Direct access to non-aromatic 1,2,3,6-tetrahydro-1,2,3,4-tetrazines via [4 + 2] cycloaddition of α-halogeno hydrazones with azodicarboxylic acid derivatives

Abstract

In the presence of K₂CO₃, the [4 + 2] cycloaddition of α-halogeno hydrazones with azodicarboxylic acid derivatives proceeded efficiently, and delivered novel non-aromatic 1,2,3,6-tetrahydro-1,2,3,4-tetrazines in moderate to excellent chemical yields.

---

1. Introduction

1,2,3,4-Tetrazines constitute a class of six-membered heterocycles bearing four contiguous nitrogen atoms, and therefore are highly enriched in nitrogen content.¹ As illustrated in Fig. 1, 1,2,3,4-tetrazines have been demonstrated to possess a wide spectrum of biological activities such as anticancer and antimicrobial properties as well as the inhibitory effects on HSP90 and H,K-adenosinetriphosphatase.² Even though the pronounced medicinal and biological importance of 1,2,3,4-tetrazines, not too many previous works focused on the synthesis of structurally diverse aromatic 1,2,3,4-tetrazines as well as non-aromatic 1,2,3,4-tetrazines.³ The chemical synthesis of the aromatic 1,2,3,4-tetrazines attracted relatively much attention from chemists because of their higher thermal stability.⁴ Moreover, the intramolecular cyclization of diazonium salts and azo compounds served as the major tool for the synthesis of the present aromatic 1,2,3,4-tetrazines.⁵ In contrast, the chemical synthesis of the non-aromatic 1,2,3,4-tetrazines did not receive considerable attention, and was not fully explored in the past decades.⁶ Therefore, it is highly needed to develop alternative protocols for the facile and efficient construction of the non-aromatic 1,2,3,4-tetrazines.

Fig. 1 Representative bioactive 1,2,3,4-tetrazines.

It has been indicated that α-halogeno hydrazones are powerful and versatile building blocks, and have found many applications in the synthesis of nitrogen-containing heterocycles. For instances, as outlined in Scheme 1, in the presence of bases such as Na₂CO₃ or K₂CO₃, the in situ generated 1,2-diaza-1,3-dienes from α-halogeno hydrazones could undergo [4 + 1],⁷ [4 + 2]⁸ and [4 + 3]⁹ cycloadditions with an array of various electron-deficient dienophiles, respectively (eqn (1)–(3)). Encouraged by these previously reported elegant examples, we designed the novel [4 + 2] cycloaddition of α-halogeno hydrazones for the efficient direct access to 1,2,3,4-tetrazines bearing potential bioactivities (eqn (4)). As compared with literature works,^6c,10 we applied the easily prepared and more stable α-halogeno hydrazones instead of the transient 1,2-diaza-1,3-butadienes¹¹ to react with azodicarboxylic acid derivatives for the preparation of the non-aromatic 1,2,3,6-tetrahydro-1,2,3,4-tetrazines. Our research results indicated that the newly designed [4 + 2] cycloaddition of α-halogeno hydrazones with azodicarboxylic acid derivatives proceeded smoothly under mild reaction conditions, and the desired 1,2,3,6-tetrahydro-1,2,3,4-tetrazines were produced in moderate to excellent chemical yields.

Scheme 1 Representative α-halogeno hydrazone involved cycloadditions.

2. Results and discussion

Initially, using DCM as solvent, we screened the effect of a series of bases on the chemical yield of the [4 + 2] cycloaddition of α-halogeno hydrazone 1a with azodicarboxylic acid derivative 2a as summarized in Table 1. Noticeably, the chemical yield was significantly affected by the bases used in the [4 + 2] cycloaddition. For instances, the use of Et₃N as base gave a trace amount of product 3aa in 48 h (Table 1, entry 4). In regard to MeONa, NaHCO₃ and KOH as bases, the chemical yields ranged from 48% to 70% (Table 1, entries 5–7). As for the other bases such as Na₂CO₃, K₂CO₃ as well as Cs₂CO₃, they delivered product 3aa in 82–93% yields, and the best chemical yield was achieved with the use of K₂CO₃ as base. Simultaneously, the solvent effect was examined in the [4 + 2] cycloaddition in the presence of K₂CO₃ as base (Table 1, entries 8–12). In MeOH solvent, 3aa was produced in low chemical yield (Table 1, entry 12). With respect to the other solvents tested, the chemical yields were changed from 82% to 99% (Table 1, entries 8–11). However, the [4 + 2] cycloaddition did not occur at all in water (Table 1, entry 13). Taking into account of the chemical yield of 3aa, the optimal reaction condition was determined as below: 1.2 : 1 molar ratio of 1a to 2a/K₂CO₃ (1.2 equiv.)/THF/room temperature.

Table 1 Optimization of reaction conditions^a

Entry	Base	Solvent	Time (h)	Yield^b (%)
a Unless otherwise noted, reactions were carried out with 1a (0.12 mmol), 2a (0.1 mmol) and base (0.12 mmol) in the solvent used (0.5 mL) at room temperature.b Isolated chemical yield.c No reaction.
1	Na2CO3	DCM	18	82
2	K2CO3	DCM	18	93
3	Cs2CO3	DCM	18	87
4	Et3N	DCM	48	Trace
5	KOH	DCM	24	70
6	NaHCO3	DCM	48	61
7	MeONa	DCM	48	48
8	K2CO3	THF	18	99
9	K2CO3	Toluene	18	97
10	K2CO3	MeCN	18	88
11	K2CO3	Et2O	18	82
12	K2CO3	MeOH	30	37
13	K2CO3	H2O	18	Nr^c

---

Subsequently, under the optimal reaction conditions, we extended the reaction scope of the [4 + 2] cycloaddition by diversifying α-halogeno hydrazones 1 and azodicarboxylic acid derivatives 2 as outlined in Table 2. In most cases, products 3 were obtained in good to excellent chemical yields (Table 2, entries 1–7, 9–17, 19 and 21). Notably, the chemical yield of the [4 + 2] cycloaddition changed differently with the used substrates 1 and 2. In the case of 2a, the chemical yield of the [4 + 2] cycloaddition ranged from 76% to 99% with the use of 1a–1l (Table 2, entries 1–12). Meanwhile, the [4 + 2] cycloaddition of 2b with the structurally different substrates 1 yielded the corresponding products 3 in 78–96% chemical yields (Table 2, entries 13–19). In comparison with the former cases, the use of 2c as substrate in the [4 + 2] cycloaddition usually gave products 3 in generally lower chemical yields (Table 2, entries 20–23).

Table 2 Extension of the reaction scope^a

Entry	1(X, R^1, R^2)	2	3	Time (h)	Yield^b (%)
a Unless otherwise noted, reactions were carried out with 1 (0.12 mmol), 2 (0.1 mmol), K2CO3 (0.12 mmol) in THF (0.5 mL) at room temperature.b Isolated yield.
1	1a(Br, Ph, Me)	2a	3aa	18	99
2	1b(Cl, Ph, Me)	2a	3aa	18	99
3	1c(Br, 4-MeOC6H4, Me)	2a	3ca	18	86
4	1d(Br, 4-MeC6H4, Me)	2a	3da	18	95
5	1e(Br, 4-ClC6H4, Me)	2a	3ea	18	91
6	1f(Br, 4-BrC6H4, Me)	2a	3fa	18	90
7	1g(Br, 4-FC6H4, Me)	2a	3ga	18	93
8	1h(Br, 4-NO2C6H4, Me)	2a	3ha	18	76
9	1i(Br, 3-ClC6H4, Me)	2a	3ia	18	91
10	1j(Cl, Ph, Ph)	2a	3ja	18	91
11	1k(Cl, Ph, O^tBu)	2a	3ka	24	82
12	1l(Cl, Ph, OMe)	2a	3la	18	89
13	1a(Br, Ph, Me)	2b	3ab	18	84
14	1d(Br, 4-MeC6H4, Me)	2b	3db	18	96
15	1f(Br, 4-BrC6H4, Me)	2b	3fb	18	90
16	1m(Br, ^tBu, Me)	2b	3mb	20	90
17	1j(Cl, Ph, Ph)	2b	3jb	18	84
18	1k(Cl, Ph, O^tBu)	2b	3kb	24	78
19	1l(Cl, Ph, OMe)	2b	3lb	18	96
20	1e(Br, 4-ClC6H4, Me)	2c	3ec	30	66
21	1a(Br, Ph, Me)	2c	3ac	30	82
22	1i(Br, 3-ClC6H4, Me)	2c	3ic	30	71
23	1l(Cl, Ph, OMe)	2c	3lc	30	50

---

Finally, the chemical structure of 3aa was unambiguously determined by single crystal X-ray analysis as depicted in Fig. 2.¹² The conformational analysis of 3aa shows that its 1,2,3,6-tetrahydro-1,2,3,4-tetrazine ring adopt twist-boat conformation. As a result, the two protons of methylene group of 1,2,3,6-tetrahydro-1,2,3,4-tetrazine ring of 3aa reside in the completely different chemical environment. This fact was strongly supported by the different ¹H NMR behaviors of two protons of methylene group of 3aa: one proton resonates at 4.38 ppm, and the other one appears at 4.93 ppm (see details in the ESI†). Noticeably, it was found that the two carboxylate moieties of 1,2,3,6-tetrahydro-1,2,3,4-tetrazine ring of 3aa orientate in the quite different spatial pattern: one adopts a pseudo-axial position; the other one has a pseudo-equatorial orientation. Simultaneously we proposed the reaction mechanism for the formation of 3aa (Scheme 2). In the presence of K₂CO₃, the elimination reaction of 1a take place to give 1,2-diaza-1,3-diene 4. Then, as Jiménez and co-workers described in their previously published work,^6c the resulted 4 undergo the [4 + 2] cycloaddition with 2a to form product 3aa through the transition state TS1 instead of the transition state TS2.

Fig. 2 X-ray single crystal structure of 3aa (with thermal ellipsoid shown at the 50% probability level).

Scheme 2 Proposed mechanism for the formation of 3aa.

3. Conclusions

In conclusion, the newly designed [4 + 2] cycloaddition of α-halogeno hydrazones with azodicarboxylic acid derivatives proceeded efficiently, and gave rise to the easy direct access to the new potentially bioactive 1,2,3,6-tetrahydro-1,2,3,4-tetrazines in moderate to excellent yields.

4. Experimental section

4.1 General information

Proton (¹H) and carbon (¹³C) NMR spectra were recorded on a Bruker AVANCE II 400 spectrometer operating at 400 MHz for proton and 100 MHz for carbon nuclei and calibrated using tetramethylsilane (TMS) as internal reference. High resolution mass spectra (HRMS) were recorded on a VG ZAB-2F under electrospray ionization (ESI) conditions. The melting point of compounds was determined by a Beijing Fukai X-5 melting point instrument. Flash column chromatography was performed on silica gel (0.035–0.070 mm) using compressed air. Thin layer chromatography (TLC) was carried out on 0.25 mm SDS silica gel coated glass plates (60F254). Eluted plates were visualized using a 254 nm UV lamp. Unless otherwise indicated, all reagents were commercially available and used without further purification. All solvents were distilled from the appropriate drying agents immediately before using. α-Chloro- or α-bromo hydrazones (1a–1m) were prepared according to literature procedures.^7b,8e,9b

4.2 Procedure for the synthesis of products 3

K₂CO₃ (1.2 equiv., 0.12 mmol) was added to a solution of α-chloro- or α-bromo hydrazone 1 (1.2 equiv., 0.12 mmol) and azodicarboxylic acid derivatives 2 (1.0 equiv., 0.1 mmol) in dry THF (0.5 mL). The mixture was monitored by TLC plate and stirred for 18–30 h at room temperature. The crude products were purified by flash column chromatography on silica gel using EtOAc–petroleum as eluent to give products 3 as white solids (50–99% yield).

Diisopropyl 3-acetyl-5-phenyl-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3aa). White solid, yield: 37.3 mg, 99%; mp = 119.0–120.2 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.74 (d, J = 5.2 Hz, 2H), 7.44–7.41 (m, 3H), 5.04–4.99 (m, 2H), 4.93 (s, 1H), 4.38 (s, 1H), 2.43 (s, 3H), 1.32–1.30 (m, 9H), 1.25 (d, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 154.2, 153.8, 133.3, 130.9, 128.7, 125.6, 73.8, 71.4, 41.5, 21.9, 21.9, 21.7, 21.6, 20.8 ppm; HRMS (ESI) calculated for C₁₈H₂₅N₄O₅ [M + H]⁺: 377.18195, found 377.18140.

Diisopropyl 3-acetyl-5-(4-methoxyphenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ca). White solid, yield: 34.9 mg, 86%; mp = 108.5–109.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.71 (d, J = 5.6 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 5.05–5.00 (m, 2H), 4.93 (d, J = 19.2 Hz, 1H), 4.34 (d, J = 15.6 Hz, 1H), 3.84 (d, J = 1.2 Hz, 3H), 2.42 (s, 3H), 1.33–1.31 (m, 9H), 1.26 (d, J = 6.0 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 161.8, 154.3, 153.8, 128.8, 127.2, 125.9, 114.1, 73.8, 71.4, 55.4, 41.3, 22.0, 21.9, 21.7, 21.7, 20.8 ppm; HRMS (ESI) calculated for C₁₉H₂₇N₄O₆ [M + H]⁺: 407.19251, found 407.19177.

Diisopropyl 3-acetyl-5-(p-tolyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3da). White solid, yield: 37.0 mg, 95%; mp = 121.7–122.4 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.65 (d J = 6.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 5.05–5.01 (m, 2H), 4.92 (s, 1H), 4.37 (s, 1H), 2.43 (s, 3H), 2.39 (s, 3H), 1.33–1.32 (m, 9H), 1.26 (d, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 154.3, 141.3, 130.6, 129.4, 128.8, 125.5, 73.8, 71.4, 41.4, 22.0, 21.9, 21.7, 21.6, 21.4, 20.8 ppm; HRMS (ESI) calculated for C₁₉H₂₇N₄O₅ [M + H]⁺: 391.19760, found 391.19696.

Diisopropyl 3-acetyl-5-(4-chlorophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ea). White solid, yield: 37.3 mg, 91%; mp = 141.1–141.8 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.70 (d, J = 7.2 Hz, 2H), 7.40–7.38 (m, 2H), 5.06–5.00 (m, 2H), 4.92 (d, J = 16.4 Hz, 1H), 4.36 (s, 1H), 2.43 (s, 3H), 1.33–1.31 (m, 9H), 1.27 (d, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 154.1, 153.7, 137.0, 131.8, 129.0, 126.8, 73.9, 71.6, 41.3, 21.9, 21.9, 21.7, 21.6, 20.8 ppm; HRMS (ESI) calculated for C₁₈H₂₄ClN₄O₅ [M + H]⁺: 411.14297, found 411.14236.

Diisopropyl 3-acetyl-5-(4-bromophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3fa). White solid, yield: 41 mg, 90%; mp = 136.8–137.7 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.63 (d, J = 6.8 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 5.07–5.01 (m, 2H), 4.92 (d, J = 17.2 Hz, 1H), 4.35 (d, J = 15.2 Hz, 1H), 2.43 (s, 3H), 1.33–1.31 (m, 9H), 1.27 (d, J = 6.0 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 154.1, 153.7, 132.2, 132.0, 127.0, 125.4, 74.0, 71.6, 41.2, 22.0, 21.9, 21.7, 21.7, 20.8 ppm; HRMS (ESI) calculated for C₁₈H₂₄BrN₄O₅ [M + H]⁺: 455.09246, found 455.09204.

Diisopropyl 3-acetyl-5-(4-fluorophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ga). White solid, yield: 36.6 mg, 93%; mp = 119.1–120.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.75 (s, 2H), 7.09 (t, J = 8.4 Hz, 2H), 5.05–4.98 (m, 2H), 4.92 (d, J = 18.0 Hz, 1H), 4.35 (s, 1H), 2.41 (s, 3H), 1.31–1.30 (m, 9H), 1.25 (d, J = 6.0 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 165.6, 163.1, 154.1, 153.7, 129.5, 127.7, 116.0, 115.8, 73.9, 71.5, 41.3, 21.9, 21.9, 21.7, 21.6, 20.8 ppm; HRMS (ESI) calculated for C₁₈H₂₄FN₄O₅ [M + H]⁺: 395.17252, found 395.17184.

Diisopropyl 3-acetyl-5-(4-nitrophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ha). White solid, yield: 32.0 mg, 76%; mp = 66.7–67.1 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.23 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H), 5.02–4.94 (m, 3H), 4.41 (s, 1H), 2.44 (s, 3H), 1.31–1.30 (m, 9H), 1.26 (d, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 153.9, 153.6, 148.9, 139.0, 126.4, 123.9, 74.2, 71.8, 41.4, 29.6, 26.9, 21.9, 21.9, 21.7, 21.6, 20.8 ppm; HRMS (ESI) calculated for C₁₈H₂₄N₅O₇ [M + H]⁺: 422.16702, found 422.16638.

Diisopropyl 3-acetyl-5-(3-chlorophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ia). White solid, yield: 37.4 mg, 91%; mp = 120.0–121.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.76 (s, 1H), 7.61 (d, J = 6.4 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 8.0 Hz, 1H), 5.05–5.02 (m, 2H), 4.91 (d, J = 15.6 Hz, 1H), 4.36 (s, 1H), 2.44 (s, 3H), 1.34–1.32 (m, 9H), 1.28 (d, J = 6.0 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.5, 154.0, 135.1, 130.8, 130.0, 125.7, 123.6, 74.0, 71.6, 41.4, 21.9, 21.9, 21.7, 21.7, 20.8 ppm; HRMS (ESI) calculated for C₁₈H₂₄ClN₄O₅ [M + H]⁺: 411.14297, found 411.14233.

Diisopropyl 3-benzoyl-5-phenyl-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ja). White solid, yield: 39.8 mg, 91%; mp = 158.4–158.8 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.92 (d, J = 7.6 Hz, 2H), 7.75 (d, J = 6.8 Hz, 2H), 7.54–7.50 (m, 1H), 7.47–7.42 (m, 5H), 5.12–5.06 (m, 1H), 4.95 (d, J = 8.0 Hz, 1H), 4.93–4.90 (m, 1H), 4.45 (d, J = 18.0 Hz, 1H), 1.35 (d, J = 6.0 Hz, 3H), 1.31 (d, J = 6.4 Hz, 3H), 1.22 (d, J = 6.4 Hz, 3H), 1.18 (d, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 165.4, 154.3, 153.9, 145.9, 133.4, 133.1, 131.2, 130.9, 129.6, 128.8, 127.6, 125.6, 73.9, 71.3, 41.8, 22.0, 21.9, 21.6, 21.5 ppm; HRMS (ESI) calculated for C₂₃H₂₇N₄O₅ [M + H]⁺: 439.19760, found 439.19708.

3-tert-Butyl 1,2-diisopropyl 5-phenyl-1,2,3,4-tetrazine-1,2,3(6H)-tricarboxylate (3ka). White solid, yield: 35.6 mg, 82%; mp = 118.5–119.2 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.77–7.75 (m, 2H), 7.41–7.38 (m, 3H), 5.05–4.97 (m, 2H), 4.91 (d, J = 18.0 Hz, 1H), 4.36 (d, J = 18.0 Hz, 1H), 1.59 (s, 9H), 1.33–1.30 (m, 9H), 1.25 (d, J = 6.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 154.3, 149.3, 144.9, 133.6, 130.5, 128.6, 125.4, 82.8, 73.1, 70.9, 41.3, 28.1, 22.1, 22.0, 21.8, 21.7 ppm; HRMS (ESI) calculated for C₂₁H₃₀N₄O₆Na [M + Na]⁺: 457.20576, found 457.20499.

1,2-Diisopropyl 3-methyl 5-phenyl-1,2,3,4-tetrazine-1,2,3(6H)-tricarboxylate (3la). White solid, yield: 34.9 mg, 89%; mp = 101.4–102.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.76–7.74 (m, 2H), 7.46–7.41 (m, 3H), 5.06–4.99 (m, 2H), 4.94 (d, J = 18.4 Hz, 1H), 4.36 (d, J = 18.4 Hz, 1H), 3.95 (s, 3H), 1.34–1.32 (m, 6H), 1.31 (d, J = 1.6 Hz, 3H), 1.27 (d, J = 6.0 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 154.2, 154.0, 151.5, 146.0, 133.4, 130.7, 128.7, 125.6, 73.5, 71.3, 67.9, 54.2, 41.6, 25.6, 21.9, 21.9, 21.7 ppm; HRMS (ESI) calculated for C₁₈H₂₅N₄O₆ [M + H]⁺: 393.17686, found 393.17612.

Diethyl 3-acetyl-5-phenyl-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3ab). White solid, yield: 29.2 mg, 84%; mp = 70.8–71.4 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.75 (d, J = 4.8 Hz, 2H), 7.46–7.41 (m, 3H), 4.99 (d, J = 17.2 Hz, 1H), 4.43–4.27 (m, 5H), 2.45 (s, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.30 (d, J = 7.2 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 154.6, 154.2, 133.2, 130.9, 129.7, 128.8, 125.5, 68.7, 65.2, 63.5, 41.4, 20.9, 14.4, 14.2 ppm; HRMS (ESI) calculated for C₁₆H₂₁N₄O₅ [M + H]⁺: 349.15065, found 349.15012.

Diethyl 3-acetyl-5-(p-tolyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3db). White solid, yield: 34.9 mg, 96%; mp = 53.6–54.7 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.66 (d, J = 5.2 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 4.98 (d, J = 16.8 Hz, 1H), 4.33–4.26 (m, 5H), 2.46 (s, 3H), 2.40 (s, 3H), 1.35 (d, J = 7.2 Hz, 3H), 1.31 (d, J = 7.6 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 154.7, 154.3, 141.3, 130.5, 129.4, 125.4, 68.6, 65.2, 63.4, 41.4, 21.4, 20.9, 14.4, 14.1 ppm; HRMS (ESI) calculated for C₁₇H₂₃N₄O₅ [M + H]⁺: 363.16630, found 363.16559.

Diethyl 3-acetyl-5-(4-bromophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3fb). White solid, yield: 38.3 mg, 90%; mp = 54.2–55.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.61 (s, 2H), 7.55 (d, J = 8.4 Hz, 2H), 4.94 (d, J = 17.6 Hz, 1H), 4.34 (s, 1H), 4.31–4.25 (m, 4H), 2.44 (s, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.30 (d, J = 6.8 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 154.5, 154.1, 132.1, 132.0, 126.9, 125.4, 65.3, 63.5, 41.3, 20.9, 14.4, 14.2 ppm; HRMS (ESI) calculated for C₁₆H₂₀BrN₄O₅ [M + H]⁺: 427.06116, found 427.06079.

Diethyl 3-acetyl-5-(tert-butyl)-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3mb). White solid, yield: 29.5 mg, 90%; mp = 64.3–65.2 °C; ¹H NMR (400 MHz, CDCl₃): δ 4.47 (d, J = 18.0 Hz, 1H), 4.29–4.22 (m, 4H), 4.02 (d, J = 17.2 Hz, 1H) 2.32 (s, 3H), 1.30–1.27 (m, 6H), 1.18 (s, 9H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 166.7, 154.7, 154.4, 68.7, 64.9, 63.2, 39.8, 37.6, 27.7, 20.8, 14.4, 14.2 ppm; HRMS (ESI) calculated for C₁₄H₂₅N₄O₅ [M + H]⁺: 329.18195, found 329.18143.

Diethyl 3-benzoyl-5-phenyl-1,2,3,4-tetrazine-1,2(3H,6H)-dicarboxylate (3jb). White solid, yield: 34.4 mg, 84%; mp = 103.3–104.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.91 (d, J = 7.2 Hz, 2H), 7.70 (d, J = 6.8 Hz, 2H), 7.55–7.51 (m, 1H), 7.48–7.40 (m, 5H), 5.00 (d, J = 18.4 Hz, 1H), 4.49 (d, J = 18.0 Hz, 1H), 4.35–4.24 (m, 4H), 1.35 (t, J = 6.8 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 165.3, 154.7, 154.3, 145.1, 133.3, 132.9, 131.3, 130.9, 129.7, 128.8, 127.7, 125.5, 65.3, 63.4, 41.7, 14.5, 14.1 ppm; HRMS (ESI) calculated for C₂₁H₂₃N₄O₅ [M + H]⁺: 411.16630, found 411.16580.

3-tert-Butyl-1,2-diethyl 5-phenyl-1,2,3,4-tetrazine-1,2,3(6H)-tricarboxylate (3kb). White solid, yield: 31.6 mg, 78%; mp = 44.6–45.1 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.77–7.75 (m, 2H), 7.42–7.40 (m, 3H), 4.93 (d, J = 18.0 Hz, 1H), 4.40 (d, J = 18.0 Hz, 1H), 4.37–4.20 (m, 4H), 1.59 (s, 9H), 1.34 (d, J = 7.2 Hz, 3H), 1.30 (d, J = 7.2 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 154.8, 154.4, 149.3, 144.7, 133.6, 130.5, 128.6, 125.3, 83.0, 64.8, 63.1, 41.4, 28.1, 26.9, 14.5, 14.2 ppm; HRMS (ESI) calculated for C₁₉H₂₆N₄O₆Na [M + Na]⁺: 429.17446, found 429.17371.

1,2-Diethyl 3-methyl 5-phenyl-1,2,3,4-tetrazine-1,2,3(6H)-tricarboxylate (3lb). White solid, yield: 34.9 mg, 96%; mp = 49.4–50.0 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.76–7.74 (m, 2H), 7.45–7.39 (m, 3H), 4.95 (d, J = 18.4 Hz, 1H), 4.40 (d, J = 18.4 Hz, 1H), 4.33–4.26 (m, 4H), 3.95 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 154.7, 151.4, 146.0, 133.3, 130.8, 128.7, 125.5, 65.1, 63.4, 54.3, 41.6, 14.4, 14.2 ppm; HRMS (ESI) calculated for C₁₆H₂₁N₄O₆ [M + H]⁺: 365.14556, found 365.14514.

(3-Acetyl-5-(4-chlorophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-diyl)bis(piperidin-1-yl-methanone) (3ec). White solid, yield: 30.4 mg, 66%; mp = 79.5–81.7 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.66 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H), 4.62 (d, J = 17.6 Hz, 1H), 4.41 (d, J = 18.0 Hz, 1H), 3.51–3.47 (m, 8H), 2.45 (s, 3H), 1.65–1.60 (m, 12H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 168.4, 158.2, 156.8, 144.5, 136.3, 132.5, 128.8, 126.5, 53.9, 46.9, 46.8, 41.6, 29.3, 25.9, 25.7, 24.4, 24.3, 21.6 ppm; HRMS (ESI) calculated for C₂₂H₂₉ClN₆O₃Na [M + Na]⁺: 483.18819, found 483.18719.

(3-Acetyl-5-phenyl-1,2,3,4-tetrazine-1,2(3H,6H)-diyl)bis(piperidin-1-yl-methanone) (3ac). White solid, yield: 34.9 mg, 82%; mp = 129.2–130.4 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.72–7.70 (m, 2H), 7.41–7.39 (m, 3H), 4.63 (d, J = 17.6 Hz, 1H), 4.44 (d, J = 18.0 Hz, 1H), 3.49 (s, 8H), 2.44 (s, 3H), 1.62–1.58 (m, 12H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 168.5, 158.4, 156.9, 145.6, 134.0, 130.3, 128.6, 125.2, 46.9, 46.8, 41.9, 25.9, 25.7, 24.4, 24.3, 21.7 ppm; HRMS (ESI) calculated for C₂₂H₃₁N₆O₃ [M + H]⁺: 427.24522, found 427.24466.

(3-Acetyl-5-(3-chlorophenyl)-1,2,3,4-tetrazine-1,2(3H,6H)-diyl)bis(piperidin-1-yl-methanone) (3ic). White solid, yield: 32.6 mg, 71%; mp = 166.1–167.4 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.72 (t, J = 1.6 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.41–7.33 (m, 2H), 4.61 (d, J = 17.6 Hz, 1H), 4.41 (d, J = 18.0 Hz, 1H), 3.51–3.48 (m, 8H), 2.46 (s, 3H), 1.67–1.60 (m, 12H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 168.4, 158.1, 156.8, 143.9, 135.8, 134.8, 130.2, 129.9, 125.3, 123.3, 46.9, 46.8, 41.7, 25.9, 25.7, 24.4, 24.3, 21.6 ppm; HRMS (ESI) calculated for C₂₂H₃₀ClN₆O₃ [M + H]⁺: 461.20624, found 461.20572.

Methyl 6-phenyl-3,4-di(piperidine-1-carbonyl)-4,5-dihydro-1,2,3,4-tetrazine-2(3H)-carboxylate (3lc). White solid, yield: 22.1 mg, 50%; mp = 76.6–77.1 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.74–7.72 (m, 2H), 7.41–7.28 (m, 3H), 4.58 (d, J = 18.4 Hz, 1H), 4.48 (d, J = 18.0 Hz, 1H), 3.92 (s, 3H), 3.51–3.44 (m, 8H), 1.64–1.58 (m, 12H) ppm; ¹³C NMR (100 MHz, CDCl₃): δ 158.6, 157.1, 153.0, 147.8, 134.1, 130.4, 128.6, 125.5, 54.3, 46.9, 46.8, 42.4, 26.9, 25.9, 25.8, 24.5, 24.3 ppm; HRMS (ESI) calculated for C₂₂H₃₁N₆O₄ [M + H]⁺: 443.24013, found 443.23978.

Acknowledgements

We thank Beijing Municipal Commission of Education (No. JC015001200902), Beijing Municipal Natural Science Foundation (No. 7102010, No. 2122008), Basic Research Foundation of Beijing University of Technology (X4015001201101), Funding Project for Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality (No. PHR201008025), Doctoral Scientific Research Start-up Foundation of Beijing University of Technology (No. 52015001200701) for financial supports.

References

1. For selected examples, see: (a) V. A. Tartakovsky, I. E. Filatov, A. M. Churakov, S. L. Ioffe, Y. A. Strelenko, V. S. Kuz'min, G. L. Rusinov and K. I. Pashkevich, Russ. Chem. Bull., 2004, 53, 2577; (b) O. Y. Smirnov, A. M. Churakov, A. Y. Tyurin, Y. A. Strelenko, S. L. Ioffe and V. A. Tartakovsky, Russ. Chem. Bull., 2002, 51, 1849; (c) D. L. Lipilin, P. A. Belyakov, Y. A. Strelenko, A. M. Churakov, O. Y. Smirnov, S. L. Ioffe and V. A. Tartakovsky, Eur. J. Org. Chem., 2002, 2002, 3821; (d) A. M. Churakov, O. Y. Smirnov, S. L. Ioffe, Y. A. Strelenko and V. A. Tartakovsky, Eur. J. Org. Chem., 2002, 2002, 2342; (e) A. E. Frumkin, A. M. Churakov, Y. A. Strelenko and V. A. Tartakovsky, Russ. Chem. Bull., 2000, 49, 482.
2. (a) D. Evans, PCT Int. Appl, WO 2015117016, 2015; (b) R. M. Kharate, P. P. Deohate, J. R. Choudhari and B. N. Berad, Indian J. Heterocycl. Chem., 2012, 22, 151; (c) A. M. Almerico, F. Mingoia, P. Diana, P. Barraja, A. Lauria, A. Montalbano, G. Cirrincione and G. Dattolo, J. Med. Chem., 2005, 48, 2859; (d) N. V. Dolgova, A. M. Churakov, O. Y. Smirnov, Y. V. Khropov, N. G. Bogdanova, N. V. Mast, S. L. Ioffe, O. D. Lopina and V. A. Tartakovskii, Russian Patent, RU 2186108, 2002Chem. Abstr. 2003, 138, 133153.
3. A. M. Churakov and V. A. Tartakovsky, Chem. Rev., 2004, 104, 2601.
4. For selected examples, see: (a) A. A. Voronin, V. P. Zelenov, A. M. Churakov, Y. A. Strelenko and V. A. Tartakovsky, Russ. Chem. Bull., 2014, 63, 475; (b) G. Chattopadhyay and P. S. Ray, Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 2013, 52, 546; (c) M. S. Klenov, A. M. Churakov, Y. A. Strelenko and V. A. Tartakovsky, Russ. Chem. Bull., 2011, 60, 2051; (d) G. Chattopadhyay, Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 2010, 49, 1229; (e) D. L. Lipilin, A. M. Churakov, Y. A. Strelenko and V. A. Tartakovsky, Russ. Chem. Bull., 2006, 55, 357.
5. For selected examples, see: (a) M. S. Klenov, O. V. Anikin, A. M. Churakov, Y. A. Strelenko, I. V. Fedyanin, I. V. Ananyev and V. A. Tartakovsky, Eur. J. Org. Chem., 2015, 2015, 6170; (b) A. A. Voronin, V. P. Zelenov, A. M. Churakov, Y. A. Strelenko, I. V. Fedyanin and V. A. Tartakovsky, Tetrahedron, 2014, 70, 3018; (c) M. S. Klenov, V. P. Zelenov, A. M. Churakov, Y. A. Strelenko and V. A. Tartakovsky, Russ. Chem. Bull., 2011, 60, 2040.
6. For selected examples, see: (a) N. A. M. Pereira, S. M. M. Lopes, A. Lemos and T. M. V. D. Pinho e Melo, Synlett, 2014, 25, 423; (b) N. A. Al-Awadi, Y. A. Ibrahim, E. John and A. Parveen, Tetrahedron, 2011, 67, 1298; (c) M. Avalos, R. Babiano, P. Cintas, F. R. Clemente, J. L. Jiménez, J. C. Palacios and J. B. Sánchez, J. Org. Chem., 1999, 64, 6297; (d) M. Avalos, R. Babiano, P. Cintas, J. L. Jiménez, M. M. Molina, J. C. Palacios and J. B. Sánchez, Tetrahedron Lett., 1991, 32, 2513; (e) H. Quast, D. Regnat, E. M. Peters, K. Peters and H. G. Von Schnering, Angew. Chem., Int. Ed., 1990, 102, 724; (f) A. M. Almerico and A. J. Boulton, J. Chem. Soc., Chem. Commun., 1985, 204.
7. (a) O. A. Attanasi, L. De Crescentini, G. Favi, F. Mantellini, S. Mantenuto and S. Nicolini, J. Org. Chem., 2014, 79, 8331; (b) J.-R. Chen, W.-R. Dong, M. Candy, F.-F. Pan, M. Jörres and C. Bolm, J. Am. Chem. Soc., 2012, 134, 6924; (c) O. A. Attanasi, P. Filippone, B. Guidi, T. Hippe, F. Mantellini and L. F. Tietze, Tetrahedron Lett., 1999, 40, 9277.
8. For selected examples, see: (a) X. Zhong, J. Lv and S. Luo, Org. Lett., 2015, 17, 1561; (b) J. Li, R. Huang, Y.-K. Xing, G. Qiu, H.-Y. Tao and C.-J. Wang, J. Am. Chem. Soc., 2015, 137, 10124; (c) M.-C. Tong, X. Chen, J. Li, R. Huang, H. Tao and C.-J. Wang, Angew. Chem., Int. Ed., 2014, 53, 4680; (d) S. M. M. Lopes, A. Lemos and T. M. V. D. Pinho e Melo, Eur. J. Org. Chem., 2014, 2014, 7039; (e) S. Gao, J.-R. Chen, X.-Q. Hu, H.-G. Cheng, L.-Q. Lu and W.-J. Xiao, Adv. Synth. Catal., 2013, 355, 3539.
9. (a) C. Guo, B. Sahoo, C. G. Daniliuc and F. Glorius, J. Am. Chem. Soc., 2014, 136, 17402; (b) X.-Q. Hu, J.-R. Chen, S. Gao, B. Feng, L.-Q. Lu and W.-J. Xiao, Chem. Commun., 2013, 49, 7905.
10. V. S. Sommer and U. Schubert, Angew. Chem., 1979, 91, 757.
11. S. M. M. Lopes, A. F. Brigas, F. Palacios, A. Lemos and T. M. V. D. Pinho e Melo, Eur. J. Org. Chem., 2012, 2012, 2152.
12. CCDC 1433224† contains the supplementary crystallographic data for compound 3aa.

---

Footnote

† Electronic supplementary information (ESI) available: Copies of NMR spectra for all products related to this article; X-ray single crystal structure analysis data for 3aa. CCDC 1433224. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra01359g

---