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

Article abstract of DOI:10.1515/hc-2013-0072

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.

Full text of DOI:10.1515/hc-2013-0072

ꢁꢁꢁꢂ
DOI 10.1515/hc-2013-0072ꢀ ꢀHeterocycl. Commun. 2013; 19(3): 171–177
Danzhu Wang, Weixuan Chena, Yueqin Zheng, Chaofeng Dai, Lifang Wang
and Binghe Wang*
A general and efficient entry to asymmetric
tetrazines for click chemistry applications
Abstract: The importance of click chemistry is widely methods for fast and efficient DNA labeling using click
recognized. Among all the known click reactions, those chemistry [5–7]; in such applications, fast reaction rates
involving tetrazines represent the fastest click reactions and their tunability are important factors to consider. We
reported and are generating a great deal of interest. How- have recently investigated the reactivity and tunability of
ever, there is no efficient entry to asymmetric tetrazines the 3,6-substituted-1,2,4,5-tetrazine and cyclooctyne reac-
and those with strong electron withdrawing groups, which tion pair [21]. A recent publication by the Bertorzzi group
limits the development of this field. Herein, we report a also elegantly exploited this issue with similar reactions
general and efficient entry to asymmetric tetrazines with [17]. Understandably, the reactivity of tetrazine deriva-
strongly electron withdrawing groups.
tives can be tuned by manipulating their electron density
through the introduction of different functional groups
Keywords: asymmetric; click chemistry; electron-with- with electron-withdrawing groups (EWGs) accelerating
drawing; tetrazine.
the reaction [22–24]. One can envision broadened applica-
tions of click reactions involving tetrazines if such com-
pounds with various substituents can be readily prepared.
(During preparation of our manuscript for submission, a
similar concept, although with a limited number of exam-
ples, was published. For details please see [15].)
aPresent address: Department of Chemistry and Biochemistry
Georgia Institute of Technology, 901 Atlantic Drive Atlanta, GA
30332-0400, USA.
*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, e-mail: wang@gsu.edu
Danzhu Wang, Weixuan Chen, Yueqin Zheng, Chaofeng Dai and
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
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 prob-
lems such as low yields and the formation of a complex
mixture. In addition, other tetrazine synthesis only allows
Introduction
In the past decade, click chemistry [1–4] has become more the preparation of those with EDGs [28, 29]. None of the
and more valuable in a wide variety of areas including above methods could be used for the efficient synthesis of
DNA modification, protein tagging, cell image study, and tetrazines with strong EWGs such as a nitro group. To the
drug discovery [5–13]. In this regard, several bioorthogonal best of our knowledge, there is no suitable method for the
click reactions have been reported for various applica- efficient synthesis of asymmetric tetrazines with strong
tions [8–12, 14, 15]. So far, inverse electron-demand Diels- EWGs despite their obvious utilities [30, 31]. Herein, we
Alder reactions involving 3,6-substituted-1,2,4,5-tetrazines report a method for the efficient synthesis of asymmetric
represent some of the fastest click reactions and are thus tetrazines with strong EWGs via substituted 1,2-dichlo-
very useful in a wide variety of applications. For example, romethylene hydrazine intermediates (Scheme 1B). The
the reactions between 3,6-substututed-1,2,4,5-tetrazine method allows separate introduction of each component,
and norborene [16], cycloalkyne [17, 18], or trans-alkene and thus leads to good yields, reproducibility, and easy
[14, 19] have high reaction rates (second order rate con- purification. Below we present the applications of this
stants up to 2000 m-1 s-1) and clean outcome [19, 20]. Our method in synthesizing both symmetric and asymmetric
laboratory has a long-standing interest in developing tetrazines with strong EWGs and asymmetric tetrazines. In
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ꢀD. Wang et al.: Asymmetric tetrazines for click chemistry applications
172ꢀ
N
NH
N
N
N
N
N
R1
R1
R2
R1
R2
R2
R1
R1
R2
R1
R2
R2
HN
Δ
Low yields
Tedious purification
Substituent dependent
R1
N
N
N2H4,
N
NH
N
N
N
N
N
A
R2
one pot
HN
1
N
NH
N
N
N
N
N
HN
O
R2
Cl
O
O
O
O
N2H4 H2O (2 eq)
THF, 0oC
4a-e
R1
R1
R2
B
R1
2a-c
X
NHNH2
3a-c
HN NH
5a-e
DCM, 0°C
High yield
Easy purification
Substituent independent
1) N2H4 H2O (1.1 eq)
CICl
N
N
N
N
Δ
EtOH,
PCl5 (5 eq)
R1
R2
R1
R2
toluene, Δ, 3h
2) NaNO2, AcOH, 0°C
N
N
6a-e
1a-e
2a: R1 = 3,5-bis(CF3)-C6H3; X = Cl
4e: R2 = 4-CF3-C6H4
: R1 = 3,5-bis(CF3)-C6H3; R2 = tBu
1a 5a 6a
1
2b
: R = 2-pyridyl; X = OMe
,
,
2c: R1 = 2-pyrimidyl; X = OMe
1b, 5b, 6b: R1 = 3,5-bis(CF3)-C6H3; R2 = 2-NO2-C6H4
4a: R2 = tBu
1c, 5c, 6c: R1 = 2-pyridyl; R2 = 4-F-C6H4
2
: R = 4-NO2-C6H4
: R1 = 2-pyridyl; R2 = 4-NO2-C6H4
, ,
: R1 = 2-pyrimidyl; R2 = 4-CF3-C6H4
1e 5e 6e
4b
1d 5d 6d
, ,
2
4c
: R = 4-F-C6H4
4d: R2 = 2-NO2-C6H4
Scheme 1ꢀSynthetic 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.
addition, the method is generally applicable for the syn-
thesis of a variety of tetrazines with superior results.
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 com-
mercially available and inexpensive acyl chlorides. Thus,
half an equivalent of hydrazine monohydrate was reacted
with acyl chlorides in tetrahydrofuran (THF) to give N,N′ -
Synthesis of tetrazines with EWGs
As discussed earlier, we have a long-standing interest in diacylhydrazine (7, Scheme 2). Without further purifica-
searching for click reactions with tunable reaction rates. In tion, N,N′-diacylhydrazine was allowed to react with PCl5
theprocess,itwasfoundthatthesynthesisoftetrazineswith in toluene under reflux to give product 8. The tetrazine
EWGs is especially difficult. Examples include 3,6-bis(4-
nitrophenyl)-1,2,4,5-tetrazine and 3,6-di(pyrimidin-2-yl)-
O
O O
N2H4 H2O (0.5 eq)
PCl5 (5 eq)
R
R
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].
R
Cl
THF, 0°C
HN NH
7a-c, f-g
toluene, Δ, 3h
4a-c, f-g
1) N2H4 H2O (1.1 eq)
ClCl
N
N
N
N
EtOH, Δ
R
R
R
R
N
N
2) NaNO2, AcOH, 0°C
8a-c, f-g
9a-c, f-g
t
c: R = 4-F-C6H4
g: R = Ph
a
: R = Bu
b: R = 4-NO2-C6H4
f: R =
3-F-C6H4
Scheme 2ꢀSynthesis of symmetrically 3,6-substituted 1,2,4,5-tetra-
zine via 1,2-dichloromethylenehydrazines.
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D. Wang et al.: Asymmetric tetrazines for click chemistry applicationsꢀ
ꢀ173
derivatives were obtained by condensation of 8 with hydra- starting with a clean intermediate is the key. The acyl chlo-
zine followed by oxidation at 0°C. 3,6-Bis(4-nitrophenyl)- ride-hydrazine approach allows for stepwise reactions
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 excit-
ing results, we extended our synthetic route to other sub-
stituted phenyltetrazines with or without EWGs attached
on the phenyl ring. For the synthesis of 9g (without sub-
stitution, 70% yield), 9f (meta-fluoro substituted), and 9c
(para-fluoro substituted), the yields were all over 60%.
and thus the preparation of a clean intermediate with only
one substituent (Scheme 1B).
Acylhydrazides 3a–c were synthesized first by react-
ing 0.7 eq. of acyl chloride with hydrazine monohydrate in
THF. Then, the addition of the second acyl chloride to acyl-
hydrazide suspension in dichloromethane led to asym-
metric 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 approxi-
mately 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%.
Synthesis of tetrazines with aliphatic
substitutions
Earlier reports stated that aliphatic tetrazines were diffi-
cult to synthesize from nitriles due to reduced electrophi-
licity of the cyano group by the alkyl group and the gen-
eration 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 sub-
stitutions. 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 synthe-
sized 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-tetra-
zine (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.
Conclusions
Synthesis of 3,6-substituted-1,2,4,5-tetrazine derivatives
was achieved by using substituted 1,2-dichlorometh-
ylene-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-tetra-
zines with aliphatic substituents and asymmetric tetra-
zines. The approach described represents a general and
convenient entry to a variety of tetrazines. We believe this
methodology will greatly help the exploration of addi-
tional applications of tetrazine-click chemistry.
Experimental
General
Synthesis of asymmetric tetrazines
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 chroma-
tography (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.
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 men-
tioned above, asymmetric tetrazines are more difficult to
make using available approaches compared with symmet-
ric 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
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
purification would be difficult. To address this problem, precipitate of 7 was collected by filtration and washed with water
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ꢀD. Wang et al.: Asymmetric tetrazines for click chemistry applications
174ꢀ
4
3
(3 × 15 mL). The crude product 7 was used for the next step without 129.8 (d, J(CF) = 3.0 Hz), 130.9 (d, J(CF) = 9.0 Hz), 143.8, 165.1 (d,
further purification.
1J(CF) = 252 Hz); HRMS (ESI): m/z Calcd for C14H8Cl2F2N2 [M+H]+
313.0105, found 313.0111.
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) drop-
wise 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.
N′-(Chloro(3-fluorophenyl)methylene)-3-fluorobenzohydrazo-
noyl chloride (8f)ꢀCompound 8f was prepared according to the
general procedure and purified by column chromatography to give
1
a yellow solid: yield 49%; H 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.
2-Picolinohydrazide (3b)/pyrimidine-2-carbohydrazide (3c)ꢀTo
a solution of methyl 2-picolinate (or methyl pyrimidine-2-carboxy-
late, 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 re-
moved under reduced pressure and the resultant white solid was col-
lected and washed with water (3 × 10 mL). The crude white solid 3b
(or 3c) was used for the next step without further purification.
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.
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 drop-
wise. This mixture was stirred at room temperature for 1 h under ar-
gon 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.
1-((3,5-Bis(trifluoromethyl)phenyl)chloromethylene)-2-(1-chlo-
ro-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.
General procedure for synthesis of 1,2-dichloromethylene hy-
drazines (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. Afer 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.
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
1
(q, J(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.
N′-(1-Chloro-2,2 dimethylpropylidene)pivalohydrazonoyl chlo-
ride (8a)ꢀCompound 8a was prepared according to the general
procedure and purified by column chromatography to give a color-
less oil: yield 45%; 1H NMR: δ 1.34 (s, 18H); HRMS(ESI): m/z Calcd for
C10H18Cl2N2 [M+H]+ 237.0920, found 237.0926.
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),
N′-(Chloro(4-nitrophenyl)methylene)-4-nitrobenzohydrazonoyl 8.80 (d, 1H, J = 4.8 Hz); 13C NMR: δ 115.8 (d, J(CF) = 22 Hz), 123.4,
2
4
3
chloride (8b)ꢀCompound 8b was prepared according to the general 125.8, 129.7 (d, J(CF) = 2Hz), 130.9 (d, J(CF) = 9.0 Hz), 136.8, 149.6,
procedure and purified by column chromatography to give a yellow 150.5, 164.1 (d, 1J(CF) = 252 Hz); HRMS (ESI): m/z Calcd for C13H8Cl2FN3
1
solid: yield 52%; H NMR: δ 8.13 (d, 4H, J = 8.0 Hz), 8.30 (d, 4H, J = [M+H]+ 296.0152, found 296.0159.
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
2-(Chloro(4-nitrophenyl)methylene)-1-(chloro(pyridin-2-yl)
366.9991.
methylene)hydrazine (6d)ꢀCompound 6d was prepared according
to the general procedure and purified by column chromatography to
N′-(Chloro(4-fluorophenyl)methylene)-4-fluorobenzohydrazo-
giveayellowsolid:yield47%;1HNMR:δ7.46–7.49(m,1H),7.84–7.88(m,
noyl chloride (8c)ꢀCompound 8c was prepared according to the 1H), 8.25 (d, 1H, J = 8.0 Hz), 8.29–8.34 (m, 4H), 8.79 (d, 1H, J = 4.4 Hz);
general procedure and purified by column chromatography to give a 13C NMR: δ 123.5, 123.7, 126.1, 129.5, 136.9, 138.9, 141.4, 144.6, 149.7,
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),
149.8, 150.2; HRMS (ESI): m/z Calcd for C13H8Cl2N4O2 [M+H]+ 323.0097,
found 323.0103.
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2-(Chloro(4-(trifluoromethyl)phenyl)methylene)-1- 3,6-Diphenyl-1,2,4,5-tetrazine (9g)ꢀCompound 9g was prepared
(chloro(pyrimidin-2-yl)methylene)hydrazine (6e)ꢀCompound according to the general procedure and purified by column chroma-
1
6e was prepared according to the general procedure and purified by tography to give a purple solid: yield 70%; H NMR: δ 7.59–7.65 (m,
column chromatography to give a yellow solid: yield 42%: 1H NMR: δ 6H), 8.65–8.67 (m, 4H); 13C NMR: δ 128.0, 129.3, 131.8, 132.7, 164.0;
7.47 (t, 1H, J = 4.4 Hz), 7.75 (d, 2H, J = 8.4 Hz), 8.24 (d, 2H, J = 8.0 Hz), HRMS (ESI): m/z Calcd for C14H10N4 [M+H]+ 235.0978, found 235.0984.
1
8.98 (d, 2H, J = 4.4 Hz); 13C NMR: δ 122.0, 124.1 (q, J(CF) = 272 Hz),
4
3
125.6 (d, J(CF) = 3.0 Hz), 125.7(d, J(CF) = 4.0 Hz), 128.8, 133.5 (d,
3-(3,5-Bis(trifluoromethyl)phenyl)-6-tert-butyl-1,2,4,5-tetrazine
(1a)ꢀCompound 1a was prepared according to the general proce-
dure and purified by column chromatography to give a purple solid:
2J(CF) = 33 Hz), 136.4, 141.1 (d, J(CF) = 2.0 Hz), 157.9, 158.7; HRMS
5
(ESI): m/z Calcd for C13H7Cl2F3N4 [M+H]+ 347.0073, found 347.0078.
1
yield 75%; H NMR: δ 1.65 (s, 9H), 8.13 (s, 1H), 9.11 (s, 2H); 13C NMR:
1
3
δ 28.2, 42.0, 122.9 (q, J(CF) = 271 Hz), 124.8, 128.1 (d, J(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.
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 or-
ange 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 chromatog-
raphy eluting with dichloromethane/hexanes (100:1) to give a purple
solid of 9a–c, f, g/1a–e.
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–
1
8.18 (m, 3H), 9.19 (s, 2H); 13C NMR: δ 122.9 (q, J(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
2
(q, J(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)ꢀCom-
pound 1c was prepared according to the general procedure and puri-
fied 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,
2
3H), 8.98 (m, 1H); 13C NMR: δ 116.7 (d, J(CF) = 22.0 Hz), 123.9, 126.4,
3,6-Di-tert-butyl-1,2,4,5-tetrazine (9a)ꢀCompound 9a was pre-
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.
pared according to the general procedure and purified by column
1
chromatography to give a purple solid: yield 42%; H NMR: δ 1.59
(s, 9H); HRMS (ESI): m/z Calcd for C10H18N4 [M+H]+ 195.1610, found
195.1618.
3-(4-Nitrophenyl)-6-(pyridin-2-yl)-1,2,4,5-tetrazine (1d)ꢀCom-
pound 1d was prepared according to the general procedure and puri-
fied 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.
3,6-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-(4-(Trifluoromethyl)phenyl)-6-(pyrimidin-2-yl)-1,2,4,5-tetra-
zine (1e)ꢀCompound 1e was prepared according to the general pro-
cedure and purified by column chromatography to give a purple solid:
3,6-Bis(4-fluorophenyl)-1,2,4,5-tetrazine (9c)ꢀCompound 9c was
prepared according to the general procedure and purified by column
1
chromatography to give a purple solid: yield 75%; H NMR: δ 7.31
1
yield 75%; H NMR: δ 7.62 (t, 1H, J = 4.8 Hz), 7.91 (d, 2H, J = 8.0 Hz),
(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.
8.88 (d, 2H, J = 8.0 Hz), 9.16 (d, 2H, J = 4.8 Hz); 13C NMR: δ 122.7, 123.6
1
3
(q, J(CF) = 271 Hz), 126.3 (q, J(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.
3,6-Bis(3-fluorophenyl)-1,2,4,5-tetrazine (9f)ꢀCompound 9f was
Acknowledgments: Financial support from the National
Institutes of Health (GM086925, GM084933, and CA159567)
and the Georgia State University Molecular Basis of Dis-
ease (MBD) Program through a fellowship to W.X.C. and
D.Z.W. is gratefully acknowledged.
prepared according to the general procedure and purified by column
1
chromatography to give a purple solid: yield 61%; H 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,
3
5
2J(CF) = 24 Hz), 119.9 (d, J(CF) = 21 Hz), 123.8 (d, J(CF) = 3.0 Hz),
4
4
1
131.1 (d, J(CF) = 9.0 Hz), 133.8 (d, J(CF) = 9.0 Hz), 163.3 (d, J(CF) =
246 Hz), 163.4; HRMS (ESI): m/z Calcd for C14H8F2N4 [M+H]+ 271.0795,
found 271.0807.
Received May 6, 2013; accepted May 7, 2013
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