Single-Step Formation of Pyrimido[4,5-d]pyridazines by a Pyrimidine-Tetrazine Tandem Reaction

Article abstract of DOI:10.1021/acs.orglett.6b01601

A straightforward synthesis of pyrimido[4,5-d]pyridazines from pyrimidines and tetrazines under basic conditions is reported. Deprotonated, substituted 5-halopyrimidines readily react with variously substituted tetrazines in a highly regioselective manner via a complex reaction pathway, which was supported by DFT calculations. This mechanism leads to the empirically observed regioisomers without going through the conceivable hetaryne intermediate. These results on 5-halopyrimidines led to development of the methodology for preparation of opposite regioisomers based on 6-halopyrimidines.

Full text of DOI:10.1021/acs.orglett.6b01601

Letter
pubs.acs.org/OrgLett
Single-Step Formation of Pyrimido[4,5‑d]pyridazines by a
Pyrimidine-Tetrazine Tandem Reaction
̌
Juraj Galeta, Michal Sala, Martin Dracínsky,* Milan Vrabel, Zdenek Havlas, and Radim Nencka*
́
̌
́
́
̌
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam
Czech Republic
́
. 2, 166 10 Prague,
S
* Supporting Information
ABSTRACT: A straightforward synthesis of pyrimido[4,5-
d]pyridazines from pyrimidines and tetrazines under basic
conditions is reported. Deprotonated, substituted 5-halopyr-
imidines readily react with variously substituted tetrazines in a
highly regioselective manner via a complex reaction pathway,
which was supported by DFT calculations. This mechanism
leads to the empirically observed regioisomers without going
through the conceivable hetaryne intermediate. These results on
5-halopyrimidines led to development of the methodology for
preparation of opposite regioisomers based on 6-halopyrimi-
dines.
eterocycles are nearly ubiquitous in modern drug design
as it is estimated that over 80% of all small-molecule
H
remedies marketed in the USA contain a heterocyclic core.1
Besides well-established procedures leading to heterocyclic
compounds, e.g., Gutknecht pyrazine synthesis,2 Pechmann
condensation leading to coumarins,3 Biginelli reaction,4 Knorr
quinoline synthesis,5 Bischler−Napieralski reaction,6 Traube
purine synthesis,7 and so forth8 that predominate in the
synthesis of heterocycles, the development of novel synthetic
strategies and improvements are of considerable interest. These
strategies are unfortunately limited, and therefore, a number of
interesting heterocyclic scaffolds remain inaccessible. This is
epitomized by the rapid recent developments in the field of
kinase inhibitors9 that demand novel synthetic protocols to
provide previously underexplored bicyclic and tricyclic hetero-
cycles.
Figure 1. Straightforward access to pyrimido[4,5-d]pyridazines via a
novel pyrimidine-tetrazine tandem reaction.
situ, could be trapped by a second suitable reacting partner in a
cycloaddition reaction to furnish the desired bicyclic product in
a single step. A robust and reliable methodology to generate the
pyrimidyne intermediate was to be the cornerstone of this
study as there are scant examples that describe the generation
of pyrimidyne and via only two processes.13 One is based on
dehydrohalogenation of the corresponding halopyrimidine in
the presence of KNH2 in liquid ammonia.13a−d The other
exploits an oxidation step promoted by Pb(OAc)4 of 1- or 3-
aminotriazolo[4,5-d]pyrimidine.13f,g Although these works
clearly show that such a hetaryne can be formed and trapped,
the highly laborious preparation of the starting aminotriazolo-
pyrimidines, the use of liquid ammonia, and low yield provide
substantial barriers to broader application. An alternative
approach of pyrimidyne generation from silyltriflate precursors
was recently found to be unsuccessful.14
We intended to use dehydrohalogenation as an alternative
method to generate synthetically useful pyrimidynes. We began
the study by using commercially available 5-bromopyrimidine 1
and two types of trapping agents: furan as an electron-rich
diene, a typical trapping agent in normal Diels−Alder reactions,
and 3,6-diphenyl-1,2,4,5-tetrazine 5 as an electron-poor diene
known15 to participate in inverse electron-demand Diels−Alder
(iEDDA) reactions. Moreover, we examined several bases to
In this study, we present the development of a novel
transformation that provides efficient access to bicyclic
heterocycles containing a pyrimidine ring in a straightforward
manner. Because the pyrimidine core is found in a myriad of
biologically active compounds10 including nucleobases and
many antiviral and anticancer drugs, the development of new
synthetic strategies is of special importance. In particular, we
show here that a class of medicinally highly relevant
pyrimido[4,5-d]pyridazines, known as antiinflammatory Jak-2
and Syk inhibitors11 or Wee-1 kinase inhibitors12 with
prospective application in anticancer therapy, can be formed
in an unprecedented reaction cascade directly from readily
accessible halopyrimidines and 1,2,4,5-tetrazines (Figure 1).
Furthermore, we provide a detailed mechanistic study and
support the data using DFT calculations.
We envisaged that expedient access to bicyclic derivatives
containing the pyrimidine core could be gained via a
precedented pyrimidyne intermediate, which when formed in
Received: June 2, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b01601
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Organic Letters
Letter
promote the initial dehydrohalogenation step that includes n-
BuLi, s-BuLi, t-BuLi, LDA, PhMgBr, i-PrMgCl·LiCl, KHMDS,
NaHMDS, LiHMDS, and NaNH2. Our initial experiments
showed that in the presence of KHMDS the reaction with furan
produces only dimeric products 2−4 in less than 15% overall
yield. All the other bases failed to give any isolatable products
and led only to decomposition of the starting material. To our
surprise, the reaction with tetrazine 5 under the same
conditions afforded the desired pyrimido[4,5-d]pyridazine 6a
in 50% yield as also confirmed by crystallographic analysis16
(Figure 2). Similar pyrimido[4,5-d]pyridazines were recently
Table 1. Reactions of Various Pyrimidines with Tetrazine 5
entry
R1
R2
X
yield (%)
product
a
1
2
MeSO2
CN
Cl
H
Br
Br
NR
a
H
NR
3
Cl
Br, Cl, F
decomp
50
4
H
H
Br
Br
Br
Br
F
6a
6b
6c
6d
6e
6e
6e
5
SMe
Cl
H
43
6
OMe
NMe2
SEt
SEt
SEt
46
7
Cl
33
b
8
Cl
44, 73
b
9
Cl
Cl
Br
34, 62
b
10
Cl
25, 34
a
b
NR = no reaction. Carried out at room temperature.
Table 2. Reactions of 5-Fluoropyrimidine with Various
Tetrazines
entry
R1
R2
yield (%)
product
1
2
Ph
Ph
73
61
55
53
64
50
49
49
50
51
6e
6f
i-Pr
i-Pr
i-Pr
Ph
Figure 2. Comparative reactions of pyrimidine 1 with furan and
tetrazine 5 in toluene. ORTEP representations of 6a are shown at 50%
probability level.
3
6g
6h
6i
4
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
Ph
NEt2
5
piperidine
morpholine
morpholine
morpholine
morpholine
morpholine
6
6j
7
6k
6l
prepared by Knorr condensation.17 Stimulated by these
unexpected results, we optimized the reaction conditions. We
found that the reaction strongly depended on the solvent
(Table S1, Supporting Information (SI)). Gentle heating
improved the yield, whereas increasing the amount of
KHMDS and/or varying the molar ratios of the reagents did
not have a significant effect.
We next performed a series of experiments to investigate the
scope of the reaction by varying the substitution pattern on the
pyrimidine scaffold. We found that only electron-rich or
-neutral pyrimidines (Table 1, entries 4−10) gave the desired
products. We noted the importance of the leaving halide on the
result (entries 8−10) and fluorine was found to be the superior
leaving group providing the best yield (entry 8). It is
noteworthy that this fluoro derivative afforded much lower
yields with weaker bases NaHMDS (18%) and LiHMDS
(16%).
With the optimized 5-fluoropyrimidine in hand, we focused
on analyzing the scope of the second reacting partner by
synthesizing a series of tetrazines bearing various substituents
(Table 2). The reaction of symmetrical tetrazines proceeded
smoothly to furnish the expected pyrimido[4,5-d]pyridazine
products in good yields (Table 2, entries 1 and 2). Most
importantly, the reaction with unsymmetrical tetrazines (Table
2, entries 3−10) yielded in all cases the desired products as
single regioisomers. To irrefutably determine the structure of
the regioisomers, we substituted the SEt group with a hydrogen
by Pd-catalyzed desulfurization (SI). Using ROESY-NMR
spectra, we observed the expected NOE interaction between
8
4-CN-Ph
2-thiophenyl
2-furyl
9
6m
6n
10
the H atom at C4 and the ortho phenolic hydrogens,
confirming the structure of the regioisomers.
In the course of the synthetic work, we started to have
doubts about the hetaryne mechanism because the structure of
some of the side products (SI) suggested that hetarynes, if
formed at all, are not the only fate of the 5-halopyrimidines in
basic environment. Therefore, we had recourse to use DFT
calculations to find support for the hetaryne mechanism or to
propose an alternative reaction pathway that would be
compatible with the experimental observations. The heat of
hydrogenation is a well-established means to explore the strains
and relative stabilities of alkynes, and it has been used recently
to predict the likelihood that a given hetaryne can be generated.
Our pyrimidyne derivatives have an energy of dehydrogenation
of ∼100 kcal/mol (SI), which is close to that of all
experimentally confirmed hetarynes.18 The second step in the
supposed hetaryne mechanism is the formation of a product
with a new cycle by iEDDA. The transition structure for the
process was found, and the calculated reaction barriers were
close to 20 kcal/mol. So far, the DFT results seemed to support
the hetaryne mechanism, but then we turned our attention to
the regioselectivity of the cyclization reaction and found the
opposite regioselectivity in the simulations then observed
experimentally. For example, the optimized transition state
structures for the reaction leading to experimentally observed
B
DOI: 10.1021/acs.orglett.6b01601
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Figure 3. Simplified mechanism proposed for the reaction of 5-fluoropyrimidines with tetrazines.
compound 6j had higher energy by 4 kcal/mol than that
leading to the opposite regioisomer.
Table 3. Reactions of 6-Chloropyrimidine with Various
Tetrazines
Because of the discrepancies described above, we abandoned
the hetaryne mechanism as unreliable and proposed an
alternative mechanism based on reaction of deprotonated 5-
halopyrimidines with the tetrazines. A one-dimensional DFT
energy scan revealed that reaction of deprotonated pyrimidine
with tetrazine is a downhill reaction and thus preferred over
dehalogenation. Additionally, we investigated the mechanism
by HPLC-MS measurements of aliquots from the reaction
mixture and by NMR analysis of the reaction mixture at various
time points (SI), and the experimental observations were
complemented with DFT calculations. An unusual spiro
compound, the structure of which was determined by NMR
spectroscopy, was observed as an intermediate of the reaction
with aryl/amino-substituted tetrazines (e.g., Table 2, entry 6),
and the conversion of this intermediate to the final product
required the addition of water. On the other hand, the reaction
with diphenyl tetrazine (Table 2, entry 1) proceeded smoothly
in the anhydrous environment. DFT calculations fully
confirmed the experimental observations. A simplified reaction
pathway is depicted in Figure 3, and full details are in the SI.
The reaction begins by nucleophilic attack of the deprotonated
pyrimidine on the tetrazine, followed by intramolecular
cyclization and nitrogen loss to form the spiro intermediate.
In the case of the diphenyl tetrazine derivative, the following
rearrangement of the spiro compound anion is a low-barrier
process, whereas the barrier is significantly higher for the aryl/
amino derivative. However, this rearrangement barrier is
reduced after protonation (neutralization) of the intermediate.
Encouraged by these findings, we speculated that the
opposite regioisomers might be accessible from the correspond-
ing 6-halo-substituted pyrimidines. To explore this possibility,
we reacted the same series of tetrazines with 2,4-dichloro-6-
(ethylthio)pyrimidine. Indeed, in all cases except isopropyl-
phenyl tetrazine, we observed the formation of the opposite
regioisomers in good yields (Table 3). These experiments
clearly show that it is possible to control the regioselectivity of
entry
R1
R2
yield (%)
product
a
1
2
3
4
5
6
7
8
9
Ph
Ph
22, 58
6e
6f
a
i-Pr
Ph
i-Pr
i-Pr
16, 57
56
b
6g
6o
6p
6q
6r
6s
6t
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
Ph
NEt2
piperidine
morpholine
morpholine
morpholine
morpholine
morpholine
56
58
42
39
46
40
4-CN-Ph
2-thiophenyl
2-furyl
10
6u
a
b
Carried out at 50 °C. Only traces observed.
the reaction by simply switching the position of the leaving
halogen atom. The ultimate observation of both regioisomers
also unambiguously discarded the hetaryne mechanism, where
only one regioisomer should be obtained regardless of the
position of the halogen atom on the starting halopyrimidine
(Figure 4).
In the case of 6-chloropyrimidines, we propose a simpler
reaction mechanism based on these steps (details in SI):
deprotonation of the pyrimidine, spontaneous downhill
reaction of the anion with tetrazine (this is the step that
governs the regioselectivity of the reaction), and cyclization of
the intermediate with simultaneous loss of the chloride anion
and nitrogen molecule. The transition state for this cyclization
with a modest energy barrier of 10 kcal/mol was found, and the
observation of single regioisomer 6g formed from both 5-
fluoro- and 6-chloropyrimidine was also supported by DFT
calculations (details in SI). Note that an analogous transition
C
DOI: 10.1021/acs.orglett.6b01601
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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.6b01601.
Experimental and computational methods, spectral data,
and supplemental data and figures (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
1968, 9, 9−13. (e) Kauffmann, T.; Nurnberg, R.; Udluft, K. Chem. Ber.
̈
*E-mail: dracinsky@uochb.cas.cz.
*E-mail: nencka@uochb.cas.cz.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was financially supported by the Czech Science
Foundation (Grants 15-09310S, 15-11223S, 15-06020Y, and
P208/12/G016) and Gilead Sciences, Inc. (Foster City, CA,
̌ ́
USA). We are grateful to Veronika Holeckova for the
(16) The crystal structure of compound 6a has been deposited at the
Cambridge Crystallographic Data Centre and allocated the deposition
number CCDC 1406413.
preparation of starting materials and to Michal Babiak for X-
ray diffraction studies with the support of the X-ray Diffraction
and Bio-SAXS Core Facility of CEITEC, Brno, Czech Republic.
We are grateful to A. Michael Downey, IOCB, Prague, for
critical reading of our manuscript. Subvention for the
development of research organization (RVO: 61388963) is
also acknowledged.
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D
DOI: 10.1021/acs.orglett.6b01601
Org. Lett. XXXX, XXX, XXX−XXX