Spin-spin coupling constants 13C-15N and 1H-15N in the investigation of azido-tetrazole tautomerism in a series of 2-azidopyrimidines

Article abstract of DOI:10.1007/s11172-013-0072-7

A new method was developed for the investigation of an azido-tetrazole equilibrium based on using a complex analysis of ¹³C-¹⁵N and ¹H-¹⁵N spin-spin coupling constants. The use of this approach became possible due to the selective inclusion of ¹⁵N isotopes into the structures of 2-azidopyrimidines and their cyclic analogs tetrazolo[1,5-a]pyrimidines.

Full text of DOI:10.1007/s11172-013-0072-7

Russian Chemical Bulletin, International Edition, Vol. 62, No. 2, pp. 521—528, February, 2013
521
Spinꢀspin coupling constants 13С—15N and 1H—15N in the investigation
of azidoꢀtetrazole tautomerism in a series of 2ꢀazidopyrimidines*
I. A. Khalymbadzha,a T. S. Shestakova,a S. L. Deev,a V. L. Rusinov,a O. N. Chupakhin,a,b
Z. O. Shenkarev,c and A. S. Arsenievc
aUral Federal University,
19 ul. Mira, 620002 Yekaterinburg, Russian Federation.
Fax: +7 (343) 374 0458. Eꢀmail: deevsl@yandex.ru
bI. Ya. Postovsky Institute of Organic Synthesis, Russian Academy of Sciences,
22 ul. S. Kovalevskoi, 620041 Yekaterinburg, Russian Federation.
Fax: +7 (343) 374 1189. Еꢀmail: chupakhin@ios.uran.ru
cM. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 ul. MiklukhoꢀMaklaya, 117997 Moscow, Russian Federation
A new method was developed for the investigation of an azidoꢀtetrazole equilibrium based
on using a complex analysis of 13С—15N and 1H—15N spinꢀspin coupling constants. The use of
this approach became possible due to the selective inclusion of 15N isotopes into the structures
of 2ꢀazidopyrimidines and their cyclic analogs tetrazolo[1,5ꢀa]pyrimidines.
Key words: azidoꢀtetrazole tautomerism, tetrazolo[1,5ꢀa]pyrimidine, spinꢀspin coupling
constants, NMR spectroscopy, 15N isotope.
Practical interest in synthesis of hetaryl azides is due to
their ability to participate in photochemical transformaꢀ
tions, which allows one to use their derivatives in biologiꢀ
cal assays. For example, the 2ꢀazidopurine derivatives are
applied as photoaffine labels1,2 for the determination of
binding sites of nucleotides with proteins and establishꢀ
ment of the role of these interactions.
The modification of nucleosides due to the insertion of
the azido group into heterocyclic bases is known.3—5 This
approach is promising, because the azido group is capable
of transforming into the amino group, and is used in synꢀ
thetic organic chemistry as well. For example, the syntheꢀ
sis of the hetarylamino derivatives of azines from the corꢀ
responding azidoazines was described.6,7
Azaaromatic compounds with the azido group in the
ꢀposition to the nitrogen atom often demonstrate azidoꢀ
tetrazole tautomerism, which is widely described in the
literature.8—12 This isomerism is characterized by the abilꢀ
ity of the azido group in the heterocyclic derivatives to
transform under the action of external factors or spontaꢀ
neously and reversibly into the corresponding tetrazoles.
Azidoꢀtetrazole tautomerism makes it possible to consider
tetrazoloazines as cyclic analogs of hetaryl azides.13,14 The
studies in the area of azidoꢀtetrazole equilibrium are necꢀ
essary for controlling the reactivity of these heterocyclic
systems classified as ambidentate polyfunctional comꢀ
pounds. So, depending on the conditions, either the tetrꢀ
azole cycle, or isomeric azido group, or the azine fragment
can undergo chemical transformation. It is important that
the direction of the reaction often depends on the form in
which the compound reacts. One of the examples of the
practical successful use of the azidoꢀtetrazole equilibrium
is the hydrogenation of tetrazolo[1,5ꢀa]pyridine.15 Under
acidic conditions where the tetrazole form predominates,
only the pyridine ring is hydrogenated, whereas the hydroꢀ
genation under basic conditions stabilizing azide affords
2ꢀaminopyridine. One of the successful examples for
the directed reduction of the tetrazole cycle is the syntheꢀ
sis of the modern antiepileptic drug lamotrigine and its
analogs.16
It is also important to know parameters of the azidoꢀ
tetrazole equilibrium for the use of photoaffine labels in
the field of molecular biology. This is related to the fact
that the azide form is photochemically more active than
the tetrazole form.17 Therefore, the maximum concentraꢀ
tion of the azide form is necessary in experiments for the
efficient application of the photoaffine label.
There is a class of hetaryl azides, whose cyclization
can give two cyclic forms. For example, 2ꢀazidopyrimꢀ
idines A (Х = CR3, R1  R3) containing various subsꢀ
tituents in positions 4 and 6 of the azine ring can transꢀ
form into tetrazole isomers Т (Х = CR3, R1  R3) and T´
(Х = CR3, R1  R3) (Scheme 1). In the most part of cases,
* Dedicated to the Academician of the Russian Academy of
Sciences S. M. Aldoshin on the occasion of his 60th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 0519—0526, February, 2013.
1066ꢀ5285/13/6202ꢀ0521 © 2013 Springer Science+Business Media, Inc.

522
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
Khalymbadzha et al.
Xꢀray diffraction analysis is used for the unambiguous deꢀ
termination of the route of azido group cyclization and
establishment of the structure of tetrazolo[1,5ꢀa]pyrꢀ
imidines T (Х = CR3, R1  R3) and T´ (Х = CR3, R1  R3).
However, this method is not suitable for studying the dyꢀ
namics of the transition of one isomeric form to another
in solutions. On the contrary, 1H and 13С NMR spectroꢀ
scopy allows one to directly observe azidoꢀtetrazole rearꢀ
rangements in a series of 2ꢀazidopyrimidines, but often
does not allow the structure of the formed isomers to be
determined unambiguously because of the low content of
hydrogen and carbon atoms in the structures of heteroꢀ
cycles of this class.
natural abundance of the 15N isotope,19 is not observed.
The selective introduction of the 15N label significantly
facilitates the measurement of longꢀrange 1H—15N SSCCs
4
(for example, JN,H through four covalent bonds), subꢀ
stantially decreases requirements for the amount of the
substance necessary for analysis, and makes it possible to
apply JN,H for the determination of structures of more
complicated organic molecules.
In this work, a complex approach based on the comꢀ
bined use of 13С—15N and 1H—15N SSCCs was proposed
with the purpose for searching for the most efficient
methods of investigation of the azidoꢀtetrazole equilibriꢀ
um for 15Nꢀlabeled 2ꢀazidopyrimidines as examples.
One of the methods for inclusion of the 15N isotope
into the structure of 2ꢀazidopyrimidines is based on the
interaction of 2ꢀhydrazinopyrimidines with labeled nitrous
acid generated from К15NO2 in an acidic medium.18
Hetaryl azide 2*A (enrichment in 15N is 86%) containing
the isotope label in the ꢀposition of the azido group was
obtained by the same method from compound 1 (Scheme 2).
Scheme 1
Scheme 2
X = CR3, N; N = 14N, 15
N
nJC,N (N = 15N, X = N)
Other examples for similar compounds are 3ꢀazidoꢀ
1,2,4ꢀtriazine derivatives A (X = N), which are capable of
cyclizing to form two cyclic isomers of tetrazolo[5,1ꢀc]ꢀ
[1,2,4]triazines T´ (X = N) and tetrazolo[1,5ꢀb][1,2,4]ꢀ
triazines T (X = N) (see Scheme 1). It has previously18
been shown that the selective introduction of the 15N isoꢀ
tope in the ꢀposition of the azido group (N = 15N) of
open form A (X = N) makes it possible to detect open and
cyclic forms and unambiguously establish the structures of
tetrazoloꢀ1,2,4ꢀtriazines from an analysis of chemical
shifts of signals in the 1D 15N NMR spectra and spinꢀspin
coupling constants (SSCCs) 13С—15N (JC,N). For examꢀ
ple, the formation of tetrazolo[1,5ꢀb][1,2,4]triazines T
(N = 15N, X = N) was confirmed by two 13С—15N conꢀ
stants in the 1D 13С NMR spectra. In the case of cyclizaꢀ
tion of compound A (N = 15N, X = N) to tetrazolo[5,1ꢀc]ꢀ
[1,2,4]triazines T´, nJC,N should be detected for all carbon
atoms of the azine fragment (see Scheme 1).
nJC,N
nJN,H
An alternative approach to the synthesis of 2ꢀazidoꢀ
azines is based on ring opening in the corresponding tetrꢀ
azole derivatives. In this case, to introduce the 15N atom
into the azide group/tetrazole fragment, it is necessary to
use labeled 5ꢀaminotetrazole, which builds up the azine
fragment.16,18 The 15N isotope can be included into posiꢀ
tion 2 of 5ꢀaminotetrazole by the treatment of aminoguaniꢀ
dine with nitrous acid or by the interaction of 15Nꢀaminoꢀ
guanidine 3* with HNO2.18 The combination of these two
In this approach, the potential of the 1H—15N SSCCs
(JN,H), which can be observed even in samples with the

Azidoꢀtetrazole tautomerism
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
523
approaches allowed one to synthesize [15N2]ꢀ5ꢀaminotetꢀ
razole 4** containing simultaneously two isotope labels in
the heterocycle. The further condensation of compound
4** with benzoylacetone 5 afforded tetrazolo[1,5ꢀa]pyrꢀ
imidine 6**T including the 15N isotope in the ꢀ and
ꢀpositions of the azido group (86% enrichment in each
15N atom, Scheme 3). It should be mentioned that earlier
[2ꢀ15N]ꢀ5ꢀaminotetrazole containing one isotope label was
applied to the preparation of unsubstituted 15Nꢀtetrazoloꢀ
[1,5ꢀa]pyrimidines and 15Nꢀtetrazolo[1,5ꢀb][1,2,4]triꢀ
azines.18,20 However, in this case, a mixture of 15N isotopoꢀ
mers was formed at the ꢀ and ꢀpositions of the azido
group. The use of compound 4** makes it possible to
obtain samples of 15Nꢀtetrazolo[1,5ꢀa]pyrimidines with
the homogeneous isotope composition.
(Table 1), which were detected in the range characteristic
of tetrazoloazines.18,21,22 An analysis of the integral intenꢀ
sities of signals in the 1D 1H and 15N NMR spectra made
it possible to monitor the mutual transformation of two
isomeric forms of compound 2*. Immediately after the
dissolution of the production of the reaction of hetarylꢀ
hydrazine 1 with labeled nitrous acid, the ratio of isomers
2*T and 2*T´ was 92 : 8 (Table 2). Further the concentraꢀ
tion of compound 2*T´ increased rapidly and changed to
49 : 51 already after incubation at 30 С within 24 h
(Table 2).
The 2D 1H—15N HMBC spectrum of a mixture of the
tetrazole isomers for compounds 2*T and 2*T´ contained
crossꢀpeaks between the labeled nitrogen atom (15N) and
protons of the azine fragment, which directly indicated
the longꢀrange 1H—15N spinꢀspin interactions between
the corresponding nuclei. However, a considerable broadꢀ
ening of the 1H NMR signals of compound 2*T probably
caused by chemical exchange processes did not allow us to
analyze the value of observed JN,H and conclude about the
structure of the tetrazole isomers formed by the cyclizaꢀ
tion of azide 2*A. Amplitude modulated 1D 1H spinꢀecho
experiments with selective inversion of the 15N nuclei were
used for the quantitative measurement of the observed
1H—15N SSCCs. The measured values of longꢀrange JN,H
(see Table 1) allowed us to establish the structures of comꢀ
pounds 2*T and 2*T´ (see Scheme 2). For example, the
1H—15N spinꢀspin interaction at the H(5) proton and the
protons of the methyl group at the С(6´) atom prove that
Pyrimidine derivatives 2*A and 6**T were studied by
the NMR method in DMSO and TFA solutions, which
make it possible to shift the azidoꢀtetrazole equilibrium
towards the tetrazole isomer or azide form, respectively.
In addition, the ringꢀchain tautomerism of sample 2*A
was additionally studied in various mixtures of DMSO
1
and TFA. The signals from the H and 13C nuclei were
assigned using 2D 1H—13С HMBC and HMQC NMR
experiments.
An analysis of the NMR spectra of sample 2*A in
a DMSO solution (Fig. 1, a) showed that the azide form
undergoes spontaneous cyclization to form simultaneousꢀ
ly two cyclic isomers 2*T´ and 2*T. This conclusion was
drawn on the basis of chemical shifts of the 15N nuclei
Scheme 3
nJC,N
nJN,H
Note. For compounds 6**T, 6**A, and 6**T´, nJC,N and nJN,H are shown for the 15N. atom only.

524
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
Khalymbadzha et al.
a
2*T´ N
2*T N
(15N)
2*A N
2*T N
b
c
2*T´ N
(15N)
2*A N
2*T N
–20
–40
–60
–80
–100
–120
–140
(15N)
d
6**T N
1JN,N = 16.8
6**T N
1JN,N = 16.8
30
20
10
0
–10
–20
–30
–40
(15N)
e
6**A N
1JN,N = 5.7
6**A N
1JN,N = 5.7
–130
–135
–140
–145
–150
–155
(15N)
Fig. 1. 1D 15N NMR spectra of compounds 2* 24 h after dissolution in DMSO (a), in a DMSO—TFА (2 : 3) mixture (b), and in
TFA (c) and of compounds 6** in DMSO (d) and ТFA (e).
the cyclization of compound 2*A to tetrazole isomer 2*T
involves the N(1) atom of the pyrimidine ring. In the case
of structure 2*T´, the annelation mode for the tetrazole
ring is confirmed by the observation of the single JN,H
between the H(5) proton and N nitrogen atom.
The conclusion about the structures of compounds 2*T
and 2*T´ is well consistent with the experimental data on
the analysis of the 13С—15N coupling constants. Thus, in
the 1D 13С NMR spectrum of a mixture of tetrazoloꢀ
[1,5ꢀa]pyrimidines for the С(2), С(4), С(5), and С(6) sigꢀ
nals of compound 2*T´ exhibits spinꢀspin interactions with
the 15N nucleus (Table 3, see Scheme 2). The interaction
between the 13С(4) and 15N atoms (2JC,N = 2.1 Hz) proves
that the N(3) atom participates in tetrazole isomer formaꢀ
tion. In the case of isomer 2*T, JC,N was observed only for
the signals of two С(6) and С(6´) atoms (see Table 3). The
expected constants at С(2) and С(5) were not detected
because of a considerable broadening of the corresponding
13C NMR signals, which is induced by the dynamic transꢀ
formation of the cyclic forms into each other and a possiꢀ
ble influence of prototropic tautomerism for compounds
2*T and 2*A. However, the presence of the spinꢀspin inꢀ
teraction between the carbon atom of the methyl group
13С(6´) and the 15N atom (3JC,N = 0.8 Hz) unambiꢀ
guously confirms the structure of cyclic isomer 2*T (see
Scheme 2).
4

Azidoꢀtetrazole tautomerism
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
525
Table 1. Chemical shifts of the 1H and 15N nuclei and the JH,Н, JN,H, and JN,N SSCC for compounds 2* and 6**
Comꢀ
pound
Solvent
1H (, J/Hz)a
15N (, J/Hz)b
N/N Other signals
H(5)
Me
Other signals
—
2*T´c
DMSO
5.94
(4JN,H = 0.6)
5.81
2.38
(4JН(5),H(6´) = 0.8)
2.33
–33.8 (N)
–259.0 (N(1));
–136.9 (N(3))
DMSO—TFA (3 : 1)
DMSO—TFA (2 : 3)
—
—
–33.8 (N)
–34.5 (N)
—
—
5.67
2.22
(4JN,H = 0.6)
6.30d
2*T
DMSO
2.62
(4JN,H = 0.2,
4JН(5),H(6´) = 1.3)
2.56
–34.7 (N)
–155.5 (N(1))
(4JN,H = 0.6)
DMSO—TFA (3 : 1)
DMSO—TFA (2 : 3)
6.17
6.05
—
—
–34.8 (N)
–34.2 (N)
—
—
2.47
(4JN,H = 0.6)
6.22
(4JN,H = 0.2)
2.51
TFA
—
—
–32.9 (N)
–154.9 (N(1))
(4JN,H = 0.7)
6.34
(4JN,H = 0.2)
2.24
2*Ae
6**T
DMSO—TFA (2 : 3)
TFA
–134.4 (N)
–131.0 (N)
—
6.33
2.24f
–228.6 (N(1));
–159.6 (N(3))
–140.8 (N(1));
–126.7 (N(3))
DMSO
8.15
2.99
8.34 (H(8));
7.65 (H(9));
–35.6 (N),
22.0 (N)
(4JN ,H = 0.82,
(4JN ,H = 0.20,


5JN ,H = 0.09)
5JN ,H = 0.08,
7.66 (H(10)) (1JN ,N = 16.8)



4JН(5),H(6´) = 1.0)
6**A
TFA
7.70
2.65f
8.15 (H(8));
7.48 (H(9));
7.62 (H(10))
–131.3 (N),
–151.9 (N)
–214.4 (N(1));
–136.6 (N(3))
(1JN ,N = 5.7)

1
a The H—15N SSCCs (JN,H) were measured using 1D experiments including amplitudeꢀmodulated spinꢀecho with 15N nuclear
selective inversion. The total time delay in the spinꢀecho experiments (the time of magnetization evolution for JN,H) was 0.6 and
1.2 s for compounds 2* and 6**, respectively. The experimental error in the measured values of JN,H does not exceed 0.05 and
0.03 Hz, respectively.
b The chemical shifts of the 15N and 15N signals were measured in the 1D 15N NMR spectra. Other signals were assigned at the
natural content of the 15N isotope using the 2D 1H—15N HMBC spectra.
c No signals of compound 2*T´ in TFA were observed.
d The signal is broadened.
e No signals of compound 2*A in DMSO and DMSO—TFA (3 : 1) were observed.
f The 4JН(5),H(6´) value was not measured because of a large halfꢀwidth of the corresponding signals in the spectrum.
In spite of the fact that isomerization of tetrazoles 2*T
and 2*T´ to each other proceeds through structure 2*A,
no azido form was observed in a DMSO solution. Thus, it
can be concluded that, under these conditions, the conꢀ
centration of isomer 2*A does not exceed 0.1%. For the
stabilization of azide 2*A, deuterated trifluoroacetic acid
was added to a solution of a mixture of compounds 2*T
and 2*T´. The DMSO : TFA volume ratio was varied from
9 : 1 to 2 : 3. The highest concentration of open form 2*A
(29%) was observed for the highest content of TFA in
a solution (see Table 2). The change in the parameters of
chemical exchange between diverse isomeric forms of comꢀ
pound 2* induced by acid addition resulted in a significant
broadening of the halfꢀwidth of the 13С(2) and 13С(5)
signals of compound 2*T´ and made it possible to meaꢀ
sure JC,N for these nuclei (see Table 3). It is noteworthy
that the chemical shifts of the 13C NMR signals of comꢀ
pounds 2*T and 2*T´ changed insignificantly upon the
addition of TFA to DMSO, which facilitated the assignꢀ
ment of the signals of these compounds at different TFA
concentrations.
An analysis of the NMR spectra of sample 2*A in a
TFA solution revealed that, under these conditions, azide
form 2*А is predominant and only 2*T, whose concentraꢀ
tion is 4%, of the tetrazole isomers exists in the solution
(see Table 2). The structure of compound 2*A was conꢀ
firmed by the observation of the spinꢀspin interaction beꢀ
tween the С(2) carbon and N nitrogen nuclei (3JC,N
=
= 0.6 Hz). In addition, the signal from the 15N atom (see
Table 1) was observed in the region characteristic of azidoꢀ
azines.18,21,22 It was impossible to detect and measure the
13С—15N coupling constants for compound 2*T in TFA
because of the low concentration of this isomeric form.
However, the amplitudeꢀmodulated spinꢀecho experiꢀ

526
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
Khalymbadzha et al.
Table 2. Azidoꢀtetrazole equilibrium of compounds 2* and 6**
nuclear selective inversion made it possible to observe the
longꢀrange JN,H between the protons of the C(6´)H3 meꢀ
thyl group and two labeled nitrogen atoms (4JN ,H

Comꢀ
pound
Solvent
Ratio of isomers

5
T : T´ : A
 0.20 Hz, JN ,H  0.08 Hz, see Table 1). The obtained

results show that the condensation of 15N2ꢀaminotetrazole
2*
DMSOa
DMSOс
192 : 8 : 0b
149 : 51 : 0d
150 : 50 : 0
149 : 51 : 0
149 : 51 : 0
151 : 47 : 2
147 : 24 : 29
1v4 : 0 : 96e
4** with benzoylacetone 5 gives tetrazolopyrimidine 6**T,
6
6
since spin—spin interactions JN ,H(6´) and JN ,H(6´)
DMSO—TFA (9 : 1)
DMSO—TFA (3 : 1)
DMSO—TFA (3 : 2)
DMSO—TFA (1 : 1)
DMSO—TFA (2 : 3)
TFA


should be absent in the case of formation of alternative
structure 6**T´ (see Scheme 3). This result also indicates
the efficiency of an analysis of 4JN,H and 5JN,H as a method
of determination of azole cycle annelation to the azine
fragment in tetrazolo[1,5ꢀa]pyrimidines.
The quantitative measurement of the 13С—15N SSCCs
in a DMSO solution for compound 6**T confirmed the
conclusions about the structure of this substance obtained
6**
DMSO
TFA
100 : 0 : 0
110 : 0 : 100
a The ratio of isomers was determined immediately after dissoluꢀ
tion.
1
by an analysis of the H—15N constants. Thus, 13С—15N
b Only one isomer 2*T has been observed earlier23 immediately
after the dissolution of the compound in DMSO.
c The ratio of isomers 1 day after dissolution.
d The ratio of isomers 2*Т : 2*Т´ equal to 52 : 48 has earlier23
been observed after incubation at 100 C in a DMSO solution
within 5 h.
spin—spin interaction was observed for the С(2), С(5),
С(6), and С(6´) atoms (see Scheme 3, Table 3). In the
case of formation of alternative structure 6**T´, the
13С—15N coupling constants should be detected for the
С(4) and С(7) signals, and the 13С—15N constants at the
С(6´) atom should be absent.
e Isomer 2*T was not earlier detected in TFA.23
The chemical shifts of the 15N and 15N signals
( –131.3 and –151.9, respectively) in a TFA solution
indicates the complete rearrangement of tetrazole isomer
6**T to open form 6**A (see Fig. 1, e, Table 1). Signal
assignment in the 1D 15N NMR spectrum of compound
6**A was performed on the basis of the earlier published
data.18,21,22 The absence of the 1H—15N SSCCs in the 1D
1H NMR spectrum and the presence of the single 13С—15N
constant in the 13С NMR spectrum unambiguously conꢀ
firms the structure of azide 6**A.
ments revealed the spinꢀspin interaction with the 15N
nucleus at the H(5) proton and the protons of the methyl
group at the С(6´) atom, which proved the structure of
tetrazolo[1,5ꢀa]pyrimidine 2*T. It should be emphasized
that the amplitudes of the longꢀrange 1Н—15N SSCCs
measured for compound 2*T in DMSO and TFA soluꢀ
tions coincide within the experimental error (see Table 1).
It is also important that the results on studying the azidoꢀ
tetrazole equilibrium of compound 2A using an analysis of
the 1Н—15N and 13С—15N SSCCs supplement the earlier
published data23 (see Table 2), and the experiments in
DMSO—TFA mixtures made it possible to reliably detect in
solutions already three tautomeric forms: 2T´, 2T, and 2А.
The obtained sample of compound 6**T was studied
by the NMR method in DMSO and TFA solutions. The
1D 15N NMR spectrum of compound 6**T recorded in
DMSO exhibits two doublets with the SSCC JN,N  16.8 Hz,
which was confirmed by the introduction of two isotope
15N labels in the N and N positions of the tetrazole
fragment (see Fig. 1, d, Table 1). In this case, the signals
from the 15N ( –35.6) and 15N ( 22.0) nuclei were
detected in the region characteristic of condensed tetrazaꢀ
loazines.18,21,22 In addition, the 2D 1H—15N HMBC specꢀ
trum of compound 6**T contained crossꢀpeaks between
the labeled nitrogen atoms and the protons of the azine
fragment caused by the longꢀrange 1H—15N coupling conꢀ
stants, which directly indicated the cyclic structure of the
compound.
Thus, we showed the efficiency of the complex analyꢀ
sis of the 13С—15N and 1Н—15N coupling constants in
studying the azidoꢀtetrazole tautomerism in selectively
15Nꢀlabeled azidoazines for examples of pyrimidines 2
and 6. It was demonstrated that the measurement and
analysis of JC,N makes it possible to unambiguously estabꢀ
lish the structure of the azide and tetrazole forms in soluꢀ
tion, although requires significant concentrations of the
studied substances. At the same time, the use of JN,H proꢀ
vides information on the structure of weakly populated
(minor) isomeric forms. For instance, an analysis of the
1Н—15N spinꢀspin interaction confirmed the structure of
isomer 2*Т, whose population in a TFA solution is ~4%.
1
The high sensitivity of H NMR spectroscopy compared
to that of 13C NMR spectroscopy at the natural abunꢀ
dance makes it possible to reliably measure the heteroꢀ
1
nuclear Н—15N SSCCs with a significantly lower amꢀ
plitude (see Tables 1 and 3) and within a considerably
shorter time that it is required for measuring the 13С—15N
SSCCs (minutes and hours, respectively). Summatꢀ
ing the aforesaid, we may conclude that the combined
The exact structure of compound 6**T in a DMSO
1
1
solution was determined by an analysis of the Н—15N
application of the 13С—15N and Н—15N SSCCs in inꢀ
SSCCs. The use of the spinꢀecho experiments with 15N
vestigation of isomerization of azidoazines to tetrazoloꢀ

Azidoꢀtetrazole tautomerism
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
527
Table 3. Chemical shifts of the 13С nuclei and the JC,N SSCC of compounds 2* and 6**
Comꢀ
pound
Solvent
13C (, J/Hz)a
С(2)
С(4)
С(5)
С(6)
Other signals
19.27 (Me)
2*T´b
DMSO
151.53
(2JC,N = 2.4)
151.31
(2JC,N = 2.4)
150.59
154.38
(2JC,N = 2.1)
154.24
(2JC,N = 2.1)
154.47
98.66
(3JC,N = 0.8)
98.39
(3JC,N = 0.8)
98.32
154.96
(4JC,N = 0.4)
154.54c
(4JC,N = 0.4)
154.70c
145.31c
(2JC,N = 3.4)
145.06
(2JC,N = 3.4)
146.15
DMSO—TFA (3 : 1)
18.65 (Me)
17.90 (Me)
DMSO—TFA (2 : 3)
DMSO
2*T
150.98c,d
161.27c,d
160.99c
161.58
109.10c,d
16.33 (Me)
(3JC,N = 0.8)
15.77 (Me)
(3JC,N = 0.8)
14.99 (Me)
14.35 (Me)
DMSO—TFA (3 : 1)
150.78
(2JC,N = 2.7)
149.64
108.91
(3JC,N = 0.7)
108.15
DMSO—TFA (2 : 3)
TFA
e
e
—
—
108.34
148.31
2*Af
DMSO—TFA (2 : 3)
TFA
157.26
157.61
(3JC,N = 0.6)g
173.55
173.10
103.81
102.86
160.73
160.84
17.54 (Me)
16.91 (Me)
6**T
DMSO
155.50
164.38
172.64
110.02
147.16
17.24 (Me)
(3JC,N = 0.7,
4JC,N = 0.2)
135.80 (C(7));
128.59 (C(8));
129.74 (C9));
132.68 (C(10))
(2JC,N = 2.6,
2JC,N = 2.5)
(3JC,N = 0.6,
4JC,N = 0.5)
(2JC,N = 3.1)
6**A
TFA
156.42
111.89
162.46
18.09 (Me);
131.36 (C(7));
128.53 (C(8));
129.23 (C(9));
135.44 (C(10))
(JCN = 0.6,g
J
CN = 0.7)g
a The 13C—15N(7) SSCCs (JC,N) were measured using the nonlinear approximation of the line shapes in the 1D 13C NMR
spectra recorded with 15N selective decoupling and without it. The experimental error in measured JC,N values does not
exceed 0.15 Hz.
b No signals of compound 2*T´ in TFA were observed.
c The signal is broadened.
d The signal was assigned upon the addition of TFA.
e The signal was not observed because of the low concentration of the corresponding isomer.
f No signals of compound 2*A were observed in DMSO and DMSO—TFA (3 : 1).
g The JC,N values were measured using 1D 13C NMR experiments including amplitudeꢀmodulated spinꢀecho with 15N
nuclear selective inversion. The total time delay in spinꢀecho experiments (the time of magnetization evolution for JC,N) was
0.4 s. The experimental error in the measured JC,N values does not exceed 0.15 Hz.
were measured at 30 C, the NMR spectra of compound 6** in
DMSOꢀd6 were recorded at 42 C, and the NMR spectra of
compounds 2* and 6** in TFAꢀd were detected at 27 C.
azines makes it possible to study these processes more
exactly and efficiently.
Two earlier developed methods18 were used to measure the
13С—15N coupling constants: the method of nonlinear approxiꢀ
mation of line shapes in the 1D 13C NMR spectra detected with
15N nuclear selective decoupling and without it and the method
of quantitative SSCCs measurement in amplitudeꢀmodulated
spinꢀecho 1D 13C experiments accumulated with selective inꢀ
version of 15N nuclear magnetization in the spinꢀecho sequence
and without it.18 The 1H—15N coupling constant values were
quantitatively measured using amplitudeꢀmodulated spinꢀecho
Experimental
1
Н, 13C, and 15N NMR spectra were recorded on an Avance
700 spectrometer (Bruker) equipped with a triple resonance probe
(1H,13C,15N) using DMSOꢀd6 and DMSOꢀd6—TFAꢀd mixtures
1
as solvents and Me4Si (13С, Н) and MeNO2 (15N) as internal
1
and external standard, respectively. Chemical shifts in the Н,
13C, and 15N NMR spectra of solutions in TFAꢀd were obtained
relative to the signals of the residual protons of the COOH group
( 11.50), Me4Si, and MeNO2, respectively. The NMR spectra
of compound 2* in DMSOꢀd6 and DMSOꢀd6—TFAꢀd mixtures
1
1D H experiments. Delays (2×) in the range from 0.4 to 1.2 s
were used in the spinꢀecho sequence (the total time of magꢀ
netization evolution for JC,N and JN,H). Adiabatic impulses

528
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 2, February, 2013
Khalymbadzha et al.
(WURSTꢀ20) with a length of 10—20 ms and a width of the
inversion range of ~1 kHz (14 ppm for 15N) were used for selecꢀ
tive decouping and 15N nuclear inversion.
Highꢀresolution mass spectra were measured with a Finniꢀ
gan LTQ FT mass spectrometer (Germany) equipped with
a superconducting magnet with a field intensity of 7 T and an Ion
Max electrosprayer.
4. T. Koudriakova, К. К. Monouilov, K. Shanmuganathan,
L. P. Kotra, F. D. Boudinot, E. CrettonꢀScott, J.ꢀP. Somꢀ
madossi, R. F. Schinazi, C. K. Chu, J. Med. Chem., 1996,
39, 4676.
5. L. P. Kotra, K. K. Manouilov, E. CrettonꢀScott, J.ꢀP. Somꢀ
madossi, F. D. Boudinot, R. F. Schinazi, C. K. Chu, J. Med.
Chem., 1996, 39, 5202.
Potassium 15Nꢀnitrite18 (with 86% 15N enrichment) and
15Nꢀaminoguanidine24 3** (with 86% 15N enrichment) were synꢀ
thesized according to earlier described procedures. Benzoylꢀ
acetone (5) was purchased from Aldrich.
6. Y. O. ElꢀKhoshien, Phosphorus, Sulfur, Silicon, 1998,
139, 163.
7. Th. Kappe, A. Pfaffenschlager, W. Stadlbauer, Synthesis,
1989, 666.
2ꢀHydrazinoꢀ6ꢀmethylpyrimidinꢀ4ꢀone (1) was synthesized by
an earlier described method.25
8. V. Ya. Pochinok, L. F. Avramenko, P. S. Grigorenko, V. N.
Skopenko, Russ. Chem. Rev., 1976, 45, 183.
9. V. Ya. Pochinok, L. F. Avramenko, P. S. Grigorenko, V. N.
Skopenko, Russ. Chem. Rev., 1975, 44, 481.
10. М. Tišler, Synthesis, 1973, 123.
11. N. B. Smirnova, I. Ya. Postovskii, N. N. Vereshchagina,
I. B. Lundina, I. I. Mudretsova, Chem. Heterocycl. Compd.
(Engl. Transl.), 1970, 4, 130 [Khim. Geterotsikl. Soedin.,
1968, 167].
12. V. A. Ershov, I. Ya. Postovskii, Chem. Heterocycl. Compd.
(Engl. Transl.), 1971, 5, 668 [Khim. Geterotsikl. Soedin.,
1971, 711].
13. H. Takeuchi, K. Watanabe, J. Phys. Org. Chem., 1998,
11, 478.
14. B. Chattopadhyay, C. I. Rivera Vera, S. Chuprakov, V. Geꢀ
vorgyan, Org. Lett., 2010, 12, 2166.
15. J. H. Boyer, M. S. Chang, R. F. Reinich, J. Org. Chem.,
1960, 25, 286.
16. E. N. Ulomskii, T. S. Shestakova, S. L. Deev, V. L. Rusinov,
O. N. Chupakhin, Russ. Chem. Bull. (Int. Ed.), 2005, 54, 726
[Izv. Akad. Nauk, Ser. Khim., 2005, 713].
17. D. E. Macfalane, D. C. B. Mills, P. C. Srivastava, Biochemꢀ
istry, 1982, 21, 544.
[15N]ꢀ2ꢀAzidoꢀ6ꢀmethylpyrimidinꢀ4ꢀone (2*A). A solution of
K15NO2 (0.08 g, 0.93 mmol in 1 mL of Н2O) was added to
a solution of compound 1 (0.1 g, 0.71 mmol in 3.00 mL of АсОН)
cooled to 5 C. The reaction mixture was stirred for 2 h at 5 C,
Н2O (10 mL) was added, and the mixture was treated with AcOEt
(3×7 mL). The organic layers were separated, dried with anhydrous
Na2SO4, and evaporated. Compound 2*A was obtained in a yield
of 0.054 g (50%), m.p. 240—242 C. Found: m/z 153.05399
[M + H]+. C5H5N415NO. Calculated: [M + H]+ = 153.0542.
[15N2]ꢀ5ꢀAminotetrazole monohydrate (4**). A 5 М solution
of K15NO2 (1 mL) was added to a suspension of [15N]ꢀaminoꢀ
guanidine carbonate 3* (0.75 g, 5.5 mmol) in 1 mL of 5 М nitric
acid at the temperature <40 C. Sodium acetate (1.5 g) was
added to the obtained yellow solution, and the mixture was reꢀ
fluxed for 5 min. The solution was cooled, and the precipitate
formed was filtered off and dried. Compound 4** was obtained
in a yield of 0.38 g (66%), m.p. 200 C. Found: m/z 88.04034
[M + H]+. CH3N315N2. Calculated: [M + H]+ = 88.04074.
[15N2]ꢀ7ꢀMethylꢀ5ꢀphenyltetrazolo[1,5ꢀa]pyrimidine (6**T).
Benzoylacetone (5) (0.39 g, 2.41 mmol) was added to a solution
of compound 4** (0.25 g, 2.38 mmol) in AcOH (2 mL). The
reaction mixture was refluxed for 1.5 h. After cooling, the preꢀ
cipitate formed was filtered off and dried. Compound 6**T was
obtained in a yield of 0.25 g (49%), m.p. 179—183 C. Found:
m/z 214.08730 [M + H]+. C11H9N315N2. Calculated: [M + H]+ =
= 214.08768.
18. S. L. Deev, Z. O. Shenkarev, T. S. Shestakova, O. N. Chupaꢀ
khin, V. L. Rusinov, A. S. Arseniev, J. Org. Chem., 2010,
75, 8487.
19. D. G. Davis, W. C. Agosta, D. Cowburn, J. Am. Chem. Soc.,
1983, 105, 6189.
20. T. S. Shestakova, S. L. Deev, E. N. Ulomsky, V. L. Rusinov,
O. N. Chupakhin, O. A. D´yachenko, O. N. Kazheva, A. N.
Chekhlov, P. A. Slepukhin, M.I. Kodess, Russ. Chem. Bull.
(Int. Ed.), 2006, 55, 2071 [Izv. Akad. Nauk, Ser. Khim.,
2006, 1993].
21. P. Cmoch, L. Stefaniak, G. A. Webb, Magn. Reson. Chem.,
1997, 35, 237.
22. P. Cmoch, J. W. Wiench, L. Stefaniak, G. A. Webb, J. Mol.
Struct., 1999, 510, 165.
This work was financially supported by the Russian
Foundation for Basic Research (Project Nos 10ꢀ03ꢀ01007ꢀa
and 12ꢀ03ꢀ31476), the Ministry of Education and Science
of the Russian Federation (agreement Nos 14.A18.21.0806
and 14.A18.21.0830), and the Program for Development
of the Ural Federal University (grants for support of young
scientists).
23. C. Temple, Jr., W. C. Coburn, Jr., M. C. Thorpe, J. A.
Montgomery, J. Org. Chem., 1965, 30, 2395.
24. O. N. Chupakhin, E. N. Ulomsky, S. L. Deev, V. L. Rusiꢀ
nov, Synth. Commun., 2001, 31, 2351.
References
1. H.ꢀJ. Schäfer, G. Rathgeber, K. Dose, Y. Kagawa, FEBS
Lett., 1989, 253, 264.
25. L. E. Brady, R. M. Herbst, J. Org. Chem., 1959, 24, 922.
2. F. Boulay, P. Dalbon, P. V. Vignais, Biochemistry, 1985,
24, 7372.
3. L. P. Kotra, P. P. Wang, M. G. Bartlett, K. Shanmugaꢀ
nathan, Z. Xu, S. Cavalcanti, M. G. Newton, C. K. Chu,
J. Org. Chem., 1997, 62, 7267.
Received December 13, 2012