1,2,3,5-Tetrazines: A General Synthesis, Cycloaddition Scope, and Fundamental Reactivity Patterns

Abstract

Despite the explosion of interest in heterocyclic azadienes, 1,2,3,5-tetrazines remain unexplored. Herein, the first general synthesis of this new class of heterocycles is disclosed. Its use in the preparation of a series of derivatives, and the first study of substituent effects on their cycloaddition reactivity, mode, and regioselectivity provide the foundation for future use. Their reactions with amidine, electron-rich, and strained dienophiles reveal unique fundamental reactivity patterns (4,6-dialkyl-1,2,3,5-tetrazines > 4,6-diaryl-1,2,3,5-tetrazines for amidines but slower with strained dienophiles), an exclusive C4/N1 mode of cycloaddition, and dominant alkyl versus aryl control on regioselectivity. An orthogonal reactivity of 1,2,3,5-tetrazines and the well-known isomeric 1,2,4,5-tetrazines is characterized, and detailed kinetic and mechanistic investigations of the remarkably fast reaction of 1,2,3,5-tetrazines with amidines, especially 4,6-dialkyl-1,2,3,5-tetrazines, established the mechanistic origins underlying the reactivity patterns and key features needed for future applications.

Graphical Abstract

INTRODUCTION

Inverse electron demand Diels–Alder reactions of electron-deficient heterocyclic azadienes have provided powerful approaches for the synthesis of highly substituted arenes or heteroaromatic systems^1,2 with widespread applications in natural product total syntheses,³ the pharmaceutical industry, and in the preparation of screening libraries.^4–7 The most widely investigated classes of these heterocycles include 1,2,4,5-tetrazines,^8–10 1,2,4-triazines,^11–13 1,3,5-triazines,^14,15 1,3,4-oxadiazoles,^16,17 1,2-diazines^18–20 and more recently 1,2,3-triazines.²¹ Among these heterocycles, 1,2,4,5-tetrazines display the greatest reactivity and broadest substate scope toward strained or electron-rich dienophiles because of their increased electron-deficient character. Their outstanding reactivity inspired the development of powerful (bio)orthogonal conjugation and labeling methods often referred to as tetrazine ligations that are widely used today in not only chemical biology,^22–32 but also material science and the polymer field.^33–36

Because of the intrinsic reactivity and widespread applications of 1,2,4,5-tetrazines, interest in the isomeric tetrazines, including 1,2,3,5-tetrazines, stimulated attempts at their preparation as well as computational investigations of their structure, stability, and electron-deficient character, the latter of which are comparable to the 1,2,4,5-tetrazines.^37–40 The reported unsuccessful attempts to prepare 1,2,3,5-tetrazines highlight the synthetic challenge of this heterocyclic system, being described as “the rarest and least studied class” of isomeric tetrazines (Figure 1A).³⁷ Although previously unknown, which is remarkable in this day and age, it was only recently that we disclosed the first synthesis of a single monocyclic aromatic 1,2,3,5-tetrazine, 4,6-diphenyl-1,2,3,5-tetrazine (1a) and established its stable existence and a reactivity suggestive of widespread utility.⁴¹ We reported its key physical, chemical, and spectroscopic properties as well as its intrinsic thermal stability. It was found that 1,2,3,5-tetrazine 1a effectively participates as the electron-deficient 4π component in inverse electron demand Diels–Alder reactions, displaying an exclusive C4/N1 cycloaddition mode, predictable regioselectivity, and a reactivity comparable to that of the isomeric 3,6-diphenyl-1,2,4,5-tetrazine with electron-rich dienophiles (Figure 1B). These studies also led to the discovery that 1,2,3,5-tetrazine 1a displays efficiencies and outstanding reaction rates, far exceeding that of the corresponding 1,2,4,5-tetrazine, in a reaction with amidines (1,2,3,5-tetrazine/amidine ligation) that proved orthogonal to that of the well-established 1,2,4,5-tetrazine-based ligation reactions. Because of this outstanding reactivity and intrinsic stability displayed by 1a in this initial work, we initiated studies to develop a general synthesis of this new class of unexplored heterocycles and expand the scope of accessible 1,2,3,5-tetrazines. Herein, we report an efficient and the first general synthesis of 1,2,3,5-tetrazines, which was used in the preparation of a series of substituted derivatives. We disclose the first study of substituent effects on the cycloaddition reactivity and scope, mode of cycloaddition (N1/C4), and cycloaddition regioselectivity, revealing the fundamental reactivity patterns and a dominant influence on cycloaddition regioselectivity of the previously unknown alkyl substituted 1,2,3,5-tetrazines. We additionally detail kinetic studies and further mechanistic investigations of the 1,2,3,5-tetrazine/amidine reaction, establishing unique features central to future applications. and providing a fundamental foundation for its future use.

Figure 1.

1,2,3,5-tetrazines, a new heterocycle class. (A) 1,2,3,5-tetrazines as an unexplored heterocycle class and isomer of the widely used 1,2,4,5-tetrazines. (B) Reactions of 4,6-diphenyl-1,2,3,5-tetrazine (1a). (C) Initial strategy for synthesis of 1,2,3,5-tetrazine 1a. (D) New, general strategy for the synthesis of substituted 1,2,3,5-tetrazines.

RESULTS AND DISCUSSION

In our initial synthesis of 4,6-diphenyl-1,2,3,5-tetrazine (1a), the substituted or protected dihydro-1,2,3,5-tetrazines 2–5 were used as key intermediates enroute to the aromatic 1,2,3,5-tetrazine (Figure 1C). This required an activating nitration step for preparation of the heavily nitrated and energetic intermediate 3 and two challenging S_NAr reactions for the removal of N-aryl substituents employed in the preparation of the dihydro-1,2,3,5-tetrazine core structure.⁴¹ Although successful in providing 1a for initial studies, it is not an approach of use to others or general for the class.⁴² Herein, we describe a now general and efficient approach that maintained the final oxidative aromatization strategy as well as Butler’s method^43,44 for the preparation of precursor N²,N⁵-diaryl 2,5-dihydro-1,2,3,5-tetrazines but now conducted with easily removed N2 and N5 substituents, overcoming the challenges in our initial synthesis. For this purpose and as detailed below, we developed a selective reductive ring-opening reaction of N²,N⁵-di(3-benzisoxazolyl)-substituted dihydrotetrazines 6, containing a weak N–O bond within the aryl substituent, which was expected to provide N²,N⁵-imidoyl-2,5-dihydrotetrazines 7 with readily removable N-substituents in a single activation step. Subsequent cleavage of the amidine with release of a nitrile was envisioned to accompany an oxidative aromatization, providing a general synthesis that include both aryl and alkyl substituted 1,2,3,5-tetrazines (Figure 1D). These efforts followed an extensive series of studies conducted over a period of years, targeting the 1,2,3,5-tetrazines following more direct but unsuccessful approaches readers themselves might also conceive of.

In the course of the work, six 4,6-disubstituted 1,2,3,5-tetrazines (1a–1f) were targeted to exemplify the generality of the new approach and to address key questions surrounding the substituent impact on the reactivity of this new heterocyclic azadiene (Figure 2). Their synthesis starts with the condensation of 1,2-benzisoxazole-3-hydrazine with the α-diketones 8a–f, bearing the varied C4/C6 substituents in the final 1,2,3,5-tetrazines, to provide the bishydrazones 9a–f in near quantitative yield (90–97%, 94% avg yield). A subsequent ceric(IV) ammonium nitrate (CAN)-mediated oxidation followed by an acid promoted ring-closure reaction generated the zwitterionic N¹,N²-substituted 1,2,3-triazolium intermediates 10a–f (70% avg yield). Notably, Pb(OAc)₄ was used as the oxidant in place of CAN for 9e to promote the slower conversion to the more sterically hindered 10e. Interestingly, 10f, which bears two different C-substituents on the triazolium ring, was isolated as an inconsequential pair of interconvertible regioisomers (4:1 mixture). Conversion to the dihydro-1,2,3,5-tetrazine system 11 was accomplished through a 1,3-dipolar cycloaddition-triggered rearrangement cascade (see Supporting Information Figure S1 or ref. 43 for detailed mechanism) where initiation of the reaction was accomplished by treatment of ylides 10a–f with N-sulfinyl 3-aminobenzisoxazole at room temperature (for 10a and 10b) or elevated temperature (60 °C, for 10c–f). The presence of the dihydro-1,2,3,5-tetrazine heterocyclic core in 11 was unambiguously established and characterized with an X-ray structure of 11a.⁴⁵ Optimization conducted on the preparation of 11a defined CH₃CN as the most effective reaction solvent of those examined and revealed that use of higher reaction concentrations improved the yield (see Supporting Information Figure S2). Dihydro-1,2,3,5-tetrazines 11 were provided in generally high yields (61–72%) although lower conversions to the desired products were observed with 11c and 11e.

Figure 2.

General synthesis of 1,2,3,5-tetrazines, providing 1a–f.

The reductive ring-opening of the benzisoxazole N-substituents on the dihydro-1,2,3,5-tetrazines 11 was accomplished by a Pd/C-catalyzed hydrogenation with little or no competing reduction of the N−N bonds in the dihydrotetrazine core. The reduction selectivity was further tuned by the choice of reaction solvent. Use of ethyl acetate was found to be effective for the reduction of the 4-aryl or 4,6-diaryl substituted dihydrotetrazines 11a, 11b and 11f, whereas a more polar reaction solvent proved more effective for the complete reduction of the 4,6-dialkyl dihydrotetrazines 11d and 11e. Additionally, a mixed solvent system (THF/t-butanol) was used for 11c where its limited solubility in both ethyl acetate and methanol was observed. Interestingly and to our delight, a clean and spontaneous in situ removal of the N⁵-benzisoxazolyl substituent was observed after the reduction, releasing 2-hydroxylbenzonitrile as the stoichiometric byproduct and providing N²-imidoyl dihydrotetrazines 12 generally in good yields (25–68%, 52% avg). A final oxidative aromatization of the dihydrotetrazines 12 was promoted by treatment with PbO₂ at ambient temperature to provide the aromatic 1,2,3,5-tetrazines 1 in good yield (58–70%, 67% avg) with the concomitant cleavage of the N²-imidoyl substituent and generation of 2-hydroxylbenzonitrile. In the oxidation of 12d–f, acetic acid was added to the reaction mixture to suppress N–N bond cleavage in the dihydrotetrazine core.

1,2,3,5-Tetrazines 1 were isolated as crystalline (1a–c) or amorphous (1d–f) yellow solids, and formation of the aromatic heterocycle was confirmed by NMR and, in the case of 1a, with a previously reported single crystal X-ray structure determination.⁴¹ The ¹H NMR spectra of 1 revealed characteristic deshielded proton signals for the ortho protons on the aryl substituents (in 1a–c, 1f) and for the α protons on the alkyl groups (in 1d–f) derived from combined inductive and deshielding effects of the aromatic 1,2,3,5-tetrazine. Tetrazines 1 also displayed characteristic downfield C4/C6 carbon peaks in ¹³C NMR, with chemical shifts of 164.6–162.1 ppm for tetrazine carbons adjacent to aryl substituents, and 173.7–170.2 ppm for tetrazine carbons adjacent to alkyl substituents.

Although predictably less stable than the isomeric 1,2,4,5-tetrazines,^37,38 all 1,2,3,5-tetrazines were found to be stable at room temperature and for long-term storage under normal lab conditions (>95% purity by NMR after storage at 4 °C for >50 days). In our original work with 1a,⁴¹ we disclosed that it is stable at 25 °C in the crystalline state, in CHCl₃, and in a protic solvent (CD₃CN/D₂O, v/v = 7:3) for >5 days after which monitoring was discontinued. It is also stable for at least 24 h at 25 °C in 70% H₂O/CH₃CN, 50% and 80% H₂O/DMSO, CH₃CN/PBS buffer (1:1), and hexafluoroisopropanol (HFIP). The only exception to the storage recommendations is 1d, where a lower temperature was used for long-term storage because it is a low melting solid (>95% purity by NMR after storage at −20 °C for >50 days).^46,47 The thermal stability of tetrazine 1d was established as a representative of dialkyl 1,2,3,5-tetrazines. When warmed at 80 °C, tetrazine 1d undergoes a retro [2+2+2] ring-opening reaction to provide 3-phenylpropionitrile in a reaction that was found to be nearly complete in several hours. ¹H NMR monitoring of the ring opening process provided first-order reaction kinetics and a quantitative determination of the reaction rate (Supporting Information Figure S16). This ring-opening reaction of 1d was found to be slightly faster than that of 4,6-diphenyl-1,2,3,5-tetrazine (1a)⁴¹ and the half-life of 1d at 80 °C (t_1/2 = 1.73 h, k = 0.40 h⁻¹ = 1.11 × 10⁻⁴ s⁻¹) was found to be shorter than that of 1a (t_1/2 = 23.6 h, k = 0.029 h⁻¹ = 8.15 × 10⁻⁶ s⁻¹). The decreased thermal kinetic stability¹⁶ of 1d may be attributed to the stabilizing tetrazine conjugation with the phenyl substituents in 1a. Although kinetically less stable than the corresponding 1,2,4,5-tetrazines in agreement with computational studies,³⁷ the observed stability of the 1,2,3,5-tetrazines permits uneventful handling and long-term storage under normal conditions and they can be warmed to elevated temperatures in solvent to promote cycloadditions with predictable limitations on the conditions used.

In our initial study on the reactivity of diphenyl-1,2,3,5-tetrazine, 1a was found to rapidly and quantitatively react with amidines under mild conditions even at dilute reaction concentrations. The superb reactivity of 1a raised the question of whether it is general to all 1,2,3,5-tetrazines and how the substituents electronically and sterically impact this reactivity. To quantitate of this reactivity, the tetrazine/amidine reaction for all six 1,2,3,5-tetrazines was monitored in a mixed solvent of CD₃CN/CDCl₃ (v/v = 1:1) by ¹H NMR with benzamidine (13a) as the reaction partner (Supporting Information Figure S3–S8). The reaction of all six tetrazines (1a–f) fitted well to second-order rate kinetics, and the measured second-order rate constants are summarized in Figure 3.

Figure 3.

Reactivity of 1,2,3,5-tetrazines 1a–f with benzamidine (13a) or phenylacetamidine (13l).

The tetrazines exhibited a remarkable reactivity with benzamidine, with rate constants in the range required for orthogonal ligation reactions (k = 10⁻² to 10⁻¹ M⁻¹s⁻¹), displaying a large electronic effect on the reactivity for the 4,6-diaryl substituted 1,2,3,5-tetrazines 1a–c. The introduction of electron-donating p-methoxy groups on the phenyl substituents slowed the reaction by 60-fold, while the presence of electron-withdrawing p-carbomethoxy substituents led to a 6-fold increase in the rate reflective of the tetrazine serving as the electron-deficient counterpart in the reaction. When compared with tetrazine 1a with two phenyl substituents, alkyl tetrazines 1d and 1e were found to be even more reactive, displaying 10-fold (for 1d) and 3-fold (for 1e) faster rates than 1a, respectively. As expected, the rate of reaction with 1f, bearing the mixed alkyl/aryl substituents, was found to lie between that of diphenyl tetrazine 1a and dialkyl tetrazine 1e. The observed trend of relative reactivity was not limited to benzamidine but it was also found to extend to alkyl amidines, using phenylacetamidine (13l) as a representative example (Supporting Information Figure S9 and S10). The rate of the reaction between tetrazine 1d and 13l (k = 1.95 M⁻¹·s⁻¹) was found to be 16-fold faster than that of 4,6-diphenyl-1,2,3,5-tetrazine (1a) (k = 1.19 × 10⁻¹ M⁻¹·s⁻¹), and both reactions were found to be 3- to 4-fold faster than the reactions with benzamidine as the reaction counterpart. Thus, 4,6-dialkyl substituted 1,2,3,5-tetrazines proved even more reactive than 4,6-diaryl substituted 1,2,3,5-tetrazines and aliphatic amidines are considerably more reactive than aryl amidines in the reaction. Aside from their use in synthesis to access 1,3,5-triazines, the measured rate constants for the more reactive 1,2,3,5-tetrazine/amidine reactions range from 10⁻¹ to 2 M⁻¹·s⁻¹, highlighting the outstanding reactivity characteristic of click ligations.⁴⁸

The reactivity of the representative 4,6-dialkyl-1,2,3,5-tetrazine 1d was examined further with a range of dienophiles, including not only a broader scope of amidines but also classic dienophiles that participate in inverse electron demand Diels–Alder reactions. All amidines examined, including aryl amidines (13a–f), heteroaryl amidines (13g–j) and aliphatic amidines (13k–n), smoothly react with tetrazine 1d (40 mM) at ambient temperature to provide the 1,3,5-triazines in uniformly high yields (>90%, Figure 4). An exclusive C4/N1 (versus N2/N5) mode of addition and a single reaction regioselectivity was observed, affording the 1,3,5-triazines (14a–n) in near quantitative yields (91–99%) within a remarkably short time (20 min) even under these mild reaction conditions. Notably, the reaction of tetrazine 1d and benzamidine 13a was also found to proceed smoothly under even more dilute reaction concentrations (4 mM, 20 min) without deterioration of the conversion or yield and without the detection of any reaction intermediates or reaction byproducts. Significantly, the same reaction was also found to be equally effective in a 50% acetonitrile/water mixed solvent, providing the corresponding 1,3,5-triazine 14a in excellent yield (93%) at a similarly dilute reaction concentration (5 mM, 20 min). Compared to the reactivity with amidines, 1,2,3,5-tetrazine 1d was found to react slower with imidate 15a, requiring an elevated reaction temperature, increased concentration, and a prolonged time to provide the 1,3,5-triazine product 14l in good yield (69%). The corresponding thioimidate 15b displayed a higher reactivity than 15a but less efficient production of the triazine 14l (22–31%) (Figure 4).

Figure 4.

Scope of the reaction of 1,2,3,5-tetrazine 1d with amidines 13a–n.

Additionally, the 4,6-dialkyl 1,2,3,5-tetrazine 1d was found to readily react with more traditional electron-rich dienophiles, including enamines (16a–d), ynamines (18a and 18b) and a ketene acetal (20) under mild reaction conditions to provide the corresponding pyrimidine products (17a–d, 19a–b, 21) (Figure 5). An exclusive C4/N1 cycloaddition mode was observed with a predictable regioselectivity where the most electron-rich atom in dienophile attaches to C4. The reactions between tetrazine 1d and cyclic (16a–c) or acyclic (16d) enamines were fast even at room temperature, but use of an elevated temperature was found to accelerate the slower, final re-aromatization step to provide the substituted pyrimidine products (17a–d, 47–83%, 66% avg). A similarly high reactivity was observed when tetrazine 1d was treated with ynamines (18a and 18b), providing the fully substituted pyrimidine products (19a and 19b) at ambient temperature with excellent yields (87–88%). The reaction between tetrazine 1d and ketene acetal 20 was found to provide pyrimidine product 21 in excellent yield (91%), although a higher reaction concentration (0.4 M) and a longer reaction time was required for complete reaction. Alternatively, a much faster reaction was observed at an elevated temperature (60 °C) in a reaction that was conducted at a more dilute reaction concentration (40 mM), albeit with a drop in the yield (61%) presumably due in part to the competitive thermal ring-opening reaction of tetrazine 1d. Enol ether 22, as a representative of a class of less electron-rich dienophiles, was found to react with 1,2,3,5-tetrazine 1d at a slower rate and required conducting the reaction neat at an increased reaction temperature (40 °C) for a prolonged reaction time to provide the pyrimidine 23 in modest yield. Combined, these studies indicate that there is little, or perhaps only subtle, distinctions in the relative reactivity of the 4,6-dialkyl-1,2,3,5-tetrazine 1d toward traditional electron-rich dienophiles from that displayed by our previously reported 4,6-diphenyl-1,2,3,5-tetrazine (1a).⁴¹

Figure 5.

Reaction of 1d with enamines, ynamines, a ketene acetal and enol ether.

A surprising orthogonal reactivity was found that distinguished 4,6-diphenyl-1,2,3,5-tetrazine (1a) and the isomeric 3,6-diphenyl-1,2,4,5-tetrazine with the former undergoing exclusive preferential reaction with amidine 13l and the latter with the strained alkyne 25.⁴¹ This prior observation with 1a led to examination of the analogous reactivity of the 4,6-dialkyl-1,2,3-5-tetrazine 1d. In order to directly compare the reactivity while minimizing the impact of the substituents, the isomeric 3,6-diphenethyl-1,2,4,5-tetrazine (24) was prepared. The comparison reactions were carried out under dilute reaction conditions (5 mM) in a 50% acetonitrile/water mixed solvent. The individual reactions between 1,2,3,5-tetrazine 1d and amidine 13a or 1,2,4,5-tetrazine 24 and cyclooctyne 25 were complete within 20 min, providing the 1,3,5-triazine product 14a (93%) or 1,2-diazine product 26 (88%), respectively, in excellent yields. In contrast, treatment of 1,2,3,5-tetrazine 1d with cyclooctyne 25 or 1,2,4,5-tetrazine 24 with amidine 13a led to no observable conversion of either tetrazine to cycloaddition products even at longer reaction times. Direct competitive experiments where a mixture of 1 equiv of each isomeric tetrazine was treated with 1 equiv of either dienophile provided the corresponding 1,3,5-triazine 14a or 1,2-diazine 26 in similarly excellent yields, with no detection of crossover products. (Figure 6A). In contrast to the fast reaction of 1,2,4,5-tetrazine 24 and cyclooctyne 25 conducted even at dilute reaction concentrations, the reaction of 1,2,3,5-tetrazine 1d with cyclooctyne 25 was only found to proceed at an increased concentration of reactants (0.4 M 1d vs 5 mM 24) with a prolonged reaction time (48 h vs 20 min) and even then provided the pyrimidine product 27 in modest yield (26%).

Figure 6.

(A) Orthogonal reactivity of 1,2,3,5-tetrazine 1d and its isomeric 1,2,4,5-tetrazine 24 with amidine 13a and cyclooctyne 25 as reaction partners. (B) Comparison of reactivity of tetrazine 1a and 1d with cyclooctyne 25.

Interestingly, and in contrast to their relative reactivities toward amidines (1d > 1a, >10-fold) or traditional electron-rich dienophiles (1d ≈ 1a), the reaction of 1d (4,6-(2-phenethyl)-1,2,3,5-tetrazine) with cyclooctyne 25 is slower and less effective than that of 1a (4,6-diphenyl-1,2,3,5-tetrazine)⁴¹ by as much as 10-fold or more (Figure 6B). Although this distinction in relative reactivities may be attributable to many different features, the most plausible explanation is that the reactions of amidines versus cyclooctynes (strained dienophile) are occurring by two different mechanisms, the latter being a concerted inverse electron demand Diels–Alder reaction. In addition, and a key element of this observation, the orthogonality of the amidine/1,2,3,5-tetrazine reaction is even more pronounced with the 4,6-dialkyl-versus 4,6-diphenyl-1,2,3,5-tetrazine, reacting both faster with amidines and even slower with cyclooctyne.

The comparison between the reactivity of isomeric tetrazines revealed a remarkable intrinsic reactivity of 1,2,3,5-tetrazines specifically with amidines (vs strained dienophiles), which is in sharp contrast to the superb reactivity of 1,2,4,5-tetrazines with strained alkenes/alkynes (vs amidines). Houk et al. systematically studied the reactivity of olefinic dienophiles with a series of heterocyclic azadiene systems in inverse electron demand Diels–Alder reactions in computational studies, and found that the outstanding reactivity of 1,2,4,5-tetrazines towards dienophiles could be attributed to both a lower distortion energy for both the diene and the dienophile as well as a large productive interaction energy (orbital overlap) in the reaction transition state.⁴⁹ Collectively, such differences underlie the higher reactivity of 1,2,4,5-tetrazine compared with 1,2,3,5-tetrazine (computational ΔE_act = 9.4 kcal/mol) towards olefinic dienophiles (computed for ethylene). The observation of the reversed reactivity of the two isomeric tetrazines with amidines as well as the unique reactivity trends between the 1,2,3,5-tetrazines 1a and 1d served as the incentive to further investigate the reaction mechanism of the newly discovered 1,2,3,5-tetrazine/amidine reaction that may explain these unusual features.

The 1,3,5-triazine products could possibly arise through a concerted, asynchronous, or stepwise addition/cyclization Diels–Alder reaction mechanism. However, formation of the products is also possible through an addition/N₂ elimination/cyclization mechanism with loss of N₂ prior to a penultimate cyclization. As a complement to computational studies conducted largely with 1,2,3-triazines⁵⁰ and to distinguish between these two possible mechanisms, reactions between tetrazines 1a–f and two doubly ¹⁵N-labeled amidines [¹⁵N]₂-13a and [¹⁵N]₂-13l were conducted as representative aryl and alkyl amidines, respectively. A complete selectivity for the generation of singly ¹⁵N-labeled 1,3,5-triazine products would be observed if the reaction progresses through either a concerted or stepwise addition/cyclization Diels–Alder reaction mechanism, while a mixture of singly and doubly ¹⁵N-labeled 1,3,5-triazine products would be expected if the reaction proceeds by stepwise addition/N₂ elimination/cyclization. The ratio of singly and doubly ¹⁵N-labeled 1,3,5-triazines in the isolated products was quantitated by two methods. Both ¹³C NMR analysis, quantitating the relative peak intensity of carbon signals split by ¹³C–¹⁵N coupling, and high-resolution mass spectrometry (HRMS) analysis, quantitating the ratio of isotopes, were utilized. The two independent methods provided reliable data that was in agreement with each other (difference <8% in all cases). The two plausible reaction pathways as well as the reaction outcomes (measured second ¹⁵N incorporation ratio and reported as the percentage of doubly ¹⁵N-labeled products) are summarized in Figure 7.

Figure 7.

¹⁵N labeling studies of the reaction between tetrazines 1a–f and amidines.

The observed ratio of singly and doubly ¹⁵N-labeled 1,3,5-triazine products (28a–f and 29a–f) was found to be highly dependent on the tetrazine substituents (R¹ and R²) as well as the labeled amidines (R³), ranging from predominant formation of singly ¹⁵N-labeled 1,3,5-triazines in products 29a–c and 29f to predominant formation of doubly ¹⁵N-labeled 1,3,5-triazines in products 28d and 28e, whereas the remaining products provided an intermediate ratio. This observation excludes a concerted or stepwise addition/cyclization Diels–Alder mechanism, since the subsequent loss of N₂ and elimination of ammonia from a bicyclic Diels–Alder cycloadduct would exclusively provide singly ¹⁵N-labeled 1,3,5-triazine products. The reaction between tetrazine 1a and amidine [¹⁵N]₂-13a provided a 1:1 mixture of singly and doubly ¹⁵N-labeled 1,3,5-triazine 28a in agreement with a stepwise addition/N₂ elimination/cyclization mechanism. In this case where all substituents are phenyl, an essentially identical pair (with the difference of isotopic nitrogens ignored) of tautomeric (6π-electro)cyclization precursors I and II would be expected to provide singly or doubly ¹⁵N-labeled 28a cyclized products with no preference. Similar observations were found in the triazine products generated from the reaction between the other diaryl substituted tetrazines 1b and 1c and labeled benzamidine [¹⁵N]₂-13a, with no significant substituent electronic impact on the reaction outcome. In sharp contrast, the predominant products formed between the same labeled amidine [¹⁵N]₂-13a and the 4,6-dialkyl-1,2,3,5-tetrazines 1d and 1e were the doubly ¹⁵N-labeled 1,3,5-triazines 28d and 28e. The reaction outcome with the alkyl amidine [¹⁵N]₂-13l and the diaryl substituted tetrazines 1a–c provided a reversed distribution of labeled 1,3,5-triazine products 29a–c, with predominant formation of singly labeled product. The observed shift in distribution revealed a trend where the (electro)cyclization-elimination favors loss of the terminal nitrogen closest to an alkyl substituent in the conjugated triazatriene system (I and II), a feature that may be attributed to preferential aryl conjugation with the triazatriene system. This selectivity in the cyclization step that is dominated by the nature of the substituents (R¹ and R³) rather than their source (from tetrazine or amidine) further supports the intermediacy of the tautomers I and II (versus a Diels–Alder adduct) as key intermediates in the reaction. The product ratio with triazines 29d and 29e was found to be in agreement with the likely reaction mechanism, and only a slight deviation from an expected 1:1 ratio between singly and doubly labeled triazines was observed.

The product distribution found in the triazines 28f and 29f provide an extension of these observed trends to unsymmetrical 1,2,3,5-tetrazines, further support the reaction mechanism, and add additional insights. Of the two possible singly ¹⁵N-labeled triazine isomers, only the isomer that arises from the labeled amidine nucleophilic attack ([¹⁵N]₂-13a or 13l) at the tetrazine carbon bearing the alkyl substituent was observed and that derived from attack at the carbon bearing an aryl substituent was found to be negligible. Thus, not only is the product distribution between singly and doubly ¹⁵N-labeled triazine products in agreement with the addition/N₂ elimination/cyclization mechanism but the ¹⁵N distribution in the labeled 1,3,5-triazine products 28f and 29f also defines the regioselectivity of such reactions. Notably, this now predictable regioselectivity with unsymmetrical tetrazine 1f is not limited to the tetrazine/amidine reaction but is also observed with electron-rich dienophiles (e.g.; ynamine 18a, Figure 8). Here, the reaction not only proceeds by a single mode of cycloaddition (only C4/N1 vs N2/N5) with a predictable regioselectivity where the dienophile nucleophilic carbon is attached to C1 (not N4), but also provides a single regioisomer in which the more nucleophilic or electron-rich dienophile atom preferentially attacks the tetrazine carbon bearing the alkyl versus aryl substituent.

Figure 8.

Regioselective cycloaddition of the unsymmetrical tetrazine 1f with ynamine 18a.

Finally, the reaction mechanism is further supported by the reaction kinetics of 4,6-diphenyl-1,2,3,5-tetrazine (1a) with a series of p-substituted benzamidines previously disclosed⁵⁰ (Supporting Information Figure S11–S14). A well-defined correlation was observed between the electronic character of the substituent (σ_p) and the reaction rate consistent with the amidine serving as the electron-rich partner in this reaction (Supporting Information Figure S15). A Hammett plot of the rate constant (log k/k_H) versus the substituent σ_p is linear with a ρ value of −1.26. This large observed electronic effect indicates substantial partial positive charge accumulation on the amidine carbon in the rate-determining step in agreement with the reaction being initiated by a rate-determining nucleophilic attack of the amidines onto the 1,2,3,5-tetrazine. Combined, the experimental evidence detailed herein support a stepwise addition/N₂ elimination/cyclization mechanism,⁵⁰ deciphering the outstanding intrinsic reactivity of 1,2,3,5-tetrazines specifically with amidines (vs strained dienophiles) as well as the underlying basis for the observed orthogonal reactivity with 1,2,4,5-tetrazines.

CONCLUSIONS

Herein, we disclose the first general approach for the efficient synthesis of a new class of previously unexplored heterocyclic azadienes, 1,2,3,5-tetrazines with C4/C6 diaryl, dialkyl, or mixed aryl/alkyl substituents from simple α-diketones. Key elements of the approach include a final oxidative aromatization, adoption of a beautiful cycloaddition cascade⁴³ for the preparation of precursor N²,N⁵-diaryl dihydro-1,2,3,5-tetrazines, and use of a N²,N⁵-benzisoxazole auxiliary and its reductive ring-opening for cleavable removal. Six 4,6-disubstituted 1,2,3,5-tetrazines were prepared, characterized, and their intrinsic and cycloaddition reactivities defined, providing the first study of the electronic and steric effects of 1,2,3,5-tetrazine substituents. A detailed study of 4,6-di(2-phenethyl)-1,2,3,5-tetrazine (1d) was conducted as a representative of alkyl substituted 1,2,3,5-tetrazines, and its cycloaddition reactivity, cycloaddition mode (C4/N1 vs N2/N5), regioselectivity, and scope were defined. In these studies, it was established that dialkyl 1,2,3,5-tetrazines are even more reactive (>10-fold) than diaryl substituted derivatives toward amidines, display qualitatively equivalent reactivity toward traditional electron-rich dienophiles, and are markedly slower with the strained cyclooctyne dienophile examined (>10-fold). The orthogonal reactivity of the 1,2,3,5-tetrazine/amidine reaction and the well-established 1,2,4,5-tetrazine-based strained alkene/alkyne cycloaddition was further generalized and the former was characterized by additional kinetic and labeling studies. A stepwise addition/N₂ elimination/cyclization mechanism elucidated first in computational studies⁵⁰ was supported by the further experimental results and was found to be general to all 1,2,3,5-tetrazines detailed herein, whereas the alternative concerted or stepwise addition/cyclization Diels–Alder mechanism proved inconsistent with the results. The proposed mechanism provides a basis for understanding the remarkable reactivity of 1,2,3,5-tetrazines with amidines (k = 10⁻¹ to 2 M⁻¹·s⁻¹) that differs from its conventional pericyclic reactions with more traditional dienophiles (e.g., strained alkenes/alkynes). This not only provides an explanation for the observed orthogonality with 1,2,4,5-tetrazine-based ligations, but also likely extends to the observed reaction orthogonality between 1,2,3-triazines versus 1,2,4-triazines.^51,52 The studies described herein provide the foundation for the effective synthesis and use of this unexplored class of heterocyclic azadienes and its powerful and now predictable reactions in organic synthesis, medicinal chemistry, material science, and (bio)orthogonal conjugation and labeling studies.

EXPERIMENTAL SECTION

General Methods.

All reagents and solvents were used as supplied without further purification unless otherwise noted. CHCl₃ was pre-treated with alumina for at least 24 h prior to use. Amidines were purchased as their hydrochloride salts and free-based by treatment with 2 M KOH (aq). Enamines and ynamines were prepared as reported in the literature.^53–58 Preparative TLC (PTLC) and column chromatography were conducted using Millipore SiO₂ 60 F₂₅₄ PTLC (0.5 mm) and Zeochem ZEOprep 60 ECO SiO₂ (40–63 μm), respectively. Analytical TLC was conducted using Millipore SiO₂ 60 F254 TLC (0.250 mm) plates. ¹H and ¹³C NMR spectra were obtained on a Bruker Avance III HD 600 MHz spectrometer equipped with either a 5 mm QCI or 5 mm CPDCH probe. IR spectra were obtained on a Thermo Nicolet 380 FT-IR with a SmartOrbit Diamond ATR accessory. Mass spectrometry analysis was performed by direct sample injection on an Agilent G1969A ESI-TOF mass spectrometer. Melting points are uncorrected. The single-crystal X–ray diffraction studies were carried out on a Bruker Kappa APEX-II CCD diffractometer equipped with Mo Kα radiation (λ = 0.71073).

Synthesis of 1,2,3,5-tetrazines 1a–f.

Intermediates 10, 12 and the final tetrazines 1 were found not to be completely stable to long-term storage at room temperature and storage at lower temperatures (4 °C or −20 °C) upon isolation and purification is suggested.

3-Amino-1,2-benzisoxazole.

3-Amino-1,2-benzisoxazole was prepared following a reported procedure⁵⁹ and detailed herein: acetone oxime (32.2 g, 440 mmol) and DMF (400 mL) were added to a 1 L round-bottom flask. t-BuOK (49.2 g, 440 mmol) was added, and the mixture was stirred at 25 °C for 30 min. The mixture was cooled to 0 °C, and 2-fluorobenzonitrile (53.2 g, 440 mmol) was added dropwise. The resulting mixture was stirred at 25 °C for 1 h and then poured into a vigorously stirred mixture of Et₂O (800 mL) and saturated aqueous NH₄Cl (800 mL). The mixture was stirred for 10 min and the organic phase was separated. The aqueous phase was extracted with Et₂O (400 mL). The organic phases were combined and dried over Na₂SO₄. The mixture was filtered and concentrated to provide 2-((propan-2-ylideneamino)oxy)benzonitrile as a yellow oil (74 g, crude yield: 97%). 2-((Propan-2-ylideneamino)oxy)benzonitrile (crude) was dissolved in a mixture of EtOH (300 mL) and aqueous 2 N HCl (300 mL) in a 1 L round-bottom flask. The mixture was stirred at reflux (oil bath) for 1 h before it was cooled to 25 °C and EtOH was removed under reduced pressure. The resulting suspension was basified with the addition of Na₂CO₃ and the suspension was extracted with EtOAc (300 mL × 3). The organic phases were combined and dried over Na₂SO₄. The mixture was filtered, concentrated, and purified by column chromatography (SiO₂, 50% EtOAc/hexanes), and recrystallized (CH₂Cl₂/hexanes) to provide 3-amino-1,2-benzisoxazole (41.2 g, 70%) as an off-white solid identical in all respects with reported material.⁵⁹

N-Sulfinyl-3-amino-1,2-benzisoxazole.

Thionyl chloride (0.37 mL, 0.60 g, 5 mmol, 0.5 equiv) was added to a stirred solution of imidazole (1.36 g, 20 mmol, 2 equiv) in anhydrous CH₂Cl₂ (20 mL) at 25 °C. The mixture was stirred at 25 °C for 10 min, filtered, and thionyl chloride (0.37 mL, 0.6 g, 5 mmol, 0.5 equiv) was added to the filtrate. The mixture was stirred at 25 °C for another 10 min and was added to a stirred suspension of 1,2-benzisoxazole-3-amine (1.34 g, 10 mmol, 1 equiv) in anhydrous CH₂Cl₂ (10 mL). The resulting mixture was stirred at 25 °C for 30 min and then filtered over Celite. The solid residue was quickly washed with anhydrous CH₂Cl₂ (10 mL). The filtrate was concentrated to provide N-sulfinyl-1,2-benzisoxazole-3-amine as a viscous brown oil which was directly and immediately used without further purification.

1,2-Benzisoxazole-3-hydrazine hydrochloride.

Although the preparation of 1,2-benzisoxazole-3-hydrazine is reported in the literature⁶⁰ by S_NAr approach starting from 3-chloro-1,2-benzisoxazole and hydazine, a relatively low yield (9–11%) was observed by the authors and the acquirement of pure sample enlisted inefficient extractions. Herein, we adopted a Japp-Klingemann-type approach developed by Norris et al.⁶¹ for this specific substrate, allowing multi-gram scale preparation of this compound from the corresponding diazonium salt. 3-Amino-1,2-benzisoxazole (8.04 g, 60 mmol) was dissolved in a mixture of EtOH (30 mL) and HBF₄ (50% aqueous solution, 21.1 g) in a 500 mL round-bottom flask. The mixture was cooled to 0 °C and t-BuONO (13.68 g, 120 mmol) was added dropwise over 10 min. The mixture was stirred at 0 °C for an additional 1 h before ice-cold Et₂O (120 mL) was added to quench the reaction. The mixture was stirred at 0 °C for another 10 min and was quickly filtered through a Buchner funnel. The yellow solid was quickly washed with ice-cold Et₂O (20 mL × 3), and dried under vacuum for 20 min to provide 1,2-benzisoxazole-3-diazonium tetrafluoroborate (6.98–7.45 g, 50–53%), which was immediately used upon preparation. Extreme caution!! The diazonium salt may be explosive upon heating or detonation, although no shock or explosion of the diazonium salt was observed by the authors in this work. Experimenter should wear full PPE and use a shield during work-up. 1,2-Benzisoxazole-3-diazonium tetrafluoroborate (7.45 g, 31.9 mmol) was suspended in CH₃CN (128 mL) at 5 °C. L-Ascorbic acid (6.08 g, 34.5 mmol) was added to the mixture at 5 °C. The mixture was stirred at 5 °C for 2 h and then at 25 °C for 18 h. The solvent was then removed to provide a yellow amorphous solid. The yellow solid was dissolved in MeOH (64 mL) and 4 N HCl in dioxane (32 mL). The mixture was stirred at reflux (oil bath) for 2 h before it was cooled to 25 °C, and Et₂O (400 mL) was added portionwise with vigorous stirring. The resulting mixture was filtered, washed with Et₂O (100 mL) and dried under vacuum to provide 1,2-benzisoxazole-3-hydrazine hydrochloride in complex with ½ dioxane (6.16 g, 83%) as an off-white solid: mp 182 °C (decomp); ¹H NMR (600 MHz, D₂O, 298K) δ 7.75 (t, J = 8.4 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 3.76 (s, 4H); ¹³C{¹H} NMR (151 MHz, D₂O, 298K) δ 163.8, 157.9, 132.3, 124.5, 121.4, 114.1, 110.8, 67.2; IR (film) ν_max 3098, 3031, 2829, 2647, 1612, 1569, 1403, 1377, 1111, 1079, 864, 804, 746, 703 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₇H₈N₃O 150.0667; Found 150.0668.

1,2-Bis(2-(benzo[d]isoxazole-3-yl)hydrazineylidene)-1,2-diphenylethane (9a).

Benzil (420 mg, 2 mmol, 1 equiv) and 1,2-benzisoxazole-3-hydrazine hydrochloride (1.01 g, 4.4 mmol, 2.2 equiv, as ½ dioxane complex) were suspended in EtOH (10 mL) in a 25 mL round-bottom flask. The mixture was stirred at reflux (oil bath) for 18 h. The mixture was cooled to 25 °C, and the product was collected by filtration and washed with EtOH (10 mL) to provide 9a (851 mg, 90%) as an off-white solid: mp 248–250 °C; IR (film) ν_max 3221, 1605, 1552, 1491, 1443, 1419, 1275, 1107, 910, 884, 744, 695, 612 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₂₁N₆O₂ 473.1726; Found 473.1720. Bis-hydrazone 9a exhibited low solubility in common NMR solvents and NMR data was not recorded.

Benzo[d]isoxazole-3-yl-(2-(benzo[d]isoxazole-3-yl)-4,5-diphenyl-1H-1,2,3-triazol-2-ium-1-yl)amide (10a).

Compound 9a (851 mg, 1.8 mmol, 1 equiv) was suspended in CH₃CN/H₂O (27 mL, v/v = 9:1) in a 100 mL round-bottom flask. A solution of ceric ammonium nitrate (CAN, 2.96 g, 5.40 mmol, 3 equiv) in CH₃CN/H₂O (27 mL, v/v = 9:1) was added to the suspension dropwise at 25 °C, and the reaction mixture was stirred at 25 °C for 30 min. The mixture was filtered to provide a brown solid, which was then resuspended in a mixture of 4 M HCl in 1,4-dioxane (1.8 mL, 4 equiv HCl) in CH₂Cl₂ (54 mL). The resulting mixture was further stirred at 25 °C for 3 h before it was quenched with the addition of saturated aqueous NaHCO₃ (50 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (50 mL × 2). The organic layers were combined, dried over Na₂SO₄, and filtered. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 80% Et₂O/hexanes) to provide 10a (709 mg, 83%) as an orange solid: mp 109–111 °C; NMR recorded in the presence of trifluoroacetic acid (TFA): ¹H NMR (600 MHz, CD₃CN, 298K) δ 8.28 (d, J = 8.2 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.83 (t, J = 8.4 Hz, 2H), 7.77 (d, J = 7.9 Hz, 2H), 7.74−7.68 (m, 5H), 7.66 (t, J = 7.5 Hz, 1H), 7.61 (t, J = 7.9 Hz, 2H), 7.58−7.55 (m, 3H), 7.44 (t, J = 7.7 Hz, 1H); ¹³C{¹H} NMR (151 MHz, CD₃CN, 298K) δ 165.8, 165.3, 157.0, 151.2, 150.4, 147.0, 134.7, 134.3, 133.1, 132.9, 130.9, 130.8, 130.5, 129.5, 128.0, 126.6, 125.5, 123.0, 122.0, 121.4, 115.1, 113.6, 112.0, 111.4; IR (film) ν_max 3053, 1609, 1496, 1432, 1332, 1266, 1240, 1089, 880, 734, 696 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₁₉N₆O₂ 471.1569; Found 471.1563.

2,5-Di(benzo[d]isoxazole-3-yl)-4,6-diphenyl-2,5-dihydro-1,2,3,5-tetrazine (11a).

N-Sulfinyl-1,2-benzisoxazole-3-amine (1.35 g, 7.5 mmol, 5 equiv) was added to a suspension of 10a (709 mg, 1.5 mmol, 1 equiv) in anhydrous CH₃CN (3.75 mL) in a reaction vessel. The mixture was stirred under Ar at 25 °C for 18 h. The solution was concentrated and the residue was purified by column chromatography (SiO₂, 75–100% CH₂Cl₂/hexanes) to provide 11a (433 mg, 61%) as a yellow solid: mp 231–233 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.30 (d, J = 8.0 Hz, 1H), 8.19 (dd, J = 7.9, 1.5 Hz, 4H), 7.62−7.58 (m, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.51 (q, J = 7.2, 6.2 Hz, 6H), 7.46−7.37 (m, 4H), 7.11 (ddd, J = 8.1, 6.0, 1.9 Hz, 1H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.5, 164.3, 159.8, 155.1, 144.5, 131.8, 130.6, 130.49, 130.47, 129.1, 128.2, 124.5, 124.1, 123.6, 121.6, 115.7, 115.5, 110.6, 110.3; IR (film) ν_max 3075, 2927, 1609, 1527, 1439, 1385, 1331, 1238, 906, 691, 616 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₁₉N₆O₂ 471.1569; Found 471.1570.

4,6-Diphenyl-2-(2-hydroxyphenylimidoyl)-2,5-dihydro-1,2,3,5-tetrazine (12a).

Palladium on carbon (10% Pd, 428 mg) was added to a suspension of 11a (428 mg, 0.91 mmol, 1 equiv) in EtOAc (43 mL). H₂ was bubbled through the mixture for 10 min and the mixture was stirred under H₂ (1 atm) at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 2.5% MeOH/CH₂Cl₂) to provide 12a (200 mg, 62%) as an orange solid: mp 96–98 °C; ¹H NMR (600 MHz, CD₃OD, 298K) δ 8.10 (s, br, 4H), 7.55 (dd, J = 7.7, 1.6 Hz, 1H), 7.51 (t, J = 7.3 Hz, 2H), 7.44 (q, J = 7.2, 6.4 Hz, 5H), 7.06−6.99 (m, 2H); ¹³C{¹H} NMR (151 MHz, CD₃OD, 298K) δ 166.8, 157.9, 135.9, 133.8, 132.4, 131.4, 129.2, 129.0, 120.5, 118.7, 118.1; IR (film) νmax 3063, 1641, 1543, 1446, 1397, 1335, 1297, 1265, 1146, 731, 691 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₁H₁₈N₅O 356.1511; Found 356.1515.

4,6-Diphenyl-1,2,3,5-tetrazine (1a).

PbO₂ (480 mg, 2 mmol, 4 equiv) was added to a solution of 12a (178 mg, 0.5 mmol, 1 equiv) in CH₂Cl₂ (25 mL) and the mixture was stirred at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 20% Et₂O/hexanes) to provide 1a (73.4 mg, 63%) as a yellow solid identical to our authentic sample.⁴¹

1,2-Bis(2-(benzo[d]isoxazole-3-yl)hydrazineylidene)-1,2-bis(4-methoxyphenyl)ethane (9b).

4,4’-Dimethoxybenzil (540 mg, 2 mmol, 1 equiv) and 1,2-benzisoxazole-3-hydrazine hydrochloride (1.01 g, 4.4 mmol, 2.2 equiv, as ½ dioxane complex) were suspended in EtOH (10 mL) in a 25 mL round-bottom flask. The mixture was stirred at reflux (oil bath) for 18 h. The mixture was cooled to 25 °C, and the product was collected by filtration and washed with EtOH (10 mL) to provide 9b (1.03 g, 97%) as a white solid: mp 234–236 °C; IR (film) ν_max 3193, 1650, 1604, 1554, 1507, 1444, 1245, 1174, 1105, 1029, 893, 878, 817, 621 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₀H₂₅N₆O₄ 533.1937; Found 533.1933. Bis-hydrazone 9b exhibited low solubility in common NMR solvents and NMR data was not recorded.

Benzo[d]isoxazol-3-yl(2-(benzo[d]isoxazol-3-yl)-4,5-bis(4-methoxyphenyl)-1H-1,2,3-triazol-2-ium-1-yl)amide (10b).

Compound 9b (1.03 g, 1.94 mmol, 1 equiv) was suspended in CH₃CN/H₂O (30 mL, v/v = 9:1) in a 100 mL round-bottom flask. A solution of CAN (3.19 g, 5.82 mmol, 3 equiv) in CH₃CN/H₂O (30 mL, v/v = 9:1) was added to the suspension dropwise at 25 °C, and the reaction mixture was stirred at 25 °C for 30 min. The mixture was filtered to provide a brown solid, which was then resuspended in a mixture of 4 M HCl in 1,4-dioxane (2 mL, 4 equiv HCl) in CH₂Cl₂ (60 mL). The resulting mixture was further stirred at 25 °C for 3 h before it was quenched with the addition of saturated aqueous NaHCO₃ (50 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (50 mL × 2). The organic layers were combined, dried over Na₂SO₄, and filtered. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 66% EtOAc/hexanes) to provide 10b (740 mg, 72%) as an orange solid: mp 88–90 °C; NMR recorded in the presence of trifluoroacetic acid (TFA): ¹H NMR (600 MHz, CD₃CN, 298K) δ 8.26 (d, J = 8.2 Hz, 1H), 7.87 (dt, J = 7.1, 3.3 Hz, 2H), 7.80 (d, J = 8.6 Hz, 1H), 7.72–7.66 (m, 6H), 7.55 (d, J = 8.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 8.8 Hz, 4H), 3.88 (s, 3H), 3.84 (s, 3H); ¹³C{¹H} NMR (151 MHz, CD₃CN, 298K) δ 165.6, 165.2, 164.8, 163.4, 157.1, 151.2, 150.1, 146.5, 134.1, 133.03, 133.00, 131.2, 127.8, 125.5, 123.1, 122.1, 116.3, 115.9, 115.1, 113.8, 113.1, 113.0, 111.9, 111.4, 56.6, 56.4; IR (film) ν_max 2932, 1609, 1495, 1463, 1434, 1301, 1248, 1178, 1107, 1028, 881, 837 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₀H₂₃N₆O₄ 531.1781; Found 531.1794.

2,5-Di(benzo[d]isoxazole-3-yl)-4,6-di(4-methoxyphenyl)-2,5-dihydro-1,2,3,5-tetrazine (11b).

N-Sulfinyl-1,2-benzisoxazole-3-amine (1.26 g, 7.0 mmol, 5 equiv) was added to a suspension of 10b (740 mg, 1.4 mmol, 1 equiv) in anhydrous CH₃CN (3.5 mL) in a reaction vessel. The mixture was stirred under Ar at 25 °C for 18 h. The solution was concentrated and the residue was purified by column chromatography (SiO₂, CH₂Cl₂) to provide 11b (484 mg, 65%) as a yellow solid: mp 197 °C (decomp); ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.29 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.8 Hz, 4H), 7.61−7.56 (m, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 6.1 Hz, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.14−7.10 (m, 1H), 6.99 (d, J = 8.8 Hz, 4H), 3.87 (s, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.4, 164.3, 162.4, 159.9, 155.3, 144.6, 130.6, 130.4, 129.9, 124.6, 124.1, 123.40, 122.7, 121.8, 115.9, 115.7, 114.5, 110.5, 110.2, 55.6; IR (film) ν_max 2923, 1606, 1511, 1439, 1312, 1257, 1172, 1092, 899, 751, 631 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₀H₂₃N₆O₄ 531.1781; Found 531.1791.

4,6-Di(4-methoxyphenyl)-2-(2-hydroxyphenylimidoyl)-2,5-dihydro-1,2,3,5-tetrazine (12b).

Palladium on carbon (10% Pd, 484 mg) was added to a suspension of 11b (484 mg, 0.91 mmol, 1 equiv) in EtOAc (48 mL). H₂ was bubbled through the mixture for 10 min and the mixture was stirred under H₂ (1 atm) at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 10% MeOH/CH₂Cl₂) to provide 12b (236 mg, 63%) as an orange solid: mp 148 °C (decomp); ¹H NMR (600 MHz, CD₃OD, 298K) δ 8.00 (d, J = 7.8 Hz, 4H), 7.55 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.04−6.98 (m, 6H), 3.85 (s, 6H); ¹³C{¹H} NMR (151 MHz, CD₃OD, 298K) δ 164.2, 163.7, 158.0, 148.4, 134.0, 131.3, 130.7, 126.5, 120.5, 119.0, 117.9, 114.7, 55.9; IR (film) ν_max 2933, 2838, 1604, 1590, 1537, 1512, 1410, 1334, 1250, 1160, 1028, 841, 770, 734 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₃H₂₂N₅O₃ 416.1732; Found 416.1732.

4,6-Di(4-methoxyphenyl)-1,2,3,5-tetrazine (1b).

PbO₂ (500 mg, 2.08 mmol, 4 equiv) was added to a solution of 12b (216 mg, 0.52 mmol, 1 equiv) in CH₂Cl₂ (25 mL) and the mixture was stirred at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue purified by column chromatography (SiO₂, CH₂Cl₂) to provide 1b (118 mg, 77%) as a yellow solid: mp 150 °C (decomp); ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.70 (d, J = 8.9 Hz, 4H), 7.09 (d, J = 8.9 Hz, 4H), 3.94 (s, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.6, 162.5, 131.5, 125.1, 114.7, 55.8; IR (film) ν_max 2929, 1607, 1479, 1429, 1312, 1265, 1166, 1024, 931, 813, 635 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₆H₁₅N₄O₂ 295.1195; Found 295.1182.

Dimethyl 4,4’-(1,2-bis(2-(benzo[d]isoxazol-3-yl)hydrazineylidene)ethane-1,2-diyl)dibenzoate (9c).

4,4’-Di(methoxycarbonyl)benzil (1.54 g, 4.7 mmol, 1 equiv) and 1,2-benzisoxazole-3-hydrazine hydrochloride (3.26 g, 14.2 mmol, 3 equiv, as ½ dioxane complex) were suspended in dioxane (24 mL) in a 50 mL round-bottom flask. The mixture was stirred at reflux (oil bath) for 18 h. The mixture was cooled to 25 °C, and the product was collected by filtration and washed with EtOH (20 mL) to provide 9c (2.73 g, 96%) as a white solid: mp >250 °C; IR (film) ν_max 3175, 3153, 1714, 1608, 1554, 1420, 1403, 1272, 1110, 901, 753, 712 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₂H₂₅N₆O₆ 589.1836; Found 589.1844. Bis-hydrazone 9c exhibited low solubility in common NMR solvents and NMR data was not recorded.

Benzo[d]isoxazol-3-yl(2-(benzo[d]isoxazol-3-yl)-4,5-bis(4-(methoxycarbonyl)phenyl)-1H-1,2,3-triazol-2-ium-1-yl)amide (10c).

Compound 9c (3.10 g, 5.27 mmol, 1 equiv) was suspended in CH₃CN/H₂O (75 mL, v/v = 9:1) in a 250 mL round-bottom flask. A solution of CAN (9.56 g, 17.4 mmol, 3 equiv) in CH₃CN/H₂O (75 mL, v/v = 9:1) was added to the suspension dropwise at 25 °C, and the reaction mixture was stirred at 25 °C for 30 min. The mixture was filtered to provide a brown solid, which was then resuspended in a mixture of 4 M HCl in 1,4-dioxane (5.3 mL, 4 equiv HCl) in CH₂Cl₂ (150 mL). The resulting mixture was warmed at 40 °C (oil bath) for 12 h before it was quenched with the addition of saturated aqueous NaHCO₃ (50 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (50 mL × 2). The organic layers were combined, dried over Na₂SO₄, and then filtered. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 5% Et₂O/CH₂Cl₂) to provide 10c (2.26 g, 73%) as an orange solid: mp 90–92 °C; NMR recorded in the presence of trifluoroacetic acid (TFA): ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.22 (d, J = 8.6 Hz, 2H), 8.19 (d, J = 8.6 Hz, 2H), 8.16 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.82−7.78 (m, 3H), 7.78 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.7 Hz, 1H), 7.68 (t, J = 8.4 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 4.03 (s, 3H), 3.99 (s, 3H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 167.7, 166.9, 164.9, 164.3, 156.4, 150.1, 145.4, 134.5, 133.2, 132.8, 132.6, 131.3, 131.0, 130.3, 129.5, 128.7, 127.1, 124.9, 124.5, 121.8, 121.7, 113.7, 112.7, 111.2, 110.4, 53.59, 53.57; IR (film) ν_max 2951, 1721, 1610, 1497, 1433, 1405, 1329, 1276, 1241, 1109, 959, 734, 705 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₂H₂₃N₆O₆ 587.1679; Found 587.1695.

2,5-Di(benzo[d]isoxazole-3-yl)-4,6-di(4-methoxycarbonylphenyl)-2,5-dihydro-1,2,3,5-tetrazine (11c).

N-Sulfinyl-1,2-benzisoxazole-3-amine (3.32 g, 18.5 mmol, 5 equiv) was added to a suspension of 10c (2.16 g, 3.7 mmol, 1 equiv) in anhydrous CH₃CN (9 mL) in a reaction vessel. The mixture was warmed under Ar at 60 °C (oil bath) for 18 h. The residue was concentrated and the residue was purified by column chromatography (SiO₂, EtOAc/CH₂Cl₂/hexanes, v/v/v = 1:3:6) to provide 11c (720 mg, 33%) as an orange solid: mp 212–214 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.28−8.23 (m, 5H), 8.17 (d, J = 8.7 Hz, 4H), 7.64−7.60 (m, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 5.9, 1.1 Hz, 2H), 7.43−7.40 (m, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.12 (ddd, J = 8.1, 5.9, 2.0 Hz, 1H), 3.95 (s, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 166.3, 164.6, 164.4, 159.3, 154.9, 143.1, 134.5, 132.9, 130.9, 130.7, 130.3, 128.0, 124.4, 124.2, 123.8, 121.1, 115.3, 115.2, 110.8, 110.4, 52.6; IR (film) ν_max 2955, 1726, 1608, 1530, 1438, 1405, 1274, 1239, 1108, 905, 764, 750, 708 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₂H₂₃N₆O₆ 587.1679; Found 587.1691.

4,6-Di(4-methoxycarbonylphenyl)-2-(2-hydroxyphenylimidoyl)-2,5-dihydro-1,2,3,5-tetrazine (12c).

Palladium on carbon (10% Pd, 300 mg) was added to a suspension of 11c (300 mg, 0.51 mmol, 1 equiv) in THF/t-BuOH (240 mL, v/v = 1:1), followed by the addition of 2,6-lutidine (165 mg, 1.53 mmol, 3 equiv). H₂ was bubbled through the mixture for 10 min and the mixture was stirred under H₂ (1 atm) at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, EtOAc/CH₂Cl₂/hexanes, v/v/v = 1:1.5:1) to provide 12c (61 mg, 25%) as a red solid: mp 190–192 °C; ¹H NMR (600 MHz, DMSO-d₆, 298K) δ 10.03 (s, 1H), 8.52 (d, J = 8.5 Hz, 2H), 8.03 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 8.6 Hz, 2H), 7.40−7.35 (m, 2H), 6.98 (d, J = 7.9 Hz, 1H), 6.94 (t, J = 8.0 Hz, 1H), 3.90 (s, 3H), 3.85 (s, 3H), 3.17 (d, J = 5.2 Hz, 1H); ¹³C{¹H} NMR (151 MHz, DMSO-d₆, 298K) δ 166.1, 165.9, 164.3, 162.4, 155.3, 143.1, 140.0, 139.9, 131.6, 131.4, 131.1, 130.1, 128.7, 128.6, 127.6, 126.8, 118.7, 117.1, 115.9, 52.3, 52.2; IR (film) ν_max 3346, 2924, 1712, 1587, 1543, 1439, 1276, 1111, 1018, 865, 736, 694, 606 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₂N₅O₅ 472.1621; Found 472.1607.

4,6-Di(4-methoxycarbonylphenyl)-1,2,3,5-tetrazine (1c).

PbO₂ (48 mg, 0.2 mmol, 4 equiv) was added to a solution of 12c (23.6 mg, 0.05 mmol, 1 equiv) in THF (2.5 mL) and the mixture was stirred at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 5% Et₂O/CH₂Cl₂) to provide 1c (10.1 mg, 58%) as a yellow solid: mp 166 °C (decomp); ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.84 (d, J = 8.7 Hz, 4H), 8.29 (d, J = 8.7 Hz, 4H), 4.00 (s, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 165.7, 162.1, 135.6, 134.6, 129.9, 128.9, 52.2; IR (film) ν_max 2956, 2926, 2857, 1726, 1475, 1434, 1278, 1104, 1013, 910, 760, 693 cm⁻¹; No observable ionization of the title compound was observed with either a ESI or APCI ionization source, while GC-MS for ionization provided only the corresponding nitrile. The structure of the tetrazine is supported by other spectroscopic data.

3,3’-((1,6-Diphenylhexane-3,4-diylidene)bis(isoxazole-1-yl-2-ylidene))bis(benzo[d]isoxazole) (9d).

1,6-Diphenyl-3,4-hexadione (1.20 g, 4.5 mmol, 1 equiv) and 1,2-benzisoxazole-3-hydrazine hydrochloride (3.10 g, 13.5 mmol, 3 equiv, as ½ dioxane complex) were suspended in EtOH (24 mL) in a 50 mL round-bottom flask. The mixture was stirred at reflux (oil bath) for 18 h. The mixture was cooled to 25 °C, and the product was collected by filtration and washed with EtOH (10 mL) to provide 9d (2.30 g, 97%) as a white solid: mp 229–231 °C; IR (film) ν_max 3205, 3107, 3049, 1607, 1558, 1494, 1470, 1433, 1240, 1124, 911, 755, 718, 612 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₂H₂₉N₆O₂ 529.2352; Found 529.2357. Bis-hydrazone 9d exhibited low solubility in common NMR solvents and NMR data was not recorded.

Benzo[d]isoxazole-3-yl(2-(benzo[d]isoxazole-3-yl)-4,5-diphenethyl-1H-1,2,3-triazol-2-ium-1-yl)amide (10d).

Compound 9d (2.30 g, 4.36 mmol, 1 equiv) was suspended in CH₃CN/H₂O (65 mL, v/v = 9:1) in a 250 mL round-bottom flask. A solution of CAN (7.12 g, 13 mmol, 3 equiv) in CH₃CN/H₂O (65 mL, v/v = 9:1) was added to the suspension dropwise at 25 °C, and the reaction mixture was stirred at 25 °C for 30 min. The mixture was filtered to provide a brown solid, which was then resuspended in a mixture of 4 M HCl in 1,4-dioxane (4.4 mL, 4 equiv HCl) in CH₂Cl₂ (130 mL). The resulting mixture was further stirred at 25 °C for 3 h before it was quenched with the addition of saturated aqueous NaHCO₃ (50 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (50 mL × 2). The organic layers were combined, dried over Na₂SO₄, and filtered. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 10% acetone/CH₂Cl₂) to provide 10d (1.77 g, 77%) as a yellow solid: mp 54–56 °C; NMR recorded in the presence of trifluoroacetic acid (TFA): ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.99 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.78−7.71 (m, 2H), 7.64 (d, J = 8.6 Hz, 1H), 7.56−7.52 (m, 2H), 7.51 (t, J = 7.7 Hz, 1H), 7.37 (t, J = 7.4 Hz, 2H), 7.32−7.27 (m, 4H), 7.16 (d, J = 7.1 Hz, 2H), 7.13 (d, J = 6.2 Hz, 2H), 3.26 (s, br, 2H), 3.07 (t, J = 7.6 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H), 2.84 (s, br, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.60, 164.55, 156.4, 151.5, 150.3, 149.7, 138.9, 137.8, 133.2, 132.8, 129.4, 129.2, 128.63, 128.61, 127.9, 127.3, 126.9, 125.2, 122.0, 121.3, 113.2, 112.8, 111.0, 110.7, 33.3, 33.1, 27.2, 26.2; IR (film) ν_max 3026, 2929, 1609, 1496, 1434, 1368, 1241, 1153, 895, 881, 749, 700, 620 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₂H₂₇N₆O₂ 527.2195; Found 527.2209.

2,5-Di(benzo[d]isoxazole-3-yl)-4,6-diphenethyl-2,5-dihydro-1,2,3,5-tetrazine (11d).

N-Sulfinyl-1,2-benzisoxazole-3-amine (3.02 g, 16.7 mmol, 5 equiv) was added to a suspension of 10d (1.77 g, 3.3 mmol, 1 equiv) in anhydrous CH₃CN (8.3 mL) in a reaction vessel. The mixture was warmed under Ar at 60 °C (oil bath) for 18 h. The solution was concentrated and the residue was purified by column chromatography (SiO₂, CH₂Cl₂) to provide 11d (1.21 g, 68%) as a yellow solid: mp 51–53 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.96 (d, J = 8.0 Hz, 1H), 7.70−7.66 (m, 3H), 7.56−7.52 (m, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.43 (dt, J = 7.9, 3.9 Hz, 1H), 7.26−7.23 (m, 5H), 7.17 (t, J = 7.4 Hz, 2H), 7.12 (d, J = 7.8 Hz, 4H), 3.09−2.97 (m, 4H), 2.72−2.61 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.7, 164.2, 160.2, 153.1, 147.1, 140.0, 131.6, 130.1, 128.7, 128.5, 126.6, 125.4, 124.7, 122.9, 120.5, 118.8, 115.8, 111.2, 110.0, 32.8, 32.6; IR (film) ν_max 3060, 1687, 1608, 1527, 1489, 1441, 1392, 1266, 1222, 1155, 905, 734, 699 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₂H₂₇N₆O₂ 527.2195; Found 527.2209.

4,6-Diphenethyl-2-(2-hydroxyphenylimidoyl)-2,5-dihydro-1,2,3,5-tetrazine (12d).

Palladium on carbon (10% Pd, 1.21 g) was added to a suspension of 11d (1.21 g, 2.3 mmol, 1 equiv) in MeOH/AcOH (121 mL, v/v = 19:1). H₂ was bubbled through the mixture for 10 min and the mixture was stirred under H₂ (1 atm) at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 20% MeOH/CH₂Cl₂ with 1% AcOH) to provide 12d (642 mg, 68%) as a yellow solid: mp 139–141 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.43 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.26 (d, J = 7.4 Hz, 4H), 7.21−7.16 (m, 6H), 7.05 (d, J = 8.2 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 2.94 (t, J = 7.5 Hz, 4H), 2.56 (t, J = 7.5 Hz, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 165.9, 160.3, 146.7, 140.8, 133.6, 129.7, 128.6, 128.5, 126.3, 121.1, 118.8, 115.4, 36.2, 32.3; IR (film) ν_max 3418, 3027, 2924, 1694, 1577, 1493, 1454, 1240, 1138, 1032, 876, 733, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₆N₅O 412.2137; Found 412.2140.

4,6-Diphenethyl-1,2,3,5-tetrazine (1d).

PbO₂ (1.13 g, 4.7 mmol, 4 equiv) was added to a solution of 12d (483 mg, 1.18 mmol, 1 equiv) and AcOH (291 mg, 4.7 mmol, 4 equiv) in CH₂Cl₂ (60 mL) and the mixture was stirred at 25 °C for 1 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 30% Et₂O/hexanes) to provide 1d (240 mg, 70%) as a yellow solid: mp 38–40 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.29 (t, J = 7.5 Hz, 4H), 7.23−7.19 (m, 6H), 3.39−3.32 (m, 4H), 3.22−3.16 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 170.2, 139.9, 128.8, 128.5, 126.7, 38.4, 33.1; IR (film) ν_max 3027, 2926, 1603, 1489, 1453, 1407, 1303, 1077, 1030, 882, 743, 698 cm⁻¹; No observable ionization of the title compound was observed upon ESI or APCI ionization, while GC-MS for ionization provided only the mass of the corresponding nitrile. The structure of the tetrazine is supported by other spectroscopic data. The title compound, a low melting solid, was found not to be stable to storage at 25 °C and storage at −20 °C is suggested upon isolation and purification.

1,2-Bis(2-(benzo[d]isoxazol-3-yl)hydrazineylidene)-1,2-dicyclohexylethane (9e).

1,2-Dicyclohexylethane-1,2-dione (2.48 g, 11.2 mmol, 1 equiv) and 1,2-benzisoxazole-3-hydrazine hydrochloride (7.73 g, 33.6 mmol, 3 equiv, as ½ dioxane complex) were suspended in EtOH (50 mL) in a 100 mL round-bottom flask. The mixture was stirred at reflux (oil bath) for 18 h. The mixture was cooled to 25 °C, and the product was collected by filtration and washed with EtOH (30 mL) to provide 9e (5.10 g, 94%) as a white solid: mp 221–223 °C; IR (film) ν_max 3197, 2926, 2852, 1612, 1565, 1494, 1434, 1242, 1110, 742, 636 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₃₃N₆O₂ 485.2665; Found 485.2662. Bis-hydrazone 9e exhibited low solubility in common NMR solvents and NMR data was not recorded.

Benzo[d]isoxazol-3-yl(2-(benzo[d]isoxazol-3-yl)-4,5-dicyclohexyl-1H-1,2,3-triazol-2-ium-1-yl)amide (10e).

Compound 9e (4.77 g, 9.86 mmol, 1 equiv) was suspended in CH₂Cl₂ (240 mL) in a 500 mL round-bottom flask. Pb(OAc)₄ (6.55 g, 14.8 mmol, 1.5 equiv) was added to the suspension at 25 °C, and the reaction mixture was stirred at 25 °C for 18 h before it was quenched with the addition of saturated aqueous NaHCO₃ (100 mL). The biphasic mixture was filtered over Celite. The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (100 mL × 2). The organic layers were combined, dried over Na₂SO₄, and filtered. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 50% Et₂O/hexanes) to provide 10e (1.85 g, 39%) as a yellow solid: mp 132 °C (decomp); NMR recorded in the presence of trifluoroacetic acid (TFA): ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.06 (d, J = 8.1 Hz, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.75−7.69 (m, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 3.28 (t, J = 12.4 Hz, 1H), 3.10 (t, J = 11.9 Hz, 1H), 2.14 (d, J = 12.5 Hz, 2H), 2.02 (d, J = 13.3 Hz, 4H), 1.93−1.87 (m, 4H), 1.82 (t, J = 11.4 Hz, 3H), 1.76−1.67 (m, 1H), 1.53 (q, J = 12.8 Hz, 2H), 1.47−1.28 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.4, 164.2, 156.3, 155.1, 151.7, 150.1, 132.8, 132.5, 126.6, 124.9, 122.00, 121.9, 113.7, 113.0, 110.9, 110.4, 36.6, 36.0, 32.5, 32.2, 29.9, 26.1, 25.7, 25.5, 25.2; IR (film) ν_max 2930, 2854, 1610, 1497, 1432, 1351, 1241, 1153, 983, 880, 748, 652 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₃₁N₆O₂ 483.2508; Found 483.2509.

2,5-Di(benzo[d]isoxazole-3-yl)-4,6-dicyclohexyl-2,5-dihydro-1,2,3,5-tetrazine (11e).

N-Sulfinyl-1,2-benzisoxazole-3-amine (3.24 g, 18 mmol, 5 equiv) was added to a suspension of 10e (1.75 g, 3.6 mmol, 1 equiv) in anhydrous CH₃CN (9 mL) in a reaction vessel. The mixture was warmed under Ar at 60 °C (oil bath) for 18 h. The solution was concentrated and the residue was purified by column chromatography (SiO₂, 15% EtOAc/hexanes) to provide 11e (330 mg, 19%) as a yellow solid: mp 75–77 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.10 (d, J = 8.0 Hz, 1H), 7.67−7.63 (m, 3H), 7.54−7.50 (m, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.42−7.38 (m, 1H), 7.28 (d, J = 7.9 Hz, 1H), 2.45 (tt, J = 11.4, 3.2 Hz, 2H), 2.12 (d, J = 12.1 Hz, 4H), 1.79 (dd, J = 10.9, 4.2 Hz, 4H), 1.61 (dt, J = 24.1, 11.8 Hz, 6H), 1.25−1.16 (m, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.2, 160.7, 152.8, 151.8, 131.2, 130.0, 125.0, 124.9, 122.8, 120.5, 118.7, 116.1, 111.0, 110.0, 39.8, 31.1, 25.9, 25.8; IR (film) ν_max 2931, 2854, 1677, 1610, 1517, 1441, 1392, 1330, 1244, 1219, 904, 864, 750 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₃₁N₆O₂ 483.2508; Found 483.2502.

4,6-Dicyclohexyl-2-(2-hydroxyphenylimidoyl)-2,5-dihydro-1,2,3,5-tetrazine (12e).

Palladium on carbon (10% Pd, 300 mg) was added to a suspension of 11e (300 mg, 0.62 mmol, 1 equiv) in MeOH/AcOH (30 mL, v/v = 4:1). H₂ was bubbled through the mixture for 10 min and the mixture was stirred under H₂ (1 atm) at 25 °C for 18 h. The hydrogenation was repeated a second time by removal of the palladium on carbon by filtration and addition of new palladium on carbon (10% Pd, 300 mg). The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 20% MeOH/CH₂Cl₂ with 2% AcOH) to provide 12e (133 mg, 58%) as a yellow solid: mp 181 °C (decomp); ¹H NMR (600 MHz, CD₃OD, 298K) δ 7.72 (dd, J = 8.1, 1.5 Hz, 1H), 7.20 (t, J = 7.7 Hz, 1H), 6.73 (d, J = 8.5 Hz, 1H), 6.52−6.47 (m, 1H), 2.22 (t, J = 11.4 Hz, 2H), 1.91 (d, J = 12.5 Hz, 4H), 1.83 (d, J = 13.0 Hz, 4H), 1.71 (d, J = 12.4 Hz, 2H), 1.47 (q, J = 14.4, 12.2 Hz, 4H), 1.37−1.23 (m, 6H); ¹³C{¹H} NMR (151 MHz, CD₃OD, 298K) δ 169.8, 161.6, 158.2, 134.7, 132.0, 122.5, 114.5, 113.4, 42.3, 30.6, 26.8; IR (film) ν_max 2929, 2854, 1689, 1605, 1573, 1475, 1386, 1259, 1136, 751, 609 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₁H₃₀N₅O 368.2450; Found 368.2453.

4,6-Dicyclohexyl-1,2,3,5-tetrazine (1e).

PbO₂ (307 mg, 1.28 mmol, 4 equiv) was added to a solution of 12e (118 mg, 0.32 mmol, 1 equiv) and AcOH (79 mg, 1.28 mmol, 4 equiv) in CH₂Cl₂ (16 mL) and the mixture was stirred at 25 °C for 6 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 15% Et₂O/hexanes) to provide 1e (55 mg, 70%) as a yellow solid: mp 42–44 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 2.95 (tt, J = 11.7, 3.5 Hz, 2H), 2.08−2.00 (m, 4H), 1.88 (dt, J = 13.4, 3.5 Hz, 4H), 1.77 (d, J = 13.0 Hz, 2H), 1.68 (qd, J = 12.6, 3.4 Hz, 4H), 1.43 (qt, J = 12.9, 3.4 Hz, 4H), 1.32 (qt, J = 12.9, 3.6 Hz, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 173.7, 45.1, 30.9, 25.9, 25.8; IR (film) ν_max 2928, 2853, 1485, 1450, 1409, 1259, 952, 880, 840 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₄H₂₃N₄ 247.1917; Found 247.1916.

3,3’-((1,4-Diphenylbutane-1,2-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(benzo[d]isoxazole) (9f).

1,4-Diphenyl-1,2-butadione (1.04 g, 4.4 mmol, 1 equiv) and 1,2-benzisoxazole-3-hydrazine hydrochloride (2.22 g, 9.6 mmol, 2.2 equiv, as ½ dioxane complex) were suspended in EtOH (24 mL) in a 50 mL round-bottom flask. The mixture was stirred at reflux (oil bath) for 18 h. The mixture was cooled to 25 °C, and product was collected by filtration and washed with EtOH (10 mL) to provide 9f (2.01 g, 91%) as a white solid: mp 214–216 °C; IR (film) ν_max 3222, 1606, 1554, 1493, 1444, 1425, 1244, 1165, 1108, 913, 756, 740, 694 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₀H₂₅N₆O₂ 501.2039; Found 501.2032. Bis-hydrazone 9f exhibited low solubility in common NMR solvents and NMR data was not recorded.

Benzo[d]isoxazol-3-yl(2-(benzo[d]isoxazol-3-yl)-5-phenethyl-4-phenyl-1H-1,2,3-triazol-2-ium-1-yl)amide (10f) and benzo[d]isoxazol-3-yl(2-(benzo[d]isoxazol-3-yl)-4-phenethyl-5-phenyl-1H-1,2,3-triazol-2-ium-1-yl)amide (10f’).

Compound 9f (2.01 g, 4.0 mmol, 1 equiv) was suspended in CH₃CN/H₂O (30 mL, v/v = 9:1) in a 100 mL round-bottom flask. A solution of CAN (6.58 g, 12 mmol, 3 equiv) in CH₃CN/H₂O (30 mL, v/v = 9:1) was added to the suspension dropwise at 25 °C, and the reaction mixture was stirred at 25 °C for 30 min. The mixture was filtered to provide a brown solid, which was then resuspended in a mixture of 4 M HCl in 1,4-dioxane (4 mL, 4 equiv HCl) in CH₂Cl₂ (60 mL). The resulting mixture was further stirred at 25 °C for 3 h before it was quenched with the addition of saturated aqueous NaHCO₃ (50 mL). The organic layer was separated, and the aqueous layer was extracted with CH₂Cl₂ (50 mL × 2). The organic layers were combined, dried over Na₂SO₄, and filtered. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 75% Et₂O/hexanes) to provide 10f (1.50 g, 75%) as an orange solid as a pair of interconverting regioisomers (major : minor = 4 : 1): mp 60–62 °C; NMR recorded in the presence of trifluoroacetic acid (TFA), proton NMR integration based on major isomer: ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.14 (d, J = 8.2 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.95 (t, J = 8.2 Hz, 0.5H), 7.78−7.75 (m, 1.25H), 7.73 (t, J = 7.9 Hz, 1H), 7.71−7.65 (m, 4.5H), 7.65−7.60 (m, 2.5H), 7.59−7.49 (m, 4H), 7.47 (d, J = 8.2 Hz, 0.25H), 7.41 (t, J = 8.0 Hz, 0.25H), 7.34 (t, J = 7.9 Hz, 0.5H), 7.28 (t, J = 8.0 Hz, 0.25H), 7.22 (d, J = 8.0 Hz, 0.5H), 7.21−7.15 (m, 3H), 7.07 (dd, J = 7.6, 1.7 Hz, 2H), 3.66 (s, br, 2H), 3.43 (t, J = 7.9 Hz, 0.5H), 3.28 (t, J = 7.9 Hz, 0.5H), 3.08 (t, J = 7.9 Hz, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.6, 164.4, 164.2, 156.4, 156.3, 150.8, 150.21, 150.15, 149.8, 148.7, 146.9, 138.9, 137.6, 133.8, 132.94, 132.89, 132.5, 132.4, 132.0, 130.0, 129.8, 129.5, 129.12, 129.07, 128.6, 128.3, 128.2, 127.5, 127.2, 126.8, 126.7, 125.5, 124.9, 124.8, 122.04, 121.97, 121.79, 121.77, 119.8, 113.8, 113.5, 113.0, 112.8, 111.0, 110.7, 110.5, 110.3, 33.4, 32.8, 28.2, 26.5; IR (film) ν_max 3061, 2930, 1609, 1495, 1434, 1335, 1241, 1006, 920, 881, 747, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₀H₂₃N₆O₂ 499.1882; Found 499.1892.

2,5-Di(benzo[d]isoxazole-3-yl)-4-phenyl-6-phenethyl-2,5-dihydro-1,2,3,5-tetrazine (11f).

N-Sulfinyl-1,2-benzisoxazole-3-amine (2.64 g, 14.6 mmol, 5 equiv) was added to a suspension of 10f (1.47 g, 2.94 mmol, 1 equiv) in anhydrous CH₃CN (7.4 mL) in a reaction vessel. The mixture was warmed under Ar at 60 °C (oil bath) for 18 h. The mixture was concentrated and the residue was purified by column chromatography (SiO₂, 75% CH₂Cl₂/hexanes) to provide 11f (1.06 g, 72%) as a yellow solid: mp 63–65 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.12 (d, J = 8.0 Hz, 1H), 7.76−7.72 (m, 2H), 7.58−7.54 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.49−7.44 (m, 3H), 7.35 (d, J = 7.1 Hz, 2H), 7.33−7.26 (m, 6H), 7.21 (t, J = 7.3 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 3.35−3.31 (m, 2H), 3.31−3.27 (m, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 164.3, 164.0, 160.2, 153.7, 148.6, 145.2, 140.2, 131.3, 130.9, 130.2, 129.8, 128.9, 128.8, 128.7, 128.2, 126.6, 124.6, 124.3, 123.1, 121.0, 117.2, 115.7, 110.6, 110.1, 34.3, 33.3; IR (film) ν_max 3061, 2926, 1667, 1609, 1520, 1487, 1440, 1394, 1323, 1238, 898, 747, 697 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₀H₂₃N₆O₂ 499.1882; Found 499.1887.

2-(2-Hydroxyphenylimidoyl)-4-phenyl-6-phenethyl-2,5-dihydro-1,2,3,5-tetrazine (12f).

Palladium on carbon (10% Pd, 1.05 g) was added to a suspension of 11f (1.05 g, 2.10 mmol, 1 equiv) in EtOAc (105 mL). H₂ was bubbled through the mixture for 10 min and the mixture was stirred under H₂ (1 atm) at 25 °C for 18 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 20% MeOH/CH₂Cl₂ with 2% AcOH) to provide 12f (283 mg, 35%) as a yellow solid: mp 152 °C (decomp); ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.02 (d, J = 7.5 Hz, 2H), 7.52−7.45 (m, 3H), 7.40 (t, J = 7.4 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 7.25 (d, J = 6.7 Hz, 2H), 7.20−7.17 (m, 1H), 7.13 (d, J = 8.2 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 3.06 (t, J = 7.8 Hz, 2H), 2.69 (t, J = 7.9 Hz, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 170.1, 164.3, 157.5, 143.2, 141.2, 133.6, 133.5, 131.9, 129.2, 128.6, 128.4, 127.7, 126.2, 121.1, 120.6, 117.0, 37.2, 32.5; IR (film) ν_max 3060, 2932, 1590, 1557, 1491, 1455, 1346, 1303, 1241, 1140, 1028, 875, 695 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₃H₂₂N₅O 384.1824; Found 384.1816.

4-Phenyl-6-phenethyl-1,2,3,5-tetrazine (1f).

PbO₂ (714 mg, 2.98 mmol, 4 equiv) was added to a solution of 12f (285 mg, 0.74 mmol, 1 equiv) and AcOH (185 mg, 1.28 mmol, 4 equiv) in CH₂Cl₂ (37 mL) and the mixture was stirred at 25 °C for 6 h. The resulting mixture was filtered over Celite, concentrated, and the residue was purified by column chromatography (SiO₂, 20% Et₂O/hexanes) to provide 1f (122 mg, 63%) as a yellow solid: mp 58–60 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.64 (dd, J = 8.4, 1.2 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.7 Hz, 2H), 7.29 (dt, J = 13.6, 7.1 Hz, 4H), 7.22 (t, J = 7.0 Hz, 1H), 3.45 (dd, J = 8.8, 6.8 Hz, 2H), 3.30 (dd, J = 8.9, 6.9 Hz, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 170.6, 162.8, 140.1, 134.3, 132.3, 129.5, 129.3, 128.8, 128.5, 126.6, 38.5, 33.1; IR (film) ν_max 3029, 1600, 1486, 1402, 1265, 1176, 895, 771, 737, 691, 624 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₆H₁₅N₄ 263.1297; Found 263.1296.

Reactivity studies of 4,6-diphenethyl-1,2,3,5-tetrazine (1d).

General procedure for the synthesis of 14a–n. All amidines 13 were purchased as their hydrochloride salts and free-based by treatment with aqueous 2 M KOH. A 40 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CH₃CN (0.5 mL) was treated with the corresponding amidine 13a–n (0.03 mmol, 1.5 equiv). The mixture was stirred at 25 °C for 20 min, and the solvent was removed under a gentle stream of N₂. The residue was purified by column chromatography (SiO₂, CH₂Cl₂, Et₂O/CH₂Cl₂, or Et₂O/MeOH) to provide 14a–n.

4,6-Diphenethyl-2-phenyl-1,3,5-triazine (14a).

Purified with CH₂Cl₂ as eluent, 7.00 mg, 96%, white solid: mp 44–46 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.53 (d, J = 7.4 Hz, 2H), 7.57 (t, J = 7.3 Hz, 1H), 7.52 (t, J = 7.5 Hz, 2H), 7.31−7.27 (m, 8H), 7.22−7.18 (m, 2H), 3.28−3.23 (m, 4H), 3.23−3.20 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.6, 171.1, 141.2, 136.0, 132.6, 129.0, 128.8, 128.61, 128.57, 126.2, 40.6, 33.6; IR (film) ν_max 3027, 1586, 1527, 1496, 1417, 1376, 1176, 1074, 1028, 778, 747, 694 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₄N₃ 366.1970; Found 366.1979.

4,6-Diphenethyl-2-(4-fluorophenyl)-1,3,5-triazine (14b).

Purified with CH₂Cl₂ as eluent, 7.61 mg, 99%, white solid: mp 74–76 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.55 (dd, J = 8.6, 5.7 Hz, 2H), 7.31−7.26 (m, 8H), 7.23−7.16 (m, 4H), 3.26−3.23 (m, 4H), 3.23−3.19 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.6, 170.1, 165.9 (d, J = 254 Hz), 141.2, 132.1 (d, J = 3 Hz), 131.4 (d, J = 9 Hz), 128.59, 128.58, 126.3, 115.9 (d, J = 21 Hz), 40.6, 33.6; IR (film) ν_max 3027, 1601, 1531, 1497, 1415, 1379, 1226, 1151, 813, 749, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₃FN₃ 384.1876; Found 384.1879.

2-(4-Bromophenyl)-4,6-diphenethyl-1,3,5-triazine (14c).

Purified with CH₂Cl₂ as eluent, 8.18 mg, 92%, white solid: mp 91–93 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.39 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.31−7.26 (m, 8H), 7.20 (t, J = 6.9 Hz, 2H), 3.27−3.22 (m, 4H), 3.22−3.18 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.8, 170.3, 141.1, 134.9, 132.1, 130.6, 128.6, 127.6, 126.3, 40.6, 33.6; IR (film) ν_max 2930, 1578, 1530, 1376, 1276, 1068, 1011, 884, 835, 802, 750, 695 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₃BrN₃ 444.1075; Found 444.1074.

4,6-Diphenethyl-2-(4-methoxyphenyl)-1,3,5-triazine (14d).

Purified with 50% Et₂O/CH₂Cl₂ as eluent, 7.48 mg, 95%, white solid: mp 51–53 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.53 (d, J = 9.0 Hz, 2H), 7.33−7.29 (m, 8H), 7.24−7.20 (m, 2H), 7.03 (d, J = 9.0 Hz, 2H), 3.93 (s, 3H), 3.26−3.21 (m, 8H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.2, 170.7, 163.5, 141.3, 131.0, 128.6, 128.6, 128.4, 126.2, 114.1, 55.6, 40.6, 33.6; IR (film) ν_max 3025, 1606, 1585, 1531, 1454, 1379, 1304, 1275, 1256, 1171, 1031, 750, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₆H₂₆N₃O 396.2076; Found 396.2073.

4-(4,6-Diphenethyl-1,3,5-triazin-2-yl)aniline (14e).

Purified with Et₂O as eluent, 7.15 mg, 94%, yellow solid: mp 58–60 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.38 (d, J = 8.8 Hz, 2H), 7.30−7.27 (m, 8H), 7.21−7.18 (m, 2H), 6.74 (d, J = 8.8 Hz, 2H), 3.23−3.17 (m, 8H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 177.3, 170.9, 151.2, 141.2, 131.4, 128.63, 128.56, 126.2, 125.3, 114.6, 40.2, 33.6; IR (film) ν_max 3387, 3213, 3026, 2926, 1621, 1605, 1521, 1407, 1375, 1306, 1176, 990, 812, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₅N₄ 381.2079; Found 381.2085.

4,6-Diphenethyl-2-(3-nitrophenyl)-1,3,5-triazine (14f).

Purified with CH₂Cl₂ as eluent, 7.22 mg, 91%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 9.38−9.35 (m, 1H), 8.87 (dt, J = 7.8, 1.3 Hz, 1H), 8.44 (ddd, J = 8.2, 2.4, 1.1 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.35−7.29 (m, 8H), 7.23 (t, J = 6.7 Hz, 2H), 3.34−3.28 (m, 4H), 3.28−3.22 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 179.2, 169.1, 148.9, 140.9, 137.9, 134.6, 129.8, 128.63, 128.60, 126.9, 126.4, 124.0, 40.5, 33.6; IR (film) ν_max 3028, 1587, 1527, 1496, 1373, 1347, 1267, 1078, 925, 807, 735, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₃N₄O₂ 411.1821; Found 411.1830.

4,6-Diphenethyl-2-(pyridin-2-yl)-1,3,5-triazine (14g).

Purified with Et₂O as eluent, 6.99 mg, 95%, white solid: mp 50–52 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.95−8.90 (m, 1H), 8.59 (dt, J = 7.9, 1.0 Hz, 1H), 7.92 (td, J = 7.7, 1.7 Hz, 1H), 7.51 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.31−7.26 (m, 8H), 7.21−7.18 (m, 2H), 3.34 (dd, J = 9.4, 6.5 Hz, 4H), 3.22 (dd, J = 9.6, 6.6 Hz, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 179.6, 170.0, 153.3, 150.4, 141.1, 137.6, 128.61, 128.58, 126.4, 126.3, 124.9, 40.7, 33.8; IR (film) ν_max 2927, 1529, 1496, 1377, 1251, 1076, 995, 790, 742, 697, 621 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₄H₂₃N₄ 367.1923; Found 367.1937.

4,6-Diphenethyl-2-(pyrimidin-2-yl)-1,3,5-triazine (14h).

Purified with 10% MeOH/Et₂O as eluent, 7.18 mg, 98%, white solid: mp 77–79 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 9.08 (d, J = 4.8 Hz, 2H), 7.50 (t, J = 4.8 Hz, 1H), 7.30−7.26 (m, 8H), 7.21−7.17 (m, 2H), 3.42−3.39 (m, 4H), 3.23−3.19 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 180.2, 169.4, 161.9, 158.4, 140.9, 128.61, 128.60, 126.3, 122.4, 40.8, 33.9; IR (film) ν_max 3027, 1534, 1496, 1453, 1374, 1208, 1029, 803, 750, 699, 631 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₃H₂₂N₅ 368.1875; Found 368.1872.

4,6-Diphenethyl-2-(thien-2-yl)-1,3,5-triazine (14i).

Purified with CH₂Cl₂ as eluent, 7.06 mg, 95%, white solid: mp 46–48 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.18 (dd, J = 3.7, 1.2 Hz, 1H), 7.60 (dd, J = 5.0, 1.2 Hz, 1H), 7.30−7.27 (m, 8H), 7.22−7.18 (m, 3H), 3.18 (q, J = 3.3, 2.8 Hz, 8H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.5, 167.4, 141.7, 141.2, 132.4, 131.7, 128.63, 128.55, 126.2, 40.4, 33.4; IR (film) ν_max 3026, 2928, 1525, 1496, 1438, 1379, 1223, 1035, 814, 720, 699 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₃H₂₂N₃S 372.1534; Found 372.1522.

4,6-Diphenethyl-2-(6-(trifluoromethyl)pyridin-3-yl)-1,3,5-triazine (14j).

Purified with CH₂Cl₂ as eluent, 8.15 mg, 94%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 9.76 (s, 1H), 8.90 (d, J = 8.2 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 7.5 Hz, 4H), 7.26 (d, J = 5.8 Hz, 4H), 7.20 (t, J = 7.1 Hz, 2H), 3.31−3.28 (m, 4H), 3.23−3.20 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 179.3, 168.4, 150.9 (q, J = 35 Hz), 150.7, 140.8, 137.8, 134.2, 128.63, 128.56, 126.4, 121.5 (q, J = 275 Hz), 120.4 (q, J = 3 Hz), 40.4, 33.5; IR (film) ν_max 3028, 2924, 1585, 1529, 1496, 1327, 1175, 1140, 1083, 1026, 862, 817, 746, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₂F₃N₄ 435.1797; Found 435.1789.

2-(2,6-Dichlorobenzyl)-4,6-diphenethyl-1,3,5-triazine (14k).

Purified with CH₂Cl₂ as eluent, 8.25 mg, 92%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.36 (d, J = 8.1 Hz, 2H), 7.23 (t, J = 7.4 Hz, 4H), 7.20 (t, J = 8.1 Hz, 1H), 7.16 (t, J = 7.4 Hz, 2H), 7.13 (d, J = 7.4 Hz, 4H), 4.55 (s, 2H), 3.12−3.07 (m, 4H), 3.06−3.02 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.4, 175.8, 141.1, 136.7, 133.5, 128.7, 128.6, 128.5, 128.0, 126.2, 40.4, 40.3, 33.3; IR (film) ν_max 3027, 2929, 1532, 1495, 1436, 1374, 1088, 928, 776, 746, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₆H₂₃Cl₂N₃ 448.1347; Found 448.1348.

2-Benzyl-4,6-diphenethyl-1,3,5-triazine (14l),

Purified with 20% Et₂O/CH₂Cl₂ as eluent, 7.50 mg, 99%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.36−7.27 (m, 4H), 7.26−7.23 (m, 5H), 7.20−7.16 (m, 6H), 4.13 (s, 2H), 3.17−3.12 (m, 4H), 3.12−3.08 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.6, 177.4, 141.0, 137.0, 129.4, 128.64, 128.58, 128.5, 126.9, 126.2, 45.5, 40.4, 33.6; IR (film) ν_max 3028, 2921, 1532, 1495, 1453, 1379, 1266, 1076, 1030, 735, 697 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₆H₂₆N₃ 380.2127; Found 380.2139.

2,4,6-Triphenethyl-1,3,5-triazine (14m).

Purified with 10% Et₂O/CH₂Cl₂ as eluent, 7.45 mg, 95%, white solid: mp 41–43 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.28 (t, J = 7.4 Hz, 6H), 7.22 (d, J = 6.9 Hz, 6H), 7.19 (t, J = 7.3 Hz, 3H), 3.17−3.13 (m, 6H), 3.12−3.08 (m, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 178.3, 141.0, 128.58, 128.56, 126.3, 40.5, 33.8; IR (film) ν_max 3026, 2924, 1603, 1534, 1495, 1453, 1420, 1382, 1180, 1029, 743, 697 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₇H₂₈N₃ 394.2283; Found 394.2296.

2-Cyclopropyl-4,6-diphenethyl-1,3,5-triazine (14n).

Purified with CH₂Cl₂ as eluent, 6.17 mg, 94%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.28 (t, J = 7.4 Hz, 4H), 7.23 (d, J = 6.8 Hz, 4H), 7.19 (t, J = 7.2 Hz, 2H), 3.09 (s, 8H), 2.10 (tt, J = 8.1, 4.6 Hz, 1H), 1.22−1.18 (m, 2H), 1.13−1.09 (m, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 180.3, 177.6, 141.2, 128.6, 128.5, 126.2, 40.4, 33.7, 18.0, 12.0; IR (film) ν_max 3026, 2927, 1534, 1496, 1450, 1384, 1328, 1029, 951, 815, 740, 697 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₂H₂₄N₃ 330.1970; Found 330.1969.

Reaction with 15a and 15b.

A 0.4 M solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CHCl₃ (50 μL) was treated with imidate 15a (16.5 mg, 0.1 mmol, 5 equiv). The mixture was warmed at 60 °C (oil bath) for 48 h in a sealed tube. The solution was cooled to 25 °C and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 25% Et₂O/hexanes) to provide 14l (5.26 mg, 69%) as a colorless oil.

A 0.4 M solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CHCl₃ (50 μL) was treated with thioimidate 15b (5.0 mg, 0.03 mmol, 1.5 equiv). The mixture was stirred at 60 °C (oil bath) for 24 h in a sealed tube. The solution was cooled to 25 °C and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 25% Et₂O/hexanes) to provide 14l (2.29 mg, 31%) as a colorless oil.

A 40 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CH₃CN (0.5 mL) was treated with thioimidate 15b (16.5 mg, 0.1 mmol, 5 equiv). The mixture was stirred at 25 °C for 48 h. The solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 25% Et₂O/hexanes) to provide 14l (1.63 mg, 22%) as a colorless oil.

General procedure for the synthesis of 17a–d.

A 40 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CH₃CN (0.5 mL) was treated with crushed 4Å molecular sieves (20 mg) and corresponding enamine 16a–d (0.024 mmol, 1.2 equiv, freshly distilled). The mixture was stirred at 25 °C for 3 h, and then warmed at 60 °C (oil bath) for 12 h. The mixture was cooled and filtered over Celite. The solvent was removed under a gentle stream of N₂ and the mixture was purified by column chromatography (SiO₂, Et₂O/hexanes) to provide 17a–d.

2,4-Diphenethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (17a).

5.45 mg, 83%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.31−7.26 (m, 4H), 7.24 (d, J = 7.6 Hz, 2H), 7.21−7.16 (m, 2H), 7.11 (d, J = 6.9 Hz, 2H), 3.30−3.22 (m, 2H), 3.15 (dd, J = 9.8, 6.3 Hz, 2H), 3.04−3.00 (m, 2H), 3.00−2.96 (m, 2H), 2.94 (t, J = 7.8 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H), 2.04−1.99 (m, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 174.4, 168.4, 164.1, 141.8, 141.3, 130.4, 128.7, 128.6, 128.5, 128.4, 126.3, 126.0, 40.9, 37.4, 35.3, 34.5, 34.3, 27.9, 22.2; IR (film) ν_max 3025, 2926, 1567, 1495, 1453, 1430, 1396, 1076, 1029, 783, 745, 699 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₃H₂₅N₂ 329.2018; Found 329.2023.

2,4-Diphenethyl-5,6,7,8-tetrahydroquinazoline (17b).

3.54 mg, 52%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.32−7.26 (m, 6H), 7.22−7.17 (m, 2H), 7.17−7.15 (m, 2H), 3.25 (s, br, 2H), 3.15 (dd, J = 9.9, 6.2 Hz, 2H), 3.05−2.95 (m, 4H), 2.87 (s, br, 2H), 2.52 (t, J = 6.3 Hz, 2H), 1.85−1.78 (m, 2H), 1.78−1.73 (m, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 168.2, 165.8, 164.8, 141.6, 141.3, 128.7, 128.58, 128.55, 128.4, 126.3, 126.0, 125.6, 40.0, 36.0, 34.9, 34.2, 32.0, 24.3, 22.4, 22.0; IR (film) ν_max 3025, 2932, 2861, 1602, 1556, 1495, 1453, 1425, 1407, 1076, 1029, 732, 699 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₄H₂₇N₂ 343.2174; Found 343.2182.

2,4-Diphenethyl-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidine (17c).

5.82 mg, 82%, colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.30−7.26 (m, 6H), 7.21−7.14 (m, 4H), 3.22−3.16 (m, 2H), 3.16−3.11 (m, 2H), 3.07 (dd, J = 9.5, 6.4 Hz, 2H), 3.00−2.91 (m, 4H), 2.73−2.66 (m, 2H), 1.86−1.80 (m, 2H), 1.70−1.64 (m, 2H), 1.54−1.48 (m, 2H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 171.2, 166.4, 165.3, 141.9, 141.3, 130.2, 128.6, 128.5, 128.4, 128.3, 126.1, 125.8, 40.7, 38.6, 36.8, 35.2, 34.9, 32.1, 27.6, 27.0, 25.9; IR (film) ν_max 3025, 2923, 2853, 1603, 1554, 1495, 1452, 1410, 1076, 1029, 954, 746, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₂₉N₂ 357.2331; Found 357.2344.

2,6-Diphenethyl-4-phenylpyrimidine (17d).

3.45 mg, 47%, white solid: mp 69–71 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.03−7.99 (m, 2H), 7.51−7.47 (m, 3H), 7.32−7.27 (m, 6H), 7.25 (s, 1H), 7.23−7.18 (m, 4H), 3.36 (dd, J = 9.6, 6.3 Hz, 2H), 3.26 (dd, J = 9.6, 6.4 Hz, 2H), 3.13−3.07 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 170.3, 170.1, 164.1, 141.9, 141.1, 137.4, 130.7, 129.0, 128.7, 128.63, 128.60, 128.5, 127.4, 126.3, 126.0, 113.5, 41.1, 39.9, 35.2, 34.7; IR (film) ν_max 3026, 2925, 1577, 1537, 1495, 1453, 1380, 1075, 1029, 772, 748, 694 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₆H₂₅N₂ 365.2018; Found 365.2020.

4-Diethylamino-2,6-diphenethyl-5-methylpyrimidine (19a).

A 40 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CHCl₃ (0.5 mL) was treated with ynamine 18a (3.3 mg, 0.03 mmol, 1.5 equiv.). The mixture was stirred at 25 °C for 1 h, and the solvent was removed under a gentle stream of N₂. The residue was purified by column chromatography (SiO₂, 50% Et₂O/hexanes) to provide 19a (6.57 mg, 87%) as a colorless oil: ¹H NMR (600 MHz, CD₃CN, 298K) δ 7.29−7.24 (m, 6H), 7.21−7.16 (m, 4H), 3.31 (q, J = 7.0 Hz, 4H), 3.13 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.6 Hz, 2H), 2.99−2.94 (m, 2H), 2.94−2.90 (m, 2H), 2.00 (s, 3H), 1.11 (t, J = 7.0 Hz, 6H); ¹³C{¹H} NMR (151 MHz, CD₃CN, 298K) δ 167.6, 166.6, 165.6, 143.3, 143.0, 129.5, 129.4, 129.22, 129.17, 126.81, 126.7, 113.8, 44.7, 40.9, 37.5, 35.0, 34.7, 14.6, 13.6; IR (film) ν_max 3025, 2968, 2928, 1553, 1495, 1453, 1414, 1347, 1188, 1069, 1029, 745, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₅H₃₂N₃ 374.2596; Found 374.2603.

4-Dibenzylamino-2,6-diphenethyl-5-methylpyrimidine (19b).

A 40 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CHCl₃ (0.5 mL) was treated with ynamine 18b (7.0 mg, 0.03 mmol, 1.5 equiv). The mixture was stirred at 25 °C for 12 h, and the solvent was removed under a gentle stream of N₂. The residue was purified by column chromatography (SiO₂, 10% EtOAc/hexanes) to provide 19b (8.52 mg, 88%) as a viscous colorless oil: ¹H NMR (600 MHz, CD₃CN, 298K) δ 7.37 (t, J = 7.4 Hz, 4H), 7.32 (t, J = 7.3 Hz, 2H), 7.27−7.12 (m, 14H), 4.79 (s, 4H), 3.16 (t, J = 7.6 Hz, 2H), 3.10−3.01 (m, 2H), 2.97 (t, J = 7.6 Hz, 2H), 2.92−2.83 (m, 2H), 2.03 (s, 3H); ¹³C{¹H} NMR (151 MHz, CD₃CN, 298K) δ 166.2, 161.6, 161.3, 141.3, 140.9, 137.8, 129.7, 129.6, 129.44, 129.42, 129.41, 128.5, 128.2, 127.4, 127.2, 112.0, 54.4, 36.6, 34.9, 33.8, 33.6, 14.8; IR (film) ν_max 3029, 2935, 1672, 1612, 1522, 1497, 1453, 1359, 1196, 1128, 1077, 1030, 825, 798, 718, 699 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₃₅H₃₆N₃ 498.2909; Found 498.2899.

2,6-Diphenethyl-4-ethoxypyrimidine (21).

A 40 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CHCl₃ (0.5 mL) was treated with ketene acetal 20 (11.6 mg, 0.1 mmol, 5 equiv). The mixture was warmed at 60 °C (oil bath) for 12 h in a sealed tube. The solution was cooled to 25 °C and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 25% Et₂O/hexanes) to provide 21 (4.25 mg, 64%) as a colorless oil. Alternatively, the reaction mixture was stirred at 25 °C for 144 h to provide 21 (6.08 mg, 92%) as a colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.29−7.26 (m, 6H), 7.21−7.16 (m, 4H), 6.29 (s, 1H), 4.37 (q, J = 7.1 Hz, 2H), 3.22−3.11 (m, 4H), 3.04−2.92 (m, 4H), 1.36 (t, J = 7.1 Hz, 3H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 169.90, 169.85, 141.8, 141.1, 128.63, 128.56, 128.5, 128.4, 126.2, 126.0, 103.7, 62.3, 40.7, 39.3, 35.0, 34.4, 14.6; IR (film) ν_max 3026, 2928, 1587, 1555, 1453, 1436, 1380, 1341, 1173, 1057, 1030, 851, 747, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₂H₂₅N₂O 333.1967; Found 333.1974.

2,6-Diphenethylpyrimidine (23).

A solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in ethyl vinyl ether 22 (72 mg, 1 mmol, 50 equiv) was warmed at 40 °C (oil bath) for 96 h in a sealed tube. The solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 50% Et₂O/hexanes) to provide 23 (1.35 mg, 23%) as a colorless film: ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.50 (d, J = 5.1 Hz, 1H), 7.31−7.26 (m, 6H), 7.20 (t, J = 6.7 Hz, 2H), 7.16 (d, J = 7.2 Hz, 2H), 6.87 (d, J = 5.1 Hz, 1H), 3.27 (dd, J = 9.7, 6.4 Hz, 2H), 3.16 (dd, J = 9.8, 6.4 Hz, 2H), 3.04 (s, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 170.3, 169.8, 156.8, 141.6, 141.0, 128.63, 128.58, 128.57, 128.5, 126.3, 126.1, 117.8, 41.2, 39.7, 35.0, 34.8; IR (film) ν_max 3026, 2924, 2857, 1575, 1555, 1496, 1450, 1396, 1261, 1075, 1029, 795, 748, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₀H₂₁N₂ 289.1705; Found 289.1707.

Comparison of reactivity of 1d and 24.

3,6-Diphenethyl-1,2,4,5-tetrazine (24).

Hydrazine hydrate (80%, 1.55 g, 25 mmol, 5 equiv) was added to a mixture of 3-phenylpropionitrile (6.55 g, 5 mmol, 1 equiv) and Zn(OTf)₂ (91 mg, 0.25 mmol, 0.05 equiv) under argon atmosphere and the mixture was warmed at 60 °C (oil bath) for 24 h. The resulting mixture was cooled to 25 °C, treated with an aqueous NaNO₂ (1 M, 25 mL, 5 equiv), acidified to pH 3 with HCl (1 M), and then stirred at 25 °C for 1 h. The mixture was extracted with CH₂Cl₂ (50 mL × 3). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography (SiO₂, 80% CH₂Cl₂/hexanes) to provide 24 (194 mg, 27%) as a purple solid: mp 35–37 °C; ¹H NMR (600 MHz, CDCl₃, 298K) δ 7.29 (t, J = 7.5 Hz, 4H), 7.25−7.19 (m, 6H), 3.65−3.60 (m, 4H), 3.30−3.25 (m, 4H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 169.6, 140.0, 128.8, 128.6, 126.7, 36.6, 34.1; IR (film) ν_max 3028, 1603, 1495, 1453, 1395, 1328, 1267, 1076, 1030, 888, 734, 698 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₁₈H₁₉N₄ 291.1610; Found 291.1620.

Reaction with amidine 13a.

A 5 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in CH₃CN/H₂O (v/v = 1:1, 4 mL) was treated with amidine 13a (3.6 mg, 0.03 mmol, 1.5 equiv). The mixture was stirred at 25 °C for 20 min, and the solvent was removed under a gentle stream of N₂. The residue was purified by column chromatography (SiO₂, CH₂Cl₂) to provide 14a (5.72 mg, 93%) as a white solid.

A 5 mM solution of 24 (5.80 mg, 0.02 mmol, 1 equiv) in anhydrous CH₃CN/H₂O (v/v = 1:1, 4 mL) was treated with amidine 13a (3.6 mg, 0.03 mmol, 1.5 equiv). No conversion was observed after 1 h.

A 5 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) and 24 (4.68 mg, 0.02 mmol, 1 equiv) in anhydrous CH₃CN/H₂O (v/v = 1:1, 4 mL) was treated with amidine 13a (2.40 mg, 0.02 mmol, 1.0 equiv). The mixture was stirred at 25 °C for 1 h, and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, CH₂Cl₂) to provide 14a (6.47 mg, 88%) as a white solid.

Reaction with cyclooctyne 25.

((6aR*,7S*,7aS*)-1,4-diphenethyl-6,6a,7,7a,8,9-hexahydro-5H-cyclopropa[5,6]cycloocta[1,2-d]pyridazin-7-yl)methanol (26).

A 5 mM solution of 24 (2.90 mg, 0.01 mmol, 1 equiv) in CH₃CN/H₂O (v/v = 1:1, 2 mL) was treated with cyclooctyne 25 (6.00 mg, 0.04 mmol, 4 equiv). The mixture was stirred at 25 °C for 20 min, and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 10% MeOH/EtOAc) to provide 26 (3.68 mg, 88%) as a white solid: mp 139–141 °C; ¹H NMR (600 MHz, DMSO-d₆, 298K) δ 7.32−7.24 (m, 8H), 7.20 (tt, J = 6.3, 1.8 Hz, 2H), 4.28 (t, J = 5.0 Hz, 1H), 3.48−3.42 (m 2H), 3.21 (dd, J = 9.3, 6.7 Hz, 4H), 3.04−2.97 (m, 4H), 2.93−2.86 (m, 2H), 2.80 (s, br, 2H), 2.23−2.09 (m, 2H), 1.52 (s, br, 2H), 0.86 (s, 1H), 0.55 (s, br, 2H); ¹³C{¹H} NMR (151 MHz, DMSO-d₆, 298K) δ 158.9, 141.5, 139.4, 128.4, 128.3, 125.9, 57.0, 34.9, 34.8, 25.8; IR (film) ν_max 3348, 2923, 2870, 1492, 1452, 1388, 1027, 730, 699, 616 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₃₃N₂O 413.2593; Found 413.2591.

A 5 mM solution of 1d (5.80 mg, 0.01 mmol, 1 equiv) in CH₃CN/H₂O (v/v = 1:1, 4 mL) was treated with cyclooctyne 25 (6.00 mg, 0.04 mmol, 4 equiv). The mixture was stirred at 25 °C for 2 h, no conversion was observed.

A 5 mM solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) and 24 (5.80 mg, 0.02 mmol, 1 equiv) in CH₃CN/H₂O (v/v = 1:1, 4 mL) was treated with cyclooctyne 25 (3.00 mg, 0.02 mmol, 1 equiv). The mixture was stirred at 25 °C for 2 h, and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, EtOAc) to provide 26 (7.77 mg, 94%) as a white solid.

((6aR*,7R*,7aS*)-2,4-diphenethyl-6,6a,7,7a,8,9-hexahydro-5H-cyclopropa[5,6]cycloocta[1,2-d]pyrimidin-7-yl)methanol (27).

A 0.4 M solution of 1d (5.80 mg, 0.02 mmol, 1 equiv) in CHCl₃ (50 μL) was treated with cyclooctyne 25 (12.0 mg, 0.08 mmol, 4 equiv). The mixture was stirred at 25 °C for 48 h, and the solvent was removed under a gentle stream of N₂. The residue was purified by PTLC (SiO₂, 10% MeOH/Et₂O) to provide 27 (2.14 mg, 26%) as a colorless oil: ¹H NMR (600 MHz, DMSO-d₆, 298K) δ 7.27−7.22 (m, 3H), 7.22−7.17 (m, 5H), 7.17−7.12 (m, 2H), 4.28 (t, J = 5.0 Hz, 1H), 3.46 (dt, J = 7.4, 4.9 Hz, 2H), 3.10−2.92 (m, 9H), 2.85−2.79 (m, 2H), 2.67 (ddd, J = 14.1, 6.5, 3.7 Hz, 1H), 2.11−2.05 (m, 2H), 1.54 (s, br, 1H), 1.46 (s, br, 1H), 0.88−0.84 (m, 1H), 0.58 (s, br, 1H), 0.44 (s, br, 1H); ¹³C{¹H} NMR (151 MHz, DMSO-d6, 298K) δ 169.5, 165.6, 165.3, 141.5, 141.4, 128.6, 128.4, 128.2, 128.1, 125.9, 125.7, 57.1, 35.8, 35.3, 34.0, 33.9, 24.1, 22.1, 21.9, 21.3, 17.9, 17.0; IR (film) ν_max 2953, 2928, 2869, 1556, 1452, 1410, 1026, 752, 699 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₈H₃₃N₂O 413.2593; Found 413.2588.

4-Diethylamino-5-methyl-6-phenethyl-2-phenylpyrimidine (30).

A 40 mM solution of 1f (5.24 mg, 0.02 mmol, 1 equiv) in anhydrous CHCl₃ (0.5 mL) was treated with ynamine 18a (3.3 mg, 0.03 mmol, 1.5 equiv). The mixture was stirred at 25 °C for 1 h, and the solvent was removed under a gentle stream of N₂. The residue was purified by column chromatography (SiO₂, 25% Et₂O/hexanes) to provide 30 (4.30 mg, 62%) as a colorless oil: ¹H NMR (600 MHz, CDCl₃, 298K) δ 8.49−8.42 (m, 2H), 7.46 (t, J = 6.7 Hz, 2H), 7.43 (d, J = 6.1 Hz, 1H), 7.30−7.25 (m, 4H), 7.20 (t, J = 6.3 Hz, 1H), 3.40 (q, J = 6.6 Hz, 4H), 3.18−3.12 (m, 2H), 3.08−3.02 (m, 2H), 2.07 (s, 3H), 1.22 (t, J = 6.3 Hz, 6H); ¹³C{¹H} NMR (151 MHz, CDCl₃, 298K) δ 167.0, 165.9, 159.7, 142.3, 139.2, 129.7, 128.7, 128.5, 128.4, 128.0, 126.0, 113.7, 44.2, 37.3, 34.6, 14.7, 13.5; IR (film) ν_max 2966, 2927, 1549, 1495, 1455, 1402, 1375, 1347, 1080, 768, 700 cm⁻¹; HRMS (ESI-TOF) m/z: [M + H]⁺ Calcd for C₂₃H₂₈N₃ 346.2283; Found 346.2293.

Data Availability Statement

The data underlying this study are available in the published article and its online supplementary material.