Nitrogen-oxygen biradical based on acrylamide connection and dynamic nuclear polarization application thereof

Abstract

The invention discloses nitrogen-oxygen double free radicals based on acrylamide connection and a dynamic nuclear polarization application thereof. The nitrogen-oxygen biradical is formed by connecting two nitrogen oxide units through a novel connecting chain based on acrylamide. The nitrogen-oxygen double-free-radical compound connecting chain has the following advantages: 1) the synthesis efficiency is nearly 2.5 times higher, and the practicability is higher; 2) the electron-electron distance is shorter, so that the generated dipole coupling (D value) effect is stronger; (3) the relative orientations of the g tensors of the two nitrogen oxide units are approximately orthogonal, so that the frequency matching of a CE mechanism is facilitated; 4) a conjugated structure is contained, and certain rigidity is maintained; 5) the electron-electron exchange coupling (J value) effect is enhanced; and 6) in a relatively high field (14.1 T), the DNP establishment time is shorter, and the depolarization effect is weakened, so that the reduced DNP enhancement factor is compensated. Therefore, compared with the water-soluble nitrogen-oxygen diradical sold in the market, the nitrogen-oxygen diradical has a better solid NMR (nuclear magnetic resonance) and MRI (magnetic resonance imaging) application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical industry, and specifically relates to nitrogen-oxygen diradicals connected based on acrylamide and dynamic nuclear polarization applications thereof. Background Art

[0002] The invention relates to novel nitrogen-oxygen diradical compounds and uses thereof, especially in nuclear magnetic resonance (NMR) technology of solid samples and medical magnetic resonance imaging (MRI) technology.

[0003] NMR technology has now developed into a powerful and versatile material analysis and characterization technology, especially solid-state NMR, which can non-invasively provide atomic-level structural and dynamic information of crystalline and non-crystalline materials, playing an important role in chemistry, biology, and medicine. However, due to the inherent low sensitivity of NMR, its scope of application will always be limited. Therefore, in the past few decades, in addition to overcoming this defect through conventional methods such as optimizing instruments, the emergence of dynamic nuclear polarization (DNP) under magic angle spinning (MAS) has enabled solid-state NMR to be used to solve previously unreachable systems, such as nanoparticles, polymers, catalysts, inorganic materials, biomolecules, and drug formulations. Theoretically, the maximum sensitivity enhancement that DNP can achieve is γ e / γ n ( 1 H is 658, 13 C is 2617).

[0004] However, DNP has not yet fully realized its full potential. Its efficiency is limited by factors such as magnetic field strength, temperature, MAS frequency, properties of the propagation medium, and polarizing agents (PAs) carrying unpaired electrons. Polarizing agents are crucial to improving DNP efficiency and have been the subject of intense recent attention. Over the years, many polarizing agents have been developed based on the described DNP polarization transfer mechanism to optimize the sensitivity enhancement obtained by DNP. Among them, CE has been shown to be the most effective polarization transfer mechanism under the high field (above 9 T) and low temperature (about 100 K) conditions commonly used in DNP. Currently, the best performing PAs for CE DNP are nitroxide diradicals or hybrid nitroxide-narrow-line radical systems. Among them, nitroxide diradicals have been widely developed due to their ease of synthesis and high performance.

[0005] In the past two decades, in the process of developing nitroxide diradical PAs with excellent DNP performance, a series of key design parameters have been proposed and confirmed, including optimizing electron-electron interactions (exchange coupling J and dipole coupling D), limiting the relative orientation of the g tensors of the two nitroxides, extending electron spin relaxation (including electron longitudinal relaxation time and transverse relaxation time), optimizing the local geometric conformation around unpaired electrons, and having favorable polarization transfer paths. For DNP applications under ultra-high fields (>9.4T), further enhancing electron-electron interactions can shorten the DNP establishment time and weaken the depolarization effect, thereby increasing the overall NMR sensitivity∑( θ is the contribution factor, including paramagnetic bleaching and depolarization).

[0006] However, it is found that the overall sensitivity of the currently better performing nitroxide diradical PAs is similar, i.e., ε, T B The three factors of θ and θ restrict each other, and the best DNP performance has not appeared. It seems that the DNP performance of nitroxide diradicals has reached a bottleneck. In addition, the synthesis efficiency of nitroxide diradicals currently available in the market is not high, and they are rarely used in China. Therefore, it is necessary to develop nitroxide diradicals with better DNP performance and higher synthesis efficiency, which can be widely used in solid-state NMR. Summary of the invention

[0007] The invention provides a nitroxide diradical based on acrylamide connection and its dynamic nuclear polarization application. The invention relates to a novel nitroxide diradical compound and its application, especially in the nuclear magnetic resonance (NMR) technology of solid samples and the magnetic resonance imaging (MRI) technology of medicine.

[0008] In order to solve the above technical problems, an object of the present invention is to provide a nitroxide diradical based on acrylamide linkage, the structural formula of which is represented by the following formula 1:

[0009]

[0010] in,

[0011] A1 is selected from one of N, CH, O, and S, preferably, A1 is N;

[0012] A2 is selected from H; substituted or unsubstituted, linear, branched or cyclic C 1-18 Aliphatic groups, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, cyclopentyl, cyclohexyl, etc.; (CH2) n -COOH, (CH2-CH2-O) n CH3, (CH2-CH2-O) nH, wherein 1≦n≦20, preferably, A2 is H, (CH2-CH2-O)4CH3 or (CH2-CH2-O)2H, more preferably, A2 is (CH2-CH2-O)4CH3 or (CH2-CH2-O)2H; when A1 is O or S, A2 does not exist;

[0013] A3 is selected from O or SO2, preferably, A3 is O;

[0014] A4 is selected from one of CH and N, preferably, A4 is CH;

[0015] A5 is selected from one of C, CH2C, (CH2)2C, preferably, A5 is C or CH2C, more preferably, A5 is C;

[0016] R1 and R2, R3 and R4, R5 and R6, R7 and R8 are the same or different and are selected from substituted or unsubstituted, linear, branched or four-membered, five-membered or six-membered cyclic alkyl groups, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, cyclopentyl, cyclohexyl, etc.; CD3; the two are connected to form a substituted or unsubstituted CHR 10 -R9-CHR 10 , CHR 10 -R9-CHR 10 -CH2, CH2-CHR 10 -R9-CHR 10 -CH2 heterocyclic structure, wherein R9 is selected from O, S, NH, NCH3, CHOH, CHSH, CHNO2, CHCN, CHCHO, CHCOOM, CHSO2OM, CHSOOM, CHOPO3M2, CHX or CHAr, R 10is selected from H; a substituted or unsubstituted, linear, branched or four-membered, five-membered or six-membered cyclic alkyl group, such as methyl, ethyl, n-propyl, n-butyl, isopropyl, cyclopentyl, cyclohexyl, etc.; OH, SH, NH2, CD3, NO2, CN, CHO, COOM, SO2OM, SOOM, OPO3M2, X or Ar, wherein M is an alkali metal selected from lithium (Li), sodium (Na), potassium (K), rubidium (Rb) and cesium (Cs), X is a halogen atom selected from fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), and Ar is a substituted or unsubstituted aryl or heteroaryl group, such as phenyl, biphenyl, One of naphthyl, pyrrolyl, pyrazolyl, imidazolyl, pyridyl, pyrimidinyl and the like, preferably, R1 and R2, R3 and R4, R5 and R6, R7 and R8 are the same or different from (CH2)2-O-(CH2)2, (CH2)2-CHOH-(CH2)2, (CH2)2-CHCOOH-(CH2)2 or CH2-CHCH3-O-CHCH3-CH2, more preferably, R1 and R2, R3 and R4, R5 and R6, R7 and R8 are the same or different from (CH2)2-CHOH-(CH2)2 or (CH2)2-CHCOOH-(CH2)2.

[0017] In the technical scheme, preferably, according to the relevant design principles of nitroxide diradicals used as DNP PAs, it is confirmed that increasing water solubility, appropriately enhancing the interaction between electrons (J and D values), limiting the relative orientation of the g tensors of the two nitroxides to be close to orthogonal, increasing the content of protons near electrons can accelerate polarization transfer, changing the local geometry around electrons to enhance solvent accessibility, and extending the electron relaxation time will affect the DNP performance of the polarizer. Therefore, combining the above key design principles, 1) modification of A1, A2, A3, and A4 can moderately adjust the relative orientation of the g tensor to be almost orthogonal; 2) modification of A2 can moderately increase water solubility; 3) modification of A4 can add a conjugated structure in the connecting chain, which can moderately increase the interaction between electrons (J and D values); 4) modification of A5 can increase the interaction between electrons (J and D values); 5) modification of R1 and R2, R3 and R4, R5 and R6, R7 and R8 groups can increase water solubility and solvent accessibility, moderately prolong the electron relaxation time, and introduce protons on the substituents to accelerate polarization transfer.

[0018] In the technical scheme, preferably, it is represented by one of the following formulae 2-7, wherein R1 and R2, R3 and R4, R5 and R6, R7 and R8 groups are represented by (CH2)2-O-(CH2)2, (CH2)2-CHOH-(CH2)2, (CH2)2-CHCOOH-(CH2)2 or CH2-CHCH3-O-CHCH3-CH2, wherein R1 and R2, R3 and R4 are the same, and R5 and R6, R7 and R8 are the same:

[0019]

[0020]

[0021] In the technical solution, more preferably, it is represented by one of the following formulas 8-15:

[0022]

[0023]

[0024]

[0025] The present invention also provides the use of the above-mentioned nitrogen-oxygen diradical compound as a polarizing agent in dynamic nuclear polarization.

[0026] The use of the present invention is the application of nitrogen-oxygen diradical compounds in nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) of solid samples.

[0027] The purpose of the present invention is to improve the sensitivity of solid-state NMR at 14.1T. 1 H DNP enhancement factor ≥ 85, overall NMR sensitivity gain ≥ 28 s -1 / 2 .

[0028] The invention discloses a nitroxide diradical based on acrylamide connection and its application in nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) of solid samples. The nitroxide diradical is formed by connecting two nitroxide units through a novel acrylamide-based connecting chain. Compared with the water-soluble nitroxide diradical AMUPol, which is the most widely used in high-field (9.4T) dynamic nuclear polarization, the connecting chain of the compound has the following advantages: 1) the synthesis efficiency is nearly 2.5 times higher and the practicability is stronger; 2) the electron-electron distance is shorter, so the dipole coupling (D value) effect is stronger; 3) the relative orientation of the g tensors of the two nitroxide units is nearly orthogonal, which is conducive to the frequency matching of the CE mechanism; 4) it contains a conjugated structure and maintains a certain rigidity; 5) the exchange coupling (J value) effect between electrons and electrons is enhanced; 6) under a higher field (14.1T), it has a shorter DNP establishment time and a weakened depolarization effect, thereby compensating for the reduced DNP enhancement factor. Therefore, this type of nitroxide diradical has better application prospects in solid NMR and MRI than the currently commercially available water-soluble nitroxide diradicals.

[0029] The beneficial effect of adopting the above technical solution is that: the present invention can design a new type of high-field DNP PAs, and this new generation of molecules uses a new type of acrylamide-based connecting chain, which can be a symmetrical or asymmetrical nitrogen-oxygen diradical. The advantage is that the connecting chain in this type of compound contains a double-bonded conjugated structure, which can provide rigidity, limit the relative direction of the g tensor of the two nitrogen oxides, and shorten the distance between the two unpaired electrons, so that the interaction strength (J and D values) between the two electrons can be moderately enhanced; the substituents on the two nitrogen oxide rings of the compound can provide a longer relaxation time, and increase water solubility through chemical modification, and can also control the geometric configuration of the substituent by changing the absolute configuration of the group on the substituent, thereby increasing solvent accessibility, and can add protons to accelerate polarization transfer; the design of this type of compound is not only to obtain a maximized DNP enhancement factor, but more to meet the requirements of shortening the polarization establishment time and reducing the influence of the depolarization effect under higher field (>9.4T) DNP conditions, so as to obtain a more accurate parameter-overall NMR sensitivity that can evaluate the DNP performance of the polarizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the room temperature EPR spectrum of compound 1 in Example 1 of the present invention;

[0031] Figure 2 is a high performance liquid phase (HPLC) chromatogram of compound 1 in Example 1 of the present invention;

[0032] Figure 3 is a high-resolution mass spectrum of compound 1 in Example 1 of the present invention;

[0033] Figure 4 is the room temperature EPR spectrum of compound 2 in Example 2 of the present invention;

[0034] Figure 5 is a high-resolution mass spectrum of compound 2 in Example 2 of the present invention;

[0035] Figure 6 is the room temperature EPR spectrum of compound 3 in Example 3 of the present invention;

[0036] Figure 7 is a high performance liquid phase (HPLC) chromatogram of compound 3 in Example 3 of the present invention;

[0037] Figure 8 is a high-resolution mass spectrum of compound 3 in Example 3 of the present invention;

[0038] Fig. 9 is the room temperature EPR spectrum of compound 4 in Example 3 of the present invention;

[0039] Fig.10is a high performance liquid phase (HPLC) chromatogram of compound 4 in Example 3 of the present invention;

[0040] Fig.11 It is a high-resolution mass spectrum of compound 4 in Example 3 of the present invention. DETAILED DESCRIPTION

[0042] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0043] Example 1

[0044] The nitrogen-oxygen diradical compound described in this embodiment has the following structural formula:

[0045]

[0046] The synthetic route of compound 1 is as follows:

[0047]

[0048] 1. Synthesis of Compounds A1 and A4:

[0049] The detailed synthesis conditions of compounds A1 and A4 have been disclosed, see references (Sauvée C et al. Angew Chem Int Ed Engl. 2013, 52(41): 10858-61.) and (Sauvée C et al.

[0050] Chemistry.2016,22(16):5598-606.).

[0051] 2. Synthesis of Compound 1

[0052] 1. Add NaH (1.2-1.6eq, 60% dispersed in mineral oil) to the reaction flask, protect with argon, dissolve with redistilled THF, and slowly dropwise add triethyl phosphoacetate (1.5-2

[0053] eq), after 1 h at room temperature, a THF solution of compound A1 (1 eq) was added dropwise, stirred overnight, and the reaction was complete. After THF was dried, the mixture was quenched with a NaHCO3 solution, the pH value was adjusted to 8-9, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to obtain a crude product, which was separated and purified by a scraper, and the developing solvent was DCM:EA=(4:1)x2. The yield was 55-75%.

[0054] 2. After adding compound A2 (1 eq) to the reaction flask, add 1M NaOH in 75% MeOH aqueous solution (0.5-1 mL), stir at room temperature for 8 h to complete the reaction. Acidify with 1M HCl, extract with DCM, remove water with anhydrous sodium sulfate, dry, filter, and concentrate in vacuo to obtain compound A3. The yield is 85-95%.

[0055] 3. Add compound A3 (1 eq) to the reaction bottle, dissolve it with heavy distilled DCM, add DIPEA (4-6 eq), HATU (1-1.2 eq), and after 5-10 min, dropwise add compound A4 (1-1.5 eq) dissolved in DCM, stir at room temperature for 4 h to complete the reaction, extract with DCM+NaHCO3 aqueous solution, dry with anhydrous sodium sulfate, filter, and concentrate in vacuo to obtain a crude product, which is separated and purified by scraping plate, and the developing solvent is DCM:MeOH=1:5%. Then dissolve the product with EtOH, add NaOH aqueous solution (0.5 mL), and stir at room temperature for 5.5 h to complete the reaction. After spinning dry EtOH, acidify with 1M HCl, adjust pH to 7, and separate and purify with C18 reverse silica gel, the mobile phase is PBS / MeOH system, and desalt by reverse phase column to obtain compound 1. The yield is 50-60%.

[0056] As attached Figure 1 is the room temperature EPR spectrum data of compound 1 in this example; Figure 2 is the high performance liquid phase (HPLC) chromatographic data of compound 1 in this example; Figure 3 This is the high-resolution mass spectrum data of compound 1 in this example.

[0057] 3. DNP experiment of compound 1

[0058] The DNP experiment was conducted by adding the nitroxide diradical compound 1 to the DNP standard solution 60 / 30 / 10 v / v / vd8-glycerol / D2O / H2O (GDH) to prepare a solution with a final concentration of 10 mM, and also containing 0.25 M 13 C, 15 N-proline. DNP experiments were performed on a 600MHz Bruker Biospin 395GHz solid-state NMR spectrometer with a magnetic field of 14.1T, a 3.2mm sapphire rotor, and a temperature of 100K.

[0059] The DNP enhancement factor of compound 1 is 115, the polarization build-up time is 4.5 s, the contribution factor is 0.52, and the overall NMR sensitivity is 28.2 s -1 / 2 .

[0060] Example 2

[0061] The nitrogen-oxygen diradical compound described in this embodiment has the following structural formula:

[0062]

[0063] The synthetic route of compound 2 is as follows:

[0064]

[0065] 1. Synthesis of Compound A5:

[0066] The detailed synthesis conditions of compound A5 have also been disclosed, see reference (Sauvée C et al. Angew Chem Int Ed Engl. 2013, 52(41): 10858-61).

[0067] 2. Synthesis of Compound 2:

[0068] The synthesis method of compound 2 is the same as that in Example 1.

[0069] As attached Figure 4 This is the room temperature EPR spectrum data of compound 2 in this example.

[0070] As attached Figure 5 This is the high-resolution mass spectrum data of compound 2 in this example.

[0071] 3. DNP experiment of compound 2

[0072] The DNP test experiment of compound 2 was carried out in the same manner as in Example 1.

[0073] The DNP enhancement factor of compound 2 is 85.2, the polarization build-up time is 2.1 s, the contribution factor is 0.51, and the overall NMR sensitivity is 29.6 s -1 / 2 .

[0074] Example 3

[0075] The nitroxide diradical compound described in this embodiment has two diastereoisomers, and the structural formula is as follows:

[0076]

[0077] The synthetic routes of compound 3 and compound 4 are as follows:

[0078]

[0079] 1. Synthesis of Compound A6:

[0080] The synthesis method of compound A6 is the same as that of compound A5 in Example 2.

[0081] 2. Synthesis of Compound 3 and Compound 4:

[0082] The synthesis method of compound 3 and compound 4 is the same as that in Example 1.

[0083] like Figure 6 It is the room temperature EPR spectrum of compound 3 in this example.

[0084] like Figure 7 It is the high performance liquid phase (HPLC) chromatogram of compound 3 in this example.

[0085] like Figure 8 It is the high-resolution mass spectrum of compound 3 in this example.

[0086] like Fig. 9 is the room temperature EPR spectrum of compound 4 in this example.

[0087] like Fig.10 It is the high performance liquid phase (HPLC) chromatogram of compound 4 in this example.

[0088] like Fig.11 It is the high-resolution mass spectrum of compound 4 in this example.

[0089] 3. DNP experiments of compounds 3 and 4

[0090] The DNP test experiment of compound 3 and compound 4 is the same as that in Example 1.

[0091] The DNP enhancement factor of compound 3 is 91.3, the polarization build-up time is 2.8 s, the contribution factor is 0.66, and the overall NMR sensitivity is 35.8 s -1 / 2 .

[0092] The DNP enhancement factor of compound 4 is 94.0, the polarization build-up time is 2.5 s, the contribution factor is 0.48, and the overall NMR sensitivity is 28.7 s -1 / 2 .

[0093] The above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention is described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A nitrogen-oxygen diradical based on acrylamide linkage, characterized in that: The nitrogen-oxygen diradical structural formula is represented by the following formula 1: in, A1 is N; A2 is H, (CH2-CH2-O)4CH3 or (CH2-CH2-O)2H; A3 is O; A4 is CH; A5 is CH2C; R1 and R2, R3 and R4, R5 and R6, R7 and R8 are the same or different and are (CH2)2-O-(CH2)2, (CH2)2-CHOH-(CH2)2 or (CH2)2-CHCOOH-(CH2)2.

2. The acrylamide-linked nitroxide diradical according to claim 1, characterized in that: The nitroxide diradical is represented by the following formulas 1, 2 and 3:

3. The acrylamide-linked nitroxide diradical according to claim 1, characterized in that: The R1 and R2, R3 and R4, R5 and R6, R7 and R8 are the same or different and are (CH2)2-O-(CH2)2, (CH2)2-CHOH-(CH2)2 or (CH2)2-CHCOOH-(CH2)2, wherein R1 and R2, R3 and R4 are the same, R5 and R6, R7 and R8 are the same.

4. Use of the acrylamide-linked nitroxide diradical according to any one of claims 1 to 3 as a polarizing agent in dynamic nuclear polarization.

5. The use of acrylamide-linked nitroxide diradicals as polarizing agents in dynamic nuclear polarization according to claim 4, characterized in that: The application is the application of nitrogen-oxygen diradical compounds in nuclear magnetic resonance and magnetic resonance imaging of solid samples.

6. The use of acrylamide-linked nitroxide diradicals as polarizing agents in dynamic nuclear polarization according to claim 4, characterized in that: The purpose is to improve the sensitivity of solid-state NMR at 14.1T. 1 H DNP enhancement factor ≥ 85, overall NMR sensitivity gain ≥ 28 s -1 / 2 .

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