Synthesis, characterization, and cycloaddition reactivity of a monocyclic aromatic 1,2,3,5-tetrazine

Article abstract of DOI:10.1021/jacs.9b07744

Herein we disclose the synthesis and full characterization of the first monocyclic aromatic 1,2,3,5-tetrazine, 4,6-diphenyl-1,2,3,5-tetrazine. Initial studies of its cycloaddition reactivity, mode, regioselectivity, and scope illustrate that it participates as the 4π-component of well-behaved inverse electron demand Diels-Alder reactions where it preferentially reacts with electron-rich or strained dienophiles. It was found to exhibit an intrinsic reactivity comparable to that of the isomeric 3,6-diphenyl-1,2,4,5-tetrazine, display a single mode of cycloaddition with reaction only across C4/N1 (no N2/N5 cycloaddition observed), proceed with a predictable regioselectivity (dienophile most electron-rich atom attaches to C4), and manifest additional reactivity complementary to the isomeric 1,2,4,5-tetrazines. It not only exhibits a remarkable cycloaddition reactivity, surprisingly good stability (e.g., stable to chromatography, long-term storage, presence of H₂O even as reaction co-solvent), and broad cycloaddition scope, but it also displays powerful orthogonal reactivity with the 1,2,4,5-tetrazines. Whereas the latter reacts at extraordinary cycloaddition rates with strained dienophiles (tetrazine ligation), the new and isomeric 1,2,3,5-tetrazine displays similarly remarkable cycloaddition rates and efficiencies with amidines (1,2,3,5-tetrazine/amidine ligation). The crossover reactivities (1,2,4,5-tetrazines with amidines and 1,2,3,5-tetrazines with strained dienophiles) are sufficiently low to indicate they may be capable of use concurrently without competitive reactions.

Full text of DOI:10.1021/jacs.9b07744

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Article
Synthesis, characterization, and cycloaddition
reactivity of a monocyclic aromatic 1,2,3,5-tetrazine
Zhi-Chen Wu, and Dale L. Boger
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b07744 • Publication Date (Web): 16 Sep 2019
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Synthesis, characterization, and cycloaddition reactivity of a monocyclic
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Zhi-Chen Wu and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
ABSTRACT. Herein we disclose the synthesis and full characterization of the first monocyclic aromatic
1,2,3,5-tetrazine, 4,6-diphenyl-1,2,3,5-tetrazine. Initial studies of its cycloaddition reactivity, mode,
regioselectivity, and scope illustrate that it participates as the 4-component of well-behaved inverse
electron demand Diels–Alder reactions where it preferentially reacts with electron-rich or strained
dienophiles. It was found to exhibit an intrinsic reactivity comparable to that of the isomeric 3,6-diphenyl-
1,2,4,5-tetrazine, display a single mode of cycloaddition with reaction only across C4/N1 (no N2/N5
cycloaddition observed), proceed with a predictable regioselectivity (dienophile most electron-rich atom
attaches to C4), and manifest additional reactivity complementary to the isomeric 1,2,4,5-tetrazines. It not
only exhibits a remarkable cycloaddition reactivity, surprisingly good stability (e.g., stable to
chromatography, long term storage, presence of H2O even as reaction co-solvent), and broad cycloaddition
scope, but it also displays powerful orthogonal reactivity with the 1,2,4,5-tetrazines. Whereas the latter
reacts at extraordinary cycloaddition rates with strained dienophiles (tetrazine ligation), the new and
isomeric 1,2,3,5-tetrazine displays similarly remarkable cycloaddition rates and efficiencies with amidines
(1,2,3,5-tetrazine/amidine ligation). The crossover reactivities (1,2,4,5-tetrazines with amidines vs 1,2,3,5-
tetrazines with strained dienophiles) are sufficiently low to indicate they may be capable of use concurrently
without competitive reactions.
INTRODUCTION
The inverse electron demand Diels–Alder cycloadditions of heterocyclic azadienes have provided
powerful methodology for the synthesis of highly substituted and functionalized heterocycles1 widely used
in organic synthesis and the pharmaceutical industry, in the divergent construction of screening libraries2
and in bioorthogonal conjugation.3,4 Our past efforts have provided systematic explorations of the reactions
of 1,2,4,5-tetrazines,5 1,2,4-triazines,6 1,3,5-triazines,7 1,3,4-oxadiazoles,8 1,2-diazines9 and most recently
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1,2,3-triazines.10 Each heterocyclic azadiene was found to possess a unique reactivity toward different
classes of dienophiles, display now predictable modes of cycloaddition, and exhibit substantial substituent
electronic effects impacting their intrinsic reactivity and cycloaddition regioselectivity. These insights
permitted their use as key steps in more than 50 natural products total syntheses11 and helped inspire the
development of the powerful 1,2,4,5-tetrazine based bioorthogonal conjugation and labeling technology
(tetrazine ligation) widely used today. Consequently, it is remarkable in this day and age that no member
of another fundamentally important heterocyclic azadiene ring system, the 1,2,3,5-tetrazines, has yet been
reported (Figure 1A).
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Limited attempts to prepare a 1,2,3,5-tetrazine have been described14 and no monocyclic aromatic
1,2,3,5-tetrazine as a discrete 6π heterocycle has been reported. Stability profiles established in
computational studies suggest a decreased kinetic stability for 1,2,3,5-tetrazine compared with the well-
known and isomeric 1,2,4,5-tetrazine (Eact 14 kcal/mol), but a greater thermodynamic stability than
1,2,4,5-tetrazine (7–8 kcal/mol).12 The principal challenge in the synthesis of 1,2,3,5-tetrazines is the
construction of two consecutive N–N bonds, which are not present in 1,2,4,5-tetrazines but are found in the
1,2,3-triazines. Although no synthesis of a monocyclic 1,2,3,5-tetrazine as a 6π aromatic system has been
reported, limited numbers of heterocyclic compounds bearing the 1,2,3,5-tetrazine core have been disclosed
(Figure 1B).13-15 The nonaromatic 1,2,3,5-tetrazine core (3) was synthesized as part of fused heterocycles
in the development of energetic materials,13 monocyclic tetrazinones have been described that were
prepared by a 6π-electrocyclization of acyclic triazene isocyanate precursors14 and the synthesis of a library
of ring-fused bicyclic tetrazinones has been reported of which a derivative (temozolomide, 4) is used
clinically.15 The methods used in this work for the construction of the core ring system as well as those used
for the preparation of 1,2,3-triazines10,16 provided valuable insights into the possible synthesis of aromatic
1,2,3,5-tetrazines. With even their stable existence in question, we explored several approaches for their
synthesis. As a result of these efforts, herein we report the synthesis, characterization, properties, and
cycloaddition reactivity of the first member of this new and previously unknown class of heterocycles.
A
N
N
Ph
N
Ph
N
N
N
N
N
1
2
)
1,2,3,5-tetrazine (
)
4,6-diphenyl-1,2,3,5-tetrazine (
B
O
H2N
N
N
N
N
Ar
O
N
O
N
N
N
N
N
NH
N
N
R
N
N
N
R
Me
H2N
N
N
High N-content
energetic material (
Non-ring fused
tetrazinone
4
Temozolomide (
)
3
)
Figure 1. (A) Structure of 1,2,3,5-tetrazine (1) and 4,6-diphenyl-1,2,3,5-tetrazine (2). (B) Reported
structures bearing a nonaromatic 1,2,3,5-tetrazine core.
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RESULTS AND DISCUSSION
5
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Synthesis and characterization of 4,6-diphenyl-1,2,3,5-tetrazine. The widely utilized syntheses
of the isomeric 1,2,4,5-tetrazines employ an oxidative aromatization of dihydro-1,2,4,5-tetrazines as an
effective final step,17 suggesting that the preparation of the 1,2,3,5-tetrazine system from a non-aromatic
dihydro precursor likely would be similarly successful. In a series of studies focused on the reactivity of
1,2,3-triazolium amides, Butler et al. reported a beautiful reaction cascade to furnish N2,N5-diaryl
substituted 2,5-dihydro-1,2,3,5-tetrazines (Scheme 1A).18 We envisioned that subsequent removal of the
N-aryl substituents by either oxidation or SNAr reactions, followed by a subsequent oxidation of the
resulting dihydrotetrazine would be a promising approach to prepare 4,6-disubstituted 1,2,3,5-tetrazines.
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Scheme 1
A
. Cascade reaction for preparation of 2,5-dihydro-1,2,3,5-tetrazines (Butler et al.):
Ph
Ph
N
Ar
Ph
Ph
Ph
N
N
N
Ar
N
N
N
N
Ar
Ar
ArNSO
N
N
O
S
N Ar
Ar
N
Ph
N
S
N
Ar
Ar
O
Ph
N
Ph
N
N Ar
N
N
N
- ArNSO
Ar
N
N
Ar
Ar
Ph
Ph
B
5
:
. Multi-gram scale preparation of 2,5-dihydro-1,2,3,5-tetrazine
O
O
NO2
Ph
Ph
H3PO4/EtOH
reflux, 24 h
PbO2/CH2Cl2
25 °C, 18 h
NHNH2·HCl
N
NH
HN
N
11.7 g, 61% over
two steps
O2N
NO2
Ph
Ph
NSO
Ph
Ph
N
N
Ph
O2N
N
NO2
NO2
N
toluene, 100 °C, 18 h
O2N
N
N
NO2
9.0 g, 78%
N
N
Ph
5
For initial studies, we chose to target the preparation of the substituted tetrazine 2, 4,6-diphenyl-
1,2,3,5-tetrazine. We began our study with development of a multigram scale preparation of 5,18 a reported
dihydro-1,2,3,5-tetrazine bearing p-nitrophenyl groups as the N-substituents (Scheme 1B). In order to
investigate the oxidative dearylation effected by reagents like cerium ammonium nitrate (CAN),
hydrogenation of 5 was conducted to yield 2,5-dihydrotetrazine 7 in which the N-substituents were reduced
to p-anilinyl as an appropriate reaction precursor (Scheme 2). The alternative SNAr reaction of 5, using
thiophenol as the nucleophile, proceeded smoothly to yield the stable N5-dearylated product 6. However,
the second dearylation of the remaining N2-substituent was found to be difficult and has not yet been
achieved even under harsher reaction conditions.
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Scheme 2
Ph
N
Ph
HN
Ph
KOH, PhSH, DMF,
80 °C, 42 h
5
6
7
8
N
N
N
N
O2N
N
NO2
N
NO2
49%
Ph
5
6
Pd/C, H2, EtOAc,
25 °C, 16 h
90%
Ph
(nBu4N)2Ce(NO3)6,
TfOH, CH2Cl2,
25 °C, 5 h,
N
9
H2N
N
N
NH2
10
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N
44%
Ph
7
Ph
N
O2N
N
N
O2N
NO2
N
Ph
O2N
8
Et3N, p-MeOC6H4SH,
DMF, 25 °C, 1 h;
then Boc2O, NaH,
DMF, -50 °C, 3 h
Ph
N
EtSH, K2CO3, DMF,
25 °C, 6 h
N
N
O2N
N
Boc
29%
70%
Ph
9
H
N
N
N
N
Boc
10
A serendipitous finding was observed when the oxidative dearylations of 5, 6, and 7 were examined.
CAN or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) mediated oxidative dearylations of 6 and most
notably 7 were not successful where oxidation and decomposition of the dihydrotetrazine system were
observed prior to the desired N-dearylations. With 5, however, treatment with (nBu4N)2Ce(NO3)6, a CAN
equivalent that is soluble in organic solvents, resulted in selective dinitration of the N2-p-nitrophenyl
substituent, affording 8 in 44% yield. The formation and structure of 8 were established by NMR and
HRMS and unambiguously confirmed with a single crystal X-ray structure determination (Scheme 2).19
CAN-mediated nitrations have been reported,20 and the presence of stoichiometric acid was needed to
effectively promote the reaction that provided 8.21 Notably, use of CAN instead of (nBu4N)2Ce(NO3)6 either
in acetonitrile or methanol resulted in little or no reaction product and CH2Cl2 was found to be the most
effective solvent for the reaction of those examined (Figure 2). Although the attempted oxidation of 5 did
not remove the aryl substituents, we viewed this transformation as an activation of the unreactive N2-aryl
moiety, permitting subsequent SNAr dearylation.
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Ph
Conditions
25 °C
N
N
N
O2N
N
NO2
Ph
N
O2N
Ph
N
N
5
O2N
N
NO2
Ph
O2N
8
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Figure 2. Discovery and optimization of the nitration reaction of 5.
With 8 in hand, two sequential SNAr dearylations were conducted. As expected, the N2-picryl group
is now more vulnerable to nucleophilic attack by an aryl thiol (p-MeOC6H4SH, Et3N) under mild conditions
(25 °C, 1 h), leaving the N5-substituent intact. The released free amine was found to be unstable at 25 °C,
which is in sharp contrast to its isomer 6 (stable at 80 °C). As a result, a subsequent in situ Boc-protection
was performed to provide 9 in 70% overall yield. Interestingly, normal protection conditions using a weak
base (Et3N) were not effective, and a strong base (NaH) was required. Conducting the reaction at low
temperature (–50 °C) was also found to be important in this protection step to avoid unproductive
consumption of the free amine. The second nucleophilic substitution reaction was more challenging due to
the instability of 9 (compared to 5) in the presence of nucleophiles. Use of an alkyl thiol as a stronger
nucleophile was found to promote the remaining SNAr reaction under mild conditions (EtSH, K2CO3, DMF,
25 °C, 6 h), providing the N2-Boc protected dihydrotetrazine 10 (29%, see Table S2 in Supporting
Information for conditions surveyed). Because limited structural information was provided by NMR, the
structure and solid-state conformation of 10 were unambiguously determined by X-ray crystallography
(Scheme 2).19 The final oxidation of 10 proceeded smoothly with a range of oxidants under mild reaction
conditions, affording the desired 4,6-diphenyl-1,2,3,5-tetrazine (2). A spontaneous loss of the Boc group
was observed to accompany the oxidation. Compared with other oxidants, including CAN or
(nBu4N)2Ce(NO3)6, MnO2 was found to be the most effective and convenient reagent of those examined,
providing 2 in high yield (80%) with simple filtration removal of the oxidant following completion of the
reaction (Figure 3). Tetrazine 2 is semi-stable on a silica gel column, although a rapid column
chromatography purification was important to provide the product in high yield.
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H
N
Oxidants
25 °C
N
N
N
N
N
N
N
Boc
10
2
7
8
9
10
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aAnhydrous solvents. bPre-mixed slurry of MnO2 in CH2Cl2 was used.
Figure 3. Oxidation of dihydrotetrazine 10 to tetrazine 2.
4,6-Diphenyl-1,2,3,5 tetrazine 2 is a yellow crystalline solid at room temperature, and crystalline
needles suitable for X-ray single crystal diffraction analysis were obtained by recrystallization from CH2Cl2,
CHCl3, or MeCN.19 The crystal structure is presented in Figure 4, alongside the structure of 2,5-dihydro-
1,2,3,5-tetrazine 10 for comparison. All atoms in 2 were found to be nearly coplanar and equalization of
the bond lengths in the central 1,2,3,5-tetrazine core was observed, with C–N bond lengths of 1.338–1.356
Å and N–N bond lengths of 1.326–1.328 Å, depicting a clear aromatic character for the delocalized 6π-
aromatic 1,2,3,5-tetrazine ring (typical bond lengths: C–N: 1.416 Å, C=N: 1.279 Å, N–N: 1.420 Å, N=N:
1.240 Å22). This is in sharp contrast to the dihydrotetrazine derivatives 8 and 10, where the dihydro-1,2,3,5-
tetrazine core adopt boat conformations, and bond lengths of 1.392–1.393 Å, 1.284–1.286 Å and 1.424–
1
1.425 Å were found for the C–N bonds, C=N bonds and N–N bonds in 10, respectively. The H NMR
spectrum of tetrazine 2 exhibited a single set of phenyl peaks, indicative of free rotation of the phenyl ring
in the solution. Notably, the ortho-protons of the phenyl substituent exhibit a downfield chemical shift of
8.8 ppm (same set of protons in 10 has a chemical shift of 7.8 ppm), resulting from a combined inductive
and deshielding effect of the aromatic tetrazine. The tetrazine was found to display a characteristic
downfield carbon peak (163 ppm) in the 13C NMR, like that of the carbon found in 3,6-diphenyl-1,2,4,5-
tetrazine (164 ppm).
Figure 4. Top and side view of single crystal X-ray structures of 10 (left) and 2 (right). The Boc-group in
10 is omitted for clarity.
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Tetrazine 2 was found to be stable at 25 °C in the crystalline state, in a chloroform solution, and
even in a protic solvent (CD3CN/D2O, V/V = 7:3), all for at least 5 days after which its monitoring was
discontinued. It was also found to be stable for at least 24 h at 25 °C in 70% H2O/CH3CN, 50% and 80%
H2O/DMSO, CH3CN/PBS buffer (1:1), and hexafluoroisopropanol (HFIP). It can be stored at 4 °C for more
than 50 days without observable decomposition. To obtain insights into its thermal stability, the kinetics of
thermal consumption of tetrazine 2 were monitored by 1H NMR at 80 °C, the lowest temperature at which
measurable conversion was readily observed. A clean transformation of tetrazine 2 to benzonitrile and N2
was found, with no observation of other byproducts or intermediates. First-order kinetics for the
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transformation were observed, with a rate constant of k = 0.0293 h-1 (8.15 × 10-6 s-1) and half-life of t1/2
=
23.6 h (8.50 × 104 s) (Supporting Information Figure S2). Although it is kinetically less stable than the
corresponding 1,2,4,5-tetrazine in agreement with earlier computational studies,12 the observed stability of
tetrazine 2 permits easy handling under normal conditions and it can be warmed to elevated temperatures
in solvent to promote cycloadditions with predictable limitations on the conditions used.21
We also examined the photophysical and electrochemical properties of 2. The UV-Vis spectrum of
2 showed a strong absorption (molar extinction coefficient ε = 2.52 × 104 L·mol-1·cm-1) peak at 275 nm,
corresponding to the π-π* transition of the phenyl rings and tetrazine core (Figure 5). A much weaker
absorption (ε = 4.90 × 102 L·mol-1·cm-1) was observed at 396 nm, corresponding to the n-π* transition of
the tetrazine. It displayed no fluorescence, being distinct from the isomeric and weakly fluorescent 3,6-
diphenyl-1,2,4,5-tetrazine ( = 1.5 x 10-4).23 Tetrazine 2 was found to undergo a quasi-reversible single-
electron reduction at –1.37 V (vs Fc+/Fc, –0.78 V vs N. H. E.) in cyclic voltammetry (CV), revealing its
electron-deficient nature (Figure 5). In contrast to its reduction potential, no observable oxidation was
observed when a positive scan was performed on 2. The reduction potential of 2 is comparable but slightly
lower than 3,6-diphenyl-1,2,4,5-tetrazine (–1.21 V vs Fc+/Fc),24 indicating a similar electron deficiency and
likely similar reactivity in inverse electron demand Diels–Alder cycloadditions.
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Figure 5. UV-Vis spectrum (top) and cyclic voltammetry data (bottom) of tetrazine 2. UV-Vis spectrum
was measured in CH3CN. CV (scan rate, 40 mV/s) measurement was carried out in a solution of nBu4NBF4
(100 mM) in CH3CN.
Cycloaddition reactivity. We previously reported the discovery of the unusually rapid inverse
electron demand Diels–Alder cycloadditions of a series of 1,2,3-triazines with amidines as
heterodienophiles.10 As a 5-aza-1,2,3-triazine, 1,2,3,5-tetrazines were anticipated to react with amidines to
provide analogous cycloaddition products. When tetrazine 2 in acetonitrile (40 mM) was treated with
benzamidine 11a, complete conversion to 12a was found after 2 h at 25 °C even at the dilute reaction
concentrations. An exclusive C4/N1 versus N2/N5 mode of cycloaddition and a single reaction
regioselectivity were observed, affording triphenyl-1,3,5-triazine 12a in near quantitative yield (97%). The
scope of this cycloaddition of tetrazine 2 with amidines was defined (Figure 6). All amidines, including
aryl amidines (11a–f), heteroaryl amidines (11g–j), and aliphatic amidines (11k–n), provided the
corresponding fully substituted 1,3,5-triazines (12a–n) in excellent yields (87–99%) under these
remarkably mild reaction conditions (40 mM, 25 °C, 2 h). Notably, the reaction proceeded effectively even
with electron-deficient (hetero)arylamidines (11f–h, 11j). To examine the compatibility of this
cycloaddition with protic solvents including water, the cycloaddition of tetrazine 2 with amidine 11l was
also conducted in CH3CN/H2O (V/V = 7:3, 2 h, 89% and 3:7, 24 h, 94%), where the reaction proceeded
smoothly even in 70% H2O/CH3CN and provided triazine 12l in the same yield observed in CH3CN alone.
In order to gain a deeper insight into the reaction mechanism and kinetics, 1H NMR was used to monitor
the reaction between tetrazine 2 (10 mM, CD3CN) and amidine 11k (2.0 equiv) at 25 °C. Fast consumption
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of tetrazine 2 and accumulation of product 12k were observed with appreciable product formation within 5
1
min, 90% conversion within 1 h, and full (>95%) conversion after 2 h. It is worth noting that H NMR
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signals from only tetrazine 2, amidine 11k, and triazine 12k were observed throughout the course of the
reaction, without detection of any reaction intermediates or byproducts (Figure 7). Therefore, it is the
cycloaddition reaction or addition-cyclization reaction between tetrazine and amidine that is the rate-
determining step, which is followed by rapid sequential elimination of N2 and NH3 from the cycloadduct.
From the NMR studies, the second-order rate constant of this reaction was determined to be k =
(5.87±0.10)*10-2 M-1·s-1 (Supporting Information Figure S3).
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Ph
Ph
N
CH3CN, 40 mM,
25 °C, 2 h
Ph
Ph
N
NH
NH2
(1.5 equiv)
4
+
N
N
1 N
N
87-99%
R
N
R
2
11a-n
12a-n
NH3
(1.0 equiv)

N
4
Ph
N
Ph
Ph
N
Ph

N2
NH
N
NH
NH2
N
R
N
1
R
NH2
F
Br
OMe
NH2
12a
12b
12c
12
12
, 97%
, 99%
, 94%
d, 98%
e, 97%
CF3
N
N
S
NO2
, 94%
N
N
12f
12g
12h
12i
12j
, 91%
, 87%
, 97%
, 94%
Cl
Ph
Ph
Cl
, 89%a (94%b)
, 98%
12k
12l
12m
12n
, 98%
, 98%
Ph
N
Ph
Ph
N
Ph
NH
CH3CN, 40 mM
N
N
+
Ph
(1.5 equiv)
N
N
X
N
12l
Ph
2
(1.0 equiv)
11l
13a
13b
X = NH2,
X = SMe,
X = OEt,
X = NH2, 25°C, 2 h, 89%
X = SMe, 25°C, 48 h, 47%
X = OEt, 80°C, 48 h, 35%
aCH3CN or CH3CN/H2O (V/V = 7:3) as solvent, 2 h.
bCH3CN/H2O (V/V = 3:7) as solvent, 24 h.
Figure 6. Scope of the cycloaddition between 2 and amidines, imidates and thioimidates.
120 min
55 min
50 min
45 min
40 min
35 min
30 min
25 min
20 min
15 min
10 min
5 min
2
2
2 12k 12k 12k 11k 12k 11k
12k
9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3
δ (ppm)
Figure 7. 1H NMR study of the reaction between tetrazine 2 and amidine 11k (2 equiv) over 120 min (10
mM, 25 °C, CD3CN).
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To further probe whether the reaction of amidines with 1,2,3,5-tetrazine 2 represents a single-step
cycloaddition or a stepwise addition-cyclization, we conducted the reaction of 2 with amidine 11l that was
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doubly labeled with N (Figure 8). The reaction produced the 1,3,5-triazine product 121 in superb yield
(94%) with incorporation of a single 15N label (1.07 ±0.01) as established by mass spectrometry (Supporting
Information Table S3) and NMR experiments (Supporting Information Figure S4). This is consistent with
a single-step cycloaddition and, while not ruling out a stepwise addition-cyclization mechanism, the latter
would have been expected to provide a near equal mixture of singly and doubly 15N labeled triazine products
(ca. 1.5 vs 1 15N). If the cycloaddition does proceed in a non-concerted fashion with generation of a
zwitterionic intermediate,25 it would appear to require tight contact ion pair association following the initial
addition with collapse to the [4+2] cycloadduct (vs proton transfer) prior to loss of nitrogen.
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N
Ph
N
N
–NH3
N
NH
NH2
Ph
Ph
singly 15N-labeled
NH2
Ph
11l
Ph
N
Ph
Ph
N
N
Ph
15
NH
N-
N
N
N
N
N = 15
N = 14
N
N
Ph
–NH3
or
CH3CN, 25 °C, 2 h, 94%
–NH3
NH
H
N
Ph
N
Ph
HN
N
N
Ph
N
Ph

N2
Ph
N
H
NH Ph NH
Figure 8. Reaction of 2 with doubly 15N labeled 11l.
Imidates and thioimidates are related heterodienophiles reported to react with some heterocyclic
azadienes, but which typically display a lower reactivity than the corresponding amidines and a similar
reactivity difference was observed with tetrazine 2 (Figure 6). Whereas the reaction of 2 with amidine 11l
proceeds smoothly under mild conditions (2 h, 25 °C, 89%), a longer reaction time was required for the
corresponding thioimidate 13a (48 h, 25 °C) and even then the formation of the same triazine 12l was
observed in a lower yield (47%). With an increase in the amount of dienophile (5 equiv) and reaction
concentration (400 mM), requiring CHCl3 as solvent for solubility purposes, the reaction provided 12l in
good yield (68%, 48 h, 25 °C). The corresponding imidate 13b exhibited an even lower reactivity and the
reaction between 2 and 13b required an elevated temperature (35%, 48 h, 80 °C) or higher concentration
(43%, 400 mM in CHCl3, 5 equiv 13b, 48 h, 25 °C) and longer reaction times, providing more modest
yields of the triazine 12l.
Additional dienophiles classes were examined, including electron-rich olefins and alkynes (Figure
9). The initial cycloaddition reaction between 4,6-diphenyl-1,2,3,5-tetrazine (2) with enamines 14a–d in
acetonitrile is fast even at room temperature and is accompanied by loss of N2. This is followed by a slower
amine elimination with rearomatization, requiring the longer reaction times and subsequent elevated
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temperatures. The same exclusive C4/N1 (vs N2/N5) cycloaddition mode was observed and occurs with a
predictable exclusive regioselectivity (dienophile most electron-rich atom attaches to C4), affording
substituted pyrimidines (15a–d) in good yields. In the case of 14b, the use of CHCl3 (41%) versus CH3CN
(43%) as an alternative solvent provided similar yields of 15b, although the impact of solvent on the slow
and occasionally problematic final aromatization was not investigated in detail. A similar reactivity was
observed for the cycloaddition of tetrazine 2 with ynamines (16a–b), where the reaction proceeded
smoothly under mild conditions. Complete conversion of tetrazine 2 to pyrimidine 17a (90% yield) was
observed after 1 h (25 °C), whereas the reaction of the sterically more hindered ynamine 16b was found to
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be slower, providing pyrimidine 17b (92% yield) after 24 h.
CH3CN, 40 mM,
25 °C, 3 h; then
60°C, 24 h
R'
R
Ph
N
R
Ph
R'
N
Ph
Ph
+
N
4 Å MS
N
N
N
N
2
14a
14b
14c
14d
, R, R' = (CH2)3
, R, R' = (CH2)4
, R, R' = (CH2)5
, R = Ph, R' = H
(1.2 equiv)
15a
, 83%
15b
, 43%
15c
, 60%
15d
, 45%
(1.0 equiv)
Ph
N
Ph
CH3CN, 40 mM,
25 °C
Ph
Ph
N
Me
+
+
N
N
N
Me
R2N
N
NR2
16a
16b
17a
, 1 h, 90%
, 24 h, 92%
, R = Et
, R = Bn
2
17b
(1.0 equiv)
(1.5 equiv)
Ph
Ph
N
N
40 mM,
60 °C, 24 h
Ph
N
Ph
EtO
OEt
N
N
CH3CN, 52%
CHCl3, 60%
toluene, 13%
1,4-dioxane, 11%
N
OEt
19
2
18
(1.0 equiv)
(5 equiv)
Figure 9. Reaction of 2 with enamines, ynamines and a ketene acetal.
Treatment of tetrazine 2 with ketene acetal 18 in warm acetonitrile (60 °C) provided the expected
pyrimidine product 19 in good yield (52%) after 24 h (Figure 9). An interesting solvent effect was observed
in this cycloaddition where the reaction in CHCl3 gave a slightly improved yield (60%), but a slower
reaction and incomplete conversion of the tetrazine 2 was observed when the reaction was conducted in
toluene (13%) or 1,4-dioxane (11%) under otherwise identical conditions.
Tetrazine 2 was also found to react with the electron-rich styrene 20 at an elevated temperature (80
°C), where cycloaddition is followed by an in situ oxidation to afford the aromatic pyrimidine product 21
(Figure 10). A slow conversion and low yield (9%) were found when the reaction was conducted in
acetonitrile, whereas an improved yield (28%) was observed when 2 was warmed with neat 20. Although
the latter results might appear modest, they do highlight the exceptional reactivity that can be expected of
the 1,2,3,5-tetrazines, rivaling that of the 1,2,4,5-tetrazines and exceeding that of the respective triazines.
Strained alkenes and alkynes are an especially interesting and important dienophile class. They
serve as an excellent group of dienophiles and their reactions with 1,2,4,5-tetrazines proceed at remarkable
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rates under extraordinarily mild conditions even at dilute reaction concentrations.4 Consequently, variations
on these reactions have been explored and adopted for powerful bioorthogonal conjugation and labeling
techniques.3,4 In order to investigate whether 1,2,3,5-tetrazine 2 possesses similar or a distinctive reactivity,
the reaction of 1,2,3,5-tetrazine 2 with the strained alkyne 22 was examined. Efficient cycloaddition with
formation of the fused pyrimidine 23 in excellent yield (97%) was observed under mild (25 °C) and
modestly concentrated reaction conditions (400 mM 2, 4 equiv dienophile) and progressively slower
conversions and decreased yields were observed under increasingly more dilute reaction concentrations (40
mM 2, 28%; 5 mM 2, no reaction) with the balance of the materials being recovered starting tetrazine 2 and
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alkyne 22 (Figure 10). Again, only a single mode of cycloaddition was observed (C4/N1 vs N2/N5).
NMe2
Ph
N
N
Ph
Ph
N
80 °C, 72 h
+
+
N
N
N
N
CH3CN, 40 mM,
N
Me2N
20
5 equiv , 9%
Ph
neat, 28%
2
20
21
23
H
H
H
Ph
N
Ph
N
Ph
25 °C, 48 h
N
OH
CH3CN, 5 mM, N. R.
CH3CN, 40 mM, 28%
CHCl3, 400 mM, 97%
OH
N
H
Ph
2
22
(4 equiv)
Figure 10. Reaction of 2 with an electron-rich styrene and a strained alkyne.
Reactivity comparison between two isomeric tetrazines: 4,6-diphenyl-1,2,3,5-tetrazine (2)
versus 3,6-diphenyl-1,2,4,5-tetrazine (24). In order to compare what became evident as distinct
cycloaddition behaviors, the reactivity of 4,6-diphenyl-1,2,3,5-tetrazine (2) and the well-known and
similarly substituted 3,6-diphenyl-1,2,4,5-tetrazines (24) was compared side-by-side and in competition
studies with representative members of the different dienophile classes. Notably, these would not be the
most reactive members of either tetrazine class, but the comparisons do provide the first opportunity to
define cycloaddition distinctions. Three dienophiles were chosen for study, amidine 11l, ynamine 16a, and
cyclooctyne 22, representing examples of a heterodienophile, an electron-rich dienophile, and a strained
dienophile, respectively. Separate reactions between each of the tetrazines and dienophiles were conducted,
followed by competition reactions where 1 equivalent of dienophile was added to an equimolar mixture of
1,2,3,5-tetrazine 2 and 1,2,4,5-tetrazine 24 (1 equiv each) (Figure 11).
Three different outcomes were observed for the three dienophiles. While amidine 11l rapidly
reacted with 1,2,3,5-tetrazine 2 under mild conditions (25 °C, 40 mM, CH3CN, 2 h, 89%), no reaction
between the same amidine 11l and 1,2,4,5-tetrazine 24 was observed under the same reaction conditions,
consistent with reports of the modest reactivity of 1,2,4,5-tetrazines toward amidines.1a Perhaps even more
remarkable, 1,3,5-triazine 12l was observed as the only product (92%) when an equimolar mixture of the
two tetrazines was treated with amidine 11l, reflecting the much greater reactivity of 2. A completely
reversed cycloaddition preference was observed when cyclooctyne was used as the dienophile. Consistent
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with prior reports, 3,6-diphenyl-1,2,4,5-tetrazine (24) possessed an outstanding reactivity toward
cyclooctyne 22, rapidly forming the pyridazine 25 (25 °C, 5 mM, CH3CN, 10 min, 95%). By contrast, the
reaction of 1,2,3,5-tetrazine 2 with the strained cyclooctyne 22 did not produce any pyrimidine 23 under
the same reaction conditions even after prolonged reaction times (48 h). Expectedly, only pyridazine 25
was isolated (98%) when an equimolar of mixture of the two tetrazines 2 and 24 were treated with
cyclooctyne 22 (1 equiv), reflecting now the greater reactivity of 24. Ynamine 16a was found to react with
either tetrazine individually (25 °C, 40 mM, 1 h), affording the corresponding pyrimidine 17a (90%) or
pyridazine 26 (95%) as products. The treatment of ynamine 16a (1 equiv) with an equimolar mixture of the
two tetrazines gave both 17a (15%) and 26 (53%) as reaction products, revealing a competitive but slightly
lower reactivity of 1,2,3,5-tetrazine 2 than 1,2,4,5-tetrazine 24 toward the ynamine dienophile (1:3.5).
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These results, along with the observations on the reactivities of tetrazines with other dienophiles,
indicates the dependence of inverse electron demand Diels–Alder reactivity on both the core structure of
the tetrazine as well as the dienophile itself. Compared to the 1,2,4,5-tetrazine 24, 1,2,3,5-tetrazine 2
exhibited an outstanding reactivity toward heterodienophiles (amidines), a comparable reactivity toward
electron-rich dienophiles (ynamines, enamines, ketene acetal), and a lower reactivity toward strained olefins
and alkynes (cyclooctyne). Houk et al. recently reported a comprehensive computational study of the
reaction of the full and systematic series of heterocyclic azadienes with olefinic dienophiles.26 A selectivity
for C–C bond formation over C–N bond formation within a given azadiene was predicted, defining a
preferred intrinsic mode of cycloaddition, and the cycloaddition reactivity was found to follow a general
trend of two C–C bond > C–C and C–N bond > two C–N bond formations. Thus, in addition to the relative
energies of the reacting LUMO energies (slightly lower LUMO for 1,2,4,5-tetrazine vs 1,2,3,5-tetrazine, E
= 0.03–0.04 eV, both of which are much lower than any of the isomeric triazines, E = 0.5–0.8 eV;
MNDO/AM1), this correspondence between cycloaddition reactivity and nature of the bond formation at
the diene termini was attributed to both a lower distortion energy of the dienes/dienophiles and productive
interaction energy (orbital overlap efficiency) in the reaction transition state, where both are more favorable
for the cycloaddition of olefinic dienophiles (ethylene) with 1,2,4,5-tetrazine versus 1,2,3,5-tetrazine (Eact
= 9.4 kcal/mol). Consequently, a 1,2,4,5-tetrazine was predicted to be more reactive than a 1,2,3,5-tetrazine
toward olefin/alkyne dienophiles. Whereas we found that the expected distinction was modest with an
ynamine (electron-rich alkyne), we found that it was remarkably pronounced with a strained alkyne.
However, the origin of the remarkably efficient and high reactivity of amidines with a 1,2,3,5-tetrazine,
especially compared with a 1,2,4,5-tetrazine, is unclear. It is possible that the polarized nature of both the
diene and dienophile and the polarity match of the reacting partners might result in a change in reaction
mechanism, with the reaction proceeding now through a non-concerted cycloaddition or asynchronous
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cycloaddition rather than a synchronous concerted cycloaddition. However, we find no evidence to support
a simple addition-cyclization reaction in direct reaction monitoring (Figure 7), 15N-labeling studies (Figure
8), or through intercepted reaction byproducts. Alternatively, it is plausible that the interaction energy of
an amidine with a 1,2,3,5-tetrazine is much more favorable than with a 1,2,4,5-tetrazine, where the latter
but not the former would suffer destabilizing electrostatic interactions of its ring nitrogens with the amidine
amine substituent in the [4+2] cycloaddition transition state. Regardless of the origin but as a result of the
remarkable distinctions in cycloaddition reactivity, a new orthogonal reaction pair was discovered, the
1,2,3,5-tetrazine/amidine pair, complementing the powerful 1,2,4,5-tetrazine/strained alkyne pair. Whereas
1,2,4,5-tetrazine displays extraordinary cycloaddition rates with strained dienophiles (tetrazine ligation),
the new and isomeric 1,2,3,5-tetrazine displays similarly remarkable cycloaddition rates and efficiencies
with amidines (1,2,3,5-tetrazine/amidine ligation). When following the reactions of 4,6-diphenyl-1,2,3,5-
tetrazine with amidines (1H NMR), only starting material and product are observed and no intermediate(s)
or byproducts are detected (see Figure 7). The crossover reactivities (1,2,4,5-tetrazines with amidines vs
1,2,3,5-tetrazines with strained dienophiles) are sufficiently low to indicate they may be used concurrently
without competitive reactions. Both reactions proceed at low reaction concentrations under mild conditions
and are compatible with H2O as the reaction co-solvent, providing potential new applications in orthogonal
or sequential ligation reactions. Such studies are presently under investigation.
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Ph
N
Ph Ph
N
N
Ph
N
N
Ph
N
Ph
N
N
+
N
N
N
N
N
N
Dienophile
N
Ph
24
Ph
N
2
2
24
(1 equiv)
(1 equiv)
(+ 1 equiv dienophile)
Ph
N
Ph
Ph
N
Ph
NH
NH2
N
N
N
N
Ph
no reaction
Ph
Ph
11l
12l
12l
, 89%
, 92%
Ph
N
Ph Ph
Me
N
Ph
N
Ph
N
Ph
Me
N
N
Me
+
N
N
Me Ph
Me
Ph
Et2N
NEt2
NEt2
NEt2
NEt2
16a
17a
17a
26
26
, 90%
, 15%
, 53%
, 95%
OH
H
OH
H
H
OH
H
H
Ph
N
Ph
H
no reaction
N
N
N
Ph
Ph
22
25
25
, 98%
, 93%
Figure 11. Comparison of the reactivity of 1,2,3,5-tetrazine 2 and 1,2,4,5-tetrazine 24 with different
dienophile classes.
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CONCLUSIONS
Herein we disclosed a novel synthesis and the full characterization of the first member of a
monocyclic aromatic 1,2,3,5-tetrazine, 4,6-diphenyl-1,2,3,5-tetrazine. It is remarkable that no member of
such a fundamental and important heterocyclic ring system had been previously described. Its synthesis and
5
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1
full characterization, including H/13C NMR, X-ray structure, intrinsic chemical stability, UV spectrum,
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and quasi reversible single-electron reduction (–1.37 V vs Fc+/Fc) are reported. Although we continue to
define its cycloaddition reactivity, mode, regioselectivity, and scope, we have found that: (1) its reactivity
is comparable to that of the isomeric 3,6-diphenyl-1,2,4,5-tetrazine, (2) it displays a single mode of
cycloaddition with reaction only across C4/N1 (no N2/N5 cycloaddition observed), (3) its cycloaddition
occurs with a predictable regioselectivity (dienophile most electron-rich atom attaches to C4), and (4) it
participates as the 4-component of well-behaved inverse electron demand Diels–Alder reactions
preferentially reacting with electron-rich or strained dienophiles. Although we are only now beginning to
explore the full cycloaddition scope, we have already found that it displays additional reactivity
complementary to the isomeric 1,2,4,5-tetrazines. It not only displays the expected remarkable
cycloaddition reactivity, a surprisingly good stability (e.g., stable to chromatography, long term storage,
presence of H2O even as reaction co-solvent), and broad cycloaddition scope, but it also exhibits a powerful
orthogonal reactivity with the 1,2,4,5-tetrazines that may be especially useful, including in the fields of
click ligation and bioconjugation. Whereas the latter displays extraordinary cycloaddition rates with
strained dienophiles (tetrazine ligation), the new and isomeric 1,2,3,5-tetrazine displays similarly
remarkable cycloaddition rates and efficiencies with amidines (1,2,3,5-tetrazine/amidine ligation). The
crossover reactivities (1,2,4,5-tetrazines with amidines vs 1,2,3,5-tetrazines with strained dienophiles) are
sufficiently low to indicate they may be capable of concurrent use without competitive reactions. Extensions
of the work to the study of additional substituted 1,2,3,5-tetrazines, the development of additional and
needed synthetic approaches to this previously unknown class of heterocyclic azadienes, and their
applications are in progress and will be reported in due time.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:10.1021/jacs.xxxxxxx. Full experimental details and characterization data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: dale.boger@outlook.com
ORCID
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Dale L. Boger: 0000-0002-3966-3317
Notes
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The authors declare no competing financial interests.
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ACKNOWLEGEMENTS
We are especially grateful to the National Institutes of Health for financial support of the studies (CA042056,
DLB) and a JITRI Fellowship (ZCW). We thank Dr. Laura Pasternack and Dr. Jason Chen for the analysis
of labeling experiments, Dr. Milan Gembicky and Dr. Arnold Rheingold of the Crystallography Facility at
the University of California, San Diego for the X-ray structure determinations of 2, 8 and 10 and Dr. Paul
Richardson (Pfizer) for the differential scanning calorimetry data on 2 and 8.
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21. Differential scanning calorimetric measurements (Supporting Information page S29) at 5 °C min-1
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and does so with a moderate thermal potential (0.177 kcal/g, 744 J/g). Intermediate 8 melts at 233 °C then
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(242–400 °C, peak at 305 °C), and releases 0.657 kcal/g (2756 J/g) of energy over the full temperature
range of decomposition. Safety precautions should be exercised especially when 8 is prepared on large scale
and conducting chemistry with 8 within 100 °C of the onset temperature should be avoided.
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Table of Contents Graphic
N
N
N
N
The first monocyclic aromatic 1,2,3,5-tetrazine:
 Synthesis and full characterization
 Reactive diene in inverse electron demand Diels-Alder reactions
 Single mode of cycloaddition (C4/N1) and predictable regioselectivity
 Orthogonal reactivity with 1,2,4,5-tetrazine without competitive reactions
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