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.


Selective Synthesis of Azoloyl NH-1,2,3-Triazoles and Azolyl Diazoketones: Experimental and Computational Insights

Here, we report that the reaction of enaminones, from a class of azole series, with sulfonyl azides leads to a difficult-to-separate mixture of two pairs of compounds: (1) 4-azoloyl-NH-1,2,3-triazoles with sulfonamides and (2) azolyl diazoketones with N-sulfonamidines, as a result of the implementation of two competing reactions. On one hand, the electron-donating methyl or methoxy group in the aryl para-position of arylsulfonyl azides favors the production of NH-1,2,3-triazoles together with sulfonamides. On the other hand, the use of highly electrophilic 4-nitrophenylsulfonyl azide promotes the formation of diazoketones and sulfonamidines. It is shown that the direction of each reaction is not only controlled by the nature of the initial enaminones and sulfonyl azides but also depends on the tested solvent. The problem of removing sulfonamides and amidines from the desired products was solved for the first time using new water-soluble enaminones. Based on the experimental and computational studies, the factors contributing to the selective course of alternative reactions were identified, and methods for the synthesis of azoloyl-NH-1,2,3-triazoles and azolyl diazoketones were developed. Density functional theory (DFT) results have shown that the 1,3-dipolar cycloaddition is totally driven toward one single regioisomer with a high asynchronous bond formation, and the introduction of an electron-deficient group in sulfonyl azides induces faster cycloaddition. Additionally, DFT calculations were used to gain further mechanistic insights on the reaction studied here.

■ INTRODUCTION
NH-1,2,3-Triazoles, linked to a second five-membered heterocycle through a carbonyl group, possess two active functional groups and are therefore promising building blocks for obtaining various 1,2,3-triazole derivatives. 1,2,3-Triazole is a heterocycle that has found wide applications as a privileged scaffold in the directed synthesis of substances with biological activity, in organic electronics for the synthesis of new materials, as a key fragment in the synthesis of substances capable of metal-catalyzed cycle transformations, and as a unique building block in the synthesis of various organic compounds. 1,2 Among NH-1,2,3-triazoles, substances with biological activity and used as new materials with interesting optical and physical properties were also found. 3−10 The primary methods of synthesis of 4-carbonyl derivatives of NHtriazoles include reactions of acylacetylenes with inorganic azides, 11−14 interaction of α-diazo-β-oxoaldehydes with aniline, ammonium, hydroxylamine, and semicarbazide, 15 and modifications of ready NH-triazoles synthesized by other methods. 16,17 All the methods mentioned above almost completely focused on the production of 4-aroyl derivatives of 1,2,3-triazole. In this aspect, another method based on the reaction of β-enaminones 1 with tosyl azide (2a) is an exception, which permitted to prepare three 4-heteroaroyltriazoles (pathway A, Scheme 1). 18 −20 Thus, the existing synthesis methods are limited in their applicability for the production of 4-heteroaroyl-NH-triazoles.
It should be noted that changing the nature of enaminones 1 and the conditions of their reaction with sulfonyl azides 2 lead to switching to other products, diazoketones 5 together with amidines 7 (pathway B, Scheme 1). 21 α-Diazoketones are widely used in organic synthesis. They are well-known precursors of carbenes and carbenoids. Being 1,3-dipoles of propargyl-allenyl type, they take part in cyclopropanations, rearrangements, cycloadditions, and electrocyclic processes. 22−26 They are valuable reagents for use in biological chemistry 27 found in naturally occurring products 28,29 and also included as a fragment into the structure of some anticancer drugs. 30 Despite a large number of methods for the synthesis of diazoketones published in the literature, 22,23,31−37 their applicability for the synthesis of heteroaroyl diazomethanes is extremely limited. All this makes us to conclude that the search for new safe and effective methods for the synthesis of both NH-1,2,3-triazoles and diazomethanes of the azoloyl series is an urgent task.
To solve this dual problem at once, the reaction of enaminones of azole series 1 with arylsulfonyl azides 2, which could undergo, depending on the structure of reactants and reaction conditions, both competing reactions A and B (Scheme 1) leading to NH-1,2,3-triazoles 4 and diazocarbonyl compounds 5, respectively, is particularly promising. It should be noted that no examples have been found in the literature when products of both types 4 and 5 were obtained and identified together in one reaction. Moreover, there is no information about the factors directing the conversion of triazolines 3 toward the formation of a particular product 4 or 5 (Scheme 1) found by either experimental or computational methods.
The reactions of sulfonyl azides 2 with heterocyclic enaminones 1 are presented in the literature to a much lesser extent than the corresponding reactions of aromatic 16,17,38,39 and alicyclic 38 enaminones. Thus, it is described 16 that the enaminones 1′a,b react with tosyl azide (2a) in water to form exclusively NH-triazoles 4′a,b (Scheme 2), which are the only described examples of NH-1,2,3-triazoles linked with the second heterocycle through a carbonyl group.
On the other hand, heterocyclic β-methylenaminones 1′c react with mesyl azide (2b) in water under microwave irradiation to form only the corresponding diazoketones 5′ (Scheme 2). 40 The method of synthesis of heteroaroyl diazo compounds 7′ by the reaction of the corresponding chloroanhydrides 6′ with diazomethane (Arndt−Eistert synthesis) has not found a wide application 41−43 due to the high explosiveness and toxicity of the latter.
Here, we demonstrate a new approach that allows one to obtain both NH-1,2,3-triazoles 4 and diazomethanes 5 of heteroaroyl series based on the use of either water-soluble sulfonyl azides or enaminones containing an N-methylpiperazine residue. This approach allowed us to develop a simple and effective method for separating the desired 4-acyl-NH-1,2,3-triazoles 4 and diazoketones 5 from byproducts, sulfonamides 6 and amidines 7 (Scheme 1). A combination of experimental and computational studies of the interaction of azolylenaminones with sulfonyl azides revealed the influence of the nature of the starting reagents and the solvents on the direction of the reaction. It was found that seemingly insignificant changes in the structure of arylsulfonyl azide, as well as a variation like the solvent, lead to drastic changes in the direction of this reaction. It is shown that the reactions of the least electrophilic tosyl azide (2a) and 4-methoxyphenyl azide (2g) in pyridine lead to the selective formation of NH-1,2,3-triazoles 4. On the contrary, the formation of diazoketones 5 is facilitated by the use of azide 2e, containing a strong electron acceptor nitro group and ethanol as a solvent. Optimal conditions were determined, and methods were developed for the selective and effective synthesis of azoloyl NH-1,2,3-triazoles 4 and diazoketones 5. As an enaminone substrate, we initially selected the earlier synthesized 44,45 N,N-dimethylenaminones 1a−o, as well as enaminone 1p [which we first obtained in two stages via 4acetyl-1,2,3-triazole 9 from Zidovudine (8)] (Scheme 3), where the substituent at position 1 of 1,2,3-triazole widely varied in electronic properties. In addition to 1,2,3-triazole derivatives 1a−p, one representative of the 1,2,3-thiadiazole 1q and isoxazole 1r 45 series were chosen for the study. Selected N,N-dimethylenaminones 1a−f,g−r were reacted with several sulfonyl azides R 3 SO 2 N 3 (2) (R 3 = 4-MeC 6 H 4 (2a), Me (2b), Ph (2c), 4-F 3 CC 6 H 4 (2d), and 4-O 2 NC 6 H 4 (2e)). As can be seen from Scheme 3, at the end of the synthesis, the reaction mixture generally contains four products, namely, two desired ones (4 and 5) and two byproducts (6 and 7). Often, non-stoichiometric ratios of reactants were used or their conversion turned out to be incomplete, which further complicates the reaction mixture due to residual amounts of reactants. The lack of differences in the components of the reaction mixture (including unpredictable chromatographic mobility), which would allow them to be easily separated, makes this task rather difficult. Indeed, in a series of experiments that we have carried out (Scheme 3), complex combinations of chromatographic and other physico-chemical techniques had to be used to isolate the desired products of reaction 4, 5; in some cases, we could not isolate pure substances at all or only partially succeeded.
We suggested that the cardinal solution of the problem could be the use of a radically different separation principle. To do this, we decided to introduce a basic fragment into the molecule of one of the reactants 1 or 2 and, consequently, into the byproducts 6 and 7, which gives the latter solubility in acidic aqueous medium. The acidity in reactants 1 or 2 as a factor in allowing the removal of byproducts 6 and 7 was not considered because one of the desired products, monosubstituted triazoles of type 4, are also quite strong acids that form stable salts with bases, for example, triethylamine.
The reactions were carried out in 1,4-dioxane and byproducts 6 and 7 were washed off after the removal of the solvent with an aqueous solution of AcOH (the use of strong acids is risky because their excess destroys diazo compounds Scheme 3. Scattered Examples of Reactions of Enaminones 1 with Sulfonyl Azides 2 5). The easy removal of water-soluble byproducts 6 and 7 made it possible to obtain mixtures consisting predominantly of NH-triazoles 4 and diazo compounds 5, which allowed to analyze them without preparative separation using 1 H NMR spectroscopy. The separation of the mixtures in order to obtain the components was carried out using column chromatography. Doing this, to increase the difference in the chromato-graphic mobility of NH-triazoles 4 and diazo compounds 5, the phenomenon of a pronounced acidity of the former was used.
Thus, the addition of triethylamine to the eluent at the beginning of chromatography drastically reduced the R f of NH-  AcOH was added to the eluent to destroy the salt, after which NH-triazoles 4 were eluted.
However, in the majority of cases, the selective formation of one of the two types of reaction products does not take place (Scheme 4). We assumed that the addition of acetic acid to the reaction mixture would shift the reaction in favor of the formation of diazo compounds 5 due to the neutralization of the basic morpholine fragment from sulfonyl azide 2f. Though the expected effect was observed in a series of experiments (cf. entry 4, Scheme 4, with entry 5 and, especially, entry 6), the desired selectivity was not achieved. Thus, the introduction of the basic fragment into the sulfonamide component of sulfonyl azides 2 did not provide the synthesis of NH-triazoles 4 and diazo compounds 5 with satisfactory selectivity, which reduces the preparative attractiveness of the tested approach.
An approach alternative to the previous one is the use of enaminones of type 10 with the basic fragment of Nmethylpiperazine, which was introduced instead of the dimethylamino group by the transamination reaction of enaminones 1. The preferred method for the synthesis of enaminones 10 was found to be heating enaminones 1 in Nmethylpiperazine with letting the releasing dimethylamine to dissipate in the atmosphere (Scheme 5). The excess Nmethylpiperazine can be regenerated to a great extent by distilling it from the reaction mixture after the end of the reaction. Thus, a series of new enaminones bearing a fragment of N-methylpiperazine of the 1,2,3-triazole series 10a−p was obtained, as well as one representative of the 1,2,3-thiadiazole 10q and isoxazole 10r series (Scheme 5).
In the synthesis of the o-(10c) and p-nitro derivative (10d), significant amounts of o-and p-nitroaniline were detected in the reaction mixture, respectively, presumably due to the decomposition of compounds 10c and 10d under the action of N-methylpiperazine. As a result, p-nitro enaminone 10d was obtained with a yield of 82%, and the yield of o-isomer 10c was only 48% with a conversion rate of the starting compound 1c of 95%. Thus, the transamination reaction is of limited applicability for the preparation of compounds 10 that are labile to N-methylpiperazine.
The synthesized enaminones 10 have a higher melting point, are more stable during storage and are significantly more polar than initial dimethyl enaminones 1, which favor their purification by crystallization or column chromatography, and altogether make them more convenient in synthetic practice.
In syntheses using N-methylpiperazine enaminones 10 (Scheme 6), it was found possible to apply a method for isolating compounds 4 and 5, similar to the one used in the reaction of enaminones 1 with azide 2f (Scheme 4).
In order to increase the practical value of the method under development, we looked for the reaction conditions leading to the predominant formation of one of the two types of reaction products, NH-triazoles 4 or diazo compound 5. Initially, the influence of the nature of sulfonyl azide 2 on the ratio of reaction products when interacting with enaminone 10h (Scheme 7) was studied. The reactions were carried out in 1,4-dioxane at room temperature.
Based on the experimental results presented in Scheme 7, it can be concluded that the electronic properties of the substituent in sulfonyl azides 2 significantly affect the direction of the reaction. Thus, the use of azides 2a and 2g containing electron-donating substituents leads to a maximum yield of NH-triazole 4h (Scheme 7, entries 1 and 6), while sulfonyl azide 2e with an electron-acceptor group affords the maximum yield of diazo compound 5h (Scheme 7, entries 1, 6). For the further optimization of the method for obtaining NH-triazoles 4 from a pair of arylsulfonyl azides 2a and 2g, which showed similar results, tosyl azide (2a) was chosen as much more available. Thus the study of the effect of the solvent nature on the yield of NH-triazoles 4 (Scheme 8) was carried out using the reaction of enaminone 10h with tosyl azide (2a).
Data of Scheme 8 demonstrate that the best yields of NHtriazole 4h are provided by the use of pyridine as a solvent (entries 9−11). Further, the effect of the reaction temperature on the yield of the desired NH-triazole 4h was studied in this solvent (Scheme 8, entries 9−11). Notably, an increase in temperature to 75°C leads to a certain decrease in the yield of the desired product 4h (Scheme 8, entry 11), while it is practically invariable in the temperature range from room to 52°C .
It should be noted that in the studies presented in Schemes 7 and 8, an attempt to introduce into the reaction an excess of enaminone 10h or sulfonyl azide 2 always led only to that after the reactant taken in deficiency completely disappeared, the reactant taken in excess was detected in the reaction mixture Thus, the optimal conditions for the synthesis of NHtriazoles 4 from enaminones 10 were recognized as the use of tosyl azide (2a) as a sulfonyl azide and pyridine as a solvent at a temperature of 25−52°C with an equimolar ratio of reactants.
Having optimal conditions in hands, we synthesized a series of compounds of the 1,2,3-triazole series 4a−p, as well as one representative of the 1,2,3-thiadiazole 4q and isoxazole 4r series; the results are given in Scheme 9.
As already noted, the highest yield of diazo compound 5h in the experiments presented in Scheme 7 is achieved using 4nitrophenylsulfonyl azide (2e). In this respect, the optimization of the method for obtaining diazo compounds 5 was carried out using reagent 2e. Thus, the effect of the solvent on the yield of reaction products was studied by analyzing a series of experiments, where it changed in the reaction of sulfonyl azide 2e with enaminone 10q. It was found that in this series (entries 1−5, Scheme 10), the use of ethanol leads to the maximum yield of diazo compound 5q. Later, we locked ethanol as a solvent and investigated the effect of the reaction temperature on the yield of diazo compound 5q (entries 5−7, Scheme 10). It was revealed that the yield of diazo compound 5q decreased in the row rt > 4°C ≈ 70°C.
Similar to the optimization of the synthesis of NH-triazoles 4 (Schemes 9 and 10), the use of an excess of enaminone 10q or sulfonyl azide 2e in the reaction led merely to their overspending. As a result, the best conditions for the synthesis of diazo compounds 5 from enaminones 10 were formulated as follows: 4-nitrophenylsulfonyl azide (2e) as a sulfonyl azide, ethanol, or methanol as a solvent, the reaction temperature was room temperature (in the case of enaminone 10o, the reaction was carried out at an elevated temperature because of its low solubility) with an equimolar ratio of reactants. In these optimal conditions, a series of diazocarbonyl compounds of 1,2,3-triazole 5a−p series were synthesized, as well as one representative of 1,2,3-thiadiazole 5q and isoxazole 5r series, which are given in Scheme 11.
NH-1,2,3-Triazoles 4 are solid colorless substances with a relatively high melting point. Their structure correlates with the data of 1 H and 13 C NMR spectroscopy, which was also confirmed by ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS) detection, and X-ray analysis was performed for a single crystal of compound 4f (Figure 1). Diazo compounds 5 are solid light yellow substances with a relatively low melting point (n-butyl derivative 5f could not be crystallized). Their structure correlates with the data of 1 H and 13 C NMR spectroscopy and UHPLC-HRMS. In the 1 H NMR spectra of diazo compounds 5, broad singlets of protons from the diazoketone fragment C(O)CHN 2 are observed at 5.04−6.76 ppm. The IR spectra of compounds 5 display the characteristic bands of the valence vibrations of the diazo group in the range of 2096−2115 cm −1 . Additionally, the structure of diazo compounds 5 is confirmed by the X-ray analysis performed for a single crystal of compound 5c ( Figure  2).
Previously, the synthesis of one of the diazo compounds, 2diazo-1-(5-methyl-1,2,3-thiadiazol-4-yl)methyl-1-one (5q), by an alternative method is described in ref 42. However, except for the melting point, no analytical characteristics are published in the article, 42 which makes it unsuitable for confirmation of the structure of compound 5q synthesized by us.
The plausible mechanism for the formation of compounds 5−7 is depicted in Scheme 12. We assumed that triazoline 3 generated by the cycloaddition reaction of enaminone 1 with sulfonyl azide 2 is a common intermediate on pathways A and B, leading to the desired products 4 and 5. Theoretically, as indicated in Scheme 12, pathway A includes the elimination of sulfonyl amide 6 and formation of triazoline 11, which is followed by the 1,3-H shift to result in final NH-triazole 5 (A1) Scheme 10. Reaction of Enaminone 10q with Azide 2e in Different Solvents Scheme 11. Syntheses of Diazo Compounds 5a−r in Optimized Conditions ACS Omega http://pubs.acs.org/journal/acsodf Article or via the elimination of dialkyl amine and generation of 1arylsulfonyl-1,2,3-triazole 12. The latter reacts with dialkyl amine to form triazole 5 accompanied by sulfonamide 4 (A2). We also assume that the formation of the second desired compound, diazo compound 5, occurs via the cycloreversion of triazoline 3. 21 Therefore, we have performed computational simulations to gain close insights into the nature of such an important transformation using density functional theory (DFT) approaches (see below). Computational Investigations. We next performed DFT simulations to gain further insights into the reaction mechanism. The calculations were performed at the (SMD)-M06-2X/6-311+G(d,p)//M06-2X/6-31G(d) level of theory (see the Computational Methods). Overall, we have calculated the mechanistic pathway for the formation of the desired product 4a and its isomers in 1,4-dioxane ( Figure 3). Despite the cycloaddition of enaminone 1a with sulfonyl azide 2a being approximately thermoneutral to give intermediate Int4a, the latter is kinetically favored over by a substantial barrier difference of more than 16 kcal/mol via TS3a although the other isomeric intermediate Int4a r is slightly thermodynamically exergonic (ΔG = −2.1 kcal/mol). This sharp preferability for the formation of intermediate Int4a is reasoned to factors indicated below.
This regiomeric preferability of the formation of intermediate Int4a has shown to be important to rationalize the  considerable difference between TSs leading to different regioisomers. Generally, the 1,3-dipolar cycloaddition is a concerted and pericyclic process as an asynchronous addition, where both electronic 47,48 and steric factors 49,50 significantly contributed to the regioselectivity. The electronic effect is a dominant combination of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), and this is clearly governed by the largest coefficients of HOMO and LUMO of the reacting atoms.
Although the steric effect is well known to compete with the electronic effects through favoring the opposite regioisomer exclusively, we have noticed a remarkably different behavior here that would lead to a considerable energy difference between TS3a and TS3a r . Our simulations have revealed three important observations that make TS3a considerably more favorable than TS3a r (Figure 3a). First, the non-covalent interaction (NCI) calculations disclose a strong bonding interaction between methyl groups of enaminone and oxygen atoms of sulfonyl azide, as shown by the absolute values of charges for atom H20 with O30 and atom H23 with O31 ( Figure 4a). Second, and in this regard, the NCI results manifest an electrostatic interaction between the hydrogen atom H5 of the triazole ring and the nitrogen atom N28 of the azide moiety. These two types of interactions are completely absent in the other TS, TS3a r . Third, the high asynchronous attack via (3 + 2) cycloaddition is clearly evidenced (Figure 4b). The intrinsic reaction coordinate (IRC) calculations showed that while the first N−C bond forms at 1.56 Å, the other N−C bond is still away in the transition state zone at 2.37 Å, consequently resulting in a high asynchronous (3 + 2) cycloaddition due to a 0.81 Å bond difference between the first and second N−C bond formation (Figure 4b, top). However, the other regiomeric (3 + 2) addition through TS3a r is calculated to be totally concerted, as indicated by the IRC calculations, by which the formation of both N−C bonds reaches 1.56 Å simultaneously (Figure 4b, bottom). Despite the fact that the 1,3-dipolar cycloaddition reaction was previously investigated using DFT simulations, 51−53 our novel (3 + 2) cycloaddition seems to be in need for further attention. The effect of substituents on the 1,3-dipolar cycloaddition is investigated and shown in Figure 4c. In this regard, in comparison with our model investigated here, R 1 and R 2 = Me ( Figure 4c, entry 1), replacing the methyl group with a phenyl group results in the formation of a five-membered ring with a slightly less barrier of ΔΔG ⧧ = 0.4 kcal/mol, making it slightly more exergonic (ΔΔG r = −1.3 kcal/mol) ( Figure 4c, entry 2). While R 2 remains a Ph group and R 1 is substituted with methyl at the para position, the calculated barrier increases to 25.5 kcal/mol but becomes more exergonic (ΔG r = −2.9 kcal/mol) (Figure 4c, entry 3). In contrast, the barrier dropped to 24.8 kcal/mol when a para-methyl group was replaced with a para-nitrophenyl group (Figure 4c, entry 4). Interestingly, in the presence of an electron-withdrawing group like NO 2 in sulfonyl azide (R 2 ), when R 1 = Ph, the fivemembered ring formation proceeded with a reduced barrier of 23.3 kcal/mol (Figure 4c, entry 6) compared to our model substrate used in this investigation ( Figure 4c, entry 1, R 1 , R 2 = Me). Overall, from these results, the rate of cycloaddition seems to be more likely higher when the electron-deficient group is involved in the azide moiety.
To complete the pathway of NH-1,2,3-triazoles 8a formation, we assumed that favorable intermediate Inta4 would undergo two separate pathways to release three different molecules (8, Int11, and 13), as shown in Figure 3. On the one hand, intermediate Int4a affords the elimination of sulfonyl amide 9 and triazoline Int11, which follows by either 1,3-H shift to form NH-triazole 13 or 1,2-H shift to form our desired product 8. However, this pathway was shown to be kinetically disallowed due to the substantial barrier of 37.8 kcal/mol that is needed to be overcome via TS10 as a thermoneutral step (ΔG = −0.8 kcal/mol) to give intermediate Int11.
Concurrently, in addition to the unfavorable sulfonyl amide 9 elimination, the intermediate Int11 will subsequently undergo either a high activation barrier of 25.7 kcal/mol via 1,2-H shift TS12 to furnish compound 13 or through a forbidden pathway 1,3-H shift TS12 with a barrier of 59.3 kcal/mol to release compound 8. Therefore, regarding the mild conditions employed in our methodology, both shifts were totally ruled out.
On the other hand, the elimination of dimethyl amine and generation of 1-methylsulfonyl-1,2,3-triazole intermediate Int6a seems to be a more sensible pathway although it still required a higher barrier of 29.7 kcal/mol through the fourmembered transition-state TS5a as a highly exergonic step of 16.8 kcal/mol to form intermediate Int6a (Figure 3). If latter intermediate Int6a is formed, it reacts with dimethylamine to form triazole 8a accompanied with sulfonyl amide 9 with a calculated exergonicity of 17.0 kcal/mol. Therefore, to overcome this high calculated barrier via TS5a and allow a smooth dimethyl amine elimination, a water molecule is needed to form a six-membered TS. Indeed, the addition of a water molecule as a catalyst lowers the barrier of HNMe 2 elimination to 23.8 kcal/mol within a relaxed TS TS5a H 2 O rather than 29.7 kcal/mol with a strained TS within TS5a. Therefore, according to the calculations performed for the model reaction of 1-(1,5-dimethyl-1H-1,2,3-triazol-4-yl)-3-(dimethylamino)prop-2-en-1-one (1a) with mesyl azide (2a), the dialkyl amine elimination is more preferable by more than 13 kcal/mol than sulfonyl amide elimination.
Further DFT simulations were performed to understand the formation of diazo compound 5a under these reaction conditions. We assume that the formation of the second desired compound occurs via the (3 + 2) cycloreversion of triazoline intermediate Int4a. Figure 5 shows an energetic comparison between the elimination and cycloreversion of intermediate Int4a calculated in 1,4-dioxane. The results presented in Figure 5 indicate that the elimination pathway is more favored than cycloreversion. Here, the cycloreversion needs a further higher barrier of 28.6 kcal/mol to release diazo compound 5a as a less exergonic pathway of 7.6 kcal/mol. Based on the experimental observations, we have seen that switching solvents from 1,4-dioxane to ethanol tunes the reaction direction in favor of diazo compound 5a formation. Accordingly, we have performed our simulations in ethanol to reveal the impact of the solvent on the pathway preferability. Table 1 presents the effect of the solvent (1,4-dioxane and ethanol) and substituents on computed free energy of activation for the elimination and cycloreversion pathways from triazoline intermediate Int4. Overall, the results point out that elimination is more likely to be favored in 1,4-dioxane despite the variety of substituents, whereas the cycloreversion comparatively becomes clearly favorable than elimination in ethanol. More importantly, our calculations reveal that 1,4dioxane lowers the barrier needed for the elimination process, whereas ethanol increases the barrier dramatically. Conversely, 1,4-dioxane increases the barrier of cycloreversion largely, whereas ethanol decreases the barrier of cycloreversion substantially. According to our calculations, the barrier for the cycloreversion of intermediate Int4 is substantially lower in ethanol for the azide moiety with electron-withdrawing R 2 = 4-O 2 N (Table 1, entry 6). Finally, these results indicate that the solvent is paramount in terms of directing the pathway of intermediate Int4 toward cycloreversion or elimination.

■ CONCLUSIONS
In conclusion, the interaction of azolyl enaminones 1 with sulfonyl azides 2 was studied. Examples of the concurrent formation of two types of products, NH-1,2,3-triazoles 4 and azolyl diazoketones 5, were discovered. The introduction of the basic fragment into the molecule of one of the reactants made it possible to significantly simplify the technology of isolating the desired products. By varying the sulfonyl azide, solvent, and reaction temperatures, factors favoring the implementation of each of the alternative pathways were found, and convenient laboratory methods for the synthesis of several hitherto unavailable azolyl-4-carbonyl-NH-1,2,3-triazoles 4 as well as azolyl diazoketones 5 were developed. DFT calculations were utilized to reveal the mechanism of the formation of NH-triazoles from enaminone 1a and azide 2a.
The DFT results have shown that the 1,3-dipolar cycloaddition is totally driven toward one single regioisomer with a high asynchronous bond formation with non-covalent interactions being considerably effective, by which the existence of an electron-deficient group on sulfonyl azides induces faster cycloaddition. Following the formation of the cycloadduct intermediate, namely, 4H-1,2,3-triazole, the reaction was found to undergo smoothly within the six-membered TS elimination of dialkyl amine, facilitated by a water molecule, which undergoes an N−S bond cleavage to release the desired product 4a. In this regard, our calculations revealed that 1,4dioxane makes the elimination process more favorable, whereas cycloreversion becomes the preferred pathway to form diazo compound 5a when ethanol is used. Finally, this work opens an avenue to the selective convenient preparation (including scale-up production) of diverse NH-1,2,3-triazoles and carbonyldiazomethanes, valuable as building blocks and for the other applications. 13 C, and 19 F NMR spectra were recorded at 400 and 600 MHz ( 1 H), 101 and 151 MHz ( 13 C), and 376 MHz ( 19 F) in DMSO-d 6 a or DCCl 3 solutions. Unless specifically mentioned, 13 C NMR spectra were recorded with proton decoupling (BB); spectra of diazo compounds 5n,o were obtained in the J-mode (attached proton test, APT). For NMR spectra on 1 H and 13 C nuclei, the residual signals DMSO-d 5 and HCCl 3 were used as an internal reference. In the 19 F NMR spectra, the chemical shifts of the signals are given relative to FCCl 3 . The analysis of compounds by UHPLC-HRMS was performed using a tandem quadrupole time-of-flight accurate mass detector, chromatographic separation was achieved using an ultra-high-performance liquid    (E)-3-(4-Methylpiperazin-1-yl)-1-(1-phenyl-1H-1,2,3-triazol-4-yl)prop-2-en-1-one (10a). Compound 10a was obtained in 80% (714 mg) yield according to the general procedure (enaminone 1a: 727 mg, 5 h). The crude product was purified with column chromatography: applied as a solution in EtOAc, eluted successively with EtOAc and EtOAc/EtOH (10:1), and finally with EtOAc/EtOH (5:1). The last portions of the eluate, containing enaminone 10a, were combined, evaporated to dryness, and the residue was crystallized from PhH/PE. Light yellow powder, mp 143−146°C. 1      The structure of compound 10g favors the trapping of residual solvents, which are difficult to get rid off. For the spectral confirmation of the structure, picrate adduct 10ga was prepared.

ACS
2-Diazo-1-(1-phenyl-1H-1,2,3-triazol-4-yl)ethan-1-one (5a). Compound 5a was obtained in 85% (181 mg) yield together with compound 4a in 9% (22 mg 1-Methyl-4-((4-nitrophenyl)sulfonyl)piperazine (6b). Aqueous acetic acid washes were combined and evaporated to dryness. The residue was treated with aqueous NH 3 , washed with water, and dried. A crude product was purified with column chromatography: applied as a solution in DCM and eluted successively with DCM and DCM/EtOAc (1:1).  2-Diazo-1-(1,5-dimethyl-1H-1,2,3-triazol-4-yl)ethan-1one (5e). Compound 5e was obtained in 89% (147 mg) yield together with compound 4e in 6% yield according to the modified general procedure (enaminone 10e: 249 mg, 3 h). Liquid phases after the removal of the main amount of amidine 7b were combined and evaporated to dryness. According to the 1 H NMR spectrum, the mixture contained NH-triazole 4e in 5% (6 mg) yield. To isolate diazo compound 5e, the mixture was separated with column chromatography: applied as a solution in DCM and eluted successively with DCM and DCM/EtOAc (1:1). Portions, containing diazo compound 5e, were combined, evaporated to dryness, and the residue was crystallized from PE to afford pure diazo compound 5e. X-ray Structural Analysis. X-ray structural analysis of compounds 4f and 5c was performed on an Xcalibur 3 singlecrystal diffractometer according to the standard method (Mo Kα radiation, graphite monochromator, 295(2) K, ω-scanning with a step of 1°). The structures were solved and refined using the SHELXTL software suite. 58 The structures were solved by the direct method with the ShelXS program, the structures were refined with the ShelXL program using a full-matrix method of least squares by F 2 in anisotropic approximation for nonhydrogen atoms. The hydrogen atoms were placed in the calculated positions and included in the refinement according to the riding model. The complete X-ray structural data sets for compounds 4f and 5c were deposited at the Cambridge Crystallographic Data Center (deposition numbers CCDC 2091552 and CCDC 2091548, respectively).

■ COMPUTATIONAL METHODS
All mechanical quantum calculations were performed using Gaussian 09. 59 The geometries were fully optimized at the hybrid meta-generalized gradient approximation M06-2X/6-31G(d). 60,61 All minima intermediates were verified by the absence of negative eigenvalues in the vibrational frequency analysis. Transition-state structures were found using the Berny algorithm 62,63 and verified by vibrational analysis. The transition states were visualized by animating the negative eigenvector coordinate. Single-point energies of the optimized geometries were evaluated using the larger basis set M06-2X/ 6-311+G(d,p) 64−67 in the presence of a solvation model based on the density (SMD) solvation model using 1,4-dioxane or ethanol as a representative solvent medium. 68 The thermal corrections evaluated from the unscaled vibrational frequencies at the M06-2X/6-31G(d) level of theory were then added to the M06-2X/6-311+G(d,p) electronic energies to obtain the free energies. In order to determine the minimum energy path on the potential energy surface, IRC calculations, by defining the phase for the transition vector motion along the path, were performed for the identified transition states using the Hessianbased predictor-corrector integrator to confirm the reaction path proceeding in both directions (reactant and product), in which the Hessian was recomputed every three predictor steps with a step size along a reaction path of 0.05 bohr. 69−71 All energies reported in this paper are Gibbs free energies at 298.15 K using unscaled frequencies. All activation free energies are quoted relative to infinitely separated reagents. Optimized structures are illustrated using CYLview. 72 ■ ASSOCIATED CONTENT
X-ray crystallographic data for compound 4f (CIF) X-ray crystallographic data for compound 5c (CIF) 1 H and 13 C NMR spectra for all new compounds, X-ray diffraction study of 4f and 5c, and calculation details (PDF) To record 13 C NMR spectra of NH-triazoles 4, in most cases, the addition of Cs 2 CO 3 was used to cause the deprotonation of NH and thus suppress prototropy.


Preparation of Polyvinyl Imine Modified Carbon Quantum Dots and Their Application in Methotrexate Detection

Objective: A sensitive and selective fluorescence-detection platform based on carbon quantum dots (CQDs) was designed and developed for the determination of methotrexate (MTX), for the purpose of minimizing the possible toxic threat of MTX in clinics. Methods: The approach was prepared for the first time by a simple, hydrothermal method, making the synthesis and modification processes realized in one step using polyethyleneimine (PEI), and the proposed PEI-CQDs were obtained with high fluorescence quantum yield (38%). Results: MTX was found highly responsive and effective in quenching the fluorescence of the PEI-CQDs, due to a suggested fluorescence resonance energy transfer mechanism or inner-filter effect. The linear range of MTX was between 1 and 600 μmol/L under optimum conditions, with a detection limit (LOD) as low as 0.33 μmol/L. Furthermore, the fluorescent method was established for the MTX assay, and satisfactory results were acquired in real-sample determination. The average labeled quantity was 98.2%, and the average added standard recovery was 100.9%. Conclusions: The proposed PEI-CQDs showed a remarkable potential for broad applications in biological molecule determination and environmental analysis.

Introduction
Currently, methotrexate (MTX) is a commonly used anti-folic acid and anti-tumor drug, mainly applicable to leukemia meningeal spinal infiltration, acute leukemia, head and neck tumors, bone tumors, breast cancer, liver cancer, lung cancer, and other diseases [1,2]. MTX has been identified as a folate antagonist capable of inhibiting dihydrofolate reductase, lowering DNA synthesis, and reducing cell proliferation and mitosis [3,4]. As an antineoplastic or cytotoxic agent, MTX also targets normal cells and tissues, which may result in alopecia, suppression of the bone marrow, and potentially fatal cirrhosis and pneumonitis [5][6][7]. Due to the increasing use and range of methotrexate drugs, monitoring the MTX level in biological samples is important for studies of efficacy, dosing schedules, different individual responses related to genetic polymorphism, and adverse drug reactions. Moreover, MTX contamination has been discovered in hospitals and sewage-treatment-plant effluent [8]. The presence of chemotherapeutic agents in the environment causes a possible detrimental impact on human health and ecological safety. As a result, contamination with chemotherapeutic agents should be monitored in both biological and environmental samples. Therefore, it is urgent to establish a rapid detection method for MTX.
Carbon quantum dots (CQDs) are a new class of carbon nanomaterials. They are quasispherical carbon nanoparticles, with a core-shell structure and a size of less than 10 nm. In conclusion, we propose a novel fluorescence probe based on PEI-CQDs for the fast and selective determination of MTX. The obtained fluorescence probes were prepared by a simple, hydrothermal method, making the synthesis and modification processes realized in one step. Moreover, the fluorescence quantum yield (QY) of the PEI-CQDs was 38%, and the PEI-CDs were used as the fluorescent probes for the determination of MTX in the phosphate buffer solution (PBS) and even in human serum, exhibiting not only good sensitivity but also satisfactory practicability.

Instrumentation
Transmission electron microscopy (TEM) (JEOL, JEM-2100) with a 200 kV operating voltage was used to characterize the microstructure and the sizes of the PEI-CQDs. Fouriertransform infrared (FTIR) spectra of pressed KBr pellets were recorded using an AVATAR-370 spectrometer (Nicolet, WI, USA) over the spectral range 400-4000 cm −1 . The surface and functional properties of PEI-CQDs were characterized using an X-ray diffractometer (XRD) equipped with a D8 advance (Bruker, Karlsruhe, Germany). The photo-luminescence (PL) spectra of the samples were recorded on a UV-2501PC spectrophotometer (Shimadzu, Kyoto, Japan) and an F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan), respectively.

Synthesis of the PEI-CQDs
PEI-CQDs were synthesized by a hydrothermal technique that utilized glycol as a source of carbon and PEI as a passivating agent for the surface. This approach was previously described in another study, with slight modifications [27]. First, 10 mL of glycol and 2 mL of ultra-pure water were mixed with an ultrasound to form a clear solution. Then, 0.54 g of PEI and 3.5 mL of 85% phosphoric acid were added to the mixed solution, and the solution was magnetically stirred at room temperature for 30 min. Finally, the mixture was added to a high-pressure, nitrifying pot (30 mL) and heated at 180 • C in a constant-temperature drying oven for 24 h to form PEI-CDs. The obtained brown liquid was centrifuged for 15 min at 5000× g rpm to remove large carbon particles and unreacted substances, and the upper solution was filtered with a 0.22 µm filter membrane, with the constant volume at 50 mL, and then stored away from light for later use.

Determination of Quantum Yield
The fluorescence quantum yield (QY) of PEI-CQDs can be determined and calculated by the following equation [28].
where quinine sulfate is used in the present work, and QY R = 54%. I and I R are the measured, integrated emission intensity of PEI-CQDs and quinine sulfate, respectively. A and A R represent the absorbance of PEI-CQDs and quinine sulfate, which are measured on a UV-Vis spectrophotometer. The quinine sulfate was dissolved in 0.1 M of H 2 SO 4 (n = 1.428), and the PEI-CQDs were dissolved in distilled water (n = 1.333).

Pretreatment and Detection of MTX Tablets
A total of 10 MTX tablets were weighed and fully ground. Then, the powder was extracted with 0.1 mol/L of NaOH solution for 30 min in an ultrasonic bath until sufficiently dissolved, transferred to a clean, 100 mL volumetric flask for constant volume, and mixed well. The solution was diluted to the exact volume with distilled water, and then filtered by a 0.22 µm filter membrane. At the end, the sample solution was 1 mg/mL, and it was stored in the dark and was used as the real sample's solution.
The following procedure was used to determine the presence of MTX in real samples: 250 µL of PEI-CQDs solution was poured into a series of colorimetric tubes (10 mL), and then 0.01 mol/L of MTX solution in different volumes was added to each colorimetric tube in turn, followed by the phosphate buffer solution (PBS) (pH 7.0) as the calibration line. After 3 min, the fluorescence intensity was detected at an excitation wavelength of 350 nm.

Structural Analysis of PEI-CQDs
During the synthesis of PEI-CQDs, the amine group in dendritic PEI can react with the hydroxyl group in glycol to form a hydrogen bond and coat the carbon point. The carbon elements on the surface of the fluorescent carbon spots form carboxyl groups under the oxidation of concentrated phosphoric acid, making them hydrophilic and providing active groups for subsequent biological applications to improve luminescence performance. The QY of PEI-CQDs was calculated using the method described, and the result was 38%, according to Section 2.4. The TEM images in Figure 2A clearly show the resultant PEI-CQDs, which have a well-defined, hyperbranched structure and a uniform particle-size distribution, with a diameter ranging between 3 and 7 nm and a polydispersity index (PDI) of 0.383, as shown in Figure 2B. CQDs have been widely reported to generate photoluminescence as a result of their band-gap transition and electron-hole recombination caused by their surface defects [29]. XRD was used to investigate the crystallinity and crystal structure of PEI-CQDs, and the resulting XRD pattern is shown in Figure 2C. The diffraction peak of amorphous carbon was observed at 2θ = 22.52 • , which was consistent with the (100) lattice spacing of the carbon-based materials, and the crystal structure of PEI-CQDs was determined to be amorphous carbon [30].

Optical Properties
UV-vis absorption and photoluminescence-emission spectroscopy were used to characterize the optical properties of PEI-CQDs. As illustrated in Figure 4, the UV-vis absorption spectrum (a) of PEI-CQDs exhibits a characteristic peak at 320 nm corresponding to the C=O n−π * transition, while PEI have nearly no absorption above 240 nm, which was a difference between PEI-CQDs and PEI [26]. Meanwhile, a strong emission peak at 460 nm (c) appeared upon excitation at 350 nm (b), which may be the result of the surface states trapping excited-state energy. All in all, the wavelengths of excitation and emission for the following experiments were set at 350 nm and 460 nm, respectively.

Analytical Performance of the PEI-CQDs for MTX
Several related factors, such as the pH and the volume of the PEI-CQDs solution, were investigated. As illustrated in Figure 5A, the fluorescence quenching of PEI-CQDs was pH-dependent and relatively low when 50 µL of 0.01 mol/L MTX was used in 10 mL of PBS under neutral and alkaline conditions. In comparison, the quenching of fluorescence by PEI-CQDs remained stable in neutral solutions with a pH of 7.0. Therefore, MTX was measured in a PBS solution with a pH of 7.0 because it is well-studied in vivo. As we know, MTX can quench the fluorescence of PEI-CQDs. In this work, the fluorescence intensity of PEI-CQDs without MTX was denoted as F 0 , and F was the fluorescence intensity when MTX was added. In Figure 5B, the fluorescence intensity was lower in the presence of 50 µM MTX than in the absence of it, regardless of the amount of PEI-CQDs added. The quenching effect was the most significant when the volume of PEI-CQDs was 250 µL because the maximum ∆F (∆F = F 0 − F) was obtained. To assess the analytical performance, MTX samples at various concentrations were mixed with PEI-CQDs under optimal conditions. The fluorescence quenching of PEI-CQDs toward MTX appeared at concentrations ranging from 1 to 600 µmol/L, as shown in Figure 6. This result is consistent with the equation log (F 0 /F) = 0.00284 C MTX + 0.04575, which has a correlation coefficient of R 2 = 0.9976. At a signal/noise ratio of 3, the limit of detection (LOD) is 0.33 µmol/L and the limit of quantitation (LOQ) is 1.0 µmol/L. This fluorescent method, as developed, can successfully be used to monitor MTX in real time. The presented method can provide a wide linear range with other previous methods (shown in Table 1).

The Reaction Mechanism of PEI-CQDs with MTX
MTX greatly quenches the fluorescence intensity of the PEI-CDs with the increase in the concentration of MTX, as shown in Figure 6A. Additionally, the mechanism by which the PEI-CQDs quench fluorescence was investigated. According to previous research, the primary quenching mechanisms for quenching fluorescence carbon dots are dynamic quenching (DQ), static quenching (SQ), photoinduced electron transfer (PET), inner-filter effect (IFE), and fluorescence resonance energy transfer (FRET) [37,38]. Carbon quantum dots are excellent electron acceptors and donors [39]. Because there are aromatic ring functional groups in the structure of methotrexate, and PEI-CQDs show that there are a large number of -NH-and -NH 2 functional groups, the reason for fluorescence quenching is due to PET, the electron transfer process between photoexcited PEI-CQDs, and the aromatic functional groups of MTX.

Selectivity of the PEI-CQDs for MTX Detection
The complexity of biological and environmental samples makes detecting chemotherapeutic drugs extremely difficult. Thus, experiments on selectivity were conducted in this work. The fluorescence intensities of PEI-CQDs were determined in the presence of a series of drugs and a group of potentially interfering counterparts. Some chemicals were added to the fluorescence-intensity-detection system (50 µL of 0.01 mol/L MTX), and the results are shown in Table 2. Most chemical compounds can coexist at higher concentrations with a relative error of less than 10%.

MTX Detection in Real Samples
The developed method's applicability was evaluated using MTX tablets purchased from a local drug store. The standard addition method was performed in human serum provided by different healthy volunteers from the infirmary of Shanghai University. The 100 µL human serum was diluted 100 times with PBS (pH 7.0), and then 100 µL supernatant containing MTX (1 mg/mL) was added. The average labeled quantity was 98.2%, with a relative standard deviation (RSD) of 4.4% (Table 3). Furthermore, the recoveries obtained from the samples spiked with standard MTX solutions range from 99.7% to 101.0%. Each sample had an RSD of 0.9%, indicating good reproducibility and precision ( Table 4). As a result, the proposed method is found to be feasible for MTX detection in drug samples.

Conclusions
In conclusion, PEI as a surface passivation agent that modified CQDs was successfully prepared using a one-step hydrothermal method with excellent water solubility and higher quantum yield. The conditions were optimized. The fluorescence quenching of PEI-CQDs by MTX was caused primarily by an inner-filter effect based on the agglomeration and electron transfer of PEI-CQDs. The as-prepared PEI-CQDs demonstrated excellent MTXdetection performance, with linear dynamic ranges ranging from 1 to 600 µmol/L and a detection limit of 0.33 µmol/L. Furthermore, the MTX spiked in drug samples monitored by the method recovered well. We anticipate that this approach can be used for developing more sensitive sensing methods in drug analysis and environmental monitoring.
Author Contributions: Conceptualization, X.S.; methodology, X.S. and H.Z.; investigation, X.W. and M.H.; data analysis, X.S.; writing-original draft preparation, X.S. and H.Z. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (32102007).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: This study does not contain experiments that involve human or animals, other than the authors who performed the work.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to school rules.

Conflicts of Interest:
The authors declare no conflict of interest.