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

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.

Pyrazolines, as noticeable, practically meaningful nitrogen-containing heterocyclic compounds, can be synthesized by a variety of methods. However, one of the most popular 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, depending 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][3][4].
Herein we report on the stability of 5-aryl-3-[β-(morpholin-1-yl)ethyl]-1,2,4-oxadiazoles 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 spiropyrazolinium 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 chloride of spiropyrazoline drug-susceptible and multidrug-resistant strains, M. tuberculosis revealed compounds with significant activity, and the results are in accordance with molecular docking studies.

Compd
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, 4b in comparison with 1,2,4-oxadiazoles with an unsubstituted phenyl ring and with a phenyl ring having electron-withdrawing substituents-4c-e (25 h). Spiropyrazolinium compounds 5a-e were obtained after evaporation of DMF in an oil pump vacuum, treatment of the residue with acetone with the isolation of rearranged products 5a-e and their recrystallization from 2-PrOH. In the case of the action of ethereal HCl solution on alcohol solutions of 1,2,4oxadiazoles 4a-e in all cases, 2-amino-8-oxa-1,5-diazaspiro[4.5]dec-1-en-5-ium chloride (6) and the corresponding benzoic acids were isolated. IR spectra view of spirocompounds 5a-e and 6 differ from the IR spectra of 1,2,4oxadiazoles 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 pronounced 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 1 H-NMR spectra of 1,2,4-oxadiazole 4a-e were recorded immediately with isolation; if they were recorded after 1-2 weeks, then the emergence and increase in the intensity of the NH 2 group signal of the rearranged spiropyrazolinium products 5a-e in the region of δ 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.
A distinctive feature of 1 H-NMR spectra of benzoates 5a-e from the spectra of 1,2,4oxadiazoles 4a-e is the presence of NH 2 proton signal with the integral intensity of 2H at δ 7.51-7.57 ppm.
Of the remarkable features of the 1 H-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 δ 3.91-3.93 ppm with a total intensity of six protons and in the other case have a multiplet signal at~δ 3.40 ppm intensity of two protons. The diastereotopicity of discussed geminal protons of compounds 4a-e, 5a-e, and 6 is associated with a dynamic cause due to slow rotation of the morpholine heterocycle. 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 1 H-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 13 C spectra of the compounds 4a-e, 5a-e and 6, all signals of aliphatic and aromatic protons were recorded in the expected regions.
So, in the 13 C NMR spectra the characteristic groups of compounds 4a-e, 5a-e and 6 include the signals of: α-methylene groups at δ 31.4 ppm; β-methylene groups at δ 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 δ 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-e are in the regions δ 167.0-168.9 ppm and δ 169.2-169.5 ppm. The carbon atoms of the C=N bond of the pyrazoline ring of compounds 5a-e have two signals at δ 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 bond of compound 6 has a chemical shift at δ 169.1 ppm. The signals of aromatic carbon atoms for compounds 4a-e and 5a-e are in the range δ 112.5-161.0 ppm; para-CН 3 Оand para-CH 3 groups of the compounds 4a, 5a and 4b, 5b, respectively, give signals at δ 55.4 ppm and δ 21.3 ppm.
As we have proved in this article, 1,2,4-oxadiazoles 4a-e are recorded spectroscopically (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-e, and 4a-e→6 can be represented as a sequence of protonation, proton transfer and nucleophilic attack steps, representing hydrolysis during the reaction of 1,2,4-oxadiazoles 4a-e with water and wet HCl in the same way as we indicated for piperidine derivatives [9].
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). A number of compounds 5a-e on DS and MDR MTB strains exhibits an average antitubercular activity of 10-100 µg/mL. Moreover, an improvement in activity is observed with a decrease in MIC to 10 µg/mL (5b); 20 µg/mL (5c) and 50 µg/mL (5a) on the DS MTB strains and up to 50 µg/mL on the MDR MTB strains for the compounds 5a-c containing donor substituents in the phenyl ring or with an unsubstituted phenyl ring. Spiropyrazolylammonium chloride 6 demonstrates high in vitro antitubercular activity 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.

X-ray Diffraction
Molecular structures of compounds 5c-e and 6 are given in Figure 1. Asymmetric units of 5c, 5e and 6 contain one water molecule besides the target ions, and the asymmetric unit 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 doesn t exceed 0.5Å (Figure 2b). The positive charge on quaternary ammonium atom causes elongation of N(1)-N(2) and 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.     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 NH 2 group, while acceptor atoms of heterocycles less readily take part in the hydrogen bonds.

Molecular Docking Studies
Our results indicate that compounds 4a-e at neutral pH in aqueous solutions undergo transformation to 5a-e, and antitubercular activity demonstrated by the latter compounds 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][25][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 derivatives with receptors, where ligands are situated on the protein surface [see, for example, 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. Overall, energies of ligand-receptor interactions vary from −35.84 to −42.84 kcal/mol, with the most prominent contribution from nonpolar C-H...π interactions. These values are close to the value of intermolecular pairwise interactions of the same cation in the crystal of 6 (−35.3 kcal/mol) estimated using UNI potentials [31,32]. The cation within 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 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 NH 2 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.

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 instrument (Waltham, MA USA) in KBr pellets. 1 H-and 13 C-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 1 H-NMR (2.50 ppm) and 13 C-NMR (39.5 ppm) spectra. Elemental analysis was carried out on a CE440 elemental analyzer (Exeter Analytical, Inc., Shanghai, 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.

Single-Crystal X-ray Diffraction
The X-ray diffraction data of 5c-e, 6 were collected at 120 K on a Bruker Apex II diffractometer (Bruker AXS, Inc., Madison, WI, USA) equipped with an Oxford Cryostream cooling unit and a graphite monochromated Mo anode (λ = 0.71073 Å). Crystal structures 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 F 2 . 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).

Molecular Docking Studies
The molecular docking studies were performed following the previously described protocol [36,37] and processed with GOLD software (version 2020. PDB ID: 4XT4) were obtained from the RCSB Protein Data Bank [29], while the 3Dstructure of cation was taken from X-ray diffraction data of 6 (two stereoisomers were analyzed independently).
To validate the molecular docking outcomes, 4,4,4-trifluoro-1-(3-phenyl-1-oxa-2,8diazaspiro [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].

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 µg/mL and 2 µg/mL on DS and MDR of M. tuberculosis strains, 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 oxidoreductase 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.

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.