Synthesis of Sterically Shielded Nitroxides Using the Reaction of Nitrones with Alkynylmagnesium Bromides

Sterically shielded nitroxides, which demonstrate high resistance to bioreduction, are the spin labels of choice for structural studies inside living cells using pulsed EPR and functional MRI and EPRI in vivo. To prepare new sterically shielded nitroxides, a reaction of cyclic nitrones, including various 1-pyrroline-1-oxides, 2,5-dihydroimidazole-3-oxide and 4H-imidazole-3-oxide with alkynylmagnesium bromide wereused. The reaction gave corresponding nitroxides with an alkynyl group adjacent to the N-O moiety. The hydrogenation of resulting 2-ethynyl-substituted nitroxides with subsequent re-oxidation of the N-OH group produced the corresponding sterically shielded tetraalkylnitroxides of pyrrolidine, imidazolidine and 2,5-dihydroimidazole series. EPR studies revealed large additional couplings up to 4 G in the spectra of pyrrolidine and imidazolidine nitroxides with substituents in 3- and/or 4-positions of the ring.


Introduction
Cyclic nitroxides with four ethyl (or more bulky alkyl) substituents adjacent to the N-O group are known to show much higher resistance to reduction with biogenic reductants and enzymatic systems than corresponding tetramethyl analogs [1]. The higher stability of these so-called "sterically shielded" nitroxides makes them favorable for those fields of application where the decay of conventional tetramethyl nitroxides is fast. The stability of the nitroxide group is of crucial importance for structural studies of biological macromolecules in their native environment inside living cells using site-directed spin labeling (SDSL) and pulsed EPR techniques [2]. For this application, reduction-resistant spin labels were prepared from sterically shielded nitroxides of pyrroline [3][4][5], isoindoline [6] and pyrrolidine series [7,8]. Sterically shielded nitroxides of piperidine [9,10] and imidazoline [11,12] series were designed for functional MRI and EPRI in vivo.
Besides that, the new 2-alkynyl-substituted nitroxides themselves may be of interest as bioorthogonal spin labels capable of binding to biomolecules, modified with azide or nitrone groups via copper-catalyzed 1,3-dipolar cycloaddition reactions [22][23][24]. These alkynyl derivatives may find even broader applications because alkynes are used in the synthesis of numerous heterocyclic systems; for recent reviews, see [25][26][27].

Nitrones
A series of sterically hindered nitrones 1-5 have been prepared to investigate their reactions with alkynylmagnesium halides. We have previously reported on the synthesis of diastereomeric pyrrolidines 6a,b and 7a,b and corresponding nitrones 8a,b from amino acids, ketones and dimethyl fumarate according to Scheme 1 [17,19]. The pyrrolidines 6c,d and 7c,d were prepared in analogy to the published procedures. The reaction afforded the mixtures of diastereomers in a 1:1 ratio (cf. [17,19]), and the individual diastereomers were isolated using column chromatography. The mixtures were subjected to reduction of the ester groups with LiAlH4 and oxidation with tungstate-hydrogen peroxide system to give the nitrones 8c,d (racemates). We noticed earlier that treatment of 3,4-bis-(hydroxymethyl)-1-pyrroline1-oxideswith Grignard reagents mightlead to THF-insoluble precipitate formation, presumably, magnesium alcoholates [17]. To avoid extra consumption of Grignard reagent and to prevent precipitation of magnesium alcoholates from the reaction mixtures, the hydroxy groups in the nitrones 8a-d were protected via treatment with 2,2-dimethoxypropane, and the resulting nitrones 1a-d were used for nitroxides syntheses. The reaction was found to give satisfactory yields of 2-ethynyl-substituted nitroxides with all these sterically hindered nitrones except for 1d. Subsequent hydrogenation of the ethynyl derivatives produced corresponding sterically shielded tetraalkylnitroxides.
Besides that, the new 2-alkynyl-substituted nitroxides themselves may be of interest as bioorthogonal spin labels capable of binding to biomolecules, modified with azide or nitrone groups via copper-catalyzed 1,3-dipolar cycloaddition reactions [22][23][24]. These alkynyl derivatives may find even broader applications because alkynes are used in the synthesis of numerous heterocyclic systems; for recent reviews, see [25][26][27].
The nitrone 2 was prepared from 9 [28,29] by successive introduction of three ethyl groups via reaction with ethylmagnesium bromide and oxidation (Scheme 2). The isomers formed in the intermediate steps were not separated, and the crude mixture was used in the second and third steps affording chiral nitrone 2 as a final product with a 25% yield.

Nitroxides
Nitrones 1-5 were treated with a 10-fold excess of ethynylmagnesium bromide in THF, then quenched with water and oxidized (Scheme 4). The reaction products, times and yields of corresponding nitroxides are given in Table 1. In the case of 1, processing of the reaction mixtures implied treatment with an aqueous acid solution to remove the The nitrone 2 was prepared from 9 [28,29] by successive introduction of three ethyl groups via reaction with ethylmagnesium bromide and oxidation (Scheme 2). The isomers formed in the intermediate steps were not separated, and the crude mixture was used in the second and third steps affording chiral nitrone 2 as a final product with a 25% yield. To prepare nitrone 4, the 2-amino-2-methylpentan-3-one oxime (10) was heated under reflux with pentane-3-one in methanol using ammonium acetate as a catalyst (cf. [30]), Scheme 3. The resulting nitrone 11was subjected to Eschweiler-Clarke alkylation in analogy to literature protocol [31]. The nitrones 3 [20] and 5 [19] were prepared according to literature protocols.

Nitroxides
Nitrones 1-5 were treated with a 10-fold excess of ethynylmagnesium bromide in THF, then quenched with water and oxidized (Scheme 4). The reaction products, times and yields of corresponding nitroxides are given in Table 1. In the case of 1, processing of the reaction mixtures implied treatment with an aqueous acid solution to remove the Scheme 2. Synthesis of nitrone 2.

Scheme 1. Synthesis of nitrones 1a-d.
The nitrone 2 was prepared from 9 [28,29] by successive introduction of three ethyl groups via reaction with ethylmagnesium bromide and oxidation (Scheme 2). The isomers formed in the intermediate steps were not separated, and the crude mixture was used in the second and third steps affording chiral nitrone 2 as a final product with a 25% yield.

Nitroxides
Nitrones 1-5 were treated with a 10-fold excess of ethynylmagnesium bromide in THF, then quenched with water and oxidized (Scheme 4). The reaction products, times and yields of corresponding nitroxides are given in Table 1. In the case of 1, processing of the reaction mixtures implied treatment with an aqueous acid solution to remove the The nitrones 3 [20] and 5 [19] were prepared according to literature protocols.

Nitroxides
Nitrones 1-5 were treated with a 10-fold excess of ethynylmagnesium bromide in THF, then quenched with water and oxidized (Scheme 4). The reaction products, times and yields of corresponding nitroxides are given in Table 1. In the case of 1, processing of the reaction mixtures implied treatment with an aqueous acid solution to remove the protecting groups. Noteworthily, the conversion of 1b and c was incomplete under these conditions, and TLC analysis of the quenched reaction mixtures showed the presence of the starting compounds. Bulky substituents at nitrone group carbon atom of 1 strongly decreased the reaction rate but didnot influence much the yield of the nitroxide. More hindered nitrone 1d was quantitatively recovered from the reaction mixture after stirring with ethynylmagnesium bromide for a week.
protecting groups. Noteworthily, the conversion of 1bandc was incomplete under these conditions, and TLC analysis of the quenched reaction mixtures showed the presence of the starting compounds. Bulky substituents at nitrone group carbon atom of 1 strongly decreased the reaction rate but didnot influence much the yield of the nitroxide. More hindered nitrone 1dwas quantitatively recovered from the reaction mixture after stirring with ethynylmagnesium bromide for a week. The addition of ethynylmagnesium bromide to 1a-c proceeded with higher selectivity than that of vinylmagnesium bromide affording only one diastereomer (cf. [19]). The structures 12a-c were assigned based on X-ray analysis data (see Figures S2-S4 in the Supplementary Information in this article "X-ray diffraction data"). Selective formation of 12a-c presumably results from the coordination of the organometallic reagent with the oxygen atom of the neighboring alkoxymethyl group. Interestingly, the reaction with chiral nitrone 2 also gave a single isomer, a chiral nitroxide 14, but the ethynyl group entered from the opposite side, trans-to the neighboring tert-butoxy group ( Figure S5 in the Supplementary Information in this article"X-ray diffraction data").
To introduce larger alkynyl groups corresponding organometallic reagents were prepared from terminal acetylenes and EtMgBr. The reaction with 1a and 3 afforded nitroxides 22a-c and 23 with 54-66% yield (Scheme 5). The stereochemistry of this reaction was similar to that of ethynylmagnesium bromide addition, see the X-ray analysis data in Figure S6-S8 in the Supplementary Information in this article"X-ray diffraction data."       We have shownearlier that careful hydrogenation of ethynyl-substituted nitroxide in THF under atmospheric pressure allows for avoiding undesired over-reduction of the nitroxide group to an amine. The resulting ethyl-substituted hydroxylamine can then be easily oxidized to the corresponding nitroxide [8]. Using this procedure, nitroxides 12a-c, 14-17 were converted to nitroxides 13a-c, 18-21 (Scheme 4 and Table 1).
It should be noted that, in agreement with our previous observations [17,19], nitroxides 13a-c, 18-20 can't be prepared via the direct addition of EtMgBr. The overall yield of 13a from 8a using the procedures described here is nearly the same as in the previously described procedure with vinylmagnesium bromide [19]. The reaction of 5 with EtMgBr gives a lower yield of 21 (45%) [15] than the above two-step procedure with ethynylmagnesium bromide (Table 1).

EPR Spectra
Parameters of the EPR spectra of the new nitroxides are given in Table 2. We have shown earlier that in nitroxides with a 3,4-disubstituted five-membered saturated ring, each pair of geminal ethyl groups at twoand fivepositions of the heterocycle produces one large (ca. 2 G) additional splitting due to hfi with one of the methylene hydrogens of the ethyl group [8,19,32]. Replacement of one of the geminal ethyls with another group may change the hyperfine structure of the spectrum greatly. The data on 13a-c in Table 2 and Figure 2 demonstrate the remarkable evolution of the EPR spectra upon the increase of the steric demand of the substituent. The spectrum of 13c follows the pattern described for 13a [19], with two additional large splittings on γ-hydrogens. Replacement of one of the ethyl groups with theisopropyl one resulted in an increase of both hfi constants (by ca. 50% and 12%). Interestingly, the tert-butyl group (in 13b) produces an opposite effect showing only one doublet splitting on one of the γ-hydrogens (cf. [17]). We have shownearlier that careful hydrogenation of ethynyl-substituted nitroxide in THF under atmospheric pressure allows for avoiding undesired over-reduction of the nitroxide group to an amine. The resulting ethyl-substituted hydroxylamine can then be easily oxidized to the corresponding nitroxide [8]. Using this procedure, nitroxides 12a-c, 14-17 were converted to nitroxides 13a-c, 18-21 (Scheme 4 and Table 1).
It should be noted that, in agreement with our previous observations [17,19], nitroxides 13a-c, 18-20 can't be prepared via the direct addition of EtMgBr. The overall yield of 13a from 8a using the procedures described here is nearly the same as in the previously described procedure with vinylmagnesium bromide [19]. The reaction of 5 with EtMgBr gives a lower yield of 21 (45%) [15] than the above two-step procedure with ethynylmagnesium bromide (Table 1).

EPR Spectra
Parameters of the EPR spectra of the new nitroxides are given in Table 2. We have shown earlier that in nitroxides with a 3,4-disubstituted five-membered saturated ring, each pair of geminal ethyl groups at twoand fivepositions of the heterocycle produces one large (ca. 2 G) additional splitting due to hfi with one of the methylene hydrogens of the ethyl group [8,19,32]. Replacement of one of the geminal ethyls with another group may change the hyperfine structure of the spectrum greatly. The data on 13a-c in Table 2 and Figure 2 demonstrate the remarkable evolution of the EPR spectra upon the increase of the steric demand of the substituent. The spectrum of 13c follows the pattern described for 13a [19], with two additional large splittings on γ-hydrogens. Replacement of one of the ethyl groups with theisopropyl one resulted in an increase of both hfi constants (by ca. 50% and 12%). Interestingly, the tert-butyl group (in 13b) produces an opposite effect showing only one doublet splitting on one of the γ-hydrogens (cf. [17]). The addition of ethynylmagnesium bromide to 1a-c proceeded with higher selectivity than that of vinylmagnesium bromide affording only one diastereomer (cf. [19]). The structures 12a-c were assigned based on X-ray analysis data (see Figures S2-S4 in the Supplementary Information in this article "X-ray diffraction data"). Selective formation of 12a-c presumably results from the coordination of the organometallic reagent with the oxygen atom of the neighboring alkoxymethyl group. Interestingly, the reaction with chiral nitrone 2 also gave a single isomer, a chiral nitroxide 14, but the ethynyl group entered from the opposite side, transto the neighboring tert-butoxy group ( Figure S5 in the Supplementary Information in this article"X-ray diffraction data").
To introduce larger alkynyl groups corresponding organometallic reagents were prepared from terminal acetylenes and EtMgBr. The reaction with 1a and 3 afforded nitroxides 22a-c and 23 with 54-66% yield (Scheme 5). The stereochemistry of this reaction was similar to that of ethynylmagnesium bromide addition, see the X-ray analysis data in Figures S6-S8 in the Supplementary Information in this article"X-ray diffraction data." protecting groups. Noteworthily, the conversion of 1bandc was incomplete under these conditions, and TLC analysis of the quenched reaction mixtures showed the presence of the starting compounds. Bulky substituents at nitrone group carbon atom of 1 strongly decreased the reaction rate but didnot influence much the yield of the nitroxide. More hindered nitrone 1dwas quantitatively recovered from the reaction mixture after stirring with ethynylmagnesium bromide for a week.

Scheme 4. Synthesis of nitroxides.
The addition of ethynylmagnesium bromide to 1a-c proceeded with higher selectivity than that of vinylmagnesium bromide affording only one diastereomer (cf. [19]). The structures 12a-c were assigned based on X-ray analysis data (see Figures S2-S4 in the Supplementary Information in this article "X-ray diffraction data"). Selective formation of 12a-c presumably results from the coordination of the organometallic reagent with the oxygen atom of the neighboring alkoxymethyl group. Interestingly, the reaction with chiral nitrone 2 also gave a single isomer, a chiral nitroxide 14, but the ethynyl group entered from the opposite side, trans-to the neighboring tert-butoxy group ( Figure S5 in the Supplementary Information in this article"X-ray diffraction data").
To introduce larger alkynyl groups corresponding organometallic reagents were prepared from terminal acetylenes and EtMgBr. The reaction with 1a and 3 afforded nitroxides 22a-c and 23 with 54-66% yield (Scheme 5). The stereochemistry of this reaction was similar to that of ethynylmagnesium bromide addition, see the X-ray analysis data in Figure S6-S8 in the Supplementary Information in this article"X-ray diffraction data." We have shown earlier that careful hydrogenation of ethynyl-substituted nitroxide in THF under atmospheric pressure allows for avoiding undesired over-reduction of the nitroxide group to an amine. The resulting ethyl-substituted hydroxylamine can then be easily oxidized to the corresponding nitroxide [8]. Using this procedure, nitroxides 12a-c, 14-17 were converted to nitroxides 13a-c, 18-21 (Scheme 4 and Table 1).
It should be noted that, in agreement with our previous observations [17,19], nitroxides 13a-c, 18-20 can't be prepared via the direct addition of EtMgBr. The overall yield of 13a from 8a using the procedures described here is nearly the same as in the previously described procedure with vinylmagnesium bromide [19]. The reaction of 5 with EtMgBr gives a lower yield of 21 (45%) [15] than the above two-step procedure with ethynylmagnesium bromide (Table 1).

EPR Spectra
Parameters of the EPR spectra of the new nitroxides are given in Table 2. We have shown earlier that in nitroxides with a 3,4-disubstituted five-membered saturated ring, each pair of geminal ethyl groups at twoand fivepositions of the heterocycle produces one large (ca. 2 G) additional splitting due to hfi with one of the methylene hydrogens of the ethyl group [8,19,32]. Replacement of one of the geminal ethyls with another group may change the hyperfine structure of the spectrum greatly. The data on 13a-c in Table 2 and Figure 2 demonstrate the remarkable evolution of the EPR spectra upon the increase of the steric demand of the substituent. The spectrum of 13c follows the pattern described for 13a [19], with two additional large splittings on γ-hydrogens. Replacement of one of the ethyl groups with theisopropyl one resulted in an increase of both hfi constants (by ca. 50% and 12%). Interestingly, the tert-butyl group (in 13b) produces an opposite effect showing only one doublet splitting on one of the γ-hydrogens (cf. [17]). Another example of an unusual hyperfine structure in the EPR spectrum is demonstrated by 20. The EPR spectrum of close analog of this nitroxide, 2,2,5,5-tetraethyl-3, 4-dimethylimidazolidine-1-oxyl (24; Figure 3), is known to contain two splitting constants (ca. 2 G) [32], while 20 showed a single additional splitting with a H = 3.64 G.  Another example of an unusual hyperfine structure in the EPR spectrum is demonstrated by 20. The EPR spectrum of close analog of this nitroxide, 2,2,5,5-tetraethyl-3,4-dimethylimidazolidine-1-oxyl (24; Figure 3), is known to contain two splitting constants (ca. 2 G) [32], while 20 showed a single additional splitting with aH = 3.64 G.   Another example of an unusual hyperfine structure in the EPR spectrum is demonstrated by 20. The EPR spectrum of close analog of this nitroxide, 2,2,5,5-tetraethyl-3,4-dimethylimidazolidine-1-oxyl (24; Figure 3), is known to contain two splitting constants (ca. 2 G) [32], while 20 showed a single additional splitting with aH = 3.64 G. Magnetic field, G Figure 3. EPR spectra of nitroxides 20 and 24. Figure 3. EPR spectra of nitroxides 20 and 24.
The above examples demonstrate that minor changes in the stricture of 2,2,5,5-tetraethylsubstituted five-membered ring nitroxides may lead to drastic changes in their EPR spectra. To the best of our knowledge, similar effects never occur for 2,2,5,5-tetramethyl nitroxides.
The IR spectra were recorded on a All the NMR spectra were acquired for 5-10% solutions in CDCl 3 , (CD 3 ) 2 SO or CDCl 3 -CD 3 OD mixtures at 300 K using the signal of the solvent as a standard. NMR spectra of nitroxides for analysis and structure assignment were recorded after reduction with Zn in CD 3 OD-CF 3 COOH at 65 • C as described in [8] or with Zn and ND 4 Cl in CD 3 OD at 5 • C. HRMS analyses were performedusinga High-Resolution Mass Spectrometer DFS (Thermo Electron, Waltham, MA, USA).
HPLC analyses were carried out using an HPLC-UV (Agilent 1100, Agilent Technologies Inc., Santa Clara, CA, USA) with a Zorbax C8 column (250 mm × 4.6 mm with 5 µm particle size; Agilent Technologies Inc., USA). The column was thermostatically controlled at 35 • C. Samples were dissolved in methanol (2 mg/mL) and 3 µL of the solutionwas injected. The mobile phase was composed of A (0.1% H 3 PO 4 in water) and B (methanol) with the following gradient elution: 0-7 min 80% B, 7-10 min 100% B, 10-12 min 100% B, the flow rate was set to 1.0 mL/min, and peaks were detected using a wavelength of 230 nm.
EPR experiments were performed on X-band (9.8 GHz) EPR spectrometer ER-200D (Bruker). All measurements were performed in 50 µL glass capillary. The radicals were dissolved in oxygen-free distilled water at a concentration of 0.2 mM. EPR settings: modulation amplitude 0.5 G, MW, power 5 mW time constant50 ms; total acquisition time 3 min. The water-insoluble radicals were dissolved in a water/ethanol mixture (50%/50%) at a concentration of 0.1 mM. Data simulation was performed with the free software Winsim.

Conclusions
The synthesis of nitroxides via reaction of 1-pyrroline 1-oxides with alkynylmagnesium halides was earlier investigated by K. Hideg et al. [34][35][36]. Recently, we suggested using this reaction for the preparation of sterically shielded 2,2,5,5-tetraalkylpyrrolidine nitroxides [8,21]. In this paper, we showed how these reagents could be applied for the preparation of various highly strained nitroxides of pyrrolidine, imidazolidine and 2,5-dihydroimidazole series. The described two-step addition & hydrogenation protocol was suitable for the introduction of an ethyl group to the carbon atom of cyclic α-ethyl-, α-isopropyland α-tert-butyl nitrones and may find broader application. Some of the new nitroxides can hardly be prepared in any other way.
The new data on the feasibility of the addition of alkynylmagnesium halides to highly hindered alkylnitrones may have another consequence. In this work, we did not utilize the synthetic potential of alkynyl groups. However, taking into account the broad application of alkynes in organic synthesis, the use of 2-alkynyl nitroxides for biorthogonal spin labeling and for the synthesis of bioactive nitroxide derivatives certainly deserves attention.
The new set of nitroxides gives impressive examples of the variability of EPR spectra of five-membered nitroxides with bulky alkyl substituents adjacent to the nitroxide group. The high additional coupling constants are highly dependent on nitroxide structure. The high sensitivity of spectral parameters to minor structural changes is a promising basis for the molecular design of functional spin probes of a new generation.