Boulton-katritzky rearrangement of 5-substituted phenyl-3-[2-(Morpholin-1-yl)ethyl]-1,2,4-oxadiazoles as a synthetic path to spiropyrazoline benzoates and chloride with antitubercular properties

Article abstract of DOI:10.3390/molecules26040967

The analysis of stability of biologically active compounds requires an accurate determination of their structure. We have found that 5-aryl-3-(2-aminoethyl)-1,2,4-oxadiazoles are generally unstable in the presence of acids and bases and are rearranged into the salts of spiropyrazolinium compounds. Hence, there is a significant probability that it is the rearranged products that should be attributed to biological activity and not the primarily screened 5-aryl-3-(2-aminoethyl)-1,2,4-oxadiazoles. A series of the 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium (spiropyrazoline) benzoates and chloride was synthesized by Boulton-Katritzky rearrangement of 5-substituted phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles and characterized using FT-IR and NMR spectroscopy and X-ray diffraction. Spiropyrazolylammonium chloride demonstrates in vitro antitubercular activity on DS (drug-sensitive) and MDR (multidrug-resistant) of MTB (M. tuberculosis) strains (1 and 2 μg/mL, accordingly) equal to the activity of the basic antitubercular drug rifampicin; spiropyrazoline benzoates exhibit an average antitubercular activity of 10-100 μg/mL on MTB strains. Molecular docking studies revealed a series of M. tuberculosis receptors with the energies of ligand-receptor complexes (?35.8-?42.8 kcal/mol) close to the value of intermolecular pairwise interactions of the same cation in the crystal of spiropyrazolylammonium chloride (?35.3 kcal/mol). However, only in complex with transcriptional repressor EthR2, both stereoisomers of the cation realize similar intermolecular interactions.

Full text of DOI:10.3390/molecules26040967

molecules
Article
Boulton-Katritzky Rearrangement of 5-Substituted
Phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles as a
Synthetic Path to Spiropyrazoline Benzoates and Chloride
with Antitubercular Properties
Lyudmila Kayukova 1,*, Anna Vologzhanina 2,* , Kaldybai Praliyev 1, Gulnur Dyusembaeva 1, Gulnur Baitursynova 1,
Asem Uzakova 1 , Venera Bismilda 3 , Lyailya Chingissova 3 and Kydyrmolla Akatan 4
1
JSC A. B. Bekturov Institute of Chemical Sciences, 106 Shokan Ualikhanov St., Almaty 050010, Kazakhstan;
praliyevkd@mail.ru (K.P.); g_gazinovna@mail.ru (G.D.); guni-27@mail.ru (G.B.); a7_uzakova@mail.ru (A.U.)
A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov St.,
2
B-334, 119991 Moscow, Russia
National Scientific Center of Phthisiopulmonology of Ministry of Health of the Republic of Kazakhstan,
3
5 Bekkhozhin St., Almaty 050010, Kazakhstan; venerabismilda@mail.ru (V.B.); lchingisova@mail.ru (L.C.)
National scientific laboratory, S. Amanzholov East Kazakhstan State University, 18/1 Amurskaya St.,
4
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Ust-Kamenogorsk 070002, Kazakhstan; ahnur.hj@mail.ru
Correspondence: lkayukova@mail.ru (L.K.); vologzhanina@mail.ru (A.V.); Tel.: +7-(705)-168-81-53 (L.K.)
ꢀꢁꢂꢃꢄꢅꢆ
*
Citation: Kayukova, L.;
Vologzhanina, A.; Praliyev, K.;
Dyusembaeva, G.; Baitursynova, G.;
Uzakova, A.; Bismilda, V.;
Abstract: The analysis of stability of biologically active compounds requires an accurate determina-
tion of their structure. We have found that 5-aryl-3-(2-aminoethyl)-1,2,4-oxadiazoles are generally
unstable in the presence of acids and bases and are rearranged into the salts of spiropyrazolin-
ium compounds. Hence, there is a significant probability that it is the rearranged products that
should be attributed to biological activity and not the primarily screened 5-aryl-3-(2-aminoethyl)-
1,2,4-oxadiazoles. A series of the 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium (spiropyrazoline)
benzoates and chloride was synthesized by Boulton–Katritzky rearrangement of 5-substituted phenyl-
3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles and characterized using FT-IR and NMR spectroscopy
and X-ray diffraction. Spiropyrazolylammonium chloride demonstrates in vitro antitubercular ac-
tivity on DS (drug-sensitive) and MDR (multidrug-resistant) of MTB (M. tuberculosis) strains (1 and
2 µg/mL, accordingly) equal to the activity of the basic antitubercular drug rifampicin; spiropyrazo-
Chingissova, L.; Akatan, K.
Boulton-Katritzky Rearrangement of
5-Substituted Phenyl-3-[2-(morpholin-
1-yl)ethyl]-1,2,4-oxadiazoles as a
Synthetic Path to Spiropyrazoline
Benzoates and Chloride with
Antitubercular Properties. Molecules
2021, 26, 967.
https://doi.org/10.3390/
line benzoates exhibit an average antitubercular activity of 10–100
docking studies revealed a series of M. tuberculosis receptors with the energies of ligand–receptor
complexes ( 35.8– 42.8 kcal/mol) close to the value of intermolecular pairwise interactions of the
same cation in the crystal of spiropyrazolylammonium chloride ( 35.3 kcal/mol). However, only
in complex with transcriptional repressor EthR2, both stereoisomers of the cation realize similar
intermolecular interactions.
µg/mL on MTB strains. Molecular
molecules26040967
Academic Editors: Vera L. M. Silva
and Artur M. S. Silva
−
−
−
Received: 21 December 2020
Accepted: 23 January 2021
Published: 12 February 2021
Keywords: 1,2,4-oxadiazoles; spiropyrazolinium compounds; in vitro antitubercular screening;
X-ray diffraction; molecular docking
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
There are pyrazoline-containing compounds that act as active pharmaceutical ingre-
dients of such commercially available drugs as aminopyrine (aminophenazone; analgesic
and antipyretic), dipyrone (metamizole, noramidopyrine; analgesic), antipyrine (benzocaine;
non-narcotic analgesic, an antipyretic and antirheumatic), zaleplon(hypnotic and sedative),
celecoxib (Aclarex, Celebrex; anti-inflammatory and antirheumatic drug), allopurinol (uri-
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
costatic agent, xanthine oxidase inhibitor) [1]. Therefore, there is always a demand for new
molecules, methodologies and improved synthetic approaches to novel pyrazoline derivatives
.
Pyrazolines, as noticeable, practically meaningful nitrogen-containing heterocyclic
compounds, can be synthesized by a variety of methods. However, one of the most popular
Molecules 2021, 26, 967. https://doi.org/10.3390/molecules26040967
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 967
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methods is the Fischer and Knoevenagel synthesis based on the reaction of
α
,
β
-unsaturated
ketones with phenylhydrazine in acetic acid under refluxing conditions. However, depend-
ing on the reactivity of molecules and the need of the chemist, they had synthesized the
pyrazolines under different solvent media and acidic or basic conditions [2–4].
Information on pyrazolinium structures with a quaternary nitrogen atom is limited.
Thus, two examples of biologically active pyrazolinium salts were found: 3-amino-1-ethyl-
1-phenyl-4,5-dihydro-1H-pyrazolinium iodide (PubChemCID: 13585073 structure, [5]) and
3-amino-1,4-dimethyl-1-phenyl-2-pyrazolinium iodide (PubChemCID: 13064197 struc-
ture, [6]) (Scheme 1):
Scheme 1. Amino-1-phenyl-4,5-dihydro-1H-pyrazolinium iodides.
In some works, we found spiropyrazolinium compounds with a quaternary nitro-
gen atom, which is common for two heterocycles. When studying the stability of 3-(2-
aminoethyl)-5-aryl-1,2,4-oxadiazoles having six-membered cyclic tertiary 2-amino groups
towards hydrolysis, we found that they were capable of rearranging to spiropyrazoline
benzoates or chlorides [7–9].
Particularly, upon keeping 3-[2-(4-phenylpiperazin-1-yl)ethyl]-5-phenyl-1,2,4-oxadiazole
recrystallized in 2-PrOH under conditions of air moisture access for nine months for
growing single crystals for X-ray structural analysis or by exposure of 3-[2-thiomorpholin-1-
yl)ethyl]-5-aryl-1,2,4-oxadiazoles in ethanol with ethereal HCl solution the above-mentioned
1,2,4-oxadiazoles underwent the rearrangement to spiropyrazolinium benzoates or chlo-
rides (Scheme 2) [7,8]:
Scheme 2. 3-(2-Aminoethyl)-5-aryl-1,2,4-oxadiazoles in the conditions of exposure to H2O and HCl.
Furthermore, at targeted exposure on 3-[2-(piperidin-1-yl)ethyl]-5-aryl-1,2,4-oxadiazoles
with: (i) water, (ii) water in DMF or (iii) ethereal HCl they underwent rearrangement with
the formation 2-amino-1,5-diazaspiro[4.5]dec-1-en-5-ium benzoates or chlorides (Scheme 2).
In the latter case, along with the formation of spiropyrazolinium chloride hydrate from
3-[2-(piperidin-1-yl)ethyl]-5-(3-chloro-phenyl)-1,2,4-oxadiazole hydrochlorides of starting
1,2,4-oxadiazoles were obtained as secondary products [9].
These facts are consistent with the known for 3,5-substituted 1,2,4-oxadiazoles with a
saturated side chain spontaneous thermally induced monomolecular Boulton–Katritzky

Molecules 2021, 26, 967
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rearrangement and provided the first examples of spirocompound formation through such
reaction. In general, a variety of Boulton–Katritzky rearrangements could be represented
as the following scheme (Scheme 3) [10,11]:
Scheme 3. Mononuclear heterocyclic Boulton-Katritzky rearrangement of 3,5-substituted
1,2,4-oxadiazoles.
In addition, spiropyrazolinium structures–2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-
5-ammonium arylsulfonates are formed at arylsulfochlorination of
(Scheme 4) [12].
β-aminopropioamidoximes
Scheme 4. Amino-4,5-dihydrospiropyrazolylammonium formation at arylsulfochlorination of β-
aminopropioamidoximes.
The practical interest in the class of
by their pronounced local anesthetic, antitubercular, and antidiabetic activities [13–16].
Herein we report on the stability of 5-aryl-3-[ -(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles
β-aminopropioamidoxime derivatives is supported
β
towards hydrolysis at: (i) DMF with the two equivalent amount of water when heated to
60–70 ◦C; (ii) alcohol/ethereal HCl mixture. A number of previously unknown spiropy-
razolinium salts were obtained and characterized using FT-IR and NMR spectroscopy
and X-ray diffraction. In vitro antitubercular screening of spiropyrazoline benzoates and
chloride was carried out, and their molecular docking was performed. It was shown
that the hydrolysis of 1,2,4-oxadiazoles with a 3-morpholinoethyl substituent leads to
spiropyrazoline compounds within 25–40 h. Acid hydrolysis of 1,2,4-oxadiazoles occurs
immediately after reagents adding. In vitro antitubercular screening of benzoates and chlo-
ride of spiropyrazoline drug-susceptible and multidrug-resistant strains, M. tuberculosis
revealed compounds with significant activity, and the results are in accordance with molec-
ular docking studies.
2. Results and Discussion
2.1. Synthesis and Spectra
The synthesis of the starting compounds
lier [17]. 5-Substituted phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles (4a
obtained by heating of О-aroyl-( -morpholin-1-yl)propioamidoximes (3a ) in DMF at
70 ◦C for several hours, evaporating off the solvent in an oil pump vacuum and treating of
1,
2a–
e
,
3a
–
e
and 4a
–e
was described ear-
–
e) were
β
–e
the residue with acetone. 1,2,4-Oxadiazoles (4a–e) are obtained as crystalline precipitates
from acetone (Scheme 5).

Molecules 2021, 26, 967
4 of 16
Scheme 5. Obtaining of 5-substituted phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles (4a–e), and 2-amino-8-oxa-1,5-
diazaspiro[4.5]dec-1-en-5-ium benzoates and сhloride (5a–e, 6).
Physicochemical, FT-IR and NMR spectral characteristics of representatives of 1,2,4-
oxadiazoles of the morpholine series (4a ) were recorded immediately after isolation
from DMF, and they correspond to the structure of 1,2,4-oxadiazoles. However, the X-ray
diffraction analysis of single crystals grown for nine months from 1,2,4-oxadiazoles 4c
recrystallized from 2-PrOH showed a complete transition of 1,2,4-oxadiazoles into rear-
ranged spiropyrazolinium compounds 5c (Section 2.3.). This indicates the hydrolysis of
1,2,4-oxadiazoles 4a by way of Boulton–Katritsky rearrangement to spiropyrazolinium
compounds 5a under the influence of air moisture. The rearrangement products 5a
–
e
–
e
–e
–e
–
e
–e
have increased values of the mobility index Rf and m.p. in comparison with the initial
compounds 4a–e (Table 1).
Table 1. Physicochemical data of 5-substituted phenyl-3-[
β-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles (4a–e) and spiropy-
razolinium compounds (5a–e) fixed in the conditions for obtaining single crystals for XRD analysis and in condition
(i).
◦
◦
Compd
X
Yield,%
m.p., C
Rf *
Compd
t, h **
Yield,%
m.p., C
Rf *
4a
4b
4c
4d
4e
p-CH3O
p-CH3
H
p-Br
m-Cl
62
70
92
83
56
230
220
216
224
190
0.71
0.62
0.66
0.67
0.62
5a
5b
5c
5d
5e
40
40
25
25
25
63
63
77
80
43
248
235
220
240
200
0.80
0.75
0.75
0.77
0.70
* Rf determined in the system ethanol:benzene, 3:1; ** the heating time of 1,2,4-oxadiazoles 4a–e in DMF + 2H2O.
Further in this work, we investigated the conditions for the Boulton–Katritsky re-
arrangement of 5-substituted phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles (4a
–e)
under the deliberate establishment of hydrolysis conditions: (i) DMF with the two equiva-
lent amount of water when heated to 60–70 ◦C; (ii) alcohol/ethereal HCl mixture in the
presence of air moisture (Scheme 5).
As can be seen from Table 1, the heating time has an increased value (40 h) for
electron-donor substituents in the phenyl ring of 1,2,4-oxadiazoles 4a
with 1,2,4-oxadiazoles with an unsubstituted phenyl ring and with a phenyl ring having
electron-withdrawing substituents—4c (25 h). Spiropyrazolinium compounds 5a were
obtained after evaporation of DMF in an oil pump vacuum, treatment of the residue with
acetone with the isolation of rearranged products 5a and their recrystallization from
2-PrOH. In the case of the action of ethereal HCl solution on alcohol solutions of 1,2,4-
oxadiazoles 4a in all cases, 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium chloride (
and the corresponding benzoic acids were isolated.
, 4b in comparison
–
e
–e
–
e
–
e
6)

Molecules 2021, 26, 967
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IR spectra view of spirocompounds 5a
–
e
and
6
differ from the IR spectra of 1,2,4-
oxadiazoles 4a–e. First, the former compounds have symmetric and asymmetric ν(N–H)
stretching bands at 3152–3485 and 3158–3457 cm−1, respectively; second, there are pro-
nounced bands of asymmetric and symmetric stretching vibrations of strong intensity
ν
(CОО–) at 1545–1557 cm−1 and 1420–1442 cm−1, respectively for 5a
–
e
and no stretching
bonds of aromatic protons for salt 6.
The 1H-NMR spectra of 1,2,4-oxadiazole 4a
–e were recorded immediately with isola-
tion; if they were recorded after 1–2 weeks, then the emergence and increase in the intensity
of the NH2 group signal of the rearranged spiropyrazolinium products 5a in the region
of 7.51–7.7.57 ppm was observed. It indicates a transition of 1,2,4-oxadiazole to the
spiropyrazolinium compounds 4a–e→5a–e in the presence of air moisture.
Comparison of the NMR spectra (1H and 13C) of compounds 4a
and 5a
most no differences in the regional characteristics of the groups of protons and carbon atoms
of the structures of 5-substituted phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles (4a
–
e
δ
–e
–e shows al-
–
e
) and of 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium benzoates (5a
tons of -CH2 and -CH2 groups in the first case give signals in the range
and 3.65–3.66 ppm and in the region 3.15–3.19 ppm and
The signals of the aromatic protons of para-substituted 1,2,4-oxadiazoles 4a
spiropyrazolinium benzoates 5a 5b 5d have the form of two symmetric doublets with the
–
δ
e
). Hence, pro-
α
β
3.15–3.17 ppm
δ
δ
δ
3.64–3.66 ppm—in the second.
4b 4d and
,
,
,
,
spin–spin coupling constant J equal 7.5 and 8.0 Hz. Aromatic protons of 1,2,4-oxadiazoles
and spiropyrazolinium benzoates with unsubstituted and meta-substituted phenyl rings
have signals at
δ 7.23–7.83 ppm (4c) and 7.22–7.80 ppm (4e) and 7.25–7.78 ppm (5c, 5e).
Proton-containing substituents CH3O and CH3 have singlet signals with an intensity of 3
protons at δ 3.73 and 2.27 ppm for compounds 4a, 4b and 5a, 5b.
A distinctive feature of 1H-NMR spectra of benzoates 5a
–e
from the spectra of 1,2,4-
oxadiazoles 4a
–
e
is the presence of NH2 proton signal with the integral intensity of 2H at
δ
7.51–7.57 ppm.
1
The H-NMR spectrum of spirocompound
6
contained the triplet proton signals
3.16 and 3.68 ppm and signals of N(+)(CH2)2(CH2)2Оand
δ 3.40 and 3.92 ppm, respectively; no aromatic proton signals
of α- and β-CH2 groups at δ
N(+)(CH2)2(CH2)2Оgroups at
were observed.
Of the remarkable features of the 1H-NMR spectra of compounds 4a
–e, 5a–e, and 6,
is that the axial and equatorial protons of the methylene groups located at the nitrogen
atom of the morpholine ring give independent multiplet signals. In one case, these signals
are superimposed with the common signal of two groups methylene protons located at
the oxygen atom of the morpholine ring at
protons and in the other case have a multiplet signal at ~
protons. The diastereotopicity of discussed geminal protons of compounds 4a
is associated with a dynamic cause due to slow rotation of the morpholine heterocycle.
δ
3.91–3.93 ppm with a total intensity of six
3.40 ppm intensity of two
5a , and
δ
–e
,
–e
6
The effect of hindered inversion of six-membered heterocycles, with a chair-like conformer
with fixed positions of the axial and equatorial protons being predominant, in the 1H-NMR
spectra is a known fact reported in reference data [18]. In addition, the diastereotopicity of
these geminal protons of compounds 5a–e and 6 is associated with asymmetry due to the
presence of the spirocyclic system.
In the 13C spectra of the compounds 4a
aromatic protons were recorded in the expected regions.
So, in the 13C NMR spectra the characteristic groups of compounds 4a
include the signals of: -methylene groups at 31.4 ppm; -methylene groups at
62.1 ppm; signals of carbon atoms of methylene groups with intensity 2C located at nitrogen
and oxygen atoms of the heterocycle are in the regions 62.4–62.5 ppm and δ 63.2–63.3 ppm
Two signals of carbon atoms of C=N bonds of 1,2,4-oxadiazoles 4a are in the regions
167.0–168.9 ppm and 169.2–169.5 ppm. The carbon atoms of the C=N bond of the
pyrazoline ring of compounds 5a have two signals at 167.0–168.8 ppm and δ 169.2 ppm,
which may be due to the existence of enantiomers A and B. The carbon atom of the C=N
–e, 5a–e and 6, all signals of aliphatic and
–e, 5a–e and
6
α
δ
β
δ
δ
.
–
e
δ
δ
–e
δ

Molecules 2021, 26, 967
6 of 16
bond of compound
atoms for compounds 4a
para-CH3 groups of the compounds 4a
55.4 ppm and δ 21.3 ppm.
6
has a chemical shift at
and 5a
δ
169.1 ppm. The signals of aromatic carbon
–
e
–
e
are in the range
5a and 4b
δ
112.5–161.0 ppm; para-CН3Оand
,
,
5b, respectively, give signals at δ
As we have proved in this article, 1,2,4-oxadiazoles 4a
ically (FT-IR and NMR spectral data), and they are the initial ones during hydrolysis to
pyrazolinium compounds. The Boulton–Katritsky rearrangement mechanism 4a
e→5a
and 4a
e→6 can be represented as a sequence of protonation, proton transfer and nucle-
ophilic attack steps, representing hydrolysis during the reaction of 1,2,4-oxadiazoles 4a
with water and wet HCl in the same way as we indicated for piperidine derivatives [9].
–e are recorded spectroscop-
–
–e,
–
–e
All these data demonstrate that the biological activity of compounds under discussion
should be associated with their spiropirazolinium form.
2.2. In Vitro Antitubercular Screening of 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium
Benzoates and Chloride (5a–e, 6)
In vitro antitubercular bacteriostatic activity of spiropyrazolinium compounds 5a
–e
and
6 on drug-sensitive (DS) and multidrug-resistant (MDR) of M. tuberculosis (MTB)
strains was studied using the method of serial dilution on the liquid Shkolnikova medium
(Table 2).
Table 2. In vitro antitubercular activity (MIC) of 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium benzoates and chloride
(5a–e, 6), µg/mL * on DS (H37Rv) and MDR (I) strains of MTB.
Compd
5a
5b
5c
5d
5e
6
Rifampicin
MIC,
µg/mL
H37Rv
I
50
50
10
50
20
50
100
100
100
100
1
2
1
2
* In the upper line of MIC values, the activity on the DS of MTB strains (H37Rv) is shown; in the lower–on the wild MDR strains (I), isolated
from the patient, resistant to rifampicin and isoniazid.
A number of compounds 5a
–
e
on DS and MDR MTB strains exhibits an average
g/mL. Moreover, an improvement in activity is observed
g/mL (5b); 20 g/mL (5c) and 50 g/mL (5a) on the
g/mL on the MDR MTB strains for the compounds 5a
containing donor substituents in the phenyl ring or with an unsubstituted phenyl ring
Spiropyrazolylammonium chloride demonstrates high in vitro antitubercular activity
antitubercular activity of 10–100
with a decrease in MIC to 10
µ
µ
µ
µ
DS MTB strains and up to 50
µ
–c
.
6
equal to the activity of the basic antitubercular drug of the first-row rifampicin: on the DS
strain as low as 1 µg/mL; on the wild MDR strain—2 µg/mL.
To rationalize the results of the antitubercular activity of 2-amino-8-oxa-1,5-diazaspiro
dec-1-en-5-ium salts, X-ray diffraction studies and molecular docking studies were carried out
.
2.3. X-ray Diffraction
Molecular structures of compounds 5c
–e and 6 are given in Figure 1. Asymmetric units
of 5c
,
5e and contain one water molecule besides the target ions, and the asymmetric unit
6
of 5d contains two cations and two symmetrically independent anions. All hydrogen atoms
could be located on residual density maps; thus, it was confirmed that the structures are
salts with deprotonated carboxylic acids. The six-membered oxo-containing cycles adopt
the chair conformation, and the five-membered aza-containing cycles realize the envelope
conformation. C(1) carbon atom deviates from the meanplane of N1=N2-C3-C2 atoms at
0.40(1)–0.49(1) Å. Although the cation is rather rigid, it can realize two conformational
isomers depending on the shift of the C(1) atom from the meanplane of the rest atoms of
the five-membered ring. In crystals of 5c
, 5e and 6, these isomers are related with each
other by an inversion center, and in acentric crystal 5d, two symmetrically independent
cations realize different conformations (Figure 2a). Nevertheless, for cations in 5c–e and 6
with similar conformations, the mean atomic deviation doesn0t exceed 0.5Å (Figure 2b).
The positive charge on quaternary ammonium atom causes elongation of N(1)-N(2) and

Molecules 2021, 26, 967
7 of 16
N(1)-C(1) bonds as it was previously demonstrated for 5-aryl-3-[2-(piperidin-1-yl)ethyl]-
1,2,4-oxadiazoles [
9], 2-amino-8-thia-1-aza-5-azoniaspiro[4.5]dec-1-ene [7], 1-(tert-butyl)-
4,5-dihydro-1H-pyrazol-1-ium [19] and 1,1,3-trimethyl-∆2-pyrazolinium [20] analogs.
Figure 1. Molecular view of 5c–e and 6 depicted in thermal ellipsoids (p = 50%).
Figure 2. Molecular conformations of (
a) two independent cations in 5d (blue and red; H atoms are omitted); (b) similar
conformations of cations in 5c (red), 5d (blue), 5e (green) and 6 (magenta).
Crystal packing of a molecule can give valuable information about the most abundant
intermolecular interactions of a polytopic molecule. The discussed cations can act as donors
of two hydrogen bonds and, theoretically, acceptors of three H-bonds (through O(1), N(2)
and N(3) atoms). However, the Full Interaction Maps tool [21
,22] implemented within the
Mercury 2020.1 package [23] undoubtedly indicates the “inertness” of this cation as an
acceptor of H-bonds (Figure 3). A monocarboxylate can only be an acceptor of 2–4 H-bonds
.
Thus, in the absence of water molecules, the cations and anions form simple H-bonded
chains (Figure 4) [9]. In 5d, these chains are further connected through halogen C-Br..O
interactions (Figure 4a). In crystals of 5c
,
5e, and 6, water molecules act as linkers between
−
the anion and cation and additionally bind two chains through O-H...O(anion) or O-H...Cl
interactions (Figure 5) so that topologically identical H-bonded chains of the ladder-type
are formed.

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Figure 3. Interaction map in the crystal of 6 probed with H-bond donors (blue) and H-bond acceptors (red).
Figure 4. Fragment of H-bonded chains in the crystal of (a) 5d and (b) 2-amino-1,5-diazaspiro[4.5]dec-1-en-5-ium
4-bromobenzoate (deposit CCDC 1496456). Color code: Br—brown, C—grey, H—white, N—blue, O—red. The H(C)
atoms are omitted. Intemolecular interactions are shown with dotted lines.
Figure 5. Fragment of H-bonded chains in the crystal of (
a) 5c, (b) 5e, (c) 6. Color code: C—grey, Cl—green, H—white,
N—blue, O—red. The H(C) atoms are omitted. Intemolecular interactions are shown with dotted lines.
To sum up, X-ray diffraction data demonstrated that molecular docking studies
should be carried out for two molecular conformations and that for stable ligand–receptor
complexes, the most likely hydrogen bond occurs through the NH2 group, while acceptor
atoms of heterocycles less readily take part in the hydrogen bonds.
2.4. Molecular Docking Studies
Our results indicate that compounds 4a
–e at neutral pH in aqueous solutions undergo
transformation to 5a , and antitubercular activity demonstrated by the latter compounds
–
e
can be referred to either anion of benzoic acids, or the cation, or a mixture of an anion
and cation. Some benzoic acids and their anions previously showed activity against
M. tuberculosis in vitro [24–26]. However, it was assumed that benzoic acids do not have
a specific cellular target for M. tuberculosis besides their general effect on disrupting the
membrane function [25], which is supported by X-ray data of some benzoic acid deriva-
tives with receptors, where ligands are situated on the protein surface [see, for example,

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PDB id 5TJZ, 5TJY, 2HRG]. Moreover, the activity of 5a
–
e
decreased in comparison with
monocarboxylate-free salt
6
allows suggesting that the antitubercular activity of these salts
should be referred to as the cation mainly.
As we mentioned above, the cation has a rigid conformation with spirocyclic motifs
known to be present in some drugs [27,28]. Thus, the mutual disposition of donor, acceptor
and hydrophobic fragments of this molecule can be compared with previously X-rayed
drugs and ligand–receptor complexes. We used CSD-Crossminer 2020.1 package to search
in the Protein Data Bank [29] ligand–receptor complexes where a ligand contains an
(i) planar heterocycle with (ii) donor amine group involved in two H-bonds and (iii)
neighboring ring, and a receptor was obtained from M. tuberculosis. The search gave three
hits with PDB id codes 1NBU, 4FOG, and 4XT4, and R.M.S.D. of pharmacophore model
from experimental ligand–receptor complexes of 0.398–0.717 Å. For all these hits,2-amino-
8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium emulates the pterin moiety, known as a
precursor of tetrahydrofolate synthesis, a coenzyme that acts as a carrier for one-carbon
units in the biosynthesis of thymidylate, purine nucleotides, and some amino acids [30].
Receptors for these complexes include dihydroneopterin aldolase, thymidylate synthase
or oxidoreductase Rv2671. The second search was carried out for structural analogs of
spiro[4.5]decane in complexes with receptors obtained from M. tuberculosis. The second
search gave two hits (PDB codes 5ICJ and 5N7O) of M. tuberculosis regulatory protein with
1-oxa-2,8-diazaspiro[4.5]dec-2-en-8-yl derivatives. These receptors were taken as targets
for molecular docking calculations together with more typical for theoretical antitubercular
screening UDP-galactopyranose mutase.
For all cases, both stereoisomers of the cation were docked independently. Docking
without constraints does not give any ligand: receptor complexes; thus, docking was
performed with H-bond constraints similar to that in the initial ligand–receptor complexes.
Sterical clashes in the binding pocket of thymidylate synthase and UDP-galactopyranose
mutase (PRB id codes, respectively, 4FOG and 4RPJ) do not allow the cation to realize
appropriate H-bonding; thus, these receptors were excluded from consideration. For 1NBU
and 4XT4, we succeeded in overcoming some steric clashes when flexible residues were
allowed to rotate freely along with single bonds. Energies of interactions between the
cations and three receptors are listed in Table 3; the closest environment of the cation is
schematically represented in Figure 6.
Table 3. Energies of interactions for the 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium
complexes with receptors [kcal/mol].
Receptor
Ligand
1NBU
4XT4
5ICJ
Type A
Type B
Type A
Type B
Type A
Type B
H-bonds
Electrostatic
Nonpolar
Repulsive
Total
−3.00
−4.37
−35.72
0.25
−2.00
−1.68
−32.51
0.35
−3.12
−6.34
−31.93
0.99
−2.00
−5.80
−34.80
0.36
−2.79
−0.51
−38.43
0.04
−3.00
−2.12
−35.43
0.01
−42.84
−35.84
−40.40
−42.24
−41.69
−40.54
Overall, energies of ligand–receptor interactions vary from
with the most prominent contribution from nonpolar C-H...
are close to the value of intermolecular pairwise interactions of the same cation in the
crystal of 35.3 kcal/mol) estimated using UNI potentials [31 32]. The cation within
−
35.84 to
interactions. These values
−
42.84 kcal/mol,
π
6
(
−
,
binding pockets is involved in two or three hydrogen bonds, and for dihydroneopterin
aldolase and thymidylate synthase complexes, the best solution for molecules type A and
type B differs in H-bonding patterns. For 1NBU, the best docking solution for molecule
type A includes three hydrogen bonds (two N-H...O interactions of amine and oxygen
atoms of Tyr52 and Glu74 residues, additionally supported with N-H...N bonds between
amide of Asn44 and heterocycle), while the solution for type B molecules includes two
H-bonds (N-H...O between amine and Tyr52 and N-H...O between ammonium of Lys99
and the oxygen atom of the cation). The latter solution with the poorest system of H-bonds

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also has the highest energy of intermolecular interactions among the six solutions under
discussion. For 4XT4, both isomers of cation form similar H-bonds with amide group
of Asn44 and carboxylate group of Asp67, and interact not only with receptor but also
cofactor. The nature of ligand-cofactor interactions differs for two isomers, as well as the
system of H-bonds. Only in 5ICJ, both isomers realize nearly similar ligand–receptor
interactions through two H-bonds between hydrogen atoms of NH2 group, and OH group
of Ser134 and COO group of Asp168 and numerous hydrophobic interactions with indolyl
fragment of Trp100 additionally supported by hydrophobic interactions with Thr138 and
Leu167. To summarize, based on molecular docking calculations, the cation is able to
bind with M. tuberculosis dihydroneopterin aldolase, thymidylate synthase and regulatory
proteins of transcription. Only in the latter case binding energy, the system of H-bonds and
contributions of various types of interactions are independent on stereoisomer of the cation.
However, other studies are needed to reveal the mechanism of antitubercular activity of
compounds under discussion.
Figure 6. Schematic representation of the interactions between 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium
and the residues within binding pockets of 1NBU (type A ( ), type B ( )), 4XT4 (type A ( ) and type B ( )), and 5ICJ ( ). The
a
b
c
d
e
colors of the residues indicate the character of bonding: pink correspond to H-bonds, green—hydrophobic interactions,
blue—electrostatic interactions. H-bonds and their distances are depicted with dotted lines. Left—column corresponds to
type A cations, and the right—to type B molecules.

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3. Materials and Methods
3.1. Synthesis
The reagents were purchased from different chemical suppliers and were purified
before use. FT-IR spectra were obtained on a Thermo Scientific Nicolet 5700 FTIR in-
1
strument (Waltham, MA USA) in KBr pellets. H- and 13C-NMR spectra were acquired
on a Bruker Avance III 500 MHz NMR spectrometer (500 and 126 MHz, respectively)
(Bruker, BioSpin GMBH, Rheinstetten, Germany). The signals of DMSO-d6 were used as
the internal reference for 1H-NMR (2.50 ppm) and 13C-NMR (39.5 ppm) spectra. Elemental
analysis was carried out on a CE440 elemental analyzer (Exeter Analytical, Inc., Shang-
hai, China). Melting points were determined in glass capillaries on a PTP(M) apparatus
(Khimlabpribor, Klin, Russia). The reaction progress and purity of the obtained products
were controlled using Sorbfil (Sorbpolymer, Krasnodar, Russia) TLC plates coated with
CTX-1A silica gel, grain size 5–17 µm, containing UV-254 indicator. The eluent for TLC
analysis was a mixture of benzene–EtOH, 1:3. The solvents for synthesis, recrystallization,
and TLC analysis (ethanol, 2-PrOH, benzene, DMF, acetone) were purified according to the
standard techniques.
3.1.1. A General Procedure for the Synthesis of
5-Aryl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles (4a–e)
A solution of О-aroyl-(
a ratio of 1 g:5 mL, respectively, was heated on an oil bath at 70 C for different times
β
-morpholin-1-yl)propioamidoximes ◦(3a
–e) in dry DMF in
characteristic for compounds 3a–e: 3.5 h (3a, 3b), 2.5 h (3c), 2 h (3d), 1.5 h (3e) with TLC
control (eluent was mixture benzene—EtOH, 1:3). The reaction mixture was evaporated to
dryness under an oil pump vacuum at 50 ◦C/1 mm Hg. The organic residue was treated
with dry acetone at a ratio of 1 g of parent compounds (3a–e): 5 mL of dry acetone. The
obtained crude precipitates of the compounds 4a–e were recrystallized from 2-PrOH.
5-(4-Methoxyphenyl)-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazole (4a). Starting from (4.93 g
,
0.01◦6 mol) of 3a in 25 mL of dry DMF to the resulting 2.89 g (62%), colorless solid 4a, m.p.
230 C, Rf 0.71. IR (KBr, cm–1): 1670 (C=N), 1597 (C=N), 1553 (C=C), 1362 (C–O). 1H-NMR
(500 MHz, DMSO-d6): 3.17 (t, J = 7.0 Hz, 2H, CH2CH2N), 3.40 [m, 2H, N(CH eq)2] and
3.93 [m, 6H (2H, N(CH ax)2 and 4H, O(CH2)2)], 3.65 (t, J = 7.0 Hz, 2H, CH2CH2N), 3.73 (s,
3H, p-CH3O), 6.75 (d, J = 8.0 Hz, 2H, o-H Ar), 7.75 (d, J = 8.0 Hz, 2H, m-H Ar). 13C-NMR
(
126 MHz, DMSO-d6): 31.4, 55.4, 62.0, 62.4, 112.5, 130.9, 136.0, 160.0, 168.9, 169.3. Anal.
Calcd for C15H19N3O3 (289,33): C, 62.27; H, 6.62. Found: C, 62.60; H, 7.02.
5-(4-Tolyl)-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazole (4b). Starting from (2.81 g, 0.0096 mol
of 3a in 15 mL of dry DMF to the resulting 1.84 g (70%), colorless solid 4b, m.p. 220 ◦C, Rf
0.62. IR (KBr, cm–1): 1648 (C=N), 1593 (C=N), 1553 (C=C), 1376 (C–O). 1H-NMR (500 MHz,
DMSO-d6):2.27 (s, 3H, p-CH3), 3.15 (t, J = 7.0 Hz, 2H, CH2CH2N), 3.40 [m, 2H, N(CHeq)2]
and 3.91 [6H, m: 2H, N(CH ax)2 and 4H, O(CH2)2], 3.66 (t, J = 7.0 Hz, 2H, CH2CH2N),
7.01 (d, J = 8.0 Hz, 2H, o-H Ar), 7.70 (d, J = 8.0 Hz, 2H, m-H Ar). 13C-NMR (126 MHz,
DMSO-d6): 21.3, 31.4, 62.1, 62.4, 63.2, 112.5, 130.9, 136.0, 160.0, 169.0, 169.3. Anal. Calcd for
C15H19N3O2 (273,33): C, 65.91; H, 7.01. Found: C, 65.50; H 7.22.
)
5-Phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazole (4c). Starting from (1.00 g, 0.0036 mol
с in 5 mL of dry DMF to the resulting 0.86 g (92%) colorless solid 4c, m.p. 216 ◦C, Rf
0.66. IR (KBr, cm–1): 1650 (C=N), 1596 (C=N), 1559 (C=C), 1381 (C–O). 1H-NMR (500 MHz
)
of
3
,
DMSO-d6): 3.18 (t, J = 7.0 Hz, 2H, CH2CH2N), 3.40 [2H, m, N(CHeq)2] and 3.93 [6H, m: 2H,
N(CHax)2 and 4H, O(CH2)2], 3.65 (2H, t, J = 7.0 Hz, CH2CH2N), 7.23–7.83 (m, 5H, C6H5).
13C-NMR (126 MHz, DMSO-d6): 31.4, 62.1, 62.4, 63.3, 127.5, 127.8, 128.9, 129.1, 129.4, 167.0,
169.2. Anal. Calcd for C14H17N3O2 (259,30):C, 64.85; H, 6.61. Found: C, 65.30; H, 7.02.
5-(4-Bromophenyl)-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazole (4d). Starting from (1.44 g,
0.00◦4 mol) of 3d in 8 mL of dry DMF to the resulting 1.12 g (83%) colorless solid 4d, m.p.
224 C, Rf 0.67. IR (KBr, cm–1): 1650 (C=N), 1596 (C=N), 1559 (C=C), 1381 (C–O). 1H-NMR
(500 MHz, DMSO-d6): 3.17 (t, J = 7.0 Hz, 2H, CH2CH2N), 3.39 [m, 2H, N(CHeq)2] and
3.93 [m, 6H: 2H, N(CH ax)2 and 4H, O(CH2)2], 3.65 (2H, t, J = 7.0 Hz, CH2CH2N), 7.40 (d,

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J = 7.5 Hz, 2H, o-H Ar), 7.74 (d, J = 7.5 Hz, 2H, m-H Ar). 13C-NMR (126 MHz, DMSO-d6):
31.4, 62.1, 62.4, 63.3, 122.7, 130.2, 131.6, 141.5, 168.3, 169.5. Anal. Calcd for C14H16BrN3O2
(338.20): C, 49.72; H, 4.77. Found,%: C, 49.29; H, 4.97.
5-(3-Chlorophenyl)-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazole (4e). Starting from (2.70 g
,
,
0.0087 mol) of 3e in 15 mL of dry DMF to the resulting 1.43 g (56%) colorless solid 4e
◦
m.p. 190 C, Rf0.62. IR (KBr, cm–1): 1680 (C=N), 1596 (C=N), 1557 (C=C), 1377 (C–O).
1H-NMR (500 MHz, DMSO-d6): 3.15 (t, J = 7.0 Hz, 2H, CH2CH2N), 3.40 [m, 2H, N(CHeq)2]
and 3.91 [m, 6H: 2H, N(CHax)2 and 4H, O(CH2)2], 3.65 (t, J = 7.0 Hz, 2H, CH2CH2N),
7.25–7.78 (m, 4H, 3-Cl-C6H4). 13C-NMR (126 MHz, DMSO-d6): 31.4, 62.1, 62.4, 63.2, 127.7,
127.8, 129.2, 132.4, 144.5, 162.1, 169.2. Anal. Calcd for C14H16ClN3O2 (293.75): C, 57.24; H,
5.49. Found,%: C, 57.55; H, 5.92.
3.1.2. A General Procedure of the Formation of
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium benzoates (5a–e)
To the solutions of 5-(3,4-substituted phenyl)-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles
( –e) in DMF in a ratio of 0.5 g: 10 mL, respectively, two equivalents of H2O was added
4a
dropwise. The reaction solutions were heated at 70 ◦C with TLC control during the time
typical for groups of compounds 4a 4a (40 h), 4c (25 h).After the disappearance
of the 1,2,4-oxadiazoles 4a spots on the silufol plate and the appearance of products
5a spots, the solvent was evaporated to dryness in an oil vacuum pump. Acetone was
added to the viscous residues of the reaction mixtures 5a at the rate of 0.5 g of the
starting compounds 4a : 15 mL of acetone. The formed precipitates of 2-amino-8-oxa-1,5-
–
e
:
,
b
–e
–
e
–e
–
e
–e
diazaspiro[4.5]dec-1-ene-5-ammonium 3,4-substituted benzoates (5a–e) were filtered off
and dried at room temperature.
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium 4-methoxybenzoate hydrate (5a).
Starting from 0.5 g (0.0017 mol) of compound 4a in 10 mL of DMF and 0.062 mL (0.0034 mol
H2O to the resulting 0.35 g (63%) colorless solid 5a, m.p. 235 ◦C, Rf 0.75. IR (KBr, cm–1):
1657 (C=N), 1594 (C=C), 1548 ( N(-H)2].
COO– as), and 1420 ( COO– sy), 3309, 3414, 3459 [
1H-NMR (500 MHz, DMSO-d6): 2.27 (s, 3H, p-CH3O), 3.17 [t, J = 7.0 Hz, 2H, CH2CH2N(+)],
3.41 [m, 2H, N(CHeq)2] and 3.92 [m, 6H: 2H, N(CHax)2 and 4H, O(CH2)2], 3.65 [t, J = 7.0 Hz
)
ν
ν
ν
,
2H, CH2CH2N(+)], 6.78 (d, J = 8.0 Hz, 2H, o-H Ar), 7.55 and 7.60 (s, 2H, NH2), 7.78 (d,
J = 8.0 Hz, 2H, m-H Ar). 13C-NMR (126 MHz, DMSO-d6): 31.4, 62.4, 63.2, 112.5, 130.9, 134.8,
159.9, 144.5, 168.8, 169.2. Anal. Calcd for C15H23N3O5 (325,36): C, 55.37; H, 7.13. Found,%:
C, 55.79; H, 7.57.
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium 4-methylbenzoate hydrate (5b).
Starting from 0.5 g (0.0018 mol) of the compound 4b in 10 mL of DMF and 0.066 mL
(
0.0036 mol) H2O to the resulting 0.35 g (63%) colorless solid 5b, m.p. 248 ◦C, Rf 0.80. IR
(KBr, cm–1): 1655 (C=N), 1597 (C=C), 1550 (COO–as), and 1442 (COO– sy), 3157, 3312, 3457
1
[N(-H)2]. H-NMR (500 MHz, DMSO-d6): 3.15 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 3.41 [m,
2H, N(CHeq)2] and 3.93 [m, 6H: 2H, N(CHax)2 and 4H, O(CH2)2], 3.64 [t, J = 7.0 Hz, 2H,
CH2CH2N(+)], 3.73 (s, 3H, p-CH3), 7.02 (d, J = 8.0 Hz, 2H, o-H Ar), 7.54 (s, 2H, NH2), 7.69
(d, J = 8.0 Hz, 2H, m-H Ar). 13C-NMR (126 MHz, DMSO-d6): 21.3, 31.38, 62.1, 62.4, 63.2,
127.9, 128.3, 129.4, 139.7, 168.8, 169.2. Anal. Calcd for C15H23N3O4. (309,36): C, 58.24; H,
7.49. Found: C, 58.29; H, 7.59.
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium benzoate hydrate (5c). Starting
from 0.5 g (0.0019 mol) of 4c in 10 mL of DMF and 0.07 mL (0.0038 mol) H2O to the resulting
◦
0.43 g (77%) colorless solid 5c, m.p. 220 C, Rf 0.75. IR (KBr, cm–1):1658 (C=N), 1596 (C=C),
1556 (COO– as), and 1426 (COO– sy), 3152, 3311, 3456 [N(-H)2]. 1H-NMR (500 MHz
,
DMSO-d6): 3.19 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 3.39 [m, 2H, N(CHeq)2] and 3.93 [m, 6H:
2H, N(CHax)2 and 4H, O(CH2)2], 3.65 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 7.22–7.80 (m, 5H,
C6H5), 7.55 and 7.64 (s, 2H, NH2). 13C-NMR (126 MHz, DMSO-d6): 31.4, 61.3, 62.4, 63.2,
127.26, 128.3, 129.4, 142.4, 168.7, 169.2. Anal. Calcd for C14H21N3O4 (295,33): C, 56.94; H,
7.17. Found: C, 56.79; H, 7.50.
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium 4-bromobenzoate (5d). Starting
from 0.5 g (0.0015 mol) of 4d in 10 mL of DMF and 0.05 mL (0.0028 mol) H2O to the

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resulting 0.43 g (80%) colorless solid 5d, m.p. 240 ◦C, Rf 0.77. IR (KBr, cm–1): 1657 (C=N),
1
1591 (C=C), 1545 (COO– as), and 1423 (COO– sy), 3302, 3411, 3485 [N(-H)2]. H-NMR
(500 MHz, DMSO-d6): 3.15 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 3.40 [m, 2H, N(CHeq)2] and
3.92 [m, 6H: 2H, N(CHax)2 and 4H, O(CH2)2], 3.65 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 7.39
(d, J = 7.5 Hz, 2H, o-H Ar), 7.55 (s, 2H, NH2), 7.73 (d, J = 7.5 Hz, 2H, m-H Ar). 13C-NMR
(
126 MHz, DMSO-d6): 31.4, 62.1, 62.4, 63.2, 121.9, 130.1, 131.6, 141.8, 167.4, 169.2. Anal.
Calcd for C14H18BrN3O3 (356,22): C, 47.20; H, 5.09. Found: C, 47.49; H, 4.97.
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium 3-chlorobenzoate hydrate (5e). Start-
ing from 0.5 g (0.0017 mol) 4e in 10 mL of DMF and 0.06 mL (0.034 mol) H2O to the resulting
◦
0.24 g (43%) colorless solid 5e, m.p. 200 C, Rf 0.70. IR (KBr, cm–1): 1657 (C=N), 1594
1
(C=C), 1557 (COO– as), and 1420 (COO– sy), 3155, 3309, 3457 [N(-H)2]. H-NMR (500 MHz,
DMSO-d6): 3.16 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 3.40 [m, 2H, N(CH eq)2] and 3.93 [6H, m:
2H, N(CHax)2 and 4H, O(CH2)2], 3.66 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 7.25–7.78 (m, 4H,
3-Cl-C6H4), 7.57 and 7.61 (s, 2H, NH2). 13C-NMR (126 MHz, DMSO-d6): 31.4, 62.1, 62.5,
63.2, 127.9, 128.2, 129.2, 129.3, 132.4, 144.7, 167.0, 169.2. Anal. Calcd for C14H20ClN3O4
(329,78): C, 50.99; H, 6.11. Found: C, 50.52; H, 6.47.
3.1.3. A General Method of the Formation of
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium chloride (6) and Substituted
Benzoic Acids
To a solution of 0.2 g (0.0007 mol) of 5-(3,4-substituted phenyl)-3-[2-(morpholin-1-
yl)ethyl]- 1,2,4-oxadiazoles (4a
dropwise to pH 2. A white precipitate of 2-amino-1,5-diazaspiro[4.5]dec-1-en-5-ium chlo-
ride hydrate ( ) in all cases was formed at once in 75–82% yields and collected by filtration.
3,4-Substituted benzoic acids were precipitated during the evaporation of mother liquors
–e) in 10 mL ethanol a HCl solution in ether was added
6
obtained after the filtration of chloride monohydrate
benzoic acids corresponded to the reference data.
6. All characteristics of substituted
2-Amino-8-oxa-1,5-diazaspiro[4.5]dec-1-ene-5-ammonium chloride hydrate (
6
)—white opaque
N(–H)2], 1362
◦
powder, m.p. 257–260 C, Rf 0.21. IR (KBr, cm–1): 1639 (
ν
C=N), [1615, δ
1
(
ν
C–O), 3158, 3310, 3457 [
ν
N(-H)2]. H-NMR (500 MHz, DMSO-d6): 3.16 [t, J = 7.0 Hz, 2H,
CH2CH2N(+)], 3.40 [m, 2H, N(CH eq)2] and 3.92 [m, 6H: 2H, N(CH ax)2 and 4H, O(CH2)2],
3.68 [t, J = 7.0 Hz, 2H, CH2CH2N(+)], 7.51 (s, 2H, NH2). 13C-NMR spectrum,
δ, ppm: 31.4,
31,5, 62.1, 62.4, 63.3, 169.2. Anal. Calcd for C7H16N3O2 (209.67): C, 40.10, H 7.69. Found: C,
40.29; H, 7.97.
3.2. Single-Crystal X-ray Diffraction
The X-ray diffraction data of 5c
diffractometer (Bruker AXS, Inc., Madison, WI, USA) equipped with an Oxford Cryostream
cooling unit and a graphite monochromated Mo anode ( = 0.71073 Å). Crystal structures
–e, 6 were collected at 120 K on a Bruker Apex II
λ
were solved using SHELXT [33] program and refined with SHELXL [34] using OLEX2
software [35]. The structures were refined by full-matrix least-squares procedure against
F2. Non-hydrogen atoms were refined anisotropically. The H(C) positions were calculated,
the H(N) and H(O) atoms were located on difference Fourier maps and refined using the
riding model. Experimental details and crystal parameters are given in Table S1 (Electronic
Supporting Information).
3.3. Molecular Docking Studies
The molecular docking studies were performed following the previously described
protocol [36,37] and processed with GOLD software (version 2020.2.0, Cambridge Crystal-
lographic Data Center, Cambridge, UK).
The three-dimensional (3D) crystal structures of M. tuberculosis regulatory protein
(PDB ID: 5ICJ), 7,8-dihydroneopterin aldolase complexed with 2-amino-6-(hydroxymethyl)
pteridin-4(3H)-one (PH2; PDB ID: 1NBO), thymidylate synthase in complex with 5-fluoro
DUMP (UFP; PDB ID: 4FOG), UDP-galactopyranose mutase from Mtb docked with UDP
(UGM; PDB ID: 4RPJ), and Rv2671 protein in complex with dihydropteridine (44W;

Molecules 2021, 26, 967
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PDB ID: 4XT4) were obtained from the RCSB Protein Data Bank [29], while the 3D-
structure of cation was taken from X-ray diffraction data of
analyzed independently).
6 (two stereoisomers were
To validate the molecular docking outcomes, 4,4,4-trifluoro-1-(3-phenyl-1-oxa-2,8-
diazaspiro[4.5]dec-2-en-8-yl)butan-1-one, PH2, UFP, UGM, and 44W were removed from
their receptors and re-docked back into their receptors. The docking results were expressed
as binding energy values (kcal/mol) of ligand–receptor complexes; these are based on
hydrogen bond, hydrophobic, and electrostatic interactions. All required docking settings,
including the preparation of mol2 files for the receptors and ligands, determination of
binding sites, the protonation state, calculations, and the overall charges, were established
as hitherto described [38].
4. Conclusions
5-Substituted phenyl-3-(2-aminoethyl)-1,2,4-oxadiazoles in the presence of moisture
and acids undergo Boulton–Katritsky rearrangement to the salts of spiropyrazolinium
compounds—2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium benzoates and chloride.
Hence, biological screening results should be associated with rearranged products and not
with the original taken on trials 5-substituted phenyl-3-(2-aminoethyl)-1,2,4-oxadiazoles. A
small library of the newly 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium benzoates and
chloride has been used in the drug design of antitubercular drugs. The tested compounds
show moderate to high in vitro antitubercular activity with MIC values of 1–100 µg/mL
.
The highest activity in 1 g/mL and 2 g/mL on DS and MDR of M. tuberculosis strains,
µ
µ
equal to the activity of the basic antitubercular drug rifampicin, was recorded for 2-amino-
8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium chloride. 3D molecular structure of the cation
extracted from crystal structure was used for molecular docking studies with various
M. tuberculosis receptors. It was demonstrated that two stereoisomers of the rigid cation
form different sets of hydrogen bonds in complexes with dihydroneopterin aldolase or oxi-
doreductase Rv2671 and similar H-bonds in complex with thymidylate synthase. However,
energies of all ligand–receptor complexes vary from −35.8 to −42.8 kcal/mol.
Supplementary Materials: The following are available online, supplementary data includingthe
NMR spectra of 5-substituted phenyl-3-[2-(morpholin-1-yl)ethyl]-1,2,4-oxadiazole and 2-amino-8-
oxa-1,5-diazaspiro [4.5]dec-1-en-5-ium benzoate and chloride associated with this article are available
online. CCDC 2049798-2049801 contains crystallographic information for 5c–e, 6. Crystallographic
information files are available from the Cambridge Crystallographic Data Center upon request
(http://www.ccdc.cam.ac.uk/structures).
Author Contributions: Conceptualization, L.K.; data curation, K.P.; formal analysis, K.P., V.B. and
K.A.; funding acquisition, L.K.; investigation, A.V., G.D., G.B., A.U., V.B. and L.C.; methodology,
L.K., G.D., G.B., A.U. and L.C.; resources, K.P., G.D., G.B., A.U., V.B., L.C. and K.A.; validation, A.V.,
K.P., G.D., G.B., A.U., V.B., L.C. and K.A.; visualization, L.K., A.V. and K.A.; writing—original draft,
L.K. and A.V.; writing—review and editing, L.K. and A.V. All authors have read and agreed to the
published version of the manuscript.
Funding: This study was supported by the Committee of Science of the Ministry of Education and
Science of the Republic of Kazakhstan (grant AP08856440). A. Vologzhanina acknowledges the
support of the Russian Science Foundation for molecular docking studies (grant 20-13-00241).
Data Availability Statement: The data presented in this study are available in this article.
Acknowledgments: Single-crystal X-ray diffraction data were measured using the equipment of the
Center for Molecular Studies of INEOS RAS.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: The samples may be available.

Molecules 2021, 26, 967
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References
1.
2.
List of Drugs–Wikipedia. Available online: https://en.wikipedia.org/wiki/List_of_drugs (accessed on 12 December 2020).
Fischer, E.; Knovenagel, O. Ueber die Verbindungen des Phenylhydrazinsmit Acroleïn, Mesityloxyd und Allylbromid. Ann. Chem.
1887, 239, 194–206. [CrossRef]
3.
4.
5.
6.
7.
Santos, C.M.M.; Silva, V.L.M.; Silva, A.M.S. Synthesis of Chromone-Related Pyrazole Compounds. Molecules 2017, 22, 1665.
[CrossRef] [PubMed]
Sharma, S.; Kaur, S.; Bansal, T.; Gaba, J. Review on Synthesis of Bioactive Pyrazoline Derivatives. Chem. Sci. Trans. 2014, 3,
861–875. [CrossRef]
1-H Pyrazolium at National Library of Medicine. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/13585073
(accessed on 12 December 2020).
3-Amino-1,4-Dimethyl-1-Phenyl-2-Pyrazolinium Iodide at National Library of Medicine. Available online: https://pubchem.
ncbi.nlm.nih.gov/compound/13064197 (accessed on 12 December 2020).
Kayukova, L.A.; Orazbaeva, M.A.; Gapparova, G.I.; Beketov, K.M.; Espenbetov, A.A.; Faskhutdinov, M.F.; Tashkhodjaev, B.T.
Rapid acid hydrolysis of 5-aryl-3-(β-thiomorpholinoethyl)-1,2,4-oxadiazoles. Chem. Heterocycl. Compd. 2010, 46, 879–886.
[CrossRef]
8.
9.
Kayukova, L.A.; Beketov, K.M.; Baitursynova, G.P. 5-Aryl-3-(β-aminoethyl)-1,2,4-oxadiazoles in Boulton-Katritzky Rearrange-
ment. In Proceedings of the International Conference Catalysis in Organic Synthesis ICCOS-2012 Book of Abstracts, Moscow,
Russia, 15–20 September 2012; p. 214; Available online: http://www.spsl.nsc.ru/FullText/konfe/ICCOS2012.pdf (accessed on 26
January 2021).
Kayukova, L.A.; Uzakova, A.B.; Vologzhanina, A.V.; Akatan, K.; Shaymardan, E.; Kabdrakhmanova, S.K. Rapid Boulton–Katritzky
rearrangement of 5-aryl-3-[2-(piperidin-1-yl)ethyl]-1,2,4-oxadiazoles upon exposure to water and HCl. Chem. Heterocycl. Compd.
2018, 54, 643–649. [CrossRef]
10. Li, J.J. Boulton–Katritzky rearrangement. In Name Reactions; Springer: Cham, Switzerland, 2014. [CrossRef]
11. Korbonits, D.; Kanzel-Szvoboda, I.; Horváth, K. Ring transformation of 3-(2-aminoaryl)-1,2,4-oxadiazoles into 3-acylaminoindazoles;
extension of the Boulton–Katritzky scheme. J. Chem. Soc. Perkin Trans. 1982, 1, 759–766. [CrossRef]
12. Kayukova, L.A.; Praliyev, K.D.; Myrzabek, A.B.; Kainarbayeva, Z.N. Arylsulfochlorination of β-aminopropioamidoximes giving
2-aminospiropyrazolylammonium arylsulfonates. Rus. Chem. Bull. 2020, 69, 496–503. [CrossRef]
13. Kayukova, L.A.; Praliev, K.D.; Akhelova, A.L.; Kemel’bekov, U.S.; Pichkhadze, G.M.; Mukhamedzhanova, G.S.; Kadyrova, D.M.;
Nasyrova, S. Local anesthetic activity of new amidoxime derivatives. Pharm. Chem. J. 2011, 45, 468–471. [CrossRef]
14. Kayukova, L.A.; Jussipbekov, U.; Praliyev, K. Amidoxime Derivatives with Local Anesthetic, Antitubercular, and Antidiabetic
Activity. In Heterocycles-Synthesis and Biological Activities; Nandeshwarappa, B.P., Sadashiv, S.O., Eds.; IntechOpen: London, UK,
17 December 2019. [CrossRef]
15. Kayukova, L.A.; Uzakova, A.B.; Baitursynova, G.P.; Dyusembaeva, G.T.; Shul’gau, Z.T.; Gulyaev, A.E.; Sergazy, S.D. Inhibition of
α-Amylase and α-Glucosidase by New β-Aminopropionamidoxime Derivatives. Pharm. Chem. J. 2019, 53, 129–133. [CrossRef]
16. The Use of a Derivative of Beta-Morpholinopropioamidoxime as an Antidiabetic Agent. Patent Owner: JSC «A.B. Institute of
Chemical Sciences». Russia Patent No. 2684779, 15 April 2019.
17. Ashenhurst, J. Homotopic, Enantiotopic, and Diastereotopic Groups: What Does It Mean? Last updated: October 16th, 2020.
Available online: https://www.masterorganicchemistry.com/homotopic-enantiotopic-diastereotopic/ (accessed on 26 January 2021).
18. Kayukova, L.A. Conditions for the Heterocyclization of O-Aroyl-β-morpholinopropioamide Oximes to 5-Aryl-3-(β-
morpholino)ethyl-1,2,4-oxadiazoles. Chem. Heterocycl. Compd. 2003, 39, 223–227. [CrossRef]
19. Usachev, S.V.; Nikiforov, G.A.; Lyssenko, K.A.; Nelyubina, Y.V.; Levkin, P.A.; Kostyanovsky, R.G. Sterically hindered and
completely arrested nitrogen inversion in pyrazolidines. Tetrahedron Asymmetry 2007, 18, 1540–1547. [CrossRef]
20. Yap, G.P.A.; Alkorta, I.; Elguero, J.; Jagerovic, N. The structure of 1,1,3-trimethyl-∆
2-pyrazolinium perchlorate: An X-ray
crystallographic and GIAO/DFT multinuclear NMR study. Spectrosc. Int. J. 2004, 18, 605–611. [CrossRef]
21. Wood, P.A.; Olsson, T.S.G.; Cole, J.C.; Cottrell, S.J.; Feeder, N.; Galek, P.T.A.; Groom, C.R.; Pidcock, E. Evaluation of molecular
crystal structures using Full Interaction Maps. Cryst. Eng. Comm. 2013, 15, 65–72. [CrossRef]
22. Vologzhanina, A.V. Intermolecular Interactions in Functional Crystalline Materials: From Data to Knowledge. Crystals 2019
9, 478. [CrossRef]
,
23. Macrae, C.F.; Sovago, I.; Cottrell, S.J.; Galek, P.T.A.; McCabe, P.; Pidcock, E.; Platings, M.; Shields, G.P.; Stevens, J.S.; Towler, M.; et al.
Mercury 4.0: From visualization to analysis, design and prediction. J. Appl. Cryst. 2020, 53, 226–235. [CrossRef]
24. French, F.A.; Freedlander, B.L. Derivatives of benzoic acid and simple phenols in the chemotherapy of tuberculosis. J. Am. Pharm. Assoc.
1949, 38, 343–346. [CrossRef]
25. Zhang, Y.; Zhang, H.; Sun, Z. Susceptibility of Mycobacterium tuberculosis to weak acids. J. Antimicrob. Chemother. 2003, 52, 56–60.
[CrossRef]
26. Maresca, A.; Vullo, D.; Scozzafava, A.; Manole, G.; Supuran, C.T. Inhibition of the β-class carbonic anhydrases from Mycobac-
terium tuberculosis with carboxylic acids. J. Enz. Inhib. Med. Chem. 2013, 28, 392–396. [CrossRef]
27. Taylor, F.F.; Faloon, W.W. The role of potassium in the natriuretic response to a steroidal lactone (SC-9420). J. Clin. Endocrinol. Metab.
1959, 19, 1683–1687. [CrossRef]

Molecules 2021, 26, 967
16 of 16
28. Brunner, H.R. The New Angiotensin II Receptor Antagonist, Irbesartan: Pharmacokinetic and Pharmacodynamic Considerations.
Am. J. Hypertens. 1997, 10, 311S–317S. [CrossRef]
29. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E. The Protein Data Bank.
Nucleic Acids Res. 2000, 28, 235–242. [CrossRef] [PubMed]
30. Hanson, A.D.; Gregory, J.F., III. Folate Biosynthesis, Turnover, and Transport in Plants. Annu. Rev. Plant Biol. 2011, 62, 105–125.
[CrossRef] [PubMed]
31. Gavezzotti, A. Are crystal structures predictable? Acc. Chem. Res. 1994, 27, 309–314. [CrossRef]
32. Gavezzotti, A.; Filippini, G. Geometry of the Intermolecular X-H . . . Y (X, Y = N, O) Hydrogen Bond and the Calibration of
Empirical Hydrogen-Bond Potentials. J. Phys. Chem. 1994, 98, 4831–4837. [CrossRef]
33. Sheldrick, G.M. SHELXT–Integrated space-group and crystal-structure determination. ActaCryst. 2015, A71, 3–8. [CrossRef]
34. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Cryst. 2015, C71, 3–8. [CrossRef]
35. Dolomanov, O.V.; Bourhis, L.J.; Gildea, R.J.; Howard, J.A.K.; Puschmann, H. OLEX2: A complete structure solution, refinement
and analysis program. J. Appl. Cryst. 2009, 42, 339–341. [CrossRef]
36. Verdonk, M.L.; Cole, J.C.; Hartshorn, M.J.; Murray, C.W.; Taylor, R.D. Improved protein-ligand docking using GOLD. Proteins
2003, 52, 609–623. [CrossRef]
37. Liebeschuetz, J.W.; Cole, J.C.; Korb, O. Pose prediction and virtual screening performance of GOLD scoring functions in a
standardized test. J. Comput. Mol. Des. 2012, 26, 737–748. [CrossRef]
38. GOLD User Guide. Available online: https://www.ccdc.cam.ac.uk/support-and-resources/ccdcresources/GOLD_User_Guide_
2020_1.pdf (accessed on 12 December 2020).