Preparation of Unsymmetrical 1,2,4,5-Tetrazines via a Mild Suzuki Cross-Coupling Reaction

Article abstract of DOI:10.1021/acs.orglett.7b02868

N-Alkyl substituted chlorotetrazines were coupled with various boronic acids under Suzuki conditions in high yield at room temperature, giving a mild and straightforward synthetic route toward diverse unsymmetrical 1,2,4,5-tetrazines, a rare heteroarene. This chemistry not only expands the known substrate scope of tetrazine cross-coupling reactions but also allows for the synthesis of novel, tetrazine-containing biologically active molecules with improved DMPK properties.

Full text of DOI:10.1021/acs.orglett.7b02868

Letter
pubs.acs.org/OrgLett
Preparation of Unsymmetrical 1,2,4,5-Tetrazines via a Mild Suzuki
Cross-Coupling Reaction
Aaron M. Bender, Trevor C. Chopko, Thomas M. Bridges, and Craig W. Lindsley*
Departments of Pharmacology and Chemistry, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville,
Tennessee 37232, United States
S
* Supporting Information
ABSTRACT: N-Alkyl substituted chlorotetrazines were
coupled with various boronic acids under Suzuki conditions
in high yield at room temperature, giving a mild and
straightforward synthetic route toward diverse unsymmetrical
1,2,4,5-tetrazines, a rare heteroarene. This chemistry not only
expands the known substrate scope of tetrazine cross-coupling
reactions but also allows for the synthesis of novel, tetrazine-
containing biologically active molecules with improved DMPK properties.
1,2,4,5-Tetrazines, or s-tetrazines, were first reported toward the
end of the 19th century, when Pinner found that combining
equimolar quantities of hydrazine and benzonitrile produced
3,6-diphenyl-s-tetrazine after oxidation.1 Since Pinner’s discov-
ery, this class of nitrogen-rich heterocycles has been of great
interest to synthetic organic chemists and materials scientists
alike, and many research groups have reported unique
syntheses and applications of s-tetrazines.2
carbanions.14−17 Despite the propensity of s-tetrazines to
participate in SNAr reactions, the generation of unsymmetrical
s-tetrazines remains limited by the narrow scope of reactions
that can be performed on the highly electron-deficient scaffold.
Our group became interested in s-tetrazines as a potential
bioisostere for other six-membered nitrogen-containing hetero-
cycles, and in particular pyridazines, as part of an ongoing
medicinal chemistry effort. Starting from commercially available
3,6-dichloro-1,2,4,5-tetrazine, we speculated whether it would
be possible to use transition metal cross-coupling reactions to
generate unsymmetrical s-tetrazines. Very few successful reports
of s-tetrazine cross-couplings currently exist in the literature,
and it is known that the highly electron-deficient core must be
stabilized with an electron-donating group if the reaction is to
be successful.16,18 Indeed, preliminary attempts in our own
laboratory to perform Pd-catalyzed cross-coupling reactions on
3,6-dichloro-1,2,4,5-tetrazine were unsuccessful. In 2003,
Carboni and Lindsey were the first to report that s-tetrazines
could undergo cycloaddition reactions,3 and a number of
groups have utilized this inverse demand Diels−Alder cyclo-
addition chemistry toward the synthesis of novel 3,6-substituted
pyridazines, a heterocyclic motif found in myriad biologically
active molecules.4 Contributions to this field by Boger and
Sauer5 have allowed for the Carboni−Lindsey cycloaddition to
find use in a number of elegant total syntheses, including
Ningalin B,6 Lycogarubin C,7 and ent-(−)-Roseophilin.8 In
addition to serving as an important building block in the total
syntheses of natural products, the cycloaddition reactions of s-
tetrazines have found applications toward the production of
other complex, nitrogen-rich heterocycles, including highly
substituted pyridazine boronic esters,9 and pyrimido[4,5-
d]pyridazines.10 This unique s-tetrazine chemistry is also
being developed as a rapid bioorthogonal coupling reaction.11
While the synthesis of symmetrical 3,6-substituted s-
tetrazines is straightforward, the generation of 3,6-unsym-
metrical analogs has proven challenging.2b A number of
interesting synthetic approaches to unsymmetrical tetrazines
have been reported. Smith and colleagues have reported
unsymmetrical macropeptides with s-substituted tetrazine
cores,12 and Devaraj and colleagues have reported Ni-catalyzed
cyclization of nitriles with hydrazine to give unsymmetrical
tetrazines.13 3,6-Bis(methylthio)-1,2,4,5-tetrazine14 and 3,6-
bis(3,5-dimethylpyrazol-1-yl)-1,2,4,5-tetrazine15 are widely
used starting materials to generate unsymmetrical tetrazines,
and such compounds are known to undergo nucleophilic
aromatic substitution (SNAr) with various amines and alkyl
́
Novak and Kotschy reported the first cross-coupling reactions
(Sonogashira and Negishi) on s-tetrazines, starting from various
3-amino-6-chloro-1,2,4,5-tetrazine scaffolds 1 to afford 3
(Figure 1).18 Despite this notable advance, yields were
moderate, and the authors were unable to isolate any desired
products under Suzuki conditions. In 2007, Leconte and
colleagues found that 3-methylthio-6-(morpholin-4-yl)-1,2,4,5-
tetrazine 4 could readily undergo Suzuki and Stille couplings
with displacement of a methylthio group to provide 6 (Figure
1), and to our knowledge this method provides the only
examples in the literature of Suzuki cross-couplings directly into
a tetrazine to give 3,6-unsymmetrical s-tetrazines.10,16 Although
encouraging as the first report of a successful Suzuki reaction
on a tetrazine, yields remained moderate for this series, and the
s-tetrazine system was limited to a morpholino group on one
side.16
Received: September 13, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b02868
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Organic Letters
Letter
a
Table 1. Optimization of the Suzuki Coupling Conditions
catalyst
loading
temp time
yield
b
entry
1
catalyst
base
K2CO3
(°C)
(h)
(%)
RuPhos Pd
G3
10
100
1
50
2
3
4
5
Pd(OAc)2
Pd(PPh3)4
Pd2(dba)3
10
10
10
10
K2CO3
K2CO3
K2CO3
K2CO3
100
100
100
100
1
1
1
1
12
53
6
Pd(dppf)Cl2·
DCM
63
6
BrettPhos Pd
G3
10
10
10
10
10
10
10
10
10
10
10
10
10
5
K2CO3
100
100
100
100
100
100
100
100
100
130
70
1
1
1
2
2
2
2
2
2
1
2
2
2
2
2
2
60
3
Figure 1. Previously reported Pd-catalyzed cross-coupling reactions of
s-tetrazines.16,18
7
tBuXPhos Pd
G3
K2CO3
8
Pd(amphos)
Cl2
K2CO3
59
71
85
92
89
65
31
63
97
94
90
90
7
We therefore wondered if the scope of this reaction could be
expanded to include other amino-substituted s-tetrazines,
specifically amines not limited to a closed, tertiary ring system
(i.e., morpholino). Additionally, we were interested in the
optimization of milder reaction conditions, as yields in the
previous report for Suzuki couplings were increased only
through microwave synthesis at 200 °C.16 For our optimization
study, we therefore selected N-butyl-6-chloro-1,2,4,5-tetrazin-3-
amine (7), synthesized under previously reported conditions
from 3,6-dichloro-1,2,4,5-tetrazine (Table 1).19 A screen of
palladium catalysts (10% loading with phenylboronic acid and
K2CO3 as base) encouragingly indicated several promising
systems, including Pd(dppf)Cl2 (entry 5) and the third
generation BrettPhos palladacycle (entry 6),20 both of which
gave moderate yields of desired product 8 after 1 h at 100 °C.
Increasing the reaction time to 2 h with both catalysts improved
yield due to the complete consumption of starting material
(entries 9 and 10), particularly in the case of the BrettPhos
palladacycle. Changing the base to Cs2CO3 further improved
yield using this catalyst (entry 11, 92% yield), and we were
encouraged to find that this reaction could be run with
comparable yields both at 70 °C and even at room temperature
(entries 16 and 18) and at a reduced catalyst loading of 5% at
room temperature (entry 19, 90% yield). A 1% catalyst loading
at room temperature (entry 20) resulted in significantly
decreased yield compared to entry 19, although a 1% catalyst
loading at 70 °C proved successful (entry 21, 81% yield). These
mild reaction conditions and high yields are a testament to the
robustness of this and other Buchwald palladacycles, partic-
ularly for otherwise problematic or unreactive coupling
partners, and the mild conditions reported for entry 19 were
selected for further substrate scope studies. Utilizing these
optimized reaction conditions, 8 was isolated in 93% yield after
column chromatography.
9
Pd(dppf)Cl2·
DCM
K2CO3
10
11
12
13
14
15
16
17
18
19
20
21
BrettPhos Pd
G3
K2CO3
BrettPhos Pd
G3
Cs2CO3
K3PO4
BrettPhos Pd
G3
BrettPhos Pd
G3
DIPEA
BrettPhos Pd
G3
CH3COONa
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
BrettPhos Pd
G3
BrettPhos Pd
G3
BrettPhos Pd
G3
50
BrettPhos Pd
G3
rt
BrettPhos Pd
G3
rt
BrettPhos Pd
G3
1
rt
BrettPhos Pd
G3
1
70
81
a
Reaction conditions: 7 (30 mg, 0.16 mmol), PhB(OH)2 (1.2 equiv),
b
base (3.0 equiv), 5:1 1,4-dioxanes/H2O (1.0 mL). Yields determined
by LCMS using anisole as internal standard.
(86%). Heteroaryl bicyclic ring systems (9g and 9h) were also
tolerated, albeit in diminished yield for 6-quinoline 9g.
The coupling of 7 with potassium vinyltrifluoroborate
(product 9i) proved sluggish at room temperature, although
this product could also be isolated in high yield by increasing
the temperature to 70 °C (73%). This result was particularly
encouraging, as vinyl-substituted s-tetrazines are of interest as a
class of precursors to highly nitrogen-rich polymers, and to our
knowledge there are no known previous examples of a
successful cross-coupling between an s-tetrazine and a vinyl
group.21 This methodology should therefore prove straightfor-
ward for the generation of new vinyltetrazines. Furthermore,
protic substrates such as 4-hydroxyphenylboronic acid (9j),
smaller aromatic systems (methylpyrazole 9k) as well as 4-Boc-
aminophenylboronic acid (9l) also proved compatible under
our conditions with substrate 7, and all were isolated in
With conditions optimized for 7 and phenylboronic acid, we
next sought to expand the scope of the boronic acid coupling
partner (Scheme 1). Under the optimized conditions, 2-, 3-,
and 4-methoxyphenylboronic acid all proved high yielding as a
coupling partner (9a−c), as did 4-methylphenylboronic acid
(9d). In cases of electron-deficient aromatic rings such as 4-
trifluoromethyl (9e) and 4-nitrile (9f), yields proved lower at
room temperature (48% for 9e and 18% for 9f), although the
total recovery of 9f could be dramatically increased by running
the reaction at 70 °C under otherwise identical conditions
B
DOI: 10.1021/acs.orglett.7b02868
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
a
Scheme 1. Scope of the Suzuki Coupling with 7
Scheme 2. Scope of the Suzuki Coupling with 10
a
a
Standard reaction conditions: 7 (30 mg, 0.16 mmol), RB(OH)2 (1.2
Standard reaction conditions: 10 (30 mg, 0.16 mmol), RB(OH)2 (1.2
equiv), base (3.0 equiv), 5:1 1,4-dioxanes/H2O (1.0 mL). Isolated
yields after column chromatography.
equiv), base (3.0 equiv), 5:1 1,4-dioxanes/H2O (1.0 mL). Isolated
yields after column chromatography. Reaction run at 70 °C.
b
moderate to high yield. Thus, the optimized conditions
afforded broad scope and general applicability to aryl,
heteroaryl, and vinyl boronic acid coupling partners to deliver
analogs 9.
Scheme 3. Synthesis of AChE Inhibitor Analog 15
We next turned our attention to the replacement of the N-
butyl substitution (10) (Scheme 2) and synthesized N-benzyl-
6-chloro-1,2,4,5-tetrazin-3-amine (10a), 3-chloro-6-(1-piperid-
yl)-1,2,4,5-tetrazine (10b), 6-chloro-N-phenyl-1,2,4,5-tetrazin-
3-amine (10c), 4-(6-chloro-1,2,4,5-tetrazin-3-yl)morpholine
(10d), and 3-butoxy-6-chloro-1,2,4,5-tetrazine (10e) in con-
ditions similar to those for the synthesis of 7 (see Supporting
Information). These substrates were selected in order to probe
the electronic requirements of the group donating electron
density to the chloro-s-tetrazine core. Our conditions proved
successful for benzylamine 11a, piperidine 11b, aniline 11c, and
morpholine 11d, although no evidence of desired product
could be detected by LCMS in the case of butoxy-s-tetrazine
11e (Scheme 2). These results indicate that our Suzuki
conditions can tolerate a variety of different amino-s-tetrazines
while maintaining good yields, and optimization efforts toward
conditions for ether-containing tetrazine substrates are ongoing
in our laboratory.
There are few reported examples of tetrazine motifs in
biologically active molecules,22 and structure−activity relation-
ship (SAR) studies that include this unusual heterocycle have
historically been limited largely due to an inability to rapidly
and consistently generate unsymmetrical substituted s-tetra-
zines. We therefore became interested in utilizing our
optimized Suzuki conditions to generate a direct s-tetrazine
analog of an existing molecule (12) and were aware of a report
describing 3,6-substituted pyridazines as acetylcholinesterase
(AChE) inhibitors, structurally related to minaprine (Scheme
3).23 Using a simple, two-step SNAr/Suzuki-coupling sequence
starting from amine 13 (Scheme 3), we were able to generate a
direct s-tetrazine analog (15) of the existing molecule (12) in
high yield under mild conditions and to compare the human
hepatic microsomal intrinsic clearance (CLint) and plasma
protein binding (PPB) as the fraction unbound (fu) of each
(Table 2), conducted as previously described.24 The tetrazine
analog 15 displayed greater than 3-fold improvement (i.e.,
Table 2. Comparison of Human CLint, Predicted CLhep, PPB,
and cLogP for Analogs 12 and 15
CLINT (hum)
mL/min/kg
CLHEP (hum)
mL/min/kg
fu (plasma)
a
compd
(%)
cLogP
12
15
46
14
14
2.5
2.6
5.5
4.4
8.3
a
Calculated using ChemDraw Professional version 16.0.
C
DOI: 10.1021/acs.orglett.7b02868
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b02868.
(21) Pican, S.; Lapinte, V.; Pilard, J.; Pasquinet, E.; Beller, L.;
Fontaine, L.; Poullain, D. Synlett 2009, 2009, 731.
1
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L.; Degnan, M. J.; Whitney, B. J. Heterocycl. Chem. 1979, 16, 881.
(b) Xu, F.; Yang, Z.; Ke, Z.; Xi, L.; Yan, Q.; Yang, W.; Zhu, L.; Lv, W.;
Wu, H.; Wang, J.; Li, H. Bioorg. Med. Chem. Lett. 2016, 26, 4580.
(23) Contreras, J.; Rival, Y. M.; Chayer, S.; Bourguignon, J.;
Wermuth, C. G. J. Med. Chem. 1999, 42, 730.
(24) (a) Wenthur, C. J.; Morrison, R.; Felts, A. S.; Smith, K. A.;
Engers, J. L.; Byers, F. W.; Daniels, J. S.; Emmitte, K. A.; Conn, P. J.;
Lindsley, C. W. J. Med. Chem. 2013, 56, 5208. (b) Engers, D. W.;
Blobaum, A. L.; Gogliotti, R. D.; Cheung, Y.; Salovich, J. M.; Garcia-
Barrantes, P. M.; Daniels, J. S.; Morrison, R.; Jones, C. K.; Soars, M.
G.; Zhuo, X.; Hurley, J.; Macor, J. E.; Bronson, J. J.; Conn, P. J.;
Lindsley, C. W.; Niswender, C. M.; Hopkins, C. R. ACS Chem.
Neurosci. 2016, 7, 1192. (c) Bridges, T. M.; Rook, J. M.; Noetzel, M. J.;
Morrison, R. D.; Zhou, Y.; Gogliotti, R. D.; Vinson, P. N.; Xiang, Z.;
Jones, C. K.; Niswender, C. M.; Lindsley, C. W.; Stauffer, S. R.; Conn,
P. J.; Daniels, J. S. Drug Metab. Dispos. 2013, 41, 1703.
Experimental procedures, characterization data, and H
and 13C NMR spectra for new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: craig.lindsley@vanderbilt.edu.
ORCID
Craig W. Lindsley: 0000-0003-0168-1445
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.W.L. thanks the William K. Warren Family and Foundation
for funding the William K. Warren, Jr. Chair in Medicine and
support of our programs.
(25) Human AChE inhibition assay by Eurofins using the method
described by Ellman (Biochem. Pharmacol. 1961, 7, 88).
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DOI: 10.1021/acs.orglett.7b02868
Org. Lett. XXXX, XXX, XXX−XXX