## 2 General Methods for the Synthesis of 1,2,4,5-tetrazines
### Pinner Synthesis Using Ininoesters  
Derivatives of 1,2,4,5-tetrazine (**6**) substituted at positions 3 and 6 are of greater importance. These compounds are prepared using a reaction developed in the late 19 th century by [PERSON] (Scheme 3) [19; 31]. In this transformation, the starting iminoester (**7**) reacts with hydrazine, yielding the corresponding amidrazone (**8**). The amidrazone then undergoes cyclization in the presence of excess hydrazine, resulting in the formation of dihydro-1,2,4,5-tetrazine derivatives (**9**). The final step involves oxidation to produce the corresponding 1,2,4,5-tetrazines (**10**) using an oxidizing agent [19]. It has been shown that performing this reaction with molecular oxygen is challenging due to high pressure requirements and its slow rate (Table 1). Significantly improved results during the oxidation of dihydro-1,2,4,5-tetrazines to 1,2,4,5-tetrazines were obtained by using nitrogen oxides generated in situ from sodium nitrite dissolved in hydrochloric or acetic acid [32; 33; 34; 35; 36; 37; 38; 39; 40; 41]. However, in some cases, side oxidation reactions affecting certain functional groups near the 1,2,4,5-tetrazine ring, particularly the amino group, have been observed [42]. Studies exploring the use of other inorganic compounds as oxidants include iron(III) chloride [43], hydrogen peroxide [44; 45], molecular bromine [31], manganese(IV) oxide [21], and chromium(VI) oxide [46]. However, low reaction yields and safety concerns, especially the carcinogenicity of chromium compounds, have limited the utility of these methods. Organic oxidants present an alternative to sodium nitrite for the oxidation of dihydro-1,2,4,5-tetrazines to 1,2,4,5-tetrazines. These include 2,3-dicyano-5,6-dichloro-1,4-benzzoquinone (**11**, DDQ) [47], [bis(acetoxy)iodo]benzene (**12**, PIDA) [48], \(m\)-chloroperoxidase acid (**13**, \(m\)-CPBA) [42], \(N\)-chloro-\(N\)-(4,4,5,5,6,6,7,7,8,8,9,9,10,11,11,11,11-heptadecafluoromedcycl)-4-methylbenzenesulfonamide (**14**, F-CAT) [49], benzoyl peroxide (**15**) [42], isopentyl nitrite (**16**) [50], or 1,4-benzoquinone (**17**) [42] (Figure 4, Table 1). The search for an effective organic oxidizing agent led to an investigation of the oxidation of 3-(5-aminopyridine-2-yl)-6-(pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazine (Scheme 4). The re  
Figure 4: Organic reagents used in the oxidation of dihydro-1,2,4,5-tetrazines to 1,2,4,5-tetrazines. action of compound **18** with 2,3-discyano-5,6-dichloro-1,4-benzoquinone (DDQ, **11**) yielded product **19**, with a yield of 81%. The authors reported challenges in isolating the target product due to the presence of the reduced form (DDQ-H2) [47]. A lower yield (38%) was obtained using [bis(acetoxy)iodo]benzene (**12**) in dichloromethane. However, this reaction was performed under milder conditions and required less time compared to the reaction with DDQ [51]. The best result (86% yield) was achieved when F-CAT (**14**) was used as the oxidizing agent, with the reaction conducted at 40 \({}^{\circ}\)C for 12 h [49]. The oxidation of dihydro-1,2,4,5-tetrazines to 1,2,4,5-tetrazines using _m_-chloroperoxybenzoic acid (**13**, _m_-CPBA) resulted in the desired products with a 55% yield. Benzoyl peroxide (**15**) and benzoquinone (**17**) were also effective in producing final products with yields of 65% and 71%, respectively [42]. Oxidation of substituted dihydro-1,2,4,5-tetrazines was also performed enzymatically using horseradish peroxidase [52]. Table 1 summarizes the inorganic and organic oxidizing agents and the conditions applied in the oxidation of dihydro-1,2,4,5-tetrazines (**1**) to the corresponding 1,2,4,5-tetrazines (**2**).