Synthesis and antimycobacterial activity of imidazo[1,2-b][1,2,4,5]tetrazines

Article abstract of DOI:10.1016/j.ejmech.2019.05.081

Tuberculosis (TB) has recently become the leading killer among infectious diseases. Multidrug and extensively drug-resistant Mycobacterium tuberculosis strains urge the need to develop anti-TB drugs with a novel mechanism of action. We describe synthesis

Full text of DOI:10.1016/j.ejmech.2019.05.081

Accepted Manuscript
Synthesis and antimycobacterial activity of imidazo[1,2-b][1,2,4,5]tetrazines
Dmitry A. Maslov, Anna V. Korotina, Kirill V. Shur, Alexey A. Vatlin, Olga B. Bekker,
Svetlana G. Tolshchina, Rashida I. Ishmetova, Nina K. Ignatenko, Gennady L.
Rusinov, Valery N. Charushin, Valery N. Danilenko
PII:
S0223-5234(19)30499-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.081
EJMECH 11391
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2018
Revised Date: 28 May 2019
Accepted Date: 29 May 2019
Please cite this article as: D.A. Maslov, A.V. Korotina, K.V. Shur, A.A. Vatlin, O.B. Bekker, S.G.
Tolshchina, R.I. Ishmetova, N.K. Ignatenko, G.L. Rusinov, V.N. Charushin, V.N. Danilenko, Synthesis
and antimycobacterial activity of imidazo[1,2-b][1,2,4,5]tetrazines, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.081.
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Synthesis and antimycobacterial activity of imidazo[1,2-b][1,2,4,5]tetrazines.
Dmitry A. Maslova,*, Anna V. Korotinab,c,*, Kirill V. Shura, Alexey A. Vatlina, Olga B. Bekkera,
Svetlana G. Tolshchinab,c, Rashida I. Ishmetovab, Nina K. Ignatenkob, Gennady L. Rusinovb,c,
Valery N. Charushinb,c, Valery N. Danilenkoa.
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a
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Laboratory of Bacterial Genetics, Vavilov Institute of General Genetics, Moscow, 119333,
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Russia
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b Laboratory of Heterocyclic Compounds, Postovsky Institute of Organic Synthesis, Ural Branch
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of RAS, Ekaterinburg, 620990, Russia.
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c Ural Federal University, Ekaterinburg, 620002, Russia.
* Corresponding authors:
Dmitry A. Maslov – 3, Gubkina str., Vavilov Institute of General Genetics, Moscow, 119333,
Russia, e-mail: maslov_da@vigg.ru;
Anna V. Korotina – 22/20, S. Kovalevskoy str./Akademicheskaya str., I.Ya. Postovsky Institute
of Organic Synthesis, Ural Branch of RAS, Ekaterinburg, 620990, Russia, e-mail:
korotina@ios.uran.ru
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Abstract.
Tuberculosis (TB) has recently become the leading killer among infectious diseases. Multidrug
and extensively drug-resistant Mycobacterium tuberculosis strains urge the need to develop anti-
TB drugs with a novel mechanism of action. We describe synthesis of 22 novel imidazo[1,2-
b][1,2,4,5]tetrazine derivatives with different substituents at C(3) and C(6) positions, and their
antimycobacterial activity in vitro. 8 compounds show activity as potential serine/threonine
protein kinase (STPK) inhibitors in M. smegmatis aphVIII+ test-system, which is characteristic
for this class. 3 compounds out of 5 most active STPK inhibitors have a prominent minimal
inhibitory concentration on M. tuberculosis H37Rv of 1 µg/ml. We were able to obtain M.
smegmatis mc2 155 mutants resistant to 4 compounds and show that they do not have cross
resistance with other drugs, but have a common mechanism of resistance among these 4
imidazo[1,2-b][1,2,4,5]tetrazines. Compound 3h seems the most promising, combining a
predicted STPK inhibitor activity, the lowest MIC on M. tuberculosis and a low frequency of
drug resistant mutants’ emergence.
Keywords: Mycobacterium smegmatis, Mycobacterium tuberculosis, imidazo[1,2-
b][1,2,4,5]tetrazine, CH-functionalization, drug discovery, drug resistance, tuberculosis,.
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1. Introduction
According to the World Health Organization, tuberculosis (TB) has recently become the
deadliest among the infectious diseases, with about 10.4 million new TB cases and over 1.8
million deaths in 2016 [1]. The number of TB cases with multidrug resistance (MDR-TB,
defined as TB resistant to rifampicin and isoniazid) is constantly growing, being a global threat
for TB treatment and leading to the necessity of using more expensive and more toxic second-
line drugs [1-3]. A custom case of MDR-TB is TB with extensive drug resistance (XDR-TB,
defined as MDR-TB with resistance to the fluoroquinolones and second-line injectables), and
recently cases of totally drug-resistance TB were also reported [4,5]. The emergence of
Mycobacterium tuberculosis strains (the causative agent of TB) with MDR, XDR and TDR leads
to an urgent need for novel anti-TB drugs with a novel mechanism of action.
A promising way of search for new drugs, including anti-tuberculosis drugs, is the synthesis and
bioscreening of polynitrogen heterocycles, in particular tetrazine derivatives. These compounds
have an acceptor character and a large number of heteroatoms in the structure, which provides
their high affinity to biotargets due to the possibility of valent and non-valent binding to electron-
donor protein groups. Azole-annulated tetrazines are of particular interest for the study of
biological activity, since they are polynitrogen-containing purine analogues. There are a number
of publications showing the different biological activities of 3,6-disubstituted and azole-
annulated 1,2,4,5-tetrazines. For example, antimicrobial activity of 1,4-dihydroimidazo[1,2-
b][1,2,4,5]tetrazines [6], antibacterial and fungistatic action of aminesubstituted 1,2,4,5-
tetrazines [7] and thiazolo[3,2-b][1,2,4,5]tetrazines [8], as well as antitumor activity of
triazolo[4,3-b][1,2,4,5]tetrazines [9] and imidazo[1,2-b][1,2,4,5]tetrazines [10]. Important
biomedical application of 1,2,4,5-tetrazines is bioorthogonal reactions of the [4+2] cycloaddition
of 1,2,4,5-tetrazines and various dienophiles (inverse electron demand Diels–Alder reaction), for
use in applications from protein labelling to cancer imaging or materials science [11,12].
Previously, tuberculostatic activity of imidazo[1,2-b]- and [1,2,4]triazolo[4,3-b]-
[1,2,4,5]tetrazines [13], as well as 3-(3,5-dimethyl-1H-pyrazole-1-yl)-1,2,4,5-tetrazines
containing in position C(6) fragments of amines [14,15], hydrazones [16] or amino acid esters
[17] was detected and described. These examples show the expediency of finding new anti-
tuberculosis drugs in a number of derivatives of 1,2,4,5-tetrazine, in particular
azolo[1,2,4,5]tetrazines.
We have previously found that substituted imidazo[1,2-b][1,2,4,5]tetrazines bearing alkylthiol
fragments at C(6) position of heterocyclic system can show the activity as serine/threonine
protein kinase (STPKs) inhibitors in the M. smegmatis aphVIII+ test-system [13].

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This paper describes the synthesis of new imidazo[1,2-b][1,2,4,5]tetrazine derivatives with
different substituents at C(3) and C(6) positions, screening of their activity as serine/threonine
protein kinase (STPKs) inhibitors in the M. smegmatis aphVIII+ test-system and investigation of
antimycobacterial activity of leading compounds on M. smegmatis and M. tuberculosis,
obtaining drug-resistant M. smegmatis mutants and their phenotypic characterization.
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2. Chemistry
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Synthesis of 3-(azol-1-yl)imidazotetrazines 1a-d was carried out by the previously described
method [18] by cyclocondensation of the corresponding 1,2,4,5-tetrazines containing 2,2-
dimethoxyethylamine fragment obtained from the available 3,6-di(azol-1-yl)-1,2,4,5-tetrazines.
To further evaluate the substituent’s in C(7) position influence on antimycobacterial activity of
imidazotetrazines we also synthesized 7-phenylimidazotetrazine 2 [19] by cyclization of 3-
amino-6-(3,5-dimethylpyrazol-1-yl)-1,2,4,5-tetrazine with aminoacetophenone by boiling the
reactants in ethanol (Scheme 1).
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Scheme 1. Synthesis of 3-substituted and 3,7-disubstituted imidazo[1,2-b][1,2,4,5]tetrazines
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Compounds 1a-d and 2 were used as starting substances for modification of imidazotetrazine
system in reactions with a wide range of S-nucleophiles (Scheme 2). Previously [20] in several
examples, we have shown that S-nucleophile, in contrast to alcohols and amines, do not replace
the leaving group in tetrazine cycle of azolotetrazines. Herewith in imidazo[1,2-
b][1,2,4,5]tetrazines instead of the expected nucleophilic attack on the electrophilic atom C(3),
an unusual replacement of the hydrogen atom in the annelated imidazole cycle is realized. In this
paper, we show that the variation of the azolyl substituent in the tetrazine cycle, as well as the
introduction of the aryl fragment to the C(7) position, has no significant effect on the direction

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and yield of substitution reactions with mercaptans. The highest yields of 6-substituted
imidazotetrazines 3 and 5 (50-75%) were achieved using alkylmercaptans, while in the reactions
with thiophenol derivatives the yields of substitution products were reduced to 15-35%. In
addition, it was observed that the small amounts of disubstituted derivatives 4 are formed along
with the desired products 3 in the reactions of imidazotetrazines 1a-d with alkylthiols. The
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structure of compounds 4 was confirmed by the H NMR and the liquid chromatography mass
spectrometry (LCMS) using products 4a,b, which were isolated by column chromatography. In
reactions with aromatic mercapto derivatives, the formation of disubstitution products was not
observed.
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Scheme 2. Synthesis of imidazo[1,2-b][1,2,4,5]tetrazines bearing alkylthiol fragments at C(6)
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position
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The interaction of imidazotetrazines with mercaptans was realized only in the presence of a base,
as which triethylamine was used in this work. The use of K2CO3 also catalyzed these reactions,
however, the yield of the target compounds was substantially reduced. When the reaction was
carried out in an argon atmosphere without oxygen access, the substitution products also formed
with a substantially lower yield. We can suppose two ways of passing the reaction of
imidazotetrazines with mercaptans (Scheme 3). Route A includes the nucleophilic addition of a
thiol to the C(6) atom in the imidazole ring, activated by the high-acceptor tetrazine core, and
subsequent rapid oxidation of the formed adduct. It should be noted that the adduct was not
possible to isolate or experimentally detect. Route B assumes a radical mechanism of the process

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where the thiol radical produced by oxidation process replaces the hydrogen atom at C (6) in
imidazotetrazine. In the case of both the A and B pathways, the acceptor molecules of the
starting azolotetrazine may act as the oxidant and can be reduced to anion radicals or dihydro
derivatives, which then oxidized back under the action of atmospheric oxygen.
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Scheme 3. Possible ways of formation of 6-alkylthioimidazo[1,2-b][1,2,4,5]tetrazines
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Using the example of the interaction of compound 3h with heptylamine and ammonia (Scheme
4) the possibility of modification of 6-alkylthioimidazotetetrazines at the C (3) position with
amine fragments has been shown. The corresponding nucleophilic substitution products of the
3,5-dimethylpyrazolyl group 6a,b were formed under mild conditions with good yields. In
addition, to further evaluation of the biological effect and the studying of reactivity of the 6-
alkylthioimidazotetrazines modification products, the alkylthiol fragment in the compound 3a
was oxidized to the sulfonic group by 3-chloroperbenzoic acid. By the example of the reaction
with heptylamine, it has been shown that the leaving group in the tetrazine ring of the sulfone 7
is easily substituted by the action of amines (Scheme 4).
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Scheme 4. Modification of 6-alkylthioimidazo[1,2-b][1,2,4,5]tetrazines
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3. Results and discussion
Mycobacterial serine-threonine protein kinases inhibition in vitro assay
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The compounds 1b and 3a have been previously shown to be able to bind to the M. tuberculosis
PknB adenine-binding socket by docking studies [13]. All synthesized imidazo[1,2-
b][1,2,4,5]tetrazine derivatives were tested for the ability to inhibit mycobacterial eukaryotic
type serine-threonine protein kinases (STPK). We used the M. smegmatis aphVIII+ test-system,
that was previously validated for mycobacterial STPK inhibitors screening, including those of M.
tuberculosis [21-23]. This test-system implies the paper-disk method: the test-compound in
subinhibitory concentration is applied on a paper-disk in combination with kanamycin and the
diameter of the growth inhibition halo is compared to the one produced by kanamycin alone.
Those compounds, that produce a larger growth inhibition halo in combination with kanamycin
than kanamycin alone, are considered active STPK inhibitors. The results are presented in Table
1.
Table 1. Study of protein kinase inhibition activity of imidazo[1,2-b][1,2,4,5]tetrazines
M. smegmatis APHVIII+
Growth inhibition halo diameter,
Compound
Subinhibitory concentration,
nmol/disk
mm
substance + kanamycin*
12.8±0.4
50
50
100
5
1b
3a
3c
3f
13.3±1.1
13.7±0.5
9.3±0.7
11.5±0.7
9.3±0.7
13.8±0.4
13.0±0.4
5
3h
3i
3m
3n
5
100
25
50
5
11.0±0.5
12.0±1.0
50
6a
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*Growth inhibition halo produced by kanamycin alone (350 µg/disk) was 9.3±0.7 mm.
The study of biological activity of synthesized compounds in the M. smegmatis aphVIII+ test-
system has shown, that STPK inhibition activity increases (showing a higher increase in the
growth inhibition halo diameter) with introduction of the alkyl(aryl)mercapto group into C(6)
position of the imidazo[1,2-b][1,2,4,5]tetrazine system. The most promising STPK inhibitors
were compounds bearing isopropylthiol (3a, 3m, 3n), benzylthiol (3c) and thiophenol (3h)

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fragment in the C(6) position of the system. Modification of imidazotetrazine at C(3) position
revealed that the most active derivatives contain imidazolyl substituent (1b, 3n, 3m). However,
further study of such derivatives may be difficult due to their low solubility.
We were able to select 5 compounds among the most active ones with decent solubility (1b, 3a,
3c, 3h and 3n) for further analysis.
Antimycobacterial activity in vitro
We measured the MICs of the compounds in both liquid and solid media on different
mycobacterial strains. The results are presented in Table 2.
The activity of the imidazo[1,2-b][1,2,4,5]tetrazines on M. smegmatis was lower than on M.
tuberculosis, with 3h and 3c being the most active compounds both in weight-per-volume and
molar concentrations. The lower activity on M. smegmatis compared to M. tuberculosis was
expected and can be explained by a wider system of intrinsic antibiotic resistance in this
bacterium [24]. However, the active concentrations of the compounds allowed us to further use
the M. smegmatis model, providing faster results without the need to use the biosafety level 3
facility, for obtaining spontaneous resistant mutants and analyzing them.
Table 2. MICs of the imidazo[1,2-b][1,2,4,5]tetrazines on mycobacterial strains.
Compound
Mw
M. tuberculosis H37Rv
M. smegmatis mc2 155
Liquid medium
Liquid medium Solid medium
µg/ml
µM
µg/ml µM
µg/ml µM
289,36
323,38
187,16
275,33
337,40
1
3.5
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221.2
99.0
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25
25
20
221.2
61.8
3a
3h
1b
3n
3c
1
3.1
25
25
1
133.6
90.8
3.0
171.0
232.4
94.8
133.6
90.8
59.3
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Obtaining spontaneous M. smegmatis mc2 155 mutants resistant to imidazo[1,2-
b][1,2,4,5]tetrazines
The acquired drug resistance may derive from mutations in genes encoding drug-targets, prodrug
activators, efflux pumps, proteins that mediate cell-wall permeability, thus it is always
determined genetically. The knowledge of the potential risk of drug-resistant mutants’
emergence, as well as potential cross-resistance with other anti-tuberculosis agents is crucial in
the development of drug-candidates for a complex treatment of tuberculosis and co-morbidities.
Thus, we investigated the frequency of emergence of spontaneous M. smegmatis mc2 155
mutants resistant to the studied compounds. We used the 3.5-4×times MIC concentrations of the
imidazo[1,2-b][1,2,4,5]tetrazines to obtain spontaneous M. smegmatis mc2 155 resistant mutants.
The actual concentrations used and the frequency of drug-resistant mutants’ emergence is shown
in Table 3.
3c was the one compound, that generated most mutants at 3,75×MIC, with 3a following it. As 3a
had the highest MIC, together with a high frequency of resistance emergence it makes this
compound the least appealing as a possible anti-TB drug candidate. 3h and 3n had similar
frequency of drug resistant mutants’ emergence. The increase of 3h compound concentration by
1×MIC (from 4× to 5×) lowered the frequency of spontaneous mutants emergence by 15,4 times,
which is usual for bacteria: one mechanism of resistance provides one MIC value, and
developing this mechanism might become less favorable when reaching this value or slightly
overcoming it, while a higher increase in MIC might need a different mechanism of resistance,
involving mutations in other genes, or even a combination of different mutations, leading to a
more dramatic change in spontaneous resistant mutants [25]. Still a dispersion in the frequency
by 10-100 times might may be observed when obtaining spontaneous mutants resistant to one
concentration of the drug in different experiments on mycobacteria [26]. We could not obtain
any spontaneous 1b resistant mutants on 3-4×MIC. We suppose that due to the fact that this

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molecule has the smallest structure, it might be less specific than the others, and might have
more than one biotarget.
We selected 3 mutants resistant to each of the compounds for further analysis: 3aR
(M. smegmatis atR8, atR9, atR10), 3hR (M. smegmatis atR1, atR2, atR11), 3nR (M. smegmatis
atR14, atR17, atR19) and 3cR (M. smegmatis atR33, atR37, atR40).
Drug resistant mutants’ phenotypic characterization
We confirmed the mutants’ resistance to imidazo[1,2-b][1,2,4,5]tetrazines by determining the
MICs in liquid medium. Moreover, we discovered that the selected mutants had cross-resistance
among all the compounds, except for 1b.
On 3a, mutants 9, 14, 17, 19 had a MIC of 256 µg/ml (4× w.t. MIC), mutants 1, 8, 10, 11, 33, 37,
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40 – 512 µg/ml (8× w.t. MIC) and mutant 2 – 1024 µg/ml (16× w.t. MIC).
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On 3h and 3c all the mutants had a MIC higher than 64 µg/ml. The exact MIC could not be
determined in liquid medium due to the crystallization of the compounds at higher
concentrations.
On 3n, mutants 8, 10 and 11 had a MIC of 256 µg/ml (4× w.t. MIC), while all the others – 128
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µg/ml (2× w.t. MIC).
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On 1b all the mutants retained the w.t. MIC of 32 µg/ml.
We used paper-disc method to further analyze the mutants’ susceptibility both to imidazo[1,2-
b][1,2,4,5]tetrazines and to antibiotics of different classes. This method allows a fast and
sensitive comparison of drug susceptibility among strains, though the results are less reliable
than MIC determination in liquid or solid medium. The results are shown on Picture 1.
We observed an increase in drug resistance that was significant according to our criterium of the
M. smegmatis atR8, atR10, atR14, atR1, atR2 and atR11 to rifampicin; M. smegmatis atR8, atR9,
atR14, atR1, atR2 and atR11 to erythromycin, M. smegmatis atR40, atR19 and atR2 to ofloxacin,
M. smegmatis atR33, atR37, atR9 and atR10 to imipenem. All the mutants showed increased

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resistance to 3a, 3h and 3c. All the mutants, except for M. smegmatis atR37 and atR2 showed
significant increase in resistance to 3n, though all of them showed increased resistance in liquid
medium. M. smegmatis atR8, atR9, atR10 and atR17 showed increased resistance to 1b, though
they had the same MIC as the w.t. strain in liquid medium. M. smegmatis atR10 and atR17
showed increased resistance to mitoxantrone. Interestingly two mutants - M. smegmatis atR37
and atR40 showed increased susceptibility to rifampicin. No significant changes in susceptibility
to kanamycin were observed.
Despite the fact that the observed differences in drug sensibility levels to rifampicin,
erythromycin, ofloxacin and imipenem were significant according to our criterion, can’t be
considered as highly increased resistance, as compared to the resistance levels of the mutants to
imidazo[1,2-b][1,2,4,5]tetrazines. Thus, we show that the selected compounds have a mechanism
of resistance, different from the drugs of comparison, however a common mechanism leads to a
resistant phenotype to 4 out of 5 compounds, that has to be elucidated by whole-genomic
sequencing.
Table 3. Frequency of spontaneous drug resistant M. smegmatis mc2 155 mutants’ emergence.
Concentration
×MIC
Drug resistance mutants’
emergence frequency
2.2*10-7
Compound
µg/ml
256
4
4
3a
3h
80
2*10-8
100
5
1.3*10-9
75-100
100
3-4
4
– (<10-9)
1b
3n
3c
2.1*10-8
75
3.75
4*10-6

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Rifampicin. 100 µg/disc
Kanamycin. 5 µg/disc
3n. 100 nmole/disc
Erythromycin. 15 µg/disc
Ofloxacin. 2 µg/disc
Imipenem. 10 µg/disc
30
20
10
M. smegmatis strains
atR1
0
3a. 300 nmole/disc
3h. 40 nmole/disc
1b. 200 nmole/disc
atR2
30
atR11
atR8
atR9
20
10
atR10
atR14
atR17
atR19
atR33
0
atR37
3c. 300 nmole/disc
Mitoxantrone. 10 nmole/disc
atR40
30
mc2 155
20
10
0
M. smegmatis strains
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Picture 1. Growth inhibition halos, produced by various antibiotics and imidazo[1,2-
b][1,2,4,5]tetrazines on w.t. and mutant M. smegmatis strains.
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The X-axis represents different antibiotics and their concentrations per disc, the legend shows
the M. smegmatis strains, the histograms represent the average values from triplicate
measurements, while the error bars represent the standard deviation.
4. Experimental
Compound 1a-d, 2, 3h have been described earlier [18-20].
All the reagents and solvents were purchased from commercial sources. Reactions were
monitored by thin layer chromatography (TLC) on Sorbfil silica gel plates, visualization done by
ultraviolet light. Column chromatography was performed on silica gel (0.040-0.063 mm, 230–
400 mesh). The eluent for TLC and column chromatography was a benzene—acetonitrile (1:1)
mixture. Preparative high-performance liquid chromatography was carried out on a device
Agilent-1200, with CH3CN-H2O (1:1) as an eluent. 1H NMR (400, 500 MHz), 13C NMR (100,

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125 MHz) spectra were recorded in CDCl3 or DMSO-d6 solvents on Bruker Avance DRX-400
and Bruker Avance II 500 spectrometer using Me4Si as the internal standard. The chemical
shifts are given in the δ scale in ppm. Spin multiplicities are given as s (singlet), br. s (broad
singlet), d (doublet), dd (doublet of doublet), t (triplet), sept (septet), and m (multiplet). Coupling
constants (J) are given in hertz. Elemental analysis was performed on a CHN РЕ 2400 Ser. II
(Perkin—Elmer) analyzer. Mass spectra were recorded on Shimadzu LCMS-2010 in the
electrospray ionization mode for solutions in MeOH. Melting points were determined using
melting point apparatus Boetius.
4.1. Chemistry
6-Substituted 3-azolylimidazo[1,2-b][1,2,4,5]tetrazines 3a-p, 5.
To a solution of imidazo[1,2-b][1,2,4,5]tetrazine 1a-d, 2 (1 mmol) in acetonitrile (5 mL), thiol
(1.1 mmol) and triethylamine (101 mg, 1 mmol) were added. The reaction mixture was stirred 1
hour for compounds 3f-i,l, 3h - 3o,p 5-7h - 3a-c, 24h - 3d,e,j,n, 48h - 3k,m,5 at room
temperature (TLC control). The solvent was concentrated. Compounds 3a-i,n-p, 5 were isolated
by column chromatography (Rf = 0.43—0.92), 3m were isolated by preparative HPLC (Rf =
0.68), 3j,k were isolated by recrystallization from methanol, 3l – from ethanol.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(isopropylthio)imidazo[1,2-b][1,2,4,5]tetrazine
(3a).
Orange solid; yield: 60% (174 mg); mp 108–109 °C, Rf = 0.73. 1H NMR (400 MHz, DMSO-d6)
δ: 1.30 (d, J = 6.7 Hz, 6H), 2.27 (s, 3H), 2.58 (s, 3H), 3.74 (sept, J = 6.7 Hz, 1H), 6.30 (s, 1H),
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8.76 (s, 1H). 13C NMR (125 MHz, CDCl3) δ: 13.8, 14.3, 23.6, 38.7, 111.1, 123.8, 143.2, 146.8,
146.9, 152.4, 153.4. Anal. calcd for C12H15N7S: C, 49.81; H, 5.23; N, 33.88. Found: C, 49.78; H,
5.30; N, 33.58.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(propylthio)imidazo[1,2-b][1,2,4,5]tetrazine
(3b).
Orange solid; yield: 51% (148 mg); mp 96–97 °C; Rf = 0.56. 1H NMR (400 MHz, CDCl3) δ:
1.06 (t, J = 7.4 Hz, 3H), 1.71 (m, 2H), 2.40 (s, 3H), 2.71 (s, 3H), 3.12 (t, J = 7.2 Hz, 2H), 6.17 (s,
1H), 8.39 (s, 1H). 13C NMR (125 MHz, CDCl3) δ: 13.1, 13.9, 14.4, 23.2, 34.8, 111.2, 125.1,

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143.3, 145.3, 146.8, 152.4, 153.4. Anal. calcd for C12H15N7S: C, 49.81; H, 5.23; N, 33.88.
Found: C, 49.87; H, 5.23; N, 34.05.
6-(Benzylthio)-3-(3,5-dimethyl-1H-pyrazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine
(3c).
Yellow solid; yield: 75% (253 mg); mp 139-140 °C; Rf = 0.85. 1H NMR (500 MHz, DMSO-d6)
δ: 2.28 (s, 3H), 2.60 (s, 3H), 4.40 (s, 2H), 6.31 (s, 1H), 7.21-7.34 (m, 5H), 8.59 (s, 1H). 13C
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NMR (125 MHz, DMSO-d6) δ: 13.4, 36.0, 110.1, 122.7, 127.5, 128.5, 128.9, 136.9, 142.8,
146.3, 146.9, 151.5, 151.7. Anal. calcd for C16H15N7S: C, 56.96; H, 4.48; N, 29.06. Found: C,
56.93; H, 4.65; N, 28.82.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(octadecylthio)imidazo[1,2-b][1,2,4,5]tetrazine
(3d).
Yellow solid; yield: 67% (335 mg); mp 86-87 °C; Rf = 0.92. 1H NMR (400 MHz, CDCl3) δ:
0.88 (t, J = 6.9 Hz, 3H), 1.25 (s, 25H), 1.38-1.48 (m, 2H), 1.62-1.72 (m, 2H), 2.39 (s, 3H), 2.71
(s, 3H), 3.12 (t, J = 7.3 Hz 2H), 6.17 (s, 1H), 8.38 (s, 1H). 13C NMR (125 MHz, CDCl3) δ: 13.8,
14.1, 14.4, 22.7, 28.4, 29.0, 29.3, 29.4, 29.5, 29.6, 29.7, 31.9, 32.9, 111.1, 125.3, 143.4, 145.3,
146.8, 152.3, 153.4. Anal. calcd for C27H45N7S: C, 64.89; H, 9.08; N, 19.62. Found: C, 64.95; H,
9.02; N, 19.65.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(2-hydroxyethylthio)imidazo[1,2-b][1,2,4,5]tetrazine
(3e). Orange solid; yield: 36% (105 mg); mp 134-135 °C; Rf = 0.43. 1H NMR (500 MHz,
CDCl3) δ: 2.36 (s, 3H), 2.75 (s, 3H), 3.28 (t, J = 5.8 Hz, 2H), 3.87 (t, J = 5.8 Hz, 2H), 6.16 (s,
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1H), 8.44 (s, 1H). 13C NMR (125 MHz, CDCl3) δ: 13.8; 14.7; 37.0, 61.6, 111.4, 123.8, 143.8,
147.0, 152.3, 153.8. Anal. calcd for C11H13N7OS: C, 45.35; H, 4.50; N, 33.65. Found: C, 45.33;
H, 4.45; N, 33.52.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(6-hydroxyhexan-3-ylthio)imidazo[1,2-
b][1,2,4,5]tetrazine (3f). Orange solid; yield: 70% (243 mg); mp 109-110 °C; Rf = 0.80. 1H
NMR (400 MHz, CDCl3) δ: 0.85 (t, J = 7.2 Hz, 3H), 1.33-1.43 (m, 1H), 1.48-1.69 (m, 5H), 2.38
(s, 3H), 2.79 (s, 3H), 3.51-3.59 (m, 1H), 3.95-4.02 (m, 1H), 4.36-4.44 (m, 1H), 5.32 (br. s, 1H),
6.18 (s, 1H), 8.46 (s, 1H). 13C NMR (125 MHz, CDCl3) δ: 13.4, 13.8, 15.0, 20.0, 38.6, 39.0,

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46.9, 58.4, 111.4, 123.8, 144.5, 147.1, 148.7, 152.5, 154.1. Anal. calcd for C15H21N7OS: C,
51.85; H, 6.09; N, 28.22. Found: C, 51.40; H, 6.04; N, 27.94.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(3-(4-hydroxy-3,5-di-tert-
butylphenyl)propylthio)imidazo[1,2-b][1,2,4,5]tetrazine (3g). Orange solid; yield: 51% (500
mg); mp 111-112 °C; Rf = 0.90. 1H NMR (500 MHz, DMSO-d6) δ: 1.34 (s, 18H), 1.85 (m, 2H),
2.26 (s, 3H), 2.56 (s, 3H), 2.59 (m, 2H), 3.18 (m, 2H), 6.28 (s, 1H), 6.70 (s, 1H), 6.87 (s, 2H),
8.74 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 13.2, 13.4, 30.4, 31.6, 31.7, 33.7, 34.4, 123.7,
124.2, 131.8, 139.2, 142.6, 145.9, 147.0, 151.3, 151.7, 151.9. Anal. calcd for C26H35N7OS: C,
63.26; H, 7.15; N, 19.86. Found: C, 63.38; H, 7.15; N, 19.66.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(phenylthio)imidazo[1,2-b][1,2,4,5]tetrazine
(3h).
Yellow solid; yield 36% (117 mg); mp 139-140 °C; Rf = 0.80. 1H NMR (400 MHz, CDCl3) δ:
2.38 (s, 3H), 2.50 (s, 3H), 6.13 (s, 1H), 7.34-7.38 (m, 3H), 7.40-7.44 (m, 2H), 8.29 (s, 1H). 13C
NMR (125 MHz, CDCl3) δ: 13.8, 14.1, 111.2, 123.9, 129.0, 129.3, 129.9, 131.6, 144.4, 146.0,
146.8, 152.2, 153.5. Anal. calcd for C15H13N7S: C, 55.71; H, 4.05; N, 30.32. Found: C, 55.61; H,
3.76; N, 30.31.
6-(2,5-Dimethoxyphenylthio)-3-(3,5-dimethyl-1H-pyrazol-1-yl)imidazo[1,2-
b][1,2,4,5]tetrazine (3i). Orange solid; yield 42% (161 mg); mp 105-106 °C; Rf = 0.74. 1H
NMR (500 MHz, DMSO-d6) δ: 2.25 (s, 3H), 2.42 (s, 3H), 3.58 (s, 3H), 3.82 (s, 3H), 6.27 (s,
1H), 6.52 (d, J = 3.0 Hz, 1H), 6.84 (dd, J1= 8.9 Hz, J2 = 3.0 Hz, 1H), 7.03 (d, J = 8.9 Hz, 1H),
8.75 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 13.0, 13.4, 55.5, 56.6, 110.1, 112.6, 112.8,
115.3, 118.5, 120.6, 142.8, 147.5, 148.3, 150.3, 151.5, 151.8, 153.7. Anal. calcd for
C17H17N7O2S: C, 53.25; H, 4.47; N, 25.57. Found: C, 53.26; H, 4.67; N, 25.46.
6-(2-Carboxyphenylthio)-3-(3,5-dimethyl-1H-pyrazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine
(3j). Orange solid; yield 25% (92 mg); mp 266-267 °C; Rf = 0.21. 1H NMR (500 MHz, DMSO-
d6) δ: 2.23 (s, 3H), 2.27 (s, 3H), 6.23 (s, 1H), 6.76 (d, J = 7.6 Hz, 1H), 7.24-7.33 (m, 2H), 8.03
(dd, J1 = 7.6 Hz, J2 = 2.0 Hz, 1H), 8.89 (s, 1H), 13.59 (br. s, 1H). 13C NMR (125 MHz, DMSO-

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d6) δ: 12.9, 13.3, 110.2, 118.7, 125.9, 127.0, 127.1, 131.4, 133.3, 137.0, 142.7, 147.9, 149.2,
151.6, 151.8, 167.5. Anal. calcd for C16H13N7O2S: C, 52.31; H, 3.57; N, 26.69. Found: C, 52.24;
H, 3.52; N, 26.65.
6-(3-Carboxyphenylthio)-3-(3,5-dimethyl-1H-pyrazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine
(3k).
Yellow solid; yield 20 % (74 mg); mp 229-230 °C; Rf = 0.10. 1H NMR (400 MHz, DMSO-d6)
δ: 2.24 (s, 3H), 2.30 (s, 3H), 6.25 (s, 1H), 7.31-7.35 (m, 1H), 7.61-7.67 (m, 1H), 7.78-7.83 (m,
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1H), 7.91-7.96 (m, 1H), 8.90 (s, 1H), 13.06 (br. s, 1H). 13C NMR (125 MHz, DMSO-d6) δ:
13.0, 13.4, 110.2, 118.7, 125.9, 128.4, 129.2, 130.0, 132.1, 132.5, 133.0, 142.8, 147.4, 148.0,
151.6, 166.4. Anal. calcd for C16H13N7O2S: C, 52.31; H, 3.57; N, 26.69. Found: C, 52.37; H,
3.65; N, 26.54.
6-(4-Carboxyphenylthio)-3-(3,5-dimethyl-1H-pyrazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine
(3l). Yellow solid; yield 18 % (65 mg); mp 243-244 °C; Rf = 0.10. 1H NMR (400 MHz, DMSO-
d6) δ: 2.24 (s, 3H), 2.30 (s, 3H), 6.25 (s, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.5 Hz, 2H),
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8.90 (s, 1H), 13.02 (br. s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 12.9, 13.4, 110.3, 117.0,
126.9, 129.1, 130.2, 138.3, 142.8, 147.6, 148.5, 151.7, 151.8, 166.6. Anal. calcd for
C16H13N7O2S: C, 52.31; H, 3.57; N, 26.69. Found: C, 52.37; H, 3.65; N, 26.54.
3-(1H-Imidazol-1-yl)-6-(isopropylthio)imidazo[1,2-b][1,2,4,5]tetrazine (3m). Orange solid;
yield 34 % (89 mg); mp 139-140 °C; Rf = 0.68. 1H NMR (400 MHz, DMSO-d6) δ: 1.33 (d, J =
6.7 Hz, 6H), 3.88 (sept, J = 6.7 Hz, 1H), 7.27-7.32 (m, 1H), 8.04-8.08 (m, 1H), 8.76-8.79 (m,
1H), 8.80 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 23.5, 38.3, 116.7, 123.0, 131.2, 136.0,
147.8, 147.9, 149.2. Anal. calcd for C10H11N7S: C, 45.96; H, 4.24; N, 37.52. Found: C, 46.02; H,
4.41; N, 37.53.
6-(Isopropylthio)-3-(4-methyl-1H-imidazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine
(3n).
Orange solid; yield 53 % (146 mg); mp 157-158 °C; Rf = 0.50. 1H NMR (400 MHz, CDCl3) δ:
1.40 (d, J = 6.8 Hz, 6H), 2.36 (s, 3H), 3.77 (sept, J = 6.8 Hz, 1H), 7.66 (s, 1H), 8.43 (s, 1H), 8.69

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(s, 1H). 13C NMR (125 MHz, CDCl3) δ: 13.2, 23.7, 38.9, 112.5, 125.2, 135.2, 140.2, 147.5,
147.7, 149.3. Anal. calcd for C11H13N7S: C, 47.98; H, 4.76; N, 35.61. Found: 47.76; H, 4.76; N,
35.56.
6-(Benzylthio)-3-(4-methyl-1H-imidazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine (3o). Yellow
solid; yield: 43 % (139 mg); mp 80-81 °C; Rf = 0.52. 1H NMR (400 MHz, DMSO-d6) δ: 2.25 (s,
3H), 4.45 (s, 2H), 7.22-7.34 (m, 5H), 7.75 (s, 1H), 8.63 (s, 1H), 8.65 (s, 1H). 13C NMR (125
MHz, DMSO-d6) δ: 13.5, 35.8, 112.5, 123.3, 127.5, 128.5, 128.9, 135.4, 136.9, 140.1, 146.7,
147.6, 149.0. Anal. calcd for C15H13N7S: C, 55.71; H, 4.05; N, 30.32. Found: C, 55.74; H, 4.07;
N, 30.26.
6-(Benzylthio)-3-(1H-indazol-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine (3p). Yellow solid; yield:
79 % (284 mg); mp 165-167 °C; Rf = 0.86. 1H NMR (400 MHz, DMSO-d6) δ: 4.47 (s, 2H),
7.19-7.30 (m, 3H), 7.31-7.37 (m, 2H), 7.43-7.49 (m, 1H), 7.68-7.76 (m, 1H), 7.99-8.04 (m, 1H),
8.56-8.61 (m, 2H), 8.71 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 36.0, 114.4, 121.8, 122.7,
123.8, 126.1, 127.5, 128.5, 128.9, 129.0, 137.0, 138.8, 140.4, 146.1, 147.2, 152.5. Anal. calcd
for C18H13N7S (FW =): C, 60.15; H, 3.65; N, 27.28 %. Found: C, 60.22; H, 3.50; N, 27.15 %.
3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6,7-bis(isopropylthio)imidazo[1,2-b][1,2,4,5]tetrazine
(4a). Orange solid; Rf = 0.79. Спектр 1Н NMR (400 MHz, CDCl3) δ: 1.32 (d, J = 6.8 Hz, 6H),
1.56 (d, J = 6.8 Hz, 6H), 2.39 (s, 3H), 2.67 (s, 3H), 3.84 (sept, J = 6.8 Hz, 1H), 4.32 (sept, J =
6.8 Hz, 1H), 6.22 (s, 1Н). MS (ESI) m/z (%) = 364.15 (100) [M + H]+ (calcd for C15H22N7S2 ,
+
364.14).
3-(1H-Imidazol-1-yl)-6,7-bis(isopropylthio)imidazo[1,2-b][1,2,4,5]tetrazine (4b). Orange
solid; yield 3 % (10 mg); mp = 104–106 °C; Rf = 0.74. Спектр 1Н NMR (400 MHz, DMSO-d6)
δ: 1.28 (d, J = 6.7 Hz, 6H), 1.51 (d, J = 6.8 Hz, 6H), 3.82 (sept, J = 6.7 Hz, 1H), 4.32 (sept, J =
6.8 Hz, 1H), 7.27 (s, 1Н), 8.03 (s, 1H), 8.71 (s, 1H). MS (ESI) m/z (%) = 336.05 (100) [M + H]+
+
(calcd for C13H18N7S2 , 336.11).

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3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(isopropylthio)-7-phenylimidazo[1,2-
b][1,2,4,5]tetrazine (5). Orange solid; yield: 66% (241 mg); mp 150–151 °C, Rf = 0.90. 1H
NMR (400 MHz, DMSO-d6) δ: 1.22 (d, J = 6.7 Hz, 6H), 2.29 (s, 3H), 2.61 (s, 3H), 3.75 (sept, J
= 6.7 Hz, 1H), 6.32 (s, 1H), 7.56-7.66 (m, 3H), 8.41-8.46 (m, 2H), 8.76 (s, 1H). 13C NMR (125
MHz, DMSO-d6) δ: 13.2, 13.4, 23.2, 110.0, 118.3, 128.8, 128.9, 130.6, 131.8, 142.6, 146.5,
151.3, 151.8, 155.2. Anal. calcd for C18H19N7S: C, 59.16; H, 5.24; N, 26.83. Found: C, 59.08; H,
5.12; N, 26.57.
3-(Heptylamino)-6-(phenylthio)imidazo[1,2-b][1,2,4,5]tetrazine (6a). To a solution of 1 mmol
(323 mg) 3-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(phenylthio)imidazo[1,2-b][1,2,4,5]tetrazine (3h)
in 1.5 ml of acetonitrile was added 1.5 ml of heptylamine, stirred for 2 hours, then the reaction
solution was evaporated, acetic acid was added to the residue, the product was precipitated with
water, and filtered. Yellow solid; yield: 60 % (206 mg); mp 89–90 °C, Rf = 0.92. 1H NMR (400
MHz, DMSO-d6) δ: 0.85 (t, J = 7.0 Hz, 3H), 1.17-1.28 (m, 8H), 1.42-1.52 (m, 2H), 3.04-3.19
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(m, 2H), 7.20-7.33 (m, 5H), 8.23 (s, 1H), 8.40 (br. s, 1H). 13C NMR (125 MHz, DMSO-d6) δ:
13.9, 22.0, 26.3, 27.5, 28.3, 31.2, 40.7, 116.4, 127.0, 128.3, 129.3, 132.5, 142.6, 146.1, 157.3.
Anal. calcd for C17H22N6S: C, 59.62; H, 6.48; N, 24.54. Found: C, 59.66; H, 6.69; N, 24.44.
3-(Amino)-6-(phenylthio)imidazo[1,2-b][1,2,4,5]tetrazine (6b). A solution of 1 mmol (323
mg) of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(phenylthio)imidazo[1,2-b][1,2,4,5]tetrazine (3h) in
dimethylformamide, ammonia was flushed for 15 minutes, the reaction was carried out in a
microwave oven for 10 minutes at a temperature of 150 ° C, the solvent was evaporated, the
product was precipitated from acetonitrile with water, filtered and washed with hexane. Orange
solid; yield: 90 % (220 mg); mp 204–205 °C, Rf = 0.78. 1H NMR (400 MHz, DMSO-d6) δ:
7.12-7.18 (m, 2H), 7.20-7.25 (m, 1H), 7.26-7.32 (m, 2H), 7.69 (br. s, 2H) 8.26 (s, 1H). 13C
NMR (125 MHz, DMSO-d6) δ: 115.3, 126.7, 127.1, 129.5, 133.4, 143.9, 146.5, 158.5. Anal.
calcd for C10H8N6S: C, 49.17; H, 3.30; N, 34.40. Found: C, 49.35; H, 3.18; N, 34.19.

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3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(isopropylsulfonyl)imidazo[1,2-b][1,2,4,5]tetrazine (7).
To solution of mmol (289 mg) 3-(3,5-dimethyl-1H-pyrazol-1-yl)-6-
a
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(isopropylthio)imidazo[1,2-b][1,2,4,5]tetrazine (3a) in acetonitrile was added 3.5 mmol (604
mg) of 3-chloroperbenzoic acid, stirred for 2 hours, the solvent was evaporated, the residue was
washed with diethyl ether and filtered. Orange solid; yield: 80% (258 mg); mp 205–206 °C, Rf =
0.85. 1H NMR (500 MHz, DMSO-d6) δ: 1.29 (d, J = 6.9 Hz, 6H), 2.29 (s, 3H), 2.58 (s, 3H),
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3.63 (sept, J = 6.9 Hz, 1H), 6.35 (s, 1H), 8.95 (s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 13.3,
13.4, 14.6, 55.3, 110.7, 121.6, 143.1, 145.0, 146.7, 151.3, 152.3. Anal. calcd for C12H15N7O2S:
C, 44.85; H, 4.70; N, 30.51. Found: C, 44.77; H, 4.88; N, 30.51.
3-(Heptylamino)-6-(isopropylsulfonyl)imidazo[1,2-b][1,2,4,5]tetrazine (8a). To a solution of
1 mmol (321 mg) of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(isopropylsulfonyl)imidazo[1,2-
b][1,2,4,5]tetrazine (7) in acetonitrile 10 mmol of heptylamine were added, after 10 minutes the
solvent was evaporated, the residue was washed with diethyl ether and filtered. Yellow solid;
yield: 82% (279 mg); mp 98-99 °C, Rf = 0.87. 1H NMR (500 MHz, DMSO-d6) δ: 0.86 (t, J =
7.0 Hz, 3H), 1.27 (d, J = 6.9 Hz, 6H), 1.28-1.40 (m, 8H), 1.63 (m, 2H), 3.24 (s, 2H), 3.64 (sept, J
= 7.0 Hz, 1H), 8.30 (s, 1H), 8.80 (br. s, 1H). 13C NMR (125 MHz, DMSO-d6) δ: 13.9, 14.6,
22.0, 26.4, 27.5, 28.4, 31.2, 40.9, 54.5, 120.3, 139.8, 145.3, 157.1. Anal. calcd for C14H24N6O2S:
C, 49.39; H, 7.11; N, 24.69. Found: C, 49.36; H, 7.18; N, 24.61.
4.2. Antimycobacterial activity testing
Bacterial strains and growth conditions
M. smegmatis mc2 155 strain was used in the study. Lemco-Tw broth (5 g/l Lemco Powder,
Oxoid, UK; 5 g/l NaCl, 5 g/l bacto peptone, Oxoid, UK, 0.05 % v/v Tween-80) and
Middlebrook 7H9 medium (Himedia, India) supplemented with OADC (Himedia, India), 0.1ꢀ%
Tween-80 (v/v) and 0.4% glycerol (v/v) were used as liquid media, while the M290 Soyabean

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Casein Digest Agar (Himedia, India) was used as solid medium. Cultures in liquid medium were
incubated in the Multitron incubator shaker (Infors HT, Switzerland) at 37 °С and 250 rpm.
Minimal inhibitory concentrations determination on M. tuberculosis
M. tuberculosis H37Rv strain was used in the study. MICs were determined in liquid medium on
the automated Bactec MGIT 960 system (Becton–Dickinson, USA) as described in [22].
Minimal inhibitory concentrations determination on M. smegmatis
Minimal inhibitory concentrations (MICs) of the studied compounds on M. smegmatis were
determined in liquid and on solid media.
For the MIC determination in liquid medium, M. smegmatis was cultured overnight in Lemco-
Tw, then diluted in the proportion of 1:200 in fresh Lemco-Tw broth. 196 µl of the diluted
culture were poured in the Greiner CELLSTAR® 96 well flat-bottom plates for suspension
cultures (Sigma-Aldrich, USA) and 4 µl of serial two-fold dilutions of the tested compounds in
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DMSO were added to the wells to final concentrations of 0.5 to 1024 µg/ml for 3a, 1b and 3n,
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and 0.5 to 64 µg/ml, for 3c and 3h, as they were not soluble in higher concentrations. 64 µg/ml
concentrations of 3c and 3h were also supplemented with 4% DMSO (v/v), that did not affect
the growth of mycobacteria. The plates were incubated at 37°С and 250 rpm for 48 hours. The
MIC was determined as the lowest concentration of the compound with no visible bacterial
growth.
For the determination of MICs on solid medium, M290 Petri dishes were prepared with serial
two-fold dilutions of the tested compounds, ranging from 8 to 64 µg/ml for 3h, 1b and 3c, and
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from 8 to 128 µg/ml for 3a and 3n, additional Petri dishes with 20 and 25 µg/ml of the tested
compound were prepared in some cases. M. smegmatis was grown in Lemco-Tw broth up to
OD600=1,8 (approximately 3×108 colony forming units per milliliter, CFU/ml). Serial ten-fold
dilutions of the culture were prepared and 3 µl of each dilution were poured on the plates. The

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MIC for each compound was determined as the minimal concentration, inhibiting the growth of
99% of CFU.
Drug resistant M. smegmatis mutants’ isolation
Serial ten-fold dilutions of M. smegmatis mc2 155 containg 107-109 were plated on Petri dishes
with M290 medium, supplemented with 3.5-4×times MIC of the tested compounds. The Petri
dishes were incubated at 30°C for 5-7 days, until the colonies were visible [27]. The number of
colonies was counted to calculate the frequency of drug resistance emergence. Twelve colonies
were plated on new Petri dishes with the same concentration of the compound, afterwards those
that have shown growth were plated on compound-free M290 medium and then plated on M290
medium supplemented with the compound again to confirm drug resistance [28]. Three mutants
with confirmed resistance to each of the corresponding compound were randomly selected for
further analysis.
Drug susceptibility testing with the paper disc method
Mycobacterial cultures were grown in Middlebrook 7H9 broth to mid-exponential phase (OD600
= 1.2). Afterwards the cultures were diluted in the proportion of 1:9:10 (culture:water:M290
medium) and 5 ml were poured as the top layer on Petri dishes with agarized M290 medium. The
plates were allowed to dry for at least 30 min, and Sensi-Discs with antibiotics (erythromycin,
ofloxacin, imipenem were plated as Sensi-Discs, while rifampicin, kanamycin, mitoxantrone and
the imidazo[1,2-b][1,2,4,5]tetrazines were impregnated on sterile paper discs and also plated.
The plates were incubated for 2–3 days at 37°C, until the bacterial lawn was fully grown.
Growth inhibition halos were measured to the nearest 1 mm. The experiments were carried out
as triplicates, the average diameter and standard deviation (SD) were calculated. Those
differences that had no intersection of the SDs with the control (M. smegmatis mc2 155) were
considered significant.

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Conclusions
The method of chemical functionalization of imidazo[1,2-b][1,2,4,5]tetrazines at C(6) position in
the reactions with alkyl and aryl mercaptanes has been developed. The possibility of further
modification of obtained products via nucleophilic substitution with amines or oxidation reaction
has been shown. These methods allowed to obtain the wide range of novel imidazo[1,2-
b][1,2,4,5]tetrazine derivatives with different substituents at C(3) and C(6) positions.
Our testing in the M. smegmatis aphVIII+ test-system [23] showed that 8 out of 28 synthesized
compounds (1b, 3a, 3c, 3h, 3m, 3n, 5 and 6a) are active mycobacterial STPK inhibitors. 5
compounds selected for further analysis (1b, 3a, 3c, 3h, and 3n) showed antimycobacterial
activity on both M. tuberculosis H37rv and M. smegmatis mc2 155, with 3 of them (3a, 3h and
3c) in a concentration as low as 1 µg/ml. MICs on M. smegmatis were higher, due to a wider
system of intrinsic antibiotics resistance [24], however this was not an obstacle to obtain
resistant mutants to 4 out of 5 compounds (3a, 3c, 3h and 3n). The investigation of the M.
smegmatis mutants’ drug resistance phenotype revealed that the 4 imidazo[1,2-
b][1,2,4,5]tetrazine have a common resistance mechanism, though it differs from those of the
tested antibiotics.
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The compound 3h seems to be the most promising compound among the studied imidazo[1,2-
b][1,2,4,5]tetrazines, due to its relatively high antimycobacterial activity on M. tuberculosis
H37Rv (1 µg/ml), as well as a relatively low frequency of drug resistance mutants’ emergence
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(2.0*10-8 at 4×MIC and 1.3*10-9 at 5×MIC).
Though the alleged target of the selected imidazo[1,2-b][1,2,4,5]tetrazines are mycobacterial
STPKs, as was shown in previous docking experiments [13] and in this study on the M.
smegmatis APHVIII+ test-system [23], the exact biotarget of these compounds, as well as the
mechanism of resistance are yet to be established by whole-genomic sequencing and “reverse-
genetics” approach, which is a usual practice in the “drug-to-target” pathway of drug
development [29].

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Conflict of interests
The authors have no competing financial interests to declare.
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Author contributions
Maslov D. A. and Korotina A. V. contributed equally to this work. Charushin V. N., Rusinov G.
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L. and Danilenko V. N. organized the collaboration. Maslov D. A. planned the biological
experiments. Maslov D.A., Shur K. V., Vatlin A. A. and Bekker O.B. carried out the in vitro
experiments. Koritna A. V., Tolshchina S. G., Ishmetova R. I. and Ignatenko N. K. synthesized
the compounds. Maslov D. A., Korotina A. V., Tolshchina S. G. and Bekker O. B. drafted the
manuscript. All authors read and approved the manuscript.
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Funding
The biological studies were supported by the Russian Science Foundation (RSF grant 17-75-
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20060).
The synthetic experiments were supported by the Russian Science Foundation (RSF grant 15-13-
00077-P).
Analytical studies were carried out using equipment of the Center for Joint Use “Spectroscopy
and Analysis of Organic Compounds” at the Postovsky Institute of Organic Synthesis of the
Russian Academy of Sciences (Ural Branch).
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Highlights
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•
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New imidazo[1,2-b][1,2,4,5]tetrazines with substituted 3 and 6 positions synthesized
6-(alkylthio)imidazo[1,2-b][1,2,4,5]tetrazines obtained by CH-functionalization
The compounds’ reactivity assessed by oxidation and interaction with N-nucleophiles
This class exhibits antibacterial activity on M. tuberculosis as STPK inhibitors
Drug resistant mutants do not show cross-resistance with several other drugs