Synthesis and Luminescent Properties of s-Tetrazine Derivatives Conjugated with the 4H-1,2,4-Triazole Ring

New derivatives obtained by the combination of unique 1,2,4,5-tetrazine and 4H-1,2,4-triazole rings have great application potential in many fields. Therefore, two synthetic few-step methodologies, which make use of commercially available 4-cyanobenzoic acid (method A) and ethyl diazoacetate (method B), were applied to produce two groups of the aforementioned heterocyclic conjugates. In both cases, the target compounds were obtained in various combinations, by introducing electron-donating or electron-withdrawing substituents into the terminal rings, together with aromatic or aliphatic substituents on the triazole nitrogen atom. Synthesis of such designed systems made it possible to analyze the influence of individual elements of the structure on the reaction course, as well as the absorption and emission properties. The structure of all products was confirmed by conventional spectroscopic methods, and their luminescent properties were also determined.


Introduction
Over the years, scientists from around the world have been keen to study heterocyclic organic compounds, and nitrogen-rich systems have proven to be particularly valuable. One of the most interesting areas of this research is the synthesis and properties of 1,2,4,5tetrazine derivatives (s-tetrazine). This unique ring contains four nitrogen atoms, which is the maximum content in a stable six-membered system. This specific structure has attracted scientists' attention as an important candidate for high energy density materials (HEDMs, A, Scheme 1), as its thermal decomposition leads to ring opening and the release of a nitrogen molecule [1][2][3]. The high nitrogen content has also encouraged research into its biological activity (B, Scheme 1), which has resulted in compounds that have anti-tubercular, anti-cancer, or anti-malarial effects [4][5][6]. Moreover, its high reactivity in Diels-Alder reactions with inverse electron demand determines its application potential in bioorthogonal chemistry (C, Scheme 1) [7][8][9][10]. Important features of the s-tetrazine ring are its low-energy n→π electronic transitions, which are especially valuable from the point of view of optoelectronics (Scheme 1). It can be used in the production of organic light-emitting diodes (OLEDs), organic field-effect transistors (OFETs), and solar cells. Due to the high electronegativity of nitrogen, the ring in question is also characterized by a high electron deficit, and thus a high electron affinity. Consequently, it is also a promising building block in ambipolar and n-type materials [11,12].
Many synthetic methods can be found in the literature for both s-tetrazine and 4H-1,2,4triazole derivatives. The five-membered heterocycle is usually obtained from acyclic compounds such as N,N'-diacylhydrazines, N-cyanoguanidine, isothiocyanates, hydrazides, aminoethylidenehydrazones, aldehydes, and semicarbazides [21]. For the six-membered s-tetrazine system, the Pinner method is the most popular: cyclization, supported by an activating agent, occurs as a result of the reaction of carbonitriles with hydrazine hydrate. The product of this transformation is the corresponding dihydro derivative that requires oxidation to give the desired ring [22,23]. This approach is distinguished by a wide range of substrates, but also the ability to synthesize both symmetrical and unsymmetrical products. Our research to date proves that, among its other uses, it is perfect for the preparation of complex conjugated systems that contain additional five-membered rings. In recent years, we have successfully synthesized s-tetrazine conjugated via a 1,4-phenylene linker with a range of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 4H-1,2,4-triazoles; however, in the latter case, we have so far only obtained symmetrical systems [24][25][26]. In a continuation of our research, we decided to use the Pinner method to prepare unsymmetrical ones. Moreover, encouraged by the improvement in the luminescent properties after the introduction of the 4H-1,2,4-triazole ring, we found that the directly connected heterocycles could be the basis of very promising products. Therefore, we focused on modifying the methodology used to prepare analogous compounds containing 1,3,4-oxadiazole and 1,3,4-thiadiazole, so as to introduce the triazole ring instead [27]. This study was planned to make it possible not only to obtain new, unknown compounds, but also to analyze the influence of their structure on their absorption and emission properties.

Synthesis
As already mentioned in previous studies, we obtained a series of symmetric s-tetrazine derivatives conjugated via a 1,4-phenylene linker with a 4H-1,2,4-triazole ring. For this purpose, it was necessary to prepare appropriate precursors for the Pinner reaction, i.e., carbonitriles containing a five-membered ring (Scheme 2, 6a-h). Initially, from the commercially available 4-cyanobenzoic acid (1), we obtained the hydrazide (2) in a two-step reaction sequence. The original assumption was to treat it with acid chlorides (3a-d) in order to obtain diacyl derivatives (4a-d), and then convert them into the corresponding imidoyl chlorides (5a-d), which, under the influence of amines, would be cyclized to the assumed products (6a-h). This approach, however, turned out to be very troublesome due to the formation of the undesirable products 7a-d. This prompted us to change the reaction path by synthesizing other imidoyl chlorides (8a-h) from the corresponding amides. These intermediates were treated with hydrazide (3), resulting in the target precursors (6a-h) in satisfactory yields [26]. Scheme 2. Synthesis of precursors containing a 4H-1,2,4-triazole ring (6a-h) [26].
The presence of the carbonitrile moiety allows the formation of a second heterocycle, which is s-tetrazine. Under the conditions of the Pinner method, the treatment of the precursors 6a-h with hydrazine hydrate, in the presence of an activating agent, leads to the formation of unoxidized derivatives of the assumed products 9a-l. One of the popular activating agents is sulfur, with the help of which we have successfully obtained symmetrical s-tetrazine derivatives connected via a 1,4-phenylene linker with a 4H-1,2,4-triazole ring, and extended systems containing 1,3,4-oxadiazole and 1,3,4-thiadiazole cores [24][25][26]. Therefore, we also began to research the synthesis of unsymmetrical compounds with the use of this methodology, which allowed us to obtain the product 10a with a yield of 42% (Entry 1, Table 1). In connection with literature reports on the possibility of improving this yield with the use of zinc catalysts [28,29], we attempted to repeat the described transformation with its participation and, as a result, the yield increased to 56% (Entry 2, Table 1). An analogous test was performed for derivatives containing an aliphatic chain attached to the triazole nitrogen atom, instead of an aromatic ring (10g). Again, the yield improved from 35% to 50% (Entries 8 and 9, Table 1). These results were an important reason to modify the previously used procedure. Such a modified approach resulted in obtaining a series of unsymmetrical systems containing both electron-donating and electron-withdrawing substituents in the terminal ring. Traces of two symmetrical products were also detected. As in the previous studies, the oxidation was carried out with hydrogen peroxide (Scheme 3). The next step was the synthesis of products in which s-tetrazine is directly linked to the 4H-1,2,4-triazole ring. As part of our previous research, we had already obtained similar compounds containing 1,3,4-oxadiazole and 1,3,4-thiadiazole, but their synthesis required the use of microwave irradiation [27]. The methodology was based on the use of commercially available ethyl diazoacetate (11), which was transformed into a dihydrazide (12) in a sequence of several transformations (Scheme 4). The product was then treated with acid chlorides to prepare bisdiacyl derivatives (13). In this case, too, we intended to convert these compounds into imidoyl chlorides (14), which could then be cyclized to triazoles (15a) under the influence of amines. However, the high reactivity of such derivatives again caused serious difficulties. Despite the maximum shortening of the reaction times, which had a beneficial effect in previous studies, the observed undesirable derivatives of 1,3,4-oxadiazole (16) were predominantly formed. Additionally, isolation of the desired product from the reaction mixture was extremely problematic and, as a result, only traces of the target compound were obtained. Based on the experience of obtaining triazole precursors for the Pinner reaction, where we encountered a similar problem, we decided to use an alternative methodology. For this purpose, the dihydrazide 12 was reacted with a range of imidoyl chlorides (8a-h) previously obtained from amides (Scheme 5). This approach was effective for both systems containing an aromatic ring (15a-d) and an aliphatic chain (15e-h) on the triazole nitrogen atom. In addition, derivatives containing both electron-donating and electron-withdrawing moieties attached to a terminal aromatic ring were obtained. Compared to the unsubstituted products, the electron-withdrawing nitro group showed a decreased yield (Entries 4 and 8, Table 2), while for the electron-donating groups (methoxy and tert-butyl) the yield was increased (Entries 2, 3, 6, 7, Table 2). The presence of an aliphatic chain also had a beneficial effect on the reaction yield (Entries 5-8, Table 2).

Scheme 5.
Synthesis of s-tetrazine derivatives directly conjugated to the 4H-1,2,4-triazole ring (15a-h). Reaction conditions: 1,2,4,5-tetrazine-3,6-dicarbohydrazide (12, 0.50 g, 2.5 mmol), imidoyl chloride (8a-h, 5.5 mmol), chloroform (20 mL), reflux, 24 h. The structure of all the obtained intermediates and final products was confirmed by 1 H-and 13 C-NMR spectroscopy. Both in the case of systems containing a 1,4-phenylene linker, and with directly conjugated heterocycles, the 13 C-NMR spectra were the most characteristic. The presence of the 4H-1,2,4-triazole ring was confirmed by signals above 140 ppm, and the presence of the s-tetrazine ring by signals above 160 ppm. The introduction of individual groups to the terminal aromatic ring conditioned the appearance of specific signals for the benzene carbon attached to them: above 160 ppm for the methoxy group, above 150 ppm for the tert-butyl group, and above 140 ppm for the nitro group. The lowest shifts corresponded to the carbon atoms of the aliphatic chain (13-45 ppm), the methoxy group (about 55 ppm), and the tert-butyl group (30-35 ppm). The 1 H-NMR spectra mainly included aromatic signals. Additionally, the protons of the aliphatic chain (butyl) gave a series of signals in the range of 0.6-4.5 ppm, the methoxy group a peak around 3.8 ppm, and the tert-butyl group a peak around 1.3 ppm.

Luminescent Properties
UV-Vis and 3D fluorescence spectra were registered for compounds 10a-l and 15a-h ( Figures S40-S64, Supplementary Materials). The fluorescence was completely quenched in the case of 15d and 15h, due to the presence of two NO 2 groups in their structure. The rest of the compounds exhibited a maximum of one emission. The range of emission wavelengths is 375-412 nm for the 10a-l series (Entries 1-12, Table 3) and 353-375 nm for the 15a-h series (Entries 13-20, Table 3). It shows that the separation of fluorophore moieties by phenyl ring leads to a bathochromic shift of fluorescence. In the tetrazine and triazole derivatives, the n→π* transitions are a source of fluorescence [30][31][32][33]. The location of emission maximum (excitation wavelength-λ ex and emission wavelength-λ em ) is dependent on substituents R 1 , R 2 , and R 3 , which indicates that both tetrazine and triazole rings are involved in the orbitals from which the excitation occurs. The influence of substituents on λ ex and λ em is the same as in previously reported symmetrically substituted analogs of the 10a-l series [26]. The R 2 affects the λ ex , whereas R 1 and R 3 affect the λ em . The Ph substituent as R 2 induces the bathochromic shift of λ ex (Entries 1-6 and 13-16, Table 3) in comparison to n-Bu (Entries 7-12 and 17-20, Table 3, red color vs. blue color in Figure S65). In the case of the 15a-h series, which consists of the symmetrically substituted compounds, the λ em increases together with the rising electron-donating strength of R 1 (H < t-Bu < OCH 3 ), which is typical for tetrazine derivatives [34,35]. A partially similar relationship is observed in the unsymmetrically substituted 10a-l series. Taking into account compounds with the same substituent as one of R 1 /R 3 , e.g., NO 2 , the λ em shifts bathochromically in line with the electron-donating properties of the second R 1 /R 3 substituent, i.e., H < t-Bu < OCH 3 . However, there are some exceptions to that rule in this series because, compared to compounds containing OCH 3 / t-Bu and OCH 3 /NO 2 substituents (10d vs. 10e and 10j vs. 10k, Entries 4, 5, 10 and 11, Table 3), those with NO 2 (which is an electron-withdrawing group) unexpectedly possess a larger λ em . This shows that the changes in the electron density distribution induced by different substituents in unsymmetrically substituted compounds are difficult to predict, thus inferring their absorption-emission properties based only on a molecular structure can be misleading. The quantum yield (Φ) is directly related to the fluorescence intensity for the studied compounds ( Figure S66). Generally, the compounds with Ph as R 2 exhibit higher Φs than those with n-Bu, which is in agreement with previous findings [26]. However, most of the studied compounds are not efficient fluorescent materials, because their Φs do not exceed 0.3 ( Table 3). The relatively favorable conjugation occurs only for three compounds, i.e., 10a, 10b, and 10d. It shows that the direct coupling of tetrazine and triazole rings, as well as n-Bu as R 2 and NO 2 as R 1 /R 3 , decreases the population of fluorescent transitions. Table 3. Spectroscopic data for the studied s-tetrazine derivatives. λ abs -wavelength of absorption maximum directly preceding λ em . λ ex and λ em -excitation and emission wavelength at global fluorescence maximum. Stokes shift was calculated as λ em − λ abs . UV-Vis absorption and 3D fluorescence spectra were registered in dichloromethane solutions (c = 5 × 10 −6 mol/dm 3 ). The quantum yields Φ were determined according to the method described by Brouwer [36] by comparison with two standards: quinine sulphate (qn-SO 4 2− ) [37] and trans,trans-1,4-diphenyl-1,3-butadiene (dpb) [38]. Summarizing the current and previous research on s-tetrazine derivatives in terms of their Φs, it can be stated that they are moderately efficient fluorescent materials. Most of the investigated tetrazine derivatives exhibit Φ no higher than 0.60, but there are some examples, which achieve Φ close to 1, which shows their great potential to use as functional materials, e.g., in optoelectronic applications. In the case of s-tetrazines conjugated via phenylene linkers with different 5-membered rings (Scheme 6, Table 4), the Φ changes approximately according to the following order, Triazole (R 2 = n-Bu) < Oxadiazole ≤ Thiadiazole < Triazole (R 2 = Ph). On the other hand, the analogical order for s-tetrazines directly conjugated with the same 5-membered rings is as follows, Triazole (R 2 = n-Bu) < Triazole (R 2 = Ph) < Oxadiazole < Thiadiazole (Scheme 7, Table 5). The greatest similarities are between oxadiazoles and thiadiazoles bearing s-tetrazine, due to small structural changes resulting from the replacement of oxygen with sulfur (atoms with similar electronic properties). Notably, the separation of tetrazine rings and triazole rings via phenylene linkers is more favorable for the fluorescence efficiency than the direct conjugation of them. This is in agreement with the study on the nature of the absorption-emission properties of tetrazine derivatives, which revealed that fluorescence is dependent on the character of HOMO and HOMO-1 orbitals [34]. Fluorescence occurs when the orbital involved in the excitation has a nonbonding n character, but if it is π orbital, the fluorescence is quenched. In this research, it was found that tetrazine derivatives directly conjugated with heteroatomic rings did not exhibit fluorescence, while diphenyl s-tetrazine was reported to be weakly fluorescent [34,39]. It showed that the conjugation with phenyl rings allows for the retention of the nonbonding n character of the excited orbitals, whereas the direct conjugation with heteroatomic rings changes its character to the π one. Scheme 6. Structure of s-tetrazine derivatives conjugated via phenylene linkers with oxadiazole, thiadiazole, and triazole rings. Table 4. Comparison of the quantum yields of s-tetrazine derivatives conjugated via phenylene linkers with oxadiazole [24], thiadiazole [25], and triazole rings (symmetrically substituted from [26], and unsymmetrically substituted from current work).

Conclusions
Two effective methodologies for the synthesis of extended systems containing 1,2,4,5tetrazine and 4H-1,2,4-triazole have been presented. The first methodology, comprising the Pinner reaction of carbonitriles bearing a 4H-1,2,4-triazole scaffold, is useful for obtaining unsymmetrical derivatives with heterocycles connected via a 1,4-phenylene linker. The second procedure, which makes use of imidoyl chloride and s-tetrazine-3,6-dicarbohydrazide, has proven to be successful for symmetrical systems with directly conjugated rings. In both cases, the approach leads to the desired products in satisfactory yields, regardless of the nature of the substituents attached to the terminal rings, as well as the type of groups on the triazole nitrogen atom. The obtained compounds exhibit mainly violet luminescence in CH 2 Cl 2 solution. Their absorption-emission properties are directly related to the compound structure. The spectroscopic investigation revealed the dependency between the electron-donating strength of substituents and the emission wavelength, as well as