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Heterocyclic Communications


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Volume 19, Issue 3

Issues

A general and efficient entry to asymmetric tetrazines for click chemistry applications

Danzhu Wang
  • Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA
  • Other articles by this author:
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/ Weixuan Chen
  • Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA
  • Present address: Department of Chemistry and Biochemistry Georgia Institute of Technology, 901 Atlantic Drive Atlanta, GA 30332-0400, USA.
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/ Yueqin Zheng
  • Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA
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/ Chaofeng Dai
  • Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA
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/ Lifang Wang
  • Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA
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/ Binghe Wang
  • Corresponding author
  • Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA
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Published Online: 2013-05-30 | DOI: https://doi.org/10.1515/hc-2013-0072

Abstract

The importance of click chemistry is widely recognized. Among all the known click reactions, those involving tetrazines represent the fastest click reactions reported and are generating a great deal of interest. However, there is no efficient entry to asymmetric tetrazines and those with strong electron withdrawing groups, which limits the development of this field. Herein, we report a general and efficient entry to asymmetric tetrazines with strongly electron withdrawing groups.

This article offers supplementary material which is provided at the end of the article.

Keywords: asymmetric; click chemistry; electron-withdrawing; tetrazine

Introduction

In the past decade, click chemistry [1–4] has become more and more valuable in a wide variety of areas including DNA modification, protein tagging, cell image study, and drug discovery [5–13]. In this regard, several bioorthogonal click reactions have been reported for various applications [8–12, 14, 15]. So far, inverse electron-demand Diels-Alder reactions involving 3,6-substituted-1,2,4,5-tetrazines represent some of the fastest click reactions and are thus very useful in a wide variety of applications. For example, the reactions between 3,6-substututed-1,2,4,5-tetrazine and norborene [16], cycloalkyne [17, 18], or trans-alkene [14, 19] have high reaction rates (second order rate constants up to 2000 m-1 s-1) and clean outcome [19, 20]. Our laboratory has a long-standing interest in developing methods for fast and efficient DNA labeling using click chemistry [5–7]; in such applications, fast reaction rates and their tunability are important factors to consider. We have recently investigated the reactivity and tunability of the 3,6-substituted-1,2,4,5-tetrazine and cyclooctyne reaction pair [21]. A recent publication by the Bertorzzi group also elegantly exploited this issue with similar reactions [17]. Understandably, the reactivity of tetrazine derivatives can be tuned by manipulating their electron density through the introduction of different functional groups with electron-withdrawing groups (EWGs) accelerating the reaction [22–24]. One can envision broadened applications of click reactions involving tetrazines if such compounds with various substituents can be readily prepared. (During preparation of our manuscript for submission, a similar concept, although with a limited number of examples, was published. For details please see [15].)

Results and discussion

Synthesis of symmetric 3,6-substituted-1,2,4,5-tetrazines with electron-donating groups (EDGs) has been reported in the literature [23, 25–27]. However, there is no easy entry to asymmetric tetrazines. This is because the most common one-pot approach (Scheme 1A) has intrinsic problems such as low yields and the formation of a complex mixture. In addition, other tetrazine synthesis only allows the preparation of those with EDGs [28, 29]. None of the above methods could be used for the efficient synthesis of tetrazines with strong EWGs such as a nitro group. To the best of our knowledge, there is no suitable method for the efficient synthesis of asymmetric tetrazines with strong EWGs despite their obvious utilities [30, 31]. Herein, we report a method for the efficient synthesis of asymmetric tetrazines with strong EWGs via substituted 1,2-dichloromethylene hydrazine intermediates (Scheme 1B). The method allows separate introduction of each component, and thus leads to good yields, reproducibility, and easy purification. Below we present the applications of this method in synthesizing both symmetric and asymmetric tetrazines with strong EWGs and asymmetric tetrazines. In addition, the method is generally applicable for the synthesis of a variety of tetrazines with superior results.

Synthetic methods for 3,6-substituted-1,2,4,5-tetrazine. (A) A one-pot method. (B) A stepwise method via a 1,2-dichloromethylene hydrazine intermediate.
Scheme 1

Synthetic methods for 3,6-substituted-1,2,4,5-tetrazine.

(A) A one-pot method. (B) A stepwise method via a 1,2-dichloromethylene hydrazine intermediate.

Synthesis of tetrazines with EWGs

As discussed earlier, we have a long-standing interest in searching for click reactions with tunable reaction rates. In the process, it was found that the synthesis of tetrazines with EWGs is especially difficult. Examples include 3,6-bis(4-nitrophenyl)-1,2,4,5-tetrazine and 3,6-di(pyrimidin-2-yl)-1,2,4,5-tetrazine. Meanwhile, aliphatic tetrazines are also difficult to make because aliphatic groups are electron-donating and thus reduce the electrophilicity of the cyano group [28]. Therefore, we set out to find a synthetic route that is not substituent-dependent and is suitable for the synthesis of various tetrazines. 1,2-Dichloromethylene hydrazines (8, Scheme 2) are known and have been used as intermediates in the synthesis of triazoles [32]. We found that the reaction of 8 with an appropriate equivalent hydrazine under neutral condition leads to the formation of dihydrotetrazine with reasonable yields independent of the substituents [33, 34].

Synthesis of symmetrically 3,6-substituted 1,2,4,5-tetrazine via 1,2-dichloromethylenehydrazines.
Scheme 2

Synthesis of symmetrically 3,6-substituted 1,2,4,5-tetrazine via 1,2-dichloromethylenehydrazines.

As the first step in testing the general applicability of this method, we studied this reaction for the synthesis of symmetrically substituted 1,2,4,5-tetrazines from commercially available and inexpensive acyl chlorides. Thus, half an equivalent of hydrazine monohydrate was reacted with acyl chlorides in tetrahydrofuran (THF) to give N,N′-diacylhydrazine (7, Scheme 2). Without further purification, N,N′-diacylhydrazine was allowed to react with PCl5 in toluene under reflux to give product 8. The tetrazine derivatives were obtained by condensation of 8 with hydrazine followed by oxidation at 0°C. 3,6-Bis(4-nitrophenyl)-1,2,4,5-tetrazine (9b) was obtained in 58% yield. This represents the first successful example of synthesizing a symmetric tetrazine with strong EWGs. With such exciting results, we extended our synthetic route to other substituted phenyltetrazines with or without EWGs attached on the phenyl ring. For the synthesis of 9g (without substitution, 70% yield), 9f (meta-fluoro substituted), and 9c (para-fluoro substituted), the yields were all over 60%.

Synthesis of tetrazines with aliphatic substitutions

Earlier reports stated that aliphatic tetrazines were difficult to synthesize from nitriles due to reduced electrophilicity of the cyano group by the alkyl group and the generation of isomerized byproduct 4-amino-1,2,4-triazoles [35–37]. Thus, we were curious about the applicability of the new method in making tetrazines with aliphatic substitutions. Therefore, the synthesis of 3,6-di-tert-butyl-1,2,4,5-tetrazine (9a) was carried out. In this case, the high steric hindrance of the t-butyl group is expected to present additional difficulties. Using the acyl chloride-hydrazine approach (Scheme 2), 9a was prepared in a modest 42% yield. Although the yield was modest, this represents for the first time that an alkyl substituted tetrazine is synthesized with ease. The relatively low yields might not be due to issues with the reaction. Rather, it is most likely due to the high sublimation rate of 3,6-di-tert-butyl-1,2,4,5-tetrazine (9a), which we observed. For example, when 9a was stored at room temperature, the purple-colored product would sublimate to the top of the storage vial.

Synthesis of asymmetric tetrazines

After successful feasibility studies, we next examined the applicability of using this new approach for the synthesis of asymmetric tetrazines. These asymmetric tetrazines are very valuable because of the possibility of putting different handles and ease for further functionalization. As mentioned above, asymmetric tetrazines are more difficult to make using available approaches compared with symmetric ones. This is because the traditional approach starts the reaction with three components (Scheme 1A) and thus would produce three products including two symmetric byproducts and one desired asymmetric product. This not only means that intrinsically yields would be low but also purification would be difficult. To address this problem, starting with a clean intermediate is the key. The acyl chloride-hydrazine approach allows for stepwise reactions and thus the preparation of a clean intermediate with only one substituent (Scheme 1B).

Acylhydrazides 3a–c were synthesized first by reacting 0.7 eq. of acyl chloride with hydrazine monohydrate in THF. Then, the addition of the second acyl chloride to acylhydrazide suspension in dichloromethane led to asymmetric substituted N,N′-diacylhydrazines 5a–e, which were easily converted to the final products by treating with PCl5 and then heating with hydrazine in ethanol under reflux followed by oxidation with NaNO2. Tetrazines with various EWGs were selected as targets. Compound 1a with aliphatic groups was obtained in modest yield of approximately 75%. The one with the strongest EWG (1e) among our examples was obtained in 75% yield. The other four combinations, 1b–d, were prepared in yields of over 50%.

Conclusions

Synthesis of 3,6-substituted-1,2,4,5-tetrazine derivatives was achieved by using substituted 1,2-dichloromethylene-hydrazines as intermediates. This method is not only suitable for the preparation of electron-deficient 1,2,4,5-tetrazines but also for the synthesis of 1,2,4,5-tetrazines with aliphatic substituents and asymmetric tetrazines. The approach described represents a general and convenient entry to a variety of tetrazines. We believe this methodology will greatly help the exploration of additional applications of tetrazine-click chemistry.

Experimental

General

All reagents and solvents were of reagent grade or were purified by standard methods before use. Column chromatography was carried out on flash silica gel (Sorbent 230–400 mesh). Thin layer chromatography (TLC) analysis was conducted on silica gel plates (Sorbent Silica G UV254). NMR spectra were recorded in CDCl3 at 1H (400 MHz) and 13C (100 MHz) on a Bruker instrument.

General procedure for synthesis of symmetrically substituted N,N′-diacylhydrazines (7a–c, f, g)

To a solution of acyl chloride 4 (2 mmol) in dried dichloromethane (20 mL) was added dropwise hydrazine monohydrate (1 mmol) at 0°C under argon atmosphere. The mixture was stirred for 1 h. The white precipitate of 7 was collected by filtration and washed with water (3 × 15 mL). The crude product 7 was used for the next step without further purification.

3,5-Bis(trifluoromethyl)benzohydrazide (3a)

To a solution of hydrazine monohydrate (2 mmol) in dry THF (10 mL) was added bis(trifluoromethyl)benzoyl chloride (1.4 mmol) in THF (2 mL) dropwise at 0°C under argon atmosphere. The mixture was stirred for 1 h. The white precipitate of 3a was collected by filtration and washed with water (3 × 15 mL). The crude solid was used for the next step without further purification.

2-Picolinohydrazide (3b)/pyrimidine-2-carbohydrazide (3c)

To a solution of methyl 2-picolinate (or methyl pyrimidine-2-carboxylate, 2 mmol) in MeOH (10 mL) was added hydrazine monohydrate (4 mmol) under argon atmosphere. The solution was heated under reflux for 3 h and then cooled to room temperature. Solvent was removed under reduced pressure and the resultant white solid was collected and washed with water (3 × 10 mL). The crude white solid 3b (or 3c) was used for the next step without further purification.

General procedure for synthesis of asymmetric substituted N,N′-diacylhydrazine (5a–e)

To a suspension of hydrazide 3a–c (1 mmol) in dichloromethane (10 mL) under argon atmosphere, acyl chloride 4a–e (1.1 mmol) in dichloromethane (2 mL) was added dropwise. This mixture was stirred at room temperature for 1 h under argon atmosphere and concentrated under reduced pressure. The white solid was collected and washed with dichloromethane (3 × 5 mL). The crude white solid 5a–e was used for the next step without further purification.

General procedure for synthesis of 1,2-dichloromethylene hydrazines (8a–c, 8f–g/6a–e)

To a suspension of 7a–c, f, g/5a–e (1.5 mmol) in 10 mL toluene, phosphorus pentachloride (7.5 mmol) was added. This mixture was stirred under reflux conditions for 3 h under argon atmosphere. After concentration under reduced pressure, the reaction mixture was directly loaded onto a silica gel column. Elution with hexanes/dichloromethane (100:1) gave a light yellow oil or solid product 8a–c, f, g/6a–e.

N′-(1-Chloro-2,2 dimethylpropylidene)pivalohydrazonoyl chloride (8a)

Compound 8a was prepared according to the general procedure and purified by column chromatography to give a colorless oil: yield 45%; 1H NMR: δ 1.34 (s, 18H); HRMS(ESI): m/z Calcd for C10H18Cl2N2 [M+H]+ 237.0920, found 237.0926.

N′-(Chloro(4-nitrophenyl)methylene)-4-nitrobenzohydrazonoyl chloride (8b)

Compound 8b was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield 52%; 1H NMR: δ 8.13 (d, 4H, J = 8.0 Hz), 8.30 (d, 4H, J = 8.0 Hz); 13C NMR: δ 122.1, 123.8, 129.5, 138.6, 140.3, 141.3, 149.7, 157.9, 158.5; HRMS (ESI): m/z Calcd for C14H8Cl2N4O4 [M+H]+ 366.9995, found 366.9991.

N′-(Chloro(4-fluorophenyl)methylene)-4-fluorobenzohydrazonoyl chloride (8c)

Compound 8c was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield 55%; 1H NMR: δ 7.16 (dd, 4H, J = 8.4 Hz, J = 8.4 Hz), 8.15 (dd, 4H, J = 7.4 Hz, J = 7.4 Hz); 13C NMR: δ 115.7 (d, 2J(CF) = 22 Hz), 129.8 (d, 4J(CF) = 3.0 Hz), 130.9 (d, 3J(CF) = 9.0 Hz), 143.8, 165.1 (d, 1J(CF) = 252 Hz); HRMS (ESI): m/z Calcd for C14H8Cl2F2N2 [M+H]+ 313.0105, found 313.0111.

N′-(Chloro(3-fluorophenyl)methylene)-3-fluorobenzohydrazonoyl chloride (8f)

Compound 8f was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield 49%; 1H NMR: δ 7.24–7.28 (m, 2H), 7.47 (dd, 2H, J = 7.2 Hz, J = 14.4 Hz), 7.86 (d, 2H, J = 6.0 Hz), 7.95 (d, 2H, J = 8.0 Hz); 13C NMR: δ 115.5 (d, 2J(CF) = 24 Hz), 118.9 (d, 3J(CF) = 21 Hz), 124.3 (d, 5J(CF) = 3.0 Hz), 130.1 (d, 4J(CF) = 8.0 Hz), 135.6 (d, 4J(CF) = 8 Hz), 143.3 (d, 5J(CF) = 3 Hz), 162.6 (d, 1J(CF) = 245 Hz); HRMS (ESI): m/z Calcd for C14H8Cl2F2N2 [M+H]+ 313.0105, found 313.0114.

N′-(Chloro(phenyl)methylene)benzohydrazonoyl chloride (8g)

Compound 8g was prepared according to the general procedure and purified by column chromatography to give a colorless oil: yield 44%; 1H NMR: δ 7.47–7.54 (m, 6H), 8.15 (d, 4H, J = 7.6 Hz); 13C NMR: δ 128.5, 128.6, 131.8, 133.7, 144.2; HRMS (ESI): m/z Calcd for C14H10Cl2N2 [M+H]+ 277.0294, found 277.0297.

1-((3,5-Bis(trifluoromethyl)phenyl)chloromethylene)-2-(1-chloro-2,2-dimethylpropylidene)hydrazine (6a)

Compound 6a was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield, 45%; 1H NMR: δ 1.40 (s, 9H), 8.00 (s, 1H), 8.48 (s, 2H); 13C NMR: δ 26.3, 38.0, 123.1 (q, 1J(CF) = 271 Hz), 125.0, 127.9 (d, 3J(CF) = 3.0 Hz), 131.8 (d, 2J(CF) = 33 Hz), 135.1, 164.6, 179.3; HRMS (ESI): m/z Calcd for C14H12Cl2F6N2 [M+H]+ 393.0354, found 393.0361.

1-((3,5-Bis(trifluoromethyl)phenyl)chloromethylene)-2-(chloro(2-nitrophenyl)methylene)hydrazine (6b)

Compound 6b was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield, 31%; 1H NMR: δ 7.69–7.84 (m 3H), 8.05–8.09 (m, 2H), 8.57 (s, 2H); 13C NMR: δ 122.9 (q, 1J(CF) = 271 Hz), 124.8, 125.4, 128.5, 129.8, 131.1, 131.8, 132.4 (q, 2J(CF) = 33 Hz), 133.3, 135.4, 141.1, 142.2, 147.7; HRMS (ESI): m/z Calcd for C16H7Cl2F6N3O2 [M+H]+ 457.9892, found 457.9899.

2-(Chloro(4-fluorophenyl)methylene)-1-(chloro(pyridin-2-yl)methylene)hydrazine (6c)

Compound 6c was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield 51%; 1H NMR: δ 7.17 (dd, 2H, J = 8.0 Hz, J = 8.0 Hz), 7.45 (dd, 1H, J = 6.2 Hz, J = 6.2 Hz), 7.84 (dd, 1H, J = 7.8 Hz, J = 7.8 Hz), 8.15 (dd, 2H, J = 7.8 Hz, J = 7.8 Hz), 8.25 (d, 1H, J = 7.6 Hz), 8.80 (d, 1H, J = 4.8 Hz); 13C NMR: δ 115.8 (d, 2J(CF) = 22 Hz), 123.4, 125.8, 129.7 (d, 4J(CF) = 2Hz), 130.9 (d, 3J(CF) = 9.0 Hz), 136.8, 149.6, 150.5, 164.1 (d, 1J(CF) = 252 Hz); HRMS (ESI): m/z Calcd for C13H8Cl2FN3 [M+H]+ 296.0152, found 296.0159.

2-(Chloro(4-nitrophenyl)methylene)-1-(chloro(pyridin-2-yl)methylene)hydrazine (6d)

Compound 6d was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield 47%; 1H NMR: δ 7.46–7.49 (m, 1H), 7.84–7.88 (m, 1H), 8.25 (d, 1H, J = 8.0 Hz), 8.29–8.34 (m, 4H), 8.79 (d, 1H, J = 4.4 Hz); 13C NMR: δ 123.5, 123.7, 126.1, 129.5, 136.9, 138.9, 141.4, 144.6, 149.7, 149.8, 150.2; HRMS (ESI): m/z Calcd for C13H8Cl2N4O2 [M+H]+ 323.0097, found 323.0103.

2-(Chloro(4-(trifluoromethyl)phenyl)methylene)-1-(chloro(pyrimidin-2-yl)methylene)hydrazine (6e)

Compound 6e was prepared according to the general procedure and purified by column chromatography to give a yellow solid: yield 42%: 1H NMR: δ 7.47 (t, 1H, J = 4.4 Hz), 7.75 (d, 2H, J = 8.4 Hz), 8.24 (d, 2H, J = 8.0 Hz), 8.98 (d, 2H, J = 4.4 Hz); 13C NMR: δ 122.0, 124.1 (q, 1J(CF) = 272 Hz), 125.6 (d, 4J(CF) = 3.0 Hz), 125.7(d, 3J(CF) = 4.0 Hz), 128.8, 133.5 (d, 2J(CF) = 33 Hz), 136.4, 141.1 (d, 5J(CF) = 2.0 Hz), 157.9, 158.7; HRMS (ESI): m/z Calcd for C13H7Cl2F3N4 [M+H]+ 347.0073, found 347.0078.

General procedure for synthesis of 3, 6-disubstituted-1,2,4,5-tetrazines (9a–c, f, g/1a–e)

A 10-mL process vessel was charged with 8a–c, f, g/6a–e (0.15 mmol) and ethanol 1 mL. Hydrazine monohydrate was added (1.1 eq.) at 0°C and the vessel was sealed with a septum and exposed under reflux condition for overnight. The vessel was cooled to room temperature and the solvent was removed under reduced pressure to yield an orange solid. The solid was treated with acetic acid (1.5 mL) followed by an aqueous solution of NaNO2 (5 eq.) at 0°C. The purple-colored tetrazine was collected and purified by silica gel column chromatography eluting with dichloromethane/hexanes (100:1) to give a purple solid of 9a–c, f, g/1a–e.

3,6-Di-tert-butyl-1,2,4,5-tetrazine (9a)

Compound 9a was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 42%; 1H NMR: δ 1.59 (s, 9H); HRMS (ESI): m/z Calcd for C10H18N4 [M+H]+ 195.1610, found 195.1618.

3,6-Bis(4-nitrophenyl)-1,2,4,5-tetrazine (9b)

Compound 9b was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 58%; 1H NMR; δ 8.51 (d, 4H, 4.8 Hz), 8.91 (d, 4H, 4.8 Hz); ESI: m/z Calcd for C14H8N6O4 [M+H]+ 325.06, found 325.06.

3,6-Bis(4-fluorophenyl)-1,2,4,5-tetrazine (9c)

Compound 9c was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 75%; 1H NMR: δ 7.31 (dd, 4H, J = 8.4 Hz, J = 8.4 Hz), 8.66–8.68(m, 4H); 13C NMR: δ 116.6 (d, 2J(CF) = 22 Hz), 127.9 (d, 4J(CF) = 3.0 Hz), 130.3 (d, 3J(CF) = 9.0 Hz), 163.1, 166.4 (d, 1J(CF) = 252 Hz); HRMS (ESI): m/z Calcd for C14H8F2N4 [M+H]+ 271.0790, found 271.0798.

3,6-Bis(3-fluorophenyl)-1,2,4,5-tetrazine (9f)

Compound 9f was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 61%; 1H NMR: δ 7.35 (dd, 2H, J = 7.8 Hz, J = 7.8 Hz), 7.60 (dd, 2H, J =7.6 Hz, J = 14.4 Hz), 8.50 (d, 2H, J = 10.0 Hz) 8.60 (d, 2H, J = 7.6 Hz); 13C NMR: δ 114.9 (d, 2J(CF) = 24 Hz), 119.9 (d, 3J(CF) = 21 Hz), 123.8 (d, 5J(CF) = 3.0 Hz), 131.1 (d, 4J(CF) = 9.0 Hz), 133.8 (d, 4J(CF) = 9.0 Hz), 163.3 (d, 1J(CF) = 246 Hz), 163.4; HRMS (ESI): m/z Calcd for C14H8F2N4 [M+H]+ 271.0795, found 271.0807.

3,6-Diphenyl-1,2,4,5-tetrazine (9g)

Compound 9g was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 70%; 1H NMR: δ 7.59–7.65 (m, 6H), 8.65–8.67 (m, 4H); 13C NMR: δ 128.0, 129.3, 131.8, 132.7, 164.0; HRMS (ESI): m/z Calcd for C14H10N4 [M+H]+ 235.0978, found 235.0984.

3-(3,5-Bis(trifluoromethyl)phenyl)-6-tert-butyl-1,2,4,5-tetrazine (1a)

Compound 1a was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 75%; 1H NMR: δ 1.65 (s, 9H), 8.13 (s, 1H), 9.11 (s, 2H); 13C NMR: δ 28.2, 42.0, 122.9 (q, 1J(CF) = 271 Hz), 124.8, 128.1 (d, 3J(CF) = 3 Hz), 132.3 (d, 2J(CF) = 33 Hz), 135.7, 138.2, 154.2; HRMS (ESI): m/z Calcd for C14H12F6N4 [M+H]+ 351.1039, found 350.1045.

3-(3,5-Bis(trifluoromethyl)phenyl)-6-(2-nitrophenyl)-1,2,4,5-tetrazine (1b)

Compound 1b was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 67%; 1H NMR: δ 7.83–8.87 (m, 1H), 7.90–7.94 (m, 1H), 8.17–8.18 (m, 3H), 9.19 (s, 2H); 13C NMR: δ 122.9 (q, 1J(CF) = 271 Hz), 125.3, 123.4 (q, 3J(CF) = 4.0 Hz), 127.6, 128.3 (d, 4J(CF) = 3.0 Hz), 132.0, 132.8 (q, 2J(CF) = 34 Hz), 133.3, 133.5, 133.6, 149.1, 161.9, 165.8; HRMS (ESI): m/z Calcd for C16H7F6N5O2 [M+H]+ 416.0557, found 416.0560.

3-(4-Fluorophenyl)-6-(pyridin-2-yl)-1,2,4,5-tetrazine (1c)

Compound 1c was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 71%; 1H NMR: δ 7.29–7.33 (m, 2H), 7.57 (m, 1H), 8.00 (m, 1H), 8.68–8.74 (m, 3H), 8.98 (m, 1H); 13C NMR: δ 116.7 (d, 2J(CF) = 22.0 Hz), 123.9, 126.4, 127.8, 130.8 (d, 3J(CF) = 9.0 Hz), 137.5, 150.2, 150.8, 163.4, 163.6, 166.1 (d, 1J(CF) = 254 Hz); HRMS (ESI): m/z Calcd for C13H8FN5 [M+H]+ 254.0836, found 254.0832.

3-(4-Nitrophenyl)-6-(pyridin-2-yl)-1,2,4,5-tetrazine (1d)

Compound 1d was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 52%; 1H NMR: δ 7.61 (s, 1H), 8.04–8.06 (m, 1H), 8.49 (d, 2H, J = 8.0 Hz), 8.50 (d, 1H, J = 7.6 Hz), 8.92 (d, 2H, J = 8.0 Hz), 9.00 (s, 1H); HRMS (ESI): m/z Calcd for C13H8N6O2 [M+H]+ 281.0787, found 281.0797.

3-(4-(Trifluoromethyl)phenyl)-6-(pyrimidin-2-yl)-1,2,4,5-tetrazine (1e)

Compound 1e was prepared according to the general procedure and purified by column chromatography to give a purple solid: yield 75%; 1H NMR: δ 7.62 (t, 1H, J = 4.8 Hz), 7.91 (d, 2H, J = 8.0 Hz), 8.88 (d, 2H, J = 8.0 Hz), 9.16 (d, 2H, J = 4.8 Hz); 13C NMR: δ 122.7, 123.6 (q, 1J(CF) = 271 Hz), 126.3 (q, 3J(CF) = 4.0 Hz), 129.1, 134.5, 134.8 (q, 2J(CF) = 33 Hz), 158.5, 159.3, 163.3, 163.7; HRMS (ESI): m/z Calcd for C13H7F3N6 [M+H]+ 305.0763, found 305.0775.

Financial support from the National Institutes of Health (GM086925, GM084933, and CA159567) and the Georgia State University Molecular Basis of Disease (MBD) Program through a fellowship to W.X.C. and D.Z.W. is gratefully acknowledged.

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About the article

Corresponding author: Binghe Wang, Department of Chemistry, Center for Diagnostics and Therapeutics, and Center for Biotechnology and Drug Design, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA


Received: 2013-05-06

Accepted: 2013-05-07

Published Online: 2013-05-30

Published in Print: 2013-06-01


Citation Information: Heterocyclic Communications, Volume 19, Issue 3, Pages 171–177, ISSN (Online) 2191-0197, ISSN (Print) 0793-0283, DOI: https://doi.org/10.1515/hc-2013-0072.

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