are each independently H, halo, CN, ORa, SRa, N(Ra)2, CORa, CO2R1, CON(Ra)2, or SO2N(Ra)2;

[0017] R4is CONRa, CO2, phenyl, biphenyl, or ethynylbenzene;

[0018] each Rais independently H, (Ci-Ce)alkyl, or (CH2CH2O)i-6O(Ci-Ce)alkyl;

[0019] R5is Formula la, lb, Ic, Id, or le:

[0020]

[0021] wherein,

[0022] the definitions of R1, R2, and R3are the same as for Formula I;

[0023] each R6is independently H or (Ci-Ce)alkyl for Formula la;

[0024] R7is CHRbor O for Formula la;

[0025] Rbis phenyl, naphthyl, OH, OP(=O)(OH)2 or H; and

[0026] 515.026WO1 2

[0027] UA 2025-035-02 each R8is independently(Ci-C6)alkyl for Formulas lb and Ic;

[0028] wherein each phenyl, biphenyl, naphthyl, and ethynylbenzene is optionally substituted; and R5is not Formula le when R4is phenyl, biphenyl, or ethynylbenzene;

[0029] or a radical thereof, or a salt thereof.

[0030] This disclosure also provides a method for high-field dynamic nuclear polarization nuclear magnetic resonance spectroscopy (DNP NMR) comprising, acquiring DNP NMR spectral data using suitable acquisition parameters of a sample comprising a compound described above or a composition thereof.

[0031] The invention provides novel compounds of Formulas I-IV, intermediates for the synthesis of compounds of Formulas I-IV, as well as methods of preparing compounds of Formulas I-IV. The invention also provides compounds of Formulas I-IV that are useful as intermediates for the synthesis of other useful compounds. The invention provides for the use of compounds and compositions Formulas I-IV for use in nuclear magnetic resonance spectroscopy.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0034] Figure 1A-E. DNP enhanced13C{1H}CP MAS NMR spectra of the TCE signal recorded at ~100 K and 600 MHz / 395 GHz DNP NMR spectrometer, using a) TEKPol, b) VerTEMPol, c) VerTEKol biradicals in 10 mM TCE at 9 kHz MAS, and d) VerTEKol biradical in 10 mM TCE at 5 kHz. The blue asterisks in panels a-c) indicate the spectra were acquired at So = 14.092 T. The red dagger in panel d) indicates the spectrum was acquired at So = 14.094 T and 5 kHz MAS. e) Computationally optimized structure of VerTEKol, visualized using Avogadro 1.2.0.

[0035] Figure 2. Comparison of solvent13C{3H} CP MAS DNP NMR enhancements (s) at high magnetic field strengths (>14.1 T) for the mono- and biradicals examined in this study (in red) and various radicals from the literature (shown in blue) with MAS frequencies ranging from 5 to 10 kHz, as discussed in the text. DNP experiments conducted on a 600 MHz / 395 GHz DNP NMR spectrometer are represented in solid bars, while the striped bars indicate experiments performed on 699 MHz / 461 GHz (white stripes) and 800 MHz / 527 GHz (black stripes) DNP NMR spectrometers. A detailed report of the experimental parameters is provided in Table 2.

[0036] 515.026WO1 3

[0037] UA 2025-035-02 Figure 3. Experimental (solid lines) and simulated (semi-transparent lines) X-band (9.62 GHz) continuous wave EPR spectra of all the mono- and biradicals listed in Table 1, recorded at 295 K.

[0038] Figure 4. a) X-band (9.62 GHz) continuous wave EPR spectra of TEKPol (in red) and VerTEKol (in blue) recorded at 295 K. b) 'H DNP field profiles of TEKPol (red circle) and hybrid biradical VerTEKol (blue square acquired at 14.1 T and 100 K; the enhancements were normalized w.r.t. the maximum enhancements (smax^H)) of 13 and 33 for TEKPol and VerTEKol, respectively.

[0039] Figure 5A-H. DNP enhanced13C{1H}CP MAS NMR spectra of the TCE signal recorded at ~100 K and 600 MHz / 395 GHz DNP NMR spectrometer, using a) TEKPol (at 5 and 7 kHz MAS on left and right, respectively), b) bTbK, c) TEKone, d) la, e) TEKone + la, f) ErythriPol, g) VerTEMPol, and h) VerTEKol in TCE (10 mM for biradicals and 20 mM for monoradicals) at 5 kHz MAS. All the DNP NMR spectra were acquired at Bo = 14.092 T, except for the spectrum of la marked with an asterisk (in red) in panel d), which was acquired at Bo = 14.112 T.

[0040] DETAILED DESCRIPTION

[0041] High-field dynamic nuclear polarization nuclear magnetic resonance (DNP NMR) spectroscopy transfers polarization from unpaired electrons in polarizing agents to nuclei of interest to boost NMR sensitivity. Verdazyl biradicals are a promising choice as polarizing agents because they have been found to generate narrower electron paramagnetic resonance (EPR) signals compared to nitroxide biradicals; an advantageous characteristic for high field DNP when operating above 400 MHz / 263 GHz. The use of verdazyl radicals as DNP polarizing agents has been very limited to date. Yet, recent numerical simulations have predicted that verdazyl-nitroxide hybrid biradicals could be more effective polarizing agents than nitroxide-nitroxide biradicals. Herein, the syntheses of a series of verdazyl mono- and biradicals, as well as verdazyl-nitroxide biradicals are described. These radicals were examined in high-field DNP NMR experiments (600 MHz / 395 GHz), by measuring 'H signal enhancements directly and through13C{JH} cross- polarization experiments. X-band EPR, 'H DNP field profiles, and experiments to determine the nuclear build-up times were performed for verdazyl-nitroxide biradicals VerTEMPol and VerTEKol. These hybrid biradicals provide enhanced signal intensities of greater than 100-fold (i.e., representing >104-fold time savings), approximately four times as high as that of the nitroxide biradical TEKPol, a commonly used polarizing agent in the field.

[0042] Definitions.

[0043] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, 515.026WO1 4

[0044] UA 2025-035-02 such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0045] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0046] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0047] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0048] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0049] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the

[0050] 515.026WO1 5

[0051] UA 2025-035-02 terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0052] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible subranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0053] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number ”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ..., 9, 10. It also means 1.0, 1.1, 1.2, 1.3, ..., 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers less than number 10, as discussed above. Similarly, if the variable disclosed is a number greater than “number 10”, it implies a continuous range that includes whole numbers and fractional numbers greater than number 10. These ranges can be modified by the term “about”, whose meaning has been described above.

[0054] The recitation of a), b), c), ... , or i), ii), iii), or the like in a list of components or steps do not confer any particular order unless explicitly stated.

[0055] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group

[0056] 515.026WO1 6

[0057] UA 2025-035-02 listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0058] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture.

[0059] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0060] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0061] This disclosure provides methods of making the compounds and compositions of the invention. The compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis. Many techniques such as etherification and esterification are well known in the art. However, many of these techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6; as well as standard organic reference texts such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, New York, 2001); Comprehensive Organic Synthesis.

[0062] 515.026WO1 7

[0063] UA 2025-035-02 Selectivity, Strategy & Efficiency in Modem Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing); Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); for heterocyclic synthesis see Hermanson, Greg T., Bioconjugate Techniques, Third Edition, Academic Press, 2013.

[0064] The formulas and compounds described herein can be modified using protecting groups. Suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Protecting Groups in Organic Synthesis, Second Edition, Greene, T. W., and Wuts, P. G. M., John Wiley & Sons, New York, and references cited therein; Philip J. Kocienski; Protecting Groups, Georg Thieme Verlag Stuttgart, New York, 1994, and references cited therein; and Comprehensive Organic Transformations, Larock, R. C., Second Edition, John Wiley & Sons, New York, 1999, and referenced cited therein).

[0065] The term "halo" or "halide" refers to fluoro, chloro, bromo, or iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0066] The term "alkyl" refers to a branched or unbranched hydrocarbon having, for example, from 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or 1-4 carbon atoms; or for example, a range between 1-20 carbon atoms, such as 2-6, 3-6, 2-8, or 3-8 carbon atoms. As used herein, the term “alkyl” also encompasses a “cycloalkyl”, defined below. Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl ( / .s -propyl), 1 -butyl, 2-methyl-l -propyl ( / .s -butyl), 2-butyl (secbutyl), 2-methyl-2 -propyl ( / -butyl), 1 -pentyl, 2-pentyl, 3 -pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3 -methyl- 1 -butyl, 2-methyl-l -butyl, 1 -hexyl, 2-hexyl, 3 -hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3 -methyl-3 -pentyl, 2-methyl-3 -pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl can be unsubstituted or substituted, for example, with a substituent described below or otherwise described herein. The alkyl can also be optionally partially or fully unsaturated. As such, the recitation of an alkyl group can include an alkenyl group or an alkynyl group. The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., an alkylene).

[0067] An alkylene is an alkyl group having two free valences at a carbon atom or two different carbon atoms of a carbon chain. Similarly, alkenylene and alkynylene are respectively an alkene and an alkyne having two free valences at two different carbon atoms, or an alkenylene can have the two free valences on the same carbon.

[0068] The term "cycloalkyl" refers to cyclic alkyl groups of, for example, from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like. The cycloalkyl can be unsubstituted or substituted. The cycloalkyl group can be monovalent or divalent and can be optionally substituted as described for alkyl groups. The cycloalkyl group can optionally include one or more cites of

[0069] 515.026WO1 8

[0070] UA 2025-035-02 unsaturation, for example, the cycloalkyl group can include one or more carbon-carbon double bonds, such as, for example, 1 -cyclopent- 1-enyl, 1 -cyclopent-2-enyl, 1 -cyclopent-3 -enyl, cyclohexyl, 1-cyclohex-l-enyl, 1 -cyclohex-2-enyl, 1 -cyclohex-3 -enyl, and the like.

[0071] The term “heteroatom” refers to any atom in the periodic table that is not carbon or hydrogen. Typically, a heteroatom is O, S, N, P. The heteroatom may also be a halogen, metal or metalloid.

[0072] The term "heterocycloalkyl" or “heterocyclyl” refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3- to 10-membered, more preferably 4- to 7-membered. Examples of suitable heterocycloalkyl substituents include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, morpholinyl, 1,3-diazapanyl, 1 ,4-diazapanyl, 1 ,4-oxazepanyl, and 1,4-oxathiapanyl. The group may be a terminal group or a bridging group.

[0073] The term "aromatic" refers to either an aryl or heteroaryl group or substituent described herein. Additionally, an aromatic moiety may be a bisaromatic moiety, a trisaromatic moiety, and so on. A bisaromatic moiety has a single bond between two aromatic moieties such as, but not limited to, biphenyl, or bipyridine. Similarly, a trisaromatic moiety has a single bond between each aromatic moiety.

[0074] The term "aryl" refers to an aromatic hydrocarbon group derived from the removal of at least one hydrogen atom from a single carbon atom of a parent aromatic ring system. The radical attachment site can be at a saturated or unsaturated carbon atom of the parent ring system. The aryl group can have from 6 to 30 carbon atoms, for example, about 6-10 carbon atoms. The aryl group can have a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like. The aryl can be unsubstituted or optionally substituted with a substituent described below. For example, a phenyl moiety or group may be substituted with one or more substituents Rxwhere Rxis at the ortho-, meta-, or ara-position, and X is an integer variable of 1 to 5.

[0075] The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. The heteroaryl can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, as described in the definition of "substituted". Typical heteroaryl groups contain 2-20 carbon atoms in the ring skeleton in addition to the one or more heteroatoms, wherein the ring skeleton comprises a 5-membered ring, a 6-membered ring, two 5-membered rings, two 6-membered rings, or a 5 -membered ring fused to a 6-membered ring.

[0076] Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, 0-carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl,

[0077] 515.026WO1 9

[0078] UA 2025-035-02 indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthenyl. In one embodiment the term "heteroaryl" denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or (Ci-Ce)alkylaryl. In some embodiments, heteroaryl denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benzo-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

[0079] As used herein, the term "substituted" or “substituent” is intended to indicate that one or more (for example, in various embodiments, 1-10; in other embodiments, 1-6; in some embodiments 1, 2, 3, 4, or 5; in certain embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group indicated in the expression using “substituted” (or “substituent”) is replaced with a selection from the indicated group(s), or with a suitable group known to those of skill in the art, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a stable compound. Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, carboxyalkyl, alkylthio, alkylsulfinyl, and alkylsulfonyl. Substituents of the indicated groups can be those recited in a specific list of substituents described herein, or as one of skill in the art would recognize, can be one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano. Suitable substituents of indicated groups can be bonded to a substituted carbon atom include F, Cl, Br, I, OR', OC(O)N(R')2, CN, CFs, OCFs, R', O, S, C(O), S(O), methylenedioxy, ethylenedioxy, N(R')2, SR', SOR', SO2R', SO2N(R')2, SOsR', C(O)R', C(O)C(O)R', C(O)CH2C(O)R', C(S)R', C(O)OR', OC(O)R', C(O)N(R')2, OC(O)N(R')2, C(S)N(R')2, (CH2)O-2NHC(O)R', N(R')N(R')C(O)R', N(R')N(R')C(O)OR', N(R')N(R')CON(R')2, N(R')SO2R', N(R')SO2N(R')2, N(R')C(O)OR', N(R')C(O)R', N(R')C(S)R', N(R')C(O)N(R')2, N(R')C(S)N(R')2, N(COR')COR', N(OR')R', C(=NH)N(R')2, C(O)N(OR')R', or C(=NOR')R' wherein R’ can be hydrogen or a carbon-based moiety (e.g., (Ci-Ce)alkyl), and wherein the carbon-based moiety can itself be further substituted. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond. When a substituent is divalent, such as O, it is

[0080] 515.026WO1 10

[0081] UA 2025-035-02 bonded to the atom it is substituting by a double bond; for example, a carbon atom substituted with O forms a carbonyl group, C=O.

[0082] Stereochemical definitions and conventions used herein generally follow S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, which form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S. are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselectivity or stereospecificity in a chemical reaction or process.

[0083] Embodiments of the Technology.

[0084] 1. A compound of Formula I:

[0085]

[0086] wherein,

[0087] each R1is independently radical, H, (Ci-Ce)alkyl, or (C3-Ce)cycloalkyl;

[0088] R2and R3are each independently H, halo, CN, ORa, SRa, N(Ra)2, CORa, CChRa, CON(Ra)2, SO2N(Ra)2, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl;

[0089] R4is CONRa, CO2, or an aromatic group such as phenyl, biphenyl, terphenyl, or ethynylbenzene;

[0090] each Rais independently H, (Ci-Ce)alkyl, or (CH2CH2O)i-6O(Ci-Ce)alkyl;

[0091] R5is Formula la, lb, Ic, Id, or le:

[0092] 515.026WO1 11

[0093] UA 2025-035-02

[0094]

[0095] wherein,

[0096] the definitions of R1, R2, and R3are the same as for Formula I;

[0097] each R6is independently H or alkyl for Formula la;

[0098] R7is CHRbor O for Formula la;

[0099] Rbis phenyl, naphthyl, OH, 0P(=0)(0H)2, H, Ns, NH2, NHalkyl, N(alkyl)2, Oalkyl, SH, Salkyl, heteroaryl, or heterocycloalkyl; and

[0100] each R8is independently (Ci-Ce)alkyl for Formulas lb and Ic;

[0101] wherein each phenyl, biphenyl, naphthyl, and ethynylbenzene is optionally substituted; and R5is not Formula le when R4is phenyl, biphenyl, or ethynylbenzene;

[0102] or radical thereof, or a salt thereof.

[0103] In some embodiments, the phenyl moiety that has a covalent bond to R4in Formula I is further substituted with one or more substituents as defined in the definitions, such as halo, alkyl, alkoxy, or hydroxy. In some embodiments, compounds of Formula I comprise verdazyl radicals and non-nitroxide radicals such as Gd-DOTA / trityl / BDPA.

[0104] 2. The compound of embodiment 1, wherein each R1is radical.

[0105] 3. The compound of embodiment 1 or 2, wherein each R2and R3is H.

[0106] 4. The compound of any one of embodiments 1-3, wherein each R2and each R3is independently in the ortho, meta, or para position.

[0107] 5. The compound of any one of embodiments 1-4, wherein R4is CONH.

[0108] In some embodiments, R4is:

[0109]

[0110] In some embodiments, R4is a polyaromatic hydrocarbon.

[0111] 6. The compound of any one of embodiments 1-4, wherein R4is CONH and R5is bonded to the nitrogen atom of CONH (e.g., -(C=O)NHR5); or wherein R4is CONH and R5is bonded to the carbon atom of CONH (e g., R5(C=O)NH-).

[0112] 515.026WO1 12

[0113] UA 2025-035-02 7. The compound of any one of embodiments 1-6, wherein R5is Formula la or Formula lb. 8. The compound of any one of embodiments 1-7, wherein R5is Formula la and each R6is H.

[0114] 9. The compound of any one of embodiments 1-8, wherein R5is Formula la and R7is CHPh. 10. The compound of any one of embodiments 1-7, wherein R5is Formula lb and each R8is CHs.

[0115] 11. The compound of any one of embodiments 1-10, wherein Formula I is represented by Formula II:

[0116]

[0117] or a salt thereof.

[0118] 12. The compound of any one of embodiments 1-10, wherein Formula I is represented by Formula III or Formula IV :

[0119]

[0120] or a salt thereof.

[0121] In some embodiments, a compound of any one of Formulas I-IV is a deuterated or fluorinated derivative thereof. In some embodiments, a compound of any one of Formulas I-IV is a tetrahydroxy derivative thereof. In some embodiments, a compound of any one of Formulas I-IV represents a monomer moiety or repeating unit in an oligomer or polymer.

[0122] 13. The compound of embodiment 1, wherein the compound is:

[0123]

[0124] 515.026WO1 13

[0125] UA 2025-035-02

[0126]

[0127] In some embodiments, the compound is:

[0128]

[0129] or a salt thereof.

[0130] 515.026WO1

[0131] UA 2025-035-02 14. The compound of embodiment 1, wherein the compound is VerTEKol or VerTEMPol:

[0132]

[0133] or a salt thereof.

[0134] 15. A composition comprising the compound of any one of embodiments 1-14 and a solvent or matrix. In some embodiments, the composition comprises an oligomer or polymer.

[0135] 16. A method for high-field dynamic nuclear polarization nuclear magnetic resonance spectroscopy (DNP NMR) comprising, acquiring DNP NMR spectral data using suitable acquisition parameters of a sample comprising a compound or composition of any one of embodiments 1-15.

[0136] In some embodiments, data acquisition is performed on a DNP NMR spectrometer configured with a gyrotron magnet that produces about 5W to about 30W of micro waves.

[0137] 17. The method of embodiment 16, wherein the composition comprises VerTEKol or VerTEMPol.

[0138] 18. The method of embodiment 16 or 17, wherein the sample comprises a substance requiring DNP NMR spectral characterization.

[0139] 19. The method of embodiment 18, wherein the substance is a protein, biomolecule, polymer, organic compound, inorganic compound, or organometallic compound.

[0140] 20. The method of embodiment 18, wherein conditions for acquiring DNP NMR spectral data of the sample results in an enhanced signal to noise ratio of the substance compared against the same conditions for a sample that does not comprise VerTEKol or VerTEMPol.

[0141] Exploring the Potential of Verdazyl-Based Radicals for High-Field Dynamic Nuclear Polarization NMR.

[0142] The most common type of organic polarizing agents for DNP are nitroxide biradicals (Chart la). Polarizing agents such as bTbK and TEKPol (s = 47 and s = 200, both at 400 MHz, respectively, Chart la) possess rigid spirocyclic backbones, which tether the nitroxide radicals together in a specific orientation and at a fixed distance, allowing for the necessary polarization transfer conditions to be met more efficiently. Since these radicals are mainly soluble in organic solvents, aqueous-soluble analogs, such as SPIROPol (s = 170, at 212 MHz, Chart la), have been developed (J. Phys. Chem. B, 2022, 126, NMTy The rigid, spirocyclic biradicals of the bTbK family can routinely provide enhancements of up to two orders of magnitude, when using

[0143] 515.026WO1 15

[0144] UA 2025-035-02 magnetic fields <400 MHz / 263 GHz (Chart la). The most commonly used water-soluble polarizing agents are AMUPol (s = 235, at 400 MHz, Chart la) and its derivatives, with HydroPol achieving enhancements of up to s = 300 (400 MHz). However, DNP at high magnetic fields still proves challenging since enhancements scale with Bo-1. This inverse relationship is mainly due to a broadening of the electron paramagnetic resonance (EPR) lines, which leads to a decreased ability to saturate the electron spin resonance signal. Lund et al. approached this problem by developing the water-soluble TinyPol radicals, which provided greater enhancements than AMUPol at high field strengths, with the most successful derivative being M-TinyPol (s = 90, at 800 MHz, Chart la). The authors attributed this improved performance to the stronger dipolar coupling between the more closely bound radicals. Similarly, asymmetrical nitroxide biradicals in the AsymPol family also contain short spacers with strong exchange and dipolar interactions between the unpaired electrons while maintaining a non-coplanar relative orientation of the nitroxides. Both organic- and water-soluble biradicals of this type have been synthesized and evaluated in DNP experiments (Chart la, AsymPol-POK and AsymPol-TEK).

[0145] Chart 1. Structures of radicals previously employed as polarizing agents for high field DNP, along with the corresponding measured enhancements and some experimental parameters: a) examples of nitroxide biradicals, b) trityl -nitroxi de hybrid biradicals, and c) a BDPA-nitroxide biradical. The enhancement values listed in this figure are not necessarily the maximum enhancement values obtained, rather, they were chosen from those reported by the respective authors, based on how closely the experimental parameters align with those from this study (see Table 3 for details).

[0146] Distances are approximated using ChemDraw 3-D, after MM2 force field optimization.

[0147]

[0148] 515.026WO1 16

[0149] UA 2025-035-02

[0150]

[0151] 515.026WO1 17

[0152] UA 2025-035-02

[0153] "

[0154]

[0155] Recently, hybrid biradicals containing a relatively narrow-line radical and a nitroxi de radical (Chart Ib-c) have become the focus of much research. These polarizing agents tend to be more effective at high fields than conventional nitroxide biradicals, because of the ability to irradiate (hole bum) on the narrow resonance, with coupling to the broad resonance for polarization transfer. So far, based on previous studies using mixtures of monoradicals, the choice of narrow-line radical moiety has been mainly restricted to carbon-centered trityl and BDPA (l,3-bisdiphenylene-2-phenylallyl) radicals, such as TEMTriPol-1 and HyTEK2, respectively (s = 65 and s = 64, at 800 MHz, Chart Ib-c). PyrroTriPol hybrid biradicals (s = 106, at 600 MHz, Chart lb) were recently introduced as adaptations from the TEMTriPol radical line, providing a more rigid spacer while also being easier to synthesize on a larger scale. In general, hybrid biradicals have shown the most promise in improving high-field DNP by producing large enhancements at high magnetic fields between 800 and 900 MHz.

[0156] Nevertheless, the choice of polarizing agent for DNP studies is very system-dependent, and sensitivity enhancements vary based on several factors. As can be seen in Chart 1, even enhancements obtained using the same polarizing agent can change quite significantly due to, for example, the field strength, solvent, micro wave power, radical concentration, magic-angle spinning (MAS) frequency, and / or sample temperature. Therefore, objectively comparing current polarizing agents between independent studies can be difficult. In general, developing new classes of polarizing agents remains an important task to accommodate a broad range of possible experimental conditions and applications within solid-state NMR spectroscopy.

[0157] Verdazyl radicals are potentially useful for high-field DNP NMR since they produce narrower EPR lines, compared to nitroxide radicals, and are relatively stable in solution. First synthesized and characterized by Kuhn and Trischmann in 1963 (Angew. Chem., 1963, 75, 294), verdazyl radicals have since been the focus of much research due to their unique magnetic

[0158] 515.026WO1 18

[0159] UA 2025-035-02 properties and stability. Nevertheless, verdazyl monoradicals have sparsely been used in liquidstate DNP and only once as a polarizing agent in solid-state DNP NMR to date. A ribose-functionalized verdazyl radical (Chart 2, verdazyl-ribose) produced up to 74-fold13C{1H } CP signal enhancements at 400 MHz / 264 GHz (in 6:3:1 r / s-glycerol / DiO / HiO, at ~30 K) with nonspinning samples and enhancements of up to 31 with MAS. Although verdazyl biradicals have previously been synthesized (Chart 2) and numerical simulations have shown that verdazyl-nitroxide hybrid biradicals may be more effective than conventional nitroxide biradicals, such radicals have never been evaluated as polarizing agents for high-field DNP NMR thus far.

[0160] Therefore, a series of nitroxide radicals (TEKone, bTbK, ErythriPol, and TEKPol, see Chart 3) were synthesized, and a series of verdazyl-containing mono- (la-b) and biradicals (2a-e, VerTEMPol, and VerTEKol, see Chart 4) was developed, to expand the current repertoire of organic polarizing agents, which consisted mainly of nitroxide and carbon-centred radicals up until now. These radicals were characterized by EPR spectroscopy and mass spectrometry, amongst other methods. The polarizing agents were then assessed in DNP NMR experiments at 600 MHz / 395 GHz, by determining their resulting13C{JH} CP MAS NMR signal enhancements (Table 1 and Figure 1), obtaining1H DNP field profiles (Figure 4), and measuring spin relaxation times (Table 1) These studies used the established nitroxide biradical TEKPol as a benchmark to compare to the verdazyl-containing radicals.

[0161] Chart 2. Examples of previously synthesized verdazyl radicals. Where applicable,13C{3H} CP MAS DNP NMR enhancements (s) are indicated along with select experimental parameters. * s = 74 was obtained with no MAS.

[0162]

[0163] Oxo -verdazyl biradical Oxo-Kuhn-ver azyl biradical 515.026WO1 19

[0164] UA 2025-035-02 Table 1. Enhancements provided by nitroxide and verdazyl radicals evaluated in this study, measured through13C{1H}CP MAS DNP NMR experiments at a 600 MHz / 395 GHz DNP NMR spectrometer at So = 14.092 T (asterisk (*) indicates the spectra recorded at So = 14.094 T).

[0165]

[0166] aThe absolute sensitivities were determined using the methodology outlined in Chem. Sci., 2012, 3, 108.

[0167] Results and Discussion.

[0168] Synthesis of Nitroxide Radicals: TEKone, bTbK, ErythriPol, and TEKPol. A short series of previously synthesized nitroxide mono- and biradicals (Chart 3) were resynthesized to metricize the verdazyl-based radicals designed and synthesized herein (see Scheme 2 in Examples). Up until the final oxidation step, the same synthetic procedure was used as was previously reported for TEKPol and its precursors. However, attempts to form TEKPol using H2O2 and the Na2WOr*2H2O catalyst were unsuccessful. It is possible that stabilizers in the H2O2 solution prevented the reaction from occurring. Therefore, we attempted a different approach using mCPBA, as this method has previously been described in the synthesis of similar nitroxide radicals. This reaction afforded TEKPol in 28% yield, which was 39% lower than previously reported for the original method (J. Am. Chem. Soc., 2013, 135, 12790). ErythriPol was synthesized using an erythritol spacer instead of pentaerythritol, in 8% yield, over two steps. This compound offers an interesting comparison to TEKPol, since it is structurally similar, but does not restrict the nitroxide radicals to an orthogonal orientation. Therefore, one would expect ErythriPol to yield lower DNP enhancements than TEKPol, since the unimpeded access to all possible molecular orientations about the central bond would decrease the probability that any

[0169] 515.026WO1 20

[0170] UA 2025-035-02 given dimer is at the optimal orientation for polarization transfer. This scaffold, in comparison to TEKPol and bTbK, can help unconfound the relative contribution of the locked orientation of the radicals and the 4-phenylcyclohexyl groups on the enhancements provided by TEKPol. TEKone was made as a monoradical equivalent of TEKPol, to evaluate the effect of tethering radicals together, and it was also a necessary precursor in the synthesis of hybrid biradicals VerTEMPol and VerTEKol. Using mCPBA, the oxidation provided TEKone in 79% yield.

[0171] Previously, this radical had been synthesized by Emsley and coworkers in 67% yield. With these radicals in hand, direct comparisons could be drawn using our 600 MHz / 395 GHz DNP NMR spectroscopy setup and sample preparation protocols.

[0172] Chart 3. Structures of nitroxide mono- and biradicals synthesized herein, which were used to metricize the verdazyl radicals.

[0173]

[0174] bTbK TEKone

[0175] Synthesis of Verdazyl Radicals: la b, 2a d. VerTEMPol, and VerTEKol. Symmetrical verdazyl biradicals with a series of spacers were synthesized (Chart 4, see Scheme 3 in Examples) based on athree step protocol adapted from Matuschek et al. (Chem. Sci., 2015, 6, 4712), who had previously synthesized mono- and biradicals l-2a. Monoradicals la-b were of sufficient solubility to utilize for DNP NMR investigation in TCE (1,1,2,2-tetrachloroethane), however, the dimeric biradical counterparts 2a-d were relatively insoluble in a suite of common organic solvents. In particular, the required radical concentrations could not be reached in TCE, even with sonication or elevated temperature. Thus, the preparation of a glassy matrix of sample and radical enhancement agent using 2a-d was not feasible. Since conventional NMR spectroscopy could not be used to analyze these compounds, establishing their purity was also

[0176] 515.026WO1 21

[0177] UA 2025-035-02 challenging. However, FTIR (Fourier transform infrared) spectra of the radicals were useful in identifying whether the product still contained N-H functional groups from the starting material or / ez / co-verdazyl impurity, first described by Kuhn and Trischmann. Based on the IR spectra, most of the isolated radicals appeared to contain some / ez / co-verdazyl impurities after recrystallization attempts, so they required further purification. The initial purification attempts (precipitation from 1 : 1 CHiCF / hexanes, or ethyl acetate, and flash column chromatography) were unsuccessful, at least in part due to the low solubility of the crude products and side products. Pure verdazyl biradicals 2a-d (as deemed by IR spectroscopy and TLC analyses) were eventually isolated, and their structures confirmed by MALDI HRMS through a combination of trituration in hot CH2CI2 and preparative TLC (2:3 CH2C12 / hexanes). Exact yields of the final step were not obtained for biradicals 2a, 2c, and 2d, as these poorly soluble synthetic targets were not pursued further. However, as a representative example, the overall reaction yield in the synthesis and purification of 2b, through preparative TLC, was approximately 69%.

[0178] Chart 4. Verdazyl mono- (la-b) and symmetrical biradicals (2a-d).

[0179]

[0180] To remedy these solubility issues, we hypothesized that increased molecular dipole and reduced packing propensity of mixed biradicals could enhance the solubility of the verdazyl-based biradical scaffolds. To investigate this supposition and since narrow-line hybrid biradicals have been successfully used in high-field DNP in the past, the synthesis of VerTEMPol and VerTEKol was undertaken (Scheme 1). Utilizing lb, EDC (1 -ethyl -3 -(3-dimethylaminopropyl)carbodiimide) coupling reactions were performed using 4-amino-TEMPO and TEKamine to afford hybrid verdazyl -nitroxi de biradicals VerTEMPol and VerTEKol, respectively. These mixed dimers were significantly more soluble in organic solvents, simplifying the purification and DNP sample preparation in TCE.

[0181] As a crude measure of their stability, a ~1 pM solution of VerTEMPol was made in TCE and stored in two cuvettes, one at room temperature and one at -30 °C. The absorption profiles of 515.026WO1 22

[0182] UA 2025-035-02 these two solutions were then compared by UV-vis spectroscopy over the course of multiple days to see if any significant changes could be seen in their spectra (at 326 nm and 546 nm). After eight days, no significant changes to the absorption spectra of the room temperature or cold sample were observed, indicating that the verdazyl -nitroxi de hybrid biradicals were relatively stable and could be safely stored over this timeframe. This result is in line with general verdazyl radical stability trends observed in the past, with compounds being stable under standard conditions for long periods. Similar studies of the stability of other verdazyl radicals and their / ez / co-verdazyl decomposition products, using UV-vis spectroscopy to monitor for decomposition, have previously been reported by Steen et al., to investigate the stability of Kuhn-type verdazyl radicals when used in batteries.

[0183] Scheme 1. Synthesis of verdazyl-nitroxide hybrid biradicals VerTEMPol and VerTEKol. TEKamine was synthesized as described by Kubicki et al. (Chem. Sci., 2016, 7, 550).

[0184] <

[0185]

[0186] 1. EDC’HCI.DMAP

[0187] CH2CI2, rt, 30 min

[0188]

[0189]

[0190] 2. 4-amino-TEMPO, or

[0191] 2) p-benzoquinone TEKamine

[0192] CH2CI2, 120 °C

[0193]

[0194] rt, 20 h

[0195] pressure vessel

[0196]

[0197] Distances are approximated using ChemDraw 3-D, after MM2 force field optimization.

[0198] Quantum Chemical Calculations. Density functional theory (DFT) calculations were performed to obtain optimized structures of the triplet biradicals. The electron-electron separation in the verdazyl- nitroxide hybrid biradicals was crudely estimated by determining the average distance between the two nitrogen atoms with the highest spin-density in the verdazyl ring (N3 and N4), and the oxygen atom from the nitroxide radical. For both VerTEKol and VerTEMPol

[0199] 515.026WO1 23

[0200] UA 2025-035-02 the unpaired electrons were found to be separated by -12.7 A (Figure le). Surprisingly, the calculated spin-spin dipolar coupling values were significantly different, with an interaction of 20 MHz for VerTEKol and 39 MHz for VerTEMPol. Additionally, the amide linkage introduces a twist angle of about 19° and about 23° between the plane of the verdazyl / phenyl groups and the amide nitrogen atom for VerTEKol and VerTEMPol, respectively (Figure le). So far, comparison of the computed structures to experimental crystal structures of these biradicals has not been possible.

[0201] Evaluation of Polarizing Agents in DNP NMR Spectroscopy at 600 MHz / 395 GHz. The low solubility of verdazyl biradicals 2a-d prevented the preparation of radical solutions of adequate concentration (-10 mM) in TCE, which has been conventionally used as the glassy matrix for polarizing agents that were soluble in organic solvents. When attempts were made to prepare the solutions at this concentration, some of the radical sample dissolved, while the rest remained suspended in the solution. DNP experiments using these radical suspensions did not provide enhancements. As was mentioned above, multiple other solvents were tested (e.g., PhMe, CHCh, CH2CI2, EtOAc, Et2O, MeCN, DMSO, DMF, DMA, and o-DCB), to assess radical solubility, however, none were suitable.

[0202] The nitroxi de radicals (TEKone, ErythriPol, and TEKPol), verdazyl monoradical la, and the verdazyl-nitroxide hybrid biradicals VerTEMPol and VerTEKol were evaluated in DNP NMR spectroscopy studies at Bo= 14.092 T, unless otherwise mentioned.13C {XH} CP MAS DNP NMR spectroscopy enhancements (a = son / oir - 1) induced by the radicals at 600 MHz / 395 GHz are provided in Table 1, and selected NMR spectra are shown in Figure 1; the remaining DNP NMR spectra are presented in Figure 5. The chosen benchmark for the other polarizing agents, TEKPol, was evaluated and provided a maximum enhancement of a = 39 at 9 kHz MAS (a = 26 at 5 kHz MAS), which, as expected, was lower than the maximum enhancement that was achieved at a lower magnetic field of 400 MHz (a = 200). This value was also lower than the previously reported maximum enhancement provided by TEKPol at 600 MHz (a = 122), which was likely due to the authors deoxygenation of TCE, through multiple freeze-thaw cycles prior to the experiments. Monoradical TEKone (at twice the concentration) and the less rigid biradical, ErythriPol, produced enhancements of a = 4 and 21 (at 5 kHz MAS), respectively. These results matched what was expected of monoradicals and less rigid biradical polarizing agents. Thus, by comparing TEKone and ErythriPol, it can be concluded that about a 4-fold increase in a could be attributed by tethering the monoradical into a biradical, which is in accordance with what has been previously found in comparing TEMPO with its biradical counterpart bTbK.

[0203] Likewise, comparing ErythriPol to TEKPol, the restricted orthogonal alignment of the two radical moieties increased a by about 20%. Also, it was observed that increasing the MAS frequency from 5 to 9 kHz led to an increase in the enhancement provided by TEKPol (a = 26 — > 515.026WO1 24

[0204] UA 2025-035-02 39). Zagdoun et al. reported a similar MAS frequency dependence with enhancements increasing as MAS frequency was increased to ~6 kHz. However, beyond 6 kHz, enhancements began to decrease, and, for reasons that were not fully understood, they increased at ~12 kHz again, with a maximum enhancement occurring at 15 kHz. The enhancement for bTbK was s = 6 (at 5 kHz MAS), almost four times less than TEKPol, analogous to the difference in enhancement previously observed at 400 MHz. Since verdazyl mono-radical la and hybrid biradicals VerTEMPol and VerTEKol were sufficiently soluble in TCE, these radicals were also tested.

[0205] Radical la initially produced an enhancement of s = -2 at 14.092 T, indicating that we were probing the radical on the negative side of its field profile. Once the field strength was adjusted (Bo= 14.112 T), the maximum enhancement obtained for la was s = 2. VerTEMPol on the other hand, produced an enhancement of s = 40 at 9 kHz MAS, matching the established polarizing agent TEKPol at the same MAS frequency, field strength, temperature, and concentration. Similar to TEKPol, the enhancements produced by VerTEMPol were lower at 5 kHz (s = 26). As shown in Figure 1, VerTEKol even surpassed TEKPol, providing an enhancement of s = 91 at 9 kHz MAS, which was greater than twice that of TEKPol at this field strength and MAS frequency, representing ~8,300x time savings. Additionally, upon adjusting the field strength (Bo= 14.094 T), the maximum enhancement for VerTEKol was achieved as s = 113 at 5 kHz MAS, representing ~13,000x time savings (Figure 1). This enhancement was roughly four times greater than TEKPol at similar experimental conditions, marking the highest enhancement observed in this study.

[0206] As previously discussed, it is difficult to compare these results to those obtained from established radicals directly. However, compared to many contemporary high-field DNP biradicals evaluated under similar conditions, VerTEKol provides comparable, if not higher,13C {XH} CP MAS DNP NMR enhancements (Table 2). At the same field strength (600 MHz / 395 GHz), similar solution matrix (10 mM in degassed TCE with h-BN), MAS frequency of 8 kHz, and temperature (105 K), the asymmetric nitroxide biradical AsymPol-TEK provided a lower enhancement of s = 74. The organic-soluble trityl -nitroxi de hybrid biradical TEMTriPol-l-OMe also produced a significantly lower enhancement (s = 32) under these conditions, while a more concentrated biradical solution (16 mM) was used. Under the same conditions as for TEMTriPol-1-OMe, DNP studies using PyrroTriPol-OMe generated an enhancement of s = 106. Direct comparison to BDPA-nitroxide hybrid biradicals, is not possible since these radicals have not been tested at a 600 MHz / 395 GHz field strength. Nevertheless, at a comparable MAS frequency (10 kHz), significantly higher radical concentration in TCE (32 mM), and higher field strength (800 MHz / 527 GHz), the best BDPA-nitroxide biradical, HyTEK2, provided a13C{JH} CP MAS DNP NMR enhancement of s = 64. The increased enhancement offered by VerTEKol compared to VerTEMPol could be attributed to longer nuclear spin relaxation times (Ti).

[0207] 515.026WO1 25

[0208] UA 2025-035-02 As has been established for bTbK and TEKPol, and is supported by our results, having more methyl groups near the nitroxide radicals can lead to quicker electron spin relaxation, due to increased molecular motion by means of the methyl group rotation. Indeed, the measured relaxation times for VerTEKol were found to be twice as long as those of VerTEMPol (Table 1), further supporting this conclusion.

[0209] Interestingly, no significant change in the13C{1H} CP enhancement of TCE, induced by VerTEKol, was observed in acquisitions run between 5 kHz and 9 kHz MAS. A 1: 1 solution of monoradicals TEKone and la in TCE, containing the same effective radical concentration, was also tested, to compare to VerTEKol. In accordance with the comparison of monoradical TEKone to TEKPol, this mixture only produced an enhancement of s = 5. Figure 2 presents a comparative plot of ’H DNP enhancements (s) for13C {XH} CP MAS NMR experiments at high magnetic field strengths (>600 MHz / 395 GHz) for the mono- and biradicals analyzed in this study, alongside various radicals reported in the literature as discussed above.

[0210] Table 2. Comparison of previously reported biradicals with VerTEMPol and VerTEKol, and the respective experimental conditions. The selected enhancements reported in the table were chosen based on the parameters that aligned best with the experimental parameters used in this study. The asterisk (*) indicates that multiple freeze-thaw cycles were used to deoxygenate the solvent.

[0211]

[0212] 515.026WO1 26

[0213] UA 2025-035-02

[0214]

[0215] ah-BN is hexagonal boron nitride, which is known to increase microwave penetration of the sample, leading to greater s.

[0216] The magnetic properties of the radicals were characterized by performing X-band (9.62 GHz) EPR spectroscopy, using all the mono- and biradicals as listed in Table 1.

[0217] Figure 3 shows the EPR spectra of the nitroxide and verdazyl-based radicals. To analyze the EPR spectra more accurately, quantum chemistry calculations were performed using the ORCA 5.0 software package with the B3LYP functional and def2-SV(P) basis set for all the calculations. The primary focus of the calculations was on extracting the principal component values of the electronic g-tensor and the hyperfine coupling (A) tensor related to the hyperfine interaction of14N with the unpaired electron spin.

[0218] Using the EPR parameters obtained from quantum chemical computations as the starting point, the EPR spectra were simulated with the EasySpin software package. The relevant experimentally simulated and computed EPR parameters are presented in Table 3. Overall, the radicals exhibited anisotropic g-tensor values and nearly axially symmetric A -tensor values, with the highest hyperfine coupling values aligned with the larger g-values. The largest g-value (gx) was found to be oriented almost perpendicularly relative to the plane of the verdazyl / phenyl rings, while the other two slightly smaller components (gyand gz) were close to coplanar with said groups. The verdazyl-based radicals exhibited smaller hyperfine couplings compared to the nitroxide radicals. Computationally, the largest spin population in the verdazyl ring was found on nitrogen atoms N3 and N4, with a smaller spin population being found on the other two nitrogen atoms, N1 and N2 (see Figure le for the labelling). Accordingly, the resulting hyperfine coupling interactions were found to be stronger to N3 / 4 than to Nl / 2. Additionally, incorporating the verdazyl component into hybrid radicals resulted in narrower spectra than those of their parent nitroxide counterparts.

[0219] 515.026WO1 27

[0220] UA 2025-035-02 Table 3. EPR parameters obtained from experimental simulations and quantum chemical calculations for the nitroxide and verdazyl-based radicals discussed in this study.a

[0221] "

[0222]

[0223] aEPR parameters from experimental simulations are presented on the first line, with those from quantum chemical calculations provided in parentheses on the second line. The uncertainties of principle component values of the g- and A-tensors are ±0.0003 and 2 MHz, respectively.

[0224] To explore the DNP mechanism of the top-performing radical in this study, VerTEKol was further characterized by determining its1H DNP field profile and comparing it to that of the standard radical, TEKPol (Figure 4b). These field profiles were generated by varying the applied magnetic field and plotting the resulting1H NMR signal enhancements. Given the distinct EPR spectra of TEKPol and VerTEKol, it is expected that their DNP field profiles would also differ. Both the radicals exhibit evidence of a cross-effect DNP mechanism, where the DNP profile for TEKPol shows the field positions of the maximum at 14.096 T and the minimum at 14.059 T, with peak-to-peak width = 0.037 T.

[0225] In contrast, VerTEKol exhibits a narrower DNP field profile, with maximum and minimum peak positions at 14.094 T and 14.074 T, respectively, resulting in a peak-to-peak width = 0.020 T, a 45% decrease from that of TEKPol. This observation can be attributed to the hybrid nature of VerTEKol, where replacing one of the nitroxide monomers (TEKone) in TEKPol with a verdazyl monomer (la) narrows the overall EPR linewidth (X-band) by 15% (Figure 4a), providing more efficient DNP matching conditions for the polarization of

[0226]

[0227] nuclei.

[0228] Additionally, VerTEKol shows an improved ratio between the positive and negative maxima intensities of 0.55, compared to 0.40 for TEKPol. Notably, the initial measured enhancement

[0229] 515.026WO1 28

[0230] UA 2025-035-02 values of VerTEKol were obtained at So = 14.092 T, prior to establishing its field profile, which in turn indicated that we were still slightly away from the highest possible

[0231]

[0232] enhancement under these initial conditions. However, by leveraging the1H DNP field profile, we set the field strength precisely at the maximum point, Bo= 14.094 T, enabling us to achieve the highest enhancement of 113 for the13C{JH} CP MAS experiment, as previously mentioned (Figure Id).

[0233] This result further supports the fact that the dominant polarization mechanism is the cross effect, consistent with other mono- and biradicals at ca. 10 mM concentrations. Furthermore, depolarization experiments were conducted on TEKPol and VerTEKol to investigate the impact of the biradical doping. Both radicals exhibit a similar depolarization factor (sdepo) of 0.45, with the signal decreasing by 45% compared to the signal from pure TCE solution. VerTEKol provides more effective high-field DNP enhancement compared to TEKPol, despite both contributing to a similar paramagnetic quenching effect on the overall signal.

[0234] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0235] EXAMPLES

[0236] Example 1. Methods.

[0237] DNP NMR Sample Preparation. 10 mM radical solutions for biradicals, and 20 mM solutions for monoradicals, were prepared in TCE (1,1,2,2-tetrachloroethane) and DNP samples were packed in 3.2 mm sapphire rotors with zirconia top caps. A silicone plug was placed between the sample solution and the zirconia top cap.

[0238] DNP NMR Spectral Acquisition.13C DNP-enhanced NMR spectra were recorded at natural abundance on a Bruker Avance NEO 600 DNP NMR spectrometer Bo = 14.1 T) equipped with a 395 GHz gyrotron microwave source. Cross-polarization (CP)13C{JH} MAS DNP NMR spectroscopy experiments were performed at spinning frequencies between 5-9 kHz using a Bruker low-temperature 3.2 mm double resonance (HX) MAS DNP probe with 1-2.5 ms contact time with a 50-100% ramp on ’H, 5-10 s recycle delays, and 4 or 8 co-added transients. Experiments were performed at temperatures at or between 98-106 K and with a microwave power of 15 W. Spectra were referenced with respect to the high-frequency peak of adamantane (8iS0(13C) = 38.56 ppm). 'H T measurements were carried out via a saturation recovery pulse sequence with a 4 ps TT / 2 pulse, 1-2 scans, 0.1 s recycle delay, 64 pre-saturation pulses, and variable delay times from 50 ms to 80 s (MW on) and 10 ms to 300 s (MW off). T curves were processed and fit using the Dynamics Center implemented in Topspin 4.3.0 using the SatRec 515.026WO1 29

[0239] UA 2025-035-02 function and manually determined integral regions. T values were extracted from the fit curve, where the equation of fit was:

[0240]

[0241] where I(t) and / (oo) represent theJH NMR peak areas recorded at time t and at infinity, respectively. Ai and A2are pre-exponential constants, while 7) and T2correspond to the relaxation times related to the TCE and the background, respectively.

[0242] Quantum Chemical Calculations. All calculations were run using ORCA 5.0.

[0243] Molecular structures were visualized on Avogadro 1.2.0. Gas-phase geometry optimizations of triplet / doublet (bi- / mono-) radicals were performed using the B3LYP functional and def2-SV(P) basis set, with dispersion correction using Grimme’s D3 approach and Becke-Johnson (BJ) damping. Vibrational frequency calculations were performed on all optimized structures to ensure they represented energy minima and no imaginary frequencies were present. EPR calculations were run using the B3LYP / def2-SVP level of theory, and auxiliary basis sets were generated with the AutoAux keyword. Hyperfine coupling interactions to all N and O atoms were calculated including the isotropic and dipolar parts. Both spin-orbit and spin-spin coupling components of the D-tensor were calculated using the default coupled-perturbed method and spin density from UHF natural orbitals (UNOs).

[0244] EPR Spectroscopy. EPR spectra were obtained at room temperature using a Bruker EMX Nano spectrometer (University of Alberta) with X-band micro waves (applied frequency = 9.621 GHz) and a microwave attenuation of 15 dB under ambient conditions. The field modulation was set to 6 G at 100 kHz, and each spectrum was recorded over 4 scans with a receiver gain of 15 dB. Microwave power was maintained at 50 mW, and the field was centered at 3425 Gauss, with a sweep width of 1000 Gauss and a sweep time of 60 s per scan. All experimental EPR spectra were fit using the EasySpin simulation package.

[0245] Example 2. Synthesis and Characterization of Radicals.

[0246] All reagents and solvents were obtained from commercial suppliers and used as received unless otherwise stated. Anhydrous solvents were obtained using a solvent purification system from LC technology solutions Inc. Purification by flash column chromatography was carried out on silica gel (SiCh, 60 A, 40-63 pm). Thin-layer chromatography (TLC) was carried out using commercially available aluminum sheets precoated with silica gel with fluorescence indicator and visualized under UV light at 254 and / or 360 nm.13C NMR spectra were recorded on a 500 MHz Varian instrument equipped with a cold probe at 126 MHz; ’H NMR spectra were recorded on a 700 MHz Agilent or 500 MHz Varian instrument. Chemical shift values are reported in ppm and coupling constants (J) in Hz are reported as observed.1H and13C NMR spectra are

[0247] 515.026WO1 30

[0248] UA 2025-035-02 referenced against the residual solvent peaks for CDCh and DMSO-^ (CHCh 3 H = 7.26, 3 C = 77.16; DMSO-c , 3 H = 2.50, 3 C = 39.52). Infrared (IR) spectra were recorded on a Thermo Nicolet 8700 FTIR spectrometer and samples were measured as cast fdms or as solids (ATR). High-resolution mass spectrometry (HRMS) spectra were recorded on aBruker 9.4 T Apex-Qe FTICR instrument (MALDI) or a Kratos MS50G instrument (El). Melting point ranges were measured on a Thomas Hoover capillary melting point apparatus.

[0249] Synthesis ofTekPol, ErythriPol, and TEKamine.

[0250] Scheme 2. Synthesis of nitroxide radicals.

[0251]

[0252] Compound SE Synthesized as previously reported by Sakai et al (Tetrahedron, 2010, 66, 2311).

[0253]

[0254] Compound S2: Synthesized as previously reported by Sakai et al.

[0255]

[0256] 515.026WO1 31

[0257] UA 2025-035-02 TEKone: To a round bottom flask was added a solution of amine S2 (103 mg, 0.266 mmol) in degassed CH2CI2 (6.5 mL). The stirred solution was cooled to 0 °C, and a solution of mCPBA (67.3 mg, 0.390 mmol) dissolved in CH2C12(0.5 mL) was added dropwise via syringe over ca. 1 min. The reaction mixture was warmed to rt. After 24 h, K2CO3 (60 mg, 0.43 mmol) and H2O (6 mL) were added, and the organic layer was separated. The aqueous phase was extracted with CH2CI2 (20 mL). The combined extract was dried over MgSCh, filtered, and the filtrate was concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, EtOH / CH2Cl21:49). Drying in vacuo afforded radical TEKone (83 mg, 79%) as a pale-yellow solid. Mp = 208-210 °C (decomposition-TLC). Rf = 0.71 (silica gel, EtOH / CH2Cl21:49). IR (CH2CI2, cast film) 3081 (w), 3022 (m), 2956 (m), 2932 (s), 2865 (s), 1720 (s), 1494 (m), 1451 (s), 714 (s) cm1. ESI HRMS m / z calcd for C27H32NO2’ (M+) 402.2428, found 402.2427. Previous syntheses of TEKone only provided low-resolution mass spectrometric characterization of the compound.

[0258]

[0259] TEKamine: Synthesized as previously reported by Kubicki et al (('hem. Sci., 2016, 7, 550). To a stirred solution of TEKone (31 mg, 0.077 mmol) in CH2CI2 (1 mL) was added dropwise NH4OAC (59 mg, 0.77 mmol) in MeOH (2 mL). After 30 min, NaBH3CN (8 mg, 0.1 mmol) was added. After 18 h, the reaction was quenched via the addition of satd. aq. NaHCO3(ca. 2 mL), concentrated under high vacuum, and extracted with CH2Q2 (2 x 3 mL), the combined organic extract was washed with water (2 x 3 mL), dried over MgSCL, filtered, and the solvent removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, EtOH / CH2Cl21:49), which afforded TEKamine (19 mg, 62%) as a pale red solid. Mp = 208-210 °C (decomposition-TLC). Rf =0.47 (silica gel, EtOH / CH2Cl23:47). IR (CH2C12, cast film) 3364 (w), 3082 (w), 3027 (w), 2939 (s), 2866 (m), 1599 (w), 1492 (w), 1451 (s) cm’1. ESI HRMS m / z calcd for C2?H36N2O' ([M + H]+) 404.2822, found 404.2821. Previous syntheses of TEKamine only reported the melting point and low-resolution mass spectrometric characterization of the compound.

[0260] 515.026WO1 32

[0261] UA 2025-035-02

[0262]

[0263] Compound S3a: Synthesized as previously reported by Zagdoun et al. (J. Am. Chem. Soc., 2013, 135, 12790) with slight modifications. To H2O (10 mL), pentaerythritol (84 mg, 0.62 mmol) and ketone S2 (529 mg, 1.36 mmol) were added, and the mixture sonicated. PhMe / H2O (4:1, 50 mL) was added, followed by TsOH»H2O (315 mg, 1.66 mmol). Once sonicated and mixed, the solution was heated to reflux and dehydrated, by means of azeotropic distillation, with a Dean- Stark apparatus. After stirring for 18 h, the reaction mixture was cooled to rt. The solvent was removed under reduced pressure, satd. aq. Na2CO2(50 mL) and CH2C12(50 mL) were added, and the solution was stirred for 2h. The aqueous layer was separated and extracted further using CH2C12(2 x 30 mL). The combined organic extracts were dried under reduced pressure, PhMe / hexanes (1: 1, 20 mL) was added, and the mixture sonicated. This solution was stored at -30 °C overnight and then filtered to isolate the resulting precipitate. The crude product was purified by flash column chromatography (silica gel, EtOH / CH2Cl21:9), which afforded the title compound S3a (340 mg, 63%, ca. 90-95% pure based onJH NMR analysis) as a white solid. Mp = 240-243 °C (partial decomposition-TLC). Rf = 0.52 (EtOH / CH2Cl21:9). IR (ATR-FTIR) 3416 (m, br), 3027 (s), 2931 (s), 2861 (s), 1602 (m), 1118 (s)cm1.1HNMR(700 MHz, CDC13) 37.29 (t, J= 8.1 Hz, 8H), 7.23 (d, J= 8.2 Hz, 8H), 7.18 (t, J= 7.3 Hz, 4H), 3.80 (s, 8H), 2.51 (tt, J= 12.2, 3.5 Hz, 4H), 2.04 (d, J= 12.2 Hz, 8H), 1.93 (s, 8H), 1.83 (d, J= 1L4 Hz, 8H), 1.62 (q, J= 12.8 Hz, 8H), 1.58-1.45 (m, 8H). ESI HRMS m / z calcd for C59H75N2O4 ([M + H]+) 875.5721, found 875.5711; calcd for C59H76N2O4 ([M + 2H]+2) 438.2897, found 438.2891. Spectral and physical data were consistent with those reported by Zagdoun et al.

[0264]

[0265] Compound S3b: Followed the analogous procedure as for S3a. PhMe / H2O (4:1, 62 mL),

[0266] 515.026WO1 33

[0267] UA 2025-035-02 erythritol (76 mg, 0.62 mmol), and S2 (529 mg, 1.36 mmol) were mixed, then TsOH’H^O (315 mg, 1.66 mmol) was added batchwise. The crude product was purified by flash column chromatography (silica gel, EtOH / CH2Cl21:19— >1:9) to yield S3b (186 mg, 34%) as a white solid. Mp = 262-264 °C (partial decomposition-TLC). Rf = 0.42 (silica gel, EtOH / CH2Cl21:19). IR (ATR-FTIR) 3392 (m, br), 3026 (m), 2924 (s), 2860 (s), 2684 (m), 2466 (m), 1602 (m), 1577 (m), 1493 (s), 1452 (s), 1367 (s), 1129 (s), 1068 (s), 753 (s), 689 (s) cm’1. ‘H NMR (700 MHz, CDC13) 37.28 (t, J = 7.4 Hz, 8H), 7.22 (d, J= 7.2 Hz, 8H), 7.18 (t, J= 7.3 Hz, 4H), 4.19-4.12 (m, 2H), 4.07-4.03 (m, 2H), 3.98-3.90 (m, 2H), 2.55-2.47 (m, 4H), 2.13 (d, J = 12.4 Hz, 4H), 2.08-2.00 (m, 4H), 1.89-1.70 (m, 16H), 1.69-1.50 (m, 16H). ESI HRMS m / z calcd for C58H73N2O4 ([M + H]+) 861.5565, found 861.5574; calcd for C58H74N2O4 ([M + 2H]+2) 431.2819, found 431.2810.

[0268]

[0269] TEKPol: Diamine S3 a (100 mg, 0.114 mmol) was suspended in CH2CI2 (3 mL), which had been sparged with N2 for ~30 min, and cooled to 0 °C. A solution of mCPBA (60 mg, 0.34 mmol) dissolved in CH2C12(0.5 mL) was added dropwise and the reaction mixture was stirred for ca. 10 min, before removing the ice-bath (the reaction mixture turned an orange-brown color). After 24 h, the reaction was quenched by the addition of K2CO3(60 mg, 0.43 mmol) and water (5 mL). The organic layer was separated, and the aqueous layer was extracted with CH2C12(3 x 5 mL). The combined extract was dried over MgSC>4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, EtOAc / hexanes 3:7), which afforded TEKPol (29 mg, 28%) as a pale yellow-orange solid. Mp = 167-173 °C (partial decomposition-TLC). Rf = 0.74 (silica gel, EtOAc / hexanes 3:7). IR (CH2C12, cast film) 3055 (w), 3024 (m), 2973 (s), 2933 (s), 2871 (s), 1601 (m), 1494 (s), 1451 (s) cm’1. MALDI HRMS (DCTB) m / z calcd

[0270]

[0271] 904.5385, found 904.5392. Spectral and physical data were consistent with those reported by Zagdoun et al.

[0272] 515.026WO1 34

[0273] UA 2025-035-02

[0274]

[0275] ErythriPol: Followed the analogous procedure for TEKPol. Diamine S3b (50 mg, 0.058 mmol) was dissolved in CH2CI2 (1.5 mL) and cooled to 0 °C. A solution of mCPBA (30 mg, 0.17 mmol) dissolvedin CH2CI2 (0.5 mL) was added. Purification by flash column chromatography (silica gel, EtOAc / hexanes 3:7) afforded biradical ErythriPol (12 mg, 23%) as an orange-amber solid. Mp = 198-202 °C (partial decomposition-TLC). Rf = 0.59 (silica gel, EtOAc / hexanes 3:7). IR (CH2C12, cast) 3059 (w), 3027 (w), 2929 (s), 2866 (m), 1601 (w), 1494 (m), 1453 (m), 1122 (m), 1058 (m), 738 (m), 700 (s) cm’1. MALDI HRMS (DCTB) m / z calcd for C58H7ON2062‘ (M+) 890.5228, found 890.5214.

[0276] Synthesis of verdazyl mono- and biradicals.

[0277] Scheme 3. Synthesis of verdazyl radicals

[0278]

[0279]

[0280]

[0281] 515.026WO1

[0282] UA 2025-035-02 General procedure A. The synthesis of amines S4a-f was adapted from Matuschek et al. (Chem. Sci., 2015, 6, 4712). To a stirred solution of phenylcarbohydrazide 3 in EtOH was added a solution of the respective aldehyde in CH2C12. The reaction mixture was heated to reflux. After 3-24 h, a precipitate formed, the heat was turned off, the reaction was cooled to rt, and the solvent was removed under reduced pressure. The crude product was suspended in MeOH, cooled to -30 °C for 15 h, and filtered. The precipitate was washed with MeOH and dried under suction to afford amine S4a-f. Note: Drying under suction for long periods results in color changes indicative of partial aerial oxidation. Products should be suction filtered under a blanket of N2to mitigate this issue.

[0283] General Procedure B. The oxidation of S4a-f to verdazyl radicals la-b and 2a-d was adapted from Matuschek et al. The tetraamine S4a-f and -benzoquinone were suspended in CH2C12in a Teflon capped, sealed tube. The reaction was heated to 120 °C and stirred for 24 h. The reaction mixture was cooled to rt. The precipitate was filtered, washed with cold CH2C12, suspended in EtOAc, and stored at -30 °C for 24 h. The resulting solid was filtered and purified further by preparative TLC (silica gel, CH2Cl2 / hexanes 2:3) to yield radical la-b / 2a-d, typically as dark red / green solids.

[0284]

[0285] Compound 3: Synthesized as previously reported by Matuschek et al. To a threaded Teflon-capped sealed tube, 1,10-phenanthroline (600 mg, 3.33 mmol), Cui (317 mg, 1.67 mmol), K3PO4 (18.5 g, 86.6 mmol), iodobenzene (8.2 mL, 73 mmol), carbohydrazide (3.00 g, 33.3 mmol), and DMF (30 mL) were added respectively. The stirred solution was heated to 60 °C for 1 h, then 90 °C for 24 h. Once cooled to rt, EtOAc (30 mL) was added, the reaction mixture was filtered through a celite plug, and rinsed through with EtOAc (2 x 5 mL). The organic layer was washed with water (5 x 100 mL), and the combined aqueous phase was back extracted with EtOAc (3 x 100 mL). The combined extract was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 100% Et2O — > 100% EtOAc), which afforded phenylcarbohydrazide 3 (2.10 g, 26%) as a pale-yellow solid after being dried in vacuo. Rf = 0.51 (silica gel, Et2O). ’H NMR (700 MHz, CDCI3) d 7.18 (t, J= 7.3 Hz, 4H), 7.13 (d, J= 7.4 Hz, 4H), 7.03 (t, J= 7.4 Hz, 2H), 4.47 (s, 4H). Spectral and physical data were consistent with those reported by Matuschek et al.

[0286] 515.026WO1 36

[0287] UA 2025-035-02

[0288]

[0289] Compound S4a: Synthesized as previously reported by Matuschek et al. According to General Procedure A the mixture of 3 (228 mg, 0.941 mmol) in EtOH (12 mL), and benzaldehyde (100 mg, 0.942 mmol) in CH2CI2 (2 mL), was heated to reflux for 3 h. Precipitation upon the addition of MeOH (ca. 2 mL), suction filtration, and washing with MeOH (3 x 3 mL) afforded compound S4a (160 mg, 51%) as a white solid. Mp = 204-206 °C (partial decomposition-TLC). IR (ATR-FTIR) 3231 (s), 3212 (s), 3064 (w), 3029 (w), 2942 (vw), 2921 (vw), 1622 (s), 1596 (s), 1494 (s), 1452 (s) cm’1. 'H NMR (700 MHz, DMSO-c4,) d 7.60 (d, J= 8.6 Hz, 4H), 7.53 (d, J= 7.2 Hz, 2H), 7.37-7.30 (m, 7H), 7.07 (t, J= 7.3 Hz, 2H), 6.38 (d, J= 9.1 Hz, 2H), 5.38 (t, J= 9.1 Hz, 1H). ESI HRMS m / z calcd for C20H19N4O ([M + H]+) 331.1553, found 331.1550; calcd for C2oHisN4NaO ([M + Na]+) 353.1373, found 353.1372. Spectral and physical data were consistent with those reported by Matuschek et al.

[0290]

[0291] Compound S4b: According to General Procedure A the mixture of 3 (524 mg, 2.16 mmol) in EtOH (5 mL), and 4-formylbenzoic acid (325 mg, 2.16 mmol) in EtOH / CH2Cl2(1:2, 15 mL), was heated to reflux for 3 h. Precipitation upon the addition of MeOH (ca. 5 mL), suction filtration, and washing with MeOH (3 x 6 mL) afforded compound S4b (540 mg, 67%) as a white solid. Mp = 256-258 °C (decomposition-TLC). R( = 0.57 (silica gel, EtOH / CH2C12 1:9). IR (CH2CI2, cast film) 3230 (s), 3218 (s), 3082 (m), 2980 (m), 2910 (m), 2672 (w), 2554 (w), 1693 (s), 1622 (s), 1611 (s), 1583 (s), 1429 (s), 1312 (s), 1298 (s), 746 (s) cm’1. ‘H NMR (500 MHz, DMSO-<76) 312.96 (s, 1H), 7.91 (d, J= 8.4 Hz, 2H), 7.66 (d, J= 8.3 Hz, 2H), 7.60 (d, J= 8.6 Hz, 4H), 7.33 (t, J= 8.5 Hz, 4H), 7.08 (t, J= 7.3 Hz, 2H), 6.48 (d, J= 8.9 Hz, 2H), 5.48 (t, J= 8.9 Hz, 1H);13C{XH} NMR (126 MHz, DMSO ) 167.0, 156.9, 142.7, 142.4, 130.5, 129.3, 128.0, 127.2, 123.3, 121.1, 72.4. ESI HRMS m / z calcd for C21H17N4O3 ([M-H]") 373.1306, found 373.1303.

[0292] 515.026WO1 37

[0293] UA 2025-035-02

[0294] <

[0295]

[0296] & &

[0297] Compound S4c: Synthesized as previously reported by Matuschek et al. According to General Procedure A the mixture of 3 (361 mg, 1.49 mmol) in EtOH (10 mL), and terephthal aldehyde (100 mg, 0.746 mmol) in CH2C12(8 mL), was heated to reflux for 7 h.

[0298] Precipitation upon the addition of MeOH (ca. 4 mL), suction filtration, and washing with MeOH (3 x 5 mL) afforded tetraamine S4c (340 mg, 78%, ca. 90-95% pure based onJH NMR analysis) as a white solid. No melt <304 °C (partial decomposition-TLC). Rf = 0.52 (silica gel, EtOH / CH2Cl21:19). IR(ATR-FTIR) 3228 (m), 3064 (w), 1624 (s), 1596 (m), 1584 (m), 1497 (m) cm1 XH NMR (700 MHz, DMSO ) d 7.58 (d, J = 8.6 Hz, 8H), 7.50 (s, 4H), 7.31 (t, J = 8.0 Hz, 8H), 7.06 (t, J= 7.4 Hz, 4H), 6.37 (d, J= 9.0 Hz, 4H), 5.36 (t, J= 9.0 Hz, 2H). ESI m / zHRMS calcd for CsrFFoNsNaCh ([M + Na]+) 605.2384, found 605.2383. Spectral and physical data were consistent with those reported by Matuschek et al.

[0299]

[0300] Compound S4d: According to General Procedure A the mixture of 3 (503 mg, 2.08 mmol) in EtOH (9.5 mL), and 4,4'-biphenyldicarboxaldehyde (136 mg, 0.649 mmol) in CH2C12(9.5 mL), was heated to reflux for 19 h. Precipitation from MeOH (ca. 4 mL), suction filtration, and washing with MeOH (3 x 5 mL) afforded tetraamine S4d (393 mg, 92%, ca. 90% pure based on ’H NMR analysis) as a white solid. Mp = 236-238 °C (partial decomposition-TLC). Rf = 0.48 (silica gel, EtOH / CH2Cl21:19). IR (ATR-FTIR) 3232 (s), 3061 (w), 3039 (w), 1625 (s), 1596 (s), 1585 (m), 1495 (s) cm’1. ‘H NMR (700 MHz, DMSO-<L,) 87.66 (d, J= 8.4 Hz, 4H), 7.61 (t, J= 7.6 Hz, 12H), 7.33 (t, J= 8.1 Hz, 8H), 7.07 (t, J= 7.3 Hz, 4H), 6.42 (d, J= 9.0 Hz, 4H), 5.43 (t, J = 9.0 Hz, 2H).I3C{JH} NMR(126 MHz, DMSO-c4,) d 157.1, 142.8, 139.4, 137.0, 128.0, 127.6, 126.5, 123.3, 121.1, 72.7. ESI HRMS m / z calcd for C4oH34N8Na02 ([M + Na]+) 681.2697, found 681.2697; calcd for C40H35N8O2 ([M + H]+) 659.2877, found 659.2870.

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[0303]

[0304] Compound S4e: According to General Procedure A the mixture of 3 (331 mg, 1.37 mmol) in EtOH (6.5 mL), and 4,4'-(ethyne-l,2-diyl)dibenzaldehyde (100 mg, 0.427 mmol) in CH2Q2 (6.5 mL), was heated to reflux for 21h. Precipitation upon the addition of MeOH (ca. 4 mL), suction filtration, and washing with MeOH (3 x 5 mL) afforded tetraamine S4e (294 mg, >99%, ca. 90% pure based on ’H NMR analysis) as a gray -purple solid. Mp = 215-220 °C (partial decomposition-TLC). 7?f= 0.68 (silica gel, EtOH / CH2Cl21:19). IR (ATR-FTIR) 3234 (m), 3067 (w), 3038 (w), 1700 (w), 1624 (s), 1596 (m), 1494 (m) cm’1. ‘H NMR (500 MHz, DMSO-<L,) d 7.60 (d, J= 8.5 Hz, 8H), 7.58 (d, J= 8.5 Hz, 4H), 7.53 (d, J= 8.5 Hz, 4H), 7.33 (t, J= 7.5 Hz, 8H), 7.08 (t, J= 7.0 Hz, 4H), 6.45 (d, J= 8.5 Hz, 4H), 5.44 (t, J = 9.0 Hz, 2H);13C {3H} NMR (126 MHz, DMSO-<76) d 156.8, 142.7,138.3,131.3, 128.0, 127.4, 123.3, 121.9, 121.2, 89.4, 72.4. ESI HRMS m / z calcd for C42H35N8O2 ([M + H]+) 683.2877, found 683.2884; calcd for C42H34NsNaO2 ([M + Na]+) 705.2697, found 705.2693.

[0305]

[0306] Compound S4f According to General Procedure A the mixture of 3 (213 mg, 0.880 mmol) in EtOH (6 mL), and 4,4'-m-terphenyldicarboxaldehyde (115 mg, 0.400 mmol) in CH2CI2 (6 mL), was heated to reflux for 24 h. Precipitation upon the addition of MeOH (ca. 3 mL), suction filtration, and washing with MeOH (3 x4 mL) afforded tetraamine S4f (246 mg, 84%, ca.

[0307] 90% pure based on1H NMR analysis) as an off-white solid. Mp = 220-223 °C (partial decomposition-TLC). 7?f= 0.56 (silica gel, EtOH / CH2Cl21:19). IR (ATR-FTIR) 3239 (s), 3207 (s), 3063 (m), 3039 (m), 2978 (w), 1624 (s), 1597 (s), 1494 (s) cm1. *HNMR (500 MHz, DMSO-r / 6) d 7.89 (t, <7=2.0 Hz, 1H), 7.76 (d, J= 8.5 Hz, 4H), 7.60-7.67 (m, 14H), 7.53 (t, <7= 7.5 Hz, 1H), 7.34 (t, J= 8.0 Hz, 8H), 7.08 (t, J= 7.5 Hz, 4H), 6.44 (d, J= 9.0 Hz, 4H), 5.45 (t, J= 9.0 Hz, 2H);13C H} NMR (126 MHz, DMSO-c / d 157.2, 142.8, 140.4, 139.9, 137.1, 129.5, 128.0, 127.5, 126.8, 125.9, 125.0, 123.2, 121.1, 72.7. ESI HRMS m / z calcd for C46H38NsNaO2 ([M + Na]+) 757.3010, found 757.3011; calcd for C46H39N8O2 ([M + H]+) 735.3190, found 735.3205.

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[0310]

[0311] Compound la: Synthesized as previously reported by Matuschek et al. In a threaded Teflon-capped sealed tube, S4a (100 mg, 0.303 mmol) and benzoquinone (50 mg, 0.47 mmol) were mixed in CH2CI2 (8 mL) and the stirred solution was heated to 85 °C. After 16 h, the heat was removed, and the flask was cooled to -30 °C for 24 h. The resulting precipitate was then suction filtered (washing with cold CH2C12, ~8 mL) and dried under suction, blanketed by N2. The resulting solid was passed through a silica plug (silica gel, CLLC^ / hexanes 1 :5) to afford la (55 mg, 55%) as a dark red solid. Mp = 204-206 °C. Rf = 0.50 (silica gel, hexanes / CH2Cl21:2). IR (CH2C12, cast film) 3092 (w), 3068 (w), 3035 (w), 1701 (s), 1590 (m), 1484 (s), 1458 (m), 1309 (m), 1251 (m), 1122 (m), 753 (s), 687 (s) cm’1. ESI HRMS m / z calcd for C2oHi5N40’ (M+) 327.1240, found 327.1240; calcd for C2oHi5N4NaO’ ([M + Na]+) 350.1138, found 350.1135. Mass spectrometric data were consistent with those reported by Matuschek et al.

[0312]

[0313] Compound lb: In a threaded Teflon-capped sealed tube, S4b (200 mg, 0.534 mmol) and benzoquinone (87 mg, 0.80 mmol) were suspended in CH2CI2 (15 mL), and the stirred suspension was heated to 85 °C. After 23 h, the heat was removed, and the flask was cooled to -30 °C for 12 h. The resulting precipitate was then suction filtered, washed with cold CH2Q2 (ca. 5 mL), and dried with suction under a blanket of N2. The crude product was dissolved in DMF (2 mL), to which CH2CI2 (50 mL) was added, and the mixture was cooled to -78 °C for 14 h.

[0314] Suction filtration of the resulting precipitate afforded lb (113 mg, 57%) as a red solid. Mp = 284-288 °C (partial decomposition-TLC). Rf = 0.73 (silica gel, EtOH / CH2C12 1 : 5). IR (CH2CI2, cast) 3230 (w), 3016 (w, br), 2897 (w, br), 2688 (w), 2560 (w), 1697 (s), 1615 (m), 1313 (m), 743 (s) cm’1. ESI HRMS m / z calcd for C2IHI4N4O3* ([M - H] ) 370.1071, found 370.1064.

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[0317]

[0318] Compound 2a: Synthesized as previously reported by Matuschek et al. According to General Procedure B using tetraamine S4c (151 mg, 0.259 mmol) and / >-benzoquinone (252 mg, 2.33 mmol) in CH2CI2 (7 mL) to afford compound 2a as a dark red solid (yield not determined). Mp = 206-210 °C (partial decomposition-TLC). Rf= 0.18 (silica gel, hexanes / CH2Cl21:3). IR (CH2C12, cast film) 3065 (w), 3048 (w), 2961 (w), 2926 (w), 2850 (w), 1689 (s), 1609 (m), 1494 (m), 1207 (s), 1121 (m), 780 (s) cm4MALDI MS (DCTB) m / z 548.2 ([M - CO]+, 100), 576.2 (M+, 38); MALDI HRMS (DCTB) m / z calcd for C34H24N8O22* (M+) 576.2017, found 576.2011. Mass spectrometric data were consistent with those reported by Matuschek et al.

[0319]

[0320] Compound 2b: According to General Procedure B using tetraamine S4d (72 mg, 0.12 mmol) and / 2-benzoquinone (106 mg, 0.981 mmol) in CH2CI2 (10 mL), 82 mg of crude solid (unknown purity) were obtained.5.4 mg of pure compound 2b were isolated as a red-gray solid (ca. 69% reaction yield) from 9.0 mg of crude product using preparative TLC (CH2C12 / hexanes 2:3). Mp =266-274 °C (partial decomposition-TLC). Rf = 0.29 (silica gel, hexanes / CH2Cl21:3). IR (CH2C12, cast) 3101 (vw), 3072 (w), 3044 (w), 1697 (s), 1742 (m), 1593 (w), 1494 (m), 1250 (m), 1124 (m), 821 (s), 744 (s), 681 (s) cm1MALDI MS (DCTB) m / z 624.2 ([M- CO]+, 100), 652.2 (M+, 37); MALDI HRMS (DCTB) m / z calcd for C4oH28N8022’ (M ) 652.2330, found 652.2323.

[0321]

[0322] Compound 2c: According to General Procedure B using tetraamine S4e (230 mg, 0.337

[0323] 515.026WO1

[0324] UA 2025-035-02 mmol) and / ?-benzoquinone (328 mg, 3.03 mmol) in CH2Q2 (9 mL) to afford compound 2c as a grey-green solid (yield not determined). No melt <300 °C (decomposition-TLC). Rf = 0.34 (silica gel, hexanes / CH2Cl21:3). IR (CH2CI2, cast) 3065 (vw), 3046 (vw), 2963 (vw), 2929 (vw), 1697 (s), 744 (s) cm1MALDI MS (DCTB) m / z 648.2 ([M - CO]+, 44), 676.2 (M+, 100); MALDI HRMS (DCTB) m / z calcd for C42H28N8O22’ (M+) 676.2330, found 676.2325.

[0325] <

[0326]

[0327] Compound 2d: According to General Procedure B using tetraamine S4f (246 mg, 0.335 mmol) and / ?-benzoquinone (326 mg, 3.01 mmol) in CH2Q2 (9 mL) to afford compound 2d as a gray -brown solid (yield not determined). Mp = 266-270 °C (partial decomposition-TLC). Rf = 0.33 (silica gel, hexanes / CH2Cl2L3). IR (CH2Q2, cast) 3060 (w), 3044 (w), 3032 (w), 1697 (s), 1493 (m), 1249 (m), 1125 (m), 837 (m), 746 (s), 680 (s) cm MALDI MS (DCTB) m / z 700.3 ([M - CO]+, 100), 728.3 (M+, 40); MALDI HRMS (DCTB) m / z calcd for C46H32N8O22’ (M+) 728.2643, found 728.2646.

[0328] Synthesis of verdazyl-nitroxide hybrid biradicals. See Scheme 1.

[0329]

[0330] VerTEMPol: To a stirred solution of EDC»HC1 (25 mg, 0.13 mmol), DMAP (16 mg, 0.13 mmol), and radical lb (24 mg, 0.065 mmol) in dry CH2C12(1 mL), was added dropwise over ca.

[0331] 1 min a solution of 4-amino-TEMPO (25 mg, 0.13 mmol) in dry CH2C12(1 mL). The resulting mixture was stirred at rt under an atmosphere of N2. After 23 h, the reaction was quenched by the addition satd. aq. NaHCO3(10 mL) and diluted with CH2C12(10 mL). The organic layer was separated, washed with satd. aq. NaHCO3(2 x 5 mL) and brine (5 mL), dried over MgSCL, filtered, and the solvent was removed under reduced pressure. Purification by flash column chromatography (silica gel, EtOH / CH2Cl21:9) and drying in vacuo afforded the biradical VerTEMPol (22 mg, 64%) as a red solid. Mp = 232-235 °C (partial decomposition-TLC). Rf = 0.75 (silica gel, EtOH / CH2Cl21:19). IR (CH2C12, cast film) 3342 (w, br), 3068 (w), 2974 (m),

[0332] 515.026WO1 42

[0333] UA 2025-035-02 2933 (m), 2850 (w), 1739 (w), 1703 (s), 1659 (m), 1538 (m) cm’1. ESI HRMS m / z calcd for C3OH32N6032’ (M+) 524.2536, found 524.2531.

[0334]

[0335] VerTEKol: To a stirred solution of EDOHC1 (3.9 mg, 0.020 mmol), DMAP (2.5 mg, 0.020 mmol), and radical lb (3.8 mg, 0.010 mmol) in dry CH2C12(3 mL) was added dropwise over ca. 1 min a solution of TEKamine (8.2 mg, 0.020 mmol) in dry CH2C12(1 mL). The resulting mixture was stirred at rt under an atmosphere of N2. After 17 h, the reaction was quenched by the addition satd. aq. NaHCO3(5 mL) and diluted with CH2C12(5 mL). The organic layer was separated, washed with satd. aq. NaHCO3(2 x 5 mL) and brine (5 mL), dried over MgSO4, filtered, and the solvent was removed under reduced pressure. Purification by flash column chromatography (silica gel, EtOH / CH2Cl21:19) and drying in vacuo afforded the biradical VerTEKol (4.6 mg, 61%) as a red solid. Mp = 168- 176 °C (partial decomposition-TLC).

[0336]

[0337] = 0.62 (silica gel, EtOAc / CH2Cl21:19). IR (CH2C12, cast film) 3337 (w, br), 3070 (w), 3028 (w), 2924 (s), 2857 (m), 1735 (w), 1704 (s), 1663 (m), 1534 (m), 1493 (m) cm ESI HRMS m / z calcd for C48H47N6O32’ ([M- H] ) 755.3715, found 755.3731.

[0338] All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0339] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

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Claims

What is claimed is:

1. A compound of Formula I:wherein,each R1is independently radical, H, (Ci-Ce)alkyl, or (Ci-Ce)cycloalkyl;R2and R3are each independently H, halo, CN, ORa, SRa, N(Ra)2, CORa, CChR1, CON(Ra)2, SO2N(Ra)2, (Ci-Ce)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, or phenyl;R4is CONRa, CO2, phenyl, biphenyl, or ethynylbenzene;each Rais independently H, (Ci-Ce)alkyl, or (CH2CH2O)i-6O(Ci-Ce)alkyl;R5is Formula la, lb, Ic, Id, or le:wherein,the definitions of R1, R2, and R3are the same as for Formula I;each R6is independently H or (Ci-Ce)alkyl for Formula la;R7is CHRbor O for Formula la;Rbis phenyl, naphthyl, OH, SH, NH2, Ns, 0P(=0)(0H)2 or H; and each R8is independently(Ci-C6)alkyl for Formulas lb and Ic;wherein each phenyl, biphenyl, naphthyl, and ethynylbenzene is optionally substituted; and R5is not Formula le when R4is phenyl, biphenyl, or ethynylbenzene;or a salt thereof.

2. The compound of claim 1, wherein each R1is radical.515.026WO1UA 2025-035-023. The compound of claim 1 , wherein each R2and R3is H.

4. The compound of claim 1, wherein each R2and each R3is in the para position.

5. The compound of claim 1, wherein R4is CONH.

6. The compound of claim 1, wherein R4is CONH and R5is bonded to the nitrogen atom of CONH.

7. The compound of claim 1, wherein R5is Formula la or Formula lb.

8. The compound of claim 1, wherein R5is Formula la and each R6is H.

9. The compound of claim 1, wherein R5is Formula la and R7is CHPh.

10. The compound of claim 1, wherein R5is Formula lb and each R8is CHs.

11. The compound of claim 1 , wherein Formula I is represented by Formula II:or a salt thereof.515.026WO1 45UA 2025-035-0212. The compound of claim 1, wherein Formula I is represented by Formula III or Formula IV:or a salt thereof.

13. The compound of claim 1, wherein the compound is:515.026WO1 46UA 2025-035-0214. The compound of claim 1, wherein the compound is VerTEKol or VerTEMPol:or a salt thereof.

15. A composition comprising the compound of any one of claims 1-14 and a solvent or matrix.

16. A method for high-field dynamic nuclear polarization nuclear magnetic resonance (DNP NMR) spectroscopy comprising, acquiring spectral data on a DNP NMR spectrometer configured with a gyrotron micro wave source powered at about 10W to about 20W and suitable acquisition parameters for a sample comprising the composition of claim 15.

17. The method of claim 16, wherein the composition comprises VerTEKol or VerTEMPol.

18. The method of claim 16, wherein DNP-NMR comprises acquiring cross polarizationMAS DNP-enhanced NMR spectra at natural abundance, wherein the DNP NMR spectrometer is configured with a low-temperature double resonance MAS DNP probe configured to have a contact time of about 1 ms to about 2.5 ms, a ramp onof about 50% to 100%, a recycle delay of about 5 s to about 10 s, a spinning frequency of about 5 kHz to about 9 kHz, and a temperature of about 98K to about 106K.

19. The method of claim 18, comprising 'H T\ measurements via a saturation recovery pulse sequence with a 4 ps ?r / 2 pulse, 1 or 2 scans, 0.1 s recycle delay, 64 pre-saturation pulses, and variable delay times of about 50 ms to about 80 s when the gyrotron microwave is powered on and about 10 ms to about 300 s when the gyrotron micro wave is powered off.

20. The method of claim 16, wherein acquiring DNP NMR spectral data of the sample results in an enhanced signal to noise ratio compared to a sample that does not comprise VerTEKol or VerTEMPol when DNP NMR spectral data is acquired under the same conditions.515.026WO1 47UA 2025-035-02

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