INTRODUCTION:

According to the World health organization, tuberculosis refers to the socially significant diseases. More than 9 million people become ill with TB and about 1,4 million people die from TB annually [1].

Insufficient efficacy and high toxicity of existing antituberculosis drugs and the emergence of resistance to them require the search and development of new more effective and safe drugs that are currently actively carry out in the world for the treatment of all varieties of tuberculosis: drug-sensitive (DS), multidrug resistant (MDR) and extremaly drug resistant (XDR) TB, that accompanies HIV infection and drugs that are suitable for patients of all ages [2].

With the aim of developing of new non-toxic, active against DS and MDR strains of M. tuberculosis anti-TB drugs in the JSC«A.B. Bekturov Institute of Chemical Sciences» and in the RSE «National Scientific Center of Phthisiopulmonology of RK»MH RK (Аlmaty, Каzakhstan) we are looking for new tuberculostatics in the class of new derivative b-aminopropioamidoximes.

The search for new antitubercular drugs in this series of compounds is interesting, as the natural antibiotics–tuberactinomycins A and N under hydrolysis produce amino acid derivatives: threo-γ-hydroxy-β-lysine and the erythro isomer of the β-alanine derivative. The derivatives of β-aminopropionamidoximes can be considered as formal analogues of β-alanine [3]. The authors found highly in vitro active on DS M. tuberculosis strains H37 Rv and MDR strains of M. tuberculosis low-toxic new compounds from the groups of β-aminopropionamidoximes derivatives [4].

In the recent years, during the search for new multi-target drugs with pleiotropic effects, are widely used methods of computer-aided drug design [5‒6].

On the basis of a computer forecast, the most promising directions of pharmacological studies of synthesized and planned to synthesize organic compounds are determined; most possible pharmacological targets are established, the effect on which leads to the desired pharmacological therapeutic effects; new fields of application of known drugs are revealed [7].

The purposes of our work are: 1) the synthesis of the new amidoxime derivatives, O-aroyl β-(piperidin-1-yl) propioamidoximes and 5-substituted phenyl 3-[β-(piperidin-1-yl) ethyl]-1,2,4-oxadiazoles, 2) analysis of potential biological activity of β-amidoxime derivatives by in silico chemoinformatics methods; and 3) an in vitro response check of antituberculosis activity. First of all, we were interested in their anti-tuberculosis potential.

MATERIAL AND METHODS:

Chemicals and reagents:

Ethanol, chloroform, DMF, K₂CO₃ and substituted benzoic acid chlorides: p-anisoyl chloride, p-toluoyl chloride, benzoyl chloride, p-bromobenzoyl chloride, m-chlorobenzoyl chloride were procured from chemical companies Sigma-Aldrich.

Instrumentation:

IR spectra were recorded on a NICOLET 5700 FT-IR spectrometer using in KBr tablets. The reaction was monitored by TLC on Sorbfil plates (CJSC Sorbopolymer) with a sorbent-loaded silica gel layer CTX-1A, with a grain size of 5‒17 μm, UV-254 UV indicator. Solvents used in the synthesis and recrystallization of compounds (ethanol, i-PrOH, and also solvents used as eluants for TLC (ethanol, benzene) are prepared according to standard procedures. NMR spectra (1H) were recorded on a JNN-ECA400 (400 MHz) spectrometer with internal standard of HMDS as solutions of V‒X compounds in DMSO-d₆.

Selection of Solvents:

Based on the good solubility of the starting β-(piperidin-1-yl)propioamidoxime (I), absolute ethanol was chosen as the solvent in the preparation step. The reaction of acylation of amidoxime I with substituted benzoic acid chlorides to form precipitating hydrochlorides II–VII was carried out in dried chloroform. Water-insoluble bases of VIII–XI were obtained by the action of K₂CO₃ on the solutions of hydrochlorides II–VII in distilled water. The dehydration stage of the bases VIII–XI is carried out in DMF, followed by its evaporation in an oil pump vacuum to isolate 1,2,4-oxadiazoles XII–XVI by the treating the reaction mixture with dried acetone.

Preparation of substances:

Preparation of β-(piperidin-1-yl)propioamidoxime («one-pot») [8].

Preparation of the base of O-anisoyl-β-(piperidin-1yl)propiamidoxime (VII). 0.37 g (0.0027 mol) of potassium was added to a solution of 0.91 g (0.0027 mol) of O-anisoyl-b-(piperidin-1-yl)propiamidoxime hydrochloride (II) in 5 mL of distilled water at room temperature. After drying and recrystallization of the technical precipitate VII from i-PrOH, 0.67 g (83%) of O-anisoyl-b-piperidinopropiamidoxime (VII) was obtained, m.p. 96 °C.

The preparation of bases VII-XI was carried out according to the above procedure.

Preparation of 5-p-methylphenyl-3-[(β-piperidin-1-yl)ethyl]-1,2,4-oxadiazole (XIII). To 0.3 g (0.001 mol) of O-p-toluoyl-β-(piperidin-1-yl)propioamidoxime (II) base was added 10 mL of dried DMF. The reaction mixture was heated in an oil bath at a temperature of 60 to 70°C with 5 hours TLC control of the reaction mixture. Then, the precipitate of 1,2,4-oxadiazole XII was filtered off. The filtrate was evaporated to dryness in an oil pump vacuum at a temperature of 50 °C/1 mm Hg. The organic residue was treated with Ac. After recrystallization of the precipitates from i-PrOH 0.15 g (56%) of 1,2,4-oxadiazole XIII was obtained (Table 1).

The remaining 5-substituted phenyl-3-[(β-piperidin-1-yl)ethyl]-1,2,4-oxadiazoles (XII, XIV‒XVI) were obtained by the same procedure.

General method of in vitro determination of bactericidal antituberculosis activity on the Soton liquid medium:

The culture of mycobacteria, on a dense egg medium under sterile conditions (14‒21 days), was removed from the cant with a one-time loop and suspended in 0.9% NaCl solution (saline). Then large particles of culture were allowed to settle, keeping the tube for 20 minutes at room temperature. The bacterial suspension was pipetted and transferred to another tube to bring it to the required optical density. The required optical density or turbidity, corresponding to standard 5, was achieved by adding saline to the tube. In 1 mL of a suspension corresponding to the 5 standard of optical density, 500 million microbial bodies (5x108 microbial bodies) are contained. A suspension of tuberculosis mycobacterium was inoculated into a liquid medium at a rate of 0.2 mL per 2 mL of medium. This method of seeding ensures the uniform introduction of seed into the samples. The stock solution of the test compound was prepared using DMSO.

Samples of substances was dissolved in a small amount of the solvent and then diluted with physiological saline to the desired concentration. For example, 1 mL of DMSO was added to a sample of 10 mg of the substance and shaken. After this, the obtained solution or homogeneous suspension was adjusted with physiological saline to the required concentration.

When using high concentration of solvents, it is necessary to control the effect of these substances on the growth of mycobacterium.

When studying the bactericidal activity of the test substances, a wide range of concentration was studied. Calculation of concentration for studying the bactericidal activity of the test substances on a Sotton liquid medium:

1-st dilution: 10 mg of substance+10 mL of liquid (one of the solvents in which the test sample dissolves: distilled water, ethyl alcohol, saline, DMSO), which corresponds to concentration of 1000 μg/mL.

2-nd dilution: 1 mL from dilution I+9 mL Sotton medium, which is the total volume in 10 mL with a concentration of 100 μg/mL.

This is the initial working dilution. The antituberculous bactericidal activity of the drugs was tested for concentrations ranging from 100 μg/mL to 0.01 μg/mL. In each test and control tube (culture medium without preparation) 0.1 mL of mycobacterium tuberculosis suspension was introduced into 0.2 mL of physiological solution. The tubes were incubated at 37°C for 10 days. After 10 days of incubation on a liquid medium, the precipitates were washed with physiological saline and inoculated on a dense medium of Levenshtein-Jensen. Accounting for the growth of crops was carried out twice–after 1 month and after 2.5 months. The experiment was carried out in two replicates.

RESULTS:

Synthesis of O-aroyl-β-(piperidin-1-yl) propioamidoximes (VII‒XI) and 5-substituted phenyl-3-[β-(piperidin-1-yl)ethyl]-1,2,4-oxadiazoles (XII‒XVII) includes: a stage of preparing of the starting β-(piperidin-1-yl) propioamidoxime (I); synthesis of O-aroyl-β-(piperidin-1-yl) propioamidoxime hydrochlorides (II‒VI) by acylation of β-(piperidin-1-yl) propioamidoxime (I) with substituted benzoic acid chlorides; preparation of O-aroyl-β-aminoethylpropioamidoxime bases (VII‒XI) as water-insoluble precipitates in the treatment of O-aroyl-β-aminoethylpropioamidoxime hydrochlorides (II‒VI) with an aqueous solution of potash and the preparation of 5-substituted phenyl 3-(β-piperidin-1-yl)ethyl-1,2,4-oxadiazoles (XII‒XVI) by heating of the bases of O-aroyl-β-aminoethylpropioamidoximes (VII‒XI) in DMF at 70°C for several hours, evaporating the solvent in vacuooil pump and treatment of the residue with acetone [2].

Synthesis of the starting compound β-(piperidin-1-yl) propioamidoxime (I) in 63% yield was performed by using the«one-pot» method. It involves the formation of an intermediate product β-(piperidin-1-yl)propionitrile upon the reaction of piperidine and acrylonitrile in absolute ethanol at room temperature, followed by the addition of equivalent amounts of hydroxylamine hydrochloride and sodium ethoxide [8].

Regardless of the electronic properties of the substituents in the acylating agent, the regiospecific course of the reaction was noted, only acylation products were obtained for the oxygen atom of the amidoxime group II–VI, isolated as hydrochlorides with yields of 77–98% [9].

The structures of the synthesized compounds were approved by the data of IR-, (¹H and ¹³C) NMR spectra. In the IR-spectra of hydrochlorides and bases of O-aroyl-(β-piperidin-1-yl) propioamidoximes (II–XI) in the region of 1712–1744 cm^-1 there is a characteristic band of stretching vibrations of ester carbonyl C(=O)-O. In the hydrochlorides of O-benzoyl-β-piperidinopropioamidoximes (II–VI) characteristic bands of ammonium-bound nitrogen are present at n 2300–2900 cm^-1. In the region n of 1564–1638 cm^-1 of propioamidoximes (II–XI) there are absorption bands of stretching vibrations of C=C double bonds of strong intensity. The remaining characteristic bands of valence and deformation vibrations, (δ_N-H, n_C=N, n_N-O, n_C-O), correspond to the accepted structure.

In the ¹H NMR spectra of hydrochlorides and bases of O-aroyl-β-(piperidin-1-yl) propioamidoximes (II–XI), there are signals of benzoyl groups (XC₆H₄CO) in the region δ 7.00–8.63 ppm, compounds (II, VII), in addition, have proton signals of para-methoxy groups at δ 2.10 and 2.50 ppm, respectively, and compounds (III, VIII)–the signals of the protons of the para-methyl group at δ 2.37 and δ 2.25 ppm, respectively. The proton signal of the NH₂ group at δ 5.35 ppm of the initial amidoxime with an intensity of two protons under acylation is shifted to δ 6.65–7.00 ppm. Also, the feature of O-benzoylation in the ¹H NMR spectra is the disappearance of the signal of NOH group proton with an intensity into one proton from the starting β-(piperidin-1-yl) propioamidoxime (I) at δ 8.18 ppm. The proton signals of the three methylene groups of the piperidine ring in the form of two multiplet signals are present in the region δ 1.40 and 1.55 ppm. The triplet signals of protons of the α-methylene group with an intensity of two protons are at δ 2.24–2.60 ppm for the bases (VII–XI) and δ 2.44–2.76 ppm–for hydrochlorides (II–VI). The triplet signals of protons of the β-methylene group with an intensity of two protons are manifested at δ 2.56–3.10 ppm for bases (VII–XI) and at 3.06–3.34 ppm for hydrochlorides (II–VI); the signals of the methylene groups of the piperidine ring bound to the nitrogen atom give a multiplet signal with an intensity of four protons at δ 2.37–3.85 ppm.

In the ¹³C NMR spectra of compounds II–XI the signals of carbonyl carbon atoms of the ester group are in the region of δ 161.9–170.0 ppm; the carbon atoms of the amidoxime groups [C(=NOCOC₆H₄X)NH₂] give the signals in the region of δ 157.3–169.2 ppm; signals of aromatic carbon atoms of the substituted benzene ring are present in the range of 125.1–144.4 ppm.

In the IR-spectra of compounds XII–XVI, there are no absorption bands for the stretching vibrations of the ester group C(=O)-O, and also the absorption bands of the valence vibrations of the amino group H-N-H. In the region of n 1584–1600 cm^-1 there are absorption bands of stretching vibrations n_C=C of double bonds of strong intensity. In the region of n 1652–1660 cm^-1 there are powerful bands of stretching vibrations of n_C=N bonds.

In the ¹H NMR spectra of the compounds XII–XVI recorded in DMSO-d₆, in the range of δ 7.01–8.76 ppm there are signals of a substituted benzene ring. The proton signals of α-methylene group are at δ 3.12–3.15 ppm; of β-methylene group–at δ 3.80–3.83 ppm; the proton signals of the methylene groups bound to the nitrogen atom of the piperidine ring give two multiplet signals with intensity of two protons belonging to the equatorial (δ 3.33 ppm) and axial protons (in the region δ 3.44–3.50 ppm), the signal of axial protons has a more large half-width.

In the ¹³C NMR spectra of compounds XII–XVI, the signals of carbon atoms C(3) and C(5) of the 1,2,4-oxadiazole ring are respectively at δ 156.3–169.2 and 163.0–170.0 ppm; signals of the α-methylene carbon atoms are located in the region δ 25.5–30.6 ppm; signals of β-methylene carbon atoms–in the region δ 54.6–63.1 ppm; the signals of the carbon atoms of the benzene ring are manifested in the range of δ 126.4–151.8 ppm [10].

In silico analysis of the biological potential of synthesized compounds:

Currently starting from the structural formula of organic compounds on the basis of large number of free and commercially available computer resources it is possible to obtain estimates of the likely biological activity. To determine the most promising directions for biological experimental studies of synthesized compounds, we decided to use the in silico one. There are several tools in it that can provide an estimate of the likely biological activity for drug-like compounds based on methods for evaluating structural similarities or machine learning methods. All these tools require structural formulas. The information is entered as MOL files (for one connection) or as an SDF file (for installation including all 10 connections). We have prepared such files with the help of the Draw ISIS program and PASS SDF Creator [11–12]. We tried to determine whether the synthesized compounds are new or they are already known and available in ChemSpider [13]. ChemSpider is freeware chemical database that provides access to more than 29 million structures from hundreds of data sources. Of the 10 synthesized compounds, no structure was found.

Secondly, we tried to find synthesized compounds among known chemicals in various chemical databases using CSLS (Chemicalstructurelookupservice) [14]. In this search, none of the compounds of O-aroyl-O-benzoyl-β-piperidinopropyamidoximes (VII-XI) and 5-substituted phenyl-3-[β-(piperidin-1-yl)ethyl]-1,2,4-oxadiazoles (XII‒XVI) in silically tested compounds, was not found. Then we tried to find similar structures inside the Integrity database (Thomson Reuters) [15]. As shown by the analysis, there are no similar structures.

The prediction of the biological activity of the 10 compounds synthesized by us (VII‒XVI) using the PASS computer program showed the following results (Tables 1, 2) [11].

According to the PASS program, antituberculous activity is determined with a low probability for analogous derivatives of O-esters of amidoximes and 3,5-disubstituted 1,2,4-oxadiazoles. According to this program, such activities as: treatment of phobic disorders, antineurotic, antidiskinetic, properties supporting the function of the kidney stimulant and anti-alcoholic biological activity were identified with good probability.

In vitro bactericidal antitubercular activity was assessed as bactericidal and bacteriostatic in the Sotton liquid medium on 2 strains: a sensitive strain of M. tuberculosis H37Rν and on wild strain of M. tuberculosis isolated from patients typified as II–multidrug-resistant (MDR)–resistant to two drugs (rifampicin and to ethambutol or streptomycin).

The bactericidal activity (MIC) of rifampicin SV on sensitive strains of M. tuberculosis H37Rν is 1 μg/mL, and on MDR wild strain of M. tuberculosis II–2 μg/mL (Table 3).

As can be seen from the table 3: samples VII and X showed pronounced bactericidal activity against DS strains of M. tuberculosis H37Rν (I) at a concentration of 2 μg/mL, while against MDR strains of M. tuberculosis–10 μg/mL and 20 μg/mL, accordingly. Samples XI and VIII exhibit bactericidal activity against DS strains of M. tuberculosis H37Rν (I) at concentrations of 5 and 10 μg/mL, accordingly, while against MDR strains of M. tuberculosis–50 and>100 μg/mL, accordingly. Bactericidal activity of the both preparations XV and XVI for the DS and MDR strains of M. tuberculosis is observed at a concentration of 0,01 μg/mL. Sample XIII demonstrates bactericidal activity against DS strains of M. tuberculosis H37Rν at a concentration of 1 μg/mL, while against MDR strains of M. tuberculosis–2 μg/mL. Samples IX, XII, XIV do not show significant antitubercular activity.

Table 1: Structures and predicted activities for the studied compounds O-benzoyl-β-piperidinopropiamidoximes (VII-XI) and 5-substituted phenyl-3-[β-(piperidin-1-yl)ethyl]-1,2,4-oxadiazoles (XII‒XVI)

No	Structure		Predicted Activity Spectra	Ра, %*	Рi, %*
VII		Х = p-CH₃O	Phobic disorders treatment Antiprotozoal Antiprotozoal (Plasmodium) Fibrinolytic Antineurotic	69 62 57 54 51	6.1 1.1 1.2 7.3 8.1
VIII		Х = p-CH₃	Phobic disorders treatment Antiprotozoal Antiprotozoal (Plasmodium) Antieczematic Kidney function stimulant	74 64 57 60 53	4.2 1 1.2 6 5.6
IX		Х = H	Phobic disorders treatment Antiprotozoal Antiprotozoal (Plasmodium) Kidney function stimulant	82 66 59 52	1.8 0.9 1 6
X		Х = p-Br	Antiprotozoal Phobic disorders treatment Antiprotozoal (Plasmodium) Kidney function stimulant	65 67 59 54	0.9 6.9 1 5
XI		Х = m-Cl	Phobic disorders treatment Antiprotozoal Antiprotozoal (Plasmodium) Antineurotic	86 68 63 54	0.9 0.7 0.8 6.9

XII

Х = p-CH₃O

Cognition disorders treatment

Antineurotic

Anxiolytic

Psychotropic

0.9

6.7

5.4

XIII

Х = p-CH₃

Cognition disorders treatment

Antidyskinetic

Antialcoholic

Phobic disorders treatment

0.8

3.8

0.5

XIV

Х = H

Cognition disorders treatment

Phobic disorders treatment

Anxiolytic

Psychotropic

0.5

1.4

2.8

Х = p-Br

Cognition disorders treatment

Antialcoholic

Anxiolytic

Antineurotic

0.8

0.5

2.3

XVI

Х = m-Cl

Phobic disorders treatment

Cognition disorders treatment

Psychotropic

Antialcoholic

2.4

0.7

3.1

0.4

Table 2: List of the most typical and most significant activities predicted for the studied compounds

Activity	Percentage of the Sample	Compound's No
Phobic disorders treatment	68	VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI
Antineurotic	49.6	VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI
Antidyskinetic	42.6	VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI
Kidney function stimulant	36.4	VII, IX, X, XI, XII, XIII, XIV, XV, XVI
Antialcoholic	35.4	VII, VIII, IX, X, XI, XIII, XIV, XV, XVI
Antiprotozoal	32.5	XII, XIII, XIV, XV, XVI
Cognition disorders treatment	30.8	VII, VIII, IX, X, XI
Antiprotozoal (Plasmodium)	29.5	XII, XIII, XIV, XV, XVI
Psychotropic	26.2	VII, VIII, IX, X, XI
Anxiolytic	25.7	VII, VIII, IX, X, XI
Diabetic neuropathy treatment	24.2	VII, VIII, IX, XI, XII, XIII, XIV, XVI
Analgesic	24	VII, VIII, IX, X, XI

Table 3: In vitro antitubercular activity of compounds II–VI and XII–XVI*

№		VII	VIII	IX	X	XI	XII	XIII	XIV	XV	XVI
Х		p-CH₃O	p-CH₃	H	p-Br	m-Cl	p-CH₃O	p-CH₃	H	p-Br	m-Cl
MIC μg/mL on strains of M. tuberculosis	Sensitive strains Н37Rν	2	10	>100	2	5	>100	1	>100	0.01	0.01
MIC μg/mL on strains of M. tuberculosis	MDR	10	>100	>100	20	50	>100	2	>100	0.01	0.01

Note: *In vitro MIC, μg/mL: for rifampicin on DS strain of M. tuberculosis is 1 μg/mL, on MDR strain of M. tuberculosis is 2 μg/mL; for isoniazid on sensitive strain of M. tuberculosis is 0,1 μg/mL, on MDR strain is 1 μg/mL.

When studying the effect of rifampicin on these strains in a liquid medium, it was found that it has bactericidal activity on DS strains at a concentration of 1 μg/mL, and on MDR strains at a concentration of 2 μg/mL.

Thus, the search for the Integrity database does not provide many initial productive ideas about which priority types of biological activity of synthesized and in silico tested compounds are most possible in the experiment. The lack of data on the necessary types of activity in bioinformatics can be considered as a deficit of experimental data on the development of new drugs in the class of amidoximes possessing of antitubercular properties.

REFERENCES:

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8. Innovative patent of Kazakhstan 28453. Method for the production of β-(piperidin-1-yl)propioamidoxime. Kayukova L.A., Praliyev K.D., Uzakova A.B., Dussembaeva G.T. Pub. 15.05.2014. Bull. № 5; https://gosreestr.kazpatent.kz/

9. Kayukova L.A., Praliyev K.D., Djumadildaeva I.S. O-Benzoyl-β-piperidinopropioamidoximes and their dehydration to 3-(β-piperidino)ethyl-5-phenyl-1,2,4-oxadiazoles. Russian Chemical Bulletin. 2002; 11: 1945–1949.

10. Uzakova A., Kayukova L., Praliyev К. Synthesis of O-aroyl-β-(piperidin-1-yl) propioamidoximes, 5-substituted phenyl-3-[β-(piperidin-1-yl) ethyl]-1,2,4-oxadiazoles and 2-amino-1,5-diazaspiro[4.5] dec-1-en-5-ium benzoates. Anatolian Conference on Synthetic Organic Chemistry, Antalya. 2015; рр. 204.

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14. http://www.cactus.nci.nih.gov/cgi-bin/lookup/search. Accessed 2018

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Received on 14.06.2018 Modified on 02.07.2018

Research J. Pharm. and Tech 2018; 11(9): 4003-4009.

DOI: 10.5958/0974-360X.2018.00736.9