¹⁵N-Labelling and structure determination of adamantylated azolo-azines in solution

Introduction

The incorporation of an adamantyl moiety in bioactive molecules and analogues of natural compounds is a widely used approach in medicinal chemistry [1]. The increased lipophilicity of adamantane-containing compounds compared with non-adamantylated derivatives [2] leads to considerably higher solubility of these compounds in blood plasma and their easier penetration through cell membranes. The conjugation of adamantane with heterocyclic compounds also provides a method to modify the pharmacological profile and frequently leads to a new type of bioactivity. For example, N-adamantyl tetrazoles 1 and 2 (Figure 1A) demonstrate lower toxicity and, simultaneously, more potent activity against influenza A virus compared with the currently used antiviral drug rimantadine (1-(1-adamantyl)ethanamine) [3]. More recently, Roberge et al. described new inhibitors of the influenza A virus M2 proton channel. Among the studied compounds, adamantyl imidazole 3 showed good activity [4].

An azolo-azine core with a bridgehead nitrogen atom is found in many natural products [5,6] and biologically active synthetic compounds [7,8]. The purine-like scaffold of these nitrogen-containing heterocycles is frequently used in medicinal chemistry and drug design. For example, 6-nitro-1,2,4-triazolo[5,1-c][1,2,4]triazine 4 (Figure 1A, Triazavirin^®) was approved in Russia for the treatment of influenza [9]. This drug targets the viral protein haemagglutinin. The incorporation of an adamantyl moiety in azolo-azine structures could lead to the development of new multifunctional antiviral drugs.

Previously, we synthesized N-adamatylated derivatives of 1,2,4-triazolo[5,1-c][1,2,4]triazines 5 and 6 by reaction with the adamantyl cation generated from 1-adamantanol in acidic medium [10]. The azolo-azine scaffold of these compounds has several nitrogen atoms that can react with alkylation reagents [11,12] (Figure 1B). For this reason, the adamantylation of compounds 5 and 6 led to mixtures of N3- and N4-adamantylated isomers, which reisomerized into each other likely via the formation of an adamantyl cation and starting NH-heterocycle. The unambiguous determination of N-adamantylation site(s) in heterocycles 5 and 6 using well-established ¹H and ¹³C NMR methods (such as 1D, 2D COSY, HMQC, HMBC, and INADEQUATE spectra) was difficult because the heterocyclic moiety was covalently attached to the adamantane tertiary carbon that had no bound hydrogen atoms. Nuclear Overhauser effect spectroscopy (NOESY or ROESY) also did not provide unequivocal structures of the N-adamantylated derivatives [13,14]. For example, the attachment of an adamantyl group to the N1 or N3 atom in the azole ring of compounds 5 and 6 could not be distinguished by NOE data. Similar problems with the unambiguous determination of the product structure were also found for N-arylation or N-alkylation with tert-butyl fragments in the series of 1,2,3-triazole [15,16], tetrazole [17-20], and purine [21] derivatives. Meanwhile, knowledge of the accurate chemical structures of N-substituted heterocycles is essential for biomedical studies and computer-assisted drug design, e.g., molecular docking techniques. Thus, the development of effective methods for the unambiguous determination of N-alkylation site(s) in the azolo-azine series is important.

The data that are required to solve this problem could be provided by ¹⁵N NMR spectroscopy. For monocyclic derivatives of azoles, the structures of N-alkylated regioisomers can be determined using 2D H-(C)-N multiple bond correlation (HCNMBC) experiments [22,23] using natural isotopic abundance. These experiments rely on the magnetization transfer through ¹³C-¹⁵N J-coupling constants (J_CN). However, the fusion of the azine ring to an azolo fragment increases the number of possible alkylation sites and considerably complicates the analysis of the J_CN patterns. This issue, together with the inherently low sensitivity of natural abundance ¹⁵N NMR spectroscopy, does not always permit the unambiguous positioning of alkyl (N-adamantyl, N-tert-butyl or N-aryl) fragments in azolo-azines.

The incorporation of ¹⁵N labels in nitrogen-containing heterocycles greatly facilitates the use of NMR spectroscopy for studies of molecular structures and mechanisms of chemical transformations [10,24-29]. The labelling enhances the sensitivity of detection and permits the quantitative measurements of J_CN and ¹H-¹⁵N J-coupling constants (J_HN) even in a mixture of tautomeric forms [24,25]. Additionally, a method based on amplitude-modulated spin-echo experiments was found to be the most efficient way to measure J_HN couplings [24]. Previously, the incorporation of a single ¹⁵N label in position 1 of the 1,2,4-triazole fragment of compounds 5 and 6 and analysis of the J_CN couplings permitted the unambiguous identification of the structures of the N3-adamantylated derivatives (Figure 1B), while the structures of the N4-adamantylated products were determined by ¹³C NMR spectroscopy via comparison with model compounds, N-methylated azolo-azines [10]. However, this preliminarily study did not evaluate the potential of the incorporation of several ¹⁵N-labels and simultaneous analysis of the J_CN and J_HN coupling constants for the determination of the N-adamantylation site(s) in heterocycles.

Herein, we report the selective incorporation of two ¹⁵N-labelled atoms in tetrazolo[1,5-b][1,2,4]triazin-7-one, 1,2,4-triazolo[5,1-c][1,2,4]triazin-7-one, and 1,2,4-triazolo[1,5-a]pyrimidin-7-one and the N-adamantylation of the obtained compounds. The combined analysis of the J_CN and J_HN couplings permitted the straightforward determination of the adamantylation sites in these azolo-azines, even when a mixture of regioisomers is formed.

Results

Synthesis. Derivatives of 1,2,4-triazolo[1,5-a]pyrimidine [30], 1,2,4-triazolo[5,1-c][1,2,4]triazinone [31] and tetrazolo[1,5-b][1,2,4]triazinone [32] can be obtained by the fusion of an azine ring to an azole fragment. This method can be used for the selective incorporation of ¹⁵N atoms in different azolo-azines. Recently, we tested this approach for the syntheses of ¹⁵N-labelled tetrazolo[1,5-b][1,2,4]triazines and tetrazolo[1,5-a]pyrimidines [25] starting from ¹⁵N-labelled 5-aminotetrazole. However, due to proton tautomerism, the use of single-labelled [2-¹⁵N]-5-aminotetrazole led to the formation of isotopomer mixtures, which complicated the subsequent NMR analysis. Meanwhile, the application of [2,3-¹⁵N₂]-5-aminotetrazole 7-¹⁵N₂ provided the single double-labelled products in the tetrazolo[1,5-a]pyrimidine series [33]. Thus, in the current work, [2,3-¹⁵N₂]-5-aminotetrazole 7-¹⁵N₂ (98% enrichment for each of the labelled ¹⁵N atoms) was used to incorporate isotopic labels in the tetrazolo[1,5-b][1,2,4]triazine core (Scheme 1). The interaction of diazonium salt 8-¹⁵N₂ derived from [2,3-¹⁵N₂]-5-aminotetrazole 7-¹⁵N₂ with ethyl α-formylphenylacetate (9) yielded compound 10-¹⁵N₂. It was expected that the cyclization of 10-¹⁵N₂ would give [1,2-¹⁵N₂]-tetrazolo[5,1-c][1,2,4]triazine 11-¹⁵N₂. Indeed, [2,3-¹⁵N₂]-tetrazolo[1,5-b][1,2,4]triazin-7-one 13-¹⁵N₂ was obtained (see below). Most likely, tetrazole 11-¹⁵N₂ underwent a ring-opening process, yielding azide 12-¹⁵N₂, and this process was followed by an alternative ring closure. This azido-tetrazole equilibrium has been previously studied in detail [25].

The coupling between compound 13-¹⁵N₂ and 1-adamantanol (14) was conducted in trifluoroacetic acid (TFA) solution under reflux. A general and convenient approach to the N-adamantylation of heterocycles involves a reaction with the adamantyl cation generated from 1-adamantanol in acidic medium [34-37]. The adamantylation of 13-¹⁵N₂ led to N2- and N1-regioisomers (15a-¹⁵N₂ and 15b-¹⁵N₂, respectively, Scheme 1). Interestingly, according to the possible resonance structures, compound 15a-¹⁵N₂ should represent a mesoionic (betaine-like) structure with positive and negative charges located at the tetrazole and triazine rings, respectively. The relative concentration of regioisomers 15a-¹⁵N₂ and 15b-¹⁵N₂ was determined from the integral intensity of the corresponding signals in the 1D ¹H and ¹⁵N NMR spectra. The regioselectivity of adamantylation depends on the reaction time. Refluxing of the 13-¹⁵N₂/14 mixture (1:1.5 mol/mol) in TFA over 5 min led to the predominant formation of N2-adamantylated derivative 15a-¹⁵N₂. The 1:2 15a-¹⁵N₂/15b-¹⁵N₂ mixture was obtained after 2 h of refluxing. This phenomenon could be explained by the reisomerization of the initially formed N2-adamantylated product (15a-¹⁵N₂). Indeed, 2 h of refluxing of isolated 15a-¹⁵N₂ in TFA with 1.5 molar equivalents of 14 yielded a mixture of compounds 15a-¹⁵N₂ and 15b-¹⁵N₂ in the same (1:2) ratio (Scheme 1).

The use of [1-¹⁵N]-3-amino-1,2,4-triazole 16-¹⁵N (98%, ¹⁵N) and labelled sodium nitrite (98%, ¹⁵N) in acidic medium allowed for the in situ production of diazonium salt 17-¹⁵N₂, which reacted with ethyl nitroacetate (18) in a sodium carbonate solution (Scheme 2). This reaction led to the formation of [1,5-¹⁵N₂]-1,2,4-triazolo[5,1-c][1,2,4]triazinone 19-¹⁵N₂. Previously, the same approach was described for the incorporation of ¹⁵N atoms in azole and azine rings of compound 4 [38]. Heterocycle 20-¹⁵N₂ was obtained by the treatment of 19-¹⁵N₂ with hydrobromic acid according to a procedure described for 6-nitro-1,2,4-triazolo[5,1-c][1,2,4]triazin-7-ones [39]. In this case, hydrogen bromide was obtained in situ by the reaction between acetyl bromide and ethanol. The adamantylation of 20-¹⁵N₂ first occurred on the N3 atom of the azole ring. It was found that 5 min reflux of 20-¹⁵N₂ in TFA with a 1.5 molar excess of 14 led to the structure 21a-¹⁵N₂ (Scheme 2). However, prolonged (6 h) refluxing of the N3-regioisomer with 1.5 molar equivalents of 1-adamantanol (14) in a TFA solution led to complete isomerization of the compound and re-attachment of adamantane to the N4-atom of the azine ring (compound 21b-¹⁵N₂).

Double-labelled [1,2-¹⁵N₂]-3-amino-1,2,4-triazole 16-¹⁵N₂ was synthesized by the interaction of ¹⁵N₂-hydrazine sulphate (98%, ¹⁵N) with S-methyl isothiourea sulphate and consecutive cyclization with formic acid (see the Supporting Information File 1). The use of 16-¹⁵N₂ in a reaction with ethyl 4,4,4-trifluoroacetoacetate (22) yielded azolo-azine 23-¹⁵N₂ containing two isotopic labels in the 1,2,4-triazole fragment (Scheme 3). The adamantylation of [1,8-¹⁵N₂]-1,2,4-triazolo[1,5-a]pyrimidine 23-¹⁵N₂ was regioselective and led to the formation of the N3-isomer 24-¹⁵N₂ only. This compound did not undergo further isomerization.

Isomerization of adamantylated derivatives. The adamantylation of 1,2,4-triazolo[5,1-c][1,2,4]triazin-7-one derivatives in acidic medium is a thermodynamically controlled reaction [10], which could explain the rearrangement of N3-isomer 21a into N4-isomer 21b and the formation of the 15a/15b mixture from compound 15a. To evaluate the relative thermodynamic stabilities of isomers 15a,b and 21a,b, we performed DFT calculations with the RB3LYP/6-31-G(d,p) approximation in the gas phase using the Gaussian 09 package [40]. Isomers 15b and 21b are thermodynamically more stable than counterparts 15a and 21a. The calculated relative energy differences were 8.3 kcal/mol and 6.4 kcal/mol for the 15a–15b and 21a–21b pairs, respectively (see the Supporting Information File 1).

To further study the mechanism of isomerization between compounds 15a and 15b, equimolar quantities of unlabelled N2-isomer 15a and its double-labelled non-adamantylated precursor 13-¹⁵N₂ (isotopic enrichment 98%) were refluxed for 2 h in TFA without the addition of 1-adamantanol (14, Scheme 4). NMR analysis of the resulting mixture revealed the compounds 13*-¹⁵N₂, 15a*-¹⁵N₂, and 15b*-¹⁵N₂ in a 5:2:3 ratio. The observed equal ¹⁵N-isotopic enrichment (≈49%) in compounds 13*-¹⁵N₂, 15a*-¹⁵N₂, and 15b*-¹⁵N₂ indicated that the equilibrium 15a15b was reached in the isomerization process. The obtained ratio between the adamantylated products confirmed the higher thermodynamic stability of compound 15b relative to isomer 15a.

NMR spectroscopy and resonance assignment. The synthesized compounds were studied by NMR spectroscopy in a dimethyl sulfoxide (DMSO-d₆) solution using samples with concentrations with range of 30–70 mM. The obtained 1D ¹⁵N NMR spectra are shown in Figure 2, and the 1D ¹H and ¹³C spectra are presented as Figures S1–S18 in Supporting Information File 1. Two signals corresponding to the ¹⁵N-labelled atoms were observed in the 1D ¹⁵N NMR spectra of all the starting azolo-azines and adamantylated products (Figure 2). The ¹⁵N spectra of compounds 13-¹⁵N₂, 15a,b-¹⁵N₂, 23-¹⁵N₂ and 24-¹⁵N₂ containing labelled nitrogens in the neighbouring positions also demonstrated 13.4–16.4 Hz splittings due to the direct ¹J_NN coupling constants (Figure 2, Table 1).

The assignments of the ¹³C and ¹⁵N signals in the synthesized compounds were obtained by analysing the 2D ¹³C-HMQC, ¹³C-HMBC and ¹⁵N-HMBC spectra and observing the ¹³C-¹⁵N and ¹H-¹⁵N spin–spin interactions (see below). The ¹³C assignment procedure for 19-¹⁵N₂, 20-¹⁵N₂, and 21a,b-¹⁵N₂ was aided by the data from a previous study of unlabelled derivatives of compound 19 [12]. The ¹³C-¹⁹F J-coupling constants (ⁿJ_CF, Table 2) observed in the 1D ¹³C spectra facilitated the assignment of the ¹³C nuclei for the heterocyclic moieties of compounds 23-¹⁵N₂ and 24-¹⁵N₂. The observations of the ³J_H2-C3a coupling constants (9.2 Hz) in the 1D ¹³C spectra of 19-¹⁵N₂ and 20-¹⁵N₂ measured without proton decoupling confirmed the assignment of C3a to the signals at 160.23 ppm and 152.32 ppm, respectively. The obtained NMR assignments are collected in Table 1 (¹H, ¹⁵N) and Table 2 (¹³C).

¹³C-¹⁵N couplings for the structure determination of N-adamantylated azoloazines. The incorporation of ¹⁵N labels into the synthesized compounds led to the appearance of ¹H-¹⁵N and ¹³C-¹⁵N J-coupling constants (J_CN and J_HN couplings, respectively). The J_CN couplings became evident from the additional splitting of the corresponding signals in the 1D ¹³C NMR spectra and were measured by nonlinear fits of the ¹³С line shapes in the 1D spectra acquired with band-selective decoupling from ¹⁵N nuclei [25] (Figure 3). This method allowed for the measurement of the ¹³C-¹⁵N spin-spin interactions of different magnitudes and ranges starting from the direct ¹J_CN couplings (magnitudes of 1.2–12.0 Hz) to long-range ⁴J_CN couplings (magnitudes of 0.2–0.8 Hz). The full list of measured J_CN couplings is collected in Table 2. The couplings between adamantane carbons and the nitrogens of the heterocycles are shown in Schemes 1–3.

The J_CN couplings observed for the C6, C7 and C8a atoms in the heterocyclic moieties of compounds 13-¹⁵N₂ and 15a,b-¹⁵N₂ confirmed the [1,5-b]-type fusion between the azole and azine rings in these structures (Table 2, Figure 3). The observation of the direct ¹J_C1'-N2 (6.5 Hz) and other ¹³C-¹⁵N interactions for the C1' (²J_C-N3 3.8 Hz), C2' (²J_C-N2 0.4 Hz and ³J_C-N3 1.2 Hz), C3' (³J_C-N2 1.6 Hz and ⁴J_C-N3 0.2 Hz) and C4' (⁴J_C-N2 0.3 Hz) atoms of the adamantane group in 15a-¹⁵N₂ indicated that the initial adamantylation of 13-¹⁵N₂ underwent a reaction with the N2 atom of the tetrazole ring (Scheme 1). However, the detection of geminal (²J_C1'-N2 2.7 Hz), vicinal (³J_C2'-N2 1.1 Hz and ³J_C1'-N3 0.3 Hz) and long-range (⁴J_C3'-N2 0.3 Hz) couplings in the 1D ¹³C NMR spectra of 15b-¹⁵N₂ revealed the attachment of the adamantane fragment to the N1 atom of the tetrazole ring.

The structures of compounds 19-¹⁵N₂, 20-¹⁵N₂, 21a,b-¹⁵N₂, 23-¹⁵N₂ and 24-¹⁵N₂ were also confirmed by the measured ⁿJ_CN patterns (Table 2). The presence of characteristic vicinal ³J_C6-N1 coupling (magnitudes of 1.1-1.3 Hz) and other coupling constants revealed that the fusions of the triazole rings with the triazine (compounds 19, 20 and 21) or pyrimidine rings (compounds 23 and 24) have [5,1-c] or [5,1-a] configurations, respectively.

The detection of a single ³J_C1'-N1 coupling (0.4 Hz) with the adamantane carbons in compound 21a-¹⁵N₂ indicated that the substituent group is attached to the N3 atom of the 1,2,4-triazole ring (Figure 2, Scheme 2). Similarly, the N3-adamantylation in compound 24-¹⁵N₂ was characterized by two weak ³J_C1'-N1/N8 couplings (0.4/0.6 Hz) detected for the C1' atom (Figure 2, Scheme 3). In contrast, the attachment of the adamantane fragment to the N4 atom of the triazine ring in compound 21b-¹⁵N₂ led to a large set of observable J_CN couplings, including geminal (²J_C1'-N5 5.0 Hz), vicinal (³J_C2'-N5 1.7 Hz) and long-range (⁴J_C1'-N1 ≤ 0.2 Hz and ⁴J_C3'-N5 0.4 Hz) couplings (Figure 2, Scheme 2).

¹H-¹⁵N couplings for the characterization of N-adamantylation sites in fused azolo-azines. The signal splittings due to the J_HN couplings were observed in the 1D ¹H spectra only in a limited number of cases (compounds 20-¹⁵N₂, 21a,b-¹⁵N₂, 23-¹⁵N₂ and 24-¹⁵N₂, see Scheme 2 and Scheme 3). In the other cases, the J_HN couplings were measured by amplitude-modulated 1D ¹H spin-echo experiments with selective inversion of the ¹⁵N nuclei [24] (Figure 4A, Table 1) or detected using the conventional 2D ¹⁵N-HMBC spectra (Figure 4B and Figures S19–S23 in Supporting Information File 1). These methods allowed for the straightforward detection and measurements of the geminal ²J_HN (values of 13.8–16.1 Hz), vicinal ³J_HN (values of 0.83–6.6 Hz) and long-range ^4/5J_HN (values of 0.04–0.9 Hz) couplings for the isotopically enriched nitrogen atoms. The ¹H-¹⁵N spin–spin interactions with the unlabelled ¹⁵N nuclei (at natural abundance) were also detected in the ¹⁵N-HMBC experiments (Table 1). The intensities of the HMBC cross-peaks for the ¹⁵N-labelled nuclei demonstrated an approximate correlation with the measured J_HN values (see Table 1). This provides a way to qualitatively estimate the J_HN magnitudes for unlabelled and ¹⁵N-labelled nuclei using the relative intensities of the HMBC cross-peaks, corrected for the degree of the isotope enrichment. The measured J_HN couplings and HMBC ¹H-¹⁵N spin–spin interactions are shown in Schemes 1–3.

Compound 15a-¹⁵N₂ was characterized by a set of J_HN couplings detected for the H2' (^3/4J_H2'-N2/N3 0.83/0.06 Hz), H3' (^4/5J_H3'-N2/N3 0.60/0.11 Hz) and H4' (⁵J_H4'-N2 0.23 Hz) atoms of the adamantane group (Figure 4A, Table 1, Scheme 1). These spin–spin interactions, with the exception of ⁵J_H3'-N3, were also observed in the 2D ¹⁵N-HMBC spectrum (Figure 4B). The observed J_HN pattern indicated that the adamantane substituent is attached to the ¹⁵N-labelled atom (N2) of the tetrazole ring. Similarly, the observation of the ⁵J_H3'-N2 coupling constant (≈0.04 Hz) and the medium intensity H2'-N1 HMBC cross-peak at natural ¹⁵N abundance revealed that compound 15b-¹⁵N₂ contains an N-adamantane moiety attached to the unlabelled N1 atom (Scheme 1). Note that the weak cross-peak corresponding to the long-range ⁵J_H2'-N3 coupling was observed in the HMBC spectrum of 15b-¹⁵N₂ (Figure S20 in Supporting Information File 1), but the magnitude of this J coupling was under the limit of reliable J_HN measurements (0.04 Hz). Thus, if the J_HN couplings are too small to be measured quantitatively, the ¹⁵N-HMBC experiment could provide useful information about the position of the adamantane substituent. However, the assignment of the ¹⁵N-labelled atoms in compounds 15a,b-¹⁵N₂ (differentiation between N2 and N3 resonances) could not be achieved using the J_HN and ¹⁵N-HMBC data alone. The absence of protons in the tetrazolo[1,5-b][1,2,4]triazine core of these compounds dictates the necessity of J_CN analysis for the unambiguous assignment of ¹⁵N-labelled nuclei.

In contrast to the situation observed for compounds 15a,b-¹⁵N₂, the J_HN interactions with the H2 proton in the 1,2,4-triazolo[5,1-c][1,2,4]triazines 19-¹⁵N₂, 20-¹⁵N₂, and 21a,b-¹⁵N₂ and the H2 and H6 protons in the 1,2,4-triazolo[1,5-a]pyrimidines 23-¹⁵N₂ and 24-¹⁵N₂ permitted the straightforward assignments of the labelled ¹⁵N atoms (see Scheme 2 and Scheme 3). The attachment of an adamantyl substituent to the N4 atom in compound 21b-¹⁵N₂ was confirmed by the measured long-range ⁴J_H2'-N5 coupling constant (0.06 Hz) and the medium intensity H2'-N4 HMBC cross-peak observed at natural ¹⁵N abundance (Table 1, Scheme 2). Notably, the weak cross-peak corresponding to the ⁴J_H2'-N5 coupling was also detected in the ¹⁵N-HMBC spectrum (Figure S22D in Supporting Information File 1).

For the adamantylated heterocycles 21a-¹⁵N₂ and 24-¹⁵N₂, the J_HN interactions between the adamantane protons and the labelled N1, N5 or N8 atoms were not detected by amplitude-modulated ¹H spin-echo or ¹⁵N-HMBC experiments. Meanwhile, the interactions between the H2' proton of the adamantane and the unlabelled N3 atom of the heterocyclic moieties of the compounds were observed in the ¹⁵N-HMBC spectra (Scheme 2 and Scheme 3). These results confirmed the coupling of the adamantane bridgehead C1' carbon with the N3 nitrogen of the azole ring in 21a-¹⁵N₂ and 24-¹⁵N₂.

The identification of adamantylation sites based on ¹⁵N-HMBC data requires the preliminary assignment of the nitrogen atoms at natural isotopic abundance. For compounds 21a,b-¹⁵N₂ and 24-¹⁵N₂, the required ¹⁵N assignment could be obtained by observing the ¹⁵N-HMBC correlations from the H2 and H6 protons. However, the detection of the corresponding cross-peaks was hindered by the presence of large (>3 Hz) J_HN couplings with the isotopically enriched ¹⁵N-nuclei. The suppression of the magnetization transfer through the geminal ²J_H2-N1 couplings by setting a delay in the ¹⁵N-HMBC experiment to 1/J_HN (62.5–71.4 ms) permitted the observation of the correlations between H2 and the unlabelled N3 and N8 atoms in compounds 21a,b-¹⁵N₂ (Figures S21 and S22 in Supporting Information File 1). Meanwhile, the presence of additional large vicinal couplings (³J_H2-N8 and ³J_H6-N8) made this strategy not applicable for compound 24-¹⁵N₂. In this case, the supposed assignment of the N3 resonance was indirectly confirmed by the similarity of its chemical shifts in compounds 21a-¹⁵N₂ and 24-¹⁵N₂.

NMR and Х-ray diffraction data revealing several rotameric configurations of adamantane substituents. The ¹³C signals of the N-adamantyl substituents in compounds 15a,b-¹⁵N₂ and 21a,b-¹⁵N₂ measured at 45 °C demonstrated additional splitting, which was not connected to the ¹H-¹³C and ¹³C-¹⁵N J-couplings. The C1' and C2' signals of adamantane (and C3' for 21a-¹⁵N₂) were split into two components with a relative intensity ratio of ≈10:7 and a frequency difference of 0.5–1.2 Hz (Figure 3, Table 2). This revealed the presence of the two structural forms of the N-adamantylated heterocycles in solution with a slow (characteristic time ≥ 1 s) exchange between them. The rotation of the N-adamantyl substituents around the N–C1' bond in the bulky bicyclic heterocycles is likely hindered, and the observed conformational heterogeneity corresponds to the different rotameric configurations of the substituent. To test this hypothesis, additional NMR measurements at elevated temperature were carried out for compound 21a-¹⁵N₂. The ¹³C 1D NMR spectrum measured at 70 °C with ¹H and ¹⁵N decoupling did not demonstrate additional splitting (Figure S25 in Supporting Information File 1). This confirmed that the studied NMR samples contained unique and chemically pure compounds, while the heterogeneity observed at 45 °C was connected to the presence of different rotameric states.

To confirm the determined positions of the N-adamantane substitutions, compounds 15a and 15b were studied by X-ray crystallography. Suitable crystals of 15a and 15b were obtained by slow evaporation from ethyl acetate solutions. The solved X-ray structures were in a full agreement with the results of the J_CN and J_HN analysis and confirmed the N2-substituted mesoionic form for compound 15a as well as the attachment of adamantane to the N1 atom in compound 15b. In accordance with expectations, the adamantane groups in the crystals of 15a and 15b were found disordered between two conformations with different rotameric configurations around the N–C1' bond (Figure 5 and Supporting Information Files 2 and 3). These forms differ by the rotation around the N–C1' bond by 40–60°; thus, in each of them, one of the C2' atoms of the adamantane substituent is located approximately in plane with the heterocyclic moiety of the compound. The populations of the two conformational forms in the single crystals of 15a and 15b (4:1 and 17:3, respectively) differ from the populations of the conformers observed by NMR spectroscopy in DMSO solution (≈10:7). Interestingly, for 15a, the major conformer corresponds to a rotameric state with a screened N1 atom, but in the major conformer of 15b, the N2 atom of the heterocycle is screened. Notably, similar structural disorder was previously observed in the crystals of adamantylated tetrazolylpyrazole derivatives [37,41].

Discussion

Comparison of different NMR approaches for the determination of N-alkylation sites in fused heterocycles. The obtained data permit a comparison of the abilities of different NMR parameters (¹³C and ¹⁵N chemical shifts, J_HN and J_CN) to provide structural information about the N-adamantylation sites in bicyclic heterocycles. The previous studies of azolo[5,1-c][1,2,4]triazin-7-ones, 1,2,4-triazolo[1,5-a]pyrimidin-7-ones and tetrazolo-azines revealed that the ¹³C chemical shifts of the nearest carbon atoms to N-alkyl fragments could be used as indicators for the formation of N-alkylated azolo-azines [12,42]. For the presently studied compounds, we can expect considerable changes in the chemical shifts of the bridgehead C3a and C8a atoms. The shifts of the other carbon atoms from the heterocyclic parts moieties of the compounds (C2, C5, C6, and C7) may also provide useful structural information.

In the studied tetrazolo-triazines and tetrazolo-pyrimidines (compounds 13-¹⁵N₂ and 15a,b-¹⁵N₂ and compounds from work [25]), the resonances of the bridgehead C8a atom were observed over a relatively narrow spectral range (144–155 ppm). In the triazolo-triazines and triazolo-pyrimidines 19-¹⁵N₂, 20-¹⁵N₂, 21a,b-¹⁵N₂, 23-¹⁵N₂, and 24-¹⁵N₂, the similar bridgehead C3a atoms are shifted slightly downfield (149–160 ppm). Here, we observed that N-adamantylation of the nitrogen atom directly attached to the C3a, C8a or C2 atoms induced up-field shifts of the corresponding ¹³C signal. The majority of these shifts had relatively small magnitudes (Δδ −0.9 to −2.8 ppm), and a large shift was only observed for the C2 resonance of 21a-¹⁵N₂ (Δδ −11.3 ppm (Table 2)). However, these up-field shifts could not be used to determine the N-adamantylation site. The attachment of the adamantane moiety to other nitrogen atoms could lead to similar ¹³C shifts. For example, similar Δδ values (−0.9 and −1.1 ppm) were observed for the C2 resonances in compounds 21b-¹⁵N₂ and 24-¹⁵N₂, where the adamantane fragments are attached to N3 and N4, respectively. Note that the C2 and N4 atoms are separated by three covalent bonds.

A similar situation was observed for the carbon atoms that are separated from the N-adamantylation site by two covalent bonds (Table 2). The attachment of adamantane to the N2 atom in compound 15a-¹⁵N₂ induced a large down-field shift (Δδ +8.6 ppm) of the C8a resonance, while modification of the N4 atom in compound 21b-¹⁵N₂ induced an up-field shift (Δδ −3.0 ppm) of the C6 resonance. Thus, the obtained data did not reveal an easily interpreted correlation between the ¹³C chemical shifts and the position of N-adamantane substituents. The same issue was previously noted in the study of N-alkylated tetrazolo[1,5-a]pyridine derivatives [43].

Similar to the situation observed for the ¹³C nuclei, a comparison of the ¹⁵N chemical shifts in the starting heterocycles 13-¹⁵N₂, 20-¹⁵N₂, and 23-¹⁵N₂ and their N-adamantylated derivatives 15a,b-¹⁵N₂, 21a,b-¹⁵N₂, and 24-¹⁵N₂ did not reveal a simple correlation with the position of the substituent group (Figure 2, Table 1). Large changes in the ¹⁵N resonance position were observed for the N2 atom in compounds 15a-¹⁵N₂ (Δδ −87.6 ppm) and 15b-¹⁵N₂ (Δδ −38.4 ppm) and for the N5 atom in compound 21a-¹⁵N₂ (Δδ +54.2 ppm). According to the data reported for tetrazolo[1,5-a]pyridines [43], the shielding of the N2 nucleus in compound 15b-¹⁵N₂ can be explained by the adamantylation of the neighbouring N1 atom in the tetrazole fragment. In contrast, the coupling of the adamantyl fragment to the N4 atom in compound 21b-¹⁵N₂ did not considerable change the chemical shift of the neighbouring ¹⁵N5 nucleus (Δδ ≈ +2.1 ppm). For clarity, we should mention that the information that could be obtained from the ¹⁵N chemical shifts is restricted by the pattern of the ¹⁵N-label incorporation. In some cases, the isotopic labels were located far from the position of the attached adamantane group. This fact could partially explain the lack of correlation between chemical shifts and structure.

The obtained data indicated that changes in the ¹³C and ¹⁵N chemical shifts could not reliably determine the adamantylation sites in azolo-azines. Therefore, we focused our study on the analysis of ¹H-¹⁵N and ¹³C-¹⁵N spin–spin interactions. Despite the relatively ‘sparse’ placement of ¹⁵N labels, in all the synthesized compounds, the bridgehead C1' atom of the adamantyl fragment demonstrated detectable J_CN couplings (Schemes 1–3, Figure 2). The observed J_CN values greatly varied in magnitude. The direct and vicinal couplings (^1,2J_CN) were relatively large (6.5–2.7 Hz), while the geminal and long-range interactions (^3,4J_CN) were small (0.6–0.2 Hz). The fact that the ¹J_CN and ²J_CN as well as the ³J_CN and ⁴J_CN couplings for the C1' atom had similar magnitudes indicated that additional data are required for the unambiguous determination of the adamantylation sites. For this purpose, we measured and analysed the ¹³C-¹⁵N and ¹H-¹⁵N spin–spin interactions for the other atoms of the adamantane groups (Schemes 1–3). These additional sets of ^2-4J_CN and ^2-5J_HN data reliably identified the N-adamantylation sites in the all studied compounds. The proposed structures of 15a,b-¹⁵N₂ were independently confirmed by Х-ray diffraction data.

One of the advantages of J_CN and J_HN data compared with chemical shift data is the usefulness of ‘negative’ information. In the majority of the cases, the absence of a detectable ¹³C-¹⁵N or ¹H-¹⁵N spin–spin interaction indicates the remote localization of the adamantane substituent and labelled nitrogen of the heterocycle. The obtained results showed that the structural information provided by the ¹H-¹⁵N spin–spin interactions (measured by 1D ¹H spin-echo experiments or detected in 2D ¹⁵N-HMBC experiments) is similar to the information obtained from the J_CN couplings. However, these approaches are not equivalent. On one hand, the acquisition of J_HN data requires less measurement time and less sophisticated equipment compared with that of J_CN data (conventional broadband probe and two-channel NMR spectrometer versus triple-resonance probe and three-channel spectrometer, respectively). On the other hand, the structural characterization of the N-adamantylation site(s) in heterocycles based on the J_HN data requires the preliminary assignment of the ¹⁵N resonances. Therefore, the combination of these approaches based on the analysis of J_CN and J_HN couplings represents the most effective NMR tool for the determination of adamantylation sites in azolo-azines.

Possible mechanisms of the isomerization of N-adamantylated derivatives 15a and 15b. The isomerization of unlabelled 15a in the presence of 13a-¹⁵N₂ (Scheme 4) elucidated the possible mechanism of the isomerization of 15a-¹⁵N₂ into 15b-¹⁵N₂. This experiment confirmed that this rearrangement occurs via the formation of adamantyl cation 25 and heterocyclic base 13-¹⁵N₂ (Scheme 5). Moreover, the equilibration of the isotope composition over the reaction products (15a*-¹⁵N₂, 15b*-¹⁵N₂ and 13*-¹⁵N₂) indicated that the transformation of 15a into 15b is reversible. Note that the protonation of compound 13 and its adamantylated derivatives probably plays an important role in the 15a15b conversion in TFA solution. The precise positions of the attached protons are unknown, and this determination requires additional investigation, but the analysis of calculated Mulliken charges in compounds 15a and 15b (see Supporting Information File 1, Scheme S2) suggests that the most negatively charged atom N8 undergoes the initial protonation. Similar mechanisms can be proposed to describe the isomerization of compounds 21a and 21b.

Conclusion

We reported the selective incorporation of two ¹⁵N atoms at different positions of 1,2,4-triazolo[1,5-a]pyrimidine, azolo-1,2,4-triazines and their N-adamantylated derivatives. The selective incorporation of the ¹⁵N-labels into the azolo and azine rings of the heterocyclic structures led to the appearance of ¹H-¹⁵N and ¹³C-¹⁵N J-coupling constants. The combined analysis of the J_HN and J_CN couplings allowed for the effective determination of the adamantylation sites in the azolo-azine series. To the best of our knowledge, the applicability of this approach for the structural determination of N-substituted heterocycles has not been previously considered. We suggest that the proposed method is generally applicable for the studies of N-alkylated heterocyclic compounds with a high abundance of nitrogen nuclei, where ¹³C chemical shifts and ¹H-¹H NOE data cannot provide reliable structural information. The incorporation of the ¹⁵N-labels also permitted the study of the mechanism of isomerization of N-adamantylated tetrazolo[1,5-b][1,2,4]triazin-7-one in TFA solution. The formation of an adamantyl cation and NH-tetrazolo-triazine during the isomerization reaction was confirmed.