OF THE ACTIVITY OF 1 . 3-DIAZINONE-4 NEW DERIVATIVES AND THEIR NON-CYCLIC PRECURSORS IN RESPECT OF ESCHERICHIA COLI

На протяжении, как минимум, последних пятнадцати лет в структуре нозокомиальных инфекций в различных странах мира лидирующее положение занимают грамотрицательные возбудители, в частности Escherichia coli. Данные Всемирной организации здравоохранения свидетельствуют о нарастающей резистентности данного патогена к антибиотикам, что обусловливает актуальность создания и разработку новых антимикробных средств. Первичный скрининг новых производных 1,3-диазинона-4 и их нециклических предшественников позволил выявить среди них соединения-лидеры, способные активно подавлять рост E. сoli, перспективные для дальнейшего изучения. Цель исследования. Исследовать активность соединений под лабораторными шифрами ПЯТs4, ПЯТs6, ПЯТs7, ПЯТd8, ПЯТd9, ПЯТd15 в отношении Escherichia coli. Материалы и методы. Исследование активности соединений проведены на коллекционных и свежевыделенных штаммах от больных с разной патологией. Все штаммы перед посевами были идентифицированы посредством программно-аппаратного комплекса BIOMIC V3 (Giles Scientific, США) для подтверждения принадлежности к роду и виду. Штаммы, использованные в работе, предварительно исследовали на чувствительность к антибактериальным препаратам методом дисков для оценки наличия или отсутствия резистентности. Активность новых соединений изучали методом серийных разведений. Результаты и обсуждение. Исследование чувствительности штаммов методом дисков к антибактериальным препаратам, применяемым при этиотропной терапии инфекций, вызываемых Escherichia coli, выявило среди них микроорганизмы с множественной лекарственной резистентностью. Анализ посевов штаммов показал, что все исследованные соединения обладают способностью подавлять рост E. сoli как коллекционных, так и свежевыделенных от больных с различной патологией. Заключение. Самыми активными в отношении изученных штаммов являются вещества под лабораторными шифрами ПЯТd8 и ПЯТs4. При этом показатели МПК50 всех соединений не превышают 10–20 мкг/мл, что позволяет считать их перспективными для дальнейшего изучения. Ключевые слова: соединения ПЯТs4, ПЯТs6, ПЯТs7, ПЯТd8, ПЯТd9, ПЯТd15, Escherichia coli, активность, МПК

Проблема растущей резистентности обусловливает актуальность создания и разработку новых антимикробных средств [25][26][27].Предварительный анализ возможной биологической активности новых производных 1,3-диазинона-4 и их нециклических предшественников, полученных посредством ком-In various countries of the world, for at least last fifteen years, in the structure of nosocomial infections the leading position has been occupied by gram-negative pathogens, Escherichia coli in particular.The data of the World Health Organization testify the increasing resistance of this pathogen to antibiotics, which determines the relevance of creating and developing new antimicrobial agents.Primary screening of new 1.3-diazinon-4 derivatives and their non-cyclic precursors made it possible to identify the leader compounds capable of active suppressing the growth of E. coli and promising for the further study.The aim of the study is to investigate the activity of the compounds under laboratory ciphers of PYaTs4, PYaTs6, PYaTs7, PJTd8, PYaTd9, PYaTd15 with respect to Escherichia coli.Materials and methods.The study of the activity of the compounds was carried out on the basis of collection and freshly isolated strains from patients with different pathologies.Before the inoculation all the strains were identified by means of the BIOMIC V3 software complex (Giles Scientific, USA) to confirm their belonging to the genus and species.The strains used in the work were previously tested for susceptibility to antibacterial drugs by the disk method to assess the presence or absence of resistance.The activity of the new compounds was studied by the serial dilution method.Results and discussion.The investigation of the sensitivity of the strains by the disk method to antibacterial drugs used in the etiotropic therapy of infections caused by Escherichia coli revealed microorganisms with multiple drug resistance.The analysis of the inoculation of the strains showed that all the studied compounds have the ability to suppress the growth of E. coli being both collectively and freshly isolated from patients with various pathologies.Conclusion.The substances under the laboratory ciphers of PYaTd8 and PYaTs4 are the most active in respect to the studied strains.At the same time, the MIC 50 (minimum inhibitory concentration) of all the compounds does not exceed [10][11][12][13][14][15][16][17][18][19][20]  Все штаммы перед посевами были идентифицированы посредством программно-аппаратного комплекса BIOMIC V3 (Giles Scientific, США) для подтверждения принадлежности к роду и виду.

INTRODUCTION.
For at least fifteen year, the gram-negative pathogens have been occupying the leading position in the structure of nosocomial infections in various countries of the world.According to the results of multicenter studies conducted in Russia and abroad, there is an increase of the etiological significance of Escherichia coli [1][2][3][4].E.coli is a habitual inhabitant of the intestine of a healthy person.Among its populations, there are highly adapted clones that have acquired vir-ulence for various reasons.This allows them to adapt to unusual conditions well and to cause a wide range of diseases: urinary tract infections, intra-abdominal infections, pneumonia, infections in the field of surgical interventions, meningitis, catheter-associated infections, osteomyelitis, skin and soft tissue infections.In this case, any infection of any localization may be accompanied by bacteremia and sepsis [5][6][7][8].
The sensitivity analysis of isolated microorganisms Фармация и фармакология Т. 6 № 1, 2018 Currently, in all regions of the World Health Organization, there is a high level of resistance of E. coli to cephalosporins and third generation fluoroquinolones -two of the most important and widely used types of antibacterial drugs, which continues to increase.There is also the occurrence of cases of resistance to carbopenems [9][10][11][12][13][14][15][16][17][18][19][20].
Antibiotic resistance has always been considered a problem for only inpatients, and currently it is also relevant for outpatient infections.The spectrum of pathogens, as a rule, is represented by E. coli (85-95%).The monitoring of the resistance of community-acquired strains shows a pronounced tendency to its growth [21][22][23][24].
The problem of growing resistance causes the urgency of creating and developing new antimicrobial agents [25][26][27].A preliminary analysis of the possible biological activity of the new 1.3-diazinon-4 derivatives and their non-cyclic precursors, obtained by means of computer prediction, suggested the presence of antibacterial activity in them [28].Primary screening of the compounds under laboratory ciphers of PYaTs4, PYaTs6, PYaTs7, PJTd8, PYaTd9, PYaTd15 with respect to E. сoli made it possible to identify the leader compounds capable of active suppressing the growth of E. coli and promising for the further study [29].
THE AIM OF THE STUDY is to investigate the activity of the compounds under laboratory ciphers of PYaTs4, PYaTs6, PYaTs7, PYaTd8, PYaTd9, PYaTd15 with respect to Escherichia coli by serial dilution method to evaluate their prospects for in vivo research.
MATERIALS AND METHODS.The study of the activity of the compounds under laboratory ciphers of PYaTs4, PYaTs6, PYaTs7, containing the "s" fragment (sulfone), and PYaTd8, PYaTd9, PYaTd15, containing the "d" fragment (dapsone), with respect to Escherichia coli, was carried out on the basis of four collection strains: Before the inoculation all the strains were identified by means of the BIOMIC V3 hardware-software complex (Giles Scientific, USA) to confirm their belonging to the genus and species.The activity of the compounds was studied by the serial dilutions method: in the first stage, the strains were planted in the meat-peptone broth (MPB) with previously diluted compounds in decrease of concentration in a geometric progression with a factor of 2: from 128 μg / ml to 0.5 μg / ml.As a control we used the inoculations with a solvent (DMSO in equivolumes), inoculations without adding substances to the medium (a positive control), control over the sterility of the medium (medium without crops and compounds).After a day of incubation in a thermostat at 37°C, the tubes were centrifuged, the precipitate was sterilely washed and then reinoculated to Endo medium.After a day of incubation, the inoculations were assessed visually then the grown colonies were counted by BIOMIC V3 apparatus.The concentration of the compounds after the incubation with which the growth of colony forming units (CFU) was inhibited with respect to control by 50% (MIC 50 ), by 90-100% (MPK 90-100 ) was determined.A comparative analysis of the activity of substances was carried out [30,31].
The strains used in the study were previously investigated for sensitivity to antibacterial drugs by the disk method to assess the presence or absence of resistance [32].A set of indicator disks DI-PLS-50-01 ("Research Center for Pharmacotherapy of CJSC", St. Petersburg) was used.The results were read on the BIOMIC V3 apparatus.
The statistical processing of the results was carried out with the help of the BIOSTAT 2009 program (Analist SoftIns., USA).The variational series were checked for normality by Kolmogorov-Smirnov's test.The reliability indicator of differences was determined by Student's t-test.Statistically, significant differences were considered at p≤ 0.05-0.01.
RESULTS AND DISCUSSION.The investigation of the sensitivity of the strains by the disk method to antibacterial drugs used in the etiotropic therapy of infections caused by Escherichia coli revealed microorganisms with multiple drug resistance (Table 1).
The analysis of the inoculations of the collection strains showed that of all the compounds containing the "s" fragment, the most active in relation to them is PYaTs4: its MIC 50 was 0.5-1.0μg / ml, the concentration of 128 μg / ml in three of four cases was bactericidal.
The MIC 50 of PYaTs6 and PYaTs 7 compounds was 2-4 times higher, no bactericidal action was observed (Table 2).
Pharmacy & Pharmacology V. 6   Among the compounds having the "d" fragment in their composition, the high activity was shown by the substance PYaTd8.Its MIC 50 corresponded to 0.5 μg / ml, high concentrations suppressed the growth of 98-100% of the population.The activity of PYaTd9 and PYaTd15, depending on the strain, varied greatly: the MIC 50 range was 1-16 μg / ml, the concentration of 128 μg / ml corresponded to MIC 88-90 (Table 2).
As for E.coli strains, isolated from patients' trophic ulcers, the most active of the compounds containing the "s" fragment was PYaTs4: it inhibited the growth of 50% of bacteria at the concentration of 1 μg / ml, and 90-95 % at 128 μg / ml.The MIC 50 range for the PYaTs6 compound was 1-2 μg / ml, for PYaTs 7 it was even higher: 4-8 μg / ml.At their highest concentration, used in this study, the growth was limited to no more than 90% of microorganisms (Table 3).
Among the compounds containing the "d" fragment, just as in the case of collection strains, the substance under the laboratory cipher of PYaTd8 was more active than others.The growth of 50% of the population was also suppressed by the compound at concentrations of 0.5 μg / ml.At a concentration of 128 μg / ml, the number of grown colonies that was less than 10% of the control.The strains of bacteria isolated from trophic ulcers were less sensitive to PYaTd9 and PYaTd15 compounds.Their MIC 50 corresponded to the range of 4-16 μg / ml, and at a concentration of 128 μg / ml they inhibited the growth of less than 90% of the population (Table 3).The analysis of the inoculation of the strains isolated from the urine of urological patients showed that all the studied compounds showed a pronounced ability to suppress the growth of microorganisms (Table 4).

Фармакология, клиническая фармакология Pharmacology, clinical Pharmacology
A high activity was shown by PYaTs4 and PYaTd8.The MIC 50 of these compounds corresponded to 0.5 μg / ml, and at the concentration of 128 μg / ml they acted bactericidally.The PYaTs6 PYaTs7and PYaTs7 compounds have an MIC 50 in the range of 1-2 μg / ml, and the incubation with these compounds at the concentration of 128 μg / ml suppresses the growth of strains by 90-95%.MIC 50 of substances PYaTd9 and PYaTd15 corresponds to 1 μg / ml, the high concentration inhibits population growth by 85-95% (Table 4).
The average statistical indices of the activity of the compounds are given in Table 5.The results show that the most active compound from the group containing the "s" fragment is PYaTs4.The MIC 50 of this compound corresponds to 0.8 μg / ml, with half of the strains studied being sensitive to the concentration of 1.0 μg / ml.
The median МIC 50 of the PYaTs6 compound is slightly higher than the median PYaTs4, the average indicators of МIC 50 are more than twice as high, but these differences are not statistically significant either.The bacteriostatic action of PYaTs7 with respect to the strains used was much weaker than in the first two compounds: МIC 50 statistically significantly exceeded the MIC 50 of PYaTs4 and PYaTs6 compounds.
CONCLUSION.Thus, all the studied compounds have the ability to suppress the growth of E. coli being both collectively and freshly isolated from patients with various pathologies.The substances under the laboratory ciphers of PYaTd8 and PYaTs4 are the most active with respect to the strains.At the same time, the MIC 50 values of all compounds do not exceed 10-20 μg/ml, which makes them promising for further investigation of their in vivo activity.

Table 2 -Activity of compounds with respect to collection strains of Escherichia coli (MIC, μg / ml)
RNote: R -resistant; I -moderately resistant; S -sensitive

Table 4 -Activity of compounds with respect to Escherichia coli strains (μg / ml) isolated from urine of patients
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